zlatić et al. 2025, biologica nyssana 16(1) 16 (1) june 2025 list of reviewers reviewer name affiliation abhay mishra senior researcher, naresuan university, thailand adisa parić department for biology, faculty of science and mathematics, university of sarajevo, bosnia and herzegovina ahmad fauzi faculty of teacher training and education, universitas muhammadiyah malang, indonesia ahmed mohran department of zoology, faculty of science, alexandria university, egypt aleksandar veselinović department of chemistry, faculty of medicine, university of niš, serbia aleksandra konjević faculty of agriculture, university of novi sad, serbia aleksandra petrović department of biology and ecology, faculty of sciences and mathematics, university of niš, serbia aleksandra zarubica department of chemistry, faculty of sciences and mathematics, university of niš, serbia alex laini department of life sciences and systems biology, university of torino, italy alexandre levi-mourao department of vegetal production and forestry science, university of lleida, spain ana džamić faculty of biology, university of belgrade, serbia ayşen çolak faculty of agriculture, uşak university, turkey bekir şan department of horticultural science, faculty of agriculture, suleyman demirel university, turkey bruno massa department of agricultural, food and forest sciences, university of palermo, italy budimir ilić department of chemistry, faculty of medicine, university of niš, serbia chavdar gussev institute of biodiversity and ecosystem research, bulgarian academy of sciences, bulgaria danijela nikolić faculty of sciences and mathematics, university of niš, serbia darja barlič-maganja faculty of health sciences, university of primorska, slovenia dawid marczak faculty of engineering and management, university of ecology and management in warsaw, poland derek engelbrecht university of limpopo, south africa dragana jugović faculty of physical chemistry, university of belgrade, serbia dragana stojičić faculty of sciences and mathematics, university of niš, serbia dubravka čerba department of assessment and aquatic ecosystems research, water research institute, slovakia emre sevindik department of agricultural biotechnology, faculty of agriculture, adnan menderes university, turkey eruthaya abisha st. mary’s college (autonomous), india evrim elçin department of agricultural biotechnology, faculty of agriculture, adnan menderes university, turkey goran anačkov department of biology and ecology, faculty of sciences and mathematics, university of novi sad, serbia gustavo costa laboratory of pharmacognosy, faculty of pharmacy, university of coimbra, portugal hanen marzouki isbm, university of monastir, tunisia biologica nyssana ● 16 (1) june 2025 list of reviewers harun özer department of horticulture, faculty of agriculture, ondokuz mayis university, turkey hassouna gouta olive tree institute, tunisia irena raca department of biology and ecology, faculty of sciences and mathematics, university of niš, serbia izet eminović faculty of science, university of sarajevo, bosnia and herzegovina jasmin mušanović faculty of medicine, university of sarajevo, bosnia and herzegovina jelena mitrović department of food technology, faculty of technology, university of niš, serbia jelena rajković department of biology and ecology, faculty of sciences and mathematics, university of niš, serbia john mendesohn raison, namibia josé blanco salas department of plant biology, ecology and earth sciences, university of extremadura, spain katarina kos department of agronomy, biotechnical faculty, university of ljubljana, slovenia ljiljana stanojević faculty of technology, university of niš, serbia m. mythri ktn college of pharmacy, india magdalena valcheva institute of biodiversity and ecosystem research, bulgarian academy of sciences, bulgaria magdalene eno effiong department of biochemistry, college of science and technology, covenant university, nigeria mahendra rai department of biotechnology, sgb amravati university, india maja vukašinović sekulić faculty of technology and metallurgy, university of belgrade, serbia marija vukelić institute of biology and human genetics, faculty of medicine, university of niš, serbia marina dimitrijević department of biology and ecology, faculty of sciences and mathematics, university of niš, serbia marko mladenović department of chemistry, faculty of sciences and mathematics, university of niš, serbia marko nikolić department of biology and ecology, faculty of sciences and mathematics, university of niš, serbia maxim trushin kazan federal university, russia milan dimitrijević department of chemistry, faculty of sciences and mathematics, university of niš, serbia milica aćimovic institute of field and vegetable crops, serbia milica pejčić department of biology and ecology, faculty of sciences and mathematics, university of niš, serbia miloš popović department of biology and ecology, faculty of sciences and mathematics, university of niš, serbia muhamed katica department of pathological physiology, veterinary faculty, university of sarajevo, bosnia and herzegovina nataša joković department of biology and ecology, faculty of sciences and mathematics, university of niš, serbia naveen thakur department of physics, career point university, india nesreen al-huwaidi department of horticulture and its technology, faculty of agriculture, foods and environment, university of sanaa, yemen nickolas kavallieratos department of crop science, agricultural university of athens, greece list of reviewers niko radulović department of chemistry, faculty of sciences and mathematics, university of niš, serbia nikola krstić faculty of medicine, university of niš, serbia nikoleta kallajxhiu department of biology, university of elbasan, albania p.v. deviswapna ktn college of pharmacy, india pauline jenifer st. mary’s college (autonomous), india peggy rigou department of agroecology and soil science, inra-montpellier supagro, university of montpellier, france perica vasiljević department of biology and ecology, faculty of sciences and mathematics, university of niš, serbia ramkumar r.p. ktn college of pharmacy, india ronit sionov institute of biomedical and oral research, israel tatjana kundakovićvasović department of pharmacognosy, faculty of pharmacy, university of belgrade, serbia tatjana mihajilov-krstev department of biology and ecology, faculty of sciences and mathematics, university of niš, serbia tlili nizar higher institute of sciences and technologies of environment, university of carthage, tunisia tuba dilmaçünal department of horticulture, faculty of agriculture, isparta university of applied sciences, turkey tuğba iduğ department of pharmacognosy, school of pharmacy, istanbul medipol university, turkey tülay aşkin çelik department of biology, faculty of arts and sciences, adnan menderes university, turkey tzenko vatchev institute of soil science, agrotechnologies and plant protection “nikola poushkarov”, bulgaria vagner de alencar de toledo department of animal science, center for agricultural sciences, universidade estadual de maringá, brazil vanja stojanović faculty of sciences and mathematics, university of niš, serbia vidak raičević faculty of medicine, university of novi sad, serbia višnja madić faculty of sciences and mathematics, university of niš, serbia vladimir cvetković faculty of sciences and mathematics, university of niš, serbia vladimir pešić department of biology, university of montenegro, montenegro vladimir žikić department of biology and ecology, faculty of sciences and mathematics, university of niš, serbia wenchao yang college of animal science (college of bee science), fujian agriculture and forestry university, china yunus korkom department of plant protection, faculty of agriculture, aydın adnan menderes university, turkey zoltán barina wwf hungary, hungary zorica stojanović-radić department of biology and ecology, faculty of sciences and mathematics, university of niš, serbia biologica nyssana ● 16 (1) june 2025 microsoft word 0103_jenackovic_et_al biologica nyssana 1 (1-2) december 2010: 23-26 jenačković, d. et al. macrophytic flora and vegetation… 23 original article ! macrophytic flora and vegetation of the rivers svrljiški and beli timok (eastern serbia) dragana jenačković*, danijela dimitrijević, vladimir ranđelović university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia * e-mail: kolibri@medianis.net abstract: jenačković, d., dimitrijević, d., ranđelović, v.: macrophytic flora and vegetation of the rivers svrljiški and beli timok (eastern serbia). biologica nyssana, 1 (1-2), december 2010: 23-26. floristic and phytocoenological investigations of macrophytic vegetation of the rivers svrljiški and beli timok in eastern serbia were performed. analysis of the collected plants showed that the hydrophilous flora contains 26 species from 17 families and 21 genuses. phytocoenological analysis showed 5 different associations from 3 alliances, 3 orders and 3 classis. aquatic vegetation is represented by the associations myriophyllo-potametum and potametum nodosi, moor vegetation by associations scirpetum lacustris and sparganietum erecti, while nitrophilous vegetation is represented by association polygono-bidentetum tripartitae. these associations have formed three clear vegetation belts: submerged, floating and emerged vegetation. key words: aquatic and moor vegetation, macrophytic flora introduction ! the rivers svrljiški and beli timok are located near the knjaževac town, between the mountains stara planina (balkan mountain), tresibaba and tupižnica in eastern serbia (fig. 1). regarding phytogeography, they belong to the eastern moesian province of the balkan floristical subregion. there are only few informations about the flora and vegetation of these rivers and they refer to the gorge of svrljig timok river (b o g o s a v l j e v i ć et al., 2007). the surrounding mountains are floristicaly and phytocoenologicaly relatively well explored , especially mt. stara planina (m i š i ć et al., 1979). macrophytic flora and vegetation of these rivers has not been investigated so far. during the period 2008-2010 we investigated the aquatic, moor and nitrophilous flora and vegetation from the riparian zone of these rivers. material and methods ! the plants collected in the field were determined using t u t i n et al., eds. (1968-1980) and j o s i f o v i ć , ed. (1970-1976) and nomenclature was updated following global plant checklist (iopi, 2005) and serb-check list (n i k e t i ć , t o m o v i ć , 2009). collected material is deposited in the herbarium of the faculty of sciences and mathematics, university of niš (hmn). life forms were determined according to s t e v a n o v i ć (1992). phytocoenological investigation was carried out following the method of b r a u n -b l a n q u e t (1965). abundance and cover of taxa was estimated using the nine-grade scale according by v a n d e r m a a r e l (1979) the nomenclature of the syntaxa is in accordance with the code of phytosociological nomenclature (w e b e r et al., 2000). 10th sfses • 17-20 june 2010, vlasina lake1 (1-2) • december 2010: 23-26 biologica nyssana 1 (1-2) december 2010: 23-26 jenačković, d. et al. macrophytic flora and vegetation… 24 fig. 1. map of investigated area a classification dendrogram has been constructed from the original table of 24 relevées (tab. 2) as a total of 26 species and infraspecies taxa (fig. 2) using the upgma algorithm and the coefficient of chord distance by means of the software flora (k a r a d ž i ć , m a r i n k o v i ć , 2009). results and discussion flora of aquatic and moor habitats along the rivers svrljiški and beli timok contains 26 species of spermatic plants, which are divided into two classes: magnoliopsida (17 species) and liliopsida (9) (tab. 1). true aquatic plants are presented with a very small number of species. species potamogeton crispus and myriophyllum spicatum belong to the group of submersed hidrophytes, while potamogeton nodosus and lemna minor belong to the group of floating hidrophytes. sparganium erectum, scirpus lacustris ssp. tabernaemontani and alisma plantago-aquatica belong to the group of emergent hydrophytes. most other plants are characteristic for wetlands, with the exception of polygonum aviculare, which is usually found in ruderal habitats. table 1. review of macrophytic flora and life forms of plants species family life form magnoliopsida berula erecta (hudson) coville apiaceae h scap bidens tripartita l. compositae t scap epilobium hirsutum l. oenotheraceae h scap lycopus europaeus l. labiatae h scap lythrum salicaria l. lythraceae h scap/emer hydg rhiz mentha aquatica l. labiatae h scap mentha longifolia (l.) hudson labiatae h scap myriophyllum spicatum l. halorrhagidaceae rad sbm hydt polygonum amphibium l. polygonaceae g rhiz-scap nat hydg polygonum aviculare l. polygonaceae t rept polygonum hydropiper l. polygonaceae t scap polygonum mite schrank polygonaceae t scap potentilla reptans l. polygonaceae h rept rumex hydrolapaum huds. rosaceae h scap scrophularia alata polygonaceae h scap symphytum officinale l. scrophulariaceae h scap veronica beccabunga l. boraginaceae h scap liliopsida alisma plantago-aquatica l. alismataceae emer hydt echinochloa crus-galli (l.) beauv. gramineae t caesp juncus effusus l. juncaceae g rhiz caesp lemna minor l. lemnaceae er nat hyd t poa palustris l. gramineae h caesp potamogeton crispus l. potamogetonaceae rad sbm hyd t potamogeton nodosus poiret potamogetonaceae rad nat hydt scirpus lacstris l. ssp. tabenaemontani (c. c. gmel.) syme cyperaceae g rhiz caesp sparganium erectum l. sparganiaceae emer hydg rhiz biologica nyssana 1 (1-2) december 2010: 23-26 jenačković, d. et al. macrophytic flora and vegetation… 25 table 2. phytocoenological table of associations (characteristics species marked with bold characters) fig. 2. dendrogram of the macrophytic vegetation reléeves biologica nyssana 1 (1-2) december 2010: 23-26 jenačković, d. et al. macrophytic flora and vegetation… 26 macrophytic vegetation is differentiated into three vegetation classes (see syntaxonomical scheme). class potametea includes aquatic vegetation, which is ecologically divided into submerged (ass. myriophyllo-potametum) and floating (ass. potametum nodosi) vegetation. submerse vegetation covers most of the bottom of rivers, while the floating vegetation develops near the coast, in places where water flow is slower. the fragmentary developed emersal vegetation is found along the coast and is presented by associations scirpetum lacustris and sparganietum erecti. in the areas which are periodically flooded with water the nitrophilic vegetation from class polygono-bidentetea is developed. this vegetation type is characterized by the presence of numerous representatives of the genus polygonum. syntaxonomical scheme potametea r. tx. et preising 1942 potametalia w.koch 1926 potamion eurosibiricum w.koch 1928 myriophyllo-potametum soó 1934 subass. potametosum crispi slavnić 1956 potametum nodosi soó (1928) 1960, segal 1964 phragmito-magnocaricetea klika in klika et novak 1941 pragmitetalia communis w.koch 1926 phragmition communis koch 1926 scirpetum lacustris (allorge 1922) chov. 1924 sparganietum erecti roll 1938 bidentetea tripartiti tx. lohm. et prsg. 1950 bidentetalia tripartiti br-bl. et tx.1943 bidentition tripartiti nordh. 1940 polygono-bidentetum tripartitae (w.koch 1926) lohm 1950 conclusion aquatic vegetation of the rivers svrljiški and beli timok is relatively poor in floristical sense. there are 26 plant species involved in the construction of five associations, two of which are typical aquatic vegetation, two belong to the vegetation of emergent plants, and one is nitrophilic vegetation in periodically flooded areas. nitrophilic vegetation developed to periodically flooded areas clearly indicates the increased content of nitrogen compounds in the water. acknowledgements. this study was supported by the project of ministry of science and technological development of republic of serbia (project no. 143015 – diversity of flora and vegetation in the central balkan peninsula – ecology, chorology and conservation). references bogosavljević, s., zlatković, b., ranđelović, v., 2007: flora klisure svrljiškog timoka, proceeding of 9th symposium on the flora of southeastern serbia and surrounding regions, niš, 41-54. braun-blanquet, j., 1965: plant sociology. hafner publishing company, new york and london. 439 p. josifović, m. (ed.) (1970-1976): flora sr srbije, iviii. sanu. beograd. karadžić b., marinković s. (2009) kvantitativna ekologija. institut za biološka istraživanja "siniša stanković". beograd. 489 str. mišić, v., jovanović-dunjić, r., popović, m., borisavljević, lj., antić, m., dinić, a., danon, j., blaženčić, ž., 1978: biljne zajednice i staništa stare planine. sanu. beograd. 389 s. ranđelović, v., zlatković, b., matejić, j., 2007: flora klisure svrljiškog timoka, proceeding of 9th symposium on the flora of southeastern serbia and surrounding regions, niš, 41-54. stevanović, v., 1992: klasifikacija životnih formi flore srbije. in sarić, m. (ed.): flora srbije, i. (drugo izdanje). sanu. beograd. 37-49. tutin, t.g., v.h. heywood, n.a. burges, d.m. moore, d.h. valentine, s.m. walters & d.a. webb, (eds.) (1964-1980): flora europaea, i-v. cambridge university press. london. van der marel, e. 1979: transformation of coverabundance values in phytosociology and its effect on community similarity. vegetatio, 39: 97-114. weber, h.e., moravec, j., theurillat, j.-p., 2000: international code of phytosociological nomenclature, 3rd edition. j. veg. sci, 11: 739768. microsoft word bn-sc-0201-10 stankovic m biologica nyssana 2 (1) september 2011: 77-81 stanković, m. rare, threatened and relict species in flora… 77 short communication ! rare, threatened and relict species in flora of snr zasavica stanković mihajlo*1 1special nature reserve „zasavica“, s. mitrovica, serbia * e-mail: zasavica@zasavica.org.rs abstract: stanković, m.: rare, threatened and relict species in flora of snr zasavica. biologica nyssana, 2 (1), september 2011: 77-81. in group of biodiversity important plant species there are 23 taxa. 20 taxa are mentioned in „preliminary red list of flora of serbia and montenegro with iucn 2001 conservation statuses“ in following categories: two as critically endangered (aldrovanda vesiculosa l. and hottonia palustris l.), four as endangered (hippuris vulgaris l., lindernia palustris hartm., ranunculus lingua l. and urtica kioviensis rogow.), five as vulnerable (achillea aspleniifolia vent., dryopteris carthusiana (vill.) h. p. fuchs, leucojum aestivum l. subsp. aestivum, stratiotes aloides l. and thelypteris palustris (schott) subsp.palustris, while 9 are with indefinite categories (cr-vu), due to data deficient (dd). special nature reserve „zasavica“ is the only habitat in serbia for aldrovanda vesiculosa l., which was until 2005. considered as extinct from serbia. key words: distribution, european adder, vipera berus, vlasina plateau introduction ! the first data on the flora and vegetation of north mačva (peripannonian serbia), and therefore the site of today's special nature reserve "zasavica" give josif p a n č i ć (1867) and ernest d o m b r o w s k i (1895). then followed a period of over half a century of sporadic research by small groups and individual researchers. in 1995 an initiative to protect river and a decree was adopted on the previous protection (official gazette rs, 51/95). at the suggestion of the institute for nature protection of serbia government of the 1997th the declared special nature reserve "zasavica (official gazette rs, 19/97). this paper aims to show the total value of the diversity of flora srp "zasavica. zasavica area is included in the ipa region of serbia s t e v a n o v i ć & š i n ž a r s e k u l i ć (2009). material and methods based on our field studies conducted on the territory of the special nature reserve “zasavica” and adjacent areas from 1997 to 2010. as well as on herbarium and literature sources, the distribution data of vascular plant taxa with great conservation value and some rare taxa were collected. voucher specimens were deposited in herbarium of institute for nature protection of vojvodina province, herbarium beou, beo, buns and herbarium of special nature reserve “zasavica” in sremska mitrovica. identification and revision of plant material were carried out according to t u t i n et al. (1968-1980; 1996), g a j i ć & k a r a d ž i ć (1991) and j á v o r k a & c s a p o d y (1975). threatened status of plant taxa is represented according to “preliminary red list of flora of serbia and montenegro with iucn 2001 conservation statuses” s t e v a n o v i ć (2002). the floristic elements are given according to the classification of g a j i ć (1980). 2 (1) • september 2011: 77-81 biologica nyssana 2 (1) september 2011: 77-81 stanković, m. rare, threatened and relict species in flora… 78 results and discussion in group of biodiversity important plant species there are 23 taxa. according to „ proclamation & regulation act of strictly protected and protected wild plant, animal and fungi species“ (official gazette rs, 36/09) 11 species are ranked as strictly protected and 46 as protected. also, 20 taxa are mentioned in „preliminary red list of flora of serbia and montenegro with iucn 2001 conservation statuses“ s t e v a n o v i ć (2002) in following categories: two as critically endangered (cr): aldrovanda vesiculosa l., hottonia palustris l.), four as endangered (en): hippuris vulgaris l., lindernia palustris hartm., ranunculus lingua l., urtica kioviensis rogow., five as vulnerable (vu): achillea asplenifolia vent., dryopteris carthusiana (vill.) h. p. fuchs, leucojum aestivum l., stratiotes aloides l. and thelypteris palustris (schott) subsp.palustris, while 9 are with indefinite categories (cr-vu), due to data deficient (dd). special nature reserve „zasavica“ is the only habitat in serbia for aldrovanda vesiculosa l., which was until 2005. considered as extinct from serbia (j a n k o v i ć & s t e v a n o v i ć 1999: 100; s t a n k o v i ć 2007: 35-37). on appendix i of convention on the conservation of european wildlife and natural habitats (strictly protected flora species) as well as on ipa criterion a, threatened species list are 3 species (aldrovanda vesiculosa l., lindernia palustris hartm., salvinia natans (l.) all.). one species (galanthus nivalis l.subsp.nivalis) is protected by cites convention (convention on international trade in endangered species of wild fauna and flora, аppendix 2). according turrill (1929) followt tertiary relicts of balkan peniusula are recorded: stratiotes aloides l., trapa natans agg., butomus umbellatus l., erythronium dens-canis l. subsp.dens-canis, humulus lupulus l., hydrocharis morsus-ranae l., isopyrum thalictroides l., loranthus europaeus jacq., tamus communis l.subsp.communis, viscum album l.subsp.album, aldrovanda vesiculosa l. some species abserved in zasavica reserve inchude tertiary relicts of southern europe wetlands as indicated by pollen analysis of peatbogs d i k l i ć (1984) include nymphaea alba l., nuphar lutea (l.) sibthorp. & sm. subsp.lutaea, salvinia natans (l.)allioni i nymphoides peltata (s.g.gmelin) o. kuntze. or hippuris vulgaris is the only representative of genus hippuris oligotips cirkumholoarctic in europe, which includes two species in america and asia. the sites in serbia are on the southern border area of its kind in europe. v u č k o v i ć & p a n j k o v i ć (1999), while hottonia palustris belongs oligotips relict genus hottonia that includes more h. inflata elliot. b u t o r a c (1999). in the area of the reserve hippuris vulgaris is in the process of withdrawal and hottonia palustris and ranunculus lingua show a tenddency to spread. by 2005. the largest population among the zasavica relic species had stratiotes aloides, which was covered with up to 90% of watercourses, and now appears with emerse belt, so that the steady increase in water level former caps (congestion) rivers have disappeared in many parts of zasavica item. floating reed islands inhabited by urtica kioviensis rogow. and schoenoplectus triqueter (l.) palla for which the peri serbian southern border area b r a n k o v i ć et al. (1996), while achillea asplenifolia vent. pannonian subendemic taxa found on the marshy meadows and are located on the southern border of its area. s o ό (1970). present u. kioviensis has riven range in eastern and central europe with the southern limit of its range in the pannonian plain and the possibility of its occurrence in neoendem wetlands pannonian plain b r a n k o v i ć et al. (1996). the patient was only present in vojvodina part. this type tends to spread within the reserve. the period of glaciation with its interglacial had significantly poorer flora in relation to the warm tertiary. by e n g l e r (1905) after the first glaciation there was a steppe interglacial stage, where in central europe rather than form a steppe tundra vegetation and some of those plants in the period represented postglacial endemic steppe periods. during the upcoming second glaciation in central and southern part of europe remained in part as glacial and partly as interglacial species ceratophyllum demersum l.subsp. demersum riparian forests are the habitat of relict species thelypteris palustris j a n k o v i ć et al. (1997). some species such as utricularia vulgaris, sparganium erectum subsp. erectum, the species myriophyllum spicatum, which were very present in marsh interglacial flora in europe, and still are widespread. four relict species that grow on zasavica are critically endangered species of flora of serbia. conclusion in group of biodiversity important plant species there are 23 taxa. according to „ proclamation & regulation act of strictly protected and protected wild plant, animal and fungi species“, 11 species are ranked as strictly protected and 46 as protected. also, 20 taxa are mentioned in „preliminary red list of flora of biologica nyssana 2 (1) september 2011: 77-81 stanković, m. rare, threatened and relict species in flora… 79 serbia and montenegro with iucn 2001 conservation statuses“ in following categories: two as critically endangered (cr) species, four as endangered (en) species, five as vulnerable (vu) species, while 9 are with indefinite categories (cr-vu), due to data deficient (dd). special nature reserve „zasavica“ is the only habitat in serbia for aldrovanda vesiculosa l., which was until 2005. considered as extinct from serbia. on appendix i of convention on the conservation of european wildlife and natural habitats (strictly protected flora species) as well as on ipa criterion a (a n d e r s o n , 2002, a n d e r s o n et al., 2005), threatened species list are 3 species. one species is protected by cites convention. to understand the importance of international protection zasavica shows and its nominations for the 2005 ipa area as well received as a member of the federation europark 2001 and the proclamation of the ramsar site in 2009. table 1. plant species significant for biodiversity conservation taxa preliminary red list of flora of serbia and montenegro st ri ct ly pr ot ec te d iucn conservation status ip a c ri te ri on b er n c on ve nt io n 1 achillea asplenifolia vent. vu 2 aldrovanda vesiculosa l. cr 3 arum orientale bieb. vu-nt (dd) 4 callitriche palustris l. en-vu (dd) 5 cyperus glomeratus l. nt-lc (dd) 6 dryopteris carthusiana (vill.) h. p. fuchs vu 7 erysimum cheiranthoides l. vu-nt (dd) 8 hesperis sylvestris crantz vu-nt (dd) 9 hippuris vulgaris l. en 10 hottonia palustris l. cr 11 leucojum aestivum l. vu 12 lindernia palustris hartm en 13 lygia passerina (l.) fassano vu-nt (dd) 14 nuphar lutea (l.) sibthorp. & sm. subsp.lutaea 15 nymphaea alba l. 16 ranunculus lingua l. en 17 salvinia natans (l.) all. 18 scirpus triqueter l. cr-vu (dd) 19 stratiotes aloides l. vu 20 thelypteris palustris (schott) subsp. palustris, vu 21 urtica kioviensis rogow. en 22 utricularia australis r. br. en-vu (dd) 23 zannichellia palustris l. vu-lc (dd) biologica nyssana 2 (1) september 2011: 77-81 stanković, m. rare, threatened and relict species in flora… 80 references anderson, s., 2002: identifying important plant areas. london, uk: plantlife international. anderson, s., kušík, t., radford, e. (eds), 2005, important plant areas in central and eastern europe. plantlife international. branković, d., budakov, lj., kovačev, n., mijović d., mikeš, b., pavkov, g., puzović, s., sekulić, n., stojšić, v., habjan-mikeš, v., garovnikov, b., stanković, m., 1996: predlog za zaštitu dobra "zasavica" kao specijalni rezervat prirode. zavod za zaštitu prirode srbije beograd -novi sad. butorac, b., 1999: hottonia palustris l.. in: v. stevanović, ed.: crvena knjiga flore srbije 1, ministarstvo za životnu sredinu r srbije, biološki fakultet univ. u beogradu i zavod za zaštitu prirode r srbije, 296-298. convention on international trade in endangered species of wild fauna and flora, аppendix 2. bonn, 1979. convention on the conservation of european wildlife and natural habitats (strictly protected flora species, appendix 1). bern, 1979. diklić, n., 1984: životne forme biljnih vrsta i biološki aspekti flore sr.srbije. in: sarić, m. (ed.), 1984: vegetacija sr srbije, i. sanu, odeljenje prirodno-matematičkih nauka, beograd, 291-317. dombrovski, e., 1895: osnovi ornitologije sjeverozapadne srbije. glasnik zemaljskog muzeja bih, sarajevo. engler, r.a., 1905: grundzüge der entwicklung der flora europas seit der tertiärzeit. bot. jahrb. b., 36, leipzig gajić, m., 1980: pregled vrsta flore sr srbije sa biljnogeografskim oznakama. glasnik šumarskog fakulteta, serie a “šumarstvo”, 54: 111-141. belgrade. gajić, m., karadžić, d., 1991: flora ravnog srema sa posebnim osvrtom na obedsku baru, šg, sremska mitrovica. janković, b., mišić v., dinić, a., vukićević, e., 1997: vegetacija srbije, ii tom, šumske zajednice 1. sanu, odeljenje prirodnomatematičkih nauka, beograd. janković, m., stevanović, v., 1999: aldrovanda vesiculosa. in: v. stevanović, ed.: crvena knjiga flore srbije 1, ministarstvo za životnu sredinu r srbije, biološki fakultet univ. u beogradu i zavod za zaštitu prirode r srbije, 299-301 jávorka, s., csapody, v., 1975: közép-európa délkeleti részének flórája képekben iconographia florae partis austro-orientalis europae centralis]. akadémiai kiadó. budapest pančić, j., 1869: građa za faunu kneževine srbije. glasnik srpskog učenog društva, beograd. službeni glasnik republike srbija br 51/95: rešenje prethodnog zaštite prirodnog dobra zasavica. službeni glasnik republike srbija br.36/09 zakon o zaštiti prirode, anex i i ii strogo zaštićene i zaštićenih divljih biljaka, životinja i gljiva. službeni glasnik rs,19/97 uredba o zaštiti zasavice kao specijalni rezervat prirode. soό,r., 1964-1970: a magyar flόra ès vegetáció rendszertani-növènyföldrajzi kèzikönyve i-iv, akadèmiai kiadó budapest. stanković m.2007. rezultati istraživanja globalno ugrožene vrste aldrovanda vesiculosa l. (fam.droseraceae) tokom 2006. godine u specijalnom rezervatu prirode zasavica, zbornik naučno-stručnog skupa „zasavica 2007“sa međunarodnim učešćem, sremska mitrovica stevanović v. (ed.) 2002. preliminarna crvena lista flore srbije i crne gore prema kriterijumima iucn-а iz 2001 godine. belgrade. (manuscr.) stevanović v., šinžar-sekulić, 2009.serbia in: conserving importand plant areas: investingin the gren gold of southeast europaea (eds.radfordea and obé b) plant life international,sa lisaury, p.63 turrill w.b.1929, the plant-life of the balkan peninsula,oxford,leipzig tutin t. g., heywood v h., burges n.a.. moore d. m.,valentine d. h,walters s. m., and webb d. a. (eds.), 1968-1980: flora europaea ii-v. cambridge university press. tutin, t.g., burges, n.a., chater, a.o., edmondson, j.r., heywood, v.h.., moore, d. m., valentine, d. h., walters, s. m. and webb, d.a. (eds.) 1993: flora europaea i, 2nd ed. cambridge university press. vučković, m. & panjković, b., 1999: hippuris vulgaris l. in: v. stevanović, ed.: crvena knjiga flore srbije 1, ministarstvo za životnu sredinu r srbije, biološki fakultet univ. u beogradu i zavod za zaštitu prirode r srbije, 299-301. microsoft word 0302_karalija_et_al biologica nyssana 1 (1) december 2010: 111-121 stojanović-radić, z. et al. antimicrobial activity and cytotoxicity of… 57 original article ! somatic embryogenesis and in vitro plantlet regeneration of lilium martagon l. var. cattaniae vis. erna karalija, sanja trbojević, adisa parić* faculty of sciences sarajevo, university of sarajevo, zmaja od bosne 33 – 35, 71000 sarajevo, bosnia and herzegovina * e-mail: adisacausevic@hotmail.com abstract: karalija, e., trbojević, s., parić, a.: somatic embryogenesis and in vitro plantlet regeneration of lilium martagon l. var. cattaniae vis.. biologica nyssana, 1 (1-2), december 2010: 57-60. in this study organogenic capacity of two different explants type (leaves and whole bulbs) of lilium martagon l. var. cattaniae vis. was examined. for induction of in vitro somatic embryogenesis and adventitive regeneration different concentrations of 2,4-dichlorophenoxyacetic acid and 6-benzilaminopurine (from 0,25 mg/l to 8,00 mg/l) added to ms basal medium were used. our results indicate that concentration of 0,5 mg/l 2,4-dichlorophenoxyacetic acid and 4 mg/l 6benzilaminopurine promoted somatic embryogenesis from leaves of lilium martagon var. cattaniae, while all other concentrations promoted direct shoot regeneration from bulb explants. root formation was induced on ms basal medium with 0,2 mg/l indole butyric acid. these plantlets were acclimatized well in a greenhouse conditions. key words: lilium martagon var. cattaniae, somatic embryogenesis, regeneration introduction ! lilium martagon l. var. cattaniae vis. is a monocotyledonous plant from the liliaceae family. this taxon is a vulnerable endemic taxon of central dinarids distributed in warmer areas of mediterranean and submediterranean. in bosnia and herzegovina this taxon is also categorised as rare and endangered, and as such is listed in the checklist of plant species for the red book of bosnia and herzegovina (š i l i ć , 1996). in recent years, there is an increasing interest in somatic embryogenesis as a method for in vitro propagation of lilies and other plant species. this is probably because somatic embryogenesis produces a large number of individuals and can be used for production of artificial seeds and for the purposes of transformation as well. the method for induction of somatic embryogenesis has been established for many lilies including lilium martagon (k e d r a & b a c h , 2005), but such methods are unknown for lilium martagon var. cattaniae. therefore, the aim of this study was to investigate the regeneration potential and possibility of somatic embryogenesis induction in this taxon. material and methods ! plant material and in vitro conditions leaves and bulbs of in vitro grown lilium martagon var. cattaniae (hindija et al., 2007) were used for induction of somatic embryogenesis and plantlet regeneration. all media were adjusted to ph 5.7-5.8 using 1 n koh/hcl and 0.8% agar was used for solidification of media. media were autoclaved at 120°c for 20 min. all cultures were kept at 24°c (±2) and relative humidity 70%. during the first 15 days of the treatment the cultures were kept in the dark while for the remainder of the experiment cultures were kept in conditions of 16 hour photoperiod provided by cool, white, fluorescent tubes (3000 lux). 10th sfses • 17-20 june 2010, vlasina lake1 (1-2) • december 2010: 57-60 biologica nyssana 1 (1-2) december 2010: 57-60 karalija, e. et al. somatic embryogenesis and in vitro plantlet … 58 in vitro treatments and acclimatisation of plantlets for induction of somatic embryogenesis and plantlet regeneration, bulbs and leaves were cultivated on ms (m u r a s h i g e & s k o o g , 1962) medium containing different concentrations of 2,4-d and bap (tab. 1), including control, with no plant growth regulators (pgr) added. embryogenic nature of cultures was confirmed by visual identification. after 30 days of pgr treatment all cultures were then transferred to pgr free ms basal medium. regenerated shoots from bulb explants were multiplied on the ms medium containing 0,5 mg/l bap, 0,2 mg/l iba and 0,1 mg/l ga3, and they were subsequently cultivated on ms medium containing 0,2 mg/l iba for induction of rhizogenesis. acclimatisation was conducted in pots containing commercial soil for plant cultivation, under greenhouse conditions, with the temperature of 25°c and relative humidity of 70%. three replicates were taken for each treatment. data from all of the replicates was statistically analyzed using statistics program spss version 7. results and disscusion induction of somatic embryogenesis out of all of the tested concentrations of 2,4d and bap only 0,5 mg/l 2,4-d and 4 mg/l bap mg/l was successful in inducting somatic embryogenesis from leaf explants. the primary sign of induction of somatic embryogenesis was swelling of explants tissue (g e o r g e et al., 2008), which in our experiment was evident on the basal parts of leaf explants (fig. 1a), 15 days after inoculation. globular embryos appeared directly from the surface of the explants (fig. 1b). it seems that this combination provides suitable conditions for progression of totipotent somatic cells into somatic embryos. all other applied concentrations showed a reverse effect on embryogenesis. embryo development reached cotyledon state (fig. 1b) a month after inoculation, and 20 embryos per explant were formed. further development and germination of embryos was accomplished after 75 days of culture, with subsequent transfer of globular embryos on pgr free medium (fig. 1c). similar results were also described for somatic embryogenesis in other lilies as well as other species, where somatic embryos developed into plantlets when plated onto ms basal medium without pgrs (t a b i r a , 1994; t r i b u l a t o et al., 1997; h o et al., 2006; g e o r g e et al., 2008). plantlets were well acclimatised in greenhouse conditions. no induction of somatic embryogenesis was noticed from bulb explants. induction of plantlet regeneration from bulb explants regeneration was successful for all tested combinations of concentrations of 2,4-d and bap but showed different rate of regeneration (tab. 1). rate of regeneration was ranging from 50 to 100%, depending on pgrs applied. bulblets regeneration varied from 7, on medium containing 1 mg/l 2,4-d + 1 mg/l bap, up to 16 bulblets per explant, on medium containing 2 mg/l 2,4-d + 1 mg/l bap (tab. 1). however, the differences were not significant. regeneration of leaves also varied, from 2 to 20 leaves per explant (tab. 1). the highest number of regenerated leaves was on the ms medium containing 0,25 mg/l 2,4-d and 8 mg/l bap with 20 regenerated leaves on average (fig. 1d). statistically significant differences on p<0,05 level (lsd) were noticed for treatment containing 2 mg/l 2,4-d and 1 mg/l bap in comparison with results obtained on ms medium containing 0,25 mg/l 2,4-d and 8 mg/l bap and in comparison with control treatment. bulbs are the most commonly used explants in lily cultures as they show the highest organogenic potential (t a k a y a m a & m i s a w a , 1979). numerous authors studied the importance of interaction between auxins and cytokinins on formation of bulbs and shoots in vitro with higher concentrations of cytokinins promoting a higher regeneration of bulblets (t a k a y a m a & m i s a w a , 1979, 1982; n i i m i , 1995). results of our research also show that with the rise in the concentration of cytokinins there is an increase in the regeneration of leaves with regeneration rate peaking on the medium with the highest concentration of bap (8 mg/ml), but for best regeneration of bulblets auxin/cytokinine ratio was in favour of auxine (treatment with 2 mg/l 2,4-d + 1 mg/l bap). in our research 2,4 d induced callus formation in only one case, specifically on the medium containing 4 mg/l bap and 0,5 mg/l 2,4 d, which produced leaves and bulbs after being transferred onto a pgr free medium. o k a z a k i & k o i z u m i (1995) reported that for shoot production from callus it is necessary to cultivate callus on pgr free medium for a period of six months or by lowering the sucrose concentration from 3% to 1%. m o r i et al (2005) later showed that for a large number of lily species shoot induction biologica nyssana 1 (1-2) december 2010: 57-60 karalija, e. et al. somatic embryogenesis and in vitro plantlet … 59 table 1. effect of 2,4-d and bap on leaf and bulblet regeneration from bulb explants of l. martagon var. cattaniae pgr treatment: 2,4 d + bap (mg/l ) rate of regeneration (%) no. of regenerated leaves no. of regenerated bulblets 0,25 + 8,00 90 20a 14 0,50 + 4,00 70 12 13 1,00 + 2,00 80 11 12 2,00 + 1,00 100 2a,b 16 1,00 + 1,00 50 5 7 0,00 + 0,00 50 18b 15 values are mean of three replicates per each treatment. same letter within the column indicates a significant differences at p<0,05 by lsd test.values with no indicators (a or b) show no significant difference. a b c d figure 1. induction of somatic embryogenesis on ms basal medium containing 0,5 mg/l 2,4-d and 4 mg/l bap; a) globular embryos; b) cotyledon phase; c) germinated embryo d) regeneration from bulb explant on ms basal medium containing 0,25 mg/l 2,4-d and 8 mg/l bap. could be successful with only two months of cultivation on a pgr free medium. in our research differentiation of shoots was successful after only a month of callus cultivation on pgr free medium. regenerated shoots were cultivated on multiplication medium containing 0,5 mg/l bap, 0,2 mg/l iba and 0,1 mg/l ga3. multiplication was successful producing up to 30 shoots per explant. after induction of rhizogenesis on the ms medium containing 0,2 mg/l iba, plants were acclimatised in the greenhouse conditions. conclusion our research showed that induction of somatic embryogenesis could be successfully induced from leaf explants by cultivation on ms medium containing 0,5 mg/l 2,4-d + 4 mg/l bap. regeneration from bulb explants was successful biologica nyssana 1 (1-2) december 2010: 57-60 karalija, e. et al. somatic embryogenesis and in vitro plantlet … 60 with differences in bulblet and leaf regeneration. best treatment for leaf regeneration contained high cytokinins level (8 mg/l), while treatment with best results for bulblet regeneration contained more auxin then cytokinin in ratio 2:1. multiplication was very successful with production of large number of shoots with good acclimatisation rate. references george, e. f., hall, m.a., de geert-jan, k. (eds) 2008: plant propagation by tissue culture 3rd edition volume 1. the background. springerverlag, berlin, heidelberg. 508 p. hindija, j., muratović, e., parić, a. 2007: in vitro regeneration of lilium martagon l. var. cattaniae vis. v symposium of agriculture, veterinary, forestry and biotechnology, vlašić, travnik, bosna i hercegovina, abstracts: 216. ho, c.w., jian, w.t., lai, h.c. 2006: plant regeneration via somatic embryogenesis from suspension cell cultures of lilium x formolongi hort. using a bioreactor system. in vitro cellular & developmental biology -plant, 42 (3): 240– 246. kedra, m., bach, a. 2005: morphogenesis of lilium martagon l. explants in callus culture. acta biologica cracoviensia / botanica, 47 (1): 6573. mori, s., adachi, y., horimoto, s., suzuki, s., nakano, m. 2005: callus formation and plant regeneration in various lilium species and cultivars. in vitro cellular & developmental biology, 41 (6): 783-788. murashige, t., skoog, f. 1962: a revised medium for rapid growth and bioassays with tobacco tissue cultures. physiologia plantarum, 15 (3): 473-497. niimi, y. 1995: in vitro propagation and post-invitro establishment of bulblets of lilium japonicum thunb. journal of japanese society of horticultural sciences, 63 (4): 843-852. okazaki, k., koizumi, m. 1995: callus formation and regeneration of some species of lilium. acta horticulturae, 392: 97–106. šilić, č. 1996: spisak biljnih vrsta (pteridophyta i spermatophyta) za crvenu knjigu bosne i hercegovine. glasnik zemaljskog muzeja bosne i hercegovine, n.s., 31:323-367. tabira, h. 1994: development of micropropagation system of gentiana triflora. xxiv international horticultural congress, august 21–27, kyoto: 186. takayama, s., misawa, m. 1979: differentiation in lilium bulbscales grown in vitro. effect of various cultural conditions. physiologia plantarum, 46: 184-190. takayama, s., misawa, m. 1982: regulation of organ formation by cytokinin and auxin in lilium bulbscales grown in vitro. plant and cell physiology, 23 (1): 67-74. tribulato, a., remotti, p.c., loffler, h.j.m., van tuyl, j.m. 1997: somatic embryogenesis and plant regeneration in lilium longiflorum thunb. plant cell reports, 17 (2): 113–118. silene noctiflora l., present in the flora of kosovo and metohija (serbia) biologica nyssana 7 (2)  december 2016: 83-86 prodanović, d. et al.  silene noctiflora l., present in the flora of… 83 original article received: 15 october 2016 revised: 21 november 2016 accepted: 29 november 2016 silene noctiflora l., present in the flora of kosovo and metohija (serbia) danijela prodanović1, miloš stanojević2 , zoran krivošej2 1university of priština, faculty of agriculture lešak, lešak, serbia. 2university of priština, faculty of natural science, kosovska mitrovica, serbia *e-mail: danijela.prodanovic@gmail.com abstract: prodanović, d., stanojević, m., krivošej, z.: silene noctiflora l., present in the flora of kosovo and metohija (serbia). biologica nyssana, 7 (2), december 2016: 83-86. the genus silene (family caryophyllaceae) comprises more than 700 species and it is one of the larger genera of the world’s flora. studying the flora of the plain part of kosovo and metohija, in the village of gračanica, on the eastern rim of the kosovo basin (approximately 10 km to the south of priština), near the gračanka stream, we identified a weed and ruderal species, silene noctiflora. the part of the stream bank is urbanised and turned into a quay. the quay is regularly maintained implying that the banks are regularly weeded and the riverbeds are cleaned as the typha latifolia species rapidly grows; it may be the reason why this species was not immediately detected and appropriately identified. except for the afore mentioned locality, the species was also reported on the neglected arable fields and tilths, around the unkempt orchards, along the new tarmac road between gračanica and laplje selo, on the locality called labura. silene noctiflora is not mentioned for the region of kosovo and metohija in the new and revised edition of the book flora of serbia 2. key words: silene noctiflora, kosovo and metohija, serbia, new chorological data apstrakt: prodanović, d., stanojević, m., krivošej, z.: silene noctiflora l., prisutna u flori kosova i metohije (srbija). biologica nyssana, 7 (2), decembar 2016: 83-86. rod silene (familija caryophyllaceae) obuhvata više od 700 vrsta i jedan je od najvećih rodova u svetskoj flori. proučavajući floru nizijskog (ravničarskog) dela kosova i metohije, u selu gračanica, na istočnom obodu kosovske kotline (oko 10 km južno od prištine), pored rečice gračanke, konstatovali smo korovsko-ruderalnu vrstu, silene noctiflora. deo obale oko rečice urbanizovan je i pretvoren u kej, koji se redovno održava, što podrazumeva i stalno košenje korova na obalama ili čišćenje korita kada se prenamnoži typha latifolia; možda je to razlog zašto ova vrsta nije ranije uočena i pravilno determinisana. vrsta je sem na navedenom lokalitetu konstatovana i na zapuštenim oranicama i njivama, oko neodržavanih voćnjaka, duž novog asfaltnog puta između gračanice i lapljeg sela, na lokalitetu koji meštani gračanice zovu labura. u novom, prerađenom i dopunjenom izdanju knjige „flora srbije 2“, silene noctiflora se ne navodi za region kosova i metohije. ključne reči: silene noctiflora, kosovo i metohija, srbija, novi horološki podatak 7 (2) • december 2016: 83-86 12th sfses • 16-19 june 2016, kopaonik mt doi: 10.5281/zenodo.200403 biologica nyssana 7 (2)  december 2016: 83-86 prodanović, d. et al.  silene noctiflora l., present in the flora of… 84 introduction silene l. is one of the larger genera of the world’s flora. in the wide sense adopted here is comprised c. 700 species, about half of which occur in the mediterranean area (g r e u t e r , 1995). there are two major centers of diversity in silene: one in the mediterranean/middle east and one in central asia. a few taxa have been introduced to other continents (m a m a d a l i e v a et al., 2014). on the balkan peninsula as many as 64 species (41%) and 98 taxa (47%) are endemic. the high level of endemism has been recorded in the flora of turkey (48%) and iran (32%) (s t e v a n o v i ć , ed., 2012). genus silene has been placed in the tribe sileneae and the subfamily caryophylloideae. the tribe sileneae has a long history of controversial taxonomy at the genus level. in recent molecular phylogenetic studies by o x e l m a n et al. (2001) the genus silene along with lychis are distinct from viscaria, ixoca (=heliosperma), and atocion which form a well supported monophyletic group. the genus silene clusters in two major clades of approximately equal size, which are tentatively classified as silene subgenus silene and silene subgenus behen (moench) bunge (m a m a d a l i e v a et al., 2014). the genus consists mainly of herbaceous plants (annuals, biennials, and perennials), and, more rarely, of small shrubs or sub shrubs. silene also includes a number of cultivated species and widespread weeds. the flowers have free petals, with each petal consisting of a usually visible limb that can be divided or entire, and a claw that is included within the synsepalous calyx (e g g e n s et al., 2007). at the representatives of the caryophyllaceae family, particularly in the genus silene, the appearance of gynodioecy, dioecy and poliploidy are frequent so they present important model for genetic studies. silene noctiflora is gynomonoecious plant, namely individuals produce two types of flowers: hermaphrodite, which often occurs and functionally female flowers, which are less frequent (p e t r o v i ć , 2015). material and methods the results are based on field work in the plain part of central kosovo, during the year 2015, as well in year 2016. besides the field survey, checking of herbarium material and relevant literature sources were used to present an overall distribution for studied taxa in serbia. identification of the collected plant material is made according to flora of serbia 2, (s t e v a n o v i ć , ed., 2012) and flora europea 1 (t u t i n et al., eds., 1993); the nomenclature was adjusted to euro+med plantbase (2006). on the basis of relevant distribution data investigated species is mapped on 10x10 sq km at utm grid system (utm zone 34t) (l a m p i n e n , 2001). results and discussion fam. caryophyllaceae silene noctiflora l. floristic element: bor-submerid; col-plan life form: a mes-meg t scap the genus silene within the flora of serbia includes 44 species. silene noctiflora species appears as a weed or ruderal species in fallow fields, by the roads, on plowed, mostly shady places. its distribution in the flora of serbia is mostly related to lower areas/altitudes of the territory of srem and banat in vojvodina province, western, central, eastern, southeastern and southern parts of serbia, as well in šumadija and pomoravlje. vascular plants adapted to arable habitats are acknowledged to be among the most vulnerable groups in national floras to the land-use change, particularly in western european states. v r b n i č a n i n et al. (2009) consider that insufficient crop management in the mountain area has also led to the appearance of some rare weed species and similar result was obtained for some endangered 'red list' species, such as silene noctiflora. in a short list of rare or threatened european arable plants species silene noctiflora is present in 26 countries, and in 13 countries the species is rare, threatened or recently extinct (s t o r k e y et al., 2012). general distribution: europe, to ireland to the west, to the peninsula coolley to the north, to central apenines and northern greece to the south; small asia and caucasus, iran, south siberia and mountains of central asia. the species has been introduced to the most of continents. distribution in serbia: vojvodina: srem: fruška gora: čortanovci, karlovčić; banat pančevo: vojlovica, pančevački rit; deliblatska peščara; western serbia: mt jelica; mt zlatibor: gmizova ćuprija; šumadija: beograd; kosmaj; pomoravlje: jagodina; northeastern serbia: đerdap: kazan (pecka bara), sip, veliki štrbac; zlot; eastern serbia: niš: gorica, seličevica, niška banja: koritnik, pirot: basara, krupac, sukovo; babušnica; south eastern biologica nyssana 7 (2)  december 2016: 83-86 prodanović, d. et al.  silene noctiflora l., present in the flora of… 85 serbia: vlasotince, kruševica; vlasina; čemernik; southern serbia vranje (s t e v a n o v i ć , ed., 2012). new chorological data in serbia (fig. 1): village gračanicagračanka river banks (near priština town): 420 35' 99,9“ n, 210 11' 66“ e (559 m a.s.l) utm 34 ten12 (leg./det. krivošej, z., prodanović, d., stanojević, m., 29-may-2015; 9-june-2016); locality labura between gračanica and laplje selo village 420 35' 581“ n, 210 10' 075“ e 580 m a.s.l (30may-2015; 9-june-2016). fig. 1. distribution of the species silene noctiflora l. in serbia according to stevanović, ed., 2012 (black squares). new record marked with red circle. according to the insight into the available references of the botanist from kosovo and metohija and the latest edition of the flora of serbia 2 (s t e v a n o v i ć , ed., 2012), silene noctiflora has not been so far recorded on the territory of kosovo and metohija. during the field work in 2015, on the territory of central part of kosovo, this species was found in two relatively close localities, near gračanica village. in the first locality the species was detected on the banks of gračanka stream about 10 kilometers south from city of priština. the part of the stream bank is urbanised and turned into a quay. the quay is regularly maintained implying that the banks are regularly weeded and the riverbeds are cleaned as the typha latifolia species rapidly grows; it may be the reason why this species was not immediately detected and appropriately identified. the floristic structure in the gračanka river locality can be seen from next phytocenological record: typha latifolia l. 4.4., polygonum lapathifolium l. +.1, arenaria serpyllifolia l. 1.1., mentha aquatica l. +, polygonum aviculare l. +, convolvulus arvensis l. +, stellaria media l. (vill). +. floristic deficiency in this locality is due to the fact that the river banks (or riverbed) are paved by stony blocks so that only a few number of species have managed to inhabit them (fig. 2). fig. 2. habitat of species silene noctiflora l., on the locality gračanka stream later the species was also reported on the neglected arable fields and tilths, around the unkempt orchards, along the new tarmac road between gračanica and laplje selo, on the locality called labura. conclusion silene noctiflora l. is rather sporadically present in serbia, and the data of its presence in gračanica village and surrounding are the only available for kosovo and metohija flora and present a new locality for the serbian flora. new reports about the distribution of silene noctiflora l. species have complemented the picture of distribution and will certainly contribute to better understanding the chorology of this relatively rare species in serbia. biologica nyssana 7 (2)  december 2016: 83-86 prodanović, d. et al.  silene noctiflora l., present in the flora of… 86 references chater, a.o., walters, s.m., akeroyd, j.r. 1993: silene l. in: tutin, t.g., burges, n.a., chater, a.o., edmondson, j.r., heywood, v.h., moore, d.m., valentine, d.h., walters, s.m., webb, d.a. (eds.): flora europaea 1. second edition, university press, cambridge. pp. 191-218. eggens, f., popp, m., nepokroeff, m., wagner, w.l., oxelman, b., 2007: the origin and number of introductions of the hawaiian endemic silene species (caryophylaceae). american journal of botany, 94: 210-218. greuter, w., 1995: silene (caryophyllaceae) in greece: a subgeneric and sectional classification. taxon, 44 (4): 543-581. lampinen, r. 2001: universal transverse mercator (utm) and military grid reference system (mgrs). http://www.luomus.fi/english/botany/ afe/map/utm.htm mamadalieva, n., lafont, r., wink, m., 2014: diversity of secondary metabolites in the genus silene l. (caryophyllaceae) structures, distribution and biological properties. diversity, 6: 415-499. niketić, m., stevanović, v., 2012: silene l. in: stevanović, v. (ed.): flora srbije 2: 372-469. srpska akademija nauka i umetnosti. beograd. oxelman, b., lidén, m., rabeler, r.k., popp, m. 2001: a revised generic classification of the tribe sileneae (caryophyllaceae). nordic journal of botany, 20: 743-748. petrović, d. 2015: efekat gustine jedinki na pojavu funkcionalno ženskih i hermafroditnih cvetova kod vrste silene noctiflora l. master rad., prirodno-matematički fakultet, departman za biologiju i ekologiju, univerzitet u nišu. niš (manuscr.) 38 pp. storkey, j., meyer, s., still, k.s., leuschner, c., 2012: the impact of agricultural intensification and land-use change on the european arable flora. proceedings of the royal society b, 279: 14211429. the euro+med plantbase (2006-)the information resource for euro-mediterranean plant diversity. available at: http://ww2. bgbm.org/europlusmed vrbničanin, s., dajić-stevanović, z., jovanovićradovanov, k., uludaǧ, k., 2009: weed vegetation of small grain crops in serbia: environmental and human impacts. turkish journal of agriculture and forestry, 33: 325-337. bogdanović et al. 2022, biologica nyssana 13(2) 13 (2) december 2022: 153-156 doi: 10.5281/zenodo.7437286 antimicrobial activity of evernia prunastri (оakmoss) resinoids original article svetlana bogdanović toplicka academy of applied studies, department of agricultural and food studies, prokuplje, serbia celebogdanovic@gmail.com (corresponding author) ivana zlatković toplicka academy of applied studies, department of agricultural and food studies, prokuplje, serbia dobrila ranđelović toplicka academy of applied studies, department of agricultural and food studies, prokuplje, serbia received: september 14, 2022 revised: november 04, 2022 accepted: november 15, 2022 abstract: infections caused by strains of bacteria that show resistance to a large number of antibiotics represent one of the leading problems today. the aim of this work is to examine new, natural resources with potential antimicrobial effects. the antibacterial activity of lichen resinoids (evernia prunastri) was tested against reference strains staphylococcus aureus atcc 25923, staphylococcus epidermidis atcc 12228, pseudomonas aeruginosa atcc 27853, escherichia coli atcc 25922 and clinical isolates of bacteria s. aureus, s. epidermidis, p. aeruginosa and e. coli, by the disc-diffusion method. each 10 µl of resinoid, of different concentrations, was applied to sterile discs with a diameter of 6 mm. the tested resinoid exhibited the most significant antibacterial activity against pseudomonas aeruginosa atcc 27853, while it was the weakest against the clinical isolate of escherichia coli. the results indicate that the tested oakmoss resinoid shows moderate antimicrobial activity against the tested strains. key words: antimicrobial activity, resinoid, lichen, bacteria apstrakt: antimikrobna aktivnost rezinoida vrste evernia prunastri (hrastova mahovina) infekcije izazvane sojevima bakterija koji pokazuju rezistenciju na veći broj antibiotika predstavljaju jedan od vodećih problema današnjice. cilj ovog rada je ispitivanje novih, prirodnih resursa sa potencijalnim antimikrobnim dejstvom. antibakterijska aktivnost rezinoida hrastove mahovine (evernia prunastri) ispitivana je na referentne sojeve staphylococcus aureus atcc 25923, staphylococcus epidermidis atcc 12228, pseudomonas aeruginosa atcc 27853, escherichia coli atcc 25922 i kliničke izolate bakterija s. aureus, s. epidermidis, p. aeruginosa i e. coli, disk-difuzionom metodom. po 10 µl rezinoida, različitih koncentracija nanošeno je na sterilne diskove prečnika 6 mm. ispitivani rezinoid ispoljio je najznačajniju antibakterijsku aktivnost prema pseudomonas aeruginosa atcc 27853, dok je najslabiji prema kliničkom izolatu escherichia coli. rezultati ukazuju da ispitivani rezinoid hrastove mahovine pokazuje umerenu antimikrobnu aktivnost na ispitivane sojeve. ključne reči: antimikrobna aktivnost, rezinoid, hrastova mahovina, bakterije introduction the introduction of antimicrobial agents in the treatment of infectious diseases saved millions of lives. antimicrobial agents have the ability to eliminate microorganisms by acting on various metabolic and structural targets such as interfering with cell wall synthesis, with nucleic acid synthesis or by other ways of inhibiting (tenover, 2006; bobbarala, 2012). the production of the first antibiotics represents the greatest achievement in medicine. however, bacterial resistance to certain antibiotics increases significantly with age (grundmann et al., 2011). tenover (2006) cites as known cases of resistance, resistance of escherichia coli to third generation cephalosporins, emergence of vancomycin-resistant staphylococcus aureus and multiresistance of pseudomonas aeruginosa, which is why there is a constant need to find new antimicrobial agents. according to data from the world health organization (2020), not a single new class of antibiotics has been developed since 1987. until now, the most clinically important classes of antibiotics are derived from molds, but we can except that basis for the identification of new antimicrobial compounds can be lichen. lichens represent a community formed by algae or cyanobacteria and fungi, developing a mutually beneficial relationship or symbiosis (calcott et al., © 2022 bogdanović et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 153 14th symposium on the flora of southeastern serbia and neighboring regions 2018). in this symbiosis, algae or cyanobacteria represent the photobiont, while fungi represent the mycobiont. nash (2008) considers lichen evernia prunastri (l.) ach. „vegetation pioneers” because of their high stress tolerance. most lichen are slowgrowing and long-lived, which indicates that they retain an unchanged morphology for a long period of time (nash, 2008). evernia prunastri (l.) ach. is widely distributed in the northern hemisphere. it belongs to the family parmeliaceae, genus evernia, and is popularly known as oak or plum moss. according to the type of structure, it belongs to foliose lichen. they have a dichotomously branched thallus, with more or less branches that are attached at one point. it reaches a width between 0.5 and 3 mm. the upper surface of the lichenis greenish-gray to pale green-yellow in color. the lower surface of the thallus in healthy lichen is the same or a shade lighter in color. if the lichen is damaged, its color becomes grayish and less intense. e. prunastri grows on the bark of deciduous trees, most often oak, while occasionally it can be found on conifers in areas of high humidity (shcherbakova et al., 2021). therefore, it is not surprising that this species is widely distributed on the territory of serbia. it likes moist, sunny areas at lower altitudes, and is very rarely found in areas higher than 1,675 m above sea level (nash, 2001). the species e. prunastriis acidophilic and sensitive to the presence of nitrogen, especially ammonia from the air. e. prunastri is characterized by usnic acid, atranorin and chloratranorin in the upper cortex, while evernic acid is found in the medulla. about 170 compounds were identified in the extract of this species, of which 47 are depsides, 25 are triterpenes and steroids (joulain & tabacchi, 2008). usnic acid is known as an acid with a broad spectrum of antimicrobial activity against gram-positive bacteria, such as streptococcus spp., bacillus spp., staphylococcus aureus (including methicillinresistant strains) and gram-negative bacteria, such as proteus vulgaris (shcherbakov et al., 2021). evernic acid, although studied to a lesser extent, has shown antimicrobial activity against bacillus mycoides and b. subtilis, escherichia coli and pseudomonas aeruginosa (shcherbakova et al., 2021). atranorin also shows some antimicrobial activity (studzinskasroka et al., 2017). according to the literature, the resinoid of e. prunastri contains monoterpenes, sesquiterpenes, diterpenes and various terpenoids. also, α-pinene, camphene, β-pinene, limonene, γ-terpinene, p-cymene, trans-pinocarveol, α-copene and α-muurolene are compounds characteristic of both the resinoids and the essential oil of e. prunastri (kahriman et al., 2011). the ancient greeks were still familiar with the healing properties of e. prunastri, from which they made salves for urogenital diseases. more recently, it has been used for diseases of the digestive tract and respiratory tract (crawford, 2015). medical use today is justified by the fact that lichens contain unique biologically active metabolites that have a wide variety of biological effects (antibacterial, analgesic, antiproliferative and cytotoxic) (kosanic et al., 2013). it is often used in research because it is easy to collect, transport and prepare for analysis. materials and methods samples of lichen e. prunastri (fig. 1) were collected in october 2021 in the village of prekopuce, at the foot of the jastrebac mountain. the thallus was dried in the air, in a shady place, at room temperature for 10 days. 154 biologica nyssana ● 13 (2) december 2022: 153-156 bogdanović et al. ● antimicrobial activity of evernia prunastri (оakmoss) resinoids fig. 1. photograph of evernia prunastri (l.) ach. extraction dried lichen (10 g) poured with 100 cm3 of 70% aqueous ethanol solution and left at a temperature of 25 °c. after a certain time, the extract was separated from the dried material by vacuum filtration. the solvent was evaporated in vacuo until a semisolid residue was obtained. the resulting semisolid residue was dried at a temperature of 60 °c biologica nyssana ● 13 (2) december 2022: 153-156 bogdanović et al. ● antimicrobial activity of evernia prunastri (оakmoss) resinoids 155 to a constant mass. the resulting dry residue is the resinoid, that is, the total alcoholic extract. disc-diffusion method to determine the antimicrobial activity of lichen resinoids, laboratory reference cultures from the american type culture collection (atcc) staphylococcus aureus atcc 25923, staphylococcus epidermidis atcc 12228, pseudomonas aeruginosa atcc 27853, escherichia coli atcc 25922 and clinical isolates of bacteria s. aureus, s. epidermidis, p. aeruginosa and e. coli, by the disc-diffusion method. clinical isolates were isolated from swabs of patients’ skin wounds. a turbidity suspension of 0.5 mcfarland containing 1.5x108 cfu/ml was made from overnight cultures of tested strains of microorganisms grown on nutrient agar (nccls national committee for clinical laboratory standards, 2003). on sterile müller-hinton agar (torlak) substrates, 0.1 ml of all prepared microorganism suspensions were inoculated. sterile cellulose discs with a diameter of 6 mm (himedia, milano, italy) were placed on the inoculated surface of the agar plate. the disks were impregnated with 10 µl of different solutions (1, 1/2 and 1/5 v/v) of resinoid in ethanol. a disc with gentamicin (10 μg/ml) was used as a positive control. petri dishes were incubated at 37 °c for 24 hours. after that, the zone of inhibition was read in mm. all growth inhibition assays were performed in triplicate. results and discussion e. prunastri resinoid inhibited the growth of all tested microorganisms; however, their antimicrobial potential was lower than the applied positive control, gentamicin. the results of the disc-diffusion method showed antimicrobial activity against reference strains p. aeruginosa atcc 27853 (depending on the resinoid concentration, the zone of inhibition was from 18 mm to 10 mm), while a significantly weaker antimicrobial activity was shown against s. epidermidis atcc 12228 (depending on the resinoid concentration, the zone of inhibition was from 15 mm to 7 mm). inhibition zone s. aureus atcc 25923 ranged from 14 mm to 9 mm. the weakest antimicrobial effect was shown by e. coli atcc 25922 with an inhibition zone ranging from 10 mm to 8 mm. it is necessary to emphasize that 1/5 v/v ethanol solution of resinoid according to s. aureus atcc 25923 and e. coli atcc 25922 did not show antimicrobial activity (tab. 1). the resinoid showed the most significant antimicrobial activity against the clinical isolate of p. aeruginosa, whose zone of inhibition ranged from 17 mm to 13 mm. it showed moderate antimicrobial activity according to s. epidermidis and s. aureus, whose inhibition zones ranged from 13 mm to 8 mm, that is, from 10 mm to 7 mm. the weakest antimicrobial activity was against e. coli, whose zone of inhibition was from 9 mm to 7 mm (tab. table 1. antimicrobial activity of e. prunastri resinoids bacterial strains gentamicin resinoid e. prunastri 10 µl 1 1/2 1/5 staphylococcus aureus atcc 25923 22 14 9 0 staphylococcus epidermidis atcc 12228 20 15 9 7 pseudomonas aeruginosa atcc 27853 28 18 16 10 escherichia coli atcc 25922 12 10 8 0 staphylococcus aureus isolate from the wound 19 13 8 0 pseudomonas aeruginosa isolate from the wound 26 17 13 0 escherichia coli isolate from the wound 10 9 7 0 156 biologica nyssana ● 13 (2) december 2022: 153-156 bogdanović et al. ● antimicrobial activity of evernia prunastri (оakmoss) resinoids 1). it is important to note that the 1/5 v/v ethanol solution of resinoid did not show antimicrobial activity against any of the tested clinical isolate, but reference strain staphylococcus epidermidis atcc 12228 had inhibition zone of 7 mm and inhibition zone of pseudomonas aeruginosa atcc 27853 was 10 mm. by comparing reference and clinical strains, it is concluded that the highest antimicrobial activity of resinoid in all concentrations is against p. aeruginosa atcc 27853, while it is the lowest against the clinical isolate of e. coli. in this study, the tested e. prunastri resinoid showed relatively low antimicrobial activity. the obtained results are in accordance with the results of testing the essential oil of e. prunastri from algeria, which showed a small effect against e.coli and a moderate antibacterial effect against p. aeruginosa (chahra et al., 2016). however, the obtained results are in contrast with the results of testing the antimicrobial activity of the essential oil of e. prunastri from turkey. in this study, the essential oil of e. prunastri did not show antimicrobial activity against p. aeruginosa, s. aureus, s. epidermidis (aslan et al., 2006). in a study conducted by kahriman et al. (2011), e. prunastri essential oil did not show antimicrobial activity against any tested bacteria. stojanović et al. (2013) showed the antimicrobial effect of e. prunastri against p. aeruginosa and e. coli. conclusions the results of the e. prunastri resinoid test indicate a relatively low antimicrobial potential. the tested microorganisms were not equally sensitive to the presence of lichen resinoids, which shows that the tested lichen possesses compounds with antimicrobial properties, and this requires further research to determine antimicrobial agents that could be used in new drugs for the treatment of infectious diseases in humans and animals, as well as diseases plants. references aslan, a., gulluce, m., sokmen, m., adiguzel, a., sahin, f., ozkan h. 2006: antioxidant and antimicrobial properties of the lichens cladonia foliacea, dermatocarpon miniatum, evernia divaricata, evernia prunastri and neofuscella pulla. pharmaceutical biology, 44: 247-252. bobbarala, v. 2012: antimicrobial agents. intech, isbn 978-953-51-0723-1. calcott, m.j., ackerley, d.f., knight, a. 2018: secondary metabolismin the lichen symbiosis. chemical societz reviews, 47: 1730-1760. chahra, d., ramdani, m., lograda, t., chalard, p., figueredo, g. 2016: chemical composition and antimicrobial activity of evernia prunastri and ramalina farinacea from algeria. biological sciences and pharmaceutical research, 4: 35-42. crawford, sd. 2015: lichens used in traditional medicine. in: ranković b. (ed.) lichen secondary metabolites: bioactive properties and pharmaceutical potential: 27-80, springer international publishing, cham. grundmann, h., de kraker, m., davey, p. 2011: clinical impact of antimicrobial resistance: design matters. the lancet infectious diseases, 11: 344. joulain, d., tabacchi, r. 2009: lichen extracts as raw materials in perfumery. part 1: oakmoss. flavour and fragrance journal, 24: 49-61. kahriman, n., yazici, k., arslan, t., aslan, a., karaoglu, s.a., yayli n. 2011: chemical composition and antimicrobial activity of the essential oils from evernia prunastri (l.) ach. and evernia divaricata (l.) ach. asian journal of chemistry, 23: 1937-1939. kosanić, m., manojlović, n., janković, s., stanojković, t., ranković, b. 2013: evernia prunastri and pseudoevernia furfuraceae lichens and their major metabolites as antioxidant, antimicrobial and anticancer agents. food and chemical toxicology, 53: 112-118. nash, th. 2008: lichen biology, 2nd ed. cambridge university press. cambridge. shcherbakova, a., strömstedt, a., göransson, u., gnezdilov, o., turanov, a., boldbaatar, d., kochkin, d., ulrich‑merzenich, g., koptina, a. 2021: antimicrobial and antioxidant activity of evernia prunastri extracts and their isolates. world journal of microbiology and biotechnology, 37: 129. stojanović, i., radulović, n., cvetković, v., mitrović, t., stamenković, s. 2013: antimicrobial activity of methanol extracts of four parmeliaceae lichen species. facta universitatis-series: physics, chemistry and technology, 11: 45-53. studzinska‑sroka, e., galanty, a., bylka, w. 2017: atranorin an interesting lichen secondary metabolite. mini-reviews medicinal chemistry, 17: 1633-1645. tenover, f.c. 2006: mechanisms of antimicrobial resistance in bacteria. american journal of infection control, 34. mihajilov-krstev, t. et al.  antimicrobial and antioxydant potential… biologica nyssana 6 (2)  december 2015: 55-58 mihajilov-krstev, t. et al.  antimicrobial and antioxydant potential… 55 original article received: 15 july 2015 revised: 12 august 2015 accepted: 01 october 2015 antimicrobial and antioxidant potential of wild growing silene baccifera (l.) roth. (caryophyllaceae) fruits juice tatjana mihajilov-krstev1*, bojan zlatković1, marija ilić2, vesna stankov-jovanović2, violeta mitić2 1university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia 2university of niš, faculty of sciences and mathematics, department of chemistry, višegradska 33, 18000 niš, serbia * e-mail: tatjanamk@pmf.ni.ac.rs abstract: mihajilov-krstev, t., zlatković, b., ilić, m., stankov-jovanović, v., mitić, v.: antimicrobial and antioxidant potential of wild growing silene baccifera (l.) roth (caryophyllaceae) fruits juice. biologica nyssana, 6 (2), december 2015: 55-58. silene baccifera is widespread plant species in europe, asia and north africa, registered in the list of medicinal plants of india. insufficiently known, biological activities of juice obtained from fresh fruits of this plant were investigated in this study. antimicrobial activity of juice was tested against pathogenic gastrointestinal microbial strains, using microwell-dilution method, while antioxidant properties were evaluated employing dpph and total phenolic and flavonoid content assays. to our knowledge, this is the first study of the juice from fruits of this plant species. key words: silene baccifera, fruit juice, antimicrobial, antioxidant activity apstrakt: mihajilov-krstev, t., zlatković, b., ilić, m., stankov-jovanović, v., mitić, v.: antimikrobni i antioksidativni potencijal soka plodova divljerastuće vrste silene baccifera (l.) roth (caryophyllaceae). biologica nyssana, 6 (2), december 2015: 55-58. silene baccifera predstavlja rasprostranjenu biljnu vrstu na području evrope, azije i severne afrike. registrovana je na listi lekovitih biljnih vrsta indije. u ovom radu je ispitivana biološka aktivnost soka dobijenog ceđenjem svežih plodova pomenute vrste. antimikrobna aktivnost je testirana mikrodilucionom metodom protiv patogena gastrointestinalnog trakta. antioksidantna aktivnost je procenjena dpph metodom i određivanjem ukupnog sadržaja fenola i flavonoida. prema našem saznanju, ovo je prva takva studija soka plodova ove biljne vrste. key words: silene baccifera, sok od plodova, antimikrobna, antioksidativna aktivnost introduction silene baccifera (l.) roth. (syn. cucubalus baccifer l.) is widely spread plant species in the area of europe, asia and north africa, especially in the regions with a temperate climate (g a j i ć , 1970). it is perennial plant with long, prostrate or ascending (50-150 cm), well-branched stems and white or 6 (2) • december 2015: 55-58 biologica nyssana 6 (2)  december 2015: 55-58 mihajilov-krstev, t. et al.  antimicrobial and antioxydant potential… 56 greenish nodding flowers sorted in lateral brunches of the inflorescence. comparatively large (up to 15 mm in diameter), subglobose, fleshy, blackish-red berries are distinctly exerted from the calyx during the ripening period. blossoms appear in june to august, fruits ripen from july to october (k o n r a d v o n w e i h e , 1972). silene baccifera is common plant species that usually grows along roadsides and weedy places in the human settlements. this plant species is registered in the list of indian medicinal plants (www.docslide.us) and is used in traditional medicine for treatment of nephritis, hydropsy, bone-fractures, pulmonary tuberculosis and scrofula (c h e n g et al., 2001). to the best of the author’s knowledge, there is no literature or field data on its use in official either traditional medicine in serbia or balkan countries. the chemical composition of the underground part of the s. baccifera has been poorly investigated. previously isolated constituents from the whole plant were oligosaccharides (c o u r t o i s & a r i y o s h i , 1960; 1962), tocopherol and tocotrienol (i v a n o v & a i t z e t m u e l l e r , 1998) and phytoecdysterones and cucubalugenin a (c h e n g et al., 2001a; 2001b). to the best of our knowledge, antioxidant and antimicrobial activity of this plant species were not investigated. in this paper, the first results of antioxidant and antimicrobial activity of the juice obtained from fresh fruits of s. baccifera are presented. material and methods plant material ripe fruits of wild growing silene baccifera were collected on the territory of serbia (kamenica village, stara planina, e serbia) and identified by dr bojan zlatković. a voucher specimens, under the acquisition number 6859, were deposited at the herbarium collection of department of biology and ecology, faculty of science and mathematics in niš (hmn). preparation of juice cleaned, ripe fruits were tap washed, followed by washing with distilled water and subsequent drying. the fruits were separated from the seeds, cut in small pieces, arranged in clean plastic containers and frozen. melted at room temperature, the softened material was grounded in mechanical blender to a fine mash prior to filtration and the juice extraction. antimicrobial assay microbial strains. in vitro antimicrobial activity of the tested samples have been investigated against the commonest human gastrointestinal pathogenic microbial strains: salmonella enteritidis (atcc 13076), shigella sonnei (atcc 25931), escherichia coli (atcc 25922), pseudomonas aeruginosa (atcc 9027), listeria monocytogenes (atcc 7644), bacillus cereus (atcc 10786), staphylococcus aureus (atcc 6538) and candida albicans (atcc 10031) using microwell-dilution method. microwell dilution method. determination of the minimum inhibitory concentration (mic) and minimum microbicidal concentration (mmc) was carried out according to the method described by clsi (2005) with some modifications. an overnight culture of tested strains were used for the preparation of 0.5 mcfarland standard turbidity suspension (corresponding 108 cfu/ml). a serial doubling dilutions of tested juice were prepared in the range 500.00-0.25 µl/ml, in a 96/well microtiter plate with inoculated mueller-hinton broth (mhb) for bacteria and sabouraud dextrose broth (sdb) for yeast. the final volume was 100 µl and the final concentration of bacterial cells was 106 cfu/ml in each well. the plates were incubated for 24 h at 37 °c. chloramphenicol at concentrations ranging from 10.00-0.002 µg/ml was used as positive control. all determinations were performed in triplicate. microbial growth was determined by adding 20 μl of 0.5% triphenyl tetrazolium chloride (ttc) aqueous solution. mic was defined as the lowest concentration of juice at which microorganisms showed no visible growth. in order to determine mmc, broth was taken from each well and inoculated on mueller-hinton agar (mha) for bacteria and sabouraud dextrose agar (sda) for yeast. the plates were incubated for 24 h at 37°c. the mmc is defined as the lowest concentration of the juice at which 99.9% of inoculated microorganisms were killed. antioxidant assay the antioxidant properties of the tested juice were evaluated employing dpph and total phenolic and flavonoid content assays. all the assays were carried out in triplicate and average value was considered. 1,1-diphenyl-2-picrylhydrasyl (dpph) radical scavenging assay. relatively stable organic radical dpph has been widely used in the determination of antioxidant activity of single compounds as well as the different plant extracts (k u l i š i ć , 2004). the dpph˙-assay was performed as described (s t o j a n o v i ć , 2010). samples of juice methanol solution (10 μl, 232.4, 232.5 and 232.6 mg/ml respectively) were mixed with 90 μmol/l dpph˙in methanol (1.0 ml), and these biologica nyssana 6 (2)  december 2015: 55-58 mihajilov-krstev, t. et al.  antimicrobial and antioxydant potential… 57 solutions were diluted up to 4.0 ml. after shaking mixtures vigorously, they were stored in darkness for 60 min at room temperature and the absorbances were measured at 515 nm (perkin-elmer lambda 15 uv-vis spectrophotometer). radical scavenging activity of the samples was calculated applying following equation: dpph rsc (%) = 100 (a0−a1/a0) where: a0-absorbance of the blank; a1absorbance of the sample. total phenolic content determination. the total phenolic concentration was determined spectrophotometrically according to modified folinciocalteu method (d i m a j o , 2008). to the samples of juice (100 µl) portions of 1 ml of folinciocalteu reagent (purchased from “mol” belgrade, serbia) and 4 ml of sodium carbonate (20% v/v) were added and diluted with distilled water up to 20 ml. the mixture was allowed to stand at room temperature in dark place for 30 min and the absorbance of the solution at 750 nm was measured with a perkin elmer lambda 15 uv/vis spectrophotometer. the total phenolic concentrations were calculated from a calibration curve using gallic acid as a standard. gallic acid was provided by sigma aldrich (darmstadt, germany). data were expressed as gallic acid equivalents per 1 mg of juice. the levels of total infusions in juice determined according to the folin–ciocalteu method are not absolute measurements of the amounts of phenolic materials, but are in fact based on their chemical reducing capacity relative to an equivalent reducing capacity of gallic acid. determination of total flavonoid content. the amount of total flavonoids was determined with aluminium chloride (alcl3) colorimetric assay according to a known method (r i c e e v a n s et al., 1996). briefly, 0.5 ml of juice was made up to a final volume of 1 ml with reaction medium (meo/h2o/ch3cooh=14:5:1). prepared solution was mixed with alcl3 reagent (4 ml, 133 mg of alcl3x6h2o and 400 mg of ch3coona dissolved in 100 ml h2o). after 5 min, the absorbance level was measured versus prepared reagent blank (containing the same chemicals, except for the sample) at 430 nm (perkin-elmer lambda 15 uv-vis spectrophotometer). total flavonoid content was calculated on the basis of the calibration curve of rutin and expressed by mg rutin/g dry extract. the total flavonoid assay was measured in triplicate. results and discussion freshly drained juice from the fruits have shown antimicrobial activity on all tested strains of patogenic microbes (tab. 1). the obtained minimal inhibitory concentrations were in the range 31.2-500.0 µl/ml, and minimal microbicidal concentrations ranged from 125.0-500.0 µl/ml. the tested juice exhibited the highest activity against p. aeruginosa and s. enteritidis (mic/mmc=31.2/125 and 62.5/125 µl/ml, respectively). also, good effect was observed against e. coli (mic=mmc=125 µl/ml). the juice was the least active against s. aureus and c. albicans (mic=mmc=500 µl/ml). table 1. antimicrobial activity of juice from s. baccifera fruits and reference antibiotics (tested strains: sb s. baccifera fruits juice, mic/mmc in µl/ml; a antibiotic, mic/mmc in µg/ml; bacterial strains: se – salmonella enteritidis; ss shigella sonnei, ec escherichia coli, pa pseudomonas aeruginosa, lm listeria monocytogenes, bc bacillus cereus, sa staphylococcus aureus; ca – candida albicans) tested strains se ss ec pa lm bc sa ca sb 62.5/125 250/250 125/125 31.2/125 250/500 125/500 500/500 500/500 a 0.035* 0.035* 0.035* 0.078* 0.035* 0.035* 0.035* 0.035** *chloramphenicol, **nystatin table 2. the antioxidant activity of juice from s. baccifera fruits tested samples rsa (ec 50 u µg/ml)** ppc (gallic acid equivalents, µg/mg of dry matter) pc (rutin equivalents, µg/mg of dry matter) silene baccifera* 0.65 6.00 15.18 *juice was prepared by dissolving fruit in water; ** ec 50 for bht as a standard is 0.63 µg/ml biologica nyssana 6 (1)  september 2015: 55-58 mihajilov-krstev, t. et al.  antimicrobial and antioxydant potential… 58 the obtained results of antioxidant activity of juices are summarized and presented in tab. 2. comparative study of antimicrobial and antioxidant activities of the fresh juices obtained from the fruits of plant species: viburnum lantana, viburnum opulus, sambucus nigra, cornus sanguinea and paliurus spina-christi, wild growing on the territory of serbia, showed that all tested juices had inhibitory concentrations in the range from 15.6500.0 μl/ml and microbicidal concentrations from mbc/mfc=62.5-500.0 μl/ml (m i h a j i l o v k r s t e v et al., 2011). generally, better activity than s. baccifera juice had only the juices from v. opulus and c. sanguinea. also, the juice of this species demonstrated the best antioxidant activity in comparison to the mentioned juices. conclusion the obtained results showed that s. baccifera fruit juice presents a natural source of antimicrobial and antioxidant components that can be applied in prevention and treatment of gastrointestinal diseases and oxidative stress in humans. in future studies, the chemical composition and biological activity of different extracts of certain parts of the plant should be investigated, as well as their cytotoxicity and acute toxicity. acknowledgements. the financial support of this work was provided by ministry of education and science of the republic of serbia (project 172047). references cheng, y.x., zhou, j., tan, n.h., ding, d.t. 2001a: indian medical plants in traditionl medicine. acta botanica sinica, 43:31. cheng, y.x., zhou j., dai, h.f., ding, d.t. 2001b: cucubalugenin a, a new triterpenoid from cucubalus baccifer. fitoterapia, 72: 848-849. clsi, 2005: clinical and laboratory standards institute, wayne, pa. m100-s22 courtois, j.e., ariyoshi, u. 1960: saccharose galactosides from the roots of cucubalus baccifer (caryophyllaceae). study of their structure. bulletin de la société de chimie biologique, 42:737-51. courtois, j.e., ariyoshi, u. 1962: the galactosides of saccharose in the roots of the carnation. bulletin de la société de chimie biologique, 44:23-30. di majo, d., guardia, m.l., giammanco, s., neve, l.l., giammanco, m. 2008: the antioxidant capacity of red wine in relationship with its polyphenolic constituents. food chemistry, 111:45–49. gajić, m. 1970: cucubalus l. [in. gajić, m.: caryophyllaceae juss.]. in: josifović, m. (ed.): flora srbije 2:240-241. srpska akademija nauka i umetnosti, beograd. ivanov, s.a., aitzetmuller, k. 1998: undersuchungen über die tocopherol-undtocotrienol zusammensetzung der samenlipide einiger arten der bulagarischen flora. fett/lipid, 100: 348-352. konrad von weihe, h. 1972: illustrierte flora. deutschland und angrenzende gebiete. gefäßkryptogamen und blütenpflanzen. begründet von august garcke. 23. auflage. paul parey, berlin/hamburg. kulišić, t., radonić, a., katalinić, v., miloš, m. 2004: use of different methods for testing antioxidative activity of oregano essential oil. food chemistry, 85:633–640. mihajilov-krstev, t., zlatković, b., ilić, m., stankov-jovanović, v., mitić, v. 2011: comparative study of antibacterial and activities activities of wild growing fruits juices. book of abstracts of international conference ''medicinal and aromatic plants in generating of new values in 21st century''. academy of sciences and arts of bosnia and herzegovina, special edition vol. cxl, department of natural sciences and mathematics, sarajevo, 9-12 november, 223. rice-evans, c.a., miller, n.j., paganga, g. 1996: structure antioxidant activity relationshipe of flavonoids and fenolic acids. free radical in biology and medicine, 20: 933-956. stojanović, g., stojanović, i., stankov-jovanović, v., mitić, v., kostić, d. 2010: reducing power and radical scavenging activity of four parmeliaceae species. open life sciences, 5(6):808–813. www.docslide.us the effect of satureja montana l. aqueous extract on soybean seedlings biologica nyssana 7 (2)  december 2016: 125-129 šućur, j. et al.  the effect of satureja montana l. aqueous… 125 original article received: 01 july 2016 revised: 18 october 2016 accepted: 24 november 2016 the effect of satureja montana l. aqueous extract on soybean seedlings jovana šućur1, dejan prvulović1, goran anačkov2, đorđe malenčić1 1 department of field and vegetable crops, faculty of agriculture, university of novi sad, trg dositeja obradovića 8, novi sad, serbia 2 department of biology and ecology, faculty of science, university of novi sad, trg dositeja obradovića 3, novi sad, serbia * e-mail: jovana.sucur@polj.edu.rs abstract: šućur, j., prvulović, d., anačkov, g., malenčić, đ.: the effect of satureja montana l. aqueous extract on soybean seedlings. biologica nyssana, 7 (2), december 2016: 125-129. the aim of this study was to examine the impact of satureja montana l. aqueous extract on soybean antioxidant properties so as to assess its possible side effects when applied as biohebicide in soybean organic production. the effects of two concentrations (0.1% and 0.2%) of s. montana aqueous extract on the activity of the antioxidant enzymes superoxide dismutase (sod) and catalase (cat) in leaves and roots of soybean (glycine max l.) seedlings were examined 24, 72 and 120 h after the treatment. our results showed that the significant increase in the catalase activity was recorded in roots of soybean treated with both concentrations of the extract used. on the other hand, both concentrations of s. montana aqueous extract stimulated the significant increase of the superoxide dismutase activity in leaves and roots of soybean. higher activity of the antioxidant enzymes in the roots of soybean compared with activity of the antioxidant enzymes in leaves showed that roots were more affected than leaves. key words: allelopathy, glycine max (l.) merr., satureja montana l. apstrakt: šućur, j., prvulović, d., anačkov, g., malenčić, đ.: efekat vodenog ekstrakta satureja montana l. na sadnice soje. biologica nyssana, 7 (2), decembar 2016: 125-129. cilj ovog rada je ispitivanje uticaja vodenog ekstrakta satureja montana l. na parametre oksidativnog stresa u sadnicama soje kako bi se procenile eventualne nuspojave nakon primene ovog ekstrakta kao bioherbicida u organskoj proizvodnji soje. ispitan je uticaj dve koncentracije (0,1% i 0,2%) vodenog ekstrakta s. montana na aktivnost antioksidantnih enzima superoksid-dismutaze (sod) i katalaze (cat) u listu i korenu sadnica soje (glycine max l.) 24, 72 i 120 časova nakon tretmana. dobijeni rezultati su pokazali statistički značajno povećanje aktivnosti enzima katalaze u korenu soje, kao i statistički značajno povećanje aktivnosti superoksiddismutaze u listu i korenu soje, u tretmanima za obe primenjene koncentracije ekstrakata. veća aktivnost antioksidantnih enzima u korenu soje, u poređenju sa aktivnošću u listu, ukazuje na veću senzitivnost korena od lista prema primenjenom ekstraktu. ključne reči: alelopatija, glycine max (l.) merr., satureja montana l. 7 (2) • december 2016: 125-129 12th sfses • 16-19 june 2016, kopaonik mt doi: 10.5281/zenodo.200409 biologica nyssana 7 (2)  december 2016: 125-129 šućur, j. et al.  the effect of satureja montana l. aqueous… 126 introduction satureja montana l. (winter savory or mountain savory), belonging to the lamiaceae family, native to the mediterranean regions, is a well-known aromatic plant which contains various biologically active constituents such as essential oils, triterpenes, flavonoids and rosmarinic acid (h a s s a n e i n et al., 2014; d u d a š et al., 2013). it is found throughout europe, russia and turkey, growing in sunny, stony and rocky regions (t r i f a n et al., 2015). owing to the active components and pleasant aroma, winter savory is often used as a medicinal plant and a spice (d u d a š et al., 2013; t r i f a n et al., 2015). the plant releases many bioactive chemicals from its various parts (leaves, stems, roots) into its surrounding environment, which are called allelochemicals (m o n d a l et al., 2015). allelopathy is an interference mechanism by which plants release allelochemicals which affect the growth of other plants (b a j a l a n et al., 2013). allelopathic interaction is either positive or negative (m o n d a l et al., 2015). released allelochemicals become stressful only when they are toxic or when they affect the growth and development of surrounding plants (c r u z – o r t e g a et al., 2007). increasing attention has been given to the role and potential of allelopathy as a management strategy for crop protection against weeds and other pests (b a j a l a n et al., 2013). the aim of this study was to examine the effect of aqueous extract of s. montana on soybean (glycine max (l.) merr.) antioxidant properties so as to assess its possible side effects when applied as biohebicide in soybean organic production. material and methods the aromatic plant, s. montana, was collected at localities near the adriatic coast in montenegro, in june, 2012. voucher specimens of collected plant was confirmed and deposited at the herbarium of the department of biology, faculty of natural sciences, university of novi sad (buns 2-1544). the air– dried plant material was ground into powder. the powdery material (10 g) was extracted with 100 ml distilled water. after 24 h, the extract was filtered through filter paper and kept at 4 °c until application. the experiment was performed at the laboratory of biochemistry, faculty of agriculture, novi sad and conducted under controlled conditions (temperature 28 °c, 60% relative humidity, 18 h photoperiod, a light intensity of 10.000 lx). soybean seedlings (g. max) cv. viktorija were grown for 30 days in plastic pots (500 ml) containing sterile sand, after which they were transferred on plastic pots (700 ml) containing the hoagland’s solution and 7 and 14 ml of s. montana aqueous extract, while 7 and 14 ml of the hoagland’s solution were added in pots of control. seedlings were harvested for determining the investigated biochemical parameters 24, 72 and 120 h after the treatments. fresh leaves and roots of soybean plants (2 g each) were homogenized in 10 ml of phosphate buffer (0.1 m, ph 7.0). homogenates were centrifuged for 20 min at 10.000 x g and filtered. the supernatants were used to test activity of the antioxidant enzymes superoxide dismutase (sod) and catalase (cat). the catalase (cat) (ec 1.11.1.6) activity was determined according to sathya & bjorn (2010). the decomposition of h2o2 was followed as a decrease in absorbance at 240 nm. the enzyme extract was added to the assay mixture containing 50 mm potassium phosphate buffer (ph 7.0) and 10 mm h2o2. the activity of the enzyme is expressed as u per 1 g of protein (u mg–1 protein). superoxide dismutase (sod) (ec 1.15.1.1) activity was assayed according to a slightly modified method of m a n d a l et al. (2008) by measuring its ability to inhibit photochemical reduction of nitro blue tetrazolium (nbt) chloride. the reaction mixture contained 50 mm phosphate buffer (ph 7.8), 13 mm l– methionine, 75 μm nbt, 0.1 mm edta, 2 μm riboflavin and 0.02 ml of the enzyme extract. it was kept under a fluorescent lamp for 30 min, and then the absorbance was read at 560 nm. one unit of the sod activity is defined as the amount of enzyme required to inhibit the reduction of nbt by 50%. the activity of the enzyme is expressed as u per 1 mg of protein (u mg–1 protein). values of the biochemical parameters were expressed as means ± standard error (se) of determinations made in triplicates and tested by anova followed by comparison of the means by the duncan multiple range test (p < 0.05). data were analyzed using statistica for windows, version 11.0. results and discussion allelochemicals can cause oxidative stress in target plants and therefore activate the antioxidant mechanism (c r u z – o r t e g a et al., 2007). accordingly, the response of plants to damaging adverse circumstances is closely related to their enzyme activity (s u n m o n u & v a n s t a d e n , 2014). enzymes, such as superoxide dismutases (sod), catalases (cat) and peroxidases, play important roles in protecting cells against reactive oxygen species (k u t h a n et al., 1986). therefore, the activity of those enzymes can be used as indicators of oxidative stress in plants (a n et al., 2005). biologica nyssana 7 (2)  december 2016: 125-129 šućur, j. et al.  the effect of satureja montana l. aqueous… 127 our results showed that a significant increase in the catalase activity was recorded in roots of soybean seedlings treated with both concentrations of the extract used 72 h after the treatment (fig. 1). on the other hand, both concentrations of s. montana aqueous extract stimulated a significant increase of the superoxide dismutase activity in roots of soybean seedlings 72 and 120 h after the treatment (fig. 2). the increases in activity of antioxidant enzymes probably occur in response to stress. a higher activity of the antioxidant enzymes in the roots of soybean seedlings compared with the activity of the antioxidant enzymes in leaves showed that roots were more affected than leaves. the activity of examined antioxidant enzymes showed a downward trend with the duration of the experiment, thus the highest activity of the catalase and superoxide dismutase was observed after 72 h. this could point to the fact that scavenging effects of superoxide dismutase and catalase could prevent an oxidative burst and the induction of lipid peroxidation process. conclusion in conclusion, our results showed that s. montana aqueous extract did not stimulate an increase of the catalase and superoxide dismutase activity in the leaves of soybean seedlings, while both tested concentrations caused a significant increase in fig. 1. activity of catalase in leaves and roots of soybean seedlings 24, 72 and 120 h after treatment with different concentrations (%) of s. montana aqueous extracts (v/v) and in control (c). fig. 2. activity of superoxide dismutase in leaves and roots of soybean seedlings 24, 72 and 120 h after treatment with different concentrations (%) of s. montana aqueous extracts (v/v) and in control (c). 0,00 5,00 10,00 15,00 20,00 25,00 30,00 c 0.1 0.2 c 0.1 0.2 c 0.1 0.2 24 h 72 h 120 h u / m g p ro te in extract concentration (%) roots leaves 0 10 20 30 40 50 60 70 80 90 100 c 0.1 0.2 c 0.1 0.2 c 0.1 0.2 24 h 72 h 120 h u /m g p ro te in extract concentration (%) roots leaves biologica nyssana 7 (2)  december 2016: 125-129 šućur, j. et al.  the effect of satureja montana l. aqueous… 128 soybean roots. this may be attributed to the permeability of allelochemicals to root tissues arising from direct contact with the phytotoxic compounds present in the extract. acknowledgements. this study was carried out within a project of the ministry of education, science and technological development, republic of serbia, grant no tr-31022. references assyov, b., denchev, c. 2015: verbascum davidoffii murb. in: biserkov, v. et al. 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(2003) reported existence of a strong additive genetic variation between and within populations in growth of oneyear old seedlings as response to drought stress. w i t h e l e y et al. (2003) also reported inerand intrapopulation variability of one-year old seedlings. examining of the four-year old plants of fraxinus angustifolia and ulmus laevis, c i c e k et al. (2011), reports influence of provenance and growth density on the development of ash seedlings, but lack of significant influence on european white elm seedlings. material and methods a total of 56 trees of europen white elm were found, marked and georeferenced on great war island, near belgrade, serbia (fig. 1). the trees are spatially divided into three subpopulations: fig. 1. spatial distribution of subpopulations european white elm (ulmus effusa willd.) at great war island table 1. coordinates and basic characteristics of 14 mother trees of european white elm (ulmus effusa willd.) at great war island subpopulation mark of tree on the field coordinates height (m) average diameter (cm) age e n i 13 7456215 4965040 23,1 48.7 26 14 7456215 4965038 7,7 15.65 13 32 7456352 4964955 20,2 38.3 23 ii 18 7456050 4965266 16,7 39.5 27 19 7456054 4965270 18,4 36.5 16 21 7456046 4965285 9,5 31.5 16 33 7456138 4965226 13,7 29.35 20 34 7456170 4965254 21 31.3 20 35 7456150 4965257 20,9 30.4 20 36 7456136 4965262 13,9 37.8 23 iii 30 7455319 4965639 13,5 23.5 15 31 7455137 4965585 12 23.5 17 the first subpopulation (i) consists of 24 trees located in the inshore area, the recent alluvial deposits with characteristics of β-gley, white willow forest type (salicetum albae issl.26) on the β -gley. the second subpopulation (ii) consists of 22 marked trees inhabiting the area with a very dense canopy and impervious. it extends in the internal part of the island, close to the canal galijaš. the third subpopulation (iii) consists of 10 trees individually dispersed throughout the forest edge or in a field. the second and the third subpopulation belong to the forest of white and black poplar (populetum albo-nigra slav.52) the mosaic of alluvial soil, which is the final stage of development of floodplain forests of soft deciduous trees. european white elm (ulmus effusa willd.) is seen here in the second floor of the trees, beside black poplar (populus nigra l.) and white poplar (populus alba l) edificators (š i j a č i ć n i k o l i ć , m i l o v a n o v i ć 2012). in the spring of 2011, during may, the seeds were collected from all trees where yield was recorded (tab. 1). seeds were collected in may 2011 and after a short drying during the same month were sown in seed beds, seeding density 1200 seeds/m 2 . seed beds were covered with a thin layer of the substrate made up of sand and soil in the ratio of 1:1 (fig. 2 and fig. 3). irrigation was initially done biologica nyssana 4 (1-2)  december 2013: 87-92 devetaković, j.,et al.  variability of morphometric caracteristic of… 89 fig. 2. seed beds with seed partialy covered daily until seedlings appeared, and then less frequently (every 3-4 days) (figure 4). each of the half-sib lines samples was taken from 50 one-year plants with bare-roots, their height (h) and root collar diameter (d) were measured. roller’s sturdiness coefficient, as the ratio of the height (cm) and diameter (mm), were calculated (r o l l e r 1977, i v e t i ć 2013). fig. 3. view on the seed beds sown and covered basic statistical analysis of the data collected included the calculation of mean values, standard deviations (sd) and width variation. clustering of the obtained mean values and the homogenity of groups were tested by tukey hsd test. the origin of variability was examined by analysis of variance (one-way anova). the interdependence of the morphological parameters was tested by calculating the pearson correlation coefficient. closeness (distance) of mother trees based on the above parameters was tested by cluster analysis. fig. 4. appearance of seedlings after germination fig. 5. the layout of rows of seedlings biologica nyssana 4 (1-2)  december 2013: 87-92 devetaković, j.,et al.  variability of morphometric caracteristic of… 90 table 2. mean values of height (h), diameter (d) and roller’s sturdiness coefficient (sq) (standard deviation (sd in parentheses), the limits for h, d, sq and hsd test of 14 tested maternal line (ml) n=700 h d sq ml mean values (sd) xmиn-xmax mean values (sd) xmиn-xmax mean values (sd) xmиn-xmax 13 20.55 (5.92) abc 12.3-40.2 3.25 (1.07) abc 1.47-7.05 6.52 (1.35) a 3.73-10.1 14 19.34 (5.64) ab 10.4-36.5 3.13 (1.17) abc 1.43-6.49 6.45 (1.29) a 3.98-9.5 18 23.34 (6.44) abc 12.2-41.2 3.46 (1.31) abc 1.67-6.57 7.18 (1.81) abc 3.81-10.6 19 23.83 (8.13) bc 9.3-46.8 3.42 (1.59) abc 1.30-7.69 7.52 (1.97) abc 4.24-15.8 21 18.87 (7.55) a 7.9-45.0 2.80 (1.35) a 0.88-7.33 7.12 (1.48) ab 4.40-10.4 29 21.93 (7.29) abc 11.0-43.4 3.52 (1.35) abc 1.55-6.97 6.51 (1.44) a 3.25-10.3 30 29.89 (9.33) d 13.4-55.7 3.91 (1.66) c 1.79-8.86 8.29 (2.80) c 2.84-21.8 31 21.67 (8.87) abc 8.9-47.1 2.91(1.35) ab 1.17-6.97 7.71 (1.53) bc 4.44-11.5 32 20.33 (5.88) abc 10.6-35.8 2.89 (0.96) ab 1.48-5.97 7.25 (1.50) abc 4.03-11.1 33 24.92 (7.94) c 7.0-43.7 3.73 (1.32) bc 1.31-7.84 6.87 (1.53) ab 4.52-10.9 34 24.73 (7.54) c 10.5-43.5 3.45 (1.34) abc 1.11-7.96 7.53 (1.64) abc 3.68-11.1 35 23.41 (6.27) abc 7.8-38.7 3.31 (1.04) abc 1.73-6.00 7.36 (1.98) abc 3.50-12.1 36 21.84 (5.94) abc 11.1-35.0 3.29 (0.98) abc 1.36-5.24 6.88 (1.45) ab 4.18-9.9 37 20.48 (7.56) abc 6.5-35.4 2.80 (1.35) a 0.97-6.96 7.86 (1.91) bc 3.46-11.6 all lines 22.5 (7.70) 6.5-55.7 3.28 (1.32) 0.88-8.86 7.22 (1.79) 2.84-21.8 results and discussion the highest average h (29.89 cm) and d (3.91 mm) was observed in half-sib seedlings line 30 of the parent tree (table 2). the maximum values of 55.7 cm h and d of 8.86 mm were recorded in the same line of seedlings at the same time. mean values in the same column followed by different letters are statistically different for p <0.05. according to the observed parameters seedlings from 30 lines segregate into a separate group. they also show the highest variability of observed parameters. thus, for h sd = 9.33 with variational width of 42.30 cm for d sd = 1.66 with the variation width of up to 7.07 mm, and for sq sd = 2.8 the variation width of 18.96. minimum value of 6.5 cm h and d of 0.97 mm were measured in seedlings of maternal line 37, while the minimum mean values of both measured parameters were recorded in the line 21. analysis of variance indicated that the variability of physiological parameters is the consequence of a mother tree, not a coincidence, and that the variability between seedlings of maternal lines (p = 0.00) is greater than the variability of plants within a maternal line (table 3). d and h showed a significant and strong positive correlation. sq shows a relatively strong, negative correlation with d, and a weak, but significant correlation with h. table 3. analysis of variance (one-way anova) height (h) and diameter (d) of 14 half-sib lines of european white elm between maternal lines својст во mean square f-ratio pvalue h 40721.00 7.74 0.00 d 1.67 3.48 0.00 table 4. correlations between the morphological parameters: the diameter (d), the height (h) and roller’s sturdiness coefficient (sq) of 14 half-sib lines of european white elm n=700 (casewise deletion of missing data) d h sq d 1.00000 h 0.79095* 1.00000 sq -0.48323* 0.10136* 1.00000* figure 6 shows a constant growth of sq with increasing of h. on the other hand, the value of sq decreases with growth of d, to certain, approximately mean values. after that, with the increase of d, sq begins to grow. biologica nyssana 4 (1-2)  december 2013: 87-92 devetaković, j.,et al.  variability of morphometric caracteristic of… 91 cluster analysis (fig. 7) shows clustering of examined maternal lines into three groups. the first group consists of the maternal lines 18, 19, 33, 34 and 35. the second group consists of lines 13, 14, 21, 29, 31, 32, 36 and 37. the third group consists of 30 lines, which are groups at the greatest distance with other maternal lines. the density of growth, as a result of planting density has a strong effect on the growth of seedlings. applied seeding density of 1,200 seeds per m 2 significantly exceeds the growth of 75 plants per m 2 , where c i c e k et al. (2011) found the highest values of h and d of one-year old seedlings of european white elm with bare-root. however, it did not significantly affect the sq, which is satisfactory and which is close to the results got by the listed authors. sq change with increasing d should be explored, especially in the second year of growth of seedlings. variability within the population of parameters observed is significant, as shown by the analysis of variance, and does not depend on the spatial distribution of mother trees. considering the number of trees in the reference population as an appropriate method may be recommended the in situ conservation of genetic resources. in endangered populations of european white elm, with a significantly reduced number of trees, may be recommended the ex situ conservation (a l e k s i c , o r l o v i c 2004). for the purpose of ex situ conservation, the level of variation within the population should be compared with the close spatial populations of european white elm of coastal areas of the danube and sava rivers, during their work, w h i t e l e y et al. (2003) found that studied populations differ in the level of genetic variation within the population. also, exploring the genetic variability of the border populations of european white elm, v a k k a r i et al. (2009) suggest that differences between populations follow the structure of isolation caused by distance (isolation by distance). conclusion the vulnerability of european white elm (ulmus effusa wild.) population on the great war island, is sufficiently explained by the record that only 56 trees of this species exist, and their age structure indicates the existence of different generations of trees in the population. according to the research of variability of morphometric caracteristics of one-year old seedlings, it can be concluded that the satisfactory variability within the popoulation exists, which is a good basis for the conservation of genepool available. special attention should be paid to the half-sib lines whose characteristics (height, diameter and roller’s sturdiness coefficient) provide the best quality seedlings, which in this case is the line halfsib seedlings of mother tree 30. but from the aspect of conservation and directed utilization the specific genotypes which manifested the minimum dimensions such as tree seedlings of lines 21 and 37 should not be ignored. for effective protection of genetic resources of european white elm on the great war island, in situ and ex situ methods should be combinated for a long time. in situ conservation is already partially realized, given that it is a protected nature reserve. to apply the ex situ methods it is necessary to continue monitoring of half-sib lines and establish progeny tests, in order to select appropriate trees for the production of plants and to expand the species population and conservation of her genetic variability in this area. 3d surface: h vs. d vs. sq (casewise deletion of missing data) z = distance weighted least squares 40 30 20 10 0 fig. 6. area of a combination of height (h) and diameter (d) as a predictor, as opposed to roller’s sturdiness coefficient (sq) as the dependent variable biologica nyssana 4 (1-2)  december 2013: 87-92 devetaković, j.,et al.  variability of morphometric caracteristic of… 92 t re e d ia g ra m fo r 1 4 c a s e s s in g le l in k a g e e u c lid e a n d is ta n c e s 0 1 0 2 0 3 0 4 0 5 0 l in k a g e d is ta n c e 3 0 3 4 3 3 1 9 3 5 1 8 3 1 3 6 2 9 2 1 1 4 3 2 3 7 1 3 fig. 7. dendrogram of the cluster analysis was done on the basis of height and root collar diameter of onehalf-sib seedlings of european white elm references aleksić, j., orlović, s. 2004: ex situ conservation of genetic resources of field elm (ulmus minor mill.) and european white elm (ulmus laevis pall.). – genetika, vol. 36, no. 3, 221-227. banković, s., medarević, m., pantić, d., petrović, n., obradović, s. 2009: šumski fond republike srbije-stanje i problemi, glasnik šumarskog fakulteta, beograd, br. 100, (7-30). collin, e. 2003. euforgen technical guidelines for genetic conservation and use for european white elm (ulmus laevis). international plant genetic resources,institute, rome, italy. 6 p. black-samuelsson s., whiteley r. e., junzhan g. 2003: growth and leaf morphology response to drought stress in the riparian broadleaved tree, ulmus laevis (pall.). silvae genetica 52, 5-6. cicek e., cicek n., tilki f. 2011: four-year field performance of fraxinus angustifolia vahl. and ulmus laevis pall. seedlings grown at different nursery seedbed densities. research journal of forestry, 5: 89-98. jovanović, b. 2007: dendrologija. šumarski fakultet univerziteta u beogradu. str. 1-536ultet, beograd: 1-77. ivetić, v. 2013: praktikum iz semenarstva rasadničarstva i pošumljavanja. univerzitet u beogradu, šumarski fakultet, 213 p.. šijačić-nikolić, m., milovanović, j. 2010: konzervacija i usmereno korišćenje šumskih genetičkih resursa, šumarski fakultet univerziteta u beogradu, beograd, 1-200. šijačić-nikolić, m., milovanović, j. 2012: conservation and sustainable use of foerst genetic resources throught an example of watland ecosystems, international conference: role of research in sustainable development of agriculture and rural areas, may 23-26, podgorica, montenegro, book of abstracts, 128. whiteley, r.e., black-samuelsson, s., jansson, g. 2003: within and between population variation in adaptive traits in ulmus laevis pall., the european white elm. forest genetics 10(4):313323. petrov & ocokoljić 2022, biologica nyssana 13(1) 13 (1) september 2022: 41-46 doi: 10.5281/zenodo.7117571 dendrometric analyses and determining biological characteristics of needles for the purpose of conservation and directed utilization of the spruce in southeastern serbia original article djurdja petrov faculty of forestry, university of belgrade, kneza višeslava 1, 11000 belgrade, serbia djurdja.stojicic@sfb.bg.ac.rs (corresponding author) mirjana ocokoljić faculty of forestry, university of belgrade, kneza višeslava 1, 11000 belgrade, serbia received: july 21, 2021 revised: september 16, 2022 accepted: september 21, 2022 abstract: this paper analyzes the morphological and dendrometric characteristics of three 97 years old european spruce trees, on čemernik mountain, in the municipality of crna trava, where european spruce is not autochthonous. the examined parameters were compared with 5 test european spruce trees, aged about 50 years, which are grown in the population, within a radius of 100 m, in identical environmental conditions. the results of this study confirm the excellent vitality of hundred-year-old trees at the researched location, which can be brought into direct correlation with the current climate changes. the selected trees have confirmed the best combinatorial ability, which is why they stand out as a good base for selection and production of planting material for establishing purpose culture, afforestation, application in landscape architecture, and also for timber production. the research confirmed that the analyzed hundred-year-old spruce trees are in excellent condition and high decorative value, with a crown formed from the ground, which is why they are proposed for protection as a natural monument of botanical character. key words: european spruce, crna trava, vitality, selection, genetic potential, conservation apstrakt: dendrometrijska naliza i utvrdjivanje bioloških svojstava četina smrče u cilju konzervacije i usmerenog korišćenja vrste u jugoistočnoj srbiji u radu su analizirane morfološke i dendrometrijske karakteristike tri stabla smrče starosti 97 godina, na čemerniku, na teritoriji opštine crna trava na kojoj smrča nije autohtona. istraživani parametri su poređeni sa pet test stabala smrče, starosti oko 50 godina, odgajenih u populaciji, u radijusu od 100 m, u identičnim uslovima. dobijeni rezultati potvrđuju odličnu vitalnost stogodišnjih stabala na istraživanoj lokaciji što se može dovesti u direktnu korelaciju sa aktuelnim klimatskim promenama. selektovana stabla potvrdila su kombinatornu sposobnost zbog čega se izdvajaju kao polazna za oplemenjivanje i proizodnju sadnog materijala, za podizanje namenskih kultura, pošumljavanje, pejzažnoarhitektonsku primenu, ali i dobijanje drvne mase. istraživanje je potvrdilo da su analizirana stogodišnja stabla smrče odličnog stanja i dekorativnosti, sa formiranom krošnjom od samog tla, zbog čega se predlažu za zaštitu kao spomenik prirode botaničkog karaktera. ključne reči: smrča, crna trava, vitalnost, selekcija, genetski potencijal, konzervacija introduction picea a. dietr. is a genus of evergreen coniferous trees in the family of pinaceae spreng. ex f. rudolphi. it comprises 37 spruce species (powo, 2022), one of which is european or norway spruce picea abies (l.) h. karst. besides european spruce, other autochthonous and allochthonous spruce species are also found in serbia. they include picea omorika (pancic) purk., picea orientalis (l.) peterm., picea pungens engelm., picea laxa (münchh.) sarg., picea engelmannii parry ex engelm. and picea sitchensis (bong.) carrière. (jovanović, 1970; schmidtvogt, 1974; matović & vujković, 1994; skrøppa, 2003; galović, 2015; cvjetićanin et al., 2016). the natural range of european spruce distribution © 2022 petrov & ocokoljić. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 41 14th symposium on the flora of southeastern serbia and neighboring regions stretches from siberia across the ural mountains and extends to northern europe and high mountain regions of central and southern europe (fukarek, 1970). fukarek (1970) stated that european spruces could not be found in the apennines and pyrenees. however, research conducted in 2016 (di pierro et al., 2016) confirmed the presence of european spruce in the far north of the apennines. in the south of europe, european spruce grows in two directions (cvjetković, 2018): eastwards (from the carpathian mountains, over the high mountains of western serbia and bulgaria to the rhodopes) and westwards (from the slopes of the eastern alps, across the dinaric alps, to the shar mountains and mount korab). however, it should be emphasized that throughout history spruce has undergone a significant shift in its range, as confirmed by pollen analyses according to which european spruce was limited to several refugia during the last glacial period, while the current range has resulted from its expansion from the refugia in the carpathians, dinaric alps and moscow region (huntley & birks, 1983). in serbia, european spruce is widely distributed in the mountains, such as tara, zlatibor, golija, zlatar, prokletije, and kopaonik, while it is not so common in the eastern and southern parts of the country: stara planina, suva planina, kučajske planine, beljanica and shar mountains (ivetić, 2004; banković et al., 2009; cvjetićanin et al., 2016). the range of distribution and genetic structure of european spruce are thought to have been shaped by historical events related to the last glaciation and postglacial colonisation processes (lagercrantz & ryman, 1990). while there was a decline in the natural distribution of european spruce in the past, there was a growing trend in the conversion of broadleaved forest habitats and abandoned agricultural land into coniferous forests. despite the high intrapopulation variability and adaptability of european spruce, abrupt distribution shifts are anticipated due to climate change. they will be reflected in the extinction of populations, primarily the edge populations growing along the distribution boundaries. according to some scenarios, numerous habitats in southern europe will be affected by climate change and european spruce will migrate north (falk & hempelmann, 2013). these predictions should cause some alarm since european spruce is one of the most common coniferous species in european forests; it is also a very important economic species; it has been widely naturalised outside its distribution range; in serbia, it covers 86,400 ha and accounts for about 5.2% of wood volume. bearing in mind the above and the fact that, due to its physical-geographical location, serbia is significantly affected by climate change, the research conducted in european spruce plantations on mount čemernik is of great importance. materials and methods according to morgenstern (1996), research on forest species can be divided into two categories: short-term and long-term tests. our research applied long-term tests of open-grown trees over 30 years of age. the suitability of the selected methods can be proved by kapeller et al. (2013) according to which the tests conducted at the age of 10 to 30 yield the most valuable research results related to species variability and adaptability. our research was conducted in a group of three solitary trees of picea abies (l.) h. karst. the trees were 97 years old (fig. 1). they were planted in the village of brod (42°84’88’’ north latitude and 22°28’39’’ east longitude) in 1925. the village is on mount čemernik, crna trava municipality, at an altitude of 856 m. the five control trees grew in a nearby spruce culture (within a 100 m radius) founded in the 1970s. 42 biologica nyssana ● 13 (1) september 2022: 41-46 petrov & ocokoljić ● dendrometric analyses and determining biological characteristics of needles for the purpose of conservation and directed utilization of the spruce in southeastern serbia fig. 1. three 100-year-old picea abies (l.) h. karst. trees 43 tree height, diameter at breast height (1.30 m), trunk girth, crown diameter, tree age, needle length, needle width, adaptability and vitality of the three analysed trees were taken as indicative values together with the differences between the group of three trees and five control trees that should facilitate the assessment of the species vitality. a total of 400 needles were collected for each tree (i.e. three 100-year-old trees and five control trees) which means that 3200 dimensions were measured for the length and another 3200 dimensions for the width of needles (fig. 2). biologica nyssana ● 13 (1) september 2022: 41-46 petrov & ocokoljić ● dendrometric analyses and determining biological characteristics of needles for the purpose of conservation and directed utilization of the spruce in southeastern serbia quantitative characteristics that included tree height, diameter at breast height, trunk girth, crown diameter and needle length and width (tab. 1, tab. 2) were biometrically processed using the statistica 10.0 software program and their limit of variability (minimum-maximum), medium values ( x ), standard deviations (s), coefficients of variation (v) and errors (s x , ss, sv) were determined. results and discussion field research in the municipality of crna trava in the village of brod recorded three trees of picea abies (l.) h. karst. of exceptional growth habit, excellent vitality and high decorative values. the three trees growing in a group stood out for their differential properties. tab. 1 shows the results of a comparative analysis of the growth elements of three investigated european spruce trees and five control trees. tree 1 reached a height of 52.9 m, a diameter at breast height of 118 cm and a crown diameter of 11.98 m. tree 2 reached a height of 53.1 m, a diameter at breast height of 103 cm and a crown diameter of 17.64 m. tree 3 reached a height of 50.94 m, a diameter at breast height of 84 cm and a crown diameter of 11.24 m. all three trees were around 100 years old and had abundant to moderately abundant yields and the highest ratings of vitality and decorativeness (5 and 5). they had broadly pyramidal crowns and shallow-cracked gray to gray-brown bark. the five control trees at the age of about 50 years old, which is the age suitable for the comparison of values, had significantly different dendrometric characteristics. the average parameters for the five control trees were as follows: trunk height – 24.16 m, diameter at breast height – 63.8 m, trunk girth – 2.01 m, and crown diameter – 8.2 m. fig. 2. analysed parameters of picea abies (l.) h. karst: length (l) and width (w) tree tree age (years) h (m) d1,30 (cm) trunk girth (m) crown diameter (m) fructification vitality (1-5) decorative value (1-5) 1 97 53.1 118 3.71 11.98 medium 5 5 2 97 52.9 103 3.24 17.64 abundant 5 5 3 97 50.94 84 2.66 11.24 abundant 5 5 4 ̴50 19.8 49 1.54 5.76 medium 3 2 5 ̴50 27.3 86 2.69 9.92 abundant 5 5 6 ̴50 26.6 74 2.33 8.74 abundant 5 4 7 ̴50 23.48 53 1.68 10.22 medium 4 4 8 ̴50 23.6 57 1.79 6.38 medium 3 3 table 1. comparative characteristics of three 100-year-old picea abies (l.) h. karst. trees and five trees planted in the 1970s 44 the analysis of statistical parameters (tab. 2) for the two characteristics of needles measured in trees at the age of 100 determined that the three investigated trees had the limit values of needle length ranging from 12 to 32 mm and from 0.8 to 2 mm for needle width. on the other hand, the five control trees aged 50 had the limit values ranging from 8 to 25 mm for needle length and from 0.5 to 1.5 mm for needle width. according to the literature (gajić & korać, 1972; ocokoljić & ninić-todorović, 2003; gebauer et al., 2011; caudullo et al., 2016; gebauer et al., 2019), spruce needles can be up to 25 mm long and more than 1 to 2 mm wide. as can be observed, the three investigated trees have significantly higher values. according to the results, the mean values of the needle length obtained for tree 1 were 22.49±0.05, tree 2 22.17±0.05, and tree 3 22.96±0, 06. the mean values of the needle width obtained for tree 1 were 1.26±0.00 for tree 2 1.11±0.00, and tree 3 1.22±0.00. on the other hand, the mean values of the needle length obtained for the five control trees (trees 4, 5, 6, 7 and 8) were 16.4±0.01 and the mean values of the needle width were 0.86±0.00. the obtained difference between the mean values was statistically significant. looking at the differential characteristics of the biologica nyssana ● 13 (1) september 2022: 41-46 petrov & ocokoljić ● dendrometric analyses and determining biological characteristics of needles for the purpose of conservation and directed utilization of the spruce in southeastern serbia table 2. statistical parameters of morphological characteristics of european spruce needles in the village of brod locality tree age (years) limit value ± s±ss v±sv (а) needle length (mm) tree 1 97 12-32 22.49 ± 0.05 3.25 ± 0.79 0.14 ± 0.01 tree 2 97 15-32 22.17 ± 0.05 3.21 ± 0.78 0.14 ± 0.01 tree 3 97 14-30.5 22.96 ± 0.06 3.25 ± 0.81 0.14 ± 0.00 tree 4 ̴50 9-21 15.19 ± 0.04 2.61 ± 0.54 0.17 ± 0.01 tree 5 ̴50 8-27 18.53 ± 0.05 3.68 ± 0.66 0.20 ± 0.01 tree 6 ̴50 10-25 18.04 ± 0.04 2.44 ± 0.64 0.13 ± 0.00 tree 7 ̴50 10-20 15.42 ± 0.04 2.20 ± 0.55 0.14 ± 0.00 tree 8 ̴50 8-20 14.83 ± 0.04 2.69 ± 0.52 0.18 ± 0.01 (b) needle width (mm) tree 1 97 0.8-2 1.26 ± 0.00 0.28 ± 0.04 0.22 ± 0.01 tree 2 97 0.9-2 1.11 ± 0.00 0.24 ± 0.04 0.22 ± 0.01 tree 3 97 0.8-15 1.22 ± 0.00 0.74 ± 0.04 0.60 ± 0.02 tree 4 ̴50 0.5-1.2 0.80 ± 0.00 0.19 ± 0.03 0.24 ± 0.01 tree 5 ̴50 0.5-2 1.02 ± 0.00 0.29 ± 0.04 0.28 ± 0.01 tree 6 ̴50 0.5-1.5 0.93 ± 0.00 0.18 ± 0.03 0.19 ± 0.01 tree 7 ̴50 0.5-1 0.77 ± 0.00 0.17 ± 0.03 0.22 ± 0.01 tree 8 ̴50 0.5-1 0.80 ± 0.00 0.21 ± 0.03 0.27 ± 0.01 group of three european spruce trees about 100 years old, it can be observed that these trees are more adaptive and ornamental than those planted 50 years later and growing in forest conditions. the data presented in tab. 1 and tab. 2 points to obvious differences in the analyzed properties between the three veteran trees and the five control trees. these differences can affect the cultivation of european spruce since the three selected veteran trees that stand out for their distinctive characteristics have considerable potential. as plus trees with a superior phenotype, they enable more successful and easier cultivation, due to better growth, higher level of adaptability, and remarkable decorativeness (compact crown formed from the ground level). further comparison of the phenotypic characteristics of the investigated 100-year-old spruce trees in the village of brod with literature data (caudullo et al., 2016; cvjetićanin et al., 2016) reveals that the analysed trees have reached maximum values, and thus can be used as starting material in the production of forest and ornamental seedlings. based on the conducted research, which included the age of trees, their vitality, decorativeness and position (sunny location for sciophilic european spruce), and their historical significance, all three trees are proposed to be protected as a botanical 45 biologica nyssana ● 13 (1) september 2022: 41-46 petrov & ocokoljić ● dendrometric analyses and determining biological characteristics of needles for the purpose of conservation and directed utilization of the spruce in southeastern serbia caudullo, g., tinner, w., de rigo, d. 2016: picea abies in europe: distribution, habitat, usage and threats. european atlas of forest tree species. publications office of the european union. luxembourg. 114-116 p. cvjetićanin, r., brujić, j., perović, m., stupar, v. 2016: dendrologija. univerzitet u beogradu, šumarski fakultet. 557 p. cvjetković, b. 2018: genetičko-fiziološka varijabilnost smrče (picea abies karst.) u testovima potomstva u bosni i hercegovini. doktorska disertacija. univerzitet u beogradu, šumarski fakultet. 351 p. di pierro, e.a., mosca1, e., rocchini, d., binelli, g., neale, d.b., la porta, n. 2016: climate-related adaptive genetic variation and population structure in natural stands of norway spruce in the southeastern alps. tree genetics & genomes, 12(2): 1-16. falk, w., hempelmann, n. 2013: species favourability shift in europe due to climate change: a case study for fagus sylvatica l. and picea abies (l.) karst. based on an ensemble of climate models. journal of climatology, 2013: 1-18. fukarek, p. 1970: areali rasprostranjenosti bukve, jele i smrče na području bosne i hercegovine. akademija nauka i umjetnosti bosne i hercegovine. knjiga 11: 235-256. gajić, m., korać, m. 1972: varijabilnost četina smrče (picea excelsa l.) u odnosu na različita staništa na planini goliji. simpozijum povodom proslave 50-godišnjice osnivanja i rada šumarskog fakulteta, beograd: 33-37. galović, v., šijačić-nikolić, m., šafhauzer, r., čortan, d., orlović, s. 2015: genetic differantion of norway spruce (picea abies (l) karst.) trees with differnet crown types from the montain golia. genetika, 47(3): 849–861. gebauer, r., volařík, d., urban, j., børja, i., nagy, n., drabløs, t., krokene, p. 2019: effects of mild drought on the morphology of sun and shade needles in 20-year-old norway spruce trees. iforest biogeosciences and forestry, 12: 27-34. gebauer, r., volarík, d., urban, j., børja, i., nagy, n., eldhuset, t., krokene, p. 2011: effect of thinning on anatomical adaptations of norway spruce needles. tree physiology, 31: 1103-1113. huntley, b., birks, h.j.b. 1983: an atlas of past and present pollen maps for europe, 0-13.000 years ago. cambridge university press. 650 p. ivetić, v. 2004: uticaj staništa i provenijencija na natural monument. conclusions in the village of brod in the municipality of crna trava, three european spruce trees were recorded at the age of 97. our research revealed great vitality and exceptional morphological characteristics of the three european spruce trees. this finding pointed out the necessity of preserving the trees so that their genetic potential can be fully exploited. the study of these 100-year-old trees also revealed their potential role in forestry. they can be of great significance in the production of planting material that would make a valuable forest resource with long-term adaptability to local environmental conditions. the analysed european spruce trees can also be greatly utilised in landscape architecture and horticulture where they can serve as starting material in breeding, pollen collection, controlled hybridisation and vegetative propagation, as well as in the identification of a new lower taxon of compacta type. the analyzed 100-year-old trees should also be protected in order to preserve the national biodiversity of species for which indigenous species are extremely important. they are also important for genetic variation, i.e. as well-adapted genotypes of specific phenotype, they greatly contribute to genetic diversity. their significance is supported by research studies that stress the issue of european spruce extinction due to climate change, and the issue of reproductive material transfer which is usually unknown, and in europe, it is mostly from geographically and ecologically undefined sources. therefore, effort must be put in to find new resistant and productive genotypes and through plant breeding, preserve the species and increase its productivity. the exquisite habit, outstanding features, high condition ratings, age, and historical significance these three 100-year-old spruce trees have for čemernik and serbia make them worthy candidates for a botanical natural monument designation. acknowledgements. the ministry of education, science and technological development is financing the scientific research work of the university of belgrade faculty of forestry in 2022 on the basis of the agreement on the realization number: 451-03-9 / 2022-14 / 200169. references banković, s., medarević, m., pantić, d., petrović, n. 2009: nacionalna inventura šuma republike srbije šumski fond republike srbije. ministarstvo poljoprivrede šumarstva i vodoprivrede republike srbje, uprava za šume. beograd. 244 p. razvoj juvenilnih kultura smrče (picea albies /l./ karst) na goliji. magistarski rad. univerzitet u beogradu, šumarski fakultet. beograd. jovanović, b. 1970: rod picea a. dietr. in: josifović m. (ed.), flora sr srbije, 1. srpska akademija nauka i umetnosti, beograd. kapeller, s., schüler, s., huber, g., božič, g., wohlgemuth, t., klumpp, r. 2013: provenance trials in alpine range – review and perspectives for applications in climate change. in: cerbu, g.a., hanewinkel, m., gerosa g., jandl, r. (eds.), management strategies to adapt alpine space forests to climate change risks: 233-256, in tech. lagercrantz, u., ryman, n. 1990: genetic structure of norway spruce (picea abies), concordance of morphological and allozymic variation. evolution, 44: 38-53. matović, m., vujković, lj. 1994: nova forma smrče (picea abies /l./ karst.) i njene ekološko-morfološke i dekorativne karakteristike. savetovanje “zelenilo u urbanističkom razvoju grada beograda”, zbornik radova, udruženje inženjera beograda: 277 284. 46 biologica nyssana ● 13 (1) september 2022: 41-46 petrov & ocokoljić ● dendrometric analyses and determining biological characteristics of needles for the purpose of conservation and directed utilization of the spruce in southeastern serbia morgenstern, e. 1996: geographic variation in forest trees, genetic basis and application of knowledge in silviculture. ubc press. vancouver. 214 p. ocokoljić, m., ninić-todorović, j. 2003: priručnik iz dekorativne dendrologije. univerzitet u beogradu, šumarski fakultet. beograd. 169 p. powo the international plant names index and world checklist of selected plant families 2022. published on the internet at http://www.ipni.org and http://apps.kew.org/wcsp/ schmidt-vogt, h. 1974: das natürliche verbreitungsgebiet der fichte (picea abies (l.) karst.) in eurasien. allgemeine forstund jagdzeitung, 145: 185–197. skrøppa, t. 2003: euforgen technical guidelines for genetic conservation and use for norway spruce (picea abies). international plant genetic resources institute. rome. 6 p. vasić et al. 2025, biologica nyssana 16(1) 129 16 (1) june 2025: 129-135 doi: 10.46793/biolnyss.16.1.9v fusarium oxysporum, the cause of fusarium wilt on cucumber (cucumis sativus l.) in serbia original article tanja vasić faculty of agriculture, university of niš, kosančićeva 4, 37000, kruševac, serbia tanjavasic82@gmail.com (corresponding author) sanja živković faculty of agriculture, university of niš, kosančićeva 4, 37000, kruševac, serbia sonja filipović faculty of agriculture, university of niš, kosančićeva 4, 37000, kruševac, serbia debasis mitra department of microbiologoy, graphic era, 566/6, bell road, clement town, dehradun, uttarakhand 248002, india darko jevremović fruit research institute, čačak, serbia received: november 13, 2024 revised: february 28, 2025 accepted: march 06, 2025 abstract: cucumber (cucumis sativus l.) is a vegetable crop with a global production of approximately 87 million tons in 2021, according to fao data. it is also an important vegetable species cultivated in both greenhouses and open fields in serbia. one of the most devastating diseases affecting cucumbers is fusarium wilt, caused by fusarium oxysporum. infected older plants may exhibit yellowing starting from the oldest leaves, stunted growth, or wilting. affected plants can develop lesions on the lower stem. the fungus infects the vascular system, and symptoms may remain unnoticed until the plant begins to bear fruit. this paper presents the results of a preliminary study on fusarium wilt in cucumbers in serbia. samples were collected in 2022 from the trebotin locality, kruševac. a total of 15 samples from mirabelle f1 hybrid cucumber plants were analyzed. our research has shown that, based on morphological, molecular, and pathogenic characteristics, cucumber wilting in serbia is caused by f. oxysporum. the morphological characteristics of the isolates (appearance, size, and septation of macroand microconidia) were as follows: microconidia dimensions ranged from 3.8–15.5 µm×2.8– 4.9 µm, macroconidia from 14.6–37.9 µm×2.4–5.6 µm, with the number of septa ranging from 1 to 4. pcr analysis using f. oxysporum-specific primers resulted in the amplification of the expected 315 bp fragment in all tested isolates. the pathogenicity test showed that symptoms of fusarium wilt initially appeared as yellowing of the oldest leaves. the disease progressed, eventually leading to plant wilting and death. the characterization and confirmation of f. oxysporum on cucumber in serbia is a significant finding, emphasizing the need for an effective disease management program to reduce yield losses. key words: cucumber, fusarium oxysporum apstrakt: fusarium oxysporum – prouzrokovač fuzarioznog uvenuća krastavca (cucumis sativus l.) u srbiji krastavac (cucumis sativus l.) je povrtarska kultura čija je globalna proizvodnja, prema podacima fao-a za 2021. godinu, iznosila oko 87 miliona tona. takođe, predstavlja značajnu povrtarsku vrstu koja se u srbiji gaji u plastenicima i na otvorenom. jedna od najrazornijih bolesti krastavca je fuzariozno uvenuće, koje izaziva fusarium oxysporum. infekcija kod starijih biljaka može izazvati žutilo, koje počinje od najstarijih listova, usporavanje rasta ili uvenuće. pogođene biljke mogu razviti lezije na donjem delu stabla. gljiva napada i sprovodne sudove, zbog čega zaražene biljke ne pokazuju simptome sve dok ne počnu da plodonose. u ovom radu predstavljamo rezultate preliminarnih istraživanja fuzarioznog uvenuća krastavca u srbiji. uzorci su prikupljeni 2022. godine na lokalitetu trebotin, kruševac. analizirano je ukupno 15 uzoraka hibridnih biljaka krastavca mirabelle f1. naše istraživanje je pokazalo da, na osnovu morfoloških, molekularnih i patogenih karakteristika, uvenuće krastavca u srbiji izaziva gljiva f. oxysporum. morfološke karakteristike izolata (izgled, veličina i septacija makroi mikrokonidija) bile su: dimenzije mikrokonidija 3,8–15,5×2,8–4,9 µm, makrokonidija 14,6–37,9×2,4–5,6 µm, dok je broj septi varirao od 1 do 4. pcr analiza sa f. oxysporum-specifičnim prajmerima rezultirala je amplifikacijom očekivanog fragmenta od 315 bp u svim testiranim izolatima. test patogenosti pokazao je da su se simptomi fuzarioznog uvenuća najpre ispoljili kao žutilo starih listova. bolest je napredovala, a zaražene biljke su venule i na kraju uginule. karakterizacija i potvrda f. oxysporum na krastavcu u srbiji predstavlja značajan nalaz za razvoj efikasnijih strategija upravljanja ovom bolešću kako bi se smanjili gubici prinosa. ključne reči: krastavac, fusarium oxysporum © 2025 vasić et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 130 introduction cucumber (cucumis sativus l.) is a herbaceous plant belonging to the cucurbitaceae family. it is widely used as a vegetable in human diets and is considered an annual plant. native to south asia, cucumber is now cultivated across most continents (sharma & shukla, 2001; akrami, 2015; hazirah et al., 2020). in serbia, cucumber is an important crop grown both in open fields and greenhouses. due to the limited availability of agricultural land and high market demand, continuous cucumber cultivation is widely practiced. a large number of pathogenic organisms parasitize cucumber plants, causing significant economic damage. the most important pathogens affecting cucumber include bacteria, viruses, and fungi. the major bacterial pathogens are pseudomonas syringae (angular leaf spot), xanthomonas campestris (bacterial leaf spot), erwinia tracheiphila (bacterial wilt) (kaiser & ernst, 2017). among viral pathogens, cucumber green mottle mosaic virus (cgmmv), squash mosaic virus (sqmv), and watermelon mosaic virus (wmv) are the most significant. phytoplasma aster yellows also causes severe economic losses in cucumber production (kaiser & ernst, 2017). however, one of the main constraints in cucumber production, leading to yield reduction and quality deterioration, are phytopathogenic fungi. the most important fungal diseases affecting cucumbers include pseudoperonospora cubensis (downy mildew), erysiphe cichoracearum (powdery mildew), botrytis cinerea (gray mold), phomopsis sclerotioides (root rot), sclerotinia sclerotiorum (white mold), didymella bryoniae (gummy stem blight and black rot), colletotrichum orbiculare (anthracnose), and fusarium oxysporum (fusarium wilt). fusarium oxysporum is a soilborne fungal pathogen that causes fusarium wilt in cucumbers, leading to significant yield losses worldwide. this disease affects the plant's vascular system, disrupting the transport of water and nutrients, ultimately resulting in wilting and plant death (sharma & shukla, 2001; al-tuwaijri, 2015). fusarium oxysporum is considered one of the most problematic and destructive pathogens in cucumber production (ye et al., 2004). it is transmitted through soil and water (sharma & shukla, 2001; shen et al., 2008; al-tuwaijri, 2015). cucumber wilt is widespread across many countries, with yield losses ranging from 40% to 70%. characteristic symptoms include plant weakening before and after the first visible signs of infection, stunted growth, chlorosis, necrosis, and eventual wilting of older leaves and entire plants. symptom development is also accompanied by vascular tissue discoloration and plant death (vakalounakis & fragkiadakis, 1999; akrami, 2015; wang et al., 2015; al-tuwaijri, 2015; hazirah et al., 2020; maymon et al., 2020; aparicio et al., 2023). given the increasing importance of cucumber cultivation in serbia, this research aimed to identify the causal agent of fusarium wilt in cucumbers to improve understanding of its impact (plant death, yield reduction, and quality deterioration) and to develop more effective disease management strategies. materials and methods the samples of cucumber plants were collected in the village of trebotin (near kruševac) on the terzić family's estate during the summer vegetation period of 2022. during sample collection, cucumber plants (mirabelle f1 hybrid) exhibiting symptoms of wilting, chlorosis of the oldest leaves, and vascular necrosis were taken (fig. 1). a total of 15 samples were collected. stem and root segments were washed under running water for 10 minutes and then cut into 1 cm long fragments. these fragments were surface disinfected by immersion in a 5% sodium hypochlorite (naocl) solution for five minutes and then rinsed three times in sterile distilled water, each time for five minutes. potato dextrose agar (pda) enriched with 300 µl/l gentamicin sulfate was used for pathogen isolation (dhingra & sinclair, 1995). on sterile media in petri dishes, cucumber plant fragments were aseptically placed, with five fragments per petri dish. the inoculated petri dishes were incubated in a thermostat at 24±2 °c in the dark until fungal colonies developed around the fragments. the mycelium completely covered the petri dish within 7 days. a total of 15 isolates were obtained, and five were selected for further research: fos1, fok2, fok3, fom6, and fot1. morphological features morphological characteristics of selected isolates of fusarium spp. were studied on a pda substrate according to the method of burgess et al. (1994). dimensions of microscopic structures were calculated based on 30 measurements for macroconidia, microconidia and chlamydospores morphology (shape, color), size (length and width) was based on the descriptions indicated by nelson et al. (1983). individual germinating conidia were selected, transferred directly to pda plates according to the procedures described by burgess biologica nyssana ● 16 (1) june 2025: 129-135 vasić et al. ● fusarium oxysporum, the cause of fusarium wilt on cucumber (cucumis sativus l.) in serbia 131 biologica nyssana ● 16 (1) june 2025: 129-135 vasić et al. ● fusarium oxysporum, the cause of fusarium wilt on cucumber (cucumis sativus l.) in serbia fig. 1. symptoms of fusarium wilt on cucumber in the field et al. (1994) and stored on pda in tubes at 4 °c. colony morphology (color, shape and growth rate) was determined after 7 days of incubation on pda at 25 °c in darkness. images were captured by an olympus sc100 color camera mounted on a cx31 microscope (olympus, japan). pathogenic characteristics in order to determine the pathogenic characteristics of selected isolates obtained from infected cucumber plants, a pathogenicity test was performed by inoculating injured cucumber plants (mirabelle f1 hybrid) (vasić, 2007). four-week-old cucumber seedlings were used. before inoculation, all plants were wounded at the base of the stem with a sterile scalpel. in this way, the injured plants were inoculated by placing small fragments of the colony of studied isolates in the wounds. inoculation was performed with isolates of the obtained fungi, 7 days old, grown on pda medium. plants inoculated in this way were stored at a temperature of 20-25 °c. the experiment was set up in two replicates with 5 plants per isolate. cucumber plants that were injured in the same way served as controls, after which they were inoculated with small fragments of the substrate without mycelia. the appearance of symptoms was monitored three, five and ten days after inoculation. molecular detection and identification colonies of tested isolates were grown on pda in the dark, at a temperature of 25 °c for 7 days. dna extraction was performed according to day & shattock (1997). in the first step, the mycelium was scraped from the surface of the substrate with a sterile spatula and placed in a 2.0 ml tube with liquid nitrogen. after evaporation of liquid nitrogen, 800 μl of 2% ctab buffer was poured into the tube and incubated for 1 h at 65 °c. during the incubation, every 15 min the contents of the tube were vigorously shaken. after incubation, 800 μl of chloroform was added into the microtube and vortexed, then centrifuged for 10 min at 11,000 rpm in a centrifuge at 4 °c (eppendorf 5804 r, germany). the supernatant (about 700 μl) was transferred by pipetting into a new 1.5 ml tube, and 0.6 volume (about 420 μl) of isopropanol was added. the mixture was centrifuged for 15 min at 11,000 rpm at 4 °c. after centrifugation, the supernatant was carefully decanted and the tubes were washed with 1 ml of ice-cold 70% ethanol and dried for 10-15 min at room temperature. after drying, the dna pellet was resuspended in 100 μl of te buffer. for the detection of five tested fusarium spp. isolates a pcr method with a f. oxysporum spe132 biologica nyssana ● 16 (1) june 2025: 129-135 vasić et al. ● fusarium oxysporum, the cause of fusarium wilt on cucumber (cucumis sativus l.) in serbia cific primers primer pairs fc-1 (5’-cataccacttgttgcctc-3’) and fc-2 (5’-attaacgcgagtcccacc3-’) was performed (zhang et al., 2010). pcr analysis was performed in a t-personal thermocycler (biometra, germany) in 20 μl reaction volume containing 1 μl of dna, 2 μl of 10× reaction buffer, 2 μl 2.5 mm dntps, 1.2 μl 25 mm of mgcl2, 0.2 μl 10 mm of fc-1 and fc-2 primer, and 0.2 μl 5u of taq dna polymerase (thermoscientific, usa) and 13.2 μl nuclease-free water. a negative control contained all pcr reaction compounds except dna template. pcr cycling conditions are as follows: initial denaturation at 94 °c for 5 min, followed by 40 cycles with denaturation for 30 s at 94 °c, annealing for 1 min at 60 °c and extension for 60 s at 72 °c, with final extension step at 72 °c for 10 min. pcr products were analyzed in 1.5% agarose gel that was stained with ethidium bromide. pcr fragments were visualized in the gel doc ez system (biorad, usa) using a uv tray. results and discussion during the vegetation period of 2022, serious symptoms of wilting were observed in cucumber plantations in the village of trebotin (near kruševac). initial symptoms in the form of yellowing were observed on older leaves, and lesions in the lower part of the tree were also observed in infected plants. on the cross-section of the stems of infected plants, necrosis of the vascular tissues was observed, which spread to the roots. infections in cucumber plants caused stunting and most often wilting. morphological characteristics morphological characteristics of selected isolates of fusarium spp. were studied on pda media with 7-day-old fungal cultures. colony growth rate of five studied isolates of fusarium spp. on the pda substrate did not differ; on average, for all five isolates, the increase was 5.5 cm after three days. the mycelium completely covers the petri dish fig. 2. appearance of 7-day-old cultures on pda medium, f. oxysporum, isolate fok2, face (a), reverse (b), chlamydospores (c), microconidia and macroconidia (d). scale bar 20 μm. 133 biologica nyssana ● 16 (1) june 2025: 129-135 vasić et al. ● fusarium oxysporum, the cause of fusarium wilt on cucumber (cucumis sativus l.) in serbia fig. 3. electrophoretic analysis of pcr with fc-1/ fc-2 primer pair. lines: negative control, 1 fos1 isolate, 2 fok2 isolate, 3 fok3 isolate, 4 fom6 isolate, 5 fot1 isolate, m marker 100 bp ladder (solis biodyne, estonia) within 5–7 days. the color of the colonies varied from creamy whitish to white to light salmon. some isolates produced salmon-colored pigments in the colonies after 15 to 20 days of incubation at 25 °c on pda medium. all studied isolates formed macroconidia, microconidia and chlamydospores on the pda medium (fig. 2). macroconidia (2.4– 5.6 x 14.6–37.9 μm) are tapered and curved to almost straight with a slight hook, thin-walled with usually three septa (1–4) and are produced from monophialides. the microconidia (2.8–4.9 x 3.8– 15.5 μm) are oval or elliptical , usually non-septate and form in false heads from short monophialides. chlamydospores (8-14 μm), both smooth and rough, thick-walled, are produced intercalary or terminal in the hyphae and form abundantly in most isolates, usually in pairs or singular (fig. 2). morphological determination of phytopathogenic fungi, in this case fusarium spp., plays a very important role in the early stage of disease detection. this approach is considered more cost-effective but requires trained personnel. therefore, the morphological identification of phytopathogenic fungi is the primary approach and the most challenging step in the identification process, especially for species of the fusarium genus (hazirah et al., 2020). these studies showed that, based on morphological characteristics, the fungi isolated from infected cucumber plants belong to the genus fusarium, more precisely to the species f. oxysporum, and are in accordance with the findings published by nelson et al. (1983), burgess et al. (1994), and leslie & summerell (2006). vakalounakis & fragkiadakis (1999), wang et al. (2015), and hazirah et al. (2020) determined that the pigmentation of f. oxysporum on pda ranged from white to light salmon. the microconidia had conical apical cells and basal cellshaped feet, and they were oval and kidney-shaped without septa, which was confirmed in our research. pathogenic characteristics on the fifth day after inoculation, all tested isolates of fusarium spp. exhibited typical symptoms, including wilting of the oldest leaves. lesions were also observed on the infected plants in the lower part of the stem. with the passage of time, the symptoms on the infected plants were more intense and brown necrosis was observed on the vascular tissue. no changes were observed in the control plants. in their research, vakalounakis and fragkiadakis (1999), akrami (2015), wang et al. (2015), al-tuwaijri (2015), hazirah et al. (2020), maymon et al. (2020), and aparicio et al. (2023) reported that various f. oxysporum formae on artificially inoculated cucumber plants caused symptoms such as chlorosis molecular detection and identification of fungi in pcr analysis with fc-1/fc-2 primer pair a fragment of 315 bp was obtained for all five tested isolates from cucumber (fig. 3). no amplification occurred in the negative control. fc-1/fc-2 primer designed by zhang et al. (2010) proved to be reliable for the detection of f. oxysporum isolates from cucumber in serbia. this primer pair was successfully applied for specific detection of f. oxysporum in different hosts. the specificity of the primers was confirmed by analyzing f. oxysporum isolates and 33 isolates of other fungal species infecting cucumbers. the primers successfully detected f. oxysporum isolates from cucumber, water melon and melon (zhang et al., 2010). also, using this primer pair f. oxysporum can be reliably detected from dna extracted from rhizosphere soil (zhang et al., 2012). conclusion based on morphological, pathogenic, and molecular characteristics, it has been determined that the fungus isolated from cucumber belongs to the species fusarium oxysporum, which is becoming an increasing problem in commercial production and of the oldest leaves, wilting, and necrosis of vascular tissue, which is consistent with our findings. this confirms that our isolates belong to the species f. oxysporum. 134 biologica nyssana ● 16 (1) june 2025: 129-135 vasić et al. ● fusarium oxysporum, the cause of fusarium wilt on cucumber (cucumis sativus l.) in serbia may threaten cucumber cultivation in serbia. in our country, these are preliminary results that highlight the significance of this issue, which is why further research will continue. the confirmed presence of f. oxysporum in cucumbers emphasizes the need for greater attention in selecting seed material. this includes the use of cucumber hybrids resistant to fusarium, avoiding continuous planting of cucumbers on the same field for multiple growing seasons, applying beneficial microorganisms such as trichoderma spp. to suppress fungal growth, ensuring proper irrigation, preventing plant injuries, and using disinfection agents. acknowledgements. this work was supported by the ministry of science, technological development and innovation of the republic of serbia, contracts 451-03137/2025-03/200383 and 451-03-136/2025-03/200215. references akrami, m. 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(2012). molecular detection of fusarium oxysporum in the infected cucumber plants and soil. pakistan journal of botany, 44(4), 1445-1451. zhang, s.m., zhao, x.y., zhang, x.c., meng, l.q, li, j., zhang, y.h., & wang, y.x. (2010). molecular detection of fusarium oxysporum in cucumber, watermelon and melon. acta phytopathologica sinica, 40(6), 636-641. morho-anatomical differentiation of the populations of daphne biologica nyssana 7 (1)  september 2016: 1-9 jušković, m. et al.  morpho-anatomical differentiation of the populations… 1 original article received: 28 june 2016 revised: 24 july 2016 accepted: 01 september 2016 morpho-anatomical differentiation of the populations of daphne cneorum l. (thymelaeaceae) from serbia marina jusković1*, perica vasiljević1, ana savić1, dragana jenačković1, branka stevanović2 1university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 nis, serbia 2 university of belgrade, faculty of biology, institute of botany and botanical garden “jevremovac”, takovska 43, 11000 belgrade, serbia * e-mail: marinaju@pmf.ni.ac.rs abstract: jušković, m., vasiljević, p., savić, a., jenačković, d., stevanović, b.: morho-anatomical differentiation of the populations of daphne cneorum l. (thymelaeaceae) from serbia. biologica nyssana, 7 (1), september 2016: 1-9. in this study was analyzed inter-population diffrentiation of the species daphne cneorum l. on the basis of morphoanatomical variability of its three different, geographically distant populations, two from eastern serbia and one from western serbia. the analyzed populations are situated in wide range of altitudes, as well as different geological substrates. the analysis of variance included 20 quantitative characters related to the leaf and stem anatomy and morphology. the results of morphoanatomical studies have shown the presence of general adaptive characteristics of the xeromorphic type. multivariate analysis have shown a clear distinction between the carbonate populations of d. cneorum from eastern serbia (mt. rtanj and mt. suva planina) and the serpentine populations from western serbia (mt. zlatibor). key words: daphne cneorum, anatomy, morphology, differentiation, serbia apstrakt: jušković, m., vasiljević, p., savić, a., jenačković, d., stevanović, b.: morfo-anatomska diferencijacija populacija vrste daphne cneorum l. (thymelaeaceae) iz srbije. biologica nyssana, 7 (1), septembar 2016: 1-9. u ovom radu analizirana je interpopulaciona diferencijacija vrste daphne cneorum na osnovu varijabilnosti morfoloških i anatomskih karakteristika listova i stabla, tri prostorno udaljene populacije, dve iz istočne i jedne iz zapadne srbije. analizirane populacije smeštene su u širokom rasponu nadmorskih visina i pokazuju preferencije prema različitim geološkim podlogama. statističkom analizom je obuhvaćeno 20 kvantitativnih karaktera anatomije i morfologije lista i stabla. analizom morfo-anatomskih karakteristika listova i stabla i staništnih uslova ustanovljeno je da d. cneorum pripada adaptivnom tipu kserofita. multivarijantne analize su pokazale da se analizirane populacije na osnovu ispitivanih karaktera klasifikuju u dve grupe. veća međusobna sličnost je prisutna između jedinki iz populacija koje rastu na karbonatu i na većoj nadmorskoj visini, u odnosu na jedinke populacije sa serpentinske podloge i niže nadmorske visine. key words: daphne cneorum, morfologija, anatomija, diferencijacija, srbija 7 (1) • september 2016: 1-9 doi: 10.5281/zenodo.158966 biologica nyssana 7 (1)  september 2016: 1-9 jušković, m. et al.  morpho-anatomical differentiation of the populations… 2 introduction daphne cneorum l. is one of seven species of the genus daphne l. native to serbia (b l e č i ć , 1972). it belongs to section daphnanthes c.a. mayer, subsection cneorum, together with d. arbuscula, d. juliae, d. petraea, d. striata (k e i s s l e r , 1898). species d. cneorum distributed in west, central and east europe, mediterranean region, south–west asia (w e b b & f e r g u s o n , 1968; m e u s e l et al., 1978). in serbia, d. cneorum is taxon with disjunct disribution and it is limited to the mountainous areas (b l e č i ć , 1972) (fig. 1). the species inhabits mountainous areas, between 700 and 1800 m a.s.l., prefers serpentinite substrate, but it was also recorded on limestone and on acid siliceous soil, usually on stony sites in the zone of seslerion rigidae, seslerio-festucion, euvaccinio-piceenion, orno-ericenion serpentinicum, poion violaceae, festucion rupicolae, ramondion nathaliae alliances (j u š k o v i ć , 2012). d. cneorum is an evergreen fruticose reptant chamaephytes (fo semp mi ch frut rept). the species d. cneroum has become one of the most popular perennial flowering shrubs, because of their desirable horticultural characteristics (b r i c k e l l & w h i t e , 1978; h a l d a , 2001). comparative morpho-anatomical studies have involved three different, geographically distant populations, two from eastern serbia and one from western serbia. the studied populations grew under different climate conditions. the habitats of populations from the area of western serbia (mt. zlatibor) are influenced by humid temperatecontinental climate (type 2.1 s t e v a n o v i ć & s t e v a n o v i ć , 1995; vi 2b w a l t e r & l e i t h , 1964), with mountain influences, or the transitional variant of temperate-continental and mountain climate of middle european type (2.1/4.1 s t e v a n o v i ć & s t e v a n o v i ć , 1995; vi 2b/x 1 w a l t e r & l e i t h , 1964). the populations that inhabit the eastern parts of serbia (mt. suva planina, mt. rtanj) are inflenced by the continental climate (2.2/4.2) this region is dominated by the sub-type of the semi-arid moderately-continental climate, also known as sub-continental climate (type 2.2 sensu s t e v a n o v i ć & s t e v a n o v i ć , 1995; vi 3 sensu w a l t e r & l e i t h , 1964). studies so far on the anatomy of thymelaeaceae and genus daphne have been limited. m e t c a l f e & c h a l k (1950) give a general description of the anatomical structure of vegetative organs from the family thymelaeaceae as well as the genus daphne. the species of genus daphne contains significant variation in native habitats, morphology and use (b r i c k e l l & w h i t e , 1978). the aim of this study was to determine variability of morpho-anatomical characteristics of the leaves and stems of the plants of species d. cneorum, as well as possible trends of differentiations among populations in serbia. material and methods the species of daphne cneorum were collected from western (mt. zlatibor -serpentinite) and from eastern serbia (mt. suva planina, mt. rtanj limestone), during the flowering season in 2008 (tab. 1). the collected plant material was either placed in the herbarium or fixed in 50% ethanol. a voucher specimen was deposited in the herbarium moesiacum niš, department of biology and ecology, faculty of sciencesand mathematics, university of niš, serbia (hmn), (tab. 1). fig. 1. distribution of the species d. cneorum in serbia. biologica nyssana 7 (1)  september 2016: 1-9 jušković, m. et al.  morpho-anatomical differentiation of the populations… 3 soil analysis soil samples were collected from 3 different sites of each of the three population localities. each analyzed sample contained 300 g of dried, sieved soil. the ph value was determined in a soil suspension with potassium chloride and in one with water (10g : 25cm3), using a potentiometer. the caco3 content was determined by the volumetric method, using the scheibler’s calcimeter. the humus content was determined after turin’s method, while that of nitrogen was calculated on the basis of humus values. the available phosphorus was measured by the method of a l l e n (1940) using a spectrophotometer, whereas the available potassium was determined according to the method of allen on a flame photometer. morpho-anatomical analysis the total of 20 quantitative characteristics subjected to statistical analysis was grouped into two categories: i morphometric characteristics: ii meristic characteristics. anatomical sections of leaves and stems was performed on permanent and temporary slides, prepared by the standard histological method for light microscopy (r u z i n , 1999). all measurements were done with on the microscope (leica dm 250-leica dfc490, leica qwin standard (leica microsystem, germany). leaf epidermis, trichomes and stomata were analyzed by a scanning electron microscope (sem, jeol 5300). statistical analysis the obtained results of measurements were analyzed in the statistical package systat 12 (systat software inc. 2007). for each quantitative character was performed basic descriptive statistics: mean, standard deviation, minimum and maximum values. one-way analysis of variance was used in order to calculate statistical significance for analyzed morpho-anatomical characters. principal component analysis (pca), canonical discriminant analysis (cda) and clustering method based on mahalanobis’ distances were used to determine the variability structure and level of importance of the anatomical differentiation of investigated populations. results soil analysis d. cneorum inhabits carbonate, silicate, as well as serpentine substrates (b l e č i ć , 1972). variations in edaphic features, the thickness of the soil, texture, ph values, concentration of minerals, are factors which table 1. ecological characteristics of the habitats and number of specimens of the analyzed populations d. cneorum. locality population biogeography coordinate habitat substratum altitude (m a.s.l.) voucher mt. rtanj, šiljak rtanj moesian province (ce) 42. 39 n, 19. 39 e subalpine pastures limestone 1450-1570 hmn-5515 mt. suva planina, devojački grob trem suva planina moesian province (ce) 43. 11 n, 22. 10 e rocky grounds limestone 16471765 hmn-5516 mt. zlatibor, šainovci zlatibor illirian province (ce) 43. 40 n, 19. 41 e pastures serpentinite 1000 hmn-5514 table 2. the results of physico-chemical analyses of the soil samples. the table contains detail informations on ph values, hardness and content of nutrients in soils on the studied habitats. locality ph caco3 (%) humus (%) n (%) p2o5 (mg/g) k2o (mg/g) mt. rtanj 6.65 15.81 11.56 0.59 12.4 >40 mt. suva planina 6.3 0.72 10.26 0.51 3.6 >40 mt. zlatibor 5.99 1.42 7.79 0.39 1.2 20.5 biologica nyssana 7 (1)  september 2016: 1-9 jušković, m. et al.  morpho-anatomical differentiation of the populations… 4 influence the distribution of plants (s t e v a n o v i ć & j a n k o v i ć , 2001). the results of soil analysis of the three different sites inhabited by d. cneorum are presented in tab. 2. the ph of soil varied from slightly acidic to neutral. soil is with low content of carbonates on mts. zlatibor and suva planina, and with high content on mt. rtanj. the greatest values of organic matter were recorded in the subalpine fig. 3. epidermal impressions of d. cneorum leaves: (a, d) mt. rtanj, (b, e) mt. suva planina, (c, f) mt. zlatibor: a-c adaxial surface, with various shapes of sinuous walls epidermal cells; d-f abaxial epidermal cells with straight or slightly sinuous anticlinal walls and anomocytic stomata (s). pastures soil in the mt. rtanj. chemical analysis shows that there is a sufficient amount of nitrogen. the amount of phosphorus in the soils from the studied localities varied from low to sufficient. concentration of potassium is sufficient. leaf shape and anatomy the leaves of all the analyzed plants species d. cneorum are evergreen, simple, narrow, oblanceolate, dark green, with a margin entire and densely arranged towards the end of the twigs. they are with a tapering base and broadly wedge-shaped apex, ending in a minute bristle-like tip. indumentum is absent from leaves (fig. 2). the leaf surface area of the analyzed plants from all localities varied, ranging between 28 mm2 and 67 mm2, whereas the leaf length was 12 and 22 mm, and the leaf width ranged between 2,5 mm and 4,6 mm (tab. 3). such microphyllous leaves reduce transpiration rate and represent an important adaptive response of the plants to the conditions of environmental water deficit (s t e v a n o v i ć & j a n k o v i ć , 2001). the highest leaf surface values were from mt. zlatibor, while the lowest were in plants from mt. suva planina (fig. 2.; tab. 3). fig. 4. cross section of the leaves of plants. (a) mt. rtanj, (b) mt. suva planina, (c) mt. zlatibor, (d) details of the transverse section of the upper leaf epidermis, (e) transverse section through the midrib, (f) details of the transverse section of the lower leaf epidermis. cut cuticule, mc mucilaginous cells, pp palisade parenchyma, vb vascular bundles, sp spongy parenchyma, s stomata, xy xylem, ph phloem. fig. 2. the leaf shapes of d. cneorum: (a) mt. rtanj, (b) mt. suva planina, (c) mt. zlatibor. biologica nyssana 7 (1)  september 2016: 1-9 jušković, m. et al.  morpho-anatomical differentiation of the populations… 5 the anticlinal cell walls of adaxial epidermis of leaves are wavy with shallow or deep amplitudes, while the anticlinal cell walls of abaxial epidermis are straight to slightly undulate (fig. 3). the undulating anticlinal walls represent a mesomorphic, while straight ones a xeromorphic characteristic (f a h n & c u t e r , 1992). the leaves are bifacial or of dorsiventral symmetry (fig. 4). their thickness in all the populations studied ranged between from 238 µm to 395 µm (tab. 3). the epidermis of the leaf is singlelayered, the adaxial epidermis is thicker than the abaxial epidermis and with well developed cuticule (fig. 4). table 3. results of one-way analysis of variance for comparing means of morphometric and meristic characteristics in three populations of d. cneorum. (* p<0.05). principal component analysis (pca) of measured parameters of the analyzed populations of d. cneorum. the first three principal components accounted for 63.28% of the variance. population mt. rtanj mt. suva planina mt. zlatibor anova p-value pca 1 pca 2 pca 3 character mean ± sd mean ± sd mean ± sd leaf thickness (µm) 324.81±45.33 316.24±27.82 319.69±32.43 0.65 0.50 0.75 -0.12 height of adaxial epidermal cells (µm) 35.27±4.66 34.98±5.49 33.70±5.11 0.45 0.44 0.74 -0.14 thickness of palisade tissue (µm) 132.75±25.19 126.18±21.28 122.33±19.23 0.19 0.39 0.69 0.03 thickness of spongy tissue (µm) 121.06±19.17 109.81±17.92 114.43±16.46 0.06 -0.39 0.22 0.12 height of abaxial epidermal cells (µm) 23.65±2.66 22.34±2.92 21.44±2.30 0.01* -0.44 0.08 0.38 surface area of adaxial epidermal cells (µm2) 2105.49±295.47 2003.01±234.59 2375.47±345.88 0.00* 0.37 0.16 -0.02 surface area of abaxial epidermal cells (µm2) 932.23±166.27 843.79±126.34 860.07±112.93 0.03* 0.47 -0.07 0.26 surface area of abaxial stomata (µm2) 1039.11±87.85 977.76±102.26 1079.10±86.38 0.00* 0.08 -0.13 0.10 leaf length (mm) 15.43±1.96 14.53±1.47 18.61±1.70 0.00* -0.07 0.44 0.37 distance between point of largest leaf width and leaf top (mm) 3.71±0.57 3.88±0.56 3.39±0.59 0.51 0.83 -0.37 0.16 largest width of leaf (mm) 3.45±0.46 3.51±0.61 3.62±0.35 0.38 0.81 -0.35 0.18 leaf surface area (mm) 43.67±9.31 39.28±7.93 51.01±7.92 0.00* 0.64 -0.22 -0.07 length of leaf nervature mm/mm2 9610.52±1162.67 9030.30±1135.57 9204.97±822.88 0.10 0.05 -0.20 -0.54 stem diameter (µm) 1405.49±202.91 1420.04±122.69 1396.40±130.73 0.84 0.19 0.24 -0.28 stem peridermis thickness (µm) 27.73±2.86 24.37±1.83 24.56±2.04 0.00* -0.04 0.36 0.44 stem cortex thickness (µm) 257.16±25.75 284.97±34.46 264.12±31.12 0.00* -0.05 0.06 -0.71 number of palisade layers 3.43±0.50 3.33±0.61 3.50±0.68 0.56 0.22 -0.31 0.41 number of abaxial stomata 131.28±18.57 163.18±23.41 163.12±22.59 0.00* 0.15 -0.41 -0.13 biologica nyssana 7 (1)  september 2016: 1-9 jušković, m. et al.  morpho-anatomical differentiation of the populations… 6 the epidermal cell walls are mucilaginous. this type of epidermis is characteristic of the family thymelaeaceae, as well as the genus daphne (m e t c a l f e & c h a l k , 1950) (fig. 4 a-d). mucilaginous epidermal cells are usually present mainly in the upper epidermis. thickened walls of the epidermal cells, well-developed cuticle and mucilaginous cells can prevent rapid water loss and are considered an ecological characteristic for plant species of sunny and dry sites (b r e d e n k a m p , 1999; b r e d e n k a m p & v a n w y k , 2001). those adaptive strategies limit water loss during periods of drought and such structural adaptations are of major importance to survival in the xerothermic habitat conditions. leaves of all studied populations are hypostomatic (fig. 4, 5). the number of stomata in leaves ranges from 95 to 223 per mm2 (tab. 3). the fig. 5. scanning electron micrographs showing epidermis of d. cneorum leaves, stomata on abaxial leaf surface. (a) mt. rtanj; (b) mt. suva planina (sem). fig. 6. cross section of the one-year old stem of d. cneorum (in primary state of growth) from mt. suva planina. (a) stem with leaves, (b) the cross section stem, (c, d) details of the transverse section of the stem. e epidermis, c stem cortex, p pith, sc sclerenchyma cells, vc vascular cylinder, xy xylem, ph phloem. biologica nyssana 7 (1)  september 2016: 1-9 jušković, m. et al.  morpho-anatomical differentiation of the populations… 7 stomata complexes are anomocytic, i.e., real subsidiary cells are absent (fig. 3). there is high wrinkled cuticule around the stomata (fig. 5). the same type of of stomata were found and described in other species of the genus daphne (j u š k o v i ć , 2010; j u š k o v i ć , 2012). the mesophyll is differentiated into spongy and palisade tissues. the palisade tissue is multilayered, composed of typically elongated, densely arranged cells, with very small intercellular spaces (fig. 4). incresead toughness of the leaves provides a considerably more efficient water transport and photosythetical light utilization. the cells of spongy parenchyma are usually irregularly shaped. the structure of leaves is involved in the general physiology of plant activity, it clearly explains the morphological adaptation of plants to specific environmental conditions (s t e v a n o v i ć & j a n k o v i ć , 2001). according to f a h n & c u t l e r (1992) reduced leaf size, increased thickness, deeply sunken stomata, and palisade developed at the expense of spongy mesophyll are common features of plants grown in xeric environments. fig. 7. the result of canonical discriminant analysis (cda). table 4. summary of canonical discriminant analysis. wilks’ lambda is the ratio of within-groups sums of squares to the total sums of squares. the f value for a variable indicates its statistical significance in the discrimination between groups. (* p<0.05) wilks's lambda lambda 0.09 df (18. 2. 87) approx. f-ratio 8.70 df (36. 140) p-value 0.00 variable f-to-remove tolerance leaf thickness 0.81 0.14 thickness of palisade tissue 0.34 0.22 thickness of spongy tissue 3.25 0.34 height of adaxial epidermal cells 0.31 0.80 height of abaxial epidermal cells 2.38 0.80 surface area of adaxial epidermal cells 2.15 0.84 surface area of abaxial epidermal cells 7.46 0.70 surface area of abaxial stomata 1.17 0.78 number of palisade layers 0.12 0.85 number of abaxial stomata 9.08 0.78 length of leaf nervature 1.41 0.73 leaf surface area 6.90 0.13 leaf length 27.38 0.18 largest width of leaf 2.03 0.35 distance between point of largest leaf width and leaf top 0.88 0.75 stem diameter 0.73 0.82 stem peridermis thickness 7.65 0.89 stem cortex thickness 2.66 0.81 biologica nyssana 7 (1)  september 2016: 1-9 jušković, m. et al.  morpho-anatomical differentiation of the populations… 8 stem shape and anatomy d. cneorum is a low-growing, evergreen shrub, with a great number of long, slender, downy branches and reddish brown bark (fig. 6). the cross-sections of one year-old stems ranged from rounded to elliptical in shape (fig. 6). stem diameter of analysis plants ranged between 1396 μm and 1420 μm (tab. 3). the epidermis is unilayered and covered by a conspicuous cuticle with unicellular trichomes (fig. 6a). underneath the epidermis, the collenchyma consists of several cell layers (fig. 6c). the cortical parenchyma consists of cells with a circular or polygonal shape and slightly thickened walls. the vascular tissue is well developed and forms a ring around pith. at the outer side of phloem, the sclerenchyma cells also form rings (fig. 6d). the parenchymatous pith has circular or polygonal shaped cells with thin walls. the correlation coefficient of the cortex and the whole stem diameter ranges from od 0,366 (mt. rtanj), 0,378 (mt. zlatibor) to 0,401 (mt. suva planina), which was within the usual values found in xeromorphic stems (f a h n & c u t l e r , 1992). these characteristics are in agreement with the data obtained from other studied daphne species (j u š k o v i ć , 2010; j u š k o v i ć , 2012). finally, leaf and stem anatomical features are closely linked to the habitat of species d. cneorum, indicating that they represent ecologically important adaptations. on the other hand, phenotypic plasticity among populations of the same plant species is one of the important mechanisms for their adaptation to heterogeneous habitats, and it is one of the major way by which plants are able to cope with environmental variability (j u š k o v i ć et al., 2010; l a k u š i ć et al., 2010; j a k o v l j e v i ć et al., 2013; g r a t a n i , 2014). there is statistical significant differences among populations according to some morpho-anatomical features: height of abaxial epidermal cells, surface area of adaxial epidermal cells, surface area of abaxial epidermal cells, surface area of abaxial stomata, leaf length, leaf surface area, stem peridermis thickness, stem cortex thickness and number of abaxial stomata among populations (tab. 3, 4). the results of pca analysis showed that the three principal components explain 63.28% of the total variability (tab. 3, 4). the largest contribution to the formation of variability provides the first principal component with 32.78%, followed second with 17.29% and the third with a 13.51% variation. the greatest significance in the formation of variability have characters that are related to leaf shape (leaf surface area, largest width of leaf, distance between point of largest leaf width and leaf top) and leaf anatomy (leaf thickness, height of adaxial epidermal cells, thickness of palisade tissue). stem cortex thickness is present in the structure of the variability in the third principal component. the results of canonical discriminant analysis of the population of the species d. cneorum show the existence of separation among the studied populations (fig. 7). population with mt. zlatibor is on the positive side of the first axis and is clearly separated from the population with mt.rtnja and mt. suva planina. less discrimination is present in populations from eastern serbia, which are the negative side of the first axis. the most important characters (tab. 4) in total discrimination among populations are characters related to the leaf shape, leaf epidermis and stem peridermis thickness. multivariate analysis (pca and cda) and cluster analysis (fig. 8) have shown that studied populations can be classified into two groups. one group includes populations (mt. rtanj and mt. suva planina) that grow on carbonate substrate, and at a higher altitude (1500-1700 m a.s.l.), while the second group includes plants from the population (mt. zlatibor), growing on serpetinite substrate and the lower altitude (1000 m a.s.l.). conclusion the investigated populations of the species daphne cneorum have shown certain differences in structure. the results of morpho-anatomical studies of the leaves and stems, and conditions on habitats, have shown the presence of general adaptive fig. 8. mahalnobius distances among three populations of daphne cneorum. biologica nyssana 7 (1)  september 2016: 1-9 jušković, m. et al.  morpho-anatomical differentiation of the populations… 9 characteristics of the xeromorphic type. the study provides useful information on the morphoanatomical and ecological characteristics of species d. cneorum. acknowledgements. the study was supported by the ministry of education, science and technological development of the republic of serbia (grant no. 173030). references blečić, v. 1972. daphne. in: josifović, m. 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(eds.), flora europaea, vol. 2: 256– 258. cambridge university press, cambridge. aleksić et al. 2024, biologica nyssana 15(1) 15 (1) june 2024: 13-21 doi: 10.5281/zenodo.11517688 anticlostridial activity of the dill seed essential oil (anetum graveolens l.): antibiofilm activity and antisporulation potential original article ana aleksić university of niš, faculty of sciences and mathematics, department of biology and ecology ana.aleksic@pmf.edu.rs (corresponding author) predrag stojanović university of niš, faculty of medicine, serbia institute for public health nis, center of microbiology, serbia zorica stojanović-radić university of niš, faculty of sciences and mathematics, department of biology and ecology received: september 26, 2023 revised: april 08, 2024 accepted: may 21, 2024 abstract: clostridioides difficile is an anaerobic, spore-forming pathogen that causes serious toxin-mediated enteric disease in humans. in addition to antimicrobial resistance, biofilm and spore formation play key roles in the persistence of c. difficile in the gut, as well as in the transmission and relapse of the disease. in this study, the antimicrobial potential of dill seed essential oil on the planktonic growth of c. difficile clinical strains (isolated from stool specimens of hospitalized patients with diarrhea and confirmed clostridioides difficile infection (cdi)) was investigated, along with its effect on biofilm and spore formation. the results showed varying degrees of antimicrobial activity, ranging from strong to weak, depending on the strain, with concentrations ranging from 0.08 to 40 mg/ml. the essential oil (eo) at concentrations of 2xmic and mic significantly reduced biofilm production in 89% and 84% of the tested strains, respectively. spore formation was also significantly reduced when treated with 0.5xmic and mic of eo. considering the anticlostridial activity of the dill seed eo, along with its inhibition of biofilm production and sporulation, this natural product is an excellent candidate for supplementary treatment of cdi. key words: dill, essential oil, clostridioides difficile, antibiofilm activity, antisporulation activity apstrakt: antiklostridijska aktivnost etarskog ulja semena mirođije (anethum graveolens l.): antibiofilmska aktivnost i antisporulativni potencijali clostridioides difficile je anaerobni sporogeni patogen koji je uzročnik ozbiljnih crevnih, toksinima uzrokovanih, bolesti kod ljudi. pored antimikrobne rezistencije, formiranje biofilma i spora može igrati ključnu ulogu u perzistenciji c. difficile u crevima, kao i u prenosu i recidivu bolesti. u ovoj studiji, ispitivan je antimikrobni potencijal etarskog ulja semena mirođije na rast kliničkih sojeva c. difficile (izolovanih iz uzoraka stolice hospitalizovanih pacijenata sa dijarejom i potvrđenom clostridioides difficile infekcijom (cdi)), kao i efekat ulja na formiranje biofilma i spora. rezultati su pokazali različite stepene antimikrobne aktivnosti, u rasponu od jake do slabe, u zavisnosti od soja, sa koncentracijama u rasponu od 0.08 do 40 mg/ml. etarsko ulje (eo) u koncentraciji 2xmic i mic značajno je smanjilo proizvodnju biofilma kod 89% i 84% testiranih sojeva, respektivno. formiranje spora je takođe značajno smanjeno, kada je tretirano sa 0.5xmic i mic eo. uzimajući u obzir antiklostridijsku aktivnost eo semena mirođije, inhibiciju proizvodnje biofilma i sporulacije, ovaj prirodni proizvod se klasifikuje kao odličan kandidat za dopunski tretman cdi. ključne reči: mirođija, etarsko ulje, clostridioides difficile, antibiofilmska aktivnost, antisporulaciona aktivnost introduction clostridioides difficile is an anaerobic sporeforming pathogen that is causative agent of serious nosocomial toxin-mediated enteric disease worldwide. the disease is manifested with a wide range of severity, from mild, self-limiting diarrhea to pseudomembranous colitis and even toxic megacolon © 2024 aleksić et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 13 which can be life-threatening and often results in death (awad et al., 2014). in a healthy individuals, human microbiota working in synergy and thus provides protection from pathogens. clostridioides difficile establish itself in the host when the gut microbiota is disturbed, usually after antibiotic treatment administered for other conditions. the bacterial species produces spores under stress conditions, which are a dormant form of the bacterium. spores are the infectious particle critical for c. difficilemediated infection and transmission (awad et al., 2014). after entering the host in the spore form, c. difficile passes through the stomach into the small intestine, where it begins the germination process, transitioning into the metabolically active vegetative cell form. toxin production then occurs, leading to host tissue damage and disease (awad et al., 2014; smits et al., 2016; schäffler & breitrück, 2018). in addition to antimicrobial therapy, which disrupts the intestinal microbiota and enables the unhindered germination of spores and release of toxins, it has been confirmed that recurrent infections can be unequivocally linked to the production of biofilms in certain isolates of c. difficile. fact that recurrence of cdi is attributed to the strain which initially caused disease (figueroa et al., 2012), indicates that the pathogen somehow avoids treatment with antibiotic therapy. biofilm and spore formation may be a key mechanisms of c. difficile persistence in the gut as well as transmission and relapse (frost et al., 2021; normington et al., 2021). antimicrobial resistance in biofilm can increase from 10 to 1000 times more compared to planktonic bacteria (wei et al., 2018). recent research suggests that c. difficile biofilms can serve as a niche for generating modified spores, which favor maintenance of a dormant cells, aiding bacterial persistence and disease recurrence (frost et al., 2021). antimicrobial drugs can lead to sporulation and biofilm formation due to unfavorable environment, so it seems that preventing development of these structures in the host intestine could be promising approach in the control of pathogen survival. to successfully control cdi, in addition to antibiotic treatment, it may be beneficial to consider new strategies and approaches aimed at countering c. difficile’s ability to develop resistance to conventional treatments and its subsequent proliferation. natural products could serve as good source of anticlostridial agents (phanchana et al., 2021). bioactive compounds derived from essential oils could provide an opportunity to discover novel potentially effective molecules to combat this problem. the diverse of chemical composition allows essential oils to exhibit multiple mechanisms of action in reducing bacterial cell viability including effect on ph homeostasis and equilibrium of inorganic ions, inhibition of nadh oxidation, and/or structural and functional damage of the cell membrane (tortajada-girbés et al., 2021). therapeutic agents that are capable of reducing c. difficile spore production (mooyottu et al., 2017) and biofilm formation (normington et al., 2021) could significantly minimize relapse of cdi and consequent transmission. anetum graveolens l. (commonly known as dill, family apiaceae), is an aromatic annual plant originated from mediterranean and west asia widely used as spice and medicinal herb. dill is commonly utilized in food and pharmaceutical industries and frequently used to treat various health problems, among other gastrointestinal disorders, flatulence and gastro-intestinal spasms (chahal et al., 2017; ozliman et al., 2021). essential oil can be extracted from various parts of plant such as seeds, leaf and flower. studies on the chemical composition of dill seed essential oil reported that major compounds were carvone and limonene whereas dill apiole, trans-dihydrocarvone and α-phellandrene were present in appreciable amounts (chahal et al., 2017). based on the reviewed published studies on the antimicrobial activity of a. graveolens seed essential oil, significant to moderate antibacterial activity was found against both gram-positive and gramnegative bacteria, as well as against fungi (elgayyar et al., 2001). to date, limited literature is available on the use of natural products against c. difficile (hammond & donkor, 2013; finegold et al., 2014; justin & antony, 2016; aljarallah, 2016; cermak et al., 2017; roshan et al., 2017; piotrowski et al., 2017; harnvoravongchai et al., 2018; wultańska et al., 2020; aleksić et al., 2022). the aim of this study was to evaluate whether natural antimicrobial components of the essential oils of well-known and widely used herb anetum graveolens l. (eng. dill) could influence growth, biofilm formation and sporulation in selected c. difficile strains isolated from patients with confirmed cdi. materials and methods test microorganisms in total 42 clinical strains (designated as cd1 – cd42) of c. difficile were isolated from stool specimens of hospitalized patients with diarrhea and cdi on the territory of serbia. reference strains, c. difficile atcc 9689 (a+b+cdt−), c. difficile atcc 43593 (a−b−cdt−) and c. difficile atcc-baa 1870 (a+b+cdt+) were used as control strains. the presence of genes that encode toxins was confirmed by multiplex pcr. based on capillary 14 biologica nyssana ● 15 (1) june 2024: 13-21 aleksić et al. ● anticlostridial activity of the dill seed essential oil (anetum graveolens l.): antibiofilm activity and antisporulation potential biologica nyssana ● 15 (1) june 2024: 13-21 aleksić et al. ● anticlostridial activity of the dill seed essential oil (anetum graveolens l.): antibiofilm activity and antisporulation potential 15 gel-based electrophoresis, 6 different ribotypes were determined. the most prevalent ribotypes were rt 001 (20/42; 48%) and rt 027 (18/42; 43%), while rts 012, 015, 020 and 205 were represented by only one isolate (2.4%). all the tested isolates are sensitive to metronidazole, vancomycin and tigecycline with mic50 values of 0.25, 0.38, and 0.016 µg/ml, respectively. among 42 strains tested, 36 showed resistance to moxifloxacin. two strains (cd4 and cd13) are resistant to rifampicin (with mics 4 and 32 µg/ml, respectively) in addition to moxifloxacin resistance (aleksić et al., 2022). essential oils dill seeds essential oil (anetum graveolens l.) (alekpharm, serbia) obtained by steam distillation method was purchased at a local health food market. as stated in the manufacturer’s specification, the main components of dill seed essential oil are carvone, limonene and (z)-dihydro carvone. determination of minimum inhibitory concentration (mic) and minimum bactericidal concentration (mbc) the minimum inhibitory concentrations (mics) of essential oil were determined using the broth microdilution method in a 96-well plate according to described methodology (wultańska et al., 2020), with slight modifications. suspensions were made from the 24 h old c. difficile culture on columbia 5% blood agar and adjusted to 3 mcfarland turbidity by using a densitometer (den-1, biosan). an initial stock solution was prepared by dissolving essential oil in 50% dimethyl sulfoxide (dmso) in concentration of 200 mg/ml. serial dilutions were made in brain heart infusion (bhi) broth (himedia, mumbai, india), with starting concentration of 40 mg/ml, which was serially diluted (dilution factor 2) to the final concentration of 0.02 mg/ml. then, bhi broth (180 µl) containing the decreasing concentrations of essential oils was inoculated with suspension of the tested c. difficile strains (20 µl, 3 mcfarland turbidity, 10-fold dilution of the 3 mcfarland in each well) and incubated anaerobically at 37 °c for 48 h. inoculated bhi broth without essential oil was used as a positive control, while the wells containing sterile bhi broth were used as negative control. the dmso was tested previously and showed no effect toward the tested strains in used concentrations (10% dmso and lower concentrations). after incubation, the mics were determined visually as the lowest concentrations without visible growth in the medium. the minimum bactericidal concentration (mbc) was determined by transferring the suspension from the wells with no visible growth (containing essential oils in mic and higher concentrations) to 5% columbia agar plates. plates inoculated in this way were incubated at 37 °c for 48 h under anaerobic conditions, and after this period the plates with the lowest concentration of oil with no grown colonies were determined as mbcs. anti-biofilm production assay to investigate the potential of the isolated strains to form biofilm, the tested isolates were first subjected to biofilm forming ability crystal violet (cv) assay as previously described (aleksić et al., 2022). the wells of the microtiter plates containing 180 µl of the bhi broth containing 0.5% yeast extract (torlak, serbia) and 1% glucose (centrohem, serbia) were inoculated with 20 µl of suspension (3 mcfarland) prepared as described for microdilution method, to achieve the final concentration of ~106 cfu/ml. the plates were incubated at 37 °c for 72 h under anaerobic conditions afterwards the well content was aspirated, and the wells were washed twice with phosphate saline buffer (ph 7.4). wells with bhi broth without the inoculums were used as the controls. then the plates were dried, stained with 1% (w/v) solution of cv for 15 min, washed and destained by addition of 250 μl of ethanol (96%, v/v). following 30 min of destaining procedure, the obtained solutions were transferred into a new microtiter plate and the absorbance of each well content was measured at 595 nm using an elisa reader (multiscan ascent, labsystems, finland). according to their biofilm producing ability, the strains were classified into the following groups: none, weak, moderate, and strong biofilm producers (stepanović et al., 2007; tijerina-rodríguez et al., 2019). the potential of dill seeds essential oils to inhibit biofilm production of the investigated c. difficile strains was done by using the same procedure. the only difference was that the wells contained three different inhibitory concentrations of the essential oil (0.5 x mic, mic and 2 x mic) together with inoculum and cultivation media. the experiment was performed in triplicate and the inhibition was detected as a decreased absorbance at 595 nm compared to positive controls (wells without essential oils). differences in the biofilm formation were calculated using two-way anova and tukey post-hoc analysis (graphpad prism v.6.0.) and considered as statistically significant for p values < 0.05. spore formation assay the effect of the dill seed eo on c. difficile spore formation was performed using a combination of the previously described protocols (garneau et al., 2014; frost et al., 2021) with slight modifications. for the 16 biologica nyssana ● 15 (1) june 2024: 13-21 aleksić et al. ● anticlostridial activity of the dill seed essential oil (anetum graveolens l.): antibiofilm activity and antisporulation potential sporulation test, the reference strains of the most abundant rts in the tested sample were selected (c. difficile atcc-baa 1870/a+b+cdt+/rt 027 and c. difficile atcc 9689/a+b+cdt−/rt001). briefly, bhi with (sub)inhibitory concentration of eo (0.5xmic and mic) was inoculated with 3 mcfarland suspension that is made from 24 h old culture of c. difficile on columbia 5% blood agar (final concentration of ~106 cfu/ml) and incubated anaerobically at 37 °c for 10 days. inoculated wells without addition of eo were used as the controls. a sample (15 μl) from each well of liquid culture was placed to microscope slide, dried at room temperature, fixed by heat and stained according to schaeffer-fulton method (hussey & zayaits, 2016). for the presence of endospores and their quantification, slides were examined using a leica dm 1000 light microscope under the oil immersion lens (1,000x). images from 3 different fields were taken on each slide and were processed with imagej program (schneider et al., 2012). vegetative cells and spores were counted in 3 fields for each dilution. the percentage of spores in each image was calculated as follows: [nspores / (nspores + nvegetative cells)] *100. an experiment was performed in triplicate. the data were analyzed using one-way anova. differences between means were considered to be significant at p<0.05. results and discussion clostridioides difficile infection (cdi) is one of the biggest concerns in current world medical practice. the infections caused by these bacteria are treated primarily with vancomycin, metronidazole, or both, and these drugs have been available for the past 30 years. however, treatment options have significant limitations. importantly, following the treatment, c. difficile infections (cdis) recur in approximately 25% of individuals treated with either vancomycin or metronidazole, and some patients experience multiple recurrences (babakhani et al., 2012). given the limited antimicrobials available for treating cdi and the increase of resistance to these drugs, an antimicrobial agent with strong inhibitory effects on both c. difficile vegetative cells and sporulation seems like a promising approach. this type of antimicrobial agent would be capable to not only treat cdi but also prevent its recurrence (sholeh et al., 2020; chiu et al., 2021). alternative therapeutic agents that can attenuate c. difficile virulence without disrupting the normal gut flora represent a viable control approach against the pathogen. as reported by semenyuk et al. (2014), biofilm cells possess 100-fold greater resistance to the antibiotic metronidazole than do planktonic cells cultured in liquid media. this report suggest that c. difficile cells and spores in biofilms have specialized properties that may facilitate infection (semenyuk et al., 2014). in this context, the bioactive components of essential oils could serve as beneficial and potentially effective alternatives to combat this problem. in recent years, there has been a growing interest in the use of natural products as an alternative in the treatment of numerous infections, primarily due to the belief that they are safe and widely available at low prices. humans have been using natural products as traditional medicine for treating illnesses for centuries. several plant extracts and plant-derived compounds possess antibacterial activity against c. difficile and their action has been investigated (phanchana et al., 2021). processed products, aloe vera gel, peppermint oil, artichoke capsules, and garlic tablets demonstrated antimicrobial activity against c. difficile (roshan et al., 2017). plantderived compounds such as allicin (derived from garlic), cinnamon powder, zingerone (derived from ginger), menthol (derived from peppermint), transcinnamaldehyde (cinnamon bark), δ-3-carene (monoterpene derived from the root of asarum heterotropoides) and some essential oils exhibited a certain anticlostridial potential (phanchana et al., 2021). numerous essential oils (eos) possess antimicrobial activity, but despite their multipurpose and widespread use, only a small fraction is used commercially. the antimicrobial activity of eos can be attributed to their composition, volume and interactions with pathogens. the essential oils affect one or more targets within the pathogen, which is related to different active compounds present in their composition. in most cases, two to three primary constituents of eo account for ≤85% of the biological activity of the oil (el-tarabily et al., 2021), but minor compounds can also be important due to synergistic interaction with the dominant ones (pejčić et al., 2021). based on the data presented in tab. 1, it can be concluded that the tested oil exhibited varying degrees of antimicrobial activity, ranging from strong to weak, depending on the strain, with concentrations ranging from 0.08 to 40 mg/ml. bactericidal activity spanned from 0.08 to complete inactivity. notably, considering the characteristics of the tested strains, as previously described (aleksić et al., 2022), it appears that strong biofilm producers exhibited greater resistance to the tested agent. the antimicrobial tolerance of biofilms has emerged as a significant challenge to medical scientists in various healthcare sectors. most recurrent episodes of cdi are attributed to the original strain/ribotype (figueroa et al., 2012), suggesting biologica nyssana ● 15 (1) june 2024: 13-21 aleksić et al. ● anticlostridial activity of the dill seed essential oil (anetum graveolens l.): antibiofilm activity and antisporulation potential 17 that c. difficile can evade antibiotic treatment and the host’s defense system, possibly by occupying a protective niche in the gut where antibiotic therapy is ineffective (normington et al., 2021). within the biofilm, c. difficile cells undergo metabolic remodeling compared to planktonic cells and have a different array of proteins/organelles on the cell surface (poquet et al., 2018). these facts indicate that production of an increased, dense biofilm could have an important clinical relevance in the treatment failure and recurrence of cdi (vuotto et al., 2016). the formation and development of biofilms is a complicated process that involves different stages which can be the target of natural antibiofilm agents for the prevention of biofilm development (mishra et al., 2020). effects of the tested eo on biofilm formation of c. difficile strains are presented in fig. 1. among the thirty-seven tested strains, thirty-four exhibited reduced biofilm production when treated with 2xmic eo. the minimum inhibitory concentration (mic) of eo effectively inhibited biofilm formation in thirty-two out of the thirty-seven tested isolates. subinhibitory (0.5xmic) concentrations enhanced biofilm formation in 7 tested strains. it is not an unexpected phenomenon, considering that antibiotic resistance in biofilms can increase from 10 to 1000 times compared to planktonic bacteria (wei et al., 2018). in the study of vuotto et al. (2016), when sub-inhibitory concentrations of metronidazole were applied, an increased biofilm production among metronidazole-susceptible c. difficile isolates has been reported (vuotto et al., 2015). this situation could be clinically relevant when there is exposure to low doses of antibiotics, as is the case at the beginning or end of antibiotic therapy, which could possibly explain ineffective treatment (petrof et al., 2013; normington et al., 2021). strains cd3 and cd11 (both strong biofilm producers), showed enhanced biofilm production in all three tested eo concentration. considering significance of spore formation in occurrence, course and prognosis of the cdi, effect of eo on sporulation in two reference strains were table 1. mics and mbcs of the dill eo strain mic mbc strain mic mbc 1cd+ 5.00 5.00 cd21 0.63 0.63 2cd2.50 2.50 cd22 5.00 5.00 3cd027 1.25 2.50 cd23 5.00 10.00 cd1 40.00 >40.00 cd24 0.63 1.25 cd2 40.00 >40.00 cd25 5.00 5.00 cd3 40.00 >40.00 cd26 5.00 10.00 cd4 40.00 >40.00 cd27 2.50 2.50 cd5 20.00 20.00 cd28 1.25 1.25 cd6 20.00 20.00 cd29 1.25 2.50 cd7 40.00 >40.00 cd30 2.50 2.50 cd8 10.00 10.00 cd31 0.08 0.08 cd9 40.00 >40.00 cd32 1.25 1.25 cd10 40.00 >40.00 cd33 0.63 2.50 cd11 20.00 20.00 cd34 0.32 0.32 cd12 10.00 10.00 cd35 2.50 5.00 cd13 20.00 20.00 cd36 0.63 1.25 cd14 5.00 10.00 cd37 2.50 5.00 cd15 5.00 10.00 cd38 1.25 5.00 cd16 2.50 5.00 cd39 2.50 2.50 cd17 20.00 20.00 cd40 2.50 5.00 cd18 10.00 10.00 cd41 1.25 2.50 cd19 5.00 5.00 cd42 2.50 5.00 cd20 2.50 5.00 biologica nyssana ● 15 (1) june 2024: 13-21 aleksić et al. ● anticlostridial activity of the dill seed essential oil (anetum graveolens l.): antibiofilm activity and antisporulation potential 18 fig. 1. biofilm formation by the tested c. difficile strains treated with 0.5xmic, mic and 2xmic of dill seed essential oil for the three defined categories of biofilm formed (strong, weak and moderate). the bars show the average values from the three measurements. the error bars show the standard deviations. an asterisk shows a statistically significant differences (*p< 0.05; **p< 0.01; ***p< 0.001; p< 0.0001) in the average od595 values when the same strain was grown in presence or absence of the essential oils; p<005) fig. 2. effect of dill seed eo on spore production biologica nyssana ● 15 (1) june 2024: 13-21 aleksić et al. ● anticlostridial activity of the dill seed essential oil (anetum graveolens l.): antibiofilm activity and antisporulation potential 19 investigated. the test included two concentrations, reference strains belonging to rt001 and rt027 and prolonged incubation (10 days). in both strains, spore formation was significantly reduced, when treated with 0.5xmic and mic, as well (fig. 2). better activity is exhibited against rt001 strain with more than 70% of reduction when treated with 0.5xmic, while mic concentration reduced the spore number to only 1% of the spore production exhibited by untreated control. up to now, only one study reported the effects of natural products on sporulation of c. difficile (roshan et al., 2018). this research tested 22 natural products and among them, only three (coconut oil, fresh onion bulb extract and fresh ginger rhizome extract) demonstrated inhibitory effects in concentrations of 6.3% (v/v), 8% and 25%, respectively. the inhibition was up to 86% for the fresh onion bulb extract, which is close to our reduction here obtained for 0.5xmic of the tested oil. conclusion clostridioides difficile infection is one of the biggest concerns in current world’s medical practice. along with the appearance of antibiotic resistance, the choice of treatment is complicated by the increase in the disease recurrence rate. the emergence of recurrent cdi could be a consequence of properties of the infectious agent related with biofilm and spores formation, together with antimicrobial resistance. considering exhibited anticlostridial activity and especially observed antibiofilm action and sporulation-reducing potential of the dill seed eo, it can be regarded as an excellent candidate for supplementary treatment of cdi. furthermore, natural extracts and natural product-based agents possess fewer side effects due to their low toxicity levels and to date, no bacterial resistance to this type of antimicrobial agents has been recorded. this is an additional reason to expand research on this subject, pinpoint the active compounds, investigate their efficacy in combination with antibiotics and then, as the final step, examine their effectiveness in vivo. acknowledgements. this work was funded by the ministry of science, technological development and innovations of serbia [no. contract 451-03-47/202301/200124]. references aleksić, a., stojanović-radić, z., harmanus, c., kuijper, e.j., & stojanović, p. 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(2020). the effect of berberine chloride and/or its combination with vancomycin on the growth, biofilm formation, and motility of clostridioides difficile. european journal of clinical microbiology and infectious diseases, 39(7), 1391–1399. https:// doi.org/10.1007/s10096-020-03857-0 mitrović et al., 2019, biologica nyssana 10(2) 10 (2) december 2019: 65-75 doi: 10.5281/zenodo.3600172 serbian spruce (picea omorika (pančić) purkyné) endemicity and advantages review article aleksandra lj. mitrović institute for multidisciplinary research, university of belgrade, kneza višeslava 1, 11000 belgrade, serbia mita@imsi.rs (corresponding author) jelena bogdanović pristov institute for multidisciplinary research, university of belgrade, kneza višeslava 1, 11000 belgrade, serbia mala@imsi.rs jasna simonović radosavljević institute for multidisciplinary research, university of belgrade, kneza višeslava 1, 11000 belgrade, serbia jasna@imsi.rs lloyd donaldson scion, private bag 3020, rotorua 3010, new zealand lloyd.donaldson@scionresearch.com ksenija radotić institute for multidisciplinary research, university of belgrade, kneza višeslava 1, 11000 belgrade, serbia xenia@imsi.rs received: july 09, 2019 revised: october 12, 2019 accepted: december 06, 2019 abstract: conifers, as a response to mechanical stress, such as wind and stem lean, form reaction wood called compression wood (cw). cw occurs in a range of gradations from near normal wood (nw) to severe cw (scw). as the severity of cw affects the mechanical and chemical properties of wood, and as cw has limited value in the forest products industry, it is desirable to be able to measure cw severity. picea omorika belong to slow-growing conifer species in which cw typically occurs in a severe form. we developed different morphometric and non-morphometric methods for estimation of cw severity tested on wood samples of p. omorika juvenile trees exposed to long term static bending. this specific review is aimed at presenting p. omorika as one of the most adaptable spruces, and as a good model for testing of methods for estimation of compression wood severity. first, we summarize main knowledge about p. omorika, features of cw, and methods for assessment of wood quality. then, we present breifly our recently published methods for estimation of compression wood severity tested on p. omorika juvenile wood samples. key words: cell wall, cellulose fibrils, compression wood, fluorescence-detected linear dichroism microscopy, double wall thickness apstract: omorika (picea omorika (pančić) purkiné) endemičnost i perspektive konifere kao odgovor na mehanički stres (vetar, savijanje) formiraju reakciono drvo koje se naziva kompresiono drvo (cw). cw se javlja u nizu gradacija od skoro normalnog drveta (nw) do jako izraženog cw (scw). s obzirom da stepen izraženosti osobina cw ima značajan uticaj na mehaničke i hemijske osobine drveta i da cw ima ograničenu vrednost za drvnu industriju, poželjno je moći odrediti stepen izraženosti osobina cw u uzorku. picea omorika spada u sporo rastuće četinarske vrste kod kojih se cw tipočno javlja u jako izraženoj formi. mi smo razvili nekoliko morfometrijskih i ne-morfometrijskih metoda za procenu izraženosti osobina cw u uzorku, testiranih na uzorcima drveta juvenilnih stabala p. omorika koja su bila izložena dugotajnom statičkom savijanju. ovaj revijski rad ima za cilj da predstavi pančićevu omoriku kao jednu od najadaptabilnijih smrča i kao dobar model za testiranje metoda za procenu izraženosti osobina cw u uzorku. u prvom delu sumiramo znanja o pančićevoj omorici, osobinama cw i metodama za procenu kvaliteta drveta, a u drugom ukratko predstavljamo naše nedavno objavljene metode za za procenu izraženosti osobina cw u uzorku, testirane na uzorcima drveta juvenilnih stabala p. omorika. ključne reči: ćelijski zid, celulozni fibrili, kompresiono drvo, fluorescentna konfokalna mikroskopija, debljina ćelijskog zida introduction picea omorika (pančić) purkynĕ is a a rare and endangered tertiary relict and endemic species (jovanović, 1970). despite its endemism, p. omorika is considered as one of the most adaptable spruces (sevill et al., 2017). conifers, as a response to mechanical stress, form reaction wood called compression wood (cw) (timell, 1986). cw occurs in a range of gradations from near normal wood (nw) to severe cw (scw). the degree of development of particular features of cw does not necessarily change in parallel to each other, so the severity of a given tracheid is represented as a function of the degrees of development of individual features, mainly lignification, helical cavi© 2019 mitrović et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 65 13th symposium on the flora of southeastern serbia and neighboring regions ties and cell wall thickness (yumoto et al., 1983). as cw has limited value in the forest products industry it is of great importance to be able to measure cw severity (altaner et al., 2009). in recent years we worked on the development of different morphometric and non-morphometric methods for distinguishing wood samples on the compression severity scale. they are based on tracheid double wall thickness (nedzved et al., 2018), cellulose microfibrils order (savić et al., 2016), or variation in lignin structure (mitrović et al., 2015). we used confocal fluorescence microscopy and spectroscopy, combined with development of additional equipment, new algorithms and statistical analysis. we tested our methods on stem samples of p. omorika juvenile trees exposed to long term static bending. picea omorika belongs to slow-growing conifer species, its wood is characterized by small, densely packed tracheids, while cw typically occurs in a severe form (timell 1986; donaldson et al., 2004). juvenile conifer wood is characterized by randomly distributed mild compression wood (mcw), nw often being absent (donaldson et al., 2004). these are the features that suggest p. omorika juvenile wood as a good choice of samples for evaluation of the precision of methods suggested for estimation of compression wood severity. our methods for distinguishing wood samples on a compression severity scale provide a fine gradation from nw to the severest form of cw, compression severity scales being partially different. these methods, alone or in combination with each other, could be a useful tool for fine gradation of wood samples on the compression severity scale, either in the estimation of wood quality or environmental influences during growth and developmental process. they confirm juvenile p. omorika stem samples as a good choice of samples for evaluation of the methods suggested for compression wood severity estimation. picea omorika: endemicity, natural range, habitat, planting outside its natural range picea omorika (pančić) purkynĕ is a slow growing endemic coniferous species and tertiary relict of the european flora. its natural habitat is fragmented and reduced to the middle and upper courses of the drina river, in western serbia and eastern bosnia and herzegovina (jovanović, 1970, sevill et al., 2017). the species was widespread in europe and asia, but after the pleistocene glaciations, this region represents species long-term, cryptic and last refugium (aleksić & geburek, 2014). an asian origin of serbian spruce has been recently confirmed (lockwood et al., 2013), grouping p. omorika with the caucasian p. orientalis, and the two japanese endemics p. alcoquiana and p. maximowiczii. serbian spruce ancestors appeared in asia at the end of the neogene, but the increasing seasonality and aridity during the late miocene led to the extinction of picea in the mid latitudes of eurasia. until the middle of the 19th century, the natural range of p. omorika was more continuous and less fragmented than it is today (sevill et al., 2017). it has been legally protected since 1964. its current distribution is on one side the result of anthropogenic factors such as general forest clearance and harvesting for timber, pastoralism and wildfires (jovanović, 1986; sevill et al., 2017). fire has perhaps been the biggest threat, and logging has been a subsidiary one (sevill et al., 2017). on the other side the limited natural range of serbian spruce is the result of the species poor competing ability. it retreats to areas less inhabitable by its competitors, predominantly picea abies and fagus orientalis (johnson, 1993; jovanović, 2000). it inhabits open habitats comprising cliffs and forest clearings, characterized by a strong northerly wind, snow, and rockfalls. the climate in its natural range is characterized by very high humidity, high precipitation, regularly distributed over the year, deep snow cover which lasts 4-5 months, and low winter temperatures. picea omorika is drought tolerant, its cold hardiness limit is between -28 °c and -23 °c, it tolerates wide soil ph range and polluted urban conditions (sevill et al., 2017). in short, it is adapted to extreme environmental conditions. planting serbian spruce outside its natural range has a long tradition in europe since the late 19th century. in addition to the initial use as ornamental plant species in parks, serbian spruce has a long tradition of use in forestry (ivetić & aleksić, 2016). it is grown to a small extent for christmas trees, timber and paper production, particularly in northern europe, although its slow growth makes it less important than sitka spruce or norway spruce (sevill et al., 2017). however, in britain p. omorika and p. orientalis are among the alternative species to sitka spruce and norway spruces particularly in areas where they might be subject to damage due to drought as the impacts of climate change (sevill et al., 2017). the great value of this species appears to be successful planting in places where other spruces are susceptible to injury by drought or spring frosts. at present, serbian spruce is of major importance only as an ornamental tree, mainly in northern europe and north america. it is regarded as one of the most attractive spruces because of its elegant form and the ability to grow on a wide range of soils (sevill et al., 2017). despite its endemism, p. omorika is considered as one of the most adaptable spruces. 66 biologica nyssana ● 10 (2) december 2019: 65-75 mitrović et al. ● serbian spruce (picea omorika (pančić) purkyné endemicity and adventages wood properties in the forest products industry and methods for estimation of wood quality forest products industry is based on wood properties, while wood properties are directly determined by cell arrangement, cell size and shape, and cell wall structure and thickness. in softwood species differences in wood structure result from genetic and abiotic factors: 1) plant age (zobel & sprague, 1998; burdon et al., 2004) juvenile wood and mature wood; 2) season of maturation within the growth ring (uggla et al., 2001) – early wood (ew) and late wood (lw); or 3) mechanical stress as a consequence of wind and stem lean (timell, 1986) compression wood, opposite wood and normal wood. in the forest products industry, juvenile wood, early wood and compression wood, generally have limited value, and therefore determination of their amounts is of great importance. in this regard, different morphometric and non-morphometric methods were developed for the evaluation of wood quality (tab. 1). the number of developed methods speak in favor of their significance for the forest products industry. compression wood reaction wood in conifers: formation, occurrence, range of gradations the resistance of trees to mechanical perturbation depends on structural modifications for mechanical biologica nyssana ● 10 (2) december 2019: 65-75 mitrović et al. ● serbian spruce (picea omorika (pančić) purkyné endemicity and adventages 67 strength. the formation of reaction wood in the stem is a reaction of the tree to leaning, as part of the geotropic response. in conifers, reaction wood is known as compression wood (timell, 1986). its formation occurs on the lower side of the leaning stem (fig. 1), resulting in eccentric growth (timell, 1986; donaldson & singh, 2013). inclination at the high angle results in severe compression wood (scw) formation (yumoto et al., 1983). cw occurs in a range of gradations from near nw to scw, mild cw (mcw) forming a continuum between nw and scw (fig. 1). wood opposite to the cw in the same growth ring is termed opposite wood (ow) (fig. 1), while wood from growth rings that do not contain any cw is termed normal wood (nw). also, in the leaning stem, compression wood severity declines from the stem base to the top of the stem (fig. 1). wood tracheid cell walls are composed of several layers containing an ordered array of cellulose microfibrils, embedded in a matrix of polysaccharides such as pectin, hemicellulose, and lignin (harris, 2006). cw is characterized by (fig. 2): increased tracheid wall thickness, reduced lumen diameter, rounder cell cross-sectional profile, presence of intercellular spaces, absence of the s3 cell wall layer and presence of helical cavities in the s2 layer, compared to nw (donaldson et al., 2004; donaldson & singh, 2013). cw is highly lignified, with the changed composition of lignin, increased amounts of p-hydroxyphenyl monomers and increased conmethods parameter for estimation of m or ph om et ric m et ho ds manual or automated measurements on micrographs (mork, 1928; brown et al., 1949; gofas & tsoumis, 1975; klisz, 2009; selig et al., 2012) automated measurements from distance maps reconstructed from digital images (travis et al., 1996; lorbach et al., 2012; nedzved et al., 2018) cell wall area radial cell wall width double wall thickness cell lumen ew/lw ratio (mork, 1928) differences between juvenile and mature wood (mitchell and denne, 1997; loustarinen, 2012) compression wood severity (andersson & walter, 1995; nyström & hagman, 1999; moëll & fujita, 2004; duncker & spiecker, 2009; nedzved et al., 2018) n on -m or ph om et ric m et ho ds fluorescence spectroscopy (donaldson et al., 2010) chemical analysis (nanayakkara et al., 2009) scanning fourier transform infrared microspectroscopy and immunolabeling (altaner et al., 2009) fluorescence-detected linear dichroism (fdld) microscopy (savić et al., 2016) confocal microscopy (donaldson et al., 2004) lignin and carbohydrate content/structure cellulose and noncellulosic polysaccharides composition and organization microfibrillar angle compression wood severity table 1. some of the methods for estimation of wood quality 68 densation of monomer units in the polymer (timell, 1986). consequently, cw contains less cellulose, with greatly increased amounts of galactan, and slightly lower amounts of mannan and xylan, together with a higher angle of cellulose microfibrils in the s2 layer of the cell wall, compared to nw (nanayakkara et al., 2009; donaldson & knox, 2012; donaldson & singh, 2013). the degree of development of particular features of cw does not necessarily change in parallel to each other, so the severity of a given tracheid is represented as a function of the degrees of development of individual features, mainly lignification, helical cavities and cell wall thickness (yumoto et al., 1983). visual detection of compression wood severity, more precisely the determination of mcw, is difficult. as the severity of cw affects mechanical and chemical properties of wood in the forest products industry, it is desirable to be able to measure cw severity (altaner et al., 2009). fig. 1. scheme of compression wood formation; compression wood occurs in a range of gradations from near normal wood to severe compression wood, mild compression wood forming a continuum between normal wood and severe compression wood; compression wood severity declines from stem base to the top of the stem; nw – normal wood, cw – compression wood. mcw mild compression wood, scw severe compression wood, ow – opposite wood fig. 2. scheme for changes in degree of development of tracheid cell wall features characterizing the transition from normal wood (nw) to severe compression wood (scw), mild compression wood (mcw) forming a continuum between nw and scw; on the left – field emission scanning electron microscopy (fesem) images of nw, on the right – fesem images of scw; nw – normal wood, mcw mild compression wood, scw severe compression wood, s1, s2, s3 – layers of secondary cell wall biologica nyssana ● 10 (2) december 2019: 65-75 mitrović et al. ● serbian spruce (picea omorika (pančić) purkyné endemicity and adventages 69 our morphometric and nonmorphometric methods for distinguishing conifer wood samples on a compression severity scale in recent years we worked on the development of different morphometric and non-morphometric methods for distinguishing wood samples on a compression severity scale. the first method is based on tracheid double wall thickness analysis (nedzved et al., 2018). the second method is based on cellulose microfibrils order (distribution and alignment of cellulose microfibrils) analysis in tracheid (double) walls (savić et al., 2016). the third one we are still developing, based on the analysis of structural modifications of lignin, and it is related to our results published a few years ago (mitrović et al., 2015). these 3 methods cover the main features of cw, related to changes in tracheid cell wall shape, lignin and cellulose organization. hence, alone or in combination with each other, they could be suggested for use in fine gradation of wood samples on the compression severity scale, either in the estimation of wood quality or in the estimation of environmental influences during growth and developmental process. we present here, in more detail, 2 methods, one morphometric (nedzved et al., 2018) and one non-morphometric (savić et al., 2016), for distinguishing wood samples on a compression severity scale. in tab. 2, the main characteristics, similarities, differences, and advantages of these methods are summarized. it is known that radial and tangential walls can vary significantly regarding different features of wood cell walls. so far, most investigations, regarding cellulose microfibrils (donaldson, 2008), or other features of wood cell walls such as cell wall thickness (mork, 1928), have been carried out on radial cell walls. accordingly, our methods confirm the selection of radial walls for the analysis as an excellent choice of tracheid cell wall region for the determination of mcw. fig. 3. reprinted by permission from: springer nature, trees 32, 1347–1356; nedzved et al. (2018) automatic image processing morphometric method for the analysis of tracheid double wall thickness tested on juvenile picea omorika trees exposed to static bending. trees 32, 1347–1356.: the clsm images (first column), corresponding binary images (second column) and distance maps (third column) of p. omorika stem samples; s1 and s2 – nw samples; s3 and s4 mcw samples; s5 and s6 – scw samples biologica nyssana ● 10 (2) december 2019: 65-75 mitrović et al. ● serbian spruce (picea omorika (pančić) purkyné endemicity and adventages 70 compression wood – reaction wood in conifers: formation, occurrence, range of gradations measurements of various anatomical characteristics of wood cells are of great importance in the research of wood structure. tracheid double wall thickness, as an important wood anatomical feature, besides being known as an indicator of transition from ew to lw within an annual ring in gymnosperms (the ratio between radial double wall thickness and cell lumen diameter, mork, 1928), also characterizes the differences between juvenile and mature wood (mitchell & denne, 1997; loustarinen, 2012), as well as between normal and compression wood (timell, 1986; plomion et al., 2001). hence, the determination of tracheid double wall thickness is of great importance in estimation of wood quality. image-processing techniques are used for estimation of the ew and lw ratio as a significant feature for forest products industry (mork, 1928; jagels & dyer, 1983; diao et al., 1999). however, such techniques for estimation of compression wood severity were more (duncker & spiecker, 2009) or less (andersson & walter, 1995; nyström & hagman, 1999; moëll & fujita, 2004) successful in recognizing mcw. for testing of our automatic image processing morphometric method for the analysis of tracheid double wall thickness (nedzved et al., 2018) we used confocal laser scanning microscopy (clsm) images of stem cross sections of juvenile p. omorika trees exposed to static bending. our algorithm (nedzved et al., 2018) was developed using imagewarp a&b software company (usa) and software for image analysis qtip developed in united institute of informatics problems (uiip), national academy of sciences (belarus). it consists of the extraction of cell patterns from the original micrograph via binarization using otsu’s threshold method (otsu, 1979) and reconstruction of the distance maps (kimmel et al., 1996). the use of euclidian distance maps for calculating the thickness of the wood cell wall was suggested earlier in estimations of pulp or paper quality (travis et al., 1996; koskenhely & paulapuro, 2005; selig et al., 2012; lorbach et al., 2012). the novelty of our fig. 4. reprinted by permission from: springer nature, trees 32, 1347–1356; nedzved et al. (2018) automatic image processing morphometric method for the analysis of tracheid double wall thickness tested on juvenile picea omorika trees exposed to static bending. trees 32, 1347–1356.: distribution of double wall thickness of a) entire tracheid walls, b) tracheid at cell corners, c) tangential walls and d) radial walls, determined by morphological processing of structural elements with different orientation from distance maps obtained from the clsm images of (s1 – s6) samples (fig. 3); a, b, c, d, e, f significant difference at 5% level of significance of the maxima positions of gaussian curves between samples s1 – s6; µ mean values, σ – standard deviation biologica nyssana ● 10 (2) december 2019: 65-75 mitrović et al. ● serbian spruce (picea omorika (pančić) purkyné endemicity and adventages 71 method is the use of morphological image processing of structural elements with different orientation (dougherty, 1992) on euclidian distance maps reconstructed from microscopic images, allowing determination of the distribution of tracheid double wall thickness separately for tangential walls, radial walls, and cell corners (it yields valuable information on circularity/rectangularity of cross-sectional profile of tracheid cell wall). we applied ibm spss software, nonlinear curve fit function, for fitting the gaussian curves on the data (distribution of double wall thickness in pixels) to show the overall variation in tracheid double wall thickness in a response to mechanical stress. for comparison of the maxima positions of gaussian curves between samples, one-way anova and duncan test were used. as a result, our nonmorphometric image processing method provides a fine gradation of p. omorika juvenile wood samples on the compression severity scale from nw to scw (fig. 4 a-d), suggesting that it could be used as a tool for estimation of compression wood severity. in addition, as different regions of p. omorika tracheid cell wall show somewhat different cell wall thickening in response to mechanical stress (fig. 4), compression severity scales based on double wall thickness distribution of entire tracheids, tracheids at cell corners, tangential and radial walls, differ to some degree. radial wallsdouble wall thickness distribution (fig. 4d) shows the additional advantage and specificity over a number of methods for estimation of compression wood severity: it groups, and consequently sharply distinguishes mcw samples from scw samples. our non-morphometric method for estimation of compression wood severity based on cellulose microfibrils order (savić et al., 2016) short report the distribution and orientation of cellulose microfibrils (mfs) in wood cell walls is determined by both, genetic and abiotic factors. genetic factors include: cell wall layer (primary wall, s1, s2, s3 layer of secondary cell wall), position (radial or tangential cell wall), plant age (juvenile or mature wood), season of maturation within the growth ring (early and late wood), while abiotic factors include wind and stem lean (compression wood, normal wood). since cellulose fibrils, as reinforcing material in conifer wood cell walls, determine tracheid cell wall properties and wood quality, one of the most frequently measured ultrastructural variables in wood cell wall is microfibrilar angle (mfa) (donaldson, 2008). various microscopic techniques have been used to study the orientation of cellulose microfibrils in the wood cell wall, using variations in polarised light techniques or directly visualizing orientation of the microfibrils (donaldson, 2008). in our method for estimation of compression wood severity (savić et al., 2016) we observed the relative order of cellulose fibrils in cell walls. fluorescence detected linear dichroism (fdld) imaging was performed using confocal laser scanning microscope (clsm) additionally equipped with constructed differential polarization extension (dplsm). fdld microscopy exploits fluorescence originating from cellulose fibrils stained specifically by congo red, enabling screening of cellulose microfibrils order in the x–y plane of the cross section, which means separately in tangential and radial walls. the method was tested on stem cross sections of juvenile p. omorika trees exposed to static bending. blue colour represents dipoles (cellulose fibers) predominantly parallel with x-axis (tangential walls), yellow colour indicates dipoles (cellulose fibers) oriented predominantly parallel with the yaxis (radial walls) (fig. 5), while the grey colour represents fibers orientated at about 45°. image processing was performed using imagej program with macros developed for this analysis. fdld images were quantified and presented as histograms (fig. 5). the decrease in cellulose fibrils order from cw to nw samples is obvious, in both radial and tangential tracheid walls (fig. 5). this is in line with xu et al. (2011) work; they showed that the characteristics of cellulose fibrils reinforcements in s2 layer of severe compression juvenile wood include lower number, abundant dislocation segments and shorter length of cellulose mf, compared to nw. we additionally showed (savić et al., 2016) that radial and tangential tracheid cell walls in p. omorika juvenile wood differ considerably regarding cellulose fibril order, and that fdld of radial walls (fig. 5 u), showing fine gradation from cw to nw, could be suggested as an easily applicable technique for estimation of cw severity. picea omorika juvenile wood samples – a model for testing of methods for estimation of compression wood severity. we tested our methods on stem samples of p. omorika juvenile trees exposed to long term static bending (bending procedure described in detail in mitrović et al., 2015). p. omorika belong to slow-growing conifer species in which cw typically occurs in a severe form (scw) (donaldson et al., 2004). juvenile conifer wood is characterized by randomly distributed mcw, nw often being absent (donaldson et al., 2004). these are the features that suggest p. omorika juvenile wood a good choice of samples for evaluabiologica nyssana ● 10 (2) december 2019: 65-75 mitrović et al. ● serbian spruce (picea omorika (pančić) purkyné endemicity and adventages 72 fig. 5. reprinted by permission from: cambridge university press, microscоpy and microanalysis 22, 361–367.; savić et al. (2016) fluorescence-detected linear dichroism of wood cell walls in juvenile serbian spruce: estimation of compression wood severity. microscоpy and microanalysis 22, 361–367.: fluorescence-detected linear dichroism (fdld) images of p. omorika sections and corresponding anisotropy histograms. a: normal wood (nw) samples; (e) mild compression wood (mcw) samples; (i,m) severe compression wood (scw) samples; (b,f,j,o) pixel values were collected in the marked areas, in tangential walls (blue boxes) and in radial walls (yellow boxes), and used to obtain anisotropy distributions; (c,g,k,p) tangential walls fdld distributions; (d,h,l,q) radial walls fdld distributions; (t) overlaid distributions (c,g,k,p) with black lines representing corresponding gaussian fits (white arrow represents gradual shifts toward gray—increasing number of disorientated fibrils); (u) overlaid distributions (d,h,l,q) with black lines representing corresponding gaussian fits (white arrows represent gradual shifts toward gray—increasing number of disorientated fibrils); (r,s) schemes of nw and cw tracheid sections, respectively. excitation at 488 nm, emission above 560 nm; image size is 64 × 64 μm biologica nyssana ● 10 (2) december 2019: 65-75 mitrović et al. ● serbian spruce (picea omorika (pančić) purkyné endemicity and adventages tion of the precision of methods suggested for estimation of compression wood severity. after testing of our methods (savić et al., 2016; nedzved et al., 2018) on stem cross sections of juvenile p. omorika trees exposed to long term static bending, we can confirm p. omorika juvenile wood samples as a good model for testing of methods suggested for estimation of compression wood severity. conclusion picea omorika, despite its endemism, and thanks to her adaptability and long tradition of planting, nowadays is present widely outside its natural range. we confirmed picea omorika juvenile wood samples as a good model for testing of methods suggested for estimation of compression wood severity. our methods, based on the analysis of tracheid double wall thickness, cellulose fibril order and structural modifications of lignin, using confocal fluorescence microscopy and spectroscopy, cover all the main features of cw. therefore, alone or in combination with each other, they could be a useful tool for fine gradation of wood samples on compression severity scale, as a valuable advantage over many other methods for the estimation of compression wood severity, as the determination of mild compression wood is difficult. hence they can be of great benefit either for estimation of wood quality in forest products industry, or for estimation of environmental influences during growth and developmental process in tree physiology. acknowledgements. this study was supported by grant 173017 of the ministry of education, science and technological development of the republic of serbia. previous version of this manuscript has been presented at 13th symposium on the 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10.46793/biolnyss.16.2.17m critical review on phytopharmacological attributes of operculina turpethum (l) silva manso original article manisha mohapatra department of biotechnology, trident academy of creative technology, bhubaneswar, odisha-751024, india orcid: 0000-0001-9883-1555 manishamohapatra7@gmail.com (corresponding author) received: august 21, 2025 revised: november 15, 2025 accepted: november 28, 2025 abstract: medicinal plants contain numerous bioactive compounds with significant therapeutic potential and are widely used in the synthesis of various drug formulations. operculina turpethum (l.) silva manso (family: convolvulaceae), commonly known as nishoth and tihudi, is one such valuable climbing plant native to india. its different plant parts have several ethno-medicinal and pharmacological attributes. since ancient times, it has been used as an anti-tumor, anti-fungal, anti-hypertensive, asthma inhibitor, anti-ulcer, and antioxidant agent. the current study is a compilation of the morphological and pharmacological activities of operculina turpethum, along with validation of its geographical distribution and phytoconstituents, to provide a brief idea of its therapeutic potential. the plant consists of several phyto-constituents, including αand β-turpethein, turpethinic acids, lanosta-5-ene, etc. the study focuses on a complete plant profile, including its availability and propagation practices. this review examines each part of o. turpethum, including its structural morphology, phytochemical composition, and therapeutic properties. key words: operculina turpethum, convolvulaceae, pharmacological properties, phyto-constituent apstrakt: kritički pregled fitofarmakoloških osobina operculina turpethum (l.) silva manso lekovite biljke sadrže brojna bioaktivna jedinjenja sa značajnim terapijskim potencijalom i široko se koriste u sintezi različitih farmaceutskih preparata. operculina turpethum (l.) silva manso (familija: convolvulaceae), poznata i pod imenima nishoth i tihudi, predstavlja jednu od značajnih penjačica koja je autohtona u indiji. različiti delovi biljke poseduju brojne etnomedicinske i farmakološke osobine. još od davnina, koristi se kao antitumorski, antifungalni, antihipertenzivni agens, inhibitor astme, antiulcerativno sredstvo i antioksidant. sadašnja studija predstavlja pregled morfoloških i farmakoloških aktivnosti operculina turpethum, zajedno sa potvrdom njene geografske distribucije i fitosastava, kako bi se dala kratka predstava o njenom terapijskom potencijalu. biljka sadrži više fitosastojaka, uključujući αi β-turpetein, turpetinske kiseline, lanosta-5-en i druge. studija se fokusira na kompletan profil biljke, uključujući njenu dostupnost i metode razmnožavanja. ovaj pregled analizira svaki deo o. turpethum, uključujući strukturnu morfologiju, fitohemijski sastav i terapijske osobine. ključne reči: operculina turpethum, convolvulaceae, farmakološka svojstva, fito-sastojak © 2025 mohapatra. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. introduction plants are the most critical source of natural therapeutic compounds that enhance longevity and quality of life, and they are gaining prominence worldwide (parekh & chanda, 2007). these are a valuable sources of a wide array of bioactive compounds, synthesized and economically beneficial for various commercial applications across several medicinal industries. egression of quest in plant-based health care nowadays paved the way for the use of medicinal flora in drug formulation processes instead of synthetic chemical compounds (gondaliya et al., 2014). this revitalization of interest in medicinal flora has led to increased global market demand and to adulteration with substitutes of minimal or no medicinal potency (sarwat et al., 2011). due to the diminishment of its natural habitat and ruthless harvesting practices, many of plant species are on the verge of extinction. the use of indigenous knowledge for healing practices is a valuable approach that promotes sustainable livelihoods by harnessing unexplored natural resources in local communities. indigenous knowledge often includes a deep understanding of medicinal plants and their healing properties. integrating traditional medicine with modern healthcare practices can provide holistic, sustainable healthcare solutions while also supporting the conservation of medicinal plant species (sarwat et al., 2011). the systematic attempt to understand the secondary metabolites of medicinal plants will definitely help to mitigate the threat status and ensure the quality of the species. scientific validation of such documentation, combined with ethnopharmacological studies of specific medicinal herbs, will facilitate the estimation of the safety and efficacy of traditional medicinal practices. in this context, operculina turpethum (l.) silva manso is one of the indispensable ret medicinal plants in the family convolvulaceae and has very diverse medicinal uses, particularly in ayurvedic and unani systems of medicine (alam et al., 2010; hoq et al., 2019). it has several common local synonyms, viz. trivrit, turbud, nishoth, dudholomo, tihudi, etc. this medicinal plant is unique in both its morphological appearance and its phyto-pharmaceutical applications. to date, several studies have been conducted on this plant; however, most findings focus either on validating specific therapeutic attributes of the plant or on the phytochemicals present in its various parts. most of these articles are based on research into specific attributes. some review articles, though available, present the plant mostly from an ayurvedic perspective. however, this article is a comprehensive compilation of all aspects of this plant, including morphological structure, taxonomic classification, types, distribution, phytochemical composition, and pharmacological properties. furthermore, this article also provides an ayurvedic perspective on the use of this plant in the preparation of many drug formulations. the above study is unique in that it not only validated the plant and compiled all its biological attributes associated with all plant parts but also examined its use in the development of ayurvedic formulations, along with available techniques suitable for plant propagation. materials and methods the review article discusses the validation of operculina turpethum (l.) silva manso for its morphological, phytochemical, and pharmacological properties. the literature search included taxonomic, pharmacognostic, and phytochemical validation of nisoth, along with validation of its pharmacological applications. for this purpose, we have considered the most relevant articles from pubmed/medline, embase, scopus, sciencedirect, scopemed, scifinder, and google scholar. the articles were selected from the database using relevant keywords and their combinations, such as operculina turpethum, plant morphology, phytochemistry, and pharmacological applications. several studies have been carried out to validate the use of the operculina turpethum plant for its morphological and phytopharmacological attributes, which are extensively used in the treatment of various diseases. statistical data from sciencedirect on the same shows only around 100 articles in this direction. however, the present study is a comprehensive compilation of the taxonomic, morphological, phytochemical, and pharmacological attributes of the plant. in this study, importance is also given to ayurvedic rasa panchak, organoleptic characteristics, and its ayurvedic properties to justify the plant’s potency in ayurvedic formulations. the inclusion criteria included only studies on the use of the plant in relation to its geographical distribution, classification, available types, current phytochemical profile, and use in the treatment of different disorders (irrespective of the plant part used), as well as all possible formulations and their impact. database searches could retrieve a total of 400-500 review papers using the aforementioned keywords. the remaining articles (around 1000-2000) were mostly research articles evaluating the specific therapeutic properties of the plant or its associated bioactive compounds. the quality screening considered parameters like relevance to the theme. the plant has been widely utilised in the ayurveda system for the treatment of many disorders. in the following sections, a brief description of the plant is provided. common name & botanical classification operculina turpethum (l) silva manso is a medicinal plant from the convolvulaceae family with several synonym names. it is an indigenous plant that is endemic to india (wealth of india, 1952). some of its common vernacular names include turpeth and/ or nisoth in hindi, trivrit in sanskrit, indian jalap or turpeth in english, tegada in telugu, turbud in bengal, and tihudi or dudholomo in oria (alam et al., 2010; hoq et al., 2019; kirtikar & basu, 2003). the scientific nomenclature of this plant is based on its morphological and taxonomic characteristics evaluated collectively (alam et al., 2010; gupta & ved, 2017) and is classified as follows: domain eukaryota, kingdom plantae, division angiosperm, class dicotyledons, order solanales, family convolvulaceae, genus operculina, and species biologica nyssana ● 16 (2) december 2025: mohapatra ● critical review on phytopharmacological attributes of operculina turpethum (l) silva manso biologica nyssana ● 16 (2) december 2025: mohapatra ● critical review on phytopharmacological attributes of operculina turpethum (l) silva manso o. turpethum. some of its common taxonomic synonyms include ipomoea turpethum (l.), ipomoea silvana (l.), ipomoea diplocalyx (l.), convolvulus anceps (l.), convolvulus turpethum (l.), convolvulus triqueter (l.), merremia turpethum (l.), and spiranthera turpethum (l.). morphological description it is a large herbaceous perennial twiner or large climber, with stems, petioles, leaves, and inflorescences that are shortly pubescent, downy, or glabrescent. when old, the stem is narrow and 3-winged (fig. 1). lower leaves are broadly ovate, cordate, often 15 x 11.8 cm, acuminate or acute and apiculate; upper ones are often oblong, obtuse, and mucronate. petioles are 1.8-7.5 cm with crisped wings above on the lower leaves (hoq et al., 2019). flowers are 3-5 cm long, peduncle cymes, 5-7.5 cm long, bracts large, oblong, 1.2-2.5 cm long, cuspidate, subpersistent, pedicels 2.5-3 cm, lengthen and become stouter in fruit. ovate sepals, oblong or broadly elliptic, 18 cm long, outer 2 sub-herbaceous, very broad, pubescent, 3 inner membranous, all enlarged, hardened, and often split in fruits. corolla white, funnel-shaped or broadly campanulate, 3-4.5 cm long, glabrous, glandular. capsule depressed globous, 1.5 cm diameter, 2-celled. seeds 4-1, obovoid, 5 mm long, brown, minutely reticulate glabrous (hoq et al., 2019; sharma & singh, 2012; ahmad et al., 2017). the leaves are also consumed as vegetables (sharma, 2006). classification of operculina turpethum the two kinds of trivrit, also known as turpeth (fig. 2), are distinguished by their color: shweta or white turpeth (botanical name: operculina turpethum, formerly known as i. turpethum) and krishna or black turpeth (botanical name: ipomoea petaloidea chois). these two types have slightly different botanical classifications (celeghini et al., 2001; mondal et al., 2006; srikantha et al., 2008). 1. shweta or white turpeth (operculina turpethum) it is found throughout india at altitudes of up to 900 meters. it is also scattered in various regions around the world including australia (northern territory, queensland), africa (kenya, tanzania, mozambique, zimbabwe; western indian ocean islands such as madagascar, mauritius), parts of the pacific (micronesia), and various asian countries (india, nepal, pakistan, sri lanka, indochina, myanmar, thailand, indonesia, malaysia, papua new guinea, philippines). it is a native herb in india (mondal et al., 2006). 2. krishna or black turpeth (ipomoea petaloidea chois) its natural range includes parts of asia, specifically china (guangdong province) and taiwan. it is also found in southern america, particularly in the west indies. it is native to the regions mentioned above (srikantha et al., 2008). results and discussion ayurveda rasa panchaka in ayurveda, rasa panchaka refers to the five basic tastes (rasa) associated with substances. these tastes are sweet (madhura), sour (amla), salty (lavana), pungent (katu), bitter (tikta), and astringent (kashaya). each taste is believed to have specific effects on the doshas (vata, pitta, and kapha). operculina turpethum is primarily associated with the bitter (tikta) taste in ayurveda (seleena et al., 2020). fig. 1. opeculina turpethum plant. (a) whole plant, (b) seeds biologica nyssana ● 16 (2) december 2025: mohapatra ● critical review on phytopharmacological attributes of operculina turpethum (l) silva manso plant is also recognized for its panduhara action, being traditionally used to support the management of anemia and liver-related disorders. finally, its udavartahara property assists in reducing bloating. (seleena et al., 2020) organoleptic characters the organoleptic characteristics of o. turpethum refer to the sensory properties perceived through human senses (seleena et al., 2020). in terms of appearance (rupa), the roots are typically thick, fleshy, and tuberous, varying in size and shape, with a generally brownish outer surface. the smell (gandha) is described as earthy, woody, or herbal, although it may be subtle and not always strongly noticeable. regarding taste (rasa), operculina turpethum is primarily characterized by a bitter taste (tikta rasa), which represents one of its most prominent organoleptic features. the texture (sparsha) of the root’s outer surface tends to be rough or slightly coarse due to its natural structure. the color (varna) of the plant powder sample typically appears yellowish brown. in terms of potency (veerya), turpeth is considered ushna in nature, meaning it produces a heating effect on the body when consumed. its qualities (guna) are primarily light, or laghu, according to ayurvedic classification. geographical distribution operculina turpethum is mostly native to asia and is distributed sporadically in tropical regions of mauritania, india, philippines, america, africa, bangladesh, australia, madagascar, pakistan, shri lanka, nepal, taiwan, bangladesh, thailand, china, malaysia, tanzania, myanmar, indonesia, and the philippines (ahmad et al., 2017; bhande et al., 2006; kohli et al., 2010). in india, the native states that have populations of operculina include rajasthan, gujarat, maharashtra, madhya pradesh, andhra pradesh, telangana, karnataka, kerala, tamil nadu, and parts of the himalayan region. however, within odisha, it is found in jajpur, bargarh, balangir, khurda, and sambalpur sporadically (hoq et al., 2019; saxena & brahmam, 1995; mohapatra & basak, 2014). phytoconstituents the root bark of operculina turpethum contains a glycosidic resin called turpethein, which is insoluble. it also contains secondary metabolites (fig. 3), including saponins, flavonoids, glycosides, phenolics, essential oils, glucose, and fructose (shastry, 2014; shankaraiah et al., 2017). the roots of o. turpethum contain different types of the taste (rasa) of operculina turpethum is described as bitter (tikta), while its other attributes (guna) are characterized as dry (ruksha) and light (laghu). its potency (veerya) is considered hot (ushna), and the post-digestive taste (vipaka) is pungent (katu). regarding its effect on the doshas, it balances kapha and pitta but may increase vata if used excessively. its prabhava (specific pharmacological effect) is classified as rechana (purgative). therapeutic actions in ayurveda in ayurveda, operculina turpethum exhibits several therapeutic actions. it functions as a laxative (sara), helping to soften and promote bowel movements, and as a purgative (rechana), exerting a stronger laxative effect that facilitates complete intestinal clearance. the plant also acts as deepana, enhancing the digestive fire and thereby improving digestion, and as pachana, supporting the digestion and metabolism of food. additionally, it demonstrates krimighna activity, indicating anti-parasitic properties beneficial in expelling intestinal worms, and vatahara action, helping to alleviate conditions associated with the vata dosha. its krumihara effect contributes further to the reduction of worm infestations. the fig. 2. types of operculina turpethum. (a) white turpeth, (b) black turpeth biologica nyssana ● 16 (2) december 2025: mohapatra ● critical review on phytopharmacological attributes of operculina turpethum (l) silva manso triterpenoids such as lanosta-5-ene, cycloartenol, and 24-methylene-δ-5-lanosterol (kirtikar & basu, 2003). additionally, it also contains various other bioactive compounds such as albumin, lignin salts, volatile oil, starch, ferric oxide, lupeol, αand β-turpethein, betulin, αand β-rhamnose, fructose, β-sitosterol, scopoletin, and some ether-soluble resin (gupta & ved, 2017). operculina turpethum is rich in various phytoconstituents, including glycosidic resin, coumarin, beta-sitosterol, reducing sugars, and essential oils, which have therapeutic benefits for treating various ailments and diseases. additionally, this plant contains resins, a mixture of alpha and β-turpethein, glycosides, coumarins, scopoletin, saponins, flavonoids, steroids, and carbohydrates (saxena & brahmam, 1995; mohapatra & basak, 2015; hoq et al., 2019). the plants in the ipomoea category (approximately 500–600 species) have the highest concentrations of the alkaloid ergoline in their seeds and are therefore utilised as medicines and ornamentals (meira et al., 2012). the methanolic extracts of leaves of o. turpethum were evaluated for phenolic compounds through high-performance liquid chromatography with a total phenol content of 1.89 mg/ml and flavonoid content of 1.0 mg/ml (jalaj & madhavan, 2016). different types of flavonoids are present in the stems and roots of operculina turpethum and exhibit maximum antibacterial activity, along with inhibition of heat shock protein production in malignant cell lines, providing a potent treatment for several types of cancer (doss et al., 2011). in one of the studies, several secondary metabolites from root extracts were evaluated. according to this study, the ethanolic root extracts of operculina turpethum (ot) contain carbohydrates, steroids, gums, and saponins. the ether and chloroform root extracts of ot contain carbohydrates, steroids, gums, flavonoids, and saponins. additionally, the ethanolic, ether, and chloroform leaf extracts of ot contain carbohydrates, steroids, gums, flavonoids, tannins, alkaloids, reducing sugars, and saponins (arif et al., 2013). fig. 3. phytoconstituents available in operculina turpethum coumarin beta-sitosterol lenosta-5-ene l-rhamnose scopoletin betulin cycloartenol lupeol operculinoside a operculinoside b operculinoside c operculinoside d biologica nyssana ● 16 (2) december 2025: mohapatra ● critical review on phytopharmacological attributes of operculina turpethum (l) silva manso propagation of operculina turpethum the vegetative macropropagation of operculina turpethum, a red-listed medicinal plant, is essential, as conventional seed propagation faces problems due to low seed viability, low germination rates, and poor survivability. these challenges make it difficult to produce adequate quantities of high-quality planting materials through seed propagation alone; hence, in one of the studies, this was achieved by applying iba (indole-3-butyric acid) at 2000 ppm and naa (naphthaleneacetic acid) at 3000 ppm. among the various types of nodal stem cuttings used as explants, those treated with this specific combination of auxins demonstrated the most promising results. interestingly, the nodal cuttings consisting of a single node and a single leaf emerged as the most suitable explant material for macropropagation of operculina turpethum (mohapatra & basak, 2014). similarly, in another study, the nodal segments were cultured in ms medium supplemented with 1.0 mg l-1 bap (benzylaminopurine). this resulted in rapid shoot bud proliferation, with an impressive success rate of 85.33% (alam et al., 2010). therapeutic applications of operculina turpethum operculina turpethum exhibited anti-diabetic, antiinflammatory, antimicrobial, antioxidant and antiarthritic activity, further affirming its therapeutic versatility. this review highlights operculina turpethum as a valuable botanical resource with a broad spectrum of therapeutic activities, making it a subject of continued research and potential pharmaceutical development, as demonstrated by its significant therapeutic potential across various traditional medicinal systems. the therapeutic applications of operculina turpethum are being validated by several researchers (alam et al., 2010; hoq et al., 2019; seleena et al., 2020; bhande et al., 2006; shastry, 2014; shankaraiah et al., 2017) and a few are depicted in tab. 1. the operculina turpethum root bark contains turpethum resin with turpethin, as the substitute ally of jalapine and convolvulin. it also contains turpethinic acids a, b, c, d, and e and has potent anthelmintic, anti-inflammatory, and analgesic activities (islam et al., 2015; hoq et al., 2019; seleena et al., 2020). the antimicrobial activity of o. turpethum has been validated by several researchers (alam et al., 2010). alam et al. (2010) validated the antimicrobial potency of ot against streptococcus, bacillus, pseudomonas, shigella sonnei, and shigella using minimum inhibitory concentration (mic) testing. apart from its use as a therapeutic ingredient, ot is also used in panchakarma treatment as virechana (shastry, 2014). operculina turpethum root is used as a potent ingredient in avipattikara churna for the treatment of hyperacidity, gastric ulcers, and other associated gastrointestinal issues (bhande et al., 2006). it is also utilised in several skin allergies, rashes, obesity, ulcers and constipation (mohapatra & basak, 2015). the other popular ayurvedic formulations, those included operculina, as one of the vital ingredients are trivrit choornam, trivritadi choornam (ignatius et al., 2013). the o. turpethum root and stem methanolic extracts showed antidiabetic activity in a streptozotocin-induced type 2 diabetic mouse model (ahmad et al., 2017). ethanolic root extract showed hepatoprotective activity against a paracetamol-induced hepatotoxic rat model by administering 100-200 mg/kg body weight of o. turpethum, reducing serum sgot, sgpt, and alkaline phosphatase levels (kumar et phyto-pharmacological properties references antihelmintic activity shankaraiah et al., 2017 antimicrobial activity alam et al., 2010; kumari et al., 2010 anti-ulcer activity bhande et al., 2006;mohapatra and basak, 2015 immunomodulatory activity venugopala et al., 2013 hepatotoxicity activity kumar et al., 2006 anti-inflammatory activity garcía-argáez et al., 2000; ignatius et al., 2013 antioxidant activity el-far and taie, 2009; kostova et al., 2011 analgesic activity prabhavathi et al., 2012; islam et al., 2015; ezeja et al., 2015 cns activity islam et al., 2015; guang‐tong et al., 2018 antidiabetic effects pulipaka et al., 2012 antidiarrheal activity shareef et al., 2014 table 1. phyto-pharmacological properties of operculina turpethum biologica nyssana ● 16 (2) december 2025: mohapatra ● critical review on phytopharmacological attributes of operculina turpethum (l) silva manso al., 2006). in another study, the antidiabetic effects of methanol extracts from o. turpethum stems and roots in streptozotocin-induced diabetic rats were validated. with administration of both extracts at a 100 mg/kg body weight dose, a reduction in fasting glucose levels by the end of the 21-day study period was comparable with glibenclamide, a standard drug (pulipaka et al., 2012). in one of the studies, different stem extracts of operculina showed antibacterial activity (kumari et al., 2010). similarly, the petroleum ether and ethanol extracts of the leaves of o. turpethum were found to have antimicrobial properties against various human pathogenic bacteria, with minimum inhibitory concentrations (mics) ranging from 0.13 to 0.75 mg ml-1, indicating their effectiveness in inhibiting pathogenic growth (alam et al., 2010). a recent study investigated the ulcer-preventive and protective properties of two different extracts derived from the stem bark of o. turpethum and validated them. the highand medium-alcohol stem extracts of o. turpethum at a dose of 100 mg/kg, b.w., orally showed enhanced ulcer-preventive activity. the study used an aspirin-induced peptic ulcer model with pylorus ligation in experimental rats (ignatius et al., 2013). in another study, the cytotoxicity of operculina turpethum stem extracts (at a dose of 100 mg/kg) was validated in a rat model of breast cancer induced by dmba (7,12-dimethylbenz [a] anthracene) at a dose of 20 mg over a period of 45 days. the results revealed a lowering in lipid peroxidation and minimal cellular damage (anbuselvam et al., 2007; ghosh et al., 2016). anticancer activity of different plant extracts including holoptelea integrifolia, operculina turpethum, cardiospermem halicacabum l, dilonix regia, and sesbania grandifora seeds, was validated, depicting operculina turpethum ethanolic extract as the one having the highest cytotoxic activity (rizwani et al., 2014). in one study, the potential protective effects of the methanolic extract of operculina turpethum stems were investigated at a dose of 100 mg/kg body weight in female sprague-dawley rats with 7,12-dimethylbenzanthracene (dmba)-induced breast cancer. the study demonstrated the potency of the plant extract by enhancing the antioxidant activities of superoxide dismutase, glutathione peroxidase, ascorbic acid, and α-tocopherol, thereby protecting against dmba-induced breast cancer (ahmad et al., 2017). operculina turpethum extract emerges as a promising candidate for cancer patients, offering cost-effectiveness and minimal side effects. the study delves into its potential as an antitumor agent against oral squamous cell carcinoma (oscc) cell lines (scc-4, kb, scc-9, and scc25). the extract demonstrated a dose-dependent inhibition of cell growth and colony formation, while also disrupting the cell cycle at the g0/g1 phase and inducing early apoptosis through p53 upregulation and a significant decrease in invasive potential of oscc cells, reducing it by up to 5560% (arora et al., 2017). the anticancer activity of operculina stem extract was validated using a 7,12-dimethylbenzanthracene-induced breast tumor model in female rats at an oral dose of 100 mg/kg, with the same 7,12-dimethylbenzanthracene as the inducer at 20 mg for at least 45 days. the study revealed potent reduction in lipid peroxidation, elevated antioxidant levels, and lowered breast tumor weight in rats (anbuselvam et al., 2007). in another study, the potential anti-arthritic activity of ethanolic root extracts of operculina turpethum was assessed using in vitro models. the ethanolic root extracts, in combination with bovine serum albumin, showed 67.22% inhibition of protein denaturation, resulting in potent anti-arthritic activity (sharma & singh, 2013). prakash et al. (2010) had evaluated the hepatoprotective effects of operculina turpethum root powder in a rat model of carbon tetrachloride-induced hepatotoxicity. the results provided valuable insights into the hepatoprotective properties of ot, suggesting its potential as a natural agent and traditional use in herbal medicine (prakash et al., 2010). the plant extract is found to be useful in snakebites, itching, fever, abdominal trouble, muscle pain, inflammation, liver disorders, and anaemia (kohli et al., 2010; mohapatra & basak, 2014). the analgesic and anti-inflammatory effects of operculina turpethum plant has been confirmed in albino mice (prabhavathi et al., 2012). in the study, they used avipattikar churna containing the potent ingredient operculina turpethum at 100 mg/ kg to validate a lower rate of rat paw edema of at least 36.45% (prabhavathi et al., 2012). in another study, the antidiarrheal activity of the root extract of operculina using ethanol as a solvent system was validated on castor oil-induced diarrhea in a mouse model, with loperamide used as a standard at a dose of 10 mg/kg, and the results validated the antidiarrheal activity of operculina root samples (shareef et al., 2014). furthermore, studies have shown that the ipomoea turpethum root extract loaded in n-vinyl pyrrolidone and acrylic acid (nipaam-vp-aa) copolymeric nanoparticles tested on wistar rats are non-toxic and efficient for therapeutic use against inflammation, cns diseases, and cancer (swami et al., 2023). studies have shown that o. turpethum exhibits analgesic and central nervous system depressant properties, in addition to demonstrating laxative and hepatoprotective effects (sudan et al., 2016), while anti-cancer activity was validated in breast cancer cell lines (mughees et al., 2019). biologica nyssana ● 16 (2) december 2025: mohapatra ● critical review on phytopharmacological attributes of operculina turpethum (l) silva manso conclusion the use of indigenous knowledge-based healing practices supported by scientific validation would not only facilitate higher success rates but also harness empirical knowledge in a properly documented format for popularization and widespread use, benefiting the livelihoods of a wide range of populations. prioritizing the ethical and sustainable sourcing of herbal ingredients from selected plants is one of the novel approaches to social responsibility and improved livelihoods. operculina turpethum, known by various vernacular names including turpeth or trivrit, embodies a rich repository of ethno-pharmacological properties rooted in traditional medicinal practices across different cultures. widely employed in ayurveda and unani systems, it exhibits a diverse array of therapeutic actions. the ethnographic study of available empirical and indigenous knowledge systems for healing practices using operculina turpethum could facilitate harnessing the available iks on this aspect. notably, it serves as a potent purgative and laxative, making it invaluable for digestive disorders. moreover, operculina turpethum has found application in alleviating arthritis, gout, sciatica, hemiplegia, epilepsy, and as an analgesic. it stands out for its potential to address peptic ulcers, counter renal carcinogenesis, and provide hepatoprotection. the plant's anti-diabetic and antiarthritic effects further amplify its significance in holistic healthcare. this compilation underscores operculina turpethum's multifaceted ethnopharmacological profile, emphasizing its enduring importance in traditional medicine and its potential as a source for modern therapeutic exploration for fully unlocking of its pharmacological potential. the above study, conducted with a focus on trivrit, underscores the extensive pharmacological validation of various extracts from different plant parts. the establishment of such validated reports is imperative to assess the effectiveness and safety of trivrit, providing scientific confidence in its traditional use in ayurveda and contributing valuable data for the development of future drugs or therapies, particularly for addressing many chronic diseases. acknowledgements: the author gratefully acknowledges the department for support in obtaining the data for the review analysis study. references ahmad, t., husain, m. k., tariq, m., siddiqui, j. i., khalid, m., ahmed, m. w., & kazmi, m. h. 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(2013). review on natural coumarin lead compounds for their pharmacological activity. biomed research international, 2013, 1–14. mohapatra ● critical review on phytopharmacological attributes of operculina turpethum (l) silva manso biologica nyssana ● 16 (2) december 2025: fadel et al. 2025, biologica nyssana 16(2) 16 (2) december 2025: doi: 10.46793/biolnyss.16.2.13f chemical composition and antioxidant activity of essential oil from algerian anacyclus clavatus original article hamza fadel research unit, valorization of natural resources, bioactive molecules and physicochemical and biological analyzes (varenbiomol), university of mentouri brothers constantine 1, ain el-bey road, 25000, constantine, algeria. meriem mermoune research unit, valorization of natural resources, bioactive molecules and physicochemical and biological analyzes (varenbiomol), university of mentouri brothers constantine 1, ain el-bey road, 25000, constantine, algeria. sabrina bicha research unit, valorization of natural resources, bioactive molecules and physicochemical and biological analyzes (varenbiomol), university of mentouri brothers constantine 1, ain el-bey road, 25000, constantine, algeria. salah akkal research unit, valorization of natural resources, bioactive molecules and physicochemical and biological analyzes (varenbiomol), university of mentouri brothers constantine 1, ain el-bey road, 25000, constantine, algeria. salah4dz@yahoo.fr (corresponding author) nabila slougui research unit, valorization of natural resources, bioactive molecules and physicochemical and biological analyzes (varenbiomol), university of mentouri brothers constantine 1, ain el-bey road, 25000, constantine, algeria. national polytechnic school of constantine hichem hazmoune research unit, valorization of natural resources, bioactive molecules and physicochemical and biological analyzes (varenbiomol), university of mentouri brothers constantine 1, ain el-bey road, 25000, constantine, algeria. received: july 15, 2025 revised: october 24, 2025 accepted: october 27, 2025 abstract: this study included the volatile composition and antioxidant potential of an algerian auresian anacyclus clavatus. anacyclus clavatus essential oil (aceo) and crude methanol extract (acme) were obtained by steam distillation and maceration, respectively. phenols and flavonoids were found in moderate amounts in acme. aceo exhibited moderate antioxidant capacity using β-carotene bleaching assays (40.09±3.7% and 78.1±5.28%, respectively). the main volatile constituents of aceo were octadecanol (33.08%), caryophyllene oxide (5.10%), cis-thujone (4.81%), tetracosane (4.36%), nonanal (4.05%), and phytone (3.66%), respectively, with nonanal and phytone as key compounds in aceo for the first time. these results will certainly enrich our knowledge of the currently limited information on the chemical composition of aceo and suggest that this species is a viable source of bioactive compounds with potential uses in medicines and nutraceuticals. key words: anacyclus clavatus, essential oil, gc-ms, phenolic content, flavonoid content, antioxidant activity apstrakt: hemijski sastav i antioksidativna aktivnost etarskog ulja anacyclus clavatus iz alžira ovo istraživanje obuhvata ispitivanje isparljivih jedinjenja i antioksidativnog potencijala anacyclus clavatus poreklom iz oblasti aures u alžiru. etarsko ulje anacyclus clavatus (aceo) i sirovi metanolni ekstrakt (acme) dobijeni su postupkom destilacije vodenom parom, odnosno maceracijom. fenoli i flavonoidi su utvrđeni u umerenim količinama u acme. aceo je pokazalo umerenu antioksidativnu sposobnost primenom testa izbjeljivanja β-karotena (40,09±3,7% i 78,1±5,28%). glavne isparljive komponente aceo bile su oktadekanol (33,08%), kariofilen oksid (5,10%), cis-tujon (4,81%), tetrakozan (4,36%), nonanal (4,05%) i fiton (3,66%), pri čemu su nonanal i fiton po prvi put identifikovani kao ključna jedinjenja u aceo. ovi rezultati svakako doprinose proširenju postojećih, ograničenih podataka o hemijskom sastavu aceo i ukazuju na to da ova vrsta može predstavljati značajan izvor bioaktivnih jedinjenja sa potencijalnom primenom u medicini i nutraceutici. ključne reči: anacyclus clavatus, etarsko ulje, gc–ms, sadržaj fenola, sadržaj flavonoida, antioksidativna aktivnost introduction people have traditionally valued aromatic and therapeutic herbs because they recognize their importance in preserving health and averting illness (mahato et al., 2025). despite the development of synthetic medicines, these natural resources remain essential in both traditional and modern medicine, providing a wide range of medicinal compounds (buragohain et al., 2025). aromatic and therapeutic species contain a wide range of secondary metabolites, including polyphenols and flavonoids, which are widely used in the culinary, cosmetics, and pharmaceutical sectors (dimitrijević et al., 2025; šovljanski et al., 2025). the asteraceae is one of the largest families, comprising 250,000 species and approximately 1,600 genera (paksoy et al., 2016). it is known © 2025 fadel et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. for its wide range of uses, not only in medicine but also in the cultivation of ornamental plants and the production of natural rubber, colorants, insecticides, and spices (mechergui et al., 2017). asteraceae plants have been distinguished by their capitula, which include closely arranged flowers on a receptacle encircled by bracts. asteraceae plants are distributed worldwide, including in subtropical, arid, and semi-arid climates, and most species have been used traditionally in conventional medicine due to their phytochemicals, including polyphenols, flavonoids, and volatile compounds (chroho et al., 2022). the asteraceae family includes a wide range of herbaceous plants, shrubs, and trees, many of which are economically and medicinally important (heywood et al., 2007). anacyclus, a species of asteraceae, is found in the mediterranean basin, including algerian regions, and is known for its adaptability to challenging, dry environments. the genus anacyclus (quezel & santa, 1963a) is distributed across the mediterranean basin, including algerian zones, and is known for its adaptability to challenging, dry environments (vitales et al., 2018). anacyclus species, encompassing both annual and perennial varieties, have historically been used to treat various ailments, including sore throat, toothache, and rhinitis (baslam et al., 2023). they have various biological capacities, including antimicrobial, anticonvulsant, anxiolytic, anabolic, aphrodisiac, immunostimulating, and antioxidant properties (sissi et al., 2024). anacyclus clavatus (desf.) pers., also known as “white anacyclus” (quezel & santa, 1963b), is an annual herb found in mediterranean countries (mifsud, 2002). its flowering season starts in march and extends to may (chroho et al., 2022). the plant grows in fields, on roads, and in wastelands. its disc achenes are winged and have large capitula. the plant is used for skin, nervous system, digestive, reproductive, and renal issues. the aerial parts are used for fever, digestive problems, and stomach pain (mechergui et al., 2017). roots are used for rheumatism, respiratory problems, diabetes, toothaches, and paralysis (manouze et al., 2017). powdered roots are also used for kidney diseases, skin problems, reproductive issues, and allergies (ouasti et al., 2024). in algeria, limited research has examined the volatile content of a. clavatus, with the main volatile chemicals being germacrene d, δ-elemene, αand β-thujone, and artemisia ketone (aliboudhar et al., 2013; aliboudhar & tigrine-kordjani, 2014). thus, the current study aimed to enrich knowledge on the algerian a. clavatus from the aures area regarding its essential oil composition by using gc-ms, and to evaluate its antioxidant capacity. materials and methods chemicals methanol (95%) was purchased from sigma-aldrich (deisenhofen, germany); dpph (sigma, st. louis, usa); h2o2 was obtained from sigma-aldrich, usa; and the analytical standards of ascorbic acid, gallic acid, folin-ciocalteu, na2co3, and α-tocopherol were obtained from sigma-aldrich, usa (purity ≥ 98%). plant material the leaves, stems, and flowers of a. clavatus were collected in april 2023 from the algerian region of aures (timgad, 35 km east of batna, 35° 29′ 05′′ north, 6° 28′ 07′′ east, altitude: 1072 m). a voucher specimen (ac/118/var/04-23) was verified and placed in the varenbiomol research unit’s herbarium at the university of constantine 1. extraction and preparation of samples the powdered, air-dried aerial part of a. clavatus (100 g) was extracted with 80% aqueous methanol for 72 hours at room temperature. following filtration through cotton, the crude methanolic extract acme was concentrated to dryness under reduced pressure (20.2 g, yield = 20.2%, w/w). the steam distillation method (yazıcı & sevgili, 2024) was used to extract the volatile components from the fresh plant material (240 g), which was broken into small pieces and processed for three hours. the obtained aceo was weighed (32.0 mg; yield = 0.01%, w/w) and stored at 4 °c until analysis. the aceo and acme yields were calculated in proportion to the plant weight using the following formula: yield (%) = (w1 × 100) / w2; where w1: weight of the obtained sample; w2: initial weight of the plant materials. gc-ms and gc-fd analyses of aceo the aceo was analyzed using a shimadzu gcms-qp2010 chromatograph with an rxi-5ms capillary column (30 m × 0.25 mm, film thickness 0.25 μm). the flow rate of the carrier gas, helium, was 1.44 ml/min. after 10 minutes at 45 °c, the oven was heated to 180 °c at a rate of 3 °c per minute and held there for 5 minutes. following that, it was heated to 280 °c at a rate of 5 °c per minute and held for 5 minutes. finally, it was heated to 330 °c at a rate of 10 °c/min for 2 minutes. the injector and detector (fid) temperatures were set at 330 °c. in split/splitless mode, 1 μl of diluted sample (in dichloromethane) was injected at a 30:1 split. for gc-ms detection, an electron ionization apparatus with an ionization energy of 70 ev was used. the biologica nyssana ● 16 (2) december 2025: fadel et al. ● chemical composition and antioxidant activity of essential oil from algerian anacyclus clavatus biologica nyssana ● 16 (2) december 2025: fadel et al. ● chemical composition and antioxidant activity of essential oil from algerian anacyclus clavatus capillary column components were identified by comparing their mass spectra fragmentation patterns and calculated retention indices (ri) with those available in commercial databases (adams, 2017; babushok et al., 2011) and literature. dpph radical scavenging assay a methanolic solution of dpph was prepared at a concentration of 0.04 mg/ml. the samples ranged in different concentrations. 50 µl of each sample was combined with 3 ml of the prepared dpph solution under the same conditions. a spectrophotometer was used to measure absorbance at 517 nm after a 30-minute incubation at room temperature. under the same circumstances, the ascorbic acid was used as a positive standard. all measurements were carried out in duplicate (n = 2), and the following formula was used to determine the anti-radical capacity: ip (%) = [(abscontrol – abssample) / abscontrol] × 100, where ip (%) is the inhibition percentage, abssample is the absorbance of the sample, and abs control is the absorbance of the negative control. the obtained values were expressed as ic50 (inhibitory concentration required to inhibit 50% of dpph) from linear regression in microsoft excel (nadji et al., 2024). β-carotene bleaching assay 0.5 mg of β-carotene was dissolved in 2 ml of chloroform with 25 μl of linoleic acid and 200 mg of tween 40 to prepare a β-carotene/linoleic acid solution. after the chloroform evaporated, 100 ml of oxygenated water was added, and the mixture was vigorously stirred until an emulsion formed. next, 0.5 ml of each aliquot was mixed with 3.5 ml of the previously prepared emulsion. the same conditions were used to test the α-tocopherol standard as a positive control. the decay of β-carotene can be rationalized in the presence of an excess of linoleic acid and o2 by assuming that a small amount of peroxyl radical generated readily reacts with both β-carotene and antioxidant (takada et al., 2006). the bleaching of β-carotene was monitored at 490 nm at regular intervals for 120 minutes at 50 °c (mahdavi et al., 2017). all determinations have been carried out in duplicate. the antioxidant activity was determined using the following formula: inhibition = [abssample t=2h / abssample t=0h] x 100, where: abssample t = 2h is the absorbance of the emulsion containing the sample after 2 hours. abssample t = 0h is the initial absorbance of the emulsion containing the sample. determination of total phenolic content the polyphenol content in acme was evaluated using the colorimetric technique published by singleton et al. (1999), with minor modifications. 250 μl of sample (1 mg/ml) was mixed with 500 μl of folin-ciocalteu (1 n) reagent. after 4 minutes of incubation at 25 °c, 20 μl of 20% sodium carbonate (na2co3) solution was added to the mixture, bringing the total volume to 770 μl. the resulting mixture was incubated in a dark environment for 120 minutes before being measured at 760 nm. gallic acid at varied amounts was used as the standard. all measurements have been carried out in duplicate. the total phenol content was estimated in μg gae/mg by extrapolating from a calibration curve of varied gallic acid concentrations. determination of total flavonoid content flavonoids were quantified using a procedure that involves the formation of a very stable combination between aluminum chloride and the oxygen atoms present on flavonoids’ carbon structures (kim et al., 2003). acme was prepared at a concentration of 1 mg/ml in methanol. 1 ml of the sample was then mixed with 1 ml of aluminum trichloride solution (alcl3, 2%), to obtain a total volume of 2 ml. following one hour of incubation, absorbance was measured at 420 nm. this experiment was performed in duplicate. the absorbance of a quercetin standard solution was measured under the same circumstances. the results were expressed as the calibration curve for quercetin in μg qe/mg. statistical analysis for the antioxidant evaluation, results are represented as mean values ± standard deviation (sd). a one-way analysis of variance (anova, p<0.05) was performed to compare group means. statistical analyses were performed using microsoft excel 2007 (microsoft, redmond, wa, usa). results and discussion gc-ms analysis aceo was isolated by steam distillation and then analyzed by gc-fid and gc-ms. thirty-seven volatile chemicals were found in the acquired data (tab. 1, fig. 1), accounting for 95.78% of the total detected oil composition. among these, 35.59% were fatty alcohols, 19.86% were sesquiterpenoids, 14.03% were monoterpenoids, and 9.43% were hydrocarbons. the main abundant compounds were octadecanol (33.08%), caryophyllene oxide (5.10%), cis-thujone (4.81%), tetracosane (4.36%), nonanal (4.05%), and phytone (3.66%), respectively. nonanal and phytone, the two main ingredients, have not been described before in the aceo, according to the literature. some anacyclus species, including a. maroccanus, a. radiatus, a. cyrtolepidioides, and a. pyrethrum, have been biologica nyssana ● 16 (2) december 2025: fadel et al. ● chemical composition and antioxidant activity of essential oil from algerian anacyclus clavatus peak n° rt kicalc ki theo component area % 1 22.992 1017 1042 artemisia ketone 1.98 2 25.476 1062 1062 cis-thujone 4.81 3 25.559 1064 1069 nonanal 4.05 4 26.137 1074 1112 trans-thujone 0.54 5 29.575 1141 1177 4-terpineol 0.97 6 30.554 1160 1194 myrtenal 0.9 7 30.656 1162 1195 myrtenol 0.66 8 34.01 1232 1233 chrysanthenyl acetate 2.55 9 39.025 1345 1365 neryl acetate 1.64 10 39.525 1357 1374 α-copaene 0.89 11 39.952 1374 1388 β-elemene 0.58 12 40.114 1404 1408 caryophyllene z 1.04 13 43.152 1444 1452 β-e-farnesene 2.44 14 44.268 1472 1485 β-copaene 2.7 15 46.068 1519 1523 δ-cadinene 0.61 16 48.382 1580 1578 spathulenol 3.39 17 48.499 1583 1582 neryl isovalerate, butanoic acid 1.96 18 48.606 1585 1587 caryophyllene oxide 5.1 19 49.036 1597 1595 salvial-4(14)-en-1-one 1.28 20 49.654 1614 1616 1,3,12-hexadecatriene 1.02 21 50.891 1648 1641 α-epi-muurolol 1.47 22 51.294 1660 1649 β-eudesmol 0.51 23 51.449 1664 1654 α-cadinol 1.13 24 53.584 1724 1701 pentadecanal 0.89 25 58.918 1864 1841 phytone 3.66 26 60.667 1906 1884 hexadecanol 0.87 27 64.266 2001 1977 hexadecanoic acid 3.32 28 66.048 2062 1999 octadecanal 0.75 29 67.989 2134 2081 octadecanol 33.08 30 68.349 2149 2102 heneicosane 0.53 31 68.763 2166 2106 phytol 1.43 32 72.85 2360 2365 heneicosanol 1.64 33 73.076 2371 2400 tetracosane 4.36 34 73.695 2404 2412 methyl-20-heneicosonoate 0.47 35 76.894 2596 2600 hexacosane 2.27 36 80.22 2822 2800 octacosane 1.35 37 83.882 3048 3000 triacontane 0.92 oil yield 0.01 total identified 95.78 fatty alcohols 35.59 sesquiterpenoids 19.86 monoterpenoids 14.03 hydrocarbons 9.43 others 16.87 table 1. chemical composition of aceo using gc-ms analysis biologica nyssana ● 16 (2) december 2025: fadel et al. ● chemical composition and antioxidant activity of essential oil from algerian anacyclus clavatus major compounds percentage (%) algeria tunisia batna boumerdes boumerdes boumerdes sousse (aliboudhar et al., 2013) (aliboudhar & tigrinekordjani, 2014) (aliboudhar et al., 2015) (hammami et al., 2013) octadecanol 33.08 0.06 0.1±0.0 to 0.2±0.1 0.8 caryophyllene oxide 5.1 1.28 1.3±0.3 to 2.5±0.6 0.2±0.2 to 0.3±0.1 cis-thujone 4.81 0.9±0.4 to 1.3±0.5 9.8 tetracosane 4.36 tr 0.5 nonanal 4.05 phytone 3.66 germacrene d 16.84 12.3±1.6 to 16.1±1.4 2 β-thujone 11.16% artemisia ketone 1.98 0.53 6.5±0.5 to 10.0±0.8 0.4±0.3 0.4 α-thujone 0.3 10.6±1.0 to 11.9±1.1 δ-elemene 0.17 0.4±0.0 to 4.0±0.7 9.1±1.1 to 10.4±1.3 trans-chrysanthenyl acetate 0.5±0.1 to 3.6±0.8 12.3 chrysanthenone 0.1 0.1±0.1 8.2 table 2. major compounds of aceo from different regions fig 1. gc-fid chromatogram of aceo shown to contain tetracosane and caryophyllene oxide in their essential oils (el baz et al., 2024; sissi et al., 2024; zardi-bergaoui et al., 2008). tab. 2 shows the presence of the main components of our sample in the aceo collected from different regions of the world. we can note variability in the volatile chemical composition of our aceo species compared to that studied in other regions. only a few published studies have reported the chemical composition of aceo in the algerian and tunisian regions (aliboudhar et al., 2013; aliboudhar & tigrine-kordjani, 2014; aliboudhar et al., 2015; hammami et al., 2013). by comparing the data (tab. 2), a significant difference aliboudhar & tigrine-kordjani; aliboudhar et al., 2015; hammami et al., 2013), were absent in our sample, except for artemisia ketone compounds. this chemical composition variation could be linked to several factors, including geographical and environmental factors (lahmar et al., 2025; sarmah & bora, 2025; burczyk et al., 2024), time of collection (hazrati et al., 2024; boaventura et al., 2025), extraction technique (acosta-vega et al., 2025; suttiarporn et al., 2025; ferraz & silva, 2024), and genetic background (jakovljević, 2025; biologica nyssana ● 16 (2) december 2025: fadel et al. ● chemical composition and antioxidant activity of essential oil from algerian anacyclus clavatus samples yield (%) total phenolic content µg gae/mg total flavonoid content µg qe/mg dpph radical scavenging ic50 (mg/ml) β-carotene (%) acme 20.2 19.28±4.93 6.53±0.70 6.96±0.06 40.09±3.79 aceo 0.01 78.1±5.28 ascorbic acid 0.22±0.02 α-tocopherol 61.42±3.64 table 3. antioxidant activity, total phenol and flavonoid contents of aceo and acme (mean values ± sd, n = 2) fig. 2. gallic acid calibration curve for tpc fig. 3. quercetin calibration graph for tfc in chemical composition was observed, where the main compounds in our sample, octadecanol, caryophyllene oxide, cis-thujone, and tetracosane, were absent or present at low concentrations in the previously mentioned samples. as for the two compounds, nonanal and phytone, they were completely absent in the previously published samples. in contrast, germacrene d, β-thujone, α-thujone, δ-elemene, trans-chrysanthenyl acetate, and chrysanthenone, which were predominant in all published samples (aliboudhar et al., 2013; ganić et al., 2025; solgi et al., 2025; mustafa et al., 2024). moreover, the high concentration of octadecanol in aceo, a fatty alcohol known as a vaginal drug-delivery vehicle (yuan et al., 2010), and caryophyllene oxide, a potent antioxidant, antiinflammatory (kumar et al., 2025), and antiparasitic (lópez-lópez et al., 2025) sesquiterpenoid, suggests this particular chemotype may exhibit an enhanced transethosomal system for enhanced transdermal delivery and therapeutic biological effects. antioxidant capacity the percentage yields (tab. 3) of aceo and acme were calculated based on the initial mass of the plant material as follows: yield (%) = m1×100 / m2, where m1 is the final obtained mass of the oil/extract, and m2 is the initial mass of the species. the equation y = 0.018x + 0.236 was used to determine phenol concentrations at 765 nm (fig. 2), with a correlation coefficient (r2) of 0.997. gallic acid was used as the standard for the linear curve. the obtained results (tab. 3) showed that the acme had a moderate amount of phenols with a tpc of 19.28±4.93 µg gae/mg. the flavonoid amount was measured using the calibration curve equation y = 0.0337x + 0.054 at 420 nm (fig. 3), with a correlation coefficient (r2) of 0.997. quercetin was used as the standard for the linear curve. the results showed that the acme had moderate flavonoid content (6.53±0.70 µg qe/mg). regarding the antioxidant evaluation of acme using the dpph assay, ascorbic acid was used as a reference, and the results were expressed as ic50 values (tab. 3). the linear curve equation y =7.208x–0.136 (r2 = 0.993) was used to determine the ic50 (fig. 4), and the linear curve equation y=240.8x+0.145 (r2=0.999) was used to determine the ic50 of the ascorbic acid (fig. 5 and fig. 6). the values indicate a weak antioxidant capacity for acme compared to ascorbic acid (ic50 = 6.96±0.06 and 0.22±0.02 mg/ml, respectively), whereas, the β-carotene assay (fig. 7), revealed a moderate antioxidant capacity (40.09±3.7%) and a high antioxidant capacity of aceo (78.1±5.28%) compared to the α-tocopherol standard with 61.42±3.64% (aceo ˃ α-tocopherol ˃ acme). this could be explained by the difference in antioxidant mechanisms (famutimi et al., 2025). several published works reported the antioxidant capacities of a. clavatus extracts and essential oils (chroho et al., 2022; bouriche et al., 2016; aliboudhar et al., 2013). furthermore, a. clavatus extracts and essential oils have been shown, according to many studies, to possess various biological activities, including anticancer, anti-inflammatory, antibacterial, antifungal, and antioxidant capacity (adiba et al., 2019; hammami et al., 2013; hasan et al., 2025). a number of published works demonstrated the direct relationship between the phenols and biological potentials, such as antioxidant capacity (vlocskó et al., 2025; hazarika et al., 2025), including the primary constituents of aceo, caryophyllene oxide (shabana et al., 2023), phytone (gao et al., 2025), and compounds that follow (tab. 1), such as spathulenol (do nascimento et al., 2018), hexadecanoic acid (ganesan et al., 2024), and chrysanthenyl acetate (di napoli et al., 2020). biologica nyssana ● 16 (2) december 2025: fadel et al. ● chemical composition and antioxidant activity of essential oil from algerian anacyclus clavatus fig. 4. linear curve of acme fig. 5. linear curve of ascorbic acid standard fig. 6. antioxidant activity by dpph assay of acme and ascorbic acid conclusion this study’s aim was to enrich knowledge of the volatile composition of anacyclus clavatus, a member of the asteraceae family collected in biologica nyssana ● 16 (2) december 2025: fadel et al. ● chemical composition and antioxidant activity of essential oil from algerian anacyclus clavatus fig. 7. antioxidant activity by β-carotene test of acme and aceo algeria’s aures zone, and to assess its crude methanol extract for total phenol and flavonoid contents. using gc-ms and gc-fid, the primary constituents of aceo were identified as octadecanol, caryophyllene oxide, thujone, tetracosane, nonanal, and phytone. the notably high antioxidant activity of aceo and moderate amounts of flavonoids and phenols found in acme imply that this species could be a useful natural resource for medicinal and nutritional uses. future phytochemical and in vivo biological studies should be focused on the isolation of major compounds, particularly octadecanol and caryophyllene oxide, to confirm their individual contributions to the observed strong antioxidant activity and to test for other potential bioactivities, such as anti-inflammatory, antiparasitic, and vaginal drug-delivery vehicle activities. acknowledgement: pr. rebbas khellaf of m’sila university and pr. bicha sabrina of constantine 1 university are acknowledged by the authors for their help in identifying the plant material. references acosta-vega, l., cifuentes, a., ibáñez, e., & galeano garcia, p. 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(2008). composition and antibacterial activity of the essential oil from flowerheads of anacyclus cyrtolepidioïdes pomel from tunisia. journal of essential oil bearing plants, 11(6), 577–585. https://doi.org/10.1080/097 2060x.2008.10643669 microsoft word 0502_lazarevic_lazarevic novo biologica nyssana 1 (1-2) december 2010: 105-109 lazarević, j., lazarević, s. possibilities for production and application of… 105 original article ! possibilities for production and application of native cyclamen neapolitanum in landscape architecture and horticulture jelena lazarević1*, slobodan lazarević2 1 biotechnical faculty department of forestry, mihaila lalića 1, 81000 podgorica, montenegro 2 kopernikova 18, 11 000 belgrade, serbia *e-mail: ena.lazarevic@gmail.com abstract: lazarević, j., lazarević, s.: possibilities for production and application of native cyclamen neapolitanum in landscape architecture and horticulture. biologica nyssana, 1 (1-2), december 2010: 105-109. cyclamen neapolitanum ten is autochthonous in montenegro and serbia in wide range of habitats, from mediterranean-seaside, to sub mediterranean and mountain ones, where it grows on fresh, porous soils, rich in humus and carbonates. it is perennial hardy herbaceous plant with underground tuber, which goes in dormancy trough the period of physical drought. c. neapolitanum flourishes during the autumn, forming after near ground rosette of leaves, which lasts to the next summer, so it is extraordinary suitable for perennials, especially alpinums, individual green areas and also for vertical greening of buildings (balconies, terraces). reproduction is generative. generative reproduction of c. neapolitanum was examined with seed collected in gorica (podgorica, mne), petrovac (mne), and palojce (grdelicka george, serbia). two different artificial substrates were used, as well as classical technologies of plant production with single seed sowing in tresset pots, which were later used to transplantation. seed germination was high (more then 85%), for the seeds originated from all three localities, and also in both of substrates. further loses in plant production were not evident, and development of seedlings as well as transplanted plants, was good. based on conducted experiments, here are given the proposals for economical commercial production, and also for cultivation of plants on green areas. key words: cyclamen neapolitanum, serbia, montenegro, seed germination, commercial production. introduction ! cyclamen l. is a genus of 23 species of flowering plants, native in mediterranean region, along with krimea, caucasus, mountains of middle europe and cappadocia. the most popular among cyclamen is c. persicum miller, originated from ciprus, syria and israel, with their cultivars, which are one of leading flower pot cultures for more then 100 years. during the long period of cultivation, numerous cultivars have been created, differing among each other according to size of entire plant, flower color and size, and petals shape. natural size of c. persicum is about 30 cm in height, and about 18-20 cm in diameter (above ground). according to size of plant, there is a whole group of selected cultivars with “small sizes”not more then 10 cm in height, and 915 cm in diameter, called mini cyclamen, which became very popular pot culture. but those mini cyclamen, as almost all c. persicum cultivars do not tolerate low temperatures, so they are appropriate only as a pot culture in interior and exterior. cyclamen neapolitanum ten is autochthonous in montenegro and serbia in wide range of habitats, from mediterranean seaside, to submediterranean and mountain ones, where it grows on fresh, porous soils, rich in humus and carbonates. it is perennial hardy herbaceous plant 10th sfses • 17-20 june 2010, vlasina lake1 (1-2) • december 2010: 105-109 biologica nyssana 1 (1-2) december 2010: 105-109 lazarević, j., lazarević, s. possibilities for production and application of… 106 with underground tuber, which, as the other cyclamen species, goes in dormancy trough the period of physical drought. c. neapolitanum flourishes during the autumn, forming after near ground rosette of leaves, which lasts to the next summer. the flowers appear in late summer and autumn before leaves, and are distinguished by the base of the petals flaring outwards into a pentagonal throat with 10 lobe-like teeth (auricles). tubers are very large, up to 10 cm in diameter, with roots growing from the upper surface only. leaves are robust and tough, very variable, usually with 5-9 shallow lobes but often rounded or lance shaped, usually toothed but not cartilaginous, blade green with silvery mottling of great variety. leaf stalks are long, creeping and then upstanding. flowers are 11.5 cm long, with varying shade of rose pink with a dark crimson blotch at the throat, with faint scent. the fruit is five-chambered capsule, containing numerous sticky seeds, about 2 mm in diameter in the base. the capsule is surrounded by sepals until fruit ripening. during the ripening, fruiting stalks are curling up in a clock-spring-like spiral and settle (condescend) to ground level, and 1-2 cm underground, taking the capsule in peat layer in depth optimal for seed germination. that’s happened in the end of plant vegetation, when the leaves start drying, i.e. when the more then half leaves are already dry. when is ripe, capsule is about 1, 5 (1-2) cm in diameter. (p a p a z o v a a n t o n o v a , 1964; g r u n e r t , 1968; p o l u n i n & h u x l e y , 1978). c. napolitanum in serbia was recorded and described by p a n č i ć (1974). it belongs to mediterranean floral element, and its native areal is widespread to middle and eastern parts of south europe, south italy and greece. in montenegro it grows in maquia, with erica arborea, spartum junceum near the adriatic coast, but also in sub mediterranean and mountain region in primarily oak forests or pine cultures, in deciduous and evergreen shrubberies and woods on fresh, porous soils, rich in humus and carbonates. in serbia is widespread in mountain regions around južna morava, in shrubberies and deciduous forests around bujanovac, predejane, lebane, vučje (leskovac), stalać, kruševac, niška banja, jelašnica (niš) and koritnik (prizren), on warm, but shaded places, on fresh, porous soils, rich in humus and carbonates (n i k o l i ć , 1972). c. neapolitanum is according to size of underground spring very close to mini cyclamen, but it withstand low temperatures, so there is a possibility for its application in exterior. it is extraordinary suitable for perennials, especially alpinums, individual green areas and also for vertical greening of buildings (balconies, terraces). during the october and november, in phase of flowering, in flower markets in vranje, leskovac, niš and podgorica, one can find c. neapolitanum planted in pots. for that purpose, tubers have been being taken directly from the native habitats. this way of popularization and cultivation of cyclamen can not be accepted from the standpoint of biodiversity protection. besides, their cultivation and plant production by generative reproduction is possible, easy and economical. we investigated the possibility of production of c. neapolitanum for commercial purposes, and first of all we were interesting in production of cyclamen as perennial plant, and for tuber production. we would like to underline that fact, because the mostly produced c. persicum cultivars are produced because of flowers, and their decorative values: as pot plants, or even as cut flowers. in that case, in regime of forced plant production, production cycles last about 12-13 months, or even 8-9 months in greenhouses, with strong fertilization and also phytohormone application. (b a c h a m , 1985; b o o d l e y , 1981). period of production of plant with aim to get tuber, which could support plant development on open, in gardens or green areas, and during the number of years, lasts much longer. material and methods seed collection generative reproduction of c. neapolitanum was examined with seed collected from 3 localities. collecting of seeds was done during the last decade of may. about 25 fruits of c. neapolitanum were collected per locality. the fruits were in ripening (5 days). the seeds are cleaned, counted and measured on 0.0001g accuracy. table 1. the main characteristics of chosen localities, according to geological base and climate locality n e altitude geological base climate petrovac (mne) 42˚15’ 18˚56’ 0 m limestone mediterranean podgorica(mne) 42˚26’ 19˚17’ 54 m limestone submediterranean predejane (serbia) 42˚50’ 22˚08’ 276 m limestone and metamorphic rocks moderate continental biologica nyssana 1 (1-2) december 2010: 105-109 lazarević, j., lazarević, s. possibilities for production and application of… 107 experiment design two different artificial substrates were used in the experiment: floradur b and floradur b: vermiculite 7:3. floradur b fine is substrate made of peat (70% of white peat and 30 % of black peat) with addition of sand and perlite, with super fine structure and with ph 5.2-6.0. it contain 70-150 mg/l n, 80-180 mg/l p2o5, 140-220 mg/l k2o and 0.5-1,1 mg/l salts. floradur b with addition of vermiculite (7:3) was also used, and vermiculite added in order to perform substrate porosity. 120 seeds per locality were separated and treated by benlate wp 50 (0.05%) and used for sowing. seed sowing and entire testing were carried out in open, with environmental conditions modified by shading: 80% during the plant dormancy (summer), and 20% during the period of plant development (autumn to spring). moderation of environmental condition was influenced by sand layer 0.03 m3/m2, placed on the bottom of bad. this sand layer contributes to fine regulation of air humidity, and consequently moderate temperature. besides, it assures good drain off substrate, and also protection against ground insects, worms and snails. classical technologies of plant production with single seed sowing in peat strips were used. strips were filled with substrate and marked. sowed strips were placed on bed previously filled with sand in 3 cm layer. seed sowing was done on jiffi strips 4x5, (product no. 30051590 ) (750 plants per m2), first transplanting in jiffy strips 6x6 was done in the september of next (second) growing year, and the second transplantation in jiffy pots 10x 8, in next september (third year of cultivation) soil moisture, and consequently air humidity, was maintained manually. phases of plant development under the examination were: from sowing to germination and plant development during the vegetation period. cultivation from sowing to germination cultivation of sowed seed have taken place in modified climate conditions, with 80-100% shading, with even watering and on temperature of 20-22˚ c. cultivation of seedlings and young plants seedlings were watered, and preventive phytopathological and entomological protection was made. in first half of september shading was reduced to 20%, and in those conditions plants have stayed until the end of vegetation period. in second half of may next year, when the 50% of assimilation leaf mass were dry, shading of 80% was established, and the watering was stopped. from the beginning of june, plants were, practically, in dormancy. first transplantation has been done during the september, second year from seed sowing. transplantation was done on jiffi strips 6x6 cm (300 plants per m2) (product no. 30052200 ). second transplantation was done in next september, third year from seed sowing. jiffi pots 10x8 (single-100 plants per m2) (product no. 30033500) were used. as a measure of plant development, the total number of developed leaves, also as general appearance and condition of plant was estimated. statistical analysis the data were analyzed by oneway analysis of variance (anova) and significant differences among treatments were separated by duncan’s test (p<0.05) for diameters of collected fruits, number of seeds per fruit and seed mass. number of days from sowing to germinations, and also all other later comparing of numbers of leaves formed per plant, also as tuber diameters, were compared on the base of mean value. we consider that those parameters, though varying in some manner, are not needed to be analyzed statistically, because they do not contribute to technological process of c. neapolitanum commercial production in frame postulated with this investigation. results and discussion average number of seeds per fruit, mass of seed and characteristics of seed germination are shown in the table 2. statistical analyses anova with duncan test (sig 0.05) showed that there were no significant differences among material collected at different localities according to diameter of fruits, number of seed per fruit, and mass of seed. seeds were sown on 10th of june. germination started on 5th of july and lasted to july 12th, i.e. germination lasted 25-32 days. seed germination was high (more then 80%), for the seeds originated from all three localities, and also in both of substrates. there were no differences according to germination in substrate 1 and substrate 2, but germination lasts shorter in substrate 2, which was more porous. as important characteristic, we would like to emphasize that the percent of technical germination was quite high according to c. persicum (p a p a z o v a -a n t o n o v a , 1964). biologica nyssana 1 (1-2) december 2010: 105-109 lazarević, j., lazarević, s. possibilities for production and application of… 108 table 2. number per fruit and mass of c. neapolitanum seed divided from different localities, and characteristics of seed germination seed origin f diam nmb of seeds mass of 10 seeds s gs gd % g petrovac (mne) 1.170 a 19.6 a 0.1701 a 1 25 7 82 2 27 4 84 podgorica (mne) 1.289 a 20.8 a 0.1803 a 1 26 4 84 2 26 3 86 predejane (serbia) 1.298 a 20.4 a 0.1753 a 1 25 6 80 2 27 5 85 f diam –diameter of collected fruits , s n number of seed per fruit, maverage mass of 10 seeds (g), s sowing substrate, gsstarting of germination (nmb. of days after sowing), dgduration of germination (nmb. of days), %-percent of germinated seeds table 3. number of leaves per plant, developed during the four year of investigation seed origin substrate number of leaves per plant sept ii may ii t r a n s p l a n t. may iii t r a n s p l a n t. may iv petrovac (mne) 1 2-3 (2.5) 4-5 (4.7) 8-9 (8.2) 13-18 (15.5) 2 2-3 (2.8) 5 (5.0) 8-9 (8.9) 15-18 (16.5) podgorica (mne) 1 2-3 (2.8) 4.5 (4.5) 8-9 (8.9) 12-17 (14.5) 2 3 (3.0) 5 (5.0) 10 (10.0) 14-18 (16,0) predejane (serbia) 1 2-3 (2.9) 4-5 (4.8) 9-10 (9.6) 14-18 (16) 2 3 (3.0) 5 (5.0) 10 (10.0) 16-17 (16.5) average number of leaves per plant developed during the four years of investigation was shown in table 3. differences in number of leaves formed in september of first year of cultivation and may of second year of cultivation were not evident. number of leaves is mostly equal independently of locality, with lightly decreased number of leaves on substrate 2. in september of second year of cultivation, there were no differences in number of formed leaves. mostly because of needs for developing above-ground spring, transplanting in single pots was done in september third year of cultivation. in that moment, the tubers were about 2 cm in diameter, independently on treatments. during the september and october the flowers were formed, 13 per plant. in regard to small number of flowers per plant, plants have not proved commercial yet. at the end of that vegetation period, in may in fourth year of cultivation, number of leaves per plant was 12-18. the diameter of tubers was about 4 cm in that moment. for following growing season (in fourth year of cultivation) the plants were fit for market: as tubers, as plantlet in peat pots, or as pot plants, transplanted in earthenware. the experiments show that the flowering starts at the same time, independently of seed origin or substrate used, in third year of cultivation, and also without differences according to developed leaf mass. average number of leaves was bigger on substrate 2. the tubers were equal independently of seed origin and substrate also in the end of vegetation season in fourth year of plant cultivation. on the manner previously described, c. neapolitanum could be generatively reproduced economically. cultivation was done under the natural environmental conditions with shading. sterile conditions of substratum and pots deplete the possibility of pests and diseases appearances. in whole cycle of plant production only 2 transplantings exist, without damaging of root system. humidity, attained by watering is applied only in first 4 months in continuum, and after that only when necessary. plants obtained this way could be used on individual and public green areas, especially in perennials because of late flowering of c. neapolitanum and retaining of leaves during the whole winter, until the end of may. cultivation measures which could be applied are the same as for the other perennes with tubers or bulbs resistant on low temperatures. by supplying market with plants produced this way, one could not only actualize it in application, but also protect their natural genofund and habitats, meaning stoppage of taking tubers from native habitats, and without harm to the wild plants and populations. biologica nyssana 1 (1-2) december 2010: 105-109 lazarević, j., lazarević, s. possibilities for production and application of… 109 conclusion c. hederifolium has a great value as a herbaceous perennial plant for use on individual or public green areas because of its late flowering and retaining of leaves during the whole winter, especially because of its withstanding on low winter temperatures. characteristics of seeds, and consequently developed plants, collecting from native populations of c. neapolitanum in montenegro (petrovac and podgorica) and serbia (predejane) were investigated on two artificial substrates (floradur b and floradur b: vermiculite 7:3). seeds collected from all tree localities have practically the same value, and can be used in generative plant reproduction. seed germination was high (more then 80%), for the seeds originated from all three localities, and also in both of substrates. further loses in plant production were not evident, and development of seedlings as well as transplanted plants, was good. plant development is better on substrate 2 in phase of plant sowing and first transplanting, so we suggest it (floradur b: vermiculite 7:3) for sowing and first transplanting, and substrate 1 (floradur b) for second transplanting. usage of jiffy strips and jiffy pots during cultivation assures good aeration of soil, which, as we believe, contributes to good results. references bacham, t. 1985: cyclamen-faster crops are here, in ball g.j. (ed)the ball red book a reston book, prentice-hall, new jersey: 440-448 boodley, j.w. 1981: the commercial greenhouse, delmar publishers inc, new york: 296-300. grunert, c. 1968: bunte blumen im garten, neumann verlag, melsungen, bez. kassel, 176177. kolić, b. 1983: šumarska ekoklimatologija sa osnovama fizike atmosfere, naučna knjiga, beograd 365-377 nikolić, v. 1972: rod cyclamen, in josifović, m. (ed), flora sr srbije iii: 500-502, srpska akademija nauke i umetnosti, beograd. pančić, j. 1976: flora kneževine srbije i dodatak flori kneževine srbije. ponovljeno izdanje, sanu, beograd papazova-antonova, g. 1964: cvetarstvo, zemzidat, sofija schacht, w. 1976: blumen europas, p.13-22, 142149. paul parey, berlin und hamburg polunin, o., huxley, a. 1978: flowers of the mediterranean, p. 140-142, chato and windus, london stojadinović, d., vidojević, d., crnobrnja-isailović, j.: variation of clutch characteristics in population of eastern hermann’s tortoises (testudo hermanni boettgeri gmelin 1789). biologica nyssana, 8 (1), september 2017 biologica nyssana 8 (1)  september 2017: 123-127 stojadinović, d. et al.  variation of clutch characteristics in population… 123 original article received: 04 january 2017 revised: 23 february 2017 accepted: 11 mart 2017 variation of clutch characteristics in population of eastern hermann’s tortoises (testudo hermanni boettgeri gmelin 1789) dragana stojadinović1, dragana vidojević1, jelka crnobrnja-isailović1,2 1university of niš, faculty of science and mathematics, department of biology and ecology, višegradska 33, niš, serbia 2 institute for biological research „sinišastanković“, university of belgrade, despotastefana 142, 11000 belgrade, serbia * e-mail: draganapenev@yahoo.com abstract: stojadinović, d., vidojević, d., crnobrnja-isailović, j.: variation of clutch characteristics in population of eastern hermann’s tortoises (testudo hermanni boettgeri gmelin 1789). biologica nyssana, 8 (1), september 2017: 123-127. we present here information on some fecundity parameters in local population of eastern hermann’s tortoise situated in the area of kunovica, at the outskirts of the city of niš. the data were collected in the field and in 97% were based on reconstruction of number and dimensions of eggs in destroyed nests what influenced on quality of information. only two of 78 nests recorded from 2010 to 2014 were found intact. other 76 clutches were found destroyed and therefore only some fecundity parameters were measurable, while the others were reconstructed by using formulas proposed in the relevant literature. on the basis of collected data we estimated average clutch size of hermann’s tortoises in kunovica as four eggs what was lower than in populations from greece. however, the most of calculated egg dimensions (e.g. maximal width, volume and mass) were higher than in populations from greece and france. key words: fecundity, clutch size, egg size, testudo hermanni boettgeri apstrakt: stojadinović, d., vidojević, d., crnobrnja-isailović, j.: varijacije karakteristika legla u populaciji istočne podvrste šumske kornjače (testudo hermanni boettgeri gmelin 1789). biologica nyssana, 8 (1), septembar 2017: 123-127. u ovom radu predstavljene su informacije o nekim parametrima fekunditeta lokalne populacije istočne podvrste šumske kornjače sa područja kunovice (okolina grada niša). svi podaci su prikupljeni na terenu i u 97% slučajeva izvršena je rekonstrukcija broja i dimenzija jaja, što utiče na kvalitet informacija. samo dva od 78 pronađenih gnezda u periodu od 2010. do 2014. su bila sa neoštećenim jajima. ostala gnezda su bila uništena, tako da su samo neki parametri mogli da budu izmereni, dok su ostali rekonstruisani pomoću formula datih u odgovarajućoj literaturi. na osnovu prikupljenih podataka, procenili smo da je prosečna veličina legla kod šumske kornjače iz kunovice 4 jaja, što je manje nego kod populacija iz grčke. ipak, većina karakteristika 8 (1) • september 2017: 123-127 doi: 10.5281/zenodo.964581 biologica nyssana 8 (1)  september 2017: 123-127 stojadinović, d. et al.  variation of clutch characteristics in population… 124 jaja ove lokalne populacije (npr. maksimalna širina, zapremina, masa) je bila veća nego kod populacija iz grčke i francuske. ključne reči: fekunditet, veličina legla, veličina jaja, testudo hermanni boettgeri introduction traditional life-history approach (g a d g i l & b o s s e r t , 1970) proposes that reproductive investment increases with age and reaches the peak in the act of terminal investment, where reproduction stops afterwards (w i l l i a m s , 1966; h i r s c h f i e l d & t i n k l e 1975; c l u t t o n b r o c k , 1984). other theory proposes that reproductive investment depends on number of characters subjected to various rates of change, what depends on age of the individual (m c n a m a r a & h o u s t o n , 1996). reptiles in general have greater and more variable fecundity in comparison to endothermous organisms (s h i n e , 2005). in general, female’s fecundity could vary on different levels: within individual female’s life, among females within the same population, among females from conspecific populations or among females of different species. in many reptiles, larger females produce bigger clutches (f i t c h , 1970). reproduction is usually delayed in females until they accumulate enough energy to produce clutch as large as their abdominal cavity is (v i t t & c o n g d o n , 1978; v i t t & p r i c e , 1982). some observations confirmed that decrease of average female body size and average clutch size, caused by environmental change, may decrease survival and growth rates (s h i n e , 2005). egg laying happens from middle may to end of june (c r u c e & r ă d u c a n , 1976; s w i n g l a n d & s t u b b s , 1985; f e r t a r d , 1992; b e r t o l e r o e t al., 2007b) and females repeatedly use the same nesting place (s w i n g l a n d & s t u b b s , 1985). incubation period varies from 90 to 124 days (c r u c e & r ă d u c a n , 1976; c h e y l a n , 1981; n o u g a r è d e , 1998). eggs are of white color, hard shelled and almost ellyptical in shape. incubation temperature varies from 23◦c, to 34◦c (e e n d e b a k , 1995). if incubation temperature keeps constantly at 25◦c or 33-34◦c, mortality of the embrios increases to approximately 50%. on the temperatures lower than 23◦c or higher than 34◦c mortality reaches 100% (e e n d e b a k , 1995). in year 2010 we established regular monitoring of one local population of eastern hermann’s tortoise in the area of village kunovica in vicinity of town of niš in south-eastern serbia. our main goal was to collect data on life history parameters and to record their variation in order to enable population viability analysis in the future. this study presents the most possible projection of female fecundity in population of hermann’s tortoises in kunovica based on data collected during first five years of monitoring. we assumed that average clutch size and egg dimensions could be in range of values reported for other analysed populations of testudo hermanni boettgeri. material and methods field procedures field work was conducted twice per year from 2010 to 2014, always in may and june and seven consecutive days each. detailed description of the studied area could be find in s t o j a d i n o v i ć et al. (2013). during the day researchers were collecting records on different aspects of biology of the species, eggs laying and nests. clutch size was estimated by reconstruction of located destroyed nests only where counting of eggs from shells was possible. direct counting of eggs was possible when females were encountered while laying eggs. in these two cases the eggs from intact nest were carefully taken from the hole, measured for mass and maximal lenght & width by pesola field balance with accuracy of 0.2 g and by digital calliper with precision of 0.01 mm, respectively. the eggs were carefully deposited back in the nest afterwards and covered with soil. we considered egg dimensions as reliable only when length and/or maximal width of egg shells were available to measure and they were included in the analysis. species description testudo hermanni (gmelin 1789) is one of three european tortoise species. two subspecies of hermann’s tortoise are currently recognized: the western subspecies -t. hermanni hermanniinhabits parts of spain, france and italy while eastern subspecies -t. hermanni boettgeriis distributed in croatia, bosnia and herzegovina, montenegro, albania, serbia, fyrm, romania, bulgaria, greece and turkey (fritz et al. 2006). statistical procedures egg volume (ev, mm³) was estimated by applying the equation ev=(π*l*w²)/6000 (longepierre et al., 2003), where π =3.14, l= longer egg axis or egg biologica nyssana 8 (1)  september 2017: 123-127 stojadinović, d. et al.  variation of clutch characteristics in population… 125 lenght, and w = shorter egg axis or egg width (c o l e m a n , 1991). egg mass (em, g) was estimated by applying the equation em=0.435*l+0.0203*w²-14.7 (h a i l e y & l o m b o u r d i s , 1990), where l = egg lenght, and w = egg width. average egg size, shape (l/w), mass and volume were estimated independently for every nest (l o n g e p i e r r e et al., 2003). all statistical analyses were done by software package statistica 7.0 (statsoft inc.). results and discussion only two of 78 hermann’s tortoise clutches recorded during four years of monitoring field site in kunovica were found intact and they contained four and five eggs. females that deposited these clutches were measured for streight carapace length (scl), midbody carapace width (mcw), maximal carapace width (maccw) and weight (w). for female that deposited four eggs measures were: scl-193.58 mm, mcw-141.38 mm, maxcw-143.42 mm, w-1347 g; for female that deposited five eggs measures were: scl184.15 mm, mcw145.93 mm, maxcw 146.13 mm, w-1390 g. average egg lenght in intact clutch was 37.40 mm ± 0.68 mm, average maximal width was 30.25 mm ± 0.39 mm, and average egg mass was 20.0 g ± 0.68 g. the reconstruction of clutch and egg size enabled us to appoximate average clutch size (4 eggs ± 1 egg) in this particular population of eastern hermann’s tortoise. clutch size varied from 1 to 7 eggs. overall number of nests recorded over years was 13 in 2010 (6 in last week of may and 7 in third week of july), 6 in 2011 (4 nests in may and 2in july), 4 in 2012 (4 nests in may), 38 in 2013 (35 in may and 3 in july) and 17 in 2014 (4 nests in may and 13 in july). using h a i l e y and l o m b o u r d i s (1990) formula for estimation of egg mass on the basis of egg dimensions we got hypothetic average egg mass value of 20.8 g ± 4.8 g. observed average egg mass recorded in the field (based on measured intact eggs) was 20.0 g ± 0.68g. additionally, we have also calculated mean values of fecundity parameters for destroyed clutches, and presented their averages in table 1. average egg length per clutch was 37.1 mm, varying from 33.4 mm to 41.3 mm. average value of maximum egg width per clutch was 29.8 mm, varying from 24.0 mm to 35.4 mm. average approximate egg mass per clutch was 19.6 g, varying from 11.8 g to 26.6 g. average egg volume per clutch was 17.5 mm³, varying from 10.3 mm³ to 24.5 mm³. clutch size in testudo hermanni varied from one to seven eggs in western (nominative) subspecies (e s t e b a n , 1987; f e r t a r d , 1992; n o u g a r è d e , 1998; l o n g e p i e r r e et al.,2003; b e r t o l e r o et al., 2007c) and from one to nine eggs in eastern (boettgeri) subspecies (s w i n g l a n d & s t u b b s , 1985; h a i l e y & l o u m b o u r d i s , 1988, 1990) (tab. 1). average clutch size varied from 3.3 in western (e s t e b a n , 1987; f e r t a r d , 1992; n o u g a r è d e , 1998; l o n g e p i e r r e et al., 2003; b e r t o l e r o et al., 2007c) to 4.3 eggs in eastern subspecies (s w i n g l a n d & s t u b b s , 1985; h a i l e y & l o u m b o u r d i s , 1988, 1990). average egg size and weight were 27.2× 34.3 mm and 15.1 g, respectively, in testudo hermanni hermanni (c h e y l a n , 1981; e s t e b a n , 1987; f e r t a r d , 1992; l o n g e p i e r r e et al., 2003; b e r t o l e r o et al., 2007a,c) and 27,9×37,4 mm and 17.1 g, respectively, in testudo hermanni boettgeri table 1. overview of fecundity parameters recorded in hermann’s tortoise populations from different parts of species range (france, greece) and from serbia. n – number of analyzed nests, nav – average clutch size, lav – average egg lenght/clutch, wavaverage maximal egg width/clutch, (l/w)av – average value of egg shape/clutch, evav average value of egg volume/clutch, emav – average value of egg mass/clutch. s.d.-standard devitiation. france 1 (longipierre, 2003) france 2 (longipierre, 2003) greece 1 (hailey, 1988) greece 2 (hailey, 1988) greece 3 (hailey, 1988) serbia (our data, destroyed clutches) serbia (our data, intact clutches) n 76 2 nav ±s.d. 3 ± 1 3 ± 1 5 ± 1 5 ± 1 5 ± 1 4 ± 1 4.5±0.71 lav±s.d. 35.6 37.1± 2.2 37.5 ± 2.2 38.8± 2.7 37.1± 2.3 37.4±0.7 wav±s.d. 27.8 27.0±1.5 28.9 ± 0.9 28.0± 1.7 29.8± 2.9 30.3±0.4 (l/w)av±s.d. 1.28 1.37± 1.5 1.3 ± 1.5 1.38± 1.5 1.25± 0.1 1.2±0.0 emav 16.5 15.8 16.24 18.57 18.09 19.6 20.0 evav 14.4 14.15 16.39 15.92 17.5 17.9 biologica nyssana 8 (1)  september 2017: 123-127 stojadinović, d. et al.  variation of clutch characteristics in population… 126 (c r u c e & r ă d u c a n , 1976; h a i l e y & l o u m b o u r d i s , 1988, 1990;). in general, reproductive traits in female hermann’s tortoises were related to body size and the biggest females have the greatest contribution to overall egg production per reproductive season (c r u c e & r ă d u c a n , 1976; h a i l e y & l o u m b o u r d i s , 1988; f e r t a r d , 1992; l o n g e p i e r r e et al.,2003; b e r t o l e r o et al., 2007c). it was not possible to test this relation in our study as it was almost completely based on data obtained from inspection of broken nests. in fact, only two of 78 recorded tortoise nests were found intact (females were spotted while laying the eggs in may 2010). nonparametric kolmogorov-smirnov two-sample test has shown that there was no significant difference between body size and mass of these two females nor between egg parameters in these two clutches. except 2014, in other years of study the most of the nests was detected in may. it could be explained by the fact that high environmental temperatures starting from july could influence on egg laying time in reptiles with multiple clutches e.g. temperature increase results in earlier nesting time in the summer. studies conducted in france and greece suggested that the first egg laying occursin the midapril, while the last was detected in july. particular flexibility was detected in beginning and the end of the nesting period what could be related to variation in environmental conditions (c r u c e & r a d u c a n , 1976). radiological surveys in those two countries confirmed that the highest number of calcified eggs in oviducts was detected during may and june (b e r t o l e r o et al., 2007b). h a i l e y and l o u m b o u r d i s (1988) were studying reproduction of hermann’s tortoise on three different localities in greece (alyki, litochoron and deskati). average clutch size was 5 eggs (table1). the lowest values of analysed fecundity parameters were recorded on the first locality – dry sandy ground. the other two localities were on fertile soil and similar regading analysed fecundity parameters. localities in france (plaine des mauresand massif des maures) were under the influence of mediterranean climate (l o n g e p i e r r e et al., 2003) what could explain their lower values of fecundity parameters in comparison to greece and serbia (tab. 1). the largest hermann’s tortoise eggs were recorded in serbia (in comparison with data from france and greece, see tab. 1) which should be taken with caution since most of the data were obtained from broken egg shells. egg size could be correlated to body size, but it is not the rule: in some populations larger female tortoises have wider eggs because egg size is constrained by width of the pelvic channel (c o n g d o n & t i n k l e , 1982). however, in some populations smaller females lay more narrow eggs but similar in weight to those of larger females (h a l i l e y & l o u m b o u r d i s , 1988). conclusion reconstruction of number and dimensions of eggs in destroyed nests of hermann’s tortoise from kunovica showed values similar to those obtained from measurement of two intact nests estimated average clutch size of female hermann’s tortoises in kunovica was lower than in populations from greece, but higher than in populations from france. maximal width, volume and mass of eggs were higher than in population in greece and france. acknowledgements. this study represents first results of long-term monitoring on eastern hermann’s tortoises in south-eastern serbia that has been established in 2010 by ds and jci as a part of national project funded by ministry of education, science and technological development of republic of serbia, grant no 173025 „evolution in heterogeneous environments: mechanisms of adaptation, biomonitoring and biodiversity conservation“. our thanks go to a number of students of biology and ecology at the faculty of sciences and mathematics university of niš for participance, especially to j. mijatović for substantal assistance in the field. moreover, logistic support of s. stojadinović and dj. milošević were highly appreciated. field work was approved by permits no 353-011134/2010-03 and no 353-01-29/2011-03 ministry of environment and spatial planning of republic of serbia, no 353-01-505/2012-03 ministry of environment, mining and spatial planning of republic of serbia, no 353-0154/2013-08 and no. 353-01-312/2014-08 ministry of energetics, development and nature protection of republic of serbia. references bertolero, a., cheylan, m., nougarède, j.p., 2007a: accroissement de la fécondité chez la tortue d’hermann testudo hermanni hermanni en condition insulaire: un contre–exemple du syndrome insulaire? revue d’écologie (terre et vie), 62:93–98. bertolero, a., nougarède, j.-p., cheylan, m., 2007b: female reproductive phenology from a population of hermann’s 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ananahirnic@gmail.com abstract: nahirnić, a.: supplements to butterfly fauna (hesperioidea and papilionoidea) of grza river gorge (eastern serbia), biologica nyssana, 2 (2), december 2011: 107-117. the aim of this article is report of butterfly fauna of grza river gorge (eastern serbia). during 2008-2009. nineteen field visits have been made, 11 localities and 12 types of habitats were investigated. a faunistic account of totally 84 recorded butterfly species is given compiling the data: 30 butterfy species previously published by other authors and 81 species from our research. we recorded 54 species new to the investigated territory. zoogeographical composition of all species is done. comparison of the data concerning two others valleys, lazarev canyon and the ibar river gorge studied by jakšić (2008), was made in order to show the specific fauna elements. key words: butterflies, grza river gorge introduction ! the butterfly fauna of grza river gorge has not been truly studied up to now. only two sources provide data on butterfly species present in the area (v a n s w a a y et al., 2007; j a k š i ć 2011). these references give us the list of only 29 species recorded at a single locality, mountain home "đorđe živković" and one more species for grza territory, respectively . grza river gorge is located 20 km eastern from paraćin, at the southern side of kučaj mountains as a part of carpatho-balkanides. examined territory includes the grza river gorge from vrelo to the mouth of čestobrodica river extending over javorački vrh mt. to the north-east, veliki kozji rog mt. to the east and pištolje to the west. grza river gorge is eight km long spreading north south and two km from east to west. this whole region is intersected by numerous brooks. the most important springs are malo vrelo and vrelo. grza river gorge has been carved during glacial and postglacial phase. the supstrate is predominantly calcareous combine with limestone, including neogene lake sediments, tertiary volcanic stones, karst and partly andesite and freshwater middle miocene sediments. the climate of the investigated region is temperate continental. climate and vegetation inversion is characteristic for the grza river gorge. in gorges air is cooler at lower altitudes. as gorge sides shelter valley from wind, atmosphere is calm and moist. this inversion provides suitable climate conditions for mesophile plants such as fagus moesiaca (k. maly) czecz which is dominant edificator of forests in this gorge. oak forests are distributed at higher altitudes above colline beech forests. the preliminary number of vascular plants comprises 346 species (z a j i ć & l a k u š i ć , 2004). according to m i š i ć (1981) relict and polydominant plant communities are typical for the grza river gorge. what makes this territory interesting for biological studies is a significant 2 (2) • december 2011: 107-117 biologica nyssana 2 (2) december 2011: 107-117 nahirnić a. supplements of butterfly fauna… 108 presence of the large number of relict and endemorelict species from the tertiary age and the elements of the sub-mediterranean flora materials and methods sampling of butterflies was done by using an entomological net, and after that the specimens were mounted and conserved. the material is in author’s collection. taxonomical order and the nomenclature were given according to k a r s h o l t & r a z o w s k y (1996) and van s w a a y et al. (2010), respectively. nineteen field examinations were done during 2008-2009, from april to september. localities on the map marked by numbers, the altitude, utm 10 x 10 km code and the habitat codes are listed on fig. 1. determination of habitats was done on the basis of eunis habitat classification l a k u š i ć (2005a) and l a k u š i ć et al. (2005), and partly h i l l et al. (2004). corresponding habitat types to d a v i e s & m o s s (2002) were identified following the l a k u š i ć (2005b). habitat types f3.242c, g1.691, g1.696 and g1.761 don’t have corresponding habitat types in international eunis habitat classification. as an alternative, these four habitat types were related to serbian national classification of habitats (l a k u š i ć & m e d a r e v i ć , 2010). serbian eunis habitat classification codes of these habitats were given together with codes and their names in english from serbian national classification. next habitat types were investigated: e grasslands and lands dominated by forbs, mosses or lichens: e1 dry grasslands: e1.2 perennial calcareous grassland and basic steppes (different than all e1.22 investigated in this study), e1.22 arid subcontinental steppic grassland (festucion valesiacae), e1.74 calamagrostis epigejos stands, e2.2 low and medium altitude hay meadows, e3.4 moist or wet eutrophic and mesotrophic grassland, e5 woodland fringes and clearings and tall forb stands: e5.21 xero-thermophile fringes, e5.22 mesophile fringes; f heathland, scrub and tundra: f3.242c b2.511 oriental hornbeam [carpinus orientalis] thicket; g woodland, forest and other wooded land: g1.691 a3.22 colline beech [fagus moesiaca] forests, g1.696 a3.22c polydominant relict colline beech [fagus moesiaca] forest, g1.761 a2.111 moesian hungarian oak [quercus frainetto] and turkey oak [quercus cerris] woods, g1.a46 southeastern european ravine forests. list of investigated localities. 1. mouth of čestobrodica to grza, 290 m a.s.l., ep 55, е2.2, e3.4, e5.21, e5.22, g1.a46. 2. weekend estate "grza", 305-337 m a.s.l., ep 56, e2.2, g1.691. 3. veliki kozji rog mt., 570 m a.s.l., ep 56, e1.74. 4. mountain home "đorđe živković", 423 m a.s.l., ep 56, e1.22, e2.2, e5.22 g1.691, g1.761. 5. pištolje, 547 m a.s.l., ep 56, e1.22, e5.22. 6. vrelski krš, 650 m a.s.l., ep 56, f3.242c. 7. vrelo, 415 m a.s.l., ep 56, g1.691. 8. vrelska padina, 679 m a.s.l., ep 56, e1.22, g1.761. 9. barski vrh мt., 785 m a.s.l., ep 56, e1.2, g1.696. 10. javoračke padine, 778 m a.s.l., ep 56, e2.2, e5.22. 11. javorački vrh mt., 930 m a.s.l., ep 56, f3.242c. figure 1. map of investigated localities results the following list includes all data of butterflies recorded in grza river gorge. all new data were made by the author. biologica nyssana 2 (2) december 2011: 107-117 nahirnić a. supplements of butterfly fauna… 109 superfamily hesperioidea family hesperiidae 1. erynnis tages (linnaeus, 1758) new data: weekend estate "grza" (e2.2), 20.04.2009; mouth of čestobrodica to grza (e5.21), 12.07.2009. 2. carcharodus alceae (esper, 1780) new data: weekend estate (e2.2), 13.06.2009. 3. pyrgus sidae (esper, 1784) new data: vrelska padina (e1.22), 14.06.2009. 4. pyrgus malvae (linnaeus, 1758) new data: weekend estate "grza" (e2.2), 20.04.2009; mouth of čestobrodica to grza (e5.21), 10.5.2009. 5. thymelicus lineola (ochsenheimer, 1808) published data: grza (380-420 m, utm: ep 46), 15.07.2005. van swaay et al. (2007). new data: weekend estate "grza" (e2.2), 13.06.2009; mountain home "đorđe živković" (e1.22), 07.07.2009; mouth of čestobrodica to grza (e5.21), 12.07.2009. 6. thymelicus sylvestris (poda, 1761) published data: grza (380-420 m, utm: ep 46), 15.07.2005. van swaay et al. (2007). new data: mouth of čestobrodica to grza (e2.2), 13.06.2009; pištolje (e1.22), 09.07.2009. 7. thymelicus acteon (rottemburg, 1775) new data: mouth of čestobrodica to grza (e2.2), 13.06.2009; mountain home "đorđe živković" (e2.2), 14.06.2009; vrelska padina (e1.22), 14.06.2009. 8. ochlodes sylvanus (esper, 1777) published data: grza (380-420 m, utm: ep 46), 15.07.2005. van swaay et al. (2007). new data: mountain home "đorđe živković" (e2.2), 28.07.2008; mountain home "đorđe živković" (e1.22), 01.08.2008; mouth of čestobrodica to grza (e2.2), 13.06.2009; vrelska padina (e1.22), 14.06.2009; mountain home "đorđe živković" (e2.2), 07.07.2009; vrelo (g1.691), 08.07.2009; pištolje (e1.22, e5.22), 09.07.2009; mouth of čestobrodica to grza (e5.21), 12.07.2009; mouth of čestobrodica to grza (e2.2), 12.07.2009; weekend estate "grza" (e2.2), 13.07.2009; vrelska padina (e1.22), 15.07.2009; barski vrh (e1.2), 15.07.2009; javoračke padine (e2.2), 15.07.2009; javorački vrh (f3.242c), 15.07.2009; mouth of čestobrodica to grza (e5.21), 02.09.2009. superfamily papilionoidea family papilionidae 9. zerynthia polyxena (denis & schiffermüller, 1775) new data: mouth of čestobrodica to grza (e2.2), 10.05.2009. 10. zerynthia cerisy (godart, 1824) new data: mouth of čestobrodica to grza (e5.21), 10.05.2010; mouth of čestobrodica to grza (e2.2) 13.06.2009, larvae. 11. parnassius mnemosyne (linnaeus, 1758) new data: vrelska padina (e1.22), 14.06.2009. 12. iphiclides podalirius (linnaeus, 1758) published data: grza (380-420 m, utm: ep 46), 15.07.2005. van swaay et al. (2007). new data: mountain home "đorđe živković" (e2.2), 28.07.2008; mountain home "đorđe živković" (e1.22), 29.07.2008; vrelska padina (e1.22), 31.07.2008; javoračke padine (e2.2), 31.07.2008; mountain home "đorđe živković" (e1.22), 01.08.2008; vrelo (g1.691), 01.08.2008; veliki kozji rog (e1.74), 02.08.2008; mouth of čestobrodica to grza (e2.2), 04.08.2008; weekend estate "grza" (e2.2), 20.04.2009; mouth of čestobrodica to grza (e5.21), 10.05.2009; mouth of čestobrodica to grza (e3.4, e5.21), 13.06.2009; weekend estate (e2.2), 13.06.2009; mountain home "đorđe živković" (e2.2), 14.06.2009; mountain home "đorđe živković" (e2.2), 07.07.2009; vrelo (g1.691), 08.07.2009; pištolje (e1.22, e5.22), 09.07.2009; mouth of čestobrodica to grza (e2.2, e5.21), 12.07.2009; javoračke padine (e2.2), 15.07.2009. 13. papilio machaon (linnaeus, 1758) new data: javoračke padine (e2.2), 31.07.2008; mouth of čestobrodica to grza (e2.2), 10.05.2009; mouth of čestobrodica to grza (e2.2), 02.09.2009. family pieridae 14. leptidea sinapis (linnaeus, 1758) published data: grza (380-420 m, utm: ep 46), 15.07.2005. van swaay et al. (2007). new data: javoračke padine (e2.2), 31.07.2008; weekend estate "grza" (e2.2), 20.04.2009; mouth of čestobrodica to grza (e3.4), 10.05.2009, 2$m; mouth of čestobrodica to grza (e5.21), 13.06.2009; biologica nyssana 2 (2) december 2011: 107-117 nahirnić a. supplements of butterfly fauna… 110 mountain home "đorđe živković" (e2.2), 14.06.2009; vrelska padina (e1.22), 14.06.2009; mountain home "đorđe živković" (e1.22, e.22), 07.07.2009; vrelo (g1.691), 08.07.2009; pištolje (e1.22, e5.22), 09.07.2009; mouth of čestobrodica to grza (e5.21), 12.07.2009; weekend estate "grza" (e2.2), 13.07.2009; mouth of čestobrodica to grza (e2.2, e3.4, e5.21), 12.07.2009; vrelska padina (e1.22), 15.07.2009; barski vrh (e1.2), 15.07.2009; javoračke padine (e2.2), 15.07.2009; mouth of čestobrodica to grza (e2.2, e3.4, e5.21), 02.09.2009. 15. antocharis cardamines (linnaeus, 1758) new data: weekend estate "grza" (e2.2), 20.04.2009; mouth of čestobrodica to grza (e3.4), 10.05.2009. 16. aporia crataegi (linnaeus, 1758) new data: mouth of čestobrodica to grza (e2.2, e5.21), 13.06.2009; weekend estate (e2.2), 13.06.2009; mountain home "đorđe živković" (e2.2), 14.06.2009; vrelska padina (e1.22), 14.06.2009. 17. pieris rapae (linnaeus, 1758) published data: grza (380-420 m, utm: ep 46), 15.07.2005. van swaay et al. (2007). new data: mountain home "đorđe živković" (e2.2), 28.07.2008; javoračke padine (e2.2), 31.07.2008; vrelo (g1.691), 01.08.2008; mountain home "đorđe živković" (e2.2), 03.08.2008; weekend estate "grza" (e2.2), 20.04.2009; mouth of čestobrodica to grza (e2.2, e5.21), 10.05.2009; mouth of čestobrodica to grza (e2.2, e5.21), 13.06.2009; weekend estate (e2.2), 13.06.2009; mountain home "đorđe živković" (e2.2), 14.06.2009; vrelska padina (e1.22), 14.06.2009; mountain home "đorđe živković" (e1.22, e2.2), 07.07.2009; vrelo (g1.691), 08.07.2009; pištolje (e1.22, e5.22), 09.07.2009; mouth of čestobrodica to grza (e5.21), 12.07.2009; mouth of čestobrodica to grza (e2.2, e3.4, e5.21), 12.07.2009; weekend estate (e2.2), 13.07.2009; vrelska padina (e1.22), 15.07.2009; barski vrh (e1.2), 15.07.2009; javoračke padine (e2.2), 15.07.2009; javorački vrh (f3.242c), 15.07.2009; mouth of čestobrodica to grza (e2.2, e3.4, e5.21), 02.09.2009. 18. pieris napi (linnaeus, 1758) published data: grza (380-420 m, utm: ep 46), 15.07.2005. van swaay et al. (2007). new data: weekend estate "grza" (e2.2), 20.04.2009; mouth of čestobrodica to grza (e2.2, e3.4, e5.21) 10.05.2009; mouth of čestobrodica to grza (e5.21, g1.a46), 13.06.2009; mouth of čestobrodica to grza (e5.21), 12.07.2009; mouth of čestobrodica to grza (e5.21), 02.09.2009. 19. colias erate (esper, 1805) new data: mountain home "đorđe živković" (e2.2), 28.07.2008; mountain home "đorđe živković" (e1.22), 01.08.2008. 20. colias croceus (fourcroy, 1785) published data: grza (380-420 m, utm: ep 46), 15.07.2005. van swaay et al. (2007). new data: mountain home "đorđe živković" (e2.2), 28.07.2008; mountain home "đorđe živković" (e1.22), 29.07.2008; mouth of čestobrodica to grza (e2.2), 04.08.2008; mountain home "đorđe živković" (e2.2), 08.07.2009; mouth of čestobrodica to grza (e2.2), 13.06.2009; mouth of čestobrodica to grza (e2.2), 12.07.2009; mouth of čestobrodica to grza (e2.2, e5.21), 02.09.2009. 21. colias hyale (linnaeus, 1758) new data: mouth of čestobrodica to grza (e5.21), 12.07.2009; mouth of čestobrodica to grza (e2.2, e5.21), 02.09.2009. 22. gonepteryx rhamni (linnaeus, 1758) published data: grza (380-420 m, utm: ep 46), 15.07.2005. van swaay et al. (2007). new data: javoračke padine (e5.22), 31.07.2008; weekend estate "grza" (e2.2), 20.04.2009, 2$m; mouth of čestobrodica to grza (e5.21, e5.22), 10.05.2009; mouth of čestobrodica to grza (e5.21), 13.06.2009; mountain home "đorđe živković" (e5.22), 14.06.2009; vrelska padina (e1.22), 14.06.2009; mountain home "đorđe živković" (e2.2), 07.07.2009; weekend estate "grza" (e2.2), 13.07.2009; mouth of čestobrodica to grza (e5.21, e5.22), 12.07.2009; javorački vrh (f3.242c), 15.07.2009. family riodinidae 23. hamearis lucina (linnaeus, 1758) new data: mountain home "đorđe živković" (e2.2), 28.07.2008; mouth of čestobrodica to grza (e2.2), 10.05.2009. family lycaenidae 24. lycaena phlaeas (linnaeus, 1761) new data: mouth of čestobrodica to grza (e2.2), 13.06.2009; mountain home "đorđe živković" (e2.2), 14.06.2009; vrelska padina (e1.22), biologica nyssana 2 (2) december 2011: 107-117 nahirnić a. supplements of butterfly fauna… 111 14.06.2009; pištolje (e1.22), 09.07.2009; mouth of čestobrodica to grza (e2.2), 02.09.2009. 25. lycaena dispar (haworth, 1802) new data: mouth of čestobrodica to grza (e2.2), 13.06.2009; vrelska padina (e1.22), 14.06.2009; mouth of čestobrodica to grza (e2.2), 02.09.2009. 26. lycaena virgaureae (linnaeus, 1758) published data: grza (380-420 m, utm: ep 46), 15.07.2005. van swaay et al. (2007). new data: vrelska padina (e1.22), 14.06.2009; weekend estate (e2.2), 13.07.2009. 27. lycaena tityrus (poda, 1761) new data: mouth of čestobrodica to grza (e3.4), 10.05.2009; mouth of čestobrodica to grza (e2.2), 02.09.2009. 28. lycaena alciphron (rottemburg, 1775) published data: grza (380-420 m, utm: ep 46), 15.07.2005. van swaay et al. (2007). new data: vrelska padina (e1.22), 14.06.2009. 29. lycaena thersamon (esper, 1784) new data: vrelska padina (e1.22), 14.06.2009. 30. favonius quercus (linnaeus, 1758) new data: vrelska padina (g1.761), 02.08.2008. 31. callophrys rubi (linnaeus, 1758) new data: vrelska padina (e1.22), 14.06.2009. 32. satyrium ilicis (esper, 1779) new data: mouth of čestobrodica to grza (e5.21, e5.22, g1.a46), 13.06.2009; vrelska padina (g1.761), 14.06.2009; mouth of čestobrodica to grza (g1.a46),12.07.2009. 33. cupido argiades (pallas, 1771) new data: mouth of čestobrodica to grza (e2.2, e3.4, e5.21), 02.09.2009. 34. celastrina argiolus (linnaeus,1758) published data: grza (380-420 m, utm: ep 46), 15.07.2005. van swaay et al. (2007). new data: mouth of čestobrodica to grza (e5.21), 13.06.2009; vrelska padina (e1.22), 14.06.2009; mountain home "đorđe živković" (e1.22, e2.2), 07.07.2009; vrelo (g1.691), 08.07.2009; mouth of čestobrodica to grza (e1.22), 13.07.2009; weekend estate "grza" (e2.2), 13.07.2009. 35. scolitantides orion (pallas, 1771) new data mouth of čestobrodica to grza (e5.21), 10.05.2009; mouth of čestobrodica to grza (e2.2, e5.21, g1.a46), 12.07.2009. 36. iolana iolas (ochsenheimer, 1816) new data: mountain home "đorđe živković" (e1.22), 39.07.2008. 37. phengaris alcon (doherty, 1891) new data: javoračke padine (e2.2), 15.07.2009. 38. plebejus argus (linnaeus, 1758) new data: mountain home "đorđe živković" (e1.22), 01.08.2008; mountain home "đorđe živković" (e2.2), 03.08.2008. 39. plebejus idas (linnaeus, 1761) new data: vrelska padina (e1.22), 14.06.2009. 40. aricia agestis (denis & schiffermüller, 1775) new data: mouth of čestobrodica to grza (e2.2, e5.21), 02.09.2009. 41. aricia artaxerxes (fabricius, 1793) new data: pištolje (e1.22), 09.07.2009. 42. polyommatus semiargus (rottemburg, 1775) new data: mouth of čestobrodica to grza (e2.2), 13.06.2009; mountain home "đorđe živković" (e2.2), 14.06.2009; mountain home "đorđe živković" (e1.22), 07.07.2009. 43. polyommatus amandus (schneider, 1792) new data: mouth of čestobrodica to grza (e2.2), 13.06.2009; mountain home "đorđe živković" (e2.2), 14.06.2009. 44. polyommatus thersites (cantener, 1835) new data: mouth of čestobrodica to grza (e2.2, e5.21), 13.06.2009; vrelska padina (e1.22), 14.06.2009. 45. polyommatus icarus (rottemburg, 1775) new data: mouth of čestobrodica to grza (e3.4), 10.05.2009; mouth of čestobrodica to grza (e2.2, e3.4, e5.21), 13.06.2009; weekend estate "grza" (e2.2), 13.06.2009; mountain home "đorđe živković" (e2.2), 14.06.2009; mountain home "đorđe živković" (e1.22), 07.07.2009; mouth of čestobrodica to grza (e2.2, e5.21), 12.07.2009; mouth of čestobrodica to grza (e2.2, e5.21), 02.09.2009. biologica nyssana 2 (2) december 2011: 107-117 nahirnić a. supplements of butterfly fauna… 112 46. polyommatus daphnis (denis & schiffermüller, 1775) new data: mountain home "đorđe živković" (e2.2), 28.07.2008; vrelska padina (e1.22), 31.07.2008; veliki kozji rog (e1.74), 02.08.2008; pištolje (e5.22), 09.07.2009; mouth of čestobrodica to grza (e5.21, g1.a46), 12.07.2009; weekend estate (e2.2), 13.07.2009; vrelska padina (e1.22), 15.07.2009; javoračke padine (e2.2), 15.07.2009. family nymphalidae 47. argynnis paphia (linnaeus, 1758) published data: grza (380-420 m, utm: ep 46), 15.07.2005. van swaay et al. (2007). new data: mountain home "đorđe živković" (e2.2), 28.07.2008; mountain home "đorđe živković" (e1.22), 29.07.2008; vrelska padina (e1.22), 31.07.2008; javoračke padine (e2.2), 31.07.2008; mountain home "đorđe živković" (e1.22), 01.08.2008; vrelo (g1.691), 01.08.2008; mountain home "đorđe živković" (e2.2), 03.08.2008; mouth of čestobrodica to grza (e2.2), 04.08.2008; mouth of čestobrodica to grza (e5.21), 13.06.2009; weekend estate "grza" (e2.2), 13.06.2009; mountain home "đorđe živković" (e2.2), 14.06.2009; mountain home "đorđe živković" (e2.2, e5.22), 07.07.2009; vrelo (g1.691), 08.07.2009; pištolje (e1.22, e5.22), 09.07.2009; mouth of čestobrodica to grza (e2.2, e5.21), 12.07.2009; barski vrh (e1.2), 15.07.2009; vrelska padina (e1.22), 15.07.2009; javoračke padine (e2.2), 15.07.2009; javorački vrh (f3.242c), 15.07.2009; mouth of čestobrodica to grza (e2.2, e5.21), 02.09.2009. 48. argynnis aglaja (linnaeus, 1758) new data: mountain home "đorđe živković" (e2.2), 07.07.2009; javoračke padine (e2.2), 15.07.2009. 49. argynnis adippe (denis & schiffermüller, 1775) published data: grza (380-420 m, utm: ep 46), 15.07.2005. van swaay et al. (2007). 50. argynnis niobe (linnaeus, 1758) new data: mountain home "đorđe živković" (e2.2), 14.06.2009. 51. issoria lathonia (linnaeus, 1758) new data: mountain home "đorđe živković" (e2.2), 28.07.2008; javoračke padine (e2.2), 31.07.2008; mountain home "đorđe živković" (e2.2), 03.08.2008; mouth of čestobrodica to grza (e2.2), 04.08.2008; mouth of čestobrodica to grza (e5.21), 10.05.2009; mouth of čestobrodica to grza (e5.21), 13.06.2009; weekend estate "grza" (e2.2), 13.06.2009; mountain home "đorđe živković" (e2.2), 14.06.2009; vrelska padina (e1.22), 14.06.2009; mountain home "đorđe živković" (e2.2), 07.07.2009; mouth of čestobrodica to grza (e2.2, e5.21), 12.07.2009; weekend estate "grza" (e2.2), 13.07.2009; barski vrh (e1.2), 15.07.2009; javoračke padine (e2.2), 15.07.2009; javorački vrh (f3.242c), 15.07.2009; mouth of čestobrodica to grza (e2.2, e5.21), 02.09.2009. 52. brenthis daphne (denis & schiffermüller, 1775) published data: grza (380-420 m, utm: ep 46), 15.07.2005. van swaay et al. (2007). new data: mouth of čestobrodica to grza (e2.2), 13.06.2009; weekend estate "grza" (e2.2), 13.06.2009; mountain home "đorđe živković" (e2.2), 14.06.2009; vrelska padina (e1.22), 14.06.2009; mountain home "đorđe živković" (e2.2), 07.07.2009; vrelo (g1.691), 08.07.2009; pištolje (e1.22), 09.07.2009; mouth of čestobrodica to grza (e2.2, e3.4, e5.21), 12.07.2009; weekend estate "grza" (e2.2), 13.07.2009; barski vrh (e1.2), 15.07.2009; vrelska padina (e1.22), 15.07.2009; javoračke padine (e2.2), 15.07.2009; javorački vrh (f3.242c), 15.07.2009. 53. brenthis hecate (denis & schiffermüller, 1775) published data: grza (380-420 m, utm: ep 46), 15.07.2005. van swaay et al. (2007). new data: mouth of čestobrodica to grza (e2.2), 13.06.2009; weekend estate "grza" (e2.2), 13.06.2009; mountain home "đorđe živković" (e2.2), 07.07.2009. 54. boloria dia (linnaeus, 1767) new data: mouth of čestobrodica to grza (e2.2), 02.09.2009. 55. vanessa atalanta (linnaeus, 1758) published data: grza (380-420 m, utm: ep 46), 15.07.2005. van swaay et al. (2007). new data: mountain home "đorđe živković" (e2.2), 28.07.2008; mountain home "đorđe živković" (e1.22), 29.07.2008; javoračke padine (e2.2), 31.07.2008; mouth of čestobrodica to grza (e2.2), 04.08.2008; mouth of čestobrodica to grza (e5.21), 13.06.2009; weekend estate "grza" (e2.2), 13.06.2009; mountain home "đorđe živković" (e2.2, e5.22), 07.07.2009; vrelo (g1.691), 08.07.2009; pištolje (e1.22, e5.22), 09.07.2009; mouth of čestobrodica to grza (e2.2, e5.21), 12.07.2009; weekend estate "grza" (e2.2), biologica nyssana 2 (2) december 2011: 107-117 nahirnić a. supplements of butterfly fauna… 113 13.07.2009; javoračke padine (e2.2), 15.07.2009; mouth of čestobrodica to grza (e2.2), 02.09.2009. 56. vanessa cardui (linnaeus, 1758) new data: mouth of čestobrodica to grza (e5.21), 13.06.2009; weekend estate "grza" (e2.2), 13.06.2009; mountain home "đorđe živković" (e2.2), 14.06.2009; pištolje (e1.22, e5.22), 09.07.2009; weekend estate "grza" (e2.2), 13.07.2009; mouth of čestobrodica to grza (e2.2), 12.07.2009; mouth of čestobrodica to grza (e2.2, e5.21), 02.09.2009. 57. aglais io (linnaeus, 1758) published data: grza (380-420 m, utm: ep 46), 15.07.2005. van swaay et al. (2007). new data: javoračke padine (e2.2), 31.07.2008; mountain home "đorđe živković" (e1.22, e2.2), 07.07.2009; weekend estate "grza" (e2.2), 13.07.2009; mouth of čestobrodica to grza (e2.2), 12.07.2009; javoračke padine (e2.2), 15.07.2009; mouth of čestobrodica to grza (e2.2), 02.09.2009. 58. aglais urticae (linnaeus, 1758) published data: grza (380-420 m, utm: ep 46), 15.07.2005. van swaay et al. (2007). new data: mouth of čestobrodica to grza (e2.2), 13.06.2009; mountain home "đorđe živković" (e2.2), 14.06.2009. 59. polygonia c-album (linnaeus, 1758) published data: grza (380-420 m, utm: ep 46), 15.07.2005. van swaay et al. (2007). new data: javoračke padine (e5.22), 31.07.2008; weekend estate "grza" (e2.2), 13.06.2009; mountain home "đorđe živković" (e5.22), 14.06.2009; vrelo (g1.691), 08.07.2009; mountain home "đorđe živković" (e5.22), 07.07.2009; mouth of čestobrodica to grza (e5.21), 12.07.2009; weekend estate "grza" (e2.2), 13.07.2009; javoračke padine (e5.22), 15.07.2009; mouth of čestobrodica to grza (e5.21), 02.09.2009. 60. araschnia levana (linnaeus, 1758) published data: grza (380-420 m, utm: ep 46), 15.07.2005. van swaay et al. (2007). new data: weekend estate "grza" (e2.2), 20.04.2009; mountain home "đorđe živković" (e5.22, g1.691), 07.07.2009; mouth of čestobrodica to grza (e2.2), 02.09.2009; mouth of čestobrodica to grza (e2.2), 02.09.2009. 61. nymphalis antiopa (linnaeus, 1758) new data: mouth of čestobrodica to grza (e5.22), 10.05.2009. 62. nymphalis polychloros (linnaeus, 1758) new data: mountain home "đorđe živković" (e5.22), 07.07.2009. 63. melitaea cinxia (linnaeus, 1758) new data: vrelska padina (e1.22), 14.06.2009. 64. melitaea arduinna (esper, 1783) published data: ep 56, grza river, 380 – 420 m, 14.06.2009. (termophile meadows in vicinity of oak forest) jakšić (2011). 65. melitaea trivia (denis & schiffermüller, 1775) new data: mouth of čestobrodica to grza (e5.21), 10.05.200; pištolje (e1.22), 09.07.2009. 66. melitaea didyma (esper, 1778) new data: mouth of čestobrodica to grza (e2.2, e3.4, e5.21), 13.06.2009; mountain home "đorđe živković" (e2.2), 14.06.2009; vrelska padina (e1.22), 14.06.2009; mountain home "đorđe živković" (e1.22, e2.2), 07.07.2009; mouth of čestobrodica to grza (e2.2), 12.07.2009; vrelska padina (e1.22), 15.07.2009; mouth of čestobrodica to grza (e2.2, e5.21), 02.09.2009. 67. melitaea aurelia (nickerl, 1850) published data: grza (380-420 m, utm: ep 46), 15.07.2005. van swaay et al. (2007). 68. melitaea athalia (rottemburg, 1775) published data: grza (380-420 m, utm: ep 46), 15.07.2005. van swaay et al. (2007). new data: mouth of čestobrodica to grza (e2.2, e3.4), 13.06.2009; weekend estate "grza" (e2.2), 13.06.2009; vrelska padina (e1.22), 14.06.2009. 69. limenitis camilla (linnaeus, 1764) new data: mountain home "đorđe živković" (e5.22), 28.07.2008; javoračke padine (e5.22), 15.07.2009. 70. neptis sappho (pallas, 1771) new data: mountain home "đorđe živković" (e5.22), 28.07.2008; vrelo (g1.691), 01.08.2008; mountain home "đorđe živković" (g1.761), 03.08.2008; mouth of čestobrodica to grza (e3.4), 10.05.2009; weekend estate "grza" (g1.691), 13.07.2009; mouth of čestobrodica to grza (e5.21, e5.22, g1.a46), 02.09.2009. biologica nyssana 2 (2) december 2011: 107-117 nahirnić a. supplements of butterfly fauna… 114 71. apatura ilia (denis & schiffermüller, 1775) published data: grza (380-420 m, utm: ep 46), 15.07.2005. van swaay et al. (2007). new data: mouth of čestobrodica to grza (e5.21), 13.06.2009; mouth of čestobrodica to grza (g1.a46), 12.07.2009. 72. apatura iris (linnaeus, 1758) new data: mountain home "đorđe živković" (g1.761), 07.07.2009. 73. kirinia roxelana (cramer, 1777) new data: barski vrh (g1.696), 15.07.2009, 1$m. 74. pararge aegeria (linnaeus, 1758) new data: vrelski krš (f3.242c), 30.07.2008; vrelo (g1.691), 01.08.2008; mouth of čestobrodica to grza (e3.4, e5.21, g1.a46), 10.05.2009; mountain home "đorđe živković" (g1.691), 14.06.2009; vrelska padina (g1.761), 14.06.2009; vrelo (g1.691), 08.07.2009; mountain home "đorđe živković" (g1.691), 07.07.2009; mouth of čestobrodica to grza (e3.4), 12.07.2009; weekend estate "grza" (g1.691), 13.07.2009; barski vrh (g1.696), 15.07.2009; javorački vrh (f3.242c), 15.07.2009. 75. lasiommata megera (linnaeus, 1767) new data: mouth of čestobrodica to grza (e5.21), 12.07.2009; mouth of čestobrodica to grza (e5.21), 02.09.2009. 76. coenonympha arcania (linnaeus, 1761) published data: grza (380-420 m, utm: ep 46), 15.07.2005. van swaay et al. (2007). new data: mouth of čestobrodica to grza (e2.2, e3.4, e5.21), 13.06.2009; mountain home "đorđe živković" (e2.2), 14.06.2009; vrelska padina (e1.22), 14.06.2009; pištolje (e1.22, e5.22), 09.07.2009; mouth of čestobrodica to grza (e3.4, e5.21, g1.a46), 12.07.2009; weekend estate "grza" (e2.2), 13.07.2009; barski vrh (e1.2), 15.07.2009; vrelska padina (e1.22), 15.07.2009; javoračke padine (e2.2), 15.07.2009. 77. coenonympha pamphilus (linnaeus, 1758) new data: mountain home "đorđe živković" (e2.2), 28.07.2008; vrelska padina (e1.22), 31.07.2008; barski vrh (e1.2), 31.07.2008; javoračke padine (e2.2), 31.07.2008; vrelska padina (e1.22), 02.08.2008; veliki kozji rog (e1.74), 02.08.2008; mountain home "đorđe živković" (e2.2), 03.08.2008; mouth of čestobrodica to grza (e2.2), 04.08.2008; mouth of čestobrodica to grza (e2.2), 10.05.2009; mouth of čestobrodica to grza (e2.2, e5.21), 13.06.2009; weekend estate "grza" (e2.2), 13.06.2009; mountain home "đorđe živković" (e2.2), 14.06.2009; vrelska padina (e1.22), 14.06.2009; mountain home "đorđe živković" (e2.2), 07.07.2009; pištolje (e1.22), 09.07.2009; weekend estate "grza" (e2.2), 13.07.2009; vrelska padina (e1.22), 15.07.2009; javoračke padine (e2.2), 15.07.2009; mouth of čestobrodica to grza (e2.2, e5.21), 02.09.2009. 78. aphantopus hyperantus (linnaeus, 1758) published data: grza (380-420 m, utm: ep 46), 15.07.2005. van swaay et al. (2007). new data: mountain home "đorđe živković" (e2.2), 28.07.2008; javoračke padine (e2.2), 31.07.2008; vrelo (g1.691), 08.07.2009; mountain home "đorđe živković" (e2.2), 07.07.2009; pištolje (e1.22, e5.22), 09.07.2009; barski vrh (e1.2), 15.07.2009; vrelska padina (e1.22), 15.07.2009; javoračke padine (e2.2), 15.07.2009. 79. maniola jurtina (linnaeus, 1758) published data: grza (380-420 m, utm: ep 46), 15.07.2005. van swaay et al. (2007). new data: mountain home "đorđe živković" (e2.2), 28.07.2008; vrelski krš (f3.242c), 30.07.2008; barski vrh (e1.2), 31.07.2008; javoračke padine (e2.2), 31.07.2008; vrelska padina (e1.22), 02.08.2008; veliki kozji rog (e1.74), 02.08.2008; mountain home "đorđe živković" (e2.2, g1.691), 03.08.2008; mountain home "đorđe živković" (e1.22), 04.08.2008; mouth of čestobrodica to grza (e2.2), 04.08.2008; mouth of čestobrodica to grza (e2.2, e3.4, e5.21, g1.a46), 13.06.2009; weekend estate "grza" (e2.2), 13.06.2009; mountain home "đorđe živković" (e2.2), 14.06.2009; vrelska padina (e1.22), 14.06.2009; mountain home "đorđe živković" (e1.22, e2.2, e5.22), 07.07.2009; vrelo (g1.691), 08.07.2009; pištolje (e1.22, e5.22), 09.07.2009; mouth of čestobrodica to grza (e2.2, e3.4, e5.21), 12.07.2009; weekend estate "grza" (e2.2), 13.07.2009; barski vrh (e1.2), 15.07.2009; vrelska padina (e1.22), 15.07.2009; javoračke padine (e2.2), 15.07.2009; javorački vrh (f3.242c), 15.07.2009; mouth of čestobrodica to grza (e2.2, e5.21), 02.09.2009. 80. erebia medusa (denis & schiffermüller, 1775) new data: vrelska padina (e1.22), 14.06.2009. biologica nyssana 2 (2) december 2011: 107-117 nahirnić a. supplements of butterfly fauna… 115 81. melanargia galathea (linnaeus, 1758) published data: grza (380-420 m, utm: ep 46), 15.07.2005. van swaay et al. (2007). new data: vrelska padina (e1.22), 31.07.2008; barski vrh (e1.2), 31.07.2008; javoračke padine (e2.2), 31.07.2008; vrelska padina (e1.22), 02.08.2008; weekend estate "grza" (e2.2), 13.06.2009; mountain home "đorđe živković" (e2.2), 14.06.2009; mountain home "đorđe živković" (e2.2, e5.22), 07.07.2009; vrelo (g1.691), 08.07.2009; pištolje (e1.22, e5.22), 09.07.2009; mouth of čestobrodica to grza (e2.2, e3.4, e5.21), 12.07.2009; weekend estate "grza" (e2.2), 13.07.2009; barski vrh (e1.2), 15.07.2009; vrelska padina (e1.22), 15.07.2009; javoračke padine (e2.2), 15.07.2009; javorački vrh (f3.242c), 15.07.2009. 82. minois dryas (scopoli, 1763) new data: mountain home "đorđe živković" (e2.2), 28.07.2008; vrelski krš (f3.242c), 30.07.2008; vrelska padina (e1.22), 31.07.2008; barski vrh (e1.2), 31.07.2008; vrelska padina (e1.22), 31.07.2008; veliki kozji rog (e1.74), 02.08.2008; mountain home "đorđe živković" (e2.2, g1.691, g1.761), 03.08.2008; vrelska padina (e1.22, g1.761), 15.07.2009; mouth of čestobrodica to grza (e2.2), 02.09.2009. 83. arethusana arethusa (denis & schiffermüller, 1775) new data: mountain home "đorđe živković" (e5.22), 03.08.2008. 84. brintesia circe (fabricius, 1775) published data: grza (380-420 m, utm: ep 46), 15.07.2005. van swaay et al. (2007). new data: javoračke padine (e2.2), 31.07.2008; veliki kozji rog (e1.74), 02.08.2008; mountain home "đorđe živković" (e1.22), 04.08.2008; mountain home "đorđe živković" (e2.2), 07.07.2009; pištolje (e1.22, e5.22), 09.07.2009; mouth of čestobrodica to grza (e2.2, e5.21), 12.07.2009; weekend estate "grza" (e2.2), 13.07.2009; barski vrh (e1.2), 15.07.2009; vrelska padina (e1.22), 15.07.2009; mouth of čestobrodica to grza (e2.2, e5.21), 02.09.2009. table 1. comparative review of specific elements of butterfly fauna of three valleys specific elements of butterfly fauna lazarev canyon grza river gorge ibar river gorge zerinthia cerisyi (godart, 1824) + + melitaea arduinna (esper, 1783) + + pyrgus sidae (esper, 1784) + iolana iolas (ochsenheimer, 1816) + maculinea alcon (d. & s., 1775) + aricia artaxerxes (fabricius, 1793) + melitaea aurelia (nickerl, 1850) + colias erate (esper, 1805) + pseudophilotes vicrama (moore, 1865) + euphidryas aurinia (rottemburg, 1775) + melanargia larissa (geyer, 1828) + disscussion and conclusion the total number of recorded species is 84. quantitative composition of established butterfly species is determined as moderate abundance, following the scale which determines the selection of prime butterfly areas in serbia (jakšić (ed.), 2008). there has been recorded 54 species of butterflies new to grza river gorge territory. compared with van swaay et al., (2007) data, all species were rerecorded, except argynnis adippe and melitaea аurelia, since they are rare in serbia. the data for melitaea arduinna is done on the basis of jakšić (2011). all species are inside the boundaries of their known area of distribution. we consider that level of fauna knowledge is satisfactory. zoogeographical division of established species is done according to jakšić (1998). in zoogeographical point of view middle-european species are dominant (30 species, 36%) followed by pontic-mediterranean group with 16 species (19%). sub-mediterranean species (12 species, 14%) are also significant members of grza river gorge fauna. biologica nyssana 2 (2) december 2011: 107-117 nahirnić a. supplements of butterfly fauna… 116 specific elements of butterfly fauna studied in this paper are compared to specific elements of butterfly fauna of lazarev canyon and the ibar river gorge on the basis of previous study (jakšić (ed.), 2008). lazarev canyon and the grza river gorge belong to moesian floristic province. ibar river gorge is under influence of illyric province. this comparision is presented in table 1. after analysing recorded species we can conclude that butterfly fauna of ibar gorge is clearly different because of the presence of species p. vicrama, e. aurinia and m. larissa and also because of the absence of z. cerisyi and m. arduinna. these elements make the main difference between illyric and moesian province. on the other side, species registered in the grza river gorge, but not in lazarev canyon: p. sidae, c. erate, i. iolas, m. alcon, a. artaxerxes and m. aurelia are species from open sunny worm habitats which are not present in covered plot of lazarev canyon. m. arduinna is the best represent of moesian province. habitat analysis is made consisting 81 species recorded by author, excluding a. adippe m. arduinna and m. aurelia. аnalysis showed that 96,3% of 81 species are recorded at e grasslands and lands dominated by forbs, mosses or lichens, 12,3% at f heathland, scrub and tundra and 30,9% at g woodland, forest and other wooded land. high percentage of butterfly species inhabit forests occurs due to dominance of forest habitats in the grza river gorge territory especially g1.69 moesian fagus forests (g1.691 and/or g1.696) where 19,7% species were recorded. e2.2 low and medium hay meadows are the most suitable habitat types for butterflies having in mind that 71,6% of species were recorded here. in e1 dry grasslands (e1.2, e1.22 and/or e1.74) were found 56,8% of species, while also high percentage 54,3% were registered in e5 woodland fringes and clearings and tall forb stands (e5.21 and/or e5.22). in e3.4 moist or wet eutrophic and mesotrophic grassland 19,7% species were found, although there is only one meadow of that type in investigated territory. g1.761 a2.111 moesian hungarian oak [quercus frainetto] and turkey oak [quercus cerris] woods and g1.a46 southeastern european ravine forests inhabits 7,4% and 11,1%, respectively. natural habitats in grza river gorge are still in good condition, but the intensive touristic development, as well as moderate traffic influence is potential risk for habitat destruction. what makes the special and much stronger reason for providing habitat protection in the grza river gorge is occurrence of 9 target species listed in prime butterfly areas in serbia (jakšić (ed.), 2008) (no. from list of species: 7, 9, 11, 25, 35, 37, 64, 67, 80) and 11 strictly protected species by low of republic serbia (anonymous, 2010) (no. from list of species: 3, 9, 11, 13, 25, 36, 37, 61, 67, 71, 72). acknowledgements. i would like to give special thanks to predrag jakšić (faculty of sciences and mathematics, university of niš) for helping me in the field examination and zoran nikolić (faculty of biology, university of belgrade) for helpful comments on an earlier version of the manuscript. the part of this study was financially supported by bid „josif pančić“ faculty of biology, university of belgrade and eko fond paraćin. i want to thank olivera košanin, teacher of english, for editing the english text. references anonymous, 2010. pravilnik o proglašenju i zaštiti strogo zaštićenih biljnih vrsta biljaka, životinja i gljiva. prilog 2. strogo zaštićene vrste. službeni glasnik rs br 5/10. davies, c.e. & moss, d. 2002: eunis habitat classification european habitats classification system, european environment agency & european topic centre on nature protection and biodiversity. hill, m.o., moss, d. & davies, c.e., 2004. revision of habitat descriptions originating from devillers et al (2001). european topic centre on nature protection and biodiversity, paris. jakšić, p., 1998. altitudinal and biogeographical division of the butterflies of balkan peninsula (lepidoptera: hesperioidea & papilionoidea). the university thought, 5(2): 77-88. jakšić, p. (ed.), 2008. prime butterfly areas in serbia. habiprot, beograd, 223 pp. jakšić, p., 2011. melitaea arduinna (lepidoptera: nymphalidae): a new species for serbia. phegea 39(1): 8-11. karsholt, o. & razowsky, j., 1996. the lepidoptera of europe, a distribution checklist, apollo books, stenstrup, 380 pp. lakušić, d., 2005a. ključ za identifikaciju staništa srbije. in: lakušić, d. (ed.), staništa srbije, rezultati projekta “harmonizacija nacionalne nomenklature u klasifikaciji staništa sa standardima međunarodne zajednice”, institut za botaniku i botanička bašta “jevremovac”, biološki fakultet, univerzitet u beogradu, ministarstvo za nauku i zaštitu životne sredine republike srbije, http://habitat.bio.bg.ac.rs/ lakušić, d., 2005b. veze između međunarodnih klasifikacija staništa i klasifikacija staništa srbije. in: lakušić, d. (ed.), staništa srbije, rezultati projekta “harmonizacija nacionalne nomenklature u klasifikaciji staništa sa standardima međunarodne zajednice”, institut za biologica nyssana 2 (2) december 2011: 107-117 nahirnić a. supplements of butterfly fauna… 117 botaniku i botanička bašta “jevremovac”, biološki fakultet, univerzitet u beogradu, ministarstvo za nauku i zaštitu životne sredine republike srbije, http://habitat.bio.bg.ac.rs/ lakušić, d., blaženčić, j., ranđelović, v., butorac, b., vukojičić, s., zlatković b., jovanović, s., šinđar-sekulić, j., žukovec, d., čalić, i., pavićević, d. 2005. staništa srbije – priručnik sa opisima i osnovnim podacima. in: lakušić, d. (ed.), staništa srbije, rezultati projekta “harmonizacija nacionalne nomenklature u klasifikaciji staništa sa standardima međunarodne zajednice”, institut za botaniku i botanička bašta “jevremovac”, biološki fakultet, univerzitet u beogradu, ministarstvo za nauku i zaštitu životne sredine republike srbije, 684 pp. http://habitat.bio.bg.ac.rs/ lakušić, d., medarević, m., 2010. tipovi staništa zastupjeni na teritoriji republike srbije. annex 1 – the rulebook on habitat types, the criteria for the selection of habitat types, on sensitive, endangered, rare and priority for protection habitat types and on the protection measures for their conservation. official gazette of rs no. 35/2010. mišić, v., 1981. the forest vegetation of gorges and canyons in eastern serbia. institut za biološka istrazivanja “siniša stanković”, beograd, 327 pp. [in serbian]. swaay, c., van, cuttelod, a., collins, s., maes, d., lópez munguira, m., šašić, m., settele, j., verovnik, r., verstrael, t., waren, m., wiemers, m. and wynhof, i. 2010. european red list of butterfies luxembourg: publications office of the european union, 47 pp. swaay, c., van, jakšić, p. & đurić, m., 2007. the notes on some summer butterflies (lepidoptera: hesperioidea & papilionoidea) of eastern serbia. acta entomologica serbica, 12(1): 1-10. beograd. zajić, v., lakušić, d., 2004. contribution to the flora of the grza river gorge (e. serbia). proceedings of the 2nd congress of ecologists of macedonia with international participation, 2529.10.2003, ohrid. special issues of macedonian ecological society 6, skopje. microsoft word bn-oa-0201-06 jaksic biologica nyssana 2 (1) september 2011: 45-50 jakšić p. butterfly species (lepidoptera: hesperioidea and papilionoidea)... 45 original article ! butterfly species (lepidoptera: hesperioidea and papilionoidea) new to the serbian fauna predrag jakšić* university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia * e-mail: jaksic@pmf.ni.ac.rs abstract: jakšić, p.: butterfly species (lepidoptera: hesperioidea and papilionoidea) new to the serbian fauna. biologica nyssana, 2 (1), september 2011: 45-50. a species pyrgus trebevicensis (warren, 1926), melitaea telona (fruhstorfer, 1908) and coenonympha orientalis (rebel, 1913) are here recorded in serbia for the first time. the key morpho-anatomical parameters for identification were done. original pictures of c. orientalis in situ and photos of genital apparatuses are given. original map of distribution examined species in serbia was made. key words: butterflies, serbia, faunistics introduction ! the first fauna list of serbian butterfly species was provided by g r a d o j e v i ć (1931), where 150 species are mentioned. for this crucial work the knowledge about the serbian butterfly fauna has been significantly increased due to activity of national and foreign lepidopterists. until now, the existence of 193 good species in serbia has been verified (j a k š i ć & đ u r i ć , 2008). on the basis of morphometric study on the male genitalia, statistic analysis of populations and the dna sequences analysis, a new contemporary approach gives us the possibility to solve old taxonomic problems, mostly among the sibling species. the „sibling species“ concept denotes morphologically similar or identical natural populations that have been reproductively isolated. soon after the formation of this concept, h e y d e m a n n (1943) published a list of 40 sibling species. recently, descimon and mallet (2009) have published a long paper about sibling species among butterflies. sibling species are easy to distinct by the differences in their habits, early stages and ecophysiology. but, their fine variations in morphology and anatomy, howewer, are also essential for their differentiation. as a rule, these differences are most dificult to discover. all the three newly established species shown here belong to a grup of sibling species. materials and methods the adults material has been collected on the field using entomological net. after preparing, we determined the specimens by the wing-patterns and in all cases the identification has been also carried out by an examination of the male genitalia. the preparations were carried out following the well known standard procedure: maceration by boiling in potash, dissecting and cleaning, clearing in xylolum and mounting in canada balsam. the photos of genital parameters were taken using the "leica dm 1000" microscope with the "camera leica dec 290"; photos of in situ specimen were taken using “olympus” sp-510uz (7,1 megapixel and 10x optical zoom). all the material (specimens and 2 (1) • september 2011: 45-50 biologica nyssana 2 (1) september 2011: 45-50 jakšić p. butterfly species (lepidoptera: hesperioidea and papilionoidea)... 46 genitalia slides) are deposited in the author's collection. figure 1. male genitalia (valva) of: p. armoricanus, serbia, šar-planina mt., brezovica, 1400 m, octob., 3rd, 1975., jakšić p. leg., prep. no. srb-5148 (a); p. alveus, serbia, kopaonik mt., žljeb, 1770 m, july, 21st, 2005., jakšić p. leg., prep. no. srb-2278 (b); p. trebevicensis, serbia, tara planina mt., kaluđerske bare, 1020 m, july, 8-9th, 1985., jakšić p. leg., prep. no. srb-638 (c). results pyrgus trebevicensis (warren, 1926) in the new list, the butterfly species of europe (s w a a y et al., 2010) this species does not exist as a distinct species separated from pyrgus alveus. as a good species within the national faunas it has been listed by the following authors: austria (r e n n i e r , 1991; g r o s & e m b a c h e r , 1998; h a b e l e r , 1999); czech republic (b e n e š et al. 2001); deutschalnd (r e n n i e r , 1991; wa g n e r , 2002); bosnia and herzegovina (s c h a w e r d a , 1918; r e n n i e r , 1991; l e l o , 2007). s h a w e r d a (1918) described this species as p. reverdini: „etwas grösser, oberseits heller braun, nicht so dunkel wie die nennform, normal stark weiss gefleckt. unterseits sind die weissen flecke grösser, das braun lichter, breitere flügelspannung meist 29 mm. 5 ♂, 2 ♀, trebevic, vucijabara. von mir im juli 1907 und 1912 erbeutet.“ wa r r e n (1926) proposed to replace the name trebevicensis into reverdini schawerda, 1918, because reverdini is a primary junior homonym of p. reverdini oberhur. he citated s c h a w e r d a (1918) that reverdini differs from the type in being slightly larger, with the ground-colour of the upperside lighter brown and not so black as in typical alveus, and with the white markings strongly pronounced. the underside has all the white markings broader and the groundcolour of the hind-wings paler yellow. all the wings are said to be a litle broader than in the type.“ new data for serbia: material examined: serbia, tara planina mt., kaluđerske bare, 1020 m (x = 430 54' 33"; y = 190 32' 55") 1m, 8-9. vii 1985., jakšić p. leg., prep. no. srb-638; serbia, užice, 670 m (x = 430 50' 44"; y = 190 49' 25") 1m, 4. vi 1982., toševski i. leg., prep. no. srb-100; serbia, divčibare, 950 m (x = 440 06' 09"; y = 190 59' 20") 1m, 27. vi 2006., đurić m., leg., prep no. srb-2323; serbia, fruška gora mt., 130 m (x = 450 12' 47"; y = 190 50' 02") 1 m, vii 1929., rogulja m. leg., prep no. srb-96; serbia, golija mt., 1800 m (x = 430 20' 20"; y = 200 16' 38") 1 m, 22. vii 1937., vagner o. leg., prep. no. srb-60; serbia, golija mt., 1800 m (x = 430 20' 20"; y = 200 16' 38") 1 m, 22. vii 1937., vagner o. leg., prep. no. srb-87; serbia, stol mt., 900 m (x = 440 10' 24"; 220 07' 20") 1 m, 28. vii 1974., zečević m. leg., prep. no. srb-82 (fig. 4: ●) melitaea telona (fruhstorfer, 1908) in the new list the butterfly species of europe (s w a a y et al., 2010) this species exists as a distinct species separated from m. phoebe. as a good species within the national faunas it has been listed by the following authors: hungary (t ó t h & va r g a , 2010); macedonia (ve r o v n i k et al., 2010). tóth and va r g a (2010) pointed their basic biologica nyssana 2 (1) september 2011: 45-50 jakšić p. butterfly species (lepidoptera: hesperioidea and papilionoidea)... 47 characteristics in male genitalia: „in males the depth of the central notch of the saccus proved to be the most important difference. except two characters all show significant differences, but if these differences are considered separately we can found major overlaping. in general we observed that in m. telona we can see a more notched saccus, and more symetric shape of processus posterior (in same side) because the inner process in the processus posterior is shorter than m. phoebe. these two characters are essentially the same as the distinctive characters in the original description of m. „phoebe“ kovacsi.“ according to p e c s e n y e et al. (2007) – cited by t ó t h & va r g a (2010) enzyme electrophoretic study of hungarian populations has also shown obvious difference between m. phoebe and m. telona without any mark of hybridisation. new data for serbia: material examined: serbia, stara planina mt., janja, 520 m (x = 430 24' 47"; 220 31' 04") 1 m, 8. vii 2010., jakšić p. leg., prep. no. srb-2484; serbia, stara planina mt., janja, 520 m (x = 430 24' 47"; 220 31' 04") 1 m, 8. vii 2010., jakšić p. leg., prep. no. srb-2485 (fig. 4: ▲) coenonympha orientalis (rebel, 1913) in the new list, the butterfly species of europe (s w a a y et al., 2010) this species exists as a distinct species separated from c. gardetta. as a good species within the national faunas it has been listed by c o u t s i s & g h a v a l á s (2005) for the territory of greece. b o i l l a t (1990) gives the first detailed presentation of arguments suppotring c. orientalis as a „bona species“. according to c o u t s i s & g h a v a l a s (2005): „... c. orientalis is being raised to specific level apparently on the basis of what is believed to be unique external characters, that do not relate to any of the other taxa under consideration, of sympatry with c. leander (though no actual syntopism is involved here), and of the supposed absence of intermediates between the two.“ in our specimens of c. gardetta the underside of fore-wing apex is grey and there is no ocelli; in c. orientalis the underside of fore-wing apex is brown-orange, with one or two ocelli. analysing the available material it can be concluded that c. gardetta is not present whereas c. orientalis is present in this part of the balkan peninsula. figure 2. melitaea telona (fruhstorfer, 1908): saccus and valva, srbija, stara planina mt., janja, 520 m, july, 8th, 2010., jakšić p. leg., prep. no. srb2484; new data for serbia: material examined: -serbia, tutin, smolućka reka, crkvine, 950 m (x = 430 02' 44"; y = 200 21' 40") 1 m, 23. vi 2006., jakšić p. leg; serbia, kopaonik mt., žljeb, 1750 – 1770 m (x = 430 18' 46"; y = 200 50' 00") 2 mm, 21. vii 2005., jakšić p. leg.; serbia, kopaonik mt., bele stene, 1760 m (x = 430 18' 30"; y = 200 49' 53") 1m, 23. vii 2010., jakšić p. leg. (fig. 4: ■). biologica nyssana 2 (1) september 2011: 45-50 jakšić p. butterfly species (lepidoptera: hesperioidea and papilionoidea)... 48 figure 3. c. orientalis (rebel, 1913), specimen from serbia, kopaonik mt., bele stene, 1760 m, july, 23rd, 2010., jakšić p. leg. discussion and conclusion the combination of morpho-anatomical, physiological-biochemical characteristics, ecological characteristics, as well as taxa biogeography present a good basis for solving the boundary taxonomic cases. even today, we are still impressed by the sensibility of the classical entomologists who were insightful enough to describe new taxa a century ago. out of three sibling species analysed here, two were raised to the status of species. in the near future combinig the morphological and dna analysis, the taxon pyrgus trebevicensis could be classified as a „bona species“ after this review 195 butterfly species have been noted in the territory of serbia: 22 species of hesperioidea and 171 species of papilionoidea (j a k š i ć , 2008) + two new species. this excludes c. gardetta, but includes the presence of c. orientalis instead. related to the butterfly fauna of europe (482 species, s w a a y et al., 2010), the species present in serbia make up 40%. references dražić beneš, j., konvička, m., & fric, z., 2001. faunistic records from the czech republic – 137 lepidoptera: hesperiidae, pyrgus trebevicensis warren, 1926. klapalekiana, 37: 152. boillat, h. 1990. coenonympha (superspecies gardetta) orientalis rebel mise au point taxinomique (lepidoptera nymphalidae satyrinae). alexanor 16(7): 395–412. figire 4. utm distribution map of examined species pyrgus trebevicensis (warren, 1926) ●; melitaea telona (fruhstorfer, 1908) ▲ and coenonympha orientalis (rebel, 1913) ■. coutsis, g.j. and ghavalás, n., 2005. a recently discovered new locality for coenonympha leander in greece, and notes about the taxonomic position of the species-group taxon coenonympha orientalis (lepidoptera: nymphalidae, satyrinae). phegea, 33(4): 121128. davenport, d. 1941. the butterflies of the satyrid genus coenonympha. bulletin of the museum of comparative zoology, harvard college 87: 215349. descimon, h. and mallet, j., 2009. bad species. in: ecology of butterflies in europe, eds. j. settele, t. shreeve, m. konvička and h. van dyck. cambridge university press. pp.: 219-249. gradojević, m., 1930-31. leptirovi srbije – diurna. 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e-mail: mita@imsi.rs abstract: mitrović, a., živanović, b., dučić, t., bogdanović-pristov, j., radotić hadži-manić, k.: chenopodium rubrum l. as a model plant for physiological and biochemical investigations of ontogenesis in vitro. biologica nyssana, 1 (1-2), december 2010: 61-64. chenopodium rubrum l. , is a suitable model plant for studying ontogenesis in vitro as an early flowering species. culture of intact plants in vitro and antioxidative enzymes detection were performed. growth pattern to the end of ontogenesis, flowering and seed development are all determined by the photoperiod seedlings experience during induction and evocation of flowering. different phases of vegetative and reproductive development are characterized by changes in antioxidative enzymes activities. we showed sequential expression of antioxidative enzymes during seed germination. prior to radicule protrusion, cat and sod showed maximal activity, while pod activity appeared later. the highest catalase (cat) activity was measured at the time of flowering while peroxidases (pods) are involved in determination of growth and development in accordance with the environmental clues. the absence of some superoxide dismutase (sod) isoforms could be the indicator of senescence. seed ageing affect changes in antioxidative status of seeds, germination, seedling growth and flowering. key words: catalase, chenopodium rubrum, germination, flowering, peroxidase, superoxide dismutase introduction ! chenopodium rubrum l. belongs to the family chenopodiaceae, genus chenopodium. this is a short-day weedy annual, widely distributed in europe, asia and northern america. ecotypes of this species differ in their photoperiodic characteristics. sel. 184 is a qualitative short-day plant with strictly defined critical night length of 8h (t s u c h i y a & i s h i g u r i , 1981). it is sensitive to photoperiodic stimulus for flowering as early as at cotyledonary stage (s e i d l o v á & o p a t r n á , 1978), when 6 adequate photoperiodic cycles are sufficient for photoperiodic flower induction. c. rubrum plants modify their growth and development in accordance with photoperiod they are exposed to (c o o k , 1975; m i t r o v i ć et al., 2007). as an early flowering species (c u m m i n g , 1967), it is a suitable model plant for studying ontogenesis in vitro. under the adequate photoperiodic conditions, plant flowers in vitro after 15 days (ž i v a n o v i ć et al., 1995), and produces seeds after 10 weeks (m i t r o v i ć et al., 2007). moreover different phases of vegetative and reproductive development are characterized by changes in antioxidative enzymes activities. thus seed germination, seedling growth, flowering, seed maturation and seed ageing are defined by the changes in their antioxidative status (m i t r o v i ć , 2007). it is well known that reactive oxygen species (ros) and their scavenging enzymes participate in protection against pathogens or abiotic stress (h e n d r y & c r a w f o r d , 1994). ros also function as signaling molecules at low 10th sfses • 17-20 june 2010, vlasina lake1 (1-2) • december 2010: 61-64 biologica nyssana 1 (1-2) december 2010: 61-64 mitrović, b. et al. chenopodium rubrum l. as a model plant… 62 concentrations in contrast to high concentrations of ros which can lead to phytotoxicity (f o y e r , 1997). the capacity of ros to serve as signaling molecules highlights the importance of antioxidants to specifically regulate different ros in various cellular compartments. the lack of data obtained from ros measuring in plants is due to technical difficulties associated with quantification of endogenous levels of these very reactive and shortlived species. so, most of the evidences for ros levels has ben provided by studies of antioxidants (d a t et al., 2000). antioxidative enzymes, catalase (cat), superoxide dismutase (sod) and peroxidase (pod) are engaged in the scavenging of ros (v a n l o o n , 1986; b o w l e r et al., 1992; k h a n & p a n d a , 2002) and therefore participate in regulation of plant growth and developmental processes or protection against patogens or abiotic stress. sod plays a crucial role in the antioxidative system by catalysing dismutation of o2 ·¯ to h2o2 and o2. cat has a high reaction rate, but a low affinity for h2o2, thereby removes the bulk of h2o2. inversely pod has a higher affinity for h2o2, allowing for the scavenging of small amounts of h2o2 in more specific locations (d a t et al., 2000). changes in cat activity are linked to desiccation during seed maturation (b a i l l y et al., 2004), seed germination (b a i l l y et al., 2002; p r o d a n o v i ć et al., 2007; b o g d a n o v i ć et al., 2008) and plant growth and development (b a i l e y & m c h a r g u e , 1943; m a t t e r s & s c a n d a l i o s , 1986; mitrović & bogdanović, 2008). pods are the most investigated enzymes since they have a role in very important physiological processes like seed germination, seedling growth (b e l a n i et al., 2002; d u č i ć et al., 2003/4; p r o d a n o v i ć et al., 2007; b o g d a n o v i ć et al., 2008), root growth (k u k a v i c a et al., 2007), plant growth and development (b a i l e y & m c h a r g u e , 1943; m i t r o v i ć & b o g d a n o v i ć , 2008), and lignin biosynthesis in cell walls (b r u c e & w e s t , 1989). this specific review summarizes some of the data obtained on model plant chenopodium rubrum l. in our laboratory in order to improve the understanding of involvement of antioxidative enzymes activities (and circumstantially ros) in regulation of c. rubrum vegetative and reproductive development. results and disscusion seed germination seed germination starts with imbibition, and ends with radicule protrusion. it is a complex process, associated with many metabolic, cellular and molecular events. accumulation of reactive oxygen species (ros), during seed imbibition, leads to germination (b a i l l y et al., 2004). therefore, antioxidant enzymes have a particular importance for the completion of germination. protein content increased during c. rubrum germination (d u č i ć et al., 2003/4), since proteins are both released from protein storage or synthesized de novo during and after the imbibition phase of germination, as building and regulatory material in emerging seedlings (r o b e r t s , 1972). during c. rubrum seed germination sequential expression of antioxidative enzymes occurred (d u č i ć et al., 2003/4). cat and sod showed the highest activity at the time preceding radicule protrusion, while significant expression of pod occurred after this term. increase in pod activity corresponds to the expression of new pod isoforms (d u č i ć et al., 2003/4). the appearance and increase of pod activity during germination could be specifically linked with final phases of seed germination or early seedling growth (s c h o p f e r et al., 2001; d u č i ć et al., 2003/4; b o g d a n o v i ć et al., 2008). sequential expression of antioxidative enzymes during c.rubrum seed germination points out that the decrease in h2o2 level coincide with final phases of seed germination and early seedling growth. the photoperiodic control of growth and development as already stated, in c. rubrum sel. 184 critical night length and sensitivity to photoperiod are well defined (t s u c h i y a & i s h i g u r i , 1981; s e i d l o v á & o p a t r n á , 1978). altering day length in such a plant, is a valuable source of information about regulation of plant development in accordance with the photoperiod. thus with the increase of day length, plant height is increased, flowering is delayed, seed development occured earlier, and plants produced more seeds (m i t r o v i ć et al., 2007). c. rubrum growth pattern to end of ontogenesis, flowering and seed development, are all determined by the photoperiod the seedlings experience during early phases of reproductive development induction and evocation of flowering (m i t r o v i ć et al., 2007; c o o k , 1975). natural c. rubrum flowering induction works in line with minimizing seed weight and maximizing seed number, favorizing physiological mechanisms that works under suboptimal photoperiods, maximizing probability to survive (c o o k , 1975). in addition to photoperiod temperature also affected seed weight, as previously reported for chenopodium quinoa (b e r t e r o et al., 1999) and c. rubrum (m i t r o v i ć et al., 2007). biologica nyssana 1 (1-2) december 2010: 61-64 mitrović, b. et al. chenopodium rubrum l. as a model plant… 63 antioxidative enzymes activities during ontogenesis in vitro by altering photoperiods in plants with well defined critical night length and sensitivity to photoperiod, it is possible to separate on the time scale different developmental phases (vegetative growth, flowering, seed development and maturation) in plants of the same age. the activities of antioxidative enzymes changes with both, phase of development and photoperiod plants are exposed to (l a l l & n i k o l o v a , 2003; m i t r o v i ć & b o g d a n o v i ć , 2008). c. rubrum flowering in vitro was associated with the highest cat activity. the intensities of 17 pod isoforms differed with day length plants were exposed to. so, it was suggested that pods are involved in determination of c. rubrum growth and development in accordance with seasonal changes of day length. pod isoform pi 4.6 could be associated with stress, induced both, by exposure to continuous light and by senescence (m i t r o v i ć & b o g d a n o v i ć , 2008). according to previously reported data for arabidopsis thaliana it was shown that ⋅o2 is involved in the induction and development of senescence (a b a r c a et al., 2001). the absence of some sod isoforms could be the indicator of c. rubrum senescence in vitro which starts during the phase of seed maturation (m i t r o v i ć & b o g d a n o v i ć , 2008). seed aging seed aging is a natural process, starting during, or shortly after harvesting due to irreversible changes leading to loss of viability (v i l l i e r s , 1972). during seed aging chromosome and membrane damage occurs, as well as the damage of the enzyme structure, caused by ros. in the same time ros have the functional significance in seed ageing and germination (s c h o p f e r et al., 2001). seeds sampled during ageing showed an increase in the time needed to start germination possibly due to the need for repair and replacement processes to occur. protein de novo synthesis starts later during imbibition in aged seeds (v i l l i e r s , 1972). protein content, as well as cat and sod activity, was lower in aged c. rubrum seeds (2.5h darkness imbibed), compared to young seeds (m i t r o v i ć et al., 2005). viability declines with seed aging (b e w l e y & b l a c k , 1982) and germination of 3 years old c. rubrum seeds was delayed compared to 3 months old ones (m i t r o v i ć et al., 2005). so, antioxidant enzyme activities may be involved in the evaluation of seed viability in seed ageing. seed age also affect seedling growth and flowering. plants derived from aged seeds shows inhibited growth in vitro (m i t r o v i ć et al., 2005), and delayed flowering (m i t r o v i ć et al., 2005; k a d m a n z a h a v i & p e i p e r , 1987). conclusion this review has summarized some of data obtained on model plant chenopodium rubrum l. grown in vitro in our laboratory during last decade. our results improved knowledge of c. rubrum sensitivity to day length and also confirmed the involvement of antioxidative enzymes in regulation of plant development, from seed germination to seed maturation. acknowledgments. this work was supported by a grant (№. 143043) from the ministry of science and technological development, republic of serbia. references abarca, d., martín, m., sabater, b. 2001: differential leaf stress responses in young and senescent plants. physiol. plantarum, 113: 409-415. bailey, l.f., mchargue, j.s. 1943: enzyme 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(ed.), seed ecology: 266-285, proceedings of the nineteenth easter school in agricultural science, university of nottingham, butterworths, london.. živanović, b., ćulafić, lj., filipović, a. 1995: the effects of hormones and saccharides on growth and flowering of green and herbicides-treated chenopodium rubrum l. plants. biol. plantarum, 37: 257-264. coumarin structure as a lead scaffold for antibacterial agents molecular docking study biologica nyssana 7 (2)  december 2016: 167-170 veselinović, j.b. et al.  coumarin structure as a lead scaffold… 167 original article received: 30 october 2016 revised: 14 november 2016 accepted: 24 november 2016 coumarin structure as a lead scaffold for antibacterial agents molecular docking study jovana b. veselinović1*, jelena s. matejić2, aleksandar m. veselinović2, dušan sokolović2 1health institution filly farm, pharmacy filly 63, cara dušana 31, 18000 niš, serbia 2faculty of medicine, university of niš, bulevar dr zorana ðinđića 81, 18000 niš, serbia * e-mail: milosavljevic.jovana@hotmail.com abstract: veselinović, j.b., matejić, j.s., veselinović, a.m., sokolović, d.: coumarin structure as a lead scaffold for antibacterial agents molecular docking study. biologica nyssana, 7 (2), december 2016: 167-170. coumarins owe their class name to “coumarou”, the vernacular name of the tonka bean (dipteryx odorata willd, fabaceae), from which coumarin was isolated in 1820. many molecules based on the coumarin structure have been synthesized utilizing innovative synthetic techniques. various synthetic routes have led to interesting derivatives including the furanocoumarins, pyranocoumarins and coumarinsulfamates which have been found to be useful in photochemotherapy, antitumor and anti-hiv therapy, as stimulants for central nervous system, antiinflammatory therapy, as anti-coagulants, etc. one of important pharmacological activity of coumarin molecules is their potential as antibacterial agents since they show inhibitory activity toward isoleucyl-transfer rna (trna) synthetase. in the presented research molecular docking studies of selected coumarin compounds inside isoleucyltransfer rna (trna) synthetase active site were performed. molecular docking scores of all studied compounds were obtained through score functions. presented results indicate that from all studied coumarin compounds the strongest interactions with studied enzyme has 7,8-dihydroxy-4-phenyl coumarin followed by 5,7-dihydroxy-4-phenyl coumarin. presented results are in accordance with in vitro obtained results for their antibacterial activity. presented findings suggest that 4-phenyl hydroxycoumarins may be considered as good molecular templates for potential antibacterial agents and can be used for further chemical modifications for improving their antibacterial activity. key words: verbascum davidoffii, endemic species, genetic diversity, conservation apstrakt: veselinović, j.b., matejić, j.s., veselinović, a.m., sokolović, d.: coumarin structure as a lead scaffold for antibacterial agents molecular docking study. biologica nyssana, 7 (2), decembar 2016: 167-170. kumarini su dobili ime od reči "coumarou", narodnom nazivu za tonku (dipterix odorata willd, fabaceae) iz koje je kumarin izolovan 1820. godine. mnogi molekuli bazirani na kumarinskoj strukturi su sintetisani primenom inovativnih hemijskih tehnika. razičiti putevi sinteze su doveli do interesantnih derivata koji uključuju furanokumarine, piranokumarine i kumarinsulfamate koji imaju primenu u fitohemoterapiji, antitumorskoj i anti-hiv terapiji, kao stimulanti za centralni nervni sistem, kao antinflamatorni i antikoagulativni agensi, itd. jedna od važnih farmakoloških aktivnosti kumarinskih jedinjenja je njihov potencijal kao antibakterijskih agenasa, jer pokazuju inhibitornu aktivnost prema izoleucil-transfer rnk sintetazi (trnk). u predstavljenom istraživanju studija molekularnog dokinga unutar aktivnog mesta izoleucil7 (2) • december 2016: 167-170 12th sfses • 16-19 june 2016, kopaonik mt doi: 10.5281/zenodo.200417 biologica nyssana 7 (2)  december 2016: 167-170 veselinović, j.b. et al.  coumarin structure as a lead scaffold… 168 transfer rnk sintetaze (trnk) je primenjena za odabrana kumarinska jedinjena. vrednosti molekularnog dokinga za sva ispitivana jedinjenja su dobijene primenom odgovarajućih skoring funkcija. predstavljeni rezultati ukazuju da od svih ispitivanih kumarinskih jedinjenja najviše interakcija sa ispitivanim enzimom ima 7,8-dihidroksi-4-fenil kumarin, a zatim 5,7-dihidroksi-4-fenil kumarin. predstavljeni rezultati su u skladu sa in vitro dobijenim rezultima za njihovu antibakterijsku aktivnost. prikazani rezultati ukazuju da se 4-fenil hidroksikumarini mogu smatrati dobrim molekuskim osnovama za potencijalne antibakterijske agense i da se mogu koristiti za dalje hemijske modifikacije u cilju poboljšanja antibakterijske aktivnosti. key words: 4-fenil hidroksikumarini, antibakterijska aktivnost, molekularni doking introduction great hopes that the discovery and the application of penicillin and other antibiotics would forever solve the problem of bacterial infections lasted for a relatively short time period. seven decades after antibiotics were introduced, infectious diseases as a cause of mortality are in third place in europe. in the european union (eu) around 2 million people are hospitalized and around 200,000 die from some infectious diseases annually. as a gloom example in south asia in every 2 minutes one infant dies due to resistance to existing antibiotics. it is considered that the main reason for this "failure" of the treatment is a large percentage of drug-resistant and multidrugresistant bacteria. today, antibacterial resistance is a growing public health threat of major concern around the world. a post-antibiotic era (in which common infections and minor injuries can kill), far from being an apocalyptic fantasy, is instead a very real possibility for the 21st century (who, 2014). bacterial resistance is a natural biological phenomenon that can be associated with their fight for survival. mechanisms of bacterial resistance to antibiotics are very different and complex and depend both on the bacteria and the structure of antibiotics. also, it involves a range of resistance mechanisms acting an ever-widening range of bacteria, most of which can cause a wide spectrum of diseases in humans and animals. factors that favor the development of bacterial resistance are: overusing prescribing antibiotics, improper use the compliance, inadvertent application of antibiotics and slow development of new effective antibiotics. of all identified bacterial strains resistant to antibiotics of particular concern are methicillinresistant staphylococcus aureus (mrsa), penicillinresistant streptococcus pneumoniae, vancomycinresistant enterococcus, and mycobacterium tuberculosis, since many of these organisms are resistant to several classes of established antibiotics and they can cause infections which are very difficult to treat. above stated facts are forcing the search for novel antibacterial agents which affect targets that are essential to bacteria (p a y n e et al., 2007; b o l l e n b a c h , 2015). in this regard, the aminoacyltrna synthetase (aars) enzymes have been a focus of recent antibacterial drug research. inhibition of these enzymes halts protein biosynthesis which in turn results in the attenuation of bacterial growth under both in vitro and infectious conditions and for this reason bacterial aarss present a vast number of opportunities for the identification of novel inhibitors that can be considered as candidates for development as antibiotics (o c h s n e r et al., 2007). today resistant bacterial strains occur significantly faster than the synthesis and the release of new effective antibiotics. it is well known fact that the development of new drugs in recent decades has become extremely expensive and time-consuming process. for this reason novel computational methods like molecular docking are used to speed up the process of drug design and development. the main aim of molecular docking is the determination of a suitable geometry and binding affinity of the tested molecule (ligand) to the active site of the target macromolecules (receptors) with the application of the “scoring” functions (k r o e m e r , 2007). coumarins are a group of molecules found extensively in plants and they show various biological and pharmacological activities including anticoagulant, estrogenic, vasodilator, hypothermic, anthelmintic, sedative, analgesic, anti-inflammatory and antiulcer. further, coumarin derivates have a wide range of structural modifications (b o r g e s et al., 2005) and they can serve as molecular templates for new drugs. coumarin derivates are also considered as potential antibacterial agents (v e s e l i n o v i ć et al., 2015). main goal of presented research was to establish the influence of 4-phenyl group addition and the number and position of hydroxyl groups within main coumarine structure on the interaction with isoleucyl-transfer rna (trna) synthetase using molecular docking study. studied interactions are considered as main influence on enzyme inhibition which can lead to antibacterial activity. material and methods molecular docking. in order to gain insight into the plausible mechanism of antibacterial action docking simulations were performed. the crystal structure of isoleucyl-transfer rna (trna) synthetase (ilers) biologica nyssana 7 (2)  december 2016: 167-170 veselinović, j.b. et al.  coumarin structure as a lead scaffold… 169 was obtained from the brookhaven protein data bank http://www.rcsb.org/pdb (pdb entry: 1qu3). the compounds were docked into enzyme binding sites using the molegrovirtual docker (mvd) and published methodology [7]. three dimensional structures of compounds (7-hydroxy-4-phenyl coumarin (7c), b) 5,7-dihydroxy-4-phenyl coumarin (57c) and c) 7,8-dihydroxy-4-phenyl coumarin (78c)) used for docking simulation were constructed using marvinsketch 6.1.0, 2013, chemaxon (http://www. chemaxon.com). geometry optimization was carried out by employing mmff94 molecular force field. fig. 1. above: chemical structures of investigated compounds: a) 7-hydroxy-4-phenyl coumarin (7c), b) 5,7-dihydroxy-4-phenyl coumarin (57c) and c) 7,8-dihydroxy-4-phenyl coumarin (78c). bellow: the structure of ilers with best docking poses for all investigated coumarins inside enzyme binding pocket. results and discussion chemical structures of investigated coumarines are presented in fig. 1. the least energy binding mode of compounds has been studied and best docking poses for all investigated coumarins inside enzyme binding pocket is presented in fig. 1. binding affinity of a ligand to the active site of the enzyme can be estimated based on the score values obtained using scoring functions form the molecular docking method. table 1 presents score values for all studied compounds. presented results indicate that of all studied compounds the highest interactions with enzyme has 78c followed by 57c. according to presented results the difference between their interaction energies is not as big as difference between their interaction energies and 7c interaction energy. it is of great importance that presented results are in accordance with in vitro obtained results for antibacterial activity. interesting result was obtained with molecular docking study – that 7c don’t form any hydrogen bond with the active site of studied enzyme. this can be one of the reasons why 7c show the lowest antibacterial activity, because number, bond length, and bond energy of hydrogen bonds formed between ligand and enzyme have an important role in ligand effect on investigated activity. two dimensional representations of formed hydrogen bonds for 57c and 78c are presented in fig. 2. it was observed from in silico studies that 57c forms one hydrogen bond oxygen atom from carbonyl group from the lacton part of molecule with lys-71 (2.80 å). 78c forms four hydrogen bonds. oxygen atom from carbonyl group from the lacton part of molecule forms one hydrogen bond with lys71 (2.79 å). hydroxyl group at position 7 forms fig. 2. two dimensional representations of the best docking pose for a) 5,7-dihydroxy-4-phenyl coumarin, and b) 7,8-dihydroxy-4-phenyl coumarin inside isoleucyl-transfer rna (trna) synthetase active site. biologica nyssana 7 (2)  december 2016: 167-170 veselinović, j.b. et al.  coumarin structure as a lead scaffold… 170 one hydrogen bond with his585 (3.23 å). hydroxyl group at position 8 forms two hydrogen bonds with his585 (3.19 å) and gly66 (2.60 å). conclusion molecular docking simulations against ilers were performed and correlation between the observed inhibitory activity and the in silico molecular docking scores of the compounds was obtained through hydrogen bonding interactions. these findings suggest that 4-phenyl hydroxycoumarins may be considered as good molecular templates for potential antibacterial agents and can be used for further chemical modifications for improving antibacterial activity what will be the main goal of our further research. acknowledgements. this work has been financially supported by ministry of education and science, republic of serbia, under project number 43012.. references bollenbach, t. 2015: antimicrobial interactions: mechanisms and implications for drug discovery and resistance evolution. current opinion in microbiology, 27: 1-9. borges, p., roleira, f., milhazes, n., santana, l., uriarte, e. 2005: simple coumarins and analogues in medicinal chemistry: occurrence, synthesis and biological activity. current medicinal chemistry, 12 (8): 887-916. kroemer, r.t. 2007: structure-based drug design: docking and scoring. current protein and peptide science, 8 (4): 312-328. ochsner, u.a., sun, x., jarvis, t., critchley, i., janjic, n. 2007: aminoacyl-trna synthetases: essential and still promising targets for new antiinfective agents. expert opinion on investigational drugs, 16 (5): 573-593. payne, d.j., gwynn, m.n., holmes, d.j., pompliano, d.l. 2007: drugs for bad bugs: confronting the challenges of antibacterial discovery. nature reviews. drug discovery, 6 (1): 29-40. veselinović, j.b, veselinović, a.m., nikolić, g.m., pešić, s.z., stojanović, d.b., matejić, j.s., mihajilov-krstev, t.m. 2015: antibacterial potential of selected 4-phenyl hydroxycoumarins integrated in vitro and molecular docking studies. medicinal chemistry research, 24 (4): 16261634. who. 2014: antimicrobial resistance: global report on surveillance. world health organization who library cataloguing-in-publication data. table 1. score values (kcal/mol) for all studied coumarin compounds. moldock rerank interaction inter. hbond docking score 78c -125.476 -108.328 -138.994 13.517 -6.0007 -127.625 57c -118.820 -101.209 -130.653 11.832 -3.8847 -124.704 7c -106.467 -88.612 -116.639 10.172 0 -104.336 karalija, e., parić, a.: effects of salicylic acid foliar application on germination, growth and antioxidant potential of basil (ocimum basilicum l.). biologica nyssana, 8 (2) biologica nyssana 8 (2)  december 2017: 145-150 karalija, e., parić, a.  effects of salicylic acid foliar application on… 145 original article received: 24 september 2017 revised: 8 december 2017 accepted: 22 december 2017 effects of salicylic acid foliar application on growth and antioxidant potential of basil (ocimum basilicum l.) erna karalija*, adisa parić biology department, faculty of natural sciences and mathematics, university of sarajevo, zmaja od bosne 33-35, 71000 sarajevo, bosnia and herzegovina * e-mail: erna.k@pmf.unsa.ba abstract: karalija, e., parić, a.: effects of salicylic acid foliar application on growth and antioxidant potential of basil (ocimum basilicum l.). biologica nyssana, 8 (2), december, 2017: 145-150. salicylic acid is one of endogenous plant growth regulators that plays a key role in many physiological processes. the present study analysed the effect of different concentrations (0, 0.01, 0.1, ad 1.0 mm) of salicylic acid on morphological parameters, photosynthetic pigments, protein, proline, total carbohydrates, and secondary metabolites content as well as peroxidase activity. one month after sowing seedlings were replanted in new pots, and salicylic acid was applied in form of a foliar spray. plants were harvested 60 days after salicylic acid application. results showed that foliar application of salicylic acid induces long-term changes in plant growth and metabolism. we recorded increase in leaf area, secondary metabolites and peroxidase activity. reduction in total sugar and proline content is also recorded. decrease in proline content is probably result of degradation of proline in stress induced conditions. key words: chlorophylls; salicylic acid; secondary metabolites; proline; peroxidases; total carbohydrates apstrakt: karalija, e., parić, a.: efekat folijarne primene salicilne kiseline na rast i antioksidativni potencijal bosiljka (ocimum basilicum l.). biologica nyssana, 8 (2), decembar, 2017: 145-150. salicilna kiselina je jedan od endogenih biljnih regulatora rasta sa ključnom ulogom u mnogim fiziološkim procesima. prezentirani rad analizira efekte različitih koncentracija (0.01, 0.1, ad 1.0 mm) salicilne kiseline na morfološke parametre, fotosintetske pigmente, proteine, ukupne ugljikohidrate i sadržaj sekundarnih metabolite kao i peroksidaznu aktivnost. mesec dana nakon setve biljke su presađene u nove posude i salicilna kiselina je aplicirana u formi folijarnog spreja. biljke su prikupljene za analizu 60 dana nakon aplikacije salicilne kiseline. rezultati su pokazali da folijarna aplikacija salicilne kiseline indukuje dugoročne promene u biljnom rastu i metabolizmu. zabeženo je povećanje površine lista, sadržaja sekundarnih metabolita i peroksidazne aktivnosti. također, zabeležena je redukcija u sadržaju ukupnih šećera i sadržaja prolina. redukcija sadržaja prolina verovatno je rezultat degradacije prolina indukovane stresnim uslovima. ključne reči: hlorofili; salicilna kiselina; sekundarni metaboliti; prolin; peroksidaze; ukupni ugljikohidrati 8 (2) • december 2017: 145-150 doi: 10.5281/zenodo.1135966 biologica nyssana 8 (2)  december 2017: 145-150 karalija, e., parić, a.  effects of salicylic acid foliar application on… 146 introduction physiological efficiency of a plant can be improved by plant growth regulators through their effect on photosynthesis, flower and fruit formation and overall productivity of the plant (a s g h a r i & a g h d a m , 2010). foliar application of plant feeding agents usually influences more plant characteristics and yield then those applied in the soil (k a z e m i , 2013). positive effects of salicylic acid (sa) have been recorded in wheat with recorded enhancement of germination rate and seedling growth (s h a k i r o v a , 2007). also activation of antioxidant enzymes such as peroxidases have been reported after plant spraying with salicylic acid, especially in drought stressed plants (h a y a t et al., 2008). although numerous studies have proven positive effects of salicylic acid it is still a subject of interest due to dependence of the salicylic acid effects upon species, developmental stage, application mode as well as concentration of sa used (v a n a c k e r et al., 2001, h o r v á t h et al., 2007). lower concentrations of salicylic acid have been beneficial for plant growth (v i c e n t e & p l a s e n c i a , 2011), while different results for metabolite content have been reported (w a n g et al., 2007, d ’ o n o f r i o et al., 2009). sweet basil (ocimum basilicum l.), member of lamiaceae family, is used as a fresh vegetable for many traditional foods, dressings and salads. its medicinal value and organoleptic quality is a result of basil aroma and essential oil composition. aim of this study was to investigate the long-term effect of short exposure to foliar application of salicylic acid on growth performances and quality of basil plants. material and methods this study was conducted in laboratory for plant physiology at faculty of natural sciences and mathematics, university of sarajevo. plants were grown in controlled growth chambers with artificial light system and main daily temperature of 22 °c and 75% humidity. plant material and treatments the experiment was conducted in a factorial arranged (2 x 2 x 2) randomised block design with five replications with sa (salicylic acid) as main factor in growth chamber of laboratory for plant physiology at department for biology, faculty of natural sciences and mathematics, university of sarajevo during 2014 and 2015. commercial obtained sweet basil (green paradise s.r.l) seeds were planted in trays. one month old seedlings with two developed leaves similar in size were selected and transferred into 10 cm in diameter plastic containers containing about 3 kg of commercial soil. when 4-6 leaves were developed sa was sprayed in ratio 0.01, 0.1 and 1.0 mm until both sides of leaves were wet. two months after salicylic acid application plant material was collected (six plants from each replication) for further analysis. plant mass and leaf area analysis all plants were carefully removed from soil, roots were washed to remove soil residues and fresh and dry weight (dried at 65 °c for 72 hours) was recorded. analysis of water content was calculated according to the formula: %𝑊𝐶 = 𝐴 − 𝐵 𝐴 where: %wc percentage of water content; a fresh mass (g); b dry mass (g) leaves were photographed on millimetre paper and leaf area was determined using imagej software. photosynthetic pigments content photosynthetic pigments were determined from 80% acetone extract by absorbance reading at 663, 646 and 440 nm according to the method of to a r n o n (1949). calculations were made according to the equations proposed by p o r r a et al. (1989) and h o l m (1954): chlorophyll a = 12.25*a663 − 2.55 ∗ a646 (µg/ml) chlorophyll b = 20.31*a646-4.91*a663 (µg/ml) total chlorophylls = 17.76*a646-7.34*a663 (µg/ml) carotenoids = 4.69*a440-0.267×(a663*a640) (µg/ml) proline content proline content was determined according to the method described by c a r i l l o et al. (2008). dried leaf material (50 mg) was homogenized in 5 ml of ethanol : water mixture (60:40) and homogenate was incubated for 24 h at +4 °c, then centrifuged at 10 000 rpm to obtain supernatant. 500 l of supernatant was mixed with 1000 ml of 1% acid ninhydrin. the reaction mixture was placed in water bath for 20 minutes at 95°c, after which was cooled to room temperature and the absorbance was measured at 520 nm. proline standard was used for calibration curve and calculation of proline content in samples and expressed as mg proline per g of dry weight (mg pro/gdw). secondary metabolite analysis phenolics were extracted from aerial parts of plants by maceration in 80% methanol (hplc grade) and biologica nyssana 8 (2)  december 2017: 145-150 karalija, e., parić, a.  effects of salicylic acid foliar application on… 147 incubation for 24h at 4 °c. extracts were centrifuged at 2000 rpm for 15 min, and supernatants were collected for further analysis. total phenolic content was analysed according to w o l f e et al. (2003) using folin-ciocalteu reagent and gallic acid as standard and it was expressed as mg of gallic acid equivalent per g of dry weight (mggae/gdw). total flavonoid content was done using aluminium chloride method (o r d o n e z et al., 2006) with catechin as standard. results were expressed as mg of catechin equivalent per g of dry weight (mgce/gdw). total flavanol content was analysed according to modified method of g a d z o v s k a et al. (2007) using 1% dmaca reagent (p-dimethyl aminocinnamaldehyde in hcl:ch3oh, 8:92) and catechin as standard. results were expressed as mg of catechin equivalent per g of dry weight (mgce/gdw). determination of soluble sugar content dry samples (0.1 g) were grinded and extracted with 2.5 ml of 80% (v/v) ethanol at 90 °c for 60 min, followed by centrifugation at 10 000 g at 4 °c for 10 minutes. the process was repeated for complete extraction. total soluble sugar content was determined using anthrone reagent and glucose as standard (r o e, 1955). results were expressed as mg soluble sugar/g dw. total protein content total protein content was determined according to the method of b r a d f o r d (1976) grinding 50 mg of fresh leaf sample in phosphate buffer (ph 7.0). the extract was centrifuged at 10 000 rpm for 30 min at +4 °c. absorbance of reaction mixture containing supernatant and diluted bradford reagent was done at 765 nm against blank. peroxidase activity extract obtained for protein analysis was used for total peroxidase activity according to g o n z a l e s et al. (1984). the reaction mixture was prepared by mixing 20 l of extract; phosphate buffer (ph 7.0); guaiacol (20 mm) and h2o2 (10 mm). the absorbance of reaction mixture was recorded continuously during 120 s against blank. statistical analysis all obtained data were analysed for significant differences using factorial analysis of variance. statistical analysis was performed using statistica 10 software and the means were compared using newman-keuls test at p<0.05. results ad discussion effect of sa on growth parameters the statistical analysis showed that application of salicylic acid increased leaf area of basil seedlings in concentration dependent manner (tab. 1). dry mass and water content did not differ between different treatments. statistically significant reduction in photosynthetic pigment contents was recorded dependent upon applied concentration indicating reducing effect of salicylic acid on photosynthesis. reduction in photosynthetic pigment content was probably the reason for increase in leaf area as a response to reduced photosynthetic activity. p a n c h e v a et al. (1996) showed that long-term treatment (7 days) of barley seedlings with sa decreased the rate of photosynthesis (chlorophyll content) and our results are in accordance with these findings indicating that this negative effect of salicylic acid on photosynthetic pigments is present also after 2 months. table 1. effect of salicylic acid on growth parameters of basil salicylic acid, mm leaf area, mm2 dry mass, g water content, % chla, mg/gdw chlb, mg/gdw chla+b, mg/gdw car, mg/gdw 0 65.825 ± 4.651d 0.095 ± 0.005a 0.919 ± 0.010a 5.404 ± 0.188a 2.341 ± 0.109a 7.827 ± 0.298a 1.068 ± 0.025a 0.01 83.739 ± 13.972c 0.092 ± 0.005a 0.931 ± 0.004a 5.280 ± 0.033b 2.235 ± 0.022b 7.596 ± 0.055b 1.059 ± 0.002b 0.1 86.749 ± 25.654b 0.107 ± 0.008a 0.925 ± 0.004a 4.650 ± 0.049c 2.026 ± 0.024c 6.746 ± 0.074c 0.935 ± 0.008c 1.0 90.650 ± 15.782a 0.081 ± 0.001a 0.943 ± 0.006a 1.560 ± 0.037d 0.783 ± 0.022d 2.367 ± 0.060d 0.413 ±0.008d values within one parameter not sharing the same letter are significantly different at p<0.05 biologica nyssana 8 (2)  december 2017: 145-150 karalija, e., parić, a.  effects of salicylic acid foliar application on… 148 reduction of chlorophyll content is usual plant response to stress conditions and since sa plays role in stress induced signalling application of sa can result in reduction of chlorophylls (f a r o o q et al., 2009). physiological and biological processes in plants are strongly regulated and sa plays significant role in this regulation. as such sa can be used to improve plant growth under environmental conditions, but the efficiency of exogenous sa can vary dependent upon the species, developmental stage, application method and sa concentration (b o r s a n i et al., 2001). effect of sa on proline content the analysis of variance for proline content showed that lower concentrations of sa induced decrease in proline content while the highest used concentration of sa (1.0 mm) induced statistically significant increase in proline content (tab. 2). in many cases it was recorded that proline content increases under stress conditions (n a y y a r & w a l i a , 2003). also considering that analysis was 2 months after sa application, low proline content in plants sprayed with 0.01 and 0.1 mm sa could be explained by the breakdown of proline upon relief of stress providing sufficient reducing agents for mitochondrial oxidative phosphorylation and atp generation for recovery from stress and alleviating stress-induced damages (h a r e & c r e s s , 1998, h a r e et al., 1998). proline can be considered as one of the protective mechanisms induced by sa application in basil plants, but no long term accumulation is achieved in this experiment. effect of sa on secondary metabolites foliar application of sa induced statistically significant (tab. 2) long-term increase in secondary metabolite content. k a b i r i et al. (2014) recorded no changes in polyphenol content after sa application in control plants presenting only short term effects of sa on polyphenols. in our experiment long-term effects of salicylic acid result in significant increase in polyphenol content dependent upon applied sa concentration similar results of increase in metabolite production was recorded by g h a r i b (2006). increase of secondary metabolite production in basil under stress condition has been previously noted (k a r a l i j a et al., 2016). soluble sugar content the results showed that sugar content decreased dependent upon applied sa concentration (tab. 2). similar sa induced reduction of soluble sugar content in plants that are not under stress conditions was previously recorded in nigella sativa plants (k a b i r i et al., 2014). our results show that this adverse effect of salicylic acid on sugar content is present also after 2 months. protein content and peroxidase activity application of sa induced significant increase in protein content dependent upon applied sa concentration (tab. 2). considering two month period from the sa application, this increase in protein content could be considered as a permanent effect of sa application. considering that protein content is usually decreased when plants are exposed to stress (k a b i r i et al., 2014) plants with higher protein content are primed for upcoming stress conditions. peroxidase activity was significantly higher in plants sprayed with 0.01 and 0.1 mm sa as compared with control plants and plants sprayed with 1.0 mm, even after 2 months (fig. 1). these differences between peroxidase activities are probably result of activation of defence mechanisms. table 2. effect of salicylic acid on total protein, secondary metabolites and total carbohydrates content in basil treatment total proteins, mg/gdw) proline, mg/gdw total flavonoids, mg/gdw total phenols, mg/gdw total flavanols, mg/gdw total carbohydrates, mg/gdw k 0.508 ±0.019a 0.069 ± 0.009b 9.504 ± 0.169c 2.672 ± 0.431cd 3.663 ± 0.146c 41.651 ± 0.774a 001sa 0.351 ± 0.001b 0.028 ± 0.004c 14.909 ± 0.866b 2.981 ± 0.327b 4.383 ± 0.478b 38.776 ± 0.887b 01sa 0.391 ± 0.031b 0.033 ± 0.002c 15.708 ± 0.867b 2.803 ± 0.532cb 4.658 ± 0.014b 38.460 ± 0.742b 1sa 0.551 ± 0.019a 0.107 ± 0.003a 17.609 ± 0.361a 3.528 ± 0.371a 6.249 ± 0.057a 29.370 ± 0.700c values within one parameter not sharing the same letter are significantly different at p<0.05 biologica nyssana 8 (2)  december 2017: 145-150 karalija, e., parić, a.  effects of salicylic acid foliar application on… 149 foliar pre-treatment of plants with abiotic elicitors induce plant resistance for different stressors. one of chemicals responsible for defence induction are phytohormones (v i c e n t e & p l a s e n c i a , 2011; m a f f e i et al., 2007, w a r et al., 2011). induction of plant defence is usually mediated through physiological, biochemical and molecular mechanisms (v i c e n t e & p l a s e n c i a , 2011). salicylic 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(lepidoptera: noctuidae). plant signaling & behavior, 6: epub. wolfe, k., wu, x., liu, r.h. 2003: antioxidant activity of apple peels. journal of agricultural and food chemistry, 51(3): 609-614. zhao, l.y., chen, j.l., cheng, d.f., sun, j.r., liu, y., tian, z. 2009: biochemical and molecular characterizations of sitobion avenae-induced wheat defense responses. crop protection, 28: 435-442. cvetković et al 2020, biologica nyssana 11(2) 11 (2) december 2020: 129-138 doi: 10.5281/zenodo.4393969 effects of different sucrose concentrations on some parameters of the life cycle in two wild drosophila species original article vladimir j. cvetković university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia biovlada@yahoo.com (corresponding autor) saša s. stanković university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia sasasta@gmail.com vladimir žikić university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia zikicvladimir@gmail.com nikola jovanović university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia nikolajov90@gmail.com jovana dimitrijević university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia jovana.dimitrijevic1@pmf.edu.rs aleksandra cvetanović university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia aleksandra.cvetanovic1@pmf.edu.rs tatjana mitrović university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia tatjanamitrovic3@gmail.com received: may 06, 2020 revised: november 19, 2020 accepted: november 30, 2020 abstract: the fruit flies collected from the wild can be easily cultured in laboratory for research purposes. on that occasion many similar recipes for cornmealbased feeding media can be used for drosophila cultivation. however, in these recipes concentrations of ingredients may differ what might imply a need to choose the recipe which is the most appropriate for culturing of wild drosophila species. the aim of this study was to check how two wild species, drosophila melanogaster and drosophila suzukii respond to different sucrose concentration in feeding media by monitoring of life cycle key parameters. the total number of oviposited eggs, formed pupae and eclosed adults as well as dynamics of pupae formation and adult eclosion were recorded. the results showed that the sucrose concentrations of 160 gl-1 caused significantly lower number of formed pupae and eclosed adults in d. suzukii than at concentrations of 40 gl-1 and 80 gl-1. prolonged and stretched dynamics of pupation and eclosion were recorded in d. melanogaster only at concentration of 160 gl-1. all tested drosophila showed no differences in life cycle parameters between sucrose concentration of 40 gl-1 and 80 gl-1. presented results can be helpful in deciding which cornmeal-based feeding media recipe to choose for cultivation of mentioned wild drosophila species. key words: fruit fly, drosophila melanogaster, drosophila suzukii, feeding media, life cycle, sucrose concentration apstract: efekat različitih koncentracija šećera na neke parametre životnog ciklusa kod dve divlje vrste drosophila voćne mušice, prikupljene u prirodi, mogu se jednostavno gajiti u laboratoriji za potrebe različitih istraživanja. za tu svrhu se može upotrebiti jedan od mnogih recepata sličnih sastojaka za pravljenje hranljive podloge, bazirane na kukuruznom grizu za gajenje drozofila. međutim, u tim receptima koncentracije sastojaka mogu da variraju što može da podrazumeva potrebu da se odabere recept koji najviše odgovara za gajenje divljih vrsta drozofila. cilj ovog istraživanja je da se praćenjem ključnih parametara životnog ciklusa proveri kako dve divlje vrste drosophila melanogaster i d. suzukii reaguju na različite koncentracije šećera u hranljivoj podlozi. praćeni su ukupan broj položenih jaja, ulutkanih i eklodiranih imaga kao i dinamike ulutkavanja i eklodiranja. rezultati su pokazali da koncentracija šećera od 160 gl-1 izaziva značajno manje ulutkavanja i eklodiranja kod d. suzukii nego što je to pri koncentracijama od 40 gl-1 i 80 gl-1. produžena i rastegnuta dinamika ulutkavanja i izleganja su zabeležene kod d. melanogaster samo pri koncentraciji od 160 gl-1. kod svih testiranih drozofila nisu detektovane značajne razlike u praćenim parametrima životnog ciklusa između tretmana sa koncentracijama šećera od 40 gl-1 i 80 gl-1. prikazani rezultati mogu biti od koristi prilikom odabira hranljivog medijuma baziranog na kukuruznom grizu za gajenje pomenutih divljih vrsta drozofila. ključne reči: voćna mušica, drosophila melanogaster, drosophila suzukii, hranljiva podloga, životni ciklus, koncentracija šećera © 2020 cvetković et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 129 introduction the common fruit fly, drosophila melanogaster meigen is a famous model organism that has very wide implementation in biological sciences, primarily in genetic researches. there are many stock centers in the world that provide an array of different drosophila stocks intended for experimental purposes. nevertheless, for some research purposes, there is a need for drosophila species that are obtained from the wild. on that occasion, the flies are caught and transferred into a laboratory ambient for further experiments. for instance, species from the genus drosophila caught in the wild are very often processed in this way, for example d. melanogaster (anagnostou et al., 2010; lin et al., 2014; gao et al., 2018). spotted wing drosophila, drosophila suzukii (matsumura) is a very serious pest that has been extensively explored in laboratory conditions (lin et al., 2014; tochen et al., 2014; silva-soares et al., 2017; fellous & xuéreb, 2017; schlesener et al., 2017). since this species is introduced worldwide, it has been already recognized as a real threat on production of certain soft fruits that are of economic importance (de ros et al., 2013; benito et al., 2016; mazzi et al., 2017). also, the presence of this fruit pest fly is confirmed for the first time in serbia in 2014 by toševski et al. (2014). thus, good laboratory practice is to provide optimal conditions including appropriate feeding media for such species to be transferred from the wild to a laboratory to obtain correct results from conducted experiments. apart from laboratory drosophila stocks, it is well-known that wild drosophila species can be successfully cultured on cornmeal-based feeding media, even d. suzukii (schlesener et al. 2017). there are many available recipes that are relevant for drosophila culturing, for example at the site of bloomington drosophila stock center (https://bdsc. indiana.edu) or in papers (lewis, 1960; schlesener et al. 2017; jovanović et al., 2018; cvetković et al., 2020). however, different recipes for cornmealbased feeding media imply different amounts of some ingredients such as carbohydrates and yeast. in addition, numerous articles are dealing with the influence of dietary yeast on d. melanogaster (anagnostou et al., 2010) and in d. suzukii on life-history traits (bellutti et al., 2018) or protein and carbohydrate ratio on larvae in d. suzukii (silva-soares et al., 2017) and dietary protein and carbohydrates balance in d. melanogaster (lee, 2015). these studies suggested that carbohydrates and proteins play very important role in drosophila lifespan so the special attention should be paid on the concentration of these two ingredients in the feeding media. according to lushchak et al., (2014) the influence of dietary carbohydrate type and dosage is still poorly understood. in addition, we believe that among different recipes, the most appropriate recipe should be selected for wild drosophila species that are transferred from natural to laboratory conditions because they cannot be adapted to artificial feeding media to the same extent as drosophila laboratory stocks. here, we ran a study to rapidly screen the degree of tolerance of the two wild species on three different sucrose concentrations in cornmeal-based feeding media at standard laboratory conditions by monitoring of the life cycle parameters. we believe that this will help to depict which cornmeal-based feeding media, in relation to sucrose concentration, is the most suitable for laboratory cultivation of wild d. melanogaster and d. suzukii collected in southeastern serbia. also, the study should contribute to overall knowledge about influence of dietary sucrose on the life cycle of two drosophila species. material and methods culturing conditions and types of feeding media the fruit flies were cultured at standard laboratory conditions which implies 60% of relative humidity, 12h/12h of the day/night light regime and constant ambient temperature at about 25±1 °c. standard feeding media based on cornmeal was used for culturing of all flies from this experiment. particularly, agar (6 gl-1), cornmeal (96 gl-1), sucrose (80 gl-1) and yeast (20 gl-1) prepared by cooking in dh2o with subsequent addition of fungicide as explained in our previous studies (cvetković et al., 2015; jovanović et al., 2016). for purposes of the experiment three different feeding media were prepared: all previously mentioned components were used in the same concentrations, except for the sucrose concentration that varied. medium with sucrose concentration of 40 gl-1 – labeled as half, medium with sucrose concentration of 80 gl-1 – labeled as normal and medium with sucrose concentration of 160 gl-1 – labeled as double, were used. fruit flies the wild d. melanogaster specimens were caught in the city of niš, near the faculty of sciences and mathematics (43°18’32.5”n; 21°55’22.9”e) in traps using grape as bait, during the september of 2018., leg. v. j. cvetković. the adult flies were immediately transferred on the standard feeding media for culturing. for the starter generation, d. suzukii adults were incubated from larvae from the infested raspberries collected in private raspberry orchard near lebane town (southeastern serbia), 130 biologica nyssana ● 11 (2) december 2020: 129-138 cvetković et al. ● effects of different sucrose concentrations on some parameters of the life cycle in two wild drosophila species 131 42°55’56.1”n; 21°45’21.6”e, during the august of 2018., leg. s. s. stanković. immediately after eclosion they were transferred onto fresh standard media for culturing (fig. 1). captured fruit fly species were determined by v. žikić and s. s. stanković. in order to establish stock, the fruit flies from the wild were cultured on standard (normal) feeding media (see culturing conditions and types of feeding media) for a dozen generations during 4 months. the referent laboratory strain of wild-type d. melanogaster oregon-r-c strain (bloomington drosophila stock center at indiana university, usa), was used in this study as referent line because this strain is adapted on artificial cornmeal-based feeding media that was used in this experiment. experimental protocol the whole experiment was performed at standard laboratory conditions (as explained in culturing conditions and types of feeding media). for the d. melanogaster – referent line and two wild species flies, three different types of vials were prepared, ten of each, that contained half, normal and double concentration of sucrose in experimental feeding media. from referent laboratory stock culture (d. melanogaster) and wild flies established stocks (d. melanogaster and d. suzukii), pairs of young flies at the same age (5-7 days old) were chosen for further analyses, similarly as it was done in our previous research (jovanović et al., 2018). in total, ten pairs were transferred into ten vials in each type of experimental feeding media to mate and lay eggs for two days. afterwards, the adult flies were discarded and the oviposited eggs were counted immediately. each day, number of formed pupae and subsequently eclosed adults was recorded. statistical analysis the statistical analysis was performed within each of the three drosophila group. the comparisons were performed by the following parameters: 1) the total numbers of oviposited eggs, 2) the total numbers of formed pupae and 3) the total numbers of eclosed adults, for each of the three sucrose concentrations. the percentage of pupation (percentage of formed pupae from hatched and survived larvae) and eclosion (percentage of eclosed adults from formed pupae) were calculated as it was described by kinjo et al. (2014). the obtained values for the same parameter (percentage of pupation or eclosion) in feeding media with different sucrose concentration were compared within each of the drosophila group. statistically significant differences were determined with the kruskall-wallis anova test for p<0.05. further, post hoc mann-whitney test was used to determine significant differences between any of two compared values. statistical analysis was conducted by using software package spss (spss inc., chicago, il, usa). results statistical analysis of the life cycle parameters i.e. the total number of oviposited eggs, formed pupae and eclosed adults are presented in tab. 1. the presented mean values and standard deviations were obtained based on 10 replicates for each strain, d. fig. 1. drosophila suzukii the starter generation. a – larvae (showed with blue arrow) from infested raspberry. b – eclosed adults, incubated from infested raspberries, transferred on standard fresh feeding media for culturing. c – pupae (showed with yellow arrows) within the feeding media, the first laboratory generation of captured d. suzukii. photos by v. j. cvetković biologica nyssana ● 11 (2) december 2020: 129-138 cvetković et al. ● effects of different sucrose concentrations on some parameters of the life cycle in two wild drosophila species 132 melanogaster-referent line, wild d. melanogaster and d. suzukii. all calculations were done for each of three feeding media types. the only significant difference in percentage of pupation between normal and double concentration were recorded in d. suzukii (fig. 2). the only significant differences in percentage of eclosion were observed between normal and double group within wild d. melanogaster and normal compared to half and double within d. melanogaster – referent line (fig. 3). in d. melanogaster – referent line, the peak of larval pupation was recorded on the fourth day, fifth and the seventh day in half, normal and double sucrose concentration, respectively (fig. 4). the peak of adult eclosion was recorded at the fifth day in half and normal and at the eighth day in double sucrose concentration (fig. 4). in addition, the peaks were higher and the pupation, as well as eclosion, lasted shorter in groups with half and normal sucrose concentration than in the group with double of the sucrose concentration. in the wild d. melanogaster, the peak of larval pupation was recorded on the fourth day in normal and double and the eighth day in half concentration of sucrose (fig. 5). the peak of adult eclosion was recorded also on the fourth day in half and normal and on the eighth day in a double concentration of sucrose (fig. 5). in addition, the peaks were higher and the pupation, as well as eclosion, species sucrose concentration oviposited eggs formed pupae eclosed adults d . m el an og as te r half 57.63 30.13 29.63 ±19.04 ±11.42 ±11.61 normal 71.5 41.1 40.7 ±21.19 ±14.49 ±14.24 double 65.87 37.88 36.38 ±29.43 ±11.51 ±11.36 d . m el an og as te r re fe re nt li ne half 71.22 62.22 59.11 ±24.54 ±20.28 ±17.88 normal 75.38 * 70.38 * 69.63 * ±20.70 ±16.49 ±16.55 double 55.4* 50.1 * 48.00 * ±19.98 ±18.54 ±17.51 d . s uz uk ii half 11.5 8.6† 8.1† ±8.22 ±5.68 ±5.5 normal 12.2 8.8‡ 8.5‡ ±6.05 ±2.3 ±3.2 double 7.1 2.9 †‡ 2.7 †‡ ±5.09 ±3.6 ±3.3 table 1. life cycle parameters in d. melanogaster-referent line, wild d. melanogaster and d. suzukii cultured on feeding media with different sucrose concentrations. mean values are presented with ± standard deviation. fig. 2. percentage of pupation in d. melanogaster, d. melanogaster-referent line and d. suzukii cultured on feeding media with half, normal and double concentration of sucrose. the lower case letter “a” marked the two values that are significantly different. differences were determined using kruskall-wallis anova and post hoc mann-whitney test. statistical significance was considered for p<0.05 -values marked with the same symbol * are significantly different within d. melanogaster – referent line -values marked with the same symbol † and/or ‡ are significantly different within d. suzukii group. -significant differences were considered for p<0.05 biologica nyssana ● 11 (2) december 2020: 129-138 cvetković et al. ● effects of different sucrose concentrations on some parameters of the life cycle in two wild drosophila species lasted shorter in groups with half and normal sucrose concentration than in double of the sucrose concentration. in d. suzukii line, the peak of larval pupation was recorded on the third, fourth and the seventh day in half, normal and double sucrose concentration, respectively (fig. 6). the peak of adult eclosion was recorded on the fourth, fifth and the eighth day in half, normal and double concentration of sucrose, respectively (fig. 6). additionaly, the peaks were higher in groups with half and normal sucrose concentration than in double of the sucrose concentration but the duration of the pupation and eclosion was mostly the same in all three groups. discussion all three parameters of the life cycle – the total number of oviposited eggs, formed pupae and 133 fig. 3. percentage of eclosion in d. melanogaster, d. melanogaster-referent line and d. suzukii cultured on medium with half, normal and double concentration of sucrose. the same lower cases letter (a, b or c) marked the two values that are significantly different. differences were determined using kruskall-wallis anova and post hoc mann-whitney test. statistical significance was considered for p<0.05. fig. 4. dynamics of pupation and eclosion in d. melanogaster – referent line. a – dynamics of larval pupation and b – dynamics of adult eclosion. half, normal and double refers to sucrose concentration in feeding media. biologica nyssana ● 11 (2) december 2020: 129-138 cvetković et al. ● effects of different sucrose concentrations on some parameters of the life cycle in two wild drosophila species 134 eclosed adults were not significantly different in the wild d. melanogaster cultivated on feeding media with different sucrose concentrations. according to these results, the wild d. melanogaster strain used in this experiment were tolerant to variation of sucrose concentration in feeding media at standard ambient conditions. in d. suzukii, at double sucrose concentration, the total number of formed pupae and eclosed adults were significantly lower (p<0.05) than in half and normal sucrose concentrations. however, the total number of oviposited eggs was not significantly different between groups with different sucrose concentrations. in some way, the higher sucrose concentrations disrupted incubation of the eggs and/or development of the hatched larvae until the pupation stage. also, in the d. melanogaster-referent line significantly lower (p<0.05) numbers of oviposited eggs, formed pupae and eclosed adults were observed in double than in normal sucrose concentration. a possible explanation is that the higher sucrose concentration in feeding media probably stimulates the increase of the products of fermentation. the higher levels of the fermentation products (ethanol, carbon dioxide etc.) may have a negative impact on larval hatching, development as well as pupation. also, it might have a negative effect on the number of oviposited eggs that was significantly lower in double concentration in d. melanogaster – referent line. this hypothesis is waiting to be confirmed or rejected in further experiments because this time we did not measure the levels of fermentation products in our experiment. a lower percentage of pupation were recorded in all three types of feeding media in the wild d. fig. 5. dynamics of pupation and eclosion in wild d. melanogaster. a – dynamics of larval pupation and b – dynamics of adult eclosion. half, normal and double refers to sucrose concentration in feeding media. biologica nyssana ● 11 (2) december 2020: 129-138 cvetković et al. ● effects of different sucrose concentrations on some parameters of the life cycle in two wild drosophila species 135 melanogaster while in the d. melanogaster – referent line was higher. this means that the laboratory drosophila strain is well adapted on applied cornmeal feeding media, as it was expected. the percentage of pupation values in d. suzukii were about 90% in average. as we were not able to count the hatched larvae, the much lower percentage of pupation could mean that many of the eggs were not fertilized or/and that many larvae have died before pupation in wild d. melanogaster. thus, these results indicate that standard laboratory conditions and applied feeding media (for medium details see section: culturing conditions and types of feeding media) influenced pupation in wild d. melanogaster. besides previous observations, in wild d. melanogaster percentage of pupations were not significantly different in all three sucrose concentrations, as in the d. melanogaster – referent line, while in d. suzukii percentage of pupation was significantly lower in double concentration compare to normal sucrose concentration. we propose one possible explanation for such a result. during pupation, larvae of d. melanogaster mostly went out of the feeding media and formed pupae on the walls of the vials where they were cultivated. in d. suzukii, pupae are formed mostly within the feeding media. thus, the explained effects of high sucrose concentrations in feeding media and the consequent presence of higher levels of fermentation products like ethanol and carbon dioxide might affect the pupation process in this case by affecting the larvae hatching or/and eggs incubation or/and larvae survival. as we did not count the number of hatched larvae, so we cannot state at which state the detrimental effect occurred, fig. 6. dynamics of pupation and eclosion in wild d. suzukii. a – dynamics of larval pupation and b – dynamics of adult eclosion. half, normal and double refers to sucrose concentration in feeding media. biologica nyssana ● 11 (2) december 2020: 129-138 cvetković et al. ● effects of different sucrose concentrations on some parameters of the life cycle in two wild drosophila species this observation remains to be tested in forthcoming analysis. gao et al. (2018) showed that both adults and larvae of d. melanogaster were more tolerant of environmental ethanol than d. suzukii (gao et al., 2018). this is consistent with our observation about the influence of possible higher levels of ethanol in double sucrose concentrated media. in addition, it has long been known that d. melanogaster was more tolerant on alcohol compared to other drosophila species (mckenzie & parsons, 1972). on the other hand, a minor difference in the percentage of eclosion in both wild drosophila species as in the d. melanogaster – referent line in any type of the tested feeding media. this could mean that the process of incubation from pupal to the adult stage was not affected in the same percentage as the pupation process by different sucrose concentrations in any experimental group since pupal mortality was very low in all drosophila groups. only significant differences showed that the percentage of eclosion was significantly affected by double sucrose concentration comparing with normal within the wild d. melanogaster group. also, within d. melanogaster – referent line, in double and half concentration percentage of eclosion was significantly affected compared to normal concentration (the percentages of eclosion were above 95%). however, these differences were minimal in both d. melanogaster groups. interestingly there were no significant differences between the percentage of eclosed flies in different feeding media within d. suzukii group, so the different sucrose concentrations did not affect d. suzukii at the pupal stage. it was already stated that in the wild d. melanogaster there were no significant differences for the total number of formed pupae and eclosed adults at different sucrose concentrations. but the differences were observed at dynamics of pupation and eclosion. the peaks for the dynamic of these parameters in half and standard concentration was on the fourth day while in double was about the eighth day. the pupation and eclosion were very prolonged and stretched at double concentration compared with the half and standard within the wild d. melanogaster. analyzing these parameters in the d. melanogaster – referent line group and d. suzukii group, a similar pattern could be observed in the double sucrose concentration. nevertheless, in d. suzukii the other two groups, half and normal, also express prolonged pupation and eclosion without pronounced peaks unlike both d. melanogaster groups. thus, due to its detrimental effect in all groups the double sucrose concentration in feeding media caused prolonged and extended pupation and eclosion. the reason might be the same, explained in previous paragraph, the detrimental effect of the higher level of fermentation products. on the other hand, half concentration did not cause any visible effect on dynamics of pupation and eclosion in any drosophila group. overall, our findings is in accordance with the conclusion of fellous & xuéreb (2017) that high sugar concentrations are detrimental for development of the d. suzukii. also, lushchak et al., (2014) stated that sucrose, commonly used for drosophila laboratory food, shorten lifespan and contribute to lower egg-laying capability in d. melanogaster. according to the results and previous observations, the detrimental effects, especially of the highest applied sucrose concentration are obvious in drosophila. even more, d. suzukii was more sensitive to higher sucrose concentration than d. melanogaster. there are many available cornmeal-based feeding media recipes that can be applied for laboratory culturing of drosophila species, but the concentrations of ingredients may vary in different recipes. despite differences in ingredients, many of them can be used for drosophila culturing. for example, some fly food recipes are available at the site of bloomington drosophila stock center from indiana university in usa (https://bdsc.indiana.edu). schlesener et al. (2017) reported modified cornmealyeast-glucose-agar medium that are suitable for d. suzukii rearing. the composition of standard feeding media that we use in this experiment were not the same but it is quite similar as in schlesener et al. (2017). we found that one of the differences between the feeding media that we used from the mentioned one is in yeast concentration. particularly, we used almost twice smaller concentration of yeast, as a protein source, what in addition to the highest applied sucrose concentration also might influenced development of the d. suzukii in our experiment. however, we did not vary yeast concentration in this experiment because it was not the subject of this research and in all treatment media was added in same amount according to one of the recipe (see section: culturing conditions and types of feeding media) that we use for laboratory drosophila stocks culturing. also, fellous & xuéreb (2017) reported that intermediate protein concentration is optimal for d. suzukii development. these observations indicate that protein concentration in feeding media should be taken into consideration when choosing appropriate feeding media for wild d. suzukii and we believe also for d. melanogaster because it is known that dietary balance of proteins and carbohydrates concentrations is important because its influence on drosophila lifespan (lushchak et al., 2014; lee, 2015). 136 biologica nyssana ● 11 (2) december 2020: 129-138 cvetković et al. ● effects of different sucrose concentrations on some parameters of the life cycle in two wild drosophila species conclusion according to our findings the sucrose concentration of 160 gl-1 in the feeding media caused significantly lower number of formed puape and eclosed adults in d. suzukii than at concentrations of 40 gl-1 and 80 gl-1. in all tested drosophila there were no significant differences in analyzed life cycle parameters between sucrose concentrations of 40 gl-1 and 80 gl-1. moreover, there were no significant differences between any of three tested sucrose concentrations for numbers of laid eggs, pupation and eclosion in wild d. melanogaster. the dynamics of pupation and eclosion were prolonged and stretched in wild d. melanogaster at 160 gl-1 sucrose concentration while in d. suzukii was mostly the same as in 40 gl-1 and 80 gl-1. we believe that the presented results might be helpful in deciding which cornmeal-based feeding media, in relation to the sucrose concentration, should be selected for the cultivation of mentioned drosophila species that originating from the wild. also, the obtained results represent contribution to the overall knowledge of the influence of dietary sucrose on lifecycle of two drosophila species. acknowledgement. this study was supported by the ministry of education, science and technological development of the republic of serbia (contract number: 451-03-68/2020-14/200124). references anagnostou, c., dorsch, m., rohlfs, m. 2010: influence of dietary yeasts on drosophila melanogaster life‐history traits. entomologia experimentalis et applicata, 136(1): 1-11. bellutti, n., gallmetzer, a., innerebner, g., schmidt, s., zelger, r., koschier, e.h. 2018: dietary yeast affects 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a geometric analysis of the macronutrient needs of drosophila suzukii larvae. drosophila information service, 100: 158-167. gao, h.h., zhai, y.f., chen, h., wang, y.m., liu, q., hu, q.l., ren, f.s., yu, y. 2018: ecological niche difference associated with varied ethanol tolerance between drosophila suzukii and drosophila melanogaster (diptera: drosophilidae). florida entomologist, 101(3): 498-504. jovanović, b., cvetković, v.j., mitrović, t.l. 2016: effects of human food grade titanium dioxide nanoparticle dietary exposure on drosophila melanogaster survival, fecundity, pupation and expression of antioxidant genes. chemosphere, 144: 43-49. jovanović, b., jovanović, n., cvetković, v.j., matić, s., stanić, s., whitley, e.m., mitrović, t.l. 2018: the effects of a human food additive, titanium dioxide nanoparticles e171, on drosophila melanogaster-a 20 generation dietary exposure experiment. scientific reports, 8: 17922. kinjo, h., kunimi, y., nakai, m. 2014: effects of temperature on the reproduction and development of drosophila suzukii (diptera: drosophilidae). applied entomology and zoology, 49: 297-304. lee, k.p. 2015: dietary protein: carbohydrate balance is a critical modulator of lifespan and reproduction in drosophila melanogaster: a test using a chemically defined diet. journal of insect physiology, 75:12-19. lewis, e.b. 1960: a new standard food medium. drosophila information service, 34: 117– 118. lin, q.c., zhai, y.f., zhang, a.s., men, x.y., zhang, x.y., zalom, f.g., zhou, c.g., yu, y. 2014: comparative developmental times and laboratory life tables for drosophlia suzukii and drosophila melanogaster (diptera: drosophilidae). florida entomologist, 97(4): 1434-1442. lushchak, o.v., gospodaryov, d.v., rovenko, b.m., yurkevych, i.s., perkhulyn, n.v., lushchak, v.i. 2014: specific dietary carbohydrates differentially influence the life span and fecundity of drosophila melanogaster. journals of gerontology 137 biologica nyssana ● 11 (2) december 2020: 129-138 cvetković et al. ● effects of different sucrose concentrations on some parameters of the life cycle in two wild drosophila species series a: biomedical sciences and medical sciences, 69(1):3-12. mazzi, d., bravin, e., meraner, m., finger, r., kuske, s. 2017: economic impact of the introduction and establishment of drosophila suzukii on sweet cherry production in switzerland. insects, 8(1):18. mckenzie, j.a., parsons, p.a. 1972: alcohol tolerance: an ecological parameter in the relative success of drosophila melanogaster and drosophila simulans. oecologia, 10: 373–388. schlesener, d.c., wollmann, j., krüger, a.p., martins, l.n., geisler, f.c., garcia, f.r. 2017: rearing method for drosophila suzukii and zaprionus indianus (diptera: drosophilidae) on artificial culture media. drosophila information service, 100: 185-189. silva-soares, n.f., nogueira-alves, a., beldade, p., mirth, c.k. 2017: adaptation to new nutritional environments: larval performance, foraging decisions, and adult oviposition choices in drosophila suzukii. bmc ecology, 17: 21. tochen, s., dalton, d.t., wiman, n., hamm, c., shearer, p.w., walton, v.m. 2014: temperaturerelated development and population parameters for drosophila suzukii (diptera: drosophilidae) on cherry and blueberry. environmental entomology, 43(2): 501-510. toševski, i., milenković, s., krstić, o., kosovac, a., jakovljević, m., mitrović, m., cvrković, t., jović, j. 2014: drosophila suzukii (matsumura, 1931)(diptera: drosophilidae): a new invasive pest in serbia. zaštita bilja, 65(3): 99-104. https://bdsc.indiana.edu 138 biologica nyssana ● 11 (2) december 2020: 129-138 cvetković et al. ● effects of different sucrose concentrations on some parameters of the life cycle in two wild drosophila species ćušterevska, r.: armerio rumelicae-potentillion micevski 1978 in south-central balkan with emphasis on galičica mountain vegetation. biologica nyssana, 8 (1) biologica nyssana 8 (1)  september 2017: 00-00 ćušterevska, r.  armerio rumelicae-potentillion micevski 1978… 61 original article received: 09 august 2017 revised: 17 august 2017 accepted: 13 september 2017 armerio rumelicae-potentillion micevski 1978 in south-central balkan with emphasis on galičica mountain vegetation renata ćušterevska institute of biology, faculty of natural sciences and mathematics, university of ss. cyril and methodius, arhimedova 5, mk-1000 skopje, republic of macedonia * e-mail: renatapmf@yahoo.com abstract: ćušterevska, r.: armerio rumelicae-potentillion micevski 1978 in south-central balkan with emphasis on galičica mountain vegetation. biologica nyssana, 8 (1), september 2017: 61-72. the article provides an overview of current knowledge about distribution of perennial grasslands supported by nutrient-poor soils on siliceous bedrocks at elevations characterized by the submediterranean climate of southcentral balkan peninsula (armerio rumelicae-potentillion). we used our own field data from galičica mountain and literature sources to compile a set of vegetation plots that have been stored in a vegetation database, data-mined and analyzed using numerical-analytical tools such as cluster analysis and ordination. five already known associations, were confirmed as well-defined floristic complexes, while the floristic composition of our data enabled recognition of ass. euphrasio-plantaginetum holostei micevski 1994 subass. festucetosum hirtovaginatae subass. nova. key words: balkan peninsula, silicicolous perennial grasslands, syntaxonomy, vegetation classification apstrakt: ćušterevska, r.: armerio rumelicae-potentillion micevski 1978 u južnom delu centralnog balkana sa posebnim osvrtom na vegetaciju planine galičice. biologica nyssana, 8 (1), septembar 2017: 61-72. članak obezbeđuje pregled postojećeg znanja o distribuciji višegodišnjih travnjaka na silikatnim zemljištima siromašnim nutrijentima i nadmorskim visinama okarakterisanim submediteranskom klimom južnocentralnog dela balkanskog poluostrva (armerio rumelicae-potentillion). korišćeni su autorski terenski podaci sa planine galičice i literaturni izvori kako bi se sastavio skup vegetacijskih plotova, koji su zatim sačuvani u vegetacijskoj bazi podataka i analizirani korišćenjem numeričko-analitičkih alata poput klaster analize i ordinacije. pet poznatih asocijacija potvrđene su kao dobro definisani floristički kompleksi, dok je iz florističkog sadržaja naših podataka prepoznata ass. euphrasio-plantaginetum holostei micevski 1994 subass. festucetosum hirtovaginatae subass. nova. ključne reči: balkansko poluostrvo, višegodišnji travnjaci na silikatima, sintaksonomija, klasifikacija vegetacije 8 (1) • september 2017: 61-72 doi: 10.5281/zenodo.964015 biologica nyssana 8 (1)  september 2017: 61-72 ćušterevska, r.  armerio rumelicae-potentillion micevski 1978… 62 introduction in the framework of a broader research of the grassland vegetation on galičica mountain, among other it was investigated the vegetation which is developing to deep decalcified soil on carbonate substrate distributed in the area between 1000-1500 m a.s.l.. whereby there was a need to define its affiliation to higher sintaxonomic categories. communities that develop on silicate surface up to 1000 m a.s.l. in south-central balkan belong to the alliance trifolion cherleri micevski 1970 (festuco-brometea), while communities that develop over this belt belong to the alliance armeriopotentillion micevski 1978. according to the original concept (m i c e v s k i , 1970, 1971, 1978), armerio-potentillion was subordinated to the order astragalo-potentilletalia. in the classification scheme, as adopted by the eurovegchecklist (m u c i n a et al., 2016), the astragalo-potentilletalia (festuco-brometea) comprises only open calcicolous grasslands of the saturejo-thymion. the trifolion cherleri represents submediterranean therophyte communities in the southern balkans, classified in the helianthemetalia guttati br.-bl. in br.-bl. & al. 1940 (helianthemetea guttati rivas goday et rivas-mart. 1963). the armerio-potentillon cannot be classified into either the astragalo-potentilletalia or in the helianthemetalia guttati. this vegetation is perennial grassland supported by nutrient-poor soils on siliceous bedrocks at elevations characterized by the submediterranean climate of macedonia, southern serbia and bulgaria. the following associations have so far been classified in the armerion-potentillon: genisto-agrostietum byzanthinae micevski 1978, koelerio-festucetum stojanovii micevski 1978, diantho-armerietum rumeliacae n. ranđelović 1978, euphrasio-plantaginetum holostei micevski 1994 and plantagini subulatae-agrostietum capillaris pedashenko et al. 2013. similar vegetation was described or reported from southeastern serbia by n. r a n đ e l o v i ć (1978), where ass. dianthoarmerietum rumelicae n. ranđelović 1978 in describing is arranged in alliance chrysopogonodanthonion kojić 1957 (order brometalia erecti br.bl. 1936 class festuco-brometea br.-bl. et tx. ex klika et hadac (1944). later, m i l o s a v l j e v i c et al. (2008), r a n đ e l o v i ć et al. (2008) and v. r a n đ e l o v i ć & z l a t k o v i ć (2010) classified this vegetation in the armerio-potentillon. v. r a n đ e l o v i ć & z l a t k o v i ć (2010) proposed classification of the armerio-potentillon into a new order – the armerietalia rumelicae, however since they failed to designate the holotypus in expressis verbis (as required by the icpn after 01.01.2002), the order name remained invalid. mucina et čarni (d i p i e t r o et al. 2015) agreed with the syntaxonomic concept of the new order suggested by r a n đ e l o v i ć & z l a t k o v i ć (2010) and therefore formally validated the armerietalia rumelicae v. randelovic et n. randelovic in v. randelovic et zlatkovic ex mucina et čarni in d i p i e t r o et al. 2015. material and methods the studied area comprised the central and southern regions of the balkan peninsula, namely territory of southeastern serbia, eastern and western macedonia and central bulgaria (fig. 1). we focused on the collection of vegetation plot data of supratemperate and submediterranean, silicicolous perennial grasslands. original vegetation plot data were collected according to the standard braun-blanquet method of field-plot sampling (b r a u n -b l a n q u e t , 1964). the sampling was done in optimal phenological period of the year – from the second half of june to mid-july during 2009 and 2010 on the galičica mountain, were investigated perennial grasslands supported by nutrient-poor soils which usually occur on silicate substrate and into this case on deep soil where carbonates are washed. the size of the sampled plots was 100 m². the choice of this sample size was motivated by the relatively homogenous vegetation at this scale and the preponderance of vegetation plot data collected at that scale in the past. fig. 1. locations (relevés numbered with ordinal numbers) of the studied area 6 3 table 1. association euphrasio-plantaginetum holostei micevski 1994 subass. festucetosum hirtovaginatae subass. nova (e-ph-fh) (g-ab genisto-agrostidetum byzanthinae; k-fs koelerio-festucetum stojanovii; e-ph euphrasio-plantaginetum holostei (bistra mt.); ps-ac plantagini subulatae-agrostietum capillaris; d-ar diantho-armerietum rumelicae) number of releve 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 e -p h -f h g -a b k -f s e -p h p sa c d -a r euphrasia pectinata + + + + 1 . 1 1 1 1 1 1 + 1 1 1 1 + + 1 + 1 1 + . + + + 1 1 1 + v v ii v plantago holosteum . . . . 1 . . 1 1 . 1 4 3 4 3 3 3 + 1 1 + 1 . . . . . 1 1 1 1 + iv v v v arenaria serpyllifolia . . + . . 1 + . . . . + . . . . . . . . . . . + . . . . . . . . i iv i subass. festucetosum hirtovaginatae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . achillea seidlii 1 + + 1 + + 1 2 1 + 1 + 1 + + + + 1 + 1 + 1 + + + 1 1 . . . . . v centaurea deusta . . . . . + + 2 1 1 1 1 2 1 + + + + 1 1 1 1 1 + + + . 1 1 1 . + iv hypericum barbatum 1 1 + 1 1 + + 1 + + + . + + . . . 1 + 1 + . + . + + 1 + . + + + iv i verbascum speciosum . . . . . . . + + 1 1 + + + 1 + + + + + + + + + + + + . . . . . iv festuca hirtovaginata . . . . 1 . . . . 1 2 + 2 2 1 2 + 4 5 4 3 4 4 4 4 4 1 . . . . . iii bupleurum falcatum subsp. cernuum + . . + . . . . . . . . . . . . . + 1 . 1 + 1 + 1 + + 1 2 2 1 1 iii armerio-potentillion agrostis castellana . . . . . . . 2 1 4 3 . + + + + + + + 1 + . 1 . . + 1 1 1 + + + iv v i leontodon hispidus + + + + . . + 1 1 1 + + + . . . . + . + + . . . . . 1 1 1 1 1 + iv iv i festuca valesiaca 3 4 4 3 4 4 4 . . . . . . . . . . . . . . . . . . . . 1 1 1 + . ii iii iv helianthemum nummularium . . . . . . . . . . . . . . . . . . . . . . . . . . + + . + . + i iii v i iii veronica austriaca subsp. austriaca . + . . . + . 1 + . + . . . . . . . . . . . . . . . . . . . . . i iii i armerietalia rumelicae , stipo giganteae-agrostietea castellanae anthoxanthum odoratum 2 1 + + 1 + 1 1 1 1 + 1 + 1 + 1 + + + 1 + + + + + + 1 1 1 1 + . v iv iii iv iii i plantago lanceolata 1 1 + + + + + 1 1 + + . + + + + + 1 . + . + + + . . + + + + + . v v v v ii v cruciata pedemontana 1 1 + + + 1 + . + . . . . . . . . 1 + 1 + + 1 1 + 1 1 . . . . . iii i i i poa bulbosa . . . . . . . + 1 1 1 1 1 1 1 1 + . . . . + + + + . . . . . . . iii v iii v ii trifolium campestre . . + . 2 2 + + 1 1 1 1 + + + + + . . . . . . . . . . . . . + . iii v i iii iii i dianthus deltoides 1 + + + . . . . . . . . . . . . . + . . . + . . . . 1 1 1 + . . ii i trifolium repens + 2 + + 2 . . + + . . . . . . . . . . . . . . . . . . + . . . . ii iv v iv ii iv veronica arvensis + . + . 2 + + . . . . . . + + + + . . . + . . + . . . . . . . . ii iii ii i i cynosurus echinatus + + + + + . . . + . . + . . . . . . . . . . . . . . . . . . . . ii ii anthemis cretica subsp. carpatica . . . . . . . 1 + . . + + + . + . . . . . . . . . . . . . . . . i arabidopsis thaliana . . . . . . . . . . . 1 . . . . . . . . . . . . . . . . . . . . i erodium cicutarium . . + + . . . . . . . . . . . . . . . . . . . . . . . . . . . . i filago arvensis . . . . . . . . . . . . . . . . . . . . + . . . . . . . . . . . i filago minima . . + + 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . i myosotis stricta . . . . . . . . . . . . . . . . 1 . . . + . . . . . . . . . . . i i i potentilla pedata . . . . . . . . . . . . . . . . . . . . . . . . . . . + 1 + + + i b i o l o g i c a n y s s a n a 8 (1 )  s e p te m b e r 2 0 1 7 : 6 1 -7 2 ć u š te re v s k a , r .  a rm e rio ru m e lic a e -p o te n tillio n m ic e v s k i 1 9 7 8 … 6 4 potentilla recta subsp. recta . . . . . . . . . . . . . . . . . + . . . . . . . . . . . . . . i i scleranthus annuus subsp. annuus . . + + . . . . . . . . . . . . . . . . . . . + . . . . . . . . i sedum urvillei . . + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i i trifolium arvense . . + . . . . . . . . . . + + + . . . . 1 . . . . . . . . . . . i iii iii vicia lathyroides . . + + . + . . . . . . . . . . . + . . . . . . . . . . . . . . i rumex acetosella + . . . . . . + + . . . . + . . . . . . . . . . . . . . . . + . i iv ii v iii scabiosa triniifolia . . . . . . . . + . . . + . . . . . . . . . . . . . . . . . . . i i i iv viola tricolor subsp. macedonica . . . . . . . . . . . . . . . . . . . . . . . . . . + . . . . . i iv other species dianthus cruentus 1 + + 1 1 2 2 1 2 1 1 + 2 1 1 1 1 + 1 + 1 + + + + + 1 + + 1 + + v ii iv galium verum 1 2 1 1 + + 1 1 1 + + + + 1 + 1 + 1 1 1 1 1 + + + + 1 1 + + 1 1 v ii iv ii iii pilosella officinarum 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 + 1 + + 1 1 1 1 1 1 v v v thymus sibthorpii 2 . 1 + 1 1 1 1 2 1 1 + + + 2 2 2 1 1 1 1 1 1 1 1 + 1 1 1 1 1 1 v v campanula patula subsp. patula 1 1 + 1 . . 1 1 1 1 1 + 1 1 + + + + + + + + 1 1 1 1 1 1 + 1 + + v iii v rhinanthus minor 1 + + 1 + . + . . . 1 + . + + + + + . 1 1 + 1 + . + + 1 . 1 + + iv v potentilla detommasii 1 1 1 + 1 1 1 1 + . 1 + + + . 1 + 1 1 + 1 + + + + + + 1 . . . . v ii stellaria graminea + + + + + + + . . . + . . . . . . + . . . + + . . . 1 1 1 + + + iii iii v trifolium pratense . + . . + . + . + . + + . . . . . + + + + . . . . . + + + + + . iii ii i i v cerastium brachypetalum subsp. roeseri 1 + 1 + + 1 1 + + . . . . . . . . . . . . . . + + . 1 + + . . . iii silene sendtneri subsp. balcanica . . . . . . . . . + + + . . + + + + . + + + + + . 1 + . . + + . iii potentilla arenaria + + + + + . . . . . . . + + . . . . . + . + 1 + . + . . . . . . ii achillea chrysocoma . . + + + . + . + . + + 1 2 1 1 1 . . . . . . . . . . . . . . . ii koeleria splendens . . . . 1 1 + 1 1 . 1 + + + + + + . . . . . . . . . . . . . . . ii genista tinctoria . . . . . . . 1 + . 1 1 + . . . 1 + . . . . + . . . 1 1 . + . + ii i stipa balcanica . . . . . . . . . . . . . . . . . . + . 1 + + . + 1 4 4 3 4 4 4 ii dichoropetalum oligophyllum . . . . . . . . + . . + . . . . . 1 . . . . . + 1 + + 1 1 + . + ii ii i lotus tenuis . . . . . . . . . . . . . + 1 1 + + . + + . . + . . . 1 + + . . ii v ii asphodelus albus . . . . . . . . . . . . . . . . . + . . . + + + + + 1 + + + + . ii cirsium eriophorum + + + + . . + . . . . . . . . . . . + . . . . + + + . + . . . . ii primulla veris subsp. columnae + + + + . . . + . . . . . . . . . . . . . . . . . . . + . + + + ii i noccaea praecox . + + + . . . . . . + + + . . . + . . . . . . . . . 1 . + . . . ii i euphorbia verrucosa . . . . . . . . . . . . . . . . . . . + . . . . 1 1 2 1 1 1 1 + ii secale strictum + . + + . . . . . . . . . . . . + . . . . + + . + 1 . . . . . . ii scorzoneroides cichoriacea . + + + 1 + + . . . . . . . . . . + . . + . . . . . . . . . . . ii silene viscaria . . . . + . . . . 2 1 . . + + 1 1 . . . . . . . . . . . . . + . ii i achillea setacea . . . . . . . . . . . . . . . . . . . + . + . . . . + 1 + + 1 + ii v v carduus tmoleus . . . . . . . 1 + . . . . + . . . . . + + . . . . + . . . . . 1 ii ii galium verticillatum . . . . . . . . + . . + + + + + 1 . . . . . . . . . . . . . . . ii knautia arvensis . . . . . . . . . . . + . . . . . . . . . . . . 1 + 1 + . . + 1 ii ii iii verbascum longifolium subsp. pannosum + . . + . . . . . . . . . . . . . . . . . . . . . . . + + + . + i ii euphorbia myrsinites . . + . + + + . . . . . . . . . . . . . + . + . . . . . . . . . i i b i o l o g i c a n y s s a n a 8 (1 )  s e p te m b e r 2 0 1 7 : 6 1 -7 2 ć u š te re v s k a , r .  a rm e rio ru m e lic a e -p o te n tillio n m ic e v s k i 1 9 7 8 … 6 5 carex halleriana . . . . . . . . . . . + 1 + 1 + + . . . . . . . . . . . . . . . i seseli peucedanoides + + + 1 . . + . . . . . . . . . . . . . . . . . . . . . . . . . i ii trifolium phleoides . + + + 1 . 1 . . . . . . . . . . . . . . . . . . . . . . . . . i muscari racemosum . + + + . . . . + . . . . . . . . . . . . . . . . . + . . . . . i ranunculus psilostachys . . + + + + + . . . . . . . . . . . . . . . . . . . . . . . . . i pimpinella tragium subsp. lithophila . . . . . . . 1 1 . . + + . . . + . . . . . . . . . . . . . . . i trisetum flavescens . . . . . . . 1 1 . . . . . . . . . . . . . . 1 + . + . . . . . i i iv i thesium arvense . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 + 1 + i i alyssum strigosum . . + + 1 + . . . . . . . . . . . . . . . . . . . . . . . . . . i festuca rubra subsp. rubra + + . . . . . + 1 . . . . . . . . . . . . . . . . . . . . . . . i erysimum kummerlei + . + + . . . . . . . . . . . . . . . . + . . . . . . . . . . . i i cruciata laevipes + . . . 1 . . . . . . . . . . . . + . . . . . . . . + . . . . . i malva moschata + . . . . . . . . . . . . . . . . . . . . . + . . . + . . . + . i i poa angustifolia + . . . . . . . . . . . . . . . . . . . . . . + 1 1 . . . . . . i i sanguisorba minor . + . . + + + . . . . . . . . . . . . . . . . . . . . . . . . . i iv i ii ii iii galium divaricatum . . + + 1 . + . . . . . . . . . . . . . . . . . . . . . . . . . i iv ii iii bromus squarrosus . . + + + . + . . . . . . . . . . . . . . . . . . . . . . . . . i trifolium alpestre . . . . . 1 . . . . . . . . . . . . . . + . . . . . . . . + + . i ii iii ii ii trifolium velenovskyi . . . . . . . . . + . . . . . . . + . . + . 1 . . . . . . . . . i iii i v briza minor . . . . . . . . . . . . . . . . . . . . . . . . . . . + + + + . i juniperus communis . . . . . . . . . . . . . . . . . . . . . . . . . . . . + + + + i iv veronica chamaedrys + 1 . . + . . . . . . . . . . . . . . . . . . . . . . . . . . . i i i i clinopodium alpinum subsp. meridionale + . + . + . . . . . . . . . . . . . . . . . . . . . . . . . . . i i teucrium chamaedrys . . 1 + 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . i ii i i phleum montanum . . . + 1 + . . . . . . . . . . . . . . . . . . . . . . . . . . i eryngium amethistinum . . . . 1 1 + . . . . . . . . . . . . . . . . . . . . . . . . . i i ranunculus oreophilus . . . . . . . 1 1 + . . . . . . . . . . . . . . . . . . . . . . i asperula aristata subsp. scabra . . . . . . . . 1 . . . . . . . . + . . + . . . . . . . . . . . i iii ornithogalum orthophyllum subsp. kochii . . . . . . . . . . . + + . . . + . . . . . . . . . . . . . . . i i stachys germanica subsp. heldreichi . . . . . . . . . . . . . . . . . . . + . + . . . + . . . . . . i polygala nicaeensis . . . . . . . . . . . . . . . . . . . + . . . . . + + . . . . . i ii polygala vulgaris . . . . . . . . . . . . . . . . . . . + . . . . . . . + . . + . i i ii iv helleborus odorus + . . . + . . . . . . . . . . . . . . . . . . . . . . . . . . . i i schedonorus pratensis + . . . . . . . . . . . . . . . . . . . . . . . . + . . . . . . i medicago lupulina . . + + . . . . . . . . . . . . . . . . . . . . . . . . . . . . i geum montanum . . . . . . . . + . . . . . . . . . . . . . . . + . . . . . . . i carthamus lanatus . . . . . . . . . + . . . . . . . . . . . . . . . . . . . + . . i thymus longicaulis . . . . . . . . . . . . . . . . . . . + + . . . . . . . . . . . i v iv ii v elytrigia intermedia . . . . . . . . . . . . . . . . . . . + . . . . . + . . . . . . i digitalis ferruginea . . . . . . . . . . . . . . . . . . . . . . + . . . + . . . . . i dactylis glomerata + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i b i o l o g i c a n y s s a n a 8 (1 )  s e p te m b e r 2 0 1 7 : 6 1 -7 2 ć u š te re v s k a , r .  a rm e rio ru m e lic a e -p o te n tillio n m ic e v s k i 1 9 7 8 … 6 6 bromopsis cappadocica . . . . 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . i orlaya daucorlaya . . . . + . . . . . . . . . . . . . . . . . . . . . . . . . . . i xeranthemum annuum . . . . + . . . . . . . . . . . . . . . . . . . . . . . . . . . i scandix australis subsp. australis . . . . . + . . . . . . . . . . . . . . . . . . . . . . . . . . i alyssum repens subsp. trychostachyum . . . . . . + . . . . . . . . . . . . . . . . . . . . . . . . . i i orlaya daucoides . . . . . . . . + . . . . . . . . . . . . . . . . . . . . . . . i prunella laciniata . . . . . . . . + . . . . . . . . . . . . . . . . . . . . . . . i i ii odontites luteus . . . . . . . . . . . . . . 1 . . . . . . . . . . . . . . . . . i silene nemoralis . . . . . . . . . . . . . . . + . . . . . . . . . . . . . . . . i cytisus austriacus subsp. heuffelii . . . . . . . . . . . . . . . + . . . . . . . . . . . . . . . . i clinopodium alpinum subsp. hungaricum . . . . . . . . . . . . . . . . . . . + . . . . . . . . . . . . i trifolium tenuifolium . . . . . . . . . . . . . . . . . . . . + . . . . . . . . . . . i delphinium fissum . . . . . . . . . . . . . . . . . . . . . + . . . . . . . . . . i silene bupleuroides subsp. staticifolia . . . . . . . . . . . . . . . . . . . . . + . . . . . . . . . . i gentiana cruciata . . . . . . . . . . . . . . . . . . . . . . . + . . . . . . . . i allium rotundum . . . . . . . . . . . . . . . . . . . . . . . . + . . . . . . . i eryngium campestre . . . . . . . . . . . . . . . . . . . . . . . . + . . . . . . . i v i ii lathyrus nissolia . . . . . . . . . . . . . . . . . . . . . . . . + . . . . . . . i rumex alpestris . . . . . . . . . . . . . . . . . . . . . . . . . . + . . . . . i i brachypodium pinnatum . . . . . . . . . . . . . . . . . . . . . . . . . . . + . . . . i stachys officinalis . . . . . . . . . . . . . . . . . . . . . . . . . . . + . . . . i veronica orbelica . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + . i b i o l o g i c a n y s s a n a 8 (1 )  s e p te m b e r 2 0 1 7 : 6 1 -7 2 ć u š te re v s k a , r .  a rm e rio ru m e lic a e -p o te n tillio n m ic e v s k i 1 9 7 8 … biologica nyssana 8 (1)  september 2017: 61-72 ćušterevska, r.  armerio rumelicae-potentillion micevski 1978… 67 the plots were located within targeted remnant patches of dry grasslands in such a way that edge effects (close forest edge, roads and settlements) were excluded. the sampled 32 relevés and the rest 108 from literature sources were entered into turboveg (h e n n e k e n s & s c h a m i n é e , 2001). classification was made using the pc-ord 4 package (m c c u n e & m e f f o r d , 1999) integrated in juice software, using flexible beta (clustering) and relative euclidean distance (known also as chord distance) as the measure of resemblance among relevés. the table sorting based on this clustering was then performed in juice 7.0 (t i c h ý & h o l t , 2006). diagnostic species of each cluster (interpreted as associations) were defined in juice by calculating the fidelity of each species to each group using the phi coefficient as the fidelity measure (c h y t r ý et al., 2002). the threshold of the phi value was selected at level 0.55. diagnostic species were identified by juice using the phi coefficient greater than 0.6. in order to assist ecological interpretation of the ordinated patterns, average bioindicator values (p i g n a t t i , 2005) for the relevés were calculated based on presence-absence data and overlaid as supplementary environmental data. we used r a u n k i a e r ’ s (1934) system of life forms and we determined the chorological spectra using data of g a j i ć (1980) and p i g n a t t i (2005). the chorological spectrum is presented as percentages of each group of species within the entire species composition. in the analytic table (tab. 1) are presented the results of the cluster analysis and subsequent juice tabular sorting. the syntaxonomic affiliation of taxa other than diagnostic ones was decided upon using expert knowledge and the database of the european vegetation checklist (m u c i n a et al., 2016). the nomenclature follows in principle the flora europaea (t u t i n et al., 1964-1993) however it was adjusted using the med-check list (g r e u t e r et al., 19841989; g r e u t e r et al., 2008) and new taxonomic and nomenclatural findings as featured in the euro+med database (www.emplantbase.org). some taxonomic concepts follow regional floras (h a y e k , 1927, 1931, 1933; j o r d a n o v , 1963-1979; j o s i f o v i ć , 1970-1986; v e l c h e v , 1982-1989; m i c e v s k i , 1985-2005; k o z u h a r o v , 1995; m a t e v s k i , 2010). formation of the names of the new syntaxa follows the international code of phytosociological nomenclature (w e b e r et al., 2000). results and discussion the optimal level of the clustering was determined by the optimclass 1 method. the cluster analysis suggested six well separated clusters (fig. 2). since they show a high degree of floristic (and geographic) integrity, five already described associations were confirmed. while, after the made extensive comparative analysis, relevés of cluster 5, showed the greatest syntaxonomic affinity with ass. euphrasioplantaginetum holostei because of the presence of its characteristic species. fig. 2. graphical representation of the classification results of the armerio rumelicae-potentillion vegetation (flexible beta clustering method, relative euclidean distance). legende: cluster 1: genisto-agrostietum byzanthinae, cluster 2: koelerio-festucetum stojanovii, cluster 3: plantagini subulatae-agrostietum capillaris, cluster 4: diantho-armerietum rumeliacae, cluster 5: euphrasio-plantaginetum holostei subass. festucetosum hirtovaginatae subass. nova., cluster 6: euphrasio-plantaginetum holostei biologica nyssana 8 (1)  september 2017: 61-72 ćušterevska, r.  armerio rumelicae-potentillion micevski 1978… 68 the association euphrasio-plantaginetum holostei has been described and for the first time registered on bistra mountain, developing exclusively on silicate substrate with a thin layer of soil, so that in some places subsoil breaks out on the surface. this association on bistra mountain is differentiated into 3 subassociations subass. typicum, subass. festucetosum nigrescentis (over the village galičnik) and subass. agrostidetosum capilaris (from kičevo side of the mountain bistra, above knežino). in the floristic composition of the vegetation relevés (cluster 5, fig. 2) made in our research on the move asan gjura-dofa, all three characteristic species of the association were registered: euphrasia pectinata, arenaria serpyllifolia and plantago holosteum. none of diagnostic taxa of existing subassociations of the ass. euphrasio-plantaginetum holostei, was registered. but therefore, with analysis "analysis of constancy columns in synoptic table" of the program package juice, were separated taxa over which the community that is developing on the galičica mountain, differs from existing community euphrasioplantaginetum holostei. diagnostic species: achillea seidlii, bupleurum falcatum subsp. cernuum, centaurea deusta, festuca hirtovaginata, hypericum barbatum, rhinanthus minor, verbascum speciosum constant species: anthoxanthum odoratum, euphrasia pectinata, galium verum, pilosella officinarum, plantago lanceolata, thymus sibthorpii dominant species: agrostis castellana, festuca hirtovaginata, festuca valesiaca, plantago holosteum, stipa balcanica because of this, the need for extraction of new subassociation festucetosum hirtovaginatae subass. nova was imposed (holotypus hoc loco: tab. 1/23) (tab. 1). subassociation is named by the species festuca hirtovaginata, which has a dominant presence and define its physiognomy. subass. festucetosum hirtovaginatae, unlike the other subassociations of ass. euphrasio-plantaginetum holostei develope on a limestone substrate, on deep soil, at the move asan gjura-dofa (fig. 3). diagnostic species of subassociation are: achillea seidlii, centaurea deusta, hypericum barbatum, rhinanthus minor, verbascum speciosum, festuca hirtovaginata and bupleurum falcatum subsp. cernuum. as far as the ecological characteristics of this community, subass. festucetosum hirtovaginatae, develops at an altitude of 1455-1500 m, in habitats with very low inclination (most vegetation relevés are made on a completely flat surface, almost without inclination) (app. 1). from the spectrum of life forms (fig. 4), it may be noted that the percentage of hemicryptophytes is 66%, which is due to the dominant presence of representatives of fam. poaceae: agrostis castellana, festuca hirtovaginata, festuca valesiaca, stipa balcanica and others. from the spectrum of floral elements it can be seen that the most common is the eurasian floral element (46%) (fig. 5), while the balkan floral elements are second group by representation (19%), fig. 3. ass. euphrasio-plantaginetum holostei micevski 1994 subass. festucetosum hirtovaginatae subass. nova – asan gjura (galičica mt.) fig. 4. ass. euphrasio-plantaginetum holostei micevski 1994. subass. festucetosum hirtovaginatae subass. nova. – spectrum of life forms biologica nyssana 8 (1)  september 2017: 61-72 ćušterevska, r.  armerio rumelicae-potentillion micevski 1978… 69 with the most dominant balkan-apennine (35%) (fig. 6). using the data from "worldclim", we received the following bioclimatic variables for the researched community: bio1 = annual mean temperature (6,6), bio2 = mean diurnal range (mean of monthly (max temp – min temp)) (9,8), bio3 = isothermality (bio2/bio7) (0,34), bio4 = temperature seasonality (standard deviation *100) (62,9), bio5 = max temperature of warmest month (22,1), bio6 = min temperature of coldest month (-6), bio7 = temperature annual range (bio5–bio6) (28.1), bio8 = mean temperature of wettest quarter (2.6), bio9 = mean temperature of driest quarter (14.7), bio10 = mean temperature of warmest quarter (14.7), bio11 = mean temperature of coldest quarter (-1.4) bio12 = annual precipitation (930), bio13 = precipitation of wettest month (117), bio14 = precipitation the driest month (48), bio15 = precipitation seasonality (coefficient of variation) (27), bio16 = precipitation of wettest quarter (311), bio17 = precipitation of driest quarter (149), bio18 = precipitation of warmest quarter (149), bio19 = precipitation of coldest quarter (272); conclusion this paper represents an oveview of the distribution and syntaxonomic position of the supratemperate and submediterranean, silicicolous perennial grasslands south-central balkan, which are covered by the alliances armerio-potentillion micevski 1978. with numerical analyses it has also been shown that own unpublished data from mountain galičica sintaxonomically have proved that they are closest to the ass. euphrasio-plantaginetum holostei micevski 1994. the fact that species festuca hirtovaginata was significantly dominanted in the reaserched stands led us to distinguish a new subassociation festucetosum hirtovaginatae subass. nova which was confirmed with additional ecological analyses. this study complements the picture of south-centarl balkan supratemperate submediterranean silicicolous perennial grasslands with additional data from the republic of macedonia, but there are still areas that are not sufficietly researched. references braun-blanquet, j. 1936: über die trockenrasengesellschaften des festucion vallesiacae in den ostalpen. berichte der schweizerischen botanischen gesellschaft, 46: 169-189. braun-blanquet, j. 1964: pflanzensoziologie: grundzuge der vegetationskunde. 3. neu bearb. aufl. springer-verlag, 866 pp., wien. braun-blanquet, j., r. molinier, wagner h. 1940: prodrome des groupements végétaux. 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[v, 2, 12, 20, 30, 41, 56, 60, 67, 68] weber, h.e., moravec, j., theurillat, j.p. 2000: international code of phytosociological nomenclature. 3rd edition. journal of vegetation sciences, 11: 739-768. appendix review of metadata. running number, locality, latitude, longitude, sampling date, area, altitude (m), aspect, inclination (%) and cover for all reléves shown in the table 1. 1. from asan gjura to krstec, 41,028498, 20,860192, 25.06.2009, 100, 1498, s, 5, 100; 2. from asan gjura to krstec, 41,028141, 20,863512, 25.06.2009, 100, 1480, nw, 3, 100; 3. from asan gjura to krstec, 41,027695, 20,860687, 25.06.2009, 100, 1480, n, 8, 96; 4. from asan gjura to krstec, 41,027817, 20,861515, 25.06.2009, 100, 1492, nw, 3, 100; 5. from asan gjura to krstec, 41,028471, 20,860517, 25.06.2009, 100, 1499, s, 10, 98; 6. from asan gjura to krstec, 41,025218, 20,866777, 25.06.2009, 100, 1483, sw, 5, 100; 7. from asan gjura to krstec, 41,024437, 20,866423, 25.06.2009, 100, 1467, ne, 3, 100; 8. from sheepfold below krstec to asan gjura, 41,068493, 20,859072, 25.07.2009, 100, 1468, w, 5, 100; 9. from sheepfold below krstec to asan gjura, 41,068493, 20,859072, 25.07.2009, 100, 1462, w, 5, 100; 10. from sheepfold below krstec to asan gjura, 41,067209, 20,858302, 25.07.2009, 100, 1460, 0, 0, 100; 11. from sheepfold below krstec to asan gjura, 41,065362, 20,857134, 25.07.2009, 100, 1455, 0, 0, 100; 12. from sheepfold below krstec to asan gjura, 41,064284, 20,856815, 25.07.2009, 100, 1461, 0, 0, 100; 13. from sheepfold below krstec to asan gjura, 41,062178, 20,856931, 25.07.2009, 100, 1457, 0, 0, 100; 14. from sheepfold below krstec to asan gjura, 41,056869, 20,856586, 25.07.2009, 100, 1455, 0, 0, 100; 15. from sheepfold below krstec to asan gjura, 41,056341, 20,856325, 25.07.2009, 100, 1451, 0, 0, 100; 16. from sheepfold below krstec to asan gjura, 41,053895, 20,855478, 25.07.2009, 100, 1458, 0, 0, 100; 17. from sheepfold below krstec to asan gjura, 41,051082, 20,855352, 25.07.2009, 100, 1460, 0, 0, 100; 18. from asan gjura to dofa, 41,031984, 20,863158, 26.07.2009, 100, 1483, se, 5, 100; 19. from asan gjura to dofa, 41,032361, 20,854084, 26.07.2009, 100, 1480, 0, 0, 100; 20. from asan gjura to dofa, 41,032862, 20,859263, 26.07.2009, 100, 1481, se, 2, 100; 21. from asan gjura to dofa, 41,032597, 20,855601, 26.07.2009, 100, 1481, 0, 0, 100; 22. from asan gjura to dofa, 41,032404, 20,851892, 26.07.2009, 100, 1475, 0, 0, 100; 23. from asan gjura to dofa, 41,033543, 20,848666, 26.07.2009, 100, 1495, 0, 0, 100; 24. from asan gjura to dofa, 41,0366, 20,847866, 26.07.2009, 100, 1481, 0, 0, 100; 25. from asan gjura to dofa, 41,041671, 20,854218, 26.07.2009, 100, 1473, 0, 0, biologica nyssana 8 (1)  september 2017: 61-72 ćušterevska, r.  armerio rumelicae-potentillion micevski 1978… 72 100; 26. from asan gjura to dofa, 41,041567, 20,855939, 26.07.2009, 100, 1468, 0, 0, 100; 27. from asan gjura to dofa, 41,039669, 20,860384, 26.07.2009, 100, 1484, e, 2, 100; 28. from asan gjura to dofa, 41,035231, 20,856573, 29.07.2009, 100, 1480, ne, 3, 100; 29. from asan gjura to dofa, 41,035491, 20,856483, 29.07.2009, 100, 1469, ne, 3, 100; 30. from asan gjura to dofa, 41,036381, 20,856884, 29.07.2009, 100, 1472, e, 8, 100; 31. from asan gjura to dofa, 41,037793, 20,857846, 29.07.2009, 100, 1467, ne, 5, 100; 32. from asan gjura to dofa, 41,038896, 20,856981, 29.07.2009, 100, 1462, ne, 3, 100; stojković piperac, m. et al.  the application of the abundance/biomas... biologica nyssana 6 (1)  september 2015: 25-32 stojković-piperac, m. et al.  the application of the abundance/biomas... 25 original article received: 24 june 2015 revised: 28 june 2015 accepted: 01 july 2015 the application of the abundance/biomass comparison method on riverine fish assemblages: limits of use in lotic systems milica stojković piperac1*, djuradj milošević1, vladica simić2 1university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia 2university of kragujevac, faculty of sciences and mathematics, department of biology and ecology, radoja domanovića 12, 34000 kragujevac, serbia * e-mail: milicas@pmf.ni.ac.rs abstract: stojković piperac, m., milošević, dj., simić, v.: the application of the abundance/biomass comparison method on riverine fish assemblages: limits of use in lotic systems. biologica nyssana, 6 (1), september 2015: 25-32. fish assemblages have been widely used as ecological indicators for assessing the level of environmental degradation and ecosystem health. environmental disturbances affect the aquatic community structure in terms of abundance and biomass. therefore, we tested the utility of abundance/biomass comparison (abc) method, originally developed for marine ecosystems, to detect the anthropogenic disturbance in lotic systems using fish community data. electrofishing was conducted in the period between 2003 and 2011 at 35 sites along the southern morava river basin. the results indicated that species richness strongly influences the utility of abc method to detect the anthropogenic disturbance in lotic systems. the warwick (w) statistic showed the positive correlation and the expected direction of response with some factors defining environmental quality, applying it on the samples with greater species richness. this approach has significant power for detecting environmental quality disturbance but may be limited due to effects of habitat variability in riverine environments. key words: abundance/biomass comparison method, lotic systems, fish community, pca analysis, southern morava river basin apstrakt: stojković piperac, m., milošević, đ., simić, v.: primena metode poređenja abundance i biomase na zajednicu riba tekućih voda: ograničenja prilikom upotrebe u lotičkim ekosistemima. biologica nyssana, 6 (1), septembar 2015: 25-32. ribe predstavljaju dobro poznate ekološke indikatore u proceni kvaliteta životne sredine. poznato je da narušavanje sredine utiče na strukturu akvatičnih zajednica u smislu abundance i biomase. zbog toga, u ovom radu testirana je korisnost metode poređenja abudance i biomase (abc method, eng. abundance/biomass comparison method), inicijalno predviđene za primenu nad marinskim ekosistema, u detekciji antropogeno izazvanih promena u lotičkim ekosistemima korišćenjem podataka o zajednici riba. procedura elektroribolova sprovedena je u periodu od 2003 do 2011 godine na 35 lokaliteta raspoređenih duž sliva južne morave. rezultati ove studije ukazuju da bogatstvo vrsta u značajnoj meri utiče na korisnost 6 (1) • september 2015: 25-32 biologica nyssana 6 (1)  september 2015: 25-32 stojković piperac, m. et al.  the application of the abundance/biomas... 26 ispitivane metode u proceni intenziteta antropogenog delovanja na lotičke ekosisteme. warwick-ova (w) statistika pokazuje pozitivnu korelaciju sa faktorima koji definišu kvalitet sredine, kao i očekivani odgovor na stres, samo prilikom primene nad lokalitetima sa većim brojem vrsta. pokazano je da ova metoda ima značajnu moć u detekciji narušenja životne sredine ali sa izvesnim ograničenjima izazvanim prirodnom varijabilnošću u lotičkim ekosistemima. key words: metoda poređenja abundance i biomase, lotički sistemi, zajednice riba, pca analiza, sliv južne morave introduction anthropogenic modifications of natural hydrology have been altering freshwater ecosystems worldwide, threatening their ecological integrity (r e h a g e & t r e x l e r , 2006). anthropogenic alteration may change the hydrological patterns of the river basin (w a r d , 1998) and modify the physical characteristics of the aquatic habitat (k a r r , 1981), strongly influencing the structure and composition of the aquatic biota. the major human activities with negative impacts on aquatic communities include: human impact on habitat morphology, intensive agriculture and urbanization, construction of channels and dams, removal of snags, and pollution (j o h n s o n et al., 1995; s p a r k s , 1995; r i c h t e r et al., 1997; e i t z m a n n & p a u k e r t , 2010). considering the fish fauna, it is also necessary to emphasise the possible changes caused by over-fishing by both anglers and poachers as an important factor that may dramatically diminish fish populations (p e n c z a k & k r u k , 1999; a n t i c a m a r a et al., 2010). however, the major threat to freshwater fish is modification of aquatic environment, which may cause decline in many species (c o l l a r e s p e r e i r a & c o w x , 2004). assessing fish community structure is one of the efficient ways of evaluating the biotic integrity in rivers in different parts of the world ( k a r r , 1981; o b e r d o r f f & h u g h e s , 1992; h u g u e n y et al., 1996; g a n a s a n & h u g h e s , 1998; b r e i n e et al., 2004; s t o j k o v i ć et al, 2014). changes in fish community composition are an important factor used to characterize environmental quality, as fishes respond to changes in the aquatic environment with great sensitivity. they play an important role in aquatic ecosystems due to their dependence on both, the physical features of their environment and the other forms of aquatic life. therefore, the health of each fish assemblage reveals conditions present in the entire aquatic community (f o l t z , 1982). fig. 1. map of the sampling sites under investigation. site codes for studied streams are the same as in table 1. biologica nyssana 6 (1)  september 2015: 25-32 stojković piperac, m. et al.  the application of the abundance/biomas... 27 environmental disturbances also affect the aquatic community structure in terms of abundance and biomass, both measured by the abundance/biomass comparison (abc) method. the abc method was initially applied on marine macrobenthic communities to detect influence of the anthropogenic activities such as pollution (w a r w i c k , 1986). in addition, many studies conducted by now, stressed that this method could be also applicable to detect the effect of anthropogenic changes on freshwater biota caused by pollution (c o e c k et al., 1993), industrial plant impact (pinto et al., 2006), over-fishing (p e n c z a k & k r u k , 1999) and water and habitat disturbance (c a s a t t i et al., 2006). however, p e n c z a k & k r u k (1999) proposed a threshold regarding a minimal number of species caught in the sample when applying abc method on riverine fish assemblages. bearing all this in mind, we here aimed to test the performance of the abc method in assessing the changes in the fish community as a response to environmental degradation in lotic systems. furthermore, we tested how species richness influences the utility of abc method to detect the anthropogenic disturbance in riverine environments. material and methods study area the source of southern morava river, also known as binačka morava, is in the skopska crna gora mountains, macedonia. this river flows in the roughly northerly direction. at the 49th kilometer it coalesces with the preševska moravica and at 295th kilometer it discharges in the morava, tributary of danube and therefore part of the black sea catchment area. the southern morava river has a catchment area of 15,469 km2, of which 14,372 km2 (92.91%) are in serbia and 1,097 km2 (7.09%) in bulgaria through its right-hand tributary the river nišava. it has 157 tributaries but most of them dry out during summer. larger, permanent left-hand tributaries are jablanica, veternica, toplica and pusta reka rivers. right-hand tributaries include vlasina, nišava (the longest) and sokobanjska moravica rivers (g a v r i l o v i c & d u k i c , 2002). sampling fish fauna was sampled along the southern morava river basin in the period between 2003 and 2011. during the investigated period, out of total number of 35 sampling sites (fig. 1), 12 were sampled ones, 18 twice, 2 three and 3 four times. since each sampling occasion was considered as separate entity in data processing, the final data matrix was consisted of 66 samples. the electrofishing procedure was conducted using the dc electrofisher “aquatech” ig 1300 (2.6 kw, 80– 470 v). a more detailed sampling procedure is described in s t o j k o v i c et al. (2013). together with the fish data collection, water and habitat quality variables were measured for each sample in order to characterize the extent of stress. water quality was expressed by five variables strongly dependent on anthropogenic disturbance (dissolved oxygen (do), electro-conductivity (ec), concentrations of ammonia nitrogen (nh4), nitrate nitrogen (no3) and orthophosphates (po4)). dissolved oxygen and electro-conductivity were estimated by a wtw multi 340i probe, while the concentrations of ammonia nitrogen, nitrate nitrogen and orthophosphates were measured using the spectrophotometer shimatzu uv–vis. habitat quality was presented by three disturbance variables: hydrological alteration (ha), channel alteration (ca) and land use intensity (lu). each site was given a score of 1, 3, or 5 if slight, moderate, or severe alteration for each habitat disturbance variable was observed (s t o j k o v i ć et al., 2014). data analysis to test how species richness influences the output of the abc method, two data matrix have been constructed. the first data matrix (a) contained all samples collected during the sampling period, which finally counts 66 samples, regardless of the number of species caught. in contrast, the second data matrix (b) was constructed to contain only samples where species richness was greater than 5, as suggested by p e n c z a k & k r u k (1999), finally presenting 28 samples. according to w a r w i c k (1986), condition of an aquatic community can be illustrated by using combined k-dominance plots of abundance and biomass, where species are ranked in the order of importance on the x-axis (logarithmic scale) with percentage dominance on the y-scale (cumulative scale). the area between the two curves is called the warwick or w statistic, calculated according to the following formula (c l a r k e , 1990). w =       150 1 11     s ajbj s j i j i j , where w is the standardized sum of the differences between each pair of species’ cumulative biomass and cumulative abundance value ranked in decreasing order. the curves    i j bj 1    i j aj 1 biologica nyssana 6 (1)  september 2015: 25-32 stojković piperac, m. et al.  the application of the abundance/biomas... 28 classify the quality of the environment (m a g u r r a n , 2004) either as undisturbed (biomass curve overlaps that of abundance, w > 0) or disturbed (abundance curve overlaps that of biomass, w < 0). w ranges from -1 to +1. w statistic was calculated using the primer v6 (c l a r k e & g o r l e y , 2006), a multivariate statistical package developed at plymouth marine laboratory. since some variables in this study were presented as ordinal, categorical principal components analysis (catpca) was used to reveal the relationship between w statistic and disturbance variables. the data matrix were consisted of 9 variables, out of with 6 were numerical and 3 categorical (given in columns), measured along the 66 and 28 samples (given in rows), depending on the data matrix used in the analysis. the catpca analysis was performed using software spss version 15.0 (spss inc, chicago, il, usa). results dominance of abundance or biomass was calculated for all 66 samples. the w statistics values, observed at the majority of sites, were positive, with an exception of a few sites, mainly situated on upper river courses (tab. 1). according to this, the abc method indicated that the majority of sites were undisturbed, as represented by positive values of w statistic. nevertheless, there were several sites having the positive values the w statistic but close to zero, where both curves roughly coincided, indicating a moderately disturbed condition. table 1. description of code, stream order and w statistic value for each sample collected along the southern morava river basin. after the each site code, abbreviation for year of sampling is included river code stream order w statistic river code stream order w statistic southern morava *1-03 2 0.428 visočica ntv1-08 1 0.109 southern morava *1-10 2 -0.031 visočica ntv1-10 1 0.536 southern morava *2-03 2 0.247 visočica ntv1-11 1 0.501 southern morava *2-08 2 0.094 visočica ntv2-06 2 0.308 southern morava *3-03 3 -0.124 visočica ntv2-10 2 -0.126 southern morava *4-03 3 0.111 dojkinačka reka ntvd-08 1 0.000 southern morava *4-08 3 0.122 jelovička reka ntvj-08 1 0.280 southern morava *5-03 4 0.090 jerma nj1-10 1 0.109 southern morava *5-08 4 0.176 jerma nj1-11 1 -0.158 southern morava *6-10 4 0.200 jerma nj2-10 1 0.380 jablanica jb1-03 2 0.217 jerma nj2-11 1 0.143 jablanica jb2-03 2 0.088 pusta reka p1-10 2 0.153 sokobanjska moravica m1-10 1 -0.020 pusta reka p2-03 2 0.232 sokobanjska moravica m2-08 1 0.053 pusta reka p2-10 2 0.634 sokobanjska moravica m3-08 1 0.162 toplica t1-03 1 0.175 sokobanjska moravica m3-10 1 0.334 toplica t1-10 1 -0.205 nišava n1-10 2 0.120 toplica t2-08 2 0.180 nišava n1-11 2 0.081 toplica t2-10 2 0.256 nišava n2-10 3 0.019 toplica t3-03 2 0.144 nišava n2-11 3 0.139 toplica t3-08 2 0.284 nišava n3-03 3 0.197 toplica t4-03 2 0.201 nišava n3-08 3 0.180 toplica t4-10 2 0.257 nišava n3-10 3 0.299 banjska reka tb-08 1 0.223 nišava n3-11 3 0.242 lukovska reka tl-08 1 -0.133 nišava n4-08 3 0.022 lukovska reka tl-10 1 0.007 nišava n4-10 3 0.160 veternica ve1-10 2 0.070 nišava n4-11 2 0.253 vlasina vl1-03 1 0.158 temska nt1-03 1 -0.160 vlasina vl1-04 1 0.000 temska nt1-08 1 -0.109 vlasina vl1-08 1 0.000 temska nt1-10 1 -0.433 vlasina vl1-10 1 -0.739 temska nt1-11 1 -0.068 vlasina vl2-10 1 0.165 temska nt2-10 2 0.264 vlasina vl3-03 2 0.291 temska nt2-11 2 -0.128 vlasina vl3-08 2 0.030 biologica nyssana 6 (1)  september 2015: 25-32 stojković-piperac, m. et al.  the application of the abundance/biomas... 29 table 2. contributions of the w statistic and water and habitat quality variables to the two fist axis of the catpca and the total variance accounted for (vaf) for a) data matrix a, b) data matrix b component loadings total vaf component loadings total vaf a) axis b) axis 1 2 1 2 w -0.431 0.230 0.189 w -0.397 0.490 0.288 no3 -0.860 -0.146 0.433 no3 0.842 0.219 0.486 po4 -0.827 -0.152 0.430 po4 0.822 0.391 0.509 nh4 -0.840 0.085 0.379 nh4 0.757 -0.193 0.380 ec -0.722 -0.335 0.913 ec 0.518 0.633 1.000 do 0.893 0.064 0.462 do 0.875 -0.064 0.478 ha -0.030 0.949 0.468 ha -0.004 -0.790 0.323 ca -0.857 0.256 0.436 ca 0.953 -0.328 0.466 lu -0.844 0.157 0.377 lu 0.764 -0.352 0.449 fig. 2. catpca plots showing relationships between w statistic and stressor gradient based on a) data matrix a and b) data matrix b the catpca analysis, conducted under the data matrix a, extracted the first and the second dimensions, which explained 56.45% and 13.46% of the total variance between 66 samples, respectively. the first axis was associated with all factors, excluding factor ha. also, the first axis had high loading for all factors except for w (tab. 2, fig. 2a). the catpca plot a indicated that w statistic is positively correlated with some factors associated with the first axis that define water and habitat quality and negatively correlated with do. according to this result, positive and high values of w statistic occur when the water and habitat quality parameters indicate stress condition (fig. 2a). on the other side, in the catpca result of the data set b, the first and second axis accounted for 49.82% and 19.31% of the observed variation, respectively (fig. 2b). the catpca plot b showed that values of w statistic increase when high values of do and the decrease of water and habitat quality variables were observed. discussion the w statistics values, according to the catpca plot a, showed correlation with factors defining stress condition but in the opposite than expected direction (fig.2a). such a result could be explained as a consequence of a low number of species found at some particular sites situated on upstream reaches (p e n c z a k & k r u k , 1999) which caused unexpected negative values of w statistic. despite the premise that the undisturbed water courses should be characterized by greater species richness and diversity indices (a r g e n t et al., 2003; g a f n y et al., 2000), fish richness and biologica nyssana 6 (1)  september 2015: 25-32 stojković piperac, m. et al.  the application of the abundance/biomas... 30 diversity are strongly influenced by stream order and longitudinal heterogeneity (p l a t t s , 1979; b a r i l a et al., 1981). longitudinal zonation in stream fish assemblages revealed that species diversity increases from upstream to downstream areas due to the greater variety of habitat diversity (g o r m a n & k a r r , 1978; f o l t z , 1982; b a i n et al., 1988). likewise, as stream order increases, the richness and total number of fish specimens also increase (p l a t t s , 1979; t h o m a s & h a y e s , 2006). similar problem was detected when using the taxonomic distinctness index applied on freshwater fish (c l a r k e & w a r w i c k , 1998; b h a t & m a g u r r a n , 2006). b h a t & m a g u r r a n (2006) found that the sites on the first and second stream order fell below the confidence limits, indicating false environmental disturbance. explanation provided in that study suggested that this may be caused by the fact that total species richness and taxonomic distinctness increase with stream order (b h a t , 2004), as downstream areas contained several upstream species as well as species unique to downstream area, whereas upstream sites tended to include closely related species. another factor considered necessary to emphasize is that the total fish biomass decrease in the upstream direction (s c h l o s s e r , 1991; t h i e l et al., 1995; h i c k s , 2003) which was also reflected on negative values of w statistic at the upper river courses. in contrast, considering the second catpca plot, where upper river courses were excluded from analysis, w statistic showed the expected direction of response to the extent of human alteration. this result unambiguously confirms presumed obstacles in applying abc method on riverine fish assemblages, which are mainly caused by natural longitudinal heterogeneity in lotic systems. consequently, we believe that abc method may be a promising tool for characterization of lotic systems under stress conditions, but may be limited to the sites with greater species richness. conclusion the results of this study have shown limited effectiveness of the abc method in pinpointing effects of the disturbance at upstream sites, since the resulting values of w statistic were highly confusing and not aligned with the real picture of the fish fauna. in some instances, the real situation in the fish community was presumably hidden by influences of longitudinal heterogeneity (b h a t & m a g u r r a n , 2006). however, we stressed that this approach has significant power for detecting environmental quality disturbance but may be limited due to effects of habitat variability in 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antimicrobial activity and cytotoxicity of commercial rosemary essential oil (rosmarinus officinalis l.) zorica stojanović-radić1*, marija nešić1, ljiljana čomić2, niko radulović3 1 university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, niš, serbia 2department of biology and ecology, faculty of sciences, university of kragujevac, radoja domanovića 12, kragujevac, serbia 3 university of niš, faculty of sciences and mathematics, department of chemistry, ćirila i metodija 2, niš, serbia * e-mail: lab3zmm@yahoo.com abstract: stojanović-radić, z., nešić, m., čomić, lj., radulović, n.: antimicrobial activity and cytotoxicity of commercial rosemary oil (rosmarinus officinalis l.). biologica nyssana, 1 (1-2), december 2010: 83-88. rosemary is well known as a spice and widely used plant in ethnomedicine worldwide. in this paper, commercial essential oil of rosemary was tested for antimicrobial and cytotoxic activity together with its effect on germination. antimicrobial activity testing showed moderate effect to both g-positive and gnegative bacteria. in order to determine its effect to the cell membrane, spectrophotometric analysis was performed. it was determined that rosemary affects the cell membrane of bacteria. cytotoxic activity of rosmarinus officinalis essential oil had been evaluated. as a plant object, germinative bulbs of allium cepa were used. cytotoxic activity that corresponded to the concentration of essential oil was determined. it had been noticed that rosemary essential oil affected mitotic phase i.e. it significantly slowed down the mitosis. also, investigation of rosemary essential oil's activity to germination was performed. it was determined that it had high effect to the germination. concentration of 5 mg/ml completely inhibited the germination of triticum vulgare. key words: essential oil, cytotoxicity, antimicrobial activity, effect on germination introduction ! essential oils are natural, concentrated, volatile aromatic compounds isolated from plants. these compounds posses a wide spectrum of pharmacological activities. the main advantage of natural agents is that they do not enhance the “antibiotic resistance”, a phenomenon encountered with the long term use of synthetic antibiotics. rosemary (rosmarinus officinalis l.) is plant belonging to the family labiateae. this plant is widely used for many purposes – it is well known as a culinary spice and frequently employed in the practice of aromatherapy (d a v i d s o n et al., 1989). also, it is used in ethnomedicine as general stimulant, for improvement of circulation, treatment of rheumatic pains, hyperglycemia and skin care (h a m e d o , 2009). rosemary is known to have antioxidant and antibacterial properties (s h a h i d i et al. 1992). p u t n a m et al. (2006) reported that rosemary essential oil inhibit osteoclast activity and increase bone density in vitro. also, cytotoxic activity of rosemary essential oil has been demonstrated by several authors (k h a f a g i et al. 2000; e l m e l e i g y et al. 2010). the essential oil of rosemary contains several compounds in high percentage and many others in traces. these major compounds determine the biological properties of the essential oil and can act 10th sfses • 17-20 june 2010, vlasina lake1 (1-2) • december 2010: 83-88 biologica nyssana 1 (1-2) december 2010: 83-88 stojanović-radić, z. et al. antimicrobial activity and cytotoxicity of… 84 in synergic manner or regulate one on another (f a i x o v a & f a i x , 2008). in the papers dealing with chemical composition of rosemary essential oil, α-pinene is reported as the major component, followed by 1,8 – cineole, camphene, β-myrcene, camphor and borneole (j a m s h i d i et al., 2009, m o g h t a d e r & a f z a l , 2009). some studies reported 1,8 – cineole as main component (f u et al., 2007, d e b e r s a c et al., 2001). rosemary is confirmed as antimicrobial agent against both gram-positive and gram-negative bacteria, as well as against fungi (v a l e r o & s a l m e r o n , 2003, p r a b u s e e n i v a s a n et al., 2006, f u et al. 2007a, m o g h t a d e r & a f z a l , 2009). in the present study, antimicrobial activity of commercially available rosemary essential oil was investigated against a panel of microorganisms for minimal inhibitory and bactericidal activity. mic and mbc were determined using broth microdilution method. the most sensitive determined bacterial strain was used to investigate the mode of action of this oil. in order to evaluate if the oil exhibits membrane damage effect, testing of the 260 nm absorbing material leakage was employed. also, cytotoxic activity of rosemary essential oil was determined. material and methods ! essential oil, bacterial strains and culture conditions essential oil of rosemary, produced by sinefarm, vršac, srbija, was purchased from the local pharmacy. bacterial strains used in this study were staphylococcus aureus atcc 25923, s. aureus (clinical isolate), escherichia coli (clinical isolate), bacillus subtilis atcc 6633 and klebsiella pneumoniae (clinical isolate). bacterial cultures were maintained on the nutrient agar (na) at the department of biology and ecology, microbiological laboratory, faculty of science and mathematics, niš. testing of antimicrobial activity the antimicrobial activity was evaluated using broth microdilution method (n c c l s , 2003). minimum inhibitory concentrations determination was performed by a serial dilution method in 96 well microtitre plates. bacterial strains were cultivated in mueller hinton agar at 37 ºc for 18 h. cultures were used for making bacterial suspensions, whose turbidity was adjusted to 0.5 mcfarland and confirmed using a spectrophotometer (uv-vis 1650 shimatzu, japan). final density of inoculum was 5 x 105. dilutions of stock rosemary essential oil solutions were made with mhb (mueller hinton broth) and inoculums were added to all wells. one inoculated well was included to allow control of the broth suitability for organism growth. one non-inoculated well, free of antimicrobial agents, was also included to ensure medium sterility. bacterial growth was determined by adding 20 µl of 0.5 % triphenyl tetrazolium chloride (ttc) aqueous solution (s a r t o r a t t o et al., 2004). minimal inhibitory concentration (mic) was defined as the lowest concentration of the oil inhibiting visible growth (red colored pellet on the bottom of the wells after the addition of ttc), while minimal bactericidal concentration (mbc) was defined as the lowest oil concentration killing 99.9 % of bacterial cells. to determine mbc, the broth was taken from each well without visible growth and inoculated in mha for 24 h at 37ºc. experiments were done in triplicate and the mean values are presented. testing the integrity of the cell membrane very important indication of disturbed membrane integrity is the release of intracellular components, such as large molecules dna and rna. since these nucleotides have strong uv absorption at 260 nm, they are described as 260 nm absorbing materials (l i u et al., 2009). leakage of 260 nm absorbing material was determined by measuring the absorbance of the culture supernatants according to the modified method of c a r s o n et al. (2002). briefly, the cultures of s. aureus were harvested and twice washed with pbs. after resuspension in pbs, cells were adjusted to absorbance of ~ 0.2. at time t = 0 min, essential oil was added to cell suspensions to give a final concentrations of mic and 2 x mic. aliquots of the treated cell suspensions were removed at regular time intervals, centrifuged at 10 000 x g for 5 min and absorbance of supernatant was measured at 260 nm and plotted against time. the experiments were done in triplicate and the mean values are presented. allium cepa test cytotoxicity was determined by calculating the mitotic index of allium cepa l. root cells after the 24 h treatment with rosemary essential oil. small onion bulbs were placed in 10 ml distilled water in petri dishes in an incubator at 25±1ºc for 24 h. after initiating growth, water had been replaced two more times (after every 24h). after 72 h, water had biologica nyssana 1 (1-2) december 2010: 83-88 stojanović-radić, z. et al. antimicrobial activity and cytotoxicity of… 85 been replaced with appropriate solution (appropriate concentration of essential oil of rosemary). distilled water and 5 % ethyl alcohol were used as controls. after the growth, root tips were collected and fixed. the fixative solution was glacial acetic acid / absolute alcohol (1/3 v/v). the root tips were kept in acetic-alcohol solution for 72 h. after fixation, the root tips were washed with water and then placed into a 70 % ethanol and stored in a refrigerator until use. for examination the root tips were hydrolyzed in 1n hcl, placed on microscopic slide in 1% acetoorcein and after a few minutes squashed. for each root tip the number of mitotic and total meristemic cells was counted in 9 fields using power (100 x) light microscope. cells manifesting different stages of mitosis i.e. interphase and prophase (p), metaphase (m), anaphase (a) and telophase (t) were recorded. the mitotic index (mi) was calculated using following formula: mitotic index = (p+m+a+t) / total cells germination test three different concentrations of essential oil were used to determine whether the oil has effect to germination of triticum vulgare seeds. twenty wheat grains were placed in small petri dishes and in an appropriate medium. distilled water and 5 % ethanol were used as controls. concentrations of rosemary essential oil were 5 mg/ml, 12.5 mg/ml, 25 mg/ml and 50 mg/ml. after 24 h of the treatment, seeds which germinated were counted and percentage of germination, compared to positive control was presented. results and disscusion the results of minimal inhibitory (mic) and minimal bactericidal concentrations (mbc) are presented in table 1. the testing showed significant antibacterial activity of rosemary with mic values ranging from 1.562 – 3.125 μl/ml, while mbc ranged from 1.562 to >25.00 μl/ml. the most sensitive strain was s. aureus with both mic and mbc values of 1.562 μl/ml, while the most resistant one was escherichia coli, whose bactericidal concentration exceeded the tested values. determined resistance of e. coli can be attributed to the outer cell envelope, the feature of all gramnegative bacteria, which makes them generally more resistant to all external agents. this is confirmed by higher mbc values of k. pneumoniae (gram negative strain). f u et al. (2007a) investigated antibacterial activity of rosemary and the results obtained in our study showed very good agreement with activity against e. coli, b. subtilis and s. aureus (1.250 μl/ml against all three bacteria) determined in that paper. m o g h t a d e r & a f z a l i (2009) reported activity of rosemary against both gram positive and negative strains. previous investigation of f u et al. (2007) reported activity of rosemary essential oil against propionibacterium acnes in the concentration of 0.56 mg/ml. in order to determine the mode of action of rosemary essential oil against the most sensitive of the tested bacteria, staphylococcus aureus atcc 25923, spectrophotometric method was employed. since damaged cell membrane increases its permeability, small molecular weight molecules such as phosphates and potassium ions first leach out from the cell, followed by larger molecules, such as dna and rna (c h e n et al, 2002). since these nucleotides have strong uv absorption at 260 nm, they are described as “260 nm absorbing materials”. the uv-vis study on the release of materials absorbing at 260 nm showed significant leakage proportional to the oil concentration (fig.1). on contrary, control did not showed any releasing of this material during 120 min period. this indicates membrane damage related to the addition of the rosemary essential oil. for testing of cytotoxicity, onion meristems (allium cepa l.) were used as the study material. they are considered as one of the best biological models for the study of cytotoxicity, genotoxicity and environmental pollutants (f i s k e s j o, 1985). allium cepa root meristems have been widely used table 1. minimal inhibitory concentrations (mic) and minimal bactericidal concentrations (mbc) of rosemary essential oil bacterial strain strain type mic (μl/ml) mbc (μl/ml) tetracycline (μg/ml) s. aureus atcc 25923 1.562 1.562 0.090 s. aureus clinical isolate 3.125 6.250 0.045 e. coli clinical isolate 3.125 >25.00 0.045 b. subtilis atcc 6633 1.562 6.250 0.090 k. pneumoniae clinical isolate 1.562 12.50 0.045 biologica nyssana 1 (1-2) december 2010: 83-88 stojanović-radić, z. et al. antimicrobial activity and cytotoxicity of… 86 for the evaluation of cytotoxicity and anti-mitotic activity of various compounds (s h e h a b , 1980; w i l l i a m s & o m o h , 1996; a l m e s h a l , 1987). in the present paper, cytotoxicity and antimitotic activity of essential oil of r. officinalis l. was tested using allium cepa test. the inhibitory effect of essential oil of rosemary on the mitotic activity of root meristems was evaluated and the effect was compared with distilled water and 5% ethyl alcohol. the results are presented in tab. 2. 0 20 40 60 80 100 120 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 ap so rb an ce a t 2 60 n m time (min) mic 2xmic control figure 1. release of 260 nm absorbing material from s. aureus atcc 25923 cells during the treatment with mic and 2 x mic concentrations of rosemary essential oil (od – optical density) table 2. mitotic index in allium cepa (l.) meristem following incubation with various concentrations of essential oil of rosmarinus officinalis (l.) concentration unit mi mi in % d h2o 0.424 42.40 etoh % 0.172 17.20 5.00 mg/ml 0.257 25.70 12.5 mg/ml 0.235 23.50 25.0 mg/ml 0.218 21.80 50.0 mg/ml 0.191 19.10 the results showed that different concentrations of essential oil have different percentage of activity, but all of them affect root growth, length and number. also, it was demonstrated that essential oil has inhibitory effect on proliferation of cells. higher concentrations of essential oil exhibit stronger inhibitory effect on mitosis (tab. 2). previous studies revealed that rosemary essential oil exhibits strong cytotoxicity against brine shrimp nauplii (k h a f a g i et al. 2000). the test which was taken is not only used for predicting cytotoxicity, but is also used as a predictor of antitumor and pesticide activity. data showed that rosemary essential oil except high cytotoxicity, might posses antitumor and pesticide potential. it is supposed that oxygenated monoterpenes of essential oil exhibit a variable degree of cytotoxicity. as a typical lipophilic substance, it passes through the cell wall and cytoplasm membranes and disrupts their structure (f a i x o v a & f a i x , 2008). k i v a n c & a k g ü l (1988) reported that the most active components of the essential oils were phenols, followed by aldehydes and ketons. one of the major compound of rosemary essential oil is 1, 8-cineole. a s a n o v a et al. (2003) demonstrated that this component has very strong cytotoxicity and pronounced antitumor action. it is possible that this component has the major role in cytotoxicity of this essential oil. 1, 8 cineole exhibits a strong toxic effect on eukaryotic cells (o b e n g o f o r i et al., 1997, s a n t o s et al., 2004). another potent compound is ß-caryophyllene. it was demonstrated that this substance may not have direct influence on cytotoxicity of essential oil, but may impact accumulation of some substances by increasing permeability of the plasma-membrane. in that way, it affects and increases cytotoxic effect of compounds with which it interacts (l e g a u l e & p i c h e t t e , 2007). according to all this facts, it is not surprising that concentration of only 5 mg/ml of rosemary essential oil totally inhibited germination of wheat. this was the lowest concentration tested. other concentrations also totally inhibited germination of triticum vulgare seeds. limonene, one of the components of rosemary essential oil, has ability to moderate proliferation of cells (m a n u e l e et al., 2008), and in that manner it affects cell mitosis. conclusion essential oil of rosemary was previously reported as an important antimicrobial agent with activity at very low concentrations. our study confirmed the previous results regarding mic and mbc values of this essential oil. for the first time, it was determined that rosemary essential oil exhibits activity by damaging the cell membrane of gram positive bacteria and further tests are necessary to explore the exact mechanism of this activity. the determined cytotoxic and antimicrobial activity of rosemary essential oil is of great importance in preservation of food products and also very important information for its pharmacological application. biologica nyssana 1 (1-2) december 2010: 83-88 stojanović-radić, z. et al. antimicrobial activity and cytotoxicity of… 87 references al-meshal i.a. 1987: mitodepressive effect of (-)cathinone, from caltha edulis (khat), on the meristematic region of allium cepa (l.) root tips. toxicon, 25 (4): 451-454. asanova, zh.k., suleimenov, e.m., atazhanova, g.a., dembitskii, a.d., pak, r.n., dar, a., adekenov, s.m. 2003: biological activity of 1, 8-cineole from levant wormwood. pharmaceutical chemistry journal, 37 (1): 2830. carson, f.c., mee, b.j., riley, t.v. 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cytologia (tokyo), 45 (1-2): 57-64. valero, m. & m.c. salmeron 2003: antibacterial activity of 11 essential oil against bacillus cereus in tyndallized carrot broth. international journal of food microbiology, 85 (1-2): 73–81. williams g.o. & l.e. omoh 1996: mitotic effects of the aqueous leaf extract of cymbopogon citratus in allium cepa (l.) root tips. cytobios, 87: 161-168. zaaboubi et al. 2025, biologica nyssana 16(2) 16 (2) december 2025: doi: 10.46793/biolnyss.16.2.9ad pcr-based investigation on transgenic maize status in corn processed products original article elham ashrafi-dehkordi nutrition research center and department of food hygiene and quality control, school of nutrition and food sciences, shiraz university of medical sciences, shiraz, iran elhamashrafi@gmail.com (corresponding author) mohsen shahdadi nutrition research center and department of food hygiene and quality control, school of nutrition and food sciences, shiraz university of medical sciences, shiraz, iran enayat berizi nutrition research center and department of food hygiene and quality control, school of nutrition and food sciences, shiraz university of medical sciences, shiraz, iran siavash babajafari nutrition research center and department of food hygiene and quality control, school of nutrition and food sciences, shiraz university of medical sciences, shiraz, iran seyed mohammad mazloomi nutrition research center and department of food hygiene and quality control, school of nutrition and food sciences, shiraz university of medical sciences, shiraz, iran received: june 09, 2025 revised: august 23, 2025 accepted: august 23, 2025 abstract: monitoring genetically modified organisms (gmos) is increasingly required by law in countries with gmo labeling regulations. efficient extraction of high-quality genomic dna is essential for detecting and quantifying gm content in foods using pcr techniques. this study developed and evaluated various dna extraction methods from corn seeds and products to ensure sufficient dna yield free of contaminants and pcr inhibitors. thirty samples, including corn seeds and derived products, were randomly purchased from supermarkets and grocery stores. genomic dna was extracted by different methods, and pcr was used to detect gmo-specific regions: nosterminator, 35s promoter, and epsps. the zein gene was also amplified to confirm the presence of the maize genome in all samples. results indicated that 14 samples contained the nos-terminator, 13 the 35s promoter, and one the epsps region, identifying 17 (57%) samples as transgenic. the methods proved effective for extracting quality dna suitable for gmo detection. many gm-positive products lacked labeling, underscoring the need for reliable detection methods and stricter labeling enforcement. key words: genetically modified organism, labeling, maize, pcr apstrakt: ispitivanje prisustva transgenog kukuruza u prerađenim proizvodima od kukuruza zasnovano na pcr metodi praćenje genetički modifikovanih organizama (gmo) sve je češće zakonski zahtevano u zemljama koje imaju propise o označavanju gmo proizvoda. efikasna ekstrakcija genomske dnk visokog kvaliteta od suštinskog je značaja za detekciju i kvantifikaciju gmo sadržaja u hrani primenom pcr tehnika. u ovom radu razvijene su i procenjene različite metode ekstrakcije dnk iz zrna kukuruza i proizvoda od kukuruza, kako bi se obezbedio dovoljan prinos dnk bez prisustva kontaminenata i inhibitora pcr reakcije. trideset uzoraka, uključujući zrna kukuruza i prerađene proizvode, nasumično je nabavljeno iz supermarketa i prodavnica. genomska dnk je ekstrahovana različitim protokolima, a pcr je korišćen za detekciju gmo-specifičnih regiona: nos terminator, 35s promotor i epsps. gen zein je amplifikovan radi potvrde prisustva genoma kukuruza u svim uzorcima. rezultati su pokazali da je 14 uzoraka sadržalo nos terminator, 13 uzoraka 35s promotor, a jedan uzorak epsps region, čime je ukupno 17 uzoraka (57%) identifikovano kao transgeno. primenjene metode pokazale su se efikasnim za ekstrakciju dnk visokog kvaliteta pogodnu za detekciju gmo. značajan broj gmopozitivnih proizvoda nije imao odgovarajuću oznaku, što ukazuje na potrebu za pouzdanim metodama detekcije i strožom kontrolom označavanja. ključne reči: genetički modifikovani organizam, označavanje, kukuruz, pcr introduction recombinant dna (rdna) technologies have been developed to improve modern farming and have provided many advantages in crops such as herbicide tolerance, biotic and abiotic stress resistance, and improved nutritional value (ashrafi dehkordi et al., 2021; dehkordi et al., 2018). many gmcs (genetically modified crops) have been approved since the mid-1990s worldwide, and the global area of gmcs was more than 190 million hectares in 2019 (isaaa, 2019) and is expected to continue to increase. more than 200 different gmcs with varying features, of which the highest cultivated © 2025 ashrafi-dehkordi et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. area belongs to soy, corn, canola, and cotton, and several products of these gmcs are available in markets (ashrafi-dehkordi et al., 2022). maize is one of the major agricultural crops and the cultivation of gm maize is increasing worldwide. the global area under gm maize is more than 60 million hectares, accounting for about 32% of global corn production (isaaa, 2019). although, maize is approved for use as feed/food in many countries based on their safety evaluation criteria. however, many consumers are demanding information and appropriate labeling for foods derived from gmos. in this regard, different labeling systems for gm foods have been introduced in different countries such as the european union (eu), korea, russia, and japan. there are different thresholds (between 0 and 5%) for accidental mixing of gm crops; for example, the thresholds in russia are 0.9%, in the european union are 0.9%, in korea, 3% and 5% in japan (chiaravutthi et al., 2022). different technologies are used for the detection of gmos in food samples, such as protein-based and dnabased methods (fraiture et al., 2015; griffiths et al., 2002; rott et al., 2004). pcr-based methods such as conventional pcr, multiplex pcr, and rt-pcr methods have become the primarily used approaches for gmo detection, which can detect even small amounts of the novel dna in both raw and processed foods, most widely used, because of its sensitivity, specificity, versatility, and high throughput application (li et al., 2022; zhang et al., 2013). on the other hand, food processing can affect the quality and quantity of dna, leading to its removal or degradation. moreover, for the successful dna amplification step, one must have an efficient dna extraction step, because there are different compounds that inhibit amplification of dna, such as lipids, polysaccharides, and polyphenols, or extraction chemicals such as phenol, ctab, and isopropanol (anklam et al., 2002; hübner et al., 1999; saghai-maroof et al., 1984). therefore, in order to be able to amplify dna, it is necessary to use an extraction method that provides dna with an acceptable quantity, quality, and purity. this step is often very time-consuming. to identify transgenic organisms, an endogenous reference gene is needed as a standard to confirm species identification and determine the quality of genomic dna. various studies have been conducted to confirm internal control genes for different plants. for example, the zein, zssiib, and invertase genes in corn (germini et al., 2004; hernández et al., 2004; pan et al., 2006), lectin in soybean (terry et al., 2001), sps, oryzain beta, and ppi-ppf in rice (chaouachi et al., 2007; hernández et al., 2005), and β-fructosidase in the solanaceae family (chaouachi et al., 2008). the elements such as the camv35s promoter (camv), nopaline synthase (nos) terminator, and selectable markers including the pat/bar gene, hpt gene, and the nptii gene are commonly derived from naturally occurring viruses, bacteria, and fungi found in many gmos (li et al., 2022). these elements are usually used for detection targets in gmo screening. this screening is a rapid and cost-efficient method for the detection of samples potentially containing one or several gmos. in this study, three dna extraction methods were employed for isolating dna from maize samples, and a pcr method was used to detect gm maize in various processed foods collected from the shiraz market. materials and methods samples collection thirty samples, including raw corn and its products (corn puff, corn nut, canned corn, corn starch, corn flour, and popcorn), were bought from supermarkets in shiraz. tab. 1 provides an overview of the corn samples collected from supermarkets in shiraz. the table lists each sample’s identification number, product name, and product type, offering a clear classification of the various corn-based foods analyzed in this study. dna extraction three different ctab-based (cetyltrimethyl ammonium bromide) methods were used for dna extraction from the samples. the first method, referred to as “method 1,” follows the standard ctab precipitation of dna protocol (gryson et al., 2004) and was applied to raw corn, corn nut, and corn flour. the second method, referred to as “method 2,” uses the ctab protocol with ethanol precipitation of dna (sisea & pamfil, 2007) and was applied to canned corn, corn puff, and popcorn. the third method, referred to as “method 3,” follows another ctab protocol (waiblinger et al., 2012) and was used for corn starch. method 1: the dna samples were extracted from raw corn, corn nuts, and corn flour using the standard ctab method. following grinding samples with a mortar and pestle, the powder was mixed with 1000 μl of buffer (containing 100 mm tris-hcl, 20 mm na2edta, 1.4 m nacl, and 20 g/l ctab, ph 8.0). the sample mixtures were transferred to 2-ml tubes and were incubated at 65 °c for 90 minutes in a water bath. the mixtures were gently rotated every 10 minutes for intermittent mixing before being centrifuged for 20 minutes at 13,000g. the resulting supernatant was transferred to a fresh 2-ml tube, followed by the addition of 500 µl of chloroform (merck, germany) and vortexing for 30 seconds. biologica nyssana ● 16 (2) december 2025: ashrafi-dehkordi et al. ● pcr-based investigation on transgenic maize status in corn processed products biologica nyssana ● 16 (2) december 2025: ashrafi-dehkordi et al. ● pcr-based investigation on transgenic maize status in corn processed products the tube was centrifuged again at 13,000 g for another 20 minutes. the upper phase was transferred to a fresh tube containing 0.7 volumes of pre-chilled isopropanol (merck, germany) and kept overnight at -20 °c. the mixture was centrifuged at 13,000 g for 10 minutes. the supernatant was discarded, and the pellet was washed with 500 μl of 70% ethanol (merck, germany). after that, the pellet was subjected to centrifugation for 10 minutes at 13,000 g. after the removal of the supernatant, the pellet was air-dried. subsequently, the dna was dissolved in 50 μl sdw. method 2: the dna sample was extracted from canned corn, corn puffs, and popcorn using the modified ctab method. the samples were ground using a mortar and pestle. before milling, liquid nitrogen was added to the popcorn, and the canned corn was heated to below 40 °c. then, the powder was mixed with 1000 μl of buffer (containing 1.4 m nacl, 0.1 m tris-hcl, 20 mm na2edta, 20 g/l ctab, and ph 8.0) in a 2-ml tube, with the addition of 25 μl of 1% (w/v) sodium dodecyl sulfate (sds) to the corn puffs and popcorns. the mixture was incubated in a water bath at 65 °c for 2-4 hours. the tubes were gently inverted every 10 minutes for intermittent mixing. after that, 20 μl proteinase k (20 mg/l) was added and incubated at 37 °c for 45 minutes. next, the tube was centrifuged at 13,000 g for 20 minutes. the supernatant was transferred to a new 2-ml tube containing 500 µl of chloroform (merck, germany) and was shaken for 30 seconds. the tube was centrifuged for 20 minutes at 13,000 g. the upper phase was transferred to a new tube, and two volumes of ctab precipitation buffer (5 g/l ctab and 0.04 m nacl) were added, and the tube was incubated at room temperature for 60 minutes. then the tube was centrifuged for 20 minutes at 13,000 g. the supernatant was discarded, and the precipitate was dissolved in 350 µl chloroform and 350 µl1.2 m nacl. the mixture was shaken for 30 seconds, and the tube was centrifuged for 20 minutes at 13,000 g. the upper phase was transferred to a new tube. the 0.7 volumes of pre-chilled isopropanol were added, and the tube was shaken and kept overnight at -20 °c. the tube was centrifuged for 20 minutes at 13,000 g. the supernatant was discarded and washed with 500 µl pre-chilled ethanol (70%) (merck, germany). the tube was shaken carefully, and then centrifugation was done for 20 minutes at 13,000 g. the pellet was air-dried, and dna was dissolved in 50 µl sdw. method 3: the dna sample was extracted from corn starch using the modified protocol of waiblinger et al. (2012). the sample (5 g) was mixed with 25 ml of ctab buffer (0.1 m tris-hcl, 1.4 m nacl, 20 mm na2edta, and 20 g/l ctab, ph 8.0), and then 50 μl of α-amylase solution (10 mg/ml) was added to the mixture. the sample mixture was transferred to a 50-ml tube and incubated at 65 °c for 4 hours in a water bath, with the tubes gently inverted every 10 minutes for intermittent mixing. the tube was centrifuged for 20 minutes at 13,000 g. the supernatant was transferred to a new 50-ml tube containing 10 ml of chloroform (merck, germany) sample code product name product type 1 corn seed nut 2 corn seed nut 3 corn seed nut 4 corn seed nut 5 corn seed nut 6 corn seed nut 7 corn seed nut 8 corn seed nut 9 corn seed nut 10 corn seed nut 11 bulk corn seed nut 12 bulk corn seed nut 13 bulk corn seed nut 14 canned corn canned 15 canned corn canned 16 canned corn canned 17 canned corn canned 18 raw seed raw corn 19 raw seed raw corn 20 raw seed raw corn 21 corn flour flour 22 corn flour flour 23 popcorn pop corn 24 popcorn pop corn 25 popcorn pop corn 26 extruded snack puff 27 extruded snack puff 28 extruded snack puff 29 corn starch starch 30 corn starch starch table 1. sample code, names of corn samples and product types collected from supermarkets in shiraz biologica nyssana ● 16 (2) december 2025: ashrafi-dehkordi et al. ● pcr-based investigation on transgenic maize status in corn processed products and was shaken for 30 s. the tube was centrifuged at 13,000 g for 20 minutes. the upper phase was transferred to a new tube with 0.7 volume of prechilled isopropanol (merck, germany) and then incubated overnight at -20 °c. the centrifugation was done at 13,000 g for 10 minutes. the supernatant was discarded, and the pellet was transferred to a 2-ml tube and washed with 1500 μl70% ethanol (merck, germany). the centrifugation was performed at 13,000 g for 10 minutes. finally, the supernatant was discarded, and the pellet was air-dried. the dna was dissolved in 50 μl sdw. dna quantification and purity measurement dna concentration was measured by an eppendorf spectrophotometer (scandrop2, analytic jena, germany), using the uv absorbance at 260 nm (nanograms of dna per microliter extract). oligonucleotide primers specific oligonucleotide primers were used for amplifying amplicons. the primers of nos, 35s, epsps, and zm were used to detect nos-terminator, camv35s promoter, epsps structure gene region, and zein gene (as an endogenous control), respectively. the sequences of oligonucleotide primers are given in tab. 2. polymerase chain reaction pcr in thermal cycler (ab applied biosystems, veriti 96 well, usa) was used in reaction mixtures (20 µl) containing 2.5 µl of 4 mm dntp, 2.5 µl of 10 x pcr buffer, 3 µl dna, 1.5 µl 25 mm mgcl2, 0.6 µl primers with 20 µm each and 5 unites/µl of taq dna polymerase (sinagene, tehran, iran). a thermal cycler was used for amplification according to the following pcr step-cycle program for all primers, as shown in tab. 3. the analysis of pcr products was done on a 1.0% agarose gel electrophoresis. results three different ctab-based dna extraction methods were applied to the corn and corn-derived food samples presented in tab. 1. a summary of the dna yields and purity ranges obtained using the three extraction methods is presented in tab. 4. the results show that there are discrepancies in the dna extraction between the three methods. following the genomic dna extraction, the pcr method was used to identify the gmo samples. firstly, the specific maize sequence, the zein gene, was employed to distinguish negative and positive outcomes resulting from amplification interference (forte et al., 2005). secondly, for the identification of gm material in corn samples, the 35s-promoter and nos-terminator were employed. name 5’-3’ sequence target amplicon (bp) refrence zm-f cgccagaaatcgtttttcat zein 139 (germini et al. 2004) zm-r ggtggtgtccttgcttccta zein 139 (germini et al. 2004) 35sf-1 gctcctacaaatgccatca p-35s 195 (cardarelli et al. 2005) 35sr-1 gatagtgggattgtgcgtca nos-f gacaccgcgcgcgataatttatcc t-nos 118 (cardarelli et al. 2005) nos-r gacaccgcgcgcgataatttatcc gmo-f atcccactatccttcgcaaga epsps 169 (cardarelli et al. 2005) gmo-r tggggtttatggaaattggaa table 2. the list of primer pairs used in this study step zein camv 35s nos epsps initial denaturation 95 °c, 3 min 95 °c, 3 min 95 °c, 3 min 95 °c, 3 min denaturation 95 °c, 45s 95 °c, 45s 95 °c, 45s 95 °c, 45s annealing 50 °c, 45s 52 °c, 45s 61 °c, 45s 57 °c, 45s extension 72 °c, 40s 72 °c, 40s 72 °c, 40s 72 °c, 40s final extension 72 °c, 10 min 72 °c, 10 min 72 °c, 10 min 72 °c, 10 min cycles 35 35 35 35 table 3. reaction conditions for zein, camv 35s, nos and epsps amplification subsequently, the samples were analyzed to identify the specific type of gmo by amplifying the roundup ready gene using specific primers (lin et al., 2000). the electrophoresis results of the pcr products showed that the zein gene (a band of about 137 bp) of all corn samples was successfully amplified. this result showed that the biologica nyssana ● 16 (2) december 2025: ashrafi-dehkordi et al. ● pcr-based investigation on transgenic maize status in corn processed products isolated dnas have good quality and contain maize dna (fig. 1). the positive detection of targetp genes in each individual corn sample is summarized in tab. 5. this table presents the sample numbers alongside the specific target detected through pcr analysis, providing a clear and detailed overview of gmo presence in the analyzed samples. electrophoresis of the pcr products showed a 118-bp band for nos (fig. 2a), a 195-bp band for p35s (fig. 2b), and a 169-bp band for the epsps gene (fig. 2c). the results showed that 13, 14, and one sample were positive for the 35s, nos, and epsps gene, respectively. discussion the quantity and integrity of dna depend on the extraction method and the type of food. contaminants present in sample matrices, including lipids, polysaccharides, polyphenols, and extraction substances, can affect dna purity (anklam et al., 2002). the food processing techniques, such as enzymatic activities, low dna extraction methods dna yield (ng/µl) dna purity a260nm/a280nm ratio a260nm/a230nm ratio method 1 123.66±39.84 1.77±0.33 1.87±0.11 method 2 122±69.70 1.89±0.47 1.67±0.13 method 3 85.49±4.90 1.95±0.04 1.78±0.03 table 4. summary of mean ±sd, dna yield and purity for corn samples using different dna extraction methods fig. 1. representative agarose gel electrophoresis of maize pcr products (137 bp for the detection of zein gene): ladder: 100 bp dna ladder, -: negative control,+: positive control, lane 1-13: corn nut, lanes 14-17: canned corn, lane 18-20: raw corn, lane 21-22: corn flour, lane 23-25: popcorn, lane 26-28: corn puff, lane 29-30: corn starch. ph, and heat, affect the integrity and quantity of dna, so extracting dna in sufficient purity and quantity is a difficult step (peano et al., 2004; vijayakumar et al., 2009). the different methods of dna extraction applied in this study are capable of extracting sufficient amounts and quality dna to identify the gm corn. a more detailed comparison of our results with previous studies further illustrates the role of extraction methods and sample matrices in dna yield and integrity. in this study, three ctab-based dna extraction methods were applied to various corn and corn-derived food samples. the results showed noticeable differences in both dna yield and purity between the methods. as shown in tab. 4, method 1 produced the highest dna yield (123.66±39.84 ng/µl) but with slightly lower purity ratios (a260/280=1.77±0.33; a260/230=1.87±0.11) compared to method 3, which yielded lower dna concentration (85.49±4.90 ng/µl) but the highest purity (a260/280=1.95±0.04; a260/230=1.780±0.03). these findings suggest that method 1 is more effective in quantity, while method 3 provides purer dna, possibly due to better removal of pcr inhibitors. that intense food processing, such as autoclaving and baking, significantly reduces dna fragment size, resulting in the complete degradation of long dna fragments and the retention of only short fragments (<500 bp). nevertheless, successful pcr amplification of short amplicons was still possible, highlighting the importance of using appropriate primer design and extraction protocols for reliable gmo detection in processed foods. in this study, pcr results using specific primers showed that 17 (57%) samples were transgenic. pcr techniques have been widely employed in several studies to detect genetically modified (gm) foods. for instance, lin et al. (2001) reported that the 35s or nos were present in 22 out of 28 commercial genetically modified crops. fifty samples of meat products processed in serbia were tested using the pcr method. the results showed that 12 out of the 50 samples contained gm soybeans (taski-ajdukovic et al., 2009). results from another study showed that 60 of 66 food samples contained rr soy (cardarelli et al., 2005). in another study, 100 food samples were randomly collected from turkish markets, and transgenic soybeans and corn were identified using pcr; 25% of the samples were transgenic (arun et al., 2013). ujhelyi et al. (2008) reported that 38% of random samples collected in hungary were transgenic and that 7 out of 80 foods contained rr soy. however, they did not contain the nos terminator. gurakan et al. (2011) reported that the 35s promoter was present in 11 out of 31 commercial turkish food products containing corn. conclusion in this study, three simple and cost-effective methods were used to extract dna from maize seeds and their products. subsequently, pcr was employed to screen for genetically modified organisms (gmos)derived products. for screening products derived from gmos, the pcr method was used. the isolated dna of all samples was confirmed by the presence of the zein gene. the 35s promoter, nos, and epsps were found in a number of samples. these results show that 17 samples (57%) are transgenic, which is consistent with the fact that iran imports the majority of its corn from countries cultivating genetically modified corn. therefore, the detection and labeling of these products is one of the demands of consumers regarding food safety and quality. funding. this work was supported by the shiraz university of medical sciences. (project no: 24647) biologica nyssana ● 16 (2) december 2025: ashrafi-dehkordi et al. ● pcr-based investigation on transgenic maize status in corn processed products sample code pro1 nos gmo 1 û ü û 2 ü û û 3 û û û 4 û û û 5 û û û 6 û û û 7 û ü û 8 ü ü û 9 û û û 10 û ü û 11 û ü û 12 û û û 13 ü û û 14 ü ü ü 15 ü ü û 16 ü û û 17 ü ü û 18 ü ü û 19 ü ü û 20 ü ü û 21 ü ü û 22 ü ü û 23 û û û 24 û û û 25 û û û 26 û û û 27 û û û 28 ü ü û 29 û û û 30 û û û table 5. summary of positive pcr detection of target in individual corn samples these results are consistent with findings by peano et al. (2004), who reported that dna yield and integrity varied widely depending on the extraction method and the complexity of the food matrix. in their study, the nucleospin food kit was most effective in extracting dna from complex matrices, while wizard and qiaamp kits performed better for simple matrices like flours. similarly, vijayakumar et al. (2009) demonstrated biologica nyssana ● 16 (2) december 2025: ashrafi-dehkordi et al. ● pcr-based investigation on transgenic maize status in corn processed products fig. 2. representative agarose gel electrophoresis of maize pcr products: ladder: 100 bp dna ladder, -: negative control,+: positive control, lane 1-13: corn nut, lanes 14-17: canned corn, lane 18-20: raw corn, lane 21-22: corn flour, lane 23-25: popcorn, lane 26-28: corn puff, lane 29-30: corn starch. a band of 118 bp for nos (fig. 2a), a band of 195 bp for p35s (fig. 2b), and a band of 169 bp for the epsps gene (fig. 2c) references anklam, e., gadani, f., heinze, p., pijnenburg, h., & van den eede, g. (2002). analytical methods for detection and determination of genetically modified organisms in agricultural crops and plant-derived food products. european food research and technology, 214(1), 3–26.https://doi.org/10.1007/s002170100415 arun, ö. ö., yılmaz, f., & muratoğlu, k. j. 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(2013). one simple dna extraction device and its combination with modified visual loop-mediated isothermal amplification for rapid onfield detection of genetically modified organisms. analytical chemistry, 85(1), 75–82. https://doi. org/10.1021/ac301640p cvetković et al. 2024, biologica nyssana 15(2) 15 (2) december 2024: 97-104 doi: 10.5281/zenodo.13842094 evaluation of wing morphology changes in drosophila melanogaster treated with pseudotsuga menziesii (pinaceae) essential oil original article vladimir j. cvetković university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia vladimir.cvetkovic@pmf.edu.rs (corresponding author) maja lazarević university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia zorica mitić university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia snežana jevtović university of niš, faculty of sciences and mathematics, department of chemistry, višegradska 33, 18000 niš, serbia gordana stojanović university of niš, faculty of sciences and mathematics, department of chemistry, višegradska 33, 18000 niš, serbia vladimir žikić university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia received: june 30, 2024 revised: september 10, 2024 accepted: september 11, 2024 abstract: the assessment of the toxicity of essential oils (eos) from conifer species in pest insects has recently gained importance. fumigant, deterrent, repellent and larvicidal activities of eos are the most evaluated in insects while studies on eos-induced changes in wing morphology are rare. this study deals with evaluation of eos derived from pseudotsuga menziesii var. menziesii (pmeo) influence on wing morphology changes in the insect model drosophila melanogaster. after treatment at the larval stage with 3% of pmeo, and finishing the development, wings were analyzed using geometric morphometrics. the results suggest that pmeo induced significant changes in wing morphology, particularly with moderate effects in wing shape. the correlation of the obtained results with available literature on moderate influence on developmental time and weak larvicidal activity in drosophila, as well as chemical composition of pmeo was discussed. we believe that this finding would significantly contribute to overall knowledge of pmeo bioactivity. key words: pseudotsuga menziesii, drosophila, essential oils, wing morphology, geometric morphometrics, toxicology apstrakt: procena promene morfologije krila kod drosophila melanogaster tretirane etarskim uljem pseudotsuga menziesii (pinaceae) ispitivanja toksičnosti etarskih ulja (eu) na insektima štetočinama su od nedavno dobila na značaju. fumigantna, odvraćajuća, obijajuća i larvicidna aktivnosti eu su najčešće ispitivane aktivnosti, dok su promene morfologije krila uzrokovane eu veoma retko istraživane na insektima. ovo istraživanje se bavi procenom uticaja eu četinara vrste pseudotsuga menziesii var. menziesii (pmeu) na promenu morfologije krila na modelu insekta drosophila melanogaster. nakon tretmana larvi sa 3% pmeu i završetka razvića, krila su analizirana pomoću metode geometrijske morfometrije. rezultati ukazuju da pmeu indukuje značajne promene u morfologiji krila izazivajući umereni efekat na oblik krila. diskutovana je korelisanost dobijenih rezultata sa prethodno publikovanim rezultatima o slaboj larvicidnoj aktivnosti, umerenom uticaju na dužinu vremena razvića i hemijskom sastavu pmeu. verujemo da će ovaj nalaz značajno doprineti sveukupnom znanju o bioaktivnostima pmeu. ključne reči: pseudotsuga menziesii, drosophila, etarska ulja, morfologija krila, geometrijska morfometrija, toksikologija introduction pseudotsuga menziesii (mirb.) franco (douglas fir) is a representative of the pinaceae family indigenous to western north america with two varieties: coastal douglas fir (var. menziesii) and inland douglas fir (var. glauca (beissn.) franco) (da ronch et al., 2016). it was introduced to europe in the 19th century as an ornamental tree, and today it is the most common non-native coniferous species in european forest plantations (schmid et al., 2014). the analyses of chemical composition of p. menziesii var. menziesii eo (pmeo) are frequently performed (buchbauer et al., 1994; gambliel & cates, 1995; jirovetz et al., 2000) while studies on biological activities are rare (han, 2017; mitić et al., 2021), especially on © 2024 cvetković et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 97 insect models. recently, anti-proliferative activity of pmeo in a human dermal fibroblast model was shown (han, 2017), fumigant toxicity against store product pest insect sitophilus oryzae and contact toxicity against aphid phyllaphis fagi (yazdgerdian et al., 2015), toxicity against brine shrimp a. salina (mitić et al., 2021) and antimicrobial properties (tešević et al., 2009; mitić et al., 2021). plant essential oils (eos) were recognized as potential eco-friendly pesticides against arthropods (park & tak, 2016). pest insects are surely the most abundant in this group and consequently most of the published results are related to larvicidal (mitić et al., 2018), fumigant (negahban et al., 2007), antifeedant (nikolić et al., 2022), repellent (nerio et al., 2010) effect of the eos against pest insects. surely, targeted toxicological studies against real threats, certain pest insect species, are the most valuable. however, screening of the bioactivities of the eos on some universal insect models such as drosophila melanogaster meigen, 1830 (drosophilidae) is also of great importance. this species has been proved as an effective model insect in different studies that involve eоs (mihajilov-krstev et al., 2014; ickovski et al., 2024). studies on alternative, ecofriendly pesticides are very important and urgent due to growing risk to environment and human health regarding the use of chemical pesticides (rattan, 2010) including developing of the resistance to chemical pesticides (pavela et al., 2016). the eos derived from conifers are known as potential biocides against insects (mitić et al., 2019, 2022; cvetković et al., 2023, 2024). considering that in most cases, high concentrations of the eos are needed for the biocide effect, consequently possible side effects against non-targeted organisms are therefore certain. thus, the evaluation of the effects of the lower, non-lethal doses of the eos is crucial in further studies of the eos. the detrimental effect of the test agents at low doses could also be reflected in the morphology of the body parts in the model organism. for example, the toxicity of the tio2 nanoparticles reflected in the wing morphology of the d. melanogaster in lower doses through 20 generations (cvetković et al., 2020) even though it was not lethal for treated flies. thus, it might be assumed that lower doses of an eos intended for pest insect controlling also have potential to disrupt morphology of the insects’ body parts, including wings what may potentially reduce insects’ pest activity as it was suggested recently (cvetković et al., 2024). hence, the potential of pmeo to disrupt wing morphology in model insect d. melanogaster was evaluated in this study. the geometrical morphometrics is reliable method that enables the visualization and evaluation of potential changes in wing morphology resulting from the application of conifers’ eos (cvetković et al., 2024). materials and methods plant material, eo isolation and characterization in this study, twigs with needles of pseudotsuga menziesii var. menziesii were used. details about collection, location and deposition of the material as well as isolation and chemical characterization of the eo, were described in mitić et al. (2021). in brief, hydrodistillation using the clevenger-type apparatus was performed, and for qualitative and quantitative analyses were conducted using gas chromatography-mass spectrometry (gc-ms), and gas chromatography-flame ionization detector (gc-fid) were used, respectively. model insect cultivation – fruit fly drosophila melanogaster a laboratory strain (oregon) of the fruit fly drosophila melanogaster was used. fruit flies were reared under standard laboratory conditions for this species, which included a temperature of 25 °c, relative humidity of 60%, and a 12-hour light-dark cycle. the flies were fed on a standard cornmeal-based feeding medium that contained agar, sucrose, water, yeast, and fungicide (4 methyl hydroxybenzoate). dietary treatment and collection of the fruit flies the adult fruit flies used in this study were treated and collected as described in a recent study by mitić et al. (2021). briefly, three-days-old d. melanogaster larvae were treated with 3% pmeo through their diet (cornmeal feeding medium) in triplicate. the control larvae were fed on media without pmeo. a total of sixty larvae were set up in three replicates, with twenty larvae per replicate. after completing their life cycle, the adults were collected, separated by sex, and preserved in 70% ethanol prior to wing dissection. since pmeo was shown to induce detrimental and significant effects in d. melanogaster, mostly at 3%, (mitić et al., 2021) we considered the 3% concentration as the most relevant for the testing of its influence on wing morphology changes. also, the 3% was the only concentration used in the study with similar methodology (cvetković et al., 2024). wing preparation and geometrics morphometrics method wing preparation and analysis were conducted following the detailed methodology described by cvetković et al. (2020). in summary, 15 male and 15 female fruit flies were randomly selected 98 biologica nyssana ● 15 (2) december 2024: cvetković et al. ● evaluation of wing morphology changes in drosophila melanogaster treated with pseudotsuga menziesii (pinaceae) essential oil biologica nyssana ● 15 (2) december 2024: cvetković et al. ● evaluation of wing morphology changes in drosophila melanogaster treated with pseudotsuga menziesii (pinaceae) essential oil 99 from both the treatment and control groups. using entomological needles and forceps, wings were detached from the flies, washed in distilled water and mounted on microscopic slides. prepared slides were photographed and processed using a leica binocular microscope dfc 320 (m2 16 a) (leica microsystems, weltzar, germany) at 40 × magnification. wing shape was depicted using 15 landmarks as described by gidaszewski et al. (2009) and the nomenclature of the wing cells was set according to morgan et al. (1919). information on wing shape as procrustes coordinates (rohlf & slice, 1990; dryden & mardia, 1998) was obtained by performing the generalized procrustes analysis (gpa) in the geomorph software package (adams et al., 2023) in rstudio (rstudio team, 2020). statistics the principal component analysis (pca) was conducted to test the differences in wing morphology caused by the exposure of fruit fly larvae to the eo of pseudotsuga menziesii var. menziesii, compared to the wings of the control group. statistical testing of changes in wing shape was performed using multivariate analysis of variance (manova) in the geomorph package. afterward, the linear discriminant analysis (lda) was conducted using the mass package (venables & ripley, 2002) incorporated in rstudio. results the morphospace displayed by pca shows that the first principal component (pc1) separates the treatment groups from the control groups, positioning the treatments in the positive part of pc1 and the controls in the negative part (fig. 1). the male control group shows a more distinct separation compared to the female control group. the wings of the treated groups of drosophila tend to be narrower. along with the second principal component (pc2), there is minimal discrimination. sexual dimorphism is observable in the control groups, while such differences are less evident in the treatment groups. cumulatively, the first two principal components (pc1+pc2) account for 51.15% of the total variation in shape and size of the tested wings, indicating a moderate level of discrimination. fig. 1. results of wing morphology changes visualized in pca morphospace defined by the first two pc axes. the ellipses represent the 90% mean confident intervals for each group. the changes in wing morphology are depicted by outline graphs; the black line for maximum/minimum values of each axis, whereas the grey line for mean wing shape 100 biologica nyssana ● 15 (2) december 2024: cvetković et al. ● evaluation of wing morphology changes in drosophila melanogaster treated with pseudotsuga menziesii (pinaceae) essential oil statistical analyses revealed highly significant differences in wing shape, both between treatment groups (f(1-55)=12.433, p=0.001) and between sexes (f(1-55)=4.948, p=0.001). significant differences in wing shape were also observed in the interaction of treatment and sex (f(1-55)=1.961, p=0.042). pairwise test of manova reveals significant differences in wing shape between control and treatment group, as well as between sexes (due to sexual dimorphism) (p<0.001). linear discriminant analysis (lda) was used to highlight the real differences in wing morphology that were already indicated by pca (fig. 2). cumulatively, ld1+ld2 account for 89.15% of the total variability in wing shape. ld1 effectively separates the control groups from the treatment groups, with the wings of the treatment groups being slightly narrower than those of the controls, while ld2 reveals that sexual dimorphism is preserved in both the control and treatment groups. discussion there are very few studies on the effects of eos on insect wings. a recent study reported that larvicidal doses of croton tetradenius eo induced changes in the wings of mosquito ae. aegypti (silva et al., 2023). but this eo was not from conifer species and the chemical composition was significantly different from the tested pmeo, so it is difficult to compare with the obtained results from this study. very recently one more study on three abies and six pinus species eos influence on wing morphology in drosophila melanogaster was published (cvetković et al., 2024). also, a few studies on the bioactivity of pmeo have been published. for example, antiproliferative activity in a human dermal fibroblast model was shown (han, 2017), but the data on pmeo bioactivity in insects are limited. in a drosophila model, pmeo did not show larvicidal activity, but only did cause a slight delay in developmental time, especially at the highest applied concentration of 3% (mitić et al., 2021). regarding these findings, pmeo showed certain but weak detrimental effects against insects. in the current study, wing morphology in drosophila was affected to some extent under the treatment of 3% of the pmeo. due to sexual dimorphism, significant differences between female and male flies were obvious. this indicates that differences in the wing morphology between females and males were maintained and did fig. 2. wing morphology analysis visualized in lda morphospace defined by the first two ld axes. the ellipses represent the 90% mean confident intervals for each group. outline graphs show wing shape changes; the black line for a maximum/minimum value of each axis, the grey line for mean wing shape biologica nyssana ● 15 (2) december 2024: cvetković et al. ● evaluation of wing morphology changes in drosophila melanogaster treated with pseudotsuga menziesii (pinaceae) essential oil 101 not change after pmeo treatment. additionally, the results indicate that the differences and significance in wing shape between the sexes are greater than the differences between the groups treated with pmeo and the corresponding controls, i.e. separately within males and within female group. therefore, it was reasonable to observe the effects on wing morphology induced by pmeo treatment separately within male and female groups. the wings of the males treated with pmeo were more affected than those of female when compared with the corresponding control, the untreated flies. generally, the wings of the treatment groups were slightly narrower than those of the controls. despite the observed significant changes in wing morphology, the general structure of the wings in all flies treated with pmeo was maintained and all were able to fly. additionally, the observed changes in the wing shape suggests that the changes were at moderate level due to overlapping of the results from the compared groups in presented morphospace in pca and lda analyses (figs. 1 and 2). this is consistent with mild effect on developmental time and weak, non-significant larvicidal effect of pmeo applied at 3% concentration in drosophila (mitić et al., 2021). considering that some abies and pinus eos with larvicidal effects, also significantly disturb wing morphology (cvetković et al., 2024) some conclusions could be drawn from the results of the present study. eos from certain abies and pinus species have demonstrated stronger larvicidal activity in drosophila (mitić et al., 2018, 2019, 2022) than pmeo (mitić et al., 2021), and also induced more pronounced and significant wing morphology alterations (cvetković et al., 2024) than pmeo in the present study. hence, larvicidal activity and probably other toxic effects in insects (fumigant effect, repellency, antifeedant effect, deterrent effect) of the eos might be correlated with the proportional level of changes in wing morphology. as it was concluded in the recent study by cvetković et al. (2024) the results from this study are important because of the potential of eos to induce morphological changes in insects’ wings might be used as agent for controlling the insect pest activities and behavior that mostly depends on wing morphology such as flight in mosquitos or serious pest drosophila suzukii, asian fruit fly. certainly, this should be evaluated in specific pest insect species. the chemical composition of the same batch of pmeo used in the presented study was presented in detail in a study by mitić et al. (2021). the dominant components in this eo are sabinene (17.9%), β-pinene (15.2%) and α-terpinolene (22.7%). these components exhibited various biological activities, such as shown in recently published studies (da silva rivas et al., 2012; matias et al., 2016; liu et al., 2019), but very few studies have focused on their effects in insects. some activities of these components, that are constituents of the pmeo, in insects have been recently published. in the red flour beetle tribolium castaneum, repellent activity of β-pinene was demonstrated (pajarocastro et al., 2017). in the rice weevils sitophilus oryzae (l.), eo of the bacopa caroliniana with the most abundant component α-terpinolene exhibited insecticidal activity (liu et al., 2019). sabinene was also shown as toxic for this species (sharma & tiwari, 2021). this study did not test the influence of the major components of pmeo, what might be potential limitation of the present study. therefore, while pmeo had a moderate effect in the wings of drosophila, its separate components such as sabinene, β-pinene and α-terpinolene could have more significant influence on wing morphology. understandably, this assumption should be verified in future studies. afterall, we believe that the results of this study are of great importance, as they provide insights into the relationship between the toxicity of the pmeo and its influence on the wing shape in d. melanogaster as potential ecofriendly agent against flying pest insects. conclusion the results from the presented study suggest that the wing morphology of fruit flies treated with pmeo was significantly changed, particularly wing shape. however, the observed changes in the wing shape were moderate, but did not impair the flies’ ability to fly. the level of changes was proportional to previously published larvicidal effects (mitić et al., 2021), which were weak and moderately delayed the developmental time. in our opinion and based on the observed effects on wing morphology of d. melanogaster, pmeo could be candidate for the potential treatment against pest insects or arthropods. especially its major components such as sabinene, β-pinene and α-terpinolene could have more significant impact on wing morphology that can impact pest activities of the treated flying insects. studies on the effect of eos on insect’s body morphology, including wings, are very rare. therefore, we believe that this finding significantly contributes to the overall knowledge of the conifer eos effect in insect models. further research is warranted to explore these correlations in depth and to clarify the mechanisms by which eos affect both the structural and functional aspects of insect wing morphology. dryden, i.l. & mardia, k.v. 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(2024). serbian artemisia species–chemical composition, acute toxicity and larvicidal activity of the essential oils. natural product research, 1-12. https://doi.org/10. 1080/14786419.2024.2334312 jirovetz, l., puschmann, c., stojanova, a., metodiev, s., & buchbauer, g. (2000). analysis of the essential oil volatiles of douglas fir (pseudotsuga menziesii) from bulgaria. flavour and fragrance journal, 15(6), 434-437. https://doi.org/10.1002/10991026(200011/12)15:6<434::aid-ffj935>3.0.co;2-0 liu, t.t., chao, l.k., hong, k.s., huang, y.j., & yang, t.s. (2019). composition and insecticidal activity of essential oil of bacopa caroliniana and interactive effects of individual compounds on the activity. insects, 11(1), 23. https://doi.org/10.3390/ insects11010023 matias, e.f., alves, e.f., silva, m.k., carvalho, v.r., figueredo, f.g., ferreira, j.v., coutinho, h.d., silva, j.m., ribeiro-filho, j., & costa, j.g. 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(2015). insecticidal effects of essential oils against woolly beech aphid, phyllaphis fagi (hemiptera: aphididae) and rice weevil, sitophilus oryzae (coleoptera: curculionidae). journal of entomology and zoology studies, 3(3), 265-271. 104 anticancer compounds from medicinal plants biologica nyssana 5 (1)  september 2014: 11-15 šarac, z. et al.  application of canonical discriminant analysis… 11 original article received: 28 june 2014 revised: 8 july 2014 accepted: 10 september 2014 application of canonical discriminant analysis in differentiation of natural populations of pinus nigra in serbia based on terpene composition zorica šarac 1* , srdjan bojović 2 , biljana nikolić 3 , bojan k. zlatković 1 , and petar d. marin 4 1 university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia 2 university of belgrade, institute for biological research “siniša stanković”, boulevard despota stefana 142, 11060 belgrade, serbia 3 university of belgrade, institute of forestry, kneza višeslava 3, 11000 belgrade, serbia 4 university of belgrade, faculty of biology, institute of botany and botanical garden “jevremovac“, studentski trg 16, 11000 belgrade, serbia * e-mail: saraczorica@gmail.com abstract: šarac, z., bojović, s., nikolić, b., zlatković, b., marin, p.: application of canonical discriminant analysis in differentiation of natural populations of pinus nigra in serbia based on terpene composition. biologica nyssana, 5 (1), september 2014: 11-15. the canonical discriminant analysis (cda) was performed in order to check the hypothesis of chemical separation infraspecific taxa of pinus nigra j.f. arnold (ssp. nigra, var. gocensis, ssp. pallasiana, and var. banatica) in serbia based on variability of the needle terpenes. the cda, which maximizes variations between a priori groups, showed division of seven native p. nigra populations into three groups, which belong to three taxonomically recognized taxa (ssp. nigra, ssp. pallasiana, and var. banatica). the most important characters in discrimination were (e)-caryophyllene, α-humulene, terpinolene, germacrene d, αpinene, and myrcene. the individuals of pallasiana group were poorer in (e)-caryophyllene and terpinolene and richer in α-humulene contrary to the individuals of nigra group. the individuals of banatica group had the highest content of α-pinene and myrcene. the obtained results were compared with recently published data on chemodiversity of p. nigra infraspecific taxa on the territory of serbia. key words: canonical, chemotaxonomy, discriminant analysis, needle terpenes, pinus nigra. apstract: šarac, z., bojović, s., nikolić, b., zlatković, b., marin, p.: primena diskriminantne kanonijske analize u diferencijaciji autohtonih populacija pinus nigra u srbiji na osnovu sastava terpena. biologica nyssana, 5 (1), september 2014: 11-15. diskriminatna kanonijska analiza (cda) izvedena je u cilju provere hipoteze o fitohemijskom razdvajanju infraspecijskih taksona pinus nigra j.f. arnold (ssp. nigra, var. gocensis, ssp. pallasiana i var. banatica) u srbiji na osnovu varijabilnosti terpena u četinama. cda, koja naglašava varijabilnost između a priori definisanih grupa, pokazala je rezdvajanje sedam autohtonih p. nigra populacija u tri grupe (ssp. nigra, ssp. 5 (1) • september 2014: 11-15 biologica nyssana 5 (1)  september 2014: 11-15 šarac, z. et al.  application of canonical discriminant analysis… 12 pallasiana i var. banatica). najvažniji karakteri u diskriminaciji bili su (e)-kariofilen, α-humulen, terpinolen, germakren d, α-pinen i mircen. individue grupe pallasiana siromašnije su u sadržaju (e)-kariofilena i terpinolena, a bogatije u sadržaju α-humulena, suprotno od individua grupe nigra. individue grupe banatica imaju veći sadržaj α-pinena i mircena. dobijeni rezultati upoređeni su sa nedavno objavljenim podacima o hemodiverzitetu p. nigra infraspecijskih taksona na teritoriji srbije. ključne reči: kanonijska, hemotaksonomija, diskriminantna analiza, terpeni u ;etinarima, pinus nigra. introduction pinus nigra j.f. arnold (pinaceae) is a species of the tertiary origin belonging to the group of taxa typical for mediterranean and submediterranean coniferous forests (b o g u n i c et al., 2007). it is one of the widespread, morphologically as well as taxonomically very variable pine species in europe (g a u s s e n et al. 1993), with highly fragmented distribution range that extends from north africa through the northern mediterranean and eastwards to the black sea. in the flora of serbia, j o v a n o v i ć (1992) recognized two subspecies of black pine that can be geographically differentiated, each further divided into several varieties. generally, in the western, southwestern and central parts of serbia, p. nigra ssp. nigra is widespread, within which a distinct variety is recognized (var. gocensis đ o r đ e v i ć ), while in eastern serbia p. nigra ssp. pallasiana (d. don) holmboe has an extremely narrow distribution range. namely, crimean black pine (p. nigra ssp. pallasiana) was only recorded in southeastern serbia (r a j e v s k i , 1950) on the mountain crnook (jarešnik). apart from this, in eastern and northeastern serbia three additional populations of black pine are known, which j o v a n o v i ć (1992) describes as var. banatica georg. et ion. within ssp. pallasiana. the chemodiversity aspect of black pine on the territory of serbia has been addressed concerning n-alkane (b o j o v i ć et al., 2012) and terpene (š a r a c et al., 2013) variability. in the second study, 58 essential-oil components were identified in the needles of 195 trees from seven populations. unlike principal component analyses (pca), which showed an overlap of all populations, cluster analyses (ca) separated analysed samples into three basic groups: the first group consisted of populations from western (banjska stena, omar, zmajevački potok), southwestern (priboj) and central serbia (goč) (considered as ssp. nigra group), the second of population from southeastern serbia (jarešnik) (ssp. pallasiana group), and the third of population from northeastern serbia (lazareva reka), which had the most distinct oil composition (ssp. banatica group) (š a r a c e t a l ., 2013). bearing this in mind, the aim of the present study was to check the hypothesis of chemical separation of infraspecific taxa of p. nigra from serbia by performing a canonical discriminant analysis (cda). this method maximizes variations between a priori defined groups and thus characterize the degree of divergence among analyzed populations (j a m e s & m c c u l l o c h , 1990). also, cda was performed in order to determine the relative importance of terpenes as discriminators between a priori groups. material and methods plant material the samples from seven typical populations of p. nigra infraspecific taxa i banjska stena, ii omar, iii zmajevački potok (ssp. nigra), iv priboj-crni vrh, v goč-gvozdac (var. gocensis), vi jarešnik (ssp. pallasiana), and vii lazareva reka (var. banatica), growing wild in serbia were analyzed. details about sampling, locations and ecological conditions of the selected populations, numbering of populations, the extraction of the essential oils, the gc-fid and gc/ms analyses, and the identification of terpenes have been reported previously (š a r a c e t a l ., 2013). statistical analysis the cda was carried out in order to check the hypothesis that the analyzed sample was composed of discrete groups, which are chemically differentiated one from other. from the total data set of 75 original compounds (for details s e e š a r a c e t a l . , 2013), 14 compounds were selected for cda, viz., α-thujene, α-pinene, camphene, β-pinene, myrcene, limonene, (e)-β-ocimene, terpinolene, (e)caryophyllene, α-humulene, γ-muurolene, germacrene d, δ-cadinene, and germacrene d-4-ol. the 61 compounds which were found in traces (content <0.5%, according to r u d l o f f e t a l . , 1975; l i e u t i e r e t a l ., 1991) were excluded from further analysis. the cda was computed on the selected data set (195 samples x 14 variables) with a priori defined groups. statistical analyses were performed using the software statistica 5.1 (s t a t s o f t , 1997). biologica nyssana 5 (1)  september 2014: 11-15 šarac, z. et al.  application of canonical discriminant analysis… 13 results and discussion the cda based on individuals from seven native p. nigra populations showed that the first two discriminant functions participated in 79.6% of the total discrimination, of which the first function with 64.7% (tab. 1). the first discriminant function is approximately equal determined by the content of terpinolene, α-thujene, (e)-caryophyllene, α-pinene, and germacrene d. the second function is mostly defined by the content of germacrene d, α-pinene, and β-pinene (tab. 1). table 1. standardized coefficients for the first two canonical axes (ca) of variation in terpene variables from the discriminant functional analysis of 7 a priori groups. variables ca1 ca2 α-thujene -1.087 0.404 α-pinene 1.017 -1.823 camphene -0.408 -0.482 β-pinene 0.829 -1.618 myrcene 0.683 0.643 limonene -0.086 -0.408 (e)-β-ocimene 0.498 -0.494 terpinolene 1.260 -0.758 (e)-caryophyllene 1.053 -0.140 α-humulene -0.880 -0.840 γ-muurolene 0.016 -0.051 germacrene d 1.010 -2.305 δ-cadinene 0.729 -0.230 germacrene d-4-ol 0.432 -0.959 eigenvalue 1.493 0.344 % explained variation 64.7 79.6 the first discriminant function mainly separated population vi while the second function separated population vii from the other populations (tab. 2, fig. 1). the cda with 7 populations was correctly classified only 53% of individuals on average (classification matrix values, data from the program). however, its results are sufficient to assume the existence of three groups of individuals: (1) populations i, ii, iii, iv, and v which can be designated as ssp. nigra, (2) population vi as ssp. pallasiana, and (3) population vii as ssp. banatica, in accordance with previous results of terpene analysis (š a r a c e t a l . , 2013). however, departing from the first group of individuals (ssp. nigra), population v was partially overlapped with the second group (ssp. pallasina) by axis 1. differentiation suggestion obtained from seven populations was the inducement for performing the cda with three a priori defined groups. table 2. means of canonical variables. population ca1 ca2 i 1.414 -0.241 ii 0.481 -0.247 iii 0.468 -0.149 iv 0.881 -0.106 v -0.654 0.041 vi -2.366 -0.273 vii -0.053 1.988 fig. 1. canonical discriminat analysis (cda) based on the content of 14 terpenes isolated from 195 pinus nigra samples of seven populations (i-vii). the cda based on individuals from three groups (taxa) showed that the first discriminant function explained 73.3% and the second 26.7% of the total discrimination (tab. 3). the first function was mostly determined by the content of (e)caryophyllene, α-humulene and terpinolene (tab 3). the second function was mainly defined by the content of germacrene d, α-pinene, and myrcene. the first discriminant function separated nigra and pallasiana group, while the second function banatica group from the other groups (fig 2). the individuals of pallasiana group had lower content of (e)-caryophyllene and terpinolene and higher content of α-humulene (considering that the majority of individuals was on the right side of the figure). the individuals of nigra group had higher content of (e)-caryophyllene and terpinolene, and lower content of α-humulene (the majority of individuals was on the left side of the figure). the individuals of banactica group had higher content of α-pinene and myrcene. the cda with 3 a priori defined gropus was correctly classified 88% of individuals on average (classification matrix values, data from the program). biologica nyssana 5 (1)  september 2014: 11-15 šarac, z. et al.  application of canonical discriminant analysis… 14 table 3. standardized coefficients for the first two canonical axes (ca) of variation in terpene variables from the discriminant functional analysis of 3 a priori groups. variables ca1 ca2 α-thujene 0,741 -0,436 α-pinene -0.548 1,682 camphene 0,496 0,411 β-pinene -0,464 1,523 myrcene -0,739 -0,621 limonene 0,329 0,281 (e)-β-ocimene -0,348 0,464 terpinolene -0,781 0,785 (e)-caryophyllene -1,069 0,294 α-humulene 0,942 0,670 γ-muurolene 0,177 -0,102 germacrene d -0,477 2,169 δ-cadinene -0,719 0,345 germacrene d-4-ol -0,128 0,832 eigenvalue 0,922 0,336 % explained variation 0,733 1,00 fig. 2. canonical discriminat analysis (cda) based on the content of 14 terpenes isolated from 195 pinus nigra samples of three groups (ssp. nigra, ssp. pallasiana and var. banatica). in accordance with previously published results of ca (š a r a c e t a l ., 2013), cda confirmed that based on the terpene variability in native populations of black pine in serbia, three discrete groups can be distinguished. the first group (nigra) consisted of populations described as var. nigra (populations i-iii) and var. gocensis (populations iv and v) within ssp. nigra according to existing morho-anatomical and phytocoenological studies (j o v a n o v i ć , 1992). also, our very recent (š a r a c et al. (2013) and present study, showed that populations determined as var. gocensis exhibited a moderate tendency of separation from the first group (nigra) (population iv in ca and population v in cda). based on n-alkanes variability, the populations assigned as var. gocensis (iv and v) showed even grater tendency of splitting (bojović et al., 2013). population v has been classified into the first group (nigra), characterized as chemotype 1 by b o j o v i ć et al. (2012), while population iv belonged to the second group (pallasiana), assigned as chemotype 2. it was assumed that populations considered as var. gocensis could be transitional form between subspecies nigra in the west and subspecies pallasiana in the southest of serbia. taxonomic position of var. gocensis within p. nigra complex is still not completely resolved. this variety was primarily described by đ o r đ e v i ć (1931) from the slopes of the mountain goč in central serbia. later, v i d a k o v i ć (1955), determined the wider distribution of this variety, and based on the leaf anatomy raised it to the subspecies level (ssp. gocensis vid.). comparing to the typical var. nigra it is different in some tree traits (bark of mature trees had prominent transverse and longitudinal furrows, resembling the bark of pinus heldreichii h. christ), as well as in needles structure. however, according to results of our analyses there is no such clear phytochemical distinction between var. gocensis and typical var. nigra. the second group is composed mainly of trees of population vi from southeastern serbia belonging to ssp. pallasiana. the presence of crimean pine in serbia was originally reported by a d a m o v i ć (1909), but without precise data on its distribution. r a j e v s k i (1950) was the first who established the position of this population within the territory of serbia. the single enclava of crimean pine is situated at the locality jarešnik in the surroundings of bosilegrad growing on the crystalline slates. the population in serbia represents the northernmost disjunction of the crimean black pine area spreading from the southern parts of balkan peninsula. based on , nalkane and terpene composition, ssp. pallasiana is clearly distinguished as separate group (taxon and chemotype) from ssp. nigra according to this and previous studies (bojović et al., 2012; šarac et al., 2013). finally, the third group consisted of trees from population lazareva reka, determined as var. banatica within ssp. pallasiana (j o v a n o v i ć , 1992). this taxon is previously described in romania (domogled valea cernei) and according to the literature data the black pine populations from northwestern romania have a very controversial taxonomic position (b o ş c a i u & b o ş c a i u , 1999). in the second edition of flora europea, g a u s s e n et al. (1993) treated these populations as an independent species, i.e., p. banatica. analysis biologica nyssana 5 (1)  september 2014: 11-15 šarac, z. et al.  application of canonical discriminant analysis… 15 of the chemodiversity of the black pine populations from serbia, based on both n-alkane and terpene composition, supports the view that var. banatica should be considered as distinct subspecies or even species (b o j o v i ć et al., 2012; š a r a c et al., 2013). conclusion the canonical discriminant analysis (cda) of recently reported terpene composition of p. nigra in serbia (š a r a c et al., 2013), also showed division of populations into three discrete groups. the cda confirmed the attitude that terpenes are good taxonomic markers at infraspecific level in the pinales. nevertheless, all these assumptions should be checked by further extending studies to the entire balkan peninsula and the mediterranean region, and especially including the classical locality (locus classicus) of p. nigra var. banatica from romania (domogled valea cernei). also, detail molecular analysis is needed to clarify relationships witin this extremely variable and complex taxon. references adamović, l. 1909: die vegetationsverhältnisse der balkanländer, leipzig, 258-262. bogunic, f., muratovic, e., ballian, d., siljakyakovlev, s., brown, s.c. 2007: genome size stability of five subspecies of pinus nigra arnold s.l. environmental and experimental botany, 59: 354-360. bojović, s., šarac, z., nikolić, b., tešević, v., todosijević, m., veljić, m., marin, p. d. 2012: composition of n-alkanes in natural populations of pinus nigra from serbia – chemotaxonomic implications. chemistry & biodiversity, 9: 27612774. boşcaiu, n., boşcaiu, m. 1999: on the presence of pinus nigra subsp. pallasiana in romania. wissenschaftliche mitteilungen aus dem niederösterreichischen landesmuseum, 12: 2124. đorđević, p. 1931: pinus nigra arn. var. gočensis, n. var. izdanje ministarstva šuma i rudnika, beograd. gaussen, h., heywood, v.h., charter, a.o. 1993. pinus l.. in: tutin, t.g., heywood, v.h., burges, n.a., valentine, d.h., walters, s.m., webb, b.a. (eds.), flora europea 1: 40-44, cambridge university press, cambridge. james, f.c., mcculloch, c.e. 1990: multivariate analysis in ecology and systematics: panacea or pandoras box?. annuual reviews of ecology and systematics, 21: 129-166. jovanović, b. 1992. p. nigra arn. in: sarić, m. (ed.), flora srbije 1: 200-202, srpska akademija nauka i umetnosti, beograd. lieutier, f., berryman, a.a., millstein, j.a. 1991: preliminary study of the monoterpene response of three pines to ophiostoma clavigerum (ascomycetes: ophiostomatales) and two chemical elicitors. annals of forest science, 48: 377-388. rajevski, l. 1950: nalazište pinus nigra arn. var. pallasiana (lamb.) u okolini bosilegrada. zbornik radova instituta za ekologiju i biogeografiju, srpska akademija nauka i umetnosti, beograd. statsoft 1997. statistica for windows, version 5.1. statsoft inc., tulsa. šarac, z., bojović, s., nikolić, b., tešević, v., đorđević, i., marin, p. d. 2013: chemotaxonomic significance of the terpene composition in natural populations of pinus nigra j. f. arnold from serbia. chemistry & biodiversity, 10: 1507-1520. vidaković, m. 1955: značenje anatomske građe iglica kod svojta crnog bora u jugoslaviji. šumarski list, zagreb, 79: 244-253. von rudloff, e. 1975: volatile leaf oil analysis in chemosystematic studies of north america conifers. biochemical systematics and ecology, 2: 131-167. biologica nyssana 5 (1)  september 2014: 11-15 šarac, z. et al.  application of canonical discriminant analysis… 16 aydin & kadioğlu 2022, biologica nyssana 13(1) 13 (1) september 2022: 33-40 doi: 10.5281/zenodo.7117537 total phenolic content and antioxidant activity of thymus vulgaris, curcuma longa, propolis and their mixtures original article sinem aydin faculty of science and arts, giresun university, department of biology, 28200, giresun, turkey sinem.aydin@giresun.edu.tr (corresponding author) gülşah kadioğlu faculty of science and arts, giresun university, department of biology, 28200, giresun, turkey received: may 23, 2022 revised: july 19, 2022 accepted: september 16, 2022 abstract: the current study, we investigated phenolic content and antioxidant activity of ethyl acetate and dichloromethane extracts of thyme, turmeric, propolis and their mixtures. the highest and the lowest phenolic contents were found in ethyl acetate extract of propolis (214.94±0.023 µg gae/ml) and dichloromethane extract of thyme (21.02±0.013 µg gae/ml). total antioxidant capacity of ethyl acetate extracts ranges from 127.15±0.031 µg aae/ml and 232.2±0.028 µg aae/ml; dichloromethane extracts ranges from 61.6±0.019 µg aae/ml and 159.95±0.035 µg aae/ml. cuprac activity and dpph radical scavenging activity of ethyl acetate extracts are higher than dichloromethane extracts. according to the obtained results, it can be said that propolis, thyme and turmeric could be an alternative to synthetic antioxidants. key words: thyme, turmeric, propolis, antioxidant activity apstrakt: ukupan sadržaj fenola i antioksidativna aktivnost thymus vulgaris, curcuma longa, propolisa i njihovih mešavina u ovoj studiji, ispitivali smo sadržaj fenola i antioksidativnu aktivnost etil acetatnih i dihlormetanskih ekstrakata majčine dušice, kurkume, propolisa i njihovih mešavina. najviši sadržaj fenola ustanovljen je u etil acetatnom ekstraktu propolisa (214.94±0.023 µg gae/ml), a najniži u dihlormetanskom ekstraktu majčine dušice (21.02±0.013 µg gae/ml). ukupna antioksidativna aktivnost etil acetatnih ekstrakata kreće se u opsegu od 127.15±0.031 µg aae/ ml do 232.2±0.028 µg aae/ml, a dihlormetanskih ekstrakata od 61.6±0.019 µg aae/ml do 159.95±0.035 µg aae/ml. cuprac aktivnost i aktivnost uklanjanja dpph radikala viša je kod etil acetatnih ekstrakata u odnosu na dihlormetanske ekstrakte. na osnovu dobijenih rezultata, može se zaključiti da bi propolis, majčina dušica i kurkuma mogli biti alternativa sintetičkim antioksidansima. ključne reči: majčina dušica, kurkuma, propolis, antioksidativna aktivnost introduction medicinal plants are utilized worldwide for the cure of many illnesses such as asthma, gastrointestinal symptoms, skin disorders, respiratory and urinary problems and cardiovascular diseases. plants synthesize various biologically active compounds which are crucial for them to survive in the natural environment and protect them against abiotic stresses derived from temperature, water and mineral nutrient supply (egamberdieva et al., 2017). plants have been utilized as therapeutic resources such as herbal teas, crude extracts or pharmaceutical preparations (tinctures, pills and capsules) for many years. the world health organization (who) predicts that 65% of the world’s population still use plants as traditional medicine (karakaş et al., 2012). medicinal plants has been investigated for their antioxidant capacities by many researchers. natural antioxidants are very effective to hinder the devastating effects caused by oxidative stress. plants, vegetables and fruits have natural antioxidants such as phenolics, flavonoids, tannins and proanthocyanidins. antioxidants present in plants may protect plants from diseases (saeed et al., 2012). © 2022 aydin & kadioğlu. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 33 thymus vulgaris l. (thyme) growing wild in turkey belongs to labiatea family and possess many advantageous effects such as carminative, antiseptic, antioxidant and antimicrobial activities. thymol and carvacrol are major components of thyme essential oil. thymol and other phenolic components in thymus inhibit microorganisms by increasing permeability of the cell membrane and reduction of vital intracellular substances or by disruption of bacterial enzyme systems (tural & turhan, 2017). curcuma longa l. (turmeric) belongs to zingiberaceae family generally utilized in indian and chinese systems of medicine. turmeric spice are obtained from plant rhizome known as “yellow root”. it has been also utilized for the treatment of many diseases. also, c. longa reduces risk of cancer (schaffer et al., 2011)and has antiinflammatory, antioxidant and wound healing properties (maheshvari et al., 2006). propolis is a natural resinous mixture produced from substances collected from some parts of plants, buds and secretions by honey bees. propolis is one of the “natural medicines” utilized since ancient times. more than 300 active compounds were defined in propolis. propolis has antibacterial, antiviral and antioxidant properties. moreover, propolis is utilized in apitheraphy, cosmetic and food industry for its antioxidant and antibacterial features (çoşkun & i̇nci, 2020). in the current study, antioxidant activity and total phenolic contents of dichlorometane and ethanol extracts of thyme, turmeric, propolis and their mixtures (thyme/propolis, turmeric/propolis) at 1/1 ratio have been evaluated. we also targeted to reveal antagonistic and synergistic effects of the combination of thyme, turmeric and propolis extracts. materials and methods providing of the samples thyme, turmeric and propolis were bought from a herbal shop in giresun, turkey. preparation of extracts 20 g of thyme, turmeric and propolis were extracted in a shaker for 24 h utilizing 200 ml ethyl acetate and dichloromethane, separetely. thyme:propolis and curcumin:propolis were extracted with 200 ml ethyl acetate and 200 ml dichloromethane in a shaker for 24 h, separetely. the extracts were filtered through whatman filter paper no. 1 and residues were evaporated (40 °c) with rotary evaporator (murugan & parimelazhagan, 2014). antioxidant activity total phenolic content total phenolic contents of the extracts were determined in accordance with the method of slinkard & singleton (1977) utilizing gallic acid standard. shortly, 0.1 ml extract was diluted with 4.5 ml distilled water. then, 0.1 ml of the folin– ciocalteu reagent (previously diluted 3‐fold with distilled water) was put into the mixture. after 3 minutes, 0.3 ml na2co3 (2%) was added. the absorbance was measured at 760 nm after incubating the mixture for 90 min. total phenolic content of the extracts was expressed as µg gallic acid equivalents (gae)/ml by using the calibration curve. the tests were performed in triplicate (slimkard & singleton, 1977). total antioxidant capacity phosphomolybdenum method was used to determine total antioxidant capacity of the extracts. 0.3 ml extract and 3000 µl reagent (contains 0.6 m sulfuric acid, 28 mm sodium phosphate and 28 m ammonium molybdate) was mixed and incubated at 95 °c for 90 min. then, absorbance was read at 695 nm. ascorbic acid was used as the standard (prieto et al., 1999). the total antioxidant capacity was expressed as µg ascorbic acid equivalent (aae)/ml. the tests were performed in triplicate. cupric reducing antioxidant capacity (cuprac) test 0.5 ml extract (250-1000 µg/ml concentration), 1.0 ml cucl2 solution (1x10-2), 1.0 ml neocuproine solution (7.5x10-3 m) and 1.0 ml ammonium acetate buffer (1.0 m, ph: 7.0) were mixed in a test tube. then, the tube was vortexed and stored in a dark place for 30 min. after this period, the absorbance was read at 450 nm. butylated hydroxytoluene (bht) was used as a standard antioxidant agent (özyürek et al., 2009). dpph radical scavenging activity dpph radical scavenging activity of the extracts was established by dpph. appropriate dilution series (250-1000 µg/ml) were prepared for ethanolic extracts in dmso. 0.75 ml of each solution was added to 1.5 ml of a 6x10-5 m methanolic solution of dpph. the mixture was stirred vigorously and allowed to stand in the dark at the room temperature for 30 min. decrease in absorbance of the solution against methanol was measured at 517 nm with a shimadzu 1240 uv-vis spectrophotometer (williams et al., 1995). rutin and butylated hydroxytoluene (bht) were used as standard antioxidants. the dpph radical scavenging activity was calculated using the following equation: 34 biologica nyssana ● 13 (1) september 2022: 33-40 aydin & kadioğlu ● total phenolic content and antioxidant activity of thymus vulgaris, curcuma longa, propolis and their mixtures 35 biologica nyssana ● 13 (1) september 2022: 33-40 aydin & kadioğlu ● total phenolic content and antioxidant activity of thymus vulgaris, curcuma longa, propolis and their mixtures dpph radical scavenging activity (%)=[(a0a1)/a0]x100 a0: absorbance of control a1: absorbance of extract or standard results and discussion total phenolic content phenolic compounds are significant plant metabolites which have redox properties responsible for antioxidant activity (aryal et al., 2019). total phenolic content was determined by utilizing the folin–ciocalteu reagent. the results were calculated from a calibration curve (y = 0.013x, r2 = 0.9934) of gallic acid and expressed as µg gallic acid equivalent (gae)/ml (tab. 1). the highest and the lowest phenolic contents were found in ethyl acetate of propolis (214.94±0.023 µg gae/ml) and dichloromethane extract of thyme (21.02±0.013 µg gae/ml). ethyl acetate extracts exhibited higher total phenolic content than dichloromethane extracts except for ethyl acetate extract of thyme/ propolis. total phenolic contents of ethyl acetate and dichloromethane extracts of thyme/propolis and turmeric/propolis were decreased when compared with ethyl acetate and dichloromethane extracts of thyme, turmeric and propolis except for dichloromethane extract of thyme/propolis. total phenolic content of thyme, turmeric and propolis was also searched by many authors. for example, bulut et al. (2020) found total phenolic content of ethanol extracts of thyme leaves as 7.01±0.13 mg gae/g (bulut et al., 2020). köksal et al. (2017) determined total phenolic content of lyophilized water extract and ethanol table 1. total phenolic contents of the extracts (µg gae/ml) extract total antioxidant capacity ethyl acetate extract of thyme 73.25±0.030 ethyl acetate extract of turmeric 175.74±0.050 ethyl acetate extract of propolis 214.94±0.023 ethyl acetate extract of thyme/propolis 129.2±0.007 ethyl acetate extract of turmeric/propolis 173.61±0.008 dichloromethane extract of thyme 21.02±0.013 dichloromethane extract of turmeric 93.66±0.013 dichloromethane extract of propolis 107.82±0.011 dichloromethane extract of thyme/propolis 174.74±0.029 dichloromethane extract of turmeric/propolis 82.89±0.025 extracts of thyme as 256 µg gae/mg and 158 µg gae/mg, respectively (köksal et al., 2017). erdoğan & erbaş (2021) stated that total phenolic content of ethanol extract of turmeric was 82.47±2.70 mg gae/g (erdoğan & erbaş, 2021). yan & asmar (2010) declared that total phenolic content of methanol extract of fresh and powder of turmeric was 348.±1.26 mg gae/100 g and 2013.09±5.13 mg gae/100 g, respectively (yan & asmah, 2010). keskin & kolayli (2019) reported that the total phenolic substance amount of anatolian propolis ranged between 16.13-178.34 mg gae/g (keskin & kolayli, 2019). özdal et al. (2019) reported that the total phenolic substance amount of propolis obtained from different regions of anatolia varies between 2,748 mg gae/100 g and 19,969 mg gae/100 g (özdal et al., 2019). collecting plants from different locations, using different extraction methods and solvents may cause discrepancy in results. total antioxidant capacity total antioxidant capacity method is based on the reduction of mo (vi) to mo (v) by the extract and subsequent formation of green phosphate/mo (v) complex at acid ph. ascorbic acid was utilized to compare total antioxidant capacity of the extracts (aliyu et al., 2012). tab. 2 shows total antioxidant capacity of extracts. while total antioxidant capacity of ethyl acetate extracts ranges from 127.15±0.031 µg aae/ml and 232.2±0.028 µg aae/ml; dichloromethane extracts ranges from 61.6±0.019 028 µg aae/ml and 159.95±0.035 µg aae/ ml. ethyl acetate extracts exhibited higher total antioxidant capacity than dichloromethane extracts except for ethyl acetate extract of thyme/propolis. total phenolic contents of ethyl acetate and dichloromethane extracts of thyme/propolis and turmeric/ propolis were decreased when compared with ethyl acetate and dichloromethane extracts of thyme, turmeric and propolis except for dichloromethane extract of thyme/propolis. this situation might be arised by the interactions among the active substances in propolis and thyme or turmeric. the presence of phenolic compounds could be attributable to the observed high total antioxidant capacity. many surveys were done by other researchers about total antioxidant capacity of propolis, thyme and turmeric. yılmaz et al. (2017) investigated total antioxidant capacity of propolis collected from sakyatan (ks) and kızılören biologica nyssana ● 13 (1) september 2022: 33-40 36 (kk) regions of konya and they found total antioxidant capacities of ks propolis and kk propolis as 2.21±0.11 mmol tes/g extract and 2.40±0.15 mmol tes/g extract, respectively (yılmaz et al., 2017). özcan & özkan (2018) investigated total antioxidant activity of different extracts of thyme and they found that total antioxidant activity of thyme ranges from 91.14±0.87 -123.34±0.95 mg aae/g. bulus et al. (2017) determined that total antioxidant capacity of butanol extract of turmeric was 370 aae/g. our results and literature results are different. this differences can be explained with collecting sample from different locations, using different solvents and extraction techniques. table 2. total antioxidant capacity of the extracts (µg aae/ml) extract total antioxidant capacity ethyl acetate extract of thyme 127.15±0.031 ethyl acetate extract of turmeric 177.16±0.021 ethyl acetate extract of propolis 232.2±0.028 ethyl acetate extract of thyme/propolis 147.52±0.031 ethyl acetate extract of turmeric/propolis 174.6±0.046 dichloromethane extract of thyme 61.6±0.019 dichloromethane extract of turmeric 113.85±0.019 dichloromethane extract of propolis 101±0.010 dichloromethane extract of thyme/propolis 159.95±0.035 dichloromethane extract of turmeric/propolis 85.04±0.038 extract concentration (µg/ml) cuprac activity ethyl acetate extract of thyme 2501.4479±0.031 5002.2108±0.0006 7502.3390±0.020 10002.3562±0.088 ethyl acetate extract of turmeric 2501.7463±0.039 5001.9936±0.003 7502.0067±0.022 10002.0151±0.017 ethyl acetate extract of propolis 2501.9856±0.073 5001.9761±0.024 7502.0374±0.019 10002.0573±0.109 ethyl acetate extract of thyme/propolis 2501.801±0.043 5001.8249±0.030 7501.9752±0.075 10002.0055±0.049 ethyl acetate extract of turmeric/propolis 2501.8199±0.018 5002.1059±0.0462 7502.0493±0.019 10001.9689±0.041 dichloromethane extract of thymus 2500.5513±0.032 5000.6634±0.049 7501.1269±0.018 10001.3479±0.037 table 3. cuprac activity of the extracts extract concentration (µg/ml) cuprac activity dichloromethane extract of turmeric 2501.3326±0.042 5001.8600±0.029 7501.8966±0.017 10001.9426±0.055 dichloromethane extract of propolis 2501.6693±0.059 5001.8658±0.005 7502.0466±0.021 10002.1393±0.013 dichloromethane extract of thyme/propolis 2501.8020±0.015 5001.9255±0.011 7501.9895±0.011 10002.0625±0.078 dichloromethane extract of turmeric/propolis 2500.9922±0.016 5001.4591±0.051 7501.7229±0.044 10001.7569±0.007 bht 2500.6635±0.023 5000.7016±0.021 7500.8283±0.024 10000.9716±0.014 aydin & kadioğlu ● total phenolic content and antioxidant activity of thymus vulgaris, curcuma longa, propolis and their mixtures aydin & kadioğlu ● total phenolic content and antioxidant activity of thymus vulgaris, curcuma longa, propolis and their mixtures biologica nyssana ● 13 (1) september 2022: 33-40 37 cuprac test tab. 3 presents cuprac activity of the extracts. ethyl acetate extracts had better cuprac activity than dichloromethane extracts at 1000 µg/ml concentration. cuprac activity of ethyl acetate and dichloromethane extracts of thyme/propolis and turmeric/propolis were decreased when compared with ethyl acetate and dichloromethane extracts of thyme, turmeric and propolis except for dichloromethane extract of thyme/propolis at concentration of 1,000 µg/ml. this situation might be a consequence of the interactions among active substances in propolis and thyme or turmeric. the results indicate a concentration dependent cuprac activity. all extracts had higher activity than bht. dpph radical scavenging activity tab. 4 demonstrates dpph radical scavenging potentials of extracts at different concentrations (250-1000 µg/ml) measured as a degree of discoloration displayed the extracts’ scavenging potential. dichloromethane extract of thyme showed no activity, while all the extracts exhibited lower activity than bht and rutin. table 4. dpph radical scavenging activity of the extracts and standards (% inhibition) extract concentration (µg/ml) dpph radical scavenging activity ethyl acetate extract of thyme 250 not activity 500 7.54±0.002 750 39.04±0.0009 1000 67.56±0.004 ethyl acetate extract of turmeric 250 10.3±0.002 500 38.53±0.0013 750 55±0.004 1000 75.25±0.006 ethyl acetate extract of propolis 250 66.4±0.005 500 67.99±0.004 750 73.36±0.001 1000 79.53±0.001 ethyl acetate extract of thyme/propolis 250 45.63±0.008 500 73.19±0.005 750 77.57±0.005 1000 81.64±0.003 ethyl acetate extract of turmeric/propolis 250 38.02±0.036 500 61.68±0.009 750 65.23±0.008 1000 74.16±0.004 dichloromethane extract of thymus 250 not activity 500 not activity 750 not activity 1000 not activity dichloromethane extract of turmeric 250 50.94±0.001 500 55.22±0.002 750 59.94±0.001 1000 70.39±0.014 biologica nyssana ● 13 (1) september 2022: 33-40 aydin & kadioğlu ● total phenolic content and antioxidant activity of thymus vulgaris, curcuma longa, propolis and their mixtures dpph radical scavenging activity of ethyl acetate and dichloromethane extracts of thyme/ propolis and turmeric/propolis were increased when compared with ethyl acetate and dichloromethane extracts of thyme, turmeric and propolis at 1,000 µg/ml concentration. the best activity was detected in ethyl acetate extract of thyme/propolis (81.64%) and the worst activity was detected in ethyl acetate extract of thyme (67.56%) concentration of 1,000 µg/ml. ethyl acetate extracts generally showed better activity than dichloromethane extracts. dpph radical scavenging activity was searched by many authors. can et al. (2015) concluded that dpph scavenging activity of propolis from azerbaijan ranges from 15±1.00-198±3.40 (can et al., 2015). köksal et al. (2017) found dpph scavenging activity (ic50 value) of lyophilized water extract and ethanol extract of thyme as 13.4 and 12.1, respectively (köksal et al., 2017). priyanka et al. (2017) investigated dpph scavenging activity (% inhibition) of turmeric cultivars and they found that activity ranges from 49.63±2.97 to 59.58±2.95 (priyanka et al., 2017). these differences might be a consequence of used solvent and different location of material collection. conclusions the results suggest that the thyme, turmeric and propolis utilized in the current study possess antioxidant properties. thyme, turmeric and propolis also can be used as ingredients for development of a new antioxidant agents. further work should be focused on the isolation and elucidation of secondary metabolites in thyme, turmeric and propolis responsible for the antioxidant activity. antioxidant activity of mixtures are lower than thyme, turmeric and propolis because of interactions of active substances in mixtures. since the antioxidant activity of plant mixtures with propolis is lower than the antioxidant activities of these plants and propolis alone, plants and propolis should be consumed individually, not as a mixture. references aliyu, a.b., ibrahim, m.a., ibrahim, h., musa, a.m., lawal, a.y., oshanimi, j.a., usman, m., abdulkadir, i.e., oyewale, a.o., amupitan, j.o. 2012: free radical scavenging and total antioxidant capacity of methanol extract of ethulia conyzoides growing in nigeria. romanian biotechnological 38 extract concentration (µg/ml) dpph radical scavenging activity dichloromethane extract of propolis 250 26.41±0.033 500 30.69±0.033 750 41.67±0.023 1000 69.08±0.005 dichloromethane extract of thyme/propolis 250 52.9±0.066 500 68.57±0.002 750 70.1±0.001 1000 74.81±0.002 dichloromethane extract of turmeric/propolis 250 59.5±0.018 500 69.81±0.014 750 71.77±0.016 1000 76.48±0.019 bht 250 88.85±0.012 500 89.55±0.005 750 90.27±0.011 1000 91.55±0.008 rutin 250 86.80±0.008 500 87.91±0.003 750 90.60±0.004 1000 91.89±0.011 biologica nyssana ● 13 (1) september 2022: 33-40 aydin & kadioğlu ● total phenolic content and antioxidant activity of thymus vulgaris, curcuma longa, propolis and their mixtures letters, 17(4): 7458-7465. aryal, s., baniya, m.k., danekhu, k., kunwar, p., gurung, r., koirala, n. 2019: total phenolic content, flavonoid content and antioxidant potential of wild vegetables from western nepal. plants (basel, switzerland), 8(4): 96. brand-williams, w., cuvelier, m., berset, c. 1995: use of a free radical method to evaluate antioxidant activity. lebensmittel-wissenschaft und technologie food science and technology, 28: 2530. bulus, t., david, s.i., bilbis, l.s., babando, a. 2017: in vitro antioxidant activity of n-butanol extract of curcuma longa and its potentıal to protect erythrocytes membrane agaınst osmotıc-induced haemolysıs. science world journal, 12(1): 13-17. bulut, m., akpolat, h., tunçtürk, y., alwazeer, d., türkhan, a. 2020: determination of optimum ethanolic extraction conditions and phenolic profiles of thyme, mint, uckun, grape seeds and green tea waste fiber. international journal of agriculture and wildlife science, 6(3): 605-614. can, z., yıldız, o., şahin, h., asadov, a., kolaylı, s. 2015: phenolic profile and antioxidant potential of propolis from azerbaijan. mellifera, 15(1): 16-28. çoşkun, p., i̇nci, h. 2020: antibacterial, antiviral, antioxidant activity and chemical content of propolis. ispec journal of agricultural sciences, 4(4): 10531070. egamberdieva, d., wirth, s., behrendt, u., ahmad, p., berg, g. 2017: antimicrobial activity of medicinal plants correlates with the proportion of antagonistic endophytes. frontiers in microbiology, 8: 199-210. erdoğan, ü., erbaş, s. 2021: phytochemical profile and antioxidant activities of zingiber officinale (ginger) and curcuma longa l. 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longa, propolis and their mixtures tural s., turhan s. 2017: antimicrobial and antioxidant properties of thyme (thymus vulgaris l.), rosemary (rosmarinus officinalis l.) and laurel (lauris nobilis l.) essential oils and their mixtures. gıda the journal of food, 42(5): 588-596. yan, s.w. asmah, r. 2010: comparison of total phenolic contents and antioxidant activities of turmeric leaf, pandan leaf and torch ginger flower. international food research journal, 17: 417-423. yılmaz, s.c., azman, z.n., kosem, k., gündüz, e., grenman, r. 2017: evaluating antioxidant capacity of different propolis samples from konya, turkey and their inhibitory effect on head and neck cancer cells. biorxiv, 183913. 40 anticancer compounds from medicinal plants biologica nyssana 2 (2)  december 2011: 00-00 nahirnić a.  supplements of butterfly fauna… 77 original article phytogeographical analysis of the flora of miljkovačka gorge in eastern serbia milica miljković, novica ranđelović, vladimir ranđelović university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia * e-mail: milica.zeka@yahoo.com abstract: miljković, m. ranđelović, n., ranđelović, v.: phytogeographical analysis of the flora of miljovačka gorge in eastern serbia. biologica nyssana, 3 (2), december 2012: 77-90. as a result of two-year investigation of the miljkovacka gorge flora, carried out during the 2010. and 2011., 331 plant taxa belonging to 245 genera and 70 families were recorded and sorted into 112 floristic elements, 15 area groups and 9 area types. phytogeographical analysis showed that the species of eurasian area type are the most abundant ones. key words: area group, area type, floristic element, miljkovačka gorge introduction one of the major biodiversity hotspots is balkan peninsula (h o r v a t et al., 1974), and the miljkovacka gorge is situated in eastern serbia, se of niš, bordered by mt kalafat (837 m), mt kamenički vis (813 m) from south and hillsides of mt devica (1187 m) from north and northeast, which belong to carpathian-balkan group of mountains (fig. 1). bela river that flows beneath mt devica and kopaljkosarska river that flows beneath mt kalafat are flowing into each other near popsica village making popsica river. during its passage through the village kravlje, it changes name to kravljanska river. on its way to south morava, it changes its name twice more: miljkovačka river and toponička river. as toponička river, it finally flows into south morava. the geological structure of the miljkovačka gorge is represented by mesozoic limestone. the pedological substrate is predominantly consisted of cambisoil and red soil. the climate of this area is humid continental with two extremely wet and cold periods (from january to may and from september to december) and one arid period during the summer. miljkovačka gorge is not well explored area. the first data concerning this area were collected by spas sotirov in 1977, who found the new locality of endemic-relict plant species, ramonda serbica in the valley of kravljanska river. m a r t i n o v i ć et al. (1985) published paper which was the first phytogeographical investigation of miljkovačka gorge. the more significant and widely extended breakthrough in the studies of the flora and vegetation of the miljkovačka gorge was made during 2010. and 2011. (m i l j k o v i ć , 2011; m i l j k o v i ć et al., 2012). materials and methods the study is based on literature data (s o t i r o v , s t a n o j e v i ć , 2012), herbarium collections (hmd), and continuous field observations carried out during the 2010 and 2011. herbarium specimens are deposited at the herbarium moesiacum (hmn). identification of the collected plants was performed according to flora 3 (2) • december 2012: 77-90 mailto:%20branko_jotic@live.com biologica nyssana 3 (2)  december 2012: 77-90 miljković m. et al.  phytogeographical analysis of the flora… 78 europaea (t u t i n et al.., 1964-1980) and the regional floras relevant for the investigated area (j o r d a n o v , 1963-1979, v e l c h e v 1982-1995, j o s i f o v i ć , 1970-1977, m i c e v s k i , 1985-1995, s a r i ć , 1986, 1992). the nomenclature follows med-checklist (g r e u t e r et al., 1984-1989), flora europaea (t u t i n et al., 1964-1980), and iopi – international organization for plant information (http://plantnet.rbgsyd.nsw.gov.au/iopi/iopihome.ht m). for the area types classification m e u s s e l et al. (1965, 1978), m e u s s e l and j ä g e r (1992) and s t e v a n o v i ć (1992) were used. results as a result of floristic investigations of the miljkovačka gorge in eastern serbia, region in the central part of the balkan peninsula, 331 plant taxa belonging to 245 genera and 70 families were recorded (tab. 1). figure 1. geographical position of investigated area table 1. list of vascular flora with area types, area groups and floristic elements taxa area type area group flostic element equisetopsida equisetaceae equisetum arvense l. hol circumcholarct. -(arct-merid) equisetum maximum lam. hol europ.-north amer.-(bor-merid) aspleniaceae polypodiopsida asplenium ceterach ea asplenium ruta-muraria l. hol asplenium trichomanes l. cosm cosm.-amfiatl. origin magnolyopsida acanthaceae acanthus balcanicus heywood & i. b. k. richardson med-smed balk http://plantnet.rbgsyd.nsw.gov.au/iopi/iopihome.htm http://plantnet.rbgsyd.nsw.gov.au/iopi/iopihome.htm biologica nyssana 3 (2)  december 2012: 77-90 miljković m. et al.  phytogeographical analysis of the flora… 79 taxa area type area group flostic element aceraceae acer monspessulanum l. med-smed acer tataricum l. ea ce-m-p-or adoxaceae adoxa moschatelina l. hol circumcholarkct.-(temp-submer) anacardiaceae cotinus coggygria scop. msm apiaceae aegopodium podagraria l. ea eurasian-(bor-merid) berula erecta (hudson) coville hol circumcholarkct.-(bor-merid) carum carvi l. ea eurasian-(temp-submer) danaa cornubiensis (l.) burnat med-smed daucus carota l. phol-ptrop central europ.-medit.-pont.orient.-turan.-east african eryngium campestre l. ea falcaria vulgaris bernh. ea heracleum sphondylium l. ce central european pastinaca sativa l. ea eurasian-(bor-merid) pimpinella saxifraga l. ea eurasian-(temp-mer) seseli rigidum waldst. & kit. med-smed skarp-balk seseli varium trev. ea ce-med torilis arvensis (hudson) link ea ce-med araliaceae hedera helix l. ea ea(w) aristolochiaceae aristolochia clematitis l. med-smed asarum europaeum l. ea eurasian asclepiadaceae cynanchum vincetoxicum (l.) pers. ea ea(w) europ.-medit.-west-asian asteraceae achillea crithmifolia waldst. & kit. msm med-pont panon.-skard.-pind.-mac.-trak.moes.-danub. achillea millefolium l. ea eurasian-(bor-submer) achillea millefolium l. forma rubra ea eurasian-(bor-submer) arctium lappa l. ea eurasian-(temp-submer) artemisia absinthium l. ea eurasian-(subbor-mer) artemisia alba l. med-smed artemisia annua l. ea artemisia scoparia waldst. & kit. ea artemisia vulgaris l. hol circumcholarkct.-(subbor-mer) bellis perennis l. ea ce-med central european-medit.submedit. bidens tripartita l. ea eurasian-(bor-temp) bombyciealena erecta (l) smolj. ea ce-med-pont carduus acanthoides l. ce central european carduus nutans l. ea carthamus lanatus l. ea ce-med-pont biologica nyssana 3 (2)  december 2012: 77-90 miljković m. et al.  phytogeographical analysis of the flora… 80 taxa area type area group flostic element centaurea solsticialis l. msm medit.-submedit.-pont.-south sibir-orijent.-turan. centaurea stoebe l. ea ea(w) chrysanthemum parthenium l. ea ea(w) cirsium palustre (l.) scop. bor eur.-west asian-(bor-temp) crepis pulchra l. ea ce-m-p-or crepis setosa haller fil. med-smed medit.-submedit. crupina vulgaris cass. msm med-pont echinops sphaerocephalus l. ea erigeron canadensis l. adv north american eupatorium cannabium l. ea ea(w) europ.-medit.-west-asian galinsoga parviflora cav. adv south american hieracium pilosella l. ea ea(w) europ.-medit.-west-asian inula conysa dc. ea ce-m-p-or sarm.-medit.-smed.-pont.-orijent. inula germanica l. ea ce-m-p-or inula helenium l. ea ce-m-p-or inula oculus-christi l. ea ce-med-pont lactuca perennis l. ea ce-med-pont matricaria trichophylla (boiss.) boiss. ea ce-med herc.-panon.-east submedit. mycelis muralis (l.) dumort. ea ea(w) europ.-medit.-west-asian petasites hybridus (l.) p. gaertner, b. meyer & scherb. ea eurasian-(bor-submer) ptilestemon afer (jacqu.) greut. med-smed south carp.-moes-mac.-trac.skard. pind. senecio jacobaea l. ea ce-med-pont senecio vernalis waldst. & kit. ea ea(w) europ.-medit.-west-asian stenactis annua (l.) less. ea ce-med-pont taraxacum officinale weber ea eurasian-(bor-temp) tussilago farfara l. ea central european-medit.submedit.-pont.-south sibir.central asian xeranthemum annuum l. med-smed submedit. xeranthemum cylindraceum szb. et sm. msm med-pont berberidaceae berberis vulgaris l. ea ea(w) boraginaceae anchusa officinalis l. ea ce-med-pont central european-east submedit.-west pont. echium italicum l. med-smed echium vulgare l. ea eurasian-(bor-merid) symphytum officinale l. ea ea(w) atlant-subatlant.-medit.submedit.-pont.-south-sibir. symphytum tuberosum l. msm med-pont medit.-submedit.-pont. brassicaceae alliaria officinalis andrz. ex bieb. ea ea(w) europ.-medit.-west-asian alyssum alyssoides l. ea ea(w) alyssum murale waldst. & kit. msm med-pont alyssum saxatile l. ea ce-med-pont arabis recta will. ea ce-med-pont biologica nyssana 3 (2)  december 2012: 77-90 miljković m. et al.  phytogeographical analysis of the flora… 81 taxa area type area group flostic element conringia orientalis (l.) dumort. ea ea(w) draba aizoides l. ea ce-med erysimum comatum pancic med-smed balk moesian-skard. pind. erysimum diffusum ehrh. ea ce-m-p-or east medit.-pont.-panon.-south sibir.-turan. hesperis matronalis l. ea ea(w) campanulaceae campanula bononiensis l. ea campanula lingulata waldst. & kit. ea ce-med karp.-panon.-balk. campanula rapunculoides l. ea ea(w) europ.-medit.-west-asian campanula trachelium l. ea eurasian-(bor-merid) cannabaceae humulus lupulus l. ea caprifoliaceae sambucus ebulus l. ea central european-medit.submedit.-pont.-south sibirorijent.-turan. caryophyllaceae cerastium brachypetalum pers. ea ce-med cerastium pumilum curtis ea ce-med cucubalus baccifer l. ea dianthus armeria l. ea ce-med dianthus petraeus waldst. & kit. ea ce-med ckarp-balk dianthus pontederae a. kerner ea ce-med herniaria hirsuta l. ea ce-m-p-or central european-submedit.-west asian-(temp-merid) holosteum umbellatum l. ea lychnis coronaria (l.) desr. ea ce-m-p-or central european-submedit.-kavk.turan. moehringia muscosa l. eam csem central-south-europ.-mont. petrorhagia illyrica (l.) p. w. ball & heywood med-smed petrorhagia saxifraga (l.) link ea ea(w) europ.-medit.-west-asian saponaria officinalis l. ea eurasian-(temp-submer) silene alba (miller) w. greuter & burdet ea eurasian-(bor-merid) silene flavescens waldst. & kit. ea ce-med silene viridiflora l. ea ce-m-p-or central european-central medit.east submedit.-orijent.-turan. silene vulgaris (moench) garcke ea eurasian-(bor-merid) stellaria holostea l. ea ea(w) europ.-medit.-west-asian stellaria media (l.) vill. cosm cosm-eurasian origin celastraceae euonymus europaeus l. ea ce-med europ.-submedit. chenopodiaceae polycnemum arvense l. ea cistaceae helianthemum nummularium (l.) miller ea ea(w) europ.-medit.-west-asian convolvulaceae calystegia sepium (l.) r. br. cosm cosm.-europ.-northamer. origin biologica nyssana 3 (2)  december 2012: 77-90 miljković m. et al.  phytogeographical analysis of the flora… 82 taxa area type area group flostic element convolvulus arvensis l. cosm cosm.mediter. origin convolvulus cantabricus l. ea ce-med cornaceae cornus mas l. ea ce-med cornus sanguinea l. ce corylaceae carpinus orientalis miller ea ce-med crassulaceae ce-med sedum acre l. ce ce-med atlant.-central europeansubmedit. sedum dasyphyllum l. med-smed sedum hispanicum l. med-smed e smed submedit. sedum ochroleucum chaix med-smed e smed sedum telephium l. ssp. maximum (l.) krocker med-smed e smed sempervivum marmoreum griseb. ea med-pont cucurbitaceae bryonia alba l. ea ce-m-p-or cuscutaceae cuscuta epithymum l. ea eur.-med.-smed.-pont.-orijent.turan.-himal. dipsacaceae cephalaria flava (sibth. & sm.) szabó med-smed balk moesian-skard. pind. dipsacus laciniatus l. ea ea(w) knautia drymeia heuffel eam csem alp.-balk. scabiosa ochroleuca l. ea ea(w) europ.-medit.-west-asian euphorbiaceae euphorbia amygdaloides l. ea ea(w) europ.-medit.-west-asian euphorbia cyparissias l. ea eurosib.-(bor-merid) euphorbia polychroma a. kerner med-smed euphorbia salicifolia host ea ce-med-pont euphorbia taurinensis all. med-smed mercurialis perennis l. ea ea(w) europ.-medit.-west-asian fabaceae astragalus cicer l. ea ce-med galega officinalis l. ea med-pont pontsko-istočnosubmedit. genista tinctoria l. ea ea(w) europ.-medit.-west-asian lathyrus venetus (miller) wohlf. ea ce-med-pont lathyrus vernus (l.) bernh. ea ce-med europ.-medit.-west-asian medicago arabica (l.) hudson ea ce-med-pont medicago falcata l. ea medicago prostrata jacq. ea ce-med-pont melilotus officinalis (l.) pallas ea eurasian ononis spinosa l. ea ce-med atlant.-central europeansubmedit. trifolium alpestre l. ea ea(w) europ.-medit.-west-asian trifolium arvense l. ea ea(w) europ.-medit.-southsib. trifolium campestre schreber ea ce-m-p-or central european-medit.submedit.-pont.-orijent.-turan. biologica nyssana 3 (2)  december 2012: 77-90 miljković m. et al.  phytogeographical analysis of the flora… 83 taxa area type area group flostic element trifolium dalmaticum vis. med-smed e smed trifolium pratense l. ea ea(w) europ.-submedit.-pont.-southsibir. trifolium repens l. hol circumcholarkct.-(arct-submer) vicia lathyroides l. ea ea(w) vicia villosa roth ea ea(w) europ.-medit.-west-asian fagaceae fagus moesiaca k.maly ce moesian quercus cerris l. med-smed quercus frainetto ten. ea ce-med quercus pubescens willd. ea ce-med gentianaceae ce-med centaurium erythraea rafin. ea ea(w) europ.-medit.-west-asian geraniaceae geranium lucidum l. ea geranium molle l. ea geranium robertianum l. cosm sec. cosm. -eurasian. origin gesneriaceae ramonda serbica panč. med-smed balk moesian-skard. pind. hypericaceae hypericum boissieri petrovic med-smed balk hypericum perforatum l. ea europ.-medit.-west-asian hypericum rumeliacum boiss. med-smed skarp-balk lamiaceae acinos arvensis (lam.) dandy ea ce-med-pont central european-submedit.pont.-south sibir. acinos hungaricus (simonkai) [ilic ea ce-med-pont herc.-panon.-south sarmat.medit.-submedit.-pont. ajuga reptans l. ea ce-med central european-submedit. calamintha sylvatica bromf. ea ce-m-p-or sublant.-central europeansubmedit.-pont.-orijantal.-turan. clinopodium vulgare l. ea ce-med-pont eurasian-(subbor-submer) glechoma hederacea l. ea eurasian-(subbor-submer) glechoma hirsuta waldst. & kit. ea ce-med-pont subatl.-ce-medit.-pont. hyssopus officinalis l. med-smed lamium amplexicaule l. hol phol-ptrop eurasian-(temp-smer)-east-afric. lamium galeobdolon l. ce central european lamium garganicum l. ea(m) csem lamium maculatum l. ea ce-med-pont central european-submedit.pont. leonurus cardiaca l. ea ce-m-p-or herc.-sarmat.-submedit.-pont.south sibir.-orijental.-turan. lycopus europaeus l. ea ea(w) europ.-medit.-west-asian marrubium peregrinum l. ea ce-med-pont melissa officinalis l. msm medit.-east submed.-orijent.turan. melittis melissophyllum l. ce mentha aquatica l. hol phol-ptrop europ.-(subbor-mer)-east.-afric.(borsubtro)-afric-(austr) biologica nyssana 3 (2)  december 2012: 77-90 miljković m. et al.  phytogeographical analysis of the flora… 84 taxa area type area group flostic element mentha longifolia (l.) hudson hol phol-ptrop europ.-west-asian-(temp-mer)east-afric.-(borstro)-afric-(austr) mentha pulegium l. ea micromeria cristata (hampe) griseb. med-smed balk moesian origanum vulgare l. ea eurasian-(temp-mer) prunella laciniata l. ea ea(w) atlant.-central-europ.-medit.orijental prunella vulgaris l. ea ea(w) europ.-medit.-west-asian salvia nemorosa l. ea ce-m-p-or central european-submedit.pont.-orijent.-turan. salvia sclarea l. ea ce-m-p-or satureja kitaibelii wierzb. med-smed scutellaria columnae all. med-smed sideritis montana l. ea ce-med stachys germanica l. msm med-pont medit.-submedit.-pont. stachys palustris l. hol circumcholarkct.-(bor-submer) stachys recta l. ea ea(w) stachys sylvatica l. ea ea(w) europ.-medit.-west-asian teucrium chamaedrys l. ea ea(w) europ.-medit.-west-asian teucrium montanum l. ea(m) csem teucrium polium l. med-smed thymus marschalianus willd. ea ce-med thymus pulegioides l. ce central european lythraceae lythrum salicaria l. cosm eurasian disjunct-(subbor-mer)southeastern austr. (austr.) malvaceae alcea rosea l. med-smed althaea officinalis l. ea oenotheraceae circaea lutetiana l. hol circumcholarkct.-(bor-merid) epilobium hirsutum l. hol phol-ptrop euras.-(subbor-mer)-afric(borsubtro-austrosubtro) epilobium lanceolatum sebastiani & mauri ea ea(w) epilobium montanum l. ea eurasian oenothera biennis l. adv oleaceae fraxinus ornus l. ea ce-med ligustrum vulgare l. ea ce-med-pont syringa vulgaris l. ea ce-med-pont paeoniaceae paeonia decora g. anderson msm med-pont papaveraceae chelidonium majus l. ea ea(w) amfiatlant.-central-europ.medit.-west-asian-(bor-merid) biologica nyssana 3 (2)  december 2012: 77-90 miljković m. et al.  phytogeographical analysis of the flora… 85 taxa area type area group flostic element corydalis bulbosa (l.) dc ea ea(w) european-west asian papaver rhoeas l. ea european-medit.-submedit.-pont.south sibir.-orijental. plantaginaceae plantago lanceolata l. ea eurasian-(subbor-temp) plantago major l. cosm cosm.-europ.-northamer. origin plantago media l. ea eurasian-(temp-submer) polygonaceae bilderdykia convolvulus (l.) dumort. ea eurasian-(bor-merid) polygonum hydropiper l. ea eurasian-(temp) portulacaceae portulaca oleracea l. adv primulaceae anagallis arvensis l. cosm cosm.mediter. origin lysimachia nummularia l. ce central european lysimachia punctata l. ea ce-pont lysimachia vulgaris l. ea eurasian-(temp-submer) ranunculaceae anemone ranunculoides l. ce central european clematis vitalba l. ea ea(w) europ.-medit.-west-asian helleborus odorus waldst. & kit. ce central european (temp-submer) hepatica nobilis schreber ce isopyrum thalictroides l. ea ce-med-pont herc.-sarmat.-pan.-medit.submedit.-pont.-southsib. nigella arvensis l. ea ce-med ranunculus millefoliatus vahl med-smed ranunculus repens l. ea eurasian-(bor-merid) ranunculus serbicus vis. eam sem apen.-balk. ranunculus stevenii andrz. ex besser msm med-pont pont.-submedit. thalictrum lucidum l. ce central european rosaceae agrimonia eupatoria ledeb. ea evropsko-mediteransko-pontskoorijentalno-južnosibirskoturanski aremonia agrimonioides (l.) dc. eam ea(w)m sem-south-west-asian-mont. filipendula hexapetala gilib. ea fragaria vesca l. cosm sekundarni kosmop. evroaz. porekla geum urbanum l. hol europ.-west asian-north amer.(bor-temp) potentilla argentea l. ce central european potentilla cinerea chaix ex vill. ea ce-med-pont potentilla detommasii ten. eam csem apen.-balk.-anatol. potentilla recta var. sulfuraea lam. ea ea(w) potentilla reptans l. ea prunus spinosa l. ea ea(w) rubus caesius l. ea eurasian-(temp-submer) sanguisorba minor scop. ea ea(w) europ.-medit.-west-asian waldsteinia geoides willd. ea ce-med-pont biologica nyssana 3 (2)  december 2012: 77-90 miljković m. et al.  phytogeographical analysis of the flora… 86 taxa area type area group flostic element rubiaceae asperula longiflora waldst. & kit. med-smed alp-balk asperula purpurea (l.) ehrend. med-smed asperula taurina l. ea ce-med south central europ.-submedit. galium album miller ce subatlant.-central european galium aparine l. ea eurasian-(bor-merid) sherardia arvensis l. cosm rutaceae dictamnus albus l. ea salicaceae salix fragilis l. ea ea(w) europ.-west-asian-(tempsubmer) salix purpurea l. ea eurasian-(bor-merid) santalaceae comandra elegans roch. med-smed southkarp.-balk. saxifragaceae saxifraga rotundifolia l. eam ea(w)m sem-anatol.-kavk. scrophulariaceae digitalis lanata ehrh. ce panon.-balk. lathraea squamaria l. ea linaria concolor gris. med-smed balk moesian-macedon.-trac. linaria genistifolia (l.) miller ea ce-med-pont central european-cmed.-pont.south-sibir. linaria vulgaris miller ea ea(w) europ.-medit.-west-asian melampyrum arvense l. ea(w) odontites rubra besser ea ce-med central european-medit.submedit. verbascum phlomoides l. ea ce-med central european-medit.submedit. veronica beccabunga l. ea ea(w) europ.-medit.-west-asian veronica hederifolia l. ea ce-med-pont central european-submedit.pont. veronica polita fries ea ea(w) veronica serpyllifolia l. cosm cosm-eurasian origin solanaceae solanum dulcamara l. ea eur.-medit.-pont.-orijent.-turan. urticaceae parietaria officinalis l. ea ce-med central european-medit.submedit. parietaria serbica pancic med-smed balk moesian urtica dioica l. hol circumcholarkct.-(bor-temp) valerianaceae valeriana officinalis l. ea eurasian-(subbor-temp) valerianella carinata loisel. ea ce-m-p-or valerianella turgida (steven) betcke med-smed verbenaceae verbena officinalis l. cosm cosm-eurasian origin biologica nyssana 3 (2)  december 2012: 77-90 miljković m. et al.  phytogeographical analysis of the flora… 87 taxa area type area group flostic element violaceae viola canina l. hol circumcholarkct.-(bor-submer) viola kitaibeliana schultes ea ce-med viola silvestris lam. p.p. ea liliopsida alysmataceae alisma plantago-aquatica l. hol circumcholarkct.-(bor-submer) araceae arum maculatum l. ea ce-med-pont ce-submedit.-west pont. cyperaceae carex bueckii wimmer ea ea(w) scirpus sylvaticus l. adv eurasian-(bor-temp) dioscoreaceae tamus communis l. ea ea(w) atlant.-central-europ..-medit.submedit.-orijental iridaceae iris pseudoacorus l. ea ea(w) lemnaceae lemna minor l. cosm cosm. liliaceae allium flavum l. msm med-pont medit.-submedit.-pont. allium rotundum l. ea ce-m-p-or sublant.-central europeansubmedit.-orijantal. allium saxatile bieb. msm med-pont asparagus tenuifolius lam. msm med-pont colchicum autumnale l. ea ce-med atlant.-central european-medit. erythronium dens-canis l. ea eurasian-(bor-merid) lilium martagon l. ea subatlant.-central europeansubmedit.-central sibir. polygonatum odoratum (miller) druce ea eurasian-(bor-submer) orchidaceae cephalanthera damasonium (miller) druce ea himantoglossum hircinum (l.) sprengel ea ce-med poaceae aegilops triaristata willd. nom. illegit. med-smed agrostis alba l. hol agrostis capillaris l. hol circumcholarkct.-(bor-merid) alopecurus pratensis l. ea eurasian-disjunct.-(bor-submer) anthoxanthum odoratum l. ea eurasian-disjunct.-(bor-submer) brachypodium sylvaticum (hudson) beauv. ea chrysopogon gryllus (l.) trin. msm med-pont dasypirum villosum (l.) cand. med-smed dichanthium ischaemum (l.) roberty cosm cosm.mediter. origin echinochloa crus-galli (l.) beauv. cosm hordeum leporinum (linc) arcangeli) hol biologica nyssana 3 (2)  december 2012: 77-90 miljković m. et al.  phytogeographical analysis of the flora… 88 taxa area type area group flostic element koeleria macrantha (ledeb.) schultes hol circumcholarkct.-(bor-submer) lolium perenne l. ea eurasian-(bor-merid) melica ciliata l. ea ea(w) europ.-medit.-west-asian orysopsis virescens (trin.) g. beck msm med-pont-or poa bulbosa l. ea eur.-medit.-submedit.-pont.south sibir.-orijent.-turan.central asian poa nemoralis l. hol circumcholarkct.-(temp) poa pratensis l. cosm sesleria rigida heuffel ex reichenb. ea(m) csem setaria viridis (l.) beauv. ea eurasian-(temp-mer) stipa capillata l. ea eurasian-(temp-mer) sparganiaceae sparganium erectum l. ea ea(w) europ.-medit.-west-asian typhaceae typha latifolia l. cosm cosm.circumcholarct. origin typha laxmanii lepechin ea ea(w) table 2. chorological spectrum of the mijkovačka gorge flora areal type (group) species % boreal (bor) 2 0.60 eurasian mountane (eam) 0.00 central south european mountane (eam/csem) 6 1.81 west eurasian mountainous (eam/ea(w)m 2 0.60 south european mountainous (eam/sem) 1 0.30 central european (ce) 16 4.83 meditteranenan-submeditteranean (med-smed)) 26 7.85 alpine-balkan (med-smed/alp-balk) 1 0.30 balkan (med-smed/balk) 8 2.42 european-submeditteranean (med-smed/e smed) 4 1.21 meridional-submeridional (msm) 0.00 mediterranean-pontic (msm/med-pont) 12 3.63 meditteranean-pontic-oriental (msm/med-pont-or) 1 0.30 meditteranean-pontic-oriental-carpathian (msm/med-pont-or-ca) 3 0.91 eurasian (ea) 72 21.75 european-west asian (ea/ea (w)) 53 16.01 central european-mediterranean (ea/ce-med) 33 9.97 mediterranean-pontic (ea/med-pont) 2 0.60 central european-mediterranean-pontic (ea/ce-med-pont) 27 8.16 central european-mediterranean-pontian-oriental-turanian (ea/ce-m-p-or) 17 5.14 central european-pontian (ea/ce-pont) 1 0.30 holarctic (hol) 18 5.44 paleoholarctic-paleotropic (hol/phol-ptrop) 5 1.51 adventive (adv) 4 1.21 cosmopolitan (cosm) 17 5.14 total 331 100.00 biologica nyssana 3 (2)  december 2012: 77-90 miljković m. et al.  phytogeographical analysis of the flora… 89 figure 2. area spectrum of the flora of the miljkovacka gorge disscusion for the phytogeographical analysis, all plant taxa were classified into 112 floristic elements, sorted in 15 area groups and 9 areal types (tab.2). as shown in fig.2, domination of eurasian area type (ea) with 205 taxa (62%) indicates the accessibility of miljkovačka gorge for colder influences coming from the northwest through the valley of the south morava river. from the other side, svrljiške mountain massif (kalafat and kamenički vis) prevent the penetration of warmer effects coming from the mediterranean by the valleys of nišava and south morava rivers. european-west asian areal group were represented with 53 taxa (16.01%), where 27 taxa belongs to european-meditteranean-west-asian floristic elements: stellaria holostea, veronica beccabunga, prunella vilgaris, cynanchum vincetoxicum, campanula rapunculoides, etc. the high presence of plants which belong to meditteranenan-submeditteranean area type (medsmed (12%), or 39 plant taxa) and meridionalsubmeridional area type (msm (5%), or 16 taxa), can be explained by the ground degradation, presence of limestone that heats easily and more easily colds, and small amount of soil covering. almost all endemic taxa, parietaria serbica, hypericum boissieri, linaria concolor, erysimum comatum, ramonda serbica, acanthus balcanicus, micromeria cristata, cephalaria flava belong to med-smed area type, while parietaria serbica and micromeria cristata represent edificatory species of ass. micromerio-pariterietum serbicae miljković, m. 2012., and ramonda serbica represents edificatory taxa of ass. ceterachi-ramondietum serbicae jovanović, r. 1953 on limestones. holarctic area type includes 23 taxa (7%) with 4 borealmeridional and 4 borealsubmeridional floristic elements, while others have disjunctive distribution area. percentage of 5% of species which belong to central european area type is product of the presence of mesophilous beach forest communities (fagetum moesiacae montanum blečić et lakušić 1970. above 700 m,, carpinio-querco-fagetum mixtum, prov., located near the stream that flows next to the monastery st. archangel gabriel in popsica) (đorđević, v. et al. 2006). low percentage of eurasian-mountain (3%) and boreal (0.6%) chorological types point to relatively low altitude of this gorge as well as to weak phytogeographical links with the high mountain region of the balkan peninsula. the significant presence in the composition of the miljkovačka gorge flora has cosmopolitan (cosm) area type with many ruderal species: plantago major, stellaria media, convolvulus arvensis, verbena officinalis, etc. small number of adventive area type taxa (4 (1%)) can be explained by the distance of the miljkovačka gorge valley and the significant migratory routes of adventive flora, which coincides with the nearby road traffic. bor 1% eam 3% med-smed 12% msm 5% eurasian (ea) 22% ea(w) 16% ce 5% ce-med 10% ce-med-pont 8% ce-m-p-or 5% hol 7% adv 1% cosm 5% biologica nyssana 3 (2)  december 2012: 77-90 miljković m. et al.  phytogeographical analysis of the flora… 90 references đorđević, v., ranđelović, n., avramović, d., lilić, a., 2006: prilog vegetaciji leskovika. ekološka istina 2006. sokobanja. greuter, w., burdet, h.m., long, g., eds., 19841989: med-checklist, 1, 3, 4. gèneve. horvat, i., glavač, v., ellemberg, h., 1974: vegetation sudosteuropa. gustav fischer verlag. stuttgart. jordanov, d. 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(ed)., 1992: flora srbije i (2.izd.) sanu. beograd. stevanović, v., 1992: floristička podela teritorije srbije sa pregledom viših horiona i odgovarajućih flornih elemenata. in sarić, m. (ed.): flora srbije, i. (drugo izdanje). sanu. beograd. 49 – 70. the euro+med plantbase the information recourse for euro-meditteranean plant diversity (http://ww2.bgbm.org/europlusmed/). the international organisation for plant information (iopi ( http://plantnet.rbgsyd.nsw.gov.au/iopi/iopihome. htm)). tutin, t.g., heywood, v.h., burges, n.a., moore, d.m., valentine, d.h., walters, s.m., webb, d.a., eds., 1964-1980: flora europaea, i-v. cambridge, university press. london. velchev, v. (ed.). 1982–1989. flora republ. popularis bulgaricae, vols 8-9. in aedibus acad. sci. bulgaricae, serdicae (in bulgarian). http://ww2.bgbm.org/europlusmed/ http://plantnet.rbgsyd.nsw.gov.au/iopi/iopihome.htm) http://plantnet.rbgsyd.nsw.gov.au/iopi/iopihome.htm) nikolić-milojević et al 2020, biologica nyssana 11(2) 11 (2) december 2020: 121-128 doi: 10.5281/zenodo.4393967 influence of the time of maceration on phenolic composition of wines produced from the indigenous variety prokupac original article natalija lj. nikolić-milojević status doo, 65a vase albanca st, svrljig, serbia lunaelumen84@gmail.com ivana s. mošić aromatika doo, jadranska st, niš, serbia ivanamosic@gmail.com ivana t. karabegović faculty of technology, university of niš, 128 bulevar oslobođenja st, leskovac, serbia ivana.karabegovic@gmail.com miodrag l. lazić faculty of technology, university of niš, 128 bulevar oslobođenja st, leskovac, serbia lmiodrag@yahoo.com nada č. nikolić faculty of technology, university of niš, 128 bulevar oslobođenja st, leskovac, serbia nadanikolic64@yahoo.com sanja r. perić academy of vocational studies south serbia, department of agricultural and food studies, 1 ćirila i metodija st, 18400 prokuplje, serbia sanjaperics@yahoo.com slađana s. golubović academy of vocational studies south serbia, department of agricultural and food studies, 1 ćirila i metodija st, 18400 prokuplje, serbia golubovicsladjana1@gmail.com dejan n. davidović academy of vocational studies south serbia, department of agricultural and food studies, 1 ćirila i metodija st, 18400 prokuplje, serbia dejandavidovic7@gmail.com dragan t. veličković academy of vocational studies south serbia, department of agricultural and food studies, 1 ćirila i metodija st, 18400 prokuplje, serbia dvelickovic7@ptt.rs (corresponding author) received: january 20, 2020 revised: september 15, 2020 accepted: september 20, 2020 abstract: the influence of the time of maceration on phenolic composition of wine (white, rosé and red wine) produced from the indigenous variety prokupac was studied in this paper. white wine was produced from free run, without maceration. in the production of rosé wine, cool-temperature extraction prior to fermentation in 2-hour period was used, while for the production of red wine, maceration was carried out at 15-20 °c for 2 weeks. with the increase of the time of maceration, the concentration of total phenolic compounds also increases, whereby, the lowest content was detected in white wine (565.4 mg/l gae). the phenolic content in red wine was almost 2.5 times higher when the time of maceration was extended to 14 days (1433.8 mg/l gae). a significant increase in total flavonoids content was detected: 153.4 mg/l ce (white wine), 179.3 mg/l ce (rosé wine), and 1205.0 mg/l ce (red wine). the total flavonoid share in total phenolic content of the wine increases with the increase of the time of maceration and its value is as follows: 27.1% (white wine), 34.3% (rosé wine) and 84.0% (red wine). key words: maceration, parameters of quality, prokupac, wine apstract: uticaj vremena maceracije na fenolni sastav vina proizvedenih od autohtone sorte grožđa prokupac u ovom radu je proučavan uticaj vremena maceracije na fenolni sastav vina (belo, roze i crveno vino) proizvedeno od autohtone sorte prokupac. belo vino je proizvedeno bez maceracije. u proizvodnji roze vina vršena je hladna maceracija pre fermentacije u dvosatnom periodu, dok je za proizvodnju crvenog vina maceracija vršena na 15-20 °c tokom 2 nedelje. sa povećanjem vremena maceracije povećava se i koncentracija ukupnih fenolnih jedinjenja, pri čemu je najmanji sadržaj otkriven u belom vinu (565,4 mg/l gae). sadržaj fenola u crvenom vinu bio je skoro 2,5 puta veći kada je vreme maceracije produženo na 14 dana (1433,8 mg/l gae). otkriven je značajan porast ukupnog sadržaja flavonoida: 153,4 mg/l ce (belo vino), 179,3 mg/l ce (vino roze), i 1205,0 mg/l ce (crveno vino). ukupan udeo flavonoida u ukupnom fenolskom sadržaju vina povećava se sa povećanjem vremena maceracije i njegova vrednost je sledeća: 27,1% (belo vino), 34,3% (vino roze) i 84,0% (crveno vino). ključne reči: maceracija, parametri kvaliteta, prokupac, vino introduction grape varieties, climate factors, soil and vineyard practices are crucial for the composition of the grapes, but the main wine attributes are highly influenced by winemaking procedures. in general, the application of different winemaking practices does not have a great effect on basic wine parameters (ethanol content, ph-value, total and volatile acidity), but significantly enhances the color, polyphenolic and volatile profile of wines (gonzález-neves et al., 2013). these characteristics are of great importance © 2020 nikolić-milojević et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work noncommercially under the same license as the original. 121 for the red wines as they are crucial for the sensorial properties, flavor, mouthfeel, aging ability of wines and value in the marketplace. also, it is well known that pre-fermentative practices, such as cold maceration or macerating enzymes application, considerably affects the extraction of compounds responsible for the phenolic composition and sensory characteristics of red wines (gonzález-neves et al., 2013; casassa et al., 2019). besides that, red wines with a higher concentration of phenolic compounds or antioxidant activity (kocabey et al., 2016; alencar et al., 2018), with better sensory properties (şener, 2018) can be produced by prolonged maceration stage which occurs simultaneously with alcoholic fermentation. according to some authors, antioxidant activity of different brazilian (alencar et al., 2018), portuguese (jordão et al., 2012) and australian (yoo et al., 2011) red wines increased with maceration time prolongation, but after reaching its maximum value, it remained stable till the end of the process. many studies showed that the basic quality parameters for teran (plavša et al., 2012), castelão (spranger et al., 2004), bombino nero (suriano et al., 2015), plavacmali (budić-leto et al., 2008) red wines do not deviate substantially with maceration time prolongation. on the other hand, the duration of maceration significantly affected the content of total anthocyanins, low molecular weight proanthocyanidins (budić-leto et al., 2008; suriano et al., 2015) and tannins (şener, 2018) in wine. furthermore, the higher antioxidant capacity of karaoglan (kocabey et al., 2016), merlot and vranec (kostadinović et al., 2012) wine was observed in samples produced with longer maceration time. sensory analysis of cabernet sauvignon (şener and yildirim, 2013), monastrell, syrah (busse-valverde et al., 2012), teran (plavša et al., 2012), karaoğlan (yilmaztekin et al., 2015), cabernet franc (cadot et al., 2012) wines showed that maceration time significantly affected sensory characteristics, but some researchers (barbará et al., 2020; plavša et al., 2012; yilmaztekin et al., 2015) highlighted that the wine produced with ten days maceration treatment gives the most harmonious wine, with the best aroma and flavor attributes. it was also shown that wine astringency and color intensity increased with longer maceration time (cadot et al., 2012; kocabey et al., 2016; şener, 2018). all these results indicate the importance of maceration treatment in winemaking, as well as the fact that desirable quality of wine can be produced by optimizing maceration duration. there are many indigenous grape varieties in serbia, but prokupac receives most of the attention in the local and international markets. thus, the objective of this study was to examine the effect of maceration duration on basic wine parameters and to verify the effects on the total content of anthocyanins, phenolic compounds and flavonoids in order to obtain premium quality wine from these grape varieties. material and methods wines three wines were used for the experiment (white, rosé and red wine) produced from the prokupac grape variety (vitis vinifera, 2016 vintage), producer from aleksandrovac (republic of serbia). the wines were made from the same grapes, where the key difference was duration of maceration. white wine was produced from wine dripping without maceration, and alcohol fermentation is carried out in the stum, i.e. in unfermented grape juice. for the production of rosé wine, short-term cold maceration was carried out for 2 hours in the press, while for the production of red wine the maceration lasted during the whole fermentation period of 2 weeks at temperature of 15°c to 20°c. relative density pycnometric analysis was used. the relative density is calculated by the formula: where: relative density of wine, mwine mass of pycnometer with wine, mwater mass of pycnometer with water, m0 mass of empty pycnometer. alcohol content pycnometric analysis with distillation was used. from the calculated relative density in the formula below, the value expressing the alcohol content of the wine is read through the tables: where: relative density of distillate, mdist mass of pycnometer with distillate, mwater mass of pycnometer with water, m0 mass of empty pycnometer. total extract after the wine has been distilled to determine the alcohol content, a residue used to determine the extract content of the wine is left in the distillation balloon. the relative density is determined, where the rinsing, quantitative transfer, refilling, thermostating and measuring are performed as in the previous cases, and the contents of the extract in g/l are read from the tables (blesić, 2016). 122 biologica nyssana ● 11 (2) december 2020: 121-128 nikolić-milojević et al. ● influence of the time of maceration on phenolic composition of wines produced from the indigenous variety prokupac 123 total acids into an erlenmeyer flask 10 ml of wine and 20 ml of distilled water are poured. a few drops of the bromo-thymol blue indicator are added. titration with a solution of naoh 0.1n is carried out. total acids (g/l, calculated on tartaric acid) are calculated as: total acids = vnaoh . 0.75 volatile acids about 300 ml of distilled water is poured into a larger normal vessel and 50 ml of wine into a smaller one. the normal water vessel is heated, and the created water vapor is introduced into the wine. when about 100 ml has been distilled, the wine is heated and at least 200 ml of wine is collected. 3-4 drops of phenolphthalein are added and titrated with a solution of naoh (0.1n). volatile acids (g/l, calculated on acetic acid) are calculated as: volatile acids = vnaoh . 0.12 . 16.67 sugar free extract sugar free extract is the difference between total dry extract and total sugars. ash into the previously measured porcelain cup 10 ml of wine is poured. annealing is carried out at the temperature which rises from 200 °c to 500 °c, and when the content is completely charred, 5 ml of distilled water is added and evaporation is carried out in a water bath, as well as re-annealing and cooling in a desiccator. for determination of the ash content (g/l), measuring was performed on an analytical scale, while the mass of the empty cup was subtracted from the obtained mass, and the value obtained was multiplied by 100. reducing sugars a fast french method based on the oxidoreduction processes of fehling’s solution and sugar was used, whereby divalent copper (cuso4) from fehling’s solution was reduced to monovalent (cu2o) and the sugar was oxidized to the corresponding acids. in an erlenmeyer flask 5 ml of fehling i and fehling ii was poured and 20 ml of distilled water were added. the content of the erlenmeyer flask is heated to boiling and the filtrate is added from the burette. during the titration, the content in the erlenmeyer flask is kept around the boiling point. the filtrate is added until the last traces of blue colour disappear, indicating that the reduction of fehling’s solution has been completed. a red precipitate is formed at the bottom of the erlenmeyer flask and the liquid above the precipitate is colorless. the content of wine sugar (g/l) was calculated as: free and total sulphur dioxide the aspiration method was used. into a flat bottom balloon 10 ml of h2o2 is added together with a few drops of methyl red and methylene blue indicator mixture. the colour should be grey, dirty blue, indigo blue or greenish. if a purple color is obtained, it should be neutralized with a solution of naoh (0.01n). as soon as the purple color disappears, adding of naoh solution is stopped. usually, only a few drops are needed for neutralization. into a round bottom balloon 10 ml of ortho-phosphoric acid and 20 ml of wine are poured. then cooling by the condenser and aspiration pumps is activated. after 15 minutes, the whole free so2 goes into the flat bottom balloon, where the colour changes to purple. titration with a solution of naoh 0.01n is performed until the colour changes to green. the consumed volume of naoh solution can be read on the burette and free so2 content (mg/l) is calculated as: free so2 = vnaoh . 16 the same sample of wine, heated for 10 minutes, is used to determine bound so2, while h2o2 and the mixture of indicators are prepared once again. after 10 minutes, titration with naoh solution is carried out until the color changes to green. the amount of bound so2 is calculated in the same way. total sulphur is the sum of bound and free sulphur. ph-value the ph-value was measured with a ph-meter, threepoint calibration. total phenols a spectrophotometric method using folin-ciocalteu reagent was used. the absorbance is measured at 765 nm. into a normal 10 ml vessel 0.1 ml of wine is poured. red wines require necessary dilution, usually at a ratio of 1:5. then 7.9 ml of distilled water is added, 0.5 ml of folin-ciocalteu reagent and 1.5 ml of na2co3 sodium-carbonate solution. the same mixture is prepared for the blank assay, only distilled water is used instead of wine. the absorbance was measured with a spectrophotometer and the results were read from a calibration curve. index of total phenols determination of total phenol index is a spectrophotometric method. the method is simple, but the results obtained do not provide the information on what phenolic compounds are present in wine and at what concentration. red wine biologica nyssana ● 11 (2) december 2020: 121-128 nikolić-milojević et al. ● influence of the time of maceration on phenolic composition of wines produced from the indigenous variety prokupac sugars = 50 a 124 requires necessary dilution of 1:100, rosé wine from 1:10 to 1:50, and white wine 1:10. the absorbance was measured at 280 nm, in quartz cuvettes with an optical path of 10 mm, in relation to distilled water as blank assay. the total phenolic index of tpi is obtained by multiplying the absorbance by the dilution factor: tpi = a280 . f total flavonoids spectrophotometric method with aluminum chloride (alcl3) was used. into a normal vessel with 4 ml of distilled water, 0.1 ml of wine is added. in case of red wines, a dilution of 1:5 is usually used. at zero moment, 0.3 ml of 5% nano2 solution is added to the normal vessel, 0.3 ml of 10% alcl3 solution is added after 5 minutes, and 2 ml of naoh 1 mol/ dm3 solution is added in the sixth minute, and the vessel is refilled with the distilled water to the index mark. the absorbance was measured at 510 nm, in comparison to the distilled water as blank assay. on the basis of the measured values from the calibration curve, the concentration of total flavonoids (mg/l) was determined. total anthocyanins the ribereau-gayon method was used. one ml of wine was mixed with 1 ml of 0.1% hcl (in 96% ethanol) and 20 ml of 2% hcl solution. the obtained mixture was stirred and 10 ml of the mixture were separated into two test tubes. into the first tube 4 ml of distilled water is added and into the second tube 20% of a solution of sodium bisulfite na2s2o3. after 20 minutes, the optical density at 520 nm was measured using a spectrophotometer. from the difference in optical density between the two tubes, the anthocyanin content (mg/l) was calculated based on a working curve for which malvidin-3 glucoside was used as a standard. results and discussion the results of testing the basic physicochemical parameters are shown in tab. 1. the alcohol content ranged from 11.43 vol% to 11.85 vol%, meaning that the maceration conditions had no effect on the alcohol content in the wine. the content of reducing sugars ranged from 1.1 g/l to 1.4 g/l, indicating that the degree of sugar overfermentation was complete, regardless of the maceration conditions. with the extension of the maceration time in wines, an increase in the content of the sugar-free extract and the ash content were determined. in this respect, the extraction of various compounds from the solid parts of the grapes (skin, seeds) into stum is prolonged, whereby the content of the sugar-free extract and the ash content in wine are increased. the lowest content of sugar-free extract was found in white wine (18.6 g/l), with virtually no maceration. with rosé wine, where the maceration lasted for two hours, the content of sugar-free extract was just slightly higher, and was 18.9 g/l. the highest content of sugar-free extract was found in red wine at 21.3 g/l. the lowest ash content was also found in white wine (1.86 g/l), while the highest content was in red wine (2.34 g/l). total acid content ranged from 4.34 g/l to 5.36 g/l and volatile acid content from 4.5 meq/l to 6.5 meq/l. duration of maceration caused decrease in the content of total acids, so the lowest content was in red wine of 4.34 g/l. the reduction of total acids during maceration is explained by the release of minerals that pass into the wine and react with tartaric acid during maceration period, resulting in table 1. basic physicochemical parameters of quality of white, rosé and red wine parameters white wine rosé wine red wine relative density, 20 °c 0.9922 0.9918 0.9927 alcohol, % v/v 11.43 11.76 11.85 total extract, g/l 19.0 19.0 21.6 reducing sugars, g/l 1.4 1.1 1.3 sugar free extract, g/l 18.6 18.9 21.3 total acids, g/l 5.36 5.16 4.34 volatile acids, meq/l 6.5 4.5 5.7 ph-value 3.24 3.26 3.75 free so2, mg/l 26.4 28.8 12.8 total so2, mg/l 148.0 110.4 28.8 ash, g/l 1.86 1.99 2.34 biologica nyssana ● 11 (2) december 2020: 121-128 nikolić-milojević et al. ● influence of the time of maceration on phenolic composition of wines produced from the indigenous variety prokupac the formation of salts. with reduction of the total acid content, the ph value of the wine increases, so that a maximum value of 3.75 was measured in red wine, while with white wine it was 3.24. according to the available results (budić-leto et al., 2008) the maceration time had no effect on the relative density, alcohol content, total extract of reducing sugars, acidity and ash in red wine. within the experimental part of this paper, the intensities and shades of wine colors were analyzed. tab. 2 shows the chromatic characteristics of the tested wines. the results are presented as the mean value out of three repetitions, with standard deviation. the color intensity of the wines (i) is the lowest in white wine; when rosé wine is concerned, short maceration led to a significant increase in colour intensity, while with red wine there is a significant rise in measured value. the shade of colour (n), which is mostly used as a parameter of aging wine, is highest in white and lowest in red wine. the colour of rosé wine is non-specific. according to glories (1984), the optimum ratio of the colour components to the type of rosé wine is 35:55:10, and in the case of the tested rosé wine is 64:33:3. it is characterized by an extremely light colour with a predominant yellow-orange tone. maceration duration changed some chemical constituents and color of red wines (kocabey et al., 2016). extended maceration duration resulted in increase in the concentration of phenolic compounds, reflecting the antioxidant activities of wine. the highest concentrations of total and individual phenolic compounds as well as antioxidant activities were found in wines macerated for 15 days. the maceration process plays an important role in the composition of the colour and sensory properties of red wine (şener, 2013, 2018). macerations carried out at low temperature ranges (10 °c to 15 °c) resulted in red wines with the highest levels of total phenolic content, anthocyanin and colour intensity, and richer fruity, flowery and spicy aroma. prolonged maceration leads to a stable red colour, too. chromatic characteristics of red wines were observed four times during the year (babincev et al., 2016). intensity, hue and brilliance of color of these wines were determined. the highest value of color intensity was observed in young wines (merlot 2.26, vranac 2.24) and the lowest value was recorded for prokupac, aged 12 months (1.18). with wine ageing the color intensity slightly decreased. the maximum value for hue in a very young wine was found in merlot (0.86) and in 12-month-old wines cabernet (0.99). brilliance of wine also decreased with wine ageing and it was most pronounced in young vranac (47.1) and the lowest value was found in 12-month old prokupac (27.7). test results for total phenols, total phenol index (tpi), total flavonoids, and total anthocyanins are shown in tab. 3 as the mean value out of three repetitions with standard deviation. 125 table 2. chromatic characteristics of white, rosé and red wine parameters white wine rosé wine red wine a420 0.098 ± 0.005 0.187 ± 0.002 1.455 ± 0.030 a520 0.030 ± 0.003 0.096 ± 0.002 1.882 ± 0.020 a620 0.001 ± 0.001 0.008 ± 0.002 0.483 ± 0.019 i 0.129± 0.006 0.291± 0.006 3.820±0.013 n 3.337± 0.388 1.955± 0.046 0.773± 0.021 a420% 76.44± 2.70 64.33± 0.88 38.09± 0.66 a520% 23.05± 1.90 32.91± 0.53 49.26± 0.61 a620% 0.51± 0.87 2.75± 0.90 12.65± 0.52 da% / / 48.49± 1.24 table 3. total flavonoids and anthocyanins in white, rosé and red wine parameters white wine rosé wine red wine total phenols, mg/l gae 565.4 ± 17.0 523.4 ± 3.5 1433.8 ± 90.9 tpi (a280) 12.29 ± 0.49 13.83 ± 0.41 31.77 ± 4.19 total flavonoids, mg/l ce 153.4±1.8 179.3±2.1 761.7±15.9 total anthocyanins, mg/l / 2.8 154.0±2.9 biologica nyssana ● 11 (2) december 2020: 121-128 nikolić-milojević et al. ● influence of the time of maceration on phenolic composition of wines produced from the indigenous variety prokupac 126 total phenols, in contrast from expected, are higher in white wine than in rosé wine. most are found in red wine and their value is 1433.8 mg/l. on the other hand, the index of total phenols in rosé wine (13.83) is slightly higher than in white wine (12.29), while it is the highest in red wine (31.77). total flavonoids are the lowest in white wine (153.4 mg/l) and the highest in red wine (761.7 mg/l). anthocyanins are below the lower detection limit in white wine, 2.8 mg/l, and in red wines they are 154 mg/l. here the influence of maceration becomes obvious. according to the results of alencar et al. (2018), the content of phenolic compounds increased up to 15th day during the process of maceration, and anthocyanins up to 20 days. extending the maceration time thus also improved the antioxidant activity of the wine. chemometric analysis of wine samples is shown in fig. 1 and 2, which were made in the program statistica v.8.0 (statsoft inc., tulsa, ok, usa). conclusion maceration has a very large influence on the physical and chemical parameters of wine quality. during the maceration process, many substances are extracted, and transferred from the solid parts into the wine. as a result, red wines have a much more complex chemical composition than white and rosé wines. in our samples, the duration of maceration had no significant effect on the relative density and alcohol content. the effect is significant on the content of the sugar-free extract and the ash content due to the higher extraction of nitrogen compounds, minerals and numerous other substances. chromatic analysis showed that the extraction of coloured substances was much higher with red wine, where the maceration lasted for two weeks at temperature ranging from 15 °c to 20 °c, compared to rosé wine, which only used short-term cold maceration. total phenols in red wine are almost three times higher than in white and rosé wines, and total flavonoids by slightly more than four times. the difference is greatest when it comes to anthocyanins extraction, which is almost nonexistent with white wine, and with rosé wine 55 times less concentrated than in red wine. the tannin concentration is also highest in red wine and lowest in white wine. the parallel examination of wine produced from the same, autochthonous prokupac variety, indicates fig. 1. dendrogram obtained by cluster analysis using means of basic physicochemical parameters of white, rosé and red wine quality single linkage euclidean distances 30 40 50 60 70 80 90 linkage distance red wine rose wine white wine fig. 2. dendrogram obtained by cluster analysis using means of chromatic characteristics and total flavonoids and anthocyanins of white, rosé and red wine biologica nyssana ● 11 (2) december 2020: 121-128 nikolić-milojević et al. ● influence of the time of maceration on phenolic composition of wines produced from the indigenous variety prokupac 127 the potential of this variety and the opportunities it offers to producers for the application of different technologies. acknowledgement. this work was supported by the ministry of education, science and technological development of the republic of serbia under the program of financing scientific re search work, number 451-032842/2019-14/172047. the authors are grateful to prof. nebojša deletić, phd, university of priština, faculty of agriculture, kosovska mitrovica lešak, for his help with the statistical processing of the results. references alencar, n.m.m., cazarin, c.b.b., corrêa, l.c., maróstica junior m.r., biasoto, a.c.t., behrens, j.h. 2018: influence of maceration time 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composition of wines produced from the indigenous variety prokupac d.d., tamborra, p. 2015: effects of skin maceration time on the phenolic and sensory characteristics of bombino nero rosé wines. italian journal of agronomy, 10(1): 21-29. yilmaztekin, m., kocabey, n., hayaloglu, a.a. 2015: effect of maceration time on free and bound volatiles of red wines from cv. karaoğlan (vitis vinifera l.) grapes grown in arapgir, turkey. journal of food science, 80(3): c556-63. yoo, y.j., prenzler, p.d., saliba, a.j., ryan, d. 2011: assessment of some australian red wines for price, phenolic content, antioxidant activity, and vintage in relation to functional food prospects. journal of food science, 76: 1355-1364. 128 biologica nyssana ● 11 (2) december 2020: 121-128 nikolić-milojević et al. ● influence of the time of maceration on phenolic composition of wines produced from the indigenous variety prokupac microsoft word bn-oa-0201-02 pehadjieva k biologica nyssana 2 (1) september 2011: 19-28 pechadjieva k. distribution of calthion palustris tüxen 1937… 19 original article ! distibution of calthion palustris tüxen 1937 in eninska river basin, central stara planina mountain kalina pachedjieva* sofia university “st. kliment ohridski” –faculty of biology, department of ecology and environmental protection, 8 dragan tzankov blvd, 1164 sofia, bulgaria * e-mail: kalina.pachedjieva@biofac.uni-sofia.bg abstract: pachedjieva, k.: distibution of calthion palustris tüxen 1937 in eninska river basin, central stara planina mountain, biologica nyssana, 2 (1), september 2011: 19-28. during a phytocoenological investigation of eninska river basin in central stara planina mountain the wetland vegetation proved to be an element of calthion alliance. this conclusion is made on the basis of cluster analysis applied by the computer program syn-tax and a detailed review of species composition of the analyzed relevés with the respective comparisons. the alliance is presented by the central european ass. filipendulo ulmariae-menthetum longifoliae. the balcan influence over the association is apparent from the balcan endemic species angelica pančičii and dactylorhiza incarnata. the association is dependent on water sufficiency and unifies mentha longifolia grasslands on nutrient soils with slightly acidic to slightly basic reaction. it is anthropogenically influenced. some stands form mentha longifolia and urtica dioica community. key words: calthion, central stara planina mountain, cluster analysis introduction ! vegetation of calthion palustris tüxen 1937 alliance has been profoundly investigated in europe by many authors (s ý k o r a , 1982, r a n d j e l o v i ć & z l a t k o v i ć , 1994, h á j k o v á et al., 2006, h á j k o v á , 2008 and others). during the last decade the studies on this type of vegetation in bulgaria according to the principles of braunblanquet phytosociological school also have increased and generalized by tzonev et al. (2009). still, there are gaps in this field especially in the conditions of continuously changing environment. the present research is a part of a phytocoenological investigation of eninska river basin situated on the southern slopes of central stara planina mountain. different types of forest and grass communities form the vegetation cover of the region. the present paper deals only with hygro and mesohygrophyte tall-herb coenosis (wetlands) distributed along and around the river and its tributaries. the aim of the investigation is put in this context: clearing up the syntaxonomy and habitat affiliation of these communities as referred in the available papers and investigations (m u c i n a , 1997; r o d w e l l et al., 2002; h á j e k et al., 2008; t z o n e v et al., 2009 and others). materials and methods object of investigation. eninska river basin covers about 7 000 ha area on the southern slopes of central stara planina mountain (fig. 1). the altitude range it takes is from 500 m a.s.l. (the lowest point) to 1450 m a.s.l. (at the main ridge). shipchenski anticlinorium is the basic geological 2 (1) • september 2011: 19-28 biologica nyssana 2 (1) september 2011: 19-28 pechadjieva k. distribution of calthion palustris tüxen 1937… 20 structure in the region. it is presented by paleozoic granitoids in the core and calcareous rocks in the mantle of triassaic, jurassic and early cretaceous age (j o r d a n o v a et al., 2002). the topographic forms are very rough because of the steep slopes and the deeply indented gorges and tributaries of the river which in its low stream forms the almost vertical and narrow eninsko zhdrelo (enina gorge). warm summer and cold winter characterize the climate of this region complimented by the following features big annual amplitude of temperature, spring-summer and winter maximum of precipitations and yearly stable snow cover (v e l e v , 2002). stara planina mountain is an example of a mountain variant of temperatecontinental climate – colder and damper. temperature decreases and wind, precipitation and clouds increasewith an increase of altitude. figure 1. geographical position of the studied area soil diversity in the region is presented mainly by brown forest soils (eutric cambisols according to fao clasiification) taking predominantly slopes at higher than 700 – 800 m a.s.l. altitudes (n i n o v , 2002). there are different types of vegetation in the investigated region normally distributed along the altitudinal gradient – temperate heathlands and grasslands at lower or higher altitude (festuco-brometea, molinioarrhenatheretea), temperate broadleaved forests and scrubs (querco-fagetea), chasmophytic vegetation and synanthropic vegetation in relation with anthropogenic pressure (m u c i n a , 1997). the basin includes two protected areas within its borders the natural reserve “kamenshtitsa” and the protected site “eninsko zhdrelo” (enina gorge). it is also a part of a nature 2000 protected zone (central balkan buffer). methods of investigation. the methods of the investigation follow the principles of the braunblanquet phytosociological school (w e s t h o f f & m a a r e l , 1973; m u e l l e r -d o m b o i s & e l l e n b e r g ; 1974 and others). the terrain work was carried out during the period from 2003 to 2006. the investigated vegetation is presented by 15 relevés made in presentable and comparatively homogeneous sites according to the requirements of the methodology (k e n t & c o k e r , 1992) at the wet lowest points of the relief. 1 relevé is removed from the analysis as transitional and complex. a cluster analysis is applied using the computer program syn-tax (p o d a n i , 2000). average linkage method (upgma) is used and floristic similarity among relevés is evaluated according to horn’s index. the diagnostic species for the revealed vegetation groups are determined after a detailed review and comparisons with the referred literature. the species determination and taxonomical nomenclature is according to flora of bulgaria (y o r d a n o v (ed.), 1963–1989, k o z h u h a r o v (ed.), 1995) and the field guide to the vascular plants in bulgaria (a n d r e e v et al., 2002). floral elements are determined according to a s s y o v (2006). raunkiaer’s life forms are also indicated (g o r u i s h i n a , 1979). the nomenclature of syntaxa and habitat diversity is according to r o d w e l l et al. (2002). results and discussion the cluster analysis resulted in the differentiation of two comparatively heterogeneous groups, both belonging to calthion palustris tüxen 1937 alliance from molinio-arrhenateretea tüxen 1937 class (fig. 2, tab. 1). the floristic similarity between relevés in the first group consisting of 9 relevés is of 35%. according to some authors (t h e u r i l l a t & m a t t h e y , 1987, t z o n e v , 2002) this is enough in order for the group to be considered one association. dominant species are mentha longifolia and deshampsia caespitosa and co-dominants are filipendula ulmaria and caltha palustris in some relevés. constant species with constancy above iii are lathyrus pratensis, filipendula ulmaria, caltha palustris, carex hirta (tab. 1). these together with ranunculus repens, biologica nyssana 2 (1) september 2011: 19-28 pechadjieva k. distribution of calthion palustris tüxen 1937… 21 myosotis scorpioides, scirpus sylvaticus, juncus effusus, lysimachia nummularia (constancy ii and i) form the characteristic species complex of ass. filipendulo ulmariae-menthetum longifoliae zlinská 1989 described in slovakia (h á j k o v á et al., 2008). calthion vegetation is comparatively well studied in central europe. great diversity at association and subassociation level was registered for slovakia (according to h á j k o v á et al., 2008) where 14 associations were distinguished in calthion alliance. six of them had been described earlier in western carpathians (h á j k o v á et h á j e k , 2005). these are cirsietum rivularis nowiński 1927, chaerophyllo hirsuti-calthetum palustris balátová-tuláčková 1985, angelico sylvestris-cirsietum palustris darimont ex balátová-tuláčková 1973, scirpetum silvatici ralski 1931, angelico sylvestris-cirsietum oleracei tüxen 1937 and scirpo silvatici-cirsietum cani balátová-tuláčková 1973. angelico-cirsietum oleracei, cirsietum rivularis and scirpetum sylvatici were recorded for hungaria (b o r h i d i , 2003) and romanian calthion vegetation (s a n d a et al., 1999). ass. angelico sylvestris-cirsietum oleracei shows some resemblance to calthion communities from central stara planina mountain – it develops on nutrient-rich, mostly alluvial, alkaline soils and forms similar physiognomy with dominant species mentha longifolia and filipendula ulmaria but is distributed at lower altitudes in areas with calciumrich bedrock. besides most of the constant species are absent in central stara planina mountain. ass. scirpetum silvatici has most common distribution in central and southeastern europe. these species poor wet grasslands dominated by scirpus silvaticus and developing on nutrient-rich acidic to alkaline soils (h á j k o v á et al., 2008) are recorded by h á j e k et al. (2005) for vitosha mountain from bulgaria. the central european origin of calthion vegetation is indirectly confirmed by r a n d j e l o v i ć & z l a t k o v i ć (2010). they treat calthion vegetation of vlasina plateau and record three associations equiseto-scirpetum silvaticae šegulja 1974, polygono-scirpetum silvaticae schwick. 1944 and the locally distributed brachythecio-menthetum longifoliae v. randj. 2001. although the euro-asian, boreal and holarctic elements prevail in the phytogeographical specters of the three associations the balkan influence is discernible by balkan endemic species in the composition of polygono-scirpetum silvaticae and brachythecio-menthetum longifoliae. in synecological respect calthion vegetation from vlasina plateau is similar to the same in central stara planina mountain – these are waterlogged wet grasslands on acidic soils with well-developed humus horizons distributed in the subalpine belt. there are more considerable differences in the constant species at association level with calthion communities from central stara planina mountain. it could be concluded that the major group of calthion communities (9 relevés) in eninska river basin shows great resemblance in ecology and species diversity with ass. filipendulo ulmariaementhetum longifolie zlinská 1989 described in slovakia (h á j k o v á et al., 2008). this association unifies grasslands dominated by mentha longifolia on nutrient-rich soils in small alluvia with slightly acidic to slightly basic groundwater reaction as described by h á j k o v á (2008) for the slovakian molinio-arrhenatheretea vegetation. here, it is marked by almost the same complex of constant species. in the context of the habitat differentiation of wet grasslands in bulgaria made by h á j e k et al. (2008) the discussed vegetation group is most likely to be a part of the sub-montane waterlogged grasslands on alkaline soils of calthion wetland vegetation. it also resembles the intermittently deschampsion alliance with diagnostic species alopecurus pratensis, carex otrubae, carex spicata, juncus compressus, potentilla reptans. but most of the diagnostic species of that alliance are either not present or occur with low constancies in the discussed group. the exposed arguments give reason to consider “our” calthion group as an element of the central european filipendulo ulmariae-menthetum longifoliae. in central stara planina mountain however this association is normally modified by the presence of angelica pančičii and dactylorhiza incarnata as balkan floral elements. thus in the present case it could be considered as a balkan variant of the central european association. it takes stands on silicate bedrock at 1200 – 1450 m a.s.l. and predominantly southern slopes of 15–30 degrees inclination. moss layer is poorly presented. the association has comparatively heterogeneous horizontal structure – mosaics are character feature (fig. 3). this is caused by the irregularity of the environmental conditions typical for this type of vegetation. being located on the watersides of the river and its tributaries it is dependent on water sufficiency. actually, mentha longifolia forms different types of communities. s ý k o r a (1982) discusses the syntaxonomy and synecology of juncomenthetum longifoliae lohmeyer 1953 and caricetum vulpinae nowinsky 1927 distributed from the northern part of europe through central to southeastern europe. the author distinguishes conglomerates in these associations on the basis of ecological variations and differences obtained by biologica nyssana 2 (1) september 2011: 19-28 pechadjieva k. distribution of calthion palustris tüxen 1937… 22 figure 2. classification dendrogram of the wetland relevés from eninska river basin made in the computer program syn-tax (podani 2002) figure 3. the heterogeneous structure gives a characteristic appearance of calthion vegetation biologica nyssana 2 (1) september 2011: 19-28 pechadjieva k. distribution of calthion palustris tüxen 1937… 23 table 1. phytosociological table of calthion palustris tüxen 1937 communities in eninska river basin, central stara planina mountain releve number 10 3 5 8 9 4 1 2 6 c on st an cy 11 7 c on st an cy relevé area (m2) 100 100 100 100 100 100 100 100 100 100 100 altitude (m) 1450 1400 1400 1200 1400 1400 1430 1400 1400 1000 1200 aspect s s s s s w s s s e s slope (degrees) 30 30 15 10 15 25 30 20 15 30 15 cover total (%) 100 100 100 100 100 100 100 100 100 100 100 ass. filipendulo ulmariae menthetum longifoliae zlinská 1989 mentha longifolia & urtica dioica com. lathyrus pratensis + + + + + + iv + iii carex hirta + + + + + iii + iii filipendula ulmaria 1 + + 3 3 iii + iii caltha palustris + + 1 2 iii ranunculus repens + + + ii myosotis scorpioides + + ii + iii scirpus sylvaticus + i juncus effusus + i lysimachia nummularia + i mentha longifolia + 2 4 + iii 2 3 v urtica dioica + + + + iii 2 2 v calthion palustris tüxen 1937 deschampsia caespitosa + 1 1 1 3 4 + iv 1 iii equisetum palustre 2 2 2 2 + + iv dactylorhiza saccifera + + + + + + + iv galium palustre + + 1 + + + iv hypericum tetrapterum + + + + + iii + + v ranunculus acris + + + + iii potentilla erecta + + + + iii dactylorhiza incarnata + + + ii 1 iii carex echinata + + + ii + iii molinietalia w. koch 1926 prunella vulgaris + + + + + + + iv + + v carex ovalis + + 1 + + + + iv juncus articulatus + + + ii + iii trifolium pratense + + ii + iii angelica sylvestris + i 2 iii mulgedio-aconitetea hadač et klika in klika 1948 angelica pancicii 1 1 + 1 + + + 2 v alchemilla glabra 1 1 + + + + + + v geum rivale + + + + + + iv molinio-arrhenatheretea tüxen 1937 juncus compressus + 1 ii poa palustris + 2 ii blysmus compressus 1 i carex otrubae + i + iii cynosurus cristatus + i potentilla reptans + i glyceria plicata + i dactylis glomerata + iii rumex crispus + iii galio urticetea passarge ex kopecký 1969 and epilobietea angustifoliae tx. & preising ex von rochow 1951 rubus idaeus + + + + iii rumex alpinus + + ii fragaria vesca + + ii + iii biologica nyssana 2 (1) september 2011: 19-28 pechadjieva k. distribution of calthion palustris tüxen 1937… 24 salix caprea + + ii + iii geum urbanum + i stachys sylvatica + i + + v nardo-callunetea preising 1949, festuco-brometea braun-blanq. et tüxen ex soó 1947, querco-fagetea braun-blanq. et vlieger in vlieger 1937 potentilla recta gr. + i stellaria graminea + i + iii bruckenthalia spiculifolia + i chamaespartium sagittale + i lerchenfeldia flexuosa + i festuca dalmatica + i hypericum perforatum + i lotus corniculatus + i cruciata laevipes + i + iii verbascum longifolium ssp. pannosum + i heracleum sibiricum + i carex sylvatica + i calystegia sepium + i dryopteris filix-mas + i calamagrostis arundinacea + iii digitalis grandiflora + iii clinopodium vulgare + iii geranium rotundifolium + iii acer platanoides + iii others athyrium filix-femina 1 + + + + + iv mentha spicata 2 + 3 1 3 iii inula helenium + + + 1 iii + iii veronica anagallis-aquatica + + + + iii epilobium nutans + + + ii + iii stachys alpina + + ii ranunculus ophioglossifolius + + ii marchantia polymorpha + + ii veronica beccabunga + + ii galium album + i carduus nutans + i + iii senecio rupestris + i glyceria maxima 1 i lapsana communis + i rumex sanguineus + i bromus secalinus + i glyceria fluitans + i carex flava + i eleocharis palustris + i cicerbita alpina + i sagina procumbens + i epilobium alpestre + i arctium lappa 1 2 v equisetum arvense + iii telekia speciosa + iii torilis japonica + iii vicia cracca + iii elymus caninus 1 iii geranium phaeum + iii pyrola rotundifolia + iii biologica nyssana 2 (1) september 2011: 19-28 pechadjieva k. distribution of calthion palustris tüxen 1937… 25 principle component analysis. the described in the present paper association resembles mentha longifolia and hypericum tetrapterum conglomerate in species composition. the last is based on relevés from former czechoslovakia at rather lower altitudes (350 – 800 m a.s.l.) and is notable with its open character. as it was already underlined much more resemblance could be found with the described calthion association equiseto-scirpetum sylvaticae šegulja 1974, subass. deschapsietosum caespitosae randjelović & zlatković 1994 in neighbouring serbia (r a n d j e l o v i ć & z l a t k o v i ć , 1994). again, these comparisons confirm the balkan modification of the accepted central european association. the second group presented in the table and dendrogram (tab. 1, fig. 2) consisting of 2 relevés belongs to the same alliance calthion but is not given a concrete syntaxonomical rank as it is highly influenced by antropophyte elements. thus it is determined as mentha longifolia and urtica dioica community. it indicates habitat degradation and is somehow a “return” in succession. in this community mentha longifolia (constancy v) could be considered as a characteristic species of the nitrophylous alliance agropyro–rumicion crispi nordh. 1940 (t z o n e v , 2009, r a n d j e l o v i ć & z l a t k o v i ć , 2010) together with rumex crispus (constancy iii) and urtica dioica (constancy v). most of the character species for calthion alliance are presented in this community (tab. 1) and it could not be affiliated to agropyro-rumicion crispi at present. future investigations will clarify its succession status. in accordance with the discussion hitherto the following classification scheme for the hygroand mesohygrophyte vegetation in eninska river basin could be proposed: class molinio-arrhenatheretea tüxen 1937 order molinietalia w. koch 1926 alliance calthion palustris tüxen 1937 ass. filipendulo ulmariae-menthetum longifoliae zlinská 1989 mentha longifolia and urtica dioica community in addition, geoelements and raunkiaer’s life forms for the species are indicated. the holarctic group of elements is well presented in calthion communities in the region by the boreal and subboreal geoelement respectively 17% and 15%. it is followed by the palearctic group including euroasian (12%) and eurosiberian elements (9%). european elements are 9%. this distribution corresponds with the central-european origin of calthion vegetation habitat conditions (climate, relief) – lowand mid-mountain. the sub-european group is presented mainly by euromediterranean elements (10%) which together with submediterranean elements (8%) indicates the south-european position of the treated vegetation. kosmopolitan species are 11%. in the biological specter the hemicryptophytes prevail – 52%, followed by the cryptophytes (helophytes) – 35%. the chamaephytes represent 2%. closeness with forest belt is apparent by phanaerophytes which take 4%. the percentage of therophytes, not typical for this kind of vegetation, is comparatively high – 7%. some of them are anthropophytes (arctium lappa, carduus nutans, lapsana communis). conclusions phytosociological analysis showed distribution of calthion alliance in eninska river basin, central stara planina mountain. the alliance is presented by ass. filipendulo ulmariaementhetum longifoliae and mentha longifolia and urtica dioica community. the described association is the optimal wetland vegetation in the subalpine belt of the region. calthion is an azonal vegetation and its structure is determined by nutrient availability, water regime and management practices (f l i e r v o e t & w e r g e r , 1985). being close to the timber-line it is influenced by mulgedioaconitetea hadač et klika in klika 1948 class. the anthropogenic pressure in the region has also influenced the species composition and the condition of calthion communities. this is marked by galio-urticetea passarge ex kopecký 1969 and epilobietea angustifoliae tüxen & preising ex von rochow 1951 species. the presented 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(ed.). handbook of vegetation science. part 5. ordination and classification of communities. junk, the hague, the netherlands: 617-726 biologica nyssana 2 (1) september 2011: 19-28 pechadjieva k. distribution of calthion palustris tüxen 1937… 28 anticancer compounds from medicinal plants biologica nyssana 6 (2)  december 2015: 75-80 radojković-kostevski, i. et al.  variations in the headspace volatile profiles… 75 original article received: 11 october 2015 revised: 12 november 2015 accepted: 20 december 2015 variations in the headspace volatile profiles of three different achillea coarctata poir. (asteraceae) populations ivana radojković-kostevski1, goran petrović1, gordana stojanović1, jelena stamenković1, bojan zlatković2 1university of niš, faculty of sciences and mathematics, department of chemistry, višegradska 33, 18000 niš, serbia 2university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia * e-mail: peca@pmf.ni.ac.rs abstract: radojković-kostevski, i., petrović, g., stojanović, g., stamenković, j., zlatković, b.: variations in the headspace volatile profiles of three different achillea coarctata poir. (asteraceae) populations. biologica nyssana, 6 (2), december 2015: 75-80. this study presents a detailed compositional analysis of six achillea coarctata poir. samples obtained by static headspace method and interrelationships based on the volatiles profiles from different plant parts, three different populations and geological substrates, using multivariate statistical analysis. the most dominant components were mutual for aerial vegetative plant parts and inflorescences collected at the same locality. main compounds differed in percentages for two localities (values in parenthesis refer to aerial plant parts and inflorescences, respectively): 1) 1,8-cineole (40.7%; 39.9%), β-pinene (29.6%; 36.4%) and α-pinene (7.2%; 3.3%); 2) 1,8-cineole (51.8%; 53.3%), β-pinene (18.0%; 28.2%) and α-pinene (5.6%; 4.5%). the most abundant constituents identified in third locality were 1,8-cineole (37.2%; 35.6%), β-pinene (18.6%; 11.7%) and ocymene (11.6%; 11,7%). samples collected on different geological substrates are qualitative and quantitative various according to agglomerative hierarchical clustering analysis and can be grouped in two clades and two subclades. key words: achillea coarctata poir., headspace, volatile profiles, statistical analysis apstrakt: radojković-kostevski, i., petrović, g., stojanović, g., stamenković, j., zlatković, b.: razlike headspace profila isparljivih komponenti tri različite populacije achillea coarctata poir. (asteraceae). biologica nyssana, 6 (2), december 2015: 75-80. u ovom radu su predstavljeni rezultati dobijeni ispitivanjem hemijskog sastava lako isparljivih komponenti iz šest uzoraka biljke achillea coarctata poir. headspace statičkom metodom, i ispitana je zavisnost varijacije hemijskih profila od ispitivanih delova biljke i geološke podloge, korišćenjem multivarijantne statističke analize. uzorci nadzemnog dela biljke i cveta prikupljani na jednom staništu sadržali su iste glavne komponente. rezultati su pokazali da je razlika između dva lokaliteta u sadržaju istih glavnih komponenti, čiji su procenti prikazani u zagradama za nadzemni deo biljke i cvet: 1) 1,8-cineol (40.7%; 39.9%), β-pinen (29.6%; 6 (2) • december 2015: 75-80 biologica nyssana 6 (2)  december 2015: 75-80 radojković-kostevski, i. et al.  variations in the headspace volatile profiles… 76 36.4%) i α-pinen (7.2%; 3.3%); 2) 1,8-cineol (51.8%; 53.3%), β-pinen (18.0%; 28.2%) i α-pinen (5.6%; 4.5%). kod trećeg staništa najdominantnije su bile sledeće komponente: 1,8-cineol (37.2%; 35.6%), β-pinen (18.6%; 11.7%) i o-cimen (11.6%; 11.7%), za nadzemni deo biljke odnosno cvet. prema rezultatima ahc analize, uzorci prikupljeni sa različitih podloga se kvalitativno i kvantitativno razlikuju i grupisani su u dva klastera i dva subklastera. key words: achillea coarctata poir., headspace, profili isparljivih komponenti, statistička analiza introduction the genus achillea l. (asteraceae) is comprised of about 115 species found in the northern hemisphere, mostly in the euro-asian continent that are commonly known as yarrows (b e n e d e k et al., 2008; nemeth & bernath, 2008; r a d u l o v i ć et al., 2010). the achillea l. species belong to the oldest medicinal plants that are used both for pharmaceutical purposes and in folk medicine. achillea species are diuretic, emmenagogue agents, used for healing wounds, curing stomachache and diarrhea, with antichloristic, antispasmodic, antiseptic and infection preventing properties. they have also been used to reduce sweating and to stop bleeding (a l s o h a i l i & a l f a w w a z , 2014). the achillea genus has a wide distributional range, and the differences in oil composition may be affected by different environmental factors such as plant genetic type, seasonality, and developmental stage, because it is a chemically polymorphic and perennial plant. terpenoids (1,8-cineole, camphor, borneol, pinenes, artemisia ketone, santolina alcohol, farnesane, caryophyllene and its oxides, cubebene, germacrenes, eudesmol, α-bisabolol and oxides, farnesene, γ-gurjunene, γ-muurolene and chamazulene) are the principle components of achillea essential oils (m o t a v a l i z a d e h k a k h k y et al., 2013). a. coarctata is a perennial herb with yellow ligules growing in dry hillsides and sandy oils, with the range restricted to the balcan peninsula, the south ukraine and the asia minor (g a j i ć , 1975; s i m i ć et al., 1999; t z a k o u et al., 2009). as far as we know, there are three reports about chemical composition of a. coarctata essential oil. different components are reported as the main constituents of the oil among these three studies. first known analysis represents caryophyllene oxide, 1,8-cineole and trans-linalool oxide as dominant constituents (s i m i ć et al., 1999). tzakou found 1,8-cineole, camphor and borneol, while toker reported 1,8cineole, camphor and viridiflorol to be major components of the essential oil (t z a k o u et al., 2009; t o k e r et al., 2003). the aims of this study were to perform a detailed compositional analysis of six a. coarctata samples obtained by static headspace method and to establish interrelationships based on the volatiles profiles from different plant parts and different populations using multivariate statistical analysis. material and methods plant material the plant material (flowering stage) was collected at three different locations in serbia (rujan mountain; preševo (mitrovac); pčinja valley (trgovište)), in june 2014. the plant materials were identified by bojan zlatković and the voucher specimens were deposited in the herbarium moesiacum niš (hmn), department of biology and ecology, faculty of science and mathematics, university of niš under the acquisition numbers 9367, 9368 and 9369. types of geological substrates were identified according to basic geological mapp 1:100.000 (k a r a j o v a n o v i ć & h r i s t o v , 1976; b a b o v i ć & c v e t k o v i ć , 1977). sample preparation 300 mg of milled fresh plant material was put into 20 ml hs vial and soaked with 2 ml of distilled water. the sample was heated at 80 °c for 20 minutes with the next mixing program: shaking for 5 seconds, pause for 2 seconds. 500 μl of vapor generated from the aerial parts was drawn out from the vial using a gas-tight syringe (90 °c) and injected directly in the chromatographic column via a transfer line (75 °c). gc and gc/ms analysis the samples were analyzed by a 7890/7000b gc/ms/ms triple quadrupole system in ms1 scan mode (agilent technologies, usa) equipped with a combi pal sampler and headspace for g6501b/g6509b. the fused silica capillary column hp-5ms (5% phenylmethylsiloxane, 30 m x 0.25 mm, film thickness 0.25 μm) was used. the injector and interface operated at 250 and 300 °c, respectively. temperature program: from 50 to 290 °c at a heating rate of 4 °c/min. the carrier gas was helium with a flow of 1.0 ml/min. 500 μl of hs vapor was injected via a transfer line (75 °c). post run: back flash for 1.89 min, at 280 °c, with helium pressure of 50 psi. ms conditions were as follows: biologica nyssana 6 (2)  december 2015: 75-80 radojković-kostevski, i. et al.  variations in the headspace volatile profiles… 77 ionization voltage of 70 ev, acquisition mass range 50-650, scan time 0.32 s. gc analysis was carried out under the same experimental conditions using the same column as described for the gc/ms. the percentage composition of the samples was computed from the gc peak areas without any corrections. identification of volatile compounds hs volatiles were identified by comparison of their linear retention indices (relative to c8-c32 n-alkanes on the hp-5ms column) with literature values and their ms with those of authentic standards, as well as those from wiley 6, nist11, agilent mass hunter workstation b.06.00 software and a homemade ms library with the spectra corresponding to pure substances and components of known essential oils by the application of the amdis software (automated mass spectral deconvolution and identification system, ver. 2.1, dtra/nist, 2011). some components were identified by co-injection of pure substances. multivariate statistical analysis the contents of the components of the a. coarctata headspace volatiles obtained in this study were analyzed by agglomerative hierarchical cluster analysis (ahc). the ahc was performed with euclidean distances as metric and using single linkage method as aggregation criterion using the “statistica, version 8.1” software. results and discussion plant materials were collected at three different locations rujan mountain (samples rn, rc), preševo (mitrovac) (samples pn, pc) and pčinja valley (trgovište) (samples tn, tc). each location has different geological substrate type: 1) serpentine (preševo), 2) silicate (rujan mountain), 3) conglomerates and molasse (pčinja valley). compositions of aerial plant parts and inflorescences headspace volatiles of a. coarctata from three different localities obtained by gc and gc/ms, are presented in tab. 1. number of identified compounds in rn, tn, pn, rc, tc and pc samples were 38, 36, 33, 34, 28 and 32, respectively (representing 98.9% (rn); 98.1% (tn); 98.3% (pn); 98.2% (rc); 98.8% (rc) and 98.6% (tc) of total hs volatiles. the most dominant components were mutual for aerial plant parts and inflorescences collected at the same locality. main compounds found in rn and rc samples were 1,8-cineole (40.7%; 39.9%), βpinene (29.6%; 36.4%) and α-pinene (7.2%; 3.3%). the same components were found as the most dominant in samples tn and tc, with following percentage: 1,8-cineole (51.8%; 53.3%), β-pinene (18.0%; 28.2%) and α-pinene (5.6%; 4.5%), for tn and tc respectively. the most abundant constituents identified in pn and pc samples were 1,8-cineole (37.2%; 35.6%), β-pinene (18.6%; 11.7%) and ocymene (11.6%; 11.7%). as far as we know, there is no any published data on headspace volatiles composition of achillea coarctata. three papers revealed no different compounds as the most abundant in obtained essential oils compositions: (1) caryophyllene oxide, 1,8-cineole and trans-linalool oxide; (2) 1,8-cineole, camphor and borneol and (3) 1,8-cineole, camphor and viridiflorol, respectively (s i m i ć et al., 1999; t z a k o u et al., 2009; t o k e r et al., 2003). content of monoterpenoids, which are more volatile compounds, is higher in headspace volatiles then in essential oil, while sesquiterpenoids, which have relatively high retention times, were only found in traces or not found at all. the results of the ahc analysis are depicted in fig. 1. table 1 lists the identified constituents with their contents of the six a. coarctata headspace volatile samples included in the ahc. the dendrogram depicted in fig. 1, obtained as the result of the ahc, indicates the existence of two statistically different classes of samples (c1–c2). it is obvious that the chemical compositions of the aerial parts and inflorescences of the samples collected at the same locations are almost identical and they are grouped in the same stocks since they have the same main components that differ only in percentages in different parts of plants. samples pc and pn are separated from the rest of the a. coarctata samples and constituted the first clade (c1). the samples tc, tn, rc and rn composed the second clade (c2). there was further subdivision within clade c2, into two more subclades consisted of the samples collected from the same sites. the second clade is more homogeneous than the first one and it is flatter on the dendrogram. samples tc, tn, rc and rn were characterized by high contents of same compounds (1,8-cineole, β-pinene and α-pinene), reason for their strong association (same clade). greater dissimilarity level (1.6) observed in this analysis separated samples pc and pn in clade c1 from the rest of the samples considering that the most abundant constituents identified in these samples were 1,8-cineole, β-pinene and o-cymene. since each population has different geological substrate: serpentine (samples pc and pn, preševo), conglomerates and molasses (samples tc and tn, pčinja valley) and biotite gneiss (samples rc and rn, rujan mountain) it can be concluded that, with respect to the sample origin, different substrates produce various chemotypes. chemical compositions biologica nyssana 6 (2)  december 2015: 75-80 radojković, i. et al.  variations in the headspace volatile profiles… 78 table 1. chemical composition of the a. coarctata volatiles achieved by gc and gc/ms relative amount % riref riexp compound rn tn pn rc tc pc class 801 802 hexanal 0.1 0.3 0.2 tr 0.4 tr o 846 851 2(e)-hexenal 0.2 1.2 0.4 0.3 0.3 tr o 850 854 3(z)-hexenol 0.2 0.7 0.3 o 859 865 2(z)-hexenol 0.1 0.2 0.8 o 863 867 n-hexanol 0.8 1.1 2.1 0.2 0.2 0.3 o 921 923 tricyclene 0.2 tr 0.2 0.2 tr 0.2 m 924 928 α-thujene 0.1 tr 0.3 0.1 0.1 0.3 m 932 935 α-pinene 7.2 5.6 11.5 3.3 4.5 4.7 m 946 951 camphene 3.5 1.6 3.2 3.0 1.7 3.1 m 952 962 benzaldehyde 0.1 0.5 tr 0.2 0.2 0.1 o 969 976 sabinene 0.7 1.0 2.8 1.5 1.5 3.5 m 974 979 β-pinene 29.6 18 18.6 36.4 28.2 25.7 m 988 992 myrcene 0.2 0.3 m 988 994 dehydro-1,8-cineole 0.1 tr 0.1 tr mo 1014 1019 α-terpinene 0.6 0.9 1.3 0.7 0.7 2.8 m 1022 1027 o-cymene 0.5 0.9 11.6 0.2 0.4 11.7 m 1024 1031 limonene 0.2 0.2 0.5 0.2 tr 0.5 m 1026 1034 1,8-cineole 40.7 51.8 37.2 39.9 53.3 35.6 mo 1054 1060 γ-terpinene 1.0 2.3 0.9 1.1 1.2 1.2 m 1065 1069 cis-sabinene hydrate 0.6 0.6 mo 1086 1092 terpinolene 0.2 0.4 0.2 0.2 0.2 0.2 m 1095 1100 linalool 0.3 0.4 0.4 mo 1107* 1108 6-ethenyldihydro-2,2,6trimethyl-2h-pyran-3(4h)-one 3.7 1.9 0.5 3.1 0.9 1.6 o 1122 1128 α-campholenal tr tr mo 1135 1142 trans-pinocarveol 0.1 mo 1141 1148 camphor 3.7 1.5 1.6 1.9 1.1 1.6 mo 1160 1166 pinocarvone tr 0.1 tr tr tr tr mo 1165 1169 borneol 0.2 0.2 0.3 0.3 0.3 0.4 mo 1170 1176 cis-linalool oxide (pyranoid) 0.1 mo 1174 1180 terpinen-4-ol 0.6 1.2 0.4 0.6 0.7 0.6 mo 1186 1193 α-terpineol 0.8 0.9 0.5 0.9 0.8 0.6 mo 1195 1199 myrtenal tr 0.3 0.1 0.1 tr mo 1288 1291 lavandulyl acetate 0.1 0.2 0.1 0.4 mo 1374 1381 α-copaene 0.2 0.9 0.4 0.6 0.2 0.3 s 1417 1427 (e)-caryophyllene 0.4 0.9 1.7 0.4 0.8 1.8 s 1454 1458 (e)-β-farnesene tr 0.1 s 1458 1466 allo-aromadendrene 0.7 0.2 0.8 0.4 s 1474 1482 10-epi-β-acoradiene 0.2 0.2 s 1484 1488 germacrene d 0.7 2.3 0.3 0.6 0.5 0.2 s 1500 1503 bicyclogermacrene 0.1 0.1 tr s 1513 1519 γ-cadinene 0.1 0.1 s 1522 1529 δ-cadinene tr 0.1 0.1 0.1 0.2 s 1582 1592 caryophyllene oxide 0.2 so 1635 1642 cis-cadin-4-en-7-ol 0.6 0.3 so total 98.9 98.1 98.3 98.2 98.8 98.6 monoterpenoids 90.7 87.8 91.3 91.3 95.3 93.5 hydrocarbons(m) 44.0 30.9 51.1 47.2 38.5 53.9 oxygenated(mo) 46.7 56.9 40.2 44.1 56.8 39.6 sesquiterpenoids 3.0 4.4 2.7 3.1 1.5 3.1 hydrocarbons (s) 2.4 4.4 2.7 2.8 1.5 2.9 oxygenated (so) 0.6 0 0 0.3 0 0.2 others (o) 5.2 5.9 4.3 3.8 2.0 2.0 compounds are listed in order of elution from a hp-5 ms column; riref: literature retention indices; riexp: experimental retention indices relative to c8-c32 n-alaknes; (*): identified by nist chemistry webbook retention indices; tr: traces (<0.1%); (-): not detectid. samples rn, tn, pnaerial plant parts (collected at rujan mountain, pčinje valley (trgovište) and preševo (mitrovac), respectively); samples rc,tc, pcinflorescences (collected at rujan mountain, pčinje valley (trgovište) and preševo (mitrovac), respectively). biologica nyssana 6 (2)  december 2015: 75-80 radojković-kostevski, i. et al.  variations in the headspace volatile profiles… 79 fig. 1. dendrogram obtained by agglomerative hierarchical clustering (constituent contents used as cases) of samples collected on biotite gneiss and conglomerates and molasses do not differ to a great extent while the sample from serpentine is slightly different. it has been already noticed that the deficiency of water and indispensable mineral elements result in numerous structural and functional adaptations of plants species that grow on a serpentine substrate (s t e v a n o v i ć et al., 2003). therefore, differences in the chemical composition of the investigated samples can probably be considered as another manifestation of the “serpentine syndrome”. acknowledgements. the authors are grateful to the ministry of education, science and technological development for financial support through the grant within frame of basic research, no 172047. references alsohaili, s. & al-fawwaz, a. 2014: composition and antimicrobial activity of achillea fragrantissima essential oil using food model media. european scientific journal, 10 (30): 156165. babović, m. & cvetković, d. 1977: trgovište sa radomirom, 34-57. in: dimitrijević, m., dragić, d., karamata, s., sikošek, b., veselinović, d. 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'hicaznar') fruits harvested at different mature periods original article civan çelik isparta university of applied sciences, agriculture faculty, department of agricultural biotechnology, isparta, türkiye civancelik@isparta.edu.tr (corresponding author) ayşe vildan pepe isparta university of applied sciences, agriculture faculty, department of horticulture, isparta, türkiye received: november 25, 2024 revised: december 03, 2024 accepted: january 10, 2025 abstract: pomegranate is a fruit species beneficial for human health due to its high antioxidant capacities and other bioactive compounds. in this study, the pomological and biochemical changes of pomegranate fruits harvested at four different ripening stages in the hicaznar variety were determined. the fruits were harvested at the stage where the seeds turned white (august 5), the stage where the color change in the seeds began (september 5), the stage where the seeds turned pink (september 29), and the stage where the seeds turned red (october 20). in line with this aim, fruit weight, fruit width, fruit length, total soluble solids, ph, titratable acidity, total phenolic content, total flavonoid content, and total antioxidant capacity were determined in fruit samples. fruit weight ranged from 132.7 to 546.49 g, fruit length ranged from 71.00 to 114.0 mm, fruit width ranged from 67.22 to 99.35 mm, total soluble solids ranged from 10.43% to 15.23%, total acidity ranged from 0.99% to 2.76%, ph ranged from 2.98 to 3.37, total phenolic content ranged from 490.40 to 570.73 mg gallic acid equivalent 100 g-1, total flavonoid content ranged from 5.85 to 7.28 mg catechin equivalent 100 g-1, and total antioxidant capacity ranged from 83.64% to 88.46%. in this study, as maturity progressed in the 'hicaznar' variety, increases in fruit weight, fruit length, fruit width, and total soluble solids content were observed. conversely, decreases in titratable acidity, total phenolic content, and antioxidant capacity were observed as maturity progressed. key words: antioxidant capacity, hicaznar, punica granatum, total phenolic content, total flavonoid content apstrakt: pomološke i biohemijske karakteristike plodova nara (punica granatum l. cv. 'hicaznar') ubranih u različitim periodima zrelosti nar je vrsta voća korisna za ljudsko zdravlje zbog visokog antioksidativnog kapaciteta i prisustva drugih bioaktivnih jedinjenja. u ovoj studiji određene su pomološke i biohemijske promene plodova nara varijeteta hicaznar, ubranih u četiri različite faze zrenja. plodovi su ubrani u fazi kada je seme bilo bele boje (5. avgust), u fazi kada je započela promena boje semena (5. septembar), u fazi kada je seme postalo ružičasto (29. septembar) i u fazi kada je seme poprimilo crvenu boju (20. oktobar). u skladu sa ovim ciljem, u uzorcima plodova određeni su masa ploda, širina i dužina ploda, ukupne rastvorljive suve materije, ph, titrabilna kiselost, ukupan sadržaj fenola, ukupan sadržaj flavonoida i ukupni antioksidativni kapacitet. masa plodova varirala je od 132,7 do 546,49 g, dužina ploda od 71,00 do 114,0 mm, širina ploda od 67,22 do 99,35 mm, sadržaj ukupnih rastvorljivih suvih materija od 10,43% do 15,23%, ukupna kiselost od 0,99% do 2,76%, ph vrednost od 2,98 do 3,37, ukupan sadržaj fenola od 490,40 do 570,73 mg ekvivalenta galne kiseline na 100 g, ukupan sadržaj flavonoida od 5,85 do 7,28 mg ekvivalenta katehina na 100 g-1, dok je ukupni antioksidativni kapacitet iznosio od 83,64% do 88,46%. u ovoj studiji je utvrđeno da su, kako je sazrevanje napredovalo kod varijeteta 'hicaznar', masa ploda, dužina, širina i sadržaj ukupnih rastvorljivih suvih materija rasli. suprotno tome, uočen je pad titrabilne kiselosti, ukupnog sadržaja fenola i antioksidativnog kapaciteta sa napredovanjem zrelosti. ključne reči: antioksidativni kapacitet, hicaznar, punica granatum, ukupan sadržaj fenola, ukupan sadržaj flavonoida © 2025 çelik and pepe. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 122 introduction pomegranate (punica granatum l.), which belongs to the lythraceae family, is part of the punica genus. it is adapted to tropical and subtropical climates, perennially deciduous, and grows in shrub form, reaching heights between 1.5 m and 5 m (onur & tibet, 1988). the fruit obtained its commercial name, „pomuni granatum”, meaning „seeded apple” in the middle ages. the carthaginians, a phoenician colony, initiated pomegranate trade in the mediterranean basin, hence its mention in ancient sources as the „carthaginian (phoenician) apple” (kurt et al., 2013). pomegranate is native to iran and afghanistan, the caucasus, the middle east, and anatolia. nowadays, cultivation occurs not only in its homeland but also in the mediterranean basin, the southern coasts of europe, the americas, the far east, and australia. the widespread dispersal of pomegranate seeds is also related to birds consuming them and spreading the seeds through their feces across vast areas (stover & mercure, 2007; glozer et al., 2008). due to being within the homeland boundaries of pomegranate, türkiye exhibits significant diversity in varieties (kelebek et al., 2010). in recent years, pomegranate production in türkiye has significantly increased from 537,847 tons (2018) to 681,460 tons (2022) (tüi̇k, 2024), marking a 21.07% increase in the last five years. currently, there are no official statistics regularly maintained by the food and agriculture organization (fao) or eurostat. pomegranate (punica granatum l.) is among the first fruits cultivated globally and is known for its health benefits, commercial value, and frequent mention in cultural contexts. with the recent increase in production, there has been a diversification in pomegranate consumption. the most well-known product is pomegranate molasses, along with its juice, wine, liqueur, concentrate, soda, syrup, jam, and jelly. apart from being a food item, it is utilized in the chemical, cosmetic, and pharmaceutical industries for citric acid, vinegar, dye, ink, and cologne production. pomegranate, containing around 28-30% tannins in its peel and being a significant source of oil from its seeds, varies in oil content from 6.6% to 19.3% depending on the variety and cultivation region (gölükçü et al., 2008). recent medical studies have revealed that pomegranate, in addition to its high antioxidant content, contains anticarcinogenic, antimicrobial, antiviral, and anti-atherosclerotic compounds that can reduce blood pressure and ldl oxidation, aid in treating alzheimer's and heart diseases, and enhance resistance against certain types of cancer; its juice has positive effects on blood parameters. particularly because of its antioxidants, pomegranate strengthens the body's defense system. studies indicate that pomegranate juice has protective effects against skin cancer and prostate cancer in men (aviram et al., 2004; reddy et al., 2007; bell & hawthorne, 2008; oğuz et al., 2011). it has been found that pomegranate juice has three times higher antioxidant activity than red wine or green tea and 2.6 to 8 times higher antioxidant activity than those found in grapes/cranberries (gil et al., 2000; rosenblat & aviram, 2006). additionally, numerous studies worldwide have been conducted on the chemical properties and health beneficial compounds of pomegranate (zarei, 2017; adiletta et al., 2018; mekni et al., 2019; el moujahed et al., 2022; parashuram et al., 2022). it has been reported that the nutritional content of fruit species significantly changes according to their ripeness level, with higher phenolic compound levels found in green fruits than in ripe fruits (choi et al., 2012; wu et al., 2012; bayar & şan, 2017). therefore, this study aimed to compare the pomological and biochemical properties of hicaznar pomegranates harvested at different maturity time. materials and methods the study was conducted in 2023. fruits of the „hicaznar” variety, which had reached twentyfive years of age and were cultivated by a private company in the serik district of antalya, were used as the material. fruit samples were collected on four different maturity time (05.08.2023. (mt1), 05.09.2023. (mt2), 29.09.2023. (mt3), 20.10.2023. (mt4)) (fig. 1), with three replicates and ten fruits per replicate, for pomological and biochemical analysis. samples for biochemical analysis were stored at -20 °c until analysis. the weight of the fruit samples (g) was measured using „hzk-210fa” precision digital scales. ten cucumber samples were weighed and selected for each genotype. additionally, the lengths (cm) and width (mm) of the fruits were measured (kılıç et al., 2022). after cleaning, the harvested fruits were peeled and filtered. the ph was measured using „adwan ad12” digital ph meter. the titratable acidity (ta) of the fruit juice was determined according to the method described by balikçi et al. (2021). pure water was added to 10 ml of fruit juice until the total volume reached 100 ml, and the mixture was titrated (with „brand titrette” brand buret) with 0.1n naoh until the ph reached 8.1. the color values were determined using „cr-400 minolta (japan)” colorimeter. the total soluble solid content (ssc) was determined using „hi 96,801” handheld refractometer (kılıç et al., 2022). biologica nyssana ● 16 (1) june 2025: 121-127 çelik and pepe ● pomological and biochemical changes in pomegranate (punica granatum l. cv. 'hicaznar') fruits harvested at different mature periods 123 biologica nyssana ● 16 (1) june 2025: 121-127 çelik and pepe ● pomological and biochemical changes in pomegranate (punica granatum l. cv. 'hicaznar') fruits harvested at different mature periods total phenolic content was determined using folin-ciocalteu’s chemical as described in the study conducted by singleton & rossi (1965). for this purpose, homogenized fruit pulp puree was subjected to extraction process in tubes for one hour using acetone, water and acetic acid (70:29.5:0.5) solution. then, folin-ciocalteu’s chemical and pure water were mixed on the extraction and waited for 8 minutes, then 7% sodium carbonate was added. after two hours of incubation, the absorbance of the solution that acquired a bluish color was read on a spectrophotometer (model t60u, pg instruments, usa) at 750 nm wavelength. the obtained results were calculated in gallic acid as μg gallic acid equivalent (gae) g-1 fresh fruit flesh. total flavonoid content was determined according to the method described by dewanto et al. (2002) and modified by çelik et al. (2023). 1.25 ml of double distilled water was added to 250 μl of extract. then, 5% sodium nitrite (nano₂) was added to the mixture and incubated for 6 min. after incubation, 150 ml of 10% aluminum chloride (alcl₃x6h₂o) solution was added to the mixture and 0.5 ml of 1 m sodium hydroxide (naoh) was added after 5 min to neutralize the solution. then, the absorbance values of the samples were measured at 510 nm using a uv spectrophotometer (model t60u, pg instruments, usa). antioxidant activity was evaluated using a method involving dpph radical scavenging. 100 μl of the extract was made up to 2 ml with 0.1 mm dpph solution and incubated for 30 min at room temperature (kumaran & karunakaran, 2006). after incubation, absorbance was measured at 517 nm using a uv spectrophotometer (model t60u, pg instruments, usa). the data obtained from the research were subjected to analysis of variance using the minitab 17 software package. tukey's multiple comparison test was used to determine significant differences between the means (p< 0.05). fig. 1. color change of fruits harvested on different maturity time (1: mt1, 2: mt2, 3: mt3, 4: mt4) 124 biologica nyssana ● 16 (1) june 2025: 121-127 çelik and pepe ● pomological and biochemical changes in pomegranate (punica granatum l. cv. 'hicaznar') fruits harvested at different mature periods results and discussion in the study, data and statistical results regarding fruit weight, fruit length, and fruit width of the hicaznar variety harvested at different ripening stages are presented in tab. 1. statistically significant differences were determined in terms of fruit weight, fruit length, and fruit width among different ripening stages (p<0.05). according to the obtained data, the highest fruit weight, fruit length, and fruit width were observed on the harvest date 'mt4' (546.49 g, 114.0 mm, and 99.35 mm, respectively), whereas the lowest fruit weight, fruit length, and fruit width were recorded on the harvest date 'mt1' (132.7 g, 71.00 mm, and 67.22 mm, respectively). fruit weight, fruit length, and fruit width of fruits harvested at four different periods in the hicaznar variety showed a significant increase with maturity. the results obtained in this study were found to be similar to those of özsayın (2012). it was determined that fruit weight increased by more than 1.5 times in the last 21 days (from september 29th to october 20th). similarly, bayar & şan (2017) reported approximately 1.5 times increase in fruit weight, 1.2 times increase in fruit width, and approximately 1.3 times increase in fruit length between september 6th and october 3rd in their study on hicaznar. similar results have been reported by different researchers (i̇kinci & dursun, 2021). was observed on the harvest date 'mt1' (2.76 g citric acid 100 g-1), while the lowest ta was observed on the harvest date 'mt4' (0.99 g citric acid 100 g-1). as a result, it was determined that the highest ph value of fruit juices (3.37) occurred during the commercial harvesting period (october 20th). the ph level in hicaznar consistently increased with maturity. consequently, it was observed that the titratable acidity content decreased consistently with the progression of ripeness. it was determined that the soluble solids content of hicaznar fruit juices consistently increased with ripening. kulkarni & aradhya (2005) and bayar & şan (2017) reported in their studies that ssc (%), and ph level increased with ripening in pomegranate, while ta (g citric acid 100 g-1) decreased. in another study, it was reported that the ssc content of hicaznar variety ranged from 14% to 18.2% at commercial harvesting time, which is consistent with our results (özsayın, 2012). the observed citric acid contents of the fruits in our study were similar to those reported by karaca (2011). karaca (2011) reported that the citric acid, malic acid, and tartaric acid contents of ripe fruits were 17,360 μg g-1, 500 μg g-1, and 590 μg g-1, respectively. mulas et al. (2013) found that the organic acid content in myrtle plants decreased with ripening. in this study, the data and statistical analysis results regarding total phenolic content, total flavonoid content, and total antioxidant content of the hicaznar variety harvested at different ripening stages are presented in tab. 3. statistically significant differences were determined in terms of total phenolic content, total flavonoid content, and total antioxidant content among different ripening stages (p<0.05). according to the obtained data, the highest total phenolic content was observed on the harvest date 'mt1' (570.73 mg gae 100 g-1), while the lowest total phenolic content was recorded on the maturity time fruit weight (g) fruit length (mm) fruit width (mm) mt1 132.70±39.40 c 71.00±12.36 d 67.22±1.10 d mt2 282.78±3.24 b 86.73±5.23 c 80.30±1.15 c mt3 354.30±83.30 b 91.21±8.42 b 90.26±0.83 b mt4 546.49±5.62 a* 114.00±7.56 a 99.35±1.41 a in this study, the data and statistical analysis results regarding ssc, ph, and ta of the hicaznar variety harvested at different ripening stages are presented in tab. 2. statistically significant differences were determined in terms of ssc, ph, and ta among different ripening stages (p<0.05). according to the obtained data, the highest ssc and ph were observed on the harvest date 'mt4' (15.23% and 3.37, respectively), while the lowest ssc and ph were recorded on the harvest date 'mt1' (10.43% and 2.98, respectively). the highest ta a,b,c values within a column with different superscripts differ significantly at p<0.05 ** the study was carried out on a total of 80 fruits, with 3 replications and 20 fruits in each replication. table 1. fruit weight, fruit length, and fruit width values of hicaznar variety at four different maturity time table 2. ssc, ph, ta values of hicaznar variety at four different maturity times maturity time ssc (%) ph ta (g citric acid 100 g-1) mt1 10.43±0.20 d 2.98±0.01 b 2.76±0.17 a mt2 13.20±0.20 b 3.07±0.02 b 2.33±0.09 b mt3 14.30±0.27 c 3.43±0.04 a 1.54±0.13 c mt4 15.23±0.15 a 3.37±0.07 a 0.99±0.04 d a,b,c values within a column with different superscripts differ significantly at at p<0.05 **the study was carried out on a total of 80 fruits, with 3 replications and 20 fruits in each replication 125 biologica nyssana ● 16 (1) june 2025: 121-127 çelik and pepe ● pomological and biochemical changes in pomegranate (punica granatum l. cv. 'hicaznar') fruits harvested at different mature periods harvest date 'mt4' (490.40 mg gae 100 g-1). the highest total flavonoid content was observed on the harvest date 'mt4' (10.67 mg cae 100 g-1), while the lowest total flavonoid content was observed on the harvest date 'mt3' (5.85 mg cae 100 g-1). the highest antioxidant content in pomegranate was observed on the harvest dates 'mt1, mt2, mt3' (88.46, 87.60% inhibition, respectively), while the lowest antioxidant content was observed on the harvest date 'mt4' (83.64% inhibition). the total phenolic content in hicaznar fruit juice was found to be higher in unripe fruits compared to ripe fruits. in the study, it was determined that the total phenolic content in fruit juice was at its highest level (530.70 mg gae 100 g-1) when the color change began (august 5th) and decreased with ripening. similarly, the total antioxidant content was higher in unripe fruits (88.46% inhibition), and it decreased during the commercial harvesting period (83.67% inhibition). in contrast, the total flavonoid content increased with ripening, and the highest value (10.67 mg cae 100 g-1) was observed during the commercial harvesting period. fadda & mulos (2010), siriamornpun et al. (2015), eşiyok (2022), and pepe et al. (2022) reported that the total phenolic content decreases with ripening. the amount of phenolic compounds and flavonoid compounds in fruit species can vary significantly depending on genotype, ecological conditions, and analysis methods, as reported in many studies (wang et al., 2010; vatansever, 2018; demir & başayiğit, 2022). in our study, it was found that there was a parallel decrease in the total phenolic content as the degree of ripeness increased. consistent with our results, al-maiman & ahmad (2002) reported a significant decrease in the total phenolic content of pomegranate fruits with ripening. similarly, siriamornpun et al. (2015) found that unripe green fruits of jujube contained more total phenolic content than ripe fruits. conclusion in conclusion, the biochemical contents of 'hicaznar' fruits significantly vary according to ripening stages. the total phenolic content, antioxidant level, and titratable acidity decrease as ripeness increases. conversely, fruit weight, fruit length, fruit width, soluble solids content (ssc), and ph increase with ripening. although the total phenolic content and antioxidant levels decrease with ripening, fruits should be harvested during the commercial harvesting period for fresh consumption. references adiletta, g., petriccione, m., liguori, l., pizzolongo, f., romano, r., & di matteo, m. (2018). study of pomological traits and physicochemical quality of pomegranate (punica granatum l.) genotypes grown in italy. european food research and technology, 244, 1427-1438. https:// doi.org/10.1007/s00217-018-3056-x al-maiman, s.a. & ahmad, d. 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'hicaznar') fruits harvested at different mature periods microsoft word bn-oa-0201-05 cavlovic et al biologica nyssana 2 (1) september 2011: 39-44 čavlović d. et al. allochthonous woody taxa in zasavica ecosystem 39 original article ! allochthonous woody taxa in zasavica ecosystem dragana čavlović, mirjana ocokoljić*, dragica obratov-petković faculty of forestry, university of belgrade, serbia * e-mail: mirjana.ocokoljic@sfb.rs abstract: čavlović, d., ocokoljić, m., obratov-petković, d.: allochtonous woody taxa in zasavica ecosystem. biologica nyssana, 2 (1), september 2011: 39-44. special nature reserve zasavica is an important wetland in serbia. therefore, it was designated as “special nature reserve of the first category“ in 1997. moreover, it has been included in the national network of ramsar sites (the ramsar convention on wetlands of international importance), in 2006. considering importance of special nature reserve zasavica and the fact that stability of ecosystems can be disturbed by introducing alien species, we analyzed extent and coverage of allochthonous woody taxa within the reserve region. researching area comprised 1821 hectares. we found 21 allochthonous woody taxa that belong to magnoliophyta subdivision. the most individuals are located within the first zone of protection. key words: alburnoides bipunctatus, cyprinidae, mesohabitat, phenotype plasticity introduction ! the ecological impact of fast spreading introduced plant species (invasive species) on natural communities has been analysed in numerous articles (e l t o n 1958; d r a k e et al. 1989; d i c a s t r i et al. 1990). some alien tree species used in commercial forestry and agroforestry cause major problems as invaders of natural and seminatural ecosystems. exotic plant species are considered as one of major threats to biodiversity (r i c h a r d s o n , 1998, r i c h a r d s o n et al., 2000). in this article we analyzed the coverage of allochthonous woody taxa within the zasavica reserve region. special nature reserve zasavica is a marshy lowland, located in north-west mačva, within the municipalities sremska mitrovica and bogatić. it is named after the watercourse zasavica which is 33.1 km long, 80 m wide. during normal water levels, the river is 2.5 metres deep on average. zasavica forms six large meanders. the terrain surrounding the meanders is marshland (p a v i ć , 2001). in 1997 zasavica was designated as “special nature reserve of the first category“ by the government of the republic of serbia and as a protected area of exceptional importance for the republic (official gazette rs, 19/97). in 2006 the reserve was included in the ramsar list of wetlands of international importance. the reserve is covered by a two-degree regime of protection: 1150 ha are the first degree, and 671 ha are the second category of protection. taking into account that, by intentional or unintentional introduction of allochthonous woody species to wet ecosystems, their stability is disturbed, this study analyses the state and the influence of allochthonous woody species to the area of the reserve (daisie, 2010). materials and methods during investigation period that covered two complete vegetation seasons (march 2009 to october 2010), we recorded each individual of 2 (1) • september 2011: 39-44 biologica nyssana 2 (1) september 2011: 39-44 čavlović d. et al. allochthonous woody taxa in zasavica ecosystem 40 allochthonous woody taxa that were determined according to š i l i ć , 1998. for each individual we assessed the age, phytopathological and entomological damage. we used separate scales to assess the vitality and ornamental value of individuals (o c o k o l j i ć , et al. 2009). we used the combined abundance-coverage scale (b r a u n -b l a n q u e t , 1965) to assess the importance of each species within investigated area. results and discussion during the research, we recorded 1850 individuals of 21 allochthonous woody taxa. ten species originate from the north american continent and ten from asia. populus x euramericana is a hybrid species. its presence in zasavica was expected because it grows in plantations (46,600 individuals) of the fe sremska mitrovica. all species belong to magnoliophyta subdivision. acer negundo l. (fam. aceraceae) this species is represented throughout the reserve with 95 individuals. their age varies from 10 to 60 years. combined abundance-coverage value of the species is 3. average mark of vitality is 4, and depending on individuals, vitality ranges from 2 to 5. average mark of ornamental value is 3, and it varies from 2 to 4. there are no phytopathological and entomological injuries on the trees. ailanthus altissima (mill.) sw. (fam. simarubaceae) this species is represented throughout the reserve with 120 individuals. tree age ranges from 10 to 20 years. coverage is evaluated by mark 3. average mark of vitality is 4, and depending on individuals, vitality ranges from 2 to 5. average mark of ornamental value is 5. there are no phytopathological and entomological injuries on any trees. amorpha fruticosa l. (fam. leguminosae) this species is represented throughout the reserve with 550 individuals. average age is 20 years. coverage is evaluated by mark 4, and at some places the abundance attains mark 5. average mark of vitality is 4, and depending on individuals, vitality ranges from 3 to 5. average mark of ornamental value is 4, and it varies from 4 to 5. no phytopathological and entomological damage is observed in any of the individuals. according to snr zasavica data, the removal of false indigo was performed in 2008. caragana arborescens lam. (fam. leguminosae) c. arborescens is recorded at the site ribnjača bara as a single individual. its age is 10 years. coverage is evaluated by mark +. vitality mark is 3, and mark of ornamental value is also 3. this specimen does not show any phytopathological and entomological damage. catalpa bignonioides walt. (fam. bignoniaceae) c. bignonioides is recorded in this area only at the locality cerik as one tree aged about 20 years. its coverage is marked +. its vitality is low (mark 1) and its ornamental value is also evaluated by the same mark. gleditsia triacanthos l. (fam. cesalpiniaceae) this species is recorded at two localities in the reserve: jovača and široka bara. there are 25 individuals on these two sites. tree age ranges from 10 to 20 years. coverage is evaluated by mark 1. average mark of vitality is 4, and depending on individuals, vitality ranges from 3 to 5. average mark of ornamental value is also 4, and it varies from 2 to 5. there are no phytopathological and entomological injuries on any trees. based on the analysis at these localities, it is concluded that gleditsia triacanthos l. occurs mainly along the roads in minor line groups. maclura aurantiaca nutt. (fam. moraceae) m. aurantiaca is recorded at two localities in the reserve: jovača and prekopac with 20 individuals. tree age ranges from 10 to 20 years. coverage is evaluated by mark 1. average mark of vitality is 3, and depending on individuals, vitality ranges from 2 to 5. average mark of ornamental value is 5. there are no phytopathological and entomological injuries on any trees. mahonia aquifolium (pursh) nutt. (fam. berberidaceae) m. aquifolium is recorded as one individual at the site jovača. the shrub age is about 10 years. its coverage is therefore marked with +. vitality and ornamental value are evaluated as 4. morus alba l. (fam. moraceae) m. alba is represented throughout the reserve with 65 individuals. tree age is from 20 to 30 years. coverage is evaluated by mark 3. all the analysed specimens show exceptional vitality and ornamental value (average mark 5). there are no biologica nyssana 2 (1) september 2011: 39-44 čavlović d. et al. allochthonous woody taxa in zasavica ecosystem 41 phytopathological and entomological injuries on any individuals. morus nigra l. (fam. moraceae) m. nigra is represented throughout the reserve with 46 individuals. tree age is 30 to 50 years. coverage is evaluated by mark 3. vitality is evaluated with 5. its average mark of ornamental value is also the highest (5). there are no phytopathological and entomological injuries on any trees. parthenocissus quinquefolia (l.) planch. (fam. vitaceae) p. quinquefolia is a climber, which is recorded at the site široka bara (1 individual). its age is about 10 years. coverage is evaluated by mark +. vitality mark is 3, and the mark of ornamental value is 4. there are no phytopathological and entomological injuries on this tree. populus alba 'pyramidalis' l. (fam. salicaceae) p. alba 'pyramidalis' is a taxon which is represented throughout the reserve with 60 individuals. tree age ranges from 20 to 30 years. coverage is evaluated by mark 1, because the trees at the study locality are solitary. average mark of vitality is 4, and depending on individuals, vitality ranges from 3 to 5. average mark of ornamental value is 4, and it varies from 3 to 5. there are no phytopathological and entomological injuries on any trees. only male individuals are represented. populus deltoides marshall (fam. salicaceae) p. deltoides is recorded at two locality in reserve: cerik and vrbovac with 11 individual. tree age ranges from 20 to 30 years. coverage is evaluated by mark 2. average mark vitality is 4, and depending on individuals, vitality ranges from 2 to 5. average mark of ornamental value is 4, and it varies from 3 to 5. there are no phytopathological and entomological injuries on any trees. prunus cerasifera ehrh. (fam. rosaceae) p. cerasifera is represented throughout the reserve with 450 individuals. this taxon grows both as a shrub and as a tree; the age is up to 20 years. coverage is evaluated by mark 4, and at the site valjevac, its coverage is 5. its average mark of vitality is 4, and depending on individuals, vitality ranges from 3 to 5. average mark of ornamental value is 5. there are no phytopathological and entomological injuries on any individuals. reduction of individuals was performed during the previous period. aerial photographs from 2008, 2009 and 2010 show the aggressive spreading and invasion of the territory of the pasture valjevac. quercus borealis michx. (fam. fagaceae) q, borealis is recorded only at the localty jovača as one individual, its age is about 20 years. coverage is evaluated by mark +. this exceptionally vital tree, with regular crown, is evaluated with mark 5, and the mark of ornamental value is also the highest (5). there are no phytopathological and entomological injuries on this tree. robinia pseudoacacia l. (fam. leguminosae) this species is represented throughout the reserve with 400 individuals. tree age varies from 10 to 30 years. coverage is evaluated by mark 4. average mark of vitality is 5. average mark of ornamental value is also 5. there are no phytopathological and entomological injuries on any trees. salix babylonica l. (fam. salicaceae) this is recorded in the reserve at the following localities: modran, gaj, cerik and zovik with 4 individuals. tree age ranges from 10 to 30 years. coverage is evaluated by mark 1. as for tree vitality, all 4 trees are evaluated with mark 5, and also their ornamental value is excellent (5). there are no phytopathological and entomological injuries on any trees. salix matsudana koidz. (fam. salicaceae) s. matsudana is recorded in the reserve at the localities: modran, cerik and zovik with three individuals. tree age ranges from 10 to 30 years. coverage is evaluated by mark +. average mark of vitality is 4, and the marks range from 3 to 5. ornamental value is evaluated 4 (all three trees). there are no phytopathological and entomological injuries on any trees. sophora japonica l. (fam. leguminosae) this is recorded at the localty modran as one individual. the observed tree age is about 20 years. coverage is evaluated +. its vitality is excellent (5), and also its ornamental value (5). ulmus pumila l.(fam. ulmaceae) this is recorded in the reserve at the localty ribnjača bara with three individuals. tree age is from 10 to 20 years. coverage is evaluated by mark +. all trees are evaluated with mark 4 both for vitality and for ornamentalness. there are no phytopathological and entomological injuries on any trees. biologica nyssana 2 (1) september 2011: 39-44 čavlović d. et al. allochthonous woody taxa in zasavica ecosystem 42 conclusion analysing trees and shrubs in snr zasavica, we recorded 1850 individuals of alien species. half of the analysed taxa occupy the second zone of protection, and 50 % grow both in the first and in the second zones of protection. total number of recorded taxa is 20 and all of them belong to the subdivision magnoliophyta. the most abundant taxon is amorpha fruticosa (550 individuals). on the other side, we recorded only one individual of caragana arborescens, catalpa bignonioides, quercus borealis and sophora japonica. the comparative analysis shows an exceptionally high vitality (4) of the alien species. this indicates on their good adaptation to environmental conditions within the study area. the species with the highest vitality (mark 5) are amorpha fruticosa, morus alba, morus nigra, quercus borealis, robinia pseudoacacia and salix babylonica. the lowest vitality (mark 1) was detected for catalpa bignonioides. high marks support the thesis of good adaptability of allochthonous taxa. the comparative analysis of the marks of ornamental value of all individuals shows that the average mark is 4. at the taxon level, the highest average mark (mark 5) is attained by: ailanthus altissima, maclura aurantiaca, morus alba, morus nigra, prunus cerasifera, quercus borealis, robinia pseudoacacia, salix babylonica and sophora japonica, and the lowest by catalpa bignonioides (mark 2). the research confirms the occurrence of spontaneous spreading in 30 % of taxa: acer negundo, ailanthus altissima, amorpha fruticosa, gleditsia triacanthos, prunus cerasifera and robinia pseudoacacia. in 20 %: acer negundo, ailanthus altissima, amorpha fruticosa and prunus cerasifera, spreading can be characterised as invasiveness. all invasive taxa are characterised by extremely good coppicing power. their spreding potential is high since their seeds are readily dispersed by wind to very distant places or zoochoriously. this study indicates that the long-term monitoring is necessary to detect the effect of allochthonous woody taxa on the stability of ecosystems in the zasavica nature reserve. acknowledgements. this paper was realized as a part of the project "studying climate change and its influence on the environment: impacts, adaptation and mitigation" (43007) financed by the ministry of education and science of the republic of serbia within the framework of integrated and interdisciplinary research for the period 2011-2014. references antić, m., jovanović, b., jović, n., avdalović, v. 1973: projekat klasifikacije šumskih zemljišta u jugoslaviji, simpozijum iz šumarske pedologije, tjentište, 1973. izdanje an b i h, knjiga 5, sarajevo, 1975. braun-blanquet,j. 1965. phlanzensoziologie. grundzuge der vegetatinskunde. 3rd ed. springer, wien, n. y. daisie 2010: handbook of alien species in europe http:// www.europe-aliens.org. di castri, f., hansen, a.j. & debussche, m. 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sredstva, beograd. tomić, z. 2004: šumarska fitocenologija, šumarski fakultet, beograd. vasin, j., sekulić, p., hadžić, v., bogdanović, d., pucarević, m. 2004: stepen zagađenja nepoljoprivrednog zemljišta u vojvodini, zbornik radova, sveska 40, naučni institut za ratarstvo i povrtarstvo, novi sad. vukićević, e. 1996: dekorativna dendrologija, šumarski fakultet, beograd. biologica nyssana 2 (1) september 2011: 39-44 čavlović d. et al. allochthonous woody taxa in zasavica ecosystem 44 anticancer compounds from medicinal plants biologica nyssana 5 (1)  september 2014: 53-61 vulić, i. et al.  taxonomic composition and community structure... 53 original article received: 14 august 2014 revised: 3 september 2014 accepted: 10 september 2014 taxonomic composition and community structure of trichoptera (insecta) on the territory of the city of niš (serbia) ivana vulić, ivana vasov, ana savić university of niš, faculty of science and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia e-mail: anka@pmf.ni.ac.rs abstract: vulić, i., vasov, i., savić, a.: taxonomic composition and community structure of trichoptera (insecta) on the territory of the city of niš (serbia). biologica nyssana, 5 (1), septemeber 2014: 53-61. the research of trichoptera community was performed within the territory of the city of niš on 10 localities. these localities have different physical and chemical characteristics ranging from hot springs to cold karst streams. some of them are with a measurable human influence (in the city of niš), while others are practically without it (dušničko vrelo). a total number of 16 genera trichoptera within 11 families were noted. number of identified species was 20. it was determined that the highest species diversity is in localities with the least human influence. during this study, the presence of the species that has the status of a strictly protected species in republic of serbia was recorded. the largest number of localities belongs to β – mesosaprobic waters. it has been found that group trichoptera is suitable for independent application in bioindicator purposes. key words: trichoptera, nišava river, saprobiological analysis, diversity, taxonomic composition, community structure apstract: vulić, i., vasov, i., savić, a.: taksonomski sastav i struktura zajednice trichoptera (insecta) na teritoriji grada niša. biologica nyssana, 5 (1), septemeber 2014: 53-61. istraživanje zajednice trichoptera sprovedeno je na teritoriji grada niša na 10 istraživanih lokaliteta. lokaliteti su različitih fizičko-hemijskih karakteristika u rasponu od termalnih izvora do hladnih kraških tekućica. neki od njih su pod značajnim antropogenim uticajem (u samom gradu nišu) dok su drugi gotovo bez njega (dušničko vrelo). konstatovano je ukupno 16 rodova trichoptera u okviru 11 familija. identifikovano je ukupno 20 vrsta. utvrđeno je da je specijski diverzitet najveći na lokalitetima sa najmanjim antropogenim uticajem. tokom istraživanja konstatovana je vrsta koja ima status strogo zaštićene vrste u republici srbiji. najveći broj lokaliteta pripada beta mezosaprobnim vodama. ustanovljeno je da je ova grupa pogodna za samostalnu primenu u bioindikatorske svrhe. ključne reči: trichoptera, diverzitet, reka nišava, saprobiološka analiza, taksonomski sastav, struktura zajednice 5 (1) • september 2014: 53-61 biologica nyssana 5 (1)  september 2014: 53-61 vulić, i. et al.  taxonomic composition and community structure... 54 introduction order trichoptera is the largest among the orders of primarily aquatic insects, which include: ephemeroptera, plecoptera, odonata and megaloptera. its diversity is similar to that of the order diptera (h o l z e n t h a l et al., 2007). trichoptera are considered to be the most common group of macro-invertebrates in the freshwater ecosystems. they are widespread and inhabit a variety of habitat types, within the multiple trophic levels (s a v i ć 2013). trichoptera larvae are a very important component of trophic dynamics and energy flows in the aquatic ecosystems they inhabit. many species of trichoptera are sensitive to pollution, so their presence and relative abundance are used in biological assessment and monitoring of water quality (h o l z e n t h a l et al., 2007). there are 23 families of trichoptera recorded in europe (including arctopsychidae and ptilocolepidae, but excluding apataniidae, because it is considered a subfamily of limnephilidae) and a total of 133 genera, including the genus alpopsyche (b o t o s a n e a n u & g i u d i c e l l i , 2004, according to w a r i n g e r & g r a f , 2013) with about 1100 recent species (i b r a h i m i et al., 2012). according to the available data, the trichoptera fauna of the balkan peninsula includes 21 families and a high number of 90 genera. within the territory of serbia, trichoptera larvae were investigated as part of macrozoobenthos studies in mountain streams and rivers. the adult forms were studied by a small number of authors, including m a r i n k o v i ć -g o s p o d n e t i ć (1975, 1980) and r a d o v a n o v i ć (1931, 1935, 1953), as cited by ž i v i ć et al. (2000). the first checklist of the trichoptera fauna in serbia included 173 species (ž i v i c et al., 2002). the trichoptera fauna of serbia currently includes 186 species, 49 genera and 18 families (ž i v i c et al., 2006). figure 1: map of the studied area with localities biologica nyssana 5 (1)  september 2014: 53-61 vulić, i. et al.  taxonomic composition and community structure... 55 table 1. database of localities and gps coordinates locality name of locality n е altitude (m) 1 niška banja spring 43° 17.468' 22° 00.749' 266 2 niška banja near jelašnica road 43° 17.903' 22° 00.498' 212 3 nišava r. near sićevo 43° 19.893' 22° 04.429' 233 4 dušničko vrelo spring 43 o 10.433’ 22° 07.033' 501 5 dušnička reka r. 43 o 10.297’ 22° 07.056’ 466 6 kutinska reka r. 43° 09.820' 22° 05.972' 395 7 banja topilo upstream from warm water discharge 43° 26.889' 21° 52.726' 256 8 banja topilo downstream from warm water discharge 43° 26.894' 21° 52.692' 253 9 nišava r. at bulevar (near the mouth of gabrovačka reka r.) 43° 19.517' 21° 55.266' 195 10 nišava r. near medoševac 43° 19.330' 21° 52.375' 189 material and methods material was collected in november and december 2012 and february and may 2013 at 10 representative sites at the nišava river and its tributaries (fig. 1). selected sites were located at points above and below the pollutant discharge. choice of sampling sites was heavily influenced by need for variety in following factors: altitude, bedrock, slope, flow rate, bottom substrate, microclimate conditions etc. localities 1 and 2 are thermal water bodies at niška banja. in contrast, localities 4 and 5 are a typical cool karst spring (locality 4) and upper course of a karst river (locality 5). locality 6 is the tributary of the nišava river-kutinska reka river. the following two localities are at the miljkovačka reka river: locality 7 is above the thermal springs of banja topilo, while locality 8 is located below the same thermal springs. localities 9 and 10 are at the nišava river, in the urban area (tab. 1). explorations were conducted once a month, during a single day in order to accurately compare chemical parameters, which change a lot faster than the biotic parameters. physical parameters were measured directly at the sampling sites. the following physical characteristics were determined: temperature, turbidity and conductivity. temperature and conductivity were measured using a device photometer system pc multidirect (lovibond ®) while turbidity of water was determined using the lovibond pc checkit device. chemical parameters were mostly measured directly, using the photometer system pc multidirect (lovibond ®) device (the saturation and oxygen concentration, ph). biochemical oxygen demand (bod5) was obtained using the standard methodology, recommended by apha (1999). sampling was conducted by using a square frame kick net (35 × 35 cm, mesh size 300 μm). the treated area of bottom surface was 35 x 35 cm. the bottom substrate was disturbed and macroinvertebrate individuals were separated from the substrate and carried by a stream as water was entering the sampling net. samples were then fixed in 4% formaldehyde, placed in labeled plastic bags and transported to the laboratory. in the laboratory, the samples were rinsed under the tap, using two screens with larger and smaller diameter of buds. a total of 40 samples were analyzed. separated materials have been sorted in transparent bottles containing 70% ethanol and later determined by use of identification keys. the determination of each individual was performed by using a binocular magnifying glass and microscopes in specially equipped laboratories at the center for biological and environmental monitoring of surface waters in the city of niš (bioekocen), at the faculty of science and mathematics, university of niš. the observation and measurements of very small body parts were done by using binocular leica mz 16a stereomicroscope with leica dfc320 digital camera and leica dm2500 microscope system with leica dfc490 digital camera. the individuals of the order trichoptera were identified by using the following keys: individuals from families glossosomatidae, beraeidae, biologica nyssana 5 (1)  september 2014: 53-61 vulić, i. et al.  taxonomic composition and community structure... 56 table 2. average values of physical-chemical parameters at the studied localities locality turbidity temperature ph conductivity oxygen mg/l oxygen % bod5 shading % 1 0.35 30.6 7.5 477 3.59 48.5 5.14 40 2 3.63 23.5 7.7 466 4.74 58 5.57 15 3 1.91 12.4 7.9 421 5.81 64.25 5.33 15 4 0.12 10.4 7.6 246 7.29 61.54 4.02 0 5 2.24 10.1 7.9 290 7.82 91.25 9.68 40 6 4.88 9.2 7.9 364 8.77 113.75 10.72 2 7 1.82 11.8 8.1 404 7.87 71 9.12 20 8 2.6 8.2 8 435 8.3 101.25 8.82 5 9 1.93 11.3 8.1 459 6.83 84.25 5.91 6 10 2.29 11.9 8.1 518 5.77 84.75 5.73 5 table 3. average abundance of trichoptera species (individuals/ m 2 ) localities 1 2 3 4 5 6 7 8 9 10 glossosomatidae agapetus ohripes curtis 1834 2.72 beraeidae beraea pullata (curtis 1834) 277.55 46.26 8.16 5.44 beraeodes minutus (linnaeus 1761) 2.72 2.72 hydropsychidae cheumatopsyche lepida (pictet 1834) 165,9 8.16 111.56 2.72 hydropsyche angustipennis (curtis 1834) 2.72 hydropsyche contubernalis mclachlan 1865 78.9 2.72 2.72 hydropsyche fulvipes curtis 1834 5.44 2.72 hydropsyche instabilis (curtis 1834) 95.25 2.72 76.2 8.16 hydropsyche pellucidula (curtis 1834) 2.72 117.01 2.72 5.44 8.16 hydropsyche sp. 2.72 limnephilidae halesus radiatus (curtis 1834) 51.7 43.54 stenophylax permistus mclachlan 1895 24.49 21.77 2.72 16.33 lepidostomatidae lasiocephala basalis (kolenati 1848) 43.54 leptoceridae setodes sp. 21.77 27.21 8.16 psychomyiidae lype reducta (hagen 1868) 2.72 tinodes unicolor (pictet 1834) 8.16 psychomyia pusilla ( fabricius 1781) 38.09 5.44 32.65 163.26 phryganeidae phryganea bipunctata retzius 1783 8.16 polycentropodidae neureclipsis bimaculata (linnaeus 1758) 2.72 rhyacophilidae rhyacophila dorsalis (curtis 1843) 2.72 2.72 2.72 rhyacophila fasciata hagen 1859 8.16 2.72 8.16 sericostomatidae sericostoma personatum (kirby & spence 1826) 2.72 5.44 biologica nyssana 5 (1)  september 2014: 53-61 vulić, i. et al.  taxonomic composition and community structure... 57 limnephilidae, lepidostomatidae, phryganeidae, psychomyiidae and leptoceridae were identified according to the w a l l a c e et al. (1990) identification key, while individuals belonging to families rhyacophilidae, polycentropodidae and hydropsychidae were identified by using the e d i n g t o m and h i l d r e w (1995) identification key. saprobity is expressed according to the saprobity index s, in accordance with the method devised by pantle buck (1955). s stands for saprobic index of the communities, h for the relative abundance and s for the saprobic value, which is characteristic for each species. for needs of saprobiological analysis, lists of indicator species by sladaček were used (s l a d a č e k 1973), while for the evaluation of the frequency of species, a modified scale of 6 degrees to rusev-in was used (r u s e v 1993). results during the analysis of the physical-chemical parameters, it was determined that locality 1 stood out from the rest due to low values of turbidity, high temperatures and minimal values of average concentration of oxygen. the lower value of turbidity was observed only at the locality 4. the highest values of turbidity were recorded at the locality 6. ph values at all localities were close to the values characteristic for neutral water (about 7). lower alkalinity values were noticed at localities 7, 8, 9 and 10 (tab. 2). the greatest number of species was recorded at the locality 4 (11 species). localities with high species diversity were locality 8 (9 species) and locality 3 (8 species). most individuals were sampled at locality 3 a total of 187. the lowest number of species was recorded at locality 2 only two species. no trichoptera individuals were recorded at localities 1 and 10 (tab. 3). the family psychomyiidae was represented by three genera, families beraeidae, hydropsychidae and rhyacophilidae by two genera, while other families were represented by one genus each. five species belonging to the genus hydropsyche were recorded, while other genera were represented by single species only. the species hydropsyche pellucidula (curtis 1834) was observed at five of the investigated localities while species cheumatopsyche lepida (pictet 1834), stenophylax permistus mclachlan 1895, hydropsyche instabilis (curtis 1834), beraea pullata (curtis 1834) and psychomyia pusilla (fabricius 1781) were recorded at four investigated localities each. the following species were recorded at only a single locality: agapetus ohripes curtis 1834 at locality 4, hydropsyche angustipennis (curtis 1834) at locality 3, lype reducta (hagen 1868) at locality 5, tinodes unicolor (pictet 1834) at locality 8, while phryganea bipunctata retzius 1783 and neureclipsis bimaculata (linnaeus 1758) were both recorded only at locality 3. the saprobity index is presented as part of autumn, winter or spring aspect. the autumn aspect included samples collected in november 2012, the winter aspect included samples collected in december 2012 and february 2013, while the spring aspect included samples collected in may 2013. in the autumn aspect, indicator species were present at localities 3, 5 and 8 (tab. 4). the saprobity index has shown that all three sites had β mesosaprobic water (β), which belongs to the class ii water. in the winter aspect, the lowest value of saprobity index was recorded at the locality 4. during this period, saprobity index values at all sites were within the limits characteristic for β mesosaprobic water, except at locality 4 where the value of saprobity index indicated oligosaprobic water. in the spring aspect, the saprobity index values at localities 5, 6 and 7 were within the range typical for oligosaprobic water, while the values recorded at localities 3 and 9 indicated β mesosaprobic water (fig. 2). following indicators of saprobity were used in this research: genus hydropsyche and species sericostoma personatum, rhyacophila septentrionis (i.e. rhyacophila fasciata), neureclipsis bimaculata and stenophylax permistus. all species recorded during this study were typical for the european continent and known for many european countries (fig. 3). all of them were also included in the checklist of the trichoptera fauna in serbia from 2006. localities where trichoptera species were recorded are also the new sites for these species in republic of serbia. biologica nyssana 5 (1)  september 2014: 53-61 vulić, i. et al.  taxonomic composition and community structure... 58 table 4. saprobity index (s) values for all aspects locality aspect 1 2 3 4 5 6 7 8 9 10 autumn 1.95 1.55 1.95 winter 1.95 1.82 1.4 1.75 1.62 1.95 spring 1.95 1.25 1.25 1.25 1.95 figure 2. saprobity index values in autumn, winter and spring aspect figure 3. comparison of the european genera and families with genera and families found within the territory of city of niš discussion the maximum value of temperature was observed at locality 1, which is a thermal spring at niška banja. at this locality, thermal pollution was dominant. the lowest values of turbidity were recorded at the locality dušničko vrelo, which may be explained by the fact that this site is located in a place still under minimal anthropogenic influence. in the period from november 2012 to may 2013, a total of 16 genera within 11 families were recorded. 20 species were identified, while two taxa were identified to genus level. the trichoptera fauna in niš contained a high percentage (47.83%) of european families and 11.63% of european genus biologica nyssana 5 (1)  september 2014: 53-61 vulić, i. et al.  taxonomic composition and community structure... 59 genera, which is a significant percentage considering that this order is insufficiently explored in our country. the greatest number of species was recorded at the locality dušničko vrelo (11 species), while the largest number of individuals was recorded at locality nišava river near sićevo. no species of trichoptera were recorded at localities niška banja spring and nišava river near medoševac. in comparison to the number of species of trichoptera previously recorded in other rivers in serbia where the trichoptera or other macrozoobenthos groups were studied, the diversity of the trichoptera assemblage in niš is greater than the diversity values observed in the following rivers: vlasina (s c h m i d t -k l o i b e r et al., 2008), ribnica and lepenica (j o v i ć et al. 2006), jablanica (s t e f a n o v i ć 2009), banja river (ž i v i ć et al. 2000), vrelska padina i ivanštica (đ u k n i ć et al. 2010), zapadna morava (n o v a k o v i ć 2013), pčinja (s i m i ć & s i m i ć 2003), južna morava (n o v a k o v i ć 2012), velika morava (m a r k o v i ć et al. 2011), pusta reka (ž i v i ć et al. 2001). the greater diversity of this order was noticed at rivers temska and visočica (ž i v i ć et al. 2005) and golijska moravica (đ i k a n o v i ć et al. 2008), with respect to the diversity of the trichoptera community in area of city of niš. the diversity of the trichoptera community in the study area is equally high as the diversity recorded along the entire flow of the nišava river (s a v i ć 2012). of all of the recorded species, beraeodes minutus (linnaeus 1761) stands out as one of the strictly protected river invertebrates in the republic of serbia (r a d u l o v i ć et al. 2012). this species was recorded in december 2012 at the locality dušničko vrelo and in february 2013 at the locality banja topilo downstream, at the place where hot water discharge enters the river. in autumn and winter, the saprobity index was within the limits characteristic of β-mesosaprobic waters. the saprobity was lower in spring due to the prevalence of indicators of oligosaprobic waters (or the species stenophylax permistus). low values of the saprobic index may be associated with variations of water level in nišava river. this river is characterized by highest water levels in spring months when rainfall and water from thawed snow are at the peak. periods of high water level lead to dilution of pollutants that continue to influence the decrease in the saprobity index. most of the studied localities at βmesosaprobic water bodies belong to class ii waters. the results obtained during this study correspond to the results collected during the hydro-biological research of the nišava river, according to the water framework directive (2000/60/ec), when it was concluded that at the studied sites the saprobic index was within the limits of β-mesosaprobic waters (b r a n k o v i ć et al. 2007). conclusion during this study it was concluded that hydropsychidae was the most common family in the study area. out of the recorded total of 16 genera, the most common genus was hydropsyche. recorded species belonging to this genus inhabited both the polluted and the minimally contaminated locations. therefore it was concluded that within the order trichoptera the taxonomic level of the genus should not be considered reliable for bio-indicator purposes. there are no literature data indicating that the sampling sites chosen for this study were previously used for studying the trichoptera species in the republic of serbia. the presence of a species with the status of a strictly protected species in the republic of serbia in studied localities provides additional significance for this area as an area of hydrobiological importance. as the sampled sites belonged to the oligosaprobic and β-mesosaprobic zone, this area may be considered to have great potential for production of high-quality drinking water. references anonymous, 2000: okvirna direktiva o vodama. water framework directive, 2000/60/ec, official journal of the european communities l327, 1-72. brajković, m., 2004: zoologija invertebrata. zavod za udžbenike i nastavna sredstva, beograd. branković, s., simić, v. & trajković, s., 2007: hidrobiološka istraživanja reke nišave prema okvirnoj direktivi o vodama. zbornik radova građevinsko-arhitektonskog fakulteta, 22: 143148. đikanović, v., jakovčev-todorović, d., nikolić, v., paunović, m. & cakić, p., 2008: qualitative composition of communities of aquatic 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the južna morava river basin. archives of biological sciences, 55(3-4): 33-34. živić, i., marković, z. & brajković, m., 2002: a contribution to the study of the trichoptera fauna in serbia over the period 1980-2001. archives of biological sciences, 52 (1-2): 15-16. živić, i., marković, z. & brajković, m., 2006: new trichoptera for the fauna of serbia. acta entomologica serbica, 11(1/2): 51-60. živić, i. & marković, z., 2006: saprobiological analysis of water of the southern morava river (a second order tributary of the danube in serbia) on the basis of macrozoobenthos as a bioindicator. proceedings 36th international conference of iad: 301-306. biologica nyssana 5 (1)  september 2014: 53-61 vulić, i. et al.  taxonomic composition and community structure... 62 valchev, v., georgiev, v., tsoneva, s.: communities of phragmites australis (cav.) trin.ex steud. in the fluvial terrace of lower danube: their diversity and structure of the above-ground phytomass. biologica nyssana, 8 (1), september 2017 53 5 3 original article received: 27 june 2017 revised: 12 august 2017 accepted: 30 august 2017 communities of phragmites australis (cav.) trin.ex steud. in the fluvial terrace of lower danube: their diversity and structure of the above-ground phytomass vladimir valchev*, valeri georgiev, sonya tsoneva institute of biodiversity and ecosystem research at the bulgarian academy of sciences, “akad. g. bonchev str.” bl. 21 bas, sofia, bulgaria * e-mail: vlado@bio.bas.bg abstract: valchev, v., georgiev, v., tsoneva, s.: communities of phragmites australis (cav.) trin.ex steud. in the fluvial terrace of lower danube: their diversity and structure of the above-ground phytomass. biologica nyssana, 8 (1), september 2017: 53-60. determination of stocks of aboveground phytomass of reed communities in bulgaria is not well studied yet. this survey is the most detailed study on the stocks of the aboveground phytomass of the reed communities (phragmites australis (cav.) trin. ex steud.) in bulgaria. the studied reed beds are formed in the floodplain part of the danube river. they are located in the fluvial terrace of the river between archar village (west) and srebarna village (east). as a result of the survey, it was found that the average stocks of the aboveground phytomass of the reed communities reached to 19.78 t/ha. moreover, 54.04% of the aboveground phytomass is concentrated in the stems of the reed plants. almost 42% of it is produced by the leaves, and the inflorescences produce less than 5% of the total amount of the phytomass. this study once again demonstrates the important role of reed beds for the normal functioning and development of riparian ecosystems. key words: reed communities, primary production, above-ground phytomass, danube apstrakt: valchev, v., georgiev, v., tsoneva, s.: zajednice phragmites australis (cav.) trin. ex steud. na fluvijalnim terasama donjeg dunava: diverzitet i struktura nadzemne biomase. biologica nyssana, 8 (1), septembar 2017: 53-60. količina nadzemne biomase zajednica trske u bugarskoj još uvek nije dovoljno proučavana. ovo istraživanje je najdetaljnija studija nadzemne biljne mase zajednice trske (phragmites australis (cav.) trin. ex steud.) u bugarskoj. ispitivane rečne zajednice su formirane u plavnom području dunava. locirane su na fluvijalnim terasama reke između sela arčar (zapad) i srebarna (istok). kao rezultat studije, utvrđeno je da su prosečne količine nadzemne biomase zajednice trske do 19,78 t/ha. štaviše, 54,04% nadzemne biomase koncentrisano je u drškama trske. skoro 42% nje je produkovano od strane lišća, a cvetovi proizvode manje od 5% ukupne količine biljne biomase. ovo istraživanje još jednom demonstrira značajnost uloge tršćaka za normalno funkcionisanje i razvoj riparijalnih ekosistema. ključne reči: zajednice trske, primarna produkcija, nadzemna biljna biomasa, dunav 8 (1) • september 2017: 53-60 doi: 10.5281/zenodo.963885 biologica nyssana 8 (1)  september 2017: 53-60 valchev, v. et al.  communities of phragmites australis… 54 introduction determination of the above-ground phytomass stocks of reed communities is a problem still pending proper investigation in bulgaria, so there are only few publications with data on this subject in the bulgarian scientific literature (k o c h e v & y o r d a n o v , 1981; k o c h e v & y u r u k o v a , 1984). in 19861987, g e r g i n a b a e v a (1994) determined the primary biological production of two species, phragmites australis (cav.) trin.ex steud. and typha angustifolia l., which formed comparatively pure associations in the so-called “reed belt” of srebarna reserve applying a method for determination of the annual growth of plant biomass (root biomass production). k o c h e v & y o r d a n o v (1981) devoted modest attention to the issues of biomass, biological productivity and total stocks of the above-ground phytomass of reed communities in their large-scale survey of the flora and vegetation of water bodies in bulgaria. however, it was not clear from their results how the dry mass was determined as a component of the primary biological production of these coenoses. some data on the bioproduction and energy stocks of common reed (phragmites australis) and lesser bulrush (typha angustifolia) in bulgaria were also published by k o c h e v & y u r u k o v a (1984), a few years latter. and these were about bulgarian publications on the surveys of reed and its communities as phytomass producers. material and methods reed coenoses (phragmitetum communis) that participate in the structure of the vegetation cover on the territory of the danube fluvial terrace are investigated in this study. dominant species of that stands, phragmites australis, was chosen for survey because its phytocoenoses are some of the widest distributed in the river valley and are the greatest producer of above-ground phytomass among the components of grassy vegetation. when growing in river shallows, they develop most powerfully at water depth between 0.2 and 0.6 m and form different in width belts. the transect method was applied, with organized sample plots, in line with the requirements of european standard en 14184 (cen, 2003a) for running waters and international standard cen tc 230/tg 3/n72 (cen, 2003b) for standing water bodies. macrophytes (higher plants) were described for each sampling plot (a n d r e e v et al., 1992). plant abundance was assessed according to the 5grade scale of braun-blanquet (g u i n o c h e t , 1973). some objects got more than one description, in order to acquire a better idea of the phytocoenotic specificities of the reed communities formed along the danube (tab. 1). determination of the above-ground phytomass stocks of the reed communities (in this case it coincides with their biological productivity) follows the quantifying method of m i l n e r & h u g h e s (1968). field samples were taken from plots sized 50×50 cm (0.25 m²), after the reed has finally formed its inflorescences. reed stems were cut close up to the soil. they were divided then into fractions (stems, leaves, inflorescences) and their fresh weight was measured, as quickly as possible. an average sample was taken from each fraction to determine the absolute dry weight (adw) in laboratory conditions. the obtained data were recalculated in kg/m², and then into t/ha of absolute dry matter. the absolute dry weight (adw) of the vegetation samples was determined following a standard method: by drying at 60 ºc in the course of 24 hours. the number of cut stems at the sampling plot and the number of inflorescences were recorded for each sample. data obtained on the quantity and distribution of the above-ground phytomass per stem and per community are published in table 2. the same table gives summarized data on the distribution of the above-ground phytomass by fractions and their quantitative participation in the formation of aboveground phytomass. similarly were determined the stocks of above-ground phytomass of the accompanying species participating in the formation of reed coenoses (tab. 3). the latin names of the plants are given according to the field guide to the vascular plants in bulgaria (a n d r e e v et al., 1992). results and discussion strong reproductive capacity is a specific characteristic of the phragmites australis (cav.) trin. ex steud. (common reed). common reed propagates by seeds and vegetatively, owing to a very good root system (of the rhizome type), which forms typical root suckers, several meters long. they give rise to the young stems and thus new, so far reed-free areas are colonized. the very mode of vegetative propagation of the species, and probably some of its allelopathic properties, precondition the creation of coenoses, which usually are monodominant and with a comparatively poor species composition of usually 6 to 10 species (tab. 1). common reed often forms mixed communities with other macrophytic species with kindred biology, namely typha latifoliа l., typha angustifolia l., bolboschoenus maritimus palla, 5 5 table 1. species composition of the common reed communities in the fluvial terrace of lower danube sites orsoya zagrazhden kaikusha kalimok malak preslavets garvan marsh srebarna frequency of occurrence species/number of transect 1 2 3 1 2 1 2 1 2 3 4 5 6 7 phragmites communis 5 5 5 4 5 5 4 4 1 5 5 5 5 4 5 3 3 17 ceratophyllum demersum + 1 + + 1 1 1 1 1 1 10 calystegia sepium + + 1 + + 1 1 1 1 9 lycopus europaeus + + + + 1 1 1 + 8 hydrocharis morsus-ranae + + + 2 1 + 6 typha angustifolia 1 + 1 1 1 + 6 urtica dioica + + 1 1 + 5 epilobium hirsutum + + + + + 5 lemna trisulca + + + 3 + 5 thelipteri spalustris 1 1 1 1 1 5 bolboschoenus maritimus + + + + 4 lemna minor + 1 + 3 4 typha latifolia + + 1 1 4 berula erecta + 1 1 1 4 mentha aquatica + + + 3 alisma plantago-aquatica + + + 3 lysimachia vulgaris + + + 3 potamogeton crispus + 1 1 3 salvinia natans 2 + 1 3 stachys palustris + + 2 cirsium arvense + + 2 sambucus ebulus + + 2 myosotis palustris + + 2 persicaria hydropiper + + 2 schoenoplectus lacustris + + 2 veronica beccabunga gr. + + 2 bidens tripartita + + 2 sparganium erectum + + 2 lythrum salicaria + 1 2 rumex hydrolapathum 1 1 2 solanum dulcamara + + 2 symphytum officinale + 1 b i o l o g i c a n y s s a n a 8 (1 )  s e p te m b e r 2 0 1 7 : 5 3 -6 0 v a lc h e v , v . e t a l.  c o m m u n itie s o f p h ra g m ite s a u s tra lis … 56 5 6 potentilla reptans + 1 bidens cernua + 1 myriophyllum spicatum + 1 centaurea stenolepis + 1 convolvulus arvensis + 1 euphorbia lucida + 1 lycopus exaltatus + 1 lapa major + 1 sanicula europaea 1 1 butomus umbellatus + 1 elodea nuttallii 1 1 spirodella polyrhiza 1 1 mentha pulegium + 1 potamogeton lucens + 1 utricularia vulgaris + 1 bidens frondosa + 1 persicaria lapathifolia 1 1 inula germanica + 1 carex sp. + 1 species richness 4 7 4 17 7 6 16 12 13 7 7 7 6 10 11 10 8 table 2. summarised data of the grass-stand samplings of phragmites australis coenoses fractions orsoya o ry a h o v o gulyantsi belene k a li m o k m .a la k p re sl a v et s g a rv a n srebarna a v er a g e v a lu es 1 2 3 milko -vitsa dolni vit kaiku sha dekov kochka island bryag 2010 stems fresh weight (g/0.25 m²) 409.7 1075.7 905.7 279.1 336.7 1400.7 168.3 424.7 669.1 1005.7 824.1 1797.1 433.1 748.4 absolute dry weight (g/0.25 m²) 307.0 461.1 450.9 150.5 193.7 749.5 84.3 228.3 312.7 509.3 344.3 812.1 225.4 371.5 absolute dry matter (%) 74.9 42.9 49.8 53.9 57.5 53.5 50.1 46.3 53.3 49.4 58.2 54.8 48.0 53.3 relative participation of the fraction (%) 82.9 57.4 51.2 46.6 46.8 64.7 74.5 53.9 54.9 69.1 67.1 58.6 52.0 58.8 leaves fresh weight (g/0.25 m²) 207.6 575.6 683.7 269.9 290.7 774.7 62.8 350.7 528.9 414.7 225.9 934.9 353.9 436.4 absolute dry weight (g/0.25 m²) 63.3 332.4 412.9 138.6 172.8 361.1 27.8 173.3 239.8 227.3 168.7 565.1 207.9 237.8 b i o l o g i c a n y s s a n a 8 (1 )  s e p te m b e r 2 0 1 7 : 5 3 -6 0 v a lc h e v , v . e t a l.  c o m m u n itie s o f p h ra g m ite s a u s tra lis … 5 7 absolute dry matter (%) 30.5 57.8 60.4 51.4 59.4 46.6 44.3 49.4 45.4 54.8 74.7 60.5 58.8 53.4 relative participation of the fraction (%) 17.1 41.4 46.9 42.9 41.8 31.2 24.6 40.9 42.1 30.9 32.9 40.8 48.0 37.6 inflorescences fresh weight (g/0.25 m²) 16.4 26.3 58.4 74.0 82.4 1.4 32.8 32.4 11.7 37.3 absolute dry weight (g/0.25 m²) 10.0 16.0 33.6 47.3 48.0 1.1 21.8 17.2 6.8 22.4 absolute dry matter (%) 61.2 60.8 57.5 63.9 58.3 76.1 66.4 53.1 57.8 62.1 relative participation of the fraction (%) 1.2 1.9 10.5 11.4 4.1 0.9 5.2 3.0 0.6 3.6 number of stalks per m² 36.0 40.0 96.0 32.0 56.0 56.0 20.0 44.0 28.0 44.0 36.0 44.0 16.0 42.2 number of inflorescences per m² 12.0 32.0 24.0 36.0 44.0 8.0 32.0 24.0 16.0 17.5 total weight for the plot (kg/m²) 1.5 2.2 3.5 1.3 1.7 4.6 0.5 1.7 2.3 2.9 2.1 5.5 1.7 2.4 yields of above-ground phytomass (t/ha) 14.8 22.2 35.2 12.9 16.6 46.3 4.5 16.9 22.8 29.5 20.5 55.4 17.3 24.2 2011 stems fresh weight (g/0.25 m²) 619.1 353.7 454.1 239.7 269.7 212.0 661.0 591.7 620.7 446.9 absolute dry weight (g/0.25 m²) 286.7 204.5 231.9 99.7 99.9 102.7 267.6 217.7 188.8 188.8 absolute dry matter (%) 46.3 57.8 51.1 41.6 37.0 48.4 40.5 36.8 30.4 54.0 relative participation of the fraction (%) 64.4 55.2 46.4 36.6 43.0 49.2 59.2 35.5 53.0 49.3 leaves fresh weight (g/0.25 m²) 395.5 320.1 471.4 282.1 224.7 211.7 381.7 545.7 472.7 367.3 absolute dry weight (g/0.25 m²) 140.8 124.2 222.6 144.9 108.5 87.9 184.2 392.4 167.7 174.8 absolute dry matter (%) 35.6 38.8 47.2 51.4 48.3 41.5 48.3 71.9 35.5 36.5 relative participation of the fraction (%) 31.6 33.5 44.6 53.2 46.7 42.1 40.8 64.1 47.1 45.6 inflorescences fresh weight (g/0.25 m²) 29.0 68.0 77.1 36.7 33.0 27.0 4.3 30.6 absolute dry weight (g/0.25 m²) 17.7 41.7 44.9 27.6 24.2 18.2 2.6 19.6 absolute dry matter (%) 61.0 61.3 58.3 75.1 73.3 67.4 60.2 50.7 relative participation of the fraction (%) 4.0 11.3 9.0 10.1 10.4 8.7 0.4 5.1 number of stalks per m² 48.0 32.0 32.0 32.0 20.0 24.0 28.0 28.0 20.0 29.3 number of inflorescences per m² 24.0 32.0 24.0 16.0 12.0 24.0 8.0 15.6 total weight for the plot (kg/m²) 1.8 1.5 2.0 1.1 0.9 0.8 1.8 2.5 1.4 1.5 yields of above-ground phytomass (t/ha) 17.8 14.8 20.0 10.9 9.3 8.4 18.1 24.5 14.3 15.3 b i o l o g i c a n y s s a n a 8 (1 )  s e p te m b e r 2 0 1 7 : 5 3 -6 0 v a lc h e v , v . e t a l.  c o m m u n itie s o f p h ra g m ite s a u s tra lis … biologica nyssana 8 (1)  september 2017: 53-60 valchev, v. et al.  communities of phragmites australis… 58 5 8 schoenoplectus lacustris palla and etc. mention deserves the fact that, as a rule, species from the reed floor participate to a much lesser extent in the formation of its grass stand. owing to the fact that reed coenoses have been studied in a relatively large region (the valley of the danube between archar and srebarna), the total species diversity of these plant communities registers 53 higher plant species, which is quite impressive floristic richness (table 1). these were reed phytocoenoses formed in marshy places in the river valleys and in other open water bodies (b a r d a t et al., 2001). they are a major component to aquatic vegetation and of vegetation around the waterbodies, attached to the floor of the fresh-waterbasins. in many places, belts and patches mainly of common reed (phragmites australis) have been formed, with the participation of rush (typha sp.), bulrush (schoenoplectus lacustris), high sedges (carex sp.), and others in the process of shallowing out, drying and filling up of the various water bodies with plant remains. this slow natural process could be stepped up by anthropogenic activity. these facts contribute to the environmental and nature-protection importance of rush coenoses. when these coenoses are formed in the river shallows or in the near-by shallow water bodies, the composition of reed communities is joined also by submerged or water floating species, such as ceratophyllum demersum, lemna minor, lemna trisulca, myriophyllum spicatum, frequently hydrocharis morsus-ranae, as well as by some representatives of the sedges (genus carex, schoenoplectus lacustris) and grasses. quite telling is the fact that ceratophyllum demersum species figures in ten out of 17 descriptions altogether. the other species identified in the reed coenoses have much lower frequency of occurrence: usually between one and four to five times (tab. 2). there is a difference in species composition in the studied descriptions of reed communities, determined by the specific conditions of the biotope. a common trait is the very strong quantitative presence of the dominant species, which forms almost pure onefloor grass stands, where the projection cover of dominant species often reaches 85-90% and over. the reed floor is 3 m to 5 m high and even higher. its characteristics (height and projection cover) determine very strongly the specific ecological conditions in these coenoses: temperature, humidity, light and air movement. the other species are mostly present on the periphery of these grass stands or in their thinned-out areas. special mention deserves a very important factor for the floristic enrichment of these phytocoenoses, namely, the presence of residual water on the surface of the terrain occupied by them, where a second (and even third) floor could be formed of the species developing in it or on its surface (lemna minor, myriophyllum spicatum, ceratophyllum demersum and others). about the formed above-ground phytomass and its distribution, the obtained data have shown that the absolutely dry matter of the different fractions of reed above-ground phytomass exceed by half its fresh weight: 53-54% for the stems, 35-53% for the leaves and 50-60% for the inflorescences (tab. 3). therefore, the plants stems contain the greatest amounts of water, which is due to the fact that most conductive tissues, which carry out the water and assimilates needed for normal existence of the plant organism, are situated there. the main part of the reed above-ground phytomass is formed by its stems: somewhere between 82.90% and 35.53%. second comes the leaves fraction: from 64.05% to 24.60%. relative participation of inflorescences does not exceed table 3. average values of fractions from different parts of reeds for the two years of investigation fractions average values stems fresh weight (g/0.25 m²) 597.64 absolute dry weight (g/0.25 m²) 280.14 absolute dry matter (%) 43.55 relative participation of the fraction (%) 54.04 leaves fresh weight (g/0.25 m²) 401.86 absolute dry weight (g/0.25 m²) 206.29 absolute dry matter (%) 44.94 relative participation of the fraction (%) 41.6 inflorescences fresh weight (g/0.25 m²) 33.93 absolute dry weight (g/0.25 m²) 21.04 absolute dry matter (%) 56.39 relative participation of the fraction (%) 4.36 number of stalks per m² 35.74 number of inflorescences per m² 16.54 total weight for the plot (kg/m²) 1.98 yields of above-ground phytomass (t/ha) 19.78 biologica nyssana 8 (1)  september 2017: 53-60 valchev, v. et al.  communities of phragmites australis… 59 11.40%, i.e. the reproductive organs of this species form less than one-twentieth of the plant aboveground phytomass (tab. 2). data obtained from the samples cut in the field have permitted calculation of the stocks of aboveground phytomass of the surveyed reed communities. apparently, the lowest stocks came from the sampling plot at belene (kaikusha locality) – 4.53 t/ha, and the highest came from the sampling plot on srebarna island – 55.36 t/ha (tab. 2). comparison of the stocks of above-ground phytomass of reed conenoses shows clearly higher data for 2010– 24.22 t/ha, against 15.33 t/ha in 2011. this is quite explicable, considering the fact that the vegetation season in 2011 was much drier than in the previous year. averaged results from the different sites during the two years of survey (tab. 3) have shown the following: ■ somewhat above half of the above-ground phytomass was concentrated in the reed stems (54.04 %). almost 42% of that mass was formed by the leaves, while the generative organs (inflorescences) contained less than 5% of the general amount of the phytomass. further more, the surveyed reed coenoses formed a main floor with a very dense grass stand: on the average, almost 36 reed stalks grew per square meter, which prompted the conclusion that reed communities make relatively very good use of the ecotope conditions. ■ the table 3 also shows that nearly half (47%) of the reed stalks in the model plot did not develop inflorescences. this could be used as a proof of the importance of vegetative propagation of this plant species. ■ the average stocks of above-ground phytomass of the plant communities amount to 19.78 t/ha, or about 4.6 times less than the data (91.7 t/ha) published by k o c h e v & y o r d a n o v (1981) from a survey of the primary biological production of reed associations in bulgaria. the figures reported by k o c h e v & y o r d a n o v (1981) were probably so high due to the fact that the authors had measured both green and dry mass in the reed floor and the latter comprised the plant substance formed during the previous vegetation season. according to k o c h e v & y u r u k o v a (1984), reed communities in the aldomir marsh form bioproduction to the tune of 36.7 t/ha, which is only twice higher than the present results. the samples taken to determine the stocks of above-ground phytomass formed by the other plant species participating in the formation of reed coenoses have shown that their proportion of the above-ground phytomass varies between 0.49 t/ha and 4.41 t/ha,or on the average is1.46 t/ha (tab. 4). thus the total stocks of above-ground phytomass of the surveyed reed coenoses amount on the average to 21.24 t/ha. ecosystematically, besides being a major phytomass producer, reed communities also play an important part in the formation of local micro climate. suitable micro climatic conditions predetermine a greater diversity of the faunistic component of these specific bio-geocoenoses: periphyton (mollusks, insects), ornithofauna, amphibians and reptiles, as well as fish stock, where there is water. table 4. data for absolute dry weight of the accompanying species in reed communities measured in 2011 sites absolute dry weight (g/0.25 m²) absolute dry weight(t/ha) orsoya 1 15.73 0.63 orsoya 2 12.24 0.49 lower vit 16.93 0.68 kaikusha 67.34 2.69 kalimok 110.24 4.41 garvan marsh 16.53 0.66 srebarna 16.16 0.65 average 36.45 1.46 conclusion the total stocks of above-ground phytomass of the surveyed reed communities averaged 21.24 t/ha. only about 8% of that amount was formed by the accompanying plant species. the average stocks of above-ground phytomass of the plant communities accounted for 19.78 t/ha. little more than half of the above-ground phytomass was concentrated in the stems of the reeds (54.04%). about 40% of it was formed by the leaves, while less than 5% of the general phytomass amount was due to the inflorescences. nearly half of the reed stalks in a model plot did not develop inflorescences, which comes to prove the importance of vegetative propagation of this plant species. considering the fact that they are among the widest distributed components along the river valley and the greatest producer of phytomass among the other components of grassy vegetation in the river’s biologica nyssana 8 (1)  september 2017: 53-60 valchev, v. et al.  communities of phragmites australis… 60 fluvial terrace, and also that reed massifs play a major part in the normal functioning and development of riparian ecosystems, it becomes clear that they are very important and determinant for the processes of mineralization of the produced phytomass and its depositing on the soil or floor of the water bodies. references andreev, n., anchev, m., kozhuharov, s., markova, m., peev, d., petrova, a., 1992: field guide to the vascular plants in bulgaria. naouka & izkoustvo, sofia (in bulgarian). 788 p. baeva, g., 1994. investigation on the overground phytomass of phragmites australis (cav.) trin. ex steud. and typha angustifolia l. in the srebarna biosphere reserve. godishnik na sofiyskiya universitet “st kl. ohridski”, biologicheski fakultet, kniga 2 – botanika, 84: 103-109. bardat, j., bioret, f., botineau, m., boullet, v., delpech, r., géhu, j. m., haury, j., lacoste, a., rameau, j. c., royer, j. m., roux, g., touffet, j. 2004: prodrome des végétations de france. muséum national d'histoire naturelle. 171 p. guinochet, m. 1973: phytosociologie. masson. paris. 227 p. cen, 2003a: water quality-guidance standard for the surveying of aquatic macrophytes in running waters, en 14184. comité européen de normalisation. bruxelles. cen, 2003b: water quality-guidance standard for the surveying of macrophytes in lakes complementary element, cen tc 230/tg 3/n72.comité européen de normalisation. bruxelles. kochev, h., yordanov, d. 1981: vegetation of bulgarian water bodies. ecology, protection and economic importance. bulgarian academy of sciences. sofia. (in bulgarian). kochev, h., yurukova, l. 1984: primary biological production and energy value of the vegetation in the aldomirovo marsh, sofia district. contemporary theoretical and applied aspects of plant ecology, 1: 166-174. (in bulgarian). milner, c., hughes, r. e. 1968: methods for the measurement of the primary production of grasslands. oxford and edinburg. abingdon, berkshire.70 p. anticancer compounds from medicinal plants biologica nyssana 2 (2)  december 2011: 00-00 ljupković r.b. et al..  removal cu(ii) ions from water... 17 original article a new species of bythiospeum bourguignat, 1882 (hydrobiidae, gastropoda) from montenegro vladimir pešić* 1 , peter glöer 2 1 department of biology, faculty of sciences, university of montenegro, cetinjski put b.b., 81000 podgorica, montenegro 2 biodiversity research laboratory, schulstraße 3, d-25491 hetlingen, germany * e-mail: vladopesic@gmail.com abstract: pešić, v., glöer, p.: a new species of bythiospeum bourguignat, 1882 (hydrobiidae, gastropoda) from montenegro. biologica nyssana, 3 (1), september 2012: 17-20. a new minute hydrobid species, bythiospeum bogici sp. nov. from one small spring near podgorica, montenegro is described. the list of subterranean species of the family hydrobiidae from montenegro is given. key words: bythiospeum, new species, subterranean, montenegro. introduction the rissooidean family hydrobiidae troschel, 1857 is one of the largest gastropod families with more than 400 recent genera assigned. the gastropods of the family hydrobiidae of montenegro have been studied by several authors during the 20th and 21th century (e.g., s c h ü t t 1960, b o l e 1961, r a d o m a n 1973, 1983, r e i s c h ü t z & r e i s c h ü t z 2008, g l ö e r & p e š i ć 2010, f a l n i o w s k i et al. 2012a, b). however, our knowledge still remains incomplete. to date, 42 hydrobioid species and subspecies belonging to 15 genera have been recorded in montenegro. it is notable that 62% of these species and subspecies are endemic for montenegro. the iucn red list of threatened species includes 18 hydrobiid species from montenegro. five of them are classified as critically endangered, six as endangered, two as data deficient and the rest as least concern (c u t t e l o d et al. 2011). some of hydrobiid gastropods are stygobiote species, i.e. living in underground waters. however, most of these species rarely been sampled in subterranean habitats and only being recorded in springs which flows directly out of the ground (see table 1). many of these springs are intermittent, and this may explain the limited recording of hydrobioid species, especially during the summer months when the freshwater springs dry up. because acess to, and sampling in hypogean habitats are difficult, subterranean hydrobiid gastropods have been described mainly from very few living animals or from empty shells only, and often collected outside the subterranean networks. consequently, ecological status (strict vs. occasional subterranean species) for most of these species are still unclear and subject to various opinions. recently, first author collected the spring in central montenegro, empty shells of one hydrobiid gastropod species which did not correspond to any described species. description of this species is given in this paper. 3 (1) • september 2012: 17-20 biologica nyssana 3 (1)  september 2012: 17-20 pešić, v., glöer, p.  a new species of bythiospeum… 18 figure 1. photo of the locus typicus (spring taban near spuž, podgorica). photo. v. pešić. materials and methods the empty shells of one minute hydrobiid gastropod from a spring taban (42º31.653 n, 19º13.145 e, 105 m asl. – fig. 1) near spuž, podgorica, were collected by hand and fixed with 80% ethanol. the spring water and sediment had a smell of hydrogen sulphide. since population abundance of this species seems to be low in the spring where it was found, only 10 specimens were collected. shell morphometric variables (namely shell height and width, aperture height and width) were measured using a stereo microscope (zeiss). shells were photographed with a leica digital camera system. the type material is stored in the zoological museum of hamburg (zmh). table 1. list of subterranean species of the family hydrobiidae from montenegro taxa sampling localities references red list category & criteria: bracenica spiridoni radoman, 1973 spring „spirov izvor“ (skadar lake area) radoman (1973) endangered b1ab(iii)+2ab(iii) ver 3.1 saxurinator orthodoxus schütt, 1960 spring of river zeta near straganik and tunjevo schütt (1960) critically endangered b1ab(iii)+2ab(iii) ver 3.1 paladilhiopsis tarae bole & velkovrh, 1987 groundwater system underlying the tara valley bole & velkovrh (1987) data deficient ver 3.1 plagigeyeria montenigrina bole, 1961 spring in obodska pećina cave near rijeka crnojevića bole (1961) critically endangered b1ab(iii)+2ab(iii) ver 3.1 plagigeyeria zetaprotogona zetaprotogona schütt, 1960 spring of river zeta near tunjevo village schütt (1960) endangered b2ab(ii,iii) ver 3.1 p.zetaprotogona pageti schütt, 1960 spring of river zeta near tunjevo village schütt (1960) endangered b2ab(ii,iii) ver 3.1 p. zetaprotogona zetadidyma schütt, 1960 spring of river zeta near tunjevo village schütt (1960) endangered b2ab(ii,iii) ver 3.1 p. zetaprotogona vitoja reischütz & reischütz, 2008 spring vitoja (skadar lake area) reischütz & reischütz, (2008) endangered b2ab(ii,iii) ver 3.1 vinodolia matjasici (bole, 1961) small spring in lipovik village (skadar lake area) bole (1961) critically endangered b1ab(iii)+2ab(iii) ver 3.1 vinodolia gluhodolica (radoman, 1973) spring at velje oko below the gluhi do village ( skadar lake area) radoman (1973) endangered b1ab(iii)+2ab(iii) ver 3.1 biologica nyssana 3 (1)  september 2012: 17-20 pešić, v., glöer, p.  a new species of bythiospeum… 19 results and disscusion systematics hydrobiidae genus bythiospeum bourguignat, 1882 bythiospeum bogici sp. nov. (fig. 2) material examined: 10 ex. from the type locality, 05.06.2012, leg. pešić & gligorović. holotype: shell height 2.3 mm, shell width 1.2 mm, zmh 79649. paratypes: 4 ex. zmh 79650; 5 ex. in coll. glöer type locality: montenegro, pogorica, spring taban, 42º31.653 n, 19º13.145 e, 105 m asl. description: the conically cylindrical shell consists of 5.5 convex whorls with a deep suture and a blunt apex. the shell is whitish and translucent. the umbilicus is slit-like. the surface is smooth and silky. the periostome in adults is bent to the outside from the lateral view. the basis of the aperture is pulled forward. shell height 1.85 – 2.3 mm, width 0.9 – 1.0 mm. etymology: named after phd student bogić gligorović who lead the first author to the spring and helped in the collecting. diferential diagnosis: due to the shape of the periostome which is bent to the outside in the adults, we ascertained the new species to the genus bythiospeum. the new species resembles bythiospeum montenegrina (schütt, 1959) (= paladilhiopsis montenegrina schütt, 1959) a species described from čeplica spring near bileća, bosnia and hercegovina (schütt, 1959). from the latter species bythiospeum bogici sp. nov. can easily be distinguished by the lower number of whorls and the aperture which is not oblique. remark: this taxon is a typical stygobiont. its very likely that bythiospeum represents a complex of closely related genera, probably with a limited distributions (e.g. balkanospeum georgiev, 2012 from bulgaria see georgiev 2012). further researches are certainly needed to collect living specimens for providing a proper anatomical description. distribution: known only from the type locality. figure 2. bythiospeum bogici sp. nov.: shell (holotype). acknowledgements. this study was supported by the research project cbfecomtg from the ministry of science, montenegro. references bole, j. 1961: nove hidrobide (gastropoda) iz podzemeljskih voda zahodnega balkana. (neue hydrobiiden (gastropoda) aus den unterirdischen gewässern westbalkans). biološki vestnik, 9: 59-69. bole, j. & velkovrh, f. 1987: nove vrste podzemeljskih polzev. jugoslavije. (new species of the subterranean snails of yugoslavia). sazu, ljubljana, 28 (3): 69-83. cuttelod a., seddon m. & neubert e. 2011: european red list of non-marine molluscs. iucn global species programme, iucn regional office for europe, iucn species survival commission, 98 pp. falniowski, a., szarowska, m., glöer, p. & pešić, v. 2012a: molecules vs morphology in the taxonomy of the radomaniola/grossuana group of balkan rissoiidea (mollusca: caenogastropoda). journal of conchology, 41 (1): 19-36 falniowski, a., szarowska, m., glöer, p., pešić, v., georgiev, d., horsák, m. & sirbu, i. 2012b: radiation in bythinella moquin-tandon, 1856 (mollusca: gastropoda: rissooidea) in the balkans. folia malacologica, 20 (1): 1-10 georgiev, d. 2012: new taxa of hydrobiidae (gastropoda: risooidea) from bulgarian cave h=2,3mm biologica nyssana 3 (1)  september 2012: 17-20 pešić, v., glöer, p.  a new species of bythiospeum… 20 and spring waters. acta zoologica bulgarica, 64 (2): 113-121 glöer, p. & pešić, v. 2010: the freshwater snails of the genus bythinella moquin-tandon (gastropoda: rissooidea: hydrobiidae) from montenegro. archives of biological sciences, 62: 441-447. radoman, p. 1983: hydrobioidea a superfamily of prosobranchia (gastropoda). i. systematics. monographs 547, serbian academy of sciences and arts, (department of science), 256 p. radoman, p. 1973: anagasta un nouveau genre prosobranchia et sa spéciation dans la bassin du lac skadar. zoologischer anzeiger, 190: 421429. reischütz, a. & reischütz, p.l. 2008: neue hydrobiiden (gastropoda, prosobranchia, hydrobiidae) aus dem becken des skutari-see (montenegro/albanien). basteria, 72: 143-145. schütt, h. 1960: neue höhlenschnecken aus montenegro. archiv für molluskenkunde, 89, 46: 11. schütt, h. 1959: zur höhlenschneckenfauna montenegros. archiv für molluskenkunde, 88 (4/6): 185-190. malićanin et al. 2024, biologica nyssana 15(2) 15 (2) december 2024: 83-90 doi: 10.5281/zenodo.14258290 evaluation of yeast derivatives on sauvignon blanc wine quality original article marko malićanin university of niš, faculty of agriculture, kosančićeva 4, 37000 kruševac, serbia bojana danilović university of niš, faculty of technology, bulevar oslobođenja 124, 16000 leskovac, serbia stojan mančić university of niš, faculty of technology, bulevar oslobođenja 124, 16000 leskovac, serbia sandra stamenković stojanović university of niš, faculty of technology, bulevar oslobođenja 124, 16000 leskovac, serbia vlada veljković university of niš, faculty of technology, bulevar oslobođenja 124, 16000 leskovac, serbia; serbian academy of science and arts, belgrade, serbia ivana karabegović university of niš, faculty of technology, bulevar oslobođenja 124, 16000 leskovac, serbia ivana.karabegovic@tf.ni.ac.rs (corresponding author) received: september 13, 2024 revised: november 19, 2024 accepted: november 20, 2024 abstract: the combined effect of the native yeast strain hanseniaspora uvarum s-2 with commercial yeast derivatives (yds) on the quality of sauvignon blanc wine was evaluated. three commercial yds were applied at two concentrations (0.2 g/l or 0.4 g/l) over a 15-day period with periodic stirring. dissolved oxygen content, browning index, total phenolic content, and sensory profile were examined. results indicated that yds effectively scavenged oxygen, with lower dissolved oxygen in treated samples than the control. the lowest oxygen content was observed in the wine treated with 0.2 g/l of commercial yd noblesse (lallemand, canada). total phenolic content did not significantly differ between yds, but treated wines exhibited more stable color. browning index was improved in samples with higher derivative doses. overall, all yds resulted in wines with comparable sensory profiles, although statistically different scores for taste attributes. the findings confirm that yd treatment mitigates oxidative browning during white wine storage and positively influences sensory aspects. key words: yeast derivatives, sauvignon blanc, wine apstrakt: uticaj derivata kvasca na kvalitet vina sorte sauvignon blanc istraživanje je imalo za cilj da ispita kombinovani uticaj nativnog soja kvasca (hanseniaspora uvarum s-2) i tri različita komercijalna derivata kvasca na kvalitet vina sauvignon blanc. komercijalni derivati su primenjeni u dve koncentracije (0.2 g/l ili 0.4 g/l) tokom 15-dnevnog perioda uz povremeno mešanje. uzorcima vina određen je sadržaj rastvorenog kiseonika, stepen potamnjivanja, ukupni sadržaj fenola i senzorne karakteristike. rezultati su ukazali da upotreba derivata kvasaca efikasno redukuje prisustvo kiseonika, što se ogleda u nižem sadržaju rastvorenog kiseonika u tretiranim vinima u poređenju sa kontrolom. najniži sadržaj kiseonika zabeležen je kod vina tretiranog komercijalnim derivatom noblesse (lallemand, kanada) u koncentraciji 0.2 g/l. sadržaj ukupnih fenola nije se značajno razlikovao između vina tretiranih različitim derivatima, ali su tretirana vina imala stabilniju boju. bolji rezultati za stepen potamnjivanja dobijeni su kod primene većih doza derivata. generalno, sva vina tretirana derivatima su rezultirala vinima sličnih senzornih profila, iako su statistički različite ocene zabeležene za ukus dobijenih vina. dobijeni rezultati potvrđuju da tretman derivatima kvasaca smanjuje oksidativno potamnjivanje tokom skladištenja belog vina i pozitivno utiče na senzorne karakteristike. ključne reči: derivati kvasca, sauvignon blanc, vino introduction wine is a complex system that undergoes numerous chemical changes during storage. while bottle storage can lead to quality improvement in some red wines (echave et al., 2021), in white wines, it mainly causes color alteration (browning), loss of fresh and fruity aromas, the appearance of the oxidized character, and unpleasant odors (kanavouras et al., 2020; vlahou et al., 2022). the main chemical reactions responsible for wine changes during bottle storage are non-enzymatic oxidation reactions responsible for an adverse change in color due to an increase in color intensity and a decrease in brightness (ricci et al., 2017). enzymatic reactions usually induce browning during the early stages of wine production (tarko et al., 2020), while nonenzymatic oxidation usually occurs in the postfermentation phase, when the active polyphenol oxidase is not present in wine, leading to the appearance of brown color and ‘‘woody” aroma. it is confirmed that the oxidative browning of white wines is mainly related to the content of flavanols © 2024 malićanin et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 83 and hydroxycinnamates. these compounds mainly participate in redox reactions that occur during wine aging (salacha et al., 2008). mostly, the formation of yellow or brown products due to the polymerization of ortho-quinones is the result of the oxidation process of (+)-catechin, (-)-epicatechin, caffeic, and other hydroxycinnamic acids (mitić et al., 2010). besides polyphenol oxidation, transition metal ions, sulphur dioxide concentration, and ascorbic acid also contribute to the oxidation of polyphenols since sulphur dioxide and ascorbic acid can reduce the concentration of ortho-quinones, and metal ions catalyze the oxidation process (salacha et al., 2008; mitić et al., 2010). conversely to red wines, white wines are susceptible to oxidation due to low total phenolic content (tarko et al., 2020). traditionally, compounds such as sulphur dioxide, ascorbic acid, tannins, or fresh lees are added to wine to prevent oxidation. more recently, inactive yeast derivatives (yds) have been proposed as an efficient tool for preventing wine oxidation due to the ability to release glutathione and other antioxidant compounds (comuzzo et al., 2015; nioi et al., 2022). yds, such as inactivated yeast, yeast autolysate, yeast protein extract, yeast cell wall, and yeast mannoprotein, refer to a fraction of yeast cells obtained in the different production processes and degrees of purification (del barrio galán et al., 2018). yds could improve the mouthfeel, aromatic profiles, and stability of the wine, reduce the perception of acidity and bitterness and prevent or reduce the oxidation of the wine. the listed effects strongly depend on yd type, characteristics, and dosage, while their protective action was mainly due to the compounds like peptides, amino acids, glutathione, and polysaccharides (nioi et al., 2022). the use of commercial yds containing these compounds has significantly increased in recent years. since color is one of the very important sensory characteristics of white wine, there are specifically formulated yd, which aims to suppress the oxidation phenomena and improve the color of wines (pozo-bayon et al., 2009; del barrio galán et al., 2018). yds are usually produced from saccharomyces cerevisiae cultivated on sugar-rich medium. afterwards, the cells are subjected to autolysis by different methods and dried to obtain the powder preparations. the application of different yds depends on their composition and covers a broad range of activities during wine production (del barrio galán et al., 2018). some yds can be used to improve alcoholic or malolactic fermentation (del barrio-galán et al., 2019), and others can act as protective agents, flavor enhancers, or aroma precursors (pons-mercadé et al., 2021). nevertheless, many types of yds present on the market for various applications in winemaking, but the mechanisms of yds actions which result in the wine’s improvements are not completely clear. accordingly, additional scientific research is needed to characterize the yds effects and establish better criteria for their oenological use. furthermore, to our knowledge, no study has investigated the combined effect of yds and native non-saccharomyces yeast strains on wine quality. however, recently, there has been a growing interest in using non-saccharomyces yeasts in many innovative wineries. therefore, the objective of the present study was to evaluate the effect of the previously characterized native yeast strain (hanseniaspora uvarum s-2) (mančić et al., 2022; karabegović et al., 2022; stamenković stojanović et al., 2023), in combination with three different commercial products based on yeast autolysates, on the white wine quality and aging process during the storage in bottles. this work constitutes a direct approach to understanding the action mode of these preparations and establishing better criteria for their use during winemaking. materials and methods wine production and treatment the grapes were harvested at technical maturity, destemmed, crushed, and pressed. after sedimentation, grape juice (21.3 brix, 5.67 g/l total acidities, and ph 3.2) was inoculated with an native yeast strain, previously isolated and identified as hanseniaspora uvarum s-2 (mančić et al., 2022; karabegović et al., 2022; stamenković stojanović et al., 2023). the inoculum was prepared by transferring a full loop of the strain h. uvarum s-2 from the plate into sabouraud dextrose broth containing 80 mg/l chloramphenicol, followed by incubation at 25 °c for 48 hours on a rotary shaker at 120 rpm. after 48 hours, the resulting biomass was centrifugated and added to the must in an amount previously calculated to achieve a final concentration of 106 cfu/ml. the fermentation was performed under a controlled temperature (16–21 °c) until dryness (reducing sugar content below 4 g/l). after fermentation, the obtained wine was divided into nine glass vessels (50 l). three commercial yds based on inactive yeast, available on the market as optimum white, opty less, and nobless (lallemand, canada), were applied in two concentrations (0.2 g/l or 0.4 g/l) for 15 days, with stirring every third day. while all three yeast derivatives are based on inactivated s. cerevisiae and aim to improve wine quality, they each offer unique benefits: noblesse focuses on enhancing sensory complexity and supporting fermentation processes, optimum white emphasizes antioxidant protection and prolonging the freshness and fruitiness of white and rosé wines, while 84 biologica nyssana ● 15 (2) december 2024: malićanin et al. ● evaluation of yeast derivatives on sauvignon blanc wine quality biologica nyssana ● 15 (2) december 2024: malićanin et al. ● evaluation of yeast derivatives on sauvignon blanc wine quality 85 optilees targets the improvement of wine texture and accelerates the maturation process for a fuller taste profile. after treatment, wine clarification was done (using bentonite, gelatin, and sio2), followed by filtration and bottling. untreated wines were used as control. after bottling, one part of the bottles was subjected to accelerated aging by heating filtered wine samples (20 ml) in screw-cap glass vials (30 ml) at 55.0±0.2 °c for 10 days (singleton & kramling, 1976), while the other part was stored in a dark place at 15–20 °c for one year under normal conditions. the browning index, dissolved oxygen content and total phenolic content of wine samples were monitored in four different production stages: before clarification and filtration (w), after clarification and filtration (cfb), after accelerated aging (acc) and after one year of aging in normal conditions (nor). dissolved oxygen content dissolved oxygen content was measured by a polarographic oxygen analyzer (ysi model 51a dissolved oxygen meter, yellow springs instruments co). determination of total phenolic content total phenolic content was determined using the folin-ciocalteu assay (ivanova et al., 2010). gallic acid was a standard, and the results were reported as mg gallic acid equivalents (gae)/l. browning index the browning (absorbance at 420 nm) was measured against 12% ethanol. the percentages of changes in browning (%δ) was calculated as follows: %∆=(an-a0)*100/an where a0 and an were the browning index (absorbance at 420 nm) at the begging of the treatment and after certain production stages (w, cfb, acc, nor), respectively. sensory evaluation the panel method was used for sensory evaluation. a panel of five judges (members of the commission of the ministry of agriculture, forestry and water management of the republic of serbia for the organoleptic assessment of wine and similar products) evaluated various smell (overall odor, spice, honey, tobacco, vegetable, citrus, tropical fruit, dry fruit, fresh fruit, flowers, complexity, duration, intensity, and typicality) and taste (overall taste, typicality, intensity, duration, complexity, fulness, astringency, acidity, harmony) attributes using a ten-point intensity scale, ranging from 0 (not detected) to 10 (very intense). the evaluations were performed in triplicate after one year of aging in normal conditions (nor), and the results were presented as the mean value. statistical analysis all experiments were conducted in triplicate and the results are expressed as the average value with standard deviation. significant differences between samples were assessed using one-way anova followed by tukey’s hsd post hoc test (ibm spss statistics software, new york, usa). differences were considered statistically significant at a p-value of less than 0.05. results and discussion oxygen content through all the phases of winemaking and aging can affect the quality of the final product. some literature data indicate that 20–50 mg o2/l is the amount of oxygen the white wine could absorb before negative oxidative defects appear (tarko et al., 2020). it implies that oxygen control during winemaking and bottling is very important in highquality wine production. oxygen content measured in all wine samples during wine production in different production stages is presented in fig. 1 (appendix 1). fig. 1. oxygen content in wine samples treated by yds (c – control, ow20 – optimum white 0.2 g/l, ow40 – optimum white 0.4 g/l, ol20 – opty less 0.2 g/l, ol40 – opty less 0.4 g/l, n20 – nobless 0.2 g/l, n40 nobless 0.4 g/l) in different production stages (w – before treatment, cfb – after clarification and filtration, acc – after accelerated aging and nor after one year of normal aging conditions) the results indicate that all used commercial yds have good oxygen scavenging properties since treated samples had lower oxygen content than the untreated wine (control sample). the most active 86 biologica nyssana ● 15 (2) december 2024: malićanin et al. ● evaluation of yeast derivatives on sauvignon blanc wine quality oxygen scavengers among the tested products were nobless, followed by opty less, while the dosage of yds had a negligible effect. after the accelerated aging test, it was also found that dissolved o2 concentrations were lower in all treated wines than in the control sample, which was particularly pronounced in the sample treated with noblesse, with a concentration of 0.2 g/l (0.79 mg/l, compared to 1.45 mg/l for the control sample). after one year in the bottle, there were similar results to those obtained after accelerated aging, with dissolved o2 concentrations in all treated wines also lower than in the control sample. although good oxygen consumption capacity was recently found for some yds (commercial pure-lees™ longevity and experimental antiox-1), the same authors claim that noblesse has no oxygen consumption capacity (pons-mercadé et al., 2021). this difference can probably be attributed to the vinification conditions, the chemical profile of the wine, the free so2 content, or the oxidative evolution during storage. however, it is important to note that noblesse, according to the manufacturer’s specification, is characterized as a derivative that, besides other benefits, can prevent oxidation. the total phenolic content (tpc) in the wine samples ranged from 402 to 362 mg gae/l (tab. 1). although the obtained results are significantly higher than the tpc found in sauvignon blanc wine (165 to 242 mg gae /l) from different wine regions of the world (new zealand, chile, south africa) (marais, 1998; olejar et al., 2015; díaz et al., 2021), the results are consistent with the tpc in slovenian sauvignon blanc wine (lužar et al., 2016) and with the previously reported average tpc for ten different serbian white wines (mitić et al., 2010). the diversity of results is probably related to the geographical region, climate, vintage, vinification, and aging conditions or the analysis methods used. regardless of yds treatment, a significant decrease (about 5%) in total phenolic content was observed in all samples after clarification and filtration. this decrease is because clarifying agents such as bentonite, apart from protein, also adsorb or interact with phenolic compounds, thus also decreasing their content (he et al., 2020). however, it can be noted that after the accelerated aging or one year of aging under normal conditions, tpc in all treated wines was significantly higher than in the control sample. such results indicate that yds treatment can mitigate polyphenols’ transformation and/or precipitation during wine aging. a good indicator of the susceptibility of wine to oxidation is the development of a brown color, which increases the color intensity and the browning index. the obtained results (fig. 2, appendix 2) show that the clarification of the wine with a ta bl e 1. t ot al p he no lic c on te nt (m g g ae /l ) i n w in e sa m pl es tr ea te d by y d s in d iff er en t p ro du ct io n st ag es * c – c on tro l, o w 20 o pt im um w hi te 0 .2 g /l , o w 40 o pt im um w hi te 0 .4 g /l , o l2 0 o pt y le ss 0 .2 g /l , o l4 0 o pt y le ss 0 .4 g /l , n 20 n ob le ss 0 .2 g/ l, n 40 n ob le ss 0 .4 g /l , w b ef or e tre at m en t, c fb – a fte r c la rifi ca tio n an d fil tra tio n, a c c – a fte r a cc el er at ed a gi ng a nd n o r – a fte r o ne y ea r o f n or m al ag in g co nd iti on s. d iff er en t l et te rs in di ca te s ta tis tic al ly s ig ni fic an t d iff er en ce s in th e sa m e ro w (p <0 .0 5) pr od uc tio n st ag es w in e tr ea tm en t c o w 20 o w 40 o l 20 o l 40 n 20 n 40 w 40 2. 02 ±4 .5 1 40 2. 02 ±4 .5 1 40 2. 02 ±4 .5 1 40 2. 02 ±4 .5 1 40 2. 02 ±4 .5 1 40 2. 02 ±4 .5 1 40 2. 02 ±4 .5 1 c fb 38 3. 68 ±3 .1 2a 38 1. 35 ±5 .2 2a 38 4. 72 ±1 .9 6a 38 2. 34 ±4 .9 0a 38 3. 48 ±1 .7 7a 38 4. 56 ±3 .8 6a 38 2. 45 ±4 .8 8a a c c 36 5. 56 ±5 .2 9a 38 4. 49 ±3 .1 8b 38 3. 33 ±6 .7 1b 38 6. 47 ±0 .9 2b 38 1. 65 ±4 .7 9b 38 2. 15 ±4 .2 9b 38 4. 63 ±4 .3 3b n o r 36 2. 84 ±6 .0 2a 38 3. 36 ±1 .0 5b 38 4. 18 ±2 .8 3b 38 3. 45 ±5 .3 3b c 38 5. 33 ±4 .8 2b c 38 6. 67 ±1 .7 9c 38 7. 33 ±1 .4 9c biologica nyssana ● 15 (2) december 2024: malićanin et al. ● evaluation of yeast derivatives on sauvignon blanc wine quality 87 fig. 2. browning index in wine samples treated by yds (c – control, ow20 – optimum white 0.2 g/l, ow40 – optimum white 0.4 g/l, ol20 – opty less 0.2 g/l, ol40 – opty less 0.4 g/l, n20 nobless 0.2 g/l, n40 – nobless 0.4 g/l) in different production stages (wbefore treatment, cfb – after clarification and filtration, acc – after accelerated aging and nor – after one year of normal aging conditions) fig. 3. results of the sensory analysis for smel (a) and taste (b) attributes of wine samples treated by yds (c – control, ow20 – optimum white 0.2 g/l, ow40 – optimum white 0.4 g/l, ol20 – opty less 0.2 g/l, ol40 – opty less 0.4 g/l, n20 – nobless 0.2 g/l, n40 – nobless 0.4 g/l) after one year of normal aging conditions et al., 2009). although nobless treatment had a slightly better effect on the browning index than other yds, the applied dose had no significant effect for all treated samples. the sensory evaluation indicates that yd treatments had a slight overall positive effect on the sensory characteristics (fig. 3). after bottling and one year of storage under standard conditions, the treated wines showed more intense smell attributes such as harmony and complexity and a more pronounced tropical fruit and honey character, compared to the control sample. as for the taste atributes, a significant improvement was observed in fullness, structure, complexity, harmony, and duration, accompanied by a decrease in astringency. conclusion in conclusion, our results indicate that yds treatment could be a suitable technique for the white wine industry to prevent or reduce oxidative processes that could negatively affect final quality. nevertheless, oenologists have to consider that the yds dose did not play a significant role, so it can be concluded that a concentration of 0.2 g/l is sufficient to achieve a satisfactory effect. acknowledgements. the paper is a part of the research done within the project of the ministry of science, technological development and innovations of the republic of serbia (451-03-65/2024-03/ 200133 and 451-0366/2024-03/ 200133), and the serbian academy of sciences 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(2022). browning development and antioxidant compounds in white wines after selenium, iron, and peroxide addition. applied sciences, 12(8), 3834, https://doi.org/10.3390/ app12083834 89 biologica nyssana ● 15 (2) december 2024: malićanin et al. ● evaluation of yeast derivatives on sauvignon blanc wine quality a pp en di ce s ap pe nd ix 1 . o xy ge n co nt en t ( m g/ l) in w in e sa m pl es tr ea te d by y d s in d iff er en t p ro du ct io n st ag es *c – c on tro l, o w 20 o pt im um w hi te 0 .2 g /l , o w 40 o pt im um w hi te 0 .4 g /l , o l2 0 o pt y le ss 0 .2 g /l , o l4 0 o pt y le ss 0 .4 g /l , n 20 n ob le ss 0 .2 g/ l, n 40 n ob le ss 0 .4 g /l , w b ef or e tre at m en t, c fb – a fte r c la rifi ca tio n an d fil tra tio n, a c c – a fte r a cc el er at ed a gi ng a nd n o r – a fte r o ne y ea r o f n or m al ag in g co nd iti on s. d iff er en t l et te rs in di ca te s ta tis tic al ly s ig ni fic an t d iff er en ce s in th e sa m e ro w (p < 0 .0 5) pr od uc tio n st ag es w in e tr ea tm en t c o w 20 o w 40 o l 20 o l 40 n 20 n 40 w 1. 45 1± 0. 02 8 1. 45 1± 0. 02 8 1. 45 1± 0. 02 8 1. 45 1± 0. 02 8 1. 45 1± 0. 02 8 1. 45 1± 0. 02 8 1. 45 1± 0. 02 8 c fb 1. 27 1± 0. 01 9a 0. 97 2± 0. 03 1b 0. 97 1± 0. 02 6b 0. 76 0± 0. 02 1c 0. 69 0± 0. 02 9d 0. 79 1± 0. 03 4c 0. 66 0± 0. 01 2d a c c 1. 10 1± 0. 02 2a 0. 95 1± 0. 02 3b 0. 90 1± 0. 03 2b c 0. 90 1± 0. 03 5b c 0. 86 1± 0. 03 0c d 0. 51 0± 0. 01 2e 0. 87 1± 0. 01 3d n o r 1. 13 1± 0. 01 4a 0. 86 1± 0. 03 3b 0. 97 0± 0. 03 6c 0. 96 1± 0. 02 1c 0. 87 1± 0. 03 0b 0. 78 0± 0. 03 0d 0. 85 1± 0. 01 5b ap pe nd ix 2 . b ro w ni ng in de x in w in e sa m pl es tr ea te d by y d s in d iff er en t p ro du ct io n st ag e pr od uc tio n st ag es w in e tr ea tm en t c o w 20 o w 40 o l 20 o l 40 n 20 n 40 w 0. 21 2± 0. 00 1 0. 21 2± 0. 00 1 0. 21 2± 0. 00 1 0. 21 2± 0. 00 1 0. 21 2± 0. 00 1 0. 21 2± 0. 00 1 0. 21 2± 0. 00 1 c fb 0. 12 7± 0. 00 4a 0. 11 9± 0. 00 9b 0. 11 5± 0. 00 1b 0. 10 9± 0. 00 2b c 0. 10 7± 0. 00 2c 0. 10 9± 0. 00 1b 0. 10 5± 0. 00 2c a c c 0. 21 2± 0. 00 2a 0. 18 4± 0. 00 2b 0. 18 4± 0. 00 2b 0. 19 9± 0. 00 2c 0. 18 0± 0. 00 2b 0. 16 5± 0. 00 1d 0. 16 5± 0. 00 2d n o r 0. 19 9± 0. 00 2a 0. 17 4± 0. 00 2b 0. 17 5± 0. 00 2b 0. 17 9± 0. 00 5b 0. 17 2± 0. 00 8b 0. 16 8± 0. 00 1c 0. 16 2± 0. 00 2d *c – c on tro l, o w 20 o pt im um w hi te 0 .2 g /l , o w 40 o pt im um w hi te 0 .4 g /l , o l2 0 o pt y le ss 0 .2 g /l , o l4 0 o pt y le ss 0 .4 g /l , n 20 n ob le ss 0 .2 g/ l, n 40 n ob le ss 0 .4 g /l , w b ef or e tre at m en t, c fb – a fte r c la rifi ca tio n an d fil tra tio n, a c c – a fte r a cc el er at ed a gi ng a nd n o r – a fte r o ne y ea r o f n or m al ag in g co nd iti on s. d iff er en t l et te rs in di ca te s ta tis tic al ly s ig ni fic an t d iff er en ce s in th e sa m e ro w (p < 0 .0 5) 90 air quality lichen monitoring at three selected urban areas biologica nyssana 7 (1)  september 2016: 19-29 stamenković, s. et al.  air quality lichen monitoring at three… 19 original article received: 09 mart 2016 revised: 21 mart 2016 accepted: 01 june 2016 air quality lichen monitoring at three selected urban areas in the southern serbia slaviša stamenković1*, tatjana djekić2, svetlana ristić1, vladica novković1, tatjana mitrović1, marija marković1 1university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia 2university of niš, faculty of sciences and mathematics, department of geography, višegradska 33, 18000 niš, serbia * e-mail: sslavisa@pmf.ni.ac.rs abstract: stamenković, s., djekić, t., ristić, s., novković, v., mitrović, t., marković, m.: air quality lichen monitoring at three selected urban areas in the southern serbia. biologica nyssana, 7 (1), september 2016: 19-29. the results of a study using epiphytic lichens as bioindicators, on teritory of three different urban areas in the southern serbia (leskovac, vlasotince, lebane) are reported. the analysis of samples from 142 investigated points indicates presence of 53 lichens taxa. the aim of the study is to present the difference in air quality over a certain time interval. using the index of atmospheric purityiap values, it has been found that there are different air pollution zones: „normal“ zone, „struggle“ zone and „lichen desert“ zone. the comparison of the results obtained in 2002 and 2013 in vlasotince indicates the presence of „normal“ zone in 2002 but, in 2013, the „normal zone“ area was completely replaced with the „struggle“ zone. in leskovac, unlike the previous studies, a slightly narrowing “lichen desert" zone is noticed. in lebane, there is a presence of all lichen zones. key words: biomonitoring, serbia, leskovac, vlasotince, lebane apstrakt: stamenković, s., djekić, t., ristić, s., novković, v., mitrović, t., marković, m.: lišajski monitoring kvaliteta vazduha određenih urbanih područja južne srbije. biologica nyssana, 7 (1), septembar 2016: 1929. istraživanja kvaliteta vazduha korišćenjem lišaja kao bioindikatora sprovedena su na teritoriji tri različita urbana područja južne srbijeleskovac, vlasotince i lebane. analiza uzoraka sa 142 istraživane tačke, ukazuje na prisustvo 53 lišajskih taksona. cilj ovog istraživanja je da prikaže promene kvaliteta vazduha koje su nastale u određenom vremenskom periodu. korišćenjem vrednosti indeksa atmosferske čistoće, konstantovano je prisustvo različitih zona zagađenja vazduha: „normalna“ zona, zona „borbe“ i zona „lišajske pustinje“. poređenje rezultata dobijenih 2002. i 2013. godine u vlasotincu, ukazuju na prisustvo „normalne“ zone u 2002. godini, međutim 2013. godine oblast „normalne“ zone je u potpunosti zamenjena zonom „borbe“. na području leskovca, razlika se ogleda u blagom suženju zone „lišajske pustinje“. u lebanu je uočeno prisustvo sve tri zone lišajske indikacije. key words: biomonitoring, srbija, leskovac, vlasotince, lebane 7 (1) • september 2016: 19-29 doi: 10.5281/zenodo.159100 biologica nyssana 7 (1)  september 2016: 19-29 stamenković, s. et al.  air quality lichen monitoring at three… 20 introduction the air quality is getting worse all over the world. the strongest effects are felt in urban areas. air pollution continuously increases, suggesting the need for timely detection, quantification and determination of levels of atmospheric pollution and introduction of monitoring system. biological monitoring is based on the assumption that changes in the environment affect the indicator organisms and these changes can be effectively used as an early warning signal for detecting of environmental changes (g a r t y , 2001). lichens are used in the biomonitoring studies because they are one of the most sensitive and valuable biomonitors of atmospheric pollution (s u j e t o v i e n e , 2015). lichens are symbiotic organisms composed of a fungus and an alga, usually either a cyanobacterium or green algae. it is estimated that there are approximately 25000 species of lichens (c h a p m a n , 2009). these unique organisms have a number of specific properties and therefore can be used for different purposes. changes in epiphytic lichen diversity are used as indicators of environmental conditions and have been widely applied in air quality assessments and monitoring programs around the world (l l o p et al., 2012). they are very good indicators of air quality. in the last decade of the twentieth century, a more intense use of epiphytic lichens as bioindicators on the territory of the republic of serbia began (m i l i ć , 1980; s a v i ć , 1991; 1997; 1998; s t a m e n k o v i ć , 1992; m i l i ć & b l a ž e n č i ć , 1993; t o d o r o v i ć , 1995; c v i j a n et al., 1995; 1997; 2008; s t a m e n k o v i ć , 1995; 1996; 1997; 1998; 2002; c v i j a n & s t a m e n k o v i ć , 1996a; 1996b; s t a n k o v i ć et al., 1999; p e j č i n o v i ć , 2000; s t a m e n k o v i ć & c v i j a n , 2002; 2003; 2004; 2010; s t a m e n k o v i ć et al., 2010; 2012; s t a m e n k o v i ć et al., 2013a; 2013b; j e v t i ć , 2013; n o v k o v i ć , 2013). this research is intended to present the results of the air quality on selected urban ecosystems and contribute the knowledge in biogeography and diversity of lichens on the territory of the republic of serbia and create the basis for the evaluation of lichens as indicators of the investigated urban territory. this is of great importance because there are no continuous physical and chemical measurements of air quality in these areas and this kind of monitoring is the only indicator of the air quality in urban conditions in the selected territory. material and methods sample location the southern serbia includes jablanica district. the administrative centre of the district is the town of leskovac, the biggest settlement in the southern serbia. this is the town with around 65289 inhabitants. the mean annual temperature is 10.7 °c and the mean annual rainfall is 591.6 mm. the most frequent winds are north, northwest and south (republic hydrometeorological service of serbia). leskovac has got a developed textile, chemical and pharmaceutical industry. the town of vlasotince is located in the southeastern part of serbia, in the area of middle and bottom basin of the river vlasina. the town has a population of 15882 people. the mean annual temperature is 11.8 °c and the mean annual rainfall is 724.3 mm, which is much higher than in the surrounding towns. east – west is the most common direction of movement of the winds (republic hydrometeorological service of serbia). fig. 1. geographical position of investigated region investigation region is framed. the town of lebane is an area which is characterized by extremely poor infrastructure, financial, economic and human resources. this is a small town having about 9272 inhabitants. the climate is moderate continental. the mean annual temperature is 10.2 °c and the mean annual rainfall in the vegetation period is 349 mm. the highest mean biologica nyssana 7 (1)  september 2016: 19-29 stamenković, s. et al.  air quality lichen monitoring at three… 21 wind speed from the north-northwest is 3.5 m/s, and the least is 1 m/s, from the northeast direction (republic hydrometeorological service of serbia). bioindication of air quality using lichens, as well as the physical and chemical measuring of air pollution have not been done in lebane until now. bioindication in vlasotince was made in 2002 and now. the institute of public health in leskovac controls physicochemical measuring of the level of air pollution since 2004. bioindication of air pollution in leskovac was made in 2002 and 2008. sample collection epiphytic lichens are determined and collected from the bark of various species of trees at the level of 1.52 m above the ground, exclusively from the trunks angled no higher than 5º. lichens were collected from 142 investigated points on the territory of the southern serbia. lichen materials were collected since 2002 to 2013. points of investigation are located in urban part of three different towns in the southern serbia. lichen samples were collected from the bark of various species of trees (tab. 1). the voucher specimens of the lichens were determined and deposited in the lichenological herbarium (under designation l; 1-9 hmn) of the department of biology and ecology, faculty of sciences and mathematics, university of niš, serbia. determination of collected lichen species was done by using a few sources (d o b s o n , 2005; w i r t h , 1995). determination of plant species was done by using “flora of serbia” (j o s i f o v i ć , ed., 1970-1977). the nomenclature of plant species follows euro+med plant base (http://www.emplantbase.org). method description the method of lichen indication of air quality means identifying, collecting, detecting and mapping the lichens and calculation of the index of atmospheric purity (iap) and it is based on determination of the zones of different degrees of air quality. investigation points have been noted and iap values for each point calculated. in this research, we were using the numeric method known as the index of atmospheric purity iap (k r i c k e & l o p p i , 2002): table 1. substrates in the three different surveys (the cells are filled with serial number of the investigated points) l o c a t i o n a n d t i m e o f c o l l e c t i o n supstrate (plant species) vlasotince 2002 vlasotince 2013 leskovac 2002 lebane 2013 acer campestre ͞ ͞ 1 ͞ carpinus betulus ͞ 2 ͞ ͞ fraxinus excelsior ͞ ͞ 6, 29, 35 ͞ fraxinus sp. ͞ ͞ ͞ 7 juglans regia 2, 5, 8, 10, 11 7 3, 4, 11, 21, 22 6, 9 malus domestica ͞ 10, 11, 13 15, 23 ͞ morus alba ͞ ͞ 18, 34 17 platanus acerifolia ͞ ͞ 33 ͞ populus alba 1 ͞ ͞ ͞ populus deltoides 4, 6, 7, 9, 13, 16 1, 4 5, 10, 19, 25, 30, 32, 36, 39 ͞ prunus armeniaca ͞ ͞ 8, 13 ͞ prunus avium ͞ 3, 12 20, 24 10 prunus domestica 3 8, 14 ͞ 4, 12, 16, 18, 19, 22 pyrus communis ͞ ͞ 12, 28, 31 ͞ quercus ceris ͞ ͞ ͞ 1, 2, 3, 8, 11, 14, 15, 24 robiania pseudoacacia ͞ ͞ 7, 27 13, 20 tilia argentea ͞ ͞ ͞ ͞ tilia plathyphyllos 12, 15 5, 6, 9 ͞ ͞ tilia sp. ̶ ̶ ͞ 5, 21, 23 biologica nyssana 7 (1)  september 2016: 19-29 stamenković, s. et al.  air quality lichen monitoring at three… 22 table 2. epiphytic lichen flora with values of frequency for each investigated point in the southern serbia taxon place/ time investigated point frequency (%) amandinea sp. leskovac/ 2002 28 2.56 amandinea punctata (hoffm.) coppins & scheid. leskovac / 2002 1, 8, 29, 30, 32, 33, 36 20.48 leskovac / 2008 25, 26, 28, 40, 41, 45, 46, 48, 49 18.37 vlasotince / 2002 7, 10, 12, 16 25.00 lebane / 2013 2, 11 8.33 arthonia punctiformis ach. lebane / 2013 19 4.16 caloplaca cerina (hedw.) th. fr. vlasotince / 2002 6, 10 12.50 caloplaca sp. leskovac / 2002 9, 14, 26 7.68 vlasotince / 2002 3, 16 12.50 candelariella aurella (hoffm.) zahlbr. leskovac / 2002 11, 14, 19, 20, 32 12.80 candelariella vitellina (hoffm.) müll. arg. vlasotince / 2013 7 7.14 candelariella xanthostigma (pers. ex ach.) lettau leskovac / 2002 1, 4, 9, 19, 26, 28, 29 12.80 leskovac / 2008 2, 9, 15, 22, 25, 26, 27, 28, 31, 36, 40, 41, 45, 46, 47, 48, 49 34.69 vlasotince / 2002 2, 13, 14, 16 25.00 vlasotince / 2013 1, 9, 12, 13 28.57 lebane / 2013 1, 3, 4, 5, 9, 11, 12, 14, 16, 17, 19, 21, 23, 24 58.33 candelariella sp. vlasotince / 2002 2 6.25 evernia prunastri (l.) ach. leskovac / 2002 1, 29, 32 12.80 leskovac / 2008 27, 28, 29, 45, 46, 47, 48, 49 16.33 vlasotince / 2002 5, 9 12.50 vlasotince / 2013 2 7.14 lebane / 2013 1, 2, 8, 11, 12, 16, 19, 21, 22, 24 41.66 flavoparmelia caperata (l.) hale leskovac / 2002 37 2.56 leskovac / 2008 47, 48 4.08 lebane / 2013 1, 2, 8, 11, 12, 14, 16, 19, 21, 22 41.66 gyalecta sp. vlasotince / 2002 16 6.25 vlasotince / 2013 10 7.14 graphis sp. vlasotince / 2002 3 6.25 hypogymnia physodes (l.) nyl. leskovac / 2002 37, 38 5.12 leskovac / 2008 36, 48 4.08 lebane / 2013 4, 6, 12 12.50 lecanora allophana (ach.) nyl. vlasotince / 2002 2, 5, 7, 8, 9, 10, 12 43.75 lecanora argentata (ach.) röhl. leskovac / 2002 9, 34, 36 10.24 leskovac / 2008 28 2.04 vlasotince / 2002 2, 4, 9, 10 25.00 lecanora carpinea (l.) vain. vlasotince / 2002 4, 9, 10, 12 25.0 lecanora glabrata (ach.) malme vlasotince / 2002 5, 10, 12 18.75 lecanora intumescens (rebent.) rabenh. leskovac / 2002 3, 9, 10, 14, 30, 34 17.92 vlasotince / 2002 2, 4, 5, 7, 8, 9, 10, 12, 13 56.25 lecanora muralis (schreb.) rabenh. vlasotince / 2002 2, 8, 9 18.75 lecanora pulicaris (pers.) ach. leskovac / 2002 22 5.12 leskovac / 2008 28 2.04 vlasotince / 2002 2, 4, 5, 8, 9 31.25 lecanora sp. leskovac / 2002 1, 8, 11, 14, 18, 19, 26, 28, 29, 32, 35, 36, 37, 39 42.18 leskovac / 2008 22, 28, 39, 45, 46 10.20 vlasotince / 2002 3, 11 6.25 vlasotince / 2013 2, 7, 9 21.43 lecidea sp. leskovac / 2008 47, 48 4.08 lecidella elaeochroma (ach.) m. choisy leskovac / 2002 1, 2, 6, 8, 16, 17, 24, 29, 30, 31 30.72 vlasotince / 2002 12, 13 12.50 lecidella sp. leskovac / 2008 47 2.04 lepraria aeruginosa (ach.) turner lebane / 2013 11 4.16 lepraria incana (l.) ach. leskovac / 2008 28 2.04 lebane / 2013 10, 16, 22 12.50 melanohalea elegantula (zahlbr.) o. blanco et al. leskovac / 2002 37, 38 5.12 melanohalea exasperata (de not.) o.blanco et al. leskovac / 2002 38, 39 5.12 vlasotince / 2002 1, 2, 8 18.75 melanohalea exasperatula (nyl.) o. blanco, a. crespo, divakar, essl., d. hawksw. & lumbsch leskovac / 2002 1, 29 7.68 melanelixia glabra (schaer.) o. blanco et al. leskovac / 2002 1 5.12 leskovac / 2008 36 2.04 melanelixia glabratula (lamy) sandler berlin & arup leskovac / 2002 1, 2, 29 7.68 leskovac / 2008 27, 28, 47, 49 8.16 vlasotince / 2002 1 6.25 biologica nyssana 7 (1)  september 2016: 19-29 stamenković, s. et al.  air quality lichen monitoring at three… 23 continuation of table 2 taxon place/ time investigated point freque ncy (%) melanelixia subargentifera (nyl.) o. blanco, a. crespo, divakar, essl., d. hawksw. & lumbsch lebane / 2013 18, 19, 21 12.50 melanelixia subaurifera (nyl.) o.blanco et al. leskovac / 2002 1 5.12 leskovac / 2008 47, 48 4.08 lebane / 2013 1, 2, 8, 11, 12, 14, 16, 24 33.33 melanelia sp. leskovac / 2002 2, 20, 28 7.68 leskovac / 2008 4 2.04 vlasotince / 2002 1, 2 12.50 vlasotince / 2013 2, 6 14.29 ochrolechia sp. vlasotince / 2013 11 7.14 parmelia sulcata taylor leskovac / 2002 1, 2, 7, 8, 16, 20, 24, 27, 28, 29, 33 33.28 leskovac / 2008 15, 36, 39, 47, 48 10.20 vlasotince / 2002 1, 3, 5, 8, 9, 10, 16 43.75 vlasotince / 2013 2, 3, 4, 5, 13, 14 42.86 lebane / 2013 1, 4, 6, 17, 18, 19, 20, 21, 22, 23, 24 45.83 parmelia tiliacea (hoffm.) ach. leskovac / 2002 1, 3, 32 7.68 leskovac / 2008 4, 14, 27, 28, 37, 48 12.24 lebane / 2013 5, 21, 23 12.50 parmelina pastillifera (harm.) hale leskovac / 2002 37, 38, 39 7.68 vlasotince / 2002 8 6.25 parmelina quercina (willd.) hale vlasotince / 2002 3, 13 12.50 lebane / 2013 3, 8, 14, 15 16.66 phaeophyscia orbicularis (neck.) moberg leskovac / 2002 1, 2, 3, 4, 5, 6, 8, 10, 11, 12, 13, 15, 16, 18, 19, 20, 21, 28, 29, 30, 35 56.32 leskovac / 2008 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 14, 15, 16, 17, 19, 21, 22, 23, 24, 25, 26, 27, 28, 31, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 79.59 vlasotince / 2002 1, 2, 3, 4, 6, 7, 8, 9, 10, 11, 12, 14, 15 81.25 vlasotince / 2013 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 100.00 lebane / 2013 1, 2, 3, 5, 8, 9, 10, 14, 16, 19, 22 45.83 physcia adscendens (fr.) h. olivier leskovac / 2002 1, 3, 8, 10, 11, 12, 19, 20, 28, 29, 30, 32, 33 40.96 leskovac / 2008 1, 2, 3, 4, 5, 6, 7, 8, 9, 12, 13, 14, 15, 16, 18, 19, 20, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 81.63 vlasotince / 2002 1, 4, 6, 8, 9, 10, 11, 13, 16 56.25 vlasotince / 2013 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 100.00 lebane / 2013 2, 5, 6, 9, 10, 11, 12, 13, 15, 17, 19, 22, 24 54.16 physcia aipolia (ehrh. ex humb.) fürnr. leskovac / 2002 29 5.12 vlasotince / 2002 8, 11 12.50 lebane / 2013 3 4.16 physcia stellaris (l.) nyl. leskovac / 2002 1, 3, 14, 20, 32 10.24 leskovac / 2008 28, 29, 48, 49 8.16 vlasotince / 2002 6, 8, 13 18.75 physcia tenella (scop.) dc. leskovac / 2002 1, 7, 20, 28, 29 17.92 leskovac / 2008 48 2.04 vlasotince / 2002 4, 8, 11 18.75 physconia enteroxanta (nyl.) poelt leskovac / 2002 1 7.68 leskovac / 2008 48 2.04 vlasotince / 2013 6 7.14 lebane / 2013 2, 4, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 19, 21, 23 62.50 physconia grisea (lam.) poelt leskovac / 2002 1, 25 10.24 leskovac / 2008 27, 28 4.08 vlasotince / 2002 1, 4, 5, 6, 10 31.25 lebane / 2013 2 4.16 pleurosticta acetabulum (neck.) elix & lumbsch leskovac / 2002 39 2.56 leskovac / 2008 28 2.04 lebane / 2013 7, 24 8.33 ramalina calicaris (l.) röhl. lebane / 2013 12, 19, 22 12.50 ramalina farinacea (l.) ach. leskovac / 2002 38 2.56 ramalina sp. leskovac / 2002 38, 39 5.12 usnea hirta (l.) weber ex f.h. wigg. leskovac / 2002 37 2.56 xanthoria parietina (l.) th. fr. leskovac / 2002 1, 2, 5, 8, 10, 14, 16, 19, 20, 25, 29, 36 35.84 leskovac / 2008 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 18, 19, 20, 22, 23, 24, 25, 26, 27, 28, 30, 32, 33, 34, 36, 37, 38, 39, 40, 41, 43, 45, 46, 47, 48, 49 83.67 vlasotince / 2002 1, 4, 5, 6, 7, 8, 10, 12, 14, 16 62.50 vlasotince / 2013 1, 3, 4, 5, 6, 8, 9, 10, 13, 14 71.43 lebane / 2013 1, 2, 3, 4, 5, 6, 8, 9, 10, 12, 13, 14, 15, 17, 18, 20, 21, 22, 23, 24 83.33 biologica nyssana 7 (1)  september 2016: 19-29 stamenković, s. et al.  air quality lichen monitoring at three… 24 𝐼𝐴𝑃 = 1 10 ∑(𝑄𝑖 × 𝑓𝑖) 𝑛 𝑖=1 where n = a number of species on the spot, f = coefficient which represents the frequency and cover of each species, and q = the ecological index of each species, i.e. the mean value of other lichen species growing with the species under study within the surveyed area. results and discussion the analysis of samples from the described localities indicate the presence of 53 lichen taxa on 142 investigated points from the territory of the southern serbia (tab. 2). the most frequent species were: phaeophyscia orbicularis, xanthoria parietina, physcia adscendens and parmelia sulcata. the highest frequency of these lichen species were found in all three investigated urban ecosystems. the least frequent species were: amandinea sp., ramalina farinacea and usnea hirta. these lichen species were found on only one of all investigated localities, in leskovac in 2002, having a low frequency 2.56%. the calculated iap values are very variable and they are between 0 and 47 (tab. 3). according to the calculated iap values, we got borders of the lichen zones of different air pollution levels in lebane (fig. 2). also, we got a picture which presents a comparison of moving the lichen zones in relation to the time of the survey in leskovac (fig. 3) and vlasotince (fig. 4). on the basis of these results and according to schulze (s c h u l z e et al., 2005), we can see that the investigated points in central part of urban ecosystems are included in “lichen desert” zone, characterized by the presence of species that are good indicators of air pollutiont. this indicates a high level of air pollution. the presence of this zone is recorded in most parts of urban areas of the southern serbia. in leskovac, we found presence of this zone in 2002 and 2008 (fig. 3). in lebane, three points belong to the “lichen desert” zone. in vlasotince, this zone did not exist in the year of 2002, but it is present in the year of 2013. (fig. 4). many investigated points belong to the “struggle” zone. in this zone, the air quality is slightly better than in the previous one. the maps clearly show that this zone usually includes peripheral parts of the city. smaller parts of the towns belong to “normal zone” which is detected in lebane and leskovac in the years of 2002 and 2008. we can also notice an interesting situation in vlasotince, where we detected the “normal zone” in the year of 2002 but, after eleven years, this zone does not exist anymore. such a picture of the different air pollution levels in the investigated areas is a logical consequence of air pollution and the mutual influence of microclimate, substrate and geophysical features, as well as the distribution of areas and objects. composition of lichens community shows certain deviations. the urban area of lebane includes all air quality zones. there is a relatively high level of air pollution, caused by the main road intersecting the center of the town and the former textile factory. agricultural land occupies 53% of the total area of the municipality of lebane, so, in this area, the presence of lichen is less probable. in 2002 and 2008, the bioindication of air quality by using lichens, in the area of leskovac was analyzed and compared. unlike in 2002, the boundaries of the “lichen desert” moved in 2008. there was a slight reduction of “lichen desert” zone. however, comparing to the year of 2002, in 2008, we can see a reduced number of lichen species of all types of the thallus morphology. other zones did not show significant changes in their limits, but the composition of the lichen species shows discrepancies. in leskovac concentrations of sulfur dioxide (so2), nitrogen oxides (nox), aero sediments and heavy metals (cd, pb, zn) in the suspended particles have been decreasing (institute of public health leskovac), which might have to do with the closing down of many industrial facilities. the reduction of the “lichen desert” zone might be correlated with a decrease of pollutants (sulfur dioxide, nitrogen oxides, aero sediments and heavy metals in suspended particles), while the reduced number of registered taxa might be linked to an increasing concentration of soot and heavy metals in the sediments. by comparing the results of bioindication of air quality by using lichens, in the vlasotince area in 2002 and in 2013, we found that the borders of the zones with different levels of air pollution using lichens as bioindicators in vlasotince are different in 2002 and 2013. investigation conducted in vlasotince in 2002 showed the presence of “struggle zone” and “normal zone”. eleven years later, in the investigated area, there aren't any records of the presence of “normal zone”. "normal" zone from 2002 is commuted into a “struggle zone" in 2013, and the "struggle zone" area has changed into "lichen desert" zone (fig. 4). compared to 2002, the absence of a number of taxa in 2013 provides the evidence for a considerably reduced diversity, which is supported by the altered frequency of some species’ presence. biologica nyssana 7 (1)  september 2016: 19-29 stamenković, s. et al.  air quality lichen monitoring at three… 25 this indicates that, in relation to the resistant species, coverage of the sensitive ones is reduced. domination of the more resistant species and a reduced total diversity of the lichen flora are indicating an increased air pollution ratio in vlasotince within the last 11 years. tabele 3. values of iap for every investigated point in the southern serbia l/t ip iap l/t ip iap l/t ip iap l/t ip iap l es k o v a c/ 2 0 0 2 1 38 l eb a n e/ 2 0 1 3 1 6 v la so ti n ce /2 0 0 2 1 17 v la so ti n ce /2 0 1 3 1 4 2 4 2 6 2 19 2 5 3 3 3 6 3 7 3 3 4 1 4 7 4 19 4 4 5 3 5 7 5 14 5 5 6 1 6 8 6 14 6 3 7 2 7 3 7 7 7 2 8 4 8 10 8 24 8 2 9 4 9 9 9 17 9 4 10 8 10 5 10 28 10 3 11 6 11 7 11 3 11 2 12 1 12 7 12 11 12 3 13 0 13 4 13 8 13 3 14 9 14 7 14 3 14 3 15 0 15 5 15 1 16 2 16 6 16 11 17 1 17 7 18 3 18 4 19 7 19 8 20 6 20 2 21 0 21 18 22 0 22 5 23 / 23 5 24 1 24 9 25 2 26 3 27 1 28 21 29 39 30 3 31 1 32 12 33 6 34 1 35 1 36 4 37 35 38 23 39 47 biologica nyssana 7 (1)  september 2016: 19-29 stamenković, s. et al.  air quality lichen monitoring at three… 26 fig. 2. zones of different levels of air pollution in lebane using lichens as bioindicators (novković, 2013). fig. 3. borders of the zones with different levels of air pollution using lichens as bioindicators in 2002 (stamenković, 2002) and 2008 (nikolić, 2008), with "wind rose" in leskovac. biologica nyssana 7 (1)  september 2016: 19-29 stamenković, s. et al.  air quality lichen monitoring at three… 27 fig. 4. the "wind rose" with the delimitation of different lichen indication zones in vlasotince from investigations in 2002 (stamenković, 2002) and 2013 (jevtić, 2013). *delimitation of different lichen zones of indication in 2002; **delimitation of different lichen zones of indication in 2013 conclusion the method used in this research on relatively simple and quick way provides an insight into the quality of the air on investigated area by calculating the index of atmospheric purity (iap) based on the lichen diversity and abundance. according to the calculated iap values, we created a map of the zones of different air pollution levels in lebane and maps which present comparison of lichen zones movement in different time periods in vlasotince and leskovac. due to the fact that during the last 11 years the level of air pollution has increased so, if we do not take measures of protection, it can be assumed that the trend of the further growth of pollution will continue. this work is a mere contribution to the biomonitoring of air pollution i.e. air quality on the territory of the southern serbia. acknowledgements. this research was supported by the ministry of education and science of the republic of serbia, project oi 171025. authors express gratitude to marina jevtić for the ceded data from its master's thesis. references chapman, a.d. 2009: numbers of living species in australia and the world. australian biological resources study (abrs). canberra. 80 p. cvijan, m., todorović, b., joksimović, v., 1995: lichens as bioindicators of air pollution in the towns of mali zvornik and aranđelovac, glasnik instituta za botaniku i botaničke bašte univerziteta u beogradu, 29: 175-186, beograd. cvijan, m., stamenković, s., 1996a: bioindication of air pollution in the niš area by use of lichens. ekologija 31 (1), 151-157. cvijan, m., stamenković, s., 1996b: lignicolous lichens in the urban area of niš town. archive of biological sciences, 48 (3-4): 115-118, belgrade. cvijan, m., savić, s., szabados, k., 1997: lichens as bioindicators of air pollution in belgrade area. ekologija, 32 (1): 99-106. cvijan, m., subakov-simić, g., krizmanić, j. 2008: monitoring of the «lichen desert» in the belgrade area (1980/81, 1991 and 2007). archive of biological sciences, 60 (2), 215-222. dobson, f. s. 2005: lichens. richmond, richmond publishing co. ltd. biologica nyssana 7 (1)  september 2016: 19-29 stamenković, s. et al.  air quality lichen monitoring at three… 28 garty, j., tamir, o., hassid, i., eshel, a., cohen, y., karnieli, a., orlovsky, l. 2001: photosynthesis, chlorophyll integrity and spectral reflectance in lichens exposed to air pollution. journal of environmental quality, 30: 884-893. gombert, s., asta, j., seaward, m. r. d. 2004: assessment of lichen diversity by index of atmospheric purity (iap), index of human impact (ihi) and other environmental factors in an urban area (grenoble, southeast france). science of the total environment, 324: 183-199. hawksworth, d. l. and rose, f. 1970: qualitative scale for estimation sulfur dioxide air pollution in england and wales using epiphytic lichens. nature, 227: 145-148. institute of public health leskovac. http://www.zzjzle.org.rs/ jevtić, m., 2013: promene lišajskih zona biološke indikacije kvaliteta vazduha u vlasotincu u periodu 2002 – 2013. god. master thesis ( in serbian). prirodno-matematički fakultet. niš. kricke, r., loppi, s. 2002: bioindication: the iap approach. in: nimis, p., scheidegger, c. & wolseley, p. 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from serbia bojan zlatković1*, stefan bogosavljević2 1 university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia 2 "filly farm pharmacy", health institution, dušanova 31, 18000 niš, serbia *corresponding autor: bojanzlat@yahoo.com abstract: zlatković, b., bogosavljević, s.: report on the new floristic data from serbia. biologica nyssana, 5 (2), december 2014: 123-129. new chorological data for 16 taxa of vascular flora of serbia (records no. 1-16) are given in the article. the taxa represented belong to the following families: asteraceae (1-8), euphorbiaceae (9), geraniaceae (10), juncaceae (13), lamiaceae (11), salicaceae (12), poaceae (14, 15) and typhaceae (16). artemisia alba var. biasolettiana is reported for the first time within the territory of serbia. key words: new chorological data, serbia, vascular flora apstrakt: zlatković, b., bogosavljević, s.: izveštaj o novim florističkim podacima iz srbije. biologica nyssana, 5 (2), decembar 2014: 123-129. prikazani su novi horološki podaci za 16 taksona vaskularne flore srbije (podaci br. 1-16). taksoni pripadaju sledećim familijama: asteraceae (1-8), euphorbiaceae (9), geraniaceae (10), juncaceae (13), lamiaceae (11), salicaceae (12), poaceae (14, 15) i typhaceae (16). prisustvo artemisia alba var. biasolettiana do sada nije bila poznato u flori srbije. ključne reči: novi horološki podaci, srbija, vaskularna flora introduction the goal of this paper is presentation of new chorological data for 16 taxa in vascular flora of serbia. according to our survey of literature, one infraspecies taxon was reported in this paper for the first time within the territory of serbia. the new records may be used as additional data for distribution of taxa presented in the edition series “the flora of sr serbia (i-x)” (j o s i f o v i ć , ed., 1970-1977; s a r i ć & d i k l i ć , 1986; d i k l i ć , 1977; n i k o l i ć et al., 1986), as well as the taxa that were reported from serbia after the monograph was published but will be included within the new edition “the flora of serbia” (s t e v a n o v i ć , 1992, 2012) and for completing the datasets on general distribution of recorded taxa. 5 (2) • december 2014: 123-129 biologica nyssana 5 (2)  december 2014: 123-129 zlatković, b., bogosavljević, s.  report for the new floristic data… 124 material and methods the collected plant material was stored at the herbarium of the institute of botany and botanical garden “jevremovac”, university of belgrade (beou), herbarium of the faculty of science and mathematics, university of niš (hmn) and herbarium of the faculty of sciences and mathematics, university of novi sad (buns). acquisition numbers are given in the brackets. distribution of the studied taxa within the territory of serbia was determined and mapped in utm grid system (10 x 10 sq. km., utm zone 34t). the nomenclature and classification of taxa was matched with the euro+med (2006). we used a geographic regionalization of serbia according to m a r k o v i ć (1970), modified by s t e v a n o v i ć (1999). results and discussion asteraceae 1. erigeron sumatrensis retz. (syn. conyza sumatrensis (retz.) e. walker, c. albida spreng.) (central): the town of niš, batušinac village, ruderal, 186 m, artemisietea vulgaris, en69, 15.09.2014, coll. b. zlatković (hmn 9245). (east): the town of gadžin han, grkinja village, ruderal, 350 m, artemisietea vulgaris, en88, 27.09.2014, coll. m. ranđelović & s. bogosavljević (hmn 9244). (east): the town of niš, gorica hill, ruderal, 358 m, artemisietea vulgaris, en79, 02.10.2014, coll. b. zlatković & s. bogosavljević (hmn 9246). (south): the town of leskovac, ruderal, 250 m, artemisietea vulgaris, en76, 21.09.2014, coll. m. ranđelović & s. bogosavljević (hmn 9243). presence of invasive species e. sumatrensis in serbia was first recorded in 2002 in vegetation of urban area of belgrade (n i k e t i ć & j o v a n o v i ć , 2002). this species originated in tropical and subtropical regions, and its presence in european countries, where it has invasive character, was first recorded in 1875 (c a s a s a y a s , 1989). spread of species e. sumatrensis in territory of serbia matches the invasive potential shown by this species in other countries of southeastern europe and balkan peninsula (a n a s t a s i u & m e m e d e m i n , 2010; v l a d i m i r o v , 2009). 2. dittrichia graveolens (l.) greuter (syn. inula graveolens (l.) desf.) (east): sićevo gorge, gradište village, limestone, ruderal places, roadside, 400 m, artemisietea vulgaris, en99, 27.09.2011, coll. b. zlatković (beou 16839). (east): the town of dimitrovgrad, srećkovac village, limestone, ruderal places, roadside, 470 m, artemisietea vulgaris, fn36, 26.10.2013. coll. b. zlatković, m. miljković & m. marković (beou 16840). dittrichia graveolens is a therophyte species with mediterranean-submediterranean range type, with additional parts of range in middle east and the atlantic region of europe (m e u s e l & j ä g e r , 1992). recently this species was introduced to north america, australia and south africa, showing a strong invasive character (b r o w n s e y et al., 2013). its range increase in central europe started in mid-20th century and continues to gain intensity (k i r á l y et al., 2014). it is assumed that spread of this species is greatly influenced by its anthropochorous type of dispersal, particularly facilitated by construction of traffic routes and formation of ruderal-type habitats. the new localities are situated along the high-frequency international traffic routes, so it is assumed that the anthropogenous factor is greatly beneficial to its range increase in serbia. it was previously recorded only in vicinity of suva reka in kosovo and metohija (g a j i ć , 1975a). 3. achillea chrysocoma friv. (southeast): vlasina plateau, vlasina rid, schists, in vegetation of mountain pastures (nardion strictae) close to the lake, 1200 m, fn03, 16.06.2013, coll m. ranđelović & s. bogosavljević (beou 16833). endemic species characteristic for vegetation of high-mountain pastures and stony ground in the central and southern part of balkan peninsula. in the territory of serbia it has been reported from high mountains of kosovo and metohija (šar-planina, koritnik), as well as besna kobila mt. (d i k l i ć , 1977; n i k o l i ć et al., 1986) and crni vrh close to vranje (a d a m o v i ć , 1908). t o m o v i ć (2007) considers the distribution data for this species in region of vlasina (gorge of masurićka reka) to be doubtful due to incomplete herbarium material in collection of beou. the new record in distribution of achillea chrysocoma in serbia supports the claim that range of this species may realistically include the area of vlasina plateau. biologica nyssana 5 (2)  december 2014: 123-129 zlatković, b., bogosavljević, s.  report for the new floristic data… 125 4. artemisia alba turra var. biasolettiana (vis.) ch. gussev (east): valley of visočica, village slavinja, carbonates, marls, 734 m, fn47, 13.08.2010, coll b. zlatković & d. jović (hmn 9247). artemisia alba is a relatively common species with pontian-mediterranean type of distribution, and within its wide range it shows a pronounced polymorphism of morphological characters. in serbia it appears in several types of thermophilous habitats, primarily on stony ground on limestone and serpentinite. besides the type variety (var. alba), this study has revealed the first record of taxon a. alba var. biasolettiana in serbia. it is characterized by presence of very thick white indumentum that completely covers the leaves, stem and inflorescences of this plant (fig. 1c, 1d). the closest known locality where this taxon was recorded is situated in western part of bulgaria (g u s s e v , 2012). 5. artemisia campestris l. (east): the town of knjaževac, rgošte village, alluvium, ruderal places near railroad, 230 m, artemisietea vulgaris, ep92, 13.10.2013, coll. b. zlatković & s. bogosavljević (beou 16832). according to g a j i ć (1975), artemisia campestris is a species distributed almost throughout serbia. however, our review has shown that this species is rare in southern and eastern parts of serbia where its distribution is insufficiently known. 6. carlina graeca heldr. & sartori (south): mt rujan, slavujevac village, silicate, dry pastures, 800 m, festuco-brometea, em68, 31.07.2008, coll. b. zlatković & n. smiljković (beou 16835). distribution of this species in serbia is still insufficiently studied and important, as it form the northern edge of its area from mediterranean region toward inner part of balkan peninsula. it was reported from the few localities from southern and southeastern region of serbia (n i č i ć , 1902; z l a t k o v i ć & r a n đ e l o v i ć , 2004; z l a t k o v i ć , 2006). 7. jurinea polycephala formánek (syn. j. bipinnata adamović) (south): mt rujan, orljak, silicate, 742 m, festuco-brometea, em67, 26.06.2009, coll. b. zlatković & n. smiljković (beou 16849). (southeast): the town of bosilegrad, izvor village, limestone, dry pastures and rocky grounds, 995 m, festuco-brometea, fc20, 21.06.2014, coll. b. zlatković & g. tomović (hmn 9372). this species inhabits steppe-like grassy habitats and thermophilous rocky ground on silicate and limestone geological substrate. records of j. arachnoidea in vicinity of sukovo (a d a m o v i ć , 1899), prizren (k o š a n i n , 1939), and bosilegrad (r a n đ e l o v i č et al., 1988), probably refers to j. polycephala. besides the new data on distribution this study has also proven the presence of this species in vicinity of bosilegrad, at the slopes of mt. rudina in serbia. 8. centaurea melanocephala pančić (fig. 1a, 1b) (central): studena planina, peak cvetalica, slopes toward brezanska reka river, serpentinite, mountain pastures and glades in oak forest, exp. nw, 1055 m, festucobrometea, dp72, 11.09.2002, coll. b. zlatković (hmn 9248). until now, this endemic species was reported from very few localities in the ibar valley and slopes of mt. stolovi in central serbia (p a n č i ć , 1874, a d a m o v i ć , 1907) and surroundings of kosovska mitrovica and đakovica in kosovo and metohija (w r a b e r , 1993). the new data offers first confirmation for existence of this extremely rare species close to its “locus classicus” (“mt. stol”= mt. stolovi) after it was described by p a n č i ć (1874). euphorbiaceae 9. euphorbia velenovskyi bornm. (east): the town of babušnica, bežište village, alluvium, 600 m, phragmitetea, fn07, 28.05.2008, coll. b. zlatković (beou 16848). (south): the town of vranje, buštranje village, silicate, wet meadows, 450 m, molinioarrhenatheretea, em79, 17.05.2008, coll. b. zlatković & n. smiljković (beou 16857). (south): the town of vranje, ribnice village, alluvium, wet meadows, 370 m, phragmitetea, en70, 17.05.2008, coll. b. zlatković & n. smiljković (beou 16834). euphorbia velenovskyi is an insufficiently studied species of serbian flora, and the only data on its distribution in serbia are from several sites in vicinity of bosilegrad in southeast serbia (u r u m o f f , 1935). certain authors consider this taxon to be a synonym for species e. palustris, but biologica nyssana 5 (2)  december 2014: 123-129 zlatković, b., bogosavljević, s.  report for the new floristic data… 126 figure 1. a, b centaurea melanocephala; c, d artemisia alba var. biasolettiana; e distribution of trachynia distachya in serbia. e. velenovskyi clearly differs by pointed tips and sharply serrated margins of leaf blades (k u z m a n o v , 1979). we believe that e. velenovskyi may have much wider distribution in serbia, but it is necessary to perform a review of data on distribution of e. palustris in vicinity of bujanovac (j a n k o v i ć & n i k o l i ć , 1972), vranje, pirot and sukovo (a d a m o v i ć , 1898). geraniaceae 10. geranium pratense l. (east): the town of dimitrovgrad, mazgoš village, alluvium, 661 m, salicetum albofragilis, fn56, 22.06.2013, coll. b. zlatković & n. stanković (hmn 9250). (east): the town of dimitrovgrad, protopopinci village, alluvium, 666 m, wet meadows, biologica nyssana 5 (2)  december 2014: 123-129 zlatković, b., bogosavljević, s.  report for the new floristic data… 127 molinio-arrhenatheretea, fn56, 22.06.2013, coll. b. zlatković & n. stanković (hmn 9249). in serbia, it was reported only for šumadija region (v e l j o v i ć , 1967). lamiaceae 11. salvia verbenaca l. (east): the town of dimitrovgrad, kozarica mt. slopes, limestone, ruderal places, roadside, 474 m, festuco-brometea, fn46, 30.04.2007, coll. b. zlatković (beou 16855). (east): the town of pirot, sarlak hill, limestone, ruderal places, roadside, 450 m, festucobrometea, fn28, 30.04.2007, coll. b. zlatković (beou 16854). (southeast): the municipality of trgovište, crnovce village, molasses, steppe-like pastures in silicate, astragalo-stipetum capillatae, 800 m, festuco-brometea, em89, 17.05.2008, coll. b. zlatković (beou 16838). presence of this species was recently recorded in several localities in eastern and southwestern parts of serbia (z l a t k o v i ć et al., 2005). monitoring of its distribution has shown spread pattern along the traffic routes as well as in ruderal habitats in human settlement, mostly in southern warmer parts of serbia. salicaceae 12. salix elaeagnos scop. (southeast): vlasina plateau, bratanov del, silicate, near stream, 1238 m, querco-fagetea, fn02, 31.07.2013, coll. b. zlatković (beou 16850). this species is distributed along the river courses of western and southwestern serbia and kosovo and metohija (j o v a n o v i ć & t u c o v i ć , 1972; j o v a n o v i ć , 1997), however the amount of substantiated data on its distribution in other regions of serbia is insufficient. juncacee 13. juncus capitatus weigel (south): rujan planina, borovac village, eroded areas along the road, 422 m, em69, 31.07.2013, coll. b. zlatković (hmn 9251). this is the second confirmed set of data on existence in serbian flora of this threatened taxon (cr b2ab(i,iii,iv)), which used to be known from a single locality at the extreme southeast of serbia (t o m o v i ć et al., 2009). at the new locality, this species appears in a localized, sparse population, within the mostly therophytic vegetation, at sandy areas around streams and springs. poaceae 14. rostraria cristata (l.) tzvelev (syn. koeleria phleoides (vill.) pers.) (east): sićevo gorge, sićevo village, limestone, 420 m, festuco-brometea, en89, 11.07.2010, coll. b. zlatković & s. bogosavljević (hmn 9254). this mediterranean grass is casual in serbia. it was reported from a few localities in eastern and southeastern parts of the country (š m a r d a , 1968; z l a t k o v i ć & s t e v a n o v i ć , 2007). 15. trachynia distachya (l.) link (syn. brachypodium distachyon (l.) p. beauv. ) (east): sićevo gorge, gradište village, arid grassy locations, en99, 05.07.1999, coll. b. zlatković (buns). (south): mt rujan, orljak, silicates, 742 m, em67, 26.06.2009, coll. b. zlatković & n. smiljković (beou 16841). (south): mt rujan, slavujevac village, arid pastures, 800 m, em68, 31.07.2008, coll. b. zlatković & n. smiljković (beou 16845). (south): preševska povija, mamince village, limestone, dry pastures, 450 m, therobrachypodietea, em58, 23.07.1998, coll. b. zlatković, n. ranđelović & v. ranđelović (beou 16843). (south): the town of bujanovac, srpska kuća village, marls, clays and sandstones, arid pastures, 421 m, en60, 31.07.2008, coll. b. zlatković & n. smiljković (beou 16846). (south): the town of vranje, buštranje village, limestone, dry pastures, 516 m, therobrachypodietea, em79, 17.05.2008, coll. b. zlatković & n. smiljković (beou 16836). (southeast): pčinja river gorge, brnjare village, marls, shales and sandstones, ephemeral pastures of dry sand area, ornithopodituberarietum guttatae, 510 m, em79, 28.06.2004, coll. b. zlatković & m. jušković (beou 16847). (metohija): the town of prizren, našec village, limestone, thermophilous pastures on shallow substrate, 390 m, thero-brachypodietea, dm77, 30.06.1996, coll. b. zlatković (beou 16837). (metohija): the town of prizren, thermophilous pastures on shallow substrate, limestone, 371 m, dm77, 30.06.1996, coll. b. zlatković (beou 16844). biologica nyssana 5 (2)  december 2014: 123-129 zlatković, b., bogosavljević, s.  report for the new floristic data… 128 mediterranean, therophyte species recorded in serbia for the first time near sredska (k r i v o š e j et al., 1993-1994), and later also in gorge of prizrenska bistrica (z l a t k o v i ć et al., 2014) at kosovo and metohija. supported by influence of warm climate from the adriatic and aegean mediterranean areas, following the valleys of larger rivers, this species has spread its range significantly in metohija, southern, southeastern and eastern serbia (fig. 1e). this species is a constituent of vegetation of thermophilous pastures and stony ground, appearing at lower altitudes. typhaceae 16. typha laxmannii lepech. (northeast): the town of zaječar, rgotina village, rgotsko lake, 185 m, fp07, 03.08.2014, coll. s. bogosavljević (hmn 9255). the first record of this species in serbia was from vojvodina (budak, 1975, 1986), while recently it was recorded at several localities in eastern and southeastern serbia (zlatković et al., 2007). at the locality in northeastern serbia typha laxmannii forms a relatively abundant population inhabiting shallow pools and ponds that dry out in summer months. the habitats are anthropogenous in origin, situated in immediate vicinity of stone quarries or gravel excavation pits. in some cases it may also appear along the stream that flows into the accumulation of artificial lake near the settlement rgotina. acknowledgements. the ministry of education, science and technological development of the republic of serbia (grant 173030) supported this research. references adamović, l., 1898: die vegetationsformationen ostserbiens. englers botanische jahrbuch, 26 (2): 124-218. adamović, l., 1899: neue beiträge zur flora von serbien. botanisches centralblatt, 78: 1-8. adamović, l., 1907: sitniji prilozi flori srbije. – nastavnik, 18 (3-4): 95-98. adamović, l., 1908: neue glieder der serbischen flora. allgemeine botanische zeitschrift, 14 (6): 85-87. anastasiu, p., memedemin, d., 2010: conyza sumatrensis: a new alien plant in romania. botanica serbica, 36 (1): 37-40. brownsey, r., kyser, g. b., ditomaso, j. m., 2013: stinkwort is rapidly expanding its range in california. california agriculture, 67 (2): 110– 115. budak, v., 1975: typha laxmannii lepech. in: sarić, m. & diklić, n. 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(eds). new floristic records in the balkans: 6. phytologia balcanica, 13(3): 433-455. zlatković, b., bogosavljević, s., smiljković, n., ranđelović, v. 2014: report on the new and insufficiently studied taxa in the flora of serbia. biologica nyssana, 5 (1): 63-69. đekić, v., milivojević, j., branković, s.: the interaction of genotype and environment on yield and quality components in triticale. biologica nyssana, 9 (1). september, 2018: 45-53. biologica nyssana 9 (1) ⚫ september 2018: 45-53 đekić et al. ⚫ the interaction of genotype and environment on yield… 45 original article received: 29 january 2018 revised: 29 mart 2018 accepted: 27 june 2018 the interaction of genotype and environment on yield and quality components in triticale vera đekić1, jelena milivojević1, snežana branković2 1small grains research centre, save kovačevića 31, 34000 kragujevac, serbia 2university of kragujevac, faculty of science, institute of biology and ecology, radoje domanović 12, kragujevac, serbia * e-mail: verarajicic@yahoo.com abstract: đekić, v., milivojević, j., branković, s.: the interaction of genotype and environment on yield and quality components in triticale. biologica nyssana, 9 (1). september, 2018: 45-53. the experiment was established at the small grains research centre in kragujevac, where two varieties of triticale were investigated. the highest three-year average of grain yield (4.910 t/ha), 1000-kernel weight (42.67 g) and test weight (69.85 kg/hl) were obtained by kg 20 variety. the highest three-year average value of protein content was found in the variety trijumf (13.157%). highly significant influence of the year on grain yield, 1000-kernel weight and protein content was established for investigated winter triticale cultivars by variance analysis, and significant influence of the year on the test weight. the influence of the cultivar on grain yield, 1000-kernel weight, test weight and protein content was not statistically significant. environmental conditions have had a significant effect on grain yield and quality in triticale. grain yield shows a tendency to increase in the years having a higher total amount and better distribution of rainfall during critical plant development stages. key words: grain yield, protein, test weight, triticale apstrakt: đekić, v., milivojević, j., branković, s.: interakcija genotipa i vegetacione sezone na prinos i kvalitet komponenti prinosa tritikalea. biologica nyssana, 9 (1). septembar, 2018: 45-53. eksperiment je izveden na oglednom polju centra za strna žita u kragujevcu, a istraživane su dve ozime sorte tritikalea. najveći trogodišnji prinos zrna (4.910 t/ha), masa 1000 zrna (42.67 g) i hektolitarska masa (69.85 kg/hl) ustanovljeni su kod sorte kg 20. najveća trogodišnja prosečna vrednost sadržaja proteina dobijena je kod sorte trijumf (13,157%). analizom varijanse ustanovljen je visoko značajan uticaj godine na prinos, masu 1000 zrna i sadržaj proteina i značajan za hektolitarsku masu. uticaj sorte na prinos, masu 1000 zrna, hektolitarsku masu i sadržaj proteina statistički nije bio značajan. vegetaciona sezona značajno je uticala na prinos i kvalitet prinosa zrna kod ispitivanih sorti tritikalea. prinos zrna pokazuje tendenciju povećanja u 9 (1) • september 2018: 45-53 doi: 10.5281/zenodo.1470850 biologica nyssana 9 (1) ⚫ september 2018: 45-53 đekić et al. ⚫ the interaction of genotype and environment on yield… 46 godinama koje imaju veću ukupnu količinu padavina i bolju distribuciju padavina tokom kritičnih faza razvoja biljaka. ključne reči: prinos zrna, sadržaj proteina, hektolitarska masa, tritikale introduction the yield per unit area is the result of the action of varieties fertility in interaction with environmental factors. therefore, the yield is relative term and is determined by the variety, environmental conditions and the level of applied technology. yield is largely dependent on the genetic potential, which could be defined as yield of variety, grown in conditions on which it had adapted, with adequate amounts of water and nutrients and efficient control of pests, diseases, weeds and other stresses (milovanovic et al., 1998; đekić et al., 2014). yields vary considerably primarily as a result of agro-ecological conditions during the growing season (tomasović, 2005; glamočlija et al., 2010; đekić et al., 2010; biberdžić et al., 2012; kondić et al., 2012; milovanović et al., 2012; janušauskaitė, 2013; kendal et al., 2014; đurić et al., 2016). the 1000-kernel weight is a direct component of grain yield and changes under the influence of environmental factors, but depends primarily on varietal characteristics. it indicates the grain size and is an important criterion in triticale breeding. milovanović et al. (2014) consider that the 1000 kernel weight in addition to the number of grains per grain heads is the most important criterion in breeding barley for yield increase. the 1000-grain weight, however, is not only a component of yield, but also a very important component of quality of grains (milovanović et al., 2011; đekić et al., 2016; kendal et al., 2016b). test weight is the weight of one hectoliter grain expressed in kilograms and is the oldest and still the most widely used method of evaluating the quality of the grains, because its assessment is quick and easy. test weight is based on physical measures of size and grain shape, which measures weight in the volume mass density. larger hectoliter means higher content of starch and protein in relation to the content of fiber and a space filled with air (milovanovic et al., 2006; đekić et al., 2014; kendal and sayar, 2016a; kendal et al., 2016b). the chemical composition and nutritive characteristics of triticale are result of huge number of varieties with a huge spectrum of characteristics (đekić et al., 2009; djekic et al., 2011; janušauskaitė, 2013; kendal et al., 2016b). milovanović (1993), emphasizes that different winter triticale genotypes have fluctuated within the two year period from 13.44 up to 16.42%. since the early 1970s, university of florida has conducted a triticalebreeding program. three spring-type cultivars that were selected from the cimmyt (international maize and wheat improvement center) nurseries have been released (barnett et al., 1999). considerable improvements in increasing grain yield and test weight have been made. the protein content of the three released cultivars is about 12% with 0.45% lysine on a dry matter basis. within the four year average values, milovanović et al. (1995), recorded protein content from 14.32% to 16.29% in different winter triticale genotypes, while for the winter wheat varieties, value of 13.70% was recorded. đekić et al. (2009) reported the protein content of 12.24% for the triticale variety kg 20, while variety favorit had 12.55% of dry matter. perišić et al. (2008) reported that another winter triticale variety, general, had 14.6% of crude proteins. within six year averages, djekic et al. (2011) recorded proteins from 11.23 to 13.87% in different winter triticale genotypes. in previous studies, grain protein content in triticale genotypes was reported by many researchers from 11.2% to 16.4% (milovanović et al., 1995; janušauskaitė, 2013; kendal and sayar, 2016a; kendal et al., 2016b). the triticale is considered a prospective crop for conditions of serbia, especially from the point of the global climate change which has not avoided the agricultural regions of serbia. the aim of this study was to determine the influence of semi-arid ecological environmental factors of central serbia on the winter triticale varieties and on their differences in stability and adaptability in regard to the grain yield and parameters of grain chemical quality, as well as specificity of explored varieties in regard to growing conditions. material and methods materials and field trials during the 2009/10, 2010/11 and 2011/12 growing seasons, two varieties of winter triticale were investigated (kg 20 and trijumf) at the small grains research centre in kragujevac (in central serbia). the experiments were conducted in randomized block systems, with a plot size of 50 m2 (5 m x 10 m) in five replications. the sowing was carried out using a machine with row spacing of 12.5 cm. the soil on biologica nyssana 9 (1) ⚫ september 2018: 45-53 đekić et al. ⚫ the interaction of genotype and environment on yield… 47 which the trial was conducted was uniform and well prepared. the amount of seed per square meter corresponded to 400-450 viable seeds, depending on the characteristics of varieties. it was sown in the third decade of october, with 400 kg/ha of fertilizer npk 15:15:15, which was added in the fall, while during the spring, fertilization soil was supplemented with 300 kg/ha (kan 27%n). the following properties were analyzed: grain yield (t/ha), 1000-kernel weight (g), test weight (kg/hl) and chemical quality of grain. grain yields were measured for each plot and converted to yield tons per hectare on the basis of 14% grain moisture. the total protein content in the grain was determined by infrared spectroscopy technique on the apparatus perten da 7000 (nir/vis spectro-photometer) employing non-destructive method. soil conditions the trial at the small grains research centre in kragujevac (in central serbia), was performed on soil that is characterized as smonitsa in process of degradation. the physical properties of the soil are very unfavorable, and it belongs to the type of heavy clays. according to the analysis, this is a soil of medium acidity, poor in humus (2.65%), with a substitution and total hydrolytic acidity that were quite high (ph in h2o=5.99, in kcl=4.91). the soil was moderately provided with total nitrogen (0.14% of n), it is poor in easily accessible phosphorus (p2o5≤12 mg/100 g of soil), a medium level of easily accessible potassium (k2o=14-23 mg/100 g of soil) was recorded. meteorological conditions this study was conducted over a three-year period in the šumadija region, central serbia, on a vertisol soil, at kragujevac location, 173-220 m a.s.l. (44° 22′ n, 20° 56′ e), in a temperate continental climate having an average annual temperature of 11.5 °c, typical for šumadija districts in serbia and a rainfall amount of about 550 mm. kragujevac area is characterized by a moderate continental climate, which in general is characterized by uneven distribution of rainfall by month. the data in tab. 1 for the investigated period (2009-2012) clearly indicate that the years in which the researches were conducted differed from the typical multi-year average for kragujevac region regarding the meteorological conditions. the data presented in tab. 1 for the analyzed triticale growing season (20092012) clearly suggest differences in weather conditions between the years of the study and the long-term mean for the region. the average air temperatures were by 1.26 °c, 1.5 °c and 2.1 °c lower in 2009/10, 2010/11 and 2011/12, respectively, as compared to the long-term mean, whereas the sums of rainfall were by 612.7 mm and 12.5 mm higher in 2009/10 and 2011/12 years and lower by 86.2 in 2010/11 year as compared to the long-term mean. compared to the long-term mean, total rainfall values, especially in the first year, second and third year, were considerably higher in february, april and may, whereas total rainfall in april 2010/11 decreased by 31.1 mm. given the high importance of sufficient rainfall amount during the spring months, particularly april and may, for triticale production, the distribution and amount of rainfall over the growing season 2010/11 were considerably more favorable, resulting in increased yields in this year. apart from the rainfall deficiency during the spring months and the non-uniform distribution of rainfall across months, an increase in average air temperatures was also observed. based on the fact that sufficient amounts of rainfall in these months are very important for the table 1. mean monthly air temperatures and precipitation in kragujevac, serbia (2009-2012), in relation to many years average (1960-2009) months interval x xi xii i ii iii iv v vi average mean monthly air temperature (oc) 2009/10 11.7 8.8 2.6 0.9 3.2 7.2 12.1 16.5 20.2 9.24 2010/11 10.2 11.4 2.4 0.9 0.5 7.2 12.0 15.8 20.9 9.0 2011/12 10.4 3.1 4.6 0.7 -3.7 8.1 12.9 16.1 23.0 8.4 average 12.5 6.9 1.9 0.5 2.4 7.1 11.6 16.9 20.0 10.5 the amount of precipitation (mm) 2009/10 102.6 77.5 194.2 57.0 150.5 43.3 142.2 116.7 196.7 1080.7 2010/11 86.9 27.9 50.1 29.1 48.5 20.4 20.8 65.8 32.3 381.8 2011/12 33.3 1.3 43.3 117.2 60.1 5.7 74.5 87.3 57.8 480.5 average 45.4 48.9 56.6 58.2 46.6 32.4 51.9 57.6 70.4 468.0 biologica nyssana 9 (1) ⚫ september 2018: 45-53 đekić et al. ⚫ the interaction of genotype and environment on yield… 48 successful production of cereal crops it can be concluded that the years in which the researches were conducted were not favorable for the triticale growing. statistical analysis on the basis of achieved research results, the usual variational statistical indicators, average values and standard deviations, were calculated. experimental data were analyzed by descriptive and analytical statistics using the statistics module analyst program sas/stat (sas institute, 2000) for windows. all evaluations of significance were made on the basis of the anova test at 5% and 1% significance levels. relative dependence was defined through correlation analysis (pearson's correlation coefficient), and the obtained coefficients were tested at the 5% and 1% levels of significance. results grain yield and chemical characteristics average values of grain yield, 1000-kernel weight, test weight and protein content of investigated kragujevac winter triticale varieties are presented in tab 2. the grain yield of triticale significantly varied across years, from 4.059 t/ha in 2009/10 to 5.615 t/ha in 2010/11 (tab. 2). in the first investigation year (2009/10), variety kg 20 achieved higher yield of grain (4.104 t/ha), while trijumf variety yielded 4.014 t/ha. in the second year (2010/11), the grain yield of variety kg 20 was the highest with 5.868 t/ha, while the slightly lower yield was realized by variety trijumf (5.362 t/ha). in the third year of research (2011/12), the grain yield of variety kg 20 was 4.757 t/ha, which was 320 kg more than the yield obtained by the variety trijumf. the thousand kernel weight of winter triticale grain was variable, depending on environmental conditions. thousand kernel weight in the test period was the highest in 2010/11 (45.34 g) but had significantly lower values in 2009/10 (by 5.72 g or 12.62%) and also significantly decreased in 2011/12 (by 3.91 g or 9%). average thousand kernel weight observed in the three-year period was the highest in kg 20 variety (42.67 g), while the trijumf cultivar had lower yield (41.59 g). tab. 2 presents average values for grain test weight across years and varieties. the average values for test weight in both cultivars were as follows: 70.34 kg/hl in 2009/10, 70.84 kg/hl in 2010/11 and 68.02 kg/hl in 2011/12, suggesting significant variations in test weight during the three years of the study. the average crude protein content of triticale significantly varied across years, from 12.345% in 2010/11 to 13.796% in 2010/11 (tab. 2). during all three years of investigation (2009/10, 2010/11 and 2011/12), the variety trijumf achieved higher average protein content (14.010%, 12.386% and 13.073%). table 2. average values of characteristics of investigated triticale varieties varieties 2009/10 2010/11 2011/12 average x s x s x s x s grain yield, (t/ha) kg 20 4.104 0.674 5.868 0.373 4.757 0.527 4.910 0.904 trijumf 4.014 0.732 5.362 0.669 4.544 0.437 4.640 0.815 average 4.059 0.665 5.615 0.576 4.651 0.470 4.775 0.857 1000-kernel weight, (g) kg 20 40.02 1.154 45.88 0.460 42.10 1.557 42.67 2.727 trijumf 39.22 0.549 44.80 1.000 40.76 3.038 41.59 2.990 average 39.62 0.951 45.34 0.929 41.43 2.383 42.13 2.864 test weight, (kg/hl) kg 20 70.11 3.171 70.89 1.699 68.56 2.533 69.85 2.557 trijumf 70.57 2.834 70.80 1.724 67.48 1.583 69.62 2.513 average 70.34 2.846 70.84 1.615 68.02 2.071 69.73 2.494 proteins content, (%) kg 20 13.581 0.361 12.300 0.454 12.919 0.637 12.935 0.709 trijumf 14.010 0.528 12.386 0.846 13.073 0.331 13.157 0.889 average 13.796 0.483 12.345 0.642 12.996 0.485 13.046 0.798 biologica nyssana 9 (1) ⚫ september 2018: 45-53 đekić et al. ⚫ the interaction of genotype and environment on yield… 49 analysis of variance between observed traits of triticale the analysis of yield variance of 1000-kernel weight, test weight and protein content of the tested winter triticale varieties grown at investigated in kragujevac during three growing seasons 2009/10, 2010/11 and 2011/12, are shown in tab. 3. based on the analysis of variance, it can be concluded that there were highly significant differences in grain yield in regard to the year of investigation (fexp=18.58659**), while among the investigated triticale varieties the differences were not significant. analysis of variance found highly significant effect of year on the 1000-kernel weight (fexp=34.44302**), protein content (fexp=18.00073**), and significant effect on the test weight (fexp=4.54129*). based on the analysis of variance, it can be concluded that there were no significant differences in grain yield, 1000-kernel weight, test weight and protein content in investigated triticale varieties relative to the cultivar of investigation (tab. 3). the interaction of the investigated factors (y x g) exhibited no statistically significant effect on grain yield, 1000-kernel weight, test weight and protein content (p > 0.05). discussion in regard to the tested winter triticale genotypes, variety kg 20 achieved higher yield of grain (4.910 t/ha). during the research, the values obtained in the second investigation year were significantly different from the first and third year. in year 2009/10, the lowest yield was achieved (4.059 t/ha), while the highest yield was achieved in year 2010/11 (5.615 t/ha). however, determined grain yields of triticale genotypes reported by kendal and sayar (2016a) ranged between 3.680 t/ha to 9.250 t/ha. in another study of kendal et al. (2016b), grain yield of genotypes ranged from 2.92 t/ha in 2013/14 to 6.61 t/ha in 2012/13 season (normal season). barnett et al. (2006) examined 22 genotypes of the winter and spring triticale in four geographically different locations (quincy, fl, usa; plains, ga, usa; bozeman, mt usa and aberdeen, id, usa) during two vegetation seasons. the same authors have also established the genetic diversity of yields of the tested triticale genotypes at different locations. the grain yield ranged from 4.709 t/ha in california to 6.133 t/ha in georgia. in the first year of the study (1997/98), grain yield of the tested triticale genotypes at different locations ranged from 2.507 t/ha to 7.245 t/ha, while in the second year of the research (1998/99) yield varied in the range of 2.341-8.033 t/ha. in these studies, a significantly higher yield of triticale in northern locations (aberdeen, bozeman) was found in relation to the southern (quincy, plains). the average grain yield values differed statistically significantly between the investigated triticale genotypes, between the investigated varieties and between the investigated sites (barnett et al., 2006). kendal et al. (2016b) have indicated that the yield of triticale genotypes in the 2012/13 season have ranged from 4.66 t/ha to 7.67 t/ha. the average grain yield was found to be 6.61 t/ha in this season. milovanovic et al. (2001) suggested that spring triticale is more prone to the negative impact of table 3. anova values of characteristics in investigated triticale varieties effect of year on the traits analyzed traits mean sqr effect mean sqr error f (2,27) p-level grain yield (t/ha) 6.167350 0.331817 18.58659** 0.000008 1000-kernel weight (g) 85.47100 2.481519 34.44302** 0.000000 test weight (kg/hl) 22.69675 4.997861 4.54129* 0.019948 protein content (%) 5.280500 0.293349 18.00073** 0.000011 effect of cultivar on the traits analyzed traits mean sqr effect mean sqr error f (2,27) p-level grain yield (t/ha) 0.545940 0.740994 0.736768 0.397987 1000-kernel weight (g) 8.640333 8.189382 1.055066 0.313136 test weight (kg/hl) 0.420083 6.425560 0.065377 0.800060 protein content (%) 0.369408 0.646858 0.571080 0.456141 effect of the year x cultivar interaction traits mean sqr effect mean sqr error f (2,24) p-level grain yield (t/ha) 0.114417 0.341012 0.335523 0.718264 1000-kernel weight (g) 0.182333 2.416500 0.075453 0.927542 test weight (kg/hl) 1.522583 5.478209 0.277935 0.759751 protein content (%) 0.083670 0.307654 0.271961 0.764201 biologica nyssana 9 (1) ⚫ september 2018: 45-53 đekić et al. ⚫ the interaction of genotype and environment on yield… 50 abiotic stress than winter triticale. milovanović et al. (2011) have observed significantly (p < 0.05) higher yield stability of hybrids compared to lines for triticale crops. the enhanced yield stability represents a major step forward, facilitating coping with the increasing abiotic stress expected from the predicted climate change. achieved statistically significantly higher yields in 2010/11 were primarily the result of heavy rainfall and their good distribution as well as favorable air temperatures during the vegetation period (tab. 1). đekić et al. (2016) stated in their research that the air temperatures and the rainfall amount and distribution during the triticale growing season have the greatest impact on high yields and grain quality. the significant difference in triticale grain yield was also indicated by barnett et al. (2006), janušauskaitė (2013) and kendal et al. (2016a). for the southeastern anatolia region, the results of the study of kendal et al. (2014), demonstrated that the triticale grain yield is higher than those of durum and bread wheat varieties under more extreme growing conditions of the kiziltepe region. milovanovic et al. (2001) stated that in the domestic production conditions, higher yields are achieved by varieties with the shorter growing season because they manage to form the largest part of the yield before the incidence of high temperatures. in this study, in both years, the triticale was not exposed to extremely high temperatures so early growth did not come into its own. variety kg 20, compared to the other genotype trijumf, in the three-year period, had significantly higher value for the thousand kernel weight (42.67 g). the thousand kernel weight of triticale significantly varied across years, from 39.62 g in 2009/10 to 45.34 g in 2010/11. highly significant influence of the year on 1000-kernel weight was established in investigated winter triticale varieties by variance analysis. milovanović (1993) stated that the 1000 grain weight, however, is not only a component of yield but also a very important component of quality of grains. milovanovic et al. (1998) reported that good quality grain needs to have the 1000 grain weight of 45-50 g. kendal et al. (2014) have found that the thousand kernel weight in the tested triticale varieties ranged from 32.9 to 42.7 g in 2010/11. the same authors indicated that the new spring varieties are more suitable than wheat for more extreme growing conditions of southeastern anatolia region. heavier grains will have more starch and less protein (filipčev et al., 2005). a number of authors (đekić et al., 2010; milovanović et al., 2012; janušauskaitė, 2013; kendal et al., 2014, 2016b; đurić et al., 2016) have underlined that 1000-grain weight is a cultivarspecific trait, with considerably higher variations being observed among genotypes than among treatments or environmental factors. previously, many researchers have reported that the thousand kernel weight values of triticale genotypes have ranged from 23.9 g to 54.9 g. (janušauskaitė, 2013; đekić et al., 2014; kendal et al., 2014, 2016b; kendal and sayar, 2016a). test weight depends on many factors such as grain filling and shape, chemical composition, the content of impurities and moisture. low test weight may be due to pre-harvest sprouting (milovanović, 1993). higher test weight means the higher content of starch and protein in relation to the content of fiber and a space filled with air (milovanović et al. 1995; filipčev et al. 2005). the genotype kg 20 had an extreme value (70.89 kg/hl) in the second year (2010/11). all three years are different in regard to the test weight. particularly in the third year (68.02 kg/hl), genotypes had significantly lower test weight compared to other years. based on the analysis of variance, it can be concluded that there were significant differences in test weight in regard to the year of investigation, which is in accordance with the results obtained by đekić et al. (2016). kendal et al. (2016b) recorded an average value of test weight between 73.0 up to 78.6 kg/hl during two growing seasons (2012/13-2013/14) in 25 genotypes, including 20 advanced lines (from cimmyt), three triticale varieties (presto, tacettinbey and karma) and two candidate lines (g15 and g10). barnett et al. (2006) have analyzed the yield and hectoliter mass of the 22 triticale genotypes in four ecologically diverse geographical locations (quincy, fl, usa (agc)=30on84ow, approximate elevation (ae)=58 m), plains, ga, usa (agc=32on84ow, ae=76 m), bozeman, mt usa (agc=45on 111ow, ae=1458 m), and aberdeen, id, usa (agc=42on 112ow, aberdeen, id, usa (agc=42on 112ow, ae=1360 m). field trials were carried out with the winter triticale, which was sowed during a two year period, 1997 and 1998, while the spring sowing of triticale was carried out in 1998 and 1999. the average value of the hectoliter mass in these studies ranged from 68.3 kg/hl to 75.0 kg/hl in the florida area. winter forms of triticale had higher average test weight (75.7 kg/hl) compared to the spring forms (73.4 kg/hl). statistically significant differences between the tested triticale genotypes and investigated sites were determined by analyzing variance of average values of the test weight. in the study reported here, considerable genetic diversity in triticale for test weight at diverse geographic areas was found indicating that selection for specific environments would be possible. generally, the test weight of triticale ranged from 63 to 72 kg/hl, while in dehulled triticale values were up to 80 kg/hl (barnett et al., biologica nyssana 9 (1) ⚫ september 2018: 45-53 đekić et al. ⚫ the interaction of genotype and environment on yield… 51 2006; janušauskaitė, 2013; đekić et al., 2014; kendal et al., 2014; kendal and sayar, 2016a). protein content differed very significantly between years and the average for all genotypes was higher in 2009/10 (13.796%) compared to 2010/11 (12.345%). kendal et al. (2014) have recorded an average value of protein content between 13.3 and 14.7% in the three different triticale genotypes (tacettinbey, karma 2000 and presto), during the growing season 2010/11. perišić et al. (2008) reported 14.6% of crude proteins in winter triticale. djekic et al. (2011) recorded values ranging from 11.23 to 13.87% of proteins in different winter triticale genotypes. in different spring triticale genotypes, kendal et al. (2016b) recorded two year (2012/13 and 2013/14) average from 12.9 to 18.4% of proteins. during the 2012/13 season, which was considered a normal season, protein content ranged from 12.0% to 14.9%. during the 2013/14 season, the protein content ranged from 16.3% to 20.8%. the protein content in triticale genotypes, as reported by researchers kendal and sayar (2016a), ranged from 12.7% to 14.7% and by researcher janušauskaitė (2013), from 11.4% to 14.3% proteins. many authors have pointed that spring triticale varieties have more proteins compared to winter varieties (djekic et al., 2011a; kendal and sayar, 2016a; kendal et al., 2014, 2016b). humid 2010/11 was more favorable year for protein synthesis. our results are consistent with the results obtained by đekić et al. (2016) and milovanovic et al. (2001), where the authors stated that the growing conditions in the observed years had a very significant impact on protein content. analysis of variance showed highly significant effect of year on the protein content (p<0.01). realized average values for these characteristics in the study were slightly higher than the values obtained by djekic et al. (2011b). janušauskaitė (2013) has recorded an increase of the yield of triticale grain achieved by fertilization by 35.7%, compared with the unfertilized control treatment. the analysis of variance of the average grain yield over the four years showed that year significantly influenced the grain yield. the influence of n rate on the grain yield was significant in basic fertilization almost in all the cases; however, the influence of n splitting regime on the yield was insignificant. utilization of fertilizers and certain supplements on extremely acid soils in certain years, particularly those less favorable for production, almost certainly had different effect on grain filling, resulting in diverse relationships between productive and qualitative traits (biberdžić et al., 2012; đekić et al., 2014; janušauskaitė, 2013). presented results confirm the opinion of many authors that the traits analyzed are genetically determined, but strongly modified by the nutrient status of the environment as well as weather conditions (tomasović, 2005; glamočlija et al., 2010; kondić et al., 2012; milovanović et al., 2012; đurić et al., 2016; kendal et al., 2016a,b). the study results indicate that under the influence of the year, highly significant differences are recorded in all of the investigated traits. also, kendal and sayar (2016a) and kendal et al. (2016b) have indicated that in the assessment of genotypes more locations and years could increase the reliability of plant breeding programs and they have drawn attention to the fact that multi-location trials were more important than multi-year trials on the same location in determining performance of tested genotypes in terms of investigated traits in plant breeding programs. conclusion based on the results obtained during the three-year investigation of the two winter triticale varieties, it can be concluded that the highest three-year average and the best adaptability in semi-arid climate of central serbia for properties grain yield and 1000kernel weight were recorded for the variety kg 20. highly significant influence of the year on grain yield, 1000-kernel weight, test weight and protein content was established in investigated winter triticale varieties by variance analysis, while genotype influence on grain yield, 1000-kernel weight, test weight and protein content was not statistically significant. acknowledgements. investigations presented in this paper are part of the project tp 31054 "development of new cereals cultivation technologies on acid soils by usage of modern biotechnology", financed by the ministry of education and science of republic of serbia. references barnett, r.d., blount, a.r., pfahler, p.l. 1999: benefits of the uf/ifas small grain breeding program to florida and the southeastern united states. soil and crop science society of florida proceedings, 58: 20-23. barnett, r.d., blount, a.r., pfahler, p.l., bruckner, p.l., wesenberg, d.m., johnson, j.w. 2006: environmental stability and heritability estimates for grain yield and test weight in triticale. journal of applied genetics, 47: 207-213. biberdžić, m., jelić, m., deletić, m., barać, s., stojković, s. 2012: effects of agroklimatic conditions at trial locations and fertilization on grain yield of triticale. research journal of agricultural science, 44(1): 3-8. biologica nyssana 9 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agroekological conditions of banja luka. genetics, 44(2): 419-428. milovanović, m. 1993: investigation of yield and technological traits of grain of the intergenus hybrids triticale (x triticosecale wittmack). rewiew of research work at the faculty of agriculture, zemun-belgrade, 38(2): 71-82. milovanović, m., kuburović, m., stojanović, s., ognjanović, r. 1995: some recent results of winter triticale breeding in kragujevac. proceedings of 1st balkan symposium “breeding and cultivation of wheat, sunflower and legume crops”, albena-iws, bulgaria, 125-129. milovanovic, m., kuburovic, m. stojanovic, s., kovacevic, b. 1998: some results of investigations and winter 6x triticale breeding in kragujevac. proceedings, internernational symposium “breeding of small grains”, kragujevac, yugoslavia, 225-231. milovanovic, s.m., rigin, v.b., xynias, n.i. 2001: genetic and breeding studies on triticale (x triticosecale wittmack). in: genetics and breeding of small grains. ari serbia, belgrade, 235-298. milovanovic, m.., perisic, v., staletic, m. 2006: favorit-new vinter triticale cultivar for intensive growing conditions. a collection of articles from the technical vocational college in pozarevac, 1-2: 93-97. milovanović, m., staletić, m., đekić, v., nikolić, o., luković, k. 2011: seed production and contribution of kg varieties to biodiversity of small grains in the period 2006-2010. 01.-02. december 2011, beograd, economics of agriculture, book ii, 58(cб/si-1): 103-111. milovanović, m, perišić, v., staletić, m., cvijanović, d., đekić, v. 2012: recent results of triticale breeding in kragujevac. proceedings, 47th croatian and 7th international symposium on agriculture, 13.-17. februar, opatija, croatia, 294-298. milovanovic, m., perishic, v., staletic, m., đekic, v., nikolic, o., prodanovic, s., lukovic, k. 2014: diallel analysis of grain number per spike in biologica nyssana 9 (1) ⚫ september 2018: 45-53 đekić et al. ⚫ the interaction of genotype and environment on yield… 53 triticale. bulgarian journal of agricultural science, 20(5): 1109-1115. perišić, v., milovanović, m., bratković, k. 2008: new cultivar of winter triticale-general. a collection of articles from the technical vocational college in pozarevac, 1-2, 59-64. tomasović, s. 2005: a newly registered winter triticale developed by the zagreb bc institute. annual wheat newsletter, 51: 23-24, kansas state university, usa. anticancer compounds from medicinal plants biologica nyssana 4 (1-2)  december 2013: 65-69 nikolova et al.  comparison of flavonoid profiles of cultivated plants… 65 original article comparison of flavonoid profiles of cultivated plants of achillea asplenifolia, achillea collina and cultivar ”proa” milena nikolova 1* , antonina vitkova 1 , malina delcheva 1 , aglika edreva 2 , emiliya gesheva 2 1 department of applied botany, institute of biodiversity and ecosystem research 23, acad. g. bonchev, str., 1113 sofia, bulgaria 2 institute of plant physiology and genetics, 1113 sofia, bulgaria * e-mail: milena_n@bio.bas.bg abstract: nikolova, m., vitkova, a., delcheva, m., edreva, a., gesheva, e.: comparison of flavonoid profiles of cultivated plants of achillea asplenifolia, achillea collina and cultivar ”proa”. biologica nyssana, 4 (1-2), december 2013: 65-69. flavonoid aglycone profiles of cultivated plants of achillea collina, a. asplenifolia and cultivar "proa" presented with different origins were compared. the evaluation was undertaken to determine intraspecific variability of flavonoid profiles when the plants are grown in the uniform conditions. eight flavonoid aglycones were detected by thin layer chromatography and comparison with known compounds. highly methylated quercetagetin derivatives predominate in the exudates. the flavonoid profiles of a. colina consist mainly of quercetagetin 3,6,4'-trimethyl ether (centaureidin), 6-hydroxykaempferol 3,6,4'-trimethyl ether and quercetagetin 3,6,7,4'-tetramethyl ether (casticin) while the profiles of a. asplenifolia of quercetagetin 3,6,7,3',4'-pentamethyl ether (artemetin), casticin and scutellarein 6,4'-dimethyl ether. in the profiles of a. asplenifolia quercetagetin 3,6,4'-trimethyl ether was not found in the extracts of one of the two origins. the extracts of cultivar exhibited a less diversified flavonoid profile with main component centaureidin. no significant difference was found in the flavonoid profiles of the extracts of the both origins of the cultivar “proa” and a. collina. key words: chemotaxonomy, external flavonoids, intraspecific variability, tlc. introduction the species of achillea millefolium group are valuable plants with wide application in the phytotherapy and cosmetics. six achillea species of the group occur in bulgaria a. collina j. becker ex reichenb, a. millefolium l., a. setaceae waldst. et kit., a. asplenifolia vent., a. pannonica scheele, a. distans waldst. et kit. ex wild. as an extension of previous study of achillea genus in bulgaria (s a u k e l et al., 2003, v i t k o v a et al., 2005) in the present research we compare the profiles of surface flavonoid aglycones of a. collina, a. asplenifolia and cultivar "proa". the most widespread species is tetraploid a. collina (4x), which probably has a hybrid origin of a. setacea (2x) and a. asplenifolia (2x). it is spread from the sea level up to 1800 m altitude, grows both in xerothermal and the mesophilic places. recently diploid achillea asplenifolia was established for bulgaria in herbaceous habitats in antropogenic influenced phytocenoses from 500 to 600 m a.s.l. only two populations have been detected in the territory of the county – near the village of opizvet and village katina, sofia region. the plants of the last population characterized by a combination of characteristics of a. asplenifolia and. a. roseoalba (v i t k o v a et al., 2005). cultivar achillea collina 11 th sfses • 13-16 june 2013, vlasina lake 4 (1-2) • december 2013: 65-69 biologica nyssana 4 (1-2)  december 2013: 65-69 nikolova et al.  comparison of flavonoid profiles of cultivated plants… 66 "proa" represents a tetraploid, rich in proazulenes (k a s t n e r et al., 1992). the examined in the present study species have been objects of phytochemical analysis regarding different secondary metabolites – sesquiterpene lactones, flavonoids, essential oils (k o n a k c h i e v et al., 2005, t o d o r o v a et al., 2005, t r e n d a f i l o v a et al., 2006, t r e n d a f i l o v a et al., 2007). the species of achillea including a. asplenifolia and a. collina have been extensively studied for content of surface flavonoids (v a l a n t v e t s c h e r a & w o l l e n w e b e r 1988, i v a n c h e v a & s t a n c h e v a , 1996, v a l a n t v e t s c h e r a & w o l l e n w e b e r 1996, v a l a n t v e t s c h e r a & w o l l e n w e b e r , 2001a). external flavonoids attract attention except for their usefulness in taxonomic studies (w o l l e n w e b e r & s c h n e i d e r , 2000, v a l a n t v e t s c h e r a & w o l l e n w e b e r 2001b, v a l a n t v e t s c h e r a et al., 2003) but also with their ecological role (o n y i l a g h a & g r o t e w o l d , 2004). the aim of present study was to compare flavonoid aglycone profiles of cultivated plants of achillea collina, a. asplenifolia and cultivar "proa"presented with different origins. the evaluation was undertaken to determine intraspecific variability of flavonoid profiles when the plants are grown in the uniform conditions. material and methods plant material. the plant material included two wild species (achillea collina and a. asplenifolia) and cultivar “proa”. each species was presented by two populations. seeds of both species were collected from their natural habitats. the material of achillea collina were collected from vitosha mountain and village gorni lozen, sofia region and those of a. asplenifolia from village bezden, sofia region and village katina, sofia region. the seeds of cultivar proa have german and poland origin. seedlings were produced in a greenhouse and then transferred to the experimental field of the institute of plant physiology and genetics, near sofia, 570 m a.s.l., in 2009. all samples are collected at full flowering stage. preparation of acetone exudates. air-dried, but not ground (1g) plant material was briefly (2-3 min) rinsed with acetone at room temperature to dissolve the lipophilic components accumulated on the surface. the obtained acetone filtrate was then dried using a rotary-evaporator to give a crude extract which was suspended in meoh and then subjected on tlc. thin layer chromatographic analysis. the acetone exudates were screened for surface flavonoids by tlc analysis. three tlc sorbents and several mobile phases were used for the analysis of the flavonoid exudates. toluene-dioxan-acetic acid (95:25:4, v/v/v) was applied for the development of the aglycones mixture on silica gel plates kiselgel 60 f254 (10x20 cm, 0.2 mm layer). toluene-methylethylketone-methanol (60:25:15, v/v/v); toluene-petrol ether-methylethylketonemethanol (60:30:10:5, v/v/v/v) and toluenemethylethylketone-methanol (30:20:15, v/v/v) were used for dc-alufolien polyamid 11 f254 plates (10x20 cm, 0.15 mm layer). acetic acid–water (30:70, v/v) was used for cellulose plates dcalufolien cellulose 5552 (10x20 cm, 0.1 mm layer). chromatograms were viewed under uv light before and after spraying with ‘‘natural product reagent a”, 1% solution of diphenylboric acid 2-aminoethyl ester complex in methanol. the identification of the compounds was achieved by co-chromatography with authentic markers obtained from prof. eckhard wollenweber. results and discussion cultivated plants of a. collina, a. asplenifolia and cultivar "proa" were analyzed for their profiles of surface flavonoid aglycones. four individuals from each origin were studied. flavonoid profile of each individual was examined inflorescences and stem areas (leaves and stem) separately. total 48 extracts were comparatively analyzed for content of surface flavonoid aglycones. eight flavonoid aglycones were identified by thin layer chromatography and comparison with known compounds (fig. 1). polymethoxy derivatives of 6hydroxyflavonol and 6-hydroxyflavone in various combinations predominate in the exudates (table 1). the simple flavonoids (apigenin and luteolin) are rather accumulated in the inflorescences than in the exudates of stem areas. the extracts of cultivar "proa“ exhibited a less diversified flavonoid profile with the main flavonoid quercetagetin 3,6,4'trimethyl ether (5) in all studied parts. quercetagetin 3,6,7,4’-tetramethyl ether (6) was detected in the stem areas in trace. the main flavonoid of the extracts of a. colina was quercetagetin 3,6,4'-trimethyl ether (centaureidin) (5). this result indicates the closeness between cultivar proa and a. colina. additionally of the extracts of a. collina 6-hydroxykaempferol 3,6,4'-trimethyl ether (3), quercetagetin 3,6,7,3',4'pentamethyl ether (7) and scutellarein 6,4'-dimethyl ether (8) were detected. the flavonoid quercetagetin 3,6,7,4’-tetramethyl ether (6) that detected in trace amounts in the cultivar while in the extracts of a. collina is presented in large quantity. flavonoid biologica nyssana 4 (1-2)  december 2013: 65-69 nikolova et al.  comparison of flavonoid profiles of cultivated plants… 67 profiles of a. asplenifolia consisted predominantly of quercetagetin 3,6,7,4'-tetramethyl ether (casticin) (6), quercetagetin 3,6,7,3',4'-pentamethyl ether (artemetin) (7) and scutellarein 6,4'-dimethyl ether (8). the identified compounds are in accordance with the data previously reported for these species (v a l a n t -v e t s c h e t a & w o l l e n w e b e r , 1988, t r e n d a f i l o v a et al., 2007). ooh 2 1 r r r ho o 3 (1,2,8) r1=h, r2=oh apigenin (1) r1=oh, r2=oh luteolin (2) r1=h, r2=och3, r3=och3 scutellarein 6,4'-dimethyl ethers (8) r r rr 1 2 oh 3 4 o r 5 o (3-7) r1=h, r2=och3,r3=och3, r4=och3, r5=oh 6-hydroxykaempferol 3,6,4'-trimethyl ether (3) r1=oh, r2=och3,r3=och3, r4=och3, r5=och3 quercetagetin 3,6,7-trimethyl ether (4) r1=oh, r2=oh,r3=och3, r4=och3, r5=och3 quercetagetin 3,6,4'-trimethyl ether (5) r1=oh, r2=och3,r3=och3, r4=och3, r5= och3 quercetagetin 3,6,7,4'-tetramethyl ether (6) r1= och3, r2=och3,r3=och3, r4=och3, r5= och3 quercetagetin 3,6,7,3',4'-pentamethyl ether(7) fig. 1. structures of the identified flavonoid aglycones (1-8) individual variability of the flavonoid composition was observed regarding flavonoids that are presented in lesser amounts. no significant differences in the flavonoid profiles of the extracts of the both origins of cultivar proa. quantitative differences were observed in the accumulation of flavonoids in the profiles of a. collina and a. asplenifolia from different origins. furthermore in the profiles of a. asplenifolia from the population of village katina quercetagetin 3,6,4'-trimethyl ether was not detected as well as the flavonoid quercetagetin 3,6,7-trimethyl ether which was abundant in the same population was found in trace in the individuals of the population of village bezden. it is possible that these quality differences of flavonoid profiles are due to large quantitative differences that tlc analysis recognizes as qualitative. also must be taken into account that there are data that the population of v. katina showed combined features of a. asplenifolia and a. roseoalba (v i t k o v a et al., 2005). the results of flavonoid variability of a. asplenifolia were consistent with conclusions of dagnon et al., 2011. the authors reported on the basis of essential oils pattern high index of similarity (94.5%) for two populations of a. collina and less similar (82.3%) for a. asplenifolia populations. more data are needed to establish whether the plants of this population deserve an independent taxonomic rank. conclusion in conclusion the comparative analysis of flavonoid profiles of examined species showed that a. asplenifolia has more variable flavonoid composition in comparison to a. colina and cultivar "proa". the extracts of the last showed the most constant and simple flavonoid profile. the results obtained in this study indicate that surface flavonoids are a reliable taxonomic feature in the target species. references dagnon, s., konakchiev, a., vitkova, a., doncheva, m., edreva, a. 2011. application of pattern recognition methods to discrimination of achillea species of bulgarian origin. international conference “food science, engineering and technologies 2011”, 1415.10.2011, university of food technologies – plovdiv. 58: 403-408. ivancheva, s., stancheva, b. 1996: exudate flavonoid aglycones of achillea sp. sect. millefolium and sect. ptarmica. phytologia balcanica, 2: 102-105. kastner, u., glasl s., jurenitsch j., kubelka w., 1992: isolation and structure elucidation of the main proazulenes of the cultivar achillea collina “proa”. planta medica, 58:718 biologica nyssana 4 (1-2)  december 2013: 65-69 nikolova et al.  comparison of flavonoid profiles of cultivated plants… 68 table1. flavonoid aglycones in the samples of achillea species samples flavonoid aglycones 1 2 3 4 5 6 7 8 cultivar proa, origin germany inflorescences 1 x x xxx inflorescences 2 tr x xxx inflorescences 3 x x xxx inflorescences 4 x x xx stem areas 1 xxx tr stem areas 2 xxx tr stem areas 3 xxx tr stem areas 4 xx tr cultivar proa, origin poland inflorescences 1 xx xx xxx inflorescences 2 tr tr xxx inflorescences 3 xx xx xx inflorescences 4 xx xx xxx stem areas 1 tr xxx stem areas 2 tr xxx stem areas 3 x x xx stem areas 4 x x xxx a.collina, origin gorni lozen, sofia region inflorescences 1 xx x x x x inflorescences 2 xx xx x xx x inflorescences 3 x x x x tr inflorescences 4 xx xx x x tr tr stem areas 1 x x x stem areas 2 x xx x stem areas 3 tr tr x xx x stem areas 4 x xx x tr tr a.collina, origin vitosha mountain inflorescences 1 xx xx tr tr x x inflorescences 2 xx xx tr tr tr tr inflorescences 3 xx xx tr xx x x x inflorescences 4 xx xx tr xx tr tr tr stem areas 1 x tr xx x x x stem areas 2 x tr xxx x x x stem areas 3 x tr xx x x x stem areas 4 x x xxx x tr tr a. asplenifolia, origin bezden, sofia region inflorescences 1 tr x tr x tr xx xx inflorescences 2 tr x x tr xx xx inflorescences 3 x x xx xx xx inflorescences 4 xx xx tr xx tr tr stem areas 1 x x xx xx stem areas 2 tr x xx xx stem areas 3 x x tr tr stem areas 4 tr xx x xx xx a. asplenifolia, origin katina, sofia region inflorescences 1 xx xx xx x xx xx inflorescences 2 x x tr x xx xx inflorescences 3 x x x x xx xx inflorescences 4 xx xx tr x xx xx stem areas 1 xx x xx xx stem areas 2 x x xx xx stem areas 3 x x xx xx stem areas 4 x x xx xx legend: apigenin (1), luteolin (2), 6-hydroxykaempferol 3,6,4'-trimethyl ethers (3), quercetagetin 3,6,7-trimethyl ethers (4), quercetagetin 3,6,4'-trimethyl ethers (5), quercetagetin 3,6,7,4'-tetramethyl ethers (6) quercetagetin-3,6,7,3',4'-pentamethyl ethers (7), scutellarein 6,4'-dimethyl ethers (8), tr. – trace konakchiev, a., mikhova, b., todorova, m., najdenski, h., tzvetkova, i., vitkova, a., duddeck, h. 2005: composition of the esentiol oil of achillea asplenifolia vent. from bulgaria. jurnal of essential oilbearning plants, 8 (3):318-323 onyilagha, j.c., grotewold, e. 2004: the biology and structural distribution of surface flavonoids. biologica nyssana 4 (1-2)  december 2013: 65-69 nikolova et al.  comparison of flavonoid profiles of cultivated plants… 69 recent research developments in plant science, 2: 53-71. saukel, j., anchev, m., guo, y.p., vitkova, a., nedelcheva, a., goranova, v., konakchiev, a., lambrou, m., nejati, s., rauchensteiner f., erendorfer f. 2003: comments on the biosystematics of achillea (asteraceae – anthemideae) in bulgaria. phytologia balcanica, 9(3): 361-400; todorova, m., mikhova, b., trendafilova, a. vitkova, a., duddeck, h., anchev, m. 2006: sesquiterpene lactones from achillea asplenifolia. 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systematics and ecology, 16 (7-8): 605-614. valant-vetschera, k., wollenweber, e. 2001b: exudate flavonoid aglycones in the alpine species of achillea sect. ptarmica: chemosystematics of a. moschata and related species. biochemical systematics and ecology, 29: 149-159. valant-vetschera, k.m., fischer, r., wollenweber, e. 2003: exudate flavonoids in species of artemisia (asteraceae-anthemideae): new results and chemosystematic interpretation. biochemical systematics and ecology, 31: 487498 vitkova, a., anchev, m., goranova, v., todorova, m., konakchiev, a. 2005: achillea millefolium group (asteraceae) in bulgaria. pharmacie, 52(1-2): 60-63. wollenweber, e., schneider, h. 2000: lipophilic exudates of pteridaceae chemistry and chemotaxonomy. biochemical systematics and ecology, 28: 751-777. nikolic et al. 2021, biologica nyssana 12(1) 12 (1) september 2021: 23-32 doi: 10.5281/zenodo.5522967 variability of headspace volatiles in native population of abies x borisii-regis from the central rhodopes original article jelena s. nikolić department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, niš, serbia jelenanikolic9311@gmail.com (corresponding author) snežana č. jovanović department of chemistry, faculty of sciences and mathematics, university of niš, višegradska 33, niš, serbia coka.sslagalica@gmail.com bojan zlatković department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, niš, serbia bojanzlat@yahoo.com gordana s. stojanović department of chemistry, faculty of sciences and mathematics, university of niš, višegradska 33, niš, serbia gordanastojanovic2411960@gmail.co zorica mitić department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, niš, serbia saraczorica@gmail.com received: march 31, 2021 revised: july 05, 2021 accepted: july 07, 2021 abstract: the present study investigates variability of headspace needle volatiles in native population of abies x boisii-regis (king boris fir) from the central rhodopes (southern bulgaria) as well as the relationships among this and seventeen previously studied fir populations from the presumed zones of natural hybridization in the balkan peninsula. according to multivariate statistical analyses, all four populations identified as a. x borisii-regis from southern bulgaria, northern and central greece included volatile profiles of both supposed parent species (a. alba and a. cephalonica), where profile frequencies changed clinally along the latitudinal gradient. considering that one of the indicators of the presence of a hybrid zone is the clinical variation of all or a larger number of characters located in the same area, the observed geographic distribution of volatile entities supports the hypothesis that studied a. x borisii-regis populations are of secondary origin due to hybridization between a. alba and a. cephalonica. key words: abies x borisii-regis, rhodopes, headspace, needle volatiles, chemodiversity apstract: varijabilnost hedspejs isparljivih jedinjenja u prirodnoj populaciji abies x borisii-regis sa centralnih rodopa ovo istraživanje ispituje varijabilnost hedspejs isparljivih jedninjenja četina prirodne populacije abies x borisii-regis (jela kralja borisa) sa centralnih rodopa (južna bugarska), kao i odnose između ove i 17 prethodno istraživanih populacija jela iz pretpostavljene zone prirodne hibridizacije na bakanskom poluostrvu. na osnovu multivarijantnih statističkih analiza, sve četiri populacije identifikovane kao a. x borisii-regis iz južne bugarske, severne i centralne grčke uključivale su ispaljive profile obe pretpostavljene roditeljske vrste (a. alba and a. cephalonica), gde su se učestalosti profila klinalno menjale po gradijentu georgrafske širine. uzimajući u obzir da je jedan od pokazatelja prisustva hibridne zone klinalno variranje svih ili većeg broja karaktera lociranih na istom području, dobijena geografska distribucija isparljivih entiteta podržava hipotezu da su ispitivane populacije a. x borisiiregis sekundarnog porekla nastale hibridizacijom a. alba i a. cephalonica. ključne reči: abies x borisii-regis, rodopi, hedspejs, isparljiva jedinjenja četina, hemodiverzitet introduction conifers of the genus abies mill. (pinaceae) play an important ecological role in forest ecosystems of the northern hemisphere (liepelt et al., 2010). most of the abies species are in the temperate zone, with a few of them on high mountains in subtropical areas (xiang et al., 2007). the mediterranean area is considered as one of the abies distribution centers with about eight species depending on particular classification scheme. mediterranean abies spp. are weakly reproductively isolated and relatively easily hybridize in natural and artificial conditions as shown by crossability studies (kormutak, 1985; 2004; moulalis, 1986). © 2021 nikolić et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 23 on the balkan peninsula, there are two native species of the section abies: a. alba mill. (silver fir), widely distributed across central europe and some parts of southern and eastern europe, and a. cephalonica loudon (greek fir), endemic to the mountain massifs of central and southern greece (panetsos, 1975). except them, a. x borisii-regis matff. (king boris fir) was described from the central rhodopes in bulgaria by mattfeld (1926) as a hybrid between a. alba and a. cephalonica. subsequently, turrill (1937) considered as equally possible that king boris fir is an ancient lineage from which a. alba diversified towards the north and a. cephalonica towards the south. in addition, there are a few more mutually exclusive hypotheses about its origin (krajmerová et al., 2015). nevertheless, in most previous classifications, a. x borisii-regis has the status of a hybrid between a. alba and a. cephalonica (chater, 1964; christensen, 1986; christensen, 1997). namely, it is considered that a. alba and a. cephalonica have experienced secondary contact in the refuges during periods of glacial expansion in pleistocene. these events probably led to the exchange of genetic material between these two species in the glacial refugia which contributed to appearance of a. x borisii-regis (liepelt et al., 2010). the reports of a. x borisii-regis distribution range 24 biologica nyssana ● 12 (1) september 2021: 23-32 nikolić et al. ● variability of headspace volatiles in native population of abies x borisii-regis from the central rhodopes fig. 1. geographical position of analyzed populations. for the description of the location and habitat conditions of the populations, cf. tab. 1. 25 are highly controversial: according to one concept a. x borisii-regis appears parapatrically along with the putative parent species, i.e. in its northern distribution with a. alba and in southern with a. cephalonica (jalas & suominen, 1973; christensen, 1997; andreev, 1992). thus, it occurs in southern albania, almost the whole greece (except the cephalonia island and the southern half of the peloponnese), the southernmost parts of northern macedonia and across southwestern and southern bulgaria (jalas & suominen, 1973). on the other hand, based on the atlas of the word’s conifers (farjon & filer, 2013), a. alba, a. x borisii-regis, and a. cephalonica are mostly allopatric, i.e., king boris fir is distributed in southern albania, northern and northeastern greece, and rhodope mt. in southern bulgaria. finally, fady (1993) considered only the populations from pindos mt. and central greece as a. x borisii-regis. the usefulness of terpenes as volatile chemophenetic markers has been addressed in detail by hanover (1992), who emphasized their importance in research of biodiversity, geographic variability, evolution, and systematics, particularly in studies of conifer order pinales. static headspace (hs) extraction is a common technique for analyzing volatile organic compounds, enabling the analyst to study a variety of sample matrices while avoiding the costly and time-consuming preparation involved with traditional gas chromatography (kolb & ettre, 2006). the advantage of combining of hs and gcms/fid (gas chromatography-mass spectrometry/ flame ionization detection) methods in rapid and effective extraction and identification of highly volatile compounds as well as for chemotyping conifers has already been reported (mitić et al., 2020; nikolić et al., 2021). in our previous study, seventeen native fir populations from the presumed zones of natural hybridization in the balkan peninsula were analyzed using hs needle volatiles as chemophenetic markers (nikolić et al., 2021). abies alba and a. cephalonica were characterized by distinct volatile profiles, while three populations from northern and central greece identified as a. x borisii-regis shared the volatile profiles with both of the supposed parent species (nikolić et al., 2021). although king boris fir was described from the central rhodopes in bulgaria (mattfeld, 1926), recent molecular study did not provide evidence on hybrid origin of any bulgarian fir populations including populations from this mountain massif (krajmerová et al., 2015). therefore, the aim of the present study was to shed more light on chemotaxonomic status of controversial bulgarian fir populations. hence, in this article we presented new results for one population of a. x borisii-regis from the southern bulgaria (the central rhodopes) regarding diversity of hs needle volatiles. for multivariate statistical analyses (canonical discriminate analysis and agglomerative hierarchical clustering), in addition to the results obtained in this study, we used our previously published data relating to the broader transition zone between a. alba and a. cephalonica (nikolić et al., 2021). in this way, hs needle volatiles of 18 populations of three abies taxa from the balkan peninsula have been analyzed for the first time (fig. 1). materials and methods plant material plant material of a. x borisii-regis mattf. was collected in the southern bulgaria (the central rhodopes, pamporovo). three-year-old needles were sampled during september 2021 from 14 individuals, which were randomly selected and distant at least 30 m. plant material, deposited in labeled polyethylene bags (sample plot, date of collection, and locality), was immediately transferred to a freezer and stored at -20 °c prior to further analysis. voucher specimen was deposited in the “herbarium moesiacum niš” (hmn) of the department of biology and ecology, faculty of sciences and mathematics, university of niš under the acquisition number 14389. locations of the presently studied population, as well as 17 populations previously analyzed by nikolić et al. (2021) are presented in fig. 1. the corresponding geographic and geologic data, date of collection, voucher information as well as the number of studied individuals for every population are listed in the tab. 1. headspace isolation three-year-old needles of every individual tree were cut into pieces of 2-3 mm length. five hundred milligrams of chopped plant material was placed into 20-ml headspace vials and 1 ml of distilled water was added in each. the prepared vials were placed into a tray for further automated procedure. each sample was heated at 80 °c for 20 minutes with the following program: shaking for 5 seconds, pausing for 2 seconds. after equilibration, the gas vapor of hs volatiles above the aliquot of volatiles was sampled with a gas syringe and subsequently injected into a gc analyzer. gc-ms/fid analyses qualitative analysis of the hs volatiles was performed by gas chromatography-mass spectrometry (gc-ms), while the quantitative data were obtained by gas chromatography-flame ionization detector (gc-fid) analysis. samples biologica nyssana ● 12 (1) september 2021: 23-32 nikolić et al. ● variability of headspace volatiles in native population of abies x borisii-regis from the central rhodopes 26 biologica nyssana ● 12 (1) september 2021: 23-32 nikolić et al. ● variability of headspace volatiles in native population of abies x borisii-regis from the central rhodopes ta bl e 1. l oc at io n an d ha bi ta t d es cr ip tio n of th e se le ct ed p op ul at io ns o f t hr ee a bi es ta xa ta xo n l oc al ity po pu la tio n co de l at itu de (n ) l on gi tu de (e ) a lti tu de (m .a .s. l.) su bs tr at um n um be r of in di vi du al s an al yz ed d at e of co lle ct io n n um be r of va uc he rs a. a lb a r om an ia , s ou th er n c ar pa th ia ns , t ra ns fã gã rã şa n a r c 45 °2 3' 30 " 24 °3 7' 56 " 95 7 si lic at e 5 a ug us t, 20 18 13 95 0 h m n a. a lb a se rb ia , m t. ta ra , p re do v k rs t a st 40 °1 1' 47 " 22 °1 0' 48 " 10 96 li m es to ne 15 ju ne , 2 01 8 13 94 9 h m n a. a lb a se rb ia , m t. k op ao ni k, k ad ije va c a sk 43 °1 9' 43 " 20 °4 5' 41 " 13 50 si lic at e 15 ju ne , 2 01 7 13 94 6 h m n a. a lb a se rb ia , m t. st ar a pl an in a, a rb in je a ss 43 °1 8' 25 " 22 °4 7' 18 " 12 95 si lic at e 15 a ug us t, 20 18 13 94 4 h m n a. a lb a n or th m ac ed on ia , m t. ša rpl an in a, l eš ni ca a m s 42 ° 1 '1 7" 20 °4 7' 04 " 15 05 li m es to ne 15 a ug us t, 20 17 13 94 5 h m n a. a lb a n or th m ac ed on ia , m t. n id že a m n 40 °5 8' 54 " 21 °4 7' 31 " 19 00 si lic at e 15 a ug us t, 20 18 13 94 8 h m n a. a lb a b ul ga ria , m t. pi rin , a bo ve b an sk o a b p 23 °2 8' 33 " 12 90 si lic at e 15 o ct ob er , 2 01 8 13 94 2 h m n a. a lb a b ul ga ria , m t. r ila , a bo ve r ila m on as te ry a b r 42 °0 9' 17 " 23 °2 4' 49 " 15 36 si lic at e 15 o ct ob er , 2 01 8 13 94 7 h m n a. x b or is iire gi s b ul ga ria , m t. r ho do pe s, pa m po ro vo b b r 41 °3 9' 01 " 24 °4 1' 40 " 16 20 si lic at e 14 se pt em be r, 20 20 14 38 9 h m n a. x b or is iire gi s g re ec e, m t. pi er ia , f te ri b g p 40 °1 1' 48 " 22 °1 1' 72 " 92 1 si lic at e 15 o ct ob er , 2 01 8 13 94 3 h m n a. x b or is iire gi s g re ec e, m t. o ly m pu s, ab ov e le pt ok ar ya b g o 40 ° 01 '5 4" 22 °3 0' 25 " 64 0 li m es to ne 15 o ct ob er , 2 01 8 13 95 1 h m n a. x b or is iire gi s g re ec e, m t. ty m fr is to s, se llo s b g t 38 °5 4' 33 " 21 °5 4' 36 " 97 1 li m es to ne 15 se pt em be r, 20 19 13 99 7 h m n a. c ep ha lo ni ca g re ec e, m t. o iti c g o 38 °4 4' 15 " 22 °2 2' 54 " 89 5 li m es to ne 15 se pt em be r, 20 19 14 00 2 h m n a. c ep ha lo ni ca g re ec e, m t. pa rn as so s, pl ei ad es c g p 38 °3 3' 06 " 22 °3 4' 10 " 16 60 li m es to ne 15 se pt em be r, 20 19 14 00 1 h m n a. c ep ha lo ni ca g re ec e, p el op on ne se , m eg a sp ila io m on as te ry c g m 38 °0 5' 17 " 22 °1 0' 23 " 86 0 li m es to ne 15 se pt em be r, 20 19 14 00 0 h m n a. c ep ha lo ni ca g re ec e, p el op on ne se , m t. c he lm os , k al av ry ta c g k 38 °0 0' 41 " 22 °0 9' 52 " 13 68 li m es to ne 15 se pt em be r, 20 19 13 99 8 h m n a. c ep ha lo ni ca g re ec e, p el op on ne se , m t. c he lm os c g c 38 °0 0' 56 " 22 °1 0' 57 " 15 50 li m es to ne 15 se pt em be r, 20 19 13 99 9 h m n a. c ep ha lo ni ca g re ec e, c ep ha lo ni a, m t. a in os c g a 38 °0 9' 37 " 20 °3 7' 13 " 10 05 li m es to ne 15 ju ly , 2 01 9 13 99 6 h m n were analyzed on agilent technologies 7890b gc equipped with a fused silica capillary column (hp5ms, 250 μm×25 m, film thickness 0.25 μm, agilent technologies, santa clara, ca, usa) and coupled with a 7890a flame ionization detector (fid) and 7000b ms/ms spectrometer (operating in ms1 scan mode) from the same company. the gc was operated under the following conditions: injector temperature 250 °c; gc-ms interface temperature 300 °c; oven temperature programmed from 50 °c for 2.25 minutes, then to 200 °c at 4 °c/minute (carrier gas he, 1.0 ml/min, constant flow mode). ms conditions were as follows: ionization voltage of 70 ev; acquisition mass range 40-440; scan time 0.32 seconds. hs volatiles were identified from tic (total ion chromatogram) by comparison of their linear retention indices relative to c8-c20 n-alkanes recorded on the same column/temperature program with literature values (nist ms search 2.0; nist chemistry webbook srd69; adams, 2007) and their mass spectra with those of standards from databases (wiley 6, nist02, adams) by the application of the amdis software (the automated mass spectral deconvolution and identification system, ver. 2.7, distributed within the software package for 78907000 bgc-ms/ms triple quadrupole system). the percentage composition of the hs volatiles was computed from the gc-fid peak areas without any corrections. statistical analyses statistical data processing was carried out by statistica 8 software (statsoft, inc., tulsa, ok, usa). statistical matrices included hs volatiles that were detected in the content ≥0.5% in at least one of the analyzed taxa (12 components in total: santene, tricyclene, α-pinene, camphene, β-pinene, myrcene, α-phellandrene, limonene+βphellandrene, terpinolene, borneol, bornyl acetate, (e)-caryophyllene). multivariate analysis included canonical discriminant analysis (cda) and agglomerative hierarchical clustering (ahc). overall differences between the compared groups are presented by mahalanobis distances. the calculated matrix distance was used for agglomerative hierarchical clustering (ahc), using the ward’s method. results and discussion variability of hs needle volatiles gc-ms/fid analyses of hs needle volatiles in a. x borisii-regis population from the central rhodopes revealed the presence of 34 compounds (tab. 2). the experimental and literary retention indices, identification methods, and classes to which identified compounds belong are also presented in tab. 2. as the most volatile compounds, monoterpene hydrocarbons represented the major compound class (95.3%), while contents of oxygenated monoterpenes and sesquiterpene hydrocarbons were significantly lower (2.2 and 2.4%, respectively). further, the phytochemical profile of the main hs volatiles for studied population could be presented as follows: β-pinene >>> α-pinene > camphene > limonene+βphellandrene (where, based on petrakis et al. (2001), =, >, >>, and >>> represent difference of 0.1-1.0%, 1.15.0%, 5.1-15.0%, and more than 15.1%, resp.). according to nikolić et al. (2021), the phytochemical profiles of the dominant hs volatiles of a. alba, a. x borisii-regis, and a. cephalonica were: a. alba: β-pinene >> limonene+β-phellandrene > camphene > α-pinene a. x borisii-regis: β-pinene >> α-pinene >> camphene > limonene+β-phellandrene a. cephalonica: β-pinene > α-pinene >>> camphene > limonene+β-phellandrene thus, presently reported volatile profile of bulgarian fir population from the central rhodopes fits well with described profile of greek a. x borisiiregis populations. multivariate analyses (cda and ahc) of hs needle volatiles at the population level the cda based on 259 individuals from 18 populations of a. alba, a. x borisii-regis, and a. cephalonica showed that the first two canonical axes participated in 62.9% of the total discrimination, of which the first axis (ca1) accounted for 44.5% (fig. 2 and tab. 3). four compounds, i.e. β-pinene, limonene+β-phellandrene, camphene, and α-pinene had significant impact on both axes, while bornyl acetate significantly affected only the ca2 (tab. 3). based on the obtained scatterplot (fig. 2), all populations of a. alba showed positive values for the ca1 (yellow ellipse), while a. cephalonica populations formed a group at the negative part of ca1 (blue ellipse). according to the results of the post-hoc test that was done in the study of nikolić et al. (2021), the “alba” profile was characterized by higher content of limonene+β-phellandrene and camphene and lower content of α-pinene compared to the second profile; vice versa the “cephalonica” profile was characterized by lower amount of limonene+β-phellandrene and camphene and higher 27 biologica nyssana ● 12 (1) september 2021: 23-32 nikolić et al. ● variability of headspace volatiles in native population of abies x borisii-regis from the central rhodopes 28 biologica nyssana ● 12 (1) september 2021: 23-32 nikolić et al. ● variability of headspace volatiles in native population of abies x borisii-regis from the central rhodopes table 2. relative percentage composition of hs needle volatiles of bulgarian a. x borisii-regis population from the central rhodopes entry compounds ri1 li2 content (%)3 n=14 class of compounds identification method 1. hexanal 801 801 tr o ri, ms 2. (2e)-hexenal 849 846 0.1±0.2 o ri, ms 3. (3z)-hexenol 850 850 tr o ri, ms 4. hexanol 866 863 tr o ri, ms 5. santene 884 884 0.5±0.2 mh ri, ms 6. tricyclene 921 921 3.3±1.2 mh ri, ms 7. α-pinene 934 932 19.7±2.5 mh ri, ms 8. camphene 949 946 16.3±4.2 mh ri, ms 9. β-pinene 980 974 40.3±5.5 mh ri, ms 10. myrcene 990 988 1.2±0.3 mh ri, ms 11. α-phellandrene 1004 1002 0.3±0.2 mh ri, ms 12. limonene+βphellandrene 1029 1024/1025 13.3±2.1 mh ri, ms 13. 2-heptyl acetate 1040 1038 tr o ri, ms 14. γ-terpinene 1057 1054 tr mh ri, ms 15. terpinolene 1087 1086 0.4±0.1 mh ri, ms 16. 2-nonanone 1089 1087 tr o ri, ms 17. borneol 1166 1165 1.0±0.9 mo ri, ms 18. linalyl acetate 1254 1254 tr mo ri, ms 19. bornyl acetate 1287 1287 1.2±1.0 mo ri, ms 20. α-longipinene 1353 1350 0.1±0.1 sh ri, ms 21. α-ylangene 1373 1373 tr sh ri, ms 22. α-copaene 1377 1374 tr sh ri, ms 23. sibirene 1404 1400 tr sh ri, ms 24. longifolene 1409 1407 tr sh ri, ms 25. (e)-caryophyllene 1420 1417 1.6±1.2 sh ri, ms 26. himachala-2,4-diene 1429 14292a 0.1±0.2 sh ri, ms 27. humulene 1456 1452 0.4±0.3 sh ri, ms 28. β-selinene 1490 1489 tr sh ri, ms 29. δ-selinene 1494 1492 0.1±0.1 sh ri, ms 30. α-selinene 1498 1498 tr sh ri, ms 31. β-himachalene 1503 1500 tr sh ri, ms 32. δ-amorphene 1509 1511 tr sh ri, ms 33. γ-cadinene 1516 1513 tr sh ri, ms 34. δ-cadinene 1525 1522 tr sh ri, ms total 100.0±0.2 monoterpene hydrocarbons (mh) 95.3±1.9 oxygenated monoterpenes (mo) 2.2±1.8 sesquiterpene hydrocarbons (sh) 2.4±1.8 others (o) 0.2±0.2 1ri: experimental linear retention indices relative to c8-c20 alkanes. 2li:literature indices-adams’ retention indices and 2aaccording to nist data base. 3contents are given as percentages (mean±standard deviation) of the total compounds content; n: the number of analyzed individuals; tr trace<0.05%; (-) not detected compounds; the most abundant hs volatiles are in boldface. amount of α-pinene compared to the “alba” profile. accordingly, eastern a. alba lineage and a. cephalonica may be undoubtedly characterized by volatile markers, while populations of a. x borisiiregis were positioned in the intermediate position. specifically, all four populations identified as a. x borisii-regis included volatile profiles of both supposed parent species, whereby profile frequencies changed clinally along the latitudinal gradient (fig. 2). in the northernmost population of a. x borisiiregis from southern bulgaria (rhodopes mt. bbr) most individuals belonged to the “alba” profile (the largest number of green diamonds is situated within the yellow ellipse), while individuals from two northern greece populations were more evenly distributed on both sides of ca1. nevertheless, in a slightly more northern population (pieria mt. bgp) a larger number of individuals belonged to the “alba” profile (green squares more abundant within the yellow ellipse), while individuals from a somewhat southern population (olympus mt. bgo) were evenly distributed on both sides of ca1, indicating approximately equal frequencies of both profiles (green circles with approximately equal representation within both ellipses). finally, in the southernmost a. x borisii-regis population from the central greece (tymfristos mt. bgt) most individuals were located in the negative part of ca1, closer to the “cephalonica” profile (the largest number of green triangles is situated within the blue ellipse). therefore, all four studied a. x borisii-regis populations included both the a. alba and a. cephalonica volatile profiles, but distributed along a latitudinal gradient connecting the ranges of presumed parent species, which confirms the hypothesis proposed in our previous study (nikolić et al., 2021). considering that one of the indicators of the presence of a hybrid zone is the clinical variation of all or a larger number of characters located in the same area, the observed geographic distribution of volatile entities supports the hypothesis that studied a. x borisii-regis populations are of secondary origin due to hybridization between a. alba and a. cephalonica. in addition, clinal (north-south) pattern in the distribution of morpho-anatomical (mitsopoulos & panetsos, 1987) and genetic traits (krajmerová et al., 2015) over the a. x borisii-regis range was previously reported. the dendrogram obtained by ahc analysis has also shown a tendency to divide analyzed fir populations in similar way (fig. 3). namely, populations of a. alba and a. cephalonica were divided in two separate clusters, while populations of a. x borisii-regis appeared heterogeneous. actually, the northernmost population of a. x borisii29 biologica nyssana ● 12 (1) september 2021: 23-32 nikolić et al. ● variability of headspace volatiles in native population of abies x borisii-regis from the central rhodopes variables ca1 ca2 santene -0.105 -0.164 tricyclene 0.827 -0.905 α-pinene 1.123 1.526 camphene 2.193 2.934 β-pinene 4.207 2.859 myrcene 0.063 0.056 α-phellandrene -0.089 0.203 limonene+β-phellandrene 3.690 2.987 terpinolene 0.066 -0.177 borneol 0.468 -0.712 bornyl acetate 0.610 1.239 (e)-caryophyllene 0.730 0.436 eigenvalue 4.197 1.729 % explained variation 0.445 0.629 table 3. standardized coefficients for the first two canonical axes (ca) of variation in 12 needle volatiles from the discriminant functional analysis of 18 a priori groups. the most significant coefficients are in boldface fig. 2. canonical discriminant analysis (cda) based on contents of 12 hs needle volatiles isolated from 259 individuals of 18 populations. abbreviations and symbols refer to populations as indicated in tab. 1. and fig 1. abbreviation colors: yellow – a. alba, green – a. x borisii-regis, and blue – a. cephalonica. the yellow and blue ellipses encompass all individuals of supposed parent species. regis from southern bulgaria (bbr) fitted within the “alba” and all greek populations of a. x borisii-regis (bgp, bgo, and bgt) within the “cephalonica” cluster. furthermore, the northernmost (bbr) and southernmost (bgt) population of a. x borisiiregis showed the greatest resemblance to the geographically closest populations of the parent species: southwestern bulgarian populations of a. alba (abr and abp) and population of a. cephalonica from central greece (cgo), respectively (fig. 3). the history of fir populations from rhodope mt. is largely unknown; fluctuating presence of fir pollen was recorded in rhodope sites since the end of the pleniglacial until the early holocene and an expansion occurred only after 8500 bp (terhürneberson et al., 2004). although king boris fir was described from the central rhodopes (mattfeld, 1926), recent molecular study did not provide evidence on hybrid origin of any of 10 analyzed populations from this mountain massif, based on maternally inherited mitochondrial (mtdna) markers (krajmerová et al., 2015). moreover, these authors found exclusively a. alba mitotype in all fir populations from bulgaria, north macedonia, and northern greece, while only two populations from central greece (tymfrostos and rentina mts.) were mixed and contained predominantly the a. cephalonica mitotype, with a lower proportion of the a. alba mitotype. even though bella et al. (2014) analyzed the same mtdna marker (nad5-4) as in the study of (krajmerová et al., 2015), they found a mixture of maternal lineages (mitotypes of both parent species) in two northern greek populations (pieria and pindos mts.). nevertheless, the second study did not include any of fir populations from central greece, bulgaria, or north macedonia. according to phytochemical markers selected in the present study, presence of both volatile profiles was recorded in a somewhat wider geographical area. i.e. four populations from the southern bulgaria, northern and central greece, where the northernmost population contained predominantly a. alba and the southernmost a. cephalonica profile (fig. 2). thus, based on this concept, a. alba and a. cephalonica were co-occurring at least in the southern bulgaria, northern and central greece, suggesting that this region may have served as a refugium for a. alba during the pleistocene glacial cycles. bearing in mind several proposed distribution concepts of king boris fir, our results are mostly in agreement with the concept given in the atlas of the word’s conifers (farjon & filer, 2013) with the addition of central greece. conclusions gc-ms/fid analyses of hs needle volatiles in population a. x borisii-regis from the southern bulgaria (the central rhodopes) revealed presence biologica nyssana ● 12 (1) september 2021: 23-32 30 nikolić et al. ● variability of headspace volatiles in native population of abies x borisii-regis from the central rhodopes fig. 3. dendrogram obtained by agglomerative hierarchical clustering (ahc) of 18 populations based on 12 hs needle volatiles. abbreviations and symbols refer to populations as indicated in tab. 1. and fig 1. abbreviation colors: yellow a. alba, green a. x borisii-regis, and blue a. cephalonica of 34 compounds. the major hs volatiles were β-pinene (40.3%), α-pinene (19.7%), camphene (16.3%) and limonene+β-phellandrene (13.3%). the presently reported phytochemical profile of the main hs volatiles fits well with previously described profile of greek a. x borisii-regis populations. based on multivariate statistical analyses, all four populations identified as a. x borisii-regis from southern bulgaria, northern and central greece included volatile profiles of both supposed parent species, where profile frequencies changed clinally along the latitudinal gradient connecting the ranges of parent species. thus, based on hs needle volatiles, a. alba and a. cephalonica were co-occurring at least in the southern bulgaria, northern and central greece, suggesting that this region may have served as a refugium for a. alba during the pleistocene glacial cycles. as a natural hybrid, populations of king boris fir can be interesting from the point of view of commercial forestry for the reason that offer the possibility to cope with the predicted forest decline in southern europe as a result of climate change and can be used for reforestation. acknowledgements. this research was supported by grants no 451-03-9/2021-14/200124 by the ministry of education, science and technological development of the republic of serbia. references andreev, n. 1992: pinophyta, in: kozuharov, s. (ed.), field guide to the vascular plants in bulgaria, 81-85, naouka & izkoustvo, sofia (in bulgarian). bella, e., liepelt, s., parducci, l., drouzas, a. 2014: genetic insights into the hybrid origin of abies x borisii-regis mattf. plant systematics and evolution, 301: 749-759. chater, a.o. 1964: abies miller. in: tutin, t.g., heywood, v.h., burges, n.a., valentine, d.h., walters, s.m., webb, d.a. (eds.), flora europaea, 1: 37-38, cambridge university press, cambridge. christensen, k.i. 1986: abies miller. in: strid, a. (ed.), mountain flora of greece, 1: 38-41, cambridge university press, cambridge. christensen, k.i. 1997: abies miller. in: strid, a., tan, k. (eds.), flora hellenica, 1: 1-3, koeltz scientific books, königstein. fady, b. 1993: caractéristiques écologiques et sylvicoles des sapins de grèce dans leur aire naturelle et en plantation dans le sud de la france. perspectives pour le reboisement en règion méditerrané enne. revue forestière française, 45: 119-133. farjon, a., filer, d. 2013: an atlas of the world’s conifers: an analysis of their distribution, biogeography, diversity and conservation status. koninklijke brill nv, leiden. 512 p. hanover, j.w. 1992: applications of terpene analysis in forest genetics. new forests, 6: 159-178. jalas, j., suominen, j. 1973: atlas florae europaeae. 2 gymnospermae (pinaceae to ephedraceae). the committee for mapping the flora of europe & societas biologica fennica vanamo, helsinki, 40 p. kolb, b., ettre, l.s. 2006: static headspace-gas chromatography: theory and practice. wileyinterscience, new york. 349 p. kormutak, a. 1985: study on species hybridization within the genus abies. acta dendrobiologica. veda, bratislava. 127 p. kormutak, a. 2004: crossability relationships between some representatives of the mediterranean, northamerican and asian firs (abies sp.).veda, bratislava. krajmerová, d., paule, l., zhelev, p., voleková, m., evtimov, i., gagov, v., gömöry, d. 2015: natural hybridization in eastern-mediterranean firs: the case of abies borisii-regis. plant biosystematics, 150: 1189-1199. liepelt, s., mayland-quellhorst, e., lahme, m., ziegenhagen, b. 2010: contrasting geographical patterns of ancient and modern genetic lineages in mediterranean abies species. plant systematics and evolution, 284: 141-151. mattfeld, j. 1926: die europäischen und mediterranen abies arten. die pflanzenareale, 1: 22-29 (in german). mitić, z.s., jovanović, s.č., zlatković, b.k., milanovici, s.j., nikolić, b.m., petrović, g.m., stojanović g.s., marin, p.d. 2020: variation of needle volatiles in native populations of pinus mugo evidence from multivariate statistical analysis. plant biosystems-an international journal dealing with all aspects of plant biology, 155(4): 700-710. mitsopoulos, d.j., panetsos, c.p. 1987: origin of variation in fir forests of greece. silvae genetica, 36: 1-15. moulalis, d. 1986: diagnosis and characterization of fir hybrids. scientific annals of the department of forestry and natural environment, aristotle university of thessaloniki, 29: 371-404. nikolić, j.s., zlatković, b.k., jovanović, s.č., stojanović, g.s., marin, p.d., mitić, z.s. 2021: needle volatiles as chemophenetic markers in 31 biologica nyssana ● 12 (1) september 2021: 23-32 nikolić et al. ● variability of headspace volatiles in native population of abies x borisii-regis from the central rhodopes biologica nyssana ● 12 (1) september 2021: 23-32 32 nikolić et al. ● variability of headspace volatiles in native population of abies x borisii-regis from the central rhodopes differentiation of natural populations of abies alba, a. x borisii-regis, and a. cephalonica. phytochemistry, 183: 112612. panetsos, c.p. 1975: monograph of abies cephalonica loudon. annals of forest science, 7: 1-18. petrakis, p.v., tsitsimpikou, c., tzakou, o., couladis, m., vagias, c., roussis, v. 2001: needle volatiles from five pinus species growing in greece. flavour and fragrance journal, 16: 249-252. terhürne-berson, r., litt, t., cheddadi, r. 2004: the spread of abies throughout europe since the last glacial period: combined macrofossil and pollen data. vegetation history and archaeobotany, 13: 257-268. turrill, w.b. 1937: xi-on the flora of the near east: xviii. new species, new records and notes. kew bulletin, 2: 79-86. xiang, x.g., cao, m., zhou, z.k. 2007: fossil history and modern distribution of the genus abies (pinaceae). frontiers of forestry in china, 2: 355365. microsoft word bn020203 gnjatovic zikic biologica nyssana 2 (2) december 2011: 119-122 gnjatović i., žikić v. new data on longhorn beetles… 119 original article ! new data on longhorn beetles for the territories of serbia and montenegro (coleptera, cerambycidae) with the detailed description of callimoxys gracilis (brullé 1832) ivan gnjatović, vladimir žikić university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia * e-mail: ivangnjatovic81@gmail.com abstract: gnjatović, i., žikić, v.: new data on longhorn beetles for the territories of serbia and montenegro (coleptera, cerambycidae) with the detailed description of callimoxys gracilis (brullé 1832), biologica nyssana, 2 (2), december 2011: 119-122. investigated sample of longhorn beetles discovers 24 species from 20 genera of 11 tribes from three subfamilies. the species vadonia moesiaca daniel 1891 apears to be a balkan endemic. also, very rare species callimoxys gracilis (brullé 1832) is recorded for the second time for the serbian fauna. the examined material was collected during the period of 2010-2011 on the territories of serbia and montenegro. key words: cerambycidae, callimoxys gracilis, sićevo gorge, torilis ucranica introduction ! the fauna of cerambycidae is more or less well investigated on the teritorry of serbia, starting from b o b i ć (1891) over the detailed monograph of the yugoslav cerambycids in three volumes by m i k š i ć at al. (1971, 1973, 1985) to the last publication of i l i ć (2005). the data from the recent studies shows that there are 259 species of longhorn beetles in serbia (p i l & s t o j a n o v i ć , 2009) and 142 species for the territory of montenegro (ć u r č i ć al., 2003). the genus callymoxys belongs to the subfamily: cerambycinae and the tribe: stenopterini comprising only two species: the blood-necked longhorn beetle, c. sanguinicollis (o l i v i e r , 1795) inhabiting north america and c. gracilis (b r u l l é , 1832) which occurs in europe. materials and methods all specimens were collected during 20102011 on the territories of serbia and montenegro. the sampling of longhorn beetles has been performed mostly by using an entomological net by sweeping over plants or picking the specimens from the ground or plants by hand. the total number of collected and examined specimens is 56. the specimens of the species callimoxys gracilis (b r u l l é , 1832), one female and three males, were found on the plant torilis ucranica spreng 1820 , apiales: apiaceae. this species had been recorded previously only once for the fauna of serbia. there has been no data for the territory of montenegro yet. identification of the sampled specimens was performed using adequate determination keys: m i k š i ć & g e o r g i j e v i ć (1973), and b e n s e (1995). additional confirmation was performed by checking the data of the fauna of longhorn beetles 2 (2) • december 2011: 119-122 biologica nyssana 2 (2) december 2011: 119-122 gnjatović i., žikić v. new data on longhorn beetles… 120 for the territory of serbia and montenegro, and also by comparing with the literature data of a l h t o f f & d a n i l e v s k y (1997), i l i ć (2005) and g n j a t o v i ć & ž i k i ć (2010). results the list of collected specimens: subfamily cerambycinae tribe clytini chlorophorus hungaricus seidlitz 1791 2♂, serbia: fruška gora mt., 01.06.2011, leg. p. jakšić. tribe stenopterini callimus angulatus (schrank 1789) 2♂, montenegro: bečići, 23.04.2011, leg. v. žikić. callimoxys gracilis (brullé, 1832) 1♀, 3♂, serbia, niš, sićevo gorge, 15.05.2011., leg. v. žikić. tribe purpuricenini purpuricenus budensis (goeze 1783) 1♀, serbia: niš, sićevačka gorge, 19.07.2011, leg. v. žikić. subfamily lamiinae tribe acanthocinini exocentrus punctipennis mulsant & guillebeau 1856, 1♂, serbia: niš, 15.07.2011, leg. v. žikić. tribe agapanthiini calamobius filum (rossi 1790) 2♀, 2♂, serbia: bovan lake, 13.06.2011, leg. v. žikić. agapanthia kirby (gyllenhal 1817) 1♀, serbia: miljkovačka gorge, 08.06.2011, leg. i. simonović; 2♂, montenegro: budva, 25.05.2011, leg. v. žikić. agapanthia intermedia ganglbauer 1884 1♂, montenegro: budva, 25.05.2011, leg. v. žikić. agapanthia dahli (richter 1821) 1♂, montenegro: bečići, 18.05.2011, leg. v. žikić. tribe lamiini leiopus nebulosus (linanneus 1758) 1♂, serbia: niš, popovac, 22.05.2011, leg. v. žikić. lamia textor (linanneus 1758) 1♀, serbia: boljevac, progor, 06.07.2011, leg. p. jakšić. tribe obereini oberea (amaurostoma) erythrocephala (schrank 1776) 1♀, montenegro: budva, 25.05.2011, leg. v. žikić. tribe phytoeciini musaria affinis (harrer 1784) 1♀, serbia: niš, 02.07.2011, leg. i. gnjatović. phytoecia caerulea (scopoli 1772) 1♀, montenegro: bečići, 25.05.2011, leg. v. žikić. tribe saperdini saperda scalaris (linanneus 1758) 1♀, 1♂, serbia: niš, sićevačka gorge, 31.05.2011, leg. v. žikić. subfamily lepturinae tribe lepturini alosterna tabacicolor (de geer 1775) 1♀, 1♂, serbia: niš, gornji matejevac, sv. jovan, 03.06.2010. leg. i. gnjatović. paracorymbia pallens (brulle 1832) 1♀, 3♂, serbia: niš, gornji matejevac, sv. jovan, 03.06.2010. leg. i. gnjatović. stictopleura scutellata (fabricius 1781) 1♀, serbia: kopaonik mt., 25.08.2010, leg. v. žikić. stenurella melanura (linanneus 1758) 2♀, 2♂, serbia: bosilegrad, dukat mt., 07.08.2010, leg. v. žikić. stenurella nigra (linanneus 1758) 5♀, 4♂, serbia: niš, gornji matejevac, sv. jovan, 03.06.2010. leg. i. gnjatović. stenurella septempunctata (fabricius 1792) 1♀, serbia: miljkovačka gorge, 08.06.2011. leg. i. simonović. vadonia moesiaca daniel 1891 1♀, serbia: niš, sićevačka gorge, 31.05.2010, leg. v. žikić. tribe rhagiini gaurotes (carilia) virginea (linanneus 1758) 3♀, 4♂, serbia: kopaonik mt., 27.08.2010. leg. v. žikić. pachyta quadrimaculata (linanneus 1758) 1♀, serbia: kopaonik mt., metođe, 20.07.2010, leg. s. stanković. disscusion identified taxa of the specimes which are listed here belong to the three subfamilies out of seven known from the fauna of serbia and montenegro, and out of eight from the fauna of europe: cerambycinae, lepturinae and lamiinae. comparing the taxonomical results for each noted subfamily, according to available data from the repesentative literature (m i k š i ć & g e o r g i j e v i ć , 1973; i l i ć , 2005), the biologica nyssana 2 (2) december 2011: 119-122 gnjatović i., žikić v. new data on longhorn beetles… 121 faunaeuropea database (a u d i s i o , 2005) and our results, we give here the total number of the recorded species: 65 from the subfamily cerambycinae, 55 from lepturinae and 77 from lamiinae. we have chosen the species callimoxys gracilis here to describe in details because of the suspicion on a new species. we were not able to find the original description of this species to compare with our specimens, therefore in this work we are not declaring this particular taxa as a new species for science. the first record of callimoxys gracilis (b r u l l é , 1832) for serbia was mentioned in the check-list of longhorn beetles by a l t h o f f & d a n i l e v s k y (1997) which encompass the whole territory of europe. there was no detailed information about habitat of this species or any other relevant data. in sićevo gorge, niš, serbia, we have found the adults of this particular longhorn species on the blossoms of torilis ucranica feeding on it. this piece of information is new about its biology. description: callimoxys gracilis (brullé, 1832) (fig. 1) original name: stenopterus gracilis brullé, 1832 material examined: 1♀, 3♂, niš, sićevo gorge, 15.05.2011. leg. i. gnjatović. the body is black, slightly greenish or bluish with metallic shine. length is about 7 (males) to 13 mm (female). head: semi-prognate. antennae: do not reach the elytra apex, quite longer in males. eyes: large, with small and fine omatidia (diurnal). pronotum is greenish to bluish in males, in female red, with black frontal and posterior margins (fig. 1). elytra are broad at the base, fine rasp punctuated, slightly narrow at the apex. hind wings: partly exposed reaching the apex of abdomen. lateral and ventral parts of both, thorax and abdomen, covered with tiny hairs. legs: all femora are slightly swollen. outer edges of hind tibia serrate (the most important taxonomic characteristic of the genus callimoxys). biology: adults apear from april to june. development is inadequately known. larvae have been found in dry branches of broadleaf trees: paliurus, prunus, quercus (š v a c h a & d a n i l e v s k y , 1987). locality: sićevo gorge, 15 km eastern of niš, serbia. territory under protection by the serbian government acts as a natural resource of great importance (t r a j k o v i ć & b r a n k o v i ć , 2007). substrate is limestone, important refuge of the arcto – tertiary flora during the last ice age. distribution: croatia (slavonia), northern bosnia and herzegovina, and montenegro (m i k š i ć & g e o r g i j e v i ć , 1973), as well in macedonia and romania, (i l i ć , 2005) and bulgaria, (g e o r g i e v at al., 2005; i l i ć , 2005). figure 1. callimoxys gracilis (brullé 1832), female (left) and male (right) biologica nyssana 2 (2) december 2011: 119-122 gnjatović i., žikić v. new data on longhorn beetles… 122 conclusion twenty four species of longhorn beetles, belonging to the three subfamilies were listed: cerambycinae (4), lepturinae (9) and lamiinae (11). the number of identified species in serbia is 18 and for the territory of montenegro it is six species. by analyzing the available literature, we deliver five species marked as rare: chlorophorus hungaricus seidlitz 1791, exocentrus punctipennis mulsant & guillebeau 1856, lamia textor (linanneus.1758), paracorymbia pallens (b r u l l e , 1832 (i l i ć , 2005) and callimoxys gracilis (b r u l l é , 1832) where we gave detailed description. the species vadonia moesiaca daniel 1891 belongs to the endemics of the balkan peninsula. acknowledgements. the authors wish to express their sincere thankfulness to saša stanković, ivan simonović and dr predrag jakšić for the loan of specimens. for the present work the authors were supported by the ministry of education and science of the republic of serbia (grant no. 43001). references audisio, p. 2005: fauna europaea: cerambycidae. u: sama g. 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2005: strižibube srbije (coleoptera, cerambycidae) – faunistički pregled. autorsko izdanje, beograd, 179 p. mikšić, r., georgijević, e., 1973: cerambycidae jugoslavije, 2. deo. akademija nauka i umjetnosti bosne i hercegovine, sarajevo, 153 p. pil, n., stojanović, d., 2009: teophilea subcylindricollis hladil, 1988 a new longhorn beetle (coleoptera, cerambycidae) for serbian fauna, acta entomologica serbica, 14(1): 125128. švácha, p., danilevsky, m., 1987: cerambycoid larvae of europe and soviet union (coleoptera, cerambycoidea). part ii. acta universitatis carolinae – biologica 31, univerzita karlova, (3-4): 121-284. trajković, s., branković, s., 2007: sićevo and jelašnica gorges environment status monitoring. institute for nature conservation of serbia, faculty of civil engineering and architecture, niš, 191 p. kajevska, savić, 2019, biologica nyssana 10(1) 10 (1) september 2019: 1-8 doi: 10.5281/zenodo.3463980 first record of neolecta vitellina (bres.) korf & j. k. rogers and sowerbyella fagicola j. moravec in serbia original article iskra kajevska department of biology and ecology, faculty of sciences and mathematics, university of niš, serbia iskra.kajevska@pmf.edu.rs (corresponding author) dragiša savić national park fruška gora, zmajev trg 1, 21208 sremska kamenica, r. serbia dragita@gmail.com received: august 28, 2018 revised: august 23, 2019 accepted: september 10, 2019 abstract: the rare ascomycetes neolecta vitellina and sowerbyella fagicola have been recently registered in serbia as new for the country. morphological description based on found specimens, along with macroand microscopic characteristics of fruiting bodies and attributes on their habitat, ecology and distribution in serbia are given. key words: neolecta vitellina, sowerbyella fagicola, first record, serbia apstract: prvi nalaz vrsta neolecta vitelina (bres.) korf. & j. k. rogers and sowerbyella fagicola j. moravec in serbia retke askomicete neolecta vitellina i sowerbyella fagicola registrovane su po prvi put u srbiji. u radu je predstavljen morfološki opis zasnovan na makroi mirokarakteristikama plodonosnih tela zabeleženih primeraka uz opis staništa i podatke o ekologiji i distribuciji navedenih askomiceta u srbiji. ključne reči: neolecta vitellina, sowerbyella fagicola, prvi nalaz, srbija introduction during fungal diversity field studies conducted from 2016-2018, in selected regions in southern and central serbia, the rare ascomycetes neolecta vitellina and sowerbyella fagicola have been registered. these specimens represent the first documented records of the species in serbia. both taxa are already taken into consideration as “relevant for assessment” in the european red list candidates of endangered fungi which is still under preparation, and only few countries (austria, germany, norway and sweden) have included these species in their national red list of fungi (eccf, c1998-2019). fungal conservation is a complex and challenging issue due to high species diversity, incomplete knowledge of degree of rarity or risk status, large number of little known species, poor understanding of their trophic or habitat requirements (molina et al., 2011). reports of neolecta vitellina and sowerbyella fagicola from europe are scarce, therefore biogeographical studies with data on local populations and habitat are necessary to better understand the nature of its distribution and further conservation approaches. this study aimed to present identification of these two rare species based on morphological description and examination of the present collections, as well as data on their habitats, ecological notes and distribution in serbia. materials and methods specimens have been collected during the period 2016-2018 in two selected regions in southern (suva planina mountain suva planina mt.) and central (kopaonik national park kopaonik np) serbia (fig. 1). a fresh material with substratum was observed for macroand micromorphological characterisation and exsiccates were deposited in the private fungal collection of the mycological society of niš (m.s.n.). part specimens were also stored in the officially unregistered fungarium of fruška gora national park. microscopic characteristics and measurements were made on multiple preparations primarily of fresh material, to obtain vital taxonomic characterisation (baral, 1992), and for comparison with dry material, using optika n-400m and leica dm 1000 light microscopes. examination and meas© 2019 kajevska, savić. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 1 uring of approximately 30 spores released from the ascus and other elements of fresh and dry material were made first in water, later in lugol’s solution (iki) and the ornamentation of the cells was observed in congo red and cotton blue. macroand microscopic descriptions are based only on the collected specimens. the species names follow index fungorum (kirk, 2018) and mycobank (stalpers & cock, 2018) and the following works were used as resources for identification of the species: moravec (1973, 1985, 1988), redhead (1977, 1979), roffler (1999), hansen & knudsen (2000), klofac & herrman (2003), landvik et al. (2003), benkert (2005), van vooren (2007), perić (2008), galán et al. (2010), chinan & hewitt (2012), friebes (2015), wieschollek (2015), ribes et al. (2016). results neolecta vitellina (bres.) korf & j.k. rogers, phytologia 21(4): 204 (1971). figs. 2 – 4. basionym: geoglossum vitellinum bres., revue mycologique 4: 212 (1882). fig 1. map of investigated areas: 1) kopaonik np, crvene bare nature reserve neolecta vitellina site; 2) suva planina mt., above gadžin han town sowerbyella fagicola site. description of studied specimen: ascomata 12-38 x 3-7 mm in diameter, slender, cylindrical, club-shaped or irregularly clavate, spathuliform or somewhat truncate, elongate or lanceolate, some flattened, smooth or wrinkled, often with longitudinal furrows, bright yellow; flesh elastic. stipe 4-25 x 2-4 mm, central, cylindric, gradually merges with the fertile part, tomentose, whitecoloured. asci 50-75(120) x 4-7 μm, cylindrical or narrowly clavate, 8-spored, mostly uniseriate, apex obtuse and not staining blue in iodine, aporynchous ascus base. paraphyses absent. ascospores 4-6(-9) x 2.5-4 μm, smooth, hyaline, thick-walled, elliptical, subglobose, amygdaliform or reniform, usually contains small globose oil drops and a nucleus most visible in lugol’s solution and congo red. excipulum of textura intricata, with septate hyaline hyphae (5)10-20(30) μm. habitat: fruiting bodies were found in a slope, scattered among the humid fallen needles of picea abies (fig. 2a, b). 2 specimen examined: kopaonik np (fig. 1), crvene bare nature reserve, 1600 m, p. abies forest, on litter, among p. abies fallen needles, attached on roots, very wet place, biologica nyssana ● 10 (1) september 2019: 1-8 fig 2. in situ photographs of neolecta vitellina; a) ascomata growing from spruce rootlets, photo i. kajevska, b) fruiting bodies at different stages of maturity. photo: m. blagojević. kajevska, savić ● first record of neolecta vitellina (bres.) korf & j. k. rogers and sowerbyella fagicola j. moravec in serbia biologica nyssana ● 10 (1) september 2019: 1-8 kajevska, savić ● first record of neolecta vitellina (bres.) korf & j. k. rogers and sowerbyella fagicola j. moravec in serbia 3 20/07/2016, i. kajeveska (legator-leg. a. trailović), m.s.n. 21/07/16-168; kopaonik np, crvene bare, cc. 1600 m, n 43°17’35.6”, e 20°48’21.81”, p. abies forest, on litter, among p. abies fallen needles, attached on roots, on a slope, 29/07/2017, i. kajevska, m.s.n. 29/07/17-240. sowerbyella fagicola j. moravec, česká mykol., 27 (2), p. 66 (1973). figs. 5 – 7. description of studied specimen: apothecia 5-23 x 3-9 mm in diameter, shallow cup-shaped, concave to fully extended in maturity; hymenium smooth, bright yellow or orange, young specimens somewhat translucent becoming dark orange in age and orange-brown when dry; excipulum concolorous with the hymenium, finely pubescent with indistinguishable white hairs; margin entire or slightly undulate, somewhat with darker color; flesh fig. 3. micrographs of neolecta vitellina, a) ascus with spores in congo red; b) asci with spores in iki; c) ascospores in iki; d) ascus with spores in iki; e) young ascus. bars: a, b, e = 10 μm; c,d = 5 μm. fig. 4. micrographs of neolecta vitellina, f) hymenium; g) asci bases; h) excipulum cells. bars: f =20 μm; g, h = 10 μm. thick, very soft, light and fragile, lubricous. stipe 10-27 x 2-4 mm, central, smooth, delicate, concolourous with the cap, often wrinkled-furrowed or somewhat moniliform, cylindrical but usually with attenuated base. asci 200-220(270) x 9-13 μm, cylindrical, 8spored, uniseriate, apex obtuse or truncate and inamyloid but asci walls somewhat blue in melzer’s reagent, ascus base pleurorynchous. paraphyses 200-240 x 2-3 μm, narrowly cylindrical, slender, septate, with yellow to orange pigments bluish in melzer’s reagent. ascospores 16-19.5(-22) x (6.5-)7.5-8.5(-9) μm, hyaline, thick-walled, fusiform, usually contains two globose oil drops (2.5-4.5 μm) accompanied with 1-2 smaller drops (< 2 μm) on the ends; ornamentation seemingly smooth but finely verrucose on oil immersion. medullary excipulum of textura intricata, with hyphae 20-45(65) x 5-7(9) μm, hyaline, with thin walls. ectal excipulum of textura subglobulosa to angularis, with hyphae of 20-40(50) x (8)15-30(45) μm. marginal hairs cylindrical, narrow, septate and hyaline to yellowish hairs of 30-150 x 4-7 μm. habitat: investigated specimens were found in the base of a gully with a lot of organic matter accumulated in a mixed deciduous forest (carpinus orientalis, quercus cerris, cornus mas, acer campesfig. 5. in situ photographs of sowerbyella fagicola; a) ascomata arising from litter composed of mixed deciduous forest, b) fruiting bodies at different stages of maturity. photos: i. kajevska. 4 biologica nyssana ● 10 (1) september 2019: 1-8 fig. 6. micrographs of sowerbyella fagicola, a) asci with spores, paraphyses; b) ascus tip; c) ascus base; d) spores with ornamentation in congo red; e) spores in ascus, congo red; f) paraphyses; g) spores in iki. bars: a, d, g = 10 μm. fig. 7. micrographs of sowerbyella fagicola, h) cells of medullary excipulum; i) cells of ectal excipulum; j) cells of ectal excipulum; k) hairs. bars: h, i, j, k = 20 μm. tre, robinia pseudoacacia, with one fagus sylvatica tree for about 20 m distance from the collection site), on a shaded place and humid for a long time. fruiting bodies scattered or gregarious, emerging from the moist forest litter (fig. 5a, b). specimen examined: suva planina mt. (fig. 1), above gadžin han town (niš), 347 m, n 43°12’25”, e 22°03’06”, mixed deciduous forest (c. orientalis, q. cerris, c. mas, r. pseudoacacia, a. campestre, f. sylvatica), on litter, attached on small fallen branches, leafs, tiny pieces of wood, etc., very wet place, 29/05/2016 and 26/05/2018, d. wieschollek (leg. i. kajevska), m.s.n. 29/05/16-162; m.s.n. 26/05/18-239. kajevska, savić ● first record of neolecta vitellina (bres.) korf & j. k. rogers and sowerbyella fagicola j. moravec in serbia discussion the species neolecta vitelina was first described in italy as geoglossum (microglossum) vitellinum bres. (bresadola, 1882). in north america, it was reported as mitrula vitellina (bres.) sacc. (durand, 1908), and lately due to the lack of paraphyses as an important distinguishing feature mains (1955) reports it under the name spragueola vitellina (bres.) nannf. and imai (1934, 1941) from japan as ascocorynium vitellinum (bres.) s. ito & imai. korf (1971) transfers m. vitellina into the genus neolecta speg. redhead (1977) reports n. vitellina in canada and finely places the genus neolecta within neolectaceae redhead due to its unique morphology. based on morphological and molecular analysis, neolecta has been placed in a separate order, neolectales landvik, o. e. erikss., gargas & gustafsson (landvik et al., 1993). subsequently, giving full morphological and ultrastructural description as well as molecular data of the genus species, landvik et al. (1996, 2001, 2003), afterwards sugiyama et al. (2006) and healy et al. (2013) confirms the unique position of neolecta (class neolectomycetes o.e. erikss. & winka, subphylum taphrinomycotina o. e. erikss. & winka) as the earliest lineage within ascomycota. neolecta vitelina so far is the only species of the genus that has been reported from europe. available published data on this species from europe come from the following countries: italy (type specimen-bresadola, 1882; carbone et al., 2015), poland (bujakiewicz, 1979), switzerland (ryman & holmåsen, 1992), germany (krieglsteiner, 1993), czech republic (svrček, 1978), fennoscandia-norway, sweden and finland (eckblad, 1963; ohenoja, 1975; hansen & knudsen 2000), romania (chinan & hewitt, 2012) and recently has been described and illustrated from austria (friebes, 2015). mycological field research held in summer 2016, at kopaonik np, revealed the first finding of n. vitellina in serbia. specimens have been preserved. another location of this species with numerous fruiting bodies was located the next year in the same area. although most reports of n. vitellina are known from northern europe, presumably, the precise geographical distribution across europe cannot be definite yet. most recent reports of this rare ascomycete like those from romania, austria and now from serbia shift its distribution range towards central and southern europe. reports of n. vitellina are scarce, therefore more focused research, monitoring and data with insights on local populations of this species are needed to better understand the nature of its distribution. our collection of n. vitellina originates from a forest slope with fruiting bodies emerging from the fallen needles in p. abies (l.) h. karst. forest at an altitude of cc.1600 m. nu5 biologica nyssana ● 10 (1) september 2019: 1-8 kajevska, savić ● first record of neolecta vitellina (bres.) korf & j. k. rogers and sowerbyella fagicola j. moravec in serbia merous fruiting bodies have been found at different stages of maturity. although this rare species has been found exclusively in spruce needle bogs, many questions regarding its morphology, phenology and its trophic affinity remain unclear. readhead (1979) indicates n. vitellina as parasite on spruce rootlets. fruiting bodies of our collections were also attached to spruce rootlets and while collecting was difficult to separate them from the substrate. macroand microscopically, the specimens collected from serbia correspond well with the descriptions presented in redhead (1977, 1979), hansen & knudsen (2000), landvik et al. (2003), chinan & hewitt (2012) and friebes (2015). until now we have only one known collection site, but it may likely occur on other sites especially within the kopaonik np where spruce forests prevail. the site where the species is registered is already within a protected area, as it is situated in a nature reserve in the national park, but it is an area of high recreational use. accordingly, we anticipate that all recreational activities on this site could be an important threat to the survival of this fungal species. sowerbyella fagicola so far is reported only from europe in the following countries: czech republic (type specimen-moravec, 1973; 1988), germany (krieglsteiner, 1979, 1993; häffner, 1989, 1993; hohmeyer et al., 1989, benkert, 2005; wieschollek, 2015), austria (rücker & wittmann, 1995; klofac & voglmayr, 2003), switzerland (rofler, 1999), poland (chmiel & ronikier, 2007), france (van vooren, 2007; moyne & petit, 2010); montenegro (perić, 2008); spain (galán et al., 2010) and recently from spain (ribes et al., 2016), bosnia and herzegovina (jukić & omerović, 2017) and bulgaria (assyov & slavova, 2017). recent reports of s. fagicola from the abovementioned three balkan countries have predicted an expected presence of this rare species also on serbian territory. thus, in may 2016 and subsequently in 2018, s. fagicola has been registered during mycological field research held in the southern part of serbia, precisely on suva planina mt., above gadžin han town. therefore, data on s. fagicola published in this paper is new for the country. sowerbyella fagicola was first described by moravec (1973). the spore ornamentation and macroscopically it reminds s. imperialis (peck) korf, but this species has smaller spores, and it is found in coniferous habitats (moravec, 1973, 1985, 1988). our collection contains fruiting bodies at different stages of maturity and macroand microscopically, the specimens collected from serbia correspond well with the descriptions presented in moravec (1973, 1985, 1988), roffler (1999), klofac & herrman (2003), benkert (2005), van vooren (2007), perić (2008), galán et al. (2010), wieschollek (2015), ribes et al. (2016) except for slightly differences in dimensions of the excipulum cells. the dimensions of the textura intricata cells in perić (2008) are 23.6-51.7 x 8.6-15.7 μm and are slightly wider than those from our collection (5-7(9)). also cells of the ectal excipulum from our collection (2040(50) x (8)15-30(45)) μm) are longer than those (11-23 x 10-22) noted by van vooren (2007). investigated specimens from our collection comprised of small amount of fruiting bodies (cca. 15) have been found in a shaded place, humid for a long time at the base of a gully where lot of organic matter is accumulated. the collection site was situated in a mixed deciduous forest comprised of c. orientalis mill., q. cerris l., c. mas l., r. pseudoacacia l., a. campestre l. and with only one f. sylvatica l. tree for about 20 m distance from the collection site. specimens were found scattered, some gregarious (with max. 3-4 fruiting bodies), barely visible among the moist layer of the forest litter. stipe base has been attached to fallen leaves, twigs, branches and other small-sized decaying organic matter. we managed to establish only leaf litter from c. orientalis, q. cerris, r. pseudoacacia and c. mas but without fagus leaf litter. it might also be worth to mention that the only herbaceous plant species on the collection site were galium trifolium michx. and hedera helix l. but poorly developed due to little sunlight unable to reach the ground in the dense forest. regarding the ecology of s. fagicola, moravec (1988) points out the presence of ammonia in the substrate as a possible condition for developing its fruiting bodies. so far this has not been confirmed. and although it is frequently related with fagus it has also been found occurring in other habitats. in our case and in reports from many countries fagus has not been always the predominant tree in the habitat but the most uncommon are the cases from france found in an oak forest (moyne & petit, 2010) and spain found in mediterranean oak forest (galán et al., 2010) and from spain found on hazel litter (ribes et al., 2016). thus, the real ecology and substrate preferences of s. fagicola, whether it is or not bound to fagus, remains unclear, but it is most likely a saprotrophic species growing on fine litter in its initial phase of decomposition. we also agree with the conviction noted by wieschollek (2015) that s. fagicola grows on a fine litter accumulated predominantly by deciduous trees. also, although suva planina mt., is also a special nature reserve and a protected area, during our continuous field studies we established various negative anthropogenic activities in this region. in 2017, on this site, we couldn’t found developed fruiting bodies of s. fagicola. namely, for the past few years, there were many actions of illegal logging and an increase in illegal landfills. these and similar activities soon biologica nyssana ● 10 (1) september 2019: 1-8 6 may be possible threat for the persistence of this and other rare species encountered in this small but regarding mycodiversity rich area. therefore, as soon as possible, this region should be an issue of concern and to be taken into consideration in all available national biodiversity monitoring and protection actions. regarding the status of s. fagicola, we consider it should be included in the preliminary national red list of fungi and for the future red book of fungi deserves a status of a critically endangered (cr) species b2b(iii) according to iucn categorisation, as well as its only known habitat to be protected. similar status for s. fagicola has been already foreseen in bosnia and herzegovina (jukić & omerović, 2017). conclusion the rare neolecta vitellina and sowerbyella fagicola have been registered during the period 2016-2018 in two selected regions in southern (suva planina mt.) and central (kopaonik np) serbia. although protected, both areas where these rare species persist are influenced by harmful anthropogenic activities which may be a possible threat to these ascomycetes. data on species with such status will increase the insights into better understanding of further fungal conservation approaches. therefore, morphological description based on found specimens, along with macroand microscopic images and biogeographical data from serbia have been fully presented in this paper. since it is already within a protected area, as it is situated in a nature reserve in the national park, it is an area of high recreational use. accordingly, we anticipate that all recreational activities on this site could be an important threat to the survival of this fungal species. acknowledgements the authors express sincere gratitude to dirk wieschollek for his useful information and generous help with the identification of s. fagicola. many thanks, we express to aleksandar trailović, danijela sokolović and marko blagojević, members of the mycological society ,,vilino kolo, petrovac na mlavi”, for providing us 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türkiye yusuf duşgun isparta university of applied sciences, faculty of agriculture, department of agricultural biotechnology, isparta, türkiye şükrü toksöz aydın adnan menderes university, faculty of agriculture, department of agricultural biotechnology, south campus, cakmar, aydın, türkiye hayrettin i̇lker özdemir aydın adnan menderes university, koçarlı vocational school, laboratory technology program, koçarlı/aydın, türkiye received: july 20, 2024 revised: september 19, 2024 accepted: september 23, 2024 abstract: in this study, phylogenetic analyses were performed using chloroplast rbcl sequences from ajuga chamaepitys, thymbra capitata, lamium moschatum, marrubium vulgare, stachys cretica, teucrium polium, and vitex agnus-castus species distributed in aydın province, türkiye. after isolating dna from the samples, pcr amplification was carried out using the rbclaf and rbclar primers. both forward and reverse sequences were edited using bioedit 7.2.3, finchtv 1.4.0, and sequencher 5.4.6 software. the corrected sequences were converted to protein sequences, and phylogenetic analyses were conducted using mega 6.0 software. the rbcl proteins have been motif analysed and their 3d structure revealed. as a result, the phylogenetic tree constructed with sequences obtained from national center for biotechnology information (ncbi) showed that lamium and marrubium species formed a clade, while sideritis and stachys species formed another one. additionally, the species of thymbra, vitex, ajuga, and teucrium each formed distinct groups. in the analyses involving only these seven species, the genetic distance matrix revealed that the closest species were ajuga chamaepitys and vitex agnuscastus (0.009), while the most distantly related species were teucrium polium and thymbra capitata (0.043). also, the nucleotide diversity was calculated to be π = 0.025278. overall, the rbcl sequence results were instrumental in elucidating the phylogenetic relationships within the lamiaceae family. key words: lamiaceae, phylogeny, rbcl, türkiye apstrakt: filogenetski odnosi između vrsta iz porodice lamiaceae iz ajdina (turska) na osnovu rbcl sekvenci u ovoj studiji su, korišćenjem hloroplastnih rbcl sekvenci ajuga chamaepitys, thymbra capitata, lamium moschatum, marrubium vulgare, stachys cretica, teucrium polium i vitex agnus-castus, vrsta prisutnih u provinciji ajdin, turska, sprovedene filogenetske analize. nakon izolacije dnk iz uzoraka, izvršena je pcr amplifikacija koristeći rbclaf i rbclar prajmere. obe sekvence (forward i reverse) uređene su korišćenjem softvera bioedit 7.2.3, finchtv 1.4.0 i sequencher 5.4.6. korigovane sekvence konvertovane su u proteinske sekvence, a filogenetske analize sprovedene korišćenjem softvera mega 6.0. rbcl proteini su analizirani na motive, a otkrivena je njihova 3d struktura. kao rezultat, filogenetsko stablo konstruisano sa sekvencama dobijenim iz nacionalnog centra za biotehnološke informacije (ncbi) pokazalo je da su lamium i marrubium formirale jednu, a sideritis i stachys drugu kladu. dodatno, thymbra, vitex, ajuga i teucrium formirale su zasebne grupe. u analizama koje su uključivale samo navedenih sedam vrsta, matrica genetičke distance je pokazala da su najbliže vrste ajuga chamaepitys i vitex agnus-castus (0,009), dok su najudaljenije vrste bile teucrium polium i thymbra capitata (0,043). takođe, izračunati diverzitet nukleotida iznosi π = 0,025278. pokazano je da su rbcl sekvence ključne u razjašnjavanju filogenetskih odnosa unutar porodice lamiaceae. ključne reči: lamiaceae, filogenija, rbcl, turska introduction türkiye is located in three phytogeographic regions and is considered a globally important center of biodiversity (başer & kırımer, 2018; yıldırım et al., 2024). the lamiaceae family in türkiye is © 2024 sevindik et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 53 represented by 787 taxa across 48 genera, including 608 species and 179 subspecies and varieties (behçet & cengiz, 2023). notable genera within this family include calamintha mill., mentha l., salvia l., satureja l., scutellaria l., stachys l., plectranthus l’hér., hyptis jacq., teucrium l., vitex l., thymus l., and nepeta l. (mamadalieva et al., 2021; kilinç et al., 2022). many members of the lamiaceae family are rich in essential oils, aromatic compounds, and secondary metabolites, making them significant in fields such as medicine, pharmaceuticals, cosmetics, and food production (agostin et al. 2009; hilooğlu et al. 2016; bekut et al. 2018). additionally, these species are known for their antimicrobial (ricci et al., 2005), antioxidant (özgen et al., 2006), antiinfluenza (protsenko et al., 2022), anticancer (kilinç et al., 2022), and antimutagenic (martínez-rocha et al., 2008) properties. the angiosperm chloroplast (cpdna) genome ranges from 107 to 218 kb and is organised as a circular dna molecule with a highly conserved tetragonal structure. most of the cpdna sequence encodes protein-coding genes, transfer rna (trna), and ribosomal rna (rrna) (lorenzana & rico, 2024). chloroplast dna sequences are effective and reliable for creating dna barcodes for plants, helping to resolve confusion in morphological identification (furan, 2024). in plants, the matk, ndhf, rbcl, rpob, rpoc1, psba-trnh, psbk-psbl, trnl-f and atpf-atph regions of cpdna sequences have been proposed as barcode gene regions (manjarres-hernández & morillo-coronado, 2023; chen et al., 2024; sevindik et al., 2024). among these gene regions, rbcl is recognized as an universal barcode gene that is ideal for plant species discrimination studies due to its high amplification efficiency and low mutation rate. this low mutation rate makes the rbcl gene suitable for detailed studies of intraspecific genetic and phylogenetic diversity (pere et al., 2023). in this study, cpdna rbcl sequences were used for the phylogenetic analysis of several lamiaceae species distributed in the aydin province of türkiye. materials and methods plant sampling and dna extraction technique ajuga chamaepitys (l.) schreb., thymbra capitata (l.) cav, lamium moschatum mill., marrubium vulgare l., stachys cretica l., teucrium polium l., and vitex agnus-castus l. species were collected from the south campus of aydın adnan menderes university, faculty of agriculture (37°45’35.85”n, 27°45’16.93”e) and brought to the laboratory for herbarium preparation. herbarium numbers of the species are given in tab. 1. genomic dna was isolated from the fresh green leaves using a commercial kit (genemark cat no: dp022). protocols and methods for polymerase chain reaction (pcr) and sequence analysis the information on the primer sequences, pcr protocol, and pcr components used in this study is provided in tab. 2. the pcr protocol was carried out according to sevindik et al. (2024). the polymerase chain reaction products were visualized using 1.0% agarose gel electrophoresis. the pcr results were sent to triogen biotechnology (istanbul, türkiye) for sanger sequencing analysis. for the analysis of both forward and reverse sequences, contig sequences were generated using bioedit 7.2.3 (hall, 1999), finchtv 1.4.0 and sequencher 5.4.6 software. the resulting sequences were then blasted at ncbi to check their similarity levels. species were uploaded to ncbi and accession numbers were obtained (tab. 1). phylogenetic and protein 3d structure analysis sequences of some species from the lamiaceae family, obtained from the national center for biotechnology information 54 biologica nyssana ● 15 (2) december 2024: sevindik et al. ● phylogenetic relationships among lamiaceae species from aydin (türkiye), based on rbcl sequences table 1. herbarium number, accession number, a+t, g+c (%) and total length (bp) of cpdna rbcl sequences in lamiaceae species species herbarium number accession number a+t g+c total ajuga chamaepitys lkr#1600 pq303556 55.8 44.2 536 lamium moschatum lkr#1606 pq303557 55.8 44.2 536 marrubium vulgare lkr#1603 pq303558 56.5 43.5 538 stachys cretica lkr#1604 pq303559 56 44 539 teucrium polium lkr#1601 pq303560 56.3 43.7 535 thymbra capitata lkr#1612 pq306792 56 44 536 vitex agnus castus e. sevindik 1203 pq303555 56.2 43.8 535 avg. 56 44 536.4 biologica nyssana ● 15 (2) december 2024: sevindik et al. ● phylogenetic relationships among lamiaceae species from aydin (türkiye), based on rbcl sequences 55 (ncbi), were aligned using the mega 6.0 program (tamura et al., 2013). a neighbourjoining (nj) phylogenetic tree (saitou & nei, 1987) was constructed using the same software. the phylogenetic tree was evaluated using the bootstrap test on a resampling of 1000 replicates (felsenstein, 1985). moreover, the nucleotide composition, genetic distance matrix, and nucleotide diversity of the rbcl sequences were determined. the rbcl sequences were converted to amino acid sequences using the dna-to-protein conversion tool in mega 6.0 software, and the amino acid content was analyzed using the same program. furthermore, the protein sequences were modeled in 3d using swiss-model (https://swissmodel.expasy.org/ interactive). to analyze all domains and conserved protein motifs, protein blast was employed in conjunction with meme (http://memesuite.org/doc/ fasta-format.html) (bailey et al., 2009). results and discussion the rbcl sequences ranged from 535 to 539 bp, with an average a+t content of 56.0% and a g+c ratio of 44.0% (tab. 1). according to the genetic distance matrix, the closest species were identified as ajuga chamaepitys and vitex agnus-castus (0.009), while the most distant species were teucrium polium and thymbra capitata (0.043) (tab. 3). tajima’s neutrality test indicated that the nucleotide diversity was π=0.025278. additionally, transition/ transversion rates for purines and pyrimidines, as well as the general transition/transversion rates independent of any base group, were calculated for the studied species and presented in tab. 4. it is shown that although the most important change is in pyrimidine bases, this situation is more balanced for purine bases than for pyrimidine bases. in this study, sequences of several species, including table 2. primers, pcr components, and pcr protocols primer name, 5’-3’ sequences and references pcr components pcr protocols rbcla-f: 5’-atgtcaccacaaacagagactaaagc-3’ (levin, 2013) 1 μl genomic dna 1 μl primer (forward), 1 μl primer (reverse), 5 μl master mix (pcr buffer, 2 mm mgcl2, dntp, 0.75 u taq dna polymerase) and 17 μl dh2o 95°c, 1 min; 35× (95°c, 30 s; 51°c, 30 s; 68°c, 1 min); 68°c, 5 min.rbcla-r: 5’-gtaaaatcaagtccaccrcg-3’ (kress et al., 2009) table 3. pairwise genetic distance matrix obtained from cpdna rbcl sequences species 1 2 3 4 5 6 7 ajuga chamaepitys lamium moschatum 0.027 marrubium vulgare 0.021 0.013 stachys cretica 0.023 0.021 0.017 teucrium polium 0.017 0.039 0.035 0.033 thymbra capitata 0.029 0.033 0.035 0.037 0.043 vitex agnus castus 0.009 0.023 0.019 0.021 0.027 0.025 table 4. maximum composite likelihood estimates of nucleotide substitution patterns a t c g a 4.1 3.11 11.1 t 4.13 19.78 3.35 c 4.13 26.07 3.35 g 13.66 4.1 3.11 lamium purpureum l., lamium maculatum (l.) l., lamium album l., lamium amplexicaule l., vitex negundo var. incisa (lam.) c.b.clarke, vitex zeyheri sond. ex schauer, vitex trifolia l., stachys byzantina k.koch, stachys germanica l., stachys alpina l., ajuga parviflora benth., ajuga bracteosa f. alba gürke, ajuga ciliata bunge, teucrium chamaedrys l., teucrium scordium l., teucrium scorodonia l., marrubium aschersonii magnus, 56 biologica nyssana ● 15 (2) december 2024: sevindik et al. ● phylogenetic relationships among lamiaceae species from aydin (türkiye), based on rbcl sequences marrubium peregrinum l., marrubium incanum desr. and thymbra spicata l. were obtained from ncbi, and the neighbor-joining (nj) phylogenetic tree was constructed. the phylogenetic analysis identified the following groups: marrubium and lamium species (bootstrap value 55%) stachys and sideritis species (bootstrap value 97%), vitex species (bootstrap value 79%), ajuga species (bootstrap value 78%), teucrium species (bootstrap value 54%), and thymbra spicata and thymbra capitata species (bootstrap value 100%) (fig. 1). (9.53%), while methionine was the least abundant (0.56%). five conserved motifs within the rbcl protein sequences were identified using the meme program (fig. 3). all species contain motifs 1, 2, 3, 4 and 5, which are associated with protein structure and function. some peptide motifs play important roles in protein protein-protein interactions (filiz & tombuloğlu, 2014). the 3d structure of rbcl proteins was modeled using the swiss-model program and is represented in fig. 4. the threedimensional structure of proteins facilitates drug fig. 1. the neighbour-joining tree generated using cpdna rbcl sequences of lamiaceae species, with sequences retrieved from ncbi the rbcl-based, phylogenetic tree showed clear genus-level clustering. the average amino acid composition of rbcl proteins was analyzed using the mega 6.0 program and graphically represented in fig. 2. glycine was the most abundant amino acid design. it helps to understand protein function and active sites (filiz & koç, 2014). in their sdspage analysis, ahmed and al-sodany (2019) identified teucrium polium along with micromeria imbricata and salvia deserti, as well as marrubium biologica nyssana ● 15 (2) december 2024: sevindik et al. ● phylogenetic relationships among lamiaceae species from aydin (türkiye), based on rbcl sequences 57 vulgare with otostegia fruticosa. in the rapd and issr dendrogram, teucrium polium and marrubium vulgare were clustered together. similarly, in their rapd-pcr analysis, topdemir et al. (2024) grouped lamium, stachys, and sideritis species together, teucrium and ajuga species together, while marrubium species formed a separate group. in our results, marrubium, lamium, sideritis and stachys were grouped together in a large group. this was supported by 81% of the bootstrap values. in the study by ayaz et al. (2020), cpdna rps14 sequence analysis placed marrubium vulgare with lamium album and lamium amplexicaule. in our study, the ncbi obtained lamium album sequence was also grouped with marrubium species. bendiksby et al. (2011) reported that the genus lamium is not monophyletic. in the phylogenetic tree constructed with the trnl intron, the trnl-f spacer, the rps16 intron and the matk sequences, the sideritis and stachys species appeared together, indicating that sideritis and stachys are not monophyletic genera. our study similarly found that stachys and sideritis species formed a single group. also, according to bendiksby et al. (2011), marrubium species were fig. 2. average amino acid composition of rbcl proteins fig. 3. combined block diagrams of conserved protein motifs in rbcl proteins, as determined by meme family. acknowledgements. this research was supported by the tubitak 2022/2209a (project no.: 1919b012223491). references agostini, f., santos, c. a., rossato, m., márcia, r. p., paula, l. s., serafini, l. a., molon, r., & patrick, m. (2009). essential oil yield and composition of lamiaceae species growing in southern brazil. brazilian archives of biology and technology, 52, 473–478. https://doi.org/10.1590/ s1516-89132009000200026 ahmed, s. m., & al-sodany, y. m. (2019). authentication of ecological, biochemical and molecular features for some lamiaceae species from saudi arabia. egyptian journal of botany, 59(3), 581-594. https://doi.org/10.21608/ ejbo.2019.6144.1246 ayaz, a., zaman, w., saqib, s., ullah, f., & mahmood, t. 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(2024). analyses of genetic diversity and population structure of endemic and endangered species sideritis gulendamii (lamiaceae) and implications for its conservation. genetic resources and crop evolution, 1-15. https://doi.org/10.1007/s10722024-01907-6 biologica nyssana ● 15 (2) december 2024: sevindik et al. ● phylogenetic relationships among lamiaceae species from aydin (türkiye), based on rbcl sequences 60 ostojić et al., 2020, biologica nyssana 11(1) 11 (1) september 2020: 9-22 doi: 10.5281/zenodo.4060283 natural values and concept of protection of the nature park “šargan-mokra gora” original article dragana ostojić institute for nature conservation of serbia, belgrade, serbia dragana.ostojic@zzps.rs (corresponding author) aleksandar dragišić institute for nature conservation of serbia, belgrade, serbia aleksandar.dragisic@zzps.rs danko jović institute for nature conservation of serbia, niš, serbia danko.jovic@zzps.rs verica stojanović institute for nature conservation of serbia, belgrade, serbia verica.stojanovic@zzps.rs bojan zlatković faculty of sciences and mathematics, university of niš, niš, serbia bojanzlat@yahoo.com vladimir nikolić ministry of agriculture, forestry and water management forest administration, belgrade, serbia vladimir.nikolic@zzps.rs miloš radaković institute for nature conservation of serbia, belgrade, serbia milos.radakovic@zzps.rs nenad sekulić institute for nature conservation of serbia, belgrade, serbia nеnad.sekulic@zzps.rs vladan bjedov institute for nature conservation of serbia, belgrade, serbia vladan.bjedov@zzps.rs mila ristić institute for nature conservation of serbia, belgrade, serbia mila.ristic@zzps.rs ana petković institute for nature conservation of serbia, belgrade, serbia ana.petkovic@ zzps.rs received: october 08, 2019 revised: april 30, 2020 accepted: may 08, 2020 abstract: this paper presents the results of the research and valorization of natural values of the šargan and mokra gora area, which were conducted by the expert team of the institute for nature conservation of serbia in 2005, 2008, 2015, 2016, and 2017. the area was protected in 2005 as outstanding natural landscape “šargan mokra gora”. in contrast, in 2008, the northwestern borders of the protected area were extended to include the sites “ljuto polje” and “dobro polje”, and the type of protected area has also been changed from the outstanding natural landscape to nature park. the paper further elaborates the results of the revision of natural values of the protected area, which has been done in 2015 for the purpose of harmonizing legal acts with the defined protection regimes according to the cadastre, and which has proposed a total protected area of 11,379.78 ha. increasing the area under protection was aimed for the conservation of natural values, sustainable development, and proper management of the protected area. key words: natural values, nature park šargan-mokra gora, protection concept, protection regimes i, ii and iii. apstract: prirodne vrednosti i koncept zaštite parka prirode „šargan-mokra gora“ u radu su prikazani rezultati istraživanja i valorizacije prirodnih vrednosti područja šargana i mokre gore, koje je uradio stručni tim zavoda za zaštitu prirode srbije tokom 2005., 2008., 2015., 2016. i 2017. godine. područje je stavljeno pod zaštitu 2005. godine kao predeo izuzetnih odlika „šargan mokra gora“, a 2008. godine proširena je granica zaštićenog područja na severozapadnoj strani lokalitetima „ljuto polje“ i „dobro polje“ i promenjena vrsta zaštićenog dobra iz predela izuzetnih odlika u park prirode. prikazani su i rezultati revizije prirodnih vrednosti zaštićenog područja urađene 2015. godine za potrebe usaglašavanja zakonskih akata sa definisanim režimima zaštite po katastru, kojom je predloženo da ukupna površina pod zaštitom bude 11.379,78 ha. povećanje površine pod zaštitom ima za cilj očuvanje prirodnih vrednosti, održivi razvoj i efikasno upravljanje zaštićenim područjem. ključne reči: prirodne vrednosti, park prirode „šargan mokra gora“, koncept zaštite, režim i, ii i iii stepena zaštite introduction the šargan mokra gora nature park is located on the territory of užice municipality, in southwestern serbia. it is situated between the massifs of tara (1,544 m) and zlatibor (1,359 m), including the mokra gora structural basin and part of the kremna structural basin, as well as the rivers kamišna, bratešina, beli rzav, and crni rzav. it is bordered by the tara national park (site zaovine) and the © 2020 ostojić et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 9 13th symposium on the flora of southeastern serbia and neighboring regions zlatibor nature park. šargan and mokra gora represent one of the phytogeographically most interesting areas of southwestern serbia and possess special natural values. based on these values, in 2005 the area was designated as an outstanding natural landscape measuring 3,678.99 ha. the revision of the natural values in 2008 resulted in the increase of the protected area to 10,813.73 ha, and the type of protected natural area was changed to the nature park, with a protection regime iii established in the expanded part of the protected area. according to the mid-term and annual program for the protection of natural resources and the spatial plan of the special purpose area of the tara national park (official gazette of the rs, no. 100/10), which also included the area of šargan and mokra gora, a legal obligation was introduced to evaluate the expanded area in detail. to revise natural values, a detailed multidisciplinary research was conducted in 2015, which slightly modified the outer boundary of the nature park. in addition, revaluation of existing sites was carried out along with the establishment of new sites with protection regimes i and ii, in comparison with those defined by the decree of 2005 and 2008. in the newly revised 2015 conservation proposal, the total area of this natural property was 11,379.78 ha (ostojić et al., 2015). the paper is based on the protection study of the outstanding natural landscape “šargan-mokra gora”, of 2005, and the protection study of the nature park “šargan-mokra gora” (ostojić et al., 2015). material and methods for valorization of natural values, field surveys were conducted to collect data and evaluate the data obtained. it is based on the national and international criteria for valorizing natural values. geological and hydrological characteristics have been selected according to general geological surveys of the area (mojsilović et al., 1977, 1978; olujić and karović, 1986a, 1986b; dimitrijevic, 1995 and others). in order to obtain as complete a picture as possible of the natural values for each area studied, existing field and literature data were used and mapped in the field. pursuant to the rulebook on the proclamation and protection of strictly protected and protected wild species of plants, animals and fungi (“official gazette of the rs”, nos. 05/10, 47/11, 32/16 and 98/16) (hereinafter: rulebook ) and the decree on the control of use and trade in wild flora and fauna (official gazette of the rs, no. 31/05, 45/05, 22/07, 38/08, 9/10 and 69/11) “strictly protected” and “protected wild species”, as well as species that are collected and traded therein have been determined. endangered taxa have been determined on the basis of field research and analysis of the threat factors and effects on populations in the wild, as well as according to the categorization of internationally accepted iucn criteria 1. the findings of the endangered taxa in the territory of serbia were also taken into account. species that are on the preliminary red list of flora of serbia have also been selected (stevanović (ed.), 2002). for the determination of forest phytocoenoses, the modern nomenclature was used with the latest principles of the syntaxonomy of forest vegetation of serbia, which were given in the editions: vegetation of serbia 2 (1) (jovanović et al., 1997) and vegetation of serbia 2 (2) (dinić et al., 2006). samplings of ichthyofauna were realized by the method of electrofishing to determine data on fish populations such as species composition, age structure, quantitative analyzes and other parameters. in the study of amphibians and reptiles fauna, sites were carefully selected for analysis, which were adapted to the weather conditions conducive to the daily activities of the herpetofauna. ornithofauna studies were based on the analysis and presence of endangered species closely related to particular habitats. they were derived according to the international standard classification of landscape types (corine, 1990), which were adapted to yugoslav conditions (stevanović, v., vasić, v., 1995). the evaluation of the investigated area as a natural resource was carried out in accordance with the rulebook on criteria for evaluation and procedure of categorization of protected areas (“official gazette of the rs”, no. 97/15), and based on based on the traits such as autochthony, rarity, representability, originality, etc. these criteria reflect the state of the natural values and their spatial distribution in the field. results and discussion the state and valorization of natural values geological characteristics the šargan-mokra gora nature park is situated in the area of the inland dinarides tectonic unit, and it is built of magmatic, metamorphic and sedimentary rocks that have been created over a long geological history. the oldest rock masses in the structure of the nature park area are sediments, mainly carbonates of the middle and upper triassic and upper cretaceous, and the most widespread are the jurassic ultramafics. 10 biologica nyssana ● 11 (1) september 2020: 9-22 ostojić et al. ● natural values and concept of protection of the nature park “šargan-mokra gora” in the long geological evolution of the carbonate mass of the karst plateau of tara and the peripheral parts of the peridotite massif of the nearby plateau of zlatibor, the tectonic movements caused the appearance of large blocks, which subsequently changed the original appearance to a large extent due to the erosion forces, and led to the formation of very dynamic relief (cvijić, j., 1924/1991). general epeirogenic ascent of the dinarides caused the appearance of deep-cut gorges and canyons in the area of šargan and mokra gora and the split in the relief of tusto brdo, putno brdo, kobilovac, vao brdo, njivičke kose, tabačke kose, skakavac, đoga, kozja stena and viogor. above these morphological units rise the peaks of kršanjska glava (1424 m) (fig. 1), carevića vis (1426 m), runjeva glava (1438 m), gavran (1453 m), iver (1478 m) and zborište (1544 m), which is the highest peak of the tara mountain. hydrological characteristics the hydrographic potential of the šargan and mokra gora areas consists of groundwater, springs and karst springs, larger and smaller river flows and thermal mineral waters, many of which are captivated for the needs of water supply. the waters of the mokra gora structural basin flow into the drina basin, whereas the waters of kremna structural basin flow into the basins of zapadna and velika morava. the watershed between the rivers kamišna and đetinja, that is, the drina and morava, is the šargan mountain that separates these two structural basins. springs and karst springs occur in the altitudinal zone of the peripheral area of šargan, tara and viogor, as well as in river valleys at the intersection of the valley side and the valley floor. the river network of the area consists of the kamišna, beli and crni rzav and their tributaries, the source-rivers of đetinja (bratešina and konjska river) the užice stream and a range of rather small rivers and streams, with numerous gorge valleys with rapids and waterfalls (veliki and mali skakavac on kamišna river), which represent interesting landscape units. the characteristics of flora the flora of this area was, for the first time, described by the famous serbian botanist josif pančić in 1860. pančić summarized his data in the capital 11 work of the serbian botany “flora of the principality of serbia” (pančić, 1874) and in the “addendum to the flora of the principality of serbia” (pančić, 1884), in which he says that mokra gora has an environment rich in rare plants. in his works, pančić described several species hitherto unknown to science. of the seven species described by pančić himself, or together with the italian professor of botany roberto visiani (18011878), as new to the science from this area (visiani et pančić 1862, 1866, 1870), five species still have the status of “good” or “undisputed” species. pančić points out that the flora and vegetation of mokra gora are distinguished by the presence of an impressive number of serpentinophytes, which is a term referring to specialized plant species of the characteristic and striking features, unique, very striking appearance, the occurrence of which is conditioned by a specific serpentinitic bedrock. the results of previous studies have shown that floristic richness and the diversity of the plant world of the mokra gora and šargan area are reflected in the presence of 722 taxa from the group of higherranked plants, excluding moss species (bryophyta) classified into 355 genera, or 92 families. it is assumed that the finite number of species is slightly higher. of the total flora species in this area, 6.2% are endemic and subendemic taxa, indicating that it is an area characterized by a diverse flora of distinct autochthonous character and originality. the largest number of endemics found in this area belongs to the group of balkan endemics. among the endemics, the most prominent are edraianthus jugoslavicus lakušić, micromeria croatica (pers.) schott, halacsya sendtneri (boiss.) dorfler, reichardia macrophylla (dc.) freyn, stachys anisochila vis. & pančić, alyssum markgrafii o. e. biologica nyssana ● 11 (1) september 2020: 9-22 ostojić et al. ● natural values and concept of protection of the nature park “šargan-mokra gora” fig 1. kršanjska glava (1424 m), photo: taken by camera drone 12 schulz ex markgraf, scrophularia tristis (k. maly) šilić, linaria rubioides vis. & pančić, helleborus serbicus adamović, euphorbia pancicii g. beck, euphorbia glabriflora vis., lathyrus binatus pančić and others. in the mokra gora area, 190 species are considered internationally and nationally important. according to the rulebook on the proclamation and protection of strictly protected and protected wild species of plants, animals and fungi, 102 species of the mokra gora area are being protected, 14 of them as strictly protected and 88 as protected species. in the mokra gora area 17 species are protected according to the cites convention on the international trade in endangered species of wild flora and fauna, and 35 plant species are protected according to the decree on the control of the use and trade in wild flora and fauna. the preliminary red list of endangered taxa of the flora of serbia (stevanović, ed. 1997) contains 49 species recorded during the research. also, the first volume of the red book of flora of serbia i lists rhaponticoides alpina (l.) m.v.agab. & greuter) (fig. 2), which grows only in ograđenica above mokra gora. this species, in accordance with the rulebook of the international union for the conservation of nature (iucn), has been categorized as the critically endangered taxon (cr) of the flora of serbia. the characteristics of vegetation in terms of vegetation, the mokra gora and šargan area belongs to the mesophilic western balkan forests of sessile oak and hornbeam (querco-carpinetum illyricum horv. et al. 1974) and beech forests of the fagenion illyricum horvat (1938) 1950 alliance. in the extended and cultivated parts of mokra gora, forests of alder and willow grows of the salicion albae soo (1930) 1940 alliance. in the serpentinitic gorges surrounding mokra gora, in addition to streams and rivers, there is the community of salicetum eleagni moor 1958 em. oberdorfer 1962, which is built by the gray willow. on thermophillic slopes, however, with a deeper soil layer, the forests of oak (quercetum cerris e. vukićević 1966) are developed, while in the humid valleys of the lower region and on the slopes rising above the streams the mesophillic forests of sessile oak and hornbeam belonging to the association querco petraea – carpinetum betuli rudski (1940) 1949 of the alliance carpinion betuli illiyrico-moesiacum ht. 1956 occur. the tree layer is composed of sessile oak (quercus petraea (mattuschka) liebl.) and hornbeam (carpinus betulus l.), while he herbaceous plant layer is mostly composed of: pulmonaria officinalis l., aposeris foetida (l.) less., corydalis solida (l.) clairv., lunaria rediviva l., epimedium alpinum l. and other forest species. the presence of valley lily (maianthemum bifolium (l.) f. w. schmidt), a species that is extremely rare in serbia and is regionally characterized as an endangered taxon (en) is of particular importance. on the mountains surrounding the mokra gora structural basin at altitudes of 700-800 m, sessile oak forests have been developed, building the quercetum montanum serpentinicum b. jovanović 1972 association. these are mostly degraded communities, developed on serpentinitic bedrock. the tree layer is dominated by sessile oak and in the ground layer semi-shrubs of winter heath (erica carnea l.) and daphne (daphne blagayana freyer) occur. steep slopes of ograđenica and tusto brdo (hill), occuring on shallow and rocky limestone, are overgrown with the ostryo carpinifoliae-fraxinetum orni aichinger 1933 community built by south european flowering ash and the european hop-hornbeam. a characteristic of this floristically rich community is the presence of peony (paeonia officinalis l.). it is a community of thermophilic, sub-mediterranean character, which in addition to edifying species is also built by an abundance of shrub species such as: acer monspessulanum l., evonimus verucosa scop., crataegus monogyna jacq., rhamnus saxatilis jacq, rosa canina l. and others. the rocky forest glades overgrown with smoke bush, especially on the ograđenica plateau, are very important since biologica nyssana ● 11 (1) september 2020: 9-22 ostojić et al. ● natural values and concept of protection of the nature park “šargan-mokra gora” fig 2. rhaponticoides alpina, photo by: m. niketić 13 they include endangered or rare species: frangula rupestris (scop.) schur, paеonia officinalis l., phyteuma orbiculare l., rhaponticoides alpina (l.) m.v.agab. & greuter, lathyrus binatus pančić, etc. the forests of european black pine develop on serpentinite, and in the mokra gora area two associations, erico-pinetum nigrae serpetinicum (z. pavlovic 1951) b. jovanović 1972 and ostryo-pinetum nigrae čolić 1965, occur. pine forests with winter heath (erico-pinetum nigrae serpetinicum) distribute all over šargan and viogor, as well as in the gorges and slopes descending towards mokra gora structural basin. these are light forests, where the ground layer is composed of winter heath (erica carnea l.) and of european blueberries (vaccinium myrtillus l.) at higher altitudes. the frequently occurring herbaceous plants in these forests are asplenium cuneifolium viv., potentilla malyana maly, stachys scardica (griseb.) hayek, veronica chamaedrys l., centaurea triumfetii all. and others. in more thermophillic and rocky sites grows spurge (euphorbia glabriflora vis.), while the steep slopes are almost 100% grown with sesleria serbica (adamovic) ujhelyi, protecting them from erosion. on the slopes of the entrance part of the beli rzav gorge, tusto brdo and vao peak, there is an another association of european black pine forests (ostryo-pinetum nigrae čolić 1965) (fig. 3). this association is composed of european black pine (pinus nigrae arn.) and hop hornbeam (ostrya carpinifolia scop.), in addition to which fraxinus ornus l., rhamnus fallax boiss., juniperus communis l. and others also occur in the community. this community is distinctively xerothermophilic, developed on limestone bedrock and on poor and shallow soil. beech communities consisting of fagetum moesiacae montanum bleč. et lakušić 1970 and fagetum submontanum moesiacum (rudski 1940) b. jovanović 1967 are developed in several places, whereby the largest areas of their distribution are the limestone soils of tusto brdo at the altitudinal range of 600 to 1100 m. the vegetation of rocky terrains and serpentinitic gorges is built by the communities of the alliances centaureo – bromion fibrosi blečić et all.1960 and potentillion visiani h. ritter-studnička 1970 of the order halacsyetalia sendtneri h. ritter-studnička 1970, which is a unique type of vegetation of serpentinitic ground. steep sites of serpentinitic gorges and steep serpentinitic rocky terrains of the mokra gora area are overgrown with the community festuco duriusculae – euphorbietum glabriflorae s. jov. et r. jov 1992., which has also been described in zlatibor (jovanović et al., 1992). the endemic, decorative euphorbia glabriflora vis. is dominant in this community, and this community consists of about one hundred plant species, the most frequent of which are festuca duriuscula l., galium purpureum l., teucrium montanum l., thymus jankae celak. and others. the particular importance of the community is reflected in the presence of endemic taxa such as halacsya sendtneri (boiss.) dorfler, stipa novakii martinovsky, genista friwaldskyi boiss, iris reichenbachii heuffel and other species. in gorges and canyons, the presence of numerous hazmophytic communities that develop in the crevices and recesses of the rocks is recorded. these areas are dominated by communities of the order asplenietea trichomanis br.-bl. 1934 corr. oberd. 1977 that are particularly well developed in serpentinitic gorges. on the other hand, the main characteristic of the vegetation of serpentinitic rocks are endemic serpentinophytes halacsya sendtneri (boiss.) dorfler and drymocallis malacophylla (borbás) kurtto. the two species are basic builders of the characteristic association halacsyo sendtneripotentilletum mollis pavlović 1955, which is particularly well developed in the kamišna river gorge and on the cliffs of kozja stena. beli rzav canyon is one of the characteristic sites where the rock vegetation is well developed (fig. 4). the vegetation of talus slopes (scree) occurs in the forest belt of oak and oak-hornbeam on limestone or serpentinitic bedrock. this vegetation consists of herbaceous plants, the roots of talus slopes grasses, or plants that look biologica nyssana ● 11 (1) september 2020: 9-22 ostojić et al. ● natural values and concept of protection of the nature park “šargan-mokra gora” fig. 3: the association of european black pine forests (ostryo-pinetum nigrae) at the šišatovac site, photo: v. nikolić 14 like low shrubs with very long semi-woody, creeping shoots that bind loose scree. it is a common occurrence that the vegetation of rocks and talus slopes is found in the same site, as is the case on ograđenica (fig. 5). the vegetation of limestone talus slopes is represented by the community of corydaletum euochroleucae b.tatić et b. atanacković 1972 and the community of saturejo montanae-achnatheretum calamagrostis r. jovanović et s. jovanović 1986. of particular importance is the occurrence of the species reichardia macrophylla (dc.) freyn, an endemic species of the southwestern part of the balkan peninsula, whereas the special characteristic of this vegetation is the occurrence of endemorelic species halacsya sendtneri (boiss.) dorfler on the limestone. the vegetation of serpentinitic talus slopes consists of roots of talus slope grasses, primarily the roots of achnatherum calamagrostis (l.) beauv. and medium-high endemic serpentinophytes: scrophularia tristis (k. maly) šilić, alyssum markgrafii o. e. schulz ex markgraf and linaria rubioides vis. & pančić. in these habitats often develop the brushwood of cotinus coggygria scop., which is formed by the suppression of forests. the characteristics of fauna within the boundaries of the protected area, a total of 6 species of fish from 3 families were recorded, namely: brown trout (salmo trutta linnaeus, 1758) from the trout family (fam. salmonidae), bleak (alburnoides bipunctatus bloch, 1782), danube barbel (barbus balcanicus kotlík, tsigenopoulos, ráb & berrebi, 2002), eurasian minnow (phoxinus phoxinus linnaeus, 1758), european chub (squalius cephalus linnaeus, 1758) from the carp family (fam. cyprinidae) and european bullhead (cottus gobio linnaeus, 1758) from the family cottidae (fig. 6). in terms of fish diversity in watercourses, the most species were recorded in beli rzav (5 species brown trout, bleak, danube barbel, european chub and bullhead), followed by kamišna river (3 species bleak, danube barbel and european chub), bratešina river (2 species – danube barbel and eurasian minnow) and dubošac (1 species danube barbel). of the total of 6 species of fish recorded in the waters of this area, only european bullhead has the status of strictly protected species according to the rulebook on the proclamation and protection of strictly protected and protected wild species of plants, animals and fungi, while 4 species have the status of protected wild species, namely: brown trout, bleak, danube barbel and european chub. the only source of data related to earlier research of batrachofauna and herpetofauna of šargan and mokra gora were data collected by radovanović (1951), as well as data collected during surveys conducted in the surrounding areas (tara national park) in 2002 and 2003. a total of 17 amphibian and reptile species (eight species from the amphibia biologica nyssana ● 11 (1) september 2020: 9-22 ostojić et al. ● natural values and concept of protection of the nature park “šargan-mokra gora” fig. 4: vegetation of rocks in beli rzav canyon, photo: taken by camera drone fig. 5: vegetation of the talus slopes and rocks on ograđenica, photo: photo documentation of the institute for nature conservation of serbia 15 class and nine species from the reptilia class) have been recorded in the šargan and mokra gora. in the lower bed of the kamišna river canyon horned viper vipera ammodytes linnaeus, 1758 has been recorded, whereas in the upper course of the river the yellow-bellied toad (bombina variegatа linnaeus, 1758) has been recorded in several sites. the twelve recorded species are strictly protected, while two species have the status of protected wild species pursuant to the rulebook on the proclamation and protection of strictly protected and protected wild species of plants, animals and fungi. of the 12 strictly protected species, 7 species are from the genus amphibia amphibians, of which two species belong to the tailed amphibians: fire salamander (salamandra salamandra linnaeus, 1758) and smooth newt (lissotriton vulgaris linnaeus, 1758), the other five species belong to the order of tailless amphibians (frogs): yellow-bellied toad (bombina variegata linnaeus, 1758), common toad (bufo bufo linnaeus, 1758), green toad (pseudepidalea viridis laurenti, 1768), greek stream frog (rana graeca boulenger, 1891), and agile frog (rana dalmatina fitzinger, 1838). the remaining 5 strictly protected species are from the genus reptilia reptiles and all species belonging to the snakes group (serpentens): aesculapian snake (zamenis longissimus laurenti, 1768), smooth snake (coronella austriaca laurenti, 1768), grass snake (natrix natrix linnaeus, 1758), dice snake (natrix tessellata laurenti, 1768) and common european viper (vipera berus linnaeus, 1758). considering the threat to these species, all species are on the list of the red book of fauna of serbia i amphibians (kalezić et al. 2015) and the red book of fauna of serbia ii reptiles (tomović et al. 2015). the initial ornithological survey of the wider area of šargan and mokra gora was carried out by the ornithologist othmar reiser during july 1899. based on the surveys conducted so far in the nature park “šargan mokra gora”, 120 bird species have been recorded, which, if compared with the bird diversity in the whole of serbia including 352 species (šćiban et al., 2015), represents 34% of the total bird diversity in serbia. most species of birds in šargan and mokra gora (101 species) are strictly protected according to the rulebook on the proclamation and protection of strictly protected and protected wild species of plants, animals and fungi, which includes the prohibition of killing, collecting eggs, disturbing nesting sites and other activities that may endanger the survival of these species. a total of 18 bird species are protected, which is regulated, in addition to the aforementioned rulebook, also by the rulebook on the closed season for protected game animal species (official gazette of rs, no. 75/2010). this group also includes game animal species such as quail, partridge, pheasant, mallard, wood pigeon and turtle dove. the importance of the area is reflected in the presence of nationally and internationally important species, as well as in the presence of vulnerable and endangered species closely related to particular habitats, which primarily refers to species such as: golden eagle (aquila chrysaetos linnaeus, 1758), the short-toed snake eagle (circaetus gallicus gmelin, 1788), peregrine falcon (falco peregrinus tunstall, 1771), capercaillie (tetrao urogallus linnaeus, 1758), corn crake (crex crex linnaeus, 1758), the collared flycatcher (ficedula albicollis temminck, 1815), the red-breasted flycatcher (ficedula parva bechstein, 1794), willow tit (parus montanus conrad von baldenstein, 1827) and the wallcreeper (tichodroma muraria linnaeus, 1758). at the international level, the birds of šargan and mokra gora are protected by various international conventions and directives. in this area, 23 species are listed in appendix i of the european birds directive (209/147/ec), according to which eu member states are required to designate special protected areas (spas) for the purpose of these birds protection. available professional and scientific papers (petrov, 1992; savić et al., 1995; paunović and milenković, 1996; kryštufek, et all., 1997; savić et all., 1997; marinović, 1997; paunović et al., 2011), as well as the results of field surveys of the institute for nature conservation of serbia in the area of šargan and mokra gora, indicate that this area is biologica nyssana ● 11 (1) september 2020: 9-22 ostojić et al. ● natural values and concept of protection of the nature park “šargan-mokra gora” fig. 6: european bullhead (cottus gobio), photo: n. sekulić permanently or occasionally inhabited by at least 49 mammal species, which makes up about half of the number of species registered so far in serbia. it is considered that this number is not definitive, given the still poor exploration of the whole space and individual groups. the largest group consists of bats (chiroptera) and rodents (rodentia), with 13 species of each order. all species of bats in serbia have the status of strictly protected species according to the rulebook on the proclamation and protection of strictly protected and protected wild species. as far as rodents are concerned, there are three strictly protected species in this area: lesser mole-rat (spalax leucodon nordmann, 1840), forest dormouse (dryomys nitedula pallas, 1778) and hazel dormouse (muscardinus avellanarius linnaeus, 1758), as well as two protected species: squirrel (sciurus vulgaris linnaeus, 1758) and edible dormouse (glis glis linnaeus, 1766). they are followed by carnivores (carnivora) with 11 species and species of the order eulipotyphla 8 of them. all these species fall into the category of “protected species”, with the carnivores such as otter (lutra lutra linnaeus, 1758) and the brown bear (ursus arctos linnaeus, 1758), and “shrew-form” (neomys fodiens pennant, 1771), which are classified as “strictly protected species” according to the same rulebook. the fauna of carnivores in the mokra gora and šargan currently includes the following species: wolf (canis lupus linnaeus, 1758), golden jackal (canis aureus linnaeus, 1758), red fox (vulpes vulpes linnaeus, 1758), brown bear (ursus arctos linnaeus, 1758), least weasel (mustela nivalis linnaeus, 1766), european polecat (mustela putorius linnaeus, 1758), european pine marten (martes martes linnaeus, 1758), beech marten (martes foina erxleben, 1777), badger (meles meles linnaeus, 1758), otter (lutra lutra linnaeus, 1758) and wildcat (felis silvestris schreber, 1775). to the smallest group belong the ungulates (artiodactyla) with 3 species: wild boar (sus scrofa linnaeus, 1758), chamois (rupicapra rupicapra linnaeus, 1758) and roe deer (capreolus capreolus linnaeus, 1758). lagomorphs (lagomorpha) are represented with 1 protected species european hare (lеpus еurоpаеus pallas, 1778). the characteristics of “šargan mokra gora” nature park the results of research and valorization of natural values of the area of šargan and mokra gora have shown that it is characterized by the following core values that account for its status of a nature park and its protection regimes: biologica nyssana ● 11 (1) september 2020: 9-22 ostojić et al. ● natural values and concept of protection of the nature park “šargan-mokra gora” • originality (autochthonous character) – the area is relatively well conserved and without significant and visible negative human intervention. a special characteristic of this area is the geological bedrock in the form of old masses of magmatic, sedimentary and metamorphic rocks, which were formed as a result of long geological history, and on which the especially rare and specific natural values of biodiversity were formed. the combination of fragments of high plains on watersheds with pastures and high pine trees of steep valley sides and deep-cut river valleys overgrown with dense centuries-old forests and here and there narrowed settlements give this area a distinct feature and specific beauty with a high degree of attractiveness. the pronounced presence of scots pine and black pine forests, in the form of pure and mixed stands, is a distinguishing feature of the autochthonous habitat, and thus more rapid and qualitative forest restoration. an example of a particularly important conserved old forest complex is the site “jelovac šišatovac”, in which a rare and endangered western capercaillie and other characteristic species of old black pine forests have been recorded. • representativeness – is reflected in the presence of large complexes of coniferous and mixed forests, certain representatives of flora, fauna and vegetation. in addition, this area is best represented by the species of plants that pančić first described here for science. on limestone rocky grounds and rocks, pančić discovered stachys anisochila vis. & pančić), and on serpentinitic rocky grounds he discovered haplophyllum boisserianum vis. & pančić, linaria rubioides vis. & pančić, euphorbia glabriflora vis. and potentilla visianii pančić. strictly protected species of ichthyofauna – european bullhead is an important resident of the waters of beli rzav. representatives of the insectivore fauna are the eurasian water shrew and mountain shrew, while the representatives of rodent fauna are: lesser mole-rat, forest dormouse and hazel dormouse. notable representatives of the carnivorous fauna are the brown bear and otter, as strictly protected species. the main brown bear habitats are in the beli rzav canyon, the sites tusto brdo and vao limestone hill. otter is a resident of mountainous, conserved, permanent watercourses of beli rzav, crni rzav and kamišna. • rareness – is expressed in highly conserved and pronounced biodiversity in a relatively small protected area. the criterion is evident in the distribution of natural black pine habitats on serpentinite, which are singled out as rare habitats in serbia. 16 biologica nyssana ● 11 (1) september 2020: 9-22 ostojić et al. ● natural values and concept of protection of the nature park “šargan-mokra gora” on the limestone massif of the site “ograđenica” the only habitat of rhaponticoides alpina (l.) m.v.agab. & greuter in serbia has been identified, which is a natural rarity in the category of critically endangered taxa (cr), the population of which is estimated at only a few specimens. in the mesophillic oak and hornbeam forests a species that is extremely rare in serbia has been recorded, namely maianthemum bifolium (l.) f. w. schmidt. rare representatives of the animal world are mountain eurasian water shrew and mountain shrew, wildcat and chamois. concerning the presence of rodents, there is forest dormouse, a rare species that inhabits thermophillic deciduous forests with a rich bush layer and rocky grounds within this layer, and with one typical habitat on the site “ograđenica”. • diversity is pronounced in landscape features deep-cut gorges, canyons, and dissected plateaus. the richness of species and ecosystem diversity is reflected by the presence of 722 species of vascular flora, classified into 355 genera or 92 families, 10 most represented and most important communities of forest ecosystems, 7 representative communities of the vegetation of rocks, talus slopes and rocky terrains. the importance of floristic diversity is further enhanced if it is known that a small area contains 22% of the total number of serbian flora, of which 6.2% of the area’s flora consists of endemic and subendemic taxa that represent a rare, specific from the point of biodiversity conservation view, and an extremely valuable group of obligate serpentinophytes. particularly valuable in terms of floristic features are the extremely rare serpentinophyte communities occurring on the rocky terrains with festuco duruusculae-euphorbetum glabriflorae and in the gorges with potentillo-halascyetum sentdneri, as well as the hazmophytic community of endemic edraiathion alliance on limestone, in which the endemic taxa form unique phytocoenological combinations. the fauna of this area is characterized by six (6) species of fish, 17 species of amphibians and reptiles, 120 species of birds and 49 species of mammals. most registered species and builders of determined communities have legal protection status, since they represent endemic and relict taxa and communities, as well as rare and endangered plant and animal species that inhabit different, mosaically distributed habitats such as: forests, pasture-meadows, rock habitats and rocky terrains, as well as aquatic habitats. • integrity – the nature park represents a whole in the geographical and geomorphological terms. it is mostly bounded by natural watersheds that separate it from adjacent river basins, which makes it a geomorphologically well-individualized area, and thus the defining of protection borders is made much easier. • beauty/aesthetics criterion – the beauty/aesthetics of the area of šargan and mokra gora is evidenced with the exceptional seasonal dynamics and vividness of colors, the mosaic shift of the diverse rural-pastoral ambiental sequences and the specific architecture of each of the railway station complexes, as well as by drvengrad. an additional beauty of this area is given by the narrow-gauge heritage railway “šarganska osmica” (šargan eight), which has become famous for its technical solution of overcoming a great altitude difference at a short distance. • conservation – natural landscapes and forest complexes in the protected area are an important factor in the conservation of ecosystem services, environmental elements, and are a significant renewable natural resource. this picture is complemented by the exceptionally conserved watercourses of the area, which is confirmed by the specific fish fauna that inhabits them (brown trout, southern barbel/gudgeon, european bullhead, chub, etc.). • the monumental heritage of šargan and mokra gora is unique in the wider region. the ethno settlement drvengrad on mećavnik, which although recently built, seems to have been here for centuries, also gives an exceptional cultural value to the area. the concept of protection and protection regimes taking into account the state in the field, protection objectives and legislation, the concept of protection of the nature park “šargan mokra gora” is based on conservation of natural and created values. for this reason, protection regimes i, ii and iii have been established in this area. the total protected area is 11.379,78 ha, within which special spatial units with protection regimes i, ii and iii have been established. protection regimes i and ii cover the most valuable and conserved natural values and specific features of the area at 3,889.70 ha. as a starting point for establishing the protected area, a study on protection was prepared as a professionally documented basis for initiating protection. the mentioned study was submitted to the ministry of environmental protection, which informed the public about the procedure for initiating protection of the natural area on the ministry’s website. with this, the area of 11,379.78 17 ha is considered protected according to the law on nature protection (official gazette of the rs, nos. 36/09, 88/10, 91/10 correction and 14/16) (fig. 7). following the revision of the natural values of the protected area, in 2015, the area under protection regime i was increased from 414.84 ha to 774.50 ha (6.81% of the total protected area). the regime of protection is prescribed in five isolated sites within the protected natural area, of which 4 sites have previously been selected. these are: site “jelovac” is situated in the eastern part of protected area. upon revision, its acreage has been 18 decreased and is now 11.31 ha. the site is overgrown with tall, medium-age, welltended forest of european black pine (erico-pinetum nigrae (z. pavlović 1951) b. jovanović 1972), which is a typical representative of the pine forest on the serpentinic bedrock of šargan mountain. “klisura dubošca-skakavac” (dubošac gorgeskakavac) covers an area of 271.34 ha. part of this site is within the previously designated site in the protection regime i “klisura dubošca” (dubošac gorge) (area 106.01 ha). the gorge is located in the southeast part of the protected area and it is deeply cut into the serpentinitic massif. the skakavac waterfall, in the kamišna river bed, about 12 m high, is particularly prominent in this area. the steep sections of the entrance to this gorge contain imposing, solitary and very old specimens of european black pine, which together with hop hornbeam build the association ostryopinetum nigrae čolić 1965. site “ograđenica” is a site that previously covered 73.12 ha and was expanded to 164.67 ha after the revision. it is located in the southern part of the protected area. it is a limestone ridge surrounded by serpentinites, and in some way, has an island character regarding the distribution of a number of flora and fauna species that are ecologically related to thermophillic limestone forests, carbonate rocky terrains and talus slopes. site “međedova ljeska” covers an area of 8.35 ha, prior to the revision of the protection regime i it was established at 123.98 ha. it is located in the southern part of the protected area and at the far southwestern point it is in contact with the site “tusto brdo” in the protection regime ii, which is in the immediate vicinity of the plateau of ograđenica, from which it differs in orographic terms and therefore in vegetation. it is a typical example of biologica nyssana ● 11 (1) september 2020: 9-22 ostojić et al. ● natural values and concept of protection of the nature park “šargan-mokra gora” fig. 7: area proposed for protection in 2015, with the external border, protection regimes and sites under certain regimes, map: d. kaličanin a combination of thermophilic oak and mesophilic beech habitats in a relatively small area. site “debela kosa” is a newly selected site under the protection regime i, which covers an area of 318.83 ha. it is located in the southeast part of the protected area. it is overgrown with tall forest of european black pine (erico-pinetum nigrae (z. pavlović 1951) b. jovanović 1972 and euphorbio glabriflorae-pinetum nigrae pavlović, 1952) on the primeval humus-silicate soils of the peridotite and serpentinite bedrock. according to the decree on the protection of outstanding natural landscape “šargan mokra gora” (“official gazette of rs”, no. 52/05), the area under protection regime ii covered 1,187.46 ha. upon revison in 2015, this area was increased to 3,124.70 ha (27.46% of the total protected area) and the protection regime ii was established at ten isolated sites within the protected area: site “šišatovac-jelovac” has previously been called šišatovac, which now covers 105.69 ha. it is located in the eastern part of the protected area, and is overgrown with high forests of black pine (ericopinetum nigrae (z. pavlović 1951) b. jovanović 1972), which are young, of an extremely dense canopy cover and with a large amount of residual felling (timber stock). site “đoga” is part of the former site “đoga kozja stena”, covering an area of 148.66 ha. this site is in the far southeast of the protected area. the steep slopes and the massive remnant rocks of the đogo ridge are dominated by giant black pine trees of umbrella-like tree crowns. site “krečnjačko brdo vao” (limestone hill vao) covers an area of 130.61 ha. it is located in the central part of the protected area and has been reduced in size upon the revision. it encompasses the deep-cut and heavily accessible valleys of the grubiševac and suvodol streams. the foothill of this site is covered with communities of hay meadows (festuco-brometea), which are characterized by the presence of different species of clover and legumes, which makes them economically important. at higher altitudes, meadow enclaves are replaced by the beech forests of fagetum moesiacae montanum bleč. et lakušić 1970 of good quality with trees of seed origin and pronounced dendrometric characteristics. site “tusto brdo” formerly part of the site “tusto brdo božurica”, which upon the revision has been divided into “tusto brdo” and “božurica”, covers 298.73 ha. it is located in the southern part of the protected area. in terms of vegetation, this site is covered with man-grown cultivated mixed stands of black pine and scots pine, which at their age of about 60 years according to structure, cenotic composition and stability, are very similar to the natural forests of scots pine and black pine (pinetum sylvestris-nigrae typicum pavlović, 1952). site “božurica” covers 64.04 ha, and it is located in the southern part of the protected area. one part of this landscape unit is covered with man-grown black pine (pinetum nigrae) stands on different brown soils, while the other part is covered by mountain beech forest (fagetum moesiacae montanum bleč. et lakušić 1970) on different brown soils. site “ljuto polje” is a newly selected site located in an extended part of the protected area, in the far northern part of the protected area, with a total area of 503.53 ha. it is a karst field with pronounced forms of karst micro-relief, the natural characteristics of which are contained in the mixed forests of spruce and scots pine that are unique in the mokra gora area. also, numerous mires are important as habitats of priority for protection at the national and international levels. site “bratešina” is a newly declared site of 1,396.52 ha. this is the largest site in the protected area, which is located in its central part, whereas in its southeastern part there is a site of the first degree of protection “jelovac”. the basic vegetation of this site consists of forests of black pine (erico-pinetum nigrae (z. pavlović 1951) b. jovanović 1972 and euphorbio glabriflorae-pinetum nigrae pavlović, 1952) on initial humus-silicate soils on peridotites and serpentinites, as well as pine forests (ornoericion horvat, 1959 and orno-pinion em, 1978) on soils formed on base rocks. site “ograđenica 2” is a new site measuring 79.93 ha. part of this site was within the previously declared site in protection regime ii “tusto brdo božurica”, and is surrounded on the north, west and southwest side by a limestone massif, that is, the site in the protection regime i “ograđenica”. considering the geomorphological, vegetational and ambient features of the part of ograđenica, it represents a buffer zone around the area under protection regime i and it physically encircles the entire area of the massif. site “kanjon belog rzava” (beli rzav canyon) is a new site of 279.77 ha. it is located in the southernmost part of the protected area. in terms of vegetation, this site is very diverse, at higher elevations, there are the specimens of magnificent, single, black pine trees, as remnants of former natural black pine forests. site “šarganska osmica” (šargan eight) is a new site of 117.22 ha. it is located in the southeast part of the protected area. this site is overgrown with pine forests (orno-ericion horvat, 1959 and orno-pinion em, 1978) on soils formed on base rocks. the forests in this site are in one part natural biologica nyssana ● 11 (1) september 2020: 9-22 ostojić et al. ● natural values and concept of protection of the nature park “šargan-mokra gora” 19 tall black pine forest, while the rest of these forests are cultivated. the protected area under the protection regime iii occupies a total of 7,480.58 ha (65.73% of the total protected area). compared to the initial protection of the area from 2005, the areas under the protection regime iii were increased from 2,138.77 ha to 5,341.81 ha. conclusion due to its geographical location, geological bedrock, hydrographic features, orography, climate and historical development, the mokra gora and šargan area is characterized by a diversity of species and ecosystems of exceptional originality. orographic plasticity and the alternation of different geological bedrocks in a rather small area are certainly the most important reasons for the pronounced richness of flora and vegetation. the relatively small area of šargan and mokra gora is inhabited by 722 species of vascular flora, of which 6.2% of taxa belong to endemic and subendemic flora. there are 14 species in this area, which represent strictly protected species, as well as 88 protected species. from a scientific point of view, particular traits of this area are very rare endemic taxa adapted to the highly unfavorable serpentinitic bedrock. the largest number of endemics found in this area belongs to the group of balkan endemics. in terms of vegetation, the šargan and mokra gora area is a natural habitat of black pine and scots pine. these pines build pure and mixed stands, significant within large forest complexes. forest ecosystems of šargan mokra gora nature park consist of 10 representative communities, namely: alder forests and willows of the salicion albae soo (1930) 1940 alliance, gray willow community (salicetum eleagni moor 1958 em. oberdorfer 1962), turkey oak forests on thermophilic slopes (quercetum cerris e. vukićević1966), mesophilic forests of sessile oak and hornbeam (querco-carpinetum moesicae rudski (1940) 1949), type of sessile oak forest (quercetum montanum serpentinicum b. jovanović 1959), as well as the community of south european flowering ash and hop hornbeam (ostryo carpinifoliae-fraxinetum orni aichinger 1933). considering the pine forests, the most important are the two associations: pine forests with winter heath (erico-pinetum nigrae serpetinicum krause 1957) on serpentinite and black pine forest with hop hornbeam (ostryo-pinetum nigrae čolić 1965) on limestone. beech forests of the types fagetum moesiacае montanum bleč. et lakušić 1970 and fagetum submontanum (rudski 1949) b. jovanović 1976 are distributed on the limestones of tusto brdo. the mokra gora and šargan area is home to a rich and diverse wildlife. in the waters of this area, a total of six (6) fish species have been recorded, with five (5) species having protection status. only european bullhead has the status of a strictly protected species, while four (4) protected species are: brown trout, spirlin/common bleak, southern barbel/gudgeon and chub. in addition, 17 species of amphibians and reptiles have been recorded in the area of šargan and mokra gora. of these, 12 species are strictly protected, two species are protected, while three species do not have protection status. the ornithofauna significance is reflected in the presence of 120 bird species, of which 101 species are strictly protected and 18 species are protected. the protected area is also characterized by 49 species of mammals, which makes up about half the number of species registered so far in serbia. the šargan-mokra gora nature park, with its conserved natural landscapes, is a recognizable landscape through which runs the narrow-gauge railway šargan eight, which is widely known for its remarkable technical solution of overcoming a great altitude difference at a short distance. 10 years upon the establishment of spatial protection, the conducted research, revision and revaluation of the natural values of šargan and mokra gora, the boundaries of the protected area were expanded, the borders under the protection regimes were revised, new sites with the protection regimes i and ii were established, with the proposed total protected area of 11,379.78 ha. with this proposal, the area under protection regime i is 774.50 ha (6.81% of the total protected area) and is located in five isolated sites. under the protection regime ii, the proposed area occupies 3,124.70 ha (27.46% of the total protected area) and is located in ten isolated sites, while the area with the protection regime iii amounts to 7,480.58 ha (65.73% of the total protected area). the concept of protection of the area is aimed at conserving the overall diversity, autochthonous character and originality of species and habitats, the sustainable and controlled use of natural values and rarities. the concept is implemented through three protection regimes and legal measures defining certain regimes. the conservation guidelines defined in the conservation study (ostojić et al., 2015) aims to preserve and enhance the authenticity and autochthonous character of this area, which requires the cooperation of public institutions and civil society sectors. all the mentioned features of the šargan – mokra gora nature park make it one of the most important natural resources for biodiversity conservation in serbia. based on the valorization of the state of natural values, the area meets the criteria of the protected natural area in the nature park category. biologica nyssana ● 11 (1) september 2020: 9-22 ostojić et al. ● natural values and concept of protection of the nature park “šargan-mokra gora” 20 references cvijić, j. 1924: geomorfologija. morphologie terrestre, državna štamparija kraljevine srba, hrvata i slovenaca, beograd. cvijić, j. 1991: geomorfologija i. reprint državne štamparije iz 1924 godine, beograd, 1-558. dimitrijević, m. 1995: geologija jugoslavije. geoinstitut i barex, beograd, 1-205. dinić, a., tomić, z., mišić, v., tatić. b., janković, m., m., jovanović, b. 2006: vegetacija srbije ii 2. sanu, odeljenje hemijskih i bioloških nauka, beograd. jovanović, b. et al. 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15: 3-21. pravilnik o proglašenju i zaštiti strogo zaštićenih i zaštićenih divljih vrsta biljaka, životinja i gljiva („službeni glasnik rs“ br. 5/2010, 47/2011, 32/2016 i 98/2016). pravilnik o kriterijumima vrednovanja i postupku kategorizacije zaštićenih područja („službeni glas rs“, br. 103/2013 i br. 97/2015). pravilnik o prekograničnom prometu i trgovini zaštićenim vrstama („službeni glas rs“, br. 99/2009 i br. 6/2014). prostorni plan područja posebne namene nacionalni parka „tara“ („službeni glasnik rs“, br. 100/2010). uredba o stavljanju pod kontrolu korišćenja i prometa divlje flore i faune („službeni glasnik rs“, br. 31/2005, 45/2005, 22/2007, 38/2008, 9/2010, 69/2011 i 95/2018-drugi zakon). uredba o zaštiti prirodnih retkosti („službeni glasnik rs“, br. 53/93, 93/93) uredba o zaštiti predela izuzetnih odlika „šargan mokra gora” („službeni glasnik rs“, br. 52/2005). uredba o zaštiti parka prirode „šargan mokra gora” („službeni glasnik rs“, br. 81/2008). zakon o divljači i lovstvu („službeni glasnik rs“, br. 18/2010). zakon o zaštiti prirode („službeni glasnik rs“, br. 36/09, 88/10, 91/10ispravka i 14/16). 22 kumar and kaul 2025, biologica nyssana 16(1) 23 16 (1) june 2025: 23-34 doi: 10.46793/biolnyss.16.1.3k role of nanotechnology in biomedical advances: a review of innovations and future directions review article pankaj kumar department of physical and mathematical science, baba farid college, bathinda, punjab, india pankajkullu22451@gmail.com (corresponding author) jasmeet kaur department of physical and mathematical science, baba farid college, bathinda, punjab, india received: november 13, 2024 revised: january 09, 2025 accepted: january 10, 2025 abstract: nanotechnology, a rapidly evolving field, enables the manipulation of materials at the nanoscale, transforming traditional manufacturing and reducing environmental impact through enhanced material efficiency. recognizing the urgent need for advanced materials in biomedical applications motivates the exploration of nanobiotechnology for drug delivery, implant design, and disease diagnostics. this study hypothesizes that nanostructured materials, including liposomes, carbon nanotubes (cnts), metallic and metal oxide nanoparticles (monps), and nanopatterned surfaces, can revolutionize biomedical technologies. using a comprehensive review methodology, the study highlights the chemical and physical properties of these nanomaterials that enable their application in drug delivery systems, biosensing, bioimaging, and bone substitute implants. our findings reveal that these materials not only enhance therapeutic efficacy but also provide avenues for safer and more efficient diagnostics, with implications for improved patient outcomes. these advancements emphasize the critical role of nanotechnology in addressing modern biomedical challenges while minimizing toxicity through insights from nanotoxicology studies. key words: inorganic particles, liposomes, nanobiotechnology, nanopatterned surfaces apstrakt: uloga nanotehnologije u biomedicini: pregled inovacija i budućih pravaca nanotehnologija, kao brzo rastuće polje, omogućava manipulaciju materijalima na nanoskopskoj skali, transformišući tradicionalnu proizvodnju i smanjujući uticaj na životnu sredinu kroz poboljšanu efikasnost materijala. prepoznavanje hitne potrebe za naprednim materijalima u biomedicini motiviše istraživanje nanobiotehnologije u isporuci lekova, dizajnu implantata i dijagnostici bolesti. ova studija postavlja hipotezu da nanostrukturisani materijali, uključujući lipozome, ugljenične nanocevčice (cnts), metalne i metal-oksidne nanočestice (monps), kao i nanostrukturisane površine, mogu značajno uticati na razvoj biomedicinskih tehnologija. sveobuhvatnim pregledom literature, studija ističe hemijske i fizičke osobine ovih nanomaterijala koje omogućavaju njihovu primenu u sistemima za isporuku lekova, biosensingu, biomedicinkom snimanju i implantatima za zamenu kostiju. naša sistematizacija otkriva da ovi materijali ne samo da poboljšavaju terapijsku efikasnost, već takođe nude mogućnosti za bezbedniju i efikasniju dijagnostiku, sa implikacijama za bolje ishode pacijenata. ovi napreci naglašavaju ključnu ulogu nanotehnologije u rešavanju savremenih biomedicinskih izazova, uz minimizaciju toksičnosti kroz uvide dobijene nanotoksikološkim studijama. ključne reči: neorganske čestice, lipozomi, nanobiotehnologija, nanostrukturisane površine introduction nanotechnology is the primary driver of economic expansion at all levels. within the nanoscale range of 1 to 100 nm, intriguing phenomena such as mechanical, optical, electrical, magnetic, and a variety of other attributes can act very differently (ebrahimi et al., 2024). a nanometer, or one billionth of a meter, is 80,000 times thinner than human hair. as a result, the nanometer domain includes sizes greater than a few atoms but less than the visible light wavelength. nanotechnology is the manipulation of 100 nm or smaller materials in at least one dimension. the physical, chemical, © 2025 kumar and kaur. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 24 colloidal, or biological characteristics differ significantly from those of the bulk material (duman et al., 2024). nanotechnology is the science and engineering of creating, synthesizing, describing, and applying materials and devices whose smallest functional organization in at least one dimension is on the nanoscale scale, or one billionth of a meter (kumar et al., 2023). the dimensionality of the nanofiller determines the classification of nanocomposites. these can be categorized as zerodimensional (nanoparticles, nps), one-dimensional (nanofibers), two-dimensional (nanolayers), and three-dimensional (nanostructured networks) (pande et al., 2024). lamellar nanocomposites can be classified as either interspersed or removed. the number of polymer layers in the interlamellar space is predetermined in intercalated nanocomposites, and the polymer chains alternate with the inorganic layers in a predetermined compositional ratio. in exfoliated nanocomposites, the layers are more than 100 å apart, and the number of polymer chains separating them fluctuates almost continually. determining and altering the structure of materials and their interfaces at the atomic and nanoscales will enable the creation of novel materials and products. in the decades ahead, nanotechnology, along with wood and woodbased products, will have significant opportunities to enhance performance, expand utility, develop new product generations, and enter new market niches (kumar et al., 2023). overview of nanotechnology in biomedical and material science nanotechnology is considered highly significant due to several key factors: (a) enhanced performance; (b) the emergence of novel phenomena and unique properties; (c) reduced consumption of materials, energy, and space; (d) an ideal scale for manufacturing precision; and (e) its ability to bridge the gap between living and non-living systems. polymer nanocomposites are formed by integrating components with one or more nanoscale dimensions into a polymeric matrix. these nanoscale materials are commonly referred to in the literature as nanofillers, nps, nanoscale building blocks, or nonreinforcements (kumar et al., 2023; santulli et al., 2024). nanocomposites, a polymer type, are stiffer, stronger, more rigid, thermally stable, barrier-like, and flame-retardant than pure polymer matrix. large surface area of these composites gives them unique properties that can be changed to produce materials with various biological traits and uses (fig. 1). nanotechnology-based products are increasingly useful in biomedicine, particularly biologica nyssana ● 16 (1) june 2025: 23-34 kumar and kaur ● role of nanotechnology in biomedical advances: a review of innovations and future directions fig. 1. changes in material characteristics at the nanoscale and the benefits of integrating various nanotechnology materials 25 biologica nyssana ● 16 (1) june 2025: 23-34 kumar and kaur ● role of nanotechnology in biomedical advances: a review of innovations and future directions in prosthetics, implants, drug administration, and diagnosis (balkrishna et al., 2021; haleem et al., 2023). common materials utilized in nanotechnology include metal oxide nanoparticles (monps), liposomes, metallic surfaces, and carbon nanotubes (cnts). properties of these materials such as surface chemistry, surface physics, surface thermodynamics, and toxicological effects influence the specific application of nms (bharathi et al., 2024). designing nanostructures by adjusting their surface characteristics is one of the ways to acquire better results for various applications. here, we focus on how medicine delivery systems, bonereplacing implants, biosensing and bioimaging devices, and diagnostics use inorganic (metal and metal oxide) and organic (cnts and liposomes) nps and nanopatterned flat surfaces. in connection with the toxicity of small particles, a novel field known as nanotoxicology is also investigated, which studies the surface effects caused by nanostructured materials (rana et al., 2024). biomedical applications of metal oxide nanoparticles monps are highly valued in the biomedical field due to their unique properties and versatility, as illustrated in fig. 2 and tab. 1. they enhance imaging as mri contrast agents, controlled drug release, and support cancer treatments through the use of photothermal, photodynamic, and magnetic hyperthermia treatments (kimta et al., 2024). monps also have antibacterial properties for infection control and induce tissue engineering, encouraging bone and nerve repair (thakur et al., 2024). because of their anti-inflammatory and antioxidant qualities, they can be utilized to treat conditions linked to oxidative stress. in addition to being studied for blood cleansing, gene therapy, and as adjuvants in vaccines, monps have demonstrated promise in a number of medical uses (kumar et al., 2024). monps have been used in the construction of numerous medical devices. among the therapeutic and diagnostic uses of iron oxide’s magnetic characteristics are magnetic particle imaging, photoacoustic imaging, magnetic particle hyperthermia (thakur et al., 2024), magnetic particle imaging, and contrast agents for magnetic resonance imaging. the electrical structure of zinc oxide (zno) is useful for medical applications; for example, the intrinsic fluorescence of zno nanowires has been exploited to image cancer cells (gupta et al., 2023). in particular, they exhibit enhanced water solubility, improved biocompatibility, and reduced cellular toxicity through surface functionalization. by modifying the zno surface with specific biomolecules, these nanostructures can be adapted for use in photosensitive biosensors. additionally, the increased surface area of nanoparticles (nps) facilitates rapid adsorption of plasma proteins, further influencing their biomedical potential (sytu & hahm, 2024). consequently, the final application of nps, as well as their interactions with other substances, is determined by their physical topography, fig. 2. metal oxide nanoparticles as a versatile platform for various biomedical applications nanoparticle type biomedical applications mechanism au-nps cancer therapy, drug delivery, imaging photothermal therapy, targeted drug delivery ag-nps antibacterial agents, wound healing reactive oxygen species generation, cell membrane damage fe-nps magnetic resonance imaging (mri), targeted drug delivery, hyperthermia treatment magnetic properties, heat induction silica-nps drug delivery, gene delivery, biosensors high surface area, porous structure table 1. a variety of nanoparticles for different biomedical uses 26 biologica nyssana ● 16 (1) june 2025: 23-34 kumar and kaur ● role of nanotechnology in biomedical advances: a review of innovations and future directions chemical composition, and the interplay of these properties. titanium oxide (tio2) has many biological applications. for instance, in bone-substituting materials, the biofluid first comes into contact with a thin layer of tio2 that spontaneously develops on the upper surface of metallic titanium (shabib akhtar et al., 2024) zirconium oxide, which has recently been utilized for dental implants, is compatible with the same kind of hard tissues as titanium (nikkerdar et al., 2024). because of their strong optical absorption linked to the surface plasmon resonance of noble metals, mnps are helpful for producing molecular contrast. absorption and scattering in the visible and near-infrared spectrums have spurred the usage of materials containing metal nanoparticles in the sensing and diagnostic fields. gold (au nps) can be deposited on appropriate substrates or added to substrate formulations to increase luminescence. the application of this technology is influenced by the particle’s size and shape, which determine its absorption and scattering properties. gold nanorods, which absorb in the near-infrared (700 nm to 2500 nm) (nir), have been used to detect blood flow in vivo via photoacoustic imaging (li et al., 2024). the literature contains examples of applications that use gold nanocages, nanoshells, and nanospheres. sulfur-containing compounds have a strong chemical affinity and can be utilized to change the surface of gold nanoparticles. modification of au nps with bio-specific compounds enhances their capacity to bind to specific tissues (hossain et al., 2024). for example, cancer cells have been targeted in vitro using surface-labeled gold nanoshells, with optical microscopy confirming the results. carbon nanotubes the physical and chemical properties of cnts have motivated their application in various scientific domains. surface modification and molecular functionalization with biological molecules have increased their use in nanobiotechnology by providing uniformly distributed samples suitable for physiological conditions. the primary biomedical applications of cnts are listed in tab. 2. in this case, nanotubes could be useful in medication delivery vehicles because of their nanoscale size, which allows them to move freely within the body. the bioavailability of methotrexate, a drug used table 2. the main biomedical uses of cnts biomedical application details references biosensing because of their high conductivity and surface functionality, cnts are utilized in electrochemical biosensors to identify biomarkers. li et al., 2024 drug delivery by encapsulating medicinal substances and improving their absorption by particular cells, functionalized cnts allow for tailored medication delivery. khan et al., 2024 cancer therapy cnts are used in targeted therapy and cancer diagnosis, either as carriers for photothermal therapy or for direct medication delivery. maghimaa et al., 2024 regenerative medicine because of their conductivity, cnts are used as scaffolds in tissue engineering to encourage cell division and proliferation. parvin et al., 2024 antibacterial therapy by strengthening microbial inhibition, cnts functionalized with antibacterial drugs are utilized to treat infections. jonathan & agvini, 2024 biomedical imaging cnts are used as contrast agents in bioimaging to improve visibility in diagnostic imaging. aher et al., 2024 neurodegenerative disease cnts can pass across the blood-brain barrier, they are being researched for drug delivery systems that target alzheimer's disease. al-zharani et al., 2024 wound healing cnt-based materials encourage tissue regeneration and cell proliferation, which speeds up wound healing. elabbasy et al., 2024 diagnostics through sophisticated biosensing platforms that identify illnesses early on, cnts make precise diagnostics possible. acharya et al., 2024 gene therapy by aiding in the minimally harmful transfer of genetic material into cells, cnts enable gene delivery. yazdani et al., 2024 27 biologica nyssana ● 16 (1) june 2025: 23-34 kumar and kaur ● role of nanotechnology in biomedical advances: a review of innovations and future directions to treat cancer, increases when it is administered following immobilization on the surface of a doublefunctionalized carbon nanotube (maghimaa et al., 2024). an active substance can be bonded to the particle’s surface or introduced to the tube to target and alter cell activity at the subcellular or molecular level. biofunctionalized singleor multi-walled cnts can also interact with dna and penetrate different cellular barriers, which can be absorbed by a range of cells (sonowal & gautam, 2024). cells can absorb and interact with cationic cnts complexed with plasmid dna, making them suitable scaffolds for osteoblast proliferation and bone repair. studies have shown that the toxicity of unmodified singleand multi-walled cnts is concentration-dependent and that they can be safely used in osteoblast cells at concentrations up to 10– 20 mg/ml (mamidi et al., 2024). synthesized cnts disrupt bacterial function through a combination of oxidative and non-oxidative stress mechanisms, including cell wall penetration, reactive oxygen species (ros) generation, and metal ion release. oxidative stress results from the production of ros such as superoxide anions, hydroxyl radicals, and hydrogen peroxide, which cause damage to cellular components, including lipids, proteins, and dna. in contrast, non-oxidative stress involves mechanical disruption, where cnts physically penetrate bacterial membranes, extract lipids, and adsorb essential biomolecules (thakur et al., 2023). their unique nanostructure enables cnts to interact with and compromise the bacterial cell wall, weakening its integrity in both gram-positive and gram-negative bacteria. cnts also enhance ros generation through electron transfer reactions, further impairing cellular respiration and metabolism. metal-doped cnts release toxic metal ions, which inhibit bacterial enzymes, disrupt membrane potential, and amplify antibacterial effects. together, these multifaceted mechanisms make cnts highly effective in combating bacterial infections. we have recently proposed using collagen-modified calcium carbonate nanotubes as a new generation of tubular structures for bone regeneration (kumar et al., 2024). liposomes and nanobiotechnology bangham and horne were the first to describe liposomes, the small synthetic lipid-bilayer spherical vesicles. liposomes, a type of nanoparticle composed of lipid bilayers that encapsulate a range of chemicals, can be used to great advantage in drug administration, gene therapy, and nanobiotechnology applications (kumar et al., 2024). their ability to encapsulate both hydrophilic and hydrophobic molecules greatly facilitates targeting and controlling the release of medicinal medications. liposomes are used in nanobiotechnology to deliver drugs directly to target cells or tissues, minimizing side effects and improving therapeutic efficacy, as seen in fig. 3. liposomes can have different properties by varying their size, composition, and surface charge. the stiffness and fluidity of the bilayer can also be fig. 3. lipid based nanoparticles towards biomedical application 28 transport the generated heat to the surrounding tissues (thakhur et al., 2024). they are helpful in therapeutic applications due to their tendency to accumulate in tumors when administered intravenously (kumar et al., 2024). cnts, au nps, magnetic nps, superparamagnetic fe2o3 nps (kumar et al., 2024), and doped fe2o3 nps (thakur & kumar, 2024) are among the materials. mnps, which produce heat and target tumor cells without harming healthy tissues, are activated by an alternating magnetic field. superparamagnetic fe2o3 nps are especially effective heat generators and are highly localized in cancers (kumar et al., 2024). doped fe2o3 nps accelerate heat generation and selective absorption in a magnetic field. additionally, superparamagnetic fe2o3, graphene oxide, and doxorubicin-incorporated nanofibers have shown efficacy in promoting tissue regeneration and preventing breast cancer recurrence (kumar et al., 2024). electrospun fibers with nps are also growing in popularity for postoperative care. unlike magnetic nps, au nps function by photothermal activation. drug delivery lipids are widely used as carriers for antiviral drugs because they are easily accessible, affordable, biodegradable, biocompatible, inert, non-toxic, and non-immunogenic (satchanska et al., 2024). their unique characteristics include better drug performance, controlled release, enhanced interface interactions, small size, large surface area, and high drug-loading capacity make them particularly helpful in drug delivery systems. antisense oligonucleotide (aso) drugs can block mrna processing and translation, which is connected to a number of illnesses (jawale & kashikar, 2024). however, when administered freely, aso therapies have limitations regarding biological stability, short circulation half-life, and low cellular absorption. nps made of human serum albumin (hsa) cross-linked with glutaraldehyde and aso significantly improves aso cellular absorption. confocal laser scanning microscope images indicate the presence of drugloaded nps in various cell types, including breast cancer cells (mda-mb-468, mcf-7, and bt-474) and lung cancer cells (a549). mda-mb-468 and mcf-7 cells exhibited the fastest absorption of nps, which were detected at an incubation temperature of 37 °c after 24 hours (aalhate et al., 2023). gene therapy gene therapy aims to replace or repair diseasecausing genes by delivering healthy genes into target cells. given that inflectionless nps coupled to dna have shown promise in transferring genes to stem cells, there are concerns with the widespread controlled by choosing specific lipids (mitsuhashi et al., 2023). these artificial membrane models can be categorized based on their diameter. small uniflagellar vesicles range from 20 to 100 nm, and large uniflagellar vesicles (luvs) range from 200 to 1000 nm (devi et al., 2024). the vesicles comprise a single lipid bilayer and an internal aqueous cavity. liposomes can also be categorized based on the quantity of lipid bilayers they contain. multiple concentric phospholipid bilayers intercalated with aqueous compartments make up multilamellar vesicles ranging in size from 400 to 3500 nm (sulthana et al., 2024). over the past 20 years, liposomes have been widely used for a number of applications, such as gene therapy, vaccines, and the administration of cancer medications. these vesicles can carry a range of bioactive compounds, such as antioxidants, antimicrobials, and anagenic proteins. these chemicals continue to operate once they are encapsulated. moreover, materials with different solubility levels can be enclosed in the aqueous cavity or at the surface of lipid bilayers (bakhshizadeh et al., 2024). since liposomes are non-immunogenic, potentially non-toxic, and degradable in physiological conditions, it is possible that they could transport drugs with a low rate of disintegration and fewer adverse effects (lee et al., 2024). nanotechnology in diagnostics nanotechnology has enormous benefit for diagnostics, particularly in imaging applications such as optical imaging and magnetic resonance imaging (mri), where nps are validated diagnostic tools (thakhue et al., 2024). quantum dots (qds) are semiconductor nanocrystals used extensively in optical imaging because they are 100 times brighter than organic dyes (palanisamy et al., 2024). the quantity of nps in the cell’s cytoplasm is essential for illuminating cells in deep tissues. despite their effectiveness as tagging materials, qds have drawbacks. more qds are required to achieve sufficient brightness, which could pose toxicity concerns (kumar et al., 2024). moreover, the blinking behavior of biomolecules labeled with qds may make tracking them more challenging. consequently, the development of fluorescent nps for in vivo imaging continues to be challenging. silicon nanocrystals are more appealing than qds because they are safe for cells and do not require a substantial surface coating to protect the nanocrystal core from the environment (kumar et al., 2024). nanotechnology in therapeutics metallic nanoparticles (mnps), known for their remarkable thermal conductivity, effectively biologica nyssana ● 16 (1) june 2025: 23-34 kumar and kaur ● role of nanotechnology in biomedical advances: a review of innovations and future directions 29 use of viral vectors for gene transfer, including the potential for cell damage. dna-coupled nps provide effective gene transfer to stem cells by shielding dna from degradation and increasing cellular absorption. surface changes to nps ensure focused distribution. once inside, they release dna for integration or expression, providing a safer, nonviral therapeutic option for regenerative medicine and stem cell engineering (alzahrani et al., 2023). by combining the benefits of gene therapy and photothermal therapy, surface-modified au nps carrying dna have recently been demonstrated to aid in photoacoustic imaging (song et al., 2024). gene transfer is a novel treatment technique that involves inserting new genes into diseased cells or surrounding tissues to kill the cells, block the spread of cancer, or correct genetic flaws to restore normal cell function. one method, called gene-directed enzyme prodrug therapy, involves directing a gene for an enzyme not naturally present in the cancerous tissue to activate a prodrug that is subsequently administered. genome editing will be a feasible cancer therapy option in the future thanks to new knowledge regarding nuclease activity and the creation of secure and efficient gene delivery vectors (ramezani et al., 2023). nanotechnology for surface engineering of metallic implants nanotechnology is also being used in tissue and implant engineering. the material’s capacity to enhance surface area and modify surface roughness at the nanometric scale should lead to improved biological responses from osteogenic cells and effective mechanical contact between tissue and implant. titanium and its alloys are considered the most attractive materials for bone replacement applications (rani et al., 2023). this metal finds extensive employment because of its superior mechanical properties, low surface reactivity, strong resistance to corrosion, and acceptable in vitro and in vivo biocompatibility. the live tissue heals in close proximity to the metal, even if there may be a thin fibrous layer separating the metallic implant from the bone, indicating a failure in the osteointegration process. the implant’s surface needs to be changed to achieve good osteointegration and strengthen the bone-implant interface in this case (bandyopadhyay et al., 2023). changing the surface should just change the topography. however, the creation of nanoscale roughness and the addition of bioactive materials appear to be more viable strategies for biomedical applications. in the following subsections, we discuss which features of implant surfaces should be modified to enhance host tissue responses. we also describe the use of nanotechnology for implant surface engineering at the nanoscale. modifying metallic surfaces improves the interface between implants and biological tissue in bone replacement applications. for orthopedic implants to be successful, bone tissue must form at the implant’s surface (zhang et al., 2024). to maximize implant attachment to the tissue, surface engineering alters properties, including topography, wettability, charge, and chemical composition. clinical research indicates that when one goes away from the implant surface rather than toward it, the rate of bone growth rises (thakur & thakur, 2024). bio-inspired surface modifications adding bioactive minerals that mimic the structure of bone is one of the most common ways to modify the metallic surfaces of implants. biomimetics is a preferred strategy since it predefines nanochemical and nanophysical structures. calcium phosphate (cap)-based coatings are often produced in biomaterials research, and their use in implants significantly affects the bone regeneration process (furko et al., 2023). however, these methods are complex and need expensive equipment and very high temperatures. this method allows for the production of continuous cap coatings with controlled surface topography despite the fact that it may need prolonged exposure times (verma et al., 2023). simulated bodily fluid is one of the most commonly used physiological solutions (sbf). sbf, composed of a supersaturated cap solution replicating the ph and ionic composition of physiological fluids, is a frequently employed method for evaluating a material’s bioactivity (suchy et al., 2021). toxicology of nanomaterials the toxicity of nanomaterials is influenced not only by the nps but also by the solvents used to disperse them. nps are frequently dissolved in solvents such as alcohol, which might influence their stability, bioavailability, and toxicity. as a result, the impact of the solvent must be carefully evaluated during toxicity studies since it may contribute to or affect the observed effects. solvent interactions with biological systems, as well as possible cytotoxicity and changes in nps behavior, can all substantially impact experimental results. to guarantee accurate and reliable results, comprehensive investigations should consider both the inherent toxicity of nps and the function of solvents. nps’ toxicological effects are poorly understood despite their potential in biomedical applications. the toxicity of nanomaterials depends on a variety of criteria, including dosage and composition, as well as physicochemical properties like size, surface charge, roughness, crystalline structure, and shape. biologica nyssana ● 16 (1) june 2025: 23-34 kumar and kaur ● role of nanotechnology in biomedical advances: a review of innovations and future directions 30 materials’ physicochemical properties are influenced by their size. consequently, the bulk material’s toxicological behavior induces hazardous events peculiar to nps. the word “nanotoxicology” has been used to better understand the physicochemical properties of nps and their detrimental effects. most nanotoxicological research is based on cell culture experiments (motamedi and tabatabaei, 2025). numerous investigations have used fundamental biomimetic cell membrane models, like langmuir monolayers and proteins in vitro. verification in vivo experiments are necessary to forecast how nanomaterials will interact with biological systems, nevertheless, because the outcomes of in vitro tests might not accurately represent their effects in life. even though multiple assays have been conducted, variable methodology has led to inaccurate toxicological results. for example, the conventional toxicological methodology, which considers exposure time and dosage, is not consistent with the toxicological consequences of nanomaterials. the selection of suitable dose measurements requires careful consideration. future of nanomedicine drug delivery and nanomedicine are rapidly growing topics with multiple clinical trials performed on 1,500 patients (raj & kaladhar, 2025). the diagnosis and treatment of illnesses like cancer have benefited immensely from nonmedical technologies. nps can be used to precisely deliver drug dosages to diseased cells, including cancer cells, without changing the cells’ normal physiology. however, the size of the nps fluctuates, and more research is needed to ensure uniform consistency and drug loading and release capabilities. mnps, including gold and silver, have shown potential for application in targeted removal techniques like radiation-based heat therapy. despite its potential, nanomedicine’s actual impact on healthcare is currently restricted because it is still in its infancy, and many crucial features are yet unknown. future research should focus on understanding disease molecular markers and developing theoretical mathematical models for regulated medication release, technology evaluation, and therapeutic benefits. the future of drug delivery technology and nanomedicine appears promising, with a focus on increasingly complex and multidisciplinary approaches. challenges and opportunities in biomedical applications, nanotechnology offers both great potential and formidable obstacles. since nms’ interactions with biological systems can provide dangers that are not fully known, one of the main problems is ensuring their biocompatibility and minimizing their toxicity. many high-precision methods used in laboratories are difficult to adapt for large-scale production while maintaining consistent quality, which makes the scaling and manufacturing of nanomaterials particularly challenging (materon et al., 2021). due to ethical and regulatory concerns, the absence of defined rules for the safety, testing, and approval of nms continues to obstruct the broad use of nanotechnology in medicine. overcoming the challenges of controlling the release of medicines to the intended location and attaining precise and efficient targeted medication delivery represent further challenges. notwithstanding these difficulties, nanotechnology presents fascinating prospects in the field of biomedicine. by providing extremely specialized drug delivery systems based on each patient’s unique genetic profile, it has the potential to completely transform personalized medicine by enhancing therapeutic results and reducing side effects. nanotechnology also holds potential for improving diagnostics because nanosensors and nps can improve the sensitivity and specificity of detection tools, allowing for early illness identification and improved patient outcomes. nanotechnology can potentially improve biosensing and bioimaging methods by offering high-resolution, real-time imaging for disease diagnosis and monitoring. lastly, by providing continuous, real-time data that may be utilized to manage chronic illnesses and identify environmental risks, nanosensors have the potential to revolutionize environmental and health monitoring. conclusion this study underscores the transformative potential of nanostructured materials in biomedical applications, demonstrating their ability to enhance drug delivery systems, biosensing, bioimaging, and bone substitute implants. novel insights include the multifunctional role of cnts, metallic and monps, and nanopatterned surfaces in improving therapeutic efficacy and diagnostic precision. these findings align with emerging trends in nanobiotechnology, addressing critical challenges in modern medicine while minimizing toxicity through nanotoxicologyguided approaches. however, limitations in the current approach must be acknowledged. the longterm biocompatibility and environmental impact of these nanomaterials remain areas of concern, necessitating further research to optimize safety profiles. additionally, scalability in synthesizing these nanostructures for industrial applications poses practical challenges. future work should focus on refining functionalization techniques to improve selectivbiologica nyssana ● 16 (1) june 2025: 23-34 kumar and kaur ● role of nanotechnology in biomedical advances: a review of innovations and future directions 31 biologica nyssana ● 16 (1) june 2025: 23-34 kumar and kaur ● role of nanotechnology in biomedical advances: a review of innovations and future directions ity and performance while ensuring sustainability. in conclusion, this work provides strong evidence for the promising role of nanostructured materials in advancing biomedical technologies, offering impactful solutions to pressing healthcare challenges. 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(2024). the antifouling mechanism and application of bioinspired superwetting surfaces with effective antifouling performance. advances in colloid and interface science, 325, 103097. https://doi. org/10.1016/j.cis.2024.103097 the ecological and floristic characteristics of natural population of micromeria juliana (l.) benth. ex rchb. in bulgaria biologica nyssana 8 (1)  september 2017: 105-111 lazarević, m. et al. morphological discrimination of the genera… 105 original article received: 25 january 2017 revised: 11 july 2017 accepted: 15 july 2017 morphological discrimination of the genera binodoxys mackauer and trioxys haliday (hymenoptera: braconidae: aphidiinae) based on the general shape of forewings maja lazarević*, marijana ilić milošević, saša s. stanković, vladimir žikić university of niš, faculty of science and mathematics, department of biology and ecology, višegradska 33, niš, serbia * e-mail: majalazarevic9@gmail.com abstract: lazarević, m., ilić milošević, m., stanković, s.s., žikić, v.: morphological discrimination of the genera binodoxys mackauer and trioxys haliday (hymenoptera: braconidae: aphidiinae) based on the general shape of forewings. biologica nyssana, 8 (1), september 2017: 105-111. the genera binodoxys and trioxys, members of the aphidiinae subfamily, are classified in the same subtribe trioxina within the tribe trioxini. these two genera are morphologically very similar, but differ in a large degree in their ecology. generally, binodoxys species are specialized in parasitizing aphids on herbaceous plants, unlike trioxys species, which have co-evolved with the tree-attacking aphids. for this study, we analyzed the right forewings of four binodoxys and two trioxys species, using the method of geometric morphometrics. sixteen specific landmarks were digitalized for each forewing of 80 parasitoid individuals. the results of two statistical methods anova and manova demonstrated statistically significant differences of the wing shapes, especially between the genera. visualizing the results of the dispersion of specimens in the morphospace via multivariate tests pca and cva, we found that the most variable character in wing structure was the radial sector, differentiating binodoxys from trioxys. key words: trioxina, geometric morphometrics, variability, parasitoids apstrakt: lazarević, m., ilić milošević, m., stanković, s.s., žikić, v.: morfološko razdvajanje rodova binodoxys mackauer i trioxys haliday (hymenoptera: braconidae: aphidiinae) na osnovu oblika krila. biologica nyssana, 8 (1), septembar 2017: 105-111. rodovi binodoxys i trioxys, članovi podfamilije aphidiinae, su klasifikovani u isti podtribus trioxina u okviru tribusa trioxini. pomenuta dva roda su morfološki veoma slična, ali se ekološki dosta razlikuju. generalno posmatrajući, vrste roda binodoxys su se specijalizovale za parazitiranje biljnih vaši koje se hrane zeljastim biljkama, za razliku od vrsta roda trioxys, koje su koevoluirale sa biljnim vašima koje žive na drvenastim biljkama. za ovu studiju, analizirana su desna krila četiri vrste roda binodoxys i dve trioxys vrste, koristeći metod geometrijske morfometrije. za svako krilo, od ukupno 80 analiziranih jedinki parazitoida, izabrano je šesnaest tačaka. rezultati dve statističke metode, anova i manova, pokazali su postojanje statistički značajnih razlika u obliku krila, naročito između rodova. vizuelizacijom rezultata multivarijantnim testovima, 8 (1) • september 2017: 105-111 doi: 10.5281/zenodo.964447 biologica nyssana 8 (1)  september 2017: 105-111 lazarević, m. et al. morphological discrimination of the genera… 106 pca i cva, uočeno je da je najvarijabilnija struktura radijalni sektor, na osnovu koga se rodovi binodoxys i trioxys razdvajaju. ključne reči: trioxina, geometrijska morfometrija, varijabilnost, parazitoidi introduction the aphidiinae subfamily can be distinguished from other members of the braconidae family for being the exclusive parasitoids of aphids (hemiptera: aphididae). they are solitary koinobiont endoparasitoids. uniquely parasitizing aphids was one of the reasons why s t a r ý (1981) recognized this group as a separate entity, raising it to the family level – aphidiidae, which can be found in older literature. nowadays, this group of parasitoids is usually considered as one of the subfamilies of braconids (q u i c k e & v a n a c h t e r b e r g , 1990; w h a r t o n et al., 1992). this is a moderately large subfamily with about 500 described species worldwide (y u et al., 2012). the subfamily is divided into five tribes, where the tribe trioxini in europe comprises nine genera: betuloxys mackauer 1960, binodoxys mackauer 1960, calaphidius mackauer 1961, falciconus mackauer 1959, harkeria cameron 1900, lipolexis föerster 1862, monoctonia starý 1962, monoctonus haliday 1833, and trioxys haliday 1833 (m a c k a u e r & s t a r ý , 1967; m a c k a u e r et al., 1968). the tribe trioxini consists of two subtribes, trioxina and monoctonina. subfamily aphidiinae have a diverse forewing venation, which is often quite a useful trait for determination of parasitoids on the generic level. among the subfamily, several members of the subtribe trioxina possess highly reduced forewing venation, with only the stigma, radial vein and radial sector vein. beside wings, which are the most useful trait, other morphological structures are important for determination of aphidiines on lower taxonomic levels, such as species (w h a r t o n et al., 1997). several genera of the subtribe trioxina contain paired prongs, which are extensions of the female’s seventh metasomal sternite. in european fauna, only the species of the genera betuloxys, binodoxys and trioxys possess those abdominal excrescences. these structures are very important for the identification of specimens on the generic and also species level; especially the shape and the curvature of prongs, the number and disposition of setae on them (s t a r ý , 1981). furthermore, european genera which have prongs can be differentiated from one another by the morphology of the second metasomal segment, petiole. while the genera betuloxys and trioxys have one pair of tubercles with spiracles, the genus binodoxys possesses two pairs, primary and secondary. in this genus, the primary tubercles are with spiracles, while the secondary are without them. the position of the secondary tubercles on petiole is variable and highly important for the identification of binodoxys at the species level (m a c k a u e r , 1959, 1960; f u l b r i g h t et al., 2007). knowing that the variability of the forewings in shape and size, and also in the wing venation can be a significant, often not conspicuous, characteristic to separate two morphologically similar species, there were a lot of studies where the geometric shape of the wing was used to accent that variability and those differences. in contrast to monoctonina, trioxina can be easily recognized by the forewing venation which is significantly reduced, but all minute differences between genera and among species of the same genus can be explained using the method of geometric morphometrics (z e l d i t c h et al., 2012). this particular method has already been successfully applied in many previous studies on aphidiinae wings (ž i k i ć et al., 2009, 2014; m i t r o v s k i -b o g d a n o v i ć et al., 2009, 2014; k o s et al., 2011; t o m a n o v i ć et al., 2014; i l i ć m i l o š e v i ć et al., 2015; s t a n k o v i ć et al., 2015), sometimes revealing a new species, such as ephedrus lonicerae tomanović, kavallieratos & starý 2009 in ž i k i ć et al. (2009). beside the morphological differences, there is also a biological dissimilarity between these genera, as their hosts feed on different plants. the species of the genus binodoxys attack aphids on herbaceous plants, while betuloxys and trioxys species are known mostly from the hosts feeding on trees and shrubs (m e s c h e l o f f & r o s e n , 1993; p i k e et al., 1996, 2000; a k h t a r et al., 2011; ž i k i ć et al., 2012). the aim of this study was to explore the variability of the shape and size of forewings, comparing the wing venation using the method of geometric morphometrics, and to determine whether there was a significant difference that would help in the further application of geometric morphometrics on the species of both investigated genera binodoxys and trioxys. material and methods sampled material the specimens for this study were collected in the period between 2010 and 2013 on the territory of serbia and montenegro (tab. 1). plant parts infested biologica nyssana 8 (1)  september 2017: 105-111 lazarević, m. et al. morphological discrimination of the genera… 107 with aphids were cut and placed into plastic containers and covered with a muslin cloth. according to the instructions given in k a v a l l i e r a t o s et al. (2010), these containers were placed in a growing cabinet and kept at the following conditions: 22.5 °c, 65% relative humidity and photo period of 16l:8d for 3-4 weeks until parasitoid emergence. thus collected parasitoids were placed in vials containing 96% ethanol. several wingless adult aphids were selected, put in 70% ethanol and sent to a specialist for identification. wing preparation, selection of landmarks and geometric morphometrics the method of geometric morphometrics was applied to explore the morphological differences in wing size and shape between the genera binodoxys and trioxys (z e l d i t c h et al., 2012). we examined 80 right forewings of females in total; four binodoxys species: b. acalephae (marshall 1896), b. angelicae (haliday 1833), b. brevicornis (haliday 1833) and b. heraclei (haliday 1833), and two species of trioxys: t. complanatus quilis perez 1931 and t. pallidus (haliday 1833) (see tab. 1). the right forewings of each specimen were detached, mounted on microscopic slides in berlese solution and photographed using leica system dm2500 microscope with leica dfc490 digital camera (leica microsystems©, wetzlar, germany). the specific landmarks from 1 to 9, together with the landmarks 11, 12 and 14, represent true landmarks (lm), while the other four (10, 13, 15 and 16) were set as semilandmarks (s-lm). these semilandmarks are used to better describe the distal part of the wing which lacks wing venation (fig. 1). the landmarks 1, 4 and 6 define the length and width of the stigma; lm 4 and 5 represent the metacarpal (r1) vein; lm 2, 6 and 7 describe the radial sector, while lm 8 represents the projection of the radial sector to the edge of the wing. the landmarks 3, 9, 10, 11 and 12, together with the s-lm 13, delineate the proximal part of the wing, while lm 5, 8, 14, and s-lm 15 and 16 define the distal part of the wing. the constellation of selected landmarks and the semi-landmarks was positioned and processed using the software makefun6 (s h e e t , 2003) and tpsdig2 (r o h l f , 2005). the general procrustes analysis was applied to eliminate variation which can be caused due to the differences in the wing size, orientation and/or positioning during the digitalization (b o o k s t e i n , 1991) and for each wing to compute the centroid size (cs) as a geometric measure of size. based on the cs, the differences in the wing size were analyzed using the analysis of variance (anova), while the shape variability was computed using the multivariate analysis of variance (manova). all statistical methods were done in statistica software package (stat soft inc. 7.0). to visualize the differences in the wing shape and size, the principal component analysis (pca) and canonical variate analysis (cva) were done using the software morphoj (k l i n g e n b e r g , 2011). table 1. the list of sampled material used in the analysis; abbreviations: srb – serbia, mne – montenegro; mj – m. janković, ss – s. stanković, zk – z. kojičić, vž – v. žikić parasitoid species no. of individuals country, locality, date, legator b. acalephae (marshall 1896) 14 srb, niš, popovac, 04.07.2010. vž b. angelicae (haliday 1833) 15 srb, sićevačka klisura (gorge) 12.05.2013. ss b. brevicornis (haliday 1833) 10 mne, durmitor mt., crno jezero (lake), 25.07.2012. vž b. heraclei (haliday 1833) 11 srb, dukat mt, 29.06.2012. ss t. complanatus quilis perez 1931 16 srb, smederevo, malo orašje, 10.06.2012. mj t. pallidus (haliday 1833) 14 srb, kruševac, 19.05.2013. zk fig. 1 set of specific landmarks and semi-landmarks on the forewing of binodoxys angelicae biologica nyssana 8 (1)  september 2017: 105-111 lazarević, m. et al. morphological discrimination of the genera… 108 results and discussion for the variability among and between the selected groups, anova test showed statistically significant differences in wing size (f(45216.25) = 3.62; p ˂ 0.006). the wing shape variability which was tested by manova also displayed statistically significant differences: wilks`λ = 0.000444; f(113631211) = 6; p ˂ 0.001. delineation of the genera in morphospace is presented over principal component analysis defined by the first two pc axes (fig. 2). the discrimination between bionodoxys and trioxys in the analysis of the forewing shape is evident, with a very small overlapping between the two genera (fig. 2), where the first two pc axes accounted for 64.76% of the total variability. the analyzed species are separated along pc1 = 34.08%, according to the length of the radial sector (fig. 1, 2) which was calculated as the most variable part of the wing. placing all binodoxys species in the negative part of pc1, this structure points a shorter radial sector than in trioxys. to a lesser extent, all other parts of the forewing contribute to the separation of the analyzed genera. as a consequence of all shape changing in the wing along the positive and the negative senses of the pc1, the wings in binodoxys species are generally shorter and broader in their distal part, lm (5, 7, 8, 14, 15 and 16), unlike trioxys species, which wings are elongated. changes in wing morphology illustrated by pc2 = 30.68% are reflected not so much in the length of the wings as much as in the width. thus, these kinds of changes explained by pc2 are more related to intragroup variability than the differences between the two genera. although there is a strong trend of delimitation of binodoxys and trioxys applying pca, to obtain a clearer view of the discrimination of these two genera, we used canonical variate analysis (cva). after applying cva, the ordination of analyzed specimens in the morphospace, which was defined by the first two canonical axes, has pointed out the interpreted morphological differences in wings much better (fig. 3). the first canonical axis (cv1) explained 53.57% of the total variability of the wing shape, thus splitting binodoxys and trioxys species in the two distinct groups. both analyzed species of the genus trioxys, t. complanatus and t. pallidus, are positioned in the negative part of the cv1, while the four species of binodoxys are distributed along the positive part of the cv1 axis. as it can be seen from the cva graph, most of the differences are caused by the variability of the distal part of the wing, where the lm 7 contributes most to the differences of the wing shape with respect to all other landmarks in this region. unlike both species of trioxys, the species of fig. 2 ordination of the binodoxys and trioxys species in the morphospace defined by the first two principal axes, for an interval of confidence of 0.8. the thin-plate transformation grids illustrate forewing changes along pc1 and pc2 biologica nyssana 8 (1)  september 2017: 105-111 lazarević, m. et al. morphological discrimination of the genera… 109 the genus binodoxys have a much shorter radial sector and rather wider wings. also, the wings of binodoxys are little larger (in average) than in trioxys. changes in wing size and shape are presented via transformation grids. the best discrimination was shown in b. acalephae, where this species is abstracted in the positive part of cv1, and the negative cv2 = 23.26% (fig. 3). the other species, b. angelicae, is clearly separated from the other three binodoxys species, being positioned in the positive part of the cv2. the other two species of binodoxys, b. brevicornis and b. heraclei, mostly overlap in the center of the morphospace defined by cv1 x cv2, sharing more or less the similar shape of the wings. in this analysis, both trioxys species are well discriminated. the reason of the partial overlapping lies in the morphology of the forewing, especially in the variation of the radial sector (f u l b r i g h t et al., 2007). since the genus trioxys is very large, comprising about 60 species (ž i k i ć et al., 2017), the radial sector can be variable. for example, it can be extremely short like in t. bonnevillensis (smith 1944), medium-sized in t. ibis (mackauer 1961) and very long in t. curvicaudus (mackauer 1967). having this in mind, it should be noted that the use of geometric morphometrics is suitable only for the species with similar wing armature. contrariwise, the majority of binodoxys species have a less variable radial sector on the forewings, and in comparison to the trioxys species listed above, we can say that it is of a medium size. therefore, we can see much greater overlapping among the binodoxys species. conclusion for a very long time, the taxonomic status of the genus binodoxys was considered as a subgenus within the genus trioxys. since m a c k a u e r (1959) separated these two genera, there was a need for the revision of both genera. most keys for determination of aphidiines are based on the morphology of petiole and shape of ovipositor and accessory prongs. adding the geometric shape of the wings to the characters listed above, it is confirmed that binodoxys and trioxys are two genera. acknowledgements. we express our gratitude to dr. željko tomanović and dr. andjeljko petrović (faculty of biology, university of belgrade, serbia) for useful comments concerning the manuscript. we would like to thank dr. olivera petrovićobradović (faculty of agronomy, university of belgrade, serbia) for the identification of the aphids. this study was funded by the ministry of education, science and technological development of the republic of serbia (iii43001). fig. 3 distribution of the species of binodoxys and trioxys in the morphospace defined by the cv1 and cv2 axes, for an interval of confidence of 0.85 biologica nyssana 8 (1)  september 2017: 105-111 lazarević, m. et al. morphological discrimination of the genera… 110 references akhtar, m.s., dey, d., usmani, m.k. 2011: a catalogue of aphid parasitoids (hymenoptera: braconidae: aphidiinae) from india. insecta mundi, 151: 1-31. bookstein, f.l. 1991: morphometric tools for landmark data: geometry and biology. cambridge university press, new york, 435 pp. 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petrović-obradović, o., kavallieratos, n. g., starý, p., tomanović, ž., 2012: aphidiinae (braconidae: hymenoptera) of serbia and montenegro – tritrophic interactions. acta entomologica serbica, 17: 83-105. žikić, v., petrović, a., ivanović, a. 2014: allometric shape changes indicate significant divergence in the wing shape between asexual and sexual lineages of lysiphlebus fabarum (marshall) (hymenoptera: braconidae: aphidiinae). acta entomologica serbica, 19: 53-62. žikić, v., lazarević, m., milošević, d. 2017: host range patterning of parasitoid wasps aphidiinae (hymenoptera: braconidae). zoologischer anzeiger – a journal of comparative zoology, 268: 75-83.doi: 10.1016/j.jcz.2016.10.001 anticancer compounds from medicinal plants biologica nyssana 2 (2)  december 2011: 00-00 ljupković r.b. et al..  removal cu(ii) ions from water... 43 short communication melanoides tuberculata (müller, 1774), a new species for the territory of serbia (gastropoda: thiaridae) miodrag milenković* 1 , vladimir gligorijević 2 1 partizanska 25, 18300 pirot, serbia 2 dragiše cvetkovića 31d/11, 18000 niš, serbia * e-mail: mjouri@gmail.com abstract: milenković, m.,gligorijević, v.: melanoides tuberculata (müller, 1774), a new species for the territory of serbia (gastropoda: thiaridae), biologica nyssana, 3 (1), september 2012: 43-45. this article relates to the first record of melanoides tuberculata (müller, 1774) for the territory of serbia. its distribution in the region of "niška banja", eastern serbia is a consequence of its release by aquarists at thermal water stream called "topla voda". short habitat description and the areal are given. key words: melanoides tuberculata, first record, niška banja, serbia, thiaridae the freshwater benthic snail m. tuberculata belongs to the family thiaridae and many of its native varieties were distributed all over the east africa, tropical asia and southeast asia (m a l e k & c h e n g , 1974). today, they are extensively dispersed in the tropical and subtropical regions of the world (b e d e , 1992; p o i n t i e r , 1993). regions that this snail invaded include north america (m u r r a y , 1964) and south america (d e m a r c o , 1999). also, the most widely used taxonomy in europe, "fauna europaea" reveals presence of mentioned thiarid snail in austria, france, germany, hungary, italy, malta, netherlands, poland, slovakia and spain (fauna europaea, 2011). according to available literature data, m. tuberculata was never recorded in serbia. usually named "malayan snail", commonly reaches a length of 30-36 mm (t h o m p s o n , 1984). he is ovoviviparous (b e n -a m i & h o d g s o n , 2005) and parthenogenetic species (p o i n t i e r and g u y a r d , 1992; p o i n t i e r et al., 1993), with diet that mostly consists of fine detritus and epiphytic algae (m a d s e n , 1992). the authors visited "topla voda" (fig. 1) stream on 27. november 2011, near "niška banja" settlement (latitude: 43, 17' 46"; latitude reference: north latitude, longitude: 22, 0' 0" longitude reference: east longitude). on this occasion, autors observed a single specimen of gastropods and realized that it represents m. tuberculata, thiarid snail who is well known to aquarists. this allochtone species was noted in a small, fast flowing, thermal stream called "topla voda" which is composed of two thermal water sources: "suvo vrelo" and "školska česma". this habitat is under the influence of temperate climate at the altitude of 210 m (d r a g i ć at al., 1997). 3 (1) • september 2012: 43-45 biologica nyssana 3 (1)  september 2012: 43-45 milenković, m., gligorijević, v.  melanoides tuberculata (müler, 1774)… 44 figure 1. "topla voda" stream, located near settlement "niška banja" photo: vladimir gligorijević the riverbed was covered in sludge and sand with some smaller rocks in the middle. the specimens were photographed on a stream bottom (fig. 2) during that survey and then fixed in alcohol for later identification according to http://el.erdc.usace.army.mil/ansrp/ansis/html/mel anoides_tuberculata_red_rimmed_melania.htm#mtu berculata_identification and http://www.gastropods.com/9/shell_11379.shtml). ten specimens, 25.3mm to 34.1mm in length were measured with vernier calliper and then collected and used for mentioned identification. the actual distribution of m. tuberculata in "niška banja" termal stream called "topla voda" is certainly the consequence of its inadequate manipulation by local acquarists. the finding of m. tuberculata increases the number of known species of gastropoda for the territory of serbia. acknowledgments. the authors want to express their gratitude to miloš popović for image processing and dr vladimir žikić. figure 2. m. tuberculata in nature habitat at "topla voda" stream., photo: miodrag milenković references bank, ruud a., fauna europaea, 2011. group coordinator audisio, p. fauna europaea version 2.4, web service available online at http://www.faunaeur.org bede, l.c., 1992. dinâmica populacional de melanoides tuberculata (prosobranchia: thiaridae) no reservatório da pampulha, belo horizonte, m.g., brasil. dissertação de mestrado. universidade federal de minas gerais, belo horizonte, 112 p. ben-ami, f. and a.n. hodgson., 2005. ovoviviparity and the structure of the brood pouch in melanoides tuberculata (gastropoda: prosobranchia: thiaridae). journal of morphology, 263:322-329. dragić, s., garovnikov, b., joksimović, a., komatina, m., marić, ž., đukanović, m., jovanović, v., čuk, m. s., mitekoš, d. 1997. predlog za utvrđivanje područja niške banje. beograd, 1-30. madsen, h., 1992. food selection by freshwater snails in the gezira irrigation channels, sudan. hydrobiologia, 228: 203-217. malek, e.a. and cheng, t.c., 1974. medical and economic malacology. academic press, london, 398 p. marco, de p. j., 1999. invasion by the introduced aquatic snail melanoides tuberculatus (müller, 1774) (gastropoda: prosobranchia: thiaridae) of http://el.erdc.usace.army.mil/ansrp/ansis/html/melanoides_tuberculata_red_rimmed_melania.htm#mtuberculata_identification http://el.erdc.usace.army.mil/ansrp/ansis/html/melanoides_tuberculata_red_rimmed_melania.htm#mtuberculata_identification http://el.erdc.usace.army.mil/ansrp/ansis/html/melanoides_tuberculata_red_rimmed_melania.htm#mtuberculata_identification http://www.gastropods.com/9/shell_11379.shtml biologica nyssana 3 (1)  september 2012: 43-45 milenković, m., gligorijević, v.  melanoides tuberculata (müler, 1774)… 45 the rio doce state park, minas gerais, brazil. studies on neotropical fauna and environment, tübinge, 34: 186-189. murray, h. d., 1964. tarebia granifera and melanoides tuberculatus in texas. american malacological union inc., annual report, pp. 1516, 1964. pointier, j.-p. and guyard, a. 1992. biological control of the snail intermediate hosts of schistosoma mansoni in martinique, french west indies. tropical medicine and parasitology, 43:98-101. pointier, j.-p., 1993. the introduction of melanoides tuberculata (mollusca: thiaridae) to the island of saint lucia (west indies) and its role in the decline of biomphalaria glabrata, the snail intermediate host of schistosoma mansoni. acta tropicalis, 54:13-18. pointier, j.-p., thaler, l., pernot, a.f. and delay, b., 1993 – invasion of the martinique island by the parthenogenetic snail melanoides tuberculata and the succession of morphs. acta ecologica, 14(1):33-42. thompson, f.g., 1984. the freshwater snails of florida: a manual for identification. university of florida press, gainesville. 94p. http://el.erdc.usace.army.mil/ansrp/ansis/html/mel anoides_tuberculata_red_rimmed_melania.htm# mtuberculata_identification http://www.gastropods.com/9/shell_11379.shtml http://www.gastropods.com/9/shell_11379.shtml 46 stanković et al. 2024, biologica nyssana 15(1) 15 (1) june 2024: 47-51 doi: 10.5281/zenodo.11517784 new records of the black soldier fly hermetia illucens (diptera: stratiomyidae) in serbia and montenegro original article saša s. stanković university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia sasasta@gmail.com (corresponding author) aleksandra trajković university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia vladimir žikić university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia received: march 07, 2024 revised: may 07, 2024 accepted: may 21, 2024 abstract: this paper presents the first report of hermetia illucens (linnaeus, 1758), commonly known as the black soldier fly, in serbia. the discovery comprises two specimens collected in the village of stari ledinci, near novi sad, captured inside a facility adhered to window glass. furthermore, we document the presence of black soldier fly specimens in montenegro, where three individuals were captured using a sweeping net. key words: black soldier fly, hermetia illucens, serbia, first report apstrakt: novi nalazi crne vojničke muve hermetia illucens (diptera: stratiomyidae) u srbiji i crnoj gori ovaj rad predstavlja prvi nalaz crne vojničke muve hermetia illucens (linnaeus, 1758) u srbiji. dva primerka crne vojničke muve su sakupljena u selu stari ledinci, blizu novog sada, pronađena unutar objekta na staklu prozora. takođe, dokumentujemo prisustvo crne vojničke muve u crnoj gori, gde su tri jedinke uhvaćene entomološkom mrežom. ključne reči: crna vojnička muva, hermetia illucens, srbija, prvi nalaz introduction hermetia illucens (linnaeus, 1758), popularly known as the black soldier fly (bsf), belongs to the subfamily hermetiinae of the dipteran family stratiomyidae. approximately 80 congeneric species are distributed mostly in the tropical regions, particularly the neotropics (fachin & carvalho-filho, 2020). although h. illucens originates from central and south america, it has become a widespread species present on all continents (demetriou et al., 2022). this cosmopolitan distribution is a result of its high tolerance to various climate conditions, as well as its plasticity in terms of food sources. the bsf has become one of the most commonly farmed insects intended for entomophagy, primarily in an attempt to substitute fish meal, the easily deteriorating and expensive protein component of traditional livestock feed. also, considerable research focuses on bsf biotransformation abilities (wang & shelomi, 2017; kumar et al., 2021) since it plays an important role in the recycling of nutrients in nature. adult black soldier flies have poorly developed mouth parts, and typically do not eat or they feed on nectar and other sugars (sheppard et al., 2002). primarily saprophagous, bsf larvae feed on decaying organic matter such as rotten plant and animal waste, manure, etc. (gobbi et al., 2013). thus, the larvae thrive on various organic waste originating from households and green markets (newton et al., 2005). the simplicity of bsf breeding led to the development of insect farms across the globe. world’s first commercial fly farm was established in 2008 in south africa, afterwards the mass production reached europe (spain, the netherlands, france, ireland), asia (malaysia, singapore), america (us, chile) (vmr, 2024) and australia (digiacomo, 2023). the profitability potential and the risks associated with the use of insects as animal feed are under scrutiny in several countries of the european union, and legislation is being actively adjusted to address the issue. these regulatory acts are also being considered in serbia (urošević et al., 2019). although bsfs originate from warm and humid habitats, they have successfully established populations in temperate regions as well (spranghers et al., 2017). the first european record dates back to early 1926, when it was discovered in malta (lindner, © 2024 stanković et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 47 1936). subsequently, the species began spreading across the western mediterranean; the iberian peninsula (leclercq, 1997), and later, the balkan peninsula, i.e., its coastal countries (beschovski & manassieva, 1996; tsagkarakis et al., 2015). the bsf has been recorded further north beyond the mediterranean basin, in france (chevin, 1986), switzerland (sauter, 1989), germany, and the czech republic, which is its northernmost distribution point in europe (roháček & hora, 2013). recently, in some databases, such as gbif (2024) it has been reported from sweden, estonia, benelux, and great britain. although bsf spreads naturally, the process is further aided by human-mediated transport. the bsf larvae’s efficient consumption of organic waste renders them valuable agents in composting processes. they can effectively convert organic waste into nutrient-rich, high-protein biomass (czekała et al., 2020). although there is no data on commercial farming in serbia nor montenegro, local attempts at rearing closed bsf colonies are probable, especially within amateur herpetoculture circles. while citizen science databases inaturalist and alciphron (alciphron database © 2014-2024 habiprot) have documented encounters with the species in the vicinity of belgrade for serbia, h. illucens has never been officially registered. similarly in montenegro, where no records have been published since beschovski & manassieva, (1996), database inaturalist reports several observations, especially along the coastal line. through reporting on the wild-caught specimens, we officially confirm the presence of bsf in serbia for the first time. additionally, we provide the first recent confirmation for montenegro. materials and methods material was collected during the year 2023 in serbia and montenegro (fig. 1). in serbia, two adult specimens of the black soldier fly were collected by hand, inside a facility on the window glass in the village of stari ledinci, near the city of novi sad. the montenegro sample originates from kotor and was collected by a sweeping net. all specimens are dry mounted, pinned, and kept in the collection at the faculty of sciences and mathematics, university of niš. legator abbreviations are as follows: vž = vladimir žikić, ss = sandra stanković, mp = mihajlo popović. results material examined: serbia: stari ledinci, 45°11’19.2”n, 19°48’24.1”e, 15.09.2023, 1 ♀, 1 ♂, leg. vž (fig. 2). montenegro: kotor, 42°25’15.9”n, 18°45’53.8”e, 05.06.2023, 1 ♀, 2 ♂♂, leg. ss & mp. remarks: hermetia illucens is a moderately large fly usually measuring around 15 mm in body length (ranging from 12 mm to 20 mm). on average, females are generally larger than males. the bsf bears some resemblance to wasps. the head is relatively small in proportion to the body, glossy black with a white stripe running along the lower part of the face. large broadly separated eyes are also black. antennae with eight flagellomere segments, with the last segment equal in size to the other segments’ combined length; usually, dark brown to black (oliveira et al., 2016). thorax is shiny black. legs are mainly black with white tarsi. the hind tibia also has white coloured basal part. the wings are smoked brown. five abdominal segments are predominately black except the posterior margin of the first segment and almost the entire second segment having a pair of translucent white oblong spots (rozkošný, 1982; oliveira et al., 2016). the white abdominal spots are one of the important and conspicuous characteristics in bsf recognition. however, the extent i.e., the size of the white markings on the female abdomen is particularly variable. 48 biologica nyssana ● 15 (1) june 2024: 47-51 stanković et al. ● new records of the black soldier fly hermetia illucens (diptera: stratiomyidae) in serbia and montenegro fig. 1. sampling sites; serbia, stari ledinci and montenegro, kotor biologica nyssana ● 15 (1) june 2024: 47-51 stanković et al. ● new records of the black soldier fly hermetia illucens (diptera: stratiomyidae) in serbia and montenegro 49 discussion since the black soldier fly has been present in europe for almost a century, starting from malta (lindner, 1936) and later spreading to the continental part (demetriou et al., 2022), it was a matter of time before it reached the entire balkan region. although bsf demonstrates the ability to adjust to the environmental conditions of many newly colonized areas (kaya et al., 2021), at this point, we cannot determine the origin of the specimens discussed in this paper. introductions from neighbouring countries, such as hungary, should not be excluded, as there are reports of both large-scale commercial farming (bsf systems kft., 2024) and small bsf pet food production. moreover, in 2022, bsf was already reported present in hungary (murányi, 2022). the specimens from serbia were caught in mid-september, which is a rather late finding, as most of the encounters in europe are during the early summer season. this suggests that the specimens might have been escaped from putative house farming. on the other hand, the locality is suburban, with a lot of small conventional livestock farms and, subsequently, plenty of available manure and organic waste. the finding from montenegro is from early june, implying that a population in this mediterranean country might have been established. according to demetriou et al. (2022) black soldier fly has the potential to spread into all countries bordering the mediterranean basin, as well as central european countries and the balkans. although the species is rather thermophilic, it has spread as far north as the eastern czech republic (roháček & hora, 2013) suggesting that it has the potential to withstand the unfavourable climate, especially in manure piles where the heat is produced by bacterial activity. adults of bsf can be easily recognized, especially by the white and translucent spots on the abdomen. although this very characteristic can vary in size and shape, it is always present. therefore, citizen science can be of the essence in reporting the spreading trajectories of bsf in the region, as well as shaping the possible introduction scenarios. although invasive, bsf is generally considered a beneficial species, especially in waste management and as a protein-rich food source (li et al, 2011; wang & shelomi, 2017), and it has even been released in nature as a biological agent against the house fly (skuhravá et al., 2010). although black soldier flies are generally harmless to humans, they have been known to cause occasional myiasis in people (rozkošný, 1982). the species’ synanthropic behaviour, quick dispersal, and adaptability to varying temperature ranges have led to its cosmopolitan distribution that is becoming increasingly pronounced over the last few decades. this raises significant concerns about its potential impact on local fauna and the ecosystem in general and emphasizes the scarcity of data regarding their role in new environments. fig. 2. appearance of the female of h. illucens sampled in serbia, stari ledinci; photo: s. stanković 50 biologica nyssana ● 15 (1) june 2024: 47-51 stanković et al. ● new records of the black soldier fly hermetia illucens (diptera: stratiomyidae) in serbia and montenegro it is therefore of great importance to gather more detailed information about the current distribution and monitor its interaction with autochthonous fauna through both citizen science and extensive ecological studies. acknowledgements. the authors would like to thank sandra stanković and mihajlo popović for providing the samples collected in montenegro. this study is partly founded by the ministry of science, technological development and innovation of the republic of serbia (contract no. 451-03-65/2024-03/200124). references beschovski, v.l. & manassieva, e.p. 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(2017). review of black soldier fly (hermetia illucens) as animal feed and human food. foods, 6(10), 91. zlatković et al. 2022, biologica nyssana 13(2) 13 (2) december 2022: 109-118 doi: 10.5281/zenodo.7437240 brometum commutati – a new halophytic plant association in central and south serbia original article ivana zlatković toplica academy of professional studies, department of agricultural and technological studies, prokuplje, serbia gajevicivana@yahoo.com (corresponding author) dragana jenačković gocić department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, niš, serbia svetlana bogdanović toplica academy of professional studies, department of agricultural and technological studies, prokuplje, serbia vladimir ranđelović department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, niš, serbia received: september 13, 2022 revised: october 25, 2022 accepted: november 15, 2022 abstract: during the phytocoenological studies in salt marshes of central and south serbia data were collected from 168 stands. after the data were entered into the vegetation database turboveg, cluster analysis performed in flora software package has shown presence of 13 associations in the study area. at the sites of oblačina and bresničić salt marshes a new plant association was recorded and phytocoenologically described under the name brometum commutati ass. new. continuous monitoring of floristic composition during an entire vegetation season has shown that this association was built by 58 species. in addition to the dominant species bromus commutatus, higher abundance and cover values were recorded for: hordeum geniculatum, puccinellia distans, cerastium dubium and trifolium lappaceum. the total cover value for stands at both localities was 100%. this association occupies a much greater surface area at oblačina salt marsh, where 11 stands were recorded. stands of this association alternate with stands of association puccinellietum limosae soó 1933. the sample also included three phytocoenologically described stands from bresničić salt marsh. although this plant community was recorded at the less halophytic soils, in the syntaxonomic sense it still belongs to the alliance puccinellion limosae soó 1933 due to the floristic similarity with other associations of that alliance. key words: saline habitats, southern serbia, plant community, oblačina salt areas, bresničić salt area apstrakt: brometum commutati nova halofitna asocijacija u centralnoj i južnoj srbiji tokom fitocenoloških istraživanja na slatinama centralne i južne srbije prikupljeni su podaci sa 168 sastojina. nakon unošenja podataka u vegetacijsku bazu turboveg i urađenu klaster analizu u flora softver paketu, na istraživanom području konstantovano je 13 asocijacija. na lokalitetima oblačinska i bresničićka slatina, konstatovana je i fitocenološki opisana nova biljna zajednica imenovana kao brometum commutati ass. nova. kontinuiranim praćenjem florističkog sastava tokom jedne vegetacione sezone, ustanovljeno je da u izgradnji zajednice učestvuje 58 vrsta. pored dominantne vrste, bromus commutatus, većom brojnošću i pokrovnošću individua ističu se: hordeum geniculatum, puccinellia distans, cerastium dubium i trifolium lappaceum. ukupna pokrovnost sastojina na oba lokaliteta iznosi 100%. asocijacija zauzima mnogo veće površine na oblačinskoj slatini, gde je zabeleženo 11 sastojina. sastojine ove asocijacije smenjuju se sa sastojinama acocijacije puccinellietum limosae soó 1933. na bresničićkoj slatini fitocenološki su opisane tri sastojine. iako je ova biljna zajednica zabeležena na zemljištu nižeg saliniteta, u sintaksonomskom smislu ipak pripada svezi puccinellion limosae soó 1933 zbog florističke sličnosti sa drugim asocijacijama pomenute sveze. ključne reči: slatine, južna srbija, biljna zajednica, oblačinska slatina, bresničićka slatina introduction halomorphic (saline) soils are relatively widespread throughout the world and occupy 10% of the earth’s surface (o’leary & glenn, 1994). these are specific types of soil covered by the general terms of salt marsh and dryland halomorphic soils. the main characteristic of saline habitats is saline or halomorphic soil suitable for development of specific flora and vegetation that is adapted to the conditions of increased salt concentration. at the same time, salt marshes are highly endangered habitats that occur in © 2022 zlatković et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 109 14th symposium on the flora of southeastern serbia and neighboring regions fragments, so the ranges of habitat-specific species are also characterized by marked fragmentation (nedeljković et al., 2014). salines are unique ecosystems and often the last refugia for a large number of endemic and relict species. plant associations represented on halomorphic soils are relatively poor in species. species combinations are specific but very diverse, so the classification of halophyte associations is quite complex. distribution of certain vegetation types is closely related to salt content, salt type and depth of soil horizons with higher salt concentration (molnar & borhidi, 2003). globally speaking, salt marsh habitats occupy significant areas and appear naturally on about one million hectares in the world. they are mostly represented within the desert, semidesert, steppe, chernozem and forest-steppe areas (yadav, 2003). in serbia, halomorphic soils are mostly present in vojvodina, while in the southern part of the country saline habitats are a real rarity and are reduced to a few small localities in the valley of rivers južna morava and toplica. they represent specific ecosystems characterized by presence of characteristic flora and vegetation which is more or less specialized to this type of habitats (zlatković et al., 2005). in that region these specific habitats develop in conditions of isolation and high anthropogenic pressure. the first studies of these important areas were carried out by pančić (1884) and petrović (1885). lalinac salt marsh is the best studied locality (niketić, 1995; milosavljević et al., 2002; ranđelović et al., 2007). a significant contribution to research on this type of vegetation in the area of vojvodina and southeastern serbia was made by slavnić (1940, 1948). there are also data on the halophytic vegetation around prokuplje (ranđelović at al., 2000) while in 2005 a detailed study on salt marshes in this part of serbia was published by the institute for nature conservation. the authors presented the results of their research on flora but also on vegetation, which was not previously described in much detail (zlatković et al., 2005). in 2014, a group of authors (zlatković et al., 2014) combined the previous study results with their own research data for all salt marsh areas of central and south serbia. they recognized presence of 333 taxa classified into 176 genera and 46 families. the ecology and ecological affinities of selected halophytes from the salt marsh areas in central and south serbia were presented in a paper from 2019 (zlatković et al., 2019a), while in the same year a new association, allio guttate-limonietum gmelinii, was described from the area of lalinac and lepaja salt marshes (zlatković et al., 2019b). materials and methods study area the study area includes localities in central and south serbia (tab. 1, fig. 1), divided into 3 groups: aleksandrovac salt marsh area, lalinac salt marsh area and the salt marsh fragments in the valley of toplica river (zlatković et al., 2014). the aleksandrovac salt marsh area includes several smaller localities (aleksandrovac, oslare, levosoje, bujanovac and neradovac) with developed halomorphic soils and very interesting flora and vegetation. the largest surface area under halomorphic soils is present at the salt marsh near aleksandrovac. the lalinac salt marsh area is situated at the foothills of mali jastrebac mt. it includes several 110 biologica nyssana ● 13 (2) december 2022: 109-118 zlatković et al. ● brometum commutati – a new halophytic plant association in central and south serbia table 1. overview of study sites with data on geographic position, altitude and general climate characteristics id locality longitude latitude altitude (m) mean annual air temperature (°c) total annual precipitation (mm) 1 levosoje 42°25'50'' 21°44'50'' 400 11.2 553 2 oslare 42°25'39'' 21°43'21'' 405 11.2 553 3 bujanovac 42°27' 21°45' 395 11.2 550 4 aleksandrovac 42°29'20'' 21°54'12'' 401 11.2 540 5 neradovac 42°31'17'' 21°52'56'' 403 11 550 6 lalinac 43°20'42'' 21°44'45'' 200 11.1 633 7 oblačina 43°18'26'' 21°40'54'' 285 11.1 636 8 lepaja 43°17'35'' 21°39'50'' 285 11.1 636 9 suva česma 43°13'58'' 21°30'43'' 257 10.9 655 10 bresničić 43°14'50'' 21°27'10'' 293 10.8 670 biologica nyssana ● 13 (2) december 2022: 109-118 zlatković et al. ● brometum commutati – a new halophytic plant association in central and south serbia 111 salt marsh fragments. lalinac salt marsh is the largest fragment while there are also oblačina and lepaja salt marsh. in addition to these salt marshes, another important fragment is situated near the village bresničić, about 1 km from the bed of river toplica, on its left bank. previously there were also several salt marsh fragments in the area between prokuplje and kuršumlija, in the alluvial plain of toplica. their soils have barely shown any halomorphic characteristics, and the flora and vegetation with halomorphic character were subsequently destroyed. the salt marsh near the village bresničić remains as one of the better preserved salt marsh fragments. the results of phytocoenological studies were fig. 1. map of study area. the main data on geographic position and climate characteristics of study sites are presented in tab. 1 used to determine the diversity of halophyte communities in the researched area and their mutual syntaxonomic relationships. for these purposes, during 2013 year, data were collected from 168 stands with individual surface areas of 10-60 m2. the phytocoenological research of the stands followed the braun-blanquet’s (1951) approach. the plant material necessary for determination was collected from the surrounding stands in order not to disrupt the qualitative and quantitative composition of the studied vegetation plots. the material was herbarized, and the herbarium specimens were stored in the herbarium collection “herbarium moesiacum” of the faculty of sciences and mathematics at the university in niš (hmn). 112 biologica nyssana ● 13 (2) december 2022: 109-118 zlatković et al. ● brometum commutati – a new halophytic plant association in central and south serbia relevant dichotomous keys (tutin et al. (ed.) 19641980; josifović (ed.) 1970-1980) and “флора на нр българия” (јорданов (ед.), 1963-1979, велчев, 1979-1995) were used for determination of plant material. nomenclature of recorded taxa was matched to the euro+med database at the following link http://ww2.bgbm.org/europlusmed/query.asp. the taxa that were not determined to species level were excluded from the analysis, as well as the mosses and lichens. the collected data were imported into the vegetation database turboveg (hennekens & schaminee, 2001). cluster analysis was performed in flora software package in order to define associations and determine the floristic similarities between associations (karadžić & marinković, 2009; karadžić, 2013). results and discussion the results of this research have shown that salt marshes of central and south serbia host 13 associations from three classes: thero-salicornietea tx. in tx. et oberd. 1958, festuco-puccinellietea soó ex vicherek 1973 and molinio-arrhenatheretea tx. they include an association with the species bromus commutatus, which was described for the first time as brometum commutati ass. new (fig. 2, appendix 1). this association was recorded at oblačina and bresničić salt marshes at altitudes of 289-308 m above sea level. vegetation cover was 100% at both sites. in addition to the dominant species bromus commutatus, following species have also shown significant values of cover: hordeum geniculatum, puccinellia distans, cerastium dubium and trifolium lappaceum. due to the grazing intensity and vicinity of cultivated areas, the association also includes a high number of ruderal species. the surface areas under this association are much greater at oblačina salt marsh, where 11 stands were recorded. the sizes of test plots were 30-50 m2. the stands of this association are interspersed with stands of association puccinellietum limosae soó 1933. at the bresničić salt marsh there were three stands for which phytocenological data were collected, with surface area of 20 m2. the species bromus commutatus was also recorded at localities: aleksandrovac, in associations puccinellietum convolutae micevski 1965 and hordeetum hystricis wendelbg. 1943; lalinac, in associations: puccinellietum limosae soó 1933, camphorosmetum monspeliacae micevski 1965, caricetum divisae slavnić 1948, juncetum compressi br.-bl. 1918 ex libb, scorzonero parviflorae-juncetum gerardii (wenzl 1934) wendelberger 1943 and allio guttate-limonietum gmelinii ass. new; and lepaja, in association allio guttate-limonietum gmelinii ass. new. at oblačina salt marsh, in addition to creating the association brometum commutati ass. new, it was also recorded within the following associations: puccinellietum limosae soó 1933, hordeo-caricetum distantis micevski 1957 and caricetum divisae slavnić 1948, while at bresničić salt marsh it was recorded within the associations puccinellietum limosae soó 1933 and caricetum divisae slavnić 1948. however, its presence is not of any significant diagnostic level in any of these associations. micevski (1965) described the association bromo-alopecuretum k. micevski 1957 from the higher, drier habitats of ovče polje as a therophyte community representing the most arid type of meadows at ovče polje. this association belongs to the alliance trifolion resupinati k. micevski 1957 and it is almost void of any halophytic plants. in addition, ranđelović & zlatković (2005) stated in the syntaxonomic overview that at the salt marshes of central and south serbia there is an association bromo-alopecuretum k. micevski 1957, composed of species bromus commutatus and alopecurus fig. 2. photos of brometum commutati ass. new. oblačina salt marsh, july 2013 113 biologica nyssana ● 13 (2) december 2022: 109-118 zlatković et al. ● brometum commutati – a new halophytic plant association in central and south serbia utriculatus, from class molinioarrhenatheretea tx. 1937 and alliance trifolion resupinati k. micevski 1957. however, in a different study several years later, presence of species alopecurus utriculatus was not recorded in stands of this association, while significantly higher number of typical halophytes (hordeum geniculatum, puccinellia distans, podospermum canum) were present, so we believe that this association belongs to the alliance puccinellion limosae soó 1933. in the dendrogram it is situated immediately next to the association puccinellietum limosae soó 1933 (fig. 3). conclusions association brometum commutati ass. new is a particularly interesting and important newly discovered halophytic association. it covers large areas at oblačina salt marsh and smaller areas at bresničić salt marsh. as the salt marsh areas in central and south serbia occupy small areas and experience strong anthropogenic impact, it is necessary to continue with appropriate conservation measures. these areas are important biodiversity centers, which is recognized as the continental salt marshes throughout europe are protected with the network natura 2000 (interpretation manual of european union habitats 1999; piernik et al. 2006). bresničić salt marsh is a protected area of the iv degree under the iucn guidelines (iucn category iv – protected area managed for conservation through management). according to classification of the national legislative, it belongs to the third category of protection, protected areas of local character, and represents a protected habitat. it was also included in the group of rare and fragile habitats (ret/frag (a)) and the list of emerald habitats, as a potential natura 2000 habitat (nedeljković et al., 2014). it is necessary to implement appropriate measures so oblačina salt marsh would be assigned a protection category as well. acknowledgements. this work was supported by the ministry of education, science and technological development fi g. 3 . d en dr og ra m o f h al op hy te v eg et at io n of c en tra l a nd s ou th s er bi a (1 -3 3 p uc ci ne lli et um li m os ae s oó 1 93 3; 3 470 c ar ic et um d iv is ae s la vn ić 1 94 8; 71 -8 3 b ro m et um c om m ut at i a ss . ne w ; 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[in serbian]. zlatković, i., zlatković, b., ranđelović, v., jenačković, d., amidžić, l. 2014: taxonomical, phytogeographical and ecological analysis of the salt marsh flora of central and southern serbia. biologica nyssana 5(2): 91-102. zlatković, i., jenačković, d., ranđelović, v. 2019a: inland salt areas of southeast serbia: ecological preferences of certain representatives of flora. biologia 74(11): 1425-1440. zlatković, i., jenačković gocić, d., ranđelović, v. 2019b: allio guttate-limonietum gmelinii new halophytic association in south serbia. 13th symposium on the flora of southeastern serbia and neighbouring regions, stara planina, june 20-23, book of abstracts, 68 p. 115 biologica nyssana ● 13 (2) december 2022: 109-118 zlatković et al. ● brometum commutati – a new halophytic plant association in central and south serbia a pp en di x a pp en di x 1. p hy to co en ol og ic al ta bl e of b ro m et um c om m ut at i a ss . n ew l oc al ity o bl ač in a sa lt m ar sh b re sn ić ič sa lt m ar sh d at e of r ec or di ng 21 m ay 23 ju n 21 m ay 23 ju n 6 ju l 27 m ay 25 se p degree of presence su rf ac e (m 2 ) 30 30 50 50 50 30 30 30 30 30 30 20 20 20 c ov er ag e (% ) 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 a lti tu de (m ) 28 9 29 1 29 1 29 0 30 8 30 5 29 2 29 5 29 2 29 2 29 1 29 9 29 8 29 8 l on gi tu de 43° 18.552' 43° 18.550' 43° 18.432' 43° 18.438' 43° 18.462' 43° 18.450' 43° 18.404' 43° 18.467' 43° 18.474' 43° 18.471' 43° 18.471' 43° 14.862' 43° 14.859' 43° 14.859' l at itu de 21° 41.078' 21° 41.076' 21° 40.954' 21° 40.952' 21° 40.939' 21° 40.961' 21° 40.989' 21° 40.996' 21° 40.998' 21° 41.005' 21° 41.018' 21° 27.247' 21° 27.250' 21° 27.250' n um be r of sp ec ie s 10 7 8 6 13 14 11 15 8 10 6 15 20 7 d ia gn os tic sp ec ie s o f c la ss m ol in io -a rr he na th er et ea br om us c om m ut at us s ch ra de r 5 5 5 5 5 5 5 5 5 5 5 5 5 5 v tr ifo liu m la pp ac eu m l . r + 1 1 2 1 r iv ju nc us c om pr es su s j ac q. r r r r r r ii i tr ag op og on p ra te ns is l . + + r r ii i po a pr at en si s l . r r r ii ra nu nc ul us sa rd ou s c ra nt z r r i ac hi lle a m ill ef ol iu m l . r i tr ifo liu m h yb ri du m l . r i sa lv ia p ra te ns is l . r i ru m ex c ri sp us l . r i biologica nyssana ● 13 (2) december 2022: 109-118 zlatković et al. ● brometum commutati – a new halophytic plant association in central and south serbia d ia gn os tic sp ec ie s o f c la ss f es tu co -p uc ci ne lli et ea h or de um g en ic ul at um a ll. 3 3 2 + + + + 1 1 + r v pu cc in el lia d is ta ns (l .) pa rl. 1 1 1 1 1 1 + 1 1 2 4 r v po do sp er m um c an um c .a . m ey er r r r r + 1 + r iv c ar ex d iv is a h ud so n r r r ii ph ol iu ru s p an no ni cu s ( h os t) tr in . r r r r ii ju nc us g er ar di i l oi se l. r i lo tu s t en ui s w al ds t. & k it. ex w ill d. r i d ia gn os tic sp ec ie s o f c la ss f es tu co -b ro m et ea tr ifo liu m c am pe st re s ch re be r 3 i tr ifo liu m n ig re sc en s v iv . r i pl an ta go la nc eo la ta l . r r i pr os pe ro a ut um na le (l .) sp et a r i xe ra nt he m um a nn uu m l . r i d ia gn os tic sp ec ie s o f c la ss p ap av er et ea rh oe ad is m at ri ca ri a ch am om ill a l. r + r r ii m yo so tis a rv en si s ( l. ) h ill + i c re pi s s et os a h al le r fi l. r i vi ci a pa nn on ic a c ra nt z + i an th em is a rv en si s l . r i d ia gn os tic sp ec ie s o f c la ss p hr ag m ite te a co m m un is ph ra gm ite s a us tr al is (c av .) st eu d. r i bo lb os ch oe nu s m ar iti m us (l .) pa lla r i el eo ch ar is p al us tr is (l .) r oe m er & s ch ul te s r i el eo ch ar is p al us tr is (l .) r i 116 117 biologica nyssana ● 13 (2) december 2022: 109-118 zlatković et al. ● brometum commutati – a new halophytic plant association in central and south serbia d ia gn os tic sp ec ie s o f c la ss a rt em is ie te a vu lg ar is c ic ho ri um in ty bu s l . + i li na ri a vu lg ar is m ill . r i c en ta ur ea so ls tic ia lis l . r i m el ilo tu s o ffi ci na lis (l .) la m . r i so nc hu s a rv en si s l . r i ru m ex p at ie nt ia l . r i d ia gn os tic sp ec ie s o f c la ss c ak ile te a m ar iti m ae at ri pl ex p ro st ra ta b ou ch er ex d c . r i d ia gn os tic sp ec ie s o f c la ss s is ym br ie te a la ct uc a se rr io la l . r r r + ii h or de um m ur in um l . r i c re pi s p ul ch ra l . r i g er an iu m d is se ct um l . + i g er an iu m m ol le l . r i d ia gn os tic sp ec ie s o f c la ss p ol yg on opo et ea a nn ua e c yn od on d ac ty lo n (l .) pe rs . r 1 i po ly go nu m a vi cu la re l . r r r + r r ii i d ia gn os tic sp ec ie s o f c la ss is öe to -n an oj un ce te a c er as tiu m d ub iu m (b as t.) o . sc hw ar tz 3 3 1 3 + r 1 iv m en th a pu le gi um l . r r i d ia gn os tic sp ec ie s o f c la ss c he no po di et ea tr ifo liu m e ch in at um b ie b. 1 + + + + ii m ed ic ag o ar ab ic a (l .) h ud . r i av en a fa tu a l. r i d ia gn os tic sp ec ie s o f c la ss t ri fo lio -g er an ie te a sa ng ui ne i c am pa nu la ra pu nc ul us l . r i biologica nyssana ● 13 (2) december 2022: 109-118 zlatković et al. ● brometum commutati – a new halophytic plant association in central and south serbia d ia gn os tic sp ec ie s o f c la ss h el ia nt he m et ea g ut ta ti tr ifo liu m st ri at um l . r i d ia gn os tic sp ec ie s o f c la ss s ed osc le ra nt he te a c er as tiu m b ra ch yp et al um pe rs . 2 i m ed ic ag o m in im a (l .) l. r i u ns pe ci fie d sp ec ie s ta ra xa cu m o ffi ci na le w eb er r r i br om us m ol lis l . r r r i bu pl eu ru m te nu is si m um l . + 1 + i ag ro st em m a gi th ag o l. r i g al iu m te nu is si m um m . b ie b. r i 118 koljanin et al. 2023, biologica nyssana 14(1) 14 (1) june 2023: 15-29 doi: 10.5281/zenodo.8027089 notes on the distribution and conservation status of some rare plants of wet habitats in bosnia and herzegovina original article dragan koljanin faculty of forestry, university of banja luka, stepe stepanovića 75a, 78 000 banja luka, bosnia and herzegovina draagaan98@gmail.com (corresponding author) jugoslav brujić faculty of forestry, university of banja luka, stepe stepanovića 75a, 78 000 banja luka, bosnia and herzegovina vladimir stupar faculty of forestry, university of banja luka, stepe stepanovića 75a, banja luka 78000, bosnia and herzegovina đorđije milanović faculty of forestry, university of banja luka, stepe stepanovića 75a, banja luka 78000, bosnia and herzegovina received: december 02, 2022 revised: december 21, 2022 accepted: december 22, 2022 abstract: wetland habitats in bosnia and herzegovina are usually small, fragmented and under numerous anthropogenic pressures. therefore species confined to such habitats can be considered being among the most threatened plant species in bosnia and herzegovina. the focus of this article is the distribution pattern and threatened status of five such plant species: achillea ptarmica, carex elongata, carex strigosa, comarum palustre and prunus padus. while these taxa can be common in other parts of europe, in bosnia and herzegovina, they are rare and usually restricted to a small number of localities. wetland flora can be found in the dinaric mountains, where it is confined to the karst fields, mountain plateaus, spring areas and banks of glacial lakes, but as well in the lowlands in the north of the country, occupying the remnants of wetland habitats. published, new and unpublished (herbarium) chorological data for bosnia and herzegovina, description of the habitat, population estimate, threats on habitats and species, and estimation of threatened status based on iucn criteria are given for each studied species. key words: achillea ptarmica, carex elongata, carex strigosa, comarum palustre, prunus padus, distribution, ecology apstrakt: o rasprostranjenju i ugroženosti nekih retkih biljnih vrsta vlažnih staništa bosne i hercegovine močvarna staništa u bosni i hercegovini su obično mala, fragmentirana i pod brojnim antropogenim pritiscima. stoga se vrste ograničene na takva staništa mogu svrstati među najugroženije biljne vrste u bosni i hercegovini. fokus ovog rada su rasprostranjenje i status ugroženosti pet takvih biljnih vrsta: achillea ptarmica, carex elongata, carex strigosa, comarum palustre i prunus padus. iako su ovi taksoni većinom uobičajeni u drugim dijelovima evrope, u bosni i hercegovini oni su retki i obično ograničeni na mali broj lokaliteta. močvarna flora se može naći i na dinarskim planinama, gde je svedena na kraška polja, planinske visoravni, izvorišta i obale glacijalnih jezera, ali i u nizijama na severu zemlje, zauzimajući ostatke močvarnih staništa. za svaku proučavanu vrstu dati su objavljeni, novi i neobjavljeni (herbarijski) horološki podaci za bosnu i hercegovinu, opis staništa, procena populacije, ugroženost staništa i vrsta, te procena ugroženosti na osnovu iucn kriterijuma. ključne reči: achillea ptarmica, carex elongata, carex strigosa, comarum palustre, prunus padus, rasprostranjenost, ekologija vrsta introduction at a broad geographical scale, species distribution is determined by macroclimate, evolutionary and migration history (huntley et al., 1995; knollová & chytrý, 2004; pearson et al., 2004; tsiftsis et al., 2012). the distribution of species is also influenced by relief, soil properties, mesoclimate and microclimate at smaller scales, as well as human activities that can introduce or cause the extinction of species in certain locations. at the border of the distribution area, a species often transits from a continuous to a disjunct distribution pattern. this is caused by the fact that favourable ecological conditions appear in disjunct patches in limited areas because of a special constellation of conditions. at the edge of their range, populations have restricted areas of occurrence; often, they are ecologically © 2023 koljanin et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 15 different and characterized by low abundance in terms of population size (lesica & allendorf, 1995; duffy et al., 2009). furthermore, plant populations that are at the edge of their distribution range are often at their ecological limits because their habitats are of declined suitability, and therefore they are more sensitive to changes in ecological conditions (koljanin et al., 2021). knowledge of distribution patterns and ecology can be considered fundamental for species conservation (heywood & iriondo, 2003). species of wet habitats can be considered to be one of the most threatened vascular plants in bosnia and herzegovina (redžić et al., 2009). due to precipitation regimes in the growing season, with long dry periods, significantly higher temperatures, and dominant types of water-permeable geological substrata (especially limestones), the balkan peninsula is less covered by wetland habitats than most of the central and northern parts of europe. therefore, some species that are common in wetlands of the northern parts of europe are rare or absent in the balkans. peatlands are of greater value in southern regions than those further on the north, as they usually have specifically distinctive fauna and flora, as well as isolated and possibly more genetically divergent populations of circumboreal species than those of northern mires (desrochers et al., 2000). in bosnia and herzegovina, mires occupy about 16,250 ha scattered throughout the country, with most of them more or less degraded in the last 150 years (milanović, 2017a). there are only two larger areas with the accumulation of peat (livanjsko polje and hutovo blato), with an area of around 13,000 ha (milanović, 2017a), while all others occupy small areas, mostly on higher elevations, where a particular constellation of ecological factors ensures the existence of these habitats (barudanović et al., 2019). since these sites are very sensitive to changes and usually have a limited area, any exploitation of these sites could have huge negative consequences for biodiversity. the northern part of bosnia and herzegovina overlaps with the edge of the pannonian plain, which is dominantly characterized by typical plain configuration influenced by slow-flowing and large alluvial rivers; this area was very rich in wetland habitats several centuries ago. due to the unregulated banks of these rivers, the surrounding lowlands were normally seasonally flooded, while rivers often changed their beds, creating conditions necessary for the development of various wetland habitats on the river terraces. the deterioration of wetlands in northern bosnia and herzegovina began with the systematic regulation of the flow of the sava river in the second half of the 18th century and continued with the construction of a reclamation system to preserve agricultural land (mrgić, 2007). it could be assumed that these changes caused the disappearance of many populations and even species confined to wetland habitats. such an example is fritillaria meleagris l. which disappeared from many localities after the construction of the drainage system (koljanin et al., 2021). going from north to south of bosnia and herzegovina, the northern lowlands gradually rise to the mountain chain of the dinaric alps, which is an ecologically very heterogeneous area. in this area, wetland flora is typically limited to karst fields, mountain plateaus and depressions, flattened small river terraces, and fragments alongside mountain brooks and around springs. these habitats are relict remnants of vegetation where some species had found a refugium and have great importance for biodiversity on a national level, as they are inhabited by many plant species that are rare in the country. a great example of such a species is liparis loeselii (l.) rich. which is found only in one locality in balkan peninsula (milanović, 2012). one of the fundamental problems in the protection of plants and habitats is the lack of data on these species. the dinaric part of the country is rich in endemic plant species, while they are absent in northern lowlands (lubarda et al., 2014), which makes it less attractive for botanical studies. this resulted in a lack of data for some species that have main distribution in the northern part of bosnia and herzegovina. on the other hand, some species in the dinaric mountains are confined to small, refugial areas; therefore, some populations went undetected. in this paper, we provide new chorological and ecological data for five species that are most common in wetlands of central european countries but occur very rarely in bosnia and herzegovina: achillea ptarmica l., carex elongata l., carex strigosa huds., comarum palustre l. and prunus padus l. also, we estimated the most appropriate red list category for all of them. materials and methods all chorological data on the occurrence of five target species (achillea ptarmica, carex elongata, carex strigosa, comarum palustre and prunus padus) in bosnia and herzegovina were collected from the available literature. all literature records were georeferenced using topographic maps with a scale of 1:25,000 in qgis software. fieldwork was conducted in the period from 2006 to 2021 in various wetlands in the northern plains of bosnia and herzegovina and in the karst fields, where such habitats are registered or 16 biologica nyssana ● 14 (1) june 2023: 15-29 koljanin et al. ● notes on the distribution and conservation status of some rare plants of wet habitats in bosnia and herzegovina biologica nyssana ● 14 (1) june 2023: 15-29 koljanin et al. ● notes on the distribution and conservation status of some rare plants of wet habitats in bosnia and herzegovina 17 expected, but also in the dinaric alps, at higher elevations, especially around glacial lakes and springs. coordinates were recorded in wgs1984 using a gps device, and digital photographs were also collected in the field. herbarium material was stored in the herbarium of the faculty of forestry of the university of banja luka and in the private collection of đorđije milanović. this material was used for identification mainly following identification keys in flora europaea (ball et al., 1968; webb & rix, 1972; richardson, 1976; chater, 1980) while the nomenclature follows the euro+med (2006) and stupar et al., (2021). target species are listed alphabetically with the following parameters: literature and new chorological data, general distribution and ecology, habitat and species threats and conservation status. published and new records are indicated by: locality name (bolded if it is a new record), wgs1984 coordinates, elevation (if available for literature data), mgrs 10×10 km grid code, plant community/habitat type (if available), soil type (if available), population characteristics (if available), date of collecting, collector and bases of record. maps of species distribution in bosnia and herzegovina are presented on a 10×10 km mgrs grid, using different symbols for published, new and unpublished (herbarium) data. short descriptions of habitats and their stability were given based on field observations. threat status for each species was estimated or reassessed following the iucn criteria and categories (iucn species survival commission, 2012a, 2012b). given that the area of occupancy (aoo) for each of the species under study is estimated to be less than 10 km2, we applied the b2 criterion to each species. additionally, considering that the likelihood of immigration from neighbouring countries is very low, based on the iucn species survival commission (2012b), we decided to maintain the original category from the initial assessment in step one for each species, as noted by the iucn species survival commission (2012a). results and discussion achillea ptarmica l. published and new chorological data achillea ptarmica is a herbaceous perennial plant easily recognizable by linear undivided leaves with serrate margins (fig. 1) and without glandular punctuations on the leaf surface (richardson, 1976). this species was previously reported only from three localities in nevesinjsko polje (sagorski, 1901; ritter-studnička, 1954; beck-mannagetta et al., 1983). new records show that species have a wider distribution in various conditions in bosnia and herzegovina (fig. 2). fig. 1. achillea ptarmica at locality of berek (© d. koljanin) fig. 2. distribution of achillea ptarmica in bosnia and herzegovina this species was recorded in the following localities: • between nevesinje and pustoljani (nevesinjsko polje); coordinates: 18.159896°e, 43.292712°n; elevation: ca 850 m; mgrs 10×10 square: bn69; habitat: wet meadows with sparganium microcarpum; population: numerous individuals; date: 1912; collector: e. sagorski; bases of record: literature (sagorski, 1901; ritter-studnička, 1954; beck-mannagetta et al., 1983), marked from a ritter-studnička, (1954) as probably extinct. • banks of dušila stream (nevesinjsko polje); coordinates: 18.15°e, 43.31667°n; elevation: ca 850 m; mgrs 10×10 square: bp60; habitat: stream banks; date: 08.1955, 09.08.1956; 18 biologica nyssana ● 14 (1) june 2023: 15-29 koljanin et al. ● notes on the distribution and conservation status of some rare plants of wet habitats in bosnia and herzegovina collector: z. devetak, hilda ritter-studnička; bases of record: as f. linearis, in herbarium sara 42214, sara 42215; corresponding literature (ritter-studnička, 1954; beck-mannagetta et al., 1983). • zlatac (nevesinjsko polje); coordinates: 18.141459°e, 43.341539°n; elevation: ca 850 m; mgrs 10×10 square: bp60; habitat: stream banks; population: unknown; date: 1954; collector: z. devetak; bases of record: literature (ritter-studnička, 1954; beck-mannagetta et al., 1983), as f. lineare. • right bank of the river sana, near village pejići (downstream from sanski most); coordinates: 16.724544°e, 44.886894°n; elevation: 147 m; mgrs 10×10 square: xk37; habitat: wet meadows; population: unknown; date: 10.08.1985; collector: č. šilić; bases of record: herbarium sara 50346-50349. • at few microlocalities on slopes of the zimomor peak towards štirinsko lake (zelengora mt.); coordinates: 18.490669°e, 43.373269°n; elevation: 1720 m; mgrs 10×10 square: bp90; habitat: mountain spring communities and calthion meadows; population: estimated about one-two thousand flowering individuals; date: 05.08.2008; collector: đ. milanović; bases of record: new – herbarium djm 180/01-001. • gredina near sajković (livanjsko polje); coordinates: 16.660997°e, 43.982725°n; elevation: 707 m; mgrs 10×10 square: xj37; habitat: seasonally flooded molinia meadows in karst fields on sandy soils; population: several hundred flowering individuals; date: 09.08.2015; collector: đ. milanović; bases of record: new – herbarium djm 180/01-002. • berek (lijevče polje); coordinates: 17.2361511°e, 45,0343937°n; elevation: 96 m; mgrs 10×10 square: xk78; habitat: wet meadows in succession; soil type: pseudogley on tertiary sediments; population: several hundred flowering individuals; date: 26.06.2021; collector: d. koljanin; bases of record: new – herbarium herb. fac. silv. 000368. • elezagići (lijevče polje); coordinates: 17.244907°e, 45.066040°n; elevation: 102 m; mgrs 10×10 square: xk79; habitat: on the edge of wet meadow, alongside the ditch; population: ca 50 adult individuals; date: 13.10.2021; collector: d. koljanin; bases of record: observation. general distribution and ecology achillea ptarmica is distributed in most of europe, reaching france and the british isles in the west, scandinavia in the north, the western bank of the odra river in poland and northern ukraine in the east. the species is relatively common in central europe. in most of southern europe, it is rare, occurring sporadically at isolated localities or is completely absent in some countries (bilz, 2012). it has boreal-subboreal eurasian distribution, reaching the southernmost limit in the balkan peninsula and can be considered a boreal relict in balkan flora (stevanović, 1999). the known populations in the balkan peninsula are rare and distant from each other, thus forming a disjunct distribution pattern. also, this species is rare in montenegro, with only one known locality from durmitor mt. (stevanović, 1999). in croatia, the species is known only from a few localities in the northern part of the country (nikolić, 2010). the species has been recorded in serbia in the past, but recent research has not confirmed its occurrence suggesting that it may be considered extinct at the national level (stevanović, 1999; bilz, 2012). in central europe, the species is noted to occur on non-calcareous and permanently moist soils in open habitats, including calthion palustris wet meadows (most often), molinion caeruleae meadows, deschampsion cespitosae alluvial meadows and other wet habitats, including even ruderal habitats (dudáš et al., 2017; kaplan et al., 2020), scrub formations and wood margins (richardson, 1976). habitat and species threats in bosnia and herzegovina achillea ptarmica inhabits various wet meadows and more/less permanently wet banks of small lowland streams and mountain springs in bosnia and herzegovina. in the northern part of the country, the populations are situated on the border of trifolion pallidi and deschampsion caespitosae mown meadows, under intense pressure from alien and/or ruderal plant species and natural succession at the same time. the further negative impact could result in the disappearance of this species in the northern part of the country. the populations on zelengora mt. and in livanjsko polje are numerous and stable, without observed negative influences. however, they still have limited spatial distribution, where some potential impacts could cause a substantial reduction in their populations. conservation status the global iucn status of achillea ptarmica is least concern (bilz, 2012). in serbia, the species has the category extinct in the wild (stevanović, 1999). šilić (1996) assessed the species as vulnerable in the list of vascular plants (pteridophyta and 19 biologica nyssana ● 14 (1) june 2023: 15-29 koljanin et al. ● notes on the distribution and conservation status of some rare plants of wet habitats in bosnia and herzegovina fraxinetum angustifoliae and genisto elataequercetum roboris; population: unknown; date: 1964; collector: m. glišić; bases of record: literature (glišić, 1964). • jakovica near the village of cerik (orig. cerje); coordinates: 18.522109°e, 44.808918°n; elevation: ca 100 m; mgrs 10×10 square: cq06; habitat: carpino betuli-quercetum roboris forest; population: unknown; date: 1975; collector: p. fukarek; bases of record: literature (fukarek, 1975). • hrastik near the village of donji žabar; coordinates: 18.656429°e, 44.941478°n; elevation: ca 85 m; mgrs 10×10 square: cq17; habitat: carpino betuli-quercetum roboris forest; population: unknown; date: 1975; collector: p. fukarek; bases of record: literature (fukarek, 1975). • burumac (orig. baranac) in the vicinity of bosanski šamac; coordinates: 18.521154°e, 45.004933°n; elevation: ca 95 m; mgrs 10×10 square: cq08; habitat: carpino betuli-quercetum roboris forest; population: unknown; date: 1975; collector: p. fukarek; bases of record: literature (fukarek, 1975). • gromiželj in semberija; coordinates: 19.311810°e, 44.866086°n; elevation: ca 82 m; mgrs 10×10 square: cq66; habitat: alnus glutinosa forest; population: numerous individuals; date: 29.05.2010; collector: đ. milanović, j. brujić, v. stupar and d. nikić; bases of record: literature (petronić et al., 2010; milanović et al., 2011;) herb. djm: 65/04–045. • han kram on sljemeska mt.; coordinates: 18.906667°e, 44.038889°n; elevation: ca 1050 m; mgrs 10×10 square: cp37; habitat: edge of pino-betuletum pubescentis; population: scattered individuals; date: 28.06.2010; collector: đ. milanović, j. brujić and j. travar; bases of record: literature (milanović et al., 2011), herb. djm: 65/04–046. • above donja pećina in bijambare; coordinates: 18.502953°e, 44.093964°n; elevation: ca 925 m; mgrs 10×10 square: cp08; habitat: tall herbs with scirpus sylvaticus on a brook terrace; population: less than ten flowering individuals; date: 13.06.2020; collector: đ. milanović; bases of record: herb. djm: 65/04-047. • podbrdo in podrašničko polje; coordinates: 16.985369°e, 44.457024°n; elevation: 730 m; mgrs 10×10 square: xk52, habitat: well preserved alnus glutinosa forest; soil type: gley on diluvial sediments; population: a dozen tussocks at spermatophyta) for the red book of flora of bosnia and herzegovina. in the red list of flora of the federation of bosnia and herzegovina, the species was also assessed as vulnerable (đug et al., 2013). considering the available data, i.e., area of occupancy (aoo) is less than 10 km2, regional population is severely fragmented, and there is continuing decline in area, extent and/or quality of habitat, we suggest regional conservation status for bosnia and herzegovina: critically endangered cr b2ab(iii). carex elongata l. published and new chorological data carex elongata belongs to the subgenus vignea and the section elongatae (kunth) kük. the species can be recognized by the following set of characteristics: densely caespitose plants often forming tussocks, all spikes (6-12) with female flowers on top and male below, utricles without wings with entire not 2-fid beak and two stigmas, lowest bract shorter than inflorescence (chater, 1980) (fig. 3). the species was known from several localities in bosnia and herzegovina scattered throughout the country (fig. 4): • under koran near pale; coordinates: 18.575645°e, 43.804779°n; elevation: ca 850 m; mgrs 10×10 square: cp05; habitat: wet meadows with scirpus sylvaticus, cardamine pratensis and scutellaria galericulata; population: unknown; date: 03.06.1934 and 03.06.1935; collector: k. malý; bases of record: sara 3932 – sara 3933; corresponding literature record (malý, 1935). remark: the area has been urbanized in the last 50 years, and the occurrence of this species is uncertain today. • gradiška; coordinates: 17.248848°e, 45.135396°n; elevation: ca 95 m; mgrs 10×10 square: xl70; habitat: cynosuretum cristati; population: unknown; date: 1959; collector: j. kovačević; bases of record: literature (kovačević 1959). • berek (lijevče polje); coordinates: 17.242484°e, 45.041149°n; elevation: ca 105 m; mgrs 10×10 square: xk78; habitat: cynosuretum cristati; population: unknown; date: 1959; collector: j. kovačević; bases of record: literature (kovačević 1959); remark: several hundred individuals have been recently confirmed but in monodominant swamp forests of quercus robur (d. koljanin, 16.06.2021. bases of record: observation). • in the vicinity of bosanska dubica; coordinates: 16.932538°e, 45.266012°n; elevation: ca 93 m; mgrs 10×10 square: xl51; habitat: leucojobiologica nyssana ● 14 (1) june 2023: 15-29 koljanin et al. ● notes on the distribution and conservation status of some rare plants of wet habitats in bosnia and herzegovina the edge of the forest; date: 29.05.2021; collector: j. brujić and d. koljanin; bases of record: herb. fac. silv: 000277. • marčete in podrašničko polje; coordinates: 16.961526°e, 44.481430°n; elevation: 740 m; mgrs 10×10 square: xk52, habitat: young alnus glutinosa forest with betula pubescens; soil type: gley on diluvial sediments; population: ca 30 tussocks were counted; date: 29.05.2021; collector: j. brujić and d. koljanin; bases of record: observation. • elezagići in lijevče polje; coordinates: 17.241189°e, 45.068918°n; elevation: 96 m; mgrs 10×10 square: xk79; habitat: swamp forest of quercus robur, in a micro-depression; soil type: pseudogley over tertiary sediments; population: several hundred tussocks; date: 26.06.2021.; collector: d. koljanin; bases of record: herb. fac. silv: 000278. • kočićevo in lijevče polje; coordinates: 17.383162°e, 45.078864°n; elevation: 92 m; mgrs 10×10 square: xk89; habitat: carici elongatae-alnetum glutinosae; soil type: gley on tertiary sediments; population: several dozens of tussocks; date: 07.08.2021; collector: d. koljanin; bases of record: observation. • bardača in lijevče polje; coordinates: 17.397036°e, 45.092282°n; elevation: 91 m; mgrs 10×10 square: xk89; habitat: carici elongatae-alnetum glutinosae; soil type: pseudogley on tertiary sediments; population: couple tussocks; date: 18.08.2021; collector: d. koljanin; bases of record: herb. fac. silv. 000367. • mašići in lijevče polje; coordinates: 17.245689°e, 45.0157962°n; elevation: 107 m; mgrs 10×10 square: xk78; habitat: along a temporary brook in a micro-depression with quercus robur and acer tataricum; soil type: pseudogley on tertiary sediments; population: ca. 30 tussocks; date: 17.04.2022; collector: d. koljanin and j. brujić; bases of record: observation. • posavine in lijevče polje; coordinates: 17.161216°e, 45.13591°n; elevation: 92 m; mgrs 10×10 square: xl70; habitat: leucojofraxinetum angustifoliae; soil type: pseudogley on tertiary sediments; population: several hundred tussocks; date: 08.05.2022; collector: d. koljanin; bases of record: observation. • stanari; coordinates: 17.821632°e, 44.750471°n; elevation: 171 m; mgrs 10×10 square: yk25; habitat: carici elongatae-alnetum glutinosae; soil type: planohistosol and pseudogley on tertiary sediments; population: several hundred tussocks; date: 22.05.2022; collector: v. stupar, j. brujić, đ. milanović & d. koljanin; bases of record: djm: 65/04-048. fig. 3. carex elongata near stanari (© đ. milanović) fig. 4. distribution of carex elongata in bosnia and herzegovina general distribution and ecology carex elongata is widespread in europe and western asia. this species is common in central europe, inhabiting various wetland habitats. the species is often a member of hygrophilous forests where stagnant water is present for a significant period of the year, and it is common in the alnus glutinosa carrs (willner & grabherr, 2007; douda, 2008; borhidi et al., 2012; slezák et al., 2014; hulík & douda, 2017). in addition, the species is also common in narrowleaved ash swamp forests (leucojo-fraxinetum angustifoliae) in balkan peninsula (glavač, 1959; glišić, 1964), monodominant swamp quercus robur forests in hungary (borhidi et al., 2012) 20 koljanin et al. ● notes on the distribution and conservation status of some rare plants of wet habitats in bosnia and herzegovina 21 and poland (sokołowski, 1972) and swamp alnus incana communities in austria (willner & grabherr, 2007). in croatia, the species is distributed mainly in the lowlands in the northern and central parts of the country (nikolić, 2020). in serbia, the species is noted to be common in swamp forests, wet meadows and channel margins from lowlands to mountainous regions (josifović, 1972). still, there are only a few precise data on the distribution and ecology of the species in this country. habitat and species threats in bosnia and herzegovina recent research shows that the species is not as rare throughout northern bosnia and herzegovina as previously considered (milanović et al., 2011). here, this species is a typical forest plant, inhabiting more or less permanently moist parts of various riparian forests, with a development optimum in preserved alnus glutinosa swamp forests and quercus robur swamp forests with +/developed peat layer. in contrast, in other forest types, where water is stagnant for a part of the vegetation season, it is restricted to micro depressions, channel margins or alongside permanent or temporal stream beds. due to largescale hydrological meliorations in the past, habitats suitable for this species are severely restricted, degraded, and fragmented, while flood prevention and changes in water regimes still negatively affect its populations. in the montane region of the dinaric alps, the species is less common, with scattered individuals on stream terraces or at the edges of peatland complexes inhabiting wet tall-herb communities and edges of mixed broadleaved-coniferous forests. various negative impacts on these habitats have been recorded there: trampling in the bijambare nature park, urbanization in koran near pale, and hydrological meliorations in han kram at sljemenska mt. conservation status global and european iucn status of carex elongata has not been estimated yet. the species is not listed in national red lists in most central european countries. in neighbouring croatia, the species is listed as data deficient (nikolić & topić, 2005). šilić (1996) has considered doubtful previously known data, and therefore the species was not included in the list of vascular plants (pteridophyta and spermatophyta) for the red book of flora of bosnia and herzegovina. however, considering the available data, i.e., aoo is less than 10 km2, the regional population is severely fragmented, and there is continuing decline in area, extent and quality of habitat, we suggest regional conservation status for bosnia and herzegovina: critically endangered cr b2ab(iii). carex strigosa huds. published and new chorological data carex strigosa belongs to the section strigosae (fries) (chater, 1980). this species was often overlooked in some parts of its distribution due to low study levels and a small percentage of suitable habitats (trčak & bačič, 2017), which could also be stated for bosnia and herzegovina. from other species of the section, it could be recognized by mainly not pendent, thin and lax female spikes, green utricles with a very short beak and acute female glumes (chater, 1980) (fig. 5). the species has been recorded from only several localities in northern bosnia and herzegovina, but recent records confirm that the species has a somewhat wider distribution in this part of the country (fig. 6): • in the vicinity of bosanska dubica; coordinates: 16.932538°e, 45.266012°n; elevation: ca 93 m; mgrs 10×10 square: xl51; habitat: genisto elatae-quercetum roboris; population: unknown; date: 1964; collector: m. glišić; bases of record: literature (glišić, 1964). note: this record is confirmed, and the species was considered rare (a few dozen individuals) at donja gradina near bosanska dubica in the well-preserved forest of fraxinus angustifolia and quercus robur, 15.05.2015, đ. milanović & v. stupar, djm: 65/04-160. • jakovica near the village of cerik (orig. cerje); coordinates: 18.522109°e, 44.808918°n; elevation: ca 100 m; mgrs 10×10 square: cq06; habitat: carpino betuli-quercetum roboris forest; population: unknown; date: 1975; collector: p. fig. 5. carex strigosa at donja gradina (© đ. milanović) biologica nyssana ● 14 (1) june 2023: 15-29 koljanin et al. ● notes on the distribution and conservation status of some rare plants of wet habitats in bosnia and herzegovina 22 biologica nyssana ● 14 (1) june 2023: 15-29 koljanin et al. ● notes on the distribution and conservation status of some rare plants of wet habitats in bosnia and herzegovina fukarek; bases of record: literature (fukarek, 1975). • lipovica forest near the village of pelagićevo; coordinates: 18.564017°e, 44.886848°n; elevation: ca 80 m; mgrs 10×10 square: cq07; habitat: carpino betuli-quercetum roboris forest; population: unknown; date: 1975; collector: p. fukarek; bases of record: literature (fukarek, 1975). • hrastik near the village of donji žabar; coordinates: 18.656429°e, 44.941478°n; elevation: ca 85 m; mgrs 10×10 square: cq17; habitat: carpino betuli-quercetum roboris forest; population: unknown; date: 1975; collector: p. fukarek; bases of record: literature (fukarek, 1975). • a forest in the village of obudovac; coordinates: 18.578385°e, 44.954521°n; elevation: ca 80 m; mgrs 10×10 square: cq08; habitat: carpino betuli-quercetum roboris forest; population: unknown; date: 1975; collector: p. fukarek; bases of record: literature (fukarek, 1975). • burumac (orig. baranac) in the vicinity of bosanski šamac; coordinates: 18.521154°e, 45.004933°n; elevation: ca 95 m; mgrs 10×10 square: cq08; habitat: carpino betuli-quercetum roboris forest; population: unknown; date: 1975; collector: p. fukarek; bases of record: literature (fukarek, 1975). • dubrave near vrbaška; coordinates: 17.157772°e, 45.137031°n; elevation: 94 m; mgrs 10×10 square: xl70; habitat: leucojo-fraxinetum angustifoliae; soil type: pseudogley over tertiary sediments; population: less than 20 flowering individuals; date: 28.05.2016; collector: đ. milanović; bases of record: observation. • suvopolje near grapska; coordinates: 18.062530°e, 44.789970°n; elevation: ca 130 m; mgrs 10×10 square: bq66; habitat: a degraded forest of white willow (salix alba); soil type: fluvisol over alluvial deposits; population: a dozen individuals scattered in the forest; date: 13.05.2017; collector: đ. milanović & v. stupar; bases of record: djm 65/04-161. • jabučik near kožuhe; coordinates: 18.096060°e, 44.859240°n; elevation: 128 m; mgrs 10×10 square: bq77; habitat: well-preserved alnus glutinosa forest; soil type: eugley river terrace; population: less than 100 individuals scattered in the forest; date: 13.05.2017; collector: đ. milanović & v. stupar; bases of record: observation. • kutlovac near podnovlje; coordinates: 18.142250°e, 44.934760°n; elevation: 116 m; mgrs 10×10 square: bq77; habitat: a wellpreserved grove of white willow; soil type: fluvisol over alluvial deposits; population: a dozen scattered individuals throughout the forest; date: 14.05.2017; collector: đ. milanović & v. stupar; bases of record: observation. • rastoka near podnovlje; coordinates: 18.114401°e, 44.919059°n; elevation: 119 m; mgrs 10×10 square: bq77; habitat: degraded grove of white willow; soil type: fluvisol over alluvial deposits; population: only a few individuals; date: 14.05.2017; collector: đ. milanović & v. stupar; bases of record: observation. general distribution and ecology this subatlantic species is widespread in a large part of western and central europe, the caucasus, and northern iran, and very rare in the iberian peninsula (laskurain et al., 2003; liendo et al., 2016). in central europe, the species is found mainly in lowlands, wet to damp places, nutrient-rich and phneutral soils, in semi-shaded to shaded habitats in the floodplains of larger rivers (kaplan et al., 2020). it usually grows in communities of poplars and tall willows, narrowed-leaved ash, european white elm and pedunculate oak which are often under the impact of flooding (borhidi et al., 2012; chytrý, 2013). in slovenia, this species is noted to grow in communities belonging to the montio-cardaminetea class but more often in lowland riparian forest communities (trčak & bačič, 2017). in croatia, this species is mostly found in the northern part of the country near the sava and drava rivers (nikolić, 2020). the species is also found in serbia in wet and fig. 6. distribution of carex strigosa in bosnia and herzegovina 23 swamp lowland forests (josifović, 1976). habitat and species threats in bosnia and herzegovina in bosnia and herzegovina, the species is restricted to northern lowlands where it occurs in remnants of riparian forests, irregularly scattered in the lower flow of major rivers of the danube catchment. these habitats are rare in bosnia and herzegovina due to intensive changes in the water regime and uncontrolled logging. historically, the species was exposed to similar negative impacts as carex elongata, but it has a wider ecological amplitude, ranging from swamps to meso-hygrophilous forests. invasive species are also a threatening factor. severe habitat changes could lead to species extinction in bosnia and herzegovina. conservation status global and european iucn status of carex strigosa is not assessed. it has different conversation statuses in european countries; nederland – vulnerable (sparrius et al., 2014), czech republic – endangered taxa (grulich, 2012), croatia – data deficient (nikolić & topić, 2005), spain – critically endangered taxa (liendo et al., 2016) etc. considering the available data, i.e., aoo is less than 10 km2, the regional population is severely fragmented, and there is continuing decline in area, extent and habitat quality, we suggest regional conservation status for bosnia and herzegovina: critically endangered cr b2ab(iii). comarum palustre l. published and new chorological data genus comarum is represented by only one species in european flora: comarum palustre. it is similar to the genus potentilla, but it has lateral and filiform style and purple petals (ball et al., 1968) (fig. 7). the species is very rare in bosnia and herzegovina, recorded only from wet refugia around glacial lakes (fig. 8): • banks of kukavičko jezero (kupreško polje); coordinates: 17.331487°e, 43.949120°n; elevation: 1202 m; mgrs 10×10 square: xj86; habitat: banks of the lake; population: numerous individuals; date: 1925.09.1924, 04.07.1955, 19.06.1990; collector: ž. protić, č. šilić, hilda ritter-studnička; bases of record: herbarium sara 17641, sara 17642, sara 0051634; corresponding literature (ritter-studnička, 1954). note: recently confirmed in alnus incana forest around a lake in about hundred flowering individuals, 25.07.2021, observation by đ. milanović, m. hayek, p. haykova & d. dite. • banks of gornje bare (zelengora mt.); coordinates: 18.607463°e, 43.320720°n; elevation: 1515 m; mgrs 10×10 square: cn09; habitat: bank of the lake; population: only one individual; date: 1969; collector: r. lakušić; bases of record: literature (bjelčić et al., 1969); stated by authors as new for bosnia and herzegovina. note: recently (19.07.2016) confirmed population numbers of a few hundred individuals in various sedge stands (carex rostrata, c. limosa, c. vesicaria and c. acuta aggr.) and as very rare in deschampsia cespitosa grasslands around the lake (milanović, 2017b). • banks of kotlaničko lake (zelengora mt.); coordinates: 18.482280°e, 43.362913°n; elevation: 1530 m; mgrs 10×10 square: bp90; habitat: comaro-menyanthetum trifoliatae on the bank of the lake; population: unknown; date: biologica nyssana ● 14 (1) june 2023: 15-29 koljanin et al. ● notes on the distribution and conservation status of some rare plants of wet habitats in bosnia and herzegovina fig. 7. comarum palustre around jugovo jezero (© đ. milanović) fig. 8. distribution of comarum palustre in bosnia and herzegovina 24 1978; collector: r. lakušić; bases of record: literature (lakušić et al., 1978; drešković et al., 2011; redžić et al., 2013). note: the record of comarum palustre around kotlaničko lake has been only indirectly gathered from a report on the vegetation map of bosnia and herzegovina for the period of 1977-1980 (was not available to the authors) as well as from prodromus of plant communities of bosnia and herzegovina (lakušić et al., 1978) where the community of comaromenyanthetum is stated but without an exact record of the species. in the same references (drešković et al., 2011; redžić et al., 2013), the community of comarum-menyanthes was also mentioned for some lakes from treskavica mt., based on the report on the vegetation map of bosnia and herzegovina for 1977, but this report was available for the authors, and this community was not stated there. more than 5000 flowering individuals of this species have been recently confirmed in floating carpets of comarum palustre-menyanthes trifoliata, carex limosa and equisetum fluviatile stands around kotlaničko lake, 18.08.2006 and 21-22.07.2016, đ. milanović, djm: 84/09-001. • crno lake (zelengora mt.); coordinates: 18.583443°e, 43.386275°n; elevation: 1440 m; mgrs 10×10 square: cp00; habitat: phragmites communis stands and floating carpets with comarum palustre; population: several hundred flowering individuals; date: 14.07.2016; collector: đ. milanović; bases of record: literature (milanović, 2017b). • jugovo lake (zelengora mt.); coordinates: 18.530804°e, 43.375774°n; elevation: 1550 m; mgrs 10×10 square: cp00; habitat: carex rostrata stands along banks of the lake, and around a source above the lake; population: a few hundred flowering individuals; date: 21.07.2016; collector: đ. milanović; bases of record: observation. general distribution and ecology comarum palustre has a wide circumboreal distribution, encompassing the temperate to arctic regions of eurasia, north america, and the southern parts of greenland (kaplan et al., 2017). in central europe, the species is found in various habitats, but most commonly on moist, acidic, and nutrient-poor soils, such as fens, transitional mires, wet moss-rich meadows, tall sedge stands, edges of fishponds, and alder carrs, less often also in bog hollows (kaplan et al., 2017). it is also usually found in salix cinerea scrub communities (borhidi et al., 2012; chytrý, 2013). the species is rarely found in southern europe. in croatia, the species can be considered rare. it has been recently confirmed in only one locality, while previous literature records stay unconfirmed (nikolić & topić, 2005). in serbia, the species is known from the eastern part of the country and could also be considered rare (josifović, 1972). in montenegro, the species occurs at three spatially close localities at the durmitor mt. (caković & stešević, 2021), all at high elevations. habitat and species threats in bosnia and herzegovina the species is recorded only in mountain depressions around glacial lakes in bosnia and herzegovina. there it occupies wet habitats on the edge of water bodies of the lakes, often forming floating carpets with other herbaceous plants (kotlaničko lake, jugovo lake, crno lake and gornje bare), while only around kukavičko lake it inhabits alnus incana forests. in spite of the fact that the recorded populations are located far from settlements and sometimes within protected areas (sutjeska national park), some of the recorded populations are exposed to strong current or potential human pressures. due to illegal off-road activities around crno lake, water from springs on the slope above the northeast bank of the lake, which provide permanent moistening of the lake terrace, is drained through the tire tracks, causing dryness during the driest part of the year. the populations around jugovo (borilovačko) lake will be even more affected by the plans for the urbanization of the area surrounding the lake. conservation status global iucn status of comarum palustre is least concern (maiz-tome, 2015). the species is considered critically endangered in croatia (nikolić & topić, 2005). šilić (1996) included the species in the list of vascular plants for the red book of bosnia and herzegovina as vulnerable. in the red list of flora of the federation of bosnia and herzegovina, the species was estimated as vulnerable (đug et al., 2013). considering the available data, i.e., aoo is less than 10 km2, the regional population is severely fragmented, and there is continuing decline in area, extent and habitat quality, we suggest regional conservation status for bosnia and herzegovina: critically endangered cr b2ab(iii). prunus padus l. published and new chorological data prunus padus is sometimes planted tree species in horticulture in bosnia and herzegovina, while its natural populations in the country are very rare (fig. biologica nyssana ● 14 (1) june 2023: 15-29 koljanin et al. ● notes on the distribution and conservation status of some rare plants of wet habitats in bosnia and herzegovina 25 9, fig. 10). only known populations are scattered on the edge of kupreško field, where they were originally considered as potentially escaped from gardens (ritter-studnička, 1958) but later confirmed as autochthonous (ritter-studnička, 1972). recently, the species was also mentioned for northern bosnia (šilić, 2005; stupar et al., 2021), potentially based on findings in some prehistoric archaeological sites (maly, 1904), but without any exact locality or coordinates. moreover, our recent research in the area did not confirm these allegations. furthermore, it should be stated that redžić & golić (1985) noted prunus padus in maoča at konjuh mt. in forest clearings of pinus nigra forests (phytosociological table). neither in the following text (redžić & golić, 1985) nor in subsequent research of the same plots (redžić, 1988) prunus padus was not mentioned again, but prunus mahaleb is regularly noted. therefore, we consider that „prunus padus” is a lapse probably referring to prunus mahaleb which is ecologically suited for such habitats. we considered these lapse as this species is not mentioned in the further text and is replaced by prunus mahaleb in further research on those permanent vegetation plots. prunus padus has been reported from the following localities: • humbat hill near prozor; coordinates: 17.616170°e, 43.816082°n; elevation: ca 770 (orig. 856 m) m; mgrs 10×10 square: yj15; habitat: unknown; population: unknown; date: 21.07.1907; collector: j. stadlmann; bases of record: literature (stadlmann, 1912) note: later indicated as erroneous record after consulting the original herbarium material (ritter-studnička, 1972) misidentification with prunus mahaleb. • near the road on slopes of kupreška vrata towards kupres; coordinates: 17.293807°e, 44.007529°n; elevation: 1200-1300 m; mgrs 10×10 square: xj87; habitat: in forests and scrubs; population: numerous individuals; date: 01.08.1958; collector: l. lažetić; bases of record: herbarium sara 18992-18993, corresponding literature (ritter-studnička, 1958, 1972, 1974). note: recently confirmed throughout the slopes of kupreška vrata and stožer mt. towards kupres, optimally developed in alnus incana forest in several hundred flowering individuals and much more in the vegetative stage as a shrub in the canopy, 30.04.2018, đ. milanović, g. tomović & k. jakovljević, djm: 84/24–001; 13.07.2021, j. brujić & d. koljanin, herb. fac. silv: 000274. • banks of kukavičko lake (kupreško polje); coordinates: 17.331487°e, 43.949120°n; elevation: 1202 m; mgrs 10×10 square: xj86; habitat: banks of the lake; population: scattered individuals; date: 1972; collector: h. ritterstudnička; bases of record: literature (ritterstudnička, 1972, 1974). note: recently confirmed in alnus incana forest around the lake – only a few individuals have been registered, 25.07.2021, đ. milanović, m. hayek, p. haykova & d. dite, djm: 84/24–002. • hrbljina; coordinates: 17.097371°e, 44.005508°n (unprecise); elevation: ca 1300 m; mgrs 10×10 square: xj67; habitat: unknown; population: scattered individuals, rare; date: 1972; collector: n. zubić; bases of record: literature (ritterstudnička, 1972). note: the record is a personal observation of novak zubić, a geographer that at the time was employed at the faculty of natural sciences in sarajevo, and having in mind that the reported locality doesn’t fit in the species ecology, biologica nyssana ● 14 (1) june 2023: 15-29 koljanin et al. ● notes on the distribution and conservation status of some rare plants of wet habitats in bosnia and herzegovina fig. 9. prunus padus in blooming stage on slopes of stožer mt. (© đ. milanović) fig. 10. distribution of prunus padus in bosnia and herzegovina 26 we consider this record as doubtful. • vitorog mt.; coordinates: 17.096455°e, 44.084158°n (unprecise); elevation: ca 1500 m; mgrs 10×10 square: xj68; habitat: unknown; population: scattered individuals, rare; date: 1972; collector: n. zubić; bases of record: literature (ritter-studnička, 1972). note: we consider this record doubtful for the same reasons as in the previous case. later research on the flora and vegetation of this mountain did not confirm this species either (redžić et al., 1984; zubić & topalić-trivunović, 2011). • podbrdo in podrašničko polje; coordinates: 16.985369°e, 44.457024°n; elevation: 730 m; mgrs 10×10 square: xj52; habitat: well preserved alnus glutinosa forest; soil type: eugley over diluvial sediments; population: several flowering individuals; date: 29.05.2021; collector: j. brujić & d. koljanin; bases of record: herb. fac. silv: 000275 & herb. fac. silv: 000276. general distribution and ecology prunus padus is a eurasian species with an almost compact distribution range that is gradually becoming disjunct on its southern border. it is widespread in most parts of europe except the southern areas (webb & rix, 1972; kurtto et al., 2013). in central europe, it typically occurs in closed forests and is a common and diagnostic taxon of alnion incanae alliance, but it can be found in a wide range of habitats (willner & grabherr, 2007; borhidi et al., 2012; chytrý, 2013). this species is common in most central european countries (rhodes & maxted, 2013), including slovenia, and northern and western croatia, which represents the edge of its compact distribution range. in the western balkan, it has a disjunct distribution, occurring mostly at higher altitudes with lower temperatures. it appears that the species is frequent in lowland forests only in colder and more humid climates, while in the balkan peninsula, the species could be considered a glacial relict. habitat and species threats in bosnia and herzegovina the species is found at higher altitudes in moist habitats on silicate substrates. on kupres, species grow in grey alder stands and in the succession of fir-beech-spruce forests. some old individuals are also found along roads. it seems that on kupres, this species does not have a narrow distribution, and it is capable of thriving in different habitats. in sitnica, species is found in only one alnus glutinosa stand, but it is likely to exist in other nearby locations. the main threat for prunus padus populations is land use change. any changes in the hydrological regime of its habitats could also be a threat causing population number and density decline. as prunus padus wood is valuable for wood carving and making lockers (nestby, 2020), there is a potential risk for the older trees. also, as some localities in bosnia and herzegovina are in succession stages, it could be expected that this natural process would be followed by a decreasing number of individuals. conservation status global and european iucn status of prunus padus supsp. padus is the least concern (sparrius et al., 2014), and it is not listed on the national red lists of the majority european countries. the species is not considered native in serbia (josifović, 1972) and, therefore, is not included in the list. species was assessed as vulnerable in a proposal for the red list of vascular flora of bosnia and herzegovina (šilić, 1996). in the red list of flora of the federation of bosnia and herzegovina, the species was also assessed as vulnerable (đug et al., 2013). considering the available data, i.e., aoo is less than 10 km2, the regional population is severely fragmented, and there is continuing decline in area, extent and quality of habitat, we suggest regional conservation status for bosnia and herzegovina: critically endangered cr b2ab(iii). references ball, p.w., pawłowski, b., & walters, s.m. 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(2011). vaskularna flora staništa velikog tetrijeba (tetrao urogallus l.) na području planine vitorog. glasnik šumarskog fakulteta univerziteta u banjoj luci, 15, 29-48. biologica nyssana ● 14 (1) june 2023: 15-29 koljanin et al. ● notes on the distribution and conservation status of some rare plants of wet habitats in bosnia and herzegovina sevindik et al. 2024, biologica nyssana 15(2) 15 (2) december 2024: 61-68 doi: 10.5281/zenodo.14258237 genetic diversity and structure analysis of ‘ak sakı’ and ‘kara sakı’ apple cultivars growing in erzincan/türkiye original article emre sevindik faculty of agriculture, department of agricultural biotechnology, south campus, aydın adnan menderes university, aydin, türkiye ph.d-emre@hotmail.com (corresponding author) filiz yangılar department of nutrition and dietetics, faculty of health sciences, erzincan binali yıldırım university, erzincan, türkiye bayram atasagun department of medical services and techniques, vocational school of health services, selcuk university, konya, türkiye erengül sofyalıoğlu faculty of agriculture, department of agricultural biotechnology, south campus, aydın adnan menderes university, aydin, türkiye selçuk alp şimşek bilkent university, department of molecular biology and genetics, ankara, türkiye muhammed ebrar çayır isparta egirdir fruit research institute, isparta, türkiye martin vivodík slovak university of agriculture, faculty of biotechnology and food sciences, institute of biotechnology, nitra, slovakia received: august 13, 2024 revised: november 13, 2024 accepted: november 15, 2024 abstract: in this study, ‘ak sakı’ and ‘kara sakı’ apple cultivars were collected from different locations in erzincan province, türkiye, and genetic diversity was determined using the start codon targeted (scot) marker technique. the scot marker technique was chosen because its gene targeting, long primer, and high annealing temperature make it more effective than other marker techniques. using ten scot primers, 60 bands were obtained, and 42 of them were polymorphic. the polymorphism rate was determined to be 70%. the upgma (unweighted pair group method with arithmetic mean) dendrogram created using the paup 4.0b10 program consists of two clades. the genetic distance between apple cultivars varies between 0.13462 and 0.45614. principal component analysis (pca) results were compatible with the upgma dendrogram. with the scot marker technique, genetic diversity among apple cultivars can be determined in a shorter time and with more reliable results. key words: apple, genetic diversity, scot, türkiye apstrakt: analiza genetske raznovrsnosti i strukture sorti jabuka ‘‘ak sakı’’ i ‘‘kara sakı’’ koje rastu u erzindžanu/turska u ovoj studiji, sorte jabuka ‘ak sakı’ i ‘kara sakı’ prikupljene su sa različitih lokaliteta u provinciji erzindžan, turska, koristeći tehniku markera startnog kodona (scot) za određivanje genetske raznovrsnosti. tehnika scot odabrana je zbog ciljanog delovanja na gene, dužine prajmera i visoke temperature vezivanja, što je čini efikasnijom od drugih tehnika markera. korišćenjem deset scot prajmera dobijeno je 60 traka, od kojih su 42 bile polimorfne. procenat polimorfizma utvrđen je na 70%. upgma dendrogram (unweighted pair group method with arithmetic mean), kreiran korišćenjem programa paup 4.0b10, sastoji se od dve klade. genetska udaljenost između sorti jabuka varira između 0.13462 i 0.45614. rezultati analize glavnih komponenti (pca) bili su kompatibilni sa upgma dendrogramom. tehnikom scot može se u kraćem vremenskom periodu i sa pouzdanijim rezultatima odrediti genetska raznovrsnost među sortama jabuka. ključne reči: jabuka, genetska raznovrsnost, scot,turska introduction the apple (malus × domestica borkh.), a member of the rosaceae family, originates from the region between the caspian sea and the black sea. it is one of the most economically and culturally significant fruit species globally (han et al., 2020; geană et al., 2021; chen et al., 2021; fotirić akšić et al., 2022). due to its ecological compatibility and high nutritional value, it is popular among both producers and consumers (eberhardt et al., 2000; boyer and liu, 2004; hyson, 2011; sarkate et al., 2017). ‘ak sakı’ is a variety grown in the erzincan province of türkiye and is the most commonly grown and readily consumed apple. in the region ‘ak sakı’ and ‘kara sakı’ ecotypes are known. it was also registered under the name ‘ak sakı’ on 03.05.1990. (öztürk et al., 2013). the ‘kara sakı’ apple is a native apple variety of erzincan, resembling the amasya apple, but with a slightly lighter and brighter color and a mildly tart taste (doğan, 2001). its aroma is the most important feature that makes it the consumer’s preference. to date, more than 300 volatile compounds have been detected in apples © 2024 sevindik et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 61 (dixon and hewett, 2000; aprea et al., 2011; yang et al., 2021). also, apples provide high amounts of carbohydrates, vitamins and bioactive compounds, such as phytosterols, β-carotene and phenolic compounds (wu et al., 2020; feng et al., 2021). various apple phytochemical compounds with antioxidant activity and anticancer properties reduce the risk of chronic and degenerative diseases (boyer and liu, 2004; inroga et al., 2021). molecular markers are widely utilized in many plant species and are one of the most essential methods for analyzing genetic diversity (yang et al., 2023). scot markers are simple and reliable markers designed to produce of gene-targeted markers. compared to many other marker systems, the scot system provides more information about the universality and biological properties of plants (yilmaz and ciftci, 2021). the scot marker approach is more effective than other random markers because of its long primer and high annealing temperatures (alzahrani et al., 2023). scot markers have been used in genetic diversity and phylogenetic relationships analysis in many plant species (guo et al., 2016; jalilian et al., 2018; etminan et al., 2018; vivodík et al., 2019; zarei and erfani-moghadam, 2021; vivodík et al., 2023). in this study, the genetic diversity and structure of the ‘ak sakı’ and ‘kara sakı’ apple cultivars, distributed in erzincan – türkiye, were analyzed using ten scot markers. materials and methods plant materials, genomic dna isolation and pcr ‘ak sakı’ and ‘kara sakı’ cultivars were collected from different localities of erzincan-türkiye. for both cultivars, the collection was made from approximately seven centers. genomic dna isolation was performed using green leaves with a commercial kit (genemark catalog no: dp022). selected ten scot primers (collard and mackill, 2009) for pcr amplifications, pcr components and the protocol used are given in tab. 1. pcr products were run on 1.0% agarose gel. in gel imaging, generuler (cat#: gmm100) between 100 bp and 3000 bp was used. gel images of pcr results using the scot 11 primer are shown in fig. 1. start codon targeted (scot) analysis following the scot analyses, the dna bands were scored by giving the value “1” in the presence of dna, “0” in the absence of dna and “?” or “9” for the missing cases. the upgma phylogenetic tree was drawn using the program paup 4.0b10 (swofford, 2001). pairwise distance was created with the same program. the genetic distance matrix between apple cultivars was calculated and shown in tab. 2. the jmp statistical software which presents the distances between individuals in a two-dimensional diagram, was used to perform the pca analysis. in addition, major allele frequency, nei’s h (gene diversity) and pic (polymorphism 62 biologica nyssana ● 15 (2) december 2024: sevindik et al. ● genetic diversity and structure analysis of ‘ak sakı’ and ‘kara sakı’ apple cultivars growing in erzincan/türkiye table 1. scot primers name, sequences and pcr components used for pcr amplification conditions scot primers dna sequences(5’-3’) tm oc pcr components pcr amplification (35 cycle) scot 1 caacaatggctaccacca 54 oc scot 2 caacaatggctaccaccc 56 oc scot 3 caacaatggctaccaccg 56 oc scot 4 caacaatggctaccacct 54 oc scot 5 caacaatggctaccacga 54 oc 1 μl genomic dna 1 μl primer, 5 μl master mix (cat. no: rp02-ii-400, rp02-ii-2000, 0.75 u of taq dna polymerase, reaction buffer, 2 mm mgcl2, 250 µm dntps and enzyme stabilizer) and 18 μl dh2o. scot 6 caacaatggctaccacgc 56 oc 95 oc/1min. scot 8 54 oc 95 oc/30 sec caacaatggctaccacgt 54-56 oc/30sec. scot 9 caacaatggctaccagca 54 oc 72 oc/1 min. scot 10 caacaatggctaccagcc 56 oc 72 oc/5 min. scot 11 aagcaatggctaccacca 54 oc biologica nyssana ● 15 (2) december 2024: sevindik et al. ● genetic diversity and structure analysis of ‘ak sakı’ and ‘kara sakı’ apple cultivars growing in erzincan/türkiye 63 information content) values were calculated using the powermarker software package version 3.25 (liu and muse, 2005). using the statistical program structure 2.3.4, a structure test was utilized to classify members of various populations (pritchard et al., 2000). results and discussion for scot analysis, ten primers were used. a total of 60 bands were obtained. 42 of these bands were polymorphic and the polymorphism rate was 70%. the highest number of bands was obtained from the scot 2 primer and the lowest number of bands was obtained from the scot 8 primer (tab. 2). “pic”, “h” and “major allele frequency” values of each primer are given in tab. 3. the mean pic value was determined at 0.2878, while the average h value and the mean major allele frequency were determined at 0.3581 and 0.7413, respectively. fig. 1. gel image of bands amplifed with scot 11 primer: 1: ‘ak sakı’ (elma village), 2: ‘ak sakı’ (yaylabaşı), 3: ‘kara sakı’ (çağlayan), 4: ‘ak sakı’ (çağlayan), 5: ‘ak sakı’ (karatuş), 6: ‘kara sakı’ (elma village), 7: ‘kara sakı’ (karakaya), 8: ‘ak sakı’ (karakaya), 9: ‘kara sakı’ (karatuş village), 10: ‘ak sakı’ (pişkidağ), 11: ‘ak sakı’ (üzümlü), 12: ‘kara sakı’ (yaylabaşı), 13: ‘kara sakı’ (üzümlü) table 2. monomorphic and polymorphic band numbers of scot primers primers total bands monomorphic bands polymorphic bands scot 1 8 6 2 scot 2 11 2 9 scot 3 7 1 6 scot 4 5 1 4 scot 5 5 1 4 scot 6 7 1 6 scot 8 1 1 0 scot 9 3 1 2 scot 10 4 1 3 scot 11 9 3 6 total 60 18 42 table 3. major allele frequency, gene diversity (h) and polymorphism information content (pic) values primers major allele frquency gene diversity (h) pic scot 1 0.6643 0.446 0.3465 scot 2 0.6014 0.4794 0.3645 scot 3 0.6783 0.4364 0.3412 scot 4 0.7692 0.355 0.292 scot 5 0.8392 0.2699 0.2335 scot 6 0.6573 0.4505 0.349 scot 8 0.9091 0.1653 0.1516 scot 9 0.8601 0.2406 0.2117 scot 10 0.8531 0.2506 0.2192 scot 11 0.5804 0.4871 0.3684 mean 0.7413 0.3581 0.2878 the upgma dendrogram was created and it consists of two main clades (fig. 2). clade a consists of ‘ak sakı’ (elma village), ‘ak sakı’ (karatuş village), ‘kara sakı’ (elma village), ‘kara sakı’ (üzümlü), ‘kara sakı’ (karatuş village), ‘kara sakı’ (yaylabaşı), ‘kara sakı’ (karakaya) apple cultivars. clade b consists of ‘ak sakı’ (yaylabaşı), ‘kara sakı’ (çağlayan), ‘ak sakı’ (çağlayan), ‘ak sakı’ (karakaya), ‘ak sakı’ (üzümlü) and ‘ak sakı’ (pişkidağ) apple cultivars. according to tab. 4, the 64 biologica nyssana ● 15 (2) december 2024: sevindik et al. ● genetic diversity and structure analysis of ‘ak sakı’ and ‘kara sakı’ apple cultivars growing in erzincan/türkiye fig. 2. the upgma tree generated using ten scot markers lowest distance (0.13462) was found between the ‘kara sakı’ (yaylabaşı) and ‘kara sakı’ (üzümlü), ‘kara sakı’ (elma village) and ‘kara sakı’ (üzümlü) cultivars. the highest distance (0.45614) was found between the ‘ak sakı’ (üzümlü) and ‘kara sakı’ (yaylabaşı) cultivars. the graphical results and eigenvalues obtained on the two-dimensional plane according to pca analysis are given in fig. 3. the first three eigenvalues explained 64.755% of the total variance in the cultivars. as a result of the analysis, 13 samples were reduced to two dimensions. the first dimension consists of seven cultivars and the second one consists of six (eigen > 1). the clustering in the two-dimensional graph obtained from the pca analysis partially showed parallelism with the dendrogram results. çokran et al. (2019) determined the genetic diversity of 30 local ‘misket’ apple genotypes using aflp, ssr, and rapd markers. in their study, aflp, ssr and rapd markers amplified a total of 423 bands and obtained 205 polymorphic bands, while 30 rapd primers obtained 207 bands, 91 of which were polymorphic (40.1%), and 10 ssr primers obtained 33 bands and 26 polymorphic (78.78%) bands. five aflp combinations obtained 183 bands, 88 of which were polymorphic (48.08%). sevindik et al. (2018) determined the genetic diversity of apple genotypes in ardahan province with the issr-pcr technique. the authors found the polymorphism rate to be 60% in their study results. kaya et al. (2015) used rapd markers to perform molecular genotyping on apples collected from van province, türkiye. as a result of their study, they found the polymorphism rate to be 89.29%. khachtib et al. (2024) determined the genetic diversity of 29 apple cultivars in different regions of morocco using issr-pcr technique. they obtained 177 bands from 15 issr primers, 156 of which were polymorphic. also, the mean values of pic, rp, i and h were determined as 0.46, 4.58, 0.43 and 0.28, respectively. in conclusion, they revealed that issr markers could be useful in detecting genetic diversity in this fruit crop. najar et al. (2023) have collected samples from the north kashmir region and screened 62 apple genotypes using ten ssr markers. in their study, they amplified a total of 77 alleles with an average polymorphism percentage of 87.5%, pic of 0.71 and resolving power (rp) of 3.58. dar et al. (2020) have determined the genetic diversity of 19 apple varieties from the kashmir region using ten rapd markers. in their study, they detected a total of 70 polymorphic bands with a polymorphism percentage of 83.33. the same study suggested that these results can be implemented in apple-related conservation and breeding programs. in our study, the cultivar structure of individuals was estimated using the structure test. structure analysis of 13 cultivars was performed using ten scot primers. our results showed a clear peak point for δk at k = 3. at the first level of clustering (δk at k = 3), although ‘ak sakı’ and ‘kara sakı’ apple cultivars were divided into three subpopulations; all 13 cultivars were considered as mixtures. (fig. 4). this suggests significant gene flow with high exchange rates and different allele combinations between populations, in agreement with the results reported by najar et al. (2023) for malus×domestica germplasm from north kashmir, india. biologica nyssana ● 15 (2) december 2024: sevindik et al. ● genetic diversity and structure analysis of ‘ak sakı’ and ‘kara sakı’ apple cultivars growing in erzincan/türkiye 65 ta bl e 4. p ai rw ise g en et ic di st an ce m at rix o bt ai ne d fro m te n sc ot p rim er s c ul tiv ar s 1 2 3 4 5 6 7 8 9 10 11 12 13 ‘a k sa kı ’ (e lm a vi lla ge ) 0. 25 0. 32 72 7 0. 36 66 7 0. 28 33 3 0. 31 57 9 0. 28 33 3 0. 4 0. 26 66 7 0. 33 33 3 0. 41 66 7 0. 24 56 1 0. 26 96 3 ‘a k sa kı ’ (y ay la ba şı ) 15 0. 16 36 4 0. 25 0. 26 66 7 0. 31 57 9 0. 26 66 7 0. 31 66 7 0. 31 66 7 0. 31 66 7 0. 3 0. 42 10 5 0. 38 46 2 ‘k ar a sa kı ’ (ç ağ la ya n) 18 9 0. 14 54 5 0. 21 81 8 0. 26 92 3 0. 30 90 9 0. 2 0. 27 27 3 0. 30 90 9 0. 21 81 8 0. 38 46 2 0. 32 69 2 ‘a k sa kı ’ (ç ağ la ya n) 22 15 8 0. 25 0. 21 05 3 0. 28 33 3 0. 23 33 3 0. 3 0. 36 66 7 0. 28 33 3 0. 35 08 8 0. 26 92 3 ‘a k sa kı ’ (k ar at uş ) 17 16 12 15 0. 17 54 4 0. 3 0. 25 0. 25 0. 28 33 3 0. 3 0. 28 07 0. 17 30 8 ‘k ar a sa kı ’ (e lm a vi lla ge ) 18 18 14 12 10 0. 26 31 6 0. 33 33 3 0. 21 05 3 0. 19 29 8 0. 17 54 4 0. 28 07 0. 13 46 2 ‘k ar a sa kı ’ (k ar ak ay a) 17 16 17 17 18 15 0. 25 0. 25 0. 31 66 7 0. 3 0. 36 84 2 0. 25 ‘a k sa kı ’ (k ar ak ay a) 24 19 11 14 15 19 15 0. 4 0. 3 0. 18 33 3 0. 43 86 0. 30 76 9 ‘k ar a sa kı ’ (k ar at uş v ill ag e) 16 19 15 18 15 12 15 24 0. 23 33 3 0. 31 66 7 0. 21 05 3 0. 17 30 8 ‘a k sa kı ’ (p iş ki da ğ) 20 19 17 22 17 11 19 18 14 0. 18 33 3 0. 33 33 3 0. 26 92 3 ‘a k sa kı ’ (ü zü m lü ) 15 18 12 17 18 10 18 11 19 11 0. 45 61 4 0. 28 84 6 ‘k ar a sa kı ’ (y ay la ba şı ) 14 24 20 20 16 16 21 25 12 19 26 0. 13 46 2 ‘k ar a sa kı ’ (ü zü m lü ) 14 20 17 14 9 7 13 16 2 14 15 7 biologica nyssana ● 15 (2) december 2024: sevindik et al. ● genetic diversity and structure analysis of ‘ak sakı’ and ‘kara sakı’ apple cultivars growing in erzincan/türkiye fig. 3. two dimensional graph and eigen values created as a result of principal component analysis with scot marker fig. 4. population structure analysis of apples conclusion the study revealed a 70% polymorphism using ten scot primers, demonstrating the genetic diversity of the ‘ak sakı’ and ‘kara sakı’ apple cultivars. the pca analysis results were consistent with the upgma dendrogram, and the cluster analysis of genetic structure indicated that the apple cultivars are best represented by three genetic groups (δk=3). in addition, the results obtained will enable the cultivars to be used more effectively in future breeding programs. references alzahrani, o.r., alshehri, m.a., alasmari, a., ibrahim, s.d., oyouni, a.a., & siddiqui, z.h. 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(2021). scot markers provide insight into the genetic diversity, population structure and phylogenetic relationships among three pistacia species of iran. genetic resources and crop evolution, 68(4), 1625–1643. https://doi.org/10.1007/s10722-020-01091-3 biologica nyssana ● 15 (2) december 2024: sevindik et al. ● genetic diversity and structure analysis of ‘ak sakı’ and ‘kara sakı’ apple cultivars growing in erzincan/türkiye 68 živković et al. 2022, biologica nyssana 13(2) 13 (2) december 2022: 141-151 doi: 10.5281/zenodo.7437274 study of eutypa lata isolates originating from serbia original article sanja živković faculty of agriculture, university of niš, kruševac, serbia zivkovic.sanja@ni.ac.rs (corresponding author) tanja vasić faculty of agriculture, university of niš, kruševac, serbia darko jevremović fruit research institute, 32102 čačak, serbia mitra debasis icar-indian institute of horticultural research, karnataka 560 089, india received: june 08, 2022 revised: october 25, 2022 accepted: november 04, 2022 abstract: the potential influence of different types of light on the growth and sporulation of eutypa lata isolates was assessed, and detection at the molecular level was also performed. by applying in vitro observations, the influence of different types of light was monitored, (24 h exposure to uv light; alternating 12 h uv and 12 h darkness; alternating 12 h artificial fluorescent light and 12 h darkness), on the radial growth and sporulation of the anamorphic stage of three isolates of eutypa lata fungus (el117, el153 and el199) by comparison with two reference isolates of eutypa lata, bx1.10 and 8f, obtained from the inra, france. after the molecular detection using of the specific primer pair lata 1/ lata 2.2, all the studied isolates were found to belong to the species eutypa lata (syn. eutypa armeniacae), anamorph libertella blepharis. it was also found that the most suitable type of light for radial growth and sporulation of the studied isolates is exposure to 24 h uv light for 30 days. key words: isolates, molecular detection, breeding traits, light types, eutypa lata apstrakt: izučavanje izolata eutypa lata poreklom iz srbije u ovom radu je proučavan jedan od uzročnika odumiranja čokota vinove loze gljiva eutypa lata. praćen je uticaj različitih tipova svetlosti na porast i sporulaciju izolata eutipa lata, a izvršena je i detekcija na molekularnom nivou. naime, u in vitro uslovima praćen je uticaj različitih tipova svetlosti (24 h izlaganje uticaju uv svetla (uv); alternativno, 12 h uv i 12 h tama (uv-t); alternativno, 12 h veštačko fluorescentno svetlo i 12 h tama (st)) na radijalni porast i sporulaciju anamorfnog stadijuma tri izolata gljive eutypa lata (el117, el153 and el199) upoređivanjem sa dva referentna izolata eutypa lata bx1.10 i 8f dobijenih sa institute national de la recherche agronomique, inra, france. takođe je urađena detekcija izolata na molekularnom nivou primenom specifičnog para prajmera lata 1/lata 2.2. nakon sprovedene molekularne detekcije, utvrđeno je da svi proučavani izolati pripadaju vrsti eutypa lata (syn. eutypa armeniacae), anamorf libertella blepharis. takođe je ustanovljeno da je najpogodniji tip svetlosti za radijalni porast i sporulaciju, proučavanih izolata izlaganje, 24 h uv svetlu u trajanju od 30 dana. ključne reči: izolati, molekularna detekcija, odgajivačke odlike, tipovi svetlosti, eutypa lata introduction eutypa dieback, esca complex and botryosphaeria dieback (sosnowski & mccarthi, 2017) cause the greatest economic damage to vines as the cause of grapevine death (gtd), affecting plant growth, reducing yield and quality of grapes and premature decay of grapevines (gramaje et al., 2018). grapevine dieback disease is caused by multiple fungal pathogens that act individually or in combination and can infect a grapevine plant at all stages of growth (fontaine et al., 2016). it must be noted that these fungi cause similar and even the same symptoms on the leaves and shoots, as well as on the trunk of the vine. this complicates the separation of symptoms and the precise identification of disease causes in vineyards (gubler et al., 2005; gramaje et al., 2018). the dieback of the grapevine occurs in almost all countries of the world where the grapevine is grown commercially. one of the causes of grapevine dieback (gtd), which will be discussed in this text, is the fungus eutypa lata. this fungus is a vascular pathogen that infects the vine through fresh pruning wounds. the disease is spread by ascospores (carter, 1994; rolshausen et al., 2015). © 2022 živković et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 141 the fungus e. lata is very difficult to identify because this fungus does not form a teleomorphic stage (ascospores) in regions where annual rainfall is less than 330 mm (ju et al., 1991), as well as on artificial nutrient media (carter, 1994; munkvold, 2001; živković, 2019). adam (1938) caused an infection on the apricot tree, for the first time, by using a suspension of conidia of this fungus. glawe et al. (1982) determined this fungus based on host symptoms, pathogenicity tests on apricot and identification of anamorphs in culture (loc cit. ju et al., 1991). fungus eutypa lata can be easily grown on conventional laboratory media, but not all isolates sporulate (ju et al., 1991; mckemy et al., 1993; munkvold, 2001). for these reasons, a number of authors have studied the anamorphic stage of the fungus e. lata (rolshusen et al., 2006; trouillas & gubler, 2010). lecomte et al. (2000) were the first to design specific primers for the determination of e. lata species. the lata 1/lata 2.2 primer pair was particularly successful in separating e. lata isolates from isolates of over fifty different species of grapevine microorganisms. the aim of this study was to determine the affiliation of three studied isolates of eutypa lata (syn. eutypa armeniacae), anamorph libertella blepharis, with the help of molecular detection, using a specific pair of primers lata 1/lata 2.2 for the species eutypa lata. also for this purpose, sporulation and radial increase of the anamorphic stage of three isolates (el117, el153 and el199) on the nutrient medium of pda under the influence of different types of light were monitored. materials and methods samples and fungal isolation examination of vineyards in several sites of grape growing in our country in the period from 2004 to 2021, revealed symptoms of dieback of grapevine plants. symptoms on the leaves of diseased grapevine plants are manifested in the form of small, chlorotic spots, distributed along the edge of the leaves, while the central part of the leaf blade has a wrinkled appearance. the edges of the leaves are worn and bent downwards, and in severe infections, the surface of the leaves is mostly covered with necrotic spots. the shoots are light green in color, have a shortened appearance and the so-called zigzag growing of internodes. in the later stages, the disease is manifested by the appearance of a large number of very short shoots that grow from the same place on the branch of the vine, very close to each other. the first signs of the disease first develop around larger pruning wounds. by removing the dead bark, necrotic parts can be seen that extend along the stem and can be several tens of centimeters long. necrosis first affects the surface, and then penetrates into the interior, affecting the central part of the vine. dead tissue is dry, light or dark brown in color. on the crosssection of the diseased tree, more or less pronounced necrosis is manifested, which is in the initial phase in the shape of the letter „v”. for isolation, 1 cm fragments of grapevine stem and cordons were used, from the junction between the healthy and diseased parts. to isolate the pathogen, diseased parts of grapevine plants were surface-sterilized with 5% sodium hypochlorite for 2 min, followed by three washes with sterile distilled water. surface sterilized tissue was transferred to sterile filter paper and placed on potato dextrose agar (pda) containing streptomycin, and incubated at 24 °c. after incubation for 3 to 5 days, colonies of fungi developed around the fragments, which were sieved on potato dextrose agar (pda) medium and served to obtain monosporial isolates. based on the color and appearance of the fungal mycelium on potato dextrose agar (pda) medium, as well as the anamorphic stage, isolates were determined to the genus level. individual conidia were selected and transferred directly to the pda plate according to the procedures described by dhingra & sinclair (1995), and stored on pda in tubes at 4 °c. in order to check the pathogenicity of the obtained monosporial isolates of the fungus, artificial inoculations of the vine were performed. the pathogenicity test of the isolates was performed by inoculation of non-rooted grapevine cuttings (peros & berger, 1994). the cabernet sauvignon variety was used for the inoculation of cuttings in the experiment. breeding traits (radial growth and sporulation under the influence of three types of light) of 3 isolates of eutypa spp. (el117, el153 and el199) isolated from serbia and two control isolates from international collections were studied after obtaining monosporial cultures and confirming the pathogenicity of fungi isolates. influence of light on the development and sporulation of the studied anamorphic phase isolates of eutypa spp. to study the influence of different variants of artificial fluorescent light and darkness on colony growth and sporulation of the studied isolates of eutypa spp. pure cultures of 3 isolates and 2 reference isolates of e. lata grown on pda at 25 °c were used. the seeded petri plates were then exposed to 3 variants of artificial fluorescent light and darkness, as follows: 142 biologica nyssana ● 13 (2) december 2022: 141-151 živković et al. ● study of eutypa lata isolates originating from serbia biologica nyssana ● 13 (2) december 2022: 141-151 živković et al. ● study of eutypa lata isolates originating from serbia 143 1. 24 h exposure to uv light (uv); 2. alternatively, 12 h uv and 12 h darkness (uvt); 3. alternating 12 x artificial fluorescent light and 12 x darkness (s-t). three 40 w bulbs served as a source of fluorescent light, and two uv bulbs, with a wavelength of 366 nm, which were about 50 cm away from the petri plate, served as a source of ultra-violet light. mycelial growth was monitored by measuring the diameter of the colony for 10 days until the first isolate completely covered the petri plate. the first measurement of colony growth was performed after three days, and then, during the next ten days, until the first isolate completely filled the petri plate, the linear growth of colonies was monitored by linear diameter measurement. the average daily colony growth was calculated according to the formula (brasier and weber, 1987): (d2-d1)/(t2-t1)=daily growth level where d1 and d2 are the diameters of the colony after the first and second measurements, and t1 and t2 days elapsed from seeding to the first and second measurements (loc. cit. živković, 2019). determination of sporulation levels, i.e. the number of conidia per ml of suspension, was performed using a hemocytometer by thom with 16 fields, whose size is 1 mm2 and depth is 0.2 mm. the number of conidia was determined in order to determine the differences in the intensity of sporulation of 3 isolates of eutypa spp. studied in this paper. for this purpose, a suspension of spores was prepared by adding 5 ml of distilled water to a petri plate with a culture of the studied isolates of eutypa spp. the resulting conidia suspension together with the mycelial fragments was poured into a clean tube and then squeezed through a polyethylene mesh to remove the mycelial fragments. the counting was performed in 2 x 16 repetitions (32 fields), and from that, the average number of conidia per 1 mm2 was calculated. the number of conidia thus obtained was taken to calculate the density of spores in 1 cm3 (1 ml) of the suspension according to the formula: n=nx5x1000 where: n number of spores in 1 ml of suspension (spore density/1ml), n – the average number of spores per 1 mm2. the density of the spores is obtained by multiplying the average number of spores by the number 5 (because the depth of the pits is 0.2 mm). to obtain the number of spores in 1 mm3, and then multiplying the obtained number by 1000, since 1 cm3 contains 1000 mm3, to obtain a spore density in a volume of 1 cm3 (1 ml). the number of conidia per 1 mm2 of the colony was determined based on the number of conidia/ml and the area of the colony, which was calculated from the proportion of paper weight on which the contours of colonies were traced and a paper weight of 1 dm2 (živković, 2019). the level of sporulation is expressed according to the scale by quesad and lopez (1980), where: +=weak sporulation (<5.000 spores/ml), ++=medium sporulation (5.000-10.000 spores/ml) and +++=abundant sporulation (>10.000 spores/ml) (loc. cit. živković, 2019). photographing the test results was performed 30 days after sowing the substrate. two reference isolates, 8f and bx1.10, were used as controls. statistical analysis statistical analysis was performed in order to determine the relationship between eutypa spp. isolate and two reference isolates 8f and bx1.10. data were analyzed by variance analysis (anova) using computer software (proc glm, sas, system, version 8.1; sas institute, cary, nc). to satisfy the assumptions of the anova, the arcsine transformation of the proportion was used (y=2xarcsine√p). the homogeneity of groups was assessed using duncan’s test with p=0.05. molecular detection detection of three isolates of eutypa spp. from serbia and 2 reference isolates of e. lata was performed using the polymerase chain reaction (pcr) method. pcr was performed using 1 pair of specific primers. visualization of the obtained products was performed by electrophoretic separation in an agarose gel. dna extraction colonies of the tested isolates were grown on pda in the dark, at a temperature of 25 °c for 7 days. dna extraction was performed according to the method described by day & shattock (1997). in the first step, the colonies of the tested isolates were scraped from the surface of the substrate with a sterile spatula and transferred to a 2 ml tube with liquid nitrogen. after evaporation of the liquid nitrogen, 800 μl of 2% ctab buffer was poured into a tube and incubated at 65 °c for 1 h. during incubation, the content of the tube was shaken vigorously every 15 min. after incubation, 800 μl of chloroform was added to each microtube and vortexed, then centrifuged for 10 min at 11000 rpm and 4 °c (eppendorf 5804 r, germany). the resulting supernatant (about 700 μl) was pipetted into a new 1.5 ml tube, about 420 μl of isopropanol was added and centrifuged for 15 min at 11000 rpm and 4 °c. after centrifugation, 144 biologica nyssana ● 13 (2) december 2022: 141-151 živković et al. ● study of eutypa lata isolates originating from serbia the supernatant was carefully drained, and 1 ml of ice-cold 70% ethanol was added to the tubes, which was then carefully drained. the open microtubes were left for 10-15 min at room temperature. after drying, the dna pellet was resuspended in 100 μl te buffer. chain amplification of nucleic acid fragments for the detection of the studied isolates of eutypa spp. the pcr reaction was applied using a specific primer pair lata 1 and lata 2.2 (tab. 1). pcr amplification of the samples was performed in a thermocycler (mastercycler® eppendorf, germany). lata 1/lata 2.2 primers enable fragment amplification in the its1 and its2 regions. this primer pair serves for the specific detection of eutypa lata species by amplifying a fragment of about 385 bp located in the its1 and its2 regions (lecomte et al., 2000) (tab. 1). the 25 μl pcr mix contained: 12.5 μl 2 x master mix, 9 μl sterile distilled h2o, 1 μl 10 μm lata 1 and lata 2.2 primer and 1.5 μl dna. pcr amplification of the samples was performed according to the same program as with primers its1-its4. as a negative control in all pcr reactions, sterile water (pcr grade) was used instead of the target dna sample (pcr mixture with rnase-free water). reference isolates of e. lata from international collections were also used in pcr reactions as positive controls. visualization and analysis of pcr reaction products analysis of pcr products was performed after electrophoretic separation of the obtained products in 1.5% agarose gel and 0.5 x tbe buffer. the agarose gel was prepared by dissolving 3.6 g of agarose (merck, germany) in 240 ml of 0.5 x tbe buffer and heating to boiling point in a microwave oven. after cooling, the gel was poured into the molds of an electrophoresis apparatus (serva, germany). a comb immersed in the appropriate mold trays is immersed in the gel. after the polymerization of the gel, the comb was removed and the gel mold was placed in an electrophoresis apparatus. 0.5 x tbe buffer was added to the apparatus to a level where the gel was completely immersed in the buffer. before pipetting into the wells, 5 μl of the pcr reaction product of each sample was mixed with 1.5 μl of infusion dye (loading dye, mbi fermentas, lithuania). in electrophoresis, a 50 bp ladder marker (sigma aldrich, germany) was used to determine the size of the product by comparison with the expected size of the dna fragments of the marker. electrophoresis was performed at 100 v for 30 min. staining was performed by immersing the agarose gel in 0.5 μg/ml ethidium bromide solution for 15 min. amplified fragments in the gel were observed under uv light using a transilluminator (biometer, uk). results samples and fungal isolation examination of vineyards in several localities where grapevine is grown in our country, in the period from 2004 to 2019, showed symptoms of dying of grapevine vines. symptoms on the leaves of diseased vines are manifested in the form of small, chlorotic spots, distributed along the edge of the leaves, while the central part of the leaf blade has a wrinkled appearance. the edges of the leaves are worn and bent downwards, and in severe infections, the surface of the leaves is mostly covered with necrotic spots. the shoots are light green in color, have a shortened appearance and the so-called zigzag growth of internodes. in the later stages, the disease is manifested by the appearance of a large number of very short shoots that grow from the same place on the branch of the vine, very close to each other. the first signs of the disease first develop around larger pruning wounds. by removing the dead bark from the vine, necrosis is noticed, which develops around older pruning wounds. necrotic parts extend along the stem and can be several tens of centimeters long. necrosis first affects the surface, and then penetrates into the interior, affecting the central part of the grapevine. dead tissue is dry, light or dark brown in color. due to the growth of a healthy part of the wood around the infected one, the cracks appear on the stem surface along the necrotic belt, whereby outside, on the dead part of the stem tissue, smaller or larger longitudinal cracks appear (fig. 1). on the cross-section of the diseased stem, more or less pronounced necrosis is manifested, which is in the initial phase in the shape of the letter „v” on table 1. specific primer pair for detection of eutypa lata targeted sequence primer name sequence 5’-3’ fragment size literal source its-its2 lata 1 gagctaccctgtagcccgctg ~385 bp lecomte et al. (2000)lata 2.2 gacgtcagccgtgacacacc 145 biologica nyssana ● 13 (2) december 2022: 141-151 živković et al. ● study of eutypa lata isolates originating from serbia to three variants of artificial light and darkness, as follows: 1) 24 h exposure to uv light (uv), 2) alternating 12 h uv and 12 h darkness (uv-t); 3) alternating 12 h of artificial fluorescent light and 12 h of darkness (s-t). the aim of this experiment was to examine which isolate has the largest radial increase, daily increase, and degree of sporulation. partially or completely dried limbs and sleeves, the bark peels and falls off over time. on a stem without bark, especially around older sections from pruning, slight surface depressions of dead tissue can be noticed. samples were taken from diseased vines by cutting fragments 10 to 20 cm long. from these fragments, pieces about 1 cm long were cut from the junction of healthy and diseased tissue and seeded on a sterile pda medium. on potato-dextrose (pda) medium, 24 h after sowing, the studied isolates of eutypa spp. form the beginning of white mycelium. the mycelium is hyaline, milky white in color, more or less airy. over time, the mycelium becomes light gray-white in color with a thick, cottony, airy mycelium adhering to the substrate, reminiscent of down feathers. all studied isolates showed pathogenic properties and caused changes in the inoculated tissue by inoculation of unrooted cuttings of cabernet sauvignon grapevine. influence of light on the development and sporulation of the studied isolates of eutypa spp. during these studies, the radial growth of colonies and sporulation of all examined isolates under the influence of 12 h uv and 12 h darkness, under the influence of 12 h light and 12 h darkness and under the influence of 24 h uv (fig. 2) in a period of 30 days were studied. to study the influence of different variants of artificial light and darkness on colony growth and sporulation of the five studied isolates, three isolates eutypa spp., from grapevine, originating from serbia, and two determined control isolates of e. lata, originating from italy and france, were used. the radial growth of colonies and sporulation of five selected isolates was studied on a potato-dextrose medium by exposing seeded petri dishes at 25 °c fig. 1. eutypa spp.: tissue cracking and deformations due to thе growth of healthy tissue radial colony growth the radial growth of colonies of all five studied isolates was statistically significantly influenced by different variants of artificial light and darkness to which the seeded cultures were exposed (tab. 2). thus, during these studies, it was found that the highest increase in colonies of all studied isolates was under the influence of alternating 12 h uv and 12 h darkness, a slightly smaller increase was under the influence of 24 h uv over a period of 30 days, and the weakest increase was under the influence of 12 h light and 12 h darkness (tab. 2). it is also observed that isolates el153, el199 and reference isolate bx1.10, under the influence of 12 h uv and 12 h darkness have a statistically significantly higher increase compared to other tested isolates. while these isolates under the influence of 12 h of light and 12 h of darkness achieve a statistically significantly smaller rise compared to other tested isolates. (tab. 2). average daily growth the average daily colony growth in all five studied isolates of different variants of artificial fluorescent light and darkness was statistically significantly fig. 2. eutypa spp.: appearance of a colony of el199 isolates on a pda medium exposed to 24 h of ultraviolet light (uv) for a period of 30 days 146 biologica nyssana ● 13 (2) december 2022: 141-151 živković et al. ● study of eutypa lata isolates originating from serbia affected. considering this trait, some isolates differed significantly. it was found that all tested isolates (el117, el153 and el199, 8f and bx1.10) achieved a statistically significantly higher daily gain when exposed to 12 light and 12 h of darkness compared to other types of light (tab. 2). after statistical analysis of the data, it can be concluded that isolate el117 has a statistically significantly higher daily yield compared to other tested isolates at all types of light, except isolate 8f, which, under the influence of treatment with light 12 h uv and 12 h darkness, achieved statistically significantly higher growth compared to isolate el117 (tab. 2). sporulation (number of conidia per mm2 of colony) the type of light greatly affects the ability of the studied eutypa spp. isolates to form conidia in greater or lesser numbers. thus, different types of light in all tested isolates influenced the level of sporulation, which can be seen from tab. 3. the number of conidia formed per mm2 of colony was statistically significantly conditioned by the variant of artificial light and darkness in all five studied isolates of eutypa spp. in terms of this trait, some isolates differed significantly. thus, under the influence of 12 h of light and 12 h of darkness after 30 days of exposure, no sporulation of the el153 and el199 isolates occurred, while in isolates el117, as well as two reference isolates 8f and bx1.10, sporulation was abundant (tab. 3). when all five studied isolates were exposed to 24 h uv light for 30 days and a combination of 12 h uv and 12 h darkness, sporulation occurred in all studied isolates of eutypa spp., as shown in tab. 3. under the influence of both types of light, the el117 isolate exhibited statistically significantly higher extent of sporulation compared to other tested isolates, as well as two reference isolates. molecular detection and identification of the studied isolates the molecular method of polymerase chain reaction has been successfully applied for the detection of the studied isolates of eutypa spp. after extraction of the total dna of the tested isolates, pcr reaction was performed with one pair of specific primers (lata 1/lata 2.2) in order to detect the tested isolates to the level of the species. the specific primer pair lata 1/lata 2.2 for the determination of e. lata species enables amplification of the its region of ribosomal dna and 5.8 rrna (its1/its2 region). by comparing the amplified fragments of the tested isolates, with the marker (m) used, the presence of amplicons of the expected size of about 385 bp was found in all isolates, which confirms that all three tested isolates belong to the e. lata species. no amplification occurred with the negative control (fig. 3). discussion during the multi-year period (2004-2021) monitoring in vineyards in 14 localities, but also on individual vines in yards in serbia, it was found that table 2. eutypa spp.: influence of light on colony growth and sporulation of studied isolates of this parasite isolates different light types 24 h uv 30 days 12 h uv+12 h darkness 12 h light + 12 h darkness colony diameter (mm) el117 88.00 a 89.00 ab 88.00 a el153 86.60 ab 90.00 a 85.40 ab el 199 88.40 a 90.00 a 87.40 a 8f 87.80 a 89.00 ab 86.00 ab bx1.10 87.00 a 90.00 a 85.40 ab daily growth (mm)** el117 5.80 a 5.80 ab 7.16 a el153 5.16 a 5.72 ab 6.48 ab el199 4.76 ab 5.70 ab 6.52 ab 8f 5.12 a 6.08 a 6.08 ab bx1.10 5.32 a 4.68 b 6.24 ab *data in columns labeled with the same letters were not statistically significantly different based on the duncan test (p=0.05); **average daily increment calculated by formula (d2-d1 )/(t2-t1 ) 147 biologica nyssana ● 13 (2) december 2022: 141-151 živković et al. ● study of eutypa lata isolates originating from serbia table 3. eutypa spp.: influence of light on colony growth and sporulation of studied isolates of this parasite isolates different light types 24 h uv 30 days 12 h uv+12 h darkness 12 h light + 12 h darkness sporulation after 30 days *** el117 +++ +++ +++ el153 +++ +++ el199 +++ +++ 8f +++ +++ +++ bx1.10 +++ +++ +++ number of conidia after 30 days per mm2 el117 40.94 a 40.75 a 33.75 a el153 12.13 bc 16.19 b 0.00 b el199 15.00 b 15.38 b 0.00 b 8f 33.94 ab 34.44 ab 33.31 a bx1.10 33.31 ab 38.12 ab 32.44 ab *data in columns labeled with the same letters were not statistically significantly different based on the duncan test (p=0.05); ***sporulation: +=weak, ++=medium, and +++=abundant fig. 3. amplified dna fragments of eutypa lata about 385 bp in size obtained using lata 1/lata 2.2 primer pairs. el117, el153, el199 studied isolates; 8f and bx1.10 reference isolates; negative control; m-50 bp dna step ladder the fungus eutypa lata causes symptoms on shoots 25 to 50 cm long, and later as the disease progresses, on the stem and branches of diseased plants. this has been proven by the isolation of pathogens, and then by artificial inoculations on the cuttings of a large number of grapevine varieties. during the mentioned period of monitoring in vineyards in 14 localities in serbia, the presence of symptoms that the species eutypa lata causes both on inflorescences and berries of grapes. according to day and carter (1976), symptoms on shoots can be observed when shoots have 3 or 4 internodes. the symptoms are very difficult to notice and detect later, during the vegetation, because the diseased leaves are sheltered by healthy shoots and leaves. diseased leaves stop growing, are chlorotic and bend towards the back. the internodes are shortened and are often zigzagged, and the clusters are poorly developed and sparse (munkvold et al., 1993, 1994). cancerous wounds are caused by the growth of healthy tissue at the junction of healthy and diseased tissue, they are brown in color and are usually covered with tree bark. the grapevine dieback when the necrosis spreads to the entire stem (péros and berger 1994; sosonowski et al. 2007; 2013; sosnowski, 2016). symptoms on grapevine shoots may indicate the presence of this fungus in the stem or branches of the vine, and as the disease progresses, the symptoms spread to the entire plant. the growth of the fungus in the stem is slow, 10-20 cm per year, which is why after infection, the symptoms of dying on herbaceous organs do not appear in the first 2 to 3 vegetation seasons (munkvold et al., 1993). the first symptoms are noticed only in the third or fourth year after the infection. such a long incubation of the fungus in the stem and branches makes this disease „insidious”, and due to the slow development of the disease, economic losses are manifested in vineyards older than eight years. also, the fungus does not infect biologica nyssana ● 13 (2) december 2022: 141-151 živković et al. ● study of eutypa lata isolates originating from serbia 148 new vines younger than 5 years, and symptoms are rarely seen on a vine younger than 8 years (gubler et al., 2005; sosnowski et al. 2007; 2013; živković et al., 2012 a, b; sosnowski, 2016; živković, 2019). tissue necrosis spreads in a circle over time, covering an increasing part of the cross-section, and at the same time, it spreads along the stem, up and down, and reaches the root neck. drying of the limbs, vines and sleeves, which occurs as a consequence of disease progression, is best observed during the winter (munkvold et al., 1993; sosnowski et al., 2013; živković, 2019). fungus eutypa lata can be easily grown on conventional laboratory media, but not all isolates sporulate. to encourage sporulation, the cultures are exposed to a light regime of 12 x uv light 12 x darkness, 12 x light 12 x darkness or 24 h uv radiation and sporulation occurs after 30 days. mckemy et al. (1993) stimulated sporulation by exposing e. lata colonies to a shift of 12 h of light 12 h of darkness. in this regime, sporulation occurs after 30 days. ju et al. (1991) stimulated the sporulation of e. lata isolates by re-seeding a 2% pda medium with 5 g/l of yeast extract. the re-seeded cultures of e. lata were kept for a period of three weeks at 20 °c and exposed to 12 h of fluorescent light 12 h of darkness. glawe & rogers (1982) and glawe et al. (1982), in order to accelerate sporulation, exposed e. lata isolates on a pda substrate to shifting of 12 h of fluorescent light 12 h of darkness for 30 days. carter (1994) stimulated sporulation of e. lata isolates on a pda medium by exposing it to 24 h of uv light for 30 days or a combination of 12 h of uv light 12 h of darkness, as well as a combination of 12 h of light 12 h of darkness. munkvold (2001) stated that sporulation of conidia of e. lata isolates on pda medium is stimulated by constant exposure to uv rays for 30 days. dye & carter (1976), glawe et al. (1982) and mckemy et al. (1993) described cotton-white colonies of e. lata isolates on a pda medium. over time, the color of the colony turns to cream. under the influence of a shift of 12 h of light 12 h of darkness and at alternating temperatures of 19 °c and 15 °c, sporulation occurs after 30 days, which agrees with the statements of carter (1994) and munkvold (2001). the same authors state that in some cultures a gray pigment is formed after 15 days and the back turns black. three to four weeks after seeding, small black pycnidia appear in the culture under the influence of constant uv light. conidia are excreted from the pycnidia in the form of a cream to an orange gelatinous mass (rolshusen et al., 2006; trouillas & gubler, 2010). the test results show that not all studied light types have a statistically significant effect on the radial growth of the tested isolates, but they do cause a significant statistical difference in their sporulation. thus, different types of light, in all tested isolates, influenced the level of sporulation. isolates el117, 8f, and bx1.10 form a statistically significantly higher number of conidia at 24 h uv light and then under the influence of 12 h uv and 12 h darkness (glawe & rogers, 1982; glawe et al., 1982; munkvold, 2001). identification of the fungus e. lata is difficult due to the large morphological similarities of related species of the genus eutypa (rolshausen et al., 2014). therefore, many authors have developed molecular methods for the detection of eutypa lata species (descenzo et al., 1999; lecomte et al., 2000; rolshausen et al., 2004, 2006, 2014; catal et al., 2007; trouillas & gubler, 2010; urbez-torres et al., 2012). thus descenzo et al. (1999) for the first time used sequences of the its region to study 115 isolates of e. lata, isolated from ten plant hosts, of different geographical origin. these authors found that in all studied e. lata isolates, the 5.8 s rdna sequences were identical, regardless of the host and origin of the isolates. then, lecomte et al. (2000) use dna sequences to identify e. lata species. based on the its region rdna, they synthesized 3 pairs of primers lata1/ lata2-1, lata1/lata2-2 and lata3/lata2-1, as well as 3 pairs of primers based on the fragment of the rapd sequence sca 10a/sca 10b, scb 02a/ scb 02b and scd 18 a/scd 18 b. using the above primer pairs, they tested 60 isolates of e. lata of different geographical origin, isolated from vines. however, the lata1/lata2-2 primer pair was found to be the most specific for the detection of e. lata species. rolshausen et al. (2004) using a universal primer pair its1-its4 and the restriction enzyme alui, by analysis of rflp products in an agarose gel, separated over 30 isolates of e. lata from different hosts and different geographical origins, from other species from the family diatrypaceae. rolshausen et al. (2006) analyzed the sequences of 46 fungal isolates representing 15 different species from the diatrypaceae family, including 25 e. lata isolates collected from different plant hosts and different geographical origins, using its rdna and the protein part of the ß-tubulin gene. catal et al. (2007) based on the its1-its2 region designed a specific pair of el1/el4 primers, the application of which proved successful for the detection of american isolates of e. lata, while in european and australian isolates there was no amplification. trouillas & gubler (2010) successfully determined 35 isolates of e. lata as well as 7 isolates of other fungi, originating from different plant hosts, 149 biologica nyssana ● 13 (2) december 2022: 141-151 živković et al. ● study of eutypa lata isolates originating from serbia using universal primers its1-its4, sequencing of products of about 566 bp in all tested isolates, and sequence analysis likewise, úrbez-torres et al. (2012) used its region, efi-α and β-tubulin for sequence analysis in order to separate the grapevine dieback fungus complex. in this way, as many as 17 different species from the family diatrypaceae have been identified, which contribute to the formation of cancer, including the species e. lata. due to the above, this paper approaches, among other things, the application of molecular detection for the determination of the studied isolates to species levels. molecular methods, as a modern approach to the study of plant pathogens, have a great advantage in the precise identification and characterization of various pathogens. due to its simplicity and reliability, the pcr method has become one of the most commonly used methods in the detection of phytopathogenic fungi. molecular detection of the studied isolates of eutypa spp. it was performed after isolation of total dna. the extracted dna was intact and suitable for further successful amplification in the pcr reaction, enabling successful detection of all isolates used in this work. using a specific primer pair lata 1/lata 2.2 (lecomte et al., 2000), a fragment of about 385 bp in length was successfully amplified in all three studied isolates. these primers were selected based on the recommendation given by lecomte et al. (2000) in a protocol for rapid and efficient detection of this pathogen. in the studied isolates, profiles corresponding to the e. lata species were obtained, which fully corresponds to the identification using the conventional and molecular methods. 8f isolates originating from italy and bx1.10 originating from france, previously identified as e. lata, were also used as reference isolates in these studies. based on the breeding and molecular characteristics of the three isolates of eutypa spp. originating from parts of the grapevine with symptoms of extinction, collected from serbia, and comparing them with two reference isolates of e. lata originating from italy and france, it was determined that grapevine dieback in our country is caused by species of the genus eutypa,eutypa lata (syn. eutypa armeniacae), anamorph libertella blepharis. conclusions based on many years of research, the results were obtained from which the following can be concluded: examination of vineyards in several localities in serbia, in the period from 2004 to 2021, showed the appearance of symptoms of death of grapevine vines. from the breeding traits to the development of colonies of the studied isolates of eutypa lata the type of light had the most significant influence. under the effect of light type 12 h of light and 12 h of darkness, the isolates el153 and el199 did not sporulate, while under the action of 24 h of uv light, and at 12 h of uv and 12 h of darkness these isolates, as well as other tested isolates, achieved abundant sporulation. molecular detection of isolates was performed using a specific primer pair lata 1/lata 2.2, which allows amplification of the its region of ribosomal dna and 5.8 rrna (its1/its2 region). using these primers in all studied isolates, as well as reference isolates from international collections, a fragment of 385 bp was multiplied, which confirmed that it belongs to e. lata species. in the studied isolates, profiles corresponding to e. lata species were obtained, which fully corresponds to the identification using the conventional method. references carter, m.v. 1994: wood and root diseases caused by fungi. eutypa dieback. in: pearson r.c. and goheen a.c. 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originating from serbia 151 šabanović et al, 2019, biologica nyssana 10(2) 10 (2) december 2019: 143-153 doi: 10.5281/zenodo.3600557 the genus orchis tourn. ex l. and its related genera in the zenica-doboj canton (bosnia and herzegovina): diversity, distribution and conservation original article elvedin šabanović public institution “regional museum“ visoko, ul. alije izetbegovića 29, visoko, bosnia and herzegovina sabanovic.elvedin@gmail.com (corresponding author) aldin boškailo university “džemal bijedić“ in mostar, teachers faculty, univerzitetski kampus bb, mostar, bosnia and herzegovina aldinboskailo@hotmail.com šemso šarić jp špd-zdk d.o.o zavidovići, alije izetbegovića 25, ba-72220 zavidovići, bosnia and herzegovina semsosumar@gmail.com sanida bektić university of tuzla, faculty of sciences and mathematics, department of biology, tuzla, bosnia and herzegovina sanida.osmanovic@untz.ba vladimir ranđelović university of niš, faculty of sciences and mathematics, department of biology and ecology, niš, serbia vladar@pmf.ni.ac.rs received: september 29, 2019 revised: december 23, 2019 accepted: december 25, 2019 abstract: this paper represents the results of the distribution study focused on the genera anacamptis, neotinea, orchis and related species belonging to orchidaceae family. the investigation was conducted in the zenica-doboj canton, during the period of four years (2016-2019). the presence of 12 species and two subspecies was noticed. all of the populations turned out to be very vulnerable, consisting of a reduced number of individuals. the most endangered populations are orchis militaris, o. pallens and o. spitzelii. key words: orchidaceae, anacamptis, neotinea, orchis apstract: rod orchis tourn. ex l. i njemu srodni rodovi u zeničko-dobojskom kantonu (bosna i hercegovina): diverzitet, rasprostranjenje i konzervacija u vom radu su dati rezultati istraživanja distribucije vrsta iz rodova anacamptis, neotinea i orchis iz familije orchidaceae na području zeničkodobojskog kantona u periodu od 2016. do 2019. godine. utvrđeno je prisustvo 12 vrsta i dve podvrste. populacije svih vrsta su izrazito malobrojne, što ih čini veoma ugroženim. najugroženije su populacije vrsta orchis militaris, o. pallens i o. spitzelii. ključne reči: orchidaceae, anacamptis, neotinea, orchis introduction the genus orchis belongs to orchidaceae family (chase, 2005; stroh, 2016), the family with the highest diversity, which comprises about 28,000 species sorted in 736 genera (christenhusz & byng, 2016). the genus is affiliated to the subtribus orchidinae, the tribus orchideae and the subfamily orchidoideae. moreover, the rest of the genera from the central part of the balkan peninsula anacamptis, dactylorhiza, gymnadenia, herminium, himantoglossum, neotinea, ophrys, platanthera, pseudorchis and traunsteinera (djordjevic, 2018) – are having the same taxonomic status. according to the flora europaea (soó, 1980), the genus orchis also included some species, recently relocated within the anacamptis and neotinea genera, as a consequence of phylogenetic analysis, based on nuclear its sequences (bateman et al., 1997). in bosnia and herzegovina, the orchidaceae family is represented by 23 genera and 69 species (šilić, 2008) – among them, 8 species belong to the genus orchis (o. italica, o. mascula, o. militaris, o. pallens, o. provincialis, o. purpurea, o. simia and o. spitzelii), 5 to the genus anacamptis (a. pyramidalis, a. coriophora, a. morio, a, laxiflora and a. palustris) and 2 to the genus neotinea (n. tridentata and n. ustullata) (back managetta, 1903; šoljan © 2019 sabanovic et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 143 13th symposium on the flora of southeastern serbia and neighboring regions et al., 2014; bucalo, 2015). “the red list of flora of the federation of bosnia and herzegovina” (đug et al., 2013) contains 4 species from the previously mentioned genera: orchis purpurea, o. simia, o. spitzelii and anacamptis pyramidalis. the same species can be found in “the red list of flora of bosnia and herzegovina“ šilić (1996). “the red list of flora and fauna of republika srpska“ (http://www. nasljedje.org/docs/crvenalista/vascular_flore.pdf), is highlighting the importance of the orchid flora, comprising all of the species from the mentioned genera. the orchid flora of the zenica-doboj canton, hadn’t been investigated previously. the data about their distribution are seriously restricted; on the one hand, most of the literature data, including 11 species from the genera orchis, anacamptis and neotinea, date from the second half of the 19th and the first half of the 20th century (kummer & sendtner, 1849; fiala, 1896, protic , 1898, beck mannagetta, 1903; maly, 1908; plavsic, 1940); on the other hand, more recent data have been reduced to individual citations (ritter-studnička, 1970; abadžić, 2012; šabanović & bektić, 2017). the morphological similarity, followed by the phylogenetic status of the genera orchis, anacamptis and neotinea, as well as the fact that species from the genera anacamptis (with a. pyramidalis as an exception) and neotinea are even nowadays represented as the members of the genus orchis (šoljan et al., 2014) in bosnia and herzegovina, are indicating the need of the thorough investigation. hence, the aim of this paper was to determine the diversity of all three genera in the area of zenica-doboj canton, to show the distribution and condition of their populations and to point out the need for conservation of habitats as the only way of preserving populations. material and methods study area the study was focused on the zenica-doboj canton (fig. 1), the territory located in the central part of bosnia and herzegovina, more precisely, bosna river basin. it covers an area of 3345 km2, between 17°44’38’’ and 18°50’11’’ longitude, and 43°54’13’’ and 44°43’41’’ latitude. it is a distinctly mountainous area, with a huge geomorphological diversity and altitudes ranging from 160 to 1472 m. the relief is characterized by three specific areas: usore river valley with its plains and hilly areas in the north, hilly-mountainous terrain in the central part and hilly relief in the south. the central area is 144 biologica nyssana ● 10 (2) december 2019: 143-153 šabanović et al. ● the genus orchis tourn. ex l. and its related genera in the zenica-doboj canton (bosnia and herzegovina) fig. 1. geographical position of investigated area al., 2019). black pine forests (erico-pinetum nigrae serpentinicum stef. 1962) and other communities preferring serpentinite represent a special feature to the vegetation (ritter-studnička, 1970a). field work the diversity and geographical distribution of the taxa from the genera orchis, anacamptis and neotinea were investigated in the period from 2016 to 2019. gps and utm (10×10 km) coordinates are determined for each population. population status was defined by counting flowering and non-flowering individuals in the square areas 50×50 m. at the same time, potential factors in taxa endangerment have been reported. the collected floristic material was deposited in the museum „visoko“ in the visoko city. considering the high degree of threatened taxa, a rich photodocumenting material was created in order to avoid the collection of the plant material. photographs are also stored in the museum. data collection and analysis in addition to the field data, literature and herbarium data (sara herbarium of the national museum of bosnia and herzegovina) were included. the identification of the taxa was performed by using keys from modern literature (soó, 1980; delforge, 2006). the nomenclature of the taxa is consistent with the euro+med plantbase (http:// ww2.bgbm.org/europlusmed/results.asp). the distribution of the taxa is shown on the grid map and zone s4t with squares of 10×10 km, based on the universal transverse mercator (utm) projection (lampinen, 2001). the different types of the data are presented as follows: the white dot the field data, the black dot the literature data, the black-white dot the field and literature data, and the grey dot the herbarium data. the affiliation to an appropriate chorological type and floral element was determined on the basis of its geographical range. the determination of the chorological type is based on the coincidence of the geographical range with the corresponding phytochoria (stevanović, 1992). the abbreviations of the chorological type, as defined in ranđelović, zlatković (2010) and đorđević (2018), are given as listed: ea eurasian, ce central european, msm mediterranean-sub-mediterranean and sem south european mountainous. the floral elements were determined according to meusel (meusel et al., 1965) modified by stevanović (1992). the endangerment of the taxa wase based on the iucn criteria. the iucn categories are presented as follows: cr critically endangered, en endangered, vu vulnerable and lc least the largest, extending between the rivers of bosnia and krivaya and consisting of the mountain massifs ravan planina, konjuh, smolin, zvijezda and čemerska planina. the highest mountain peak of karasanovina (1472 m asl) is located on zvijezda mountain (goletić, ed. 2016). from the aspect of the geological substrates, the study area is quite diverse, including silicates, carbonates (goletić, ed. 2016) and, in particular, serpentinite (ritter-studnička, 1930). the entire area is characterized by a large number of river flows belonging to the bosna river basin. the lowland and hilly parts of the canton are characterized by a continental to a temperate continental climate, while with the altitude increases, the influence of the mountain climate is getting more pronounced (goletić, ed. 2016). in a domain of phytogeography, zenica-doboj canton belongs to the illyrian subregion of the central european region and holarctic floristic kingdom (horvat et al., 1974). this area is characterized by high diversity of flora, including endemic species, e.g. daphne blagayana, hesperis dinarica, viola beckiana, euphorbia gregersenii, e. montenegrina, dianthus croaticus and others. many plant species are declared as endangered, such as halacsya sendtneri, notolaena maranthe, eranthis hiemalis, kitaibela vitifolia and others. the orchid species contained in this study are included in the group of highly sensitive species. this area is characterised by the pronounced vegetation diversity as well, unfortunately enormously degraded due to the anthropogenic factor. on the banks of rivers and streams riparian gallery forests (classis alno glutinosae-populetea albae p. fukarek et fabijanić 1968) have been developed. the mountain belt is overgrown with the oak-hornbeam and mesic oak forests (ordo carpinetalia betuli p. fukarek 1968). the climatogenic vegetation of this area is represented with the sessile oak and the common hornbeam forest (querco-carpinetum illyricum ht. et al. 1974), whereas the sessile oak forests (quercetum montanum čer. et jov. 1968) prefer thermophilic habitats. forest vegetation of the mountain belt is seriously degraded, giving a place to the mountain grasslands as a secondary type of vegetation. above this belt, at higher altitudes, beech forests (fagetum illyricum ht. 1938) gradually turn into mixed deciduous and coniferous forests (abieti-fagetum illyricum ht. 1938). coniferous spruce forests (piceetum illyricum montanum ht. 1950) are growing on the small areas of the mountains zvijezda and ravna planina (horvat et al. 1974, fukarek, jovanović, ed., 1983). in surrounding of vareš, forests with peat moss and spruce (sphagno-piceetum montanum stefanović 1964) are developed in the depressions of the mountain belt (barudanović et 145 biologica nyssana ● 10 (2) december 2019: 143-153 šabanović et al. ● the genus orchis tourn. ex l. and its related genera in the zenica-doboj canton (bosnia and herzegovina) 146 biologica nyssana ● 10 (2) december 2019: 143-153 šabanović et al. ● the genus orchis tourn. ex l. and its related genera in the zenica-doboj canton (bosnia and herzegovina) fig. 2. investigated species: 1. anacamptis coriophora, 2. a. morio, 3. a. palustris, 4. a. pyramidalis, 5. neotinea tridentata, 6. n. ustulata, 7. orchis mascula, 8. o. militaris, 9. o. pallens, 11. o. purpurea, 11. o. simia, 12. o. spitzelii 147 fig. 3. distribution of investigated species: 1. anacamptis coriophora, 2. a. morio, 3. a. palustris, 4. a. pyramidalis, 5. neotinea tridentata, 6. n. ustulata, 7. orchis mascula, 8. o. militaris, 9. o. pallens, 11. o. purpurea, 11. o. simia, 12. o. spitzelii biologica nyssana ● 10 (2) december 2019: 143-153 šabanović et al. ● the genus orchis tourn. ex l. and its related genera in the zenica-doboj canton (bosnia and herzegovina) concern. relative frequency, as defined in vuković et al. (2011), was given as listed: a abundant, f frequent, r rare, rr extremely rare. the species and subspecies with the corresponding data (chorological type, iucn category, relative frequency, localities and habitats if available) are presented alphabetically in the list as follows: name of species chorological type/floral element iucn category/abundance distribution: literature and herbarium data: locality, altitude, utm 10×10, habitat (reference or collector, dd.mm.yyyy, herbarium). field data: locality, altitude, utm 10x10, habitat (collector, dd.mm.yyyy). results the presence of 12 orchid species and 2 subspecies from genera anacamptis (4 species, 1 subspecies), neotinea (2 species) and orchis (6 species, 1 subspecies) has been identified in the zenica-doboj canton. list of species anacamptis coriophora (l.) r. m. bateman, pridgeon & m. w. chase (fig. 2-1) ea/central european-mediterranean-sub-mediterranean-southwest asian vu/f (small populations, only the population near village porječani is more than 10 individuals) distribution (fig. 3-1): literature and herbarium data: oko vareša, bp89 (protić, 1898); oko vareša, bp89 (beck, 1903); pajtov han, bp88 (plavšić, 11/6/1940, sara). field data: stipin han, 350 m, bq91, mesophilous grassland (šabanović, 10/5/2018); prhinje, 465 m, bp77, hygrophilous grassland by the mining pit lake (šabanović, 27/5/2018); visočica, 690 m, bp77, mesophilous grassland from alliance arrhenatherion elatius (šabanović, 27/5/2018); porječani, 445 m, bp78, mesophilous grassland from alliance arrhenatherion elatius (šabanović, 3/6/2019); jelaške, bq90 (šarić, 14/5/2016; 17/5/2018); milankovići, cp09 (šarić, 6/6/2016); smailbegovići, breza, bp87 (šarić, 29/5/2018); drecelj, olovo, cp19 (šarić, 8/6/2017); ajdinovići, cp08, (šarić, 30/6/2017). anacamptis morio (l.) r.m.bateman, pridgeon & m.v.chase (fig. 2-2) ea/european-mediterranean-west asian lc/a distribution (fig 3-2): literature and herbarium data: borovica, bp79; vranduk, yk30 (kummer & sendtner, 1849); in grassland around pobrin han, bp89 (protić, 1898); around vranduk, yk30; between žepče and golubinje, yk31; near visoko, bp77; near vareš, bp89 (beck, 1903); kruškovica, bp99 (plavšić, 19/6/1940, sara); kondžilo, bp68 (plavšić, 9/6/1939, sara); visočica, bp77 (plavšić, 25/6/1939, sara). field data: sokolina, 870 m, bq90, mesophilous pasture in a belt of mixed deciduous and coniferous forests of the abieti-fagion type (šabanović, ranđelović, šarić, 30/4/2018); župa-jelaške, 585 m, bq90, mesophilous grassland (šabanović, ranđelović, šarić, 30/4/2018); visočica, 670 m, 685 m, bp77, mesophilous grassland (šabanović, 13/5/2018); visočica, 640 m, bp77, mesophilous grassland from alliance arrhenatherion elatius (šabanović, 11/5/2018); visočica, 710 m, bp77, dry grassland festuco-brometea type (šabanović, 11/5/2018); visočica, 790 m, bp77, mesophilous grassland from alliance arrhenatherion elatius (šabanović, 11/5/2018); dvor, 585 m, bp76, mesophilous grassland (šabanović, 9/5/2018); ribnica, 350 m, bq91, mesophilous grassland from alliance arrhenatherion elatius (šabanović, 12/5/2018); pogar, 1055 m, bp89, mesophilous pasture (šabanović, 12/5/2018); gorani, 635 m, bp77, thermophilic rocky meadows (šabanović, 13/5/2018); dvor, 515 m, bp76, mesophilous grassland (šabanović, 17/5/2018); porječani, 445 m, bp78, mesophilous grassland from alliance arrhenatherion elatius (šabanović, 3/6/2019); karići, 1190 m, bp98 (šabanović, 18/5/2019); žepče, 220 m, bq62, mesophilous grassland from alliance molinion caeruleae (šabanović, 25/5/2019); perun, 1330 m, bp88, mesophilous pasture (šabanović, 9/6/2019); brezik, zavidovići, bq82 (šarić, 21/4/2016); jelaške, bq90 (šarić, 21/4/2016; 25/4/2018); dištica, zavidovići, bq90 (šarić, 27/4/2018); bukov dol, bq90 (šarić, 29/5/2019). anacamptis palustris (jacq.) r.m. bateman, pridgeon & m.w. chase (fig. 2-3) ea/atlantic-european-euxinian-caucasianpontian-irano-turian vu/r (population near lupoglav is very rich and counts over 500 individuals, while other populations are with few individuals) distribution (fig 3-3): literature and herbarium data: između trbuka i maglaja, (beck, 1903); tajan, bq70 (maly, 22/5/1898; 24/6/1906; 21/5/1908; 30/5/1912; 148 biologica nyssana ● 10 (2) december 2019: 143-153 šabanović et al. ● the genus orchis tourn. ex l. and its related genera in the zenica-doboj canton (bosnia and herzegovina) 29/6/1912); tajan, bq70 (maly, 6/6/1921, sara); ljeskovica, bq72 (maly, 29/5/1930, sara); klek-zavidovići, bq72 (fiala, 16/6/1892, sara). a. palustris subsp. elegans (heuff.) r.m. bateman, pridgeon & m.w. chase distribution (fig. 3-3): field data: stošnica, 260 m, bq81, swamp meadow (šabanović, 30/5/2018); lupoglav, 230 m, bq62, swamp meadow (šabanović, 4/6/2019); stošnica, bq81 (šarić, 12/5/2016; 29/5/2018); žepče, bq62 (šarić, 5/6/2019); vozuća, bq81 (šarić, 2/6/2016). anacamptis pyramidalis (l.) richard (fig. 2-4) msm/mediterranean-sub-mediterranean-central european-pontian-euxinian-caucasian-iranian vu/f (populations are with few individuals) distribution (fig. 3-4): literature and herbarium data: in grasslands near pobrin han, bp89 (protić, 1898); between tolović and zenica, yj29; near vareš, bp89 (beck, 1903); raspotočje, yj39 (ritter, 26/6/1939, sara); sutjeska, bp78 (plavšić, 5/7/1939, sara); visoko, bp77 (laschnigg, 28/5/1930, sara); dabravine, bp88 (maly, 8/6/1930, sara); kula banjer, bp77 (maly, 8/6/1930, sara); vardište, bp88 (bjelčić, 24/6/1907, sara). field data: kula banjer, 570 m, bp77, thermophilic shrub (šabanović, 23/5/2018); prhinje, 510 m, 520 m, 530 m, bp77, thermophilic shrub and grassland (šabanović, 27/5/2018); porječani, 560 m, bp78, mesophilous grassland (šabanović, 1/6/2018); lipnica, 1210 m, bp79, mesophilous pasture (šabanović, 15/7/2018); bajrići, maglaj, 300 m, bq64 (šabanović, 25/5/2019); klupe-stog zavidovići, bq81 (šarić, 1/6/2016, serpentin); potoci near vareš, bp89 (šarić, 28/7/2016); lovnica, zavidovići, bq71 (šarić, 11/6/2016); magulica, bq90 (šarić, 13/6/2016); ćude, olovo, cp19 (šarić, 8/6/2017); smailbegovići, breza, bp87 (šarić, 27/5/2018). neotinea tridentata (scop.) r.m. bateman, pridgeon & m.v. chase (fig. 2-5) msm/mediterranean-sub-mediterranean-central european-euxinian-caucasian-pontian en/f (populations are with few individuals, a number rarely exceeding 10 individuals) distribution (fig. 3-5): literature and herbarium data: osova, bq62 (fiala, 1896); around vranduk, yk30; around vareš, bp89 (beck, 1903); on the mt. macat near vradište, bp88 (maly, 1912); visoko, bp77 (plavšić, 23.5.1939, sara). field data: magulica, near the franciscan church, 830 m, bq90, by the mountain road (šabanović, ranđelović, šarić, 30/4/2018); dvor, 590 m, bp76, mesophilous grassland (šabanović, 9/5/2018); visočica, 640 m, 650m, 670m, 680m, bp77, mesophilous grassland (šabanović, 11/5/2018); visočica, 790 m, bp77, mesophilous to thermophilous grassland (šabanović, 13/5/2018); gorani, 630 m, bp77, thermophilous rocky grassland (šabanović, 13/5/2018); prhinje, 530 m, bp77, dry grassland (šabanović, 27/5/2018); ajdinovići, 870 m, cp08, thermophilous grassland (šabanović, 5/6/2019); perun, 845 m, bp88, mesophilous grassland (šabanović, 9/6/2019); perun, 1340 m, bp88, mesophilous pasture (šabanović, 9/6/2019); ćude near olovo, cp19 (šarić, 12/5/2016); crni potok, bp99 (šarić, 19/5/2018); magulica, bq90 (šarić, 17/5/2016); bukov dol, bq90 (šarić, 29/5/2019). neotinea ustulata (l.) r.m. bateman, pridgeon & m.v. chase (fig. 2-6) ce/central european-pontian-south sibirian lc/f (populations are with few individuals, a number rarely exceeding 10 individuals) distribution (3-6): literature and herbarium data: borovica, bp79 (kummer & sendtner, 1849); around osova, bq62 (fiala, 1896); near pobrin han, bp89; on the slopes above vareš, bp89 (protić, 1898); between tolović and zenica, yj29; vareš, bp89; osova, bq62 (beck, 1903); banjer near visoko, bp77 (maly, 1933b); swamp bistrik, haljinići, bp78 (abadžić, 2012); čatići kod kaknja, bp68 (brandis, 19/7/1896, sara); vranjkovci, bp89 (plavšić, 19/6/1940; 7/7/1940, sara). field data: jelaške, 500 m, bq90, ruderal habitat (šabanović, ranđelović, šarić, 30/4/2018); visočica, 640 m, bp77, mesophilous grassland (šabanović, 11/5/2018); ribnica, 350 m, bq91, mesophilous grassland (šabanović, 12/5/2018); visočica, 685 m, bp77, mesophilous grassland (šabanović, 13/5/2018); ginje, 540 m, bp76, mesophilous grassland (šabanović, 16/5/2018); lipnica, 840 m, bp79, mesophilous grassland in a belt of mixed deciduous and coniferous forests of the abieti-fagion type (šabanović, 15/7/2018); brezik, zavidovići, 250 m, bq73, mesophilous grassland (šabanović, 25/5/2019); near borik resort, maglaj, 215 m, bq63, mesophilous grassland (šabanović, 25/5/2019); 149 biologica nyssana ● 10 (2) december 2019: 143-153 šabanović et al. ● the genus orchis tourn. ex l. and its related genera in the zenica-doboj canton (bosnia and herzegovina) lipnica, 860 m, bp79, mesophilous grassland (šabanović, 26/5/2019); perun, 1310 m, bp88, mesophilous pastures (šabanović, 9/6/2019); jelaške, bq90 (šarić, 21/4/2016; 21/5/2016; 7/5/2017; 13/5/2017; 29/5/2017; 23/4/2018; 28/4/2018; 14/5/2019; 29/5/2019); ribnica, zavidovići, bq91 (šarić, 7/5/2018); vozuća near zavidovići, bq81 (šarić, 14/5/2016). orchis mascula (l.) l. (fig. 2-7) ea/mediterranean-sub-mediterranean-central european-euxinian-caucasian-iranian vu/f (populations are with few individuals, a number rarely exceeding 20 individuals) distribution (fig. 3-7) literature and herbarium data: around pajtov han, bp88 (protić, 1898); around vareš, bp89; near osova, bq62 (beck, 1903). orchis mascula subsp. speciosa (vest) soo literature and herbarium data: tajan, 1296 m, bq70 (hilda ritter-studnička, 1970); sutjeska, bp78 (plavšić, 19/6/1940, sara). field data: jelaške 610 m, bq90, mesophilous grassland (šabanović, 10/5/2018); duboštica, 770 m, bq80, mesophilous grassland (šabanović, 12/5/2018); ponijeri, 1090 m, bp79, mesophilous pasture (šabanović, 20/5/2018); from ponijeri to lipnica, 830 m, bp79, mesophilous grassland (šabanović, 26/5/2019); lipnica, 815 m, bp79, mesophilous grassland (šabanović, 26/5/2019); perun, 1436 m, bp88, mesophilous grassland (šabanović, 9/6/2019); duboštica, bq80 (šarić, 19/5/2016; 13/5/2018); jelaške, bq90 (šarić, 14/5/2017); kopališta in vareš, bp89 (šarić, 23/5/2016); ponijeri, bp79 (šarić, 28/5/2017). orchis militaris l. (fig. 2-8) ea/central european-caucasian-pontian-south sibirian en-cr/rr (occurs single or with 2 to 3 individuals) distribution (fig. 3-8) literature and herbarium data: near pobrin han and village potoci near vareš, bp89 (beck, 1903); vardište, 900 m, bp88 (maly, 30/5/1918, sara). field data: duboštica, 770 m, bq80, mesophilous grassland (šabanović, 12/5/2018); duboštica, bq80 (šarić, 12/5/2018). orchis pallens l. (fig. 2-9) ce/central european-caucasian-pontiansouthwest sibirian en-cr/rr (occurs single or with 2 to 3 individuals) distribution (fig. 3-9) literature and herbarium data: on the hill bogoš, bp88 (protić, 1898); on the hill bogoš near vareš, bp88 (beck, 1903); visočica, bp77 (plavšić, 8/5/1939, sara); vardište, bp88 (maly, 30/5/1911, sara). field data: jelaške, stojčići, 600 m, bq90, roadside (šabanović, 28/04/2019); vranduk, yk30 (šarić, 29/10/2016); jelaške, bq90 (šarić, 29/10/2016; 27/04/2017; 23/4/2018); stojčići, bq90 (šarić, 14/5/2019). orchis purpurea huds. (fig.2-10) ce/central european-mediterranean-submediterranean-caucasian-pontian vu/f (small populations) distribution (fig. 3-10) literature and herbarium data: oko sela potoci prema pobrinom hanu, bp89 (protić, 1898); between tolovići and zenica, yj29; around vareš, bp89 (beck, 1903). field data: kamensko, 915 m, bq90, in mixed deciduous and coniferous forests of the abietifagion type (šabanović, ranđelović, šarić, 30/4/2018); perun, 990 m, bp88, in mixed deciduous and coniferous forests of the abietifagion type (šabanović, 9/6/2019); kamensko, bq90 (šarić, 29/5/2017); sokolina, bq90 (šarić, 17/5/2016); jelaške, bq90 (šarić, 14/5/2019). orchis simia lam. (fig. 2-11) msm/mediterranean-sub-mediterraneancaucasian-crimean vu/f (small populations) distribution (fig. 3-11) literature and herbarium data: kod zenice, yj39 (beck, 1903); oko vareša, bp89 (beck, 1903); oko vareša, bp89 (protić, 1898); visočica, bp77 (plavšić, 8/5/1939, sara); sutjeska, bp78 (plavšić, 22/5/1940, sara). field data: visočica, 660 m, bp77, forest carpino-quercetum petreae, roadside (šabanović, 11/5/2018); pobrin han, 1105 m, bp89, coniferous forests, roadside (šabanović, 12/5/2018); gorani, 630 m, bp77, forest carpinoquercetum petreae, roadside (šabanović, 13/5/2018); lipnica, 820 m, bp79, mesophilous grassland (šabanović, 26/5/2019); bobovac, 780 m, bp89, mesophilous grassland (šabanović, 8/6/2019); road from dabravine to kariće, 660 m, bp88, roadside (šabanović, 18/05/2019); boganovići, ćude, cp19 (šarić, 12/5/2018); bukov dol near olovo, bq90 (šarić, 28/4/2018; 29/5/2019); milankovići, cp09 (šarić, 150 biologica nyssana ● 10 (2) december 2019: 143-153 šabanović et al. ● the genus orchis tourn. ex l. and its related genera in the zenica-doboj canton (bosnia and herzegovina) 21/5/2017); jelaške, bq90 (šarić, 17/5/2018); žeravice, olovo, cp29 (šarić, 6/6/2016). orchis spitzelii saut. ex w.d.j.koch. (fig. 2-12) sem/pyrenean-alpine-apennine-dinaric-balkancarpathian-anatolian en-cr/rr (occurs single or with 2 to 3 individuals) distribution (fig. 3-12) literature and herbarium data: zavidovići (ritter studnička, 1970); visočica, bp77 (goul, 30/4/1936, sara); vardište, 900 m. bp88 (maly, 30/5/1911, sara). field data: kamensko, 900 m, bq90, šume bukve, jele i smrče (šabanović, ranđelović, šarić, 30/4/2018); vareš, bp89 (šarić, 13/5/2016); sokolina, kamensko, bq90 (šarić, 1/5/2018). discussion the study revealed the presence of 12 species and one subspecies of the genera anacamptis, neotinea and orchis. compared to the data available in the literature (protic, 1898; back mannagetta, 1903 and other), one of the subspecies found, anacamptis palustris ssp. elegans, is new to the zenica-doboj canton. additionally, after the close check of the all potential localities listed in the literature sources, the presence of orchis mascula, described as the type species for this area, has not been confirmed. since the type species wasn’t recorded in the neighboring sarajevo canton, but its mentioned subspecies under the name orchis ovalis (šoljan et al, 2014), it‘s almost certain that subspecies o. mascula ssp. speciosa (which has been found in several localities) is the only one growing in the investigated region. compared to the neighboring sarajevo canton (šoljan et al., 2014), it‘s just the subspecies anacamptis morio ssp. picta (loisel.) jacquet & scappat that is lacking. on the other hand, the study area is characterized by the species that are not recorded in sarajevo canton, including anacamptis palustris, orchis militaris and o. spitzelii. the wild orchids are protected by cites convention (appendice ii), which is implying the additional importance of this study. considering the species diversity in the investigated region as well, the necessity to propose the continuous monitoring system of all the recorded populations (including the remaining genera from the orchidaceae family throughout the canton) is obvious. according to the iucn red list of threatened species, anacamptis palustris categorized as the least concern (lc) represents the only one globally threatened species among all the others included in this study. a. coriophora, a. pyramidalis, neotinea tridentata, orchis mascula, o. militaris, o. pallens, o. purpurea and o. simia are having the same status at the european level. anacamptis morio and orchis spitzelii are categorized as near threatened (nt) species in europe. the proper conservation mechanism of the investigated genera is still lacking from the territory of bosnia and herzegovina. more precisely, “the red list of flora of bosnia and herzegovina“ (đug et al., 2013) contains only 4 species of these genera: orchis spitzelii as critically endangered (cr), orchis purpurea and o. simia as vulnerable (vu) and anacamptis pyramidalis with the near threatened (nt) status. the same species were mentioned by šilić (1996) in his list of plant species for the red book of bosnia and herzegovina, but all defined as vulnerable (vu). surprisingly, both of these lists do not contain anacamptis palustris (present on the iucn red list of threatened species as global endangered) and all the species from the european red list. consequently, the red list of flora of bosnia and herzegovina requires a thorough revision in order to ensure the adequate red list categories and criteria for all the representatives of the orchidaceae family, followed by defining measures for their conservation. references abadžić, s. 2012: stanje biodiverziteta prirodnog močvarnog područja bistrik. kakanj. barudanović, s., mašić, e., macanović, a., hatibović, e. 2019: state of peatland ecosystems in bosnia and herzegovina. fondeco science, sarajevo, 1(1): 48-60. bateman, r.m., pridgeon, a.m., chase, m.w. 1997: phylogenetics of subtribe orchidinae (orchidoideae, orchidaceae) based on nuclear its sequences. 2. infrageneric relationships and 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(eds.): bosna i hercegovina – zemlja raznolikosti. izvješće bosne i hercegovine za konvenciju o biološkoj raznolikosti. federalno ministarstvo okoliša i turizma. sarajevo. šoljan, d., muratović, e., abadžić, s. 2014: orhideje planina oko sarajeva. dobra knjiga d.o.o. sarajevo. sarajevo. tutin t.g., heywood v.h., burges n.a., moore d.m., valentine d.h., walters s.m., webb d.a. 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ain defla, with 15.1 million quintals, and mostaganem, with 9.1 million quintals. carrot cultivation plays a significant role in vegetable production programs in algeria. it is widely © 2025 djebara et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. practiced in coastal regions (alger, boumerdes, and mostaganem), the high plateaus (m’sila, oum el bouaghi, and sétif), and the southern areas (adrar, biskra, ghardaia, and el oued). the most commonly cultivated varieties include super muscade, muscade, touchon, maestro, carlo, and bolero. thrips are among the most significant pests affecting vegetable crops, particularly carrots, due to the recurrent attacks by native and invasive species. these attacks often directly or indirectly limit crop yields, leading to economic losses (ryckewaert & rhino, 2017). thrips cause direct damage when they feed on plant organs. the saliva injected during feeding can be toxic to plant tissues (bournier, 1983). thrips are also known to be vectors of viruses. according to mound (2004), over 50 species cause harm to cultivated plants, and 10 species act as vectors for tospoviruses worldwide. thrips exhibit high adaptability and polyphagy. additionally, fungi transmitted by thrips can cause diseases such as gray rot and white mold (ebratt, 2013). the lesions caused by thrips feeding on leaves, flowers, and fruits reduce the leaf area available for photosynthesis, potentially resulting in significant damage (fraval, 2006). following a case study on thrips affecting carrot crops in m’sila province, our objective is to conduct a qualitative and quantitative assessment of the key thrips species associated with carrot varieties cultivated in open fields within the region, and analyze their population fluctuations over the study period. this evaluation aims to inform the development of effective pest management strategies, particularly those targeting thrips that impact carrot production, for application in future studies. materials and methods study area the m’sila region is situated in a strategic location in northeast algeria. it is bordered to the northeast by the wilayas of bordj bou-arreridj and sétif, to the northwest by médéa and bouira, to the east by batna, to the west by djelfa, and to the southeast by biskra. geographically, m’sila lies in the algerian high plateaus, at coordinates 35°42’07’’n and 4°32’43’’e. historically, the region was classified a semi-arid and arid zone; however, the effects of climate change have significantly altered its climatic characteristics (zedam et al., 2016). the area is characterized by a mediterranean arid bioclimatic zone with mild winters (q2 = 17.02– 19.84; m = 3.37–4.10°c; m = 38.87–40.06°c; p = 182.94–201.86 mm). the rainy season extends from october to may, while the dry season typically lasts until august. the mediterranean climate of the region is influenced by the sahara (mimeche et al., 2018). m’sila is also distinguished by its extensive agricultural land, primarily dedicated to cereal crops, vegetable production, and arboriculture. the study on thrips population fluctuations on daucus carota l. cultivated in open fields was carried out in the el sagiya region (m’sila region). the area dedicated to carrot cultivation covers 1.5 hectares within a total farm area of 10 hectares, managed as a private farm. the soil is clayey, and carrots (daucus carota l.) had previously been cultivated in these fields. the farm supports a variety of agricultural practices, including greenhouse vegetable production (tomatoes, chili peppers, bell peppers, melons, eggplants, zucchinis, and cucumbers), fruit orchards (apple, pomegranate, apricot, pear, fig, and citrus trees), and limited cereal crops (durum wheat and bread wheat). sampling technique sampling was conducted from february to june 2024 using sticky traps and basin traps as trapping tools. the sticky traps were suspended above the crops with wire; new traps were installed every 15 days, while the old ones were collected and preserved carefully with cellophane. basin traps of different colors (blue and yellow) were placed on the soil surface. each basin was filled to one-third of its capacity with water, and a detergent was added as a surfactant to prevent insects from escaping. thrips collection and counting thrips were collected by filtering the contents of the basin traps using a strainer. the collected specimens were placed in vials containing 96% alcohol, with each vial labeled to indicate the date, collection site, and trap color. in the laboratory, the contents of the vials were transferred to petri dishes to facilitate the separation of thrips using entomological pins. the identification of the thrips on the sticky traps was conducted directly using a binocular magnifying glass and various identification keys. the identification process also relied on the works of several authors, including wang et al. (2010), mound & marullo (1996), bournier (2002), and moritz et al. (2004). the results were analyzed using ecological indices (shannon diversity index, pielou’s evenness index, simpson’s index of diversity and fisher’s alpha index) with the software past (version 2001). for better graphical representation, we applied a logarithmic transformation to the abundance data. biologica nyssana ● 16 (2) december 2025: djebara et al. ● diversity and population fluctuations of thrips (thysanoptera: thripidae) on carrot crops in m’sila region (algeria) biologica nyssana ● 16 (2) december 2025: djebara et al. ● diversity and population fluctuations of thrips (thysanoptera: thripidae) on carrot crops in m’sila region (algeria) results inventory of thrips species the results of the faunistic inventory and the fluctuations of thrips species recorded on carrots crops during our study are summarized in tab. 1 below. the inventory of thrips species conducted on a carrot crop in the m’sila region in 2024 identified seven species, distributed across four families and four subfamilies. the thripidae family was the most dominant, represented by three species: frankliniella occidentalis, thrips tabaci, and liothrips sp. this was followed by the aeolothripidae family, with two species: aeolothrips fasciatus and aeolothrips intermedius. lastly, the melanthripidae and phlaeothripidae families each contained a single species: melanthrips pallidior and gynaikothrips ficorum, respectively. abundance of thrips populations on carrot crops the results of thrips trapping using sticky traps and basins traps on carrot crops in the m’sila region during 2024 are summarized in tab. 2 below. the analysis of thrips captures across the two traps revealed that aeolothrips fasciatus, frankliniella occidentalis, thrips tabaci, and liothrips sp. were consistently captured in all traps. in contrast, gynaikothrips ficorum was detected exclusively in basins traps. this finding suggests that g. ficorum is not closely associated with vegetable crops. according to ziouani et al. (2016), this species is primarily associated with ficus retusa. its occurrence in the traps was likely incidental, with no more than four individuals recorded. the highest number of thrips captures was recorded on sticky traps, with 4,370 individuals collected on blue traps and 1,703 on yellow traps, representing 71.96% and 28.04% of the total population, respectively. in contrast, captures from basins traps were negligible, comprising no more than 62 individuals in blue basins and only 34 in yellow basins. aeolothrips fasciatus and frankliniella occidentalis exhibited notable activity in carrot crops. aeolothrips fasciatus was predominantly attracted to yellow traps, where 955 individuals were recorded compared to 419 on blue traps. conversely, f. occidentalis was primarily captured on blue families sub-families species of thrips aeolothripidae aelothripinae aelothrips fasciatus (linnaeus, 1758) aeolothrips intermedius (bagnall, 1934) melanthripidae melanthripinae melanthrips pallidior (priesner, 1919) phalaeothripidae phalaeothripinae gynaikothrips ficorum (marchal, 1908) thripidae thripinae frankliniella occidentalis (pergande, 1895) thrips tabaci (lindemann, 1888) liothrips sp. table 1. thrips species collected from carrots using sticky traps and basin traps thrips species sticky traps basins traps total yellow blue yellow blue yellow blue aelothrips fasciatus 955 419 8 19 1374 27 aeolothrips intermedius 0 0 1 5 0 6 melanthrips pallidior 33 105 2 0 138 2 gynaikothrips ficorum 0 0 3 4 0 7 frankliniella occidentalis 488 3717 16 19 4205 35 thrips tabaci 142 65 1 5 207 6 liothrips sp. 85 64 3 10 149 13 total 1703 4370 34 62 6073 96 table 2. abundance of thrips populations identified on carrot crops in m’sila biologica nyssana ● 16 (2) december 2025: djebara et al. ● diversity and population fluctuations of thrips (thysanoptera: thripidae) on carrot crops in m’sila region (algeria) captures was observed starting in late may. during the final three sampling periods, the number of thrips captured ranged between 0 and 26 individuals. the thrips population captured on blue sticky traps was the highest, representing 71.96% of the total individuals recorded. two major peaks were observed during the study: the first on april 21, with 616 individuals, and the second, more significant peak on may 20, with 3,291 individuals. following this, a sharp decline in captures occurred during june, with numbers ranging from 0 to 111 individuals. basins traps the thrips population captured using basins traps was significantly lower compared to the numbers recorded on sticky traps, accounting for less than 2% of the total captures. blue basins consistently attracted the highest number of thrips, with 62 individuals captured, compared to 36 in yellow basins. for blue basins, the curve in fig. 1 indicates a peak of 19 individuals on may 20, followed by a sharp decline, reaching a minimum of 6 individuals traps, with 3,717 individuals compared to 488 on yellow traps. other thrips species, including thrips tabaci, liothrips sp., and melanthrips pallidior, were observed only occasionally, with populations not exceeding 142 individuals. lastly, thrips showed minimal attraction to basins traps, with only isolated individuals captured, totaling no more than 19 in all. performance of various traps sticky traps the initial thrips captures in the carrot crops were recorded at the start of the sampling. on blue sticky traps, 18 individuals were captured, compared to only 8 on yellow traps. an analysis of the curves in fig. 1 reveals a gradual increase in thrips captures on yellow traps throughout april and may. the first peak of 741 individuals was recorded on april 21, followed by a slight population decline, leading to a second peak of 673 individuals on may 5. as the carrot crop cycle approached its end, a sharp decline in thrips fig. 1. temporal dynamics of thrips populations recorded on carrot crops using sticky and basins traps in m’sila, 2024 blue trap yellow trap yellow basins blue basins biologica nyssana ● 16 (2) december 2025: djebara et al. ● diversity and population fluctuations of thrips (thysanoptera: thripidae) on carrot crops in m’sila region (algeria) by the end of may. in yellow basins, the total thrips population did not exceed 8 individuals, with the highest count recorded on may 5. by late may, captures in yellow basins decreased to a minimum of 5 individuals, signaling the end of the carrot crop’s cycle. evolution of the main thrips species recorded on carrot crops aeolothrips fasciatus on yellow sticky traps, aeolothrips fasciatus was first detected at the end of february, with a very low population of no more than 8 individuals. a gradual increase in captures was observed throughout april and may, with the first peak of 442 individuals recorded on april 21. this increase was followed by a slight decline in early may, leading to a second peak of 403 individuals. subsequently, a sharp decrease in captures occurred, with a minimum of 30 individuals recorded on may 20. by june, a. fasciatus was completely absent, marking the ending of the carrot crop’s vegetative cycle. the thrips population in the blue basins was low, with a maximum of 172 individuals captured. there were two significant peaks in captures: the first peak occurred on april 21 with 172 individuals, and the second, smaller peak was on may 20 with 119 individuals. notably, there were no thrips observed in june, coinciding with the ending of the carrot crop cycle (fig. 2). frankliniella occidentalis unlike the species a. fasciatus, the thrips f. occidentalis is significantly more attracted to the blue color of the sticky traps. as a result, a total of 3,717 individuals (88.39%) were counted on the blue traps, compared to only 788 individuals (11.61%) on the yellow traps. the first thrips were captured on april 21, marking an initial peak of 305 individuals. following a decline in population at the beginning of may, there was a notable increase in both population and captures, reaching a second peak of 3,144 individuals on may 20. thrips populations declined throughout fig. 2. temporal dynamics of aeolothrips fasciatus populations recorded on carrot crops in m’sila, 2024 blue trap yellow trap yellow basins blue basins diversity indices to characterize the species diversity of thrips populations observed on carrot crops in m’sila, we calculated several ecological parameters, including dominance, shannon’s diversity index (h’), pielou’s equitability index (e), simpson’s index, and fisher’s alpha index. the results are presented in tab. 3 below. the results summarized in tab. 3 indicate that the highest numbers of thrips individuals were recorded on may 20 and april 21, with 3,484 and 1,480 individuals, respectively. shannon’s diversity index was generally low, remaining below 2.00 bits. it ranged from 0.30 to 1.32 bits, with the highest value observed on february 29. when the diversity index is less than 2, the population is considered to have low diversity. overall, equitability ranged from 0.18 on may 20 to 0.82 on may 30, approaching 1 in some cases. when equitability nears 1, it indicates that the population is composed of a similar number of individuals across species, with no single june, with captures not exceeding 116 individuals. on yellow sticky traps, the first frankliniella occidentalis individuals were captured around april 7, with a total of only 20 recorded. this was followed by a population increase, reaching a first peak of 206 individuals on april 21. a smaller second peak was observed in early may, with 175 individuals. however, beginning on april 20, the f. occidentalis population experienced a significant decline, with captures ranging from 3 to 94 individuals (fig. 3). thrips tabaci our findings indicate that yellow sticky traps are significantly more effective in attracting thrips tabaci populations. a total of 142 individuals (68.60%) were captured on yellow traps, compared to 65 individuals (31.40%) on blue traps. the first individuals were recorded on april 21, with 49 captured. a slight increase in population was observed in early may, with in a peak of 60 individuals. this was followed by a sharp decrease in captures throughout june, with only 9 individuals recorded during the entire month (fig. 4). biologica nyssana ● 16 (2) december 2025: djebara et al. ● diversity and population fluctuations of thrips (thysanoptera: thripidae) on carrot crops in m’sila region (algeria) fig. 3. temporal dynamics of frankliniela occidentalis populations recorded on carrot crops in m’sila, 2024 blue trap yellow trap yellow basins blue basins species dominating. simpson’s index measures the probability that two randomly selected individuals belong to the same species within a population. the closer the index is to 1, the more homogeneous the population. in this study, simpson’s index ranged from 0.12 in may to 0.68 on april 21. it is worth noting that simpson’s diversity index gives greater weight to abundant species than to rare ones, reflecting the co-dominance of multiple species as the index approaches 1. fisher’s alpha index (α) is an abundance model based on a logarithmic series, calculated using only the number of species (s) and the total number of individuals (n). higher α values indicate lower biologica nyssana ● 16 (2) december 2025: djebara et al. ● diversity and population fluctuations of thrips (thysanoptera: thripidae) on carrot crops in m’sila region (algeria) sampling period 29.02. 2024 23.03. 2024 07.04. 2024 21.04. 2024 05.05. 2024 20.05. 2024 30.05. 2024 08.06. 2024 19.06. 2024 individuals 30 62 106 1480 983 3484 11 137 16 dominance_d 0.39 0.62 0.51 0.31 0.37 0.87 0.47 0.70 0.78 shanon_h 1.32 0.75 1.04 1.29 1.16 0.30 0.90 0.56 0.37 equitability_j 0.68 0.47 0.53 0.80 0.72 0.18 0.82 0.51 0.54 simpson 1-d 0.60 0.37 0.48 0.68 0.62 0.12 0.52 0.29 0.21 fisher_alpha 2.87 1.28 1.68 0.64 0.68 0.57 1.35 0.54 0.60 table 3. abundances and diversity indices of thrips species on carrot crops in m’sila by sampling period in 2024 fig. 4. temporal dynamics of thrips tabaci populations recorded on carrot crops in m’sila, 2024 blue trap yellow trap yellow basins blue basins dominance by a single species. in this study, fisher’s alpha values were moderate, ranging from 0.54 on june 8 to 2.87 on february 29. these values suggest that the ecosystem is relatively unbalanced, with at least one species dominating the population at the study site. discussion thrips are highly adaptable insects that can thrive on a wide range of crops, including vegetables, cereals, fruit trees, and ornamental plants (ullman et al., 1997). they can cause both direct and indirect damage to crops through their feeding habits and their potential role as vectors of plant diseases (bournier, 1968; bournier, 1983). the study of thrips diversity conducted for the first time in algeria on carrot crops (daucus carota l.) in 2024, in the el sagiya region (m’sila province), identified seven species belonging to four families and four subfamilies. in a separate study conducted in the batna region, djerah (2022) documented 19 thrips species associated with six species of fruit trees from the rosaceae family. djebara et al. (2018) found four thrips species in greenhouse-grown tomatoes in algiers (north algeria). additionally, milat-bissaad et al. (2011) were the first to investigate the diversity of thrips associated with vineyards in algeria. their inventory revealed four species: frankliniella occidentalis, drepanothrips reuteri, aeolothrips fasciatus and liothrips sp. toudji & benrima (2022) highlighted the widespread presence of thrips on various plants in algeria, highlighting the importance of continuous monitoring to prevent damage caused by these pests. in the biskra region (southern algeria), during the 2010/2011 agricultural season, laamari & houamel (2015) reported, for the first time in algeria, the presence of thrips tabaci and frankliniella occidentalis on greenhouse crops. our results show that the species aeolothrips fasciatus, frankliniella occidentalis, thrips tabaci, and liothrips sp. were captured in all traps. in contrast, gynaikothrips ficorum was only captured in the basins. ziouani (2019) reports that this thrips species is primarily associated with ficus retusa. the highest number of thrips captures was recorded in the sticky traps, with 4,370 individuals in the blue traps and 1,703 individuals in the yellow traps, accounting for 71.96% and 28.04% of the total population, respectively. the monitoring of the overall thrips population fluctuations during our study revealed the presence of two main peaks. the first was recorded on april 21 with 616 individuals, while the second, even more significant peak, was observed on may 20, with no fewer than 3291 individuals. for the species aeolothrips fasciatus, the first individuals were captured towards the end of february. the species showed intense activity from late april to early may, with a peak of 442 individuals recorded on april 21 in the yellow sticky traps. in contrast to a. fasciatus, the thrips frankliniella occidentalis is much more attracted to the blue color of the sticky traps. the highest number of captures was noted on may 20 in the blue traps, totaling 3,144 individuals. the fluctuations of thrips tabaci were much less pronounced than the two previous species. the number of captures did not exceed 60 individuals, regardless of the type of trap. thrips tabaci was notably more attracted to the yellow sticky traps. the two notable peaks in thrips activity that we observed on april 21 and may 20 align with the typical seasonal patterns of these insects. research has shown that thrips populations often follow distinct seasonal cycles, which are closely tied to environmental factors like temperature, humidity, and the availability of plants they feed on (ullman et al., 1997). in this case, the peaks in april and may likely reflect the ideal conditions for thrips development and reproduction. this is especially true in temperate regions, where the warming temperatures of spring create an environment that allows their populations to grow rapidly. such seasonal fluctuations are common among various thrips species, including f. occidentalis and a. fasciatus (loomans & lenteren, 1995). we observed that f. occidentalis is attracted to blue sticky traps while a. fasciatus prefers yellow ones supports what previous research has shown: different thrips species are attracted to specific colors. studies have consistently found that f. occidentalis has a strong preference for blue traps (gillespie & vernon, 1990), finding them much more appealing than other colors like yellow or white (ullman et al., 1997). this preference may be linked to the resemblance of blue to their favored host plants or to its high contrast with the surrounding background, enhancing detectability. on the other hand, our results show that a. fasciatus favors yellow traps, which aligns with findings from other studies as well (nguyen et al., 2017). the early appearance of aeolothrips fasciatus in late february fits its usual springtime behavior, as this species is often among the first thrips to appear as the season changes. its noticeable activity in late april and early may, particularly in yellow sticky traps, suggests that a. fasciatus may be one of the most common thrips species found in carrot crops during this period, especially when spring conditions are ideal for their development. similarly, the peak in f. occidentalis populations on biologica nyssana ● 16 (2) december 2025: djebara et al. ● diversity and population fluctuations of thrips (thysanoptera: thripidae) on carrot crops in m’sila region (algeria) may 20 aligns with its typical late-spring to earlysummer activity. this species tends to reach its highest numbers around this time, due to its quick reproductive cycle and preference for specific crops (morse & hoddle, 2006). the relatively low fluctuations in the population of thrips tabaci, with a maximum of just 60 individuals captured across all traps, suggest that this species might be less attracted to carrot crops or possibly less common in the area during the observation period. this is consistent with research indicating that t. tabaci is generally less abundant in certain crops compared to others, with its activity affected by factors such as the availability of host plants and competition with other thrips species (perring et al., 1993). our study noted a preference for yellow traps, which is typical for t. tabaci; this species tends to be more attracted to yellow than to other colors (bournier, 1968). conclusion the dominance of thrips as pests in carrot crops highlights their remarkable adaptability and economic significance. these tiny insects not only inflict direct damage by feeding on the plants but also pose a serious threat as potential carriers of plant pathogens. this study underscores the need for integrated pest management (ipm) strategies. by combining regular monitoring with blue and yellow sticky traps, along with cultural practices like crop rotation and removing plant residues, farmers can better manage thrip populations. in situations where it’s necessary, adding biological or chemical control methods can also be beneficial. this research marks the first comprehensive assessment of thrips in carrot crops in algeria, laying the groundwork for future investigations. there is great potential to explore biological control options, such as using predatory mites or examine how thrips operate as vectors of plant diseases. additionally, studying the effects of climate variability on thrips populations could enrich our understanding and lead to better long-term management strategies. in summary, this study provides valuable insights for farmers, agronomists, and pest management professionals. the data gathered can help develop more effective strategies to safeguard carrot crops and minimize economic losses due to thrips in algeria. the results confirm that at least two species, aeolothrips fasciatus and frankliniella occidentalis, dominate the population, while thrips tabaci plays a less significant but still has notable role in the community structure. references bournier, a. 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(2019). population fluctuations of gynaikothrips ficorum (marchal, 1908) (thysanoptera: tubulifera) and natural enemies on leaf gall of ficus retusa in algeria. arxius de miscel·lània zoològica, 17, 59-71. djebara et al. ● diversity and population fluctuations of thrips (thysanoptera: thripidae) on carrot crops in m’sila region (algeria) biologica nyssana ● 16 (2) december 2025: uzelac et al. 2022, biologica nyssana 13(2) 13 (2) december 2022: 191-203 doi: 10.5281/zenodo.7476250 structure and chemistry of glandular trichomes of selected micromeria and clinopodium species (lamiaceae): in vitro culture approach original article branka uzelac department of plant physiology, institute for biological research “siniša stanković” national institute of the republic of serbia, university of belgrade, bulevar despota stefana 142, 11060 belgrade, serbia branka@ibiss.bg.ac.rs (corresponding author) snežana budimir department of plant physiology, institute for biological research “siniša stanković” national institute of the republic of serbia, university of belgrade, bulevar despota stefana 142, 11060 belgrade, serbia dragana stojičić department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, niš, serbia received: november 14, 2022 revised: november 30, 2022 accepted: december 07, 2022 abstract: many of the species belonging to the lamiaceae family are considered aromatic plants due to the presence of glandular trichomes, which have a distinct ability to synthesize, secrete or store large amounts of specialized metabolites that play a crucial role in mediating the plant–environment interactions. these compounds often have marked bioactive properties, rendering a commercial value to the plants that produce them. a number of biological effects have been associated with the main monoterpenoids detected in investigated micromeria spp. and clinopodium spp. essential oils. one alternative for the production of these bioactive metabolites is in vitro plant tissue culture. the present study was initiated to investigate the effects of in vitro culture on the secretion of leaf glandular trichomes, the main structures involved in the essential oil production. the glandular indumentum was studied by means of light microscopy and scanning electron microscopy in an attempt to correlate the phytochemical traits with the glandular trichome morphotypes of selected lamiaceae species. key words: glandular trichomes, histochemistry, lamiaceae, micropropagation, morphology apstrakt: struktura i hemija žlezdanih trihoma odabranih vrsta rodova micromeria i clinopodium (lamiaceae): primena kulture in vitro mnoge vrste familije lamiaceae su označene kao aromatične biljke zahvaljujući prisustvu žlezdanih trihoma, koje odlikuje karakteristična sposobnost da sintetišu, luče ili skladište velike količine specijalizovanih metabolita koji posreduju u interakciji između biljaka i njihove životne sredine. ova jedinjenja često imaju značajna bioaktivna svojstva, koja biljkama koje ih produkuju daju komercijalnu vrednost. veliki broj bioloških efekata dovodi se u vezu sa glavnim monoterpenoidima detektovanim u etarskim uljima ispitivanih vrsta micromeria spp. and clinopodium spp. kultura biljaka in vitro predstavlja jednu od alternativa za proizvodnju ovih bioaktivnih metabolita. prikazano istraživanje je započeto sa ciljem da se ispitaju efekti kulture in vitro na sekreciju žlezdanih trihoma listova, osnovnih struktura uključenih u proizvodnju etarskih ulja. žlezdani pokrivač je proučavan primenom svetlosne i skenirajuće elektronske mikroskopije, u pokušaju da se ustanovi korelacija između fitohemijskih odlika i morfotipova žlezdanih trihoma odabranih biljnih vrsta familije lamiaceae. ključne reči: histohemija, lamiaceae, mikropropagacija, morfologija, žlezdane trihome secretion refers to the complex phenomena of separation of secreted substances from the protoplast and their removal either to the plant surface or into internal spaces, or their accumulation in some compartment of the cell (evert, 2006). the secreted substances are produced by the secretory tissues of diverse structure and topographic position that occur in most vascular plants. glandular trichomes are the most recently evolved secretory structures found on the surface of about 30% of all vascular plants. they vary in their structure, in the chemical composition of the substances they produce and the mode of their secretion, and in their function. many of the species belonging to the lamiaceae © 2022 uzelac et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 191 14th symposium on the flora of southeastern serbia and neighboring regions family are considered aromatic plants due to the presence of glandular trichomes, a valuable source of biologically active volatile compounds. glandular trichomes in lamiaceae can produce a variety of specialized metabolites, among which terpenoids are particularly well represented and have been used by humans in a variety of industries (tissier et al., 2013). glandular trichomes secreting lipophilic substances generally consist of a group of glandular cells at the apex of a stalk of one or more cells in length. the storage compartment of glandular trichomes is part of the glandular cell, or cells, which are metabolically active. the present taxonomy ascribes great value to the structure (shape, cell number) and distribution of trichomes, as well as to the type of the secretion and its storage, and mode of release (giuliani & maleci bini, 2008). lamiaceae members are characterized by a great variety of trichomes. from a functional viewpoint and in accordance with their mode of secretion, the glandular trichomes of lamiaceae species can be classified into two main types, peltate and capitate trichomes (hallahan, 2000; werker, 2000). peltate trichomes have common characteristics in structure and morphology across genera and mostly produce and store biogenic volatile or semi-volatile organic compounds related to plant abiotic or biotic stress responses (corsi & bottega, 1999; turner et al., 2000; machado et al., 2006). they are the main sites of essential oil production and storage, as revealed by their ultrastructure and histochemistry (werker, 1993; turner et al., 2000). peltate trichomes are considered long-term trichomes, given that the accumulation of the secreted material continues during the growth of the organs that bear them (werker, 1993). there are various types of capitate trichomes that differ significantly in both structure and size (werker, 1993; werker et al., 2000), and whose primary function is thought to be the repelling of the pests (lin & wagner, 1994). capitate trichomes are specialized to produce and store a large amount of diterpenes and a wide array of nonvolatile or poorly volatile compounds that are directly exuded onto trichome surface and are particularly active at the early stages of organ development. they are considered short term trichomes because the secretory materials are extruded to the outside soon after their production, and the entire process of secretion is soon terminated (werker, 1993). the plant family lamiaceae, one of the largest among the dicotyledons, comprises more than 7200 species across approximately 240 genera. the vast number of genera and species, especially those placed in subtribe menthinae (tribe menthae, subfamily nepetoideae), are spices and medicinal herbs of great economic importance (brauchler et al., 2010). the genus micromeria benth. comprises more than 70 perennial herbs, sub-shrubs and shrubs, rarely annual herbs, distributed throughout the temperate belt (harley et al., 2004; erhardt et al., 2014). micromeria spp. are more or less aromatic species producing a small quantity of essential oils dominated by various terpene compounds. closely related genus clinopodium l. comprises ca. 100 (braüchler et al., 2010) to 135 (erhardt et al., 2014) perennial herbs, including taxa that were recently transferred from the polyphyletic genus micromeria sect. pseudomelissa based on molecular and morphoanatomical evidence (braüchler et al., 2006). clinopodium species are distributed worldwide, with the mediterranean being the main center of species diversity and sectional diversity and are also aromatic. they produce variable quantities (depending on the sample collection site) of essential oils dominated by oxygenated monoterpenes. essential oils of micromeria and clinopodium species exhibit substantial antimicrobial (marinković et al., 2002; duru et al., 2004; šavikin et al., 2010; vuko et al., 2012) and antioxidant activities (vladimirknežević et al., 2011), thus protecting the plant from pathogen attacks. it is for these properties that the aboveground plant parts of some micromeria and clinopodium species are used for medical, insecticidal, herbicidal, and culinary purposes (duru et al., 2004; šavikin et al., 2010; vladimir-knežević et al., 2015). plant sources are increasingly being exploited for new drug development, and there is a growing interest in validating traditional medicines and herbal remedies (verpoorte, 2000). natural sources of bioactive compounds used as pharmaceuticals, agrochemicals, flavors, fragrances, food additives and biopesticides are often endangered due to increasing industrial demands. plant cell, tissue, and organ cultures have emerged as potential sources of structurally complex and high-value natural products, especially if the plant source material is an overexploited, slow-growing, or low-yielding plant. through the application of various in vitro approaches and strategies, plant cell, tissue, and organ culture techniques permit manipulation of growth and production of medicinally or commercially important plant metabolites in the microenvironment of in vitro cultures, independently of geographical or seasonal variation (isah et al., 2018). a valuable method for the multiplication of selected genotypes and chemotypes of many medicinal and aromatic plants is in vitro propagation from wild-growing plants through axillary shoot formation (fig. 1). the present study was initiated to investigate the effects of in vitro culture on the secretion of leaf 192 biologica nyssana ● 13 (2) december 2022: 191-203 uzelac et al. ● structure and chemistry of glandular trichomes of selected micromeria and clinopodium species (lamiaceae): in vitro culture approach biologica nyssana ● 13 (2) december 2022: 191-203 uzelac et al. ● structure and chemistry of glandular trichomes of selected micromeria and clinopodium species (lamiaceae): in vitro culture approach 193 glandular trichomes, the main structures involved in the essential oil production, in an attempt to correlate the phytochemical traits with the glandular trichome morphotypes of selected lamiaceae species. to that aim, we carried out observations on micromorphology, morphoanatomy, and secretion of the glandular trichomes in shoot cultures of micromeria croatica (pers.) schott, micromeria graeca (l.) benth. ex rchb., clinopodium pulegium (rochel) bräuchler and clinopodium thymifolium (scop.) kuntze. general aspects of plant trichomes the plant trichomes are specialized unior multicellular epidermal projections of diverse form, structure, and functions, which cover most surfaces of most plants (esau, 1953). they display greatly variable morphology depending on the organ and the species, which has often been used in plant classification (wagner, 1991). in lamiaceae, the fig. 1. micropropagation of selected micromeria and clinopodium species. in vitro plantlets of micromeria graeca cultured on plant growth regulator-free medium after 4 weeks of culture (a-c); note numerous axillary shoots and well-developed adventitious roots in c. in vitro plantlet of micromeria croatica (d). axillary bud proliferation of clinopodium pulegium (e). acclimatized c. pulegium plantlets regenerated via axillary shoots (f). type, size and density of trichomes vary within genera, within species, and between different organs of the same plant, but most differences are speciesspecific and can often be of taxonomic significance (bhatt et al., 2010). the physiological functions of trichomes are diverse, as their morphological and mechanical features (size, shape, density, orientation) influence many aspects of plant physiology and ecology (wagner et al., 2004). two general types of trichomes can be discerned: non-glandular trichomes, which mainly differ in their morphology, and glandular (secreting) trichomes, which typically differ in the substances that they secrete. non-glandular trichomes play an important role in mechanical defense against biotic and abiotic stresses, and generally do not produce or secrete phytochemicals. these trichomes are diverse in morphology, anatomy and microstructure. nonglandular trichomes are abundant in the early phases of leaf development, but their density decreases with 194 biologica nyssana ● 13 (2) december 2022: 191-203 uzelac et al. ● structure and chemistry of glandular trichomes of selected micromeria and clinopodium species (lamiaceae): in vitro culture approach leaf maturity (corsi & bottega, 1999; ascensão et al., 1999). glandular trichomes are the specific sites for the biosynthesis, accumulation and excretion of secondary metabolites, i.e. pestor pollinatorinteractive chemicals that are stored or volatilized from the plant surface (keene & wagner, 1985; turner et al., 2000; werker et al., 2000). they not only accumulate and store what is often phytotoxic secretory material in a compartment that is virtually outside the plant body, but also position these compounds as an apparent first line of defense at the surface of the plant (wagner, 1991). glandular trichomes contain cells that are highly specialized for the biosynthesis and secretion of copious amounts of particular secretory products (lange & turner, 2013). glandular trichomes are the most recently evolved secretory structures, and are characteristic feature of many angiosperms (fahn, 1988; lange & turner, 2013). they are suggested to have developed phylogenetically from non-glandular trichomes, because both trichome types are initiated and develop similarly up to the stage of a three-celled primordium, after which the differences between the two types begin to appear (fahn & shimony, 1977). from a functional viewpoint, based on the mode and timing of secretion, glandular trichomes can be classified into two types: long-term glandular trichomes, which gradually accumulate their secretion in a subcuticular space, and short-term glandular trichomes, which start and end their secretion rapidly (werker, 1993). the former corresponds to peltate trichome morphotype, whereas the latter correspond to capitate trichome morphotype. peltate trichomes do not exhibit considerable variations in their basic morphology, although they can differ significantly in their size (ascensão et al., 1999; corsi &bottega, 1999; turner et al., 2000; machado et al., 2006; stojičić et al., 2022). various types of capitate trichomes differ significantly in both structure and size (werker et al., 1985; ascensão & pais, 1998; kolb & müller, 2004; amrehn et al., 2014). apart from morphology, peltate and capitate trichomes also differ in metabolites that they produce. besides volatile or semi-volatile stress-related organic compounds that are secreted and stored in extracellular storage space, capitate trichomes produce a wide array of nonvolatile or poorly volatile compounds that are directly exuded onto trichome surface. trichome types, functions, and distribution in micromeria and clinopodium spp. in vitro the aerial parts of in vitro grown plants of all examined species are covered with indumentum that appears colorless to the naked eye and consists of non-glandular and glandular trichomes. the most conspicuous are numerous sharply pointed, unbranched non-glandular trichomes, which are particularly elongated and abundant on leaf petioles and midribs (fig. 2a). uniseriate non-glandular trichomes with warty surfaces (fig. 2b) are found on both leaf sides and are especially abundant along the margins and veins and at the leaf base (fig. 3a). their morphology and distribution on plants in vitro fig. 2. stem and leaf indumentum of in vitro grown micromeria graeca plantlets (a). cross section of pubescent leaf, showing many non-glandular and glandular trichomes on both leaf surfaces (b) 195 biologica nyssana ● 13 (2) december 2022: 191-203 uzelac et al. ● structure and chemistry of glandular trichomes of selected micromeria and clinopodium species (lamiaceae): in vitro culture approach concentrated on the intervein areas in all examined species (fig. 3). trichome production and maturation is limited to short periods early in leaf development (hülskamp et al., 1994). in all examined in vitro plants, glandular trichomes are initiated very early during leaf ontogeny, with trichome initials and young developing trichomes present already on the youngest leaf primordia (fig. 4). glandular trichomes arise from a single expanding protodermal cell undergoing a periclinal division. peltate and capitate trichomes morphotypes cannot be easily distinguished at their inception, both being first discernible as protruding protodermal cells with an asymmetrical cytoplasmic distribution (fig. 4a). after enlarging and extending above from the leaf surface, the expanded protodermal cell is partitioned by a periclinal, asymmetrical cell division, resulting in the two-celled stage trichome comprised of an apical, more meristematic cell and a basal, more vacuolated cell (fig. 4b). slightly older, three-celled stage trichomes are further partitioned by periclinal cell divisions, separating an apical initial and a stalk cell, atop a vacuolated basal cell (fig. 4c). the apical initial may remain unicellular, as is the case in capitate trichomes of all investigated species or give rise to multicellular secretory head of peltate trichomes, as a result of several rounds of anticlinal divisions, depending on the species. further changes on the ultrastructural level occur in accordance with the chemistry of glandular trichome secretion (ascensão & pais, 1998; turner et al., 2000; giuliani & maleci bini 2008; amrehn et al., 2014; uzelac et al., 2015). the main types of glandular trichomes observed in micropropagated plants are peltate and capitate, which is a characteristic feature of lamiaceae species (ascensão et al., 1999). peltate trichomes are less abundant than capitate trichomes in all examined species. they are present on both leaf surfaces but are more abundant on the abaxial side (fig. 3, fig. 5). two types of capitate trichomes, differing in size and structure, could be distinguished in all examined species: type 1 (c1) and type 2 (c2) capitate trichomes (fig. 3c). both capitate types are found on both abaxial and adaxial surfaces of leaves, on stems and fully correspond to wild-growing plants (kremer et al., 2012; dunkić et al., 2017). the length of these trichomes varies from very short on the leaf lamina, to very long on leaf petioles and stems, which display the densest indumentum of non-glandular trichomes. glandular trichomes are constant features of many plant species and develop over the aerial vegetative and reproductive plant organs without external stimuli (fahn, 1988). although covering all the aboveground organs of in vitro propagated plants, glandular trichomes are particularly abundant on the leaves, where they appear on both sides but at different proportions and are particularly fig. 3. sem micrographs showing distribution and types of trichomes on vegetative and reproductive organs of in vitro grown plants. young, not fully developed leaf of micromeria graeca, with many non-glandular trichomes at the leaf margins and along the veins on the abaxial surface (a). mature glandular trichomes on calyx of clinopodium thymifolium. inset: detail of b, showing c2 trichomes at full secretion (bar=25 μm) (b). abaxial side of fully developed leaf of micromeria croatica. p – peltate trichome; c – capitate trichomes type 1 (c1) and type 2 (c2); ng – nonglandular trichome (c) 196 biologica nyssana ● 13 (2) december 2022: 191-203 uzelac et al. ● structure and chemistry of glandular trichomes of selected micromeria and clinopodium species (lamiaceae): in vitro culture approach on the abaxial side of calices (figs. 2, 3b-c, 5b). on leaves of in vitro grown plants, c1 trichomes were the most abundant trichome type, randomly distributed over the entire surface (predominantly abaxial). c1 trichomes are the most commonly occurring capitate trichome type in lamiaceae, reported in nearly all the species examined (werker et al., 1985; ascensão et al., 1999; mota et al., 2013). they were found in all investigated micromeria and clinopodium sp. plants in vitro, as well as in their wild-growing counterparts (kremer et al., 2012; marin et al., 2013; dunkić et al., 2017; kremer et al., 2021). compared fig. 4. glandular trichome initiation on leaves of in vitro cultured micromeria croatica. a transverse section through shoot apex, showing two youngest pairs of leaf primordia with trichomes at different developmental stages, mostly on the abaxial side. b detail of a. note trichome initial (arrowhead) adjacent to two-celled stage trichome (arrow) on the youngest leaf primordium. c early-stage glandular trichome following periclinal division of protodermal cell, comprising a more vacuolated basal cell (asterisk) and two densely cytoplasmatic cells; later during ontogeny, a smaller proximal meristematic cell (arrow) will give rise to the stalk cell, whereas larger apical cell (double arrow) will give rise to glandular head 197 biologica nyssana ● 13 (2) december 2022: 191-203 uzelac et al. ● structure and chemistry of glandular trichomes of selected micromeria and clinopodium species (lamiaceae): in vitro culture approach fig. 5. peltate trichomes of in vitro grown plants. a-b sem micrographs showing peltate trichomes at different secretory stages: immature, presecretory to early secretory stage trichomes of micromeria graeca (a) and fully mature, secretory stage trichome of clinopodium thymifolium (b). longitudinal section of post secretory stage peltate trichome of micromeria croatica (c). note multicellular secretory head (h), comprising 4 central cells surrounded by 12 peripheral cells arranged in a shield, subtended by unicellular stalk (s) and vacuolated basal cell (b). upper view of glandular head of fully developed peltate trichome of m. graeca, with 4 central cells surrounded by 16 peripheral cells arranged in a shield (d). note cuticular cap (arrowhead) detached from the secretory cell lateral walls. positive nile blue a reaction in the subcuticular space (pink droplet) and head cells (intense blue staining) of clinopodium pulegium (e). secretion in the subcuticular space stained positively (dark violet) for terpenes with nadi reagent in m. croatica (f) biologica nyssana ● 13 (2) december 2022: 191-203 uzelac et al. ● structure and chemistry of glandular trichomes of selected micromeria and clinopodium species (lamiaceae): in vitro culture approach 198 to dominant c1 trichomes, c2 trichomes were less abundant in all examined in vitro plants and occurred more frequently on the adaxial leaf side. morphoanatomy of glandular trichomes in micromeria and clinopodium spp. in vitro in micropropagated plants of all examined species, peltate trichomes at different secretory stages were observed (fig. 5). immature glands with wrinkled surface (fig. 5a), indicative of the close attachment of the cuticle to the upper secretory cells’ walls are predominantly located at the leaf base and closer to the midrib. during gland maturation, the secretory material is gradually secreted from the head cells into developing subcuticular space, formed by detachment and elevation of the cuticle, where the secretion was shown to accumulate in many lamiaceae species (werker, 1993; serrato-valenti et al., 1997; zuzarte et al., 2010). therefore, mature glands appear balloon shaped, and their surface becomes smoother, displaying occasional droplets of secreted material (fig. 5b). peltate trichomes exhibited rather uniform morphology across examined species and consisted of a broad basal cell embedded in the epidermis, a short unicellular stalk with cutinized walls and a round multicellular secretory head (ca. 50-60 μm in diameter, at secretory stage), with a variable number (8-20) of secretory cells, depending on the species (fig. 5c-d). different types of capitate trichomes, differing in stalk length and secretory head structure, have been described (werker et al., 1985; giuliani & maleci bini, 2008). in vitro grown plants of examined micromeria and clinopodium species displayed two types of capitate trichomes, differing in size and structure (fig. 3c). differences in the morphological characteristics of various capitate trichome types reflect different secretory processes within trichomes, and probably distinct functions (ascensão et al., 1999). type 1 capitate trichomes (c1) were positioned at an angle to the leaf surface (fig. 6). they were composed of one basal epidermal cell, short unicellular stalk with cutinized lateral walls and unicellular ellipsoidal head of ca. 20-25 μm in size (fig. 6a). no cuticle elevation was observed, and the secretory product accumulated within the secretory cell. as suggested by fahn (1988), the thickenings of the cuticle on the lateral walls of the neck cell, functioning also as a short stalk, probably prevent the backflow of secreted products into mesophyll tissue. in this way, trichomes remain structurally (and biosynthetically) isolated from the rest of the leaf, and can therefore, unlike leaf, produce large quantities of secretory material that is often phytotoxic (nielsen et al., 1991; werker, 2000). type 2 capitate trichomes (c2) are positioned upright and composed of conical, elongated, and highly vacuolated basal cell, unito bicellular stalk, and unicellular, usually elongated secretory fig. 6. capitate trichomes type 1 on leaves of in vitro grown plants. a-b longitudinal sections of c1 trichomes, showing large basal cell, short unicellular stalk and ellipsoidal unicellular head bent to the surface of micromeria graeca leaf, after staining with sudan iv (a) and nile blue a (b); c-d longitudinal sections of c1 trichomes showing faint staining with nadi reagent of the secretion in the head cell of clinopodium thymifolium (c) and micromeria croatica (d). note intense staining of the stalk cell lateral walls 199 biologica nyssana ● 13 (2) december 2022: 191-203 uzelac et al. ● structure and chemistry of glandular trichomes of selected micromeria and clinopodium species (lamiaceae): in vitro culture approach head of ca. 10 μm in diameter (fig. 7). immature c2 trichomes (fig. 7a, fig. 7d) appear finger-like since elongated head cell is of the same diameter as the stalk cell(s). during maturation, secretory head of c2 trichomes develops spherical subcuticular space where the secretion accumulates (fig. 7b, fig. 7e, fig. 7f), rendering trichome head rounded appearance (fig. 3c). in the post-secretory phase, after cuticle rupture, subcuticular elevation is no longer visible, so that the glandular head of c2 trichomes no longer appears round but regains fig. 7. capitate trichomes type 2 on leaves of in vitro grown plants. longitudinal sections of c2 trichomes, showing vacuolated pyramidal basal cell, subtending cylindrical unicellular stalk and unicellular head, both of approximately same diameters, after staining with sudan iv (a, d), nile blue a (b, e) and nadi reagent (c, f). note secretion accumulated in the subcuticular space of the early (b) and late secretory-stage trichomes (e, f), rich in essential oils (b, e) and terpenoids (f); in post secretory stage trichome (c), following rupture, the cuticle remains firmly attached to the cell wall in the basal part of the secretory cell, but no subcuticular elevation is visible. mg – micromeria graeca, mc – micromeria croatica, cp –clinopodium thymifolium finger-like shape (fig. 7c). type 2 capitate trichomes of in vitro grown plants correspond to those described by werker et al. (1985) for the lamiaceae, and are very similar to those detected in salvia officinalis (corsi & bottega, 1999) or lavandula pedunculata (zuzarte et al., 2010). nevertheless, kremer et al. (2021) distinguished 4 subtypes of capitate trichomes in studied micromeria and clinopodium spp. besides c1 capitate subtype, present in all investigated micromeria and clinopodium species, the authors biologica nyssana ● 13 (2) december 2022: 191-203 uzelac et al. ● structure and chemistry of glandular trichomes of selected micromeria and clinopodium species (lamiaceae): in vitro culture approach 200 observed c2 subtype trichomes (a rounded head cell) only in micromeria species studied, whereas roundish-head c3 and finger-like c4 (an elongated head cell, as narrow as the stalk cells, and only slightly enlarged above) subtypes were observed only in clinopodium taxa. based on these findings, and since the investigated taxa of the genus clinopodium belong to the former micromeria section pseudomelissa, the authors concluded that micromorphological traits also support the recent transfer of micromeria sect. pseudomelissa to the genus clinopodium (kremer et al., 2021). however, these claims were based solely on scanning electron micrographs, which do not always allow precise determination of the cell number or shape. in our study, both sem and light micrographs showed that the appearance of c2 trichomes differed depending on trichome developmental stage and secretory phase, as well as on sectioning plane. capitate trichomes type 2 that we observed in both investigated micromeria species fully corresponded to those observed in clinopodium species. fingerlike capitate trichomes were observed in both taxa, and were indicative of early secretory-stage trichomes, with still developing subcuticular space. as the secretion continued, and more secretory material accumulated in this space, the morphology of the glandular head changed from roundish to spherical, as observed in m. croatica (fig. 3c) as well as in c. thymifolium (fig. 3b) and c. pulegium (fig. 7e). histochemical characterization of secretions of glandular trichomes in micromeria and clinopodium spp. in vitro histochemical studies enable preliminary identification of the classes of bioactive compounds in tissues and cell compartments and are often employed to localize in situ the main classes of secondary metabolites present in plant secretions. together with morphological and structural investigations, histochemical studies provide data on secretory modes and secretions, thus allowing us to postulate a functional role for different glandular trichome types in plant interactions with its abiotic and biotic environment. the main classes of metabolites secreted by glandular trichomes of micropropagated plants were characterized by the histochemical tests in plant exudates in situ (figs. 5-7). in all examined species, peltate trichome secretion contained lipophilic substances, as revealed by sudan iv, and was for the most part composed of essential oil (tab. 1). nile blue a staining for neutral and acidic lipids gave positive reaction in the subcuticular space and to a lesser degree in secretory cells and was particularly intense in clinopodium species (fig. 5e, tab. 1). the presence of terpenoid compounds in this trichome morphotype was evidenced by intense dark-violet staining, with nadi reagent, of the secretory product within subcuticular space of all examined species (fig. 5f). the use of nadi reagent demonstrated the presence of terpenoids (resiniferous acids and essential oils) in the trichome glandular cells or in their secretion in a number of plant species belonging to lamiaceae (corsi & bottega, 1999; ascensão et al., 1999; marin et al., 2013). in all examined species, capitate trichomes type 1 of in vitro grown plants stained weakly positive for lipophilic secretion (fig. 6, tab. 1). on the other hand, type 2 capitate trichomes exhibited much stronger histochemical reactions (fig. 7, tab. 1). lipophilic secretory products accumulated in the subcuticular space of c2 trichomes, and small amounts could also be observed within secretory cells. that the majority of those lipophilic substances were the essential oils was shown by nile blue a staining (fig. 7b, e). nadi reaction resulted in an intense dark-violet staining of the secretory product, thus confirming the presence of terpenoid compounds in type 2 capitate trichomes (fig. 7c, f). although it is generally assumed that the bulk of the essential oil in lamiaceae is produced by peltate trichomes, studies showed that capitate trichomes in some species seem to produce significant amounts of essential oils (ascensão et al., 1999; mota et al., 2013; stojičić et al., 2016). reports on the histochemical detection of different secondary metabolites carried out in plants table 1. histochemical characterization of secretions of leaf glandular trichomes of selected micromeria and clinopodium species chemical compounds micromeria graeca micromeria croatica clinopodium thymifolium clinopodium pulegium p c1 c2 p c1 c2 p c1 c2 p c1 c2 lipids ++ ± + ++ ± + ++ + ± + + ++ essential oils + ± + + ± + ++ + ± ++ + ++ terpenoids ++ ± + ++ ± + ++ + ± ++ + ++ 201 biologica nyssana ● 13 (2) december 2022: 191-203 uzelac et al. ● structure and chemistry of glandular trichomes of selected micromeria and clinopodium species (lamiaceae): in vitro culture approach grown in vitro are scarce (uzelac et al., 2015; stojičić et al., 2016; dantas et al., 2017; tošić et al., 2019). defined environment of in vitro culture provides plants with nearly optimal growth conditions, in contrast to marked variations of growth conditions of wild-growing plants, which may contribute to increased secondary metabolite production in vitro reported for a number of species (fraternale et al., 2003; affonso et al., 2009; tošić et al., 2019). shoot cultures of many medicinal and aromatic plants have been shown to accumulate secondary metabolites to a greater extent compared to natural plants (bassolino et al., 2015; makowczyńska et al., 2016). since in vitro cultured plants are by definition considered as juvenile-stage plants (croteau et al., 1981), the reported quali-quantitative differences in the volatile profile between wild-growing plants and those grown in vitro could be the consequence of different ontological stage of the plants. conclusions in recent decades, considerable research has focused on understanding and exploiting glandular trichomes ability to secrete phytochemicals that might improve plant resistance to pests, modify trichome metabolism towards improving exudate levels and quality traits and allow commercial production of useful compounds. plant cell, tissue and organ culture is a valuable method for the multiplication of selected genotypes and chemotypes, enabling efficient clonal propagation regardless of the season as well as quantitative and qualitative modifications in the production of plant secondary metabolites by different physical and chemical factors. acknowledgements. this research was financially supported by the ministry of education, science and technological development of the republic of serbia through contracts nos. 451-03-68/2022-14/200007 and 451-03-68/2022-14/200124. references affonso, v.r., bizzo, h.r., salguiero lage, c.l., sato, a. 2009: influence of growth regulators in biomass production and volatile profile of in vitro plantlets of thymus vulgaris l. journal of agricultural and food chemistry, 57: 6392–6395. amrehn, e., heller, a., spring, o. 2014: capitate glandular trichomes of helianthus annuus (astearceae): ultrastructure and cytological development. protoplasma, 251: 161-167. ascensão, l., mota, l., castro, m. de m. 1999: glandular trichomes on the leaves and flowers of plectranthus ornatus: morphology, distribution and histochemistry. annals of botany, 84: 437-447. ascensão, l., pais, m.s. 1998: the leaf capitate trichomes of leonotis leonurus: histochemistry, ultrastructure and secretion. annals of botany, 81: 263-271. bassolino, l., giacomelli, e., giovanelli, s., pistelli, l., casetti, a., damonte, g., bisio, a., ruffoni, b. 2015: tissue culture and aromatic profile in salvia dolomitica codd. plant cell, tissue and organ culture, 121: 83-95. bhatt, a., naidoo, y., nicholas, a. 2010: an investigation of the glandular and non-glandular foliar trichomes of orthosiphon labiatus n.e.br. 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(eds.), advances in botanical research, 31: 1-35, academic press, new york. werker, e., ravid, u., putievsky, e. 1985: structure of glandular hairs and identification of the main components of their secreted material in some species of the labiatae. israel journal of botany, 34: 31-45. zuzarte, m.r., dinis, a.m., cavaleiro, c., salgueiro, l.r., canhoto, j.m. 2010: trichomes, essential oils and in vitro propagation of lavandula pedunculata (lamiaceae). industrial crops and products, 32: 580-587. oxidative stress in bromus (bromus mollis l.) seedlings treated with clary sage (salvia sclarea l.) aqueous extract biologica nyssana 7 (2)  december 2016: 141-144 šućur, j. et al.  the effect of satureja montana l. aqueous… 141 original article received: 01 july 2016 revised: 11 november 2016 accepted: 24 november 2016 oxidative stress in bromus (bromus mollis l.) seedlings treated with clary sage (salvia sclarea l.) aqueous extract jovana šućur1, dejan prvulović1, goran anačkov2, đorđe malenčić1 1 department of field and vegetable crops, faculty of agriculture, university of novi sad, trg dositeja obradovića 8, novi sad, serbia 2 department of biology and ecology, faculty of science, university of novi sad, trg dositeja obradovića 3, novi sad, serbia * e-mail: jovana.sucur@polj.edu.rs abstract: šućur, j., prvulović, d., anačkov, g., malenčić, đ.: oxidative stress in bromus (bromus mollis l.) seedlings treated with clary sage (salvia sclarea l.) aqueous extract. biologica nyssana, 7 (2), december 2016: 141-144. extensive use of synthetic pesticides has negative effects on the environment and on human and animal health. knowledge on allelopathic interactions could provide effective tools for a better exploitation of natural resources in the management of weeds without using herbicides. one of highly resistant weed species is bromus. the effects of two concentrations (0.1% and 0.2%) of salvia sclarea l. aqueous extract on the activity of the antioxidant enzymes, superoxide dismutase (sod) and catalase (cat) in leaves and roots of bromus (bromus mollis l.) seedlings, were examined. our results showed that both concentrations of the extract used (0.1% and 0.2%) stimulated the significant increase of the superoxide dismutase activity in leaves and roots of bromus 72 hours and 120 hours after the treatment. the significant increase of the catalase activity was recorded in roots of bromus 72 h after the treatment. two tested extract concentrations affected activity of the antioxidant enzymes in the same way, but the higher activity was observed in the roots treated with higher concentration (0.2%). the increase of the activities of antioxidant enzymes, in response to stress induced by s. sclarea aqueous extract, indicate that the plant extract possesses allelopathic activity on treated plant. key words: allelopathy, biopesticides, bromus mollis l., salvia sclarea l. apstrakt: šućur, j., prvulović, d., anačkov, g., malenčić, đ.: oksidativni stres u sadnicama klasače (bromus mollis l.) tretiranim vodenim ekstraktom muskatne žalfije (salvia sclarea l.). biologica nyssana, 7 (2), decembar 2016: 141-144. upotreba sintetičkih pesticida ima negativan efekat na životnu sredinu, kao i zdravlje ljudi i životinja. poznavanje alelopatskih interakcija može da obezbedi efikasniji način eksploatacije prirodnih resursa u suzbijanju korova bez upotrebe sintetičkih herbicida. jedna od otpornijih vrsta korova je korov klasača. u ovom radu je ispitan uticaj dve koncentracije (0,1% i 0,2%) vodenog ekstrakta s. sclarea na aktivnost antioksidantnih enzima superoksid-dismutaze (sod) i katalaze (cat) u listu i korenu klasače (bromus mollis l.). dobijeni rezultati su pokazali statistički značajno povećanje aktivnosti enzima superoksid-dismutaze u 7 (2) • december 2016: 141-144 12th sfses • 16-19 june 2016, kopaonik mt doi: 10.5281/zenodo.200411 biologica nyssana 7 (2)  december 2016: 141-144 šućur, j. et al.  the effect of satureja montana l. aqueous… 142 listu i korenu klasače, 72 i 120 časova nakon tretmana za obe primenjene koncentracije ekstrakata (0,1% i 0,2%). statistički značajno povećanje aktivnosti katalaze uočeno je u korenu klasače 72 časa nakon tretmana. obe ispitane koncentracije vodenih ekstrakata uticale su na aktivnost antioksidantnih enzima, pri čemu je veća aktivnost uočena u korenu klasače tretirane sa višom koncentracijom (0,2%). povećanje aktivnosti antioksidantnih enzima, kao odgovor na stres prouzrokovan vodenim ekstraktom s. sclarea, pokazuje da ekstrakt poseduje alelopatski uticaj na tretiranu biljku. ključne reči: alelopatija, glycine max (l.) merr., satureja montana l. introduction the flowering plant family of lamiaceae is very important since its highly diverse species are potent source of secondary metabolites (f a k o o r z i b a et al., 2014). numerous species of the genus salvia, belonging to lamiaceae, have been used in traditional medicine (n a s e r m o a d e l i & r o w s h a n , 2013). salvia sclarea l., known as clary sage, a plant native to southern europe, is one of the most important aromatic plants cultivated world-wide as a source of essential oils and many other compounds derived from different parts of the plant (h u d a i b et al., 2001, n a s e r m o a d e l i & r o w s h a n , 2013). plant-derived chemical compounds are also involved in communication between plants (w e i r et al., 2004). allelopathy is an interference mechanism in which plants release chemical substances, called allelochemicals, from leaves, stems, roots, flowers and seeds which inhibit or stimulate plant growth (g e l l a et al., 2013). accordingly, allelopathy is a natural and an environment-friendly technique which may prove to be also a unique tool for weed control (s h a r m a & s a t s a n g i , 2013). allelochemicals are highly attractive as new classes of herbicides due to a variety of advantages. most of them are water-soluble which makes them easier to apply and their chemical structures are more environmentally friendly than synthetic ones (s o l t y s et al., 2013). the aim of this study was to examine the effect of two concentrations (0.1% and 0.2%) of salvia sclarea l. aqueous extract on the activity of the antioxidant enzymes superoxide dismutase (sod) and catalase (cat) in leaves and roots of bromus (bromus mollis l.) seedlings and to explore the biopesticidal potential of this species in weed control. material and methods the wild, aromatic plant, s. sclarea l., was collected on mt rujan, in the south of serbia (n 42º22ʹ40.44ʹʹ, e 21º53ʹ09.23ʹʹ, 494 m.a.s.l.) in july of 2012. voucher specimens of collected plant was confirmed and deposited at the herbarium of the department of biology, faculty of natural sciences, university of novi sad (buns 2-1545). the aqueous extract of s. sclarea was prepared with air-dried plant material (10 g) in boiling distilled water (100 ml). after 24 h, the extract was filtered through whatman no.4 filter paper and kept at 4 °c in the fridge until application. the experiment was performed at the laboratory of biochemistry, faculty of agriculture, novi sad and conducted under controlled conditions (28 oc, 60% relative humidity, a photoperiod of 18 h, and a light intensity of 10.000 lx). the bromus (bromus mollis l.) seeds were grown in plastic pots containing sterile sand. after 30 days, the plants were transplanted in plastic pots containing 700 ml of hoagland’s solution and 7 and 14 ml of s. sclarea aqueous extract, while pots of control contained just the same volume of hoagland’s solution (10% mgso4 x 7h2o, 10% ca(no3)2 x 4 h2o, 10% kh2po4, 10% kno3, microelements, 7.5% feedta). plants were harvested for further biochemical analyzes 24, 72 and 120 h after the treatments. fresh leaves and roots of bromus plants (2 g each) were homogenized in 10 ml of phosphate buffer (0.1 m, ph 7.0). homogenates were centrifuged for 20 min at 10.000 x g (boeco, germany) and filtered. the supernatants were used for determining the investigated biochemical parameters (activity of the antioxidant enzymes superoxide dismutase (sod) and catalase (cat)). biochemical analyses were carried out spectrophotometrically using an uv/vis spectrophotometer model thermo scientific evolution 220 (usa). the catalase (cat) (ec 1.11.1.6) activity was determined according to s a t h y a & b j o r n , (2010). the decomposition of h2o2 was followed as a decrease in absorbance at 240 nm. the enzyme extract was added to the assay mixture containing 1 ml of 50 mm potassium phosphate buffer (ph 7.0) and 10 mm h2o2 (centrohem, serbia). the activity of the enzyme is expressed as u per 1 g of protein (u mg–1 protein). superoxide dismutase (sod) (ec 1.15.1.1) activity was assayed according to a slightly modified method of mandal et al. (2008) by measuring its ability to inhibit photochemical reduction of nitro blue tetrazolium (nbt) chloride. the reaction mixture contained 50 mm phosphate buffer (ph 7.8), 13 mm l-methionine (sigma (st. biologica nyssana 7 (2)  december 2016: 141-144 šućur, j. et al.  the effect of satureja montana l. aqueous… 143 louis, mo.)), 75 μm nbt (sigma (st. louis, mo.)), 0.1 mm edta, 2 μm riboflavin (sigma (st. louis, mo.)) and 0.02 ml of the enzyme extract. it was kept under a fluorescent lamp for 30 min, and then the absorbance was read at 560 nm. one unit of the sod activity is defined as the amount of enzyme required to inhibit the reduction of nbt by 50%. the activity of the enzyme is expressed as u per 1 mg of protein (u mg–1 protein). values of the biochemical parameters were expressed as means ± standard error (se) of determinations made in triplicates and tested by anova followed by comparison of the means by the duncan multiple range test (p < 0.05). data were analyzed using statistica for windows, version 11.0. the results are presented as histograms (values marked with different letter differ significantly at p < 0.05). results and discussion in the leaves of bromus seedlings, the significant increase in activity of cat was detected in the treatment with lower concentrations of the s. sclarea aqueous extract 120 h after the treatment (fig. 1). the highest activity of the sod was observed in plants 120 h after the treatment with 0.2% s. sclarea aqueous extract (fig. 2). in the roots of bromus, the significant increase in cat activity was recorded 72 and 120 h after the treatment (fig. 1). the activity of sod was significantly increased 72 and 120 h after the treatments with s. sclarea aqueous extract (fig. 2). numerous growth inhibitors identified in some plants are responsible for their allelopathic properties and may be a useful source for the future development of bioherbicides (x u a n et al., 2005). in the research of s h a r m a & s a t s a n g i (2013) fig. 1. activity of cat in leaves and roots of bromus seedlings 24, 72 and 120 h after the treatment with different concentrations (%) of s. sclarea aqueous extracts (v/v) and in control (c). fig. 2. activity of sod in leaves and roots of bromus seedlings 24, 72 and 120 h after the treatment with different concentrations (%) of s. sclarea aqueous extracts (v/v) and in control (c) 0 5 10 15 20 25 30 c 0.1 0.2 c 0.1 0.2 c 0.1 0.2 24 h 72 h 120 h u /m g p ro te in s extract concentration (%) leaves roots a a a,b a,b a a c b c b c a a b c a a,b c 0 10 20 30 40 50 60 70 80 c 0.1 0.2 c 0.1 0.2 c 0.1 0.2 24 h 72 h 120 h u /m g p ro te in s extract concentration (%) leaves roots a b b c b b c b a a a,b c b c c b c c biologica nyssana 7 (2)  december 2016: 141-144 šućur, j. et al.  the effect of satureja montana l. aqueous… 144 the aqueous leaf extract of the sunflower (helianthus annuus l.) showed an inhibitory effect on germination and growth of parthenium hysterophorus. this negative effect came from allelochemicals present in the aqueous extract of the sunflower. h u s s a i n & r e i g o s a (2011) reported that allelochemical cinnamic acid, widespread phenolic acid, was phytotoxic to c3 perennial plant species (dactylis glomerata, lolium perenne and rumex acetosa). the toxicity of the extracts varied with the concentration and soaking periods. the extract with higher concentration and longer soaking periods were more inhibitory then lower (h u s n a et al., 2016). further, lower concentrations of plant’s aqueous extracts could be more inhibitory in sterile soil than in nonsterile soil (b a r n e s & p u t n a m , 1986). our results showed that both concentrations of s. sclarea extract used (0.1% and 0.2%), stimulated the significant increase of the superoxide dismutase activity in leaves and roots of bromus 72 and 120 h after the treatment. the two tested extract concentrations affected the activity of the catalase in the bromus roots in the same way, but a higher activity was observed in the treatment with the higher concentration of the s. sclarea aqueous extracts (0.2%). the increases in the activities of antioxidant enzymes probably occur in response to stress induced by s. sclarea aqueous extract. conclusion in conclusion, our results showed that s. sclarea aqueous extract stimulates an increase of the catalase activity in the roots of bromus seedlings while both tested concentrations caused a significant increase of the superoxide dismutase activity in bromus leaves and roots. the results indicate that the plant extract possesses allelopathic activity and increases in activity of antioxidant enzymes probably occur in response to stress. acknowledgements. this study was carried out within a project of the ministry of education, science and technological development, republic of serbia, grant no tr-31022. references barnes, j.p., putnam, a.r. 1986: evidence for allelopathy by residues and aqueous extracts of rye (secale 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exploiting plant allelopathy: an overview. crop protection, 24: 197–206. jakšić et al., 2019, biologica nyssana 10(1) 10 (1) september 2019: 43-48 doi: 10.5281/zenodo.3464006 further notes on zygaenidae (lepidoptera) from montenegro original article predrag jakšić čingrijina 14/25, zvezdara, 11000 beograd, serbia jaksicpredrag@gmail.com gerhard m. tarmann tiroler landesmuseen, ferdinandeum, naturwissenschaftliche abteilung, sammlungsund forschungszentrum, krajnc-straße 1, 6060 hall, austria g.tarmann@tiroler-landesmuseen.at ana nahirnić national museum of natural history, tsar osvoboditel blvd. 1, 1000 sofia, bulgaria ananahirnic@nmnhs.com (corresponding author) received: april 23, 2019 revised: july 17, 2019 accepted: august 13, 2019 abstract: faunistic data on 11 species of zygaenidae from seven pre-selected research areas in montenegro obtained during several trips in the year 2017 are presented. photographs of adults of jordanita notata, zygaena punctum, z. viciae, z. ephialtes and z. filipendulae are shown. interspecific mating attempts by males of z. filipendulae with females of z. ephialtes and z. viciae are discussed. key words: zygaenidae, interspecific mating, montenegro apstract: novi podaci o ziganidama (insecta, zygaenidae) crne gore predstavljeni su faunistički podaci o 11 vrsta zygaenidae iz sedam prethodno odabranih i istraženih područja u crnoj gori, dobijeni tokom više obilazaka terena 2017. godine. prikazane su fotografije adulta vrsta jordanita notate, zygaena punctum, z. viciae, z. ephialtes i z. filipendulae. diskutovani su pokušaji interspecijskog parenja mužjaka z. filipendulae sa ženkama z. ephialtes i z. viciae. ključne reči: zygaenidae, interspecijsko parenje, crna gora introduction as montenegro intends to become a member of the eu, data about regional species distributions are of great importance, specifically as regards annex ii species of the habitats directive. on the other hand, species of the lepidopterous family zygaenidae are excellent indicators of environmental conditions. during the last decades more than 300 papers were published dealing with the moths and butterflies of montenegro (jakšić & nahirnić, 2017). according to nahirnić & tarmann (2014) there are about 30 publications known which contain data about zygaenidae for montenegro. in 2017 & 2018 the first author had the opportunity to participate in the ipa project ‘establishment of natura 2000 network, montenegro’. some of the results are presented in this paper. materials and methods specimens were collected with a butterfly net. research was done in preselected areas (key biodiversity areas kbas) some of which should be proposed as potential natura 2000 sites. the coordinates of the localities where zygaenidae were collected were determined by using garmin e-trex vista gps device (tab. 1). the photos in situ of specimens were taken using nikon camera with af-s micro nikkor lens. after preparation, we determined the specimens on the base of habitus and male genitalia. the preparations were carried out following the well-known standard procedure: maceration by boiling in potash, dissecting and cleaning, clearing in xylolum and mounting in canada balsam. abdomen and genitalia of zygaena purpuralis were mounted in euparal on glass slide or were after cleaning placed in glycerol © 2019 jakšić et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 43 filled microvials. all the material is deposited in the collection of p. jakšić. the taxonomic order follows nieukerken et al. (2011). results zygaenoidea latreille, 1809 zygaenidae latreille, 1809 subfam. procridinae boisduval, 1828 jordanita notata (zeller, 1847) podgorica, cijevna (restaurant “niagara”), 38 m, 9. may 2017, 1♂, jakšić p. leg. et coll. genitalia checked, slide: cg-2871. (fig. 1); ćemovsko polje, 40 m, 2. june, 2017, 2♀, jakšić p. leg et coll. third record for montengro! so far, this species was only mentioned once by jakšić (1990) on durmitor (crno jezero, čeline). it has been recollected on durmitor in 2015 (tepca, tara river, 6. june 2015; leg. p. jakšić, 1♂, genitalia checked, slide: cg 2684). an early flying green “forester moth” (= procridinae). the preferred habitats are semidry and dry grassy places with centaurea-species, the larval host-plants. this species is not common but widespread throughout southern europe and can be found from the sea shore up to lower mountain meadows. adscita geryon (hübner, 1813) durmitor mt., žabljak, meždo, 1376 m, 21 july, 2017, 1♂, jakšić p. leg et col., genitalia checked: slide cg-2912. a widespread species that is most common in higher elevations. the preferred habitats are open mountain grassland, grassy ravines, often on steep, rocky ground and semidry and dry meadows. the larvae feed on helianthemum spp. (cistaceae). adscita mannii (lederer, 1853) bukovica, in the vicinity of tušina, 999 m, 21 july 2017, 1♂, jakšić p. leg. et coll. genitalia checked, slide: cg-2906; bistrica, đalovića klisura gorge, 780 m, 20 july, 2017, 1♀, jakšić p. leg et col. gen44 habitats and localities elevation (m) coordinate latitudeφ (n) longitude λ (e) 4070 bushes with pinus mugo and rhododendron hirsutum (mugo-rhododendretum hirsuti) prutaš, durmitor mt. 2262 43° 07' 47,3'' 19° 00' 04,9'' veliki štuoc, durmitor mt. 1880 43° 11' 28'' 19° 03' 25'' 6210 semi-natural dry grasslands and scrubland facies on calcareous substrates (festuco-brometalia) meždo, žabljak, durmitor mt. 1376 43° 09' 54,8'' 19° 09' 02,0'' 6520 mountain hay meadows tepca village, tara river canyon 880-920 43° 12' 19'' 19° 04' 37'' 8210 calcareous rocky slopes with chasmophytic vegetation todorov do, durmitor mt. 1820 – 1900 43° 07' 40'' 18° 59' 15'' 9110 luzulo-fagetum beech forests (meadow on forest edge) monastery st. nikola, bistrica, đalovića klisura 780 43° 04' 14'' 19° 54' 12'' vrelo bukovice, durmitor mt. 1350 43° 03' 28,8'' 19° 06' 37,4'' bukovica, in the vicinity of tušina village 999 42° 57' 07'' 19° 10' 19,7'' sušičko jezero lake sušica river canyon 1180 43° 11' 46'' 19° 00' 18'' komarnica river, before nevidio canyon 981 42° 59' 17'' 19° 04' 02'' 9280 quercus frainetto woods cijevna river, podgorica (restaurant “niagara”) 38 42° 22' 50'' 19° 16' 28'' 9530 (sub-) mediterranean pine forests with endemic black pines sušičko jezero, sušica river canyon, mala crna gora 1554 43° 11' 45,8'' 19° 00' 18,5'' 6410 molinia meadows on calcareous, peaty or clayeysilt-laden soils (molinion caeruleae) veliko pošćensko jezero, pošćenska jezera lakes 1010 42° 59' 03'' 19° 04' 04'' table 1. list of annex i habitat types according to the “catalogue of habitat types of eu importance of montenegro” (petrović et al., 2012) and collecting localities in montenegro 2017 biologica nyssana ● 10 (1) september 2019: 43-48 jakšić et al. ● further notes on zygaenidae (lepidoptera) from montenegro 45 fig. 1. jordanita notata (zeller, 1847), male, podgorica, cijevna (restaurant “niagara”), 38 m, 9. may 2017, photo p. jakšić biologica nyssana ● 10 (1) september 2019: 43-48 jakšić et al. ● further notes on zygaenidae (lepidoptera) from montenegro italia checked, slide: cg-2913. this adriato-mediterranean species is widespread along the adriatic coast inhabiting various types of biotopes like mediterranean bushland near the sea, semidry and dry meadows, forest clearings and rocky ground with grassy spots between the rocks. in montenegro this species occurs at the coast and in the mountains. the larvae feed on helianthemumand cistus-species (cistaceae). only a few records are known for this species in montenegro but it is expected to be more widespread. subfam. zygaeninae latreille, 1809 zygaena punctum ochsenheimer, 1808 podgorica, cijevna (restaurant “niagara”), 38 m, 9. may 2017, 4♂, 2♀, jakšić p. leg. et coll. (fig. 2). all populations of zygaena punctum in montenegro belong to ssp. dalmatina boisduval, 1834. this population group is distributed from southern croatia and western bosnia and herzegovina southwards as far as greece. one of the main characters of this subspecies is its small size (compared with the nominotypical z. punctum punctum from eastern central europe) and the tendency to confluence of the red pattern on the forewing upperside. the larva of z. punctum feeds on eryngium species, mainly on e. campestre (apiaceae). the preferred habitats are dry, undisturbed meadows, rocky slopes with grassy spots and abandoned cultures with low or medium high vegetation. zygaena purpuralis (brünnich, 1763) bukovica near tušina village, 900 m, 6. june, 2017, 2♀, jakšić p. leg et col.; meždo, žabljak, 1376 m, 21-24. july 2017, 2♂, 1♀, jakšić p. leg. et coll.; veliki štuoc, 1880 m, 22. july 2017, 1♂, 1♀, jakšić p. leg et col.; mala crna gora, right bank of sušica river canyon, 1554 m, 22. july, 2017, 2♂, 5♀, jakšić p. leg et coll., genitalia checked: glycerin and slide anz573♀; sušičko jezero (sušica lake), 1180 m, 22. july 2017, 3♂, 2♀, jakšić p. leg. et coll.; todorov do, 1900 m, 23. july, 2017, 2♂, 2♀, jakšić p. leg et coll., genitalia checked: glycerin and slide anz574♂; prutaš, 2262 m, 22. july 2017, 1♂, 1♀, jakšić p. leg et coll. in montenegro only z. purpuralis lathyri boisduval, 1828 can be found. it inhabits various biotopes in hilly and mountainous areas. zygaena minos ([denis & schiffermüller], 1775), a species which is indistinguishable from z. purpuralis by habitus of imago, occurs in montenegro as well (nahirnić et al., 2013). correct determination of these species must be based on genitalia or larvae (nahirnić & tarmann, 2016). fig. 2. zygaena punctum ochsenheimer, 1808, female, podgorica, cijevna, 38 m, 9. may 2017, photo p. jakšić 46 zygaena carniolica (scopoli, 1763) meždo, žabljak, 1376 m, 21-24. july 2017, 3♂, 2♀, jakšić p. leg. et coll.; tepca village, 1260 m, mt., 22. july 2017, 6♂, 2♀, jakšić p. leg. et coll.; komarnica: nevidio and pošćenska jezera, 1010 m, 24. july 2017, 3♂, 2♀, jakšić p. leg. et coll.; vrelo bukovice, 1350 m, 24. july 2017, 3♂, 1♀, jakšić p. leg. et coll. the populations of z. carniolica of montenegro belong to the nominotypical subspecies z. carniolica carniolica. this population group is distributed in the balkans from the south-eastern alps and the karst areas in slovenia throughout the dinaric arc to northern albania. the larva feeds mainly on onobrychis spp. (fabaceae). the preferred habitats are undisturbed and unfertilized semidry and dry meadows. this species is known as a perfect indicator for air pollution by pesticides. it has disappeared in many agricultural and industrial areas in europe where it was still common half a century ago (tarmann, 2009). zygaena viciae ([denis & schiffermüller], 1775) bukovica, in the vicinity of tušina, 999 m, 21. july 2017, 1♀ in copula with ♂ of z. filipendulae, jakšić p. leg. et coll. (fig. 5); veliki štuoc mt., 1880 m, 22. july 2017, 3♂, jakšić p. leg. et coll. all populations of z. viciae in montenegro belong to ssp. bosniensis reiss, 1922. this subspecies differs from the nominotypical z. viciae viciae from central europe by its much darker appearance and the broad dark margin on the hindwing. zygaena viciae has so far only been found in the mountains of montenegro around durmitor. its preferred habitats are humid meadows. the larval host-plants in europe are fabaceae (preferably vicia and lathyrus species) but nothing is known on the life history of the populations of montenegro up to this date. zygaena ephialtes (linnaeus, 1767) monastery st. nikola, bistrica, đalovića klisura, 780 m, 20. july 2017, 12. ♂, 7♀, jakšić p. leg. et coll.; bukovica, in the vicinity of tušina, 999 m, 21. july 2017, 12♂, 4♀, jakšić p. leg. et coll.; 1554 m, 22. july 2017, 9♂, 2♀, jakšić p. leg. et coll.; komarnica: nevidio, pošćenska jezera, 1010 m, 24. july 2017, 4♂, 3♀, jakšić p. leg. the specimens of zygaena ephialtes collected during this project belong to ssp. istoki silbernagel, 1944. this subspecies, consisting of black ephialtoid morphs only, with a mixture of red and yellow individuals that can be 5or 6-spotted, is distributed throughout large parts of the southern balkans from bosnia and herzegovina and southern serbia to greece and eastwards to the black sea. there is another subspecies of z. ephialtes known from montenegro, viz. z. ephialtes rauchi hofmann, 2003. this population group is restricted to the surroundings of the bay of kotor and the coastal areas of montenegro. it is significantly different from ssp. istoki (see hofmann, 2003). zygaena ephialtes prefers bushy habitats with presence of the larval hostplant securigera varia l. (lassen) (fabaceae) and good nectar resources for the adults. it is often found on clearings, near forest roads and in river valleys with flowering rubus bushes where the adults obtain nectar. zygaena angelicae ochsenheimer, 1808 bistrica, đalovića klisura gorge, 780 m, 20. july, 2017, 1♀, jakšić p. leg et coll., durmitor mt., todorov do, 1900 m, 23. july 2017, 1♀, jakšić p. leg et col. a widespread species on the whole balkans from lowland to elevations above 2000 meters. larvae on fabaceae, mainly on hippocrepis comosa l. this species is represented in montenegro by its subspecies z. angelicae herzegowinensis reiss, 1922 that inhabits the whole mountainous part of the balkans from slovenia to greece and the black sea. zygaena filipendulae (linnaeus, 1758) bukovica, in the vicinity of tušina, 999 m, 21. july 2017, 4♂, 3♀ , jakšić p. leg.; meždo, žabljak, 1376 m, 21-24. july 2017, 6♂, 3♀, jakšić p. leg. et coll.; 1554 m, 22. july 2017, 8♂, 4♀., jakšić p. leg. et coll.; vrelo bukovice, 1350 m, 24. july 2017, 1♂, 1♀, jakšić p. leg. et coll. zygaena filipendulae is common on the whole balkans and occurs here in a number of different subspecies. montenegro is inhabited by z. filipendulae illyrica holik, 1943. this species has a wider ecological tolerance than most other zygaena species and can inhabit various biotopes from wetlands to dry environment, open meadows to forest clearings and roadside habitats. it can be observed in the lowlands but also in the mountains up to more than 2000 meters. the larvae live on various fabaceae, mainly on lotus species. zygaena lonicerae (scheven, 1777) monastery st. nikola, bistrica, đalovića klisura, 780 m, 20. july 2017, 4♂, 1♀, jakšić p. leg. et coll.; mala crna gora, right edge of sušica river canyon, 1554 m, 21. july, 2017, 1♂, jakšić p. leg et coll. zygaena lonicerae is widespread and common in the balkans and in montenegro. its preferred habitats are meadows near forest or forest clearings. the larvae feed on lotus and trifolium (fabaceae). biologica nyssana ● 10 (1) september 2019: 43-48 jakšić et al. ● further notes on zygaenidae (lepidoptera) from montenegro 47 fig. 4. interspecific mating attempt by male of zygaena filipendulae and female of z. ephialtes in the wild. bukovica river, near tušina village, 1006 m, 21. july 2017. photo p. jakšić fig. 3. zygaena filipendulae male x z. viciae female, found in copula on durmitor mt., veliki štuoc, 1930 m, 22. july, 2017. jakšić p. leg. fig. 5. interspecific mating attempt by male of zygaena filipendulae and female of z. ephialtes in the wild. mala crna gora village, right bank of sušica river canyon, 1554 m, 22. july 2017. photo p. jakšić. discussion and conclusion in all seven pre-selected research areas zygaenidae species could be found. this is an important proof that these biotopes are in a good environmental condition. as stated by šašić, nahirnić & tarmann (2016), zygaenidae are extremely stenoecious insects and are therefore good indicator species for environmental changes. they can show us perfectly whether a biotope is in a good‚ natural condition or somehow disturbed or even contaminated. special studies on this topic have been undertaken in northern italy in val venosta (vinschgau) (huemer & tarmann, 2001; tarmann, 2000, 2009, 2016). it has been shown that destructive poisons from apple plantations (some of them commonly used plant protection substances) were transported by wind into the environment over kilometres into earlier habitats and that zygaenidae were amongst the first insects that disappeared from seemingly completely intact habitats. several of such habitats are annex i habitat types according to the habitats directive. more intensive studies on montenegrin zygaebiologica nyssana ● 10 (1) september 2019: 43-48 jakšić et al. ● further notes on zygaenidae (lepidoptera) from montenegro 48 nahirnić, a., tarmann, g.m. 2016: on the early stages of species of the zygaena purpuralis – complex on the balkan peninsula and adjacent regions (zygaenidae, zygaeninae). xv international symposium on zygaenidae, mals/malles, südtirol/alto adige, italy: 30. nahirnić, a., tarmann, g.m., jakšić, p. 2011: a review of faunistical data on zygaenidae (lepidoptera) in the central-western balkan peninsula. in: bulić z. (ed.), international conference, nature protection in xxi century, proceedings of the conference, thematic sessions, abstracts, poster presentations. book no 2: 451–455. nahirnić, a., tarmann, g.m., jakšić, p. 2013: new data on rare zygaenidae from the balkan peninsula. xviii european congress of lepidopterology, blagoevgrad, bulgaria: 58. petrović, d., hadžiablahović, s., vuksanović, s., mačić, v., lakušić, d. 2012: katalog tipova staništa crne gore značajnih za evropsku uniju. podgorica – beograd zagreb. 116 p. šašić, m., nahirnić, a., tarmann, m.g, 2016: zygaenidae (lepidoptera) in the lepidoptera collections of the croatian natural history museum. natura croatica, 25(2): 233-248. tarmann, g. 1999: tagfalter und widderchen in südtiroler wiesen. ein fallbeispiel einer schleichenden umweltkatastrophe. in gepp, j., entomologische forschung in den alpen, öeg-fachgespräch 16. oktober 1999, dornbirn. kurzfassung der vorträge: 15–16. tarmann, g.m. 2009: die vinschger trockenrasen – ein zustandsbericht auf basis der bioindikatoren tagfalter und widderchen (lepidoptera: rhopalocera, zygaenidae). wissenschaftliches jahrbuch der tiroler landesmuseen, 2: 306–350. tarmann, g.m. 2016: the decline of zygaenidae in the valleys of the alps during the last 100 years, p. 39. in: tarmann, g. m., tremewan, w. g. & spalding, a. (eds), abstracts of the xv international symposium on zygaenidae, mals, 11–18 september 2016: i–xx, 1–47. tremewan, w.g. 2006: ecology, phenotypes and the mendelian genetics of burnet moths (zygaena fabricius, 1775). gem publishing company, wallingford. 190 p. nidae and especially their ecology and biology are needed as there is little known about their exact distribution in the country, local larval food plants, their nectar resources, preferred habitat requirements, parasites and predators. during this study three interspecific copulae could be observed. this is a well-known phenomenon in zygaenidae and has been studied by various authors. the reason is a somehow unspecific pheromone system that leads to the attraction of males of various species by females. however, hybrids can only be produced between extremely closely related species. such hybrids are known only for a few species. they can not only be produced by artificial rearing experiments but are also found in nature. the species combinations found in copula in montenegro in 2017 (figs. 3-5) cannot produce fertilized eggs as they are not very closely related species. in his monographic study, tremewan (2006) provided data for 200 literature sources that provide examples of this phenomenon. in these data 73 different combinations of species are listed. references hofmann, a. 2003: zygaena (zygaena) ephialtes (linnaeus, 1767) in südlichen balkan peninsula nebst anmerkungen zur entstehung von polymorphismus sowie melanistischer zygaena-formen im mittelmeerraum (lepidoptera: zygaenidae). entomologische zeitschrift mit insekten-börse, 2: 50-54; 3: 75-86; 4: 108-120. huemer, p., tarmann, g. 2001: artenvielfalt und bewirtschaftungsintensität: problemanalyse am beispiel der schmetterlinge auf wiesen und weiden südtirols. gredleriana, 1: 331–418. jakšić, p. 1990: zygaenidae (insecta, lepidoptera). in: nonveiller, g. (ed.), fauna durmitora. crnogorska akademija nauka i umjetnosti, titograd 3: 203– 232. jakšić, p., nahirnić, a. 2017: istorijat lepidopteroloških (insecta, lepidoptera) istraživanja u crnoj gori sa bibliografijom. tokovi, 37–81. nahirnić, a., tarmann, g.m. 2014: zygaena brizae (esper, 1800), a new species for montenegro (lepidoptera: zygaenidae). acta entomologica serbica, 19(1/2), 79-82. biologica nyssana ● 10 (1) september 2019: 43-48 jakšić et al. ● further notes on zygaenidae (lepidoptera) from montenegro zaaboubi et al. 2025, biologica nyssana 16(2) 16 (2) december 2025: doi: 10.46793/biolnyss.16.2.2z sustainable management of the codling moth cydia pomonella (lepidoptera: tortricidae) using sugar in northeast of algeria original article siham zaaboubi institute of veterinary and agronomic sciences, department of agronomy, 05000 batna, algeria; lapapeza laboratory, department of agricultural sciences, institute of veterinary and agricultural sciences, university of batna 1, algeria imene brahim institute of veterinary and agronomic sciences, department of agronomy, 05000 batna, algeria; lapapeza laboratory, department of agricultural sciences, institute of veterinary and agricultural sciences, university of batna 1, algeria imene.brahim@univ-batna.dz (corresponding author) abdelghani djerah institute of veterinary and agronomic sciences, department of agronomy, 05000 batna, algeria; lapapeza laboratory, department of agricultural sciences, institute of veterinary and agricultural sciences, university of batna 1, algeria smail chafaa department of ecology and environment, laboratory of cellular and molecular physiotoxicologybiomolecules, faculty of science of nature and life, university of batna 2, algeria fateh mimeche department of agricultural sciences, university of m’sila, algeria iness brahim iness brahim, barika, directorate of agricultural services, subdivision of agriculture, batna received: april 16, 2025 revised: july 14, 2025 accepted: july 14, 2025 abstract: the research aimed to test whether foliar sugar sprays are effective in promoting anna apple tree resistance to the codling moth (cydia pomonella l.), a key pest in algeria. the sucrose, fructose, and blend mixture (100 ppm) treatments at low concentrations were evaluated in an experiment conducted in batna from 2017 to 2019. treatments were applied 21 days apart, starting after flowering and continuing until harvest, to stimulate trees’ innate immunity through “sugar immunity.” results indicated a significant reduction in infestation levels, from 42.62% to 12.24%, with sucrose alone being the most efficient (11.57%–14.91%). reductions were also obtained with fructose and the mixture. infestation levels were lower in 2018 and 2019 compared to 2017, which is attributed to climatic factors. the study emphasizes the effects of climate change on pest ecology and encourages the use of sugar-induced immunity rather than chemical insecticides, recognizing the need to carefully manage application times and dosages amid global warming challenges key words: anna variety, biocontrol, enhanced defence, infestation rate, sugar signalization apstrakt: održivo suzbijanje jabukinog smotavca cydia pomonella (lepidoptera: tortricidae) primenom šećera u severoistočnom alžiru istraživanje je imalo za cilj da ispita da li folijarne aplikacije šećera mogu da povećaju otpornost stabala jabuke sorte anna na jabukinog smotavca (cydia pomonella l.), jednog od glavnih štetočina u alžiru. tretmani saharozom, fruktozom i njihovom mešavinom (100 ppm) pri niskim koncentracijama ispitani su u ogledu sprovedenom u batni od 2017. do 2019. godine. tretmani su primenjivani na svakih 21 dan, počev od završetka cvetanja pa sve do berbe, u cilju stimulacije urođene imunosti stabala putem „šećerne imunosti“. rezultati su pokazali značajno smanjenje nivoa infestacije, sa 42,62% na 12,24%, pri čemu je saharoza pokazala najveću efikasnost (11,57%– 14,91%). smanjenje infestacije zabeleženo je i kod fruktoze i mešavine. niži nivoi infestacije u 2018. i 2019. godini u poređenju sa 2017. pripisuju se klimatskim faktorima. studija ističe uticaj klimatskih promena na ekologiju štetočina i podstiče upotrebu imunosti indukovane šećerom umesto hemijskih insekticida, uz naglašavanje potrebe za pažljivim upravljanjem vremenom i dozama primene u uslovima globalnog zagrevanja. ključne reči: sorta anna, biološka kontrola, pojačana odbrana, stopa infestacije, šećerna signalizacija introduction the domesticated apple tree, malus domestica borkh., is one of the most cultivated fruit species in the world, second only to citrus in demand and third only to bananas and grapes in production, with a production of 87.82 million tons in 2019 (fao, 2021). apple cultivation in algeria covered an area of 32,989 hectares in 2019, yielding an output of 5,588,300 quintals (fao, 2021). apple cultivation around the batna region was increased from 3,288.27 hectares in 2009, to 4,537.01 hectares during 2019, whereas production increased from 167,094 to 1,012,240 quintals over the same period (dsa, 2020). from nearly 6,000 cultivated apple varieties, six make up 90% of the national production in algeria (madr, 2019). despite the expansion of cultivated areas, pro© 2025 zaaboubi et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. duction levels have remained low due to factors such as cultivation practices, orchard age, fertilization practices, soil management, and pest control. apple trees are affected by numerous diseases and insect pests, among which the codling moth (cydia pomonella l.) is the most damaging pest of apples worldwide (boivin et al., 2001; 2003; mcguffin et al., 2014; reyes et al., 2015). if left unchecked, codling moth can cause yield loss of up to 100% (beers et al., 2003; sauphanor et al., 2009). the larvae damage the fruit by boring into it, resulting in the loss of marketable apples, and even more than 1% damage is considered unacceptable in commercial orchards (benoît et al., 2009; belkair, 2018). fruit growers are forced to treat their orchards annually for pest management and rely solely on chemical management with 7 to 8 applications per year on average. the overuse of insecticides has led to immense loss of biodiversity of beneficial insects (ismail & albittar, 2016). further, synthetic pesticides have been found to be responsible for neurological diseases, and it has become a concern whether they affect health or contribute to environmental problems (baldi et al., 2013; choinard et al., 2016). therefore, it is a critical necessity to develop management strategies that provide sustainable, biocompatible, and effective alternatives for chemical pest control. innovative methods for stimulating the plant’s natural defenses are also being developed. although this view has become quite popular, researchers have chosen the path of indirect defense by either empowering plants to defend themselves or changing the plant’s own defense processes against the aggressor. an elicitor representing the newly explored and promising avenue is sucrose, which acts in the insecticidal action by stimulating plant immunity and defense mechanisms (bolouri moghaddam & van den ende, 2013; itab, 2018). the use of these water-soluble plantderived compounds (such as sugars) allows them to penetrate the leaf and serve as genuine signals that trigger a cascade of defensive reactions in the plant. furthermore, these molecules present in the plant can cross the cuticle and, at the plant’s surface, act as signals perceived by insects through contact, thereby influencing their behavior and host plant selection for oviposition (derridj et al., 2011a). the concept of “sweet immunity” or “sugar-enhanced defense” is based on accumulating evidence that endogenous sugars may function as signaling molecules activated by stress exposure, thereby triggering a more rapid and robust amplification of defense mechanisms, immunity, and stress tolerance (tarkowski et al., 2019). in recent years, sugars like glucose, fructose, and sucrose have developed several significant regulatory roles and hence are increasingly recognized as signaling molecules in plants, modulating the expression of genes related to plant metabolism, resistance to stress, and development. this creates an opening for soft priming, a defined physiological process preparing plants for a quicker and/or stronger defense response to future stress conditions without incurring the costs of implementing a fully induced defense response (arnault et al., 2015; ceusters et al., 2017). this introduces a new concept of sugar-related defense (arnault et al., 2015). foliar spraying of low doses of sugars induces a response in the plant, resulting in increased resistance to biotic stressors. the application of sugars has shown promising results in apple and grapevine models (arnault et al., 2015; 2016; 2021). the objective of this study is to test the effect of ultra-low doses of commercial sugar sprays during the period from 2017 to 2019, in order to reduce attacks by the codling moth on apples and pears, as well as to decrease the number of c. pomonella larvae in an apple orchard of the anna variety, located in the tilatou region (batna province, algeria). materials and methods the experiment was conducted in an apple orchard (anna variety) located in the tilatou region (35°19′57.30″ n, 5°47′57.23″ e), about 45 km southwest of batna province, at an altitude of 900 meters above sea level. the study lasted three years, from 2017 to 2019. the region is characterized by an arid climate (q2=34.68), with average annual rainfall ranging from approximately 169 to 338.8 mm, and average temperatures from -1.5 °c to 38.0 °c. according to the orchard owner, codling moth infestations have been observed since 2013, with damage reaching an average of 80% in this untreated orchard. in addition to apple trees, the orchard includes plots of pear trees, apricot trees, olive trees, pomegranate trees, and a small number of fig trees. the experiment was conducted from 2017 to 2019, using a randomized complete block design (fisher) with four replications. the four treatments were randomly assigned within each block, and each block contained 12 trees. the four tested modalities and the doses applied for each modality are presented in table 1. it should be noted that the sugars used are sourced from cevital company (algeria). the choice of this dose is based on studies of sugar sprays (sucrose, fructose, glucose) applied to crops at concentrations ranging from 0.1 g to 10 g/100 l, targeting various bioaggressors such as the corn borer (ostrinia nubilalis hbn.), codling moth (c. pomonella l.), melon powdery mildew (oidium biologica nyssana ● 16 (2) december 2025: zaaboubi et al. ● sustainable management of the codling moth cydia pomonella (lepidoptera: tortricidae) using sugar in northeast of algeria biologica nyssana ● 16 (2) december 2025: zaaboubi et al. ● sustainable management of the codling moth cydia pomonella (lepidoptera: tortricidae) using sugar in northeast of algeria table 1. tested modalities and doses used year modalities active substance formula dose 2017-2019 1 control water / 2 commercial sucrose (succ) sugar 100 ppm 3 commercial frucrose (frucc) sugar 100 ppm 4 mixture (succ+ frucc) sugar 100 ppm +100 ppm neolycopersici l. kiss), and gray rot (botrytis cinerea pers.). these studies have shown greater activity at extremely low concentrations compared to those above 100 ppm. it should be noted that increasing the dose beyond 100 ppm (10 g per 100 l) does not enhance resistance induction effects and may even negate them (ferré, 2008; derridj et al., 2009; 2012). in addition, higher doses may cause side effects, such as altering insect feeding behavior and potentially affecting the epiphytic flora or the epiphytic phase of certain pathogens prior to contamination, as well as impacting bioaggressors on the plant surface (derridj et al., 2011a; arnault et al., 2016). applications began at the end of flowering (april) in all three blocks and were repeated every 21 days until harvest in august (ondet & gorski, 2015; arnault et al., 2016; 2021). this interval is based on trials conducted in france on two lepidopteran species (ostrinia nubilalis and cydia pomonella) (derridj et al., 2009; 2012; ondet & gorski, 2015; arnault et al., 2015; 2016; 2021). a total of six treatments were applied each year (table 2). treatments are conducted very late in the day, starting at 5:00 pm, because at that time the leaves naturally contain less sugar (the apoplast or intercellular space is poor), which facilitates penetration and modifies the balances on the surface of the plant, inducing systemic resistance through a “sugar” signal. the female codling moth poorly recognizes the treated plant and does not lay her eggs on it (derridj et al., 2011b). the treatments were applied using a 16 l backpack sprayer (main material: plastic; power source: pressure; type: hand compression; non-drip spraying) with four sprayers used, one for each modality, to avoid the risk of sugar contamination), attempting to spray the entire tree. spraying on the bark should also allow for the penetration of sugar into the plant (derridj et al., 2009). the solution was applied through foliar spraying in a non-dripping v-shaped jet across the entire plant to facilitate the absorption of sugar. the volume of water is suited for vegetative development to ensure proper wetting of the vegetation. the sugars were put into solution and dissolved just before pulverization (ferré et al., 2008). the observations focused on counting the fruits attacked by the codling moth and were conducted on the fruits in each elementary plot (the fruits examined on the tree as well as the fruits that fell to the ground). the fruits examination was conducted every 7 days until harvest. during the season, the fallen fruits under the identified trees were examined, and after each examination, these fruits were removed from the trial because the apples rot, and the damage caused by the codling moth can no longer be seen (derridj et al., 1999). for all the remaining fruits on the tree, the assessment was carried out at harvest. table 2. treatment dates treatment no. year 2017 2018 2019 1 13.04. 26.04. 13.04. 2 03.05. 06.05. 03.05. 3 23.01. 27.06. 23.05. 4 12.01. 17.07. 12.06. 5 02.11. 06.08. 04.07. 6 22.01. 10.08. 24.07. bands of corrugated cardboard (15 cm wide, 60 cm long) were placed in may of each study year on tree trunks to trap larvae (l5), males, and females coming down to the base of the tree to enter diapause. each band consisted of two superimposed sheets of corrugated cardboard covered with a polyethylene mesh. the three layers were secured with wire, and the mesh was designed to prevent birds from feeding on the larvae. the trap bands were positioned around the base of the experimental tree trunks, close to the ground. bands were removed at the end of october each year, coinciding with the end of the third generation of this pest in the area (brahim, 2010; srpv, 2017). all analyses were performed using statistica 08 software. for each modality, we compared the means of each variable, the overall mean infestation rate, average number of larvae captured, and the biologica nyssana ● 16 (2) december 2025: zaaboubi et al. ● sustainable management of the codling moth cydia pomonella (lepidoptera: tortricidae) using sugar in northeast of algeria abbott efficacy treatment. the abbott efficacy equation (1925) is: efficacy = 100 × [(to-tt)/to] where tₒ is the total percentage of damaged fruits in the control block, and tₜ is the total percentage of damaged fruits in the treated block. comparisons were made using analysis of variance (anova), followed by post hoc analysis with fisher’s lsd test. prior to analysis, the assumptions for using this test were verified, including the normality of distribution with the shapiro–wilk test. results infestation rate caused by c. pomonella the infestation rate caused by the codling moth in 2017 was higher compared to that recorded in 2018 and 2019, ranging from 42.62%±5.75 to 12.24%±2.14, regardless of the treatment modality tested on the anna variety (table 3). the attack rate in treated plots was reduced and appeared to be two to three times lower than that recorded in the control. among the treatments, the sacc (100 ppm) modality consistently showed the lowest infestation rate across all study years, with values ranging from 11.57%±2.02 to 14.91%±2.43, compared to the frucc modality at 100 ppm (13.46%±2.35 to 18.14%±2.93) and the mixture (100 ppm) modality, which recorded rates between 16.16%±2.77 and 21.57%±3.10. analysis of variance (anova) followed by fisher’s lsd test (p<0.05) for the average percentages of attacked and fallen fruits showed a highly significant difference for all three years studied (table 3). estimation of diapausing larvae the data analysis reveals several significant trends. sucrose showed a more pronounced inhibitory effect than fructose during 2017–2018, as evidenced by the values for males in 2017 (2.75±0.48 vs. 2.25±0.75, p<0.05) and females in 2018 (6.0±2.45 vs. 8.75±2.10, p<0.01). however, this difference was greatly diminished in 2019 (8.75±2.39 vs. 7.25±1.11, p>0.1). the sugar mixture had an intermediate profile, leaning more towards fructose in 2017 (2.0±0.91 vs. 2.25±0.75, p>0.05) and more towards sucrose in 2019 (10.25±1.65 vs. 8.75±2.39, p>0.05). the pupal stage appeared to be particularly sensitive, with almost complete inhibition under sucrose in 2017 (0±0 vs. 1.25±0.48, p<0.001) and a paradoxical effect of the mixture in 2018 (3.0±1.15 vs. 1.75±0.63, p<0.05). low standard errors (±0 to ±1.15) attest to the reproducibility of these observations (fig. 1). abbott efficacy treatments the results obtained for this parameter show a relatively moderate to high efficacy for all tested modalities on the anna variety, ranging from 21.87%±4.27 to 44.71%±7.60. it is noteworthy that all tested modalities achieved their highest effectiveness in 2017. among these, sacc (100 ppm) demonstrated the greatest efficacy, while the mixture modality (100 ppm) showed the lowest effectiveness over the three years of study (fig. 2). analysis of variance (anova) followed by fisher’s lsd test (p<0.05) for the total number of attacked fruits on the anna variety revealed a significant difference in effectiveness between the study years only in 2019 for the mixture modality (fig. 2). discussion the present study evaluated the efficacy of very low concentrations (100 ppm) of industrial sugars (sucrose, fructose, and their mixture) in providing rapid sugar-induced immunity against the codling moth through foliar treatments applied at 21day intervals from post-flowering to harvest over three growing seasons (2017, 2018, and 2019) in the tilatou area (batna, algeria). results showed significant year-to-year fluctuations in codling moth infestation intensity, which were strongly associated with certain climatic conditions and were more evident in 2018 and 2019 compared to 2017. infestation intensity declined considerably following the application of the test treatments, with medium to high efficacies (44.08% to 68%) compared to the control (p<0.05, anova followed by tukey’s test). the wide confidence intervals (±7.02 in 2017) indicate extreme variance. these results confirm anova, fisher’s lsd tests, p<0.05; different letters show significant differences between treatments within each experiment. ****: p≤0.0001 table 3. average infestation rate caused by larvae on apple fruit years control sucrose (100ppm) fructose (100ppm) mixture (100ppm) 2017 42.62%±5.75a 14.91%±2.43b**** 18.14% ± 2.93b**** 21.57 % ±3.10b**** 2018 34.09% ± 5.05a 11.57% ±2.02b**** 13.46% ± 2.35b**** 17.14% ± 2.80b**** 2019 25.54% ± 3.99a 12.24% ±2.14 b**** 13.84% ± 2.46b**** 16.16 % ± 2.77b**** biologica nyssana ● 16 (2) december 2025: zaaboubi et al. ● sustainable management of the codling moth cydia pomonella (lepidoptera: tortricidae) using sugar in northeast of algeria fig. 1. average number of captured larvae and pupae. anova, fisher’s lsd test, p<0.05; different letters indicate significant differences between treatments within each experiment (*p<0.05, **p<0.01) the viability of sugar immunity as an integrated pest control strategy, potentially reducing the use of chemical pesticides. in 2017, the region experienced a wet and chilly spring followed by a hot summer, with maximum temperatures exceeding 35 °c for 51 days, resulting in a codling moth infestation rate of 42.62%±5.75. spring rains likely created favorable conditions for larval development, while summer heat accelerated their life cycle. this supports the observations of hermans (2018), who stated that warmer and longer summers lead to higher pest pressure. in contrast, 2018 experienced moderate temperatures, with a maximum of 27 °c and only 44 days exceeding 35 °c, leading to a lower infestation rate of 34.09% ± 5.05. this finding is consistent with kleckova & klecka (2016), who reported that moderate temperatures limit pest reproduction. in 2019, the region recorded 71 days with temperatures above 35 °c, while spring rains occurred less frequently than in 2017, resulting in an intermediate infestation rate of 25.54%±3.99. this reflects the combined influence of higher temperatures and lower rainfall. additionally, milder winters can hinder fruit tree vernalization, making them more susceptible to pests and diseases, as noted by coureau (2020), who found that weather-stressed trees were more vulnerable to pests such as the codling moth. hermans (2018) also observed that climate change accelerates codling moth generations, thereby increasing infestation levels. the findings showed that sucrose was the most efficient treatment, reducing infestation rates to 11.57%–14.91%, followed by fructose (13.46%–18.14%) and the sugar blend (16.16%–21.57%). these results align with those of bounahouche (2017) and dafri (2017), who recorded high infestations of 77%–80% on another apple variety (royal gala) under similar conditions, further highlighting the influence of climatic and yearly variations on pest pressure. notably, the lowest infestation levels were observed in 2018 and 2019, while the conditions in 2017 favored codling moth development, leading to higher infestations. sugars applied as foliar sprays have a dual effect: enhancing plant defenses and disrupting codling moth larval feeding. cabbanat (1999) demonstrated complex interactions between sugars, such as a potential antagonistic effect in mixtures and a loss of specificity in 2019. this shows the possibility of sugar-based systems managing pest infestations as captures were significantly lower in treatment than control in 2017. this shows that the sugars could be affecting larval behavior or ability to pupate. this agrees with knight & light (2005), who showed that sugars affected codling moth larval movement and feeding and reduced survival. however, effectiveness of sugars treatment also appears to decline with years, as evidenced by the increase in captures for 2018 and 2019, either due to larval adaptation or environmental factors on the effectiveness of treatment. witzgall et al. (2008) have indicated a long-term trend towards reduced control operation effectiveness based on repellent or attraction regimes and claimed that an integrated approach would have to be employed to effectively manage codling moth infestations. the use of a blend of sugars in 2019 was effective with sub-control captures, demonstrating synergism between components, concurring with varela et al. (1993) that blends of components increase the efficiency of treatment by acting on different insect behavior. this research confirmed that sugar control was able to effectively suppress the population of codling moths but its efficiency is dependent on local condition and treatment duration. chouinard et al. (2016) further included that traps and bands supplemented by attractants or repellents such as that polyols and sugars affect c. pomonella larval feeding by inhibiting their ability to penetrate fruit. similarly, ondet (2019) reported reduced codling moth infestations on the akane variety in france following sugar applications. recent studies, such as ceusters et al. (2016), have examined sugar-induced immunity mechanisms, showing that sucrose and fructose activate defense genes and strengthen plant resistance. additionally, derridj et al. (2012) found that sugars disrupt insect perception of plants and their oviposition behavior, confirming their role as anti-phagostimulants that limit crop damage. the study also revealed a significant reduction in the number of codling moth larvae captured in corrugated cardboard trap bands treated with sucrose and fructose compared to the control. inhibitory activity analysis showed notable differences between sugars, with sucrose outperforming fructose in 2017 and 2018, as indicated by male counts in 2017 (2.75±0.48 vs. 2.25±0.75, p<0.05) and female counts in 2018 (6.0±2.45 vs. 8.75±2.10, p<0.01). however, this difference diminished in 2019 (8.75±2.39 vs. 7.25±1.11, p>0.1), indicating annual fluctuations in larval response. the sugar mixture showed an intermediate pattern—similar to fructose in 2017 (2.0±0.91 vs. 2.25±0.75, p>0.05) and to sucrose in 2019 (10.25±1.65 vs. 8.75±2.39, p>0.05). overall, statistical analyses confirmed significant inhibitory effects of sugar solutions (most p values <0.05), pronounced interannual variability requiring improved experimental controls, and biologica nyssana ● 16 (2) december 2025: zaaboubi et al. ● sustainable management of the codling moth cydia pomonella (lepidoptera: tortricidae) using sugar in northeast of algeria fig. 2. abbott efficacy of treatments. anova, fisher’s lsd test, p<0.05; different letters indicate significant differences between treatments within each experiment (*p<0.05) sugars monitor and control effectively orchard insect’s pests. these results illustrate the complexity of interaction between different types of sugars and their variation in effect from year to year, but together supporting the greater inhibitory effect of sucrose over fructose throughout the first two years of study. arnault et al. (2021) evaluated the effectiveness of biocontrol strategies, including sweet immunity, within an integrated pest management program. results of their evaluation show that the use of sugar in combination with other biocontrol strategies had the potential to effectively control codling moth infestations and reduce environmental degradation. the results of this study carry promising potential in the context of climate change, where pests become more resistant to conventional means of control, thereby making sustainable pest control possible. sweet immunity as a method of biocontrol is an efficient alternative to chemical pesticides and is safe for human health and the environment. conclusion this research demonstrates that treating apple trees (anna variety) with low doses of a sucrose and fructose mixture enhances their resistance to cydia pomonella, effectively reducing infestation rates and larval populations under dry conditions. the findings validate the feasibility of sugar-induced immunity as a green pest management strategy that helps conserve biodiversity. sucrose proved to be the most effective treatment and the most attractive to farmers, as it is cost-effective, easy to apply, and environmentally friendly. however, treatment efficacy is influenced by climatic conditions, underscoring the role of climate change in shaping pest dynamics. this approach offers a pathway toward integrated crop management strategies that combine induced resistance with sustainable agricultural practices to address present and future agricultural challenges. nevertheless, the observed interannual variability highlights the need to develop protocols tailored to local climatic conditions, which should be a priority for the large-scale adoption of this technique. references abbott, w. s. 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(2008). codling moth management and chemical ecology. annual review of entomology, 53(1), 503– 522. biologica nyssana ● 16 (2) december 2025: zaaboubi et al. ● sustainable management of the codling moth cydia pomonella (lepidoptera: tortricidae) using sugar in northeast of algeria trends in biological activity research of wild-growing aromatic plants from central balkans biologica nyssana 7 (2)  december 2016: 61-73 džamić, a. et al.  trends in biological activity research of wild… 61 review article received: 10 october 2016 revised: 02 november 2016 accepted: 07 november 2016 trends in biological activity research of wild-growing aromatic plants from central balkans ana m. džamić1*, jelena s. matejić2, petar d. marin1 1university of belgrade, faculty of biology, institute of botany and botanical garden “jevremovac”, 11.000 belgrade, serbia 2university of niš, faculty of medicine, 18.000 niš, serbia * e-mail: simicana@bio.bg.ac.rs abstract: džamić, a.m., matejić, j.s., marin, p.d.: trends in biological activity research of wild-growing aromatic plants from central balkans. biologica nyssana, 7 (2), december 2016: 61-73. flowering plants consists of more than 300.000 species around the world, out of which a small percentage has been sufficiently investigated from phytochemical and biological activity aspects. plant diversity of the balkans is very rich, but still poorly investigated. the aim of this paper is survey of current status and trends in research of wild-growing aromatic plants from central balkans. many aromatic plants are investigated from morphological, physiological, ecological, systematic and phytochemical aspects. however, traditionally used medicinal and aromatic plants can also be considered from applicative aspects, concerning their health effects, and from wide range of usage in cosmetics, and as food, agrochemical and pharmaceutical products. in order to achieve all planned objectives, following methodology has been applied: field research, taxonomic authentication and, comparative biologically assayed phytochemical investigations. the total herbal extracts, postdistillation waste (deodorized) extracts, essential oils and individual compounds of some autochthonous plants have been considered as potential source of antibacterial, antifungal, anti-biofilm, antioxidant and cytotoxic agents. in this manuscript, composition of essential oils and extracts were evaluated in a number of species, from the apiaceae, lamiaceae, rosaceae and asteraceae families. extracts which were rich in phenols mostly of flavonoids, often showed high antioxidant potential. also, phenolic compounds identified in essential oils and extracts were mostly responsible for expected antimicrobial activity. current worldwide demand is to reduce or, if possible, eliminate chemically synthesized food additives. plant-produced compounds are becoming of interest as a source of more effective and safe substances than synthetically produced antimicrobial agents (as inhibitors, growth reducers or even inactivators) that control growth of microorganisms. many different pathogens have developed resistance toward synthetic antibiotics and mycotics, so, there is a need for discovering a new antimicrobials. it is worth noting that synergistic effect of components found in essential oils or in various extracts may pay key role in its biological activities. key words: antibacterial, antifungal, antioxidant, cytotoxic activity, plant extracts, essential oil 7 (2) • december 2016: 61-73 12th sfses • 16-19 june 2016, kopaonik mt doi: 10.5281/zenodo.200401 biologica nyssana 7 (2)  december 2016: 61-73 džamić, a. et al.  trends in biological activity research of wild… 62 apstrakt: džamić, a.m., matejić, j.s., marin, p.d.: trendovi u istraživanjima biološke aktivnosti divljerastućih aromatičnih biljaka centralnog balkana. biologica nyssana, 7 (2), decembar 2016: 61-73. broj cvetnica u svetu se procenjuje na više od 300.000 vrsta, od kojih je mali procenat u potpunosti proučen sa fitohemijskog i sa aspekta biološke aktivnosti. biljni diverzitet balkana je bogat, ali je i dalje slabo proučen. cilj ovog rada je pregled trenutnog stanja i trendova u proučavanje samoniklih aromatičnih biljaka sa područja centralnog balkana. mnoge aromatične biljke su proučavane sa morfološkog, fiziološkog, ekološkog, sistematskog i fitohemijskog aspekta. međutim, lekovite i aromatične biljke koje se koriste u narodnoj medicini mogu se razmatrati i sa stanovišta njihove primenljivosti, imajući u vidu njihov efekat na zdravlje i široku upotrebu u kozmetici, kao hrane, i u agrohemijskim i farmaceutskim proizvodima. u cilju postizanja planiranih zadataka, primenjena je sledeća metodologija istraživanja: terenski rad, taksonomska autentifikacija i komparativna biološka i fitohemijska istraživanja. ukupni biljni ekstrakti, ekstrakti postdestilacionih ostataka (deodorisani), etarska ulja i pojedinačne komponente autohtonih biljka su razmatrani kao potencijalni izvori antibakterijskih, antifungalnih, anti-biofilm, antioksidativnih i citotoksičnih agensa. u ovom radu prikazan je hemijski sastav etraskih ulja i ekstrakata brojnih vrsta iz familija apiaceae, lamiaceae, rosaceae and asteraceae. ekstrakti bogati fenolima, naročito flavonoidima često poseduju visok antioksidativni potencijal. takođe, fenolne komponente idetifikovane u etarskim uljima i ekstraktima se smatraju odgovornim za antimikrobnu aktivnost. širom sveta se trenutno radi na smanjenju ili potpunom eliminisanju hemijski sintetisanih aditiva u hrani. biljni proizvodi su postali značajni kao izvor efikasnijih i bezbednih supstanci nego što su sintetički antimikrobni agensi koji kontrolišu rast mikroorganizama (inhibitori, regulatori rastenja ili inaktivatori rasta). mnogi različiti patogeni su postali rezistentni na sintetičke antibiotike i mikotike, pa je potrebno pronaći nove efikasne antimikrobne supstance. potrebno je istaći da sinergistički efekat između komponenti u etarskom ulji ili različitim ekstraktima može imati ključni efekat za njegovu biološku aktivnost. ključne reči: antibakterijska, antifungalna, antioksidativna, citotoksična aktivnost, biljni ekstrakti, etarska ulja introduction plant kingdom, with over 300.000 higher species, represents a source of new chemical agents for active pharmaceutical ingredients and lead compounds. only a small percent 5% to15% of these plants have been chemically and pharmacologically investigated. approximately 10.000 to 15.000 of the world’s plants have documented medicinal uses and roughly 150-200 have been incorporated in western medicine (k r a u s e & t o b i n , 2013). the balkan flora is not only the richest in europe, but comprises also many endemics. according to contemporary assessments, vascular flora of the balkans comprises almost 8.000 taxa (s t e v a n o v i ć et al., 2007). in flora of sebia 3.562 species and subspecies are recorded and among them 32.12% are protected (i g i ć et al., 2010). plants which contain bioactive compounds are often categorised as medicinal or poison plants, and their consumation should have adverse or beneficial result which often depends on the dose of taking. secondary metabolites are small organic molecules and specific biologically active compouds that are produced by plants. they pay important role in the function of plant organisms and are important for long-term survival and defence. nowadays, it is known that production of secondary metabolites are the rule rather than exception. secondary metabolites represent essential source for discovery of a novel compounds because of great diversity of their structures (b e r n h o f t , 2010). modern approach adopted to explore plant and its bioactive constituents involve interdisciplinary work in botany, pharmacognosy, chemistry and toxicology (h o s t e t t m a n n et al., 1995) and should take following steps: 1. selection, collection, botanical identification, preparation of plant material 2. extraction with suitable solvents and preliminary analysis 3. biological and pharmacological screening of crude extracts 4. chromatographic separation of pure bioactive constituents guided by bioassay 5. structure determination 6. analyses and pharmacological profile of pure compounds 7. toxicological testing if plants show no signs of being attacked by pests and neither has pieces eaten out of the leaves, there is a good chance that some metabolites are present which act as insecticides or antimicrobial agents. the evaluation of plant extracts to ensure efficacy and safety is followed by identification of active principles, dosage formulations, efficacy and pharmaco-kinetic profile of the new drug. many plants have been used because of their antibacterial, antifungal, anti-biofilm, antioxidant, cytotoxic, antiinflammatory, antidiabetic, anti-cholinesterase, antinociceptive, anti-proliferative, genotoxic, antigenotoxic, neuroprotective, herbicidal, enzyme biologica nyssana 7 (2)  december 2016: 61-73 džamić, a. et al.  trends in biological activity research of wild… 63 inhibition, anti-alzheimer properties and have been investigated by a number of researchers worldwide. ethnopharmacologists, botanists, microbiologists, natural product chemists, and many others are searching for phytochemicals which could be developed for some applications (d a s et al., 2010). it has to be pointed out that biological activity data for majority of chemically investigated wild growing plants are still missing (č a v a r z e l j k o v i ć & m a k s i m o v i ć , 2014). this study gives an overview of current status and trends in research of potential of wild-growing aromatic plants of different biological aspects and methods in vitro. extracts and essential oils plants are complex matrices, which produce a wide range of secondary metabolites with different functional groups and polarities. categories of natural products commonly encountered include waxes and fatty acids, polyacetylenes, terpenoids (e.g. monoterpenoids, iridoids, sesquiterpenoids, diterpenoids, triterpenoids), steroids, essential oils (lower terpenoids and phenylpropanoids), phenolics (simple phenolics, phenylpropanoids, flavonoids, tannins, anthocyanins, quinones, coumarins, lignans), alkaloids and glycosidic derivatives (e.g. saponins, cardiac glycosides, flavonoid glycosides). for preliminary investigation of biological properties the most important is to test complex mixture as total plant extracts. several approaches can be employed to extract the plant material. although water is used as an extractant in many traditional protocols, organic solvents of varying polarities are generally selected in modern methods of extraction to exploit a solubility of plant constituents. solvent extraction procedures applied for the initial extraction plant natural products include maceration, percolation, soxhlet extraction, pressurized solvent extraction, ultrasound assisted solvent extraction, and steam distillation. terpenoids and essential oils are secondary metabolites that are highly enriched in compounds based on an isoprene structure. the most abundant terpenes in essential oils are monoterpenes (c10) and sesquiterpenes (c15) while diterpenes (c20), as well as aliphatic hydrocarbons, acids, alcohols, aldehydes, acyclic esters or lactones (s e i d e l , 2006; b o h l i n et al., 2012) are often present. antimicrobial activity current standard antimicrobial methods approved by various organizations such as clinical and laboratory standards institute (clsi), and the european committee for antimicrobial susceptibility testing (eucast) exist, for guidelines of antimicrobial susceptibility testing of convenient drugs, these might not be exactly applicable to plant extracts and modifications have to be made. in general, antimicrobial methods are broadly classified into diffusion and dilution methods. diffusion tests include agar well diffusion, agar disk diffusion, poison food technique, and bioautography, while dilution methods include agar dilution, broth microdilution and broth macrodilution technique (d a s et al., 2010). agar well diffusion and disk diffusion have been applied in the first screening, where extract or test substance are placed into agar or applied on the filter disc. zone of inhibition have been measured. bioautography is also employed as a preliminary phytochemical screening technique, by bioassay guided fractionation, to detect active components. bioautography is a very convenient way of testing plant extracts and pure phytochemical compounds for their effect on both human pathogenic and plant pathogenic microorganisms. it can be employed in the target directed isolation of active constituents (c h o m a & g r z e l a k , 2011). however, the most preferred method is broth microdilution assay (or micro-well dilution assay), using microtitration plates, by making the serial twofold dilutions of test substances in corresponding medium. using this method, minimum inhibitory concentrations (mic) and minimum bactericidal/fungicidal concentrations (mbc/mfc) are recorded. minimal inhibitory concentration (mic) is defined as the lowest concentration of the samples inhibiting visible growth. minimal bactericidal/fungicidal concentration (mbc/mfc) is defined as the lowest sample’s concentration that kills 99.9% of bacterial/fungal cells. the main advantages of broth methods, especially microdilution which is carried out in microtiter trays are lower workloads for a larger number of replicates and the use of small volumes of the test substance and growth medium. in this method dilution of the oil is better and there is no agar in the medium, which both enable better diffusion through the liquid medium (s o k o v i ć et al., 2011). antimicrobial activity of wild growing apiaceae species the in vitro antimicrobial activity of essential oils and various extracts of wild growing apiaceae has been investigated by the microdilution method, where a panel of pathogenic bacteria and microfungi was tested (m i l o s a v l j a v i ć et al., 2007; s o k o v i ć et al., 2009; s t o j k o v i ć et al., 2009; biologica nyssana 7 (2)  december 2016: 61-73 džamić, a. et al.  trends in biological activity research of wild… 64 g l a m o č l i j a et al., 2011; m a t e j i ć et al., 2012, 2013, 2014, 2015; m a r č e t i ć et al., 2014a; 2014b; m i l e s k i et al., 2014, 2015; p o p o v i ć et al., 2015a, 2015b) (tab. 1). essential oil analyses of inflorescence and aerial parts of endemic species, ferulago macedonica showed α-pinene as the main compound (43% and 23%, respectively). both oils demonstrated strong antibacterial activity. the essential oil of echinophora spinosa, containing δ-3carene (60.86%) as the dominant component, was the most prominent against escherichia coli and pseudomonas aeruginosa while the most resistant bacterial species against the oil was the staphylococcus aureus (g l a m o č l i j a et al., 2011). essential oil of echinophora sibtorpiana (characterized by presence of methyl eugenol (60.40%), followed by p-cymene (11.18%) and αphellandrene (10.23%) demonstrated considerable antimicrobial activity. the lowest antimicrobial activity was detected for aqueous extracts. the most resistant were fungus aspergillus niger and bacteria micrococcus flavus and escherichia coli (m i l e s k i et al., 2014). essential oil of seseli rigidum, with dominance of α-pinene (48.5%), showed antimicrobial activity. the most resistant bacterial strains were micrococcus flavus and staphylococcus epidermidis, and the most resistant micromycete was aspergillus niger (s t o j k o v i ć et al., 2009). methanol extracts of various seseli taxa revealed activity against different bacteria and yeast candida albicans (with mics of 0.78-12.5 mg/ml) (matejić et al., 2012). methanol and ethyl acetate extracts of cacrys cristata rare and critically endangered species in the flora of serbia, exhibited similar antibacterial activity. methanol extract from aerial parts, inflorescence and fruits of opopanax hispidus showed significant antimicrobial effect (m a t e j i ć et al., 2014, 2015). antimicrobial activity of wild growing lamiaceae species the in vitro preliminary antimicrobial tests were carried out by disc diffusion method. the results of the antimicrobial activity obtained by disc-diffusion assay showed that the diethyl ether extracts of stachys inhibited the growth of all the tested bacteria, and s. plumosa exhibited significant antimicrobial activity against pathogens e. coli, p. aeruginosa and s. aureus (comparable to antibiotics used as the positive controls (l a z a r e v i ć et al., 2010). in last decade majority of antimicrobial experiments used microdilution method. it is well known, that essential oils from lamiacae family showed high antimicrobial potential. antibacterial and antifungal activity of essential oils of wild growing thymus, mentha, mellisa, hyssopus, salvia, satureja, sideritis and stachys species were analyzed (tab. 1). essential oils of salvia sclarea (linalyl acetate and linalool) and mentha longifolia with dominance of cisand trans-dihydrocarveol and piperitone, were analyzed for their antifungal potential. the most susceptible was phytopathogen phomopsis helianthi, while the most resistant was trichoderma viride (d ž a m i ć et al., 2008, 2010). essential oil of satureja kitaibelii characterized by dominance of pcymene showed strong antimicrobial activity (mic values of 0.10-25 mg/ml) (k u n d a k o v i ć et al., 2011; m i h a j i l o v -k r s t e v et al., 2011), while essential oil of hyssopus officinalis subsp. pilifer (1,8-cineole, β-pinene and isopinocamphone) showed lower antimicrobial potential (d ž a m i ć et al., 2013). methanolic extract of satureja kitaibelii and its major compound rosmarinic acid possessed strong antimicrobial activity against tested bacteria and microfungi (s t a n o j k o v i ć et al., 2013). the components with phenolic structure are known to be highly active antimicrobial agents. thymol, a main constituent of various thymus essential oils, was responsible for wide spectrum of antibacterial and antifungal activity (s o k o v i ć et al., 2009; v l a d i m i r -k n e ž e v i ć et al., 2012; p e t r o v i ć et al., 2016). carvacrol and thymol are recognized as strong antimicrobial agents able to disintegrate the outer membrane of gram-negative bacteria, releasing lipopolysaccharides and increasing the permeability of the cytoplasmic membrane. antimicrobial activity is associated with chemical structure of compounds. the most important is lipophilic character of hydrocarbon skeleton and hydrophilic character of functional group. antimicrobial activity should be arranged in this order: phenols > ketones > alcohols > ethers > aldehydes > hydrocarbons (dorman & deans, 2000). antimicrobial activity of wild growing rosaceae species antibacterial activity of three wild fruits, red wild berry fruit (cornus mas), blackthorn (prunus spinosa) and wild blackberry (rubus fruticosus) extracts was analyzed using disc-diffusion and microdilution methods against 12 bacterial strains (tab. 1). the antimicrobial activity of wild berry fruits extracts was high against almost all the tested bacterial strains (r a d o v a n o v i ć et al., 2013). antimicrobial activity of wild growing asteraceae species essential oil of helichrysum arenarium in bioautography assay showed clear inhibition zones which were indication of strong antimicrobial activity gram (+) (micrococcus luteus, biologica nyssana 7 (2)  december 2016: 61-73 džamić, a. et al.  trends in biological activity research of wild… 65 staphylococcus aureus, staphylococcus epidermidis) and gram (-) bacteria (escherichia coli, pseudomonas tolaasii, salmonella enteritidis, salmonella typhimurium) and yeast candida albicans (r a n č i ć et al., 2005). the most rarely used was micro-atmosphere assay. the essential oil evaporated and the activity by vapor contact has been measured. due to the high lipophilic nature of mycelia coupled with a large surface area relative to the volume of a fungus, vapours of essential oils may act mainly by accumulation on mycelia than in the agar (i n o u y e et al., 2000). s t u p a r et al. (2014) investigated antifungal activity of the essential oil of helichrysum italicum using micro-atmosphere method. the main components of the oil were γcurcumene, α-pinene, and neryl acetate. the essential oil of h. italicum showed moderate antifungal activity against fungi isolated from cultural heritage objects. the most susceptible fungi to oil treatment were epicoccum nigrum and penicillium sp., while the most resistant was trichoderma viride. pathogenic strain pseudomonas aeruginosa causes pathological changes on vegetables, and it is considered as conditional parasite of plants, but also as a human parasite because it causes infections of urinary and respiratory tract. the effect of the water and ethanol plant extract was tested against pathogenic strains pseudomonas aeruginosa and p. fluorescens using disc diffusion method. water and ethanol extract of achillea millefolium and salvia officinalis showed the greatest inhibitory effect on pseudomonas aeruginosa, (the water extract of achillea). great inhibitory effect of the ethanol extract had helichrysum arenarium, rosmarinus officinalis, satureja montana (p e t r o v i ć et al., 2003). in general, microdilution assay is the most rewarding method for different analyses. the several strains of microorganisms should be tested on one plate, and small amount of tested sample could be used. nowadays this technique is the most frequently used. the chemical composition and the potential to have antimicrobial activity depend highly on the plant species, habitat and ontogeny phase as well as of methods of processing, extraction solvent and extract dose. many times the values of the inhibition zones were proportional to the concentration of the extracts and essential oils. sometimes dominant compounds are crucial for the biological activity. syinergistic effect of the compounds are also important. antioxidant activity compounds with antioxidant activity are mainly phenolic acids, flavonoids and polyphenols (d i l l a r d & g e r m a n , 2000). phenols provide the plants with defense mechanisms to neutralize reactive oxygen species (ros) in order to survive and prevent molecular damage and damage by microorganisms, insects, and herbivores. flavonoids show a wide range of biological activities such as inhibition of cell-proliferation, induction of apoptosis, inhibition of enzymes and other antibacterial and antioxidant effects (p a n d e y & g u p t a , 2014). antioxidants may directly react with the reactive radicals to destroy them by accepting or donating electron(s) or they may indirectly decrease the formation of free radicals by inhibiting the activities or expressions of free radical generating enzymes or by enhancing the activities and expressions of other antioxidant enzymes. many research models have been established in chemical and/or biological systems for studying the mechanisms of action of antioxidants and for identifying new antioxidants (lu et al., 2010) and it is suggested to test potential antioxidants by different methods. important step for determination of the antioxidant capacity is quantification of phenolic compounds in samples by using total phenol content (tpc) assay with folin–ciocalteu reagent, as well as quantification of total flavonoids (tfc). the most frequently used in vitro antioxidant methods are: 1,1-diphenyl-2-picrylhydrazyl (dpph) radical scavenging assay, 2,2-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid (abts) radical scavenging activity, ferry reducing power (frap) assay, total reducing power (trp) assay, β-carotenelinoleic acid assay etc. available literature data of in vitro antioxidant activity of wild growing species from central balkans are given in tab. 1. antioxidant activity of wild growing apiaceae species methanolic extract of seseli taxa tested for its antioxidant potential (total phenols, total flavonoids dpph and abts assays) revealed seseli libanotis ssp. libanotis for its highest reducing power (m a t e j i ć et al., 2012). essential oil of echinophora sibthorpiana possessed high radical scavenging capacity in dpph and abts assays. aqueous extract of aerial parts was the strongest tested extract. extracts of aerial parts expressed higher radical scavenging activity in comparison to the root extracts. all extracts had lower antiradical activity compared to bha and vitamin c (m i l e s k i et al., 2014). in the case of various extracts of ferulago macedonica, it was found that there is no strong relationship between total phenolic and flavonoid contents and radical scavenging activities biologica nyssana 7 (2)  december 2016: 61-73 džamić, a. et al.  trends in biological activity research of wild… 66 (dpph, abts). these results can be explained since the antioxidant property of a plant extract is generally considered as the result of the combined activity of a wide range of compounds including, not only phenols, but also peptides, organic acids and other components. both, phenols and flavonoids were present in higher amounts in f. macedonica extracts of inflorescence in comparison to the aerial parts (m i l e s k i et al, 2015). antioxidant activity of wild growing lamiaceae species extracts of waste water after hydrodistillation of satureja montana and s. cuneifolia, showed better ability to reduce stable dpph radical than the standards essential oils or thymol, carvacrol, and thymoquinone. this is probably due to the high concentration of other active compounds, such as (e)-coniferyl alcohol. to compare these results, total antioxidant activity of these two satureja species was also tested using the phosphomolybdenum method. this test is based on the reduction of mo(vi) to mo(v) by the extract and subsequent formation of a green phosphate/mo(v) complex at acid ph. total antioxidant activity of the phosphomolybdenum model evaluates both water-soluble and fat-soluble antioxidants, i. e. total antioxidant capacity. the highest activity in this method was found for essential oil of s. cuneifolia (č a v a r et al., 2013). for scavenging ability on dpph radicals, various extracts of hissopus officinalis subsp. pilifer were effective in the order of: deodorized aqueous extract > deodorised metanol extract > deodorised ethyl acetate extract. the total phenol content, determined by folin–ciocalteau reagent was in the same order. essential oil of h. officinalis subsp. pilifer possessed the lowest activities compared to other extracts and control substances (d ž a m i ć et al., 2013). antioxidant potential of melittis melissophyllum various hydro alcoholic, ethanol and methanol extracts revealed a high scavenging activity by dpph and abts methods. reducing power of extracts were evaluated through the frap assay. the highest phenol and flavonoid content were identified in the 96% ethanol extract and the lowest were in the ethanol 10% solution. also, the highest correlation was observed between total phenol and flavonoid contents. statistically significant correlations were registered for dpph and abts assays which is logical due to their similar mechanism of action as radical scavengers. a hierarchical cluster analysis (hca) was performed on the total phenols and total flavonoids content dpph, abts, and frap assay. the dendrogram (fig. 1) shows that total phenols and total flavonoids content are quite homogeneous, while dpph was placed in separate cluster (g r u j i ć et al., 2014). tree diagram for 5 variables ward`s method euclidean distances 0 10 20 30 40 50 60 70 80 90 100 110 (dlink/dmax)*100 ic50 dpph, (mg/ml) frap, (µmolfe/mg) abts, mg vit c/g total flavonoids (que/g) total phenols (gae/g) fig. 1. dendrogram of applied assays for antioxidant characteristics evaluation for melittis melissophyllum extracts (g r u j i ć et al., 2014). biologica nyssana 7 (2)  december 2016: 61-73 džamić, a. et al.  trends in biological activity research of wild… 67 table 1. antimicrobial and in vitro antioxidant activity of extracts and essential oils of wild-growing species from central balkans fam species extracts eo composition antimicrobial methods antioxidant methods references a p ia ce a e cachrys cristata dc. methanol, ethyl acetate, acetone, aqueous aerial parts, fruits antibacterial antifungal microdilution total phenols total flavonoids dpph abts matejić et al.,2012, 2014 daucus carota l. fruits, flowers, root, leaves and stem antibacterial antifungal microdilution soković et al., 2009 echinophora sibthorpiana guss. echinophora spinosa l. methanol, ethanol, aqueous inflorescence aerial parts antibacterial antifungal microdilution total phenols total flavonoids dpph abts mileski et al., 2014 glamočlija et al., 2011 eryngium palmatum pančić and vis. methanol chloroform root microdilution antibacterial anticandidal total phenols dpph frap marčetić et al., 2014a; 2014b ferulago macedonica micevski and e. mayer methanol, ethanol, aqueous antibacterial antifungal microdilution total phenols total flavonoids dpph abts mileski et al., 2015 laserpitium latifolium l. l. zernyi hayek l. ochridanum micevski chlorophorm underground parts aerial parts fruits antibacterial antifungal microdilution antibiofilm popović et al, 2015 a,b opopanax hispidus (friv.) griseb. methanol, ethyl acetate antibacterial anticandidal microdilution total phenols total flavonoids dpph abts matejić et al., 2015 seseli pallasii besser, seseli libanotis (l.) koch ssp. libanotis seseli libanotis ssp. intermedium (rupr.) p. w. ball seseli rigidum waldst. & kit. seseli globiferum vis. seseli annuum l. methanol aerial parts fruits antibacterial antifungal microdilution total phenols total flavonoids dpph abts frap matejić et al., 2012 ilić et al., 2014; 2015 janacković et al, 2011 stojković et al, 2009 milosavljević et al., 2007 tordylium maximum l. methanol, aqueous antibacterial anticandidal microdilution total phenols total flavonoids dpph abts matejić et al., 2013 l a m ia ce a e hyssopus officinalis ssp. pilifer deodorized aqueous, methanol and ethyl acetate aerial parts antifungal microdilution total phenols dpph džamić et al., 2013 mentha longifolia l. aerial parts antifungal microdilution dpph džamić et al., 2010 mellisa officinalis l. aerial parts antibacterial antifungal microdilution dpph oh radical mimica-dukić et al., 2004 melittis melissophyllum l. methanol, ethanol (hydroalcoholic) total phenols total flavonoids dpph abts frap grujić et al., 2014 salvia nemorosa l. ssp. nemorosa salvia sclarea l. aerial parts antibacterial antifungal microdilution dpph phosphomolybd enum božin et al., 2011 džamić et al., 2013 satureja montana l. satureja cuneifolia ten satureja montana ssp. pisidica (wettst.) šilić satureja montana l. ssp. montana aerial parts antibacterial antifungal microdilution dpph orac trp apf čavar et al., 2013 kundaković et al., 2014 mihajilov-krstev et al., 2014; 2011 biologica nyssana 7 (2)  december 2016: 61-73 džamić, a. et al.  trends in biological activity research of wild… 68 table 1. continuation of the table fam species extracts eo composition antimicrobial methods antioxidant methods references l a m ia ce a e satureja kitaibelii wierzb. ex heuff. satureja horvatii šilić bukvički et al., 2014 lakušić et al., 2008 sideritis montana l. aerial parts antibacterial microdilution miladinović et al., 2012 stachys germanica ssp. heldreichii (boiss) hayek stachys iva griseb. stachys plumosa griseb. stachys scardica griseb. diethyl ether, ethyl acetate antibacterial antifungal disk-diffusion phosphomolybd enum lazarević et al., 2010 thymus praecox opiz ssp. polytrichus thymus longicaulis c. presl thymus serpillum l. thymus tosevii velen supercritical extracts methanol, ethanol, aqueous aerial parts antibacterial antifungal microdilution macrodilution disk-diffusion dpph petrović et al., 2016 a, b vladimir-knežević et al., 2012 soković et al., 2009 r o sa ce a e crataegus oxyacantha l. antibacterial disk-diffusion total phenols anthocyan content flavonoid content dpph kostić et al., 2012 cornus mas l. prunus spinosa l. acidified methanol solution antibacterial disk-diffusion microdilution dpph radovanović et al., 2013 rosa canina l. methanol antibacterial antibiofilm microdilution živković et al., 2015 rubus caesius l. var. aquaticus weihe and nees rubus fruticosus l. methanol, ethanol, acetone, aqueous acidified methanol solution antibacterial disk-diffusion microdilution total phenols total flavonoids dpph abts frap trc veličković, et al., 2015; radovanović et al., 2013 a st er a ce a e achillea collina becker ex heimerl s.l. achillea pannonica scheele aerial parts disk-diffusion dpph božin et al., 2008 ambrosia artemisiifolia l. 70% aqueous acetone extracts total phenols total flavonoids dpph frap maksimović, 2008 artemisia alba turra artemisia absinthium l. ethanol aerial parts antibacterial anticandidal microdilution total phenols total flavonoids dpph frap abts đorđević et al., 2013 mihajilov-krstev et al., 2014 helichrysum arenarium (l.) moench helichrysum italicum (roth) g. don aerial parts antibacterial antifungal microdilution bioautography rančić et al., 2005 stupar et al., 2014 hieracium pillosela l. methanol, dychlorometane, ethyl acetate, dychlorometane:me thanol antibacterial disk-diffusion stanojević et al., 2008 xeranthemum annuum l methanolic acetone ethyl acetate antibacterial antifungal microdilution total phenols total flavonoids dpph stanković et al., 2011 biologica nyssana 7 (2)  december 2016: 61-73 džamić, a. et al.  trends in biological activity research of wild… 69 antioxidant activity of wild growing rosaceae species r a d o v a n o v i ć et al. (2013) found that in polyphenolic extracts of fruits from tree rosaceae species main compounds were gallic acid, caffeic acid, p-coumaric acid, ferulic acid, (+)-catechin, procyanidin b2, (-)-epicatechin, quercetin, rutin and quercetin-3-glucoside. all extracts showed high scavenging effect on dpph radical. extracts obtained from hieracium pilosella have significant free radical scavenging activity on stable dpph• and high reactive hydroxyl radical. the data suggest that aqueous, ethanolic and methanolic extracts of h. pilosella from southeastern serbia are a potential source of natural antioxidants. chlorogenic acid was detected in the highest quantities in all investigated extracts (s t a n k o v i ć et al., 2009). antioxidant activity of wild growing asteraceae species according to b o ž i n et al. (2008) the main detected compounds in essential oil of achillea callina were β-pinene, chamazulene and e-caryophyllene, while in essential oil of a. pannonica 1,8-cineole and camphor were the dominant components. authors represented that both oils possess strong antioxidant effects in vitro and in vivo assays. antioxidant capacities are influenced by many factors, which cannot be fully described with a single method. the most commonly used method for assessment of antioxidant properties of natural products is dpph radical assay. the dpph radical assay overcomes the limitations of monitoring the activity of the numerous samples over a specified period of time. it is reproducible and strongly correlated with phenolic compounds (m i l i a u s k a s , 2004). cytotoxic activity brine shrimp, artemia salina, is the test organism that is used for evaluation of possible cytotoxic activity of plant extracts or essential oils. brine shrimp assay is simple, inexpensive method and results are obtained quickly. in the study represented by j a n a ć k o v i ć et al. (2008) a brine shrimp assay was used to examine potential cytotoxic activity of different centaurea species, measuring lethal concentration (lc). cytotoxic activity of methanol and ether extracts was tested. the methanol extract of c. arenaria showed very significant activity while the lowest activity was found with c. chrysolepis methanol extract. among ether extracts, the most active was c. splendens (and the lowest activity was recorded for the ether extract of c. scabiosa. toxicity test on drosophila melanogaster showed that the essential oil of a. absinthium is toxic for developing insect larvae. starting with the concentration of 0.38 % of essential oil in medium, significant mortality of larvae exposed to the oil was noted when compared to the control. the essential oil also affected the development of d. melanogaster larvae and significantly delayed achievement of the pupa stadium (m i h a j i l o v -k r s t e v et al., 2014). many plant extracts and natural products, especially phenols, with high antioxidant activity have shown cytotoxic effects in different cell lines. flavonoid anticancer activities include inhibition of cell growth, inhibition of protein kinase activities, and induction of apoptosis. popular and broadly used for cytotoxity examination is the mtt (3-[4,5dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay which is based on the conversion of mtt into formazan crystals by living cells, which determines mitochondrial activity. methanolic extract of origanum vulgare showed significant antiproliferative activity in both colon cancer (hct116) and breast cancer (mda-mb-231) cell lines. inhibitory activity appeared to be particularly conspicuous in treated hct-116 cell lines, whereas at the highest concentration (500 µg/ml), only 10% of the cells remained viable. the results indicate that o. vulgare is considered to be a particularly valuable source of effective anti-proliferative and cytotoxic substances (g r b o v i ć et al., 2013). the methanol extract of satureja kitaibelii exhibited strong activity against fem-x human malignant melanoma cells, and moderate activity against breast cancer cell lines (s t a n o j k o v i ć et al., 2013). the cytotoxic effect of the essential oils from satureja montana ssp. pisidica from two localities (mountains korab and galičica) was tested against cancer cell lines mdamb-361, mda-mb-453, hela, ls174 and mrc5 cells. the essential oil from korab demonstrated significantly better results than the oil from galičica, particularly against hela and mda-mb-453 cell lines, while the oil from galičica was the most active on the human epithelial cervical cancer hela cells (k u n d a k o v i ć et al., 2014). other activities most studies describe the action of essential oil and extracts for other biological activities. strong inhibition of human serum cholinesterase by essential oil of satureja montana, suggests this as a natural source of compounds that can be used in the treatment of foodborne and neurological diseases, wound and other infections, as well as for general health improvement (m i h a j i l o v -k r s t e v et al., 2014). considering human and horse serum biologica nyssana 7 (2)  december 2016: 61-73 džamić, a. et al.  trends in biological activity research of wild… 70 cholinesterase inhibition, both essential oils acted as strong inhibitors, even higher than effect of their dominant constituent α-pinene, leading to the conclusion that the other constituents of the essential oil manifested their action toward cholinesterase (ilić et al., 2015). thyme essential oil, as well as its major phenolic constituent’s thymol and carvacrol were researched for application in alzheimer’s disease treatment or as remedies for cognitive disorders. it was noted that phenolic chemotypes possess higher antioxidant activity than non-phenolic ones, as well as acetylholinesterase inhibitory activity (č a v a r z e l j k o v i ć & m a k s i m o v i ć , 2014). conclusion the reported results from in vitro studies hint the potential antimicrobial and antioxidant value of the wild growing plants from central balkan. it was prepared based on plenty literature search of biological activity of extracts and essential oils. the microdilution method was found as the mostly used in antimicrobial assay. dpph method is the most frequently used in antioxidant assay. the traditional use of many aromatic species leads to discovering the unknown biological potential of related species. the most important approach is combination of phytochemical investigation and bioassay. essential oil and extracts without identifying of compounds responsible for this activity are also insufficient for preclinical trials. in spite the fact that a lot of studies have been reported so far, many of wild-growing plants are still waiting to be explored. acknowledgements. this work was founded by the ministry of education, science and technological development of the republic of serbia for 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(2)  december 2014: 113-121 ilić milošević, i. et al.  tritrophic accociations of lysiphlebus fabarum… 113 original article received: 5 december 2014 revised: 18 december 2014 accepted: 20 december 2014 tritrophic associations of lysiphlebus fabarum (marshall) (hymenoptera, braconidae, aphidiinae) in serbia marijana ilić milošević1*, maja lazarević1, olivera petrović-obradović2, vladimir žikić1 1university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia 2faculty of agriculture, nemanjina 6, 11081 belgrade – zemun, university of belgrade, serbia * corresponding author: marijanailic83@yahoo.com abstract: ilić milošević, m., lazarević, m., petrović-obradović, o., žikić, v.: tritrophic associations of lysiphlebus fabarum (marshall) (hymenoptera, braconidae, aphidiinae) in serbia. biologica nyssana, 5 (2), december 2014: 113-121. one hundred and eighty-six tritrophic parasitoid-aphid plant associations were established between lysiphlebus fabarum and 64 aphids with 148 different plant species from the territory of serbia. we identified 18 aphid genera of which the genus aphis was represented by 38 species. the most frequent interactions between the parasitoid and the hosts were the ones of aphis fabae, a. craccivora and a. ruborum on various plants comprising one third of the total trophic interactions listed here. key words: parasitoids, trophic chains, distribution, aphids, host plant apstrakt: ilić milošević, m., lazarević, m., petrović-obradović, o., žikić, v.: tritrofičke asocijacije vrste lysiphlebus fabarum (marshall) (hymenoptera, braconidae, aphidiinae) u srbiji. biologica nyssana, 5 (2), decembar 2014: 113-121. na teritoriji srbije je sada ustanovljeno 186 tritrofičkih asocijacija između parazitoida, biljnih vaši i biljaka. vrsta lysiphlebus fabarum je registrovana na 64 vrste biljnih vaši koje napadaju 148 biljnih vrsta. utvrđeno je da se biljne vaši grupišu u 18 različitih rodova, od kojih je najzastupljeniji bio rod aphis sa 38 vrsta. najčešće parazitirane vrste biljnih vaši od strane l. fabarum bile su aphis fabae, a. craccivora i a. ruborum prikupljene sa mnogih biljaka, formirajući oko jednu trećinu svih zabeleženih trofičkih interakcija. key words: parazitoidi, trofički lanci, distribucija, biljne vaši, biljke introduction the subfamily aphidiinae (hymenoptera: aphidiidae) represents a small group of solitary endoparasitoid wasps of aphids including many important species acting as biological control agents (b r e w e r & e l l i o t t , 2004; l e v i e et al., 2005). they are very useful in crop fields, as well as in greenhouses and non-arable fields such as meadows and forests reducing the number of pest aphids 5 (2) • december 2014: 113-121 biologica nyssana 5 (2)  december 2014: 113-121 ilić milošević, i. et al.  tritrophic accociations of lysiphlebus fabarum… 114 (s t a r ý , 1970, 1974; h a g v a r & h o f s v a n g , 1991; k a v a l l i e r a t o s et al., 2004). one genus in subfamily aphidiinae which has been studied in detail is lysiphlebus foerster, which comprises about 25 species worldwide, 16 occuring in the european fauna (y u et al, 2013; van achterberg, 2013). on the territory of serbia, ten species were recorded including the two that have already been introduced here: l. orientalis starý & rakhshani, 2006 and l. testaceipes (cresson, 1880) (p e t r o v i ć et al., 2013; ž i k i ć et al., 2015). according to the wing venation, this genus could fall into two species groups. the “fabarum” group includes the species with a long metacarpal vein while in “testaceipes” group the species have a short metacarpal vein (p e t r o v i ć et al., 2015). lysiphlebus fabarum (marshall, 1896) is a polyphagous aphid parasitoid that attacks more than a hundred of aphid species in various habitats (y u et al., 2013). they often exploit small aphids, usually from the genus aphis l., including some serious pests, e.g. the black bean aphid, a. fabae scopoli, the cowpea aphid, a. craccivora koch and the cotton aphid, a. gossypii (glover) (k a v a l l i e r a t o s et al., 2004; s t a r ý , 2006) but also the target aphid genus for l. fabarum is brachycaudus van der goot, e.g. the plum-thistle aphid, b. cardui (l.) and the leaf-curling plum aphid, b. helichrysi (kaltenbach). likewise, the species l. fabarum has a tendency to attacks the antprotected aphid colonies. its distribution is very wide, including the whole palaearctic, as well as australia and oceania. in europe, l. fabarum is found in the fauna of andorra, bulgaria, the canary islands, corsica, croatia, czech republic, france, germany, great britain, greece, hungary, italy, latvia, lithuania, madeira, montenegro, poland, russia, sicily, slovakia, slovenia, spain, and the netherlands. there are few studies regarding tritrophic associations of l. fabarum, and its diversity in agroecosystems in serbia (t o m a n o v i ć & b r a j k o v i ć , 2001; k a v a l l i e r a t o s et al., 2004; ž i k i ć et al., 2012). in this study we present the parasitoid-aphidplant trophic associations of l. fabarum registered on the territory of serbia. the knowledge about parasitoid-aphid-plant associations appears to be necessary for a better implementation of programs of biological control of aphids as an economically important group of pests. also, host-plant associations are important for better knowledge of biodiversity of species l. fabarum. material and methods we used the data from the collections of lysiphlebus fabarum sampled during the period 1989-2014 from numerous localities throughout serbia. our samples represent cut branches infested by aphids, sometimes with visible signs of parasitization. this plant material., with or without mummified aphids, was put in small plastic meshcovered boxes to enable ventilation, and then placed in a growth cabinet (22.5°c, 65% relative air humidity, 16:8 h, light:dark photoperiod) until the parasitoid emergence. before leaving the samples in the growth cabinet, some live adult aphids were preserved in 90% ethyl-alcohol for identification. parasitoid material and plants were deposited at the faculty of sciences and mathematics, department of biology and ecology, university of niš, serbia and the faculty of biology, university of belgrade, serbia. in order to obtain some data relevant literature was used (ž i k i ć et al., 2012). the aphids used in this study were from the collection of dr olivera petrović-obradović (faculty of agriculture, university of belgrade, serbia). the nomenclature of aphids is given according to r e m a u d i è r e and r e m a u d i è r e (1997), whereas the nomenclature of plants is based on flora europaea (1964, 1968, 1972, 1976, 1980) and g r e u t e r et al. (1984, 1986, 1989). results and discussion the results shown in appendix 1 refer to the long time period of investigation of lysiphlebus fabarum and all the interactions with aphids infesting analysed plants on the territory of serbia. analysing established trophic chains, we found 186 tritrophic parasitoid-aphid-plant associations including 64 hosts on 148 plants. the aphids belong to 18 genera. it is evident that the species belonging to the genus aphis, were most frequently recorded in this investigation, about 38 taxa. the species a. fabae and a. craccivora made a great number of associations with different plants. we registered 57 trophic chains for a. fabae together with two subspecies, a. fabae cirsiiacanthoidis scopoli and a. fabae solanella theobald, species a. fabae forming 75 interactions with different plant species. as far as the species aphis craccivora is concerned, we counted 30 associations with plants. the third most commonly encountered host species was a. ruborum, forming 11 associations. biologica nyssana 5 (2)  december 2014: 113-121 ilić milošević, i. et al.  tritrophic accociations of lysiphlebus fabarum… 115 conclusion based on such a large number of recorded associations of the parasitoid lysiphlebus fabarum and the previous studies regarding this species, l. fabarum is a suitable candidate for the biological control programs of various aphid species. future investigations should be focused on interactions between many aphid species and l. fabarum, especially the very close relation between l. fabarum and polyphagous aphid, aphis fabae as its host. through different methods, such as the level of parasitism success, field campaign, and the introduction or detection of bacterial symbionts using suitable molecular markers have already been applied in the pioneering work by v o r b u g e r et al. (2009). acknowledgements. we wish to thank dr. željko tomanović and dr. andjeljko petrović (faculty of biology, university of belgrade) for the loan of specimens; saša stanković (faculty of sciences and mathematics, university of niš) for his help in sampling material and dr. nickolas kavallieratos (benaki phytopathological institute, athens, greece). this study was supported by the ministry of education, science, and technological development of the republic of serbia (iii43001). references brewer, m. j., elliott, n. c. 2004: biological control of cereal aphids in north america and mediating effects of host plant and habitat manipulations. annual review of entomology, 49: 219–242. greuter, w., burdet, h. m., long, g. 1984: medchecklist 1. conservatoire et jardin botaniques de la ville de gèneve, med-checklist. trust of optima, gèneve, 330 pp. greuter, w., burdet, h. m., long, g. 1986: medchecklist 3. conservatoire et jardin botaniques de la ville de gèneve, med-checklist. trust of optima, gèneve, 395 pp. greuter, w., burdet, h. m., long, g. 1989: medchecklist 4. conservatoire et jardin botaniques de la ville de gèneve, med-checklist. trust of optima, gèneve, 458 pp. hågvar, e. b., hofsvang, t. 1991: aphid parasitoids (hymenoptera: aphidiidae): biology, host selection and use in biological control. biocontrol news and information, 12 (1): 13-42. kavallieratos, n. g., tomanović, ž., starý, p., athanassiou, c. g., sarlis, g. p., petrović, o., niketić, m. a., anagnou-veroniki, m. 2004: a survey of aphid parasitoids (hymenoptera: braconidae: aphidiinae) of southeastern europe and their aphid-plant associations. applied entomology and zoology, 39 (3): 527–563. levie, a., legrand, m. a., dogot, p., pels, c., baret, p., hance, t. 2005: mass releases of aphidius rhopalosiphi (hymenoptera: aphidinae), and strip management to control of wheat aphids. agriculture, ecosystems and environment, 105:17–21. petrović, a., mitrović, m., ivanović, a., žikić, v, kavallieratos, n. g., starý, p., mitrovskibogdanović, a., tomanović, ž., vorburger, c. 2015: genetic and morphological variation in sexual and asexual parasitoids of the genus lysiphlebus – an apparent link between wing shape and reproductive mode. bmc evolutionary biology (in press). petrović, a., mitrović, m., starý, p., petrovićobradović, o., žikić, v., tomanović, ž., vorburger, c. 2013: lysiphlebus orientalis (hymenoptera: braconidae), a new invasive aphid parasitoid in europe – evidence from molecular markers. bulletin of entomological research, 103 (4): 451-457. remaudière, g., remaudière, m. 1997: catalogue des aphididae du monde – of the world’s aphididae. homoptera aphidoidea. inra editions, paris, 473p. starý, p. 1970: biology of aphid parasites (hymenoptera: aphidiidae) with respect to integrated control. dr. w. junk, the hague. 643 pp. starý, p. 1974: taxonomy, origin, distributionand host range of aphidius species(hymenoptera, aphidiidae) in relation to biological control of the pea aphid ineurope and north america. zeitschrift furangewandte entomologie, 77 (2): 141-171. starý, p. 2006: aphid parasitiods of the czech republic. academia, prague, czech republic. tomanović, ž., brajković, m. 2001: aphid parasitoids (hymenoptera, aphidiidae) of agroecosystems of the south part of the pannonian area. archives of biological sciences, 53 (1-2): 57-64. tutin, t. g., heywood, v. h., burges, n. a., moore, d. m., valentine, d. h., walters, s. m., webb, d. a. (eds.), 1964-1980: flora europaea, i-v. cambridge university press. london. van achterberg, c., 2013. fauna europaea: braconidae. in: fauna europaea: hymenoptera: symphyta + ichneumonoidea, fauna europaea, version 2.6.2. available through: http://www.faunaeur.org vorburger, c., sandrock, c., gouskov, a., castañeda, l. e., ferrari, j. 2009: genotypic variation and the role of defensive biologica nyssana 5 (2)  december 2014: 113-121 ilić milošević, i. et al.  tritrophic accociations of lysiphlebus fabarum… 116 endosymbionts in an all-parthenogenetic host– parasitoid interaction. evolution, 63 (6):1439-50. yu, d. s., van achterberg, c., horstmann, k. 2013. world ichneumonoidea 2011. taxonomy, biology, morphology and distribution. taxapad (scientific names for information management), interactive catalogue, ottawa available on: www.taxapad.com. žikić, v., ilić-milošević, m., stanković, s., petrović, a., petrović-obradović, o., kavallieratos, n., starý, p., tomanović, ž. 2012: aphidiinae (hymenoptera: braconidae) of serbia and montenegro – tritrophic interactions. acta entomologica serbica, 17 (1/2): 83-105. žikić, v., stanković, s. s., ilić milošević, m., petrović-obradović, o., petrović, a., starý, p., tomanović, ž. 2015: first detection of lysiphlebus testaceipes (cresson) (hymenoptera: aphidiinae) in serbia: an introduced species invading europe?. northwestern journal of zoology, 11 (1): 97-101. appendix 1. list of registered tritrophic associations plant aphid finding althaea officinalis l. aphis fabae novi sad, 10.06.1997, žt aphis davletshinae hille ris lambers surčin, 20.05.1989, op aphis fabae glover zemun, galenika, 07.06.2011, ap amaranthus retroflexus l. aphis fabae belgrade, crveni krst, 05.07.1996, žt; lebane, konjino, 06.07.2010, ss. arctium lappa l. aphis fabae suva planina mt., vetanska reka river, 20.07.1997, žt; sićevačka klisura gorge, ostrovica, 23.06.2012, ss; 28.05.2013, mđ; tara mt., mitrovac, 25.06.2013, vž atriplex patula l. aphis craccivora palićko jezero lake, 06.09.1995; žt zemun, 09.07.1996, op; ballota nigra l. aphis balloticola szelegiewicz belgrade, avala mt.,01.06.1996, žt; obedska bara swamp, 07.06.1997, žt; stara pazova, 10.06.1997, žt; belgrade, radmilovac, 18.06.1999, žt; niš, popovac, 25.06.2010, vž; niš, bubanj, 05.06.2013, mđ aphis spp. zemun, izbis, 30.05.2011, ap; beta vulgaris l. aphis fabae zemun, 27.09.1990, žt; capsella bursa pastoris (l.) medik. aphis fabae čemernik, 04.08.2011, ss; capsicum annuum l. myzus persicae kikinda, 23.06.1997, žt; carduus acanthoides l. aphis fabae stara planina mt., balta berilovac, 22.07.1997, žt; slankamen, 24.06.2011, žt; brachycaudus cardui (l.) zrenjanin, 20.06.1996, žt; užice, trešnjica, 23.07.1996, žt; beljanica mt., 03.08.1996, žt; bečej, 03.08.1996, žt; mol, 03.08.1996, žt; rusko selo, 03.08.1996, žt; sefkerin, 03.08.1996, žt; perlez, 23.06.1997, žt; stara planina mt., glogovac, 16.07.1997, žt; stara planina mt., balta berilovac, 20.07.1997, žt; stara planina mt., ćuštica, 20.07.1997, žt; beljanica mt., 12.07.1998, op; pčinja canyon, prohor pčinjski, 10.06.2014, vž; uroleucon sp. mordvilko surduk, 21.06.2011, žt; carduus crispus l. brachycaudus carduii (l.) vlasina plateau, čemernik, 04.08.2011, ss; carduus nutans l. brachicaudus carduii (l.) niš, popovac, 23.06.2012, ss; carduus sp. aphis fabae kragujevac, ilićevo, 06.06.2011, amb;valjevo, petnica, 12.06.2011, ap; brachycaudus carduii (l.) vlasinsko jezero lake, 06.08.2010, ss;đerdap, veliki kazan, 28.05.2011, žt; valjevo, petnica, 12.06.2011, ap; dukat mt., 06.08.2011, ap; carlina acaulis l. aphis carlinae (borner 1940) šara mt., ošljak, 04.07.1995, op; centaurea cyanus l. aphis celastrii matsumura divčibare, 08.08.2000, op; lebane, konjino, 31.05.2014, ss; aphis fabae lebane, konjino, 31.05.2014, ss; centaurea sp. brachycaudus cardui (l.) kopaonik mt., brzeće, 29.07.2010, ss; cephalaria transsylvanica (l.) schrad. aphis craccivora niš, duvanište, 13.06.2013, pm; chamaecytisus sp. aphis craccivora dukat mt., 29.06.2012, ss; biologica nyssana 5 (2)  december 2014: 113-121 ilić milošević, i. et al.  tritrophic accociations of lysiphlebus fabarum… 117 plant aphid finding chaenopodium album l. aphis fabae zemun, 30.10.1990, op; belgrade, botanical garden, 29.06.1996, žt; niš, kunovica, 16.10.1998, vž; niš, popovac, 22.05.2010, vž; niš, popovac, 22.05.2010, ss; niš, popovac, 10.07.2010, vž; novi belgrade, block 62, 28.05.2011, žt; sićevačka klisura gorge, sićevo, 04.06.2011, ss; zemun, galenika, 07.06.2011,ap; slankamen, 24.06.2011, žt; lebane, konjino, 01.06.2013, ss; sićevačka klisura gorge, sićevo, 06.06.2013, vž; tara mt., derventa canyon, 25.06.2013, vž; sićevačka klisura gorge, sićevo, 17.07.2013, ss; aphis spp. majur, 21.06.2011, žt; chrysanthemum sp. aphis fabae glover kanjiža, 08.06.1995, žt; cichorium intibus l. aphis intybi koch bela crkva, 13.06.1996, žt; kragujevac, 05.07.2011, amb; niš, matejevac, 23.06.2012, vž; lebane, konjino, 31.05.2014, ss; aphis spp. negotin, 01.07.2011, žt; aphis craccivora tara, derventa canyon, 03.07.2012, vž; cirsium acanthoides l. aphis fabae lebane, konjino, 31.05.2014, ss; brachycaudus cardui (l.) lebane, konjino, 31.05.2014, ss; cirsium arvense (l.) scop. aphis fabae vlasinsko jezero lake, 11.08.2006, vž; vlasinsko jezero lake, 06.08.2010, vž; belgrade, ostružnica, 06.07.2011, žt; vlasinsko jezero lake, 03.08.2011, vž; vlasinsko jezero lake, 28.06.2012, vž; vlasinsko jezero lake, 15.06.2013, ss; vlasinsko jezero lake, 21.07.2013, ss; merošina, oblačinsko jezero lake, 15.06.2014, ss; aphis fabae cirsiiacanthoidis scopoli surčin, 29.05.1993, žt; vlasinsko jezero lake, 01.08.1996, žt; vlasina plateau, čemernik, 24.07.1997, žt; belgrade, kovilovo, 11.06.1999, žt; belgrade, radmilovac, 18.06.1999, žt; suva planina mt., glogovac, 16.07.1999, žt; zrenjanin, ečka, 31.05.2001, žt; kopaonik mt., brzeće, 29.07.2010, vž; vlasinsko jezero lake, 06.08.2010, vž; vlasina plateau, čemernik, 07.08.2010, ss; vlasinsko jezero lake, 21.07.2013, ss; aphis fabae solanella theobald valjevo, petnica, 12.06.2011, ap; uroleucon aeneum (hille ris lambers) kopaonik, 27.07.2010, vž; zemun, 02.06.2011, žt; vlasina plateau, čemernik, 1400 m, 04.08.2011, vž; bajina bašta, 06.07.2012, vž; merošina, oblačinsko jezero lake, 15.06.2014, ss; cirsium eriophorum (l.) scop. aphis fabae cirsiiacanthoidis scopoli vlasinsko jezero lake, 1250 m, 21.07.2013, ss; brachycaudus cardui (l.) suva planina mt., korube, 17.07.1997, žt; vlasinsko jezero lake, 1250 m, 21.07.2013, ss; cirsium lanceolatum (l.) scop. brachycaudus cardui (l.) beljanica mt., 12.07.1997, žt; aphis fabae cirsiiacanthoidis scopoli stara planina mt., balta berilovac, 20.07.1997, žt; cirsium palustre (l.) scop. aphis fabae cirsiiacanthoidis scopoli goč mt., 13.07.2011, žt; brachycaudus cardui (l.) vlasinsko jezero lake, 21.07.2013, ss; cirsium sp. aphis fabae cirsiiacanthoidis scopoli kopaonik mt., 17.07.2013, ss; cirsium vulgare (savi) ten. brachycaudus cardui (l.) bosilegrad, jarešnik, 22.07.2013, ss; clematis sp. aphis spp. užice, djetinja canyon, 26.06.1999, žt; coreopsis verticillata l. aphis fabae zemun, 17.10.1991, op; coronilla varia l. aphis craccivora niš, pantelej, 28.06.2012, vž; niš, trošarina, 26.04.2014, vž; corydalis sempervirens (l.) pers. aphis fabaei glover niš, pantelej, 01.06.2013, vž; crepis biennis l. aphis crepidis (börner) mol, 23.06.1997, žt; aphis spp. žitište, 22.07.1997, žt; crepis foetida l. uroleucon cichorii (koch) niš, jasenovik, 12.06.2011, mđ; vlasinsko jezero lake, 1250 m, 21.07.2013, ss; aphis gr craccivora niš, pantelej, 01.06.2013, vž; crepis sp. aphis spp. niš, bubanj, 31.05.2013, mđ; aphis craccivora niš, donji matejevac, 05.06.2013, vž; niš, tvrdjava, 05.06.2013, mđ; aphis crepidis (börner) vlasinsko jezero lake, 1250 m, 28.06.2012, ss; cucumis sativus l. aphis fabaei glover užice, carina, 05.08.1996, žt; biologica nyssana 5 (2)  december 2014: 113-121 ilić milošević, i. et al.  tritrophic accociations of lysiphlebus fabarum… 118 plant aphid finding daucus carota l. semiaphis dauci (fabricius) carska bara, 02.07.1996, op; daucus sp. semiaphis dauci (fabricius) kruševac, slobodište, 13.07.2012, zk; digitalis ambigua murray aphis fabae tara, derventa canyon, 03.07.2012, vž; dorycnium herbaceum vill. aphis craccivora zlatarsko jezero lake, 20.06.1998, žt; epilobium angustifolium l. aphis spp. kopaonik mt., 27.07.2010, vž; epilobium sp. aphis spp. sombor, 16.07.1996, op; euphorbia cyparissias l. aphis spp. niš, bubanj, 11.05.2013, mđ; euonymus japonicus l.f. aphis fabae lebane, konjino, 31.05.2014, ss; aphis fabae solanella theobald lebane, konjino, 31.05.2014, ss; euonymus europaeus l. aphis fabae belgrade, botanical garden, 17.06.1997, žt; filipendula ulmaria (l.) maxim. macrosiphum cholodkovskyi (mordvilko) dukat mt., 29.06.2012, vž; foeniculum vulgare mill. cavariella sp. del guercio sićevačka klisura gorge, sićevo, 28.05.2013, zk; galium aparine l. aphis craccivora vlasinsko jezero lake, 06.06.2014, ss; aphis fabae belgrade, dušanovac, 20.05.1993, žt; niš, tvrdjava, 05.06.2013, mđ; vlasinsko jezero lake, 06.06.2014, ss; genista tinctoria l. aphis genistae (börner) belgrade, ostružnica, 17.06.2010, žt; gentiana asclepiadea l. aphis gentianae (börner) stara planina mt., babin zub, 19.07.1994, op; geranium sp aphis spp. zemun, galenika, 07.06.2011, ap; hedera helix l. aphis hederae kaltenbach zemun, 13.06.2011, op; zemun, 14.06.2011, op; niš, trošarina, 18.05.2013, mđ; niš, pantelej, 01.06.2013, vž; heracleum sphondylium l. aphis fabae belgrade, botanical garden, 29.06.1996, žt; suva planina mt., toponički put, 19.07.1997, žt; hypochaeris radicata l. uroleucon sp. mordvilko adžine livade, žeželj, 01.06.2011, žt; nasonovia sp. mordvilko adžine livade, žeželj, 01.06.2011, žt; iris germanica l. aphis newtoni theobald belgrade, 13.10.1997, žt; knautia drymeia heuff. macrosiphum knautiae holman sićevačka klisura gorge, ostrovica, 28.05.2013, mđ; kochia scoparia (l.) schrad. aphis sp. lebane, konjino, 06.07.2010, ss; lactuca sp. aphis craccivora lebane, konjino, 01.06.2013, ss; lamium sp. aphis spp. zemun, 23.06.1995, op; smederevo, lugavčina, 06.06.1999, op; laserpitium halleri crantz aphis spp. zemun, altina, 09.06.2011, ap; dobanovci, 17.06.2011, ap; levisticum officinale w. d. j. koch aphis fabae užice, 14.08.1999, žt; lilium sp. aphis spp. belgrade, zvezdara, 10.05.1997, žt; lolium italicum a. braun sitobion avenae (fabricius) obrenovac, 15.06.1989, op; lolium perene l. aphis fabae lebane, konjino, 06.06.2010, ss; lotus corniculatus l. aphis craccivora kruševac, slobodište, 22.06.2013, zk; lycopus europaeus l. aphis spp. valjevo, petnica, 17.05.1998, žt; lythrum salicaria l. aphis sp. dobanovci, 17.06.2011, žt; sićevačka klisura gorge, sićevo, 28.05.2013, zk; malus domestica borkh. aphis pomi de geer novi belgrade, 15.11.1992, op; niš, donji matejevac, 05.06.2013, vž; dysaphis plantaginea (passerini) niš, pasi poljana, 27.05.2013, vž; malus sp. aphis pomi de geer zemun, galenika, 18.06.2011, ap; malva sylvestris l. aphis umbrella (börner) belgrade, kalemegdan, 29.09.1990, op; novi belgrade, 23.11.1991, op; zemun, 07.12.1991, op; belgrade, radmilovac, 22.06.1995, op; višnjica, 15.06.2011, op; niš, mediana, 21.05.2013, ss; niš, quay, 02.07.2013, mđ; niš, trošarina, 28.09.2013, vž; niš, mediana, 25.10.2013, vž; matricaria chamomilla l. pro parte aphis fabae lebane, konjino, 01.06.2013, ss; matricaria inodora l. aphis fabae surčin, 12.05.1989, op; matricaria perforata mérat aphis fabae vlasinsko jezero lake, 05.08.2011, ss; medicago lupulina l. aphis craccivora užice, djetinja canyon, 26.06.1999, op; medicago minima (l.) bartal. aphis craccivora vlasinsko jezero lake, 05.08.2011, ss; medicago sativa l. aphis craccivora kosmaj mt., 24.05.1995, žt; palićko jezero lake, 06.09.1995, žt; bečej, 20.06.1996, žt; zrenjanin, 20.06.1996, žt; žabalj, biologica nyssana 5 (2)  december 2014: 113-121 ilić milošević, i. et al.  tritrophic accociations of lysiphlebus fabarum… 119 plant aphid finding 20.06.1996, žt; belgrade, besni fok, 10.06.1997, žt; kač, 10.06.1997, žt; perlez, 23.06.1997, žt; senta, 23.06.1997, žt; valjevo, petnica, 28.06.1997, ap; zemun, 20.05.2011, ap; novi belgrade, block 45, 06.06.2011, zk; bešnjaja mt., 14.06.2011, žt; zemun, galenika, 17.06.2011, ap; slankamen, 24.06.2011, žt; kragujevac, ilićevo, 06.07.2011, amb; melilotus album medik. aphis craccivora vlasinsko jezero lake, 28.06.2012, vž; melilotus officinalis (l.) pall. aphis craccivora žitište, 22.06.1997, žt; negotin, 01.07.2011, op; mentha aquatica l. aphis affinis del geurcio surčin, 20.05.1989, op; belgrade, radmilovac, 12.06.1995, op; užice, duboko, 18.06.1995, žt; belgrade, radmilovac, 09.06.2011, op; mentha longifolia (l.) huds. aphis afinis del geurcio vlasinsko jezero lake, 15.06.2013, ss; mentha piperita l. aphis affinis del geurcio kruševac, 18.06.2013, zk; musa sapientia cavendish aphis spiraecola patch kruševac, 21.06.2013, zk; aphis fabae kruševac, 21.06.2012, zk; nepeta nuda l. aphis nepetae kaltenbach bosilegrad, jarešnik, 22.07.2013, vž; aphis frangulae kaltenbach dukat mt., 06.08.2011, ap; aphis origani passerini beljanica mt., 12.07.1998, žt; oenanthe stenoloba schur aphis fabae vlasinsko jezero lake, 05.08.2011, mim; oenothera biennis l. aphis oenotherae oestlund novi belgrade, block 45, 05.06.2002, žt; onopordum acanthium l. brachicaudus carduii (l.) preševo, slavujevac, 10.06.2014, ss; papaver somniferum l. aphis fabae kanjiža, 08.06.1995, žt; pastinaca sativa l. aphis fabae belgrade, radmilovac, 22.08.1996, op; aphis spp. vlasinsko jezero lake, 05.08.2011, mim; phaseolus vulgaris l. aphis fabae kovačica, 22.06.1996, žt; aphis craccivora užice, duboko, 18.06.1995, žt; phragmites australis (cav.) trin. hyalopterus pruni (geoffroy) belgrade, radmilovac, 10.05.1995, op; picris hieracioides l. hyperomyzus picridis (börner & blunck) palićko jezero lake, 06.09.1995, žt; plantago lanceolata l. aphis fabae vlasinsko jezero lake, 05.08.2011, mim; polygonum aviculare l. myzus persicae suva planina mt., toponički put, 19.07.1997, žt; aphis polygonata (nevsky) zemun, 05.06.2011, ap; zemun, galenika, 09.06.2011, ap; polygonum lapathifolium l. capitophorus sp. van der goot niš, pantelej, 13.10.3013, vž; populus tremula l. capitophorus sp. van der goot niš, popovac, 22.05.2010, vž; prunus cerasifera ehrh. hyalopterus pruni (geoffroy) belgrade, karađorđev park, 08.06.1996, žt; phorodon humuli novi belgrade, 17.06.1993, op; prunus cerasifera pisardi (carrière) l. h. bailey phorodon humuli niš, mediana, 21.05.2013, vž; prunus cerasus l. myzus cerasi niš, pmf, 10.05.2013, vž; brachycaudus helichrysi belgrade, radmilovac, 10.05.1995, op; prunus persica (l.) batsch brachycaudus schwartzi belgrade, 02.05.1995, op; belgrade, radmilovac, 10.05.1995, op; smederevo, lugavčina, 24.05.1995, op; niš, pasi poljana, 27.05.2013, vž; punica granatum l. aphis nasturtii kaltenbach belgrade, crveni krst, 17.07.1996, žt; ranunculus sp. aphis fabae sićevačka klisura gorge, ostrovica, 28.05.2013, mđ; ribes sp. aphis schilderi (börner) belgrade, konjarnik, 06.06.2011, žt; robinia pseudacacia l. aphis craccivora deliblatska peščara sands, devojački bunar, 29.06.1992, op; niš, pmf, 02.06.2012, vž; slankamen, 24.06.2011, žt; rosa canina l. aphis spp. brestovik, 27.05.2011, žt; aphis spp. smederevo, 27.05.2011, žt; rosa sp. aphis spp. koštunići, 17.06.2011, žt; rubus sp. aphis ruborum bešnjaja mt., 14.06.2011, žt; sićevačka klisura gorge, ostrovica, 29.05.2010, vž; zemun, 06.06.2011, op; koštunići, 17.06.2011, žt; sićevačka klisura gorge, sićevo, 01.06.2012, ss; sićevačka klisura gorge, ostrovica, 12.05.2013, vž; tara mt., derventa canyon, 26.06.2014, ss; rubus caesius l. aphis ruborum kragujevac, čumić, 14.05.2011, amb; sićevačka klisura gorge, sićevo, 04.06.2011, vž; ivanovići, 05.06.2011, žt; kragujevac, biologica nyssana 5 (2)  december 2014: 113-121 ilić milošević, i. et al.  tritrophic accociations of lysiphlebus fabarum… 120 plant aphid finding grošničko jezero lake, 05.06.2011, amb; ljig, 05.06.2011, žt; kragujevac, ilićevo, 06.06.2011, amb; kragujevac, košutnjačko brdo, 13.06.2011, amb; aphis idaei sićevačka klisura gorge, sićevo, 23.06.2012, vž; rubus discolor boiss. aphis ruborum preševo, slavujevac, 10.06.2014, ss; aphis idaei tara mt., derventa canyon, 03.07.2012, ss; rubus fruticosus l. aphis idaei niš, matejevac, 23.06.2012, vž; rubus idaeus l. aphis ruborum čačak, zdravljak, 24.05.2000, žt; aphis idaei valjevo, petnica, 18.05.1998, žt; banja vrujci spa, berkovac, 30.04.2000, žt; rumex acetosella l. aphis fabae kač, 10.06.1997, žt; rumex obtusifolius l. aphis fabae belgrade, dušanovac, 29.05.1993, žt; rumex sp. aphis fabae tara mt., derventa canyon, 03.07.2012, vž; niš, trošarina, 22.05.2013, mđ; sićevačka klisura gorge, sićevo, 28.05.2013, zk; salix cinerea l. aphis farinosa j. f. gmelin šara mt., 21.07.1995, op; aphis farinosa j. f. gmelin zlatar mt., aljinovići, 12.07.1991, op; salvia nemorosa l. aphis salviae walker uzdin, 22.06.1997, žt; salvia officinalis l. aphis salviae walker sićevačka klisura canyon, ostrovica, 28.05.2013, mđ; salvia sclarea l. aphis craccivora niš, pantelej, 23.06.2012, vž; salvia sp. aphis craccivora niš, bubanj, 05.06.2013, mđ; salvia verticillata l. aphis spp. l. beljanica mt., resava, 11.07.1998, žt; crna trava, 22.07.2001, op; dukat mt., 29.06.2012, ss; sanguisorba minor scop. aphis sp. l. preševo, 05.06.2011, op; kalna, 11.06.2011, op; aphis sanguisorbae (schrank) niš, donji matejevac, 05.06.2013, vž; pčinja canyon, gornji starac, 10.06.2014, ss; scabiosa argentea l. aphis sp. l. pčinja canyon, prohor pčinjski, 10.06.2014, vž; macrosiphum sp. passerini pčinja canyon, prohor pčinjski, 10.06.2014, vž; solanum nigrum l. aphis fabae solanella theobald obedska bara swamp, 15.06.1996, žt; apatin, prigrevica, 27.09.1997, žt; srbobran, 27.09.1997, žt; vršac, 02.10.1997, žt; sonchus oleraceus l. uroleucon sonchi (l.) brestovik, 27.05.2011, op; symphytum officinale l. aphis fabaei glover obedska bara swamp, 02.06.2011, op; sićevačka klisura gorge, 28.05.2013, mđ; aphis spp. ečka, 31.05.2001, žt; valjevo, petnica, 20.05.1996, ap; tamarix sp. brachyunguis tamaricis (lichtenstein) niš, niška banja spa, 06.06.2013, vž; tanacetum parthenium (l.) sch. bip. aphis fabae šara mt., muržička reka river, 21.07.1995, op; beljanica mt., suvaja, 11.07.1997, op; tanacetum vulgare l. metopeurum fuscoviride stroyan vlasinsko jezero lake, 06.08.2010, ss; vlasinsko jezero lake, 15.06.2013, ss; jerma canyon, 19.07.2013, ss; vlasinsko jezero lake, 21.07.2013, ss; taraxacum officinale weber aphis taraxicicola blackman belgrade, dušanovac 08.05.1993, žt; belgrade, dušanovac, 29.05.1993, žt; belgrade, crveni krst, 14.06.1997, žt; tecoma radicans (l.) juss. aphis spiraecola patch niš, pmf, 21.05.2013, ss; thymus sp aphis serpylli koch zlatar mt., 13.07.1991, op; tordylium maximum l. aphis spp. suva planina mt., vetanska reka river, 16.07.1997, žt; tragopogon sp. aphis spp. belgrade, botanical garden, 24.06.1999, žt; trifolium sp. aphis craccivora surčin, 15.06.2011, op; therioaphis trifolii monell surčin, 15.06.2011, op; trifolium lagopus pourr. aphis craccivora pčinja canyon, prohor pčinjski, 10.06.2014, bz; trifolium pratense l. aphis craccivora beljanica mt., 31.07.1996, op; kruševac, slobodište, 13.07.2012, zk; trigonella procumbens (besser.) rchb. aphis craccivora uzdin, 22.06.1997, žt; kostolac, 02.06.2001, žt; triticum aestivum l. anoecia corni (fabricius) suva planina mt., vetanska reka river, 17.06.1997, žt; triticum vulgare vill. sitobion avenae (fabricius) leskovac, 01.06.2013, vž; typha latifolia l. rhopalosiphum nymphaeae (l.) niš, niška banja spa, 23.07.2013, vž; urtica dioica l. aphis fabae brestovik, 27.05.2011, op; smederevo, 27.05.2011, op; aphis sp. niš, popovac, 22.05.2010, vž; sićevačka klisura gorge, sićevo, 29.05.2010, ss; biologica nyssana 5 (2)  december 2014: 113-121 ilić milošević, i. et al.  tritrophic accociations of lysiphlebus fabarum… 121 plant aphid finding aphis urticata j. f. gmelin smederevo, lugavčina, 15.04.1990, op; kokin brod, 12.07.1991, žt; belgrade, dušanovac, 08.05.1993, žt; surčin, 23.05.1993, žt; surčin, 02.05.1995, žt; kanjiža, 08.06.1995, žt; belgrade, radmilovac, 20.05.1996, žt; omoljica, 13.06.1996, žt; čenta, 20.06.1996, žt; užice, trešnjica, 23.07.1996, žt; belgrade, radmilovac, 10.06.1997, žt; novi sad, 10.06.1997, žt; belgrade, radmilovac, 13.05.1998, žt; niš, popovac, 10.05.2010, vž; lebane, konjino, 06.06.2010, ss; stara planina mt., bojanine vode, 11.07.2010, ss; sićevačka klisura gorge, sićevo, 04.06.2011, vž; rogojevac, 16.06.2011, op; sićevačka klisura gorge, sićevo, 23.06.2012, ss; kruševac, pakašnica, 11.05.2013, zk; sićevačka klisura gorge, sićevo, 12.05.2013, vž; sićevačka klisura gorge, sićevo, 28.05.2013, zk; niš, bubanj, 31.05.2013, mđ; lebane, konjino, 01.06.2013, ss; sićevačka klisura gorge, sićevo, 06.06.2013, vž; sićevačka klisura gorge, sićevo, 22.04.2014, vž; urtica urens l. aphis fabae kumane, 20.06.1996, žt; valeriana officinalis l. aphis fabae vlasinsko jezero lake, 21.07.2013, ss; verbascum nigrum l. aphis verbasci schrank vlasinsko jezero lake, 05.08.2011, vž; verbascum sp. aphis verbasci schrank gornjane, 06.07.1990, op; belgrade, 12.10.1990, op; kopaonik mt., brzeće, 25.08.1998, op; lebane, konjino, 01.07.2012, ss; niš, popovac, 08.06.2013, vž; viburnum opulus l. aphis viburni scopoli belgrade, žarkovo, 19.04.1990, op; vicia cracca l. aphis craccivora vlasinsko jezero lake, 05.08.2011, mim; lebane, konjino, 31.05.2014, ss; prokuplje, bresničić, 15.06.2014, vž; vicia cordata wulfen aphis craccivora niš, mediana, 21.05.2013, ss; zea mays l. aphis fabae beljanica mt., 30.07.1996, op; stojkovic piperac et al. 2025, biologica nyssana 16(1) 297 16 (1) june 2025: 297-305 doi: 10.46793/biolnyss.16.1.17sp the impact of pond origin on species and functional diversity of fish communities original article milica stojković piperac university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18 000 niš, serbia milica.piperac@pmf.edu.rs (corresponding author) vladica simić institute of biology and ecology, faculty of science, university of kragujevac, radoja domanovića 12, 34000 kragujevac, serbia djuradj milošević university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18 000 niš, serbia received: march 24, 2025 revised: april 18, 2025 accepted: april 22, 2025 abstract: ponds are standing freshwater ecosystems, that play a significant role in supporting biodiversity and ecological functions. despite their small size, they support high species diversity, particularly fish communities, that are influenced by variations in environmental factors such as hydroperiod, size of the pond, and water quality. this study investigates the differences in fish species composition and functional diversity between natural and artificial ponds in southeastern serbia. the research reveals that functional diversity is more sensitive indicator of ecological differences between these ponds than species diversity. artificial ponds, formed through human activity, tend to harbor opportunistic fish species, often resulting in lower functional diversity, while natural ponds exhibit greater species richness and more specialized fish communities. the findings underscore the importance of functional diversity in maintaining ecosystem health and highlight the distinct ecological roles played by ponds of different origins in sustaining freshwater biodiversity. understanding these dynamics is essential for effective conservation and management strategies. key words: fish community, natural ponds, artificial ponds, species richness, functional diversity apstrakt: uticaj porekla bara na specijski i funkcionalni diverzitet ribljih zajednica bare predstavljaju stajaće slatkovodne ekosisteme koji imaju značajnu ulogu u očuvanju biodiverziteta i ekoloških funkcija. iako su male površine, karakteriše ih visoka raznovrsnost vrsta, posebno ribljih zajednica, koje su pod uticajem različitih ekoloških faktora kao što su hidroperiod, veličina bare i kvalitet vode. ovo istraživanje se bavi ispitivanjem razlike u sastavu ribljih vrsta i funkcionalnom diverzitetu između prirodnih i veštačkih bara u jugoistočnoj srbiji. rezultati ukazuju na to da je funkcionalni diverzitet bolji pokazatelj ekoloških razlika između ovih tipova bara u poređenju sa specijskim diverzitetom. veštačke bare, nastale pod uticajem ljudskih aktivnosti, često sadrže oportunističke riblje vrste, što dovodi do nižeg funkcionalnog diverziteta, dok prirodne bare karakteriše veće bogatstvo vrsta i prisustvo specijalizovanijih zajednica. dobijeni rezultati ističu značaj funkcionalnog diverziteta za očuvanje vodenih ekosistema i ukazuju na različite ekološke uloge koje bare različitog porekla imaju u očuvanju slatkovodnog biodiverziteta. razumevanje ove dinamike od suštinskog je značaja za efikasno planiranje strategija očuvanja i upravljanja vodenim ekosistemima. ključne reči: riblje zajednice, prirodne bare, veštačke bare, bogatstvo vrsta, funkcionalni diverzitet introduction ponds are small, standing aquatic ecosystems that play a critical role in supporting biodiversity and ecological processes such as nutrient cycling, primary production, water purification, and habitat provision. these ecosystems are characterized by their relatively small size, shallow depths, and the absence of significant water flow. although ponds are relatively small in size, they are highly productive environments that host a variety of organisms, including aquatic plants, invertebrates, amphibians, fish, and birds (biggs et al., 2005). ponds play a crucial role in regional biodiversity, particularly in supporting diverse and often rare fish communities (wezel et al., 2014). the high β diversity of ponds, driven by variations in features such as hydroperiod, size, origin, and physical and chemical properties, © 2025 stojković piperac et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 298 creates a mosaic of habitats that support a diverse array of aquatic life (de meester et al., 2005). these unique environmental conditions foster complex fish communities, often characterized by species that are adapted to the specific conditions of pond ecosystems (wezel et al., 2014). as such, ponds are not only essential for freshwater biodiversity but also serve as critical habitats for maintaining and enhancing the resilience of fish populations in fragmented landscapes (wetzel, 2001). ponds can vary significantly in terms of their origin, and these variations influence the biological communities that inhabit them (biggs et al., 2005). the formation process of a pond influences its physical and chemical properties, such as water depth, temperature, and nutrient levels, which in turn affect the types of species that can thrive in it. natural ponds are typically formed through geological or climatic processes over long periods. they vary in origin depending on local geography, climate, and ecological interactions (brönmark & hansson, 2017). artificial ponds are created by human activity, and their biological communities can differ considerably from those of natural ponds. the way they are constructed and their intended use (e.g., ornamental, agricultural, or stormwater management) influence key aspects of their biological structure, including species richness, community composition, and the presence of sensitive or specialist species (oertli et al., 2005). it has been shown that ponds differ significantly in terms of species richness and composition, meaning they possess high β biodiversity, thereby making a disproportionate contribution to regional biodiversity (oertli et al., 2002; williams et al., 2004). this is, of course, facilitated by their high variability in physicochemical properties and hydroperiod duration, which causes even spatially close ponds to exhibit unique flora and fauna (de meester et al., 2005). recent studies propose two main approaches for the evaluation ecosystem health, stability, and resilience using aquatic communities (pease et al., 2012; villéger et al., 2017; martini et al., 2021). one approach focuses on the taxonomic composition of the community and associated metrics, while the other emphasizes the functional characteristics of the observed community. according to the first approach, changes in water quality can lead to a noticeable shift in the taxonomic structure of aquatic communities, with only a limited number of tolerant species from the local pool likely to persist (blonder et al., 2014). this shift in taxonomic composition, and the subsequent simplification of community structure, can further drive alterations in functional diversity (petchey and gaston, 2002). functional diversity offers valuable insights into changes in the functional niches of species, as organisms are selected based on traits such as feeding behavior, physiology, and ecological role. bearing all this in mind, the objectives of our study were to: (i) test the differences between artificial and natural ponds in terms of fish species and functional diversity, and (ii) examine how environmental factors influence species and functional diversity in ponds of different origins. materials and methods study area the investigation was conducted in southeastern serbia, around 10 kilometers west of the city of niš (coordinates: 43°29′–43°30′n, 21°79–21°80′e). the study area includes six ponds, located along the south morava river, between the villages of batušinac and mramor. two sets of ponds were included in the study, each composed of three ponds, differing in terms of origin (natural and artificial) (fig. 1). artificial ponds are located along the right bank (pond 1, 2, 3) of the south morava river and were created by excavating sand and gravel during highway construction in the 1980s. on the other side, the set of ponds of the left side of the river (ponds 4, 5, 6) are natural and represent the remains of the former river channel. the bottoms of all three natural ponds are covered with submerged macrophytes, with significantly greater coverage in comparison to artificial ponds. in addition, littoral zone and shallower parts of the natural ponds are covered with emergent vegetation, which is, on the other hand, negligibly developed in the ponds created by gravel excavation. the sediment of natural ponds was dominated by a muddy substrate with organic material, while the substrate in the artificial ponds was mainly composed of gravel. sampling field work was performed during september 2016. fish communities were surveyed using the en 14011:2003 (ec, 2003) methodology. fish were sampled from a boat on three transect per each pond, using a dc aquatech ig 1300 electrofisher (2.6 kw, 80–470 v). the constant catch-per-unit-effort (cpue) of time (10 min) was provided. the fish density is expressed as the number of individuals caught per 1 min. each individual fish was identified to the species level according to its relevant morphological and anatomic features (kottelat and freyhof, 2007). water quality was assessed using the basic water quality parameters. dissolved oxygen content (do), biologica nyssana ● 16 (1) june 2025: 297-305 stojković piperac et al. ● the impact of pond origin on species and functional diversity of fish communities 299 biologica nyssana ● 16 (1) june 2025: 297-305 stojković piperac et al. ● the impact of pond origin on species and functional diversity of fish communities fig. 1. map of the study area with the position of the study sites. arabic numbers indicate artificial (1-3) and natural ponds (4-6). rs stands for republic of serbia oxygen saturation (do%), electroconductivity (ec), and temperature (t) were measured by a wtw multi 340i probe. the concentrations of ammonia nitrogen, nitrate nitrogen and orthophosphates were estimated using the spectrophotometer shimatzu uv–vis, while biological oxygen demand (bod) was measured using standard methods (apha, 1999). finally, the percentage of the total organic matter in the sediment (c-tot) was measured according to standard methodology given by walkley and black (1934). data analysis to assess differences between artificial and natural ponds in terms of fish species and functional diversity, specific diversity metrics were established. first, each fish species found in the present study is categorized with regard to trophic and reproductive guilds. the assignment of fish species into ecological guilds was based on the data developed for the efi (fame consortium, 2004), as well as our own observations (tab. 1). the proportional abundance for each group of fish was calculated as the number of individuals within this group divided by the number of all individuals in the sample. we constructed a functional guild matrix assigning each taxon to their associated reproductive (lithophils, phytophils, phyto-lithophils, ostracophils) and trophic group (omnivores, insectivores, or carnivores). based on the data regarding trophic and reproductive requrements, we assessed functional diversity (fd), functional evenness (feve), and functional dispersion (fdis) using the bat package (mammola and cardoso, 2020). following the methodology of mammola and cardoso (2020), we constructed a community functional hypervolume for each sample using the hypervolume package (blonder et al., 2014). alpha functional diversity was determined using the "kernel.alpha" function, which calculates the total volume of the functional hyperspace occupied by the species in the community (mammola and cardoso, 2020). functional evenness was evaluated with the "kernel.evenness" function, which measures the regularity in the distribution of functional elements across the guild space (mammola and cardoso, 2020; mason et al., 2005). we calculated functional dispersion using the "kernel.dispersion" function to determine the average distance between the guild space centroid and randomly chosen points within the hypervolume. functional dispersion serves as a measure of functional divergence, indicating how species abundance is distributed in niche space, and reflects the degree of divergence in the community's functional guilds (mason et al., 2005). finally, six species richness/diversity measures were calculated to estimate differences between groups of ponds: total taxa richness, number of individuals, margalef diversity index, evenness, shannon and simpson index (magurran, 2003). as the normality assumption was not met for 300 biologica nyssana ● 16 (1) june 2025: 297-305 stojković piperac et al. ● the impact of pond origin on species and functional diversity of fish communities table 1. fish species classification into ecological guilds according to their origin, trophic group and reproduction strategy family species origin trophic group reproduction strategy cyprinidae alburnus alburnus (linnaeus, 1758) native omnivore phyto-lithophilic carassius gibelio (bloch, 1782) invasive omnivore phytophilic chondrostoma nasus (linnaeus, 1758) native lithophilic cyprinus carpio linnaeus, 1758 native omnivore phytophilic squalius cephalus (linnaeus, 1758) native omnivore lithophilic pseudorasbora parva (temminck & schlegel, 1846) invasive omnivore phytophilic rhodeus amarus (bloch, 1782) native omnivore ostacophilic rutilus rutilus (linnaeus, 1758) native omnivore phyto-lithophilic cobitidae cobitis taenia linnaeus, 1758 native phytophilic centrarchidae lepomis gibbosus (linnaeus, 1758) invasive insectivore phyto-lithophilic esocidae esox lucius linnaeus, 1758 native carnivore phytophilic percidae perca fluviatilis linnaeus, 1758 native omnivore phyto-lithophilic siluridae silurus glanis linnaeus, 1758 native carnivore phytophilic ictaluridae ameiurus nebulosus (lesueur, 1819) invasive omnivore lithophilic tested parameters, we applied the mann-whitney post hoc test to compare whether the parameters differed across the pre-defined groups of ponds. a distance-based redundancy analysis (dbrda; anderson et al., 2008) was conducted to examine the relationships between the fish community, represented by 1) species diversity and 2) functional diversity, and relevant environmental variables at the investigated groups of ponds. before performing the dbrda, bray-curtis similarity matrices were generated for both the species and functional diversity datasets. environmental variables were tested for normality and log-transformed when necessary to meet normality assumptions. functional diversity and related parameters were calculated using r version 4.1.1 (r core team, 2016). univariate tests were conducted using the software spss version 19.0, while multivariate analyses (dbrda) ware carried out with primer version 6 and the permanova+ for primer package (anderson et al., 2008). results during the sampling campaign, a total of 372 individuals were collected from 6 surveyed ponds, represented by 7 families, 14 genera, and 14 species (tab. 1). the highest diversity was recorded within the family cyprinidae (8 species), while only one species was recorded within the other families. out of total number of species, four invasive fish species were detected. a total of 10 species and functional diversity measures were tested (fig. 2). mann–whitney tests confirmed that the medians of all tested parameters, except for the number of individuals, differed significantly between the previously defined pond groups (tab. 2, fig. 2). specifically, both functional and species diversity measures were significantly higher in natural ponds compared to artificial ones. in contrast, the only measure found to be higher in artificial ponds than in natural ones was the number of invasive species. the dbrda ordination plots clearly demonstrated that the separation of groups based on the functional diversity of the fish community was considerably more pronounced than that based on species diversity. specifically, the dbrda analysis of species diversity explained 73.5% of the variation in the fitted model and 57.4% of the total variation. in contrast, the dbrda analysis of functional diversity explained 91.1% of the variation in the fitted model and 70.6% of the total variation (fig. 3a, fig. 3b). 301 biologica nyssana ● 16 (1) june 2025: 297-305 stojković piperac et al. ● the impact of pond origin on species and functional diversity of fish communities fig. 2. box plots of species and functional diversity parameters. the middle solid lines in boxes are medians, box ends are first and third quartiles, whiskers represent maximum and minimum values table 2. medians (ranges) of selected species and functional diversity measures. asterisks in the column “u test” indicate significant result of mann-whitney u test for comparison of variables (*p<0.05, **p<0.01, ***p<0.001, ns=non-significant difference) measured parameter artifical ponds natural ponds u test functional richness 0.340 (0.1121-0.6975) 0.892 (0.5160-1.0525) *** functional dispersion 0.590 (0.5473-0.6721) 0.677 ( 0.5905-0.7262) ** functional evenness 0.347 (0.2886-0.5328) 0.440 (0.4160-0.4808) ** species richness 4 (3-5) 6 (3-9) ** number of individuals 23 (10-159) 28 (14-39) ns margalef index 0.834 (0.5880-1.6681) 1.384 (0.6379-2.5173) * species eveness 0.577 (0.2650-0.8787) 0.863 (0.6629-0.9687) ** shannon diversity 0.800 (0.2911-1.4143) 1.624 (0.8762-2.0046) *** simpson diversity 0.462 (0.1310-0.7818) 0.809 (0.5336-0.8804) *** % of invasive species 84.61 (63.6364-100) 54.16 (16.2162-93.5484) * discussion our findings indicate clear difference in species and functional diversity of fish assemblages in two groups of ponds differing in origin (natural and artificial) (tab. 2, fig. 2). indeed, artificial ponds, that appears after excavating gravel or other materials are usually colonized by fish species with opportunistic strategies (maday et al., 2023). early colonizers tend to be species that are highly adaptable to changing environmental conditions and capable of exploiting the newly available resources. these species are often r-strategists, characterized by high reproductive rates, short life spans, and a capacity to rapidly populate available habitats. they thrive in disturbed or unstable environments, where the competition is low, and conditions are unpredictable (begon et al., 2006). among the first to colonize are typically generalist species that can survive a range of conditions, including variable oxygen levels, temperature fluctuations, and the presence of few other competitors. opportunistic species, such as small cyprinids or livebearers, can quickly establish themselves due to their high reproductive output and ability to tolerate environmental variability (ricklefs & schluter, 1993). ultimately, the diversity of fish in newly excavated ponds is shaped by a combination of colonization events, environmental conditions, and species’ life history traits, with opportunistic species playing a crucial role in the early stages of ecological succession (connell & slatyer, 1977). for instance, the prussian carp is an opportunistic species known 302 biologica nyssana ● 16 (1) june 2025: 297-305 stojković piperac et al. ● the impact of pond origin on species and functional diversity of fish communities fig. 3. ordination plots of distance-based redundancy analysis (dbrda) of a) species and b) functional diversity parameters of fish community for its rapid reproduction and ability to expand into available niches. its gynogenetic reproduction allows it to thrive in environments with low genetic diversity, and it can adapt to conditions like low oxygen, high pollution, and fluctuating temperatures (tomljanović et al., 2012). this adaptability, combined with its high reproductive rate, enables the prussian carp to quickly colonize new habitats, often outcompeting native species and causing ecological damage (tomljanović et al., 2012). thus, the absolute constancy and high abundance of this species at all sites withing group of artificail ponds, contributed to the significantly higher abundance of invasive species as well as total number of individuals in comparison to natural ponds (fig. 2). the diversity of ponds that have formed as remaining of old river channels is often characterized by fish community structure shaped by the historical connection to the river system. these ponds are usually dynamic environments with a variety of fish species due to their physical and hydrological connection to the main river. over time, these habitats develop their own ecological characteristics, often containing a suit of species that are adapted to both the slow-moving waters of the pond and the more turbulent conditions of the river that once fed them (junk et al., 1989). those communities often include both riverine species that are capable of adapting to lentic ecosystems, as well as species that are typically found in standing waters. our results are consistent with this, since significantly higher species and functional richness were observed on sited from the natural ponds (tab. 2, fig. 2). common fish found in these habitats may include species such as cyprinids, perch, and catfish, which are well-adapted to both slow-flowing and still waters. these species are often more tolerant of changes in water quality, temperature fluctuations, 303 biologica nyssana ● 16 (1) june 2025: 297-305 stojković piperac et al. ● the impact of pond origin on species and functional diversity of fish communities and lower oxygen levels compared to those in more stable riverine habitats (junk et al., 1989; penczak et al., 2000). from a functional perspective, the fish community in natural ponds typically includes a combination of opportunistic species and specialist species. opportunistic species such as small cyprinids and livebearers tend to dominate during the early stages of succession, rapidly exploiting the available resources and reproducing in abundance. over time, however, the pond may support a more diverse functional community, including predators (e.g., larger piscivorous fish) and detritivores (e.g., bottom-feeding species), which contribute to the cycling of nutrients and the overall health of the ecosystem (penczak et al., 2000). species diversity indices describe the structure of an assemblage, but their connection to ecological functions, such as productivity or stability, are still not well understood (kwak & peterson, 2007). species diversity indices have been criticized for their limited biological relevance and should be seen as just one of many tools for describing assemblage structure they are not a replacement for thorough analysis. nevertheless, cardinale et al. (2012) emphasized that increased species diversity in freshwater ecosystems may lead to the enhanced ecosystem stability. despite the fact that some authors encourage the utility of species diversity indices, functional diversity has been considered to be the most effective measure of diversity for detecting the positive impact of biodiversity on ecosystem functioning and services (balvanera et al., 2006; díaz et al., 2006). indeed, the results of this study shows that functional structure of fish community appears to be more sensitive and robust in discriminating origin-divided pond groups, in comparison to species diversity measures (fig. 3a, b). conclusion in conclusion, functional diversity is critically important for maintaining ecosystem functioning and services. the complex relationship between species traits and ecosystem services demonstrates that the loss of species with specific functional traits can lead to biased impacts on ecosystem processes. given that species with certain traits are more vulnerable to biodiversity loss, conserving functional diversity is essential to preserving the integrity and resilience of ecosystems. the origin of a pond profoundly shapes the biological communities within it. natural ponds tend to support diverse, often specialized species that have adapted to the particular conditions of the pond, such as floodplain ponds that support species adapted to fluctuating water levels. artificial ponds, on the other hand, tend to have a more controlled ecosystem, which can limit biodiversity but allows for human intervention in managing species and water quality. understanding the origin of a pond is crucial for understanding its ecological function, biodiversity, and the challenges it faces in a changing environment. acknowledgements. this research was supported by the ministry of science, technological development and innovations of the republic of serbia [grant no.: 451-03137/2025-03/200124]. references apha (1999). standard methods for the examination of water and wastewater (20th ed.). american public health association, washington, dc. balvanera, p., pfisterer, a. b., buchmann, n., he, j.s., nakashizuka, t., raffaelli, d., & schmid, b. 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(2004). comparative biodiversity of rivers, streams, ditches and ponds in an agricultural landscape in southern england. biological conservation, 115, 329–341. doi:10.1016/s0006-3207(03)00153-8. grigorov & pavlovic 2021, biologica nyssana 12(2) 12 (2) december 2021: 123-129 doi: 10.5281/zenodo.5759850 quality control, phenolic content and in vitro antioxidant potential of orange mullein flower and leaf original article maja grigorov department of pharmacy, faculty of medicine, university of niš, bulevar dr zorana ðinđića 81, 18000 niš, serbia maja.grigorov@gmail.com (corresponding author) dragana pavlović department of pharmacy, faculty of medicine, university of niš, bulevar dr zorana ðinđića 81, 18000 niš, serbia received: august 23, 2021 revised: november 24, 2021 accepted: december 02, 2021 abstract: verbascum species are traditionally used in the treatment of respiratory disorders. orange mullein (verbascum phlomoides l.) is one of three species that are the official source of drug mullein flower (verbasci flos). our study aimed to determine the overall quality of the v. phlomoides flos and v. phlomoides folium, the contents of polyphenols, tannins, flavonoids, and phenylpropanoid derivatives alongside the antioxidant capacity of their ethanolic extracts. quality control tests were conducted according to monographs of ph. eur. 10.0, secondary plant metabolites were quantified spectrophotometrically while the antioxidant potential was estimated by two complementary methods (dpph free radical scavenging and β-carotene bleaching assay). the higher contents of examined compounds were determined in flower extract although the leaf extract showed a stronger antioxidant capacity. we can conclude that verbascum phlomoides is a rich source of biologically valuable phenolics, it possesses considerable antioxidant capacity and investigated herbal drugs fulfill pharmacopeia’s quality requirements. key words: verbascum phlomoides, overall quality, polyphenols, flavonoids, antioxidant activity apstrakt: ispitivanje opšteg kvaliteta, sadržaj fenola i in vitro antioksidativni potencijal cveta i lista krupnocvetne divizma vrste roda verbascum se tradicionalno koriste u lečenju respiratornih poremećaja. krupnocvetna divizma (verbascum phlomoides l.) je jedna od tri vrste koje se koriste za dobijanje biljne droge cvet divizme (verbasci flos). cilj naše studije bio je da se utvrdi opšti kvalitet droge v. phlomoides flos i v. phlomoides folium, kao i sadržaj polifenola, tanina, flavonoida i derivata fenilpropanoida i ispita antioksidativna aktivnost etanolnih ekstrakata. opšti kvalitet droge ispitan je u skladu sa ph. eur. 10.0, sekundarni biljni metaboliti su određeni spektrofotometrijski, dok je antioksidativna aktivnost ispitivana pomoću dve metode (dpph metoda i test inhibicije obezbojavanja β-karotena). veći sadržaji ispitivanih jedinjenja utvrđeni su u ekstraktu cveta, iako je ekstrakt lista pokazao jači antioksidativni kapacitet. možemo zaključiti da je verbascum phlomoides bogat izvor biološki vrednih fenola, da poseduje značajan antioksidativni kapacitet i da ispitani biljni lekovi ispunjavaju zahteve farmakopeje u pogledu kvaliteta. ključne reči: verbascum phlomoides, ispitivanje opšteg kvaliteta, polifenoli, flavonoidi, antioksidativna aktivnost introduction orange mullein (verbascum phlomoides l.) belongs to the genus verbascum from the family scrophulariaceae (georgiev et al., 2011). this genus comprises about 360 species around the world. they are mostly represented in europe (95 species, 23 of which are also found in serbia), asia, and north america (georgiev et al., 2011; mihailović et al., 2016). plant species of this genus are known as mullein (georgiev et al., 2011). mulleins are flowering biennial plants (rarely perennial or annual), which form a rosette of leaves in the first year and flowering stems in the second year (georgiev et al., 2011; marian et al., 2018). they reach a height of 0.3-2 m (georgiev et al., 2011). yellow flowers are grouped under bracts. they bloom from june to august, sometimes in september (marian et al., 2018). traditionally, verbascum species are used in © 2021 grigorov & pavlović. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 123 the treatment of respiratory disorders, such as dry cough, bronchitis, tuberculosis, asthma (georgiev et al., 2011; selseleh et al., 2019). yet, these species show beneficial effects in hemorrhoids, wounds, rheumatism, diarrhea, eczema, and other types of inflammatory conditions (luca et al., 2019), burns and, according to some literature data, are also effective against headache (mihailović et al., 2016). a mild sedative, as well as mild diuretic and soothing effects on the ureter, have been noticed. verbascum species are sometimes used to reduce urinary inflammation (süntar et al., 2010; selseleh et al., 2019), and as a tea to reduce abdominal pain (süntar et al., 2010). mulleins have been used since ancient times and in recent years their popularity is growing (georgiev et al., 2011). nowadays, herbal products for tea preparation from flowers of v. thapsus, v. densiflorum and v. phlomoides can be found in american and european health stores (mihailović et al., 2016). according to data from 2010, verbascum spp. is used in six european union countries as traditional herbal supplements or herbal medicinal products (mihailović et al., 2016). verbascum phlomoides is one of three species of the genus verbascum (the other two are: v. densiflorum bartol, and, rarely used due to some reported deficiencies, v. thapsus l. (georgiev et al., 2011)) that are the source of the drug mullein flower (verbasci flos) (ema, 2007). it is a biennial plant, known as a very rich phytochemical source (paun et al., 2016). the most common compounds of the drug verbasci flos are iridoid glycosides (aucubin, catalpol, 6-xylosilaukubin, and 6-xylosylcatalpol), flavonoids (tamarixetine 7-rutinoside, tamarixetin 7-glucoside, apigenin, drimolphein and luteolin), phenylethanoid glycosides (verbascoside), triterpene saponins (verbascosaponin), polyphenols, polysaccharides, phytosterols, oleic acid (ema, 2007). due to such complex composition, verbascum species show many beneficial effects on human health. numerous studies have confirmed that the fractions of saponins, iridoids, and phenylethanoids are responsible for their anti-inflammatory effect (mihailović et al., 2016). in numerous developing countries, traditional medicines are preferred over conventional ones. the interest in herbal products is in constant increase in developed countries too. consequently, the quality, safety and efficacy of herbal products must be at the highest level (he et al., 2015; van wyk & prinsloo, 2020). that is why a comprehensive specification must be developed for each herbal substance even if the starting material for the manufacture of the finished product is a herbal preparation. the specification should be established based on recent scientific data and the monographs of the european pharmacopoeia (sarfaraz, 2007) and other pharmacopeias (like german pharmacopoeia, swiss pharmacopoeia, british herbal pharmacopoeia). considering the mentioned pharmacological potential of verbascum species, our study aimed to determine the overall quality of the verbasci flos and verbasci folium as well as the content of polyphenols, tannins, flavonoids, and phenylpropanoid derivatives in their ethanolic extracts. estimation of antioxidant activity of these extracts was added into the study since this activity could be linked to many beneficial health outcomes. materials and methods plant material and extracts the plant material (flowers and leaves) was collected in the vicinity of bosilegrad during august 2019. botanical identification was performed by prof. bojan zlatković (faculty of science and mathematics, university of niš). the voucher specimens are stored in the herbarium of the faculty of science and mathematics, university of niš (herbarium code: 14506). after drying at room temperature, in a dark place with a constant flow of air, the plant material was ground using a laboratory mill and stored in glass bottles until the examination. extracts were prepared by percolation with 50% ethanol (vpf – v. phlomoides flower extract and vpl – v. phlomoides leaf extract) according to european pharmacopeia 10.0 (2019) (european pharmacopeia, 2019), followed by total removal of the solvent in a rotary vacuum evaporator at 40 °c. yields after evaporation are expressed in % of dry plant material used and as der (drug:extract ratio specifies the initial amount of drug used for the preparation of a certain amount of extract). the dry extracts were preserved for one month in the refrigerator until examinations. the determinations were performed in extracts dissolved in 50% alcohol. chemical reagent and instrumentation all reagents used in this study were of analytical grade. rutin and gallic acid were purchased from sigma-aldrich co. (sigma-aldrich co., st louis, mo), and catechin from extrasynthese (lyon, france). spectrophotometric measurements were performed on an evolution 60 thermo scientific spectrophotometer (fisher scientific, loughborough, u.k.) and multiskan ascent no354 (thermo labsystems, finland) elisa microplate reader. for incubation incuterm raypa® trade (catalonia, spain) was used. 124 biologica nyssana ● 12 (2) december 2021: 123-129 grigorov & pavlović ● quality control, phenolic content and in vitro antioxidant potential of orange mullein flower and leaf 125 quality control the overall quality examinations of herbal drugs included a swelling index, loss on drying, total ash, ash insoluble in hydrochloric acid following the regulations of ph. eur. 10.0 (2019) and by the monograph of the examined herbal drug verbasci flos (european pharmacopeia, 2019). these parameters are listed in the part tests of the monograph. total polyphenols and total tannins the content of total polyphenols was determined by folin–ciocalteu colorimetric method (makkar et al., 2000). the absorbances of the samples and standards were measured at 725 nm, and the results were expressed as gallic acid equivalents per g of extract. the total tannin content was determined by the same folin-ciocalteu procedure (makkar et al., 2000) after removal of tannins using insoluble binder (polyvinylpolypyrrolidone, pvpp). the test was performed with a clear supernatant, and the results were also expressed as gallic acid equivalents per g of extract. phenylpropanoid derivates the content of total phenylpropanoid derivatives was determined spectrophotometrically, according to the arnow method (arnow, 1937). this method is based on the reaction of derivatives of o-dihydroxycinnamic acid with sodium molybdate. absorbance was measured at 525 nm, and the phenylpropanoid content was expressed as the content of the corresponding hydroxycinnamic acid derivative. total flavonoids the total flavonoid content was determined spectrophotometrically by the alcl3 method at 430 nm (lamaison & carnat, 1990). results were expressed as a microgram of rutin per milligram of the sample (mg ru/mg). antioxidant activity radical scavenging activity was determined using stable free radical 1,1-diphenyl-2-picrylhydrazyl (dpph). the determination was done according to the method described by chang et al (2010) (chang et al., 2010) with certain modifications. dpph free radical inhibition in percent was calculated according to: % dpph = (ab as / ab) x 100, where ab presents the absorbance of the control reaction (containing ethanol instead of the test solution) and as is the absorbance of the sample. a regression curve was constructed, and the obtained results were presented as the ic50 value, which indicates the sample concentration required to remove 50% of dpph free radicals. rutin was used as a reference compound. concentrations are expressed in µg/ml. β-carotene bleaching test (bcbt) was done according to the method described by koleva et al. (2002) with slight modifications. the absorbance was measured using an elisa reader at a wavelength biologica nyssana ● 12 (2) december 2021: 123-129 grigorov & pavlović ● quality control, phenolic content and in vitro antioxidant potential of orange mullein flower and leaf ta bl e 1. r es ul ts o f t he o ve ra ll qu al ity te st in g of o ra ng e m ul le in fl ow er a nd le af sw el lin g in de x l os s o n dr yi ng (% ) t ot al a sh (% ) a sh in so lu bl e in hy dr oc hl or ic a ci d (% ) % o f d ry pl an t m at er ia l us ed d e r * ph . eu r.1 0 ph . eu r.1 0 ph . eu r.1 0 ph . eu r.1 0 ve rb as ci flo s 9. 20 ±1 .3 2 >9 9. 06 ±0 .4 7 <1 2 3. 5± 0. 92 <6 0. 97 ±0 .1 2 <2 40 .8 2% 2. 45 :1 ve rb as ci fo liu m 14 .7 5± 1. 86 >9 7. 28 ±0 .3 1 <1 2 6. 05 ±0 .0 6 <6 1. 76 ±0 .2 3 <2 12 .9 3% 7. 73 :1 * d ru g/ ex tra ct ra tio 126 of 450 nm after shaking the plate on a microplate shaker. the initial absorbance was measured, the plate was placed in an incubator at 47 °c for 120 minutes, afterward, the absorbance was measured again. the ability to inhibit β-carotene decolorization is expressed as a percentage of inhibition according to the formula: % inhibition = (a120 / a0) x 100, where a120 is the absorbance measured at t=120 min and a0 is the absorbance measured at t=0 min. after the regression curve was constructed, the ic50 value (concentration required to inhibit β-carotene bleaching to 50%) was calculated and expresses as the average value of three independent experiments in µg/ml. statistical analysis all tests and experiments were performed in triplicates, and the experimental results were presented as the mean of three consecutive measurements ± standard deviation. results results of the overall quality testing of dry flowers and leaves alongside pharmacopoeial demands are presented in tab. 1. tab. 2 shows the contents of total polyphenols, total tannins, phenylpropanoid derivatives, and total flavonoids and the results of in vitro antioxidant activities of ethanolic extracts vpf and vpl. discussion the obtained results of the overall quality testing of the herbal drug are in accordance with the recommendations of the ph. eur. 10.0 (european pharmacopeia, 2019). the extract yields were 40.82% and 12.93% for flower and leaf extract, respectively. many herbal drugs are of specific therapeutic or pharmaceutical utility because of their swelling properties (e.g., gums and those containing an appreciable amount of mucilage, pectin or hemicellulose) (who, 1998). the swelling index of v. phlomoides flower and leaf define mucilage content and present a valuable parameter of drug quality. in our study, verbasci folium showed a higher swelling index than verbasci flos, although both parameters are in accordance with pharmacopoeial demand (>9). polyphenols show numerous biological effects, such as anti-inflammatory, antioxidant, immunomodulatory, anticancer, cardioprotective effects (yahfoufi et al., 2018). their content was determined by the colorimetric folin–ciocalte method, which is based on the reducing ability of the hydroxyl groups of polyphenolic compounds. the polyphenolic compounds are oxidized, and the resulting phenoxide anion reduces the folinciocalteu reagent to a blue-colored ion. based on the results, shown in tab. 2, the plant species v. biologica nyssana ● 12 (2) december 2021: 123-129 grigorov & pavlović ● quality control, phenolic content and in vitro antioxidant potential of orange mullein flower and leaf phlomoides is rich in polyphenols and their higher content was determined in the vpf compared to the vpl. these results are close to the results of the determination of polyphenols obtained by mihailović et al. (2016) in the aqueous extract (51.3±0.3 mg gallic acid equivalents (gae)/g extract), while content in the methanol extract in the mentioned study was slightly higher (88.2±1.6 mg gae/g extract) (mihailović et al., 2016). the total polyphenol content expressed as a percentage of gallic acid is 4.91, a bit higher than the 4.18%, which is the content of polyphenols given by ema (ema, 2007). our results are also similar to the results of determining polyphenols in the butanol extract of v. phlomoides (grigore et al., 2013). numerous epidemiological data indicate beneficial effects of tannins in the treatment of inflammation and skin injuries, as well as in preventing the development of chronic diseases (badal mccreath & delgoda, 2017). flavonoids are a group of polyphenols, with a lot of beneficial effects on human health. their antioxidant effect is most often discussed, but their anti-inflammatory, table 2. contents of total polyphenols, total tannins, phenylpropanoid derivatives and total flavonoids in ethanolic extracts of orange mullein flower and leaf and ic50 values of dpph test and bcbt total polyphenols (mg ga1/g extract) total tannins (mg ga1/g extract) phenylpropanoid derivates (ha2/g extract) total flavonoids (mg r3/g extract) ic50 in dpph test (µg/ml) ic50 in β-carotene bleaching test (µg/ml) vpf 49.15±2.70 25.19±4.17 6.32±0.19 16.71±0.91 257.61±2.62 164.24±7.11 vpl 39.70±4.12 16.56±1.82 7.10±0.01 12.81±0.49 157.04±3.86 132.59±5.08 1ga – gallic acid, 2ha – hlorogenic acid, 3r – rutin 127 biologica nyssana ● 12 (2) december 2021: 123-129 grigorov & pavlović ● quality control, phenolic content and in vitro antioxidant potential of orange mullein flower and leaf anticancer, antimutagenic effects are equally important (panche et al., 2016). the results of the determination of tannins and flavonoids show that the vpf is richer in these compounds than the vpl. this could be one of the explanations why is the mullein flowers rather than leaves used in folk medicine. our results of total flavonoids determination are close to the results obtained by mihailović et al. (2016) for the aqueous extract but lower than their results for the methanol extract (mihailović et al., 2016). armatu et al. (2011) determined the chemical compositions of two extracts of v. phlomoides collected in the southeastern part of romania. their results for the total flavonoid content are close to ours, with the butanol extract being richer in flavonoids, compared to the ethanol (armatu et al., 2011). paun et al. (2016) in their work determined the content of polyphenols and flavonoids in aqueous and ethanol extract of v. phlomoides and reported a higher content of polyphenols and flavonoids compared to ours (paun et al., 2016). the content of polyphenols and flavonoids, as well as other compounds in plant species, is influenced by numerous factors, such as the region in which the species grows, the conditions in which it grows, the time in which plant material is collected, storage methods and many others (elmastaş et al., 2017). additionally, the determination of phenylpropanoids was included in this study since this class of secondary metabolites besides anti-inflammatory, antimicrobial, cytoprotective and wound healing effects could be beneficial for skin regeneration (pavlović et al., 2013). the total phenylpropanoid derivatives content in vpl was slightly higher than in vpf (tab. 2). there are several different methods for assessing the antioxidant capacity of natural products. however, given the range of different types of free radicals that could be generated under in vivo conditions and the different reaction mechanisms involved in the antioxidant capacity of antioxidants, there is still no universal method to determine antioxidant capacity accurately and qualitatively (mihailović et al., 2016). in our work, we used the dpph and the bcbt tests. the obtained results of the dpph test show that the vpf has a lower antioxidant capacity (ic50=257.61±2.62 µg/ml) compared to the vpl (ic50=157.04±3.86 µg/ml). the antioxidant activity tested in the bcbt test was also higher for vpl extracts compared to vpf extracts, although the difference in ic50 values is not so pronounced (132.59±5.08 µg/ml and 164.24±7.11 for vpl and vpf, respectively). it is important to underline that the antioxidant capacity was dosedependent (higher doses of extract demonstrated higher effects which was the base for the construction of regression curves). since plant extracts are a mixture of different compounds, more different antioxidant tests are needed for a full understanding of the properties of one particular extract (pavlović et al., 2013). as expected, antioxidant activities of rutin, used as reference substance (positive control), were much lower (ic50 values in dpph and bcbt: 2.68±0.25 µg/ml and 33.29±4.06 µg/ml, respectively) compared with antioxidant activities of tested extracts. conclusions based on the presented results, we can conclude that the plant species verbascum phlomoides contains significant amounts of polyphenols, tannins, and flavonoids, and due to the well-known positive effects of these compounds on human health, this species deserves to remain in the focus of our study. also, the contents of these compounds were higher in flowers compared to leaves. this could be one of the explanations why is the mullein flowers rather than leaves used in folk medicine for topical applications. yet, the leaf extract showed a stronger antioxidant capacity in applied tests. further investigations and detailed chemical profiling are needed to assess the relevance of ethnopharmacological application and define the potential health benefits of orange mullein. references armatu, a., bodirlau, r., nechita, c.b., niculaua, m., teaca, c.a., ichim, m., spiridon, i. 2011: characterization of biological active compounds from verbascum phlomoides by chromatography techniques. i. gas chromatography. romanian biotechnological letters, 16(4): 6297-6304. arnow, e. 1937: colorimetric determination of the components of 3,4-dihydroxyphenylalaninetyrosine mixtures. journal of biological chemistry, 118: 531-537. badal mccreath, s., delgoda, r. 2017: pharmacognosy: fundamentals, applications and strategies. elsevier science. 2246 p. chang, l.c., zhang, j.l., chen, y.r., kuo, y.l.m., huang, p.j., huang, c.h., lee, h.k., wu, c.y., kuo, h.y. 2010: antioxidant and antiinflammatory phenylpropanoid derivatives from calamus quiquesetinervius. journal of natural products, 73: 1482–1488. council of europe 2019: european pharmacopeia. 10th edition. council of europe: european directorate for the quality of medicines and healthcare, strasbourg. dimitrova, p., alipieva, k., grozdanova, t., simova, s., bankova, v., georgiev, m.i., popova, m.p. 2018: new iridoids from verbascum nobile and their effect on lectin-induced t cell activation and proliferation. food and chemical toxicology, 111: 605–615. elmastaş, m., demir, a., genç, n., dölek, ü., güneş, m. 2017: changes in flavonoid and phenolic acid contents in some rosa species during ripening. food chemistry, 235: 154-159. ema 2018: assessment report on verbascum thapsus l., v. densiflorum bertol. 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medicinal plant materials. who, geneva. 187 p. 129 biologica nyssana ● 12 (2) december 2021: 123-129 grigorov & pavlović ● quality control, phenolic content and in vitro antioxidant potential of orange mullein flower and leaf yahfoufi, n., alsadi, n., jambi, m., matar, c. 2018: the immunomodulatory and antiinflammatory role of polyphenols. nutrients, 10(11): 1618. ćirković et al. 2023, biologica nyssana 14(1) 14 (1) june 2023: 31-38 doi: 10.5281/zenodo.8027114 results of the first analysis of tadpoles’ diet and determination of microplastics presence of rana, bufo and bufotes species from different localities in serbia original article glorija ćirković department of biology and ecology, faculty of science, university of kragujevac, radoja domanovića 12, kragujevac, serbia glorija.cirkovic@pmf.kg.ac.rs (corresponding author) aleksandra rakonjac department of biology and ecology, faculty of science, university of kragujevac, radoja domanovića 12, kragujevac, serbia rastko ajtić department of biology and ecology, faculty of science, university of kragujevac, radoja domanovića 12, kragujevac, serbia received: october 14, 2022 revised: april 19, 2023 accepted: may 02, 2023 abstract: nutrition is one of the main processes in living beings because it provides energy. amphibians are an essential link in food chains, but they have not been sufficiently studied. it is believed that most anuran larvae and tadpoles are omnivorous, but the trophic status of many tadpoles has not been precisely determined. microplastics (mps) are one of the major threats to aquatic ecosystems and can be ingested by various groups of aquatic organisms, such as plankton, shellfish, fish, and amphibians, but current knowledge of mps occurrence in these aquatic organisms is still scarce. in this research, the intestinal contents of tadpoles of four anuran species were analyzed to assess nutrition and the presence of microplastics. the results show that the largest percentage of tadpoles’ diet consists of diatoms from the genera achnanthidium and navicula, and detritus. the remains of some invertebrates were also present in the material. microplastic analysis proved the presence of mps in all investigated samples. the data obtained in this research represent the first concrete results due to which it is necessary to continue extensive research at several levels. key words: tadpoles, nutrition, microplastics, rana, bufo, bufotes apstrakt: rezultati prve analize ishrane punoglavaca i utvrđivanje prisustva mikroplastike kod vrsta iz rodova rana, bufo i bufotes sa različitih lokaliteta u srbiji ishrana je jedan od ključnih procesa živih bića jer predstavlja glavni izvor energije. vodozemci su važna karika u lancima ishrane, ali nisu dovoljno proučavani. veruje se da je većina larvi bezrepih vodozemaca, odnosno punoglavaca, omnivorna, ali trofički status mnogih punoglavaca nije precizno utvrđen. mikroplastika (mp) je jedan od glavnih zagađivača u vodenim ekosistemima pa je različite grupe vodenih organizama, kao što su plankton, školjke, ribe i vodozemci mogu uneti u organizam, ali trenutno znanje o prisustvu mp u ovim vodenim organizmima je još uvek nedovoljno. u ovom istraživanju analiziran je sadržaj creva punoglavaca četiri vrste bezrepih vodozemaca kako bi se procenila ishrana i prisustvo mikroplastike. rezultati pokazuju da najveći procenat ishrane punoglavaca čine silikatne alge iz rodova achnanthidium i navicula, kao i detritus. takođe, u sadržaju creva bili su prisutni i ostaci nekih beskičmenjaka. analize mikroplastike dokazale su da su čestice mp prisutne u svim ispitivanim uzorcima. podaci dobijeni u ovom istraživanju predstavljaju prve konkretne rezultate zbog kojih je neophodno nastaviti dodatna istraživanja na više nivoa ključne reči: punoglavci, ishrana, mikroplastika, rana, bufo, bufotes introduction knowing a species’ trophic status and spectrum of nutrition is of particular importance, given that food is the primary link between the species and its habitat. nutrition is an ongoing process and therefore can be crucial to the ecology of a particular species. also, feeding is the most important activity of living beings, primarily because of obtaining the energy needed to perform life activities. amphibians © 2023 ćirković et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 31 are vertebrates that can be at the top of food chains, although they are more often found in the middle of the food chains and form the link between lower taxa, such as insects, and higher ones, such as birds and mammals (cogălniceanu et al., 2000). they are a very important link in food chains because, due to their life history, they connect aquatic and terrestrial ecosystems. adults are, in most cases, carnivores. they feed on most invertebrates, and on some vertebrates. in contrast to adults, larvae of tailed and anuran amphibians show much greater variability in diet types. some feed on plankton, some are microphagous, herbivorous, or detritivorous, and there are also predatory larvae (mcdiarmid & altig, 2010). even though they are very important, they have been insufficiently investigated and the trophic status of many species of amphibians, both larvae and adults, has not been sufficiently studied. in the literature related to the balkan peninsula, there is very little information about the diet of representatives of the order anura (popović et al., 1992; simić et al., 1992; bjelić et al., 1996; paunović, 2000; paunović et al., 2010; paunović, 2011; crnobrnja-isailović et al., 2012; čađenović, 2014), and the diet and morphology of the larval anuran, i.e. tadpoles, have been particularly poorly researched (sidorovska et al., 2002; ilić, 2020). most aquatic tadpoles are primary consumers that metamorphose into terrestrial adults, thereby transferring energy from aquatic habitats to food chains in terrestrial ecosystems (trakimas et al., 2011). because of their different habitats and the variety of life forms and feeding structures, different species of anuran amphibian larvae use several food sources including periphyton and detritus (ranvestel et al., 2004). many tadpoles are benthic in their habitats, spending most of their time at the bottom of ponds, and relatively little in free water, so the composition of the benthos in the aquatic ecosystems they inhabit is important for their nutrition. tadpoles from the bufonidae and ranidae families are, among others, with these characteristics. however, some species from these families sometimes, except for benthic organisms, feed on organisms from the surface of the water (bacterial film) (rana dalmatina), scraping periphyton, corpses, and eggs of the same or other species or plankton (rana temporaria, bufo bufo) (wells, 2007). species r. dalmatina, r. temporaria and b. bufo in serbia can be syntopic. they enter aquatic habitats only during a short reproductive period in early spring, where tadpoles develop after hatching from eggs. the species r. dalmatina, b. bufo and bufotes viridis inhabit the entire territory of serbia, while the species r. temporaria is found on 10-50% of the entire territory of serbia (vukov et al., 2013). the increased use of plastic products in human activities has caused concern for the environment. when plastic reaches (directly or indirectly) aquatic ecosystems, it undergoes degradation, after which there is a change in particle size and density. however, most plastic products are very resistant to biochemical degradation and therefore persist for a long time in the environment. over time, they break down into smaller and smaller fractions called microplastics (from 1 to 5 μm in size) (pastorino et al., 2022). there are two types of microplastics: primary and secondary. the primary represents plastic particles that are produced and that enter the composition of various products (for example cosmetics), and the secondary is created by the decomposition of macroplastics or during the use and disposal of plastic products such as plastic packaging (pastorino et al., 2022). microplastics (mps) are one of the major threats to aquatic ecosystems and can be ingested by various groups of aquatic organisms, such as plankton, shellfish, fish, and amphibians, but current knowledge of mps occurrence and impact on these aquatic organisms is still scarce (hu et al., 2018; kolenda et al., 2020). also, despite the key role of amphibians in the food webs and the potential to transfer contaminants through trophic levels and from freshwater to terrestrial ecosystems, the data on their capacity to accumulate mps are very scarce compared to other vertebrates (pastorino et al., 2022). there are only a few published field studies about mps accumulation by tadpoles (hu et al., 2018; karaoğlu & gül, 2020; kolenda et al., 2020). the main goal of this research is to obtain results on the nutrition of tadpoles from different habitats in serbia and thus supplement knowledge about the nutrition of anuran larvae and contribute to determining their trophic status. another goal of this study was to determine whether tadpoles digest microplastics which can serve as a good initial step for further analyses of whether the mps affect the development and metabolism of tadpoles. materials and methods sampling we used for the analysis 130 tadpoles of rana dalmatina, r. temporaria, bufo bufo and bufotes viridis from 11 localities in serbia (tab. 1; fig. 1). the tadpoles were collected using standard nets for amphibians and then preserved in alcohol. the collection was obtained during spring (april, may) 2021. and 2022. the 70 tadpoles were used to investigate tadpoles’ diet, and 60 tadpoles 32 biologica nyssana ● 14 (1) june 2023: 31-38 ćirković et al. ● results of the first analysis of tadpoles’ diet and determination of microplastics presence of rana, bufo and bufotes species from different localities in serbia biologica nyssana ● 14 (1) june 2023: 31-38 ćirković et al. ● results of the first analysis of tadpoles’ diet and determination of microplastics presence of rana, bufo and bufotes species from different localities in serbia 33 were analyzed to estimate the presence of microplastics (tab. 1). the selected four species for this study were the most adequate for analysis because they were the most abundant in investigated localities. for future research, we plan to include other species and more localities. the tadpoles were from different developmental stages (most of them were 25 and 27), according to gosner’s simplified table for staging anuran embryos and larvae (gosner, 1960). determination of species tadpoles can be identified using the details of the overall body proportions, but the position of the spiraculum and the arrangement of tooth rows and other mouthpart details allow a confident identification to be made. for the identification of the tadpole samples, we used an identification key (ajtić & ćirković, in prep.) based on the specific arrangement of tooth rows and other mouthpart details. nutrition analysis to assess nutrition, intestinal contents were analyzed. a whole intestine was removed from each tadpole and contents were placed in ethanol (96%) and homogenized. depending on the abundance of particles, either the entire sample or a subsample was observed under the microscope at table 1. localities and a number of tadpoles used for intestinal content analysis, and developmental stages according to gosner (1960) species localities number of specimens for nutrition analysis number of specimens for microplastic analysis developmental stages rana temporaria golijska river 10 20 34, 38 trešnjica river (povlen) 5 10 25 srednji povlen 5 / 29 rana dalmatina medvednik 5 / 24,25 cer 5 5 25,38 golija 5 / 36 bufo bufo tometino polje 5 / 37 cer 5 5 27 zlatibor 5 10 26 besna kobila 5 / 25 bufotes viridis starac 10 10 28 zasavica 5 / 34 fig. 1. localities 34 biologica nyssana ● 14 (1) june 2023: 31-38 800x magnification. the observation was made by transects, photographed and, if possible, identified. algae were identified to the genera level using standard identification keys (krammer & langebertalot, 1986, 1988, 1991; john et al., 2011; jüttner et al., 2015; wehr et al., 2015). microplastics analysis the presence of microplastics was analyzed using the method described by hu et al. (2018) with some modifications. the specific number of tadpoles from each investigated species was separated in glass petri dishes. then, they were washed three times with distilled water and digested using h2o2 (30%) for no more than 72 h. the digested solutions were filtered through 47 mm diameter polycarbonate filters with a pore size of 5 µm. all filters were incubated at 60 °c for 4 h in a dryer and stored in dry glass petri dishes until further observation. mps were then searched for by means of a stereomicroscope. to avoid contamination, a cotton laboratory coat was worn during procedures, and before analysis, all liquids (pure water and h2o2) were filtered using filter paper (5 µm porosity), and all the equipment used during analysis was rinsed in distilled water. the procedural blanks were performed to assess whether the materials, solutions, equipment, or workspace were contaminated with microplastics. results and discussion the intestinal content analysis confirmed that the most significant percent of tadpoles’ nutrition were diatoms, i.e., algae from class bacillariophyceae, and the most present were specimens from the genera achnanthidium and navicula, and in a smaller amount, specimens from the genera surirella, pinnularia, and nitzschia (tab. 2). this may be a consequence of the period in which the tadpoles were feeding, as not all algae develop at the same time. their development depends on the season, the physical properties of substrata, and different environmental factors (murdock & dodds, 2007). the presence of algae from cl. xanthophyceae, especially genus tribonema was also noted. in some intestinal content, the representatives from the genus phacus (cl. euglenophyceae, phylum euglenozoa; specimens from srednji povlen) (tab. 2) were found as well. detritus was also found in intestinal content, as well as the remains of invertebrates that could not be determined. our results showing that the diet of tadpoles is not limited to algae is consistent with other studies (altig et al., 2007), where it was reported that tadpoles consume a range of different particles in their diet, including benthic or planktonic algae, vegetation, bacteria, fungi, zooplankton, and animal flesh, as well as inorganic particles, such as sand. it is assumed that the diverse diet of most tadpoles actually reflects an indiscriminate diet. the type and dynamics of tadpole nutrition are related to the structure and morphology of the oral apparatus, but also to the availability and diversity of nutritional resources in the habitats where metamorphosis takes place. a smaller number of studies (peterson & boulton, 1999) have focused on tadpoles that feed on algae by scraping from the substrate, but herbivorous tadpoles have been shown to alter the structure and/or biomass of algae in permanent lentic aquatic ecosystems. some researchers claim that the contents of tadpoles’ guts represent an inexhaustible sample of algae (huang et al., 2003; skelly & golon, 2003), which is confirmed by our results. it is believed that tadpoles, many of which are primary consumers, greatly influence on the structure and functioning of ecosystems by changing the composition of algae, i.e. the composition of primary producers, as well as on the dynamics of organic matter in many aquatic habitats. previous research has shown that primary production, nutrient cycling, plant residue decomposition, and invertebrate populations change when tadpoles are removed from habitats or reduced in numbers (whiles et al., 2006). the substrate in aquatic ecosystems is a significant source of food for tadpoles. the tadpoles use organic matter from detritus in their nutrition, benthic algae from phyla bacillariophyta and chlorophyta, and remains of all other algae, invertebrates, and vertebrates. this refers to tadpoles of many species in the families bufonidae, discoglossidae, hylidae, leptodactylidae, megophryidae, myobatrachidae, pelobatidae, ranidae, and rhacophoridae (wells, 2007). the results of the skelly & golon (2003) study confirm that tadpoles are affected by variations in the benthic particles as a food source. the same authors showed that tadpoles are nevertheless selective when it comes to nutrition and that the variety and availability of nutrient sources in and between aquatic habitats significantly impact their development. in serbia, there are no studies of microplastics’ influence on anurans, especially not on their larvae. there is some information about the presence of mps in some fish species (nikolić et al., 2022) which suggests that there are particles of mps in our aquatic ecosystems and that’s why we could assume that it can also be found in tadpoles because they are extremely sensitive to changes in the environment. after examination of the remains of tadpoles used for microplastic analyses, it was confirmed that ćirković et al. ● results of the first analysis of tadpoles’ diet and determination of microplastics presence of rana, bufo and bufotes species from different localities in serbia ta bl e 2. a lg ae c om po si tio n in ta dp ol es ’ i nt es tin e b uf o bu fo b uf ot es v ir id is r an a da lm at in a r an a te m po ra ri a c er to m et in o po lje b es na ko bi la zl at ib or st ar ac za sa vi ca m ed ve dn ik c er g ol ija g ol ijs ka r iv er tr eš nj ic a r iv er (p ov le n) sr ed nj i po vl en c l. b ac ill ar io ph yc ea e a ch na nt hi di um ++ + ++ + ++ + ++ ++ ++ + ++ + ++ + ++ + ++ + ++ + n av ic ul a ++ + ++ + + ++ + ++ + ++ + ++ + ++ + ++ + ++ + m el os ir a + + ++ d en tic ul a ++ + ++ su ri re lla + + + + + + ++ n itz sc hi a ++ + ++ + + + + + + pi nn ul ar ia ++ + + + + + + + ++ + + ++ + g om ph on em a ++ ++ + + + + + u ln ar ia + + + + + + f ra gi la ri a ++ + + + h an na ea + ++ + + c l. c hl or op hy ce ae m ic ro sp or a ++ + c l. x an th op hy ce ae tr ib on em a + ++ + + ++ + ++ + + + + + ++ c l. e ug le no ph yc ea e ph ac us + + ++ + biologica nyssana ● 14 (1) june 2023: 31-38 ćirković et al. ● results of the first analysis of tadpoles’ diet and determination of microplastics presence of rana, bufo and bufotes species from different localities in serbia ++ + p re se nt in e ac h sa m pl e; ++ p re se nt in e ve ry s am pl e, b ut in a s m al le r p er ce nt ag e; + p re se nt in le ss th an 5 0% o f s am pl es ; n ot p re se nt 35 36 biologica nyssana ● 14 (1) june 2023: 31-38 ćirković et al. ● results of the first analysis of tadpoles’ diet and determination of microplastics presence of rana, bufo and bufotes species from different localities in serbia that mps particles are present in tadpoles (karaoğlu & gül, 2020). pastorino et al. (2022) showed that mps can be found in the digestive tract of adult specimens of rana temporaria. the presence of microplastics in tadpole samples from different localities in serbia represents the first concrete results, so it is necessary to continue the elements of microplastics were present in all investigated species. the mps in most cases were fibrils in different colors (tab. 3, fig. 2). this finding provides novel insights into the accumulation of mps in tadpoles from different localities in serbia. as early life stages of amphibians, tadpoles show extensive feeding activity that exposes them to mps table 3. results of microplastic analysis species localities number of specimens presence of microplastics characteristics of microplastics (shape, color) bufo bufo cer 5 + black particles bufotes viridis starac 10 + red filaments rana dalmatina cer 5 + black filament rana temporaria golijska river 20 + blue and red filaments trešnjica (povlen) 10 + black filaments fig. 2. microplastic filaments in tadpole samples + present elements present in the aquatic environment (hu et al., 2018). our results match with the results of da costa araújo et al. (2019) which have shown that mps can be ingested by physalaemus cuvieri (fam. leptodactylidae) tadpoles. mps can accumulate in their organs and cause morphological, cytotoxic, and mutagenic changes that may affect their health and development. kolenda et al. (2020) study showed that tadpoles of pond-breeding anurans from central europe are exposed to mps pollution. the same authors also suggest that tadpoles can be a significant vector of mps transfer from aquatic to terrestrial ecosystems. investigation of mps in tadpoles of species pelophylax ridibundus and rana macrocnemis (fam. ranidae) from turkey confirmed extensive research at several levels. for future analysis, we plan to include other species and more localities. tadpoles are extremely sensitive to changes in the environment and can give scientists valuable insight into how an ecosystem is functioning, and because they have an important role in food chains, many other animals are affected by them. the investigation of mps in amphibians is of critical importance due to their endangerment and decline, and the accumulation of mps in amphibians can be a pathway for transfering these important pollutants between freshwater and terrestrial ecosystems (pastorino et al., 2022). 37 biologica nyssana ● 14 (1) june 2023: 31-38 acknowledgements. this work was funded by the ministry of science, technological development and innovation of the republic of serbia. agreement no. 451-03-68/2022-14/ 200122. references altig, r., whiles, m.r., & taylor, c.l. 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(2013). amphibians in serbia: distribution and diversity patterns. bulletin of the natural history museum, 6, 90-112. wehr, j.d., sheath, r.g., & kociolek, j.p. (2015). freshwater algae of north america, ecology and classification (1066 p.). academic press, san diego. wells, k.d. (2007). the ecology and behaviour of amphibians. (1141 p.) the university of chicago press, chicago. whiles, m.r., lips, k.r., pringle, c.m., kilham, s.s., bixby, r.j., brenes, r., connelly, s., colongaud, j.c., hunte-brown, m., huryn, a.d., montgomery, c., & peterson, s. (2006). the effects of amphibian population declines on the structure and function of neotropical stream ecosystems. frontiers in ecology and the environment, 4(1), 2734. ćirković et al. ● results of the first analysis of tadpoles’ diet and determination of microplastics presence of rana, bufo and bufotes species from different localities in serbia anticancer compounds from medicinal plants biologica nyssana 5 (1)  september 2014: 63-69 zlatković, b. et al.  report on the new and insufficiently studied taxa… 63 original article received: 3 september 2014 revised: 6 september 2014 accepted: 10 september 2014 report on the new and insufficiently studied taxa in the flora of serbia bojan zlatković 1 *, stefan bogosavljević 2 , nenad smiljković 3 , vladimir ranđelović 1 1 university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia 2 the pharmaceutical chamber of serbia, dečanska 8a, belgrade, serbia 3 serbian herpetological society "milutin radovanovic", despota stefana 142, belgrade, serbia *e-mail: bojanzlat@yahoo.com abstract: zlatković, b., bogosavljević, s., smiljković, n., ranđelović, v.: report on the new and insufficiently studied taxa in the flora of serbia. biologica nyssana, 5 (1), septemeber 2014: 63-69. several important floristic records were established during our recent floristic investigations of southeastern serbia and province kosovo-metohija. minuartia mediterranea (caryophyllaceae) and arabis glabra subsp. pseudoturritis (brassicaceae) are reported for the first time from the territory of serbia, while presence of neglected species symphytum bulbosum (boraginaceae) and valerianella microcarpa (valerianaceae) is reevaluated, as they were reported from new localities. key words: vascular flora, new and neglected taxa, distribution, serbia, balkans. apstrakt: zlatković, b., bogosavljević, s., smiljković, n., ranđelović, v.: izveštaj o novim i manje poznatim taksonima za floru srbije. biologica nyssana, 5 (1), septemeber 2014: 63-69. tokom florističkih istraživanja jugoistočne srbije i kosova i metohije, zabeležen je veći broj značajnih podataka za floru srbije. minuartia mediterranea (caryophyllaceae) i arabis glabra subsp. pseudoturritis (brassicaceae) su po prvi put ustanovljeni na teritoriji srbije. rasprostranjenje symphytum bulbosum (boraginaceae) i valerianella microcarpa (valerianaceae) dopunjeno je novim lokalitetima, čime je potvrđeno prisustvo ovih, u flori srbije, zanemarenih taksona. ključne reči: vaskularna flora, novi i manje poznati taksoni, distribucija, srbija, balkan introduction the territory of serbia, in common with the other regions of balkan peninsula, is characterized by relatively high floristic richness compared to the rest of europe (t u r r i l l , 1929, s t e v a n o v i ć et al. 1995; 1999). flora of serbia has been studied since g r i s e b a c h (1843) and p a n č i ć (1874, 1884), and this long period of continuous research has significantly increased the number of reported floristic data from this country. the largest amount of data from the recent period is included in the 8volume publication flora of sr serbia (j o s i f o v i ć , ed., 1970-1976), summarizing the 5 (1) • september 2014: 63-69 biologica nyssana 5 (1)  september 2014: 63-69 zlatković, b. et al.  report on the new and insufficiently studied taxa… 64 overall knowledge collected by the date of publication of each volume. however, as a large amount of new floristic data was reported for the region during the last decades, additional papers were published either simultaneously or after the publication date of the volume dealing with those taxa (d i k l i ć 1977; n i k o l i ć et al. 1986). even though the flora of serbia presently comprises accounts on more than 3600 species and subspecies, including data in two more recent volumes (s t e v a n o v i ć 1992, 2012), the number of newly reported taxa still fluctuates in the positive direction. during the most recent period of floristic research of serbia (and particularly its southern provinces) many taxa new or poorly known for this area have been registered and reassessed on the topic of their distribution, ecology and conservation (z l a t k o v i ć et al. 2009; 2011). the aim of this paper is to report findings of two recently discovered plant taxa in the flora of serbia, and to emphasize presence of two other neglected species recently established at new localities in serbia. resuming their distribution at the local scale would be useful as an addition to the forthcoming volumes of “the flora of serbia (i-x)” (j o s i f o v i ć , ed., 1970-1977; s a r i ć & d i k l i ć 1986), offering newly obtained floristic data. material and methods distribution of the studied taxa within the territory of serbia was determined and mapped in utm grid system (10 x 10 sq. km., utm zone 34t). in addition to the field survey, herbarium collections and literature sources were also checked for supplementation of distribution data. recent volumes of atlas florae europae (j a l a s & s u o m i n e n 1986; j a l a s et al. 1999) and comparative chorology of the central european flora for selected taxa (m e u s e l et al. 1965; 1978; m e u s e l & j ä g e r 1992) were used in order to discuss and compare recent and previously published data on distribution. in order to align plant distribution with geology substrate, adequate sections of geology map of yugoslavia (k 34-56, 34-57, 34-68) were used at scale 1:100,000 (b a b o v i ć & c v e t k o v i ć 1968; k a r a j o v a n o v i ć & h r i s t o v 1972; t e r z i n et al. 1970). descriptions of taxa match those in the selected volumes of the flora of europe (h a l l i d a y 1993; j o n e s 1964; p a w l o w s k i 1972; e r n e t et al. 1976). the main taxonomic characters of enumerated taxa were briefly compared with other similar representatives of their genera from serbian flora. in order to provide explanation of the ecological conditions in relation to the new parts of their range, short notes on ecological conditions of their habitats are added. nomenclature and synonyms are aligned with the euro+med (http://ww2.bgbm.org/europlusmed/) databases and adequate volumes of the flora of europe (e r n e t et al. 1976). voucher specimens are deposited in the herbarium of the institute of botany and botanical garden “jevremovac”, university of belgrade (beou), with acquisition numbers given in the brackets. results and discussion minuartia mediterranea (link) k. malý general distribution: algeria, albania, bosnia and herzegovina, bulgaria, croatia, cyprus, egypt, france [incl. corse], greece [incl. crete & aegean islands], spain [incl. balearic islands], israel, jordan, italy [incl. sardinia & sicily], libya, lebanon, portugal, syria, malta, macedonia, montenegro, morocco, slovenia, tunisia, turkey. new distribution data: prizrenska bistrica gorge (rečane), xeric pastures (thero-brachypodietea) on shallow soil, limestone, 430 m, 26-05-1997, dm77, coll./det. b. zlatković (beou 16853); prizrenska bistrica gorge (st. arhangeli monastery), rocky ground and screes, limestone, 390 m, 26-05-1997, dm87, coll./det. b. zlatković (fig. 1). figure 1. distribution of minuartia mediterranea in serbia. biologica nyssana 5 (1)  september 2014: 63-69 zlatković, b. et al.  report on the new and insufficiently studied taxa… 65 description: annual plant, with erect stems branching from the base or growing upward. stem 3-15 cm tall, glabrous or subglabrous. leaves linear, 10-12 mm long. inflorescence dense, almost crowded. pedicels usually shorter than sepals. sepal segments 3-5 mm long and 0.5-0.7 mm wide, 3veined, linear-lanceolate. petals shorter than sepals or absent. capsule usually shorter than sepals, with numerous almost smooth seeds, 0.4 mm in diameter (h a l l i d a y 1993). in the flora of serbia this is the fourth reported species from sect. sabulina (rchb.) graebner of genus minuartia, after m. verna, m. hybrida and m. mesogitana (diklić & stevanović 2012). among the enumerated group of species, the newly reported m. mediterranea is most similar to m. hybrida. main differences are in inflorescences, as cymes of m. mediterranea are denser and more crowded, while pedicels are shorter than sepals in the second species. this species inhabits xeric and rocky pastures on shallow limestone soils, mainly occupied by annual plants. it was also recorded in warm positions of limestone screes in the gorges of upland region. it grows mainly as a constituent of communities in thero-brachypodietea alliance, together with phleum exaratum, trachynia distachya, helianthemum salicifolium, valerianella coronata, pterocephalus plumosus etc. arabis glabra (l.) bernh. subsp. pseudoturritis (boiss. & heldr.) maire general distribution: algeria, italy [c & s, incl. sicily], albania, bulgaria, greece [incl. aegean islands], macedonia, malta, morocco. new distribution data: kozjak mt. (delinovački rid), querco-carpinetum orientalis, granite and migmatites, 560 m, 16-05-2004, em78, coll./det. b. zlatković (beou 16883), subnom. turritis pseudoturritis; starac mt. (gornji starac), ostryocarpinion aegeicum, migmatites, 800 m, 02-052005, em78, coll./det. b. zlatković (beou 16882), subnom. turritis pseudoturritis; pčinja river gorge (village barbace), querco-carpinetum orientalis, fine-grained biotite and biotite-muscovite gneiss, 530 m, 23-04-2003, em79, coll./det. b. zlatković; rujan mt. (orljak), querco-fagetea, silicate, 742 m, 26-06-2009, em67, coll./det. b. zlatković & n. smiljković (beou 16884), subnom. turritis pseudoturritis (fig. 2). description: biennial to perennial plant with usually erect and non-branching stems (40-70 cm), pubescent on ventral side. basal leaves lanceolate, sinuate-dentate to lyrate, with stellate hairs. cauline leaves usually ovate, sagittate in the basal part, figure 2. distribution of arabis glabra subsp. pseudoturritis in serbia. glabrous and glaucous. petals 4-8 mm, cream or whitish, compound in lax, elongate inflorescence. siliqua 50-80 (100) x 0.8-1.2 mm, erect, somewhat compressed and with prominent median veins. seeds in a single row in each locus (j o n e s , 1964). the taxonomic position of this taxon is still uncertain as various authors have different opinions. in the earlier floristic literature it was either regarded as a well-defined species: turritis pseudoturritis (j o n e s , 1964, a s e n o v , 1970, t r i n a j s t i ć , 1976) or a. pseudoturritis (m i c e v s k i , 1995), or in the later period as a variety or synonym for a. glabra (j o n e s & a k e r o y d , 1993; a n č e v , 2007). according to several morphological traits, subsp. pseudoturritis is distinguishable from the typical subspecies of a. glabra, in our opinion indicating subspecies level. the most prominent difference from subsp. glabra is uniseriate seed alignment within the fruit and generally longer siliquae (up to 110 mm). some of the authors considered both shape and venal prominence of the fruit wall as valuable additional characters for making difference of two taxa more clear. the valves of siliquae are more compressed and prominently three-veined in subsp. pseudoturritis, compared to the single central vein in subsp. glabra. the typical subspecies is distributed throughout europe except for extreme north, while subsp. pseudoturritis is confined to southern europe and northern africa, overlapping biologica nyssana 5 (1)  september 2014: 63-69 zlatković, b. et al.  report on the new and insufficiently studied taxa… 66 the southernmost part of the species range. a. glabra subsp. pseudoturritis may be confused with a. laxa which inhabits southern balkans and transcaucasia. this last species is distinguished from a. glabra subsp. pseudoturritis by oblong, sparsely ciliate, cauline leaves cordate in the base, and petals that are rather white or lilac in color. it is also similar with a. pauciflora, which is possibly erroneously reported for southern serbia (a d a m o v i ć , 1899). a. glabra subsp. pseudoturritis inhabits forest edges and stony places on silicate soils in the mountains of study region. records of this taxon in the neighboring countries also show its sporadic distribution in mountains, but usually at higher altitudes. it was recorded in bulgaria between 1400 and 2200 m above sea level (a s e n o v , 1970), at higher altitude than records from serbia. on the other hand, in macedonia it was reported from a much wider altitudinal range, from 800 to 2300 m above sea level (m i c e v s k i , 1995), similar to the recorded positions in serbia. this subspecies has probably wider distribution in other mountain regions of serbia that should be investigated in the future. symphytum bulbosum k. f. schimp. general distribution: algeria, albania, bulgaria, france [corse], croatia, germany, greece, switzerland, italy [incl. sardegna and sicily], slovenia, turkey. new records: pčinja river gorge (village jablanica), humid and shady places in the woods (quercofagetea), fine-grained biotite and biotite-muscovite gneiss, 560 m, 15-05-2005, em78, coll./det. b. zlatković (beou 16856); literature data for serbia: bosilegrad (village gornja ljubata) – fn00, bosilegrad (village musulj) – fn00 (u r u m o f f 1937), (fig. 3). description: perennial plant with slender and creeping stock, producing subglobose underground tubers. stems up to 50 cm, poorly branched in the upper part or simple, covered with short hooked hairs and (about 1.5 mm long) setae like the remaining part of the plant. basal leaves large, with ovate to elliptical-lanceolate lamina, tapered to a distinct, long petiole. the cauline leaves elliptical to lanceolate with short petioles, the upper sessile and narrowly decurrent. corolla about 8 – 11 mm long, pale yellow, with short and erect lobes. tubular scales acute, lanceolate-subulate, papilose and exerted about 1-4 (5) mm from the tube. stamens small and included, with minutely apiculate 2-2.5 mm long anthers (p a w l o w s k i , 1972). according to the phenotype, this plant is similar to s. tuberosum, and may be confused because both species have swollen subterranean parts, especially in the vegetative stage. however, s. bulbosum is well distinguished according to several flower traits, first of all longer and exerted scales. corolla is generally smaller, with terminal lobes that are not deflexed like in s. tuberosum. figure 3. distribution of symphytum bulbosum in serbia. the reconstruction of its occurrence in serbia is based at the data by u r u m o f f (1937), who reported this species for two localities in the serbian part of krajište region, formerly regarded as bulgarian territory. the species inhabits different types of deciduous, termophilous forests mainly at lower altitudes of the montane region, in the zone of oak forests. it is sporadically recorded in the lower part of pčinja valley, close to the river at somewhat damp and shady habitats in the oak woods, together with lathyrus laxiflorus, primula vulgaris, arum orientale, cardamine bulbifera, helleborus odorus, galanthus nivalis, corydalis solida and other species of the herb layer. valerianella microcarpa loisel. general distribution: bulgaria, france [incl. corse], greece [incl. crete & aegean islands], spain [incl. balearic islands], italy [incl. sardinia & sicily], portugal, turkey [european part], northern africa. biologica nyssana 5 (1)  september 2014: 63-69 zlatković, b. et al.  report on the new and insufficiently studied taxa… 67 new records in serbia: prizren (village našec), thero-brachypodietea, xerophilous pastures on shallow soil, limestone, 390 m, 26-05-1997, dm77, coll./det. b. zlatković (beou 16852); prizrenska bistrica gorge (st. arhangeli monastery), therobrachypodietea, thermophilic pastures on shallow surface, limestone, 390 m, 26-05-1997, dm87, coll./det. b. zlatković (beou 16851); literature data for serbia: bosilegrad – fn20 (u r u m o f f 1937); (fig. 4). figure 4. distribution of valerianella microcarpa in serbia. description: dwarf plant with stems 5-15 (35) cm long, in upper part dichotomously branched. lower cauline and basal leaves ovate-spatulate, obtusate, entire to sinuate. inflorescence dichotomous, dense, crowded. bracts more or less auriculate, green, with a narrow scarious margin, the lower linear to spatulate, obtusate, the upper narrowly triangular acuminate. fruits ovate to connate, densely hairy or glabrous, c. 1-1.5 mm long, numerous, arranged in fasciculate terminal clusters. sterile loculi of the fruit reduced to tiny ribs, separated by ovate flat area. calyx reduced to an indistinctly dentate, narrow rim (e r n e t & r i c h a r d s o n , 1976). as one of the representatives with smallest fruit size, this species is easily recognized among the species of genus valerianella in serbian flora. according to the shape of the fruit it might be confused with v. dentata and v. muricata, but both have larger fruits. after v. costata, this is the second report of the presence of neglected species of this genus in serbia (z l a t k o v i ć et al., 2011) after the reports by k o j i ć (1973) and r a n đ e l o v i ć (1975). occurrence of v. microcarpa was first reported by u r u m o f f (1937) from the former territory of bulgaria close to bosilegrad in se serbia. remarkable flora of this area was consequently investigated in the later period (r a n d ž e l o v i č & s t a m e n k o v i č , 1986; 1987; r a n d ž e l o v i č et al., 1988), but presence of v. microcarpa has not been confirmed there yet. as in the case of v. costata, existence of this mediterranean species may be expected from several other new sites influenced by mild climate in southern serbia. the species is recorded in dense plant communities at hill slopes and along the roadsides, formed at slightly eutrophic soils, consisting mainly of grasses and annuals, e.g. poa bulbosa, vulpia cilliata, trifolium dalmaticum, medicago minima, parentucellia latifolia. conclusion according to the recent floristic studies, presence of two completely new and two neglected taxa for the flora of serbia is reported from its southern regions and the province of kosovometohija. to the best of our knowledge, presence of minuartia mediterranea and arabis glabra subsp. pseudoturritis has not been reported from this region before. reports on their occurrence from the new, remote sites in the central part of balkan peninsula are especially thought-provoking. the new records of these two taxa extend their ranges in northward direction, out of their known distribution in southern areas. the presence of neglected species symphytum bulbosum and valerianella microcarpa in the flora of serbia is confirmed by adding new floristic data on their distribution. all new records contributed to knowledge of their general distribution and are important for the comprehensive publications that consider diversity of vascular plants of serbia. details on habitat preferences for the reported taxa offer additional information on their ecology in the newly established parts of their range in balkan peninsula. acknowledgements. the ministry of education, science and technological development of the republic of serbia (grant 173030) supported this research. we are grateful to dr marjan niketić for his valuable suggestions considering arabis glabra subsp. pseudoturritis. biologica nyssana 5 (1)  september 2014: 63-69 zlatković, b. et al.  report on the new and insufficiently studied taxa… 68 references adamović, l., 1899: neue beiträge zur flora von serbien. botanisches centralblatt, 78: 1-8. ančev, m., 2007: catalogue of the family brassicaceae (cruciferae) in the flora of bulgaria. phytologia balcanica, 13(2): 153-178. asenov, i., 1970: turritis l. in: jordanov, d. 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(ed.). analitička flora jugoslavije 2(2). 237-240. institut za botaniku sveučilišta u zagrebu, zagreb. turrill, w.b., 1929: the plant-life of the balkan peninsula. a phytogeographical study. clarendon, oxford. 490 p. urumoff, i. k., 1935: florata na kjustendilskij okržja (flora des kustendiler kreises). sbornik na b’lgarskata akademija na nauki. 30: 1-235. zlatković, b., tomović. g., ranđelović, v., vukojičić, s., niketić, m. 2009: distribution and conservation status of several new and neglected vascular plants in serbia. phytologia balcanica, 15(1): 95-105. zlatković, b., ranđelović, v., lakušić, d., stevanović, v., 2011: novelties for the vascular flora of serbia. botanica serbica, 35(2):103-111. http://ww2.bgbm.org/europlusmed/ biologica nyssana 5 (1)  september 2014: 63-69 zlatković, b. et al.  report on the new and insufficiently studied taxa… 70 damjanović-vratnica, b. et al.  composition and antimicrobial studies… biologica nyssana 6 (2)  december 2015: 67-73 damjanović-vratnica, b. et al.  composition and antimicrobial studies… 67 original article received: 23 november 2015 revised: 17 december 2015 accepted: 20 december 2015 composition and antimicrobial studies of essential oil of thymus vulgaris from montenegro biljana damjanović-vratnica1*, danka caković2, svetlana perović2 1faculty of metallurgy and technology, university of montenegro, bul. džordža vašingtona bb., 81000 podgorica, montenegro 2faculty of natural sciences and mathematics, university of montenegro, bul. džordža vašingtona bb., 81000 podgorica, montenegro * e-mail: biljanad@ac.me abstract: damjanović-vratnica, b., caković, d., perović, s.: composition and antimicrobial studies of essential oil of thymus vulgaris from montenegro. biologica nyssana, 6 (2), december 2015: 67-73. chemical composition of the hydrodistilled essential oil of thymus vulgaris l. (thyme) from montenegro was analyzed by gas chromatography-mass spectrometry and its antimicrobial activity was evaluated against 10 microorganisms, including reference and clinically isolated strains. t. vulgaris essential oil yield was 0.42% (v/w, based on the dry leaves weight) whereas the analysis showed that major components, amongst 22 identified in the oil, were geraniol (25.66%), geranyl-acetate (20.34%), linalool (10.89%) and caryophyllene oxide (9.89%). the results of the antimicrobial activity tests revealed that the essential oil of t .vulgaris from montenegro has rather strong antimicrobial activity, especially against staphylococcus aureus, escherichia coli, candida albicans and klebsiella pneumoniae. these results confirm the potential use of t. vulgaris essential oil in food products the as well as for therapeutic applications. key words: thymus vulgaris l., essential oil, chemical composition, antimicrobial activity apstrakt: damjanović-vratnica, b., caković, d., perović, s.: sastav i antibakterijska studija etarskog ulja thymus vulgaris iz crne gore. biologica nyssana, 6 (2), december 2015: 67-73. hemijski sastav etarskog ulja thymus vulgaris l. (timijan) sa područja crne gore analiziran je gasno-masenom spektrometrijom i ispitano je antimikrobno dejstvo ulja na 10 mikroorganizama, uključujući referentne i klinički izolovane sojeve. prinos etarskog ulja t. vulgaris bio je 0.42% (v/w, računato na težinu suvog lišća), dok su, od ukupno 22 identifikovanih komponenti, najzastupljenije: geraniol (25.66%), geranil acetat (20.34%), linalol (10.89%) i kariofilen oksid (9.89%). dobijeni rezultati antimikrobnog ispitivanja pokazuju da etarsko ulje t. vulgaris sa područja crne gore ima prilično snažno antimikrobno dejstvo, posebno na staphylococcus aureus, escherichia coli, candida albicans i klebsiella pneumoniae. ovi rezultati potvrđuju mogućnost korišćenja etarskog ulja t. vulgaris u prehrambenoj industriji kao i u terapeutske svrhe. key words: thymus vulgaris l., etarsko ulje, hemijski sastav, antimikrobna aktivnost 6 (2) • december 2015: 67-73 biologica nyssana 6 (2)  december 2015: 67-73 damjanović-vratnica, b. et al.  composition and antimicrobial studies… 68 introduction the genus thymus, member of the lamiaceae family, contains about 400 species of perennial aromatic, evergreen or semi-evergreen herbaceous plants with many subspecies, varieties, subvarieties and forms (d e m a r t i n o et al., 2009). t. vulgaris, also known as common thyme, is indigenous in the mediterranean region and has long history as a source of the essential oil (thyme oil) and other constituents (e.g. thymol, flavonoids, caffeic acid and labiatic acid) derived from the different parts of the plant (hudaib et al., 2002). it has been grown commercially in a number of countries for the production of the dried leaves, thyme oil, thyme extracts, and oleoresins. thyme oil is among the world’s top 10 used essential oils, which are also utilized as a preservative for food. thymus species are commonly used as herbal tea, flavoring agents (condiments and spices) and for medicinal purposes (s t a h l b i s k u p & s a e z , 2002). studies have found that the main chemical compositions and the concentrations of essential oil of thyme are highly variable (j o r d á n et al., 2006). seven thyme chemotypes have been described whose principal volatile components are: 1,8-cineole, lanolool, α-terpineol, geraniol, trans-thujan-4-ol, terpinen-4-ol, thymol and carvacrol (d i a z m a r o t o et al., 2006). many researchers investigated chemical composition and biological properties of t. vulgaris essential oil and extracts from various origins (c o s e n t i n o et al., 1999; l e t c h a m o et al., 1999; d o r m a n & d e a n s , 2000; a z a z et al., 2004; d e b u r t , 2004; h u d a i b & a b u r j a i , 2007; b a k k a l i et al., 2008; s o k o v i ć et al., 2008; i m e l o u a n e et al., 2009; l i s i et al., 2011; r o b y et al., 2013; s t o j k o v i ć et al., 2013; b o r u g ă et al., 2014; m a r t i n s et al., 2015). it was found that, both the isolation yield and the chemical composition of the eos can be influenced by environmental and management factors such as temperature, water stress, soil fertility, light, pest pressure, cutting date and plant maturity (l e t c h a m o et al., 1999). in the mediterranean environment, there are several ecotypes of wild-growing thyme, which differ in morphological characteristics, distinguished by a strong and penetrating odor and sometimes a very evident balsamic and spicy flavor (d e l i s i at al., 2011). the genus thymus is represented by 15 wildgrowing species in the flora of montenegro (r o h l e n a , 1942; p u l e v i ć , 2005). essential oils composition of two species were determined (c o u l a d i s et al., 2004; s l a v o v s k a et al., 2006), but there are no studies on the chemical profile of the best known species of this genus. the aim of this study is to determine the chemical composition together with the antimicrobial properties of the essential oil from leaves of cultivated t. vulgaris l. from montenegro, as natural sources of antiseptics with potential applications in the pharmaceutical and food industry. material and methods preparation of herb material: fresh leaves of t. vulgaris were collected manually from the collection site in the podgorica region (komani n 42°27'43.40'', e 19°06'27.46''; central part of montenegro) in june 2014. the initial water inherent in the herb leaves found to be 8.9% (w/w) using a dean and stark apparatus with n-heptane as the reflux solvent. herb material was milled in a domestic coffee mill and, after sieving in erweka set of sieves, sample with a mean particle diameter size of 0.9 mm was obtained. a prepared batch was kept in an airtight resalable polypropylene bag and stored at 6 ºc for maximum 3 days before use, in order to avoid losses of volatile compounds. essential oil preparation: herb material (70 g) was submitted to hydrodistillation in a clevenger-type apparatus for 2 hours according to yugoslav pharmacopoeia iv. the obtained oil was dried over anhydrous sodium sulphate, measured, poured in hermetically sealed dark-glass containers and stored in refrigerator at 4 c until analyzed by gc-ms. gas chromatography mass spectrometry (gcms): the gc-ms analyses were carried out using a shimadzu 2010+ gas chromatograph-mass spectrometer equipped with a zb-5 ms (30 m x 0,25 mm x 0,25 μm) capillary column. the column temperature was programmed from 35 c (5 min) to 300 c at 5 c/min. the injection port temperature was 260 c, while the interface temperature was 305 c. the samples of oil were injected by splitting and the split ratio was adjusted to 1:100. helium was used as the carrier gas at a flow rate of 1.2 ml/min and 61.8 kpa inlet pressure. the ms conditions were: the ionisation voltage 70 ev, scanning interval 1.5 s, detector voltage 1.0 kv and m/z range 40 500. the components were identified by comparing their mass spectral data with those in the wiley229 and the nist107 mass spectra libraries, as well as by comparison of the fragmentation patterns of the mass spectra with those reported in the literature and whenever possible, by co-injection with authentic standards (fluka, great britain). biologica nyssana 6 (2)  december 2015: 67-73 damjanović-vratnica, b. et al.  composition and antimicrobial studies… 69 microbial strains: in order to evaluate the activity of the essential oil of t. vulgaris, the following microorganisms were used: reference strains staphylococcus aureus atcc 25923, escherichia coli atcc 25922, pseudomonas aeruginosa atcc 27853 and candida albicans atcc 10231 (torlak, belgrade); and clinical isolates of staphylococcus aureus, escherichia coli, pseudomonas aeruginosa, klebsiella pneumoniae and candida albicans. the microorganisms were isolated from clinically treated or hospitalized patients of medical health centre (podgorica). antimicrobial screening: the agar disc diffusion method was employed to determine the antimicrobial activity of the essential oil (e r e l et al., 2012). briefly, fresh overnight cultures of the tested microorganism were adjusted with sterile saline to a concentration of approximately 1106 cfu/ml (spectrophotometry was used to determinate optical density of cultures) and spread on the solid media plates. the above-mentioned bacteria were cultured on nutrient broth (torlak, belgrade) at 370.1 c while the fungi was grown on sabouraud dextrose broth (merck, germany) at 200.1 c. filter paper discs (6 mm in diameter) were individually impregnated with undiluted t. vulgaris essential oil (4.5, 9 and 18 g) and placed on the incubated plates. the plates were kept at room temperature for 30 min and then incubated at 37 c for 20 h (for bacterial strains) and 30 c for 72 h (for fungi). reading of the results was carried out by measuring diameters of zones of inhibition, in mm. in addition, reference antibiotic discs (provided by the institute for serums, vaccines and diagnostic preparations torlak, belgrade): ampicillin (10 g), ceftriaxone (30 g), erythromycin (15 g), amykacine (30 g), tetracycline (30 g), for bacteria and nystatin for fungi (100 g), were used for comparison, at the same condition as in the essential oil experiment. all tests of inhibitory activity were carried out in duplicate and the developing inhibition zones were compared with those of reference disks. the essential oil was also subjected to the test of sterility and was found to be free of microorganisms. results and discussion hydrodistillation of the leaves of t. vulgaris yielded 0.42% of essential oil (v/w, based on the dry weight of the adult leaves) with a spicy aromatic odor. the results obtained are higher than essential oil yield reported in the literature 0.09% in wild growing thyme in southern italy (m a n c i n i et al., 2015), and rather similar to those reported in the literature where yield was 0.15-1.2% in cultivated thyme from italy (h u d a i b et al., 2002) and 0.75% in cultivated thyme in iran (p i r b a l o u t i et al., 2013). obtained essential oil yield was smaller compared to the results reported in the literature where yield was 0.9% in wild-growing thyme in romania (g r i g o r e et al., 2010), 1.0% in wild-growing thyme in morocco (i m e l o u a n e et al., 2009), and significantly smaller compared to yield of 2.0% in wild-growing thyme in spain (a r r a i z a et al., 2009) and 3.7-5.6% and 1.1-2. 0% in wild-growing and cultivated thyme, respectively, in jordan (h u d a i b & a b u r j a i , 2007). table1. chemical composition of t. vulgaris essential oil (mi method of identification) no compound % mi* 1 α-pinene 0.15 2,3 2 camphene 0.23 2,3 3 sabinene 0.9 2,3 4 -pinene tr 2,3 5 α-terpenine 0.43 2 6 p-cymene 1.67 2,3 7 cis-ocimene 0.22 2 8 γ-terpenene 0.69 2,3 9 linalool 10.89 1,2,3 10 camphor 0.28 2,3 11 borneol 1.21 2,3 12 terpene-4-ol 5.21 1,2,3 13 α-terpineol 0.34 2,3 14 nerol 2.75 2,3 15 neral 0.48 2 16 geraniol 25.66 1,2,3 17 geranial 2.82 2,3 18 carvacrol 0.78 2 19 thymol 0.08 2,3 19 neryl-acetate 1.12 2 20 geranyl-acetate 20.34 1,2,3 21 -caryophyllene 1.05 2,3 22 bicyclogermacrene tr 2 23 elemol 5.36 2,3 24 caryophyllene oxide 9.89 1,2,3 25 viridiflorol 0.52 2,3 26 -eudesmol 2.87 2,3 total 95.94 monoterpene hydrocarbons 4.29 oxygenated monoterpenes 71.96 sesquiterpene hydrocarbons 1.05 oxygenated sesquiterpenes 18.64 * 1 – co-injection with authentic compounds; 2 – ms; 3 – literature comparison; tr < 0.1 it is acknowledged that yield and chemical composition of herbal extracts are determined by a series of factors including herb genetic, climate, elevation, and topography as well as by interaction of various factors (p i r b a o i l i t i et al., 2013). thus, biologica nyssana 6 (2)  december 2015: 67-73 damjanović-vratnica, b. et al.  composition and antimicrobial studies… 70 yield and composition of aromatic plants essential oil are influenced by harvesting time, ecological and climatic conditions. it was previously found that many species of the thymus genus present different intraspecific chemotypes and that chemical composition of the essential oils is variable in relation to the harvesting time, to the stage of development of the plant, and to the field environmental conditions (d e l i s i et al., 2011). the chemical composition of the hydrodistilled thyme essential oil is shown in tab. 1. gc-ms analyses revealed the presence of 26 compounds representing 95.94% of the total oil. the major components were geraniol (25.66%), geranyl-acetate (20.34%), linalool (10.89%) and caryophyllene oxide (9.89%). other major compounds (1.0% of the identified portion) in the gained oil were geranial (2.82%), elemol (5.36%), terpinen-4-ol (5.21%), -eudesmol (2.87%), nerol (2.75%), p-cymene (1.67%), caryophyllene (1.05%), borneol (1.21%) and nerylacetate (1.12%). it was previously reported than t. vulgaris species has seven genetically distinct chemotypes that can be distinguished on the basis of the dominant monoterpene produced in glandular trichomes on the surface of the leaves (d i a z m a r o t o et al., 2006). in southern france, six chemotypes were found and named after its dominant monoterpene: geraniol (g), α-terpineol (a), thuyanol-4 (u), linalool (l), carvacrol (c), and thymol (t) (t h o m p s o n et al., 2003). the six monoterpenes are all produced from geranyl pyrophosphate through a series of changes in configuration and have quite similar molecular structures. a major distinction is the phenolic nature of carvacrol and thymol, and the nonphenolic nature of the four other monoterpenes. it was found that the mean values of the proportion of dominant monoterpenes in the α-terpineol, geraniol, and linalool chemotypes exceeded those of the thuyanol, carvacrol, and thymol chemotypes (t h o m p s o n et al., 2003). m a n c i n i et al. (2015) found that the most abundant compounds in t. vulgaris from southern italy were thymol (46.2%–67.5%), carvacrol (5.7%–7.3%) and caryophyllene oxide (1.7%–7.3%), however de lisi et al. (2011) reported that the predominant compounds of essential oil were geraniol, thymol and linalool (d e l i s i et al., 2011). o z c a n & c h a l c h a t (2004) found that essential oil from wild-growing t. vulgaris in turkey has high content of thymol (46.2%). it was previously reported (i m e l o u a n e et al., 2009) that the oil of t. vulgaris from morocco contained camphor (38.54%), camphene (17.19%), α-pinene (9.35%) while thyme oil from jordan was characterized by high content of phenolic monoterpenoids (mainly thymol and carvacrol) in the range 70.8-89.0% (h u d a i b & a b u r j a i , 2007). g r o s s o et al. (2010) found that in thyme essential oil from spain the most abundant compounds were thymol (35.441.6%) and p-cymene (28.9-34.8%). chemical composition of obtained t. vulgaris essential oil from montenegro showed that the most abundant compounds were geraniol and geranylacetate, thus examined t. vulgaris belongs to chemotype geraniol. the presence of phenolic compounds thymol and carvacrol in the oil was insignificant, while content of p-cymene was only 1.58%, which significantly deferred from most results from literature survey. the thyme oil from montenegro consisted mostly of oxygenated monoterpenes (71.96%) and oxygenated sesquiterpenes (18.64%). table 2. antimicrobial activity of the t. globulus essential oil and some standard antibiotics inhibition zone (mm)* t. vulgaris oil standard antibiotics (g)** (g) amp ctr er amyx te ny microorganism 4.5 9 18 10 30 15 30 30 100 staphylococcus aureus 28 37 50 22 14 31 24 13 s. aureus atcc 25923 30 43 56 26 32 31 28 40 escherichia coli 29 37 43 36 26 e. coli atcc 25922 34 43 56 27 34 22 30 29 pseudomonas aeruginosa 14 17 16 21 16 p. aeruginosa atcc 27853 18 22 26 19 31 candida albicans 31 35 41 18 c. albicans atcc 10231 33 37 43 20 klebsiella pneumoniae 38 41 52 15 35 15 26 25 * includes diameter of disc; (-) not active; **amp: ampicillin; ctr: ceftriaxone; er: erythromycin; amyk: amykacine; te: tetracycline; ny: nystatin. biologica nyssana 6 (2)  december 2015: 67-73 damjanović-vratnica, b. et al.  composition and antimicrobial studies… 71 the antimicrobial activity of t. vulgaris essential oil is due to the presence of a mixture of monoterpenes and oxygenated monoterpenes and sesquiterpenes (most of the antimicrobial activity in the oils has been attributed to the oxygenated monoterpenes). identification of such compounds with wide biological activity is critical for mankind. it helps in the search for chemical structures that should assist in designing new drugs as therapeautics against human pathogens (d a m j a n o v i ć v r a t n i c a et al., 2011).the antimicrobial plate diffusion assay for thymus vulgaris essential oil, as summarized in the tab. 2, showed that different microorganisms tested had different susceptibility to the same essential oil. the essential oil activities against tested microorganisms were increased in both cases (for atcc strains and clinically isolated strains) with increased amount of investigated essential oil. thyme oil was very potent against all chosen microorganisms, except pseudomonas aeruginosa (at the lowest oil concentrations), whether as clinically isolated strain or as atcc strain. since pseudomonas species are known to have ability to metabolize a wide range of organic compounds and for this fact is used extensively in bioremediation, this may explain their high level of resistance. they may simply metabolize the compounds in the oils that are inhibitory to many of the other bacteria (chao et al., 2000). because of that fact, the obtained activity of 18 g of thyme oil against p. aeruginosa for clinically isolated and atcc strains is noteworthy (17 and 22 mm, respectively). for tested atcc strains, t. vulgaris essential oil showed the strongest antimicrobial activity against e. coli atcc 25922, followed by s. aureus atcc 25923 and fungi c. albicans atcc 10231. regarding clinically isolated bacterial strains, the highest inhibition zone values were observed against medically important pathogens staphylococcus aureus and escherichia coli, ranged from 28 to 50 mm and from 29 to 43 mm, respectively. for comparison, used standard antibiotics diameters of growth inhibition zones ranged from 13 to 31 mm (for s. aureus) and 26 to 36 mm (for e. coli). very high antibacterial activity thyme essential oil showed against klebsiella pneumoniae, which were significantly susceptible to the essential oil at concentration of 9 and 18 g, with significant diameters of growth inhibition zones (29 and 38 mm), respectively. oil exhibited very strong activity against fungi candida albicans at all concentrations, which could be significant since c. albicans invades different areas of the human body causing cutaneous, mucocutaneous and opportunistic infections. in our experiment, diameters of growth inhibition zones ranged from 29 to 40 mm, giving two times higher effect in comparison to nystatin. it is probably due to the high amount of monoterpene alcohols and aldehydes, already known as components that inhibit the growth of fungi, especially c. albicans (d a l l e a u at al., 2008; l e i t e et al., 2015). most of the antimicrobial activity of the essential oils has been attributed to the oxygenated monoterpenes (b a k k a l i et al., 2008). in the literature, it was reported that various chemical compounds have direct activity against many species of bacteria, such as terpenes and a variety of aliphatic hydrocarbons (alcohols, aldehydes and ketones). the lipophilic character of their hydrocarbon skeleton and the hydrophilic character of their functional groups are of the main importance in the antimicrobial action of essential oils components (m a n c i n i et al., 2015). in addition, the components present in lower amount in thyme essential oil, such as p-cymene, carvacrol, camphor and terpinen-4-ol, could also contribute to the antimicrobial activity of the oil. it was previously found that terpinen-4-ol exhibits very high antimicrobial effect (b a r e l et al., 1991, c a r s o n & r i l e y , 1995; c a r s o n et al., 2006; p a z y a r et al., 2013). the antimicrobial activity of the thyme essential oil could also be associated with presence of borneol and linalool, well-known chemicals with their pronounced antimicrobial properties (v i l j o e n et al., 2003). in fact, it is also possible that the components which are present in lower amount might be involved in some type of synergism with the other active compounds. antibacterial activity of essential oils from many herb species has been extensively surveyed in last decades (r i o s & r e c i o , 2005), but their antimicrobial mechanism has not been reported in excessive details. it was found that most active antimicrobial compounds of essential oils are terpenes and phenolics and, thus, their mode of action might be similar to that of other phenolic compounds (s h u n y i n g et al., 2005). individual essential oil contains complex mixtures of such compounds however, a little is known about the effect of interaction between individual constituents on antimicrobial activity. interactions between constituents may lead to additive, synergistic or antagonistic effects (d e l a q u i s et al., 2002). conclusion the obtained results revealed that examined thymus vulgaris from montenegro belongs to chemotype geraniol. hydrodistillation of the leaves of t. vulgaris yielded 0.42% of essential oil (v/w, biologica nyssana 6 (2)  december 2015: 67-73 damjanović-vratnica, b. et al.  composition and antimicrobial studies… 72 based on the dry weight of 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(1984). national institute for health protection, belgrade, yugoslavia, vol. i: 126-128. biologica nyssana 6 (2)  december 2015: 67-73 damjanović-vratnica, b. et al.  composition and antimicrobial studies… 74 anticancer compounds from medicinal plants biologica nyssana 5 (2)  december 2014: 75-82 huseinović, s. et al.  morphological and ecological differentiation of the fruit … 75 original article received: 12 june 2014 revised: 10 october 2014 accepted: 20 december 2014 morphological and ecological differentiation of the fruit of fragaria vesca l. (rosaceae) from different habitats in bosnia and herzegovina samira huseinović1*, sanida osmanović1, zerina terzić1, marizela šabanović2 1university of tuzla, faculty of science, univerzitetska 4, 75000 tuzla, bosnia and herzegovina 2university of tuzla, faculty of pharmacy, univerzitetska 7, 75000 tuzla, bosnia and herzegovina * corresponding author: samira.huseinovic@untz.ba abstract: huseinović, s., osmanović, s., terzić, z., šabanović, m.: morphological and ecological differentiation of the fruit of fragaria vesca l. (rosaceae) from different habitats in bosnia and herzegovina. biologica nyssana, 5 (2), december 2014: 75-82. 30 individuals from each population of the fragaria vesca in the fruiting phenophase were collected in the ecologically different habitats. morphological variation was investigated using 10 populations and 300 individuals. all specimens were properly preserved in a formalin-acetic acid mixture and stored in herbarium. the fruits of fragaria vesca were investigated in order to determine the most variable and the most consistent morphological character, depending on the environmental factors under which they developed. each population and its belonging individuals were labeled with special numbers. the percentage of variability for the height of fruit is 24.338% and for the width of fruit is 25.747%. however, considering high resource values of strawberries, it is necessary to emphasize that the size of the fruit follows the overall size of an individual. key words: fragaria vesca l., fruits, morphological and ecological variability apstrakt: huseinović, s., osmanović, s., terzić, z., šabanović, m.: morfološka i ekološka diferencijacija plodova vrste fragaria vesca l. (rosaceae) na različitim staništima u bosni i hercegovini. biologica nyssana, 5 (2), decembar 2014: 75-82. na ekološki različitim staništima prikupljeno je po 30 individua iz svake populacije vrste fragaria vesca u fenofazi plodonošenja. morfološka varijabilnost je sagledana na osnovu 10 populacija i 300 jedinki. sve jedinke su konzervirane u fo-a smjesu ili pohranjene u herbarijumu. plodovi odabranih vrsta roda fragaria (fragaria vesca) su istraženi s ciljem da se odredi najvarijabilniji i najkonzistentniji morfološki karakter, u zavisnosti od ekoloških faktora pod kojima se razvijao. utvrđen je stepen varijabilnosti morfometrijskog karaktera za parametar: maksimalna širina ploda (p1), kretao se u rasponu 24,338%, a za širinu ploda (p2) iznosi 25,747%. na osnovu statističkih analiza utvrđeno je da plod jagode ne spada u grupu onih biljnih delova koji najviše variraju. ključne reči: fragaria vesca l., plodovi, morfološka i ekološka varijabilnost 5 (2) • december 2014: 75-82 biologica nyssana 5 (2)  december 2014: 75-82 huseinović, s. et al.  morphological and ecological differentiation of the fruit … 76 introduction strawberry, genus fragaria l., is a plant from the family rosaceae and subfamily rosoideae (p o t t e r et al., 2007). it has great economic importance and therefore it has been investigated from different aspects morphological (h a r r i s o n et al., 1997), systematic and biogeographic (s t a u d t , 1989, 2009), demographic (a n g e v i n e , 1983), genetic (g a l l e t t a & m a a s , 1990), molecular (d i m e g l i o et al., 2014) and other. the genus fragaria includes 20 wild species, three described naturally occurring hybrid species, and two cultivated hybrid species important to commerce (h u m m e r et al., 2011). the wild species are distributed in the north temperate and holarctic zones. the only species which is widespread in europe, asia and america is fragaria vesca l. (s t a u d t , 1989, 2009). in europe it is represented with 6 species (t u t i n et al., 1968; euro+med, 2006). the genus fragaria is represented by 3 species in bosnia and herzegovina f. vesca l., f. moschata weston and f. viridis weston (b e c k -m a n n a g e t t a , 1927; euro+med, 2006). fragaria vesca is the most widely distributed species. it is growing in a different natural habitats (forests, meadow, pasture and other) (b e c k m a n n a g e t t a , 1927) and therefore it is very variable. up to now, the morphological studies of this species included only vegetative parts of the plant (h u s e i n o v i ć & o s m a n o v i ć , 2010). the objective of this study was to investigate morphological and ecological differentiation of the fruits of fragaria vesca from different habitats in bosnia and herzegovina. material and methods plant material of the fragaria vesca was collected in the fruiting phenophase from ten sites around tuzla during 2013 (tab. 1). during the selection of the research sites, efforts were made to cover populations developed in different plant communities. field research the field research consisted of a determination of biotic habitat factors of each of the studied populations, establishing phenological data and collecting samples and herbarium material. during the fieldwork we also made an adequate photographic documentation. based on literature data (r e d ž i ć , 1997) we determined type of plant communities on each site. collecting samples of plant material on the selected ecologically diverse habitats, living material of the species fragaria vesca was collected. at least 30 individuals from the population in the fruiting phenophase were sampled from each locality. morphological variability was observed on the basis of 10 populations and 300 individuals. during the collection of plants in the field, keys for determination of plant species have been used (d o m a c , 2002; a i c h e l e , 2004). all specimens were properly preserved in a formalin acetic acid mixture, or stored in the herbarium. as the collection of plant material was run at the time of fruiting, each population and its dependent individuals were marked with special numbers. laboratory studies in addition to the morphometric and statistical processing of materials and data, the laboratory part of the study assumed also numerous activities: consulting the relevant literature, collecting and processing eco-climate data, creation of original illustrations, etc. table 1. list of plant collection sites site locality type of vegetation 1 kladanj (muška voda) abieti-fagetum fragarietosum vescae 2 gornja tuzla arrhenatherion elatioris 3 cerik-ljubače arrhenatherion elatioris 4 husino querco-pinetum nigrae 5 ilinčica ii agropyro-rumicion in zone tilio-quercetum petraeae 6 krojčica agrimonyo-fragarietum 7 simin han arrhenatherion elatioris 8 ilinčica i fragarion vescae in zone querco-carpinetum betuli 9 zlaća bridge fagetum montanum serpentinicum 10 zlaća road fagetum montanum serpentinicum biologica nyssana 5 (2)  december 2014: 75-82 huseinović, s. et al.  morphological and ecological differentiation of the fruit … 77 morphological studies of the species fragaria vesca on the localities of gornja tuzla, simin han, ilinčica i, ilinčica ii, krojčica, husino, cerik, zlaća (bridge), zlaća (road) and kladanj (muška voda), were performed in the laboratories of the faculty of science in tuzla. the following morphological characters were determined: maximum width of the fruit (p1) and maximum height of the fruit (p2). characters of the fruit besides all the above mentioned characters we studied the fruit as well, as an essential characteristic of the gene expression. according to its shape (p3), the fruit was placed into one of the following groups: rounded, globose, conical or elongated. statistical analysis evaluation and interpretation of individual character’s variability, the degree of correlation or relationship between individual characters and ecological parameters, as well as analysis of the significance of the obtained results were performed in accordance with the relevant statistical and biological and ecological patterns contained in the available software packages (anova, 2006; statistics spss). by methods of descriptive statistics we determined: the arithmetic mean, range, variance, standard deviation and coefficient of variation of individual characters in specific patterns. the significance of the identified differences between the arithmetic means was determined by the method of testing the difference between the arithmetic means of small independent samples (p e t z , 1983). results fragaria vesca has a pronounced ecological valence in relation to a complex of environmental factors. it inhabits a variety of volcanic and sedimentary rocks, as well as the different stages and types of automorphic soils. it was noted also at the serpentine peridotic substrate (locality m. voda, kladanj). its populations equally well developed on bare parent substrate, cleared land, fire sites, and at developed soil in hornbeam, pine and pine-oak forests. so far it has been recorded in all regions of the former yugoslavia, including bosnia and herzegovina, and the first varieties have been created in north america and chile, from where they were transferred to europe in the 17th and 18th century. variability of morphometric characters comparative morphological analysis of plant material collected from different habitats showed a significant degree of variability in a series of characters of the fragaria vesca. variability in character of the fruit observing these two characteristics (height and width of the fruit) on the total sample (whose number is 300 individuals for all ten sites) it can be seen that for the first property, the minimum and maximum values range from 4 to 18 mm, and for the second property they range from 4 to 14 mm. percentage of variability for the fruit height is 24.338% and for the width of the fruit is 25.747% (tab. 2). fig. 1: variations in height of the fragaria vesca fruits by locality (1-10) the height of the fruit (fig. 1) is the largest in populations from the localities ilinčica ii (5), krojčica (6), simin han (7) and zlaća road (10). populations from the sites (6) and (7) represent communities on cleared land. at the given localities, in forest conditions, the light was limited. after cutting down the forest, there was a rapid expansion of the strawberries population due to the optimization of conditions of its ecological valence. fruit width (fig. 2) is the largest in populations from the localities krojčica (6), simin han (7) and zlaća road (10). these sites represent communities of open habitats and forest edges suggesting that these are the communities which by their ecological characteristics provide optimum conditions for the growth of strawberries. observing present forms of fruit by localities, it can be seen that the globose ones were present in the largest proportion at the site krojčica (6), which is cleared land of a forest. the round fruits were biologica nyssana 5 (2)  december 2014: 75-82 huseinović, s. et al.  morphological and ecological differentiation of the fruit … 78 table 2. statistical data of the analyzed fragaria vesca characters in all localities location variable n x xs minx maxx range s v (%) total p1 386 8.486 0.105 4 18 14 2.065 24.338 p2 386 7.459 0.098 4 14 10 1.920 25.747 table 3. significance in variations of fruit characters significance site 1 p1 p2 p3 p1 0.066 0.009 p2 0.066 0.000 p3 0.009 0.000 site 2 p1 p2 p3 p1 0.000 0.068 p2 0.000 0.127 p3 0.068 0.127 site 3 p1 p2 p3 p1 0.098 0.130 p2 0.098 0.163 p3 0.130 0.163 site 4 p1 p2 p3 p1 0.218 0.026 p2 0.218 0.000 p3 0.026 0.000 site 5 p1 p2 p3 p1 0.000 0.284 p2 0.000 0.427 p3 0.284 0.427 site 6 p1 p2 p3 p1 0.000 p2 0.000 p3 site 7 p1 p2 p3 p1 0.049 0.989 p2 0.049 0.124 p3 0.989 0.124 site 8 p1 p2 p3 p1 0.002 0.528 p2 0.002 0.194 p3 0.528 0.194 site 9 p1 p2 p3 p1 0.020 0.461 p2 0.020 0.040 p3 0.461 0.040 site 10 p1 p2 p3 p1 0.043 0.005 p2 0.043 0.728 p3 0.005 0.728 fig. 2: variations in width of the fragaria vesca fruits by locality (1-10) fig. 3: variations in shape of the fragaria vesca fruits by locality (1-10) present at all sites, with the exception of the site (6) and they prefer forest edges and meadows. the conical were present most in localities kladanj (1), zlaća road (10) and simin han (7), which leads to the conclusion that they prefer cooler places. the strawberries with elongated fruits in the greatest proportion inhabit the site ilinčica ii (fig. 3, 4). correlation in variations of fruit characters we can conclude that in most cases there is a correlation between the width and shape of the fruit (tab. 3, fig. 5). ecological differentiation differences between morphological characteristics of populations originate from biologica nyssana 5 (2)  december 2014: 75-82 huseinović, s. et al.  morphological and ecological differentiation of the fruit … 79 differences in environmental factors prevailing in their habitats. determination of a high degree of variability of the analyzed characters indicates high interpopulation variability, which further points to the possible influence of the present environmental factors. the basis of the high degree of variability are genetic determinants of the species fragaria vesca. wide ecological valence in relation to the complex environmental factors resulted with spreading of this species in an environmentally very dynamic habitats, usually ecotones, where environmental factors of two different habitats collide (e.g. forests and meadows). as a specific answer to this kind of dynamic (changeable) value of environmental factors, there is expressed variability of morphological and meristic characters. these features have the function of population’s adaptation within the present environmental factors in order to achieve the best use of capacities and potentials of the habitats. given the very different types of habitats, and very different conditions in which the population can survive, the question is which habitat factors favor, and which limit the population to achieve its biological / ecological functions. in the aim of shedding light on the role of individual factors from the complex, a cluster analysis was performed on the basis of the observed morphometric characters. based on the character of the fruit fig. 6 shows high morphological similarity of populations from localities 8 and 9, i.e. from populations of the community habitats of fragarion vescae in the zone querco-carpinetum betuli and fagetum montanum serpentinicum. morphological characteristics of individuals of these populations are similar: individuals are of average height and have average values of the other monitored characters. fig 4. presence of the four basic groups of the fruit shape by locality (n – number of individuals) fig. 5: correlations for the height (p1), width (p2) and shape (p3) of the fruit fragaria vesca 0 5 10 15 20 25 30 35 40 n 45 kladanj muška voda gornja tuzla cerik ljubače husin o ilinčica ii krojčic a simin han ilincica i zlaća bridge zlaća road conical rounded globose elongate biologica nyssana 5 (2)  december 2014: 75-82 huseinović, s. et al.  morphological and ecological differentiation of the fruit … 80 fig. 6: cluster analysis of the populations of fragaria vesca in the investigated area based on the observed character of the fruit the next group of very similar populations is made up of the populations from sites 1 and 4, and the related population from locality 10. the complex of factors that govern the habitats of these populations is heterogeneous. at the localities 1 and 10, vegetation of the ecosystem of beech-fir forests on serpentine geological substrate has been developed. in contrast to that, the site 4 has developed vegetation of the ecosystem of pine and oak. although considering the temperature these are very different habitats, in ecological sense the sites are linked by having poor supply of water. in relation to the appearance of individuals in this group of populations (individuals are small, as well as most of individual characters), it can be assumed that the supply of water to habitats plays a major role in the possibilities of biomass production and dissemination of strawberries population. the group of populations of meadow ecosystems of the alliance arrhenatherion elatioris shows similarity with the populations developed in vegetation of fern colonies. individuals are high in growth (stems are elongated), the leaves are large and petioles are small. in the dense meadow complexes, individuals of strawberry, looking for sufficient quantities of light and heat, in a competitive fight, "invest" their energy in stem elongation and increase in all vegetative parts of the plant body (sites 3, 7 and 2). however, the greatest in growth are individuals that grow on the so-called cleared land (site 6, cleared land of the forest tilio-quercetum petraeae) and on the very edge of the forest (site 9, the edge of the forest fagetum montanum "serpentinicum.") this fact indicates that the strawberry as a species is very capable that through rapid expansion occupy new habitats before other species use nutrients that forest land offers. however, this also points to the fact that the lack of light and heat in a given period of development (conditions prevailing in assembled forest communities), stand out as the limiting factor for the expansion of its populations. a similar differentiation is shown also by the observed characters of the fruit. discussion variability in character of the fruit (i) the largest coefficient of variation in the height of the fruit was recorded at the site 10 and amounts 26.618%, the smallest on the site 7 and amounts 14.573%. (ii) in the variation of the width of the fruit, the largest coefficient of variation was recorded at the site 2 (23.863%) and the smallest amounts 14.667%, being recorded at the site 6. (iii) the coefficient of variation of the height of the fruit was 24.338%, and of the width of the fruit was 25.747%. thus, the fruit does not fall into the group of those plant parts that vary the most. however, due to the high resource values of strawberries, it is necessary to emphasize that the size of the fruit follows the overall size of individuals. as seen from the previous findings, the highest growth was achieved by individuals from the cleared land of the community tilio-quercetum petraeae, which indicates that strawberries in our conditions optimally thrive on mesophilic, well lit to somewhat shaded and relatively warm habitats of the mountain zone. biologica nyssana 5 (2)  december 2014: 75-82 huseinović, s. et al.  morphological and ecological differentiation of the fruit … 81 morphological differentiation the populations of species fragaria vesca in the study area were not morphologically uniform, but they contain more or less differentiated morphoeco-types (r e d ž i ć , 1990). the fact that in the populations of different geo-biocenosis the same morphological types of plants occured, and that these populations differ among themselves, however, shows that the quantitative participation of individual types in them is different (b a š i ć , 2004; k o v a č i ć , 2006). at the site of muška voda, kladanj, the size of specimens is relatively small compared to individuals from cleared land and open meadow habitats. the flowers are small, as well as the fruits, which are mostly elongated. individuals of the population at the locality of gornja tuzla developed within the community of arrhenatherion elatioris. flowers are also relatively small, as well as the fruits, which are mostly elongated. variations within the population are relatively large. individuals of the population at the site cerik ljubače (in the zone of the forest community querco-carpinetum) are characterized by the lowest average length and width of a rosette leaf midrib. individuals sampled at the site husino developed within the community sambucetum nigrae and are characterized by the fact that there have not been reported extremely high or low values for the observed characters. population individuals of the site ilinčica ii developed within the community fragarion vescae by the edge of querco-carpinetum betuli. the specimens are large, but still far less than those on the site krojčica, where maximum values for the most of the studied characters were recorded. individuals of the population at the site simin han developed within the community pteridiofragarietum vescae and are very variable among them. on the site ilinčica i, population has been developed within the community agropyrorumicion in anthropogenized conditions in the zone of mesophilic meadows. they do not stand out by the high growth, nor by the size of the fruit. individuals of the population zlaća (road) developed on the anthropogenized eutric cambisol within the community of dark deciduous forests, as well as their neighboring population. individuals of both populations are characterized by relatively low growth. ecological differentiation group of populations from meadow ecosystems of the alliance arrhenatherion elatioris showed similarity with the population developed in the vegetation of fern colonies and all other populations that are found in the open (nonforested) habitats (r e d ž i ć , 1987, 1988). individuals were high in growth (stems are elongated), the leaves were large, while petioles were small. in dense meadow complexes, individuals of strawberry, looking for sufficient quantities of light and heat, in a competitive fight, "invest" their energy in stem elongation and increase in all vegetative parts of the plant body (sites 2, 3 and 7). however, the greatest growth had the individuals that grew on the so-called cleared land (site 6, cleared land of the forest tilio-quercetum petraeae) and on the very edge of the forest (site 9, the edge of the forest fagetum montanum "serpentinicum"). this fact indicates that the strawberry as a species, is very capable to occupy new habitats by rapid expansion and before other species use nutrients that forest lands offer. (i) as a specific answer to this kind of dynamic (changeable) values of environmental factors there is an expressed variability of morphological and meristic characters, which has the function of adaptation of populations to present environmental factors, that is, the function of the best use of habitat's capacities and potentials (e r i k s s o n et al., 2003). (ii) the investigated morphologic characters are changing, and the emerged changes and differences among populations are not stable. further, stronger environmental isolation does not lead to a stronger morphological differentiation of the same character or group of characters. greater morphological differentiation is achieved by increasing the number of differentiating characters. in the second case, individual differential characters can lose that property and differentiation among populations is accomplished through other characters. (iii) since ecologically distant populations are simultaneously morphologically remote too, it could be assumed that those changes are still at the level of phenotypic variation. however, within the same population clearly separated groups of individuals (groups of clones) can be observed, among which there are no observed larger and clearer environmental differences. similar clones are located in ecologically different populations. therefore, a detailed knowledge of morphological variability and ecological differentiation of populations within the species is biologica nyssana 5 (2)  december 2014: 75-82 huseinović, s. et al.  morphological and ecological differentiation of the fruit … 82 essential condition for the full overview of biological potential and evolutionary status in which the analyzed group of plants is located. references aichele, d., 2004: was blűht denn da? kosmos naurfűhrer. stuttgart. angevine, m.w., 1983: variations in the demography of natural populations of the wild strawberries fragaria vesca and f. virginiana. journal of ecology, 71 (3): 959-974. bašić, n., 2004: morfološko – taksonomska istraživanja glogova (crategus l.) na području bosne i herzegovine. magistarki rad. sarajevo. beck-mannagetta, g., 1927: flora bosne, hercegovine i oblasti novog pazara. zemaljska štamparija; 6 – 8.sarajevo. dimeglio, l.m., staudt, g., yu, h., davis, t.m., 2014: a phylogenetic analysis of the genus fragaria (strawberry) using intron-containing sequence from the adh-1 gene. plos one, 9 (7), e102237. domac, r. 2002: flora hrvatske. školska knjiga. zagreb. 504p. eriksson, t., malin, s., hibbs., anne, d., yoder, s., charles, f., delwiche., michael, j. 2003: the phylogeny of rosoideae (rosaceae) based on sequences of theinternal transcribed spacers (its) of nuclear ribosomal dna and the trnl/f region of chloroplast dna. international journal of plant science, 164 (2): 197 – 211. galletta, g.j., maas, j.l. 1990: strawberry genetics. hortscience, 25 (8): 871-879. harrison, r., lubz, j., furnier, g., hancock, j., 1997: morphological and molecular variation among populations of octoploid fragaria virginiana and f. chiloensis (rosaceae) from north america. american journal of botany, 84 (5): 612-620. hummer, k.e., bassil, n, njuguna, w., 2011: fragaria, 17-44. in: c. kole (ed.), wild crop relatives: genomic and breeding resources, temperate fruits. springer-verlag berlin heidelberg. 247p. huseinović, s., osmanović, s. 2010: morphometric and meristic characteristics of the wild strawberry (fragaria vesca l.) on konjuh mountain. acta agriculturae serbica, 15 (30): 133-140. kovačić, s. 2006: srodstveni odnosi i korologija izofilnih i heterofilnih zvončića (campanula l., campanulaceae) primorskih dinarida. doktorska disertacija, zagreb. petz, b., 1983: osnovne statističke metode za nematematičare. sveučilišna naklada liber, zagreb. potter, d., eriksson, t., evans, r.c., oh, s., smedmark, j.e., morgan, d.r., kerr, m., robertson, k.r., arsenault, m., dickinson, t.a., campbell, c.s. 2007: phylogeny and classification of rosaceae. plant systematics and evolution, 266: 5-43. redžić, s., 1987: djelovanje faktora čistih sječa na spektar sistematske pripadnosti vrsta viših biljaka u nekim šumskim ekosistemima bosne. godišnjak biološkog instituta univerziteta u sarajevu, 40: 89-100. redžić, s., 1988: šumske fitocenoze i njihova staništa u uslovima totalnih sječa. godišnjak biološkog instituta univerziteta u sarajevu, 41: 1-260. redžić, s., 1990: morfološka diferencijacija populacija taksona potentilla malyana borbas. bilten društva ekologa bih, serija b, 5: 93-99. redžić s., 1997: karakteristike biljnog svijeta bosne i hercegovine. prirodno-matematički fakultet univerziteta u sarajevu. staudt, g., 1989: the species of fragaria, their taxonomy and geographical distribution. acta horticulturae, 265: 23–33. staudt, g., 2009: strawberry biogeography, genetics and systematics. acta horticulturae, 842: 71-84. tutin, t.g., heywood, v.h., burges, n.a., moore, d.m., valentine, d.h., walters, s.m., webb, d.a. (ed.), 1968: flora europaea, ii. cambridge, university press. london. anticancer compounds from medicinal plants biologica nyssana 4 (1-2)  december 2013: 93-96 petrović, s.  a contribution to the knowledge of the neuroptera … 93 original article a contribution to the knowledge of the neuroptera (insecta) fauna of serbia sonja petrović university of nis, faculty of science and mathematics, department of biology and ecology, višegradska str. 33, 18000 niš, serbia * e-mail: sonjichka@gmail.com abstract: petrović, s.: a contribution to the knowledge of the neuroptera (insecta) fauna of serbia. biologica nyssana, 4 (1-2), december 2013: 93-96. we present a record of a rare species libelloides lacteus brullè, 1832 (ascalaphidae, ascalaphinae) inhabiting jelašnica gorge, formerly known to be present only in one single site located inside serbia's province kosovo and metohija. existing literature data regarding balkan peninsula distribution of this species is listed here. jelašnica gorge's adult and larval stage specimen findings are illustrated. morphoanatomical parameters essential for species determination are listed. species habitat conditions presented here, have a closer determination according to vegetation units and according to eunis classification system. it is concluded that a jelašnica gorge population has the most continental distribution on balkan peninsula. further species investigation is recommended, especially the one regarding it's ecology and it's relationship to other populations. key words: libelloides lacteus brullè, 1832, jelašnica gorge, serbia. introduction serbian neuroptera fauna is not researched sufficiently enough, and it's study is rather fragmented. first written data is to be found in p o n g r a c z (1923) work that refers to fauna belonging to this group in albania which states 12 more species in kosovo and metohija. researching an insect fauna on university land near majdanpek, ž i v o j i n o v i ć (1950) affirmed the existence of 7 more neuroptera species among large number of others. in a contribution about kosovo and metohija's neuroptera species, d e v e t a k and j a k š i ć (2003) are listing findings of a 51 species of this group. j o n e s and d e v e t a k (2009) gave new fauna data on nevrorthus apatelios species. as part of an ecological study on macro-invertebrates in ivanštica river, had been noted a finding of neuroptera osmylus fulvicephalus (scopoli, 1763) (đ u k n i ć et al., 2010). beside this faunistic works, there is existent literature data where neuroptera is not the object of primary faunistic research, but mentioned and regarded to as a predator of other groups of insects (g r a o r a , 2009; j e r i n i ć -p r o d a n o v i ć , 2010; t o m a n o v i ć , 2008, and others). summing the results of data in this literature, we are free to conclude that there are 54 species of neuroptera described in serbia. in balkan peninsula neuroptera fauna, there are 244 species known (p o p o v & l e t a r d i , 2010). genus libelloides is occurring in europe by the number of 9 species. looking into an overview of a literature quoted here, we can conclude that serbian fauna recognizes only two species of libelloides: libelloides macaronius (scopoli, 1763) (syn.: ascalaphus macaronius) and libelloides lacteus brullè, 1832 [syn.: ascalaphus ottomanus (germar, 1817)]. however, this work present a newly discovered finding of libelloides lacteus brullè, 1832 species. 11 th sfses • 13-16 june 2013, vlasina lake 4 (1-2) • december 2013: 93-96 biologica nyssana 4 (1-2)  december 2013: 93-96 petrović, s.  a contribution to the knowledge of the neuroptera … 94 material and methods field research on jelašnica gorge domain, had been done during 2011 and 2012. material concerning this species had been registered on the site, habitat and species pictures photographed using a digital camera. due to population preservation concerns, only a few of evidential specimens had been collected. morphologic and anatomical parameter analysis used for determination, had been done in a standard procedure in a form of classic microscopic slide preparation. photographs of specimen in situ had been taken using “olympus” sp-510uz (7.1 megapixel and 10x optical zoom), photographs of mandible had been taken using "leica dm 1000" microscope with "camera leica dec 290”. all of the material (specimens and mandible slides) are deposited in the author's collection. results and discussion in a few recent years of fieldwork in jelašnica gorge area, species l. macaronius had been found rather regularly. in early may of 2012, however, we had found a few adult specimens of a. lacteus species, clearly discerned by it's charachteristic habitus of white base colored wings with blue tones heading toward an apex area, as opposed to l. macaronius whose wings are dominated by a yelow. we have sampled evidential specimens and made our snapshots in situ (fig.1). fig. 1. libelloides lacteus in jelašnica gorge (photo s. petrović) one month later and according to our expectations, during a careful field inspection we have noticed a number of characteristic sand funnels (fig. 2), on bottoms of which we had found and sampled a few adult larvae. fig. 2. characteristic habitat (sand funnels) of the larvae of libelloides lacteus (photo s. petrović) conserved larvae have been photographed previously (fig. 3), followed by dissection of a mandible under a binocular and preserved in a form of a permanent microscopic slide preparation (fig. 4). comparison of larvae habitus and morphological details of mandible to the same parameters found on other related species, led to an unambiguous conclusion of the matter being a larval species listed (g e p p, 1984). mandible is characterised by three pronounced teeth on the inside, with presence of an accompanying hairs over a whole surface. fig. 3. larva of libelloides lacteus (photo s. petrović) ecological conditions of the habitat, and the species that exist on it, fully correspond to those in which the species had been found in bulgaria (b u r e s c h , 1936) or on the adriatic side of balkan peninsula (d e v e t a k , 1998). we may charachterise them as being an exeptionally arid climate habitats, exposed to a south wind, and being poor in vegetation. in jelašnica gorge we have established the species exactly on such kind of a site, inscribed in a geographical literature by the toponym ploča (fig. 5). this site's vegetation is represented by a plant associations of species poterio-festucetum valesiacae danon 1969, and biologica nyssana 4 (1-2)  december 2013: 93-96 petrović, s.  a contribution to the knowledge of the neuroptera … 95 sedo-potentilletum arenariae ružić 1978. according to eunis habitat classification system, habitats being registered on this site are : e1.224 (perennial calcareous grassland and basic steppes) dominated by festuca valsiaca s. lat. and e1.5 (mediterranean-montane grassland). fig. 4. mandubula of adult larva of libelloides lacteus (photo i. gnjatović) summary review shown on a map (fig.6), displays a distribution of this species on a balkan peninsula according to a i s t l e i t n e r (2007), supplemented with our own finding in jelašnica gorge. it is shown that the find of this anatolian ponto mediterranean species, is being the most continental one. b u r e s c h ' s (1936) findings are comming from thrace part of european turkey and bulgaria (patlejna monastery near preslav, st. todor monastery near bodom, dede agač) . place of discovery in jelašnica gorge, is located 350 km further away from that teritory. it is certain that a communication to populations on the east of bulgaria is achived over a valley of nišava river. due to non existent, inadequate habitats and mountain barriers (kukavica mt., radan mt., jastrebac mt., kopaonik mt.) which do not permit populations mixing, a communication with populations from kosovo and metohija is a less certain one. bearing these facts in mind, we may infer that further ecological and molecular-genetic research would complement a comprehensive biologycal knowledge on this species. fig. 5. site pleče in the jelašnica gorge fig. 6. distribution of libelloides lacteus on balkan penninsula, italy and turkey (dashed line is the boundary of the distribution of species on the balkan peninsula according to aistleitner, 2007) conclusion on a basis of morphological parameters in adults and on a basis of a larvae structure details, a pressence of a libelloides lacteus brullè, 1832 (neuroptera, ascalaphidae) in jelašnica gorge, near niš, has been proven. this is by far the most continental finding of this species on balkan peninsula. references aistleitner, e., 2007. zur taxonomie und chorologie des schmetterlingshaftes libelloides biologica nyssana 4 (1-2)  december 2013: 93-96 petrović, s.  a contribution to the knowledge of the neuroptera … 96 lacteus (brullè, 1832). entomofauna, 28(26): 357-368. buresch, i., 1936. prinos' k'm' izučvaneto na mrežokrilnata fauna na b'lgaria. izvestija na b'lgarskoto entomologično družestvo, ix: 135150. devetak, d,.1998. libelloides ottomanus (germar, 1817) in the northwestern part of the balkan peninsula (neuroptera, ascalaphidae). ent.croat., (1997) 1998. 3(1-2): 45-48. devetak, d. and jakšić, p. 2003. neuroptera of kosovo and metohija (serbia). z. arb. germ. ost. ent.öst.ent., 55: 45-53. đuknić, j., bjelanović, k., durutović, a. and jovanović, j. 2010. ecological survey of macroinvertebrate communities in the vrelska padina and the ivanštica rivers (eastern serbia). balwois 2010 ohrid, republic of macedonia. pp.: 1-12. davies, c., moss, d., hill, o.m., 2004. eunis habitat classification revised 2004. http://www.searchmesh.com/pdf/gmhm1%20e unis_habitat_classification_revised_2004.pdf gepp, j., 1984. erforschungsstand der neuropterenlarven der erde. progress in world's neuropterology. gepp j., h. aspöck & h. hölzel ed, 265 pp 1984, pp.: 183-239, graz. graora, d. 2009. parazitoidi i predatori štitastih vašiju iz familije diaspididae u nekim voćnjacima u srbiji. pestic. fitomed. (beograd), 24(4), 2009, 295-301. jerinić-prodanović, d., 2010. predatori i parazitoidi cacopsylla pyri (l.) (hemiptera: psyllidae) u srbiji. pestic. fitomed. (beograd), 25(1), 2010, 29-42 jones, r. and devetak, d. 2009. first record of nevrorthidae from slovenia. acta entomologica slovenica 17(2): 99-106. pongracz, s. 1923. recesszarnyuak. neuropteroiden. – in: csiki erno allattani kutatasai albaniaban. explorationes zoologicae ab e. csiki in albania perectae. ix. a. magyar tudomanyos akademia balkan-kutatasainak tudomanyos eredmenyei. budapest, 1(1): 143166. popov, a., letardi, a., 2010. comparative zoogeographical analysis of neuropterida of the apennine and balkan peninsulas. proceedings of the tenth international symposium on neuropterology. piran, slovenia, 2008. devetak, d., lipovšek, s. & arnett, a.e. (eds). maribor, slovenia, 2010. pp. 239–256. tomanović, ž., kavallieratos, n.g., stary, p., petrović-obradović, o., athanassiou, c.g., stanisavljević, lj.ž., 2008: cereal aphids (hemiptera: aphidoidea) in serbia: seasonal dynamics and natural enemies. eur. j. entomol. 105: 495–501. živojinović, s. 1950. fauna insekata šumske domene majdanpek. san, knjiga clx (2), beograd. jakšić, p., zlatković, b.  distribution of burnets & foresters species in... biologica nyssana 6 (1)  september 2015: 33-39 jakšić, p., zlatković, b.  distribution of burnets & foresters species in... 33 original article received: 11 april 2015 revised: 15 june 2015 accepted: 01 july 2015 distribution of burnets & foresters species in plant associations and habitats of special nature reserve „jelašnička klisura gorge“ (serbia) (lepidoptera: zygaenidae) predrag jakšić*, bojan zlatković university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia * e-mail: jaksicpredrag@gmail.com abstract: jakšić, p., zlatković, b.: distribution of burnets & foresters species in plant associations and habitats of special nature reserve „jelašnička klisura gorge“ (serbia) (lepidoptera: zygaenidae). biologica nyssana, 6 (1), september 2015: 33-39. general geographic and biogeographic features of special nature reserve „jelašnička klisura gorge“ in eastern serbia are represented. the lists of plant communities and habitat types for the area are given. faunistic account of zygaenidae species recorded in period 2009-2014. is analised. distribution of zygaenidae species within plant assotiations and habitat types is presented for the first time. key words: zygaenidae, habitats, jelašnička klisura gorge apstrakt: jakšić, p., zlatković, b.: distribucija vrsta familije zygaenidae (lepidoptera) u biljnim zajednicama i staništima specijalnog rezervata prirode „jelašnička klisura“ (srbija). biologica nyssana, 6 (1), septembar 2015: 33-39. prikazane su opšte geografske i biogeografske odlike specijalnog rezervata prirode „jelašnička klisura“, smeštenog u istočnoj srbiji. navedene su liste biljnih zajednica i tipova staništa konstatovanih na podrućju klisure. sumirani su rezultati faunističkog istraživanja predstavnika familije zygaenidae realizovanog u periodu od 2009. do 2014. godine. po prvi put je prikazana distribucija utvrđenih vrsta familije zygaenidae na primeru biljnih zajednica i staništa specijalnog rezervata prirode „jelašnička klisura“. key words: zygaenidae, staništa, jelašnička klisura introduction adopting rio declaration on environment and development (1992), serbia significantly initiated activities in protection of biodiversity and nature in general (a n o n y m o u s , 1992). within numerous protected natural resources there are 67 nature reserves in the country. one of them is jelašnička klisura gorge, situated 15 km se of the town of niš, between jelašnica village and čukljenik village (fig. 1). geographic location, geological, geomorphological, climatic and other natural features have made jelašnička klisura gorge 6 (1) • september 2015: 33-39 biologica nyssana 6 (1)  september 2015: 33-39 jakšić, p., zlatković, b.  distribution of burnets & foresters species in... 34 one of the most interesting areas in eastern serbia from the aspect of biodiversity and nature conservation. the main hidrological phenomena among several, periodical streams of the area is studena river, that forms small but magnificent gorge, with narrowed sometimes closed sides. geological base is made of limestone. the gorge is strongly influenced by a temperate continental climate (semi-arid temperate continental (subcontinental) climate) with ethesian influences from sub-mediterranean region. however, largest part of the gorge is situated at lower altitudes (ranged from 293 to 580 m.a.s.l.) and therefore opened to the influence of the continental and southern climate that strongly influenced its plant life and species composition in general. typical climate type is additionally underlined by characteristic of limestone ground and prominent karst geomorphology, making the special mesoclimatic conditions in the gorge. the climate of the area is depicted by climate diagram according to w a l t e r & l e i t h (1967) for niška banja spa as the nearest location (fig. 2). from the phytogeographycal point of view investigated area belongs to the west-moesian floristic province of the balkan sub-region, and central european floristic region. due to a strong presence of steppe floristic region to the gorge, some of its parts can be understood as an enclave of pontic-south-sibirian floristic region. the role of refugia that have gorges and canyons of the central balkan peninsula explains species richness, originality and the antiquity of their flora and vegetation (m i š i ć , 1981). the most important elements of flora are tertiary relicts ramonda serbica pančić and r. nataliae pančić & petrović, as well as balkan endemic and sub-endemic taxa such as satureja kitaibelii wierzb. ex heuff., dianthus noeanus boiss., parietaria lusitanica subsp. serbica (pančić) p. w. ball, micromeria cristata (hampe) griseb., ect. (l a z a r e v i ć et al., 2007). the protected area of of the nature reserve, established in 1995., is around 116 ha (a n o n y m o u s , 1995). investigated area is positioned in the close vicinity of large sićevačka klisura gorge and surrounded by large massif of mt. suva planina, also important from the point of biodiversity and nature conservation. material and methods during the period of six years of field work (2009.-2014.) representatives of burnets & foresters (family zygaenidae) were notified in certain habitat types of jelašnička klisura gorge. the adult specimens has been collected on the field using fig. 1. jelašnička klisura gorge, habitat detail, photo p. jakšić fig. 2. climatic diagram of niška banja spa according to w a l t e r & l e i t h (1967) biologica nyssana 6 (1)  september 2015: 33-39 jakšić, p., zlatković, b.  distribution of burnets & foresters species in... 35 entomological net. after preparing, we determined the specimens by the wing-patterns and in all cases the identification has been also carried out by an examination of the male genitalia. the preparations were carried out following the well known standard procedure: maceration by boiling in potash, dissecting and cleaning, clearing in xylolum and mounting in canada balsam. the photos of genital parameters were taken using the "leica dm 1000" microscope with the "camera leica dec 290"; photos terrain, habitats and in situ specimen were taken using “olympus” sp-510uz (7.1 megapixel and 25x optical zoom). all the material (specimens and genitalia slides) are deposited in the first author's collection. the list of plant communities is based on field survey as well as relevant literature sources given in the article. the list of syntaxonomic units is aligned with j o v a n o v i ć et al. (1986), k o j i ć et al. (1998). habitat types are defined by immediate field study. the habitat types are in accordance with “natura 2000“ classification system (a n o n y m o u s , 2000). their equivalents for the territory of serbia according to l a k u š i ć (2005) are shown in the tab. 2. and 3. fieldwork on protected areas was done on the basis of permits provided by the ministry of environment, mining and spatial planing, republic of serbia, no. 353-01-1559-2011-03, dated from 8. 6. 2011; no. 353-01-1070/2012-03, dated from 12. 06. 2012. and no. 353-01-916/2014-08, dated from 29.05.2014. results and discussion on the base of the literature data given by several authors (j o v a n o v i ć d u n j i ć , 1955; j o v a n o v i ć , 1980; m i š i ć , 1981; s t e v a n o v i ć et al., 1987; l a z a r e v i ć et al., 2007) as well as field survey, the list of plant communities in jelašnička klisura gorge has been established (tab. 1). the presence of 23 plant associations belonging to 16 alliances and 9 classes is recorded. table 1. syntaxonomic review of vegetation in jelašnička klisura gorge syntaxonomic unit (classis, order, alliantia, assotiation) querco-fagetea br.-bl. et vlieger 1937 quercetalia pubescentis br.-bl. 1932 ostryo-carpinion orientalis horvat 1954 ass.: carpinetum orientalis serbicum rudski 1940 em. b. jov. 1953 quercion frainetto ht. 1954 ass.: quercetum frainetto-cerris rudski (1940) 1949 subass. carpinetosum orientalis (knapp) b. jovanović 1953 syringo-carpinion orientalis jakucs 1959 ass.: carpino orientalis-quercetum mixtum mišić 1967 ass.: syringo-carpinetum orientalis (greb. 1950.) mišić 1967 prunetalia spinosae r. tx. 1952 prunion spinosae soó (1930) 1940 ass.: pruno spinosae-crataegetum (soó 1927) hueck 1931 populetalia albae br.-bl. 1931 salicion albae soó (1930) 1940 ass.: salicetum albae-fragilis soó (1933) 1958 paliuretea trinajstić 1978 paliuretalia trinajstić 1978 paliurion moesiacum b. jov. 1985 ass.: botryochloo-paliuretum b. jov. 1973 asplenietea trichomanis br.-bl. 1926 potentilletalia caulescentis br.-bl. 1926 edraiantho graminifolii-erysimion comatae mucina et al. 1990 ass.: ceterachi-ramondetum serbicae r. jovanović 1953 subass.: ramondetosum nathaliae v. stevanović et al. ass.: parietarietum serbicae niketić 1986 micromerion cristatae n. & v. ranđ. 1998 ass.: campanulo velebiticae-micromerietum cristatae v. et n. ranđ. 1999 festuco-seslerietea barbero et bonim 1969 seslerietalia juncifoliae ht. 1930 seslerion rigidae zolyomy 1939 ass.: seslerietum filifoliae zolyomy 1939 subass. scabiosetsoum fumarioides niketić et lakušić 1988 ass.: anthyllo-seslerietum rigidae r. jovanović 1955 biologica nyssana 6 (1)  september 2015: 33-39 jakšić, p., zlatković, b.  distribution of burnets & foresters species in... 36 festuco-brometea br.-bl. et r. tx. 1943 scorzonero-chrysopogonetalia horvatić et horvat (1956) 1958 chrysopogoni-satureion horvat et horvatić 1934 ass.: euphorbio myrsiniti-bothryochloetum r. jovanović 1955 festucetalia valesiacae br.-bl. et tx. 1943 festucion valesiacae klika 1933 ass.: poterio-festucetum valesiacae danon 1960 ass.: potentillo-caricetum humilis r. jovanović 1955 ass.: galio-festucetum valesiacae r. jovanović 1955 ass.: andropogono-danthonietum calycinae danon et blaženčić 1975 ass.: festuco-agrostidetum vulgaris danon et blaženčić 1978 satureion kitaibelii horvat 1962 ass.: sedo-potentilletum arenariae ružić 1978 phragmitetea communis r. tx. et preising 1942 magnocaricetalia pignatti 1953 caricion gracilis-vulpinae e. bálatová-tulačková 1963 ass.: junco articulatae-caricetum vulpinae danon et blaž. 1978 bidentetea tripartiti tx., lohm. et prsg. 1950 bidentetalia tripartitii br.-bl. ex tx. 1943 bidention tripartiti nordh 1940 ass.: polygono-rumicetum conglomerati v. ranđ. 1990 plantaginetea majoris tx. et preising 1950 plantaginetalia majoris tx.et preising 1950 polygonion avicularis br.bl.1931 ass.: lolio-plantaginetum majoris beger 1930 artemisietea vulgaris lohm., preising et r. tx. 1950 artemisietalia vulgaris lohm. apud r.tx.1947 arction lappae tx.(1937) 1942 em. gutte 1972 ass: arctio-artemisietum vulgaris (r.tx.1942) oberd. et al.1967 table 2. survey of “natura 2000“ habitat types in jelašnička klisura gorge. codes for their equivalents according to lakušić (2005) given in the brakets temperate heath and scrub 40a0 *subcontinental peri-pannonian scrub (f3.24) semi-natural dry grasslands and scrubland facies 6210 semi-natural dry grasslands and scrubland facies on calcareous substrates (festuco-brometalia) (*important orchid sites) (e1.21, e1.22, e1.55) rocky slopes with chasmophytes vegetation 8210 calcareous rocky slopes with chasmophytic vegetation (h3.2a) forests of temperate europe 9180 *tilio-acerion forests of slopes, screes and ravines (g1.a4) 91e0 *alluvial forests with alnus glutinosa and fraxinus excelsior (alno-padion, alnion incanae, salicion albae) (g1.11) 91m0 pannonian-balkanic turkey oak-sessile oak forests (g1.76) *priority habitat summarizing literature data (l a k u š i ć et al., 2005; s e k u l i ć et al., 2010), as well as personal terrain insight, the list of most important habitats for species of family zygaenidae has been made (tab. 2). field research established that fauna of burnets and foresters in special nature reserve “jelašnička klisura gorge” are represented with total number of 8 species: adscita (1 species), jordanita (2 species), and zygaena (5 species). following species have been notified: adscita mannii (8 records), jordanita chloros (2 records), j. graeca (4 records), zygaena carniolica (1 record), z. ephialtes (1 record), z. filipendulae (1 record), z. loti (1 record) and z. purpuralis (6 records), listed in tab. 3. the largest number of representatives, actually the presence of entire 8 identified species have been notified at habitats type 6210 in accordance with classification “natura 2000”. at habitat type 8210 in accordance with previously mentioned classification, only 3 species have been notified (a. mannii, j. graeca and j. chloros), while at the rest of habitat types in the gorge, only 1 species of burnets and foresters has been presented and notified. biologica nyssana 6 (1)  september 2015: 33-40 jakšić, p., zlatković, b.  distribution of burnets & foresters species in... 37 table 3. distribution of zygaenidae species within the main habitats of jelašnička klisura gorge and presence of their larval foodplants species, date of collection and genitalia slides number habitat type according to „natura 2000“, (eunis); larval foodplants and related plant species adscita (tarmannita) mannii (lederer, 1853) 5.vi 2013., 1♂ (prep. no. sr-2556); 14. v 2014., 1♂ (prep. no. sr-2571); 20. v 2014., 2♂♂ (prep. no. sr-2578 and sr-2589) and 27.v 2014., 1♂ (prep. no. sr-2576) 6210 (e1.21, e1.22, e1.55), 8210 helianthemum spp. (including h. nummularium, h. canum and h. salicifolium) jordanita (jordanita) graeca (jordan, 1907) 6. vii 2010., 1♂; (prep. no. sr-2464); 2. vi 2011., 2♀♀. 6210 (e1.21, e1.22, e1.55), 8210 centaurea spp. (including c. salonitana, centaurea scabiosa subsp. apiculata and c. cyanus) jordanita (jordanita) chloros (hűbner, [1813]) 6. vii 2010., 2♂♂, 1♀. (prep. no. sr-2473 and sr-2474). 6210 (e1.21, e1.22, e1.55), 8210 centaurea spp. (including c. salonitana, centaurea scabiosa subsp. apiculata and c. cyanus) zygaena (mesembrynus) purpuralis (brünnich, 1763) 2. vi 2011., 1♂; 14. v 2014., 3♂♂ (prep. no. anz-164 and anz-165); 20.v 2014., 1♂ (prep. no. anz-166); 5.vi 2014., 1♂ (prep. no. anz167). 6210 (e1.22), 9180 (g1.a4), 91m0 (g1.76) thymus sl. (including thymus praecox subsp. jankae, and t. glabrescens); eryngium spp. as larval foodplants of z. minos (e. palmatum, e. campestre) zygaena (agrumenia) carniolica (scopoli, 1763) 6. vii 2010, 1♀. 6210 (e1.21, e1.22, e1.55) onobrychis arenaria (including o. alba) zygaena (zygaena) loti ([denis & schifferműller], 1775) 6. vii 2010., 1♂. 6210 (e1.22, e1.55) onobrychis spp., (including onobrychis arenaria and o. alba) coronilla varia (including c. elegans), lotus corniculatus zygaena (zygaena) ephialtes (linnaeus, 1767) 6. vii 2010., 1♂, 1♀. 6210 (e1.22, e1.55) coronilla varia (including c. elegans) zygaena (zygaena) filipendulae (linnaeus, 1758) 23. vi 2011., 1♂. 6210 (e1.22) dorycnium pentaphyllum (including d. pentaphyllum subsp. germanicum and d. pentaphyllum subsp. herbaceum) semi-natural dry grassland and scrubland facies on calcareous substrates (6210) are developed throughout research area, so the fact that they have a great significance in terms of species diversity of family zygaenidae, is not unexpected. they are mainly dry, termophilic steppe alike habitats, occupying large areas on shallow soil of eroded and moderately steep slopes of the gorge. importance of this habitat type is also multiple, according to the presence of several protected orchid species e.g. orchis purpurea hudson and himantoglossum hircinum (l). sprengel on the slopes of the gorge. in terms of vegetation point of view they belong to a vegetation of dry pastures of rocky continental which is characterized by extreme presence of steppe and submediterranian plant species. in syntaxonomic sense, these are associations of class festuco-brometea, presented in jelašnička klisura gorge with large numbers of phytocoenoses, where by the most significant are communities of festucion valesiacae and satureion kitaibelli alliances. supported by anthropogenic activities this vegetation type has been widespread throughout east and south-east serbia. it is of secondary character and it is developed under subaride climate conditions on destroyed forest communities, mainly oak forest communities with termophilic character (d i k l i ć & n i k o l i ć , 1964). calcareous rocky slopes with chasmophytic vegetation (8210) habitat types have far less importance compared to represents of family zygaenidae in terms of distribution and diversity of the research area. although in terms of spatial representation, this habitat type represents one of the area characteristic, and it is one of the most interesting in the gorge, from the phytogeographical point of view, but has secondary role in terms of diversity of burnets and foresters. this type of habitat includes rocky, limestone cliffs, that are overgrown by vegetation from the class asplenietea biologica nyssana 6 (1)  september 2015: 33-39 jakšić, p., zlatković, b.  distribution of burnets & foresters species in... 38 fig. 3. position of examined habitat types on topographic map (sheet 583_1_3_bela palanka, 1: 25000) fig. 4. male genitalia slide of adscita (tarmannita) mannii (lederer, 1853), jelašnička klisura gorge, 27.v 2014., genitalia slide no. sr-2576, x25 fig. 5. zygaena purpuralis (brünnich, 1763), jelašnička klisura gorge, 14. v 2014., (genitalia slide prep. no. anz-164), photo p. jakšić trichomanis on slopes and canyon parts of the gorge. the greatest significance in the context of this type of vegetation on the research area are phytocoenoses of alliances edraiantho graminofolii-erysmion comatae and micromerion cristatae, comprising community of endemo-relict species of the genus ramonda, as well as community of parietaria lusitanica subsp. serbica, which represents specific vegetation of this part of europe. related to two mentioned habitat types, all other types of habitats, listed in tab. 2, as well as plant communities, listed in tab. 1, have much smaller role in terms of distribution and diversity of family zygaenidae. conclusion after several years of monitoring of distribution of the species of zygaenidae family in jelašnica gorge we have established their priority habitats and shed light to their diversity in this area (tab. 3). analysing distribution of certain types we can conclude that main criteria in selection of habitats was presence of their larval foodplants, followed by the number of zygaenidae species recorded. from that point of view, the most important habitats in the gorge are semi-natural dry grasslands and scrubland facies on calcareous substrates and calcareous rocky slopes with chasmophytic vegetation. according to the parameters that calculated richness of zygaenidae in the gorge those habitats are particularly important, diserving special attention in further aspects of conservation within deserving area of the gorge. acknowledgements. we express our thanks to ivan gnjatović for the photo of genitalia and to ana nahirnić for photo treatment in photoshop and determination of zygaena purpuralis specimens. the fieldwork was supported by the scientific society biodat alpin (innsbruck, austria). references anonymous, 1992: agenda 21, rio declaration, united nations. anonymous, 1995: uredba o zaštiti specijalnog rezervata prirode „jelašnička klisura“. službeni glasnik rs, 9/95. anonymous, 2000: reference list of habitat types and species present in the region. continental region. natura 2000. european environmental agency, european topic centre of nature conservation. doc. cont/b/fin. 3. anonymous, 2010: the rulebook on habitat types, the criteria for the selection of habitat types, biologica nyssana 6 (1)  september 2015: 33-39 jakšić, p., zlatković, b.  distribution of burnets & foresters species in... 39 on sensitive, endangered, rare and priority for protection habitat types and on the protection measures for their conservation. official gazette of rs 35/2010. beograd diklić, n., nikolić, v., 1964: o nekim zajednicama pašnjaka i livada na svrljiškim planinama. glasnik prirodnjačkog muzeja u beogradu. b18: 65-88. jakšić, p., 2014: 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slavnić 1964; bjelčić & stefanović, 1986; abadžić, 1986/87). however, only since the verification of cbd (convention on biological diversity) in 2002, monitoring ias has become mandatory. according to the national cbd report (strategija i akcioni plan za zaštitu biološke raznolikosti bosne i hercegovine 2015-2020. podrška bosni i hercegovini za revidiranje strategije i akcionog plana za zaštitu biološke raznolikosti i izradu petog nacionalnog izvještaja prema konvenciji o biološkoj raznolikosti, 2016), the plan was to observe all ias and identify pathways of spreading by 2020. so far, no official list of invasive flora and fauna exists. in that light, every investigation which goes into the direction of study the ias is of great importance. every year, high biodiversity and ecological value of kozara national park attract many visitors, who can potentially endanger certain eco© 2020 hasanović et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 65 systems. it is worth noting that bucalo et al. (2007) detected 657 vascular plants in a relatively small area of the park (3910 ha). with this in mind, the main purpose of the study was to contribute to better understanding of distribution of invasive flora in the kozara national park. the investigated area included the central and south part of kozara massif. the fieldwork was performed on 6 locations through original phytocoenological research during the summer 2018. the analysis of the floristic composition was executed with the standard method zurich-montpellier school braun-blanquet (1964). description of each site was made using garmin gps and topographic, geological and pedological maps (tab. 1.) preliminary list of invasive plant flora for b&h (maslo, 2016) was used as a primary database. for the plant identification, we used following identification keys: domac (1979), javorka and csapody (1991), šarić (1991), šilić (2005), nikolić et al. (2014). the nomenclature 66 table 1. general locality information no. locality coordinates altitude (m) slope (º) exposition bedrock soil type 1 zečiji kamen viewpoint n 45° 1'3.13" e 16°52'16.15" 655 45 sw limestone calcomelanosol 2 ski plateau mrakovica n 45° 0'50.75" e 16°54'13.38" 785 5 w eocene flysch deposol 3 gumline viewpoint n 44°59'43.29" e 16°54'43.37" 520 10 sw limestone calcomelanosol 4 road for kotlovača n 45° 00'23.9" e 16°52'42.5" 655 0 w gabbro deposol 5 sledding site mrakovica n 45° 0'56.59" e 16°54'18.73" 790 5 se eocene flysch deposol 6 mountain hut mrakovica n 45° 00'50.8" e 16°54'20.6" 650 50 se eocene flysch deposol table 2. recorded invasive flora in kozara national park taxon family detected in community origin life ambrosia artemisiifolia l. asteraceae artemisietalia lohm. prsg. et tx. in tx. 1950; querco-ostryetum carpinifolie ht. 1938 north america t erigeron canadensis (l.) cronquist asteraceae artemisietalia lohm. prsg. et tx. in tx. 1950 north america h erigeron annus (l.) pers. asteraceae artemisietalia lohm. prsg. et tx. in tx. 1950; querco-ostryetum carpinifolie ht. 1938 north america h galinsoga parviflora cav. asteraceae artemisietalia lohm. prsg. et tx. in tx. 1950 north and south america t juncus tenuis willd. juncaceae artemisietalia lohm. prsg. et tx. in tx. 1950 north america h parthenocissus quinquefolia (l.) planch. vitaceae fagetalia sylvaticae pawlowski in pawlowski et al. 1928 north america ph phytolacca americana l. phytolaccaceae artemisietalia lohm. prsg. et tx. in tx. 1950 north america g reynoutria japonica houtt. polygonaceae adenostyletalia br.-bl. 1931 east asia g robinia pseudoacacia l. fabaceae fagetalia sylvaticae pawlowski in pawlowski et al. 1928 north america ph solidago gigantea aiton asteraceae artemisietalia lohm. prsg. et tx. in tx. 1950 northen america g sorghum halepense (l.) pers. poaceae artemisietalia lohm. prsg. et tx. in tx. 1950 middle east g veronica persica poir. artemisietalia lohm. prsg. et tx. in tx. 1950 south-western asia t xanthium strumarium l. subsp. italicum (moretti) d. love asteraceae artemisietalia lohm. prsg. et tx. in tx. 1950 north america t biologica nyssana ● 11 (1) september 2020: 65-69 hasanović et al. ● contribution to the knowledge of invasive flora in kozara national park 67 follows the euro+med plantbase (2006) and the international organization for plant information (iopi) database (2017). syntaxonomic position of the analyzed communities was determined based on the list of plant communities in bosnia and herzegovina (prodromus) (lakušić 1978, barudanović et al., 2015) and international code of phytosociological nomenclature (weber et al., 2000). the taxa are listed alphabetically and accompanied by life form data (ph-phanerophyte, ch-chamaephyte, h-hemicryptophyte, g-geophyte, hy-hydrophyte, t-therophyte) (raunkiaer, 1934). out of the 119 identified plant species in the kozara national park, 13 (10.9%) were ias (tab. 2). maslo (2016) listed 50 ias in the preliminary list of ias in bosnia and herzegovina. it is important to state that the overall number of ias in the park may not be definitive due to the scope of research. all identified plants are neophytes, originated from north america (76.92%) and asia (23.08%). the majority of ias belong to asteraceae (46%) which is, regarding invasiveness, one of the most abounding in the world (pyšek, 1998). the most abundant life forms in the examined flora of the investigated area are therophytes (30.76%) and geophytes (30.76%) followed by hemicryptophytes (23.1%) and phanerophytes (15.38%). even though the studied area is characterized by mesophilic and hydrophilic habitats with the larger portion of phanerophytes and hemicryptophytes (lakušić et al., 1991; bucalo et al., 2007), continuous anthropogenic impacts in the form of degradation resulted in expansion of nitrified, xerophytic and stomped habitats. hard and compact soil, high insolation and temperature, along with weak soil transparency enabled the development and domination of therophytes and geophytes (jovanović, 1993). the relatively high percentage of hemicryptophytes (23.1%) is not surprising because these plant species have extraordinary regenerative potential and can endure stomping and breaking (topalić-trivunović, 2006). continuous construction projects and expansion of tourist offer allow undisturbed spreading of invasive flora, both alien and native. with five marked recreational trails and the accompanying infrastructure, invasive flora is spreading on the ski and trim tracks near mrakovica where we detected nine ias: ambrosia artemisiifolia, erigeron canadensis, erigeron annuus, galinsoga parviflora, juncus tenuis, reynoutria japonica, robinia pseudoacacia, veronica persica and xanthium strumarium subsp. italicum. along the roads from mrakovica to gola planina, three ias were found, ambrosia artemisiifolia, reynoutria japonica and erigeron annuus. by the trails from mrakovica to kozarački kamen, benkovac and zečiji kamen we identified ambrosia artemisiifolia, erigeron canadensis, erigeron annuus, robinia pseudoacacia, solidago gigantea, galinsoga parviflora and veronica persica. phytolacca americana was detected in the northwestern and sorghum halepense in the southern part of the park. the gathered data suggest that all ias continuously spread alongside the roads and trails. many studies confirm that ias spread near roads (hansen & clevenger, 2005; meunier & lavoie, 2012; brisson et al., 2017). of the detected invasive flora, the most important is the spreading of ambrosia artemisiifolia and reynoutria japonica given their negative impact on human health (leru et al., 2015) and biodiversity (aguilera et al., 2010). the high abundance of a. artemisiifolia on the mrakovica plateau is of special concern. even though it was detected in only two localities near mrakovica, reynoutria japonica populations could possibly continue the spreading around the nearest pathways. growing in high-density populations (naiman et al., 2005), japanese knotweed disables the development of any other plant species through strong allelopathic effect (murrell et al., fig. 1. map of detected invasive flora in the kozara national park biologica nyssana ● 11 (1) september 2020: 65-69 hasanović et al. ● contribution to the knowledge of invasive flora in kozara national park 68 2011). the spreading of this species would be harmful particularly in the zone of pašini konaci due to unique swamp habitats with communities glycerietum plicatae kulczynski 1928 and lemnetum minoris (oberd.1957) th. mull. gors.1960. acknowledgements. this research was supported by bosnia and herzegovina federal ministry of education and science and embassy of the united states in bosnia and herzegovina. references abadžić, s. 1986-1987: prilog poznavanju horologije i ekologije dviju adventivnih vrsta – echinocystis lobata (michx) torrey et gray i bidens bipinnata l. u flori bosne i hercegovine. 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lavoie, c. 2012: roads as corridors for invasive plant species: new evidence from smooth bedstraw (galium molugo). invasive plant science and management, 5(1): 92-100. murrell, c., gerber, e., krebs, c., parepa, m., schaffner, u., bossdorf, o. 2011: invasive knotweed affects native plants through allelopathy. american journal of botany, 98 (1): 38-43. naiman, r.j., décamps, h., mclain, n. 2005: riparia: ecology, conservation, and management of streamside communities. elsevier academic press, burlington, mass: 448 p. nikolić, t., mitić, b., boršić, i. 2014: flora hrvatske: invazivne biljke. udžbenici sveučilišta u zagrebu, zagreb: 295 p. pyšek, p. 1998: is there a taxonomic pattern to plant invasions? oikos 82: 282-294. raunkiaer, c. 1934: the life form of plants and statisti-cal plant geography. clarendon press, oxford: 729 p. biologica nyssana ● 11 (1) september 2020: 65-69 hasanović et al. ● contribution to the knowledge of invasive flora in kozara national park biologica nyssana ● 11 (1) september 2020: 65-69 hasanović et al. ● contribution to the knowledge of invasive flora in kozara national park slavnić, ž. 1960: o useljavanju, širenju i odomaćivanju nekih adventivnih biljaka u bosni i hercegovini. godišnjak biološkog instituta univerziteta u sarajevu, 8 (1-2): 117-146. sarajevo. slavnić, ž. 1964: rod bidens l. u flori bosne i hercegovine. radovi naučnog društva sr bih, 25 (7): 155-162. šarić, t. 1991: atlas korova: 100 najvažnijih vrsta korovskih biljaka u jugoslaviji. svjetlost, sarajevo: 221 p. šilić, č. 2005: atlas dendroflore (drveće i grmlje). matica hrvatska, zagreb: 575 p. the catalogue of life partnership 2017: iopi global plant checklist in the catalogue of life. checklist dataset. https://doi.org/10.15468/ topalić-trivunović, lj. 2006: ruderalna flora i vegetacija područja banje luke, phd thesis, prirodno-matematički fakultet univerziteta u banjoj luci weber, h.e., moravec, j., theurillat, j.-p. 2000: international code of phytosociological nomenclature. 3rd edition. journal of vegetation science, 11: 739-768. 69 the ecological and floristic characteristics of natural population of micromeria juliana (l.) benth. ex rchb. in bulgaria biologica nyssana 7 (2)  december 2016: 91-99 aneva, i. et al.  the ecological and floristic characteristics… 91 original article received: 12 october 2016 revised: 08 november 2016 accepted: 29 november 2016 the ecological and floristic characteristics of natural population of micromeria juliana (l.) benth. ex rchb. in bulgaria ina aneva1*, petar zhelev2, milena nikolova1, ivan evtimov2 1institute of biodiversity and ecosystem research, bulgarian academy of sciences, 1113 sofia, bulgaria 2university of forestry, 10 kliment ohridsky blvd., 1797 sofia, bulgaria * e-mail: ina.aneva@abv.bg abstract: aneva, i., zhelev, p., nikolova, m., evtimov, i.: the ecological and floristic characteristics of natural population of micromeria juliana (l.) benth. ex rchb. in bulgaria. biologica nyssana, 7 (2), december 2016: 91-99. micromeria juliana is a rare species in bulgaria. it is included in the red data book of the country with conservation status “endangered” and is protected by the biodiversity act. the present report focuses to the study of natural populations and plant communities of m. juliana in the two regions of its occurrence in bulgaria – eastern rhodopes and the valley of mesta river. in the first locality the species grows on steep stony slopes and on walls of a medieval fortress, and the second one – on a steep calcareous slope, and on abandoned agricultural land. we present results of a survey on the species composition of plant communities of m. juliana together with analyses of floristic elements and ecological forms. the floristic composition indicates that the participation ratio of elements with mediterranean origin is high. the ecological factors that have the highest impact on the floristic composition are intensive light, air temperature and humidity. the area occupied by the species is limited due to its very specific requirements and low competition ability. key words: medicinal plant, species composition, floristic elements apstrakt: aneva, i., zhelev, p., nikolova, m., evtimov, i.: ekološke i florističke karakteristike prirodnih populacija vrste micromeria juliana (l.) benth. ex rchb. u bugarskoj. biologica nyssana, 7 (2), decembar 2016: 9199. micromeria juliana je retka vrsta u bugarskoj. nalazi se u crvenoj knjizi ove zemlje sa konzervacionim statusom “ugrožena” i zaštićena je zakonom o biodiverzitetu. ovde prezentovano istraživanje je bilo usmereno na proučavanje prirodnih populacija i biljnih zajednica vrste m. juliana unutar dva regiona u kojima se ova vrsta pojavljuje u bugarskoj – istočnim rodopima i dolini reke mesta. na prvom lokalitetu vrsta raste na strmim kamenitim padinama, kao i na zidinama srednjevekovne tvrđave, dok na drugom – na strmim krečnjačkim padinama i napuštenim poljoprivrednim zemljištima. mi smo prezentovali rezultate pregleda sastava vrsta biljnih zajednica m. juliana, zajedno sa analizom florističkih elemenata i ekoloških formi. floristički sastav ukazuje da je udeo mediteranskih elemenata visok. intenzivna svetlost, temperature vazduha 7 (2) • december 2016: 91-99 12th sfses • 16-19 june 2016, kopaonik mt doi: 10.5281/zenodo.200405 biologica nyssana 7 (2)  december 2016: 91-99 aneva, i. et al.  the ecological and floristic characteristics… 92 i vlažnost su ekološki faktori koji imaju najznačajniji uticaj na floristički sastav. oblast koju zauzima vrsta je ograničena zbog veoma specifičnih prohteva i niske kompetitivne sposobnosti vrste. ključne reči: lekovite biljke, sastav vrsta, florni elementi introduction genus micromeria benth. (lamiaceae) is represented in bulgaria by four species (a n č e v , 1989), two of them (m. juliana (l.) benth. ex rchb. and m. cristata (hampe) griseb.) belonging to section micromeria and two (m. dalmatica benth. and m. frivaldszkyana (degen) velen.) – to section pseudomelissa benth. the species of the latter section were transferred to genus clinopodium based on the evidence of recent phylogenetic studies (b r ä u c h l e r et al., 2005, 2006; r y d i n g 2006). according to b r ä u c h l e r et al. (2008) there are still a lot of taxonomic problems to be solved, despite of the success achieved using modern cladistic analyses based on molecular data (w a g s t a f f et al., 1995; b r ä u c h l e r et al., 2005; see b r ä u c h l e r et al., 2008, for synopsis and review). many species of the genus possess substantial interest as medicinal and aromatic plants. there were some studies on the cytology, morphology and chemical composition of the essential oil of some micromeria representatives in balkans and at least part of them addressed also the question of antibacterial and antifungal activity of the species (m a r i n k o v i c et al., 2002; s l a v k o v s k a et al., 2005; k o s t a d i n o v a et al., 2007; m a r t i n et al., 2011; k a r o u s o u et al., 2012; k r e m e r et al., 2014, m a r i n et al., 2015). bulgarian species of the genus are considered important as medicinal and aromatic plants and also from conservation point of view. two of them – m. juliana (fig. 1) and m. frivaldszkyana – are listed in the different editions of the red data book of bulgaria with the category “endangered” (a n č e v , 1984; a p o s t o l o v a , 2015; s t o y a n o v , 2015). even though not an endemic, m. juliana is a rare species and occurs only in two small localities in the eastern rhodopes (a n č e v , 1984, 1989; s t o y a n o v , 2015). however, t a s h e v (2015) reported another occurrence in the valley of river mesta. the information about biological peculiarities, habitat requirements and natural resources of micromeria species is very important for designing of proper strategy for their conservation and sustainable use. therefore, the objective of the present study was to characterize the natural localities of micromeria juliana as a part of a program for conservation and sustainable use of the species of genus micromeria in bulgaria. material and methods the objects of the study included the two known localities of the species in bulgaria. the first one is located in the eastern rhodopes, locality called “kaleto”, close to gugutka village, ivaylovgrad district (41°23'52.60", 25°57'17.96"), which will be referred to hereafter as rhodopes. the second one is in the valley of river mesta, locality called “dimanska stena”, close to the village godeshevo (41°27'57.41", 24° 3'31.53") and will be referred to hereafter as mesta. the field observations took place in the period april-july 2016. full floristic inventory was performed in the two natural localities of the species. taxonomic treatment followed a s s y o v & p e t r o v a (eds., 2012). phytogeographic affinity of the species was determined according to w a l t e r (1985), summarized by a s s y o v & p e t r o v a (eds., 2012), and the life forms – according to r a u n k i a e r (1934). fig. 1. micromeria juliana biologica nyssana 7 (2)  december 2016: 91-99 aneva, i. et al.  the ecological and floristic characteristics… 93 results and discussion in the locality “kaleto”, eastern rhodopes, m. juliana was found mostly on the wall of the medieval fortress (fig. 2), and also one small spot was found on the road ledge close to the fortress. the populations of m. juliana are represented by small number of individuals. the main reason can be traced in its ecological requirements to specific type of habitats – stony and rocky places or open soils free of turf grasses. usually on balkans m. juliana occurs in the plant communities known as aegean phrygana (eunis 2016), and these habitats are characterized by open stony and sandy spots. however, even though the habitats in the studied locality resemble the phrygana, they are not identical. the potential vegetation on the place could be classified as quercus pubescens forests (b o n d e v , 1991), and these forests, even rather dry, often form dense herbaceous cover, thus preventing the establishment of m. juliana individuals. therefore, the most visible occurrence of the species was on the walls of the fortress. it occurred also on the rocky places and on the slopes free of turf grasses. occurrence of the species on walls is not unprecedented, as reported by l a g i o u et al. (1998). m. juliana is heat demanding species and is therefore found predominantly on eastern and southern expositions. the area occupied by the species in rhodopes is about 0.1 ha and the population size is about 40 individuals. further studies are necessary to reveal whether there are more spots of this rare species in the rocky slopes of byala reka river. in the second locality, in the valley of river mesta, the species was found on a steep (3040°) rocky slope in the place called “dimanska stena”, but also few isolated and smaller spots were found in the adjacent abandoned arable land (fig. 3). all these spots were bulked together when analyzing the locality. the area occupied by the species in the valley of mesta river is about 0.4 ha. the population size is larger and consists of about 90 individuals, whose dimensions tend to be larger than those in rhodopes. m. juliana is not collected by people, and direct anthropogenic pressure cannot be considered as a threat, with the exception of some potential incidental fire, as suggested by s t o y a n o v (2015). total 124 species were recorded in the two localities – 47 species in the eastern rhodopes and 89 species in the valley of mesta river. it should be noted that these values represent a minimum species number, because some short-lived spring annuals and/or geophytes could have terminated their vegetation at the time of the field study. also, some species with insufficiently developed diagnostic traits at that moment could have been overlooked or misidentified. the higher number of species recorded in the locality mesta is due to its larger occupied area on the rocky slope and on the abandoned arable land. there were 4 species of ferns, one gymnosperm, and 119 angiosperm species – 107 dicots and 12 monocots (tab. 1). the species recorded belong to 39 families and 103 genera. the most numerous family is asteraceae – 13 genera and 14 species, followed by lamiaceae – 10 genera and 13 species, poaceae – 9 genera and 11 species, and rosaceae – 7 genera and 9 species. eighteen families are represented by only one species. the flora in these two localities had not been studied in detail previously. t a s h e v (2015) mentioned few species occurring in the community of mesta locality together with m. juliana, like asyneuma limonifolium (l.) janch,, inula aschersoniana janka, teucrium polium l., euphorbia myrsinites l., and dichanthium ischaemum (l.) roberty, all of them recorded during our field survey. classification of species regarding their light dependence, or luminosity of the environment, fig. 2. locality 1 (eastern rhodopes) – the medieval fortress fig. 3. locality 2 (mesta) biologica nyssana 7 (2)  december 2016: 91-99 aneva, i. et al.  the ecological and floristic characteristics… 94 fig. 4. (see table 1 for legend.) a. species distribution according to their light dependence b. species distribution according to their dependence on humidity c. species distribution according to their biological type d. species distribution according to their life form fig. 5. distribution of the floristic elements. see table 1 for legend. biologica nyssana 7 (2)  december 2016: 91-99 aneva, i. et al.  the ecological and floristic characteristics… 95 revealed that 119 species (96%) are heliophytes, only one can be classified as sciophyte and four species are of transitional character (fig. 4a). xerophytes represent the most numerous type regarding dependence on water availability (104 species, 84%). the other species are classified as mesophytes (7 species), xero-mesophytes (8 species) and 5 species belong to other categories (fig. 4b). perennial species predominate – 65 (52%) followed by annuals – 26 (21%), plus 3 species (2%), which are of transitional type annual-biennial. thirteen species (10%) are shrubs and 9 species (7%) are trees (fig. 4c). this fact reflects the potential vegetation type of the locality, which can be defined as quercus pubescens forests, like in eastern rhodopes. currently, trees and shrubs are represented by a few individuals and even though they do not shape the physiognomy of the plant community, they show the natural trend towards restoring the indigenous vegetation. classification by life forms could be predicted by the biological type. hemi-cryptophytes are the predominating type – 63 species (52%), which is expected because most perennial plants fall into this category (fig. 4d). the second most numerous group is the one of therophytes – 32 (26%) and the third one – of phanerophytes – 18 (15%). distribution of floristic elements reflects the mediterranean character of the flora. the predominant elements are: sub-mediterranean – 29 species (23%), euro-mediterranean – 18 (14.5%), typical mediterranean – 11 (9%), and euro-asian – 10 (8%). however, if all elements having mediterranean component are considered (for example, eur-med, eur-submed, med, med-as, pont-med etc.) they would amount to 80 species (65%). the trends are the same in the two studied localities, in spite of some minor differences (fig. 5). the results of the study underline the submediterranean character of the flora and vegetation of the two localities (p a v l o v a et al., 2004; p e t r o v a , 2004; g o r a n o v a et al., 2013). conclusion species composition and characteristics of the two natural localities of m. juliana in bulgaria revealed that the plant communities the species is part of are typical for the dray and hot habitats in the zone of strong mediterranean influence. the locality in the valley of mesta river is larger both in terms of area occupied and population size. the status of the species can be evaluated as relatively stable. it is practically not affected by the human activities and only some incidental fire could pose a threat. acknowledgements. the authors are grateful to the financial support provided by program for career development of young scientists, bulgarian academy of sciences– grant № dfnp-67_a1. references ančev, m. 1984: micromeria juliana. in: velchev v. 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(english translation), springer-verlag, berlinheidelberg, 318 p. biologica nyssana 7 (2)  december 2016: 91-99 aneva, i. et al.  the ecological and floristic characteristics… 97 table 1. list of plant taxa recorded in the two localities, together with their characteristics (er-eastern rhodopes, mr-mesta river valley) taxon light dependence humidity dependence biological type life form floristic element er mr polypodiophyta polypodiopsida aspleniaceae asplenium adianthum-nigrum l. sc m/hg p h subboreal + asplenium ruta-muraria l. h x p h boreal + + ceterach officinarum dc. h x p h submed + magnoliophyta hypolepidaceae pteridium aquilinum (l.) kuhn h m p h kos + pinophytina pinopsida cupressaceae juniperus deltoides r.p. adams h x s ph eur-med + + magnoliophytina magnoliopsida aceraceae acer campestre l. h x/m t ph eur-ot + acer monspessulanum l. h x/m t ph submed + anacardiaceae pistacia terebinthus l. h x t ph pont-med + rhus coriaria l. h x t ph med-as + apiaceae eryngium campestre l. h m/x p h pont-med + scandix pecten-veneris h x a th eur-as + asteraceae carlina vulgaris l. h m/x a th eur-med + + centaurea stoebel. h x p h submed + centaurea salonitana vis. h x p h pont-med + chondrilla juncea l. h x a th eur-sib + cirsiumligulareboiss. h x a-b th med + crepis biennisl. h x a th submed + crupina vulgariscass. h x a th submed + inula aschersonianajanka h x p h bal-anat + jurinea consanguinea dc. h x p h submed-sib + leontodon hispidusl. h x p h eur-med logfia arvensis (l.) holub h x a th eur-med + picnomon acarna (l.) cass. h x p h med + scorzonera laciniata l. h x p h med + tragopogon pratensis l. h x a th eur-med + betulaceae carpinus betulusl. h x/m t ph eur-submed + carpinus orientalis miller h x/m t ph submed + boraginaceae buglossoides purpurocaerulea (l.) i. m. johnst. h x p h eur-as + myosotis arvensis (l.) hill h x a th eur-as + + onosma echioides l. h x p h med + brassicaceae aethionema saxatile (l.) r. br. h x p h submed + alyssum alyssoides (l.) l. h x p h eur-med + alyssum murale waldst. & kit. h x p h eur-submed + clypeola jonthlaspi l. h x a th med + erysimum diffusum ehrh. h x/m a-b h/th eur + campanulaceae asyneuma limonifolium (l.) janchen h x p cr ap-bal + caryophyllaceae cerastium bulgaricum uechtr. h x a th bul + herniaria incana lam. h x a th eur-med + minuartia caespitosa (ehrh.) degen h x p h eur-med + minuartia hirsuta (m. bieb.) hand.mazz. h x p h submed + scleranthus perennis roch. ex baumg. h x p h eur-med + silene conica l. h x a th submed-as + + biologica nyssana 7 (2)  december 2016: 91-99 aneva, i. et al.  the ecological and floristic characteristics… silene italica (l.) pers. h/sc m/x p h eur-med + stellaria graminea l. h x p h eur-as + cistaceae cistus incanus l. h x s ch med + fumana procumbens (dunal) gren. &godr. h x s ch pont-med + + helianthemum numularium (l.) mill. h x s ch alp-med + rhodax canus (l.) fuss h x s ch pont + convolvulaceae convolvulus cantabrica l. h/sc x p h pont + crassulaceae sedum anopetalum dc. h x p ch submed + sedum hispanicum l. h x p h eur-med + dipsacaceae scabiosa rotata m. bieb. h x p h med + euphorbiaceae euphorbia cyparissias l. h/sc m p h eur + euphorbia myrsinites l. h x p h submed + euphorbia plathyphyllos l. h x a th eur-med + fabaceae astragalus onobrychis l. h x p h eur-as + coronilla cretica l. h x p h med coronilla scorpioides (l.) c. koch h x a th submed dorycnium herbaceum vill. h x p h eur-med + lathyrus nissolia l. h x p h eur-submed + lotus aegaeus (griseb.) boiss. h x p h med + medicago lupulina l. h x p h eur-as + medicago minima (l.) bartal. h x a th eur-as + + onobrychis arenaria (kit.) dc. h x p h pont + ononis pusilla l. h x a th submed + vicia dalmatica a. kern. h x p h submed + fagaceae quercus pubescens willd. h x/m t ph eur-submed + geraniaceae erodium cicutarium (l.) l'hér. h x a th subboreal + geranium robertianum l. h x a th subboreal + + geranium rotundifolium l. h x a th eur-as + + hypericaceae hypericum cerastoides (spach) n.k.b. roloson. h m p h submed + hypericum olympicum l. h x/m p h submed + lamiaceae acinos suaveolens (sm.) don h x p h submed + + ajuga chamaepitys (l.) schreber h x a-b h/th pont-med + calamintha nepeta (l.) savi h x p h eur-med + clinopodium vulgare l. h x p h subboreal + micromeria dalmatica bentham ssp. bulgarica (velen.) guinea h x p h bal + micromeria juliana (l.) benth. ex rchb. h x p h med + satureja coerulea janka h x s ch submed + satureja cuneifolia ten. h x s ch submed + sideritis montana l. h x a th submed + stachys germanica l. h x p h eur-submed + teucrium chamaedrys l. h/ sc x/m p h submed + teucrium polium l. h x p h pont-med + + thymus atticus čelak. h x s ch bal-anat + linaceae linum hologynum rchb. h x p h submed + moraceae ficus carica l. h x t ph adv (med) + oleaceae fraxinus ornus l. h x t ph submed + jasminum fruticans l. h x s ph pont-cas + plantaginaceae plantago lanceolata l. h m p h kos + primulaceae anagalis arvensis l. h x a th kos + biologica nyssana 7 (2)  december 2016: 91-99 aneva, i. et al.  the ecological and floristic characteristics… 99 ranunculaceae clematis vitalba l. h x s ph eur + resedaceae reseda lutea l. h x p h subboreal + rhamnaceae paliurus spina-christi mill. h x s ph eur-as + rosaceae agrimonia eupatoria l. h m p h eur-med + crataegus monogyna jacq. h x s ph subboreal + potentilla argentea l. h x p h pont + potentilla sulphurea lam. h x p h submed + prunus spinosa l. h x s ph pont + rosa myriacantha dc.ex lam. & dc. h x s ph submed + rubus discolor weihe & nees h x s ph submed + rubus sanguineus friv. h x s ph pont-med + + sanguisorba minor scop. h x p h subboreal + + rubiaceae cruciata pedemontana (bellardi) ehrend h x a th med-as + + galium pseudoaristatum schur. h x p h pann-bal + santalaceae thesium linophyllon l. h m p h submed + scrophulariaceae linaria vulgaris mill. h m p h eur-sib + misopates orontium (l.) raf. h x a th eur-med + verbascum densiflorum bertol. h x a th submed + veronica triphyllos l. h x a th eur-med + urticaceae parietaria lusitanica l. h x a th med-as + valerianaceae valerianella carinata loisel. h x a th eur-med + liliopsida cyperaceae carex otrubae podp. h m/x p h eur + poaceae aegilops neglecta req. ex bertol. h x a th submed + bromus squarrosus l. h x a th submed + bromus sterilis l. h x a th boreal + chrysopogon gryllus (l.) trin. h x p h pont-med + dichanthium ischaemum (l.) roberty h x p h submed-as + echinaria capitata (l.) desf. h x a th med + festuca valesiaca scheicher ex gaudin h x p h pont + koeleria nitidula velen. h x p h pont + melica ciliata l. h x p h eur-submed + poa bulbosa l. h x p h eur-as + + poa pratensis l. h x p h kos + legend: light dependence: h heliophyte; h/sc – helio-sciophyte; sc/h – scio-heliophyte; sc sciophyte. humidity dependence: x xerophyte; m mesophyte; m/x – meso-xerophyte; x/m – xero-mesophyte; m/hg – meso-hygrophyte. biological type: a – annual; a-b – annual to biennial; b – biennial; p – perennial; s – shrub; t – tree. life form: th therophyte; н hemicryptophyte; ch chamaephyte; ph – phanerophyte. floristic element: adv – adventive; anat – anatolian; ap – appenine; as – asian;bal – balkan; boreal; bul – bulgarian; eur – european; kos – cosmopolitan; med– mediterranean; ot – oriental-turanian; pann – pannonian; pont – pontic; sib – siberian; additional abbreviation: s – southern; e – eastern; w – western; n – northern; c – central. biologica nyssana 7 (2)  december 2016: 91-99 aneva, i. et al.  the ecological and floristic characteristics… sevindik et al. 2021, biologica nyssana 12(2) 12 (2) december 2021: 131-139 doi: 10.5281/zenodo.5759841 assessment of phylogenetic relationships of some worldwide-cultivated cotton genotypes (gossypium hirsutum l.) by using issr and rapd markers original article emre sevindik faculty of agriculture, department of agricultural biotechnology, adnan menderes university, south campus, cakmar, aydin, turkey ph.d-emre@hotmail.com (corresponding author) muhammed ebrar çayir faculty of agriculture, department of agricultural biotechnology, adnan menderes university, south campus, cakmar, aydin, turkey tülay emrebaş ankara university, faculty of agriculture, department of field crops, turkey ertuğrul filiz duzce university, department of crop and animal production, cilimli vocational school, 81750, cilimli, duzce, turkey received: june 09, 2021 revised: september 07, 2021 accepted: october 03, 2021 abstract: cotton belongs to the genus of the malvaceae family and presents an economically and biologically highly valuable multi-purpose crop grown as a source of fiber, food and feed worldwide. in this study, phylogenetic relationships of cotton genotypes of the thirteen countries (twelve gossypium hirsutum and one gossypium barbadense (giza 75 egypt) as outgroup) taken from the directorate of nazilli cotton research institute genetic stock were analyzed by using rapd and issr markers. rapd–pcr analysis obtained total of 53 dna bands with the highest number of bands for opa-02 and opa-13 primers, no bands for ope-08 primer and rate of polymorphism of 90.56%. issr –pcr analysis showed a total of 81 dna bands, where the highest number of bands was obtained with ubc-811, ubc-834 and ubc836 primers, no bands with ubc-853 primer and rate of polymorphism of 77.77%. phylogenetic tree was obtained by using the upgma algorithm and the tree consisted of two large clades. analysis of the results suggests that rapd and issr markers are useful tools to demonstrate phylogenetic relationships between cotton genotypes. key words: gossypium hirsutum, rapd, issr, molecular markers, phylogenetic apstrakt: utvrđivanje filogenetskih odnosa nekih genotipova pamuka (gossypium hirsutum l.) kultivisanih širom sveta korišćenjem issr i rapd markera pamuk pripada rodu gossypium iz familije malvaceae i predstavlja ekonomski i biološki veoma vredan višenamenski usev koji se gaji kao izvor vlakana i hrane širom sveta. u ovom istraživanju, analizirani su filogenetski odnosi genotipova pamuka iz trinaest različitih zemalja (dvanaest gossypium hirsutum i jedna gossypium barbadense (giza 75 egypt) kao zasebna grupa) uzetih iz banke genotipova nazili instituta za istraživanje pamuka, a proučavani su korišćenjem rapd i issr markera. rapd–pcr analizom dobijeno je ukupno 53 dnk traka uz najveći broj traka sa opa-02 i opa-13 prajmerima, nije bilo trake za ope-08 prajmer, a stopa polimorfizma iznosila je 90.56%. issr –pcr analiza pokazala je ukupno 81 dnk traka, gde je najveći broj traka dobijen sa ubc-811, ubc-834 i ubc-836 prajmerima, nije bilo traka za ubc-853 prajmer, a stopa polimorfizma iznosila je 77.77%. filogenetsko drvo je dobijeno korišćenjem upgma algoritma i sadržalo je dve velike klade. analiza rezultata sugeriše da rapd i issr marker predstavljaju korisne alate za demonstriranje filogenetskih odnosa između genotipova pamuka. ključne reči: gossypium hirsutum, rapd, issr, molekularni markeri, filogenetski introduction the malvaceae family consists of 243 genera with 4,225 known species, and contains various economically valuable species such as gossypium (cotton), corchorus (jute), hibiscus, alcea, theobroma cacao (cacao), abelmoschus esculentus (okra) and malva in tropical and subtropical regions (pravin et al., 2018; ben-simchon et al., 2019; wang et al., 2020). the main spreading center of these family members is south america. besides, it spreads everywhere except for the very cold regions of the earth (uzunhisarcıklı & vural, 2009). the members of the family malvaceae include herbs, © 2021 sevindik et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 131 subshrubs, shrubs, trees and even woody climbers (ţîţei & teleuță, 2018). in addition, the malvaceae family has mucilage, fixed oils and essential oils and is used for medicinal purposes. plants of this family are used publicly for the treatment of respiratory and digestive system irritations and inflammations as expectorant, bronchodilator, diuretic and for the treatment of gastric ulcers (erarslan & koçyiğit, 2019). cotton belongs to the gossypium genus of the malvaceae family and contains 51 species, among which 45 are diploid (2n = 26) and 6 are allotetraploid (2n = 52) (elçi & hançer, 2016). in addition, this genus includes only four cultivated cottons that are morphologically distinct and geographically widespread, namely: g. arboreum l. and g. herbaceum l. known as the old world african-asian cottons, g. barbadense l. (sea island cotton) and g. hirsutum l. and or others known as upland cotton (ibrahim et al. 2007). eighty-seven percent of cotton growth area in the world occurs in developing countries (nix et al., 2017). cotton is an economically and biologically highly valuable multipurpose crop grown as a source of fiber, food and feed worldwide. the cottons of gossypium hirsutum l. and gossypium barbadense l., are leading natural fiber crops as well as oilseed crops important to bioenergy, feed and food industries (zhang et al. 2015; noman et al., 2016; bilval et al., 2017). the primary product of the cotton plant is mohair, which covers the seeds in the seed or cocoon. this mohair has been used for thousands of years to dress the people of ancient india, asia, america and africa (abdellatif et al., 2012). molecular markers have developed a wide range of applications in the field of molecular biology, which may include phylogenetic study, genetic diversity, evolution, ecology, population genetics, and the study of complex genomic properties in both plants and animals (atasagun et al., 2018; raza et al., 2019). genetic diversity can be estimated from pedigree analysis or from multivariate analysis carried out on a large number of plant attributes (sharma et al., 2018). dna markers offer a convenient way to study polymorphisms, taxonomy, physiology, embryology, genetic engineering and their use in product development (kesawat & kumar, 2009; shaheen et al., 2013). pcr (polymerase chain reaction) based molecular markers such as issr, rapd, ssr, and aflp were found to be useful in various applications of genetic variation and plant breeding (dongre et al., 2004; cui et al., 2017). for the use in detecting genetic variation, the rapd (random amplified polymorphic dna) marker has a number of advantages such as technical simplicity, speed of testing, minimum dna requirements, and low costs. in addition, no prior knowledge about the sequence under investigation is required. rapd markers have been used to analyze genetic relations and diversity in plant populations and cultivars (ghasemi et al., 2014; saha et al., 2019; akinro et al., 2019). issr (inter simple sequence repeats) technique is a pcr based technique, reported by ztetkiewicz et al. (1994), which can quickly show the differences of individuals with close relationships (jafari et al., 2019). this marker is based on determining the random distribution of dna nucleotides in plant genomes, independent of chromosome regions. issr markers are usually dominant markers, so it is difficult to determine whether individuals are heterozygous. this technique is much more sensitive and reproducible than rapd technique (gülşen & roose, 2001; vijayan, 2005; güngör et al., 2020). in this study, genetic diversity of cotton genotypes of the thirteen countries (twelve gossypium hirsutum and one gossypium barbadense as outgroup) taken from the directorate of nazilli cotton research institute genetic stock was analyzed by using rapd and issr markers. materials and methods plant materials and genomic dna isolation the cotton varieties of the thirteen countries 132 biologica nyssana ● 12 (2) december 2021: 131-139 sevindik et al. ● assessment of phylogenetic relationships of some worldwide-cultivated cotton genotypes (gossypium hirsutum l.) by using issr and rapd markers fig. 1. three-week appearance of thirteen different cotton varieties after planting fig. 2. genotypes of gdna isolation gel image 133 were obtained from nazilli directorate of cotton research institute and this study was carried out in the biotechnology laboratory of the directorate. in addition, the 13 cotton varieties were planted in two repeats and three seeds (fig. 1). the seedlings were grown for about three weeks until they had four leaves. those who did not germinate to yield four leaves were waited or replanted until they grown four leaves. the cottons, which were sown in three seeds and two repeats, were diluted into the best growing single plant after they had four leaves. two or threedays old leaves were collected by observing the selected plants every day. after being washed with pure water and dried, the leaf samples were stored at -86 ºc for genomic dna isolation. power plant dna isolation kit (qiagen) was used for genomic dna isolation from the specimens. the obtained genomic dna samples were made ready for use and stored at -20 ºc. for preparation of the gdna (genomic dna) gel image, the mixture was prepared by using 1 µl gdna, 8 µl dh20 and 1 µl loading dye. then 1% agarose gel electrophoresis was prepared and the samples were loaded. gdna gel images of the 13 cotton varieties are shown in fig. 2. the purity of the genomic dna was assessed with a nanodrop spectrophotometer (implen nanophotometer biologica nyssana ● 12 (2) december 2021: 131-139 sevindik et al. ● assessment of phylogenetic relationships of some worldwide-cultivated cotton genotypes (gossypium hirsutum l.) by using issr and rapd markers table 1. primers used in the rapd-pcr reactions and their tm degrees primers dna sequences(5’-3’) tm (°c) amplifications pcr protocol (35 cycles) opa-02 5' tgccgagctg 3' 34 °c + 94 oc/2 min 94 oc/1 min 32-34 oc/1 min 72 oc/1 min 72 oc/10 min opa-05 5' aggggtcttg 3' 32 °c + opa-13 5' cagcacccac 3' 34 °c + opa-15 5' ttccgaaccc 3' 32 °c + opa-16 5' agccagcgaa 3' 32 °c + opa-18 5'aggtgaccgt 3' 32 °c + opa-20 5' gttgcgatcc 3' 32 °c + ope-08 5' tcaccacggt 3' 32 °c opa-07 5’-gaaacgggtg-3’ 32 °c + opa-03 5’-agtcagccac-3’ 32 °c + table 2. primers used in the issr-pcr reactions and their tm degrees issr primers dna sequences(5’-3’) tm (°c) amplifications pcr protocol (35 cycles) ubc-831 5’-ctctctctctctctctt-3’ 50 oc + 94 oc/1 min 94 oc/1 min 48-53 oc/1 min 72 oc/1 min 72 oc/10 min ubc-830 5’-tgtgtgtgtgtgtgtgg-3’ 52 oc + ubc-807 5'-agagagagagagagagt-3' 50 oc + ubc-819 5' gtgtgtgtgtgtgtgta -3 50 oc + ubc-808 5’-agagagagagagagagc-3’ 52 oc + ubc-836 5'-agagagagagagagagya-3’ 52 oc + ubc-856 5'–acacacacacacacacya-3’ 52 oc + ubc-853 5' tctctctctctctctcrt -3' 52 oc ubc-855 5'-acacacacacacacacyt-3' 52 oc + ubc-810 5' -gagagagagagagagat-3' 50 oc + ubc-826 5’-acacacacacacacacc-3’ 52 oc + ubc-811 5’-gagagagagagagagac-3’ 53 oc + ubc-834 5’-agagagagagagagayt-3’ 52 oc + ubc-873 5’-gacagacagacagaca-3’ 48 oc + 134 p330). blanking was applied with the pd7 solution we used in the last stage of the dna isolation kit. the amount of dna was determined by taking 1 µl of each sample and reading of the od260 and od280 nm in a nanodrop spectrophotometer. the purity amount of dna was determined by od260/od280 ratio. according to nanodrop spectrophotometer measurements after isolation, the lowest dna amount was obtained from brown egyptian and the highest dna amount was obtained from ujchi 2 uzbek. according to od260/od280 ratios which are the indications of purity, the highest value was obtained from haridost and the lowest value was obtained from bulgarian 73. pcr amplifications rapd and issr primers chosen for pcr amplifications are shown in tab. 1 and tab. 2. ready mixes were used as an alternative to manual pcr loadings. amplification was carried out by adding 2 μl genomic dna, 1 μl primers and 5 μl of master mix (cat. no: rp02-ii-400, rp02-ii-2000 0.75u of taq dna polymerase, reaction buffer, 2mm mgcl2, 250µm dntps and enzyme stabilizer) and 17 μl dh2o into pcr tube. amplification products were analyzed by electrophoresis on 1% agarose gel buffered with 1.0x tae (tris-acetate-edta), stained with ethidium bromide and photographed under ultraviolet light. as a results of rapd-pcr, some of the gel images were shown in fig. 3a, fig. 3b, fig. 3c and fig. 3d. data and phylogenetic analysis following the pcr analysis, dna bands were scored as “1” in the presence of dna, “0” in the absence of dna and a ‘’?’’ for missing data. genetic relationship of the some gossypium genotypes used in the study was analyzed using paup 4.0b10 (swofford, 2001), and the genetic distance matrix between the populations was calculated by drawing the upgma (unweighted pair group method) phylogenetic tree in the same program in accordance with the arithmetic mean of the pedigree trees. the phylogenetic trees were evaluated with bootstrap test on 10,000 resamplings (felsenstein, 1985) and these values were added to the upgma tree. results and discussion molecular markers developed over the last 30 years have an important role in breeding programs in agriculture (kalkışım et al., 2016). dna markers based on pcr are versatile tools in different aspects of genomic studies. it is possible to analyze closely related genera to evaluate phylogenic and genetic relationships using these markers (zarei et al., 2017). as a result of rapd-pcr analysis, a total of 53 bands were obtained. of these, 48 were polymorphic and the rate of polymorphism was about 90.56%. most of the bands were obtained from opa-02 and opa-13 primers and no band was obtained from ope-08 primer. phylogenetic tree and genetic distances between genotypes were calculated using the paup 4.0b10 analysis program. according to the paup analysis, the closest genetic distance was 0.06 (tam c66 16 els and sc-2079), while 1.0 was the farthest genetic distance (arcota-129 and ziroatkar-81, biologica nyssana ● 12 (2) december 2021: 131-139 sevindik et al. ● assessment of phylogenetic relationships of some worldwide-cultivated cotton genotypes (gossypium hirsutum l.) by using issr and rapd markers fig. 3. gel image of rapd-pcr bands amplified with opa-02 (a) and opa-13 (b). gel image of issr-pcr bands amplified with ubc-807 (c) and ubc-836 (d) 135 biologica nyssana ● 12 (2) december 2021: 131-139 sevindik et al. ● assessment of phylogenetic relationships of some worldwide-cultivated cotton genotypes (gossypium hirsutum l.) by using issr and rapd markers bulgar 73 and ziroatkar-81) (tab. 3). the upgma tree constructed using the paup 4.0b10 phylogenetic analysis program consists of the two large clades (fig. 4). clade 1 was subdivided into multiple subclades. subclade a1, consisted of tam c66-16 els, sc-2079, tropical 225, azgr-7711, fibermax 832, eva and giza-75 (gossypium barbadense) genotypes. subclade a2, consisted of brown egyptian, arcota-129 and bulgar 73 genotypes and this subclade has a 53% bootstrap value. clade 2 consisted of ujchi 2 uzbek, ziroatkar-81 and haridost table 3. pairwise genetic distance matrix obtained from rapd primers genotypes 1 2 3 4 5 6 7 8 9 10 11 12 13 tam c66 16 els 0.14 0.37 0.21 0.40 0.40 0.14 0.25 0.22 0.58 0.58 0.42 0.06 tropical 225 7 0.35 0.10 0.60 0.60 0.17 0.14 0.22 0.64 0.55 0.47 0.21 brown egyptian 15 14 0.32 0.20 0.20 0.37 0.42 0.20 0.58 0.55 0.47 0.40 azgr-7711 10 5 13 0.36 0.27 0.10 0.08 0.34 0.64 0.52 0.33 0.27 arcota-129 2 3 1 4 0.09 0.40 0.60 0.54 0.60 1.00 0.54 0.60 bulgar 73 2 3 1 3 1 0.40 0.60 0.63 0.60 1.00 0.45 0.60 eva 7 8 15 5 2 2 0.10 0.32 0.64 0.52 0.47 0.17 fibermax 832 12 7 17 4 3 3 5 0.32 0.64 0.52 0.47 0.23 giza 75 9 10 8 16 6 7 13 13 0.47 0.58 0.40 0.20 ujchi 2 uzbek 10 11 10 11 3 3 11 11 8 0.30 0.23 0.58 ziroatkar-81 21 20 20 19 5 5 19 19 21 4 0.19 0.55 haridost 9 10 10 9 6 5 10 10 11 3 4 0.38 sc-2079 3 10 16 13 3 3 8 11 8 10 20 8 fig. 4. the upgma tree generated using rapd data genotypes genotypes with bootstrap value of 83%. as a result of issr-pcr analysis, a total of 81 bands were obtained. of these, 63 were polymorphic and the rate of polymorphism was 77.77%. most of the bands were obtained from ubc-811, ubc834 and ubc-836 primers and no band was obtained from ubc-853 primer. according to the paup analysis, the closest genetic distances were 0.00 (ujchi 2 uzbek and ziroatkar-81, haridost and ziroatkar-81), while the farthest genetic distances were 0.77 (arcota-129 and fibermax 832, arcota-129 and sc2079, azgr-7711 and arcota-129) (tab. 4). the upgma tree constructed using the paup 4.0b10 phylogenetic analysis program consists of two large clades (fig. 5). clade one was subdivided into multiple subclades. subclade a1 consisted of tam c66 16 els, eva, sc-2079, tropical 225, azgr-7711 and fibermax 832 genotypes. subclade a2 is composed of brown egyptian and giza-75 (gossypium barbadense) genotypes. this subclade has 81% bootstrap value. clade two was subdivided into multiple subclades and this subclade has a 73% bootstrap value. subclade b1 is composed of arcota-129, ujchi 2 uzbek and bulgar 73 genotypes.subclade b2 is composed of ziroatkar-81 and haridost genotypes. in our study, both rapd and issr results showed that tam c66 16 els, eva, sc-2079, tropical 225, azgr-7711 and fibermax 832 genotypes belong to the same group. ujchi 2 uzbek, ziroatkar-81 and haridost genotypes appeared in the same group. in addition, these three genotypes have been geographically supported. in rapd results, bulgar 73, arcota-129 and brown egyptian appeared 136 biologica nyssana ● 12 (2) december 2021: 131-139 sevindik et al. ● assessment of phylogenetic relationships of some worldwide-cultivated cotton genotypes (gossypium hirsutum l.) by using issr and rapd markers table 4. pairwise genetic distance matrix obtained from issr primers genotypes 1 2 3 4 5 6 7 8 9 10 11 12 13 tam c66 16 els 0.07 0.23 0.11 0.66 0.55 0.04 0.08 0.28 0.57 0.24 0.31 0.06 tropical 225 6 0.28 0.13 0.66 0.60 0.07 0.13 0.28 0.71 0.27 0.36 0.08 brown egyptian 18 22 0.20 0.44 0.53 0.24 0.22 0.20 0.28 0.32 0.31 0.24 azgr-7711 9 11 16 0.77 0.53 0.11 0.07 0.33 0.14 0.19 0.25 0.07 arcota-129 6 6 4 7 0.33 0.66 0.77 0.44 0.00 0.44 0.44 0.77 bulgar 73 36 39 35 35 3 0.53 0.52 0.60 0.00 0.38 0.33 0.56 eva 4 6 19 9 6 35 0.08 0.32 0.57 0.25 0.32 0.06 fibermax 832 7 11 17 6 7 34 7 0.32 0.42 0.20 0.27 0.07 giza 75 22 22 16 26 4 39 25 25 0.51 0.40 0.41 0.32 ujchi 2 uzbek 4 5 2 1 0 0 4 3 4 0.00 0.00 0.57 ziroatkar-81 19 21 25 15 4 25 20 16 31 0 0.09 0.23 haridost 24 28 4 20 4 22 25 21 32 0 7 0.32 sc-2079 5 7 19 6 7 37 5 6 25 4 18 25 fig. 5. the upgma tree generated using issr data 137 biologica nyssana ● 12 (2) december 2021: 131-139 sevindik et al. ● assessment of phylogenetic relationships of some worldwide-cultivated cotton genotypes (gossypium hirsutum l.) by using issr and rapd markers together, while issr results showed only brown egyptian genotype in a separate group. in our study, both rapd and issr results were consistent with each other. in previous studies, hossain et al. (2017) in bangladesh, determined the genetic diversity of the 4 varieties of the gossypium hirsutum through the rapd technique. five rapd primers were used in the study; they yielded 53 bands in total and determined the polymorphism rate to be 90.56%. maleia et al. (2010) determined the genetic difference between african and american varieties and inbred cotton lines (gossypium hirsutum l. race latifolium h.) through rapd molecular markers. 24 rapd primers were used in the study; 166 bands were obtained in total, and the rate of polymorphism was determined to be 90.96%. consequently, the use of rapd molecular markers has been effective for genetic differentiation in cotton varieties and genotypes. surgun et al. (2012) determined the identification and genetic diversity of some turkish cotton (gossypium hirsutum l.) genotypes through rapd-pcr analysis. in the study, genetic difference of nine cotton genotypes was determined through rapd-pcr technique. among the genotypes, a total of 42 rapd primers were used to detect polymorphism; assessable results were obtained from 34 of these primers, and a total of 319 rapd-pcr bands were obtained. the rate of polymorphism among the studied genotypes was determined as 18.1% and genetic similarity between any 2 genotypes ranged from 90.2% to 96.5%. the results revealed that genotypes can be separated at the molecular level with the help of rapd markers. in the study conducted by khan et al. (2000), 45 rapd primers were used in 35 gossypium species in total containing 31 species, 3 subspecies and 1 interspecific hybrid. in the study, a total of 579 dna bands were obtained and the rate of polymorphism was 99.8%. the results obtained in the study provide good correspondence to the taxonomic sections recognized within the genus gossypium. these results show that the rapd technique produces reliable results to create the phylogenetic history of the gossypium genus. in a study carried out in turkey, şahin et al. (2020) determined the genetic relationship between 30 cultivated cotton varieties (gossypium hirsutum l.) through issr-pcr method. 24 issr primers were used to determine polymorphism in 30 cotton varieties; while creating a total of 41 bands from the primers, it was determined that an average of 22.3 of these bands were polymorphic. the polymorphic information content values for issr primers ranged from 0.19 to 0.68 and it was found as 0.49 in average. as a result of the study, they concluded that issr molecular marker technique can be successfully applied in the studies of determining genetic diversity in cotton; in addition, due to its high polymorphism and reproducibility, it can be successfully applied in many laboratories with limited laboratory facilities. bilval et al. (2017) analyzed genetic variation among nine cotton (gossypium) genotypes by using rapd, issr and ssr markers. in the study, 25 rapd, 22 issr and 24 ssr primers were used. the number of polymorphic amplicons was found to be 156 (rapd), 142 (issr) and 24 (ssr) accounting for polymorphism of 91.22% (rapd), 67.94% (issr) and 72.72% (ssr), respectively. as a result of their work, they explained the suitability and reliability of the molecular markers (rapd, issr and ssr) to predict genetic diversity and genetic relationships between cotton genotypes. bardak and bolek (2012) used 5 issr and 39 ssr primers to determine the genetic relationship between diploid and tetraploid 25 cotton genotypes (gossypium spp.) cultivated in different regions of the world. they found that 155 (89.60%) out of 173 alleles obtained as a result of the study were polymorphic. the average number of alleles per ssr and issr markers was 3.93, ranging from 1 to 8 alleles. genetic diversity ranged from 0.04 to 0.58 among all the genotypes inspected. this ratio was 0.04-0.23 within g. hirsutum and 0.07-0.26 within g. barbadense species. as a result of the study, the use of wild cotton species has been suggested to increase the diversity in the gene pool and it has been stated that it will be useful in the selection of the desired properties. in conclusion, rapd and issr markers were used in this study to investigate the genetic diversity of some worldwide cultivated cotton genotypes. the results of the present research have confirmed that these molecular markers can detect polymorphism among gossypium hirsutum genotypes, predict if they are related, and identify genotype-specific rapd and issr markers. according to the results obtained with rapd markers, polymorphism was found to be high and this rate is 90.56 and 77.77% respectively. in addition, the findings obtained from this study are thought to contribute to breeding programs, development of germplasm sources and future marker studies. 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(malvaceae) species cited in the data deficient (dd) category. biodicon, 2(2): 90-95. vijayan, k. 2005: inter simple sequence repeat (issr) polymorphism and its application in mulberry genome analysis. international journal of industrial entomology, 10(2): 79-86. wang, d., fan, w., guo, x., wu, k., zhou, s., chen, z., danyang, l., wang, k., zhu, y., zhou, y. 2020: magendb: a functional genomics hub for malvaceae plants. nucleic acids research, 48(1): 1076-1084. zarei, a., erfani-moghadam, j., mozaffari, m. 2017: phylogenetic analysis among some pome fruit trees of rosaceae family using rapd markers. biotechnology & biotechnological equipment, 31(2): 289-298. zhang, m., rong, y., lee, m.k., zhang, y., stelly, d.m., zhang, h.b. 2015: phylogenetic analysis of gossypium l. using restriction fragment length polymorphism of repeated sequences. molecular genetics and genomics, 290(5): 1859-1872. zietkiewicz, e., rafalski, a., labuda, d. 1994: genome fingerprinting by simple sequence repeat (ssr)-anchored polymerase chain reaction amplification. genomics, 20:176183. anticancer compounds from medicinal plants biologica nyssana 5 (1)  september 2014: 1-10 stojković, m. et al.  antioxidant potential of tanacetum vulgare l. … 71 short communication received: 14 august 2014 revised: 26 august 2014 accepted: 10 september 2014 several records of tachinidae (diptera) reared from their hosts in serbia and montenegro saša s. stanković1, vladimir žikić1, boženka hric2, hans-peter tschorsnig3 1department of biology and ecology, faculty of science and mathematics, university of niš, 33 višegradska street, 18 000 niš, serbia 2jovana popovića 72, 22300 stara pazova, serbia. 3staatliches museum für naturkunde, rosenstein 1, 70191 stuttgart, germany. * e-mail: sasasta@gmail.com abstract: stanković, s.s., žikić, v., hric, b., tschorsnig, h.p.: several records of tachinidae (diptera) reared from their hosts in serbia and montenegro. biologica nyssana, 5 (1), septmeber 2014: 71-73. during the year 2013 over fifty tachinid flies were reared. nine tachinid species form two subfamilies were reported for the territory of serbia and montenegro. species erynniopsis antennata and phryxe hirta are recorded for the first time in serbia. key words: tachinidae, parasitoids, serbia, montenegro. sažetak: stanković, s.s., žikić, v., hric, b., tschorsnig, h.p.: nekoliko zapisa iz familije tachinidae (diptera) odgajene sa domaćina na teritoriji srbije i crne gore. biologica nyssana, 5 (1), septmeber 2014: 71-73. tokom 2013. godine sakupljeno je i odgajeno preko pedeset jedinki tahinida. devet vrsta tahinida iz dve potfamilije su zabeležene na teritoriji srbije i crne gore. vrste erynniopsis antennata i phryxe hirta su po prvi put registrovane u srbiji. ključne reči: tachinidae, parazitoidi, srbija, crna gora. introduction the family tachinidae is one of the most diverse group among diptera order, so far over 10 000 species have been described worldwide (i r w i n e t a l ., 2003). all members of the family are endoparasitoids of other insects, but few genera attack centipedes, scorpions and spiders (w i l l i a m s e t a l ., 1990; v i n c e n t , 1985). many species parasitize economically important insects such are many species of lepidoptera, coleoptera and symphyta (e g g l e t o n & b e l s h a w , 1993) therefore, they have been often used as biocontrol agents against phytophagous pest insects (g r e n i e r , 1988). most tachinid species parasitize larval stage of their hosts. they deposit eggs on or near the host since they lack an ovipositor, unlike parasitic hymenoptera to which they share similar life history. the family tachinidae comprises four subfamilies: exoristinae, 5 (1) • september 2014: 71-73 biologica nyssana 5 (1)  september 2014: 71-73 stanković, s. et al.  several records of tachinidae (diptera) … 72 dexiinae, phasiinae and tachininae (h e r t i n g & d e l y d r a s k o v i t s 1993) according to fauna europaea, internet database 877 species have been recorded so far. from that number, 242 species are recorded for the territory of ex serbia and montenegro state. during the year 2013 several tachinids were reared in serbia and montenegro from a number of lepidopterous, hymenopterous, and coleopterous hosts. the material was bred by b. hric, z. kojičić, a. petrović, s. stanković and v. žikić. the annotated results are listed below. the remarks are, as far as they are not referenced in detail, based on a catalogue on palaearctic tachinid-host records which is currently compiled by the last author (tschorsnig). two new species are newly reported for the investigated territory, and they are marked by an asterisk (*). the arrangement and nomenclature of the tachinids follow herting & dely-draskovits (1993). the data mean the date of collection of the host material. the material is stored at the department of biology and ecology, faculty of science and mathematics, university of niš. species list subfamily dexiinae voria ruralis (fallén, 1810) 3 ♂, 1 ♀, serbia, stara pazova, 4.vii 2013, ex autographa gamma linnaeus [lep. noctuidae], leg. b. hric. – remark: voria ruralis is a common parasitoid of noctuidae-plusiinae, with a. gamma as a very often reared host. subfamily exoristinae ceromasia rubrifrons (macquart, 1834) 1 ♂, serbia, tara, mitrovac, 27.vi 2013, ex p. aporia crataegi linnaeus [lep. pieridae], leg. v. žikić. – remark: ceromasia rubrifrons typically develops in caterpillars of zygaena spp. [lep. zygaenidae]. other lepidopterous host families are only rarely parasitized by this species. among the pieridae, a. crataegi was not yet known as host, but pieris brassicae linnaeus was. compsilura concinnata (meigen, 1824) 12 ♂, 14 ♀, montenegro, visitor, 8–10.vii 2013, ex l. nymphalis antiopa linnaeus [lep. nymphalidae] on salix caprea, leg. a. petrović. – remark: compsilura concinnata is the tachinid species with the largest number of hosts; many records are known from n. antiopa. drino inconspicua (meigen, 1830) 2 ♂, 3 ♀, serbia, jelašnička gorge, 14.v 2013, ex neodiprion sertifer geoffroy [hym. diprionidae] on pinus sp., leg. v. žikić. – 1 ♂, montenegro, visitor, 10.vii 2013, ex nymphalis antiopa linnaeus [lep. nymphalidae] on salix caprea, leg. a. petrović. – 4 ♂, 2 ♀, montenegro, plužine, 8.vii 2013, ex nymphalis polychloros linnaeus on salix caprea, leg. v. žikić. – remark: neodiprion sertifer is, among other diprionidae, a usual host of drino inconspicua. this tachinid is also known from many lepidopterous hosts, but the two often reared nymphalidae, n. antiopa and n. polychloros are untypical. only a single rearing from another nymphalid host, vanessa cardui linnaeus, was known before (richter, 1996). *erynniopsis antennata (rondani, 1861) 3 ♂, 2 ♀, serbia, sićevačka gorge, 28.v 2013, ex xanthogaleruca luteola müller [col. chrysomelidae], leg. z. kojičić. – remark: erynniopsis antennata is an important parasitoid of the elm leaf beetle (see silvestri, 1910: under the erroneous name erynnia nitida). this tachinid fly was not yet known from serbia (compare hubenov, 2008). *phryxe hirta (bigot, 1880) 2 ♀, serbia, vlasina lake, 15.vi 2013, ex p. heterogynis sondereggeri de freina [lep. heterogynidae] on chamaecytisus heuffelii, leg. v. žikić. – remark: phryxe hirta is well-known as a tachinid specialized on the genus heterogynis (see de freina & tschorsnig, 2005), but h. sondereggeri as a new described moth for the world fauna has recorded only once (žikić et al., unpublished). p. hirta is herewith also recorded for the first time from serbia. senometopia separata (rondani, 1859) 1 ♀, serbia, bovansko lake, 9.vi 2013, ex lymantria dispar linnaeus [lep. lymantriidae] on prunus domestica, leg. v. žikić. – remark: s. separata is a common parasitoid of l. dispar. records from this host were also already known from serbia (sisojević, 1959). sturmia bella (meigen, 1824) 1 ♀, serbia, stara pazova, 5.vii 2013, ex vanessa atalanta linnaeus [lep. nymphalidae], leg. b. hric. – 1 ♀, serbia, stara pazova, 2.vii 2013, ex vanessa cardui linnaeus [lep. nymphalidae], leg. b. hric. – remark: sturmia bella is a common parasitoid of nymphalidae. abundant records exist for the hosts v. atalanta and biologica nyssana 5 (1)  september 2014: 71-73 stojković, m. et al.  antioxidant potential of tanacetum vulgare l. … 73 v. cardui, for the latter also from serbia (lehrer & dobrivojević, 1967). winthemia quadripustulata (fabricius, 1794) 1 ♀, serbia, vlasina lake, 21.vii 2013, ex l. cucullia lanceolata villers [lep. noctuidae] on verbascum thaspus, leg. s. stanković. – 1 ♂, serbia, jerma canyon, 19.vii 2013, ex cucullia sp., leg. v. žikić. – remark: cucullia spp. belong to the preferred hosts of w. quadripustulata, but the species c. lanceolata becomes known for the first time. here we report nine tachinid species form two subfamilies for the territory of serbia and montenegro. species erynniopsis antennata and phryxe hirta are recorded for the first time in serbia. also, for two species of tachinidae we report new hosts. cucullia lanceolata is a new host for winthemia quadripustulata, while aporia crataegi is new host for ceromasia rubrifrons. acknowledgements. thanks are due to daniel bartsch (stuttgart) who identified the host pupa of aporia crataegi. this study is supported by the ministry of education and science of the republic of serbia (iii43001). references de freina, j. j., tschorsnig, h. p. 2005: raupenfliegen (diptera: tachinidae) aus heterogynis spp. (lepidoptera: heterogynidae). nachrichtenblatt der bayerischen entomologen, 54: 95-100. eggleton, p., belshaw, r. 1993; comparisons of dipteran, hymenopteran and coleopteran parasitoids: provisional phylogenetic explanations. biological journal of the linnean society, 48: 213-226. grenier, s. 1988: applied biological control with tachinid flies (diptera, tachinidae): a review. anzeiger für schädlingskunde, pflanzenschutz, umweltschutz, 61 (3): 49-56. herting, b., dely-draskovits, a. 1993. family tachinidae. in: soós, a., papp, l. (ed.), catalogue of palaearctic diptera 13: 118-624, hungarian natural history museum, budapest. hubenov, z. 2008. composition and zoogeographical characteristics of the family tachinidae (diptera: insecta) in serbia and bulgaria. in: makarov, s. e., dimitrijević, r. n. (ed.), advances in arachnology and developmental biology pp. 375-394, vienna, belgrade and sofia. irwin, m. e., schlinger, e. i., thompson, f. c. 2003. diptera, trueflies. in: goodman, s. m., benstead, j. p. (ed.), the natural history of madagascar, pp. 692-702.: univ. chicago press. chicago/london 1728pp. lehrer, a. z., dobrivojević, k. 1967: fichier bioécologique et morphologique de diptères entomophages obtenus d’élevage, vii–xiii. bulletin et annales de la société royale d’entomologie de belgique, 103: 53-62. richter, v. a. 1996: on the fauna of tachinids (diptera, tachinidae) of the crimea. entomologiceskoe obozrenie 75: 908-929 [in russian; english translation in entomological review 76 (7): 900-918]. silvestri, f. 1910: contribuzioni alla conoscenza degli insetti et dei loro simbionti. i. galerucella dell’olmo (galerucella luteola f. müll.). bollettino del laboratorio di zoologia generale e agraria della facoltà agraria in portici, 4: 246-288. sisojević, p. 1959: ecological studies of tachinid parasites of the gipsy moth. preliminary communication (annual report for 1958). zaštita bilja, 10: 165-166 (in serbo-croatian with english summary). vincent, l. s. 1985: the first record of a tachinid fly as an internal parasitoid of a spider (diptera: tachinidae; araneae: antrodiaetidae). panpacific entomologist, 61: 224-235. williams, s. c., arnaud, p. h., lowe, g. 1990: parasitism of anuroctonus phaiodactylus (wood) and vaejovis spinigerus (wood) (scorpiones: vaejovidae) by spilochaetosoma californicum smith (diptera: tachinidae), and a review of parasitism in scorpions. myia, 5: 11-27. žikić, v., stanković, s. s, hric, b., mitroiu, m. d., schwartz, m., tschorsnig, h. p.: first records of parasitoids (hymenoptera: ichneumonidae and pteromalidae; diptera: tachinidae) of heterogynis sondereggeri de freina (lepidoptera: heterogynidae), (unpublished). simić & zlatković 2022, biologica nyssana 13(2) 13 (2) december 2022: 89-107 doi: 10.5281/zenodo.7437214 flora around slavujevac on rujan mountain (se serbia) original article milica simić faculty of applied sciences in niš, university “union nikola tesla”, dušana popovića 22a, belgrade, serbia milican92s@gmail.com milica.stankovic2@pmf.edu.rs (corresponding author) bojan zlatković department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, niš, serbia received: september 15, 2022 revised: october 09, 2022 accepted: october 22, 2022 abstract: this paper presents the results of floristic research conducted in the surrounding of village of slavujevac on rujan mt. in se serbia. the presence of 592 taxa from the group of vascular plants was ascertained in the investigated area, with 566 species and subspecies recorded for the first time. out of a total of 26 species listed in the literature for the flora of the slavujevac area, the presence of 11 species has been confirmed. the presence of 45 plant species was recorded in the category of protected taxa, and 13 in the category of strictly protected taxa. the most common life form in the flora of the studied area is the form of hemicryptophytes present with 40.47% of the total number of recorded taxa. in the phytogeographical sense, the flora of the investigated area is dominated by species of the mediterranean-sumediterranean (31.29%) and eurasian (26.36%) area types, also including several balkan endemic taxa. key words: flora, species diversity, floristic composition, slavujevac, rujan mt., serbia apstrakt: flora okoline slavujevca na rujan planini (jugoistočna srbija) u ovom radu su prikazani rezultati florističkog istraživanja sprovedenog u okolini sela slavujevac na planini rujan u jugoistočnoj srbiji. na istraživanom području konstatovano je prisustvo 592 taksona iz grupe vaskularnih biljaka, pri čemu je prvi put zabeleženo 566 vrsta i podvrsta. od ukupno 26 vrsta navedenih u literaturi za floru ovog područja, potvrđeno je prisustvo 11 vrsta. u kategoriji zaštićenih taksona zabeleženo je prisustvo 45, a u kategoriji strogo zaštićenih 13 biljnih vrsta. najčešća životna forma biljaka u flori istraživanog područja su hemikriptofite prisutne sa 40.47% od ukupnog broja evidentiranih taksona. u fitogeografskom smislu, u flori istraživanog područja dominiraju vrste mediteransko-sumediteranskog (31.29%) i evroazijskog (26.36%) areal tipa, uključujući i veći broj balkanskih endemičnih taksona. ključne reči: flora, specijska raznovrsnost, floristički sastav, slavujevac, rujan planina, srbija introduction the part of the balkan peninsula that is characterized by a high diversity of flora and vegetation primarily refers to its southern part, including the southern parts of serbia and the republic of northern macedonia (nikolić et al., 2007). along the land border of the two mentioned countries, there is the mountain region including rujan mt., whose northern slopes include a specific area covered by our research. weakened climatic as well as florogenetic influences from the aegean mediterranean area reach the southern parts of serbia, including the investigated area, creating a characteristic composition and diversity of flora. the specificity of the richness of the plant world is especially noticeable in the thermophilic and xerophilous habitat types, which dominate the entire surroundings of the village of slaujevac, located in the hilly and mountainous belt of the rujan mt. the first data on the flora around slavujevac come from ranđelović and stamenković (1984), who in the second half of the 20th century presented the first data on the presence of certain plant species in this area. two years later, emphasizing the botanical significance of this area, the same authors published the work “flora i vegetacija rujan planine u jugoistočnoj jugoslaviji” with a large number of important data, some of which relate to the vicinity of slavujevac (ranđelović & stamenković, 1986). thanks to the great contribution of prof. n. ranđelović, d.a. hill and prof. v. ranđelović, a new species of the genus crocus was discovered in the area of this mountain, named crocus rujanensis (ranđelović et al., 1988). mount rujan, with the © 2022 simić & zlatković. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 89 surroundings of slavujevac, as an area of distinct floristic richness, has always been an attractive space for various botanical research, including research for the needs of the conservation of the flora of serbia. the aspect of endangerment and protection of rare species that occur in the area of slavujevac, such as: cachrys cristata, opopanax hispidus and alkanna pulmonaria, is discussed in detail in the “crvena knjiga flore srbije 1” (ranđelović et al., 1983; ranđelović et al., 1999; niketić, 1999). later, in the period from 2007-2018, more data on the flora of the investigated area appear, with some data again referring to newly discovered taxa in the flora of serbia (nikolić et al., 2007; diklić & stevanović, 2012; stevanović & gajić, 2012; buzorović et al., 2013; zlatković & bogosavljević, 2014; bogosavljević & zlatković, 2018; nikolić et al., 2018). the latest data from the researched area appears in 2021, when a new taxon (knautia orientalis) was discovered for the flora of serbia (niketić et al., 2021). although the flora of the rujan mt. has been completely processed (ranđelović & stamenković, 1984), a specific analysis of the flora around slavujevac, for which a small number of specific floristic data is stated, was not done until 2015 (stanković, 2015). also, bearing in mind that more than 30 years have passed since the last thorough research of the flora of rujan mt., the aim of this paper is to present a list of flora, but also to indicate whether there have been some changes in the flora of this area which in geographical and phytogeographical terms. materials and methods field research of flora covered the period from march to september 2014 and 2015, i.e. the period from march to september 2021 and from march to june 2022. the area covered by our research includes parts of the rujan mt. located near the village of slavujevac (amedan, dedovica, lismider,majden, mali orljak,veliki orljak,), as well as several localities that make up its rural areas (dolinska mahala, garska mahala and karadačka mahala). the investigated part of the area covers a total area of about 20 km2. the results of field research were documented with herbarium material that was herbarized, labeled and deposited in the herbarium of the faculty of science in niš (hmn). the collected plant material was identified using the keys of contemporary european monographs, including “flora europea” (tutin et al., ed. 19681980), as well as literature sources reference for the researched area (josifović, ed. 1970-1977, sarić, ed. 1986, 1992). the nomenclature is largely in line with the latest checklists, papers, monographs and databases (tutin et al., ed. 1968 – 1980; https://www. europlusmed.org; http://www.worldfloraonline.org). the affiliation of taxa to the appropriate life form was also determined according to the raunkiaer system (1934), which was supplemented by mueller-dombois, ellenberg (1974), and developed for taxa at the level of serbia by stevanović (1992a). the method of meusel et al. (1965, 1978), meusel, jäger (1992) was used to define the range of types and their adequate classification, which according to stevanović (1992) was modified for the territory of serbia. the legend for abbreviations of life forms and flora elements are shown in tab. 1. results and discussion the presence of 592 species from the group of vascular plants was ascertained in the investigated area. all recorded taxa were classified into 312 genera and 73 families. the number of 566 species was recorded for the first time in the vicinity of slavujevac. out of a total of 26 species listed in the literature for the flora of the slavujevac area, the presence of 11 species was confirmed (alkanna pulmonaria, centaurea benedicta, colchicum doerfleri, crocus olivieri subsp. olivieri, crocus pallasii subsp. pallasii, crocus rujanensis, jurinea polycephala, knautia orientalis, petrorhagia velutina, trifolium cherleri, vinca herbacea). the 90 biologica nyssana ● 13 (2) december 2022: 89-107 simić & zlatković ● flora around slavujevac on rujan mountain (se serbia) table 1. abbreviations of life forms and floral elements of plant taxa ch chamaephytes adv adventitive g geophytes ea eurasian h hemicryptophytes eap eurasian mountain hyd hydrophytes hol holarctic p phanerophytes kosm cosmopolitan par parasitophytes (parasitic or semiparasitic plants) med mediterranean s scandentophytes pont pontic t therophytes se central european table 2. overview of the 10 richest families in the flora around slavujevac biologica nyssana ● 13 (2) december 2022: 89-107 simić & zlatković ● flora around slavujevac on rujan mountain (se serbia) 91 presence of 15 species listed in the literature for a given area has not been confirmed by our field research. the group pteridophyta (ferns and horsetails) includes 3 genera, 3 families and 3 species (0.51% of the total number of species) of the flora of the study area. within the group spermatophyta (seeds), the class coniferopsida (gymnosperms) is represented by 1 genus, 1 family and one species (0.17%). the species seeds from the class dicotyledones are by far the most numerous, comprising representatives of 251 genera, 59 families and 486 species (82%). the class monocotyledones is represented by 57 genera, 10 families and 102 species (17.32%). in relation to the number of species with which they are represented in the flora of the researched area, the following families have the highest species richess: compositae (73 species), leguminosae (67) and gramineae (45). families represented with one genus and one species are: aspleniaceae, athyriaceae, alismataceae, araceae, amaranthaceae, anacardiaceae, asclepiadaceae, corylaceae, cupressaceae, dioscoreaeceae, equisetaceae, globulariaceae, haloragaceae, onagraceae, orobanchaceae, protulaceae, saxifragaceae, thymelaceae, tiliaceae and verbenaceae. the richness of the flora around slavujevac as well as the total flora of hilly and lower mountain region of the southern parts of serbia is associated with the fact that the investigated area has a transitional phtogeographical position, i.e. that it is located in the transitional zone between the mediterranean-submediterranean and pontic-south siberian floristic region (zlatković, 2011). tab. 2 shows an overview of 10 by the number of species family n (number of species) % compositae (asteraceae) 73 12.42 leguminosae (fabaceae) 67 11.39 gramineae (poaceae) 45 7.65 caryophyllaceae 36 6.12 labiatae (lamiaceae) 27 4.59 scrophulariaceae 27 4.59 brassicaceae 26 4.42 rosaceae 23 3.91 umbelliferae (apiaceae) 21 3.57 liliaceae 16 2.72 of the richest families in the flora of the investigated area. from the aspect of species conservation, taxa that enjoy one of the two forms of legal protection at the national level on the basis of the law on nature protection are of importance in the researched area (official gazette of the republic of serbia, no. 36/09). the first group is represented by plants that are on the list of strictly protected wild species of plants, animals and fungi, the collection of which is prohibited on the territory of the whole of serbia, i.e. possible only with the permission of the ministry responsible for nature protection. this group refers to 16 species present in the flora of slavujevac. the second group consists of species that are not critically endangered, and whose collecting is allowed only in limited quantities, i.e. controlled on the basis of their presence on the list of protected wild species of plants, animals and fungi. the mentioned group is significantly larger in relation to the previous one and includes a number of 47 species present in the flora of slavujevac. lists of protected species from the two mentioned categories are shown in tab. 3 and tab 4. table 3. list of strictly protected taxa of flora around slavujevac (official gazette of the republic of serbia, no. 36/09) alkanna pulmonaria griseb. astragalus wilmottianus stoj. campanula scutellata griseb. crocus olivieri gay subsp. olivieri crocus pallasii goldb. subsp. pallasii crocus rujanensis randelovic & d. a. hill gladiolus communis l. subsp. communis groenlandia densa (l.) fourr. himantoglossum caprinum subsp. rumelicumh. baumann & r.lorenz pyrus elaeagrifolia pallas ranunculus illyricus l.(literature data) sideritis montana l. subsp. montana spiranthes spiralis (l.) chevall. tuberaria guttata (l.) fourr. vinca herbacea waldst. et kit. haplophyllum suaveolens (dc.) g. don fill. species belonging to the category of endemic taxa are also present in the study area. data on the affiliation of such a category are presented according to the list of endemic taxa of the flora 92 biologica nyssana ● 13 (2) december 2022: 89-107 simić & zlatković ● flora around slavujevac on rujan mountain (se serbia) of serbia provided by tomović et al. (2014). this group includes 12 plant taxa of the flora around table 4. list of protected plant species of flora around slavujevac (official gazette of the republic of serbia no. 36/09) achillea millefolium l. subsp. millefolium herniaria hirsuta l. acinos hungaricus (simonkai) šilić herniaria incana lam. subsp. incana ajuga laxmannii (l.) bentham hypericum perforatum l. anthyllis vulneraria l. limodorum abortivum (l.) schwarz arum orientale bieb. subsp. orientale linum corymbulosum reichenb. campanula lingulata waldst. & kit. ononis spinosa l. subsp. spinosa campanula phrygia jaub. & spach orchis purpurea hudson centaurea cuneifolia sibth. & sm. subsp. pallida (friv.) hayek orchis simia lam. centaurium erythraea rafin. subsp. erythraea ornithogalum montanum cyr. cephalanthera damasonium (miller) druce prunus spinosa l. cephalanthera rubra (l.) l. c. m. richard rosa canina l. colchicum autumnale l. saxifraga bulbifera l. comandra elegans (rochel ex reichenb.) reichenb. fil. scabiosa fumarioides vis. & pančić cornus mas l. scorzoneroides cichoriacea (ten.) greuter crataegus monogyna jacq. subsp. monogyna sedum caespitosum (cav.) dc. centaurea napulifera rochel subsp. tuberosa (vis.) dostál trifolium cherleri l. cyclamen hederifolium subsp. hederifolium aiton trifolium physodes steven ex bieb. equisetum arvense l. trifolium tenuifolium ten. gagea pusilla (f. w. schmidt) schultes & schultes fil. subsp. pusilla trifolium trichopterum pančić galium flavescens borbás tussilago farfara l. galium verum l. subsp. verum valeriana tuberosa l. geranium macrorrhizum l. viola odorata l. gymnadenia conopsea (l.) r. br. subsp. conopsea eryngium palmatum pančič & vis helianthemum salicifolium (l.) miller table 5. list of endemic taxa in the flora around slavujevac (according to tomović et al., 2014) alkanna pulmonaria griseb. dianthus cruentus griseb. subsp. cruentus armeria rumelica bieb. subsp. rumelica dianthus stenopetalus griseb. astragalus wilmottianus stoj. pulsatilla montana (hoppe) reichenb. subsp. bulgarica rummelspecher campanula scutellata griseb. stachys scardica (griseb.) hayek crocus rujanensis ranđelović & d. a. hill trifolium trichopterum pančić centaurea napulifera rochel subsp. tuberosa (vis.) dostál eryngium palmatum pančić & vis. slavujevac. the list of endemic taxa recorded in the vicinity of slavujevac is shown in tab. 5. 93 biologica nyssana ● 13 (2) december 2022: 89-107 simić & zlatković ● flora around slavujevac on rujan mountain (se serbia) specific floristic data from the literature related to this relatively unexplored area include data from only 9 literature references. it should be emphasized that all 26 taxa listed in the literature for these sites have been critically reviewed. if no evidence has been found for a particular taxon listed in the scientific literature during field research, a free assessment of the correctness of the literature citation or possible correction of the citation is given on the basis of a comprehensive analysis of the taxon distribution. in a small number of cases, a dilemma remained regarding the correctness of the literature citation, with such data marked as suspicious or negative. the published data on the critical taxa are compared with our field observation from this study in tab. 6. table 6. list of taxa represented in the published sources compared to data from this study literature data this study observation alkanna pulmonaria griseb. orljak (niketić, 1999) veliki orljak, lismider, dedovica arabis glabra(l.) bernh.subsp. pseudoturritis(boiss. & heldr.) maire orljak (zlatković et al., 2014) not recorded, probably refers to pseudoturritis turrita aristolochia lutea desf. rujan mt., orljak (bogosavljević and zlatković, 2018) not recorded, presence expected astraglus wilmottianus stoj. orljak (ranđelović and stamenković, 1984., 1986; nikolić et al., 2007) not recorded, possibly locally extinct centaurea benedicta (l.) l. rujan mt., slavujevac (zlatković and bogosavljević, 2014) amedan cnicus bulgaricus p. pan. orljak (ranđelović and stamenković, 1984, 1986) not recorded, presence expected colchicum doerfleri halácsy rujan mt., veliki orljak (niketić i saradnici, 2018) veliki orljak crocus olivieri kitanov & drenkovski subsp. olivieri veliki orljak (ranđelović and stamenković, 1986; ranđelović and ranđelović 1999; ranđelović and hill 1986) amedan, veliki orljak crocus pallasii goldbach subsp.pallasii veliki orljak (ranđelović and stamenković, 1986; ranđelović and ranđelović 1999; ranđelović and hill 1986) veliki orljak crocus rujanensis ranđelović & d. a. hill veliki orljak (randjelovic et al., 1988) amedan, dedovica, veliki orljak goniolimon collinum (griseb.) boiss. rujan mt., orljak (ranđelović and stamenković, 1986) not recorded (buzurović et al., 2013) goniolimon tataricum (l.) boiss. rujan mt., orljak, (ranđelović and stamenković, 1984., 1986; buzurović i et al., 2013) not recorded, possibly locally extinct hippocrepis bifloraspreng. rujan mt., orljak (ranđelović and stamenković, 1984.,1986; ranđelović et al., 1986) probably refers to h. glauca 1984.,1986 jurinea polycephala formánek orljak (zlatković and bogosavljević, 2014) veliki orljak knautia orientalis l. rujan mt., mali orljak (niketić et al., 2021) mali orljak 94 biologica nyssana ● 13 (2) december 2022: 89-107 simić & zlatković ● flora around slavujevac on rujan mountain (se serbia) literature data this study observation minurtia glomerata m. bieb orljak (diklić and stevanović, 2012) not recorded, presence expected moenchia graeca boiss. & heldr. orljak (gajić and stevanović, 2012) not recorded, presence expected petrorhagia velutina (guss.) p. w. ball & heywood rujan mt., slavujevac (zlatković et al., 2011; stevanović and gajić, 2012) lismider, amedan ranunculus illyricus l. rujan mt., orljak (ranđelović and stamenković, 1986) not recorded, presence expected scabiosa fumarioides vis. & pančić rujan mt. (ranđelović and stamenković, 1984) not recorded, presence expected trachynia distachya (l.) link rujan mt., slavujevac (zlatković and bogosavljević, 2014) not recorded, presence expected tremastelma palestinum (l.) janch. orljak (ranđelović and stamenković, 1984) not recorded, cer, đeren (zlatković, pers. comm.) trifolium cherleri l. orljak, (ranđelović and stamenković, 1986, 1978) majden, orljak trifolium trichopterum pančić rujan mt., slavujevac (ranđelović and stamenković, 1984) not recorded, presence expected valerianella costata (steven) betcke rujan mt., slavujevac (zlatković et al., 2011) not recorded, presence expected vinca herbacea waldst. & kit. (ranđelović and stamenković, 1984) veliki orljak, dedovica the most common life form in the flora of the investigated area (fig. 1) is the form of hemicryptophytes (h) present with about 40.47% of taxa. with a slightly smaller share in the total flora follows the group of therophytes (t), about 35.37% of the total number of species. since the two mentioned groups of plants have an almost codominant relationship in the overall ecological spectrum of the flora of slavujevac, it can be said that it has a hemicryptophytic-terophytic character. the life forms of geophytes (g), phanerophytes (p) are also relatively well represented, while other life forms are far less represented in the studied area. hydrophytes (hyd) are the least represented (0.51%). such a small number of hydrophytic species is explained by the lack of habitats suitable for the development of aquatic plants. the flora of slavujevac is characterized by the largest share of hemicryptophytes, which is, in general, a characteristic of the flora of the largest number of areas in the temperate climate zone. however, the flora of slavujevac is characterized by only a 5% lower share of therophytes in relation to the life form of hemicryptophytes. the conspicuous percentage of the mentioned life form in the flora of this area indicates significant climatic zone from the mediterranean submediterranean area, and above all the pronounced dry period during the vegetation season which favors the appearance of annual plants as the best adapted ecological groups of plants to hot and dry habitat conditions. based on the horological analysis of the flora slavujevac area, 8 basic habitat types were singled out. in the area spectrum of the flora of the investigated fig. 1. representation of life forms (%) in the biological spectrum of the flora of slavujevac (ch– chamaephytes; g–geophytes; h–hemicryptophytes; hyd–hydrophytes; p– phanerophytes; t–therophytes) 95 biologica nyssana ● 13 (2) december 2022: 89-107 simić & zlatković ● flora around slavujevac on rujan mountain (se serbia) area (fig. 2), the mediterranean-submediterranean area type (31.29%) and the eurasian area type (26.36%) are dominant. these two area types make up a total of 57.65%, i.e. more than half of the total flora of slavujevac. the explanation for the dominance of the mentioned range types lies in the relatively low altitude of the area, while the emphasis of the mediterranean-submediterranean elements is explained by the strong florogenetic influence from the aegean mediterranean area through the lowland area of macedonia and northern macedonia. a significant percentage of the flora of the investigated area belongs to both the central european (12.92%) and the pontic area type (12.58%). indicating strong anthropogenic influences, the group of cosmopolitan plants is also well represented (10.03%). other range types are represented by a much smaller number of plant taxa. fig. 2. representation of types (%) of slavujevac flora (adv–adventive; ea – eurasian; eap–eurasian mountain; hol–holarctic; kosm–cosmopolitan; med–mediterranean; pont–pontic; se–central european) list of recorded taxa* classis: pteridophyta fam: aspleniaceae 1. asplenium trichomanes l. (ch, kosm) fam: athyriaceae 2. cystopteris fragilis (l.) bernh. (ch, kosm) fam: equisetaceae 3. equisetum arvense l. (g, hol) classis: gymnospermae fam: cupressaceae 4. juniperus oxycedrus l. (p, med) classis: monocotyledones fam: alismataceae 5. alisma plantago-aquatica l. (g, kosm) fam: araceae 6. arum orientale bieb.subsp. orientale (g, se) * class, family, and species are given in alphabetical order. the notation (*) in front of the taxon name indicates data from the literature, and the bibliographic data is given after the taxon name. after the bibliographic data in brackets is an abbreviation for life form and area type fam: cyperaceae 7. carex caryophyllea latourr. (h, ea) 8. carex vulpina l. (h, ea) 9. eleocharis palustris (l.) roemer & schultes (h, kosm) 10. heleocharis palustris l. (h, kosm) 11. holoschoenus vulgaris link (h, ea) 12. scirpus setaceus l. (t, kosm) fam: dioscoreaceae 13. tamus communis l. subsp. communis (g/s, med) fam: gramineae (poaceae) 14. aegilops geniculata roth (t, med) 15. aegilops triuncialis l. subsp. triuncialis (t, med) 16. aira elegantissima schur (t, med) 17. alopecurus rendlei eig. (t, med) 18. anthoxanthum odoratum l. (h, se) 19. avena fatua l. (t, ea) 20. bothriochloa ischaemum (l.) keng (h, ea) 21. briza media l. (h, kosm) 22. bromus arvensis l. (t, ea) 23. bromus commutatus schrader subsp. commutatus (t, se) 24. bromus hordeaceus l. subsp. hordeaceus (t, se) 25. bromus racemosus l. (t, ea) 26. bromus scoparius l. (t, ea) 27. bromus squarrosus l. (t, ea) 28. bromus sterilis l. (t, ea) 29. bromus tectorum l. (t, ea) 30. chrysopogon gryllus (l.) trin. (h, pont) 31. cynodon dactylon (l.) pers. (g, kosm) 32. cynosurus cristatus l. (h, kosm) 33. cynosurus echinatus l. (t, kosm) 34. dactylis glomerata l. (h, kosm) 35. dasypyrum villosum (l.) p. candargy (t, med) 36. echinochloa crus-galli (l.) beauv. (t, kosm) 37. elymus hispidus hispidus (opiz) melderis subsp. hispidus (g, med) 38. festuca valesiaca schleicher ex gaudin (h, pont) 39. holcus lanatus l. (h, kosm) 40. holcus mollis l.subsp.mollis (h, se) 41. hordeum murinum hudson subsp. murinum (t, ea) 96 biologica nyssana ● 13 (2) december 2022: 89-107 simić & zlatković ● flora around slavujevac on rujan mountain (se serbia) 42. koeleria nitidula velen. (h, pont) 43. koeleria simonkai adamović (k. glaucovirens aggr.) (h, pont) 44. leersia oryzoides (l.) sw. (h, kosm) 45. lolium perenne l. (h, kosm) 46. lolium temulentum l. (t, kosm) 47. melica altissima l. (h, pont) 48. phleum montanum montanum c. koch subsp. montanum (h, eap) 49. phleum pratense l. subsp. pretense (h, ea) 50. poa annua l. (t, kosm) 51. poa bulbosa l. (h, ea) 52. poa compressa l.(h, ea) 53. poa nemoralis l. (h, ea) 54. poa pratensis l. (h, hol) 55. sclerochloa dura (l.) beauv. (t, ea) 56. taeniatherum caput-medusae (l.) nevski subsp. asperum (simonkai) melderis (t, med) 57. *trachynia distachya (l.) link (t, med) 58. vulpia muralis (kunth) nees (t, med) fam: iridaceae 59. *crocus olivieri kitanov & drenkovski subsp. olivieri (g, med) 60. *crocus pallasii goldbach subsp. pallasii (g, med) 61. *crocus rujanensis ranđelović & d. a. hill (g, med) 62. gladiolus communis l. subsp. communis (g, med) 63. iris germanica l. (g, adv) 64. iris graminea l. (g, pont) 65. iris suaveolens boiss. & reuter (g, med) fam: juncaceae 66. juncus articulatus l. (g, kosm) 67. juncus bufonius l. (t, kosm) 68. juncus effusus l. (g, kosm) 69. juncus inflexus l. (h, kosm) 70. juncus thomasii ten. (g, eap) 71. luzula campestris (l.) dc. (h, se) 72. luzula forsteri (sm.) dc. subsp. forsteri (h, se) fam: liliaceae 73. allium scorodoprasum l. (g, med) 74. allium sphaerocephalon sphaerocephalon l. subsp. sphaerocephalon(g, med) 75. colchicum autumnale l. (g, se) 76. colchicum doerfleri halacsy (g, med) 77. fritillaria gussichiae (degen & dörfler) rix (g, med) 78. gagea pratensis (pers.) dumort. (g, pont) 79. gagea pusilla (f. w. schmidt) schultes & schultes filsubsp. pusilla (g, pont) 80. muscari comosum (l.) miller (g, med) 81. muscari neglectum guss. ex ten (g, med) 82. ornithogalum comosum l. (g, med) 83. ornithogalum kochii parl. (g, pont) 84. ornithogalum montanum cyr. (h, eap) 85. ornithogalum pyramidale l. (g, pont) 86. polygonatum latifolium (jacq.) desf. (g,se) 87. polygonatum odoratum (miller) druce subsp. odoratum (g, se) 88. scilla autumnalis l. (g, med) fam: orchidaceae 89. cephalanthera damasonium (miller) druce (g, se) 90. cephalanthera rubra (l.) l. c. m. richard (g, se) 91. gymnadenia conopsea (l.) r. br. subsp. conopsea (g, ea) 92. himantoglossum caprinum (biebl) c. koch subsp. rumelicum (g, med) 93. limodorum abortivum (l.) swartz (g/par, med) 94. ophrys scolopax cav. subsp. cornuta (steven) camus (g, med) 95. orchis coriophora l. subsp. fragrans (pollini) sudre (g, med) 96. orchis laxiflora lam. subsp. laxiflora (heuffel) soó (g, med) 97. orchis mascula (l.) l.subsp. signifera (vest) soó (g, se) 98. orchis morio l. subsp. picta (loisel.) arcangeli (g, med) 99. orchis papilionacea l. subsp. grandiflora (boiss.) nels (g, med) 100. orchis purpurea hudson (g, med) 101. orchis simia lam. (g, med) 102. orchis tridentata scop. subsp. commutata (tod.) nyman (g, med) 103. platanthera chlorantha (custer) reichenb. (g, se) 104. spiranthes spiralis (l.) chevall. (g, pont) fam: potamogetonaceae 105. groenlandia densa (l.) fourr. (hyd, ea) 106. potamogeton natans l. (hyd, kosm) classis: dicotyledones 97 biologica nyssana ● 13 (2) december 2022: 89-107 simić & zlatković ● flora around slavujevac on rujan mountain (se serbia) 139. sambucus nigra l. (p, se) fam: caryophyllaceae 140. agrostemma githago l. (t, ea) 141. arenaria serpyllifolia l. (t, ea) 142. cerastium banaticum (rochel) heuffel subsp. banaticum (ch, eap) 143. cerastium brachypetalum subsp. brachypetalum (t, med) 144. cerastium glomeratum thuill. (t, ea) 145. cerastium semidecandrum l. (t, ea) 146. dianthus corymbosus sibth. & sm. (t, med) 147. dianthus cruentus griseb. subsp. cruentus (h, eap) 148. dianthus giganteiformis borbás subsp. pontederae (kerner) soó (h, pont) 149. dianthus gracilis sibth. & sm. (ch, med) 150. dianthus stenopetalus griseb. (h, med) 151. gypsophila muralis l. (t, ea) 152. herniaria glabra l. subsp. glabra (h, ea) 153. herniaria hirsuta l. (t, ea) 154. herniaria incana lam. subsp. incana (t, med) 155. holosteum umbellatum l. subsp. glutinosum (bieb.) nyman (t, med) 156. lychnis flos-cuculi l. subsp. flos-cuculi (h, ea) 157. *minurtia glomerata m. bieb (t, pont) 158. *moenchia graeca boiss. & heldr. (t, med) 159. moenchia mantica (l.) bartl. (t, med) 160. petrorhagia illyrica (l.) p. w. ball & heywood subsp. haynaldiana (janka) p. w. ball & heywood (h, med) 161. petrorhagia prolifera (l.) p. w. ball & heywood (t, med) 162. *petrorhagia velutina (guss.) p. w. ball & heywood (t, med) 163. scleranthus perennis l. subsp. dichotomus (schur) stoj. & stefanov (t, pont) 164. scleranthus polycarpos l. (t, med) 165. silene armeria l. (t, med) 166. silene gallynii heuffel ex reichenb. (t, med) 167. silene latifolia poiret subsp. alba (miller) w. greuter & burdet (t, ea) 168. silene nemoralis waldst. & kit. (h, eap) 169. silene rhodopaea janka (h, med) 170. silene subconica friv. (t, pont) 171. silene vulgaris (moench) garcke (h, ea) fam: aceraceae 107. acer campestre l. (p, se) 108. acer tataricum l. (p, pont) fam: amaranthaceae 109. amaranthus albus l. (t, adv) 110. amaranthus retroflexus l. (t, adv) fam: anacardiaceae 111. cotinus coggygria scop. (p, ea) fam: apocynaceae 112. *vinca herbacea waldst. & kit. (ch, pont) 113. vinca minor l. (ch, adv) fam: aristolochiaceae 114. aristolochia clematitis l. (g, med) 115. *aristolochia lutea desf. (g, med) fam: asclepiadaceae 116. vincetoxicum fuscatum (hornem.) reichenb. fil. (h, med) fam: boraginaceae 117. aegonychon purpurocaeruleum (l.) holub (h, pont) 118. *alkanna pulmonaria griseb. (h, med) 119. anchusa barrelieri (all.) vitman (h, pont) 120. anchusa officinalis l. (h, pont) 121. myosotis stricta link ex roemer & schultes (t, ea) 122. buglossoides arvensis (l.) i. m. johnston subsp. arvense (t, kosm) 123. cynoglossum officinale l. (h, ea) 124. echium vulgare l. (h, ea) 125. myosotis arvensis (l.) hill (t, ea) 126. myosotis ramosissima rochel (t, med) 127. myosotis sicula guss. (t, med) 128. myosotis sparsiflora mikan ex pohl (t, ea) 129. myosotis stricta link ex roemer & schultes (t, ea) fam: campanulaceae 130. campanula lingulata waldst. & kit. (h, med) 131. campanula phrygia jaub. & spach (t, med) 132. campanula rapunculoides l. (h, ea) 133. campanula rapunculus l. (h, med) 134. campanula scutellata griseb. (t, med) 135. jasione heldreichii boiss. & orph. (h, eap) 136. legousia speculum-veneris (l.) chaix (t, med) fam: caprifoliaceae 137. lonicera caprifolium l. (ch/s, se) 138. sambucus ebulus l. (h, ea) biologica nyssana ● 13 (2) december 2022: 89-107 98 simić & zlatković ● flora around slavujevac on rujan mountain (se serbia) 172. spergula arvensis l. (t, kosm) 173. stellaria graminea l. (h, ea) 174. stellaria media (l.) vill. (t, kosm) 175. viscaria vulgaris bernh. (h, se) fam: celastraceae 176. evonymus europaeus l. (p, se) 177. evonymus latifolius (l.) miller (p, se) fam: chenopodiaceae 178. atriplex patula l. (t, ea) 179. chenopodium album l. (t, kosm) fam: cistaceae 180. fumana procumbens (dunal) gren. & godron subsp. procumbens (ch, med) 181. helianthemum ledifolium (l.) miller (t, med) 182. helianthemum nummularium (l.) miller subsp. nummularium (ch, se) 183. helianthemum salicifolium (l.) miller (t, med) 184. tuberaria guttata (l.) fourr. (t, med) fam: compositae (asteraceae) 185. achillea coarctata poiret (h, pont) 186. achillea crithmifolia waldst. & kit. (h, pont) 187. achillea millefolium l. subsp. millefolium (h, hol) 188. anthemis arvensis l. subsp. arvensis(t, ea) 189. arctium lappa l. (h, ea) 190. bellis perennis l. (h, se) 191. carduus acanthoides l. (h, kosm) 192. carduus nutans l. subsp. leiophyllus (petrović) stoj. & stefanov (h, pont) 193. carlina corymbosa l. subsp. graeca (boiss.) nyman (h, med) 194. carthamus creticus l. (t, med) 195. *centaurea benedicta (l.) l. (t, med) 196. centaurea calcitrapa l. subsp. calcitrapa (h, kosm) 197. centaurea cuneifolia sibth. & sm. subsp. pallida (friv.) hayek (h, med) 198. centaurea cyanus l. (t, med) 199. centaurea jacea l. (h, ea) 200. centaurea salonitana vis. (h, med) 201. centaurea stoebe stoebe l. subsp. stobe (h, pont) 202. chondrilla juncea l. (h, ea) 203. cichorium intybus l. (h, kosm) 204. cirsium arvense (l.) scop. (h, ea) 205. cirsium ligulare boiss. (h, eap) 206. cirsium vulgare (savi) ten. (h, kosm) 207. *cnicus bulgaricus p. pan. (t, med) 208. cota tinctoria (l.) j. gay (t, ea) 209. crepis biennis l. (h, se) 210. crepis foetida l. subsp. rhoeadifolia (bieb.) čelak (t, ea) 211. crepis sancta (l.) bornm. (t, med) 212. crepis setosa haller fil. subsp. setose (t, med) 213. crupina vulgaris cass. (t, ea) 214. echinops sphaerocephalus l. (h, med) 215. filago arvensis l. (t, ea) 216. filago minima (sm.) pers. (t, ea) 217. galinsoga parviflora cav. (t, kosm) 218. hypochaeris maculata l. (h, hol) 219. inula britannica l. (h, ea) 220. inula germanica l. (h, ea) 221. inula hirta l. (h, pont) 222. inula oculus-christi l. (h, pont) 223. inula salicina l. subsp. aspera (poiret) hayek (h, ea) 224. jacobaea vulgaris gaertn. (h, ea) 225. *jurinea polycephala formánek (h, pont) 226. lactuca saligna l. (h, ea) 227. laphangium luteoalbum (l.) tzvelev (t, kosm) 228. lapsana communis l. subsp. adenophora (boiss.) rech. fil. (t, ea) 229. leontodon biscutellifolius dc. (h, med) 230. leontodon hispidus l. (h, se) 231. leucanthemum ircutianum dc.(h, ea) 232. matricaria chamomilla l. (t, kosm) 233. onopordum acanthium l. subsp acanthium (t, ea) 234. pilosella bauhinii (schultes) arv.-touv. subsp. thaumasia (peter) soják (h, se) 235. pilosella macrantha f. w. schultz & schultz bip. fratt. (h, ea) 236. ptilostemon afer (jacq.) w. greuter (h, med) 237. scorzonera hispanica l. (h, pont) 238. scorzonera mollis bieb. subsp. mollis (g, pont) 239. scorzoneroides cichoriacea (ten.) greuter (g, med) 240. senecio leucanthemifolius poiret subsp. vernalis (waldst. & kit.) greuter (t, pont) 241. senecio vulgaris l. subsp. vulgaris (t, kosm) simić & zlatković ● flora around slavujevac on rujan mountain (se serbia) biologica nyssana ● 13 (2) december 2022: 89-107 99 242. sonchus arvensis l. (t, ea) 243. sonchus asper (l.) hill subsp. asper (t, hol) 244. tanacetum corymbosum (l.) schultz bip. (h, ea) 245. tanacetum macrophyllum (waldst. & kit.) schultz-bip. (h, eap) 246. tanacetum vulgare l. (h, ea) 247. taraxacum erythrospermum besser (h, ea) 248. taraxacum haussknechtii hausskn. (h, med) 249. taraxacum officinale weber (h, kosm) 250. tragopogon dubius scop. subsp. major (jacq.) vollm. (t, pont) 251. tragopogon pratensis l. subsp. pratensis (h, ea) 252. tripleurospermum inodorum (l.) schultzbip. (t, ea) 253. tussilago farfara l. (g, ea) 254. xanthium strumarium l. subsp. italicum (moretti) d. löve (t, adv) 255. xeranthemum annuum l. (t, med) 256. xeranthemum cylindraceum sm. (t, med) fam: convolvulaceae 257. calystegia sepium (l.) r. br. (t/s, ea) 258. convolvulus arvensis l. (t/s, kosm) 259. convolvulus cantabrica l. (h, med) fam: cornaceae 260. cornus mas l. (p, med) 261. cornus sanguinea l. subsp. australis (c.a. meyer) jáv. (p, se) fam: corylaceae 262. carpinus orientalis miller subsp. orientalis (p, med) fam: crassulaceae 263. sedum caespitosum (cav.) dc. (t, med) 264. sedum hispanicum l. (t, med) 265. sedum rubens l. (t, med) 266. sedum urvillei dc. (ch, med) fam: cruciferae (brassicaceae) 267. alliaria petiolata (bieb.) cavara & grande (t, ea) 268. alyssum alyssoides (l.) l. (t, med) 269. alyssum minutum schlecht. ex dc. (t, pont) 270. alyssum montanum l. subsp. reiseri (velen.) hayek (t, pont) 271. alyssum turkestanicum regel & schmalh. ex regel (t, ea) 272. arabidopsis thaliana (l.) heynh. (t, ea) 273. *arabis glabra (l.) bernh. subsp. pseudoturritis (boiss. & heldr.) maire (t, med) 274. arabis sagittata (bertol.) dc. (h, med) 275. berteroa incana (l.) dc. subsp. stricta (boiss. & heldr.) stoj. & stef. (h, ea) 276. bunias erucago l. (t, med) 277. calepina irregularis (asso) thell. (t, ea) 278. camelina rumelica velen. (t, med) 279. capsella bursa-pastoris (l.) medicus (t, kosm) 280. cardamine bulbifera (l.) crantz (g, se) 281. cardamine hirsuta l. (t, ea) 282. descurainia sophia(l.) webb ex prantl (t, ea) 283. erophila verna verna (l.) chevall. subsp. verna (t, ea) 284. erysimum diffusum ehrh. (h, ea) 285. lepidium campestre (l.) r. br. (t, pont) 286. lepidium ruderale l. (h, ea) 287. raphanus raphanistrum l. (t, adv) 288. rorippa prolifera (heuffel) neilr. (h, se) 289. rorippa pyrenaica (lam.) reichenb. (h, med) 290. rorippa sylvestris (l.) besser subsp. sylvestris (h, se) 291. sisymbrium officinale (l.) scop. (t, ea) 292. thlaspi perfoliatum l. subsp. perfoliatum(t, ea) fam: dipsacaceae 293. dipsacus laciniatus l. (h, ea) 294. knautia arvensis (l.) coulter subsp. rosea (baumg.) soó (h, ea) 295. knautia orientalis l. (t, med) 296. scabiosa argentea l. (h, pont) 297. *scabiosa fumarioides vis. & pančić (h, eap) 298. *tremastelma palestinum (l.) janch. (t, med) fam: euphorbiaceae 299. euphorbia cyparissias l. (h, se) 300. euphorbia esula l. subsp. thommasiniana (bertol.) nyman (h, ea) 301. euphorbia helioscopia l. (t, kosm) 302. euphorbia seguierana necker subsp. niciciana (h, med) fam: fagaceae 303. quercus cerris (k. maly) czecz. (p, med) 304. quercus frainetto ten. (p, med) 305. quercus pedunculiflora k. koch (p, med) biologica nyssana ● 13 (2) december 2022: 89-107 simić & zlatković ● flora around slavujevac on rujan mountain (se serbia) 306. quercus pubescens willd. (p, med) fam: gentianaceae 307. centaurium erythraea rafin. subsp. erythraea (t, ea) 308. centaurea napulifera rochel subsp. tuberosa (vis.) dostál (h, med) 309. centaurium pulchellum (swartz) druce (t, ea) fam: geraniaceae 310. erodium cicutarium (l) l’her. (t, kosm) 311. geranium columbinum l. (t, se) 312. geranium lucidum l. (t, ea) 313. geranium macrorrhizum l. (g, eap) 314. geranium molle molle l. subsp. molle (t, kosm) 315. geranium sanguineum l. (h, se) fam: globulariaceae 316. globularia bisnagarica l. (h, ea) fam: guttiferae (hypericaceae) 317. hypericum perforatum l. (h, ea) 318. hypericum rumeliacum boiss. (h, med) 319. hypericum tetrapterum fries (h, se) fam:haloragaceae 320. myriophyllum spicatum l. (hyd, hol) fam: labiatae (lamiaceae) 321. acinos arvensis (lam.) dandy subsp. villosus (gaudin) sojak (t, ea) 322. acinos hungaricus (simonkai) šilić (ch, med) 323. ajuga chamaepitys (l.) schreber subsp. chia (schreber) arcangeli (t, ea) 324. ajuga genevensis l. (h, se) 325. ajuga laxmannii (l.) bentham (h, pont) 326. ballota nigra l. subsp. foetida hayek (h, se) 327. clinopodium vulgare l. (h, hol) 328. lamium amplexicaule l. subsp. amplexicaule (t, ea) 329. lamium maculatum l. (h, se) 330. lamium purpureum l. subsp. purpureum (t, ea) 331. marrubium peregrinum l. (h, pont) 332. mentha aquatica l. (h, ea) 333. mentha longifolia (l.) hudson subsp. longifolia (h, ea) 334. nepeta nuda l. subsp. nuda (h, ea) 335. origanum vulgare l. subsp. vulgare (h, ea) 336. prunella laciniata (l.) l. (h, pont) 337. prunella vulgaris l. (h, hol) 338. salvia amplexicaulis lam. (h, med) 339. salvia verticillata l. subsp. verticillata (h, pont) 340. scutellaria columnae all. subsp. columnae (h, se) 341. sideritis montana l. subsp. montana (t, med) 342. stachys annua (l.) l. (h, ea) 343. stachys germanica l. (h, ea) 344. stachys officinalis (l.) trevisan subsp. officinalis (h, se) 345. stachys scardica (griseb.) hayek (h, med) 346. teucrium chamaedrys l. (h, med) 347. teucrium polium l.subsp. capitatum (ch, med) 348. thymus glabrescens willd. (ch, pont) fam: leguminosae (fabaceae) 349. anthyllis vulneraria l. (h, se) 350. astragalus glycyphyllos l. (h, se) 351. astragalus onobrychis l. subsp. onobrychis (ch, pont) 352. *astragalus wilmottianus stoj. chamaecytisus tommasinii (vis.) rothm. (h, pont) 353. chamaecytisus triflorus (lam.) scalicka (ch, eap) 354. colutea arborescens l. (p, med) 355. coronilla scorpioides (l.) koch (t, med) 356. coronilla varia l. (h, se) 357. dorycnium germanicum (gremli) rikli (ch, se) 358. genista ovata waldst. & kit.(ch, pont) 359. *hippocrepis biflora spreng. the species has not been confirmed by field research, therefore, the data only refer to the species hippocrepis glauca (h, med) 360. hippocrepis glauca ten. (h, med) 361. lathyrus hirsutus l. (t, med) 362. lathyrus latifolius l. (h, med) 363. lathyrus laxiflorus (desf.) o. kuntze (h, se) 364. lathyrus pratensis l. (h, ea) 365. lathyrus sativus l. (t, ea) 366. lathyrus tuberosus l. (g, ea) 367. lembotropis nigricans (l.) griseb. (ch, pont) 368. lotus angustissimus l. (t, med) 369. lotus corniculatus l. subsp. corniculatus 100 biologica nyssana ● 13 (2) december 2022: 89-107 simić & zlatković ● flora around slavujevac on rujan mountain (se serbia) (h, ea) 370. medicago arabica (l.) hudson (t, kosm) 371. medicago falcata (l.) hudson subsp. falcate (h, kosm) 372. medicago lupulina (l.) hudson (h, kosm) 373. medicago orbicularis (l.) bartal. (t, ea) 374. medicago rigidula (l.) all. (t, med) 375. medicago sativa l.(h, adv) 376. onobrychis alba (waldst. & kit.) desv. subsp. alba (h, med) 377. ononis spinosa l.subsp. spinosa (ch, se) 378. ornithopus compressus l. (t, med) 379. robinia pseudacacia l. (p, adv) 380. trifolium alpestre l. subsp. alpestre (h, se) 381. trifolium angustifolium l. (t, med) 382. trifolium arvense l. (t, ea) 383. trifolium campestre l. (t, ea) 384. *trifolium cherleri l. (t, med) 385. trifolium echinatum bieb. (t, med) 386. trifolium fragiferum l.subsp. bonannii (h, ea) 387. trifolium glomeratum brot. (t, med) 388. trifolium hirtum all. (t, med) 389. trifolium incarnatum l. subsp. mollineri (balbis ex hornem.) syme (t, med) 390. trifolium montanum l. (h, pont) 391. trifolium nigrescens viv. subsp. nigrescens (t, med) 392. trifolium ochroleucon hudson (h, med) 393. trifolium pallidum waldst. & kit. (h, med) 394. trifolium patens schreber (t, med) 395. trifolium physodes steven ex bieb. (h, med) 396. trifolium pratense l. (h, ea) 397. trifolium purpureum loisel. (t, med) 398. trifolium repens l. (h, kosm) 399. trifolium resupinatum l. (t, med) 400. trifolium retusum l. (t, med) 401. trifolium striatum l. (t, se) 402. trifolium strictum l.(t, med) 403. trifolium sylvaticum gerard sec. c. visioso (t, med) 404. trifolium tenuifolium ten. (t, med) 405. *trifolium trichopterum pančić (t, med) 406. trigonella monspeliaca l. (t, med) 407. vicia cracca l. subsp. incana(h/s, med) 408. vicia grandiflora scop. (t/s, pont) 409. vicia hirsuta(l.) s. f. gray (h/s, ea) 410. vicia lathyroides l. (h/s, med) 411. vicia melanops sibth. & sm. (t/s, med) 412. vicia pannonica pannonica crantz subsp. pannonica (t/s, pont) 413. vicia peregrina l. (t/s, ea) 414. vicia tenuifolia roth subsp. dalmatica (a. kern.) greuter (h/s, med) fam: linaceae 415. linum austriacum l. (h, pont) 416. linum corymbulosum reichenb. (t, med) 417. linum hirsutum l. subsp. hirsutum (t/h, pont) 418. linum tenuifolium l. (h, med) 419. linum trigynum l. (t, med) fam: lythraceae 420. lythrum hyssopifolia l. (t, kosm) 421. lythrum salicaria l. (h, kosm) fam: malvaceae 422. alcea pallida (willd.) waldst. & kit. subsp. pallida (h, pont) 423. alt ha ea can na bi na l. (h, med) 424. hibiscus trionum l. (t, ea) 425. malva neglecta wallr. (h, ea) 426. malva sylvestris l. (h, kosm) fam: oleaceae 427. fraxinus ornus l. (p, med) 428. ligustrum vulgare l. (p, se) fam: onagraceae (oenotheraceae) 429. oenothera biennis l. (h, adv) fam: orobanchaceae 430. orobanche minor sm. (g/par, med) fam: papaveraceae 431. corydalis solida (l.) clairv. (g, se) 432. chelidonium majus l. (t, ea) 433. fumaria petteri reichenb. subsp. thuretii (t, med) 434. fumaria rostellata knaf (t, pont) 435. papaver albiflorum (besser) pacz (t, med) 436. papaver dubium dubium l. subsp. dubium (t, ea) 437. papaver hybridum l. (t, med) 438. papaver rhoeas l. (t, med) fam: plantaginaceae 439. plantago holosteum scop. (h, eap) 440. plantago lanceolata l. (h, kosm) 441. plantago major l. subsp. major (h, ea) 442. plantago media l. (h, ea) 101 biologica nyssana ● 13 (2) december 2022: 89-107 simić & zlatković ● flora around slavujevac on rujan mountain (se serbia) fam: plumbaginaceae 443. armeria rumelica bieb. (h, eap) 444. *goniolimon tataricum (l.) boiss. (h, pont) 445. *goniolimon collinum (griseb.) boiss. (h, pont) fam: polygalaceae 446. polygala comosa schkuhr (h, ea) 447. polygala vulgaris l. (h, se) fam: polygonaceae 448. fallopia convolvulus (l.) å. löve (t/s, kosm) 449. persicaria maculosa s.f.gray (t, kosm) 450. persicaria mitis delarbre (t, ea) 451. polygonum aviculare l. (t, kosm) 452. polygonum persicaria l. (t, ea) 453. rumex acetosa l. (h, hol) 454. rumex acetosella l. subsp. acetosella (h, se) 455. rumex patientia l. (h, ea) 456. rumex pulcher l. subsp. pulcher (h, med) fam:portulacaceae 457. portulaca oleracea l. subsp. oleracea (t, kosm) fam: primulaceae 458. anagallis arvensis l. (t, kosm) 459. cyclamen hederifolium aiton subsp. hederifolium (g, med) 460. lysimachia nummularia l. (h, se) 461. lysimachia punctata l. (h, med) 462. lysimachia vulgaris l. (h, ea) 463. primula veris l. subsp. suaveolens (h, eap) fam: ranunculaceae 464. consolida regalis s.f. gray subsp. paniculata (host) soó (t, pont) 465. helleborus odorus waldst. & kit. subsp. odorus (g, se) 466. nigella arvensis l. subsp. arvensis (t, med) 467. pulsatilla montana (hoppe) reichenb. subsp. bulgarica rummelspecher (g, pont) 468. ranunculus acris l.subsp. acris (h, ea) 469. ranunculus arvensis l. (h, ea) 470. ranunculus ficaria l. subsp. calthifolius (g, se) 471. *ranunculus illyricus l. (g, pont) 472. ranunculus lanuginosus l. (h, se) 473. ranunculus laterifolius dc. (t, ea) 474. ranunculus millefoliatus vahl (g, med) 475. ranunculus psilostachys griseb. (g, med) 476. ranunculus repens l. (h, ea) 477. thalictrum lucidum l. (h, se) 478. thalictrum minus l. subsp. minus (h, ea) fam: rosaceae 479. agrimonia eupatoria lebed. (h, ea) 480. aremonia agrimonoides (l.) dc. subsp. agrimonoides (h, se) 481. crataegus monogyna jacq. subsp. monogyna (p, se) 482. filipendula vulgaris moench (h, ea) 483. fragaria viridis duchesne subsp. viridis (h, ea) 484. geum urbanum l. (h, ea) 485. potentilla chrysantha trev. (h, pont) 486. potentilla detommasii ten. (h, med) 487. potentilla inclinata vill. (h, ea) 488. potentilla laciniosa waldst. & kit. ex nestler (h, pont) 489. potentilla neglecta baumg. (h, ea) 490. potentilla pedata l. (h, med) 491. potentilla recta l. (h, pont) 492. potentilla reptans l.(h, kosm) 493. prunus spinosa l. (p, se) 494. pyrus communis l. (p, se) 495. pyrus elaeagrifolia pallas (p, med) 496. rosa canina l. (p, ea) 497. rosa dumalis bechst. (p, se) 498. rosa gallica l. (p, se) 499. rubus caesius l. (ch, ea) 500. sanguisorba minor scop. subsp. muricata briq. (h, ea) 501. sorbus domestica l. (p, med) fam: rubiaceae 502. asperula arvensis l. (t, med) 503. asperula cynanchica l. (ch, pont) 504. crucianella angustifolia l. (t, med) 505. cruciata glabra (l.) ehrend. (h, ea) 506. cruciata laevipes opiz. (h, se) 507. galium album miller subsp. album (h, se) 508. galium aparine l. (t, ea) 509. galium flavescens borbás (h, med) 510. galium spurium l. (h, se) 511. galium tenuissimum bieb. (t, ea) 102 biologica nyssana ● 13 (2) december 2022: 89-107 simić & zlatković ● flora around slavujevac on rujan mountain (se serbia) 512. galium vernum scop. (h, ea) 513. galium verum l. subsp. verum (h, ea) 514. sherardia arvensis l. (t, kosm) fam: rutaceae 515. dictamnus albus l. (ch, pont) 516. haplophyllum suaveolens (dc.) g. don fill. (h, pont) fam:salicaceae 517. populus alba l. (p, ea) 518. populus nigra l. subsp. nigra (p, ea) 519. salix amplexicaulis bory (p, med) 520. salix fragilis l. (p, ea) fam: santalaceae 521. comandra elegans (rochel ex reichenb.) reichenb. fil. (ch/par, pont) 522. thesium arvense horvátovszky ( h / p a r , pont) fam: saxifragaceae 523. saxifraga bulbifera l. (g, med) fam: scrophulariaceae 524. antirrhinum orontium l. (t, ea) 525. digitalis lanata ehrh. (h, med) 526. euphrasia pectinata ten. (t, pont) 527. euphrasia stricta d. wolff ex j. f. lehm. (t/par, ea) 528. gratiola officinalis l. (h, hol) 529. linaria genistifolia genistifolia (l.) miller subsp. genistifolia (h, pont) 530. linaria pelisseriana (l.) miller (t, med) 531. melampyrum arvense l. (t/par, se) 532. melampyrum cristatum l. (t, se) 533. parentucellia latifolia (l.) caruel (t/par, med) 534. rhinanthus angustifolius c.c. gmelin (t/ par, ea) 535. rhinanthus rumelicus velen. (t/par, eap) 536. verbascum banaticum schrader (h, pont) 537. verbascum blattaria l. (t, ea) 538. verbascum lychnitis l. (h, pont) 539. verbascum macrurum ten. subsp. pannosiforme (stoj.) murb. (h, med) 540. verbascum phlomoides l. (h, pont) 541. verbascum phoeniceum l. subsp. phoeniceum (h, ea) 542. veronica agrestis l. (t, se) 543. veronica anagallis-aquatica l. subsp. anagallisaquatica (h, kosm) 544. veronica austriaca l. subsp. jacquinii (baumg.) maly (h, pont) 545. veronica chamaedrys l. subsp. vindobonensis m. fischer (h, se) 546. veronica hederifolia l. (t, se) 547. veronica persica poiret (t, adv) 548. veronica polita fries (t, ea) 549. veronica serpyllifolia l. (h, hol) 550. veronica verna l. (t, ea) fam:solanaceae 551. datura stramonium l.(h, kosm) 552. lycium europaeum l. (p, adv) 553. solanum nigrum l. subsp. schultesii (opiz) wessely (t, ea) fam: thymelaeaceae 554. thymelaea passerina (l.) cosson & germ. (t, ea) fam: tiliaceae 555. tilia platyphyllos scop. (p, se) fam: ulmaceae 556. ulmus minor miller (p, se) fam: umbelliferae (apiaceae) 557. anthriscus caucalis bieb. (t, se) 558. anthriscus cerefolium (l.) hoffm. (t, med) 559. anthriscus nemorosa (bieb.) sprengel (h, ea) 560. bifora radians bieb. (t, med) 561. caucalis platycarpos l. (t, med) 562. chaerophyllum temulum l. (t, se) 563. conium maculatum l. (h, kosm) 564. daucus carota l.(t, sea) 565. daucus guttatus sibth. & sm. subsp. zahariadii heywood (t, pont) 566. eryngium campestre l. (h, med) 567. eryngium palmatum pančić & vis. (h, med) 568. falcaria vulgaris bernh. (h, ea) 569. ferulago sylvatica (besser) reichenb. (h, med) 570. heracleum sphondylium l. subsp. sibiricum (l.) simonkai (h, ea) 571. oenanthe banatica heuffel (h, se) 572. orlaya grandiflora (l.) hoffm. (t, med) 573. physocaulis nodosus (l.) koch (t, med) 574. scandix pecten-veneris l. (t, ea) 575. tordylium maximum l. (t, med) 576. torilis japonica (houtt.) dc. (t, ea) 577. trinia glauca (l.) dumort. (h, pont) fam: urticaceae 103 biologica nyssana ● 13 (2) december 2022: 89-107 simić & zlatković ● flora around slavujevac on rujan mountain (se serbia) 104 578. urtica dioica l. (h, hol) 579. urtica urens l. (h, hol) fam: valerianaceae 580. valeriana tuberosa l. (g, pont) 581. valerianella carinata loisel. (t, med) 582. *valerianella costata (steven) betcke (t, med) 583. valerianella locusta (l.) laterrade (t, med) 584. valerianella rimosa bast. (t, pont) fam: verbenaceae 585. verbena officinalis l. (h, kosm) fam: violaceae 586. viola alba (ten.) w. becker subsp. dehnhardtii (jord.) nyman (h, med) 587. viola arvensis murray subsp. arvensis (t, ea) 588. viola hirta l. (h, ea) 589. viola jordanii hanry (h, med) 590. viola kitaibeliana schultes (t, med) 591. viola odorata l. (h, se) 592. viola tricolor l. subsp. macedonica (boiss. & heldr.) a. schmidt (h, med) conclusions floristic influences from the southern part of the balkan peninsula strongly influenced the flora of southern and southeastern serbia. for that reason, the flora of rujan mt., including the surroundings of the village of slavujevac is largely specific and diverse and requires more detailed botanical research. the presence of 592 plant species, 312 genera and 73 families of vascular plants was established by recent floristic research in the area of slavujevac. only a small number of taxa are listed in the available literature for the vicinity of slavujevac. of that number, only the presence of 11 taxa was confirmed during our field research. at the same time, arabis glabra subsp. pseudoturritis, goniolimon collinum and hippocrepis biflora are incorrectly listed for the studied area. the species of pteridophyta, as well as gymnosperms, are represented by a poor number of species. taxa from the group dicotyledones are by far the most numerous with about 82% of the total number of flora in the investigated area. taxonomic analysis shows that the families compositae, leguminosae and gramineae are the richest in species and subspecies. analysis of the biological spectrum indicates the dominance of the hemicryptophytes and therophytes in the flora. in the phytogeographical sense, the species of the mediterranean-submediterranean and eurasian area type prevail, including 10 species belonging to the group of balkan endemic taxa. a large presence of plants from the group of endangered taxa was found in the investigated habitats. this area is floristically rich, but it is also exposed to numerous negative anthropogenic factors. it is necessary to educate the local population in order to reduce the rate of destruction of natural habitats in the area. also, it is necessary to valorize rujan mt. by the organizations for nature conservation in order to preserve this floristically rich and diverse mountain for a longer period of time. references bogosavljević, s., zlatković, b. 2018: report on the new floristic data from serbia ii. biologica nyssana, 9 (2): 63-75. buzurović, u., stevanović, v., niketić, m., jakovljević, k., tomović, g. 2013: on the distribution of goniolimon tataricum (plumbaginaceae) in serbia. botanica serbica, 37 (2): 67-172. diklić, n., stevanović, v. 2012: cayophylaceace. in: stevanović, v. 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(2011+): chenopodiaceae (pro parte majore). – in: euro+med plantbase the information resource for euro-mediterranean plant diversity. published at http://www.europlusmed.org zlatković, b. 2011: flora i fitogeografska pripadnost doline reke pčinje u jugoistočnoj srbiji. doktorska disertacija. biološki fakultet. univerzitet u beogradu (manuskript). beograd. zlatković, b., bogosavljević, s. 2014: report on the new floristic data from serbia. biologica nyssana, 5 (2): 123-129. zlatković, b., bogosavljević, s., smiljković, n., ranđelović, v. 2014: report on the new and insufficiently studied taxa in the flora of serbia. biologica nyssana, 5 (1): 63-69. zlatković, b., ranđelović v., lakušić, d., stevanović, v. 2011: novelties for the vascular flora of serbia. botanica serbica, 35 (2): 103-110. biologica nyssana ● 13 (2) december 2022: 89-107 simić & zlatković ● flora around slavujevac on rujan mountain (se serbia) 107 appendix appendix 1. selected representatives of the flora of slavujevac on rujan mt. (crocus rujanensis (a), colchicum doerfleri (b), spiranthes spiralis (c), pulsatilla montana subsp. bulgarica (d), himantoglossum caprinum subsp. rumelicum (e), gladioulus communis subsp. communis (f), fritillaria gussichiae (g), iris suaveolens (h), aristolochia lutea (i)). authors of photographs: a-f, milica simić; g-i, bojan zlatković. nikolić et al., 2019, biologica nyssana 10(1) 10 (1) september 2019: 9-16 doi: 10.5281/zenodo.3463982 morphometric analysis of vegetative and reproductive organs of the fragaria species original article danijela nikolić department of biology and ecology, faculty of sciences and mathematics, university of niš, serbia danid@pmf.ni.ac.rs (corresponding author) milena veličković department of biology and ecology, faculty of sciences and mathematics, university of niš, serbia milenavelickovic9890@gmail.com irena raca department of biology and ecology, faculty of sciences and mathematics, university of niš, serbia racairena@gmail.com dragana jenačković gocić department of biology and ecology, faculty of sciences and mathematics, university of niš, serbia draganaj@pmf.ni.ac.rs marina jušković department of biology and ecology, faculty of sciences and mathematics, university of niš, serbia marinaju@pmf.ni.ac.rs vladimir ranđelović department of biology and ecology, faculty of sciences and mathematics, university of niš, serbia vladar@pmf.ni.ac.rs received: july 29, 2019 revised: september 9, 2019 accepted: september10, 2019 abstract: this paper presents results of a study on morphological variability of fragaria vesca, f. moschata and f. viridis populations collected in their natural habitats in serbia, macedonia and montenegro. the goals of the study were to determine the degree of morphological variability as well as the morphological characters with the most significant impact on differentiation of these species. the morphometric study was carried out on 7 populations (142 individuals x 19 morphological characters). analyses included standard univariate (descriptive, anova) and multivariate (principal component analysis, canonical discriminant analysis, upgma classification) analyses. the obtained results indicate moderate to high variability in most of the analysed characters. fragaria moschata has shown the highest level of variability for almost all characters in comparison to f. vesca and f. viridis. the most significant differentiation in fragaria species was present in following characters: leaf-l, lam-cent-l, lam-cent-w, lam-lat-l, ped-l and sep-int-l. key words: fragaria, morphological characters, variability pattern apstract: morfometrijska analiza vegetativnih i reproduktivnih organa vrsta roda fragaria u ovoj studiji istraživana je morfološka varijabilnost populacija vrsta fragaria vesca, f. moschata i f. viridis sakupljenih na njihovim prirodnim staništima u srbiji, makedoniji i crnoj gori. ciljevi ovog rada su bili da se ustanovi nivo morfološke varijabilnosti kao i da se utvrde morfološki karakteri koji najviše doprinose u diferencijaciji ovih vrsta. morfometrijska studija je sprovedena na 7 populacija (142 individua x 19 morofoških karaktera). urađene su standardne statističke metode univarijantne (deskripitivna, anova) i multivarijantne (analiza glavnih komponenti (pca), kanonijska diskriminantna analiza (cda), upgma klasifikacija) analize. dobijeni rezultati ukazuju na umerenu do visoku varijabilnost većine analiziranih karaktera. fragaria moschata je pokazala najveći stepen varijabilnosti za skoro sve karaktere u poređenju sa f. vesca i f. viridis. karakteri koji su najviše doprineli u diferencijaciji istraživanih fragaria vrsta su: leaf-l, lam-cent-l, lam-cent-w, lam-lat-l, ped-l and sep-int-l. ključne reči: fragaria, morfološki karakteri, varijabilnost introduction genus fragaria l. includes 24 species (dimeglio et al., 2014) distributed in eurasia, north america and south america. flora europea (tutin et al., 1968) lists 5 species: fragaria vesca l., f. moschata weston, f. viridis weston, f. virginiana mill. and f. í ananassa (weston) rozier, while euro+med plantbase also includes f. chiloensis (l.) weston. in serbia, this genus is represented with three wild species: f. vesca, f. moschata and f. viridis (gajić, 1972). these species are also present in macedonia and montenegro (micevski, 1998; euro+med plantbase). fragaria vesca has the eurasian origin and the most extensive native range among all fragaria species, as it is distributed from the west of the urals throughout northern europe and across the north american continent (hummer et al., 2011). in serbia it has a wide distribution (gajić, 1972) and may be found in meadows and forests in following vegeta© 2019 nikolić et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 9 tion associations: quercetum frainetto-cerris, querceto-carpinetum, quercetum montanum, fagetum montanum. fragaria moschata is distributed in central and southern europe and the central part of russia and ukraine. in serbia this species grows in forests (querceto-fraxinetum, quercetum farnetto-cerris and querceto-carpinetum), at forest edges, burned areas, open grassy hillsides and meadows, usually in shadowy places and tall grass, preferring saturated fertile soils. fragaria viridis is distributed almost throughout europe, as well as in siberia and at the canary islands. in serbia it is recorded at meadows (galietofestucetum vallesiacae, chrysopogonetum pannonicum), forests and burned areas (gajić, 1972). morphological recognition among species is complicated within the genus fragaria, due to high levels of morphological variations and similarities (nosrati et al., 2015). diploids, tetraploids, hexaploids, octaploids and decaploids have been reported, as well as several intermediate ploidy levels of interspecific hybrids of fragaria species (nosrati et al., 2015). fragaria vesca and f. viridis are diploids, while f. moschata is the only hexaploid species (dimeglio et al., 2014). the best-studied form is f. vesca, included in many studies dealing with morphological variability caused by ecological conditions (hancock & bringhurst, 1978; jenson & hancock, 1981; huseinović & osmanović, 2010; malinikova et al., 2013; huseinović et al., 2014) or by genetic diversity fig 1. some morphological characters of the vegetative and reproductive organs of the analysed species: a-fragaria moschata, b-f. vesca, c-f. viridis (1-leaf-l, 2-peti-l, 3-lam-cent-l, 4-lam-cent-w, 5-lam-lat-l, 6-lam-lat-w, 7-loblam-cent-n, 8-scap-l, 9-ped-l, 10-sep-ext-l, 11-sep-ext-w, 12-sep-int-l, 13-sep-int-w, 14-pet-l, 15-pet-w). (labokas & bagdonaitë, 2005; zhang et al., 2014). the quantification of variation at morphological and molecular levels within and between populations of f. vesca and the american octoploid species f. chiloensis and f. virginiana have been described by harisson et al. (1997, 2000). phenotypic variation within the diploid section of genus fragaria was also a subject of several studies (sargent et al., 2004; labokas & bagdonaitë, 2005). however, no quantitative studies of morphological variability have been carried out to measure the phenotypic variation between diploid and hexaploid fragaria species. this study aimed to research the morphological variability of vegetative and reproductive organs of two diploid fragaria species (f. vesca and f. viridis) and a hexaploid species f. moschata and to determine the morphological characters with the most significant impact on differentiation between these species. materials and methods sample collections the seven populations of fragaria species (f. vesca, f. viridis and f. moschata) were collected for morphological analysis. the samples were taken from serbia, montenegro and macedonia (tab. 1) during two vegetation seasons (2017 and 2018). the voucher specimens were deposited in the herbarium moesiacum niš (hmn) at department of biology and ecology, university of niš. identification of collected material was made by using literature sources 10 biologica nyssana ● 10 (1) september 2019: 9-16 nikolić et al. ● morphometric analysis of vegetative and reproductive organs of the fragaria species (tutin et al., 1968; gajić, 1972; markova, 1973). morphometric analyses the 7 morphological characters of vegetative organs and 12 morphological characters of reproductive organs were selected for analyses. vegetative morphological characters included: length of leaf (leaf-l ), length of petioles (peti-l), length of central lamina (lam-cent-l), width of central lamina (lam-cent-w), number of lobes on central lamina (lob-lam-cent-n), length of lateral lamina (lamlat-l) and width of lateral lamina (lam-lat-w). the reproductive morphological characters included: length of scape (scap-l), length of peduncles (pedl), length of external sepal (sep-ext-l), width of external sepal (sep-ext-w), number of lobes on external sepal (lob-sep-ext-n), number of external sepals with lobes (sep-ext-lob-n), length of internal sepal (sep-int-l), width of internal sepal (sepint-w), length of petal (pet-l), width of petal (petw), number of flowers (flow-n), number of flower branches (flow-branch-n) (fig. 1). the plant material was first digitised by the camera (sony 20 megapixels) and then measured using imagej software. statistical analysis descriptive statistics (mean, max, min, standard deviation and coefficient of variation) were computed to evaluate the range of variation for morphological traits. the coefficient of variability was used to describe biologica nyssana ● 10 (1) september 2019: 9-16 nikolić et al. ● morphometric analysis of vegetative and reproductive organs of the fragaria species 11 the morphological variability of characters. moderately variable characters are those with cv=20-40%, highly morphologically variable had cv higher than 40%, while stable characters are those with a coefficient of variation below 10%. analysis of variance (anova) was performed to identify levels of significant variation for each character. multivariate analyses (principal component analysis (pca), canonical discriminant analysis (cda) and cluster analyses (upgma) based on mahalanobis distances) were performed to examine relationships between the studied species and to find the best characters that facilitate discrimination between the species. statistical analyses were performed using the package statistika 8.0 (statsoft 2007). results and discussion morphological variability of vegetative and reproductive characters descriptive analysis the results of the descriptive statistics have shown significant variability for almost all morphological characters (tab. 2) generally, characters of vegetative and reproductive organs of fragaria species have shown moderate to high degrees of variability. following characters may be considered most variable within f. moschata populations, characterised by large-scale deviation of maximum and minimum values from the mean values, and with highest standard deviation values: ped-l (23.40 ± 9.01), leaf-l (22.55 ± no. taxon locality number of individuals voucher number (hmn) legators 1. fragaria viridis stara planina mt. (serbia) 20 13697 veličković, m. nikolić, d. 2. fragaria moschata rtanj mt. (serbia) 20 13703 veličković, m. nikolić, d. 4. fragaria moschata vlasina (serbia) 20 13701 veličković, m. nikolić, d. 3. fragaria vesca stol mt. (serbia) 20 13707 veličković, m. nikolić, d. 5. fragaria vesca kraljevica (serbia) 12 13710 veličković, m. nikolić, d. 6. fragaria vesca jablanica (macedonia) 20 13958 raca i. ranđelović, v. 7. fragaria vesca prokletije (montenegro) 30 13959 raca, i., ljubisavljević, i. jenačković, d. ranđelović, v. table 1. the list of analysed populations of the fragaria species 12 biologica nyssana ● 10 (1) september 2019: 9-16 nikolić et al. ● morphometric analysis of vegetative and reproductive organs of the fragaria species f ra ga ria v es ca fr ag ar ia m os ch at a fr ag ar ia v iri di s c ha ra ct er n m ea n m in . m ax . st d. d ev . c v % n m ea n m in . m ax . st d. d ev . c v % n m ea n m in . m ax . st d. d ev . c v % le af -l (c m ) 82 12 .0 2 4. 83 22 .6 9 3. 91 32 .5 3 35 22 .5 5 8. 67 39 .9 6 7. 06 31 .2 9 20 11 .5 0 6. 09 16 .7 3 2. 54 22 .0 4 pe til (c m ) 82 8. 39 2. 60 16 .7 7 3. 22 38 .4 2 35 16 .4 9 5. 40 28 .8 3 5. 72 34 .7 0 20 8. 29 3. 06 12 .7 1 2. 24 27 .0 1 la m -c en t-l (c m ) 82 3. 34 1. 99 5. 06 0. 76 22 .6 7 35 5. 32 2. 68 10 .4 9 1. 62 30 .5 5 20 2. 90 2. 02 4. 02 0. 66 22 .9 0 la m -c en t-w (c m ) 82 2. 47 1. 31 3. 81 0. 53 21 .4 3 35 3. 44 1. 74 6. 52 0. 93 26 .9 4 20 2. 06 1. 19 2. 86 0. 47 22 .9 2 lo b la m -c en t-n 82 7. 44 5. 00 11 .0 0 1. 32 17 .6 9 35 7. 86 6. 00 10 .0 0 1. 06 13 .5 1 20 7. 60 6. 00 10 .0 0 1. 19 15 .6 3 la m -la t-l (c m ) 82 2. 74 1. 37 4. 91 0. 86 31 .5 5 35 4. 48 2. 42 8. 00 1. 16 25 .8 5 20 2. 74 1. 81 3. 76 0. 54 19 .7 9 la m -la t-w (c m ) 82 2. 19 1. 06 3. 90 0. 63 30 .5 7 35 3. 03 1. 58 5. 06 0. 81 26 .7 6 20 1. 92 1. 37 2. 70 0. 40 20 .6 1 sc ap -l (c m ) 81 2. 34 0. 70 10 .1 1 1. 70 72 .7 6 35 4. 25 1. 31 8. 60 2. 09 49 .2 2 20 3. 00 1. 35 6. 00 1. 13 37 .5 9 pe dl (c m ) 81 11 .9 7 4. 06 20 .5 5 3. 72 31 .1 0 35 23 .4 0 9. 16 41 .0 6 9. 01 38 .5 2 20 9. 22 6. 16 13 .2 1 1. 79 19 .4 5 se pex t l (c m ) 82 0. 40 0. 23 0. 80 0. 10 24 .9 5 34 0. 49 0. 25 0. 91 0. 15 31 .3 7 20 0. 28 0. 21 0. 42 0. 06 21 .5 4 se pex t-w (c m ) 82 0. 13 0. 06 0. 23 0. 04 29 .4 5 34 0. 11 0. 04 0. 19 0. 04 39 .6 7 20 0. 08 0. 06 0. 13 0. 02 21 .1 4 lo bse pex t-n 73 2. 00 2. 00 2. 00 0. 00 0. 00 22 2. 00 2. 00 2. 00 0. 00 0. 00 20 2. 00 2. 00 2. 00 0. 00 0. 00 se pex t-l ob -n 43 1. 37 1. 00 3. 00 0. 62 45 .0 5 22 1. 45 1. 00 5. 00 0. 91 62 .6 8 20 1. 55 1. 00 2. 00 0. 51 32 .9 3 se pin t-l (c m ) 82 0. 42 0. 26 0. 71 0. 08 20 .3 5 35 0. 59 0. 26 0. 97 0. 17 27 .9 6 20 0. 33 0. 26 0. 45 0. 06 18 .7 4 se pin t-w (c m ) 82 0. 19 0. 09 0. 40 0. 05 24 .9 9 35 0. 19 0. 11 0. 38 0. 05 26 .6 6 20 0. 14 0. 10 0. 21 0. 03 22 .8 6 pe t-l (c m ) 71 0. 49 0. 28 0. 88 0. 14 28 .8 8 34 0. 68 0. 29 0. 94 0. 17 25 .1 0 20 0. 38 0. 25 0. 66 0. 12 32 .0 0 pe t-w (c m ) 71 0. 48 0. 21 0. 90 0. 16 32 .8 2 34 0. 58 0. 24 1. 00 0. 17 29 .9 8 20 0. 32 0. 18 0. 50 0. 08 25 .7 6 fl ow -n 82 3. 15 1. 00 6. 00 1. 33 42 .1 2 35 4. 09 1. 00 8. 00 1. 67 40 .8 6 20 2. 50 1. 00 5. 00 1. 05 42 .0 5 fl ow -b ra nc hn 82 1. 40 1. 00 4. 00 0. 66 47 .3 5 35 1. 00 1. 00 1. 00 0. 00 0. 00 20 1. 50 1. 00 3. 00 0. 61 40 .4 7 ta bl e 2. r es ul ts o f d es cr ip tiv e st at is tic s fo r t he m or ph ol og ic al c ha ra ct er s of th e fr ag ar ia s pe ci es . 13 7.06), peti-l (16.49 ± 5.72). the number of lobes on central lamina (lob-lam-cent-n) ranged from 6 to 10 lobes. the least variable character of the vegetative region within f. moschata populations was lam-lat-w (3.03 ± 0.81). of characters of the flowering parts, characters sep-ext-w (0.11 ± 0.04) and sep-int-w (0.19 ± 0.05) were distinguished as the least variable. the number of lobes on external sepal (lob-sep-ext-n) was 2, while number of external sepals with lobes (sep-ext-lob-n) ranged from 1 to 5. the number of flowers (flow-n) was represented by a range from 1 to 8, while number of flower branches (flow-branch-n) was 1. slightly smaller variability of characters was recorded within f. vesca populations in comparison to f. moschata populations. the most variable characters of the vegetative region were leaf-l (12.02 ± 3.91) and peti-l (2.60 ± 16.77). the number of lobes on the central lamina (lob-lam-cent-n) ranged from 5 to 11. the least variable characters of the vegetative region in f. vesca populations were lam-cent-w (2.47 ± 0.53) and lam-lat-w (2.19 ±0.63). among the characters of the flowering parts, characters sepext-w (0.13 ± 0.04), sep-int-w (0.19 ± 0.05) and sep-intl (0.42 ± 0.08) were distinguished as the least variable. lobes on external sepals (lob-sep-ext-n) was 2, while external sepals with lobes (sep-ext-lob-n) ranged from 1 to 3. flowers (flow-n) was from 1 to 6, while flower branches (flow-branch-n) ranged from 1 to 4. the morphological characters of vegetative and reproductive organs of f. viridis individuals have shown the least variability when compared with f. moschata and f. vesca populations. the most variable characters of the vegetative region were leaf-l (11.5 ± 2.54) and peti-l (8.29 ± 2.24). the number of lobes on central lamina (lob-lam-cent-n) ranged from 6 to 10. the least variable characters of the vegetative region within f. viridis populations were lam-lat-w (1.92 ±0.40) and lam-cent-w (2.06 ± 0.47). within the characters of the flowering parts, characters such as sep-ext-w (0.08 ± 0.02) and sep-int-w (0.14 ± 0.03) were determined to be the least variable. lobes on external sepals (lob-sep-ext-n) was 2, while external sepals with lobes (sep-ext-lob-n) ranged from 1 to 2. flowers (flow-n) was reprebiologica nyssana ● 10 (1) september 2019: 9-16 nikolić et al. ● morphometric analysis of vegetative and reproductive organs of the fragaria species acronyms f p pca 1 pca 2 pca 3 leaf-l 64.718 0.000 -0.932 -0.160 -0.080 peti-l 56.213 0.000 -0.898 -0.129 -0.104 lam-cent-l 52.904 0.000 -0.919 -0.173 0.066 lam-cent-w 37.295 0.000 -0.862 -0.150 0.176 lam-lat-l 47.633 0.000 -0.907 -0.257 -0.116 lam-lat-w 31.539 0.000 -0.873 -0.230 -0.012 lam-centlob-n 1.407 0.249 -0.398 -0.092 0.225 scap-l 65.050 0.000 -0.898 -0.131 -0.028 sep-int-l 45.775 0.000 -0.672 0.477 -0.137 sep-int-w 8.180 0.000 -0.194 0.644 0.334 pet-l 29.814 0.000 -0.616 0.450 -0.226 pet-w 17.237 0.000 -0.499 0.605 -0.072 sep-ext-l 21.148 0.000 -0.578 0.552 -0.147 sep-ext-w 14.970 0.000 -0.079 0.701 0.378 lobsep-ext-n 11.130 0.000 0.398 0.413 -0.520 flow-branch-n 7.503 0.001 0.407 0.050 0.328 flow-n 9.115 0.000 -0.427 0.246 0.488 ped-l 15.185 0.000 -0.573 -0.202 -0.301 sep-ext-lob-n 3.837 0.025 0.234 0.263 -0.661 table 3. results of anova and pca analysis for quantitative characters of individuals of the fragaria species. (ffisher’s coefficient, pthe level of significance (p<0.05); pca axescomponent with loadings >0.700 is highlighted in bold) sented by a range from 1 to 5, while flower branches (flow-branch-n) ranged from 1 to 3. according to values of the coefficient of variation, the most variable character (cv>70%) within the f. vesca populations was scap-l. high level of variability was shown by characters sep-extlob-n, flow-n and flow-branch-n. one character had no detected variability: lob-sep-ext-n. all the other characters have shown moderate variability (20%<cv>40%). morphological characters with high variability within f. moschata populations were: sep-ext-lob-n, scap-l, and flow-n. all the other characters have shown moderate variability, except character lob-lam-cent-n with low variability and lob-sep-ext-n and flow-branch-n, which did not show any variability (tab. 2). the characters with low variability (cv<20%) within population of f. viridis were lob-lam-cent-n, lam-lat-l, pedl and sep-int-l. only flow-n and flow-branch-n have shown high variability (cv>40%), while all other investigated characters have shown a moderate degree of variability (tab. 2). analysis of variance (anova) the anova test has shown that all characters, except lam-cent-lob-n, were statistically significant (p< 0.005) (tab. 3). according to fisher’s coefficient (f), almost all vegetative characters may be treated as the most significant (f>30). among the characters of flowering region, scap-l (f = 65.05) and sep-int-l (f = 45.78) were the most significant. this set of characters with the highest f values is the most suitable for describing morphological differentiation between fragaria species, based on the results of this analysis. morphological differentiation of analysed fragaria species the first three principal components accounted for 57.44% of the total observed variance (tab. 2). the characters which contributed the most to variability along the first axis (36.19%) are the following: leaf-l, lam-cent-l, lam-cent-w, lam-lat-l, pedl and sep-int-l. the second principal component (13.17%) had high contributing factor loadings from sep-ext-w. the third principal component accounted for 8.08% of the total variation. the pca ordination diagram of population samples (fig. 2) indicated a slight differentiation of f. moschata and f. viridis populations along the first pc axis. the individuals of f. vesca are overlapping with individuals of f. moschata and f. viridis. canonical discriminant analyses have shown the separation of the three morphological groups of populations that match the analysed species (fig. 2). populations of f. viridis and f. moschata were differentiated along the first da axis, while the populabiologica nyssana ● 10 (1) september 2019: 9-16 nikolić et al. ● morphometric analysis of vegetative and reproductive organs of the fragaria species 14 fig 2. results of the principal component analysis (pca) and canonical discriminant analysis (cda) based on morphometric characters of the fragaria species biologica nyssana ● 10 (1) september 2019: 9-16 nikolić et al. ● morphometric analysis of vegetative and reproductive organs of the fragaria species 15 tion of f. viridis was slightly differentiated along the second axis. cluster analysis of morphometric data has shown differentiation into two clusters, the first consisted of f. vesca and f. viridis, and the second included only f. moschata (fig. 3). fig 3. results of cluster analysis (upgma) for morphological characters of the fragaria species based on mahalanobis distances our study confirmed the presence of a high degree of variability in vegetative and reproductive characters of f. vesca populations, which is in agreement with the previous studies on this species (sargent et al., 2004; labokas & bagdonaitë, 2005; huseinović & osmanović, 2010; maliníková et al., 2013). the study of phenotypic diversity of f. vesca and f. viridis by labokas & bagdonaitë (2005) recognised some differences in the number of flowers, length of petioles, length and width of leaf between f. vesca and f. viridis. in our sample, inflorescences of f. vesca populations were composed of 6-12 flowers while f. viridis produced a lower number of flowers (4-7). in comparison to populations of f. viridis, f. vesca was characterised by shorter petioles, longer inflorescences and a higher ratio of peduncle to rosette leaf length (labokas & bagdonaitë, 2005). our study has shown different results regarding the range of variability of mentioned characters in f. vesca and f. viridis populations. namely, the number of flowers in f. vesca populations ranged from 1 to 7 and in f. viridis populations from 1 to 5. also f. vesca individuals had longer petioles than f. viridis. according to the available literature, morphological variability of f. moschata was never studied before. this species has shown the highest level of variability for almost all characters. f. moschata individuals had the highest values for characters pertaining to leaves (leaf-l, leaf-w, lam-cent-l, lam-cent-w, lam-lat-l, lam-lat-w) and almost all morphological characters of the flowering part of the plant. conclusion the morphological characters of vegetative and reproductive organs of fragaria species have shown high to moderate levels of variability. the most variable morphological characters of vegetative organs were peti-l and leaf-l, while the most variable morphological characters of reproductive organs were scap-l, flow-branch-n, flow-n, and sepext-lob-n. analysis of variance has shown that all morphometric characters except lam-cent-lob-n contributed significantly to differentiation of analysed fragaria species. morphological characters responsible for differentiation of analyzed fragaria species included: leaf-l, lam-cent-l, lam-cent-w, lam-lat-l, ped-l and sep-int-l. acknowledgements. this investigation is supported by the ministry of education, science and technological development of the republic of serbia grant number 173030. references dimeglio, l.m., staudt, g., yu, h., davis, t.m. 2014: a phylogenetic analysis of the genus fragaria (strawberry) using intron-containing sequence from the adh-1 gene. plos one. 9(7): e102237. published 2014 jul 31. doi:10.1371/journal. pone.0102237 gajić, m. 1972: fragaria l. in: josifović, m. (ed.), flora sr srbije, 4: 118-122, srpska akademija nauka i umetnosti. beograd. hancock, j.f., bringhurst r.s. 1978: inter-populational differentiation and adaptation in the perennial diploid species fragaria vesca l. american journal of botany 65: 795-803. harrison, r.e., luby, j.j., furnier, g.r., hancock, j.f. 1997: morphological and molecular variation among populations of octoploid fragaria virginiana and fragaria chiloensis (rosaceaea) from north america. american journal of botany 84: 612-620. harrison, r.e., luby, j.j., furnier, g.r., hancock, j.f. 2000: differences in the apportionment of molecular and morphological variation in north american strawberry and the consequences for genetic resource management. genetic resources and 16 biologica nyssana ● 10 (1) september 2019: 9-16 nikolić et al. ● morphometric analysis of vegetative and reproductive organs of the fragaria species crop evolution 47: 647-657. huseinović, s., osmanović, s. 2010: morphometric and meristic characteristics of the wild strawberry (fragaria vesca l.) on konjuh mountain. acta agriculturae serbica 15 (30): 133-140. huseinović, s., osmanović, s., terzić, z., šabanović, m. 2014: morphological and ecological differentiation of the fruit of fragaria vesca l. (rosaceae) from different habitats in bosnia and herzegovina. biologica nyssana 5(2): 75-82. jenson r.j., hancock, j.f. 1981: multivariate relationships among california strawberries. bulletin of the torrey botanical club 109: 136-147. labokas, j., bagdonaite, e. 2005: phenotypic diversity of fragaria vesca and f. viridis in lithuania. laboratory of economic botany, institute of botany 3: 19-22. maliníková, e., kukla, j., kuklová, m., balážová m. 2013: altitudinal variation of plant traits: morphological characteristics in fragaria vesca l. (rosaceae). annals of forest research 56(1): 79-89, 2013. micevski k. 1998. flora na republika makedonija, tom i, sv. 4. skopje: makedonska akademija na naukite i umetnostite. hummer, e.k., bassil, n., njuguna, w. 2011: fragaria, in: kole, c. (ed.), wild crop relatives: genomic and breeding resources, temperate fruits: 17-44, springer-verlag berlin heidelberg. nosrati, h., price, h.a., gerstberger, p., wilcock c.c. 2015: characterization of an allotriploid strawberry fragaria × bifera duchesne (rosaceae) from europe. natura sloveniae 17(1):5-15. sargent, d.j., geibel, m., hawkins, j.a., wilkinson, m.j., battey, n.h., simpson, d.w. 2004: quantitative and qualitative differences in morphological traits revealed between diploid fragaria species. annals of botany 94: 787-796. tutin, t.g., heywood, v.h., burges, n.a., moore, d.m., valentine, d.h., walters, s.m., webb, d.a. (eds.) 1964-1980: flora europaea, i-v. cambridge university press. london. zhang, q., folta, k.m., davis, t.m. 2014: somatic embryogenesis, tetraploidy, and variant leaf morphology in transgenic diploid strawberry (fragaria vesca subspecies vesca ‘hawaii 4’). bmc plant biology, 14: 1-11. anticancer compounds from medicinal plants biologica nyssana 4 (1-2)  december 2013: 19-33 jotić et al.  the vascular flora of the tepoš… 19 original article the vascular flora of the tepoš plateau around pirot city (eastern serbia) branko jotić, milica miljković, marija marković, bojan zlatković, vladimir ranđelović university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia * e-mail: marijam@pmf.ni.ac.rs abstract: jotić, b., miljković, m., marković, m., zlatković, b., ranđelović, v.: the vascular flora of the tepoš plateau around pirot city. biologica nyssana, 4 (1-2), december 2013: 19-33. the flora of the karst plateau tepoš, which is the part of vidlič mountain (eastern serbia) was studied. based on the analysis of the collected material and data from the literature, it was noted that the flora of tepoš plateau is represented by 486 species, distributed in 284 genera and 68 families of vascular plants. the biological spectra was shown. the hemicriptophytic-terophytic character was established by analyzing the presence of plant life forms. by phytogeographical analysis the presence of 25 area types was established. the euroasian florystic chorion with 258 species of eurasian area type is the most abundant, followed by mediterranean-submediterranean (58 species), central-european (47 species), cosmopolitan (27 species) and pontian area type (24 species). in the flora of tepoš plateau the 9 balkan endemic taxa were recorded, which is 3,14% of total balkan endemic taxa in flora of serbia. a small number of endemic taxa is expected, considering the low diversity of habitats. balkan endemic taxa recorded in this area are: acer hyrcanum subsp. intermedium, bupleurum flavicans, crucianella graeca, eryngium palmatum, pastinaca hirsuta, hypericum rumeliacum subsp. rumeliacum, thymus praecox subsp. jankae, linaria rubioides subsp. nissana and viola tricolor subsp. macedonica. key words: flora, pirot city, tepoš plateau introduction karst plateau tepoš (650-690m) is situated in eastern serbia, 14 km se of pirot city, above the krupac village (fig. 1). this is the part of mt. vidlič, westernmost part of the mountain system stara planina. the karst plateau is elongated in the southeastern-northwestern direction. in contrast to other regions of mountain system stara planina, mt. vidlič are represented by unique geological structure and climate conditions. the geological structure of the most part of mt. vidlič is represented by limestone, while other serbian parts of stara planina are built of silicate (v i d a n o v i ć , ed., 1960). the pedological substrate is consisted of reddish soil tiller and mountain soil. the climate is transitional humid continental, between dry climate of pirot region and wet climate on mt. stara planina (v i d a n o v i ć , ed., 1960). although the flora of this part of mt. stara planina was investigated by pančić (1884) and adamović (1911), it has not been well known. the tepoš plateau is not investigated up to now. material and methods the flora of tepoš plateau were studies in the period between 2009 and 2011. herbarium specimens are deposited at the herbarium moesiacum, university of niš (hmn). determination of plant material was carried out using the keys from the regional flora reference 11 th sfses • 13-16 june 2013, vlasina lake 4 (1-2) • december 2013: 19-33 biologica nyssana 4 (1-2)  december 2013: 19-33 jotić et al.  the vascular flora of the tepoš… 20 (j o s i f o v i ć , ed. 1970-1977; в е л ч е в , ed. 19821989). the nomanclature folows med-checklist (greuter et al., 1984-1989) and flora europaea (tutin et al., eds., 1964-1980). for the area types classification meusel et al. (1965, 1978), meusel and jäger (1992) and stevanović (1992) were used. life forms were determined according to mueller-dombois and ellenberg (1974) and s t e v a n o v i ć (1992a). results as a results of the floristic investigations of tepoš plateau in eastern serbia, 486 plant taxa belonging to 284 genera and 68 families were recorded (tab. 1). figure 1. geographical position of investigated area localities: l1 – road from village krupac to village visočki odorovci (500 – 750 m), l2 – ″kragujevac″ (810 m), l3 – ″kurjasi vrh″ (934 m), l4 – ″kočino″ (857 m), l5 – ″strči krak″ (861 m), l6 – ″begova rudina″ (651 m) table 1. list of vascular flora with life forms and area-types taxa life form area type locality polypodiopsida aspleniaceae asplenium ruta-muraria l semp ch herb caesp hol l5 asplenium trichomanes l. semp ch herb semiros csm l5 woodsiaceae cystopteris fragilis (l.) bernh. fo dec ch herb semiros csm l2 magnoliopsida acanthaceae acanthus balcanicus heywood & i.b.k.richardson a meg-alt h scap semiros eas l2 biologica nyssana 4 (1-2)  december 2013: 19-33 jotić et al.  the vascular flora of the tepoš… 21 taxa life form area type locality aceraceae acer campestre l. fo dec mes p scap eas l1 acer hyrcanum fischer & c. a. meyer subsp. intermedium (pančić) bornm. fo dec mes p scap me l1 acer monspessulanum l. fo dec mi-mes p scap med l1 acer platanoides l. fo dec mes p scap me l3 acer pseudoplatanus l. fo dec mes p scap me l3 acer tataricum l. fo dec mi-mes p scap eas l2 amaranthaceae amaranthus retroflexus l. a mes-alt t scap adv l1 anacardiaceae cotinus coggygria scop. fo dec mi p caesp eas l6 apiaceae aegopodium podagraria l. v-a mes-meg h scap/g rhiz eas l1 bifora radians m. bieb. a meg t scap eas l1 bupleurum commutatum boiss. & bal. a mac-meg t scap p l1 bupleurum flavicans boiss. & heldr. a mi-mes t scap med l6 bupleurum praealtum l. v-a mi-mes t scap med l1 carum rigidulum (viv.) koch ex dc. a mes h scap eamt l1 caucalis platycarpos l. v-a mes t scap eas l1 chaerophyllum temulentum l. a mes t scap/h bienn eas l1 daucus carota l. a meg t/h scap hol l1 eryngium campestre l. v-a mes-meg h semiros eas l6 eryngium palmatum pančić et vis. a mes-meg h scap semiros med l5 ferulago sylvatica (besser) reichenb. a meg-alt h scap eamt l2 orlaya grandiflora (l.) hoffm. a mes t scap eas l1 pastinaca hirsuta pančić a mes-meg h scap/t scap bienn eamt l1 peucedanum alsaticum l. a-aut mes-alt h scap eas l6 physocaulis nodosus (l.) koch a mes-meg t scap med l1 seseli peucedanoides (bieb.) kos. pol. a mes-meg h scap eas l2 smyrnium perfoliatum l. a mes-meg h scap med l2 tordylium maximum l. a meg t scap med l1 torilis leptophylla (l.) reichenb. a mes t scap eas l1 apocynaceae vinca herbacea waldst. et kit. v-a mi-mes ch suff rept p l6 araliaceae hedera helix l. aut fo semp alt s lig me l5 aristolochiaceae aristolochia pallida willd. v-a mes g rad med l1 asarum europaeum l. fo semp ch herb rept eas l5 asclepiadaceae vincetoxicum hirudinaria medicus a mes-alt h scap eas l2 asteraceae achillea clypeolata sibth. & sm. a mes-meg h scap med l2 achillea crithmifolia waldst. et kit. a mes-meg h scap msm l5 achillea millefolium l. a meg h scap hol l1 anthemis arvensis l. a mes-mac t scap med l1 arctium lappa l. a mes-alt h scap bienn eas l1 arctium tomentosum miller a-aut meg-alt h scap bienn eas l6 artemisia alba turra aut meg fo dec ch suffr caesp eamt l5 artemisia vulgaris l. aut meg-alt h scap hol l1 bellis perennis l. v-a mi-mes h ros eas l1 carduus acanthoides l. a mac h scap me l1 biologica nyssana 4 (1-2)  december 2013: 19-33 jotić et al.  the vascular flora of the tepoš… 22 taxa life form area type locality carduus candicans waldst. et kit. subsp. globifer (velen.) kazmi a mes-meg h scap msm l6 carduus nutans l. a mes-meg h scap me l1 carthamus lanatus l. a mes-mac t scap eas l6 centaurea biebersteinii dc. a mes-meg h scap eas l1 centaurea calcitrapa l. a mes-meg h caesp eas l1 centaurea phrygia l. a mes-meg h scap me l3 centaurea salonitana vis. a mac h scap med l2 centaurea scabiosa l. a meg h scap eas l2 centaurea solsticialis l. a alt t scap msm l1 chamomilla recutita (l.) rauschert a mes-meg t scap csm l1 chondrilla juncea l. a mes-meg t scap bienn med l6 cichorium intybus l. a mac h scap csm l1 cirsium eriophorum (l.) scop. a mes-alt t/h scap bienn eamt l1 cirsium vulgare (savi) ten. a mes-alt t/h scap bienn eas l1 conyza canadensis (l.) cronquist a-aut mes-alt t scap adv l1 crepis biennis l. v-a mac h scap bienn me l1 crepis foetida l. a mes-meg t ros eas l6 crepis sancta (l.) babcock v mi-mes t scap eas l6 doronicum columnae ten. v mes-meg h scap/g rhiz eamt l2 echinops ritro l. subsp. ruthenicus (bieb.) nyman a-aut mac-meg h scap eas l1 erigeron annuus (l.) pers. v-a mes-meg t scap adv l1 hieracium pilosella l. a mi-mes h ros eas l6 hieracium praealtum vill. ex gochnat subsp. bauhinii (besser) petunnikov a meg-alt h scap me l6 inula britannica l. a mes-meg h scap eas l1 inula conyza dc. a mes-meg h scap eas l1 inula oculus-christi l. a mes-meg h scap p l3 lapsana communis l. a meg-alt t scap eas l1 leontodon crispus l. a mi-mes h ros med l3 leontodon hispidus l. a mi-mac h ros eas l2 leucanthemum vulgare lam. a mes-meg h scap eas l2 logfia minima (sm.) dumort. a mi-mes t scap eas l6 onopordum acanthium l. a meg-alt h scap bienn eas l1 ptilostemon afer (jacq.) w. greuter a mac-meg h scap bienn med l1 pulicaria dysenterica (l.) bernh. a mes-mac h scap eas l1 scorzonera hispanica l. a mac-meg h scap p l2 senecio vernalis waldst. et kit. v mes t scap eas l1 sonchus asper (l.) hill a-aut meg-alt t scap bienn eas l1 tanacetum corymbosum (l.) schultz bip. a meg-alt h scap eas l5 taraxacum officinale weber v-aut mes h ros eas l1 tragopogon balcanus velen. a mes-meg h scap bienn med l1 tragopogon pratensis l. a mes-meg h scap eas l1 xanthium spinosum l. a mes-meg t scap adv l1 xanthium strumarium l. a meg-alt t scap csm l1 xeranthemum annuum l. a mes-meg t scap med l6 boraginaceae anchusa barrelieri (all.) vitman a mac-alt h scap bienn eas l6 anchusa officinalis l. a alt h scap bienn eas l1 buglossoides arvensis (l.) i. m. johnston v-a mes t scap eas l1 buglossoides purpurocaerulea (l.) i. m. johnston v-a mac h scap eas l1 cerinthe minor l. a mi-meg h scap bienn eas l1 echium italicum l. v-a mac-meg h scap bienn med l1 biologica nyssana 4 (1-2)  december 2013: 19-33 jotić et al.  the vascular flora of the tepoš… 23 taxa life form area type locality echium vulgare l. a mac h scap eas l1 heliotropium europaeum l. a mes-meg t scap eas l6 myosotis arvensis (l.) hill v-a mi-mes t scap hol l6 nonea pulla (l.) dc. v-a mes-mac h scap bien eas l3 pulmonaria officinalis l. v mes-mac h scap me l2 symphytum tuberosum l. v mes-mac h scap/g tub msm l2 brassicaceae alliaria petiolata (bieb.) cav. & grande v-a mes-meg t/h scap eas l1 alyssum alyssoides (l.) l. v mi-mes t scap eas l1 alyssum montanum l. v mi-mes fo dec ch suffr rept eas l1 alyssum repens baumg. v mi-mes fo dec ch suffr p l1 arabis hirsuta (l.) scop. a mes-meg h ros bienn csm l1 arabis recta vill. v meg h scap bienn eas l1 arabis sagittata (bertol.) dc. a mes-meg h ros bienn eas l1 arabis turrita l. v-a meg h scap semiros eas l1 berteroa incana (l.) dc. a mes-mac t/h scap eas l1 calepina irregularis (asso) thell. a mes-meg t scap eas l1 capsella bursa – pastoris (l.) medicus v-aut mi-meg t/h ros bienn csm l1 cardamine bulbifera (l.) crantz v-a mes-mac g rhiz eas l2 cardamine graeca l. v mes t scap ros med l1 cardamine hirsuta l. a mi-mes t scap ros eas l1 draba muralis l. v mi-meg t scap csm l1 erophila verna (l.) besser v mi-mes t ros eas l2 erysimum cuspidatum (bieb.) dc. a mac t/h ros bienn p l1 erysimum diffusum ehrh. a mes-meg t/h scap eas l1 lepidium campestre (l.) r. br. a meg t/h scap bienn eas l1 rorippa pyrenaica (lam.) reichenb. a mes-mac h ros eas l1 sinapis arvensis l. v-a mes-mac t scap eas l1 sisymbrium officinale (l.) scop. a mes-mec t scap eas l1 sysimbrium orientale l. a meg t scap bienn eas l1 syrenia cana (piller & mitterp.) neilr. a mes t scap ros/h scap ros p l1 thlaspi alliaceum l. v-a mes-meg t semiros med l1 thlaspi goesingense halascy a mes t scap eas l1 thlaspi kovatsii heuff. a mes-alt h scap p l1 thlaspi perfoliatum l. v mi-mes t scap semiros eas l1 campanulaceae asyneuma anthericoides (janka) bornm. a mes-meg h scap me l2 asyneuma canescens (w. et k.) gris. et sch. a mes-meg h scap eas l2 campanula bononiensis l. a mes-meg h scap eas l5 campanula cervicaria l. a mes-meg h scap eas l3 campanula patula l. subsp. abietina (griseb.) simonkai a mes-meg h scap me l3 campanula persicifolia l. a mes-meg h scap me l2 campanula rapunculus l. a mes-meg h scap eas l5 campanula sparsa friv. a mes-mac t scap me l5 caprifoliaceae lonicera xylosteum l. fo dec mi p caesp eas l2 sambucus ebulus l. a alt g rad/h scap eas l1 sambucus nigra l. fo dec mi p scap eas l1 viburnum lantana l. fo dec n-mi p caesp eas l1 caryophyllaceae agrostemma githago l. a meg t scap eas l6 arenaria serpyllifolia l. v-a mi-mes t scap eas l1 cerastium glomeratum thuill. v-a mi-mes t scap csm l1 biologica nyssana 4 (1-2)  december 2013: 19-33 jotić et al.  the vascular flora of the tepoš… 24 taxa life form area type locality cerastium pumilum curt. a mes h scap eas l1 dianthus diutinus kit. a mes-meg h scap msm l1 herniaria incana lam. a mi h caesp rept eas l6 holosteum umbellatum l. a mi t scap eas l1 lychnis coronaria (l.) desr. a mes-meg h scap eas l2 minuartia verna (l.) hiern. a mi-mes h caesp hol l1 moenchia mantica (l.) bartl. a mes t scap eas l5 petrorhagia prolifera (l.) p. w. ball & heywood a mes t scap eas l6 petrorhagia saxifraga (l.) link a mes-mac ch caesp eas l6 silene italica (l.) pers. a mes-meg h scap ros med l2 silene viridiflora l. a mes-meg h scap eas l2 silene vulgaris (moench.) garcke a mes-meg h scap/g rad eas l1 stellaria graminea l. a mes-mac h scap eas l2 stellaria holostea l. a mes-mac h scap eas l1 stellaria media (l.) vill. a mes-meg h scap rept csm l1 celastraceae evonymus europaeus l. fo dec n-mi p caesp eas l2 evonymus latifolius (l.) miller fo dec n-mi p caesp eas l2 chenopodiaceae atriplex patula l. a-aut mes-meg t scap hol l1 chenopodium album l. a mes-meg t scap csm l1 cistaceae fumana procumbens (dunal) gren. & gordon v-a mi-mes ch frut eas l1 helianthemum nummularium (l.) miller a mes-meg fo dec ch suffr caesp eas l3 helianthemum salicifolium (l.) miller v mi-mes t scap med l5 convolvulaceae convolvulus arvensis l. a mes-alt s herb eas l1 convolvulus canthabrica l. a mes-meg h scap eas l6 cornaceae cornus mas l. fo dec mi p caesp eas l2 cornus sanguinea l. fo dec mi-mes p caesp me l2 corylaceae carpinus betulus l. fo dec meg-alt p caesp scap eas l3 carpinus orientalis miller fo dec mi-mes p caesp med l2 corylus avellana l. fo dec meg-alt p caesp eas l1 corylus colurna l. fo dec mes p scap med l1 crassulaceae sedum acre l. a mi-mes ch herb scap succ me l1 sedum album l. a mi-mes ch herb scap succ med l1 sedum annuum l. a n-mi ch herb scap succ ab l1 sedum hispanicum l. a mi t/h succ eas l1 sedum ochroleucum chaix. a mes ch herb succ med l1 sedum sexangulare l. a mi-mes ch herb succ me l1 dipsacaceae dipsacus laciniatus l. a mes-alt t scap bienn eas l1 knautia integrifolia (l.) bertol a mes-meg t scap med l2 knautia macedonica griseb. a mes-meg h scap msm l2 scabiosa argentea l. a mes-meg h scap p l2 euphorbiaceae euphorbia amygdaloides l. a mes-mac t scap eas l2 euphorbia cyparissias l. v-a mes-mac h scap eas l1 euphorbia esula l. a meg-alt h scap me l1 euphorbia esula l. subsp. tommasiniana a mes-meg h scap eas l6 biologica nyssana 4 (1-2)  december 2013: 19-33 jotić et al.  the vascular flora of the tepoš… 25 taxa life form area type locality (bertol.) nyma euphorbia helioscopia l. a mi-meg t scap eas l1 euphorbia seguierana necker subsp. niciciana (borbás ex novák) rech. fil. a mes-meg h scap eas l1 fabaceae anthyllis vulneraria l. a mes-meg h scap eas l1 astragalus glycyphyllos l. a mes-meg h scap rept eas l2 astragalus onobrychis l. a mes fo ch suffrut rept eas l1 chamaecytisus glaber (l. fil.) rothm. v-a mes-meg fo dec ch frut caesp eamt l1 chamaecytisus rochelii (wierzb.) rothm. v-a mes-meg fo dec ch frut caesp msm l2 chamaespartium sagittale (l.) p. gibbs a mes fo dec ch suffrut caesp eas l1 colutea arborescens l. fo dec mi p caesp eas l2 coronilla scorpioides (l.) koch v mi-mes t scap med l2 coronilla varia l. a mes-meg h scap eas l2 cytisus procumbens (waldst. & kit. ex willd.) sprengel a mes fo dec ch suffrut caesp med l2 dorycnium pentaphyllum scop. subsp. germanicum (gremli) gams a mes fo dec ch suffrut caesp msm l2 genista tinctoria l. a mes-mac fo dec ch suffrut caesp eas l2 gleditsia triacanthos l. fo dec mes p scap adv l1 lathyrus aphaca l. a mes t/s herb eas l1 lathyrus niger (l.) bernh. a mes-meg h scap me l2 lathyrus nissolia l. a mes-meg t scap eas l1 lathyrus pratensis l. a mes-meg h scap eas l5 lathyrus tuberosus l. a meg h scap/g tub eas l1 lathyrus venetus (mill.) wohlf. a mes-meg h scap p l3 lotus corniculatus l. a mes h scap hol l6 medicago lupulina l. a mes t/h scap hol l6 medicago minima (l.) bartal v mi-mes t scap eas l6 medicago rigidula (l.) all. v mi-mes t scap eas l1 medicago sativa l. a mes-meg h scap adv l1 medicago sativa l. subsp. falcata (l.) arcangeli a mes-meg h scap eas l6 melilotus officinalis (l.) pallas a meg t/h scap bienn eas l1 onobrychis alba (waldst. et kit.) desv. a mes-meg h scap med l1 onоnis pusilla l. a-aut mi-mes fo dec ch suffrut caesp eas l1 ononis spinosa l. a mes-meg fo dec ch suffrut caesp eas l1 robinia pseudoacacia l. fo dec mes p scap adv l2 trifolium alpestre l. a mes-mac h scap eas l2 trifolium arvense l. a mes t/h scap bienn eas l3 trifolium badium schreber a mes-meg h scap eamt l3 trifolium campestre schreber a mes-mac t scap eas l2 trifolium incarnatum l. a mes-meg t scap eas l2 trifolium medium l. v-a mes-mac h scap eas l2 trifolium montanum l. a mes h scap eas l3 trifolium pratense l. a mes h scap eas l1 trifolium repens l. a mi h rept hol l1 trifolium scabrum l. a mi-mes t scap med l5 vicia cracca l. a meg-alt h/s scap herb eas l1 vicia grandiflora scop. v-a mes-mac h scap/sh herb eas l1 vicia hirsuta (l.) s. f. gray a mes-meg t/s scap herb eas l5 biologica nyssana 4 (1-2)  december 2013: 19-33 jotić et al.  the vascular flora of the tepoš… 26 taxa life form area type locality vicia lathyroides l. v-a mes-meg t scap eas l2 vicia pannonica crantz v-a mes t/s scap herb p l1 vicia sativa l. subsp. cordata wulfen ex hoppe v-a mes-meg t scap bienn eas l1 fagaceae fagus moesiaca (k. maly) czecz. fo dec meg-alt p scap me l2 quercus cerris l. fo dec mes p scap med l2 quercus petraea (mattuschka) liebl. fo dec meg-alt p scap me l2 quercus pubescens willd. fo dec mes p scap med l2 fumariaceae corydalis solida l. v mes g tub me l2 gentianaceae gentiana cruciata l. a mes-meg g rad scap eas l3 geraniaceae erodium ciconium (l.) l’her. v-a mi-mes t scap msm l1 geranium dissectum l. a mi-meg t scap eas l1 geranium lucidum l. a mi-meg t scap eas l1 geranium molle l. v mes t scap eas l1 geranium purpureum vill. a mi-meg t scap med l5 geranium pyrenaicum burm. fil. a mes-meg h scap eas l2 geranium sanguineum l. v-a mes h semiros eas l1 hypericaceae hypericum hirsutum l. a mes-meg h scap eas l1 hypericum perforatum l. a mes-meg h scap eas l1 hypericum richeri vill. a mes-meg h scap eamt l2 hypericum rumelicum boiss. a mi-mes h scap med l1 lamiaceae acinos alpinus (l.) moench a mes t scap/g rhiz eas l1 acinos arvensis (lam.) dandy a mi-mes t scap/g rhiz eas l6 ajuga chamaepytus (l.) shreber subsp. chia (schreber) arcangeli a mi-mes t scap med l5 ajuga genevensis l. v-a mes-mac h scap ros eas l1 ajuga laxmannii (l.) bentham v-a mes h scap/g rhiz p l2 ballota nigra l. a meg h scap eas l1 clinopodium vulgare l. a mes-meg h scap hol l1 glechoma hirsuta waldst. et kit. a mes h rept eas l3 lamiastrum galeobdolon (l.) ehrend. & polatschek v-a mes-mac h scap me l2 lamium amplexicaule l. v mi-mes t scap hol l1 lamium maculatum l. v mes-mac h scap eas l1 lamium purpureum l. v mi-mes t scap eas l1 marrubium peregrinum l. a mes-meg h scap eas l1 marrubium vulgare l. a meg h scap eas l2 mentha longifolia (l.) hudson a mes-meg h scap hol l1 nepeta nuda l. a mac-meg h scap eas l1 origanum vulgare l. a mes-meg h scap eas l2 prunella laciniata (l.) l. a mes-mac h scap eas l3 prunella vulgaris l. a mi-mes h scap semiros eas l1 salvia aethiopis l. a mes-mac h scap p l1 salvia austriaca jacq. a mes-mac h scap p l1 salvia nemorosa l. a mes-meg h scap eas l1 salvia pratensis l. a mes-meg h scap msm l2 salvia sclarea l. a mes-mac h scap med l1 salvia verticillata l. a mes-mac h scap eas l1 satureja montana l. subsp. kitaibelii (wierzb.) p.w. ball a mes ch suffrut caesp eamt l1 biologica nyssana 4 (1-2)  december 2013: 19-33 jotić et al.  the vascular flora of the tepoš… 27 taxa life form area type locality scutellaria columnae all. a mes-mac h scap med l1 scutellaria galericulata l. a mes-meg h scap bor l2 sideritis montana l. a mi-mes t scap eas l6 stachys germanica l. a meg h scap msm l6 stachys officinalis (l.) trevisan a mes-meg h scap eas l3 stachys recta l. a mes-meg h scap eas l6 stachys sylvatica l. a mes-meg h scap eas l5 teucrium chamaedrys l. a mes ch suffrut caesp eas l1 teucrium montanum l. a mes ch suffrut caesp eamt l1 thymus glabrescens willd. a mi-mac ch herb rept eas l1 thymus pannonicus all. a mi-mac ch rept me l1 thymus praecox opiz subsp. jankae (čelak.) jalas a mi ch herb rept pulv med l1 linaceae linum catharticum l. a mi-mes t scap eas l2 linum hologynum reichenb. a mes-mac h caesp med l2 malvaceae althaea officinalis l. a meg h scap bienn eas l5 hibiscus trionum l. a mes t scap bien hol l1 lavatera thuringiaca l. a meg-alt h scap eas l3 malva sylvestris l. v-a mes-alt h scap eas l1 oleaceae fraxinus excelsior l. fo dec mes p scap eas l3 fraxinus ornus l. fo dec mi-mes p scap eas l2 ligustrum vulgare l. fo dec mi p caesp eas l1 syringa vulgaris l. fo dec mi p caesp med l3 papaveraceae papaver dubium l. a meg t scap eas l1 papaver rhoeas l. a mes-meg t scap eas l1 plantaginaceae plantago argentea chaix a mi-meg h ros eamt l6 plantago lanceolata l. a mi-meg h ros eas l1 plantago major l. a mes-meg h ros csm l1 plantago media l. a mes-meg h ros eas l1 polygalaceae polygala comosa schkuhr a mi-mes h scap eas l1 polygonaceae rumex acetosa l. a meg h scap hol l1 rumex arifolius all. a meg h scap p l2 rumex conglomeratus murray a meg h scap csm l1 rumex sanguineus l. a meg h scap hol l1 primulaceae lysimachia nummularia l. a n-mes ch herb rept me l1 lysimachia vulgaris l. a mes-meg h scap eas l3 primula veris l. v mi-mes h ros eas l1 ranunculaceae adonis aestivalis l. a mes-mac t scap eas l1 adonis vernalis l. v mes-meg g rhiz p l1 anemone ranunculoides l. v mi-mes g rhiz me l5 clematis integrifolia l. v mes-meg g rhiz scap p l2 clematis vitalba l. a dec s lig eas l1 consolida regalis s. f. gray a mes-meg t scap eas l1 delphinium fissum waldst. et kit. a meg-alt g tub med l3 helleborus odorus waldts. & kit. v meg g rhiz me l1 isopyrum thalictroides l. v mes g rhiz eas l2 biologica nyssana 4 (1-2)  december 2013: 19-33 jotić et al.  the vascular flora of the tepoš… 28 taxa life form area type locality nigella arvensis l. a-aut mes t scap eas l1 ranunculus bulbosus l. a mes-meg h scap eas l2 ranunculus ficaria l. v mi-mes g tub me l1 ranunculus millefoliatus wahl. v-a mes h scap semiros med l2 thlalictrum minus l. a meg-alt h scap eas l5 resedaceae reseda lutea l. a mes-meg t/h scap bienn eas l1 rosaceae agrimonia eupatoria ledeb. a meg h scap eas l1 aremonia agrimonioides (l.) neck. v mi-mes h scap ros eamt l2 crataegus monogyna jacq. fo dec mi p caesp eas l1 cydonia oblonga mill. fo dec np caesp adv l1 filipendula vulgaris moench a mes-meg h scap eas l1 fragaria vesca l. a mes h rept csm l1 fragaria viridis duchesne a mes h rept eas l1 geum urbanum l. a mes-meg h scap hol l1 malus pumila miller fo dec mi-mes p scap p l1 potentilla argentea l. a mes-meg h scap me l1 potentilla cinerea chaix ex vill. v mi-mes h caesp rept eas l1 potentilla micrantha ramond ex d.c. v mi-mes h scap med l2 potentilla recta l. a mes-meg h scap eas l2 prunus avium l. fo dec mes p scap eas l2 prunus spinosa l. fo dec n p caesp eas l1 pyrus amygdaliformis vill. fo dec n p caesp med l1 pyrus pyraster burgsd. fo dec mes p scap eas l1 rosa canina l. fo dec n p caesp eas l1 rosa micrantha borrer ex sm. fo dec n p caesp eas l1 rubus caesius l. fo dec np rept eas l1 rubus canescens dc. fo dec np rept eas l1 sanguisorba minor scop. a mes-meg h scap eas l1 sorbus aria (l.) crantz fo dec mes p scap me l3 sorbus torminalis (l.) crantz fo dec mes p scap eas l2 rubiaceae asperula cynanchica l. v-a mes-mac h scap eas l1 asperula taurina l. v-a mes-mac h scap eas l5 crucianella angustifolia l. a mi-mes t scap med l1 crucianella graeca boiss. a mi-mes t scap med l1 cruciata glabra (l.) ehrend. v-a mi-mes h scap eas l1 cruciata laevipes opiz v-a mes-mac h scap eas l1 galium album miller a meg-alt h/s scap herb me l1 galium aparine l. v-a mes-meg t/s herb csm l1 galium sylvaticum l. a mes-mac h scap/g rhiz scap me l2 galium verticillatum danth. v-a m-mes t scap med l1 galium verum l. a mes-meg h scap eas l1 sherardia arvensis l. v-a mi-mes t scap med l1 rutaceae dictamnus albus l. v-a mes-alt ch suffrut caesp eas l2 salicaceae populus tremula l. fo dec mes p scap eas l1 santalaceae thesium alpinum l. a mes-mac h scap eamt l6 saxifragaceae saxifraga tridactylites l. v-a mi t ros me l1 scrophullariaceae biologica nyssana 4 (1-2)  december 2013: 19-33 jotić et al.  the vascular flora of the tepoš… 29 taxa life form area type locality digitalis grandiflora miller a mac-alt h scap eas l5 digitalis lanata ehrh. a mac-alt h scap me l6 euphrasia pectinata ten. a mi-mes t scap/ep semipar eas l1 euphrasia salisburgensis funck a mi-mes t scap/ep semipar eamt l1 kickxia elatine (l.) dumort. a mes t rept eas l1 lathraea squamarria l. a mes-meg g par eas l2 linaria rubioides vis. & pančić subsp. nissana (petrović) niketić & tomović a mes h scap med l5 linaria vulgaris l. a mes-mac h scap eas l1 melampyrum arvense l. a mes-mac t scap/ep semipar eas l2 melampyrum cristatum l. a mes-meg t scap/ep semipar eas l2 rhinanthus angustifolius c. c. gmelin a mi-mac t scap/ep semipar me l2 rhinanthus rumelicus velen. a mi-mac t scap/ep semipar eas l2 verbascum densiflorum bertol. a mac-alt t scap ros bienn eas l6 verbascum lychnitis l. a mac-alt t/h scap ros bienn eas l1 verbascum phlomoides l. v-a mes-alt h ros eas l6 verbascum phoeniceum l. v-a mac-meg h ros eas l6 veronica arvensis l. a mes-mac t scap eas l1 veronica austriaca l. a mes-mac h scap eas l2 veronica beccabunga l. a mes-mac h scap eas l1 veronica chamaedrys l. v-a mi-mes h scap eas l2 veronica hederifolia l. v mi-mes t scap eas l1 simaroubiaceae ailanthus altissima (miller) svingle fo dec mes p scap adv l6 solanaceae datura stramonium l. a-aut meg-alt t scap hol l1 hyascyamus niger l. v-a mes-meg t scap bienn eas l1 lycium barbarum l. fo dec np caesp med l1 solanum dulcamara l. a meg-alt s lig eas l1 solanum nigrum l. a-aut mes-meg t scap hol l1 staphyleaceae staphylea pinnata l. fo dec mi p caesp/mi p scap me l1 tiliaceae tilia tomentosa moench fo dec mes p scap med l3 urticaceae urtica dioica l. a mes-meg t/h scap hol l1 valerianaceae valeriana officinalis l. a meg-alt h scap eas l2 valerianella locusta (l.) laterrade v mes t scap csm l1 valerianella rimosa bast. v mes t scap eas l1 verbenaceae verbena officinalis l. a mes-meg h scap eas l1 violaceae viola alba besser v mi-mes h rept semiros eas l2 viola arvensis murray v-aut mi-mac t scap csm l1 viola jordanii hanry v mes h scap ros p l1 viola kitaibeliana schultes v n-mi t scap eas l6 viola odorata l. v mi-mes h rept ros eas l1 viola tricolor l. subsp. macedonica (boiss. & heldr.) a. schmidt v mi-mes t/h scap med l1 liliopsida araceae arum maculatum l. v mes-meg g rhiz me l6 biologica nyssana 4 (1-2)  december 2013: 19-33 jotić et al.  the vascular flora of the tepoš… 30 taxa life form area type locality cyperaceae carex caryophyllea latourr. a mes-meg h caesp eas l1 eleocharis palustris (l.) roem. & schult. a mes-meg emer hyd g rhiz csm l1 dioscoreaceae tamus communis l. v alt s/g herb rhiz med l3 iridaceae gladiolus illyricus w. d. j. koch a mes-meg g tub scap med l5 iris graminea l. v-a mes-mac g rhiz eas l1 juncaceae juncus effusus l. a mes-meg g rhiz caesp csm l1 luzula campestris (l.) dc. v mes-meg h caesp csm l1 liliaceae allium albidum fischer ex bieb. a mes g bulb p l6 allium flavum l. a mes-mac g bulb msm l1 allium scorodoprasum (l.) subsp. rotundum (l.) stearn a mes-meg g bulb eas l6 anthericum ramosum l. a mes-meg g rhiz eas l2 asparagus officinalis l. a meg-alt g rhiz p l6 asparagus tenuifolius lam. a meg-alt g rhiz eas l3 colchicum autumnale l. aut mes g bulb eas l6 erythronium dens – canis l. v mes g bulb eas l2 lilium martagon l. a meg-alt g bulb me l2 muscari comosum (l.) miller v mes g bulb eas l2 muscari neglectum guss. ex ten. v mes g bulb eas l1 muscari tenuiflorum tausch v mes g bulb p l1 ornithogalum ortophyllum ten. subsp. kochii (parl.) zahar. v mi-mes g bulb eas l1 ornithogalum pyramidale l. v mi-mes g bulb eas l1 polygonatum odoratum (miller) druce a mes-meg g rhiz eas l2 veratrum nigrum l. a meg-alt g rhiz eas l5 orchidaceae cephalanthera damasonium (miller) druce a mes-mac g tub scap eas l1 epipactis atrorubens (hoffm.) besser a mes g tub scap p l5 epipactis helleborine (l.) crantz a mes g tub scap eas l2 epipactis microphylla (ehrh.) swartz a mes g rhiz scap p l2 himantoglossum hircinum (l.) sprengel v-a mac-meg g tub med l1 limodorum abortivum (l.) swartz a mes-meg g rhiz scap p l2 neottia nidus-avis (l.) l. c. m. richard a mes-mac sapr g rhiz eas l2 ophrys apifera hudson a mes g tub scap eas l6 ophrys cornuta steven a mes g tub scap eas l6 orchis morio l. v-a mes g tub scap eas l6 orchis purpurea hudson v-a mac-meg g tub med l6 platanthera chlorantha (custer) reichenb. a mes g tub scap eas l1 poaceae aegilops geniculata roth. a mi-mes t caesp med l1 alopecurus pratensis l. a mes-meg h caesp eas l1 antoxanthum odoratum l. a mes-meg h caesp eas l1 arrhenatherum elatius (l.) beauv. ex j. & c. presl a mes-alt h caesp eas l1 avena versicolor vill. a meg h scap eamt l6 brachypodium pinnatum (l.) beauv. a mac-alt h caesp eas l1 brachypodium sylvaticum (hudson) beauv. a mac-alt h caesp eas l2 briza media l. a mes-meg h caesp me l1 bromus racemosus l. a mes-meg t caesp me l6 bromus sterilis l. a mes-meg t caesp eas l6 biologica nyssana 4 (1-2)  december 2013: 19-33 jotić et al.  the vascular flora of the tepoš… 31 taxa life form area type locality chrysopogon gryllus (l.) trin. a alt h caesp eas l1 dactylis glomerata l. a meg h caesp eas l1 danthonia alpina vest a mes-meg h caesp eamt l6 dasypyrum villosum r. br. ex fresen. a mes-meg t caesp med l1 dichanthium ischaemum (l.)roberty a mes-meg h caesp eas l6 elymus repens (l.) gould a mes-meg g rhiz caesp csm l1 festuca valesiaca schleicher ex gaudin a mes-mac h caesp eas l6 holcus lanatus l. a mes-mac h caesp eas l6 hordeum murinum l. subsp. leporinum (link) arcangeli a mes t caesp med l1 koeleria glaucovirens domin a mes-meg h caesp med l6 koeleria nitidula velen. a mes-meg h caesp med l6 melica ciliata l. a mes-meg h caesp eas l6 melica transsilvanica schur a mes-meg h caesp p l6 melica uniflora retz. a mes-mac h caesp me l2 phleum pratense l. a mes-alt h caesp me l1 poa annua l. a-aut mi-mes t/h caesp csm l1 poa bulbosa l. a mes-meg h caesp eas l1 poa compressa l a mes-mac h caesp csm l1 poa nemoralis l. a mes-meg h caesp hol l2 poa pratensis l. a mes-meg h scap eas l1 setaria italica (l.) beauv. a mes-meg t scap hol l1 setaria pumila (poiret) roem. & schultes a mes-meg t caesp csm l1 setaria viridis (l.) beauv. a mes-mac h caesp eas l1 stipa capillata l. a mac-meg h caesp eas l1 trisetum flavescens (l.) beauv. a mes-meg h caesp eamt l1 potamogetonaceae potamogeton obtusifolius mert. & koch a rad sbm hyd t hol l1 typhaceae typha latifolia l. a alt emer hyd g rhiz csm disscusion taxonomic analysis divisio pteridophyta was represented by single class polypodiopsida with only 3 species. divisio magnoliophyta was represented by 481 species, from which 408 are classified as dicotyledons (magnoliopsida) and 73 as monocotyledons (liliopsida). in taxonomic spectrum of the flora of investigated area the most numerous in genera were asteraceae (34), poaceae (24), lamiaceae (19), fabaceae (18), apiaceae (17), brassicaceae (16). families with the most number of species were asteraceae (55), fabaceae (46), lamiaceae (38) and poaceae (35). the analysis of the particular genera occurrence in flora of the tepoš plateau has shown domination of representatives of genera trifolium (10), following by the genuses acer, campanula, centaurea, euphorbia, geranium, lathyrus, salvia, sedum, vicia and viola with 6 species in each genus. phytogeographical analisys for the phytogeographical analysis, all plant taxa were classified into 25 area types (fig. 3) according to the classification by s t e v a n o v i ć (1992). eurasian area type (eas) was the most dominant in the flora of tepoš plateau with 258 taxa (56%). the second one was mediterraneansubmediterranean area type (med) with 58 species (12 %). there were 9 balcan endemic taxa in the flora of the tepoš plateau. these represents 3,14% of the total number of balkan endemics in serbian flora, which includes 287 taxa in rank of species and subspecies (s t e v a n o v i ć e t a l , 1999). small number of endemics are expected due to uniformity of habitats in the wider area of tepoš plateau. the recorded balkan endemics are: acanthus balcanicus, acer hyrcanum subsp. intermedium, bupleurum flavicans, eryngium palmatum, pastinaca hirsuta, hypericum rumeliacum, thymus biologica nyssana 4 (1-2)  december 2013: 19-33 jotić et al.  the vascular flora of the tepoš… 32 praecox subsp. jankae, linaria rubioides subsp. nissana, viola tricolor subsp. macedonica. me 10% med 12% p 5% hol 5% csm 6% adv 2% eam 3% eas 57% msm 4% ab 0% figure 2. area spectrum of the flora of vučje hill: csm – cosmopolitan, hol – holarctic, ab – arctoalpian + boreal, me – middle-european, eas – euroasian, eamt – euroasian mountain, p – pontic, msm – merridional-submerridional, med – mediterranean-submediterranean, adv adventitious biological spectrum the analysis of plant life forms (fig. 2) showed that flora of investigated area was of hemicryptophyta type, as well as the flora of serbia (diklić, 1984) and balkan penninsula (turill, 1929). the hemicryptophytes were represented with 45%; therophytes followed with 27% and geophytes with 11%. the climatics characteristics of this area caused the high percentage of hemicryptophyta. h 45% g 11% hyd 1% t 27% p 9% ch 7% figure 3. biological spectrum of the flora of tepoš plateau: p – phanerophyta, ch – chamaephyta, h – hemycryptophyta, g – geophyta, t – terophyta, hydhydrophyta conclusion as a results of the floristic investigations of the tepoš plateau between 2009 and 2011, 486 plant taxa belonging to 284 genera and 68 plant families were recorded. the flora of tepoš plateau has similar taxonomic characters as the serbian and european flora. phytegeographical analysis showed that the species of eurasian area type are the most abundant ones. there were 9 endemic taxa in the flora of investigated area. the hemicriptophytic character of flora with significant contribution of therophytes and geophytes, is established by analyzing the presence of plant life forms. although the flora of this region is studied in detail, the expected number of taxons of karst plateau tepoš must be higher, presenting a base for a future studies in this area. acknowledgements. the authors are grateful to ministry of education and science of republic of serbia for partial support (projects number 173030 and 171025). references adamović, l., 1911: flora jugoistočne srbije. jazu. zagreb. diklić, n., 1984: životne forme biqnih vrsta i biološki spektar flore sr srbije. in: sarić, m., ed: vegetacija sr srbije, i: 291-316. sanu, posebna izdanja. beograd. greuter, w., burdet, h.m., long, g., eds., 19841989: med-checklist, 1,3,4. gèneve. josifović, m., ed.,1970-1976: flora sr srbije, i-ix. sanu. beograd. meussel, h., jäger, e., weinert, e., 1965: vergleinchende chorologie der zentraleuropaischen flora. veb. gustav fischer verlag, 1. jena. meussel, h., jäger, e., raischert, s., weinert, e., 1978: vergleinchende chorologie der zentraleuropaischen flora. veb. gustav fischer verlag, 2. jena. meussel, h., jäger, e., 1992: vergleinchende chorologie der zentraleuropaischen flora. veb. gustav fischer verlag, 3. jena. mueller – dombois, d., ellenberg, h. (1974.): aims and methods of vegetation ecology. john wiley and sons. new york. pančić, j., 1884.: dodatak flori kneževine srbije. kraljevska srpska državna štamparija, beograd. stevanović, v. ed., 1999: crvena knjiga flore srbije. 1, iščezli i krajnje ugroženi taksoni. ministarstvo za životnu sredinu republike srbije, zavod za zaštitu prirode srbije. beograd. stevanović, v., 1992: floristička podela teritorije srbije sa pregledom viših horiona i odgovarajućih flornih elemenata. in sarić, m. biologica nyssana 4 (1-2)  december 2013: 19-33 jotić et al.  the vascular flora of the tepoš… 33 (ed.): flora srbije, i. (drugo izdanje). sanu. beograd. 49 – 65. stevanović, v., 1992a: klasifikacija životnih formi flore srbije. in sarić, m. (ed.): flora srbije i. (drugo izdanje). sanu. beograd. 39 – 46. turill, w.b., 1929: the plant life of balkan peninsula. a phytogeographical study. oxford. tutin, t.g., heywood, v.h., burges, n.a., moore, d.m., valentine, d.h., walters, s.m., webb, d.a., eds., 1964-1980: flora europaea, i-v. cambrige university press. london. велчев в., ед., 1982-1989: флора на народна република българия, т. i-x, българската академия на науките, софия. vidanović, g., 1960: vidlič – zabrđe, prilog poznavanju privrednog tipa, ekonomsko – geografska studija, srpska akademija nauka, posebna izdanja geografskog instituta, knjiga 15, beograd. microsoft word bn020203 milosevic et al biologica nyssana 2 (2) december 2011: 123-128 milošević dj. et al. checklist of the family chironomidae… 123 original article ! checklist of the family chironomidae (diptera) of southern morava river basin, serbia djuradj milošević1, vladica simić2, ivan todosijević1, milica stojković1 1university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia 2university of kragujevac, faculty of sciences and mathematics, institute of biology and ecology, radoja domanovića 14, 34000 kragujevac, serbia * e-mail: djuradj@pmf.ni.ac.rs abstract: milošević, dj., simić, v., todosijević, i., stojković, m.: checklist of the family chironomidae (diptera) of southern morava river basin, serbia. biologica nyssana, 2 (2), december 2011: 123-128. this paper presents the first checklist of the family chironomidae (diptera) in serbia, based on literature citations in two periods (1981/82 and 2010/11) and the unpublished data as well as material examined by the authors. our survey ranges over the central, eastern, southern and southeastern parts of serbia (southern morava river basin). we have listed 110 taxa distributed in following five subfamilies: chironominae (39), diamesinae (3), orthocladiinae (48), prodiamesinae (2) and tanypodynae (16). key words: checklist, chironomidae, serbia, southern morava river introduction ! chironomidae represent the group of flies in the suborder nematocera (diptera). this group is usually the most abundant macroinvertebrate group in number of species and individuals, encountered in the majority of fresh-water aquatic habitats. the fauna of family chironomidae currently consists of 11 described subfamilies and nominally, 22 tribes with 339 genera counting 4147 (sub)species. (f e r r i n g t o n , 2008). the family chironomidae plays the most important role in detritus processing in freshwater ecosystems, recycling natural and introduced organic matter (h i r a b a y a s h i & w o t t o n , 1998; j o n e s & g r e y , 2004). also, they present an important part of the food chain (a r m i t a g e et al., 1995). considering the characteristics mentioned above, chironomidae presents a proper candidate for the water quality assessment. regardless their importance in aquatic ecosystems, little information on qualitative composition of the chironomid fauna in serbia is available at the moment. this is particularly true for lotic systems in central, eastern, southern and southeastern parts of serbia, where research on this topic has not been conducted for 29 years (1981 2010). the first investigation was those of j a n k o v i ć (1985), who sampled across the entire drainage basin of the southern morava river, serbia, in 1981. recently, m i l o š e v i ć et al. (2012) repeated the survey of j a n k o v i ć (1985) over the same range, with the aim of examining the changes in the southern morava river basin. according to the investigations of the authors mentioned above, as well as unpublished data examined by author from the southern morava range, we presented the first chironomid check list of central, eastern, southern and southeastern parts of serbia. the checklist should contribute to the research on this unexplored group of insects in 2 (2) • december 2011: 123-128 biologica nyssana 2 (2) december 2011: 123-128 milošević dj. et al. checklist of the family chironomidae… 124 serbia which have often been proved as useful in the water quality assessment. materials and methods the southern morava river, also known as the binačka morava, has its source in the skopska crna gora mountain, macedonia, and flows roughly towards the north. at km 49 it joins up with the preševska moravica and at km 295 it flows into the morava, which itself eventually flows into the black sea. the southern morava river has a catchment area of 15,469 km2 of which 14,372 km2 (92.91%) are situated in serbia and 1,097 km2 (7.09%) in bulgaria, through this river’s right-hand tributary, the river nišava. the southern morava has 157 tributaries most of which dry out during summer. larger, permanent left-hand tributaries are the jablanica, veternica, toplica and pusta reka rivers. right-hand tributaries are the vlasina, nišava (the longest) and sokobanjska moravica rivers (g a v r i l o v i c & d u k i c , 2002). we studied the chironomid faunal composition once in the each season of 2010 at 28 localities, chosen across the southern morava range to include different stream orders and a variety of habitats (fig. 1). specimens were identified up to the genusor species-level with established keys (s m i t h & d i s t l e r , 1981, w i e d e r h o l m , 1983, p i l l o t , 1984a, 1984b, 2009, v a l l e n d u u k & p i l l o t , 2007). the taxonomy used here is that of s p i e s & s æ t h e r (2004), with adjustments following h. m. p i l l o t (pers. comm.). our survey ranges over the central, eastern, southern and southeastern parts of serbia (m a r k o v i c , 1970). figure 1. sampling localities over the southern morava river. squares indicate localities studied in 1981 and circles indicate localities studied in 2010 biologica nyssana 2 (2) december 2011: 123-128 milošević dj. et al. checklist of the family chironomidae… 125 results we have listed 110 taxa distributed into the following five subfamilies: chironominae (39), diamesinae (3), orthocladiinae (48), prodiamesinae (2) and tanypodynae (16). the review of taxa subfamily chironominae 1. beckidia zabolotzkyi (goetghebuer 1938) site:bob, 2. chernovskiia orbicus (townes 1945) site:bim, sta 3. chironomus sp. site:grd, mez, sta, bob, vet, 2 ,3, 6, 7, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 25, 26, 27 4. cladotanytarsus sp. site: bim, mez, bre, jab, top, 1, 3, 4, 5, 6, 9, 13, 15, 16, 17, 18, 19, 20, 21, 26 5. cryptochironomus sp. site: bim, grd, mez, bre, jab, 1, 2, 3, 5, 6, 7, 9, 10, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 25, 26, 27 6. demicryptochironomus vulneratus (zetterstedt 1838) site: bim, bob, 2, 5, 6, 7, 8, 9, 12, 13, 21 7. dicrotendipes nervosus (staeger 1839) site: 10, 15, 16, 17, 19, 20, 23, 26 8. dicrotendipes notatus (meigen 1818) site: 23 9. endochironomus albipennis (meigen 1830) site: 14, 26 10. endochironomus dispar (meigen, 1830) site: 12 11. glyptotendipes cauliginellus (kieffer 1913) site: vlh, 13 12. harnischia fuscimanus kieffer 1921 site: bob, 1, 10, 13, 18, 19, 21, 27 13. microchironomus tener (kieffer, 1918) site: 19 14. micropsectra bidentata goetghebuer 1921 site: 4, 5, 6, 11, 12, 14, 16, 22, 23, 28 15. micropsectra sp. site: put, 4, 5, 6, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18, 22, 23, 25, 27 16. microtendipes pedellus (de geer 1776) site:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 13, 14, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 27 17. parachironomus frequens (johannsen 1905) site: 4 18. paracladopelma laminatum (kieffer 1921) site: 1, 2, 3, 4, 5, 6, 8, 9, 10, 13, 14, 19, 20, 21, 22, 23, 25, 26, 27, 28 19. paracladopelma nigritulum (goetghebuer 1942) site: mpv, 10, 12, 14, 17, 18 20. paralauterborniella nigrohalteralis (malloch 1915) site: 1, 2, 16, 17 21. paratanytarsus austriacus (kieffer 1924) site: 19 22. paratanytarsus dissimilis (johannsen 1905) site: bre, put, vet, 1, 10, 13, 16, 17, 18, 20, 25, 26, 27 23. paratendipes albimanus (meigen 1818) site: grd, 1, 2, 4, 5, 6, 7, 8, 9, 11, 13, 14, 16, 17, 18, 19, 20, 21, 22, 23, 25, 26, 27, 28 24. phaenopsectra sp. site: grd, 10, 11, 12, 13, 14, 16, 17, 19, 20, 21, 25, 26, 27 25. polypedilum albicorne (meigen 1838) site: 1, 4, 7, 12, 13, 14, 17, 18, 19, 21, 22, 23, 24, 25, 27, 28 26. polypedilum convictum (walker 1856) site: mez, bre, sta, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 27. polypedilum cultellatum goetghebuer 1931 site: 13, 25 28. polypedilum laetum (meigen 1818) site: miv, 1, 2, 3, 4, 5, 7, 8, 9, 10, 12, 13, 14, 16, 19, 20, 21, 25, 27, 28 29. polypedilum pedestre (meigen 1830) site: grd, mez, bre, 3, 7, 11, 12, 13, 17, 18, 19, 20, 23, 25, 27, 28 30. polypedilum scalaenum (schrank 1803) site: bim, miv, mpv, grd, mez, bre, sta, bob, 1, 2, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 biologica nyssana 2 (2) december 2011: 123-128 milošević dj. et al. checklist of the family chironomidae… 126 31. polypedilum uncinatum goetghebuer 1921 site: 5, 12 32. pseudochironomus prasinatus (staeger 1839) site: sta 33. rheotanytarsus sp. site: jab, sta, 1, 2, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 16, 17, 18, 19, 20, 21, 22, 23, 25, 26, 27 34. saetheria reissi jackson 1977 site: 2, 6, 10, 13, 17, 18, 19, 20, 21 35. saetheria sp. site: mpv, bre, jab, 1 36. stempellinella brevis (edwards 1929) site: 11, 12, 28 37. stictochironomus maculipennis (meigen 1818) site: 5, 6, 10, 11, 12, 26, 28 38. stictochironomus pictulus (meigen 1830) site: 1, 4, 7, 14, 16, 17, 18, 20, 21, 22, 25 39. tanytarsus sp. site: grd, mez, bre, jab, sta, bob, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 subfamily diamesinae 40. diamesa sp. site: miv, 1, 2, 4, 8, 9, 13, 24, 28 41. potthastia gaedii (meigen 1838) site: 1, 2, 3, 4, 5, 6, 7, 14, 20, 22, 23, 25, 27 42. potthastia longimanus kieffer 1922 site: 1, 3, 4, 5, 6, 7, 9, 12, 13, 14, 16, 17, 18, 19, 20, 21, 22, 23, 25, 26, 27, 28 subfamily orthocladiinae 43. brillia flavifrons (johannsen 1905) site: 9, 10, 13, 17, 18, 21, 23, 27 44. brillia bifida (kieffer 1909) site: 1, 2, 4, 5, 6, 11, 12, 14, 16, 19, 24, 26, 28 45. bryophaenocladius subvernalis (edwards 1929) site: grd 46. cardiocladius fuscus kieffer 1924 site: 3, 8, 13, 18, 19, 21, 27 47. corynoneura celeripes winnertz 1852 site: grd, mez 48. corynoneura lobata edwards 1924 site: 2, 4, 5, 6, 7, 12, 14, 28 49. corynoneura scutellata winnertz 1846 site: grd, mez, 27 50. cricotopus triannulatus agg. sensu moller pillot 1984 site: miv, vlh, grd, mez, bre, sta, vet, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16, 17, 18, 19, 21, 22, 23, 25, 26, 27, 28 51. cricotopus bicinctus (meigen 1818) site:vlh, grd, mez, bre, jab, sta, bob, vet, top, 1, 5, 6, 8, 9, 10, 13, 14, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 52. cricotopus gr. sylvestris sensu hirvenoja 1973 site: bre, 10 53. cricotopus tremulus (linnaeus 1758) site: vlh, bre, jab, vet, 3, 4, 13, 24, 25 54. cricotopus trifascia edwards 1929 site: mez, jab, 3, 8, 13, 14, 17, 20, 21, 24, 25, 27 55. epoicocladius ephemerae (kieffer 1924) site: 1, 2, 3, 4, 5, 6, 7, 9, 11, 12, 14, 16, 28 56. eukiefferiella brevicalcar (kieffer 1911) site: mor, 4, 5, 6, 8, 12, 20, 28 57. eukiefferiella claripennis (lundbeck 1898) site: bre, 22 58. eukiefferiella clypeata (kieffer 1923) site: 4, 5, 8, 9, 10, 12, 14, 18, 19, 26, 27, 28 59. eukiefferiella gracei (edwards 1929) site: miv, 1, 2, 3, 6, 7, 8, 10, 11, 13, 14, 17, 18, 19, 25, 27 60. eukiefferiella ilkleyensis (edwards 1929) site: 1, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 17, 18, 19, 20, 21, 23, 24, 25, 26, 27 61. eukiefferiella lobifera goetghebuer 1934 site: vlh, bre, 2, 13, 17, 20, 21, 23, 25, 26, 27 62. eukiefferiella minor (edwards 1929) site: 4 63. eukiefferiella sp. site: miv 64. heleniella ornaticollis (edwards 1929) site: 3, 4, 5, 6, 12, 28 biologica nyssana 2 (2) december 2011: 123-128 milošević dj. et al. checklist of the family chironomidae… 127 65. hydrobaenus sp. site: mor, miv, bre, jab 66. limnophyes sp. site: grd, 2, 4, 5, 8, 13, 14, 16, 19, 27 67. nanocladius bicolor (zetterstedt 1838) site: 6, 7, 16, 26 68. nanocladius rectinervis (kieffer 1911) site: mez, bre, jab, top, 4, 5, 8, 10, 13, 14, 17, 18, 20, 24, 25, 26, 28 69. orthocladius (euorthocladius) sp. site: miv, vlh, mez, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 70. orthocladius (orthocladius) sp. site: miv, bre, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 71. orthocladius frigidus (zetterstedt 1838) site: 2, 3, 4, 5, 6, 8, 11, 12, 13, 14 72. orthocladius rivulorum kieffer 1909 site: 2, 3, 4, 5, 6, 7, 8, 13, 14, 16 73. paracladius conversus (walker 1856) site: 1, 2, 3, 6, 9, 16, 18, 19, 21, 25, 27 74. paracricotopus niger (kieffer 1913) site: 4, 22 75. parakiefferiella gracillima (kieffer 1922) site: mor 76. parametriocnemus stylatus (spaerck 1923) site: 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, 26, 27, 28 77. paratrichocladius rufiventris (meigen 1830) site: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 78. paratrissocladius excerptus (walker 1856) site: 2, 3, 6, 7, 11, 12, 14, 16, 23, 28 79. psectrocladius calcaratus (edwards 1929) site: mez, 1, 6 80. pseudosmittia danconai (marcuzzi 1947) site: vlh, top 81. rheocricotopus chalybeatus (edwards 1929) site: 1, 8, 10, 13, 15, 16, 17, 18, 19, 20, 21, 23, 25, 26, 27 82. rheocricotopus effusus (walker 1856) site: jab, 2, 4, 5, 6, 12, 23, 28 83. rheocricotopus fuscipes (kieffer 1909) site: 3, 4, 5, 6, 7, 9, 11, 12, 14, 22, 24, 28 84. rheosmittia spinicornis (brundin 1956) site: 5, 11, 28 85. smittia sp. site: bre, 20 86. synorthocladius semivirens (kieffer 1909) site: mor, 1, 2, 4, 5, 6, 7, 8, 9, 12, 13, 14, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 27 87. thienemanniella clavicornis (kieffer 1911) site: 2, 3, 4, 5, 6, 9, 11, 12, 14, 16, 20, 21, 24, 25, 27, 28 88. thienemanniella majuscula (edwards 1924) site: jab, 1, 3, 8, 10, 13, 17, 18, 19, 21, 22, 23, 26 89. tvetenia bavarica (goetghebuer 1934) site: 4, 5, 6, 7, 11, 12, 14, 22, 28 90. tvetenia calvescens (edwards 1929) site: 1, 2, 3, 8, 9, 10, 13, 16, 17, 18, 19, 20, 21, 23, 24, 25, 27 91. tvetenia discoloripes (goetghebuer & thienemann 1936) site: 2, 3, 4, 5, 6, 10, 11, 12, 17, 19, 23, 25, 28 subfamily prodiamesinae 92. odontomesa fulva (kieffer 1919) site: 2, 3, 4, 6, 7, 11, 12 93. prodiamesa olivacea (meigen 1818) site: grd, 1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15 ,16 ,17 ,18 ,19 , 20, 21, 22, 23, 24, 25, 27, 28 subfamily tanypodinae 94. ablabesmyia longistyla fittkau 1962 site: mez, 10, 16, 20 95. ablabesmyia phatta (egger 1864) site: 18 96. anatopynia plumipes (fries 1823) site: 4 97. apsectrotanypus trifascipennis (zetterstedt 1838) site: 4, 6, 11, 14, 23 biologica nyssana 2 (2) december 2011: 123-128 milošević dj. et al. checklist of the family chironomidae… 128 98. arctopelopia barbitarsis (zetterstedt 1850) site: 5, 12 99. conchapelopia melanops (meigen 1818) site: mez, jab, sta, put, 1, 2, 3, 5, 6, 7, 9, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21, 24, 25, 26, 27 100. krenopelopia sp. site: 1, 4 101. larsia curticalcar (kieffer 1918) site: mez, bre, jab 102. macropelopia adaucta kieffer 1916 site: 6, 22, 23 103. macropelopia nebulosa (meigen 1804) site: 2, 3, 4, 5, 6, 7, 8, 10, 12, 14, 15, 18, 23, 27, 28 104. nilotanypus dubius (meigen 1804) site: 1, 2, 4, 5, 6, 7, 8, 9, 14, 16, 22, 24, 25, 28 105. procladius sp. site: grd, 2, 3, 4, 6, 7, 10, 12, 13, 14, 16, 17, 18, 19, 20, 21, 22, 23, 25, 26, 27 106. rheopelopia sp. site: 1, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 107. tanypus punctipennis meigen 1818 site: grd, sta, bob, 19, 27 108. thienemannimyia sp. site: mez, jab, sta, put, 4, 5, 6, 12, 22, 28 109. trissopelopia flavida kieffer 1923 site: mez, sta 110. zavrelimyia sp. site: 7 acknowledgements. this work was supported by grant # 043002 ‘biosensing technologies and global system for long-term research and integrated management of ecosystems’ by the ministry of science of the republic of serbia. we want to thank h. m. pillot (tilburg, the netherlands) for the great support and useful advice about this work. references armitage, p. d., cranston, p., & pinder, l., 1995: the chironomidae: biology and ecology of nonbiting midges. chapman & hall, london. ferrington, l. c. 2008: global diversity of nonbiting midges (chironomidae; insecta-diptera) in freshwater. hydrobiologia 595: 447-455 gavrilovic, l. & dukic, d. 2002: reke srbije. zavod za udžbenike i nastavna sredstva, beograd. hirabayashi, k. & wotton, r. s. 1998: organic matter processing by chironomid larvae (diptera: chironomidae). hydrobiologia 382: 151-159. jankovic, m. 1985: the investigation of the chironomid fauna in the south morava and its components rivers. proceedings on the fauna of sr serbia 3: 71-110. jones, r. i. & grey, j. 2004: stable isotope analysis of chironomid larvae from some finnish forest lakes indicates dietary contribution from biogenic methane. boreal environment research 9: 17-24. markovic, j., 1970: geografske oblasti socijalisticke federativne republike jugoslavije. – zavod za izdavanje udžbenika i nastavna sredstva srbije, beograd. milošević, dj., simić, v., stojković, m., živić, i., 2012: chironomid faunal composition represented by taxonomic distinctness index reveals environmental change in a lotic system over three decades. hydrobiologia 683: 62-82 pillot, h. k. m. m. 1984a: de larven der nederlandse chironomiae (diptera). 1a: inleiding, tanypodinae en chironomini. st. e.i.s. nederland, leiden. pillot, h. k. m. m. 1984b: de larven der nederlandse chironomiae (diptera). 1b: orthocladiinae sensu lato. st.e.i.s nederland, leiden. pillot, h. k. m. m. 2009: chironomidae larvae of the netherlands and adjacent lowlands: biology and ecology of the chironomini. knnv publishing, zeist. smith, d. l. & distler, d. a. 1981: recovery of a benthic macroinvertebrate community following a toxic chemical discharge in a sandy plains stream. the southwestern naturalist 25: 547-551. spies, m. & sæther, o. a. 2004: notes and recommendations on taxonomy and nomenclature of chironomidae (diptera). zootaxa 752: 1-90. vallenduuk, h. j. & pillot, h. k. m. m. 2007: chironomidae larvae of the netherlands and adjacent lowlands: general ecology and tanypodinae. knnv publishing, zeist. wiederholm, t. 1983: chironomidae of the holarctic region: keys and diagnoses. publishing house of the swedish research councils, stockholm. stojković et al. 2023, biologica nyssana 14(2) 14 (2) december 2023: 97-106 doi: 10.5281/zenodo.10209805 morphological variability and differentiation of selected achillea species (sections achillea sensu lato and anthemoideae) from serbia original article jelena p. stojković department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, 18000 niš, serbia jelenapstojanovic93@gmail.com (corresponding author) zorica s. mitić department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, 18000 niš, serbia bojan k. zlatković department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, 18000 niš, serbia received: september 26, 2023 revised: october 22, 2023 accepted: october 27, 2023 abstract: in the present study, morphological variability and differentiation of 13 achillea species, including 11 from the achillea section achillea sensu lato (a. chrysocoma, a. clypeolata, a. coarctata, a. collina, a. crithmifolia, a. distans, a. grandifolia, a. millefolium, a. nobilis, a. pseudopectinata and a. vandasii) and two species from the achillea section anthemoideae (a. ageratifolia and a. lingulata) from serbia were analyzed. morphometric analysis included a total of 9 quantitative characteristics from the vegetative and reproductive plant parts. multivariate statistical analyses reflected almost clear differentiation between species from two studied sections whereby ligulate floret length and stem leaf segment length had the greatest influence for their distinction. thus, the section achillea sensu lato was characterized by significantly shorter ligulate florets and longer segments of stem leaf compared to the section anthemoideae. furthermore, a. grandifolia featured the longest segments of stem leaf and showed considerable separation from all other species within the section achillea sensu lato. it could be concluded that the selected set of morphological characteristics in the present study mainly positioned the studied species in accordance with the current infrageneric classification of achillea. key words: achillea, morphometry, multivariate statistical analysis, balkan peninsula apstrakt: morfološka varijabilnost i diferencijacija odabranih vrsta roda achillea (sekcija achillea sensu lato i anthemoideae) iz srbije u ovom istraživanju analizirana je morfološka varijabilnost i diferencijacija 13 vrsta roda achillea, uključujući 11 iz sekcije achillea sensu lato (a. chrysocoma, a. clypeolata, a. coarctata, a. collina, a. crithmifolia, a. distans, a. grandifolia, a. millefolium, a. nobilis, a. pseudopectinata i a. vandasii) i dve vrste iz sekcije anthemoideae (a. ageratifolia i a. lingulata) iz srbije. morfometrijskom analizom obuhvaćeno je ukupno 9 kvantitativnih karaktera iz vegetativnih i reproduktivnih delova biljaka. multivarijantne statističke analize pokazale su gotovo jasnu diferencijaciju izmedju vrsta iz dve istraživane sekcije, pri čemu su dužina jezičastog cveta i dužina režnja lista stabla imale najveći uticaj na njihovo razlikovanje. tako se sekcija achillea sensu lato odlikovala znatno kraćim jezičastim cvetovima i dužim režnjevima lista stabla u odnosu na sekciju anthemoideae. osim toga, a. grandifolia je imala najduže režnjeve lista stabla i pokazala značajno odvajanje od svih ostalih vrsta u okviru pomenute sekcije. može se zaključiti da je odabrani skup morfoloških karaktera u ovoj studiji pozicionirao istraživane vrste uglavnom u skladu sa aktuelnom infrageneričkom klasifikacijom roda achillea. ključne reči: achillea, morfometrija, multivarijantne statističke analize, balkansko poluostrvo introduction genus achillea l. (yarrows) is one of the most polymorphic and taxonomically complicated genera in the family asteraceae, including more than 100 species, distributed in europe, temperate climate asia and few species in north america (saukel et al., 2003; ehrendorfer & guo, 2006; guo et al., 2006). in the flora of serbia, 27 achillea species have been recognized (euro+med, 2006). © 2023 stojković et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 97 the genus is characterized by the ability to adapt in different ecological amplitudes and ranges in extreme environmental conditions, such as deserts to water-logged habitats and from sea coastal areas to the high mountains (ehrendorfer & guo, 2006). consequently, several widespread achillea species show extremely high morphological variability and complexity. in addition, the presence of spontaneous hybrids, alloand autopolyploids, aneuploids and phenocopies results in a great cytological, morphological and phytochemical variability both at interand at intraspecific level. therefore, taxonomic evaluation, identification of species and classification has been a difficult task for decades (inotai et al., 2016). taxonomical grouping within the genus has been rearranged several times in the past and intensively researched even today (kidlovits & nemeth, 2012). based on previous morpho-anatomical and ecogeographical analyses, many classification schemes of the genus achillea have been proposed (afanasyev & bochantsev, 1961; huber-morath, 1975). however, the most recent multidisciplinary studies recognized five sections within this genus: achillea sensu lato, anthemoideae (dc.) heimerl, babounya (dc.) o. hoffm., ptarmica (mill.) w. d. j. koch and otanthus (hoffmanns. & link) ehrend. & y. p. guo (saukel et al., 2003; guo et al., 2004; ehrendorfer & guo, 2005). certain morphological characteristics, including characteristics of leaves, florets and pollen grains may be useful for morphological characterization and classification of achillea taxa (saukel & länger, 1992; valant-vetschera & kastner, 2000; rauchensteiner et al., 2002; nedelcheva, 2008; azani et al., 2009; akyalcin et al., 2011). the main aims of the present study were: 1) determination of highly variable morphological characteristics as well as those of low variability in two sections, achillea sensu lato and anthemoideae, and 2) assessment of the degree of differentiation of the studied species based on selected set of morphological characteristics. materials and methods plant material plant material (whole plants) of 13 achillea species, including 11 from the section achillea sensu lato (a. chrysocoma friv., a. clypeolata sm., a. coarctata poir., a. collina (becker ex rchb. f.) heimerl, a. crithmifolia waldst. & kit., a. distans waldst. & kit. ex willd., a. grandifolia friv., a. millefolium l., a. nobilis l., a. pseudopectinata janka and a. vandasii velen.) and two species from the section anthemoideae (a. ageratifolia (sm.) benth. & hook. f. and a. lingulata waldst. & kit.) were collected in their natural habitats in serbia. additionally, plant material of one population of a. pseudopectinata was collected in north macedonia. the voucher specimens of each population were deposited in the „herbarium moesiacum niš” (hmn) of the department of biology and ecology, faculty of sciences and mathematics, university of niš. the map of the studied area, with locations of the selected populations, is shown in fig. 1. furthermore, the original data on position, habitat and voucher information for each population are listed in tab. 1. plant material was identified by dr. b. k. zlatković, dr. z. s. mitić and m.sc. j. p. stojković. 98 biologica nyssana ● 14 (2) december 2023: 97-106 stojković et al. ● morphological variability and differentiation of selected achillea species (sections achillea sensu lato and anthemoideae) from serbia fig. 1. geographical position of studied populations. for the description of the location and habitat conditions of the populations, cf. tab. 1 morphometric analysis a total number of 384 individuals from 26 populations and 13 species of achillea, from two different sections, were compared at the level of 9 quantitative morphological characteristics. quantitative traits such as rosette leaf segment length (rlsl) / rosette leaf tooth length (rltl), stem leaf segment length (slsl) / stem leaf tooth length (sltl), ligulate floret length (lfl), tubular floret length (tfl) and involucral bract length (ibl) of each examined individual were mounted as permanent microscope slides and photographed after preparation with a leica mz-16a binocular magnifier (leica microsystems©, wetzlar, germany). as a background, graph paper was used in the photography process. each characteristic was measured with three consecutive measurement repetitions. all measurements of the mentioned properties were performed using digimizer image analysis software (medcalcsoftware©, belgium), biologica nyssana ● 14 (2) december 2023: 97-106 stojković et al. ● morphological variability and differentiation of selected achillea species (sections achillea sensu lato and anthemoideae) from serbia 99 ta bl e 1. p os iti on a nd h ab ita t d es cr ip tio n of s tu di ed p op ul at io ns o f a ch ill ea s pe ci es se ct io n sp ec ie s l oc al ity l at itu de (n ) l on gi tu de (e ) a lti tu de (m a. s.l .) su bs tr at um n um be r of in di vi du al s an al yz ed d at e of co lle ct io n n um be r of va uc he r (h m n ) ac hi lle a se ns u la to a. c hr ys oc om a se rb ia : m t. b es na k ob ila 42 °3 1' 05 " 22 °1 3' 05 " 19 23 si lic at e 15 ju ly 2 02 0 14 30 0 a. c ly pe ol at a se rb ia : s ić ev ač ka go rg e 43 °1 8' 36 " 22 °1 0' 49 " 27 6 li m es to ne 15 m ay 2 01 9 13 95 7 a. c ly pe ol at a se rb ia : v lk ov ija 43 °0 5' 02 " 22 °5 4' 24 " 10 20 li m es to ne 15 ju ne 2 02 1 14 31 0 a. c oa rc ta ta se rb ia : p či nj a riv er va lle y, b ud ov ija 42 °2 0' 23 " 21 °5 3' 26 " 44 0 si lic at e 15 ju ne 2 01 8 13 68 1 a. c oa rc ta ta se rb ia : s ić ev ač ka go rg e 43 °3 3' 42 " 22 °0 6' 95 " 36 5 li m es to ne 15 ju ne 2 01 8 13 68 5 a. c oa rc ta ta se rb ia : t em sk a 43 °1 7' 06 " 22 °3 5' 06 " 44 0 si lic at e 15 ju ly 2 02 0 14 30 3 a. c ol lin a se rb ia : p či nj a riv er va lle y, b ud ov ija 42 °2 0' 23 " 21 °5 3' 26 " 68 0 si lic at e 15 ju ne 2 01 9 13 95 2 a. c ri th m ifo lia se rb ia : p či nj a riv er va lle y, b ud ov ija 42 °2 0' 23 " 21 °5 3' 26 " 44 0 si lic at e 22 ju ne 2 01 8 13 68 4 a. c ri th m ifo lia se rb ia : k un ov ic a 43 °1 7' 30 " 22 °0 6' 34 " 46 7 li m es to ne 15 ju ne 2 01 8 13 68 0 a. d is ta ns se rb ia : m t. st ar a pl an in a, b ab in z ub 43 °2 3' 04 " 22 °3 5' 35 " 16 00 si lic at e 15 ju ly 2 02 0 14 30 5 a. d is ta ns se rb ia : k op ao ni k 43 °1 8' 51 " 20 °5 1' 01 " 13 70 si lic at e 15 ju ly 2 02 0 14 30 6 a. g ra nd ifo lia se rb ia : j er m a go rg e 42 °5 8' 18 " 22 °3 7' 23 " 54 8 li m es to ne 15 ju ne 2 02 0 14 30 2 a. g ra nd ifo lia se rb ia : t em sk a 43 °1 6' 56 " 22 °3 5' 04 " 44 0 si lic at e 5 ju ly 2 02 0 14 61 4 a. m ill ef ol iu m se rb ia : s ić ev ač ka go rg e 43 °2 0' 04 " 22 °0 4' 09 " 24 0 li m es to ne 15 ju ne 2 01 8 13 68 2 a. m ill ef ol iu m se rb ia : n iš 43 °1 8' 19 " 21 °5 3' 50 " 26 0 n eo ge ne se di m en ts 15 ju ne 2 01 9 13 95 5 a. m ill ef ol iu m se rb ia : l al in ač ka sa lt m ar sh 43 °2 0' 42 " 21 °4 4' 45 " 20 0 n eo ge ne se di m en ts 15 m ay 2 01 9 14 40 0 a. m ill ef ol iu m se rb ia : p či nj a riv er va lle y, b rn ja re 42 °2 3' 30 " 21 °5 5′ 01 " 50 7 n eo ge ne se di m en ts 15 ju ne 2 01 9 13 96 1 100 biologica nyssana ● 14 (2) december 2023: 97-106 stojković et al. ● morphological variability and differentiation of selected achillea species (sections achillea sensu lato and anthemoideae) from serbia ac hi lle a se ns u la to a. n ob ili s se rb ia : s ić ev ač ka go rg e 43 °2 0' 04 " 22 °0 4' 09 " 36 5 li m es to ne 15 ju ne 2 01 8 13 68 3 a. n ob ili s se rb ia : l al in ač ka sa lt m ar sh 43 °2 0' 42 " 21 °4 4' 45 " 20 0 n eo ge ne se di m en ts 15 ju ne 2 02 0 14 29 8 a. p se ud op ec tin at a n or th m ac ed on ia : k aj m ak ča la n 40 °5 9' 12 " 21 °4 0' 06 " 71 8 si lic at e 12 ju ne 2 02 0 14 30 9 a. p se ud op ec tin at a se rb ia : o bl ač in a 43 °1 8' 35 " 21 °4 0' 15 " 33 3 si lic at e 15 ju ne 2 02 0 14 30 1 a. v an da si i se rb ia : v ra nd ol 43 °1 6' 41 " 22 °1 5' 31 " 42 6 c ar bo na te 15 ju ne 2 02 0 14 30 4 an th em oi de ae a. a ge ra tif ol ia se rb ia : b or , m t. v el ik i k rš 44 °1 0' 47 " 22 °0 4' 30 " 99 5 c ar bo na te 15 ju ne 2 01 9 13 95 4 a. a ge ra tif ol ia se rb ia : j el aš ni čk a go rg e 43 °1 6' 37 " 22 °0 4' 09 " 36 0 li m es to ne 15 m ay 2 02 1 14 54 2 a. li ng ul at a se rb ia : m t. st ar a pl an in a, b ab in z ub 43 °2 2' 06 " 22 °3 5' 42 " 16 00 si lic at e 15 ju ne 2 01 9 13 95 3 a. li ng ul at a se rb ia : m t. b es na k ob ila 42 °3 1' 45 " 22 °1 3' 40 " 18 50 si lic at e 15 ju ly 2 02 0 14 29 9 on previously taken photographs. characteristics of large plant parts, such as rosette leaf length (rll), and stem leaf length (sll), were determined using a ruler. in contrast, rounded structures, capitulum length (cl) and capitulum width (cw), were measured with a high-precision digital meter (mahr federal 410710716u, esslingen, germany). statistical analysis the statistical analysis of the obtained data was performed using the statistica 8 software (statsoft, inc., tulsa, ok, usa). statistical data processing included descriptive statistics and multivariate statistical analyses: canonical discriminant analysis (cda), and agglomerative hierarchical clustering (ahc). unweighted pair group average (upgma) was used as a criterion for the clusters development, and euclidean distances as diversity assessment criteria in ahc analysis. results and discussion variability of morphological characteristics of the studied achillea species results of descriptive statistics for 9 quantitative morphological characteristics of 13 achillea species were presented in tab. 2. according to the obtained results, most of the studied characteristics were within a moderate degree of variability (10<cv%<50). nevertheless, in almost all species, some of the characteristics of the reproductive plant parts stood out as low variable (cv%<10): involucral bract length (a. chrysocoma, a. clypeolata, a. coarctata, a. collina, a. distans, a. grandifolia, a. millefolium, a. nobilis, a. pseudopectinata and a. vandasii), tubular floret length (a. ageratifolia, a. chrysocoma, a. collina, a. crithmifolia, a. distans, a. grandifolia, a. millefolium and a. vandasii), ligulate floret length (a. ageratifolia, a. collina, a. distans, a. grandifolia and a. pseudopectinata), capitulum length (a. collina, a. distans and a. pseudopectnata) and capitulum width (a. collina and a. grandifolia). there is a relatively limited amount of literature data which is related to the degree of variability of morphological characteristics of achillea species. morphological characteristics such as the height of plant, length, width and shape of leaf, shape and color of involucrum, as well as length and color of flowers were mainly commented in a general sense (gajić, 1975; saukel et al., 2003; guo et al., 2004, 2006). in agreement with our data, mottaghi et al. (2015) reported that involucrum length and width and ligulate florets length were the most stable characteristics, while leaf width, capitulum number and seed weight were the most variable characteristics of six iranian achillea species. biologica nyssana ● 14 (2) december 2023: 97-106 stojković et al. ● morphological variability and differentiation of selected achillea species (sections achillea sensu lato and anthemoideae) from serbia 101 sp ec ie s d es cr ip tiv e st at is tic s c ha ra ct er is tic s r os et te le af le ng th (r l l ) r os et te le af se gm en t l en gt h/ ro se tt e le af to ot h le ng th (r l sl /r l t l ) st em le af le ng th (s l l ) st em le af se gm en t l en gt h/ st em le af to ot h le ng th (s l sl / sl t l ) c ap itu lu m le ng th (c l ) c ap itu lu m w id th (c w ) l ig ul at e flo re t le ng th (l fl ) t ub ul ar flo re t le ng th (t fl ) in vo lu cr al br ac t le ng th (i b l ) a . c hr ys oc om a m 1 47 .7 3. 7 21 .8 2. 8 4. 2 3. 1 5. 7 4. 2 4. 9 m in 2 37 .2 2. 5 16 .0 2. 2 3. 6 2. 3 4. 5 3. 6 4. 1 m ax 3 61 .7 4. 7 29 .2 3. 6 4. 9 3. 7 6. 4 4. 7 5. 6 c v 4 13 .2 16 .4 18 .1 14 .4 10 .1 12 .6 11 .7 7. 2 9. 0 a . c ly pe ol at a m 21 7. 0 9. 7 60 .8 5. 0 3. 6 2. 3 2. 7 2. 5 2. 9 m in 15 3. 9 6. 7 40 .4 2. 2 2. 8 1. 8 2. 1 2. 0 2. 5 m ax 28 8. 6 12 .9 87 .9 9. 1 4. 4 2. 8 3. 6 3. 1 3. 3 c v 15 .6 14 .6 19 .2 37 .1 12 .6 10 .0 16 .8 12 .6 6. 3 a . c oa rc ta ta m 14 2. 2 4. 5 52 .1 3. 1 3. 2 2. 3 2. 0 2. 2 2. 6 m in 63 .4 3. 0 25 .3 2. 0 2. 4 1. 8 1. 4 1. 7 2. 3 m ax 21 6. 4 6. 8 95 .7 4. 9 4. 7 3. 3 3. 7 3. 3 3. 3 c v 24 .6 20 .3 30 .6 20 .7 12 .7 16 .5 24 .2 14 .7 7. 7 a . c ol lin a m 14 0. 4 4. 3 33 .0 2. 4 3. 3 1. 8 3. 4 2. 6 3. 8 m in 10 3. 4 3. 2 21 .3 1. 8 2. 8 1. 6 2. 8 2. 2 3. 3 m ax 17 2. 8 5. 4 45 .7 3. 7 3. 7 2. 1 3. 6 2. 9 4. 3 c v 18 .1 13 .4 18 .7 19 .6 7. 3 7. 8 6. 9 7. 6 7. 1 a . c ri th m ifo lia m 75 .7 5. 3 29 .7 3. 9 2. 9 1. 8 3. 4 2. 6 3. 3 m in 34 .6 2. 9 14 .5 2. 1 1. 6 1. 2 2. 8 2. 1 2. 5 m ax 12 2. 5 7. 5 48 .7 6. 4 3. 2 2. 2 4. 4 3. 0 4. 3 c v 30 .5 22 .7 28 .7 30 .3 11 .5 11 .8 11 .1 7. 5 13 .0 a . d is ta ns m 22 2. 8 12 .9 67 .8 9. 6 4. 4 2. 4 3. 9 3. 1 3. 8 m in 13 9. 8 8. 0 51 .6 6. 1 3. 6 1. 6 3. 1 2. 5 3. 0 m ax 30 4. 9 18 .7 95 .1 12 .7 5. 0 3. 0 4. 5 3. 5 4. 2 ta bl e 2. r es ul ts o f d es cr ip tiv e st at is tic s fo r s tu di ed q ua nt ita tiv e m or ph ol og ic al c ha ra ct er is tic s of s tu di ed a ch ill ea s pe ci es 102 biologica nyssana ● 14 (2) december 2023: 97-106 stojković et al. ● morphological variability and differentiation of selected achillea species (sections achillea sensu lato and anthemoideae) from serbia a . d is ta ns c v 19 .2 18 .5 17 .5 17 .0 8. 5 11 .5 8. 0 7. 9 7. 0 a . g ra nd ifo lia m 13 4. 4 24 .7 11 1. 0 25 .5 3. 6 2. 4 3. 8 2. 8 3. 4 m in 10 5. 8 16 .7 86 .1 18 .2 3. 0 2. 1 3. 3 2. 4 3. 0 m ax 19 1. 3 30 .1 13 8. 2 33 .7 4. 5 2. 8 4. 5 3. 1 3. 8 c v 20 .2 17 .2 14 .6 16 .8 10 .9 9. 3 9. 3 6. 8 7. 3 a . m ill ef ol iu m m 17 1. 9 6. 5 36 .3 3. 8 3. 1 1. 9 3. 2 2. 5 3. 3 m in 62 .7 4. 2 21 .3 2. 0 2. 2 1. 3 2. 3 2. 0 2. 6 m ax 28 2. 4 10 .5 50 .8 5. 4 3. 8 2. 4 4. 0 3. 0 4. 1 c v 32 .1 20 .0 20 .4 16 .9 12 .1 12 .2 12 .1 9. 7 8. 9 a . n ob ili s m 76 .9 8. 9 30 .7 6. 3 2. 2 1. 7 1. 9 1. 8 2. 1 m in 39 .0 4. 8 18 .9 3. 7 1. 8 1. 3 1. 3 1. 3 1. 8 m ax 13 0. 2 12 .9 42 .6 10 .4 3. 3 2. 3 2. 6 2. 3 2. 4 c v 27 .7 23 .6 20 .3 25 .8 15 .4 16 .1 19 .0 16 .3 6. 3 a . p se ud op ec tin at a m 25 .7 2. 5 33 .0 3. 6 3. 1 2. 2 2. 8 2. 9 3. 0 m in 18 .3 1. 7 24 .5 2. 4 2. 6 1. 7 2. 3 2. 4 2. 6 m ax 34 .9 4. 0 42 .3 5. 0 3. 7 2. 7 3. 2 3. 5 3. 4 c v 13 .6 24 .3 13 .4 19 .4 9. 2 16 .1 7. 3 12 .0 6. 9 a . v an da si i m 15 6. 6 4. 5 56 .6 3. 3 3. 1 2. 3 2. 3 2. 5 2. 9 m in 12 4. 1 3. 0 37 .4 2. 6 2. 6 1. 8 1. 8 2. 3 2. 4 m ax 20 8. 3 6. 0 75 .1 4. 8 3. 7 2. 7 2. 8 2. 8 3. 2 c v 17 .0 20 .9 16 .9 18 .0 11 .0 11 .5 10 .5 6. 1 7. 5 a . a ge ra tif ol ia m 39 .3 0. 3 14 .4 0. 2 5. 5 5. 2 8. 8 3. 7 4. 2 m in 22 .9 0. 2 9. 7 0. 0 4. 3 3. 8 7. 3 2. 9 2. 8 m ax 54 .8 0. 4 24 .1 0. 4 6. 6 6. 6 10 .2 4. 5 5. 6 c v 19 .2 23 .0 22 .9 32 .5 13 .1 13 .3 9. 3 8. 4 16 .0 a . l in gu la ta m 12 2. 1 0. 4 41 .3 0. 4 5. 3 4. 2 6. 5 3. 6 4. 5 m in 83 .7 0. 3 29 .9 0. 2 4. 3 3. 3 5. 2 2. 9 3. 7 m ax 18 2. 0 0. 9 56 .7 0. 7 6. 8 5. 5 9. 1 4. 2 5. 8 c v 26 .0 28 .5 18 .1 33 .9 14 .0 12 .4 14 .9 10 .8 11 .4 1 m : m ea n (m m ). 2 m in : m in im um (m m ). 3 m ax : m ax im um (m m ). 4 c v: c oe ffi ci en t o f v ar ia tio n (% ). 103 biologica nyssana ● 14 (2) december 2023: 97-106 stojković et al. ● morphological variability and differentiation of selected achillea species (sections achillea sensu lato and anthemoideae) from serbia nevertheless, differences in leaf shapes among achillea taxa are stable and often used in taxonomy as diagnostic characteristics (sha et al., 2018). multivariate analyses (cda and ahc) of morphological characteristics of the studied achillea species the cda based on 9 morphological characteristics of 13 species of achillea showed that the first two canonical axes participated in 80.26% of the total discrimination, of which the first axis (ca1) accounted for 53.23% (fig. 2, tab. 3). the obtained scatter plot showed the tendency of differentiation of species from two studied sections along the ca1 that explained the highest percentage of discrimination (fig. 2). namely, individuals of most species from the section achillea sensu lato were positioned at the negative part of ca1, while individuals of two studied species from the section anthemoideae formed the group on the positive part of this axis. despite the positioning of individuals of species from the section achillea sensu lato at the negative part of ca1, they formed two groups clearly separated along the ca2. the first group, with positive values for ca2, included only individuals of a. grandifolia, while individuals of other species from this section formed the second group showing mostly negative table 3. standardized coefficients for the first two canonical axes (cas) of variation in quantitative morphological characteristics from the discriminant functional analysis of studied achillea species. significant coefficients are in boldface variables ca1 ca2 rll -0.004 -0.332 rlsl -0.393 0.359 sll -0.074 0.068 slsl -0.41 0.514 cl 0.042 0.195 cw 0.354 0.175 lfl 0.822 0.819 tfl -0.386 -0.406 ibl -0.058 -0.245 eigenvalue 28.038 14.236 cumulative percentage of variance 0.532 0.803 values for ca2. besides that, individuals of one species from the section achillea sensu lato (a. chrysocoma) were positioned at the positive part of ca1 approaching the section anthemoideae. fig. 2. canonical discriminant analysis (cda) based on morphological characteristics of studied achillea species 104 biologica nyssana ● 14 (2) december 2023: 97-106 stojković et al. ● morphological variability and differentiation of selected achillea species (sections achillea sensu lato and anthemoideae) from serbia characteristic ligulate floret length (lfl) had significant impact on both axes, while stem leaf segment length (slsl) significantly affected only the ca2 (tab. 3). thus, the basic differences between observed groups were related to fact that the section achillea sensu lato was characterized by significantly shorter ligulate florets and longer segments of stem leaf compared to the section anthemoideae (tab. 2). in addition, a. grandifolia featured the longest segments of stem leaf and showed considerable separation from all other species of the section achillea sensu lato. on the other hand, intermediate position of a. chrysocoma between the studied sections was probably the consequence of the fact that this species had the longest ligulate florets within the section achillea sensu lato, i.e., almost like a. lingulata from the section anthemoideae. finally, if we compare differentiation of achillea species on the scatter plot (fig. 2) and the geographical position of their populations included in the analysis (fig. 1), it is obvious that there is no correlation between them. this result suggested a stronger differentiation between morphologically identified species than between geographical regions. the dendrogram obtained by ahc has also shown a tendency to divide analyzed achillea species in a similar way but indicating the closer position of a. chrysocoma to a. lingulata than to other species of the section achillea sensu lato (fig. 3). specifically, the dendrogram suggested the division of the studied species into four main groups: 1) a. grandifolia; 2) a. ageratifolia; 3) a. lingulata and a. chrysocoma; and 4) other studied species. thus, a. grandifolia again showed considerable separation from all other species of the section achillea sensu lato, and a. chrysocoma was in the same cluster with a. lingulata from the section anthemoideae. both multivariate analyses were used to determine the level of morphological differentiation of the studied achillea species and sections. apparently, characteristics ligulate floret length (lfl) and stem leaf segment length (slsl) had the greatest influence on the differentiation of the two investigated sections. moreover, within the section achillea sensu lato some separation of a. grandifolia and a. chrysocoma was evident. in agreement with our findings, saukel & länger (1992) reported that the characteristics of leaf segment and ligulate fig. 3. dendrogram obtained by agglomerative hierarchical clustering (ahc) of studied achillea species based on morphological characteristics biologica nyssana ● 14 (2) december 2023: 97-106 stojković et al. ● morphological variability and differentiation of selected achillea species (sections achillea sensu lato and anthemoideae) from serbia 105 floret were the most significant features for the morphological characterization of taxa within a. millefolium aggregate from central europe. however, azani et al. (2009) showed that some quantitative and qualitative morphological characteristics did not position iranian achillea species in agreement with the infrageneric classification. conclusion most of the studied morphological characteristics of 13 species of achillea from the central balkans showed moderate level of variability. nevertheless, in almost all species, some of the characteristics of the flowering region stood out as low variable. multivariate statistical analyses reflected almost clear morphological differentiation between species from two studied sections whereby ligulate floret length and stem leaf segment length had the greatest influence for their distinction. thus, the section achillea sensu lato was characterized by significantly shorter ligulate florets and longer segments of stem leaf compared to the section anthemoideae. furthermore, a. grandifolia featured the longest segments of stem leaf and showed considerable separation from all other species of the section achillea sensu lato. on the other hand, a. chrysocoma was characterized by the longest ligulate florets within the section achillea sensu lato and thus showed some tendency to approach the section anthemoideae. nevertheless, it could be concluded that the selected set of morphological characteristics in the present study mainly positioned the studied species in accordance with the current infrageneric classification of achillea. acknowledgements. this research was supported by grant no 451-03-47/2023-01/200124 by the ministry of education, science and technological development of the republic of serbia. the authors would like to thank ivan gnjatović for kindly producing the distribution map. references afanasyev, k.s. & bochantsev, v.p. 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(1992). die achillea millefolium gruppe (asteraceae) in mitteleuropa 1. phyton (horn), 31(2), 185–207. radulović et al. 2022, biologica nyssana 13(2) 13 (2) december 2022: 179-189 doi: 10.5281/zenodo.7437345 the essential oil composition of different parts of artemisia absinthium and its antibacterial activity against phytopathogenic bacteria original article maja radulović faculty of biology, university of belgrade, studentski trg 16, 11000 belgrade, serbia maja.radulovic@bio.bg.ac.rs (corresponding author) milan gavrilović faculty of biology, university of belgrade, studentski trg 16, 11000 belgrade, serbia nemanja rajčević faculty of biology, university of belgrade, studentski trg 16, 11000 belgrade, serbia tamara janakiev faculty of biology, university of belgrade, studentski trg 16, 11000 belgrade, serbia ivica dimkić faculty of biology, university of belgrade, studentski trg 16, 11000 belgrade, serbia pedja janaćković faculty of biology, university of belgrade, studentski trg 16, 11000 belgrade, serbia received: october 04, 2022 revised: december 01, 2022 accepted: december 02, 2022 abstract: the composition of essential oil (eo) from different aerial parts of artemisia absinthium l. (asteraceae), from serbia, was analyzed. shoots without leaves and inflorescences (swli), leaves (l), and inflorescences (i) were subjected to hydrodistillation using the clevenger-type apparatus. analysis of eo was done by gas chromatography with ms of flame ionization detection (gc/ms and gc/fid). microdilution method was used to determine the minimum inhibitory concentration (mic) and minimum bactericidal concentration (mbc). organoleptic characteristics and quantitative and qualitative differences were documented between examined eos. in total, 17 compounds were identified in swli, 11 in l and 18 in i. the principal constituent in all eos was trans-thujone (swli 23.56%, l 60.41%, and i 46.16%). the most susceptible strains were xanthomonas campestris pv. campestris, erwinia amylovora and rathayibacter tritici. the strongest activity was shown for swli eo against e. amylovora (0.03 mg/ml). the same mic values were observed for l eo against e. amylovora and r. tritici (0.09 mg/ ml). the equal activity for all eos tested was detected against x. campestris (0.13 mg/ml). key words: wormwood, asteraceae, hydrodistillation, volatile terpenes, antibacterial activity apstrakt: sastav etarskog ulja iz različitih delova vrste artemisia absinthium i njegova antibakterijska aktivnost protiv fitopatogenih bakterija u ovom radu je analiziran sastav etarskog ulja (eu) iz različitih nadzemnih delova vrste artemisia absinthium l. (asteraceae) iz srbije. izdanci bez listova i cvasti (iblc), listovi (l) i cvasti (c) podvrgnuti su hidrodestilaciji pomoću klevendžerove aparature. analiza eu je izvršena upotrebom gh/ ms (gasne hromatografije i masene spektrometrije). mikrodilucionom metodom određena je minimalna inhibitorna koncentracija (mik) i minimalna baktericidna koncentracija (mbk). utvrđeni su prinos (%, w/w), organoleptičke karakteristike (boja i miris), kao i kvantitativne i kvalitativne razlike između ispitivanih etarskih ulja. ukupno je identifikovano 17 komponenti u eu iblc, 11 u eu l i 18 u eu i. dominantno jedinjenje u svim etarskim uljima je trans-tujon (iblc 23,56%, l 60,41%, i c 46,16%). najosjetljiviji sojevi bili su xanthomonas campestris pv. campestris, erwinia amylovora i rathayibacter tritici. najveću aktivnost pokazalo je eu iblc protiv e. amylovora (0,03 mg/ml). iste mik vrednosti dobijene su za eu l protiv e. amylovora i r. tritici (0,09 mg/ml). sva testirana etarska ulja pokazala su istu aktivnost protiv x. campestris (0,13 mg/ml). ključne reči: pelin, asteraceae, hidrodestilacija, isparljivi terpeni, antibakterijska aktivnost introduction artemisia absinthium l. (anthemideae, asteraceae), commonly known as wormwood, is a perennial semi-shrubby plant up to 120 cm high (gajić, 1975; bora & sharma, 2011) which grows in dry habitats in eurasia, north and south america (deans & kennedy, 2001). this herb is the main ingredient of one of the most popular alcoholic beverages – absinthe (padosch et al., 2006). wormwood is known for its medical properties both in traditional medicine and in modern pharmacology (nguyen et al., 2018; © 2022 radulović et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 179 ahamad, 2019). as a remedy, wormwood is used for stomachache, kidney and gallbladder diseases, insomnia, and for the treatment of many other diseases and disorders (judzentiene et al., 2012). in serbia, a few ethnobotanical studies showed that it has been traditionally used as a diuretic and against respiratory, urinary, and digestive system disorders (jarić et al., 2015; živković et al., 2020). the most significant active constituents of the wormwood are volatile compounds and bitter substances which have attracted the interest of many researchers and producers throughout the world. numerous studies have shown that wormwood displays significant intraspecific variation in the terpene constituents of the eo (basta et al. 2007; mohammadi et al., 2015). many previous researchers have examined eos from herba of a. absinthium and have shown that the main component was bicyclic monoterpene – thujone (juteau et al., 2003; blagojević et al., 2006). both isomeric forms, cisand trans-thujone were present, but the eo was characterized by a higher percentage of trans-thujone (benkhaled et al., 2020). besides thujone, which is convulsant and has toxic activity (olsen, 2000), some other natural products were found in the oil: (z)-epoxyocimene, chrysanthenyl acetate (juteau et al., 2003; nguyen et al., 2017), caryophyllene oxide, p-cymene, 1,8-cineole (basta et al., 2007), sabinene, sabinyl acetate, α-phellandrene (mihajilov-krstev et al., 2014), camphor, and chamazulene (benkhaled et al., 2020). the chemical composition of wormwood eo varies significantly not only depending on the individual genetic variability, but also according to phenological stage and the plant part used (nguyen & németh 2016; nguyen et al., 2018). however, data regarding eo composition of separated plant parts are scarce. there are few papers dealing with variability in volatile composition regarding plant organ in wormwood, mainly aerial parts and roots (blagojević et al., 2006), or leaves and flowers (judzentiene & budiene, 2010; riahi et al., 2013), while shoots without leaves and inflorescences have not been analyzed to date. the wormwood eo has a broad spectrum of biological properties: antioxidant, antimicrobial (mihajilov-krstev et al., 2014; riahi et al., 2015), antiparasitic (yildiz et al., 2011), cytotoxic activity (taherkhani, 2014), and insecticidal and repellent effect (mihajilov-krstev et al., 2014). many previous studies investigated the antibacterial activity of wormwood eo, but they have been mainly focused on human pathogens (blagojević et al., 2006; mihajilov-krstev et al., 2014), while only a few studies were focused on phytopathogens (kordali et al., 2005). unfortunately, crop loss represents a problem due to plant diseases caused by insects and plant pathogens. since widely used synthetic chemicals in plant disease control are associated with undesirable effects and some toxic residues in the products (isman, 2000), there has been a growing interest in research concerning alternative pesticides and active antimicrobial compounds, including the plant extracts and eos. to date, there are no data regarding the antimicrobial activity of separated parts of wormwood against phytopathogens, but there are only few studies against human pathogens (joshi, 2013; vieira et al., 2016). investigation of the eo composition of different plant parts as well as their antimicrobial activity deserves special attention, bearing in mind that a specific chemical composition could significantly affect biological activities. therefore, the objectives of the present work were to (1) isolate and analyze the composition of eo of separated parts of a. absinthium; (2) investigate their antibacterial properties against selected phytopathogenic bacterial strains; and (3) highlight their potential future importance in chemophenetics and agriculture. materials and methods plant material plant material of artemisia absinthium was collected during the flowering period in village reka, near kladovo (eastern serbia, 44°29’35’’n, 22°24’55’’ e) in august 2021. the plants were identified using appropriate professional literature (gajić, 1975). a voucher specimen was deposited at the herbarium of the university of belgrade – faculty of biology, institute of botany and botanical garden „jevremovac” (beou 17804). the collected material was dried at room temperature and then divided into three groups: (1) shoots without leaves and inflorescences (swli), (2) leaves (l), and (3) inflorescences (i). isolation of essential oil separated plant material (swli – 172 g, l – 72 g and i – 124 g) was chopped and subjected to hydrodistillation using a clevenger type apparatus for 3 h, according to the procedure described in ph. eur. 6. (european directorate for the quality of medicines, 2007). the obtained eos were stored at 4 °c before the analysis by gas chromatography with ms of flame ionization detection (gc/ms and gc/ fid). gc-fid and gc/ms analysis the gc-fid and gc/ms analyses were carried out with an agilent 7890 a apparatus equipped with 180 biologica nyssana ● 13 (2) december 2022: 179-189 radulović et al. ● he essential oil composition of different parts of artemisia absinthium and its antibacterial activity against phytopathogenic bacteria biologica nyssana ● 13 (2) december 2022: 179-189 radulović et al. ● he essential oil composition of different parts of artemisia absinthium and its antibacterial activity against phytopathogenic bacteria 181 a 5975 c mass-selective detector (msd), a flame ionization detector (fid), and an hp-5 msi fusedsilica cap (column length 30 m, diameter 0.25 mm, film thickness 0.25 mm). the oven temperature was programmed linearly, rising from 60 °c to 240 °c at 3 °c/min; the injector temperature was 220 °c; the detector temperature was 300 °c, and the transferline temperature was 240 °c. the carrier gas was he (flow rate: 1.0 ml/min at 210 °c, constant pressure mode) at an injection volume of 1 μl and a split ratio of 10:1. electron impact mass spectra (ei-ms; 70 ev) were acquired over the m/z range 40–550. library search and mass spectral deconvolution and extraction were performed using the nist amdis (automated mass spectral deconvolution and identification system) software, version 2.64.113.71, with the retention index (ri) calibration data analysis parameters set to the strong level and a 10% penalty for compounds without a ri. the ris were experimentally determined using the standard method involving retention times (rt) of n-alkanes, which were injected after the essential oil under the same chromatographic conditions. the search was performed against our home-made library, containing 4972 spectra. the relative contents of identified compounds were computed from the gc peak areas. tested bacterial strains and growth conditions antibacterial activity was tested using one gram-positive (rathayibacter tritici), and five gram-negative phytopathogenic bacterial strains previously identified: agrobacterium tumefaciens, erwinia amylovora, pantoea alli, pseudomonas oryzihabitans, xanthomonas campestris pv. campestris. the bacterial strains were cultured in lb medium (composition g/l: tryptone 10, yeast extract 5, nacl 5) for 24 h at 30 °c. suspensions were prepared in phosphate saline buffer (1×pbs, sigma aldrich, usa) in the final concentration of 106 cfu/ml. mic assay microdillution method (mic assay) described in a previous study (ristivojević et al., 2016) was used to determine the minimum inhibitory concentrations (mic) and minimal bactericidal concentrations (mbc) of the a. absinthium essential oils. twofold serial dilutions with lb medium in 96-well microtiter plates were performed. except for the sterility control, each well was inoculated with 20 μl of bacterial suspensions (1×106 cfu/ml), reaching a final volume of 200 μl. besides a negative control, a sterility control, and control for the solvent (meoh), the antibiotics gentamicin and kanamycin were tested as positive controls in the final concentration of 0.025 mg/ml. the final concentration of methanol (meoh) as a solvent was 10%. all dilutions were done in duplicate, and the results were expressed in mg/ml. after reaching the final volume, 22 μl of resazurin as an indicator was added, and the 96well microtiter plates were incubated for 24 h at 30 °c. according to the resazurin reaction, the lowest concentration, which showed no change in color was defined as the mic. the lowest concentration that after sub-culturing did not show bacterial growth overnight was defined as the mbc value. results and discussion organoleptic characteristics, yield and composition of investigated eos the yield and organoleptic characteristics of the eos of separated parts of a. absinthium are given in tab. 1. the lowest yield (w/w) was recorded for swli eo (0.05%), while the highest was recorded for l eo (0.29%). this may be in relation with the abundance of glandular trichomes and the presence of secretory canals in the plant parts. although secretory canals are found only in the stem, while glandular trichomes were found both on the stem and leaves of a. absinthium (janaćković et al., 2019), it can be considered that certain mass of leaves, used for distillation, contain more glandular trichomes which contribute to higher eo yield. riahi et al. (2013) recorded variation between yields of the flowers and leaves eos. the highest yield was recorded for flowers eo (2.98%) whereas leaves eo exhibited a lower yield (1.87%) (riahi et al. 2013). on the contrary, we have shown that leaves produced more eo than inflorescences (tab. 1). it was noted that the color of obtained eos had changed over time. the l and i eo samples were light orange at the beginning of distillation but became dark brown at the end of distillation. the swli eo was orange-brown at the beginning and during distillation, but then changed color and became brown at the end of distillation. after 24 h in the refrigerator, the color of this oil was greenish, and after a few days, it was light yellow (tab. 1). wormwood eos, obtained from aerial parts of the plant, has a dark green to orange or dark brown color (sensitive to ultraviolet/visible light) (judzentiene, 2016). dark blue eo was reported by riahi et al. (2013) and msaada et al. (2015). on the other hand, according to pino et al. (1997), the eo obtained from the dried leaves and flowering tops of wormwood varied from dark green to brown or dark brownish green. different colors of eo can be a consequence of the presence/absence of different constituents. besides the color, the smell of eos was also specific. all of the obtained eos had a specific and 182 biologica nyssana ● 13 (2) december 2022: 179-189 radulović et al. ● he essential oil composition of different parts of artemisia absinthium and its antibacterial activity against phytopathogenic bacteria unsavory odor. swli eo had an odor very similar to the smell of tobacco. l eo showed the strongest and most unpleasant fragrance in comparison with the other two samples. this smell was described as „typically wormwood”, which reminds of bitter constituents. i eo was also recognized as fragrance with bitter characteristics. because of high concentrations of volatile terpenes, especially in leaves and inflorescences, the eo of this species has strong aromatic smell (nguyen & németh, 2016). phytochemical analyses of investigated eos showed some qualitative and quantitative differences. the conducted gc-fid and gc/ ms analyses detected and identified a total of 27 compounds (17 in the swli eo, 11 in l eo, and 18 in i eo). all compounds are listed in tab. 2. more compounds were identified in i eo, than in l eo, which is in agreement with the investigation done by riahi et al. (2013). the dominant group of compounds in the l eo and i eo were oxygenated monoterpenes (81.80% and 62.63%, respectively). on the other hand, swli eo was not dominated by terpenes (other compounds including fatty acid derivates, esters, were found in higher abundance, 62.15%). this finding is important as there are no studies dealing with eo from shoots without leaves and inflorescences. the l eo was also rich in monoterpene hydrocarbons (15.26%), while i eo and swli eo contain these compounds in smaller quantities (8.94% and 7.85%, respectively). sesquiterpenes were present only in two analyzed eos, but in low amounts (2.94% in l eo and 1.19 in i eo). thus, monoterpenes were found in higher abundance than sesquiterpenes both in l eo and i eo, which also showed nguyen et al. (2018). on the other hand, swli eo lacks sesquiterpenes, while l eo lacks other compounds. blagojević et al. (2006) documented differences between aerial parts eo and root eo, where monoterpenes (84.6%) dominated in the aerial parts eo, while aliphatic esters (64.5%) were major compounds of the root eo. in addition, a predominance of oxygenated monoterpenes (81.489.1%) was documented in aerial parts eo, while the root eo showed high ratios of hydrocarbon monoterpenes (43.8–55.1%) and monoterpene esters (36.6–41.5%) (llorens-molina & vacas 2015). the dominant compound in all three eos was trans-thujone (in swli eo 23.6%, in l eo 60.4%, in i eo 46.2%). wormwood is usually known and reported to be rich in thujone (meschler & howlett, 1999; juteau et al., 2003). the concentration of trans-thujone was the highest in the l eo (60.4%), which is in agreement with riahi et al. (2013). however, judzentiene & budiene (2010) determined higher ratios of thujone in flowers eo (5.3-10.4%) than in leaves eo (0.0-8.9%). it should be noted that for the praxis the ratio of plant parts may play an important role in reaching lower thujone levels of the drug. in our study, less thujone content was detected in inflorescences eo, thus a. absinthium drug should contain more flowers than leaves which was also concluded by nguyen et al. (2019). on the other hand, it was shown that thujone is not necessarily the main compound of the essential oil of wormwood (nguyen et al., 2017). in addition, nguyen et al. (2017) showed that trans-thujone is the major compound, however, in some cases, cisthujone reached comparable percentages, while no sample was found where this latter one would have been the only isomer. in this study, we documented that swli eo and i eo lack cis-thujone. other dominant compounds were as follows: sample dry plant material (g) obtained oil (g) yield (%, w/w) smell color start of distillation end of distillation after 24 h after few days swli1 172 0.09 0.05 strong; unpleasant; like tobacco orangebrown dark brown green light yellow l 72 0.21 0.29 strong; very unpleasant; bitter; typically wormwood light orange dark brown dark brown dark brown i 124 0.26 0.21 less unpleasant; bitter light orange dark brown dark brown dark brown table 1. yield and organoleptic characteristics of eos of separated parts of a. absinthium 1swli shoots without leaves and inflorescences; l leaves; i inflorescences 183 biologica nyssana ● 13 (2) december 2022: 179-189 radulović et al. ● he essential oil composition of different parts of artemisia absinthium and its antibacterial activity against phytopathogenic bacteria table 2. composition of eos of different parts of a. absinthium no ri* compounds % swli1 l i 1 923 α-thujene 1.12 1.01 2 943 α-fenchene 1.65 3 971 sabinene 1.51 6.23 4.17 4 1024 p-cymene 4.68 7.08 2.62 5 1031 1,8-cineole 2.67 4.28 6 1059 γ-terpinene 0.83 1.14 7 1104 linalool 4.54 8 1110 cis-thujone 1.12 9 1122 trans-thujone 23.56 60.41 46.16 10 1134 (z)-epoxyocimene 2.93 15.23 5.22 11 1137 iso-3-thujanol 0.99 12 1178 terpinen-4-ol 1.38 2.42 13 1228 nerol 3.51 14 1291 lavandulyl acetate 3.49 15 1425 lavandulyl isobutanoate 9.81 1.27 16 1488 β-selinene 1.19 17 1491 neryl isobutanoate 5.73 2.44 18 1511 geranyl isobutanoate 11.65 3.15 19 1513 lavandulyl 2-methylbutanoate 4.03 1.52 20 1578 geranyl butanoate 8.94 7.97 21 1584 caryophyllene oxide 2.94 22 1585 neryl isovalerate 5.29 6.40 23 1603 fatty acid ester 4.25 24 1609 geranyl isovalerate 2.35 25 2008 fatty acid ester 2.47 1.78 26 2014 fatty acid ester 2.72 27 2082 fatty acid ester 4.16 total monoterpenes 37.85 97.06 71.57 monoterpene hydrocarbons 7.85 15.26 8.94 oxygenated monoterpenes 30.00 81.80 62.63 total sesquiterepenes 0.00 2.94 1.19 sesquiterpene hydrocarbons 0.00 0.00 1.19 oxygenated sesquiterpenes 0.00 2.94 0.00 other 62.15 0.00 27.24 total 100.00 100.00 100.00 no. of compounds 17 11 18 contents of the total essential oil composition are given as percentages; -: not detected; *linear retention index was calculated for all compounds using the following formula: lri=100•(trs-trn)/((trn+1-trn)+100•n; other non terpenoid compounds, i.e. alyphatic and aromatic hydrocarbons; ms spectra of unidentified fatty acids are given in the appendix 1. 1swli shoots without leaves and inflorescences; l leaves; i inflorescences 184 biologica nyssana ● 13 (2) december 2022: 179-189 radulović et al. ● he essential oil composition of different parts of artemisia absinthium and its antibacterial activity against phytopathogenic bacteria geranyl isobutanoate and lavandulyl isobutanoate (11.65%, 9.81%, respectively) in swli eo; (z)epoxyocimene and p-cymene (15.23%, 7.08%, respectively) in l eo; and geranyl butanoate and neryl isovalerate (7.97%, 6.40% respectively) in i eo. some constituents were specific for certain examined eo. for example, six compounds were found only in swli eo: α-fenchene, nerol, lavandulyl acetate, geranyl isovalerate, and two fatty acid esters; three were found only in l eo: cis-thujone, iso-3-thujanol and caryophyllene oxide; while three were found only in i eo: linalool, β-selinene and one fatty acid ester. nguyen et al. (2018) also showed qualitative differences in eo profiles in a. absinthium thujone chemotype, e.g. geranyl-p-cymene, nuciferol esters and two unknown compounds were present only in the flower oil. also, neryl-isobutanoate, nerylisovalerate and caryophyllene oxide were much more present in flower oil than in the leaves oil (nguyen et al., 2018). however, we have shown herein that neryl-isobutanoate was in a higher amount in swli eo (5.73%) than in i eo (2.44%), while l eo lacks this compound. on the other hand, the amount of neryl-isovalerate was slightly lower in swli than in i eo (5.29% and 6.40, respectively). caryophyllene oxide was found only in the l eo. the relative amount of sabinene was higher in the l eo compared to i eo (6.23% and 4.17%, respectively), which is in accordance with the results obtained by riahi et al. (2013). blagojević et al. (2006) showed that the main compound in the aerial parts eo of wormwood was trans-thujone (β-thujone), while α-fenchene was the main compound found in root oil. α-fenchene was recorded only in swli eo. in addition, llorens-molina & vacas (2015) documented (z)epoxyocimene, (z)-chrysanthenyl acetate and linalool as the main compounds in aerial parts eo, while β-myrcene and α-fenchene were the main compounds in the root oil. (z)-epoxyocimene was recorded in all three eos, (z)-chrysanthenyl acetate and β-myrcene were not recorded, while linalool was found only in i eo. significant differences in eo composition were observed also between leaves and flowers. ariño et al. (1999) reported some quantitative differences in eo profile from leaves and flowers, e.g., cis-epoxyocimene was found in higher ratios in leaf oil, which is in accordance with our study. also, ariño et al. (1999) showed that cischrysanthnyl acetate, which we have not detected, was the dominant compound in the flower oil. in addition, riahi et al. (2013) showed that camphor was present only in flower oil but not in the leaves oil. on the other hand, bornan-2-one, was found in leaf oil, whereas flower oils were lacking in this compound. neither camphor nor bornan-2-one have been detected in our examined eos. differences in the composition of the essential oils of each part of the plant, especially in the flower heads, may have chemophenetic significance in future research, given that their components are very significant and genetically stable due to their repellent role in flower protection. antibacterial activity of a. absinthium eo the results of mic and mbc are given in tab. 3. it was shown that examined eos exhibited various degrees of antibacterial activities depending on tested bacterial strains (tab. 3). in general, tested oils had moderate antibacterial activity. mic assay indicates that the most sensitive bacterial strains were erwinia amylovora, rathayibacter tritici, and xanthomonas campestris pv. campestris, while pseudomonas oryzihabitans and pantoea alli were the most resistant. the strongest effect was shown for swli eo against e. amylovora (mic 0.03 mg/ ml). also, eo from l demonstrated high activity against e. amylovora and r. tritici (mic 0.09 mg/ ml). equal activity against x. campestris (mic 0.13 mg/ml) was detected for all three tested eos. antibacterial activity of wormwood eo against human pathogens was documented in several studies. it was shown that a. absinthium eo had a significant antimicrobial effect against human pathogenic bacteria isolated from wounds and stools of patients (mihajilov-krstev et al., 2014) with minimal inhibitory/bactericidal concentrations ranging from <0.08 to 2.43 mg/ml and from 0.08 to 38.80 mg/ml, respectively. riahi et al. (2015) reported that majorly gram-positive bacterial strains showed more sensitivity against the wormwood eos. in another study, the wormwood eo has shown very pronounced antibacterial activity, with inhibitory and bactericidal concentrations ranging from 4.72 against gram-negative to 37.80 mg/ml against grampositive bacterial strains (stanković et al., 2016). the study by kordali et al. (2005) demonstrated by disk diffusion method that a. absinthium eo at 600-1200 μ/disk concentrations was active against a limited number of bacterial strains, including phytopathogens: curtobacterium flaccumfaciens, pseudomonas syringae pv. maculicola, p.syringae pv. syringae (rk-470), xanthomonas axanopodas pv. vesicatoria, and x. pelargonii. different inhibitory effects of tested eos may be attributed to the differences in the biological properties of the main compounds, differences in composition in the oil and their synergistic effects. this result is in agreement with delaquis et al. (2002), who reported that the biological activity of different eos was the result of the interaction between their total chemical compounds, mainly with additive and 185 biologica nyssana ● 13 (2) december 2022: 179-189 radulović et al. ● he essential oil composition of different parts of artemisia absinthium and its antibacterial activity against phytopathogenic bacteria synergistic effects. the action mode of antimicrobial agents essentially depends on the type of the treated microorganism in relation to the structure of their cell wall and the outer membrane (shan et al., 2007). considering that there are a large number of different compounds in eo, it is most likely that their antibacterial activity is a result of targeting different cell components and consequently causing several types of damage (cha et al., 2005; sadaka et al., 2013). that could explain the broad antibacterial activity of tested a. absinthium eo against both gram-negative e. amylovora and x. campestris pv. campestris and gram-positive r. tritici were observed in the present study. it was confirmed that monoterpene ketones, α –and β-thujone, could be responsible for antibacterial potential against gram-positive and gram-negative bacteria (juteau et al., 2003; blagojević et al., 2006; tsiri et al., 2009; mihajilov-krstev et al., 2014). according to these, high amounts of thujone in all tested eos could be responsible for antibacterial activity. also, these substances can be important in agriculture as potential agents in pest control. conclusions herein, we presented a detailed analysis of the eo composition of the a. absinthium different parts (shoots without leaves and inflorescences, leaves, and inflorescences) and their antimicrobial activity. trans-thujone was the dominant compound in all three eos. on the other hand, the organoleptic characteristics, yield, number of identified components, the dominant group of compounds, the amount of the dominant compound, as well as the presence and amount of other compounds differed among the tested eos. these differences indicate the different biological functions of different components in different parts of the plant. moreover, the strongest effect was shown for swli eo, which may be in relation to the dominance of non-terpene compounds present in the eo. although the biochemical and physiological processes behind these differences in eo profiles still await explanation, other artemisia species should be included in future similar studies in order to get better insight into eo variability and antimicrobial activity of different plant parts. acknowledgements. financial support was provided by the ministry of education, science and technological development of the republic of serbia (451-03-68/202214/200178). references ahamad, j. 2019: a pharmacognostic review on artemisia absinthium. international research journal of pharmacy, 10(1): 25-31. ariño, a., arberas, i., renobales, g., arriaga, s., domínguez, j.b. 1999: seasonal variation in wormwood (artemisia absinthium l.) essential oil composition. journal of essential oil research, 11: 619–622. basta, a., tzakou, o., couladis, m., pavlović, m. 2007: chemical composition of artemisia absinthium l. from greece. journal of essential oil research, 19: 316-318. benkhaled, a., boudjelal, a., napoli, e., baali, f., ruberto, g. 2020: phytochemical profile, table 3. antibacterial activity of different parts of a. absinthium eos phytopathogenic strains artemisia eos (mg/ml) gentamicin (mg/ml) kanamycin (mg/ ml)swli l i mic mbc mic mbc mic mbc mic mbc mic mbc xanthomonas campestris pv. campestris 0.125 0.5 0.125 0.25 0.125 0.25 0.008 0.006 0.008 0.006 pseudomonas oryzihabitans 1 >1 0.75 1 1 >1 0.016 0.013 0.016 0.0125 erwinia amylovora 0.031 0.5 0.094 0.0625 0.25 0.5 0.004 0.003 0.008 0.006 rathayibacter tritici 0.125 0.25 0.094 0.0625 0.031 0.008 0.006 0.003 0.025 pantoea alli >1 0.75 1 >1 0.006 0.025 0.006 0.025 agrobacterium tumefaciens 0.5 1 0.75 1 0.5 1 >0.400 >0.400 not detected in the range of tested concentrations biologica nyssana ● 13 (2) december 2022: 179-189 radulović et al. ● he essential oil composition of different parts of artemisia absinthium and its antibacterial activity against phytopathogenic bacteria 186 antioxidant activity and wound healing properties of artemisia absinthium essential oil. asian pacific journal of tropical biomedicine, 10(11): 496. blagojević, p., radulović, n., palić, r., stojanović, g. 2006: chemical composition of the essential oils of serbian wild-growing artemisia absinthium and artemisia vulgaris. journal of agricultural and food chemistry, 54(13): 4780-4789. bora, k.s., sharma, a. 2011: the genus artemisia: a comprehensive review. pharmaceutical biology, 49: 101-109. cha, j.d., jeong, m.r., jeong, s.i., moon, s.e., kim, j.y., kil, b.s., song, y.h. 2005: chemical composition and antimicrobial activity of the essential oils of artemisia scoparia and a. capillaris. planta medica, 71(2): 186–190. deans, s.g., kennedy, a.i. 2001: artemisia absinthium. in: wright c.w. 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lri2082 anticancer compounds from medicinal plants biologica nyssana 5 (1)  september 2014: 17-30 papović, o. et al.  analysis of the flora of rogozna… 17 original article received: 14 july 2014 revised: 21 august 2014 accepted: 10 september 2014 analysis of the flora of rogozna mountain in southwestern serbia olivera papović 1* , milica miljković 2 , novica ranđelović 2 , vladimir ranđelović 2 1 university of pristine, faculty of sciences and mathematics, department of biology, ive lole ribara 29, 38220 kosovska mitrovica, serbia 2 university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia * e-mail: olja.bio@open.telekom.rs abstract: papović, o., miljković, m., ranđelović, n., ranđelović, v.: analysis of the flora of rogozna mountain in southwestern serbia. biologica nyssana, 5 (1), september 2014: 17-30. as a result of the two-year investigation of the rogozna mt. flora, 795 plant taxa (species and subspecies) belonging to 337 genera and 77 families were recorded. floristic analysis of investigated area was performed in comparison to the floristic data of serbia, balkan peninsula and neighboring region of rogozna mt. (ibar river valley and high mountain region of kopaonik mt). key words: flora, floristic analysis, floristic spectrum. apstrakt: papović, o., miljković, m., ranđelović, n., ranđelović, v.: analiza flore planine rogozne u jugozapadnoj srbiji.. biologica nyssana, 5 (1), september 2014: 17-30. kao rezultat dvogodišnjih istraživanja flore planine rogozne utvrđeno je 795 biljnih taksona (vrsta i podvrsta) iz 337 rodova i 77 familija. floristička analiza istraživanog područja je sprovedena u poređenju sa florističkim podacima koji postoje za srbiju, balkansko poluostrvo i susedna područja planine rogozne (dolina reke ibar i visokoplaninski region kopaonika). ključne reči: flora, floristička analiza, floristički spektar. introduction rogozna mt. is situated in southwestern serbia in the triangle between the cities of kosovska mitrovica, raška and novi pazar. it is elongated in the northwestern-southeastern direction, bordered by the golija mt. massif on the northwest, slopes of the mokra mt. on the south, a great massif of kopaonik mt. on the east and northeast and stari kolašin on the west. on the south, east and northeast, rogozna mt. is mostly bordered by the ibar river, while on the northwest and west by raška and jošanica river. the main ridge of the mountain is slightly sinuous and starts from the highest mountain peak crni vrh (1479 m) 5 (1) • september 2014: 17-30 biologica nyssana 5 (1)  september 2014: 17-30 papović, o. et al.  analysis of the flora of rogozna… 18 figure 1. geographical position of investigated area followed by čador (1354 m), ravna glava (1369 m), smilov laza (1302), kašalj (1081 m), šanac (1292 m), bar (1207 m), zminjac (1111 m) and vinorog (1225 m). the geological structure of rogozna mt. is mostly represented by serpentine and eruptive stones. the pedological substrate consists of loamy soil. the climate of this area is between temperate and subalpine (b o ž a n i ć , 2006). up to date, rogozna mt. has not been well investigated and therefore, this paper gives a significant contribution to the knowledge of the flora of this mountain and serbia in general. biologica nyssana 5 (1)  september 2014: 17-30 papović, o. et al.  analysis of the flora of rogozna… 19 material and methods herbarium specimens collected during the two-year investigation are deposited at the herbarium of faculty of science and mathematics, university of niš (hmn). identification of the collected plants was performed according to flora europaea (t u t i n et al., 1964-1980) and the regional floras relevant for the investigated area (j o s i f o v i ć , ed., 1970-1977, v e l c h e v , ed., 1982-1989). the nomenclature follows med-checklist (g r e u t e r et al., 1984-1989), flora europaea (t u t i n et al., 1964-1980), and international organization for plant information (iopi) (http://plantnet.rbgsyd.nsw.gov.au/iopi/iopihome.htm). taxonomic structure of the rogozna mt. flora was compared to the flora of the balkan peninsula (t u r r i l l , 1929), serbia (s t e v a n o v i ć et al. 1994), kopaonik mt. (l a k u š i ć , 1993) and ibar river valley (p r o d a n o v i ć , 2007). generic coefficient was calculated using the formula ng/ns x 100, where ng is the number of genera and ns the number of species (jaccard, 1912, a л ë х и н , 1944, j a n k o v i ć , 1985). results and discussion list of the recorded vascular flora of rogozna mt equisetopsida equisetaceae equisetum arvense l. equisetum telmateia ehrh. polypodiopsida aspidiaceae dryopteris filix-mas (l.) schott polystichum setiferum (forskål) woynar polypodiaceae polypodium vulgare l. adiantaceae notholaena maranthae (l.) desv. aspleniaceae asplenium adiantum-nigrum l. asplenium ceterach l. asplenium cuneifolium viv. asplenium ruta-muraria l. asplenium trichomanes l. asplenium septentrionale (l.) hoffm. woodsiaceae cystopteris fragilis (l.) bernh. pinopsida cupressaceae juniperus communis l. magnoliopsida aristolochiaceae asarum europaeum l. ranunculaceae anemone nemorosa l. clematis vitalba l. consolida regalis s. f. gray helleborus odorus waldst. & kit. helleborus serbicus adamović hepatica nobilis schreber ranunculus acris l. ranunculus bulbosus l. ranunculus illyricus l. ranunculus platanifolius l. ranunculus polyanthemos l. ranunculus repens l. ranunculus sardous crantz ranunculus strigulosus schur thalictrum aquilegiifolium l. berberidaceae epimedium alpinum l. papaveraceae fumaria officinalis l. papaver dubium l. papaver rhoeas l. ulmaceae ulmus glabra hudson fagaceae fagus sylvatica l. quercus cerris l. quercus petraea (mattuschka) liebl. subsp. medwediewii (a. camus) menitsky quercus petraea (mattuschka) liebl. subsp. petraea quercus pubescens willd. betulaceae alnus alnobetula (ehrh.) hartig corylaceae carpinus betulus l. carpinus orientalis miller http://plantnet.rbgsyd.nsw.gov.au/iopi/iopihome.htm biologica nyssana 5 (1)  september 2014: 17-30 papović, o. et al.  analysis of the flora of rogozna… 20 corylus avellana l. corylus colurna l. ostrya carpinifolia scop. caryophyllaceae agrostemma githago l. arenaria leptoclados (reichenb.) guss. arenaria serpyllifolia l. atocion armeria fourr. cerastium brachypetalum pers. cerastium decalvans schlosser & vuk. cerastium decalvans schlosser & vuk. cerastium fontanum baumg. subsp. vulgare (hartm.) greuter & burdet cerastium grandiflorum waldst. & kit. cerastium transsilvanicum schur cerastium sylvaticum waldst. & kit. dianthus armeria l. dianthus carthusianorum l. dianthus carthusianorum l. dianthus ciliatus guss. dianthus cruentus griseb. dianthus petraeus waldst. & kit. dianthus pinifolius sibth. & sm. subsp. pinifolius dianthus pinifolius sibth. & sm. subsp. serbicus wettst. dianthus serotinus waldst. & kit. dianthus sylvestris wulfen herniaria glabra l. herniaria hirsuta l. lychnis coronaria (l.) desr. lychnis flos-cuculi l. minuartia graminifolia (ard.) jáv. minuartia hirsuta (bieb.) hand.-mazz. subsp. falcata (griseb.) mattf. minuartia montana l. minuartia verna (l.) hiern. minuartia viscosa (schreber) schinz & thell. misopates orontium (l.) rafin. moehringia trinervia (l.) clairv. moenchia mantica (l.) bartl. paronychia cephalotes (bieb.) besser petrorhagia illyrica (ard.) p. w. ball & heywood petrorhagia saxifraga (l.) link sagina saginoides (l.) karsten saponaria glutinosa bieb. scleranthus annuus l. scleranthus perennis l. subsp. perennis scleranthus perennis l. subsp. dichotomus (schur) nyman scleranthus polycarpos l. silene bellidifoliajacq. silene bupleuroides l. subsp. bupleuroides silene bupleuroides l. subsp. staticifolia (sibth. & sm.) chowdhuri silene conica l. silene gallinyi heuffel ex reichenb. silene italica (l.) pers. silene noctiflora l. silene otites (l.) wibel silene paradoxa l. silene sendtneri boiss. silene viridiflora l. silene vulgaris (moench) garcke stellaria graminea l. stellaria holostea l. stellaria media (l.) vill. viscaria vulgaris bernh. amaranthaceae amaranthus crispus (lesp. & thév.) n. terracc. chenopodiaceae bassia prostrata (l.) a. j. scott chenopodium album l. chenopodium botrys l. chenopodium hybridum l. chenopodium polyspermum l. chenopodium vulvaria l. polygonaceae fallopia convolvulus (l.) å. löve fallopia dumetorum (l.) j. holub persicaria maculosa s. f. gray polygonum arenarium waldst. & kit. polygonum aviculare l. rumex acetosella l. plumbaginaceae goniolimon incanum (l.) hepper limonium gmelinii (willd.) o. kuntze hypericaceae hypericum annulatum moris hypericum barbatum jacq. hypericum elegans stephan ex willd. hypericum hirsutum l. hypericum humifusum l. hypericum montanum l. hypericum perforatum l. violaceae viola aetolica boiss. & heldr. subsp. kopaonikensis pančić ex tomović & niketić, ined. viola arvensis murray viola canina l. viola kitaibeliana schultes viola mirabilis l. viola sylvestris lam. viola tricolor l. biologica nyssana 5 (1)  september 2014: 17-30 papović, o. et al.  analysis of the flora of rogozna… 21 cistaceae fumana bonapartei maire & petitmengin helianthemum alpestre (jacq.) dc. helianthemum canum(l.) baumg. helianthemum nummularium (l.) miller brassicaceae aethionema saxatile (l.) r. br. subsp. graecum (boiss. & spruner) hayek alyssum alyssoides (l.) l. alyssum bertolonii desv. alyssum markgrafii o. e. schulz ex markgraf alyssum montanum l. subsp. serbicum novák alyssum murale waldst. & kit. subsp. murale alyssum repens baumg. arabis alpina l. subsp. caucasica (willd. ex schlecht.) briq. arabis turrita l. aurinia saxatilis (l.) desv. aurinia saxatilis (l.) desv. subsp. orientalis (ard.) t. r. dudley berteroa incana (l.) dc. berteroa mutabilis (vent.) dc. calepina irregularis (asso) thell. capsella bursa-pastoris (l.) medicus cardamine bulbifera (l.) crantz cardamine hirsuta l. cardamine resedifolia l. draba muralis l. erophila verna (l.) chevall. erucastrum gallicum (willd.) o. e. schulz erysimum carniolicum dolliner erysimum cuspidatum (bieb.) dc. erysimum diffusum ehrh. erysimum kuemmerlei jáv. fibigia clypeata (l.) medicus lepidium draba l. rorippa pyrenaica (all.) reichenb. rorippa sylvestris (l.) besser thlaspi alliaceum l. thlaspi arvense l. thlaspi perfoliatum l. resedaceae reseda lutea l. subsp. lutea salicaceae populus tremula l. salix fragilis l. salix purpurea l. salix viminalis l. ericaceae andromeda polifolia l. bruckenthalia spiculifolia (salisb.) reichenb. primulaceae anagallis arvensis l. anagallis foemina miller lysimachia nemorum l. lysimachia nummularia l. tiliaceae tilia platyphyllos scop. tilia tomentosa moench malvaceae malva moschata l. euphorbiaceae euphorbia barrelieri savi subsp. thessala (form.) bornm. euphorbia cyparissias l. euphorbia epithymoides l. euphorbia falcata l. euphorbia glabriflora vis. euphorbia helioscopia l. euphorbia salicifolia host euphorbia stricta l. euphorbia subhastata vis. & pančić euphorbia taurinensis all. euphorbia waldsteinii (soják) a. r. sm. mercurialis ovata sternb. & hoppe mercurialis perennis l. rosaceae agrimonia eupatoria ledeb. amelanchier ovalis medicus aremonia agrimonoides (l.) dc. cotoneaster tomentosus lindley crataegus monogyna jacq. cydonia oblonga miller filipendula ulmaria (l.) maxim. filipendula vulgaris moench fragaria vesca l. geum urbanum l. potentilla argentea l. potentilla erecta (l.) räuschel potentilla heptaphylla l. subsp. australis (krašan ex nyman) gams potentilla hirta l. potentilla incana p. gaertner, b. meyer & scherb. potentilla leucopolitana p. j. mueller potentilla recta l. potentilla reptans l. potentilla tommasiniana f. w. schultz potentilla visianii pančić prunus avium l. prunus spinosa l. pyrus communis l. rosa canina l. biologica nyssana 5 (1)  september 2014: 17-30 papović, o. et al.  analysis of the flora of rogozna… 22 rosa pendulina l. rosa spinosissima l. rubus hirtus waldst. & kit. rubus idaeus l. rubus ulmifolius schott sanguisorba minor scop. sanguisorba officinalis l. sorbus aria (l.) crantz sorbus graeca (spach) kotschy sorbus torminalis (l.) crantz spiraea cana waldst. & kit. spiraea media franz schmidt crassulaceae hylotelephium maximum (l.) holub sedum acre l. subsp. acre sedum hispanicum l. sedum ochroleucum chaix sedum rupestre l. sedum sartorianum boiss. sedum serpentini janchen sempervivum heuffelii schott sempervivum marmoreum griseb. saxifragaceae saxifraga rotundifolia l. parnassiaceae parnassia palustris l. fabaceae anthyllis vulneraria l. astragalus glycyphyllos l. astragalus onobrychis l. astragalus onobrychis l. colutea arborescens l. cytisus hirsutus l. subsp. ciliatus (wahlenb.) ascherson & graebner cytisus hirsutus subsp. hirsutus cytisus jankae velen. cytisus nigricans (schur) nyman cytisus pseudoprocumbens markgraf cytisus supinus l. dorycnium germanicum (gremli) rikli dorycnium herbaceum vill. genista depressa bieb. genista germanica l. genista januensis viv. genista ovata waldst. & kit. hippocrepis comosa l. hippocrepis emeroides (boiss. & spruner) czerep. lathyrus hallersteinii baumg. lathyrus latifolius l. lathyrus niger (l.) bernh. lathyrus nissolia l. lathyrus pratensis l. lathyrus sphaericus retz. lathyrus sylvestris l. lathyrus venetus (miller) wohlf. lathyrus vernus (l.) bernh. lens nigricans (bieb.) godron lotus corniculatus l. medicago arabica (l.) hudson medicago carstiensis jacq. medicago falcata l. medicago lupulina l. medicago minima (l.) bartal. medicago prostrata jacq. medicago sativa l. melilotus officinalis (l.) pallas onobrychis alba (waldst. & kit.) desv. onobrychis arenaria (kit.) dc. onobrychis viciifolia scop. ononis spinosa l. securigera elegans (pančić) lassen securigera varia (l.) lassen trifolium alpestre l. trifolium arvense l. trifolium aureum pollich trifolium badium schreber trifolium campestre schreber trifolium dalmaticum vis. trifolium diffusum ehrh. trifolium hirtum all. trifolium medium l. subsp. balcanicum velen. trifolium montanum l. trifolium ochroleucon hudson trifolium pannonicum jacq. trifolium pignantii fauché & chaub. trifolium pratense l. subsp. pratense trifolium pratense l. subsp. serotinum (witte) holub trifolium repens l. trifolium scabrum l. trifolium striatum l. trifolium sylvaticum gerard sec. c. visioso trifolium trichopterum pančić trifolium velenovskyi vandas vicia cassubica l. vicia cracca l. subsp. cracca vicia cracca l. subsp. incana (gouan) rouy vicia dumetorum l. vicia lathyroides l. vicia pannonica crantz vicia pisiformis l. vicia sativa l. subsp. sativa vicia sativa l. subsp. nigra (l.) ehrh. vicia sepium l. vicia tetrasperma (l.) schreber lythraceae lythrum salicaria l. biologica nyssana 5 (1)  september 2014: 17-30 papović, o. et al.  analysis of the flora of rogozna… 23 oenotheraceae circaea lutetiana l. epilobium angustifolium l. epilobium palustre l. epilobium dodonaei vill. epilobium hirsutum l. epilobium lanceolatum sebastiani & mauri epilobium montanum l. epilobium parviflorum schreber epilobium roseum schreber anacardiaceae cotinus coggygria scop. rutaceae dictamnus albus l. haplophyllum boissieranum vis. & pančić aceraceae acer campestre l. acer heldreichii orph. ex boiss. subsp. visianii k. malý acer hyrcanum fischer & c. a. meyer subsp. intermedium (pančić) bornm. acer platanoides l. acer pseudoplatanus l. acer tataricum l. linaceae linum austriacum l. linum catharticum l. linum flavum l. linum hirsutum l. linum hologynum reichenb. linum tauricum willd. subsp. serbicum (podp.) petrova linum tenuifolium l. balsaminaceae impatiens noli-tangere l. geraniaceae geranium columbinum l. geranium dissectum l. geranium lucidum l. geranium phaeum l. geranium purpureum vill. geranium pyrenaicum burm. fil. geranium robertianum l. geranium sanguineum l. geranium sylvaticum l. polygalaceae polygala alpestris reichenb. polygala comosa schkuhr polygala major jacq. polygala supina schreber cornaceae cornus mas l. cornus sanguinea l. apiaceae aegopodium podagraria l. angelica verticilaris l. astrantia major l. bifora radians bieb. bupleurum apiculatum friv. bupleurum falcatum l. subsp. cernuum (ten.) arcangeli bupleurum falcatum l. subsp. falcatum bupleurum praealtum l. bupleurum rotundifolium l. bupleurum veronense l. carum carvi l. cervaria rivinii gaertner chaerophyllum aureum l. chaerophyllum hirsutum l. daucus carota l. eryngium palmatum pančić & vis. eryngium serbicum pančić falcaria vulgaris bernh. ferulago sylvatica (besser) reichenb. geocaryum cynapioides (guss.) l. engstrand hacquetia epipactis (scop.) dc. holandrea carvifolia (vill.) reduron, charpin & pimenov laserpitium siler l. orlaya daucoides (l.) greuter orlaya grandiflora (l.) hoffm. pastinaca hirsuta pančić peucedanum austriacum (jacq.) koch peucedanum officinale l. peucedanum oreoselinum (l.) moench physospermum cornubiense (l.) dc. pimpinella saxifraga l. pimpinella serbica (vis.) bentham & hooker fil. ex drude sanicula europaea l. smyrnium perfoliatum l. tordylium apulum l. tordylium maximum l. torilis japonica (houtt.) dc. trinia glauca (l.) dumort. trinia ramosissima (fischer ex trev.) koch celastraceae evonymus europaeus l. evonymus verrucosus scop. rhamnaceae frangula dodonei ard. biologica nyssana 5 (1)  september 2014: 17-30 papović, o. et al.  analysis of the flora of rogozna… 24 rhamnus saxatilis jacq. salntalaceae thesium arvense horvátovszky thesium bavarum schrank thesium divaricatum jan. ex mert. & koch thesium linophyllon l. asclepiadaceae vincetoxicum hirundinaria medicus gentianaceae centaurium erythraea rafin. gentiana asclepiadea l. gentiana cruciata l. gentiana lutea l. gentiana pneumonanthe l. gentiana utriculosa l. oleaceae fraxinus ornus l. rubiaceae asperula cynanchica l. asperula hungarorum borbás asperula purpurea (l.) ehrend. subsp. apiculata (sibth. & sm.) ehrend. asperula purpurea (l.) ehrend. subsp. purpurea crucianella angustifolia l. cruciata laevipes opiz cruciata laevipes opiz cruciata pedemontana (bellardii) ehrend. galium album miller galium aparine l. galium mollugo l. galium odoratum (l.) scop. galium rivale (sibth. & sm.) griseb. galium rubrum l. galium sylvaticum l. galium verum l. rubia tinctorum l. caprifoliaceae lonicera caprifolium l. sambucus ebulus l. sambucus nigra l. valerianaceae valeriana officinalis l. dipsacaceae cephalaria laevigata (waldst. & kit.) schrader cephalaria leucantha (l.) roemer & schultes knautia dinarica (murb.) borbás knautia dipsacifolia kreutzer subsp. lancifolia (heuffel) ehrend. knautia drymeia heuffel scabiosa argentea l. scabiosa columbaria l. scabiosa fumarioides vis. & pančić scabiosa ochroleuca l. succisa pratensis moench convolvulaceae convolvulus arvensis l. convolvulus cantabricus l. cuscuta epithymum (l.) l. boraginaceae anchusa officinalis l. cerinthe minor l. echium vulgare l. halacsya sendtneri (boiss.) dörfler lithospermum purpureocaeruleum l. myosotis arvensis (l.) hill myosotis nemorosa besser myosotis scorpioides l. myosotis sparsiflora mikan ex pohl myosotis sylvatica hoffm. onosma echioides l. pulmonaria officinalis l. symphytum tuberosum l. solanaceae atropa bella-donna l. physalis alkekengi l. solanum nigrum l. scrophulariaceae chaenorhinum minus (l.) lange digitalis ferruginea l. digitalis grandiflora miller digitalis laevigata waldst. & kit. digitalis lanata ehrh. euphrasia picta subsp. kerneri (wettst). yeo euphrasia stricta d. wolff ex j. f. lehm. euphrasia tatarica fischer ex sprengel kickxia elatine(l.) dumort. linaria genistifolia (l.) miller linaria genistifolia (l.) miller subsp. sofiana (velen.) chater & d. a. webb linaria rubioides vis. & pančić linaria vulgaris miller melampyrum arvense l. melampyrum bihariense a. kerner melampyrum cristatum l. melampyrum fimbriatum vandas melampyrum heracleoticum boiss. & orph. melampyrum hoermannianum k. malý melampyrum nemorosum l. melampyrum pratense l. melampyrum scardicum wettst. biologica nyssana 5 (1)  september 2014: 17-30 papović, o. et al.  analysis of the flora of rogozna… 25 odontites luteus (l.) clairv. odontites vernus (bellardi) dumort. pedicularis comosa l. rhinanthus angustifolius c. c. gmelin scrophularia canina l. scrophularia nodosa l. scrophularia scopolii hoppe ex pers. scrophularia tristis (k. malý) šilić verbascum banaticum schrader verbascum lychnitis l. verbascum phlomoides l. verbascum thapsus l. veronica anagalloides guss. veronica austriaca l. veronica beccabunga l. veronica chamaedrys l. veronica cymbalaria bodard. veronica incana l. veronica jacquinii baumg. veronica officinalis l. veronica spicata l. orobanchaceae orobanche nowackiana markgraf orobanche reticulata wallr. acanthaceae acanthus hungaricus (borbás) baenitz plantaginaceae plantago altissima l. plantago argentea chaix subsp. liburnica v. ravnik plantago holosteum scop. subsp. holosteum plantago media l. verbenaceae verbena officinalis l. lamiaceae acinos alpinus (l.) moench subsp. albanicus (kümmerle & jáv.) niketić acinos arvensis (lam.) dandy acinos hungaricus (simonkai) šilić ajuga genevensis l. ajuga laxmannii (l.) bentham ballota nigra l. calamintha officinalis moench clinopodium menthifolium merino clinopodium thymifolium (scop.) kuntze clinopodium vulgare l. galeopsis speciosa miller glechoma hirsuta waldst. & kit. lamium galeobdolon (l.) l. lamium garganicum l. lamium maculatum l. lamium purpureum l. leonurus cardiaca l. lycopus europaeus l. melittis melissophyllum l. subsp. albida (guss.) p. w. ball mentha × piperita l. mentha aquatica l mentha longifolia (l.) hudson nepeta cataria l. origanum vulgare l. prunella laciniata (l.) l. prunella vulgaris l. salvia glutinosa l. salvia nemorosa l. salvia sclarea l. salvia verticillata l. scutellaria altissima l. scutellaria columnae all. scutellaria galericulata l. sideritis montana l. stachys alpina l. stachys alpina l. subsp. dinarica murb. stachys cretica l. subsp. cassia (boiss.) rech. fil. stachys officinalis (l.) trevisan stachys recta l. stachys recta l. subsp. baldaccii (k. malý) haye stachys recta l. stachys scardica (griseb.) hayek teucrium chamaedrys l. teucrium montanum l. thymus glabrescens willd. thymus lykae degen thymus praecox opiz subsp. jankae (čelak.) jalas thymus pulegioides l. ziziphora capitata l. campanulaceae asyneuma anthericoides (janka) bornm. asyneuma limonifolium (l.) janchen campanula bononiensis l. campanula cervicaria l. campanula glomerata l. campanula grossekii heuffel campanula lingulata waldst. & kit. campanula moesiaca velen. campanula patula l. campanula persicifolia l. campanula rapunculoides l. campanula rapunculus l. campanula sparsa friv. campanula sparsa friv. subsp. sphaerothrix (griseb.) hayek campanula trachelium l. legousia speculum-veneris (l.) chaix phyteuma orbiculare l. biologica nyssana 5 (1)  september 2014: 17-30 papović, o. et al.  analysis of the flora of rogozna… 26 asteraceae achillea crithmifolia waldst. & kit. achillea distans waldst. & kit. ex willd. achillea grandifolia friv. achillea millefolium l. anthemis arvensis l. anthemis ruthenica bieb. arctium lappa l. artemisia alba turra artemisia vulgarisl. aster alpinus l. aster amellus l. bidens cernua l. bidens tripartita l. bombycilaena erecta (l.) smolj. carduus candicans waldst. & kit. carduus personata (l.) jacq. carlina vulgaris l. centaurea jacea l. centaurea jacea l. subsp. angustifolia (dc.) gremli centaurea phrygia l. centaurea phrygia l. subsp. stenolepis a. kerner centaurea scabiosa l. centaurea stoebe l. subsp. australis (a. kerner) greuter centaurea stoebe l. subsp. stoebe cichorium intybus l. subsp. intybus cirsium arvense (l.) scop. cirsium eriophorum (l.) scop. cirsium grecescui rouy cirsium vulgare (savi) ten. cota austriaca (jacq.) schultz-bip. cota tinctoria (l.) j. gay crepis biennis l. crepis foetida l. subsp. rhoeadifolia crepis pulchra l. crepis sancta (l.) bornm. crepis setosa haller fil. crepis vesicaria l. subsp. taraxacifolia (thuill.) thell. crupina vulgaris cass. cyanus triumfettii (all.) á. löve & d. löve doronicum columnae ten. echinops exaltatus schrader echinops ritro l. subsp. ruthenicus (bieb.) nyman erigeron acris l. subsp. acris erigeron annuus (l.) desf. erigeron canadensis l. eupatorium cannabinum l. filago arvensis l. filago minima (sm.) pers. galatella albanica degen galatella linosyris (l.) bernh. gnaphalium sylvaticum l. gnaphalium uliginosum l. hieracium bifidum hornem. hieracium murorum l. hieracium racemosum willd. hieracium sabaudum l. hieracium tommasinianum k. malý hypochaeris illyrica k. malý hypochaeris maculata l. hypochaeris radicata l. inula britannica l. inula conyzae dc. inula ensifolia l. inula hirta l. inula oculus-christi l. inula salicina l. jurinea mollis (l.) reichenb. lactuca muralis (l.) gaertner lactuca saligna l. lactuca viminea (l.) j. presl & c. presl lapsana communis l. leontodon biscutellifolius dc. leontodon crispus vill. leontodon hispidus l. leucanthemum vulgare lam. picris hieracioides l. pilosella bauhinii (schultes) arv.-touv. pilosella sabina (sebastiani & mauri) f. w. schultz & schultz bip. fratt. pilosella cymosa (l.) f. w. schultz & schultz bip. fratt. pilosella hoppeana (schultes) f. w. schultz & schultz bip. fratt. pilosella officinarum f. w. schultz & schultz bip. fratt. podospermum canum c. a. meyer podospermum laciniatum (l.) dc. prenanthes purpurea l. pulicaria dysenterica (l.) bernh. reichardia dichotoma (dc.) freyn scorzonera austriaca willd. scorzonera hispanica l. senecio leucanthemifolius poiret subsp. vernalis (waldst. & kit.) greuter senecio squalidus l. subsp. squalidus senecio squalidus l. subsp. rupestris (waldst. & kit.) greuter ined. senecio vulgaris l. serratula tinctoria l. solidago virgaurea l. tanacetum corymbosum (l.) schultz bip. tanacetum macrophyllum (waldst. & kit.) schultz-bip. tanacetum parthenium (l.) schultz-bip. taraxacum officinale weber tephroseris crassifolia (schultes) griseb. & schenk biologica nyssana 5 (1)  september 2014: 17-30 papović, o. et al.  analysis of the flora of rogozna… 27 tephroseris papposa (reichenb.) schur tragopogon dubius scop. tragopogon pterodes pančić ex petrović tripleurospermum inodorum (l.) schultz-bip. tripleurospermum tenuifolium (kit.) freyn xeranthemum annuum l. liliopsida liliaceae anthericum liliago l. anthericum ramosum l. colchicum autumnale l. convallaria majalis l. lilium martagon l. ornithogalum gussonei ten. paris quadrifolia l. polygonatum multiflorum (l.) all. polygonatum odoratum (miller) druce allium carinatum l. subsp. pulchellum bonnier & layens allium flavum l. allium moschatum l. allium paniculatum l. allium scorodoprasum l. allium senescens l. subsp. montanum (f. w. schmidt) holub allium sphaerocephalon l. tulipa scardica bornm. tulipa serbica tatić & krivošej iridaceae iris graminea l. dioscoreaceae tamus communis l. orchidaceae cephalanthera damasonium (miller) druce cephalanthera longifolia (l.) fritsch epipactis helleborine (l.) crantz epipactis microphylla (ehrh.) swartz gymnadenia conopsea (l.) r. br. gymnadenia odoratissima (l.) l. c. m. richard neottia nidus-avis (l.) l. c. m. richard orchis ustulata l. platanthera bifolia (l.) l. c. m. richard juncaceae juncus articulatus l. juncus atratus krocker juncus bufonius l. juncus conglomeratus l. juncus effusus l. juncus inflexus l. juncus thomasii ten. luzula campestris (l.) dc. luzula luzuloides (lam.) dandy & wilmott luzula pilosa (l.) willd. luzula sylvatica (hudson) gaudin cyperaceae carex digitata l. carex distans l. carex divisa hudson carex divulsa stokes carex echinata murray carex hirta l. carex ovalis good. carex pairae f. w. schultz carex pallescens l. carex spicata hudson carex vulpina l. cyperus fuscus l. eleocharis palustris (l.) roemer & schultes scirpus sylvaticus l. poaceae achnatherum calamagrostis (l.) beauv. agropyron cristatum (l.) gaertner agrostis capillaris l. alopecurus pratensis l. apera spica-venti (l.) beauv. bothriochloa ischaemum (l.) keng brachypodium pinnatum (l.) beauv. brachypodium sylvaticum (hudson) beauv. bromus commutatus schrader bromus hordeaceus l. bromus pannonicus kummer & sendtner bromus ramosus hudson bromus riparius rehmann bromus squarrosus l. bromus sterilis l. calamagrostis epigejos (l.) roth chrysopogon gryllus (l.) trin. cynosurus cristatus l. cynosurus echinatus l. dactylis glomerata l. danthonia alpina vest. dasypyrum villosum (l.) p. candargy elymus repens (l.) gould festuca pratensis hudson festuca valesiaca schleicher ex gaudin festuca valesiaca schleicher ex gaudin subsp. parviflora (hack.) tracey glyceria notata chevall. holcus lanatus l. koeleria glauca (schrader) dc. lolium perenne l. melica ciliata l. melica uniflora retz. biologica nyssana 5 (1)  september 2014: 17-30 papović, o. et al.  analysis of the flora of rogozna… 28 molinia arundinacea schrank phleum bertolonii dc. phleum montanum c. koch phleum phleoides (l.) karsten phleum pratense l. piptatherum virescens (trin.) boiss. poa badensis haenke ex willd. poa bulbosa l. poa bulbosa l. subsp. pseudoconcinna (schur) domin poa compressa l. poa nemoralis l. poa pratensis l. setaria viridis (l.) beauv. stipa pennata l. stipa pulcherrima c. koch trisetum flavescens (l.) beauv. araceae arum maculatum l. taxonomic diversity flora of investigated area contains even 24.30% of the plant species recorded for the territory of serbia. significant quantitative indicator of floristic richness and taxonomic diversity is generic coefficient (42.51%). this value indicates a relatively low diversity of habitats on one hand, and relatively low level of autochthonous florogenesis tendency on the other hand. this phenomenon is a consequence of a pretty uniform geological structure and relatively small size of the investigated area. the most abundant family of the investigated area is, as expected, the most abundant family of the holarctic kingdom (in both species and genera), family asteraceae. at the investigated area, 106 species from 47 genera were recorded, which represents 13.33% of the total flora. beside the asteraceae family, the most numerous families considering the number of the species were fabaceae (75), caryophyllaceae (59), lamiaceae (49), poaceae (48) and scrophulariaceae (44), together with apiaceae (39), rosaceae (36) and brassicaceae (33). regarding the number of genera, the most numerous was, once again, family asteraceae, followed by poaceae (28), apiaceae (26), lamiaceae (22), caryophyllaceae (29), fabaceae (17) and rosaceae (17). floristic spectrum of the rogozna mt. deviates somewhat from the spectra of serbia and balkan peninsula (tab.1). the increased presence of the boreal and arctic families rosaceae and poaceae indicates a significant impact of these two horions on the rogozna mt. flora genesis. the presence of the families characteristic for mediteranneansubmediterannean region (fabaceae, caryophyllaceae) is increased. this can be explained by the geographical position of investigated area, floristic and florogenetic mediterranean impacts and the relatively low average elevation of the rogozna mt. the flora of rogozna mt. is differentiated from the flora of serbia by centraleuropean and temperate-boreal elements owing to reduced percentage of the families brassicace and poaceae (fig.2). table 1. comparative review of taxonomical structure of the most abundant families of rogozna mt., serbia (stevanović et al., 1995) and the balkan peninsula (turill, 1929) rogozna mt. serbia balkan peninsula family n % n % n % asteraceae 106 13.33 366 11.19 913 13.52 fabaceae 75 9.43 250 7.64 545 8.07 caryophyllaceae 59 7.42 205 6.27 418 6.19 lamiaceae 49 6.16 148 4.52 371 5.49 poaceae 48 6.04 250 7.64 358 5.30 scrophulariaceae 44 5.53 161 4.92 311 4.65 apiaceae 39 4.91 142 4.34 334 4.95 rosaceae 36 4.53 111 3.39 188 2.78 brassicaceae 33 4.15 194 5.93 344 5.09 rubiaceae 18 2.26 49 1.50 129 1.91 biologica nyssana 5 (1)  september 2014: 17-30 papović, o. et al.  analysis of the flora of rogozna… 29 figure 2. percentage deviation of the most abundant families in the flora of rogozna mt. from the families spectra of serbia and the balkan peninsula. taxonomical spectrum of the rogozna mt. flora was compared with spectra of neighboring kosovo’s part of ibar river valley (p r o d a n o v i ć , 2007) and the high mountain flora of kopaonik (l a k u š i ć , 1993). altitudinal range of the ibar river valley investigation area was 500-900 m and of the rogozna mt. was 800-1473, where similarity reflected in almost identical schedule of the 9 most abundant families is obvious (fig. 3). just like on rogozna mt., mediterranean impacts are more pronounced in ibar river valley in contrast to the high mountain area of kopaonik. on kopaonik, the second place in taxonomical spectrum belongs to the family of arctic and boreal regions, poaceae (9.34%) (l a k u š i ć , 1993), which is another proof of weaker mediteranian impact. investigation of the taxonomical spectrum showed that trifolium genera is the most abundant with 21 species (2.71%), followed by campanula with 13 species, dianthus and silene with 12 species, carex, euphorbia, potentilla, vicia with 11 species, melampyrum with 10 species, geranium, lathyrus and veronica with 9 species (tab. 2). the dominance of the genera trifolium is probably caused by the increased presence of xerothermic meadows and pastures. hieracium, the most abundant genera on the balkan peninsula, is even on the 33 rd place in the rogozna mt. taxonomical spectrum, which can only be explained by insufficient taxonomical research of this genera (jaccard, 1912). figure 3. overview of the most abundant families on the rogozna mt. and neighboring regions table 2. taxonomic structure of the most abundant genera in the flora of rogozna mt. genera species n° % trifolium 21 2.71 campanula 13 1.68 dianthus 12 1.55 silene 12 1.55 carex 11 1.42 euphorbia 11 1.42 potentilla 11 1.42 vicia 11 1.42 melampyrum 10 1.29 geranium 9 1.16 lathyrus 9 1.16 veronica 9 1.16 centaurea 8 1.03 epilobium 8 1.03 galium 8 1.03 ranunculus 8 1.03 stachys 8 1.03 references aлëхин, в.в., 1944: география растений.-изд. советская наука, москва. 455 p. božanić, s., 2006: ibarsko jezgro svetostefanskog vlastelinstva. filozofski fakultet novi sad-ia “srem”, novi sad-sremska mitrovica. 494 p. greuter, w., burdet, h.m., long, g. (ed.), 19841989: med-checklist, 1, 3, 4. gèneve. jaccard, p., 1912: the distribution of flora in the alpine zone. new phytologist, 11: 37-50. janković, m.m., 1985: fitogeografija. univerzitet u beogradu. 425 p. josifović, m. (ed.), 1970-1976: flora sr srbije, iix. sanu. beograd. biologica nyssana 5 (1)  september 2014: 17-30 papović, o. et al.  analysis of the flora of rogozna… 30 lakušić, d., 1993: visokoplaninska fl ora kopaonika ekološko fi togeografska studija. magistarski rad. biološki fakultet, beograd. prodanović, d., 2007: serpentinska flora kosovskog dela ibarske doline. doktorska disertacija. univerzitet u prištini, prirodno matematički fakultet, kosovska mitrovica. stevanović, v., vasić, v. (ed.), 1995: biodiverzitet jugoslavije sa pregledom vrsta od međunarodnog značaja. biološki fakultet i ekolibri, beograd. the international organisation for plant information http://plantnet.rbgsyd.nsw.gov.au/iopi/iopihome. htm)). turrill, w.b., 1929: the plant life of the balkan peninsula. a phytogeographical study. clarendon, oxford. tutin, t.g., heywood, v.h., burges, n.a., moore, d.m., valentine, d.h., walters, s.m., webb, d.a. (ed.), 1964-1980: flora europaea, i-v. cambridge, university press. london. velchev, v. (ed.), 1982–1989: flora reipubl. popularis bulgaricae, vols 8-9. in aedibus acad. sci. bulgaricae, serdicae (in bulgarian) http://plantnet.rbgsyd.nsw.gov.au/iopi/iopihome.htm) http://plantnet.rbgsyd.nsw.gov.au/iopi/iopihome.htm) pepłowska-marczak et al., 2019, biologica nyssana 10(1) 10 (1) september 2019: 63-64 doi: 10.5281/zenodo.3464012 new observations of the woodchat shrike lanius senator linnaeus, 1758 in southeastern serbia short communication danuta pepłowska-marczak kampinos national park, department of science and monitoring, tetmajera street 38, izabelin, poland sowa.ruda@gmail.com dawid marczak university of ecology and management in warsaw, olszewska street 12, warsaw, poland dawid.marczak@gmail.com saša s. stanković university of niš, department of biology and ecology, faculty of sciences and mathematics, višegradska 33, 18000 niš, serbia sasasta@gmail.com (corresponding author) received: december 10, 2018 revised: mart 21, 2019 accepted: august 13, 2019 abstract: during the period from 2013 to 2018 three observations of the woodchat shrike (lanius senator linnaeus, 1758) were made. one pair and two single males were observed in the southeastern serbia on three localities. localities of the three records were marked on the utm map of serbia and utm codes were given. key words: lanius senator, woodchat shirke, birds of serbia apstrakt: nova opažanja crvenoglavog svračka lanius senator linnaeus, 1758 u jugoistočnoj srbiji u periodu od 2013. do 2018. godine zabeležena su tri zapažanja crvenoglavog svračka (lanius senator linnaeus, 1758). jedan par i dva samca su primećeni u jugoistočnoj srbiji na tri lokaliteta. lokaliteti ovih zapisa označeni su na utm mapi srbije i date su utm šifre. ključne reči: lanius senator, crvenoglavi svračak, ptice srbije woodchat shrike lanius senator linnaeus, 1758 is a rare breeding species recorded in several regions of serbia (puzović et al., 2015, šćiban et al., 2015). this species occurs in open areas with shrubs, usually on the hills (šćiban et al., 2015). in serbia the size of population is estimated at 580-790 nesting pairs, out of which in serbia without kosovo i metohija 280-390 nesting pairs respectively. it is estimated that its population is increasing (puzović et al., 2015). woodchat shrike breeds in west palearctic in middle and lower middle latitudes mainly in mediterranean climatic zone (snow & perrins, 1998). the most northern distribution is in northern poland, in gdansk pomerania (neubauer, 2013). in this short note we report three new observations of this species in southeastern serbia, from 2013 to 2018. observations were made using leica 10x50 binoculars, while birdwatching in serbia. during this period, l. senator individuals were spotted on the following localities (fig 1): 1. one pair observed in suitable nesting habitat in breeding season. 17.07.2013. milovanci, near vlasina lake, southeastern serbia. utm grid code: fn12 (42°40’50.54’’ n, 22°21’52.98’’ e) on the map marked with black triangle (fig 1). obs. danuta pepłowska-marczak, dawid marczak. 2. one adult male observed in breeding season in possible nesting habitat. 26.07.2017. čar near the border between serbia and fyr macedonia, southeastern serbia. utm grid code: em57 (42°15’43.27’’ n, 21°42’45.40’’ e) on the map marked with black square (fig 1). obs. danuta pepłowska-marczak, dawid marczak. 3. one adult male observed in breeding season in possible nesting habitat. 10.05. 2018. ivanje, near leskovac, southeastern serbia. utm grid code: en46 (43°00’11.54’’ n, 21°35’07.96’’ e). on the map marked with black circle (fig 1). obs. danuta pepłowska-marczak, dawid marczak, saša stanković. observed individuals occurred in the open area mostly dry meadows covered with individual low bushes. the presence of thorny shrubs, mainly from the family rosaceae such as pyrus spp., crataegus monogyna jacq., prunus spinosa l. and others was notable (fig 2). the males in all cases showed territorial behaviors, with breeding status: possible breeder or probable breeding. © 2019 pepłowska-marczak et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work noncommercially under the same license as the original. 63 biologica nyssana ● 10 (1) september 2019: 63-64 pepłowska-marczak et al. ● new observations of the woodchat shrike lanius senator linnaeus, 1758 in southeastern serbia these observations of l. senator represent a contribution to the distribution and population census of this species, and also the need of protection of its habitat. fig 1. utm map of serbia with tree localities where woodchat shrike specimens were spotted. locality 1: milovanci, near vlasina lake; black triangle. locality 2: čar, serbia and fyr macedonia border; black square. locality 3: ivanje, near leskovac; black circle fig 2. typical habitat of woodchat shrike. ivanje, southeastern serbia. photo s. stanković references neubauer, g. 2013. recent records of lesser grey shrike lanius minor and woodchat shrike l. senator in gdańsk pomerania. ptaki pomorza, 4: 135147. puzović, s., radišić, d., ružić, m., rajković, d., radaković, m., pantović, u., janković, m., stojnić, n., šćiban, m., tucakov, m., gergelj, j., sekulić, g., agošton, a., raković, m. 2015. ptice srbije: procena veličina populacija i trendova gnezdarica 2008-2013. društvo za zaštitu i proučavanje ptica srbije, novi sad. 159 p. snow, d. w., perrins, c. m. 1998. the birds of the western palearctic, concise edition. vol. 2, passerines. oxford university press, oxford, new york. 1697 p. šćiban, m., rajković, d., radišić, d., vasić, v., pantović, u. 2015. ptice srbije kritički spisak vrsta. pokrajinski zavod za zaštitu prirode, društvo za zaštitu i proučavanje ptica srbije, novi sad. 196p. 64 stefanović et al. 2021, biologica nyssana 12(2) 12 (2) december 2021: 167-176 doi: 10.5281/zenodo.5759829 melatonin affects gene expression of noradrenaline transporter and enzymes in the hearts of stressed rats original article bojana stefanović laboratory of molecular biology and endocrinology, vinča institute of nuclear sciences, the national institute of the republic of serbia, university of belgrade, mike petrovića alasa 12-14, 11351 belgrade, serbia stefanovic_bojana@vin.bg.ac.rs (corresponding author) nataša spasojević laboratory of molecular biology and endocrinology, vinča institute of nuclear sciences, the national institute of the republic of serbia, university of belgrade, mike petrovića alasa 12-14, 11351 belgrade, serbia predrag jovanović cedars-sinai, centre for neural science and medicine, 8700 beverly blvd., los angeles, ca 90048, usa harisa ferizović laboratory of molecular biology and endocrinology, vinča institute of nuclear sciences, the national institute of the republic of serbia, university of belgrade, mike petrovića alasa 12-14, 11351 belgrade, serbia milica janković laboratory of molecular biology and endocrinology, vinča institute of nuclear sciences, the national institute of the republic of serbia, university of belgrade, mike petrovića alasa 12-14, 11351 belgrade, serbia perica vasiljević department of biology and ecology, faculty of sciences and mathematics, university of nis, višegradska 33, 18000 niš, serbia slađana dronjak laboratory of molecular biology and endocrinology, vinča institute of nuclear sciences, the national institute of the republic of serbia, university of belgrade, mike petrovića alasa 12-14, 11351 belgrade, serbia received: april 28, 2021 revised: september 01, 2021 accepted: november 01, 2021 abstract: the role of stress is important in the etiology of depression which subsequently affects cardiovascular regulation. there has been a lot of evidence of the beneficial effects of melatonin in various cardiovascular pathologies. we examined the effect of chronic melatonin treatment on noradrenaline content, synthesis, transport and degradation in the left atria and ventricle of rats exposed to chronic mild unpredictable stress (cums). our results show that cums decreased expression of mrna th and net in the left atria and comt in the left ventricles, whereas increased mao-a enzymes in the left ventricles. melatonin treatment induced a significant increase in gene expression of net in the atria and a decrease in mao-a in the ventricle. the observed beneficial effects of enhanced uptake and reduced degradation during melatonin treatment were most probably a compensatory mechanism which protects cardiomyocytes from the deleterious effects of noradrenaline overstimulation. key words: melatonin, noradrenaline, heart, stress, transporter, enzymes apstrakt: efekat melatonina na ekspresiju gena za noradrenalinski transporter i enzime degradacije u srcu hronično stresiranih pacova uloga stresa je važna u etiologiji depresije koja posledično može uticati na kardiovaskularnu regulaciju. postoji mnogo dokaza o blagotvornom dejstvu melatonina u različitim kardiovaskularnim oboljenjima. iz tih razloga smo ispitivali uticaj hroničnog tretmana melatoninom na količinu noradrenalina, njegovu sintezu, transport i degradaciju u levoj pretkomori i komori pacova izlaganih hroničnom blagom nepredvidivom stresu (cums). naši rezultati pokazuju da je cums smanjio količinu irnk za th i net u levoj pretkomori i comt u levoj komori, dok je povećao gensku ekspresiju mao-a enzima u levoj komori. tretman melatoninom je značajno povećao gensku ekspresiju net u pretkomorama i smanjio količinu enzima mao-a u komori. uočeni korisni efekti melatonina na ponovno preuzimanje i smanjenu degradaciju noradrenalina predstavljaju kompenzacione mehanizme koje štite kardiomiocite od štetnih efekata njegove prekomerne stimulacije. ključne reči: melatonin, noradrenalin, srce, stres, transporter, enzimi introduction stress is an underlying trigger that leads to the development of depression as well as cardiovascular disease. depression is found in 20– 30% of cardiovascular patients (whooley, 2006). depression is associated with the autonomic nervous system dysfunction which could subsequently lead to cardiovascular disregulation (carney et al., 2005). several studies have demonstrated an exaggerated cardiac noradrenaline response in major depression (veith et al., 1994; yehuda et al., 1998; mausbach et al., 2005). the methods inducing depression-like states in experimental animals are generally accepted as valuable tools for the studies of affective disorders. among these methods, chronic unpredictable mild stress (cums) model of depression has been frequently employed (willner et al. 1992). grippo et al. (2006) reported that cums model of depression is characterized by © 2021 stefanović et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 167 anhedonia and elevated sympathetic cardiac tone. functional noradrenergic transmission consists of a balance between noradrenaline synthesis, secretion and reuptake. noradrenergic activity is dependent on the synthesis of noradrenaline as determined by the rate limiting enzyme tyrosine hydroxylase (th). noradrenaline released from cardiac sympathetic nerve terminals is removed from the neuroeffector junction by the neuronal noradrenaline transporter (net) and metabolized to dihydroxyphenylglycol (dhpg) via monoamine oxidase-a (mao-a) (esler et al., 1990). jovanović et al. (2019) reported that exposure of the animals to social isolation for 12 weeks induced hypertrophy of cardiomyocytes in the wall of the left ventricle and a significant increase in noradrenaline levels in the left atria and the ventricle. melatonin (n-acetyl-5-methoxytryptamine) is a neuroendocrine hormone, which is synthesized primarily by the pineal gland (hardeland et al., 2006). melatonin easily crosses all morphophysiological barriers, and it can enter all cells of the body, affecting the function of a variety of tissues (tan et al. 1999). additionally, by virtue of its ability to detoxify free radicals and related oxygen derivatives, melatonin affects the molecular physiology of cells and contributes to improved cellular and organismal physiology (reiter et al., 2010). the experimental data obtained from rodent studies suggest that melatonin has beneficial effects in the treatment of cardiovascular diseases (reiter et al., 2010). melatonin is also a well-established antioxidant molecule with many beneficial effects in various cardiovascular pathologies (sun, et al., 2016). in humans, melatonin production not only diminishes with age (sack et al., 1986) but is also significantly lower in many age-related diseases, including cardiovascular disease (sakotnik et al., 1999; altun et al., 2002; dominguez-rodriguez et al., 2002; yaprak et al., 2003). the link between the heart and melatonin and the effect of endogenous melatonin on cardiovascular function is well established (dominguez-rodriguez et al., 2010). melatonin interacts with the heart and blood vessels indirectly via the nervous system (sewerynek, 2002; pechanova et al., 2014), and directly, through its receptor-dependent and independent activities as a signaling molecule and a free radical scavenger (paulis et al., 2012; galano & reiter, 2018). there is also an increasing amount of evidence regarding the beneficial effects of melatonin via exogenous administration (tarocco et al., 2019). recently, we have reported that melatonin treatment enhanced noradrenaline content and protein expression of th, dbh, pnmt, and net in the adrenal medulla of rats exposed to cums (stefanovic et al., 2019). although many studies have indicated its cardioprotective actions, the underlying mechanistic links remain unclear. hence, it is important to examine the regulation of more specific variables, such as the gene expression of noradrenaline biosynthetic and degrading enzymes and transporter in the left atria and ventricles of rats exposed to cums model of depression. materials and methods animals and treatment the experiments were performed on adult (twomonth-old) male wistar rats. the weight of males at the beginning of the experiment was 250-310 g. all animals were housed in a temperature-controlled room (20±2.0 °c) and synchronised to a 12-hour light/dark regime. four rats were kept in a standard plastic cages and given ad libitum access to standard laboratory food (commercial rat pellets) and tap water. care was taken to minimise the pain and discomfort of the animals in accordance with the guide for the care and use of laboratory animals (8th edition, national academies press). all procedures with animals were approved by the ethical committee for the use of laboratory animals of the vinča institute and the ministry of agriculture and environmental protection, authority for veterinary, permission no. 323-07-04657/2015-05/02. in this experiment, we used 24 animals, which were allocated to control (unstressed) and chronic unpredictable mildly stressed groups. these groups were further divided into two subgroups each and were receiving daily injection of vehicle (5% ethanol) or melatonin in dose of 10 mg kg-1 body weight by intraperitoneally (i.p.) route one hour before the dark phase (bassani et al., 2014). this time was chosen to avoid disruption of circadian rhythms that occurs if melatonin is administered throughout the day. melatonin (q-1300, bachem, switzerland) was dissolved in nacl (0.9%) containing 5% ethanol. the dose of 10 mg kg-1 is a supraphysiological dose chosen with the aim of assessing the drug’s therapeutic effect and not for replacing or restorative purposes. the dosage of 10 mg/kg/day was regularly adjusted according to the body weight for the entire period of treatment. exposure to cums and the vehicle, i.e. drug administration started on the same day and continued for 4 weeks. the stressors were presented for 1 week and repeated during the following 4 weeks. the same stress method was not continuously applied to ensure the rats would be unable to anticipate the next type of stress that would be applied. control animals were left undisturbed in their home cages with the exception of general handling (i.e. regular 168 biologica nyssana ● 12 (2) december 2021: 167-176 stefanović et al. ● melatonin affects gene expression of noradrenaline transporter and enzymes in the hearts of stressed rats 169 cage cleaning and body weight measuring). chronic unpredictable mild stress the cums procedure was carried out according to the methods described by grippo et al. (2002), and it was designed to maximise the unpredictable nature of the stressors. the cums groups of animals were exposed to the following stressors in random order: forced running (15 mins., 10 m/ min); soiled cage (500 ml 22 °c water spilled into bedding for 5 h); 45° cage tilt along the vertical axis for 7 h; 17 h food deprivation, 5 h cold room (4 °c) and water deprivation (water bottles were removed from cage during this time), 5 h grouped housing (8 rats per cage) and individual housing (48 h). control animals were left undisturbed in the home cage with the exception of general handling (i.e. regular cage cleaning and body weight measuring), which was matched to that of the cums group. immediately after the cums procedure, all animals were sacrificed, between 10:00–11:00 a.m., by rapid decapitation with a guillotine (harvard-apparatus, usa). left atria and ventricles were extracted quickly on ice, frozen in liquid nitrogen and stored at -70 °c until sample preparation for further molecular analysis. high-performance liquid chromatography assay ethylene glycol tetraacetic acid (egta) and dlnorepinephrine hydrochloride (na) were purchased from sigma–aldrich. ammonium formate was supplied by fisher scientific (loughborough, uk); formic acid (49–51%) by fluka analytical (switzerland); methanol by j.t. baker (deventer, the netherlands), perchloric acid (70%) by sigma– aldrich and magnesium chloride (mgcl2×5h2o) by sigma–aldrich. purified water was obtained via a blueclearro600p reverse osmosis water cleaning system with integrated bluesoft07-mb mixed bed salt remover (euro-clear ltd., hungary). the stock standard solution of na [1 mg/ ml] was prepared in methanol and kept at -20 °c. standard solutions in concentrations of 1, 2, 4, 8, 16, 32, 40 and 50 µg/ml were prepared by dilution of the stock standard solution in deprot (2% egta; 0,1n hclo4; 0.2% mgcl2). the ammonium formate buffer (100 mm, ph 3.6) was used as one of the mobile phase components. tissue samples were homogenised in deprot (1 mg:10 µl) using an ultra-turrax homogeniser (t10 basic), sonicated (branson sonic power company, danbury, connecticut; 3×10 s) and centrifuged (sorvall supert21; 30 mins, 18.000 rpm, +4 °c). supernatants were transferred in separate tubes and placed in the autosampler of the hplc system. data were obtained using a thermo scientific (dionexultimate 3000) hplc system consisting of degasser unit, binary pump (hpg3200sd), autosampler (wps-3000 splitloop), column oven (col. comp. tcc-3000) and rs electrochemical detector (ecd-3000rs) equipped with the 6041rs ultra amperometric analytical cell and glassy carbon working electrode. a hibar 125-4 licrospher100 rp-18 (5 µm) hplc column (merck millipore, darmstadt, germany) was used. instrument control and data acquisition was carried out by the chromeleon7 chromatography data system (thermo scientific). the following chromatographic conditions were obtained after the method optimisation. the mobile phase consisting of the ammonium formate buffer (100 mm, ph 3.6) as an a solution and methanol as a b solution was pumped at a flow rate of 500 µl/mins with the following step gradient. the run started with a mobile phase consisting of 98% a and 2% b solution. at the 9th minute of the run the part of b solution started to rise, reaching the 50% in the 15th mins and 80% in the 18th mins. starting from the 20th mins until the end of the run (30th mins) the column was re-equilibrated with the initial mixture of mobile phase solutions (2% of a and 98% of b solution). the applied potential for electrochemical measurements was +850 mv and the separation temperature was set at 25 °c. the 50 µl of samples and standard solutions were applied to the system. rna isolation and real-time rt-pcr the total rna from left atria and ventricles was extracted using trizol® reagent (invitrogen, ca, usa). total rna was isolated using chloroformisopropanol extraction and quantification and rna quality was carried out using spectrophotometer (nanodrop 1000 spectrophotometer, thermo scientific). tissue samples were homogenised in 1 ml trizol® reagent per 100 mg of tissue, using electrical homogeniser (ika-werke, gmbh & co, germany). reverse transcription was performed using ready-to-go you-prime first-strand bead (ge healthcare life sciences, pa usa) and pd(n)6 primer according to the manufacturer’s protocol. real-time rt-pcr assay was done exactly as previously described by gavrilović et al. (2008). pcr reaction was performed in the abi prism 7000 sequence detection system at 50 °c for 2 mins., 95 °c for 10 mins., followed by 40 cycles at 95 °c for 15 s and 60 °c for 1 min. taqman pcr reaction was carried out using assay-on-demand gene expression products (thermo fischer scientific, ma usa) for th (id: rn00562500_m1), for net (id: rn00580267_m1), for comt (id:rn01404927_g1) and for mao-a (id:rn01430950_m1). a reference endogenous control was included in each analysis to biologica nyssana ● 12 (2) december 2021: 167-176 stefanović et al. ● melatonin affects gene expression of noradrenaline transporter and enzymes in the hearts of stressed rats 170 correct the differences in the inter-assay amplification efficiency and all transcripts were normalised to cyclophilin a (id:rn00690933) expression. the obtained results were analyzed by rq study add on software for 7000 v 1.1 sds instrument (abi prism sequence detection system) with a confidence level of 95% (p<0.05). the relative expression of the target gene was normalized to cyclophyline a and expressed in relation to the calibrator, i.e. the control sample. western blot analysis left atria and ventricles were homogenised in ripa lysis buffer system (santa cruz biotechnology, inc., santa cruz, ca, sc-24948). after centrifugation (12 000 r.p.m., 20 mins at 4 °c), the supernatant was taken and protein concentration was determined by the method of lowry et al. (1951). samples were incubated for 5 mins at 100 °c in the appropriate amount of denaturing buffer according to laemmli (1970). 30 µg of protein extract from tissue samples separated by 12% sds-poly-acrylamide gel electrophoresis were transferred to a supported pvdf membrane (immobilon-p membrane, merck millipore, massachusetts, usa). the membranes were blocked in 5% non-fat dry milk in tris-buffered saline-tween 20 (tbst) for 1 h. all following washes (three times for 15 mins.) and antibody incubation (overnight at 4 °c for primary antibody and 1 h at ambient temperature for secondary antibody) were also performed in tbst at ambient temperature on a shaker. for measuring th, net, comt and mao-a, a rabbit polyclonal anti-th primary antibody (ab51191, dilution 1:1000, abcam, cambridge, uk), a rabbit polyclonal anti-net primary antibody (ab41559, dilution 1:1000, abcam, cambridge, uk), a rabbit polyclonal anti-comt primary antibody (ab126618, dilution 1:5000, abcam, cambridge, uk) and a rabbit polyclonal anti-mao-a primary antibody (ab126751, dilution1:1000, abcam, cambridge, uk), was used, respectively. washed membrane was further incubated in the horseradish peroxidaseconjugated secondary anti-rabbit antibody for luminol-based detection (ab6721, dilution 1:5000, abcam, cambridge, uk). the secondary antibody was then visualized by immobilon western chemiluminescent hpr substrate (merck millipore, massachusetts, usa) and exposed to x-ray film for western blot detection. image j analysis pc software (nih, bethesda, md) was used for quantification densitometry of protein bands on x-ray film. amounts of all analysed proteins were normalised to β-actin levels. statistical analysis the results are reported as means ± s.e.m. the significance of the differences in the catecholamine concentration, gene expression and protein levels of the examined th, net, comt and mao-a in the left atria and ventricles of the four groups of rats was estimated by a two-way anova test. the tukey post-hoc test was used to evaluate the differences between the groups. statistical significance was set at p<0.05. results the concentration of noradrenaline in the left atria and ventricles the results (fig. 1) indicate that chronic exposure of rats to unpredictable stressors, as well as melatonin treatment, did not alter the noradrenaline concentration in the left atria and ventricles. the gene expression of th enzyme in the left atria and ventricles two-way anova showed that chronic stress biologica nyssana ● 12 (2) december 2021: 167-176 stefanović et al. ● melatonin affects gene expression of noradrenaline transporter and enzymes in the hearts of stressed rats fig. 1. the effect of chronic melatonin treatment on noradrenaline content in the left atria (a) and ventricle (b) of controls and rats exposed to cums. the results are presented as mean ± s.e.m. for a sample of 6 rats 171 biologica nyssana ● 12 (2) december 2021: 167-176 stefanović et al. ● melatonin affects gene expression of noradrenaline transporter and enzymes in the hearts of stressed rats fig. 2. the effect of chronic melatonin treatment on th mrna (a, b) and protein level (c, d) in the left atria and ventricle of controls and rats exposed to cums. the results are presented as mean ± s.e.m. for a sample of 6 rats. statistical significance: *p<0.05 control vs. cums (tukey test) fig. 3. the effect of chronic melatonin treatment on net mrna (a, b) and protein level (c, d) in the left atria and ventricle of controls and rats exposed to cums. the results are presented as mean ± s.e.m. for a sample of 6 rats. statistical significance: **p<0.01 control vs. cums; #p<0.05 placebo vs. melatonin (tukey test) affects th mrna levels (f(1.23) = 4.72, p<0.05) in the left atria (fig. 2a). exposure to cums significantly reduced mrna levels for th (by 55%, p<0.05), but did not alter the protein levels of this enzyme in the left atria. there was not a significant effect of melatonin treatment on the gene expression of th enzyme in the left atria. cums did not change the expression of the th gene in the left ventricles. the analysis of the results showed no significant effect of melatonin treatment on the th mrna and protein levels in the left ventricles. the gene expression of net transporter in the left atria and ventricles the analysis of the results showed in fig. 3 indicated the significant effect of both stress and melatonin treatment on the net mrna levels (f(1.23) = 16.77, p<0.001), as well as the protein content of this transporter (f(1.23) = 4.63, p<0.05) in the left atria. chronic stress led to a decrease in both the mrna level (by 55%, p<0.01), as well as the protein level of this transporter (by 30%, p<0.01). on the other hand, melatonin treatment induced an increase in net gene expression (p<0.05) in animals exposed to cums, returning mrna levels to control values. the obtained results in fig. 3 showed that neither cums nor melatonin changed net gene expression in the left ventricles. the gene expression of mao-a enzyme in the left atria and ventricles changes as a result of the interaction between chronic stress and melatonin treatment on mao-a mrna (f(1.23) = 48.13, p <0.001) and protein levels (f(1.23) = 69.28, p<0.001) in the left ventricles are presented in fig. 4. cums increased mao-a gene expression in the left ventricles (p<0.01), while melatonin decreased mrna (p<0.01) and protein levels (p<0.05) of mao-a enzyme in the left ventricles of stressed rats. the gene expression of comt enzyme in the left atria and ventricles the results showed in fig. 5 indicate that chronic exposure to unpredictable stressors, as well as melatonin treatment, did not significantly alter comt gene expression in the left atria of examined animals. the analysis of the results showed that cums 172 biologica nyssana ● 12 (2) december 2021: 167-176 stefanović et al. ● melatonin affects gene expression of noradrenaline transporter and enzymes in the hearts of stressed rats fig. 4. the effect of chronic melatonin treatment on mao-a mrna (a, b) and protein level (c, d) in the left atria and ventricle of controls and rats exposed to cums. the results are presented as mean ± s.e.m. for a sample of 6 rats. statistical significance: **p<0.01 control vs. cums; ##p<0.01 placebo vs. melatonin (tukey test) reduced comt mrna levels (by 45%, p<0.05), while melatonin failed to change the mrna levels of comt in the left ventricles. there was also no significant change in comt protein levels in the left ventricles under the stress protocol and melatonin treatment. discussion the changes in autonomic regulation of the heart, such as the activation of the sympathetic nervous system, the reduction in vagal tone and the increase in heart rate (hr) is often observed in depressed patients. the cums model of stress increases the sympathetic tone (grippo et al., 2002), suggesting that autonomic changes may mediate changes in the heart rate of rats exposed to cums. noradrenaline contribution to an increase in cardiac output is well established. our results showed that cums as well as melatonin treatment does not change the concentration of noradrenaline in the left atria and ventricles (fig. 1). ganguly et al. (1997) also reported that the level of noradrenaline remained unchanged in rats with myocardial infarction. induction of gene expression of the noradrenalinesynthesizing enzyme has a very important role in responding to stress and in the regulation of the cardiovascular system. our results show that cums decreased the expression of mrna th in the left atria whereas gene expression of th remained unchanged in the ventricle (fig. 2). these findings are in agreement with gavrilović et al. (2009) who have demonstrated that long-term social isolation stress also produced a decrease in th mrna level in left atria. the cardiac neuronal net in sympathetic neurons is responsible for the uptake of released noradrenaline from the neuroeffector junction (esler et al., 1990). there are studies that indicated reduced uptake in the diseased heart (habecker et al., 2006). we showed a decrease in the gene expression of the net transporter in the left atria, while the expression of this transporter is unchanged in the left ventricle under stress protocol (fig. 3). in the study doca-salt hypertension, net mrna was reduced in the left atria (wehrwein et al., 2013). decreased sympathoneural uptake of noradrenaline has been associated with cardiovascular disease while faulty net activity is a contributing factor to the increase in cardiac pathology (shanks et al., 2013). catechol o-methyltransferase (comt) and mao-a contributes to the removal of noradrenaline in the myocardium and it is believed to exert degradative action at high noradrenaline levels. in our experimental model there was no change in the 173 biologica nyssana ● 12 (2) december 2021: 167-176 stefanović et al. ● melatonin affects gene expression of noradrenaline transporter and enzymes in the hearts of stressed rats fig. 5. the effect of chronic melatonin treatment on comt mrna (a, b) and protein level (c, d) in the left atria and ventricle of controls and rats exposed to cums. the results are presented as mean ± s.e.m. for a sample of 6 rats. statistical significance: *p<0.05 control vs. cums (tukey test) level of mrna and the comt protein in the atria but the level of mrna for the same enzyme decreased in the left ventricles (fig. 5). these effects of cums were not observed at the levels of comt protein in the left ventricle (fig. 5). changes caused by stress in comt degrading enzyme certainly depend on the type and duration of the stressor and numerous other factors. kuroko et al. (2005) have shown that comt contributes to the inactivation of high levels of noradrenaline during myocardial ischemia and it has been assumed that this degrading enzyme is more active in more severe heart damages. namely, higher levels of catecholamine are necessary for the activation of comt. in the study of kaludercic et al. (2014), mao activity has been pointed out as an another mechanism potentially implicated in heart failure. in peripheral tissues, mao is involved in oxidative catabolism of amines, which generates aldehyde, which is in its turn metabolized to the corresponding acid by aldehyde dehydrogenase, and also ammonia and h2o2, products that are toxic in high concentration (kaludercic et al., 2011). in our experimental cums model, the gene expression of mao-a enzymes in the left ventricles has been increased (fig. 4). mao-a contributes to heart failure progression via enhanced noradrenaline catabolism and oxidative stress. based on our results we could suggest that enhanced gene expression of mao-a might results in augmented ros generation, contributing to left ventricular dysfunction in hearts rats subjected to cums. among endogenous substances with great success against cardiac illness certainly is melatonin. that is also a hormone with marked antioxidant properties. studies reports that circulating levels of melatonin and his synthesis are reduced in patients with different cardiovascular diseases (dominguezrodriguez et al., 2012). effects of melatonin on noradrenaline biosynthesis, uptake and degradation in the heart of depression model rats are still poorly understood. the melatonin treatment in our experiment induced a significant increase in gene expression of net in the atria (fig. 3) and a decrease in mao-a in the ventricle (fig. 4). we failed to observe any effects of the melatonin treatment in the gene expression of th (fig. 2) and comt (fig. 5) in the heart of cums rats. given that net is responsible for rapidly eliminating noradrenaline from the cardiac synaptic cleft, the elevated expression of net with the melatonin treatment may explain the greater capacity of noradrenaline uptake in the left atria; a fact which may contribute to the maintenance of atrial function in a stressful situation. an aberrantly high level of cytoplasmic noradrenaline has been linked to oxidative stress and neuronal toxicity. increased sequestration and reduced deamination of noradrenaline by mao-a during melatonin treatment produced a decreased generation of reactive oxygens species. thus, the observed beneficial effects of enhanced uptake and reduced degradation during melatonin treatment were most probably a compensatory mechanism which protects cardiomyocytes from the deleterious effects of noradrenaline overstimulation. conclusions additional experiments are needed for a better understanding of the role of melatonin, by identifying the molecular markers involved in chronic stress and thereby ensuring the use of melatonin for therapeutic purposes. the implications of this study may provide insights into the mechanisms of cardiovascular dysfunction that frequently accompany depression. although melatonin is an effective cardioprotective agent, further investigation is needed to expand its range of therapeutic applications. these findings may provide an 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levengood, r.a., gerber, d.k., schmeidler, j., yang, r.k. 1998: plasma norepinephrine and 3-methoxy4-hydroxyphenylglycol concentrations and severity of depression in combat posttraumatic stress disorder and major depressive disorder. biological psychiatry, 44(1): 56-63. živković et al. 2020, biologica nyssana 11(1) 11 (1) september 2020: 59-64 doi: 10.5281/zenodo.4060302 yield of the tomato farmed by organic principles in the greenhouse with the application of retort beach charcoal original article sanja živković agriculture faculty, kruševac, university of niš, serbia gajicsanja43@gmail.com (corresponding author) tanja vasić agriculture faculty, kruševac, university of niš, serbia tanjavasic82@gmail.com biljana mihajlović trayal korporacija ad, kruševac, serbia biljanamih037@gmail.com snežana andjelković institue for forage crops, kruševac, serbia snezana.andjelkovic@ikbks.com biljana anđelić agriculture faculty, kruševac, university of niš, serbia andjelic.biljana@ni.ac.rs ivana stanojević agriculture faculty, kruševac, university of niš, serbia stanojevic.ivana85@gmail.com sonja filipović agriculture faculty, kruševac, university of niš, serbia sonjafilipovic86@yahoo.com received: december 06, 2019 revised: february 04, 2020 accepted: march 23, 2020 abstract: the experiment was established in the greenhouse designed by random block system with tomato genotype optima, treated with cow manure and retort beech charcoal, while the control treatment comprised of cow manure only. the aim was to determine the influence of the applied material on the number of fruits per plant and the weight of the fruit directly affecting the yield in the organic growing system. in the treatment with retort beech charcoal, the optima genotype had an average yield 23.88% higher than the control plants. key words: fruit, organic cultivation, retort beach charcoal, tomato, yield apstract: prinos paradajza uzgajanog po organskim principima u stakleniku uz primenu retortnog bukovog uglja eksperimentalni ogled bio je postavljen po dizajnu slučajni blok sistem u plasteniku sa sortom paradajza optima, tretmanom sa kravljim stajnjakom i retortnim bukovim ugljem i kontrolom sa kravljim stajnjakom bez retortnog bukovog uglja. cilj je bio da se utvrdi uticaj retortnog bukovog uglja na broj ploda po biljci i masu ploda koji direktno utiču na prinos u organskom sistemu gajenja. u tretmanu, sorta optima je u proseku imala 23,88% veći prinos u odnosu na biljke iz kontrole. ključne reči: plod, organsko gajenje, retortni bukov ugalj, prinos introduction tomato (lycopersicon esculentum mill.) is the most widely grown vegetable in the world, with a very wide range of distribution. it is grown on 4,725,416 ha with a yield of about 35 t ha-1 (fao, 2013). the world’s largest producers of tomatoes are china, india, the united states, turkey, egypt, russia, italy and mexico. in 2018, 8,629 ha were sown under these vegetables in serbia, with an average yield of 15.3 t ha-1 (webrzs.stat.gov.rs, 2018). tomato accounts for 11.6% of total vegetable consumption in serbia 15.2 kg per capita per year (vlahović & puškarić, 2012). tomato is one of the most used and widespread vegetable species used as a fresh vegetable, ripe fruit and in the form of wide-range products (de sousa et al., 2008). annually, over 40 million tons of tomatoes are processed worldwide to produce canned tomatoes, ketchup, tomato juice, sauce and many other products (wptc, 2015). in particular, the consummation of tomato and its products has been shown to be associated with a reduced risk of prostate, lung, and gastric cancer (hwang & bowen, 2005; palozza et al., 2011; yang et al., 2013). depending on the type of growth, tomato can be produced in different ways and used for different © 2020 živković et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 59 purposes. seeds and planting material, especially in the vegetable industry, represent an important factor for high quality production (popović et al., 2015). organic farming combines tradition, innovation and science in order to produce healthy product and keep the environment protected. the use of activated carbons in organic production is one of the possibilities to preserve and raise the soil quality and therefore the yield of the cultivated plants (lehman et al., 2005; biederman & harpole, 2013). soil biochar amendment is based on two thousand years old experience, which in recent decades has been renewed because of proven multiple benefits (chan et al., 2007). this importance is largely long-term, but also reveals the short-term effects (mann, 2005). both biochar and activated carbon are pyrogenic carbonaceous materials (pcm). they are produced by thermochemical conversion of carbonaceous feedstock (pyrolysis or/and activation). biochar is produced from sustainably sourced biomass and is used for non-oxidative applications in agriculture (e.g., in the soil) and is also discussed as a raw material for industrial processes. by definition, it is used for carbon sequestration. hence, if “biochar” is used as a fuel, it is burned and the carbon is transformed (oxidized) into co2, it is actually classified as charcoal. activated carbon is produced from any carbon source (fossil, waste or renewable) and engineered to be used as sorbent to remove contaminants from both gases and liquids. both materials have their distinct history, widely separated scientific communities and separated bodies of literature. unfortunately, a generally accepted terminology and definition is lacking (hagemann et al., 2018). however, as the proposed applications of biochar and activated carbon increasingly overlap, awareness of the “other” domain in each case can be beneficial. nowadays both biochar and activated carbon are used for soil remediation, which before has been solely an application of activated carbon. when the activated carbon is not removed after the application and if this activated carbon was produced from renewable feedstock and is complying to further specifications, it can be considered as biochar (laird 2008; woolf et al., 2010; hagemann et al., 2018; yadav et al., 2018). many studies confirmed that soil incorporated with biochars can improve plant growing (šeremešić et al., 2015; tian et al., 2018; yadav et al., 2018). according to tian et al. (2018) biochar incorporation induces soil alkalization which can increase soil nitrification and nitrogen (n) levels. increases in soil ph are likely to affect electrical conductivity (ec), cation exchange capacity (cec) and increase alkaline metal (mg2+, ca2+ and k+) oxides. likewise, it reduces soluble forms of aluminum, which is suggested as the most significant biochar factor affecting p solubility (de luca et al., 2009; tian et al., 2018; yadav et al., 2018). beneficial effects of biochar have been elaborated in studies word wide. however, there is a lack of experimental confirmation of the biochar application in our agricultural science. researches of biochar use have been mainly conducted on soils under tropical and humid climatic conditions, which are more degraded and have a lack of soil organic carbon (šeremešić et al., 2015; tian et al., 2018; yadav et al., 2018). therefore, the aim of this study is to research the charcoal application in vegetable farming under organic conditions. the parameter that is followed is tomato yield under temperate climatic conditions. for the organic production, it is very important to choose the right genotype to be grown according to ecological principles (vasić, 2016). the total area under certification (taking into account the organic status of the plots and plots in the conversion period) in serbia is 7,998 ha, plus meadows and pastures of 1,549 ha. in 2014, vegetables accounted for only 2% of the certified plant species. material and methods the experiment was set up at trmčare locality, kruševac municipality in a greenhouse according to a random block system in 2 replicates, planting 23 plants treated with cow manure and retort beech charcoal and 23 plants treated only with cow manure as a control. the row spacing was 70 cm, while the plant spacing in one row was 40 cm. the production of tomato optima genotype seedlings has been started on 20 march, 2019 in the glasshouse, and the seedling has been done on 20 may, 2019 in the greenhouse. before sowing, the soil was prepared by adding cow manure in the amount of 200 kg per 60 m2 at the depth of the sowing layer. seven days before planting, a mixture of cow manure in the amount of 200 g/plant and 200 g/plant of retort beech charcoal was introduced at the depth of the sowing layer. the control was not treated with retorted beech charcoal, but only with cow manure in the amount of 200 g/ plant. the activated charcoal used for this purpose was produced from natural raw materials and obtained by carbonation of beech, selected according to strictly defined technical requirements by activation of steam in a static furnace. due to its organic origin and production method, charcoal has a certain degree of activity (iodine number of 233-750 mg/g), which allows it to retain water reserves and thus provide the moisture needed by the plant. the use of activated carbon in organic production is very useful because of the introduc60 biologica nyssana ● 11 (1) september 2020: 59-64 živković et al. ● yield of the tomato farmed by organic principles in the greenhouse with the application of retort beach charcoal 61 tion of n, p, k, trace elements (mg/kg: ca-8590, mg-1260, k-7400, p-380, s-350 mn-32, fe-230, zn-20, cu-23), organic substances, humic acids and amino acids that help the plant’s level and improve their health. the granulometric range of the material ensures that the soil is loose because it does not dissolve in the soil. activated carbon granulometry 0-2.5 mm was used in this experiment. the ashes of the material thus obtained have a high level of elements such as oxides of potassium, calcium and magnesium. also, activated charcoal contains a higher percentage of charcoal so it has phosphates in ash, which is an excellent source of this microelement for the plant. due to the origin and content of alkali metals, the ph of this material is 9-11. tomatoes were grown by a support (pillar) on a single tree. the following parameters were monitored in the experiment: fruit yield per plant and number of fruits per plant. analysis of variance (anova) in statistica 8 statistical program was used to examine differences in the measured characteristics between treated and untreated tomato plants and their interaction, using the student’s ttest of significance level 0.05. the results were presented in a graph and table. results the yield of treated plants averaged 3,535.65 g per plant and in the control plants averaged 2,690.87 g (fig. 1). fruit mass is a genotype characteristic and is one of the factors that determine its purpose. analysis of variance (anova) for fruit yield (grams per plant) indicated a statistically significant difference between plants treated with retorted beech charcoal and non-treated plants as well as their interactions. specifically, it was found that the plants from the treatment had a statistically significantly higher fruit yield (in grams per plant) than the plants from the control. it was also found that there was a statistically significant difference between the total fruit yield and the number of fruits on the tomato plants under treatment compared to the control plants (fig. 2 and 3, tab. 1 and 2). treatment and control varied, both in total fruit table 1. t-test for dependent samples marked differences are significant at p<0.05 for fruit yield and fruit size (in grams per plant) t-test for dependent samples marked differences are significant at p<0,05000 variable mean std. dv. n diff. std. dv. diff. t df p test 242,6221 38,4676 test 242,6221 38,4676 172 -0,0000 0,00000 0,00000 171 1,00000 test 239,1909 36,8420 cont. 316,2818 123,1446 110 -77,0909 120,4985 -6,70993 109 0,00000 cont. 316,2818 123,1446 test 239,1909 36,8420 110 77,0909 120,4985 6,70993 109 0,00000 cont. 310,4830 118,5543 cont. 310,4830 118,5543 147 -0,0000 0,00000 0,00000 146 1,00000 * the statistically significant differences in treatment and control are indicated in red. fig. 1. effect of treatment and control on yield of the friut (grams per plant) fig. 2. total fruit yield (grams) of treated and untreated tomato plants with retorted beech charcoal biologica nyssana ● 11 (1) september 2020: 59-64 živković et al. ● yield of the tomato farmed by organic principles in the greenhouse with the application of retort beach charcoal 62 yield per plant, harvest time and fruit yield over time. in plants treated with charcoal the first fruits were harvested on june 24, 2019 and the first harvest for plants in control was on july 7, 2019. it was also observed that the plants in the treatment had more even distribution of fruits for harvest than the plants in the control. plants treated with activated charcoal began to bear fruits earlier than plants in control. it was also found that the plants under treatment had a larger number of smaller and uniform fruit sizes compared to control plants that had a smaller number of larger fruits, of unequal size (tab. 1, fig. 3). the differences in yield between treatment and control within each individual harvest were statistically significant, with the smallest difference within the last harvest. on average, in treatment and control, the highest yield was recorded at the last harvest (tab. 1 and 2). the total fruit yield for the tomato plants under treatment was 81,320 g and for the control plants 61,890 g. we also monitored the dimension of the fruits and after statistical data processing it was observed that the fruits from the treatment were smaller than the fruits from the control (tab. 1, fig. 4). discussion tomato yield is positively correlated with the number of fruits per plant and the weight of the fruit (popović et al., 2015). the yield of the treated and control plants ranged from 3,535.62 g/plant and 2,690.87 g/plant, respectively (tab. 1). today, there are genotypes of large (120 g 250 g), medium (80 g 120 g), small fruits (60 g 80 g), and more recently genotypes of cocktail type (30 g 50 g) and mini (cherry) tomatoes (10 g 30 g) (đurovka et al., 2006). optimal temperatures and brightness in the early stages of development determine the yield and quality of the fruit (rylski et al., 1994). in this study, it was found that plants treated with beech retort charcoal had a higher total fruit yield per plant as well as compared to control plants. thus, for the plants from the treatment the first harvest was already on june 24, while for the plants from the control the first harvest was on july 7, 2019. fruit yield is conditioned primarily by genetic polygenic factors, but is also dependent on the external environment (zhuchenko, 1973). it is clear from this study that beech retort charcoal has a positive effect on tomato yield in the greenhouse. by growing tomatoes on five or six floors or growing on two trees, the yields would be higher by about 30-35%. there are no data in the literature on the effect of retorted beech charcoal on tomato yield. positive crop and biomass yield was found for biochar produced from wood, paper pulp, wood chips and poultry litter. yadav et al. (2018) reviewed published data from 59 pot experiments and 57 field experiments from 21 countries and found crop productivity increased by 11% on average. also, yadav et al. (2018) found benefits at field application rates typically below 30 t ha-1 field application and biologica nyssana ● 11 (1) september 2020: 59-64 živković et al. ● yield of the tomato farmed by organic principles in the greenhouse with the application of retort beach charcoal fig. 3. effect of treatment and control on total number of fruits fig. 4. effect of treatment and control on the dimension of tomato fruit effect degr. of freedom test test test test control control control control intercept 1 6293352 6293352 4636,567 0,00 11003761 11003761 725,6221 0,00 error 109 147949 1357 1652940 15165 total 109 147949 1652940 table 2. analysis of variance (anova) for fruit yield (in grams per plant) reported that increases in crop productivity varied with crop type with greater increases for legume crops (30%), vegetables (29%), and grasses (14%) compared to cereal crops corn (8%), wheat (11%), and rice (7%). these data are consistent with the results of this paper. according to yamato et al. (2006) maize production was significantly increased after the application of bark charcoal under a fertilized condition in an infertile soil environment. a positive effect of biochar addition on maize dry biomass could be ascribed to higher soil n-retention that was observed by baronti et al. (2010). these are only preliminary results. further detailed investigations should be undertaken in order to find the most optimal amount and time of application of beech retort charcoal in crops under the climatic conditions of serbia. conclusion the highest fruit yield was achieved on activated carbon plants, while the yield on control plants was 23.88% lower. this study indicates the positive impact of retorted beech charcoal on the yield of tomato plants in the greenhouse. references baronti, s., alberti, g., vedove, g.d., gennaro, f.d., fellet, g., genesio, l, miglietta, f., peressotti, a., vaccari, f.p. 2010: the biochar option to improve plant yields: first results from some field and pot experiments in italy, italian journal of agronomy. 5: 3–12. biederman, l.a., harpole, w.s. 2013: biochar and its effects on plant productivity and nutrient cycling: a meta-analysis, gcb bioenergy, 5: 202– 214. chan, k.y., van zwieten, l., meszaros, i, downie, a., joseph, s. 2007: agronomic values of greenwaste biochar as a soil amendment, australian journal of soil research, 45: 629–634. de luca, t.h., mac kenzie, m.d., gundale, m.j. 2009: biochar effects on soil nutrient transformation. in: j lehmann, s joseph (ed.), biochar for environmental management science and technology. earthscan, london, pp 251–280. de sousa, a.s., borges, s.v., magalhaes, n.f., ricardo, h.v., azevedo, a. d. 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za prirodne nauke, n 129: 17-25. rajković, j., joković, n.  probiotic properties and safety assesment… biologica nyssana 6 (2)  december 2015: 81-89 rajković, j., joković, n.  probiotic properties and safety assesment… 81 original article received: 02 december 2015 revised: 14 december 2015 accepted: 20 december 2015 probiotic properties and safety assessment of lactic acid bacteria isolated from kajmak jelena rajković, nataša jokovića* university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia * e-mail: jole35@mts.rs abstract: rajković, j., joković, n.: probiotic properties and safety assessment of lactic acid bacteria isolated from kajmak. biologica nyssana, 6 (2), december 2015: 81-89. one hundred forty-one strains of lactic acid bacteria (lab) were tested for probiotic features such as antimicrobial activities, autoaggregation ability, hydrophobicity of bacterial cell walls and ability to grow on and hydrolyze bile salts. additionally, resistance to antibiotics and production of biogenic amines were performed as safety assessment of lab strains. five lc. lactis ssp. lactis and six lc. raffinolactis strains showed antibacterial activities against used indicator strains. protein nature of antibacterial compounds was proven by the addition of pronase. enterococcus durans strains had the highest degree of autoaggregation and hydrophobicity while the rest of the strains showed low degree for both characteristics. lb. paracasei, lb. plantarum, ln. mesenteroides, en. faecium, en. faecalis and en. durans strains could growth on bile salts and some of them were able to hydrolyse them. safety assessment of lab indicates that many strains of enterococci were resistant to chloramphenicol, tetracycline, cephalosporin and to the lowest used concentration of erythromycin. the strains from other lab genera were resistant to antibiotics used in lower concentration and in smaller numbers when compared to enterococci. key words: lactic acid bacteria, probiotic properties, safety assessment apstrakt: rajković, j., joković, n.: probiotska svojstva i bezbednost upotrebe mlečno kiselinskih bakterija izolovanih i kajmaka. biologica nyssana, 6 (2), december 2015: 81-89. probiotska svojstva mlečno kiselinskih bakterija kao što su antimikrobna aktivnost, autoagregacija, hidrofobnost i sposobnost da rastu na žučnim solima i vrše njihovu hidrolizu ispitivana su na sto četrdeset jednom bakterijskom izolatu. pored toga, ispitivana je rezistentnost ovih bakterijih izolata na antibiotike i sposobnost produkcije biogenih amina u okviru procene bezbednosti izolata za upotrebu u starter kulturama. pet lc. lactis ssp. lactis i šest lc. raffinolactis izolata pokazala su antimikrobno delovanje prema korišćenim indikatorskim sojevima. proteinska priroda antimikrobnih jedinjenja je potvrdjena dodavanjem pronaze. najveći stepen hidrofobnosti i autoagregacije imali su en. durans izolati, dok su kod ostalih izolata ova probiotska svojstava bila izražena u manjem stepenu. lb. paracasei, lb. plantarum, ln. mesenteroides, en. faecium, en. faecalis i en. durans izolati su pokazali rast na podlogama sa žučnim solima, dok su neki od njih imali sposobnost da vrše njihovu hidrolizu. procenom sigurnosti upotrebe izolata utvrđeno je da su mnogi izolati enterokoka bili rezistentni na hloramfenikol, tetraciklin, cefalosporin i na najniže korišćene koncentracije eritromicina. izolati iz ostalih rodova mlečno kiselinskih bakterija su bili rezistentni na niske koncentracije antibiotika, ali u manjem broju u poređenju sa enterokokama. key words: mlečno kiselinske bakterije, probiotska svojstva, procena bezbednosti 6 (2) • december 2015: 81-89 biologica nyssana 6 (2)  december 2015: 81-89 rajković, j., joković, n.  probiotic properties and safety assesment… 82 introduction lactic acid bacteria (lab) are a heterogeneous group of bacteria, including gram-positive cocci and bacilli, with common feature to produce lactic acid as the end-product of metabolism. they are mainly isolated from the various fermented foods produced from milk, meat or vegetables but also present a normal microflora of the nasopharyngеal, intestinal and vaginal mucosa of humans and animals (s t i l e s & h o l z a p f e l , 1997). various strains of lab are widely used in the industry as a part of starter cultures for improving the quality and shelf life of fermented foods in different food processes. in recent years, lab has been intensively studied as probiotic bacteria. probiotics are defined as "live microorganisms which when administered in adequate amounts confer a health benefit to the host'' (fao/who, 2002). in dairy products bifidobacteria and lactobacilli are commonly used as probiotic bacteria, but other lab, such as enterococci, lactococci or streptococci, also may possess probiotic properties (p r i n g s u l a k a et al., 2015). intake of probiotic bacteria could lead to the establishment of intestinal microbial balance, prevention of allergies and alleviation of certain intolerances. antimutagenic, anticarcinogenic, hypocholesterolemic, antihypertensive, antiosteoporosis, and immunomodulatory effects on the host have been also reported for some probiotic bacteria (c h i a n g & p a n , 2012). considering the fact that beneficial effects of lab are strain-dependent, screening of lab isolated from different sources for probiotic properties is a common way for searching new probiotic bacteria. synthesis of antimicrobial compounds, adhesion potential to the intestinal epithelium and ability to survive conditions in the gastrointestinal tract are the characteristics that have been studied for preliminary selection of potential probiotics (b a u t i s t a g a l l e g o et al., 2013; p r i n g s u l a k a et al., 2015; a n g m o et al., 2016). the strains of lab which have capacity to produce different antimicrobial compounds could inhibit the pathogenic bacteria in gastrointestinal tract, thus reducing the risk of gastrointestinal diseases. in order to achieve the effect in intestine, probiotic bacteria must have ability to pass through upper parts of gastrointestinal tract and adhere to intestinal epithelial cells. conditions in the upper gastrointestinal tract, such as ph values and bile salts, may adversely affect the probiotic bacteria. therefore, it is necessary to examine the possibility of survival of the probiotic bacteria in the presence of bile salts. aggregation ability and hydrophobicity of bacterial cell walls are correlated to adherence properties of probiotic bacteria (d u a r y et al., 2011; j a n k o v i ć et al., 2012). required property of potential probiotic bacteria is the safety for human consumption. resistance to different antibiotics, the presence of virulence factors and synthesis of biogenic amines are commonly tested undesirable characteristics of probiotic bacteria (c h a m b a & j a m e t , 2008). traditional fermented products are the source of the new probiotic bacteria that can be used in the production of various types of functional foods. the aim of current study was screening of lab isolated from serbian traditional dairy product, kajmak, for some probiotics features such as antimicrobial activities, autoaggregation ability, hydrophobicity of bacterial cell walls and the ability to grow on and to hydrolyze bile salts. additionally, lab strains were tested for resistance to antibiotics and production of biogenic amines as safety assessment of strains. material and methods bacterial strains, media, and growth conditions one hundred forty-one strains of lab isolated from different samples of kajmak were used in this study. all strains were previously identified by molecular methods to the species level as lactococcus lactis ssp. lactis (19), lactococcus raffinolactis (6), streptococcus thermophilus (4), enterococcus faecalis (9), enterococcus faecium (19), enterococcus durans (10), leuconostoc mesenteroides (50), lactobacillus plantarum (15), lactobacillus paracasei (6), lactobacillus satsumensis (1), lactobacillus kefiri (1) and lactobacillus kefiranofaciens (1) (j o k o v i c et al., 2008; joković et al., 2014). the strains were stored at -20 c and activated by triple streaking on appropriate media. mrs agar (merck, gmbh, darmstadt, germany) was used for growth of leuconostoc and lactobacillus strains whereas lactococcus, streptococcus and enterococcus strains were cultured on m17 agar (merck, gmbh, darmstadt, germany) supplemented with glucose (0.5%, w/v; gm17 broth). the inoculated media were incubated overnight at 30 c for mesophilic and 37 c for thermophilic strains. tryptic soy agar (himedia, india) was used for the growth of non-lab strains (escherichia coli, bacillus subtilis and listeria innocua) which were incubated at 37 c for 24 h. yeast (candida albicans) was cultured on sabouraud maltose agar (torlak, serbia) at 30 c for 48 h. biologica nyssana 6 (2)  december 2015: 81-89 rajković, j., joković, n.  probiotic properties and safety assesment… 83 antimicrobial activity of strains the lab strains were screened for antimicrobial activity by the agar-well diffusion method (t a g g & m c g i v e n , 1971) using various indicator strains that are listed in table 1. asssay was performed by overlaying solid mrs or gm17 medium in petri dishes with 5 ml of soft (0.7% agar) mrs, gm17, tryptic soy and sabouraud maltose media inoculated with 105-106 cells of indicator strain/ml medium. the wells, 5 mm in diameter, were made in soft agar and filled with 100 l of overnight strain culture with a potential of bacteriocin production. the presence of antimicrobial substances was detected by the appearance of clear zones around wells as a result of growth inhibition of sensitive bacterial strains. the protein nature of bacteriocins was confirmed by adding a cristal of pronase e near to the edge of the well. absence of a zone of inhibition in the place where pronase e crystals were added indicates the protein nature of bacteriocins. adhesive properties of strains bacterial adhesion to hydrocarbons was determined by the method described by f o r t i n a et al. (2008). cells from overnight culture were struck down by centrifugation at 5000 rpm for 15 min, washed twice in pbs buffer and resuspended in 0.1 m kno3 (ph 6.2). the initial absorbance of the cell suspension was adjusted to 0.5-0.6 at 600 nm (a0). then, 3 ml of cell suspension was mixed with 1 ml of nhexadecane. the mixture was incubated at room temperature for 10 min and mixed well on vortex for 2 min. after 20 min of incubation at room temperature, the aqueous phase was carefully separated and the optical density was measured at 600 nm (at). the percentage of bacterial adhesion to n-hexadecane was expressed as: % hydrophobicity = 1(at/a0) x100, where a0 and at represent absorbance values of aqueous phase before and after contact with n-hexadecane. aggregation of strains was monitored in phosphate buffered saline (pbs) according to f o r t i n a et al. (2008). bacterial cells from overnight cultures were centrifuged at 5000 rpm for 15 min, washed twice in pbs and resuspended in 4.0 ml of pbs to give an od600nm of 1.0. the cell suspensions were further well mixed on a vortex for 10 s and autoaggregation was determined during 5 h of incubation at room temperature. every hour, 100 l from the surface of the suspension was transferred to new microtube containing 900 l pbs buffer and od600nm was measured. the percentage of aggregated cells was calculated as 1-(at/a0) x100 where a0 represents absorbance values at time t=0, while at represents absorbance values at time t= 5 h. the growth of strains on bile salts the growth of strains in the presence of bile salts was monitored on laptg medium consisted of: 15 g/l peptone, 10 g/l tryptone, 10 g/l yeast extract, 10 g/l glucose, 15 g/l agar, 0.01 ml/l of tween 80 and 0.3 g/l bovine bile (f o r t i n a et al., 2008). the ability of strains to hydrolyse bile salts was detected by precipitation of deconjugated bile acids around the colony that grew on the laptg medium without bovine bile and with the addition of 0.05 g/l bile salts no. 3 (torlak, belgrade, serbia) and 0.2 g/l cacl2. a positive reaction for the presence of hydrolase is the appearance of a precipitate around the colonies. safety assessment the resistance of strains to antibiotics was tested by spot agar test on antibiotic-containing gm17 and mrs agar media optimal for lab growth. tests were done for the following antibiotics and concentration: chloramphenicol (10 g/ml, 20 g/ml), tetracycline (10 g/ml, 20 g/ml, 30 g/ml) and erythromycin (5 g/ml, 10 g/ml). table 1. list of strains used in antimicrobial activity assay bacterial strains source or reference lactococcus lactis ssp. lactis biovar. diacetylactis, s50 (kojic et al., 2005) lactococcus lactis ssp.cremoris, ns1 (kojic et al., 1991) lactococcus lactis ssp. lactis, bgmn1-596 (gajic et al., 1999) lactococcus lactis ssp. lactis, np45 lab. kolekcija lactobacillus paracasei ssp. paracasei, bgbuk2-16/k4 (lozo et al., 2004) lactobacillus plantarum, a112 (vujcic & topisirovic, 1993) escherichia coli, atcc 25923 atcc bacilus subtilis, atcc 6633 atcc listeria innocua, atcc 33090 atcc candida albicans, atcc 10231 atcc biologica nyssana 6 (2)  december 2015: 81-89 rajković, j., joković, n.  probiotic properties and safety assesment… 84 the ability of lab isolates to produce biogenic amines was determined according to the method described by b o v e r -c i d and h o l z a p f e l (1999). histidine, tyrosine, ornithine and lysine were used as precursors for the biogenic amines synthesis. the color change of colonies to purple or occurrence of precipitates around the colonies in case of tyramine synthesis indicated the decarboxylation activities of isolates. results and discussion antimicrobial activity lab produce different substances that have antimicrobial activity, such as an organic acid (lactic, formic, acetic acid etc.), co2, h2o2, diacetyl, ethanol, and bacteriocins. among these products, bacteriocins are the most important because of their possible application in the food industry as biopreservatives (d e v u y s t & l e r o y , 2007). since bacteriocins have the highest inhibitory effect against related gram positive bacteria, bacteria from genus lactococcus, lactobacillus, bacillus and listeria were used as indicator strains (tab. 1). additionally, gram negative bacteria e. coli and yeast c. albicans were also used in antimicrobial assays. results of antimicrobial assays showed that a small number of strains could synthesize bacteriocins (tab. 2). five lc. lactis ssp. lactis, and six lc. raffinolactis strains produced bacteriocins with inhibitory effect on the growth of lactococcal indicator strains lc. lactis ssp. cremoris ns1, lc. lactis ssp. lactis bgmn1-596 and lc. lactis ssp. lactis np45 (tab. 2). lactococcal strains formed table 2. antimicrobial effect of tested strains shown through the zones of inhibition species n o . o f is o la te s indicator strains lactococcus lactis ssp. lactis bgmn1-596 lactococcusl actis ssp. cremoris ns1 lactococcus lactis ssp. lactis np45 lactococcus lactis ssp. lactis biovar. diacetylactis s50 escherichia coli atcc2592 listeria innocua atcc 33090 lc. lactis ssp. lactis 3 1 mm s 1 mm s 1 mm s lc. lactis ssp. lactis biovar. diacetylactis 4 7 mm s 7 mm s 3 mm s lc. raffinolactis 6 5 mm s 5 mm s 3 mm s 2 mm s 1 mm s 2 mm s en. faecium 1 2 mm s 5 mm s en. faecalis 1 4 mm s 2 mm s 0.5 mm s mm – zone width; s – clear zone. table 3. autoaggregation, hydrophobicity, growth on bile salts and hydrolysis of bile salts of tested lab strains l c. l a ct is ss p . la ct is ( 1 9 )1 l c. r a ff in o la ct is ( 6 ) s t. t h er m o p h il u s (4 ) e n . fa ec a li s (9 ) e n . fa ec iu m ( 1 9 ) e n . d u ra n s (1 0 ) l n . m es en te ro id es ( 5 0 ) l b . p a rc a se i ( 6 ) l b . p la n ta ru m ( 1 5 ) l b . sa ts u m en si s (1 ) l b . ke fi ri ( 1 ) l b . ke fi ra n o fa ci en s (1 ) hydrophobicity (%)2 19 2 10 14 28 45 17 6 6 27 2 3 autoaggregation (%)2 39 31 36 31 44 71 36 50 27 36 8 37 growth in the presence of bile salts3 4 10 10 3 3 9 hydrolysis of bile salts 3 1 4 5 3 3 8 1 -numbers in parenthesis represent the number of isolates 2 -results are presented as mean value 3 -results are shown as the number of isolates that has a certain property biologica nyssana 6 (2)  december 2015: 81-89 rajković, j., joković, n.  probiotic properties and safety assesment… 85 fig. 1. inhibitory effects of lc. lactis ssp. lactis and lc. raffinolactis strains on lc. lactis ssp. cremoris ns1 as indicator strain clear zones of inhibition, which lacked on the points where pronase e was added (fig. 1). lc. raffinolactis strains also inhibited the growth of lc. lactis ssp. lactis biovar. diacetylactis s50, e. coli atcc 2592 and l. inoccua atcc 33090 but zones of inhibition were smaller in these cases (tab. 2). one strain of en. faecium formed clear zones of inhibition against lc. lactis ssp. lactis biovar. diacetylactis s50, and l. innocua atcc 33090 while en. faecalis strain had inhibitory effects on lc. lactis ssp. cremoris ns1, lc. lactis ssp. lactis bgmn1-596 and l. inoccua atcc 33090 (tab. 2). zones of inhibition formed by enterococci were not sensitive to pronase e, so it can be concluded that the antimicrobial substance doesn’t have protein nature. strains which have been found to have antimicrobial activity against any of the above mention indicator strains, did not act inhibitory against lb. plantarum a112, lb. paracasei ssp. paracasei bgbuk2-16 / k4, b. subtilis atcc 6633 and c. albicans atcc 10231 indicator strains. synthesis of bacteriocins is significant characteristics of probiotic bacteria because bacteriocin producing strains could inhibit pathogenic bacteria in the gastrointestinal tract. except probiotic effects that strains with antimicrobial activities could have, bacteriocin producing lab strains are a part of starter cultures for improving the microbiological quality and safety of dairy products, or for accelerating the ripening of fermented foods (a y a d et al., 2004; m o h a m m e d et al., 2009). adhesive properties of strains autoaggregation and hydrophobicity, as surface characteristics of the lab strains, indicate potential adhesion ability of probiotic lab strains to epithelial cells of human intestine (f o r t i n a et al., 2008). the high degree of autoaggregation indicates better adhesive capability of isolates, while physicochemical properties of the cell wall of lab may probably affect autoaggregation and adhesion (f o r t i n a et al., 2008). according to m a l d o n a d o et al. (2012), three different groups of lab strains can be distinguished on the basis of autoaggregation and hydrophobicity degrees: high (60–100%), medium (30–60%) and low (0–30%). the average values of these characteristics of tested lab species are summarized in table 3. enterococcus durans strains showed the highest autoaggregation and hydrophobicity degrees with the average values of 71% and 45%, respectively. among six en. durans strains, three of them aggregated quickly after 15 min, forming a precipitate and clear solution. these strains also expressed hydrophobicity values higher than 60%. lc. lactis, lc. raffinolactis, st. thermophilus, en. faecalis, en. faecium, ln. mesenteroides, lb. paracasei, lb. satsumensis and lb. kefiranofaciens strains showed a medium degree of autoaggregation. en. faecium and lb. paracasei strains had the highest degree of autoaggregation in this group (44% and 50%, respectively). all strains in this group had very low level of binding to n-hexadecane and only en. faecium strains had average degree of hydrophobicity higher than 20%. lb. plantarum strains had low degree of autoaggregation and hydrophobicity (27% and 6%, respectively) while one lb. kefiranofaciens strain had the lowest degree of both properties among tested strains. high intraspecies variability in autoaggregation and hydrophobicity degrees was observed for lab strains evidencing that these properties seems to be strain specific. similar findings have been reported by other authors (t o d o r o v et al., 2008; g i a o u r i s et al., 2009; i y e r et al., 2010). growth in the presence of bile salts the growth of the lab strains in the presence of bile salts is a significant characteristic of probiotic lab strains, which indicates the possibility of their survival in gastrointestinal tract (d u n n e et al., 2001). biologica nyssana 6 (2)  december 2015: 81-89 rajković, j., joković, n.  probiotic properties and safety assesment… 86 table 4. resistance of lab strains to different antibiotics antibiotic chloramphenicol, g/ml tetracycline,g/ml erythromycin, g/ml cephalosporin,g/ml lab species 10 20 10 20 30 5 10 10 20 30 lc. lactis ssp. lactis (19)1,2 5 19 18 8 19 18 18 lc. raffinolactis (6) st. thermophilus (4) 4 2 1 4 2 2 en. faecalis (9) 9 6 9 9 3 8 9 9 9 en. faecium (19) 19 13 19 19 5 19 19 19 19 en. durans (10) 8 10 10 7 10 10 10 ln. mesenteroides(50) 50 45 4 40 50 40 9 lb. parcasei (6) 6 6 4 6 6 4 lb. plantarum (15) 15 14 14 4 15 14 12 lb. satsumensis (1) 1 1 1 1 1 1 lb. kefiri (1) 1 1 1 1 1 lb. kefiranofaciens (1) 1 -numbers in parenthesis represent the number of isolates of the certain strain 2 -the results are presented as number of isolates which are resistant to a particular antibiotic the ability of bile salts hydrolysis is desirable probiotic characteristics of lab strains, because the bile salts deconjugation contributes to the intestinal microflora balance (m a t h a r a et al., 2008). selection of probiotic strains based on their deconjugation activities is also important, because of the correlation between this activity and the ability of lab strains to decrease cholesterol level (p e r e i r a et al., 2003). in our study, both characteristics were detected only in strains of genus enterococcus, leuconostoc and lactobacillus (tab. 3). the most of the strains that could grow in the presence of bile salts belonged to species en. faecium, en. durans and lb. plantarum while half of the lb. plantarum strains could hydrolyse bile salts. the growth of lb. plantarum, lb. paracasei and en. faecium strains on the media with the addition of bile salts was also found in earlier studies (x a n t h o p o u l o s et al., 2000; s a a v e d r a et al., 2003; t o d o r o v et al., 2008). certain lb. plantarum, lb. paracasei and en. faecium strains were found to be able to deconjugate bile salts (s a a v e d r a et al., 2003; m a r a g k o u d a k i s et al., 2006; n g u y e n et al., 2007). safety assessment although lab has gras (generally recognized as safe) status, the strains isolated from fermented foods that can be used as starter culture should be tested for their potentially adverse effects on human health. antibiotic resistance and the synthesis of biogenic amines are mostly analyzed characteristics of lab strains important for their safe use in starter cultures (f r a n c i o s i et al., 2009). antibiotic resistance of lab strains is a major problem in the food industry after the discovery that certain strains of lactococci, lactobacilli and enterococci have genes for antibiotics resistance located on plasmids and transposons. thus, antibiotic resistance can be transferred to other bacterial species (k a s t n e r et al., 2006). in our work, resistance to antibiotics was determined by growing the isolates on the appropriate medium with various antibiotics (chloramphenicol, tetracycline, erythromycin, and cephalosporin) added in certain concentrations (tab. 4). cephalosporin belongs to the group of antibiotics which inhibit the synthesis of the cell wall, while tetracycline, erythromycin, chloramphenicol affect the protein synthesis in the bacterial cell. results from the present study showed that the most of the tested lab strains were resistant to cephalosporin (tab. 4). all enterococci, lb. satsumensis strain, lb. kefiri strain and the most of lc. lactis ssp. lactis, st. thermophilus, lb. paracasei and lb. plantarum strains grew well on the media where cephalosporin was added in the concentration of 30 µg/ml. strains belonging to species ln. mesenteroides showed the highest sensitivity to cephalosporin with only 9 strains resistant to the highest concentration of cephalosporin used (tab. 4). biologica nyssana 6 (2)  december 2015: 81-89 rajković, j., joković, n.  probiotic properties and safety assesment… 87 the most of the lab strains were resistant to tetracycline when it was added in media in low concentration (10 g/ml and 20 g/ml). increasing of tetracycline concentration in media reduced the number of resistant strains for all lab species but some strains of enterococci were resistant to this high concentration of tetracycline. when chloramphenicol was added in the concentration of 10 g/ml, all enterococcal strains and a small number of lactococcal strains were resistant. increasing concentrations of chloramphenicol at 30 g/ml did not affect growth of the most of en. faecium and en. faecalis strains. addition of erythromycin in a concentration of 5 µg/ml in media did not inhibit the growth of en. faecium, en. faecalis, ln. mesenteroides, lb. paracasei, lb. plantarum and lb. satsumensis strains, but some of lc. lactis, all st. thermophilus and en. durans strains and one lb. kefiri strain were sensitive to addition of this antibiotic in low concentration. only four lb. plantarum strains were resistant to the higher concentration of erythromycin (10 µg/ml). the most sensitive to the tested antibiotics were lc. raffinolactis and lb. kefiranofaciens strains that showed no growth on any of the medium with the addition of antibiotics. antibiotic resistant strains from dairy environment were also found by other researchers (f l ó r e z et al., 2005; d e v i r g i l i i s et al., 2010). data on proposed minimum inhibitory concentrations of antibiotics used for lab isolates from dairy products are different in the literature due to the differences between selected methods (ammor et al., 2007). overall, lab isolates from dairy products are the most sensitive to erythromycin, while their resistance is most pronounced to cephalosporins (k a t l a et al., 2001; t e m m e r m a n et al., 2003). resistance to chloramphenicol and tetracycline is distinguished among lab isolates from different genus and it is usual to obtain strains with a much higher resistance compared to the proposed minimal inhibitory concentration for a particular species (d a n i e l s e n & w i n d , 2003; f l ó r e z et al., 2005; m a r a g k o u d a k i s et al., 2006). in fermented food, biogenic amines formed by bacterial decarboxylation of amino acids histidine, tyrosine, lysine or ornithine, lead to the undesirable flavor of final product and may have negative impact on human health (l a n d e t e et al., 2007). among analyzed lab strains, all en. faecalis strains, 12 strains of en. faecium and 5 strains of en. durans synthesized tyramine from tyrosine. production of tyramine is commonly detected characteristics of enterococci from dairy products (m a r t í n p l a t e r o et al., 2009). conclusion traditional fermented products, such kajmak is, are new sources of lab strains with interesting characteristics. in recent years, lab strains that have probiotic properties are particularly popular because they can be used for the production of new types of functional foods. the results of the present research indicate that among analyzed lab strains isolated from kajmak, a few strains had interesting probiotic characteristics. five 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mathematics, department of biology and ecology, niš, serbia maja.jovanovic1@pmf.edu.rs (corresponding author) dmitar lakušić university of belgrade, faculty of biology, institute of botany and botanical garden “jevremovac”, takovska 43, 11000 belgrade, serbia dlakusic@bio.bg.ac.rs chavdar gussev institute of biodiversity and ecosystem research, department of plant and fungal diversity and resources, bulgarian academy of science, sofia, bulgaria chgussev@gmail.com predrag lazarević university of belgrade, faculty of biology, institute of botany and botanical garden “jevremovac”, takovska 43, 11000 belgrade, serbia predrag.lazarevic@bio.bg.ac.rs bojan zlatković university of niš, faculty of sciences and mathematics, department of biology and ecology, niš, serbia bojanzlat@yahoo.com received: november 12, 2019 revised: december 23, 2019 accepted: december 24, 2019 abstract: sempervivum ciliosum belongs to the group of yellow-flowered houseleek species (s. ciliosum complex) endemic for the territory of the balkan peninsula. concerning the traditional taxonomy of genus sempervivum, there are several taxa included in the above-mentioned group (s. ciliosum, s. jakucsii, s. klepa, s. octopodes and s. galicicum). however, due to highly expressed phenotypic variability, it isn’t possible to certainly establish whether all taxa from that group, including s. ciliosum as a type species, are morphologically clearly defined. the aim of this study was to analyze the level of variability of morphological traits within the populations of s. ciliosum, taken in a narrow sense. measurements of 36 quantitative traits of the vegetative and the flowering region of s. ciliosum subsp. ciliosum, including populations from bulgaria, greece and north macedonia, were processed in statistica 8.0. results of descriptive statistics, correlation analysis and univariate variance analysis (anova) indicate that most of the analyzed traits express moderate to high variability within all examined populations. key words: morphological traits, phenotypic plasticity, variability, balkans apstract: morfološka varijabilnost endemične vrste sempervivum ciliosum subsp. ciliosum (carassulaceae) sa balkanskog poluostrva sempervivum ciliosum pripada grupi žutocvetnih vrsta čuvarkuća (s. ciliosum complex) endemičnih za teritoriju balkanskog poluostrva. u odnosu na tradicionalno shvatanje taksonomije roda sempervivum, nekoliko taksona je uključeno u gore pomenutu grupu (s. ciliosum, s. jakucsii, s. klepa, s. octopodes i s. galicicum). međutim, zbog veoma izražene fenotipske varijabilnosti, nije moguće sa sigurnošću utvrditi da li su svi taksoni ove grupe, uključujući s. ciliosum kao tipsku vrstu, morfološki jasno definisani. cilj ove studije bila je analiza stepena varijabilnosti morfoloških karaktera u okviru populacija s. ciliosum s.s. merenja 36 kvantitativnih karaktera vegetativnog i cvetnog regiona s. ciliosum subsp. ciliosum, kod jedinki populacija iz bugarske, grčke i severne makedonije, obrađena su u programu statistica 8.0. rezultati deskriptivne statistike, korelacione analize i univarijantne analize varijanse (anova) ukazuju da većina analiziranih karaktera ispoljava umerenu do visoku varijabilnost u okviru analiziranih populacija. ključne reči: morfološki karakteri, fenotipska plastičnost, varijabilnost, balkansko poluostrvo introduction genus sempervivum l. belongs to a relatively large family crassulaceae j.st.-hil. the family includes 1300-1500, mostly leaf and stem succulent species divided into 33-35 genera (berger, 1930; thiede & eggli, 2007; mort et al., 2010). although some of the species of this family are extensively studied, highly expressed phenotypic variability as well as the ability of hybridization obscures the already vague picture of their morphological variability, taxonomy, as well as the number of taxa within the family. sempervivum l. is represented by perennial, herbaceous, rosulate, hardy leaf and partially stem succulent plants. according to an earlier, more widely accepted understanding, the genus sempervivum encompasses 40 to 60 species (‘t hart et al., 2005; thiede & eggli, 2007). however, the latest studies indicate that there are 46 species within the genus (karaer et al., 2011; klein & kadereit, 2015; klein © 2019 jovanović et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 113 13th symposium on the flora of southeastern serbia and neighboring regions & kadereit, 2016) as well as 16 heterotypic infraspecific taxa and 16 described natural hybrids sensu ‘t hart et al. (2003). blurred picture of the total number of species within this genus is a consequence of the difficult identification of the representatives as well as the fact that currently there is an extremely high number of the names pertaining to species rank (karaer et al., 2011). the natural habitat of sempervivum species includes rocky and dry terrains of the high mountains of europe and asia (‘t hart et al., 2003). the only outlier of this genus, located outside the main area in europe and asia, is s. atlanticum (hook.f.) ball, endemic of northern africa (karaer et al., 2011). the taxonomic problems within this genus primarily arise from pronounced phenotypic plasticity, reflected through the manifestation of morphological similarity, which is often neglected or overemphasized, when describing species. sempervivum ciliosum taken in a broader sense represents a group of taxa, described from the territory of the central and southern parts of the balkan peninsula. although intriguing taxonomical complex, s. ciliosum is still an under-researched group that includes s. ciliosum craib as a type species, s. jakucsii pénzes, s. klepa micevski, s. octopodes turrill and s. galicicum (a. c. sm.) micevski. sempervivum ciliosum s.s. is a perennial, leafy succulent plant with closed or semi-open rosettes, 2-4 cm in diameter. rosette leaves are oblong-oblanceolate to oblong-lanceolate, acute, characterized by the presence of long trichomes, both on the abaxial and adaxial surface as well as on the margin of leaves which can often be interwoven with each other and with trichomes of adjoining leaves. the flowering stem is erect, 4-10 cm long, cauline leaves are slightly wider than those in the rosette. inflorescence is compact, composed of 9to 14-merous flowers with acute, reddish-brown sepals, lemon yellow to yellow petals (8-12 × 1.5-2 mm), pale green carpels and pale yellow stamens. within s. ciliosum two subspecies have been described (‘t hart, 2002; ‘t hart et al., 2003): sempervivum ciliosum subsp. ciliosum characterized by lemon yellow, 10-12 × 1.5 mm, petals, and s. ciliosum subsp. octopodes recognizable by pale yellow at base purple or lilac petals and purplish filaments. according to ‘t hart et al. (2003) the first subspecies also includes s. ciliosum var. galicicum a. c. sm., described from mt. galičica in north macedonia. howewer, taxonomical position of this taxon is still not completely resolved since micevski (1998) considers it as a separate taxon (s. galicicum) at the species rank. due to highly expressed phenotypic plasticity and scarce literature data available of the morphological features, it is not possible to certainly establish whether all described species of this group are, so-called, valid or well-defined taxa. therefore, the possibility of having more or fewer taxa, than the number already reported in the literature is present. furthermore, taxonomic relations between taxa, nomenclature, and status of species within the complex are extremely vague. studies of the morphological variability of vegetative traits and traits from flowering region, despite the need for more detailed studies, have not been conducted for the taxa within s. ciliosum s.s. in accordance with current taxonomic concept (‘t hart et al., 2003), the species s. ciliosum here was considered in the narrow sense, as s. ciliosum subsp. ciliosum (incl. s. ciliosum var. galicicum a. c. smith). aim of this study was to examine the degree of variability of selected quantitative morphological traits from both previously mentioned regions of individuals from s. ciliosum subsp. ciliosum populations originated from different parts of the balkan peninsula. therefore, the purpose of this study in theoretical terms was to determine the level of morphological variability of s. ciliosum and accordingly to clarify the taxonomic relationships both in the s. ciliosum complex and throughout the genus sempervivum. the practical purpose was to provide additional information for a valid description of the taxa analyzed and to improve the keys for identifying species, and subspecies of this genus. material and methods plant material morphological traits from the vegetative and the flowering region were analyzed on 45 individuals from three populations collected from the territory of central and southern balkan peninsula. from each population, individuals were deposited in the herbarium of the department of biology and ecology, faculty of sciences and mathematics, university of niš (hmn) and herbarium of the university of belgrade (beou). detailed information of habitat characteristics, legators and voucher numbers, as well as localities from where populations were collected, are given in tab. 1 and fig. 1. during the field research, s. ciliosum was collected at a much smaller number of localities than expected because many of the reported distribution data for this species refer to other, morphologically similar species from the genera jovibarba and sempervivum. finally, this species spontaneously hybridizes with other species of the genus sempervivum, resulting in the large presence of hybrid individuals in most of the studied populations, also excluded from the study. in order to eliminate the influence of ecological factors on the variability of morphological traits of selected populations in their natural environment, all individuals, prior to morphometric analysis, were subjected to a period of growing, for a minimum 114 biologica nyssana ● 10 (2) december 2019: 113-123 jovanović et al. ● morphological variability of endemic sempervivum ciliosum subsp. ciliosum (crassulaceae) from the balkan peninsula riod. measurements of morphological traits from the inflorescence and flower (tab. 3) were performed in several ways. quantitative traits such as the number of flowers in the main inflorescence (nf), as well as number of sepals, petals, stamens and carpels (ns, np, sn, nc), as meristic characters, are counted within all examined individuals during the flowering period. the angle of branching of the main inflorescence (abi) was determined using a protractor. measurements of length and width of the main inflorescence (li, wi), length of the pedicel (lp) and diameter of flower (df) are conducted by using a digital caliper. length and width of parts of different segments of the flower are measured on scanned pictures of appropriate permanent slides, using digimizer image analysis software (medcalc software©, belgium). from all examined individuals, to prepare permanent slides, one flower was selected from a different region of the same inflorescence (central, middle and outer). each permanent slide was prepared after floral structures of each flower were carefully dissected and placed on slides. after preparation, slides were scanned on a resolution of 600 dpi on hp scanjet 200. as background, during the process of scanning, blue paper was used in order to provide high-contrast with yellowish floral parts. obtained values of measured floral parts for a flower, from each region of the inflorescence, were statistically processed to obtain mean values of analyzed traits. this way of analyzing floral parts allowed us to reduce differences in dimensions of floral parts between flowers from a different region of an inflorescence. it should be also emphasized that, except for height of the plant (ph), diameter of the stem (ds), length of internodes in the lower and upper part of the stem (lil, liu), the number of flowers in the main inflorescence (nf) and angle of branching of the main inflorescence (abi), measurements of all selected quantitative traits were performed in triplicate. of one year, in a greenhouse. collected individuals from each population were planted in pots of the same diameter, on floradur® substrate (floragard, vertriebs gmbh für gartenbau, germany). after a growing period, 15 individuals from each population were morphometrically processed. morphometric procedures a total of 36 quantitative morphological traits were analyzed for this study i.e., 18 morphological traits each from the vegetative and the flowering region. all morphological traits of the vegetative part (tab. 2), except the height of the plant (ph), which was determined using the ruler, were measured using high precision digital caliper (mahr federal 4107107 16u, esslingen, germany) during the flowering pebiologica nyssana ● 10 (2) december 2019: 113-123 115 jovanović et al. ● morphological variability of endemic sempervivum ciliosum subsp. ciliosum (crassulaceae) from the balkan peninsula table 1. localities, habitat characteristics, legators name and voucher numbers of individuals from analyzed populations of sempervivum ciliosum subsp. ciliosum. locality altitude (m) habitat subtratum legator voucher no. bulgaria, mt. rila, rila 580 cliffs silicate zlatković, b., gussev, ch. hmn 13899 greece, mt. orvilos 1737 subalpine rocky grasslands (daphnofestucetea) limestone lakušić, d., kuzmanović, n., janković, i. beou 46354 north macedonia, mt. galičica, trpejca 1570 rocks limestone zlatković, b., lazarević, p. hmn 13991 fig. 1. locations of analyzed populations of sempervivum ciliosum subsp. ciliosum. statistical analysis for the purposes of this study, variability of selected quantitative morphological traits was analyzed using basic statistical analysis (descriptive statistics), correlation analysis and univariate analysis of variance (anova). all values of measured traits were statistically processed using statistica 8.0 (statsoft, inc., tulsa, usa). 116 results descriptive statistics based on obtained results of descriptive statistics, quantitative morphological traits from the vegetative region manifest a low to moderate degree of variability (tab. 2, fig. 2). morphological traits which stand out by a higher degree of variability, based on highest values of standard deviation, which is the table 2. results of descriptive statistics and anova of morphological traits from the vegetative region of analyzed individuals of sempervivum ciliosum subsp ciliosum. character abbreviation, name and unit n mean min. max. std. dev. cv % f p ph height of the plant (cm) 45 23.97 16.50 36.30 4.99 21 6.33 0.0040** dr diameter of the rosette (cm) 45 3.33 2.15 4.68 0.56 17 4.08 0.0240* llr length of the lower part of the rosettes leaf (mm) 45 21.62 9.05 40.99 7.17 33 0.0001*** lur length of the upper part of the rosettes leaf (mm) 45 3.88 2.41 5.49 0.71 18 4.67 0.0137* wlr width of leaves from the middle part of the rosette (mm) 45 6.48 4.68 9.17 1.06 16 0.15 0.8575 n.s. tlr thickness of leaves from the middle part of the rosette (mm) 45 2.55 1.68 3.58 0.55 22 0.0002*** ds diameter of the stem (mm) 45 4.09 2.05 6.72 1.15 28 0.0000*** lil length of internodes in the lower part of the stem (mm) 45 12.19 7.89 17.35 2.22 18 1.44 0.2475 n.s. liu length of internodes in the upper part of the stem 45 8.24 4.77 13.68 2.31 28 2.12 0.1327 n.s. lll length of the lower leaf of the stem (mm) 45 27.27 11.14 40.76 6.08 22 7.33 0.0019** wll width of the lower leaf of the stem (mm) 45 6.88 4.07 9.05 1.11 16 2.25 0.1181 n.s. tll thickness of the lower leaf of the stem (mm) 45 2.92 1.18 4.05 0.57 19 2.54 0.0912 n.s. lml length of the middle leaf of the stem (mm) 45 25.23 15.37 36.88 4.70 19 3.19 0.0512 n.s. wml width of the middle leaf of the stem (mm) 45 6.51 3.40 8.93 1.26 19 1.58 0.2181 n.s. tml thickness of the middle leaf of the stem (mm) 45 2.51 1.23 3.59 0.52 21 4.55 0.0162* lul length of the upper leaf of the stem (mm) 45 19.83 12.69 28.77 3.52 18 5.76 0.0061** wul width of the upper leaf of the stem (mm) 45 4.66 3.01 6.72 0.92 20 2.94 0.0640 n.s. tul thickness of the upper leaf of the stem (mm) 45 1.61 1.03 2.10 0.27 17 0.16 0.8513n.s. (n – number of individuals, mean – mean value, min. – minimum value, max. – maximum value, std. dev. – standard deviation, cv% – coefficient of variation, f – fisher’s coefficient, p – level of significance, ***extremely high statistical significance, **high statistical significance, *low statistical significance; n.s.not significant) biologica nyssana ● 10 (2) december 2019: 113-123 jovanović et al. ● morphological variability of endemic sempervivum ciliosum subsp. ciliosum (crassulaceae) from the balkan peninsula 117 consequence of large scale differences of minimum and maximum values from mean values, are: length of the lower part of the rosettes leaf (llr, 21.62 ± 7.17), length of the lower leaf of the stem (lll, 27.27 ± 6.08), height of the plant (ph, 23.97 ± 4.99) and length of the middle leaf of the stem (lml, 25.23 ± 4.70). values of the most variable trait of the vegetative region, length of the lower part of the rosettes leaf (llr), ranges from 9.05 mm, as a minimum, up to 40.99 mm, as a maximum value. less variable trait, the height of the plant (ph), range from 16.50 cm to 36.30 cm. furthermore, lengths of the leaves of the stem are distributed in the range of minimal and maximal values of 11.14-40.76 mm for the lower leaves (lll) and 15.37-36.88 mm for the middle leaves (lml). morphological traits from the vegetative region exhibiting the lowest degree of variability are the following: thickness of the upper leaf of the stem (tul, 1.61 ± 0.27) with a range of minimal and maximal values of 1.03-2.10 mm, thickness of the middle leaf of the stem (tml, 2.51 ± 0.52) with values in range of 1.23-3.59 mm, thickness of the leaves from the middle part of rosette (trl, 2.55 ± 0.55) which is within a range of 1.683.58 mm, diameter of the rosette (dr, 3.33 ± 0.56) characterized by a minimum value of 2.15 cm and maximal value of 4.68 cm, and thickness of the lower leaf of the stem (tll, 2.92 ± 0.57) with values within a range of 1.18-4.05 mm. obtained values of the coefficient of variation indicate that only length of the lower part of the rosettes leaf is highly variable (llr, cv=33%) while remaining of analyzed morphological traits from vegetative region show moderate degree of variability (cv=10-30%). quantitative morphological traits from flowering region, generally, and according to obtained results, are less variable regarding to traits of the vegetative region. although traits of the inflorescence and flower show a low to a high degree of variability, the number of traits with low variability is significantly higher in regards to highly variable ones. as the most variable traits stand out: length of the main inflorescence (li, 65.92 ± 25.66), width of the main inflorescence (wi, 70.38 ± 24.95), number of flowers in the main inflorescence (nf, 37.75 ± 18.37) and angle of branching of the main inflorescence (abi, 46.20 ± 14.94). the most variable trait, length of the main inflorescence (li) has a range of minimal and maximal values from 20.41 mm to 121.49 mm. thus, the width of the main inflorescence (wi) is within the range of 29.43-137.35 mm, number of flowers in the main inflorescence (nf) varies from 7 to even 103 flowers per inflorescence and minimal value of angle of branching of the main inflorescence (abi) is 24.00° while the maximal value is 90.00°. the morphological traits of lowest variability are as follows: width of carpels (cw, 2.08 ± 0.25), width of petals (wp, 2.11 ± 0.31) width of sepals (ws, fig. 2. box and whisker plots of basic statistic parameters of analyzed vegetative morphological traits of sempervivum ciliosum subsp. ciliosum: height of the plant (ph); dr-diameter of the rosette; dsdiameter of the stem; lillength of internodes in the lower part of the stem; liulength of internodes in the upper part of the stem; llrlength of the lower part of the rosettes leaf; lur-length of the upper part of the rosettes leaf; wlrwidth of leaves from the middle part of the rosette; tlrthickness of leaves from the middle part of the rosette; lll length of the lower leaf of the stem; wllwidth of the lower leaf of the stem; tllthickness of the lower leaf of the stem; lmllength of the middle leaf of the stem; wmlwidth of the middle leaf of the stem; tmlthickness of the middle leaf of the stem; lullength of the upper leaf of the stem; wulwidth of the upper leaf of the stem; tulthickness of the upper leaf of the stem (middle point – mean value; box – mean ± sd; whisker – minimum value maximum value). biologica nyssana ● 10 (2) december 2019: 113-123 jovanović et al. ● morphological variability of endemic sempervivum ciliosum subsp. ciliosum (crassulaceae) from the balkan peninsula 1.58 ± 0.33), length of longer filaments of the flower (llf, 5.13 ± 0.48) and length of shorter filaments of the flower (lsf, 4.63 ± 0.49). based on the values of the coefficient of variation most of the analyzed traits from the flowering region are in the extent of moderate variability, while only four of analyzed traits show a high degree of variability: number of flower in the main inflorescence (nf, cv=49%), length of the main inflorescence (li, cv=39%), width of the main inflorescence (wi, cv=35%) and angle of branching of the main inflorescence (abi, cv=32%). these same traits, as already explained, have been characterized as the most variable, based on the values of standard deviation. thus, length of longer filaments of the flower (llf) and length of petals (lp) with the values of coefficient of variation of 9%, number of carpels (nc) with coefficient of correlation of 8%, number of sepals (ns), as well as number of petals (np) and number of stamens (sn), all characterized with the value of 7% of the same coefficient, are presented as the least variable. results for minimum and maximum values, as well table 3. results of descriptive statistics and anova of morphological traits from the inflorescence and flower of analyzed individuals of sempervivum ciliosum subsp. ciliosum. character abbreviation, name and unit n mean min. max. std. dev. cv % f p li length of the main inflorescence (mm) 45 65.92 20.41 121.49 25.66 39 6.01 0.0050** wi width of the main inflorescence (mm) 45 70.38 29.43 137.35 24.95 35 2.78 0.0738* nf number of flowers in the main inflorescence 45 37.75 7.00 103.00 18.37 49 13.15 0.0000*** abi angle of branching of the main inflorescence ( ̊ ) 45 46.20 24.00 90.00 14.94 32 12.48 0.0001*** pl length of the pedicel (mm) 45 1.82 0.92 2.83 0.51 28 13.41 0.0000*** df diameter of the flower (mm) 45 21.53 16.65 27.12 1.85 9 9.26 0.0005*** ns number of sepals of the flower (mm) 45 11.58 10.00 13.67 0.82 7 16.55 0.0000*** ls length of sepals of the flower (mm) 45 4.66 3.45 7.37 0.68 15 18.29 0.0000*** ws width of sepals of the flower (mm) 45 1.58 1.10 3.12 0.33 21 15.86 0.0000*** np number of petals of the flower 45 11.59 10.00 13.67 0.84 7 17.63 0.0000*** lp length of petals of the flower (mm) 45 9.87 8.27 12.11 0.91 9 31.91 0.0000*** wp width of petals of the flower (mm) 45 2.11 1.49 3.10 0.31 15 15.36 0.0000*** sn number of stamens of the flower 45 23.09 20.33 27.33 1.71 7 14.06 0.0000*** lsf length of shorter filaments of the flower 45 4.63 3.40 6.14 0.49 11 0.72 0.4936n.s. llf length of longer filaments of the flower 45 5.13 4.09 6.47 0.48 9 2.42 0.1011n.s. nc number of carpels of the flower 45 11.47 10.00 13.67 0.86 8 20.66 0.0000*** lc length of carpels of the flower (mm) 45 5.70 4.12 6.93 0.72 13 46.39 0.0000*** cw width of carpels of the flower (mm) 45 2.08 1.53 2.67 0.25 12 15.66 0.0000*** (n – number of individuals, mean – mean value, min. – minimum value, max. – maximum value, std. dev. – standard deviation, cv% – coefficient of variation, f – fisher’s coefficient, p – level of significance, ***extremely high statistical significance, **high statistical significance, *low statistical significance) 118 biologica nyssana ● 10 (2) december 2019: 113-123 jovanović et al. ● morphological variability of endemic sempervivum ciliosum subsp. ciliosum (crassulaceae) from the balkan peninsula as mean values, standard deviation and coefficient of variation, of all analyzed morphological traits of flowering region, are given in tab. 3 and fig. 3. correlative variability results from correlation analysis of analyzed morphometric traits have shown that all three degrees of linear correlation were represented, both as negative and positive form of correlations. majority of analyzed traits, 54.60%, show a weak positive linear correlation with values of correlative coefficient (r) between 0.00 and 0.50. a weak negative correlation (-0.50 < r < 0.00) is represented in 32.06% pairs of morphological traits. moderate, positive (0.50 < r < 0.80) or negative (-0.50 < r < -0.80), correlation is less represented, i.e. in 10.63% as positive correlation form, respectively, 0.79% as negative correlation form. thus, strong linear connectivity was established only as a positive form of correlation (0.80 < r < 1.00), in three pairs of traits from vegetative and nine pairs of traits from the flowering region (1.90% in total). the pairs of traits with highest degree of correlation (r > 0.90) are: length of shorter filaments of the flower/length of longer filaments of the flower (lsf/llf), number of sepals/ number of petals (ns/np), length of sepals/width of sepals (ls/ws), number of petals/number of carpels (np/nc), number of petals/number of stamens (np/ sn), number of sepals/number of carpels (ns/nc), number of stamens/number of carpels (sn/nc), number of sepals/number of stamens (ns/sn) and thickness of the middle leaf of the stem/thickness of the lower leaf of the stem (tml/tll) (tab. 4). the correlation among pairs of traits from the vegetative region, as well as pairs of traits from the vegetative and the flowering region is mostly represented by weak negative and moderate positive linear correlation, while a correlation between pairs of traits from the flowering region is mostly represented by weak negative correlation. analysis of variance (anova) the significance of mean values differences were tested using analysis of variance (anova) for all selected morphological traits. based on p values (*** p < 0.001), most of the analyzed traits are extremely statistically significant, and as such may be considered as the most significant for general morphological variability of analyzed populations. as the most variable morphological traits, from the vegetative region, stand out: diameter of the stem (ds) with p=0.0000, length of the lower part of the rosettes leaf (llr) with p=0.0001 and thickness of leaves from the middle part of the rosette (tlr) with p=0.0002 (tab. 2). statistically significant traits at extremely high level, from the flowering region, are: diameter of the flower (df, p=0.0005), the angle of branching of the main inflorescence (abi, p=0.0001), as well as all morphological traits of flower parts, all characterized by p=0.0000, except for the length of shorter filaments of the flower (lsf) and length of longer filaments of the flower (llf), which proves to be not significant (tab. 2). among previously mentioned morphological traits, according to the highest obtained values of fisher’s coefficient (f), the fig. 3. box and whisker plots of basic statistic parameters of analyzed morphological traits from flowering region of sempervivum ciliosum subsp. ciliosum: lilength of the main inflorescence; wiwidth of the main inflorescence; nfnumber of flowers in the main inflorescence; abiangle of branching of the main inflorescence; pllength of the pedicel; df-diameter of the flower; nsnumber of sepals; lslength of sepals; wswidth of sepals; npnumber of petals; lplength of petals; wpwidth of petals; snnumber of stamens; lsflength of shorter filaments; llflength of longer filaments; ncnumber of carpels; lclength of carpels; cwwidth of carpels (middle point – mean value; box – mean ± sd; whisker – minimum value maximum value). 119 biologica nyssana ● 10 (2) december 2019: 113-123 jovanović et al. ● morphological variability of endemic sempervivum ciliosum subsp. ciliosum (crassulaceae) from the balkan peninsula following traits can be considered as the most significant: length of carpels (lc, f=46.39), length of petals (lp, f=31.91) number of carpels (nc, f=20.66), length of sepals (ls, f=18.29), and number of petals (np, f=17.63). this morphological traits may be considered as those that best indicate morphological differences between analyzed populations of s. ciliosum. furthermore, traits statistically not significant for delamination of analyzed populations from vegetative region (tab. 2), based on high p values (and extremely low f values), are as follows: width of leaves from the middle part of the rosette (wlr, p=0.8575), width of the middle leaf of the stem (wml, p=0.2181), length of internodes in the lower part of the stem (lil, p=0.2475), length of internodes in the upper part of the stem (liu, p=0.1327), width of the lower leaf of the stem (wll, p=0.1181), thickness of the lower leaf of the stem (tll, p=0.0912), width of the upper leaf of the stem (wul, p=0.0640) and length of the middle leaf of the stem (lml, p=0.0512). above-mentioned set of morphological traits should not be considered as relevant for describing main morphological differences between the analyzed populations of s. ciliosum subsp. ciliosum. discussion morphological variability of any taxa from s. ciliosum complex, including s. ciliosum s.s has not been extensively researched. relatively limited amounts of scientific data, which indicate the degree of variability of morphological traits of previously mentioned taxon, especially those from the flowering region, are available in the current floristic literature (hagemann, 1986; parnell & favarger, 1993; ‘t hart, 2002; ‘t hart et al., 2003). from the other side, understanding of degree and pattern of the variability of morphological traits is the first step leading to an understanding of taxonomical relation between all related yellowflowered taxa from balkan peninsula belonging to s. ciliosum group. obtained results in this study indicate that morphological traits from the vegetative region are characterized by a greater degree of variability than those from the flowering region. a high degree of variability, pronounced within the vegetative morphological traits, especially the leaf traits, may be, to some extent, justified by the possibility of these plants to store water in leaves. but, regardless of the growing ta bl e 4. v al ue s of c or re la tio n co effi ci en t ( r) fo r pa irs o f m or ph ol og ic al tr ai ts w ith s tro ng p os iti ve c or re la tio n (b ol d va lu es ) w ith in a na ly ze d po pu la tio ns o f s em pe rv iv um c ili os um s ub sp . c ili os um . ll l w ll tl l lm l tm l lu l n s ls w s n p lp sn ls f ll f n c lc ll l 1. 00 0. 81 0. 37 0. 76 0. 28 0. 65 -0 .1 7 -0 .0 1 0. 00 -0 .1 9 0. 39 -0 .1 2 -0 .2 6 -0 .1 5 -0 .3 0 0. 55 w ll 0. 81 1. 00 0. 63 0. 74 0. 56 0. 62 -0 .1 9 0. 14 0. 21 -0 .1 8 0. 27 -0 .1 5 -0 .1 7 -0 .1 6 -0 .2 5 0. 41 tl l 0. 37 0. 63 1. 00 0. 66 0. 91 0. 57 -0 .2 0 0. 15 0. 17 -0 .1 7 -0 .2 1 -0 .1 8 -0 .1 2 -0 .2 1 -0 .1 5 -0 .1 3 lm l 0. 76 0. 74 0. 66 1. 00 0. 67 0. 85 -0 .3 8 -0 .0 5 -0 .0 4 -0 .3 8 0. 07 -0 .3 1 -0 .1 3 -0 .1 3 -0 .4 0 0. 22 tm l 0. 28 0. 56 0. 91 0. 67 1. 00 0. 61 -0 .3 0 0. 01 0. 04 -0 .2 5 -0 .2 8 -0 .2 6 -0 .1 7 -0 .2 6 -0 .1 9 -0 .2 2 lu l 0. 65 0. 62 0. 57 0. 85 0. 61 1. 00 -0 .4 0 -0 .1 7 -0 .1 8 -0 .4 0 0. 04 -0 .3 5 -0 .1 6 -0 .1 5 -0 .4 4 0. 23 n s -0 .1 7 -0 .1 9 -0 .2 0 -0 .3 8 -0 .3 0 -0 .4 0 1. 00 0. 49 0. 39 0. 96 0. 23 0. 92 0. 09 0. 10 0. 94 -0 .1 7 ls -0 .0 1 0. 14 0. 15 -0 .0 5 0. 01 -0 .1 7 0. 49 1. 00 0. 95 0. 48 0. 28 0. 50 0. 12 0. 07 0. 51 0. 01 w s 0. 00 0. 21 0. 17 -0 .0 4 0. 04 -0 .1 8 0. 39 0. 95 1. 00 0. 38 0. 27 0. 43 0. 14 0. 07 0. 42 0. 08 n p -0 .1 9 -0 .1 8 -0 .1 7 -0 .3 8 -0 .2 5 -0 .4 0 0. 96 0. 48 0. 38 1. 00 0. 20 0. 93 0. 08 0. 09 0. 93 -0 .1 9 lp 0. 39 0. 27 -0 .2 1 0. 07 -0 .2 8 0. 04 0. 23 0. 28 0. 27 0. 20 1. 00 0. 22 0. 44 0. 57 0. 08 0. 84 sn -0 .1 2 -0 .1 5 -0 .1 8 -0 .3 1 -0 .2 6 -0 .3 5 0. 92 0. 50 0. 43 0. 93 0. 22 1. 00 0. 08 0. 08 0. 90 -0 .1 8 ls f -0 .2 6 -0 .1 7 -0 .1 2 -0 .1 3 -0 .1 7 -0 .1 6 0. 09 0. 12 0. 14 0. 08 0. 44 0. 08 1. 00 0. 96 0. 07 0. 33 ll f -0 .1 5 -0 .1 6 -0 .2 1 -0 .1 3 -0 .2 6 -0 .1 5 0. 10 0. 07 0. 07 0. 09 0. 57 0. 08 0. 96 1. 00 0. 05 0. 48 n c -0 .3 0 -0 .2 5 -0 .1 5 -0 .4 0 -0 .1 9 -0 .4 4 0. 94 0. 51 0. 42 0. 93 0. 08 0. 90 0. 07 0. 05 1. 00 -0 .3 4 lc 0. 55 0. 41 -0 .1 3 0. 22 -0 .2 2 0. 23 -0 .1 7 0. 01 0. 08 -0 .1 9 0. 84 -0 .1 8 0. 33 0. 48 -0 .3 4 1. 00 120 biologica nyssana ● 10 (2) december 2019: 113-123 jovanović et al. ● morphological variability of endemic sempervivum ciliosum subsp. ciliosum (crassulaceae) from the balkan peninsula procedure that was conducted, morphological traits of the leaves pronounced high degree of variability, which indicates that even under the same condition leaves of populations of s. ciliosum subsp. ciliosum are very variable. furthermore, in this study majority of analyzed traits of flowering region manifest low level of variability with exception of length of the main inflorescence (li), width of the main inflorescence (wi) and number of flowers in the main inflorescence (nf) as well as the angle of branching of the main inflorescence (abi) with higher pronounced degree of variability. low extent of degree of morphological variability within individuals of the analyzed populations is characteristic for flower parts, such as width and length flower parts. it is interesting to emphasize that the width of the sepals (ws) is one of the least variable morphological trait, although very often sepals may exhibit a feature of succulence. one of the reasons for the explained difference in variability between morphological traits of vegetative and the flowering region is probably that morphology of vegetative parts is more ecologically determined, vegetative parts are more prone to store water, which, ultimately, affects on the variability of their morphology. values of the morphological traits of s. ciliosum s.s. represented in the literature (hagemann, 1986; parnell & favarger, 1993; ‘t hart, 2002; ‘t hart et al., 2003) are compared with the corresponding morphological traits of s ciliosum subsp. ciliosum obtained in this study (tab. 5). in comparison with data given in the literature, the obtained values of vegetative traits in this study are significantly higher, whit the exception of values diameter of the rosette (dr) which differs in narrower scale. the differences are most noticeable in the traits height of the plant (ph) and length of rosette leaves (expressed as a sum of the length of the lower part of the rosettes leaf (llr) and length of the upper part of the rosettes leaf (lur)). those variations may be partially explained due to stable growth conditions in which analyzed individuals developed during the period of growing, i.e. amount of water that the plants received during this period were the same. but it is important to note that this is certainly not the only, and most important, cause of such differences. regarding the morphological traits from the flowering region, more traits are available for comparison. table 5. a comparative review of values of morphological traits of s. ciliosum subsp. ciliosum from this study and corresponding traits of s. ciliosum s.s. represented in literature (hagemann, 1986; parnell & favarger, 1993; 't hart, 2002, 't hart et al., 2003). character name and abbreviation this study hagemann, 1986 parnell & favarger, 1993 't hart, 2002 't hart et al., 2003 height of the plant (ph) 23.97-36.30 cm 12 cm 4-10 cm – 13 cm diameter of the rosette (dr) 2.15-4.68 cm 2-3.5 cm 2-3(-5) cm 2-5 cm 2-5 cm length of rosette leaves 11.46-46.48 mm* 15-20 mm 10 mm 7-10(-25) mm 7-10(-25) mm width of leaves from the middle part of the rosette (wrl) 4.68-9.17 mm 3-4(-6) mm 4 mm 3-4(-6) mm 3-4(-6) mm diameter of the flower (df) 16.65-27.12 mm 16-25 mm – – 16-25 mm length of the sepals (ls) 3.45-7.37 mm 3-4 mm – 5-7 mm 5-7 mm length of petals (lp) 8.27-12.11 mm 8-10 mm 10-12(-15) mm 8-12 mm – width of the petals (wp) 1.49-3.10 mm 1.5-2 mm – 1.5-2 mm – length of shorter filaments (lsf) length of longer filaments (llf) 4.63-6.14 mm 5.13-6.93 mm – – ⁓ 6 mm – length of carpels (lc) 4.12-6.93 mm 6-7 mm – 6-7 mm – *expressed as a sum of the length of the lower part of the rosettes leaf (llr) and length of the upper part of the rosettes leaf (lur). 121 biologica nyssana ● 10 (2) december 2019: 113-123 jovanović et al. ● morphological variability of endemic sempervivum ciliosum subsp. ciliosum (crassulaceae) from the balkan peninsula generally, measured values of traits of flower parts are slightly higher, except for the length of carpels (lc) which proves to have lower values, than those presented in the literature (tab. 5). values of the diameter of the flower (df), given in this study, do not deviate significantly, in a morphological sense, from those given in scientific sources. measured values of length of petals (lp), as well as width of the petals (wp), are within the range, or could vary in narrower scale regarding the maximum value, of values given in scientific sources (hagemann, 1986; parnell & favarger, 1993; ‘t hart, 2002). values of the length of filaments are difficult to compare to those stated in literature reports, because in this study variability of filaments was observed through two traits: length of shorter filaments (lsf) and length of longer filaments (llf), while in literature this segregation is nonexistent i.e. all filaments were considered as equals. regardless of this, maximal values of length of the shorter filaments (lsf) and length of the longer filaments (llf) are slightly higher than those suggested by ‘t hart (2002). it is interesting to note that, regarding morphological variability between analyzed populations, values of morphological traits from the flowering region are generally lower in individuals from mt. galičica while values of traits from the vegetative region are higher regarding values of traits of individuals from mt. rila and mt. orvilos. this morphological variation may be due to the fact that the population from mt. galičica belongs to s. ciliosum var. galicicum. as already explained micevski (1998) considers previously mentioned taxon as s. galicicum but to confirm this statement more detailed studies are needed. the number of morphological traits analyzed in this study is much greater than those presented in the referent scientific sources. as explained in the previous chapter, in the current literature, for the purposes of distinguishing s. ciliosum from related species, a greater number of characters of the flowering region are in use. those traits are recognized as more stable i.e. traits in which morphological variability is less expressed, which was also confirmed by this study. obtained differences in values of analyzed morphological traits in this study, from those mentioned in the available literature, may arise from the following reasons: the impact of growing conditions, especially on traits from vegetative region, enhanced phenotypic plasticity, insufficiency of more data of variability as well as that many of data present in literature are outdated and unresolved taxonomical position of taxa within s. ciliosum complex. therefore, further research is necessary. correlative analysis proved similarity in the variation in specified pairs of morphological traits. among analyzed traits, all degrees, weak, moderate and strong, as well as forms, negative and positive, of correlation are expressed. a weak positive correlation is represented by the largest number of pairs, especially between pairs of traits from flowering region, followed by traits from vegetative part. this degree of linear correlation indicates that changes in morphology, i.e. increasing or decreasing, in a dimension of one morphological trait does not greatly affect the dimensions of correlated morphological trait, therefore those traits can be potentially used in examination purposes of morphological differentiation among analyzed populations. strongest linear connection, according to results of this study is established in pairs of traits from the flowering region, such as numbers, lengths and widths of particular parts of a flower. this connection in morphological variation between traits of floral parts, may characterize them as ineligible for purposes of examining the extent of morphological variability between populations of s. ciliosum. but bearing in mind that the numbers of flower segments, sepals, petals, stamens, and carpels, usually changes simultaneously, values of those traits should not be considered as less important in determining degree of variability between analyzed populations, regardless to their high correlation. from vegetative region, high correlations are established between morphological traits of stem leaves, which, to some degree, can be expected. as the length of leaves increases, width and thickness of the leaves also increase, since leaves accumulate larger quantities of water. to the best of our knowledge, analysis of variance (anova), same as correlative analysis, was not conducted within researches of morphological variability of s. ciliosum. according to anova results, the majority of morphological traits from the flowering region are highly statistically significant. the most important, for morphological differentiation, are traits: length of carpels (lc), length of petals (lp), number of carpels (nc), length of sepals (ls) and number of petals (np). it is interesting to point out that parnell & favarger (1990) stated that within population of s. ciliosum s.s., great variation, especially in filaments size occurs. as demonstrated by this study as well, length of filaments, based on the differences in their length among individuals, is characterized as statistically not significant for delamination of analyzed populations. from a vegetative region, traits which highly reflect morphological differences among analyzed population are diameter of the stem (ds), length of the lower part of rosette leaf (llr) and thickness of the leaves from the middle part of rosette (tlr). previous claims again point to the significance of morphological traits from the flowering region, as more reliable to use in purposes of establishing the morphological differences be122 biologica nyssana ● 10 (2) december 2019: 113-123 jovanović et al. ● morphological variability of endemic sempervivum ciliosum subsp. ciliosum (crassulaceae) from the balkan peninsula tween populations of s. ciliosum subsp. ciliosum. previously explained results of this study indicate the existence of difference in morphology within populations of typical subspecies of s. ciliosum from the balkan peninsula, which ultimately can be used to explain pronounced morphological variability of this and related species within s. ciliosum complex. in order to confirm this, the additional analysis (e.g. more detailed morphological, anatomical and other studies) should be conducted in the future. acknowledgements: this study was funded by the ministry of education, science and technological development of the republic of serbia, grant no. 173030. references berger, a. 1930: crassulaceae. in: engler, a., prantl, k. (eds.), die natürlichen pflanzenfamilien. 2nd edition, 18 a: 352–483. leipzig. hagemann, i. 1986: sempervivum l. in: strid, a. (ed.), mountain flora of greece, 1: 338. cambridge university press, cambridge. karaer, f., celep, f., eggli, u. 2011: a taxonomic revision of the sempervivum davisii complex (crassulaceae). nordic journal of botany, 29: 49– 53. klein, j., kadereit, j. 2015: phylogeny, biogeography, and evolution of edaphic association in the european orophytes sempervivum and jovibarba (crassulaceae). international journal of plant sciences, 176 (1): 44–71. klein, j., kadereit, j. 2016: allopatric hybrids as evidence for past range dynamics in sempervivum (crassulaceae), a western eurasian high mountain oreophyte. alpine botany. swiss botanical society. micevski, k. 1998: flora na republika makedonija, tom i, sv. 4. makedonska akademija na naukite i umetnostite. skopje. 1043 p. mort, m., o’leary, t., carrilo-reyes, p., nowell, t., archibald, j., randle, c. 2010: phylogeny and evolution of crassulaceae: past, present, and future. schumannia, 6: 69–86. parnell, j., favarger, c. 1990: notes on sempervivum l. and jovibarba opiz. in: chater, a. o. (ed.), flora europaea notulae systematicae, ser. 2, no. 3. botanical journal of the linnean society, 103: 216–220. parnell, j., favarger, c. 1993: sempervivum l., jovibarba opiz. in: tutin, t. g., burges, n. a., chater, a. o., edmondson, j. r., heywood, v. h., moore, d. m., valentine, d. h., walters, s.m., webb, d.a. (eds.), flora europaea-psilotaceae to platanaceae, 1: 426. cambridge university press, cambridge. ‘t hart, h. 2002: sempervivum l. in: strid, a., tan, k. (eds.), flora hellenica, 2: 309. ruggell (d): a.r.g. gantner verlag k.g. ‘t hart, h., bleij, b., zonneveld, b. 2003: sempervivum. in: eggli, u. (ed.), illustrated handbook of succulent plants, vol 5: 338. springer, berlin. thiede, j., eggli, u. 2007: crassulaceae. in: kubitzki, k. (ed.), families and genera of vascular plants. vol. 9. flowering plants eudicots, 83-118. hamburg: springer. 123 biologica nyssana ● 10 (2) december 2019: 113-123 jovanović et al. ● morphological variability of endemic sempervivum ciliosum subsp. ciliosum (crassulaceae) from the balkan peninsula pejčić et al. 2021, biologica nyssana 12(1) 12 (1) september 2021: 47-54 doi: 10.5281/zenodo.5522995 antimicrobial efficacy of basil and sage essential oils against pseudomonas aeruginosa: time-lapse kinetics and type of interaction with ciprofloxacin original article milica pejčić department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, niš, serbia milicapejcic1991@gmail.com (corresponding author) zorica stojanović-radić department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, niš, serbia zstojanovic@pmf.ni.ac.rs marina dimitrijević department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, niš, serbia dimitrijevicmarina92@yahoo.com niko radulović department of chemistry, faculty of sciences and mathematics, university of niš, višegradska 33, niš, serbia nikoradulovic@yahoo.com received: october 21, 2020 revised: february 16, 2021 accepted: february 17, 2021 abstract: the increasing resistance of pseudomonas aeruginosa strains to used antibiotics is a big problem of modern medicine. finding antimicrobial substances which will increase the antibiotics effects is necessary. therefore, the aim of the present study was to evaluate the time-lapse activity of the basil and sage oils, to evaluate the combined oils effect against p. aeruginosa clinical isolates and to determine the oils potential to enhance the activity of ciprofloxacin. the obtained growth curves showed a reduction in the number of bacterial cells in the range of 10.5-94% when basil oil was applied. in the case of the sage oil, higher reduction has been observed (48.5-100%). the basil oil achieved a synergistic interaction with ciprofloxacin in 8 isolates, while the same effect for sage/ciprofloxacin combination was observed in 6 isolates. indifferent effect was noticed in 64% of the tested isolates for basil/sage combination. these data showed potential benefits of the oils and ciprofloxacin combination therapy in the treatment of p. aeruginosa infections. key words: basil oil, sage oil, time-lapse activity, synergy, ciprofloxacin, pseudomonas aeruginosa apstract: antimikrobna efikasnost etarskih ulja bosiljka i žalfije na pseudomonas aeruginosa: vremenska kinetika i tip interakcije sa ciprofloksacinom povećanje rezistencije sojeva pseudomonas aeruginosa na dosad korišćene antibiotike predstavlja veliki problem u medicini. neophodno je pronaći antimikrobne supstance koje će povećati efikasnost antibiotika. zbog toga je cilj ovog istraživanja bio analiza vremenske kinetike ulja bosiljka i žalfije, analiza kombinovanog efekta ispitivanih ulja na kliničke izolate p. aeruginosa i određivanje potencijala ulja u povećanju aktivnosti ciprofloksacina. dobijene krive rasta pokazale su smanjenje broja bakterijskih ćelija u rasponu od 10,5-94% prilikom primene ulja bosiljka. u slučaju ulja žalfije primećeno je smanjenje broja ćelija u rasponu 48,5-100%. ulje bosiljka pokazalo je sinergističku interakciju sa ciprofloksacinom na 8 testiranih izolata, dok je isti efekat kombinacije žalfija/ciprofloksacin primećen kod 6 izolata. indiferentni efekat uočen je kod 64% testiranih izolata za kombinaciju bosiljak/žalfija. ovi rezultati su pokazali potencijalne koristi kombinovane terapije ulja i ciprofloksacina u lečenju infekcija izazvanih vrstom p. aeruginosa. ključne reči: ulje bosiljka, ulje žalfije, vremenska kinetika, sinergizam, ciprofloksacin, pseudomonas aeruginosa introduction the increase of resistance between the bacterial strains caused by incorrect or over used antimicrobial therapy is becoming a big problem in modern medicine these days. antimicrobial resistance is a natural evolutional phenomenon where microbes develop resistance against commonly used antibiotics or antimicrobial drugs by different adaptation mechanisms. owing to increased spreading, the world health organization placed antimicrobial resistance among the top 10 urgent threats for the year 2019 (yu et al., 2020). among these bacteria, pseudomonas aeruginosa is one of the most frequent multiresistant species and becomes a major concern in public health (el-hosseiny et al., 2014; alhazmi, 2015). therefore, it is necessary to find same antimicrobial substances which will increase © 2021 pejčić et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 47 the antimicrobial potential of existing drugs, and help in overcoming the bacterial resistance. the last few decades are marked by a large number of studies on antimicrobial (akthar et al., 2014; calo et al., 2015; tariq et al., 2019), antibiofilm and anti-virulence (artini et al., 2018; reichling, 2020) activity of essential oils (eos) and their components. the use of drug combinations in antibacterial treatment rather than single antimicrobial agents provides better results, because the use of a single agent is highly associated with resistance occurrence. due to the complexity in chemical composition, no particular bacterial adaptation to essential oils has been described (mikulášová et al., 2016). owing to the existing antimicrobial potential, combinations between different antibiotics and other antimicrobial substances, such as eos, represent one of the most promising advances against drugresistant microorganisms. the interaction between antimicrobials in combinations can result in three different outcomes: synergistic, additive, or antagonistic effect (chouhan et al., 2017). in recent years, there have been many studies that investigated the increasing effect of eos and their components to antimicrobial potential of conventional drugs. these studies have been performed on a number of pathogenic bacteria and p. aeruginosa was among them (hemaiswarya & doble, 2009; van vuuren et al., 2009; langeveld et al., 2014; yap et al., 2014; aelenei et al., 2016; chouhan et al., 2017). the antimicrobial potential of salvia officinalis (sage) and ocimum basilicum (basil) essential oils against different pathogenic bacteria is already well known (delamare longaray et al., 2007; bassolé et al., 2010; fournomiti et al., 2015; tardugno et al., 2018). furthermore, different interactions between the sage oil and some conventional antibiotics (elhosseiny et al., 2014; adrar et al., 2016), as well as interactions between the basil oil and antibiotics (mahmoud et al., 2016; silva et al., 2016) against p. aeruginosa have been reported. based on our previous work (pejčić et al., 2020), we decided to broaden our investigations on the basil (ocimum basilicum l.) and sage (salvia officinalis l.) essential oils, which demonstrated the antibacterial efficacy, as well as potential to reduce the virulence factors (biofilm formation, mature biofilm resistance, motility, and pyocyanin production) of the p. aeruginosa clinical isolates. therefore, the aim of the present study was: a) to evaluate the time-lapse activity of the tested oils; b) to evaluate the combined effects of basil and sage essential oils, used in mixture against p. aeruginosa clinical isolates; and c) to determine the potential of the tested oils to enhance the activity of antibiotic ciprofloxacin. materials and methods essential oils the essential oils of sage (salvia officinalis l. (lamiaceae), montes, leskovac) and basil (ocimum basilicum l. (lamiaceae), mygreenway, niš) were purchased at a local herbal pharmacy (beyond, niš). the minimal inhibitory concentrations (mics) and chemical composition of the oils were studied in detail and compared to the literature data in our previous work (pejčić et al., 2020). used microorganisms strains used in this study were isolated from different human material, identified as p. aeruginosa, phenotypically characterized and presented in our previous work (pejčić et al., 2020). total of 13 isolated p. aeruginosa strains were stored at the microbiology laboratory, department of biology and ecology, faculty of sciences and mathematics, university of niš. for the experiments, the investigated strains were grown on nutrient agar (na) for 18 h and used to prepare suspensions in physiological saline (0.85% nacl) that corresponded to the 0.5 mcfarland turbidity standard (108 cells/ ml). the suspension turbidity was adjusted by using a mcfarland densitometer (den-1 mcfarland densitometer, biosan). time-kill curve the activity of essential oils against the bacterial strains over time was determined by using the time-kill assay. the initial bacterial suspension was adjusted in mueller hinton broth to achieve the final cell concentrations of 5x106 cfu/ml. the effect of oil in mics concentrations previously determined (pejčić et al., 2020) on bacterial growth was monitored via measurement of turbidity changes 37 °c during 48 h at 600 nm (stojanović-radić et al., 2012). checkerboard assay the antimicrobial agents (basil oil, sage oil and ciprofloxacin) were combined with each other and their interaction against p. aeruginosa clinical isolates was investigated using a checkerboard method (stojanović-radić et al., 2020). three combinations were tested: basil oil/ciprofloxacin; sage oil/ciprofloxacin, and basil oil/sage oil. each antibacterial agent was tested in mics (previously determined) and several concentrations below the mics. the test was performed in 96-well microtiter plates, where serial dilutions of each agent were made in horizontal and vertical rows, which resulted in various combinations of both agents. the stocks of tested essential oils were prepared in 100% dmso 48 biologica nyssana ● 12 (1) september 2021: 47-54 pejčić et al. ● antimicrobial efficacy of basil and sage essential oils against pseudomonas aeruginosa: time-lapse kinetics... 49 and then diluted with mhb. the combination of essential oils and antibiotics was prepared by adding 25 µl of each to the same well of microtiter plate and inoculating with 150 µl of bacterial suspension (5x106 cfu/ml), afterwards the plates were incubated at 37 °c for 24 h. the interaction between the two antimicrobial agents was estimated by calculating the fractional inhibitory index (fici). the calculation of fici was done using the following equations: fica = mica-b/ mica ficb = micb-a/ micb fici = fica + ficb where fica is the fractional inhibitory concentration of antimicrobial agent a, mica-b is mic of the agent a in combination with agent b, mica is mic of agent a alone, ficb is the fractional inhibitory concentration of agent b, micb-a is mic of agent b in combination with the agent a, and micb is mic of agent b alone. fici was interpreted as a synergistic effect when its value was ≤ 0.5, as an additive 0.5 < fici ≤ 1, indifferent effect when fici was 1 < fici ≤ 2, and as an antagonistic interaction when it was < 2 (stojanović-radić et al., 2020). statistical analysis all experiments were done in triplicate and data are given as average values ± standard deviations. statistical comparison was done using two way anova followed by tuckey post hoc test (graphpad prism version 5.03, san diego, ca, usa). probability values (p) less than 0.05 were considered to be statistically significant. results and discussion as a continuation of our previous investigation (pejčić et al., 2020), where the basil and sage oils antibacterial and anti-virulence potential were determined, the present work reports the timelapse activity of the tested oils and interactions of combined antimicrobial agents (basil, sage oil and ciprofloxacin) against p. aeruginosa clinical isolates. growth curve our previous investigation (pejčić et al., 2020) of the herein tested oils antimicrobial activities showed the moderate action of both oils in the range of 5.0–20.0 mg/ml, depending on the isolated strain. the mics were further tested to investigate the activities of oils during the total incubation period of 48 h, and the results are presented as a growth curve (fig. 1). inhibition of bacterial growth was observed during the first 21 h of the treatments, after which very slight recovery was detected, but the oils significantly reduced total population number. following the incubation period of 48 h in total, the basil oil exhibited growth inhibition in the range 10.5-94%. the highest activity has been achieved against isolate no. 7, where reduction of 93.95% was observed after 48 h. on the other hand, the isolate no. 4 showed somewhat higher resistance in comparison to the others, where inhibition of only 10.5% was measured. in the case of the sage oil, higher reduction has been observed (48.5100%). the most prominent action of the sage oil was detected against several isolates, namely no. 4, no. 7, no. 11 and no. 13, where 100% of growth reduction was measured. the sage oil effectively inhibited regrowth of isolates no. 3, no. 4, no. 6, no. 7 and no. 11 (bacteriostatic effect). after total incubation period, ld50 (reduction over 50% of the total population size) was achieved in 92% (sage oil) and 53% (basil oil) of the tested isolates, which demonstrated higher time-lapse efficacy of the sage oil. according to our previous results, the oils showed similar potential considering antimicrobial action, as well as virulence factors-inhibiting potential (pejčić et al., 2020). however, in the present study, higher effect over time is visible in the case of the sage essential oil. studies reported that the antimicrobial activities of the essential oils were due to the presence of some major and minor constituents and some type of interaction between many components (miticculafic et al., 2005; van vuuren & viljoen, 2007; bassolé et al., 2010). salvia officinalis essential oils containing α-thujone (24%) and camphor (10%) (delamare longaray et al., 2007) or 1,8 cineol as a major compounds (el-hosseiny et al., 2014) failed to show activity against p. aeruginosa. in another study, where time-lapse effect of the sage oil was investigated, a very low efficacy against pseudomonas aeruginosa (atcc 27853) was observed after the total incubation period of 24 h. each tested concentration (5, 10 and 20 µl) affected only limited number of cells after 10 minutes and 1 h, which resulted in unchanged population size after total incubation period (khalil & li, 2011). the mentioned oil was characterized by 1,8cineole (62%) as a major component while the herein tested oil had different chemical composition with α-thujone (29.7%), camphor (16.6%), and 1, 8 cineole (9.2%) as the dominant compounds (pejčić et al., 2020). this is probably the reason for better activity of the herein tested oil. on the other hand, salvia officinalis essential oil with very similar content (α-thujone 37%, camphor 14%, and 1,8 cineole 14%) to the oil used in the present study biologica nyssana ● 12 (1) september 2021: 47-54 pejčić et al. ● antimicrobial efficacy of basil and sage essential oils against pseudomonas aeruginosa: time-lapse kinetics... 50 showed promising results on the kinetics of survival of staphylococcus aureus and bacillus subtilis. sage oil and fractions with high content of α-thujone exhibited a strong bactericidal effect on s. aureus, and within 4 hours the bacterial population was completely inactivated. on the other hand, fractions containing other dominant compounds (camphor and 1,8 cineole) exhibited lower effect, demonstrating that α-thujone presents the carrier of antimicrobial activity (mitic-culafic et al., 2005). previous studies on the basil oil efficacy against p. aeruginosa reported mics from 5-40 mg/ml, (hammer et al., 1999; niculae et al., 2009; pejčić et al., 2020). however, these studies have not performed the time kill assays which would allow us to compare the basil’s efficacy against p. aeruginosa strains over the time. on the other hand, data for the time-kill kinetic against one s. aureus mrsa clinical isolate in the presence of basil oil showed decreased cell number in the first 30 min and this reduction continues until the end of incubation period of 24 h (opalchenova & obreshkova, 2003). the same results were obtained for basil oil in mic concentration against candida albicans attc 10231, where after 2 h of incubation the oil reduced the number of living cells (gucwa et al., 2018). when compared to the literature data, the commercial essential oils tested in the present study showed higher activity during a longer application time. also, having in mind that the oils that the oils showed efficiency against clinical isolates with higher resistance to antibiotics, it is another confirmation of their higher activity. type of interaction between antimicrobial agents oils combination with ciprofloxacin resistance of p. aeruginosa to fluoroquinolones, the group of antibiotics that ciprofloxacin belongs to, can be due to mutations in regulation of the efflux systems, or to mutations of the topoisomerase targets (gyra and also parc). efflux and target mutations can cooperate to increase the resistance level to other drugs (rossolini & mantengoli, 2005). the use of combined therapy has been shown as a better solution in comparison to the use of individual antimicrobial agents in the treatment of bacterial resistance. to investigate the potential of the tested oils for enhancing the activity of ciprofloxacin and to determine the type of interaction between the tested essential oils against p. aeruginosa clinical isolates, checkerboard assay was performed. the three combinations were tested: basil/sage oil (bs), basil oil/ciprofloxacin (bc), sage oil/ciprofloxacin (sc) and the results are presented in tab. 1. the synergy was found in 61.5% of the tested isolates and additive effect against the two tested isolates when bc combination was applied. isolates no. 7, no. 11 and no. 13 expressed somewhat higher resistance to the tested combination in comparison to other isolates, where calculated ficis showed indifferent biologica nyssana ● 12 (1) september 2021: 47-54 table 1. interaction between antimicrobial agents against clinical isolates of pseudomonas aeruginosa basil oil/ciprofloxacin sage oil/ciprofloxacin basil oil/sage oil strains fici outcome fici outcome fici outcome no.1 0.38 s 0.38 s 2.00 i no.2 0.25 s 2.00 i 2.00 i no.3 0.09 s 0.16 s 0.56 ad no.4 0.13 s 0.17 s 0.63 ad no.5 0.12 s 0.15 s 0.53 ad no.6 0.53 ad 0.53 ad 0.75 ad no.7 1.03 i 0.53 ad 0.53 ad no.8 0.38 s 1.25 i 2.00 i no.9 0.38 s 0.38 s 1.50 i no.10 0.75 ad 0.56 ad 2.00 i no.11 1.06 i 1.13 i 2.00 i no.12 0.13 s 0.16 s 2.00 i no.13 1.03 i 0.53 ad 2.00 i fici -fractional inhibitory index s-synergistic effect ≤ 0.5; ad-additive effect 0.5 >1; i-indifferent effect 1 ≥2; a-antagonistic effect >2. pejčić et al. ● antimicrobial efficacy of basil and sage essential oils against pseudomonas aeruginosa: time-lapse kinetics... 51 biologica nyssana ● 12 (1) september 2021: 47-54 fig. 1. growth of pseudomonas aeruginosa clinical isolates in the presence of salvia officinalis and ocimum basilicum commercial essential oils. data are given as mean ± sd. the analysis was done using two way anova followed by tukey’s post hoc test (p < 0.05) vs. control. pejčić et al. ● antimicrobial efficacy of basil and sage essential oils against pseudomonas aeruginosa: time-lapse kinetics... interaction between the basil oil and ciprofloxacin. on the other hand, sc indicated somewhat weaker activity in comparison to the bc combination. the sc combination expressed synergistic effect in 46.15% and additive in 30.7% of tested isolates. isolates no. 8 and no. 11 manifested the same sensitivity to sc combination in comparison to the results obtained for these agents alone. these results are very promising and point to the very high potential of basil and sage essential oil as agents that would increase the effectiveness of ciprofloxacin used in antipseudomonal therapy. the somewhat lower potential of herein tested sage oil in enhancing the ciprofloxacin activity in comparison to the basil may be due to the chemical composition of the oil. the results of chemical characterization of herein tested oils showed α-thujone (29.7%), camphor (16.6%), and eucalyptol (9.2%) as the major constituents of the sage oil and (e)-anethole (39.4%), linalool (34.3%), eugenol (5.8%) and longifolene (6.2%) of the basil oil (pejčić et al., 2020). it has been reported that terpenoid ethers (1,8-cineole) have a lower capacity to sensitize the pathogen to the antibiotic activity as they lack free hydroxyl groups in comparison to both phenolic (eugenol) and alcoholic terpenoids (linalool) which are characterized by the presence of free hydroxyl groups. the hydroxyl group of eugenol is responsible for the inhibition of some enzymes, such as atp-ase, histidine decarboxylase or proteases (griffin et al., 1999). herein obtained results for basil oil are in agreement with silva et al. (2016) who reported the synergism for o. basilicum oil associations with ciprofloxacin against the clinical isolate p. aeruginosa 1662339, while effect of the same combination against p. aeruginosa attc 25852 was classified as indifferent. mahmoud et al. (2016) also reported that the basil/ciprofloxacin combination was effective in 88.89% of the total of 9 tested p. aeruginosa isolates. the only study on the sage oil in combination with antibiotics activity reported its potential to significantly increase the efficacy of several conventional antibiotics against p. aeruginosa (el-hosseiny et al., 2014). in the mentioned study (el-hosseiny et al., 2014) although the sage oil displayed inactivity against the p. aeruginosa strain, it potentiated the activity of antibiotics piperacillin, meropenem, and gentamicin, while in combination with antibiotic norfloxacin displayed antagonistic interaction. another study showed the potential of the sage oil to enhance the activity of ciprofloxacin in control of infections caused by mrsa strains (milenković et al., 2015). sage/basil oil combination sb combination expressed indifferent effect in 61.54% and additive in 38.46% of tested isolates. there was not synergistic effect noticed between the basil and sage oils. on the other hand, calculated ficis showed values very close to antagonistic interaction in 7 of the total 13 tested isolates. according to the chemical compositions of investigated oils, these results are expected. the study of faleiro et al. (2003) reported both antagonistic and synergistic effects of the pure essential oil components in combination, depending on the tested microorganisms. the mixture of linalool plus 1,8-cineole showed antagonistic effect in the case of the escherichia coli strain, while the same mixture showed synergistic interaction when applied to c. albicans. similar results were reported by adrar et al. (2016), where the sage oil when combined with thymus numidicus essential oil (reach in linalool (8.62–9.26%)), expressed antagonistic effect against e. coli 161. conclusions the observed results showed time-lapse efficacy of the basil and sage oils. however, higher effect over time is visible in the case of the sage essential oil. tested oils have also shown promising effect on increasing activity of antibiotic ciprofloxacin against p. aeruginosa clinical isolates. on the other hand, basil and sage oils used in combination require caution due to possible antagonism, and therefore additional research needs to be done on this topic. according to the presented results, basil and sage commercial essential oils can be used as an adjunctive therapy in combination with ciprofloxacin for the treatment of chronical infections caused by pseudomonas aeruginosa. references adrar, n., oukil, n., bedjou, f. 2016: antioxidant and antibacterial activities of thymus numidicus and salvia officinalis 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(anethum graveolens l.) seeds essential oil as a potential biologica nyssana 7 (1)  september 2016: 31-39 stanojević, lj.p. et al.  dill (anetum graveolens l.) seeds essential oil… 31 original article received: 18 january 2015 revised: 19 february 2016 accepted: 01 mart 2016 dill (anethum graveolens l.) seeds essential oil as a potential natural antioxidant and antimicrobial agent ljiljana p. stanojević*, mihajlo z. stanković, dragan j. cvetković, bojana r. danilović, jelena s. stanojević university of niš, faculty of technology, bulevar oslobođenja 124, 16000 leskovac, serbia * e-mail: stanojevic@tf.ni.ac.rs abstract: stanojević, lj.p., stanković, m.z., cvetković, d.j., danilović, b.r., stanojević, j.s.: dill (anethum graveolens l.) seeds essential oil as a potential natural antioxidant and antimicrobial agent. biologica nyssana, 7 (1), september 2016: 31-39. synthetic antioxidants and antimicrobial agents can induce many undesired side effects, which attracts interest of food producers and consumers in finding ingredients of natural origin. the antioxidative and antimicrobial activity of essential oil from dill (anethum graveolens l.) seeds was investigated in terms of its possible application as natural antioxidant and antimicrobial agent. dpph test and frap method have been used for the investigation of antioxidative activity of essential oil. disc-diffusion method has been used for investigation of oil antimicrobial activity on following microorganisms: staphylococcus aureus, listeria monocytogenes, bacillus subtilis, escherichia coli, salmonella enteritidis and candida albicans. essential oil, in concentration of 29 mg/ml, incubated for 60 minutes has shown the highest degree of dpph radicals’ neutralization (79.62%). frap activity of oil was 40.63 μmol fe2+/g of essential oil. essential oil showed the best antimicrobial activity on staphylococcus aureus. furthermore, there was a significant antimicrobial activity on all investigated microorganisms. key words: anethum graveolens l., dill seeds, essential oil, antioxidant activity, antimicrobial activity apstrakt: stanojević, lj.p., stanković, m.z., cvetković, d.j., danilović, b.r., stanojević, j.s.: etarsko ulje semena mirođije (anethum graveolens l.) kao potencijalni prirodni antioksidans i antimikrobni agens. biologica nyssana, 7 (1), septembar 2016: 31-39. sintetski antioksidansi i antimikrobni agensi mogu dovesti do brojnih neželjenih efekata, pa je zato sve veće interesovanje proizvođača i potrošača hrane za sastojcima prirodnog porekla. proučavana je antioksidativna i antimikrobna aktivnost etarskog ulja semena mirođije (anethum graveolens l.) u cilju moguće primene kao prirodnog antioksidansa i antimikrobnog agensa. antioksidativna aktivnost etarskog ulja je određena primenom dpph-testa i frap metode.antimikrobna aktivnost ulja je određena disk-difuzionom metodom, na sledeće mikroorganizme: staphylococus aureus, listeria monocytogenes, bacilus subtilis, escherichia coli, salmonela enteritidis i candida albicans. najveći stepen neutralisanja dpph radikala (79,62%) pokazuje ulje inkubirano 60 minuta, u koncentraciji 29 mg/ml. frap vrednost etarskog ulja iznosi 40,63 μmol fe2+/g etarskog ulja. ulje pokazuje najbolje antimikrobno dejstvo na staphylococcus aureus. takođe, postoji značajna antimikrobna aktivnost na sve ispitivane mikroorganizme. key words: anethum graveolens l., seme mirođije, etarsko ulje, antioksidativna aktivnost, antimikrobna aktivnost 7 (1) • september 2016: 31-39 doi: 10.5281/zenodo.159101 biologica nyssana 7 (1)  september 2016: 31-39 stanojević, lj.p. et al.  dill (anetum graveolens l.) seeds essential oil… 32 introduction there are an increasing number of scientific investigations to find natural products that exhibit different biological activities, and antioxidant, antimicrobial and anti-inflammatory activities are the most commonly studied (t e p e et al., 2004; b a k k a l i et al., 2008; m i š i ć et al., 2008). one of the most important trends in food industry is discovery of natural antioxidants from plant material (d e l a q u i s , 2002). one of the most efficient ways of lipid peroxidation inhibition is the addition of synthetic antioxidants to the oils and foods, such as ascorbyl palmitate (ap), tert-butyl-4hydroxyanisole (bha), tert-butyl-4-hydroxytoluene (bht), propyl gallate (pg), butyl gallate (bg), octyl gallate (og), dodecyl gallate (dg). but, synthetic antioxidants have some undesirable side effects wherefore natural antioxidants are increasingly used (m a e s t r i et al., 2006). plants have long been used for various infectious diseases treatment and some of these traditional medicines are still involved in the treatment of various diseases (m e n d o n ç a f i l h o , 2006). essential oils and herbal extracts, as sources of natural products, have become interesting in recent decades. they represent an alternative to synthetic antioxidants and antimicrobial agents in food industry (t e p e et al., 2004; h i n n e b u r g et al., 2006) as well as in pharmaceutical industry, alternative medicine and natural therapy (b u r t , 2004; t e p e et al., 2004; m i š i ć et al., 2008). anethum graveolens l., commonly known as dill, is an annual and sometimes biennial medicinal plant from the family apiaceae (umbelliferae). dill is one of the most significant spices in food industry (o r h a n et al., 2013; l e u n g & f o s t e r , 2003). dill is native plant to mediterranean region, southeastern europe and central southern asia (k a u r & a r o r a , 2010). dill herb and dill seeds have been used as flavoring agent in food industry for sauces, salads and seafood (p i n o et al., 1995; k a u r & a r o r a , 2010). the food industry often uses essential oil instead of dill leaves and seeds (p i n o et al., 1995) due to its characteristic aroma and flavor (j i r o v e t z et al., 2003). it has been reported that dill has antimicrobial, antihyperlipidemic, diuretic, hypotensive, antispasmodic, antiemetic, laxative effect (k o p p u l a & c h o i , 2011; h o s s e i n n z a d e h et al., 2002; t u c a k o v , 1997) and anticancer activity (p e e r a k a m et al., 2014). bioactive components of dill are: essential oil, fatty oil, proteins, carbohydrates, fiber, mineral elements (potassium, calcium, magnesium, phosphorous, sodium), vitamin a and niacin (k a u r & a r o r a , 2010). essential oil is present in all parts of plant, but its content is the highest in the seeds (2-5%) (l e u n g & f o s t e r , 2003). the major component in dill seeds essential oil is carvone (20-60%) (l e u n g & f o s t e r , 2003; r a d u l e s c u et al., 2010, d e l a q u i s et al., 2002). besides carvone, there are also present: limonene, phelandrene, -pinene, -terpinene, apiole, dill apiole, 1,8-cineole, dihydro carvone and p-cymene (l e u n g & f o s t e r , 2003; p i n o et al., 1995). in their study, s t a n o j e v i ć et al. (2015) and coworkers have found a high content of carvone (about 90%) in dill seeds essential oil from the territory of southeast serbia. since dill seeds are one of the most commonly used spices in serbian traditional cuisine and food industry, and, at the same time, dill is a plant with many medicinal properties, the aim of this study was to investigate antioxidant activity of essential oil from dill seeds by two antioxidant assay: dpph and frap as well as antimicrobial activity against some intestinal pathogens. material and methods plant material the commercial sample of non-disintegrated dill seeds (anethi fructus) was purchased („planta mell“, svrljig, southeast serbia) and used for investigations. chemicals and reagents ethanol, 96% (centrochem, zemun, serbia), 2,4,6tris (2-pyridyl)-1,3,5-triazine (tptz reagent), 1,1diphenyl-2-picrylhydrazyl (dpph radical), butylated hydroxy toluene (bht), iron (iii) chloride hexahydrate, iron (ii) sulfate heptahydrate (sigma chemical company, st. louis, usa), dimethyl sulfoxide (dmso; bdh, milan, italy). all other chemicals were analytical-grade. isolation of essential oil essential oil from dill seeds was isolated by classic clevenger-type hydrodistillation (cohobation) according to ph. jug. v (2000). dill seeds (15 g) were immersed in 300 ml of water in round bottom flask, and the oil was isolated using a clevenger-type apparatus for 3 h. the obtained essential oil was dried over anhydrous sodium sulfate and used for analysis (s t a n o j e v i ć et al., 2015). antioxidant activity dpph assay antioxidant activity of essential oil was deterimined by the use dpph test (a q u i n o et al., 2002; c h o i et al., 2002; s a n c h e z -m o r e n o , 2002). biologica nyssana 7 (1)  september 2016: 31-39 stanojević, lj.p. et al.  dill (anetum graveolens l.) seeds essential oil… 33 the essential oil was dissolved in ethanol (96%) and a series of different concentration solutions were prepared (0.23 to 29 mg/ml). the ethanol solution of dpph radical (1 ml, 3×10-4 mol/l) was added to 2.5 ml of each essential oil solutions. absorbance of one sample was immediately measured at 517 nm, while the other samples were incubated at room temperature in the dark, for 20, 30, 45 and 60 minutes, and the absorbance was also measured at 517 nm (au). the absorbance at 517 nm was measured for pure ethanol solution of dpph radical prepared as described above – 1 ml of the dpph radical (3×10-4 mol/l) diluted with 2.5 ml of ethanol, ak), as well as for the essential oil before treatment with dpph radical (2.5 ml of essential oil diluted with 1 ml of ethanol, ab). free radical scavenging capacity was calculated by the following equation (s t a n o j e v i ć et al., 2015a): dpph radicals scavenging capacity (%) =          k bu a aa 100 100 (1) essential oil concentration needed for the neutralization of 50% of the initial dpph radical concentration is called ec50 value. this value was determined by interpolation from the linear regression analysis in the concentration range between 0.23 and 29 mg/ml of essential oil added to the reaction mixture. bht was used as the reference compound (ec50 = 0.021 mg/ml). frap assay the antioxidant activity of essential oil by frap assay is determined using benzie and strain method with some modifications (b e n z i e & s t r a i n , 1996). frap reagent was prepared from acetate buffer (300 mmol/l, ph = 3.6), tptz reagent (10 mmol/l in 40 mmol/l hcl) and fecl3×6 h2o (20 mmol/l) in 10:1:1 ratio. ethanol solution of essential oil (0.1 ml, concentration 9.25 mg/ml) and 3 ml of frap reagent were added in a test tube. absorbance was measured at 593 nm after 30 minutes of incubation at 37 °c against blank control. the calibration curve for frap values determination was obtained by measuring the absorbance of fe2+ (0.2 to 1 mmol/l fe2so4×7h2o) standard solution, which was treated in the same way as the essential oil samples. frap value was expressed as mol fe2+/g of the essential oil. antimicrobial activity microorganisms and substrates. six microorganisms were selected to determine the antimicrobial activity: staphylococcus aureus (atcc 25923), bacillus subtilis (atcc 6633), escherichia coli (atcc 25922), listeria monocytogenes (atcc 19166), salmonella enteritidis (atcc 13076) and candida albicans (atcc 10259). mediums used for the growth of the microorganisms: antibiotic agar no. 1 for microbiology (merck, darmstadt, germany) for bacteria and sabouraud dextrose agar (torlak, belgrade) for fungi. microorganisms are from the collection of the microbiological laboratory of the faculty of technology, leskovac. disc-diffusion method. the agar disc-diffusion method was used for testing antimicrobial activity of dill seeds essential oil (k i e h l b a u c h et al., 2000). the mediums were sterilized for 15 minutes in an autoclave at 121oc. the suspension was prepared with overnight culture and adjusted to 0.5 mcfarland standard. the inoculum of 0.1 ml of suspension was added to 10 ml of medium and poured into the petri dishes. for screening, sterilized filter paper disks (12.7 mm dia., schleicher & schuell) were placed on the surface of inoculated mediums and impregnated with 60 l of essential oil. plates were incubated for 24 hours at 37 °c for bacteria, and 48 hours at 25 °c for yeast. antimicrobial activity was expressed as the diameter of inhibition zones (mm) obtained by investigated sample. standardized discs of amoxicillin (30 μg/disc, hemofarm, a.d. vršac), cephalexin (30 μg/disc, panfarma, beograd), amracin (30 μg/disc, galenika, a.d. zemun) and nystatin (100 u/disc, bioanalyse) served as positive controls. all experiments were carried out in three replications. data were expressed as mean ± standard deviation. the obtained data were analyzed by microsoft excel 2007 and origin 7 trial. results and discussion essential oil composition the moisture content and initial oil content in dill seeds were 7.33% and 4.0 ml/100 g of dry plant material, respectively. the yield of essential oil was 2.80 ml/100 g dry plant material (s t a n o j e v i ć et al., 2015). in the essential oil twenty nine components have been identified (99.9% of all components). these results are presented in our previous studies where the influence of the technique on the yield, composition and kinetics of essential oil hydrodistillation from dill seeds have been investigated (s t a n o j e v i ć et al., 2015). it has been found by gc-ms analysis of essential oil that carvone has the highest content (85.9%). carvone content in dill essential oil is usually 20 to 60% (l e u n g & f o s t e r , 2003; d e biologica nyssana 7 (1)  september 2016: 31-39 stanojević, lj.p. et al.  dill (anetum graveolens l.) seeds essential oil… 34 c a r v a l h o et al., 2006). besides carvone, limonene (5.1%), cis-dihydrocarvone (3.0%), transdihydrocarvone (2.7%), cis-carveol (1.8%) and trans-carveol (1.4%), all other components of oil were identified in much lower concentrations (s t a n o j e v i ć et al., 2015). the higher content of carvone in oil from serbia compared to its content in oils from other areas (bulgaria, canada, india and romania) (d e l a q u i s et al., 2002; j i r o v e t z et al., 2003; s i n g h et al., 2005) is probably due to different climatic conditions, as well as genetic characteristics of seeds (s t a n o j e v i ć et al., 2015). antioxidant activity antioxidant activity of essential oil investigated by dpph test (ability of oil in different concentrations to scavenge free dpph radical) is shown on fig. 1 (results for not-incubated and 20, 30, 45 and 60 min incubated samples are represented). fig. 1. dpph radicals scavenging activity by dill (anethum graveolens l.) seeds essential oil it can be noticed that the degree of dpph neutralization depends on incubation time, for all investigated concentrations of oil. the highest degree of dpph radicals’ neutralization is for 60 minutes incubation, in concentration of 29 mg/ml (79.62%). ec50 values of essential oil are shown in tab. 1. non-incubated samples of essential oil have not achieved ec50 value in the investigated range of concentrations. the tested synthetic antioxidant has showed better antioxidant activity compared to the essential oil. ec50 values of essential oils are lower than the ec50 values of synthetic antioxidant, bht. these synthetic antioxidants are used in food industry despite their documented undesirable side effects (t e p e et al., 2005). based on these results (fig. 1 and tab. 2) it can be concluded that the incubation time has effect on dpph radicals’ neutralization. dill seeds essential oil from thailand has shown lower extent of dpph neutralization (ec50 = 128.49 mg/ml) than oil obtained in our investigation (n a n a s o m b a t & w i m u t t i g o s o l , 2011). table 1. ec50 values of dill (anethum graveolens l.) seeds essential oil incubation time, min ec50, mg/ml* 20 20.27 ± 0.811 30 18.20 ± 1.019 45 13.45 ± 0.807 60 12.20 ± 0.464 *all data represent the mean of tree replications ± standard deviation (mean  sd). removal of free radicals is very important in food and food products preservation (h i n n e b u r g et al., 2006; t e p e et al., 2004). essential oil of dill seeds can be an alternative to dill as spice in the form of powdered plant (whole or some parts). oil is also a potential source of natural antioxidants, as a possible alternative to synthetic antioxidants. but, for this potential application of dill oil it is necessary to perform in vivo tests, which will be the aim of our further investigation. essential oil of dill seeds has lower dpph neutralization activity compared to acetone extracts of dill seeds (s i n g h et al., 2005). better activity of extract compared to unstable oil is probably due to presence of nonvolatile phenol compounds. in addition, some of the compounds with a different polarity, which are present in very small amounts in the extract, are also able to contribute to better antioxidative activity of extract. some compounds can originate in extract during hydrolysis or other processes of decomposition. some chemical reactions initiated by heating can also drive up to activities changes of complex extract, composed of a number of compounds with different chemical and physical properties (s i n g h et al., 2005). s i n t i m et al. (2015) reported on the significant effect of the antioxidant capacity of the dill seed essential oil using the oxygen radical absorbance capacity (orac) method. in addition, it was found that dill essential oil from the aerial parts of the plant had antioxidant activity. kazemi reported that dill oil exhibited a high activity in each antioxidant system with a special attention for β-carotene bleaching test and reducing power (k a z e m i , 2015). frap value, as a measure of essential oil antioxidant activity, was 40.63 μmol fe2+/g of essential oil. l a d o et al. (2004) determined the antioxidant properties of commercially purchased essential oils using the frap assay (cumin, biologica nyssana 7 (1)  september 2016: 31-39 stanojević, lj.p. et al.  dill (anetum graveolens l.) seeds essential oil… 35 coriander, dill, chamomile, hyssop, lavender, parsley, rosemary, sage and yarrow). frap value of the investigated dill essential oil was 42.64 μmol/g. the essential oil, investigated in our work, was incubated for 30 min at 37 c with frap reagent, while l a d o et al. (2004) have been incubated the oil only for 5 minutes. due to the long incubation time our oil should express greater frap value. however, lower frap value of our essential oil is probably the result of different chemical composition compared to the oil used by lado and coworkers. different origin of plant material can be also the reason of such differences in obtained results. tab. 2 shows the frap values of dill essential oil as well as literature frap values of some volatile components of oil (carvone, limonene and linalool). table 2. frap value of dill (anethum graveolens l.) seeds essential oil as well as literature frap values of some volatile oil components essential oil or volatile component frap value, μmol fe2+/g essential oil dill seeds essential oil 40.63 ± 3.23* dill seeds essential oila 42.64a carvonea 15.77a linaloola 25.73a limonenea 37.44a a l a d o et al. (2004) *all data represent the mean of tree replications ± standard deviation (mean  sd). the dill essential oil has the highest frap value (tab. 2), but the individual components identified in the essential oil also have the reductive capacities (l a d o et al., 2004). frap value obtained in our study is most likely the result of reducing ability of components present in the oil, especially carvone and limonene, which are present in the highest content. such activity of oil is due to the presence of minor component too, such as dihydrocarvone, cisand trans-carveol and linalool. so, frap value of oil is a result of synergistic effect of all present components since reducing ability of oil is higher than reducing ability of individual components (oil shows the highest frap activity). a significant number of natural products investigations suggest that essential oils have antioxidant activity. it is believed that antioxidants are directly responsible for antimutagenic and anticarcinogenic activity due to their radical scavenging properties. essential oil with antioxidant activity could be beneficial for human health (b a k k a l i et al., 2008). antimicrobial activity antimicrobial activity of dill essential oil as well as activity of reference antibiotics are shown in tab. 3. essential oil of dill has effect on all tested microorganisms. the highest effect was observed on s. aureus. tested antibiotics showed much less activity on this bacterium. it has also a significant effect on b. subtilis and e. coli. staphylococcus aureus is a gram-positive bacterium that is, among many other harmful effects to humans, one of the most frequent mastitis agents in herds of dairy cows (s i n g & p r a k a s h , 2008; m i l a n o v et al., 2010). milk contaminated with s. aureus, as well as products from such milk can cause a variety of infections, by bacteria itself and by their enterotoxins (s a m a r ž i j a et al., 2007). the relatively high effect of dill essential oil upon these bacteria suggests the potential use of oil as a natural antimicrobial agent in milk products. however, in order to use the obtained oil in such way, detailed studies are necessary to establish its minimum inhibitory concentration as well as its acute toxicity in particular concentration which would be the goals of our further studies. it is significant that investigated essential oil has a higher effect on b. subtilis compared to widely used cephalexin and amoxicillin antibiotics. bacillus species most likely cause alimentary toxic infections in humans. toxic infections are consequence of various food products consumption, in which starch and proteins are dominating, such as rice, meat and meat products, desserts, and other canned food. they are very often present as contaminants in food of animal and vegetable origin since they can survive various physical and chemical conditions because of resistant spores. in addition to alimentary infection, bacillus causes a number of other diseases: septic meningitis, cellulitis, gangrene, and many eye infections (k o t i r o n t a et al., 2000). based on this results it can be concluded that dill seeds essential oil can be used for natural antimicrobial formulation production. essential oil from dill seeds shows higher antimicrobial activity on e. coli (zone diameter 38 mm) compared to cephalexin and amoxicillin antibiotics. e. coli is considered as a dominant bacterium species in the digestive tract. the presence of this bacterium in water and food is a reliable indicator of fecal contamination. this bacterium commonly contaminates meat and dairy products, as well as fruit and vegetables (m a r k o v et al., 2009). presence of enteropathogenic e. coli in the food products can cause vomiting and diarrhea in infants and young children (s i n g h & p r a k a s h , 2008). http://www.ncbi.nlm.nih.gov/pubmed/10742691 biologica nyssana 7 (1)  september 2016: 31-39 stanojević, lj.p. et al.  dill (anetum graveolens l.) seeds essential oil… 36 the good antimicrobial activity of essential oil against e. coli probably mainly originates from carvone, which is present in the amount of 86% in the oil. this result is in accordance with studies of naigre and coworkers who determined the effect of carvone on enterococcus faecium, escherichia coli and aspergillus niger (n a i g r e et al., 1996). salmonella are gram-negative bacteria from enterobacteriaceae family and they are the most common causes of food poisoning. salmonella are natural inhabitants of animals’ gastrointestinal tract; they are widespread in soil, water and plants. all representatives of salmonella genus are potential human pathogens. they cause three types of disease in humans enteric fever, sepsis and gastroenteritis. about 95% of salmonella are ingested through food and the most common sources of infection are milk and dairy products, eggs, meat and meat products (c o x , 2000; m a r k o v et al., 2009). there are scientific papers on antimicrobial activity of commercial dill seeds essential oil on salmonella typhimurium (d e l a q u i s et al., 2002). there is no data about the effect of dill seeds essential oil from the southeast serbia region on salmonella species. the investigated dill seeds oil had a significant effect against s. enteritidis which is in the range with the effect of the commercial antibiotics. l. monocytogenes is a pathogenic bacterium that leads to listeriosis disease after consumption of food. this is a particularly dangerous pathogen since it can survive at low temperatures (foods that are kept in the fridge). listeriosis is one of the most common diseases with a fatal outcome (30%) (m a r k o v et al., 2009). there are studies of essential oils effects on l. monocytogenes (d e l a q u i s et al., 2002). dill seeds essential oil has less effect on bacteria compared to fractions of oils that are rich in carvone and limonene (d e l a q u i s et al., 2002). carvone, as a major component of the oil isolated in our study exhibited antimicrobial activity against l. monocytogenes (d e c a r v a l h o et al., 2006), so it is probably the most responsible for the effect of oil on these bacteria. isolated essential oil showed antifungal activity against c. albicans, which is, again, most probably due to the high content of carvone in the oil. carvone and limonene are the main components of caraway essential oil which showed strong antifungal activity against c. albicans. limonene is a carvone precursor and it is mainly present in mentha species being toxic to most microorganisms. carvone is widely used in the manufacture of aromas and fragrances and it has antifungal activity (p i n a et al., 2012). commercial dill seeds essential oil, with content of limonene and carvone of 46.3% and 49.5% respectively, shows antimicrobial activity against pseudomonas fragi, escherichia coli, salmonella typhimurium, listeria monocytogenes, staphylococcus aureus and saccharomyces cerevisiae. the fractions of essential oils with high content of limonene (more than 90%), and fractions with high content of carvone (67-99%) have shown better activity against gram-positive and gramnegative bacteria. thereby, the fractions rich in carvone have showed weaker activity in some extents compared to fractions rich in limonene (d e l a q u i s et al., 2002). essential oils are complex mixtures and their biological properties are result of synergistic effects of all components or major compounds. in most cases, biological activity of main components only, like thymol, carvacrol, linalool, terpineol, eugenol, carvone, geraniol, citronellol, nerol, safrole, eucalyptol, limonene, cinnamaldehyde, were analyzed. generally, the main components of table 3. antimicrobial activity of dill (anethum graveolens l.) seeds essential oil microorganism essentail oil antibiotic cephalexin amracin amoxicillin nystatin inhibition zone diameter, mm escherichia coli 38.0 ± 1.52 26.0 ± 1.04 n.t. 28.0 ± 0.84 n.t. staphylococcus aureus 80.0 ± 2.16 26.0 ± 0.78 n.t. 27.0 ± 0.97 n.t. salmonela enteritidis 28.0 ± 0.97 30.0 ± 1.08 25.0 31.0 ± 0.90 n.t. listeria monocytogenes 20.0 ± 0.83 34.0 ± 0.88 n.t. 36.0 ± 0.72 n.t. bacillus subtilis 52.0 ± 1.38 48.0 ± 0.96 52.0 ± 0.99 37.0 ± 0.59 n.t. candida albicans 18.0 ± 0.57 n.t. n.t. n.t. 17.0 ± 0.32 n.t.-not treated; all data represent the mean of tree replications ± standard deviation (mean  sd). biologica nyssana 7 (1)  september 2016: 31-39 stanojević, lj.p. et al.  dill (anetum graveolens l.) seeds essential oil… 37 essential oils are the ones on which biophysical and biological properties of oils depend (b a k k a l i et al., 2008). antimicrobial activity of dill seeds essential oil in our study probably originates from carvone, having in mind literature data about antimicrobial effect of carvone on a large number of bacteria and fungi (s i n g h et al., 2005). there are also data of limonene antimicrobial activity, a component that is represented with about 5% in isolated oil in this study (d e l a q u i s et al., 2002; s i n g h et al., 2005). other components of the oil probably also contribute to this activity. the high content of carvone, monotherpenic ketone, with numerous biological properties, indicates the possible application of dill seeds essential oil for medical purposes beside food industry, as a bioactive product of natural origin. antimicrobial activity of dill seeds essential oil is significant for human and animal pathogens as well as for food protection (b a k k a l i et al., 2008). conclusion essential oils are the source of natural products with different pharmacological activities. these oils represent the complex mixture of number components what is the reason for difficult explanation of their pharmacological activities. the presented data on antimicrobial and antioxidant activities of dill seeds essential oil showed that the isolated oil (southeast serbia, svrljig), with a high content of carvone is a potential source of natural antioxidants and antimicrobial agents. the results indicate possible application of essential oils in food and pharmaceutical industry as a safer alternative to synthetic antioxidants and antimicrobial agents. bearing in mind the results obtained in this work, dill seeds essential oil should be considered for further investigation with practical applications in different food and pharmaceutical systems. acknowledgements. this research is a part of project tr34012 which is supported by ministry of education, science and technological development of the republic of serbia. references aquino, r., morelli, s., tomaino, a., pellegrino, m., saija, a., grumetto, l., puglia, c., ventura d., bonina, f., grumetto, l. 2002: antioxidant and photoprotective activity of a crude extract of culcitium reflexum h. b. k. leaves and their major flavonoids. journal of ethnopharmacology, 79: 183–191. bakkali, f., averbeck, s., averbeck, d., idaomar, m. 2008: biological effects of essential oils a review. food and chemical toxicology, 46 (2): 446–475. benzie, i.f.f., strain, j.j. 1996: the ferric reducing ability of plasma (frap) as a measure of ‘‘antioxidant power’’: the frap assay. analytical biochemistry, 239 (1): 70–76. burt, s. 2004: essential oils: their antibacterial properties and potential application in foods – a review. international journal of food microbiology, 94 (3): 223–253. choi, w.c., kim, c.s., hwang, s.s., choi, k.b., ahn, j.h., lee, y.m., park. h.s., kim, k.s., lee, y.m. 2002: antioxidant activity and free radical scavenging capacity between korean medicinal plants and flavonoids by assay-guided comparison. plant science, 163: 1161–1168. cox, j. 2000. salmonella. in: robinson, r.k., batt, c.a., patel, p.d. 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engineering, 66 (4): 447–454. tucakov, j. 1997: lečenje biljem, rad-beograd. beograd (in serbian). 717 p. rajković, vračarić 2020, biologica nyssana 11(2) 11 (2) december 2020: 103-107 doi: 10.5281/zenodo.4393959 breeding range extension of the peregrine falcon (falco peregrinus l.) in the pannonian plain original article draženko rajković šumadijska 18/30, 21000 novi sad, seerbia strix.draze@gmail.com (corresponding author) miroslav vračarić novi sad, serbia miroslav.vracaric@gmail.com received: may 21, 2020 revised: september 18, 2020 accepted: november 30, 2020 abstract: during three consecutive winter/spring seasons in the period 2018-2020, the breeding territory of peregrine falcon was monitored at an abandoned quarry on fruška gora mt., (utm dr 00; 45.165527 n, 19.803396 e) at vojvodina province, northern serbia. described record represents the first confirmed nesting of peregrine falcon in this part of the country and provides evidence of the further expansion of breeding distribution range of this species in the pannonian plain. key words: falco peregrinus, fruška gora, nesting, peregrine falcon, vojvodina province apstract: širenje areala gnežđenja sivog sokola (falco peregrimus l.) u panonskoj niziji u periodu zima/proleće tokom tri uzastopne godine (2018-2020), praćeno je gnežđenje sivog sokola u napuštenom kamenolomu na fruškoj gori (utm dr 00; 45.165527 n, 19.803396 e) u vojvodini (severna srbija). opisani nalaz predstavlja prvi potvrđen slučaj gnežđenja sivog sokola u ovom delu zemlje koji pripada panonskoj niziji, a koji ukazuje na dalje širenje gnezdećeg areala ove vrste. ključne reči: falco peregrinus, fruška gora, sivi soko, vojvodina, gnežđenje introduction the peregrine falcon (falco peregrinus) has a widespread, nearly a cosmopolitan distribution, except extreme polar and tropical regions including new zealand as well as high mountain ranges and many oceanic islands (cade, 1982; ferguson-lees & christie, 2001; white et al., 2013). depending on geographical location and environmental conditions, it establishes their eyries in a variety type of habitats including cliffs, rocky outcrops, trees, ground mounds and, in a recent time artificial structures (cade, 1982; ratcliffe, 1993; banks et al., 2003). since the dramatic decline after world war ii caused by the widespread use of organochlorine pesticides, its populations has recovered in most parts of former distribution range (white et al., 2002; ratcliffe, 1993; rizzolli et al., 2005; prommer & bagyura, 2018). the current european nesting population is composed of three subspecies: f. p. calidus in remote tundra areas of north-eastern europe, f. p. peregrinus in most parts of the continent, including the british islands and f. p. brookei situated mostly in the mediterranean, including interior balkan peninsula (white, 1994; ferguson-lees & christie, 2001). however, there were no clear boundaries between f. p. peregrinus and f. p. brookei and in some areas, hybridisation could occur (zuberogoitia © 2020 rajković, vračarić. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 103 et al., 2009; wink, 2018). according to the most recent data, the european population extent lies between 14,900 and 28,800 breeding pairs with an increasing trend (birdlife international, 2015). in serbia, during the first half of the 20th century, peregrine falcon sporadically bred in the rocky areas, mainly in the western part of the country (matvejev, 1950, 1964). the similar situation persisted during the rest of 20th century with slight changes in population abundance and density (marinković & grubač, 2000; puzović et al., 2003; grubač, 2018). historically, in northern serbia (vojvodina province), there are no evidence-based breeding records, although few authors reported occasional breeding in low numbers (marčetić & medaković, 1954; marčetić, 1957; antal et al., 1971), but with insufficient information and lack of irrefutable proofs (pelle et al., 1977; mikuska, 1982; pelle, 1990; šćiban et al., 2015; grubač, 2018). in the recent period, peregrine falcon breeds in serbia only in hilly and mountain regions with cliffs and gorges south of danube and sava rivers (grubač, 2018). the population is estimated to 51-63 breeding pairs with a moderate decline (puzović et al., 2015). results since 2010, we have been visiting central and eastern parts of fruška gora mt. near novi sad (vojvodina province, northern serbia) for birds mapping. on 4th march 2019, central parts of fruška gora mt. was surveyed, more precisely, the area between main ridge road (partizanski put) and stari ledinci village. near one forest clearing, in close vicinity of abandoned trachyte quarry “srebro” (utm dr 00; 45.165527 n, 19.803396 e) one specimen of peregrine falcon, was unintentionally disturbed by our appearance. the peregrine perched on the lateral branch of european beech (fagus sylvatica) close to the quarry edge and feeding on a feral pigeon (columba livia f. domestica). the abandoned trachyte quarry “srebro” is situated around 1 km south from village stari ledinci (petrovaradin municipality) in the territory of national park “fruška gora” at approximately 274 m above sea level. the quarry has an erratic shape; most resemble an ellipse with approximate dimensions 450 × 140 m. the height of the vertical cliffs varies from 15 to 80 m. deep-water cover the bottom of the quarry, forming the ledinačko lake (fig. 1). the observed individual had a dark cap, whitish breast and bluishgrey mantle, which are characteristics of an adult bird. shortly after, another larger individual of peregrine falcon was repeatedly calling from the opposite, south-faced cliff. dark brown plumage and profoundly marked breast were showed that this specimen was in their second year. the bird was sitting on the protruding rock in the middle of the cliff, around 40 m high from the water surface. the observed peregrines were probably a pair in suitable habitat within the breeding season. thus, in the following weeks, the pair was monitored using a spotting scope and binoculars. these visits were conducted every 3 to 10 days, usually in the morning hours until the end of may. the observations have been obtained from a distance of 250-400 m away from peregrines to avoid disturbance. the pair spent most of their time on the western part of a quarry where the vertical cliffs are broadest and highest. both individuals were usually sitting and preening or occasionally aggressively chasing away the common buzzards (buteo buteo) and common ravens (corvus corax) from the vicinity of the quarry. between 8th and 17th march of 2018, a peregrine’s copulation was observed on nine occasions. the copulation took 8.5 sec on average (ranged 6-12 sec). the act of copulation in 66% of cases took place on the lateral branch of a partially dead, around 10 m tall tree, which grew out from one of the cracks between cliffs. during our visit on 10th april, the female of peregrine falcon was observed sitting low on a cliff shelf. therefore, we suspected that the female was incubating, although we did not observe eggs. at the same time, male patrolled in soaring flight above the quarry and defended eyrie from one specimen of common buzzard and two 104 fig. 1. trachyte quarry “srebro” near village stari ledinci – breeding eyrie of peregrine falcon (falco peregrinus). the yellow circle indicates the position of the nest from 2020. biologica nyssana ● 11 (2) december 2020: 103-107 rajković, vračarić ● breeding range extension of the peregrine falcon (falco peregrinus l.) in the pannonian plain 105 common ravens. the height of the cliff with potential nest ledge was around 60 m above the lake level. the scrape was approximately 20 m from the clifftop with estimated dimensions of 30 × 20 cm. the cliff with potential nest had a southwest aspect and with an angle of approximately 80°. during visits in april, it seemed that potential nesting attempt had failed apparently due to frequent human disturbance or less likely that no eggs were laid at all. however, the scrape was empty with only a few body feathers from peregrine’s female. in 2018, the pair of peregrine falcons was noticed the last time on 28th april. meanwhile, due to significant improvement of weather conditions from april onwards, approximate 50-100 visitors were regularly hiking, camping and even burning fire in the proximity of potential nesting scrape. as the peregrine falcon is a strictly protected species in serbia, the provincial institute for nature conservation was informed about the situation. unfortunately, no conservation measures have been made for the restriction of visitors since march of 2020. during two short visits in late may, the specimens of peregrine falcon were not observed inside the quarry or in its surroundings. during two our visits in march and three visits in april of 2019, only adult male was observed on the described location. the fate of the immature female remains unknown. proven eggs laying and incubation on the abandoned quarry finally happened in 2020. on 7th march, peregrines were sitting close to each other, watching the surrounding and periodically preening. both birds clearly showed adult morphological features. from time to time, they showed aggressive behaviour towards the pair of common raven who, in the end, left the quarry. later in the same morning, the male brought a small passerine bird as prey to the female. that was the start of courtship behaviour with many display flights, which continued at least 45 minutes and culminated in copulation on four occasions. copulations lasted from seven to 13 seconds and occurred on the tree at the western part of the quarry. shortly after that, the female took off from the copulation tree and landed on abandoned raven’s nest, after a circular flight in the northern slopes of the quarry. it was the first sign that a pair of peregrines had selected an old stick nest for laying eggs instead of cliff crevice or crag. the position of the nest was in the shade on the northern cliffface, below rock vault, around 22-25 m from water level. one week later, on 14th march, a female was observed on incubation posture at the same raven’s nest (fig. 2). when the female was briefly absent from the nest, it was possible to see at least two eggs inside the nest. hence, it was assumed that she laid the first egg between 8th and 10th march. on 17th april, the female was sitting on the old raven’s nest. however, she was not in such a low posture like at the beginning of the incubation period. therefore, we supposed that the chicks had already hatched, which was confirmed an hour later when the female shortly left the nest to pick up prey from the male. three chicks, 4-6 days old, were observed. accordingly, the incubation lasted approximately 29-31 days. after taking a prey (feral pigeon) female landed on the nest and fed the chicks and herself around 15 minutes. shortly after that, she continued brooding on puny chicks. at the same time, male spent most of its time outside of the quarry. however, between 25th april and 3rd may, the nesting attempt had failed, but reason stays unclear. the nest depression was empty, and no adult birds were observed. during the study period, specimens of peregrine falcon were observed hunting a different prey, mostly small or medium-sized birds. however, because of long-distance and unfavourable environmental conditions (fog and intense sunlight), the prey was rarely identified to the genus or species level. in three occasions that was a feral pigeon, in two occasions common blackbird (turdus merula) and once the prey was starling (sturnus vulgaris), common chaffinch (fringilla coelebs), song thrush (turdus philomelos) and european green woodpecker (picus viridis). according to the moustache, breast colour and spot density, it appears fig. 2. incubating peregrine falcon (falco peregrinus) female in the occupied raven nest in northern cliffs of trachyte quarry “srebro” during march of 2020 biologica nyssana ● 11 (2) december 2020: 103-107 rajković, vračarić ● breeding range extension of the peregrine falcon (falco peregrinus l.) in the pannonian plain 106 that all three observed specimens of peregrine falcon phenotypically corresponded to the nominal subspecies falco peregrinus peregrinus, which is typical subspecies in most parts of central europe. discussion the findings described in this article present the first documented evidence of a peregrine falcon nesting in vojvodina province and at the same time the northernmost breeding site of this species in serbia. consequently, the territory defending and breeding attempts at the same locality in three consecutive years indicated presumably satisfactory habitat quality and suggested site fidelity. the colonisation of the fruška gora mt. by breeding individuals of peregrine falcon has been expected for more than one decade (stojnić & puzović, 2008), but without concrete evidence until 2018. thus, these nesting attempt(s) represent evidence of extension of the species nesting range in the pannonian basin and serbia. the described nesting site is approximately 140 km east from slavonski brod in croatia (budinski, 2013), 130-160 km south from pairs in the baranya county in hungary (https://www.pecsiujsag.hu), and around 100 km from the breeding location near valjevo in central serbia (stojnić & puzović, 2008). in hungary, the first breeding pair was confirmed 23 years ago, after at least 33 years of absence (bagyura 1997). in croatia, single data was collected in 2009, and few pairs are breeding near the capital city of zagreb (budinski, 2013). recently, there are no new published data from continental parts of croatia. however, the situation of the peregrine falcon in hungary is much better, and recently has more than 70 active eyries (prommer & bagyura, 2018). there is no breeding evidence from the romanian part of the pannonian plain, although some pairs nest at the border of the area at apuseni mt. (komáromi et al., 2009). the laying dates, landscape, location and other notes presented in this paper generally coincide with previous data reported across pannonian plain and europe (ratcliffe, 1993; carlier, 1995; moore et al., 1997; prommer & bagyura, 2018). dimensions of potential nesting crag from the year 2018 are significantly smaller than reported earlier (ratcliffe, 1993). this female choice may be related to the insufficient supply of crags and crevices, which is the case at this quarry. although the diet sample is small, the combined observations from different years suggest notable flexibility and wide trophic niche by peregrines on the fruška gora mt. as peregrine falcon established first pair and fruška gora mt. and also regularly hosts floaters from increasing central european stock (prommer & bagyura, 2018), we expect that breeding population would continue to rise in the future and expand its range through other suitable locations on fruška gora and rest of the vojvodina province (e.g. titel hills, vršac mt). together with the discovery of a new breeding site, it is necessary to implement strict conservation measures to protect its breeding territory. acknowledgements. authors are warmly grateful to ivan đorđević, dragomir damnjanović, nikola stanojević and jovana koturov for their help during field visits. mátyás prommer kindly provided references from hungary. additionally, we are grateful for all comments and suggestions of two anonymous reviewers and editor-inchief of the journal. references antal, l., fernbach, j., mikuska, j., pelle, i., szlivka, l. 1971: namenverzeichnis der vögel der autonomen provinz vojvodina. 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(eds.). handbook of birds of the world: new world vultures to guinea fowl. 2. barcelona: lynx edicions. pp. 216–275, plates 24–28. white, c.m., cade, t.j., enderson, j.h. 2013: peregrine falcons of the world. lynx edicions, barcelona. white, c.m., clum, n.j., cade, t.j., hunt, w.g. 2002: peregrine falcon (falco peregrinus), version 2.0. in the birds of north america (a. f. poole and f. b. gill, editors). cornell lab of ornithology, ithaca, ny, usa. wink, m. 2018: phylogeny of falconidae and phylogeography of peregrine falcons. ornis hungarica 26(2): 27–37. zuberogoitia, i., azkona, a., zabala, j., astorkia, l., castillo, i., iraeta, a., martínez, j.a., martínez, j.e. 2009: phenotypic variations of peregrine falcon in subspecies distribution border in: sielicki j., mizera t. (eds.). peregrine falcon populations – status and perspectives in the 21st century. turul & poznan university of life sciences press, poznan, pl. ht tps : / /www.pecs iu jsag .hu/pecs /h i r /he ly i hireink/tiz-fiokat-neveltek-fel-a-vandorsolymok baranyaban?fbclid=iwar20zgp-mk4lyc-l353ed m711xreo2apznkliv2ka7yknjkljlr1sepq1dm 107 biologica nyssana ● 11 (2) december 2020: 103-107 rajković, vračarić ● breeding range extension of the peregrine falcon (falco peregrinus l.) in the pannonian plain jovanović et al 2020, biologica nyssana 11(2) 11 (2) december 2020: 139-147 doi: 10.5281/zenodo.4393973 optimizing conditions for mgmt promoter methylation status analysis in glioblastoma ffpe samples original article nikola m. jovanović university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia nikolajov90@gmail.com (corresponding autor) vesna nikolov university of niš, faculty of medicine, clinic of neurosurgery, clinical center, 18000 niš, serbia v.novak@yahoo.com nataša vidović university of niš, faculty of medicine, pathology and pathological anatomy center, 18000 niš, serbia vidovic.patologija@gmail.com jelena vitorović university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia jelena.rajkovic@gmail.com svetlana tošić university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia tosicsvetlana59@yahoo.com vladimir j. cvetković university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia biovlada@yahoo.com tatjana mitrović university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia tatjanamitrovic3@gmail.com tatjana jevtović-stoimenov university of niš, faculty of medicine, department of biochemistry, 18000 niš, serbia tjevtovic@yahoo.com received: may 14, 2020 revised: september 15, 2020 accepted: november 30, 2020 abstract: the methylation status of the mgmt promoter represents the valuable prognostic and predictive marker in glioblastoma (gbm) patients undergoing treatment with alkylating agents such as temozolomide. although formalinfixed and paraffin-embedded tissue (ffpe) signifies the most commonly used source for tissue-based molecular testing, its use in methylation-specific polymerase chain reaction (msp) analysis manifests certain limitations due to low dna integrity. our study aimed to identify the optimal mgmt promoter msp reaction conditions concerning the utilization of bisulfite-converted ffpe-derived template dna. several optimizing reactions were conducted and subjected to imagej software analysis. as a result, 4u of hotstartaq and 125 ng of template dna were specified as necessary for successful msp reactions. the confirmation of optimization success was obtained through comparison of semi-quantitative values of dna methylation levels between reference fresh frozen tissue and corresponding ffpe sample obtained from the same gbm patient. key words: glioblastoma, ffpe, methylation, mgmt, msp, optimization, taq polymerase apstract: optimizacija uslova msp reakcije za promotorni region mgmt gena kod ffpe uzoraka glioblastoma status metilacije promotornog regiona mgmt gena predstavlja jedan od najvažnijih prognostičkih i prediktivnih markera pacijenata obolelih od glioblastoma (gbm) podvrgnutih lečenju alikirajućim agensima poput temozolomida (tmz). iako uzorci tkiva fiksiranih u formalinu i ukalupljenih u parafinske blokove (ffpe) predstavljaju najuobičajeniji izvor dnk materijala prilikom molekularno-bioloških analiza, njihova upotreba pri metodi lančane reakcije polimeraze specifične za metilaciju (msp) ispoljava određena ograničenja usled niskog integriteta dnk. cilj ovog rada predstavljalo je definisanje optimalnih uslova msp reakcije za mgmt promotorni region prilikom korišćenja bisulfitno-konvertovane dnk izolovane iz ffpe uzoraka kao matrice za msp reakciju. izvedeno je nekoliko reakcija optimizacije, a dobijeni rezultati su obrađeni u imagej programu. kao rezultat, hotstartaq polimeraza u količini od 4u i dnk matrica u količini od 125 ng izdvojene su kao neophodne za uspešnu msp reakciju. potvrda uspešnosti reakcija optimizacije dobijena je upoređivanjem semi-kvantitativnih vrednosti nivoa metilacije dnk između referentnog sveže-zamrznutog uzorka (ff) i odgovarajućeg ffpe uzorka poreklom od istog gbm pacijenta. ključne reči: glioblastomi, ffpe, metilacija, mgmt, msp, optimizacija, taq polimeraza © 2020 jovanovic et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 139 introduction glioblastoma (gbm) refers to the highly infiltrative type of gliomas, malignant brain tumors. owing to great morphological and genetic heterogeneity, it is characterized by extremely low five-year survival rates – only 5%, and an annual incidence of 5.26 per 100,000 people (soomro et al., 2017). although it is widely accepted that gliomas originate from normal glial cells, increasing evidence supports the role of various cell types such as glial or neural precursors and stem cells as their possible origin (chen et al., 2017). recent findings 140 in the molecular biology of glioma have led to the novel, improved system of classification which was established by the world health organization (who) in 2016 (louis et al., 2016). namely, the shortcomings of histopathology based classification were exceeded through the incorporation of molecular diagnostic criteria testing for isocitrate dehydrogenase (idh) mutation, chromosome 1p/19q deletion, and histone mutations. in 2010, the cancer genome atlas (tcga) classification of gbms had identified 4 subtypes of glioblastomas based upon gene expression profiles and genomic clustering – proneural, neural, classical, and mesenchymal subtypes (verhaak et al., 2010). the methylation of the cytosine-phosphate-guanine (cpg) islands in the promoter region of o6-methyl guanine-dna methyltransferase gene (mgmt) represents one of the most common epigenetic alterations in gbm. it is present in both primary and secondary gbm in 42% and 79% patients respectively (soomro et al., 2017). the mgmt enzyme is an excision repair enzyme that removes alkyl adducts from the o6position of the guanine. thus, it protects normal cells from carcinogens by repairing double-strand breaks and base mispairing which leads to apoptosis and cell death. during that process it is being irreversibly deactivated, which is why mgmt is referred to as “suicidal” enzyme (thon et al., 2013; soomro et al., 2017). regarding the mgmt role of counteracting the activity of alkylating agents, its inactivation through epigenetic silencing was recognized as an important and clinically relevant factor in gbm patients undergoing treatment with alkylating agents such as temozolomide (tmz). the positive methylation status of the mgmt promoter represents a strong and independent predictive factor of favorable survival in gbm. the median survival was significantly longer in patients with a methylated mgmt promoter (21.7 months) in comparison with the patients lacking methylation (12.7 months). additionally, a high frequency of mgmt promoter methylation was documented in long-term gbm survivors undergoing tmz treatment (thon et al., 2011; thon et al., 2013). given the fact that methylated cytosine in cpg islands exhibits the same base-pairing interactions as unmethylated cytosine, the evaluation of methylation status using conventional hybridization-based methods, i.e. microarrays and pcr, is not suitable (holmes et al., 2014). to address that, frommer et al. have designed a protocol that utilizes bisulfite– induced modification of genomic dna resulting in the conversion of unmethylated cytosine to uracil, while 5-methylcytosines remain intact (frommer et al., 1992). such bisulfite-converted dna could be subsequently analyzed via pcr in order to acquire desired epigenetic information. with the numerous commercially available kits and several technological advances, current bisulfite-treatment protocols are more convenient and user friendly in comparison with the original 16 hours protocol (holmes et al., 2014). however, the success of bisulfite-conversion may vary considerably depending on the quality of dna samples and the choice of tissue samples (tournier et al., 2012). there are strong suggestions for avoiding formalin-fixed and paraffin-embedded tissues (ffpe) samples for bisulfite conversion and following methylation-specific polymerase chain reaction (msp). these are supported by evidence that the use of ffpe induces non-reproducible bisulfite conversion leading to unreliable and inconsistent results for methylation levels (tournier et al., 2012). in contrast with ffpe, the use of fresh frozen tissue (ff) brings reproducible and satisfactory results, owing to the process of cryopreservation which provides the adequate dna preservation. given that the ffpe samples consist of degraded dna generally less than 300 bp, the main challenge with managing ffpe samples is to provide an efficient cell lysis which releases dna of sufficient quality and quantity for further analysis (holmes et al., 2014). what makes it even more difficult, formalin-fixation induces the formation of dna-protein crosslinks, which often could not be completely removed by common lysis protocols. nevertheless, as ffpe samples are widely available, this type of tissue sample is the most commonly used source for tissue-based molecular testing (dietrich et al., 2013). alongside the low pricing of long term storage, ffpe is often the only available material for retrospective studies and the most important material for standard routine diagnostics in the era of personalized medicine (dietrich et al., 2013). therefore, several commercially available kits for ffpe dna isolation and bisulfite conversion were developed to ensure results as reliable as possible (holmes et al., 2014; de ruijter et al., 2015; ludgate et al., 2017; kint et al., 2018). this study aimed to define optimal pcr conditions for evaluation of mgmt promoter methylation status in the ffpe sample obtained from gbm patient, considering the recommendations from the previous studies (dietrich et al., 2013). also, to investigate the validity of the results, they were compared with mgmt promoter methylation status obtained from the ff sample of the same gbm patient. material and methods patient and tumor specimens both ff and ffpe tissue tumor specimens were collected from the gbm patient (male, 63 years old) operated on at the neurosurgery clinic (the biologica nyssana ● 11 (2) december 2020: 199-147 jovanović et al. ● optimizing conditions for mgmt promoter methylation status analysis in glioblastoma ffpe samples 141 biologica nyssana ● 11 (2) december 2020: 199-147 jovanović et al. ● optimizing conditions for mgmt promoter methylation status analysis in glioblastoma ffpe samples clinical centre of niš, serbia) in 2013. the patient underwent total resection of the tumor and had a karnofsky score of ≥80%. the diagnosis of glioblastoma who grade iv was confirmed by an expert neuropathologist (n.v and m.k). the written informed consent of study participation was obtained from the gbm patient. the ethics committee of the faculty of medicine, niš, serbia, approved the informed consent form and study protocol (01-211310). dna isolation and bisulfite conversion extraction of genomic dna was performed using qiaamp® dna mini kit (qiagen, hilden, germany) from 25 mg of ff sample and qiaamp dna ffpe tissue kit (qiagen, hilden, germany, catalogue no. 56404) from 8 freshly cut sections with a thickness of 10 µm from ffpe sample. a total of 2 µg of genomic dna was modified by sodium bisulfite using epitect® bisulfite kit (qiagen, hilden, germany) for the ff dna sample and epitect plus ffpe bisulfite kit (qiagen, hilden, germany, catalogue no. 59144) for ffpe dna sample. biospec–nano uv–vis spectrophotometer (shimadzu, kyoto, japan) was utilized for the determination of quantity and quality of isolated dna and bisulfite converted samples. isolated dna samples were inspected for degradation and visualized by running the dna samples on 2% agarose gel. methylation-specific polymerase chain reaction (msp) all of the msp reactions were carried out in a total volume of 20 µl containing 0.2 μm dntp mix, 1 × pcr buffer with 1.5 mm mgcl2 (qiagen, hilden, germany) and 10 pm of appropriate forward and reverse primer (tab. 1). amplification reactions were performed in a mastercycler gradient (eppendorf) using the following program: 95 °c for 15 min, then 35 cycles of 95 °c for 50 s, 59 °c for 50 s and 72 °c for 50 s, and a final extension at 72 °c for 10 min. msp optimization reactions with four different amounts of bisulfite-converted template dna (31.25 ng, 62.5 ng, 125 ng, and 250 ng) in combination with 1u and 4u hotstartaq dna polymerase (qiagen, hilden, germany, catalogue no. 203203) were conducted according to suggestions from the previous study (dietrich et al., 2013). following the determination of template dna optimal concentration, another set of optimization reactions was designed regarding the investigation of the optimal concentration of hotstartaq polymerase for successful mgmt msp reactions using ffpe dna isolates. therefore, msp reactions with 1u, 2u, and 4u of hotstartaq polymerase and unmethylated set of mgmt primers were conducted simultaneously with msp reaction including template dna obtained from the ff sample and 1u of hotstartaq polymerase. for comparison of methylation level evaluation between ffpe and ff dna isolates, another set of msp reactions was carried out using both sets of mgmt primers and ffpe and ff isolates as dna templates, followed by agarose gel electrophoresis. msp reactions using ffpederived bisulfite-converted template dna were conducted in duplicate. analysis of methylation data after each optimization, msp gel images were subjected to imagej software analysis (national institute of health, bethesda, md, usa) with the aim of measuring the fluorescence intensity of methylated (m) and unmethylated (u) msp bands. m/u intensity ratio values represent the common approach of semi-quantitative evaluation of the mgmt promoter methylation level (christians et al., 2012). results ff and ffpe dna isolates concentrations and purity of genomic dna isolates and bisulfite-converted dna samples are presented in tab. 2. gene primer sequence (5’-3’) amplicon size (bp) mgmt unmethylated (u) f: tttgtgttttgatgtttgtaggtttttgt r: aactccacactcttccaaaaacaaaaca 93 mgmt methylated (m) f: tttcgacgttcgtaggttttcgc r: gcactcttccgaaaacgaaacg 81 table 1. primer sequences and amplicon size total yield of dna (ng) od 260/280 yield of bisulfite-converted dna per single conversion (ng) ffpe dna isolate (from 8 sections with a thickness of 10 µm) 4,537.5 1.94 2,265.8 ff dna isolate (50 mg of tissue) 35,209.2 1.87 2,094.4 table 2. quantity and quality of isolated dna and bisulfite converted samples (biospec–nano uv–vis spectrophotometer) 142 total yield of isolated dna from ffpe sample was 4,537.5 ng and 35,209.2 ng from ff sample, with od 260/280 values of 1.94 and 1.87, respectively. recorded yield of bisulfite-converted dna per single conversion was 2,265.8 ng for ffpe and 2,094.4 ng for ff dna sample. agarose gel images representing the integrity of isolated dna are shown in fig. 1. agarose gel images of ff dna samples displayed that most of the dna fragments have migrated conjointly with the largest fragments of the dna ladder marker (~9000 bp) thereby forming a noticeable band. in contrast, highly degraded ffpe dna samples were presented on agarose gels as characteristic large smears originating from dna fragments of various sizes, mostly shorter than 1,264 bp. mgmt msp template dna concentration optimization in ffpe samples agarose gel image of msp optimization of optimal template dna quantity is presented in fig. 2. with the clear difference in fluorescence intensity of both methylated and unmethylated mgmt msp products for 1u and 4u of hotstartaq polymerase, the most appropriate m and u msp products were recorded for 125 ng quantity of template dna and 4u of hotstartaq polymerase. biologica nyssana ● 11 (2) december 2020: 199-147 jovanović et al. ● optimizing conditions for mgmt promoter methylation status analysis in glioblastoma ffpe samples fig. 1. agarose gel electrophoresis images of isolated dna samples (~400ng) a) high-quality dna isolates obtained from ff samples (4000-9000 bp fragments). lane 1 – marker λ dna-bst eii (new england biolabs® inc. #n3014s), lane 2ff dna sample (first dna elution), lane 3ff dna sample (second dna elution) b) low-quality ffpe dna samples with highly degraded dna fragments: lane 1 – marker λ dnabst eii, lanes 2,3,4 ffpe dna samples. fig. 2. msp optimization for ffpe dna isolates a) agarose gel image b) legend 143 biologica nyssana ● 11 (2) december 2020: 199-147 jovanović et al. ● optimizing conditions for mgmt promoter methylation status analysis in glioblastoma ffpe samples fig. 3. results of msp optimization regarding defining the optimal amount of hotstartaq polymerase. a) original agarose gel image with a legend on the right, presenting ffpe unmethylated mgmt promoter msp products with 1u, 2u and 4u of hotstartaq polymerase alongside with ff unmethylated mgmt msp with 1u of hotstartaq polymerase product as a positive control. b) imagej software processed image with fluorescence intensity analysis of specific unmethylated mgmt msp product bands, with corresponding chart representing their relative values. c) imagej fluorescence intensity analysis of primer-dimer presence in msp reactions. 144 biologica nyssana ● 11 (2) december 2020: 199-147 jovanović et al. ● optimizing conditions for mgmt promoter methylation status analysis in glioblastoma ffpe samples mgmt msp hotstartaq polymerase concentration optimization in ffpe samples regarding further determination of the optimal amount of hot start taq polymerase, agarose gel image was acquired following msp optimization and processed with imagej software analysis (fig. 3). fluorescence intensity values acquired using the imagej software have confirmed the 4u of hotstartaq polymerase as an optimal amount for successful msp mgmt reaction in ffpe samples. among tested concentrations, the highest fluorescence intensity value of unmethylated msp products was recorded for 4u of hotstartaq polymerase. it was equivalent to 57,43% of the recorded fluorescence intensity value of the reference ff dna mgmt msp product. furthermore, the fluorescence intensity value of primer-dimers was the lowest for the 4u concentration, being even lower than the one recorded for the reference ff dna template msp 37.16% of the reference value. finally, only for the 4u hotstartaq polymerase concentration was documented that the mgmt product/primer-dimer fluorescence intensity ratio exceeds 100% of the reference value (113.12 %), suggesting optimal reaction conditions (tab. 3 and fig. 4). mgmt methylation assesment in ffpe and ff sample figure 5 (a) shows the agarose gel image of mgmt msp reactions which included bisulfite-converted template dna of the same patient originating from both ffpe and ff samples, alongside with randomly chosen ffpe sample from another gbm patient. m/u ratio values of ff and ffpe samples were acquired via imagej software analysis fig. 5 (b). as a result of imagej software analysis, both the ffpe and ff samples were assessed as strongly methylated semi-quantitative category of mgmt promoter methylation (m/u ratio <1). the observed difference in m/u ratio values was 8.47% (tab. 4). discussion results from our study have confirmed the great difference in dna fragmentation level between ff and ffpe dna isolates (fig. 1). in contrast with the ff sample, the ffpe sample from our study was in great portion consisted of dna fragments shorter than ~1,000 bp, which were forming a smear and lacking a distinguishable band on the agarose gel. this observation is consistent with previously described ffpe dna isolate properties originating from the tissue preparation and fixation process. namely, it was shown that mechanical stress resulting from cross-linking during the fixation process, the concentration of formalin, ph, and salt, the temperature, and tissue type have a great impact on the quality of the ffpe-derived dna (ludgate et al., 2017). among such factors, the deparaffinization was emphasized as the crucial process which reduces the quality of the isolated dna (sengüven et al., total amount of hotstartaq polymerase units in msp reaction 1u 2u 4u fluorescence intensity of unmethylated mgmt mps product compared to ff refference (%) 48.27 34.67 57.43 fluorescence intensity of primer-dimers compared to ff refference (%) 200.60 219.85 37.16 product / primer-dimer intensity ratio compared to ff refference (%) 49.10 36.30 113.12 table 3. fluorescence intensity values of 3 different concentrations of hotstartaq polymerase obtained using imagej software analysis fig. 4. mgmt product / primer-dimer fluorescence intensity ratios detected for 125 ng of template dna and 3 different concentrations of hotstartaq polymerase in ffpe and reference ff dna bisulfiteconverted samples 145 2014). given the fact that bisulfite conversion further degrades dna, the challenges which arise in dna methylation analysis are no surprising (patterson et al., 2011). as a consequence, msp reaction conditions concerning methylation analysis may significantly differ in case of using the ffpe instead of the ff bisulfite-converted sample as template dna. considering the significance of ffpe tissues as a valuable dna source for mgmt methylation analysis in gbm patients, as well as for clinical and cancer research in general, our main goal was to reveal the optimal mgmt promoter methylation msp reaction conditions which could bring the results as valid as those involving ff-derived dna. the most significant msp reaction parameters – taq polymerase and template dna concentrations were selected considering the findings of improved pcr performance in ffpe tissue samples presented by dietrich et al. (2013). primarily, our study tested 4 concentrations of template dna (31.25 ng, 62.5 ng, 125 ng, and 250 ng) and 2 concentrations of hotstarttaq polymerase (1u and 4u) for the capability of alleviating pcr inhibition and amplifying methylated mgmt promoter (81 bp) and unmethylated mgmt promoter (93 bp) msp products. in concordance with previous findings, this optimization reaction revealed the 4u of hotstarttaq polymerase, alongside with 125 ng of template dna as optimal concentrations for both methylated and unmethylated msp products. as seen in fig. 2, the gradual increase of template dna amount enhances the msp product intensity, both in 1u and 4u reaction subgroups. this overcoming of pcr inhibition occurs through increasing the probability of the presence of template molecules of proper length and integrity. as a result, higher template concentrations and 1u concentration of hotstartaq polymerase emerged as one of the possible solutions for optimal amplification conditions. furthermore, a 4-fold increase of the hotstarttaq polymerase clearly showed the successful amplification in replicates containing low amounts of template dna. the listed observations were suggesting the optimal amount of 125 ng and 4u of hotstarttaq polymerase in both methylated and unmethylated mgmt products. however, the presence of additional primer-dimer bands on agarose gel could lead to misinterpretation of methylation level analysis. to address that issue and further elucidate optimal msp conditions, another optimization reaction was conducted. given the relatively short length of the mgmt promoter msp products, this optimization introduced the 2u concentration of hotstarttaq polymerase as a possible solution for economizing enzyme consumption. for that purpose, optimization reaction was performed including 3 concentrations of hotstartaq polymerase (1u, 2u, and 4u), 125 ng amount of template dna, and unmethylated mgmt promoter primer set (fig. biologica nyssana ● 11 (2) december 2020: 199-147 jovanović et al. ● optimizing conditions for mgmt promoter methylation status analysis in glioblastoma ffpe samples fig. 5. differences in mgmt promoter methylation assessment between ff and ffpe samples: a) agarose gel image presenting methylated and unmethylated mgmt promoter msp products with the corresponding legend on the right side. lanes 3 and 4 methylated mgmt promoter msp products involving ffpe samples of the previously listed gbm patient as template dna; lanes 8 and 9 unmethylated mgmt promoter msp products involving ffpe samples of the previously listed gbm patient as template dna; lanes 12 and 13 reference ff sample mgmt promoter msp products (methylated and unmethylated); lanes 5,6,10,11 mgmt promoter msp products (methylated and unmethylated) assessed with ffpe bisulfite-converted sample of different gbm patient. b) fluorescence intensity levels of methylated (m) and unmethylated (u) msp bands obtained using imagej software. products and ffpe-derived template dna. although the above-mentioned pcr conditions refer to the amplification of specific mgmt promoter pcr products using the end-point pcr method, they could also be taken into consideration while performing ms-qpcr reactions. however, it should be noted that due to the small sample size and a relatively small number of measurements, the presented results were not additionally supported by some form of statistical analysis. acknowledgement. this study was supported by the ministry of education, science and technological development of the republic of serbia (contract number: 451-03-68/2020-14/200124). references chen, r., smith-cohn, m., cohen, a.l., colman, h. 2017: glioma subclassifications and their clinical significance. neurotherapeutics, 14: 284297. christians, a., hartmann, c., benner, a., meyer, j., von deimling, a., weller, m., wick, w., weiler, m. 2012: prognostic value of three different methods of mgmt promoter methylation analysis in a prospective trial on newly diagnosed glioblastoma. plos one, 7(3): e33449. de ruijter, t.c., de hoon, j.p., slaats, j., de vries, b., janssen, m.j., van wezel, t., aarts, m.j., van engeland, m., tjan-heijnen, v.c., van neste, l., veeck, j. 2015: formalin-fixed, paraffinembedded (ffpe) tissueepigenomics using infinium humanmethylation450beadchip assays. models and techniques, 95: 833-842. dietrich, d., uhl, b., sailer, v., holmes, e.e., jung, m., meller, s., kristiansen, g. 2013: improved pcr performance using template dna from formalin-fixed and paraffin-embedded tissues by overcoming pcr inhibition. plos one, 8(10): e77771. esteller, m., garcia-foncillas, j., andion, e., goodman, s.n., hidalgo, o.f., vanaclocha, v., baylin, s.b., herman, j.g. 2000: inactivation of the dna-repair gene mgmt and the clinical response of gliomas to alkylating agents. the new england journal of medicine, 343(19): 1350-1354. frommer, m., mcdonald, l.e., millar, d.s., collis, c.m., watt, f., grigg, g.w., molloy, p.l., paul, c.l. 1992: a genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual dna strands. proceedings of the national academy of sciences of the united states of america, 89(5): 1827-1831. holmes, e.e., jung, m., meller, s., leisse, a., 146 biologica nyssana ● 11 (2) december 2020: 199-147 jovanović et al. ● optimizing conditions for mgmt promoter methylation status analysis in glioblastoma ffpe samples 3). ff dna bisulfite-converted sample served as a control msp reaction. despite successful amplification of the specific product in 1u and 2u replicates, an imagej software analysis showed the significant presence of primer-dimer bands in 1u and 2u, but not in 4u replicate, confirming the 4u concentration of hotstartaq polymerase as the optimal one (fig. 4). since the original study concerning mgmt promoter methylation in gbm patients, there were several common approaches of methylation status assessment (esteller et a., 2000). in order of improving the qualitative assessment results obtained by the end-point pcr method, the semiquantitative approach which utilizes additional imagej software analysis of gel images was proposed (dietrich et al., 2013). thus, through the measuring of fluorescence intensity ratios of methylated and unmethylated mgmt promoter product bands, patients could be sorted into three groups – unmethylated (m/u ratio =0), with weak promoter methylation (m/u ratio between 0 and 1) and strongly methylated (m/u ratio >1). although this approach is widely replaced by qpcr methods of methylation status assessment, for this study it presents a valuable method for validation of optimizing msp reaction results. by comparing m/u ratio values with those of ff samples, ffpe mgmt promoter msp reaction conditions could be tested. for that purpose, the final set of mgmt promoter msp reactions in our study have included both reference ff and ffpe dna isolates, performed in duplicate with previously defined pcr conditions (4u hotstarttaq polymerase and 125 ng of template dna). indeed, m/u ratios obtained for corresponding ff and ffpe samples in our study were very similar, differing only in 8.47 percent (tab. 4). in that manner both of the samples were assessed as strongly methylated (tab. 4), although the absolute intensity values were almost 3-fold different (fig. 5b). this observation suggests that the optimizing conditions for mgmt promoter msp using ffpe-derived dna samples presented in this study were properly defined. conclusion the results acquired in our study were in line with previous findings concerning the use of ffpe tissue in msp reactions. thus, gel electrophoresis analysis of the ff and ffpe dna isolates confirmed the high level of degradation in ffpe isolates. based on several optimizing reactions, 4u hotstartaq polymerase and 125 ng of template dna could be singled out with high certainty as suitable concentrations for successful msp reaction concerning methylated and unmethylated mgmt promoter amplification biologica nyssana ● 11 (2) december 2020: 199-147 jovanović et al. ● optimizing conditions for mgmt promoter methylation status analysis in glioblastoma ffpe samples sailer, v., zech, j., mengdehl, m., garbe, l.a., uhl, b., kristiansen, g., dietrich, d. 2014: performance evaluation of kits for bisulfiteconversion of dna from tissues, cell lines, ffpe tissues, aspirates, lavages, effusions, plasma, serum, and urine. plos one, 9(4): e93933. kint, s., de spiegelaere, w., de kesel, j., vandekerckhove, l., van criekinge, w. 2018: evaluation of bisulfite kits for dna methylation profiling in terms of dna fragmentation and dna recovery using digital pcr. plos one, 13(6): e0199091. louis, d.n., perry, a., reifenberger, g., von deimling, a., figarella-branger, d., cavenee, w.k., ohgaki, h., wiestler, o.d., kleihues, p., ellison, d.w. 2016: the 2016 world health organization classification of tumors of the central nervous system: a summary. acta neuropathologica, 131(6): 803-820. ludgate, j.l., wright, j., stockwell, p.a., morison, i.m., eccles, m.r., chatterjee, a. 2017: a streamlined method for analysing genome-wide dna methylation patterns from low amounts of ffpe dna. bmc medical genomics, 10(1): 54. patterson, k., molloy, l., qu, w., clark, s. 2011: dna methylation: bisulphite modification and analysis. journal of visualized experiments, (56): 3170. sengüven, b., baris, e., oygur, t., berktas, m. 2014: comparison of methods for the extraction of dna from formalin-fixed, paraffin-embedded archival tissues. international journal of medical sciences, 11(5): 494-499. soomro, s.h., ting, l.r., qing, y.y., ren, m. 2017: molecular biology of glioblastoma: classification and mutational locations. the journal of the pakistan medical association, 67(9): 14101414. thon, n., eigenbrod, s., grasbon-frodl, e.m., lutz, j., kreth, s., popperl, g., belka, c., kretzschmar, h.a., tonn, j.c., kreth, f.w. 2011: predominant influence of mgmt methylation in non-resectable glioblastoma after radiotherapy plus temozolomide. journal of neurology, neurosurgery, and psychiatry, 82(4): 441–446. thon, n., kreth, s., kreth, f.w. 2013: personalized treatment strategies in glioblastoma: mgmt promoter methylation status. onco targets and therapy, 6: 1363-72. tournier, b., chapusot, c., courcet, e., martin, l., lepage, c., faivre, j., piard, f. 2012: why do results conflict regarding the prognostic value of the methylation status in colon cancers? the role of the preservation method. bmc cancer, 12: 12. verhaak, r.g., hoadley, k.a., purdom, e., wang, v., qi, y., wilkerson, m.d., miller, c.d., ding, l., golub, t., mesirov, j.p., alexe, g., lawrence, m, o’kelly, m.,temayo, p., weir, b.a., gabriel, s., winckler, w., gupta, s., jakkula, l., feiler, h.s., hodgson, j.g., james, c.d., sarkaria, j.n., brennan, c., kahn, a., spellman, p.t., wilson, r.k., speed, t.p., gray, j.w., meyerson, m., getz, g., perou, c.m., hayes, dn.; cancer genome atlas research network. 2010: integrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities in pdgfra, idh1, egfr, and nf1. cancer cell, 17(1): 98-110. 147 djebbouri & terras 2022, biologica nyssana 13(1) 13 (1) september 2022: 1-9 doi: 10.5281/zenodo.7117398 community structure with particular reference to the effect of grazing in forest formations of saïda (algeria) original article mohammed djebbouri laboratory of water resources and environment, department of biology, faculty of science, university of saïda dr. tahar moulay, saida, 20000, algeria conservation of forests in the wilaya of saïda general directorate of forestry (gdf), algeria mohammeddjebbouri@yahoo.com (corresponding author) mohamed terras laboratory of water resources and environment, department of biology, faculty of science, university of saïda dr. tahar moulay, saida, 20000, algeria received: august 16, 2021 revised: may 04, 2022 accepted: may 24, 2022 abstract: to better understand and manage the forest vegetation of the saïda district (types of vegetation present, floristic compositions, possible types of degradation), a total of 215 phytoecological surveys were carried out in the study area. in each survey, the intensity of the grazing and the presence or absence of regeneration of the tree layer were noted. the plant groups were determined using a modified twinspan classification. for each vegetation group, the diagnostic species were selected based on their fidelity index (phi coefficient). we identified 9 plant groups and selected 147 diagnostic species. the results of this study showed that in the surveys carried out, there were more than 80% of moderately to heavily grazed, while in more than 70% of the studied plots, there was no regeneration of the main tree species. this alarming situation implies the rapid and effective implementation of protection and conservation measures. key words: structure, forest formations, grazing, saïda, algeria apstrakt: struktura zajednica sa posebnim osvrtom na efekat ispaše u šumskim formacijama saïda (alžir) kako bi se bolje razumelo i upravljalo šumskom vegetacijom okruga saïda (prisutni tipovi vegetacije, floristički sastav, mogući tipovi degradacije), u oblasti istraživanja prikupljeno je ukupno 215 fitocenoloških snimaka. za svaku sastojinu utvrđen je intenzitet ispaše i prisustvo ili odsustvo regeneracije sprata drveća. biljne grupe su utvrđene korišćenjem modifikovane twinspan klasifikacije. za svaku vegetacijsku grupu, dijagnostičke vrste su odabrane na osnovu vrednosti indeksa “vernosti” (phi koeficijenta). identifikovali smo 9 biljnih grupa i selektovali 147 dijagnostičkih vrsta. rezultati ove studije su pokazali da u više od 80% izvršenih istraživanja postoji prekomerna ili umerena ispaša, dok u više od 70% ispitivanih lokaliteta nema regeneracije glavnih vrsta drveća. ova alarmantna situacija zahteva brzu i efikasnu implementaciju mera zaštite i konzervacije. ključne reči: struktura, šumske formacije, ispaša, saïda, alžir introduction algeria is part of the mediterranean basin, which has been described as one of the 34 “hotspots” on the planet (myers et al., 2000). indeed, myers et al. (2000) consider that the mediterranean countries hold almost 4.5% of the world’s flora. in recent decades, a number of stakeholders have threatened important habitats and their associated plants. an estimate of the extinction rate of higher mediterranean plants is 0.15 percent of the total, representing 37 species presumed extinct, while 4,251 plant taxa are threatened (greuter, 1994). it is therefore urgent to improve vegetation management. the identification and description of plant groups, as well as their relationship to the environment, is a vital step in understanding arid environments (al harthy & grenyer, 2019). first, different plant communities are considered as substitutes for ecosystem delineation, and community definitions therefore help to understand the macro-ecological differences between biosphere components (al harthy & grenyer, 2019). indeed, using ecological species groups and the presence-absence or coverage of their indicator species can help with the development of management plans and the selection of preferred practices for the long-term management of ecosystems, such as monitoring environmental © 2022 djebbouri & terras. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 1 changes, assessing management effectiveness, and providing warning signals for impending ecological changes (barnes et al., 1982; siddig et al., 2016). effective conservation planning also includes identifying and describing plant communities (kent, 2011; brown et al., 2013). in algeria, knowledge on the structure of forest and preforest vegetation is still incomplete (meddour, 2012). in this context, the present study aims to improve our knowledge on the structure of forest and preforest groupings in the saïda region and to obtain an accurate inventory and a detailed diagnosis of existing plant groupings, possibly for mapping plant groups (elaboration of the typology). the final objective was always to better understand and manage natural areas (types of vegetation present, floristic compositions, conservation issues, possible types of degradation...). the results are intended for biodiversity specialists, managers of natural areas and/or naturalists. materials and methods study area the study area (forest formations and preforests), located in the district of saïda in the northwest of algeria with an area of about 174,361 hectares, between 35°6’23.68” and 34°35’9.14” latitudes north and 0°25’10.89” west at 0°47’28.58” longitudes east (fig. 1). vegetation sampling the inventory of flora is based on the 215 floristic surveys carried out through the forests area of the saïda region during the years 2017-2018-2019 2 biologica nyssana ● 13 (1) september 2022: 1-9 djebbouri & terras ● community structure with particular reference to the effect of grazing inforest formations of saïda (algeria) fig. 1. location of study area. projection system is utm zone 30-n, wgs84 3 between the periods of march to mid-june. the sampling plan used to analyze the vegetation is subjective. this is the simplest and most intuitive form of choosing as samples areas that appear particularly homogeneous and representative (gounot, 1969; terras, 2011). the identification of the inventoried plant species was carried out using the new flora of algeria and the southern desert regions (quézel & santa, 19611962). in this study, the synonymic index of north africa was used for the botanical nomenclature of species inventoried (dobignard & chatelain, 20102013). a total of 215 records were collected in the turboveg database (hennekens & schaminée, 2001). floristic classification and statistical analysis the phytosociological data were analyzed using the modified twinspan (two-way indicator species analysis) method (hill, 1979; roleček et al., 2009), integrated into the program juice 7.0 (tichý, 2002). the minimum group size has been set at 3 and the coverage thresholds have been set at 0%, 5% and 25%. the whittaker beta diversity index (whittaker, 1972) was used for the analysis of the heterogeneity of groups, because it provides balanced classifications, respecting the size and heterogeneity of the group, but also because of its robustness (roleček et al., 2009). to find an optimal cluster depth in the dendrogram produced by the modified twinspan algorithm, we used the optimclass procedure (tichý et al., 2010), which is also implemented in juice. optimclass was applied in mode 1. in naming plant groupings, we followed the informal approach of menz et al. (2012) and ik-gürsoy et al. (2016) meaning that the nomination of plant groups always includes the name of the most diagnostic species and the species with the highest coverage. grazing intensity to obtain an estimate of the grazing pressure in the study area, we used the following scale (medjahdi, 2001; medjahdi, 2010): 0 absent; 1 present but without causing damage other than on the herbaceous layer (very low); 2 fairly significant damage to the herbaceous layer and to the foliage of the bushy layer (low); 3 significant damage to the bushy layer (moderately); 4 the heavily degraded forest undergrowth (height); 5 almost total disappearance of the bushy layer (heavily). biologica nyssana ● 13 (1) september 2022: 1-9 djebbouri & terras ● community structure with particular reference to the effect of grazing inforest formations of saïda (algeria) calculation of diversity indices for each floristic survey the following indices were calculated using the juice 7.0 software (tichý, 2002): richness-index, shannon-index, eveness index and simpson-index. species richness (s), shannon-wiener diversity index (h′) and eveness index were calculated to describe the pattern of species diversity for each floristic record (i.e. alpha diversity α), as well as for each plant group identified by twinspan (that is to say the beta diversity β). the differences between the mean values of the diversity indices between the plant groups were evaluated using a nonparametric analysis of variance with the kruskal-wallis test and the multiple comparison of the mean ranks. the differences were considered significant if p<0.05. this analysis was performed using spss 24.0 (statistical package for the social sciences). to study species diversity and its relationship to the variables measured in this study, the data collected was examined using the pearson correlation coefficient, which aims to draw a brief conclusion about the factors likely to affect species diversity. stratification and recouvrement to bring out the main lines of the vertical structure of the vegetation, only three strata were retained: arborescent, shrub and herbaceous. these three strata are identified according to the scale: tree stratum: (3 meters and more), shrub stratum (between 0.50 m and 3 m), herbaceous stratum (includes all nonwoody species, these species do not exceed 0.50 m in general). in each floristic survey we noted: the percentage of the overall recovery and the recoveries of the three identified strata, the presence or absence of the natural regeneration of the tree layer. results and discussion plant groups and diagnostic species free based on the twinspan analysis, and after the optimclass1 analysis, nine plant groups were identified. considering a cut-off value of 0.30 for the fidelity coefficient f, 58 diagnostic species were selected. these groups each have a particular floristic composition (tab. 1). in its study in the same area terras (2011) identified seven plant groups. this indicates the wide variation in the results of this study and the study conducted by terras (2011). there are several reasons why a gap exists. this could be the difference in scale of the surveys, the difference in methods and style of assessment as well as the variation in anthropozoic factors such as fires and grazing (fig. 2). the overall recovery rate and the contribution of 4 biologica nyssana ● 13 (1) september 2022: 1-9 djebbouri & terras ● community structure with particular reference to the effect of grazing inforest formations of saïda (algeria) table 1. synoptic table of vegetation sampling and species fidelity values in the nine groups group n 1 2 3 4 5 6 7 8 9 number of floristic surveys 6 47 5 16 34 4 39 11 53 total number of species 15 83 35 54 136 23 99 62 144 group 1: asparagus albuspistacia atlantica asparagus albus l. 87.5 -- -- -- -- -- -- -- --pistacia atlantica desf. 75 -- -- -- -- -- -- -- --ballota hirsuta benth. 69.9 -- -- -- -- -- -- -- --psychine stylosa desf. 55.5 -- -- -- -- -- -- -- --capsella bursa-pastoris (l.) medik. subsp. ábursa-p 55.5 -- -- -- -- -- -- -- --calendula arvensis (vaill.) l. 54.8 -- -- -- -- -- -- -- --group 2: olea europaea l. subsp. europaea tetraclinis articulata olea europaea l. subsp. europaea --64 -- -- -- -- -- -- --drimia fugax (moris) stearn --41.4 -- -- -- -- -- -- --group 3: papaver rhoeas hordeum murinum papaver rhoeas l. -- --94.2 -- -- -- -- -- --clypeola jonthlaspi subsp. microcarpa (moris) arca -- --73.5 -- -- -- -- -- --avena sterilis l. -- --69 -- -- -- -- -- --carduus getulus pomel -- --67.9 -- -- -- -- -- --hordeum murinum subsp. leporinum (link) arcang -- --61.4 --18.1 -- -- -- --galium viscosum vahl -- --61 -- -- -- -- -- --hohenackeria exscapa (steven) koso-pol. -- --61 -- -- -- -- -- --coronilla scorpioides (l.) w.d.j. koch -- --61 -- -- -- -- -- --dasypyrum hordeaceum (coss. & durieu) p. -- --61 -- -- -- -- -- --lolium perenne l. -- --61 -- -- -- -- -- --valerianella coronata (l.) dc -- --54 -- -- -- -- -- --group 4: juniperus oxycedrus subsp. oxycedrus quercus ilex juniperus oxycedrus l. subsp. oxycedrus -- -- --46.6 28.7 -- -- -- --ferula communis l. --26.4 --34.6 28.8 -- -- -- --group 5: neatostema apulum macrochloa tenacissima neatostema apulum (l.) i.m. johnst. -- -- -- --50 -- -- -- --carthamus pinnatus desf -- -- -- --46.3 -- -- -- --paronychia arabica subsp. cossoniana j. gay ex ba -- -- -- --44.6 -- -- -- --plantago albicans l. -- -- -- --40 -- -- -- --atractylis cancellata l. -- -- -- --36.4 -- -- -- --anacyclus pyrethrum (l.) link -- -- -- --34.2 -- -- -- --echinaria capitata (l.) desf. -- -- -- --34.2 -- -- -- --scandix australis l -- -- -- --32.6 -- -- -- --group 6: quercus faginea quercus faginea lam. -- -- -- -- --100 -- -- --each stratum to the overall recovery are summarized in fig. 3. the overall recovery rate of the identified groups is high. this importance is mainly due to the strong contribution of the shrub layer. these values confirm once the openness of the environment and the role played by the shrub layer in the fight against erosion, the stability of the plant cover and the protection of the phytocenosis despite the attacks to which it is subjected (medjahdi, 2010; terras, 2011). if we consider only the floristic richness (tab. 2), we notice that the obtained results are logical, that is to say that the richness is decreased when the intensity of grazing is increased. in a recent study (herrero‐jáuregui & oesterheld, 2018), it was found that the effect of grazing intensity on species 5 biologica nyssana ● 13 (1) september 2022: 1-9 djebbouri & terras ● community structure with particular reference to the effect of grazing inforest formations of saïda (algeria) richness and diversity was negative for arid systems. according to our results, the correlation between grazing intensity and tree cover was absent, which also showed the high grazing intensity for all the plant groups identified in this study. a negative correlation was recorded between richness and tree cover rate (tab. 2). the average wealth index was higher in groups 3, 5, 8 characterized by a low tree cover which is between 0% and 14% (fig. 3, fig. 4). according to quézel (2000), the matorrals constitute the vegetation structures undoubtedly the most remarkable of the maghreb, because of their floristic richness. similarly, medjahdi (2010) indicates for the mountains of traras that the floristic richness of smilax aspera l. -- -- -- -- --84.1 -- -- --nerium oleander l. -- -- -- -- --79 -- -- --dittrichia viscosa (l.) greuter -- -- -- -- --68.6 -- -- --mentha suaveolens ehrh. -- -- -- -- --68.6 -- -- --geranium molle l. -- -- -- -- --68.6 -- -- --oxalis pes-caprae l. -- -- -- -- --68.6 -- -- --group 7: pinus halepensis pinus halepensis mill. -- -- -- -- -- --53.5 --23 helianthemum cinereum subsp. rotundifolium -- -- -- -- -- --33.9 -- --group 8: ziziphora hispanica macrochloa tenacissim ziziphora hispanica l. -- -- -- -- -- -- --58.6 --alyssum simplex rudolphi -- -- -- -- -- -- --58 --globularia alypum l. subsp. alypum -- -- -- -- -- -- --55.2 29.4 reseda collina müll. arg. -- -- -- -- -- -- --52.6 --teucrium polium l. -- -- -- -- -- -- --51.4 18.7 erysimum grandiflorum desf. -- -- -- -- -- -- --50 --achillea santolinoides lag. -- -- -- -- -- -- --50 --ebenus pinnata aiton -- -- -- -- -- -- --48 --erodium botrys (cav.) bertol. -- -- -- -- -- -- --45 --group 9: rosmarinus eriocalyx tetraclinis articulata rosmarinus eriocalyx jord. & fourr. -- -- -- -- -- -- -- --56.7 cistus creticus l. -- -- -- -- -- -- -- --43.9 quercus coccifera l. -- -- -- -- -- -- -- --37.6 cistus clusii dunal -- -- -- -- -- -- -- --34.5 macrochloa tenacissima (l.) kunth -- -- -- -- -- -- -- --34 lavandula stoechas l. -- -- -- -- -- -- -- --31.9 genista quadriflora munby -- -- -- -- -- -- -- --30.3 thymus munbyanus subsp. ciliatus (desf.) g -- -- -- -- -- -- -- --30.2 tetraclinis articulata (vahl) mast. --56.3 -- -- -- -- -- --35.5 quercus ilex l. -- -- --55.7 43.8 -- -- -- --asphodelus ramosus l. -- -- --32.5 38.6 -- -- -- --the habitats is maximum for the matorrals more or less open, it decreases in the case of closure of the medium. the values of the shannon index are less strong overall (2.8 bits) and the equitability varies between 0.4 and 0.9, but they are higher in wooded matorrals than in closed formations (fig. 3, fig. 4). these results are confirmed by the negative correlation that has been recorded between the values of the shannon index and the coverage rate (p<0.05) (tab. 2). according to orth & girard (1996), the shannon index has high values for species with similar recoveries and takes low values when a few species have high recoveries, whereas equitability tends towards 0 when a species has a very strong overlap and tends towards 1 when all species have the same importance. this is due to the presence of rare and infrequent taxa. no quantitative studies on pastures have already been carried out in the saïda region, so it is difficult to measure changes in vegetation composition and diversity. nevertheless, based on the results of this study, it was evident that most of the groups identified in this study are severely disturbed by grazing. this is one of the main threats, indicated as more than 80% of the sites surveyed were moderately to heavily grazed and the rest showing signs of disturbance. during the period of study and continuous observation of the vegetation, in more than 70% of the sites studied there was no regeneration of the main tree species such as pistacia atlantica, 6 biologica nyssana ● 13 (1) september 2022: 1-9 djebbouri & terras ● community structure with particular reference to the effect of grazing inforest formations of saïda (algeria) fig. 2. some of the anthropogenic actions in the pre-forest regions of saïda (photos: djebbouri. m). a: burnt stand of aleppo pine (djebel aoun); b: pasture (hssasna); c: misdemeanors and illegal cuts; d: land clearing. (tendfelt) massif; e: aleppo pine groups spared by the fires 7 biologica nyssana ● 13 (1) september 2022: 1-9 djebbouri & terras ● community structure with particular reference to the effect of grazing inforest formations of saïda (algeria) fig. 3. the overall recovery rate and the contribution of each stratum to the overall recovery in each group of plants richness index shannon index simpson index eveness index grazing intensity tree cover (%) richness index 1 0.830** 0.634** 0.499** -0.269** -0.367** shannon index 1 0.940** 0.824** -0.176* -0.657** simpson index 1 0.833** -0.129 -0.733** eveness index 1 0.032 -0.697** grazing intensity 1 -0.025 tree cover (%) 1 table 2. pearson correlation between measured variables fig. 4. mean values of diversity indices (means ± standard error) in the identified groups of plant species. different letters in each column indicate a significant difference at the 0.05 level according to the kruskal wallis test biologica nyssana ● 13 (1) september 2022: 1-9 8 djebbouri & terras ● community structure with particular reference to the effect of grazing inforest formations of saïda (algeria) tetraclinis articulata and olea europaea, quercus ilex subsp. ballota (desf.) samp., pinus halepensis, quercus faginea, which is indisputably due to grazing (χ2 = 18.129, p = 0.0001). this has already been underlined by benabedli (1996) who specifies that: “the course in forest formation constitutes a very degrading factor by its aggressiveness and the damage it causes to the vegetation and to the soil. source of partial or total removal of plant cover, the unregulated route imposes the following degradation process: total exploitation of the herbaceous layer, consumption of young shoots, seedlings and suckers, grazing of the palatable bushy layer, pruning of the shrub layer”. louni (1994) also indicates that overgrazing thus has the consequence of eliminating by browsing young regenerations, low branches and suckers and the effects of trampling on the ground are also serious. indeed, this phenomenon of overgrazing has never ceased to endanger the entire forest heritage of the saïda region. the excess livestock destroys the protective plant cover, while by trampling the surface of the soil powdery and by compacting it, it reduces the permeability of the soil, and therefore its water reserves (ayoub et al., 2019). conclusions the forest vegetation of the saïda region has not been described in detail before. the need for classification and detailed description has become necessary. the present study allowed a better knowledge of the floristic composition and the structural characteristics through the 09 identified plant groups. the vegetation of the study area is quite rich and consists of 406 plant species. nine (9) groups were discriminated against and the g9 group (rosmarinus eriocalyx tetraclinis articulata) is the richest in terms of number of species with 136 species. the diversity and equity indices show that there is a high diversity of species within the groupings and that many species participate in the recovery. the structure of the stands shows the dominance of the shrub character of the woody stratum in the groups. this testifies to the state of degradation and disturbance of the vegetation in this area subject to overgrazing and arid climatic conditions and further reveals the regressive trends in the overall dynamics of the vegetation. this alarming situation requires the rapid and effective implementation of protection and conservation measures. in order to maintain this biological wealth and safeguard this diversity of natural habitats, which remain strongly threatened due to the accentuation of the constraints of anthropogenic origins in these regions. references al harthy, l., grenyer, r. 2019: classification and ordination of the main plant communities of the eastern hajar mountains, oman. journal of arid environments, 169:1-18. ayoub, a., habib, b.a., abelkrim, k., mohamed, z., raphael, g. 2019: effects of overgrazing on the physico-chemical and biological properties of semiarid forest soils in western algeria. indian journal of ecology, 46(4): 745-750. barnes, b.v., pregitzer, k.s., spies, t.a., spooner, v.h. 1982: ecological forest site classification. journal of forestry, 80(8): 493-498. benabdeli, k. 1996: mise en évidence de l’importance des formations basses dans la sauvegarde des écosystèmes forestiers: cas des monts de dhaya (algérie occidentale). ecologia mediterranea, 22(3): 101-112. brown, l.r., du preez, p.j., bezuidenhout, h., bredenkamp, g.j., mostert, t.h., collins, n.b. 2013: guidelines for phytosociological classifications and descriptions of vegetation in southern africa. koedoe, 55(1): 1-10. djebbouri, m., terras, m. 2020: effects of aleppo pine (pinus halepensis mill) plantation on plant biodiversity in the high plains of saïda (algeria). asian journal of research in biosciences, 29-36. dobignard, a., chatelain, c. 2010-2013: index synonymique de la flore d’afrique du nord. conservatoire et jardin botanique de genève. gounot, m. 1969: méthodes d’étude quantitative de la végétation. masson et cie, paris. 314 p. greuter, w. 1994: extinctions in mediterranean areas. philosophical transactions of the royal society b: biological sciences, 344(1307): 41-46. hennekens, s. m., schaminée, j. h. 2001: turboveg, a comprehensive data base management system for vegetation data. journal of vegetation science, 12(4): 589-591. herrero‐jáuregui, c., oesterheld, m. 2018: effects of grazing intensity on plant richness and diversity: a meta‐analysis. oikos, 127(6): 757-766. hill, m.o. 1979: a fortran program for arranging multivariate data in an ordered twoway table by classification of the individuals and attributes. twinspan. cornel university press, ithaca, ny, usa. 48 p. ik-gürsoy¸ i.d., uğurlu, e., oldeland, j. 2016: plant communities, diversity and endemism of the kula volcano, manisa, turkey. plant biosystems-an international journal dealing with all aspects of plant biology, 150(5): 1046-1055. kent, m. 2011: vegetation description and data analysis: a practical approach. john wiley & sons, new jersey, états-unis. 448 p. louni, d. 1994: les forêts algériennes. forêt méditerranéenne.t: xv, 7: 59–63. meddour, r. 2012: bioclimatologie, phytogeographie et phytosociologie en algerie: exemple des groupements forestiers et preforestiers de kabylie djurdjureenne. phd thesis. universite mouloud mammeri. algerie. medjahdi, b. 2001: réponse de la végétation du littoral des monts des trara (ouest algérien) aux différents facteurs de dégradation. mém. magistère: université de tlemcen. algerie. medjahdi, b. 2010: reponse de la vegetation du littoral oranais aux perturbations: cas des monts des trara (nord-ouest de l’algerie). phd thesis. université de tlemcen. algerie. menz, g., alvarez, m., möseler, b. m., mogha, n.g., handa, c., oyieke, h.a., misana, s.b., boehme, b., mwita, e.j., kamiri, h.w. 2012: floristic classification of the vegetation in small wetlands of kenya and tanzania. biodiversity & ecology, 4: 63–76. myers, n., mittermeier, r.a., mittermeier, c.g., da fonseca, g.a., kent, j. 2000: biodiversity hotspots for conservation priorities. nature, 403(6772): 853. orth, d., girard, c.m. 1996: espèces dominantes et biodiversité: relation avec les conditions édaphiques et les pratiques agricoles pour des prairies des marais du cotentin. ecologie, 27(3): 171. quézel, p. 2000: réflexions sur l’évolution de la flore et de la végétation au maghreb méditerranéen. paris edition, ibis.117 p. quézel, p., santa, s. 1961-1962: nouvelle flore de l’algérie et des régions désertiques. centre nat edition, paris. 1170 p. roleček, j., tichý, l., zelený, d., chytrý, m. 2009: modified twinspan classification in which the hierarchy respects cluster heterogeneity. journal of vegetation science, 20(4): 596-602. siddig, a.a., ellison, a.m., ochs, a., villarleeman, c., lau, m.k. 2016: how do ecologists select and use indicator species to monitor ecological change? insights from 14 years of publication in ecological indicators. ecological indicators, 60: 223-230. terras, m. 2011: typology, mapping of forest stations and modeling of forest stands. for forests in the province of saida. phd thesis. université of tlemcen. algeria. tichý, l. 2002: juice, software for vegetation classification. journal of vegetation science, 13(3): 451-453. tichý, l., chytrý, m., hájek, m., talbot, s.s., botta‐dukát, z. 2010: optimclass: using species‐ to‐cluster fidelity to determine the optimal partition in classification of ecological communities. journal of vegetation science, 21(2): 287-299. whittaker, r.h. 1972: evolution and measurement of species diversity. taxon: 213-251. djebbouri & terras ● community structure with particular reference to the effect of grazing inforest formations of saïda (algeria) biologica nyssana ● 13 (1) september 2022: 1-9 9 yaman et al., 2020, biologica nyssana 11(1) 11 (1) september 2020: 23-29 doi: 10.5281/zenodo.4060285 the endemic plants in bartın (turkey), and their conservation status original article barbaros yaman bartın university faculty of forestry department of forest botany, turkey yamanbar@gmail.com (corresponding author) zafer kaya bartın university faculty of forestry department of forest botany, turkey zkaya23@hotmail.com bilge tunçkol bartın university ulus vocational school department of forestry and forest products program, turkey bilgetunckol@gmail.com halil barış özel bartın university faculty of forestry department of silviculture, turkey halilbarisozel@gmail.com received: february 04, 2020 revised: april 01, 2020 accepted: april 14, 2020 abstract: bartın province located in the western black sea region of turkey has been selected as a floristic research unit. bartın, previously a district of zonguldak, has earned a provincial status in 1991. compared to other provinces of turkey, the flora and vegetation studies in bartın has been started quite late in detail since the second half of the 1990s. the present study aims to determine endemic taxa in bartın province and to evaluate their conservation status on the basis of iucn, cites and bern criteria. turkey has numerous laws, regulations, and programs on biodiversity conservation, but there are many deficiencies and problems in the implementation of these guidelines. our study was the first report on the endemic taxa and their conservation status in bartın province of turkey. we have determined that 36 of turkey’s endemic taxa are also found in bartın’s flora. we state that 3 of bartın’s endemic plant taxa are endangered (en), 2 is critical (cr), 1 is vulnerable (vu), 7 are near threatened (nt) and the others are least concern (lc) categories. key words: endemic taxa, flora, vegetation, conservation status apstract: endemične biljke bartina, turska, i njihov conservacijski status provincija bartın, koja se nalazi u zapadnom crnomorskom regionu turske, izabrana je za florističku istraživačku jedinicu. bartın, prethodno okrug zonguldak, stekao je status provincije 1991. godine. u poređenju sa drugim provincijama turske, detaljnije studije flore i vegetacije u bartınu započete su prilično kasno, od druge polovine 1990-ih. cilj ove studije je bio utvrđivanje endemičnih taksona u provinciji bartın i procena njihovog statusa u zaštiti na osnovu iucn, cites i bern kriterijuma. turska ima brojne zakone, propise i programe o očuvanju biodiverziteta, ali postoje mnogi nedostaci i problemi u njihovoj primeni. naša studija predstavlja prvi izveštaj o endemičnim taksonima i njihovom statusu očuvanosti u provinciji bartın u turskoj. odredili smo da se 36 turskih endemičnih taksona nalazi u flori bartına. utvrđeno je da su na području barrtina 3 endemična biljna taksona ugrožena (en), 2 kritično ugrožen (cr), 1 ranjiv (vu), 7 su skoro ugroženi (nt), a ostali su u kategoriji poslednje brige (lc). ključne reči: endemični taksoni, flora, vegetacija, conservacijski status introduction in terms of biogeography, turkey is at the crossroads of three different flora region within holoarctic ecozone (ketenoğlu et al., 2014) and three biodiversity hotspots (caucasus, irano-anatolian, and mediterranean) (şekercioğlu et al., 2011). because of these and other traits related to its geomorphological and climatic characteristics, turkey has one of the richest floras of the temperate zone in the world (özhatay et al., 2003). ekim (2014) stated that the number of native vascular plant species in turkey is 9753, and 3035 of them (31.12%) is endemic to the country. bartın, previously a district of zonguldak located in the western black sea region of turkey, earned a provincial status in 1991. flora and vegetation studies in bartın province has been started in detail since the second half of the 1990s. the first studies in this regard were carried out by yatkın (1996), başaran (1999), sarıbaş et al., (2002) and sarıbaş et al., © 2020 yaman et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 23 (2007). after these small-scale studies carried out in the restricted areas in bartın, the first flora list for the whole province was published by kaya & basaran (2006), and later first biotope maps for coastal habitats between amasra and inkum in bartın were constituted by nayim (2010). indeed, kaya & basaran (2006) have stated that there are 672 different plant taxa belonging to 368 genera under 97 families in bartın, and the authors stated that 7 of them (1.04 %) were endemic for turkey. afterward, within the scope of bartın’s biodiversity project, kaya & yaman (2017) have worked on the flora of the whole province in detail. according to kaya & yaman (2017), 35 of turkey’s endemic taxa are also found in bartın’s flora, and 9 of them are geophyte taxa. recently, kure mountains national park (bartın section) has been also examined in terms of its flora and vegetation by tunçkol & aksoy (2018). however, in 2017, a study related to the distribution of endemic taxa locations in turkey’s flora according to provinces has shown that any endemic taxon location doesn’t present in bartın (şenkul & kaya, 2017). although some studies on the province’s partial flora, this review by the authors may be due to the lack of a comprehensive article published on bartın flora available until then. the red-list criteria and conservation status of plant species in the world have been determined by different organizations such as iucn, cites, and bern. based on the iucn criteria, the plant species of turkey have been evaluated by the turkish association for the conservation of nature & van centennial university (ekim et al., 2000). the present study aims to determine the endemic taxa in bartın, and to evaluate their conservation status on the basis of iucn, cites and bern criteria. material and methods bartın province determined as a floristic research unit is divided into grids on the map (fig. 1). during the year 2016, 2017 and 2018, we went to the grids representing different habitats to collect plant specimens every week. the photographs of the plants were also taken during the field work. the specimens were pressed and dried between the folds of old newspapers, and the dried specimens are mounted on herbarium sheets of standard size (41x29 cm). the taxa were identified on the basis of flora of turkey and the east aegean islands (davis (ed.), 1965-1982). the nomenclature was harmonized 24 fig. 1. the map of bartın province located on the western black sea region of turkey. biologica nyssana ● 11 (1) september 2020: 23-29 yaman et al. ● the endemic plants in bartin (turkey), and their conservation status 25 family latin name/locality, altitude, legators cites iucn cupressaceae juniperus oxycedrus f. yaltirikiana avci & ziel. a4 bartın: hatipler coast,10 m., z.kaya & b.yaman * pinaceae abies nordmanniana subsp. equi-trojani (asch. & sint. ex boiss.) coode & cullen a4 bartın:ulus,uluyayla plateau, 1000 m., z.kaya & b.yaman nt amaryllidaceae allium kastambulense kollmann a4 bartın: kurucuşile coast, 20 m., z.kaya & b.yaman nt amaryllidaceae allium olympicum boiss. a4 bartın: ulus, drahna valley, 500-950 m., z.kaya & b.yaman lc apiaceae astrantia maxima subsp. haradjianii (grintz.) rech.f. a4 bartın: ulus, uluyayla, gendarme cemetery, 1500 m, z.kaya & b.yaman lc apiaceae ferulago platycarpa boiss. & balansa a4 bartın: kaynarca, slopes across the municipal park, 30 m., z.kaya & b.yaman lc apiaceae seseli resinosum freyn & sint. a4 bartın: ulus, ulukaya waterfall, 310 m., z.kaya & b.yaman vu araceae arum hygrophilum subsp. euxinum (r.r.mill) alpınar a4 bartın: kurucaşile, kapusu coast, 10 m., z.kaya & b.yaman lc asparagaceae bellevalia clusiana griseb. a4 bartın: güzelcehisar, the field edge on the way to the castle ruins, 70 m.,z.kaya & b.yaman lc asteraceae centaurea cadmea subsp. pontica köse & ocak a4 bartın: ulus, ulukaya waterfall, 310 m., z.kaya & b.yaman lc asteraceae centaurea inexpectata wagenitz a4 bartın: uluyayla plateau, 970 m., z.kaya & b.yaman lc asteraceae centaurea kilaea boiss. a4 bartın: mugada, coast dune, 5 m., z.kaya & b.yaman en asteraceae helichrysum arenarium subsp. aucheri (boiss.) davis & cupicha a4 bartın: ulus, uluyayla plateau, 970 m., z.kaya & b.yaman lc asteraceae inula helenium subsp. orgyalis (boiss.) grierson a4 bartın: arıt, zoni plateau, 841 m., z.kaya & b.yaman nt asteraceae turanecio hypochionaeus (boiss.) hamzaoğlu * a4 bartın: güzelcehisar, lava columns, 10 m., z.kaya & b.yaman cr boraginaceae onosma armena dc. a4 bartın: arıt, zoni plateau, roadside, 800m., z.kaya & b.yaman lc boraginaceae onosma intertexta hub.-mor. a4 bartın: ulus, drahna valley, 770 m., z.kaya & b.yaman nt brassicaceae alyssum pateri nyár. subsp. pateri a4 bartın: ulus, border between bartın and karabük, 1305 m.,z.kaya & b.yaman lc brassicaceae aubrieta canescens subsp. canescens a4 bartın: ulus, drahna, in front of kemerli cave, 850 m., z.kaya & b.yaman lc brassicaceae hesperis bicuspidata (willd.) poir. a4 bartın: arıt, çöpbey,yapıkayası hill, 600 m., z.kaya & b.yaman lc campanulaceae campanula grandis fisch. & c.a.mey. subsp. grandis a4 bartın: ulus,ulukaya village, 350 m., z.kaya & b.yaman lc caprifoliaceae cephalaria paphlagonica bobrov. a4 bartın: kurucaşile, kapısuyu village, 80 m., b.tunçkol nt caryophyllaceae minuartia mesogitana subsp. flaccida mcneill a4 bartın: arıt, roadside to zoni plateau, 864 m., z.kaya & b.yaman en celastraceae euonymus latifolius subsp. cauconis coode & cullen a4 bartın: from ulus to arıt, near to şahin village, 775 m., z.kaya & b.yaman nt fabaceae astragalus bartinense aytaç, tunçkol & n. aksoy a4 bartin: ulus, küre mountains national park, above abdurrahman village, 550 m, tunçkol cr iridaceae crocus ancyrensis (herb.) maw a4 bartın: ulus, uluyayla plateau, near to inonu cave, 950 m., z.kaya & b.yaman lc table 1. endemic taxa in bartın province and their conservation status. biologica nyssana ● 11 (1) september 2020: 23-29 yaman et al. ● the endemic plants in bartin (turkey), and their conservation status 26 plants have strong adaptation capabilities to environmental changes, anthropogenic global warming and climate change have a strong effect on plant life cycles and species’ interactions (settele et al., 2014). in spite of worldwide climate crisis, turkey has not a comprehensive conservation program for its own endemic plants yet. the most of endemic plant species in turkey has been threatened by biodiversitydamaging activities such as coal-fired power plant, dam construction, stone and marble quarry, gold mining, clearing grounds for fields, overgrazing, reform of barren lands, urbanization, tourism, wild fires, and afforestation without biodiversity base (bulut & yılmaz, 2010, şekercioğlu et al., 2011). in terms of conservation importance of each grid square and its threatened endemic plant taxa number in turkey, a4 grid square has high conservation importance (türe & böcük, 2010), and bartın province is on this grid square. recently also in bartın as well as many provinces in turkey, a coal-fired power plant in amasra district and a stone and marble quarry in ulus district have been planned by some private enterprises (atmiş, 2016, atmiş & günsen, 2017, yaman, 2019). in the province, the potential threats to biodiversity can result in the extinction of endemic plant species as well as habitat loss, habitat fragmentation and habitat degradation. in addition, intense tourism pressure on the extremely limited and exclusive coastal habitat of centaurea kilaea (en) and turanecio hypochionaeus (cr), which are bartın’s two of five endangered species, can result in the loss of these two species and their habitats. other endangered plant species in bartın are minuartia mesogitana subsp. flaccida and corydalis caubiologica nyssana ● 11 (1) september 2020: 23-29 yaman et al. ● the endemic plants in bartin (turkey), and their conservation status with ngbb electronic herbarium database in turkey (http://bizimbitkiler.org.tr) and the plant list database (http://www.theplantlist.org/). after taxonomic identification, they were labeled and stored in the herbarium of bartın faculty of forestry at the foundation stage. results and discussion bartın province which is determined as a floristic research area is located in the western black sea region of turkey, and mainly it shows bio-geographically the characteristics of euro-siberian region; however, some plant species belonging to the irano-turanian and mediterranean regions occur in the province. according to kaya & yaman (2017) and our recent field studies, the number of plant taxa in bartın province is approximately 1000. 36 of the turkey’s endemic taxa are also found in bartın’s flora, and 9 of them are geophyte taxa. the most of endemic taxa in bartın is euro-siberian element (21 taxa), 7 of them are irano-turanian element, and the others are mediterranean (1 taxon) or common elements (6 taxa) of two or three flora regions. we state that 3 of bartın’s endemic plant taxa are endangered (en), 2 critically endangered (cr), 1 vulnerable (vu), 7 near threatened (nt) and most of the other least concern categories (lc). the endemic taxa of bartın province and their conservation status and the photographs of some of them are given tab. 1 and fig. 2 respectively. today there is a concept on a biodiversity crisis worldwide related to global warming and climate change. it is known that more than 30.000 species are threatened with extinction (url-1). although family latin name/locality, altitude, legators cites iucn iridaceae crocus bolensis (ruksans) ruksans a4 bartın:uluyayla plateau, near to marble quarry, 970 m., z.kaya & b.yaman * iridaceae iris kerneriana asch. & sint. ex baker a4 bartın: ulus, border between bartın and karabük, 1298 m., z.kaya & b.yaman lc lamiaceae phlomis russeliana (sims.) lag. ex benth. a4 bartın: ulus, sarıçiçek hill, 1500 m., z.kaya & b.yama lc lamiaceae sideritis dichotoma huter a4 bartın: ulus, ulukaya waterfall, 310 m., z.kaya & b.yaman lc orchidaceae dactylorhiza nieschalkiorum h.baumann & künkele a4 bartın: ulus, sarıçiçek hill, 1500 m., z.kaya & b.yaman + lc orobanchaceae melampyrum arvense var. elatius boiss. a4 bartın: ulus, near to çerçi village, 560m., z.kaya & b.yaman nt papaveraceae corydalis caucasica subsp. abantensis lidén a4 bartın: arıt, zoni plateau, 900 m., z.kaya & b.yaman en plantaginaceae digitalis lamarckii ivanina a4 bartın: ulus, kavakseydibaşı, 910m., z.kaya & b.yaman lc ranunculaceae delphinium fissum subsp. anatolicum chowdhuri & p.h.davis a4 bartın: ulus, kumluca, road side after kızıllar, 493m., z.kaya & b.yaman lc rubiaceae asperula pestalozzae boiss a4 bartın: ulus, near to abdurrahman village, 710 m., z.kaya & b.yaman lc 27 biologica nyssana ● 11 (1) september 2020: 23-29 yaman et al. ● the endemic plants in bartin (turkey), and their conservation status fig. 2. some of threatened endemic taxa in bartın, turkey, a. seseli resinosum, b. centaurea kilaea, c. turanecio hypochionaeus, d. corydalis caucasica subsp. abantensis, e. minuartia mesogitana subsp. flaccida casica subsp. abantensis. one of vulnerable species in the province, seseli resinosum, is threatened by the planned stone and marble quarries in ulus district. the project in a high plateau (uluyayla) of the district, also threats the habitats of endemic and nonendemic geophyte species such as crocus ancyrensis and galanthus elwesii, respectively as well as crocus bolensis which is a relatively common endemic species (ruksans, 2017). in addition, dactylorhiza nieschalkiorum, which is not in any threat category based on iucn, might be under the risk of illegal plant collection due to its commercial ornamental value, thus d. nieschalkiorum is on the cites appendix ii (url-2). however, in bartın, there is not any endemic species included in bern convention appendix i (url-3). environmental impact assessment (eia) has been applied legally in the industrial projects in turkey since 1993. however, only 1.58 percent of all eia decisions (67040) were negative (url-4). although turkey has numerous laws, reg28 ulations, and programs on biodiversity conservation, there are many deficiencies and problems in the implementation of these guidelines (kaya & raynal, 2001). in practice, although non-governmental organizations oppose, these rules are often stretched by central and local governments with the idea of economic development and profit (atmiş & günsen, 2017, atmiş, 2018). conclusion our investigation was the first report on the endemic taxa and their conservation status in bartın province of turkey. based on a long-term conservation program, in situ conservation systems should be established urgently for turkey’s plant taxa in the cr, en and vu categories of iucn. in addition, while developing the provincial conservation programs for plant species, critical (cr), endangered (en) and vulnerable (vu) endemic species in the provinces should be considered primarily. biodiversity-damaging activities cited above must not be allowed in habitats having critical (cr), endangered (en) and vulnerable (vu) endemic species in bartın province. references atmiş, e. 2016: yerelde dikkate değer bir örnek: bartın platformu’nun termiksiz yaşam mücadelesi. in: yıldırım, d., haspolat, e. (eds.), değişen karadeniz’i anlamak, 501-522, phoenix, ankara. atmiş, e., günsen, b. 2017: türkiye’de orman yıkımına karşı mücadelelerin analizi. in: uydacı, m. (ed.), turkish studies from different perspectives, 315-336, athens institute for education and research, athens. atmiş, e. 2018: a critical review of the (potentially) negative impacts of current protected area policies on the nature conservation of forests in turkey. land use policy, 70: 675-684. başaran, s. 1999: kirazlık (bartın) barajı florası. phd thesis. zkü fen bilimleri enstitüsü. zonguldak. bulut, z., yılmaz, h. 2010: the current situation of threatened endemic flora in turkey: kemaliye (erzincan) case. pakistan journal of botany, 42(2): 711-719. davis, p.h. (ed.) 1965-1982: flora of turkey and east aegean islands, i-ix. university press, edinburg. ekim, t., koyuncu, m., vural, m., duman, h., aytaç, z., adıgüzel, n. 2000: red data book of turkish plants (pteridophyta and spermatophyta). turkish association for the conservation of nature & van centennial university, barışcan ofset, ankara. 246 p. ekim, t. 2014: damarlı bitkiler. in: güner, a., ekim, t. (eds.), resimli türkiye florası (illustrated flora of turkey), 1: 159-162, ali nihat gökyiğit vakfı, flora araştırmaları derneği ve türkiye i̇ş bankası kültür yayınları, i̇stanbul. kaya, z., raynal, d.j. 2001: biodiversity and conservation of turkish forests. biological conservation, 97: 131-141. kaya, z., başaran, s. 2006. bartın florasına katkılar. gazi üniversitesi orman fakültesi dergisi, 6(1): 40-62. kaya, z., yaman, b. 2017: bartın i̇linin karasal ve i̇ç su ekosistemleri biyolojik çeşitlilik envanter ve i̇zleme projesi (flora bölümü). orman ve su i̇şleri bakanlığı doğa koruma ve milli parklar (dkmp) 10. bölge müdürlüğü bartın i̇l şube müdürlüğü. ankara. 526 p. ketenoğlu, o., vural, m., kurt, l., körüklü, t. 2014: vejetasyon. in: güner, a., ekim, t. (eds.), 2019: resimli türkiye florası (illustrated flora of turkey), 1:163-224, ali nihat gökyiğit vakfı, flora araştırmaları derneği ve türkiye i̇ş bankası kültür yayınları, i̇stanbul. özhatay, n., byfield, a., atay, s. 2003: türkiye’nin önemli bitki alanları. wwf türkiye (doğal hayatı koruma vakfı). i̇stanbul. 88 p. nayim, s.y. 2010: amasra-i̇nkum (bartın) arasında yer alan önemli biyotopların haritalanması. phd thesis. i̇stanbul üniversitesi fen bilimleri enstitüsü. i̇stanbul. ruksans, j. 2017: the world of crocuses. latvian academy of sciences. latvia. 568 p. sarıbaş, m., kaya, z., başaran s., yaman, b. 2002: batı karadeniz bölgesi’nde doğal olarak yetişebilen bitkilerden peyzaj uygulamalarında kullanılabilecek bitkilerin saptanması. in: yahyaoğlu, z. (ed.), 2. ulusal karadeniz ormancılık kongresi, 520-537, kafkas üniversitesi artvin orman fakültesi, artvin. sarıbaş, m., kaya, z., başaran, s., yaman, b., sabaz, m. 2007: the use of some natural plant species from the western black sea region of turkey for landscape design. fresenius environmental bulletin 16(2): 193-205. şekercioğlu, ç. h., anderson, s., akçay, e., bilgin, r., can, ö.e., semiz, g., tavşanoğlu, ç., yokeş, m.b., soyumert, a., kahraman, i̇., sağlam, i̇.k., yücel, m., dalfes, h.n. 2011: turkey’s globally important biodiversity in crisis. biological conservation, 144: 2752–2769. biologica nyssana ● 11 (1) september 2020: 23-29 yaman et al. ● the endemic plants in bartin (turkey), and their conservation status 29 settele, j., scholes, r., betts, r, et al., 2014: terrestrial and inland water systems. in: field, c.b., barros, v.r., dokken, d.j. et al., (eds.), climate change: impacts, adaptation, and vulnerability. part a: global and sectoral aspects, contribution of working group ii to the fifth assessment report of the intergovernmental panel on climate change, 271–359, cambridge university press, cambridge. şenkul, ç., kaya, s. 2017: geographical distribution of endemic plants of turkey. türk coğrafya dergisi, 69: 109-120. tunçkol, b., aksoy, n. 2018: flora of küre mountains national park (bartın section). journal of forestry, 14(2): 80-113. tunçkol, b., aytaç, z., aksoy, n. and fişne, a., 2020. astragalus bartinense (fabaceae), a new species from turkey. acta botanica croatica, 79(2): 131-136. türe, c., böcük, h. 2010: distribution patterns of threatened endemic plants in turkey: a quantitative approach for conservation. journal for nature conservation, 18: 296–303. url-1. https://www.iucnredlist.org/ [accession date: 30 december 2019]. url-2. http://checklist.cites.org/ [accession date: 21 january 2020]. url-3. https://www.coe.int/en/web/conventions/ full-list/-/conventions/treaty/104 [accession date: 21 january 2020]. url-4. https://ced.csb.gov.tr/ [accession date: 21 january 2020]. yaman, b. 2019: bartın’ın gözbebeği uluyayla mermer ocaklarına feda edilebilir mi? orman ve av, 97(5): 10-12. yatkın, h. 1996: amasra yöresi floristik kompozisyonu. msc thesis, zkü fen bilimleri enstitüs. zonguldak. biologica nyssana ● 11 (1) september 2020: 23-29 yaman et al. ● the endemic plants in bartin (turkey), and their conservation status new locality of centaurea pichleri (asteraceae) in bulgaria biologica nyssana 7 (2)  december 2016: 87-90 bancheva, s., delcheva, m.  new locality of centaurea pichleri… 87 original article received: 01 september 2016 revised: 10 november 2016 accepted: 24 november 2016 new locality of centaurea pichleri (asteraceae) in bulgaria svetlana bancheva, malina delcheva institute of biodiversity and ecosystem research, bulgarian academy of sciences, acad. g. bonchev str., bl. 23, 1113-sofia, bulgaria * e-mail: sbancheva@yahoo.com abstract: bancheva, s., delcheva, m.: new locality of centaurea pichleri (asteraceae) in bulgaria. biologica nyssana, 7 (2), december 2016: 87-90. centaurea pichleri boiss. belongs to subgenus cyanus of asteraceae and has a very complex taxonomy. it is distributed in turkey (mainly in central anatolia), greece, bulgaria and lebanon. in the bulgarian flora the species has a conservation value being protected by the national biodiversity act and listed in the red list of vascular plants in bulgaria. during field investigations in the spring of 2016 a new locality of the species in toundzha hilly country (se bulgaria) was found. the new population of c. pichleri is in a good state and numbers over then 1000 individuals. from the spatial location of individuals it can be assumed that the reproduction of the species in this locality combined vegetative and seed model. in the new locality, no threats to the species' population were registrated. key words: asteraceae, centaurea, bulgarian flora, rare plant species apstrakt: bancheva, s., delcheva, m.: novi lokalitet vrste centaurea pichleri (asteraceae) u bulgarskoj. biologica nyssana, 7 (2), decembar 2016: 31-34. centaurea pichleri boiss. pripada podrodu cyanus porodice asteraceae i ima veoma kompleksnu taksonomiju. rasporostranjena je u turskoj (uglavnom u centralnoj anatoliji), grčkoj, bugarskoj i libanu. ova vrsta ima konzervacionu vrednost u bugarskoj flori, jer je zaštićena nacionalnim zakonom o biodiverzitetu i nalazi se na crvenoj listi vaskularnih biljaka bugarske. tokom terenskih istraživanja vršenih u proleće 2016. godine, nov lokalitet ove vrste je nađen u tundža oblasti (bugarska). nova populacija c. pichleri je u dobrom stanju i broji preko 1000 jedinki. iz prostornog rasporeda jedinki može se pretopstaviti da je reprodukcija vrste na ovom lokalitetu kombinacija vegetativnog i generativnog načina razmnožavanja. na novom lokalitetu nisu uočene pretnje populaciji ove vrste. ključne reči: asteraceae, centaurea, flora bugarske, retka biljna vrsta 7 (2) • december 2016: 87-90 12th sfses • 16-19 june 2016, kopaonik mt doi: 10.5281/zenodo.200404 biologica nyssana 7 (2)  december 2016: 87-90 bancheva, s., delcheva, m.  new locality of centaurea pichleri… 88 introduction during field investigations in the spring of 2016 a new locality of centaurea pichleri boiss. in toundzha hilly country (se bulgaria), was found. previously, the species was known from only three floristic regions of the country up to 800 m alt. (b a n c h e v a & d e n c h e v , 2000). a s s y o v & p e t r o v a (2012) erroneously indicate it also from thracian valley. centaurea pichleri belongs to subgenus cyanus of asteraceae and has a very complex taxonomy. cyanus was first mentioned by m i l l e r (1754) as a genus. de candolle (1838) was the first to considered it a section within the genus centaurea. in light of the molecular evidence, the latest compilations of the cardueae (s u s a n n a & g a r c i a -j a c a s 2007, 2009) suggest that cyanus should be left within centaurea. according to b o r š i ć et al. (2011) c. pichleri is a species of controversial delineation. the populations of this taxon appear to be diversely associated with other eastern relatives and collectively do not form a supported clade. bulgarian populations of the species seem to have less variability and they are more clearly identifiable. the species is distributed in turkey (mainly in central anatolia), greece, bulgaria and lebanon (w a g e n i t z , 1975; b o r š i ć et al., 2011). in bulgaria the species is rare and has conservation value – it is protected by the national biodiversity act and included in the red list of vascular plants in bulgaria as “vulnerable” (b a n c h e v a , 2009). material and methods this investigation was based on material collected by the authors in toundzha hilly country floristic region (se bulgaria) and on specimens deposited in the following herbaria: bgbm, iste, ma, p, prm, som, w. results and discussion new record centaurea pichleri boiss.: toundzha hilly country (se bulgaria), near to kostur village, svilengrad municipality, haskovo district, on the periphery of dry, stony meadows in termophilous oak forest, n 41,977788, e 26,270822, 572 m alt., 29 april 2016, coll. s. bancheva & m. delcheva, som – 172833. distribution and ecology the species is distributed in four (out of 20) floristic regions in bulgaria – strandzha mt, black sea coast, the rhodopes (east) and toundzha hilly country up to 800 m alt. the new population of c. pichleri inhabits the periphery of dry, stony meadows in termophilous oak forest and brushwood in the natural landmark "habitat of wild peony" fig. 1. habit of c. pichleri fig. 2. c. pichleri in its natural habitat biologica nyssana 7 (2)  december 2016: 87-90 bancheva, s., delcheva, m.  new locality of centaurea pichleri… 89 proclaimed in 1976 (figs 2, 3). the species grows together with: quercus frainetto ten., q. pubescens willd., fraxinus ornus l., carpinus orientalis mill, acer monspessulanum l., anthemis arvensis l., linaria genistifolia (l.) mill., paeonia peregrina mill., muscari botryoides (l.) mill., saxifraga rotundifolia l., veronica austriaca l. subsp. jacquinii (baumg.) eb. fisch., geranium sanguineum l., verbascum phoeniceum l., dactylis glomerata l., ranunculus illyricus l., lamium purpureum l., cornus mas l., potentilla neglecta baumg., centaurea thirkei sch. bip., achillea millefolium l., lychnis coronaria (l.) desr., plantago lanceolata l., hypericum perforatum l., ajuga laxmanii (l.) benth., lathyrus aphaca l., ranunculus neapolitanus ten., silene conica l. etc. this territory is a part of a site of community importance bg0000212 “sakar” from the ecological network natura 2000 in bulgaria. this habitat type named “pannonian-balkanic turkey oak – sessile oak forests – 91m0” is included in annex i of the “habitat” directive 92/43/eec. another specimen of centaurea pichleri from toundzha hilly country, south of the village of iglika, bolyarovo municipality (som 162724), is stored in som, which was collected about 50 km east of the new population. this is also unpublished record of the species. population observations the new population of c. pichleri is in a good state and numbers over then 1000 individuals. although finding a new population that is in good condition we consider regrettable that the conservation status of the species should not be changed. from the spatial location of individuals it can be assumed that the reproduction of the species in this locality combined vegetative and seed model. in the new locality no threats to the species' population were noticed. conclusion newly established population represents the northern boundary of the distribution of the species. acknowledgements. this research received support from the synthesys project http://www.synthesys.info/ which is financed by european community research infrastructure action under the fp7 integrating activities programme (es-taf-3669, fr-taf-3913, de-taf4725, cz-taf-4824). the field studies were supported by the financial mechanism of the european economic area 2009-2014, contract № д-33-88/28.08.2015 “mapping and assessment of sparsely vegetated land ecosystem services in bulgaria (spa-ecoservices)”. the authors thank vladimir trifonov from the regional inspectorate of fig. 3. map of distribution of c. pichleri biologica nyssana 7 (2)  december 2016: 87-90 bancheva, s., delcheva, m.  new locality of centaurea pichleri… 90 environment and water, haskovo for providing information about the newly established population. references assyov, b. & petrova, a. (eds). 2012. conspectus of the bulgarian vascular flora. bulgarian biodiversity foundation sofia. 452 p. bancheva, s. 2009. cyanus pichleri. in: petrova, a. & vladimirov, v. (eds). 2009. red list of bulgarian vascular plants. phytologia balcanica, 15 (1): 83. bancheva, s. & denchev, c. 2000. occurrence and taxonomic investigation of centaurea pichleri boiss. (asteraceae) in bulgaria. phytologia balcanica, 6 (2-3): 167-175. boršić, i., susanna, a., bancheva, s., garcia-jacas n. 2011. centaurea sect. cyanus: nuclear phylogeny, biogeography and life-form evolution. international journal of plant sciences, 172 (2): 238-249. de candolle, a.p. 1838: prodromus systematis naturalis regni vegetabilis. vol 6. treuttel & würtz, paris. miller, p. 1754: the gardeners dictionary. abridged ed. 4. privately published, london. wagenitz, g., 1975: genus centaurea l. pp. 465585. in: davis, p. h. (ed.): flora of turkey and the east aegean islands, 5. edinburgh university press, edinburgh. alex et al. 2025, biologica nyssana 16(1) 267 16 (1) june 2025: 267-276 doi: 10.46793/biolnyss.16.1.23a formulation and characterization of nanoparticles loaded with curcumin and amantadine as a promising neuroprotective agent original article aksa alex department of pharmaceutics, faculty of pharmacy, karpagam academy of higher education, coimbatore, tamilnadu, india apsara unni research scholar, department of pharmaceutical chemistry, jss college of pharmacy, ooty, tamilnadu, india gayathri kumararaja department of pharmaceutics, faculty of pharmacy, karpagam academy of higher education, coimbatore, tamilnadu, india gayubpharm@gmail.com (corresponding author) received: january 30, 2025 revised: april 25, 2025 accepted: april 28, 2025 abstract: parkinson's disease (pd) is regarded as the second most common neurodegenerative disease and is characterized by observable motor dysfunction and loss of dopaminergic neurons. to date, there is no cure for pd, nor the treatment that stops its progression. nanotechnology is very useful for controlling drug release, thus improving drug pharmacokinetic and pharmacodynamic properties. in this study, we fabricated curcumin and amantadine-loaded nanoparticles using bovine serum albumin (bsa) as a nanocarrier using the desolvation method. encapsulating curcumin and amantadine were beneficial in improving its aqueous solubility and bioavailability. the prepared nanoparticles had the particle size and zeta potential of 257 nm and -29 mv, respectively. the in vitro neuroprotective effect was determined using shsy5y cell lines through the mtt assay, which showed strong neuroprotective activity. key words: bovine serum albumin, curcumin, amantadine, desolvation, neuroprotective activity apstrakt: formulisanje i karakterizacija nanočestica koje sadrže kurkumin i amantadin kao obećavajućeg neuroprotektivnog agensa parkinsonova bolest (pb) se smatra drugom najčešćom neurodegerativnom bolešću i karakteriše se izraženim motornim disfunkcijama i gubitkom dopaminergičkih neurona. do danas ne postoji lek za pb, niti terapija koja može zaustaviti njeno napredovanje. nanotehnologija se pokazala kao veoma korisna u kontroli oslobađanja lekova, čime se poboljšavaju farmakokinetička i farmakodinamička svojstva lekovitih supstanci. u ovoj studiji, metodom desolvatacije formirali smo nanočestice napunjene kurkuminom i amantadinom, koristeći albumin goveđeg seruma (bsa) kao nosač. inkapsulacija kurkumina i amantadina doprinela je poboljšanju njihove rastvorljivosti u vodi i bioraspoloživost. pripremljene nanočestice su imale veličinu čestica od 257 nm i zeta potencijal od -29 mv. in vitro neuroprotektivno dejstvo ispitano je na ćelijskim linijama shsy5y korišćenjem mtt testa, koji je pokazao izraženu neuroprotektivnu aktivnost. ključne reči: albumin goveđeg seruma, kurkumin, amantadin, desolvatacija, neuroprotektivna aktivnost introduction parkinson’s disease (pd) is a severe and debilitating neurodegenerative disorder that predominantly affects neurons in the brain. it is characterized by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta (snpc), leading to a deficiency of dopamine. motor symptoms include bradykinesia, resting tremors, postural instability, and muscular rigidity. in addition to motor impairments, non-motor symptoms such as dementia, autonomic dysfunction, sleep disturbances, sensory abnormalities, depression, and anxiety are also frequently observed in patients (poewe et al., 2017). pd is the second most prevalent neurodegenerative disorder after alzheimer’s disease (ad), affecting approximately 35 to 100 individuals per 100,000 people globally (simon et al., 2020). the worldwide prevalence of pd is estimated at 7 to 10 million individuals, with an average incidence of around © 2025 alex et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 268 41 per 100,000 people. notably, in populations aged 80 years and older, the prevalence increases significantly to 1,900 per 100,000 (parkinson’s news today, 2021). projections suggest that this number will rise to approximately 12.9 million by 2040 (dorsey et al., 2018). pd is a progressive and neurodegenerative disease first systematically described by james parkinson, who referred to it as „shaking palsy” in his early observations (parkinson, 2002). while parkinson’s terminology laid the groundwork, subsequent research has focused on understanding the clinical manifestations and slowing disease progression. since the degeneration of dopaminergic neurons cannot currently be halted or reversed, existing treatments for pd primarily aim to alleviate both motor and non-motor symptoms. a major limitation of current pharmacological therapies is their restricted ability to cross the blood-brain barrier (bbb), which limits central nervous system localization and often necessitates low-dose administrations (tonda et al., 2018). many treatments also suffer from issues such as low stability, potential toxicity of degradation by-products, high production costs, and limited neuroprotective efficacy—highlighting the need for further research. consequently, there is a pressing need for the development of novel drug delivery systems that can prolong and regulate drug release. emerging trends in nanotechnology have demonstrated that nanoparticles (nps) can enhance drug delivery by improving cellular uptake, reducing issues of low bioavailability, minimizing pharmacokinetic side effects, reducing the dosing frequency, and increasing drug concentration in targeted brain regions (alonso et al., 2012; yadav et al., 2025). among these, controlled drug release systems are particularly promising due to their simplicity, reproducibility, and effectiveness. nanotechnologyassisted delivery can significantly enhance both the pharmacokinetic and pharmacodynamic profiles of therapeutic agents. curcumin, a well-known polyphenolic compound with broad pharmacological activity (gayathri et al., 2023), exhibits notable neuroprotective effects (tsai et al., 2011; nair et al., 2014). its mechanisms of neuroprotection may include inhibition of reactive oxygen species (ros) production due to its potent antioxidant and anti-inflammatory properties. curcumin also affects glial cell function, α-synuclein aggregation, neuronal apoptosis, and ferroptosis. however, clinical application is limited by its poor oral bioavailability and low solubility (young et al., 2014). these challenges may be mitigated through nano-based drug delivery systems (takahashi et al., 2009; cheng et al., 2013; mourtas et al., 2014). studies in drosophila models of pd have shown that nanocurcumin significantly reduces pd symptoms, and it demonstrates higher bioavailability and neuroprotective activity in murine brains compared to conventional curcumin (nazari et al., 2014). amantadine originally approved as an antiviral agent for influenza a (staničová et al., 2001), has demonstrated antiviral activity against sarscov-2 (rascol et al., 2021) and anti-parkinsonian properties (maksymiuk et al., 2021; espay et al., 2024). it has also shown antiproliferative effects in various melanoma cell lines. this study aims to develop and evaluate nanoparticles co-loaded with curcumin and amantadine for their neuroprotective potential in parkinson’s disease. the combination of curcumin and amantadine within a nanoparticle formulation may produce increased therapeutic effects, enhancing their individual benefits while reducing side effects. improving bbb penetration and bioavailability through nanocarrier systems can help achieve sustained drug release, prolonged therapeutic action, and reduced dosing frequency. materials and methods amantadine and curcumin were purchased from yarrow chem products (mumbai), bovine serum albumine (bsa) from sigma, and ethanol and glutaraldehyde from nice chemicals pvt ltd (kochi). preparation of albumin nanoparticles desolvation involves the addition of organic solvents to an aqueous solution of albumin, followed by chemical cross-linking with glutaraldehyde. the required amount of lyophilized bsa powder was dissolved in distilled water to make a bsa solution. the drug was dissolved in either ethanol or acetone separately. the ph was adjusted to between 7 and 8. a magnetic stirrer at 500 rpm was used for stirring until turbidity was observed by adding ethanol at a 1 ml/min rate. glutaraldehyde was then added for cross-linking the bsa nanoparticles. specifically, 0.1 ml of 4% glutaraldehyde solution was added, and the mixture was stirred for 2 hours. the stirring process was continued for 24 h, followed by five cycles of differential centrifugation (3000 rpm for 30 minutes), refining of the suspension, and resuspension of the pellet in a final volume of 10 ml distilled water. finally, the nanoparticles were lyophilized and stored at 4 °c (kim et al., 2010). a total of eight formulations (f1–f8) were prepared with varying concentrations of carrier and drug ratio. biologica nyssana ● 16 (1) june 2025: 267-276 alex et al. ● formulation and characterization of nanoparticles loaded with curcumin and amantadine as a promising neuroprotective agent 269 biologica nyssana ● 16 (1) june 2025: 267-276 alex et al. ● formulation and characterization of nanoparticles loaded with curcumin and amantadine as a promising neuroprotective agent characterization drug-excipient compatibility studies (ftir) compatibility between the drug and excipients used in the preparation of nanoparticles was confirmed by infrared spectroscopy. the nature of the interactions was identified. the samples were scanned using a fourier transform infrared (ft-ir) spectrophotometer in the range of 4000 to 400 cm⁻¹. ir spectra of all individual drugs and synthesized nanoparticles were recorded. furthermore, changes in appearance and peak positions were studied in the spectra to determine the possibility of chemical and physical interactions. particle shape and surface morphology a scanning electron microscope (sem; zeiss evo40) was used to examine the shape and morphology of the prepared nanoparticles. samples were spread onto a slide using double-sided sticky tape, and the gold coating was done using a vacuum evaporator in an argon atmosphere assisted by gold flakes. samples were scanned at various magnifications, and photomicrographs were captured. particle size and zeta potential a concentrated solution of 1 ml albumin nanoparticles was diluted with 10 ml of water. the samples were analyzed using dynamic light scattering (dls) with a malvern zetasizer. the polydispersity index (pdi) and average particle size were determined. zeta potential was measured to assess formulation stability. the surface charge potential of the samples was analyzed in the zetasizer chamber, and peaks were recorded to obtain zeta potential values. when interpreting the data, a monodisperse character is considered more desirable than a polydisperse one. drug entrapment efficiency 10 ml of the nanoparticle solution (equivalent to 10 mg of the drug) was transferred into a centrifuge tube. the mixture was centrifuged using a refrigerated centrifuge (remi motors, c-24 plus) at 1000 rpm for 15 minutes at 25 °c. the supernatant was collected, and drug content was quantified using a calibration curve established by uv-visible spectrophotometry. entrapment efficiency was calculated using the formula: encapsulation efficiency (%ee)=[(total drug added–free drug in supernatant)/total drug added]×100 in vitro drug release study the drug's release behavior from albumin nanoparticles was evaluated using the dialysis bag method. a predetermined volume of aqueous nanoparticle suspension was sealed in a dialysis bag and immersed in phosphate-buffered saline (pbs, ph 7.4) at 37 °c with stirring at 100 rpm. samples were withdrawn from the release medium at specific intervals and replaced with fresh pbs. samples were centrifuged, and the drug content in the supernatant was determined using hplc or fluorescence/uvvisible spectrophotometry. a graph of cumulative drug release versus time was plotted. release kinetics study in hydrophilic matrices, polymers swell and erode simultaneously, contributing to drug release. the resulting release profile often reflects zero-order kinetics. this study prepared a nanoformulation loaded with curcumin and amantadine for sustained release. the drug release data were fitted to various kinetic models: zero-order (cumulative % drug released vs. time), first-order (log % drug remaining vs. time), higuchi's model (cumulative % drug released vs. square root of time), and korsmeyer– peppas model (log % drug released vs. log time). the physicochemical properties, effectiveness, and quality of the nanoparticles were assessed using these models. in vitro neuroprotective effect determination by mtt assay sh-sy5y (neuroblastoma) cell line was procured from the national centre for cell sciences (nccs), pune, india, and maintained in dulbecco’s modified eagle’s medium (dmem; sigma aldrich, usa). cells were cultured in 25 cm² tissue culture flasks with dmem supplemented with 10% fetal bovine serum (fbs), l-glutamine, sodium bicarbonate (merck, germany), and antibiotics: penicillin (100 u/ml), streptomycin (100 µg/ml), and amphotericin b (2.5 µg/ml). cultures were maintained at 37 °c in a humidified 5% co₂ incubator (nbs eppendorf, germany). cell viability was assessed by inverted phase-contrast microscopy and further confirmed via mtt assay. cytotoxicity evaluation once cells reached sufficient growth, rotenone (10 µm) was added to induce cytotoxicity and incubated for 1 hour. after incubation, the medium was removed, and freshly prepared compounds were added in concentrations of 25, 12.5, 6.25, 3.1, and 1.5 µg/ml. each concentration was added in triplicate to the respective wells. control and rotenone-only wells were maintained for comparison. microscopic observation after 24 hours of treatment, plates were observed 270 under an inverted phase contrast microscope (olympus ckx41 with optika pro5 ccd camera). morphological changes such as cell rounding, shrinkage, granulation, and vacuolization were noted as indicators of cytotoxicity. mtt assay fifteen mg of mtt (sigma, m-5655) was reconstituted in 3 ml pbs and sterilized by filtration. after 24 hours of incubation, 30 µl of mtt solution was added to each well and incubated for 4 hours at 37 °c in a humidified 5% co₂ incubator. after incubation, the supernatant was removed, and 100 µl of dmso (sigma aldrich, usa) was added to dissolve the formazan crystals. absorbance was read using a microplate reader at 540 nm (talarico et al., 2004). the percentage of growth inhibition was calculated using the formula: % of viability = (mean od samples x 100)/ mean od of control group results and discussion particle size, pdi, and zeta potential the particle size and pdi of the nanoparticles were measured using the dls technique and a computerized zetasizer. a zetasizer cell was loaded with nanoformulation, and the measurements were taken. values of all particle sizes in the different formulations (tab. 1) were at the nanometer scale. particle size, zeta potential, and pdi for formulation f7 (fig. 1a, fig. 1b) were 257.8 nm, -29 mv, and 0.05, respectively. the size of the nanoparticles was 255.5 nm to facilitate uptake into the cell and delivery to the appropriate site. another important characteristic parameter of nanoparticles is pdi. for example, a pdi below 0.05 shows that the population is very monodisperse, and a pdi of greater than 0.7 biologica nyssana ● 16 (1) june 2025: 267-276 alex et al. ● formulation and characterization of nanoparticles loaded with curcumin and amantadine as a promising neuroprotective agent fig. 1. particle size distribution graph and zeta potential distribution graph of f7 formulation table 1. particle size, zeta potential and pdi values of optimized nanoformulation formulation code particle size (nm) zeta potential (mv) pdi f1 1349 -12.6 0.13 f2 857.5 -21.4 0.71 f3 906.4 -17.7 0.62 f4 589.1 -11.5 0.91 f5 496.8 -21.6 0.47 f6 272.4 -28.4 0.32 f7 255.5 -29.1 0.05 f8 257.8 -21.4 0.52 means the size distribution is wide. the result of this experiment also indicated a pdi of 0.05, signifying no significant variation in particle sizes, along with a zeta potential of -29 mv that is adequate electrostatic repulsion, stabilizing the suspension and preventing agglomeration of the particles. although the particle size of 257.8 nm may seem relatively large, it falls within the optimal range (100–300 nm) for effective brain-targeted delivery. such sizes are known to cross the blood-brain barrier via transcytosis mechanisms. additionally, the narrow pdi (0.05) and stable zeta potential (–29 mv) indicate a uniform and stable formulation. larger nanoparticles in this range can also offer advantages such as higher drug-loading capacity and sustained release, which are beneficial for neuroprotective applications. entrapment efficiency the entrapment efficiency of the formulation is based on the properties of the polymer, drug, and desolvating agent. the prepared nanoparticles were investigated using a refrigerated centrifuge method to determine this parameter. the nanoparticles 271 biologica nyssana ● 16 (1) june 2025: 267-276 alex et al. ● formulation and characterization of nanoparticles loaded with curcumin and amantadine as a promising neuroprotective agent were centrifuged at 3000 rpm for 30 minutes. the supernatant, containing the un-entrapped drug, was collected and quantified using a uv spectrophotometer at a wavelength of 425 nm (fig. 2). the entrapment efficiency across all batches varied between 45.61% and 72.92%, with formulation f7 showing the highest efficiency. the high entrapment efficiency of 72.92% indicates significant drug loading, suggesting that this formulation has excellent potential for achieving a good therapeutic payload. high concentrations of polymer and ethanol enhanced the entrapment efficiency, as the drug is lipophilic. the increased lipid content improved the solubilization of the lipophilic drug, creating additional space within the matrix for drug entrapment. in vitro release study the in vitro drug release profile of the formulations is shown graphically. the drug release profiles indicate different release rates over a period of 8 hours. in the formulations, f1 to f5 showed relatively higher cumulative drug release percentages, and around 40–45% were released at the end of the study. formulations f6, f7, and f8 show slow release, but f7 showed a prolonged drug release profile, with the lowest cumulative drug release among all the formulations. the formulation composition of f7 would be responsible for the sustained release behavior due to its possible enhancement of drug encapsulation and minimization of rapid diffusion (fig. 3). such characteristics are advantageous for controlled drug delivery systems in ensuring prolonged therapeutic effects and reducing the number of administrations. this highlights the significant role of formulation variables in adjusting the release kinetics to meet the therapeutic requirements. release kinetics study the drug release is in zeroorder kinetics since the r² of 0.917 shows that it is constant with time and does not depend on the drug concentration within the nanoparticles; this behavior displays the system's controlled-release properties, such that it offers predictable, consistent drug release and, hence, sustainable therapeutic levels for an extensive period. this suggests that the mechanism is diffusioncontrolled and further indicates that the diffusion in the nanoparticle matrix controls the release rate of the drug rather than any other controlling mechanism. the r² value of 0.9245 for the higuchi model further confirms this since the drug release varies as a square root function of time. the korsmeyer–peppas model with an r² of 0.9735 shows a non-fickian or anomalous release mechanism, meaning that drug release that is subjected to diffusion within the polymeric matrix, along with the relaxation of polymer chains, will depict such a mechanism (tab. 2, fig. 3. in vitro drug release graph of f1 to f8 nanoformulation fig. 2. entrapment efficiency graph of nanoformulations 272 biologica nyssana ● 16 (1) june 2025: 267-276 alex et al. ● formulation and characterization of nanoparticles loaded with curcumin and amantadine as a promising neuroprotective agent table 2. release kinetics of f7 formulation fig. 4). thus, it exemplifies an interplay between diffusion-driven and matrix relaxation-controlled processes, thereby offering more complex, reliable drug delivery systems targeted for sustained therapeutic outcomes. in vitro neuroprotective effect determination by mtt assay the neuroprotective potential of the sample combination (curcumin+amantadine) was assayed by carrying out the mtt assay with the neurotoxic agent rotenone. rotenone was selected as the neurotoxic agent in this study due to its well-established ability to mimic parkinson’s disease by inducing oxidative stress and dopaminergic neuron damage through mitochondrial complex i inhibition in in vitro and in vivo models. hence, it is an appropriate choice for modeling pd-related neurotoxicity. cells without treatment were kept aside to control. od values for the diverse concentrations of samples within 1.5 to 25 µg/ml are obtained, and respective percentages of cell viability are calculated. the control group had the highest average od value, 0.8725, which means 100% cell survival. however, rotenone-treated cells displayed a drastic reduction in viability, and the average od value was 0.4179, which translated to 47.90%. this confirms the potent neurotoxic effect of rotenone. the curcumin+amantadine formulation increased the cell viability in a concentration-dependent manner. the sample with the lowest concentration of 1.5 µg/ml had an average od of 0.4703 and a model name zero order kinetics first order kinetics higuchi model korsmeyer peppas model r2 value 0.917 0.8512 0.9245 0.9735 fig. 4. release kinetics of f7 formulation (a zero order kinetics; b first order kinetics; c higuchi plot; d korsmeyer peppas plot) 273 biologica nyssana ● 16 (1) june 2025: 267-276 alex et al. ● formulation and characterization of nanoparticles loaded with curcumin and amantadine as a promising neuroprotective agent cell viability of 53.90%. a slight recovery from rotenone-induced cytotoxicity was noted. with the increase to 3.1 µg/ml, the average od became 0.5484, and cell viability reached 62.85%, meaning that the protective effect is moderate. the average od at 6.25 µg/ml increased further to 0.6011, corresponding to 68.90% cell viability. this trend continued well up to 12.5 µg/ml, where at that concentration, the average od reached 0.6591, with the equivalence of 75.54% viability. the maximal protective effect was obtained with an average od of 0.7301 and 83.68% cell viability, with the highest concentration tested being at 25 µg/ ml (tab. 3). (od), percentage viability, standard deviations (stdev), and standard error (se). the statistics that accompanied the experiment verified the results and validated the effectiveness of the sample with various concentrations. the control received no treatment, and cell viability remained 100% at all the time points, with an average od of 0.8725, showing no variability (stdev=0, se=0). results will form the comparison control. the results from cytotoxicity for the samples that were treated with rotenone showed a very high statistical significance for the remaining average percentage of cell viability at about 47.91% as opposed to the control average. this would mean table 3. od values of curcumin+amantadine nanoparticles sample concentration(µg/ml) od value i od value ii od value iii average od percentage viability control 0.8748 0.8817 0.8609 0.8725 100 rotenone 0.4208 0.4131 0.4198 0.4179 47.9 sample code: curcumin + amantadine 1.5 0.4643 0.4729 0.4737 0.4703 53.9 3.1 0.5514 0.5413 0.5525 0.5484 62.85 6.25 0.5956 0.6068 0.601 0.6011 68.9 12.5 0.656 0.6639 0.6574 0.6591 75.54 25 0.7324 0.7229 0.7351 0.7301 83.68 these results indicate that curcumin and amantadine formulations indeed possess a good neuroprotective potential as they rescue rotenoneinduced cytotoxicity in a concentration-dependent manner. the progressive increase in percentage viability as concentration increases implies that the formulation could perhaps neutralize oxidative stress and neuronal damages, probably due to the synergism of curcumin through antioxidant properties and amantadine's neuroprotective mechanisms. the potential of this combination of therapies can be a bright prospect as a therapeutic measure for oxidative stress-related and neuronal damage-related neurodegenerative conditions. the findings suggest significant neuroprotective effects of the drug from an increase in percentage viability and betterment of cell morphology. statistical analysis of in vitro neuroprotective effect using mtt assay the neuroprotective activity of the curcumin+amantadine combination was investigated on the sh-sy5y cell line. data were obtained from the mtt assay, which used optical density that neurotoxicity would have a significantly high degree. the two variables, stdev=0.97015 and se=0.32338 illustrate the variation in the cytotoxicinduced response (tab. 4). in this study, the curcumin+amantadine treatment demonstrated a concentration-dependent effect on cell viability improvement. on 1.5 µg/ml, the mean percentage viability was 53.91% (stdev=1.00335, se=0.33445), representing partial protection from rotenone-induced damage. successively higher concentrations showed increasingly increased percent viability, such as up to 3.70% at 25 µg/ ml (stdev=1.69913, se=0.56638). this significant recovery reveals a synergistic action between curcumin and amantadine against neuronal damage. the trend for cell viability at a higher concentration of curcumin+amantadine was statistically analyzed to determine the decreasing variability at intermediate concentrations: 6.25 µg/ ml, stdev=0.86632, se=0.28877; thus, there may be uniform protection at such doses. the highest dose, 25 µg/ml, yielded the highest variability at stdev=1.69913, possibly because the high dose of the sample triggered an increase in metabolic 274 biologica nyssana ● 16 (1) june 2025: 267-276 alex et al. ● formulation and characterization of nanoparticles loaded with curcumin and amantadine as a promising neuroprotective agent activity and/or variability in cellular response. the bar graph represents the neuroprotective action (fig. 5). control and rotenone-treated samples are two extremes wherein the viability was highly different. the bars related to curcumin-amantadinetreated samples represent a considerable positive trend in the recovery process, indicating that the increase is concentration-dependent. the error bars depict that at middle concentrations (6.25–12.5 µg/ ml), the values are more accurate. statistical analysis confirms that the neuroprotective effects of the combination of curcumin and amantadine are indeed dose-dependent. percent viabilities are high regarding protection against cytotoxicity from rotenone at concentrations of 12.5–25 µg/ml; data approach near-control levels. the consistency and reproducibility of these data at key concentrations, evidenced by low standard errors, support the therapeutic potential for this combination (fig. 6a-6f). these results support further exploration of curcumin and amantadine as a synergistic neuroprotective strategy in oxidative stress and neurodegeneration models. conclusion in this study, we fabricated curcumin and amantadine nanoparticles using bovine serum albumin (bsa) as a nanocarrier using the desolvation method for antiparkinson activity. eight nanoformulations were prepared by varying the concentration of ethanol, glutaraldehyde, and stirring time. the nanoparticles were acquired in the particle size of 255.5 nm, within the optimal range for cellular growth and targeted delivery. pdi was found to be 0.05, indicating a highly uniform size distribution, and a zeta potential of -29 mv is generally considerably stable, indicating a sufficient electrostatic repulsion between particles to prevent aggregation. nanoparticles showed a sustained release profile, and both diffusion and relaxation-controlled processes influence the mechanism of drug release. the results of in vitro antiparkinson activity confirmed that the optimized nanoparticle has significant neuroprotective effects of the drug from an increase in percentage viability and betterment of cell morphology. hence, a combination of curcumin and amantadine nanoparticles will be a better choice for neuroprotective effects. acknowledgements. i would like to thank the centre for food nanotechnology, karpagam academy of higher education, coimbatore for helping me to perform particle size analysis and zeta potential characterization. references alonso, m.j. & couvreur, p. 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(2014). oral administration of nano-emulsion curcumin in mice suppresses inflammatory-induced nfκb signaling and macrophage migration. plos one, 9(11), e111559. doi: 10.1371/journal.pone.0111559. vashishth et al. 2025, biologica nyssana 16(2) 16 (2) december 2025: doi: 10.46793/biolnyss.16.2.19v exploring the applications of allelopathy in agriculture: a review original article deepti singh vashishth department of environmental science, graphic era (deemed to be university), dehradun, india rinky yadav department of environmental science, alpine group of institutes, dehradun, uttarakhand, india rakesh kumar bachheti centre of molecular medicine and diagnostics, saveetha dental college & hospitals, saveetha institute of medical and technical sciences, saveetha university, chennai, india ashish kumar arya department of environmental science, graphic era (deemed to be university), dehradun, india archana bachheti department of environmental science, graphic era (deemed to be university), dehradun, india baccheti.archana@gmail.com (corresponding author) received: august 16, 2025 revised: november 26, 2025 accepted: november 28, 2025 abstract: this review delves deep into the world of allelopathy and its applications in agriculture, offering a detailed examination of its environmental, physiological, and ecological aspects. the review begins by dissecting the intricate factors that influence allelochemical production, including environmental and physiological determinants. it also explores the mechanisms underlying allelochemical activity, shedding light on how these compounds disrupt fundamental plant functions and signalling pathways. the review underscores the pivotal role of allelopathy in agriculture, emphasizing its dual impact on crop yields and weed management. allelopathic interactions between crop plants and weeds are explored, revealing the intricate ways in which they influence each other. the review also highlights the effects of common plants on both crops and weeds, showcasing allelopathy's potential as a sustainable agricultural practice and an effective tool for weed control. the review provides valuable insights into the multifaceted applications of allelopathy in modern agriculture. another crucial aspect discussed in the review is the persistence and degradation of allelochemicals in soil and their effects on soil nutrient dynamics. understanding how these compounds interact with soil and impact nutrient availability is vital for optimizing their use in agriculture. this section emphasizes the need for a holistic approach to sustainable farming, taking into account the complex interplay between allelopathy and soil health. key words: allelochemical, agriculture, nutrients, weed control, soil health apstrakt: istraživanje primena alelopatije u poljoprivredi ovaj pregled dubinski ulazi u svet alelopatije i njenih primena u poljoprivredi, nudeći detaljnu analizu njenih ekoloških, fizioloških i ekoloških aspekata. pregled započinje razlaganjem složenih faktora koji utiču na proizvodnju alelohemikalija, uključujući ekološke i fiziološke determinante. takođe istražuje mehanizme koji stoje u osnovi aktivnosti alelohemikalija, osvetljavajući načine na koje ova jedinjenja narušavaju osnovne biljne funkcije i signalne puteve. pregled naglašava ključnu ulogu alelopatije u poljoprivredi, posebno njen dvostruki uticaj na prinos useva i kontrolu korova. istražene su alelopatske interakcije između useva i korova, otkrivajući složene načine njihovog međusobnog delovanja. pregled takođe ističe efekte uobičajenih biljaka na useve i korove, ukazujući na potencijal alelopatije kao održive poljoprivredne prakse i efikasnog alata za suzbijanje korova. na taj način, pregled pruža dragocene uvide u višestruke primene alelopatije u savremenoj poljoprivredi. još jedan ključni aspekt obrađen u tekstu jeste postojanost i razgradnja alelohemikalija u zemljištu, kao i njihov uticaj na dinamiku hranljivih materija u tlu. razumevanje načina na koji ova jedinjenja stupaju u interakciju sa zemljištem i utiču na dostupnost hranljivih materija od suštinske je važnosti za optimizaciju njihove primene u poljoprivredi. ovaj deo naglašava potrebu za holističkim pristupom održivoj poljoprivredi, uzimajući u obzir složenu međuigru između alelopatije i zdravlja zemljišta. ključne reči: alelohemikalija, poljoprivreda, hranljive materije, suzbijanje korova, zdravlje zemljišta © 2025 vashishth et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. introduction for almost two thousand years, scientists have been studying plants and their relationship with other organisms. many examples described by botanists, farmers, and horticulturalists provide strong evidence for allelopathic interactions among plants. japanese literature also reports instances of plants harming other plants due to the formation of bane compounds in sediments, especially in pinus densiflora, commonly known as japanese red pine (rice, 1985). in 1800, agronomists began to record repeated problems in a few perennial cropping systems (weston, 2005). the most fascinating fact was that most plant species with therapeutic value for humans are also reported to exhibit potential allelopathic effects later (wink, 2018). the generation of allelochemicals from plant residues and the role of microbial interactions in their degradation are significant (putnam & weston, 1986). after the development of various techniques, such as extraction, bioassays, chemical isolation, and identification, in the 20th century, curiosity in the field of allelopathy developed (willis, 1997). first, muller and his associates reported many compositions of volatile compounds induced by plants in the us, california, and elroy rice reported allelopathy in prairie-type ecosystems (usa, oklahoma). he described the effects of allelochemicals on nitrogen-fixing and nitrifying microorganisms in soil (rice, 1985). research in this field can bring together various disciplines to build sustainable agriculture successfully. in the 21st century, many weed scientists, physiologists, natural product chemists, and soil scientists began to take an interest in this challenging field of allelopathy (macias, 2002). studies have revealed crucial examples of allelochemicals in seed and growth suppression that alter vegetation patterns, order of plant succession, weed plethora, agricultural yield, and challenges in transplanting fruits and crops (el-darier & youssef, 2022; hatata & el-darier, 2009). these substances are produced by plants and microbes, referred to as “donor species,” while species whose growth and development are affected are referred to as “recipient plants.” allelopathic interactions include those between plants, microorganisms, viruses, insects, soil, and plants, as well as interactions between plants and chemicals. the nature of the active chemicals and the targeted species defines whether allelopathic effects will be stimulatory or suppressive (keating, 1999) (fig. 1). secondary metabolites are substances emitted by plants, and some have allelopathic effects. the majority of allelochemicals are labelled as secondary metabolites from the shikimic acid or acetate pathways (inderjit & bhowmik, 2002). allelopathic interference has been mainly linked to all kinds of secondary plant products (weston & duke, 2003). the plant kingdom contains hundreds of allelochemicals; many are phytotoxic, and several have been isolated from plant tissue and soil and are suspected of suppressing germination and growth. biological and abiotic soil factors modulate the phytotoxic action of allelochemicals once they leave the plant. some allelochemicals are water-soluble and can be washed away from leaves by rain, mist, dew, or fog drip (kobayashi, 2004). plant parts that produce these chemicals are flowers, inflorescences, stems, leaves, fruits, roots, rhizomes, and seeds (rice, 2012). allelochemicals are synthesized in plant parts above, below, or both above and below the soil surface, and they cause allelopathic effects on many plant communities. donor plant species that produce allelochemicals, such as lignins, salts, and tannins, often store them in an inactive form within plant cells. these hazardous compounds are produced by plant enzymes or by environmental stress (weston, 1996). allelochemicals are discharged through different mechanisms: (a) release of chemicals from the aerial parts (leaves and stems) by rainfall and dew, (b) release of chemicals from roots called root exudation through diffusion vesicle transport, and (c) disintegration by microorganisms (keating, 1999). allelochemicals are the plant’s secondary compounds or breakdown products of degrading plant tissue. these compounds are frequently retained in vacuoles to protect the generating plant but are frequently extruded or filtered out of the tissues (hall & henderlong, 1989). phytotoxic effects are shown to depend on the density of phytochemicals. plant growth declines due to resource constraints, although phytotoxicity is highest at low plant densities (weidenhamer et al., 1989). in most cases, early in the disintegration process, plant remains have the highest phytotoxic potential. phytotoxicity lessens or is even eliminated as decomposition progresses. activities in the soil, both biotic and abiotic, can influence the amount and activity of phytotoxins biologica nyssana ● 16 (2) december 2025: vashishth et al. ● exploring the applications of allelopathy in agriculture: a review fig. 1. path of allelochemicals from a donor plant to recipient plant biologica nyssana ● 16 (2) december 2025: vashishth et al. ● exploring the applications of allelopathy in agriculture: a review (sampietro et al., 2007). the concentration of allelochemicals in the plant tissue may fluctuate with time. for instance, bark leachates of a species of eucalyptus globulus are more harmful to particular food crops than foliar leachates and leaf litter leachates (rizvi et al., 1999). materials and methods literature data the data were collected from different types of secondary sources, including a systematic review of many web-based searches, and analysed published scientific articles and books on the allelopathic activity of several plants. the literature was searched by using google scholar, research gate, pubmed, scopus, and web of science with particular search phrases like ‘allelopathy’, ‘phytochemicals’, ‘agriculture’, ‘bioherbicides’, ‘secondary metabolites’, ‘aromatic plants’, ‘weeds’, and ‘abiotic factors’. the remaining literature was searched using the names of allelochemicals known to confer herbicidal activity to identify publications not found with these search terms. additional searches were carried out for technical reports, student theses, government publications, agency reports, websites, and synthesis articles or book chapters. the collected material was published from 2000 to 2022. a total of 196 articles on allelopathy, allelochemical mechanisms, and allelopathy in agriculture were collected and included in this review (fig. 2). although it is difficult to compile all of the material from studies on allelopathy, we focused on the positive aspects of allelopathy that can be employed in weed management for sustainable agriculture. finally, we examined the data to investigate the allelopathic activity of common aromatic plants in weed management. results and discussion factors influencing the production of allelochemicals secondary metabolite deficiency, unlike primary metabolite deficiency, results in prolonged deterioration of the creature’s survival, prolificacy, aesthetics, or possibly nothing noteworthy changed. they are also crucial in plant defence against herbivory and other interspecies protection. they have a relatively restricted range in the plant kingdom (jain et al., 2019). they differ in quantity and quality for specific plant species that thrive in different locations. they are frequently produced in smaller quantities by specific cell types at various stages of development. secondary metabolites are a diverse set of molecules found in plants, including alkaloids, flavonoids, phenolics, glycosides, terpenes, amines, and steroids, that have been widely exploited in the pharmaceutical industry (sharanabasappa et al., 2007). climatic variables climatic variables have a significant impact on the generation of allelochemicals. numerous allelochemicals are impacted by variations in light intensity, quantity, and duration. the majority are produced during protracted photoperiods and uv light exposure (escobar-bravo et al., 2019). plants exposed to high ambient temperatures produced more allelochemicals. water is essential to the phenomenon of allelopathy, as it serves as a solvent and a transporter of allelochemicals and leachate from plant aerial sections and the soil. soil microorganism action is affected by soil moisture (aliotta et al., 2006). allelochemicals are composed and concentrated differently, depending on the maturity, organ, and plant species (qasem & foy, 2001). environmental factors can influence allelopathy expression. the plant’s allelopathic capability in nature is likely to differ by location owing to edaphic and climatic conditions. seasonal variables, such as soil temperature, air temperature, and soil moisture, significantly influence allelopathy. the separation and identification of molecules with biological activity from donor plants does not prove that these compounds interact in nature fig. 2. search strategy for the review biologica nyssana ● 16 (2) december 2025: vashishth et al. ● exploring the applications of allelopathy in agriculture: a review determining the concentration at which each distinct reaction takes place is essential if allelopathic interactions are to be used in weed control programmes. moreover, the allelopathic potential of different plant components may differ (chon & kim, 2002). the plants’ leached metabolites contained various materials, including carbohydrates, amino acids, mineral nutrients, and more organic compounds. depending on the season, leachability, concentration, and plant age, these substances can either promote or inhibit plant growth (reganold, 2016). biochemicals with complex structures are among the molecules plants use to interfere with each other, and inorganic elements may also be exploited in an allelopathic manner. in soil, elements like salts and heavy metals may build up due to hyperaccumulation, breakdown of litter, and changes in the chemistry of the rhizosphere (morris, 2009). in the ground, allelopathic plant residues can remain on the soil surface, and the subsequent crop can be planted into the remnants without tillage. furthermore, entire leftovers may be incorporated into the planting region’s soil via strip-till, allowing conventional equipment to be used. biotic and abiotic variables, such as soil physical, chemical, and microbiological properties, may affect the phytotoxicity of chemicals released from integrated remnants (popa et al., 2008). thus, soil microorganisms can inactivate a phytotoxic chemical, make it more active, and transform it into new poisons (kobayashi, 2004). mechanisms of action of allelochemicals it can be classified as a direct or indirect mechanism. the direct action of allelochemicals involves biochemical and physiological effects on numerous critical plant developmental and metabolic processes. the indirect action is represented by changes in soil characteristics, nutritional status, and changes in the population or activity of microorganisms and nematodes. the allelochemicals should be soaked to have a direct effect on the target plant. allelochemicals are absorbed mainly by recipient plants through their root network, either actively or passively, and the allelochemicals travel by way of the xylem by mass flow (inderjit, 2005). when an allelochemical is absorbed, it alters the target plant’s physiological activities. however, investigations have found that the allelochemical reaction might differ depending on concentration. those allelochemicals that restrict the growth of one species at a particular concentration might promote the growth of a different species at other concentrations (muzell et al., 2016). previously, allelopathy was known for its negative impacts on neighbouring plants, but in through allelopathic mechanisms. the retention and movement of allelochemicals in soil and the constituents of soil all affect the destiny of allelochemicals (inderjit, 2001; inderjit, 2005). physical, chemical, and biological variables physical and chemical parameters determine the quality, quantity, and number of allelopathic chemicals; soil texture, particularly, has a considerable impact on the manifestation of allelopathy in natural systems. sandal loam soils absorb fewer phenolic chemicals than fine-textured soils. allelopathic expression is influenced by soil chemical and biological factors, including nutrients, soil consistency, microbes, moisture, ph, and organic matter. they impact soil adsorption and transport, as well as allelochemical metabolism. the ph of the soil is vital for the absorption and immobilization of inorganic ions, as well as for the resulting nutrient buildup, and a high ph can enhance microbial activity (kobayashi, 2004). similarly, soil chemical properties frequently change with the inclusion of plant waste, leaves, roots, or donor plant leachate, and these alterations have been shown to influence allelochemical activity. in soil settings, allelochemical interactions are heavily influenced by allelochemical turnover rates in the rhizosphere, and their interactions with organic matter, clay, and other soil variables further alter soil biological and physicochemical properties (scavo et al., 2019). blum reported that soil ph, texture, organic carbon, and nitrogen are all significant in regulating allelochemical absorption and persistence when soil microorganisms are present. as well as allelochemical phytotoxicity, it can also be influenced by the dynamics of soil moisture. according to data reported, increased evaporationtranspiration and reduced soil water content would result in the decline of allelochemical phytotoxicity of plants in the soil mixture (blum, 2002) allelopathy is also influenced by soil microorganisms, which can alter outcomes by decomposing hazardous chemicals or producing toxic chemicals (inderjit, 2001). they have the capacity to alter the availability of soil nutrients, as well as the release of chemical substances bound to soil particles. allelopathic chemicals can exist in three states: irreversibly bound, reversibly bound, and free. in general, the last two types are relevant to allelopathy (inderjit et al., 1999). allelopathic plants enhance the synthesis of phytotoxic secondary metabolites when exposed to environmental challenges, such as excessive light levels, severe temperatures, mineral deficiencies, moisture stress, and exposure to fungicides, insecticides, herbicides, and plant growth regulators (pgrs) (aliotta et al., 2006). biologica nyssana ● 16 (2) december 2025: vashishth et al. ● exploring the applications of allelopathy in agriculture: a review recent years, many researchers have adopted a more positive perspective, and the mechanisms underlying both perspectives are described in fig. 3. allelochemicals can affect the growth and seed germination of nearby plants. when plants come into contact with soil-based allelochemicals, these effects primarily affect plant roots. exposure to these compounds increases the physiological responsiveness of target plants, inducing oxidative stress and potentially leading to cell death. lipid peroxidation, alterations in the cell membrane, protein abnormalities, and elevated protease activity are early indicators of cell damage. the plant’s protoplast (the living component of the plant cell enclosed by the plasma membrane, excluding the cell wall) may degenerate through hydrolysis or necrosis if antioxidants are unable to eliminate these reactive oxidants, leading to the loss of cell organelle integrity and function. moreover, variations in the structure and function of the apical meristem impact root development and water absorption. these allelopathic substances could provide a less harmful alternative to chemical pollution for controlling weeds and invasive plants (šoln et al., 2022). the mechanisms and modes of action of allelochemicals, which have since been studied, were identified by rice (2012). allelochemicals have several mechanisms of action to suppress and alter plant growth and development. allelochemicals are known to target the following sites or processes: cell division, plant hormone balance and production, membrane permeability and stability, germination of pollens, uptake of minerals, movement of stomata, synthesis of pigments, respiration, photosynthesis, nitrogen fixation, amino acid synthesis, specific enzyme activities, nitrogen-fixing bacteria, and modification of nucleic acids. allelochemicals may operate selectively, or plants may respond selectively. these problems are exacerbated by the presence of several active compounds in a single plant. for example, sorghum spp. include flavonoids, cyanogenic glycosides, tannins, phenolic acids, and quinones; all of these have repressive activities, and most cause various biological effects (weir et al., 2004). the suppressive actions of most of the artemisia spp. foliar extracts appear to contain a variety of chemicals, including terpenoids, polyacetylenes, and coumarins (weston & duke, 2003). sesquiterpenes are a broad family of naturally occurring chemical compounds that are represented by several physiologically active molecules, including those with intriguing herbicidal potential. other studies have shown that trichomes on the leaf surface generate substantial amounts of camphene, cineole, and camphor, as well as considerable amounts of artemether and artemisinin, among other compounds, with camphor proving to be the most effective in seed suppression, i.e., inhibition of seed growth (barney & weston, 2002). a sesquiterpene endoperoxide lactone known as artemisinin, separated from artemisia species, was originally found to have phytotoxic action. artemisinin has the greatest impact on chlorophyll content and root network development (duke et al., 2000). in the meristem of wheat root tips, romagni discovered that monoterpenes such as 1,8-cineole impede the respiration of mitochondria and substantially inhibit all phases of mitosis. camphor possesses mitotic and respiration effects comparable to those of 1,8-cineole (romagni, 2000). fig. 3. mechanism of action of allelochemicals that can influence receptor plants harmfully or beneficially biologica nyssana ● 16 (2) december 2025: vashishth et al. ● exploring the applications of allelopathy in agriculture: a review amylase activity regulates the breakdown of starch and is necessary for providing substrates for respiratory metabolism, which in turn affects the germination of cereals. the leachates of eucalyptus globosus leaf inhibited germination by decreasing amylase activity in finger millet (eleusine coracanta) seeds (padhy et al., 2000). comparable results were achieved with lepidium sativum seeds due to the presence of mboa (6-methoxy-2benzoxazolinone), which is widely found in cereals (kato-naguchi & macias, 2004). mobilization of lipids was inhibited in the presence of p-coumaric and ferulic acids, while the seeds of brassica napus and helianthus annus were germinated in the presence of datura stramonium (baleroni et al., 2000). allelopathic chemicals affected isocitrate lyase activity and the expression of the isocitrate lyase gene. thus, isocitrate lyase seems to be a highly sensitive enzyme in response to allelopathic pressure, and its reduced performance may cause the retardation of seed germination or delay. furthermore, it has been proposed that the observed reduction in enzyme activity is a side effect of allelochemicals, which damage proteins. as a result, allelochemicals’ impacts on seed germination are mediated by disturbances in basic cellular metabolism rather than by organelle destruction. reserve mobilization, which normally occurs rapidly during the early phases of seed germination, appears to be slowed or reduced under allelopathic stress (weir et al., 2004). disruption of photosynthesis is among the most frequently documented physiological consequences of several allelochemicals. allelopathic chemicals have the potential to be widely employed in organic agriculture, for example, as natural herbicides in sustainable weed control (gniazdowska & bogatek, 2005). in lactuca sativa treated with artemisinin and its sesquiterpene, the amount of chlorophyll was reduced, and the quantity of carotenoid was also reduced (dayan et al., 1999). when exposed to helianthus annuus allelochemicals, leaves of sinapis alba exhibited lower photosynthetic rate. however, it coincided with a decreased transpiration rate, suggesting restricted co2 absorption in the chloroplasts owing to stomatal closure (bernat et al., 2004). the proposed reason for the interruption of bine growth under allelopathic stress is a change in mitochondrial respiration, leading to a lower atp supply for all energy-dependent activities. coumarin decreased the pace at which mitochondria in allium cepa root cells respired; monoterpenes are the allelochemicals that reduce the respiration of mitochondria by enhancing the rate of electron transport via an alternate pathway. cinnamic acid and pinene similarly reduced oxygen consumption in glycine max cotyledons while increasing the relative splitting of electrons toward the alternate route (cheng & cheng, 2015). the monoterpenes inhibited mitochondrial respiration in zea mays main roots, resulting in complete inhibition of respiratory regulation. it was proposed that such chemicals may operate as oxidative phosphorylation uncouplers. the capacity of allelochemicals, such as monoterpenes, hydroxamic acids, and coumarins, to permeate plant tissue may be responsible for their inhibitory influence on o2 uptake (abrahim, 2000). the hydroxamic acid can also influence gene expression and chromatin modification in target plants by inhibiting histone deacetylases (venturelli et al., 2015). the helianthus annuus leaf extract impeded seed germination of sinapis alba (bogatek et al., 2006), thereby reducing the respiration rate of seeds during the initial three days of seedling development. this demonstrates a connection between dark respiration suppression and germination delay when allelopathic substances are present (gniazdowska & bogatek, 2005). seedling growth inhibition under allelopathy stress circumstances may therefore be due to reduced ion uptake. the impact of allelochemicals on onion uptake is crucial, as the root is the rhizosphere organ that initially comes into contact with them. cucumis sativus root discharge reduced ion uptake by cucumber seedlings (no3 -, k+, ca2+, mg2+, fe2+) (mushtaq, 2020). salicylic acid, a phenolic compound, has been shown to inhibit potassium absorption in plants. both salicylic acid and ferulic acid inhibited k+ absorption by the roots of oats, especially when the ph is lower. various additional investigations using entire plants and cell cultures have found that the absorption of macro and micronutrients is decreased when phenolic acids are present (sahu, 2013). phenolic acid inhibits the absorption of no3 -, k+, po4 3-, and mg+2 ions, and altered mineral ion uptake affects plant growth and development. (einhellig et al. 2002). vanillic and p-coumaric acids were more harmful when the seedlings of barley were low in phosphorus or nitrogen. they determined that the toxicity of phenolic compounds depends upon the nutritional quantities. as a result, the impact of allelochemicals on anion absorption may be due to a lowered respiration rate and an inadequate quantity of adenosine triphosphate generated in cells of roots (gniazdowska & bogatek, 2005). terpenoids are known to be emitted from plants in drought-stricken areas. volatile organic compounds have been found to have dual effects on germination and plant growth, both promoting and inhibiting. volatile allelochemicals may impede the competitiveness of weed species and have piqued biologica nyssana ● 16 (2) december 2025: vashishth et al. ● exploring the applications of allelopathy in agriculture: a review public interest (xie et al., 2021). many secondary metabolites, such as terpenoids, flavonoids, phenolic compounds, and alkaloids, operate as plant allelochemicals. in natural and agroecosystems, these phenolic acids are recognized as allelopathic agents. they have been observed to affect seed germination, chlorophyll content, respiratory activity, seedling growth, enzyme activity, and cell division. however, there is little information on how phenols and other substances interact and alter seed germination and seedling growth; evidence suggests that combining phenols enhances their degrading activity. it is phytotoxic to various weedy plants and can be used as both a pre and post-emergent herbicide (bachheti et al., 2020). allelopathy’s most frequent phenolic chemicals include benzoic and cinnamic acid derivatives, coumarins, tannins, other polyphenolic complexes, and some flavonoids. the amount of these chemicals produced and released by plants varies greatly. the various coumarins, tannins, and phenolic acids appear to have identical modes of action, limiting plant development through a variety of physiological effects that impart broad cytotoxicity (macias et al., 2004). the mechanisms of action of flavonoids are less understood than those of phenolic acid. some flavonoids powerfully regulate energy metabolism and inhibit chloroplast and mitochondrial processes (mierziak et al., 2014). generally, the phenolic acid concentration necessary to impede seed germination is larger than that required to prevent growth in plant seedlings (einhellig, 2002). a reduction in photosynthetic efficiency is a persistent side effect of phenolics. sorgoleone, a p-benzoquinone, was found to block oxygen evolution in leaf discs and isolated chloroplasts, leading to decreased growth and photosystem ii electron transport (zhou & yu, 2006). bioassays in allelopathy the initial step for investigating probable allelopathic involvement is a laboratory bioassay. bioassays are valuable tools for assessing the allelopathic potential of plant extracts and for monitoring their activity during the purification and identification of allelochemicals. most allelopathic papers describe some form of bioassay procedure to demonstrate allelopathic activity (macias et al., 2000). bioassays for this type of study often involve tests of seed germination and growth, seedling, radicle, and coleoptile length, seedling fresh weight, and photosynthetic activity under controlled laboratory conditions. these tests can detect potential allelopathic effects (gawronska et al., 2006). although bioassays using plant aqueous extracts are valuable for demonstrating the presence of allelopathic chemicals in plants, these effects often disappear in the field due to adsorption onto soil particles, degradation, and leaching. screening bioassays using entire plant seedlings have been developed in response to complaints about aqueous extract bioassays. allelopathy research has employed plants at the seedling stage (wu et al., 2000). the biosynthetic pathways of the major allelopathic substances are shown in fig. 4. role of allelopathy in agriculture in india’s agroecosystems, weeds are predicted to cause a 13.2% loss in the eight most important cash crops worldwide. to manage weeds, hand weeding, like mechanical approaches, is used, which takes a lot of effort and time. at the same time, excessive herbicide use can lead to environmental contamination, hazardous agricultural products, and a risk to human health (kordali et al., 2009). allelopathy is crucial in agriculture, resulting in diverse interactions among crop–weed, crop– crop, and tree–crop systems (singh et al., 2001). some plants hinder weed growth. they are an fig. 4. biosynthetic pathways of the major allelopathic substances (seigler, 2006) biologica nyssana ● 16 (2) december 2025: vashishth et al. ● exploring the applications of allelopathy in agriculture: a review excellent, strategic source of natural compounds for developing natural herbicides. plant allelopathic properties can be efficiently used for biological weed management in agricultural production (vashishth et al., 2023). the allelochemicals in these plants can be the same or distinct, and when combined, they may have synergistic or additive effects. allelopathic crops can be used to influence weeds in a variety of ways, including surface mulching (cheema et al., 2000), spraying of aqueous extract (cheema et al., 2002), blending with the soil (sati et al., 2004), and intercropping and mixed cropping (hatcher & melander, 2003). a number of papers have been published on screening plants for allelopathic potential in ecosystems. grewia optiva, acacia spp., eucalyptus spp., albizzia lebbeck, glircidia sepium, moringa oleifera, leucaena leucocephala, and populus deltoides are examples of tree species that affect crop production through allelopathy (singh et al., 2001). the variety of weeds, particularly those in agroecosystems, is also known to exhibit allelopathic traits that increase their competitiveness and, consequently, negatively impact crops (weston & duke, 2003). allelopathic effect of crop plants on other crop plants and weeds the majority of research has focused on the impact of cover crops on weed control, and crop growth research has focused on legumes or winter cereals (dhima et al., 2006). the extract of the root, seed, seed coat, and aerial parts of arachis hypogaea can hinder the development of rootlets and hypocotyls and the germination of seeds of lactuca sativa. the separation of ethanol-based seed extract into dichloromethane, n-hexane, butanol, ethyl acetate, and aqueous residue also exhibits positive allelopathic potential. in some large-scale crops, the dichloromethane fraction of ethanolic seed extract can be used as an eco-friendly herbicide to control weeds (casimiro et al., 2017). the effective field treatment of purple nutsedge using plant extracts from a variety of allelopathic crops (sorghum, brassica, and sunflower) was reported by iqbal and cheema (2007). the brassica spp. water-based extracts significantly inhibited physalis angulata, a troublesome weed in turkey’s cotton, maize, and soybean fields (uremis et al., 2005). sorghum mulches dramatically decreased the dry biomass and density of cyperus rotundus, one of the worst weeds in the world (mahmood & cheema, 2004), and an alfalfa aqueous extract inhibited the germination and seedling growth of lepidium sativum (el-darier & youssef, 2000). the findings indicated that alfalfa pellets effectively prevented the development of rice weeds, suggesting that they could be used as a natural pesticide in rice fields (xuan & tsuzuki, 2001). the leaves, roots, and stems extract of coriandrum sativum at the flowering and maturity stage was applied in petri plates on seeds of triticum aestivum, and some weeds (like lolium multiflorum, sinapis alba, and amaranthus retroflexus), and the leaves extract showed more allelopathic activity, followed by stem and root extracts, but only on weeds seeds and not on t. aestivum seeds. also, the extract of mature c. sativum was less allelopathic than the one obtained from the flowering stage (pannacci et al., 2025) allelopathic effect of weeds on crop plants and other weeds seed germination and plant growth of t. aestivum, cucumis sativus, and sinapis arvensis were tested when treated with different concentrations (5%, 10%, 15%, and 20%) of the aqueous extract of euphorbia heterotophylla. it was reported that the germination percentage of sinapis arvensis and t. aestivum was reduced. at the 20% concentration, sinapis arvensis showed the highest inhibition, i.e., approximately 44.44%, whereas at the 5% concentration, germination in cucumis sativus seeds was stimulated and did not affect 10%, 15%, or 20%. in the case of growth, the peduncle length of t. aestivum was inhibited at all concentrations, the peduncle length of sinapis arvensis was inhibited only at 15% and 20% and was stimulated at 5% and 10%, and the peduncle length of cucumis sativus showed stimulation at all concentrations, with the highest stimulation noted at 10%, i.e., 66.90%. while the growth of rootlets at 5% concentration of t. aestivum and sinapis arvensis showed stimulation, while inhibition at all other concentrations, and rootlets of cucumis sativus showed inhibition at all concentrations, the highest inhibition was noted in t. aestivum at 20% concentration, i.e., 44.71% (fandah et al., 2020). the allelopathic and antioxidant activities of euphorbia heterophylla against the weed cenchrus echinatus were investigated, and it was found that shoot growth was reduced by approximately 57.8%, root growth by 84.6%, and germination by 93.95% at a concentration of 100 μl/l of the essential oils. hence, essential oils from euphorbia heterophylla can be used as a biocontrol agent against the weed (elshamy et al., 2019). on the other hand, lactuca sativa and sorghum bicolor are indicator plants whose root and shoot growth at the early seedling stage are inhibited by 100% with the methanol extract of euphorbia heterophylla at 2.0 mg ml/l. gc–ms analysis is performed using methanol-derivatized extraction, yielding the first study of the roots of euphorbia heterophylla in a series of allelopathy interactions biologica nyssana ● 16 (2) december 2025: vashishth et al. ● exploring the applications of allelopathy in agriculture: a review and the metabolic activity of their organic acids (da silva et al., 2018). the allelopathic effect of euphorbia hirta and parthenium hysterophorus leaf extract on seed germination and seed elongation of capsicum annum. they treated the seeds of capsicum annum with different concentrations (25, 50, 75, and 100%) and found that an increase in the concentration of plant extract of euphorbia hirta showed a stimulatory impact on germination of seed and seedling elongation of capsicum annum, and a fresh and dry weight of seeds showed a positive allelopathic effect. similarly, the plant extract of parthenium hysterophorus also shows a stimulatory effect on the weight, both fresh and dried seeds of capsicum annum (pathare et al., 2014). the allelopathic effects of the parts of euphorbia thiamifolia on seedling growth and pigeon pea germination were studied. the inflorescence, root, stem, and leaf parts of euphorbia thiamifolia are extracted in water, and research is performed to comprehend the impact of this extract on the dry weight of the shoot and plant germination after 4 days, and the root length of cajanus cajan over a range of different concentration levels. seeding growth and pigeon pea seed germination are relatively lower in all aqueous extractions compared with the standard distilled water control; also, these factors are significantly affected by extract concentration, as growth is lower at higher concentrations. during the study, it was also demonstrated that seedling length and weight, and pigeon pea germination, are more inhibited by euphorbia thiamifolia aqueous extract from stems than from leaves. the root of euphorbia thiamifolia on the 6th day has a relatively lower effect on growth than the leaf and inflorescence parts of the weeds (kumbhar et al., 2011). an enhanced concentration of leachate of euphorbia hierosolymitana caused a substantial decrease in the length of the roots and shoots, the dry and fresh weight of wheat (triticum durum) seedlings, and a decrease in total protein and chlorophyll content (abu-romman et al., 2010). furthermore, the herbicidal activity of peganum harmala residue on seedling growth of avena fatua and convolvulus arvensis, as well as the tendency of its phytotoxins to degrade in soil, was investigated. they also found that c. arvensis showed greater declines in plant growth metrics, and that p. harmala residue exhibited strong herbicidal activity and could be used as a natural pesticide for weed control (sodaeizadeh et al., 2010). the aqueous extract derived from lantana camara was observed to promote the growth of cucumis sativus, brassica juncea, raphanus sativus, phaseolus mungo, cicer arietinum, and vigna unguiculata. the inhibitory effect on germination, root, and shoot length was reported because of aqueous leaf extracts of l. camara. this was proportional to the extract concentrations, with higher concentrations having stronger inhibitory effects and lower concentrations having a stimulatory effect in some circumstances. the inhibitory impact is significantly stronger on primary and lateral root growth than on shoot and germination development (ahmed et al., 2007). using a lemna (lemna aequinoctialis) bioassay, allan and adkins (2007) examined the ability of allelochemicals from medicinal herbs to inhibit plant growth. the aqueous extracts from plant components of acacia farnesiana, acacia melanoxylon, ageratum conyzoides, castanospermum australe, chamaesyce hyssopifolia, alphitonia excelsa, melaleuca quinquenervia, and phyllanthus virgatus hindered the growth of l. aequinoctialis, in which leaf, stem, and bark extracts inhibited the most growth. the water extract of cirsium arvense and ageratum conyzoides can decrease the germination and growth of seedlings of various weeds associated with wheat (akhtar et al., 2001). allelopathic effect of common plants on crop plants and weeds however, little is known about the allelopathic effects of therapeutic and aromatic herbs on weeds, crop germination, and seedling growth (qasem, 2002). allelopathic medicinal and aromatic plants have recently been proposed as feasible solutions for managing weeds in sustainable agriculture (mekky, 2008). the compounds derived from medicinal or aromatic plant residue may aid in the reduction of the use of synthetic herbicides for the control of weeds, resulting in lesser pollution and better agricultural services (khanh et al. 2007). the effects of aromatic plants on crops and weeds, whether employed as cover crops, absorbed into the soil as green manure, used in the soil as mulch, or used as an aqueous extract, are sparse in the literature. the ethanolic extracts of tephrosia purpurea, prosopis juliflora, abutilon indicum, and cassia occidentalis were tested for their potential bioherbicidal activities on parthenium hysterophorus, and it was reported that prosopis juliflora and cassia occidentalis had stronger phytotoxic activity than tephrosia purpurea and abutilon indicum. however, all four plants had phytotoxic properties and can act as natural biodegradable herbicides against weeds such as parthenium hysterophorus (andhare, 2019). the wheat yield is adversely affected by allelopathic interactions; hence, shah et al. (2018) suggested that wheat should not be sown near quercus coccifera, moringa oleifera, prosopis juliflora, eucalyptus camaldulensis, acacia nilotica, and acacia salinga to prevent destructive effects on various developmental stages of the crop. the effect of six aromatic and therapeutic plants, basil (ocimum basilicum), chamomile (matricaria chamomilla), common mallow (malva sylvestris), greater celandine (chelidonium majus), lemon balm (melissa officinalis), and lovage (levisticum officinale koch), on the germination and growth of the scentless mayweed (tripleurospermum inodorum schultz). weed seed germination was reduced by 32.2% when lovage seeds were used in petri dishes. extracts from fresh plant biomass inhibited weed germination and development at 5% and 10% concentrations, with extracts from chamomile and common mallow having the greatest inhibitory effect. the inhibitory impact of extracts from dry plant biomass was greater. weed seed emergence and growth were reduced when dried plant wastes were introduced into the soil at rates of 10 and 20 g/kg. the greatest allelopathic impact was observed with lovage and higher celandine residues (baličević et al., 2015). the allelopathic effect of extracts of roots and leaves of donor species (lonicera maackii, ranunculus ficaria, and alliaria petiolata) on recipient species (elymus hystrix, anemone virginiana, and blephilia hirsuta) was studied, and it was found that leaf extracts had more germination inhibition than root extracts and that increasing the concentration of extract also increased the effects. the leaf extract of alliaria petiolata had the highest inhibition of germination. lonicera maackii and ranunculus ficaria extracts reduced the germination of anemone virginiana and blephilia hirsute, while lonicera maackii and ranunculus ficaria leaf and root extracts had minimal impact on elymus hystrix (cipollini et al., 2013). the seed germination and seedling growth of avena fatua, phalaris minor, rumex dentated, and amaranthus retroflexu is hindered by the allelopathic activity of methanolic extracts of ocimum basilicum, artemisia annua, and eucalyptus globulus (mekky et al., 2008). the essential oil isolated from sweet basil and oregano populations inhibited barnyard grass and common lamb’s quarters’ germination and root development (vasilakoglou et al., 2007). these results implied that aromatic plants capable of producing phytotoxic essential oils may be important for controlling weeds in sustainable agricultural systems, whether applied as green manure or as mulch (dhima et al., 2009). the acetone extracts (n-hexane soluble fraction, acetone soluble fraction, and water soluble fraction) of the shoot of azadirachta indica prevented the germination and development of roots and shoots of digitaria sanguinalis, cirsium arvense, amaranthus rotundus, lactuca sativa, sinapis arvensis, and lolium ultiforum. the watersoluble fractions had the highest inhibitory activity in all bioassays. as the extract concentration increased, germination and root and hypocotyl growth were significantly reduced. allelochemicals may be present in all three fractions, based on the test plants’ concentration-dependent responses, with the water-soluble fraction showing the most promise. these findings suggested that azadirachta indica residue or aqueous extract might effectively control weeds (ashrafi et al., 2009). the phenolic chemicals found in the fruits of artemisia herba-alba have the capacity to hinder the seedling growth and germination of anabasis setifera (modallal & al-charchafchi, 2006). the azadirachta indica (neem) severely suppresses the development and germination of numerous crops, including bean (vigna angularis), alfalfa (medicago sativa), radish (raphanus sativus), carrot (daucus carota), sesame (sesamum indicum), and rice (oryza sativa). the aqueous extract of eucalyptus camaldulensis could inhibit the development of echinochloa crusgalli, rumex acetosella, and avena fatua (moradshahi et al., 2003). similarly, in a bioassay experiment, assaeed (2003) found that the water extract of artemisia monosperma leaves and flowers reduced seed germination of some plant species from sandy areas, including pennisetum divisum, lasiurus scindicus, scrophularia hypericifolia, and plantago boissieri. the aqueousacetone extract of melissa officinalis decreased the germination and growth of seeds and seedlings in lepidium sativum, amaranthus caudatus, phleum pratense, digitaria sanguinalis, lolium multiflorum, and lactuca sativa. it was stated that the efficiency of the extract on the roots is larger than that on the shoots of the test plants and that the inhibitory effect is proportional to the extract concentration (katonoguchi, 2001). an aqueous extract of artemisia herba-alba had an inhibitory impact on the germination percentage of seeds of helianthemum squamatum escudero et al. (2000). such inhibitors significantly slow or impair the development, production, and physiology of the agricultural plants linked with them (el-darier & youssef, 2007). interaction of allelochemicals in the ecosystem several aspects, such as the physicochemical and physiological transformations of volatile organic compounds and water-soluble phytochemicals into the soil matrix, necessitate more innovative experimental approaches. reference models are also needed to understand how plants naturally interact with their biotic and abiotic surroundings across various ecosystems, including crop ecosystems. conventionally, the hunt for novel herbicides with several modes of action and little risk of resistance vashishth et al. ● exploring the applications of allelopathy in agriculture: a review biologica nyssana ● 16 (2) december 2025: vashishth et al. ● exploring the applications of allelopathy in agriculture: a review biologica nyssana ● 16 (2) december 2025: development is part of the strategy to leverage these powerful synergies for comprehensive weed control (i.e., crop rotation, intercropping, green manuring, or using plant residue) (serino et al., 2021). the fate of allelochemicals in nature and the complexity of the multiple and multiscale interactions can be explained using the example of the study of pardo-muras (2022), in which the efficiency of cytisus scoparius leaf in controlling weeds, and likely that of other allelopathic plants, depends on intricate, many synergistic interactions involving soil-preferred volatile and water-soluble allelochemicals. the allelopathic activity of artemisia species varied with extract concentration. with an increase in extract concentration in the soils, both the chlorophyll content and biomass production rapidly decreased. soil obtained beneath plants of various artemisia genera or from contaminated areas (the area around the treated area) inhibited the growth and germination of a range of test species. the allelopathic activities of artemisia spp. have been related to the phenolic compounds and a few flavonoids found in aqueous preparations (kong et al., 1999). several published studies show varying levels of organic carbon, fertility, and nutrient content across vegetation diversity (adiputra, 2022). complex agroforests increase soil organic carbon (saputra et al., 2020), while matos et al. (2020) reported that litter quality supports soil restoration. according to the findings, soil fertility and organic carbon levels increase with plant population diversity. the interaction of allelochemicals and both inorganic and biological materials between plants and the soil environment is a complex process (fig. 5). herbicides are harmful to plants and soil quality because of their direct action on the soil surface. allelopathy in the sorghum crop significantly impacted weeds and improved soil quality. ullah et al. (2022) observed that the water extract of sorghum residue can suppress weed growth. implementing sorghum residue may improve weed control, soil health, and seed yield of spring-planted mung beans. future research should concentrate on how micronutrients interact with one another in the soil environment when using various allelopathic weed control methods in the working environment. furthermore, there are yet problems with managing weeds using different allelopathic techniques and with monitoring the allelopathic relationship between the treated crop and crop leftovers. conclusion and future aspects in conclusion, this review sheds light on the complex world of allelopathy, in which plants communicate with their neighbours via allelochemicals to influence them. the complex interplay between allelopathy and crop plants and weeds underscores its promise as a natural tool for sustainable weed management and increased crop output. furthermore, the destiny of allelochemicals in soil highlights the complexities of allelopathic interactions in ecosystems. this review gives an in-depth look into allelopathy’s potential to influence future agricultural practices and ecosystem management. further avenues of study emerge fig. 5. cycle of allelochemicals, organic and inorganic matter between soil and plant species (scavo et al., 2022) vashishth et al. ● exploring the applications of allelopathy in agriculture: a review biologica nyssana ● 16 (2) december 2025: from this review. mechanistic research could delve deeper into the specific molecular mechanisms by which allelochemicals affect plant physiology and signalling. understanding the genetic basis of allelopathy, as well as the potential to develop crop varieties with improved allelopathic traits, could revolutionise weed management tactics. exploring the ecological dynamics of allelopathy across various contexts, as well as its interactions with other factors such as climate change and soil health, offers significant potential. using modern approaches such as metabolomics and molecular profiling could yield new insights into allelochemical diversity and relationships. finally, as knowledge progresses, the potential for allelopathy to revolutionise agricultural practices, improve weed management, and contribute to ecological sustainability remains a viable route for future research and innovation. acknowledgments: the author wishes to thank santosh arya, shiv kishore, and manoj singh for their support. references abrahim, d., braguini, w. l., kelmer-bracht, a. m., & ishii-iwamoto, e. l. 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(2001). effects of alfalfa pellets on weeds. journal of crop production, 4(2), 303–312. microsoft word 0304_nesic_et_al biologica nyssana 1 (1-2) december 2010: 65-69 nešić, m. et al. total content of organic acid in plants… 65 original article ! total content of organic acids in plants from fire affected forest marija nešić1*, marija marković1, radmila trajković2, dragana pavlović3, marija ilić4, violeta mitić4, vesna stankov-jovanović4 1 university of niš, faculty of science and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia 2 faculty of science and mathematics, university of kosovska mitrovica, serbia 3 faculty of science and mathematics, university of kragujevac, department of biology and ecology, radoja domanovića 12, 34000 kragujevac, serbia 4 university of niš, faculty of science and mathematics, department of chemistry, ćirila i metodija 1, 18000 niš, serbia *e-mail: nesic_marija@yahoo.com abstract: nešić, m., marković, m., trajković, r., pavlović, d., ilić, m., mitić, v., stankov-jovanović, v.: total content of organic acids in plants from fire affected forest. biologica nyssana, 1 (1-2), december 2010: 65-69. in 2007 catastrophic fire on vidlič mountain had been occurred. it had been burned down nearly 1000 hectares of forest. that year vegetation was totally destroyed. ecosystems affected by fire are those with great changes in variety of ecological parameters and they can recover by natural succession. post fire areas are being occupied by pioneer plants which start one natural cycle. total organic acids content in plants from fire affected forest was studied and the same plant species from forest which had not been affected by fire were taken as a control. total organic acids content for all plants from forest affected by fire was higher than for plants from fire non affected forest except one plant (aegopodium podagraria). key words: organic acids, forest affected by fire, pioneer plants, vidlič mountain introduction ! fire is one of the basic natural forces that influence plant communities over evolutionary period of time. certain plant communities require periodic fires to maintain their position in the ecosystem (m u t c h 1970). according to hypothesis of m u t c h (1970) fire-dependent plant communities burn more readily than fire-nondependent communities, because natural selection has favored development of characteristics that make them more flammable. but, not every forest fire is consequence of natural disturbance. some of them are appeared after human activity and have more sever consequences on landscape and ecosystems. one of such had been appeared on vidlič mountain near pirot town in june 2007 and had last for 10 days and almost 1000 hectares of forest were burnt down. after expiration of fire only grate amount of dust was remained. forest fires create the conditions which are favorable for certain plant species that were not present in that ecosystem earlier or which were presented but with very few number, and they are known as pioneer plants. these plants are specific because they face with many unfavorable ecological factors such as: high illumination, high temperature, low moisture, increased evaporation and finally significant changes in soil composition. fire affected landscapes are very specific according their ecological factors and soil compositions. so, it is very interesting to examine 10th sfses • 17-20 june 2010, vlasina lake1 (1-2) • december 2010: 65-69 biologica nyssana 1 (1-2) december 2010: 65-69 nešić, m. et al. total content of organic acid in plants… 66 biochemical and physiological parameters in plants which inhabit that kind of landscapes. the total amount of organic acids in pioneer plants from fire affected forest has been investigated, taking into consideration their importance as indicators of physiological status of plants and effects of ecological factors on them. material and methods the plant material had been collected one year after forest fire. four plant species have been chosen. control group of plants was collected in the same time but from forest which hadn’t been affected by fire. tested plants were: fagus moesiaca (k.malỳ) czeczott, tussilago farfara l., doronicum columnae ten. and aegopodium podagraria l.. the method by p l e s h k o v (1985) was applied for determination of total organic acids content. accurately weighted mass (20.00 g) of fresh plant material was extracted in blender with 150 ml of distillated water, transferred into erlenmeyer flask and heated in water bath at 70ºc for 30 minutes. after that extract was filtered and total volume of filtrate was adjusted to 200 ml. the filtrate aliquot of 50 ml was transferred into erlenmeyer flask and titrated with 0.1 mol/l standard solution of naoh. the titration endpoint was registered potentiometrically. acidity was calculated by formula: x=axtx200x10/nx50 where: x=content of acids in analyzed solution in mekv/g of fresh plant material, a = volume of 0.1 mol/l standard solution of naoh used for titration in ml, t = factor of 0.1 mol/l standard solution of naoh, 200 = total filtrate volume in ml, 10 = factor for calculating mekv of acids n = mass of plant material used for extract preparation, 50 = volume of filtrate aliquote results and discussion organic acids are secondary metabolites, produced by degradation of carbohydrates, fats and proteins and mostly include: oxalic, formic, citric, fumaric, malic, succinic, acetic and phosphoric acids as well as many others which arise in lower amount (r i v a s s e a u et al., 2006). accumulation of some specific organic acids in plants is in relationship with their enzymatic reactions, growing and development. also, their abundance is under effect of different ecological factors. biochemical function of organic acids is dependent on different ecological conditions. they are donors of protons in some oxido-reduction reactions, for example -conversion of malic to oxalic acid. some earlier studies showed that content of organic acids in plants is dependent on respiration, transpiration, various biochemical processes in plants, plant’s phenophase (g a š i ć , 1992). fig. 1. doronicum columnae ten. fig. 2. aegopodium podagraria l. biologica nyssana 1 (1-2) december 2010: 65-69 nešić, m. et al. total content of organic acid in plants… 67 fig. 3. total content of organic acids in some plant species (ap c= aegopodium podagraria control; ap t= aegopodium podagraria test plant; tf c= tussilago farfara control; tf t= tussilago farfara test plant; fm c= fagus moesiaca control; fm t= fagus moesiaca test plant; dc c= doronicum columnae control; dc t= doronicum columnae test plant) obtained results indicate that the content of organic acids in plants are different in analyzed plant species but, in general, they are higher for plants from fire affected areas than in control plants. the only exception is aegopodium podagraria which has lower total content of organic acids compared with control plant and it is 0.05 mekv/g of fresh weigh in test plant and 0.162 mekv/g of fresh weigh (tab. 1). the highest total content of organic acids in tested plants compared with control had been found in f. moesiaca and it is 115.38% in comparison to the control. the content total content of organic acids in plants from post fire area was 0.270 mekv/g, while in control plants that amount was 0.234 mekv/g (tab. 1) common beech is perennial plant and seedlings which were examined were one year old shoots. acorns had been produced in the same year when forest fire had occurred or one year before that. they had been exposed to heat, fire, fume, dust, high illumination and low humidity which are unfavorable conditions for germination and development of seedlings and so they can be considered as stressed. in those situations plants activate defense mechanisms which are numerous. some of them are species specific, while the others are common for huge number of species (trajković et al., 2007). plant’s organic acids have very important role in plant detoxification caused by heavy metals (j o n e s , 1998; m a , 2000; m a et al., 2001). the lowest level of total organic acids had been found in a. podagraria, and it was supposed that it has different defense mechanisms as well as specific morphological characteristics that are very important for adaptations and survivor in stress conditions (t r a j k o v i ć , 1995; t r a j k o v i ć et al., 2007). in d. columnae the total content of organic acids was 0.202 mekv/g in tested plants and 0.189 mekv/g in control plants. in t. farfara the content in tested plants is 0.108 mekv/g and 0.103 mekv/g in control plants. it had been established that content of organic acids can vary in underground and up ground plant organs in the same species (t r a j k o v i ć , 1995; t r a j k o v i ć et al., 2007). they are of different anatomy, morphology and defenses mechanisms which are genetically caused. it was supposed that plant organs have different activities, different needs and different active processes. analysis of soil indicates that soil which was affected by fire was mineralized and enriched with mineral elements (tab. 2) and some of them are not favorable for plant grow and development (j o n e s , 1998). organic acids are needed for detoxification from heavy metals. they make helate complexes with them and enable their circulation in plant. in the same time that complexes are accumulated in plant organs which later can be excreted (j o n e s , 1998; m a , 2003; a r n e t o l i et al., 2008.). on the other hand it had been demonstrated that iron deficiency causes a substantial accumulation of organic acids in root tissues and also includes a large increase in h+ and organic acids excretion (d e v o s et al., 1986; g u e r i n o t & y i , 1994; o h w a k i & s u g a h a r a , 1997). except that, deficiency of phosphorus (h o f f l a n d et al., 1989; j o h n s o n et al., 1996; d a k o r a & p h i l l i p s , 2002) or elevated concentrations of al3+ ions lead to increasing exudation of organic acid anion in some species (l i et al., 2002; p i r e n o s et al., 2002). it is reported that higher content of organic acids have plants which are tolerant to particular metal (d e l h a i z e et al., 1993; b a s u et al., 1994). conclusion according all this facts and results which were presented one conclusion is irrevocably: synthesis of organic acids is one of the major defense mechanism in plant reaction on stress conditions. it is known that high concentrations of heavy metals cause increase content of organic acids biologica nyssana 1 (1-2) december 2010: 65-69 nešić, m. et al. total content of organic acid in plants… 68 table 1. comparison of total content of organic acids in some plants from post fire region from vidlic mountain with control plant group (mekv/g fresh weigh) plant species control test plant % compared with control aegopodium podagraria l. 0.162 0.050 30.86 tussilago farfara l. 0.103 0.108 104.85 doronicum columnae ten. 0.189 0.202 106.88 fagus moesiaca (k. malý) czeczott 0.234 0.270 115.38 table 2. total content of heavy metals in soil from fire affected and fire non affected forest areas metal ( ppm) plant species pb cd cu zn fe fr om fi re af fe ct ed so il a. podagraria 113.12 0.21 15.78 50.93 9893.64 f. moesiaca 1.94 0 3.92 0 7367.97 t. farfara 202.79 3.545 35.25 130.14 12840.17 d. columnae 146.23 1.89 25.91 56.22 5236.11 fr om no n fir e af fe ct ed so il a. podagraria 30.92 0 13.60 18.10 10764.98 f. moesiaca 14.18 0 18.94 0 16813.46 t. farfara 10.14 2.29 12.32 0 10687.54 d. columnae 34.33 3.50 16.07 37.01 14401.91 in plants (v e r g a n a & g a b r i e l l i , 1987; t r a j k o v i ć , 1995; t r a j k o v i ć et al., 2007). plants on fire affected forest region are high adaptive plants and have mechanism for survival. they can change environment and make favorable conditions for natural succession and invasion of 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article fatma alan ankara universty, kalecik vocational school, department of plant and animal production, ankara, türkiye falan@ankara.edu.tr (corresponding author) ayşen melda çolak uşak university, faculty of agriculture, department of horticulture, uşak, türkiye civan çelik isparta university of applied science, agriculture faculty, department of agricultural biotechnology, isparta, türkiye received: may 08, 2025 revised: june 04, 2025 accepted: june 09, 2025 abstract: date vinegar is a traditional functional food that, thanks to the rich nutritional content of dates and the bioactive compounds formed through natural fermentation, exhibits beneficial effects, particularly on the digestive system, metabolism, and metabolic health. traditional vinegar production stands out as an alternative source, as the fruits used in the process have a high natural sugar content and rich nutritional profile, making it a natural and economical healthsupporting product. in this study, the production method and phytochemical profile of date vinegar were thoroughly examined, including total phenolic content, total flavonoid level, dpph antioxidant capacity, and organic acid composition through hplc analysis. the results revealed that date vinegar contains a high amount of antioxidant compounds (total phenolic content of 429.00 mg gae l⁻¹, total flavonoid content of 302.00 μg qe ml⁻¹), and exhibits a strong dpph free radical scavenging activity of 89%. in terms of organic acid composition, tartaric acid (556.33 mg 100 g⁻¹) and oxalic acid (354.33 mg 100 g⁻¹) were detected at the highest concentrations. the presence of these organic acids contributes not only to the taste of the vinegar but also to its positive biological effects on health. the findings indicate that date vinegar has a complex and diverse phytochemical structure and possesses significant potential as a functional food due to its strong antioxidant capacity. moreover, these results are in parallel with similar studies reported in the literature, supporting the use of date vinegar as a traditional product with health-promoting biological activities. in conclusion, the functional properties and rich organic acid content of date vinegar make it a valuable natural healthsupporting and functional food product. key words: phoenix dactylifera l., date vinegar, functional foods, fermentation, antioxidant, health effects apstrakt: određivanje i procena nekih osnovnih fitohemijskih svojstava urminog sirćeta urmino sirće je tradicionalna funkcionalna hrana koja, zahvaljujući bogatom nutritivnom sastavu urmi i bioaktivnim jedinjenjima nastalim prirodnom fermentacijom, pokazuje povoljne efekte, naročito na digestivni sistem, metabolizam i metaboličko zdravlje. tradicionalna proizvodnja sirćeta ističe se kao alternativa, jer plodovi korišćeni u procesu imaju visok prirodan sadržaj šećera i bogat nutritivni profil, čineći ga prirodnim i ekonomičnim proizvodom pozitiivnim po zdravlje. u ovoj studiji detaljno su ispitani način proizvodnje i fitohemijski profil urminog sirćeta, uključujući ukupni sadržaj fenola, ukupan nivo flavonoida, dpph antioksidativni kapacitet i sastav organskih kiselina putem hplc analize. rezultati su pokazali da urmino sirće sadrži visoku količinu antioksidativnih jedinjenja (ukupni sadržaj fenola 429,00 mg gae l⁻¹, ukupni sadržaj flavonoida 302,00 μg qe ml⁻¹) i pokazuje snažnu sposobnost hvatanja slobodnih radikala od 89%. što se tiče sastava organskih kiselina, najviše su prisutne vinska kiselina (556,33 mg na 100 g) i oksalna kiselina (354,33 mg na 100 g). prisutnost ovih organskih kiselina doprinosi ne samo ukusu sirćeta, već i njegovim pozitivnim biološkim efektima na zdravlje. nalazi ukazuju da urmino sirće ima složenu i raznovrsnu fitohemijsku strukturu i poseduje značajan potencijal kao funkcionalna hrana zbog snažnog antioksidativnog kapaciteta. štaviše, ovi rezultati su u skladu sa rezultatima sličnih publikovanih literaturnih podataka, podržavajući upotrebu urminog sirćeta kao tradicionalnog proizvoda sa zdravstveno-promotivnim biološkim aktivnostima. u zaključku, funkcionalna svojstva i bogat sadržaj organskih kiselina čine urmino sirće vrednim prirodnim proizvodom koji podržava zdravlje i funkcionalnom hranom. ključne reči: phoenix dactylifera l., urmino sirće, funkcionalna hrana, fermentacija, antioksidans, zdravstveni efekti © 2025 alan et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 200 introduction date palm (phoenix dactylifera l.) has been cultivated in hot climates and arid and semiarid regions, especially in the middle east and north africa, for 5,000 years. it has always played an important role in these regions' economic and social life and is considered one of the most important fruits of the arabian peninsula. date palm (phoenix dactylifera l.), from the arecaceae (palmae) family, is a dioecious fruit-bearing tree. the arecaceae family contains more than 200 genera and 2500 species. it is a functional fruit species that attracts attention with its rich carbohydrate content, vitamins (especially b group vitamins), minerals (potassium, magnesium, iron), and high phenolic compound content (al-bulushi et al., 2017; hamden et al., 2022; barakat & alfheeaid, 2023; pepe et al., 2024; çolak & alan, 2025). while a low-quality date with dark fruit color, small, rotten, and undesirable taste cannot be marketed, its high sugar content strengthens its use as a raw material in the production of various value-added products such as date syrup, date paste, liquid sugar, and vinegar (mohamed & fennir, 2017). food safety, healthy nutrition, and sustainable food production are among the issues that are becoming increasingly important today. therefore, functional foods obtained by traditional fermentation techniques have become the focus of attention again due to their contributions to health and natural production methods. vinegar is an important fermented product used as both a food additive and a natural treatment tool for centuries. while fruits and vegetables with high sugar or starch content are generally used in vinegar production, in recent years, tropical fruits with high nutritional value, such as dates, have also begun to be evaluated in this field (al-farsi & lee, 2008). it is a liquid product containing alcohols, acids, esters, aldehydes, and ketones, the main volatile component of which is acetic acid, which gives vinegar its sour taste and defines it as vinegar (ho et al., 2017). dates constitute a suitable carbon source substrate for alcohol fermentation in vinegar production since they contain 70-80% natural sugar (glucose, fructose, and sucrose) (sahari et al., 2014). date vinegar, which is obtained by subjecting date juice to alcoholic fermentation with yeast species such as saccharomyces cerevisiae and then to a second fermentation with acetic acid bacteria such as acetobacter aceti, attracts attention due to both its sensory and functional properties (ali et al., 2018). historically, numerous plant species have been studied and used in treating or preventing various diseases. factors such as the increase in population, insufficient drug supply, side effects of various synthetic drugs, and the emergence of new diseases due to resistance to currently used drugs have led to an increasing emphasis on the use of plant materials as a source of medicine for a wide variety of human ailments (siddiqui, 2011). thus, palm vinegar, which can exhibit antimicrobial, antioxidant, hypoglycemic, and cholesterol-lowering effects thanks to the organic acids (especially acetic acid), flavonoids, phenolic compounds, and volatile compounds it contains, is also reported to have positive effects on the digestive system, balancing stomach acidity, and supporting intestinal microbiota (al-hooti et al., 2002). palm vinegar, which has a wide range of uses in traditional medicine from stomach disorders to detoxification, is currently being studied, with scientific studies supporting this traditional knowledge, and it especially reveals its potential as a functional food and natural preservative due to its high antioxidant capacity (al-hilfy et al., 2020). this article compiles the production process, chemical components, and potential effects of date vinegar on health in accordance with the existing literature and evaluates its potential for use as a functional food. materials and methods materials in this study, a sample of date (phoenix dactylifera l.) vinegar, which was fermented at home using traditional methods, was used. this vinegar is not widely available commercially but is available through online sales platforms. vinegar samples were stored in the refrigerator at +4 °c until the analysis day. all analyses were carried out using standardized methods in a laboratory environment. in the study, the phytochemical properties of date vinegar produced from tropical fruit species were investigated. the applied analysis methods are explained in detail below. method vinegar installation the production of date vinegar used in this study used the traditional method (akarca et al., 2020; öztürk, 2022). after the supplied fruits were washed, the date fruits were dried without any pretreatment, and their seeds were removed. the fruits prepared for vinegar production were added to 5 l glass jars at a rate of one-third of the jar volume. in addition, 0.5% honey was added to each sample to assist the fermentation process. following this process, the jars were filled using spring water taken from its natural source, and the jars were closed with a cheesecloth. the prepared samples were left to ferment at room biologica nyssana ● 16 (1) june 2025: 199-206 alan et al. ● determination and evaluation of some basic phytochemical properties of date vinegar 201 biologica nyssana ● 16 (1) june 2025: 199-206 alan et al. ● determination and evaluation of some basic phytochemical properties of date vinegar temperature and in a dark environment by utilizing the natural microbiota of the fruits, and the samples were mixed every day during the fermentation period. the fermentation process was carried out for 60 days, during which a sufficiently large vinegar mother was formed on the surfaces of the jars (approximately 0.5 cm). following fermentation, the vinegar samples were filtered and transferred to new glass jars to separate the fruit pulp. following this process, the jars were closed tightly, and the vinegar samples were stored in a dark environment at 25 °c until analysis was performed. determination of total phenolic content this study used the folin-ciocalteu method to determine the total phenol content (tpc) (kähkönen et al., 1999). a 200 μl volume of vinegar samples, obtained by the conventional method, and 500 μl of folin-ciocalteu reagent (diluted 10-fold with water) were added to a 10 ml test tube. the solution was left in the dark for 5 min before adding 1000 μl of sodium carbonate solution (7.5% w/v in water). the tubes were closed, shaken, and then left in the dark for 1 h again. uv spectrophotometric analyses for all absorbance measurements were carried out in a double-beam spectrophotometer at 765 nm using 1.0 cm quartz cells. they were compared with the gallic acid calibration curve. the results were expressed as mg gae l–1. each experiment was carried out in triplicate. determination of total flavonoid content the total flavonoid content of vinegars was determined by the aluminum chloride colorimetric method (chang et al., 2002). briefly, a 50 μl vinegar sample was placed in a 10 ml test tube. it was mixed with 950 μl of methanol and 4 ml of distilled water, then with 300 μl of sodium nitrite solution (5% in water). after incubation, 300 μl of aluminum chloride solution (10% in water) was added, and the mixture was left to stand for 6 min. then, 2 ml of sodium hydroxide solution (1 m, in water) was added, and the mixture was adjusted to a final volume of 10 ml with distilled water. the mixture was left to stand for 15 min. uv spectrophotometric analyses were carried out on a double-beam spectrophotometer at 510 nm using 1.0 cm quartz cells for all absorbance measurements. total flavonoid content was calculated from the quercetin calibration curve, and the result was expressed as mg quercetin equivalent per liter. dpph (2,2-diphenyl-1-picrylhydrazyl) method the dpph (2,2-diphenyl-1-picrylhydrazyl) experiment was performed by thaipong et al. (2006) with minor modifications. dpph stock solution: 24 mg dpph was weighed and dissolved in methanol. it was transferred to a 100 ml flask, and the volume was completed with 100 ml of methanol. it was stored at -18 °c until use. the working solution was obtained by mixing 20 ml of the stock solution with 90 ml of methanol. an absorbance value of 1.1 +/ 0.02 was obtained at 515 nm using a spectrophotometer. three hundred μl of the vinegar sample was filtered and taken into a tube, and then 5700 μl of the dpph working solution was added and mixed. it was allowed to react for one hour in a dark place. then, the absorbance of this solution was measured at 515 nm on a spectrophotometer. antioxidant activity was calculated as the decrease in absorbance value using the following formula: antioxidant activity (%)=(a0 -a1)/a×100 where a0 is the absorbance value of the control solution without a sample. a1: absorbance of the mixture containing the sample. organic acid analysis vinegar samples were homogenized with heidolph silent crusher m (heidolph, germany) and mixed on a shaker (heidolph unimax 1010, germany) for 1 h, and then centrifuged at 14,000 rpm for 15 min. the supernatant was filtered through a 0.45 μm membrane filter (millipore millex-hv hydrophilic pvdf; millipore, usa). this separation was carried out using a reverse-phase ace-c18 column (4 mm x 150 mm, 5 μm). the mobile phase was 10 mm potassium dihydrogen phosphate aqueous solution adjusted with ortho-phosphoric acid. the mobile phase was vacuum filtered through a 0.45 μm nylon filter and degassed online with a micro vacuum degasser. chromatographic separation of these compounds was carried out at room temperature. the analysis was carried out at a flow rate of 1 ml/ min; the run time was 10 min. the detector for ascorbic acid was set at λ=245 nm and for other organic acids, the detector was set at λ= 210 nm. the injection volume was 10 μl (büyüktuncel et al., 2017). each experiment was performed three times, and the results were expressed in g l–1 fresh weight. quantification of organic acids and phenolic compounds by hplc-uv vinegar samples were first shaken for 1 h and then centrifuged at 14,000 rpm for 15 min. then, the supernatant was filtered using a 0.45 µm membrane filter. the filtered vinegar samples were analyzed with an agilent 1260 hplc instrument (agilent technologies, ca, usa) equipped with chemstation software, a quaternary pump, an autosampler, and a uv detector. 202 biologica nyssana ● 16 (1) june 2025: 199-206 alan et al. ● determination and evaluation of some basic phytochemical properties of date vinegar table 1. chromatographic conditions of hplc method parameters conditions mobile phase a: ultrapure water containing 0.1% acetic acid. b: acetonitrile containing 0.1% acetic acid. mobile phase flow rate 1.0 ml min−1 column ace-c18 (4.6 mm × 150 mm, 5 µm) column temperature 25 °c injection volume 10 µl run time 30 min. detection wavelengths 280 nm for syringic acid, protocatechic acid, and gallic acid. 225 nm for vanillic acid. 305 nm for p-coumaric acid. 330 nm for caffeic acid and chlorogenic acid. elution gradient time min. b% (volume) 0.00 8 3.25 10 8.00 12 15.00 25 15.8 30 25 90 25.40 100 30.00 100 organic acids were determined using an ace-c18 column (4 mm × 150 mm, 5 µm; hichrom ltd., theale, united kingdom). the mobile phase consisted of a 10 mm potassium phosphate aqueous solution (ph 2.2, with ortho-phosphoric acid) with a 1 ml/min flow rate. the injection volume was 20 µl, and the detector was set at 245 nm for ascorbic acid and 210 nm for all other organic acids (fu et al., 2015). an ace-c18 (4.6 mm × 150 mm, 5 µm; hichrom ltd., theale, united kingdom) column was used for the chromatographic separation of phenolic compounds. details of the chromatographic conditions are given in tab. 1. the mobile phase flow rate was kept at 1.0 ml min-1. mobile phase a was ultrapure water, while mobile phase b was acetonitrile; both contained 0.1% acetic acid. gradient conditions were 0–3.25 min, 8–10% b; 3.25–8 min, 10–12% b; 8–15 min, 12–25% b; 15–15.8 min, 25–30% b; 15.8–25 min, 30–90% b; 25–25.4 min, 90–100% b; 25.4–30 min, 100% b. the injection volume was 10 µl and the column temperature was kept at 25 °c. detection wavelengths were selected according to the wavelengths where the phenolic compounds to be analyzed had maximum absorption. syringic acid, protocatechuic acid, ferulic acid, ellagic acid, quercetin, and gallic acid were detected at 280 nm. caffeic acid was detected at 330 nm and p-coumaric acid was detected at 305 nm (wen et al., 2005). statistical analysis the data obtained were analysed using the minitab software package. basic descriptive statistics were calculated using the 'basic statistics' command. results and discussion this study summarizes the quantitative analysis results of various phytochemical components in date vinegar. these analyses were conducted to reveal the chemical composition of date vinegar and to characterize the product phytochemically. the study determined total phenolic and flavonoid contents, along with dpph activity reflecting antioxidant capacity; in addition, levels of different phenolic acids (protocatechuic, gentisic, caffeic, p-coumaric, and ellagic acids) and organic acids (oxalic, tartaric, acetic, and citric acids) were analyzed. the data indicate that date vinegar possesses a complex and rich phytochemical composition (tab. 2). the results showed that the total phenolic content was 429.00 mg gae l⁻¹, the total flavonoid content 203 biologica nyssana ● 16 (1) june 2025: 199-206 alan et al. ● determination and evaluation of some basic phytochemical properties of date vinegar table 2. phytochemical analysis results of date vinegar variable mean±stdev se mean±stdev coefvar minimum maximum total phenolic (mg gae l-1) 429.00±1.00 0.58±1.00 0.23 428.00 430.00 total flavonoids (μg qe ml-1) 302.00±1.00 0.58±1.00 0.33 301.00 303.00 dpph (%) 89.00±1.00 0.58±1.00 1.12 88.00 90.00 protocatechic acid (mg 100g-1) 6.10±0.08 0.05±0.08 1.31 6.02 6.18 gentisic acid (mg 100g-1) 56.20±0.14 0.08±0.14 0.25 56.06 56.34 cafeic acid (mg 100g-1) 1.50±0.22 0.13±0.22 14.67 1.28 1.72 coumaric acid (mg 100g-1) 1.82±0.16 0.09±0.16 8.84 1.67 1.99 ellagic acid (mg 100g-1) 9.20±0.15 0.09±0.15 1.63 9.05 9.35 oxalic acid (mg 100g-1) 354.33±14.98 8.65±14.98 4.23 342.00 371.00 tartaric acid (mg 100g-1) 556.33±14.64 8.45±14.64 2.63 543.00 572.00 acetic acid (mg 100g-1) 167.67±7.37 4.26±7.37 4.40 162.00 176.00 citric acid (mg 100g-1) 1.13±0.23 0.13±0.23 20.38 1.00 1.40 was 302.00 μg qe ml⁻¹, and the dpph activity was 89.00%. among the organic acids, the highest values were detected in tartaric acid (556.33 mg 100 g⁻¹) and oxalic acid (354.33 mg 100 g⁻¹), indicating that date vinegar possesses a rich and diverse phytochemical profile and suggesting that these components may have significant biological activities in addition to contributing to the product’s potential antioxidant capacity. the health benefits of the organic acids identified in this study were discussed in light of the literature and related to our own findings. hegazy et al. (2024) aimed in their study to determine the total phenolic content (tpc) and total flavonoid content (tfc) of four different commercial date-based vinegars marketed in saudi arabia (date, date and garlic, date and pomegranate, date and turmeric), reported that tpc values varied from 240.81 ± 34.71 to 2228.79 ± 81.24 mg gae l⁻¹ in different vinegar samples. it was noted that these high phenolic and flavonoid contents increased the antioxidant capacity of the vinegars. additionally, tests conducted on eight different bacterial strains emphasized the high antimicrobial potential of the vinegar samples. similarly, in our study, the presence of high phenolic and flavonoid contents and 89% dpph activity supports the fact that date vinegar possesses potent antioxidant and potential health-protective properties. akarca et al. (2020) determined the total phenolic content of vinegar produced by traditional methods from iranian mazafati dates as 231.37±44.44 mg gae l⁻¹. they also reported that the vinegar contains beneficial components for human health such as carotenoids, phytosterols, b-group vitamins, and phosphorus, which may contribute to the prevention of various chronic diseases. the phenolic and flavonoid levels detected in our study are higher compared to those in this study, which may be due to differences in date variety, production method, and geographical factors. this finding provides an important basis for considering date vinegar as a functional food. tang et al. (2024) reported that date vinegar has high total acidity and phenolic contents, with acetic, l-malic, and oxoglutaric acids being dominant. our data found tartaric and oxalic acids at the highest concentrations. this diversity of organic acids contributes both to the taste and aroma characteristics of the vinegar and its health benefits. for example, the antioxidant and antimicrobial effects of tartaric acid have been supported in the literature (kumar et al., 2018). in this context, our findings support the positive health effects of organic acids in date vinegar. boonsupa et al. (2023) reported that the antioxidant activity of vinegar produced from different date varieties in thailand was at the level of 24.96 mg ml⁻¹, emphasizing that these values correlated with the phenolic content in the vinegar. the 89% dpph activity observed in our study indicates that date vinegar possesses strong antioxidant activity. measuring antioxidant activity is critically important for determining the product’s capacity to neutralize free radicals and thus evaluating its potential health benefits. considering that oxidative stress plays a significant role in the development of chronic diseases, the antioxidant capacity of date vinegar offers functional properties that may support the prevention of such diseases (lobo et al., 2010). 204 biologica nyssana ● 16 (1) june 2025: 199-206 alan et al. ● determination and evaluation of some basic phytochemical properties of date vinegar nosratabadi et al. (2024) investigated the changes in microflora during fermentation and demonstrated that phenolic compounds increased while the ph value decreased as fermentation progressed. this supports the fact that date vinegar's chemical and biological properties are closely related to the fermentation process. the high phenolic contents obtained in our study indicate that appropriate fermentation conditions enhance the beneficial components in the vinegar. benhammadi et al. (2025) detailed the acidic ph, dry matter, and sugar contents of date vinegars; our findings are consistent with these data and provide information about the chemical stability and flavor profile of date vinegar. habiba et al. (2024) reported that vinegar obtained from wild date palm fruit exhibited high nutritional value, acidity, and phenolic content; additionally, it demonstrated strong free radical scavenging capacity. this confirms that date vinegar is rich in health-beneficial compounds. al-malki et al. (2023) stated that traditional date vinegars are safe regarding microbial contamination. similarly, our study suggests that healthy and hygienic production conditions contribute to preserving phenolic and antioxidant components. in conclusion, the phenolic compounds, flavonoids, and antioxidant values obtained in our study are consistent with many studies in the literature, with some being higher or at moderate levels than others. the main reasons for these differences include the type of date and its growing region, fermentation duration and conditions, vinegar production method, analytical techniques, and additives. conclusion date vinegar stands out as a functional food obtained through traditional production methods, with its rich chemical content and positive effects on health. date vinegar, prepared from fermented dried date fruit, is widely used due to its nutritional and health benefits, ability to act as an antimicrobial agent, and ability to add flavor to foods. although dates are produced in many countries and date vinegar has been consumed since ancient times, scientific studies on date vinegar have increased in recent years. in particular, the production techniques of date vinegar produced by natural fermentation methods should be standardized. additionally, its usability in industrial production, microbiological quality, shelf life, microbiological reliability, and bioavailability should be increased. in this direction, contributing to the academic literature by examining the production process, chemical composition and health effects of date vinegar in detail and developing both traditional and alternative functional food sources can make significant contributions to the functional food sector. references akarca, g., tomar, o., çağlar, a., & i̇stek, ö. 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(2005). a universal hplc method for the determination of phenolic acids in compound herbal medicines. journal of agricultural and food chemistry, 53(17), 6624–6629. https://doi.org/10.1021/jf0511291 harpke, d., kerndorff, h., raca, i., pasche, e.: a new serbian endemic species of the genus crocus (iridaceae). biologica nyssana, 8 (1), september 2017 biologica nyssana 8 (1)  september 2017: 07-13 harpke, d. et al.  a new serbian endemic species of the genus crocus… 7 original article received: 02 july 2017 revised: 18 july 2017 accepted: 06 september 2017 a new serbian endemic species of the genus crocus (iridaceae) dörte harpke1, helmut kerndorff2, irena raca3, erich pasche4 1 leibniz institute of plant genetics and crop research (ipk), gatersleben, germany 2 casa da eira, são romão, são bras de alportel, portugal 3university of niš, faculty of science and mathematics, department of biology and ecology, višegradska 33, niš, serbia 4 feldstraße, velbert, germany * e-mail: raca.irena@gmail.com abstract: harpke, d., kerndorff, h., raca, i., pasche, e.: a new serbian endemic species of the genus crocus (iridaceae). biologica nyssana, 8 (1), september 2017: 07-13. recent research within the genus crocus (iridaceae) let us doubt that crocus adamii gay from serbia represents the same taxon as c. adamii s. str. of the locus classicus in the caucasian mountains. the latter belongs to a group of crocuses, which is distributed from the anatolian diagonal, a mountain belt in inner anatolia, to iran and the caucasian mountains. to infer (i) if the serbian c. adamii represents a new species and (ii) its taxonomical and phylogenetic affiliation within the genus we combined morphological and molecular investigations. the results show the presence of a morphologically and molecularly differentiated lineages, which both share a close relationship e.g. to c. alexandrii, c. chrysanthus, and c. weldenii but not to c. adamii s. str., which indicates a new species. as a result, we here describe c. randjeloviciorum to honor the serbian botanists novica and vladimir ranđelović. key words: crocus, new species, serbia apstrakt: harpke, d., kerndorff, h., raca, i., pasche, e.: nova endemična vrsta roda crocus (iridaceae) u srbiji. biologica nyssana, 8 (1), septembar 2017: 00-00. skorašnja istraživanja u okviru roda crocus (iridaceae) dovela su u sumnju teoriju da crocus adamii gay iz srbije predstavlja takson istovetan c. adamii s. str. čiji je locus classicus na kavkazu. drugi navedeni takson pripada grupi šafrana, distribuiranih od planinskog pojasa u centralnom delu anatolije, do irana i kavkaskih planina. da bi se zaključilo o tome (i) da li takson c. adamii iz srbije predstavlja novu vrstu, te (ii) sudilo o njegovom taksonomskom i filogenetskom položaju unutar roda, kombinovana su morfološka i molekularna istraživanja. rezultati ukazuju na postojanje morfološki i molekularno diferenciranih linija, srodnim c. alexandrii, c. chrysanthus i c. weldenii, ali ne i c. adamii s. str., što sugeriše da je c. adamii iz srbije nova vrsta za nauku. kao rezultat, u ovom radu biće opisana vrsta c. randjeloviciorum, čiji je naziv dodeljen u čast srpskih botaničara novice i vladimira ranđelovića. ključne reči: crocus, nova vrsta, srbija 8 (1) • september 2017: 07-13 doi: 10.5281/zenodo.962907 biologica nyssana 8 (1)  september 2017: 07-13 harpke, d. et al.  a new serbian endemic species of the genus crocus… 8 introduction crocus l. (iridaceae) comprises currently about 200 species. within the last few years, over 50 new species were described (e r o l et al., 2012; r a n đ e l o v i ć et al., 2012; k e r n d o r f f et al., 2013; s c h n e i d e r , 2014; h a r p k e et al., 2014, 2015; e r o l et al., 2015; r u k s a n s , 2015; m i l j k o v i ć et al., 2016). taxonomically the genus is currently in a complicated situation as many of the newly described taxa are unplaced in the system. moreover, recent phylogenetic analyses proved several units within the genus crocus to be paraor polyphyletic. as a consequence of ongoing revisions of taxonomical groups, with the aim to define monophyletic units, several other taxa are unplaced, too. one of those groups of currently unplaced taxa comprises c. adamii gay and its allies (k e r n d o r f f et al., 2013). it is a very homogenous group and occupies the sister group position to a large clade comprising series aleppici b. mathew, flavi b. mathew, speciosi b. mathew, reticulati b. mathew (h a r p k e et al., 2016). the c. adamii group comprises about 20 taxa (k e r n d o r f f et al., 2013). the taxa of this group are characterized e.g. by toothless basal tunic rings (with the exception of c. aerius herb. which has no rings), silvery bract and bracteole (in some taxa they turn brown with aging), glabrous throat, trifurcate style and vernal flowers. the species of the c. adamii group are inhabitants of the anatolian diagonal, a mountain belt dividing inner anatolia, and occur north-, east-, and south-east of it. considering that despite extensive field studies c. adamii and its allies were never found west of the anatolian diagonal, the presence of c. adamii in serbia became questionable. the serbian plant was mentioned by r a n d j e l o v i ć et al. (1990), located in serbia in the regions of timok, niš, and south morava. from the photograph shown in this work it has obviously some affinity to c. adamii but also to some forms of c. nubigena (h a r p k e et al., 2016). furthermore, its chromosome number (2n) is 18, members of the c. adamii group from the mentioned eastern areas have mostly 2n = 20. material and methods morphological analyses leaf-parameters were measured on fresh material (45 specimens for number, 10 specimens for white stripe, diameter, colour of cataphylls, bract and bracteoles, and ribs underneath). corm and flower parameters were measured on 36 randomly collected and dried specimens from the type-locality hkep 1328. for the investigation of leave anatomy, samples were collected during flowering time (06.03.2016) and preserved in 50% alcohol. anatomical studies were done in the laboratory of plant systematics and ecology, faculty of science and mathematics, university of niš. thirty transverse leaf cross sections were made by manual microtome (g l i g o r i j e v i ć & p e j č i n o v i ć , 1983) and stained with safranin alcian blue. the slides with coverslips were then photographed using a leica dm 1000 microscope. nineteen anatomical features were obtained of leaf cross sections: section height, section length, arm length, white stripe width, lacuna area, adaxial epidermis cells height and width, palisade cells height and width, palisade tissue height, spongy cells height and width, spongy tissue height, abaxial epidermis cells height and width, sclerenchyma area, phloem area, xylem area and number of vascular bundles. measurements of the listed parameters were done in imagej. molecular and phylogenetic analyses molecular analyses were carried out using 3 individuals of one c. cf. adamii populations. extraction of genomic dna and amplification of the nuclear rdna its were conducted according to h a r p k e et al. (2014). both strands of the pcr products were directly sequenced with applied biosystems’ bigdye terminator technology on an abi 3730xl automatic dna sequencer using either the primers from pcr amplifications. forward and reverse sequences were manually checked, edited where necessary, and combined in consensus. the newly obtained sequence was submitted to the embl nucleotide database and is accessible through accession numbers mf766260. sequences were aligned manually. if sequences were found to be identical within the same species or population they were included only once in the analyses. the nuclear data were subjected to phylogenetic analyses using bayesian phylogenetic inference (bi) with mrbayes 3.2 (r o n q u i s t et al., 2012). for bi 2 times 4 chains were run for 2 million generations under the appropriate models of sequence evolution (nuclear data set: gtr+γ+i), sampling a tree every 1000 generations. converging log-likelihoods, potential scale reduction factors for each parameter and inspection of tabulated model parameters in mrbayes suggested that stationary had been reached in all analyses. the first 25% of trees of each run were discarded as burn-in. two independent runs of bi analysis were performed to confirm that separate analyses converged on the same biologica nyssana 8 (1)  september 2017: 07-13 harpke, d. et al.  a new serbian endemic species of the genus crocus… 9 result. in each of the 2 analyses the same topology and similar posterior probabilities (pp) of nodal support resulted. results and discussion affiliation within the genus. crocus cf. adamii from serbia groups within a polytomic clade comprising c. puncatus and its relatives with strong support (pp 1.0; fig. 1). its its sequence differs in at least three positions from its closest relatives c. alexandri (serbia), c. weldenii (ne-italy) and five from c. adamioides (nw-turkey). the seeds (fig. 2d) have more similarity with the latter species than to those of the relatives of the c. adamii-group (k e r n d o r f f et al. 2016). further morphological characters like corm tunics (fig. 2a, fig. 3) also clearly show that it does not belong to the c. adamiigroup. table 1. crocus randjeloviciorum leaf anatomy characters measurements height(µm) width (length)(µm) area(µm2) mean±sd min-max mean±sd min-max mean±sd min-max section 563±75 446-692 3154±311 2644-3668 arm 1393±170 1164-1726 white stripe 434±91 323-599 lacuna 133157±37620 70665-234620 adaxial e. cell 16±2 14-19 16±1 13-18 palisade cell 46±6 34-61 17±1 13-19 palisade tissue 68±11 51-90 spongy cell 18±2 14-22 26±3 21-33 spongy tissue 50±8 37-68 abaxial e. cell 16±2 12-20 18±2 13-22 sclerenchyma 5478±945 3022-7019 phloem 549±123 351-954 xylem 673±128 448-903 mean±sd min-max v. bundles no. 18±3 11-21 sd – standard deviation fig. 1. phylogenetic tree obtained by bayesian phylogenetic inference of the nuclear rdna its regions. numbers along branches give posterior probabilities. sequences obtained by cloning are indicated by a small letter. crocus randjeloviciorum is indicated in bold. biologica nyssana 8 (1)  september 2017: 07-13 harpke, d. et al.  a new serbian endemic species of the genus crocus… 10 description of new species the serbian crocus considered as c. adamii is phylogenetically and morphological quite distinct from c. adamii s. str.. it represents a new species. crocus randjeloviciorum kernd., pasche, harpke & raca sp. nova type: east serbia, tupižnica mt., 950m a.s.l., hkep 1328 (holotype 23042 gat!) diagnosis. crocus randjeloviciorum differs from other species by intermediate outer corm tunic between coriaceous and membranous (e.g. in c. biflorus it is coriaceous, in c. tauri membranous), the 2 ribs in the grooves of the leaves (none in c. biflorus, 2 in c. tauri), no dark spot at the basis of the outer segments, unusually long lobes of the anthers (1.7-3 mm) and very broad connectives. crocus randjeloviciorum has also a very rare property in the genus. the styles are compared to the stamens in more or less the same proportion. this means approximately in 1/3 of the specimens they are shorter, in 1/3 they are equal and in 1/3 they are longer. in the majority of species there is either a tendency to have longer or to have shorter styles compared to the stamens. description. corm sub-globose about 9-11 mm in diameter. outer and inner tunics coriaceous to membranous, the inner ones softer; tunic splits mainly into segments of 2-5 mm rarely into >5 mm, sub-splits absent. neck around 5 mm long consistent of medium-based “triangles”, caps membranous, medium-sized (k e r n d o r f f et al. 2015). rings narrow to normal-sized coriaceous to membranous mostly smoothedged, very rarely with tiny teeth <0.5 mm. basal tunics small plates of 5-7 mm in diameter. cataphylls silvery, skinny, sometimes slightly brownish towards tips. leaves 2 – 3.1 – 4, green, glabrous, 1.5-2.5 mm in diameter with two ribs underneath of both sides of the keel. leaves reach or slightly overtop the flowers at anthesis. white stripe <1/3 to 1/3 of leaf diameter. flowers 1(-2). the outer segments are between 21 and 30 mm but usually 25 mm long, between 6 and 14 mm mostly 9.5 mm wide. the inner segments are between 18 and 28 mm but usually 23 mm long and between 6 and 14 mm in average 10.2 mm wide. segment proportion of length/width of the outer segments is 2.6 (n = 36). the insides of all of segments are light to deep violet blue rarely whitish without markings, the inner ones also at outside with an indistinct brownish-greyish zone or spot near the basis, overlaid by the yellow of the throat which shines through. the outside of the outer segments has either the inside flower colour or can be white or yellowish (buff-coloured) dominated by different numbers of intense brownish-violet stripes, vertically orientated to the tips, occasionally accompanied by many thin ones also upwards orientated but more to the edges than to the tips of the segments, sometimes the whole outside is suffused deep violet. there is no dark blotch or area at the bases of the segments, the violet stripes merge into the perianth tube which is otherwise colourless (white) especially near the ground. prophyll absent. bract and bracteole present, subequal, mostly silverywhite and skinny but sometimes with brownish tips. filaments deep yellow, hairy, 4-5.2-6.5 mm long; anthers yellow 7-11.5-15.2 mm long, having remarkably long lobes of about 1.7-2-3 mm (n = 15), connective very broad and conspicuous, colourless. throat deep yellow, glabrous. pollen yellow. the orange to orange-red red styles are divided into three branches sometimes papillous in the upper part. the branches are 3.6-5.6-8.5 mm long mostly expanded and fringed towards at the apex. the styles are 29% shorter, 37% equal and 34% longer compared to the stamen. capsule small, about 1 cm long, broadly ellipsoid and peaky at top. seeds light brown with an orange tint, main body ellipsoid about 1.6 -2 mm long and 1.3-1.4 mm wide (fig. 2d), caruncle and raphe distinct. chromosome number 2n=18. fig. 2. photographs of corm tunics (a), hairy filaments (b, 9x), leaves with papillae (c, 9x) and seeds (d). biologica nyssana 8 (1)  september 2017: 07-13 harpke, d. et al.  a new serbian endemic species of the genus crocus… 11 distribution and habitat: serbia, in hornbeam-lilac scrub (syringo-carpinetum). etymology: prof. dr. novica ranđelović (1937–) and his son prof. dr. vladimir ranđelović (1965–) are among the most prominent serbian botanists. they extensively studied the flora of the balkan peninsula. the results of their investigations were published in numerous papers. they both are crocus enthusiasts and described several new crocus species and they published a monograph about serbian crocuses (r a n đ e l o v i ć et al., 1990) and authored and co-authored several publications about the genus (r a n đ e l o v i ć et al., 2007, 2011, h a r p k e et al., 2014, 2015, m i l j k o v i ć et al., 2016). phenology: flowering period from february to april. taxonomic relationships: crocus randjeloviciorum belongs to a group of crocuses comprising species e.g. from ne–italy (c. weldenii), the balkan peninsula (c. alexandri) and west turkey (c. adamioides). leaf cross sections leaf blade. leaf cross sections of crocuses are of a unique shape consisting of a central part called the “keel” and two lateral “arms”. the keel is squared, almost rectangular while the arms are re-curved towards the keel (r u d a l l & m a t h e w , 1990). keel corners, length and curving degree of arms vary between different species. the investigated crocus cf. adamii is characterized by four well defined ribs (fig. 4a) on the abaxial side of the keel and arms. v e l e n o v s k y (1907) typified crocus leaf type as bifacial. leaf surface. finger – like papillae can be found on the adaxial side at the end of the arms (fig. 4a). adaxial epidermis cells are square or rectangular, in contrast to the abaxial cells which are more oval. anomocytic stomata occurs in the abaxial epidermis (e r o l et al., 2007). mesophyll. the central part of the keel consists of round parenchyma cells, these cells are mostly broken down forming air space called the lacuna. it can fig. 3. photos of plants from the type population of crocus randjeloviciorum. biologica nyssana 8 (1)  september 2017: 07-13 harpke, d. et al.  a new serbian endemic species of the genus crocus… 12 be noticed as a central white stripe all along the leaf (e r o l et al., 2007; y e t i ş e n et al., 2013) (fig. 4a). the mesophyll of the arm consists of palisade (oriented to the adaxial side) and a spongy tissue below. the palisade parenchyma contains polygonal cells organized in two layers. two or three layers of the oval or irregular shaped cells are present in the spongy parenchyma (fig. 4b). vascular bundles. leaf cross sections have 11-21 collateral vascular bundles positioned in one row all along the mesophyll. four of these bundles are larger. two of them are located in distal parts of the arms and another two in the keel base near corners. the xylem is oriented to the adaxial and phloem to the abaxial side of the leaf (bundles in the end of the arms have opposite orientation of vascular tissues). the fig. 4. anatomic characters of leaves of crocus randjeloviciorum: a-leaf cross sections (5x), b-arm detail (20x), c-big bundle (40x) (ad: adaxial side, ab: abaxial side, la: lacuna area, r: rib, pp: papillae, e: epidermis, pp: palisade paremchyma, sp: spongy parenchyma, st: stomata, sc: sclerenchyma cap, ph: phloem, xy: xylem) table 1. crocus randjeloviciorum leaf anatomy characters measurements height(µm) width (length)(µm) area(µm2) mean±sd min-max mean±sd min-max mean±sd min-max section 563±75 446-692 3154±311 2644-3668 arm 1393±170 1164-1726 white stripe 434±91 323-599 lacuna 133157±37620 70665-234620 adaxial e. cell 16±2 14-19 16±1 13-18 palisade cell 46±6 34-61 17±1 13-19 palisade tissue 68±11 51-90 spongy cell 18±2 14-22 26±3 21-33 spongy tissue 50±8 37-68 abaxial e. cell 16±2 12-20 18±2 13-22 sclerenchyma 5478±945 3022-7019 phloem 549±123 351-954 xylem 673±128 448-903 mean±sd min-max v. bundles no. 18±3 11-21 biologica nyssana 8 (1)  september 2017: 07-13 harpke, d. et al.  a new serbian endemic species of the genus crocus… 13 sclerenchyma tissue is well developed and in a form of a “cap” (fig. 4c). measurements for leaf anatomy are given in tab. 1. acknowledgements. the work was funded by the ministry of education, science and technological development of republic of serbia (project no. 173030). references erol, o., can, l., şık, l. 2012: crocus demirizianus sp. nov. from northwestern turkey. nordic journal of botany, 30: 665-667. erol, o., harpke, d., yıldırım, h. 2015: a new crocus l. (iridaceae) species from se turkey, based on morphological and molecular data. phytotaxa, 239: 223-232. erol, o., küçüker, o. 2007: leaf anatomy of some endemic crocus l. (iridaceae) taxa from western anatolia. international journal of botany, 3 (3): 290-295. gligorijević, s., pejčinović, d. 1983: contribution to the methodology of anatomical sections sreparation. acta biologiae et medicinae experimentalis, 8: 43-45. harpke, d., kerndorff, h., pasche, e., peruzzi, l. 2016: neotypification of the name crocus biflorus mill. (iridaceae) and its consequences in the taxonomy of the genus. phytotaxa, 260: 131143. harpke, d., meng, s., kerndorff, h., rutten, t., blattner, f.r 2013: phylogeny of crocus (iridaceae) based on one chloroplast and two nuclear loci: ancient hybridization and chromosome number evolution. molecular phylogenetic and evolution, 66: 617-627. harpke, d., peruzzi, l., kerndorff, h., karamplianis, t., constantinidis, t., ranđelović, v., ranđelović, n., jušković, m., pasche, e., blattner, f.r. 2014: phylogeny, geographic distribution, and new taxonomic circumscription of the crocus reticulatus species group (iridaceae). turkish journal of botany, 38: 11821198. kerndorff, h., pasche, e. harpke, d. 2015: the genus crocus (liliflorae, iridaceae): life-cycle, morphology, phenotypic characteristics, and taxonomical relevant parameters. stapfia 103: 27–65. kerndorff, h., pasche, e., blattner, f.r., harpke, d. 2013: crocus biflorus miller (liliiflorae, iridaceae) in anatolia-part iv. stapfia, 99: 159186. mathew, b. 1982: the crocus. a revision of the genus crocus (iridaceae). b.t. batsford, london. miljković, m., ranđelović, v., harpke, d. 2016: a new species of crocus (iridaceae) from southern albania (sw balkan peninsula). phytotaxa, 265: 39-49. peruzzi, l., carta, a. 2011: crocus ilvensis sp. nov. (section crocus, iridaceae), endemic to elba island (tuscan archipelago, italy). nordic journal of botany, 29: 6-13. ranđelović, n., ranđelović, v., hill, d.a. 1990: the genus crocus l. in serbia. srpska akademija nauka i umetnosti , beograd. ranđelović. n., ranđelović, v., hristovski, n. 2012: crocus jablanicensis (iridaceae), a new species from the republic of macedonia, balkan peninsula. annales botanici fennici, 49: 99-10. ronquist, f., teslenko, m., van der mark, p., ayres, d.l., darling, a., hohna, s., larget, b., liu, l., suchard, m.a., huelsenbeck, j.p. 2012: mrbayes 3.2: efficient bayesian phylogenetic inference and model choice across a large model space. systematic biology, 61: 539-542. rudall, p., mathew, b. 1990: leaf anatomy in crocus (iridaceae). kew bulletin, 45 (3): 535-544. rukšāns, j. 2015: some new crocus taxa (iridaceae) from western turkey and east aegean islands. international rock gardener, 64: 1-38. schneider, i. 2014: crocus brachyfilus(iridaceae), a new species from southern turkey. willdenowia, 44: 45-50. velenovsky, j. 1907: vergleichende morphologie der pflanzen. teil ii prag yetişen, k., şen, u., yıldırım, t., özdemir, c. 2013: morphological and anatomical study on endemic crocus olivieri gay subsp. istanbulensis mathew subspecies (iridaceae). anadolu university journal of science and technology-c life sciences and biotechnology, 3 (1): 31-37. anticancer compounds from medicinal plants biologica nyssana 2 (2)  december 2011: 00-00 ljupković r.b. et al..  removal cu(ii) ions from water... 21 review article checklist of the genus bracon (hymenoptera: braconidae) in serbia vladimir žikić 1* , saša s. stanković 1 , marijana ilić 1 1 faculty of science and mathematics, department of biology and ecology, university of niš, višegradska 33, 18000 niš, serbia * e-mail: vzikic@yahoo.com abstract: žikić, v., stanković, s., ilić, m.: checklist of the genus bracon (hymenoptera: braconidae) in serbia, biologica nyssana, 3 (1), september 2012: 21-29. this is the first checklist of the genus bracon for the territory of serbia. here we present 80 species of this cosmopolitan genus of the subfamily braconinae which is one of the cyclostome braconid groups. we connected the parasitoids with their hosts where the data about them were available. there are 290 host species in total belonging to 39 families of four insect orders: diptera, coleoptera, hymenoptera and lepidoptera. key words: cyclostome braconidae, bracon, serbia, checklist introduction braconinae belongs to the cyclostome group of the family braconidae. this subfamily assembles ectoparasitoid wasps which lay their eggs on the host body. according to their biology, ectoparasitic braconid wasps belong to the group of idiobionts; which means that they do not permit the host to continue its development after oviposition (m a t h e w s , 1974). oviposition in this group is split in two acts: paralisation of the host, than laying eggs which is opposite in koinobions. as a cosmopolitan group of parasitoids they represent a large and powerful biological weapon (eg. b a i r d , 1958; l e w i s et al., 1990) against various pests from the following holometabolous insect orders: diptera, coleoptera, hymenoptera and lepidoptera. the species of the genus bracon show different degree of the host specificity, ranging from a strict specialization to only one host species, whereas other can be polyphagous, when parasitism occurs in more than a hundred hosts in different types of habitats and geographical areas (s h a w & h u d d l e s t o n , 1991). because of their important role in biological control, biology of some species is very well studied: for b. intercessor (van a c h t e r b e r g et al., 1990; g e o r g i e v et al., 2005), for b. brevicornis (t e m e r a k , 1982), or many publications for b. hebetor and its host plodia interpunctella (pyralidae) (r e i n e r t et al., 1971; t a y l o r , 1988a, b; y u et al., 1999; m i l o n a s , 2005; d w e c k et al. 2010). according to the fauna europaea (v a n a c h t e r b e r g , 2012), about 250 species of the genus bracon are recorded in europe. the present publication stemmed from the need to make the list of all registered bracon species on the territory of serbia after many years of investigation and also to connect the parasitoids with their hosts. investigation of the genus bracon in serbia in different aspects is presented in following publications by the authors b e l o k o b y s k i j & ž i k i ć (2009); b r a j k o v i ć (1989); b r a j k o v i ć et al. (1991, 1994); s t a n k o v i ć et al. (2010) and ž i k i ć et al. (2000, 2010, 2012). 3 (1) • september 2012: 21-29 biologica nyssana 3 (1)  september 2012: 21-29 žikić, v. et al.  checklist of the genus bracon (hymenoptera: braconidae)… 22 materials and methods the majority of analyzed specimens were taken from the collection of the faculty of biology, university of belgrade and the faculty of sciences and mathematics, university of niš, serbia. a part of specimens is deposited at the zoological institute ras (st. petersburg, russia) and the natural history museum of budapest, hungary. the data about braconid hosts are compiled using the available literature; the list of references is bellow. the basic key for the identification of the species we used is one by tobias et al. (1986), updated fauna europaea (http://faunaeur.org) and the database taxapad (y u et al. 2012). a lot of information about the hosts and the distribution of the investigated genus we collected from p a p p (1973, 1977, 2008) and b e y a r s l a n et al. (2005). results we present 80 species of the genus bracon registered on the territory of serbia. the data regarding parasitoids are connected with the adequate data about their hosts. the species of bracon are listed alphabetically according to their subgenera. for 25 braconid species there were no host records for serbia nor for other parts of europe. in addition, we give the geographical distribution of all registered parasitoids in continental part of europe and russia. bracon (bracon) alutaceus szepligeti 1901 – no host data. distribution: austria, great britain, bulgaria, cyprus, czech republic, denmark, germany, hungary, lithuania, poland, russia (northwestern), slovakia, spain, sweden, ukraine, serbia. bracon (bracon) crassiceps thomson 1892 – no host data. distribution: austria, finland, germany, hungary, russia (northwestern), sweden, switzerland, serbia. bracon (bracon) crassungula thomson 1892 – no host data. distribution: austria, hungary, italy, sweden, switzerland, serbia. bracon (bracon) filicornis thomson 1892 – no host data. distribution: austria, sweden, serbia. bracon (bracon) flagellaris thomson 1892 – no host data. distribution: croatia, germany, hungary, romania, russia (south), slovenia, sweden, switzerland, ukraine, serbia. bracon (bracon) fulvipes nees 1834 on apion minimum, cistapion cyanescens (apionidae); curculio villosus, mononychus punctumalbum (curculionidae); tetramesa brevicornis t. hyalipennis (eurytomidae); coleophora coronillae (coleophoridae); paranthrene tabaniformis (sesiidae). distribution: austria, belgium, great britain, bulgaria, croatia, turkey, finland, france, germany, greece, hungary, ireland, italy, latvia, lithuania, poland, russia (central and south), slovakia, slovenia, spain, sweden, switzerland, netherlands, serbia. bracon (bracon) grandiceps thomson 1892 on bembecia scopigera (sesiidae). distribution: austria, croatia, turkey, finland, france, germany, greece, hungary, italy, macedonia, poland, russia (south), slovenia, sweden, serbia. bracon (bracon) intercessor nees 1834 on apion opeticum (apionidae); agapanthia villosoviridescens (cerambycidae); anthonomus pedicularius, a. pomorum, a. sorbi, lixus brevirostris, l. incanescens, l. junci, l. scabricollis, microlarinus lareynii, m. lypriformis (curculionidae); rhynchites bacchus (rhynchitidae); tetramesa hyalipennis, t. rossica (eurytomidae); haplochrois theae (agonoxenidae); augasma atraphaxidellum (coleophoridae); synanthedon culiciformis, paranthrene tabaniformis (sesiidae); sparganothis pilleriana (tortricidae). distribution: albania, austria, great britain, bulgaria, croatia, cyprus, turkey, france, germany, greece, hungary, italy, latvia, lithuania, macedonia, norway, poland, romania, russia (central, east, northwestern and south), slovakia, slovenia, spain, sweden, switzerland, netherlands, ukraine, serbia. bracon (bracon) laetus wesmael 1838 on anthonomus pomorum, magdalis nitida (curculionidae); amblypalpis tamaricella (gelechiidae); argyresthia chrysidella, prays citri; prays oleae (yponomeutidae). distribution: belgium, great britain, bulgaria, croatia, cyprus, czech republic, france, germany, greece, hungary, spain, serbia. bracon (bracon) leptus marshall 1897 on metzneria lapella (gelechiidae). distribution: austria, great britain, bulgaria, croatia, cyprus, germany, greece, hungary, lithuania, macedonia, romania, russia (central and south), slovakia, slovenia, spain, switzerland, netherlands, serbia. bracon (bracon) longicollis wesmael 1838 on chlorops pumilionis (chloropidae). distribution: austria, belgium, great britain, bulgaria, croatia, cyprus, turkey, finland, france, germany, greece, hungary, italy, lithuania, macedonia, norway, poland, russia (central, northwestern and south), biologica nyssana 3 (1)  september 2012: 21-29 žikić, v. et al.  checklist of the genus bracon (hymenoptera: braconidae)… 23 slovenia, spain, sweden, switzerland, netherlands, serbia. bracon (bracon) longulus thomson 1892 on oscinella frit (chloropidae). distribution: austria, bulgaria, croatia, cyprus, czech republic, denmark, finland, france, germany, greece, hungary, italy, macedonia, norway, slovenia, spain, sweden, netherlands, serbia. bracon (bracon) luteator spinola 1808 on urophora solstitialis (tephritidae); metzneria aestivella, m. lappella (gelechiidae). distribution: austria, great britain, bulgaria, croatia, cyprus, turkey, france, greece, hungary, italy, latvia, macedonia, russia (central, northwestern and south), slovakia, spain, switzerland, ukraine, serbia. bracon (bracon) mariae dalla torre 1898 on chaetorellia jaceae (tephritidae). distribution: bulgaria, croatia, germany, hungary, italy, russia (south), slovakia, spain, sweden, switzerland, ukraine, serbia. bracon (bracon) mediostriatus papp 1968 – no host data. distribution: hungary, serbia. bracon (bracon) nigratus wesmael 1838 on chaetostomella cylindrica (tephritidae); coleophora millefolii (coleophoridae); grapholita compositella (tortricidae); zygaena loti (zygaenidae). distribution: austria, belgium, great britain, croatia, czech republic, france, germany, hungary, ireland, italy, lithuania, romania, russia (central and north), slovakia, slovenia, spain, sweden, switzerland, netherlands, serbia. bracon (bracon) pallicarpus thomson 1892 – no host data. distribution: great britain, bulgaria, finland, germany, hungary, italy, spain, sweden, serbia. bracon (bracon) pectoralis wesmael 1838 on chrysobothis affisinis, sphenoptera gossypii, s. lobicollis, s. montana (buprestidae); plagionotus arcuatus, plagionotus bobelayei (cerambycidae). distribution: austria, belgium, great britain, bulgaria, croatia, cyprus, turkey, france, germany, greece, hungary, italy, macedonia, romania, russia (central and south), slovakia, spain, switzerland, serbia. bracon (bracon) scabriusculus dalla torre 1898 – no host data. distribution: austria, germany, hungary, italy, romania, russia (central), spain, serbia. bracon (bracon) scutellaris wesmael 1838 on anobium abietis (anobiidae); euura mucronata, pontania pedunculi, p. viminallis (tenthredinidae). distribution: belgium, great britain, finland, france, hungary, ireland, italy, poland, sweden, netherlands, serbia. bracon (bracon) speerschneideri schmiedeknecht 1897 on argyresthia thuiella, ocnerostoma sp. (yponomeutidae). distribution: austria, great britain, france, germany, norway, sweden, switzerland, serbia. bracon (bracon) subglaber szepligeti 1901 on noeeta pupillata, tephritis leontodontis, terellia serratulae, urophora cardui, u. quadrifasciata, u solstitialis (tephritidae); metzneria lappella (gelechidae); epichnopterix sieboldi (psychidae); paranthrene tabaniformis, pennisetia hylaeiformis (sesiidae); aethes williana (tortricidae). distribution: croatia; germany; hungary; italy, kazakhstan, moldova, russia (russia-krasnodar, yaroslavl oblast), slovenia, switzerland, ukraine, serbia. bracon (bracon) trucidator marshall 1888 on metzneria lappella (gelechiidae). distribution: albania, austria, belgium, great britain, bulgaria, croatia, denmark, turkey, finland, france, germany, greece, hungary, italy, latvia, poland, romania, russia (central, northwestern and south), slovenia, spain, switzerland, netherlands, serbia. bracon (bracon) variegator spinola 1808 on ernobius abietis (anobiidae); coleophora iutipennella (coleophoridae); agonopterix propinquella, (depressariidae); anarsia lineatella, recurvaria nanella, teleiodes luculella (gelechiidae); phyllonorycter klemannella, ph. mespillela (gracillariidae); endrosis sarcitrella (oecophoridae); acrobasis obsutella (pyralidae); archips rosana, cydia strobilella, tortrix viridana (tortricidae); yponomeuta padella (yponomeutidae). distribution: albania, austria, croatia, cyprus, france, hungary, italy, macedonia, russia (south), sardinia, spain, switzerland, ukraine, serbia. bracon (cyanopterobracon) sabulosus szepligeti 1896 on tephritidae. distribution: bulgaria, croatia, cyprus, greece, hungary, macedonia, romania, russia (south), serbia. bracon (cyanopterobracon) urinator (fabricius 1798) on larinus saussureae, l. sturnus, rhinocyllus conicus (curculilonidae); tephritis pulchra (tephritidae). distribution: albania, austria, belgium, bosnia and herzegovina, great britain, bulgaria, croatia, cyprus, turkey, france, germany, greece, hungary, italy, macedonia, portugal, romania, russia (central, east, biologica nyssana 3 (1)  september 2012: 21-29 žikić, v. et al.  checklist of the genus bracon (hymenoptera: braconidae)… 24 northwestern and south), slovakia, spain, sweden, switzerland, netherlands, ukraine, serbia. bracon (cyanopterobracon) uromelas costa 1888 – no host data. distribution: albania, austria, croatia, cyprus, france, hungary, italy, macedonia, russia (south), sardinia, spain, switzerland, ukraina, serbia. bracon (glabrobracon) abbreviator nees 1834 on anthonomus pomorum (curculionidae); lipara lucens (chloropidae); cephus pygmaeus (cephidae); coleophora trogldytella (coleophoridae); mesapamea secalis, oria musculosa, sesamia nonagrioides (noctuidae); pennisetia hylaeiformis (sesiidae); cochylis posterna, pandemis heparana, rhyacionia buoliana (tortricidae). distribution: austria, great britain, croatia, cyprus, czech republic, finland, germany, hungary, poland, romania, slovakia, spain, sweden, switzerland, netherlands, serbia. bracon (glabrobracon) arcuatus thomson 1892 – no host data. distribution: austria, great britain, bulgaria, croatia, cyprus, czech republic, germany, greece, hungary, slovenia, sweden, netherlands, serbia. bracon (glabrobracon) atrator nees 1834 on omphalapion buddebergi (apionidae); gymnetron villosulum (curculionidae); amauromyza flavifrons (agromyzidae); tephritis conura, t. separata (tephritidae); coleophora coronillae (coleophoridae). distribution: austria, belgium, great britain, bulgaria, croatia, cyprus, turkey, finland, france, germany, greece, hungary, ireland, italy, latvia, lithuania, macedonia, poland, russia (central, northwestern and south), slovakia, spain, sweden, switzerland, netherlands, serbia. bracon (glabrobracon) caudatus ratzeburg 1848 on hylesinus fraxini (curculionidae). distribution: austria, great britain, bulgaria, czech republic, germany, greece, hungary, poland, serbia. bracon (glabrobracon) caudiger nees 1834 on coleophora obscenella (coleophoridae); aphelia amplana, cydia splendana, c. strobilella (tortricidae). distribution: austria, belgium, czech republic, turkey, france, germany, hungary, italy, poland, romania, russia (central, north, northwestern and south), sweden, the netherland, serbia. bracon (glabrobracon) colpophorus wesmael 1838 on coleophora sp. (coleophoridae). distribution: austria, belgium, great britain, czech republic, france, germany, hungary, italy, moldova, poland, romania, russia (northwestern), netherlands, ukraine, serbia. bracon (glabrobracon) coniferarum fahringer 1928 on millieria dolosalis (choreutidae); coleophora gallipennella, c. serratella (coleophoridae). distribution: great britain, bulgaria, czech republic, germany, italy, poland, switzerland, netherlands, serbia. bracon (glabrobracon) delibator haliday 1833 on gymnetron campanulae, hylobius piceus (curculionidae); olibrus aeneus (phalacridae); urophora cuspidata (tephritidae); cydia strobilella (tortricidae). distribution: austria, belgium, bosnia and herzegovina, great britain, bulgaria, croatia, cyprus, czech republic, turkey, finland, france, germany, greece, hungary, ireland, italy, macedonia, poland, russia (central and south), slovakia, slovenia, spain, sweden, switzerland, netherlands, ukraine, serbia. bracon (glabrobracon) delusor spinola 1808 – no host data. distribution: austria, croatia, france, germany, italy, latvia, poland, russia (northwestern), serbia. bracon (glabrobracon) dichromus wesmael 1838 on gymnetron asellus (curculionidae); platyedra subcinerea (gelechidae); grapholita funebrana (tortricidae). distribution: austria, belgium, bulgaria, croatia, cyprus, france, germany, hungary, italy, poland, romania, slovakia, spain, serbia. bracon (glabrobracon) discoideus wesmael 1838 on saperda populnea (cerambycidae); anthonomus pomorum, a. spilotus, ceutorhynchus assimilis, c. quadridens, curculio crux, c. nucum, c. pyrrhoceras, c. salicivorus, pissodes notatus (curculionidae); byctiscus betulae, b. populi, rhynchites pauxillus (rhynchitidae); chlorops pumilionis (chloropidae); biorhiza pallida (cynipidae); euura mucronata, pontania bridgmanii, p. collactanea, p. foetidae, p. pedunculi, p. proxima, p. vesicator, p. viminalis (tenthredinidae); anticlea badiata (geometridae); adoxophyes orana, rhyacionia buoliana (tortricidae). distribution: austria, belgium, great britain, bulgaria, czech republic, france, germany, hungary, ireland, italy, poland, romania, russia (northwestern), slovakia, slovenia, sweden, switzerland, netherlands, serbia. bracon (glabrobracon) fumatus szepligeti 1901 – no host data. distribution: austria, croatia, cyprus, turkey, france, greece, hungary, russia (south), spain, switzerland, serbia. bracon (glabrobracon) fuscipennis wesmael 1838 on gastrophysa polygoni, g. viridula, plagiodera versicolora (chrysomelidae). distribution: austria, http://www.faunaeur.org/full_results.php?id=11049 http://www.faunaeur.org/full_results.php?id=11175 biologica nyssana 3 (1)  september 2012: 21-29 žikić, v. et al.  checklist of the genus bracon (hymenoptera: braconidae)… 25 belgium, croatia, france, germany, hungary, italy, poland, slovenia, serbia. bracon (glabrobracon) hyalinipennis szepligeti 1901 – no host data. distribution: hungary, serbia. bracon (glabrobracon) immutator nees 1834 on anthonomus rubi, ceutorhynchus pleurostigma, cryptorhynchus lapathi, curculio villosus, pissodes notatus (curculionidae); urophora stylata (tephritidae); biorhiza pallida, (cynipidae). distribution: austria, belgium, bosnia and herzegovina, great britain, croatia, cyprus, czech republic, finland, france, germany, hungary, italy, macedonia, poland, romania, russia (central, north, and northwestern), slovenia, sweden, switzerland, netherlands, ukraine, serbia. bracon (glabrobracon) larvicida wesmael 1838 – no host data. distribution: austria, belgium, great britain, bulgaria, croatia, turkey, france, germany, greece, hungary, italy, poland, romania, russia (central, northwestern and south), slovakia, slovenia, serbia. bracon (glabrobracon) marshalli szepligeti 1901 on trachys sp. (buprestidae); myopites inulaedyssentericae (tephritidae); cydia nigricana (tortricidae). distribution: great britain, bulgaria, croatia, cyprus, denmark, finland, germany, greece, hungary, italy, norway, portugal, romania, slovakia, slovenia, spain, sweden, switzerland, serbia. bracon (glabrobracon) minutator (fabricius 1798) on anthonomus pomorum (curculionidae); lipara lucens (chloropidae); trypeta jaceae (tephritidae); cephus pygmaeus (cephidae); caloptilia syringella (gracillariidae); mesapamea secalis, oria musculosa, sesamia nonagrioides (noctuidae); pennisetia hylaeiformis (sesiidae); cochylis posterana, leguminivora glycinivorella, rhyacionia buoliana (tortricidae). distribution: albania, austria, belgium, great britain, bulgaria, cyprus, czech republic, denmark, turkey, finland, france, germany, greece, hungary, ireland, italy, latvia, lithuania, macedonia, norway, poland, russia (central and south), slovakia, slovenia, spain, sweden, switzerland, netherlands, serbia. bracon (glabrobracon) obscurator nees 1811 on trachys pumilus, t. troglodytes (buprestidae); hylesinus fraxini, ips typographus, orthotomicus suturalis, trypodendron domesticum, t. signatum (curculionidae); coleophora caespititiella (colephoridae); epermenia fulviguttella (epermeniidae); homoeosoma sinuella (pyralidae). distribution: austria, belgium, bosnia and herzegovina, great britain, bulgaria, croatia, cyprus, czech republic, denmark, turkey, finland, france, germany, greece, hungary, italy, macedonia, poland, romania, russia (central, northwestern and south), slovakia, slovenia, spain, sweden, switzerland, netherlands, serbia. bracon (glabrobracon) osculator nees 1811 on millieria dolosalis (choreutidae); coleophora albicostella, c. alticolella, c. caespititiella, c. colutella, c. discordella, c. flaviella, c. frischella, c. gallipennella, c. lutipennella, c. milvipennis, c. obscenella, c. ochrea, c. saponariella, c. serenella, c. serratella, c. solitariella, c. spinella, co. spiraeella, c. therinella, c. trifariella, c. vibicella, c. vicinella, c. vulpecula, coleophora sp.1, coleophora sp.2 (coleophoridae); cosmopterix eximia (cosmopterigidae); elachista contaminatella, elachista sp. (elachistidae); phyllonorycter sp. (gracillariidae); mompha conturbatella (momphidae); ectoedemia agrimoniae (nepticulidae); retinia perangustana (tortricidae). distribution: austria, belgium, bosnia and herzegovina, great britain, bulgaria, croatia, cyprus, czech republic, denmark, turkey, finland, france, germany, greece, hungary, ireland, italy, latvia, lithuania, macedonia, poland, romania, russia (central, east, northwestern and south), slovakia, slovenia, spain, sweden, switzerland, netherlands, serbia. bracon (glabrobracon) peroculatus wesmael 1838 on cnephasia chrysantheana (tortricidae). distribution: belgium, finland, france, germany, hungary, italy, russia (central), serbia. bracon (glabrobracon) picticornis wesmael 1838 on clytus sp., plagionotus arcuatus (cerambicidae); nematus bipartitus, n. salicis, pontania pedunculi, p. proxima, p. vesicator, p. viminallis (tenthredinidae); cochylis pallidana (tortricidae). distribution: austria, belgium, great britain, bulgaria, turkey, finland, france, germany, greece, hungary, ireland, italy, lithuania, poland, russia (central, northwestern and south), slovakia, spain, sweden, switzerland, netherlands, serbia. bracon (glabrobracon) pineti thomson 1892 on ernobius abietis, e. longicornis (anobiidae); cydia strobilella (tortricidae). distribution: austria, bulgaria, croatia, czech republic, finland, germany, hungary, lithuania, moldova, poland, russia (central, north, northwestern and south), slovakia, sweden, serbia. bracon (glabrobracon) ratzeburgii dalla torre 1898 on ceutorhynchus maculaalba, hylesinus fraxini, scolytus rugulosus (curculionidae). distribution: great britain, germany, italy, serbia. biologica nyssana 3 (1)  september 2012: 21-29 žikić, v. et al.  checklist of the genus bracon (hymenoptera: braconidae)… 26 bracon (glabrobracon) terebella wesmael 1838 on miarus campanulae (curculionidae); cephus pygmaeus, trachelus tabidus (cephidae). distribution: austria, belgium, great britain, denmark, finland, germany, hungary, italy, lithuania, poland, romania, russia (central,northwestern and south), slovakia, sweden, switzerland, serbia. bracon (glabrobracon) titubans wesmael 1838 – no host data. distribution: austria, belgium, bulgaria, finland, france, germany, hungary, italy, poland, russia (northwestern), slovakia, sweden, switzerland, netherlands, serbia. bracon (glabrobracon) tornator marshall 1885 – no host data. distribution: austria, great britain, bulgaria, croatia, finland, germany, greece, hungary, italy, sweden, switzerland, serbia. bracon (glabrobracon) variator nees 1811 on bruchidius lividimanus, bruchus atomarius, b. laticollis, b. lentis, b. viciae (bruchidae); anthonomus pomorum, baris chlorizans, b. cuprirostris, b. laticollis, ceutorhynchus punctiger, gymnetron asellus, g. tetrum, larinus jaceae, l. turbinatus, magdalis rufa, miarus campanulae, pissodes validirostris, sibinia viscariae, sitona longulus (curculionidae); chaetostomella cylindrica, noeeta pupillata, sphenella marginata, tephritis leontodontis (tephritidae); hoplocampa brevis, h. flava (tenthrenidae); colephora coronillae, c. medelichenis (coleophoridae); mesophleps corsicella, pexicopia malvella, platyedra subcinerea, (gelechiidae); phyllonorycter mespilella (gracillariidae); hadena bicruris (noctuidae); dioryctria abietella, etiella zinckenella, myelois circumvoluta (pyralidae); synathedon andrenaeformis (sesiidae); barbara herrichiana, cydia strobilella, eucosma cana, grapholita funebrana, g. lunulana, pandemis cerasana, retinia resinella (tortricidae). distribution: austria, belgium, bosnia and herzegovina, great britain, bulgaria, canary is., croatia, cyprus, czech republic, turkey, finland, france, germany, greece, hungary, ireland, italy, latvia, lithuania, macedonia, moldova, poland, romania, russia (central, east, north, northwestern and south), slovakia, slovenia, spain, sweden, switzerland, netherlands, ukraine, serbia. bracon (habrobracon) brevicornis wesmael 1838 on sesamia cretica (noctuidae). distribution: belgium, great britain, bulgaria, czech republic, france, germany, hungary, italy, poland, portugal, romania, russia (central and south), slovakia, spain, switzerland, netherlands, serbia. bracon (habrobracon) hebetor say 1836 on ostrinia nubilalis (crambidae); pexicopia malvella (gelechiidae); helicoverpa armigera, heliothis peltigera, spodoptera littoralis (noctuidae); ephestia elutella, e. kuehniella, etiella zinckenella, galleria mellonella, plodia interpunctella (pyralidae); adoxophyes orana, lobesia botrana (tortricidae). distribution: austria, belgium, great britain, bulgaria, croatia, cyprus, czech republic, turkey, finland, france, germany, greece, hungary, italy, lithuania, macedonia, poland, russia (central and south), slovakia, slovenia, spain, netherlands, serbia. bracon (lucobracon) apricus schmiedeknecht 1897 – no host data. distribution: germany, greece, serbia. bracon (lucobracon) exarator marshall 1885 – no host data. distribution: great britain, germany, serbia. bracon (lucobracon) femoralis brullé, – no host data. distribution: cyprus, greece, hungary, spain, switzerland, serbia. bracon (lucobracon) fortipes wesmael 1838 on plagionotus floralis (cerambycidae); lixus anguinus (curculionidae); gortyna xanthenes (noctuidae); bembecia scopigera (sesiidae). distribution: belgium, bulgaria, croatia, cyprus, france, germany, greece, hungary, italy, poland, slovenia, switzerland, serbia. bracon (lucobracon) fumarius szepligeti 1901 – no host data. distribution: austria, croatia, cyprus, hungary, romania, serbia. bracon (lucobracon) hungaricus (szepligeti 1896) on clytus arietis (cerambycidae). distribution: bulgaria, croatia, turkey, hungary, russia (central and south), slovakia, ukraine, serbia. bracon (lucobracon) hylobii ratzeburg 1848 on hylobius abietis, h. piceus, pissodes harcyniae, p. notatus, p. piceae, p. pini, polygraphus polygraphus (curculionidae). distribution: austria, great britain, bulgaria, czech republic, denmark, germany, hungary, italy, lithuania, poland, russia (central and northwestern), sweden, switzerland, netherlands, serbia. bracon (lucobracon) megapterus wesmael 1838 on glaphyra umbellatarum (cerambycidae); chaetorellia jaceae (tephritidae). distribution: belgium, bulgaria, czech republic, finland, france, germany, sweden, serbia. bracon (lucobracon) nigriventris wesmael 1838 on plagionotus floralis (cerambycidae); hylobius piceus, pissodes pini (curculionidae). distribution: biologica nyssana 3 (1)  september 2012: 21-29 žikić, v. et al.  checklist of the genus bracon (hymenoptera: braconidae)… 27 albania, austria, belgium, bulgaria, croatia, cyprus, turkey, germany, hungary, italy, poland, romania, russia (south), slovakia, switzerland, ukraine, serbia. bracon (lucobracon) ochraceus szepligeti 1896 – no host data. distribution: croatia, hungary, russia (south), spain, switzerland, serbia. bracon (lucobracon) planiceps telenga 1936 – no host data. distribution: russia (north), serbia. bracon (lucobracon) romani fahringer 1927 on chamaesphecia bibioniformis, paranthrene tabaniformis, pennisetia hylaeiformis, sesia apiformis, s. bembeciformis, synanthedon culiciformis, s. spheciformis (sesiidae). distribution: greece, hungary, slovakia, slovenia, sweden, netherlands, serbia. bracon (lucobracon) thuringiacus schmiedeknecht 1896 – no host data. distribution: great britain, germany, greece, italy, serbia. bracon (lucobracon) triangularis nees 1834 on synanthedon tipuliformis (sesiidae). distribution: great britain, bulgaria, czech republic, finland, germany, hungary, ireland, italy, poland, slovakia, switzerland, netherlands, serbia. bracon (orthobracon) epitriptus marshall 1885 on cryptorhynchus lapathi, hylobius transversovittatus, pissodes spp. (curculionidae); agromyza flaviceps (agromyzidae); iteomyia capreae (cecidomyiidae); fenusa pumila (tenthredinidae). distribution: austria, great britain, bulgaria, germany, greece, hungary, italy, lithuania, poland, russia (central, northwestern and south), slovakia, slovenia, switzerland, netherlands, serbia. bracon (orthobracon) erraticus wesmael 1838 on gastrophysa viridula, phaedon cochleariae, phyllotreta nemorum (chrysomelidae); chaetostomella cylindrca, urophora eriolepidis, terellia serratulae (tephritidae); coleophora laricella, c. lutipennella (coleophoridae); metzneria lappella (gelechiidae); pennisetia hylaeiformis (sesiidae); acleris rhombana (tortricidae). distribution: austria, belgium, bosnia and herzegovina, great britain, bulgaria, croatia, cyprus, turkey, finland, france, germany, greece, hungary, ireland, italy, lithuania, macedonia, poland, russia (central, east, north, northwestern, and south), slovakia, slovenia, spain, sweden, switzerland, netherlands, ukraine, serbia. bracon (orthobracon) exhilarator nees 1834 on omphalapion hookerorum (apionidae); nanna sp. (scatophagidae); acleris rhombana (tortricidae). distribution: belgium, great britain, bulgaria, cyprus, turkey, finland, france, germany, hungary, italy, latvia, lithuania, norway, poland, russia (central, north, northwestern and south), slovakia, sweden, switzerland, netherlands, serbia. bracon (orthobracon) laevigatissimus dalla torre 1898 on plagionotus arcuatus (cerambycidae); pontania pedunculi, p. proxima, p. vesicator, p. viminalis (tenthredinidae). distribution: austria, great britain, finland, france, germany, italy, poland, romania, switzerland, netherlands, serbia. bracon (orthobracon) ochropus nees 1834 – no host data. distribution: austria, france, germany, italy, serbia. bracon (orthobracon) roberti wesmael 1838 on synanthedon andrenaeformis (sesiidae); cydia strobilella (tortricidae). distribution: austria, belgium, great britain, turkey, france, germany, ireland, russia (northwestern), netherlands, serbia. bracon (orthobracon) subcylindricus wesmael 1838 – no host data. distribution: belgium, great britain, france, poland, russia (northwestern), serbia. bracon (orthobracon) tenuicornis wesmael 1838 on phloetribus scarabaeoides (curculionidae). distribution: belgium, great britain, france, hungary, russia (south), sweden, netherlands, serbia. bracon (striobracon) idrianus fahringer 1934 on hylesinus oleiperda (curculionidae). distribution: serbia. discussion the present account is the first summary of all available data about the genus bracon and its hosts until now on the territory of serbia. out of total number 250 bracon species in europe, 80 species are present in serbian fauna. we have registered 290 hosts for 55 parasitoid species connected in 409 trophic associations. the hosts come from four insect orders. the most frequent hosts belong to lepidoptera and coleoptera, whereas diptera and hymenoptera are less parasitized. numbers in brackets refer to the number of registered host species. 1. diptera (28) with five families: agromyzidae (2); chloropidae (3); cecidomyiidae (1) scatophagidae (1) and tephritidae (21). 2. coleoptera (109) with nine families: anobiidae (3); apionidae (5); bruchidae (5); buprestidae biologica nyssana 3 (1)  september 2012: 21-29 žikić, v. et al.  checklist of the genus bracon (hymenoptera: braconidae)… 28 (7); cerambycidae (12); chrysomelidae (6); curculionidae (66); phalacridae (1) and rhynchitidae (4). 3. hymenoptera (26) with four families: cephidae (2); cynipidae (1); eurytomidae (3) and tenthredinidae (20). 4. lepidoptera (128) with 21 families: agonoxenidae (1); choreutidae (1); coleophoridae (37); cosmopterigidae (1); crambidae (1); depressariidae (1); elachistidae (1); epermeniidae (1); gelechiidae (11); geometridae (1); gracillariidae (4); momphidae (1); nepticulidae (1); noctuidae (5); oecophoridae (1); psychidae (1); pyralidae (9); sesiidae (14); tortricidae (24); yponomeutidae (6) and zygaenidae (1). the most suitable host species in these trophic relations parasitoid-host, belongs to the coleopteran family curculionidae and lepidopteran family coleophoridae especially the genus coleophora which has 11 parasitoids registered in this work. the torticid moth cydia strobilella has remarkably wide range of parasitoids, six bracon species were registered here as parasitoids of this moth (b. (bracon) variegator, b. (glabrobracon) caudiger, b. (glabrobracon) delibator, b. (glabrobracon) pineti, b. (glabrobracon) variator and b. (orthobracon) roberti). we noted that bracon idrianus is present only on the territory of serbia. this basic research shows better insight into the trophic interactions about each parasitoid species which can help in further biological control programs. nonetheless, it can ease the identification of some parasitoid species according to their hosts. acknowledgements. the authors wish to express their deepest gratitude to dr. sergey a. belokobylskij from the zoological institute ras (st. petersburg, russia) and dr. jenő papp from the natural history museum of budapest, hungary for the identification of parasitoid specimens and to dr. aleksandar ćetković from the faculty of biology, university of belgrade for the loan of some specimens from his collection. for the present work the authors were supported by the ministry of science and environment protection of the republic of serbia (grant no. iii43001). references achterberg, c. van 2012: fauna europaea: braconidae. in: van achterberg (ed.): fauna europaea: hymenoptera: symphyta + ichneumonoidea. fauna europaea, version 2.5. http://www.faunaeur.org achterberg, c. van, hemminga, m.a., soelen, j. van 1990: new host record of bracon intercessor nees f. megasomides strand (hymenoptera: braconidae), a parasite of agapanthia villosoviridescens degeer (coleoptera: cerambycidae) in salt marshes. zoologische mededelingen, 64 (3): 25-29. achterberg, c. van. 1985: notes on braconidae iiv. zoologische mededelingen, 59 (15): 163167. baird, a.b. 1958: biological control of insects and plant pests in canada. in: baecker, e.c., (ed.), proceedings of 10 th international congress of entomologist, ottawa, 4: 483-485. belokobylskij, s.a., žikić, v. 2009: new data on cyclostome braconid subfamilies doryctinae, exothecinae, rogadinae and braconinae (braconidae: hymenoptera) of serbia and neighbouring territories. acta entomologica serbica, 14 (1): 65-71. beyarslan, a., çetin erdoğan, ö., aydoğdu, m. 2005: a survey of braconinae (hymenoptera, braconidae) of turkish western black sea region. linzer biologische beiträge, 37 (1): 195–213. brajković, m., 1989: knowledge of the braconidae (hymenoptera) fauna in yugoslavia. glasnik prirodnjačkog muzeja, b (43/44): 127-138. brajković, m., krunić, m., tomanović, ž., 1991: a contribution to the braconid fauna (braconidae: hymenoptera) of yugoslavia. in: hawksworth, d.l. (ed.): proceedings of the fourth european congress of entomology and the xiii internationale symposium für entomofaunistik mitteleuropas, hungarian natural history museum, budapest, (2): 424-428. brajković, m., krunić, m., tomanović, ž., 1994: research of braconids (braconidae: hymenoptera) of deliblatska peščara. in: marinković, p. (ed.): proceedings of deliblatski pesak, srbijašume, 6: 197-502 dweck, h.k., svensson, g.p., gündüz, e.a., anderbrant, o. 2010: kairomonal response of the parasitoid, bracon hebetor say, to the maleproduced sex pheromone of its host, the greater waxmoth, galleria mellonella (l.). journal of chemical ecology, 36: 171-178. fischer, m. 1959: neue und wenig bekannte braconiden aus jugoslawien (hymenoptera). acta musei macedonici scientiarum naturalium, 6 (1): 53: 1-25. georgiev, g. 2005: bioecological characteristics of bracon intercessor nees (hymenoptera: braconidae) as a parasitoid of the poplar clearwing moth, paranthrene tabaniformis http://www.faunaeur.org/full_results.php?id=11049 biologica nyssana 3 (1)  september 2012: 21-29 žikić, v. et al.  checklist of the genus bracon (hymenoptera: braconidae)… 29 (rott.) (lepidoptera: sesiidae) in bulgaria. journal of pest science, 78: 161-165. lewis, w.j., vet, l.e.m., tumlinson, j.h., van lenteren, j.c., papaj, d.r. 1990: variations in parasitoid foraging behavior: essential element of a sound biological control theory. environmental entomology, 19: 1183-1193. mathews, r.w. 1974: biology of braconidae. annual review of entomology, 19: 15-32. milonas, p.g. 2005: influence of initial egg density and host size on the development of the gregarious parasitoid bracon hebetor on three different host species. biocontrol, 50: 415-428. papp, j. 1973: contributions to the braconid fauna of yugoslavia (hymenoptera, braconidae) i. acta musei macedonici scientarium naturalium, skopje, 14 (1: 119): 1-23. papp, j. 1977: contribution to the braconid fauna of yugoslavia. (hymenoptera: braconidae) iii. folia entomologica hungarica (series nova), 30: 105-117. papp, j. 2008: a revision of the bracon (subgenera bracon s. str., cyanopterobracon, glabrobracon, lucobracon, osculobracon, subgen. n., pigeria) species described by szépligeti from the western palaearctic region. biologia, 65 (1): 110-112. reinert, j.a., king, e.w. 1971. action of bracon hehetor say as a parasite of plodia interpunctella at controlled densities. annals of the entomological society of america, 64: 1335 1340. shaw, m.r. and huddleston, t. 1991: classification and biology of braconid wasps (hymenoptera: braconidae). royal entomological society of london, london. 126p. stanković, s., žikić, v., ilić, m. 2010: betula species as host plants for various insects parasitized by braconids (hymenoptera: braconidae) in serbia. biologica nyssana, 1 (12): 117-122. taylor, a.d. 1988a: host effects on larval competition in the gregarious parasitoid bracon hehetor. journal of animal ecology, 57: 163172. taylor, a.d. 1988b: host effects on functional and ovipositional responses of bracon hebetor. journal of animal ecology 57: 173-184. temerak, a.s. 1982: laboruntersuchungen zur wirtshabitat-wahl von bracon brevicornis wesm. (hym., braconidae), einem parasitoiden der raupen von sesamia cretica led. (lep., noctuidae) in sorghum-stengeln. anzeiger für schädlingskunde, berlin und hamburg, 55: 152154. tobias, v.i., belokobylskij, s.a., kotenko, a.g. 1986: family braconidae. in: medvedev g.s. (ed.). a key to insects of the european part of the ussr. hymenoptera 3: 4. leningrad: nauka. 500 p. [in russian]. yu, d.s., achterberg, c. van, horstmann, k. 2012: world ichneumonoidea 2011. taxonomy, biology, morphology and distribution (braconidae). taxapad (scientific names for information management) interactive catalogue on dvd/cdrom. vancouver. yu, s.-h., ryoo, m.ii., na, j.h. 1999: life history of bracon hebetor (hymenoptera: braconidae) on plodia interpunctella (lepidoptera: pyralidae) on a dried vegetable commodity. journal of asia-pacific entomology, 2 (2): 149-152. žikić, v., achterberg, c. van, stanković, s. 2010: a contribution to braconidae, hybrizontidae (ichneumonoidea: hymenoptera) and stephanidae (stephanoidea: hymenoptera) from the southwest balkans. acta entomologica serbica, 15 (2): 227-235. žikić, v., brajković, m., tomanović, ž. 2000: preliminary results of braconid fauna research (braconidae: hymenoptera). acta entomologica serbica, 5 (1/2): 95-111. žikić, v., stanković, s.s., ilić, m., kavallieratos, n.g. 2012: braconid parasitoids (hymenoptera: braconidae) on poplars and aspen (populus spp.) in serbia and montenegro. north-western journal of zoology [in press]. biologica nyssana 3 (1)  september 2012: 21-29 žikić, v. et al.  checklist of the genus bracon (hymenoptera: braconidae)… 30 mašić et al. 2020, biologica nyssana 11(2) 11 (2) december 2020: 93-101 doi: 10.5281/zenodo.4393955 new data on distribution of hydrurus foetidus (villars) trevisan in freshwater habitats on vranica mountain (bosnia and herzegovina) original article ermin mašić university of sarajevo, faculty of science, department of biology, zmaja od bosne 33-35, 71000 sarajevo, bosnia and herzegovina erminmasic@hotmail.com (corresponding author) senka barudanović university of sarajevo, faculty of science, department of biology, zmaja od bosne 33-35, 71000 sarajevo, bosnia and herzegovina sebarudanovic@gmail.com sabina žero university of sarajevo, faculty of pharmacy, department of natural sciences in pharmacy, zmaja od bosne 8, 71000 sarajevo, bosnia and herzegovina sabina_zero@hotmail.com emina ramić university of sarajevo, faculty of science, department of biology, zmaja od bosne 33-35, 71000 sarajevo, bosnia and herzegovina emina.ramich@gmail.com armin macanović university of sarajevo, faculty of science, department of biology, zmaja od bosne 33-35, 71000 sarajevo, bosnia and herzegovina arminecology@gmail.com senaid fejzić secondary technical school „kemal kapetanovic“ kakanj, šehida 32, 72240 kakanj, bosnia and herzegovina senaidfejzic@yahoo.com received: april 08, 2020 revised: august 13, 2020 accepted: october 11, 2020 abstract: freshwater habitats such as springs, streams and lakes at mountain area represent extremely fragile ecosystems. these types of habitats occur in the wide area of vranica mountain. during our research (august and september 2018) of freshwater habitats in this area, rare golden macroalgae hydrurus foetidus (villars) trevisan was found. it has been already detected in bosnia and herzegovina, but in this study new localities are presented. water temperature, ph, dissolved oxygen, specific electrical conductivity, turbidity and total dissolved solids were measured on sampling sites. in addition to the analysis of ecological characteristics of habitats, diatom assemblages were analysed. in this study a total of 48 diatom taxa belonging to 25 genera were identified. the results of diatom indices confirmed the good water quality of the studied sites on which h. foetidus was found. this study represents a contribution to algological research in bosnia and herzegovina. key words: bioindicators, conservation ecology, macroalgae, springs, streams, water quality apstract: novi podaci o rasprostranjenosti hydrurus foetidus (villars) trevisan u slatkovodnim staništima na planini vranici (bosna i hercegovina) slatkovodna staništa kao što su izvori, potoci, reke i jezera na planinskim područjima predstavljaju izuzetno osetljive ekosisteme. ovi tipovi staništa su zastupljeni na širem području planine vranice. tokom istraživanja (avgust i septembar, 2018) slatkovodnih tipova staništa na ovom području, utvrđena je retka zlatna makroalga hydrurus foetidus (villars) trevisan. vrsta je već konstatovana u bosni i hercegovini, ali u ovom istraživanju predstavljeni su novi lokaliteti. temperatura vode, ph, rastvoreni kiseonik, elektroprovodljivost, mutnoća i ukupna količina rastvorenih organskih materija su izmereni na mestu uzorkovanja. pored analize ekoloških karakteristika staništa, analiziran je i sastav diatomeja. tokom ove studije ukupno je identifikovano 48 dijatomeja iz 25 rodova. rezultati dijatomskih indeksa potvrdili su dobar kvalitet vode na lokalitetima gdje je utvrđen h. foetidus. ova studija predstavlja značajan doprinos algološkim istraživanjima bosne i hercegovine. ključne reči: bioindikatori, konzervacijska ekologija, makroalge, izvori, potoci, kvalitet vode introduction the species hydrurus foetidus (villars) trevisan is a macroscopic golden alga with mucilaginous, branched and bushy to feathery, dark brown thallus up to 30 cm long. the thallus and other characteristics are well described in various publications (john et al., 2003; wehr et al., 2015). this species inhabit mountain streams during early spring and lowland rivers at latitudes where seasonal conditions are appropriate for this coldwater species (klaveness et al., 2011; klaveness, 2017). according to guiry & guiry (2019), this alga is widely distributed in europe and it’s presence was reported in austria (pfister, 1992), britain (kristiansen, 2002; whitton et al., 2003; john et al., 2011), croatia (stanković & leitner, 2016; koletić et al., 2017), georgia (barinova & kukhaleishvili, 2017), germany (mauch & schmedtje, 2003), © 2020 mašić et al.. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 93 norway (klaveness et al., 2011; klaveness & lindstrøm, 2011; klavness, 2012), romania (caraus, 2017), russia (europe) (voloshko, 2007; patova & demina, 2007; patova et al., 2014), serbia (krizmanić et al., 2008), slovakia (hindák & hindáková, 2016) and turkey (europe) (aysel, 2005). on the territory of bosnia and herzegovina h. foetidus was reported only from a few localities (fig. 1), and these data are found in various publications (protić, 1904; blagojević, 1966; kosorić, 1977; aganović, 1981; ratković, 1985; redžić, 1988; hafner, 1991; redžić, 1991). until now, several algological studies were performed on vranica mountain concerning different algal groups and habitats (protić, 1926; kapetanović & hafner, 2007; barudanović et al., 2017) but did not demonstrate the occurrence of h. foetidus in any studied sites. the main aim of this study was to present new localities where h. foetidus occurs considering the ecological characteristics of habitat in which the species was found. material and methods study area vranica mountain has very heterogeneous geology and petrography. the area is rich in numerous springs, brooks and small rivers, which are active throughout the year. unfortunately, in recent years due to growing tourism activity anthropogenic impact has increased. these effects are manifested mostly through the construction and expansion of road communication, drainage of wet habitats, springs capturing and the construction of new accommodation facilities (redžić, 2007). sampling sites are listed in tab. 1. sampling and algae analysis physical and chemical parameters of water were measured directly on sampling sites. water temperature (°c), ph, dissolved oxygen (mgl-1) and specific electric conductivity (μscm-1) were measured with portable multimeter orion star a329, while turbidity (ntu) was measured with portable turbidimeter aq3010 and total dissolved solids (ppm) with pce-cm 41. live algological material was collected from six microlocalities in 2018, during summer and autumn seasons, photographed and transported to the laboratory of algae and fungi at faculty of science, university of sarajevo. phytobenthos was collected 94 table 1. the main physical characteristics of the sampling sites (sp – spring; st – stream) biologica nyssana ● 11 (2) december 2020: 93-101 mašić et al. ● new data on distribution of hydrurus foetidus (villars) trevisan in freshwater habitats on vranica mountain (bosnia and herzegovina) site habitat types date coordinate [n] coordinate [e] altitude [m] anthropogenic disturbance 1. sp1 11/08/2018 43.95353 17.75757 1682 moderate 2. st1 11/08/2018 43.95353 17.75757 1682 moderate 3. st2 11/08/2018 43.95440 17.75760 1677 moderate 4. st3 25/08/2018 43.95731 17.74518 1727 moderate 5. st4 20/10/2018 43.95563 17.75786 1465 moderate 6. st5 20/10/2018 43.95544 17.75767 1652 moderate fig. 1. utm map of distribution of hydrurus foetidus in bosnia and herzegovina 95 from small stones by scraping or by squeezing of mosses growing on rocks and stones. the collected material was fixed with a 4% formalin. small portion of h. foetidus was collected and observed under the microscope from temporary slides under 40 x magnification. the identification of h. foetidus was supported by the following references: starmach (1985), john et al. (2003) and wehr et al. (2015). laboratory processing of diatoms was carried out applying methods used by hustedt (1930). light microscope observation was conducted using best scope 2020 microscope. species composition and the quantitative relationship of diatoms are estimated from the permanent slides under 1000 x magnification. the identification of diatoms was done according to lange-bertalot & metzeltin (1996) and cantonati et al. (2017). the nomenclature of identified algae was adjusted according to the internet base of guiry & guiry, 2019. at least 300 valves in each slide were counted for all samples. species abundance of diatoms are estimated on a five-degree scale as follows: 1-rare (single valve or frustule), 2-sparse (up to 10% of the sample), 3-frequent (11-15% of the sample), 4-very frequent (51-75% of the sample), 5-common (in more than 75% of the sample) (tab. 3). omnidia software (lecointe et al., 1993) version 6.0.8, was used to calculate diatom indices, including ecological and taxonomic data. water quality of the localities where h. foetidus was assessed based on the following diatom indices: ips (coste, 1982), slad (sládeček, 1986), tid (rott et al., 1999) and sid rott (rott et al., 1997). range of diatom indices varied from 1 to 20 and corresponding to the water quality as follows: bad (1-4), poor (5-8), moderate (9-12), good (13-16) and very good (1720) (solak et al., 2020). results description of sampling sites the species of h. foetidus (chrysophyceae) was found near a reocren spring of mountain stream and on five localities downstream. it lives as a benthic species attached to the mesolithic and megalithic stones on the bottom of the water body an is often associated with mosses. although it tolerates occasional water fluctuations, in most cases it has been found in running water, but also in places where the water is a little deeper and slower. the altitude of the sites where the h. foetidus was recorded, ranged from 1465 to 1727 meters. the spring and mountain stream are covered with dense macrophytes and helophytes vegetation. two types of habitats where h. foetidus is recorded are showed on fig. 2, while in fig. 3 and fig. 4 the macroscopic and microscopic appearance of the thallus are shown. physical and chemical parameters of water a comparison of the physical and chemical parameters of water in the mountain spring and stream revealed the following: the water temperature on sampling sites during the investigated period varied from 6.2 to 19.9 °c. the lowest ph of the water was 7.92 while the highest ph value was 8.50. the highest concentration of dissolved oxygen in water was measured at the site 2 (10.82 mgl-1), while the smallest value was measured at the site 4 (7.56 mgl-1). value of specific conductivity varied from 162 to 330 μscm-1. the lowest value of turbidity was measured at the site 4 (0.45 ntu), while at the site 6 the highest value of turbidity was measured (2.81 ntu). fig. 2. two types of habitats where hydrurus foetidus was recorded: mountain spring (left) and mountain stream (right) biologica nyssana ● 11 (2) december 2020: 93-101 mašić et al. ● new data on distribution of hydrurus foetidus (villars) trevisan in freshwater habitats on vranica mountain (bosnia and herzegovina) 96 values of physical and chemical parameters are presented in tab. 2. characteristics of habitats of hydrurus foetidus and diversity of diatoms hydrurus foetidus has been identified at six sites located in the area of vranica mountain. it inhabits mainly spring and fast and clean mountain streams. together with mosses and other groups of algae, it grows on mesolithic and megalithic bases, and represents a significant product component of these freshwater habitats. the presence of this species indicates good water quality and good ecological status of freshwater ecosystems. in addition to h. foetidus, diatoms are second the most prominent group of algae which have been found in this mountain freshwater habitats. a total of 48 diatom taxa belonging to 25 genera were determined in the collected material (tab. 3). the number of recorded species of diatoms in the study sites ranged from 11 to 22. genera with the highest number of species were pinnularia (5), gomphonema (5), diatoma (4) and cocconeis (4). altogether 47 diatom taxa mostly belonging to pennate diatoms were found. the centric species, aulacoseira crenulata (ehrenberg) thwaites, appeared in only one sample. the most common species were odontidium mesodon (ehrenberg) kützing, cocconeis lineata ehrenberg, meridion circulare (gréville) c. agardh, cocconeis placentula ehrenberg, gomphonema minusculum krasske and achnanthidium minutissimum (kützing) czarnecki. water qualities of investigated sites according to diatom indices values of ips diatom index varied from 15.0 to 18.3. according to the ips diatom index, all studied sites have very good water quality. saprobic index at four sites indicates good, while on the other studied sites it indicates very good water quality. trophic index at all investigated sites indicates moderate water quality. saprobic index according to rott et al. (1997) ranged from 16.4 to 17.1. high values of diatom indices correspond to a very good water quality of investigated freshwater habitats on vranica mountain (tab. 3). discussion hydrurus foetidus was found in the mountain spring (1682 m a.s.l.) and in the mountain streams with table 2. value of the water temperature, ph, dissolved oxygen, specific electric conductivity, turbidity and tds [t1 – water temperature, do – dissolved oxygen, sc – specific electric conductivity, t2 – turbidity, tds – total dissolved substance]. site group t1 [°c] ph do [mgl-1] sc [μscm-1] t2 [ntu] tds [ppm] 1. sp1 6.8 7.92 10.35 330 0.80 n/a 2. st1 6.2 7.97 10.85 162 1.12 n/a 3. st2 7.4 8.23 10.25 208 0.73 n/a 4. st3 19.9 8.31 7.56 171 0.45 121 5. st4 8.1 8.47 10.51 n/a 0.91 267 6. st5 7.4 8.50 10.29 219 2.81 161 *n/a-not available fig. 3. thallus of h. foetidus (villars) trevisan fig. 4. microscopic appearance of the thallus of h. foetidus (villars) trevisan (40 x) biologica nyssana ● 11 (2) december 2020: 93-101 mašić et al. ● new data on distribution of hydrurus foetidus (villars) trevisan in freshwater habitats on vranica mountain (bosnia and herzegovina) 97 table 3. records of hydrurus foetidus and diatom taxa at sampling sites no. taxa / site 1 2 3 4 5 6 h hydrurus foetidus (villars) trevisan + + + + + + 1. odontidium mesodon (ehrenberg) kützing 4 6 4 1 1 3 2. cocconeis lineata ehrenberg 1 2 5 2 3 4 3. meridion circulare (gréville) c. agardh 1 2 1 2 1 1 4. cocconeis placentula ehrenberg 1 3 2 1 . 3 5. gomphonema minusculum krasske 1 1 1 1 1 . 6. achnanthidium minutissimum (kützing) czarnecki 6 1 . 1 1 1 7. encyonema minutum (hilse) d.g. mann 4 . 1 1 1 . 8. tetracyclus rupestris (braun) grunow in van heurck . 1 1 1 1 . 9. diatoma ehrenbergii kützing . . 1 4 4 4 10. ulnaria ulna (nitzsch) compère . . 1 6 1 1 11. encyonema silesiacum (bleisch) d.g. mann . 1 1 1 . . 12. hannea arcus (ehrenberg) r.m.patrick in patrick & reimer . . 5 . 4 3 13. encyonema ventricosum (c.agardh) grunow in a. schmidt et al. 1 1 . . . . 14. pinnularia borealis ehrenberg 1 1 . . . . 15. planothidium lanceolatum (brébisson ex kützing) lange-bertalot 1 1 . . . . 16. gomphonema parvulum (kützing) kützing . 1 2 . . . 17. denticula tenuis kützing . 1 1 . . . 18. caloneis alpestris (grunow) cleve . 1 . 1 . . 19. diploneis krammeri lange-bertalot et reichardt . 1 . 1 . . 20. epithemia turgida (ehrenberg) kützing . 1 . 1 . . 21. gomphonema acuminatum ehrenberg . . 1 . 1 . 22. cocconeis pediculus ehrenberg 1 . . . . . 23. cymbopleura naviculiformis (auerswald) krammer 1 . . . . . 24. gomphonema productum (grunow) lange-bertalot et reichardt in l-b 1 . . . . . 25. navicula lanceolata (c.agardh) ehrenberg 1 . . . . . 26. nitzschia fonticola grunow in cleve & möller 1 . . . . . 27. pinnularia viridis (nitzsch) ehrenberg 1 . . . . . 28. diatoma vulgaris bory . 1 . . . . 29. diploneis ovalis (hilse) cleve . 1 . . . . 30. eunotia sp. 1 . 1 . . . . 31. diatoma elongatum (lyngbye) agardh . 1 . . . . 32. pinnularia rupestris hantzsch in rabenhorst . 1 . . . . 33. amphora copulata (kützing) schoemann et archibald . 1 . . . . 34. aulacoseira crenulata (ehrenberg) thwaites . . 1 . . . 35. cymbella aspera (ehrenberg) h. peragallo . . 1 . . . 36. navicula tripunctata (o.f. müller) bory . . 1 . . . 37. cymbella neolanceolata w.silva . . . 1 . . 38. cymbopleura amphicephala (nägeli) krammer . . . 1 . . 39. diatoma moniliformis (kützing) williams . . . 1 . . 40. gomphonema subclavatum (grunow) grunow . . . 1 . . 41. pinnularia neomajor krammer . . . 1 . . 42. staurosirella leptostauron (ehrenberg) d.m. williams et round . . . 1 . . 43. staurosirella pinnata (ehrenberg) d.m. willams et round . . . 1 . . 44. surirella spiralis kützing . . . 1 . . 45. nitzschia acidoclinata lange-bertalot . . . . 1 . 46. pinnularia subrupestris krammer . . . . 1 . 47. cocconeis pseudolineata (geitler) lange-bertalot . . . . . 1 48. cymbopleura austriaca (grunow) krammer . . . . . 1 a total number of taxa (n) 16 22 17 22 13 10 b ips/20 18.3 18.3 17.7 15.0 17.0 17.0 c sla/20 16.4 17.1 17.7 14.7 17.2 16.5 d rott ti/20 (tid) 11.6 8.5 11.5 6.5 11.6 12.0 e rott ti/20 (sid) 16.5 16.9 16.5 16.7 16.4 17.1 biologica nyssana ● 11 (2) december 2020: 93-101 mašić et al. ● new data on distribution of hydrurus foetidus (villars) trevisan in freshwater habitats on vranica mountain (bosnia and herzegovina) altitude ranged between 1465 and 1727 meters a.s.l. in all studied sites, the species was well developed. according to literature data (klaveness, 2017, 2019) h. foetidus appears under different ecological conditions. this species is a rheophile, preferring swiftly flowing water. it is also a psychrophile and dependent on low temperature. the species hydrurus foetidus is visibly a large freshwater alga found in cold rivers. generally, the species may be found during late winter months, sometimes forming dense cover on pebbles and rocks at the river bottom and less prominent in the spray zone. the speci es is usually well developed during the colder months of the year but disappears in summer with the increasing flow (klaveness, 2019). according to klaveness (2017) this species is an indicator of clean water and good ecological status, but there is a various observation that some population tolerate mesosaprobic or oligomesosaprobic condition. in some occasions, h. foetidus thrives well under seasonal climatic conditions, where there is snow in the winter, cold meltwater and moderate summer temperatures (klaveness, 2019). by comparing our results, with the results of other literature data, a certain similarity of ecological characteristics of habitas can be obtained. the results of our study are correlated with other research conducted in the balkan peninsula dealing with ecology of h. foetidus (blagojević & hafner, 1979; redžić, 1988; krizmanić et a., 2008; stanković & leitner, 2016) especially in terms of habitats characteristics and physical and chemical parameters of water. on the site in the river neretva (redžić, 1988) water temperature ranged from to 11.5 – 14.9 °c, ph value varied between 7.5 – 8.3 and dissolved oxygen ranged between 10.6 – 12.3 mgl-1. the distribution and ecological characteristics of h. foetidus in serbia are given by the authors krizmanić et al. (2008). at two investigated sites, water temperature ranged from 4.6 to 9.6 °c, while the oxygen concentration ranged from 11.0 to 15.1 mgl-1. by summarizing a large number of data, but also on the basis of the study carried out, the authors concluded that the measured physical and chemical parameters of water confirm the rheophilic nature of this species. stanković & leitner (2016) recorded h. foetidus in croatia for the first-time during the environmental study of the bijela rijeka located within the plitvice lakes national park. the water temperature on the studied site was 9.0 °c, ph value 8.3 while the specific electrical conductivity was 441 μscm-1. the concentration of oxygen was 11.02 mgl-1, cod 1.32 mgo2l -1, and the alkalinity had the values of 259.5 mgcaco3l -1. in terms of measured nutrients, ammonium was not found in the studied sites, the nitrates had low values (0.003 mgnl-1), while the nitrates had somewhat higher values (0.759 mgnl1). on the studied site orthophosphates were also measured, and their value was 0.025 mgpl-1. the average water velocity was 0.60 ms-1 and the water depth ranged from 20 to 15 cm. during investigation of algal assemblages in springs of the konjuh mountain golden algae phaeodermatium rivulare hansgirg (chrysophyceae) was identified (kamberović et al., 2019). unfortunately, species which are very common in alpine springs, such as h. foetidus was not recorded in this study. the main reason for absence of this species was due to the lower water velocity and different geological substratum (kamberović et al., 2019). in addition to h. foetidus which indicates clean water and good ecological status of freshwater habitats according to literature data (klaveness, 2019), ascertained diatoms indicate the same or similar environmental conditions. the results of diatom indices confirmed the good water quality of the studied sites on which h. foetidus was found. along with h. foetidus, the authors stanković & leitner (2016) found certain diatoms and bluegreen algae in the bijela rijeka which is located in the plitvice lakes national park. in addition to h. foetidus same authors identified two macroalgae, namely zygnema sp. and vaucheria sp. during complex ecological studies on algal assemblages in the krivaja river (bosnia and herzegovina) authors blagojević & hafner (1979) noted the presence of h. foetidus. in all investigated samples 40 taxa are common and the most prominent are chamaesiphon incrustans, nostoc sphaericum, phormidium favosum, phormidium autumnale, vaucheria sp., diatom ehrenbergii, cocconeis pediculus, achnanthidium minutissimum, navicula tripunctata and encyonema ventricosa. despite previously conducted research on vranica mountain, the presence of rare and endangered species h. foetidus has not been noted. the main reason lies in the fact that the weather conditions that prevail in the area of the vranica mountain are very severe, and that earlier research was carried out during favourable climatic conditions and at a time when the species was no longer present. the species h. foetidus inhabits freshwater habitats in high altitudes that are under weak or moderate anthropogenic impacts. as it indicates the good ecological status of mountain springs and streams it can be used in bioindication of oligotrophy and good ecological status of freshwater habitat types 98 biologica nyssana ● 11 (2) december 2020: 93-101 mašić et al. ● new data on distribution of hydrurus foetidus (villars) trevisan in freshwater habitats on vranica mountain (bosnia and herzegovina) in the mountain area. in this respect, the habitats that provide conditions for the development of this alga have great conservation importance. according to the red list of slovakia, the species h. foetidus is classified into endangered categories (en), while the category of vulnerability is not included in the iucn red list (feráková et al., 2001). hydrurus foetidus develops thick mat on solid river bottom, therefore, it is a valuable food source for emerging waterborne insect larvae (milner et al., 2001, 2009; zah et al., 2001 in klaveness, 2017). hydrurus foetidus appears to be of specific importance for the early emerging aquatic larvae of chironomids (klaveness, 2019). in a study conducted by the authors moog & janecek (1991) in the rivers of the central alps, it was determined that the distribution of biomass macrozoobenthos varies significantly if different substrates are compared, and that a positive correlation is determined by the biomass of the benthic macroalgae h. foetidus, and negative correlation is determined by the flow of water. conclusions the presented study suggests that the species h. foetidus prefers freshwater habitats located at higher altitudes. it usually occurs during the cold months, but if the conditions permit, it can occur even during the warm months when the thallus starts to disintegrate slightly. the physical and chemical characteristics measured on the investigated sites corresponded to the literature data. habitats providing conditions for the development of h. foetidus on vranica mountain have a good water quality. in our study distributional data are presented, and also environmental characteristics of habitats in which h. foetidus usually occures. in order to expand knowledge about the ecological characteristics of macroalgae in the future, it is necessary to conduct more detailed research not only in the area of vranica mountain but also on the other mountains of bosnia and herzegovina. in addition to measuring the basic physical and chemical parameters of water, it is necessary to take into account other parameters, such as nutrients and heavy metals. this study contributes to algological research in bosnia and herzegovina and also give good basis for future investigation of macroalgae in this area. acknowledgements. this study is a part of a project entitled as: “conservation of freshwater habitat types on vranica mountain and establishment of long-term monitoring of biodiversity” which is funded by rufford foundation (application id 24578-1). we would like to thank the two anonymous reviewers for their suggestions and comments. references aganović, m. 1981: istraživanje ekosistema na području srednjeg toka vrbasa područje uticaja “he bočac”. blagojević, s., hafner, d. sastav algi. elaborat. biološki institut univerziteta u sarajevu. aysel, v. 2005: check-list of the freshwater algae of turkey. journal of black sea/mediterranean environment, 11: 1-124. barinova, s., kukhaleishvili, l. 2017: diversity and ecology of algae and cyanobacteria in the aragvi river, georgia. elixir bio sciences, 104: 45934-45947. barudanović, s., mašić, e., macanović, a. 2017: tresetišta na bosanskim planinama. prirodnomatematički fakultet. sarajevo. 1-184. blagojević, s. 1966: prilog poznavanju algi kraških izvorišta u bosni i hercegovini. i. chrysophyceae, xanthophyceae, bacillariophyceae. godišnjak biološkog instituta, sarajevo, 29: 5-22. blagojević, s., hafner, d. 1979: ekološka istraživanja na cijanofitama i algama rijeke krivaje. godišnjak biološkog instituta, sarajevo, 32: 13-31. cantonati, m., kelly, g. m., lange-bertalot, h. 2017: freshwater benthic diatoms of central europe: over 800 common species used in ecological assesment. english edition with updated taxonomy and added species. koeltz botanical book. caraus, i. 2017: algae of romania. a distributional checklist of actual algae. version 2.4. studii si cercetari biologie, 7: 1-1002, 1 map. coste, m. 1982: étude des méthodes biologiques d’appréciation quantitative de la qualité des eaux. rapport cemagref qe lyon-af bassin rhône méditerranée corse. feráková, v., maglocký, š., marhold, k. 2001: červený zoznam paprodostov a semenných raslín slovenska (december 2001) – in: baláž, d., marhold, k. & urban, p. eds., červený zoznam rastlín slovenska, ochrana prírody, 20 (suppl.): 48-81. guiry, m. d., guiry, g. m. 2019: algaebase. worldwide electronic publication, national university of ireland, galway. http://www.algaebase.org; 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(anon. eds), 57-68. wehr, j. d., sheath, r. g., kociolek, j. p. 2015: freshwater algae of north america: ecology and classification. elsevier. whitton, b. a., john, d. m., kelly, m. g., haworth, e. y. 2003: a coded list of freshwater algae of the british isles. second edition. worldwide web electronic publication. zah, r., burgherr, p., bernasconi, s. m., uehlinger, u. 2001: stable isotope analysis of macroinvertebrates and their food sources in a glacier stream. freshwater biology, 46: 871–882. 101 biologica nyssana ● 11 (2) december 2020: 93-101 mašić et al. ● new data on distribution of hydrurus foetidus (villars) trevisan in freshwater habitats on vranica mountain (bosnia and herzegovina) dimitrov, d.s.: floristic and habitat diversity of dospat dere in the dabrash mountain (west rhodopes), bulgaria. biologica nyssana, 8 (1), september 2017 biologica nyssana 8 (1)  september 2017: 39-45 dimitrov, d.  floristic and habitat diversity of dospat dere… 39 original article received: 09 april 2017 revised: 17 july 2017 accepted: 02 september 2017 floristic and habitat diversity of dospat dere in the dabrash mountain (west rhodopes), bulgaria dimitar s. dimitrov national natural history museum, 1 tsar osvoboditel blvd., sofia 1000, bulgaria * e-mail: dimitrov.npm@gmail.com abstract: dimitrov, d.s.: floristic and habitat diversity of dospat dere in the dabrash mountain (west rhodopes), bulgaria. biologica nyssana, 8 (1), september 2017: 39-45. a research has been done of the flora and natural habitats of the dospat dere area, which is located on the right bank of the dospat river, between the tuhovitsa and zhizhevo villages, next to the border with greece. the systematic specter of the flora contains 197 species of vascular plants (excluding the moss species). these vascular plants are referred to 143 genera and 57 families. as a result of this research, 5 habitats were established: 10 е1 pseudo-steppe with grasses and annual of the thero-brachypodietea, 08h3 calcareous rocky slopes with chasmophytic vegetation, 21g1 supra mediterranean hop-hornbeam woods, 02g1 southern helleno-balkanic swamp alder woods and 07g1 helleno-balkanic riparian plane forests. key words: floral analysis, floristic elements, natural habitats apstrakt: dimitrov, d.s.: floristički i stanišni diverzitet dospat derea na planini dabraš (zapadni rodopi), bugarska. biologica nyssana, 8 (1), septembar 2017: 39-45. izvršeno je istraživanje flore i prirodnih staništa oblasti dospat dere, koja se nalazi na desnoj obali reke dospat, između sela tuhovica i žiževo, pored granice sa grčkom. sistematski spektar flore sadrži 197 vrsta vaskularnih biljaka (sa izuzećem mahovina). ove vaskularne biljke se svrstavaju u 143 roda i 57 familija. kao rezultat ovog istraživanja, utvrđeno je 5 staništa: 10 e1 psedostepe sa travama i jednogodišnjim biljkama therobrachypodietea, 08h3 krečnjačke stene sa hazmofitskom vegetacijom, 21g1 supramediteranske šume crnog graba, 02g1 južne grčko-balkanske močvarne šume jove i 07g1 grčko-balkanske riparijalne ravničarske šume. ključne reči: floristička analiza, florni elementi, prirodna staništa introduction the dabrash mountain is the westernmost part of the rhodopes mountain range, which happens to be the oldest piece of land on the balkan peninsula. its highest peak is located in the northern part of this area: beslet peak (1937 m height). its geological makeup is rather variable. there are predominantly silicate rocks: granite and granite gneiss. however, there are also basic and ultrabasic rocks, especially in the southern part, where the investigated area is located. the rocks are marble dating back to the 8 (1) • september 2017: 39-45 doi: 10.5281/zenodo.963435 biologica nyssana 8 (1)  september 2017: 39-45 dimitrov, d.  floristic and habitat diversity of dospat dere… 40 proterozoic eon (s t e f a n o v , 1981). the highest peaks here are pobit kamak 1086 m and chukata 1010 m (fig. 1). there are several caves in this area: mecha dupka and gargina dupka. the climate is transitional continental, since the valley of the dospat river follows a meridional direction and thus allows for strong mediterranean climate influence. the average annual temperature here is 10 °с. the average temperature in january is between 0 °с and 7 °с. there are predominantly autumn-winter and spring-summer rainfalls. the composition of the rocks is similar to the one in northern and southern pirin, stargach, slavyanka and trigradsko zhdrelo, which of course has an impact on the flora in this area. until now, the flora of the dabrash mountain has not been researched specifically in details. there are some brief notifications about some new and rare species for the bulgarian flora (k i t a n o v , 1936) and also about some new localities of macedonian pine (j o r d a n o v , 1939, к i t a n o v , 1939b). there are new chorological data for 25 new species of the floristic area in the west rhodopes (dabrash) which are reported by d i m i t r o v & v u t o v (2016). material and methods the researched area is 1.35 кm2. the species determination was done according to: j o r d a n o v (1963-1979), v e l c h e v (1982-1989), k o z u h a r o v (1992), d e l i p a v l o v & c h e s h m e d i j e v (2004), p e e v (2012), t u t i n et al. (1964-1980). the transect and trial sites methods were used for the determination of the vegetation in this case. the habitats were determined according to b i s s e r k o v et al. (2015). the geoelemental makeup was analyzed according to a s s y o v & p e t r o v a (2012). the species` category with conservation status was determined according to p e t r o v a & v l a d i m i r o v (2009). the protected species` status is based on the law for biological diversity of the republic of bulgaria. the herbarium specimens have been deposited in the herbarium of the institute of biodiversity and eco systematic research (som). results and discussion the systematic specter of the flora of dospat dere is comprised mainly with specimens from phylum magnoliophyta – 52 families (91.2%), 137 genera (90.6%) and 188 species (95.4%). from class dicotyledonae – 45 families (78.8%), 109 genera (76.2%), and 152 species (77.1%). from class monocotyledonae – 7 families (12.2%), 28 genera (19.5%) and 36 species (18.2%). from phylum gymnospermae there are 2 families (3.5%), 2 genera (1.3%) and 4 species (2.03%). from phylum pteridophyta – 3 families (5.2%), 4 genera (2.7%) and 5 species (2.5%). out of the examined families, the ones with the biggest number of species are: fabaceae (23 species; 11.6% of the total number of species), poaceae (17; 8.6%), asteraceae (15; 7.6%), caryophyllaceae (14; 7.1%) and lamiaceae (11; 5.5%). other families contain less than 10 species: apiaceae, rosaceae, scrophulariaceae (7; 3.5%), orchidaceae (6; 3.0%), liliaceae, brassicaceae, cyperaceae (5; 2.5%), campanulaceae, dipsacaceae, linaceae (4; 2.03%), polygalaceae, ranunculaceae, aspleniaceae, betulaceae (3; 1.5%), juniperaceae, pinaceae, boraginaceae, cornaceae, cistaceae, crassulaceae, euphorbiaceae, geraniaceae, hyperica fig. 1 – a geographical position of the investigated territory in western rhodopes (tourist map, domino, stara zagora, isbn 978-954-651-2055, m= 1:100 000) and on the balkan peninsula (down left) biologica nyssana 8 (1)  september 2017: 39-45 dimitrov, d.  floristic and habitat diversity of dospat dere… 41 ceae, loranthaceae, oleaceae, orobanchaceae, rubiaceae (2; 1.02%), polypodiaceae, equisetaceae, aceraceae, amarillidaceae, anacardiaceae, asclepiadaceae, cannabaceae, convolvulaceae, dioscoreaceae, fagaceae, globulariaceae, iridaceae, plantaginaceae, platanaceae, plumbaginaceae, polygonaceae, rutaceae, salicaceae, santalaceae, thymeleaceae, ulmaceae, urticaceae, valerianaceae, violaceae (1; 0.5%). according to biological type, there are predominately perennial herbaceous species (120 species; 60.8% of the total number of species), followed by annual species (29; 10.1%), arboreal species (10; 5.07%), bushes (10; 5.07%), annual to biennial species (9; 4.51%), semi-bushes (7; 3.5%), biennial (4; 2.02%), biennial to perennial (4; 2.07%) and annual to perennial species (4; 2.07%). based on the conducted research, the following floristic elements were determined: there are predominantly mediterranean elements (34 species; 17.7% of the total number of species), followed by euro-asiatic (23; 11.6%), euromediterranean (19; 9.6%), pontic-mediterranean (16; 8.1%), mediterranean (14; 7.1%), balkan endemics (10; 5.07%), euro-siberian (9; 4.5%), boreal, european (8; 4.5%), subboreal (6; 3.4%), euro-submediterranean elements (5; 2.53%), subpontic and pontic elements (4; 2.03%), mediterano-central asiatic, apennino-balcanic and balcano-anatolian (3; 1.5%), submediterranoasiatic, euro-central asiatic, mediterrano-asiatic, euro-orientalo turanian, subbalcanic, kosmopolitian (2; 1.01%), mediterano-orientalo turanian, ponto-submediterranean, ponto-balcanic, alpo-mediterranean, south siberian, bulgarian, carpato-balcanic and ponto-submediterranean elements (1; 0.5%). the genera represented by the biggest number of species are: trifolium, silene: (5 species; 2.5% of the total number of species), carex, scabiosa, vicia, linum (4; 2.03%), scorzonera, dianthus, astragalus, coronilla, ajuga, festuca, polygala, veronica (3; 1.5%). the floristic complex of the investigated flora includes 1 bulgarian endemite cerastium velenovskyi and 13 balkan endemites: anthemis macedonica, chondrilla urumoffii, onosma thracica, trachelium rumelianum, dianthus cruentus, dianthus drenovskyanus, scabiosa triniifolia, hypericum umbellatum, micromeria dalmatica, linum thracicum, orobanche esulae, festuca penzesii and silene frivaldszkyana. there are also 20 tertiary relics, some of them being: trachelium rumelianum, chondrilla urumoffii, onosma thracica, festuca penzesii. the research has found 15 species with conservation status: galanthus elwesii hook f. en b1ab(ii,iii,iv,v)+2ab(ii); c2a(i) (e v s t a t i e v a , 2009), bda, annex 2, 3. chondrilla urumoffii degen vu b2ab(ii,iii) (d i m i t r o v a , 2009), bda, annex 3 trachelium rumelianum hampe vu b1ab(ii,iii,iv) (g o r a n o v a & a n c h e v , 2009), bda, annex 2, 3. romulea bulbocodium (l.) sebast. & mauri vu b2ab(ii,iv); c2a(i) (m e s h i n e v , 2009), bda, annex 2, 3. limodorum abortivum (l.) schwartz vu b2b(iv)c(iv) (p e t r o v a , 2009), bda, annex 2, 3. ophrys cornuta steven vu b2b(ii,iv)c(iv) (p e t r o v a 2009), bda, annex 2, 3. lotus aegaeus (griseb.) boiss. lc (s o p o t l i e v a , 2009) arabis collina ten. bda, annex 2, 3. trinia glauca (l.) dumort. ssp. carniolica (a. kerner ex janchen) h. wolff. bda, annex 2. veronica multifida l. bda, annex 2, 3. dianthus drenowskyanus rech. f. bda, annex 3. lilium martagon l. bda, annex 4 asparagus tenuifolius lam., bda, annex 4. stipa tirsa steven, bda, annex 4. pulsatilla montana (hoppe) rchb. bda, annex 4. out of these species, 7 are protected by the law for biological diversity of the republic of bulgaria (annex 2, 3): galanthus elwesii hook. f., trachelium rumelianum hampe, romulea bulbocodium (l.) sebast. & mauri, limodorum abortivum (l.) schwartz, ophrys cornuta steven, arabis collina ten. and veronica multifida l. one species trinia glauca (l.) dumort. ssp. carniolica (a.kerner ex janchen) h.wolff is reported in annex 2. two species chondrilla urumoffii degen and dianthus drenowskyanus rech. f. are in annex 3 and 4 species lilium martagon l., asparagus tenuifolius lam., stipa tirsa steven and pulsatilla montana (hoppe) rchb. are in annex 4. three species from the researched flora are reported in the red data book of the republic of bulgaria, v.1 plants and fungi (p e e v et al., 2015): dianthus drenovskyanus rech. f. cr b2b(ii,v)c(ii,iv); d (d e n c h e v & a s s y o v , 2015), chondrilla urumoffii degen vu b2ab(ii,iii) (d i m i t r o v a , 2015) and trachelium rumelianum hampe vu b1ab (ii,iii,iv) (g o r a n o v a & a n c h e v , 2015). six species are also under the protection of the cites convention: cephalanthera rubra (l.) rich., epipactis atrorubens (hoffm.) besser, limodorum biologica nyssana 8 (1)  september 2017: 39-45 dimitrov, d.  floristic and habitat diversity of dospat dere… 42 abortivum (l.) schwarz, ophrys cornuta steven, orchis coriophora l. and platanthera chlorantha (custer) rchb. out of all the determined species in this research, there are 75 (38% of the total number) medicinal plants. regarding the natural habitats here, there are: 10 е1 pseudo-steppe with grasses and annual of the thero-brachypodietea, 08h3 calcareous rocky slopes with chasmophytic vegetation, 21g1 supra mediterranean hop-hornbeam woods, 02g1 southern helleno-balkanic swamp alder woods and 07g1 helleno-balkanic riparian plane forests. in general, the habitat 10 е1 pseudo-steppe with grasses and annual of the thero-brachypodietea has a conservation status: vulnerable [vua1,2 b1 c1 d2 e2 f2 g2 h2 i l2] (g u s s e v , 2015). however, this habitat in the west rhodopes has not been reported in the literature (b i s e r k o v et al., 2015). at the moment, the habitat is being used as a grazing land for domestic sheep and goats. the habitat 08н3 calcareous rocky slopes with chasmophytic vegetation has a conservation status: vulnerable [vu – a1, 2 b2 d2 h2 i j] (g u s s e v & r u s s a k o v a , 2015). the habitat 21g1 supra mediterranean hophornbeam woods has a conservation status: vulnerable [vua1 b2 c1 d2 e2 f1 g1 h2i l2] (r u s s a k o v a & t a s h e v , 2015). it is located on the steep and vertical slopes above the right bank of the dospat river. this habitat has not been reported either according to b i s e r k o v et al. (2015). the habitat 02g1 southern helleno-balcanic swamp alder woods has a conservation status: endangered [en – a1, 2 c2 d2 e2 f2 g2 h2 i j l2] (d i m i t r o v & t a s h e v , 2015). this habitat also has not been reported according to b i s e r k o v et al. (2015). the habitat 07g1 helleno-balcanic riparian plane forest has a conservation status: endangered [en – a1, 2 c1 d1 e2 f2 g2 h2 l3] (g o g o u s h e v , 2015). this habitat has not been reported according to b i s e r k o v et al. 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(eds.). 2009: red list of bulgarian vascular plants. phytologia balcanica, 15 (1): 63-94. sopotlieva, d. 2009: lotus aegaeus. in: petrova, a. & vladimirov, v. (eds.). red list of bulgarian vascular plants. phytologia balcanica, 15 (1): 92. stefanov, p. 1981: peculiarities of karst in dabrash. in: national congress of geographs in bulgaria. 4. collection of summary and reports. tutin, t. g., heywood, v. h., burges, n. a., moore, d. m., valentine, d. h., walters, s. m., webb, d. a. (eds.). 1964-1980: flora europaea, 1-5. cambridge, university press. london. appendix list of determined plants from the researched area pteridophyta aspleniaceae 1. asplenium ruta-muraria l. 2. a. trichomanes l. 3. ceterach officinarum dc. polypodiaceae 4. polypodium vulgare l. equisetophyta equisetaceae 5. equisetum palustre l. pinophyta juniperaceae 6. juniperus communis l. 7. j. oxycedrusl. pinaceae 8. pinus nigra arnold 9. p. sylvestris l. magnoliophyta aceraceae 10. acer campestre l. ssp. campestre amaryllidaceae 11. galanthus elwesii hook.f. en apiaceae 12. eryngium campestre l. 13. ferulago sylvatica (besser) rchb. 14. orlaja daucoides (l.) greuter 15. o. grandiflora (l.) hoffm. 16. peucedanum alsaticum l. 17. torilis japonica (houtt.) dc. 18. trinia glauca (l.) dumort. anacardiaceae 19. pistacia terebinthus l. asclepiadaceae 20. vincetoxicum hirundinaria medicus ssp. nivale (boiss. et heldr.) markgr. asteraceae 21. achillea setacea waldst. & kit. 22. anthemis macedonica boiss. ssp. orbelica (balkan endemic) 23. carlina acanthifolia all. 24. chondrilla urumoffii degen balcan end. vu, bda 25. crepis sancta (l.) babc. 26. inula aschersoniana janka (balkan endemic) 27. leontodon crispus vill. 28. i. ensifolia l. 29. jurinea mollis (l.) rchb. ssp. anatolica (boiss.) stoj.et stef. 30. scorzonera hispanica l. 31. s. laciniata l. 32. s. purpurea l. 33. senecio vernalis waldst.& kit. 34. tanacetum vulgare l. 35. tragopogon dubius scop. betulaceae 36. alnus glutinosa (l.) gaertn. 37. carpinus orientalis mill. 38. ostrya carpinifolia scop. boraginaceae 39. buglossoides purpurocaerulea (l.) i.m.johnst. 40. onosma thracica velen. balcan.end. brassicaceae biologica nyssana 8 (1)  september 2017: 00-00 dimitrov, d.  floristic and habitat diversity of dospat dere… 44 41. aethionema saxatile (l.) r.br. 42. alyssum alyssoides (l.) l. 43. arabis collina ten. 44. a. sagittata (bertol.) dc. 45. erysimum diffusum ehrh. campanulaceae 46. asyneuma canescens (waldst. & kit.) griseb. et schenk 47. campanula lingulata waldst. & kit. 48. c. persicifolia l. 49. trachelium rumelianum hampe (balkan endemic) vu, bda cannabaceae 50. humulus lupulus l. caryophyllaceae 51. cerastium luridum guss. 52. c. velenovskyi hayek bul. end. 53. dianthus cruentus griseb. (balkan endemic) 54. d. drenowskyanus rech. f. (balkan endemic) 55. d. petraeus waldst. & kit. 56. herniaria hirsuta l. 57. lychnis coronaria (l.) desr. 58. minuartia mesogitana (boiss.) hand.-mazz. 59. petrorhagia prolifera (l.) p.w.ball & heywood 60. silene conica l. 61. s. flavescens waldst. & kit. 62. s. frivaldszkyana hampe (balkan endemic) 63. s. italica (l.) pers. 64. s. radicosa boiss. & heldr. convolvulaceae 65. convolvulus cantabrica l. cornaceae 66. cornus mas l. 67. cornus sanguinea l. cistaceae 68. fumana procumbens (dunal) gren. & godr. 69. rhodax alpestris (jacq.) fuss. crassulaceae 70. sedum alpestre vill. 71. s. anopetalum dc. cyperaceae 72. blysmus compressus (l.) panz. ex link 73. carex distans l. 74. c. divisa huds. 75. c. hirta l. 76. c. liparocarpos gaudin dipsacaceae 77. scabiosa argentea l. 78. s. columbaria l. 79. s. ochroleuca l. var. ochroleuca 80. s. triniifolia friv. (balkan endemic) dioscoreaceae 81. tamus communis l. euphorbiaceae 82. euphorbia myrsinites l. 83. e. niciciana borbas f. niciciana fabaceae 84. anthyllis vulneraria l. ssp. pulchella (vis.) bornm. 85. astragalus glycyphyllos l. 86. a. monspessulanus l. 87. a. onobrychis l. 88. coronilla emerus l. 89. c. scorpioides (l.) c. koch 90. c. varia l 91. dorycnium herbaceum vill. 92. genista lydia boiss. 93. lotus aegaeus (griseb.) boiss. lc 94. medicago minima (l.) bartal 95. onobrychis gracilis besser 96. ononis adenotricha boiss. 97. o. pusilla l. 98. trifolium alpestre l. 99. t. arvense l. 100. t. campestre schreb. var. campestre 101. t. incarnatum l. 102. t. scabrum l. ssp. scabrum 103. vicia cracca l. 104. v. grandiflora scop. 105. v. varia host 106. v. villosa roth fagaceae 107. quercus pubescens willd. geraniaceae 108. geranium robertianum l. 109. g. sanguineum l. globulariaceae 110. globularia aphyllanthes crantz hypericaceae 111. hypericum rumeliacum boiss. 112. h. umbellatum a. kern. (balkan endemic) iridaceae 113. romulea bulbocodium (l.) sebast. & mauri, vu, bda juglandaceae 114. juglans regia l. lamiaceae 115. ajuga chamaepitys (l.) schreb. 116. a. genevensis l. 117. a. laxmannii (l.) benth. 118. calamintha sylvatica bromf. 119. micromeria dalmatica benth. (balkan endemic) 120. salvia glutinosa l 121. sideritis montana l. 122. stachys recta l. 123. teucrium polium l. 124. thymus callieri borb.ex velen. 125. th. striatus vahl. liliaceae 126. allium moschatum l. 127. anthericum liliago l. biologica nyssana 8 (1)  september 2017: 39-45 dimitrov, d.  floristic and habitat diversity of dospat dere… 45 128. asparagus tenuifolius lam. 129. lilium martagon l. 130. muscari comosum (l.) mill. linaceae 131. linum austriacum l. 132. l. tenuifolium l. 133. l. nervosum waldst. & kit. 134. l. thracicum (griseb.) degen (balkan endemic) loranthaceae 135. arceutobium oxycedri (dc.) m.bieb. 136. viscum laxum boiss. & reuter ssp. laxum oleaceae 137. fraxinus ornus l. 138. ligustrum vulgare l. orchidaceae 139. cephalanthera rubra (l.) rich. 140. epipactis atrorubens (hoffm.) besser 141. limodorum abortivum (l.) schwarz, vu, bda 142. ophrys cornuta steven vu, bda 143. orchis coriophora l. 144. platanthera chlorantha (custer) rchb. orobanchaceae 145. orobanche elatior sutton 146. o. esulae pancic plantaginaceae 147. plantago lanceolata l. platanaceae 148. platanus orientalis l. plumbaginaceae 149. armeria rumelica boiss. (balkan endemic) poaceae 150. aegilops neglecta req. ex bertol. 151. bromus intermedius guss. 152. b. sterilis l. 153. chrysopogon gryllus (l.) trin. 154. festuca nigrescens lam. 155. f. penzesii(acht. )markgr.-dann. (balkan endemic) 156. f. spectabilis jan. ssp. affinis (boiss. & heldr. ex hack.) hack. 157. koeleria nitidula velen. 158. melica ciliata l. 159. molinia coerulea (l.) moench 160. phleum pratense l. 161. sclerochloa dura (l.) p.beauv. 162. sesleria alba sm. 163. stipa tirsa steven 164. trachynia distachya (l.) link 165. vulpia ciliata dumort. 166. v. myurus (l.) c.c.gmel. polygalaceae 167. polygala comosa schkuhr 168. p. major jacq. 169. p. rhodopea (velen.) janch. (balkan endemic) polygonaceae 170. rumex acetosella l. ranunculaceae 171. pulsatilla montana (hoppe) rchb. 172. ranunculus sardous crantz 173. thalictrum aquilegifolium l. rosaceae 174. agrimonia eupatoria l. 175. crataegus monogyna jacq. 176. filipendula vulgaris moench 177. potentilla argentea l. 178. prunus spinosa l. 179. rosa micrantha borrer ex sm. 180. sanguisorba minor scop. ssp. muricatum briq. rubiaceae 181. asperula tenella heuff. ex deg. in kerner 182. cruciata laevipes opiz rutaceae 183. dictamnus albus l. salicaceae 184. salix alba l. santalaceae 185. thesium arvense horv. scrophulariaceae 186. euphrasia rostkoviana hayne 187. melampyrum sylvaticum l. 188. parentucellia latifolia (l.) caruel 189. rhinanthus wagneri degen ssp. wagneri 190. veronica annagalis-aquatica l. 191. v. chamaedrys l. 192. v. multifida l. thymeleaceae 193. thymelaea passerina (l.) coss. & germ. ulmaceae 194. ulmus minor mill. urticaceae 195. urtica dioica l. valerianaceae 196. valerianella rimosa bast. violaceae 197. viola alba besser ssp. scotophylla (jordan) nyman microsoft word 0402_stanisavljevic_et_al ispravljeno biologica nyssana 1 (1-2) december 2010: 89-93 stanisavljević, d.m. et al. effects of different drying methods… 89 original article ! effects of different drying methods on the yield and the composition of essential oil from herb mentha longifolia (l.) hudson dragana m. stanisavljević1, sofija m. đorđević2, mihailo s. ristić2, dragan t. veličković1, novica v. ranđelović3 1 college of agriculture and food technology, ćirila and metodija 1, 18400 prokuplje, serbia 2 institute for medicinal plant research "dr josif pančić", tadeuša košćuška 1, 11000 belgrade, serbia 3 university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia * e-mail: draganastanisavljevic72@gmail.com abstract: stanisavljević, d.m., đorđević, s.m., ristić, m.s., veličković, d.t., ranđelović, n.v.: effects of different drying methods on the yield and the composition of essential oil from herb mentha longifolia (l.) hudson. biologica nyssana, 1 (1-2), december 2010: 89-93. this paper discusses the impact of different methods of drying on the content and chemical composition of the essential oil from the herb mentha longifolia (l.) hudson. drying of plant material was carried out naturally in the shade of draughty place, in the laboratory oven at the temperature 45°c and absorptive low temperature condensation drying oven at 35°c (low temperature drying). isolation of essential oil from dried samples in three different ways was conducted by hydrodistillation, whilst chemical analysis was carried out by gc/fid and gc/ms methods. the highest yield of the essential oil was obtained from the herb which was dried at low temperature (1.1%) and the lowest from that dried in the laboratory oven (0.6%). the biggest content of the dominant component of essential oils, piperitone, was recorded in the oil from low temperature dried herb (71.7%), while those isolated from naturally dried drug and in from the laboratory oven contained piperitone in lower concentrations (50.8% and 43.1%, respectively). key words: drying, essential oil, mentha longifolia l., piperiton introduction mentha longifolia (l) hudson, (wild mint, english horsemint) belongs to the genus mentha, lamiaceae family. inhabits wet habitats, the rivers, lakes, ponds and channels, wet meadows, forested terrain, and even drier places along the roads and arable land. it is widespread in serbia except in vojvodina, where it can be found only on fruška gora. in south serbia it is widespread from the regions of lowlands, where it can be found along the roads and on the banks of rivers, to the hilly, near the streams in the community junco-menthetum longifolia. it is widespread throughout the mediterranean, central and northern europe, asia minor, africa (j a n k o v i ć , 1974; s t a m e n k o v i ć , 1995). wild mint is an aromatic and melliferous plant. it is used in the pharmaceutical, tobacco, food industry (in the development of various liqueurs and sweets), and especially in cosmetology. essential oil of english horsemint has pleasant and refreshing odour. according to the pdr exhibits carminative 10th sfses • 17-20 june 2010, vlasina lake1 (1-2) • december 2010: 89-93 biologica nyssana 1 (1-2) december 2010: 89-93 stanisavljević, d.m. et al. effects of different drying methods… 90 and stimulant properties of the gastrointestinal tract. relieves colds, respiratory inflammation, headaches, pain in muscles and joints. internally it is used in the form of infusion, and externally as bath additive (pdr, 2004). there are phytopreparations on the basis of the active ingredients of essential oils of mint (menthol, menthone, pinene, and cineole) in the form of cream, the solution of rubbing (alcohol, oil), syrup, capsule, coated tablet (rote liste, 1997). chemical composition of the essential oil of wild mint herb is very variable depending on the habitat and climate where the species grow. fortyfive constituents were identified in the essential oil of m. longifolia from turkey, with the cis-epoxy piperitone, pulegone and piperitenone oxide as main components, and studied oil exhibits strong antimicrobial activity (g u l l u c e et al., 2007). analysis of essential oil of moroccan m. longifolia showed interesting relative quantities of piperitenone oxide and piperitone oxide (ghoulami et al., 2001). in the essential oil of wild mint from south africa, 31 components were identified. menthone (50.9%), pulegone (19.3%) and 1,8cineole (11.9%) were the main ingredients of the oil (oyedeji and afolayan, 2006). analysis of oil of m. longifolia from italy and israel revealed piperitenone oxide as the main component, while the essential oil from sinai contained 1,8-cineole (28.8%), piperitone oxide (15.4%) and piperitone (13.8%) (m a f f e i , 1988; f l e i s h e r & f l e i s h e r , 1998). there is more then 70% of pulegone in the oil that grows in the desert of jordan (f l e i s h e r & f l e i s h e r , 1991). the dominant components of essential oil of wild mint herb in vojvodina are menthone, isomenthone and 1,8cineole, and the oil exhibits a strong antimicrobial and significant fungicidal effect (m i m i c a et al., 2003). wild mint from croatia contains carvone, piperitenone oxide, limonene and β-caryophyllene as the main ingredients (m a s t e l i ć & j e r k o v i c , 2002). the main components of oil types which were collected on zlatar are cisand transdihidrocarvone (15.9% and 30.6%) (m a t o v i ć & l a v a d i n o v i ć , 1999). piperitone oxide was found as the main component in nine populations of m. longifolia essential oils from greece (k o k k i n i & p a p a g e o r g i o u , 1988). drying is the easiest way of preservation of raw plant material. drying procedures are different and have an impact on the content of active substances in drugs. in the process of drying the plant material moisture content is reduced, but the amount and composition of volatile compounds are changed, too (m o y l e r , 1994). the way of drying has a significant impact on qualitative and quantitative composition of essential oils of aromatic plants. this paper presents the results of the impact of different methods of drying on the yield and the composition of essential oil of m. longifolia herb originating from southern serbia. material and methods plant material plant material was collected in the flowering stage in july 2009 from the municipality of prokuplje, at the rastovnica village, on the mountain pasjača. samples of collected plants are deposited in the herbarium hmd (herbarium moesiacum doljevac) № 233. plants were collected in the morning hours, in dry times and dried in three ways: 15 days in the shade of a draughty place, natural drying (nd), in the laboratory oven at the temperature of 45°c (lod) and in the low temperature absorptive condensation dryer (ntks/60s) at temperature of about 35°c (ltd). the drying air used was of low humidity and a minimally heated. dried plant material was packed in paper bags and kept in a dry and cool place. isolation and determination of the content of the essential oil isolation of the essential oil from dried and cut m. longifolia herb was carried out by water distillation in the clevenger type apparatus, according to procedure ph. jug. iv (ph. jug. iv, 1984). distillation lasted about 2 hours at the boiling temperature. pure essential oils were kept in dark glass ampoules at +4°c. these are colourless liquids, with the characteristic sharp odour. results of determination of the contents of the essential oils in the three studied samples of the herb m. longifolia represent the average value of three comparative analyses and refer to the dry samples. qualitative and quantitative analysis of the essential oils qualitative and quantitative analysis of essential oils was conducted by gc and gc/ms methods. analytical gas chromatography (gc/fid) gc/fid analysis of the oils was carried out on a hp-5890 series ii gc apparatus [hewlettpackard, waldbronn (germany)], equipped with split-splitless injector and automatic liquid sampler (als), attached to hp-5 column (25 m · 0.32 mm, 0.52 µm film thickness) and fitted to flame ionisation detector (fid). carrier gas flow rate (h2) was 1 ml/min, split ratio 1:30, injector temperature biologica nyssana 1 (1-2) december 2010: 89-93 stanisavljević, d.m. et al. effects of different drying methods… 91 table 1 the chemical composition of essential oils of herb m. longifolia dried by different procedures (%) constituents kie kil drying method nd lod ltd α-pinene 932.8 932 0.8 0.6 0.6 sabinene 973.2 969 0.7 0.5 0.4 β-pinene 975.6 974 1.3 0.9 0.8 myrcene 993.0 988 0.7 1.2 0.5 3-octanol 1000.2 988 0.3 0.2 limonene 1029.0 1024 6.3 1.6 2.4 1,8-cineole 1030.9 1026 3.9 3.6 3.5 cis-β-ocimene 1039.6 1032 1.3 0.8 1.2 trans-β-ocimene 1049.2 1044 0.4 linalool 1102.8 1095 0.4 menthone 1154.6 1148 0.6 17.5 iso-menthone 1165.1 1158 8.3 ocimenol 1169.4 n/a 0.3 menthol 1175.3 1167 0.3 cis-isopulegone 1177.1 n/a 0.7 α-terpineol 1193.4 1186 1.1 0.3 0.5 cis-dihydro carvone 1198.2 1191 3.5 0.6 0.3 trans-dihydro carvone 1205.4 1200 2.3 1.1 neoiso-dihydro carveol 1231.8 1226 0.8 cis-3-hexenyl isovalerate 1238.8 1232 0.5 pulegone 1240.7 1233 1.4 carvone 1247.1 1239 20.0 2.9 5.0 piperitone 1257.7 1249 50.8 43.1 71.7 dihydroedulan i 1288.6 1289 0.3 piperitenone oxide 1369.0 1366 2.7 β-bourbonene 1385.1 1387 0.8 β-elemene 1392.9 1389 0.3 trans-caryophyllene 1420.1 1417 4.3 4.1 5.4 α-humulene 1454.1 1452 0.3 γ-muurolene 1482.3 1478 3.1 4.3 3.6 bicyclogermacrene 1497.6 1500 0.4 1.1 0.8 sum of contents % 100.0 100.0 100.0 number of constituents 18 24 20 kie=kovats (retention) index experimentally determined (amdis), kil=kovats (retention) index literature data (a d a m s , 2007), n/a=not available, nd-natural drying , lod-laboratory drying oven, ltd-low temperature drying was 250°c, detector temperature 300°c, while column temperature was linearly programmed from 40-260°c (at rate of 4°/min). solutions of essential oils (~1%) were consecutively injected by als (1 µl, split mode). area percent reports, obtained as result of standard processing of chromatograms, were used as base for the quantification purposes. gas chromatography/mass spectrometry (gc/ms) the same analytical conditions as those mentioned for gc/fid were employed for gc/ms analysis, along with column hp-5ms (30 m · 0.25 mm, 0.25 µm film thickness), using hp g 1800c series ii gcd system [hewlett-packard, palo alto, ca (usa)]. instead of hydrogen, helium was used as carrier gas. transfer line was heated at 260°c. mass spectra were acquired in ei mode (70 ev), in m/z range 40-450. solutions of the essential oils (~1%) were injected by als (200 nl, split mode). the constituents were identified by comparison of their mass spectra to those from wiley275 and nist/nbs libraries, using different search engines. the experimental values for retention indices were determined by the use of calibrated automated mass spectral deconvolution and identification system software (amdis ver.2.1.), compared to those from available literature biologica nyssana 1 (1-2) december 2010: 89-93 stanisavljević, d.m. et al. effects of different drying methods… 92 (adams, 2007), and used as additional tool to approve ms findings. results and disscusion the content of the essential oil from aboveground part of m. longifolia, which has been dried on the three described ways, was variable. the highest yield is obtained from the herb dried at low temperature (1.1%), and then the herb which was dried naturally (0.9%), while the lowest yield was obtained in the laboratory oven (0.6%). chemical composition of essential oils is also variable (tab. 1). twenty-four components were identified in the essential oil obtained from wild mint herb that was dried in the laboratory oven. content of dominant component (piperitone) was 43.1%, along with 1,8-cineole, 3.6%, limonene 1.6%, menthone 17.5%, isomenthone 8.3%, carvone 2.9%, transcaryophyllene 4.1% and γ-muurolene 4.3%. in the essential oil obtained from the naturally dried herb 18 components were identified. content of piperitone was 50.8%, limonene 6.3%, 1,8cineole 3.9%, menthone only 0.6%, carvone 20.0 %, trans-caryophyllene 4.3%, γ-muurolene 3.1%. isomenthone was not recorded. in the essential oil from herb dried in low temperature oven 20 components were identified. content of piperitone was 71.7%, 1,8-cineole 3.5%, limonene 2.4%, carvone 5.0%, trans-caryophyllene 5.4% and γ-muurolene 3.6%. menthone and isomentone were not registered. changes in the concentrations of volatile compounds during drying depend on several factors, such as drying methods and classes of plants. mint belongs to the family lamiaceae, which is known to have storage of essential oil on or near the surface of the leaf (m o y l e r , 1994). pulegone has been proved to be a powerful hepatotoxin, even at low concentrations. it is metabolized by the liver to menthofuran, which is highly reactive metabolite, and it has adverse effect on the liver (c h e n et al., 2001, g o r d o n et al., 1987), and it can also destroy the rat cytochrome p450 (m o o r t h i , 1991). in our tests we can find pulegone only in the oil which was isolated from wild mint herb dried in laboratory oven. conclusion from the results it can be concluded that the highest essential oil content was found in plant material dried at low temperature, then dried naturally, and the lowest content was found in the plant material dried in laboratory oven. the content of the major constituent, piperitone, is reduced in the same order (71.7%, 50.8%. 43.1%). pharmacologically active menthol, cineole, limonene and pinene are the most represented in the oil from the naturally dried herb. so, drying of plant material for isolation of essential oils at low temperatures is could be marked as the best method. simultaneously, draying in the natural way is quite acceptable. pulegone is present only in the oil obtained from the dried herb in a laboratory oven, and from the security aspects this method of drying should be avoided. acknowledgements. the authors wish to thank serbian ministry of science for financial support project № tr 20049. references adams, r. 2007: identification of essential oil components by gas chromatography/mass spectrometry, 4th ed., allured publishing corp., carol stream, il 60188 usa. chen, lj., lebetkin, eh., burka, lt. 2001: metabolism of (r)-(+)-pulegone in f344 rats. drug metabolism and disposition, 29(12): 1567– 1577. farmakopeja sfrj, pharmacopoea jugoslavica, editio quatra (ph. jug. iv), 1984, savezni zavod za zdravstvenu zaštitu, beograd. fleisher, a., fleisher, z. 1991: the essential oils from mentha longifolia growing in sinai and israel aromatic plants of the holy land and the sinai. part iv. journal of essential oil research, 3(1): 57-8. 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(1995): neškodljive lekovite biljke. i izdanje, solaris, leskovac. williams, ac., barry, bw. 1991: terpenes and the lipid-protein-partitioning theory of skin penetration enhancement. pharmaceutical research, 8:17–24. vasić et al., 2019, biologica nyssana 10(1) 10 (1) september 2019: 17-21 doi: 10.5281/zenodo.3463990 phytopathogenic fungi causers fungal diseases of the faba bean (vicia faba l.) in serbia original article tanja vasić faculty of agriculture, university of niš, kosančićeva 4, 37000 kruševac, serbia tanjavasic82@gmail.com (corresponding author) sanja živković faculty of agriculture, university of niš, kosančićeva 4, 37000 kruševac, serbia gajicsanja43@gmail.com jordan marković institute for forage crops, 37251 kruševac, serbia jordan.markovic@ikbks.com ivana stanojević faculty of agriculture, university of niš, kosančićeva 4, 37000 kruševac, serbia stanojevic.ivana85@gmail.com sonja filipović faculty of agriculture, university of niš, kosančićeva 4, 37000 kruševac, serbia sonjafilipovic86@yahoo.com dragan terzić institute for forage crops, 37251 kruševac, serbia dragan.terzic.agro@gmail.com received: june 24, 2019 revised: september 8, 2019 accepted: september 9, 2019 abstract: faba bean (vicia faba l.) is one of the oldest legume crops mainly grown as a valuable protein-rich food, both for human and animal consumption, where, in latter case, it provides an alternative to soybean meals in the temperate regions. there has not been systematic research of faba bean mycoflora in serbia. this paper aims to present the results of preliminary investigation of mycopopulation of 10 different genotypes of faba bean. total of 400 plant parts were examined, and 6 genera of fungi were isolated: fusarium, rhizoctonia, phoma, sclerotinia, alternaria and ascochyta. the results indicate that faba bean is vulnerable to a large number of phytopathogenic fungi that can have a significant impact on reducing its yield and quality. key words: faba bean, mycopopulation apstract: fitopatogene gljive izazivači gljivičnih oboljenja boba (vicia faba l.) u srbiji bob (vicia faba l.) je najstarija kultura koja se uzgaja kao hrana bogata proteinima, kako za ljudsku, tako i za stočnu ishranu, predstavljajući alternativu sojinim obrocima u umerenim područjima. s obzirom da nije bilo sistematskog istraživanja mikoflore boba u srbiji, cilj ovog rada je da se predstave preliminarni rezultati mikopopulacije 10 različitih genotipova boba. ispitano je ukupno 400 biljnih delova i izolovano je 6 rodova gljiva: fusarium, rhizoctonia, phoma, sclerotinia, alternaria i ascochyta. dobijeni rezultati ukazuju da je bob osetljiv na veliki broj fitopatogenih gljiva koje mogu imati značajan uticaj na smanjenje prinosa i kvaliteta boba. ključne reči: bob, mikopopulacija introduction faba bean is an annual plant from the genus of vetches (vicia), the family of legumes (fabaceae). it is originally of african-asian origin, and it is grown all over the world. faba bean was known as an annual field legume since ancient times. faba bean was cultivated in ancient egypt, greece, and rome, as confirmed by numerous records. it was used for human nutrition as well as for green fertilisation. today, faba beans, both garden and fodder, are grown in many countries of the world. in europe it is cultivated in england, belgium, scandinavian countries, germany, russia and italy (mišković, 1986). in our country faba bean is grown in a low capacity, especially as a fodder. faba bean is a versatile fodder crop. the content of nutrients in the grain of the faba bean and the plant as a whole is significant. the grain contains about 85% dry matter, 25.1% of crude proteins, 46.8% of bem, 1.6% of crude fat, 9.4% of raw cellulose and about 3.5% of crude ash. because of this composition, faba bean serves as an excellent concentrate feed for most species of livestock. also, fabae bean grain is used in human nutrition, like stew, as it has a high nutritional value (vučković, 1999; aleksić et al., 2015). vicia faba l. can fix nitrogen through symbiosis with rhizobium leguminosarum in its root nodules (stoddard et al., 2010). the most important and widespread fungal diseases observed at all locations in the world are: rust (uromyces fabae), chocolate spot (botrytis fabae and b. cinerea), ascochyta blight (ascochyta fabae), leaf © 2019 vasić et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 17 spots (alternaria alternata and cercospora fabae) and wilt/root rot complex (fusarium oxysporum and macrophomina phaseolina dominating). other diseases of apparent minor importance recorded at low incidence levels at most locations in the world are sclerotinia blight (sclerotinia sclerotiorum), downy mildew (peronospora viciae) (akem and bellar, 1999; sillero et al., 2010; stoddard et al., 2010). in ethiopia chocolate spot (botrytis fabae and b. cinerea) and rust (uromyces fabae) are the major diseases which can reduce yield by about 61 and 21%, respectively (tegen, 2017). in croatia two fusarium species were determined on the seed vetch, f. verticillioides and f. proliferatum (miličević et al., 2013). since faba bean has become very important in recent years as a fodder crop in serbia, the aim of this paper was to determine the phytopathogenic fungi disease causative agents in faba bean for more precise understanding of the problems (plant die-off, reduction of yield and quality, etc.) that arise as a result of the presence of phytopathogenic fungi in faba bean. materials and methods the samples were collected between march and june 2016-2017 at the location of the institute for forage crops in globoder. the samples of faba bean seed for the study of mycoflora were taken from different sites: the rasina region (kruševac 1, kruševac 2, gaglovo 2), zaječar region (crni kao), nišava region (praskovče, rujevica, šarbanovac, sokobanja) and pomoravlje region (jagodina 3, oparić). parts of plants are carefully washed under running water. after washing, the parts of stem and roots were cut to pieces of 0.5-1 cm in size. prepared samples of roots and stems were disinfected with 96% ethanol for 10 seconds and with 1% sodium hypochlorite (naocl) for 1 minute and then washed three times in sterile distilled water. they were then dried on sterile filter paper and placed on potato dextrose agar (pda) with streptomycin. five pieces of the plant parts (root and stem) were each placed in petri dish in four replications. they were kept in a thermostat at 25°c in 12 h light / 12 h dark regime. the observations were performed every 3 days, and the majority of mycelium samples were developed up to 14 days. developed mycelia were screened to a new pda substrate and, after an initial grow, the peak part of the mycelium was reseeded on pda again. microscopic examination was performed using microscopes olympus cx31. morphological identification of fungi to the genus was carried out using a standard key. the frequency of isolation was calculated in percents according to the formula by vrandečić et al. (2011): results and discussion in these studies, the mycopopulation of faba bean genotypes was examined in a total of 400 plant parts. the isolation frequency of the pathogens from diseased samples largely confirmed the field diagnosis based on visual symptoms. there were clearly expressed symptoms on the leaves in the form of necrotic spots and lesions in all the plants where fungi were isolated. fungi of genera ascochyta and alternaria were isolated from these plants (tab. 1). likewise, necrosis with the presence of white, aerial mycelium was observed on stems in a large number of plants. fungi from the genus sclerotinia were isolated from the lower third of the stems of these (tab. 1). symptoms in the form of light to dark brown necrosis were present at the plant root system, and from these plants were isolated fungi from the genera fusarium, phoma and rhizoctonia (tab. 1). in these studies, there was a difference in the isolation frequency of some genera of phytopathogenic fungi in faba bean genotypes originating from different regions of serbia. in the samples from the rasina region (krusevac 1, krusevac 2, gaglovo 2), on the leaves, the infection by genus alternaria was 10-40%, and by genus ascochyta was represented in all tested samples at 30% (tab. 1). fungi from genera fusarium with 66.67% and phoma with 40% were dominant in the crowns of the root in the faba bean plants from rasina region (tab. 1). in the zaječar region (crni kao), the most represented fungi were ascochyta with 40% in leaves, and sclerotinia with 43.33% it roots (tab. 1). in the nišava region (praskovče, rujevica, šarbanovac, sokobanja), the prevalent species were from the genus alternaria with 80% in leaves, while in the root system, fungi from genus rhizoctonia were the most abundant with 46.67%. genera alternaria with 80% and sclerotinia with 60% were the most prevalent in pomoravlje region (jagodina 3, oparić) (tab. 1). the genera fusarium, phythophthora, rhizoctonia, phoma, verticillium, alternaria and sclerotinia were dominant in annual and perennial legumes in the world (tivoli et al., 2006; villegas-fernández and rubiales, 2011; salam et. al., 2011; sillero et al., 2014; o’sullivan and angra, 2016). miličević et al. (2013) determined two fusarium species, f. verticillioides and f. proliferatum in vetch seed in croatia. rhizoctonia solani kühn is a soil parasite that can 18 biologica nyssana ● 10 (1) september 2019: 17-21 vasić et al. ● phytopathogenic fungi causers fungal diseases of the faba bean (vicia faba l.) in serbia (%) isolation frequency number of segments containing the fungal species total number of segments used in the isolation x 100= cause serious problems on many legumes, especially on faba bean (assunção, 2011). in canada, 304 faba bean genotypes were tested for resistance to r. solani and only five were identified with high resistance (rashid and bernier, 1993). sclerotinia stem rot, a fungal disease caused by sclerotinia trifoliorum, is often a serious problem in faba beans (vicia faba) in greece (lithourgidis et al., 2005). chocolate spot is caused by botrytis cinerea pers. and b. fabae sard., the latter being the most important since its action can result in serious plant damage, which is not usually the case with b. cinerea. chocolate spot is especially severe in humid areas, having been reported to be the cause of heavy reductions in yields in places such as the maghreb, southern china, egypt, uk or france (tivoli et al., 2006). the type of alternaria tenuissima was detected on the broad bean in japan. the disease was found in all surveyed fields. the initial lesion was brown, water-soaked, circular to slightly irregular. then the lesion enlarged and became concentric. mature 19 leaves had coalescing necrosis surrounded by yellowing. older leaves of the plant were particularly affected. in a later stage of the disease, the leaves became blighted from the margin to the centre and most of the diseased plants defoliated (rahman et al., 2002). ascochyta blight, caused by ascochyta fabae speg., is a common and destructive disease of faba bean (vicia faba l.) in the middle east, europe, canada, new zealand (díaz-ruiz et al., 2009). in syria ascochyta blight were frequently isolated from infected samples showing typical chocolate spot symptoms (akem and bellar, 1999). symptoms occur on leaves, stems and pods of infected plants, and can be confused with the early stages of chocolate spot (botrytis fabae). on leaves, small, circular, dark-brown spots appear first. as the disease develops, lesions enlarge and turn light and then change to dark grey. they become irregular in shape, often zonate, and may coalesce to cover most of the leaf surface. leaf tissue next to the lesions may become black and necrotic. within the lesions, numerous pinhead-sized black fruiting bodies (pycnidia) of genotypes number of samples (plant part) fungi species (leaf) (%) isolation frequency fungi species (root) (%) isolation frequencyleaf root 3/ii jagodina 3 10 30 alternaria sp. 80 sclerotinia sp. 60 19 red rujevicasokobanja 10 30 alternaria sp. 80 sclerotinia sp. rhizoctonia sp. 26.67 20 41 red kruševac 1 10 30 alternaria sp. ascochyta sp. 30 30 fusarium sp. 66.67 7/ii oparić 10 30 alternaria sp. ascochyta sp. 30 10 fusarium sp. sclerotinia sp. 23.33 6.67 15 red šarbanovacsokobanja 10 30 alternaria sp. ascochyta sp. 60 10 fusarium sp. sclerotinia sp. 43.33 16.67 20 red crni kao 10 30 alternaria sp. ascochyta sp. 20 40 sclerotinia sp. 43.33 kruševac 2 10 30 alternaria sp. ascochyta sp. 10 30 phoma sp. rhizoctonia sp. 40 23.33 10 red gaglovo 2 10 30 alternaria sp. ascochyta sp. 40 30 rhizoctonia sp. fusarium sp. 33.33 26.67 4 red sokobanja 10 30 alternaria sp. ascochyta sp. 50 20 rhizoctonia sp. 46.67 13 red praskovče 10 30 alternaria sp. ascochyta sp. 30 30 fusarium sp. sclerotinia sp. rhizoctonia sp. 16.67 13.33 13.33 table 1. frequency of fungal isolation on vicia faba l. biologica nyssana ● 10 (1) september 2019: 17-21 vasić et al. ● phytopathogenic fungi causers fungal diseases of the faba bean (vicia faba l.) in serbia 20 the fungus develop. these appear only under moist conditions and are often concentrically arranged (elkomy, 2014). ascochyta blight is a common disease that causes up to 90% yield losses in susceptible cultivars when environmental conditions are favourable for disease development (díaz-ruiz et al., 2009). conclusion this paper presents preliminary results of mycopopulation of 10 experimental faba bean genotypes. the obtained results indicate that faba bean is susceptible to the attack by a large number of phytopathogenic fungi that can significantly reduce its yield and affect its quality. faba bean has more and more significance in our country, becoming an important fodder crop as livestock feed. this paper is the beginning of a more comprehensive study of phytopathogenic fungi in faba bean. so far, there has been no significant research in this field in serbia, so future investigations related to the selection of genotypes with increased tolerance to the disease-causing agents will be conducted. selection to disease resistance can be used as part of the integral plant protection program (ipp), which can include biological and agronomic measures, as well as cultivation of resistant cultivars and hybrids, and which aims to prevent economically significant damage and preserve the environment. principles and practice of ipp include the monitoring or other detection methods, accurate identification of the target pest (phytopathogenic fungi), population monitoring, rotation of products with different mechanisms of action, and the use of plant protection products when the target pest population (phytopathogenic fungi) threshold at the local level reaches economically significant level. references akem, c., bellar, m. 1999: survey of faba bean (vicia faba l.) diseases in the main faba bean-growing regions of syria. arab journal of plant protection, 17 (2): 113-116. aleksić, j., banović, b., miljuš-đukić, j., jovanović, ž., mikić, a., ćupina, b., zlatković, b., anđelković, s., spanu, i., jelić, m., maksimović, v. 2015: a rapid and cost-effective procedure for delineation and 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kostić kokić et al. 2025, biologica nyssana 16(2) 16 (2) december 2025: doi: 10.46793/biolnyss.16.2.1kk effects of freezing vegetables on nitrate content and their health-related risks original article ivana kostić kokić university of niš, faculty of sciences and mathematics, department of chemistry, višegradska 33, 18000 niš, serbia ivana.chem@outlook.com (corresponding author) tatjana anđelković university of niš, faculty of sciences and mathematics, department of chemistry, višegradska 33, 18000 niš, serbia danica bogdanović university of niš, faculty of sciences and mathematics, department of chemistry, višegradska 33, 18000 niš, serbia received: may 30, 2025 revised: june 12, 2025 accepted: july 30, 2025 abstract: vegetables are the major source of nitrate entry into the human body. the present study was conducted to investigate the influence of freezing on nitrate content in 15 species of vegetables from three commonly used groups: leafy, root and fruit vegetables. determination of nitrate ions in sample extracts was performed using ion chromatography. the highest level of nitrate content before freezing was determined in samples of radish, while the lowest content of nitrate in raw vegetables was determined in samples of pepper. in raw samples of leafy celery and cucumber, nitrates were not detected. results obtained by analyzing samples after freezing showed that this process has a different influence on the investigated samples. some samples contained a higher level of nitrate after freezing, while others contained less. part of the investigated vegetables showed an increased health risk if consumed in an amount of 400 g per day. key words: nitrate, vegetables, freezing, health risk, ion chromatography apstrakt: uticaj zamrzavanja povrća na sadržaj nitrata i njihov zdravstveni rizik povrće je glavni izvor unosa nitrata u ljudski organizam. ovo istraživanje je sprovedeno sa ciljem ispitivanja uticaja zamrzavanja na sadržaj nitrata u 15 vrsta povrća iz tri najčešće korišćene grupe: lisnato, korenasto i voćno povrće. određivanje nitratnih jona u ekstraktima uzoraka izvršeno je jonskom hromatografijom. najviši nivo sadržaja nitrata pre zamrzavanja određen je u uzorcima rotkve, dok je najniži sadržaj nitrata u sirovom povrću određen u uzorcima paprike. u sirovim uzorcima lisnatog celera i krastavca nisu detektovani nitrati. rezultati dobijeni analizom uzoraka nakon zamrzavanja pokazali su da ovaj proces ima različit uticaj na ispitivane uzorke. pojedini uzorci su sadržali viši nivo nitrata nakon zamrzavanja, dok su drugi sadržali manji. deo ispitivanog povrća pokazao je povećan zdravstveni rizik ako se konzumira u količini od 400 g dnevno. ključne reči: nitrati, povrće, zamrzavanje, zdravstveni rizik, jonska hromatografija introduction vegetables are a rich source of minerals, vitamins, and biologically active compounds and play an important role in human nutrition (prasad & chetty, 2008). they are necessary for human wellbeing, and the world health organization (who) and food and agricultural organization (fao) have recommended a minimum intake of 400 g of fruits and vegetables per day to prevent chronic diseases (who, 2003; qasemi et al., 2024). in recent years, rapid population growth, as well as industrialization, have increased need for more food production. this leads to extensive use of chemical fertilizers, which are nitrogen-based. in these conditions, vegetables can accumulate a notable amount of nitrate and nitrite (kounnoun et al., 2024; wang et al., 2024). nitrogen uptake depends on many biological and environmental factors, including the composition of soil, light intensity, air temperature and moisture, duration of growth period, etc. (bahadoran et al., 2016). vegetables represent the major source of nitrate intake in the human diet, and it has been estimated that 75–80% of the total daily intake (tdi) comes from vegetables (chang et al., 2013a and 2013b). both inorganic compounds, nitrite (no2 -) and nitrate (no3 -), are involved in the nitrogen (n) cycle (kyriacou et al., 2019). these nitrogen forms are present in edible plants produced endogenously as byproducts of the nitrate-nitrite-nitric oxide (no) metabolic pathway in humans (pinaffi-langley © 2025 kostić kokić et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. et al., 2025). nitrate can easily be reduced in the mouth and stomach to nitrite, which can react with secondary amines and then form cancerogenic n-nitrosamines. also, nitrite has a high affinity for haemoglobin in the blood and can combine vigorously to form methemoglobinemia, known as blue baby syndrome (kiani et al., 2022). analysis of nitrate in vegetables is certainly necessary to assess the exposure to nitrate for adverse or beneficial effects (yu et al., 2018). according to the scientific risk assessment on nitrate in vegetables, requested by the european commission and adopted by the european food safety authority (efsa), the fao/ who expert committee on food additives set the acceptable daily intake of 3.7 mg nitrate/kg body weight (fao/who, 2003, 2005a, and 2005b). numerous methods have been recommended for determining nitrate in fresh vegetables, but three techniques have been validated by international standards: iso 6635, ion chromatography (ic) and cadmium column reduction (hasheminasab et al., 2025). the aims of this research were to determine the amount of nitrate in different types of vegetables that are commonly represented in the human diet in serbia, the effects of the freezing process on nitrate concentration and the assessment of the health risks of nitrates in vegetables. materials and methods sampling this study was performed during the summer in 2022. vegetables are classified into three groups: root vegetables, leafy, and fruit vegetables. the population consisted of carrot (daucus carota subsp. sativus (hoffm.) schübl. & g.martens), radish (raphanus sativus l.), celery (apium graveolens l.), parsley (petroselinum crispum (mill.) fuss), parsnip (pastinaca sativa l.), lettuce (lactuca sativa l.), chard (beta vulgaris l.), spinach (spinacia oleracea l.), parsley leaves, celery leaves, tomato (solanum lycopersicum l.), bell pepper (capsicum annuum l.), blue tomato, zucchini (cucurbita pepo l.), and cucumber (cucumis sativus l.) cultivated on niš vegetable fields. all samples were purchased on the local open market. chemicals and instruments all chemicals used (sodium tetraborate acid, potassium hexacyanoferrate (iii) for carrez i solution and zinc acetate dihydrate for carrez ii solution) were purchased from sigma aldrich (germany). all solutions were prepared using ultradeionized water. whatman quantitative filter paper (no. 41) and membrane filters with 0.22 µm pore size were purchased from merck (germany). an analytical balance (abt 100-5m, kern, germany) with precision at ±0.00001 g was used to measure chemicals and samples. ultra-deionized water was obtained using the smart2pure system (thermo scientific, usa). nitrate determination in extracts was done using an ion chromatograph (dionex aquion, thermo scientific, usa). sample preparation five samples of all the mentioned vegetables were prepared for analysis. at first, non-edible parts were removed, and vegetables were washed with tap water. then, vegetables were washed with distilled water, followed by deionized water. the vegetables were dried at room temperature for 24 h. one half of each vegetable was put in polypropylene storage bags, separately, and samples were stored in a freezer for 45 days at -20 °c. a 10 g portion of each vegetable was homogenized by a mixer (bosh, germany). from each homogenised sample, three subsamples of 1.0 g were weighed out by analytical balance and placed into a 100 ml glass. next, 1.25 ml of disodium tetraborate (na2b4o7x10h2o) solution and 25 ml of hot ultrapure deionized water (80 °c) were added. the beaker was placed into a boiling water bath for 15 min. after this time, the beaker was removed from the water bath and 0.5 ml of carrez (i) solution was added and the solution was shaken. then, 0.5 ml of carrez (ii) solution was added, and it was shaken once more. this solution was allowed to cool down to room temperature. after cooling, the solution was transferred into a 25 ml volumetric flask. the beaker was rinsed with ultra deionized water, and the rinse water was transferred to the same volumetric flask. the ultrapure water was added to fill up the volumetric flask. solutions were filtered through the whatman no. 41. then, the obtained filtrates were filtered through a 0.22 µm cellulose filter prior to chromatographic analysis. a dilution with ultrapure water was necessary due to high concentrations of ions. the final filtrate was diluted to one twentyfifth to match the dilution for analysis on an ion chromatograph. each of the five filtrate samples was separated into a replication for nitrate analysis. instrumentation the three replications were analysed for nitrate concentrations as follows. volume of 5 ml of each filtrate sample was placed into the dionex as 50 model autosampler vials. each of the samples was loaded automatically into the ion chromatograph. separation was achieved using a dionex ionpac as22 column (4×250 mm) with the guard column dionex ionpac ag22 (4×50 mm). the ion chromatograph biologica nyssana ● 16 (2) december 2025: kostić kokić et al. ● effects of freezing vegetables on nitrate content and their health-related risks biologica nyssana ● 16 (2) december 2025: kostić kokić et al. ● effects of freezing vegetables on nitrate content and their health-related risks used a mix of 4.5 mm sodium carbonate and 1.4 mm sodium bicarbonate as an eluent at a flow rate of 1.2 ml/min. the concentration of nitrate anions in samples was detected by the dionex aers 500, carbonate, 4 mm, conductivity detector. dionex seven anion standard (product no. 056933) was used for preparing standard solutions. the nitrate concentration values were calculated automatically based on a previously made processing method using chromeleon 7 software. human health risk assessment non-cancerogenic risk assessment was performed by the target hazard quotient (thq) method provided by the us environmental protection agency (epa) (usepa, 2001). the estimated daily intake (edi) was calculated using equation 1, and then thq was calculated using equation 2. (1) (2) where ef is exposure frequency (365 days/year), ed is exposure duration (70 years), irf is average daily intake (g/day), c is concentration in vegetable (mg/kg), lt is average length of life (70 years for non-cancerogenic effect), bw is average body weight (70 kg) and rfd is chronic referent dose for contaminant (3.7 mg/kg of body weight per day). according to the report provided by milešević et al. (2024), the vegetable consumption in serbia is 312 g/day. a one-way anova test was used to examine the significance relationship between vegetable nitrate levels in raw and frozen samples. the difference between nitrate levels obtained from raw and frozen vegetables was compared to a critical value to determine if the difference is significant. the post-hoc test, tukey’s test, was performed, and the test compares the difference between each pair of mean values with appropriate adjustment for the multiple testing. values of hsd (honest significant difference) for each pair were computed by the origin© program. comparing was performed in case p<0.05. results and discussion in total, 15 species of vegetables were examined. vegetables are divided into three categories: leafy, root, and fruit vegetables. nitrate concentrations were determined using ion chromatography, based on a previously defined processing method. calibration curve showed a good correlation coefficient, 0.99899, in the concentration range 0.25 to 10 mg/l (fig. 1). fig. 1. calibration curve for nitrate ion at 0.25–10 mg/l concentration range biologica nyssana ● 16 (2) december 2025: kostić kokić et al. ● effects of freezing vegetables on nitrate content and their health-related risks figs. 2 and 3 present the chromatograms obtained after analyzing the standard solution and the extract of the radish sample after freezing. mg/kg, while the lowest nitrate level was in parsley, 315.56 mg/kg. in the same vegetable category, the highest level of nitrates in frozen samples was determined in samples of radish, too (2335.90 mg/kg), as well as the lowest in parsley (126.61 mg/kg). in the category of leafy vegetables, the highest nitrate level in raw samples was determined in lettuce (851.42 mg/kg), as well as in frozen samples (865.52 mg/kg), while in raw samples of celery, nitrates were not detected. the lowest level of nitrate in frozen samples from the category of leafy vegetables was determined in spinach (173.68 mg/kg), while in raw samples, it was detected in samples of parsley (269.37 mg/kg). in the last category, fruit vegetables, the highest level was determined in raw samples of zucchini (2578.33 mg/kg), as well as in frozen samples (1309.76 mg/kg), but in raw samples of cucumber, nitrates were not detected. in frozen samples from fruit vegetables, nitrate level was not detected in tomato, but the lowest determined concentration was in samples of pepper (43.64 mg/kg). in 10 of 15 vegetables, the nitrate concentrations in frozen samples were lower than in raw samples. results of tukey’s test showed that there is a significant difference in nitrate level between all raw and frozen samples, except in the samples of lettuce. the average nitrate level in all raw vegetables was 928.18 mg/kg in raw samples, with the lowest and highest nitrate levels in vegetables being 96.40 mg/kg in pepper and 3406.66 mg/kg in radish. in case of frozen vegetables, the average nitrate level was 592.80 mg/g and the lowest and highest nitrate levels were found in the same vegetables, 43.64 mg/kg in pepper and 2335.90 mg/kg in radish. the mean level of nitrate in lettuce samples was lower than values reported in literature (eichholzer & gutzwiller, 1998; chung et al., 2011; ziarati & arbabi-bidgoli, 2014; yu et al., 2018). the amount of nitrate in raw tomato was higher than the results reported in some other studies (zhou et al., 2000; ali fig. 2. chromatograph of standard solution at nitrate concentration 2.5 mg/l fig. 3. chromatogram of frozen radish sample the present study found that vegetable nitrate concentrations were variable. tab. 1 shows the nitrate levels (mg/kg) in all investigated samples of vegetables, raw and frozen. the highest nitrate level in the raw sample of the root vegetables category was in samples of radish with an average of 3406.66 biologica nyssana ● 16 (2) december 2025: kostić kokić et al. ● effects of freezing vegetables on nitrate content and their health-related risks et al., 2021). in another study in mashhad (zendehbad et al., 2022) reported mean nitrate levels in carrot and cucumber, 355.88 and 221.36 mg/kg, respectively, and these levels were lower for carrot and higher for cucumber. in a study by bahadoran et al. (2016), nitrate concentrations in radish and celery were significantly higher than concentrations determined in our study (6250 mg/kg and 2610 mg/ kg, respectively). also, investigation of nitrate concentration in samples of chard showed that the obtained results are lower compared to some studies which reported 1690 and 1728 mg/kg (efsa, 2008; roila et al., 2018) and for parsley 526 mg/kg (salehzadeh et al., 2020). the average amount of any kind of vegetable in the serbian food basket is not clear. according to data in the literature, 58 g/day of leafy vegetables and 68 g/day of some fruit vegetables, and 39 g/day of root vegetables are consumed from some household baskets (salehzadeh et al., 2020). nitrate risks in case of consuming 58, 68, and 39 g/day are presented in tab. 2 and tab. 3. tab. 2 reveals that the highest health table 1. nitrate content in each type of investigated vegetable vegetable nitrate content (mg/kg) raw frozen root carrot 918.87±4.11a 127.57±1.56b radish 3406.66±7.41a 2335.90±8.70b celery 421.56±14.52a 831.78±3.39b parsley 315.56±5.73a 126.61±2.83b parsnip 887.26±1.50a 0b leafy lettuce 851.42±4.70a 865.52±8.89a chard 647.53±6.23a 266.64±2.06b spinach 468.29±6.35a 173.68±3.09b parsley 269.37±3.28a 258.24±1.09b celery 0a 274.93±2.26b fruit tomato 579.74±2.28a 0b pepper 96.40±3.20a 43.64±2.08b blue tomato 625.39±4.34a 838.19±3.33b zucchini 2578.33±7.94a 1309.76±3.49b cucumber 0a 253.89±5.69b table 2. health risk of nitrates in raw vegetables based on the food basket vegetable edi for raw vegetables health risk (thq) carrot 0.5119 0.1403 radish 1.8980 0.5200 celery 0.2349 0.0643 parsley 0.1758 0.0482 parsnip 0.4943 0.1354 lettuce 0.7055 0.1933 chard 0.5365 0.1470 spinach 0.3880 0.1063 parsley 0.2232 0.0611 celery 0.0000 0.0000 tomato 0.5632 0.1543 pepper 0.0936 0.0257 blue tomato 0.6075 0.1664 zucchini 2.5047 0.6862 cucumber 0.0000 0.0000 values with the same letter (a-b) within a row are not statistically significantly different at the p<0.05 level table 3. health risk of nitrates in frozen vegetables based on the food basket vegetable edi for frozen vegetables health risk (thq) carrot 0.0711 0.0195 radish 1.3014 0.3566 celery 0.4634 0.1270 parsley 0.0705 0.0193 parsnip 0.0000 0.0000 lettuce 0.7171 0.1965 chard 0.2209 0.0605 spinach 0.1439 0.0394 parsley 0.2140 0.0586 celery 0.2278 0.0624 tomato 0.0000 0.0000 pepper 0.0424 0.0116 blue tomato 0.8142 0.2231 zucchini 1.2723 0.3486 cucumber 0.2466 0.0676 conclusion in this study, nitrate concentration in vegetables and the effect of freezing on nitrate levels were investigated. also, the health risk assessment was done. the obtained results indicate that the average nitrate concentration in root vegetables is the highest, then in fruit vegetables, and the lowest nitrate concentration is determined in leafy vegetables. the results of this study indicate that the freezing process reduces the nitrate amount in all investigated root vegetables except celery. in a group of leafy vegetables, nitrate concentration after freezing and storing was higher in samples of lettuce and celery, while in a group of fruit vegetables, it was the case in samples of blue tomato and cucumber. the health risk is directly related to the nitrate concentrations in vegetables. therefore, the highest average health risk showed root vegetables, then fruit vegetables, and at the end, the lowest health risk showed leafy vegetables. it should be emphasized that consuming vegetables is only one way to intake nitrate. it means that if the value of health risk is lower than 1, it cannot alone indicate a healthy level of nitrate intake. other nitrate sources, such as other foodstuffs and drinking water, should be considered to determine the nitrate health risk. therefore, the risk in raw vegetables can be ascribed to zucchini at 0.6862, and the lowest health risk in pepper at 0.0257, excluding leaf celery and cucumber in which samples nitrates were not detected in raw samples. also, considering the data from tab. 3, the highest health risk in frozen vegetables can be ascribed to radish at 0.3566. the lowest health risk in frozen samples was obtained for pepper, as in raw vegetables, at 0.0116. in frozen samples, nitrates were not detected in tomato and parsnip. the obtained results showed that health risk values for most of the investigated samples are far below the maximum allowed value (thq<1). but the much higher nitrate risk of consuming vegetables is in the case of the consumption of 400 g/day, which results are presented in tab. 4 and tab. 5. in some cases, the health risk is very close to the maximum permissible health risk (raw tomato), while in some cases it is higher than 1. increased health risk, in case of consuming 400 g/day, was obtained for the following raw samples: carrot, radish, parsnip, lettuce, chard, and zucchini, while in the group of frozen samples, those were: radish, root celery, lettuce, blue tomato, and zucchini. in this study, health risk regarding nitrates in vegetables decreases in the following order: root vegetables, fruit vegetables, and leafy vegetables, respectively. biologica nyssana ● 16 (2) december 2025: kostić kokić et al. ● effects of freezing vegetables on nitrate content and their health-related risks table 4. health risk of nitrates in raw vegetables in accordance with the who standard (400 g of vegetable daily intake) table 5. health risk of nitrates in frozen vegetables in accordance with the who standard (400 g of vegetable daily intake) vegetable edi for raw vegetables (400 g) health risk (thq) carrot 5.2507 1.4385 radish 19.4666 5.3333 celery 2.4089 0.6600 parsley 1.8032 0.4940 parsnip 5.0701 1.3891 lettuce 4.8653 1.3329 chard 3.7002 1.0137 spinach 2.6759 0.7331 parsley 1.5393 0.4217 celery 0.0000 0.0000 tomato 3.3128 0.9076 pepper 0.5509 0.1509 blue tomato 3.5737 0.9791 zucchini 14.7333 4.0365 cucumber 0.0000 0.0000 vegetable edi for frozen vegetables (400 g) health risk (thq) carrot 0.7290 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(2014). investigation of cooking method on nitrate and nitrite contents in crops and vegetables and assess the associated health risk. international journal of plant animal and environmental sciences, 4(2), 46–52. biologica nyssana ● 16 (2) december 2025: kostić kokić et al. ● effects of freezing vegetables on nitrate content and their health-related risks milenković et al. 2024, biologica nyssana 15(1) 15 (1) june 2024: 29-35 doi: 10.5281/zenodo.11517757 the effect of extraction techniques on the mineral content of black pepper fruit ethanolic extracts original article aleksandra milenković university of niš, faculty of technology, bulevar oslobođenja 124, 16000 leskovac, serbia aleksandra.milenkovic@student.ni.ac.rs (corresponding author) sanja petrović university of niš, faculty of technology, bulevar oslobođenja 124, 16000 leskovac, serbia saša savić university of niš, faculty of technology, bulevar oslobođenja 124, 16000 leskovac, serbia jelena stanojević university of niš, faculty of technology, bulevar oslobođenja 124, 16000 leskovac, serbia ljiljana stanojević university of niš, faculty of technology, bulevar oslobođenja 124, 16000 leskovac, serbia received: november 01, 2023 revised: december 24, 2023 accepted: march 29, 2024 abstract: black pepper is one of the most used spices around the world, with numerous biological activities. precisely because of the frequent use of this spice, the aim of this research was to determine the elemental composition of black pepper fruit ethanol extracts (bpees). the presence of heavy metals in spices is of particular importance because it can lead to the accumulation of these elements in human organs, which can further cause various health problems. the preparation of samples was done by dilution of the initial extracts with distilled water to a concentration of 0.5 mg/cm3. in the group of spice macroelements, the concentration of potassium is the highest of all the tested elements (20.412-26.370 mg/g of dry extract). from the group of heavy metals, bismuth was the most present in the range of 0.134-0.156 mg/g of dry extract, while lead, cadmium, and mercury were detected in smaller amounts. the extracts that contained elevated levels of certain heavy metals should be consistently monitored. however, it is crucial to develop effective procedures for removing already-extracted heavy metals from bpees. key words: black pepper extract, extraction techniques, macro-elements, micro-elements, heavy metals. apstrakt: uticaj tehnika ekstrakcije na mineralni sadržaj etanolnih ekstrakata plodova crnog bibera crni biber je jedan od najčešće korišćenih začina širom sveta, sa brojnim biološkim aktivnostima. upravo zbog česte upotrebe ovog začina, cilj ovog istraživanja bio je da se utvrdi elementarni sastav etanolnih ekstrakata ploda crnog bibera. prisustvo teških metala u začinima je od posebnog značaja jer može dovesti do akumulacije ovih elemenata u ljudskim organima, što dalje može izazvati razne zdravstvene probleme. priprema uzoraka je vršena razblaživanjem početnih ekstrakata destilovanom vodom do koncentracije od 0,5 mg/cm3. u ekstraktima je iz grupe makroelemenata, koncentracija kalijuma bila najveća od svih ispitivanih elemenata (20,412-26,370 mg/g suvog ekstrakta). iz grupe teških metala, bizmut je bio najzastupljeniji u opsegu koncentracija od 0,134-0,156 mg/g suvog ekstrakta, dok su olovo, kadmijum i živa detektovani u manjim količinama. neophodno je kontinuirano pratiti ekstrakte koji sadrže neke teške metale u visokim koncentracijama. međutim, ključno je razviti efikasne procedure za uklanjanje već ekstrahovanih teških metala iz etanolnih ekstrakata ploda crnog bibera. ključne reči: ekstrakt crnog bibera, tehnike ekstrakcije, makroelementi, mikroelementi, teški metali introduction black pepper (piper nigrum l.) is a very important species from the family piperaceae, not only for its taste and aromatic properties but also for its potential health benefits. it is a perennial plant native to the tropic areas of india and southeast asia and is commercially cultivated in 26 countries around the world. in the balkan countries, it is most often imported from vietnam, brazil, indonesia, china, and thailand, which are the major cultivating countries (yogesh & mokshapathy, 2013). as one of the most widespread spices in the world, it is traditionally used in cuisines around the world. in addition to its culinary applications, black pepper has been investigated for its medicinal and therapeutic © 2024 milenković et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 29 properties (nahak & sahu, 2011; nirwane & bapat, 2012; zarai et al., 2013; tasleem et al., 2014; prashant et al., 2017). the chemical composition of black pepper is diverse, and this plant, in addition to the alkaloid piperine, also contains essential oil, phenolic compounds, fatty oil (saturated and unsaturated fatty acids), starch, proteins, vitamins (a, c, e, k, vitamin b complex, etc.), and minerals (ca, k, cu, fe, mg, zn, etc.) (meghwal & goswami, 2012; hossain et al., 2014). overall, black pepper extracts contain a variety of minerals and vitamins that can contribute to a healthy diet. however, the exact mineral content may vary depending on the specific extract and the method of analysis used. black pepper extracts are important because of the variety of active compounds that contribute to their numerous biological activities, such as antioxidant (nahak & sahu, 2011), antimicrobial (zarai et al., 2013), anticancer (prashant et al., 2017), hepatoprotective (nirwane & bapat, 2012), anti-inflammatory (tasleem et al., 2014), etc. given the widespread usage of black pepper fruit in the traditional serbian food industry and cuisine, it is important to extensively analyze the medicinal potential of this plant. minerals, in their various forms, are essential elements that play an irreplaceable role in human nutrition and health. their presence in food and extracts of plant origin can significantly affect the taste, nutritional value, and potential health effects of these products. black pepper contains a wide range of minerals that contribute not only to its sensory characteristics but also to its health benefits. several studies have investigated the mineral composition of black pepper pericarp and extracts (nwofia et al., 2013; delowar et al., 2014; ameh et al., 2016; olalere et al., 2019). the minerals that are typically more abundant in black pepper extracts and pericarp include potassium, calcium, and magnesium. however, even though black pepper is an excellent source of nutrients, it can also contain accumulated heavy metals or harmful chemicals from the process of production or improper storage. prolonged exposure to heavy metals can lead to detrimental effects on human health, such as fetal illnesses, premature labor, cognitive impairments in children, as well as fatigue, hypertension, and renal issues in adults (inam et al., 2013). heavy metals are described as those metals whose specific gravity is greater than or equal to 5 g/cm3. the most common heavy metals are copper, nickel, chromium, lead, cadmium, mercury, and iron. however, some heavy metals, such as iron, nickel, and copper, are essential for survival in low concentrations (olayiwola et al., 2017), and because of that, they are classified as micro-elements in this study. however, heavy metals such as lead, cadmium, and mercury are toxic to living organisms even in small quantities, causing anomalies in the metabolic functions of the organism, especially in larger amounts (olayiwola et al., 2017). the use of black pepper as a spice, which contains heavy metals, can lead to the accumulation of these elements in human organs and cause various health problems. for these reasons, interest in harmful metals present in food has increased in the last two decades. to date, comprehensive studies dealing with the mineral content of black pepper, especially in ethanolic extracts, are limited. although previously used methods for mineral identification were expensive or did not allow the simultaneous assessment of the micro-elements present in the samples, a precise method for multielement analysis and isotope ratio determination, inductively coupled plasma with optical emission spectrometry (icp-oes), has been established (savić et al., 2019). this method enables the simultaneous analysis of a wide range of trace elements in the sample and was therefore used in this study. the tendency of modern society to use natural raw materials and products has increased interest in research. therefore, the aim of this study was to analyse the influence of extraction techniques (maceration-m, reflux extraction-re, soxhlet extraction-se and ultrasonic extraction-ue) on the content of macro(p, k, ca, mg, na) and microelements (fe, mn, cr, cu, li, ni, zn, si) in black pepper ethanolic extracts (bpees) using the icpoes method. also, the objective was to detect some heavy metals (cd, co, pb, and hg) in the obtained bpees. considering that black pepper fruit is often used in traditional serbian cuisine, this study can be of great importance when it comes to comparing the effect of extraction technique on the mineral content of bpees, which are a potentially important source of nutrients for humans, natural raw materials for the development of dietary supplements, and can be used in the food and pharmaceutical industries. materials and methods plant material the commercial sample of black pepper fruit (piper nigrum l.; country of origin: vietnam; packed by thessaloniki gyros and spices cannon; ugrinovački put, part 25-no 32, zemun, altina) was used in this investigation. the plant material was protected from light and refrigerated in its original package. right before the analysis, the plant material was ground in an electric mill (laboratory electric mill „braun 30 biologica nyssana ● 15 (1) june 2024: 29-35 milenković et al. ● the effect of extraction techniques on the mineral content of black pepper fruit ethanolic extracts biologica nyssana ● 15 (1) june 2024: 29-35 milenković et al. ● the effect of extraction techniques on the mineral content of black pepper fruit ethanolic extracts 31 aromatic ksm2”). reagents and chemicals ethanol 96% v/v (reachem d.o.o.), multistandard iv (multielement standard solution, merck, concentration of 1000 ppm), standard solutions of si, p, hg and s (reagecon, ireland) and argon 5.0 (99.999% purity) as the carrier gas were used for icp-oes analysis. all other chemicals are of analytical reagent grade (p.a.). preparation of extracts extraction techniques ground and homogenised plant material (black pepper fruit) was used for extraction in all applied extraction techniques. for maceration, 2 g of plant material, and the solvent ethanol 70% v/v in the ratio 1:10 m/v were used, and the extraction process was carried out for 120 minutes at room temperature. reflux extraction followed a similar process but was carried out at boiling temperature. ultrasonic extraction lasted 60 minutes at room temperature in a thermostated ultrasonic bath, and involved pouring 70% v/v ethanol over 2 g of plant material at a ratio of 1:10 m/v. finally, soxhlet extraction was performed under specific conditions using 25 g of plant material and 250 cm3 of 70% v/v ethanol for a 240-min extraction. the obtained liquid extracts were separated by filtration using a „buchner” funnel. icp-oes analysis reagents and standard solutions distilled purified water from fisher chemical (hplc grade) was used to dilute the extracts. argon 5.0 (99.999% purity) was used as the carrier gas. in addition to the samples for analysis, calibration standards were prepared earlier. for the preparation of the calibration solution, multistandard iv multielementary standard solution (merck) was used, which contains ag, b, ba, bi, ca, cd, co, cr, cu, fe, in, k, li, mg, mn, na, ni, pb, sr, and zn, as well as additional standard solutions of si, p, hg, and s (reagecon, ireland) in a concentration of 1000 ppm. the preparation of standard solutions for testing selected elements was performed by diluting multistandard iv so that the concentrations of standards for making calibration diagrams were in the range of expected concentrations of tested elements. sample preparationy the evaporated ethanolic extracts of black pepper fruit were diluted with distilled water to a concentration of 0.5 mg/cm3. the samples were filtered through cellulose filters (0.45 μm), and after preparation subjected to icp-oes analysis. all measurements were performed on icp-oes (inductively coupled plasma optical emission spectrometry, arcos fhe12, spectro, germany) by the method of quantitative analysis. the operating conditions of the icp-oes instrument and the parameters for determining the concentration of the selected elements are given in tab. 1. table 1. operating conditions for icp-oes analysis icp-oes operating parameter parameter value plasma power (w) 1400 gas flow (l/min) -coolant -auxiliary 13 0.80 nebulizer type cross flow nebulizer flow rate (l/min) 0.95 pump speed (rpm) 30 stabilization time (s) 0 number of probes for each measuring 3 plasma observation axial results and discussion mineral composition of bpees the presence of 24 elements in the extracts was determined, and their content is given in tab. 2. table 2. content of macroand micro-elements in bpees (mg/g d.e.) mg/g d.e. 1 2 3 4 macro-elements (p, k, ca, mg, na, s) p 11.696 7.796 11.738 5.876 k 26.370 22.790 24.850 20.412 ca n.i. n.i. n.i. n.i. mg 0.650 0.534 0.744 0.246 na 2.850 5.206 3.080 3.450 s 2.730 3.120 2.440 2.756 micro-elements (ag, fe, mn, cr, cu, li, ni, zn, and si) ag n.i. n.i. n.i. n.i. fe n.i. n.i. n.i. n.i. mn n.i. n.i. n.i. n.i. cr n.i. n.i. n.i. n.i. cu 0.042 0.044 0.040 0.044 li 0.008 0.008 0.008 0.008 ni n.i. n.i. n.i. n.i. zn 0.102 0.182 0.110 0.208 si 2.472 2.490 2.462 2.268 elements with unknown benefits (b, ba, in, sr) b n.i. n.i. 0.098 n.i. ba 0.050 0.052 0.05 0.054 in 0.030 0.032 0.030 0.032 sr 0.012 0.034 0.016 0.038 heavy metals (bi, cd, co, pb, hg) bi 0.156 0.144 0.134 0.152 cd 0.006 0.006 0.008 0.006 co n.i. n.i. n.i. n.i. pb n.i. 0.016 0.008 n.i. hg 0.072 0.092 0.100 0.090 *1-maceration; 2-reflux extraction; 3-soxhlet extraction; 4-ultrasonic extraction; n.i.-not identified the obtained results vary in the analysed samples depending on the extraction technique by which the extracts were obtained. based on the presented results, it can be concluded that the presence of silver, boron, calcium, cobalt, chromium, iron, manganese, and nickel was not detected in any of the analysed samples, i.e. their content in the samples was below the detection limit (tab. 2). of the macro-elements whose content was determined in four extracts (p, k, ca, mg, na, and s), the highest content of potassium (20.412-26.370 mg/g of dry extract) was detected in all samples, and the most of it was in the m extract. these results agree with the studies of özcan & akbulut (2008), al-jasass & al-jasser (2012), lee et al. (2020), where potassium was the most present of all macroelements with values of 663-2377 mg/100 g. the study by lee et al. (2020) showed that the pericarp of black pepper contains a much higher level of potassium compared to white beans (463.1 mg/100 g), one of the best sources of k. potassium is a highly important electrolyte for maintaining the acid-base balance in the body as well as the osmotic pressure. it has a similar role in the body as sodium, calcium, and magnesium, and is important for the nerve impulse transmission, the proper functioning of the intestines and muscles, and heart contractions. also, potassium participates in carbohydrate metabolism and membrane transport (petrović et al., 2015). calcium and magnesium are essential elements that help in bone formation, heart strengthening function, muscle relaxation, memory function, and glucose metabolism. although the samples in the mentioned works contained a high level of calcium, it was not detected in our research. the mg level in bpees was detected in the range of 0.246-0.744 mg/g of dry extract, with the highest content of it in se extract. the me and se extracts were better sources of mg compared to tuna (64 mg/100 g) and brown rice (44 mg/100 g). the recorded concentrations of mg in bpees from this study were similar to concentrations in black pepper pericarp from the study of al-jasass & al-jasser (2012). when it comes to sodium, it is the most common cation that participates in water metabolism, muscle contraction, and allows the transfer of carbon dioxide to the lungs. the sodium content of bpees was in the range of 2.850-5.206 mg/g of dry extract (the highest value re extract), which is higher than the sodium content of black pepper pericarp according to previous studies (özcan & akbulut, 2007; al-jasass & al-jasser, 2012; ameh et al., 2016; lee et al., 2020). sulphur is the third most abundant mineral in our body after calcium and phosphorus, with no recommended intake. it is essential for the biosynthesis of chondroitin in bone and cartilage, heparin, and insulin. also, sulphur is an important component of the enzyme glutathione peroxidase (nimni et al., 2007). current data is insufficient to establish a tolerable upper intake level (institute of medicine, 2006). in the bpees, the content of sulphur was detected in the range of 2.440-3.120 mg/g of dry extract (the highest content was detected in re extract). the presence of lithium, zinc, copper, and silicon was detected in the micro-elements in all bpees samples. the study by cicero et al. (2022) showed the presence of chromium, iron, and manganese which were not detected in the present investigation, as well as potentially dangerous minerals such as cadmium, lead, mercury, and aluminum (cicero et al., 2022). the higher quantities of zinc were detected in current research (0.102 to 0.208 mg/g biologica nyssana ● 15 (1) june 2024: 29-35 milenković et al. ● the effect of extraction techniques on the mineral content of black pepper fruit ethanolic extracts 32 biologica nyssana ● 15 (1) june 2024: 29-35 milenković et al. ● the effect of extraction techniques on the mineral content of black pepper fruit ethanolic extracts d.e.) than in the studies of özcan & akbulut (2008) (0.93 mg/100 g), al-jasass & al-jasser (2012) (0.9 mg/100 g), and lee et al. (2020) (1.02 mg/100 g). zinc is a micro-element that is required for the effective functioning of the brain and biomembrane, as well as the treatment of mental diseases. it is also required for protein and carbohydrate metabolism, prostate function, dna synthesis, and is important in the development and function of reproductive organs (savić et al., 2019). copper, vitamin c, and zinc play an important role in preserving skin elasticity and providing essential support for the structural integrity of the skin. copper deficiency can lead to conditions such as anemia, hair depigmentation, and bone deformities. the world health organisation (who) recommends a daily intake of 1 mg of copper (who, 1989). a diet lacking essential elements like zinc (zn), iron (fe), and copper (cu) can potentially lead to the accumulation of toxic metals, including cadmium (cd) or lead (pb) (who, 1996). in the current study, the concentration of copper in the bpees samples ranged from 0.040 to 0.044 mg/g (as indicated in tab. 2). it’s important to note that the who has set a maximum permissible limit for copper in edible plants at 3.00 mg/kg, and based on this recommendation, all analysed samples (bpees) in this study have copper above the permissible limit (fao/who, 1984). of the analysed micro-elements, the highest silicon (2.268-2.490 mg/g of dry extract) content was detected in all samples, and iron, manganese, chromium, and nickel were not identified in extracts. silicon is an essential element for growth of crops, but also has a role in human health. the bioavailability of silicon in the human diet, for example, strengthens bones and improves the immune response, as well as the health of neurons and connective tissue (farooq & dietz, 2015). the literature data doesn’t provide any specific information on the silicon content of black pepper. environmental pollution with heavy metals and long-term cumulative health effects are among the leading health problems worldwide. in this study, the presence of heavy metals was detected in bpees. cobalt was not identified in extracts. of the all analysed heavy metals, the highest bismuth (0.1340.156 mg/g of dry extract) content was detected in all samples. there is not much information about the direct toxic effects of this element (sano et al., 2005), but the toxicity of bismuth and its compounds occurs mainly when used as a therapeutic agent during prolonged administration, which mainly leads to kidney failure or mental disorders (michalke et al., 2008). bioaccumulation of lead (pb) in the human body interferes with the functioning of mitochondria, which affects breathing and causes swelling of the brain, paralysis, and eventual death. lead was detected only in re and se extracts with values that exceed the maximum allowable levels set for this metal by the who—10 mg/kg (who, 2007). studies by savić et al. and al-eed et al. showed lower values than this study and no presence of lead, respectively (al-eed et al., 2002; savić et al., 2019). the presented results also correspond with the analyses of spice samples conducted in other european countries, for example in serbia, where the lead concentration in black pepper samples varied in the range of 0.280 mg/kg to 0.42 mg/kg. however, it should be emphasised that black pepper is not grown in these countries, but is exclusively imported (blagojević et al., 2015). the cadmium was detected in all bpees in the concentration range of 0.006-0.008 mg/g d.e., which exceeded the limit value set by the world health organisation (who) at the level of 0.3 mg/kg, i.e. 0.0003 mg/g (who, 2007). based on the mineral content of bpees and also taking into account the content of heavy metals in the samples, the best extract is the one obtained by maceration. why is it so? this extract contains the highest amount of potassium, which is the most abundant of the macro-elements, but also the lowest amount of heavy metals compared to the other extracts. however, m extract cannot be considered an ideal choice, but in the future, greater attention should be directed to quality control of imported raw materials on the soil of serbia and other balkan countries. the differences in the obtained results, and those reported in previous studies may be due to environmental factors that prevail in production areas, cultivars used to produce seeds, and due to the different methods used to prepare samples (fatoki, 2000). also, some techniques of remediation is ex-situ soil washing using chelating agents or purification of already isolated extracts (tandy et al., 2004; shyam sunder et al., 2020). conclusion the use of the icp-oes method provides a simple and efficient procedure for determining the nutritional profile of spices and spice extracts. the obtained results showed that the analysed extracts of black pepper (bpees) are a good source of potassium, phosphorus, sodium, and sulphur and therefore represent an important source of nutrients for humans. from the group of macro-elements, the most common element was potassium (20.41226.370 mg/g of dry extract), while the lowest content was recorded for magnesium (0.246-0.744 mg/g of dry extract). the analysed bpees showed the presence of heavy metals, among which the highest 33 biologica nyssana ● 15 (1) june 2024: 29-35 milenković et al. ● the effect of extraction techniques on the mineral content of black pepper fruit ethanolic extracts content of bismuth was detected (0.134-0.156 mg/g of dry extract), lower quantities of lead, cadmium, and mercury, while the presence of cobalt was not detected. the human need for spices is very small on a daily basis, so there should be no danger of using them in the diet or food industry in that quantity. also, if isolated extracts were to be used as a source of minerals in another industry, it is important to analyse and find a way to eliminate harmful extracted minerals. in the future, it is important that extracts containing some toxic metals at a relatively high level, should be under continuous monitoring. however, it is essential to find appropriate and efficient methods for the removal of already extracted heavy metals from bpees. acknowledgements. this work was supported by the ministry of education, science and technological development of the republic of serbia under the program of financing scientific research work, number 451-0347/2023-01/200133. references al-eed, m.a., assubaie, f.n., el-garawany, m.m., el-hamshary, h., & eltayeb, z.m. 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(2013). antioxidant and antimicrobial activities of various solvent extracts, piperine and piperic acid from piper nigrum. lwtfood science and technology, 50(2), 634-641. https://doi.org/10.1016/j.lwt.2012.07.036 biologica nyssana ● 15 (1) june 2024: 29-35 milenković et al. ● the effect of extraction techniques on the mineral content of black pepper fruit ethanolic extracts 35 joković et al. 2025, biologica nyssana 16(1) 215 16 (1) june 2025: 215-223 doi: 10.46793/biolnyss.16.1.2j physicochemical properties, antioxidant and antimicrobial activity of seven honey samples collected in the nišava district original article nataša joković university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia natasa.jokovic@pmf.edu.rs (corresponding author) nikola jovanović university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia jelena matejić faculty of medicine, university of niš, bulevar dr zorana đinđića 81, 18000 niš, serbia zorica stojanović-radić university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia jelena vitorović university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia received: october 07, 2024 revised: november 30, 2024 accepted: december 23, 2024 abstract: honey is a functional food used in traditional medicine to treat numerous human ailments. the health-promoting properties of honey depend on its physicochemical characteristics, chemical composition, and antioxidant potential, which result from the botanical and geographical origin of honey. in this study, the physicochemical properties, antioxidant and antimicrobial potential of seven honey samples from the nišava region were analyzed. the honeydew sample had the highest content of phenols and flavonoids and the best scavenging and antimicrobial activities (mic 6.25-25%). the content of flavonoids was also high in meadow honey, which demonstrated excellent scavenging activity against the dpph radical. acacia and lavender honey samples showed good antibacterial activity (mic 12.5-25%), but only honeydew inhibited the growth of yeast canida albicans (mic 12.5%). the results presented in this paper revealed differences in the biological activity of honey samples, indicating the necessity of analyzing each honey sample individually before using it as a functional food. key words: blossom honey, honeydew, antioxidant, antimicrobial, physicochemical properties apstrakt: fizičko-hemijska svojstva, antioksidativna i antimikrobna aktivnost sedam uzoraka meda prikupljenih na teritoriji nišavskog okruga med je funkcionalna hrana koja se koristi u tradicionalnoj medicini za lečenje brojnih ljudskih bolesti. biološka aktivnost meda zavisi od njegovih fizičkohemijskih karakteristika, hemijskog sastava i antioksidativnog potencijala, koji proizilaze iz botaničkog i geografskog porekla meda. u ovoj studiji analizirana su fizičko-hemijska svojstva, antioksidativni i antimikrobni potencijal sedam uzoraka meda iz nišavskog regiona. uzorak medljike je imao najveći sadržaj fenola i flavonoida i najbolju antioksidativnu i antimikrobnu aktivnost (mik 6,25-25%). sadržaj flavonoida je takođe bio visok u livadskom medu, koji je pokazao odličnu aktivnost neutralizacije dpph radikala. bagremov med i med od lavande su pokazali dobru antibakterijsku aktivnost (mik 12,5-25%), ali je samo medljika inhibirala rast kvasca canida albicans (mik 12,5%). rezultati prikazani u ovom radu su pokazali velike razlike u biološkoj aktivnosti uzoraka meda, što ukazuje na neophodnost analiziranja svakog uzorka meda pojedinačno pre njegove upotrebe kao funkcionalne namirnice. ključne reči: cvetni med, medljika, antioksidativna aktivnost, antimikrobna aktivnost, fizičkohemijske karakteristike introduction honey is a bee product that is one of the most important functional foods in the world, with an annual global production of 1.83 million tons in 2022 (faostat, 2024). blossom (floral) honey and honeydew are the two types of honey, classified according to the kind of sugar sources collected by the bees (bergamo et al., 2019). floral honey is made from the secretions of living plants (nectar), while honeydew is the product of collecting the excretions of insects that live on plants. these two types of honey differ in their physicochemical, chemical, and biological properties (pita-calvo & vázquez, 2017). the consumption of honey is popular with consumers all over the world for its nutritional and health-promoting properties, which have been known since ancient times in many traditional © 2025 joković et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 216 medicines (nikhat & fazil, 2022). various studies have reported antimicrobial, antioxidant, antiinflammatory, antidiabetic, and anticarcinogenic effects of different honey samples (erejuwa et al., 2012; meo et al., 2017; pasupuleti et al., 2020; tafere, 2021). clinical studies have confirmed that honey positively affects human health in healing wounds and burns, as well as cardiovascular, gastrointestinal, and diabetes diseases (samarghandian et al., 2017; palma-morales et al., 2023). however, the chemical composition and biological activities of honey vary greatly from sample to sample and depend primarily on the geographical, seasonal, and botanical origin of the samples as well as the conditions under which the honey is collected, processed, and stored (da silva et al., 2016; obey et al., 2022). the aim of the present study was to compare the physicochemical properties, antioxidant and antimicrobial activity of seven honey samples collected in the nišava district. one of the selected samples was honeydew and the other six were blossom honey. the blossom honey samples had different botanical origins. acacia and lavender honey were selected as monofloral honey samples, while mixed acacia and sage honey and meadow honey were selected as polyfloral honey samples. materials and methods honey samples this study analyzed seven honey samples collected on the territory of the nišava district. the samples were obtained from the association of beekeepers „suva planina” from niška banja. table 1 lists the types of honey and the localities where the samples were collected. merck (darmstadt, germany). na2co3, c2h3ko2, k2o8s2, and l(+)-ascorbic acid were purchased from analar normapur (vwr, leuven, belgium) and al(no3)3x9h2o from fluka chemie ag (buchs, switzerland). abts and quercetin hydrate (qu) were obtained from tci europe nv (boerenveldsweg, belgium). all chemicals were of analytical grade. physicochemical analysis the physicochemical analysis of the honey samples was carried out according to the methods listed in the harmonized methods of the international honey commission (international honey commission, 2009). the water content was determined two spaces with a refractometer at 20 °c using wedmore tables. the ph value and acidity of honey samples were determined by dissolving 10 g of honey in 75 ml of distilled water. a ph meter (llg ph meter 7, llg, germany) was used to measure the ph values of the samples at room temperature. the total acidity of honey samples was determined as the sum of free and lactone acidity. the free acidity was determined by adding 0.05m naoh to ph 8.5. the lactone acidity was determined by adding 10 ml of 0.05m naoh to ph 8.5 and subsequent titration with 0.05m hcl to ph 8.3. a honey solution for the analysis of the hydroxymethylfurfural (hmf) content was prepared by dissolving 5 g of honey in 25 ml of water. subsequently, 0.5 ml of carrez reagents i and ii were added to the solution for clarification and the samples were additionally diluted with water. alcohol was also added to prevent foaming. the absorbance of the sample was measured at 284 nm and 336 nm using a uv-vis spectrophotometer (shimadzu 1650 pc, kyoto, japan). the hmf content is expressed in mg/kg and was calculated using the following equation: hmf=(a284-a336)x149.7. diastase activity was determined using a buffered solution of soluble starch and honey incubated at 40 °c. the absorbance of 1 ml of solution was measured at 660 nm at 5-minute intervals. the amount of reducing sugars and sucrose was determined by titration of fehling’s solution containing methylene blue with a honey solution. antioxidative activity to test the antioxidant potential of honey samples, a honey solution was prepared by dissolving 10 g of honey in 10 ml of distilled water. the solution was then filtered, and the resulting samples were used to determine the total phenolic and flavonoid contents and to test the potential of the samples to neutralize dpph (2,2-diphenyl-1-picrylhydrazil) and abts (2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) radicals. biologica nyssana ● 16 (1) june 2025: 215-223 joković et al. ● physicochemical properties, antioxidant and antimicrobial activity of seven honey samples collected in the nišava district table 1. honey samples used in the study samples abv. localities acacia honey a1 zaplanje (g. barbeš) acacia honey a2 sićevo acacia honey (crystallized) ac sićevo acacia and sage honey as sićevo lavender honey l tamnjanica meadow honey m niš surroundings honeydew honey h niš surroundings chemicals and reagents butylated hydroxyanisole (bha) and dpph were obtained from sigma chemicals co (st louis, mo, usa) and folin-ciocalteu phenol reagent from 217 biologica nyssana ● 16 (1) june 2025: 215-223 joković et al. ● physicochemical properties, antioxidant and antimicrobial activity of seven honey samples collected in the nišava district total phenolic content the total phenolic content (tpc) was determined using the folin-ciocalteu method (singleton et al., 1999). concentrated commercial folin-ciocalteu reagent (merck, germany) was diluted with distilled water at a ratio of 1:10. 1500 μl of the reagent was added to 300 μl of a diluted honey sample with concentrations of 1 and 0.5 g/ml. after the mixture was kept in the dark for 6 minutes, a 7.5% na2co3 solution was added. the contents were mixed with a vortex, the test tubes were covered and left in a dark place for 2 hours. the absorbance of the mixtures was measured with a spectrophotometer at a wavelength of 740 nm. the tpc is expressed in mg gallic acid equivalents (ga)/kg honey calculated using the standard curve for gallic acid. total flavonoid content the total flavonoid content (tfc) was measured according to the method described by woisky and salatino (woisky & salatino, 1998). the reaction mixture was prepared by adding 4.1 ml of 80% c2h5oh, 0.1 ml of 10% al(no3)3x9h2o and 0.1 ml of 1m c2h3ko2 to 600 μl of a diluted honey sample with a concentration of 1 and 0.5 g/ml. the contents were mixed with a vortex, the test tubes were covered and left in a dark place for 40 minutes. the absorbance of the mixtures was measured with a spectrophotometer at a wavelength of 415 nm. the tfc was calculated using the standard curve for quercetin and expressed in mg equivalents of quercetin (qe)/kg honey. dpph scavenging activity the determination of the potential antioxidant capacity by dpph assay was performed spectrophotometrically using the blois method (blois, 1958). dpph dissolved in methanol at a 0.04 mg/ml concentration was used as the reagent. the concentrations of the honey solution were selected based on experiments with the dpph solution according to the procedure indicated. the reaction mixture, prepared by mixing 200 µl of the tested sample with 1800 µl of the dpph solution, was gently shaken and kept at room temperature in the dark for 30 minutes. the absorbance of the solution was then measured at 517 nm. the percentage of dpph radical scavenging was calculated according to the following equation: scavenging activity (%) = (a0-a1) x 100/a0 where a0 represents the absorbance of the initial dpph solution and a1 represents the absorbance of the samples. the results are presented as the ic50 value, which indicates the concentration of the sample that scavenges 50% of the dpph radicals. two known antioxidants, vitamin c and butylated hydroxyanisole (bha), were used as positive controls. abts radical scavenging activity the abts radical scavenging test was performed according to the modified method of miller and rice-evans (miller & rice-evans, 1997). the abts reagent was prepared by dissolving 19.2 mg of abts in 5 ml of 2.46 mm k2o8s2, and the solution was allowed to stand in the dark at room temperature for 12-16 hours. the reaction mixture was prepared by mixing 75 μl of diluted honey sample and 3 ml of abts reagent. the contents were mixed with a vortex, then the test tubes were covered and allowed to stand for 30 minutes at 30 ºc in a water bath. the absorbance was measured at a wavelength of 734 nm. abts scavenging activity was calculated based on the calibration curve for vitamin c and expressed in mg vitamin c equivalents (vitc)/g honey. antimicrobial activity microbial strains the antimicrobial activity of honey samples was determined against gram-positive bacteria kocuria rhizophila atcc 9341 and staphylococcus aureus atcc 6538, and gram-negative bacteria escherichia coli atcc 8739, proteus mirabilis atcc 12453 and pseudomonas paraeruginosa atcc 9027. two fungal strains, the yeast candida albicans atcc 10231 and the mold aspergillus brasiliensis atcc 16404 were also used for the antimicrobial test. the cultures of the bacterial strains were grown on nutrient agar (torlak, serbia) at 37 °c, while the fungal cultures were incubated on sabouraud dextrose agar (torlak, serbia) at 30 °c. antimicrobial assay the antimicrobial activity of the honey samples was tested using the micro/well dilution method (clinical and laboratory standards institute, 2012.). the bacterial cultures were incubated at 37 °c on mueller-hinton agar, while the fungi were incubated at 30 °c on sabouraud dextrose agar. the bacterial suspensions were prepared in mueller-hinton broth, fungal suspensions were made in sabouraud dextrose broth, and their turbidity was standardized using a mcfarland densitometer (den-1b, biosan). the final density of the bacterial and yeast inocula was 5x105, while the final density of the mold inoculum was 1x104. the stock solutions of the honey samples were prepared in distilled sterile water (initial concentration 0.5 g/ml = 50%) and then serially diluted (dilution factor 2). the final concentration of the tested samples in the medium was 250.00-0.1 mg/ ml (25-0.01%). after dilution, 218 biologica nyssana ● 16 (1) june 2025: 215-223 joković et al. ● physicochemical properties, antioxidant and antimicrobial activity of seven honey samples collected in the nišava district the inoculum was added to all wells of the microtiter plates and then the plates were incubated at 37 °c for 24 hours (bacterial strains) and at 30 °c for 48 hours (fungi). chloramphenicol and nystatin were used as a positive control, while a non-inoculated well without an antimicrobial substance was used to confirm the sterility of the medium. artificial honey prepared by dissolving 40 g fructose, 30 g glucose, 8 g maltose and 2 g sucrose in 100 ml distilled water and sterilized at a temperature of 121 °c for 15 minutes served as a control (al-waili et al., 2013). the minimum inhibitory concentration (mic) is defined as the minimum concentration of honey that inhibits the visible growth of cell cultures. all experiments were done in triplicate. statistical analysis all measurements in antioxidant activity tests were performed in triplicate, and the results are presented as averages ± standard deviation (sd). the results were statistically analyzed using one-way analysis of variance (anova) followed by tukey’s hsd test (p≤0.05) using the minitab®17 software (minitab, llc, state college, pennsylvania, usa). results and discussion physicochemical properties of honey samples the physicochemical properties of honey as important indicators of the quality and authenticity are determined by european and serbian regulations (codex alimentarius commission, 2001; vranić et al., 2017). tab. 2 shows the values of physicochemical parameters for all analyzed honey samples and the reference values prescribed by serbian regulations (vranić et al., 2017). most of the physicochemical parameters of the analyzed honey samples complied with the regulations (tab. 2), which indicates the good quality of the collected honey samples. only the content of hydroxymethylfurfural (hmf), an organic compound formed during the dehydration of sugars in honey, was higher in one sample of crystallized acacia honey ac (68.47 mg/kg) than the maximum value of 40 mg/kg specified in the codex alimentarius standard. in the ac sample, the activity of the enzyme diastase, which converts starch into sugars with shorter chains, was also the lowest (tab. 2). the obtained values of diastase activity and hmf content of the ac sample could indicate improper preservation of the honey and its possible exposure to sunlight or another heat source during processing and storage. the sample of acacia and sage honey (as) had an extremely low hmf content and the lowest water content so this honey could be described as the freshest of all analyzed honey samples according to the codex alimentarius standard. the diastase activity was the highest in the as sample, which also indicates the exceptional quality of this honey sample. several physicochemical parameters are important for the differentiation between blossom honey and honeydew (pita-calvo & vázquez, 2017). manzanares et al. (2011) concluded that acidity, ph, electrical conductivity, proline, invertase, and glucose are the most important differentiating factors between these two kinds of honey based on a multivariate analysis of the physicochemical parameters and sugar composition of 77 honey samples. the honeydew honey in our study had the highest ph and acidity, but the lowest reducing sugar content (tab. 2). in addition, the color of sample h was darker compared to the other honey samples (data not shown), which is also characteristic of this honey type (kesić et al., 2020). antioxidant activity one important parameter for the biological activity of honey is its antioxidant activity, which table 2. physicochemical properties of analyzed honey samples parameters samples referent valuea1 a2 ac as l m h moisture (%) 15.64 15.24 16.73 14.9 16.32 15.43 15.06 <20 diastase activity (degrees of the goethe scale) 23.75 25.69 11.63 32.78 39.45 30.9 37.71 >8 hmf (mg/kg) 4.79 7.84 68.47 1.87 6.99 4.8 9.11 <40 ph 4.15 4.23 3.79 4.12 3.54 4.33 4.51 acidity (meq/kg) 10.55 7.36 10.41 13.44 32.88 23.8 33.25 <50 reducing sugar content (%) 76.4 73.95 70.19 72.94 74.05 67.15 66.71 >60 sucrose content (%) 2.15 2.03 2.82 2.92 2.48 2.62 2.77 <5 219 biologica nyssana ● 16 (1) june 2025: 215-223 joković et al. ● physicochemical properties, antioxidant and antimicrobial activity of seven honey samples collected in the nišava district was evaluated in this study by determining the content of total phenols (tpc) and flavonoids (tfc) and measuring the ability of honey samples to trap dpph and abts radicals. total phenol and flavonoid contents were significantly different (p<0.05) for most honey samples (fig. 1). the highest tpc and tfc values were determined in the honeydew sample (sample h), while the acacia honey sample a2 had the lowest phenol and flavonoid content. apart from sample h, tpc was quite high in samples of acacia crystal (ac), lavender (l), and meadow (m) honey (fig. 1a). on the other hand, tfc was two or ten times higher in sample h compared to other analyzed honey samples (fig. 1b). high tfc was also determined in the samples of meadow honey (55.78±0.018) and acacia crystal honey (36.54±0.038). values were also found for ac and as samples (0.87 mg/ml and 0.89 mg/ml, respectively), while the a2 sample exhibited the weakest dpph scavenging activity with an ic50 value of 3.45 mg/ml. phenolic compounds from various biological sources are one of the most important radical scavengers, so the dpph scavenging activity of the sample is directly correlated with tpc (lewoyehu & amare, 2019). the sample of honeydew and meadow honey, which in this work had the lowest ic values, also had high tpc and tfc, indicating that flavonoids are the main carriers of scavenging activity. honey is rich in various flavonoids among which pinocembrin, apigenin, kaempferol, quercetin, pinobanksin, luteolin, galangin, hesperetin, and isorhamnetin are the most important compounds that affect the antioxidant activity of honey (da silva et al., 2016). fig. 1. total phenol (a) and flavonoid (b) contents in honey samples. results are presented as mean±sd. values marked with the same letters are not significantly different (p>0.05) phenolic acids and flavonoids are components found in honey in varying amounts depending on the biological origin of the honey and the geographical area from which the honey was collected (becerril‐ sánchez et al., 2021). however, the amount of these biologically active compounds is higher in honeydew than in floral honey samples (pita-calvo & vázquez, 2017). also, polyfloral honey such as meadow honey had higher tpc and tfc and better antioxidant activity compared to monofloral honey (atanacković krstonošić et al., 2019). the results obtained in this study for tpc and tfc are similar to previously published results for different honey samples collected on the territory of serbia (savatović et al., 2011; gašić et al., 2014; čanadanović-brunet et al., 2014; velimirović et al., 2023). the results of the scavenging activity of honey samples are presented in fig. 2. honeydew and meadow honey showed the best antioxidant activity in the dpph method with ic50 values of 0.26 mg/ml and 0.44 mg/ml, respectively. low and similar ic50 the obtained values for antioxidant activity in the abts method showed that all honey samples, except sample a1, had a better antioxidant potential than the positive control bha (fig. 2b). the difference between the samples was small but statistically significant between certain honey samples (fig. 2b) and the best activity was found in samples as and h (3.81 mgvitc/g). sample a1 had the lowest activity in the abts method of 1.46 mgvitc/g. in the abts assay, there was no such clear correlation between the content of phenols and flavonoids and the abts scavenging activity as in the dpph assay. stagos et al. (2018) showed by examining the antioxidant activity of honey samples from mount olympus in greece that there was no positive correlation between tpc and scavenging activity in dpph and abts assays although some honey samples with high tpc had the best dpph activity (stagos et al., 2018). apart from phenolic acids and flavonoids, ascorbic acid and other vitamins, carotenoids, catalase, peroxidase, 220 e. coli. the crystallized acacia honey (ac) showed the weakest antimicrobial activity, exhibiting the same pattern of antimicrobial activity as the control sample of artificial honey (ah). the antimicrobial activity of honey can be attributed to various chemical components and enzymes contained in honey and to its specific physico-chemical properties. low water content, high osmolarity due to a high sugar concentration, low ph due to organic acids in honey, and the concentration of hydrogen peroxide are considered the most important antimicrobial factors (almasaudi, 2021). phenolic compounds, including phenolic acids and polyphenols, or some specific compounds, such as methylglyoxal in manuka honey or the peptide bee-defensin-1 in revamil honey, can also contribute to the antimicrobial properties of honey (kwakman et al., 2011; majtan et al., 2021). sample h, which showed the best antimicrobial activity, had the highest acidity, the highest content of phenols and flavonoids, and the best scavenging activity for dpph and abts radicals. the synergistic effect of all these potential antimicrobial factors probably influenced the good antimicrobial activity of this honey sample. the total phenolic content was also high in lavender honey (sample l), and this sample had the lowest measured ph (3.54), suggesting that phenolics and acidity are probably the main contributors to antimicrobial activity. on the other hand, the sample of acacia honey a1 with the best antibacterial activity had a low content of phenols and flavonoids but the highest concentration of reducing sugars. the weakest antimicrobial effect of all honey samples analyzed was against the mold a. brasiliensis, while only sample h had an inhibitory effect on the growth of the yeast c. albicans. the better antimicrobial effect of honey on bacteria and maillard reaction products found in honey can contribute to the antioxidant activity of honey (chua et al., 2013; pauliuc et al., 2020). antimicrobial activity of honey the antimicrobial activity of honey is a well-known therapeutic property of this functional food which has been used in many parts of the world since ancient times (majtan et al., 2021). considering that honey is an antimicrobial agent with a wide spectrum of action, it was used in clinical studies for the treatment of postoperative infection, burns, necrotizing fasciitis, infected and nonhealing wounds and ulcers, boils, pilonidal sinus, venous ulcers, and diabetic foot ulcers (al-waili et al., 2011). the antimicrobial activity of seven honey samples was evaluated against gram-positive and gram-negative bacteria, the yeast c. albicans and the mold a. brasiliensis (tab. 3). sample h (honeydew) showed the best antimicrobial activity, acting on three bacterial strains (k. rhizophila, p. mirabilis, p. paraeruginosa) with mic values of 6.25-12.5% and was the only honey sample that inhibited the growth of the yeast c. albicans. this sample had the lowest mic of 6.25% against two bacterial strains, k. rhizophila and p. mirabilis. the better antimicrobial activity of honeydew compared to blossom honey samples was also evident in other studies (nedić et al., 2022). sample a1 (acacia honey) also had good antibacterial activity (mic 12.5%) against all three tested strains of gram-negative bacteria (e. coli, p. mirabilis, p. paraeruginosa) and the gram-positive bacterium k. rhizophila. similar mic values as in this work were reported for honey samples of different botanic and geographic origins (basson & grobler, 2008; živković et al., 2019; obey et al., 2022). lavender honey also showed similar activity to sample a1 but was less effective against biologica nyssana ● 16 (1) june 2025: 215-223 joković et al. ● physicochemical properties, antioxidant and antimicrobial activity of seven honey samples collected in the nišava district fig. 2. antioxidant activity measured by the dpph (a) and abts (b) methods in honey samples. the antioxidant activity was compared to butylated hydroxyanisole (bha) and vitamin c as standards. results are presented as mean±sd. values marked with the same letters are not significantly different (p>0.05) 221 compared to fungi has been established in many studies (basson & grobler, 2008; al-waili et al., 2013; kolayli et al., 2020) among the bacterial strains, s. aureus atcc 6538 showed the highest resistance, while p. mirabilis atcc 12453 was the most sensitive bacterial strain, which was affected by all honey samples except the ac sample. conclusion the results presented in this paper show that the seven honey samples collected in the nišava district were of good quality. the honeydew sample differed from the other honey samples regarding its physicochemical properties and had the best antioxidant and antimicrobial activity. the content of phenols and flavonoids, the scavenging activity of dpph and abts radicals, and the antimicrobial activity of the other honey samples were diverse, indicating the need to test each honey sample individually before using it as a functional food. acknowledgements. this research was supported by the ministry of education, science and technological development of the republic of serbia (contracts no 451-0365/2024-03/200124, no 451-03-66/2024-03/200027). references almasaudi, s. 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(2019). total phenolic and flavonoid contents, antioxidant and antibacterial activities of selected honeys against human pathogenic bacteria. acta poloniae pharmaceutica drug research, 76(4), 671–681. https://doi. org/10.32383/appdr/105461 perišić, v., perišić, v., vukajlović, f., pešić, s., predojević, d., đekić, v., luković, k.: feeding preferences and progeny production of rhyzopertha dominica (fabricius 1792) (coleoptera: bostrichidae) in small grains. biologica nyssana, 9 (1). september, 2018: 55-61. biologica nyssana 9 (1) ⚫ september 2018: 55-61 perišić et al. ⚫ feeding preferences and progeny production of rhyzopertha… 55 original article received: 3 may 2018 revised: 3 june 2018 accepted: 6 july 2018 feeding preferences and progeny production of rhyzopertha dominica (fabricius 1792) (coleoptera: bostrichidae) in small grains vladimir perišić1, vesna perišić1, filip vukajlović2, snežana pešić2, dragana predojević2, vera đekić1, kristina luković1 1center for small grains, save kovačevića 31, 34000 kragujevac, serbia 2university of kragujevac, faculty of science, radoja domanovića 12, 34000 kragujevac, serbia * e-mail: vperisic@kg.ac.rs abstract: perišić, v., perišić, v., vukajlović, f., pešić, s., predojević, d., đekić, v., luković, k.: feeding preferences and progeny production of rhyzopertha dominica (fabricius 1792) (coleoptera: bostrichidae) in small grains. biologica nyssana, 9 (1). september, 2018: 55-61. the lesser grain borer, rhyzopertha dominica (fabricius, 1792) (coleoptera: bostrichidae) is a primary pest of stored wheat. many authors studied its development on this cereal. the wheat is commonly stored together with other grain, especially barley, rye, oats and triticale. the objectives of this study are to assess the feeding preferences for the small grains (wheat, barley, rye, oat and triticale) and to evaluate their susceptibility/resistance to progeny production of r. dominica. the mean survival rate of r. dominica adults, progeny emergence and amounts of insect-damaged grains and dockage in various small grain species were determined. also, influence of r. dominica feeding on chemical properties (moisture protein and ash contents in grains) was found out. the influence of the grain species on the development of r. dominica was significant. the highest mortality of parents and the lowest progeny counts in oats, while triticale is very suitable for development of r. dominica, much more then wheat, as a primary host. the biggest amount of damage kernals and dust were found in triticale. the contents of moisture, protein and ash in the grain has been changed due to feeding of r. dominica. key words: rhyzopertha dominica f., small grains, progeny, damaged kernels, dust apstrakt: perišić, v., perišić, v., vukajlović, f., pešić, s., predojević, d., đekić, v., luković, k.: preferencija u ishrani i pojava potomstva rhyzopertha dominica (fabricius 1792) (coleoptera: bostrichidae) u strnim žitima. biologica nyssana, 9 (1). septembar, 2018: 55-61. žitni kukuljičar (rizoperta) rhyzopertha dominica (fabricius, 1792) (coleoptera: bostrichidae) je primarna štetočina uskladištene pšenice. njen razvoj na ovoj vrsti strnih žita proučavali su brojni autori. pšenica se 9 (1) • september 2018: 55-61 doi: 10.5281/zenodo.1470852 biologica nyssana 9 (1) ⚫ september 2018: 55-61 perišić et al. ⚫ feeding preferences and progeny production of rhyzopertha… 56 obično skladišti sa drugim strnim žitima, posebno ječmom, raži, ovsom i tritikaleom. ovo istraživanje je sprovedeno u cilju ispitivanja preferencija u ishrani r. dominica u strnim žitima (pšenica, ječam, raž, ovas i tritikale) i ocene osetljvosti/otpornosti ovih žita na pojavu potomstva r. dominica. utvrđen je prosečan broj preživelih jedinki r. dominica, pojava potomstva i broj zrna oštećenih insektima, kao i količina prašine u različitim vrstama strnih žita. takođe, utvrđen je uticaj r. dominica na hemijske osobine strnih žita (sadržaj vlage, proteina i pepela u zrnu). uticaj vrste žitarica na razvoj r. dominica bio je značajan. najveća smrtnost roditelja i najmanja pojava potomstva utvrđena je kod ovsa, dok je tritikale veoma povoljan za razvoj r. dominica, mnogo više nego pšenica, kao primarni domaćin. najveća količina oštećenih zrna i prašine nađeni su u tritikaleu. ishrana r. dominica je dovela do promene sadržaja vlage, proteina i pepela u zrnu. ključne reči: rhyzopertha dominica, strna žita, potomstvo, oštećena zrna, prašina introduction the lesser grain borer, rhyzopertha dominica (fabricius, 1792) (coleoptera: bostrichidae) is a primary pest of stored grain, with the great economic importance in the republic of serbia and many regions of the world (kljajić, 2008). it feeds mostly on grains from families poeaceae (e.g. rice, wheat, sorghum, oats, pearl, millet, malt, barley) and fabaceae (e.g. chickpeas, peanuts, beans) (edde, 2012). a lot of research was conducted which dealt with the influence of the species and variety of the plant on the development of r. dominica and occurrence of the progeny (arthur et al., 2012, 2013; astuti et al., 2013; metwaly et al., 2015; pires, 2016). although it is one of the most important stored-grain insect pests in the world, most of research was conducted on wheat and rice (chanbang et al., 2008; mebarkia et.al, 2009; nawrot et al., 2010). wheat is commonly stored together with other grain, especially barley, rye, oats and triticale. however, there are no studies in which adults of r. dominica have been directly exposed to small grains and assessed the influence of these species on progeny production and feeding preferences. the larvae and adults of r. dominica spend most of their life inside the kernel, feeding on both the germ and endosperm, directly causing damages and changes in grain physicochemical properties (rees, 2004; edde, 2012). insect-damaged kernels are characterized by the presence of irregularly shaped holes (about 1 mm in diameter) of increasing depth that extends from the point of larval entry, around the edge of the kernel to the pupation chamber (rees, 2004). adult feeding activities produce large amount of dust, which often leads to the reduction of grain kernel to the pericarp. jood & kappor (1993), mebarkia et al. (2009) and nawrot et al. (2010) investigated the amount of grain consumed by r. dominica under experimental conditions, but the losses of nutrient value varied among the authors. small grains contain different quantities of main nutrition groups, which are very important for development and progeny production of r. dominica. the most commonly grown grain species in serbia are wheat, barley, rye, oats and triticale. according to serna-saldivar (2010a), this species differed in amount of proteins, starch and other nutrients. furthermore, above-mentioned species express differences in morphological properties, for example, presence/absence of a hull. nitrogen and phosphorus are particularly mismatched when herbivorous insects are compared with their host plants (huberty & dennno, 2006). the objectives of this study are to assess feeding preferences of r. dominica for small grains (wheat, barley, rye, oats and triticale) through determination of some grain properties, i.e. mass of infested-damaged kernels (idk) and dust, contents of moisture, proteins and ashes in grains infested with r. dominica and to evaluate their susceptibility/ resistance to progeny production of r. dominica. material and methods examination of the influence of different small grain species (wheat, barley, oats, rye and triticale) on the emergence of the progeny of r. dominica, on stored small grains, as well as effect of their presence on chemical properties of small grains were conducted in laboratories of center for small grains kragujevac, serbia during 2016. tested insects adults of r. dominica of both sexes and 2-4-weeks old were used during the experiment, reared in the center for small grains on the whole wheat kernels, in laboratory conditions with temperature (t) 26±1°c and relative humidity (rh) 60±5%. tested cereal species r. dominica was reared on five commercially available small grain varieties (wheat variety vizija, barley variety rekord, oat variety vranac, rye variety raša and triticale variety favorit), originating from the center for small grains kragujevac, serbia. different cereal species with the grain moisture 1112% were used during the testing. this value was biologica nyssana 9 (1) ⚫ september 2018: 55-61 perišić et al. ⚫ feeding preferences and progeny production of rhyzopertha… 57 chosen because it has been proposed level of the moisture for stored small grains (the official gazette of rs, 2016). the moisture content was determined with motomco moisture meter (motomco inc, 919, canada) according to aacc method 44-11 (aacc, 1995). examination of the influence of different small grain species (wheat, barley, oats, rye and triticale) on the emergence of the progeny of r. dominica, on stored small grains was based on the modified method according to arthur et al. (2013). after the harvest, grains were put in the refrigerator at 4 °c. before the incorporation of adults of r. dominica, grains were sieved on the 2 mm sieve and cleaned by hand. then, plastic vessels of 200 ml were filled with 50 g of grains and put in the incubator with controlled t 26±1 °c and rh 60±5% (incubator xo 1450 special, iskra, loka, slovenia). twenty-five adults of r. dominica were released into each vessel. vessels were then closed with cotton cloths fixed with a rubber band. for each small grain species, eight replications were conducted. hatching period is about seven days or less (crombie, 1940; cit.loc. edde, 2012), while we decided to determine the influence of commodity on feeding preferences and survival of r. dominica for a period of three weeks. parental adults were removed from the vessels after three weeks and classified as alive or dead. afterwards, damaged grains and dust (the feeding damage) were separated and their mass was measured on analytical balance (mettler 609-b6, zürich, switzerland) for each sample (50 g) of grain types, and then the contents were put back into the vessels. since egg-to-adult development of r. dominica could be shorter than five-six weeks on wheat (edde, 2012), the period of seven weeks was chosen for incubation, as duration of development was expected to be longer in non-preferred commodities. after seven weeks at the constant temperature and humidity, vessels with grains were opened and the progeny emergence was determined by counting insects separated from grains. damaged grains and dust were weighed again, after progeny counting. the whole procedure was repeated twice. determination of the influence of r. dominica feeding on chemical properties of small grains. infested and damaged kernel, as well as dust, were returned to the vessel after weighing. for this research, uninfested samples from the same year of production were used as a standard (control). all samples were milled and prepared for chemical analysis of moisture, total protein and ash contents. the moisture content was determined based on the loss of samples weight after drying on t 130-133 °c (icc standard no. 110/1: 1976). the total protein content of grains (n x 5.7 for wheat, rye and triticale, and n x 6.25 for oats and barley) was determined according to modified kjeldahl’s method (icc standard no. 105/2:194). ash content was estimated by burning a sample at 900 °c and measuring the residue (icc standard method no.104/1: 1990). results were expressed as percent (%) from the sample weight and represented as a dry matter. statistical analysis. results of examination were expressed in percent of mortality (%) with computed standard error (se). before analysis, results for the progeny number was transformed with formula log(x+1), while formula sqrt(x) was used for the amount of damaged grain and dust. recorded data were analyzed by one-way or two-way analysis of variance (anova) depending on the trial design. the significance of mean differences was determined according to duncan test (for p=0.05). pearson's coefficient at p=0.005 was used to determine linear correlation between the survival parents or the number of progeny and the number of damaged kernels, and correlation between mass of damaged kernels and the parameters of chemical analysis (moisture, proteins and ash contents). all data were processed on statsoft version 7.1 (statsoft inc., tulsa, oklahoma). results survival of r. dominica parents on different small grain species three weeks after the infestation ranged from 91.25% on oat to 99.25% on rye (tab. 1) and was significantly smaller in oat than in other examined cereals. the significantly smaller amount of insect-damaged grains and dust after two weeks was found in oats. the highest mass of damaged grains and dust was found in the triticale (3.87 g/1.96 g). mean r. dominica parent survival and feeding damage were compared using a linear correlation analysis with pearson’s coefficient and significant positive correlations were not detected for p<0.005, n=16 (wheat r=-0.123, p=0.650; barley r=0.287, p=0.281; rye r=-0.257, p=0.925; oats r=-0.564, p=0.836 and triticale r=0.232, p=0.386). based on the data given in tab. 2, our analyses show that the production of r. dominica progeny and feeding damage varied considerably in dependence of cereal type. the influence of the grain species on the development of r. dominica was significant. the highest progeny was recorded in triticale (430.35 in average), followed by rye (221.42 in average), wheat (150.2 in average), barley (91.1 in average) and the smallest in oats (35.15 in average). biologica nyssana 9 (1) ⚫ september 2018: 55-61 perišić et al. ⚫ feeding preferences and progeny production of rhyzopertha… 58 the amounts of insect-damaged grains and dust after the removal of the progeny on various grains is presented in tab. 3. the dust weight after progeny count was significantly lower in oats (0.45 g). the highest damage and dust were detected in the triticale, compared to the wheat, as primary host of r. dominica (9.60 g vs. 4.52 g). pearson’s correlation coefficient revealed a significant positive correlation between progeny production and damaged grain in wheat (r=0.948, p<0.005 ), barley (r=0.677, p<0.005 ), rye (r=0.807, p<0.005) and triticale (r=0.880, p<0.005), while no correlation was found between the two data in oats (r=-0.194, p=0.470). the infested samples, with an exception of oats, had significantly higher moisture content, compared to un-infested sample. moisture content and the weight of damaged grain (tab. 2) were compared using a linear correlation analysis with pearson’s coefficient and significant positive correlations were detected for wheat (r=0.564, p<0.005), triticale (r=0.847, p<0.005) and rye (r=0.867, p<0.005), the lowest for barley (r=0.135, p<0.005), while no correlation was found between the two data for oats (r=0.319, p=0.229). total content of proteins was significantly lower in infested sample in all types of grain, except in oats, where no difference was found (tab. 3). pearson’s correlation coefficient revealed a significant negative correlation between protein content and damaged grain in wheat (r=-0.738, p<0.005), barley (r=-0.786, p<0.005) rye (r=-0.909, p<0.005) and triticale (r=-0.483, p<0.005 ), while no correlation was found between the two data in oats (r=-0.189, p=0.317). significant differences in ash content between the tested variants were recorded only for rye and triticale where ash contents were significantly increased. pearson’s linear correlation coefficient between ash content and damaged grain revealed significant positive correlations in rye (r=0.647, p<0.005) and triticale (r=0.847, p<0.005), while no correlation was recorded for wheat (r=0.198, p=0.038), barley (r=0.119, p=0.201) and oats (r=0.194, p=0.104). table 1. mean survival (% ± se) of rhyzopertha dominica adults and amounts of insect-damaged grains and dockage (g ± se) after three weeks of infestation of various cereal grains species. grain type adult survival (% ± se) damaged kernels (g ± se) dust (g ± se) wheat 98.25±0.11 b* 1.84±0.03d 0.43±0.05c barley 96.75±1.28 b 0.97±0.12b 0.18±0.06b rye 99.25±0.08 b 1.58±0.18c 0.89±0.09d oat 91.25±0.15 a 0.09±0.09a 0.02±0.01a triticale 98.25±0.89 b 3.87±0.23e 1.96±0.11e f 11.163 59.56 74.22 p < 0.05 < 0.05 < 0.05 * within each column, means followed by the same letter are not significantly different, in all cases df=4.75; duncan test at p>0.05. table 2. progeny emergence ( x ± se) and amounts of insect-damaged grains and dockage (g ± se) after the removal of the progeny from various cereal grains species grain type number of progeny ( x ± se) damaged kernels (g ± se) dust (g ± se) wheat 150.2±0.67c* 5.42±0.05c 1.69±0.06c barley 91.1±0.40b 2.61±0.09b 0.95±0.04b rye 221.42±0.79d 5.17±0.10c 3.33±0.09d oats 35.15±0.19a 0.45±0.03a 0.21±0.02a triticale 430.35±0.42g 9.60±0.11d 4.52±0.08e f 72.08 43.42 50.12 p < 0.05 < 0.05 < 0.05 * within each column, means followed by the same letter are not significantly different, in all cases df=4.75; duncan test at p>0.05. biologica nyssana 9 (1) ⚫ september 2018: 55-61 perišić et al. ⚫ feeding preferences and progeny production of rhyzopertha… 59 discussion mechanisms of tolerance of cereal grains are complex and depend on many factors, such as the physicochemical and biochemical grain properties as well as the insects’ capacity for biochemical and physical adaptation to post-harvest conditions (nawrot et al., 2006). it should be noted that physical and chemical properties of cereal grain have a fundamental influence on the developmental rate of r. dominica. the physical properties significantly affect the percentage of grain infestation of r. dominica (kavallieratos et al., 2010). keskin & ozkaya (2013) stated that grain hardiness represents one of the main reasons for stronger or weaker infestation of r. dominica. in our study, the highest progeny was found in triticale, which grain is classified as the softest (perišić et al., 2009a) and rye, which is significantly softer than wheat, while significantly smaller number of progeny was found in the barley, whose grain is harder than wheat (serna-saldivar, 2010b). also, triticale has the biggest 1000-kernel mass (am ≈ 45 g), compared to the oats and rye (am ≈ 29.35 and 36 g, respectively) (perišić et al., 2009b). according to kavallieratos et al. (2010), smaller size and kernel shape of grain could discourage the lesser grain borer from laying eggs in clusters on the kernels. edde (2012) claims that the texture of larval food can have significant influence on the rate of brood development. our data confirmed results reported by nawrot et al. (2010), who established high resistance of some varieties of wheat to sitophilus granarius (linnaeus, 1758) infestation. this resistance was associated with vitreosity-grain hardness and lower 1000-kernel mass and kernel diameter. it must be emphasized that kernel of barley and oats have a hull as an outer layer, namely hull stayed intact on the grain of this species after the harvest. hull of barley is completely concrescence with pericarp of kernel, while oats’ hull could be mechanically removed from the grain. mebarkia et al. (2009) noted that barley grain covered with its cellulose envelopes is unfavorable for s. granarius, but it is not disadvantage for r. dominica. the development of progeny in our study on barley can therefore be explained only by the greater hardness and with some chemical properties (content of proteins). table 3. moisture, protein and ash content (% ± se) in various cereal grains species after rhyzopertha dominica progeny count. sample moisture, protein and ash contents (% ± se) in various grain types wheat barley rye oats triticale moisture content infested sample 14.4±±0.05b* 12.3±0.05b 13.4±0.05b 11.3a 14.2±0.05b un-infested sample (control) 12.1 ±0.05a 11.7±0.05a 11.7±0.05a 10.8±0.05a 11.8±0.05a f 56.49 68.04 15.23 1.8 72.91 p < 0.05 < 0.05 < 0.05 0.204 < 0.05 protein content infested sample 12.5±0.05 b 11.73±0.05b 15.37±0.05b 13.26±0.05a 10.90±0.05b un-infested sample (control) 13.21±0.05a 12.04±0.05a 15.78±0.05a 13.30±0.05a 12.34±0.05a f 95.3 42.6 33.36 2 195.94 p < 0.05 < 0.05 < 0.05 0.149 < 0.05 ash content infested sample 1.83a 2.45b 2.08b 3.41a 2.81b un-infested sample (control) 1.71a 2.36a 1.91a 3.38a 2.25a f 1.21 3.745 22.05 0.42 98.91 p 0.280 0.07 < 0.05 0.52 < 0.05 * for each parameter separately, means within columns followed by the same letter are not significantly different, in all cases df=1.30; duncan test at p>0.05. un-infested sample (control) dockage and insect free sample biologica nyssana 9 (1) ⚫ september 2018: 55-61 perišić et al. ⚫ feeding preferences and progeny production of rhyzopertha… 60 in our study, the highest mortality of parents and the low progeny count was determined in oats. among all studied species, only oats have a hull which is not grown up with grain and which is covered with hair. the hull is unpalatable, dry, and brittle outer layer which accounts for approximately 25–36% of the total dry weight of the oat (welch, 1995). according to nawrot et al. (2010), the hull in oats is composed mainly of silica, which protects the kernel against insects and can slow down the infestation by these pests. these authors also claim that an additional factor which can be taken into consideration is the compact structure of the lemma and palea. reproduction insect ecology postulate is that the oviposition preference of a female should be correlated with the host’s suitability for the development of its offspring. hull on oats covers its grain as well as the clusters on the grain where r. dominica lays eggs which significantly reduces this phase of development. the amounts of damaged kernel and dust were in correlation with the emergence of progeny. since the highest number of progeny was determined in triticale, it was quite expected that aforementioned parameters were greatest in this species. mebarkia et al. (2009) examined the influence of grain species (soft and hard wheat, barley, rice, maize) on the development and the way of feeding of r. dominica. their results were similar to our study, with established differences between examined species for progeny number, length of development and the loss of dry matter generated after feeding of this pest. furthermore, loss of a dry matter as a consequence of the development of r. dominica progeny was different in examined species of small grains (wheat, barley, rice, and maize). all samples showed a significant change in moisture content, except in oats. as more damage was found in those grains, the increase in moisture was expected. pearson’s correlation coefficient also confirmed the association of these two parameters. as r. dominica mostly feed on seed endosperm and germ, it is able to reduce the grain merely to its pericarp (edde, 2012) and increased infestation and higher grain damage are expected to reduce total protein contents in untreated grains. jood & kapor (1993) also reported reduced protein contents in wheat seeds but only under intensive r. dominica infestation of 75%, while ozkaya et al. (2009) determined significant increase of protein, moisture and ash contents. the explanation for these opposites between different researches is based on the presence of impurities and uric acid, while the real content of protein was decreased as a consequence of r. dominica feeding. a significant difference in ash content was detected in triticale and rye. changes in ash content were caused by r. dominica presence and feeding, which was confirmed by pearson’s correlation coefficient. our data are in agreement with those reported by jood et al. (1992), who found that r. dominica influence the content of mineral matter in grain by increasing it under strong infestation. keskin & ozkaya (2013) confirmed the same effect of s. granarius as this insect also feeds mostly on seed endosperm and germ. these experiments were conducted only on wheat, with no results on the other small grain. small grains wheat, barley, rye and triticale can be considered as ''nutritionally suitable'' for r. dominica development. triticale is the most susceptible to attacks and suitable for development of r. dominica. it is clearly demonstrated that oats is less infested and is not a suitable host for the strain of r. dominica used in the present study. the presence of the hull in oats proved to be important morphological characteristic of a kernel which has an effect on development and feeding of the examined pest. the role of the hull can be objective of future research, i.e. is it represents a physical barrier only or possesses some chemical substances which act as a repellent against r. dominica. acknowledgements. this study was carried out within a project supported by the ministry of education, science and technological development of the republic of serbia, grant no oi 173038. references arthur, f.h., ondier, g.o., siebenmorgen, t.j. 2012: impact of rhyzopertha dominica (f.) on quality parameters of milled rice. journal of stored products research, 48: 137-142. arthur, f.h., laura starkus, l., smith, m.c., phillips, t.w. 2013: methodology for determining susceptibility of rough rice to rhyzopertha dominica and sitotroga cerealella. journal of pest science, 86: 499–505. astuti, l.p., mudjiono, g., rasminah, ch.s., rahardjo, b.t. 2013: susceptibility of milled rice varieties to the lesser grain borer (rhyzopertha dominca f.). journal of agricultural science, 5: 145-149. chanbang, y., arthur, f.h., wilde, g.e., throne, j.e. 2008: hull characteristics as related to susceptibility of different varieties of rough rice to rhyzopertha dominica (f.) (coleoptera: bostrichidae). journal of stored product research, 44: 205–212. biologica nyssana 9 (1) ⚫ september 2018: 55-61 perišić et al. ⚫ feeding preferences and progeny production of rhyzopertha… 61 edde, p., 2012: a review of the biology and control of rhyzopertha dominica (f.). the lesser grain borer. journal of stored products research, 48: 1-18. huberty, a.f., denno, r.f. 2006: plant water stress and its consequences for herbivorous insects: a new synthesis. ecology, 85: 83-98. icc standard methods, international association for cereal science and technology (1976): determination of the moisture content of cereals and cereal products (practical method). icc standard method no. 110/1. icc, vienna, austria. icc standard methods, international association for cereal science and technology (1990): determination of ash in cereals and cereal products. icc standard method no. 104/1. icc, vienna, austria. icc standard methods, international association for cereal science and technology (1994): determination of crude protein in cereals and cereal products for food and for feed. icc standard method no. 105/2. icc, vienna, austria. jood, s., kapor, a., singh, r., 1992: mineral content of cereal grains as affected by storage and insect infestation. journal of stored products research, 28 (3): 147-151. jood, s., kapor, a.c., 1993: protein and uric acid contents of cereal grains as affected by insect infestation. food chemistry, 46: 143-146. kavallieratos, n.g., athanassiou, c.g, vayias, b.j., kotzamanidis, s., synodis, s. 2010: efficacy and adherence ratio of diatomaceous earth and spinosad in three wheat varieties against three stored-product insect pests. journal of stored products research, 46: 73-80. keskin, s., ozkaya, h. 2013: effect of storage and insect infestation on the mineral and vitamin contents of wheat grain and flour. journal of economic entomology, 106 (2): 1058-1063. kljajić, p. 2008: suzbijanje štetnih insekata uskladištenog žita. in. kljajić, p. (ed.), zaštita uskladištenih biljnih proizvoda od štetnih organizama. 67-100 p. institut za pesticide i zaštitu životne sredine, beograd. mebarkia, a., guechi, a., mekhalif, s., mekhalif, m. 2009: biochemical composition effect of the some cereal species on the behavior of sitophilus granarius l. and rhyzoperta dominica f. species in semi-arid zone of setif, algeria. journal of agronomy, 8 (2): 60-66. metwaly, m.r., abou-ghadir, m. f., abdu-allah, g. m., abdel-nasser, m. k. 2015: susceptibility of certain wheat varieties to the infestation by rhyzopertha dominica (f.) and tribolium confusum (du val). journal of phytopathology and pest management, 2 (3): 1-8. nawrot, j., warchalewski, j.r., piaseckakwiatkowska, d., niewiada, a., gawlak, m., grundas, s.t., fornal, j. 2006: the effect of some biochemical and technological properties of wheat grain on granary weevil (sitophilus granarius l.) (coleoptera: curculionidae) development. 9th international working conference on stored product protection, 400407 p. nawrot, j., gawlak, m., szafranek,j., szafranek, b., synak, e., warchalewski, j.r., piaseckakwiatkowska, d., b1aszczak, w., jelinski, t., fornal, j. 2010: the effect of wheat grain composition, cuticular lipids and kernel surface microstructure on feeding, egg-laying, and the development of the granary weevil, sitophilus granarius (l.). journal of stored products research, 46: 133-141. ozkaya, h., ozkaya, b., colakoglu, a. 2009: technological properties of a variety of soft and hard bread wheat infested by rhyzopertha dominica (f.) and tribolium confusum du val. journal of food, agriculture and environment, 7 (3-4): 166-172. pires, m., nogueira, r., pina, d., manica, c., faroni, r., moreira, p. 2016: walking stability of rhyzopertha dominica (fabricius, 1792) (coleoptera: bostrichidae). brazilian journal of biology, 76 (3): 568-576. perišić, v., milovanović, m., staletić, m., nikolić, o. 2009a: delija new spring triticale cultivar. the iv congress of the serbian genetic society, tara, 285-289 p. perišić, v., milovanović, m., ćulaković, b., janković, s., staletić, m. 2009b: produktivnost kragujevačkih sorata ozime pšenice, ječma i jarog ovsa. poljoprivredne aktuelnosti, 3-4: 5-14. rees, d.p. (ed.). 2004: insects of stored products. csiro publishing, australia. 181 p. serna-saldivar, o. 2010a: chemical composition of cereal grains. in: serna-saldivar, o. (ed.), cereal grains: properties, processing and nutritional attributes, 3: 81-108, crc press, taylor & francis group, new york. serna-saldivar, o. 2010b: physical properties, grading, and specialty grains. in: sernasaldivar, o. (ed.), cereal grains: properties, processing and nutritional attributes, 2: 43-80, crc press, taylor & francis group, new york. the official gazette of the republic of serbia. 2016: pravilnik o kvalitetu žita, mlinskih i pekarskih proizvoda, 68/16. welch, r. 1995: the chemical composition of oats. in: welch, r. (ed.), the oat crop, 279–320. springer, the netherlands. http://www.scielo.br/scielo.php?script=sci_serial&pid=1519-6984&lng=en&nrm=iso http://www.scielo.br/scielo.php?script=sci_serial&pid=1519-6984&lng=en&nrm=iso genomic dna from rat blood biologica nyssana 7 (1)  september 2016: 47-52 takić miladinov, d.  genomic dna from rat blood… 47 original article received: 22 january 2016 revised: 23 february 2016 accepted: 01 mart 2016 genomic dna from rat blood: a comparison of two extraction methods dijana takić miladinov1,2* 1department of molecular biology, institute for biological research, university of belgrade, bulevar despota stefana 142, 11060 belgrade, serbia 2department of biology and ecology, faculty of science and mathematics, university of niš, višegradska 33, niš, serbia * e-mail: takicdijana@gmail.com abstract: takić miladinov, d.: genomic dna from rat blood: a comparison of two extraction methods. biologica nyssana, 7 (1), september 2016: 47-52. in this study, two methods for dna extraction from fresh rat blood were compared. one is based on the use of cetyltrimethylammonium bromide (ctab method), while the other one is well-known salting out method. spectrophotometric analysis was employed to assess yield and purity of isolated dna, while agarose gel electrophoresis was carried out to evaluate dna integrity. the results have clearly demonstrated that the extraction method has significantly influenced the quantity and purity of isolated dna. by using the ctab method, a larger quantity of high-molecular weight dna with good purity is obtained which, along with time and cost-efficiency of the procedure, makes this method more suitable for the extraction of dna from rat whole blood. key words: dna extraction; ctab; salting out; rat whole blood apstrakt: takić miladinov, d.: genomska dnk iz krvi pacova: poređenje dveju metoda ekstrakcije. biologica nyssana, 7 (1), septembar 2016: 47-52. u ovoj studiji su upoređivane dve metode za ekstrakciju dnk iz sveže krvi pacova. jedna je zasnovana na upotrebi cetil trimetil amonijum bromida (ctab metoda), dok je druga dobro poznata metoda isoljavanja. za procenu prinosa i čistoće izolovane dnk korišćena je spektrofotometrijska analiza dok je agarozna gel elektroforeza korišćena za procenu integriteta dnk. rezultati su jasno pokazali da je način ekstrakcije značajno uticao na količinu i čistoću izolovane dnk. korišćenjem ctab metode dobijena je veća količina dnk velike molekulske težine i dobre čistoće što, zajedno sa efikasnošću u pogledu trajanja i ekonomičnosti procedure, čini ovu metodu pogodnijom za ekstrakciju dnk iz pune krvi pacova. key words: dnk ekstrakcija; ctab; isoljavanje; puna krv pacova 7 (1) • september 2016: 47-52 doi: 10.5281/zenodo.159103 biologica nyssana 7 (1)  september 2016: 47-52 takić miladinov, d.  genomic dna from rat blood… 48 introduction deoxyribonucleic acid (dna) isolation is a process of extraction of dna from wide variety of sources. the first dna was isolated from white blood cells in 1869 by swiss physician and biologist friedrich miescher (d a h m , 2008). currently, it is a routine procedure in molecular biology, forensic science and medical diagnostics. the preparation of high quality dna samples from various sources is the first and vital step for subsequent molecular biological techniques such as polymerase chain reaction (pcr), restriction fragment length polymorphism (rflp), mutation detection, cloning as well as genotyping (p h i l l i p s et al., 2000; w a n g et al., 2 0 0 3 ; t a n r i o v e r et al., 2010). although in some cases pcr can be performed without previously dna isolation step (k o v a c e v i c g r u j i c i c et al., 2012), it is usual to employ pure genomic dna in determination of genetic disorders, epigenetic studies and diagnostic tests (a n g e l i n i et al., 2000; p h i l l i p s et al., 2000; w a n g et al., 2003; l e w i s et al., 2005). various methods for dna isolation from blood samples have already been established (m i l l i g a n , 1998; a n g e l i n i et al., 2000) and many dna isolation kits are commercially available today. the choice of a method for preparing dna depends on the source as well as quantity, quality and purity of the dna desired for downstream applications. an ideal extraction technique should optimize yield, purity and integrity of isolated dna, and also be efficient in terms of cost, time and safety. the cetyltrimethyl ammonium bromide (ctab) method is successfully used for extraction of high quality genomic dna from wide variety of sources (g u s t i n c i c h et al., 1991; t h o m a s et al., 1997; d e s l o i r e et al., 2006; j a s b e e r et al., 2009; c h e n et al., 2010; f i l h o & a l m e i d a , 2013). also, a rapid salting out dna extraction method is well documented in the literature (m i l l e r et al., 1988; o l e r u p & z e t t e r q u i s t , 1992; a l j a n a b i & m a r t i n e z , 1997). the aim of this study was to determine whether these two methods are suitable for isolation of high-molecular weight dna from fresh rat blood. the two methods were compared in terms of yield, purity and quality of isolated dna as well as in terms of cost/time efficiency and environmental safety. material and methods dna extraction published protocols with slight modifications were performed for the ctab (g u s t i n c i c h et al., 1991) and salting out (o l e r u p & z e t t e r q u i s t , 1992) extraction methods. samples of fresh whole blood were obtained from rat tail vein and aliquoted in 1.5 or 2 ml tubes containing edta. for both methods, dna was extracted from four samples of fresh blood. in the final step of isolation, dna was resuspended in 50 µl of te buffer (10 mm tris-hcl, 1mm edta, ph 7.6) and stored at -20 °c until analysis by agarose gel electrophoresis. ctab method solution i: 8% ctab, 1.5 m nacl, 100 mm tris ph 8.5, 50 mm edta ph 8. solution ii: 5% ctab, 0.1 m nacl 500µl of solution i was pre-warmed at 68 °c, mixed with 250 µl of fresh blood and further incubated at 68°c for 30 min. afterwards, 750 µl of chloroform was added, inverted several times to mix and samples were centrifuged at 13 000 rpm for 5 min at room temperature (rt). dna containing upper phases were transferred into a new tubes, with no disturbing of proteins containing medium phase, and theirs volumes (v) were determined. aqueous phases were mixed with solution ii (v/50) and ddh2o (v) and centrifuged at 13 000 rpm for 5 min at rt. supernatants were discarded and pellets were resuspended in 250 µl 1.2 m nacl. to precipitate dna, 750 µl of ice cold absolute ethanol was added and samples were centrifuged at 13 000 rpm for 5 min at +4 °c. the pellets were washed twice with 750 µl of 70% ethanol, and centrifuged at the above mentioned conditions to remove residual salt. the pellets were air-dried at rt. salting out extraction method (se method) red blood cell lysis buffer (rbcl): 0.5 gr of khco3 and 4.14 gr of nh4cl were diluted in 500 ml of dh2o. digestion buffer: 100 mm nacl, 10 mm tris-hcl ph 8, 25 mm edta ph 8, 0.3 mg/ml proteinase k, 0.05% triton x-100. adjust final volume to 5 ml with dh2o. fresh blood (500 µl) was aliquoted in four sterile 1.5 ml tubes and centrifuged at 3 000 rpm for 10 min at rt. supernatants were discarded and pellets of the cells were resuspended in 1.5 ml of rbcl buffer. following incubation for 10 min at rt with occasional inverting of the tubes, the mixtures were centrifuged at the same conditions mentioned above. the lysis step was repeated for 3-4 times until the supernatants were nearly clear and the pellets were mostly white. after lysis, the pellets were resuspended in 100 µl of digestion buffer. two tubes with samples were digested at 37 °c overnight, while two others were digested at 65 °c for one hour. after digestion was complete, 240 µl of saturated 5m nacl was added to all samples and, after centrifugation at biologica nyssana 7 (1)  september 2016: 47-52 takić miladinov, d.  genomic dna from rat blood… 49 3 000 rpm for 10 min, precipitated protein pellets were left at the bottom while supernatants containing dna were transferred to the new tubes. ice-cold absolute ethanol was added in a double volume and, after centrifugation at 13 000 rpm for 20 min at +4 °c, supernatants were discarded and pellets were washed with 1 ml of 70% ethanol. after final centrifugation under the same conditions, the pellets were air-dried at rt. spectrophotometric analysis – concentration and purity determination concentration and purity of extracted dna samples were determined by biospectrometer (eppendorf, germany). two microliters of each dna sample was mixed with 48 µl of 1 × te buffer and put in a quartz cuvette. absorbance was measured at a wavelengths of 260, 280 and 230 nm (a260, a280 and a230) which are max absorption points for bases in nucleic acids, proteins and polyphenols/polysaccharides, respectively. dna was quantified by measuring uv absorbance at 260 nm, while the purity of genomic dna was evaluated on the basis of uv absorption ratios at 260 and 280 nm (a260/a280) and 260 and 230 nm (a260/a230). agarose gel electrophoresis – integrity determination the quality and integrity of all dna samples were assessed by agarose gel electrophoresis. about 650 ng of the isolated dna was analyzed on 0.8% agarose gel (applied biosystems, usa) in 1 × trisborate-edta buffer containing 0.5 µg/ml ethidium bromide, under conditions: 80v and 120 ma for one hour. the bands were visualized using chemidoc xrs uv transilluminator (biorad, usa). results the two different dna extraction methods were analyzed according to the following criteria: yield, purity, quality, environmental safety, time and cost efficiency. dna yield rates and purity table 1 summarizes the yield and purity ranges of dna samples extracted by two different methods. ctab method resulted in high mean yield of dna (1130.25 ng/µl; 22.6 µg of dna per 100 µl of blood). the dna yields for the se method were dependent on duration of digestion step. samples 1 and 2 were incubated in digestion buffer just 1 hour at 65 °c while samples 3 and 4 were digested overnight at 37 °c. prolonged digestion yielded 236.65 ng/µl dna (2.37 µg of dna per 100 µl of blood), while approach with short digestion resulted in approximately six times lower mean dna yield (39 ng/µl; 0.39 µg of dna per 100 µl of blood). according to data from the literature, it is common to table 1. dna yields, absorbance ratios, environmental safety, and estimated duration of the two different methods for dna extraction from fresh rat blood. concentration (ng/µl) a260/280 a260/230 yield (µg) per 100 µl of blood e n zy m es c h lo ro fo rm d u ra ti o n ( h ) ctab method no yes ~ 1 sample 1 456 1.77 2.07 sample 2 1577 1.8 2.31 sample 3 1093 1.81 1.93 sample 4 1395 1.82 1.9 mean ± s.e.m. 1130.25 ± 245 1.8 ± 0.01 2.05 ± 0.09 22.6 se method (digestion for 1 h at 65 °c) yes no ~ 3.5 sample 1 40 1.76 1.57 sample 2 38 1.66 2.1 mean ± s.e.m. 39 ± 1 1.71 ± 0.05 1.83 ± 0.26 0.39 (digestion overnight at 37 °c) ~ 16 sample 3 220.9 1.51 1.12 sample 4 252.4 1.41 1.2 mean ± s.e.m. 236.65 ± 15.75 1.46 ± 0.05 1.16 ± 0.04 2.37 biologica nyssana 7 (1)  september 2016: 47-52 takić miladinov, d.  genomic dna from rat blood… 50 extract approximately 2 µg of genomic dna per 100 µl of rat/mouse whole blood by employing various extraction methods (h o f s t e t t e r et al., 1997; x i n g et al., 2007; c h a c o n -c o r t e s , 2012) and about 6 µg of dna by using commercial kits (n o e t h & d a s o v i c h -m o o d y , 1997; c h a c o n -c o r t e s et al., 2012). a260/a280 and a260/a230 absorbance ratios were employed to evaluate the purity of extracted dna samples. according to s a m b r o o k et al. (1989) pure dna extracts have 1.8 < a260/a280 < 2 and a260/a230 > 2. the data in table shows that there are some differences in dna extracts’ purity dependent on the method of extraction. the ctab method produced a260/a280 ratios ranged from 1.77 to 1.82, with a mean value of 1.8. such results indicate the pure dna samples with no protein or rna contaminations. furthermore, a260/a230 ranged from 1.9 to 2.31, with a mean value of 2.05, indicating the high purity of samples 1 and 2. samples 3 and 4 had slight lower a260/a230 ratios which means certain contamination with carbohydrates, salts or organic solvents. on the other side, se method produced dna samples with poor a260/a280 and a260/a230 ratios. all dna samples have been contaminated with proteins, salts or organic solvents. sample 3 was the only one which met criteria a260/a230 > 2, but had a poor a260/a280 ratio of 1.66 indicating protein contamination. the two techniques were found to be very different in terms of yield and purity of dna recovered from blood. ctab method resulted in much higher yield of pure dna compared to se method which produced dna samples of poor yield and purity. since source effect was reduced by using the same samples, variations in yield and purity can be attributed to the effects of the extraction methods. high dna yield and good purity obtained by ctab method were previously described in the literature for a variety of extraction sources (j a s b e e r et al., 2009; c h e n et al., 2010; f i l h o & a l m e i d a , 2013). on the contrary, salting out method often results in an insufficient purity of dna extracts (f i l h o & a l m e i d a , 2013). to improve purity, s a m b r o o k et al. (1989) suggested taking a special care when dispart supernatant, containing dna, from the sediment, containing so many contaminants. also, in a case of contamination of dna extract with salts, it is suggested to re-precipitate dna with ethanol. gel electrophoresis analysis of isolated dna the quality and integrity of genomic dna samples isolated by two different methods were examined by 0.8% agarose gel electrophoresis. this technique has been widely used for determining the size of dna samples (z i m m e r m a n n et al., 1998). as can be seen in figure 1, the main bands of dna were slowmigrating and located high above 3 kb. all samples contained high-molecular weight dna with no lowmolecular weight smear tails. although all lines should contain the same quantity of dna (650 ng), the lane 4 in se method had much lower dna content on the gel (fig. 1). one possible reason could be over-estimation of dna concentration in the spectrophotometric analysis. proteins, rna, salts, lipids and other contaminants can increase the spectrophotometric estimation of dna concentration (h a q u e et al., 2003). except rna molecules, the other contaminants can not be visualized on gel by staining with ethidium bromide, but one can notice the lower amount of dna on a gel in regard to the expected amount. the data from dna agarose gel electrophoresis supplement the data from absorbance readings indicating compatibility of the two techniques. fig. 1. agarose gel electrophoresis of total genomic dna samples isolated from rat fresh blood by two different extraction methods. l – 100 bp dna ladder (fermentas). environmental safety, time and cost efficiency the ctab method required approximately 1 h compared to 3.5 h or overnight incubation required for the se method. moreover, the use of proteinase k makes se method less cost-effective than ctab method. despite time and cost efficiency, ctab method employs chloroform, well-known hazardous organic solvent. conclusion yield, purity and 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u. 1998: quantitative and qualitative evaluation of nine different extraction methods for nucleic acids on soy bean food samples. zeitschrift fur lebensmitteluntersuchung und forschung a, 207: 81-90. anticancer compounds from medicinal plants biologica nyssana 4 (1-2)  december 2013: 1-7 sadakovic, k., kuštera, m..  fungal conservation: protected species… 35 original article fungal conservation: protected species of fungi in south serbia region dušan sadiković , marjan kuštera mycological socety of niš, somborska 81 a/9, 18000 niš, serbia * e-mail: dusan.sadikovic@gmail.com abstract: sadiković, d., kuštera, m.: fungal conservation: protected species of fungi in south serbia region. biologica nyssana, 4 (1-2), december 2013: 35-40. protection and conservation of fungi has only recently became an issue of concern. main motives for increased attention are uncontrolled, mass collecting of edible wild mushrooms and environmental pollution which leads to the rapid decline of their natural habitats, some of which are rich with rare and endangered species. by serbian nature conservation law 2010. there are 38 strictly protected fungal species of which 17 species are recorded in this paper. 11 of those recorded species are on european and/or national red list of endangered fungal species. all investigated territories were in south serbia region. this study is a contribution to conservation of protected and threatened fungi and their respective habitats in serbia. key words: red list, protected species, south serbia, macrofungi. introduction fungi play vital roles in most of the ecosystems on the biosphere. as they are the dominant decomposers of the lignocellulose plant debris and knowing that cellulose is the most abundant organic polymer on the earth (k l e m m , 2005), it is obvious that fungi are integral factors in processes of recycling carbon and reprocessing of nutrients in general. they have established mutualistic symbioses with a wide range of organisms: cyanobacteria and green algae (in lichens), bryophytes, pteridophytes, gymnosperms and angiosperms (in mycorrhizae), and coleopteran, dipteran, homopteran, and isopteran insects (k e n d r i c k , 2011) . also with some hymenopterian insects like fungus-growing ants (attini: formicidae) engage in an obligate mutualism with fungi they cultivate for food (c a l d e r a e t a l ., 2009). in europe more than 15000 species of macroscopic fungi had been identified. this is a conservative estimate because many morphological species concepts are proving to be species complexes. also identifying species of concern has been hampered by the sporadic occurrence of the sexual fruit bodies (sporocarps) of the fungi, the cryptic nature and the lack of morphological characters of the vegetative state, and the difficulty in culturing most species for lab work (l i l l e s k o v e t a l ., 2010). in serbia the official number of recorded species, presented by institute for nature conservation of serbia, is 1300 but it’s estimated that it’s closer to 3000, a figure which would represent about 20% of total european mycodiversity. biogeographical studies on fungi are relatively scarce, due mainly to methodological problems (a r n o l d s , 1997). the awareness that fungi can be threatened came recently, in the early 1980s, when it was noticed that heavy acidification of soil due to acid rains has a receding effect, both qualitatively and quantitatively. acid rain results into acidification of soil, which increases the exchange between 11 th sfses • 13-16 june 2013, vlasina lake 4 (1-2) • december 2013: 35-40 biologica nyssana 4 (1-2)  december 2013: 35-40 sadakovic, k., kuštera, m..  fungal conservation: protected species… 36 hydrogen ion and nutrient cations like potassium (k), magnesium (mg) and calcium (ca) in the soil. these cations are liberated into soil and can be rapidly leached out in soil solution along with sulphate from acid input. acid induced leaching leads to nutrient deficiency in the affected soils and thus negatively affecting microbial processes (s i g h & a g r a w a l l , 2008). also higher levels of nitrogen caused by anthropogenic activities has a reducing effect on mycofloral diversity as it was proven that with increasing the n deposition, ectomycorrhizal root tip abundance and mycelial production can be decreased five and 10-fold. in addition, fungal community can be changed and the species richness decreased (k j ø l l e r e t a l ., 2012). recognizing this problem, national red lists started to emerge, first being in germany in 1982. today more than 35 countries have some form of red list for fungi. evaluations of those red lists in almost every european country, together with many studies regarding fungal conservation, indicate that a large portion, probably in the range of 2000 –3000 species of macrofungi throughout europe are declining and their future is uncertain. european mycologists, through the european council for conservation of fungi (eccf), have prepared list of fungi for possible inclusion in the bern convention. these 33 species represent only a small fraction of all threatened fungal species, but by including them in the convention appendix, the need for conservation of fungi and their habitats would be officially recognized (k j ø l l e r et al., 2012). in serbian legislation, first species of fungi were put under protection in 1991, as “natural rarities threatened by exploitation and trade”, under the decision on amending the decision on putting plant species under protection as natural rarities (1991) but it had inadequate protection measures as well as cardinal taxonomic mistakes. because yugoslavia was in the first half of the last decade the largest exporter of bolets in the, of which the largest quantities were from serbia, the protection only included a few edible species world (for example in 1993. 5 186 100kg of boletus edulis was purchased in serbia) (i v a n c e v i c e t a l ., 2012). currently by the nature conservation law (2009) and regulation on the proclamation and protection of strictly protected and protected wild species of plants, animals and fungi (2010), 38 species of macroscopic fungi are treated as strictly protected. serbian preliminary national red list of endangered species was comprised in 1998 (i v a n c e v i c , 1998) and it contained 96 species. in 2004 the list was corrected and amended to iucn standards. the nprl of endangered species was not fully or not at all considered when making the list of strictly protected species and methods used in comprising this list were unclear. material and methods the areas were investigated between 2011. and 2013. all mycological investigations were conducted in south serbia region (fig. 1.). the fungal specimens were identified on the basis of macroscopic and microscopic morphological characteristics and specific chemical reactions of fruiting bodies according to specific identification keys: (božac 1978; foht 1986; breitenbach & kränzlin 1995; maca et al., 1995; mcknight et al., 1998; jordan 2004; uzelac 2009). author citations for each taxon are abbreviated according to the index fungorum(2013). for the analysis of the legal protection of fungi in serbia and europe, following material was used: guidance for the conservation of mushrooms in europe. convention on the conservation of european wildlife and natural habitats. document prepared by beatrice senn-irlet, jacob heilmannclausen and anders dahlberg for the european council for conservation of fungi (eccf) within the european mycological association (ema). strasbourg, 17 october 2007. одлука о изменама и допунама одлуке о стављању под заштиту биљних врста као природних реткости. службени гласник срс 49, 15. 08. 1991; [decision on amending the decision on putting plant species under protection as natural rarities, 1991] закон о заштити природе. службени гласник републике србије 36, 12.05.2009. i 88/2010; [nature conservation law, 2009 – actual] правилник о проглашењу и заштити строго заштићених и заштићених дивљих врста биљака, животиња и гљива. службени гласник републике србије 5, 05. 02. 2010; [regulation on the proclamation and protection of strictly protected and protected wild species of plants, animals and fungi, 2010 – actual]. the purpose of this study is presenting to the scientific community precise bio geographical data on rare and endangered species of macromycetes in serbia and thus helping in process of making more accurate national red list and national checklist. this is the beginning of more extensive investigations which will revel yet unexplored south serbian mycodiversity. biologica nyssana 4 (1-2)  december 2013: 35-40 sadakovic, k., kuštera, m..  fungal conservation: protected species… 37 figure 1. map of invastigated area jsmt.mali jastrabac, dd-debeli del hill, kf-mt.kalafat, komt.koritnik, jk-jelašnica gorge, se-mt.seličevica, bg-mt.babička gora, gh-mt.suva planina(sw foothill), ds-mt.suva planina, sd-mt.stara planina(sedlar), tdmt.stara planina(arbinje), temt.stara planina(temska area), vs-mt.veliki stol, vp-mt.vlaške planine, mc-mt.mali čemernik, go-mt.goljak, pc-mt.pljačkovica, bk-mt.besna kobila, du-mt.dukat results and discussion of 38 species of macromicetes protected by nature conservation law we located and indentified 17 species, belonging to 13 genera, 7 families and 5 orders. the list of species with number of sightings, location and status is presented in the table (table 1. list of species found with number and localities of findings and status). phylloporus pelletieri (lév.) quél., sarcosphaera coronaria (jacq.) j. schröt. and hericium erinaceus (bull.) pers. are listed on european preliminary red list and are regarded as highly endangered species. p.pelletieri is widespread in europe (very rare in european russia, also in ukraine and moldova), from the lowland up to the montane zone, sometimes subalpine. it is rare in most regions, very local and in low numbers, but more widespread in the alps, e.g. in switzerland. the species is spreading into asia. it is in national lists of endangered fungi in austria, denmark, germany, hungary, moldova, norway, poland, sweden, and the netherlands. in total it has 926 localities in europe and to our knowledge, other than founding in this paper, it has only few unofficial locations in serbia. s.coronaria (syn. s.crassa) is distributed in north europe and mountains of central and south europe up to 1700 m altitude. it is rare in most regions but in some regions more widespread and even numerous in places, for instance in the swiss alps and central italy. it is in national lists of endangered fungi in austria, bulgaria, denmark, estonia (also protected by law), finland, germany, great britain, hungary, norway, poland, and sweden. it has 691 localities in europe with official 2 localities in serbia according to (dahlberg et al., 2006). h.erinaceus is widespread in europe, from the lowland up to the montane zone, north to southern scandinavia; generally rare, for instance in switzerland (5 records between 220 and 930 m) and austria (4 records from the vienna region, altitude 150m). it is locally more common, e.g. in southern england and the central part of the netherlands. it is found on national lists of endangered fungi in armenia, austria, bulgaria, denmark, germany, great britain, greece, macedonia, poland, sweden (also protected by law), switzerland, the biologica nyssana 4 (1-2)  december 2013: 35-40 sadakovic, k., kuštera, m..  fungal conservation: protected species… 38 netherlands (eccf, 2001). it has 435 localities in europe with 3 official in serbia. the rest of the fungal species recorded in this research are also all on other european countries red lists and/or leagaly protected, but because of many lists are still in proces of forming and their data deficiency, we are unable to produce the precise data on the number of their localities and state of populations fully yet. table 1. list of species found with number and localities of findings and status (pnrl-preliminary national red list) taxa total number of recorded localities listed in location pezizales pezizaceae sarcosphaera coronaria (jacq.) j. schröt 1 perl mt. stara planina agaricales hygrophoraceae hygrocybe punicea (fr.) p. kumm 1 pnrl dukat mountain hygropophrous marzuolus (fr.) bres. 6 pnrl mt. suva planina mt.suva planina sw foothill mt.babicka gora mt. seličevica mt. mali jastrebac debeli del hill mt.golemi stol tricholomataceae leucopaxillus giganteus (quel.) singer 1 pnrl mt. stara planina boletales boletaceae boletus impolitus fr. 4 pnrl mt.kalafat mt.seličevica mt.suva planina mt.babicka gora boletus regius kromb. 8 pnrl mt.besna kobila mt. pljackovica mt.selicevica mt.selicevica (gabrovac) mt.mali jastrebac mt.suva planina mt. golemi stol mt.koritnik boletus rhodoxanthus (krombh.) kallenb 2 pnrl mt.vlaška planina debeli del hill boletus satanas lenz pnrl mt. stara planina leccinum crocipodium (letell.) watling 1961 3 mt.seličevica mt.besna kobila jelašnica gorge phylloporus pelletieri (lév.) quél. 1 perl mt.mali čemernik strobilomyces strobilaceus (scop.) berk. 1 pnrl mt.goljak geastrales geastraceae geastrum fornicatum (huds.) hook. 1 mt.dukat geastrum melanocephalum (czern.) v.j. staněk 2 mt.veliki stol debeli del hill phallales phallaceae mutinus caninus (huds.) fr. 1 pnrl mt.veliki stol phallus hadriani vent. 1 pnrl mt. stara planina russulales hericiaceae hericium coralloides (scop.) pers. 1 mt.veliki stol hericium erinaceus (bull.) pers. 1 pnrl perl mt.stara planina (arbinje) biologica nyssana 4 (1-2)  december 2013: 35-40 sadakovic, k., kuštera, m..  fungal conservation: protected species… 39 only 20 species from the red list one found on the strictly protected list and knowing that on the preliminary red list also contained some internationally significant and protected wild species which serbian government had promise to protect (biodiversity strategy of the republic of serbia for the period 2011 – 2018) it is evident that forming of the list of protected species was not based on the preliminary red list data, rather in superficial, subjective and unscientific manner. the fact that in 2007 the author of nprl has presented to ministry of environment and spatial planning project proposition for making the red book of fungi in serbia, which was highly acclaimed by the ministry, was not yet accepted furthermore shows the gap between serbian mycological experts and the government (personal email communication with author). conclusion if a species is rare or endangered in one region or in whole country it doesn’t mean that its state is homogeneous throughout europe and vice versa so biogeographical information on fungi is crucial for understanding biodiversity patterns, evolutionary processes and monitoring. unfortunately these kinds of publications are scarce in serbia, especially for south serbian region due to lack of experts and it’s vastly unexplored, but rich terrains. deficient number of this sort of research may also be contributed to the annual fluctuations of fungal fruiting bodies due to micro and macro climate conditions, uncontrolled wild mushrooms picking, stochastic events and effemeral properties of fungal fruiting bodies. in order to better protect natures rearities, serbia must adopt less anthropocentric and more ecocentric laws. wild mushroom picking itself does not effect the numbers of emerging fruiting bodies but soil trampeling of the mushroom pickers can destroy pre-fruit body primordia formed at the soil surface and thus reduce the frequency of occurrence and reduce their quantity (egli et al., 2006). beside the prevention of picking young and immature specimens and specimens less in size then size allowed for respective species, the state should specify time annually and daily when picking of certain species is allowed. also instead of allowing only picking 2/3 of total number of sporocarps found at the picking site, which is very difficult to assess, government should restrict maximum allowed quantity of collected sporocarps per person and for groups like in some european countries legislations (e.g. in luxembourg one person can pick up to 1kg of sporocarps per day and for a group, the allowed maximum is 3kg). with these measures the disturbance made by trampling of forest floor would be significantly reduced and mushroom picking for sale purposes would be significantly decreased at the same time. in order to bring better nature protecting laws, actual state of serbian mycodiversity must be evaluated. this is only possible trough national and regional checklists, red lists and more extensive biogeographical research. for this to be possible, far better state cooperation with scientific community and experts is imperative. acknowledgments the authors would like to express gratitude to all members of the mycological society of niš for helping in research for this paper. references arnolds, e. 1997: biogeography and conservation. in: wicklow, d., soderstrom, b. (ed.), the mycota iv: environmental and microbial relationships: (2): 115-131, springer-verlag, berlin. biodiversity strategy of the republic of serbia for the period 2011 – 2018. ministry of environment and spatial planning, belgrade 2011. božac, romano. 1978: gljive naših krajeva. grafički zavod hrvatske. breitenbach, j., kränzlin, f. 1995: pilze der schweiz. band 4. verlag mykologia, luzern. 371p. cladera, e., poulsen, e., suen, g., currie, c., 2009: insect symbioses: a case study of past, present, and future fungus-growing ant research. environmental entomology, 38(1):78-92. dahlber, a., croneborg, h., 2006: the 33 threatened fungi in europe. council of europe publishing, belgium. egli, s., peter, m., buser, c., stahel, w., ayer, f., 2006: mushroom picking does not impair future harvests – results of a long-term study in switzerland. biological conservation, 129: 271276. focht, i., 1986: ključ za gljive: ilustrirani uvod u gljivarstvo. naprijed, zagreb. index fungorum. http://www.indexfungorum.org/names/names.asp ivančević, b., 1998: a preliminary red list of the macromycetes of yugoslavia. in: perini, c. (ed.), conservation of fungi in europe: 57-61, università degli studi, siena. ivančević, b., matavulj, m., vukojević, j., karaman, m., 2012: fungi in the legislation of biologica nyssana 4 (1-2)  december 2013: 35-40 sadakovic, k., kuštera, m..  fungal conservation: protected species… 40 the republic of serbia. zbornik matice srpske za prirodne nauke, 123: 51–64 jordan, m., 2004: the encyclopedia of fungi: of britain and europe. frances fincoln ltd., london. kendrick. b. (2011). fungi: ecological importance and impact on humans. wiley online library. kjøller, r ., nilsson, l., hansen, k., schmidt, i., vesterdal, l., gundersen, p., 2012: dramatic changes in ectomycorrhizal community composition, root tip abundance and mycelial production along a stand-scale nitrogen deposition gradient. new phytologist , vol 194(1): 278-286. klemm, d., 2005: cellulose: fascinating biopolymer and sustainable raw material. chemlnform. vol. 36(issue 36), lilleskov, e., hobbie, e., horton, t., 2011: conservation of ectomycorrhizal fungi: exploring the linkages between functional and taxonomic responses to anthropogenic n deposition. fungal ecology, 4: 174-183. mcknight, k., mcknight v., 1998: a field guide to mushrooms: north america. houghton mifflin harcourt, boston. rikardo, m., ileš, m., unković, s., 1995: kako da raspoznate pečurke: jestive gljive i njihovi otrovni dvojnici. evro, beograd. sigh, a., agrawal, m., 2008: acid rain and its ecological consequences. journal of environmental biology, 29 (1): 15-24. the european council for conservation of fungi (eccf). 2001: datasheets of threatened mushrooms of europe, candidates for listing in appendix i of the convention. strasbourg, 13 june 2001. uzelac, b., 2009: gljive srbije i zapadnog balkana. bgv logik, beograd. microsoft word bn-sc-0201-11 crnobrnja-isailovic et al biologica nyssana 2 (1) september 2011: 81-84 crnobrnja-isailović, j. et al. occurrence of european adder… 81 short communication ! occurrence of european adder (vipera berus, viperidae, ophidia) on vlasina plateau (southeastern serbia) jelka crnobrnja-isailović*1, 2, jelena dinov3, & vladimir ranđelović1 1faculty of sciences and mathematics, university of niš, višegradska 33, 18000 niš, serbia; 2institute for biological research „siniša stanković“, despota stefana 142, 11000 beograd, serbia; 3biological society “dr sava petrović “, 18000 niš, serbia * e-mail: jelka@pmf.ni.ac.rs abstract: crnobrnja-isailović, j., dinov, j., ranđelović, v.: occurrence of european adder (vipera berus, viperidae, ophidia) on vlasina plateau (southeastern serbia). biologica nyssana, 2 (1), september 2011: 81-84. the european adder (vipera berus) is among the most widespread reptile species in europe, but it’s distribution at the balkan peninsula seems to be scarce and fragmented. as going toward the south, specimens were more frequently found in the vegetational zone of boreal forests and high alpine pastures. in the south-eastern serbia, recent occurrence of european adder was confirmed on the vlasina plateau, in june 2010, in a mosaic-complex of peat bogs and marsh vegetation. recent engagement of ecology students from university of niš in mapping actual local distribution of adder will help locating key spots for its conservation in this area. key words: distribution, european adder, vipera berus, vlasina plateau ! the european adder (vipera berus (l. 1758) viperidae) is among the most widespread reptile species in europe (g a s c et al., 1997). its distribution goes from the contact zone between pyrenean peninsula and central europe on the west; southern border follows the northern parts of the apennine peninsula and highlands of western, central, southern and eastern balkans. eastern part of species area spreads deeply into the central and western asia, over large part of former soviet republics, with southern border running through moldavia, central ukraine and southern russia (n i l s o n & a n d r e n , 1997). the european adder is concerned as boreal relict at the balkan peninsula. it was supposed to be widely distributed in lowlands during pleistocene glaciations, but only isolated refugial mountain populations persisted during interglacial periods, as it is in recent time (u r s e n b a c h e r et al., 2006). phylogeographic structure of this species undoubtedly points that the balkan population group was important source of the species genetic diversity, as one of three major mitochondrial lineages probably originated in the balkans (u r s e n b a c h e r et al., 2006). despite being evaluated globally as least concerned (lc) on the iucn red list of threatened species (c r n o b r n j a i s a i l o v i ć et al., 2008), european adder has been assigned as „scarce“ in serbia as far back as in r a d o v a n o v i ć (1951) book on amphibians and reptiles of yugoslavia. national conservation status of european adder in serbia is defined in anonymous (2010) as „strictly protected species“, what means prohibition of any collecting, killing or harming of specimens. on that way, detailed 2 (1) • september 2011: 81-84 biologica nyssana 2 (1) september 2011: 81-84 crnobrnja-isailović, j. et al. occurrence of european adder… 82 knowledge on distribution of this species in republic of serbia is necessary for appropriate design of further national conservation plans. historical records confirmed presence of european adder in vojvodina, but restricted on its hilly parts covered with mainly oak forests quercocarpinetum sensu lato (fruška gora mountain and the vršačke planine mountain – d ž u k i ć & p u r g e r , 1988) and relatively intact marshy areas (obedska bara – d ž u k i ć & p u r g e r , 1988). in the balkan part of serbia, published historical records of adder’s presence exist for the avala mountain (d ž u k i ć , 1972), but there is no confirmation about species recent occurrence there. as far as we know, european adder inhabits the kopaonik mountain (c r n o b r n j a & r o h a l j , 1988), the stara planina mountain (i v a n č e v i ć et al., 2007) and the zlatibor mountain (c r n o b r n j a i s a i l o v i ć , unpublished data), but many new records are still unpublished or deposited in manuscripts, as well. it is reliable to consider potential presence of this species on all the mountains in serbia where habitats typical for boreal woods and alpine pastures exist. specimens were more frequently found in the vegetational zone of boreal forests and high alpine pastures as going more toward the southern parts of the state. in southern serbia, communities potentially suitable for the european adder are distributed mostly on the altitudes higher than 1000 m. intensive degradation of natural potential vegetation communities stretch the area of potential occurrence of this species even more. on the vlasina plateau potential vegetation is represented by beech forests, but they were quite degraded. however, phytogeographical analysis shows that boreal has made a strong impact on the genesis of the flora and vegetation, which was particularly reflected in the significant presence of peat bogs (r a n đ e l o v i ć & z l a t k o v i ć, 2010). presence of european adder on the vlasina plateau was recently confirmed within utm square fn03 in the second decade of june 2010 (n42o43’ 9.9“, e22o19’ 59.1“, 1227 m altitude – figure 1.). one juvenile specimen was found dead on the magistral road, obviously killed by car while crossing the road. according to our knowledge, it was one year old juvenile female (number of supracaudal scales sc – less than 30), with 187 mm of total lenght (l) and 169 mm of snout-to-vent lenght (svl). natural habitat of european adder at the site near village vlasina rid is a mosaic-complex of peat bogs and marsh vegetation consisted of associations equisetoscirpetum silvaticae, deschampsietum caesoitosum, caricetum gracilis and caricetum goodenowii (r a n đ e l o v i ć & z l a t k o v i ć , 2010). sintopic amphibian species recorded there were triturus vulgaris, t. alpestris, bufo bufo and rana synklepton esculenta, while reptile species detected in the area were lacerta agilis and anguis fragilis. published records of occurrence of the european adder on the vlasina plateau, up to our knowledge, are restricted on information occurring in publication of institute for nature conservation of republic of serbia (a n o n y m o u s 2006). there was no detailed description of species distribution or about its preferable habitat types within the area of the vlasina plateau, that points to the lack of details important for future conservation strategies in situ. department of biology and ecology of faculty of sciences and mathematics in niš is already conducting conservation studies and monitoring in figure 1. new record of vipera berus on the vlasina plateau biologica nyssana 2 (1) september 2011: 81-84 crnobrnja-isailović, j. et al. occurrence of european adder… 83 the area, so recent engagement of ecology students there, under the scope of realisation of their master theses, will help facilitation of mapping actual local distribution of the european adder. the main objectives of their research will be evaluation of importance and suitability of different habitats on the vlasina plateau for the maintenance of viable populations of the european adder, as well as recording of most plausible threats that occur in the area. acknowledgements. we are grateful to our colleagues imre krizmanić and danko jović who kindly provided us information about literature data on herpetofauna of the vlasina plateau. this work was supported by faculty of sciences and mathematics of university of niš, touristic enterprise „vlasina“ and grant 173025 ministry of education and science of republic of serbia. references anonymous, 2006: predeo izuzetnih odlika vlasina, studija zaštite, beograd. anonymous, 2010: pravilnik o proglašenju i zaštiti strogo zaštićenih i zaštićenih biljnih vrsta biljaka, životinja i gljiva. prilog 2. strogo zaštićene vrste. službeni glasnik rs br 5/10. crnobrnja, j., rohalj, a., 1988: prilog poznavanju herpetofaune kopaonika. zbornik radova bid "josif pančić", beograd: 59 76. crnobrnja isailović, jelka, milan vogrin, claudia corti, valentin pérez mellado, paulo sa-sousa, marc cheylan, juan m. pleguezuelos, ljiljana tomović, bogoljub sterijovski, urlich joger, a. westerström, bartosz borczyk, benedikt schmidt, andreas meyer, roberto sindaco, dušan jelić 2008. vipera berus. in: iucn 2010. iucn red list of threatened species. version 2010.4. <www.iucnredlist.org>. džukić, g., 1972: herpetological collection of the belgrade museum of natural history. bull. mus.hist.natur. ser. b, liv. 27: 165-180. džukić, g., purger, j., 1988: significance of adder vipera berus (linnaeus, 1758) presence in vojvodina. arh.biol.sci, 40 (1-4): 13p-14p. gasc, j-p., cabela, a., crnobrnja-isailović, j., dolmen, d., grossenbacher, k., haffner, p., lescure, j., martens, h., martinez-rica, j.p., maurin, h., oliveira, m.l., sofianidou, t.s., veith, m., zuiderwijk, a., eds., 1997: atlas of amphibians and reptiles in europe. societas europaea herpetologica & museum nationall d' histoire naturelle (iegb/spn), paris, 496 p. ivančević, b., savić, s., sabovljević, m., niketić, m., tomović, g., zlatković, b., ranđelović, v., lakušić, d., ćetković, a., pavićević, d., krpoćetković, j., crnobrnja-isailović, j., puzović, s., paunović, m., 2007: pregled vrsta stare planine u srbiji. in: d. lakušić, a. ćetković, eds.: biodiverzitet stare planine u srbiji rezultati projekta: "p r e k o g r a n i č n a saradnja kroz upravljanje zajedničkim prirodnim resursima promocija umrežavanja i saradnje između zemalja jugoistočne evrope". regionalni centar za životnu sredinu za centralnu i istočnu evropu, kancelarija u srbiji, beograd: 159-219. nilson, g., andren, c. 1997: vipera berus. in: gasc, j-p., cabela, a., crnobrnja-isailović, j., dolmen, d., grossenbacher, k., haffner, p., lescure, j., martens, h., martinez-rica, j.p., maurin, h., oliveira, m.l., sofianidou, t.s., veith, m., zuiderwijk, a., eds.: atlas of amphibians and reptiles in europe. societas europaea herpetologica & museum nationall d' histoire naturelle (iegb/spn), paris: 388 389. radovanović, m., 1951: vodozemci i gmizavci naše zemlje. narodna knjiga, beograd. ranđelović, v. n., zlatković, b. k. 2010. flora i vegetacija vlasinske visoravni. prirodnomatematički fakultet, niš. ursenbacher, s., carlsson, m., helfer, v., tegelström, h., fumagalli, l., 2006: phylogeography and pleistocene refugia of the adder (vipera berus) as inferred from mitochondrial dna sequence data. molecular ecology, 15: 3425-3437. microsoft word bn-oa-0201-08 stojkovic m_et_al biologica nyssana 2 (1) september 2011: 59-66 stojković, m. et al. ichthyological integral indices, the history of development… 59 original article ! ichthyological integral indices, the history of development and possible application on rivers in serbia milica stojković*1, djuradj milosević1, vladica simić2 1university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia 2university of kragujevac, faculty of sciences and mathematics, institute of biology and ecology, radoja domanovića 12, 34000 kragujevac, serbia * e-mail: milicastojkovic83@gmail.com abstract: stojković, m., milošević, đ., simić, v.: ichthyological integral indices, the history of development and possible application on rivers in serbia. biologica nyssana, 2 (1), september 2011: 59-66. based on a literature review, the different approaches in the water quality assessment using fish communities in freshwaters are summarized. fish assemblage indicators, developed throughout the world, were reviewed and the main differences in methodologies, number of metrics and values are summarized. we have drawn attention to the methods used for designing a fish-based index with a particular focus on original developments in north america and its adaptations in many different regions and habitat types. the main obstacles for ecological assessment are scarce ecological information and the problem with the classification. the lack of knowledge is especially true for species assemblages in the relatively unexplored river basins of europe, e.g. the balkans peninsular. key words: biotic indices, fish, multimetric approach, water quality assessment introduction ! monitoring the ecological status of aquatic systems is in use by water resource managers worldwide. historically, aquatic systems were assessed primarily through chemical measuring, providing a snapshot of water quality conditions. the anthropogenic capacity to alter the natural world usually overcomes our ability to assess the impacts of these alterations on the biological inhabitants within the water ecosystems (s i m o n , 1999). fish are an unavoidable element for such assessments because of their biological and socioeconomic status. fish are considered as a reliable indicators in water quality assessment for various reasons: fish are present in most surface waters; the identification of species is relatively easy; the sensitivity to disturbance is well known for many species; as well as their responses to environmental stressors; different species represent distinct trophic levels; fish occupy a variety of habitats in rivers; diminished growth and recruitment are easily assessed and reflect exposure to stress (fame consortium, 2004). however, there are a few disadvantages: manpower needs a three person crew is required to effectively and safely sample fish communities; migration of fish may provide misleading data; overlapping the effects of overfishing with pollution effects may lead to erroneous conclusions (g r a b a r k i e w i c z & d a v i s , 2008; s i m i ć & s i m i ć , 2009) various fish-based indices have been developed worldwide for assessing the ecological status of rivers. most of them include a reference condition approach and appropriate biological variables or metrics (n o b l e & c o w s , 2007; r o s e t et al., 2007), describing the fish assemblage 2 (1) • september 2011: 59-66 biologica nyssana 2 (1) september 2011: 59-66 stojković, m. et al. ichthyological integral indices, the history of development… 60 attributes and quantifying the impact of anthropogenic activities on the biota. the index of biotic integrity (ibi) was initially developed by dr. james karr for the purpose of evaluating and describing the condition of small warm water streams in central illinois and indiana (k a r r , 1981). as the ibi became widely used, different versions were developed for different regions and ecosystems. new versions, generally, retained most of the original metrics but the some of them have been modified for application in a particular region or type of stream. the aim of this paper is to compare different approaches for assessing the ecological condition using fish communities. likewise, we want to propose the most applicable metrics for rivers in serbia. material and methods the comparison was made between different methods used for designing a fish-based index according to a literature review from a period 19812009. the different approaches, used in north america, according to k a r r (1981), l e o n a r d & o r t h (1986), m o y l e et al. (1986), h u g h e s & g a m m o n (1987), m i l l e r et al. (1988), s t e e d m a n (1988), s i m o n (1991), ly o n s (1992), s i m o n & l y o n s (1995), ly o n s et al. (1996), o v e r t o n (2001), t e e l s et al. (2004), barbour et al. (2002), n i e m e l a & f e i s t (2000), were described. in addition, different modifications of ibi applied for water quality assessment in europe (b e l l i a r d et al., 1999; b r e i n e et al., 2004; a n g e r m e i e r & d a v i d e a n u , 2004; fame consortium, 2004; l e n h a r d t et al., 2009) were compared. the original version had 12 metrics that refer to fish species richness and composition, number and abundance of species, trophic organization and function, reproductive behavior, fish abundance, and condition of individual fish (tab. 1). k a r r (1981) proposed a rating system for each metric based upon the degree of deviation (5 (none to slight), 3 (moderately) and 1 (significantly)) from the appropriate ecoregional reference conditions. the metrics were scored and summed to arrive at an index ranging from 60 (best) to 12 (worst). metrics, which are defined in original version of ibi (k a r r , 1981) and envisaged for illinois area, have been changed for application in many different regions worldwide. in every other version (k a r r , 1981; l e o n a r d & o r t h , 1986; m o y l e et al., 1986; h u g h e s & g a m m o n , 1987; m i l l e r et al., 1988; s t e e d m a n , 1988; s i m o n , 1991; l y o n s , 1992; s i m o n & l y o n s , 1995; l y o n s et al., 1996; o v e r t o n , 2001; te e l s et al., 2004; b a r b o u r et. al., 2002; n i e m e l a & f e i s t , 2000), list of metrics has been modified according to features of target region (tab. 1). some of them are commonly used such as: total number of fish species, % individuals as omnivores, % individuals with anomalies; but most of them was adjusted according to assemblage of investigated area (n o b l e & c o w s , 2007). results and discussion according to the metrics from the list above and its variations, it can be concluded that each of them assess the same aspect of functional community but in a different way. before making a choice which metrics are more appropriate, it should be drawn attention to their applicability in the investigated area. the potential metrics have to be changed in a predictive manner according to anthropogenic influence. likewise, they have to be sensitive to stressors and to provide the response that can be discriminated from natural variation (n o b l e et al., 2007). the utilization of the fish-based indices in europe is less widespread than in the united states of america. recently, scientists in france, belgium and romania have endeavored to adjust ibi for usage in their own countries (vi d a l , 2008). b e l l i a r d et al. (1999) have suggested 10 metrics for water quality assessment in france, applying them on the seine river. in belgium, from the total of 28 candidate metrics, nine had been selected (breine et al., 2004). in romania, angermeier & davideanu (2004) made distinction between hilly and montane region, and have chosen 7 metrics for each region to include in preliminary multimetric indices (pmis). k a r r ’ s (1981) original ibi was also adapted in serbia. this version related to lentic system, analyzed changes in the fish assemblage compared to sediment deposition in the reservoir. it contains 10 suitable metrics (l e n h a r d t et al., 2009). in each of these four versions, scoring system was used but with different scores applied. b e l l i a r d et al. (1999) kept the original scoring system (k a r r , 1981) using score values: 5, 3, 1 for slightly, moderately and the most impacted, respectively. in the second case, ibi score of an investigated site is the mean of scores for all metrics, and varies between 0 and 5. in dependence of the final ibi score, a given site will be classified in an appropriate water class according to water framework directive (b r e i n e et al., 2004). values for metric scoring classes in romania were assigned for each metric at each site as 3, 2, 1 for biologica nyssana 2 (1) september 2011: 59-66 stojković, m. et al. ichthyological integral indices, the history of development… 61 table 1. metrics of the original ibi and other versions used for designing ibi in different regions category metrics of the original ibi (karr, 1981) version of metrics used in different regions species richness total number of species 1. total number of native species (karr et al., 1986, leonard & orth, 1986, moyle et al., 1986), hughes & gammon, 1987, miller et al., 1988, steedman, 1988, simon, 1991, lyons, 1992, simon & lyons, 1995, lyons et al., 1996, teels et al., 2004) 2. salmonid age classes (moyle et al., 1986, hughes & gammon, 1987) species composition number of darter species 1. number of darter and sculpin species (steedman, 1988) 2. number of sculpin species (hughes & gammon, 1987) 3. number of benthic species (karr et al., 1986, leonard & orth, 1986, moyle et al., 1986, hughes & gammon, 1987, miller et al., 1988, steedman, 1988, simon, 1991, lyons, 1992, simon & lyons, 1995, lyons et al., 1996) 4. number of darter species, excluding tolerant species (gatz & harig, 1993) number of sunfish species 1. number of sunfish species, including genus micropterus (karr et al., 1986, simon, 1991, hoefs and boyle, 1992, lyons, 1992) 2. number of cyprinid species (hoefs and boyle, 1992) 3. number of sunfish and trout species (steedman, 1988) 4. number of water column species (miller et al., 1988, oberdorff & hughes, 1992) 5. number of headwater species (simon, 1991) number of sucker species 1. number of sucker and cyprinid species (hoefs and boyle, 1992) 2. number of sucker and catfish species (steedman, 1988) 3. number of minnow species (hughes & gammon, 1987, simon, 1991, hoefs and boyle, 1992) tolerance guilds number of intolerant species 1. number of amphibian species (moyle et al., 1986) 2. % individuals as brook trout (langdon, 1989) 3. number of trout species (moyle et al., 1986, simon, 1991) 4. number of sensitive species (karr et al., 1986, leonard & orth, 1986, moyle et al., 1986), hughes & gammon, 1987, miller et al., 1988, steedman, 1988, simon, 1991, lyons, 1992, simon & lyons, 1995, lyons et al., 1996) % individuals as green sunfish 1. % individuals as common carp (hughes & gammon, 1987) 2. % tolerant species (karr et al., 1986, leonard & orth ,1986, moyle et al., 1986), hughes & gammon, 1987, miller et al., 1988, steedman, 1988, simon, 1991, lyons, 1992, simon & lyons, 1995, lyons et al., 1996) 3. % introduced species (hughes & gammon, 1987) 4. % individuals as common roach (oberdorff & hughes, 1992) trophic guilds % individuals as omnivores 1. % individuals as omnivores and herbivores (overton, 2001) 2. % individuals as generalists (karr et al., 1986, leonard & orth, 1986, moyle et al., 1986), hughes & gammon, 1987, miller et al., 1988, steedman, 1988, simon, 1991, lyons, 1992, simon & lyons, 1995, lyons et al., 1996) % individuals as insectivorous cyprinids 1. % individuals as insectivorous (hughes and gammon, 1987, simon, 1991, miller et al., 1988, lyons, 1992, hoefs and boyle, 1992, oberdorff and hughes, 1992) 2. % specialized insectivores (leonard & orth ,1986) 3. % individuals as benthic insectivorous (teels et al., 2004) % individuals as piscivorous 1. % catchable salmonids karr et al., 1986, leonard & orth ,1986, moyle et al., 1986), hughes & gammon, 1987, miller et al., 1988, steedman, 1988, simon, 1991, lyons, 1992, simon & lyons, 1995, lyons et al., 1996) 2. % pioneering species (karr et al., 1986, leonard & orth ,1986, moyle et al., 1986), hughes & gammon, 1987, miller et al., 1988, steedman, 1988, simon, 1991, lyons, 1992, simon & lyons, 1995, lyons et al., 1996) biologica nyssana 2 (1) september 2011: 59-66 stojković, m. et al. ichthyological integral indices, the history of development… 62 abundance number of individuals 1. density of individuals (karr et al., 1986, leonard & orth ,1986, moyle et al., 1986), hughes & gammon, 1987, miller et al., 1988, steedman, 1988, simon, 1991, lyons, 1992, simon & lyons, 1995, lyons et al., 1996) 2. biomass of fish (hughes and gammon, 1987) reproduction and condition % individuals as hybrids 1. % introduced species (karr et al., 1986, leonard & orth ,1986, moyle et al., 1986), hughes & gammon, 1987, miller et al., 1988, steedman, 1988, simon, 1991, lyons, 1992, simon & lyons, 1995, lyons et al., 1996) 2. % simple lithophils (simon, 1991, lyons, 1992, hoefs and boyle, 1992) 3. number of late maturing species (teels et al., 2004) 4. percentage of species with multiple age groups (ncdehnr, 1997) % individuals with anomalies 1. percent of individuals with heavy infestation of cysts of the parasite neascus (steedman, 1988) slightly, moderately and the most impacted, respectively (a n g e r m e i e r & d a v i d e a n u , 2004). finally, in serbia, for each metric, data were sorted into quartiles, according to l o n g & wa l k e r (2005). a site had score of 1 where the observed value for the metric fell below the first quartile threshold. a score of 5 was attributed to those sites for which the observation occurred above the fourth quartile. sites with the observation values falling within the second and third quartiles scored 3. european fish index (efi) has been developed as a result of european union project, named fame (development, evaluation and implementation of the standardized fish-based assessment method for the ecological status of european rivers). generally, efi is based on a predictive model that derives reference condition for the each site and quantifies the deviation between predicted and observed condition of the fish fauna (fame consortium, 2004). efi employs 10 metrics (table ii.) and their response to human pressures has been already known. theoretical values are predicted for each metric using environmental variables by means of a multilinear regression model. in order to quantify a level of degradation, the residuals, calculated as difference between observed and predicted metric values, are used. the ecological status is expressed as an index ranging from 1 (high ecological status) to 0 (bad ecological status). it has been recommended that efi should not be applied in areas where a fish fauna significantly deviates from those of the tested regions e.g. rivers of the south-eastern part of europe (fame consortium, 2004). in order to adjust ibi for application on rivers in serbia, a great amount of ecological information is required for each species, which is the base for their classification in ecological guilds. the election of metrics is essential to make ibi strong enough to represent the condition of the river ecosystems in serbia. we have suggested the preliminary list of metrics (table iii.) which can be potentially used in serbia. the total number of species represents a reliable indicator of the target population condition. healthy ecosystem contains a great number of species (k a r r et al., 1986). however, invasive and introduced species should be considered as a separate metric, because of their negative influence on the status of natural populations. according to fame (fame consortium, 2004), metrics, which refer to tolerance guilds, are represented as a number of intolerant species and a number of tolerant species. in addition, fame proposed to employ metrics related to reproductive requirements: relative abundance of lithophilic species and density of the phytophilic species. the evaluation of these characteristics is based on the fact that the increase of habitat degradation reduces the possibility of finding a specific substrate for spawning. the percentage of species with multiple age groups simultaneously may assess suitability of habitat conditions for reproduction and the degree of reproductive success. from the parameters related to the assessment of trophic structure, the percentage of: omnivorous individuals; specialized insectivores; obligate piscivores can be applied as the appropriate metrics. this recommendation can made a difference between species specialized for the certain type of food from those within a wide spectrum. each species has its own specific requirements according to the habitat type (reophilic, limnophilic and euritop). according to k a r r (1981), species are divided in two groups: the species which lives nearby the bottom and the species which lives in the water column (percidae, centrarchidae, respectively). this kind of division, biologica nyssana 2 (1) september 2011: 59-66 stojković, m. et al. ichthyological integral indices, the history of development… 63 table 2. metrics used by european fish index (efi) and their response to anthropogenic influence selected metrics response to anthropogenic influence 1. density of the insectivorous species decrease 2. density of the omnivorous species increase 3. density of the phytophilic species increase 4. relative abundance of lithophilic species decrease 5. number of benthic species decrease 6. number of rheophilic species decrease 7. relative number of intolerant species decrease 8. relative number of tolerant species increase 9. number of species migrating over long distances decrease 10. number of potamodromous species decrease table 3. preliminary list of metrics for application on rivers in serbia selected metrics response to anthropogenic influence 1. total number of native species decrease 2. total number of alien species increase 3. number of intolerant species decrease 4. number of tolerant species increase 5. % individuals as omnivores increase 6. % individuals as specialized insectivorous decrease 7. % individuals as obligate piscivorous decrease 8. number of rheophilic species decrease 9. number of euritop species increase 10. percentage of species with multiple age groups decrease 11. % individuals with anomalies increase beside habitat type, takes trophic requirements into account and introduces the problem of stratification in shallow waters. therefore, it would be recommented more suitable metrics such as: number of rheophilic species (specialized of habitat type), and number of euritop species (tolerant of habitat type). metrics related to migration guilds are not acceptable for applying on rivers in serbia, because there are very few species which can be classified as diadromous (acipenseridae and clupeidae). the total number of individuals, as a rule, should be reduced with increasing the environmental degradation. in some instances, although the environmental conditions are degraded, the number of individuals increase in term of abundance. increased abundance of individual species is an outcome of their tolerance to the changes in the environment. percentage of diseased fish, the presence of tumors and other abnormalities, in most cases has low value or it is absent, but may have a great importance for identifying areas with high concentration of toxic substances. conclusion classification of fish into ecological guilds is a prerequisite for the development of an ibi. regional modifications of ibi are based on a different metrics which are employed. choice of metrics requires the great ecological knowledge of the local fish population. the chosen list of metrics should be suitable for the target fish assemblage and strong enough to detect changes in functioning biologica nyssana 2 (1) september 2011: 59-66 stojković, m. et al. ichthyological integral indices, the history of development… 64 community. formulation of an index, such as index of biotic integrity in serbia, based on fish assemblages, and its affiliation to the already existing balkan biotic index (s i m i c & s i m i c , 1999), based on macrozoobenthos, may contribute to the development of a new approach in the environmental condition assessment. such a comprehensive assessment, including a majority of freshwater biota, may lead to the formulation of an advanced strategy for conservation of ecosystem health. acknowledgments. this work was supported by ministry of education and science, serbia, grant no.043002. references angermeier, p.l. and davideanu, g. 2004: using fish communities to assess streams in romania: initial development of an index of biotic integrity. hydrobiologia, 511: 65-78. barbour, m.t., gerritsen, j., snyder, b.d., stribling, j.b. 2002: rapid bioassessment protocols for use in streams and wadeable rivers: periphyton, benthic, macroinvertebrates and fish. environmental 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southern ontario. canadian journal of fisheries and aquatic science 45:492-501. teels, b.m., mazati, l.e. and rewa, c.a. 2004: using an ibi to assess effectiveness of mitigation measures to perlace loss of wetland-stream ecosystem. wetlands 24: 375-384. vidal, l.b. 2008: fish as ecological indicators in mediterranean freshwater ecosystems. ph.d. thesis. institute of aquatic ecology and department of environmental science. university of girona. biologica nyssana 2 (1) september 2011: 59-66 stojković, m. et al. ichthyological integral indices, the history of development… 66 jugović et al. 2023, biologica nyssana 14(1) 14 (1) june 2023: 39-46 doi: 10.5281/zenodo.8027130 the frequency and type of mutations in the egfr gene of colorectal carcinoma patients in southeastern serbia original article dragana jugović laboratory for immunology and genetics, center for medical and clinical biochemistry, university clinical center niš, serbia dragana.jugovic@live.com (corresponding author) marija vukelić nikolić faculty of medicine, research center for biomedicine, university of niš, serbia višnja madić department of biology and ecology, faculty of sciences and mathematics, university of niš, serbia aleksandar milićević pathology and pathological anatomy center, university clinical center niš, serbia perica vasiljević department of biology and ecology, faculty of sciences and mathematics, university of niš, serbia received: february 28, 2023 revised: april 28, 2023 accepted: april 28, 2023 abstract: colorectal carcinoma (crc) represents a global health problem. the egfr signaling pathway is very important in the initiation of different human epithelial cancers and plays a key role in colorectal carcinogenesis. considering the role of egfr as a mitogenic inducer, this study aimed to determine the frequency and type of mutations in the egfr gene and its correlation with clinicopathological characteristics of patients with crc on the territory of southeastern serbia. the genomic dna of patients was isolated from formalin-fixed and paraffin-embedded tissues. the presence of mutations in exons 18, 19, 20, and 21 was determined by the real-time pcr method. the frequency of mutations in the egfr gene in crc patients of southeastern serbia was 7.9%. all detected mutations were g719x. no statistically significant correlation was found between mutational status in the egfr gene and the clinicopathological characteristics of patients. key words: colorectal carcinoma, egfr, mutations apstrakt: učestalost i tip mutacija u egfr genu kod pacijenata sa kolorektalnim karcinomom u jugoistočnoj srbiji kolorektalni karcinom (krk) predstavlja globalni zdravstveni problem. egfr signalni put je veoma važan u inicijaciji različitih humanih epitelnih karcinoma i ima ključnu ulogu u kolorektalnoj kancerogenezi. s obzirom na ulogu egfr-a kao mitogenog inducera, ovo istraživanje imalo je za cilj da utvrdi učestalost i tip mutacija u genu za egfr, kao i hjihovu korelaciju sa kliničkopatološkim karakteristikama pacijenata sa krk-om na teritoriji jugoistočne srbije. genomska dnk pacijenata izolovana je iz tkiva fiksiranih u formalinu i ukalupljenih u parafin. prisustvo mutacija u egzonima 18, 19, 20 i 21 određivano je real-time pcr metodom. učestalost mutacija egfr gena kod pacijenata sa krk-om na teritoriji jugoistočne srbije iznosila je 7,9%. sve otkrivene mutacije bile su tipa g719x. nije pronađena statistički značajna korelacija između mutacionog statusa u egfr genu i kliničkopatoloških karakteristika pacijenata. ključne reči: kolorektalni karcinom, egfr, mutacije introduction colorectal carcinoma (crc) is a malignant epithelial tumor that represents one of the major health problems in the world. namely, it is the third most common cancer in men and the second most common cancer in women, accounting for about 11% of all diagnosed cancers (ferlay et al., 2020). usually, it is diagnosed in the older population, however, recent research shows an increased incidence of crc in the younger population as well (campos, 2017; connel et al., 2017). colorectal carcinogenesis is a gradual process where the clonal accumulation of genetic changes results in the loss of cell cycle control, absence of the cells’ natural response to genomic damage, further accumulation of mutations and, finally, in general genomic instability, which represents the basic characteristic of malignant cells (mármol et al., 2017). the main role in the evolution of crc has the dysregulation of several intracellular signaling pathways such as mitogen-activated protein kinase (mapk) and phosphatidylinositol 3 kinase (pi3k)/protein kinase b (akt) signaling pathway activated by epidermal growth factor receptor © 2023 jugović et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 39 (egfr) bounded with the ligand. dysregulation of these pathways leads to the activation of various transcription factors whose disruption can lead to malignant cell transformation and tumor progression through increased cell proliferation, anti-apoptosis, angiogenesis, and invasion (wee et al., 2017; koveitypour et al., 2019). the egfr gene is located on chromosome 7p12– 13. it encodes a 170-kda transmembrane receptor with an intracellular tyrosine kinase domain. it is a member of the erbb family of receptors that has four closely related members: erbb-1bb-1 (her1/ egfr), erbb-2 (her2), erbb-3 (her3), and erbb-4 (her4). all these receptors contain an extracellular ligand-binding domain, a membranespanning region, and a cytoplasmic region which contains a tyrosine kinase (hirsh, 2018). after epidermal growth factor (egf) ligand binding, egfr forms a functionally active dimer (homodimer or heterodimer) that causes phosphorylation of tyrosine kinases in the intracellular domain of egfr. this phosphorylation initiates different complex intracellular signals in the cytoplasm followed by the activation of a variety of transcription factors responsible for controlling multiple cellular functions including proliferation, differentiation, apoptosis, and cell migration (koveitypour et al., 2019). the presence of mutations in some genes involved in these signaling pathways often represents a negative predictive biomarker of response to current therapy (mármol et al., 2017). the principal mechanism of egfr dysregulation in crc is egfr overexpression. protein upregulation is found in 60-80% of crc, making it an adequate target for anti-egfr therapy (porru et al., 2018). nowadays, in addition to the standard chemotherapy, there are two other therapeutic approaches in the treatment of cancer, i.e., administration of tyrosine kinase inhibitors (tki) and administration of monoclonal antibodies (mabs). although the mechanism of these two therapies is similar, i.e., blocking of the egfr autophosphorylation, the outcome of the therapy may differ depending on the mutational status of egfr. to wit, studies showed that the administration of tki failed to improve the outcome of the treatment in crc patients with egfr mutations in the tk domain located in exons 8,19, 20 and 21 (saletti et al., 2015). on the other hand, the study of cho and associates showed that cell lines that express g719s and g724s mutations in exon 18 are sensitive to the cetuximab monoclonal antibody (cho et al., 2014). thus, patients that are carriers of these mutations might benefit from the use of cetuximab, even though it is still mainly used only in the treatment of metastatic crc patients with wild-type (wt) kras. moreover, although somatic mutations are rare in crc, some of them can cause acquired resistance to anti-egfr therapy (montagut et al., 2012; cho et al., 2014). the most common somatic mutations that lead to the oncogene activation of egfr are missense mutations, insertions, or deletions, mainly in the tyrosine kinase (tk) domain (liu et al., 2020). according to the study of metzger and associates, the frequency of mutations in the egfr tk domain of crc patients in europe is 3.8% in exon 21 (codons 847, 836), 2.6% in exon 18 (codons 707, 710, 711, 712, 725), 0.8% in exon 20 (codons 795, 796, 787), and only 0.5% in exon 19 (codon 742) (metzger et al., 2011). namely, egfr mutants bypass the mechanisms that initiate termination of the signal transduction by avoiding receptor endocytosis and degradation (mosesson et al., 2008). studies have indicated that egfr mutations not only act as oncogenic drivers of crc but can also serve as genomic predictors of response to anti-egfr therapy in crc patients (kim et al., 2020). having in mind such an important role of egfr as a mitogen inducer, the aim of this study was to determine the frequency and type of mutations in the tyrosine kinase domain of egfr in starting (t1/ t2) and final (t4) stages of tumor infiltration and its correlation with clinical characteristics of patients with crc on the territory of southeastern serbia. materials and methods this study investigated the mutational status in egfr of 38 crc patients. the analysis was performed on the 5-10 μm thick formalin-fixed paraffin-embedded (ffpe) sample tissue sections of patients with confirmed crc who were referred to the laboratory for immunology and genetics, the center for medical and clinical biochemistry, university clinical center niš, serbia during 20192021. ethical approval has been obtained from the ethics committee of the clinical center, niš, serbia no 24722/6. the following clinical data were collected for each patient: sex, age, tumor location. stage of tumor infiltration, nodal status, and the presence of distant metastases. the stage of the tumor was determined in accordance with tumor-nodes-metastasis (tnm) classificational system recommended by the american joint committee on cancer staging system (ajcc), where t1 and t2 denote starting stages (t1, when tumor invades submucosa, and t2, when tumor invades muscularis propria) and t4 denotes the final stage of tumor infiltration, when tumor perforates visceral peritoneum and/or directly 40 biologica nyssana ● 14 (1) june 2023: 39-46 jugović et al. ● the frequency and type of mutations in the egfr gene of colorectal carcinoma patients in southeastern serbia biologica nyssana ● 14 (1) june 2023: 39-46 jugović et al. ● the frequency and type of mutations in the egfr gene of colorectal carcinoma patients in southeastern serbia 41 following cycling conditions initial denaturation step at 95 °c for 2 min, then running 40 cycles of 95 °c for 10 seconds/58 °c for 60 seconds. the mutation analysis was carried out in relation to the amplification of positive and negative control tests provided by the manufacturer and according to the included protocol. statistical analysis statistical analysis was done using microsoft excel 2010 (microsoft corporation, usa). results of egfr mutational analysis were used as categorical variables (presence or absence of the mutation). comparison between clinical characteristics and mutational status was done using fisher’s exact test or χ2 test where appropriate. statistical significance was accepted if p<0.05. results this study included samples of 38 patients with crc, collected during 2019-2021. there were 24 male and 14 female patients, aged from 42 to 79 years (tab. 1). of all 38 tested patients, 35 of them (92.1%) did not have any detectable mutations in the egfr. egfr mutations (g719x) were observed in exon 18 in 3 (7.9%) patients. the g719x mutation in egfr refers to point mutations that result in substitutions of the glycine at position 719 to other residues, primarily alanine (g719a), cysteine (g719c), and serine (g719s) (fig. 1). no mutations were detected in exons 19, 20, and 21. two out of the three patients with detected mutations have the t2 stage of crc, located in the left colon, and with no metastases in lymph nodes. the third case was a patient with the t4 stage of the tumour, located in the right colon and with metastases in lymph nodes. mutation in egfr (g719x) was detected in one patient with distant metastases in the lungs, one patient with distant metastases in both lungs and liver, and in one patient with distant metastases in the pelvis. there was no statistically significant correlation between the mutational status in the egfr and the clinical characteristics of patients (tab. 1). discussion in serbia, colorectal carcinoma is the third most common cancer, as well as the second most common cause of cancer-related death, right after lung cancer (ferlay et al., 2020). namely, according to the cancer register for republic of serbia, there were even 4646 new crc cases registered in serbia in 2018. more than a thousand of these new crc cases invades other organs or structures (tong et al., 2018). according to the location, the tumors were divided into the left-sided colon (lcc) and rightsided colon (rcc). lcc included splenic flexure, descending colon, sigmoid, and rectum, while the rcc included caecum, ascending colon, hepatic flexure, and transverse colon. dna isolation genomic dna was extracted from the ffpe tumour tissue sections using a qiaamp dna ffpe tissue kit (ce-ivd-marked; qiagen, hilden, ger many), according to the manufacturer’s protocol. the dna quality was determined with 260/280 optical density (od) ratios in all samples, which were stored at -20°c until use. real-time pcr the analysis of the egfr mutation was performed using the „easy® egfr” kit (diatech pharmacogenetics, italy) with a rotorgene 6000-corbett rt-pcr device (diatech pharmacogenetics, italy), where the list detectable mutations were: egfr exon 18: g719s (2155g>a), g719c (2155g>t), g719a (2156g>c) not distinguishable between them; egfr exon 19: e746_a750del (2235_2249del15), e746_a750del (2236_2250del15), l747_p753>s (2240_2257del18), l747_a750>p (2239_2248ttaagagaag>c), e746_s752>v (2237_2255>t), l747_t751del (2240_2254del15), l747_s752del (2239_2256del18), e746_t751>a (2237_2251del15), l747_t751del (2239_2253del15), l747_t751>p (2239_2251>c), l747_e749del (2239_2247del9), e746_e749del (2235_2246del12), l747_p753>q (2239_2258>ca), l747_t751>s (2240_2251del12), e746_s752>a (2237_2254del18), l747_a750>p (2238_2248>gc), e746_s752>d (2238_2255del18), e746_ t751>i (2235_2252>aat), l747_t751>q (2238_2252>gca), e746_t751del (2236_2253del18) not distinguishable between them. egfr exon 20: v769_d770insasv ( 2 3 0 7 _ 2 3 0 8 i n s g c c a g c g t g ) , d 7 7 0 _ n771insg (2310_2311insggt), h773_v774insh (2319_2320inscac) not distinguishable between them, t790m (2369c>t), s768i (2303g>t); egfr exon 21: l858r (2573t>g), l861q (2582t>a). egfr sequence was amplified using a mixture of 10 μl taq premix, 4 μl double distilled (dd) water, 1 μl mutation primers, and 5 μl dna template. each run included at least one amplification of the negative control (dd water) and one amplification of the positive control („easy® egfr” kit positive control). egfr sequence was amplified using the 42 biologica nyssana ● 14 (1) june 2023: 39-46 were patients from southern and eastern serbia, with 640 males, and 371 females. at the same time, the total number of deaths caused by crc was 479, with 397 male and 182 female patients (institute of public health „dr. milan jovanovic batut”, 2020). a key role in colorectal carcinogenesis has the egfr signaling pathway, which is involved in the initiation of human epithelial cancers. the most common pathological changes of egfr are overexpression of egfr protein and egfr mutations that activate the kinase. based on the hypothesis that a monoclonal antibody against egfr can inhibit receptor tyrosine kinase activity and cancer cell proliferation, proposed in 1980 by dr. john mendelsohn and gordon sato, years of studies led to the discovery of anti-egfr treatments for specific carcinomas such as crc and non-small-cell lung cancer (nsclc) (mendelsohn et al., 2015). it has been observed that egfr-directed therapy such as cetuximab was more successful in crc patients with highly expressed egfr than in patients with low egfr expression (liu et al., 2016). many studies evaluated the connection between over-expressed egfr with a variety of carcinomas such as breast cancer (silva rocha et al., 2021), table 1. correlation between egfr mutation status and clinicopathological parameters of crc patients jugović et al. ● the frequency and type of mutations in the egfr gene of colorectal carcinoma patients in southeastern serbia parameters n=38 egfr status n=38 p value n % mutated wt sex male 24 63.2 2 22 0.89 female 14 36.8 1 13 age at diagnosis ≤50 6 15.8 / 6 51-60 6 15.8 / 6 61-70 18 47.4 2 16 ≥71 8 21.0 1 7 location left colon 23 60.5 2 21 0.82 right colon 15 39.5 1 14 tumor extent t1/t2 14 36.8 2 12 0.26 t4 24 63.2 1 23 nodal status without metastasis 14 36.8 2 12 0.26 with metastasis 24 63.2 1 23 distant metastasis liver 19 50.0 / 19 lung 6 15.8 1 5 liver and lung 5 13.2 1 4 other 8 21.0 1 7 wt-wild type fig. 1. g719x mutated sample and amplification plot detected by „easy® egfr” kit biologica nyssana ● 14 (1) june 2023: 39-46 jugović et al. ● the frequency and type of mutations in the egfr gene of colorectal carcinoma patients in southeastern serbia stomach adenocarcinoma (wang et al., 2017), prostate cancer (hashmi et al., 2019), nsclc (tasdemir et al., 2019), head and neck cancer (barnes et al., 2020). in all these cancers, the level of expression was different with a tendency to increase, but in all cases, egfr expression was recognized as an unfavorable prognostic biomarker of the disease. furthermore, many scientists have been researching overexpression and abnormal activation of the egfr in crc patients. these studies have shown the prognostic value of egfr expression visible as the key factor in the invasion and development of metastasis in crc patients (du et al., 2017; del carmen et al., 2020; uhlyarik et al., 2020). in addition, many studies evaluated the frequency of mutations in the egfr gene in a variety of cancers. for example, it has been observed that in nsclc patients mutations of the gene were highly frequent in exons 19 (over 50%) and less frequent in exons 18 and 20 (8.1% and 3.5%) (skřičková et al., 2020). interestingly, nsclc patients with a mutation in exon 19 had higher overall survival (os) rate compared to nsclc patent with mutations in exons 18 and 20 (syrigos et al., 2018). also, a high frequency of mutations was found in glioblastoma multiforme (26.8%), lung adenocarcinoma (14.4%), while a low frequency of mutations was found in breast invasive carcinoma, kidney renal papillary cell carcinoma, and prostate adenocarcinoma (liu et al., 2020). the results of this study showed that of 38 patients only 3 of them had mutations in the kinase domain of the egfr. two out of these three patients had t2 stage of crc, located on the left side of the colon and the absence of metastasis in lymph nodes. similarly to our results, the study of oh and associates showed that the presence of egfr mutations is more frequent in the earlier stages and the absence of lymph node metastasis (oh et al.,2011). moreover, our results showed that all these three patients had the same mutation, i.e., in exon 18 (g719x). ruhe and colleagues showed that one of the three mutations in exon 18 (g719s) identified in the sw48 colon cancer cell line had oncogenic potential (ruhe et al., 2007). at the same time, according to kim and associates, inhibitors of egfr tyrosine kinases, such as cetuximab and panitumumab, efficiently block the growth of the same cell line with the same mutation (kim et al. 2020, cho et al., 2014). having in mind all these, it might be concluded that crc carriers of egfr mutation in exon 18 might benefit from cetuximab or panitumumab applied as first-line therapy. the incidence of mutated egfr in crc patients included in our study was only 7.9%, while its frequency in the korean population was even 22.41%. on the contrary to the population of southeast serbia, their population had mutations only in exon 20 (oh et al., 2011). additionally, the frequency of mutations of in exon 18 (codon 719) in our study was higher than the frequency of mutations in the same exon in the european population (2.6%, codons 707, 710, 711, 712, 725) (metzger et al., 2011). our results are different from the study of phua and associates who did not detect any mutations of egfr in codons 18, 19, and 21 in a group of 45 patients with crc (phua et al. 2015). similarly, the study that included 31 caucasians did not detect any mutations in exons 18 and 19, while only one of them had a missense heterozygous mutation in exon 21 (gly857arg) (moroni et al., 2005). on the other hand, barber and colleagues found only one carrier of g719s out of 293 examined crc patients (0,34%) (barber et al., 2004), while the study of ogino and associates showed that the incidence of this mutation was 3.33% (1/30) (ogino et al., 2005). in romanian population, the frequency of egfr mutation in exon 19 was 5% (3/60), and it was positively correlated with lymphovascular invasion, perineural invasion, tumor budding of all grades as well as with the invasion of tissues in the proximity of the tumor (liver, visceral, parietal pleura, etc.) (ionescu et al., 2022). in our study, we did not identify any mutation in exon 19. however, we noticed a trend of egfr mutation appearance in the left-sided crc (2/3), while the study of randon and associates observed a statistically important correlation between the amplification of egfr and wt ras/braf in leftsided crc (randon et al., 2021). over 60% of crc patients develop metastases in the liver, while the second most common site of metastasis is the lungs (uhlyarik et al., 2020). the presence of distant metastases greatly reduces the five-year survival rate, and the development of these metastases is associated with the mutation of many genes (tanaka et al., 2019, hao et al., 2022). according to the results of our study, the presence of egfr mutation was detected in patients with distant metastases in both lungs and liver, metastasis only in lungs, and metastasis in pelvis. however, contrary to the study of zou and associates that detected a g735c mutation of egfr in one patient with liver metastasis, we found no mutation in patients with metastasis in the liver only (zou et al., 2018). to the best of our knowledge, this is the first report on the evaluation of frequency and type of mutations in the gene and its correlation with clinicopathological characteristics of patients with 43 biologica nyssana ● 14 (1) june 2023: 39-46 jugović et al. ● the frequency and type of mutations in the egfr gene of colorectal carcinoma patients in southeastern serbia garcía, j., bengoechea, o., muñoz-bellvis, l., sayagués, j.m., & abad, m. 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(2018). the gene mutational discrepancies between primary and paired metastatic colorectal carcinoma detected by next-generation sequencing. journal of cancer research and clinical oncology, 144(11), 2149– 2159. jugović et al. ● the frequency and type of mutations in the egfr gene of colorectal carcinoma patients in southeastern serbia stamenković et al. 2021, biologica nyssana 12(1) 12 (1) september 2021: 63-70 doi: 10.5281/zenodo.5523027 benthic macroinvertebrate community structure in batušinac ponds (serbia) relative to the distance from a river original article olivera stamenković department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, niš, serbia olivera.stamenkovic@pmf.edu.rs (corresponding author) vladica simić institute of biology and ecology, faculty of science, university of kragujevac, radoja domanovića 12, 34000 kragujevac, serbia vladica.simic@pmf.kg.ac.rs djuradj milošević department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, niš, serbia djuradj@pmf.ni.ac.rs ana petrović institute of biology and ecology, faculty of science, university of kragujevac, radoja domanovića 12, 34000 kragujevac, serbia ana.petrovic@pmf.kg.ac.rs milica stojković piperac department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, niš, serbia milicas@pmf.ni.ac.rs received: december 04, 2020 revised: march 30, 2021 accepted: april 02, 2021 abstract: batušinac ponds are situated in south-eastern serbia, near the južna morava river. in this study we compared benthic macroinvertebrate community structures of the river and three ponds located at contrasting distances from the river in order to assess the impact of the river proximity on benthic macroinvertebrate community structures in batušinac ponds. only 7 out of 43 taxa were common for the river and the study ponds. based on the nmds analysis, the community structures of the river and the study pond situated closest to the river were the most similar. the results of an anosim analysis showed that community structures of the river and all study ponds were significantly different (r=0.901; p=0.003). given the observed differences between the river and pond macroinvertebrate communities, it can be concluded that batušinac ponds contribute significantly to freshwater diversity of the study area. key words: macrozoobenthos, species composition, lotic taxa, lentic taxa, spatial proximity apstract: struktura zajednice bentosnih makroinvertebrata batušinačkih bara (srbija) u odnosu na udaljenost od reke batušinačke bare su smeštene u jugoistočnoj srbiji, u blizini reke južne morave. u ovoj studiji smo upoređivali strukturu zajednica bentosnih makroinvertebrata reke i tri bare smeštene na različitoj udaljenosti od reke, s ciljem da procenimo uticaj blizine reke na strukturu zajednica bentosnih makroinvertebrata u batušinačkim barama. od ukupno 43 taksona, svega je 7 bilo zajedničko za reku i istraživane bare. rezultati nmds analize su pokazali da su zajednice reke i bare smeštene najbliže reci najsličnije po strukturi. rezultati anosim analize su pokazali da se strukture zajednica reke i svih bara međusobno značajno razlikuju (r=0.901; p=0.003). uzimajući u obzir uočene razlike između zajednica makroinvertebrata reke i bara, može se zaključiti da batušinačke bare značajno doprinose slatkovodnom diverzitetu istraživanog područja. ključne reči: makrozoobentos, sastav vrsta, lotički taksoni, lentički taksoni, prostorna bliskost introduction ponds are small and shallow (surface area 1 m2–2 ha; max depth 8 m), natural or man-made water bodies that can be permanently or seasonally filled with standing water (biggs et al., 2005). despite their small size, ponds represent a valuable source of freshwater biodiversity, since they host more rare and threatened species than other freshwater ecosystems (williams et al., 2004; davies et al., 2008). ponds contribute disproportionately to regional biodiversity due to their high β diversity, which is related to the existence of a wide variety of pond types that differ in their features (e.g., length of their hydroperiod, size, or physical and chemical properties), even on a small spatial scale (de meester et al., 2005). macroinvertebrates constitute an important part of animal diversity in small water bodies, such as ponds (davies et al., 2008). it has been recognized that community structure (species composition and © 2021 stamenković et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 63 abundances) of macroinvertebrate communities in ponds is strongly driven by local environmental and biotic factors (e.g., water permanence, water chemistry, macrophyte cover, and fish predation) (trigal et al., 2007; hentges & stewart, 2010; walker et al., 2013; gleason & rooney, 2018). in addition, evidence has accumulated that macroinvertebrate communities in ponds are structured by dispersal processes (leibold et al., 2004; van de meutter et al., 2007). therefore, besides local, regional factors are also of huge importance for structuring macroinvertebrate communities in ponds. among structuring processes that operate at regional level, connectivity between habitats (e.g., small distance among habitats) is of huge importance for macroinvertebrate communities in ponds (oertli et al., 2008). in this study we compared the community structures of benthic macroinvertebrates between a part of the river and three ponds situated at contrasting distances from the river. we aimed to assess the impact of the river proximity on benthic macroinvertebrate community structures in ponds. we hypothesised that a pond situated closest to the river and the river have the most similar benthic macroinvertebrate community structures. materials and methods study area batušinac ponds are located in south-eastern serbia, about 10 km west of the city of niš. study area encompasses three permanent ponds situated on the left bank of the južna morava river, at distances of 17.5 m, 212 m, and 386 m (fig. 1). study ponds are the remains of the former river channel that were isolated, as a result of the redirection of the river flow during the construction of a nearby highway (fig. 1). the ponds are connected to the current riverbed both by groundwater supplies and by river flooding (ranđelović et al., 2007). pond 1 is the smallest (surface area of 429.42 m2; maximum depth 120 cm). it is situated nearest to the river (fig. 1), and it is consequently the most intensively flooded. pond 2 has a surface area of 6340.88 m2 and maximum depth of 250 cm. due to their spatial proximity pond 1 and pond 2 become a connected system during the flood events (fig. 1). pond 3 is the largest (surface area of 14206.05 m2; maximum depth 200 cm) and it is farthest from the river (fig. 1). in addition, pond 3 is isolated from the rest of the two study ponds by the nearby highway (fig. 1). sampling macroinvertebrate sampling was performed in september 2016. within each pond, we developed three sampling transects (study sites) starting from the shore towards the deeper water. sampling transects were distributed across the ponds to cover all mesohabitats within a pond. one sampling transect was developed within the river, encompassing river banks and a midstream. the part of the river investigated represents the river’s middle rich. benthic macroinvertebrates were sampled using a kick-net of 250 µm mesh size. three benthic subsamples were taken from the most common substrate types along each transect. all three benthic subsamples were merged into a single sample. the material collected was sorted out of sediment and preserved in 70% ethanol. macroinvertebrates were identified to the lowest possible taxonomic level using relevant taxonomic keys (elliot et al., 1988; wallace et al., 1990; edington & hildrew, 1995; nilsson, 1997; waringer & graf, 1997; gerken & sternberg, 1999; timm, 1999; pfleger, 2000; bauernfeind & humpesch, 2001; glöer, 2002; eiseler, 2005; elliot & humpesch, 2010). most of the taxa were identified to species or genus level, except the families whose individuals were damaged: aphelocheiridae (hemiptera), heptageniidae (ephemeroptera), hydrophilidae, elmidae (coleoptera), or whose identification requires taxonomic expertise, such as chironomidae (diptera). data analysis family chironomidae was present at all study sites, and due to its ubiquitous nature, was excluded from the analyses. 64 biologica nyssana ● 12 (1) september 2021: 63-70 stamenković et al. ● benthic macroinvertebrate community structure in batušinac ponds (serbia) relative to the distance from a river fig. 1. map of the study area with the position of the study sites 65 biologica nyssana ● 12 (1) september 2021: 63-70 stamenković et al. ● benthic macroinvertebrate community structure in batušinac ponds (serbia) relative to the distance from a river taxon abbreviation p1a p1b p1c p2a p2b p2c p3a p3b p3c river radix auricularia (linnaeus, 1758) raur x x x x x x radix labiata (rossmässler, 1835) rlab x x physella acuta (draparnaud, 1805) pacu x x x x x x x gyraulus sp. gyr x acroloxus lacustris (linnaeus, 1758) alac x viviparus acerosus (bourguignat, 1862) vace x unio pictorum (linnaeus, 1758) upic x limnodrilus hoffmeisteri claparède, 1862 lhoff x x x x x limnodrilus claparedeanus ratzel, 1868 lcla x x limnodrilus undekemianus claparède, 1862 lund x tubifex tubifex (müller, 1774) ttub x x x x x erpobdella octoculata (linnaeus, 1758) eoct x x helobdella stagnalis (linnaeus, 1758) hsta x haementeria costata (fr. müller, 1846) hcos x heamopis sanguisuga (linnaeus, 1758) hsan x gammarus balcanicus schäferna, 1922 gbal x pelocoris sp. pel x aphelocheiridae aph x platycnemis pennipes (pallas, 1771) ppen x x x x x coenagrion puella (linnaeus, 1758) cpue x x x ischnura elegans (vander linder, 1820) iele x x pyrrhosoma nymphula (sulzer) pnym x x x sympetrum fonscolombii (selys, 1840) sfon x x x x anax imperator leach, 1815 aimp x calopteryx splendens (harris, 1780) cspl x table 1. list of benthic macroinvertebrate taxa recorded across the study sites. x indicates the presence of a taxon 66 to visualise differences/similarities in the benthic macroinvertebrate community structures of study ponds and the river and spatial patterns of benthic macroinvertebrate taxa we used non-metric multidimensional scaling (nmds) with a braycurtis dissimilarity matrix. to test the statistical significance of the nmds results on benthic macroinvertebrate community structures we used an analysis of similarities (anosim). prior to analyses abundance data were square-root transformed. all analyses were performed using primer version 6 (clarke & gorley, 2006). results overall, 43 taxa (including the family chironomidae) were recorded across the study sites (tab. 1). a total of 31 taxa were recorded across the study ponds, of which 9 taxa were recorded in pond 1; 20 taxa in pond 2, and 17 taxa in pond 3. the recorded taxa belonged to 22 families, 10 orders, 3 classes and 3 phyla. benthic macroinvertebrate community of the river was represented with 20 taxa, belonging to 18 families, 12 orders, 5 classes and 3 phyla. insects were the taxonomically dominant class, both in the river and the study ponds, of which the order ephemeroptera was the taxonomically dominant in the river (represented with 4 taxa), while the order odonata was the taxonomically dominant in the study ponds (represented with 6 taxa). limnodrilus hoffmeisteri claparède, 1862 (annelida, oligochaeta) was the most abundant species both in the river and in pond 1. the most abundant species in pond 2 was radix auricularia (linnaeus, 1758) (mollusca, gastropoda), followed by pyrrhosoma nymphula (sulzer) (insecta, odonata) (fig. 2). pyrrhosoma nymphula was the most abundant species in pond 3 (fig. 2). overall, 23 taxa were recorded exclusively in the study ponds, while 13 taxa were recorded exclusively in the river (tab. 1; fig. 3). besides the chironomidae family, common taxa of both pond and the river were: radix labiata (rossmässler, 1835) (mollusca, gastropoda); caenis sp. (insecta, ephemeroptera); culicoides sp. (insecta, diptera); l. hoffmeisteri, tubifex tubifex (müller, 1774) (annelida, oligochaeta), and erpobdella octoculata (linnaeus, 1758) (annelida, hirudinea) (tab. 1; fig. 3). the study sites were grouped in the nmds plot in two dimensions based on the similarities of their benthic macroinvertebrate community structures biologica nyssana ● 12 (1) september 2021: 63-70 stamenković et al. ● benthic macroinvertebrate community structure in batušinac ponds (serbia) relative to the distance from a river gomphus vulgatissimus (linnaeus, 1758) gvul x onychogomphus forcipatus (linnaeus, 1758) ofor x ecnomus sp. ecn x athripsodes aterrimus (stephens, 1836) aate x leptocerus sp. lep x hydropsyche contubernalis mclachlan, 1865 hcon x caenis sp. cae x x x x cloeon dipterum cdip x baetis rhodani (pictet, 1843) brho x heptageniidae hep x potamanthus luteus (linnaeus, 1767) plut x hydrophilidae hydr peltodytes caesus (duftschmid, 1805) pcae x dytiscus sp. dyt x elmidae elm x culex sp. cule x culicoides sp. culic x x x x x x chironomidae x x x x x x x x x x with stress value 0.06 (fig. 4). the study sites from pond 1 and the river were grouped in the same cluster, while sites form pond 2 and pond 3 were clearly separated from the river and pond 1 (fig. 4). the results of the anosim analysis showed that benthic macroinvertebrate structures of the river and all three ponds were significantly different (r = 0.901; p = 0.003). discussion as previously reported in the literature, ponds and rivers, as distinct freshwater habitats, usually support distinct invertebrate assemblages, even at the genusfamily level (céréghino et al., 2008). this was the case in our study, since only 7 out of 43 recorded taxa were common for the river and the study ponds. moreover, our results are similar to previous research showing that rivers make a higher contribution than ponds to the taxonomic richness of ephemeroptera, trichoptera, plecoptera, and mollusca, while ponds make a higher contribution to the taxa richness of odonata, heteroptera, and coleoptera (céréghino et al., 2008). it has been previously shown that the species we recorded both in the river and in the ponds are generalists, inhabiting both lotic and lentic ecosystems. for instance, e. octoculata is usually dominant among leeches in both lotic and lentic habitats (kubová et al., 2013). the snail r. labiata is adapted to inhabit both lotic and lentic environments by having larger and wider but shorter (globular) shells in lotic versus lentic habitats (lam & calow, 1988). oligochaetes, such as l. hoffmeisteri and t. tubifex inhabit a wide variety of freshwater body types, and the latter one is known to tolerate pollution and can be found in very different conditions (timm, 1999). similarly, the biting midges from the genus culicoides inhabit various aquatic, but also semiaquatic habitats (mullen & murphree, 2019). on the other hand, most of the species we recorded exclusively in the river or in the study 67 biologica nyssana ● 12 (1) september 2021: 63-70 stamenković et al. ● benthic macroinvertebrate community structure in batušinac ponds (serbia) relative to the distance from a river fig. 2. partitioning of the taxa recorded at the study ponds and the river by their abundances. for taxa name abbreviations see tab. 1 ponds have been previously found to prefer to a certain extent either lotic or lentic aquatic habitats. this is especially true for dragonflies and damselflies (odonata) whose clear preferences for specific habitat types are mainly determined by the presence of water flow, water regime, vegetation structure, and physicochemical water properties (corbet, 1999). species that were recorded in ponds in our study, such as: coenagrion puella (linnaeus, 1758), ischnura elegans (vander linder, 1820) (coenagrionidae), sympetrum fonscolombii (selys, 1840) (libellulidae) and anax imperator leach, 1815 (aeshnidae) typically inhabit standing waters (rajkov, 2013). in contrast, the species we recorded in the river, such as: calopterix splendens (harris, 1780) (calopterigidae), gomphus vulgatisimus (linnaeus, 1758) and onychogomphus forcipatus (linnaeus, 1758) (gomphidae) almost exclusively inhabit running waters (rajkov, 2013). similarly, the mayfly species (ephemeroptera) that we recorded in the river, such as baetis rhodani (pictet, 1843) and potamanthus luteus (linnaeus, 1767), are typical lotic species whose larval development is restricted to running waters (eliot et al., 1988). the similar situation is with caddisflies (trichoptera) recorded in our study. namely, hydropsyche contubernalis mclachlan, 1865, the species we recorded in the river, is regarded as a species of the large rivers (edington & hildrew, 1995). on the other hand, the distribution of the species we recorded in the ponds, such as athripsodes aterrimus (stephens, 1836), is determined by slow flowing water, fine sediments and presence of macrophytes, and it is typical for standing waterbodies (wallace et al., 1990; previšić et al., 2010). although l. hoffmeisteri has low dispersal capacity, a possible reason for high abundance of this species both in the river and pond 1 could be its passive dispersion via river floods. it has been shown that an increase in water exchange by flooding events can increase exchange of macroinvertebrates (siziba et al., 2011), and that passive dispersers can spread by direct water connections in flooded areas (van leeuwen et al., 2013). on the other hand, spatial proximity and connectivity of the ponds in our study were not crucial for structuring their benthic macroinvertebrate communities. the observed differences between macroinvertebrate communities of pond 1 and pond 2, despite their connectedness and spatial proximity, suggest that local factors were more important for structuring benthic macroinvertebrates in the study ponds. for instance, similar depth or water quality could be the reasons for the observed similarity of benthic macroinvertebrate communities between pond 2 and pond 3, even though these ponds are separated by the nearby highway. overall, by taking into account that benthic macroinvertebrate communities of the river and all study ponds were statistically significantly different, it may be concluded that river proximity does not affect macroinvertebrate 68 biologica nyssana ● 12 (1) september 2021: 63-70 stamenković et al. ● benthic macroinvertebrate community structure in batušinac ponds (serbia) relative to the distance from a river fig. 3. non-metric multidimensional scaling (nmds) plot showing spatial patterns of benthic macroinvertebrates from the river and study ponds. p and r denote pond and river respectively. for taxa name abbreviations see tab. 1 fig. 4. non-metric multidimensional scaling (nmds) plot showing similarities among the study sites based on their benthic macroinvertebrate community structures communities in the study ponds and that batušinac ponds contribute to 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and ponds in an agricultural landscape in southern england. biological conservation 115(2): 329–341. biologica nyssana ● 12 (1) september 2021: 63-70 70 stamenković et al. ● benthic macroinvertebrate community structure in batušinac ponds (serbia) relative to the distance from a river anticancer compounds from medicinal plants biologica nyssana 4 (1-2)  december 2013: 71-74 sućur, j.,et al.  oxidativ e stress in soy bean seedings treated with… 71 original article oxidative stress in soybean seedlings treated with thymus serpyllum aqueous extract jovana šućur, milan popović, dejan prvulović, đorđe malenčić, biljana kiprovski faculty of agriculture, university of novi sad, trg dositeja obradovića 8, 21000 novi sad, serbia * e-mail: bkiprovski@polj.uns.ac.rs abstract: šućur, j., popović, m., prvulović, d., malenčić, đ., kiprovski, b.: oxidative stress in soybean seedlings treated with thymus serpyllum aqueous extract . biologica nyssana, 4 (1-2), december 2013: 71-74. the effect of different concentrations (0.05, 0.1 and 0.2%) of thymus serpyllum l. aqueous extracts on lipid peroxidation process (lp), as well as reduced glutathione content (gsh) in leaves and roots of soybean seedlings were examined 24 and 72 h after the treatment. our results showed that only highest concentration of the extract used (0.2%) enhanced process of lipid peroxidation, while concentration of 0.1% stimulated gsh accumulation in soybean seedlings. key words: allelopathy, antioxidants, biopesticides, glycine max l., thymus serpyllum l. introduction the genus thymus l. (lamiaceae) consists of about 215 species of herbaceous perennial and subshrubs. thymus species are considered as medicinal plants due to their pharmacological and biological properties (h u s s a in et al., 2013). it is commonly used as tonic, herbal tea and flavouring agent. thymus oils and extracts are widely used in pharmaceutical, cosmetic and perfume industry also for flavouring and bio-preservation of several food products (v e r m a et al., 2011). in the mediterranean region, which can be described as the centre of this genus, annual aromatic herb, thymus serpyllum l. inhabits cultivated and uncultivated lands including wastelands (v e r m a et al., 2011). research on allelopathy of t. serpyllum on weeds shown that water extract of t. serpyllum promoted test plants' growth at low concentration but inhibited them at high, and inhibition became stronger as concentration increased (z h a n g , 2009). the use of allelochemicals as weed control agents, becomes widely investigated, however the impact of these bioherbicide on cultivated plants is less known. since ros (reactive oxygen species)generation resulting in oxidative damage has been proposed as one of the modes of action of allelochemicals, the aim of this study was to examine the impact of thymus serpyllum l. aqueous extract on soybean antioxidant properties so as to assess its possible side effects when applied as bioherbicide in soybean organic production. material and methods the wild, aromatic plant, thymus serpyllum l., was collected in northern serbia (vojvodina province) in may of 2010. voucher specimens of collected plant was confirmed and deposited at the herbarium of the department of biology, faculty of natural sciences, university of novi sad. seeds of soybean (glycine max l.) cv. sava were obtained 11th sfses • 13-16 june 2013, vlasina lake 4 (1-2) • december 2013: 71-74 biologica nyssana 4 (1-2)  december 2013: 71-74 sućur, j.,et al.  oxidativ e stress in soy bean seedings treated with… 72 from the institute of field and vegetable crops, novi sad, serbia. the aqueous extract of t. serpyllum was prepared with air-dried plant material (10 g) in boiling distilled water (100 ml). after 24 h, the extract was filtered through filter paper and kept at 4 °c until application. the experiment was performed at the laboratory of biochemistry, faculty of agriculture, novi sad and conducted under controlled conditions (temperature 25 °c, 60-70% relative humidity, 16 h photoperiod). soybean seedlings were grown for 14 days in plastic pots (500 ml) containing sterile sand, after which they were transferred on plastic pots (700 ml) containing the hoagland’s solution and 3.5, 7 and 14 ml of t. serpyllum aqueous extract, while 3.5, 7 and 14 ml of the hoagland’s solution were added in pots of control. seedlings were harvested for determining the investigated biochemical parameters 24 and 48 h after the treatments. biochemical analyses were carried out spectrophotometrically using an uv/vis spectrophotometer model 6105 (jenway, uk). lipid peroxidation (lp) was measured at 532 nm using the thiobarbituric acid (tba) test. the total amount of tbars (tba-reactive substances) is given as nmol malondialdehyde (mda) equivalents mg -1 proteins (m a n d a l et al., 2008). reduced glutathione (gsh) was determined according to s e d la k a n d l in d s a y (1968) and expressed as µmol gsh mg -1 proteins. values of the biochemical parameters were expressed as means  standard error of determinations made in triplicates and tested by anova followed by comparison of the means by duncan’s multiple range test (p<0.05). data were analysed using statistica for windows version 11.0. results and discussion the toxicity of many allelochemicals can largely be attributed to the formation of semiquinone radicals that donate electrons to molecular oxygen, forming reactive oxygen species (ros), such as superoxide anions (o2 .), and more reactive hydroxyl ( . oh) or hydroperoxyl (ho2 ) radicals (h a m m o n d k o s a k & j o n e s , 1996). this plant-plant allelopathic interaction generates a cascade of signaling events-including ca 2+ influx and proton efflux that actives the nadph-oxidase complex (generating . o2 ) and ph-sensible cell wall peroxidases (producing h2o2), which produces an oxidative burst (d o s s a n t o s et al., 2008). enhancement of ros production during the oxidative burst is one of the earliest reactions elicited in response to various abiotic and biotic stimuli. some allelochemicals rapidly depolarize the cell membrane, increasing membrane permeability, inducing lipid peroxidation and causing a generalized cellular disruption that ultimately leads to cell death (d e v i & p r a s a d , 1996; z e n g et al., 2001; y u et al., 2003). the peroxidation of unsaturated lipids of biological membranes is the most prominent symptom of oxidative stress in animals and plants (m a n d a l et al., 2008). malondialdehyde (mda) content, an end-product of lipid peroxidation process, is used as oxidant biomarker. it is suggested that different concentrations of allelochemicals induce production of mda, however due to their antagonistic and synergistic effect mda content is not always in reciprocity with these chemicals (d in g et al., 2007; h a d d a d c h i & g e r iv a n i , 2009). our results showed that 24 h after the treatment with t. serpyllum aqueous extracts no significant difference in the lp intensity in soybean leaves and roots between plants form control and treatments were recorded. the significant increase was recorded 72 h after the treatment only in leaves of soybean plants treated with the highest concentration of t. serpyllum aqueous extract (fig. 1). allelochemicals might directly inhibit oxidizing enzymes in some way, leaving the plant vulnerable to oxidative damage, similar to the reports of q ia n et al. (2009). it has been documented that gsh can remove ros and also protect proteins from the oxidation of protein thiol groups into denaturation (n o c t o r , 2002). t. serpyllum aqueous extract with concentration of 0.1% induced production of gsh in leaves of soybean plants 24 h, and in leaves and roots 72 h after treatment (fig. 2). also, 24 h after the treatment with t. serpyllum aqueous extract with concentration of 0.2%, roots of soybean had highest level of gsh content (fig. 2). conclusion in conclusion, our results showed that low concentration of thymus serpyllum l. aqueous extract did not induce lipid peroxidation in soybean seedlings, while the highest concentration used (0.2%) enhanced lipid peroxidation process 72 h after the treatment. furthermore, concentration of 0.1% of t. serpyllum aqueous extract stimulated gsh accumulation in soybean seedlings which propose its possible stimulative effect on antioxidant system. biologica nyssana 4 (1-2)  december 2013: 1-7 balian, d., mujagić-pašić, a..  morphological v ariability of the f ruit… 73 fig. 1. lipid peroxidation level in leaves and roots of soybean seedlings 24 and 72 h after treatment with different concentrations (%) of t. serpyllum aqueous extracts (v/v) and in control (c). values marked with different letter differ significantly at p<0.05 (duncan’s test). fig. 2. reduced glutathione content in leaves and roots of soybean seedlings 12 and 48 h after treatment with different concentrations (%) of t. serpyllum aqueous extracts (v/v) and in control (c). values marked with different letter differ significantly at p<0.05 (duncan’s test). acknowledgements. this study was carried out within a project of the ministry of education, science and technological development, republic of serbia, grant n o tr-31022. references devi, s.r., prasad, m.n.v. 1996: ferulic acid mediated changes in oxidative enzymes of maize seedlings: implications in growth. biology of plants, 38 (3):387-395. ding, j., sun, y., xiao, c. l., shi, k., zhou, y. h., yu, j. q. 2007: physiological basis of different allelopathic reactions of cucumber and figleaf gourd plants to cinnamic acid. journal of experimental botany, 58 (13): 3765-3773. dos santos, w.d., ferrarese, m.m.l., ferraresefilho, o. 2008: ferulic acid: an allelochemical troublemaker. functional plant science and biotechnology, 2 (1): 47-55. haddadchi, g.r., gerivani, z. 2009: effects of phenolic extracts of canola (brassica napuse l.) biologica nyssana 4 (1-2)  december 2013: 71-74 sućur, j.,et al.  oxidativ e stress in soy bean seedings treated with… 74 on germination and physiological responses of soybean (glycine max l.) seedlings. international journal of plant production, 3 (1): 63-74. hammondkosak, k.e., jones, j.d.g. 1996: resistance gene-dependent plant defense responses. plant cell, 8 (1):1773-1791. hussain a.i., anwar f., chatha s.a.s., latif s., sherazi s.t.h., ahmad a., worthington j., sarker s.d. 2013: chemical composition and bioactivity studies of the essential oils from two thymus species from the pakistani flora. food science and technology, 50: 185-192. mandal, s., mitra, a., mallick, n. 2008: biochemical characterization of oxidative burst during interaction between solanum lycopersicum and fusarium oxysporum f. sp. lycopersici. physiological and molecular plant pathology, 72 (1): 56-61. noctor, g., gomez, l., vanacker, h., foyer, c.h. 2002: interactions between biosynthesis, compartmentation and transport in the control of glutathione homeostasis and signalling. jornal of experimental botany, 53 (372): 1283–304. qian, h., xu, x., chen, w., jiang, h., jin, y., liu, w., fu, z. 2009: allelochemical stress causes oxidative damage and inhibition of photosynthesis in chlorella vulgaris. chemosphere, 75 (3): 368-375. sedlak, j., lindsay, h. 1968: estimation of total protein bound and non protein sulphydryl groups in tissue with ellman’s reagent. analytical biochemistry, 25: 192-205. verma r.s., verma r.k.,chauhan a., yadav a.k. 2011: seasonal variation in essential oil content and composition of thyme, thymus serpyllum l. cultivated in uttarakhand hills. indian journal of pharmaceutical sciences, 73 (2): 233-235. yu, j.q., ye, s.f., zhang, m.f., hu, w.h. 2003: effects of root exudates and aqueous root extracts of cucumber (cucumis sativus), and allelochemicals on photosynthesis and antioxidant enzymes in cucumber. biochemical systematics and ecology, 31 (2):129-139. zeng, r.s., luo, s.m., shi, y.h., shi, m.b., tu, c.y. 2001: physiological and biochemical mechanism of allelopathy of secalonic acid f on higher plants. agronomy journal, 93 (1):72-79. zhang y., m.sc., may 2009: studies of thymus serpyllum l. var. asiaticus kitag. allelopathy on weeds. http://www.globethesis.com/?t=2143360245465855 anticancer compounds from medicinal plants biologica nyssana 4 (1-2)  december 2013: 9-14 nikolić, lj. et al.  analysis of weed flora in conventional… 9 original article analysis of weed flora in conventional and organic potato production ljiljana nikolić, olivera ilić, dejana džigurski, branka ljevnaić-mašić faculty of agriculture, university of novi sad, trg dositeja obradovića 8, 21 000 novi sad, serbia * e-mail: ljnik@polj.uns.ac.rs abstract: nikolić, lj., ilić, o., džigurski, d., ljevnaić-mašić, b.: analysis of weed flora in conventional and organic potato production. biologica nyssana, 4 (1-2), december 2013: 9-14. composition of weed flora is highly dynamic and depends upon great number of factors, of which cultural practices that are applied by humans in certain crops are the most important. one of the most frequently grown plants in the world and in our country is potato (solanum tuberosum l., solanaceae), due to its high biological and nutritive value. therefore, in the paper was presented taxonomic analysis of weed flora in potato grown conventionally and according to the principles of organic agricultural production, with the intention to point out to eventual differences between present weeds. of the total number of identified species, from phylum equisetophyta and class equisetopsida, in organic potato crop, was determined only one, equisetum arvense. of remaining 38 weeds from phylum magnoliophyta., classified into two classes, magnoliopsida and liliopsida. on both of potato growing systems, 39 weed species were found, classified into 16 families and 32 genus. of the total number, 31 species was identified in conventional potato crop, and only 23 species in potato crop grown according to organic principles, which is for about quarter less. biological spectrum of weed flora in both potato growing systems is pronouncedly of terrophytic – geophytic type. in the spectrum of area types were recorded differences, i.e. in the conventional potato crop represented are only widely distributed species, while in the organic crop, beside species of wide distribution are also present elements of pontic group. key words: taxonomic analysis, weeds, potato introduction floristic diversity, as well as overall biodiversity of natural and anthropogenic ecosystems, is a subject of numerous studies. fundamental research comprising of direct verification and systematization of biodiversity forms the foundations for biodiversity conservation in general. biodiversity changes due to anthropogenic factors pose numerous risks, while yielding paucity of immediate benefits (s t e v a n o v i ć & v a s i ć 1995). hence, research focusing on studying and preserving the biodiversity of agroecosystems that are under direct human influence is of particular importance, as reflected in the development of a european-level action plan (the 2001 biodiversity action plan for agriculture /com/2001/0162). since the early nineties, interest in organic farming has significantly increased in europe, with particular attention to agroecosystems and their biodiversity, emphasizing the differences between conventional crops and those grown according to organic principles (r o s c h e w i t z et al., 2005; w e i b u l l et al., 2003; h y v ö n e n et al., 2003; h y v ö n e n , 2007). the main difference between conventional and organic farming principles is reflected in the fact that the latter completely exclude the use of chemicals and fertilizers. it is this specificity that, 11 th sfses • 13-16 june 2013, vlasina lake 4 (1-2) • december 2013: 9-14 mailto:ljnik@polj.uns.ac.rs biologica nyssana 4 (1-2)  december 2013: 9-14 nikolić, lj. et al.  analysis of weed flora in conventional… 10 on one hand, raises the question of whether organic farming could contribute to the restoration and preservation of biodiversity (h y v ö n e n , 2007). however, on the other hand, control of weed proliferation—which, as a regular crop companion, has a significant negative impact on the quality and quantity of agricultural products—is a major problem in organic production, as it almost completely excludes chemical weed control measures (b à r b e r , 2002; k r i s t i a n s e n , 2003). to address these issues, this paper presents the analysis of the weed flora accompanying potato (solanum tuberosum l.), one of the most widely cultivated plants in the world and in our country, grown both conventionally and according to the organic farming principles, in order to identify potential differences between the outcomes of the two systems. the reported results provide a foundation for long-term monitoring and study of weed flora as an important component of agroecosystem biodiversity. material and methods floristic weed analysis in conventional potato production was carried out during the 2008-2010 vegetative period at the experimental plots in the vicinity of bečej. the two-factor experiment was carried out, whereby conventional potato production with and without herbicide application was conducted in the varying cultivation conditions (number of inter-row potato earthing repetitions). this paper reports only on the weed flora found in the control part of the experiment, which included cultivation and planting, with no other crop care or weed protection measures. floristic weed analysis in organic potato production was carried out during 2012, on the localities registered for organic farming—family "biofarm" in kelebija and biofarm "mamudžić" in ljutovo—where weed proliferation is suppressed by mechanical means only. determination of plant material was performed according to josifović (1970-1977), whereas taxonomy conformed to takhtajan (2009), life-form categorization followed ujvárosi (1973), and floral elements were determined according to gajić (1980). results and discussion the following sections present the analysis of weed flora in potato crops grown conventionally and according to organic principles. in both potato cultivation systems, 39 weed species were identified, grouped into 32 genera, 16 families, 12 orders, 5 subclasses, 3 classes and 2 divisions. more specifically, the control variant of conventional potato crops showed the presence of 31 weed species, classified into 25 genera, 11 families, 7 orders, 5 subclasses, 2 classes and 1 division. on the other hand, in the organic potato crops, only 23 weed species were recorded, which were classified in 22 genera, 15 families, 12 orders, 5 subclasses, 3 classes and 2 divisions (table 1 and 2). of the total number of identified species, equisetum arvense l. (2.56%) from the equisetophyta division and equisetopsida class was found in organic potato crops only. the remaining 38 weeds from the magnoliophyta division were noted in varying numbers in both conventional and organic potato crops. these were divided into two classes, whereby the magnoliopsida (82.05%) class was represented by four subclasses (caryophyllidae, dillenidae, lamiidae and asteridae) and the liliopsida class (15.38%) was represented by the commelinidae subclass only (table 1, 2). as noted above, greater floristic diversity (31 species) and more significant weed proliferation was found in the control variant of the conventionally grown potatoes, while nearly 25% (23 species) reduction in floristic diversity is evident in organically grown potato crops. the analysis revealed presence of 15 common species, as well as 8 specific to organic and 16 to conventional potato crops. in the conventionally grown crops, the following were found in significant numbers: panicum crus-galli l., cirsium arvense (l.) scop., amaranthus retroflexus l., sorghum halepense l. and polygonum lapathifolium l., while only individual weeds were generally recorded in organic potato crops, except for equisetum arvense l. and polygonum convolvulus l. in ljutovo, both of which were more abundant. although the findings reported by some authors (hald, 1999; menalled et al., 2001; boguzas et al., 2004) indicate greater species wealth in organic crops compared to conventional cultivars, data obtained in this work point to the greater weed floristic diversity in conventional potato crops. this discrepancy can be explained by the fact that our analyses were conducted on the control variant of the experiment, which did not permit any herbicide application. however, it should be noted that, as the soil on which the experiment was conducted had been previously used for conventional agricultural production, the previously applied agrotechnical practices certainly affected the obtained results. biologica nyssana 4 (1-2)  december 2013: 9-14 nikolić, lj. et al.  analysis of weed flora in conventional… 11 table 1. taxonomic review of weed flora in potato crops p h y lu m c la ss s u b cl a ss order family genus plant species life form floristic elements c o e q u is et o p h y ta e q u is et o p si d a equisetales equisetaceae equisetum e. arvense l. g1 cirk. + m a g n o li o p h y ta m a g n o li o p si d a c a ry o p h y ll id a e caryophyllales portulacaceae portulaca p. oleracea l. t4 cosm.. + + caryophyllaceae stellaria s. media l. t1 cosm.. + amaranthaceae amaranthus a. retroflexus l. t4 adv. + + chenopodiaceae chenopodium c. album l. t4 cosm.. + + c. hybridum l. t4 subcirk. + + polygonales polygonaceae polygonum p. convolvulus l. t4 subeur. + + p. lapathifolium l. t4 subcirk. + p. persicaria l. t4 eur. + p. aviculare l. t4 cosm.. + d il le n id a e capparales brassicaceae capsella c. bursa-pastoris (l.) med. t1 cosm.. + + sinapis s. arvensis l. t3 subeur. + malvales malvaceae hibiscus h. trionum l. t4 pont.ea.subm. + malva m. silvestris l. ht-h4 eur. + euphorbiales euphorbiaceae euphorbia e. helioscopia l. t4 subeur. + l a m ii d a e solanales solanaceae datura d. stramonium l. t4 cosm.. + + solanum s. nigrum l. t4 cosm.. + + convolvulales convolvulaceae convolvulus c. arvensis l. g3 cosm.. + + cuscutaceae cuscuta c. epithymum l. t4 subeur. + c. europea l. t4 eur. + boraginales boraginaceae heliotropium h. europaeum l. t4 pont.-subm. + lamiales lamiaceae stachys s. annua l. t4 subpont. subm. + lamium l. amplexicaule l. t1 subeur. + a st er id a e asterales asteraceae ambrosia a. artemisiifolia l. t4 adv. + + cirsium c. arvense (l.) scop. g3 subeur. + + galinsoga g. parviflora cav. t4 adv. + erigeron e. canadensis l t4 adv. + matricaria m. chamomilla l. t2 eur. + m. inodora l. t4 eur. + senecio s. vulgaris l. t1 eur. + sonchus s. arvensis l. g3 eur. + + s. oleraceus l. t4 subeur. + xanthium x. strumarium l. t4 adv. + l il io p si d a c o m m el in id a e poales poaceae agropyrum a. repens beauv. g1 eur. + cynodon c. dactylon pers. g1 cosm.. + digitaria d. sanguinalis scop. t4 cosm.. + panicum p. crus-galli l. t4-t4(h) cosm.. + + setaria s. glauca p.b. t4 cosm.. + + sorghum s. halepense l. g1 cosm.. + + 2 3 5 12 16 32 39 31 23 biologica nyssana 4 (1-2)  december 2013: 9-14 nikolić, lj. et al.  analysis of weed flora in conventional… 12 table 2. the number of weed species of the higher taxonomic categories in potato crop division class subclass family genus species no % equisetophyta equisetopsida 1 1 1 2,56 magnoliophyta magnoliopsida 4 14 25 32 82,05 liliopsida 1 1 6 6 15,38 ukupno 2 3 5 16 32 39 100 the 25% reduction in weed variety in the organic potato crops, when compared to the conventional production, could be potentially explained by the still not fully balanced ecological conditions in these plots, their partial isolation, as well as regular mechanical weed control. still, some authors (weibull et al., 2003) maintain that the cultivation (farming) system is yet to be established as a factor affecting the floristic diversity, while roschewitz et al. (2005) reported that the floristic diversity of conventional crops, especially their peripheral parts, is significantly influenced by dissemination of various species from the surrounding area. as can be seen in table 3, the total floristic spectrum of weed flora found in potato crops is dominated by the asteraceae family with 10 species (25.65%), and poaceae family with 6 species (15.39%). this relationship is not surprising, given that these vascular plant families are already very rich in species, among which the representatives from the weed category are very numerous. table 3. floristic range of families according to species number familija broj vrsta % asteraceae 10 25,65 poaceae 6 15,39 polygonaceae 4 10,26 chenopodiaceae 2 5,13 brassicaceae 2 5,13 malvaceae 2 5,13 solanaceae 2 5,13 cuscutaceae 2 5,13 lamiaceae 2 5,13 equisetaceae 1 2,56 portulacaceae 1 2,56 caryophyllaceae 1 2,56 amaranthaceae 1 2,56 euphorbiaceae 1 2,56 convolvulaceae 1 2,56 boraginaceae 1 2,56 ukupno 39 100 polygonaceae family is represented by 4 species (10.26%), whereas chenopodiaceae, brassicaceae, malvaceae, solanaceae, cuscutaceae and lamiaceae families are represented by 2 species (each with 5.13% participation). finally, only one species (2.56%) each was found for equisetaceae, portulacaceae, caryophyllaceae, amaranthaceae, euphorbiaceae, convolvulaceae and boraginaceae families. life-form analysis of the identified potato crop weed flora indicated its terophytic-geophytic character (fig. 1). in conventional potato crops, terophytes comprised 80.64% (25 species), and were dominated by t4 terophytes—annual weeds whose seeds germinate in spring and bear fruit in summer—with 64.52% (20 species). the remaining 19.36% (6 species) were of geophytic character, of which three species (9.68%) belonged to the g1 category, with developed rhizomes, and the remaining three (9.68%) to the g3 category, characterized by adventitious root buds. in the organic potato crops, the ratio is similar, as the terophytes predominate with 73.91% (17 species), albeit with a slightly lower share compared to the conventional crops. the participation of geophytes is also similar, with 21.74% (5 species), of which three (13.05%) are in g3 and two (8.69%) in the g1 category. only one species (malva silvestris) belongs to the hemiterophyte-hemicryptophyte category (fig. 1). this life-form ratio is characteristic of agroecosystems, where due to intensive agricultural practices, the conditions are most favorable for development of terophytes (kojić et al., 1998; knežević & baketa, 1990; gabriel & tscharntke, 2007). the spectrum of areal types, as is characteristic of agroecosystems, is dominated by species capable of wide spatial distribution, due to their wide ecological valence, and adaptation to intense anthropogenic impacts (kojić et al., 1998). although they cover relatively small surface and comprise only a few species, we note a few differences. namely, in the conventional potato crops, only highly proliferative species are present, among which, the cosmopolitan floral elements predominate with 41.94% (13 species). in contrast, biologica nyssana 4 (1-2)  december 2013: 9-14 nikolić, lj. et al.  analysis of weed flora in conventional… 13 in the organic crops, in addition to proliferative species, the conditions are favorable for the development of markedly continental representatives of pontic groups (ponticsubmediterranean – heliotropium europaeum l., pontic-eastern submediterranean – hibiscus trionum l., and subpontic-submediterranean – stachys annua l.) (fig. 2). fig. 1. biological spectrum of weed flora in potato crop, a – conventional, and b organic these results point to slight differences in the structure of the weed flora found in potato crops grown conventionally and according to organic principles, probably due to differences in applied agrotechnical practices. it should be noted that, although the weed flora plays a significant role in the overall biodiversity and the functioning of these anthropogenic ecosystems, their optimal operation still requires maintaining a correct balance between cultivated and wild plants, as weeds contribute to better regulation of environmental conditions (bound & grundy, 2001; boguzas et al., 2004). protection and conservation of sensitive weed species can be achieved by different means, and given their increasing endangerment, the focus should be on the intensification of organic agriculture, with reduced use of herbicides and fertilizers. however, conservation of these species in botanical gardens is also one of the possible measures (znaor, 1996; moss et al., 2004; hulina, 2005). fig. 2. area type spectrum of weed flora in potato crop, a – conventional, and b – organic conclusion in both organic and conventional potato crops, the presence of 39 weed species was noted, which were grouped into 16 families and 32 genera. greater floristic diversity (31 species) and greater weed infestation was found in the control plots of conventionally grown potatoes, while nearly 25% (23 species) reduction in floristic diversity was established in organically grown crops. the two cultivation methods had 15 species in common, while 8 were specific to organic and 16 to conventional potato crops. in the total floristic spectrum of potato crops weed flora, asteraceae family predominates with 10 species, followed by poaceae family with 6 species. biological spectrum of weed flora in both potato production systems is of predominantly terophytic-geophytic character, with a slightly lower participation of terophytes in organic potato crops. biologica nyssana 4 (1-2)  december 2013: 9-14 nikolić, lj. et al.  analysis of weed flora in conventional… 14 in terms of the spectrum of areal types, only species capable of wide distribution are present in conventional potato crops, while in organic crops, elements of pontic group are also found. the results reveal small differences in the weed flora structure between conventionally and organically grown potato crops, most likely due to agrotechnical practices. acknowledgements. this study is part of the project tr – 31027 «organic agriculture: improvement of production by use of fertilizers, biopreparations and biological measures« funded by subsidized by the ministry for science and technological development of the republic of serbia. references bàrberi, p. 2002: weed management in organic agriculture: are we addressing the right issues? weed research, 42(3): 177-193. boguzas, v., marcinkeviciene, a., kairyte, a. 2004: quantitative and qualitative evaluation of weed seed bank in organic farming. agronomy research, 2(1): 13-22. bound, w., grundy, a.c. 2001: non-chemical weed management in organic farming systems. agronomy research, 41: 383-405. gabriel, d., tscharntke,t. 2007: insect pollinated plants benefit from organic farming. agriculture, ecosystems and environment, 118: 43-48. gajić. m. 1980: pregled vrsta flore sr srbije sa biljnogeografskim oznakama. glasnik šumarskog fakulteta, 54(a): 111-141. hald, a.b. 1999: weed vegetation (wild flora) of long estabilished organic versus conventional cereal fields in denmark. annals of applied biology, 134: 307-314. http://europa.eu/legislation_summaries/agriculture/e nvironment/l28024_en.htm the 2001 biodiversity action plan for agriculture /com/2001/0162). hulina, n. 2005: list of threatened weeds in continental part of croatia and their possible conservation. agriculturae conspectus scientificus, 70 (2): 37-42. hyvönen, t., ketoja, e., salonen, j., jalli, h., tiainen, j. 2003: weed species diversity and community composition in organic and conventional cropping of spring cereals. agriculture, ecosystems and environment, 97: 131-149. hyvönen, t. 2007: can conversion to organic farming restorethe species composition of arable weed communities? biological conservation, 137: 382-390. josifović, m. (ed.), 1970-1977: flora sr srbije, iix, sanu, beograd. kojić, m., popović, r., karadžić, b. 1998: sintaksonomski pregled vegetacije srbije. institut za biološka istraživanja »siniša stanković«, beograd, 1-218. knežević, m., baketa, e. 1990: phytocenological characteristics of the buckwheat weed community in north-eastern croatia. fagopyrum, 11: 15-18 kristiansen, p. e. 2003: sustainable weed management in organic herb and vegetable production. monash, new england, australia, university of new england, phd thesis, archived at http://orgprints.org/4829 menalled, f.d., gross, k.l., hammond, m. 2001: weed aboveground and seedbank community response to agricultural management systems. ecological applications, 9: 634-641. moss, r.s., storkey, j., cussans, w.j., perryman, a.m.s., hewitt, v.m. (2004): simposium the broadbalk long-term experiment at rothamsted: what has it told us about weeds? weed science, 52: 864-873. roschewitz, i., gabriel, d., tscharntke, t., this c. 2005: the effects of landscape complexity on arable weed species diversity in organic and conventional farming. journal of applied ecology, 42: 873-882. stevanović, v. , vasić, v. 1995. o biodiverzitetu. in: stevanović, v. & vasić, v. (eds.). biodiverzitet jugoslavije sa pregledom vrsta od međunarodnog značaja, 1-9, ecolibri, beograd, biološki fakultet, beograd. weibull, a.c., östman, ö., granqvist, a. 2003: species richnes in agroecosystems; the effect of landscape, habitat and farm management. biodiversity and conservation, 12: 1335-1355. takhtajan, a. 2009: flowering plants. second edition. springer. ujvárosi, m. 1973: gymnövények. mezőgazdasági kiado, budapest. znaor, d. 1996: ekološka poljoprivreda. nakladni zavod globus, zagreb, 469pp. http://europa.eu/legislation_summaries/agriculture/environment/l28024_en.htm http://europa.eu/legislation_summaries/agriculture/environment/l28024_en.htm http://orgprints.org/4829 febrianti et al. 2025, biologica nyssana 16(1) 259 16 (1) june 2025: 259-265 doi: 10.46793/biolnyss.16.1.21f the effects of dopamine at physiological concentration on the growth and biofilm formation of staphylococcus aureus, staphylococcus epidermidis, and escherichia coli original article anissa febrianti biology department, institut teknologi sepuluh nopember, surabaya, indonesia endry nugroho prasetyo biology department, institut teknologi sepuluh nopember, surabaya, indonesia enny zulaika biology department, institut teknologi sepuluh nopember, surabaya, indonesia arif luqman biology department, institut teknologi sepuluh nopember, surabaya, indonesia arif.luqman@its.ac.id (corresponding author) received: march 14, 2025 revised: april 06, 2025 accepted: april 14, 2025 abstract: iron is an essential macronutrient for almost all living organisms, including pathogenic bacteria. pathogenic bacteria have developed iron acquisition mechanisms due to the low bioavailability of iron inside the host. one of them is ability to utilize dopamine as a xenosiderophore. in this study we investigated the effects of dopamine at physiological concentrations (0.1–2 μm) on the growth and biofilm formation of bacteria grown in iron-limited and iron-enriched media. the significant growth enhancement was observed in staphylococcus aureus usa300 grown in iron-enriched media. dopamine at the concentration of 1 μm increased biofilm formation in s. aureus usa300, staphylococcus epidermidis o47, and escherichia coli k12 in both iron-limited and iron-enriched media after 3 days of incubation. these findings provide an insight into the effects of dopamine on bacteria at the physiological concentrations. key words: dopamine, siderophore, biofilm, bacteria, microorganisms apstrakt: efekat dopamina u fiziološkoj koncentraciji na rast i formiranje biofilma kod vrsta staphylococcus aureus, staphylococcus epidermidis i escherichia coli gvožđe je osnovni makronutrijent za gotovo sve žive organizme, uključujući i patogene bakterije. zbog niske bioraspoloživosti gvožđa unutar domaćina, patogene bakterije su razvile mehanizme za njegovo usvajanje. jedan od tih mehanizama je sposobnost korišćenja dopamina kao ksenosiderofora. u ovoj studiji ispitivali smo uticaj dopamina u fiziološkim koncentracijama (0,12 μm) na rast i formiranje biofilma kod bakterija gajenih na podlogama sa ograničenim i obogaćenim sadržajem gvožđa. značajno poboljšanje rasta uočeno je kod soja staphylococcus aureus usa300 gajenog u podlozi sa obogaćenim sadržajem gvožđa. dopamin u koncentraciji od 1 μm povećao je formiranje biofilma kod sojeva s. aureus usa300, staphylococcus epidermidis o47 i escherichia coli k12, i u uslovima sa ograničenim i obogaćenim sadržajem gvožđa, nakon tri dana inkubacije. ovi nalazi pružaju uvid u delovanje dopamina na bakterije u fiziološkim koncentracijama. ključne reči: dopamin, siderofor, biofilm, bakterije, mikroorganizmi introduction iron (fe) is an essential element for almost all living organisms, including pathogenic bacteria. iron plays crucial roles in various processes, such as in cellular respiration (blake et al., 1993; levi & rovida, 2009), dna synthesis (puig et al., 2017), as well as in several redox enzymes activities (vigani & murgia, 2018). although iron is abundant in the environment, the bioavailability of iron inside the host is very limited due to the most of the iron is in complex form with proteins, such as hemoglobin, transferrin, and ferritin (krzyminiewski et al., 2011), which makes it difficult for bacteria to access (bullen, 1981). in order to overcome this limitation, bacteria have developed an iron acquisition mechanism through siderophore production by chelating iron from the environment thus making iron accessible by the bacteria through specific transport protein (ellermann & arthur, 2017; kramer et al., 2020). © 2025 febrianti et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 260 staphylococcus aureus, staphylococcus epidermidis, and escherichia coli are commensal bacteria that are naturally present in the human body (escobar-paramo et al., 2004; luqman et al., 2018; luqman et al., 2020). under normal conditions, these three bacteria play an important role in maintaining the balance of the host microbiota. however, when immunity decreases or environmental changes occur, these bacteria can transform into opportunistic pathogens that cause a variety of infections, including skin infections, urinary tract infections, bacteremia, and medical device-associated infections due to biofilms (otto, 2009; dey & ray chaudhuri, 2023). biofilms are bacterial communities embedded in an extracellular matrix, providing protection from the host immune system and antibiotic therapy (otto, 2006; hanke & kielian, 2012; le et al., 2018). iron availability plays a key role in biofilm formation, with iron deficiency inhibiting this process (greenberg & banin, 2008; avidan et al., 2010). to overcome iron deficiency, bacteria use siderophores to obtain iron from the environment. however, in addition to siderophores, several small molecules such as catecholamines, including dopamine, are also known to play a role in increasing iron availability to bacteria (dichtl et al., 2019; perraud et al., 2022). dopamine is an important neurotransmitter found in human body fluids, and its catechol group allows dopamine to interact with iron ions, forming a dopamine-iron complex that can be used as an iron source by bacteria (luqman, 2023). this study aimed to investigate the effect of dopamine on iron binding, growth, and biofilm formation by s. aureus, s. epidermidis, and e. coli in iron-limited and iron-enriched media conditions. we used dopamine concentrations that mimic physiological levels in the human body (0.1–2 μm) to ensure the relevance of the results to the in vivo situation. thus, the results of this study provide new insights into the interaction of dopamine with bacteria as well as its implications for bacterial colonization and biofilm formation in the human body. materials and methods chrome azurol s (cas) assay the cas assay was performed according to previous publications (schwyn & neilands, 1987; murakami et al., 2021). we used cas solution which containing chrome azurol s (0.15 mm) (sigma), hexadecyltrimethylammonium bromide (0.6 mm) (sigma), and fecl3 (1.25 mm) (sigma). the siderophore activity of dopamine was investigated by mixing the cas solution with dopamine at ratio 1:1 and the absorbance was measured using microplate reader at a wavelength of 630 nm. the amount of iron bound was calculated by using a standard curve which was prepared prior the assay by measuring the absorbance of cas solution containing various concentrations of fecl3. bacterial growth curve bacterial strains, staphylococcus aureus usa300, staphylococcus epidermidis o47, and escherichia coli k12, were grown in tryptic soy broth (tsb) (himedia) overnight at 37 ºc with agitation and used as preculture. the precultures were pelleted, washed, and resuspended in sterile phosphate buffer. the resuspended bacterial cells were then inoculated into mkb medium (2.5 g/l k2hpo4 (sigma); 2.5 g/l mgso4·7h2o (sigma); 15 ml/l glycerin (sigma); 5.0 g/l casamino acids (himedia) and the ph was adjusted to 7.2) (gu et al., 2021). the iron limited mkb medium was supplemented with 10 μm of fecl3 (sigma) while the iron enriched mkb medium was supplemented with 50 μm of fecl3. dopamine was added to the media at final concentration of 0.1, 0.2, 1, and 2 μm. the bacterial growth curve measurement was performed in a 48 well plates where we added the bacterial culture at a volume 0.3 ml/well with initial od600 of 0.1. the plates were incubated at 37 ºc with agitation for 5 days. the growth was measured at certain important hours (1, 2, 3, 4, 5, 6, 7, 8, 24, 27, 30, 33, 48, 51, 54, 57, 72, 75, 78, 81, 96, 99, 102, 105, and 120 h) at od600. bacterial biofilm assay bacterial strains grown in tsb overnight at 37 ºc with agitation were used as preculture. the resuspended bacterial cells in sterile phosphate buffer were then re-inoculated into mkb at initial od 0.1 medium supplemented with 10 μm or 50 μm of fecl3. the cultures were grown in 96 well plate with 200 μl culture of each well and incubated at 37 ºc without agitation for 72 h. after 72 h, the biofilm formation was measured according to previous publication (o'toole, 2011). briefly, after incubation, the supernatant was discarded carefully and the biofilm formed in the well was washed using phosphate buffer. crystal violet solution (0.1%) was then added (200 μl) to stain the biofilm and incubate at room temperature for 1 h. the biofilm was then washed carefully to remove the unbound crystal violet. methanol (200 μl) was added to the well to solubilize the bound crystal violet to the biofilm and the absorbance was measured at 550 nm. statistical analysis the obtained growth curve data were analyzed statistically using repeated measures (rm) one-way biologica nyssana ● 16 (1) june 2025: 259-265 febrianti et al. ● the effects of dopamine at physiological concentration on the growth and biofilm formation of staphylococcus aureus, staphylococcus epidermidis, and escherichia coli 261 biologica nyssana ● 16 (1) june 2025: 259-265 febrianti et al. ● the effects of dopamine at physiological concentration on the growth and biofilm formation of staphylococcus aureus, staphylococcus epidermidis, and escherichia coli anova followed by tukey’s multiple comparison test for the grouping. the data obtained from biofilm assays were analyzed using 2-way anova and followed by tukey’s multiple comparison test. results and discussion dopamine at low concentration enhances the growth of s. aureus and s. epidermidis in order to investigate the effects of dopamine on the iron availability and bacterial growth, we measured the siderophore activity of dopamine. the experiments showed that dopamine is able to chelate iron. at the concentration of 0.5 μm, dopamine can release fe3+ from the cas approximately 0.45 mm. however, although we added higher concentrations of dopamine, the released fe3+ from cas was never higher (fig. 1a). we then analyzed the effects of dopamine on bacterial growth by supplementing dopamine into bacterial culture in iron-limited and iron-enriched media and observed the growth regularly for 5 days. in this study we used s. aureus usa300, s. epidermidis o47, and e. coli k12 as bacterial models representing human commensals. growth curve experiments showed that dopamine supplementation significantly enhanced the growth of s. aureus usa300 only in iron-enriched media, but not under iron-limited conditions. (fig. 1b). this tendency was not observed in either s. epidermidis o47 nor e. coli k12 (fig. 1c & 1d). dopamine increases the biofilm formation we also investigated the effect of dopamine supplementation on biofilm formation of these strains. in general, dopamine supplementation enhanced biofilm formation in all three bacterial strains under both iron-limited and iron-enriched conditions after 3 days of incubation. in s. aureus usa300, dopamine increased biofilm formation at a concentration of 1 μm in iron-limited media and 2 μm in iron-enriched media (fig. 2a). staphylococcus epidermidis o47 was more sensitive to dopamine, where in iron-limited media, 0.2 μm significantly increased biofilm formation, while in iron-enriched media, a significant increase was observed at 1 μm (fig. 2b). escherichia coli k12 showed similar tendency, where 1 μm of dopamine enhanced biofilm formation in both iron-limited and iron-enriched media (fig. 2c). many studies reported the capability of dopamine in chelating iron (porter et al., 2010; dichtl et al., 2019). given that dopamine concentrations in the human body range from 10 to 1000 nm (liu & liu, 2021), we investigated the extent of iron binding by dopamine at these physiological levels. our results showed that, even at the physiological concentrations, dopamine showed significant iron fig. 1. dopamine increases the growth of some bacteria due to its siderophore activity. (a) the cas assays showed the capability of dopamine in chelating iron thus releasing iron from the cas with the maximum released iron from the cas was around 0.45 mm. the supplementation of dopamine enhances the growth of the iron enriched mkb medium in s. aureus usa300 (b) but no clear growth enhancement in s. epidermidis o47 (c) and e. coli k12 (d). for all graphs, each data point is the mean value ± sd (n=3). the statistical grouping was obtained by analyzing the growth data using repeated measures (rm) one-way anova followed by the tukey’s multiple comparisons test. dop: dopamine. 262 biologica nyssana ● 16 (1) june 2025: 259-265 febrianti et al. ● the effects of dopamine at physiological concentration on the growth and biofilm formation of staphylococcus aureus, staphylococcus epidermidis, and escherichia coli by s. epidermidis. no clear effect on s. epidermidis growth was observed with varying concentrations of fecl₃ and dopamine in the media, likely due to its efficient ability to overcome severe iron limitation by utilizing multiple mechanisms for iron acquisition (oliveira et al., 2017). a similar trend was observed in e. coli, which also demonstrates redundant iron acquisition systems (garcia et al., 2011; garenaux et al., 2011). biofilm formation is also greatly influenced by iron. the presence of dopamine in the media increased the biofilm formation at the concentration of 1 μm for all tested bacteria, which is still in the range of physiological dopamine level in human body (liu & liu, 2021). this implies that dopamine in human body might increase biofilm formation during bacterial infection. this could, in turn, increase the persistence of the infection (soto et al., 2006; sanchez et al., 2013) due to biofilm properties that help bacteria to resist antibiotic treatment and evade host immune responses (otto, 2006; hanke & kielian, 2012; le et al., 2018). fig. 2. dopamine increases biofilm formation of bacteria. the biofilm formation assays of bacteria grown in iron-limited and enriched mkb medium and incubated for 72 h, showed that dopamine increased biofilm formation in general at 1 μm for all three species: s. aureus usa300 (a), s. epidermidis o47 (b), and e. coli k12 (c). for all graphs, each data point is the mean value ± sd (n=3); *p<0.05; **p<0.01; ***p<0.001; and ****p<0.0001, 2-way anova followed by tukey’s multiple comparisons test were used to analyze the data. chelating capability. however, the iron-binding capacity did not increase proportionally with higher dopamine concentrations, likely due to saturation of available iron, equilibrium between soluble and bound iron, kinetic limitations of iron binding to siderophores, and potential interference from dopamine autooxidation (herlinger et al., 1995; leventhal et al., 2016; schiessl et al., 2017; umek et al., 2018). thus, the exposure of dopamine to human commensals or pathogenic bacteria in the case of infection could pose a significant effect. the physiological concentration of dopamine increased the growth of s. aureus usa300 significantly in iron-enriched media but not in iron-limited media. this is most probably due to the capability of s. aureus to produce its own siderophore, such as staphyloferrin (hammer & skaar, 2011; luqman & ohlsen, 2024), which is sufficient to bind most of the available iron in the media. the presence of dopamine increased the iron availability for s. aureus in the iron-enriched media which in turn caused s. aureus to grow even more. moreover, s. aureus has been reported to be able to utilize xenosiderophore for iron acquisition (endicott et al., 2017). different tendency was shown 263 biologica nyssana ● 16 (1) june 2025: 259-265 febrianti et al. ● the effects of dopamine at physiological concentration on the growth and biofilm formation of staphylococcus aureus, staphylococcus epidermidis, and escherichia coli by the human pathogen staphylococcus aureus. acs infectious diseases, 3(7), 542-553. https://doi. org/10.1021/acsinfecdis.7b00036 escobar-paramo, p., grenet, k., le menac'h, a., rode, l., salgado, e., amorin, c., gouriou, s., picard, b., rahimy, m.c., andremont, a., denamur, e., & ruimy, r. (2004). largescale population structure of human commensal escherichia coli isolates. applied and environmental biology, 70(9), 5698-5700. https://doi.org/10.1128/ aem.70.9.5698-5700.2004 garcia, e.c., brumbaugh, a.r., & mobley, h.l. (2011). redundancy and specificity of escherichia coli iron acquisition systems during urinary tract infection. infection and immunity, 79(3), 1225– 1235. https://doi.org/10.1128/iai.01222-10 garenaux, a., caza, m., & dozois, c.m. (2011). the ins and outs of siderophore-mediated iron uptake by extra-intestinal pathogenic escherichia coli. veterinary microbiology, 153(1–2), 89–98. https://doi.org/10.1016/j.vetmic.2011.05.023 greenberg, e.p., & banin, e. (2008). ironing out the biofilm problem: the role of iron in biofilm formation. in n. balaban (ed.), control of biofilm infections by signal manipulation (pp. 141–156). berlin, heidelberg: springer. https://doi. org/10.1007/7142_2007_014 gu, s., wan, w., shao, z., & zhong, w. (2021). high-throughput method for detecting siderophore production by rhizosphere bacteria. bioprotocol, 11(9), e4001. https://doi.org/10.21769/ bioprotoc.4001 hammer, n.d., & skaar, e.p. (2011). molecular mechanisms of staphylococcus aureus iron acquisition. annual review of microbiology, 65, 129–147. https://doi.org/10.1146/annurevmicro-090110-102851 hanke, m.l., & kielian, t. (2012). deciphering mechanisms of staphylococcal biofilm evasion of host immunity. frontiers in cellular and infection microbiology, 2, 62. https://doi.org/10.3389/ fcimb.2012.00062 herlinger, e., jameson, r.f., & linert, w. (1995). spontaneous autoxidation of dopamine. journal of the chemical society, perkin transactions, 2(2), 259–263. https://doi.org/10.1039/p29950000259 kramer, j., ozkaya, o., & kümmerli, r. 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(2018). iron-requiring enzymes in the spotlight of oxygen. trends in plant science, 23(10), 874-882. https://doi.org/10.1016/j. tplants.2018.07.005 çolak and alan 2025, biologica nyssana 16(1) 111 16 (1) june 2025: 111-119 doi: 10.46793/biolnyss.16.1.4c evaluation of pomological and chemical properties of naturally growing hawthorn (crataegus spp.) genotypes original article ayşen melda çolak usak university, faculty of agriculture, department of horticulture, uşak, turkey aysenmelda.colak@usak.edu.tr (corresponding author) fatma alan ankara university, kalecik vocational school, department of plant and animal production, ankara, turkey received: november 27, 2024 revised: february 24, 2025 accepted: february 26, 2025 abstract: hawthorn (crataegus spp.), a wild fruit whose consumption has increased in recent years, is abundant in turkey’s ecology. this fruit is particularly important due to its aroma, color, partial taste formation, and antimicrobial effects. the aim of this study was to determine some pomological and chemical characteristics of hawthorn fruits collected from ankara province and the districts of melikgazi, hacılar, and sarız in kayseri province during 2022-2023. the pomological characteristics of the selected hawthorn genotypes included fruit weight, fruit width, fruit length, and seed weight, while the chemical characteristics examined were ph, soluble solids content (ssc), titratable acidity (ta), vitamin c, phenolic compounds, flavonoids, antioxidant capacity (measured by dpph), and anthocyanin content. the study found that the maximum fruit weight was 2.80 grams, with a fruit width of 18.80 mm and a fruit length of 16.01 mm. the seed weight was 0.99 grams. regarding chemical characteristics, the ph was 3.91, titratable acidity was 2.5%, soluble solids content (ssc) was 14.3%, and the vitamin c content was 71.03 mg/100 g. the total phenolic content was 773.32 µg gae/100 g, while the total flavonoid content was 23.87 mg ce/100 g. the antioxidant activity was determined to be 74.40%, and the anthocyanin content was found to be 1.11 μg cy-3-glu/g ta. key words: chemical, hawthorn (crataegus spp.), pomological apstrakt: procena pomoloških i hemijskih karakteristika prirodno rastućih genotipova gloga (crataegus spp.) glog (crataegus spp.), divlje voće čija je potrošnja poslednjih godina u porastu, zastupljen je u turskoj. ovo voće je posebno važno zbog svoje arome, boje, delimično formiranog ukusa i antimikrobnog dejstva. cilj ovog istraživanja bio je da se utvrde neke pomološke i hemijske karakteristike plodova gloga prikupljenih iz provincije ankara i okruga melikgazi, hacilar i sarız u provinciji kayseri tokom 2022-2023. pomološke karakteristike odabranih genotipova gloga obuhvatale su masu ploda, širinu ploda, dužinu ploda i masu semena, dok su od hemijskih karakteristika ispitivane ph, sadržaj rastvorljivih čvrstih materija (ssc), titrabilna kiselost (ta), vitamin c, fenolna jedinjenja, flavonoidi, antioksidativni kapacitet (izražen kao dpph) i sadržaj antocijana. studijom je utvrđeno da maksimalna težina ploda iznosi 2,80 grama, širina ploda 18,80 mm, a dužina ploda 16,01 mm. težina semena iznosila je 0,99 grama. što se tiče hemijskih karakteristika, vrednost ph iznosila je 3,91, titraciona kiselost 2,5%, sadržaj rastvorljivih čvrstih materija (ssc) bio je 14,3%, a sadržaj vitamina c 71,03 mg/100 g. ukupan sadržaj fenola bio je 773,32 µg gae/100 g, dok je ukupan sadržaj flavonoida bio 23,87 mg ce/100 g. utvrđeno je da antioksidativna aktivnost iznosi 74,40%, a sadržaj antocijanina 1,11 mg ci-3-glu/g ta. ključne reči: hemijske karakteristike, glog (crataegus spp.), pomološke karakteristike introduction anatolia is an important gene center and natural distribution area for many fruit species. turkey is a country rich in plant genetic resources due to its phytogeographic location (european siberian, irano-turanian and mediterranean phytogeographic regions). with approximately 12,000 plant species, © 2025 çolak and alan. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 112 turkey has a plant diversity equal to the european continental flora alone (kutbay et al., 2014; demir et al., 2017). in turkey, most fruit species are cultivated, and only a few grow naturally. hawthorn (crataegus spp.) is one of these few species that anatolian people have used for many years. in turkey, it grows spontaneously in its natural ecological environment in bushes, river banks, rocky places, and mountainous regions (anonymous, 2022). hawthorn, which has local names such as may thorn, white thorn, quince hawthorn, bird nuts, edran in the popular language, is botanically a plant species belonging to the order rosales, family rosaceae, species crataegus, generally in the form of thorny shrubs or trees, deciduous in winter (davis et al., 1972; kayacık, 1981; seçmen et al., 1989), and its homeland is spread over a wide area such as europe, asia and north america. there are 50 species of hawthorn in the world and more than 20 species in turkey. geographically, crataegus orientalis, crataegus monogyna, crataegus oxyacanth and crataegus aroniaen are known as the most widespread species (davis et al., 1972). in turkey, hawthorn grows intensively in natural ecological environments in the aegean, east anatolian, and mediterranean regions (kıral, 2022). hawthorn (crataegus spp.) fruits are highly nutritious, containing a variety of minerals that promote human health, including potassium (k), calcium (ca), phosphorus (p), iron (fe), and magnesium (mg). in addition to these minerals, hawthorn fruits are also rich in sugars, carbohydrates, and vitamin c (özcan et al., 2005). hawthorn, known for its ability to inhibit low-density lipoprotein (ldl) cholesterol and its antioxidant properties, offers several health benefits due to its richness in phenolic compounds. it plays a particularly significant role in medicine for supporting heart health, including the prevention of hypertension and cardiovascular diseases, and is also recognized for its anti-carcinogenic effects (batu, 2012). hawthorn fruit is consumed as molasses, jam, paste, concentrated juice, soft drinks, sauce, nectar, hawthorn yogurt, turkish delight, and dried fruit. hawthorn powder is also produced and marketed as cakes, biscuits, sweets, and ice cream (batu, 2012). this study was carried out to determine some pomological (fruit weight, fruit width, fruit length, and kernel weight) and chemical (soluble solids content (ssc), ph, titratable acidity (ta), phenolic content, flavonoid content, antioxidant capacity (dpph), anthocyanin and vitamin c) properties of fruits of naturally growing hawthorn genotypes. various genotypes of hawthorn were collected from melikgazi (m), hacılar (h), and sarız (s) districts of kayseri province and ankara province during the years 2022/2023. materials and methods plant material fruit samples were collected from the melikgazi, hacılar, and sarız districts of kayseri and ankara provinces and stored in a refrigerator at 4 °c. the pomological (average fruit weight, fruit width, fruit length, and kernel weight) and chemical (soluble solids content (ssc), ph, titratable acidity (ta), phenolic content, flavonoid content, antioxidant capacity (dpph), anthocyanin and vitamin c) characteristics of selected hawthorn genotypes were analyzed. pomological measurements and chemical analyses for each genotype, 20 fruits were weighed and the fruit samples were weighed on a digital balance with a sensitivity of 0.05 g. pomological measurements and examinations of the sample fruits from the genotypes were conducted by measuring the widest part of the fruit, the length between the top and bottom of the fruit, as well as the width and length of the fruit in millimeters (mm) using a digital caliper. the soluble solids content of the fruit was determined using a mettler-toledo 30 p digital refractometer at 22 °c, and the acidity was determined using the titrimetric method according to cemeroğlu (1992). ten ml of juice was made up to 100 ml with distilled water and titrated with 0.1 n sodium hydroxide (naoh) until the ph was 7.0. the resulting titration acidity was calculated as a percentage of malic acid. the ph values of the hawthorn genotypes were determined by measuring with a digital ph meter (şen & güneş, 1996). for the determination of vitamin c (ascorbic acid) content (mg/100 g), 5 g of fruit flesh was weighed and immediately made up to 50 ml by using 0.4% oxalic acid solution, and the samples were filtered with filter paper and read in a spectrophotometer at a wavelength of 520 nm (kılıç et al., 1991). the spectrophotometric method was used to determine total phenolic content (tpc) (spanos & wrolstad, 1992). 100 µl of the sample was transferred into a tube, to which 900 µl of water, 5 ml of 0.2 n folin-ciocalteau reagent, and 4 ml of saturated sodium carbonate solution (75 g/l) were added. the tubes were then vortexed and kept in the dark for 2 hours. calculation was then made by reading the values at a wavelength of 765 nm in the spectrophotometer and using the graph prepared with gallic acid. the spectrophotometric method described by karadeniz et al. (2005) was used for the colorimetric determination of total flavonoid content (tfc) with aluminum chloride. one ml sample was placed in a 10 ml glass bottle, to which 4 ml of distilled water and 0.3 ml of 5% nano2 were biologica nyssana ● 16 (1) june 2025: 111-119 çolak and alan ● evaluation of pomological and chemical properties of naturally growing hawthorn (crataegus spp.) genotypes 113 acidity (%), ph, phenolic content (µg gae/100 g), flavonoid content (mg ce/100 g), antioxidant capacity (%), anthocyanin content (μg cy-3-glu/g ta) and vitamin c (mg/100 g) are given in tab. 2. as shown in tab. 1, the highest values of fruit weight 2.80 g, fruit width 18.80 mm, fruit length 16.01 mm, and kernel weight 0.99 g were recorded in the s genotype. in a study conducted in 2021, it was found that the fruit weights of the hawthorn genotypes were between 0.89-1.80 g, fruit length was 11.4813.62 mm, fruit width was 10.92-15.68 mm, and the fruit width/length ratio was between 0.95-1.15 mm (i̇kinci et al., 2022). in some studies carried out in different regions of turkey, the mean fruit weight of the investigated genotypes of hawthorn (crataegus spp.) was between 0.29-7.67 g, the mean fruit width was 6.56-28.10 mm, and the mean fruit length was 5.86-24. 23 mm (karadeniz & kalkışım 1996; gazioğlu, 2000; asma & birhanlı, 2003; özcan et al., 2005; balta et al., 2006; yanar et al., 2011; balta et al., 2015; gürsoy, 2016; yavıç et al., 2016; bektaş et al., 2017; okatan et al., 2017; bağran, 2018; gürlen, 2018; keles, 2018). caliskan et al. (2018) determined the average fruit weight of yellow hawthorn genotypes as 15.03 g, fruit width as 32.03 mm and fruit length as 26.88 mm. okatan et al. (2017) found that the fruit width and length of the genotypes were between 12.53-19.94 and 10.4817.43 mm, respectively. özgen & sorgun (2010) found the mean fruit width between 14.28-20.87 mm and mean fruit length between 14.38-17.43 mm among the genotypes. in a study conducted in the van region, türkoğlu et al. (2005) reported that the average fruit height was between 12.45-12.89 mm, and the average fruit width was between 13.44-14.48 mm among the genotypes. okatan et al. (2017) found the average fruit weight to be between 0.96 and 4.03 g in genotypes in their research. balta et al. (2015) reported the average fruit weight between 1.54-4.72 g, and karadeniz and kalkamış (1996) reported it between 0.81-2.14 g in genotypes they investigated in their research. okatan et al. (2017) found that the mean kernel weight of the genotypes they examined was between 0.230.98 g, yavıç et al. (2016) 0.39-0.86 g, and özgen & sorgun (2010) found that the mean kernel weight among the genotypes was between 0.310.83 g for each fruit. öztürk et al. (2023) determined the highest fruit weight as 5.75 ± 0.14 g and the lowest fruit weight as 2.12 ± 0.10 g from some pomological characteristics of hawthorn fruits. ercişli et al. (2015) reported that the fruit weight of biologica nyssana ● 16 (1) june 2025: 111-119 çolak and alan ● evaluation of pomological and chemical properties of naturally growing hawthorn (crataegus spp.) genotypes added and mixed. after 5 min, 0.6 ml of 10% alcl3 x 6 h2o and 2 ml of 1 mol l-1 naoh were added and the total volume was made up to 10 ml with distilled water. after homogenization, readings were taken at a wavelength of 510 nm in a spectrophotometer, and calculations were made using the graph prepared with catechin. the antioxidant activity (aa) of the extracts was measured using 2,2-diphenyl-1picrylhydrazyl (dpph) according to the method modified by lafka et al. (2007). to 0.1 ml of the sample solution prepared at different concentrations with methanol, 3.9 ml of dpph solution prepared in methanol (25 mg l-1) was added, stirred for 30 seconds with a vortex, and kept for 30 min in a dark room at room temperature. the absorbance of the samples was measured against methanol at 515 nm using a uv spectrophotometer. the % inhibition values obtained were plotted, and the effective concentration (ec50) and the % effect that reduced the effect of dpph by 50% were calculated for each sample. the total amount of anthocyanins in the fruits was determined by the ph difference method (giusti & wrolstad, 2005). the extracts were measured at 531 and 700 nm wavelength by preparing solutions at ph 1.0 and 4.5. total anthocyanin content (molar extinction coefficient of 28000 cyanidin-3-glycoside) was calculated as absorbances [(a531-a700) ph 1.0(a531-a700) ph 4.5] μg anthocyanin/g dry matter. statistical analyses all analyses were repeated 3 times, and spss 16.0 statistical analysis package program was used to evaluate the data obtained after the experiment. differences between groups were determined using the duncan multiple comparison test at a significance level of p<0.05 (spss, 2007). results and discussion results of fruit weights (g), fruit width and length (mm) and kernel weights (g) of the genotypes are given in tab. 1; soluble solids content (%), titratable table 1. some pomological characteristics of hawthorn genotypes genotype fruit weight (g) fruit width (mm) fruit length (mm) kernel weight (g) m 2.31b 17.02b 15.08b 0.87bc h 2.11b 17.75b 15.46b 0.85c s 2.80a 18.80a 16.01a 0.99a a 1.92c 16.84c 13.56c 0.90b * differences between means marked with different letters in the same column are significant at the p≤0.05 level 114 biologica nyssana ● 16 (1) june 2025: 111-119 çolak and alan ● evaluation of pomological and chemical properties of naturally growing hawthorn (crataegus spp.) genotypes hawthorn species varied from 0.76 to 4.27 g. gürlen et al. (2020) found that the fruit length and width of hawthorn genotypes grown in turkey varied from 8.43 ± 0.12 to 17.58 ± 0.23 mm and from 6.56 ± 0.93 to 20.71 ± 1.22 mm, respectively. according to five-year average data, caliskan et al. (2018) determined the average fruit weight to be 15.03 g, fruit width to be 32.03 mm, and fruit length to be 26.88 mm in yellow hawthorn genotypes. they also reported that there were 2.4 seeds in each fruit and the average weight of these seeds was 0.7 g. karadeniz & kalkışım (1996) reported that the fruit weights of hawthorn varieties naturally distributed in the edremit and gevaş districts of van province were between 0.81-2.14 g, and the seed weights were between 0.17-0.55 g. guo & jiao (1995) reported that the fruit weights of hawthorn varieties grown in china varied between 10-16 g. gündoğdu et al. (2014) found fruit weight between 0.58-3.48 g, seed number between 1.0-5.0, seed weight between 0.130.68 g, and water-soluble dry matter (brix) content between 2.35-20.0% in hawthorn species selected from erzincan province. in other studies, balta et al. (2015) found fruit weight between 1.54-4.72 g and seed weight between 0.32-0.90 g, ercişli et al. (2015) found fruit weight between 0.76-4.27 g, and özderin and fakir (2015) found average fruit weight between 0.45-3.88 g. the values obtained in our study and those of other studies were found to be close to each other. the water-soluble solids (ssc) value of the hawthorn genotypes was 14.3%, the titratable acid (ta) ratio was 2.5%, the ph value was 3.91, the vitamin c content was 71.03 mg/100 g, the tpc 773.32 µg gae/100 g, the tfc 23.87 mg ce/100 g, the anthocyanin 1.11 μg cy-3-glu/g ta, and the highest values were obtained in the s genotype (tab. 2). rüzgar & yazıcı (2022), as a result of their measurements on the production of hawthorn puree suitable for industry from hawthorn fruit, measured the soluble solids content amounts of hawthorn fruit samples obtained from erzincan and sivas su city district as 8.47±0.02 and 6.79±0.02obx, ph values of 3.45±0.03 and 3.44±0.01, total acidity (malic acid equivalent) of 0.690±0.03 and 0.345±0.02 g/100 g, whereas for puree they found soluble solids content values of 7.37±0.01obx, ph values of 3.66±0.01 and total acidity (malic acid equivalent) of 0.517±0.01 g/100 g. in another study, soluble solids content of hawthorn samples was reported to be in the range of 12.6-17.7obx, ph and total acidity (malic acid equivalent) values were 3.6-4.0 and 0.9-1.7 g/100 g, respectively (güzel, 2021). in tunisia, bahrisahloul et al. (2009) conducted a study with 14 hawthorn genotypes collected from three locations. they determined the soluble solids content of hawthorn samples in the range of 16.3-21.8obx, ph in the range of 3.2-4.2, and acidity (malic acid equivalent) in the range of 0.9-1.9. caliskan et al. (2018) reported ph values between 3.0-3.6 and titratable acid content (in terms of malic acid) between 1.3-1.6 g/100 g; gündoğdu et al. (2014) reported these values between 4.22-5.99 and 0.222.40% and soluble solids content between 2.3520.0%, respectively. öztürk et al. (2023) reported that the highest soluble solids content of hawthorn samples in their study was 16.99°bx. in some studies conducted in different regions of turkey, yanar et al. (2011) determined the total soluble solids content and ph of hawthorn genotypes to be 11.66% and 24.00% and 3.12 and 4.09, respectively. alireza et al. (2020) found ph 3.03-4.35, titration acidity 0.75-1.17%, and soluble solids content 14.99-23.43% for 15 species of the genus crataegus. radi et al. (2023) found ph values of 5.12±0.00 and 4.51±0.00 and titration acidity in terms of citric acid of 0.202±0.00 and 0.1±0.02, respectively. in other studies conducted in previous years, karadeniz and kalkışım (1996) reported that the soluble solids content of hawthorn cultivars grown in the edremit and gevaş districts of van province ranged between genotype ssc (%) ta (%) ph (%) vitamin-c (mg/100 g) total phenolic content (µg gae/100 g) total flavonoid content (mg ce/100 g) total antioxidant capacity (%) total anthocyanin (μg cy-3-glu/g ta) m 12.5c 2.4ab 3.89ab 65.03c 705.06c 18.78c 70.75b 0.73d h 13.2b 2.3b 3.84b 67.84b 742.191b 20.05b 70.9b 0.93b s 14.3a 2.5a 3.91a 71.03a 773.32a 23.87a 67.76c 1.11a a 10.3d 2.4ab 3.82b 62.59d 670.43d 13.69d 74.40a 0.89c table 2. some chemical properties of hawthorn genotypes * differences between means marked with different letters in the same column are significant at the p≤0.05 level 115 biologica nyssana ● 16 (1) june 2025: 111-119 çolak and alan ● evaluation of pomological and chemical properties of naturally growing hawthorn (crataegus spp.) genotypes 12.20 and 27.20%. guo & jiao (1995) reported that hawthorn cultivars grown in china had dark red bark and their soluble solids content ranged from 4.9% to 13.8%. similarly, ercişli et al. (2015) found that the soluble solids content of hawthorn cultivars ranged from 6.71% to 14.85%. murathan et al. (2022), after comparing the bioactive constituents, antioxidant and mutagenic properties of different crataegus species growing in elazig, found that the bioactive constituent content and antioxidant activity of c. sinaica species were high and determined that the tpc in the samples was between 670.43 and 773.32 mg/100 g, the tfc was between 345.52 and 452.46 mg/100 g, and the total ascorbic acid content was between 17.7 and 60.3 mg/100 g. this value was reported by ercişli et al. (2015) as 660 to 3460 mg/100 g wet weight, by çalışkan et al. (2012) as 26.6 to 57.1 mg/g dry weight, and by mraihi et al. (2013) as 60.89 to 122.26 mg/100 g dry weight in different hawthorn species. güzel (2021) determined the total flavonoid content of hawthorn samples from çorum province in the 78.2 to 272.6 mg/100 g range. ebrahizadeh and bahramian (2009) reported total flavonoid content between 10.56 and 23.98 mg/g, and edwards et al. (2012) between 4.46 and 147.3 mg/g. previous studies on the antioxidant capacity of hawthorn samples showed that keser et al. (2012) measured 50.76% in the aqueous extract and 52.50% in the ethanolic extract. additionally, the dpph radical scavenging activity was 27.63% in the aqueous extract and 33.24% in the ethanolic extract. okatan et al. (2017) reported that the dpph radical scavenging activity of hawthorn samples grown in uşak province ranged from 19.24% to 59.24%. caliskan et al. (2012) found that the dpph radical scavenging rate of hawthorn genotypes grown in the eastern mediterranean region ranged between 21.433.2%. keser (2012) found that the dpph radical scavenging activity of c. monogyna fruit ethanol extract was 88.22%, 87.22% in water extract; abts radical scavenging activity was 52.50% in ethanol extract and 50.76% in water extract. kiselova et al. (2006) observed that the abts radical scavenging capacity of water extracts of c. monogyna fruits was 50.76%. çınar et al. (2020), in their study to determine the amount of phenolic/flavonoid substances, antioxidant and antimicrobial activity values in crataegus spp. taxa from the western mediterranean region, found the highest antioxidant activity value to be 20% in fruit samples. in a study on crataegus oxyacantha species, which has an important medicinal use in europe, the antioxidant activity (dpph) was determined to be 78.9%, and the total phenolic content was determined to be 244.3 mg g-1 (mraihi et al., 2013). çoklar & akbulut (2016) found the total phenolic content of 1245.3 mg gae 100 g-1 (12.45 mg gae g-1), 12.89-16.54 mg gae g-1 and 2.56-63.02 mg 100 g-1 in konyabeyşehir region, respectively. demir (2021), in his study on the determination of antioxidant contents of some crataegus taxa naturally distributed in afyonkarahisar, found the gallic acid value to be 30.32 mg g-1, 1,2-dihydroxybenzene value to be 178.63 mg g-1 and catechin amount to be 163.08 mg g-1. the antioxidant activity ranged from 0.255 mg g-1 to 0.082 mg g-1, and the tpc ranged from 0.75 mg g-1 to 0.621 mg g-1. in addition, the highest phenolic compound detected was 3,4-dihydroxybenzoic acid and the lowest was the naringenin compound. bahorun et al. (2003), in a study on hawthorn genotypes, determined the amount of total phenolics in the samples as 47.40 mg/g and total anthocyanin as 2.38-6.12 µg siy3-glk/g, while kostić et al. (2012) determined the amount of total phenolics as 2.12-30.63 mg gae g-1 and total anthocyanin as 0.3207-3.168 mg. öztürk et al. (2023) determined the total phenolic content in their samples to be 13.81±0.93, 2.86±0.24 and 13.43±0.89 mg gae/g dw, respectively. according to the different results obtained in the studies investigating the antioxidant activity and total phenolic content of fruits of different hawthorn species, čopra-janićijević et al. (2018) reported that the total phenolic content in the extract for c. microphylla fruits varied between 2.47 and 8.63 mg gae/g dw, çoklar et al. (2018) reported that the total phenolic content in the extract for c. orientalis species was 13.36 mg gae/g dw, and tahirović and bašić (2014) determined that the total phenolic content of the fruit varied between 2.02 and 4.60 mg gae/g dw in their extraction for c. monogyna l. using different proportions of water and different solvents. radi et al. (2023) investigated the amount of polyphenols and flavonoids present in the aqueous extracts of c. monogyna and c. laciniata fruits. they reported that the aqueous extract of c. monogyna fruits contained the most abundant phenolic compounds, 1.04 ± 2.4 eag/g, while the aqueous extract of c. laciniata fruits contained only 0.9 5 ± 0.21 eag/mg extract. total flavonoids were 0.19 ± 0.02 eq/g and 0.13 ± 0.04 eq/g for c. laciniata and c. monogyna, respectively. they also determined the antioxidant values of aqueous extracts of c. monogyna and c. laciniata as 0.34 ± 0.03 mg/ml and 0.47 ± 0.04 mg/ml and the ascorbic acid values as 0.13 ± 0.03 mg/ml and 0.05 ± 0.03 mg/ml, respectively. çoklar and akbulut (2016) conducted a study to determine the changes in antioxidant activity, total phenolic content, and phenolic profile of naturally grown hawthorn 116 references abacı, z.t., sevindik, e., & selvi, s. (2014). ardahan’da yetişen bazı erik (prunus domestica l.) genotiplerinde toplam fenolik içerik, toplam antosiyanin ve askorbik asit içeriğinin belirlenmesi. journal of tekirdağ agricultural faculty, 11, 3. alirezalu, a., ahmadi, n., salehi, p., sonboli̇, a., alirezalu, k., khaneghah, a.m., barba, f.j., munekata, pes., & lorenzo, j.m. (2020). physicochemical characterization, antioxidant activity, and phenolic compounds of hawthorn (crataegus spp.) fruits species for potential use in food applications. foods, 9: 436-451. anonymous. (2022). alıç. erişim linki: https:// yemek.com/alic-nedir-faydalari/ (erişim tarihi:05.11.2022) asma, b., & birhanli, o. (2003). malatya ve çevresinde doğal olarak yetişen alıçlarda seleksiyon çalışmaları. türkiye iv. ulusal bahçe bitkileri kongresi. akdeniz üniversitesi, ziraat fakültesi, bahçe bitkileri bölümü, antalya, s: 61–62. bağran, c. (2018). orta kelkit vadisinde doğal olarak yetişen alıç genotiplerinin (crataegus spp.) seleksiyon yolu ile ıslahı. yüksek lisans tezi, abant i̇zzet baysal üniversitesi, fen bilimleri enstitüsü, bahçe bitkileri anabilim dalı, bolu, 46 s. bahri-sahloul, r., ammar, s., grec, s., harzallah, & skhiri f. (2009). chemical characterisation of crataegus azarolus l. fruit from 14 genotypes found in tunisia. journal of horticultural science biotechnology, 84(1), 23–28. doi:10.1080/ 14620316.2009.1151247. bahorun, t., aumjaud, e., ramphul, h., rycha, m., luximon-ramma, a., trotin, f., & aruoma, oi. (2003). phenolic constituents and antioxidant capacities of crataegus monogyna (hawthorn) callus extracts. die nahrung, 47(3), 191–198. balta, m.f., çelik, f., türkoğlu, n., özrenk, k., & özgökçe, f. (2006). some fruit traits of hawthorn (crataegus sp.) genetic resources from malatya, turkey. research journal of agriculture and biological sciences, 2(6), 531-536. balta, m.f., karakaya, o., & kaptan ekici, g. (2015). çorum’da yetişen alıçların (crataegus spp.) fiziksel özellikleri. ordu üniversitesi bilim teknik dergisi, 5(2), 35-41. batu, a. (2012). alıç meyvesinin fonksiyonel gıda olarak değerlendirilmesi ve i̇nsan sağlığı bakımından önemi. türk bilimsel derlemeler dergisi, 5(2), 01-05. (crataegus orientalis) fruits in konya-beyşehir. as a result, they determined the total phenolic content as 818.3, 974.2, and 1957.4 mg gae 100 g-1 and the antioxidant activity as 52.85±1.81, 31.74±1.09 and 22.75±2.26 mmol trolox equivalent dry weight, respectively, according to the 2,2-diphenyl-1picrylhydrazyl (dpph) antioxidant activity method. according to the results of vitamin c content in previous years, gürsoy (2016) reported 17.91-35.17 mg/100 g, koşar (2017) reported 17.00 mg 100 g-1 to 64.00 mg 100 g-1, keles (2018) reported 19.5767.19 mg/100 g, yanar (2021) reported 19.1 mg/100 g-0.9 mg/100 g and guo & jiao (1995) reported that vitamin c content of hawthorn cultivars grown in china varied between 52-74 mg/100 g in fresh fruit flesh. conclusion there are many hawthorn populations in different regions of turkey. each region has hawthorn genotypes with their own characteristics. these areas should be studied and hawthorn genotypes with superior characteristics should be protected. in order to popularise the consumption of hawthorn fruit, it is very important to produce products suitable for the industry. in this way, products with high added value will be developed, and the attention of the producers will be attracted. the nutritional value of hawthorn is impressive, and its vinegar, in particular, has become a highly valuable product. hawthorn fruit is very convenient for fresh consumption due to its rich content of bioactive components. for this reason, hawthorn, which is consumed in the form of various products, especially vinegar, is also valued as a nutrient with its rich content of many minerals and vitamins. due to the phytochemical content of hawthorn and its ease of industrial processing, it is necessary to work with genotypes with the highest phytochemical content in future studies, which have been preserved by appropriate methods and whose content characteristics can be preserved. in this context, the study to determine some pomological and chemical properties of hawthorn genotypes grown in kayseri and ankara ecology was carried out in 2022-2023. soluble solids content, ph, titratable acidity, total phenolics, total flavonoids, vitamin c, antioxidants, and anthocyanins analyses were carried out on the samples of all genotypes, and the highest values were obtained from genotype s. the genotype that was found to be promising in our research can be valuable as gene source material for future breeding studies. biologica nyssana ● 16 (1) june 2025: 111-119 çolak and alan ● evaluation of pomological and chemical properties of naturally growing hawthorn (crataegus spp.) genotypes 117 bektaş, m., bükücü, ş.b., özcan, a., & sütyemez, m. 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(2012). bazı alıç (crataegus spp.) genotiplerinin pomolojik, kimyasal ve moleküler karakterizasyonu. çukurova üniversitesi, fen bilimleri enstitüsü, biyoteknoloji anabilim dalı, 161s. adana. yavıç, a., taylan, a., balcı, h., & encu, t. (2016). biochemical and pomological characteristics of hawthorn (crataegus spp.) fruits grown in şemdinli, hakkâri. yüzüncü yıl üniversitesi tarım bilimleri dergisi, 26(4), 500-504. tusevski et al., 2019, biologica nyssana 10(2) 10 (2) december 2019: 159-168 doi: 10.5281/zenodo.3600195 antioxidant activity and phenolic compounds in hypericum perforatum l. wildgrowing plants collected in the republic of macedonia original article oliver tusevski department of plant physiology, institute of biology, faculty of natural sciences and mathematics, university „ss. cyril and methodius“, arhimedova str. 3, 1000 skopje, macedonia oliver.tusevski@pmf.ukim.mk (corresponding author) marija todorovska department of plant physiology, institute of biology, faculty of natural sciences and mathematics, university „ss. cyril and methodius“, arhimedova str. 3, 1000 skopje, macedonia todorovskabt96@gmail.com mirko spasenoski department of plant physiology, institute of biology, faculty of natural sciences and mathematics, university „ss. cyril and methodius“, arhimedova str. 3, 1000 skopje, macedonia mirkoms@pmf.ukim.mk sonja gadžovska simić department of plant physiology, institute of biology, faculty of natural sciences and mathematics, university „ss. cyril and methodius“, arhimedova str. 3, 1000 skopje, macedonia sonjag@pmf.ukim.mk received: september 29, 2019 revised: december 11, 2019 accepted: december 17, 2019 abstract: the aim of this study was to evaluate phenolic compounds composition and antioxidant activity in roots (ro), non-flowering shoots (nfs) and flowering shoots (fs) of hypericum perforatum l. wild-growing plants collected in the republic of macedonia. the analyses of total phenolic compounds included quantification of phenolics, flavonoids, catechin derivatives, flavan-3-ols, condensed tannins and hypericin. antioxidant activity was determined by the cupric ions reducing antioxidant capacity, phosphomolybdenum test, reducing power and dpph scavenging. the contents of phenolics in fs and nfs were significantly higher than those found in ro extracts. hypericin content in fs was 2-fold increased in comparison to nfs, while ro showed low capability for the accumulation of naphthodianthrones. also, fs and nfs exhibited markedly higher antioxidant activities compared to ro. in summary, aerial parts of h. perforatum accumulated significant levels of antioxidant phenolic compounds that were characterized with hydrogen atom donation, radical scavenging and participation in redox reactions. key words: antioxidant activity, flowering shoots, hypericum perforatum l., non-flowering shoots, phenolic compounds, roots apstract: antioksidativna aktivnost i fenolna jedinjenja u divljerastućim biljkama hypericum perforatum l. sakupljenim u republici makedoniji cilj ove studije bio je da utvrdi sastav fenolnih jedinjenja i antioksidativnu aktivnost korena (ro), necvetajućih ((nfs) i cvetajućih izdanaka (fs) divljerastućih biljaka hypericum perforatum l., sakupljenih u republici makedoniji. analize totalnih fenolnih jedinjenja uključivale su kvantifikaciju fenola, flavonoida, derivata katehina, flavan-3-ola, kondenzovanih tanina i hipericina. antioksidativna aktivnost određivana je kapacitetom redukcije bakarnih jona, fosfomolibdenskim testom, redukcionim potencijalom i dpph testom. sadržaj fenola u fs i nfs bili su značajno viši nego onaj nađen u ekstraktima ro. sadržaj hipericina u fs bio je dvostruko veći u poređenju sa nfs, dok je ro pokazao nisku sposobnost akumulacije naftodiantrona. takođe, fs i fns su ispoljili značajno veću antioksidativnu aktivnost u poređenju sa ro. u zaključku, nadzemni delovi h. perforatum akumulirali su značajni nivo antioksidantnih fenolnih jedinjenja okarakterisanih dinacijom vodonikovih atoma, hvatanjem slobodnih radikala i učešćem u redoks reakcijama. ključne reči: antioksidativno delovanje, cvetajući izdanci, hipericum perforatum l., necvetajući izdanci, fenolna jedinjenja, korenovi introduction hypericum perforatum (st john’s wort) is an important medicinal plant with long and worldwide usage in traditional medicine. the crude drug (hyperici herba) consists of dried flowering tops or aerial parts of the wild-growing plants. the phytochemistry and pharmacology of this species have been extensively studied (greeson et al., 2001; nahrstedt & butterweck, 2010). phenolic compounds (naphthodianthrones, acyl-phloroglucinols and flavonoids) that contributed to the biological activities of h. perforatum are usually accumulated in the leaves and flowers. naphthodianthrones are chemotaxonomic marker compounds of the genus hypericum that is © 2019 tusevski et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 159 13th symposium on the flora of southeastern serbia and neighboring regions mainly localized in the dark glands dispersed over the margins of leaves and flower petals (zobayed et al., 2006). the naphthodianthrones presented by hypericin and pseudohypericin, as well as their protoforms (protohypericin and protopseudohypericin) are included in the analytical term “total hypericins” (williams et al., 2006). the cytotoxic and photodynamic properties of hypericin are responsible for antiviral, antitumor and antibacterial effects of h. perforatum extracts (delaey et al., 2001). two closely related acyl-phloroglucinol derivatives, hyperforin and its homolog adhyperforin have been detected in the translucent glands of the leaves, flowers and fruits of h. perforatum (soelberg et al., 2007). it has been considered that hyperofrin is the major constituent responsible for the antidepressant activity of h. perforatum extracts through the inhibition of synaptic re-uptake of neurotransmitters (chatterjee et al., 1998). the h. perforatum is widely known for its richness in flavonoids especially by the flavonols quercetin and kaempferol, as well by the catechin derivatives represented with monomeric flavan-3ols and oligomeric proanthocyanidins (nahrstedt & butterweck, 2010). flavonoids are mainly presented in the leaves as water-soluble glycosides in the vacuoles of epidermal cells due to their uv-protective function (germ et al., 2010). the flavonoid-containing fractions of h. perforatum have been shown to exhibit powerful antioxidant activity and radical scavenging capacity (barnes et al., 2001). even that flowers from h. perforatum wildgrowing plants have been the subject of many research studies concerning the production of total phenolics, flavonoids and hypericins (germ et al., 2010; altun et al., 2013; becker et al., 2016), the biosynthesis of various groups of phenolic compounds in different plant parts are not yet fully understood. recent phytochemical investigations revealed that roots from h. perforatum wild-growing plants represented a promising source of phenolics with potential biological activities (crockett et al., 2011; tusevski et al., 2018). however, the roots of h. perforatum wild-growing plants have never been evaluated for their capacity for the production of total phenolic compounds and antioxidant activities. therefore, it would be of great interest to evaluate the distribution of certain groups of phenolic compounds and antioxidant properties in various plant organs of h. perforatum. the main objective of this study was to determine the contents of six groups of phenolic compounds and antioxidant capacity in h. perforatum roots (ro), non-flowering shoots (nfs) and flowering shoots (fs). for the realization of this objective, the following topics were included: (1) quantification of total phenolics, flavonoids, catechin derivatives, flavan-3-ols, condensed tannins and hypericin, (2) evaluation of antioxidant activity by cupric ion reducing antioxidant capacity, reducing power, phosphomolybdenum assay and dpph radical scavenging and (3) determination of relationship between phenolic compounds contents and antioxidant activities. material and methods phenolic compound contents wild-growing plants of hypericum perforatum (voucher number 060231) collected in the national park pelister (1394 m a.s.l.) were separated into three plant sections: roots (ro), non-flowering shoots (nfs) and flowering shoots (fs). phenolic compounds were extracted from powdered plant material (0.2 g) with 80% (v/v) ch3oh in ultrasonic bath for 30 min at 4 ºc. thereafter, methanolic extracts were centrifuged at 12.000 rpm for 15 min, and the supernatants were used for the determination of phenolic compound contents. the phenolic compound contents in plant extracts included determination of total phenolics (tp), flavonoids (tf), catechin derivatives (tcd), flavan3-ols (tfa), condensed tannins (tct) and hypericin (th). spectrophotometric analyses were performed on spectramax 190 microplate reader (molecular devices corp., sunnyvale, ca) supported with softmax pro (v. 5.4.1) software. total phenolics (tp). the tp contents were determined when plant extracts were mixed with folin–ciocalteau reagent and 0.7 m na2co3 (singleton & rossi, 1965). the samples were incubated at 50 ºc for 15 min and then cooled at room temperature. then, absorbance was measured spectrophotometrically at 765 nm. the results for tp concentration were expressed as milligrams of gallic acid equivalents (ga) per gram of dry weight (mg ga·g-1 dw). total flavonoids (tf). the tf contents in plant extracts were determined using the assay described by zhishen et al. (1999). an aliquot of the extract was mixed with 5% nano2 and allowed to react for 5 min. following this, 10% alcl3 was added and the mixture stood for further 5 min. then, 1 m naoh and distilled water were added to the sample and the absorbance was measured spectrophotometrically at 510 nm. the concentration of tf was expressed as milligrams of catechin equivalents (c) per gram of dry weight (mg c·g-1 dw). total catechin derivatives (tcd). the tcd contents in plant extracts were determined by the vanillin method (sun et al., 1998). a diluted plant extract was mixed with 1% vanillin-hcl solution. the samples were incubated at room temperature 160 biologica nyssana ● 10 (2) december 2019: 159-168 tusevski et al. ● antioxidant activity and phenolic compounds in hypericum perforatum l. wild-growing plants collected in the republic of macedonia) room temperature and the absorbance was recorded at 450 nm. the molar extinction coefficient of trolox (ε535=1.67x104 l·mol-1·cm-1) was used for the determination of cuprac. the cuprac values were expressed as micromoles of trolox equivalents (t) per gram of dry weight (μmol t·g-1 dw). reducing power (rp). the rp of plant extracts was determined according to the method of oyaizu (1986), with the following modifications. the reaction mixture contained plant extract, phosphate buffer (0.2 m kh2po4/k2hpo4, ph 6.6) and 1% k3[fe(cn)6]. after incubation at 50 ºc for 20 min, an aliquot of 10% tca was added to the mixture followed by centrifugation at 1000 rpm for 10 min. finally, the collected supernatant was mixed with ch3oh and 0.1% fecl3 and incubated at room temperature for 10 min. the absorbance of the samples was measured at 700 nm. the rp was expressed as micromoles of ascorbic acid equivalents (aa) per gram of dry weight (μm aa·g-1 dw). phosphomolybdenum (pm) assay. the pm assay was evaluated using the procedure of prieto et al. (1999). the plant extract was mixed with pm reagent [0.6 m h2so4, 28 mm na3po4 and 4 mm (nh4)2moo4] and the samples were incubated at 95°c for 90 min. after cooling at room temperature, the absorbance of the green pm complex was measured at 695 nm. the pm capacity was expressed as milligrams of ascorbic acid equivalents (aa) per gram of dry weight (mg aa·g-1 dw). dpph radical scavenging activity. the ability of plant extracts to scavenge stable free radical 2,2-diphenyl-1-picrylhydrazyl (dpph•) was determined with the method of brand-williams et al. (1995). the reaction mixture consisted of plant extract and 0.25 mm dpph in ch3oh. in the control sample, the extract was replaced with ch3oh. the reaction for dpph• scavenging was carried out at room temperature in the dark for 10 min, and then decrease in absorbance was recorded at 518 nm. the dpph radical scavenging activity was expressed as micromoles of trolox equivalents (t) per gram of dry weight (μm t·g-1 dw). results phenolic compound contents and antioxidant activities in h. perforatum wild-growing plants the contents of total phenolic compounds in h. perforatum extracts (ro, nfs and fs) are summarized in tab. 1. the tp contents in fs and nfs were about 2.9-fold higher than that of ro. the amounts of tf were significantly higher in nfs and fs (2.7and 2.4-fold, respectively), compared to ro extracts. also, fs and nfs showed higher tcd contents (2.7and 2.3-fold, respectively) in comparison to ro sample. similar amounts of for 30 min and the absorbance was measured spectrophotometrically at 500 nm. the concentration of tcd was expressed as milligrams of catechin equivalents (c) per gram of dry weight (mg c·g-1 dw). total flavan-3-ols (tfa). the tfa in plant extracts were estimated by dmaca (4-dimethylaminocinnamaldehyde) assay (arnous et al., 2002). the 0.1% dmaca reagent in ch3oh/ hcl (91.4/8.6, v/v) was added to the extracts. samples were incubated for 10 min at room temperature and the absorbance was measured at 640 nm. the tfa concentration was expressed as milligrams of catechin equivalents (c) per gram of dry weight (mg c·g-1 dw). total condensed tannins (tct). the tct contents in plant extracts were determined by the method of porter et al. (1985) with the following modifications. the samples were prepared by mixing the diluted plant extracts with n-butanol/hcl (95:5, v/v) and 2% nh4fe(so4)2 x 12h2o. the mixture was incubated at 95°c for 50 min and the absorbance was measured at 535 nm. the molar extinction coefficient of cyanidin-3-glucoside (ε535=34700 l·mol-1·cm-1) was used for determination of tct in the extracts. the results were expressed as milligrams of cyanidin-3glucoside equivalents (cg) per gram of dry weight (mg cg·g-1 dw). total hypericin (th). the th contents in plant samples were determined according to european pharmacopoeia (2008). the powdered plant sample was extracted with 80% tetrahydrofuran at 65 ºc for 30 min. samples were centrifuged at 3000 rpm for 10 min and the collected supernatant was lyophilized under vacuum (0.22 mbar). the dry residue was dissolved with ch3oh in an ultrasonic bath and then centrifuged at 12.000 rpm for 10 min. the absorbance of the supernatant was measured at 590 nm. the concentration of th was expressed as milligrams of hypericin equivalents (h) per gram of dry weight (mg h·g-1 dw). antioxidant capacity assays the antioxidant capacity of plant extracts was determined by the following methods: cupric ion reducing antioxidant capacity (cuprac), reducing power (rp), phosphomolybdenum assay (pm) and dpph radical scavenging activity. those methods were performed in the same extracts used for the determination of phenolic compound contents. cupric ion reducing antioxidant capacity (cuprac). the cuprac assay of plant extracts was determined by the method of apak et al. (2004). the reaction mixture consisted of plant extract, 10 mm cucl2, 7.5 mm neocuproine and 1 m ch3coonh4 buffer (ph 7.0). samples were incubated 30 min at 161 biologica nyssana ● 10 (2) december 2019: 159-168 tusevski et al. ● antioxidant activity and phenolic compounds in hypericum perforatum l. wild-growing plants collected in the republic of macedonia) tfa and tct were observed between nfs and fs, (about 2.5-fold higher) compared to ro extracts. the th concentration in fs was about 2-fold higher than that found in nfs extracts, while ro showed significantly lower capability for the accumulation of hypericin derivatives. the antioxidant activities in h. perforatum ro, nfs and fs extracts are presented in tab. 2. the results for cuprac assay showed that fs and nfs extracts had significantly higher antioxidant activities (3-fold) compared to ro. similar results were observed for rp, where nfs and fs had markedly higher values for antioxidant activity (4.4and 3.8-fold, respectively) compared to ro extracts. the antioxidant activity measured by pm assay was significantly higher in nfs and fs (2.2 and 1.9-fold, respectively) than ro extracts. also, fs and nfs showed markedly higher dpph radical scavenging activity (3.1and 2.6-fold, respectively) in comparison to ro extracts. principal component analysis of phenolic compounds production and antioxidant activities in h. perforatum wild-growing plants principal component analysis (pca) was performed to define the correlation between total 162 phenolic contents and antioxidant activities among different parts of h. perforatum wild-growing plants. in order to find out if any ordination of the analyzed ro, nfs and fs extracts exists, a pca (fig. 1) was applied using the following variables: total contents of phenolics (tp, tf, tcd, tfa, tct, th) and antioxidant activities (cuprac, rp, pm, dpph). two main principal components (pc) were used to characterize phenolic compound composition and antioxidant activities in ro, nfs and fs extracts. the pca data showed that the pc1 and pc2 explained 100% of the total variation. the pca plot (fig. 1) indicated that pc1 explained the variance of 96.19% that was positively related to all variables concerning the phenolic compound contents and antioxidant activities. the pc2 showed a variance of 3.81% and it was not connected to the tested parameters. the pca plot showed that tested h. perforatum samples are well separated on pc1 (fig. 1). in this context, fs and nfs extracts exhibited positive scores on pc1 and they were characterized with high contents of phenolic compounds and antioxidant activities. on the other hand, ro extracts had a negative score on pc1 and possessed a low capacity for phenolic compound production and antioxidant table 1. the content of total phenolic compounds in h. perforatum wild-growing plants* phenolic compounds ro nfs fs tp (mg ga·g-1 dw) 36.51±1.02 a 103.88±4.97 b 107.38±0.90 b tf (mg c·g-1 dw) 28.14±1.56 a 76.70±7.66 b 68.59±0.26 b tcd (mg c·g-1 dw) 15.97±0.46 a 36.42±4.58 b 42.52±1.10 b tfa (mg c·g-1 dw) 9.98±0.78 a 22.78±2.09 b 24.82±0.45 b tct (mg cg·g-1 dw) 6.52±0.51 a 15.58±3.03 b 17.60±0.57 b th (μg h·g-1 dw) 7.00±0.25 a 581.86±14.45 b 1150.28±7.86 c * ro: roots; nfs: non-flowering shoots; fs: flowering shoots; tp: phenolics; tf: flavonoids; tcd: catechin derivatives; tfa: flavan-3-ols; tct: condensed tannins; th: hypericin; ga: gallic acid; c: catechin; cg: cyanidin-3-glucoside; h: hypericin; dw: dry weight. a the values in one row marked with lower-cases denoted significant differences between samples at p<0.05 (duncan's multiple range test). table 2. the antioxidant activities in hypericum perforatum wild-growing plants* antioxidant activities ro nfs fs cuprac (μм т·g-1 dw) 230.11±19.24 a 685.60±17.33 b 708.30±12.58 b rp (μм aa·g-1 dw) 425.05±13.91 a 1865.21±78.81 b 1584.39±163.80 b pm (mg aa·g-1 dw) 57.89±3.55 a 108.96±4.99 b 128.46±0.51 c dpph (μм т·g-1 dw) 158.81±0.10 a 407.86±2.36 b 496.67±11.67 c * ro: roots; nfs: non-flowering shoots; fs: flowering shoots; cuprac: cupric ions reducing antioxidant capacity; rp: reducing power; pm: phosphomolybdenum test; dpph: dpph radical scavenging activity; t: trolox; aa: ascorbic acid; dw: dry weight. a the values in one row marked with lower-cases denoted significant differences between samples at p<0.05 (duncan's multiple range test). biologica nyssana ● 10 (2) december 2019: 159-168 tusevski et al. ● antioxidant activity and phenolic compounds in hypericum perforatum l. wild-growing plants collected in the republic of macedonia) activities. in addition, fs and nfs extracts were located on the opposite side on pc2 and the major parameters responsible for the separation of these two aerial plant samples were th content with positive score on pc2, as well tf content and antioxidant activity measured by rp assay with negative scores on pc2. according to these results, fs extracts showed significantly higher th contents, but lower tf amount and rp value in comparison to nfs extracts. pearson’s correlation matrix (tab. 3) demonstrated significant positive correlations between phenolic compound contents and antioxidant activities in tested samples. in this view, the tp contents were in significant positive correlation with cuprac assay. a significant positive correlation was also found between tf and rp methods. similarly, tcd amounts were positively related to the antioxidant activity measured by pm and dpph assays. these results clearly indicated that phenolic compounds greatly contributed to the observed antioxidant activities of plant extracts. discussion production of phenolic compounds in h. perforatum wild-growing plants present data showed that aerial plant parts of h. perforatum were an excellent source of phenolics and flavonoids, while root tissues showed lower capability for accumulation of these phenolic compounds. outgoing results for total phenolics and flavonoids are in accordance with those previously reported for aerial parts of h. peforatum and other hypericum species (germ et al., 2010; hernandez et al., 2010). for instance, gioti et al. (2009) have noticed that shoots, non-flower and flower branches of h. peforatum possess significant amounts of phenolic compounds. taking into account literature data, kalogeropoulos et al. (2010) reported lower amounts of total phenolics and flavonoids for h. peforatum plants collected in greece in comparison to the results presented here. on the other hand, altun et al. (2013) observed higher values of these compounds for hyperici herba from turkey. the variation of phenolic and flavonoid contents in h. fig. 7. canonical discriminant analysis of the six g. pratensis populations from serbia and montenegro table 3. correlation analysis between phenolic compounds production and antioxidant activities in hypericum perforatum wild-growing plants (n = 3 samples)* r tp tf th tcd tfa tct cuprac rp pm tf 0.980 th 0.889 0.779 tcd 0.984 0.929 0.956 tfa 0.996 0.960 0.924 0.996 tct 0.992 0.946 0.940 0.999 0.999 0.999 0.980 0.888 0.984 0.996 0.992 rp 0.974 0.999 0.761 0.919 0.952 0.937 0.974 pm 0.974 0.910 0.969 0.998 0.990 0.995 0.974 0.898 dpph 0.978 0.916 0.965 0.999 0.992 0.996 0.977 0.904 0.999 * r: pearson’s coefficient; tp: phenolics; tf: flavonoids; tcd: catechin derivatives; tfa: flavan-3-ols; tct: condensed tannins; th: hypericin; cuprac: cupric ions reducing antioxidant capacity; rp: reducing power; pm: phosphomolybdenum test; dpph: dpph radical scavenging activity. the values in bold indicate significance at p < 0.05. 163 biologica nyssana ● 10 (2) december 2019: 159-168 tusevski et al. ● antioxidant activity and phenolic compounds in hypericum perforatum l. wild-growing plants collected in the republic of macedonia) peforatum could be attributed to the genotypic and environmental factors, various plant organs, as well harvesting time (gioti et al., 2009; germ et al., 2010). however, the direct comparison of phenolic and flavonoid contents is a complex task due to the differences in solvents and procedures for plant extraction, the usage of reference compounds for quantification and the methods for determination of phenolic compounds. even that some non-phenolics (ascorbic acid, amino acids, proteins, organic acids and sugars) could react with folin-ciocalteu reagent (prior et al. 2005), the procedure for h. peforatum plant extraction used in this study eliminated the possibility for interference of these compounds. taking into account the richness of h. perforatum extracts with monomeric flavan-3-ols, as well as oligomeric and polymeric tannins (nahrstedt & butterweck, 2010), the different assays were applied for determination of catechin derivatives (tcd, tfa and tct) in plant samples. present results clearly demonstrated that aerial parts of h. perforatum have greater capability for accumulation of those catechin derivatives compared to root samples. the literature data for total contents of flavan-3-ols or tannins in h. perforatum are rather scarce. in this view, germ et al. (2010) have shown higher values for catechin derivatives in h. perforatum leaves and flowers than those reported here. the heterogeneity in the results for total catechin derivatives may be due to the high sensitivity of vanillin-hcl assay, as well the solvents, reaction time, temperature and vanillin concentrations used for analysis (sun et al., 1998). the ratio tcd/tct could be used as a rough estimation of the polymerization degree of monomeric flavan-3-ols into oligomeric and polymeric tannins (ribéreau-gayón & stonestreet, 1966). in this study, the ratio tcd/tct in analyzed samples was from 2 to 3 indicating that polymeric tannins in h. perforatum are probably represented by proanthocyanidin dimers and trimers. moreover, the summation of total flavan-3-ol and condensed tannin contents accurately represented the amounts of total catechin derivatives in the analyzed samples. therefore, vanillin-hcl assay for total catechin derivatives could be proposed as a routine procedure for determination of total catechin derivatives including flavan-3-ol monomers and polymeric condensed tannins in h. perforatum. the measurement of total hypericin is commonly used for quality control of h. perforatum standardized extracts and phytopharmaceuticals (williams et al., 2006). the results for total hypericin contents showed that flowering shoots accumulated markedly higher contents of hypericin compared to non-flowering shoots, while minor amounts were noticed for roots. in this view, naphthodianthrone accumulation has been connected with the growth and development of h. perforatum reproductive parts (southwell & bourke, 2001). in addition, the correlation between dark glands formation and hypericin levels in leaves, stems, flower petals, stamens, carpels and sepals of h. perforatum has been reported (zobayed et al., 2006). therefore, the dark glands could be considered as the limiting factors for hypericin accumulation due to necessity of these glandular structures for deposition of hypericin (pasqua et al., 2003). even that aerial parts of h. perforatum in full bloom have been proposed as excellent sources of hypericin (southwell & bourke, 2001), the roots have never been explored as a hypericin-producing tissues. however, recent histochemical analysis showed the presence of darkred globules with hypericin in h. perforatum leaves, stems and roots (qian et al., 2012). these findings suggested that dark glands are not the only site of naphthodianthrone accumulation and hypericin is probably synthesized either in mesophyll, root or stem cells and subsequently is transported to the dark glands. all these observations suggested that aerial parts of h. perforatum accumulated higher contents of phenolic compounds (flavonoids, catechin derivatives and hypericin) compared to roots. antioxidant activity of h. perforatum wild-growing plants the cuprac assay has been widely used to determine the antioxidant capacity of plant extracts as it requires relatively standard equipment and delivers fast and reproducible results (apak et al., 2004). the cuprac assay is useful for determination of antioxidant capacity in a wide variety of antioxidant compounds, such as phenolic acids, flavonoids, carotenoids, anthocyanins, as well for thiols (glutathione), synthetic antioxidants, vitamins c and e. results from this study demonstrated very strong cuprac activity for the aerial parts, while root extracts possessed the lowest cuprac value. the relevance of cuprac method for evaluation of antioxidant potential of aerial parts from h. perforatum and other hypericum species has also been reported (maltas et al., 2013; boga et al., 2016). the results obtained from cuprac in vitro measurements could be efficiently extended to the in vivo antioxidant activity because the cuprac reaction is carried out at physiological ph (apak et al., 2004). the non-flowering and flowering shoots of h. perforatum demonstrated high antioxidant activity measured by rp assay, thereby could be proposed as efficient reductones. the reductones terminated free radical chain reactions and thus, rp could be related to the antioxidant activity of plant extracts 164 biologica nyssana ● 10 (2) december 2019: 159-168 tusevski et al. ● antioxidant activity and phenolic compounds in hypericum perforatum l. wild-growing plants collected in the republic of macedonia) (gordon, 1990). phenolic compounds as effective reductones have previously been confirmed for h. perforatum (zou et al., 2004; gioti et al., 2009), as well as other hypericum species (şerbetçi et al., 2012; rainha et al., 2013). in this study, aerial parts showed high rp values, which indicated that h. perforatum is characterized by abundance of antioxidant compounds that reduce fe3+/ferricyanide complex into ferrous form. present results showed that flowering shoots exhibited the highest value for pm assay, followed by non-flowering shoots, while the lowest antioxidant capacity was found for root extracts. these results for aerial parts were consistent with those previously reported for h. perforatum flower, leaf and stem extracts (radulović et al., 2007). in contrast, raghu chandrashekhar et al. (2009) showed that roots have greater antioxidant capacity determined by pm method than those found for aerial parts of h. hookerianum wild-growing plants. the discrepancy in the results for pm assay among different plant organs could be related to the interspecies variability concerning the distribution of antioxidant compounds. despite polyphenols, many other non-phenolic hydrophilic and lipophilic compounds could significantly contribute to the antioxidant activity of plant extracts measured by pm assay (prieto et al., 1999). in this study, dpph method was selected as the most effective assay for evaluation of radical scavenging capacity of plant extracts by chainbreaking mechanism (tusevski et al., 2014). present results demonstrated that flowering and non-flowering shoots were better dpph scavengers compared to root extracts. accordingly, there are many studies that confirm the strong dpph radical scavenging activity of h. perforatum extracts (silva et al., 2008; gioti et al., 2009; kalogeropoulos et al., 2010; becker et al., 2016). taken together, the variation in the results for antioxidant activities of h. perforatum extracts could be due to the structural features, antioxidant mechanism, as well as synergistic effects of different classes of phenolic compounds. it is widely accepted that the antioxidant activity of h. perforatum extracts is correlated to the content of phenolic compounds. although present results were focused on this topic, there is still a lack of evidence to determine the contribution of various classes of phenolic compounds to the antioxidant activity of h. perforatum. since the methods used in this study enabled the quantification of various groups of phenolic compounds, it was possible to establish the correlation between those metabolites and antioxidant activity in h. perforatum. correlation between phenolic compound contents and antioxidant activity in h. perforatum wild-growing plants the establishment of a relationship between antioxidant activity and phenolic contents is a very difficult approach because plant extracts represented complex mixtures of various metabolites with antioxidant and prooxidant properties that exhibit synergistic actions. in this study, the principal component analysis (pca) as a statistical tool showed that flowering shoot and non-flowering shoot extracts represented the richest source of phenolics with strong antioxidant activity. on the other side, roots were characterized by low capacity for phenolic compound production and antioxidant activity. the correlation analyses performed in this study demonstrated significant positive correlation between tp and cuprac. these results suggested that phenolics in h. perforatum wild-growing plants could be proposed as preeminent constituents that exhibit cupric ion reducing capacities. in this view, antioxidant activity of h. perforatum and other hypericum species measured with cuprac assay has been well correlated with total phenolic and flavonoid contents (maltas et al., 2013; moein et al., 2015). moreover, results presented here showed a significant correlation between tf and rp in h. perforatum extracts indicating that high reducing power could be attributed to the total flavonoid contents. such a correlation between total flavonoids and reducing power of h. perforatum extracts has also been reported by zou et al. (2004). the strong positive correlations of tcd with dpph and pm assays found in this study were expected since monomeric flavan-3-ols and polymeric proanthocyanidins have already been shown as the most active dpph scavengers (froehlicher et al., 2009). in addition, raghu chandrashekhar et al. (2009) confirmed that total flavan-3-ol contents in h. hookerianum correlated with total antioxidant capacity measured by pm assay. outgoing results showed a significant positive correlation between pm and dpph indicating that these assays could be proposed as suitable and reliable methods for evaluation of antioxidant activity of h. perforatum extracts. the th contents did not show any significant correlation with antioxidant methods used in this study. these data could be ascribed to the small percentage distribution of hypericin (up to 1%) in phenolic profile that resulted in their minor contribution to the antioxidant activity of h. perforatum extracts. 165 biologica nyssana ● 10 (2) december 2019: 159-168 tusevski et al. ● antioxidant activity and phenolic compounds in hypericum perforatum l. wild-growing plants collected in the republic of macedonia) conclusion in this study, the capability for the production of phenolic compounds and antioxidant activities of roots, non-flowering shoots and flowering shoots of h. perforatum wild-growing plants was presented as detailed for the first time. the aerial plant samples represented the richest source of phenolics, flavonoids, catechins (flavan-3-ols and condensed tannins) and hypericin with strong antioxidant activities. noteworthy, root extracts from h. perforatum demonstrated satisfactory amounts of various groups of phenolics with moderate antioxidant activities that make them potential candidates as a natural source of antioxidant and radical scavenging compounds. the correlation analysis used in this study indicated that antioxidant activity of h. perforatum wild-growing plants is related to the phenolic compounds that are characterized by hydrogen atom donation, radical scavenging and participation in redox reactions. the phytochemical profiling and initial screening of antioxidant activity of h. perforatum extracts could be a step forward in the preparation of new phytoproducts in food and pharmaceutical industry. references altun, m.l., yılmaz, b.s., orhan, i.e., citoglu, g.s. 2013: assessment of cholinesterase and tyrosinase inhibitory and antioxidant effects of hypericum perforatum l. 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faculty of biology, institute of botany and botanical garden “jevremovac”, takovska 43, 11000 belgrade, serbia * e-mail: veljicm@bio.bg.ac.rs abstract: veljić, m., rajčević, n., bukvički, d.: a revision of the moss collection of the university of belgrade herbarium (beou) from the ostrozub mountain in serbia. biologica nyssana, 7 (1), september 2016: 11-17. during the revision of moss collection of the university of belgrade herbarium from the ostrozub mountain which was collected in period from 1950 to 1953 it has been found that numerous specimens had not been determined. in total, 132 species were recorded: 23 liverwort, 109 moss species were found on the mt. ostrozub. out of those, 38 taxa (6, 25 and 7 species belonging to jungermanniopsida, bryopsida and sphagnopsida, respectively) represent new record for the mt. ostrozub. nineteen species belong to the endangered species, of different endangerment degree, on the red lists of european and serbian mosses. key words: liverworts, mosses, peat moss, conservation apstrakt: veljić, m., rajčević, n., bukvički, d.: revizija kolekcije mahovina iz herbarijuma univerziteta u beogradu (beou) sa planine ostrozub u srbiji. biologica nyssana, 7 (1), septembar 2016: 11-17. tokom revizije kolekcije mahovina iz herbarijuma univerziteta u beogradu prikupljene na planini ostrozub u periodu od 1950. do 1953. godine utvrđeno je da brojni primerci nisu određeni. ukupno je na ostrozubu pronađeno 132 vrste, odnosno 23 marchantiophyta i 109 bryophyta. u materijalu su zabeležena 38 taksona do sada nepoznata na ostrozubu, i to 6 jungermanniopsida, 25 bryopsida i 7 sphagnopsida. na crvenim listama mahovina evrope i srbije u različitim stepenima ugroženosti nalazi se 19 vrsta. key words: jetrenjače, mahovine, bele mahovine, konzervacija introduction this work includes the review of the research results on the mosses from the mt. ostrozub and the revision of the herbarium collection made by ilić, popović et al., 1950 and 1953. the material from 1950 was collected on localities: zeleničje (1), village ostrozub (2), near road predejane – ostrozub (3), the spring srebrni kladenac (4), near road ostrozub – ruplje (5), village ruplje (6), near the stream golema dolina (7) and peat near village stojimirovo (8). the collection from 1953 is mainly from peatlands from the following villages: selište (9), preslap (10), dobro polje (11), krstićevo (12), ruplje (6), stojimirovo (8), kolibište (13), čobanac (14) and zeleničje (1). only part of this collection 7 (1) • september 2016: 11-17 doi: 10.5281/zenodo.159099 biologica nyssana 7 (1)  september 2016: 11-17 veljić, m. et al.  a revision of the moss collection… 12 was published in the papers cited bellow, the rest remained undetermined. investigation of the mt. ostrozub moss flora was performed in parallel with phytocenological research. however, the only specified article on the bryoflora is the publication on mosses in reserves and protected areas where 84 taxa have been listed (15 liverworts and 69 mosses) in the community of prunus laurocerasus (p o p o v i ć , 1966). m i š i ć et al. (1984) mentioned 38 (10 liverworts and 28 mosses), while il ić (1951, 1952/53), mentioned several moss taxa within the community with cherry laurel. the mt. ostrozub is located in the southeast of serbia (fig. 1), within the rhodope mountain range (a v r a m o v i c et al., 2005). the area is linked to vlasina complex in the south and babička gora in the north. the peaks of čuklenik (1280 m.a.s.l.), ostrozubska čuka (1546 m.a.s.l.), čobanac (1496 m.a.s.l.) and title (1487 m.a.s.l.) extend in the southwest-northeast direction. hydrographic network consists of the rivers ostrozubska, rupska, kozačka and bistrička, and rivulets and streams srebrni, kladenac and zelenički. the largest area is covered with the silicate surface. the most widely spread of soil is skeletal brown forest soil, with a weak acid reaction. potential zonal vegetation is fagetum montanum b. jov. 1967. (jovanović et al. 1997), i.e. subtype lauroceraso – fagetum b. jov. 1967. (j o v a n o v i ć , 1967). regular shift of vegetation zones was observed from the foothill to the peak of the mt. ostrozub. the mt. ostrozub drew attention of many botanists primarily because of cherry laurel (prunus laurocerasus l.) discovered by p a n č i ć (1887). after his discovery, a series of papers was created on this species and the beech forests, with which it forms a community lauroceraso-fagetum b. jov. (k o š a n i n , 1913; i l i ć , 1951; p o p o v i ć , 1952; j o v a n o v i ć , 1952/53, 1967, 1973; r a n đ e l o v i ć et al., 1983; m i š i ć , et al., 1984; j o v a n o v i ć et al., 1997). zeleničje area, a part of the mt. ostrozub, is now protected as a nature reserve. a rich hydrographic network and varied geological substrate enable formation of a large number of different microclimate habitats on the mt. ostrozub. all of the listed characteristics, as well as many others not mentioned here, suggest good conditions for the development of very diverse bryoflora on this mountain. material and methods the bryophyte samples were collected from fourteen localities in 1950 and 1953 in mt. ostrozub. for identification of taxa, the relevant bryology literature was used (p e t r o v , 1975, l a n d w e h r & b a r k m a n , 1966; l a n d w e h r & g r a d s t e i n , 1980; s m i t h , 1990, 2006). the nomenclature is in accordance with g r o l l e & l o n g (2000) and h i l l et al. (2006). the lists of the moss taxa are summarized according to classification by j o n a t h a n & k a r e n (2004) while genera are given in the alphabetical order. literature sources (*) and new data on localities (**) are given for each fig. 1. map of the mt. ostrozub with the study sites and its position. biologica nyssana 7 (1)  september 2016: 11-17 veljić, m. et al.  a revision of the moss collection… 13 table 1. list of bryophyte species on the mt. ostrozub with the literature and new data. species *literature data (1,2,3,4) **new data marchantiophyta marchantiopsida conocephalum conicum (l.) dum. (3) zeleničje marchantia polymorpha l. (3,4) zeleničje, peatland near stojimirova jungermanniopsida apometzgeria pubescens (schrank) kuwah. (3,4) bazzania trilobaia (l.) gray. (3,4) blepharostoma trichophyllum (l.) dum. (3) zeleničje calypogeia azurea stotler & crotz (4) •c. mulleriana (schiffn.) mull. frid. zeleničje, stream golema dolina chiloscyphus pallescens (ehrh. ex hoffm.) dum. (3) c. polyanthos (l.) corda var. polyanthus (3,4) near road ostrozub ruplje •frullania dilatata (l.) dum. zeleničje •jungermannia leiantha grolle zeleničje, srebrni kladenac lophocolea bidentata (l.) dum. var. bidentata (3) zeleničje metzgeria furcata (l.) dum. (3) •pedinophyllum inerruptum (nees.) kaal. zeleničje pellia epiphylla (l.) corda. (3,4) srebrni kladenac plagiochila asplenioides (l. emend. taylor) dum. (3,4) •p. porelloides (torrey ex nees) lindeb. zeleničje, stream golema dolina •porella cordaeana (hub.) moore zeleničje p. platyphylla (l.) pfeiff. (3,4) radula complanata (l.) dum. (3) near road ostrozub ruplje scapania nemorea (l.) grolle (4) zeleničje s. undulata (l.) dum. (3) zeleničje trichocolea tomentella (ehrh.) dum. (1,3,4) zeleničje bryophyta bryopsida abietinela abietina (hedw.) m. fleisch. (4) •amblistegium serpens (hedw.) schimp. near road predejane ostrozub antitrichia curtipendula (hedw.) brid. (3,4) zeleničje atrichum angustatum (brid.) bruch & schimp. (3) a. undulatum (hedw.) p. beauw. (4) zeleničje, near road predejane ostrozub barbula bicolor (bruch & schimp.) lindb. (3) b. convoluta hedw. (3) b. unguiculata hedw. (3) bartramia ityphylla brid. (3) stream golema dolina b. pomiformis hedw. (3,4) •brachythecium albicans (hedw.) schimp. peak of ostrozub •b. rivulare schimp. zeleničje, stream golema dolina b. rutabulum (hedw.) schimp. (3) near road predejane ostrozub bryum alpinum huds. ex with. (3) b. caespiticium hedw. (3) zeleničje, stream golema dolina b. capillare hedw. (3) near road predejane ostrozub, stream golema dolina b. archangelicum bruch & schimp. (3) b. intermedium (brid.) blandow (3) •b. pallescens schleich. ex schwarg stream golema dolina •b. pseudotriquetrum (hedw.) p. gaertn. & al. zeleničje calliergonella cuspidata (hedw.) loeske (3) peatland near stojimirovo ceratodon purpureus (hedw.) brid. (3) stream golema dolina climacium dendroides (hedw.) f. weber & d. mohr. (3,4) zeleničje •cratoneuron filicinum (hedw.) spruce zeleničje ctenidium molluscum (hedw.) mitt. (3,4) zeleničje, near road predejane ostrozub dichodontium palustre (dicks.) m. stech (3) dicranella crispa (hedw.) schimp. (3) zeleničje d. heteromalla (hedw.) schimp. (3) zeleničje, stream srebrni izvor dicranum scoparium hedw. (2,3,4) zeleničje, near road predejane ostrozub, ruplje, stream golema dolina biologica nyssana 7 (1)  september 2016: 11-17 veljić, m. et al.  a revision of the moss collection… 14 d. viride (sull. & lesq.) lindb. (3) diphyscium foliosum (hedw.) d. mohr (3) ditrichum flexicaule (schwagr.) hampe (3) •d. pallidum (hedw.) hampe stream golema dolina drepanocladus aduncus (hedw.) warnst. (3) zeleničje •eurhynchium angustirete (broth.) t. j. kop. zeleničje e. striatum (hedw.) schimp. (3,4) fissidens adianthoides hedw. (3) fontinalis antipyretica hedw. (3,4) funaria hygrometrica hedw. (3,4) zeleničje, stream golema dolina •grimmia hartmanii schimp. peak of ostrozub g. ovalis (hedw.) lindb. (3) •g. pulvinata (hedw.) sm. village ostrozub hedwigia ciliata (hedw.) p. beauv. (3) •herzogiella seligeri (brid.) z. iwats. stream golema dolina homalothecium lutescens (hedw.) h. rob. (3,4) •h. philippeanum (spruce) schimp. zeleničje hylocomium splendens (hedw.) schimp. (2,3,4) zeleničje, ruplje, stream golema dolina •hymenoloma crispulum (hedw.) ochyra stream golema dolina hypnum cupressiforme hedw. (3,4) zeleničje, stream golema dolina isothecium alopecuroides (lam. ex dubois) isov. (4) zeleničje, near road predejane ostrozub, stream golema dolina •kindbergia praelonga (hedw.) ochyra zeleničje •leskea polycarpa hedw. near road predejane ostrozub, stream golema dolina leucobrium glaucum (hedw.) ĺngstr. (2) leucodon sciuroides (hedw.) scwaegr. (3) zeleničje mnium marginatum (dicks.) p. beauv. (3) neckera crispa hedw. (4) stream golema dolina orthothecium rufescens (dicks. ex brid.) schimp. (4) orthotrichum pulchellum brunt. (3) •o. speciosum nees. village ostrozub •oxyrrhynchium schleicheri (r. hedw.) roll near road predejane ostrozub palustriela commutata (hedw.) ochyra var. commutate (3) zeleničje philonotis fontana (hedw.) brid. (3) peatland near stojimirovo plagiomnium cuspidatum (hedw.) t. j. kop. (3,4) near road predejane ostrozub p. undulatum (hedw.) t. j. kop. (1,3,4) zeleničje, near road predejane ostrozub, stream golema dolina plagiothecium denticulatum (hedw.) schimp. (3,4) zeleničje p. nemorale (mitt.) a. jaeg. (3) platyhypnidium riparioides (hedw.) dixon (3) pleurozium schreberi (willd. ex brid.) mitt. (3) pogonatum aloides (hedw.) p. beauv. (3) zeleničje p. urnigerum (hedw.) p. beauv. (3) zeleničje, stream golema dolina polytrichastrum formosum (hedw.) g. l. sm. (3) zeleničje polytrichum commune hedw. (2,3,4) zeleničje, near road predejane ostrozub, peatland near stojimirovo, selište, kolibište p. juniperinum hedw. (3) zeleničje p. piliferum hedw. (3) psedoleskea incurvata (hedw.) loeske (3) zeleničje •pseudoleskeella nervosa (brid.) nyholm stream golema dolina pseudoscleropodium purum (hedw.) m. fleich. (3,4) zeleničje pterigynandrum filiforme hedw. (3) zeleničje rhizomnium punctatum (hedw.) t. j. kop. (1,3,4) zeleničje, stream golema dolina rhytidiadelphus loreus (hedw.) warnst. (3,4) zeleničje •r. squarrosus (hedw.) warnst. zeleničje, stream golema dolina r. triquetrus (hedw.) warnst. (2,4) zeleničje, stream golema dolina, ruplje •sanionia uncinata (hedw.) loeske stream golema dolina schistidium apocarpum (hedw.) bruch & schimp. (3) zeleničje, stream golema dolina •sciuro-hypnum populeum (hedw.) ignatov & huttunen zeleničje •s. reflexum (hedw.) ignatov & huttunen zeleničje syntrichia latifolia (bruch ex hartm.) huebener (3) biologica nyssana 7 (1)  september 2016: 11-17 veljić, m. et al.  a revision of the moss collection… 15 s. montana nees (3) s. ruralis (hedw.) f. weber & d. mohr (3) tetraphis pellucida hedw. (3) •thamnobryum alopecurum (hedw.) gangulee stream golema dolina •thuidium recognitum (hedw.) lindb. near road predejane ostrozub, stream golema dolina t. tamariscinum (hedw.) schimp. (4) zeleničje tortella humilis (hedw.) jenn. (3) t. tortuosa (hedw.) limpr. (3) tortula lanceola r. h. zander (3) t. muralis hedw. (3) •t. subulata hedw. stream golema dolina, near road predejane ostrozub warnstorfia fluitans (hedw.) loeske (4) sphagnopsida •sphagnum angustifolium (c. e. o. jens. ex russ.) c. e. o. jens. selište •s. capillifolium (ehrh.) hedw. stojimirovo s. cuspidatum ehrh. ex hoffm. (3) ruplje •s. fallax (h. klinggr.) h. klinggr. selište, stojimirovo •s. flexuosum dozy & molk. selište, stojimirovo •s. inundatum russ. selište, under preslapa, čobanac, krstićevo, zeleničje, dobro polje, s. palustre l. (3) selište, under preslap, kolibište, dobro polje, krstićevo, stojimirovo •s. squarrosum crome under preslap, kolibište s. subsecundum nees (3) zeleničje, čobanac, krstićevo •s. teres (schimp.) angstr. golema dolina * literature data: 1 (i l ić , 1951, 1952/53), 2 (jovanović , 1952/53), 3 (popović, 1966), 4 (mišić et al., 1984) ** data from the herbarium material collected in 1950 and 1953 • new species in the flora of the mt. ostrozub taxon. new species on the mt. ostrozub are marked with the dot (•) in tab. 1. herbarium collection has been deposited at the university of belgrade – faculty of biology herbarium (beou). results and discussion in this paper, we report new data on the bryoflora of the mt. ostrozub obtained during the revision of the material collected in the mt. ostrozub in 1950 and 1953, as well as available literature data (tab. 1). in total, 132 taxa of mosses were recorded, which makes this small area bryologically very important for serbia. twenty-three taxa were recorded from the marchantiophyta: two from classis marchantiopsida and twenty-one from classis jungermanniopsida. six of this species were recorded for the first time on the mt. ostrozub: calypogeia mulleriana, frullania dilatata, jungermannia leiantha, pedinophyllum inerruptum, plagiochila porelloides and porella cordaeana. bryophyta (class bryopsida and sphagnopsida) is represented with 109 taxa. the class bryopsida includes 99 taxa of which 25 species are recorded for the first time for the bryoflora of the mt. ostrozub: amblistegium serpens, brachythecium albicans, b. rivulare, bryum pallescens, b. pseudotriquetrum, cratoneuron filicinum, ditrichum pallidum, eurhynchium angustirete, grimmia hartmanii, g. pulvinata, herzogiella seligeri, homalothecium philippeanum, hymenoloma crispulum, kindbergia praelonga, leskea polycarpa, ortotrichum speciosum, oxyrrhynchium schleicheri, pseudoleskeella nervosa, rhytidiadelphus squarrosus, sanionia uncinata, sciuro-hypnum populeum, s. reflexum, thamnobryum alopecurum, thuidium recognitum, tortula subulata. ten peat moss (class sphagnopsida) species were recorded, out of which seven were recorded for the first time on the mt. ostrozub (s. angustifolium, s. capillifolium, s. fallax, s. inundatum, s. flexuosum, s. squarrosum, s. teres). the genera with the highest number of species for mosses this study were sphagnum (10 taxa), bryum (7 taxa) and barbula, brachythecium, grimmia, polytrichum, rhytidiadelphus, syntrichia, tortula (3 taxa each). the most diverse bryoflora (54 taxa) is recorded in the reserve area, which includes zeleničje locality (36). in locality number 7 (stream biologica nyssana 7 (1)  september 2016: 11-17 veljić, m. et al.  a revision of the moss collection… 16 golema dolina), great number of taxa (30) was also recorded, making this locality a bryologycally rich area. significantly lower diversity was recorded in all other localities, possibly due to the less intense research in these localities by the researchers. it is remarkable that on the mt. ostrozub peatlands, ten sphagnum species were recorded. it is also one of the localities with the greatest diversity of this group. on the mt. ostrozub, nineteen recorded taxa are classified in the red book of europe (iucn, 1994) and serbia (s a b o v l j e v i ć et al., 2004) at different levels of vulnerability (tab. 2). bern convention annex 1 (council of europe, 1979) declares the species dicranum viride as a strictly protected species. according to the ordinance on the proclamation and protection of the wild species of plants, animals and fungi, sphagnum species are strictly protected by local legislation (sl. glasnik rs, 2009). calypogeia muelleriana and bazzania trilobata are also protected according to this document. a great number of species on the red lists justifies preserving maximum protection of habitats on the mt. ostrozub. it is also necessary to put the special emphasis on the protection of the peatlands outside the reserve in order to maintain the diversity of the genus sphagnum. acknowledgements. this research was supported by a grant from the ministry of education, science and development of serbia (project no. 173029). references avramović, d., zlatković, b., ranđelović, n., 2005: zaštićena prirodna dobra jugoistone srbije. viii symposium on flora of southeasteren serbia and neighbouring regions, niš. proceding, 223-227. council of europe, 1979: convention on the conservation of european wildlife and natural habitats. bern. grolle, r., & long, d. g., 2000: an annotatated check-list of the hepaticae and anthocerotae of europe and macronesia. journal of bryoogy, (22), 103-140. hill, m. o., bell, n., bruggeman-nannenga, m. a., brugues, m., cano, m. j., enroth, j., flatberg, k. i., frahm, j. p., gallego, m. t., garilleti, r., guerra, j., hedenäs, l., holyoak, d. t., hyvönen, j., ignatov, m. s., lara, f., mazimpaka, v., muñoz, j., and l. söderström., 2006: an annotated checklist of the mosses of europe and 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doi: 10.5281/zenodo.5523045 bats assemblages in awasian water forest reserve, tandag city, surigao del sur, philippines original article allyka eve g. balo college of teacher education, surigao del sur state university-main campus, rosario, tandag city, surigao del sur, philippines allykaevebalo@gmail.com mark lee o. sabandal college of teacher education, surigao del sur state university-main campus, rosario, tandag city, surigao del sur, philippines malesab017@gmail.com johnny b. maglasang college of teacher education, surigao del sur state university-main campus, rosario, tandag city, surigao del sur, philippines johnnymaglasang12345@gmail.com lovely jean b. cosmiano college of teacher education, surigao del sur state university-main campus, rosario, tandag city, surigao del sur, philippines cosmianolovelyjean14@gmail.com arturo g. gracia jr. college of teacher education, surigao del sur state university-main campus, rosario, tandag city, surigao del sur, and department of natural sciences and mathematics, college of arts and sciences, surigao del sur state university-main campus, rosario, tandag city, surigao del sur, philippines artzgracia@gmail.com (corresponding author) received: mart 19, 2020 revised: october 11, 2020 accepted: june 23, 2021 abstract: bats are an integral part of the ecosystem that plays a vital role in its stability. however, their existence is being threatened by uncontrollable anthropogenic activities, and its basic ecological information, especially in eastern mindanao, is limited. thus, the study was conducted to evaluate the assemblage of bats in one of the underexplored sites of eastern mindanao biodiversity corridor. mist-netting for a total of 9 net nights was carried out to document the species. a total of 10 species was observed. among these species, cynopterus brachyotis had the highest relative abundance comprising 53% of the population. species diversity was relatively higher in the secondary mixed-dipterocarp forest with a shannon-weiner diversity index (h’) of 1.41 compared to the primary dipterocarp forest (h’=1.19). the species ptenochirus jagori and rhinolophus subrufus were the only bats observed with aggregated population distribution pattern. among the recorded species, six (60%) were assessed as endemic comprising 5 philippine and 1 mindanao endemic, while eonycteris robusta and rhinolophus subrufus were the near-threatened species recorded. based on the results, awasian water forest reserve houses an array of bat species with a high percentage of endemicity. key words: assessment, diversity, endemism, volant mammals apstract: zajednica slepih miševa u rezervatu awasian water forest, tandag, surigao del sur, filipini slepi miševi su integralni deo ekosistema koji igraju vitalnu ulogu u njegovoj stabilnosti. ipak, njihovo postojanje je ugroženo nekontrolisanim antropogenim aktivnostima i osnovne ekološke infomacije o njima su ograničene, posebno u istočnom minadau. zbog toga, sprovedeno je istraživanje kako bi se procenio raspored slepih miševa na jednoj od neistraženih lokacija koridora biodiverziteta u istočnom minadau. kako bi se dokumentovale vrste, izvršeno je postavljanje mreže tokom ukupno 9 noći. utvrđeno je ukupno 10 vrsta. među njima, cynopterus brachyotis imao je najveću relativnu učestalost sačinjavajući 53% populacije. diverzitet vrsta bio je relativno veći u sekundarnoj mešovitoj dipterokarpnoj šumi sa shannon-weiner indeksom diverziteta (h) od 1,41 u poređenju sa primarnom dikterokarpnom šumom (h=1,19). vrste ptenochirus jagori i rhinolophus subrufus su jedini slepi miševi čije su populacije bile grupisanog rasporeda distribucije. među zabeleženim vrstama, šest (60%) procenjene su kao endemične od koji 5 pripadaju filipinskim a 1 mindanao endemitima, dok vrste eonycteris robusta i rhinolophus subrufus predstavljaju skoro ugrožene vrste. na osnovu rezultata, rezervat awasian water forest je stanište za spektar vrsta slepih miševa sa visokim procentom endemizma. ključne reči: procena, diverzitet, endemizam, leteći sisari © 2021 balo et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 71 introduction the clade chiroptera includes two extant clades, megachiroptera (old world fruit bats) and microchiroptera (echolocating bats). bats are considered as an ecological indicator because they control pests, pollinate fruit trees, and regenerate forests (kasso and balakrishnan, 2013). in the philippines, bats are deemed as one of the most diverse mammals with at least 80 total extant species comprised of 26 megachiropteran and 54 microchiropteran species (sedlock et al., 2020). of these, approximately 40% are reported to be endemic. however, uncontrolled anthropogenic activities, habitat destruction, and disturbances are among the threats that threaten these taxa (quibod et al., 2019). the philippines is previously known for its dense forest in the early 20th century, with a reported forest cover of 95% of the total landmass. however, the continuous deforestation resulted in massive forest denudation, which is estimated to be around 80 to 90% (butler, 2014). bats are highly forest-dependent animals, and changes in their habitat could decrease their population and lead to eventual extinction (mickleburgh et al., 2002). this report is further supported by murphy and romanuk (2014) that mammalian species diversity, abundance, and along its mean biomass tend to decrease with increasing human disturbance and continuous habitat loss. for these reasons, the philippines has been identified as an important area for bat diversity that prioritizes conservation efforts globally (tanalgo and hughes, 2018). awasian water forest reserve is one of the watersheds of mt. hilong-hilong, a biodiversity corridor that lies between the provinces of agusan and surigao in the north eastern mindanao. although reported to exhibit rich flora and fauna, these claims are mainly based on scientific expeditions done on the western side of the mountain ecosystem (agusan); and only a few to none scientific data can be retrieved to represent the eastern side (surigao). the availability of ecological data is considered vital since it serve as a basis for setting conservation priorities and guidelines (goodman and benstead, 2005; mallari, 2009). thus, this study was conducted to address the scarcity of scientific information on bat community in the underexplored area of mt. hilong-hilong; and provide pragmatic findings that can be utilized for a better understanding of bat assemblage in the area. materials and methods place and duration of the study the study was conducted in the awasian water forest reserve located at barangay awasian, tandag city surigao del sur philippines (fig. 1). the site is geographically located at 9°04’28.9”n and 126°08’34.7”e. the site is one of the watersheds of mt. hilong-hilong, situated in the south-eastern part of the mountain ecosystem. the study was conducted from october 1 to 9, 2017, covering nine days of sampling. the site’s topography is generally plain, rolling, and gently sloping. the climatic condition in the area falls under the philippines’ type ii climate condition, which is characterized by rainfall distributed throughout the year, with a negligible short dry season (pagasa, 2011). establishment of sampling stations and habitat assessment two 1-km transect lines were established in the study site with an aerial distance from each other of 200 meters. the first transect was considered as station 1 that cut-across the dipterocarp forest. it was situated at an elevation that extends from 50 to 150 meters above sea level (masl). the dominant plant species were mixtures of shorea spp., ficus spp., and cultivated fruit trees like lansium domesticum, durio zibithenus, and artocarpus odoratissimus. the soil-litter was thin and dry. the area’s distance to the nearest water body (stream) was around 5 to 100 meters. during the sampling period, the temperature ranges from 24 to 30 °c, with a general weather condition of calm-night with a clear sky. the second transect was designated as station 2, established along the secondary mixed-dipterocarp forest. it was situated at an elevation of 100 to 250 m a.s.l. with a slightly-closed to closed canopy cover. the station’s dominant plant species were shorea spp. of the family dipterocarpaceae and ficus spp. of family moraceae. soil-litter was dry but thicker as compared with station 1. the area’s distance to the nearest stream was around 700 m, and 10 to 100 m from the small spring and stagnant canals. the tem72 biologica nyssana ● 12 (1) september 2021: 71-78 balo et al. ● bats assemblages in awasian water forest reserve, tandag city, surigao del sur, philippines fig. 1. location map of mt. hilong-hilong and spot map of the study site. (birdlife international, 2020) 73 perature during the sampling period ranged from 20 to 24 °c, with a general weather condition of gloomy with intermittent rain. data collection and identification along these transects, 21 mist-nets with a dimension of 4.5 meters long and 12 meters wide with a mesh size of 33 mm were established to capture the bats. checking of nets was performed from 6:00 pm to 5:00 am with 30 minutes to 1-hour interval to monitor if there were entangled bats and avoid possible mortality. captured species were placed into a cloth bag to prevent further stress on the organism and were brought to the processing area for taxonomic examination. the morphological metrics, which include ear (e), forelimb (fl), hindfoot (hf), tail (t), total length (tl), total body length (tbl), and teeth formation, were noted. these morphological metrics served as the bases for the identification of bat species. the dichotomous key for philippine bats by ingle and heaney (1992) was used for species identification and classification. after taking all the taxonomic information, bats were fed with a sucrose solution and were released back to their captured habitat. however, upon the release, markings for each individual were considered to avoid count duplication if recaptured. on the other hand, dead bat individuals were prepared as voucher specimens and were preserved directly in a 10% formalin solution. data analysis the shannon-weiner diversity index (h’) was used as the primary index to represent bats’ diversity. along with this, the species diversity equitability (hmax) or the highest possible diversity index that can be obtained based on the given data set was also determined to assess how close the h’ index to the theoretical value. moreover, the simpson diversity index (1-d) and species evenness (j’) were calculated to assess the level of species dominance and how evenly distributed the community’s population. with an interpretation that if the obtained 1-d and j’ values are closer to the value of one, means more diverse, and if it is closer to zero, indicates the opposite. the species rarefaction curve was generated to represent the estimated species richness of bats in the sampled area and assess the bat sampling adequacy. for determining the species distribution, the statistical analysis – chi-square was used to evaluate if the bat species’ populations are either random or aggregated. the level of significance used was 0.05 percent. the distribution probability values that are greater than the level of significance was given a random remark. in contrast, those with below the significance value are considered to have an aggregated distribution pattern. all these analyses were done using the biodiversity professional software 2.0 by macleece (1997). all ecological and conservation assessments were based on the international union conservation for nature (iucn) redlist of 2020. results and discussion species richness and diversity a total of 196 individuals of bats belonging to three families and nine genera representing ten species were recorded. the species richness is comparatively lower than the records in loboc watershed, mt. irid, mt. kanlaon, mt. malindang, mt. matutum, mt. palali, polilo islands, and sikatuna protected landscape. the discrepancy of the number of species observed is attributed to the sample size. the aforementioned studies had a larger sample area that crossed a broader elevational gradient and various vegetation types. on the other note, the result is observed to have a higher richness as compared to the records in bagumbayan, bega watershed, lowland forest of upland cavite, mt. apo, mt. kalatungan, mt. kitanglad, mt. natib, and cagayan de oro river. the findings even equate with the number of species observed in the expansion site of mt. hamiguitan in davao oriental and clarin river in misamis occidental even though these studies also covered a more extensive sampling area with at least four sampling stations (tab. 1). with this, it could entail that awasian water forest reserve houses more bats with respect to the ratio and proportion between the species observed and the sampled area. the findings could also imply that the area has better ecological support for bat assemblage since it can hold various species that even surpasses some of those major mountain ecosystems in the philippines like mt. apo and mt. kalatungan. a condition that conforms with the findings of mohagan et al. (2015) that bat richness tends to be higher in a forest ecosystem with more evident ecological support. not to mention that probable new species could be added to the list if the sampling effort was extended as the species rarefaction depicts (fig. 2). however, the chance is narrowing down as the data saturation is gradually flattening. as for diversity measures, the secondary mixeddipterocarp forest showed to have a consistently better result in all the calculated diversity analysis compared to the dipterocarp forest (fig. 3). this result is attributed to a more complex plant structure that results in broader food options for bats. it was noted that at the course of the study, the explicitly observed fruit trees in the secondary mixed-dipterocarp forest biologica nyssana ● 12 (1) september 2021: 71-78 balo et al. ● bats assemblages in awasian water forest reserve, tandag city, surigao del sur, philippines 74 like l. domesticum, d. zibithenus, and a. odoratissimus were fruiting, thus, possibly attracting more species and population of bats in the said habitat. not to mention that the dominant bat-guild observed was fruit bats; hence, the likelihood for the species to congregate in the area is plausible. the observation aligns with the report of hodgkison and balding (2004) and shafie et al. (2011) that bat diversity in a particular area is highly influenced by food availability. meanwhile, the overall all bat diversity in the area was revealed to be moderate. among the species, cynopterus brachyotis and ptenochirus jagori were the well-documented bats, wherein the two species comprised 53 and 25 percent of the population, respectively (fig. 4). the result conforms with various studies conducted in the philippine forests (tab. 1). according to tan et al. (1998), c. brachyotis is widely distributed in the southeast asian region and is common in the philippine forest. the species also has a wide range of habitat preferences, extending from primary forest to disturbed habitats. while the species p. jagori is a common philippine endemic bat observed in the lowland forests (sedlock et al., 2008; iucn, 2020). table 1. summary table of some of the bat studies conducted in the philippines site species richness endemism (%) dominant species observed references bagumbayan, sultan kudarat 8 38 cynopterus brachyotis tanalgo (2017) bega watershed, agusan del sur 8 50 cynopterus brachyotis monteclaro and nuñeza (2015) cagayan de oro river, misamis oriental 8 25 cynopterus brachyotis lobite et al. (2013) clarin river, misamis occidental 10 40 ptenochirus jagori del socorro et al. (2018) loboc watershed, bohol 15 40 cynopterus brachyotis joe et al. (2012) lowland forest, upland cavite 6 67 * lagat and causaren (2018) mt. apo, north cotabato 8 38 cynopterus brachyotis achondro et al. (2014) mt. hamiguitan, davao oriental 10 40 cynopterus brachyotis amoroso et al. (2019) mt. irid, southern sierra madre 16 19 cynopterus brachyotis balete et al. (2013) mt. kalatungan, bukidnon 6 50 haplonycteris fischeri mohagan et al. (2018) mt. kanlaon, negros occidental 23 35 cynopterus brachyotis deligero et al. (2015) mt. kitanglad, bukidnon 4 75 alionycteris paucidentata mohagan et al. (2015) mt. malindang, misamis occidental 19 47 cynopterus brachyotis nuñeza et al. (2006) mt. matutum, saranggani 15 47 cynopterus brachyotis nuñeza et al. (2015) mt. natib, bataan province 9 33 cynopterus brachyotis rickart et al. (2013) mt. palali, caraballo mountains 12 42 otopteropus cartilagonodus alviola et al. (2011) polilo islands, calabarzon 25 24 * alviola (2010) sikatuna protected landscape, bohol 11 44 cynopterus brachyotis van vegchel (2003) fig. 2. species rarefaction plot in awasian water forest reserve biologica nyssana ● 12 (1) september 2021: 71-78 balo et al. ● bats assemblages in awasian water forest reserve, tandag city, surigao del sur, philippines species distribution pattern across habitats, endemicity, and conservation status out of the ten bat populations, the population of p. jagori (fruit bat) and r. subrufus (insectivorous bat) were noted as aggregated (p<0.05). on the other hand, the rest of the bat populations were noted as randomly distributed (p>0.05) (tab. 2). the results could suggest two major points: first, for the case of p. jagori and r. subrufus, necessary ecological support like food source is only found in a particular area or habitat and hence, the reason why the populations of the two bats tend to cluster. secondly, as for those bat populations with random distribution, the ecological factors that support the population are sporadically distributed in the area; hence, its population is also randomly distributed. this observation conforms with the report of de jong and ahlen (1991) and kush et al. (2004) that food source and supply are essential indicators for bat’s regional distribution, especially for insectivorous bats. among the ten documented species, five bats are assessed as philippine endemic, and one is noted as mindanao endemic. the assessment leads to com75 table 2. distribution pattern of bat’s population across habitats taxon variance mean chi-sq df. probability remarks pteropodidae cynopterus brachyotis 24.5 52.5 0.4667 1 0.501796 random eonycteris robusta** 4.5 1.5 3 1 0.079335 random haplonycteris fischeri** 0.5 2.5 0.2 1 0.659200 random harpyionycteris whiteheadi** 0.5 1.5 0.3333 1 0.571066 random macroglossus minimus 12.5 7.5 1.6667 1 0.193468 random ptenochirus jagori** 420.5 24.5 17.1633 1 0.000066 aggregated ptenochirus minor* 18 5 3.6 1 0.054660 random rhinolophidae rhinolophus subrufus** 12.5 2.5 5 1 0.023993 aggregated vespertilionidae miniopterus schreibersi 0.5 0.5 1 1 0.318676 random pipistrellus sp. 0.5 0.5 1 1 0.318676 random note: names with a double asterisk (**) are philippine endemic bat species. while names with a single asterisk (*) are mindanao endemic fig. 3. comparative bar graph of shannon-weiner diversity index (h’), maximum diversity index value (hmax), species diversity equitability (h’/hmax), and simpson diversity index (1-d) biologica nyssana ● 12 (1) september 2021: 71-78 balo et al. ● bats assemblages in awasian water forest reserve, tandag city, surigao del sur, philippines prehensive documentation of 60% (6/10) total bat endemicity in the area. this total endemism is observed to be relatively higher than the bat endemism reported in various philippine forests (tab. 1), with an endemism percentage difference that ranges from 10 to 41 percent. thus, suggesting that the area is an ideal abode to various endemic species. meanwhile, among the bats, two species (e. robusta and r. subrufus) are evaluated to be under the near-threatened category based on the iucn 2017 and 2020 assessments. under this criterion, the species are considered to have a decreasing population and are subject to a threatened status if pressures on its existence will be unaddressed. according to iucn (2020), the primary threat that affects these species’ populations is habitat loss. an evident threat in the buffer zones of the watershed. conclusions awasian water forest reserve harbors various species of bats with high species endemicity. the presence of bat species with random distribution patterns is advantageous to the area, for it contributes to a wider dispersion of ecological services like pollination and seed dispersal. near-threatened species’ presence suggests further conservation efforts considering that threats, especially from anthropogenic pressures, are evident. acknowledgment. the authors would like to express its heartfelt gratitude to the department of environment and natural resources (denr), tandag water district (twd), and local government unit of tandag for the permits and logistical support. to mr. gerry bernadas, aldrin dua, jose buaya, jesus verano, and all the local guides for the extended support during the study’s conduct. references achondo, m.j.m.m., casim, l.f., tanalgo, k.c., agduma, a.r., lloyd, b., bretaña, p., mancao, l.s., salem, j.g.s., bello, v.p. 2014: occurrence and abundance of fruit bats in selected conservation areas of north cotabato, philippines. asian journal of conservation biology, 3(1): 3-7. alviola, p.a. 2000: the distribution and ecology of bats in the polillo islands, philippines. wildlife of polillo island, philippines. university of oxford-university of the philippines at los banos polillo, 99. alviola, p.a., duya, m.r.m., duya, m.v., heaney, l.r., rickart, e.a. 2011: mammalian diversity patterns on mount palali, caraballo mountains, luzon. fieldiana life and earth sciences, 2011(2): 61-74. amoroso, v., mohagan, a., coritico, f., laraga, s., lagunday, n., domingo, k. l., colong, r.d., ponce, r. 2019: status of mammals in the expansion sites of the 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journal of biodiversity and environmental sciences, 6(6): 377-390. philippine atmospheric geophysical and astronomic services administration (pagasa)department of science and technology. 2011. climate change in the philippines. funded under the mdgf-1656 strengthening the philippines institutional capacity toadapt to climate change. quibod, m.n.r.m., alviola, p.a., de guia, a p.o., cuevas, v.c., lit jr, i.l., pasion, b.o. 2019: diversity and threats to cave-dwelling bats in a small island in the southern philippines. journal of asiapacific biodiversity, 12(4): 481-487. rickart, e.a., heaney, l.r., balete, d.s., alviola, p.a., duya, m.r.m., duya, m.v., sedlock, j.l. 2013: the mammals of mt. natib, bataan province, luzon, philippines. national museum of the philippines journal of natural history, 1: 31-44. shafie, n.j., sah, s.a. m., latip, n.s.a., azman, n.m., khairuddin, n.l. 2011: diversity pattern of bats at two contrasting habitat types along kerian river, perak, malaysia. tropical 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ecological questions, 25: 85-93. van vegchel, f.j.p. 2002. a survey of the bats of rajah sikatuna protected landscape, bohol island, philippines and local attitudes towards them. sylvatrop, the technical journal of philippine ecosystems and natural resources, 13(1-2): 1-14. 78 biologica nyssana ● 12 (1) september 2021: 71-78 balo et al. ● bats assemblages in awasian water forest reserve, tandag city, surigao del sur, philippines anticancer compounds from medicinal plants biologica nyssana 4 (1-2)  december 2013: 1-7 stešević, d. caković, d.  contribution to the alien flora of montenegro … 97 short communication first record of erpobdella concolor (annandale, 1913) (hirudinida: erpobdellidae) from greece clemens grosser 1 and vladimir pešić 2 1 am wasserturm 20, 04523 elstertrebnitz, germany; 2 department of biology, university of montenegro, cetinjski put bb, 81000 podgorica, montenegro * e-mail: vladopesic@gmail.com abstract: grosser, c., pešić, v.: first record of erpobdella concolor (annandale, 1913) (hirudinida: erpobdellidae) from greece. biologica nyssana, 4 (1-2), december 2013: 97-98. erpobdella concolor (annandale, 1913) was recorded for the first time from a spring in the peloponnese, greece. this locality represents the westernmost spot of the species range known so far. key words: hirudinida, erpobdellidae, erpobdella concolor, greece, first record introduction erpobdella concolor (a n n a n d a l e , 1913) is presumably considered as the east-mediterranean species (n e s e m a n n & n e u b e r t 1999), but the current knowledge of the geographical distribution of this species is still far from complete. recently, g r o s s e r & p e š i ć (2006) revealed possibly the eastern border of range of this species in the kerman province in iran. however, it has not been clear how far the range extends to the west in the mediterranean region. this research deals with the finding of erpobdella concolor in the peloponnese, greece, and establishes the westernmost locality of the species known so far. material and methods on 4 th july, 2017, one leech specimen was collected by hand under stones within 10-50 cm off the shore of the small spring (sentenikos spring, 37°11'31.06"ν, 22°21'53.61"ε, close to zoros spring, region of sparti town) in the central peloponnese. the specimen was collected by junior author and preserved in 96% ethanol. the specimen is deposited in the collection of senior author. results and discussion the specimen was identified as erpobdella concolor (a n n a n d a l e , 1913). the anterior part of the body is cylindrical; posteriorly, there are two blunt keels. the body is unicoloured without paramedian stripes. the male genital pore is situated in furrow b2/a2, the female in furrow b5/c11. the genital pores are separated by two annuli. the size of the leech is 13 mm in the length and 3 mm in the width. the single specimen of erpobdella concolor (a n n a n d a l e , 1913) from the peloponnese agree well with the description of n e s e m a n n & n e u b e r t (1999). this species was a long time treated as subspecies of dina lineata (m ü l l e r , 1774). however, recent phylogenetic studies (t r o n t e l j & s k e t 2000; s i d d a l l 2002) based on morphology and dna sequence data showed that a revision of the family was necessary because the morphological characters (first the annulation) used to distinguish former erpobdellid 4 (1-2) • december 2013: 97-98 biologica nyssana 4 (1-2)  december 2013: 97-98 grosser, c. pešić, d.  first record of erpobdella concolor … 98 genera are not informative. for this reason, s i d d a l l (2002) after morphogenetic analysis synonymized all the genera of erpobdellidae with erpobdella. furthermore, s i d d a l l (2002) elevated erpobdella concolor to full species status. according to the most comprehensive revision of the european leeches by nesemann & n e u b e r t (1999), erpobdella concolor is known from cyprus, southern turkey, syria, iraq, israel, jordan and the northern part of saudi arabia. the stream in village sirch in the kerman province (se iran) represent the easternmost locality of this species (g r o s s e r & p e š i ć 2006). on other side, our finding in the peloponnese represent the westernmost spot of the species range known so far. no informations on the ecology of this species are available. we found this species in the peleponnese under stones of a spring with a stream outflow (fig. 1). the accompaining fauna includes: planorbis atticus b o u r g u i g n a t , 1852 (gastropoda), eylais mutila k o e n i k e , 1897 (hydrachnidia) and scarodytes roberti f e r y , 2011 (coleoptera). in greece, two erpobdellid species have been found (see: n e s e m a n n & n e u b e r t 1999): erpobdella testacea (s a v i g n y , 1822) and e. latestriata (n e s e m a n n & n e u b e r t , 1995). additional field work is highly needed for appropriate evaluation of leech biodiversity in greece as well the extant distribution of erpobdella concolor. references grosser, c. & pešić, v. (2006) on the diversity of iranian leeches (annelida: hirudinea). archives of biological sciences, 58 (1): 21-24. nesemann, h. & e. neubert (1999): annelida, clitellata: branchiobdellida, acanthobdellea, hirudinea. – in: schwoerbel, j., & p. zwick (eds): süßwasserfauna von mitteleuropa. begründet von a. brauer 6/2, 178 pp., (spektrum) heidelberg siddall, m. e. (2002). phylogeny of the leech family erpobdellidae (hirudinea: oligochaeta). invertebrate systematics, 16: 1–6. trontelj, p. & sket. b. (2000) molecular reassessment of some phylogenetic, taxonomic and biogeographic relationships between the leech genera dina and trocheta (hirudinea: erpobdellidae). hydrobiologia, 438: 227-235. fig. 1. the westernmost locality of erpobdella concolor (annandale, 1913): spring in region of sparti town, peloponnese, greece stojanović-radić et al. 2021, biologica nyssana 12(1) 12 (1) september 2021: 33-45 doi: 10.5281/zenodo.5522981 antistaphylococcal activity of thymus vulgaris and origanum vulgare essential oils: timelapse kinetics, antibiofilm activity and synergistic potential original article zorica stojanović-radić department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, niš, serbia zorica.stojanovic-radic@pmf.edu.rs (corresponding author) marina dimitrijević department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, niš, serbia dimitrijevicmarina92@yahoo.com ana aleksić department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, niš, serbia 90anna.aleksic@gmail.com nikola stanković department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, niš, serbia nikola.stankovic@pmf.edu.rs received: may 21, 2021 revised: june 23, 2021 accepted: september 01, 2021 abstract: staphylococcus aureus is one of the most frequent human pathogens, whose high virulence and severity of chronic infections are related to their ability to produce biofilms. in the present study, antimicrobial potential of oregano and thyme essential oils on the planktonic growth of s. aureus clinical strains (isolated from nose, throat and wound), as well as the effect of the oils on the biofilm production, compared with conventional antibiotic streptomycin were investigated. in addition, time-lapse kinetics and combinations of individual oils with streptomycin were investigated for synergism against the selected staphylococcal strains. the results showed high potential of both oils where minimal inhibitory values ranged between 0.078 and 2.50 mg/ml, while biofilms were reduced up to 96%. in the case of biofilms, the reverse concentration dependency has been observed. time-lapse kinetics showed recovery of some strains after 4 h of contact with the oils, but these strains demonstrated significantly reduced growth in comparison to the control. all mentioned assays showed slightly higher efficacy of oregano essential oil. calculated fics showed either synergistic or additive effect of all tested combinations (thyme-oregano, thyme-streptomycin, oregano-streptomycin), with the thyme-streptomycin as the most efficient one. all mentioned results point to a very high potential of both oils to be used as an adjuvant agent for control of human staphylococcal infections. key words: thyme, oregano, staphylococcus aureus, antibiofilm activity, synergism, clinical isolates apstract: antistafilokokna aktivnost etarskih ulja thymus vulgaris i origanum vulgare: vremenska kinetika, antibiofilm aktivnost i sinergistički potencijal staphylococcus aureus is one of the most frequent human pathogens, whose hstaphylococcus aureus je jedan od najčešćih humanih patogena, čija se visoka virulencija i ozbiljnost hroničnih infekcija povezuju sa sposobnošću produkcije biofilmova. u ovom istraživanju, ispitivani su antimikrobni potencijal etarskih ulja origana i timijana na planktonski rast kliničkih sojeva s. aureus (izolovanih iz nosa, grla i rane), kao i efekat ulja na produkciju biofilma u poređenju sa konvencionalnim antibiotikom streptomicinom. u dodatku, u odnosu na odabrane sojeve stafilokoka istraživani su vremenska kinetika i kombinovan efekat etarskih ulja sa streptomicinom u smislu sinergizma. rezultati su pokazali visok potencijal oba ulja gde je minimalna inhibitorna aktivnost bila u opsegu između 0,078 i 2,50 mg/ml, dok su biofilmovi bili smanjeni za do 96%. u slučaju biofilmova, uočena je obrnuta koncentraciona zavisnost. vremenska kinetika je ukazala na oporavak nekih sojeva nakon 4 h kontakta sa uljem, ali su ovi sojevi imali značajno umanjen rast u odnosu na kontrolu. svi pomenuti testovi pokazali su nešto značajniji efekat ulja origana. preračunati fik-ovi pokazali su ili sinergistički ili aditivni efekat svih testiranih kombinacija (timijan-origano, timijan-streptomicin, origanostreptomicin), dok je najefikasnija bila timijan-streptomicin. svi pomenuti rezultati ukazuju na veoma visok potencijal oba ulja za primenu u vidu dopunskih agenasa za kontrolu humanih infekcija izazvanih stafilokokama. ključne reči: timijan, origano, staphylococcus aureus, antibiofilm aktivnost, sinergizam, klinički izolati © 2021 stojanović-radić et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work noncommercially under the same license as the original. 33 introduction in the past few decades, increased number of bacterial infections, reduced antimicrobial efficacy and newly emerged microbial resistance have become global public concern, leading to improving interest in the examination of the antimicrobial activity of plantbased therapeutics (xiao et al., 2019; zhang et al., 2020). among all alternative natural antimicrobial agents, essential oils display the most significant antimicrobial potential. it is important to note that no tolerance or resistance to essential oils has been discovered yet, which can be explained by the great complexity of their structure and action on several target places at the same time, rather than conventional antibiotics (man et al., 2019). in the contemporary experimental research, an increasing interest is directed to the modulation of virulence factors of microorganisms, in terms of avoiding resistance development (zhang et al., 2020). biofilm is considered to be a major virulence factor that contributed to >80% of all human infections. biofilms are highly organized multicellular bacterial communities consisting of a complex matrix composed of polysaccharide, proteins, lipids and extracellular dna. the best way of controlling biofilms is to prevent their development (abdallah et al., 2020). staphylococcus aureus (s. aureus) is one of the most dominant human pathogen, responsible for a variety of chronic and severe infections including simple soft skin infections, endocarditis, bacteremia, and severe pneumonia (xiao et al., 2019). this bacteria is the member of the upper respiratory microbiome in more than 30% of healthy individuals (prince, 2013). once hosts defense mechanisms are disrupted, s. aureus can proliferate and the host begin to be a nasal carrier (najem, 2020). there are substantial epidemiological data indicating an association between nasal (or other mucosal site) colonization and subsequent s. aureus infection. surgical wounds contamination with skin staphylococci is not a rare case, and the results of the one study confirmed the molecular similarity of strains isolated from wounds and those found in the nose of the same patients (prince, 2013; najem, 2020). since 1960, with the emergence of mrsa-strains (methicillin-resistant s. aureus), s. aureus has become a key pathogen associated with many health-care infections, that are usually difficult to treat (uzair et al., 2017). staphylococcus aureus possesses a variety of virulence factors and their ability to form biofilm plays a critical role in chronic infections. a negative modulation or complete inhibition of biofilm formation may represent an important strategy for infection control and is considered as a major target for the development of novel therapeutic agents (papa et al., 2020). aromatic plants from the lamiaceae family present great sources of biologically active substances with wide use and proven antimicrobial and antioxidant properties (pecarski et al., 2016; uzair et al., 2017; man et al., 2019; zhang et al., 2020). anti-virulence activity of these essential oils have been well investigated and some of them such as cinnamon, oregano, clove, and ginger oil (zhang et al., 2020) are classified as gras (generally recognized as safe) by the u.s. food and drug administration (fda). anti-biofilm activity of these oils and its main compounds (thymol and carvacrol) against food-born (bilge et al., 2010; vázquez-sánchez et al., 2014) and ocular infections isolates (nostro et al., 2007) or mrsa strains (abdallah et al., 2020) has been investigated in several research papers. in the recently published study, the effectiveness of the essential oils (eucalyptus, cinnamon, clove, and tea tree) on the biofilm-producing ability of s. aureus nasal isolates was demonstrated (najem, 2020). in the present study we investigated antimicrobial potential of oregano and thyme essential oils on the planktonic growth of s. aureus clinical strains (isolated from nose, throat and wound), as well as the effect of the oils on the biofilm production, compared with conventional antibiotic streptomycin. in addition, time-lapse kinetics analysis was performed to determine the time required for exhibiting antimicrobial activity and the time necessary for recovery of the cells at sub/inhibitory concentrations. finally, to explore the potential of these two oils in enhancing the effect of other antimicrobials, the combined oils and also combinations of individual oils with streptomycin were tested for synergism against the selected staphylococcal strains. materials and methods isolation of essential oils plant material of origanum vulgare l. and thymus vulgaris l. in pulverized form (aerial parts) was purchased at the local herb market and used for extraction of the essential oils. isolation of the essential oils was performed by using the original clevenger-type apparatus and the obtained oils were separated by extraction with diethyl ether, dried over anhydrous mgso4 and stored at -20 °c before use. test microorganisms the essential oils were tested against a total of 12 strains of staphylococcus aureus clinical isolates of different origin (nose, throat, wound and atcc – american type culture collection strain 6538). 34 biologica nyssana ● 12 (1) september 2021: 33-45 stojanović-radić et al. ● antistaphylococcal activity of thymus vulgaris and origanum vulgare essential oils: time-lapse kinetics... 35 after isolation on blood agar and identification (selective media-msa agar and coagulase test), the cultures were maintained on nutrient agar until used for testing. determination of minimal inhibitory concentration (mic) sensitivity of the staphylococcal strains to the action of oregano and thyme essential oils was determined by a broth microdilution method as previously described (stojanović-radić et al., 2020). overnight cultures (18 h, exponential phase) were used for making suspensions of bacteria in sterile physiological saline (0.85% nacl). the obtained suspensions were adjusted to 0.5 mcfarland turbidity (den-1, biosan densitometer) and further used to inoculate the wells of a 96-well microtiter plates. stock solutions of the essential oils made in 100% dmso were serially diluted in the microtiter plate in the concentration range from 0.01-5.00 mg/ ml and then inoculum was added to each well. the final density of bacterial inoculum was 1-5 x 106 cfu (colony forming unit)/ml in each well. the plates included growth control (mueller hinton broth + 10% dmso + inoculum), sterility control (mhb + 10% dmso + test oil) as well as positive control (mhb + inoculum + serial double dilutions of streptomycin). after incubation of the plates at 37 °c for 24 h, the bacterial growth was determined by adding 20 µl of 0.5% (w/w) 2,3,5-triphenyltetrazolium chloride aqueous solution. minimal inhibitory concentration of the oil was defined as the lowest concentration of the tested samples where visible growth was not detected after the addition of the growth indicator. anti-biofilm assay to investigate the potential of the isolated strains to form biofilm, the tested isolates were first subjected to biofilm forming ability crystal violet (cv) assay as previously described (pejčić et al., 2020). the wells of the microtiter plates containing 200 μl of tryptone soy broth containing 0.5% (w/v) glucose were inoculated with suspensions prepared as described for microdilution method, to achieve the final concentration of ~106 cfu/ml. the plates were incubated 24 h at 37 °c afterwards the well content was aspirated, and the wells were washed twice with phosphate saline buffer (ph 7.4). then the plates were dried, stained with 0.5% (w/v) solution of cv for 20 min, washed and distained by addition of 250 μl of ethanol (96%, v/v). following 45 min of distaining procedure, the obtained solutions were transferred into a new microtiter plate and the absorbance of each well content was measured at 595 nm using an elisa reader (multiscan ascent, labsystems, finland). according to their biofilmproducing ability, the strains were classified into the following groups: none, weak, moderate, and strong biofilm producers (stepanović et al., 2007). the potential of oregano and thyme essential oils to inhibit biofilm production of the investigated s. aureus strains was done by using the same procedure. the only difference was that the wells contained serial doubling dilutions of the essential oils in the desired concentration range (0.01-5.00 mg/ml) together with inoculum and cultivation media. the percentage inhibition of biofilm formation caused by oregano and thyme essential oils was calculated according to the following equation: percentage inhibition= 100[(a595 of the well containing the essential oil / a595 of the well without essential oil) * 100]. the results are presented on the graph as absorbances of the control and experimental wells, where absorbance of the control represents 100%. time-lapse kinetics of inhibitory action to investigate whether the oils achieve long term inhibitory action and the amount of obtained inhibition against each strain, the modified timekill assay has been performed in microtiter plates. the assay was done by inoculation of the media (final cell density was ~106 cfu/ml) containing the essential oils in mic and mic/2 concentrations and subsequent incubation of the plates for total of 48 h at 37 °c. after inoculation, the absorbance of each well was measured at 600 nm using an elisa reader (multiscan ascent, labsystems, finland) at the following incubation periods: 0, 2, 3, 4, 22, 24, 46 and 48 h. the control wells contained only inoculated medium without essential oils. checkerboard assay the type of interaction between the two essential oils and also between the single oil and antibiotic streptomycin was tested using a microdilution checkerboard assay (stojanović-radić et al., 2020). the testing was performed against the three strains, two isolates and one atcc strain. briefly, the medium containing various combinations of antibacterial agent in question (essential oil or antibiotic), obtained by two-fold serial dilutions, were inoculated to achieve the final concentration of ~106 cfu/ml per well. the plates were incubated for 24 h at 37 °c and the combined effects of essential oils with antibiotic or with other essential oil were calculated. the obtained results are expressed as fractional inhibitory concentrations (fics) and the calculation was done using the following equations: ficeo= miceo-s/miceo fics=mics-eo/mics biologica nyssana ● 12 (1) september 2021: 33-45 stojanović-radić et al. ● antistaphylococcal activity of thymus vulgaris and origanum vulgare essential oils: time-lapse kinetics... 36 fici= ficeo + fics ficeo1=miceo1-eo2/miceo1 ficeo2=miceo2-eo1/miceo2 fici=ficeo1 + ficeo2 where ficeo is the fractional inhibitory concentration of essential oil (oregano or thyme), miceo-s is mic of the oil in combination with streptomycin, miceo is the mic of the oil alone; fics is the fractional inhibitory concentration of streptomycin, mics-eo is mic of the streptomycin in combination with the oil, mics is the mic of the streptomycin alone; ficeo1 is the fractional inhibitory concentration of oregano, miceo1-eo2 is mic of the oregano oil in combination with thyme oil, miceo1 and miceo2 is the mic of the oregano and thyme oil alone, respectively; ficeo2 is the fractional inhibitory concentration of thyme, and miceo2-eo1 is mic of the thyme oil in combination with oregano oil. fici was interpreted as synergistic when the value was ≤0.5, as an additive or indifferent when fici was >0.5 and ≤2, and as antagonistic interaction when it was ≥2. the antimicrobial activity of o. vulgare and t. vulgaris essential oils was exhibited against all tested strains and minimal inhibitory values ranged between 0.078 and 2.50 mg/ml (tab. 1). the oregano essential oil showed higher antistaphylococcal potential by inhibiting the growth of the tested panel of staphylococci in the range 0.156-0.625, with the average mic of 0.45 mg/ml. on the other hand, thyme essential oil exhibited inhibition in the broader mic range (0.078 2.50 mg/ml, micavrg=1.1 mg/ ml) and mostly at much higher concentrations in comparison to oregano essential oil. among the strains of staphylococci, the strain no. 7 showed the highest resistance, while the most sensitive one has been the strain no. 4. these data are not corresponding to those for antibiotic, since the two strains showed the same sensitivity to streptomycin. this is one more proof that the oils possess different mode of action then the antibiotic, used as a control antibacterial agent. previous studies on the t. vulgaris essential oils antimicrobial efficacy reported variable active concentrations against s. aureus, which were highly dependent on the essential oil composition and ranged from ˂0.2 µl/ml to 4 mg/ml (rota et al., 2008; kazemi et al., 2012; kon & rai, 2012; de carvalho et al., 2015; pesavento et al., 2015; benabed et al., 2016; valizadeh et al., 2016; fani & kohanteb, 2017; gedikoğlu et al., 2019; pourazar et al., 2018). studies on the antimicrobial activity of oregano essential oil showed very high efficacy against many bacterial species due to high content of a well-known antibacterial compounds, carvacrol and thymol, also present in the thyme essential oil (sakkas & papadopoulou, 2017). it has been reported that oregano essential oil inhibited the growth of mrsa (methicillin resistant s. aureus strains) at concentrations of 0.16 and 0.32 mg/ml (boskovic et al., 2015; lu et al., 2018). in other studies where efficacy of oregano has been investigated against s. aureus strains by microdilution method, inhibitory concentrations ranged from 0.15 µg/ml to 0.8 mg/ml (nostro et al., 2007; özkalp et al., 2010; boskovic et al., 2015; marques et al., 2015; pesavento et al., biologica nyssana ● 12 (1) september 2021: 33-45 stojanović-radić et al. ● antistaphylococcal activity of thymus vulgaris and origanum vulgare essential oils: time-lapse kinetics... no. strains thymus vulgaris mic *mg/ml origanum vulgare mic *mg/ml streptomycin mic *mg/ml origin 1 staphylococcus aureus 1.250 0.625 0.00625 nose 2 staphylococcus aureus 1.250 0.312 0.00156 nose 3 staphylococcus aureus 1.250 0.625 0.00078 throat 4 staphylococcus aureus 0.078 0.312 0.00078 nose 5 staphylococcus aureus 1.250 0.312 0.0001 wound 6 staphylococcus aureus 0.625 0.312 0.00039 nose 7 staphylococcus aureus 2.500 0.625 0.00078 nose 8 staphylococcus aureus 1.250 0.625 0.00078 atcc 9 staphylococcus aureus 0.625 0.625 0.0001 nose 10 staphylococcus aureus 1.250 0.625 0.00312 nose 11 staphylococcus aureus 1.250 0.312 0.00078 nose 12 staphylococcus aureus 0.625 0.156 0.00078 nose table 1. antistaphylococcal activity of the thymus vulgaris and origanum vulgare essential oils (mic: minimum inhibitory concentration; atcc: american type culture collection) 37 biologica nyssana ● 12 (1) september 2021: 33-45 stojanović-radić et al. ● antistaphylococcal activity of thymus vulgaris and origanum vulgare essential oils: time-lapse kinetics... fig. 1. antistaphylococcal activity of the thymus vulgaris and origanum vulgare essential oils *the results are presented as the percent of formed biofilms, where the control biofilm absorbance represents 100%. ovabsorbance of the wells containing origanum vulgare essential oil; tv absorbance of the wells containing thymus vulgaris essential oil; str streptomycin; c control biofilm. 38 biologica nyssana ● 12 (1) september 2021: 33-45 stojanović-radić et al. ● antistaphylococcal activity of thymus vulgaris and origanum vulgare essential oils: time-lapse kinetics... fig. 2. antibiofilm activity of oregano and thyme essentail oils against the isolates of staphylococcus aureus no. 7-12 *the results are presented as the percent of formed biofilms, where the control biofilm absorbance represents 100%. ovabsorbance of the wells containing origanum vulgare essential oil; tv absorbance of the wells containing thymus vulgaris essential oil; str streptomycin; c control biofilm. 2015; araujo & longo, 2016; evangelista-martínez et al., 2018; simirgiotis et al., 2020; xiao et al., 2020). in comparison to these results, the oregano essential oil investigated in this paper fits into the previous range of active concentrations. biofilm producing ability and antibiofilm activity the results on the biofilm producing ability of investigated s. aureus strains demonstrated that only one strain was a weak biofilm producer (no. 5), three strains were moderate producers (no. 7, 9, 11), while the remaining strains showed strong biofilm producing ability. the testing of the two essential oils showed that oregano oil had higher antibiofilm activity and that this activity was the most efficient at half-mics (fig. 1 and fig. 2). the double and four-time increment of the oils concentration (mic, 2 x mic) did not cause higher reduction. in contrast, reduction was lower with higher oil concentrations. the only exception was the action of this oil against isolate no. 5, a weak biofilm producer. the observed reductions were higher than 90% of the control biofilm for strains no. 1, 2, 4, 6 and 8 and more than 80% in strains no. 3, 7, 9 and 10. in contrast to these strains, the strain no. 5 demonstrated direct concentration-dependency where the reduction ranged from 39% (half mic) to 48% (2 x mic). in strain no. 11, the reductions were concentration dependent and ranged from 55 to 60%. finally, the strain no. 12 demonstrated very unusual effect of oregano essential oil, where half mic reduced biofilm by 72%, but higher concentrations promoted biofilm formation. similar results were obtained when these three concentrations were tested against p. aeruginosa in the study of pejčić et al. (2020) where in many cases, higher concentrations exhibited a lower reduction of biofilm formation. this was explained by an enhanced production of exopolysaccharides (eps) as a response of the cells to stress caused by high concentrations of antimicrobials. by this production, eps interfere with diffusion and reduce their toxicity. also, these high concentrations can cause decreased interaction with cells and/or activation of some resistance mechanisms in the biofilm cells (pejčić et al., 2020). therefore, herein obtained results suggest that similar processes occur in s. aureus biofilms as well. in the study of nostro et al. (2007), oregano essential oil antibiofilm efficacy was tested at three sub-inhibitory concentrations (mic/2, mic/4 and mic/8). their results showed increased reduction with higher concentrations, which is opposite to our results. the explanation might be that in present work, we tested only one subinhibitory concentration and it obtained very high efficiency. therefore, only subinhibitory concentrations should be tested in the future. lu et al. (2017) also confirmed high efficacy of oregano essential oil against 24-h-old multidrugresistant (mrsa) staphylococcal biofilms at concentration of 0.4 mg/ml. in the case of the thyme essential oil, the mentioned reverse concentration dependence has also been observed. here, in almost half of the strains (no. 1, 7, 8, 9, 11) the mentioned concentration dependency was observed, while in others the reductions exhibited by all three concentrations were statistically insignificant (no. 2, 3, 4 and 10). only in strain no. 12, the twice mic demonstrated the highest efficiency. up to now, several studies investigated potential of the thyme essential oil against staphylococcal biofilms (kavanaugh & ribbeck, 2012; vázquez-sánchez et al., 2014; ben abdallah et al., 2020). when the thyme essential oil was tested against sc-01, which is a biofilm-forming, oxacillinand methicillin-resistant clinical isolate, it has been efficient at ∼ 0.8% (v/v) concentration against its biofilm (kavanaugh and ribbeck, 2012). in the study of vazquez-sanchez et al. (2014), the thyme oil showed the highest efficacy with mic of 0.04% (v/v) among several tested essential oils and the most efficiently eradicated 48-h-old biofilms formed on stainless steel. finally, the recent research od abdallah et al. (2020) showed that the essential oil of thymus zygis reduced the biofilms of mrsa isolates up to 91%. in the present study, the reductions exhibited by the thyme essential oil ranged from 31% to 96%. time-lapse kinetics of inhibitory action the present study also dealt with kinetics of inhibitory activity in order to investigate the efficiency of the two oils during a time period of 48 h and the results are presented in fig. 3 and 4. in these experiments, two concentrations were tested, mics and halfmics. the strains 7, 8 and 9 were completely inhibited throughout the entire incubation period by both concentrations of both essential oils. in the case of oregano oil, it has been observed that in all strains the inhibition of growth was complete during the first four hours, afterwards the cultures started recovery and exponential growth in strains 1, 2, 3, 5, 6, 10 and 12. however, this growth was significantly reduced in comparison to the control and also the observed growth was noticed only at half mics (no. 1, 2, 3, 10, 11 and 12) or were concentration dependent (no. 5 and 6). also, in isolates no. 11 and 12, it is notable that the recovery has been prolonged, and the growth started after 24 h for both oils. the thyme essential oil exhibited similar action, where complete inhibition during the entire period of incubation has been observed in strains no. 1, 7, 8, 9 and 12, while some strains recovered 39 biologica nyssana ● 12 (1) september 2021: 33-45 stojanović-radić et al. ● antistaphylococcal activity of thymus vulgaris and origanum vulgare essential oils: time-lapse kinetics... but only at the lower tested concentration (no. 2, 3, 10 and 11). finally, in strains no. 4, 5 and 6, the recovery was observed at both mic and mic/2, but the final growth of bacterial populations has been significantly reduced in comparison to the control values. according to this, the efficiency against planktonic cells is the highest during the first day of incubation and this information might be used for application of some synergistic antimicrobials during this period to achieve the complete killing effect at lower oil/antimicrobial agent concentrations. biologica nyssana ● 12 (1) september 2021: 33-45 40 stojanović-radić et al. ● antistaphylococcal activity of thymus vulgaris and origanum vulgare essential oils: time-lapse kinetics... fig. 3. time-lapse kinetics of oregano and thyme essential oils inhibitory action against staphylococcus aureus clinical isolates (strains no. 1-6) 41 biologica nyssana ● 12 (1) september 2021: 33-45 stojanović-radić et al. ● antistaphylococcal activity of thymus vulgaris and origanum vulgare essential oils: time-lapse kinetics... fig. 4. time-lapse kinetics of oregano and thyme essential oils inhibitory action against staphylococcus aureus clinical isolates (strains no. 7-12) checkerboard assay to find out which is mutual relationship between the oils and also of the oils alone with the reference antimicrobial drug, the testing of sinergism was done by checkerboard assay. calculated fics showed either sinergistic or additive effect of all tested combinations (tab. 2). when the essential oils were combined together, mics of both oils reduced from two to eight times. in combination with streptomycin, sinergistic and additive effect was observed for both oils, with reductions of mics up to eight times for both participants in the combination. biologica nyssana ● 12 (1) september 2021: 33-45 42 stojanović-radić et al. ● antistaphylococcal activity of thymus vulgaris and origanum vulgare essential oils: time-lapse kinetics... c om bi na tio ns st ap hy lo co cc us a ur eu s a tc c 6 53 8 st ap hy lo co cc us a ur eu s * n o. 1 st ap hy lo co cc us a ur eu s * n o. 1 2 m ic al on e m ic co m bi ne d fi c m ic al on e m ic co m bi ne d fi c m ic al on e m ic co m bi ne d fi c 1 th ym e + or eg an o th ym e 1. 25 0. 15 6 0. 12 5 1. 25 0. 62 5 0. 5 0. 62 5 0. 31 2 0. 5 or eg an o 0. 62 5 0. 07 8 0. 12 5 0. 62 5 0. 31 2 0. 5 0. 15 6 0. 07 8 0. 5 *f ic i in te ra ct io n 0. 25 sy n 1 a dd 1 a dd 2 th ym e + st re pt om yc in th ym e 1. 25 0. 15 6 0. 12 5 1. 25 0. 15 6 0. 12 5 0. 62 5 0. 15 6 0. 25 st re pt om yc in 0. 00 07 8 0. 00 02 4 0. 31 3 0. 00 62 5 0. 00 09 0. 15 6 0. 00 07 8 0. 00 04 8 0. 62 6 *f ic i in te ra ct io n 0. 43 8 sy n 0. 28 1 sy n 0. 87 6 a dd 3 or eg an o + st re pt om yc in or eg an o 0. 62 5 0. 07 8 0. 12 5 0. 62 5 0. 15 6 0. 25 0. 15 6 0. 07 8 0. 5 st re pt om yc in 0. 00 07 8 0. 00 04 8 0. 62 5 0. 00 62 5 0. 00 09 0. 15 6 0. 00 07 8 0. 00 09 7 1. 25 *f ic i in te ra ct io n 0. 75 a dd 0. 40 6 sy n 1. 65 a dd ta bl e 2. m in im um in hi bi to ry co nc en tra tio ns (m g/ m l) an d fra ct io na l in hi bi to ry co nc en tra tio n va lu es o f t he m ut ua l c om bi na tio ns o f e o s a nd w ith st re pt om yc in fi c : f ra ct io na l i nh ib ito ry c on ce nt ra tio n; f ic i: fr ac tio na l i nh ib ito ry c on ce nt ra tio n in de x; m ic : m in im um in hi bi to ry c on ce nt ra tio n; a tc c : a m er ic an ty pe cu ltu re c ol le ct io n; s yn : s yn er gi sm ; a dd : a dd iti ve the results pointed to the thyme-streptomycin as the most efficient combination, where sinergism was demonstrated against two out of the three tested strains of staphylococci. also, important to mention is the fact that these combinations were sinergistic for the strains with higher mics of oils. in the third strain, which was more susceptible, additive effect has been observed for all tested combinations. up to now, it has been reported that the thyme oil exhibits sinergistic potential against staphylococci when combined with garlic (garcía-díez et al., 2017), myrtus communis essential oil (sadiki et al., 2014) and with mustard (reyes-jurado et al., 2016). on the other hand, van vuuren et al. (2009) demonstrated the predominance of antagonism against staphylococci (and some other microbial species) when the thyme essential oil was combined with cyprofloxacin. in the case of oregano, it has been reported that it possesses an additive effect in combination with acetic acid (de souza et al., 2009), basil or bergamot essential oils (lv et al., 2011), silver nanoparticles (scandorieiro et al., 2016), as well as with mustard (reyes-jurado et al., 2016). the study of rejes-jurado et al. (2016) demonstrated that in combination, thyme and mexican oregano exhibited additive effect only. our study partially confirms these results since additive effect has been observed for one of the three tested strains. together with this, a recent study of xiao et al. (2019) revealed that oregano when combined with quinolone drugs (tosufloxacin, levofloxacin, ciprofloxacin) and rifampin completely eradicated all stationary phase s. aureus cells. based on the herein presented results, we can suggest that both oils might be used in combination or for an effective enhancement of the streptomycin efficacy against human infections caused by staphylococci. conclusion the present study tested the antimicrobial potential of the thyme and oregano essential oils against clinical isolates of s. aureus. the results showed moderate to high action against planktonic cells and significant antibiofilm potential of both oils. oregano exhibited higher activity in all tests in comparison to the thyme essential oil. time-lapse kinetics study revealed that the action of both oils is strain-dependent and that in most cases, the mics very significantly reduced or even completely eradicated the growth of the cells. also, inhibitory effect in recovering cultures is the most 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received: may 18, 2025 revised: june 10, 2025 accepted: june 11, 2025 abstract: soil health is vital for the sustainability of agriculture. therefore, samples were collected from agricultural lands of 5 different districts of aydın province. in this context, it was determined that the ph values of 61 soil samples collected were between 7.08 and 9.37. when the variation analysis of electrical conductivity (ec) values was analyzed, it was seen that the soils showed a high degree of variability. when the soil samples were analyzed in terms of microbial loads, it was observed that 14.75% of the soil samples were below the healthy soil reference values (6-8 log cfu/g), 3.27% had microbial loads close to the lower limit and 81.98% were microbially healthy. the number of halotolerant bacteria was found to be in the range of 4.3-6.89 log cfu/g in the soil samples. halotolerant bacteria are important microorganisms in aydın agriculture because they are stress-resistant bacteria that can promote plant growth under extreme conditions. key words: agricultural lands, microbial load, soil health, soil sample apstrakt: procena plodnosti poljoprivrednog zemljišta u provinciji ajdin, turska zdravlje zemljišta je od suštinskog značaja za održivost poljoprivrede. zbog toga su uzorkovani uzorci sa poljoprivrednih površina u 5 različitih okruga provincije ajdin. u tom kontekstu, utvrđeno je da su ph vrednosti 61 prikupljenog uzorka zemljišta bile u rasponu od 7.08 do 9.37. varijacionom analizom vrednosti električne provodljivosti (ec) utvrđeno je da zemljište pokazuje visok stepen varijabilnosti. kada su uzorci zemljišta analizirani sa aspekta mikrobnog opterećenja, primećeno je da se 14,75% uzoraka nalazi ispod referentnih vrednosti za zdravo zemljište (6-8 log cfu/g), 3,27% ima mikrobno opterećenje blizu donje granice, dok je 81,98% uzoraka mikrobiološki zdravo. broj halotolerantnih bakterija u uzorcima zemljišta bio je u rasponu od 4.3-6.89 log cfu/g. halotolerantne bakterije predstavljaju važne mikroorganizme u poljoprivredi provincije ajdin jer su to bakterije otporne na stres koje mogu podsticati rast biljaka u ekstremnim uslovima. ključne reči: poljoprivredno zemljište, mikrobno opterećenje, zdravlje zemljišta, uzorak zemljišta introduction while it takes thousands of years for bedrock to turn into soil, it only takes a few decades for our agricultural land to degrade (hillel, 2003). it is a matter of concern that the productivity of agricultural land is decreasing day by day despite the rapid increase in the world population. although alternative crop cultivation methods such as cellular agriculture are being developed, soils will be an indispensable resource for many years to come. for this reason, determining the current status of our indispensable soil, improving it and ensuring sustainability are very important issues. the unconscious pollution of agricultural land by human beings has unfortunately led to irreversible disasters. salinity and alkalinity problems cause salinization and soil loss. salinity and erosion are the main constraints on the productivity of turkish soils (dinç et al., 1991, 2001). eighty percent of türkiye's salt-affected soils are saline or mildly salty. soil salinity is measured by a soil's electrical conductivity (ec), which has a direct impact on plant growth and development. the ec of the solution rises in the presence of ions, or salts. the low ec in the absence of salts suggests that the soil solution has poor electrical conductivity. the quantity and timing of rainfall, internal soil drainage, and irrigation techniques are some of the variables that determine salts. rainfall generally has a tendency to dilute the © 2025 yamaner and okumuş. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 158 salts in the soil. during drought, water is lost from the soil through evaporation and salts are effectively concentrated. in addition to soil salinity, soil ph is also important for plant health. soil ph affects plant growth primarily through nutrient availability. plant-essential nutrients are most available at neutral ph. in addition, soil ph affects nutrient toxicity and microbial activity and has a direct effect on the protoplasm of plant root cells (larcher, 1980; marschner, 1986). a soil ph between 5.5 and 8 is ideal for most plants (brady & weil, 1996). in acidic soils, plants cannot utilize some elements such as potassium, magnesium, calcium and phosphorus, while in basic soils, elements such as copper, zinc, boron, manganese and iron are not easily absorbed by plants (neina, 2019). healthy soil is the basis for productive and sustainable agriculture. in healthy soil, parameters such as salinity and ph, as well as the number and diversity of soil microorganisms are vital. soil microorganisms support soil structure, water infiltration and plant root growth. they also contribute significantly to plant growth by fixing atmospheric nitrogen (datta et al., 2015), increasing the amount of soluble phosphorus in the soil (etesami & maheshwari, 2018), activating plant hormones (ahmad et al., 2008) and producing siderophores (mia et al., 2012; baber et al., 2018). however, overuse of synthetic fertilizers, pesticides and herbicides can damage soil ph and ec as well as beneficial soil organisms. today, pesticides account for nearly one-third of the global chemical input market, which is approximately usd 1.7 trillion. these chemicals negatively affect the diversity and number of soil microorganisms that are effective in soil health. the deterioration in soil quality means much more than a reduction in food production. this represents a broken link in the complex web of life in which every organism, regardless of size, plays a role. a healthy soil ecosystem is filled with billions of microorganisms, including rhizosphere bacteria, all contributing to the soil's nutrient cycling. when this system is disrupted, a domino effect is triggered, jeopardizing the entire ecosystem. it is therefore clear that this issue is vital. maintaining healthy soils is essential to halt desertification and land degradation and even for the balance of the entire ecosystem. in order to monitor the productivity of agricultural lands, it is necessary to monitor the current salinity, alkalinity status and determine the soil microbial load in shorter periods. for these reasons, ph, ec, total number of microorganisms and the number of bacteria that can survive at 6% salt content were determined in soil samples taken from agricultural areas of 5 different districts of aydın province. with these results, it was aimed to have information about the current status of agricultural soils in aydın province. materials and methods collection of soil samples in june and july 2024, 61 soil samples were collected from agricultural lands in 5 different districts [söke (akçakonak, güllübahçe and argavlı neighborhoods), koçarlı, i̇ncirliova, nazilli, bozdoğan] of aydın province in the aegean region (fig. 1). soil samples were taken from the surface to a depth of 25 cm. while sampling from agricultural lands, care was taken to represent the entire area by zigzagging. the collected samples were first placed in paper bags and then in plastic bags and transferred to the laboratory in cold chain. determination of microorganism counts in soil samples microorganism counts in soil samples collected from agricultural fields were carried out according to atlas ronald (1984). each of the collected soil samples was passed through a soil sieve with a hole size of 0.2 mm. from the homogeneously mixed and sieved soil samples, 10 g of soil samples were shaken in 90 ml physiological saline (fts-0.85% nacl) for two hours at room temperature. dilution series were prepared in tubes containing 9 ml fts by taking 1 ml of the suspension and 100 μl of -3 -4, -5 and -6 dilutions were taken and sown on nutrient agar (na) (merck, germany) in 3 replicates. after incubation at 28 °c for 48 hours, colony forming units per gram of soil (cfu/g) were calculated. determination of the number of microorganisms resistant to 6% salt in soil samples each of the collected soil samples was passed through a soil sieve with a hole size of 0.2 mm. homogenously mixed, 10 g of the sieved soil samples were shaken in 90 ml fts for two hours at room temperature. taking 1 ml of the prepared suspension, dilution series were prepared in tubes containing 9 ml fts and 100 μl of the -3 -4, -5 and -6 dilutions were transferred to na containing 6% nacl (merck, germany) in 3 replicates and the sample was spread on the petri surface by smear inoculation method. petri dishes were incubated at 28 °c for 48 hours. after the incubation period, colony forming units per gram of soil (cfu/g) were calculated. measurement of ph and electrical conductivity 20 g of each soil sample was weighed and mixed with 50 ml of deionized water and stirred for biologica nyssana ● 16 (1) june 2025: 157-164 yamaner and okumuş ● evaluation of the fertility status of agricultural lands in aydın province, türkiye 159 biologica nyssana ● 16 (1) june 2025: 157-164 yamaner and okumuş ● evaluation of the fertility status of agricultural lands in aydın province, türkiye 120 minutes. the soil solutions were then filtered through filter paper. the resulting liquid was used to determine ph and electrical conductivity (ec). the ph and ec of the samples were measured using an ad8000 ph/mv/ec/tds & temperature meter. results and discussion in the study, 5 different districts of aydın were visited (fig. 1). sixty-one soil samples were collected from agricultural lands. the ph, electrical conductivity (ec), total number of microorganisms in 1 gram of soil and the number of microorganisms that can survive in 6% salt were determined for each sample. the minimum and maximum values, arithmetic mean, standard deviation and coefficient of variation values of ph and ec values of soil samples are given in tab.1 the ph values of soil samples collected from agricultural lands were found to vary between 7.08 and 9.37. in the same samples, ec values were found to vary between 71-3450 μs/cm. while the coefficient of variation between the ph values of the soil samples collected from agricultural lands was below 10%, the coefficient of variation in ec values was above 50% (tab.1). total mesophilic aerophilic bacteria counts of agricultural soils collected from 5 different districts of aydın were 5.27 8.89 log cfu/g (tab. 2). in 9 of 61 soil samples, the number of bacteria was below 6 log cfu/g (fig. 2). in the same samples, the number of bacteria that can survive in 6% saline environment was 4.3 6.89 log cfu/g (tab. 2). in 31.15% of these soil samples, the number of bacteria that can survive in 6% saline environment was above 6 log cfu/g (fig. 3). coefficient of variation (cv) is the most discriminating factor compared to other parameters to describe the variability of soil properties (zhang et al., 2007). wilding (1985) and mulla & mc bratney (2000) classified the coefficient of variation, which is accepted as an important indicator in explaining the changes in soil properties, according to its values, those less than 15% are classified as less variable, those between 15-35% as moderately variable and those greater than 35% as highly variable. tab. 1 shows that the coefficient of variation of soil ph values is less than 15%. although soil samples did not show variability in terms of ph values, they were found to be highly variable in terms of ec values. according to moasheri & foroughifar (2013), the low coefficient of variation of ph is due to the composition of the main substance in the soil. the high coefficient of variability may be due to land management factors such as fertilization and land use type. richards (1954) reported that the electrical fig. 1. districts (indicated by arrows) where the soil samples used in the study were collected 160 biologica nyssana ● 16 (1) june 2025: 157-164 yamaner and okumuş ● evaluation of the fertility status of agricultural lands in aydın province, türkiye table 1. ec and ph values of the collected soil samples soil sampling site parameter min. max. ao s.s. v.k. söke ec (μs/cm) 519 3450 1661.82 905.97 54.52 ph 7.15 8.08 7.51 0.38 5 i̇ncirliova ec (μs/cm) 174 1220 562.13 322.36 57.35 ph 7.5 9.37 8.26 0.57 6.84 koçarlı ec (μs/cm) 71 580 204.78 150.54 73.51 ph 7.44 8.32 8.03 0.28 3.53 nazilli ec (μs/cm) 109 2100 521.85 638.83 122.42 ph 7.66 8.9 8.22 0.27 3.34 bozdoğan ec (μs/cm) 560 1625 828 597.42 72.15 ph 7.08 7.37 7.34 0.27 3.62 min: minimum value, max: maximum value, ao: arithmetic mean, s.s: standard deviation, vk: coefficient of variation (%), ec: electrical conductivity table 2. total numbers of mesophilic aerophilic bacteria and bacteria viable at 6% nacl in soil samples soil sampling site bacteria counts (log cfu/g) parameter min. max. ao söke mab 5.27 8.48 6.61 %6 nacl mab 4.3 6.89 5.24 i̇ncirliova mab 6.93 7.59 7.27 %6 nacl mab 5.48 6.35 5.92 koçarlı mab 5.66 7.57 6.65 %6 nacl mab 4.6 6.07 5.48 nazilli mab 6.18 8.89 7.31 %6 nacl mab 5.02 6.2 5.89 bozdoğan mab 7.06 7.24 7.15 %6 nacl mab 6.18 6.44 6.35 mab: mesophilic aerophilic bacteria, %6 nacl mab: 6% salt viable mesophilic aerophilic bacteria conductivity value of soil is unsaline when it is less than 2 ds/m, very slightly saline when it is between 2-4 ds/m, moderately saline when it is between 4-8 ds/m, very saline when it is between 8-16 ds/m and extremely saline when it is more than 16 ds/m. the ec values of the soil samples taken from the agricultural lands in akçakonak neighborhood of söke district were lower than güllübahçe and argavlı neighborhoods. it was determined that the soil samples taken from argavlı neighborhood of söke district had the highest ec value (3450 μs/ cm). among 61 soil samples, the highest number of bacteria that can survive at 6% salt (6.89 log cfu/g) was observed in argavlı samples. this suggested that microorganisms adapted to soil salinity (tab. 1 and 2). nazilli soil samples were generally unsaline, but only one sample contained salts in the range of 2-4 ds/m and one sample was close to this range. soil samples from i̇ncirliova, koçarlı and bozdoğan districts were generally classified as saline soils (tab. 1). according to kadam (2016), soil can be classified into three main categories based on ph: acidic soil: ph<6.5 (low ph), normal soil: ph 6.5-7.8 (medium ph) and alkaline soil: ph>7.8 (high ph). according to these criteria, the ph values of the soil samples taken from incirliova were found to be very strongly alkaline (ph:9.37) compared to other districts. when the soils of söke district were analyzed in terms of ph, it was observed that the ph values shifted from normal ph values to alkaline soil (highest ph: 8.08), while similar situations were found in other districts except bozdoğan (tab. 1). başar (2001) reported that the ph values of soil samples obtained 161 biologica nyssana ● 16 (1) june 2025: 157-164 yamaner and okumuş ● evaluation of the fertility status of agricultural lands in aydın province, türkiye monitoring and management of soil ecosystems in the context of changing agricultural practices and land use (semenov et al., 2025). measuring the number of soil bacteria is an indicator of soil health, e.g. if there are 6-8 log cfu/g culturable bacteria per gram of soil, this soil is considered a healthy soil. a count of less than 6 log cfu per gram indicates poor soil health due to nutrient deficiency found in soils with low organic matter, abiotic stress from excessive soil ph values (<5 or >8), or toxicity from organic/inorganic anthropogenic pollutants (ratnakar & shikha, 2018). the number of culturable bacteria in soil samples collected from söke district was between 5.27 and 8.48 log cfu/g (tab. 2). while 45.4% of from soils where different crops were grown in bursa region were slightly and strongly alkaline, taşova & akın (2013) reported that the soils of the marmara region were slightly alkaline. demirekin & erdal (2015) reported that the agricultural soils of hakkâri-çukurca region were slightly alkaline, while yorulmaz et al. (2020) reported that the ph value of söke plain soils was 8.32 on average. it is noticeable that the alkalinity problem seen in soils is general. increasing soil ph negatively impacts its physical properties by disrupting structure and thereby reducing water permeability. integrating microbiological indicators into soil investigations offers a more comprehensive approach to soil health assessment, enabling better fig. 2. total number of mesophilic aerophilic bacteria in soil samples fig. 3. number of mesophilic aerophilic bacteria viable in the presence of 6% nacl in soil samples 162 biologica nyssana ● 16 (1) june 2025: 157-164 yamaner and okumuş ● evaluation of the fertility status of agricultural lands in aydın province, türkiye the soil samples contained less than 6 log cfu/g, 18.2% were close to the lower limit and 36.4% were microbially healthy (fig. 2). the ph and ec values of the soils with low numbers of microorganisms were not very high, suggesting the presence of organic and inorganic pollutants in this region. a similar situation was observed in soil samples collected from koçarlı district. in 44.4% of the soil samples collected from this district, the number of microorganisms in 1 g soil sample was less than 6 log cfu/g (fig. 2). the number of microorganisms in 1 g of soil samples from other districts was within the normal range (fig. 2). increased microbial activity enhances their contribution to soil biochemical processes, hence allowing a reduction in the use of inorganic fertilizers (solihin & fitriatin, 2017; octaprama et al., 2020). soil microorganisms fulfill various functions, including nutrient provision, organic matter decomposition, enhancement of plant growth, and regulation of plant pests and diseases. the quantity of microbial populations in soil might signify soil fertility, since a substantial microorganism population reflects the presence of suitable organic matter, sufficient water availability, optimal temperature, and good soil ecological circumstances. in this context (fig. 1), the soils of i̇ncirliova, bozdoğan, and nazilli districts are presumably more productive than those of söke and koçarlı. in addition to determining the total number of bacteria in 1 g of the collected soil samples, the number of bacteria that can survive in the presence of 6% nacl (halotolerant bacteria) was also determined (tab. 2, fig. 3). the number of halotolerant bacteria in the samples ranged from 4.3 to 6.89 log cfu/g. the highest number of halotolerant bacteria in the soil sample from söke can be explained by the high ec value of the same sample. if we look at the other soil samples, although the ec values are not high, the number of halotolerant bacteria is close to 6 log cfu/g. perhaps there are microenvironments within the general soils that have high salinity at certain times under certain environmental conditions. according to howell et al. (2022), while much of the soil has little to no measurable salinity, pores in soil particles can trap moisture. over time, salts can leach from minerals in the soil to form separate salt-enriched phases. when these evaporites are wetted by dissolution in or direct contact with water, separate brines can form within the soil particles. since saline solutions are a restrictive growth medium, the soil can become enriched in halotolerant microorganisms. since many soils can be quite dry during certain seasons, this competitive advantage may increase the survival of microorganisms under unfavorable conditions. from this point of view, the high number of halotolerant bacteria despite low salinity in samples collected in aydın districts during a season without rainfall for a long time can be considered normal. moreover, the ability of halotolerant bacteria to tolerate variable salinity concentrations, i.e. 3-30% nacl, makes them a suitable candidate for their application in stress agriculture (ventosa et al., 1998). recently, banik et al. (2018) isolated halotolerant bacillus and halobacillus sp. from saline media. they reported that these bacteria supported peanut growth in both saline and metal-contaminated conditions. conclusion the soil food web is a complex and interconnected system of soil-dwelling organisms. soil microorganisms perform important functions such as nutrient cycling, organic matter decomposition, disease suppression, and plant symbiosis. a thriving soil provides numerous benefits to plant nutrition, agriculture and the environment. in general, the soil samples used in the study were found to be moderately to strongly alkaline with high variability in ec values and a wide range between unhealthy and very healthy soils from the microbial point of view. considering the climatic conditions of aydın, the high number of halotolerant bacteria, resistant to harsh conditions such as high ph and arid climate, positively affects the yield and quality in agricultural areas. considering these results, it can be recommended to pay attention to the organic matter ratio in order to increase the microbial activity in the soil. acknowledgements. this research was financially supported by aydın adnan menderes university research fund. project number: zrf-24002. references ahmad, f., ahmad, i., & khan, m. 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(2007). spatial variability of nutrient properties in black soil of northeast china. pedosphere, 17(1), 19–29. microsoft word 0104_stesevic_petrovic biologica nyssana 1 (1-2) december 2010: 35-42 stešević, d., petrović, d.. preliminary list of plant invaders… 35 original article ! preliminary list of plant invaders in montenegro danijela stešević, danka petrović faculty of natural sciences and mathematics, university of montenegro, džordža vašingtona bb, 81 000 podgorica, montenegro * e-mail: denist@t-com.me abstract: stešević, d., petrović, d.: preliminary list of plant invaders in montenegro. biologica nyssana, 1 (1-2), december 2010: 35-42. due to the fact that invasive alien species (ias) are considered to be the second cause of global biodiversity loss after direct habitat destruction and have adverse environmental, economic and social impacts from the local level upwards, in last decades investigations of alien flora of montenegro are intensified. in this paper we are presenting a preliminary list of ias, with the aim to provide a basic data on ias in montenegro, to enable future monitoring and to draw attention on the problems which expansion of ias is bringing with itself. the list consists of 50 plant taxa species and supspecies level. key words: invasive alien plants, montenegro abbrevations: iasinvasive alien species, pphanaerophytes, chchamaephytes, h hemicriptophytes, ttherophytes, ggeophytes, namnorth america, samsouth america, cam & samcentral and south america, asasia, afrafrica, eas-euroasia. introduction ! unlike some worldwide regions where invasive ecology is well developed, in montenegro the interest for this a rather new field in ecology, that study a human mediate transfer of organisms to areas outside their natural dispersal range and the consequences of such transfer, has appeared in last few years: s t e š e v i ć & j o v a n o v i ć (2005, 2008), s t e š e v i ć (2005), steš ević & j o g a n (2006, 2007), t o m o v i ć & s t e š e v i ć (2007), s t e š e v i ć et al. (2009). due to the concept of r i c h a r d s o n et al. (2000), later supplemented by p y š e k et al. (2004), richardson & pyšek (2004), plants taxa in a given area whose presence there is due to intentional or unintentational human involvement, or which have arrived there without the help of people from an area in which they are alien are known as alien plants (exotic, non-native, non-indigenous). invasive plants are naturalized plants that produce reproductive offspring often in very large numbers, at considerable distance from parent plants (approximate scales: > 100m; <50 years for taxa spreading by seeds and other propagules; > 6m/3 years for taxa spreading by roots, rhizomes, stolons, or creeping stem) and thus have a potential to spread over considerable area. on global level invasive species are recognized as a major factor of environmental change, and are considered one of the most important causes of biodiversity loss worldwide (w a l k e r & s t e f f e n , 1997; m c n e e l y , 1999, 2001, issg 2000, millennium ecosystem assessment 2005 etc.). these species have invaded and affected native biota in virtually every ecosystem type on earth and ecological cost is the irretrievable loss of native species and ecosystems. it is estimated that the direct economic cost of alien invasive species runs into many billions of dollars annually (issg 2000). beside mentioned impacts, invasive alien plants sometimes have a negative impact on humans health, such as ambrosia artemisiifolia, heracleum mantegazzianum etc. 10th sfses • 17-20 june 2010, vlasina lake1 (1-2) • december 2010: 35-42 biologica nyssana 1 (1-2) december 2010: 35-42 stešević, d., petrović, d.. preliminary list of plant invaders… 36 considering habitat types, many invasive species seem to do best in urban und urban-fringe environments where long histories of human disturbance have created vacant niches and abundant bare ground. cities also tend to be the focal points of the global economy and the entry points for many invasives (k o w a r i k 1990). pathways, as the process by which alien species are introduced from one location to another are different and it is possible to distinguish six ways: i) deliberate release, ii) escape (from gardens, aquaculture), iii) contamination, iv) stowaway, v) corridor (transportation infrastructure), vi) unaided from neighboring countries (the natural spread of an alien species from another region where it is not native) (h u l m e et al. 2008). compared to the other pathways ornamental horticulture is therefore the main pathway for plant invasions worldwide (h u l m e et al. 2008), although its importance compared to other pathways has found the attention of ecologists rather late (m a c k 1991; r e i c h a r d & w h i t e 2001; m a c k & e r n e b e r g 2002). according to j e n k i n s (1996); l e v i n e & d ' a n t o n i o (2003) an increasing amount of species inadvertently introduced into areas outside their natural range is directly linked with increases in trade. due to specific history, long period of isolation, low level of industrial and economic development, bad road infrastructure as well as international commerce and travel, montenegro for a long time hesitated to introduction of alien species. but, in last last decade fast urban, trade and tourism development facilitated their spread. although alien flora of montenegro has never been systematically explored, first records of some alien species dates from the beginning of xix century (p a n č i ć , 1875, p a n t o c z e k , 1874 etc.). these historical notes consist only of information about the presence of a species, so population size or ecological impact of its spreading on natural communities was not documented. during the field excursions conducted in last decade, some interesting results in monitoring of selected alien species are obtained. in this paper we are presenting these results and giving an overview of a species that should be considered as invasive in montenegro. in addition we are giving taxonomic, biologic and geographic analysis of invasive alien flora. material and methods ! preliminary check list of invasive alien species (ias) is mainly created according to authors own field observations, while the list of alien species is extracted from: r o h l e n a (1942), p u l e v i ć (2005) and s t e š e v i ć et al. (2008). invasiveness of species is estimated according to r i c h a r d s o n et al. (2000). life forms follows raunkier’s system (r a u n k i e r , 1943, e l l e n b e r g et al., 1967), while origin of species is given according to p i g n a t t i (1982), pyšek et al. (2002), t u t i n et al. (1964-1980, 1993). results and disscusion due to the to the sheme proposed by r i c h a r d s o n et al. (2000), p y š e k et al. (2004), p y š e k & r i c h a r d s o n (2004), 50 taxa of alien plants of montenegro can be classified as invasive. the list of ias is given in table 1, while one taxa from the list carpobrotus edulis is for the first time reported for alien flora of montenegro. first finding of this species dates from 2006, when rather poor population was noted near the side shore in bijela (boka kotorka bay). in the meantime species is recorded on numerous locations near the coast, where compose rather dense patches. the list of ias of montenegro consists of 50 species and subspecies belonging to 36 genera and 20 families. majority of families (18), genera (31) and species/subspecies (42) belongs to dicotyledons, while other representative belongs to monocotyledons. it is not surprising that two of the largest families worldwide poaceae and asteraceae (h e y w o o d , 1993) provide the highest number of invasive alien species in montenegro: asteracee (16), poaceae (7) (tab.2). similar to the world’s list of leading invaders families (see c r a w l e y , 1987, w e b e r , 1997, p y š e k , 1998) fabaceae and brassicaceae are near to the top. due to c r o n q u i s t (1981) asteraceae is considered as one of the evolutionarily most advanced families. possessing a number of features advantageous in the invasion process, e.g. high reproductive rate, specialized dispersal structures, diversity of metabolic products providing protection from grazing, high level of apomixes etc. (h e y w o o d , 1989, p y š e k , 1997). similarly, successful dispersal mechanisms in poaceae and fabaceae, together with a highly evolved inflorescence in the former, and an ability to fix atmospheric nitrogen as well as remarkably successful pollination system in the latter may serve to explain why these families among the world’s leading invaders. advanced features that might be considered as a clue for successful invasion are: high reproductive rate, long biologica nyssana 1 (1-2) december 2010: 35-42 stešević, d., petrović, d.. preliminary list of plant invaders… 37 table 1. list of ias in montenegro: taxonomy, biology, origin, first record and habitat type. legend: p phanaerophytes, chchamaephytes, hhemicriptophytes, ttherophytes, ggeophytes, namnorth america, samsouth america, cam & samcentral and south america, asasia, afrafrica, easeuroasia. taxon family life form origin 1st record/author habitat type acer negundo l. aceraceae p nam 2005 (steševič&jovanović) riverine forest, ruderalis/mainly waste places ailanthus altissima (mill.) swingle simaroubaceae p as 1971 (popović & sterniša) ruderalis/widespread, vegetation near the road sides, railways, waste places etc., shrubland of carpinus orientalis, rocklands alcea rosea l. malvaceae h as 1900 (baldacci) ruderalis/vegetation near the road sides, railways, waste places, etc. amaranthus hybridus l. amaranthaceae t nam 2005 (steševič&jovanović) city lawns, ruderalis/vegetation near the road sides, waste placeas, cultivated soils amaranthus retroflexus l. amaranthaceae t nam 1875 (pančić) ruderalis/wate places, vegetation near the road sides, cultivated land , city lawns … ambrosia artemisiifolia l. asteraceae t nam 2009 (stešević) ruderalis/vegetation near the road sides, gravely river banks, city lawns amorpha fruticosa l. fabaceae p nam 1973 (pulević) river banks, sandy dunes artemisia verlotiorum lamotte asteraceae g as 2006 (stešević & jogan) ruderalis/ vegetation near the road sides, railways, waste places …. asclepias syriaca l. asclepiadaceae g as 2005 (steševič&jovanović) wet meadows aster squamatus (spreng.) hieron asteraceae t cam, sam 2004 (hadžiablahović) ruderalis/ vegetation near the road sides and railways, waste places.. broussonetia papyrifera (l.) vent. moraceae p as 2005 (steševič&jovanović) ruderalis/ waste places, vegetation near the road sides, roofs … bidens subalternans dc. asteraceae t sam 1993 (trinajstić) ruderalis/ waste places, vegetation near the road sides, railways etc. bidens frondosa l. asteraceae t nam 2005 (steševič&jovanović) river banks, rudelralis carpobrotus edulis (l.) n.e.br. aizoaceae ch afr 2006 stešević (leg.) ruderalis/vegetation near road side, costal rocks commelina communis l. commelinaceae h as 1997 (karaman) ruderalis/ waste places, city lawns chamomilla suaveolens (pursh.) rydb. asteraceae t as 1986 (vasić) ruderalis, vegetation near the road sides conyza albida willd. asteraceae t sam 2005 (steševič&jovanović) ruderalis/ widespread conyza bonariensis (l.) cronq. asteraceae t sam 2005 (steševič&jovanović) ruderalis/ widespread conyza canadensis (l.) cronq. asteraceae t nam 1874 (pantoczek) ruderalis/ widespread cuscuta caesattiana bertol. cuscutaceae t nam 1949 (černjavski et al.) ruderalis, (sub)mediterranean shrublands and rocklands cuscuta campestris yuncker. cuscutaceae t nam 1997 (karaman) ruderalis, (sub)mediterranean shrublands and rocklands datura stramonium l. solanaceae t nam 1875 (pančić) ruderalis, widespread eleusine indica (l.) gaertn. poaceae t afr 1959/1960 (hodak) ruderalis/ vegetation near the road sides, trampled habitats, city lawns etc. eleusine tristachya (lam.) lam. poaceae t sam 1998 (lakušić) ruderalis/ trampled habitats, city lawns erigeron annuus (l.) pers. asteraceae t nam 1972 stanković-tomić ruderalis/widespread, seminatiral habitats, meadows, shurbalnd erigeron annuus subsp. serpentionalis asteraceae t nam 2005 (steševič&jovanović) ruderalis/widespread, seminatiral habitats, meadows, shurbalnd euphorbia maculata l. euphorbiaceae t nam 1979 (obradović & budak) ruderalis/ trampled habitats euphorbia prostrata aiton. euphorbiaceae t nam 1984 (pulević) ruderalis/trampled habitats galinsoga parviflora cav. asteraceae t nam 1968 (blečić) ruderalis, widespread helianthus xlaetiflorus pers. asteraceae g nam 1980 (obradović) ruderalis/waste places, river banks biologica nyssana 1 (1-2) december 2010: 35-42 stešević, d., petrović, d.. preliminary list of plant invaders… 38 helianthus tuberosus l. asteraceae g nam 2005 (steševič&jovanović) ruderalis/waste places, river banks impatiens parviflora dc. balsaminaceae t as 2010 (steševič & drescher) river bank lepidium virginiacum l. brassicaceae t sam 1936 (rohlena) ruderalis/ vegetation near the road sides and railways, roofs medicago sativa l. fabaceae h as 1874 (pantoczek) widespread oenothera biennis l. onagraceae h nam 1976 (pulević) ruderalis oenothera glazioviana micheli onagraceae h nam 2008 (rakaj & krzysztof) sandy dunes, ruderalis oenothera fallax renner et rostanski onagraceae h nam 2008 (rakaj & krzysztof) sandy dunes, ruderalis opuntia vulgaris mill. cactaceae h nam 1930 (in pulević 2005) mediterannean rocklands, vegetation near the roadside paspalum dilatatum poir. poaceae h sam 1986 (ilijanić & topić) ruderalis,/trampled habitats, lawns, wet meadows paspalum paspaloides (michx.) schribn. poaceae h cam, sam 1949 (černjavski et al.) ruderalis, city lawns, wet meadows reynoutria japonica houtt. polygonaceae g as 2005 (stešević&jovanović) natural forests, river banks, ruderalis/vegetation near the road sites robinia pseudacacia l. fabaceae p nam 1911 (rohlena) natural forests, river banks, ruderalis/vegetation near the road sites solanum elaeagnifolium cav. solanaceae h sam 2003 (hadžiablahović et al.) ruderalis, wet meadows sorghum halepense (l.) pers. poaceae h eas 1874 (pantoczek) widespread sporobolus poirettii (r.et s.) hitche poaceae h nam 1998 (niketić in greuter) ruderalis, vegetation near the road sides, city lawns sporobolus vaginiiflorus (torrey) wood poaceae t nam 2006 (stešević & jogan) ruderalis/ vegetation near the road sides, city lawns tamarix dalmatica baum. tamaricaceae p afr 1942 (rohlena) river banks, sandy dunes veronica persica poir. scrophulariaceae t as 1900 (baldacci) widespred xanthium strumarium l. italicum (moretti) d.löve asteraceae t nam 1900 (horak) ruderalis, sandy dunes xanthium spinosum l. asteraceae t sam 1874 (pantoczek) ruderalis, widespread viability of seeds or c4 photosynthetic pathways like in amaranthaceae (c r o n q u i s t , 1981, h e y w o o d , 1989). majority of families (13) are represented with only one species (tab. 2). genera represented with highest number of ias are conyza (3) oenothera (3), erigeron (2), euphorbia (2), amaranthus (2), bidens (2), xanthium (2), eleusine (2), paspalum (2), sporobolus (2). up to now only two subspecies of erigeron annuus are recorded in the flora of montenegro: ssp. annuua and ssp. serpentionalis, but considering the fact that third ssp. strigosus is reported for the flora of neighboring countries (croatian i k o l i ć (2010), serbiaj o v a n o v i ć (1994)) we expect to find it soon, so taxonomic spectrum of genera will look a bit different. as it was expected considering life forms therophytes shows predominance with 26 species fig. 1. they are followed by hemicriptophytes (12), than phanaerophytes (6), geophytes (5) and chamaephytes (1). hydrophytes are not present at all. feature that are enabling therophytes to become very successful invaders are short life cycle (annuals), high reproductive rate, easily dispersed seeds etc. due to its native range, exactly half of ias originates from north america (25), while second dominant are asian (11) (fig.2). these species were mainly introduced as ornamental plants (robinia pseudoacacia, acer negundo, ailanthus altissima, reynoutria japonica, etc.) and very soon they started to escape into the wild, invading native ecosystems with disastrous results and become invasive species. from the other side, greatly improved transport that enables traders to move goods from distant destinations provided ideal opportunities for the accidental introduction of ais (paspalum spp., eleusine spp.). considering the patterns of habitat invasion, our results are similar to c h y t r ý et al. (2008). despite large differences in species, it is possible to notice regularity in the patterns of habitat invasion. a general trend is: in harsh climatic conditions and nutrient-poor habitats, invasion levels are low. alien biologica nyssana 1 (1-2) december 2010: 35-42 stešević, d., petrović, d.. preliminary list of plant invaders… 39 plants tend to thrive in nutrient-rich and man-made habitats. mountains, cliffs, bogs, dry grasslands and coniferous woodlands tend to resist alien invasion, while coastal and riverine habitats, where nutrient availability and disturbance can be high, are more prone to invasion by alien plants. human made habitats such as farmland and urban landscapes also facilitate the spread of alien plants. table 2. taxomonic spectrum of families of ias in montenegro family no of taxa % asteraceae 16 32 poaceae 7 14 fabaceae 3 6 brassicaceae 3 6 onagraceae 3 6 amaranthaceae 2 4 cuscutaceae 2 4 euphorbiaceae 2 4 aceraceae 1 2.0 aizoaceae 1 2.0 asclepiadaceae 1 2.0 balsaminaceae 1 2.0 cactaceae 1 2.0 commelinaceae 1 2.0 malvaceae 1 2.0 moraceae 1 2.0 polygonaceae 1 2.0 scrophulariaceae 1 2.0 simaroubaceae 1 2.0 solanaceae 1 2.0 tamaricaceae 1 2.0 due to the favorable climate and higher level of disturbance mediterranean part of the country hosted significantly higher number of invasive species than continental-mountainous. ias typical for mediterranean part of the country are: carpobrotus edulis, opuntia vulgaris, oenothera glazioviana, o. fallax, conyza bonariensis, c. albida, aster squamatus, asclepias syriaca, acer negundo, amaranthus hybridus, lepidium virginicum, bidens subalternans, helianthus tuberosus, h. laetiflorus, xanthium spinosum, eleusine indica, e. tristachya, sporobolus poiretti, s. vaginiiflorus, commelina communis, solanum elaeagnifloium, tamarix dalmatica, amorpha fruticosa, paspalum paspaloides, p. dilatatum. up to now only one species (impatiens parviflora) has distribution restricted only to continental part of the country (riverine forests). this is explained by its ecological preferences of species: very susceptible to water stress and a shade tolerant, mostly found at 5-40% relative daylight, prefer nitrogen rich stands, with ph range between 4.5 and 7.6 (c o m b e , 1956). fig. 1. structure of invasive alien flora with respect to life forms. 52% 24% 12% 10% 2% t h p g ch fig. 2. structure of invasive alien flora with respect to origin. 50% 16% 4% 22% 6% 2% nam sam sam&cam as afr eas other species, such as robinia pseudoacacia, erigeron annuus, amaranthus retroflexus, veronica persica, xanthium strumarium ssp. italicum, ailanthus altissima, reynoutria japonica are distributed in both part of the country. considering ailanthus and reynoutria, at the beginning of our field study we noticed that first species mainly invaded mediterannean part of the country, while the second one is restricted to continental part. but during the last year both species started to move its borders. along the roadsides ailanthus moved towards the north and reynoutria along roadsides and riverbanks towards the south. success of this “movement” is strongly supported by increase of a disturbance. as it can be seen in table 1, majority of ias are for the first time recorded in our flora in last few decades, but we can’t exactly state when these species were really introduced, by which pathways and when expansion of its populations has really started. with this study we intended to do a zero stage for a future monitoring of ias, but also to draw attention to the problems which expansion of ias is bringing with itself. biologica nyssana 1 (1-2) december 2010: 35-42 stešević, d., petrović, d.. preliminary list of plant invaders… 40 majority of species listed above are also recognized as invasive in our first neighboring mediterranean country croatia (b o r š i ć , 2008). conclusion preliminary list of ias in montenegro consists of 50 flowering plants belonging to 36 genera, 20 families and 2 classes. carpobrotus edulis is for the first time reported for montenegro. the highest number of ias has families asteraceae and poaceae, while between genera dominant are conyza and oenothera. in the spectrum of life forms therophytes are prevailing, while due to its native range, majority of ias has north american and asian origin. considering the patterns of habitat invasion, highly disturbed, man made and nutrient rich habits are more prone to invasion by alien plants than natural and nutrient-poor habitats. due to the favorable climate and higher level of disturbance mediterranean part of the country is more favorable for plant invaders than continentalmountainous. the aim of this study was to provide a basic data on ias in montenegro, to enable future monitoring and to draw attention on the problems which expansion of ias is bringing with itself. references baldacci, a. 1900: controbutio la conoscenza della flora montenegrino-albanese. memoria letta ala r. accademia delle scienze 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(budva), p. 203 walker b.h., steffen w. 1997: an overview of the implications of global change for natural and managed terrestrial ecosystems. conservation ecology 1, http://www.consecol.org/vol1/iss2/art2/index.ht ml (online journal) weber, e.f. 1997: the alien flora of europe: a taxonomic and biogeographic overview? journal of vegetation science, 8: 565-572. kopij 2025, biologica nyssana 16(1) 307 16 (1) june 2025: 307-316 doi: 10.46793/biolnyss.16.1.14k inter-annual variation in the structure of avian communities in zambezi rural riparian forest original article grzegorz kopij department of vertebrate ecology, wrocław university of environmental & life sciences, ul. kożuchowska 5b, 51-631 wroclaw, poland grzegorz.kopij@upwr.edu.pl (corresponding author) received: january 08, 2025 revised: february 19, 2025 accepted: march 17, 2025 abstract: in terms of biodiversity, riparian forests are among the richest habitats in the world, but in the tropical regions of the world, these forests are understudied. in the present study, the avian community was quantified in the zambezi rural riparian forest in ne namibia for two years, with a similar rainfall (423 mm in 2013 vs. 428 mm in 2014). in total, 135 breeding bird species were recorded in the study area. sörensen similarity index was i=0.79. there were two dominant species: quelea quelea and euplectes orix. the group of subdominants were represented by 11 species. the species composition and population densities only slightly differed between the years 2013 and 2014. the proportions of three main feeding guilds, insectivores, granivores, and frugivores were similar in the riparian forests. this feature distinguishes this community from others studied so far in southern africa, where either granivores or insectivores are the dominant guilds. key words: tropical riparian forests, avian community, population density apstrakt: međugodišnja varijacija u strukturi zajednica ptica u ruralnim riparijalnim šumama reke zambezi u pogledu biodiverziteta, riparijalne šume spadaju među najbogatija staništa, ali su u tropskim oblastima nedovoljno proučene. u ovoj studiji kvantifikovana je zajednica ptica u ruralnim riparijalnim šumama reke zambezi na severoistoku namibije, tokom dve godine, sa sličnim godišnjim vrednostima padavina (423 mm u 2013. i 428 mm u 2014.). ukupno je zabeleženo 135 vrsta gnezdarica. sörensenov indeks sličnosti iznosio je i=0,79. dominantne vrste bile su quelea quelea i euplectes orix, dok je grupu subdominantnih činilo 11 vrsta. sastav vrsta i gustine populacija su blago varirali između analiziranih godina, 2013. i 2014. u smislu ishrane, udeo pripadnika tri glavne gilde (insektivori, granivori i frugivori) bio je sličan u riparijalnim šumama. ova karakteristika razlikuje ovu zajednicu od drugih u južnoj africi, gde preovladavaju granivorne ili insektivorne vrste. ključne reči: tropske riparijalne šume, zajednica ptica, gustina populacije introduction among different biomes in southern africa, the tropical riparian forests appear to be especially rich in terms of biodiversity (hockey et al., 2005). in namibia, most of these forests lay in zambezi, limpopo and okavango river valleys (mucina & rutherford, 2024). despite this, little is known about animal communities occupying these forests. it is also true with respect to avian communities, which are often the most intensively studied animal communities in other biomes. in southern africa, the structure of avian communities has been investigated only in the acacia savanna in eswatini (monadjem, 2003; monadjem, 2005), acacia savanna along vaal river in south africa (seymour & simmons, 2008) and in the tamarix vegetation in karoo, south africa (brooke, 1992). in other parts of the world, birds associated with tropical riparian forests are also understudied. species diversity and community structure of birds were studied along paraiba do sul river in atlantic forests, sao paulo state, brazil (de paula laurenço & de toledo, 2019); in alta foresta, mato grosso, brazil (lees & peres, 2008); gallery forests in costa rica (seaman & schultze, 2010); rainforest in new guinea (korejs et al., 2023); monsoonal forest in hong kong (chan et al., 2008); oil palm-forest mosaic in malaysia (azman et al., 2011; michell © 2025 kopij. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 308 et al., 2018); savanna in australia (woinarski et al., 2000), and forests in south-western australia (palmer & bennett, 2006). the purpose of this study is to determine the structure of an avian community in a rural riparian forest, in terms of: 1) species diversity, 2) dominance structure, 3) guilds proportions, and 4) population densities of particular species making up the community. study area studies were conducted in the zambezi riparian forest in a rural setting. the area is situated near katima mulilo, zambezi region, ne namibia. a transect was established along the zambezi river between 29°83's, 18°93'e and 29°25's, 21°30'e (fig. 1). the riparian forest is interspersed with some traditional rural homesteads with gardens; small fields, pastures for sheep and cattle, water canals (backwaters), grassy depressions flooded almost on an annual basis, and afforested areas with main tree species such as acacias (acacia spp.), african teak (pterocarpus angolensis), albizias (albizia spp.), apple leaves (lonchocarpus nelsii), burkea (burkea africana), combretum (combretum spp.), camel-thorn (acacia erioloba), jackal berry (diospyros mespiliformis), mopane (colophospermum mopane), pod mahogany (afzelia quanzensis), silver cluster-leaf (terminalia sericea), sausage tree (kigelia africana), sycamore fig (ficus sycomorus), white bauhinia (bauhinia petersiana), zambezi teak (baikiaea plurijuga), and silver tree (terminalia sericea). biologica nyssana ● 16 (1) june 2025: 307-316 kopij ● inter-annual variation in the structure of avian communities in zambezi rural riparian forest fig. 1. the location of the transect (yellow line) along the zambezi river fig. 2. monthly rainfall in katima mulilo in 2013 and 2014 309 biologica nyssana ● 16 (1) june 2025: 307-316 kopij ● inter-annual variation in the structure of avian communities in zambezi rural riparian forest the monthly distribution of rainfall in 2013 and 2014 is shown in fig. 2. the total amount of rainfall was similar in both years (423 mm in 2013 vs. 428 mm in 2014). methods the line transect method in the american version (sutherland, 1996; bibby et al., 2012) has been employed to quantify avian assemblages (frequency of occurrence and relative abundance of each species) along the designed transect. counts on transects were conducted three times (february/ march, may, september), both in 2013 and 2014. the transect was 6 km long. counts were conducted in the mornings by walking slowly from c. 6 a.m. till c. 11 a.m. and recording all birds seen and heard. for resident birds, a breeding pair was a census unit, while for nonresident species, the census unit was an individual. a bird calling and/or showing other territorial or breeding behaviour was regarded as an actual breeder, i.e. it represented a breeding pair. caution was taken to not register the same individuals by noting the movements of counted birds in the field and by paying special attention to simultaneously calling birds. following assumptions of the line transect method, the maximal number of breeding pairs on the transect in whatever of the three counts conducted in a given year was assumed as the number of all potentially breeding birds (bibby et al., 2012). the dominance of each species is expressed as the percentage of the total number of pairs of a given species in relation to the total number of all pairs of all species recorded. dominant species: >5%, subdominant: 2-4.99%. the following guilds were distinguished: diet: g – granivorous, i – insectivorous, f – frugivorous, n – nectarivorous, r – carnivorous. nesting: t – in trees or shrubs, h – in holes, b – in/ on buildings, v – herbaceous vegetation. habitat: f – forest interior, e – ecotone (forest/open area), o – „open” area (grassland/savanna). residency: r – resident throughout the year, a – intra-african migrant, c – nomad. the following indices were used to characterize the diversity and evenness of the communities: 1) shannon’s diversity index: h’=-∑ pi ln pi where: pi is the proportion of breeding pairs belonging to the ith species 2) simpson’s diversity index: d=((∑n(n-1))/n(n-1) where: n is the total number of breeding pairs belonging to a given species, n is the total number of breeding pairs of all species 3) pielou’s evenness index: j’=(-∑ pi ln pi)/ln s, where: pi is the proportion of breeding pairs belonging to the ith species; s is the total number of species. j’ varies between 0 and 1. the less variation between species in a community, the higher j’ is. 4) community dominance index: di=(n1+n2)/n where: n1, n2 is number of pairs of two most abundant species, n is total number of pairs of all species. 5) sörensen’s coefficient: i=2c/a+b where: a is the number of bird species in one breeding season, b is the number of bird species in another breeding season, and c is the number of bird species common to both breeding seasons. the ch2-test was used to test differences in population densities between 2013/14 and 2015/16. for statistical testing, only those species with at least 10 breeding pairs in both seasons were included. systematics and nomenclature of bird species follow hockey et al. (2005), with later nomenclatorial changes. results in 2013-2014, a total of 135 breeding bird species were recorded in the study area: 103 species in 2013, 118 species in 2014 (tabs. 1 and 2); 87 species were common for 2013 and 2014. sörensen similarity index was i=0.79. in addition, 7 non-breeding palearctic species were recorded: 3 in 2014 and 6 in 2015. there were two dominant species: red-billed quelea quelea quelea and southern red bishop euplectes orix. both are nomad breeders; the redbilled quelea was recorded as dominant in 2013 and was not recorded at all in the next year. on the other hand, the southern red bishop was recorded as dominant in 2014, while in 2013 it was uncommon (0.2%). the group of subdominants were represented by 11 and 9 species in 2013 and 2014 respectively, as listed in tab. 1. the following species were subdominants in both years: cercotrichas leucophrys, lybius torquatus, phyllastrephus terrestris, prinia subflava, streptopelia capicola, turtur chalcospilos, and uraeginthus angolensis. the following species were more common (statistically significant difference) in 2013 than in 2014: cercotichas leucophrys, francolinus sephaena, lamprotornis australis, mirafra rufocinnamomea, nettapus auratus, telophorus sulfureopectus, trachyphonus vaillantii, and vidua macroura. on the other hand, more common in 2014 than in 2013 were dicrurus adsimilis, euplectes orix, ploceus velatus, spilopelia senegalensis, streptopelia semitorquata, and turtur chalcospilos. the abundance of 26 species was not significantly different in 2013 and 2014. for 19 species the numbers of breeding pairs were identical in both years (tab. 2). 310 biologica nyssana ● 16 (1) june 2025: 307-316 kopij ● inter-annual variation in the structure of avian communities in zambezi rural riparian forest table 1. characteristics of the breeding bird community parameter 2013 2014 number of species and breeding pairs number of spp. 103 118 number of pairs 823 801 overall density 137.2 133.5 dominance structure dominance index 0.17 0.19 number of dominant species 1 1 percentage of dominants 12.2 14.4 number of subdominant species 11 9 percentage of subdominants 34.5 27.7 diversity indices h’ 3.95 4.06 j’ 0.85 0.85 d 0.97 0.97 table 2. number of breeding pairs, linear density and dominance of birds breeding in a riparian forest (6 km) in a rural setting in 2013 and 2014. explanations: n – number of breeding pairs, d – linear density (pairs per km), dom – dominance species 2013 2014 x2-test n d dom n d dom accipiter spp. 0 0 0.0 1 0.2 0.1 actophilornis africanus 6 1 0.7 6 1 0.7 0.0 alopochen aegyptiacus 1 0.2 0.1 0 0 0.0 anastomus lamelligerus 1 0.2 0.1 0 0 0.0 anthoscopus caroli 0 0 0.0 1 0.2 0.1 apalis flavida 10 1.7 1.2 10 1.7 1.2 0.0 apus affinis 0 0 0.0 2 0.3 0.2 ardea ibis 2 0.3 0.2 9 1.5 1.1 4.1* ardeola ralloides 1 0.2 0.1 1 0.2 0.1 batis molitor 5 0.8 0.6 10 1.7 1.2 1.9 bostrychia hagedash 1 0.2 0.1 1 0.2 0.1 bucorvus leadbeateri 1 0.2 0.1 0 0 0.0 buphagus erythrorhynchus 3 0.5 0.4 0 0 0.0 butorides rufiventris 2 0.3 0.2 0 0 0.0 butorides striatus 0 0 0.0 1 0.2 0.1 bycanistes bucinator 2 0.3 0.2 2 0.3 0.2 camaroptera brevicaudata 0 0 0.0 1 0.2 0.1 campephaga flava 0 0 0.0 1 0.2 0.1 most species were breeding, either as african residents or infra-african migrants. seven species (merops apiaster, acrocephalus arundinaceus, acrocephalus palustris, hirundo rustica, lanius collurio, muscicapa striata, and phylloscopus trochilus) were non-breeding palearctic migrants. in 2013, the proportion of palearctic migrants (159 individuals of three species) was much higher than in 2014 (18 individuals of six species) because there was an influx of c. 150 individuals of merops apiaster in 2013 (none recorded 2014). among breeding birds, most were african residents; very few were intra-african migrants (0.6% and 0.2% in 2013 and 2014 respectively). the proportions of main feeding guilds were much the same in 2013 and 2014 (fig. 3). the main nesting guild was tree/shrub nesting birds, with similar proportions in 2013 and 2014 (fig. 3). however, the proportion of hole-nesting birds was much higher in 2013 than in 2014, while in the case of the guild nesting in herbaceous vegetation, the reverse was recorded (much higher proportion in 2014 than in 2013). 311 biologica nyssana ● 16 (1) june 2025: 307-316 kopij ● inter-annual variation in the structure of avian communities in zambezi rural riparian forest campethera abingoni 3 0.5 0.4 2 0.3 0.2 centropus cupreicaudus 5 0.8 0.6 5 0.8 0.6 0.0 centropus senegalensis 4 0.7 0.5 4 0.7 0.5 centropus spp. 0 0 0.0 4 0.7 0.5 centropus superciliosus 10 1.7 1.2 6 1 0.7 2.0 cercotrichas leucophrys 33 5.5 4.0 22 3.7 2.7 4.1* ceryle rudis 2 0.3 0.2 8 1.3 1.0 3.4 chlorocichla flaviventris 9 1.5 1.1 9 1.5 1.1 0.0 chrysococcyx cupreus 0 0 0.0 1 0.2 0.1 cinnyricinclus leucogaster 0 0 0.0 5 0.8 0.6 circaetus cinerascens 1 0.2 0.1 1 0.2 0.1 circus ranivorous 1 0.2 0.1 0 0 0.0 cisticola chiniana 17 2.8 2.1 14 2.3 1.7 0.5 cisticola juncidis 0 0 0.0 8 1.3 1.0 cisticola spp. 0 0 0.0 2 0.3 0.2 coracias caudata 2 0.3 0.2 2 0.3 0.2 corvinella melanoleuca 1 0.2 0.1 0 0 0.0 corvus albus 2 0.3 0.2 2 0.3 0.2 corythaixoides concolor 11 1.8 1.3 14 2.3 1.7 0.5 crithagra atrogularis 5 0.8 0.6 1 0.2 0.1 dendrocygna viduata 0 0 0.0 1 0.2 0.1 dicrurus adsimilis 1 0.2 0.1 13 2.2 1.6 8.3* dondropicos fuscescens 2 0.3 0.2 3 0.5 0.4 dryoscopus cubla 7 1.2 0.9 7 1.2 0.9 0.0 egretta garzetta 0 0 0.0 1 0.2 0.1 emberiza flaviventris 0 0 0.0 2 0.3 0.2 eyrystomus glaucurus 0 0 0.0 1 0.2 0.1 erythropygia leuconotus 10 1.7 1.2 10 1.7 1.2 0.0 estrilda astrild 12 2 1.5 7 1.2 0.9 2.7 euptectes orix 2 0.3 0.2 115 19.2 14.4 83.3** falco dickinsoni 2 0.3 0.2 0 0 0.0 falco rupicoloides 0 0 0.0 1 0.2 0.1 francolinus sephaena 12 2 1.5 3 0.5 0.4 20.3** francolinus swainsonii 2 0.3 0.2 3 0.5 0.4 halcyon albiventris 0 0 0.0 2 0.3 0.2 halcyon leucocephala 1 0.2 0.1 1 0.2 0.1 halcyon senegalensis 2 0.3 0.2 3 0.5 0.4 haliaeetus vocifer 5 0.8 0.6 3 0.5 0.4 hedydipna collaris 2 0.3 0.2 1 0.2 0.1 hirundo abyssinica 6 1 0.7 18 3 2.2 312 biologica nyssana ● 16 (1) june 2025: 307-316 kopij ● inter-annual variation in the structure of avian communities in zambezi rural riparian forest hirundo smithii 0 0 0.0 4 0.7 0.5 indicator indicator 1 0.2 0.1 0 0 0.0 indicator minor 1 0.2 0.1 1 0.2 0.1 kaupifalco monogrammicus 1 0.2 0.1 0 0 0.0 lagonosticta senegala 11 1.8 1.3 7 1.2 0.9 1.7 lagonosticta nitidula 0 0 0.0 1 0.2 0.1 lamprotornis australis 14 2.3 1.7 1 0.2 0.1 126.8** lamprotornis nitens 6 1 0.7 8 1.3 1.0 0.4 laniarius bicolor 14 2.3 1.7 11 1.8 1.4 0.6 lophaetus occipitalis 0 0 0.0 1 0.2 0.1 lybius torquatus 26 4.3 3.2 18 3 2.2 2.7 matacilla aguimp 1 0.2 0.1 1 0.2 0.1 merops bullockoides 1 0.2 0.1 6 1 0.7 merops hirundineus 1 0.2 0.1 0 0 0.0 merops nubicoides 1 0.2 0.1 6 1 0.7 merops pusillus 7 1.2 0.9 5 0.8 0.6 micronisus gabar 0 0 0.0 1 0.2 0.1 milvus eagyptius 3 0.5 0.4 1 0.2 0.1 mirafra rufocinnamomea 13 2.2 1.6 1 0.2 0.1 108.0** muscicapa caerulescens 0 0 0.0 1 0.2 0.1 myioparus plumbeus 0 0 0.0 1 0.2 0.1 nattapus auritus 14 2.3 1.7 4 0.7 0.5 18.8* nectarinia mariquensis 12 2 1.5 7 1.2 0.9 2.7 nectarinia senegalensis 0 0 0.0 2 0.3 0.2 nectarinia talatala 4 0.7 0.5 3 0.5 0.4 nectarinidae spp. 17 2.8 2.1 17 2.8 2.1 0.0 nilaus afer 7 1.2 0.9 5 0.8 0.6 0.6 oriolus auratus 0 0 0.0 4 0.7 0.5 oriolus larvatus 1 0.2 0.1 1 0.2 0.1 ortygospiza atricollis 1 0.2 0.1 2 0.3 0.2 parus niger 1 0.2 0.1 4 0.7 0.5 passer diffusus 2 0.3 0.2 3 0.5 0.4 phalacrocorax africanus 7 1.2 0.9 8 1.3 1.0 0.1 phoeniculus purpureus 2 0.3 0.2 6 1 0.7 phyllastrephus terrestris 31 5.2 3.8 21 3.5 2.6 3.6 plectropterus gambensis 0 0 0.0 9 1.5 1.1 ploceus velatus 18 3 2.2 5 0.8 0.6 25.4** pogoniulus chrysocomus 8 1.3 1.0 7 1.2 0.9 0.1 poicephalus meyeri 5 0.8 0.6 3 0.5 0.4 prinia subflava 32 5.3 3.9 23 3.8 2.9 2.6 prinia flavicans 0 0 0.0 1 0.2 0.1 prionops plumatus 2 0.3 0.2 2 0.3 0.2 prionops retzii 0 0 0.0 5 0.8 0.6 313 biologica nyssana ● 16 (1) june 2025: 307-316 kopij ● inter-annual variation in the structure of avian communities in zambezi rural riparian forest pytilia afra 1 0.2 0.1 1 0.2 0.1 quelea quelea 100 16.7 12.2 0 0 0.0 rhinopomastus cyanomelas 2 0.3 0.2 3 0.5 0.4 spermestes cucullatus 0 0 0.0 1 0.2 0.1 spilopelia senegalensis 2 0.3 0.2 14 2.3 1.7 7.7* streptopelia capicola 20 3.3 2.4 31 5.2 3.9 2.9 streptopelia decipiens 0 0 0.0 4 0.7 0.5 streptoperia semitroquata 10 1.7 1.2 21 3.5 2.6 4.3* sylvietta rufescens 6 1 0.7 3 0.5 0.4 tachybaptus ruficollis 1 0.2 0.1 0 0 0.0 tauraco schalowi 13 2.2 1.6 12 2 1.5 0.1 tchagra australis 1 0.2 0.1 1 0.2 0.1 tchagra senegala 0 0 0.0 3 0.5 0.4 tchagra spp. 1 0.2 0.1 3 0.5 0.4 telophorus sulfureopectus 14 2.3 1.7 7 1.2 0.9 5.3* thalassornis leuconotus 4 0.7 0.5 0 0 0.0 tockus erythrorhynchus 1 0.2 0.1 0 0 0.0 tockus nasutus 8 1.3 1.0 10 1.7 1.2 0.3 trachyphonus vaillantii 18 3 2.2 10 1.7 1.2 4.8* treron calva 4 0.7 0.5 1 0.2 0.1 tringa glareola 1 0.2 0.1 0 0 0.0 turdides jardineii 8 1.3 1.0 1 0.2 0.1 turdoides hartlaubii 3 0.5 0.4 8 1.3 1.0 2.3 turdus libonyanus 0 0 0.0 1 0.2 0.1 turnix sylvatica 1 0.2 0.1 1 0.2 0.1 turtur chalcospilos 20 3.3 2.4 36 6 4.5 5.3* upupa epops 16 2.7 1.9 5 0.8 0.6 18.2** uraeginthus angolensis 38 6.3 4.6 31 5.2 3.9 1.2 urocolius indicus 19 3.2 2.3 13 2.2 1.6 2.1 vanellus armatus 2 0.3 0.2 2 0.3 0.2 vanellus senegallus 6 1 0.7 4 0.7 0.5 0.8 vidua halybeata 0 0 0.0 1 0.2 0.1 vidua macroura 30 5 3.6 4 0.7 0.5 126.8** zosterops senegalensis 1 0.2 0.1 1 0.2 0.1 total number of pairs 823 100.0 801 100.0 discussion in total, 135 bird species were recorded in the riparian forest (this study, tab. 2). in the neighbouring mopane-terminalia forest and kalahari woodland mixed with rural areas, the numbers were much lower, i.e. 51 and 56, respectively (tab. 3). in the mopane woodland, situated in north-central namibia, the number of breeding species was 85 (kopij, 2013a and 2013b), while in the kaokoland savanna, in 314 nw namibia, it was 64 (kopij, 2014b). it is clear, therefore, that in namibia, the number of breeding bird species declines westwards, i.e. with declining precipitation (mendelsohn et al., 2009). however, within the same region, it may differ markedly depending on the soil type, inundation and topography. all these factors affect, in turn, variance in vegetation type. there were more breeding species in the riparian forest than in other forest types situated in the zambezi region. also the population densities of many species were much higher in the riparian forests than in the other forest types (tab. 3). out of 32 species compared, only grey-backed camaroptera (camaroptera brevicaudata), southern grey-headed sparrow (passer diffusus), lilac-breasted roller (coracias caudatatus), and red-billed hornbill (tockus erythrorhynchus) were less numerous in the riparian forests than in other forest types. in terms of the number of breeding pairs, the proportions of the three main feeding guilds: insectivores, granivores, and frugivores were similar in the riparian forests. however, in terms of the number of species, insectivores comprised almost half of this assemblage. this feature distinguishes this community from others studied so far in southern africa, where either granivores or insectivores were the dominant guilds (kopij, 2000, 2001a, 2001b, 2006, 2013a, 2013b, 2014a, 2014b, 2015, 2016, 2017, 2018, 2021, 2022). in riparian forests of africa, the fruit trees are usually abundant and may benefit frugivorous birds (especially bulbuls), while seeds may not be so abundant, as these are in more open savanna or grassland biomes. typical granivores, such as doves and sparrows, may therefore breed in lower densities in riparian forests than in the neighbouring more open habitats dominated by grasses. the species composition only slightly differed between the years 2013 and 2014. there was also no shift in species diversity, or in the community structure. this interannual stability can be linked to almost the same level biologica nyssana ● 16 (1) june 2025: 307-316 kopij ● inter-annual variation in the structure of avian communities in zambezi rural riparian forest fig. 2. proportions of feeding (columns in the upper row) and nesting guilds (columns in the lower row) in 2013. and 2014 table 3. linear population densities (pairs per 1 km of transect) of selected bird species (=32) in different forest types in rural settings in the zambezi region. habitat types: 1) riparian forest (this study), 2) mopane/terminalia forest (kopij, 2022), 3) kalahari woodland (kopij, 2021) species habitat types 1 2 3 batis molitor 1.7 0.2 1.3 camaroptera brevicaudata 0.2 0 1.3 cercotrichas leucophrys 5.5 0.9 0 chlorocichla flaviventris 1.5 0 1.1 cisticola chiniana 2.8 0 0.7 cisticola juncidis 1.3 0 0.2 315 biologica nyssana ● 16 (1) june 2025: 307-316 kopij ● inter-annual variation in the structure of avian communities in zambezi rural riparian forest of precipitation in these two years compared. the amount of rainfall is usually the main limiting factor governing both the distribution and population densities of most bird species in africa (maclean, 1990). a high number of species and high densities of their populations compared to other forest types in the zambezi region (tab. 3) suggest that riparian forests play an important role as breeding and feeding habitat for birds, especially frugivores and nectarivores. it may also play a role as a corridor allowing dispersal, migration and free movements within a mosaic of natural and human-modified environments (seaman & schulze, 2010). the riparian forest corridor may be therefore considered as a main instrument to offset the negative effects of habitat loss and fragmentation (turner, 1996; lees & peres, 2008), and for that reason, it deserves special protection. references azman, n.m., latip, n.s.a., sah, s.a.m., akil, m.a.m.m., shafie, n.j., & khairuddin, n.l. (2011). avian diversity and feeding guilds in a secondary forest, an oil palm plantation and a paddy field in riparian areas of the kerian river basin, perak, malaysia. tropical life sciences research, 22(2), 45-64. bibby, c.j., burgess, n.d., hill d.a. & mustoe, s. (2012). bird census techniques (2nd ed.). london: academic press brooke, r.k. (1992). the bird community of tamarix-clad drainages, northwestern karoo, cape province. ostrich journal of african ornithology, 63(1), 42-43. chan, e.k., yu, y.t., zhang, y., & dudgeon, d. (2008). distribution patterns of birds and insect prey in a tropical riparian forest. biotropica, 40(5), 623-629. de paula laurenço, a.c.p. & de toledo m.c.b. (2019). effects of proximity to urban areas on a riparian bird community in remnant atlantic forest in southeastern brazil. ambiente e agua an interdisciplinary journal of applied science, 14(7), 1-18. hockey, p.a., dean, w.r.j., ryan, p.g., maree, s., & brickman, b.m. (eds.) (2005). robert’s birds of southern africa. cape town: trustees of the john voelcker bird book fund kopij, g. (2000). birds of maseru. national university of lesoto journal of research, 8, 104151. kopij, g. (2001a). atlas of birds of bloemfontein. lesotho, roma / republic of south africa, bloemfontain: department of biology, national university of lesotho / free state bird club kopij, g. (2001b). birds of roma valley, lesotho. lesotho, roma: department of biology, national university of lesotho kopij, g. (2006). the structure of assemblages and dietary relationships in birds of south african coracias caudatus 0.3 0.2 0.7 corythaixoides concolor 2.3 0.7 1.3 dicrurus adsimilis 2.2 1.2 1.3 dondropicos fuscescens 0.5 0.1 0.2 dryoscopus cubla 1.2 0 0.7 laniarius bicolor 2.3 0 1.1 lybius torquatus 4.3 0.1 1.0 nilaus afer 1.2 0.1 0.2 passer diffusus 0.5 1.2 0.2 phyllastrephus terrestris 5.2 0 0.7 ploceus velatus 3.0 0.3 0 pogoniulus chrysocomus 1.3 0 0.2 poicephalus meyeri 0.8 0 1.6 prinia subflava 5.3 0.2 0.4 spilopelia senegalensis 2.3 0 2.0 streptopelia capicola 5.2 2.6 6.7 streptopelia decipiens 0.7 0 0 streptopelia semitroquata 3.5 0.1 1.3 telophorus sulfureopectus 2.3 0.1 0.4 tockus erythrorhynchus 0.2 0.7 0 tockus nasutus 1.7 1.1 0.9 trachyphonus vaillantii 3.0 0.3 0 turtur chalcospilos 6.0 0.3 3.1 upupa epops 2.7 0.3 0 uraeginthus angolensis 6.3 2.0 5.6 urocolius indicus 3.2 0.2 0 number of all pairs 823 409 338 number of all breeding species 135 56 51 316 biologica nyssana ● 16 (1) june 2025: 307-316 kopij ● inter-annual variation in the structure of avian communities in zambezi rural riparian forest grasslands. wrocław: wydawnictwo akademii rolniczej. kopij, g. (2013a). avian assemblages in natural and modified kaokoland (mopane) savanna in the cuvelai drainage system, north-central namibia. lanioturdus, 46(5), 22-33. kopij, g. (2013b). seasonal changes in avian assemblages in kaokoland (mopane) savanna in the ogongo game reserve, north-central namibia. international science & technology journal of namibia, 2(1), 44-58. kopij, g. (2014a). avian assemblages of urban habitats in north-central namibia. international science & technology journal of namibia, 3(1): 64-81. kopij, g. (2014b). avian communities of a mixed mopane-acacia savanna in the cuvelai drainage system, north-central namibia, during the dry and wet season. vestnik zoologii, 48(4), 269-274. kopij, g. (2015). avian diversity in an urbanized south african grassland. zoology & ecology, 25(2), 87-100. kopij, g. (2016). birds of katima mulilo town, zambezi region, namibia. international science & technology journal of namibia, 7, 85-102. kopij, g. (2017). structure of avian assemblages in zambezian baikiaea woodlands, northern namibia. zoology & ecology, 27(1), 1-10. kopij, g. (2018). provisional atlas of breeding birds of swakopmund in the coastal namib desert. lanioturdus, 51(2), 2-12. kopij, g. (2021). effect of farming and rainfall on the species diversity, population density and community structure of birds breeding in the kalahari woodland, ne namibia. zoodiversity, 55(6), 451-458. kopij, g. (2022). effect of a road on avian diversity in kalahari woodland. zoology and ecology, 32(1), 1-8. korejs, k., riegert, j., kigl, m., & novotny, v. (2023). differences in bird community structure between riparian and upland zones in a new guinean rainforest. australian field ornithology, 40, 179195. lees, a.c. & peres, c.a. (2008). conservation value of remnant riparian forest corridors of varying quality for amazonian birds and mammals. conservation biology, 22(2), 439-449. maclean, g.l. (1990). ornithology for africa. pietermaritzburg: university of kwazulu natal press mendelsohn, j., jarvis, a., roberts, c., & robertson, t. (2009). atlas of namibia. a portrait of the land and its people. cape town: sunbird publishers michell, s.l., edwards, d.p., bernard, h., coomes, d., jucker, t., davies, z.g., & struebig, m.j. (2018). riparian reserves help protect forest bird communities in oil palm dominated landscapes. journal of applied ecology, 55(6), 2744-2755. monadjem, a. (2003). population densities and community structure of birds in riverine forest in the lowveld of swaziland. ostrich journal of african ornithology, 74(3-4), 173-180. monadjem, a. (2005). associations between avian communities and vegetation structure in a low-lying woodland-savanna ecosystem in swaziland. ostrich journal of african ornithology, 76(1-2), 45-55. mucina, l. & rutherford, m.c. (2024). zonal biomes of southern africa. switzerland, cham: springer palmer, g.c. & bennett, a.f. (2006). riparian zones provide for distinct bird assemblages in forest mosaics of south-east australia. biological conservation, 130(3), 447-457. seaman, b.c. & schulze, c.h. (2010). the importance of gallery forests in the tropical lowlands of costa rica for understorey forest birds. biological conservation, 143(2), 391-398. seymour, c.l. & simmons, r.e. (2008). can severely fragmented patches of riparian vegetation still be important for arid-land bird diversity. journal of arid environment, 72(12), 2275-2281. sutherland, w.j. (1996). ecological census techniques: a handbook. united kingdom, cambridge: cambridge university press turner, i.m. (1996). species loss in fragments of tropical rain forest: a review of the evidence. journal of applied ecology, 33, 200-209. woinarski, j.c.z., brock c., armstrong m., hempel, c., cheal, d., & brennan, k. (2000). bird distribution in riparian vegetation in the extensive natural landscape of australia’s tropical savanna: a broad-scale survey and analysis of a distributional data base. journal of biogeography, 27(4), 843-868. jogan, n.: morphometric recognition of hordeum murinum l. subspecies in slovenia. biologica nyssana, 8 (1), september 2017 biologica nyssana 8 (1)  september 2017: 23-30 jogan, n.  morphometric recognition of hordeum murinum… 23 original article received: 28 june 2017 revised: 26 july 2017 accepted: 08 september 2017 morphometric recognition of hordeum murinum l. subspecies in slovenia nejc jogan university of ljubljana, biotechnical faculty, department of biology, večna pot 111, ljubljana, slovenia * e-mail: nejc.jogan@bf.uni-lj.si abstract: jogan, n.: morphometric recognition of hordeum murinum l. subspecies in slovenia. biologica nyssana, 8 (1), september 2017: 23-30. morphometric analysis of slovenian material belonging to hordeum murinum ssp. leporinum and type subspecies confirmed some of already reported distinguishing characters, but majority of them were not measured precisely before. after revision of herbarium material based on results, about 9% of sheets are still somehow »intermediate«, h. murinum ssp. leporinum is more common in submediterranean part with some scaterred populations in continental slovenia and the type subspecies occurr all over slovenia in lowland ruderal places. detailed determination key is provided based on results. key words: hordeum murinum, subspecies delimitation, slovenia apstrakt: jogan, n.: morfometrijsko raspoznavanje podvrsta hordeum murinum l. u sloveniji. biologica nyssana, 8 (1), septembar 2017: 23-30. morfometrijske analize materijala vrste hordeum murinum sa područja slovenije potvrdile su prisutnost dve podvrste: h. murinum ssp. leporinum i tipske podvrste. potvrđeni su neki već ranije upotrebljeni karakteri za razlikovanje i pored toga preciznije su obrađeni neki od karaktera, koje su prethodni autori pretežno navodili samo deskriptivno. između ostalog, revizija herbarijumskog materijala je jedan od rezultata, koji potvrđuje veliku prisutnost h. murinum ssp. leporinum u obalnom submediteranskom području slovenije sa nekoliko lokalnih populacija i u unutrašnjosti, a tipska podvrsta se javlja po čitavoj sloveniji na ruderalnim mestima u nizijama. oko 9% materijala nije identifikovano. . prikazan je ključ za determinaciju pomenute dve podvrste. ključne reči: hordeum murinum, razdvajanje podvrsta, slovenija introduction grasses themselves are often neglected even by serious field botanists, particularly so when they are part of ruderal vegetation. this is also the case of hordeum murinum complex, which is probably one of the best studied groups in the genus, however, the taxonomic delimitation of subordinated taxa has always been controversial (n e v s k i , 1941; c u a d r a d o et al., 2013). in central and se europe, hordeum murinum complex represents a taxonomically interesting polyploid group with 4 8 (1) • september 2017: 23-30 doi: 10.5281/zenodo.963149 biologica nyssana 8 (1)  september 2017: 23-30 jogan, n.  morphometric recognition of hordeum murinum… 24 taxa recognized at the level of subspecies (or sometimes species). two of them are rare in the mentioned territory, h. murinum ssp. glaucum (steud.) tzvelev confined to the warmest areas of southern europe (e.g. greece, cyprus, s italy, spain) and a peculiar h. murinum ssp. setariurum h. scholz & raus known only from few localities in n greece and s macedonia (s c h o l z & r a u s , 1997; j o g a n , 2005). hordeum murinum ssp. leporinum (link) arcang. and the type subspecies are reported in much wider area with "leporinum" being more thermophilous and particularly common in mediterranean europe and the type subspecies widespread but with overlapping range reaching as far south as peloponnese and n aegean (s t r i d , 2016). there are three ploidy levels recognized in the group, diploids (2n=14), tetraploids (2n=28) and hexaploids (2n=42) but the taxonomic delimitation is not congruent with ploidy levels so at least two different ploidy levels are reported for each subspecies, e.g. mostly 2n=28 but also 2n=42 or 2n=14 for both widespread subspecies (c v e l e v , 1976; k a n k a n p a a et al., 1996; t i s o n & f o u c a u l t , 2014; c u a d r a d o & al., 2013). there is no completely reliable morphological characteristic that distinguishes the recognized subspecies, so several authors refer to the "murinum complex" (c u a d r a d o & al., 2013). despite several studies evolution of the polyploid h. murinum complex is still not well understood (ibid.) and we can still agree with n e v s k i (1941), that h. leporinum is an ancestor of h. murinum s. str., whose spread towards north had been connected to man made ruderalization. in the territory of slovenia (and also neighbouring contries) only the the two widespread subspecies are reported, namely h. murinum ssp. murinum (further hmm) and h. murinum ssp. leporinum (hml). in the past, their distribution had been simply recognized as allopatric: hml reported only for the coastal submediterranean region of slovenia and hmm as being excluded from that region and scaterred in all other parts (m a r t i n č i č , 1984). but some field records blurred that picture with hml records in the continental slovenia and also hmm recorded in some localities in the coast (j o g a n & al., 1999; j o g a n , 2007). using determination keys it is not always easy to distinguish the two subspecies so our aim was to test the usefulness of morphometric characters for delimitation. in european floristic works approach in delimitation of the studied two taxa is slightly diverse, either taxa are recognized as independent species (n e v s k i , 1941; p i g n a t t i , 1983; m a r t i n č i č , 1984; l a m b i n o n & al., 1992; j o g a n , 2007) or mostly as subspecies (a m a r a l f r a n c o & r o c h a a f o n s o , 1998; b o l o s & v i g o , 2001; c i n c o v i ć & k o j i ć , 1976; c i o c i r l a n , 1990; c o n e r t , 2000; c s i k y , 2009; c u a d r a d o & al., 2013; f i s c h e r & al., 2008; i l i j a n i ć & t o p i ć , 2000; l a u b e r & w a g n e r , 1998; p o l d i n i & al., 2002; s t o h r , 2002; t i s o n & f o u c a u l t , 2014). both were mentioned in countries with at least some submediterranean influence, but in central and w europe hml mostly as only casual plant of ruderal sites (c o n e r t , 2000; l a m b i n o n & al., 1992). determination keys are mostly using 1-3 characters for delimitation between hmm and hml, only some up to 5 characters (n e v s k i , 1941; b o l o s & v i g o , 2001; j o g a n , 2007; l a u b e r & w a g n e r , 1998). in majority of keys difference in spikelet (and awn) lenght between central and lateral spikelets of triple is mentioned and quite often also differences in shape of both glumes of lateral spikelet. often the only mentioned distinguishing measure is lenght of central spikelet's "stalk". this stalk is a peculiar characteristic in some hordeum species and is developed above glumes and below lemma, so in fact this is a prolonged internode of spikelet axis. reported gap of this measurment between the subspecies is between 0.6 and 1 mm, in hmm central spikelet stalk is shorter and in hml longer, mostly with no overlapping reported (n e v s k i , 1941; a m a r a l f r a n c o & r o c h a a f o n s o , 1998; b o l o s & v i g o , 2001; c i o c i r l a n , 1990; f i s c h e r & al., 2008; j o g a n , 2007; s t o h r , 2002; t i s o n & f o u c a u l t , 2014). in practice it is often not so easy to use this character. other distinguishing characters used in some of the determination keys were: inflorescence width (b o l o s & v i g o , 2001; l a u b e r & w a g n e r , 1998) and colour (l a u b e r & w a g n e r , 1998; t i s o n & f o u c a u l t , 2014), lateral lemma width (n e v s k i , 1941; j o g a n , 2007) and hairiness (c v e l e v , 1976; j o g a n , 2014). material and methods herbarium material available in herbarium lju (university of ljubljana, biotechnical faculty) and personal herbarium collection of the author (hsnj, hortus siccus n. jogan) enriched by some more systematic sampling for h. murinum have been taken as the main source for selection of representative sample of well preserved specimens, together 86 sheets served as operational taxonomic unis (otus). in each selected herbarium sheet one well preserved and complete plant served as an otu and all the measurements taken on that plant. in the measuring phase, one inflorescence was broken apart biologica nyssana 8 (1)  september 2017: 23-30 jogan, n.  morphometric recognition of hordeum murinum… 25 so that all spikelet measurements were done on the triple of spikelets from the central part of inflorescence. remaining spikes are enclosed in herbarium capsulae attched to the sheet. macroscopic measurements were done by a ruler and all smaller parts measured under stereo microscope euromex at 20x magnification and with an ocular measuring scale divided to 1/20 mm. all the distinguishing characters used in above mentioned literature were measured (one measurement per each character per otu) and in addition to them about as big number of other potentially useful characters as follows: vsn: total plant height (dm), cpp: palea length of central spikelet (cm), cr: length of cental lemma's awn (cm), cpc: length of central "stalk" (mm), ckp: width of central lemma (mm), ant: length of ripe anther (mm), lpp: palea length of lateral spikelet (cm), lr: length of lateral lemma's awn (cm), lpc: length of lateral "stalk" (mm), lkp: width of lateral lemma (mm), dpp: central palea hairy (y/n), dstr: margin of outer lateral spikelet glume ciliate (y/n), dnot: margin of inner lateral spikelet glume ciliate (y/n), kod: length of central spikelet axis prolongation (mm), kos: width of central spikelet axis prolongation (mm), kob: axis prolongation yellow (y/n). palea lengths were taken as equal to lemma lengths, so the lemma's awn lenghts were measured from the palea tip to the end of awn. some peculiar character measurements are shown in fig. 1. input matrix of 86 otus with 12 measured characters were used for all subsequent analyses starting with simple univariate statistics and further on different multivariate approaches (using past 1.74, http://palaeo-electronica.org/2001_1/past/) to recognize texonomic structure. a priori formed groups of 2 taxa (hmm and hml) were slightly modified after resuls of numerical analyses so at the end a small "transitional" group of otus with unclear taxonomic position had been recognized as the third entity. after removing that group from analyses, the taxonomic structure is much more clear-cut, but in the field we have to be aware of such populations. studied material is listed in supplementum with locality, mtb grid code (n i k l f e l d , 1971), author, date, herbarium acronym and accession number. results and discussion majority of otus was determined using the mentioned determination keys but some of them (9%) remain at the level of h. murinum s. lat. those were temporarily excluded from the data matrix for first univariate statistical analyses. from all measured characters, some that showed a potential usefulness for distinguishing studied taxa are represented by box plots in fig. 2. usefulness of already reported distinguishing characters has mostly been confirmed for following characters: central stalk length (fig. 2a), central awn length (fig. 2b), lateral awn length (fig. 2c), lateral palea length (fig. 2d), lateral lemma width (fig. 2e) and plant height (fig. 2f). several of them are reported in determination keys without measurements, only describing their relative length. some of the recorded atributive characters were more randomly distributed among studied taxa and results are not presented here because of limited usability for distiguishing taxa. several conducted multivariate analyses helped us to recognize the most useful distinguishing characters and also to recognize the overall taxonomic structure. with exclusion of mentioned small group of «intermediates« separation of two clusters representing both subspecies was clear with only slight overlapping as shown in fig. 3. obviously delimitation of subspecies will remain uneasy in some populations. interesting results turned to be comparison of total lengths of spikelets (fig. 4), quite often roughly mentioned as »lateral awn exceeding central awn« or »... not exceeding ...«. total lengths were calculated from 3 measurements for each spikelet (cpc+cpp+cr and lpc+lpp+lr respectively). indeed in hmm central awn is mostly slightly fig. 1. three of the mentioned characters that are specific for hordeum spikelet triple: cpc (length of central "stalk"), lpc (length of lateral "stalk") and kod (length of central spikelet axis prolongation) biologica nyssana 8 (1)  september 2017: 23-30 jogan, n.  morphometric recognition of hordeum murinum… 26 fig. 2. box plots of some of the useful distinguishing characters. box comprising 2nd and 3rd quartile, whiskars 1st and 4th quartile, some outliers marked with *, hmm on the left (grey), hml on the right (white). a) central stalk length; b) central awn length; c) lateral awn length; d) lateral palea length; e) lateral lemma width; f) plant height fig. 3. results of pca. input data matrix with 12 characters and 86 otu grouped in hmm (green squares), hml (red crosses) and intermediate group (blue triangles) mur lep 0,0 0,2 0,4 0,6 0,8 1,0 1,2 1,4 m m central "stalk" length mur lep 0 2 4 6 c m central awn length mur lep 0 2 4 6 c m lateral awn length mur lep 0,0 0,2 0,4 0,6 0,8 1,0 1,2 1,4 1,6 1,8 2,0 c m lateral palea length mur lep 0,0 0,2 0,4 0,6 0,8 1,0 1,2 1,4 1,6 1,8 2,0 m m lateral lemma width mur lep 0 2 4 6 8 10 d m plant height -0,24 -0,16 -0,08 0 0,08 0,16 0,24 0,32 coordinate 1 -0,25 -0,2 -0,15 -0,1 -0,05 0 0,05 0,1 0,15 c o o rd in a te 2 biologica nyssana 8 (1)  september 2017: 23-30 jogan, n.  morphometric recognition of hordeum murinum… 27 exceeding the laterals, but in hml where it is often stated, that laterel awns are exceeding central one, situation is more diverse with only about half of otu having laterel awns reaching or exceeding the central one. but mostly dimensions of spikelets are distinctly bigger than in hmm, so probably better distinguishing character for quick orientation in the field would be: total length of spikelets 3-5 cm vs. 57 cm in hmm and hml respectively. in fact all the average measured values of hmm and hml differ mostly 20-35%, an interesting situation that is often observed in polyploids when compared to ancestral diploids, but in discussed group both subspecies are predominantly tetraploid so interpretation must be different. our results can be compiled in a determination key which use much more characters than the others mentioned above and some of the measurements are now more precisely measured. but of course we have to bear in mind that slovenia is a tiny territory where distribution range of both discussed taxa overlap and so populations far from studied area can be slightly different. statistics of the useful distinguishing characters are presented as minimum and maximum (in brackets) and an interval between 1st and 3rd quartile. 1 total spikelet lengths (including awns) (2.5) 3.54.6 (5.9) cm, central spikelet of a triple with »stalk« (0.3) 0.6-1 (1.4) mm long, palea (0.8) 0.85-1 (1.2) cm long and lemma's awn (1.8) 2.4-3.5 (4) cm long, lateral spikelets with lemma's awn (1.4) 2-3 (3.5) cm long, palea (0.75) 0.9-1.1 (1.2) cm long and lemma (1) 1.2-1.6 (1.8) mm wide. anthers (0.6) 0.7-1 (1.4) mm, plant (20) 25-45 (60) cm ........................ h. murinum ssp. murinum 1* total spikelet lengths (including awns) (4) 5-6.5 (8) cm, central spikelet of a triple with »stalk« (0.5) 0.9-1.2 (1.5) mm long, palea (0.9) 1-1.15 (1.3) cm long and lemma's awn (2.8) 3.4-4.7 (5.9) cm long, lateral spikelets with lemma's awn (2.8) 3.5-4.8 (5.7) cm long, palea (1.1) 1.2-1.4 (1.6) cm long and lemma (1.2) 1.6-1.9 (2.1) mm wide. anthers (0.7) 0.9-1.3 (1.6) mm, plant (25) 40-50 (90) cm ...................... h. murinum ssp. leporinum and finally here is the updated distribution map of the studied taxa in slovenia (fig. 5). with light grey dots not only "intermediate" otus were represented but also other reported occurrences of h. murinum complex (cf. j o g a n et al., 2001) for which no herbarium material had been available so it is better to recognize them on that taxonomic level. obviously the group is represented only in lowland slovenia mostly below 300 m a.s.l., only few scattered localities can reach up to 600 m a.s.l. along the main highways. in the extreme sw part along the adriatic coast and in vipava valley hml is the common taxon, but also some populations of hmm can be found. in central and e slovenia hmm is present in majority of sampling sites, but there are also some records of hml populations. as it has been expected that both studied taxa are mostly tetraploids, possible hybridization could not be excluded as a cause for "intermediate" populations, but detailed study of those "intermediate" otus was beyond our scope. it is important to stress that in the discussed territory both studied taxa are sympatric with slighly bigger frequency of hml populations in the sw coastal part of slovenia and hmm present in all lowland parts of the country. in interpreting »intermediates« also a hypothesis of n e v s k i (1941), that hml is an ancestral relative of hmm can be roughly taken into consideration, but it seems that the evoultion of the »murinum complex« had been more complicated (c u a d r a d o & al., 2013) distribution pattern matches quite well pattern in adjacent friuli-venezia giulia (ne italy, fig. 4. comparison of total spikelet lengths of central vs. lateral spikelet of triple. hmm represented with grey, hml with empty symbols. equal lengths at dashed diagonale, longer central spikelet below total spikelet lengths 0 2 4 6 8 0 2 4 6 8 10 central spikelet (cm) la te ra l s p ik e le t (c m ) hmm hml biologica nyssana 8 (1)  september 2017: 23-30 jogan, n.  morphometric recognition of hordeum murinum… 28 p o l d i n i 2002) but maybe also in mentioned region hml can be expected with some local populations further from the coast. in croatia situation is similar, hml reported in the coastal region with only one locality in continental part and hmm scattered (n i k o l i ć , 2015): in continental adjacent countries, austria and hungary, hml ocurrence is very local to ephemeral (c s i k y , 2009; f i s c h e r et al., 2008). in the future presence of hml in continental slovenia is to be studied in detail as it would not be possible to exclude the probability of mostly ephemeral occurrence of this taxon in extremely dry ruderal places as e.g. along the railways or in abandoned gravel sites in bigger cities where "urban heat island" phenomenon can impact local mesoclimatic conditions. on the other hand it would be interesting to study populations of hmm in the coastal part of slovenia. at least the impression after the sampling is that hmm is more linked to shadowy, slightly wet ruderal places whereas hml is common in very dry ruderal communities. conclusion as a result we can confirm, that both previously mentioned subspecies of h. murinum are present in slovenia, there are more useful distinguishing characters than reported before but still it is not always easy to recognize the subspecies. hordeum murinum ssp. murinum is present all-over slovenian lowland and h. murinum ssp. leporinum predominantly in the coastal region, but with some scaterred populations also inland. ecological preferences in the areas where both co-ocurr remained to be studied. references amaral franco, j. do, rocha afonso, m. 1998: nova flora de portugal 3/ii, gramineae. escolar editora, lisboa. 283 p. bolos, o. de, vigo, j. 2001: flora dels paisos catalans iv. editorial barcino, barcelona. 750 p. cincović, t., kojić, m. 1976: poaceae. in: josifović, m. (ed.), flora sr srbije 8: 259-472, sanu, beograd. ciocirlan, v. 1990: flora ilustrata a romaniei ii. editura ceres, bucuresti. 598 p. conert, h. j. 2000: pareys graeserbuch. parey bucherlag, berlin. 592 p. csiky, j. 2009: hordeum l. arpa. in: g. kiraly (ed.), uj magyar fuveszkonyv 522-523 aggteleki nemzeti park igazgatosag, josvafo. cuadrado, a., carmona, a., jouve, n. 2013: chromosomal characterization of the three subgenomes in the polyploids of hordeum murinum l.: new insight into the evolution of this complex. plos one, 8 (12). fig. 5. distribution of discussed taxa in slovenia: green squares hmm, black dots hml, only revised records presented, grey circles: h. murinum s. lat. partly as a result of revision, mostly based on literature and unpublished field records hordeum murinum 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 s. lat. ssp. murinum ssp. leporinum © n e jc j o g a n 46°n 15°e a i cro h biologica nyssana 8 (1)  september 2017: 23-30 jogan, n.  morphometric recognition of hordeum murinum… 29 cvelev, n.n. 1976: zlaki sssr. nauka, leningrad. 788 p. fischer, m.a., adler, w., oswald, k. 2008: exkursionsflora. österreich, liechtenstein, suedtirol. biologiezentrum der oberoesterreichischen landesmuseen, linz. 1380 p. ilijanić, l., topić, j. 2000: poaceae. in: nikolić, t. (ed.), index florae croaticae 3, natura croatica, 9 (suppl. 1): 130-149. jogan, n. (ur.), bačič, t., frajman, b., leskovar, i., naglič, d., podobnik, a., rozman, b., strgulc krajšek, s., trčak, b. 2001: gradivo za atlas flore slovenije. center za kartografijo favne in flore, miklavž na dravskem polju. 443 p. jogan, n. 1999: skupina mišjega ječmena (hordeum murinum) v sloveniji. in: jogan, n. (ed.), flora in vegetacija slovenije 1999, zbornik izvlečkov referatov simpozija. botanično društvo slovenije, ljubljana. jogan, n. 2005: some interesting records of grasses in the flora of r macedonia. in: ranđelović, n. (ed.): symposium on the flora of se serbia and neighbouring regions, niš 20.-24.06.2005. abstracts. niš: university of niš, faculty of sciences and mathematics. p. 46. jogan, n. 2007: poaceae (gramineae) trave. in: martinčič, a., t. wraber, n. jogan, a. podobnik, b. turk, b. vreš, v. ravnik, b. frajman, s. strgulc krajšek, b. trčak, t. bačič. m. a. fischer, k. eler & b. surina: mala flora slovenije. 4 izd. 826-932, tehniška založba slovenije, ljubljana. jogan, n. 2014: poaceae barnhart – suesgraeser. in: rottensteiner, w. k.: exkursionsflora fuer istrien. naturwissenschaftlicher verein fuer kaernten. 679-751. jogan, n., bačič, t., vreš, b. 1999. prispevek k poznavanju flore okolice ormoža. natura sloveniae, ljubljana 1(1): 5-27. kankanpää, j, schulman, a. h, & mannonen, l., 1996: the genome sizes of hordeum species show considerable variation. genome, 39 (4): 730-735. lambinon, j., de langhe, j. e., delvosalle, l., duvigneaud j. & al. 1992: nouvelle flore de la belgique, du grand-douche de luxembourg, du nord de la france et des regions voisines. edition du jardin botanique national de belgique, meise. lauber, k., wagner, g. 1998: flora helvetica. haupt verlag, bern, stuttgart, wien. martinčič, a. 1984: poaceae trave. in: martinčič, a. & f. sušnik: mala flora slovenije: praprotnice in semenke. dzs, ljubljana. nevskij, s. a. 1941: materiali k poznaniju dikorastuščih jačmenej v svjazi s voprosom proishoždenija hordeum vulgare l. i hordeum distichon l. flora i sistematika vyšsih rastenij, 5: 64-255. moskva, leningrad. niklfeld, h., 1971: bericht über die kartierung der flora mitteleuropas. taxon, 20: 545–571. nikolić, t. (ed.), 2015: flora croatica baza podataka; http://hirc.botanic.hr/fcd. prirodoslovno-matematički fakultet, sveučilište u zagrebu; access: 21.06.2017 pignatti, s. 1983: flora d'italia 1-3. edagricole, bologna. poldini, l. 2002: nuovo atlante corologico delle piante vascolari nel friuli venezia giulia. regione autonoma friuli venezia giulia, azienda parchi e foreste regionali & universita degli studi di trieste. scholz, h., raus, t. 1997: zwei neue unterarten des hordeum murinum (gramineae) aus griechenland und spanien. feddes repertorium, 108: 527-531. stohr, g. 2002: poales (graminales). in: rothmaler, w. & al.: exkursionsflora. spektrum akademischer verlag, berlin. strid, a. 2016: atlas of the aegean flora. bgbm, berlin. 700-878. tison, j. m., de foucault, b. 2014: flora gallica. biotope editions, meze. 1196 p. supplemetal data: specimina visa: hordeum murinum ssp. leporinum sežana (10249/3), leg. r. justin, 16.3.1905, lju 10130753 (17315); strunjan (10447/4), leg. t. wraber, 21.7.1972, lju 10130751 (33548); koper (10448/3), leg. t. wraber, 19.5.1973, lju 10130750 (24142); biljenski griči (10148/1), leg. g. seljak, 22.5.1987, lju 10130766 (120321); prade (10448/4), leg. b. mozetič, 5.5.1989, lju 10130785 (121541); sežana-vrhovlje (10249/3), leg. v. debevec, 10.6.1989, lju 10130784 (121095); dane pri sežani (10249/3), leg. m. frelih, 10.6.1989, lju 10130786 (121751); piran, mogoron (10447/3), leg. g. planinc, 9.5.1993, lju 10130772 (124994); osp (10449/1), leg. n. dolenc, 16.4.1994, lju 10130743 (59976); strunjan (10447/4), leg. s. toth, 24.4.1994, lju 10130748 (59992); škocjan (10448/3), leg. a. zajko, 18.6.1994, lju 10130747 (60006); koper, norbedi (10448/2), leg. b. toškan, 20.6.1994, lju 10130749 (60051); sežana, kopriva (10249/1), leg. biologica nyssana 8 (1)  september 2017: 00-00 jogan, n.  morphometric recognition of hordeum murinum… 30 m. vrabec, 20.5.1995, lju 10130757; avber pri sežani (10249/1), leg. m. pegan žvokelj, 22.5.1995, lju 10130774 (126998); lozice (10250/1), leg. m. žvanut, 15.6.1995, lju 10130759; brkini, sv. pavel (10551/1), leg. n. jogan, 29.7.1996, lju 10130760; vrtovin, šateji (10048/4), leg. u. jelenc, 25.5.1997, lju 10130768 (127748); dane (10249/3), leg. m. šebart, 31.5.1997, lju 10130767 (127788); turjak (10153/2), leg. p. presetnik, 26.6.1997, lju 10130770 (127743); panovec, strelišče (10047/2), leg. n. jogan, 19.7.1997, lju 10130761 (63481); vel. žablje (10149/1), leg. k. vodopivec, 19.6.1999, lju 10130763 (63403); haloze, borl (9662/1), leg. n. jogan, 12.7.2002, lju 10143040; sp. škofije (10448/2), leg. p. glasnović, 2.5.2003, lju 10134768; sp. škofije, bonifika (10448/2), leg. p. glasnović, 8.5.2004, lju 10134770; bertoki (10448/2), leg. p. glasnović, 15.5.2004, lju 10134769; ankaran (10448/1), leg. p. glasnović, 20.5.2005, lju 10134771; (9957/4), leg. n. jogan, hsnj l1028; (9952/2), leg. n. jogan, hsnj l993; (10447/4), leg. n. jogan, hsnj p1134; (10449/3), leg. n. jogan, hsnj p1277; (10449/1), leg. n. jogan, hsnj p1293; (10148/2), leg. n. jogan, hsnj p1296; (10447/4), leg. n. jogan, hsnj p1350; (10447/4), leg. n. jogan, hsnj p1379; (10448/1), leg. n. jogan, hsnj p1423; (10448/1), leg. n. jogan, hsnj p1424; (10449/1), leg. n. jogan, hsnj p1459; (10447/4), leg. n. jogan, hsnj p1505; (10447/4), leg. n. jogan, hsnj p1623; (10447/4), leg. n. jogan, hsnj p1633; (10447/4), leg. n. jogan, hsnj p1763; (10149/4), leg. n. jogan, hsnj p562; (10349/2), leg. n. jogan, hsnj p578; (10448/1), leg. n. jogan, hsnj p879; hordeum murinum ssp. murinum vremski britof (10350/1), leg. r. justin, 30.6.1905, lju 10130752 (17314); gornje ležeče (10350/1), leg. r. justin, 2.7.1905, lju 10130746 (17313); ljubljana (9953/1), leg. r. justin, 30.5.1918, lju 10130745 (17310); šmarna gora (9852/4), leg. r. justin, 6.7.1936, lju 10130744 (17308); ljubljana, ilirska cesta (9953/1), leg. b. turk, 15.9.1985, lju 10130739; ljubljana, bežigrad (9953/1), leg. n. jogan, 24.5.1988, lju 10130756; solkan (10047/2), leg. l. merljak, 16.6.1989, lju 10130783 (121108); novo mesto (10257/1), leg. d. kovačec, 10.6.1993, lju 10130778 (125060); lendava, sv. trojica (9464/2), leg. n. jogan, 12.7.1994, lju 10130755; preserje pri domžalah (9853/2), leg. t. bačič, 1.6.1995, lju 10130776 (126581); belveder-ronek (10447/4), leg. j. france, 17.7.1995, lju 10130775 (126532); ormož (9562/4), leg. n. herga, 8.6.1996, lju 10130782 (127498); pobegi (10448/4), leg. k. sedmak, 17.6.1996, lju 10130780 (127448); sežana (10249/3), leg. b. tavčar, 18.6.1996, lju 10130781 (127524); vel. badin (10549/2), leg. n. jogan, 11.5.1997, lju 10130769; ormož, ž. p. (9562/4), leg. n. jogan, 29.7.1997, lju 10130758; grosuplje (10053/2), leg. d. simonič, 18.5.1998, lju 10130762 (54571); metlika, radovica (10358/1), leg. b. frajman, 1.8.2001, lju 10130864; valdoltra (10448/1), leg. p. glasnović, 21.4.2003, lju 10134767; izola (10447/4), leg. u. & w. starmuhler, 24.4.2006, lju 10135027; portorož (10447/4), leg. v. mikolaš & al., 15.5.2010, lju 10142708; dovje (9549/2), leg. v. plemel, 4. 8. 1873, lju 10130740; (9848/1), leg. n. jogan, hsnj a910; (9852/4), leg. n. jogan, hsnj l1033; (9953/1), leg. n. jogan, hsnj l1035; (9953/1), leg. n. jogan, hsnj l818; (9953/2), leg. n. jogan, hsnj l867; (10349/1), leg. n. jogan, hsnj p1241; (10249/1), leg. n. jogan, hsnj p1266; (10547/2), leg. n. jogan, hsnj p1346; (10449/3), leg. n. jogan, hsnj p1385; (10448/2), leg. n. jogan, hsnj p1408; (10447/4), leg. n. jogan, hsnj p1477; (10448/3), leg. n. jogan, hsnj p1630; (10349/4), leg. n. jogan, hsnj p1820; (10548/1), leg. n. jogan, hsnj p27; (10349/2), leg. n. jogan, hsnj p571; (9363/1), leg. n. jogan, hsnj š769; (9462/2), leg. n. jogan, hsnj š902; (9359/2), leg. n. jogan, hsnj š917; (9360/3), leg. n. jogan; hordeum murinum s. lat. celje (9757/4), leg. a. knap, 7.8.1938, lju 10130741; žalec (9757/1), leg. s. grobelnik, 14.6.1993, lju 10130777 (125058); ljubljana, bs3 (9953/1), leg. h. krečič, 19.6.1994, lju 10130742 (60076); lendava, železnica (9464/2), leg. n. jogan, 12.7.1994, lju 10130754; dane-šmarje (10249/3), leg. a. lisjak, 30.6.1995, lju 10130779 (126939); hrvatinski hrib (10448/2), leg. k. marc, 25.7.1996, lju 10130773 (127831); ig-pod strahom (10053/1), leg. j. zajc, 26.6.1997, lju 10130764 (63775); zg. hrušica (9953/3), leg. m. kukec, 31.7.1997, lju 10130771 (127911); tolmin (9848/1), leg. š. glišovič, 22.5.1999, lju 10130765 (63422); (10547/2), leg. n. jogan, hsnj p1345; anticancer compounds from medicinal plants biologica nyssana 6 (1)  september 2015: 41-48 žikić, v. et al.  new data on microgastrinae in serbia and ... 41 original article received: 27 august 2015 revised: 30 august 2015 accepted: 01 september 2015 new data on microgastrinae in serbia and montenegro (hymenoptera: braconidae) and their hosts vladimir žikić, maja lazarević * , saša s. stanković, marijana ilić milošević university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia * e-mail: majalazarević@gmail.com abstract: žikić, v., lazarević, m., stanković, s.s., ilić milošević, m.: new data on microgastrinae in serbia and montenegro (hymenoptera: braconidae) and their hosts. biologica nyssana, 6 (1), september 2015: 4148. the fauna of microgastrinae wasps was investigated on the territory of serbia and montenegro. eighteen species were recorded and most of them emerged from the previously identified hosts. the species belong to seven genera: apanteles (1), cotesia (7), dolichogenidea (2), glyptapanteles (3), microgaster (1), microplitis (2) and pholetesor (2). for the territory of montenegro there is one newly reported species, cotesia tetricus, while for serbia seven species are new: cotesia ofella, dolichogenidea breviventris, glyptapanteles compressiventris, g. porthetriae, microgaster bicolor, microplitis pellucida, m. varipes and pholetesor elpis. apart from microgastrine wasps, some hyperparasitoids were also identified. key words: microgastrinae, parasitoids, lepidoptera, trophic associations apstrakt: žikić, v., lazarević, m., stanković, s.s., ilić milošević, m.: novi podaci o fauni mikrogastrina u srbiji i crnoj gori (hymenoptera: braconidae) i njihovih domaćina. biologica nyssana, 6 (1), septembar 2015: 41-48. istraživana je fauna paraziskih osa iz potfamilije microgastrinae na teritoriji srbije i crne gore. identifikovano je ukupno 18 vrsta, od čega je većina dobijena iz odgajenog i identifikovanog domaćina. registrovane vrste parazitoida pripadaju sledećim rodovima: apanteles (1), cotesia (7), dolichogenidea (2), glyptapanteles (3), microgaster (1), microplitis (2) i pholetesor (2). za teritoriju crne gore registrovana je jedna nova vrsta cotesia tetricus, dok je za teritoriju srbije: cotesia ofella, dolichogenidea breviventris, glyptapanteles compressiventris, g. porthetriae, microgaster bicolor, microplitis pellucida, m. varipes i pholetesor elpis. pored mikrogastrina registrovano je više vrsta hiperparazitoida. key words: microgastrinae, parasitoidi, lepidoptera, trofičke asocijacije introduction members of the subfamily microgastrinae are koinobiont endoparasitoids of larvae lepidoptera and represent one of the most numerous and very important group of braconidae. most of the species are usually small, about 2-5 mm in length, black coloured, sometimes with yellowish parts, 6 (1) • september 2015: 41-48 biologica nyssana 6 (1)  september 2015: 41-48 žikić, v. et al.  new data on microgastrinae in serbia and ... 42 especially on legs. some of the species are solitary parasitoids, in which case only one microgastrine larva develops inside the host body. however, some exhibit different way of parasitism which is gregarious, when several parasitoid individuals develop from only one egg laid by a single female; in which case all parasitoids have the same genotype (s h a ѕ w & h u d d l e s t o n , 1991). beside lepidoptera as hosts for microgastrines larvae, there are several records of other insect groups such as coleopterans, dipterans or hymenopterans (symphyta) (y u et al., 2012). a total of 2000 species of this subfamily have been reported worldwide and of that number about 500 species inhabit the palaearctic (w h i t f i e l d et al., 2002; p é r e z r o d r í g u e z et al., 2013). phylogenetically microgastrinae belongs to the microgastroid complex of subfamilies along with cardiochilinae, cheloninae, ichneutinae, khoikhoiinae, miracinae (w h a r t o n et al., 1992) which is confirmed by later analysis using molecular markers for 18s rrnk, 28s rrnk, protein-coding genes (cad54-405), and the coding gene for acetyl-coenzyme a carboxylase (acc) (s h a r a n o w s k i et al., 2011). classification of microgastrinae is still very confusing because many authors prefer keeping their own systems of identification and separation of some genera and subgenera to new ones. another important fact is that this large subfamily constantly reveals its richness in diversity of species and genera further forcing new reclassifications. the maximum accepted number of genera is 31, which are reported in y u et al. (2012). the biggest problems occur in very large, “traditional genera”, firstly in apanteles förster, cotesia cameron, than in microgaster latreille, microplitis foerster and protapanteles ashmead. the fauna of microgasterinae in serbia and montenegro is poorly investigated. there are few publications that are directly related to the taxonomy of this subfamily: p a p p (1973, 2009), b r a j k o v i ć (1989) and ž i k i ć et al. (2000); whereas most of the cited publications predominantly refer to serbia. sporadically, some records could be found in papers by other authors e.g. (w h i t f i e l d , 1997) but, certainly, the most important are in t o b i a s (1986) and in the global database taxapad by y u et al. (2012). in s a n t o s et al. (2009) there are some data on microgastrinae in citrus orchards. as a result of rather poor and limited research of microgastrinae fauna on the territory of serbia and montenegro, the main aim of the present study was to present some new findings of these wasps through tritrophic associations. material and methods for the purposes of this study the specimens of microgastrinae were collected on the territory of serbia and montenegro during the period of 20012013. material was sampled mainly by collecting caterpillars from plant leaves, both from trees and herbaceous plants. some of the wasp specimens were collected using sweep net and malaise trap. caterpillars were put in specially made cages and they were fed with adequate food plants in the following 10 days. some of the parasitoids emerged directly from caterpillars, while some of them appeared after host pupation (larva/pupal parasitism). most of the collected parasitoids were kept dry and mounted on paper cards and pinned. partly, specimens of each species were conserved in 96% ethanol for further dna analyses. all analysed material has been deposited in the insect collection of the department of biology and ecology, faculty of sciences and mathematics, university of niš, serbia. identification for the identification of feeding plants botanists from the department of biology and ecology, faculty of sciences and mathematics, university of niš, serbia were consulted. larvae of butterflies were identified by boženka hric. parasitoid specimens were identified by dr. jenő papp from hungarian natural history museum, budapest. in this particular study we followed the classification given in the keys proposed by p a p p (1978, 1981, 1982, 1983, 1987). investigated localities all the localities are given the altitude value. all measurements are given in meters above sea level. serbia: bosilegrad, 780 m; deliblatska peščara, 160 m; dukat mt., 1150-1400 m; kruševac, 165 m; lebane, konjino, 275 m; niš, gornji matejevac, 425 m; niš, niška banja 250 m, niš, pantelej, 220 m; sićevačka klisura (gorge), 225 m; tara mt., 1090 m; vlasinsko jezero 1250 m; zasavica, valjevac, 75 m. montenegro: durmitor, žabljak, 1450 m; visitor mt. 1700 m. results the identified species are listed alphabetically. for each species the locality, date, legator’s name and the number of males and females collected were given and those were marked with universal symbols. in cases where there was the host and the plant the information was updated by adding the full names of these members of tritrophic biologica nyssana 6 (1)  september 2015: 41-48 žikić, v. et al.  new data on microgastrinae in serbia and ... 43 associations. additional information and remarks concerning parasitoid biology/strategy and morphology of cocoons are presented in the notes. finally, before the species name new findings for the investigated territories were marked using the following symbols: (*) new to serbia, ( # ) new to montenegro. apanteles lenea nixon, 1986 srb, sićevačka klisura (gorge), 04.06.2011, leg. v. žikić, 1♀; srb, vlasinsko jezero (lake), 05.06.2014, leg. v. žikić, 1♂ on centaurea jacea l. ex larvae verbascum thapsus l. notes: solitary parasitoid. hosts: tortricidae: olethreutes arbutella (l.), oncocera semirubella (scopoli), sparganothis pilleriana (denis & schiffermüller). distribution: austria, bulgaria, czech republic, france, germany, hungary, italy, south korea, romania, russia (chita oblast, primorskye kray, sakhalin oblast), serbia, slovakia, spain, sweden, switzerland, turkey, united kingdom. cotesia jucunda (marshall, 1885) srb, niš, gornji matejevac, 25.05.2014, leg. v. žikić, 25♂, 21♀ on linaria dalmatica subsp. macedonica (griseb.) d. a. sutton ex larvae melitaea didyma (esper). notes: gregarious, separately spun cocoons randomly around dead caterpillar. hosts: geometridae: abraxas grossulariata (l.), alsophila aceraria (denis & schiffermüller), campaea margaritaria (l.), cyclophora linearia (hübner), c. punctaria (l.), c. ruficiliaria (herrichschäffer), epirrita autumnata (borkhausen), erannis defoliaria (clerck), eupithecia dodoneata guenée, operophtera brumata (l.), o. fagata (scharfenberg), phigalia pilosaria (denis & schiffermüller); pieridae: pieris brassicae (l.); pterophoridae: stenoptilia veronicae karvonen; nymphalidae: vanessa atalanta (l.), curculionidae: anthonomus pomorum (l.). distribution: armenia, austria, bulgaria, croatia, czech republic, denmark, estonia, finland, france, germany, greece, hungary, iran, ireland, moldova, mongolia, poland, romania, russia (primorskye kray), serbia, slovakia, spain, sweden, switzerland, turkey, united kingdom. *cotesia ofella (nixon, 1974) srb, kruševac, 03.05.2014, leg. m. lazarević, 65 ♀. notes: gregarious, from unknown yellowish group cocoon associated with chamaecyparis lawsoniana (a. murray) parl. hosts: erebidae: spilosoma lubricipeda (l.); lasiocampidae: euthrix potatoria (l.); noctuidae: acronicta aceris (l.), a. rumicis (l.), simyra dentinosa freyer. distribution: belgium, czech republic, finland, germany, hungary, iran, italy, netherlands, poland, slovakia, spain, switzerland, turkey, ukraine, united kingdom. cotesia sp. 1 srb, bosilegrad, dukat mt., 07.08.2011, leg. v. žikić, 1♂. notes: sweep net. cotesia sp. 2. srb, vlasinsko jezero (lake), 06.06.2014, leg. s. stanković, 3♂, 21♀. notes: gregarious in shared cocoon. hyperparasitized with gelis sp. (ichneumonidae: cryptinae). # cotesia tetricus (reinhard, 1880) mne, durmitor, žabljak, 03.08.2005, leg. v. žikić, 25♀. notes: gregarious from unknown yellowish group cocoon. hyperparasitized by mesochorus sp. (ichneumonidae: mesochorinae). hosts: erebidae: parasemia plantaginis (l.), spilosoma lubricipeda; geometridae: erannis defoliaria, eupithecia exiguata (hübner); nymphalidae: erebia aethiops (esper), lasiommata megera (l.), maniola jurtina; zygaenidae: zygaena filipendulae. distribution: austria, croatia, czech republic, finland, france, germany, greece, hungary, iran, ireland, italy, kazakhstan, latvia, norway, poland, romania, russia (yaroslavl oblast), sweden, switzerland, turkey, united kingdom. cotesia tibialis curtis, 1830 srb, sićevačka klisura (gorge), 13.04.2014, leg. v. žikić, 13♀; srb, vlasinsko jezero (lake), 06.06.2014, leg. s. stanković, 3♂, 21♀. notes: gregarious in shared cocoon. hyperparasitized with gelis sp. (ichneumonidae: cryptinae). hosts: arctiidae: arctia tigrina (villers); bombicidae: bombyx mori l.; cossidae: dyspessa ulula (borkhausen); erebidae: diaphora mendica (clerck), ocnogyna baetica (rambur), o. loewii (zeller), spilosoma lubricipeda (l.); gelechiidae: pexicopia malvella (hübner); geometridae: eupithecia abbreviata stephens, e. innotata (hufnagel), e. tripunctaria herrich-schäffer, phigalia pilosaria (denis & schiffermüller); lasiocampidae: dendrolimus pini (l.), eriogaster lanestris (l.); lycaenidae: plebejus loewii (zeller); biologica nyssana 6 (1)  september 2015: 41-48 žikić, v. et al.  new data on microgastrinae in serbia and ... 44 noctuidae: acronicta aceris, a. euphorbiae (denis & schiffermüller), a. leporina (l.), a. rumicis, agrotis clavis (hufnagel), a. rectangular (denis & schiffermüller), a. segetum (denis & schiffermüller), anarta myrtilli (l.), antoculeora ornatissima walker, autographa gamma (l.), euclidia mi (clerck), catocala nupta (l.), cerastis rubricosa (denis & schiffermüller), cucullia argentea (hufnagel), c. artemisiae (hufnagel), c. asteris (denis & schiffermüller), c. lucifuga (denis & schiffermüller), diachrysia chrystis (l.), discestra trifolii (hufnagel), eupsilia transversa (hufnagel), eurois occulta (l.), euxoa temera (hübner), e. tritici (l.), graphiphora augur (fabricius), lacanobia oleracea (l.), l. pisi (l.), mamestra brassicae (l.), noctua pronuba (l.), orthosia gothica (l.), o. gracilis (denis & schiffermüller), o. incerta (hufnagel), xestia cnigrum (l.), x. exoleta leech, x. stigmatica (hübner), x. triangulum (hufnagel), xylena exsoleta (l.), x. vetusta (hübner); notodontidae: clostera anastomosis (l.), phalera bucephala (l.); nymphalidae: aglais urticae (l.), euphydryas aurinia (rottemburg), aglais io (l.), maniola jurtina (l.), pararge aegeria (l.), pyronia tithonus (l.), vanessa indica herbst; pieridae: aporia crataegi (l.), leptidea sinapis (l.), pieris brassicae; plutellidae: plutella xylostella (l.); pyralidae: loxostege sticticalis (l.), ostrinia nubilalis (hübner), pyrausta cingulata (l.), p. purpuralis (l.), p. sanguinalis (l.); tortricidae: ancylis laetana (fabricius), tortrix viridana l.; yponomeutidae: yponomeuta malinellus zeller; zygaenidae: zygaena filipendulae (l.). distribution: afghanistan, armenia, austria, azerbaijan, belgium, bulgaria, canary islands, china (shanxi, xinjiang), croatia, czech republic, estonia, finland, france, georgia, germany, greece, hungary, iran, israel, italy, japan, kazakhstan, kyrgyzstan, latvia, lithuania, f.y.r. of macedonia, moldova, mongolia, montenegro, netherlands, poland, romania, russia (adygeyskaya, chita oblast, irkutsk oblast, kaliningrad oblast, krasnodor kray, krasnoyarsk kray, moscow oblast, primorskye kray, sankt petersburg, tatarskaya respublika, vladimir oblast, yaroslavl oblast), serbia, slovakia, spain, sweden, switzerland, turkey, turkmenistan, ukraine, united kingdom, uzbekistan. cotesia zygaenarum (marshall, 1885) srb, sićevačka klisura (gorge), 15.05.2012, leg. v. žikić, 155♂ 167♀ ex 5th instar larvae of zygaena filipendulae. notes: gregarious. associated with ulmus sp. host: geometridae: phigalia pilosaria; lycaenidae: polyommatus icarus (rottemburg); noctuidae: diloba caeruleocephala (l.); nymphalidae: euphydryas aurinia; pieridae: colias hyale (l.); zygaenidae: zygaena brizae (esper), z. ephialtes (l.), z. filipendulae, z. laeta (hübner), z. lonicerae (scheven), z. trifolii (esper), z. viciae (denis & schiffermüller). distribution: albania, armenia, austria, azerbaijan, china (hubei), czech republic, finland, france, germany, greece, hungary, iran, italy, japan, korea, f.y.r. of macedonia, moldova, mongolia, poland, romania, russia (dagestanskaya respublika, krasnodar kray, omsk oblast, primorskye kray, ryazan oblast, voronezhskaya oblast), serbia, slovakia, switzerland, turkey, united kingdom. *dolichogenidea breviventris (ratzeburg, 1848): srb, sićevačka klisura (gorge), 13.04.2014, leg. v. žikić, 13 ♀. notes: gregarious. hosts: coleophoridae: coleophora anatipennella (hübner), c. betulella heinemann, c. flavipennella (duponchel), c. gryphipennella (hübner), c. lutipennella (zeller), c. prunifoliae doets, c. serratella (l.); noctuidae: orthosia miniosa (denis & schiffermüller). distribution: canada (newfoundland & labrador), czech republic, egypt, finland, germany, hungary, italy, korea, moldova, netherlands, poland, romania, russia (voronezhskaya oblast), slovakia, sweden, switzerland, turkey, united kingdom. dolichogenidea candidata (haliday, 1834) = d. longicauda (wesmael, 1837) srb, niš, pantelej, 29.05.2014, leg. v. žikić, 1♀ on euonymus europaeus l. ex pupa yponomeuta cagnagella (hübner). notes: solitary parasitoid. hosts: chimabachidae: diurnea lipsiella (denis & schiffermüller); erebidae: euproctis chrysorrhoea (l.); gelechidae: recurvaria leucatella (clerck), r. nanella (denis & schiffermüller); geometridae: abraxas grossulariata; gracillaridae: gracillaria syringella (fabricius); lasiocampidae: malacosoma neustria (l.); tortricidae: hedya nubiferana (haworth), pandemis cerasana (hübner), spilonota ocellana (denis & schiffermüller); yponomeutidae: paraswammerdamia lutarea (goeze), swammerdamia caesiella (hübner). distribution: azerbaijan, bulgaria, cape verde islands, germany, greece (crete), hungary, ireland, f.y.r. of macedonia, mongolia, romania, russia (khabarovsk kray, krasnodar kray, primorskye biologica nyssana 6 (1)  september 2015: 41-48 žikić, v. et al.  new data on microgastrinae in serbia and ... 45 kray, sakhalin oblast), serbia, sweden, tajikistan, turkmenistan, united kingdom, uzbekistan. [d. longicauda (wesmael, 1837)] host: bucculatridae: bucculatrix cristatella (zeller); chimbachidae: diurnea salicella (hübner); choreutidae: choreutis nemorana (hübner), c. pariana (clerck); elachisitdae: agonopterix ocellana (fabricius), blastodacna atra (haworth); erebidae: euproctis chrysorrhoea; gelechiidae: anarsia eleagnella kuznetsov, a. lineatella zeller, dichomeris derasella (denis & schiffermüller), exoteleia dodecella (l.), mirificarma lentiginosella (zeller), recurvaria leucatella, r. nanella, teleiodes paripunctella (thunberg), t. proximella (hübner), t. saltuum (zeller); gracillariidae: acrocercops brongniardella (fabricius), caloptilia populetorum (zeller), gracillaria syringella, phyllonorycter corylifoliella (hübner), p. elmaella doğanlar & mutuura; lasiocampidae: malacosoma neustria; momphidae: mompha subbistrigella (haworth); peleopodidae: carcina quercana (fabricius); plutellidae: plutella xylostella; praydidae: atemelia torquatella (lienig & zeller); psychidae: dahlica lichenella (l.); pyralidae: acrobasis consociella (hübner), a. marmorea (haworth); elegia fallax (staudinger), etiella zinckenella (treitschke), tortricidae: acleris holmiana (l.), a. laterana (fabricius), a. quercinana (zeller), adoxophyes orana (fischer von röslerstamm), archips oporana (l.), a. rosana (l.), choristoneura murinana (hübner), c. rosaceana (harris), ditula angustiorana (haworth), epinotia nigricana (herrich-schäffer), eudemis profundana (denis & schiffermüller), exapate duratella heyden, hedya nubiferana, h. pruniana (hübner), pandemis cerasana, parapandemis chondrillana (herrich-schäffer), rhopobota naevana (hübner), rhyacionia buoliana (denis & schiffermüller), spilonota ocellana, tortricodes alternella (denis & schiffermüller), tortrix viridana, zeiraphera griseana (hübner), z. rufimitrana (herrichschäffer); yponomeutidae: paraswammerdamia albicapitella (scharfenberg), p. lutarea (haworth), swammerdamia caesiella, yponomeuta cagnagella (hübner), zelleria parnassiae braun; ypsolophidae: ypsolopha alpella (denis & schiffermüller); cecidomyiidae: dasineura pyri; tenthredinidae: phyllocolpa benson. distribution: afghanistan, armenia, austria, azerbaijan, belarus, belgium, bulgaria, canada (british colombia), czech republic, estonia, finland, france, georgia, germany, hungary, italy, korea, latvia, lithuania, moldova, mongolia, netherlands, poland, romania, russia (buryatskaya respublika, krasnodar kray, sankt petersburg), serbia, slovakia, slovenia, spain, switzerland, turkey, ukraine, united kingdom. *glyptapanteles compressiventris (muesebeck, 1921) srb, niš, pantelej, 26.04.2014, leg. v. žikić, 42♂, 62♀, ex larva arctia villica (l.). notes: gregarious, separately spun cocoons. hosts: erebidae: amata phegea (l.), diaphora mendica, arctia villica, ocnogyna baetica, o. loewii, o. pierreti (rambur), phragmatobia fuliginosa (l.), spilosoma lubricipeda; noctuidae: amphipyra perflua (fabricius); tortricidae: archips xylosteana (l.). distribution: armenia, azerbaijan, canada (manitoba, northwest territories, nunavut, quebec), croatia, czech republic, finland, germany, hungary, italy, italy (sicily), kazakhstan, lithuania, f.y.r. of macedonia, moldova, netherlands, romania, russia (kamchatka oblast, primorskye kray, sakhalin oblast, sankt petersburg, voronezhskaya oblast), slovakia, spain, switzerland, turkey, usa (new hampshire), united kingdom. glyptapanteles liparidis (bouché, 1834) srb, tara mt., 25.06.2014, leg. v. žikić, 2♀. on ostrya carpinifolia. ex 3rd larval instar lymantria dispar (l.). notes: gregarious. hosts: erebidae: calliteara abietis (denis & schiffermüller), c. pudibunda (l.), arctia villica, euproctis chrysorrhoea, e. taiwana (shiraki), orgyia antiquiodes (hübner), o. australis walker, o. thyellina butler; lasiocampidae: dendrolimus albolineatus matsumura, d. pini, d. punctatus (walker), d. spectabilis (butler), d. superans (butler), eriogaster lanestris, malacosoma neustria; lymantriidae: dasychira pseudabietis butler, ivela auripes butler, lymantria dispar, l. obfuscate walker; noctuidae: acronicta rumicis; notodontidae: clostera anastomosis; tortricidae: rhyacionia buoliana. distribution: austria, belarus, bulgaria, china (beijing, heilongjiang, hunan, jilin, liaoning, nei menggu, shaanxi, taiwan, zhejiang), czech republic, finland, france, germany, hungary, india, iran, italy, japan, kazakhstan, lithuania, moldova, mongolia, poland, romania, russia (chita oblast, irkutsk oblast, kaliningrad oblast, khabarovsk kray, krasnodar kray, novosibirsk oblast, primorskye kray, sakhalin oblast, sankt petersburg, saratov oblast, tomsk oblast, voronezhskaya oblast, yaroslavl oblast), serbia, biologica nyssana 6 (1)  september 2015: 41-48 žikić, v. et al.  new data on microgastrinae in serbia and ... 46 slovakia, spain, sweden, switzerland, ukraine, united kingdom. *glyptapanteles porthetriae (muesebeck, 1928) srb, deliblatska peščara, 17.05.2001, a. ćetković, 1♀. notes: mal. trap. srb, niš, niška banja, 23.05.2014; leg. s. stanković on acer monspssulanum l. and ulmus sp. ex 2nd instar larvae of l. dispar; srb, sićevačka klisura (gorge), 21.05.2014, leg.v. žikić, 2♂, 2♀. notes: solitary, white cocoon underside leaf, on populus tremula l. ex 2nd instar larvae of l. dispar; srb, lebane, konjino, 03.05.2014, leg. s. stanković, 7♂, 10♀ on prunus domestica l. ex 2nd instar larvae of l. dispar; 3♂, 3♀ on cydonia oblonga mill. ex 2nd instar larvae of l. dispar. notes: solitary, white cocoons. hosts: geometridae: alcis repandata (l.), phigalia titea cramer; lymantridae: lymantria dispar, l. obfuscate. distribution: armenia, austria, azerbaijan, bulgaria, china (jilin), croatia, czech republic, finland, france, georgia, germany, greece, hungary, india, iran, italy, sicily, korea, moldova, morocco, poland, portugal, romania, russia (chita oblast, dagestanskaya respublika, moscow oblast, primorskye kray, voronezhskaya oblast, yaroslavl oblast), serbia, slovakia, spain, switzerland, turkey, ukraine, united kingdom. *microgaster bicolor (nees 1834) srb, deliblatska peščara, 17.05.2001, leg. a. ćetković, 1♂, 1♀. notes: mal. trap. hosts: elachistidae: elachista gleichenella (fabricius); erebidae: euproctis similis (fuessly), orgyia thyellina; gelechiidae: chrysoesthia drurella (fabricius); gracillariidae: aspilapteryx tringipennella (zeller), caloptilia semifascia (haworth), gracillaria syringella, parornix fagivora (frey), phyllonorycter acaciella (duponchel), p. acerifoliella (zeller), p. agilella (zeller), p. blancardella (fabricius), p. cavella (zeller), p. celtidella (rondani), p. comparella (duponchel), p. connexella (zeller), p. corylifoliella (hübner), p. cydoniella (denis & schiffermüller), p. emberizaepennella (bouché), p. froelichiella (zeller), p. harrisella (l.), p. heegeriella (zeller), p. junoniella (zeller), p. kleemannella (fabricius), p. lantanella (schrank), p. mespilella (hübner), p. millierella (staudinger), p. nicellii (stainton), p. parisiella (wocke), p. populifoliella (treitschke), p. robiniella (clemens), p. spinicolella (zeller), p. strigulatella (lienig & zeller), p. tenerella (joannis); lyonetiidae: leucoptera malifoliella (o. costa), lyonetia rajella (l.); noctuidae: melanchra persicariae (l.); plutellidae: plutella porrectella (l.); psychidae: acanthopsyche atra (l.); pyralidae: ephestia elutella (hübner); tineidae: psychoides verhuella bruand; tischeriidae: tischeria ekebladella (bjerkander); tortricidae: eucosma aemulana (schläger); cimbicidae: trichiosoma betuleti. distribution: belgium, bulgaria, canary islands, china, china (ningxia), croatia, finland, france, georgia, germany, greece, greece (crete), hungary, iran, ireland, italy, japan, lithuania, moldova, mongolia, new zealand, poland, romania, russia (kamchatka oblast, moscow oblast, sankt petersburg, yaroslavl oblast), serbia, slovakia, spain, switzerland, turkmenistan, ukraine, united kingdom. *microplitis pellucida telenga, 1955 srb, zasavica, valjevac, 19.05.2010, leg. a. ćetković, 5♂. notes: mal. trap; srb, sićevačka klisura, 12.05.2013, leg. v. žikić, 2♂, 3♀. on various plants: populus alba l., ulmus sp., malus domestica borkh. ex lymantria dispar 2nd larval instar. notes: solitary, white cocoon. hosts: no data distribution: denmark, germany, hungary, korea, netherlands, russia (altayskiy kray, primorskye kray). *microplitis varipes (ruthe, 1860): srb, niš, pantelej, 14.09.2014, leg. v. žikić, 1♀. notes: sweep net hosts: crambidae: loxostege sticticalis; noctuidae: cucullia scopariae dorfmeister, hecatera bicolorata (hufnagel), omphalophana antirrhinii (hübner); chrysomelidae: chrysolina didymata. distribution: austria, azerbaijan, china (qinghai, xinjiang), czech republic, finland, germany, hungary, italy, kazakhstan, moldova, mongolia, montenegro, netherlands, poland, russia (chita oblast, krasnodar kray, ryazan oblast, sankt petersburg, yaroslavl oblast), switzerland, turkey, ukraine. *pholetesor elpis (nixon 1973) srb, niš, g. matejevac, 24.05.2014, leg. v. žikić, 30♂, 5♀. notes: gregarious; yellow cocoons distributed in 3 rows, one over another forming kind of cluster. hosts: coleophoridae: coleophora serratella (l.); elachistidae: elachista cingillella herrich-schäffer), e. subnigrella (douglas); gracillariidae: caloptilia rufipennella (hübner), calybites auroguttellus biologica nyssana 6 (1)  september 2015: 41-48 žikić, v. et al.  new data on microgastrinae in serbia and ... 47 (stephens), phyllonorycter blancardella, p. comparella. distribution: austria, azerbaijan, bulgaria, croatia, czech republic, finland, france, greece, hungary, korea, mongolia, netherlands, poland, russia (magadanskaya oblast, primorskye kray, sakhalin oblast), slovakia, ukraine, united kingdom. pholetesor sp. mne, visitor mt, 11.07.2013, leg. v. žikić, 7♀, ex euproctis similis; notes: gregarious, from small white group cocoon. discussion in this study 18 microgastrinae species emerged forms caterpillars from different larval instars or from pupae. the identified species belong to seven genera: apanteles (1), cotesia (7), dolichogenidea (2), glyptapanteles (3), microgaster (1), microplitis (2) and pholetesor (2). although there is a limited number of species reported here, it should be pointed out that eight of the total identified numbers are recorded for the first time on the investigated territories; one for montenegro and seven for the territory of serbia. as a result it is evident that a better knowledge about the diversity of this group of braconidae is needed. concerning the role of microgastrine in ecosystems as a very important factor of biological control, the importance of investigation of this group is even higher. it needs to be added that, beside the primary parasitoids such microgastrinae are, our study revealed various hyperparasitoids which will be considered in the next step of this investigation aiming at connecting them with other members in tetratrophic associations. as for dolichogenidea longicauda the data on hosts and the distribution are presented separately because there are two opinions on its taxonomical status; it is also considered as a different species d. candidate. acknowledgements. we are thankful to dr. jenő papp from hungarian natural history museum, budapest, hungary for his great help in the organisation of this manuscript. we also want to thank to dr. andjeljko petrović for collecting some material for this study. this research was supported by the “ministry of education, science and technological development of the republic of serbia”, no. iii43001. references brajković, m.m. 1989: knowledge of the braconidae (hymenoptera) fauna in yugoslavia. bulletin of the natural history museum, b (43/44): 127-138. [in serbian with english s.]. papp, j. 1973: contributions to the braconid fauna of yugoslavia (hymenoptera, braconidae) i. acta musei macedonici scientiarum naturalium, 14: 1-23. papp, j. 1978: a survey of the european species of apanteles foerster (hym.: braconidae: microgastrinae) ii. the leavigatus group 1.s annales historico-naturales musei nationalis hungarici, 70: 265–300. papp, j. 1981: contributions to the braconid fauna of hungary, ii. microgasterinae (hymenoptera: braconidae). folia entomologica hungarica, 42 (1): 129–141. papp, j. 1982: a survey of the european species of apanteles foerster (hymenoptera: braconidae: microgastrinae) vi. the laspeyresiella, merula, falcatus and validus group. annales historiconaturales musei nationalis hungarici, 74: 255– 267. papp, j. 1983: contributions to the braconid fauna of hungary, iv. microgastrinae (hymenoptera: braconidae). folia entomologica hungarica, 44 (1): 125–138. papp, j. 1987: a survey of the european species of apanteles foerster (hymenoptera: braconidae: microgastrinae). x. the glomeratus group 2. annales historico-naturales musei nationalis hungarici, 79: 207–258. papp, j. 2009: contribution to the braconid fauna of the former yugoslavia, v. ten subfamilies (hymenoptera: braconidae). entomofauna, 30 (1): 1-35. pérez-rodríguez, j., oltra-moscardó, t., perisfelipo, f.j., jiménez-peydró, r. 2013: microgastrinae (hymenoptera: braconidae) in the forest state of artikutza (navarra: spain): diversity and community structure. insects, 4: 493-505. santos, j.p. dos, redaelli, l.r., dal soglio, f.k., foelkel, e., costa, v.a. 2009: variacao sazonal de lepidopteros minadores e seus parasitoides em plantas de crescimento espontaneo em pomar organico de citros em montenegro, rs, brasil. arquivos do instituto biologico (sao paulo), 76 (3): 381-391. sharanowski, b. j., dowling, a. p. g., sharkey, m. j. 2011: molecular phylogenetics of braconidae (hymenoptera: ichneumonoidea), based on multiple nuclear genes, and implications for classification. systematic entomology, 36 (3): 549-572. shaw, m. r. & huddleston, t. 1991: classification and biology of braconid wasps, pp. 80–86. in: handbook for the identification of british biologica nyssana 6 (1)  september 2015: 41-48 žikić, v. et al.  new data on microgastrinae in serbia and ... 48 insects, vol. 7, part 11, royal entomological society, london, england. tobias, v.i. 1986. acaeliinae, cardiochilinae, microgastrinae, miracinae. supplement. pp. 336501. in: medvedev g.s. (ed.) 'opredelitel nasekomych evrospeiskoi tsasti sssr 3, peredpontdatokrylye 4. opr. faune sssr.' 145:1-501. pp. 336-501. [keys to the insects of the european part of ussr. hymenoptera.] wharton, r.a., shaw, s.r., sharkey, m.j., wahl, d.b., woolley, j.b., whitfield, j.b., marsh, p.m., johnson, w. 1992: phylogeny of the subfamilies of the family braconidae (hymenoptera: ichneumonoidea): a reassessment. cladistics, 8 (3): 199–235. whitfield, j.b. 1997: subfamily microgastrinae. in manual of the new world genera of the family braconidae (hymenoptera). the international society of hymenopterists: washington, dc, usa, pp. 339-371. whitfield, j.b., mardulyn, p., austin, a.d., dowton, m. 2002: phylogenetic analysis of relationships among microgastrine braconid wasp genera based on data from the 16s, coi and 28s genes and morphology. systematic entomology, 27: 337-359. yu, d.s., achterberg, c. van, horstmann, k. 2012: taxapad 2012 – world ichneumonoidea 2011. taxonomy, biology, morphology and distribution. on usb flash drive. ottawa, ontario, canada. www.taxapad.com žikić, v., brajković, m., tomanović, ž. 2000: preliminary results of braconid fauna research (hymenoptera: braconidae) found in sićevo gorge, serbia. acta entomologica serbica, 5 (12): 95-110. aydin 2024, biologica nyssana 15(1) 15 (1) june 2024: 23-27 doi: 10.5281/zenodo.11517719 the investigation of total phenolic content and antioxidant properties of some essential oils in turkey original article sinem aydin giresun university, faculty of science and arts, department of biology, 28200, giresun, turkey sinem.aydin@giresun.edu.tr (corresponding author) received: september 25, 2023 revised: december 19, 2023 accepted: march 29, 2024 abstract: this study targeted to determine the total phenolic content and antioxidant activity of essential oils cinnamomum zeylanicum, vitis vinifera, allium cepa and sesamum indicum. total phenolic content was measured by folin-ciocalteu assays. the antioxidant potential of the tested extracts was evaluated using dpph, cuprac and total antioxidant capacity (tac) assays. the total phenolic compounds in essential oils varied widely, ranging from 80.26±0.007 µg gae/ml to 268.20±0.008 µg gae/ml. cinnamon oil exhibited higher dpph scavenging activity and cuprac activity. this study revealed that essential oils of c. zeylanicum, v. vinifera, a. cepa and s. indicum are good sources of natural antioxidants. key words: essential oil, plant, antioxidant activity apstrakt: istraživanje ukupnog sadržaja fenola i antioksidativnih svojstava nekih etarskih ulja u turskoj ova studija je imala za cilj da odredi ukupan sadržaj fenola i antioksidativnu aktivnost etarskih ulja vrsta cinnamomum zeylanicum, vitis vinifera, allium cepa i sesamum indicum. ukupan sadržaj fenola je meren pomoću folin-ciocalteu testova. za procenu antioksidativnog potencijala testiranih ekstrakata, korišćeni su dpph, cuprac i tac (total antioxidant capacity) testovi. ukupna količina fenolnih jedinjenja u etarskim uljima je varirala u širokom rasponu, od 80,26±0,007 µg gae/ml do 268,20±0,008 µg gae/ml. ulje cimeta je pokazalo veću dpph sposobnost vezivanja slobodnih radikala i cuprac aktivnost. ova studija je pokazala da su etarska ulja c. zeylanicum, v. vinifera, a. cepa i s. indicum dobar izvor prirodnih antioksidanasa. ključne reči: etarska ulja, biljka, antioksidativna aktivnost introduction under stress, our bodies produce more reactive oxygen species than enzymatic antioxidants can counteract. this imbalance causes cell damage and health issues. a lack of antioxidants facilitates the development of degenerative diseases such as cardiovascular diseases, neurodegenerative disorders, alzheimer’s disease, inflammatory diseases, and cancer. one way to address this problem is to supplement the diet with antioxidant compounds found in natural plant sources (krishnaiah et al., 2011). plants are widely used as nutritional supplements because they are cheaper and safer, gaining attention among researchers as natural antioxidant sources (el-miziani et al., 2015; stanković et al., 2016). antioxidant components have the ability to scavenge free radicals and prolong shelf life by delaying the lipid peroxidation process, which leads to the deterioration of food and pharmaceutical products. antioxidants play important roles in chain-breaking reactions, reducing the concentration of reactive oxygen species, and chelating transition metal catalysts (erdoğan, 2022). essential oils are volatile, oily mixtures obtained from plants through hydrodistillation of water or water vapor, remaining liquid at room temperature. they are called “essential oils” or “etheric oils” because they can evaporate even at room temperature. essential oils are secondary metabolites produced in plants (erdoğan, 2022). cinnamomum zeylanicum, known as ‘ceylon cinnamon’ or ‘true cinnamon,’ is an evergreen tree © 2024 aydin. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 23 that has been used as a medicinal plant. it is utilized in ayurveda and folklore medicine as concoctions and decoctions. this plant has been used to treat inflammation of the eyes, dyspnea, leucorrhea, rheumatism, neuralgia, wounds, toothache, and diabetes (weerasekera et al., 2021). vitis vinifera (grape) has been utilized for many years due to its nutritional and medicinal properties. the seeds and leaves of v. vinifera are used in herbal medicine, and its fruits are consumed as dietary supplements. fresh grapes can be eaten or processed into wine and juice. grape seeds are used in soaps and as a substitute for linseed. the sap of young branches is used as a remedy for skin diseases (kanagarla et al., 2013). allium cepa, known as onion, is a perennial herb with an underground bulb stem. onion peel can cure hypertrophic scars and prevent keloids. studies also revealed that onion extract helps remove hypertrophic wounds. regular consumption of onions lowers the risk of various cancers, including colorectal, lung, liver, brain, stomach, ovarian, prostate, and breast cancers (chakraborty et al., 2022). sesamum indicum seeds have been used worldwide since prehistoric times. sesame seeds are a rich source of protein and are used as ingredients in soap, cosmetics, lubricants, and medicines. sesame seeds also contain two significant substances: sesamin and sesamol. these substances have cholesterol-lowering effects and can help prevent high blood pressure. sesame oil acts as a laxative, emollient, and demulcent. it has been utilized in the treatment of several chronic diseases, including hepatitis, diabetes, and migraines, by ayurvedic physicians in holland (anilakumar et al., 2010). in the literature, many studies have been conducted on the antioxidant activity of essential oils from these plants, but location might affect the phytochemical composition of the plants and their essential oils. the current research was carried out to reveal the antioxidant activity and total phenolic content of essential oils of c. zeylanicum (cinnamon), v. vinifera (grape seed), a. cepa (onion), and s. indicum obtained from a herbal shop in turkey. materials and methods providing of the samples essential oils of cinnamon, grape seed, onion and sesamum were bought from herbal shop. total phenolic content total phenolic contents of the essential oils were determined in accordance with the method of slinkard & singleton (1977) utilizing gallic acid standard. total phenolic content of the extracts was expressed as µg gallic acid equivalents (gae)/ ml by using the calibration curve. the tests were performed in triplicate. antioxidant activity dpph radical scavenging activity appropriate dilution series (250-1000 µg/ml) of essential oils were prepared in dmso. the absorbance of the solutions was measured using a spectrophotometer (shimadzu uv mini-1240) at 517 nm (brand-williams et al., 1995). rutin and butylated hydroxytoluene (bht) were used as standard antioxidants. the tests were performed in triplicate. the dpph radical scavenging activity was calculated using the following equation: dpph radical scavenging activity (%)=[(a0a1)/ a0]x100 a0: absorbance of control a1: absorbance of extract or standard cupric reducing antioxidant capacity (cuprac) essential oils were prepared in 250-1000 µg/ ml concentration. the absorbance was read by a spectrophotometer (shimadzu uv mini-1240) at 450 nm. butylated hydroxytoluene (bht) was used as a standard antioxidant agent (özyürek et al., 2009). the tests were performed in triplicate. total antioxidant capacity the phosphomolybdenum method was used to determine the total antioxidant capacity of the extracts. absorbance was measured using a spectrophotometer (shimadzu uv mini-1240) at 695 nm. ascorbic acid was used as the standard (prieto et al., 1999). the total antioxidant capacity was expressed as µg ascorbic acid equivalent (aae)/ ml. the tests were performed in triplicate. results and discussion total phenolic content and total antioxidant capacity phenolic substances are among the most crucial groups of natural antioxidants in plants (öztürk et al., 2022). phenolic compounds have redox properties responsible for their antioxidant capabilities and facilitating free radical scavenging. the phenolic content in each essential oil was determined using the folin–ciocalteu reagent (aryal et al., 2019). the total phenolic compounds in the essential oils varied widely, ranging from 80.26±0.007 µg gae/ml to 268.20±0.008 µg gae/ml. the total phenolic contents of the essential oils increased in the following order: grape seed < sesame < onion < 24 biologica nyssana ● 15 (1) june 2024: 23-27 aydin ● the investigation of total phenolic content and antioxidant properties of some essential oils in turkey biologica nyssana ● 15 (1) june 2024: 23-27 aydin ● the investigation of total phenolic content and antioxidant properties of some essential oils in turkey 25 table 1. total phenolic content and total antioxidant capacity of esssential oils essential oil total phenolic content (µg gae/ml) total antioxidant capacity (µg aae/ml) grape seed 80.26±0.007 56.03±0.002 cinnamon 268.20±0.008 91.57±0.009 onion 123.36±0.025 30.23±0.006 sesamum 90.76±0.002 23.84±0.007 data are presented as mean ± sd of triplicate determinations cinnamon (tab. 1). in general, about 90% of the total amount of grape seed oil comprises monoand polyunsaturated fatty acids, with linoleic acid (58–78%) being the most abundant, followed by oleic acid. the second largest group of lipophilic molecules in grape seeds are vitamins. phytosterols, another important lipophilic molecule in grape seed oil, are biologically significant due to their antioxidant activity (kapcsándi et al., 2021). sesame seed oil is among the most expensive and coveted edible oils globally. it is one of the healthiest oils, containing 38.84% oleic acid and 46.26% linoleic acids. additionally, the high content of phytoestrogen and lignin in sesame seed oil can significantly impact oxidative stress and lipid profiles (oboulbiga et al., 2023). the major compounds present in onion essential oil are dipropyl disulfide and dipropyl trisulfide, which have been reported to possess antimicrobial and antioxidant activities (mnayer et al., 2014). the essential oil of cinnamon is composed of cinnamaldehyde (56–78%), eugenol (4–10%), and other compounds such as pinene, linalool, epicatechin, and quercetin. cinnamaldehyde, the main compound in cinnamon essential oil, has also been reported to have antioxidant activity (unalan et al., 2021). total phenolic contents of the tested essential oils have been studied by other researchers. for instance, aktaş et al. (2018) found the total phenolic content of grape seed essential oil to be 437.84 mg gae/g. behbahani et al. (2020) reported the total phenolic content of cinnamon oil as 106.19±0.63 mg gae/g. bopitiya & madhujith (2013) concluded that the total phenolic content of sesame oil collected from sri lanka was 26.00±0.14 mg gae/g. the total antioxidant capacity (tac) was determined by the reduction of mo(vi) to mo(v) by the essential oils and the subsequent formation of a green phosphate/mo(v) complex at acidic ph (aliyu et al., 2013). the tac of the extracts was determined using the phosphomolybdenum method as described by prieto et al. (1999). cinnamon oil exhibited the highest total antioxidant capacity (91.57±0.009 µg aae/ml), while sesame oil had the lowest (23.84±0.007 µg aae/ml) (tab. 1). cuprac and dpph radical scavenging activity antioxidants also have the ability to reduce higher valence elements such as copper and iron. the redox potential of an antioxidant is a significant indicator of its activity. the cuprac assay is a redox potentialbased method in which the copper(ii)-neocuproine complex is reduced to copper(i)-neocuproine chelate by antioxidants (arıtuluk et al., 2016). in the cuprac method, cinnamon oil showed a remarkable reducing capacity, even better than bht, which is used as a standard antioxidant agent. the reducing capacities decreased in the order: cinnamon oil > bht > grape seed oil > sesame oil > onion oil at 1000 µg/ml concentrations (tab. 2). the hydrogen atom donating capacity of the essential oils was revealed by the decolorization of 2,2-diphenyl-1-picrylhydrazyl (dpph). in a methanol solution, dpph produces a violet/purple color that fades to yellow in the presence of an antioxidant in the essential oil (rahman et al., 2015). among the oils, cinnamon oil possessed the highest activity. at a concentration of 1000 µg/ml, the scavenging activity of grape seed oil, cinnamon oil, allium cepa oil, and sesame oil was 30.38±0.031, 93.27±0.005, 29.65±0.013, and 16.09±0.011, respectively. cinnamon oil exhibited higher activity than bht and rutin, which are standard antioxidant agents. the other essential oils showed lower effects than bht and rutin. the dpph scavenging activities of the essential oils and standards were dose-dependent and increased with increasing concentrations (tab. 2). there are many studies in the literature about the dpph scavenging activity of essential oils from grape seed, cinnamon, onion, and sesame. aktaş et al. (2018) reported the dpph scavenging activity of grape seed oil to be 79%. behbahani et al. (2020) reported the dpph scavenging activity of cinnamon essential oil to be 71.12±0.77%. shalaby et al. 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(2021). cinnamomum zeylanicum: morphology, antioxidant properties and bioactive compounds. microsoft word 0602_stankovic_et_al biologica nyssana 1 (1-2) december 2010: 117-122 stanković s. et al. betula species as host plants for various insects… 117 original article ! betula species as host plants for various insects parasitized by braconids (hymenoptera: braconidae) in serbia saša stanković*, vladimir žikić, marijana ilić university of niš, faculty of science and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia e-mail: sasasta@gmail.com abstract: stanković, s., žikić, v., ilić, m.: betula species as host plants for various insects parasitized by braconids (hymenoptera: braconidae) in serbia. biologica nyssana, 1 (1-2), december 2010: 117-122. this work presents braconid wasps which can be found on insects attacking birches, especially betula alba and b. pubescens (betulaceae) which are autochthonous in serbia. we have found 49 braconid species from 26 genera on 40 phytophagous insects; one from the hemimetabolous order: homoptera: homoptera and three from the holometabolous orders: coleoptera, hymenoptera and lepidoptera. registered braconid species belong to the subfamilies: aphidiinae, braconinae, doryctinae, euphorinae, exothecinae, microgastrinae, orgilinae, rhysipolinae and rogadinae. although most of the registered phytophagous insects pose a significant threat to betula species, the two species: epirrita autumnata (geometridae) and lymantria dispar (noctuidae) are the most important, because they can defoliate entire forests when their populations are in gradation. also, there are two buprestid pests agrilus anxius native to north america and a. planipennis (buprestidae) from central asia which are considered as potentially invasive species. key words: betula, braconidae, hosts, tritrophic associations, serbia introduction ! braconidae wasps comprise at least 50.000 species of solitary or gregarious ectoand endoparasitoids that parasitize larvae of their hosts, as well as eggs and very rarely recorded, the adult stage. the body size of braconid wasps is in range from 0.5 mm to over 20 cm including the “fake ovipositor” (s t a r ý , 1970, 1988; s h a w & h u d d l e s t o n , 1991). there are two strategies among braconidae wasps: idiobionts that permanently parasitize their host before laying eggs and konobionts that allow the host to continue developing while the larvae feed within the host’s body. the great majority of braconidae are cosmopolitan and polyphagous insects. some species show different degrees of host specificity. there are programs of utilization of braconid wasps and other insects as biological control agents starting from b a i r d (1958), m a t h e w s (1974), l e w i s et al. (1977, 1990), over the fieldworks such as biocontrol of olive fruit flies (s i m e et al., 2006; y o k o y a m a et al., 2008), to brand-new, k u l a et al. (2010). not much data exists about the insect pests feeding on birch and their parasitoids from the family braconidae in europe, especially in serbia. some sporadically records exist in the following journals: (t o m a n o v i ć & k a v a l l i e r a t o s , 2002; k a v a l l i e r a t o s et al., 2002, 2004). world fauna of braconidae which attack pests in the birch communities is poorly investigated as well. birches are autochthones for the greater part of europe, as well as for the territory of serbia. we have chosen to present this work in this journal because the first issue is dedicated to vlasina lake, which is the place full of native birches: betula alba and b. pubescens (betulaceae). most important parasitoids 10th sfses • 17-20 june 2010, vlasina lake1 (1-2) • december 2010: 117-122 biologica nyssana 1 (1-2) december 2010: 117-122 stanković s. et al. betula species as host plants for various insects… 118 in biological control of the species from the genus agrilus (coleoptera: buprestidae), could be spathius agrili yang 2005, (braconidae) found in the usa on european birch betula alba (yang et al. 2008). the host range of s. agrili is presented by 18 wood boring insects belonging to the genus agrilus, who are present on the other host plants such as fraxinus, prunus and alianthus. material and methods the examined parasitoid wasps for this study were collected from the hosts feeding on betula alba and b. pubescens all over serbia. in the tritrophic associations: parasitoid-host-plant include all hosts found in europe. one part of specimens is deposited at the faculty of sciences and marthematics, department of biology and ecology, university of niš, serbia while the rest of specimens are at the faculty of biology, university of belgrade, serbia. identification of birch species was done according to v u k i ć e v i ć (1970). results and discussion the parasitoid subfamilies, genera and species and their hosts are arranged in four tables as trophic associations connected by (x). the total number of 49 braconids species from 26 genera belong to the following subfamilies: aphidiinae, braconinae, doryctinae, euphorinae, exothecinae, microgastrinae, orgilinae, rhysipolinae and rogadinae. we have found 42 host species belonging to four insect orders: coleoptera, homoptera, hymenoptera and lepidoptera (tab. 1). all registered host species are phytophagous insects feeding on leaves or they are xylophagous on birches. the trophic relations between aphidiinae and aphid hosts (homoptera: aphididae) are presented in the table 2. the species callaphis flava (aphididae) is registered as the host of four aphidiinae wasps: aphidius aquilus mackauer, 1961, praon flavinode (haliday, 1833), trioxys betulae (marshall, 1896) and t. cirsii (curtis, 1831). parasitoid wasp aphidius aquilus attack three of four recorded aphids. the ectoparasitoid groups of braconidae witch parasitize coleopteran and hymenopteran host larvae are shown in the table 3. the subfamily braconinae is presented by five parasitoids of coleoptra species coming from curculionidae and cerambycidae. bracon obscurator nees 1811 was found as parasitoid of the genus trypodendron: t. domesticum and t. signatum (curculionidae). for buprestid chrysobothris affinis (buprestidae) we noted four natural enemies: glyptomorpha pectoralis (brullé 1832), pseudovipio castrator (fabricius 1798), vipio appellator (nees 1834) from the subfamily braconinae and doryctes leucogaster (nees 1834) from the subfamily doryctinae. the species iphiaulax impostor (scopoli 1763) parasitizes aegomorphus clavipes and leiopus nebulosus from the family of lonhorned beetles (cerambycidae). braconidae attacking the caterpillars of various lepidopteran families found on birches belong to the genus bracon are b. abbreviator nees 1834, b. discoideus wesmael 1838, b. hebetor say 1836, b. intercessor nees 1834, b. romani fahringer 1927 and b. variegator spinola 1808 (tab. 4). among bracon species the most important is polyphagous b. intercessor which attacks various insects from coleopteran families, especially curculionidae and lepidoptera, sessidae synanthedon culiciformis, paranthrene tabaniformis. another important species is bracon hebetor recorded from many larvae of crambidae, gelechiidae, noctuidae and tortricidae (milonas, 2005); an important natural enemy of indian meal moth plodia interpunctella, (pyralidae), common as pest of stored products. subfamily doryctinae is presented by six species from four genera attacking different hosts form the order coleoptera: dendrosoter protuberans (nees 1834), doryctes leucogaster (nees 1834), ontsira ignea (ratzeburg 1852), o. imperator (haliday 1836), spathius curvicaudis ratzeburg 1844 and s. rubidus (rossi 1794), tab. 3, and from lepidoptera, only one but the most important and extremely polyphagous species, lymantiria dispar from the family noctuidae. it is interesting that the presence of any spathius parasitoid of agrylus cynaescens –species group (buprestidae), native in europe, but an invasive alien pest in north america is not reported in europe (j e n d e k & g r e b e n n i k o v , 2009). one of the candidates for the biological control of a. cynaescens is spathius rubidus known as polyphagous on various coleopteran families: buprestidae, cerambycidae, curculionidae, and rhizophagidae as well as xiphydriidae from the order of hymenoptera. in addition to this species, we report it as parasitoid of two species of agrylus: a. angustulus and a. hastulifer (buprestidae) and leiopus nebulosus and rhagium mordax from the family of cerambycidae. diversity and some parasitoid-host data are the best known for this subfamily among ectoparasitic (b e l o k o b y l s k i j & ž i k i ć , 2009). from the ectoparasitoid subfamily exothecinae we registered only two species: colastes braconius haliday 1833 parasitizes on heterarthrus vagans sawfly (tenthredinidae) and on another extremely polyphagous species epirrita autumnata geometridae), biologica nyssana 1 (1-2) december 2010: 117-122 stanković s. et al. betula species as host plants for various insects… 119 table 1. the list of pests on betula alba and b. pubescens order family species homoptera aphididae callaphis flava mordvilko, 1928 euceraphis betulae (koch 1855). euceraphis punctipennis (zetterstedt 1828) symydobius oblongus (von heyden, 1837). coleoptera buprestidae agrilus angustulus (illiger 1803) agrilus cyanescens ratzeburg 1837 agrilus hastulifer ratzeburg 1837 chrysobothris affinis (fabricius 1794) dicerca berolinensis (herbst 1779) curculionidae scolytus ratzeburgi janson 1856 trypodendron domesticum (linnaeus 1758) trypodendron signatum (fabricius, 1787) cerambycidae aegomorphus clavipes (schrank 1781) leiopus nebulosus (linnaeus 1758) rhagium mordax (de geer 1775) hymenoptera tenthredinidae fenusa pumila leach 1817 fenusella nana (klug 1816) heterarthrus vagans (fallen 1808) scolioneura betuleti (klug 1816) xiphydriidae xiphydria prolongata (geoffroy 1785) lepidoptera geometridae cyclophora pendularia (clerck 1759) epirrita autumnata (borkhausen 1794) hypagyrtis unipunctata (haworth 1809) gracillariidae caloptilia betulicola (hering 1928) heliozelidae heliozela betulae stainton 1890 noctuidae euproctis chrysorrhoea (linnaeus 1758) hyphantria cunea (drury 1773) lymantria dispar (linnaeus 1758) orthosia gothica (linnaeus 1758) orthosia incerta (hufnagel 1766) lasiocampidae malacosoma neustria (linnaeus 1758) psychidae megalophanes viciella (denis & schiffermuller 1775) sesiidae synanthedon culiciformis (linnaeus 1758) tortricidae acleris hastiana (linnaeus 1758) adoxophyes orana fischer von rösslerstamm 1834) apotomis sororculana (zetterstedt 1839) archips rosana (linnaeus 1758) pandemis cerasana (hubner 1786) pandemis heparana (denis & schiffermuller 1775) geometridae cyclophora pendularia (clerck 1759) hypagyrtis unipunctata (haworth 1809) coleophoridae coleophora serratella (linnaeus 1761) and shawiana catenator (haliday 1836) on caterpillars of caloptilia betulicola (gracillariidae). subfamily rogadinae assembles eight species of aleiodes: a. circumscriptus (nees 1834), a. gastritor (thunberg 1822), a. nigricornis wesmael 1838, a. pallidator (thunberg 1822), a. rossicus (kokujev 1898) and a. sanctihyacinthi (provancher 1880), distributed mostly in whole europe (aydogdu & beyarslan, 2006) and petalodes compressor (herrich-schäffer 1838). the species a. pallidator has been recorded as parasitoid of four lepidopteran larvae: cyclophora pendularia (geo table 2. subfamily aphidiinae and the aphid hosts c al la ph is fla va e uc er ap hi s be tu la e e uc er ap hi s pu nc tip en ni s s ym yd ob iu s ob lo ng us aphidius aquilus x x x betuloxys compressicornis x x praon flavinode x trioxys betulae x x trioxys cirsii x biologica nyssana 1 (1-2) december 2010: 117-122 stanković s. et al. betula species as host plants for various insects… 120 metridae), lymantria dispar, megalophanes viciella (psychidae) and apotomis sororculana (tortricidae) (tab. 4). the last registreted ectoparasitic braconidae subfamily is rhysipolinae, with only one species, rhysipolis meditator (haliday 1836) on caloptilia betulicola (gracillariidae). table 3. subfamilies of ectoparasitoid braconidae on coleopteran and hymenopteran hosts. tr yp od en dr on d om es tic um tr yp od en dr on s ig na tu m s co ly tu s ra tz eb ur gi a gr ilu s cy an es ce ns a gr ilu s an gu st ul us a gr ilu s ha st ul ife r c hr ys ob ot hr is a ffi ni s d ic er ca b er ol in en si s a eg om or ph us c la vi pe s le io pu s ne bu lo su s r ha gi um m or da x fe nu sa p um ila fe nu se lla n an a h et er ar th ru s va ga ns s co lio ne ur a be tu le ti x ip hy dr ia p ro lo ng at a braconinae bracon obscurator x x glyptomorpha pectoralis x iphiaulax impostor x x pseudovipio castrator x vipio appellator x doryctinae dendrosoter protuberans x x doryctes leucogaster x ontsira ignea x ontsira imperator x x spathius curvicaudis x spathius rubidus x x x x x exothecinae colastes braconius x shawiana catenator x x x x table 4. subfamilies of ectoparasitoid braconidae on lepidopteran hosts c yc lo ph or a pe nd ul ar ia h yp ag yr tis u ni pu nc ta ta c al io ph ili a be tu lic ol a h el io ze la b et ul ae e pi rr ita a ut um na ta ly m an tri a di sp ar e up ro ct is s im ili s h yp ha nt ria c un ea o rth os ia g ot hi ca o rth os ia in ce rta m al ac os om a ne us tri a m eg al op ha ne s vi ci el la s yn an th ed on c ul ic ifo rm is a cl er is h as tia na a do xo ph ye s or an a a po to m is s or or cu la na a rc hi ps ro sa na p an de m is c er as an a p an de m is h ep ar an a braconinae bracon abbreviator x bracon discoideus x bracon hebetor x bracon intercessor x bracon romani x bracon variegator x x doryctinae doryctes leucogaster x exothecinae colastes braconius x rogadinae aleiodes circumscriptus x aleiodes gastritor x x aleiodes nigricornis x x aleiodes pallidator x x x x aleiodes rossicus x x x aleiodes sanctihyacinthi x aleiodes signatus x aleiodes testaceus, x petalodes compressor x rhysipolinae rhysipolis meditator x biologica nyssana 1 (1-2) december 2010: 117-122 stanković s. et al. betula species as host plants for various insects… 121 in this study we have found the data of three endoparasitic braconid subfamilies: euphorinae, microgastrinae and orgilinae (tab. 5). as bracon obscurator, cosmophorus regius niezabitowski 1910 (euphorinae) shows the same affinity to the species of trypodendron (tab. 2). two species of meteorus, m. pulchricornis (wesmael 1835) and m. meteorus, m. pulchricornis (wesmael 1835) and m. versicolor (wesmael 1835) attack the most common pest lymantria dispar. polyphagous epirrita autumnata is the host of solitary endoparasitoid zele deceptor (wesmael 1835). mostly gregarious, microgastrinae are presented by nine members on betula spesies that eight of them attack lymantria dispar: three species of apanteles: a. lacteicolor viereck 1911, a. melanoscelus (ratzeburg 1844) and a. xanthostigma (haliday 1834), one species of cotesia: c. lomerata (linnaeus 1758), microgaster hospes marshall 1885, and three species of protapanteles: p. fulvipes (haliday 1834), p. liparidis (bouche 1834) and p. immunis (haliday 1834). larvae of epirrita autumnata is parasitized by cotesia jucunda (marshall 1885). there is only one species of orgilinae, orgilus punctulator (nees 1811) has been found as pest of coleophora serratella (coleophoridae). table 5. subfamilies of euphorinae, microgastrinae and orgilinae and their hosts. tr yp od en dr on d om es tic um tr yp od en dr on s ig na tu m ep irr ita a ut um na ta ly m an tri a di sp ar c ol eo ph or a se rra te lla euphorinae cosmophorus regius x x meteorus pulchricornis x meteorus versicolor x zele deceptor x microgastrinae apanteles lacteicolor x apanteles melanoscelus x apanteles xanthostigma x cotesia glomerata x cotesia jucunda x microgaster hospes x protapanteles fulvipes x protapanteles liparidis x protapanteles immunis x orgilinae orgilus punctulator x conclusion we noted that pest insects make no difference on which betula species they feed. the most important pests on betula as well as other trees are polyphagous species lymantria dispar and epirrita autumnata, because they can defoliate entire forests when their populations are in gradation. we have found that larvae of lymantria dispar on birches are attacked by 11 parasitoid species from the subfamilies braconinae, rogadinae, euphorinae and microgastrinae. the moth epirrita autumnata can be very dangerous in birch forests. the example from northern sweden shows that this pest’s caterpillars can defoliate birch forests, causing death of stems of the polycormic trees as a result (t e n o w , et al., 2004). there are three buprestid pests of the genus agrilus found on betula alba and b. pubescens: a. angustulus, a. cyanescens, a. hastulifer. they are extremely polyphagous wood borers. for now there is no data of the natural enemies of a. cyanescens. agrilus anxius, which is native to north america and a. planipennis from central asia are considered as potentially invasive species. acknowledgements. the authors wish to express their sincere thankfulness to dr. ž. tomanović (faculty of biology, belgrade, serbia). for the present work the authors were supported by the ministry of science and environment protection of the republic of serbia (grant no. 143006b). references aydogdu, m., beyarslan, a. 2006: first records of aleiodes wesmael, 1838 species in east marmara region of turkey (hymenoptera: braconidae: rogadinae). acta entomologica slovenica, 14(1): 81–88 baird, a. b. 1958: biological control of insects and plant pests in canada. prc 10th int. cong. ent. 4: 483-485. belokobylskij, s. a., žikić, v., 2009: new data on cyclostome braconid subfamilies doryctinae, exothecinae, rogadinae and braconinae (braconidae: hymenoptera) of serbia and neighbouring territories. acta entomologica 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emerald ash borer. biological control, 47: 216–221. yokoyama, v.y., rendo, p.a., sivinski, j., 2008: psyttalia cf. concolor (hymenoptera: braconidae) for biological control of olive fruit fly (diptera: tephritidae) in california. environ. entomol. 37(3): 764-773. tenow, o., bylund, h., karlsson, p. s., hoogesteger, j., 2004: rejuvenation of a mountain birch forest by an epirrita autumnata (lepidoptera: geometridae) outbreak. acta oecologica, 25(1/2): 43-52. madić, v., jovanović, j., stojilković, a., jušković, m., vasiljević, p.: evaluation of cytotoxicity of ‘anti-diabetic’ herbal preparation and five medicinal plants: an allium cepa assay. biologica nyssana, 8 (2) biologica nyssana 8 (2)  december 2017: 151-158 madić, v. et al.  evaluation of cytotoxicity of „anti-diabetic“ herbal… 151 original article received: 14 september 2017 revised: 21 november 2017 accepted: 22 december 2017 evaluation of cytotoxicity of ‘anti-diabetic’ herbal preparation and five medicinal plants: an allium cepa assay višnja madić*, jelena jovanović, aleksandar stojilković, marina jušković, perica vasiljević university of niš, faculty of science and mathematics, department of biology and ecology, višegradska 33, niš, serbia * e-mail: visnja.madic@gmail.com abstract: madić, v., jovanović, j., stojilković, a., jušković, m., vasiljević, p.: evaluation of cytotoxicity of ‘antidiabetic’ herbal preparation and five medicinal plants: an allium cepa assay. biologica nyssana, 8 (2), december, 2017: 151-158. traditional medicine is often used as the treatment for diabetes mellitus, but little is known about potential toxicity of medicinal plants used for this purposes. the cytotoxic effect of aqueous extracts of a traditional ‘anti-diabetic’ herbal preparation, as well as its constituents: rubus fruticosus, vaccinium myrtillus, potentilla erecta, geum urbanum and phaseolus vulgaris was evaluated using the allium cepa assay. onion bulbs were exposed to 400 μg/ml, 800 μg/ml and 1200 μg/ml concentrations of each extract. there was concentration dependent inhibition of root growth by the extracts when compared with the control. all the tested extracts had mitodepressive effect on cell division. ‘anti-diabetic’ herbal mixture (1200 µg/ml) and v. myrtillus (400 µg/ml) caused surprisingly high prophase accumulation. these events manifested the cytotoxic effect of this herbal preparation as well as all the tested medicinal plants at some of the tested concentrations. key words: diabetes, ‘anti-diabetic’ herbal preparation, cytotoxicity apstrakt: madić, v., jovanović, j., stojilković, a., jušković, m., vasiljević, p.: ispitivanje citotoksičnosti “antidijabetičkog biljnog preparata“ i pet medicinskih biljaka: allium cepa test. biologica nyssana, 8 (2), decembar, 2017: 151-158. tradicionalna medicina se često koristi u terapiji dijabetes melitusa, ali malo toga se zna o potencijalnoj toksičnosti medicinskih biljaka korišćenih u ove svrhe. allium cepa testom ispitali smo citotoksični efekat vodenih ekstrakata tradicionalnog “antidijabetičkog” biljnog preparata, kao i njegovih sastojaka: rubus fruticosus, vaccinium myrtillus, potentilla erecta, geum urbanum i phaseolus vulgaris. lukovice su bile izložene trima koncentracijama svakog od ovih ekstrakata, i to: 400 μg/ml, 800 μg/ml and 1200 μg/ml. svi testirani ekstrakti pokazali su mitodepresivni efekat na ćelijsku deobu. “antidijabetički” biljni preparat (1200 µg/ml) i v. myrtillus (400 µg/ml) su, uz to, izazvali iznenađujuće veliku akumulaciju ćelija u profazi. ovi događaji su manifestacija citotoksičnog efekta ovog biljnog preparata i ovih medicinskih biljaka u nekim od ispitivanih koncentracija. ključne reči: dijabetes,”antidijabetički” biljni preparat, citotoksičnost 8 (2) • december 2017: 151-158 doi: 10.5281/zenodo.1135968 biologica nyssana 8 (2)  december 2017: 151-158 madić, v. et al.  evaluation of cytotoxicity of „anti-diabetic“ herbal… 152 introduction the use of medicinal plants has always been part of the human tradition. according to the world health organisation, 80% of the world population relies on the traditional medicine (f a r n s w o r t h et al., 1985). in recent years there has been an increased popularity of traditional medicine globally, mostly due to the high costs of nursing care and international commercial medicines. however, some constituents of medicinal plants have been found to be potentially toxic, mutagenic, carcinogenic and teratogenic (p i n g et al., 2012). also, potentional contraindications between herbal preparations and modern pharmaceutical products are not sufficiently examined, both for short-term and long-term use. for all this, the purpose of this study is to evaluate the toxicological effects of some of the herbal preparations used in traditional medicine. traditional medicine is used as the treatment for a variety of diseases, including diabetes mellitus, commonly referred to as diabetes. in 2015, there were 415 milion adults and half a million children with diabetes globally, most in lowto middle-income countries, predicted to rise to over 642 milion in the next 23 years. every 6 s someone dies from diabetes (international diabetes federation (idf), 2015). uk diabetologist edwin gale’s quoted, ’what is the commonest cause of death in a child with diabetes? the answer – from a global perspective – is a lack of access to insulin’ (g a l e & w a g n e r , 2006). although herbal substituents can not replace insulin, traditional treatments can lead to the development of new hypoglycemic drugs, especially for t2d. there is a variety of herbal mixtures intended for maintaining normal glycemic values, and one of the most commonly used in europe, primarily in the balkan countries, is made of blackberry (rubus fruticosus l., rosaceae) leaves (rfl), blueberry (vaccinium myrtillus l., ericaceae) leaves (vml), tormentil (potentilla erecta uspenski ex ledeb., rosaceae) roots (per), st. benedict's herb (geum urbanum l., rosaceae) aerial parts (gua), and kidney bean (phaseolus vulgaris l., fabaceae) pods (pvp). there is a long history of using these medicinal plants in traditional therapy for diabetes. their hypoglycemic effect was examined by a number of studies (s w a n s t o n -f l a t t et al. 1990; e d d o u k s et al., 2002; t o m c z y k & l a t t é , 2009; k y z n i e t s o v a et al., 2015; k o u p ý et al., 2015; p a u n et al., 2015; j o h n s o n & d e m e j i a , 2016; s i d o r o v a et al., 2017), but a little is known about their potential toxicity. for the most of the substances on this planet there are doses which are toxic and which are nontoxic on the cell level. for this reason, the aim of this study was to investigate cytotoxic effect of aqueous extracts of the entire ‘anti-diabetic’ herbal preparation as well as its individual ingredients and predict their ec50 on the root meristem cells of allium cepa l., fam. amaryllidaceae. allium cepa test is often used in assessing cytological and cellular effects of medicinal plant’s extracts (a k i n b o r o & b a k a r e , 2007; ç e l i k & a s l a n t ü r k , 2010; p i n g et al., 2012). it is a shortterm test with many advantages, such as low cost and good chromosome conditions for the study of disturbance of cell division or chromosome aberrations (f i s k e s j o , 1985). some researchers show certain restriction in regards to using plant test systems for evaluating cytotoxicity of medicinal plants. however, a. cepa test, as a plant test systems in vivo, is validated by several researches, which jointly performed animal testing in vitro and the results obtained were similar (c a m p a r o t o et al., 2002; t e i x e i r a et al., 2003), providing valuable information for human health. moreover, r a n k & n i e l s e n (1994) showed a correlation of 82% between the a. cepa test and the carcinogenicity test in rodents and concluded that the same was even more sensitive than the ames test. material and methods preparation of extracts the medicinal plants utilized in this study, i.e. blackberry (r. fruticosus) leaves (rfl), blueberry (v. myrtillus) leaves (vml), tormentil (p. erecta) roots (per), st. benedict's herb (g. urbanum) aerial parts (gua), and kidney bean (p. vulgaris) pods (pvp), were purchased immediately before evaluation in the certified herbal pharmacy in niš, serbia. originally, these medicinal plants were collected on the mountains of south-eastern serbia. r. fruticosus, v. myrtillus, g. urbanum, p. vulgaris, were collected on the rtanj mt., near soko banja, while p. erecta was collected on the stara planina mt. pirot. rubus fruticosus, v. myrtillus, g. urbanum and p. erecta were found in the wild, while p. vulgaris was organically grown, therefore, the possible impact of pesticide use on results of the a. cepa test was excluded. dried plant material was finely grinded, and 0.48 g of each tested material was separately boiled in 800 ml of distilled water until half of the liquid evaporated. the extract was then filtered and used as a stock solution. stock solutions and dilutions were made immediately before use. three concentrations of each extract, viz: 400 μg/ml, 800 μg/ml and 1200 μg/ml were considered. biologica nyssana 8 (2)  december 2017: 151-158 madić, v. et al.  evaluation of cytotoxicity of „anti-diabetic“ herbal… 153 allium cepa assay small bulbs (1 – 2 cm in diameter) of the common onion, a. cepa (2 n = 16) were purchased at the agricultural pharmacy in niš, serbia. the base of each of the bulbs was suspended on distilled water for 24 h. after that period, the bulbs with satisfactory root lengths were exposed to the tested materials, 5 bulbs per every concentration, for 48 h. test extracts were changed daily. the negative control was 5 bulbs suspended on distilled water for 72 h. the positive control was 5 bulbs suspended 24 h on distilled water, and afterwards, 48 h on 500 µg/ml toluene diluted in 2% dmso. the experiment was performed at 25 ± 1 ºc in the dark. at the end of the exposure period, in order to prevent drying of the material, root length of the whole root bundle was measured outside of the test tube by the ruler, as shown on fig. 1. this method gives one value for each bulb, and permits the experiment to be continued (f i s k e s j o , 1985). fig. 1. measuring the root length of the whole root bundle. root length of the whole root bundle was measured outside of the test tube by the ruler. this method gives one value for each bulb, and permits the experiment to be continued. root tips were cut by scalpel, fixed in methanol / glacial acid (3:1, v/v) for 24 h at 4 ºc. the next day they were placed in 70% ethanol alcohol and refrigerated until used. slides were prepared immediately before microscopic analysis. the root tips were hydrolyzed in 1 n hcl at 60 ºc for 13 minutes and washed three times in distilled water. after that, they were stained with 1% acetocarmine at room temperature for 15 minutes or, alternatively, on a bunsen burner for 1 2 minutes and washed three times in distilled water. the root tips were afterwards transferred onto the cold slides, squashed by cover slips and heat fixed. the following parameters were used for determination of cytotoxicity: treated root growth inhibition (trg), mitotic index (mi) and phase index (pi). for the treated root growth inhibition, from the length of bundle for each concentration (ltr) the percentage of root growth inhibition in relation to the negative control (lnc), i.e. 𝑇𝑅𝐺 = 𝐿𝑇𝑅 (𝑐𝑚) 𝐿𝑁𝐶 (𝑐𝑚) 𝑥100% and the ec50 (the effective concentration where root growth amounts to 50% of the controls) for each extract was determined (f i s k e s j o , 1985). for the mi and pi analysis, for every concentration, as well as for the controls, 5 microscopic slides were made, and 500 cells per slide, i.e. 2500 cells per concentration were observed by leica microscope with 400 x (fig. 2.) and 1000 x magnification (fig. 3.), and analyzed by the same microscope with 400 x magnification. mi was calculated as the total number of cells in prophase (p), metaphase (m), anaphase (a), and telophase (t) in relation to the number of observed cells (t e d e s c o & l a u g h i n g h o u s e iv, 2012). 𝑀𝐼 = 𝑃 + 𝑀 + 𝐴 + 𝑇 500 𝑥100% the proportion of mitotic phases was determined as a percent ratio between cells in specific phase and the dividing cells (f i s k e s j o , 1985). fig. 2. mitosis of a. cepa root meristems. magnification 400 x. leica light microscope. normal stages of mitotic division. (a) interphase (b) prophase (c) metaphase (d) anaphase (e) telophase. statistical analysis statistical analysis was carried out using microsoft excel 2010 and systat 13 with five replicates for each group. data were expressed as mean ± standard deviation (sd) calculated by one way analysis of variance (anova). differences between controls and the individual dosage group of each extract were analyzed by means of the paired t-test, with 95% confidence limits where significance was accepted at p<0.05. biologica nyssana 8 (2)  december 2017: 151-158 madić, v. et al.  evaluation of cytotoxicity of „anti-diabetic“ herbal… 154 results ad discussion the effects of the aqueous extracts of ‘anti-diabetic’ herbal mixture, r. fruticosus, v. myrtillus, p. erecta, g. urbanum and p. vulgaris on root growth of a. cepa are shown on tab. 1. good root growth was achieved in the negative control, while the positive control inhibited root growth. at tested concentrations, root growth was the highest at the 400 µg/ml concentration of all the extracts while it was the lowest at 1200 µg/ml, with the exception of p. vulgaris extract, where the highest root growth was in exposure to 800 µg/ml concentration of extract. inhibition of root growth implying toxicity was concentration dependent and statistically significant (p<0.05) at all tested concentrations of p. erecta and g. urbanum extracts, while extracts of ‘anti-diabetic’ herbal mixture, r. fruticosus and v. myrtillus triggered concentration dependent and statistically significant (p<0.05) inhibition of root growth at higher concentrations (800 µg/ml and 1200 µg/ml). the ec50 for the extracts of ‘anti-diabetic’ herbal mixture, r. fruticosus, v. myrtillus, p. erecta, g. urbanum and p. vulgaris were 1400 µg/ml, 800 µg/ml, 990 µg/ml, 690 µg/ml, 550 µg/ml, and 1590 µg/ml, respectively. the effects of tested extracts on cell division of a. cepa root meristem cells are presented in tab. 2. mitotic index in the negative and the positive control were 20.28 ± 2.17 and 3.88 ± 0.92, respectively. with increasing concentration of the all the tested extracts there was concentration dependent decrease in the mitotic index with the exception of p. erecta extract, where 400 µg/ml concentration did not cause statistically significant decrease of mi compared to the positive control. of all the lowest concentration of tested extracts (400 µg/ml), the fig. 3. stages of mitotic division in cells of a. cepa treated with aqueous extracts of ‘anti-diabetic’ herbal mixture, r. fruticosus, v. myrtillus, p. erecta, g. urbanum and p. vulgaris. magnification 1000 x. leica light microscope. normal stages of mitotic division. (a) interphase (b) prophase (c) metaphase (d) anaphase (e) telophase. table 1. the effects of aqueous extracts of ‘anti-diabetic’ herbal mixture, r. fruticosus, v. myrtillus, p. erecta, g. urbanum and p. vulgaris on root growth of a. cepa. rg (%) of the control, treated root growth expressed as % of the control. mix, ‘anti-diabetic’ herbal mixture. rfl, leaves of r. fruticosus. vml, leaves of v. myrtillus. per, roots of p. erecta. gua, aerial parts of g. urbanum. pvp, pods of p. vulgaris. nc, negative control. pc, positive control. * significant compared to its negative control at 0.05 level. º significant compared to its positive control at 0.05 level. mix rfl vml per gua pvp treatment mean root length (cm) ±sd trg (%) of control mean root length (cm)±sd trg (%) of control mean root length (cm) ±sd trg (%) of control mean root length (cm) ±sd trg (%) of control mean root length (cm) ±sd trg (%) of control mean root length (cm) ±sd trg (%) of control nc 3.04±0.59 100 3.04±0.59 100 3.04±0.59 100 3.04±0.59 100 3.04±0.59 100 3.04±0.59 100 400 µg/ml 2.82±0.46 92.8 º 2.7±0.48 88.8 º 2.44±0.59 80.1 º 2.3±0.21 75.7 * 1.56±0.45 51.3 * 2.9±0.22 95.4 º 800 µg/ml 2.1±0.24 69.1 * 1.52±0.53 50 * 1.7±0.47 55.1 * 1.3±0.41 42.8 * 1.48±0.53 48.7 * 3.12±0.25 102.6 º 1200 µg/ml 1.82±0.49 59.8 * 0.84±0.17 27.6 * 1.5±0.47 49.3 * 1.04±0.11 34.2 * 1±0.14 32.9 * 2.16±0.18 71 * pc 1.38±0.7 45 1.38±0.7 45 1.38±0.7 45 1.38±0.7 45 1.38±0.7 45 1.38±0.7 45 ec50 1400 µg/ml 800 µg/ml 990 µg/ml 690 µg/ml 550 µg/ml 1590 µg/ml biologica nyssana 8 (2)  december 2017: 151-158 madić, v. et al.  evaluation of cytotoxicity of „anti-diabetic“ herbal… 155 highest decrease of mitotic index was influenced by v. myrtillus extract (10.96 %). allium cepa root meristem cells of the control groups displayed the existence of all phases of mitosis. the different concentrations of the tested extracts caused different changes in the proportion of mitotic phase's distribution in comparison to the positive and negative controls, as shown on tab. 3. compared to the positive and negative control, influence of ‘anti-diabetic’ herbal mixture, r. fruticosus, v. myrtillus extracts on phase index was statistically significant concentration depended. prophase index was, as expected, the most frequent one in all of the tested groups. extracts of ‘anti-diabetic’ herbal mixture (400 µg/ml and 800 µg/ml concentrations) and r. fruticosus (800 µg/ml and 1200 µg/ml concentrations) decreased the prophase index of a. cepa root meristem cells, while 400 µg/ml and 800 µg/ml extract of v. myrtillus and 1200 µg/ml of ‘anti-diabetic herbal mixture’ increased the prophase index. the extracts of p. erecta, g. urbanum and p. vulgaris, with the exception of 400 µg/ml concentrated p. erecta which increased prophase index, had no influence on it. extract of ‘anti-diabetic’ herbal mixture did not change metaphase index with statistical significance, while higher concentrations of r. fruticosus (1200 µg/ml) and v. myrtillus (800 µg/ml) increased it. extracts of p. erecta, g. urbanum and p. vulgaris produced no change in metaphase index. none of the extracts had any influence on anaphase index. as expected, telophase index was the least frequent one in all the tested groups. extracts of r. fruticosus (400 µg/ml and 1200 µg/ml), v. myrtillus (all the tested concentrations), g. urbanum (800 µg/ml) and p. vulgaris (1200 µg/ml) decreased this index. higher plants such as a. cepa are accepted as admirable genetic models to evaluate genotoxic effects. results of the current study reflected the utility of root tips of cells of a. cepa for monitoring the cytotoxic effects of medicinal plant extracts. data on the effects of the extracts on root growth of a. cepa showed that there was concentration dependent decrease in root growth. based on the ec50 the order of induction of root growth inhibition was pvp < mix < vml < rfl < per < gua, as shown on tab. 1. as it is already said, little is known about their potential toxicity. our results are consistent with what has been done so far, i.e. a blackberry extract has cytotoxic capabilities as it can induce apoptosis in human leukemia hl-60 cells (s u n et al., 2002); p. erecta rhizome extracts is considered safe with respect to acute toxicity when applied to humans (s h u s h u n o v et al, 2009), and g. urbanum is considered non-toxic as well (p a u n et al., 2015). there is a linear relationship between macroscopic and microscopic parameters for all the extracts. in a. cepa, whenever there is root growth inhibition, there is always reduction in the number of dividing cells (f i s k e s j o , 1985). mitotic index was characterized by the total number of dividing cells in the cell cycle. mitotic index is used as an indicator of cell proliferation biomarkers which measures the proportion of cells in the mitotic phase of the cell cycle. therefore, the decrease in the mitotic index of a. cepa meristem cells could be interpreted as cellular death. low mitotic index may be reflecting a direct cytotoxic effect of the tested extract. (s a s i d h a r a n et al., 2012.) the cells of a. cepa root tips after treatment with extracts of all the tested extracts showed decrease in mitotic index with increasing concentration. there were significant differences (p table 2. cytological effects of aqueous extracts of ‘anti-diabetic’ herbal mixture, r. fruticosus, v. myrtillus, p. erecta, g. urbanum and p. vulgaris on meristematic cells of a.cepa. mi, mitotic index. mix, ‘anti-diabetic’ herbal mixture. rbl, leaves of r. fruticosus. vml, leaves of v. myrtillus. per, roots of p. erecta. gua, aerial parts of g. urbanum. pvp, pods of p. vulgaris. nc, negative control. pc, positive control. * significant compared to its negative control at 0.05 level. º significant compared to its positive control at 0.05 level. mix rfl vml per gua pvp treatment mitotic index (%) ± sd mitotic index (%) ± sd mitotic index (%) ± sd mitotic index (%) ± sd mitotic index (%) ± sd mitotic index (%) ± sd nc 20.28±2.17 20.28±2.17 20.28±2.17 20.28±2.17 20.28±2.17 20.28±2.17 400 µg/ml 13.96±0.64 * º 16.12±1.36 * º 10.96±0.78 * º 16.76±2.67 º 16.92±2.61 * º 16.16±1.38 * º 800 µg/ml 9.8±0.55 * º 12.2±0.6 * º 10.04±1.91 * º 14.68±0.84 * º 12.76±0.48 * º 9.68±0.48 * º 1200 µg/ml 6.4±0.93 * º 9.56±1.08 * º 7.72±1.4 * º 11.84±0.73 * º 9.12±1.47 * º 7.96±0.68 * º pc 3.88±0.92 3.88±0.92 3.88±0.92 3.88±0.92 3.88±0.92 * º 3.88±0.92 * º biologica nyssana 8 (2)  december 2017: 151-158 madić, v. et al.  evaluation of cytotoxicity of „anti-diabetic“ herbal… 156 < 0.05) between treated groups and a control group in mitotic index, as shown on tab. 2. observed mitodepressive effect suggests that tested extracts had some effects on cell division of a. cepa. this may be due to abnormal conditions of the cells induced by the treatments. the reduction of the mitotic index might be explained as being due to the obstruction of the onset of prophase, the arrest of one or more mitotic phases, or the slowing of the rate of cell progression through mitosis (b r i a n d & k a p o o r , 1989). of all the tested aqueous extracts, only ‘antidiabetic’ herbal preparation, r. fruticosus, v. myrtillus showed an influence on the mitotic phases, and ‘anti-diabetic’ herbal preparation (1200 µg/ml) and v. myrtillus (400 µg/ml) caused surprisingly high prophase accumulation. this highly prophase accumulation could be correlated to the blockage of the dividing cells at chfr point which prevents prophase-metaphase transition as proved by s c o l n i c k and h a l a z o n e t i s (2000) who explained that chfr protein delays chromosome condensation and nuclear envelope breakdown in response to drug such as taxol and nocodazole that disrupt microtubule structure. although chromosome abnormalities were not analyzed in this paper, it is observed that ‘anti-diabetic’ herbal mixture, r. fruticosus, v. myrtillus and the positive control caused spindle disturbance, especially c-mitosis, as the most common type of abnormalities, which indicates that these extracts not only caused disruption in the spindle structure, but also caused inhibition of spindle formation and prevented its polymerization (s a l m o n et al., 1984). conclusion the diabetes problem is a global epidemic, mostly in lowto middle income countries. costs of international commercial medicines are high, which caused an increased popularity of traditional medicine. however, some constituents of medicinal plants have been found to be potentially toxic (p i n g et al., 2012). according to our literature review, a little is known about potential toxicity of r. fruticosus, v. myrtillus, p. erecta, g. urbanum and p. vulgaris, medicinal plants used as a traditional remedy for diabetes. table 3. phase index of a. cepa root meristem cells treated with different concentrations of aqueous extracts of ‘anti-diabetic’ herbal mixture, r. fruticosus, v. myrtillus, p. erecta, g. urbanum and p. vulgaris. mix, ‘antidiabetic’ herbal mixture. rfl, leaves of r. fruticosus. vml, leaves of v. myrtillus. per, roots of p. erecta. gua, aerial parts of g. urbanum. pvp, pods of p. vulgaris. nc, negative control. pc, positive control. * significant compared to its negative control at 0.05 level. º significant compared to its positive control at 0.05 level. tested extract phase index (%) ± sd treatment nc 400 µg/ml 800 µg/ml 1200 µg/ml pc mix prophase 52.02±4.17 44.47±5.05 * º 48.91±3.84 º 62.46±6.41 * º 84.0±16.04 metaphase 20.61±4.39 22.06±2 º 22.47±2.3 º 14.93±2.63 º 4.29±4.72 anaphase 14.07±6.41 16.47±5.6 16.76±5.23 13.49±3.8 5.34±5.29 telophase 13.36±2.88 16.99±6.34 11.87±6.48 7.86±4.78 6.29±11.23 rfl prophase 52.02±4.17 55±3.59 * º 48.55±3.59 º 49.36±3.17 * º 84.0±16.04 metaphase 20.61±4.39 24.1±1.56 º 24.85±2.27 º 30.18±2.25 * º 4.29±4.72 anaphase 14.07±6.41 13.41±4.03 22.19±6.42 17.17±3.47 º 5.34±5.29 telophase 13.36±2.88 7.4±3.474 * 7.42±3.61 3.28±1.69 * 6.29±11.23 vml prophase 52.02±4.17 77.18±3.22* º 59.57±1.95 * º 49.66±7.05 º 84.0±16.04 metaphase 20.61±4.39 9.44±1.99 * º 30.84±3.98 * º 17.34±11.53 4.29±4.72 anaphase 14.07±6.41 7.15±2.8 8.44±2.27 23.8±10.91 º 5.34±5.29 telophase 13.36±2.88 6.23±1.75 * 1.14±1.07 * 9.19±2.9 * 6.29±11.23 per prophase 52.02±4.17 58.25±2.55* º 51.42±7.98 º 48.62±10.12 º 84.0±16.04 metaphase 20.61±4.39 18.26±3.12 º 19.56±3.47 º 26.11±14.82 º 4.29±4.72 anaphase 14.07±6.41 13.14±2.21 º 15.13±3.04 º 12.15±4.89 º 5.34±5.29 telophase 13.36±2.88 10.34±3.49 13.9±5.18 13.13±5.88 6.29±11.23 gua prophase 52.02±4.17 53.28±5.81 º 56.17±6.35 º 50.24±4.65 º 84.0±16.04 metaphase 20.61±4.39 19.31±3.19 º 18.14±5.1 º 22.49±4.1 º 4.29±4.72 anaphase 14.07±6.41 15.07±5.89 º 17.88±3.13 º 17.45±3.31 º 5.34±5.29 telophase 13.36±2.88 12.23±4.58 8.78±2.97 * 9.82±4.35 6.29±11.23 pvp prophase 52.02±4.17 58.12±4.96 º 55.46±3.92 º 65.94±10.26 84.0±16.04 metaphase 20.61±4.39 23.23±1.69 º 21.09±2.61 º 16.29±5.38 º 4.29±4.72 anaphase 14.07±6.41 9.95±2.87 13.62±2.16 º 10.79±4.29 5.34±5.29 telophase 13.36±2.88 8.7±3.78 9.82±4.88 6.97±1.67 * 6.29±11.23 biologica nyssana 8 (2)  december 2017: 151-158 madić, v. et al.  evaluation of cytotoxicity of „anti-diabetic“ herbal… 157 the results presented in this paper are therefore important since they suggest the cytotoxic effect of these medicinal plants in some concentrations. exposure to the high concentrations of all the tested extracts, especially to ‘anti-diabetic’ herbal mixture, r. fruticosus and v. myrtillus, initiates a cascade of deleterious changes within a. cepa root cells, visible as the inhibition of root growth and the reduction of the mitotic index. moreover, ‘anti-diabetic’ herbal preparation (1200 µg/ml) and v. myrtillus (400 µg/ml) caused surprisingly high prophase accumulation as well as spindle disturbance, especially c-mitosis, indicating that these extracts at these concentrations have a toxic effect. further cytogenetic studies dealing with clastogenicity and genotoxicity of these extracts with more comprehensive genotoxicity assessment in animal model may reveal 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26 (4): 551-555. tomczyk, m., latté, k. p. 2009: potentilla a review of its phytochemical and pharmacological profile. journal of ethnopharmacology, 122 (2): 184– 204. stanković, n., joković, n., mihajilov krstev, t., pejčić, m., dimitrijević, m.: frequency and antibiotic resistance of bacteria in urinary tract infections in south serbia. biologica nyssana, 8 (2) biologica nyssana 8 (2)  december 2017: 137-144 stanković, n. et al.  frequency and antibiotic resistance of bacteria… 137 original article received: 20 november 2017 revised: 22 december 2017 accepted: 23 december 2017 frequency and antibiotic resistance of bacteria in urinary tract infections in south serbia nikola stanković*, nataša joković, tatjana mihajilov krstev, milica pejčić, marina dimitrijević university of niš, faculty of science and mathematics, department of biology and ecology, višegradska 33, niš, serbia * e-mail: nikola.stankovic@pmf.edu.rs abstract: stanković, n., joković, n., mihajilov krstev, t., pejčić, m., dimitrijević, m.: frequency and antibiotic resistance of bacteria in urinary tract infections in south serbia. biologica nyssana, 8 (2), december 2017: 137-144. urinary tract infection (uti) refers to the presence of microbial pathogens within the urinary tract. the aim of this study was to determine frequency and antibiotic resistance of bacteria that cause urinary tract infections in south serbia during the year 2015. from 4784 analyzed urine samples 2367 isolates of pathogenic bacteria were obtained. the most frequent cause of uti was escherichia coli (43.0%) followed by enterococcus spp. (31.0%). also, there was a significant number of proteus mirabilis (11.0%) and klebsiella spp. (7.0%) isolates. in addition, remaining 8.0% of isolated bacteria belonged to genera staphylococcus spp., enterobacter spp., citrobacter spp., providencia spp., acinetobacter spp. and to species pseudomonas aeruginosa and proteus vulgaris. gram-negative bacterial isolates were the most resistant to penicillin antibiotics, and the most sensitive to cephalosporins. enterococcus spp., which are gram-positive bacteria showed sensitivity to ampicillin in significant percentage and also to vancomycin (glycopeptide antibiotic), while fosfomycin (quinolone antibiotic) showed the lowest potency against these isolates. key words: antibiotic resistance, urinary tract infections, escherichia coli, enterococcus spp. apstrakt: stanković, n., joković, n., mihajilov krstev, t., pejčić, m., dimitrijević, m.: učestalost i antibiotska rezistencija bakterija izazivača urinarnih infekcija u južnoj srbiji. biologica nyssana, 8 (2), decembar 2017: 137-144. infekcije urinarnog trakta (uti) predstavljaju prisustvo patogenih mikroorganizama u urinarnom traktu. cilj ovog rada je bio da se utvrdi učestalost i rezistencija na antibiotike uzročnika urinarnih infekcija na teritoriji južne srbije u toku 2015. godine. ispitivanje je vršeno u mikrobiološkoj laboratoriji poliklinike “human” u nišu. iz ukupno analiziranih 4784 uzoraka urina bilo je 2367 bakterijskih izolata. najčešći uzročnik urinarnih infekcija je bila bakterija escherichia coli (43.0%), a zatim enterococcus spp. (31.0%). u značajnijem broju je bilo i izolata proteus mirabilis (11.0%) i klebsiella spp. (7.0%). pored navedenih bakterija izolovane su i staphylococcus spp., pseudomonas aeruginosa, proteus vulgaris, enterobacter spp., citrobacter spp., providencia spp., acinetobacter spp., koje zajedno čine 8.0% izolata iz analiziranih uzoraka. gram-negativni 8 (2) • december 2017: 137-144 doi: 10.5281/zenodo.1135959 biologica nyssana 8 (2)  december 2017: 137-144 stanković, n. et al.  frequency and antibiotic resistance of bacteria… 138 bakterijski izolati su bili najrezistentniji na penicilinske antibiotike, dok su najveću osetljivost pokazali na cefalosporine. enterococcus spp., koje su inače gram-pozitivne bakterije, su u velikom procentu pokazale osetljivost na ampicilin, kao i vankomicin (glikopeptidni antibiotik), dok je primećena najveća rezistencija na fosfomicin (hinolonski antibiotik). ključne reči: antibiotska rezistencija, infekcije urinarnog trakta, escherichia coli, enterococcus spp. introduction urinary tract infections (uti) are among the most common infectious diseases occurring in either the community or healthcare settings. uti are caused by bacteria which, in case that they are present in significant number in urinary system, result by occurrence of different symptoms in patients (h o o t o n , 2000; n i c o l l e , 2005). according to f o x m a n (2000), utis are up to 10 times more frequent in women than in men. among the bacterial species escherichia coli account to 80% to 85% of the infection (v a s u d e v a n , 2014). except of e. coli, other bacteria can be the cause of uti including klebsiella spp., proteus spp., enterococcus spp., staphylococcus spp., pseudomonas aeruginosa (l i n h a r e s et al., 2013). resistance to antibiotics is a rising problem worldwide and many studies reported results on the main causative agents and their antibiotic resistance patterns in different regions (c h o w , 2000; a i b i n u et al., 2004; r u d y et al., 2004; v e l i č k o v i ć r a d o v a n o v i ć et al., 2009; h e i n t z et al., 2010; s w a m i n a t h a n & a l a n g a d e n , 2010; e l b o u a m r i et al., 2014). local antimicrobial susceptibility patterns of urinary isolates should be known in order to achieve a satisfactory therapeutic effect (h u a n g et al., 2014). usually, the first step in treating uti is an empiric antibiotic selection, partially determined by local resistance patterns. because of increasing resistance of bacteria to most common agents used in empiric therapy (trimethoprim/sulfamethoxazole), it is necessary to isolate, identify and make a susceptibility test on bacterial causative agent of infection for an adequate treatment of uti (n i c o l l e , 2005; h a n d e et al., 2005). the aim of this research was to to isolate and identify bacterial species that are the most common causes of uti in south serbia, as well as to determine whether the incidence of infection is related to gender. resistance to antibiotics of obtained isolates was also done in order to determine the local resistance patterns. material and methods samples urine samples from patients of polyclinic “human” in niš, were investigated in this study. a total of 4784 urine samples collected in the period january – december 2015. were analyzed. mediums and antibiotics cultivation and isolation of pathogenic bacteria from urine samples were performed on following microbiological culture media: uti agar, sheep blood agar, macconkey agar, mannitol salt agar base, esculin bile agar, kligler iron agar, peptone water, simmons citrate agar, christensen urea agar, parisian mannitol, mueller-hinton agar (titan media, india). kovacs reagent and gram staining reagents were used for identification purposes. testing the resistance of isolates was done using antibiotic discs (himedia, india), by list and concentration recommended by clsi standards (clsi, 2010). gram-negative isolates were tested to following antibiotics: ampicillin (10 µg), amoxicillin/clavulanic acid (30 µg), cefuroxime (30 µg), cefotaxime (30 µg), cefixime (30 µg), gentamicin (10 µg), nitrofurantoin (300 µg), ciprofloxacin (5 µg), and trimethoprim/ sulphamethoxazole (25 µg). isolates of enterococcus spp. were tested to: ampicillin (10 µg), levofloxacin (15 µg), doxycycline (30 µg), fosfomycin (50 µg), gentamicin (120 µg), ciprofloxacin (5 µg), nitrofurantoin (300 µg), and vancomycin (30 µg). isolation and identification of bacteria isolation of bacteria from urine samples was performed by plating samples on the uti agar followed by 24 h incubation on 35-37 °c. bacterial colonies with different macromorphological characteristics were further transferred on different selective and differential media in order to identify isolates on the basis of their biochemical characteristics. small blue-green colonies on uti agar were plated on blood and esculin agar. bacteria which had hemolytic activity on blood agar and decomposed bile salts on esculin agar were identified as biologica nyssana 8 (2)  december 2017: 137-144 stanković, n. et al.  frequency and antibiotic resistance of bacteria… 139 enterococcus spp. large white colonies on uti agar were further transferred on mannitol salt agar. isolates that formed yellow colored colonies and were positive on coagulase test (rabbit plasma coagulase test) were identified as s. aureus (k a r a k a š e v i ć , 1989). small white-light blue colonies on uti agar were transferred on blood agar in order to examine hemolytic activity. confirmation of streptococcus spp. was done by using agglutination strepto-kit (microgen, bioproducts). on uti agar, gram-negative bacteria form a large purple, light to dark blue, or white to cream colored, mostly slimy colonies. identification of these bacteria was performed using standard biochemical tests. susceptibility testing of bacteria agar plates were inoculated with a standardized inoculum (mcfarland standard 0.5) of the bacteria and antimicrobial disks were placed on the inoculated agar plate according to guidelines of the clinical and laboratory standards institute (clsi, 2010). the disks used for a disk diffusion assay contains a standardized known amount of an antimicrobial agent (table 1 and 2), which diffuses into the agar when is in contact with the agar surface. the plates were incubated inverted at 36±1°c for 18 to 24 h. following incubation, the diameter of this zone was measured, and the results were interpreted as resistant (r), intermediate (i), or susceptible (s) using standard guidelines. the isolates that were intermediary sensitive to certain antibiotics were classified into sensitive, because intermediate represents the sensitivity to a particular antibiotic in a smaller extent. results frequency of bacteria in uti in order to examine the dominant groups of bacteria in patients with uti, 2367 bacterial strains from the 4784 urine samples were isolated and identified. the most common pathogen in urine samples was e. coli (43.0%), while significant percentage of enterococcus spp. (31.0%), proteus mirabilis (11.0%) and klebsiella spp. (7.0%) was observed. also, staphylococcus spp., pseudomonas aeruginosa, proteus vulgaris, enterobacter spp., citrobacter spp., providencia spp., acinetobacter spp. were isolated but in smaller percentage (8.0% in total). considering that gender of the patients is the most common factor that affects the urinary infections, an analysis of the frequency of isolation of the pathogen from urine in relation to this factor was performed. analyses related to the gender of the patients were done for four most frequent bacteria causing uti and the results are presented in tab. 1. from a total of 2367 isolates from urine samples,1737 (73.4%) isolates were isolated from female patients, 630 (26.6%) isolates from male patients. e. coli was significantly represented in females than males (82.4% and 17.6% respectively). other pathogens were also more common in females than in males (tab. 1). in male patients, enterococcus spp. were most frequent (n=225, 35.7%), followed by e. coli (n=181, 28.7%), p. mirabilis (n=128, 20.3%) and klebsiella spp. (n=37, 5.9%). when it comes to female patients, e. coli was most frequent (n=846, 48.7%), followed by enterococcus spp. (n=503, 29.1% ), p. mirabilis (n=142, 8.2%), and klebsiella spp. (n= 127, 7.3%). antibiotic resistance of isolates antibiotic susceptibility testing was performed for e. coli, p. mirabilis, klebsiella spp., and enterococcus spp. (tab. 2). the following groups of antibiotics were tested: penicillins (apicillin and amoksicillin/clavulanic acid), sulfonamides (trimethoprim/sulphamethoxazole), quinolones (ciprofloxacin and levofloxacin), cephalosporins (cefixime, cefotaxime and cefuroxime), aminoglycosides (gentamicin), nitrofuran derivates (nitrofurantoin), glycopeptide (vancomicin), tetracyclines (doxycycline), and fosfomicin. table 1. number and frequency of isolates considering gender of the patients male female bacteria total number frequency number % of isolates by gender number % of isolates by gender escherichia coli 1027 43.0% 181 17.6 846 82.4 enterococcus spp. 728 31.0% 225 30.9 503 69.1 proteus mirabilis 270 11.0% 128 47.4 142 52.6 klebsiella spp. 164 7.0% 37 22.6 127 77.4 other bacteria 178 8.0% 59 33.1 119 66.8 biologica nyssana 8 (2)  december 2017: 137-144 stanković, n. et al.  frequency and antibiotic resistance of bacteria… 140 among the penicillins all isolates were more resistant to ampicillin in comparison to amoxicillin/clavulanic acid. klebsiella species showed the highest resistance to ampicillin (98.1%), followed by e. coli (72.7%) and p. mirabilis (58.2%). the same order of frequencies for amoxicillin clavulanic acid was observed, but with a much smaller percentage of resistant isolates (53.4%, 36.5%, and 24.9% respectively). it was noticed that resistance to trimethoprim/sulfamethoxazole was significant. resistance to trimethoprim/sulfamethoxazole for p. mirabilis was 49.6%, for klebsiella spp. 48.4%, and for e. coli 41.8%. resistance to ciprofloxacin for klebsiella spp. was 40.7%, for e. coli 25.0%, and for p. mirabilis 16.5%. in enterococcus spp. resistance to ciprofloxacin was 35.7%. table 3. antibiotic resistance of enterococcus spp. antimicrobials % resistant enterococcus spp. ampicillin 2.5 ciprofloxacin 35.7 levofloxacin 29 doxycycline 32 vancomycin 1.1 nitrofurantoin 7.7 fosfomycin 79.5 gentamicin 38.4 in relation to cephalosporins resistance was: to cefixime in klebsiella spp. 41.4%, in e. coli 18.5%, and p. mirabilis 13.4%; to cefuroxime in klebsiella spp. 44.4%, in p. mirabilis 16.2%, and in e. coli 15.6%; to cefotaxime in klebsiella spp. 45.3%, in p. mirabilis 16.0%, and in e. coli 14.7%. resistance to gentamicin in klebsiella spp. was 41.4%, in p. mirabilis 25.0%, and in e. coli 22.9%. resistance to nitrofurantoin in klebsiella spp. was 53.7%, in p. mirabilis 47.6%, and in e. coli 7.7%. resistance to pipemidic acid was identified in 47.4% of klebsiella spp., in p. mirabilis 41.0%, and in e. coli 36.3%. enterococcus spp. isolates were also tested to fosfomicyn, doxycycline, levofloxacin, and vancomycin, and resistance to named antibiotics were 79.5%, 32%, 29%, and 1.1% respectively (tab. 3). discussion infections of the urinary tract are one of the most common bacterial infections and one of the most common reasons for prescribing antimicrobial drugs. antibiotics are a group of effective and commonly used drugs. unfortunately, bacteria have developed extremely genetic mechanisms of antibiotic resistance. the most important factor that leads to the development of bacterial resistance to antibiotics is their overuse, especially in cases where their use is not necessary (f o x m a n , 2010). in our study, of total 2378 isolated bacterial strains from urine samples obtained of patients from south serbia, the most common cause of uti was e. coli, as expected. our study showed that e. coli was less frequent than in most european regions (s c h i t o et al., 2009; m a l m a r t e l & g h a s a r o s s i a n , 2015) but more frequent than in ivory coast (m o r o h et al., 2013). the frequency of p. mirabilis was 11%, which is similar to bosnia (m a h m u t o v i ć v r a n i ć & u z u n o v i ć , 2016). unlike the other members of enterobacteriaceae, p. mirabilis is not a common pathogen that causes table 2. antibioticresistance of gram-negative pathogens, (%) resistant antimicrobials e. coli p. mirabilis klebsiella spp. ampicillin 72.7 58.2 98.1 amoxicillin clavulanic acid 36.5 24.9 53.4 trimethoprim/sulphamethoxazole 41.8 49.6 48.4 ciprofloxacin 25 16.5 40.7 cefixime 18.5 13.4 41.4 cefuroxime 15.6 16.2 44.4 cefotaxime 14.7 16 45.3 gentamicin 22.9 25 41.4 nitrofurantoin 7.7 47.6 53.7 biologica nyssana 8 (2)  december 2017: 137-144 stanković, n. et al.  frequency and antibiotic resistance of bacteria… 141 urinary tract infections in normal hosts (c h e n at al., 2012). the prevalence of klebsiella spp. infection was similar to studies carried out in portugal (6.013.45%), reported by l i n h a r e s et al. (2013). other pathogens (staphylococcus spp., p. aeruginosa, p. vulgaris, enterobacter spp., citrobacter spp., providencia spp., acinetobacter spp.) that we isolated were not investigated because they all together accounted 8 percent of the total number of isolates. our research has shown that urinary infections are three times more common in women. that was expected because studies worldwide reported that uti is more common in females (f o x m a n et al., 2000; a y e g o r o et al., 2007; o m o r e g i e et al., 2008). these data on the frequency of the gender coincide with the data obtained in the other regions worldwide (c u n h a et al., 2016). the lowest in vitro efficiency on pathogens has been shown by ampicillin, where only 1 of 164 klebsiella spp. isolates were susceptible to this antibiotic. escherichia coli, as the most prevalent pathogen had also high rate of resistance to ampicillin, followed by p. mirabilis. resistance of gram negative pathogens to ampicillin observed in this study was like those observed in south africa, israel, hong-kong, philippines, iran and bosnia, where range of resistance were 62.0 – 84.0% (d e s e n c l o s et al., 1988, k a z e m n i a et al., 2014; m a h m u t o v i ć -v r a n i ć & u z u n o v i ć , 2016). minor resistance of enterococcus spp. to ampicillin was observed. that was expected, because enterococci are typically susceptible to ampicillin, because of the lack of of beta-lactamase (h o l l e n b e c k & r i c e , 2012). resistance of e. coli and other gram-negative pathogens to trimethoprim/sulphametho-xazole was similar, in the range of 41.8 –49.6%. high percentage of resistance to this antibiotic was reported in european countries and brazil, in the range of 38.9% 50.6% (n i c k e l , 2007; c u n h a et al., 2016). the reason for this high resistance may be due to the wide use of these antimicrobials in the treatment of community-acquired infections (c u n h a et al., 2016). guidelines of the american infectious diseases society and the european society for microbiology and infectious diseases suggest that antimicrobials with a resistance rate above 20% should not be prescribed empirically to patients with uncomplicated cystitis, unless susceptibility is determined by priorisolation in culture (g u p t a et al., 2011). fluoroquinolones are widely used for empirical treatment of uti (r o c h a et al., 2012). it can be noticed that resistance to ciprofloxacin in our study is dramatically high comparing with studies from region and another european countries. in bosnia, the most common pathogen e. coli was resistant to ciprofloxacin in 4.3% of cases, comparing with our study where 25% of e. coli isolates were resistant. in 9 european countries its reported that e. coli was resistant in 8.8% of cases (n i c k e l , 2007). the highest percentage of resistance to ciprofloxacin was reported in nigeria, with 65.7% (o l u r u n m o l a et al., 2013). it is considered that ciprofloxacin has only modest activity against enterococci (p e r r y et al., 1994). results for the resistance of enterococcus spp. isolates are in accordance with with results from other studies (a b d u l l a & a b d u l l a , 2006; g i l h o , 2013). overuse of one of the fluoroquinolone leads to the development of resistance to the whole group of quinolone antibiotics (m a h m u t o v i ć -v r a n i ć & u z u n o v i ć , 2016). resistance of gram-negative pathogens to cephalosporins of second (cefuroxime) and third generation (cefixime and cefotaxime) was relatively low comparing with other antibiotics. there was no significant diference in a resistance patern within bacterial species based on cephalosporin clasification. the most resistant were klebsiella spp. isolates, and the least resistant were p. mirabilis. the most common pathogen e. coli was resistant to cephalosporins in range of 14.7-18.5%. m e y e r et al. (2010), reported dramatic increase of third generation cephalosporin-resistant e. coli in german intensive care units in a period of 8 years, where this pathogen developed resistance from 1.2% to 19.7%. gentamicin was only aminoglycoside antibiotic used in this study. klebsiella spp., p. mirabilis and e. coli have shown significant resistance. rate of resistance presents double value comparing with brazil (c u n h a et al., 2016). compared with region, resistance rate of e. coli in bosnia was 2.15%, which presents 10 times lower value (m a h m u t o v i ć -v r a n i ć & u z u n o v i ć , 2016). higher rate of resistance in e. coli to gentamicin was observed in egypt and iran, 40% and 36% respectively (g a d et al., 2011; k a z e m n i a et al., 2014). in our study, nitrofurantoin was the most efficient antibiotic against two most common pathogens, e. coli and enterococcus spp.. similar data were obtained in bosnia, brazil, nigeria, and portugal (o l u r u n m o l a et al., 2013; l i n h a r e s et al., 2013; m a h m u t o v i ć -v r a n i ć & u z u n o v i ć , 2016; c u n h a et al., 2016). the rising prevalence of vancomycin-resistant enterococci (vre) is of particular concern within many institutions because of its association with increased mortality and health care costs, as well as limited treatment options (h e i n t z et al., 2010). biologica nyssana 8 (2)  december 2017: 137-144 stanković, n. et al.  frequency and antibiotic resistance of bacteria… 142 present study detected vre but in a very small percentage. fosfomycin (monural) is very frequently prescribed at first symptoms of utis, but high resistance to this agent was observed, showing unjustified use of this tretment. resistance to gentamicin was also significant, and explanation of rising level of resistance to amynoglicosides at general is that enterococci have acquired aminoglycoside resistance genes that mediate production of aminoglycoside-modifying enzymes (c h o w , 2000). conclusion the results obtained in this study suggest that e. coli is the most common cause of urinary tract infections, followed by enterococcus spp. urinary tract infections occur more often in women than in men. nitrofurantoin is the most efficient agent against bacterial uropathogens and represent effective option for empirical therapy. ampicillin presents the least effective antibiotic against gram-negative uropathogens, but still effective against enterococcus spp. maximum resistance to all applied antibiotics showed klebsiella spp. isolates, wherein the resistance to ampicillin was almost absolute. it can be concluded that the cephalosporins in vitro are more efficient than other groups of antibiotics to gram-negative uti pathogens, and it can be assumed that cephalosporins are the most effective in the treatment of urinary tract infections. also, conclusion is that ampicillin (penicillins) is still highly efficient to enterococcus spp., while fosfomycin and aminoglycosides cannot be used as the first-choice treatment due to low efficiency against these isolates. we reported unique local pattern of frequency and resistance to antibiotics of bacterial uropathogens in south serbia, which can help in definition of empirical treatment for uti. besides, it is more efficient to perform culture and susceptibility tests on isolated pathogen prior to treat, in order to avoid failure of therapy. references abdulla, f.e., abdulla, e.m. 2006: antibiotic options for enterococcus faecalis infections. pakistan journal of medical sciences, 22 (3): 286-290. aibinu, i., aednipekun, e., odugbemi, t. 2004: emergence of quinolone resistance amongst escherichia coli strains isolated from clinical infections in some lagos state hospitals in nigeria. nigerian journal of health and biomedical sciences, 3 (2): 73-78. ayegoro, o.a, igbinosa, o.o., ogunmwonyi, i.n., odjadjare, e.e., igbinosa, o.e., okoh a.i. 2007: incidence of urinary tract infections (uti) among children and adolescents in ile-ife, 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sex, drugs, bugs, and age: rational selection of empirical therapy for outpatient urinary tract infection in an era of extensive antimicrobial resistance. brazilian journal of infectious diseases, 16 (2): 115-121. rudy, m., zientara, m., bek, t., martirosian, g. 2004: occurrence of antibiotic resistant enterococci in clinical specimens from a pediatric hospital. polish journal of microbiology, 54 (1): 77-80. schito, g.c., naber, k.g., botto, h., palou, j., mazzei, t., gualco, l., marchese, a. 2009: the aresc study: an international survey on the antimicrobial resistance of pathogens involved in uncomplicated urinary tract infections. international journal of antimicrobial agents, 34 (5): 407-413. swaminathan, s., alangaden, g. j. 2010: treatmant of resistant enterococcal urinary tract infections. current infectious disease reports, 12 (6): 455464. vasudevan, r. 2014: urinary tract infection: an overview of the infection and the associated risk biologica nyssana 8 (2)  december 2017: 137-144 stanković, n. et al.  frequency and antibiotic resistance of bacteria… 144 factors. journal of microbiology & experimentation, 1 (2): 00008. veličković-radovanović, r., petrović, j., kocić, b., antić, s., ranđelović, g. 2009: correlation between antibiotic consumption and bacterial resistance as quality indicator of proper use of these drugs in inpatients. vojnosanitetski pregled, 66 (4): 307–312. anticancer compounds from medicinal plants biologica nyssana 3 (2)  december 2012: 105-107 georgiev, d.  new species of stygobiotic belgrandiella… 105 original article new species of stygobiotic belgrandiella from north bulgaria (gastropoda: hydrobiidae) dilian georgiev department of ecology and environmental conservation, university of plovdiv, tzar assen str. 24, bg-4000 plovdiv, bulgaria * e-mail: diliangeorgiev@abv.bg abstract: georgiev, d.: new species of stygobiotic belgrandiella from north bulgaria (gastropoda: hydrobiidae). biologica nyssana, 3 (2), december 2012: 105-107. a new stygobiotic species of belgrandiella from a cave in north bulgaria was described. nine shells were collected by the author on 09.03.2013 at the entrance of the water cave (vodnata) near village of musina, north bulgaria, n43 13 14.9 e25 25 39.6, 198 m alt. the systematic position of the genus belgrandiella was also discussed. key words: aquatic, bulgaria, endemic, subterranean, snail introduction the genus belgrandiella wagner, 1927 (gastropoda: risooidea) consists of minute snails inhabiting the springs and the subterranean waters of south-west and central europe to the caucasus (k a b a t , h e r s l e r , 1993; a r c o n a d a , r a m o s , 2003). this is one of the most diverse genera from the risooidea superfamily in bulgaria having 11 species known till now (g e o r g i e v , 2011a, g e o r g i e v , 2013). there are two ecological groups of species from the genus: stygobiotic (belgrandiella hessei wagner, 1927, belgrandiella pusilla angelov, 1959, belgrandiella bulgarica angelov, 1972, belgrandiella bureschi angelov, 1976, belgrandiella stanimirae georgiev, 2011, belgrandiella maarensis georgiev, 2013) and spring-living species (belgrandiella angelovi pintér, 1968, belgrandiella zagoraensis glöer & georgiev, 2009, belgrandiella dobrostanica glöer & georgiev, 2009, belgrandiella bachkovoensis glöer & georgiev, 2009, belgrandiella pandurskii georgiev, 2011, the last one also found in cave waters) (w a g n e r , 1927; a n g e l o v , 1959, 1972, 1976; p i n t é r , 1968; h u b e n o v , 2005, 2007; g l ö e r , g e o r g i e v , 2009; g e o r g i e v , 2011b, c, d). in this paper i describe a new stygobiotic species of belgrandiella from a cave in north bulgaria showing that the subterranean waters of the country are still poorly known considering their fauna and many new species could be found in further research. material and methods the shells were collected by sieving the cave river deposits by 1x1 and 2x2 mm width of the mesh sieves. the material from the smaller meshed sieve was then brought to the laboratory and dried. after that it was again put into water and the floating shells were collected by a strainer and small brush. the measurements were carried out by means of ceti stereo microscope and an eye-piece micrometer, and photographs were made with camera system with a digital adapter. the material is stored in the national museum of natural history, sofia, bulgaria. abbreviations used: h shell height, w shell width, ah aperture height, aw aperture width, lh last whorl height. 3 (2) • december 2012: 105-107 biologica nyssana 3 (2)  december 2010: 105-107 georgiev, d.  new species of stygobiotic belgrandiella… 106 table 1. shell measurements of belgrandiella hubenovi n. sp. (for abbreviations see “materials and methods”) n h w ah aw lh w/h ah/h aw/ah lh/h 1 1.49 0.76 0.54 0.54 1.06 0.51 0.37 1.00 0.71 2 1.49 0.89 0.56 0.56 1.02 0.60 0.38 1.00 0.69 3 1.67 0.89 0.63 0.59 1.12 0.53 0.38 0.95 0.67 4 1.73 0.99 0.71 0.63 1.19 0.57 0.41 0.88 0.69 5 1.55 0.89 0.56 0.59 1.12 0.57 0.36 1.06 0.72 6 1.62 0.86 0.63 0.59 1.12 0.53 0.39 0.95 0.69 7 1.49 0.87 0.56 0.59 1.09 0.59 0.38 1.06 0.73 8 1.68 0.87 0.63 0.59 1.19 0.52 0.37 0.95 0.71 average 1.59 0.88 0.60 0.59 1.11 0.55 0.38 0.98 0.70 fig. 1. front and side view of the holotype of belgrandiella hubenovi n. sp. results and discussion genus belgrandiella wagner, 1927 type species: belgrandia kusceri wagner, 1914 belgrandiella hubenovi n. sp. material examined: 9 shells, 09.03.2013, the water cave (vodnata) near village of musina, north bulgaria, n43 13 14.9 e25 25 39.6, 198 m alt., d. georgiev leg. holotype: h = 1.49 mm, w = 0.76 mm, ah = 0.54 mm, aw = 0.54 mm, lh = 1.06 paratypes: 3 shells, national museum of natural history, sofia, bulgaria, 5 shells coll. d. georgiev. locus typicus: water cave near village of musina, north bulgaria, n43° 13' 14.9'' e25° 25' 39.6'', 198 m alt. differential diagnosis: by its bent periostome the new species differs from all known bulgarian species from the genus belgrandiella with an exception of b. hessei – another stygobiont known from temnata dupka cave near lakatnik town, about 167 km to the west. from this species b. hubenovi n. sp. differs by its smaller size (h max = 1.73 mm versus 2.3 mm in b. hessei), and not so thick and developed aperture lip. description: shell: the shell is ovate-conic to ovate-cylindrical, white, whorls are 3-4, regularly growing with, shining surface, with deep suture, fine growth lines sometimes forming small, irregular smooth ribs. the umbilicus is slit-like, the aperture is egg-shaped with sharp periostome which is bent to the outside from the lateral view. the operculum and the soft body are not known. h = 1.49-1.73 mm, w = 0.76-0.99 mm, ah = 0.54-0.71 mm, aw = 0.54-0.63 mm, lh = 1.02-1.19 mm, w/h = 0.510.60, ah/h = 0.36-0.41, aw/ah = 0.88-1.06, lh/h = 0.67-0.73. etymology: named after assoc. prof. dr zdravko hubenov (national museum of natural history, sofia, bulgaria) who contributed so much in the studies of the bulgarian aquatic malacofauna and paid me attention of some springs as potential localities of freshwater snails. distribution: known only from the type locality, possibly local endemic species. habitat and ecology: a stygobiotic species found only as empty shells in the river deposits at the entrance of vodnata cave near village of musina. this cave can be explored only by boat and experienced divers for searching living snails on the bottom substrate of the inner stream. the systematic position of the genus belgrandiella wagner, 1927 is unclear. it is referred by various authors to different family groups as hydrobiidae, orientalinidae or belgrandiellidae (radoman, 1983; kabat & hersler, 1993; angelov, 2000; arconada & ramos, 2003). the genus consists of minute risooid snails with different shell shapes, mainly ovoid-conical but also conical or ovoid and different penis morphology (simple long, biologica nyssana 3 (2)  december 2012: 105-107 georgiev, d.  new species of stygobiotic belgrandiella… 107 simple short, conical or triangular, or same shape but with one small lobe on the left side). it can be supposed (as for other extremely subterranean genera, for example bythiospeum bourguignat, 1882) that belgrandiella is a complex of more still undefined genera. however it is a very hard task and sometimes is impossible for the scientists to reach the habitats of some of the stygobiotic species. references angelov, a. 1959. neue gastropoden aus den unterirdischen gewässern bulgariens. archiv für molluskenkunde, 88(1/3): 51-54. angelov, a. 1972. neue hydrobiidae aus höhlengewässern bulgariens. archiv für molluskenkunde, 102(1/3): 107-112. angelov, a. 1976. ein neuer vertreter der gattung belgrandiella a. wagner, 1927 (gastropoda, hydrobiidae) von grundwassern bulgariens. acta zoologica bulgarica, 4: 78-80. angelov, a. 2000. mollusca (gastropoda et bivalvia) aquae dulcis, catalogus faunae bulgaicae. – pensoft & backhuys publ., sofia, leiden, 54 p. arconada, b., ramos, m. 2003. the ibero-balearic region: one of the areas of highest hydrobiidae (gastropoda, prosobranchia, risooidea) diversity in europe. graellsia, 59 (2-3): 91-104. georgiev, d. 2011a. check list of the bulgarian minor freshwater snails (gastropoda: risooidea) with some ecological and zoogeographical notes. zoonotes, 24: 1-4. georgiev, d. 2011b. a new species of belgrandiella (wagner 1927) (mollusca: gastropoda) from caves in northern bulgaria. acta zoologica bulgarica, 63 (1): 7-10. georgiev, d. 2011c. new species of snails (mollusca: gastropoda: risooidea) from cave waters of bulgaria. buletin shkenkor, ser. shkenkat natyrore, 61: 83-96. georgiev, d. 2011d. new localities of four bulgarian hydrobiidae species (gastropoda: risooidea). zoonotes, 24: 1-4. georgiev, d. 2013. catalogue of the stygobiotic and troglophilous freshwater snails (gastropoda: rissooidea: hydrobiidae) of bulgaria with descriptions of five new species. ruthenica, 23 (1): 59-67. glöer, p., georgiev, d. 2009. new rissooidea from bulgaria (gastropoda: rissooidea). – mollusca, 27 (2): 123-136. hubenov, z. 2005. malacofaunistic diversity of bulgaria. in: current state of bulgarian biodiversity – problems and perspectives, petrova a. (ed.) bulgarian bioplatform, sofia, 199-246. (in bulgarian). hubenov, z. 2007. fauna and zoogeography of marine, freshwater, and terrestrial mollusks (mollusca) in bulgaria. in: fet, v., a. popov (eds.). biogeography and ecology of bulgaria. springer, dodrecht, 141-198. kabat, a., hersler, r. 1993. the prosobranch family hydrobiidae (gastropoda: rissooidea): review of classification and supraspecific taxa. smithsonian contributions to zoology, 547: 194. pintér, l. 1968. eine neue wasserschnecke aus bulgarien. archiv für molluskenkunde, 98 (1/2): 61-63. radoman, p. 1983. hydrobioidea a superfamily of prosobranchia (gastropoda). i. systematics. monographs 547, serbian academy of sciences and arts, (department of science), 256 pp. wagner, a. 1927. studien zur molluskenfauna der balkanhalbinsel mit besonderer berücksichtigung bulgariens und thraziens, nebst monographischer bearbeitung einzelner gruppen. annales zoologici muzei polonici hist. nat., 6 (4): 263-399. subburaj et al. 2025, biologica nyssana 16(2) 16 (2) december 2025: doi: 10.46793/biolnyss.16.2.14s simarouba glauca leaves as a natural arsenal: gc-ms-based proximate and phytochemical insights with antimicrobial efficacy original article vanitha subburaj psg college of pharmacy, the tamil nadu dr. m. g. r. medical university, coimbatore, tamil nadu umaa kuppuswamy psg college of pharmacy, the tamil nadu dr. m. g. r. medical university, coimbatore, tamil nadu umaagangadhararao@gmail.com (corresponding author) sankar veintraimunthu psg college of pharmacy, the tamil nadu dr. m. g. r. medical university, coimbatore, tamil nadu syamala g. psg college of pharmacy, the tamil nadu dr. m. g. r. medical university, coimbatore, tamil nadu saran v. psg college of pharmacy, the tamil nadu dr. m. g. r. medical university, coimbatore, tamil nadu manoj prabhakar saravanan psg college of pharmacy, the tamil nadu dr. m. g. r. medical university, coimbatore, tamil nadu manju velmurugan karpagam faculty of medical sciences and research, the tamil nadu dr. m. g. r. medical university, coimbatore, tamil nadu received: june 02, 2025 revised: june 17, 2025 accepted: june 24, 2025 abstract: simarouba glauca, commonly known as lakshmi taru, is a medicinal plant rich in bioactive compounds with diverse therapeutic potential. this study aimed to investigate the phytochemical profile, elemental composition, and antimicrobial efficacy of its leaf extracts. gc-ms analysis of methanol and chloroform extracts revealed the presence of key constituents, including flavonoids, alkaloids, glycosides, and quinic acid. proximate and elemental analyses confirmed high nutritional content and minimal heavy metal contamination, with all detected metals within who safety limits. the antimicrobial activity was evaluated against staphylococcus aureus, escherichia coli, and candida albicans, and the chloroform extract exhibited the largest inhibition zones (up to 22 mm). these findings highlight the plant’s potential as a safe natural source of antimicrobial agents and dietary supplements. the comprehensive profile of s. glauca supports its use in traditional medicine and paves the way for its integration into modern phytopharmaceutical applications. key words: simarouba glauca, phytochemical analysis, gc-ms, antimicrobial activity, elemental composition apstrakt: listovi simarouba glauca kao prirodni arsenal: gc-ms zasnovana analiza osnovnog hemijskog sastava i fitokonstituenata simarouba glauca, poznata i kao lakshmi taru, predstavlja lekovitu biljku bogatu bioaktivnim jedinjenjima sa širokim spektrom terapijskog potencijala. ova studija imala je za cilj da ispita fitohemijski profil, elementarni sastav i antimikrobnu efikasnost ekstrakata njenih listova. gc-ms analiza metanolnog i hloroformskog ekstrakta otkrila je prisustvo ključnih sastojaka, uključujući flavonoide, alkaloide, glikozide i kininsku kiselinu. proksimalna i elementarna analiza potvrdile su visok nutritivni sadržaj i minimalnu kontaminaciju teškim metalima, pri čemu su svi detektovani metali bili u granicama bezbednosti koje propisuje szo. antimikrobna aktivnost ispitivana je protiv staphylococcus aureus, escherichia coli i candida albicans, a hloroformski ekstrakt pokazao je najveće zone inhibicije (do 22 mm). ovi nalazi ukazuju na potencijal ove biljke kao bezbednog prirodnog izvora antimikrobnih agenasa i dijetetskih suplemenata. sveobuhvatan profil vrste s. glauca podržava njenu upotrebu u tradicionalnoj medicini i otvara mogućnost za njenu integraciju u savremene fitofarmaceutske primene. ključne reči: simarouba glauca, fitohemijska analiza, gc-ms, antimikrobna aktivnost, elementarni sastav introduction simarouba glauca (abbreviated as s. glauca), commonly known as lakshmi taru or the paradise tree (simaroubaceae family), is a tropical plant species native to the rainforests of south america, particularly regions of brazil, colombia, and peru. known for its diverse medicinal properties (armour., 1959), this plant has been widely used in traditional medicine to treat a range of ailments, including fever, dysentery, gastrointestinal disorders, and malaria (fiaschetti et al., 2011, jose et al., 2018). recent studies have focused on its vast pharmacological potential of simarouba glauca, with increasing interest in its bioactive compounds, namely quassinoids (vieira & braz-filho., 2006) and therapeutic efficacy in managing chronic diseases such as cancer, diabetes, and inflammatory conditions significant due to the rich content of bioactive phytochemicals including quassinoids, alkaloids, flavonoids, terpenoids, saponins, and tannins in various parts of this plant (hussain et al., 2021). as © 2025 subburaj et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. the therapeutic potential of plant-based medicines becomes more widely recognized, the safety of herbal products has come into focus (anne et al., 2020). one of the critical safety concerns is heavy metal contamination in medicinal plants, which can pose significant health risks if consumed over long periods. heavy metals such as lead (pb), arsenic (as), cadmium (cd), and mercury (hg) are often absorbed from soil and water and accumulate in plant tissues. therefore, it is essential to assess heavy metal levels in simarouba glauca to ensure its safety for therapeutic applications (anne et al., 2020). furthermore, evaluating the nutritional elements in the s. glauca plant is vital for understanding its potential health benefits, particularly in the context of dietary supplements. a scientific investigation of phytochemicals through gc-ms analysis, microbiological studies, and elemental analysis of s. glauca focused on highlighting the need for further research into its medicinal and safety profiles. in particular, research focusing on the leaf extracts of s. glauca, which are readily accessible and commonly used in folk medicine, is critical for a better understanding of its pharmacological mechanisms (niketh et al., 2022). the objective of this research is to provide a comprehensive evaluation of simarouba glauca leaf extracts through multiple analytical approaches. the phytochemical composition of s. glauca leaf extracts will be identified using techniques such as gc-ms (gas chromatography-mass spectrometry), enabling the detection of volatile and non-volatile compounds and the identification of functional groups. in addition, energy-dispersive x-ray spectroscopy (edx) will be employed to determine the elemental composition, providing a detailed analysis of the nutritional elements and trace metals present in the leaves. to assess the plant’s safety profile, heavy metal analysis will be conducted to measure toxic metal concentrations. lastly, the antimicrobial activity of s. glauca leaf extracts will be evaluated against a variety of fungal and bacterial strains to confirm its traditional use as an antimicrobial agent (ramasamy et al., 2022). by combining phytochemical, elemental, and microbiological analyses, this research provides valuable insights into the medicinal efficacy and safety profile of simarouba glauca leaf extracts. the findings of this study will contribute to understanding s. glauca as a promising natural resource for pharmaceutical applications while ensuring its safety for human consumption. furthermore, this research could guide the formulation of standardized herbal products derived from simarouba glauca, thereby advancing its potential in modern medicine and phytotherapy. materials and methods collection and authentication of simarouba glauca leaves fresh, healthy leaves of simarouba glauca were collected from the psgcp herbal garden, psgimsr hospital campus, peelamedu, coimbatore, tamil nadu, india, and a voucher specimen was prepared and stored at the psg college of pharmacy. simarouba glauca (sg) leaves were identified and authenticated (no.: bsi/src/5/23/2021/tech./328) by plant taxonomist dr. m. u. sharief, botanical survey of india (bsi), tnau campus, coimbatore-641 004. collected leaves were washed with tap water to remove dust and adherent materials, shade-dried, and pulverized (preethi aries mg 216 mixer, 750 watts, green) into coarse powder for the extraction process. preparation of simarouba glauca leaf extracts shade-dried, pulverized s. glauca leaves were defatted using petroleum ether (40–60 °c), and then extracted with chloroform and methanol using the ultrasonic-assisted extraction technique (tatke & rajan., 2016). the resulting s. glauca leaf extracts were then concentrated using a rotary evaporator, and the percentage yield of sg leaf extracts was determined. following gc-ms analysis, the antibacterial activity of the obtained sg leaf extracts was investigated. additionally, an icp-oes analysis was performed on leaf powder of s. glauca to detect both nutritious and hazardous elements, such as lead, arsenic, mercury, and cadmium (sundharamoorthy et al., 2025). phytochemical analysis of simarouba glauca leaf extracts the prepared sg leaf extracts (chloroform and methanol) were subjected to qualitative phytochemical analysis in order to determine the presence of secondary metabolites such as proteins, alkaloids, sterols, terpenoids, flavonoids, glycosides, and tannins as per the standard protocol (rao et al., 2023). proximate analysis of simarouba glauca the proximate content of air-dried simarouba glauca leaf powder was determined in accordance with who guidelines, including moisture content (lod), ash value, extractive value, and elemental analysis (who., 2007). moisture content determination: the standard protocol was followed to determine moisture content or loss on drying, with reference to the air-dried sample (sg leaf) as per the who guideline. two biologica nyssana ● 16 (2) december 2025: subburaj et al. ● simarouba glauca leaves as a natural arsenal: gc-ms-based proximate and phytochemical insights with antimicrobal efficacy biologica nyssana ● 16 (2) december 2025: subburaj et al. ● simarouba glauca leaves as a natural arsenal: gc-ms-based proximate and phytochemical insights with antimicrobal efficacy grams of powdered sg leaf sample were precisely weighed into a petri dish covered with a lid, and the sample was then dried for 1 hour at 110 °c in the hot-air oven. after cooling in a desiccator, it was weighed once again. the amount of air-dried powder was used to calculate the moisture content of the s. glauca leaf sample (schubnell et al., 2020). determination of ash value: the ash remaining after ignition of plant material was determined to assess the potency and quality of the herbal drug, with reference to the amount of air-dried crude drug, using three distinct approaches: total ash, acid-insoluble ash, and water-soluble ash (who., 2007). total ash value: the whole quantity of substance left over after ignition was measured using the total ash method. as per the guideline, 2-4 g of air-dried s. glauca leaf material was weighted and ground in a tarred silica crucible. after the material was evenly distributed, the temperature was gradually increased from 450 to 600 °c until the material turned white, indicating complete carbon removal. the sample was then weighed after cooling in a desiccator. if carbon-free ash could not be obtained, the residue was moistened with 2 ml of water, dried in a water bath or on a hot plate, and then re-ignited to constant weight. after allowing the residue to cool in a desiccator for 30 minutes, the weight was measured without delay. the percentage of total ash in mg/g with reference to the air-dried material was calculated. acid-insoluble ash value: the residue left after heating the remaining insoluble material and boiling the entire ash in diluted hydrochloric acid is known as acid-insoluble ash. this quantifies the amount of silica, particularly in the form of siliceous earth and sand. 25 ml of dilute hydrochloric acid was added to the total ash, and the mixture was gently boiled for five minutes. following filtration through ashless filter paper, the sample was thoroughly rinsed with warm water. the filter paper containing the insoluble material was transferred back to the original crucible and ignited to a constant weight. after leaving the residue to cool for 30 minutes in an appropriate desiccator, it was weighed without delay. the amount of acid-insoluble ash (mg) per gram of air-dried substance was subsequently calculated. water-soluble ash value: water-soluble ash value refers to the portion of ash from a substance that dissolves in water, indicating the amount of soluble mineral content and inorganic matter present. total ash was boiled with 25 ml of water, the insoluble matter filtered and ignited to a constant weight. to calculate the water-insoluble ash value, the weight of the insoluble matter was subtracted from the total ash weight and then the difference was expressed as a percentage of the original air-dried sample. determination of extractive values: chloroform, methanol, and aqueous soluble extractive values were determined in accordance with the recommended protocols, and the results are documented (agrawal et al., 2021) methanol-soluble extractive value: 5 g of the finely powdered air-dried sg leaf sample was macerated in 100 ml of methanol in a closed flask for 24 hours, with frequent shaking during the first 6 hours. the sample was then allowed to stand for 18 hours before being quickly filtered to prevent solvent loss. 25 ml of the filtrate was dried to constant weight and then evaporated to dryness in an evaporating dish. the methanol-soluble extractive value was calculated with reference to the air-dried drug. water-soluble extractive value: in a closed flask, 5 g of air-dried, finely powdered sg leaf sample was macerated with 100 ml of chloroform-water for 24 hours, with frequent shaking during the first 6 hours and standing undisturbed for the remaining 18 hours. the mixture was quickly filtered with precautions to minimize solvent loss. then, 25 ml of the filtrate was evaporated to dryness in a tarred flat-bottomed evaporating dish, dried at 105 °c to constant weight, and weighed. the percentage of water-soluble extractive value (% w/w) was calculated with reference to the air-dried sample. chloroform soluble extractive value: five grams of air-dried powdered sg leaf sample was macerated in 100 ml of chloroform for 24 hours in a covered flask, shaken regularly for the first 6 hours, and then allowed to stand for 18 hours before being quickly filtered to prevent solvent loss. 25 ml of the filtrate was evaporated to dryness in a tarred, flat-bottomed dish to obtain a constant weight. chloroform soluble extractive value was calculated with reference to the air-dried material. heavy metal analysis of simarouba glauca leaf by the icp-oes method: the detection of heavy metals is mandatory for all plant samples undergoing research, as the presence of organic and inorganic heavy metals in the sg plant samples may cause many side effects. the icp-oes (inductively coupled plasma–optical emission spectrometry) is the technique in which the elements/heavy metals present in the sg leaf sample could be determined using perkin elmer optima 5300 dv icp-oes equipped with a concentric torch with alumina injector, scott spray chamber, and segmented charge coupled device detector. the observed limits were compared with the normative limits stated in the biologica nyssana ● 16 (2) december 2025: subburaj et al. ● simarouba glauca leaves as a natural arsenal: gc-ms-based proximate and phytochemical insights with antimicrobal efficacy who recommendation. (parvathy et al., 2020). acid digestion procedure: a dried sg leaf powder sample (0.1012 g) was accurately weighed and transferred into a clean digestion vessel. then, 8 ml of concentrated nitric acid and 2 ml of concentrated hydrochloric acid were added. the mixture was digested using a microwave digestion system at 180 °c for 30 minutes, after which it was allowed to cool. the digested sample was filtered through whatman no. 41 filter paper, and the filtrate was made up to volume in a clean 50 ml volumetric flask. the resulting solution was analyzed using an icp-oes instrument. an ideal blank solution was also prepared (momen., 2006). the chemical contaminants in medicinal plants can be classified into toxic heavy metals and nonmetals, with heavy metal contamination a major concern. among toxic heavy metals, mercury (hg), lead (pb), arsenic (as), and cadmium (cd) are considered major contaminants. the heavy metal analysis was performed using a perkin elmer optima 5300 dv icpoes equipped with a concentric torch, alumina injector, scott spray chamber, and a segmented charge-coupled device detector. the following parameters were set: nebulizer flow, 0.61/ min (nebulizer: gem tip cross flow); radio frequency power, 40 mhz/ 1500 watts; sample introduction, 1.5 ml/min; wavelength range, 165-782 nm; view, axial/radial; auxiliary flow, 1.0 l/min; plasma flow, 15 l/min; equilibration time, 30 sec; a series of multi element standards (1.5 and 10 ppm) were prepared using a 1000 ppm stock solution. the standard was aspirated into an argon plasma operating at 11,000 k, and a calibration graph was constructed using standard concentration (x-axis) and emission intensity (y-axis). unknown values were obtained by interpolating the emission intensity with the concentration axis. weight percentages of the elements present in the samples were calculated as follows. wt % (g/100g) = [instrument value (ppm)] x [volume in ml] x [dilution factor] x [10 -4] weight of sg leaf sample in grams the multi-acid digestion technique was used to digest the samples for lead (pb), cadmium (cd), copper (cu), zinc (zn), nickel (ni), and chromium (cr). as mercury (hg) and arsenic (as) are volatile, they were digested using a nitric acid-hydrochloric acid-potassium permanganate system before analysis. lead (pb), cadmium (cd), arsenic (as), mercury (hg), copper (cu), zinc (zn), nickel (ni), and chromium (cr) standards were bought from merck in germany and used for constructing the calibration curves for these metals (mohammed et al., 2017). analysis of nutritional elements by edax method (energy-dispersive x-ray spectroscopy): energy-dispersive x-ray (edx) microprobe analysis detects the presence of chemical elements in plant tissues as they are viewed under an electron microscope (em). the detection depends on an element’s atomic number and cannot distinguish between ionized, bonded, or free atoms. standard edx analyzers can detect elements with atomic numbers 11 (sodium) and higher. the windowless edx analyzer can detect elements from boron (atomic number 5) upwards, including the major elements present in tissues (carbon, nitrogen, and oxygen). in plant tissues, elements such as sodium, magnesium, potassium, aluminum, silicon, phosphorus, sulfur, chlorine, and calcium are often present at sufficiently high concentrations to be detected and quantified by edx analysis. elements at trace concentrations, such as those associated with proteins at enzyme active sites, are usually not detectable. semi-quantitative analyses compare spectra or the numbers of x-rays emitted by particular elements to draw limited conclusions about differences in the absolute or relative amounts of elements between specimens or regions of a specimen. many researchers are involved in the ongoing development of edx analysis and associated techniques to increase spatial resolution, reduce the risk of detecting trace elements, and accurately quantify composition. the present study provides information on the basic principles and applications of edx analysis, along with guidance on some of the technical difficulties encountered with plant tissues. in this technique, at low resolution for qualitative or semi-quantitative analysis of plant specimens, em-edx analysis is extremely critical. normally, plant tissue is dehydrated or frozen before analysis, and is not alive, although some fresh tissues can be analyzed quickly, while they are dehydrating in a vacuum. the techniques for preparing samples for edx analysis must be specifically designed to avoid artifacts that are common due to element mobility or loss during preparation and sampling. under this study the relative error of chemical analysis was as follows: if the content of the element was from 1 to 5%, the error was less than 10%; from 5 to 10% less than 5%; if the content was more than 10%, the error was less than 2% (goldstein et al., 2003; scimeca et al., 2018). gc-ms analysis of simarouba glauca leaf extracts: prepared simarouba glauca leaf extracts to inoculate nutrient agar plates, ensuring even distribution of the inoculum. after allowing the surface moisture to evaporate, wells of 6 mm diameter were made in the agar, and 30 µl of leaf extracts at concentrations of 25, 50, 75, 100, and 125 µg/ml were applied to each well. the plates were incubated at 37 °c for 24 hours, and the zone of inhibition around each well was measured to assess antibacterial activity (bhalodia et al., 2011; ramya et al., 2019). antifungal activity: for antifungal testing, candida albicans was subcultured on potato dextrose agar plates and incubated at 25 °c for 4-5 days. the fungal inoculum was spread evenly across fresh pda plates using a sterile swab. after allowing excess moisture to evaporate, disks containing the sg leaf extracts at varying concentrations (25, 50, 75, 100, and 125 µg/ ml) were placed on the plates, which were then incubated at 30 °c for up to 7 days. the inhibition zones around the disks were measured to determine the antifungal activity. results for both antibacterial and antifungal tests were recorded as the diameter (in mm) of the zoi (zones of inhibition) for staphylococcus aureus and escherichia coli, and for antifungal activity against candida albicans, respectively (liu et al., 2002; berkow et al., 2020). results and discussion collection and preparation of simarouba glauca leaf extracts healthy fresh leaves of simarouba glauca were procured from the psgcp herbal garden and authenticated by plant taxonomist dr. m. u. sharief of the botanical survey of india (bsi), tnau campus (no.: bsi/src/5/23/2021/tech./328). fig. 1(a) and 1(b) displayed an image of an sg leaf along with its authenticity certificate. the collected sg leaves were shade-dried and defatted before extraction using an ultrasonic-assisted method with methanol and chloroform. the percentage yields of methanol and chloroform in the resulting sg leaf extracts were determined to be 15.84±0.03 % w/w and 9.18±0.01 % w/w, respectively. phytochemical analysis of simarouba glauca leaf extracts the qualitative phytochemical analysis revealed that the sg methanol leaf extract contained proteins, alkaloids, glycosides, flavonoids, terpenoids, and steroids. however, there were no flavonoids, saponins, or carbohydrates in the sg chloroform leaf extract. the results of the observed phytochemical tests are displayed in tab. 1. biologica nyssana ● 16 (2) december 2025: subburaj et al. ● simarouba glauca leaves as a natural arsenal: gc-ms-based proximate and phytochemical insights with antimicrobal efficacy (chloroform and methanol) were analyzed by gcms to identify the chemical constituents. the gc-ms analysis was performed on a combined gc-ms instrument (agilent 8890: version: 20220823-2154-09279), which provides comprehensive analytical capabilities, allowing users to achieve sensitive, robust, and reliable gc-ms analysis of any sample in a routine setting through an hp-5ms agilent column with dimensions of 30 m x 250 μm x 0.25 μm. the initial flow rate was 1.2 ml/min, the post-run flow rate was 1 ml/min, and the holdup time was 1.2376 minutes. the initial temperature for the gc/msd transfer (gas chromatography/mass spectrometry detector) was set to 280 °c. this method was designed to analyze phytochemicals through gc-ms. 1 µl of prepared sample aliquot was injected into the column (syringe size: 10 μl) using an injector with a temperature set at 280 ºc, with an air gap of 0.2 μl. the oven initial temperature was set to 75 °c, with a maximum temperature of around 350 °c and a postrun temperature of 50 °c with a 0.5-minute hold time. the gc program was initiated with a column temperature set to -60 °c to 325 °c (350 °c), a holdup time of 1.2376 min, and a pressure of about 11.367 ps. the average velocity of the column, 40.402 cm/ sec, was applied. helium was used as the carrier gas (1.5 ml/min). the mass spectrometer was operated in ei mode (electron energy: 70 ev) with the mass source set at 230 °c for 53 min. the chromatogram and spectrum of the peaks were scanned three times, and the peaks were observed from 50 to 600 °c at a scanning speed of approximately 1,562 [n=2]. the phytocompounds present in the sg leaf extracts (chloroform, methanol, and aqueous) were identified by matching their mass spectral fragmentation patterns of the respective peaks in the chromatogram with those stored in the national institute of standards and technology mass spectral database (nist-ms, 1998) library (shettar et al., 2024). evaluation of antimicrobial activity: antibacterial activity: the antibacterial and antifungal activities of sg leaf extracts were assessed using the kirby-bauer method. for antibacterial testing, the bacterial strains of staphylococcus aureus (gram-positive) and escherichia coli (gram-negative) were cultured on agar plates, and well-isolated colonies of similar morphology were selected. these colonies were transferred into a broth medium and incubated at 35 °c for 2-6 hours to achieve sufficient turbidity. the culture turbidity was then adjusted with sterile saline or broth to obtain a suspension containing approximately 1-2 × 108 cfu/ml. a sterile cotton swab was used biologica nyssana ● 16 (2) december 2025: subburaj et al. ● simarouba glauca leaves as a natural arsenal: gc-ms-based proximate and phytochemical insights with antimicrobal efficacy the amount of active phytochemical components contained was determined using the extractive values of sg leaves following extraction with the appropriate solvents. following that, it was calculated using the amount of air-dried crude medicine. methanol, chloroform, and aqueous sg extracts all contained several phytochemical components, with extractive values of 18.40% w/w, 11.28% w/w, and 5.00% w/w, respectively. the result are shown in tab. 2 as a percentage (%) w/w. heavy metal analysis of simarouba glauca leaf by the icp-oes method since ancient times, phytometal elements have been used in a variety of pharmaceutical approaches to support and maintain the body’s health. based on our research, the leaf sample of simarouba glauca is a rich source of essential elements, including fe2+, zn2+, cu2+, and mg2+. in the current investigation, icp-oes analysis was used to detect the presence of metal ions and heavy metals in the sg leaf sample. cadmium (0.002 g/ml), lead (0.028 g/ml), nickel (0.085 g/ml), and chromium (0.279 g/ml) were detected, whereas mercury, potassium, and arsenic were below the detection limits (bdl). other elements, such as copper (0.134 mg/l), iron (1.768 mg/l), magnesium (7.592 mg/l), sodium (0.099 mg/l), zinc (0.072 mg/l), calcium (28.23 mg/l), and aluminum (0.221 mg/l), were also detected. the concentrations of all detected heavy metals (as, cd, pb, ni, and hg) were found to be within the permissible limits recommended by the world health organization (who, 1996), as presented in tab. 3. proximate analysis of simarouba glauca the proximate composition of air-dried simarouba glauca leaf powder, including moisture content (lod), ash value, extractive value, and elemental analysis, was analysed according to who guidelines. the present study’s results help to evaluate the potency and quality of the herbal drug relative to the quantity of air-dried crude drug. the quantity of moisture lost during drying was calculated to be 1.12±0.12 % w/w, accounting for the amount of drugs that were airdried. as indicators of the presence of contaminants in the leaf sample, such as sand, dirt, organic and inorganic residues, and other foreign elements, the total ash value (7.6±0.15%), water soluble ash value (10.2±0.19% w/w), and acid-insoluble ash value (4.0±0.13% w/w) have been identified. s.no phytochemicals test sg chloroform extract sg methanol extract 1 alkaloids + + 2 glycosides + + 3 flavonoids + 4 terpenoids + + 5 steroids + + 6 saponins 7 carbohydrates 8 tannins + + 9 proteins + + table 1. phytochemical analysis of sg chloroform and methanol leaf extracts sg simarouba glauca, (+) presence, (-) absence fig. 1. (a) simarouba glauca leaf and (b) authentication certificate biologica nyssana ● 16 (2) december 2025: subburaj et al. ● simarouba glauca leaves as a natural arsenal: gc-ms-based proximate and phytochemical insights with antimicrobal efficacy gc-ms analysis of simarouba glauca leaf extracts the combined gc-ms instrument (agilent 8890: version: 2022-0823-2154-09279) was used to analyse the phytochemicals in simarouba glauca leaf extracts (chloroform and methanol). the study’s findings showed that the sg chloroform leaf extract contained 14 chemical compounds, specifically carbohydrates and amino acids, such as 1,2,3 trihydroxypropane (11.76%), 2,4-dihydroxy-2,5-dimethyl-3(2h)-furan-3one (2.17%), cyclohexanamine, n-3-butenyl-n-methyl (2.36%), dl arabinose (2.42%), 4h-pyran-4-one,2,3physicochemical parameter values (%) w/w moisture content determination (lod at 105°c) 1.12±0.12% total ash value 7.6±0.15% acid-insoluble ash value 4.0±0.13% water soluble ash value 10.2±0.19% chloroform soluble extractive value 5.00% methanol soluble extractive value 11.28% aqueous soluble extractive value 18.40% table 2. proximate analysis of simarouba glauca leaf sophisticated analytical instrument facility, iit madras, chennai-36 perkin elmer optima 5300 dv icp-oes who permissible limits for heavy metals in plant (mg/kg) source: who (1996) sample code element symbol & wavelength (nm) weight of sample in gm / volume in ml dilution factor concentration dried leaf powder (sg) al 308.215 0.1012 g/50 ml 1 0.221 mg/l as 188.979 ,, ,, bdl 0.05 mg/kg ca 317.933 ,, ,, 28.23 mg/l cd 228.802 ,, ,, 0.002 mg/l 0.02 mg/kg cr 267.716 ,, ,, 0.279 mg/l 1.6 mg/kg cu 327.393 ,, ,, 0.134 mg/l 10 mg/kg fe 238.204 ,, ,, 1.768 mg/l hg 253.652 ,, ,, bdl 0.03 mg/kg k 334.532 ,, ,, bdl mg 285.213 ,, ,, 7.592 mg/l na 259.393 ,, ,, 0.099 mg/l ni 231.604 ,, ,, 0.085 mg/l 10 mg/kg pb 220.353 ,, ,, 0.028 mg/l 2 mg/kg si 251.611 ,, ,, 0.679 mg/l zn 213.857 ,, ,, 0.072 mg/l 0.60 mg/kg bdl below the detection limit table 3. elemental/heavy metal analysis of simarouba glauca leaf powder analysis of nutritional elements by edax method (energy-dispersive x-ray spectroscopy) edax analysis provides a fast, high-resolution, and simple method for simultaneously examining the amounts and locations of many different elements within a tissue. it can be used at many different levels of sophistication, although care is always required in specimen preparation and data analysis. from this method, qualitative and semi-quantitative results can be easily obtained, as shown in fig. 2 and tab. 4. biologica nyssana ● 16 (2) december 2025: subburaj et al. ● simarouba glauca leaves as a natural arsenal: gc-ms-based proximate and phytochemical insights with antimicrobal efficacy fi g. 2 . e le m en ta l a na ly si s of s im ar ou ba g la uc a by e d ax m et ho d biologica nyssana ● 16 (2) december 2025: subburaj et al. ● simarouba glauca leaves as a natural arsenal: gc-ms-based proximate and phytochemical insights with antimicrobal efficacy dihydro-3,5-dihydroxy-6-methyl (5.40%), 5-hydroxy methyl furfural 1(1.31%), tetraethyl ammonium nitrate (2.55%), 1,3-diazacyclooctane-2-thione (1.81%), 3-o-methyl-d-glucose (5.23%), 6-deoxy-d-mannono-4lactone (1.98%), 3-o-methyl-d-glucose (5.23%), lactose (11.72%), 1, 2, 3-benzenetriol (13.67%), 3, 4-altrosan (3.89%), and quinic acid (33.75%). tab. 5 shows the detected compounds’ chemical formulas and molecular weights, while fig. 3 displays their mass spectra. the sg methanol leaf extract’s gc-ms analysis revealed the presence of 12 chemical compounds including 2-ethyl-n-[(2s)-1-methoxypropan-2-yl], 2,4-dihydroxy-2,5-dimethyl-3(2h)-furan-3-one (1.407%), 6-methylaniline (1.70%), 1,2,3 trihydroxypropane (12.77%), decane (1.76%), 4h-pyran-4-one 2,3-dihydro3,5-dihydroxy-6-methyl (4.56%), isosorbide dinitrate (1.72%), 3-o-methyl-d-glucose (5.07%), 6-deoxyd-mannono-4-lactone (4.36%), 1, 2, 3-benzenetriol (13.67%), melezitose (13.91%), d-allose (5.50%), 1,2,3,5-cyclohexanetetrol (1α,2β,3α,5β) (23.99%). fig. 4 displays the mass spectrum, while tab. 6 lists the chemical formula and molecular weight of the chemical compounds found in the methanol leaf extract of simarouba glauca. evaluation of antimicrobial activity simarouba glauca, commonly known as laxmi taru or the paradise tree, belongs to the simaroubaceae family. the leaf and bark extracts of sg are known to have a bioactive components with considerable medicinal uses. quassinoids are among the s. no elements weight (%) atomic (%) 1 nak 0 0.01 2 mgk 4.25 6.4 3 alk 0.81 1.1 4 sik 41.99 54.7 5 mol 3.02 1.15 6 pbm 0.12 0.02 7 cdl 0.26 0.09 8 kk 8.83 10.35 9 snl 3.41 1.05 10 cak 22.59 20.62 11 crk 0.28 0.2 12 mnk 0.68 0.45 13 fek 0.58 0.38 14 cok 0.68 0.43 15 nik 0.81 0.5 16 cuk 1.58 0.91 17 znk 0.4 0.22 18 hgl bdl 0.83 19 ask 0.02 0.01 20 sek 3.97 3.97 table 4. the percentage of nutritional elements present in simarouba glauca leaves s. no chemical compound name molecular formula molecular weight m/z (g/mol) retention time (min) peak area (%) 1 1,2,3-trihydroxypropane c3h8o3 92.09 3.712 11.76 2 2,4-dihydroxy-2,5-dimethyl-3(2h)-furan-3-one c6h8o4 144.12 4.019 2.17 3 cyclohexanamine, n-3-butenyl-n-methylc11h21n 167.29 5.619 2.36 4 dl arabinose c5h10o5 150.13 6.769 2.42 5 4h-pyran-4-one 2,3-dihydro-3,5-dihydroxy-6-methyl c6h8o4 144.12 7.094 5.4 6 5-hydroxymethylfurfural c6h6o3 126.11 9.026 1.31 7 acetic acid, 2,2'[oxybis(2,1-ethanediyloxy)] bis c8h20n2o3 192.26 9.32 2.55 8 1,3-diazacyclooctane-2-thione c6h12n2s 144.24 10.132 1.81 9 3-o-methyl-d-glucose c7h14o6 194.18 10.201 5.23 10 6-deoxy-d-mannono-4-lactone c6h10o5 162.14 11.076 1.98 11 1,2,3-benzenetriol c6h6o3 126.11 12.633 13.67 12 lactose c₁₂h₂₂o₁₁ 342.3 13.827 11.72 13 3,4-altrosan c6h10o5 162.14 15.002 3.89 14 quinic acid c7h12o6 192.17 17.0865 33.75 table 5. gc-ms analysis of the chloroform leaf extract of simarouba glauca biologica nyssana ● 16 (2) december 2025: subburaj et al. ● simarouba glauca leaves as a natural arsenal: gc-ms-based proximate and phytochemical insights with antimicrobal efficacy most important active phytochemicals in the triterpene family, with pharmacological activities such as antibacterial, anticancer, antipyretic, and haemostatic. sg phytochemicals (tannins, terpenoids, alkaloids, phytosterols, and flavonoids) are more effective and less toxic, making them an alternative treatment for common diseases caused by bacterial and fungal species. antimicrobial properties of sg leaf extracts (chloroform and methanol) were determined as per the kirby-bauer method using staphylococcus aureus, escherichia coli, and candida albicans. the spherical, gram-positive bacterium staphylococcus aureus is commonly found on the skin and in the upper respiratory tract. warmblooded creatures’ lower intestines are frequently home to the rod-shaped, facultatively anaerobic, gram-negative coliform bacteria escherichia coli. common e. coli infections include meningitis in newborns, pneumonia, diarrhoea, bacteremia, and urinary tract infections (utis). the standard drug teicoplanin (formerly known as teichomycin a2) is a semisynthetic glycopeptide antibiotic with a spectrum of activity similar to that fig. 3. gas chromatogram–mass spectrum chromatogram of chloroform leaf extract of simarouba glauca fig. 4. gas chromatogram–mass spectrum chromatogram of methanol leaf extract of simarouba glauca biologica nyssana ● 16 (2) december 2025: subburaj et al. ● simarouba glauca leaves as a natural arsenal: gc-ms-based proximate and phytochemical insights with antimicrobal efficacy of vancomycin, which was used as a positive control. glycopeptide antibiotics inhibit bacterial cell wall formation by inhibiting peptidoglycan synthesis. they are used to treat multidrug-resistant staphylococcus aureus and enterococcal infections, including utis, bacteremia, and infective endocarditis, and rarely cause intra-abdominal infections and meningitis. candida albicans is a yeast-like fungus naturally found in oral cavity, skin, and intestines. the overgrowth of yeast can cause infections like candidiasis, which is treated with synthetic antifungal medication (amphotericin). both natural and synthetic antibiotics kill or harm bacteria (atef et al., 2019). a prolonged course of synthetic antibiotic treatment can lead to antibiotic resistance and further damage to the body’s immune system. in recent years, the hunt for medications and phytochemicals derived from natural sources has increased. traditional healers have long utilized plants to prevent or cure infectious diseases. plants contain a wide range of secondary metabolites, including tannins, terpenoids, alkaloids, phytosterols, and flavonoids, all of which have antibacterial activities in vitro. the current study aimed to identify phytochemicals in sg leaf extracts (chloroform and methanol) through gc-ms analysis and to investigate their potential antimicrobial properties. the sg extract showed variable antibacterial and antifungal activity against the tested bacterial and fungal strains. the largest zone of inhibition was observed for sg chloroform leaf extract against s. aureus (19 mm) and e. coli (22 mm). in contrast, sg methanol leaf extract showed 15 mm and 19 mm, respectively, compared with the reference drug (23 mm and 25 mm) (tab. 7 and fig. 5). the sg leaf extracts (chloroform and methanol) were found to be efficient in inhibiting the growth of a fungal strain (candida albicans), with inhibition zone sizes of 11-14 mm, 6-12 mm, and 6-10 mm, respectively (tab. 8 and fig. 6). the above findings were more susceptible than fungi. terpenoids, steroids, alkaloids, tannins, and flavonoids, among other active phytochemicals found in sg, are responsible for antimicrobial effects. s. n o c he m ic al c om po un d n am e m ol ec ul ar w ei gh t m ol ec ul ar w ei gh t m /z (g /m ol ) r et en tio n ti m e (m in ) pe ak a re a (% ) 1 1, 2, 3tri hy dr ox yp ro pa ne c 3h 8o 3 92 .0 9 3. 71 2 11 .7 6 2 2, 4d ih yd ro xy -2 ,5 -d im et hy l-3 (2 h )fu ra n3on e c 6h 8o 4 14 4. 12 4. 09 9 1. 4 3 l-a la ni ne n p ro pa rg yl ox yc ar bo ny l, is oh ex yl e st er 2 -e th yl -n -[ (2 s) -1 -m et ho xy pr op an -2 -y l]6m et hy la ni lin e c 13 h 21 n o 20 7. 31 5. 61 9 1. 7 4 d ec an e c ₁₀h ₂₂ 14 2. 28 6. 12 5 1. 76 5 4h -p yr an -4 -o ne , 2 ,3 -d ih yd ro -3 ,5 -d ih yd ro xy -6 -m et hy l c 6h 8o 4 14 4. 12 7. 1 4. 56 6 is os or bi de d in itr at e c 6h 8n 2o 8 23 6. 14 9. 32 6 1. 72 7 3o -m et hy l-d -g lu co se c 7h 14 o 6 19 4. 18 10 .2 07 5. 07 8 6d eo xy -d -m an no no -4 -la ct on e c 6h 10 o 5 16 2. 14 11 .0 82 4. 36 9 1, 2, 3b en ze ne tri ol c 6h 6o 3 12 6. 11 12 .6 7 21 .9 9 10 m el ez ito se c 18 h 32 o 16 50 4. 4 13 .7 95 13 .9 1 11 d -a llo se c 6h 12 o 6 18 0. 16 14 .9 96 5. 5 12 1, 2, 3, 5c yc lo he xa ne te tro l, (1 α, 2β ,3 α, 5β ) c 6h 12 o 4 14 8. 16 17 .8 52 23 .9 9 ta bl e 6. g c -m s an al ys is o f t he m et ha no l l ea f e xt ra ct o f s im ar ou ba g la uc a biologica nyssana ● 16 (2) december 2025: subburaj et al. ● simarouba glauca leaves as a natural arsenal: gc-ms-based proximate and phytochemical insights with antimicrobal efficacy antibacterial assay details zone of inhibition (mm) bacterial strains name staphylococcus aureus escherichia coli sg leaf extract concentrations (µg/ml) 25 50 75 100 125 25 50 75 100 125 standard*(teicoplanin-standard drug) 23 mm 25 mm negative control 0 mm 0 mm sample a & c: sg chloroform leaf extract 11 11 10 15 19 9 12 12 20 22 sample b & d: sg methanol leaf extract 12 17 18 16 15 10 14 15 18 19 table 7. antibacterial activity of sg leaf extracts (chloroform and methanol) *teicoplanin-tei30 susceptibility test disc 30 µg fig. 6. zone of inhibition (mm) of sg leaf extracts against candida albicans. (a) chloroform leaf extract against candida albicans, (b) methanol leaf extract against candida albicans. antifungal assay details zone of inhibition (mm) fungal strain name candida albicans sg leaf extracts concentrations (µg/ml) 25 50 75 100 125 control-amphotericin (standard drug) 18 mm negative control 0 mm sample e: sg chloroform leaf extract 0 mm 0 mm 11 mm 14 mm 14 mm sample f: sg methanol leaf extract 0 mm 6 mm 9 mm 10 mm 12 mm table 8. antifungal activity of sg leaf extracts (chloroform and methanol) fig. 5. zone of inhibition (mm) for sg leaf extracts (a) chloroform leaf extract against staphylococcus aureus, (b) methanol leaf extract against staphylococcus aureus, (c) chloroform leaf extract against escherichia coli, (d) methanol leaf extract against escherichia coli conclusion in conclusion, the study of simarouba glauca (lakshmi taru) leaf extracts revealed significant phytochemical and proximate composition, as well as antimicrobial properties. the sg leaf extracts contain various bioactive compounds, including flavonoids, alkaloids, glycosides, and terpenoids, which contribute to its potential medicinal benefits. the physicochemical analysis confirmed a low moisture content and notable extractive values, suggesting the presence of active compounds. the results of the heavy metal analysis showed that the leaf extracts remained within the who guidelines’ acceptable limits. additionally, the antimicrobial activity showed that the extracts were highly effective against both bacterial and fungal strains. because sg leaf contains a variety of phytochemicals and nutrients, the chloroform extract exhibited the most potent antibacterial and antifungal activity. these biologica nyssana ● 16 (2) december 2025: subburaj et al. ● simarouba glauca leaves as a natural arsenal: gc-ms-based proximate and phytochemical insights with antimicrobal efficacy current medicinal chemistry, 18(3), 316–328. https://doi:10.2174/092986711794839205. goldstein, j., newbury, d., joy, d., lyman, c., et al. 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(solanaceae) as a new invasive species in algeria original article lahouaria mounia mansouri department of ecology and environment, university of batna 2, batna 05078, algeria laboratory of biodiversity, biotechnology and sustainable development, university of batna 2, batna 05078, algeria abdenour kheloufi department of ecology and environment, university of batna 2, batna 05078, algeria laboratory of biodiversity, biotechnology and sustainable development, university of batna 2, batna 05078, algeria laboratory of biotechnology for food and energy security, university of oran 1, oran 31000, algeria a.kheloufi@univ-batna2.dz (corresponding author) madjda razane rihane department of ecology and environment, university of batna 2, batna 05078, algeria received: september 07, 2025 revised: december 01, 2025 accepted: december 01, 2025 abstract: solanum bonariense l. (solanaceae), a shrub native to south america, is here recorded for the first time as a naturalized and potentially invasive species in algeria. field surveys and herbarium investigations (2022–2025) revealed its occurrence at multiple coastal and peri-urban sites across the eastern, central, and western regions, with the largest stands in the western region. populations were established in ruderal habitats such as agricultural lands, roadsides, railways, and abandoned lots, as well as in semi-natural scrublands and rocky slopes, illustrating a broad ecological amplitude. the species was frequently observed co-occurring with invasive congeners (s. elaeagnifolium, s. linnaeanum) and ruderal taxa (s. nigrum, s. villosum), suggesting similar invasion dynamics. a comparative morphological analysis and an identification key are provided to improve recognition and management of invasive solanum species in algeria. the documented westward range extension, coupled with evidence of viable, regenerating populations, signals an emerging phytosanitary threat. early detection, systematic monitoring, and targeted control measures are urgently required to prevent further spread and mitigate ecological, livestock, and economic impacts. key words: solanum bonariense, invasive plants, mediterranean flora, weed risk, solanaceaea apstrakt: pojava vrste solanum bonariense l. (solanaceae) kao nove invazivne vrste u alžiru solanum bonariense l. (solanaceae), žbun poreklom iz južne amerike, ovde se po prvi put navodi kao naturalizovana i potencijalno invazivna vrsta u alžiru. terenska istraživanja i herbarske analize (2022–2025) otkrile su njeno prisustvo na više obalnih i prigradskih lokaliteta širom istočnog, centralnog i zapadnog regiona, sa najvećim populacijama u zapadnom regionu. populacije su utvrđene u ruderalnim staništima kao što su poljoprivredna zemljišta, putevi, železničke pruge i zapuštene parcele, kao i u poluprirodnim šikarama i stenovitim padinama, što ukazuje na široku ekološku amplitudu. vrsta je često uočavana u zajednici sa invazivnim srodnicima (s. elaeagnifolium, s. linnaeanum) i ruderalnim taksonima (s. nigrum, s. villosum), što sugeriše sličnu dinamiku invazije. data je uporedna morfološka analiza i identifikacioni ključ radi boljeg prepoznavanja i upravljanja invazivnim vrstama roda solanum u alžiru. dokumentovano širenje areala ka zapadu, u kombinaciji sa dokazima o održivim, regenerišućim populacijama, ukazuje na novu fitosanitarnu pretnju. rano otkrivanje, sistematsko praćenje i ciljane mere kontrole hitno su potrebni kako bi se sprečilo dalje širenje i ublažili ekološki, stočarski i ekonomski uticaji. ključne reči: solanum bonariense, invazivne biljke, mediteranska flora, rizik od korova, solanaceae © 2025 mansouri et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. introduction the genus solanum l., one of the largest within the family solanaceae, comprises over 1200 species globally, with its primary center of diversity in south america (motti, 2021). these species occupy diverse habitats across temperate to tropical regions, including africa, asia, and australia (ramsay & bryan, 2011; samuels, 2015). solanum species range morphologically from herbaceous to subshrubby and shrubby forms, typically presenting simple leaves that may be entire, sinuate, or lobed (särkinen & knapp, 2016). floral characteristics include a five-lobed calyx, a rotate corolla, and a distinctive conical anther formation resulting from five appressed anthers, while fruits are multiovulate berries, generally two-locular. the genus includes economically significant taxa such as s. lycopersicum l. (tomato), s. melongena l. (eggplant), and s. tuberosum l. (potato) (quézel & santa, 1963; ganaie et al., 2018). in algeria, both native and non-native solanum species are present. common annuals like s. nigrum l. and s. villosum mill. inhabit disturbed and agricultural landscapes (quézel & santa, 1963; idrissi et al., 2024). more concerning are introduced congeners such as s. elaeagnifolium cav. and s. linnaeanum hepper & p.-m.l.jaeger, which aggressively invade pastures, roadsides, and orchards across northern algeria (meddour et al., 2020). these taxa exemplify the ecological plasticity and high colonizing potential of the genus in mediterranean ecosystems (karmezi et al., 2023; khamar et al., 2024). at the global scale, several solanum species pose serious invasion threats (fensham et al., 2019). in particular, s. elaeagnifolium is reported from every continent except antarctica and is recognized as an aggressive invader, especially in the mediterranean region. it thrives in disturbed habitats and environments affected by human activity-roadsides, agricultural lands, and urban environs-fueled by prolific sexual and vegetative reproduction, remarkable drought and salinity tolerance, and broad dispersal pathways including soil, water, animals, and machinery (roberts & florentine, 2022). this example underscores recurring traits favoring invasiveness within the genus: high reproductive output (both sexual and vegetative), tolerance to abiotic stresses (drought, salinity, disturbance), phenotypic and genetic plasticity, and effective dispersal mechanisms (tataridas et al., 2023). against this backdrop, solanum bonariense l., a south american perennial shrub, has been the subject of extensive investigation and is now urgently recorded in algeria. known from its native range in argentina, uruguay, paraguay, and southern brazil (palchetti et al., 2020), s. bonariense has established invasive populations in countries such as south africa, australia, and new zealand, where it invades pastures, grasslands, and forest edges (galasso et al., 2019). according to verdes et al. (2006), s. bonariense poses serious risks to livestock: grazing cattle exposed to this shrub may suffer from cerebellar cortical degeneration, manifesting as ataxia, hypermetria, hyperesthesia, head and limb extension, nystagmus, and falls, with microscopic lesions including purkinje cell loss and white-matter degeneration in the cerebellum (verdes et al., 2015). although mechanisms remain under investigation, these neurotoxic effects can impair animal health and productivity. ecologically, s. bonariense is classified as an invasive alien plant in some regions, capable of altering habitat structure, reducing native plant recruitment, and thereby undermining biodiversity through competitive displacement (khamar et al., 2024). herbarium and field surveys conducted between 2022 and 2025 confirmed its first algerian record, with specimens from coastal and peri-urban sites in northern algeria; no prior records were found in national floristic references or herbarium collections. given the documented invasiveness of congeners worldwide, the discovery of s. bonariense in northern algeria represents a significant emerging bioinvasion risk. its ecological adaptability, reproductive strategies, and establishment in anthropogenically disturbed environments portend similar dynamics to those of s. elaeagnifolium and other invasive solanum species. consequently, proactive investigation, ecological impact assessment, and integrated management measures are warranted to mitigate its potential spread and ecological consequences. materials and methods study area field surveys were carried out between 2022 and 2025 across coastal and peri-urban localities of northern algeria, spanning the provinces of jijel, skikda, annaba, bejaia, algiers, and oran. the regions are characterized by a mediterranean climate, with mild, wet winters and hot, dry summers. according to the algerian national office of meteorology, the mean annual temperature is 18.2 °c, ranging from 12.0 °c in january to 26.8 °c in august, while the mean annual precipitation is 345 mm, with most falling between october and april. field surveys and specimen collection populations of solanum bonariense were located and georeferenced using a garmin etrex 10 gps receiver. for each site, data on habitat type (roadsides, abandoned fields, coastal scrublands, peri-urban margins), disturbance level, associated vegetation, and potential dispersal pathways were recorded. voucher specimens were collected, pressed, and prepared following standard herbarium techniques. specimens were subsequently deposited at the herbarium of the university of oran (huo). herbarium and literature analysis taxonomic verification of s. bonariense and comparison with related solanum taxa were performed using morphological descriptions and biologica nyssana ● 16 (2) december 2025: mansouri et al. ● emergence of solanum bonariense l. (solanaceae) as a new invasive species in algeria biologica nyssana ● 16 (2) december 2025: mansouri et al. ● emergence of solanum bonariense l. (solanaceae) as a new invasive species in algeria diagnostic keys from dobignard & chatelain (2013). additional reference material was examined from collections at the national museum of natural history, paris, and the university of vienna herbarium (wu). historical floristic records (quézel & santa, 1963) and regional invasive species databases were consulted to confirm the presence or absence of prior reports of s. bonariense in algeria. morphological analyses and identification key morphological characterization included stem habit, leaf morphology (shape, size), floral traits, and fruit dimensions. these features were documented for s. bonariense and compared with those of four congeners present in algeria: s. elaeagnifolium, s. linnaeanum, s. nigrum, and s. villosum (tab. 1). mapping and data processing occurrence data for s. bonariense were processed using arcgis 10.8 (esri, redlands, ca, usa) to produce a georeferenced distribution map (fig. 2). gps locality points were integrated with geospatial layers from the algerian national geodetic database. habitat data were summarized to describe ecological conditions favoring the establishment of the species. biosecurity protocols to prevent inadvertent spread of s. bonariense during fieldwork, standard biosecurity measures were implemented. equipment, including gps devices, sampling tools, and footwear, was thoroughly cleaned between sites to avoid seed transfer. plant material collected for herbarium deposition was bagged character s. bonariense s. nigrum s. villosum s. elaeagnifolium s. linnaeanum habitat ruderal, roadsides, disturbed soils, pasture, scrublands, coastal areas cultivated fields, gardens, waste places, disturbed areas, agricultural lands cultivated fields, gardens, waste places, disturbed areas, agricultural lands, ruderal sites, open ground dry sandy soils, roadsides, abandoned fields, disturbed soils, wastelands, pastures, coastal areas scrub, field margins, mediterranean maquis, roadsides, rocky and coastal areas height (m) 1–2.5 0.2–0.6 0.3–0.7 0.3–1 0.5–2.5 leaves shape elliptic to lanceolate, entire or slightly wavy, ovate, entire margins ovate to lanceolate, heart-shaped, wavy-margined ovate, thinner, wavy-margined, entire or shallowly lobed deeply lobed, graygreen, tomentose, lanceolate, wavy edges deeply lobed, prickly, pubescent, lobed margins, persistent, alternate leaf length (cm) 5–12 2–7 3–8 5–15 5–12 leaf petiole present, short present, 1–3 cm present, slender present, 2–5 cm present, stout, often winged and prickly flowering time spring to late autumn summer to autumn summer to early autumn spring to autumn spring to autumn flower color purple, with yellow anther white, greenish to whitish petals, with yellow anthers white, or purpletinged purple, blue to pale lavender, occasionally white purple corolla corolla diameter (mm) 15–20 8–12 10–15 20–30 15–25 corolla shape star-shaped, spreading lobes, rotatepentagonal star-shaped star-shaped star-shaped, deeply divided star-shaped fruit diameter (cm) 1–1.2 0.6–1.3 0.5–0.9 1–1.5 2–5.5 table 1. morphological differences among solanum species in algeria biologica nyssana ● 16 (2) december 2025: mansouri et al. ● emergence of solanum bonariense l. (solanaceae) as a new invasive species in algeria elaeagnifolium cav. 6. fruits green-striped turning yellow at maturity; plants armed with prickles on stems and leaves – s. linnaeanum hepper & p.-m.l. jaeger the morphological analysis highlights that s. bonariense differs markedly from both annual and perennial congeners in growth form, leaf architecture, flowering phenology, and reproductive traits. these features, combined with its broad habitat tolerance and extended flowering period, underscore its potential for rapid colonization of disturbed and semi-natural habitats in northern algeria. distribution of solanum bonariense in algeria solanum bonariense was recorded at several localities along the northern mediterranean coastal region of algeria, spanning from the east (annaba, skikda, jijel, béjaïa) to the central (algiers) and to the western (oran) parts of the country (fig. 2). these occurrences are confined to the temperate coastal belt (20–25 °c average annual temperatures), where both anthropogenic disturbance and semi-natural habitats provide suitable conditions for establishment. across sites, populations were observed in ruderal habitats such as transport corridors and abandoned lots, as well as in semi-natural scrublands, underscoring the species’ ecological plasticity. northeastern algeria: jijel (36°49′22″ n, 5°45′41″ e). a roadside separately and handled to minimize seed dispersal into surrounding environments. all field personnel were trained in identification and containment protocols, ensuring that survey activities did not contribute to further naturalization of the species. results and discussion morphological differentiation solanum bonariense can be readily distinguished from other solanum species present in northern algeria by its perennial, woody habit and extended flowering period, from spring to late autumn. several additional morphological traits further separate s. bonariense from co-occurring congeners (tab. 1). to facilitate field identification, we provide a dichotomous key for the solanum species recorded in northern algeria: 1. plants woody, perennial; leaves entire, glabrous or sparsely pubescent; purple flowers with yellow anther cone; fruits yellow to orange at maturity – s. bonariense l. (fig. 1). 2. plants spiny or unarmed, perennial or annual; leaves often lobed, grey-green to pubescent; fruits variously colored at maturity 3. fruits black at maturity; annual herbs – s. nigrum l. 4. fruits red or orange at maturity; annual herbs – s. villosum mill. 5. fruits yellow, berries persistent; stems and leaves often covered with stellate hairs – s. fig. 1. solanum bonariense l., oran (nw-ageria), july 2025 (photo by lm mansouri) biologica nyssana ● 16 (2) december 2025: mansouri et al. ● emergence of solanum bonariense l. (solanaceae) as a new invasive species in algeria embankment with ~20 mature shrubs across ~30 m². the soil was sandy-loam and moderately disturbed, with cooccurring species including atriplex halimus l., dittrichia viscosa (l.) greuter, and sonchus oleraceus (l.). skikda (36°52′48″ n, 6°55′04″ e). the largest eastern population (~40 shrubs across ~200 m²) occurred along abandoned lots. associated taxa included erigeron bonariensis l., chenopodiastrum murale (l.) fuentes, uotila & borsch, and anethum foeniculum (l.). numerous seedlings indicated active recruitment and potential spread along linear transport networks. annaba (36°53′07″ n, 7°45′01″ e). approximately 25 shrubs were recorded in degraded scrubland on clay-sandy soils, accompanied by olea europaea l., asphodelus tenuifolius cav., and cynodon dactylon (l.) pers. proximity to a watercourse suggests hydrochorous dispersal pathways. béjaïa (36°46′44″, 5°00′49″ e). a small population (<10 shrubs) was observed on a rocky slope bordering cultivated terraces, within semi-natural vegetation dominated by pistacia lentiscus l. and pinus halepensis (mill.). north-central algeria: algiers (36°45′58″ n, 3°20′30″ e). a newly recorded central population of 20 mature shrubs was found within ruderal habitats adjacent to peri-urban scrublands. the site lies close to reghaia lake, favoring dispersal and persistence. northwestern algeria: oran, cap falcon (35°46′05″ n, 0°47′50″ w). the largest western coastal population (~100 shrubs on an area of 300 m2) was established on clay sandy loam soil within coastal scrub, accompanied by atriplex halimus l., hyoscyamus albus l., and ficus carica (l.). the extent and density suggest a well-established stand with significant reproductive capacity. oran, ain turk (35°45′16″ n, 0°47′34″ w). another dense population (~100 shrubs) was recorded in degraded scrubland near a cultivated area. cooccurring species included tamarix africana poir., retama monosperma (l.) boiss., and hordeum murinum (l.). both coastal sites (cap falcon and ain turk) represent major invasion fronts. oran, misserghin (35°37′24″ n, 0°43′58″ w). approximately 50 shrubs were recorded in abandoned agricultural fields with sandy-clay soils, surrounded by scolymus hispanicus l., hordeum murinum l., and malva sylvestris (l.). oran, bir el djir (35°43′56″ n, 0°32′57″ w). a moderate population (~30 shrubs) was observed near cultivated areas along transport corridors and urban wastelands, indicating tolerance to disturbed urban environments. these records demonstrate that s. bonariense has successfully established across both ruderal and semi-natural habitats in algeria, forming viable populations with evidence of persistence and regeneration. its occurrence along roadsides, railways, riparian corridors, and coastal areas suggests fig. 2. distribution map of solanum bonariense l. in algeria, with the highest records in northwestern region (oran) biologica nyssana ● 16 (2) december 2025: mansouri et al. ● emergence of solanum bonariense l. (solanaceae) as a new invasive species in algeria multiple dispersal vectors, including anthropogenic, hydrochorous, and possibly zoochorous mechanisms. compared with other naturalized solanum species in the mediterranean basin, s. bonariense exhibits ecological strategies consistent with those of successful invaders. for instance, s. elaeagnifolium is widespread in north africa, dominating agricultural fields and disturbed soils, while s. nigrum and s. villosum are ubiquitous in ruderal habitats with nitrophilous flora. the establishment of s. bonariense in similar environments, combined with dense coastal populations in oran, indicates a strong potential to follow a comparable invasion trajectory if left unmanaged. the updated distribution map (fig. 2) confirms that s. bonariense extends across northern algeria, representing a significant range expansion, with high records in oran (nw algeria). invasive potential and ecological implications the successful establishment of solanum bonariense in algerian coastal habitats highlights a suite of functional traits that underpin its invasive potential. these include dual reproductive strategies (sexual reproduction via seeds and vegetative propagation), physiological tolerance to drought and moderate salinity, and morphological resistance to grazing (zhang et al., 2024). such characteristics are well recognized as invasion-promoting syndromes in mediterranean-type ecosystems, where species must tolerate seasonal water deficits, nutrient-poor soils, and recurrent disturbance regimes (jan et al., 2024). in agricultural contexts, its spread can lead to diminished rangeland quality and potential grazing losses, as cattle avoid or are harmed by the plant, reducing grazing efficiency and increasing management challenges (barry & huntsinger, 2021). the invasion trajectory of s. bonariense is consistent with patterns already observed in other alien solanum species in the mediterranean basin. solanum elaeagnifolium is now among the most problematic weeds in north africa and southern europe, where it invades cereal fields, vineyards, and pastures, forming persistent seed banks and reducing crop yields (roberts & florentine, 2022; mou et al., 2025). in algeria and morocco, dense infestations of s. elaeagnifolium have been linked to reduced forage quality and increased management costs (menioui, 2021). similarly, s. linnaeanum (apple of sodom) has naturalized widely in tunisia and coastal morocco, particularly in scrublands and along roadsides, where its spiny morphology and high tolerance to aridity favor its spread (idrissi et al., 2024). together with s. bonariense, these taxa exemplify a broader trend of multiple solanum invasions facilitated by ecological versatility, endozoochorous dispersal, and human-mediated disturbance. the climatic context of northern algeria-mild, wet winters and long, dry summers (mean annual temperature ~18 °c; annual rainfall ~350 mm)provides a niche particularly favorable to the establishment of invasive solanaceae. comparable climates across the western mediterranean (e.g., southern spain, sicily, coastal greece) have facilitated the naturalization and expansion of s. elaeagnifolium and s. linnaeanum (krigas et al., 2023). their co-occurrence with s. bonariense raises concerns about cumulative ecological impacts, including the displacement of native shrubs (pistacia lentiscus, olea europaea), disruption of successional dynamics in scrublands, and reductions in agricultural productivity due to direct competition and increased management burdens. similar ecological impacts have been reported in greece and portugal, where s. elaeagnifolium has significantly altered farmland ecology (tataridas et al., 2022). given the early stage of s. bonariense invasion in algeria, proactive management remains feasible. early detection and rapid eradication of nascent populations are essential to prevent the formation of persistent seed banks. mechanical removal can be effective for small, isolated stands, provided it is followed by repeated monitoring. high-risk habitats such as ports, road verges, and abandoned farmland should be prioritized for surveillance due to their role as invasion gateways. integrated approaches combining soil seed bank management, reduction of livestock-mediated dispersal, and rotation of less susceptible crops are required. public awareness campaigns may further reduce inadvertent spread through contaminated machinery, fodder, or soil transport. the emergence of s. bonariense alongside s. elaeagnifolium signals an accelerating trend of solanum-driven biotic homogenization in mediterranean north africa. without coordinated and regionally integrated interventions, these species are likely to intensify ecological and economic pressures on both natural ecosystems and agroecosystems. this study provides the first record of solanum bonariense naturalized in coastal northern algeria. its drought and salinity tolerance, extended flowering, and efficient dispersal allow it to establish across diverse habitats, from transport corridors to semi-natural ecosystems. in algeria, solanum bonariense shows a notable ability to colonize a broad spectrum of habitats, ranging from highly disturbed to semi-natural environments. beyond its frequent occurrence in ruderal sites such as road verges, abandoned urban lots, and agricultural margins, the species is increasingly penetrating semi-natural mediterranean scrublands (maquis biologica nyssana ● 16 (2) december 2025: mansouri et al. ● emergence of solanum bonariense l. (solanaceae) as a new invasive species in algeria and garrigue formations), phrygana-like xeric shrublands, and rocky calcareous slopes. these habitats correspond broadly to the alliances oleo– ceratonion siliquae and rosmarinus officinalis l. (quézel & santa, 1963), which characterize large portions of the mesoand thermo-mediterranean bioclimatic zones. the presence of established and regenerating populations within these semi-natural vegetation units indicates an ongoing transition from ruderal naturalization to incipient invasion of native plant communities. this ecological expansion raises concerns regarding potential competition with native shrubs and herbs that dominate these syntaxa. together with s. elaeagnifolium, a major agricultural pest, and s. linnaeanum, s. nigrum, and s. villosum, which invade pastures and shrublands, s. bonariense illustrates a growing trend of solanum invasions in algeria with profound ecological and socio-economic implications. lessons from other mediterranean countries show that once solanum species become widespread, eradication is extremely difficult and costly. prevention and rapid eradication of small populations are therefore critical, particularly in high-risk sites such as ports, roadsides, abandoned fields, and riverbanks. for well-established species, integrated management combining mechanical, cultural, and awareness measures is required. the establishment of s. bonariense adds urgency to coordinated monitoring and management efforts, as unchecked expansion of invasive solanum species will further threaten biodiversity, land use, herbivorous and rural economies in mediterranean algeria. medicinal and agrochemical prospects the bioprospecting of endophytes associated with the solanum genus represents a promising frontier for discovering novel bioactive agents with dual applications in medicine and agriculture. a recent review by mili (2023) highlights that endophytes function as significant repositories of chemically diverse secondary metabolites. although only 13 solanum species have been extensively surveyed to date, 98 bioactive compounds (comprising 64 from fungi and 34 from bacteria) have been identified. these metabolites, including sterols, flavonoids, and volatile organic compounds, exhibit a broad spectrum of biological activities, ranging from anticancer and antiparasitic effects to potent antioxidant properties. of particular significance is the capability of these endophytes to synthesize antimicrobial compounds that mitigate phytopathogenic threats. within this context, solanum bonariense emerges as a species of distinct interest. the bioactive profile of its associated endophytes suggests significant potential to develop biofungicides and bactericides capable of naturally controlling plant diseases. consequently, the study of s. bonariense not only contributes to the search for new pharmaceutical leads but also offers sustainable strategies for crop protection against phytopathogens, leveraging the host-endophyte symbiotic machinery. in addition to its antimicrobial potential, s. bonariense has been recognized for possessing significant insecticidal properties, attributed to its rich secondary metabolite profile. in a screening of wild flora from southeastern spain, pascualvillalobos and robledo (1999) identified s. bonariense as one of the most active species against the stored grain pest tribolium castaneum (herbst) (coleoptera). the study demonstrated that acetone extracts from the aerial parts induced a high larval mortality rate of 78.7%. furthermore, methanolic extracts exhibited significant growth-regulating activity in pupae, resulting in 51.4% mortality and distinct hormonal disruptions. conclusion this study provides the first detailed assessment of solanum bonariense in algeria, revealing its clear morphological distinctiveness and its successful naturalization across the northern coastal region. its presence in both ruderal and semi-natural mediterranean habitats, combined with traits such as perennial growth, extended flowering, and tolerance to drought and disturbance, indicates a strong potential for further spread. dense, regenerating populations (particularly in oran, nw algeria) indicate that the species is already transitioning toward invasive behavior. the ecological trajectory of s. bonariense parallels that of other invasive solanum species in the mediterranean basin and highlights the need for early detection, rapid removal of small populations, and targeted monitoring of high-risk corridors. without timely management, the species is likely to contribute to habitat homogenization, competition with native shrublands, and increased impacts on agriculture and rangelands. at the same time, s. bonariense holds notable biotechnological value. this dual ecological and bioprospecting significance underscores the importance of integrating invasion management with continued investigation of the species’ medicinal and agrochemical potential. acknowledgments: the authors are grateful to the ministry of higher education and scientific research, algeria. the research was conducted as part of the prfu research project d00l02un050220220001 (university of batna 2, algeria). biologica nyssana ● 16 (2) december 2025: mansouri et al. ● emergence of solanum bonariense l. 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(2024). invasion stage and competition intensity co-drive reproductive strategies of native and invasive saltmarsh plants: evidence from field data. science of the total environment, 954, 176383. https://doi.org/10.1016/j. scitotenv.2024.176383 trajchevski et al. 2025, biologica nyssana 16(2) 16 (2) december 2025: doi: 10.46793/biolnyss.16.2.11k the association of vitamin d, vitamin d-binding protein, and ferritin levels in iraqi men with type 2 diabetes original article mohammed jawad kadhim department of chemistry, institute of graduate studies, near east university, nicosia, cyprus mohammedjawadkd@hotmail.com (corresponding author) süleyman aşir department of chemistry, institute of graduate studies, near east university, nicosia, cyprus amer hasan department of chemistry, college of science, almustansiriya university, baghdad, iraq received: july 19, 2025 revised: october 01, 2025 accepted: october 02, 2025 abstract: this study investigated the relationship between vitamin d, vitamin d-binding protein (vdbp), and ferritin in iraqi men with type 2 diabetes (t2d). a total of 120 participants, comprising 60 diabetic patients and 60 healthy controls, aged 30 years and older, were matched for normal body mass index. glucose levels were significantly elevated in the diabetic group (p=0.001), confirming impaired glycemic regulation. liver function markers, including total serum bilirubin (tsb) and glutamate oxaloacetate transaminase (got), were also significantly higher (p=0.015 and p=0.009, respectively), suggesting hepatic dysfunction. although vitamin d3 concentrations did not differ significantly, reduced vitamin d bioactivity may result from liver impairment and significantly lower vdbp levels in diabetic individuals (p=0.001), compromising its transport to target tissues. no significant differences were found in ferritin and renal markers. these findings highlight the potential role of disrupted vitamin d metabolism, particularly in transport and activation— in the pathogenesis of type 2 diabetes (t2d). key words: vitamin d, vitamin d binding protein (vdbp), ferritin, type 2 diabetes apstrakt: povezanost nivoa vitamina d, proteina koji vezuje vitamin d i feritina kod iračkih muškaraca sa dijabetesom tipa 2 ova studija je ispitivala odnos između vitamina d, proteina koji vezuje vitamin d (vdbp) i feritina kod iračkih muškaraca sa dijabetesom tipa 2 (t2d). ukupno je učestvovalo 120 ispitanika — 60 pacijenata sa dijabetesom i 60 zdravih kontrola starijih od 30 godina, sa normalnim indeksom telesne mase. nivoi glukoze bili su značajno povišeni u grupi sa dijabetesom (p=0.001), što potvrđuje poremećenu regulaciju glikemije. markeri funkcije jetre, uključujući ukupni serumski bilirubin (tsb) i glutamat-oksaloacetat transaminazu (got), takođe su bili značajno viši (p=0.015 i p=0.009), što ukazuje na disfunkciju jetre. iako koncentracije vitamina d3 nisu pokazale značajne razlike, smanjena biološka aktivnost vitamina d može biti posledica oštećenja jetre i značajno nižih nivoa vdbp kod dijabetičara (p=0.001), što narušava njegov transport do ciljnih tkiva. nisu pronađene značajne razlike u nivou feritina i bubrežnim markerima. ovi nalazi naglašavaju potencijalnu ulogu poremećenog metabolizma vitamina d, posebno njegovom transportu i aktivaciji, u patogenezi dijabetesa tipa 2 (t2d). ključne reči: vitamin d, vezujući protein vitamina d (vdbp), feritin, dijabetes tipa 2 introduction type 2 diabetes (t2d) has become one of the most prevalent metabolic disorders in modern society, driven by sedentary lifestyles, poor dietary habits, and urbanization (kakil & meena, 2020). globally, it presents significant public health challenges due to its progressive nature and multi-organ complications (antar et al., 2023). in iraq, the rising incidence— particularly among men—underscores the urgent need for deeper investigation into contributing physiological and biochemical factors (world health organization, 2020). recent studies have highlighted a strong correlation between vitamin d deficiency and the onset of type 2 diabetes (t2d) (zhao et al., 2024). vitamin d plays a pivotal role in regulating immune responses, promoting calcium absorption, and supporting the differentiation and protection of various cell types (janoušek et al., 2022). as a fatsoluble hormone, its bioavailability and metabolic function heavily rely on vitamin d binding protein © 2025 kadhim et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. (vdbp), which transports vitamin d metabolites throughout the bloodstream (wu et al., 2023; abu-shana et al., 2024). evidence suggests that inadequate levels of vitamin d can impair glucose metabolism and insulin sensitivity, two key processes in the pathophysiology of t2d (zhang et al., 2020). furthermore, deficiencies in vitamin d have been linked to reduced pancreatic β-cell activity, which compromises insulin secretion and exacerbates insulin resistance (szymczak-pajor & śliwińska, 2019). despite iraq’s abundant sunshine, many individuals—especially older males—exhibit low serum levels of vitamin d, a paradox that may reflect underlying metabolic or organ-specific dysfunction, such as impaired liver hydroxylation or kidney conversion of inactive vitamin d into its biologically active form (salim et al., 2023). the current study aims to evaluate the levels of serum vitamin d, vdbp, and ferritin in iraqi males diagnosed with t2d, compared to healthy controls matched for age, body mass index (bmi), and lifestyle factors. the study excludes individuals with existing kidney, liver, or thyroid disorders, ensuring a focused analysis on diabetes-related variables. through this investigation, we aim to generate region-specific biochemical data that may guide preventive, diagnostic, and therapeutic approaches. an enhanced understanding of vitamin d’s role and transport mechanisms could provide novel strategies for managing type 2 diabetes (t2d), particularly in populations with limited access to specialized care. moreover, findings may serve as a foundation for future research into the interplay between micronutrient metabolism and chronic disease. materials and methods this study employed a comparative design to investigate biochemical variables associated with t2d, with a focus on vitamin d, vdbp, and ferritin. a total of 120 iraqi male participants, all aged ≥30 years, were carefully selected based on stringent inclusion criteria. among them, 60 were clinically diagnosed with t2d and were managing their condition with oral hypoglycemic medication. the control group consisted of 60 healthy males who were matched in age and maintained a bmi between 19 and 24 kg/m². all individuals underwent preliminary interviews to confirm eligibility and received written information outlining study details. informed consent was obtained from each participant in accordance with research ethics protocols. blood samples were collected from each subject after an overnight fast ranging from 8 to 12 hours, at al-qanah medical laboratory in baghdad, iraq, between 15 january and 1 march 2025. samples were processed promptly following standard clinical guidelines. laboratory analyses approximately 5 ml of blood was drawn from each participant using sterile needles. the collected blood was placed in gel tubes designed to separate serum through centrifugation. after separation, the serum was transferred using micropipettes into cuvette tubes for analysis. serum biomarker levels were assessed using standardized clinical analyzers and kits. insulin, ferritin, and vitamin d concentrations were determined using the cobas e 411 analyzer and diagnostic kits manufactured by roche (germany). vitamin d binding protein (vdbp) levels were measured via immunoassay kits obtained from shanghai coon koon biotech co., ltd (china). glucose levels were evaluated using glucosespecific test kits, while renal function indicators— blood urea and serum creatinine—were analyzed using renal function kits. liver function biomarkers, including total serum bilirubin (tsb), glutamate pyruvate transaminase (gpt), glutamate oxaloacetate transaminase (got), and alkaline phosphatase (alp), were quantified using liver function kits. all biochemical reagents for glucose, renal, and hepatic assessments were provided by elitech clinical systems sas (france). participant height and weight were recorded using the anker eufy smart scale p2 pro, which includes an integrated tape measure for height calculations. bmi was computed using the equation: bmi = weight in kilograms /height in square meters. insulin resistance was measured using the homa-ir equation: homa-ir = [glucose (mg/dl) × insulin (mu/l)] ÷ 405. for reference, 18 mg/dl ≈ 1 mmol/l. a homa-ir value ≥2.5 typically indicates insulin resistance, as it is associated with impaired insulin sensitivity (crook & godsland, 1998; porchia et al., 2024). it should be noted that individual factors such as age, weight, and health status may influence reference values (polonsky & burant, 2015). as for determining vdbp concentrations, absorbance was measured using a uquant bio-tek microplate spectrophotometer (usa). temperature stability and sample preservation were maintained using a laboratory refrigerator and incubator. statistical analysis all data obtained were analyzed using spss, medcalc, and graphpad prism for descriptive statistics and inferential comparisons. the gen5 – biotek software was used in conjunction with the microplate reader to record optical density and biologica nyssana ● 16 (2) december 2025: kadhim et al. ● the association of vitamin d, vitamin d-binding protein, and ferritin levels in iraqi men with type 2 diabetes biologica nyssana ● 16 (2) december 2025: kadhim et al. ● the association of vitamin d, vitamin d-binding protein, and ferritin levels in iraqi men with type 2 diabetes calculate vdbp concentrations using a quadratic fit model. a p-value of <0.05 was considered statistically significant across tests. the rigorous design and use of matched samples enhance the validity and reproducibility of the findings, aligning with established research protocols and literature standards. results and discussion the demographic and anthropometric characteristics of the participants are presented in tab. 1. the mean age of the control group was 35.52 years (±2.81), and 35.25 years (±3.24) for the t2d group. no significant difference was observed (p=0.631), indicating age homogeneity between groups. mean body weight also showed similarity, measured at 65.65 kg (±4.96) in controls and 65.07 kg (±4.78) in t2d patients (p=0.513). in terms of height, however, a statistically significant difference was noted (p=0.020), with mean values of 1.741 m (±0.047) in controls and 1.717 m (±0.047) in the diabetic group. bmi values were closely aligned, with means of 21.62 kg/m² (±1.26) and 22.06 kg/ m² (±1.51) respectively, and the difference was not statistically significant (p=0.085), in accordance with the study’s inclusion criteria (bmi 19–24 kg/m²). fig. 1. illustrates a near-balanced bmi distribution, with 49% of participants belonging to the control group and 51% to the diabetic group. the second graph shows that participant distribution across ages was evenly split, with both control and diabetic groups comprising 50% of the sample. tab. 2. summarizes t2d biochemical variables. glucose levels were significantly elevated in the t2d group, with a mean of 152.32 mg/dl (±40.00) compared to 87.67 mg/dl (±5.94) in the control group (p=0.001). this confirms the expected glycemic dysregulation in patients with diabetes. insulin levels showed no significant difference between groups, with values of 12.60 mu/l (±5.23) in the t2d group and 11.63 mu/l (±3.89) in the control group (p=0.252). however, insulin resistance was markedly higher in diabetic subjects, control group, n=60 t2d patients, n=60 parameters mean sd mean sd p-value age (year) 35.52 2.8134 35.25 3.245 0.631 weight (kg) 65.65 4.957 65.067 4.779 0.513 height (m) 1.741 0.047 1.717 0.047 0.02* bmi (kg/m2) 21.618 1.258 22.058 1.508 0.085 table 1. the statistical disparities among control and t2d patients for age and anthropometric measurements *significant at p≤0.05, **highly significant p<0.001, ns: non-significant with a mean value of 4.72 (±2.34) compared to 2.52 (±0.85) in controls (p=0.001), underscoring its role in the pathophysiology of t2d. ferritin levels did not differ significantly between groups; control group had a mean of 61.32 ng/ml (±16.08), figure 1. distribution of age and bmi between t2d patients and the control group biologica nyssana ● 16 (2) december 2025: kadhim et al. ● the association of vitamin d, vitamin d-binding protein, and ferritin levels in iraqi men with type 2 diabetes 2 diabetes (152.32±40.00 mg/dl) compared to the non-diabetic control group (87.67±5.94 mg/dl), with the difference reaching high statistical significance (p<0.001). this hyperglycemic state is indicative of impaired glucose homeostasis, primarily driven by insulin resistance, a central mechanism in the pathogenesis of t2d. the finding aligns with established literature recognizing persistently elevated glucose levels as a key clinical marker in the diagnosis and progression of type 2 diabetes. although insulin levels did not differ significantly between the diabetic and control groups (12.60±5.23 mu/l vs. 11.63±3.89 mu/l; p=0.252), this does not reflect practical metabolic function in t2d patients. despite near-normal circulating insulin levels, the elevated blood glucose observed in the diabetic group suggests underlying insulin resistance. this condition impairs the hormone’s ability to facilitate glucose uptake in peripheral tissues, a hallmark of type 2 diabetes. previous studies have similarly reported that individuals with t2d may exhibit normal or even elevated insulin concentrations due to compensatory β-cell activity. however, the presence of insulin alone is not indicative of glycemic control in the context of resistance. consistent with this, the current study demonstrated a significant rise in insulin resistance among diabetic participants (4.72±2.34) compared to controls (2.52±0.85), with a p-value < 0.001. these findings reinforce the clinical understanding that insulin resistance plays a central role in the pathogenesis and progression of t2d (american diabetes association, 2018). no statistically significant difference in serum ferritin levels was observed between type 2 diabetes patients and the control group (64.65±19.32 ng/ ml vs. 61.32±16.08 ng/ml; p=0.307). this result while t2d patients had a mean of 64.65 ng/ml (±19.32) (p=0.307), suggesting limited relevance in differentiating disease status. tab. 3. presents the serum concentrations of vitamin d3 and vdbp parameters in both study groups. the mean vitamin d3 level in the control group was 26.63 ng/ml (±14.68), slightly higher than in the t2d group, which averaged 22.11 ng/ml (±12.55). although the diabetic group demonstrated lower levels, the difference was not statistically significant (p=0.072). vdbp levels were significantly reduced in the diabetic group, averaging 135.71 ng/ ml (±20.29) compared to 159.66 ng/ml (±28.36) in controls (p=0.001), suggesting impaired vitamin d transport capacity. liver function results (tab. 4.) indicate that tsb was significantly higher in t2d patients (0.83 mg/dl ± 0.087) compared to the control group (0.79 mg/dl ± 0.090), with a p-value of 0.015. no significant difference was observed in gpt levels (t2d: 30.42±5.91 iu/l; control: 31.38±6.02 iu/l; p=0.377). however, got levels were significantly elevated in t2d patients (29.38±5.52 iu/l) compared to controls (26.73±5.46 iu/l), with a p-value of 0.009. alp levels did not differ significantly between the groups (t2d: 78.70 iu/l ±9.97; control: 76.63 iu/l ±10.84; p=0.279). kidney function assessments (tab. 5.) showed no significant differences. blood urea levels were comparable between t2d (31.85±4.70 mg/dl) and control subjects (32.05±4.25 mg/dl; p=0.807). similarly, serum creatinine values were consistent, with t2d patients recording 0.76 mg/dl (±0.12) and controls at 0.73 mg/dl (±0.13), yielding a non-significant p-value of 0.185. a substantial elevation in fasting blood glucose was identified in individuals with type control group, n=60 t2d patients, n=60 parameters mean sd mean sd p-value glucose (mg/dl) 87.666 5.942 152.316 40.004 0.001** insulin (mu/l) 11.633 3.894 12.601 5.229 0.252 insuline resistance 2.516 0.852 4.722 2.338 0.001* ferritin (ng/ml) 61.316 16.082 64.648 19.315 0.307 table 2. the statistical disparities among control and t2d patients for t2d parameters *significant at p≤0.05, **highly significant p<0.001, ns: non-significant control group, n=60 t2d patients, n=60 parameters mean sd mean sd p-value vita. d3 (ng/ml) 26.632 14.682 22.106 12.554 0.072 vdbp (ng/ml) 159.66 28.362 135.71 20.286 0.001** table 3. the statistical disparities among control and t2d patients for vitamin d3 and vdbp *significant at p≤0.05, **highly significant p<0.001, ns: non-significant biologica nyssana ● 16 (2) december 2025: kadhim et al. ● the association of vitamin d, vitamin d-binding protein, and ferritin levels in iraqi men with type 2 diabetes in diabetic populations and correlates with poorer clinical outcomes, supporting its role in both disease pathogenesis and therapeutic monitoring (arafat et al., 2020). the current study revealed a highly significant reduction in vdbp concentrations among patients with type 2 diabetes (135.71±20.29 ng/ml) compared to the control group (159.66±28.36 ng/ ml), with a p-value of 0.001. this decline in vdbp may compromise the transport efficiency of vitamin d metabolites in the bloodstream, potentially reducing their bioavailability and limiting cellular uptake at target sites. such disruption could contribute to the metabolic dysfunction associated with t2d, despite the presence of measurable vitamin d levels. notably, these findings diverge from those reported, which found no significant differences in vdbp levels between individuals with diabetes and those without diabetes. the discrepancy may be attributed to population-specific factors or differences in study design, underscoring the need for further investigation into vdbp’s role in vitamin d metabolism and diabetes pathogenesis (arafat et al., 2020). analysis of liver function parameters revealed several biochemical distinctions between diabetic patients and control subjects. tsb levels were modest but significantly elevated in the diabetic group (0.833±0.087 mg/dl) compared to controls (0.793±0.090 mg/dl), with a p-value of 0.015. this aligns with findings that elevated bilirubin may be linked to a higher risk of developing type 2 diabetes. similarly, got levels were significantly higher in t2d patients (29.38±5.52 iu/l) than in the control group (26.73±5.46 iu/l; p=0.009), suggesting potential hepatic stress or dysfunction, which has been implicated in the pathogenesis of diabetes. aligns with the findings, which reported that ferritin concentrations were not associated with glycemic control in t2d patients (al argan et al., 2023). previous research has demonstrated a correlation between elevated ferritin levels and an increased risk of t2d. however, it remains unclear whether this elevation plays a causal role in the onset of diabetes or merely reflects the progression of insulin resistance, a recognized precursor to t2d (podmore et al., 2016). additionally, it was found that individuals with t2d exhibited increased ferritin levels, which may be linked to prolonged hyperglycemia, showing a strong positive correlation with glycemic markers such as fasting blood glucose and hba1c (bayih et al., 2024). taken together, these findings suggest that while ferritin may play a role in metabolic disturbance, its diagnostic value in t2d requires further clarification. although the observed differences in serum vitamin d3 concentrations between the diabetic group (22.11±12.55 ng/ml) and the control group (26.63±14.68 ng/ml) did not reach statistical significance (p=0.072), this does not preclude a clinical impact. the biological activity of vitamin d3 depends on its conversion to the active form, a process influenced by vdbp, tsb, and liver enzyme levels such as got. disruptions in these factors—common in individuals with t2d—may contribute to reduced levels of active vitamin d3, thereby impairing glucose metabolism and insulin sensitivity. a study reported that vitamin d deficiency is associated with deteriorating glycemic control (pittas et al., 2007). furthermore, longitudinal data suggest that low vitamin d status may predict the future onset of t2d (forouhi et al., 2008). it was also highlighted that vitamin d deficiency is prevalent control group, n=60 t2d patients, n=60 parameters mean sd mean sd p-value tsb (mg/dl) 0.793 0.09 0.833 0.087 0.015* gpt (iu/l) 31.38 6.023 30.417 5.907 0.377 got (iu/l) 26.73 5.455 29.383 5.521 0.009* alp (iu/l) 76.63 10.84 78.7 9.968 0.279 table 4. the statistical disparities among control and t2d patients for liver function parameters *significant at p≤0.05, **highly significant p<0.001, ns: non-significant control group, n=60 t2d patients, n=60 parameters mean sd mean sd p-value urea (mg/ml) 32.05 4.252 31.85 4.697 0.807 creatinine (mg/ml) 0.73 0.125 0.76 0.121 0.185 table 5. the statistical disparities among control and t2d patients for renal function parameters *significant at p≤0.05, **highly significant p<0.001, ns: non-significant progression and highlight that diminished vitamin d bioavailability, despite normal serum levels, may stem from compromised liver function and reduced vdbp. together, these findings underscore the importance of evaluating not only vitamin d levels but also its transport and activation pathways in understanding and managing t2d. references abu-shana, j. h., abdulkareem, n. g., jasim, a. o., & abbood, y. h. 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(1998). safety evaluation by contrast, no statistically significant differences were found in gpt and alp levels between the two groups. nonetheless, previous research indicates that elevated gpt and alp may arise from liver conditions such as hepatitis or biliary disorders. it may also be linked to abnormalities in bone metabolism or long-term hyperglycemia (rafaqat et al., 2023). these parameters, relevant in specific metabolic or comorbid contexts. importantly, liver function plays a crucial role in the hydroxylation and activation of vitamin d. any hepatic impairment can disrupt this conversion, potentially reducing active vitamin d availability and contributing to the progression of t2d. the observed elevations in tsb and got levels may reflect subtle liver dysfunction in diabetic individuals, underscoring the interconnected nature of hepatic health and vitamin d metabolism. renal function analysis revealed no statistically significant differences in urea or creatinine levels between participants with diabetes and those without diabetes. urea concentrations were comparable between the t2d group (31.85±4.70 mg/dl) and the control group (32.05±4.25 mg/ dl; p=0.807). similarly, creatinine levels showed minimal variation, with the diabetic group averaging 0.76±0.12 mg/dl and the control group averaging 0.73±0.13 mg/dl (p=0.185). these findings suggest that renal impairment was not apparent within the study population. however, this contrasts with prior research indicating that elevated urea and creatinine are commonly observed in patients with t2d, reflecting progressive kidney damage (lad & kava, 2019). a high prevalence of renal dysfunction has also been documented among individuals with diabetes, highlighting the importance of early screening and ongoing monitoring of kidney health in this population (shahwan et al., 2019). it is essential to note that the kidneys play a crucial role in converting vitamin d into its active form. any decline in renal function may compromise vitamin d activation, thereby reduce its physiological effectiveness and increase susceptibility to metabolic disturbances, such as type 2 diabetes. conclusion in conclusion, this study demonstrates that while vitamin d3 levels did not differ significantly between diabetic and non-diabetic iraqi men, functional disruptions in vitamin d transport and activation—reflected in altered vdbp concentrations and liver functions—may contribute to impaired metabolic regulation in type 2 diabetes. the data affirm the central role of insulin resistance in disease biologica nyssana ● 16 (2) december 2025: kadhim et al. ● the association of vitamin d, vitamin d-binding protein, and ferritin levels in iraqi men with type 2 diabetes of modern oral contraceptives: effects on lipoprotein and carbohydrate metabolism. contraception, 57(3), 189–201. https://doi.org/10.1016/s00107824(98)00018-3 forouhi, n. g., luan, j., cooper, a., boucher, b. j., & wareham, n. j. 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(2020). effects of vitamin d supplementation on prevention of t2d in patients with prediabetes: a systematic review and meta-analysis. diabetes care, 43(7), 1650–1658. https://doi.org kadhim et al. ● the association of vitamin d, vitamin d-binding protein, and ferritin levels in iraqi men with type 2 diabetes biologica nyssana ● 16 (2) december 2025: perišić et al. 2023, biologica nyssana 14(2) 14 (2) december 2023: 117-124 doi: 10.5281/zenodo.10203102 differences in the relationship of the most important traits of bread wheat depending on the different agro-ecological conditions original article vesna perišić faculty of agriculture, university of niš, kosančićeva 4, kruševac, serbia vperisic@kg.ac.rs (corresponding author) vera rajičić faculty of agriculture, university of niš, kosančićeva 4, kruševac, serbia kristina luković institute for vegetable crops smederevska palanka, karađorđeva 71, smederevska palanka, serbia filip vukajlović faculty of science, university of kragujevac, radoja domanovića 12, kragujevac, serbia vladimir perišić institute for vegetable crops smederevska palanka, karađorđeva 71, smederevska palanka, serbia received: september 25, 2023 revised: october 18, 2023 accepted: november 04, 2023 abstract: different agro-ecological conditions have a great influence on the phenotypical expression of yield components and grain yield. besides this, their relationships are changed, too. in extreme wet conditions of the year, with large amounts of precipitations during the most important development stages, phenotypic differences and the relationship between the most important yield components and grain yield are less pronounced. coefficients of variation, correlation analysis, and cluster analysis were used to determine the performances of advanced wheat genotypes under different agro-climatic conditions of research locations. in this research, the highest degree of variation, i.e. coefficients of variation for spikes mass and grain mass per plant from localities kragujevac, kruševac, and sombor were expressed. compared with the other two localities, the locality sombor is distinguished by a strong and positive degree of correlations between yield components and grain yield. key words: wheat, yield components, grain yield, correlations apstrakt: međuzavisnost najvažnijih osobina hlebne pšenice u različitim agroekološkim uslovima različiti agroekološki uslovi, u velikoj meri, utiču na fenotipsku ekspresiju komponenti rodnosti i prinosa zrna. pored toga, odnosi između njih se menjaju, takođe. u godini sa velikim količinama padavina i ekstremno vlažnim uslovima tokom najvažnijih faza razvoja, fenotipske razlike i odnosi između najznačajnijih komponenti rodnosti i prinosa zrna su manje izraženi. koeficijent varijacije, korelaciona analiza i klaster analiza su korišćene u cilju određivanja ponašanja perspektivnih genotipova pšenice u različitim agroklimatskim uslovima ispitivanih lokaliteta. u ovom istraživanju, najviši stepen varijacije, tj. koeficijent varijacije, utvrđen je za masu klasa i masu zrna po biljci na lokalitetima kragujevac, kruševac i sombor. u poređenju sa ostala dva lokaliteta, lokalitet sombor se odlikovao najvećim brojem pozitivnih i negativnih korelacija između komponenti rodnosti i prinosa zrna. ključne reči: pšenica, komponente rodnosti, prinos zrna, korelacije introduction compared to other plant species, bread wheat (triticum aestivum l.) is characterized by a genetically complex constitution, composed of three homologous genomes (abd), with a high number of repetitive sequences and homologous dna copies (guan et al., 2020). the hexaploid genomic nature of wheat with expressed multiple allelism for the most quantitative traits distinguishes a high degree of interactions between homoeologous genes and chromosomes. the result is a wide range in the degree of gene expression, which provides wide adaptability of production features in different agroecological conditions (dubcovsky & dvorak, 2007). the wheat breeding process is usually focused on genetic gain responsible for grain yield and yield components (spike number per surface unit, spike length, spikelets number per spike, kernel number per spike, 1000 grain weight, etc.). future selection is mostly determined by current and upcoming climatic © 2023 perišić et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 117 changes, where the drought represents one of the key factors. on the other hand, an extreme amount of precipitation in certain years, during intensive wheat development, can represent a limiting factor for yield and technological quality of grain (luković et al., 2020). the wheat yield is a complex character representing a function of several component features and their interaction with the environment. examination of yield structure involves the assessment of relationship among various characters contributing to the yield (kadam et al., 2022). analysis of correlations among traits with a potential impact on grain yield is of great importance for the process of selection that takes place within a breeding program. among the many methods potentially utilized for this purpose, the analysis of correlation coefficients is one of the most widely used ones (hristov et al., 2011; nukasani et al. 2013). a study was conducted to estimate the relationship between the most important yield components and grain yield of advanced wheat genotypes under different agro-climatic conditions of research locations and the degree of their variability. the wheat genotypes chosen during the experiment would be used as initial material for further improvement of grain yield. materials and methods the experimental part of the study was performed during 2018/2019 in three localities: institute of forage crops kruševac, agroinstitute in sombor, and centre for small grains kragujevac. the plant material consisted of 14 perspective lines of winter wheat created in the center from kragujevac (1 kg-27/6; 2 kg-244/4; 3 kg-199/4; 4 kg-307/4; 5 kg-331/4; 6 kg-28/6; 7 kg-162/7; 8 kg191/5-13; 9 kg-40-39/3; 10 kg-52/23; 11 kg60-3/3; 12 kg-1/6; 13 kg-52/3; 14 kg-47/21 and 15 cultivar „pobeda”). the experiment was conducted in field conditions with the application of a completely random block system, in three replications with the size of the basic plot of 5 m2 (5 x 1 m). the machine sowing was conducted with 600-650 germinated grains per m2 depending on the characteristics of the genotype. a total of 45 plants (three replications with 15 plants) was selected from each examined genotype per locality, in order to analyze the following morphological and productive traits: number of productive spikes per m2 (nps), plant height (ph), spike length (sl), number of spikelets per spike (nss), spike mass (sm), spikes mass per plant (smp), grain number per spike (gns), grain mass per spike (gms), grain mass per plant (gmp), hectoliter mass (hm), thousand-grain weight (tw), and grain yield (y). after the harvest, grain yield (y) was measured for each plot in three replications and then converted to t/ha. specificities of all three localities used in the research were represented by the most important climatic characteristics average air temperatures and precipitation values during the period of investigation, as well as in the period of 15 years (fig. 1). temperature degrees were not a limiting factor during the development of examined genotypes, since small differences in their average values were recorded between the investigation period and multi-year period. the main climate characteristic was an uneven arrangement of precipitation amounts during different months. during february, march and april, low values were recorded in all three locations, while extremely large precipitation values, compared with 15-year average ones, were noticed in kragujevac and sombor during may and june. coefficients of variation of examined traits were calculated in microsoft excel representing a percentage relationship between the average and standard deviation value of the analyzed trait. relative dependencies between yield components and grain yield were defined through correlation analysis (pearson’s correlation coefficient), while the obtained coefficients were tested at the 5% and 1% levels of significance. in order to group similar genotypes, the euclidean distance was used as a measure of the similarity of potential clusters. data were standardized (reduced to zero mean and unit deviation) and statistically processed by minitab 20 and spss 20 software packages. results and discussion between examined genotypes from the different localities, wheat genotype kg-331/4 achieved the highest value of average grain yield in kragujevac, ranging from 3.870 t/ha to 5.250 t/ha. the highest average grain yield values of the genotype kg-199-4 were found in kruševac, with a yield range of 3.130 5.400 t/ha. the best wheat genotype in sombor was kg-60-3/3 with grain yield values ranging from 4.100 t/ha to 7.200 t/ha. average values and coefficients of variation (%) of examined yield components and grain yield in investigated locations (kragujevac ‒ 1, kruševac ‒ 2, and sombor ‒ 3) are represented in tab. 1. most yield components had the highest average values in kragujevac (spike length, spike mass, spikes mass per plant, grain number per spike, grain mass per spike, grain mass per plant). this statement can be explained by the fact that kragujevac represents the 118 biologica nyssana ● 14 (2) december 2023: 117-124 perišić et al. ● differences in the relationship of the most important traits of bread wheat depending on the different agro-ecological conditions biologica nyssana ● 14 (2) december 2023: 117-124 perišić et al. ● differences in the relationship of the most important traits of bread wheat depending on the different agro-ecological conditions 119 revealing of genetic determinants of plant development becomes urgent, due to global climate change, which can seriously affect and even disrupt the locally adapted developmental patterns (horvath et al., 2023). the polygenic nature of grain yields as well as the large influence of the environment and the complex relationship among yield component traits represent challenges in wheat selection (cao et al., 2020). the impact of different management practices on yield components and their role in yield modulation are not well understood (jaenisch et al., 2022). the highest degree of variation, i.e. coefficients of variation (cv) for spikes mass per plant (16.81, 23.87, and 17.10, respectively) and grain mass per plant (16.38, 22.17, and 18.02, respectively) on localities kragujevac, kruševac, and sombor were expressed since a large amount of precipitation was recorded during grain filling phase. similar findings were pointed out by sokoto et al. (2012), nukasani et al. (2013), and haydar et al. (2020), while kozdój et al. (2015) recorded higher values for coefficients of variation. as a consequence of large soil heterogeneity and differences in the water table 1. average values and coefficient of variation (%) of examined yield components and grain yield in investigated locations fig. 1. average values of air temperatures and precipitations during 2018/2019 and 15-year period (20052019) in kragujevac (a), kruševac (b), and sombor (c) place of selection of all examined wheat genotypes. since environmental factors (soil especially) had the most limited influence in this locality (luković et al., 2020), examined genotypes stabilized their expression of yield components and grain yield at similar levels. on the contrary, in the other two localities, where the conditions were nearest to the optimal ones, a high degree of differentiation of the wheat genotypes was noticed, especially in a grain yield domain. sombor genotype was distinguished by the highest average values of the number of productive spikes per m2 (626) and grain yield (5.111 t/ha), influenced by optimal fertility and better water regimes of the soil. the highest value of the plant height (93.06 cm) is measured in plants from the locality of kruševac. unlike the other two localities with large amounts of precipitation, in this locality, wheat genotypes had optimal conditions of soil humidity during the phase of stem elongation, in april and may especially. besides having an impact on ecological adaptation, the dynamics of plant development also contribute to the realization of genetically determined yield potentials in various environments. location nps* ph (cm) sl (cm) nss sm (g) smp (g) gns gms (g) gmp (g) hm (kg/ hl) tw (g) y (t/ha) average values ( ) 1 580 91.01 10.23 21.58 2.41 7.66 47.67 1.82 5.33 75.67 40.57 4.438 2 594 93.06 9.99 21.60 1.82 5.51 41.75 1.36 3.97 75.67 39.44 4.449 3 626 88.22 9.92 20.68 1.91 5.84 44.21 1.52 4.48 75.61 40.51 5.111 coefficient of variation 1 10.80 9.57 10.67 8.06 13.34 16.81 17.44 21.04 16.38 1.87 4.58 12.61 2 9.00 9.55 10.39 5.09 12.36 23.87 9.94 13.34 22.17 2.68 3.96 14.51 3 10.78 9.96 10.86 6.12 13.22 17.10 9.90 14.56 18.02 3.33 4.97 17.57 *nps number of productive spikes per m2; ph plant height; sl spike length; nss number of spikelets per spike; sm spike mass; smp spike mass per plant; gns grain number per spike; gms grain mass per spike; gmp grain mass per plant; hm hectoliter mass; tw – thousand-grain weight; y grain yield 120 biologica nyssana ● 14 (2) december 2023: 117-124 regime, grain mass per spike and grain number per spike expressed higher cv values in the locality of kragujevac. besides this, the highest cv for grain yield is recorded in sombor, while the smallest variation was noticed in kragujevac. hectoliter mass was the character that showed the lowest value of coefficients of variations. matković et al. (2022) examined the variability of grain yield and its components on different soil types, and they recorded the lowest values for the coefficient of variation for thousand-grain weight and number of grains per spike. on the other hand, coefficient of variation values were higher for the number of grains per spike on the less fertile soil, due to the more pronounced influence of ecological variance. amin et al. (2015) pointed out that phenotypic selection of the number of grains per spike may be unsuccessful due to the pronounced influence of the non-additive source of variation. in the locality of kragujevac (tab. 2), none of the examined yield components showed a statistically significant degree of correlation with grain yields. the primary effect of precipitation change on grain yield is expressed through growth limitations, with a positive relationship between average yields and precipitation at all sites (pirttioja et al., 2015). this phenomenon was strongly emphasized at temperature levels close to the baseline and progressively reduced under the conditions of increased warming. compared to the other examined localities, soil represented the most limiting factor in kragujevac. spike mass was expressed with spike mass per plant (0.78), grain mass per spike (0.76), and grain mass per plant (0.77). plant height revealed a modest, statistically significant, positive correlation with spike mass and hectoliter mass (0.57 and 0.52, respectively). a statistically highly significant coefficient of correlation (0.98) was confirmed between the characters spike mass per plant and grain mass per plant. in the locality of kruševac, grain yield expressed a statistically significant positive correlation with the number of productive spikes per m2 (0.53). the highest coefficient of correlation (0.97), statistically very significant, is recorded between spike mass and grain mass per spike, as well as between spike mass per plant and grain mass per plant. the results of the investigation are in accordance with those published by mohammadi et al. (2012), philipp et al. (2018), and baye et al. (2020). hectoliter mass and thousand-grain weight showed moderate positive correlation with spike mass, spike mass per plant, grain mass per spike, and grain mass per plant. thousand-grain weight expressed a modest, statistically significant, positive correlation with spike mass, spike mass per plant, grain mass per spike, and grain mass per plant. the number of spikelets per spike revealed a similar, statistically significant correlation with spike length and grain mass per plant. stressful environmental conditions reduced values of all traits in all analyzed genotypes, at the same time reducing their genetic potential. in favorable environmental conditions, grain weight per spike, followed by the number of grains per spike and spike weight, had the greatest and most direct positive effect on grain yield (matković stojšin et al., 2022). compared with the other two localities, sombor is distinguished by a great number of strong and positive degrees of correlation between yield components and grain yield. it can be explained by the fact that in the optimal environmental conditions as in the locality of sombor, examined wheat genotypes expressed the maximum of their genetic potential. grain yield showed a statistically very significant, positive correlation with grain mass per plant (0.89), spike mass per plant (0.87), grain mass per spike (0.75), number of spikelets per spike (0.68), number of productive spikes per m2 (0.67), and spike mass (0.67). thousand-grain weight revealed a very significant positive linkage compared to spike mass and grain mass per spike (0.79), grain mass per plant (0.61), spike mass per plant (0.59), and plant height. at the same locality, the highest degree of correlation (0.99) is recorded between spike mass and grain mass per spike, as well as between spike mass per plant and grain mass per plant. similar results were published by ayer et al. (2017), ullah et al. (2021) and matković et al. (2022) as well. correlative dependence between traits showed large variation depending on the investigated year, resulting from the interaction within the genetic background of each genotype, as well as their interactions with environmental factors (terzić et al., 2018). the authors found a more positive, stronger, and statistically significant correlation during the year with optimal monthly temperature and more proper arrangements of precipitation. this data is in accordance with our findings in the locality of sombor, where yield components expressed a strong and positive correlation with grain yield in the majority of cases. cluster analysis was used for grouping the examined genotypes of winter wheat according to phenotypic similarity of grain components and grain yield (fig. 2). the additional objective of cluster analysis was to provide a general picture of the genotype’s interaction with their environment, while selecting those with a high average value of an analyzed trait (hristov et al., 2011). examined genotypes of winter wheat from all investigated localities were grouped into four major perišić et al. ● differences in the relationship of the most important traits of bread wheat depending on the different agro-ecological conditions biologica nyssana ● 14 (2) december 2023: 117-124 perišić et al. ● differences in the relationship of the most important traits of bread wheat depending on the different agro-ecological conditions 121 table 2. coefficients of correlation of examined yield components and grain yield in kragujevac, kruševac, and sombor traits kragujevac nps ph sl nss sm smp gns gms gmp hm tw ph 0.45 sl -0.06 0.03 nss -0.19 -0.08 0.47 sm 0.38 0.57* -0.01 0.02 smp 0.45 0.34 -0.32 -0.26 0.78** gns 0.26 -0.18 0.16 0.46 0.27 0.40 gms 0.03 0.44 -0.10 -0.05 0.76** 0.43 -0.26 gmp 0.29 0.30 -0.35 -0.29 0.77** 0.98** 0.33 0.52* hm 0.26 0.52* 0.25 -0.33 0.57* 0.54* 0.06 0.25 0.50 tw -0.10 0.24 -0.26 -0.43 -0.10 -0.12 -0.61* 0.24 -0.01 -0.14 y 0.01 0.17 -0.19 -0.01 0.36 0.31 0.08 0.30 0.34 0.20 0.22 kruševac nps ph sl nss sm smp gns gms gmp hm tw ph -0.05 sl -0.34 0.05 nss -0.35 -0.08 0.65** sm 0.14 0.48 0.08 -0.24 smp 0.23 0.63* -0.34 -0.51 0.55* gns 0.40 -0.22 0.073 0.17 0.40 -0.22 gms 0.19 0.43 -0.07 -0.39 0.97** 0.54* 0.36 gmp 0.27 0.52* -0.43 -0.67** 0.53* 0.97** -0.24 0.57* hm 0.07 0.40 0.08 -0.19 0.50 0.48 -0.15 0.44 0.46 tw 0.24 0.23 -0.22 -0.25 0.53* 0.53* 0.28 0.56* 0.53* 0.06 y 0.53* -0.05 -0.35 -0.21 0.34 0.15 0.42 0.38 0.14 0.18 0.46 sombor nps ph sl nss sm smp gns gms gmp hm tw ph 0.15 sl -0.63* 0.02 nss -0.50 0.02 0.71** sm 0.32 0.36 -0.12 -0.47 smp 0.52* 0.40 -0.43 -0.56* 0.84** gns 0.04 -0.33 0.25 0.23 0.22 0.10 gms 0.37 0.32 -0.22 -0.56* 0.99** 0.88** 0.16 gmp 0.53* 0.35 -0.49 -0.63* 0.83** 0.99** 0.06 0.88** hm -0.12 -0.10 0.03 -0.37 0.30 0.22 -0.33 0.36 0.27 tw 0.32 0.52* -0.15 -0.45 0.79** 0.59* -0.07 0.79** 0.61* 0.30 y 0.67** 0.06 -0.56* -0.68** 0.67** 0.87** 0.17 0.75** 0.89** 0.36 0.42 *nps number of productive spikes per m2; ph plant height; sl spike length; nss number of spikelets per spike; sm spike mass; smp spike mass per plant; gns grain number per spike; gms grain mass per spike; gmp grain mass per plant; hm hectoliter mass; tw – thousand-grain weight; y grain yield 122 biologica nyssana ● 14 (2) december 2023: 117-124 perišić et al. ● differences in the relationship of the most important traits of bread wheat depending on the different agro-ecological conditions fig. 2. cluster analysis of examined wheat genotypes in the investigated localities kragujevac (a), kruševac (b), and sombor (c) (*genotypes: 1 kg-27/6; 2 kg-244/4; 3 kg-199/4; 4 kg-307/4; 5 kg-331/4; 6 kg-28/6; 7 kg-162/7; 8 kg-191/5-13; 9 kg-40-39/3; 10 kg-52/23; 11 kg-60-3/3; 12 kg-1/6; 13 kg-52/3; 14 kg-47/21; 15 pobeda) clusters, showing the significant genetic diversity among them for all analyzed traits. in the localities of kragujevac and kruševac, most of the high-yielding genotypes, followed by the highest trait values, are located in the fourth cluster; on the other hand, the dispersion of genotypes throughout different clusters was more equal in sombor. genotypes 1 and 6 as low-yielding genotypes were grouped into the same cluster in all examined localities, pointing out their great similarity and stability in phenotypic expression. the most yielding perspective genotypes of bread wheat were mostly positioned in the third and fourth clusters (kg-60-3/3, pobeda, kg-199/4, and kg-40-39/3). conclusion genetic background determines phenotypic expression of yield components and grain yield as well as interactions between genotype and environmental conditions. wheat genotype kg-331/4 achieved the highest value of average grain yield in kragujevac, ranging from 3.870 t/ha to 5.250 t/ha. genotype kg-199-4 expressed the highest average grain yield in kruševac, with a yield range between genotypes 3.130 5.400 t/ha. most of the yield components had the highest average values in kragujevac (spike length, spike mass, spike mass per plant, grain number per spike, grain mass per spike, grain mass per plant). since kragujevac represents the place of selection, examined genotypes stabilized their expression of yield components and grain yield on similar levels. the highest degree of variation, i.e. coefficients of variation (cv), was confirmed for the characters spike mass per plant (16.81, 23.87, and 17.10, respectively) and grain mass per plant (16.38, 22.17, and 18.02, respectively) in investigated localities (kragujevac, kruševac, and sombor, respectively). compared with the other two localities, sombor wheat is distinguished by a great number of strong and positive degrees of correlation between yield components and grain yield. acknowledgement. the research is financially supported by the ministry of science, technological development, and innovations of the republic of serbia (contract no. 451-0347/2023-01/ 200216). 123 biologica nyssana ● 14 (2) december 2023: 117-124 references amin, m.f., hasan, m., barma, n.c.d., rahman, m.m., & hasan, m.m. 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(2021). grain yield and correlated traits of bread wheat lines: implications for yield improvement. saudi journal of biological sciences, 28, 5714–5719. perišić et al. ● differences in the relationship of the most important traits of bread wheat depending on the different agro-ecological conditions matijević, m., popović, i., stepić, m., nešić, m., radoičić, m., stanković, m., šaponjić, z., petković, m.: inorganic nanoparticles in biology: drug carriers and auxiliary tools in bioimaging and bioanalytics. biologica nyssana, 9 (1). september, 2018: 1-19. biologica nyssana 9 (1) ⚫ september 2018: 1-19 matijević et al. ⚫ inorganic nanoparticles in biology: drug carriers… 1 review article received: 13 april 2018 revised: 27 june 2018 accepted: 22 august 2018 inorganic nanoparticles in biology: drug carriers and auxiliary tools in bioimaging and bioanalytics milica matijević1, iva popović1, milutin stepić1, maja nešić1, marija radoičić1, maja stanković2, zoran šaponjić1, marijana petković1 1“vinča” institute of nuclear sciences, university of belgrade, belgrade, serbia; 2faculty of natural sciences, university of niš, niš, serbia * e-mail: milica.m@vin.bg.ac.rs abstract: matijević, m., popović, i., stepić, m., nešić, m., radoičić, m., stanković, m., šaponjić, z., petković, m.: inorganic nanoparticles in biology: drug carriers and auxiliary tools in bioimaging and bioanalytics. biologica nyssana, 9 (1). september, 2018: 1-19. among various nano-scaled materials composed from a spectrum of chemical compounds, inorganic nanoparticles are very attractive due to their physico-chemical properties, as well as their availability, simplicity, possibility of modifications, stability and biocompatibility. they are, on the one hand, an useful tool in advanced analytical chemistry, in particular for studying of biologically-relevant processes, but also important as functional parts of the systems designed for controlled and targeted delivery of medicaments for treatment of a variety of diseases and for imaging. so far, thousands of compounds and systems have been developed for the above-mentioned purposes, but there are only a few reviews dealing with these topics. the aim of this review is, thus, to summarize recent applications of nano-structured inorganic materials in the field of drug delivery, bioimaging and bioanalytics, and to give a prospective from the standpoint of biology-related applications. key words: inorganic nanoparticles, photodynamic therapy, imaging apstrakt: matijević, m., popović, i., stepić, m., nešić, m., radoičić, m., stanković, m., šaponjić, z., petković, m.:: neorganske nanočestice u biologiji: nosači lekova i pomoćni alat u bioimidžingu i bioanalitici. biologica nyssana, 9 (1). septembar, 2018: 1-19. među različitim nanomaterijalima, neorganske nanočestice privlače posebnu pažnju zbog svojih fizičkohemijskih osobina, kao i dostupnosti, jednostavnosti, mogućnosti modifikacije, stabilnosti i biokompatibilnosti. neorganske nanočestice su korisna pomoćna sredstva u analitičkoj hemiji, posebno za ispitivanje biološki-relevantnih procesa, ali su i značajan funkcionalni deo sistema za kontrolisanu i ciljanu dostavu medikamenata za terapiju raznih oboljenja, kao i za medicinsku imidžing dijagnostiku. do sada je razvijen veliki broj jedinjenja i sistema za gore-navedenu svrhu, ali se može naći samo nekoliko preglednih članaka na ovu temu. cilj ovog revijalnog rada je da dâ zbirni pregled primene nano-strukturnih neorganskih 9 (1) • september 2018: 1-19 doi: 10.5281/zenodo.1470841 2 materijala u oblasti dostave medikamenata, bioimidžinga i bioanalitike, kao i da pruži buduće smernice sa stanovišta primene u oblasti biologije. ključne reči: neorganske nanočestice, fotodinamička terapija, imidžing introduction nanomaterials are attracting more and more attention for use in biological systems. the number of their potential applications in biology causes an expansion of new scientific discipline, so called nanobiology. this term refers not only to the application of nanomaterials for investigations of important biological processes in health and in pathological states but also to the utilization of various nanomaterials as auxiliary tools in different analytical methods for investigations of biological systems and their components. in this work, we are focusing on the application of inorganic nanoparticles for drug delivery, imaging and analytical chemistry, to cover the units which are interconnected and can be merged in practical work. apart from those, there are also other numerous applications of nanoparticles, which are important from the standpoint of advances in technology, medicine and science in general. for instance, nanoparticles are widely used in diagnostics (imaging diagnostics, laboratory diagnostics, genetic diseases` and tumor diagnostics), radiosensitization, tissue engineering, drug delivery, therapeutic drugs, to the specific medical and other instrumentations such are nanoprobes, nanosensors and nanorobots (su h. et all., 2017). other application areas that are not covered in this review, but should be mentioned are the areas of the molecular biology as a bio-labels (kumar & sophia, 2018), surface chemistry, protein corona and intracellular nanoparticles pathways. the literature data suggest that term “nano” in biology mostly refers to the particles smaller than 5 fig. 1. trajectory of peptide-conjugated gold nps (gnps) through the cell. a, schematic of a functionalized gnp used in the study b. trajectory of peptide-conjugated gnps through the cell. c-f, path of the nps was captured using tem images and is as follows: c, gnp-peptide complex bound to the plasma membrane for entry into the cell via the endocytosis process, d, internalized nps were localized in vesicles, such as, endosomes and lysosomes, e, escaping of nps from vesicles into the cytoplasm, f, entering the nucleus through nuclear pore complex (npc) (scale bars = 100nm). reproduced with permission from yang et al. (2014). biologica nyssana 9 (1) ⚫ september 2018: 1-19 matijević et al. ⚫ inorganic nanoparticles in biology: drug carriers… 3 µm (singh & lillard, 2009), which can be, with minimum risk, administered into the blood circulation. comparing with the smallest human cells, i.e. erythrocytes, the dimension of a typical nanoparticle is about 5 times smaller, whereas in comparison to oocytes, the largest nanoparticle could be more than 120 times smaller. that indicates that nanoparticles (nps), speaking in physical terms, can be ingested by a cell, reach cellular interior and become available for the interaction with intracellular organelles or to target specific biomolecules. nps can be ingested by a cell through utilizing the existing cellular pathways for ingestion, and/or diffusion through the membrane in the case of smaller nps. another way, i.e. endocytosis, or a receptor-mediated pathway is illustrated in fig. 1, on the example of a peptide-bound to the np (yang et al., 2014). based on that, nps can be designed for intracellular imaging, to target particular biomolecule or an organelle and to interfere with the intracellular processes/signaling, either for the diagnostic or the therapeutic processes. in terms of a therapy, nps as carriers for targeted and controlled drug delivery have attracted much attention. they have functions to preserve a drug until it reaches target tissue/organ and to secure targeted delivery and control a drug concentration (pandey, 2017). after activation by various mechanisms, the drug is released and can act on desired site, thus minimizing side effects on healthy tissues. although numerous medicaments have been designed to use intrinsic properties of diseased tissues, such as low intrinsic ph, due to a low oxygen concentration in tumor cells (casciari et al., 1992), nanoparticle-based carriers are still attractive and promising approach for targeted therapy. in analytical (bio)chemistry, nps are used as auxiliary tools in various techniques. thanks to their properties, they enable better precision of the method (frigerio et al., 2012; will et al., 2006; lucena et al., 2011), and its increased selectivity and sensitivity (ling et al., 1991; arakawa & kawasaki, 2010; thompson et al., 2008). a waste amount of biologically important molecules are qualitatively and quantitatively analyzed with the assistance of nps (kawasaki et al., 2007; chiang et al., 2010a; popović et al., 2016; popović et al., 2016a; popović et al., 2016b). some examples of methods which exploit the advantageous properties of nps are mass spectrometry, chromatography, but also mass spectrometry imaging, and their application will be discussed in more details in this review. physical and chemical properties of nanoparticles nps can be fabricated from various materials, and coarse classification involves organic and inorganic, and commonly used types are shown at the fig. 2. based on their size and composition, there are several groups of nano-structured materials: nps (1-100 nm), nanocapsules (nanoscale shells made of a polymer), fig. 2. types of nps commonly used for biomedical applications: (a) polymer, (b) liposomes, (c) amphiphilic cyclodextrines, (d) dendrimers, (e) gold nps, (f) micelles, (g) carbon nanotubes and (h) quantum dots (qds), modified form (mccarthy et al., 2014). 4 fullerenes (made of a carbon and with various shapes), dendrimers (repetitively branched molecule), quantum dots (qds), (a semiconductor nanostructures), nanostructures (size between intermediate and microscopic particles) and nanopores (with tiny hole in a thin membrane) (akeson et al., 1999). in line with the topic of this review, the following part will summarize some biologically-relevant properties of organic vs. inorganic nps. each of them, organic and inorganic, has its own advantages and disadvantages. organic materials are made mostly from biodegradable materials, and they can be metabolized in the organism, finally resulting with co2 and water. as disadvantage, there is always a risk of premature degradation, which leaves the medicament exposed to enzymes and immunological system, causing too early activation of a medicament, which leads to the expressed side effects. this disadvantage could be avoided, or minimized, through the encapsulation of a medicament in the nps (dobrovolskaia & mcneil, 2007; singh & lillard, 2009). monitoring of the destiny/biodistribution of organic nps in organism is sometimes difficult, due to limited possibilities to distinguish them in the surrounding organic milieu. the labeling techniques, for instance by some voluminous fluorescence dyes, might prevent the interaction with target molecules/tissue leading to the misinterpretation of the data. the application of inorganic materials for fabrication of nps gives much more possibilities for surface functionalization, and much broader spectrum of methods which can be used for the monitoring of nps` biodistribution. modification of nps allows controlled changes of the interface properties, which affects the dissolution and degradation rates, catalytic activity and possibility of stabilization through the adsorption of macromolecules (stark, 2011). on the other hand, precaution is needed because inorganic material must be selected by making a compromise between biological inertness, availability and potential of surface modification. for biological applications, selection of inorganic materials for fabrication of nps should be based on the information of their potential biological role. for instance, selected material should not contain a metal which might be a co-factor for any enzyme in the cells. in other case, the final concentration of metal should be under the enzyme-activating level (chen et al., 2012; lee & yeo, 2015). the presence of higher concentrations of the enzyme cofactors, might lead to the enzyme activation and the interference with the intracellular signaling processes. inorganic materials made of non-biological metals enable also easier detection by instrumental methods, which utilize either properties of their atoms (magnetic or paramagnetic properties, for instance) or intrinsic fluorescence, i.e. optical properties. for example, titanium is not biologically abundant, and it can be detected with methods which differentiate inorganic from organic materials, i.e. nmr (padro et al., 2000), or other approaches. some of most frequently used nps in medicine are those made of gold, which could be visualized by microscopy, or fluorescence labeling, or other method (gonciar, 2014). having in mind that the inorganic nps are the focus of this review, the summarized list of the different types of inorganic nps that are relevant for this review, is shown in the tab. 1. metal oxide semiconductor-based nanoparticles a number of metals can form oxides that can conduct an electrical current, and nps made of them are used as drug carriers, for imaging purposes and in analytical chemistry. the most commonly used are zno, cuo, tio2, zro2 or mgo. besides electroconductivity, properties which are advantageous for the variety of applications are photo-responsivity, magnetism, possibility of surface modification, etc. metal nps have large surface-to-volume ratio and due to their high density and limited size of corner or edge on the surface site, metal oxide nps exhibit unique chemical and physical properties. for instance, for the application in anticancer drug delivery systems, the size of metal nps should be larger than 10 nm in order not to be cleared by the kidney excretion and captured by macrophages (lee & yeo, 2008), and on the other hand, to be smaller than 100 nm to reach the tumor tissue and enter the cell (cho et al., 2008). even within the range of 10100 nm, small alteration in size of nps makes a big difference regarding their properties, i.e., as the size decreases, the number of surface and interface atoms generates strain or stress and concomitant structural perturbations (gleiter, 1995; trudeau & ying, 1996; valden et al., 1998; rodriguez et al., 2002; song et al., 2003; schoiswohl et al., 2004). in terms of the nps uptake by mammalian cells, oxides of zinc, iron, manganese, and cobalt exhibit nonclassical interaction – the trojan-horse-type, which enables diffusion of the toxins by packing it in the small particles (stark, 2011). surface of inorganic nps can be easily modified, and thanks to that property, they are often considered for drug discovery and therapy development. they can be modified for targeted drug biologica nyssana 9 (1) ⚫ september 2018: 1-19 matijević et al. ⚫ inorganic nanoparticles in biology: drug carriers… 5 delivery for various significant biomolecules (nucleic acids, proteins or peptides) or for specific tissues. such targeted nanocarriers decrease overal drug toxicity and offer more effective biodistribution, as discussed above. the conjugate of a drug and nanocarrier can pass some natural body barriers, such as blood or brain barriers (rawat et al., 2006) and deliver drug to the target tissue. there are several external activators, which employ inner properties of inorganic nanoparticle as a carrier, eventually leading to cytotoxic effects at the target tissue, which will be discussed more in the next chapter. stimulus or activators can be high magnetic field or ultrasound applied locally, which increase the temperature of diseased tissue, due to the accumulated nps in the tissue (arruebo et al., 2007; singh & lillard, 2009; lee and yeo, 2015). light as activator attracted much attention in later decades, because it can be easily table 1. overview of different types of inorganic nps from cited literature in this review system structure characteristics references aunps semiconductor nps: cdse or cdte doped oxide materials, y2o3 fe2o3, conps colloidal effective phototermal destruction of cancer cells and tissues photoluminiscence in the form of fluorescence phosphorescence magnetic moment sperling & parak, 2010 feridex colloid with low molecular weight dextran coating, with a particle size of 120180 nm magnetic moment diagnostic agent in use cherukuri et al., 2010 cobalt ferrite, cofe2o4 magnetic moment binding to serum albumin proteins amiri et al., 2017 fe3o4 nps fe3o4-peg nps naked, 80±5 nm functionalization with polyethylene glycol 600 diacid, 46±0,6 nm magnetic moment higher accumulation in the lungs due to their in vivo agglomeration magnetic moment potential in hyperthermia based cancer treatmens radović et al., 2015 platinum-tethered gold nps-peg au nps functionalized with thiolated poly(ethylene glycol) (peg monolayer capped with a carboxylate group of [pt(r,r-dach)] nps functionalized with peg do not make aggregate higher uptake than pt(r,r-dach) nps are capable of delivering [pt(r,r-dach)] to the cell and then releasing it brown et al., 2010 scg8-auag nanoroads gold-silver-(auag-) nanoroads labeled with molecular aptamers higher hyperthermia efficiency and selectivity to cem cells than aptamer alone conde et al., 2012 carbon nanotubes carbon cylinders composed of benzene ring linked to the methotrexate attached to medicament-methotrexate water-soluble and biocompatible through chemical modification pastorin et al., 2006 s1mps, s2mps mesoporous sio2 nps, s1mps which large pores are loaded with s2mps high biocompatibility high surface areas and pore volumes controlled release dynamic of medicament tasciotti et al., 2008 tio2 nps colloidal tio2 nps attached to potentional medicament – ru bis-bipyrydil complex minimal dark cytotoxicity high surface to volumes relation the surface of tio2 can be modified controlled release dynamic of medicament nešić et al., 2016, 2017 6 controlled and focused to minimize influence on the healthy tissue. light can be used in a combination of photo-responsive drugs or nanoparticle/drug system, which is then called a photosensitizer. among inorganic nps, we have selected for our investigations, those made of tio2, due to their availability, stability, non-toxicity, potential to modify particles surface, but also photo-responsivity, and its properties will be discussed in the next paragraph, with the focus on the photodynamic therapy. the inorganic nps, which are photoresponsive, can serve as a source of photogenerated charges that interact with the electronic properties of the biomolecules. the linking of inorganic nps with (bio)molecules facilitate hole transfer across the interface, establishing efficient crosstalk between the (bio)molecule and metal oxide nps. these photoactive bioinorganic conjugates have properties that make them good for light induced manipulation of biomolecules and their switching functions (rajh et al., 2004). tio2, which will be presented also later in this review, is typical n-type semiconductor material, with many good properties, such as costeffectiveness, chemical and photo-stability, and excellent biocompatibility in the dark. besides photo catalysis, energy storage system development and energy conversion, sun screening and sensor research, tio2 has a wide application in medical fields, too. it can be applied as anti-cancer agent, implant and a substrate for stem cell expansion (hamidi et al., 2017; oliveira et al., 2017; sims et al., 2017). quantum dots qds are semiconductors nanocrystals with size between 1-10 nm, which have unique and tunable absorption and emission properties, depending on their size and shape. for instance, they have large transition dipole moment, so can be precisely tuned from the uv to the infrared region (lucky et al., 2015). some of them exhibit photoluminescence or fluorescence (semiconductor quantum dots, e.g., cdse, cdte, cdtese/zns) (gozuacik et al., 2014). structurally, qds consist of a metalloid crystalline core and a “cap” or “shell” that shields the core, and further assignation of coatings or functional groups to the qd core–shell can give qds a desired bioactivity. qds` core consists of the metal complex which defines the structure group that particular qd presents. for example, the group iii–v series qds are composed of indium phosphate (inp), indium arsenate (inas), gallium arsenate (gaas) and gallium nitride (gan) metalloid cores, and group ii–iv series qds, of zinc sulfide (zns), zinc–selenium (znse), cadmium–selenium (cdse), cadmium–tellurium (cdte) cores and heavier structures such are cdte/cdse, cdse/znte) and hybrids composed of lead–selenium (pbse). regarding qd toxicity, it depends on multiple factors: qd size, charge, concentration, outer coating bioactivity (capping material, functional groups), and oxidative, photolytic, and mechanical stability have each been shown to be determining factors in qd toxicity (hardman, 2006). in biological systems, qds have been investigated for the application mostly in cancer treatment, theranostics, cancer drug delivery, photothermal and photodynamic therapy. the ability of fluorescence emission of qds, make some of them good imaging agents. furthermore, photoluminescent properties of qds have been exploited in the other fields of application, such as in analytical chemistry in environmental monitoring, pharmaceutical and clinical analysis and food quality control (frigerio et al., 2012). magnetic nanoparticles the advances in the synthesis of biocompatible magnetic nps in a reproducible way, allowed extensive research and further development of drug delivery systems based on magnetic field as an external driving and control force. applications of magnetic nps in biomedical areas, on the other hand, require the use of magnetic colloids, which consist of a suspension of magnetic particles of nano sizes in a carrier liquid like water, with usual particle concentrations in the range of 1021-1023 particles/m3 (goya et al., 2008). magnetic iron oxide nps are the most widely used nps with magnetic properties, and there are various trade names for super oxide and ultra-small super oxide variations in structure (shadab et al., 2015). usually, inorganic nanoparticle core is coated by a suitable coating material, which increases the stability and solubility of the nano-drug conjugate, leading to higher rates of biocompatibility and aqueous stability in the saline environment of biological tissues. feridex is an example of this commercially available iron nps, which are coated with sugars (e.g. dextran) (cherukuri et al., 2010). the enrichment of magnetic np surface, for instance with sio2, can allow direct functionalization making it more suitable for biomedical application (radović et al., 2015). the other group of compounds with magnetic properties, facile synthesis and chemical stability, are ferrites of the general formula mfe2o4 where m = fe, mn and co. some ferrites exhibit additional biologica nyssana 9 (1) ⚫ september 2018: 1-19 matijević et al. ⚫ inorganic nanoparticles in biology: drug carriers… 7 properties making them even more suitable for application in the biological systems, for instance, cobalt ferrite, cofe2o4 binds strongly to serum albumin proteins (amiri et al., 2017). inorganic nanoparticles controlled drug delivery systems: applications for anticancer therapy in recent years, a combined nanotechnology – drug delivery systems have been developed by two components within the general formulation, nanocarrier/drug complex. the purpose of the nanodrug conjugation is to allow modification of the pharmacokinetics and metabolism of the drugs, particularly regarding reducing the side effects, such as non-selective toxicity to tumor cells and inactivity against drug-resistant cell lines (llevot & astruc, 2012). the drug delivery system with nanocarrier has even more advantageous properties, such as better encapsulation, bioavailability, lower toxic effects to the healthy tissues and possibility for controlled release (pandey, 2017). in such manner, some of these features are related to each other. as instance, the encapsulation of the drug in the nanocarrier prevents quick elimination by the reticuloendothelial system, which consequently leads to higher levels of blood circulation and to increased rates of the transport through biological barriers. the final outcomes of these processes are better availability of drug at the targeted tissue and lower toxic effects to the healthy tissues. moreover, in the focus of the researches is to develop the drug delivery system which is capable of maintaining constant concentration gradients by continuous and controlled release of therapeutic drugs. these unique chemical and biological properties of the nanocarriers are linked to the nps large surface area-to-volume ratio, as discussed above. this property allows them to combine, absorb and hold other compounds like drugs molecules, dna, rna, proteins (shadab et al., 2015). additionally, surface modification can enable the increase of the circulation time in the body, cell specific targeting and membrane permeability. by interacting with biomolecules placed on the cells surface and inside the cells, penetration into the tissues is promoted with the higher levels of specificity. furthermore, it is possible to develop the advanced nps drug delivery systems for the spatially and temporally controlled release of drugs in response to specific stimuli within the tumor microenvironment (yin et al., 2013). a stimuli-responsive delivery system improves the efficacy of the drug, and can be designed to react to the disease specific property (ph level, redox property or enzyme levels). drug accumulation and release at the tumor site can be additionally intensified by external forces like magnetic field (arruebo et al., 2007; shadab et al., 2015), light (mari et al., 2014; el-hussein et al., 2015; nešić et al., 2017) and/or heat (cherukuri et al., 2010). to date, there are only a few clinically approved nanocarriers that incorporate molecules to selectively bind and target cancer cells, and these drug delivery systems are mostly based on organic nanocarriers, such as liposomes and polymers, free or attached to proteins (lópez-dávila et al., 2012). inorganic nanocarriers, on the other hand, are much smaller, and the active form is usually simple metal based structure with gold (brown et al., 2010), silver (conde et al., 2012), carbon (pastorin et al., 2006; cho et al., 2008), oxides (mesoporous sio2, tio2, fe2o3, graphene oxide) (goya et al., 2008; tasciotti fig. 3. schematic representation of various stages involved in pdt. reproduced with permission from the authors avirah et al. (2012) 8 et al., 2008; nešić et al., 2016; nešić et al., 2017), or in the form of qds (selenides, sulfides or tellurides of metals like cadmium, lead or zinc) (sperling & parak, 2010; chen et al., 2014). different designs varying in size, shape, and porosity allow easy conjugation with drugs for cancer therapy (bhattacharyya et al., 2011) and they are more stable over large ranges of ph and temperature (shadab et al., 2015). photo-sensitive systems for drug delivery optical energy can trigger a variety of photochemical processes useful for therapies. it is the raising field in the development of new therapeutic approaches in the last decades. photodynamic therapy (pdt) involves the administration of a tumor-localizing photosensitizer (ps), usually np, which undergoes reversible changes upon light exposure of a specific wavelength. the induced changes in such manner, can lead to the release of therapeutic drugs, thereby providing spatio-temporal control of drug release (shadab et al., 2015), or the excited ps can transfer its energy to molecular oxygen, thus generating cytotoxic reactive oxygen species (ros) and singlet oxygen, which can modify cellular macromolecules leading to tumor cell ablation (lucky et al., 2015). simplified schematic representation of the stages involved in pdt, is shown in fig. 3. inorganic, metallic, or composite nps have been investigated as multifunctional carriers for pdt due to their unique characteristics such as optical properties and tunability in its shape, size, porosity, and that they may not degrade readily in the biological systems. it is also reported that the degree of drug resistance is far less than those encountered in chemotherapy, and that there is no cross-resistance between pdt and chemotherapy (kim et al., 2015) the most promising pss are nanosized oxides, which exhibit distinctive optical absorption properties, transparency of the matrix to light absorption and chemical inertness. they can be easily attached to therapeutic medicament in the form of nanocomposite system, which can exhibit greater cytotoxicity (nešić et al., 2017) and higher therapeutic effectiveness for cancer than the corresponding free drugs (chen et al., 2011; wang, y. et al., 2015). the most investigated nps/metal oxides are mesoporous silica (teng et al., 2013), titanium dioxide (nešić et al., 2016) and zinc oxide (zhang et al., 2014). tio2 nps are specially interesting as, upon ultraviolet (uv) light excitation, they absorb the energy higher than its own band gap energy (3.2 ev), which leads to excitation of the electrons from the valence to the conduction band, creating the electron/hole pairs and further generating active free radicals, thus allowing oxidation/reduction of species in surrounded medium. tio2 itself was reported to have a good anticancer effect in response to the light, owing to the production of active free radicals under uv irradiation (wang, t. et al., 2015). in addition, the incomplete coordination sphere of the surface metal atoms exhibits the high affinity of tio2 nps for linking with biomolecules and oxygen-containing ligands (rajh et al., 2004). while tio2 nps with the anatase crystalline phase absorb uv light and consequently generate ros, great chemical stability and minimal cytotoxicity of tio2 nps are detected in the dark. in comparison with traditional organic photosensitizers, tio2 nps can be maintained for a longer time in the body. even tio2 nps possess high photosensitization activity toward various cancer cell lines, for the suppression of in vivo tumors there are characteristics that need to be improved, as low penetration into tissue and the damage to normal tissue (hou et al., 2015). in the study of nanocomposite system based on tio2 nps and daunorubicin, mtt assay have confirmed significant cytotoxicity when leukemia cell line, k562, was treated with the nanocomposite system, while low cytotoxicity was shown when k562 cells were treated solely with tio2 nps, and thus tio2 nps demonstrated important properties of a drug carrier and showed good potential for the applications in cancer therapy (zhang et al., 2012). in a similar study, it was demonstrated that the nanocomposite system made of pt(nh3)4cl2 transitional metal complex and tio2 nps, as well as individual components, lead to a significant decrease in c6 glioma cell proliferation; the reduction in the tumor volume was also confirmed (lópez et al., 2008). in our previous work we studied in vitro light stimuli-responsive properties of nanocomposite system based on the colloidal tio2 nps and transitional metal complex, cis-dichlorobis(2,2′bipyridyl-4,4′-dicarboxylic acid)ruthenium(ii). the system had the light controlled release properties, as the release of the complex was facilitated upon illumination with uv light, and sustained upon illumination with visible light. results obtained by the investigation of the nanocomposite system cellular cytotoxicity towards melanoma cancer cells line a375 were in agreement with in vitro drug release test: while the nanocomposite system and its components exhibited the significant decrease of the cell viability upon uv irradiation, red light irradiation showed an extremely low anti-cancer effect (nešić et al., 2017). biologica nyssana 9 (1) ⚫ september 2018: 1-19 matijević et al. ⚫ inorganic nanoparticles in biology: drug carriers… 9 inorganic nanoparticles and tissue imaging inorganic nps including semiconductor qds, iron oxide nps, and gold nps have been developed as contrast agents for diagnostics by molecular imaging (huang et al., 2011; yu & zheng, 2015). compared to traditional contrast agents, nps offer several advantages: their optical and magnetic properties can be tailored by engineering the composition, structure, size, and shape; their surfaces can be modified with ligands to target specific biomarkers of disease, as mentioned in the introduction section. the contrast enhancement provided can be equivalent to millions of molecular counterparts; and they can be integrated with a combination of different functions for multimodal imaging (cho et al., 2010). broadly, the field of biomedical imaging can be divided into categories based upon the electromagnetic spectrum as shown in fig. 4. molecular imaging is a new frontier of biomedical research for visualizing, characterizing, and monitoring biological processes in cells, tissues, and organisms using sensitive instrumentation and contrast mechanisms. molecular imaging differs from traditional imaging in that way in which the contrast agents are typically utilized to help identify particular biomarkers or pathways with high sensitivity and selectivity (cho et al., 2010), and for that purposes, inorganic nps is an actively explored technology for the development of contrast agents. this imaging function based on interrogation of biological processes to report on and reveal the molecular abnormalities that might point to a disease, being thus a powerful tool for the diagnosis of cancer, cardiovascular syndrome, or neurological disorders. imaging based on ionizing radiation generally refers to the detection of high frequency emissions from radioactive elements such as the gamma ray emitters 111in or 99mtc (metastable nuclear isomer of technetium-99) or the passage of x-rays through the body. the main technologies involved are positron emission tomography (pet), single-photon emission computed tomography (spect), and x-ray computed tomography (ct). magnetic resonance imaging (mri) tends to operate on the other end of the spectrum in the mhz frequency range, relying upon contrast agents such as gadolinium or superparamagnetic iron oxide to modify the relaxivity of water molecules to provide soft tissue contrast (pansare et al., 2012). compared with other imaging approaches, optical imaging is a versatile, economic and efficient modality for bio-imaging with high sensitivity, great choice for probe selection and it does not require precaution, like in the case of application of x-rays or other ionizing radiation. various fluorescent molecules or nanomaterials, such as conventional organic fluorescein, semiconductor qds, rare-earth co-doped up-conversion nanophosphors, etc., have been explored to label and image cancer cells both in vitro and in vivo. integrating mesoporous silica with fluorescent materials towards multifunctionalization could endow them with simultaneous optical bioimaging and drug transport capabilities (chen et al., 2013). there are numerous efforts to combine the unique advantages which nanoprobes have in cancer imaging, and to create a “universal” nanoprobe. such nanoprobe should be multimodal, have a strong epr (electron paramagnetic resonance) effect, able to circulate in a body for a long time (bouccara et al., 2015), be stable in physiological conditions, specific for target tissue, and not to interfere with physiological processes. most inorganic nps can meet some criteria, but one of the most challenging is the avoidance of renal clearance, which enables the prolonged blood circulation, and afterwards, accumulation in target, or tumor tissues. some examples of inorganic nps for various molecular imaging purposes are given below in the text, classified according to their purpose, i.e. imaging technique (yu & zheng, 2015). fig. 4. types of medical imaging: ionizing radiation (x-ray and ct), magnetic resonance imaging (mri), ultrasound and optical imaging 10 major inorganic nanoparticles for molecular imaging contrast agents for various imaging purposes should be easy to design but it is required for them to have particular electrical, magnetic and optical properties. their properties can be fine-tuned by tailoring nps` composition, geometry and structure. therefore, particles can be composed of metals, metal oxides, semiconductors, they can vary in size and shape, and be solid, or hollow. some of nps used in imaging techniques are iron oxide nps, qds, gold nps, and others, which can be used in different imaging approaches (cho et al., 2010; huang et al., 2011; chen et al., 2013; bouccara et al., 2015; yoon et al., 2017), and most inorganic nps possess great potential for noninvasive and real-time in vivo diagnosis of disease (chen et al., 2013). in terms of magnetic resonance imaging (mri), both, magnetic or superparamagnetic nps can be used as contrast agents. among them, iron/based nps are routinely applied because they were approved by fda for human use (yoon et al., 2017). however, a caution is needed regarding the size of nps for mri. in general, reducing a size of ferroor ferrimagnetic particles below critical, could lead to randomization of the magnetic dipoles in a short period of time, as a thermal energy becomes comparable to what is needed for spins to flip. those nps are superparamagnetic, because they do not have permanent magnetic moments in the absence of an external field, but they can quickly respond to it when applied (cho et al., 2010). qds are currently replacing fluorescence dyes, because they have high absorption coefficients and broad emission range in visible and near infrared region (cho et al., 2010). gold nps are also utilized in optical imaging, thanks to their optical properties, i.e. presence of strong extinction peaks in visible and near ir region (pansare et al., 2012). those extinction peaks are caused by the collective oscillations of conduction electrons in the presence of an incident light. this phenomenon is known as localized surface plasmon resonance. in addition, gold nps can be used as contrast agents for ct imaging, due to their absorption coefficient for the xrays, but also, they are capable of photoluminescence. finally, gold nps can enhance the intensity of raman-active molecules and be used for the surface-enhanced raman spectroscopy (cho et al., 2010; huang et al., 2011). a particular class of nps are rare-earth doped nps, which exhibit sharp emission peaks, long fluorescence lifetimes, high quantum yields, and excellent photostability (cho et al., 2010; bouccara et al., 2015). applications of inorganic nanomaterials in analytical (bio)chemistry nps have been used as an auxiliary tool to increase sensitivity and specificity of standard analytical methods. one of earlier applications of inorganic particles, was the use of magnetic particles (beads) in the affinity chromatography, in which the antigen was bound to the surface of a particle, and after specific and selective interaction with a component from a complex solution magnetic nps could be easily collected and separated from the mixture (chen & chen, 2005). also, nps are used as the stationary phase in several chromatographic techniques, where they increase the active surface available for the interaction with analytes. in this chapter, the overview of inorganic nps and their application in chromatography and mass spectrometry will be given and discussed. chromatography the application of nps and nanomaterials in chromatography has been of great interest in recent years due to the unique physical properties of these substances, their large surface area-to-volume ratios, and their ability to be employed with a variety of surface chemistries (guihen, 2013; tang et al., 2014; zhang & qiu, 2015; castillo-garcía et al., 2016). a number of carbon-based nanomaterials and inorganic nanomaterials have been considered for use in lc, some examples are: carbon nanotubes, fullerenes and nanodiamonds (valcárcel et al., 2007; scida et al., 2011; guihen, 2013; pyrzynska, 2013; speltini et al., 2013; zhang et al., 2013), metal nps and nanostructures based on silica, because of their relatively high surface area, availability in various particle sizes and pore diameters, and the ease with which surface can be modified to contain a wide range of functional groups to increase specificity and sensitivity of detection and separation (zhang et al., 2006). these properties have led to the use of silica nps and related materials (e.g., silica nanofibers) in making stationary phases or supports for such methods as reversed-phase chromatography, hilic, and utlc (ge et al., 2006; jim et al., 2011; newsome & olesik, 2014; aydoğan & el rassi, 2016), alumina, zirconia and titanium dioxide (zhang et al., 2006; nesterenko et al., 2013). application of these nanomaterials have included the reversed-phase, normal-phase, ionexchange, and affinity modes of lc, as well as related methods such as chiral separations, ion-pair chromatography and hydrophilic interaction liquid chromatography. biologica nyssana 9 (1) ⚫ september 2018: 1-19 matijević et al. ⚫ inorganic nanoparticles in biology: drug carriers… 11 mass spectrometry small molecules – metabolites (molecular mass less than 1000 da) are significant for understanding of cellular processes and metabolism, and their importance is reflected in the development of relatively new discipline, called metabolomics. the metabolome represents the large scale qualitative and quantitative changes of all metabolites in a biological cell, tissue, organ or organism, which are the end products of cellular processes. this is of high importance for the understanding of biological processes. metabolomics combine strategies for identification and quantification of metabolites with different analytical approaches and statistical methods, and the great efforts are made for their development. metabolites are involved in different cell processes e.g. cell signaling which controls cell division, growth and differentiation. another very important and diverse group of small molecules are drugs. qualitative and quantitative analysis of drugs (especially their biodistribution) is essential for the drug discovery, monitoring and understanding of their mechanism of action and pharmacological and toxicological effects (monro, 1994; mizojiri et al., 1996). method that is routinely applied for detection and analysis of drugs is autoradiography, which for qualitatively and quantitatively investigation of the drugs in the tissues requires radioactive materials, and the procedure is rather time consuming. other methods that are in use for the detection of small molecules are: high-performance liquid chromatography, hplc (araki & sako, 1987; rafii et al., 2007), capillary electrophoresis, се (caussé et al., 2000; bayle et al., 2002), gas chromatography/mass spectrometry, gc/ms (larsson & lindgren, 2005), sers (pietzsch et al., 1997) and fluorescence spectroscopy (ling et al., 1991). these techniques are time consuming, and additional methods for the sample preparation and additional derivatization are needed. some of these methods have additional disadvantages such as low selectivity and low sensitivity for the detection of small molecules in biological samples. matrix assisted laser desorption and ionization time of flight mass spectrometry (maldi tof ms) is powerful technique that is traditionally used for the analysis of large, non-volatile, and termolabile biomolecules and biopolymers (proteins, dna, saccharides). in this method, organic matrices are traditionally in use, like 2,5-dihydroxybenzoic acid (dhb), 9-aminoacridine (9-aa), 2′,4′,6′trihydroxyacetophenone (thap), α-cyano-4hydroxycinnamic acid (chca), sinapic acid (sa). in theory, maldi tof can be used for the semiquantitative mass analysis of all kind of molecules, but in practice that is not a case because of numerous disadvantages which are consequence of the use of organic matrices. firstly, inhomogeneous distribution of samples molecules between matrix molecules (hot spots, cold spots) (tholey et al., 2006; jaskolla et al., 2009; kuzema, 2011) lead to the mass spectra with poor quality, and eventually the reproducibility and the repeatability of a method is low. second drawback refers to the analysis of small molecules; organic matrices have low molecular masses and the high number of signals in the low mass range (usually less than 500 da) which interfere with sample signals. matrices are usually organic acids and they have intensive signals in negative ion mode, so it is very difficult to detect the negative ions of samples (petković et al., 2001a; petković et al., 2001b). another disadvantage is that the organic matrices do not tolerate high concentrations of salts. biological samples have high concentrations of inorganic salts, and because of that the great number of matrix signals are present in spectra and the signals arising from the analytes are with underestimated intensity if not completely suppressed. to overcome listed disadvantages of organic matrices, the inorganic matrices/substrates are much more in use. wei et al. (1999), were synthesized porous nanostructures based on silicon (desorption and ionization on silicon, dios). the term saldi the first was used by sunner et al. (1995), when replaced organic matrices with graphite, and refers to the techniques that use nanostructured substrates instead of organic matrices. different inorganic materials which absorb ultraviolet light were used as substrates for saldi: carbon (chen & chen, 2006; kawasaki et al., 2009; tang et al., 2009; dong et al., 2010), silicon (hu et al., 2007; northen et al., 2007; teruyuki et al., 2007; guénin et al., 2009; walker et al., 2010; cheng et al., 2012), nanomaterials based on metals and metal oxide (chen & chen, 2004; chen & chen, 2005; mclean et al., 2005; okuno et al., 2005; chen & chen, 2006; huang & chang, 2006; wu et al., 2007; kailasa et al., 2008; kawasaki et al., 2008; sherrod et al., 2008; chen et al., 2009; chiang et al., 2010b). substrates based on metals and metal oxides have additional advantages compared to substrates based on silicon and carbon. these substrates are more chemically stable in the air and have high conductivity (chen & chen, 2004; arakawa & kawasaki, 2010), the samples are nps based on au, pt, ag, zno, fe and mno2/mno3. different inorganic nps are in use in analytics of different biological molecules and samples: sherrod et al. (2008) describe selective ionization of olefin compounds (cholesterol and carotenoids) direct from 12 the mixture with silver nps. wu et al. (2007) were analyzed and quantified small, neutral carbohydrates from urine with gold nps. lee at al. (2007) found that тio2 nps with diameter less than 20 nm interact with enediol compounds, which have an impact on increase of absorptivity in uv-vis spectra of light. chiu et al. (2008) analyzed urine samples for the detection of estrogen hormones – estrone, estradiol and estriol with silver nps of a diameter (34 ± 3) nm. watanabe et al. (2008) were used zno nps that have anisotropic shapes, such as cubic or rectangular parallelepiped, for the detection of verapamil hydrochloride, testosterone, phospholipids, oligosaccharides and synthetic polymers. semiconductor zns nps with surface modification with different functional groups were used for the analysis of cyclodextrin and small proteins (kailasa et al., 2008). arakawa & kawasaki, (2010) used oxidized graphitized carbon black (gcb) particles for the analysis of pharmaceutically active compounds. a common pharmaceutical compound, propranolol, was successfully extracted from baltic sea blue mussels and quantified using oxidized gcb. tio2 is a good candidate for saldi mass spectrometry, because it is readily available, nontoxic and efficiently absorb uv light of n2 laser in maldi instrument. it is first shown applicability of sol-gel tio2 films (chen & chen, 2004), and then applicability of tio2 nps (radisavljević et al., 2012), nanotubes (lo et al., 2008) and nanowires (kim et al., 2014) in saldi analysis of biomolecules. the example of positive ion spectra obtained by saldi method in which tio2 served as a substrate is shown in fig. 5. scarce data were found in literature about tolerance of substrate on high concentration of salts in samples solutions. popović et al. (2015) have shown that the quality of mass spectra of transition metal complexes obtained with tio2 nps was idependent of the high concentration of sodium and potassium ions. size and shape of nanocrystals have impact on analytical performances and ionization efficiency in saldi mass spectrometry (popović et al., 2016; popović et al., 2016a; popović et al., 2016b). tio2 nanocrystals with various shapes and sizes, such colloidal tio2 nps (nps, average diameter ~ 5 nm), prolate nanospheroids (pnss, length: 40–50 nm, the lateral dimension: 14–16 nm) and nanotubes (nts, length: 100-150 nm, average diameter 11 nm), were investigated substrates for saldi-tof ms for quantitative analysis of small biomolecules such as various sex hormones (testosterone and progesterone), vitamins (vitamin a and e), carbohydrates and others (popović et al., 2016; popović et al., 2016a). in general, the best reproducibility was obtained with the larger nanocrystals, pnss and nts, making them good candidates for the quantitative determination of small molecules. however, for analysis of citric acid, dexasone, vitamins e and a, pnss provided the highest sensitivity and reproducibility (popović et al., 2016b). future use of nanomaterials in analytics of (bio)molecules – a summary inorganic particles often exhibit improved and some novel properties as their size approaches nanometer scale dimensions. the unique electronic and optical properties of nanocrystals may lead to future advances of analytical (bio)chemistry, biology, for investigation of biological processes and in medicine. their additional and important application is the development of novel therapeutical approaches. in terms of therapy, novel nps and activators are expected to be exploited. the challenge is to further minimize the side effects and involve fig. 5. positive ion saldi tof mass spectra of inorganic complexes: the upper spectrum represents the spectrum of rucomplex, whereas the bottom is the spectrum of pt-complex. the spectra are acquired with the assistance of tio2 nps with diameter of 5 nm. transmission electron micrograph of tio2 nps is given in the upper left corner of the figure. reproduced with permission (popović et al., 2016). biologica nyssana 9 (1) ⚫ september 2018: 1-19 matijević et al. ⚫ inorganic nanoparticles in biology: drug carriers… 13 light (and potentially other stimuli) in therapy of deep tumors. in terms of imaging, the future challenge is to target specifically a single molecule by a np. one of advanced imaging approaches is mass spectrometry imagining which is a rapidly growing field, which uses nanotechnology (either for production of targets or for the interaction with molecules within the cell/tissue) for the visualization of molecules in tissues, based on the identification of molecules on ions generated. one of the problems with which is this approach facing is lateral resolution in cells and still, despite of the creativity of scientists, is not easy to visualize the 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belgrade, serbia * e-mail: vladar@pmf.ni.ac.rs abstract: ranđelović, v., zlatković, b., dimitrijević, d., vlahović, t.: phytogeographical and phytocoenological analysis of the threatened plant taxa in the flora of the vlasina plateau (se serbia). biologica nyssana, 1 (1-2), december 2010: 1-7. in analysing the flora of the vlasina plateau, it was determined that this area contains 956 species, 23 subspecies, 32 varieties and 28 forms of vascular plants. the data shows the exceptional floristic riches of this territory. among these species, there are 91 species (9.52%) which may be categorized as under threat in the flora of serbia. critically endangered taxa (cr) are especially interesting because their sanctuary in serbia is only in the vlasina plateau. this group includes the following species: betula pubescens, elatine triandra, utricularia minor, cirsium helenioides and carex limosa. all these species have the boreal type of distribution and live in wetlands. key words: flora, serbia, threatened plant taxa, vlasina plateau introduction ! the vlasina plateau is situated in southeastern serbia between vardenik, čemernik, plana and bukova glava mountains (fig. 1). geologically, the greater part of vlasina plateau is uniform area and it is made of metamorphic rocks, the most frequent of them being schists, rarely gneisses, andesites and dacites (p e t k o v i ć et al., 1977, d r a g i š i ć , 1997). vlasina plateau is in the zone of brown forest land (dystric cambisol), but characteristic soil is peat, which is differentiated in four types: phragmitetum-type, caricetum-type, equisetetumtype and mossy peat (r a n đ e l o v i ć 1994). the climate of this plateau is a typical continental one (đ u k a n o v i ć , 1967, r a n đ e l o v i ć & z l a t k o v i ć , 2010). floristically and phytocoenologicaly, vlasina plateau is relatively well documented in the monograph "flora and vegetation of the vlasina plateau" (r a n đ e l o v i ć & z l a t k o v i ć , 2010). also, in this monograph threatened plant taxa are processed. however, phytogeographical and phytocoenological analysis of vlasina plateau is not made. material and methods list of flora of the vlasina plateau from r a n đ e l o v i ć & z l a t k o v i ć (2010) was used for the analysis. the nomenclature follows medchecklist (g r e u t e r et al., 1984-1989) and flora europaea (t u t i n et al., eds. 1964-1980). all taxa that occur in the area of the vlasina are assessed according to iucn version 3.1 (iucn, 2001) methods for determining vulnerability at the local level and in serbia. iucn red list criteria were used to classify species according to one of the 10th sfses • 17-20 june 2010, vlasina lake1 (1-2) • december 2010: 1-7 biologica nyssana 1 (1-2) december 2010: 1-7 ranđelović, v. et al. phytogeographical and phytocoenological… 2 five categories of threat: extinct in serbia (ew), critically endangered (cr), endangered (en), vulnerable (vu) and near threatened (nt). if there was insufficient information to make an assessment, the category data deficient (dd) was assigned, especially in cases of taxonomic uncertainty. area-types, area-groups and life forms are taken from r a n đ e l o v i ć & z l a t k o v i ć (2010). phytocoenological relevance of taxa is determined by r a n đ e l o v i ć & z l a t k o v i ć (2010), h o r v a t et al. (1974) and a p o s t o l o v a & s l a v o v a (1997). results and discussion flora of vlasina plateau contains 956 species, 23 subspecies, 32 varieties and 28 forms of vascular plants. the data shows the exceptional floristic riches of this territory. among these species, there are 90 species (9.41%) which may be categorized as under threat in the flora of serbia (tab. 1) (r a n đ e l o v i ć & z l a t k o v i ć , 2010). the high degree of presence of endangered species that make flora of the vlasina plateau and the fact that every eleventh species in some way is endangered and that sooner or later it is in danger of disappearing from the territory of serbia, shows the biodiversity importance of this area. among other things, it was one of the reasons for classifying the vlasina plateau in botanical significant areas of europe(ipa) (r a n đ e l o v i ć in s t e v a n o v i ć , ed. in r a d f o r d , o d é , eds. 2009). the red data list of flora of serbia ( s t e v a n o v i ć , ed. 1997) states 53 taxa from the vlasina plateau. the first volume of the red data book of flora of serbia (s t e v a n o v i ć , ed. 1999), which contains extinct and critically endangered taxa, lists 11 taxa from the vlasina plateau. as extinct species from the flora of serbia, the following species are processed: polemonium coeruleum (r a n đ e l o v i ć , 1999a), dracocephalum ruyschiana (d i k l i ć , 1999), caldesia parnassifolia (v u k o j i č i ć , j a n k o v i ć , 1999) and juncus capitatus (r a n đ e l o v i ć , 1999). however, later investigations showed that species juncus capitatus (t o m o v i ć et al, 2009) and dracocephalum ruyshiana (l a z a r e v i ć et al., 2009) are not extinct, because they were found on the other localities in serbia. the species ranunculus lingua (s t o j š i ć , p a n j k o v i ć , 1999), betula pubescens (j o v a n o v i ć , 1999), elatine triandra (b l a ž e n č i ć , b l a ž e n č i ć , 1999), utricularia minor (b l a ž e n č i ć , b l a ž e n č i ć , 1999a), cirsium helenioides (r a n đ e l o v i ć , 1999c), carex limosa (r a n đ e l o v i ć , 1999b) and sparganium natans (b l a ž e n č i ć , b l a ž e n č i ć , 1999b) are classified as critically endangered. phytogeographical analysis according to phytogeographical analysis (fig. 2), highest proportions of threatened taxa have boreal (21 taxa) or euroasian mountain (27) type of distribution. within euroasian mountain area-type, middle-south-european mountain area-group (19) stands out with the number of taxa, which includes most of the balkan endemic species (11). phytocoenological analysis the highest proportion of threatened taxa is characteristic of peat vegetation (21), and another 39 for other types of wet vegetation. high presence of threatened taxa (11) is found in the forest vegetation. (fig. 3). analysis of life forms according to analysis of life forms (fig. 4) the dominance of hemicryptophytes (33 taxa) is established. fig 1. geographical position of investigated area biologica nyssana 1 (1-2) december 2010: 1-7 ranđelović, v. et al. phytogeographical and phytocoenological… 3 0 5 10 15 20 25 csm hol pho l-p tro p aa bor eamt msemt semt eas easw mem mem p msm me med n um be r o f t ax a fig. 2. phytogeographical analysis of the threatened plant taxa (balkan endemic taxa (b) are marked in black). area types and area groups: csm – cosmopolitan, hol – holarctic, phol-ptrop – palaeoholarcticpalaeotropic, aa arcto-alpian, bor boreal, eamt euroasian mountain, msemt middle-southeuropean mountain, semt south-european mountain, eas euroasian, easw european west asian, mem middle european-mediterranean, memp middle european-mediterranean-pontic, msm merridional-submerridional, me middle-european, medp mediterranean pontian, med mediterranean 0 5 10 15 20 25 cha r le mn pota phra iso e moli mulg mon t sch e fest call ju nc asp l vac c que r rud e n um be r o f t ax a fig. 3. phytocoenological analysis of the threatened plant taxa (balkan endemic taxa are marked in black). vegetation classis: char charetea, lemn lemnetea, pota potametea, phra phragmitetea, isoe isoeto-nanojuncetea, moli molinio-arrhenatheretea, mont montio-cardaminetea, mulg mulgedioaconitetea, sche scheuchzerio-caricetea fuscae, fest festuco-brometea, call calluno-ulicetea, junc juncetea trifidi, aspl – asplenietea trichomanis, vacc vaccinio-piceetea, quer querco-fagetea, rude ruderalis biologica nyssana 1 (1-2) december 2010: 1-7 ranđelović, v. et al. phytogeographical and phytocoenological… 4 0 2 4 6 8 10 12 14 16 p ch hs hc hrp hro hsr hsg hrog gb gr gt hyd g hyd t t th n um be r o f t ax a fig. 4. life forms of the threatened plant taxa (balkan endemic taxa are marked in black): p phanerophyta, ch chamaephyta, hs hemicryptophyta scaposa, hc hemicryptophyta caespitosa, hrp hemycryptophyta reptosa, hro hemycryptophyta rosulata, hsr hemycryptophyta semirosulata, hsg hemycryptophyta scaposa/geophyta, hrog hemicryptophyta rosulata/geophyta, gb geophyta tuberosa, gb geophyta bulbosa, gr geophyta rhyzomatosa, hydg hydrogeophyta, hydt hydrotherophyta, t therophyta (including: tc therophyta caespitosa, ts therophyta scaposa, tsb therophyta scaposa biennis), th therophyta/hemicryptophyta scaposa table 1. contribution of threatened taxa in the flora of vlasina plateau with categories of endangered (ce) in the flora of serbia, area-type (at), area-group (ag), life form (lf) and phytocoenological relevance of taxa (p) (abbreviations are shown in the legends of appropriate figures) name of taxon ce at ag lf p caldesia parnassifolia ew csm csm hydg pota polemonium coeruleum ew bor borea hs mulg trisetum sibiricum ew bor borh hc sche betula pubescens cr bor borea p sche cirsium helenioides cr bor borea hs mulg eleocharis mammillata cr bor borea hydg isoe sparganium natans cr bor borh hydg pota utricularia minor cr bor borh hydt pota carex limosa cr bor borh gr sche elatine triandra cr bor borh tsb isoe hypericum humifusum cr csm csm hrp isoe ranunculus lingua cr eamt eamt hydg phra ranunculus auricomus var. biformis cr eas eas hrp sche dracocephalum ruyschiana cr eas eas hs fest gentiana pneumonanthe cr eas memp hs moli sisyrinchium montanum cr hol hol gt moli juncus capitatus cr hol phol-ptrop tc isoe campanula hemschinica cr me meb tsb fest sagina saginoides en aa aa hc mont pirola chlorantha en bor borh hsr quer lycopodium clavatum en bor borh ch mont cardamine raphanifolia ssp. acris en eamt msemt hro mont alchemilla cinerea en eamt msemt hrog fest alchemilla connivens en eamt msemt hrog sche biologica nyssana 1 (1-2) december 2010: 1-7 ranđelović, v. et al. phytogeographical and phytocoenological… 5 table 1. continued alchemilla crinita en eamt msemt hrog fest alchemilla heterophylla en eamt msemtb hrog fest alchemilla viridiflora en eamt msemtb hrog mulg geum rivale x rhodopaeum en eamt msemtb gr mulg pedicularis hoermaniana en eamt msemtb hs fest gentiana acaulis en eamt semt hro call pirola rotundifolia en eas eas hsr quer dactylorhiza sambucina en eas mem gt quer scleranthus neglectus en eas mem tc aspl eriophorum gracile en hol hol gr sche stellaria alsine en hol hol hc mont potentilla palustris en hol hol hsg sche potamogeton obtusifolius en hol hol hydt pota ranunculus fontanus en med med ths mont ranunculus ophioglossifolius en msm msm ths isoe pseudorchis albida vu aa aa gt sche sagittaria sagittifolia vu bor borea hydt pota arctostaphylos uva-ursi vu bor borh ch vacc corallorhiza trifida vu bor borh gr quer drosera rotundifolia vu bor borh hro sche orthilia secunda vu bor borh hsr quer zanichellia palustris vu csm csm hydt pota salix rosmarinifolia vu eamt eamt p sche dactylorhiza cordigera vu eamt msemtb gt sche pseudorchis friwaldii vu eamt msemt gt sche geum rhodopaeum vu eamt msemtb gr mulg alchemilla glabra vu eamt msemt hrog mulg alchemilla reniformis vu eamt msemt hrog moli silene asterias vu eamt semtb hs sche salix pentandra vu eas eas p sche cephalanthera longifolia vu eas eas gt quer epipactis palustris vu eas eas gt sche dianthus superbus vu eas eas hs sche stellaria palustris vu eas eas hs sche dactylorhiza incarnata vu eas easw gt sche trollius europaeus vu eas eas hs moli callitriche verna vu hol hol hydt lemn menyanthes trifoliata vu hol hol hrp sche ranunculus aquatilis vu hol hol hydg pota najas minor vu hol phol-ptrop hydt char saxifraga stellaris ssp. alpigena nt aa aa hro mont schoenus ferrugineus nt bor bore hc phra pirola media nt bor borea hsr quer pinguicula vulgaris nt bor boren hro sche pirola minor nt bor borh hsr quer pedicularis palustris nt bor borh tsb sche alchemilla acutiloba nt eamt eamt hrog call alchemilla gracilis nt eamt eamt hrog mulg allium melanantherum nt eamt msemtb gb call silene lerchenfeldiana nt eamt msemtb hc aspl peucedanum oligophyllum ssp. aequiradium nt eamt msemtb hs junc trifolium trichopterum nt eamt msemtb ts fest biologica nyssana 1 (1-2) december 2010: 1-7 ranđelović, v. et al. phytogeographical and phytocoenological… 6 table 1. continued verbascum adamovicii nt eamt msemtb tsb call achillea lingulata nt eamt semt hro call butomus umbelatus nt eas eas hydg phra euphorbia villosa nt eas eas hs rude ribes alpinum nt eas easw p quer cardamine amara nt eas easw hs mont ranunculus flammula nt hol hol hydg isoe gratiola officinalis nt hol hol hs moli lilium jankae dd eamt msemt gb call ranunculus serbicus dd eamt semt hsg mulg silene viridiflora dd eas memp hs quer anagalis minimus dd me me ts mont malus praecox dd msm msm p quer conclusion phytogeographical analysis showed that representatives of the boreal flora are the most endangered, which indicates that the vlasina plateau is one of the most important refuges of postglacial floristic elements characteristic for far north of europe and asia. in support of this statement is the fact that the largest number of threatened taxa (60) occurs in wet habitats, especially in the peat vegetation (21). of 18 extinct and extremely vulnerable taxa, 16 are characteristic of wetlands. the highly fragile are forest ecosystems which are the habitat for 11 endangered taxa. based on the performed analysis we can conclude that it is necessary to protect all wetlands on the vlasina plateau, especially peat vegetation. also, it is necessary to prevent the irrational exploitation of forest cover, which is partly achieved by the declaration of the entire area of outstanding natural landscape features. acknowledgements. this study was supported by the project of ministry of science and technological development of republic of serbia (project no. 143015 – diversity of flora and vegetation in the central balkan peninsula – ecology, chorology and conservation). references апостолова, и., славова, л., 1997: конспект на растителните съобщества в българия (compendium of 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zaštitu prirode r srbije, 62-63. jakšić, p.: a contribution to the knowledge of the lepidoptera fauna of eastern serbia. biologica nyssana, 8 (1), september 2017 biologica nyssana 8 (1)  september 2017: 113-122 jakšić, p.  a contribution to the knowledge of the lepidoptera… 113 original article received: 02 july 2017 revised: 18 august 2017 accepted: 30 august 2017 a contribution to the knowledge of the lepidoptera fauna of eastern serbia predrag jakšić university of niš, faculty of science and mathematics, department of biology and ecology, višegradska 33, niš, serbia * e-mail: jaksicpredrag@gmail.com abstract: jakšić, p.: a contribution to the knowledge of the lepidoptera fauna of eastern serbia. biologica nyssana, 8 (1), september 2017: 113-122. the faunistic review of lepidoptera, heterocera in the eastern serbia area, with ecological characteristics and zoogeographical distribution is presented. author gives details of lepidoptera species collected in the years 1998, 2014 and 2016 respectively. in total, 44 species are registered, of which there were 1tineidae, 1 limacodidae, 1 cossidae, 1 pterophoridae, 1 thyrididae, 2 lasiocampidae, 1 sphingidae, 1 drepanidae, 13 geometridae, 4 notodontidae, 8 erebidae, 8 noctuiodae and 1 nolidae species. some of them show narrow distribution, of which 15 are recorded for the second, or third, time in serbia. illustration of some habitats, adults and genitalia slides are given. key words: lepidoptera, heterocera, eastern serbia apstrakt: jakšić, p.: prilog poznavanju faune lepidoptera u istočnoj srbiji. biologica nyssana, 8 (1), septembar 2017: 113-122. prikazani su rezultati faunističkih istraživanja lepidoptera, heterocera u istočnoj srbiji, sa njihovim ekološkim i zoogeografskim osobenostima. detalji se odnose na vrste lepidoptera sakupljenih tokom 1998., 2014. i 2016. godine. ukupno je prikazano prisustvo 44 vrste, među kojima su brojčano po familijama predstavnici 1tineidae, 1 limacodidae, 1 cossidae, 1 pterophoridae, 1 thyrididae, 2 lasiocampidae, 1 sphingidae, 1 drepanidae, 13 geometridae, 4 notodontidae, 8 erebidae, 8 noctuiodae i 1 nolidae vrsta. neke od utvrđenih vrsta pokazuju usku distribuciju, a 15 njih pronađeno je po drugi ili treći put u srbiji. date su i ilustracije staništa, pojedinih utvrđenih vrsta in situ i genitalne armature mužjaka. ključne reči: lepidoptera, heterocera, istočna srbija introduction the basin of nišava river in eastern serbia, with the associated mountain ranges, is very interesting biogeographically. on the other site, basin is still insufficiently explored when it comes to its lepidoptera fauna. the first field research from this area were compiled by m. hilf in 1894-1896. it is the area between niš and bela palanka (then called akpalanka). hilf collected a total of 109 species of 8 (1) • september 2017: 113-122 doi: 10.5281/zenodo.964535 biologica nyssana 8 (1)  september 2017: 113-122 jakšić, p.  a contribution to the knowledge of the lepidoptera… 114 lepidoptera, of which 58 species are moths and 51 are butterflies. the material was processed and published by r e b e l (1903, 1904). the following contribution to the knowledge of the fauna was given than by the hungarian natural history museum's preparator józsef uhl who collected material in the vicinity of niš and pirot. the results were published in 1903. t o d o r o w a & p e t k o f f (1915) published faunal studies of dimitrovgrad (then named caribrod) and its environment. r a d o s a v l j e v i ć (1924) gives a contribution to the knowledge of harmful lepidoptera species in this area. after the world war ii v a s i ć (1948) published a contribution of gradation (outbreaks) of harmful moths. the works of those authors can be considered as the pioneer contributions. material and methods specimens were collected with butterfly net and light trap, using “philips” mercury vapor lamp, 125 w. the localities and coordinates where the lepidoptera were collected were obtained using garmin-trex vista gps device. the material was sampled on the following localities: pirot, vidlič mt., hotel “stara”, 1040 m, fn38, 43° 10' 46" (n), 22° 41' 30" (e); pirot, dojkinci, ponor, 1550 m, fn49, 43° 15' 24" (n), 22° 48' 34" (e) (fig. 1); pirot, vidlič mt., crni vrh, 1116 m, fn38, 43° 11' 08" (n), 22° 38' 58" (e); pirot, vidlič, kitka – čuka, 957 m, fn38, 43° 11' 16" (n), 22° 38' 35" (e) (fig. 2); suva planina, bojanine vode, 850 m, en88, 43° 13' 19" (n), 22° 06' 39" (e); suva planina, devojački grob, 1317 m, en88, 43° 11' 59" (n), 22° 08' 14" (e) and knjaževac, staro selo village, dvoja vrata cave, 451 m, fp13, 43° 40' 34" (n), 22° 22' 55" (e). fig. 1. stara planina mt. – ponor near dojkinci the photos in situ of specimens were taken using nikon camera with af-s micro nikkor lens. after setting, we determined the specimens by the wing-patterns. male genitalia were examined for species that cannot be reliably identified based exclusively on wing morphology. the preparations were carried out following the well-known standard procedure: maceration by boiling in potash, dissecting and cleaning, clearing in xylolum and mounting in canada balsam. the photographs of the genital structure were taken using a "leica dm 1000" microscope with a "camera leica dfc 290". all the material (specimens and genitalia slides) is deposited in the author's collection. fig. 2. vidlič mt. near pirot the taxonomic order is done according to n i e u k e r k e n et al. (2011) and f i b i g e r et al. (2012). nomenclature and id number before the species follows k a r s h o l t & r a z o w s k i (1996) ecological preferences and biotopes were done according to c a r l et al. (2005). estimation by analogy for missing species was done by author. fieldwork in protected areas was realized in agreement with permits provided by the ministry of environment, mining and spatial planning, republic of serbia, no. 353-01-1559-2011-03, dated from 8. 06. 2011; no. 353-01-1070/2012-03, dated from 12. 06. 2012.; no. 353-01-916/2014-08, dated from 29.05.2014.; no. 353-01-356/2015-17, dated from 27. 04. 2015 and no. 353-01-389/2016-17, dated from 08. 04. 2016. results ordo lepidoptera fam. tineidae 724. euplocamus anthracinalis (scopoli, 1763) material examined: pirot, vidlič mt., crni vrh, 1130 m, 30. v 2016., 1 male. (fig. 3). this species is well known in serbia (j a k š i ć , 2016).the larva is mycophagous, inhabit rotting wood, which they eat bracket fungi growing on it and dead wood. habitat: e. anthracinalis inhabit humid woodland, as well as biologica nyssana 8 (1)  september 2017: 113-122 jakšić, p.  a contribution to the knowledge of the lepidoptera… 115 thermophilous forests of fagus, quercus and crataegus. fig. 3. euplocamus anthracinalis (scopoli, 1763), pirot, vidlič mt., crni vrh, 1130 m, 30. v 2016. fam. limacodidae 3907. apoda limacodes (hufnagel, 1766) material examined: pirot, vidlič mt., hotel “stara”, 1040 m, 6.vii 2016., 1 male, 5 females. this species is well known in serbia (j a k š i ć , 2016). caterpillars is oligophagous, feed on quercus and fagus species. according to ecological amplitude this species is eurytope (wide ecological amplitude). habitat: a. limacodes inhabit deciduous forests. fam. cossidae 4151. cossus cossus (linnaeus, 1758) material examined: pirot, vidlič mt., hotel “stara”, 1040 m, 6.vii 2016., 1 male. this species is well known in serbia (j a k š i ć , 2016). the larva is oligophagous, feed in the branches of a wide variety of trees. ecological amplitude: eurytope. habitat: woodland biotope, deciduous forests. fam. pterophoridae 5378. gillmeria ochrodactyla ([denis & schiffermüller], 1775) (syn.: g. tatradactyla (linnaeus, 1758)) material examined: pirot, vidlič mt., crni vrh, 1130 m, 6. vi 2016., 1 male (fig. 4). so far, this species is known in serbia only from žljeb mt. (r e b e l , 1917b). vidlič mt. is second known locality of g. ochrodactyla in serbia. this is a monophagous species, larval food-plant is tanacetum vulgare. m a r k o v i ć (2014) reported both t. vulgare, t. corymbosum and t. macrophyllum on vidlič mt. they have restricted ecological amplitude. this species inhabit woodland biotopes along edges of thermophilous forest. fig. 4. gillmeria ochrodactyla denis & schiffermüller, 1775, pirot, vidlič mt., crni vrh, 1130 m, 6. vi 2016 fam. thyrididae 5562. thyris fenestrella (scopoli, 1763) material examined: suva planina mt., devojački grob, 1317 m, 4. vii 2016., 1 male. this species is well known in serbia (j a k š i ć , 2016). the larva is monophagous, feed on clematis vitalba. j o v a n o v i ć (1980) reported this plant species on suva planina mt. this is a species with restricted ecological amplitude. habitat: hedgerows and related biotopes. fam. lasiocampidae 6744. malacosoma castrensis (linnaeus, 1758) material examined: pirot, vidlič mt., hotel “stara”, 1040 m, 6.vii 2016., 1 male. so far, b e s h k o v (2015c) reported this species for western serbia, prijepolje region, zvijezda village, savina voda near jabuka pass, 1117 m. the caterpillars are oligophagous and feed on caryophyllaceae, chenopodiaceae, betulaceae, plantaginaceae et cetera. malacosoma castrensis is eurytope, it has wide ecological amplitude. it inhabits woodland biotopes, beech dominated forest. 6834. hyloicus pinastri (linnaeus, 1758) material examined: pirot, vidlič mt., hotel “stara”, 1040 m, 6.vii 2016., 1 male. this species is well known in serbia (z e č e v i ć , 1996). hyloicus pinastri is polyphagous species, larval food plants are different conifer species. they have wide ecological biologica nyssana 8 (1)  september 2017: 113-122 jakšić, p.  a contribution to the knowledge of the lepidoptera… 116 amplitude, inhabit pine forest, fir-tree dominated forest, spruce forests, et cetera. fam. sphingidae 6843. macroglossum stellatarum (linnaeus, 1758) material examined: suva planina mt., devojački grob, 1317 m, 4. vii 2016., 1 male. this species is well known in serbia (z e č e v i ć , 1996). for this polyphagous species larval food plants are galium, rubiatinctoria and stellaria. j o v a n o v i ć (1980) reported g. aparine, g. mollugo, g. sylvaticum, g. aristatum, s. holostea, and others on suva planina mt. ecological amplitude: eurytope, wide ecological amplitude. they inhabit woodlands biotopes. fam. drepanidae 7505. watsonalla cultraria (fabricius, 1775) material examined: pirot, vidlič mt., hotel “stara”, 1040 m, 6.vii 2016., 1 male (genitalia slide sr2855). so far, only g r a d o j e v i ć (1926) reported this species for serbia, south kučaj mt. the larvae is monophagous, feed on fagus species. ecological amplitude: eurytope, wide ecological amplitude. biotope: woodland biotopes, deciduous forests. fam. geometridae 7522. abraxas grossulariata (linnaeus, 1758) material examined: pirot, vidlič mt., hotel “stara”, 1040 m, 6.vii 2016., 1 male. this species is well known in serbia (z e č e v i ć , 1996). recorded foodplants for their caterpillars are ribes rubrum, r. nigrum, prunus spinosa, crataegus, corylus, salix and euonymus europaeus, this is a polyphagous species. ecological amplitude: eurytope, wide ecological amplitude. biotope: woodland biotopes, deciduous forests. 7537. heliomata glarearia ([denis & schiffermüller], 1775) material examined: pirot, vidlič mt., hotel “stara”, 1040 m, 6.vii 2016., 1 male. r e b e l (1904) reported this species on the basis of hilf's material. the caterpillars are olygophagous, feed on different fabaceae (trifolium, medicago, lathyrus, hippocrepis). heliomata glarearia is an stenotope species, with restricted ecological amplitude. they inhabit meadows and pastures biotopes. 7559. narraga tessularia (metzner, 1845) material examined: pirot, vidlič mt., crni vrh, 1100-1116 m, 30.v 2016., 1 female (genitalia slide sr-2850); 6.vi 2016., 3 males. so far, only beshkov (2017) reported this species for the same area, as a new for serbia. the species is locally distributed in the forest belt, up to 1000 m above sea level, occurring in forest margins. it is monophagous, larva feed on artemisia maritima and a. campestris. our photo shown adult specimens on a. alba turra (fig. 5a). variation in the signum shape in female genitalia (fig. 5b) is significant, our specimen is closely related to female from austria (s k o u & s i h v o n e n , 2015). they have restricted ecological amplitude. this species inhabit meadows biotopes along edges of thermophilous forest. fig. 5. narraga tessularia (metzner, 1845): a-adult specimens (pirot, vidlič mt., crni vrh, 1100-1116 m, 30.v 2016) (photo p. jakšić); b-the signum (genitalia slide sr-2850, photo ivan gnjatović) biologica nyssana 8 (1)  september 2017: 113-122 jakšić, p.  a contribution to the knowledge of the lepidoptera… 117 7790. cleorodes lichenaria (hufnagel, 1767) material examined: vidlič mt., hotel “stara”, 1040 m, 6.vii 2016., 1 male, 1 female. this species is well known in serbia (z e č e v i ć , 1996). cleorodes lichenaria is lichen-eating moth. ecological amplitude: stenotope, restricted ecological amplitude. this species inhabit woodland biotopes. 7961. aplasta ononaria (fuessly, 1783) material examined: pirot, vidlič mt., hotel “stara”, 1040 m, 6.vii 2016., 1 male (genitalia slide sr-2857, fig. 6). male genitalia are illustrated in c a n (2010), our specimen belong to the same type. this species is well known in serbia (z e č e v i ć , 1996). the larva is oligophagous, feed on different papilionoceae: sarothamnus scoparius and ononis repens. m a r k o v i ć (2014) reported ononis pusilla, o. spinosa and o. arvensis on vidlič mt. ecological amplitude is wide. they inhabit woodland biotopes of mesophilous forests. fig. 6. aplasta ononaria (fuessly, 1783), pirot, vidlič mt., hotel “stara”, 1040 m, 6.vii 2016., (genitalia slide sr-2857, photo ivan gnjatović) 8269. catarhoe putridaria (herrich-schäffer, 1852) material examined: pirot, vidlič mt., hotel “stara”, 1040 m, 6.vii 2016., 1 male (genitalia slide sr-2856, fig. 7). in serbia this species is known only from the same locality, described by b e s h k o v (2015) as a new for serbia. the larval food-plants are different rubiaceae, this is oligophagous species. according to m a r k o v i ć (2014) there are 21 rubiaceae species on vidlič mt. ecological amplitude: eurytope. biotope: wetland biotopes, woodland biotopes with deciduous forests and hedgerows and related biotopes. fig. 7. catarhoe putridaria (herrich-schäffer, 1852), pirot, vidlič mt., hotel “stara”, 1040 m, 6.vii 2016. (genitalia slide sr-2856, photo ivan gnjatović) 8272. epirrhoe tristata (linnaeus, 1758) material examined: pirot, vidlič mt., hotel “stara”, 1040 m, 6.vii 2016., 1 male. this species is well known in serbia (z e č e v i ć , 1996). the larval foodplants are galium species, like previous moth species. hibernates as pupa in a cocoon on the ground. ecological amplitude: eurytope. biotope: woodland biotopes with deciduous forests. 8319. cosmorhoe ocellata (linnaeus, 1758) material examined: pirot, vidlič mt., hotel “stara”, 1040 m, 6.vii 2016., 1 male, 1 female. this species is well known in serbia (z e č e v i ć , 1996). caterpillars are monophagous, feeding on a single genus – galium species, like previous moth species. ecological amplitude: eurytope species. biotope: wetland biotopes, woodland biotopes, meadows and pastures, as well as intensively used agricultural areas. 8350. cidaria fulvata (forster, 1771) material examined: vidlič mt., hotel “stara”, 1040 m, 6.vii 2016., 1 male, 1 female. this species is well known in serbia (z e č e v i ć , 1996). c. fulvata is monophagous species, the larval food-plants are different rosaceae species. ecological amplitude: eurytope. biotope: woodland biotopes and cultural biotopes (gardens, parks, groves). 8427. triphosa sabaudiata (duponchel, 1830) material examined: knjaževac, staro selo village, dvoja vrata cave (pećina dvoja vrata), 9.ix 1998., 2 males, 2 females. the adults hibernate and live from july to may. hibernation takes place in rather dry caves, like dvoja vrata cave. this species is well biologica nyssana 8 (1)  september 2017: 113-122 jakšić, p.  a contribution to the knowledge of the lepidoptera… 118 known in serbia (z e č e v i ć , 1996). the larval-food plants are rhamnus cathartica, r. saxatilis, r. orbiculata, r. alpina and frangula alnus, this is monophagous species. ecological amplitude: stenotope. biotope: areas with rock formations. 8457. perizoma hydrata (treitschke, 1829) material examined: pirot, vidlič mt., hotel “stara”, 1040 m, 6.vii 2016., 1 male. this species is well known in serbia (z e č e v i ć , 1996). oligophagous species, the larval food-plants are silene nutans, s. inflata, lychnis viscaria and melandrium species, well presented in the flora of vidlič mt. (m a r k o v i ć , 2014). ecological amplitude: stenotope. biotope: rock formations, scree formations. 8620. aplocera plagiata (linnaeus, 1758) material examined: pirot, vidlič mt., hotel “stara”, 1040 m, 6.vii 2016., 1 female. this species is well known in serbia (z e č e v i ć , 1996). the larva of this polyphagous species feed on solidago virgaureae, calluna vulgaris, silene vulgaris, echium vulgare centaurea species and cirsium species. ecological amplitude: eurytope. biotope: deciduous forests and coniferous forests. 8624. aplocera praeformata (hübner, 1826) material examined: pirot, vidlič mt., hotel “stara”, 1040 m, 6.vii 2016., 1 female. this species is well known in serbia (z e č e v i ć , 1996). aplocera praeformata is monophagous species. the larva feed on different hypericum species. according to m a r k o v i ć (2014) there are 8 hypericum species on vidlič mt. ecological amplitude: eurytope. biotope: hedgerows and related biotopes, as well as meadows and pastures. fam. notodontidae 8709. furcula bicuspis (borkhausen, 1790) material examined: pirot, vidlič mt., hotel “stara”, 1040 m, 6.vii 2016., 1 male. d o d o k (1997) reported that f. bicuspis has been found at užice and jelova gora mt. this is oligophagous species: species feeding on a single family. larval food-plants are betula pendula and alnus glutinosa. m a r k o v i ć et al. (2010) reported both species for area of eastern serbia. ecological amplitude: eurytope. biotope: beech dominated forests, mountainous green alder shrubs. 8738. ptilodon capucina (linnaeus, 1758) material examined: stara planina mt., dojkinci, ponor, 1550 m, 5. vii 2016., 1 female. this species is well known in serbia (z e č e v i ć , 1996). caterpillars of this olygophagous species feed on carpinus, corylus, quercus, fagus, acer, betula, alnus and other. ecological amplitude: eurytope. biotope: woodland biotopes and cultural biotopes (gardens, parks, groves). 8739. ptilodon cucullina ([denis & schiffermüller], 1775) material examined: pirot, vidlič mt., hotel “stara”, 1040 m, 6.vii 2016., 1 female. l a z a r e v i ć (1898) reported this species for the vicinity of beograd. p. cucullina is a polyphagous species. larval foodplants are acer pseudoplatanus and acer campestre, both present on explored area (m a r k o v i ć , 2014). ecological amplitude: eurytope. biotope: deciduous trees/shrubs. 8750. phalera bucephala (linnaeus, 1758) material examined: pirot, vidlič mt., hotel “stara”, 1040 m, 6.vii 2016., 1 female. this species is well known in serbia (z e č e v i ć , 1996). caterpillars live gregariously and feed on a number of different deciduous trees (tilia, quercus, corylus, salix spp.). ecological amplitude: eurytope. biotope: deciduous trees/shrubs. fam. erebidae 8874. catocala nupta (linnaeus, 1767) material examined: knjaževac, staro selo village, dvoja vrata cave (= pećina dvoja vrata), 9.ix 1998., 1 female. this species is well known in serbia (z e č e v i ć , 1996). the larval food-plants are salix alba, s. fragilis and populus nigra. it is polyphagous species – species feeding on a single family. ecological amplitude: eurytope. biotope: wetland biotopes, woodland biotopes. 8882. catocala promissa(schiffermüller, 1775) material examined: pirot, vidlič mt., hotel “stara”, 1040 m, 6.vii 2016., 1 female. this species is well known in serbia (z e č e v i ć , 1996). the caterpillars are monophagous, feed on quercus species. ecological amplitude: eurytope. biotope: woodland biotopes, deciduous forests: thermophilous forests. 8888. catocala nymphagoga (esper, 1787) material examined: pirot, vidlič mt., hotel “stara”, 1040 m, 6.vii 2016., 1 female. b e s h k o v (2015c) recently reported this species from suva planina mt., bojanine vode. the caterpillars are monophagous, feed on quercus species. ecological amplitude: eurytope. biotope: woodland biotopes, deciduous forests: thermophilous forests. biologica nyssana 8 (1)  september 2017: 113-122 jakšić, p.  a contribution to the knowledge of the lepidoptera… 119 8984. scoliopteryx libatrix (linnaeus, 1758) material examined: knjaževac, staro selo village, dvoja vrata cave (pećina dvoja vrata), 9.ix 1998., 2 females. the adults hibernate in caves. scoliopteryx libatrix is well known in serbia (z e č e v i ć , 1996). during the winter s. libatrix hibernates in dark, cool places. the larval food-plants are salix and populus species, this is oligophagous species. ecological amplitude: eurytope. biotope: woodland biotopes: floodplain forests, moorland forests, as well as cultural biotopes (gardens, parks, groves). 10375. lymantria monacha (linnaeus, 1758) material examined: pirot, vidlič mt., hotel “stara”, 1040 m, 6.vii 2016., 1 male. this species is well known from western serbia (z e č e v i ć , 1996). lymantria monacha is a major pest of broadleaved and coniferous trees in europe and asia (pinus, picea, larix and abies are preferred hosts), feed on needles or leaves and can defoliate host trees. this is polyphagous species. ecological amplitude: stenotope. biotopes: woodland biotopes. 10489. eilema lurideola (zincken, 1817) material examined: pirot, vidlič mt., hotel “stara”, 1040 m, 6.vii 2016., 2 males (genitalia slides sr2745 and sr-2746). this species is well known from western serbia (z e č e v i ć , 1996; j a k š i ć & d i m o v i ć , 2000). the caterpillars are mycophagous, feeds on various lichens, including parmelia. ecological amplitude: eurytope. biotopes: woodland biotopes. 10579. rhyparia purpurata (linnaeus, 1758) material examined: suva planina mt., devojački grob, 1317 m, 25. vii 2014., 1 female. b e s h k o v (2015c; b e s h k o v & n a h i r n i ć , 2016) reported this species from pirot, kamenolom kitka and pirot, krupac village, as well as from starac mt., preševo district. the caterpillars feed calluna and other herbaceous plants and deciduous trees, they are polyphagous. ecological amplitude: stenotope. biotopes: woodland biotopes, hedgerows and related biotopes. 10603. callimorpha dominula (linnaeus, 1758) material examined: pirot, vidlič mt., hotel “stara”, 1040 m, 6.vii 2016., 1 female. this species is well known from western serbia (z e č e v i ć , 1996). the caterpillars feed on herbaceous plants, especially symphytum spp. m a r k o v i ć (2014) reported s. tuberosum and s. officinale on vidlič mt. ecological amplitude: stenotope. biotopes: this species inhabits humid clearings in wet forests, bog margins, beech dominated forests et cetera. fam. noctuidae 9307. amphipyra pyramidea (linnaeus, 1758) material examined: pirot, vidlič mt., hotel “stara”, 1040 m, 4. vii 2016., 1 male and 6.vii 2016., 1 female. this species is well known in serbia (zečević, 1996). the larva is polyphagous, feeds on a variety of trees and schrubs: fraxinus, ligustrum, lonicera, malus, quercus, rhododendron, rosa, sorbus, syringa et cetera. ecological amplitude: eurytope. biotope: woodland biotopes: floodplain forests, moorland forests, deciduous forests. 9453. hoplodrina respersa ([denis & schiffermüller], 1775) material examined: pirot, vidlič mt., hotel “stara”, 1040 m, 6.vii 2016., 1 male. so far, only v a s i ć (1969) reported this species from deliblato sands. the caterpillars are active at night and can be found on hippocrepis comosa. a d a m o v i ć (1909) reported this species for eastern serbia. ecological amplitude: stenotope. biotope: thermophilous forests, pine forests, scree formations and xerophilous meadows and pastures. 9515. actinotia polyodon (clerck, 1759) material examined: suva planina mt., bojanine vode, 850 m, 4.vii 2016., 1 female. so far, this species is known only from deliblato sands (g r a d o j e v i ć , 1963). the caterpillar feed on hypericum perforatum and astragalus glycyphyllos, they are polyphagous. j o v a n o v i ć (1980) reported this plant species for suva planina mt.ecological amplitude: stenotope. biotope: hedgerows and related biotopes. 9550. cosmia trapezina (linnaeus, 1758) material examined: pirot, vidlič mt., hotel “stara”, 1040 m, 6.vii 2016., 1 male (genitalia slide sr2780). this species is well known in serbia (z e č e v i ć , 1996). the caterpillars are polyphagous, feed on carpinus, betula, quercus, fagus, ulmus, pyrus, corylus avellana, and others. ecological amplitude: eurytope. biotope: woodland biotopes: floodplain forsts, moorland forests, deciduous forests. 9642. brachylomia viminalis (fabricius, 1777) material examined: pirot, vidlič mt., hotel “stara”, 1040 m, 6.vii 2016., 1 male, 2 females. this species is well known from western serbia (z e č e v i ć , biologica nyssana 8 (1)  september 2017: 113-122 jakšić, p.  a contribution to the knowledge of the lepidoptera… 120 1996). the larvae are monophagous, feed on salix species. ecological amplitude: eurytope. biotope: woodland biotopes. 9781. oligia versicolor (borkhausen, 1792) material examined: pirot, vidlič mt., hotel“stara”, 1040 m, 6. vii 2016., 3 males, 1 female. b e s h k o v (2015c) reported this species from suva planina, bojanine vode; pirot, stone-pit kitka and pirot, krupac village. the larvae is polyphagous, feed on deciduous tree and schrubs, brachypodium sylvaticum, carex sp., poaceae, luzula luzuloides et cetera. ecological amplitude: stenotope. biotope: wetland biotopes, floodplain forests, moorland forest. 10100. noctua fimbriata (schreber, 1759) material examined: pirot, vidlič mt., hotel “stara”, 1040 m, 6.vii 2016., 1 female. this species is well known from western serbia (z e č e v i ć , 1996). the larvae are polyphagous and feed on ligustrum, primula, rubus, rumex, salix, trifolium and urtica species. ecological amplitude: eurytope. biotope: woodland biotopes, meadows and pastures. 10372. colocasia coryli (linnaeus, 1758) material examined: pirot, vidlič mt., hotel “stara”, 1040 m, 6.vii 2016., 2 males, 1 female. this species is well known from western serbia (z e č e v i ć , 1996). the larval food-plants are carpinus betulus, corylus avellana, fagus sylvatica, quercus robur, et cetera, this is polyphagous species. ecological amplitude: eurytope. biotopes: floodplain forests, moorland forests, deciduous forests, hedgerows and related biotopes. fam. nolidae 10427. nola cucullatella (linnaeus, 1758) material examined: pirot, vidlič mt., hotel “stara”, 1040 m, 6.vii 2016., 1 males (genitalia slide sr2860). male genitalia are illustrated in h a c k e r et al. (2012). this species is well known from western serbia (z e č e v i ć , 1996). the caterpillars feed on crataegus sp., malus sp. and prunus spinosa. ecological amplitude: eurytope. biotopes: woodland biotopes. discussion the results are related to geographical area of east serbia that gravitates to catchment of nišava river basin. the area is located in the contact zone of three mountain systems: rhodopy mountain system, carpathian mountain system and balkan mountain range. climate impact also varies from continental climate to the sub-mediterranean climate. nišava river basin is influenced by pontic and mediterranean region through struma river valley. its position suggests faunal richness but its lepidoptera fauna has not been sufficiently explored to the present day. results given by b e s h k o v (2017), as well as results shown here suggest existence of significant faunal richness. presence of 43 species of lepidoptera (heterocera) from 13 families is shown. among the established species 14 (35%) are rare and found only in 2-3 locations in serbia so far: gillmeria ochrodactyla, malacosoma castrensis, watsonalla cultraria, heliomata glarearia, narraga tessularia, catarhoe putridaria, epirrhoe tristata, furcula bicuspis, ptilodon cucullina, catocala nymphagoga, hoplodrina respersa, actinotia polyodon, oligia versicolor and rhyparia purpurata. rich fauna is significant part conditioned by the existence of differentiated biotope and ecological niches. the good examples are the species related to the calcareous rocky habitats. besides there is noticeable connection with neighboring mountain areas such as dinarides (which are also limestone surface). although geographically distant, the two systems have common elements of lepidoptera fauna. for example we can mention species of adscita albanica distributed on mountain paštrik mt. (dinarides) and suva planina mt. (rhodope) (n a h i r n i ć et al., 2016); gillmeria ochrodactyla distributed on žljeb mt. (dinarides) and vidlič mt. (rhodope); malacosoma castrensis distributed on prijepolje, zvijezda (dinarides) and vidlič mt. (rhodope). it is interesting to point out that listed species in geographical area close to kopaonik mt. and šar-planina mt. have not been found because it is not the limestone bedrock. biogeographical connection of the tested area exists with carpathian system based again on the limestone. examples of such species are watsonalla cultraria present in kučaj mt. and vidlič. thanks to erosion processes in the limestone cavernicolous facilities are developed. therefore the list of these species are also petrophilous species: triphosa sabaudiata, catocala nupta and scoliopteryx libatrix. all these species are troglophile. however the real number of species of environmental group is much higher. in neighboring bulgarian caves b e s h k o v and p e t r o v (1996) have determined the presence of 32 species of lepidoptera. special environmental group is comprised by species whose caterpillars are fed by lichen, lichen eating moths. typical representatives are: cleorodes lichenaria and eilema lurideola. environmentally biologica nyssana 8 (1)  september 2017: 113-122 jakšić, p.  a contribution to the knowledge of the lepidoptera… 121 specific species is also euplocamus anthracinalis, whose caterpillars are mycophagous. to summarize we can say that shown fauna species represent significant contribution to the knowledge of its distribution in serbia. also these findings indicate a high potential for faunal species of this area. this indicates that further faunistic research will result in new and significant findings. acknowledgements. i am thankful to dr bojan zlatković for the primary identification of artemisia alba turra. i also thank to ana nahirnić and ivan gnjatović for their valuable assistance and useful advices. this investigation was partly supported by scientific society biodat alpin from innsbruck, austria, as well as university of maribor, faculty of natural sciences and mathematics (project biodiversity of the green lacewing in serbia, grants nos. rp biodiv chryser 2015). references adamović, l. 1909: die vegetationsverhältnisse der balkanländer (mösische länder). in: engler, a. & drude, o. 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(istočna srbija) nađene u periodu 1954 – 1992. godine. razvitak, xxxiii (1–2): 22–30. zaječar. zečević, m., 1996: pregled faune leptira srbije. nauka i institut za istraživanja u poljoprivredi srbija. pp. 1–157. beograd. zečević, m., radovanović, s. 1974: leptiri timočke krajine (makrolepidoptera). zavod za poljoprivredu zaječar i novinska ustanova “timok”. str. 1–183. zaječar. bakić et al. 2023, biologica nyssana 14(2) 14 (2) december 2023: 107-116 doi: 10.5281/zenodo.10259712 pollen of allergenic plants in honey samples from bosnia and herzegovina original article velida bakić department of biology, faculty of science, university of sarajevo, zmaja od bosne 33, sarajevo, bosnia and herzegovina velida.durmic@yahoo.com (corresponding author) sabina trakić department of biology, faculty of science, university of sarajevo, zmaja od bosne 33, sarajevo, bosnia and herzegovina edina muratović department of biology, faculty of science, university of sarajevo, zmaja od bosne 33, sarajevo, bosnia and herzegovina samir đug department of biology, faculty of science, university of sarajevo, zmaja od bosne 33, sarajevo, bosnia and herzegovina received: april 30, 2023 revised: november 06, 2023 accepted: november 26, 2023 abstract: honey plants are characterized by the specific chemical composition of nectar and pollen grains, which depends on the physiological characteristics of plant species, ecological conditions, and anthropogenic pressure. in total, 100 honey samples were analyzed according to the rulebook on methods for the control of honey and other bee products of bosnia and herzegovina. the melissopalynological analysis included plant identification based on the micromorphological characters of pollen grains and the number of pollen grains for each identified plant. in total, 31,183 pollen grains were detected in melissopalynological profiles. based on the micromorphological features, 48 plant species, including seven with allergenic potential, were identified. the proportion of pollen grains of allergenic plants was 18.01% (5,616 pollen grains). both the quality and health of honey are reduced due to presence of allergenic pollen in it which can cause allergic reactions. hence the choice of bee pasture concerning its floristic composition and its distance from the pollution source are crucial parameters for the honey quality. key words: allergy, honey, melissopalinology, pollen apstrakt: polen alergenih biljaka u uzorcima meda iz bosne i hercegovine medonosne biljke karakteriše specifičan hemijski sastav nektara i zrnaca polena, što zavisi od fizioloških karakteristika biljnih vrsta, ekoloških uslova i antropogenog pritiska. analizirano je ukupno 100 uzoraka meda prema pravilniku o metodama kontrole meda i drugih pčelinjih proizvoda bosne i hercegovine. melisopalinološka analiza uključivala je identifikaciju biljaka na osnovu mikromorfoloških karakteristika zrnaca polena i broja zrnaca polena za svaku identifikovanu biljku. ukupno je detektovano 31.183 zrnaca polena u melisopalinološkim profilima. na osnovu mikromorfoloških karakteristika identifikovano je 48 biljnih vrsta, uključujući sedam sa alergenim potencijalom. proporcija zrnaca polena alergenih biljaka bila je 18,01% (5.616 zrnaca polena). kvalitet i zdravstvena bezbednost meda smanjuju se zbog prisustva polena koji može izazvati alergijske reakcije. izbor pčelinje paše, u pogledu florističkog sastava i udaljenosti od izvora zagađenja, ključan je parametar za kvalitet meda. ključne reči: alergija, med, melisopalinologija, polen introduction honey plants’ nectar, honeydew and pollen grains represent an obligatory source of food for bees and ensure their existence, work, and community development (dujmović prugar & hulina, 2007; zima, 2007). plant pollen is the dominant source of protein and fat, but also of various vitamins and minerals for the honeybee, and its origin is crucial for the physiological processes of the bee colony (diniz frias et al., 2016; ljevnaić-mašić et al., 2019). honey plants are characterized by a specific combination of physical and chemical properties of pollen grains and nectar, which depend on the systematic affiliation of the species, physiological characteristics, but also the ecological parameters in which the species exists (evelin et al., 2011). precisely because of its chemical specificity, many authors state that the pollen grains of some types of plants can be one of the strongest natural allergens (d’amato et al., 1991; oswalt et al., 2008; mansouritorghabeh et al., 2019). the allergenic potential of pollen depends on the type of water-soluble proteins and glycoproteins, whose molecular mass ranges from 5 to 70 kda (peternel, © 2023 bakić et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 107 2011; kostić, 2015). numerous studies have shown that allergens, in just a few seconds, leave the pollen grain after contact with the moist mucous membrane (chen et al., 2018). the apiflora of bee grazing is the result of environmental, biological, and anthropogenic factors. for these reasons, the selection of the floristic composition of the honey pasture, as well as its distance from various sources of pollution, is crucial in defining the botanical origin and the physicochemical properties of honey (ball, 2007; sari & ayyildiz, 2013; alibabić et al., 2017). this research aimed to determine a) whether there is and b) how much allergenic plant pollen is present in honey samples originating from bosnia and herzegovina. materials and methods as part of the research, 100 samples of different types of honey from bosnia and herzegovina were collected and analyzed (fig. 1). melissopalynological preparations followed the rulebook (rulebook on methods for the control of honey and other bee products of bosnia and herzegovina, official gazette, 37/09, 2009). according to the rulebook, making of the preparation included the following steps: a solution of 10 g honey and 20 ml distilled water was incubated in a water bath at a temperature of 45 ˚c (julabo sw 22). then the solution was centrifuged for 15 minutes at 3500 rpm. after draining off the supernatant, the sediment was transferred to a glass slide with a micropipette and embedded in glyceringelatin with fuchsin and the preparation was covered 108 biologica nyssana ● 14 (2) december 2023: 107-116 bakić et al. ● pollen of allergenic plants in honey samples from bosnia and herzegovina fig. 1. map of the distribution of the analyzed samples biologica nyssana ● 14 (2) december 2023: 107-116 bakić et al. ● pollen of allergenic plants in honey samples from bosnia and herzegovina 109 detected. the analysis of honey plants in the melissopalynological spectrum identified the pollen of seven allergenic plants. the identified plant species whose pollen has different allergic potentials are ambrosia artemisiifolia l., artemisia vulgaris l., ailanthus altissima (mill.), zea mays l., tilia sp., poa sp., and the moderately allergen species medicago sativa l. (yakhlef et al., 2021) (tab. 1). the total number of pollen grains with allergenic potential is 5.616, or 18.01% of the absolute number of pollen grains in the research. greater number of allergenic plants was observed in the palynological profiles of polyfloral (seven species) types of honey compared to monofloral (six species). in the melissopalynological profiles of polyfloral samples, pollen grains of poa sp., ambrosia artemisiifolia and artemisia vulgaris represent the most important biological and health hazard. as part of the research, a total of 106 allergenic and non-native ragweed pollen grains were identified. ambrosia artemisiifolia pollen grain has a very high allergic potential due to a combination of specific antigens (peternel, 2011). eleven different allergenic proteins found in pollen grains have been identified and named amb a 1 to amb a 11, whose molecular weights are in the range of 9 to 38 kda, of which amb a 1 is the strongest allergen (chen et al., 2018). in addition to allergenic proteins, lactone compounds, flavonoids, monoterpenes, and bitter flavonoids were also detected in pollen grains (kanter et al., 2013). ambrosia artemisiifolia pollen grains were detected in the highest number in the palynological profiles from bosanska krupa (19.23%), sanski most (13.66%), and bihać (6.25%). in this part of the country, this allergenic and invasive plant is marked with a coverslip. for each sample of honey, two parallel preparations were prepared according to the instructions of the official protocol. the method of analysis of melissopalynological preparations acts by the rulebook, as well as the methods proposed by icbb (international commission for bee botany) (von der ohe et al., 2004; rulebook on methods for the control of honey and other bee products of bosnia and herzegovina, official gazette, 37/ 09, 2009). all melissopalynological preparations were analyzed using a wild m20 phase-contrast microscope. the plant species was determined according to the pollen grains’ micromorphological characteristics (hesse et al., 2009). the qualitative analysis included a list of determined honey plants, while the quantitative analysis involved recording the exact number of pollen grains in the sample. samples were classified as monoor polyfloral according to the aforementioned rulebook. based on the results of these analyses, melissopalynological profiles for each slide were created. marking of allergenic species in the palynological spectrum of honey samples was carried out based on the list of allergenic plants (redžić & mehić, 2009). results and discussion in the melissopalinological analysis of 100 honey samples, a total number of 31.183 pollen grains was counted. in the botanical sense, 24 plant families were identified, with the most numerous pollen grains for the families: fabaceae (8.303), fagaceae (4.683) and asteraceae (3.792) (fig. 2). analyzed micromorphological features showed 48 different types of pollen grains. based on the frequency of plant species in the palynological spectrum, 46 polyfloral and 54 monofloral types of honey were fig. 2. total number of pollen grains within families 110 biologica nyssana ● 14 (2) december 2023: 107-116 bakić et al. ● pollen of allergenic plants in honey samples from bosnia and herzegovina table 1. the percentage of pollen of allergenic plants in the investigated localities locality honey type a m br os ia ar te m is io fo lia a rt em is ia vu lg ar is ti lia sp . po a sp . ze a m ay s a ila nt us al tis im a m ed ic ag o sa tiv a drenova glavica m 17% cazin, koprivna m 1% 4% cazin, koprivna m 1% bihać m 1% 1% zalin m 48% kozarac m 2% bosanska krupa m 63% 4% crno jezero m 64% bužim , radoč m 1% 2% prijedor m bihać p buturović polje m 1% bratunac p 35% gračanica p 23% 11% majevica m 22% 10% bosanska krupa p 19% 27% 5% 3% gračanica m 41% 7% gračanica m 6% trebinje, bobani m 12% 6% gradačac m 8% tuzla p 11% 19% trebinje, bobani m bosanski dubačac m 7% bosanska dubica, babinac m bosansko grahovo m bosanka dubica, međeda p 24% cazin m 9% bosanski brod i derventa m popovo polje, ravno m bratunac m 2% prijedor i sanski most m 2% 3% ljubuški m blagaj, kamena m 1% gradačac m 55% 4% nevesinje m 4% modriča m 3% bratunac m 8% bosanski brod, svilaj m 2% biologica nyssana ● 14 (2) december 2023: 107-116 bakić et al. ● pollen of allergenic plants in honey samples from bosnia and herzegovina 111 gradačac m 6% gradačac, novalići p 1% zenica m bosanska krupa m 11% 12% srebrenica m 8% stolac m 9% 10% kakanj m 4% sanski most m 14% 1% 44% ključ, sanica m 50% 4% bosanska krupa, suvaja p 19% bosanska krupa, vranjska m 41% 6% bosanska krupa, benkovac m 63% nevesinje p 5% 25% goražde, milanovići m 33% 10% derventa, zborišta i bosanski brod p 18% 7% 6% olovo p 5% 25% višegrad p 5% bijeljina p 6% 9% cazin p 6% ustiprača, radič p 1% 6% 11% foča m 3% romanija p 4% zenica, nemila m 12% 4% goražde p čelebići m 1% žepa , begići p 4% 2% 10% travnik, karaula p 10% travnik, bijelo buče m 2% travnik, karaula, krčevine p 8% bosanska krupa, jasenica m 17% bosanska krupa m 58% 2% velika kladuša m 2% 4% 1% bosanska krupa, veliki dubovik m 30% 2% bihać p 5% 6% bosanska krupa, zalin m 84% 5% prijedor, petrov gaj p 3% 11% bosanski petrovac p 3% bihać p 6% 6% 1% kostajnica m 29% bosanska krupa m 35% 5% nevesinje p 1% 3% 11% jablanica p 112 biologica nyssana ● 14 (2) december 2023: 107-116 bakić et al. ● pollen of allergenic plants in honey samples from bosnia and herzegovina *m– monofloral, *p– polyfloral pofalići p 7% 7% bosanska krupa p 20% 31% gradačac p 14% 15% majevica p 13% srebrenica, brežani p 10% 9% zavidovići p 12% 11% srebrenica, osmanovići p 1% 15% 11% bosanska krupa p 18% 6% breza, bukovik p 5% bosanski kobaš p 14% 6% trebinje, bobani p brezići p 7% bosanski brod p 6% 10% livno p 20% sarajevo p 4% 3% 8% pofalići p 17% 7% konjic, mostar p 10% 8% bosanska krupa p 23% 11% 3% tarčin p 8% derventa i bosanski brod, zborišta p 8% 8% 3% with code a2 (djug et al., 2019; eppo, 2023). the spread of ragweed represents an important biological problem. its aggressive spreading on honey pastures suppresses native apiflora and reduces diversity or prevents the development of competitively weaker native honey species (šoljan & muratović, 2004; bašić et al., 2017). the pollen of this allergenic plant species, apart from samples from bosnia and herzegovina, was confirmed in numerous honey samples from croatia (štefanić et al., 2013; rašić et al., 2018), albania (pupuleku et al., 2016) and serbia (nedić et al., 2022) which, according to the european food safety agency (efsa, 2010), represents a serious health problem. a total of 178 mugwort (a. vulgaris) pollen grains were also determined. mugwort pollen has a high allergenic potential and causes the development of various allergic symptoms. according to gao et al. (2019), seven allergen components were identified. the most important are art v 1 and art v 3 located in the wall of the pollen grain. allergenic art v 2 is present in the cytoplasm or the pollen coat, while art v 7 is located around the nucleus (gao et al., 2019). the allergenic protein art v 6 shows approximately 58% similarity with the mentioned amb a 1, so there is a cross-reaction between ragweed and mugwort allergens (celenk, 2019). mugwort pollen was detected in ten polyfloral honey samples, with the largest number in samples from bosanka krupa (27.35%), derventa (18.33%) and bosanski brod (6%). it is specific that in the palynological spectrum of the samples, in addition to mugwort pollen, ragweed was also identified as an accessory species, which is an indicator that honey grazing was carried out in places with intense anthropogenic pressure. as part of the research, 1.767 grass (poa sp.) pollen grains were identified. grass pollen has a high allergenic potential due to the presence of 13 different groups of allergens. due to the amount and existence of pollen grains, groups 1 and 5 are considered immunodominant among grass pollen allergens. besides them, there are allergens from group 12 (profilins) and 7 (polcalcin), which belong to the group of proteins responsible for cross-allergic reactions between grass, tree, and other weed pollens (andersson & lidholm, 2003; garcıa-mozo, 2017). grass pollen grains appeared in 62 samples, predominantly in polyfloral (76%), but also in some monofloral (24%) honeys. given that the honey-bearing grazing of polyfloral honey is most desirable in meadows where the grass family generally has its ecological optimum, they are often accessory species in the palynological profiles of this type of honey. the presence of grass family 113 biologica nyssana ● 14 (2) december 2023: 107-116 pollen has been established in numerous studies of honey (silici et al., 2007; mačukanović-jocić & jarić, 2015; pupuleku et al., 2016; alty et al., 2018; rašić et al., 2018), but in individuals with pollen allergies it can trigger allergic or even anaphylactic reactions (martín-muñoz et al., 2010; aguiar et al., 2017). in contrast to polyfloral, monofloral honey was dominated by linden (tilia sp.) and tree of heaven (a. altissima) pollen. a total of 3.433 linden (tilia sp.) pollen grains were identified. it was dominantly represented as an allergenic plant in the research. linden pollen grains condition the development of moderate to strong allergic reactions in humans, and there is also a cross-reaction between linden and known allergenic plants such as birch and olive (ognjenović et al., 2012; ognjenović, 2013). in 2001, the presence of three protein allergens with a molecular weight of 10,23 and 50 kda was confirmed for pollen grains of linden (mur et al., 2001), and in 2021, two more (molecular wight 40 and 80 kda) that can cause sensitization in humans were discovered (tivsiz et al., 2021). linden is one of the most honey-bearing woody plants, it provides generous amounts of pollen and nectar, so bees visit it very often (umeljić, 2015). argoti (2016) detected mannose in the nectar, together with phenols and the alkaloid nicotine, which can have a very toxic, and in some cases lethal, effect on bees (singaravelan et al., 2006; tiedeken et al., 2014; baracchi et al., 2015). however, jacquemart et al. (2018) state that concentrations of mannose and nicotine in linden nectar may not be lethal to bees and indicate that other volatile secondary metabolites may be dangerous. linden pollen, as a primary and accessory honey-bearing species, was found in an significant number of honey samples, but due to its chemical characteristics, has to be consumed with precaution. the pollen of the tree of heaven (a. altissima) was not recognized as dangerous for provoking allergic reactions in recent years. however, several studies have shown that over 125 of its proteins, of which the most active are enolase and pectate lyase, can cause allergic reactions (mousavi et al., 2017; mousavi et al., 2019). considering that it is an invasive plant species, in addition to the allergenic potential of pollen in honey, it represents a great danger for native flora (djug et al., 2019). the presence of pollen from this allergenic, and invasive species, was detected in the melissopalynological profiles of honey from the area of mostar, konjic (8.33%) and stolac (10%). according to boškailo et al. (2017), it is intensively spreading throughout the mediterranean and submediterranean areas. the pollen of the tree of heaven is also detected in honey samples originating from croatia (rašić et al., 2018; zima et al., 2018) and bulgaria (atanassova et al., 2009; tashev et al., 2015). thanks to its eurivalence and allelopathy, the tree of heaven aggressively and quickly conquers new areas and suppresses honey-bearing plant species from their natural habitats, thus reducing the biodiversity of the autochthonous flora of bih (gómez-aparicio et al., 2008). conclusion after the analysis of 100 melissopalynological profiles, a total of 5.616 (18.02%) pollen grains of allergenic plants were found in the honey samples. out of the total number of analyzed samples, pollen from seven allergenic plant species was identified in 88 (88%) palynological profiles. the detected pollen grains of ragweed, mugwort, and the tree of heaven were bioindicators of the area burdened by anthropogenic pressures. honey-bearing grazing in localities with such floristic composition reduces the biological quality of honey due to the presence of allergenic pollen. the selection of the floral composition in honey pastures is crucial to guarantee a botanical origin suitable for the honey we consume. references aguiar, r., duarte, f.c., mendes, a., bartolomé, b. & barbosa, m.p. 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(2018). analiza medonosnosti invazivnih biljnih vrsta požeške bakić et al. ● pollen of allergenic plants in honey samples from bosnia and herzegovina 115 biologica nyssana ● 14 (2) december 2023: 107-116 kotline. 53rd croatian & 13th international symposium on agriculture, 18 -23 february 2018, vodice, croatia, book of proceedings. bakić et al. ● pollen of allergenic plants in honey samples from bosnia and herzegovina 116 biologica nyssana ● 14 (2) december 2023: 107-116 cvetković, v. et al.  toxicity of dimethyl sulfoxide against drosophila… biologica nyssana 6 (2)  december 2015: 91-95 cvetković, v. et al.  toxicity of dimethyl sulfoxide against drosophila… 91 original article received: 11 july 2015 revised: 17 august 2015 accepted: 01 october 2015 toxicity of dimethyl sulfoxide against drosophila melanogaster vladimir j. cvetković1*, tatjana lj. mitrović1, boris jovanović2, slaviša s. stamenković1, miljana todorović1, miljana đorđević3, niko radulović3 1university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia 2chair for fisheries biology and fish diseases, ludwig maximilian university of munich, kaulbachstrasse 37, 80539, munich, germany 3university of niš, faculty of sciences and mathematics, department of chemistry, višegradska 33, 18000 niš, serbia * e-mail: biovlada@yahoo.com abstract: cvetković, v.j., mitrović, t.lj., jovanović, b., stamenković, s.s., todorović, m., đorđević, m., radulović, n.: toxicity of dimethyl sulfoxide against drosophila melanogaster. biologica nyssana, 6 (2), december 2015: 91-95. dimethyl sulfoxide (dmso) is commonly used as a solvent for organic compounds but its toxic properties can affect both in vitro and in vivo studies. thus, evaluation of dmso toxicity must be performed on the model organism before its application as a solvent in the target studies. present study aimed to determine the lethal concentration required to kill 50% of the population in a given period of time (lc50), no observed effect concentration (noec), and lowest observed effect concentration (loec) values for dmso on drosophila melanogaster which is frequently used as a model for toxicity studies. twelve different concentrations of dmso were tested on three-day old d. melanogaster larvae for 12 days. at the end of the life cycle, number of live hatched adults and un-hatched pupae were counted. based on probit analysis 12 days loec was 0.04% v/v, 12 days noec was ˂ 0.04% v/v and 12 days lc50 was 0.42% v/v. the results indicate that dmso may be more toxic to d. melanogaster than it was initially considered. key words: drosophila melanogaster, noec, loec, lc50, dmso, toxicity apstrakt: cvetković, v.j., mitrović, t.lj., jovanović, b., stamenković, s.s., todorović, m., đorđević, m., radulović, n.: toksičnost dimetil sulfoksida na modelu drosophila melanogaster. biologica nyssana, 6 (2), december 2015: 91-95. dimetil sulfoksid (dmso) se često koristi kao organski rastvarač ali njegova toksičnost može da utiče na ishode in vitro i in vivo istraživanja. zato se procena toksičnosti dmso-a mora ispitati na model organizmu pre njegove primene kao rastvarača u željenom istraživanju. ovo istraživanje ima za cilj da se za dmso ustanovi letalna koncentracija koja ubija 50% populacije u posmatranom vremenskom periodu (lethal concentration 50 lc50), koncentracija koja ne uzrokuje detektabilne promene (no observed effect concentration noec) i koncentracija koja uzrokuje najmanje detektabilne promene (lowest observed effect concentration loec) na modelu drosophila melanogaster koji se veoma često koristi u toksikološkim 6 (2) • december 2015: 91-95 biologica nyssana 6 (2)  december 2015: 91-95 cvetković, v. et al.  toxicity of dimethyl sulfoxide against drosophila… 92 istraživanjima. larve d. melanogaster stare tri dana tretirane su sa dvanaest različitih koncentracija dmso-a u periodu od 12 dana. na kraju životnog ciklusa, prebrojani su adulti koji su se izlegli i lutke iz kojih se adulti nisu izlegli. prema probit analizi, za 12 dana tretmana loec je 0.04% v/v, noec je ˂ 0.04% v/v i lc50 je 0.42% v/v. rezultati ukazuju da je dmso toksičniji za d. melanogaster nego što se prvobitno smatralo. key words: drosophila melanogaster, noec, loec, lc50, dmso, toksičnost introduction dmso originates from 19th century as a by-product in the process of paper production from a wood pulp (c a p r i o t t i & c a p r i o t t i , 2012; h o r i t a & w e b e r , 1964). since then, colorless liquid dmso is characterized as a good aprotic solvent which can easily dissolve various non-polar and polar molecules (c a p r i o t t i & c a p r i o t t i , 2012; h o r i t a & w e b e r , 1964). the concern regarding the toxicity of solvents is always a topical issue in toxicology studies. thus, the pre-testing of solvents on certain model organism, which is planned to be used in further toxicology studies, is an imperative. one of the widest and frequently used model organisms in toxicity and genotoxicity studies is fruit fly, drosophila melanogaster (s t a m e n k o v i ć r a d a k et al., 2008; p a t e n k o v i ć et al., 2009; v a l e s et al., 2013). drosophila is a remarkable model for in vivo assays in toxicology because of high number of progeny and its short lifespan. in addition, culturing and maintaining of drosophila is more economical compared to other model organisms. it was demonstrated that administration of the test substances through the feeding medium is simple (n a z i r et al., 2003; s t a m e n k o v i ć r a d a k et al., 2008) making toxicology assays on drosophila even easier. the adverse effects of dmso in drosophila were noticed in the 1980's (m a s s i e et al., 1985; b r o d b e r g et al., 1987) when the authors recommended that dmso must be used carefully as a solvent. therefore, present study aimed to determine the lethal concentration required to kill 50% of the population in a given period of time (lc50), no observed effect concentration (noec), and lowest observed effect concentration (loec) values for dmso on a three-day-old d. melanogaster larvae of the wild-type strain. material and methods test organism in this experiment the d. melanogaster oregon-rc (wild-type flies, stock no. 5) ("bloomington drosophila stock center", indiana university, usa) were used. flies were cultivated on a standard feeding medium for drosophila (9% sugar, 10% corn meal, 2% agar, 2% yeast) with addition of fungicide 2.50 mg ml-1 methyl 4-hydroxybenzoate (nipagin®, alfa aesar gmbh & co kg, germany) diluted in 95% etoh (ethanol). the flies were cultivated in a standard cultivating 200 ml vials on 25 oc, 60% humidity, with 12 h/12 h day-night cycle. adults were allowed to lay eggs during the eight hour period, after that they were removed from the vials. after three days larvae which were 72±4 h old, have been removed from the cultivating medium, washed with distilled water and used in further procedure. test substance and treatment schedule twelve concentrations of dmso (carl roth, germany) were tested in duplicates and each experimental group consisted of 30 three-day-old larvae. therefore, final concentrations of dmso in 3ml of standard feeding medium were: 0.16%, 0.32%, 0.48%, 0.64%, 0.80%, 0.97%, 1.13%, 1.29%, 1.45%, 1.61%, 2.42% and 3.23% v/v. negative, water control, was also included. at the end of incubation time, which lasted up to 12 days, numbers of live hatched units (adults) and un-hatched units (at pupal stadium) were counted. statistical analysis mean values were calculated and used for further analysis. risk assessment software tool (ra v1.0; pensacola, florida, usa) and probit analysis were utilized in order to calculate values for lc50, loec, and noec. also, the confidence intervals (95%) were calculated. results and discussion the toxic effects of dmso, particularly antimicrobial and virucidal properties, were noticed in the 1960's. dmso was shown to inhibit bacterial and fungal growth at concentration of 30-50% and at concentration of 80% inactivated infectivity of some rna and dna viruses (b a s c h & g a d e b u s c h , 1968; c h a n & g a d e b u s c h , 1968; w a g n e r et al., 2002). even nowadays, the adverse properties of dmso, which is used as a solvent in toxicology studies of aquatic model organisms, was noticed (m á c h o v á et al., 2009). m á c h o v á et al. (2009) tried to establish general value for the non-toxic concentration of dmso as a solvent that could be applied across different conditions in aquatic model organism such as fish. but they found that effect of biologica nyssana 6 (2)  december 2015: 91-95 cvetković, v. et al.  toxicity of dimethyl sulfoxide against drosophila… 93 dmso on fish model depends on fish age, species, type of test and indicators observed so they concluded that general value for the non-toxic concentration of dmso can't be found and every experiment should include solvent control (m á c h o v á et al., 2009). it implies that dmso must be pre-tested on certain model organism if it is planned to be used in experiment as solvent. the fig. 1a represents survival plot of d. melanogaster units exposed to different concentration of dmso which unambiguously shows that number of hatched flies decreased in dosedepended manner. fig. 1b represents probability of d. melanogaster units mortality exposed to dmso which was calculated by risk assessment software tool. 12 days lc50 value is 0.42% v/v (tab. 1) and total number of adults hatched from pupal stadium drastically decreased from 0.32% v/v where almost every unit developed into adult to 0.97% v/v where none of the adults hatched. in addition, pupal mortality (un-hatched units) increased drastically in this concentration range, respectively (fig 2). concentration of dmso in substrate, from 0.97% v/v up to the 3.23%, v/v caused increase of larval mortality (fig. 2). concentrations above 0.32% v/v decreased hatchability of adults. these observations are in accordance with similar study (n a z i r et al., 2003). in genotoxicity and toxicity studies larvae, or adults, of d. melanogaster, as a model organisms, are widely used (n a z i r et al., 2003; a d a m s k i et al., 2009; c a s t a ñ e d a p a r t i d a et al., 2011; m i y a z a w a et al., 1998; 2000; g r a f et al., 1984). there are many references on dmso toxicity, but only a few papers are on dmso toxicity in drosophila. for instance, g r a f et al. (1984) used dmso in final concentration of 2% in feeding medium for drosophila in somatic mutation and recombination test (g r a f et al., 1984). but recent research by n a z i r et al. (2003) showed that dmso should be used very cautiously as solvent because it could results with false conclusions in the screening fig 1. survival plot of d. melanogaster units (a) and probability of the units mortality exposed to dmso with 95% confidence intervals (b). table 1. probit analysis: loec, noec and lc50 values with confidence intervals after 12 days of exposure. % of population response concentration in % (v/v) lower limit 95% upper limit 95% loec 12 days 0.01% 0.04555 0.00780 0.09803 0.10% 0.06642 0.01461 0.12884 1.00% 0.10502 0.03122 0.18004 5.00% 0.15806 0.06122 0.24374 10.00% 0.19656 0.08741 0.28734 20.00% 0.25598 0.13387 0.35252 30.00% 0.30967 0.18096 0.41093 40.00% 0.36435 0.23254 0.47149 lc50 12 days 50.00% 0.42404 0.29133 0.54068 biologica nyssana 6 (2)  december 2015: 91-95 cvetković, v. et al.  toxicity of dimethyl sulfoxide against drosophila… 94 fig 2. percentage of hatched (alive adults), un-hatched (non-survived units at pupal stadium) and non-survived units at larval stage (larval mortality) influenced with different concentration of dmso (showed in % v/v) in feeding media and in water (negative) control. of test substances because of its toxicity to d. melanogaster (n a z i r et al., 2003). our results confirmed observation that dmso is toxic and can impact normal larval development in concentration greater than 0.04% v/v (loec value) in feeding medium. the present study was performed with twelve concentrations which provided better resolution of obtained results than in the study by n a z i r et al. (2003) where six concentrations were tested. also, we treated three-day-old larvae of the wild-type strain of d. melanogaster which differs from the study by n a z i r et al. (2003) whose treatment started at the egg stadium of the transgenic strain of d. melanogaster and lasted for 10-14 days. finally, probit analysis was performed as a common analysis in toxicology, in order to determine noec, loec and lc50. n a z i r et al. (2003) considered dmso at 0.3% v/v as noael (no observed adverse effect level) value which can be used as dietary concentration in toxicity studies with d. melanogaster. however, according to present research 12 days loec is 0.04% v/v so the absolutely safe dietary concentration of dmso in feeding medium for d. melanogaster larvae of wildtype strain, should be less than 0.04% v/v. one of the reasons for higher toxicity (and lower value of loec) observed in the present study compared to the n a z i r et al. (2003) may be the lower sensitivity of d. melanogaster eggs to dmso since the eggs do not actively feed on the exposure media thus the cumulative toxicity during same exposure time period is lower. therefore, a more realistic scenario would be to start the exposure during the larval stage, as it was performed in the present study. conclusion the probit analysis showed that 12 days loec was 0.04% v/v, noec was ˂ 0.04% v/v and lc50 was 0.42% v/v. pupal mortality reached maximum at 0.97% v/v of dmso in substrate while concentration greater than 0.32% v/v reduced hatchability of adults at the end of lifespan. these results indicate that dmso is more toxic to d. melanogaster larvae than it was initially thought. obtained lc50, loec and noec values for dmso in this experiment should be considered seriously in further toxicity testing with d. melanogaster larvae as experimental model. acknowledgements. this work was supported by the ministry of education, science and technological development of the republic serbia during activity on project oi171025 and iii41018. also, authors want to thank dr. aleksandra patenković and dr. zorana kurbalija-novičić (institute of biological research, university of belgrade, serbia) who kindly provided drosophila melanogaster oregon-rc strain which was used in this experiment. references adamski, z., błoszyk, j., musiał, j., łysiak, m., urbaniak, l., ziemnicki, k. 2009: diflubenzuron inhibits reproduction of different strains of drosophila melanogaster. insect science, 16 (4): 305-309. 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% p er ce n ta ge o f u n it s concentration of dmso in treatments hatched un-hatched (pupal mortality) larval mortality biologica nyssana 6 (2)  december 2015: 91-95 cvetković, v. et al.  toxicity of dimethyl sulfoxide against drosophila… 95 aguilar, j.s., roy, d., ghazal, p., wagner, e.k. 2002: dimethyl sulfoxide blocks herpes simplex virus-1 productive infection in vitro acting at different stages with positive cooperatively. bmc infectious diseases, 2 (9): 1–10. basch, h. & gadebusch, h.h. 1968: in vitro antimicrobial activity of dimethyl sulfoxide. applied microbiology, 16: 1953-1954. brodberg, r.k., mitchell, m.j., smith, s.l., woodruf, r.c. 1987: specific reduction of n,ndimethylnitrosamine mutagenicity in drosophila melanogaster by dimethyl sulfoxide. environmental and molecular mutagenesis, 10: 425–432. capriotti, k. & capriotti, aj. 2012: dimethyl sulfoxide: history, chemistry, and clinical utility in dermatology. journal of clinical and aesthetic dermatology, 5 (9): 24–26. castañeda-partida, l., heres-pulido, m.e., guzmánrincón, j., hernández-portilla, l.b., dueñasgarcía, i.e., durán-díaz, á., delfín-alcalá, i. 2011: lead acetate does not inhibit dimethylnitrosamine activation and interacts with phenobarbital which is genotoxic in the st cross of the drosophila wing spot test. food and chemical toxicology, 49 (9): 2172-2179. chan, j.c. & gadebusch, h.h. 1968: virucidal properties of dimethyl sulfoxide. applied microbiology, 16: 1625-1626. graf, u., würgler, f., katz, a., frei, h., juon, h., hall, c., kale, pg. 1984: somatic mutation and recombination test in drosophila melanogaster. environmental mutagenesis, 6 (2): 153-188. horita, a. & weber, lj. 1964: skin penetrating property of drugs dissolved in dimethyl sulfoxide (dmso) and other vehicles. life sciences, 3: 1389-1395. máchová, j., prokeš, m., kroupová, h., svobodová, z., mácová, s., doleželová, p., velíšek., j. 2009: early ontogeny, growth and mortality of common carp (cyprinus carpio) at low concentrations of dimethyl sulfoxide. acta veterinaria brno, 78: 505-512. massie, h.r., williams, t.r., lodice, a.a. 1985. influence of antiinflammatory agents on the survival of drosophila. journal of gerontology, 40: 257–260. miyazawa, m., nakamura, y., ishikawa, y. 2000: insecticidal sesquiterpene from alpinia oxyphylla against drosophila melanogaster. journal of agricultural and food chemistry, 48 (8): 36393641. miyazawa, m., yoshio, k., ishikawa, y., kameoka, h. 1998: insecticidal alkaloids against drosophila melanogaster from nuphar japonicum dc. journal of agricultural and food chemistry, 46 (3): 1059-1063. nazir, a., mukhopadhyay, i., saxena, d., chowdhuri, d.k. 2003: evaluation of the no observed adverse effect level of solvent dimethyl sulfoxide in drosophila melanogaster. toxicology mechanisms and methods, 13 (2): 147-52. patenković, a., stamenković-radak, m., banjanac, t., andjelković, m. 2009: antimutagenic effect of sage tea in the wing spot test of drosophila melanogaster. food and chemical toxicology, 47 (1): 180-183. stamenković-radak, m., kalajdzić, p., savić, t., savić, m., kurbalija, z., rasić, g., andjelković, m. 2008: the effect of lead on fitness components and developmental stability in drosophila subobscura. acta biologica hungarica, 59 (1): 47-56. vales, g., demir, e., kaya, b., creus, a., marcos, r. 2013: genotoxicity of cobalt nanoparticles and ions in drosophila. nanotoxicology, 7: 462-468. http://www.sciencedirect.com/science/article/pii/s0278691511002456 biologica nyssana 6 (2)  december 2015: 91-95 cvetković, v. et al.  toxicity of dimethyl sulfoxide against drosophila… 96 živković et al., 2019, biologica nyssana 10(1) 10 (1) september 2019: 29-34 doi: 10.5281/zenodo.3464000 susceptibility of different grapevine cultivars to eutypa lata isolate, causing agent of eutypa dieback, originating from serbia original article sanja živković faculty of agriculture, university of niš, kosančićeva 4, 37000 kruševac, serbia gajicsanja43@gmail.com (corresponding author) tanja vasić faculty of agriculture, university of niš, kosančićeva 4, 37000 kruševac, serbia tanjavasic82@gmail.com jordan marković institute for forage crops, 37251 kruševac, serbia jordan.markovic@ikbks.com received: april 25, 2019 revised: may 17, 2019 accepted: august 13, 2019 abstract: the experiment was established in the greenhouse during the two years. the grapevine cultivars used in experiment were riesling blanc, chardonnay, banatski muskat and gamay noir. the susceptibility of grapevine cultivars to the isolate of the fungi eutypa lata, causing agent of eutyposis, was analysed. one isolate el17 was used in the experiment. the highest susceptibility was found in riesling blanc, banatski muskat and gamay noir cultivars, while chardonnay cultivar was less susceptible to the isolates of fungus eutypa lata. key words: grapevine, eutypa lata, cultivar susceptibility apstract: osetljivost različitih sorti vinove loze na izolat eutypa lata, prouzrokovača eutipoze, poreklom iz srbije eksperiment je izveden tokom dve godine u stakleniku. u eksperimentu su korišćene sorte vinove loze rajnski rizling, šardone, banatski muskat i game crni. ispitivana je osetljivost sorti na izolat gljive eutypa lata prouzrokovača odumiranja čokota vinove loze. u ogledu je korišćen jedan izolat el17. najosetljivijom se pokazala sorta rajnski rizling, banatski muskat i game crni, a sorta šardone pokazala se manje osetljivom na izolate gljive eutypa lata. ključne reči: vinova loza, eutypa lata, osetljivost sorti introduction the eutypa dieback is one of the most harmful mycoses on the grapevine (vitis vinifera l.) worldwide caused by fungal pathogen named eutypa lata. this fungus can significantly reduce the yield and quality of the grapevines, but above all, it significantly reduces the longevity of the vine, as it causes partial or complete dying of the trunk (bolay and moller, 1977; carter et al., 1985, 1994; živković et al., 2012a, b). in serbia, during the period from 2010 to 2012, the occurrence the eutypa dieback symptoms on the grapevines were observed. the symptoms first appear in the form of small, chlorotic and necrotic spots along the periphery of the leaves, deformation of the leaves, the appearance of shortened shoots, often with the so-called zig-zag internodes. in time, there is a partial or complete dying of the trunk of the vine (carter at al., 1985; živković et al., 2012a, b). fungus e. lata is a vascular pathogen that infects the grapevine through fresh pruning wounds. the infection occurs when askospores arrive at fresh cross cut sections and in the presence of water droplets penetrate the vascular tissue. the wounds are particularly sensitive immediately after pruning, although infections may occur even after seven weeks following the cut (munkvold, 2001; sosnowski et al., 2007a, b; pitt et al., 2013). phytosanitary and chemical measures are used in the vineyards to suppress the degradation of grapevine. the use of preventive measures is reflected in the selection of the cultivars, the selection of the breeding form of the trunk, the time of the cutting and the removal and burning of diseased parts of the trunk. in a region where there are more hosts of e. lata, control of the disease cannot be done only by sanitary measures. regular cutting and destruction of diseased trunk parts provide control of the inoculum within limits suitable for grapevine. given that © 2019 živković et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 29 there are no resistant grape cultivars to e. lata, planting of vineyards, it is recommended to plant tolerant cultivars, while avoiding sensitive cultivars. as a preventive measure of the fight, it is recommended to select one of the breeding forms, which involves the formation of a lower trunk and mixed cutting (rolshausen et al., 2015; sosnowski, 2016). the main aim of this study was to determine the susceptibility of the grapevine cultivars to isolate of e. lata and finding potential sources of resistance for different grapevine cultivars. for the investigation, the four grapevine cultivars, the one domestic (banatski muskat) and three foreign (riesling blanc, chardonnay, and gamay noir) were selected. a test of the sensitivity of investigated grapevine cultivars to e. lata isolate was carried out by inoculation of unrooted cuttings. materials and methods samples and fungal isolation during the period from 2010 to 2012, symptoms on the grapevine from 14 locations in serbia, it was noticed a plant with dieback. sampling was done in the second half of may and early june in vineyards aged 11 to 22 years. during the collection of samples of diseased plants in the field, fragments of the trunk and cordon of the grapevine, including diseased and healthy tissue, in size from 10 to 20 cm, were taken. symptoms initially appeared as small, chlorotic and necrotic spots along the rim of the leaves, deformation of leaves and the appearance of shortened shoots, often with the so-called zig-zag internodes. over time, partial or complete dying of the vines developed. to isolate the pathogen, diseased parts of grapevine plants were surface-sterilised with 5% sodium hypochlorite for 2 min, followed by three washes with sterile distilled water. surface sterilised tissue was transferred to sterile filter paper and placed on potato dextrose agar (pda) containing streptomycin and incubated at 24 °c in the 24 h uv light for 30 days. single conidia were selected and transferred directly to the pda plate according to the procedures described by choi et al. (1999) and stored on pda in tubes at 4 °c. of the total number of isolates obtained for this study, one isolate el 17 (from cv. cabernet sauvignon) was selected. cultivar test cultivar test was performed according to the methods of peros and berger (1994). unrooted cuttings of four cultivars riesling blanc, chardonnay, banatski muskat and gamay noir wereinoculated with mycelium of one isolate (el17) in a randomised block design with four replication on eight cuttings. the day before the inoculation, 2-node cuttings, with the basal bud eliminated, were prepared and soaked in water overnight. a hole (5 mm in diameter, 5 mm deep) was drilled 4 cm below the upper bud, and a plug (5 mm diameter) of agar and mycelium, cut from a fresh culture on pda medium was inserted into the hole, with the mycelium towards the wood. plugs of pda medium were used for the control. just after inoculation, cuttings were inserted into pieces of rock wool, dimensions 50 x 70 x 30 cm. five and ten weeks after inoculation, the plants were observed using the following scale: 0 (plants without symptoms), 1 (plants with few small and dried leaves), 2 (plants with higher number of small and dried leaves), 3 (plants with barely present leaves) and 4 (plants with dried shoots). after observing, the pieces were put into drained plastic containers, dimensions 12 x 12 x 30 cm, filled with substrate (class 1) (without feeding) and finally placed on benches in a greenhouse. the plants were trimmed in february of the next vegetation cycle. experiments were arranged in a complete randomised design. there were nine replicates of eight cuttings per treatment. five and ten weeks after bud break, plants were observed in the second vegetative growth cycle for each treatment of the cultivar experiment, evaluating the appearance of deformations and necrosis on leaves of inoculated plants. calculation of the intensity of the disease on the shoots was done by direct proportion of the number of diseased leaves on inoculated cuttings and the number of leaves on control cuttings (péros and berger, 1994). statistical analysis statistical analysis was performed to determine the relationship between e. lata isolate and grapevine cultivar. data were analysed by analyse of variance (anova) using the computerised software (proc glm, sas, system, version 8.1; sas institute, cary, nc). to satisfy the assumptions of the anova, the arcsine transformation of the proportion was used (y=2x arcsine √p). homogeneity of groups was assessed using duncan’s test with p=0.05. results by examining the vineyards in 14 locations where grapevine is cultivated in our country in the period from 2010 to 2012, symptoms of eutypa dieback of grapevine have been observed. samples were collected from 14 locations in serbia. during the vegetation, the diseased parts of the vines in vineyards become covered with a new leaf mass, so that the symptoms are more difficult to notice. symptoms on the leaves of diseased plants are ex30 biologica nyssana ● 10 (1) september 2019: 29-34 živković et al. ● susceptibility of different grapevine cultivars to eutypa lata isolate, causing agent of eutypa dieback, originating from serbia) pressed in the form of tiny, chlorotic spots, arranged on the periphery of the leaves. the edges of the leaf are often spiked and bent toward the back, and with stronger infections, the leaf surface is mostly covered with necrotic spots. the central part of the leaf has a wobbly look. over time, the leaves were dried and fall off early. the susceptibility/resistance of different grapevine cultivars, to the selected isolate of e. lata, originating from serbia, under greenhouse conditions was determined. characterisation of morphological and molecular levels had previously been done on the fungi isolate. assessment of susceptibility/resistance was carried out using methods of inoculation of unrooted cuttings of different cultivars of grapevine. this research aimed to determine the susceptibility of different cultivars of grapevines to the isolate based on the appearance of symptoms on leaves and shoots, from the movement of growers on grapevine cuttings. based on the results of this study, the method of inoculation of grape cuttings according to peros and berger (1994) will be used in the future. four grapevine cultivars were selected for testing: riesling blanc, chardonnay, banatski muskat, and gamay noir. tested isolate caused eutyposis symptoms on grapevine cuttings. the reaction of the tested grapevine cuttings, as well as the estimated level of development of the disease after inoculation to selected e. lata isolate, are shown in tab. 1. the foliar expression of eutypa dieback was compared between four cultivars of grapevine following inoculation by e. lata isolate el17 (tables 1). the numbers of cuttings showing foliar symptoms and other abnormalities were recorded five and ten weeks after inoculation in the first vegetative growth cycle and five and ten weeks after bud break in the second cycle vegetative growth cycle in cultivar experiments. all the cultivars inocu31 lated exhibited foliar symptoms of the disease and the other abnormalities. in experiment, the effect of cultivar was highly significant. some of the shoots showing necrotic leaves five weeks after inoculation showed normal growth ten weeks after inoculation and this phenomenon explained the decrease in some percentage. the differences between cultivars were less acute ten weeks after inoculation in first and ten weeks after bud break in second vegetative growth cycle (tab. 1) indicating that the effects of the fungus appeared faster for some cultivars than for others. the foliar symptoms occurred during the vegetative growth cycle on the inoculated unrooted cuttings. the smaller and necrotic leaves were similar to that observed in shoots of infected vines in the vineyard. the cultivars had different responses in the test, indicating that cultivars differ in their susceptibility to fungal toxins and fungal invasion. the high susceptibility of cultivars riesling blanc, banatski muskat and gamay noir was confirmed. cultivars riesling blanc, banatski muskat and gamay was considerably more affected than the chardonnay cultivar, and the inoculation of cuttings caused a faster appearance of abnormal plants for riesling blanc, banatski muskat and gamay noir than for the cultivar chardonnay (tab. 1). the infected plants exhibited the typical symptoms of eutyposis on the shoots and leaves. thus, on the inoculated cuttings of all grapevine cultivars studied, the intensity of the disease was similarly caused by isolate el17 (tab. 1). discussion eutypa lata is one of the most important phytopathogenic fungi that threaten vineyards, causing signifitable 1. effect of grapevine cultivar on percentage of plants showing foliar symptoms and abnormalities after inoculation with e. lata isolate el17 a-c: means in a column followed by the same letter are not significantly different according to duncan’s multiple range test at the p=0.05. biologica nyssana ● 10 (1) september 2019: 29-34 živković et al. ● susceptibility of different grapevine cultivars to eutypa lata isolate, causing agent of eutypa dieback, originating from serbia) i s o l a t e e l 1 7 year n year n+1 5th week 10th week 5th week 10th week cultivar f o l i a r s y m p t o m s ( % ) riesling blanc 82.03 a 76.57 ab 80.21 a 65.63 ab chardonnay 44.53 b 48.44 b 56.25 b 61.46 b banatski muskat 86.72 a 79.69 a 72.92 ab 78.48 a gamay noir 74.22 ab 82.03 a 71.79 ab 77.08 a control 0.00 c 0.00 c 0.00 c 0.00 c 32 cant economic damage (pitt et al., 2013; travadon et al., 2013). according to munkvold et al. (1994) the eutypa dieback is a very dangerous disease of the grapevine because there is no effective chemical protection, and after the advancement of this disease there is a decrease in the yield and dying of parts or whole infected trunks, leading to significant losses in production (rolshausen et al., 2015). in the world, there is a limited number of works on testing the susceptibility of different grapevine cultivars to this disease. earlier studies of the susceptibility of different grapevine cultivars were based on apricot research that was very susceptible to e. lata, and very little was known about the colonisation of grapevine tissue by this fungus (carter et al., 1985; munkvold, 2001). for these reasons, péros and berger (1994) examined the aggressiveness of twelve e. lata isolates and the susceptibility of fifteen cultivars of grapevines in their experiments. sosnowski et al. (2007a) studied the virulence of twenty-eight e. lata isolates isolated from the grapevine, originating from australia, and the susceptibility of three cultivars of grapevines cabernet sauvignon, grenache and merlot. travadon et al. (2013) observed the susceptibility of seven commercial grapevine varieties (cabernet franc, cabernet sauvignon, chardonnay, merlot, riesling, shiraz and thompson seedless) as well as the susceptibility of the interspecific hybrid of vitis labrusca variety concord by inoculation of one-year rooted cuttings during the dormant season. comparison of the obtained symptoms in this study with the symptoms of natural infections in serbia, as well as with the descriptions of this phenomenon in literature, points to their exceptional similarity in the appearance and dynamics of development. the pathogenic isolate of e. lata was used in the sensitivity tests on grapevine cultivars, and it has been demonstrated that this species is capable of contaminating the unrooted cuttings of the grapevine and causing the appearance of symptoms characteristic of the dieback of the grapevine. after the inoculation of unrooted cuttings under greenhouse conditions during the dormant season, after five and ten weeks, in the first vegetation cycle, the symptoms occurred. symptoms were observed on inoculated plants as stunted, chlorotic leaves with curved edges, and chlorotic and necrotic patches along the periphery, which later spread over the entire surface, and subsequently caused drying and falling leaves. the shoots were significantly shortened due to slow growth and have a bright green color, often with the so-called zig-zag internodes. symptoms on shoots which appeared on leaves under greenhouse conditions were similar to symptoms found in grapevines in nature. according to the literature data, leaf necrosis appearing on inoculated cuttings is identical to the necrosis of the leaf-induced fungi culture or to the eutypine toxin obtained by extracting leaves from infected grapevine plants from the vineyard (tey-rulh et al., 1991, loc. cit. péros and berger, 1994; mahoney et al., 2005; jimenez-teja et al., 2006; trouillas and gubler, 2010). according to petzold et al. (1981) and mur (1988), the appearance of leaf symptoms is a consequence of toxic products that move from the inoculation point to the point of growth (travadon et al., 2013). the studied e. lata isolate proved to be the most virulent on grapevine cultivars riesling blanc cultivar, banatski muskat, and gamay noir compared to chardonnay. on the other hand, the chardonnay cultivar exhibits statistically significant resistance to the tested isolate. péros and berger (1994) observed the onset of symptoms five, seven and ten weeks after inoculation in the susceptibility of the cultivars. they concluded that the percentage of symptoms occurring during the fifth to seventh week increased by 70% and remained unchanged in the period from the seventh to the tenth week. cultivars are variously susceptible to the toxin that fungus produce and invasion of fungi (rumbos, 1985; travadon et al., 2013). many inoculated cuttings that exhibited symptoms in the first year did not exhibit symptoms in the second year. this is explained by the fact that the number of toxic products can vary and dilute depending on the development or growth of the shoots (péros and berger, 1994, 1999, 2003). also, this phenomenon can be illustrated by the variation in the intensity of the observed symptoms in some cultivars, the ability to begin to develop regular shoots that overlap deformed shoots after the onset of symptoms. foliar symptoms are initially confined to one span of infected vines, however, as the disease progresses symptoms my spread throughout the entire vine (péros and berger, 1994, 1999, 2003; sosnowsli et al., 2007b; živković et al, 2012a, b; pitt et al., 2013; travadon et al., 2013). the artificial inoculation of the grapevine with isolate of e. lata gave preliminary information on the different susceptibility of four grapevine cultivars. namely, by comparing the reactions of four grapevine cultivars to artificial e. lata infections, it was found that there were statistically very significant differences in the response of inoculated cuttings, which indicates the existence of a difference in the level of susceptibility. péros and berger (1994) in their studies suggest that the tested grapevine cultivars exhibited a different susceptibility to the e. lata toxin fungi and its invasion, which reflects on the intensity of the symptoms on their span. also, by analysing the sensitivity of fifteen grapevine cultivars to e. lata, péros and berger (1994, 1999, 2003) biologica nyssana ● 10 (1) september 2019: 29-34 živković et al. ● susceptibility of different grapevine cultivars to eutypa lata isolate, causing agent of eutypa dieback, originating from serbia) 33 found that the cultivars cabernet sauvignon, ugni blanc, and chasla were the most susceptible. a bit lower degree of susceptibility was revealed by the merlot cultivar. travadon et al. (2013) have found in their experiments that the thompson seedless cultivar has exhibited a high degree of susceptibility to the e. lata isolates examined. likewise, there was no significant difference in the expressed susceptibility between the cultivars of cabernet sauvignon and merlot. unfortunately, currently, there are no developed programs for selection of vines for disease resistance in our country. one of the contributions of this is the examination of the reaction of domestic commercial cultivars, as well as cultivars from the environment, to susceptibility to e. lata under conditions of artificial inoculation for the purpose of preventing the production of vineyard. under experimental conditions, investigated isolate was able to cause typical symptoms on the tested grapevine cultivars. conclusion based on the results during the two-year investigation on susceptibility of four cultivars of the grapevines to the inoculation of e. lata, it can be concluded that the cultivars riesling blanc, banatski muskat and gamay noir manifest high susceptibility compared to cultivar chardonnay. all the cultivars inoculated exhibited foliar symptoms of the disease and the other abnormalities. the differences between cultivars were less acute ten weeks after inoculation in first and ten weeks after bud break in second vegetative growth cycle. references bolay, a. and moller, w.j. 1977: eutypa armeniacae hansf. & carter, agent d’un grave dépérissement de vignes en production. revue suisse de viticulture, arboriculture, horticulture, 9: 241-251. carter, m. v., bolay, a., english, h., rumbos, i. 1985: variation in the pathogenicity of eutypa lata (= e. armeniacae). australian journal of botany, 33(3), 361-366. carter, m.v. 1994: wood and root diseases caused by fungi. eutypa dieback. in: r. c. pearson, and a.c. goheen (ed.), compendium of grape diseases, 3rd ed. aps press. st. paul, minnesota: 32-34. carter, m. v., bolay, a., english, h., rumbos, i. 1985: variation in the 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assess the aggressiveness of e. lata isolates and the susceptibility of grapevine cultivars to eutypa dieback. agronomie, 14: 515-523. peros, j.p., berger, g. 1999: diversity within natural progenies of the grapevine dieback fungus eutypa lata. current genetics, 36, 301-309. peros, j.p., berger g. 2003: genetic structure and variation in aggressivness in european and australian populations of the grapevine dieback fungus, eutypa lata. european journal of plant pathology, 109: 909-919. petzoldt, c.h., moller, w.j., sall, m.a. 1981: eutypa dieback of grapevine: seasonal differences in infection and duration of susceptibility of pruning wounds. phytopathology, 71: 540-543. pitt, w.m., trouillas, f.p., gubler, w.d., savocchia, s., sosnowski, m.r. 2013: pathogenicity of diatrypaceous fungi on grapevine in australia. plant disease, 97: 749-756. rolshausen, p.e., sosnowski, m., trouillas, f.p., gubler, w.d. 2015: diseases caused by fungi and oomycetes. eutypa dieback. in w. f. wilcox, w. d 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originating from serbia) i̇nanir et al. 2024, biologica nyssana 15(2) 15 (2) december 2024: 69-74 doi: 10.5281/zenodo.13902146 biochemical contents of some jujube genotypes original article veli i̇nanir isparta university of applied sciences, faculty of agriculture, department of horticulture, isparta, türkiye adnan nurhan yildirim isparta university of applied sciences, faculty of agriculture, department of horticulture, isparta, türkiye civan çelik isparta university of applied sciences, faculty of agriculture, department of agricultural biotechnology, isparta, türkiye civancelik@isparta.edu.tr (corresponding author) received: august 20, 2024 revised: september 20, 2024 accepted: september 23, 2024 abstract: this study aimed to evaluate the biochemical content of 15 jujube genotypes with superior characteristics from the denizli/çivril region over a two-year period. significant differences were found among the genotypes regarding their biochemical properties. genotypes 10, 11, and 13 consistently exhibited higher total phenolic content compared to the others in both years. additionally, genotypes 10, 2, and 11 showed higher total flavonoid content than the other genotypes. genotypes 2, 10, and 1 demonstrated greater total antioxidant content, while genotypes 10, 2, and 11 had elevated vitamin c levels. overall, genotypes 10, 11, and 2 consistently exhibited higher biochemical values compared to the others across both years. key words: ziziphus jujuba, total antioxidant, total phenolic content, total flavonoid content, vitamin c apstrakt: biohemijski sastav nekih genotipova jujube ova studija je imala za cilj da proceni biohemijski sadržaj 15 genotipova urme sa superiornim karakteristikama iz oblasti denizli/čivril tokom dvogodišnjeg perioda. uočene su značajne razlike među genotipovima u pogledu njihovih biohemijskih svojstava. genotipovi 10, 11 i 13 dosledno su pokazivali viši ukupni sadržaj fenola u poređenju sa ostalim genotipovima tokom obe godine. takođe, genotipovi 10, 2 i 11 imali su veći ukupni sadržaj flavonoida u odnosu na druge genotipove. genotipovi 2, 10 i 1 su pokazali viši ukupni antioksidativni sadržaj, dok su genotipovi 10, 2 i 11 imali povećan nivo vitamina c. generalno, genotipovi 10, 11 i 2 su dosledno pokazivali veće biohemijske vrednosti u poređenju sa ostalim genotipovima tokom obe godine. ključne reči: ziziphus jujuba, ukupni antioksidansi, ukupni saržaj fenola, ukupni sadržaj flavonoida, vitamin c introduction jujube is one of the oldest known fruit species cultivated for over 5,000 years, especially in china and india. therefore, its use for medical and food purposes has become widespread (mengjun, 2003; yildirim et al., 2015; san et al., 2016). jujube makes a significant contribution to human nutrition due to its rich nutrient and phytochemical profile. it is particularly abundant in vitamins, organic acids, sugars, minerals, polyphenols, amino acids, carbohydrates, oleic acid, and linoleic acid (san & yildirim, 2010; jat et al., 2013; san et al., 2016; wojdyło et al., 2016; gündoğmuş & taşçı, 2017; liu et al., 2021). tepe et al. (2022) stated that jujube fruit has a high phenolic content. they also emphasized that jujube extracts can have a natural antioxidant effect due to their high phenolic content. the antioxidant, anti-inflammatory, anti-obesity, hepatoprotective, antidiabetic, antimicrobial, and anticancer properties of these components have been reported to have protective effects on human health (san & yildirim, 2010; ji et al., 2017; gowd et al., 2020; feng et al., 2021; kahraman et al., 2022; hançer et al., 2023; aoudeh et al., 2024; pepe et al., 2024; yıldırım et al., 2024). in addition, according to the world health organization, an adult can meet the daily requirement of vitamins c and b from a single jujube fruit (jat et al., 2013). in addition to its effects on human health, its high adaptability, long and economic life, various consumption methods, and ease of cultivation have made it an increasingly popular fruit worldwide (mengjun, 2003). in türkiye, the jujube planting area has increased by 512% and the production quantity by 1,072% in the last 10 years. china meets more than 90% of © 2024 i̇nanir et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 69 the world’s jujube production (jat et al., 2013), and today it produces approximately 8 million t in a 2 million ha area (liu et al., 2021). as natural habitats in türkiye, the coruh valley basin, demirci district of manisa, and çivril district of denizli are among the leading regions where jujube cultivation is widespread (yasa, 2016). this study aimed to determine the biochemical contents of different 15 jujube genotypes with superior fruit characteristics in the denizli/çivril region. materials this study was conducted in an orchard with 15-yearold jujube (ziziphus jujuba mill.) trees planted on seedlings at a spacing of 7 × 3.5 m in the çivril district of denizli province. the research area is situated at an altitude of 915 m, at coordinates of 38°14´ north, and 29°59´ east. fruits were harvested at least 50% of the outer skin colour turned from green to golden yellow (hussain et al., 2021). the fresh fruits were harvested on september 12, 2021, for the first year, and the second year’s harvest was on september 5, 2022. fifty fruit samples were collected from each jujube genotype in 2021 and 2022, representing the tree from four different directions. fruits were placed in perforated bags, transported to the laboratory and analyses were conducted immediately. methods determination of total phenolic content the total phenolic content was determined using the folin-ciocalteu method as described by singleton & rossi (1965). homogenized fruit pulp was subjected to extraction in tubes for 1 h using a solution of acetone, water, and acetic acid (70:29.5:0.5). accordingly, the samples were mixed with 0.1 ml of extract with di-ionized water and 0.1 ml (2n) folin-ciocalteu indicator and kept at room temperature for 6 min. then 0.5 ml of 20% sodium carbonate solution was added and incubated at room temperature for 30 min for color development. after color development, the absorbance values of the samples were read in a spectrophotometer at 760 nm wavelength (model t60u, pg instruments, and usa). determination of total flavonoid content the total flavonoid content was determined according to the method described by kim et al. (2003). first, 1 ml of each sample was mixed with 0.3 ml of 5% sodium nitrite (nano2) solution and incubated for 5 min. then, 0.3 ml of 10% aluminium chloride (alcl3) solution was added and incubated for an additional 6 min. afterwards, 2 ml of 1 m sodium hydroxide (naoh) was added, and the mixture was allowed to stand for 2 min. subsequently, 4 ml of distilled water was added, and thoroughly mixed, and the absorbance values of the samples were read at 510 nm wavelength. determination of total antioxidant the total antioxidant capacity was determined using the method reported by kumaran & karunakaran (2006) employing 1,1-diphenyl-2-picrylhydrazyl (dpph). first, 2 g of each sample were grounded and incubated in 20 ml of 80% methanol at -20 °c for 2 h. then, the mixture was centrifuged at 2,000 rpm for 5 min, and the supernatant was collected for analysis. following this step, 2 ml of 0.1 mm dpph dissolved in methanol was added to 100 µl of the supernatant. after 30 min of incubation, the absorbance was measured at 517 nm wavelength. determination of vitamin c the vitamin c content in the fruits was determined according to namdar (2005). fruit juice was obtained from a portion of each sample, and the vitamin c content was determined by titrimetric method. specifically, 10 ml of fruit juice was mixed with 50 ml of meta-phosphoric acid solution and filtered. then, 10 ml of the filtrate was titrated with a dye solution. the obtained values were used to calculate the vitamin c content according to the formula provided by the researcher. statistical analysis the data obtained in the study were subjected to the analysis of variance using the minitab software package. tukey’s multiple comparison test was used to determine significant differences among the genotypes. results and discussion the results of the biochemical measurements of the genotypes in 2021 are presented in tab. 1. significant differences were observed among the genotypes only in terms of total phenolic content (p≤0.05). statistically significant differences were not found among the genotypes in other characteristics. in the study, the highest total phenolic content was obtained in genotype 10 with 3.131 mg gae 100 g-1. this was followed by genotype 11 with 2.445 mg gae 100 g-1 and genotype 13 with 2.362 mg gae 100 g-1, respectively. the lowest total phenolic content was found in genotype 5 with 0.669 mg gae 100 g-1. the highest total flavonoid content was obtained in genotype 10 with 0.885 mg ce 100 g-1. this was followed by genotype 2 with 0.884 mg ce 100 g-1 and genotype 11 with 0.738 mg ce 100 g-1, respectively. the lowest total flavonoid content 70 biologica nyssana ● 15 (2) december 2024: i̇nanir et al. ● biochemical contents of some jujube genotypes biologica nyssana ● 15 (2) december 2024: i̇nanir et al. ● biochemical contents of some jujube genotypes 71 1.055 mg ce 100 g-1. the lowest total flavonoid content was observed in genotype 7 with 0.728 mg ce 100 g-1. in terms of total antioxidant content, the highest value was recorded in genotype 2 with 1.719 mmol te g-1, followed by genotype 10 with 1.580 mmol te g-1, and genotype 1 with 1.434 mmol te g-1. the lowest total antioxidant content was detected in genotype 7 with 0.836 mmol te g-1. the highest vitamin c content was obtained in genotype 10 with 342.00 mg 100 g-1, followed by genotype 2 with 341.60 mg 100 g-1, and genotype 11 with 285.30 mg 100 g-1. the lowest vitamin c content was found in genotype 7 with 196.70 mg 100 g-1. li et al. (2007) reported varying levels of vitamin c content ranging from 192 mg/100 g to 359 mg 100 g-1 and total phenolic content ranging from 5.18 mg gae g-1 to 8.53 mg gae g-1 in five chinese jujube varieties. they indicated that both nutrient and biochemical contents varied among different varieties. san et al. (2009) reported vitamin c contents ranging from 271.30 mg 100 g-1 to 366.00 mg 100 g-1 in some promising jujube genotypes, suggesting their safe use in human nutrition. san et al. (2016) stated that jujube fruits are rich in vitamin c and phenolic compounds, with vitamin c content varying from 32 mg 100 g-1 to 375 mg 100 g-1 across varieties. gao et al. (2011) reported vitamin c contents ranging from 77.20 mg 100 g-1 to 102.3 mg 100 g-1, total phenolic content ranging table 1. biochemical contents of the genotypes in 2021 the genotypes total phenolic content (mg gae 100 g-1)* total flavonoid content (mg ce 100 g-1) total antioxidant (mmol te g-1) vitamin c (mg 100 g-1) 1 1.091±0.36 de 0.645±0.33 0.713±0.28 174.30±89.70 2 1.337±0.60 cde 0.884±0.40 0.855±0.37 238.90±19.00 3 1.852±0.09 bcd 0.685±0.04 0.634±0.06 185.05±12.67 4 1.233±0.02 cde 0.718±0.16 0.693±0.20 194.10±44.10 5 0.669±0.10 e 0.521±0.07 0.484±0.04 140.90±21.10 6 1.378±0.24 cde 0.515±0.09 0.451±0.08 139.30±26.00 7 1.584±0.12 bcde 0.509±0.08 0.415±0.09 137.60±23.40 8 2.105±0.43 bc 0.577±0.04 0.488±0.04 155.90±11.05 9 1.302±0.46 cde 0.737±0.05 0.681±0.08 199.18±15.01 10 3.131±0.05 a 0.885±0.22 0.786±0.22 239.10±60.60 11 2.445±0.59 ab 0.738±0.11 0.621±0.12 199.50±31.00 12 2.157±0.06 bc 0.663±0.10 0.560±0.11 179.30±27.20 13 2.362±0.35 ab 0.704±0.20 0.637±0.13 190.30±54.50 14 2.131±0.07 bc 0.565±0.06 0.447±0.05 152.67±17.15 15 1.784±0.24 bcd 0.704±0.04 0.569±0.01 190.19±12.94 * the difference between means indicated by different letters is significant at the p≤0.05 level was found in genotype 7 with 0.509 mg ce 100 g-1. in the study, the highest total antioxidant content was determined in genotype 2 with 0.855 mmol te g-1. this was followed by genotype 10 with 0.786 mmol te g-1 and genotype 1 with 0.713 mmol te g-1, respectively. the lowest total antioxidant content was found in genotype 7 with 0.415 mmol te g-1. the highest vitamin c content was obtained in genotype 10 with 239.10 mg 100 g-1. this was followed by genotype 2 with 238.90 mg 100 g-1 and genotype 11 with 199.50 mg 100 g-1, respectively. the lowest vitamin c content was found in genotype 7 with 137.60 mg 100 g-1. the biochemical measurements of the genotypes in 2022 are presented in tab. 2. similar to the findings in 2021, statistically significant differences were observed among the genotypes only in terms of total phenolic content (p≤0.05). no statistically significant differences were found among the genotypes in other characteristics. the highest total phenolic content was obtained in genotype 10 with 3.914 mg gae 100 g-1. this was followed by genotype 11 with 3.057 mg gae 100 g-1 and genotype 13 with 2.953 mg gae 100 g-1. the lowest total phenolic content was found in genotype 5 with 0.837 mg gae 100 g-1. the highest total flavonoid content was achieved in genotype 10 with 1.266 mg ce 100 g-1, followed by genotype 2 with 1.264 mg ce 100 g-1, and genotype 11 with 72 biologica nyssana ● 15 (2) december 2024: i̇nanir et al. ● biochemical contents of some jujube genotypes from 428.50 mg gae 100 g-1 to 600.40 mg gae 100 g-1, and total flavonoid content ranging from 159.30 mg rutin eq. 100 g-1 to 230.30 mg rutin eq. 100 g-1. chen et al. (2018) reported vitamin c contents ranging from 162.50 mg 100 g-1 to 244.57 mg 100 g-1 and total flavonoid contents ranging from 41.21 mg g-1 to 62.72 mg g-1 across different jujube varieties. wojdyło et al. (2016) indicated total phenolic content ranging from 1442 mg 100 g-1 to 3432 mg 100 g-1 and vitamin c content ranging from 387 mg 100 g-1 to 555 mg 100 g-1. the researchers attributed these variations among varieties to genetic differences. ivanišová et al. (2017) determined total flavonoid content ranging from 1.49 to 11.57 g qe g-1, suggesting that variation in fruit properties stems from genetic diversity. reche et al. (2019) reported vitamin c contents of 0.49 g 100 g-1 for organically grown jujubes and 0.50 g 100 g-1 for conventionally grown jujubes, total phenolic contents of 452.2 mg gae 100 g-1 for organically grown jujubes and 433.7 mg gae 100 g-1 for conventionally grown jujubes, and total flavonoid contents of 83.10 mg eq. rutin 100 g-1 for organically grown jujubes and 111.30 mg eq. rutin 100 g-1 for conventionally grown jujubes. tepe & ekinci (2021) examined the effect of different drying temperatures on total phenolic and antioxidant content in jujube fruits. the researchers reported that the total phenolic and antioxidant content of fresh jujube fruits were 911.4±47.32 mg gae 100 g-1 dw and 0.214±0.001 mmol te g-1 dw. they found that phenolic and antioxidant contents in fruits decreased with the increase in drying temperature. riaz et al. (2021) determined the total phenolic contents of jujube fruits to be on average 284.6 mg gae 100 g-1 and total flavonoid contents to be on average 98.7 mg qe 100 g-1. our research findings showed similarities with previous studies in some values while deviating in others. the reasons for these discrepancies include the wide variation in physical, chemical, and functional properties among jujube varieties cultivated worldwide, as well as the influence of ecological factors, variety/genotype characteristics, genetic traits, and storage and cultural-care conditions on jujube fruit quality (gao et al., 2011; gündüz & saraçoğlu, 2014; almansa et al., 2016; abdel-sattar et al., 2024). in fact, cultural practices can be so influential that fruits obtained from regularly irrigated trees exhibit higher vitamin c content compared to those from trees subjected to limited irrigation (cui et al., 2008). conclusion in conclusion, jujube is an excellent natural antioxidant source due to its high content of vitamin c and total phenolic compounds. it is believed that the information obtained in this study will increase consumers’ interest in jujube fruits. the research results have revealed the significant influence of the genotype on bioactive compounds. in terms of biochemical contents, genotypes 10, 11, and 2 table 2. biochemical characteristics of the genotypes in 2022 the genotypes total phenolic content (mg gae 100 g-1)* total flavonoid content (mg ce 100 g-1) total antioxidant (mmol te g-1) vitamin c (mg 100 g-1) 1 1.364±0.44 de 0.923±0.47 1.434±0.57 249.30±12.40 2 1.671±0.75 cde 1.264±0.57 1.719±0.74 341.60±15.70 3 2.315±0.12 bcd 0.979±0.06 1.275±0.13 264.70±18.10 4 1.541±0.02 cde 1.028±0.23 1.392±0.40 277.60±63.10 5 0.837±0.13 e 0.745±0.11 0.974±0.08 201.50±30.30 6 1.722±0.30 cde 0.737±0.13 0.907±0.17 199.30±37.10 7 1.980±0.16 bcde 0.728±0.12 0.836±0.18 196.70±33.50 8 2.631±0.53 bc 0.825±0.05 0.981±0.01 222.98±15.89 9 1.627±0.57 cde 1.054±0.07 1.370±0.17 284.80±21.40 10 3.914±0.06 a 1.266±0.32 1.580±0.45 342.00±86.60 11 3.057±0.74 ab 1.055±0.16 1.248±0.24 285.30±44.30 12 2.696±0.08 bc 0.949±0.14 1.126±0.23 256.40±38.90 13 2.953±0.44 ab 1.007±0.28 1.281±0.27 272.20± 77.90 14 2.664±0.09 bc 0.808±0.09 0.899±0.11 218.40±24.60 15 2.229±0.30 bcd 1.006±0.06 1.144±0.03 272.00±18.50 * the difference between means indicated by different letters is significant at the p≤0.05 level biologica nyssana ● 15 (2) december 2024: i̇nanir et al. ● biochemical contents of some jujube genotypes 73 consistently stood out with the highest values in both years, while genotype 7 consistently exhibited the lowest values. promising genotypes with high biochemical contents are expected to play a significant role in a variety of breeding programs. references abdel-sattar, m., al-obeed, r.s., rihan, h.z., & el-badan, g.e. 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(2009). mineral composition of leaves and fruits of some promising jujube (zizyphus jujuba miller) genotypes. asian journal of chemistry, 21(4), 2898. biologica nyssana ● 15 (2) december 2024: i̇nanir et al. ● biochemical contents of some jujube genotypes 74 göse, hacıoğlu doğru, 2019, biologica nyssana 10(2) 10 (2) december 2019: 169-173 doi: 10.5281/zenodo.3600197 antibiofilm activity of verbascum pinnatifidum vahl. ethanolic extract original article mehmet göse department of plant physiology, institute of biology, graduate school of natural and applied sciences, çanakkale onsekiz mart university, çanakkale, turkey m_gose@hotmail.com nurcihan hacıoğlu doğru department of biology, faculty of arts and sciences, çanakkale onsekiz mart university, çanakkale, turkey nurcihan.n@gmail.com (corresponding author) received: july 1, 2019 revised: september 9, 2019 accepted: september 13, 2019 abstract: microbial biofilms pose health risks in clinical environments, food industry and drinking water systems. microorganisms within biofilms are more resistant to antibiotics and chemical agents than planktonic cells in suspension. new alternatives for controlling infections have been proposed focusing on the therapeutic properties of medicinal plants and their antibiofilm activities. here, we investigated in vitro antibiofilm activity of ethanol extract of verbascum pinnatifidum vahl. against escherichia coli nrrl b-3704, pseudomonas aeruginosa atcc 27853, proteus vulgaris atcc 13315, acinetobacter baumanii atcc 19606, bacillus subtilis atcc 6633, staphylococcus aureus atcc 25923, s. haemolyticus atcc 43252 and candida albicans atcc 10231 test microorganisms based on crystal violet binding assay. the highest antibiofilm activity was shown against biofilm formed by b. subtilis atcc 6633 and s. haemolyticus atcc 43252 at the lowest mic values of 2.5 and 10 µg/ml, respectively. the current findings indicated that bacterial biofilm formation can be potentially managed using v. pinnatifidum plant extracts. key words: antibiofilm activity, verbascum pinnatifidum apstract: antibiofilm aktivnost etanolnog ekstrakta verbascum pinnatifidum vahl. biofilmovi mikroorganizama predstavljaju zdravstveni rizik u kliničkim uslovima, prehrambenoj industriji i sistemima pijaće vode. unutar biofilmova, mikroorganizmi su rezistentniji na antibiotike i hemijske agense od planktonskih ćelija u suspenziji. predložene su nove alternative za kontrolisanje infekcija, koje su fokusirane na terapeutske osobine medicinskih biljaka i njihovu antibiofilm aktivnost. ovde je, korišćenjem kristal violet testa, vršeno istraživanje in vitro antibiofilm aktivnosti etanolnog ekstrakta verbascum pinnatifidum vahl. u odnosu na vrste escherichia coli nrrl b-3704, pseudomonas aeruginosa atcc 27853, proteus vulgaris atcc 13315, acinetobacter baumanii atcc 19606, bacillus subtilis atcc 6633, staphylococcus aureus atcc 25923, s. haemolyticus atcc 43252 i candida albicans atcc 10231. najveća antibiofilm aktivnost, sa najnižim mik vrednostima od 2,5 i 10 µg/ml, utvrđena je za biofilmove formirane od strane vrsta b. subtilis atcc 6633 i s. haemolyticus atcc 43252, respektivno. ovi nalazi ukazuju na to da stvaranje bakterijskih biofilmova potencijalno može biti sprečeno korišćenjem ekstrakta v. pinnatifidum. ključne reči: antibiofilm aktivnost, verbacum pinnatifidum introduction microbial biofilms are communities of bacteria, embedded in a self-producing matrix, forming on living and nonliving solid surfaces (vasudevan, 2014). they are considered as an important virulence factor that causes persistent chronic and recurrent infections; they are highly resistant to antibiotics and host immune defenses (grant and hung, 2013). biofilm resistance is due to several reasons, like restricted diffusion of antibiotics into biofilm matrix, expression of multidrug efflux pumps, type iv secretion systems, decreased permeability, and the action of antibiotic-modifying enzymes (alekshun and levy, 2007). the increased biofilm resistance © 2019 göse, hacıoğlu doğru. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work noncommercially under the same license as the original. 169 13th symposium on the flora of southeastern serbia and neighboring regions to conventional treatments enhances the need to develop new control strategies (simões et al., 2007; sanchez et al., 2016). verbascum plants have been used medicinally since ancient times in folk medicine as a remedy for respiratory problems such as bronchitis, dry coughs, whooping cough, tuberculosis, and asthma. the leaves, roots and the flowers have been used also as anodyne, sedative, diuretic, sudorific, expectorant and antidiarrheal agents in traditional world and turkish medicine (baytop, 1999; georgiev et al., 2011). therefore verbascum plant species have been also previously reviewed for their antiviral, antimicrobial, antimalarial, antioxidant, anti-inflammatory, antinociceptive, antitumor, anticancer, cytotoxic, immunomodulatory, anticholinesterase, antiulcerogenic, antihepatotoxic, antihyperlipidemic, anthelminthic, antitussive and antigermination activities (tatli and akdemir, 2006; dulger and hacioglu, 2009; kahraman et al., 2010; kahraman et al., 2011; kozan et al., 2011; ozcan et al., 2011; boğa et al., 2016). however, there is no any literature about verbascum plants antibiofilm activity, except moghaddam et al. (2015), which evaluated the antibiofilm activity of verbascum pinnatifidum vahl. ethanol extract. material and methods plant materials verbascum pinnatifidum was collected from canakkale, kumkale near the lantern d.s. sandy area, (40.008101 n, 26.203127 e) in 2018 and identified with the aid of flora of turkey (davis et al., 1988) by dr. ersin karabacak. voucher specimens were deposited in the biology department at çanakkale onsekiz mart university, çanakkale, turkey. preparation of plant extracts the plant parts were air-dried. dry powdered plant material (10 g) was extracted with 300 ml of 80% ethanol (merck, darmstadt, germany) for 24 h by using soxhlet equipment (khan et al., 1988). the extracts were filtered using whatman filter no.1, and the filtrates were then evaporated under reduced pressure and dried using a rotary evaporator at 55 oc. dried extracts were stored in labelled sterile screwcapped bottles at + 4 oc. test microorganisms gram negative bacteria escherichia coli nrrlb 3704, pseudomonas aeruginosa atcc 27853, proteus vulgaris atcc 13315, acinetobacter baumanii atcc 19606 and gram positive bacteria – bacillus subtilis atcc 6633, staphylococcus aureus atcc 6538, s. haemolyticus atcc 43252 and yeast culture candida albicans atcc 10231 were used as test microorganisms. minimum inhibitory concentration assay to determine the plant extract doses to be used in biofilm inhibition study, minimum inhibitory concentration (mic) values of all samples were determined. mic was investigated as recommended instruction of the clinical and laboratory standards institute (clsi, 2006). the lowest concentration of extract inhibiting the visible growth of each test microorganisms was taken as the mic. the medium, 0.1% (w/v) streptomycin (st), nystatin (nys100) and 10% dmso were used as the nontreated, positive and negative controls, respectively (teanpasian et al., 2017). biofilm inhibition assay microplate biofilm method (merrit et al., 2005) was used to evaluate the inhibition of biofilm formation by v. pinnatifidum plant ethanol extract against test microorganisms. cultures were incubated in 5 ml tryptic soy broth (tsb) medium containing 5% glucose. cultures were diluted 1:100 in tsb and loaded into each well in 4 sterile microplates. different concentrations of plant extract (mic and sub-mic concentrations: 50, 25, 12.5% of mic) were prepared and transferred to each microplate well. after incubation at 37 ± 0.1°c for 48 h planktonic bacteria were removed from the wells and wells were washed twice with distilled water. 200 μl of 0.1% crystal violet solution was added to each well (20 minutes). the crystal violet bounded extracts were poured and washed until the crystal violet removed. the microtiter plates were inverted, and the remaining liquid was drained and dried in room heat. finally, the adhered biofilm bounded crystal violet was eluted in ethanol (95%), and the absorbance was measured at 550 nm by using an automated elisa reader. all experiments were repeated thrice in triplicate. the measurement of the antibiofilm effect of the extract was made by the percentage reduction formulation. % inhibition = (acontrol – asample / acontrol) x 100 acontrol: absorbance of the control (containing 100 µl tbs instead of plant extract) reaction asample: absorbance of the test compound results and discussion ethanol extract of v. pinnatifidum was tested against eight clinical bacterial and fungal strains (tab. 1). ethanol was observed as the best solvent for extracting antimicrobial substances in a previous study (jonathan and fasidi, 2003). results of mic were quite variable between each test microorganisms ranging from 2.5 to 20.0 µg/ 170 biologica nyssana ● 10 (2) december 2019: 196-173 göse, hacıoğlu doğru ● antibiofilm activity of verbascum pinnatifidum vahl. ethanolic extract biofilm is considered to be one of the essential virulence factors that cause persistent chronic and recurrent infections, because of their high resistance to antibiotics and host immune defensive system (grant and hung, 2013). bacteria are more resistant to antibiotics than planktonic cells because they are preserved by exopolysaccharide. this necessitated the screening of new and natural antibiotic sources in the fight against biofilm. new alternatives for controlling infections have been proposed focusing on the therapeutic properties of medicinal plants and their antibiofilm activities. many studies showed that the biofilm formation by pathogens leads to an increase in their virulence (vuong et al., 2004; antunes et al., 2010). therefore, if the biofilm formation is inhibited, the bacterial infection can be prevented (erdönmez et al., 2018). biofilm formation can be controlled by quorum sensing, a bacterial communication system which causes a rapid and coordinated change of expression pattern in the bacterial population in response to population density (pratiwi et al., 2015). the fact that at mic and sub-mic concentrations, v. pinnatifidum extract is capable of disturbing biofilm formation and causes biofilm breakdown, suggests that this disturbance may have been caused by the presence of compounds that inhibit quorum sensing. other plant compounds could attenuate biofilm development by inhibiting bacterial peptidoglycan synthesis (ogunlana et al., 1987), disrupting the permeability barrier of microbial membrane structures, causing the cell to leak out (cox et al., 2000), modify bacterial membrane structure hydrophobicity (türi et al., 1997; das, 2014), or disturbing the extracellular polymeric matrix in the biofilm to release biofilm from the surface of the solid substratum (traba and liang, 2011; pratiwi et al., 2015). further studies need to be performed ml (tab. 1). it was observed that almost all tested microorganisms were sensitive towards the ethanol extract of v. pinnatifidum. there is no data about v. pinnatifidum antimicrobial activity in the literature. our findings about mic ranges confirmed the observations of some other researchers, which stated that some verbascum species have antimicrobial activity against gram positive and negative bacteria and yeast and mold cultures (dulger and hacioglu, 2008;2009; ozcan et al., 2010; morteza-semnani et al., 2012; noori et al., 2012; anil et al., 2016; dulger and dulger, 2018). however, we found that v. pinnatifidum extract was also effective against e. coli nrrlb 3704 and c. albicans atcc 10231, contrary to some studies with the other verbascum species in the literature (dulger and hacioglu, 2008; morteza-semnani et al., 2012; amin et al., 2015). the results of potential inhibition of test microorganism’s biofilm formation by ethanol extract of v. pinnatifidum were shown in tab. 1. the results indicated that v. pinnatifidum extract in 2.5 to 10.0 µg/ml concentrations could inhibit the biofilm formation of all the tested microorganisms except e. coli nrrlb 3704 and c. albicans atcc 10231. throughputs also showed that antibiofilm function of extract was in a dose-dependent manner. the highest antibiofilm activity was noticed against biofilm formed by b. subtilis atcc 6633 and s. haemolyticus atcc 43252 at the lowest mic values of 2.5 µg/ml and 10 µg/ml, respectively. verbascum pinnatifidum has not been investigated in terms of antibiofilm activity. in a study conducted by moghaddam et al. (2015), ethanol extract of v. thapsus had inhibitory effect on biofilm formation of streptococcus mutans, s. sanguinis, and s. salivarius. the current findings indicated that biofilm forming of bacteria could be potentially managed using v. pinnatifidum plant extracts. 171 biologica nyssana ● 10 (2) december 2019: 169-173 göse, hacıoğlu doğru ● antibiofilm activity of verbascum pinnatifidum vahl. ethanolic extract table 1. the percentage of inhibition of biofilm structure of test microorganisms mic (µg/ml) % inhibition of biofilms formation test microorganisms plant extract control st/ ny100 mic mic/2 mic/4 mic/8 e.coli nrrlb 3704 20.0 4.0 7.27±0.33 3.34±0.08 p. aeruginosa atcc 27853 10.0 1.0 74.65±6.23 65.33±5.67 43.56±4.22 34.76±1.76 p. vulgaris atcc 13315 10.0 4.0 70.50±0.54 60.03±1.55 45.54±1.00 40.01±0.23 a. baumanii atcc 19606 10.0 2.0 57.42±0.12 43.12±0.10 b. subtilis atcc 6633 2.5 4.0 90.02±0.01 70.01±0.10 55.78±1.23 43.26±1.43 s. aureus atcc 6538p 10.0 4.0 70.54±0.60 s. haemolyticus atcc 43252 10.0 5.0 76.12±3.11 58.41±3.12 44.45±4.11 23.56±1.56 c.albicans atcc 10231 2.5 2.5 10.45±1.14 5.45±0.99 -: no inhibition of biofilm formation was observed due to the lack of antibiofilm effect. to confirm the actual mode of action of anti-biofilm activity from these extracts. conclusion our investigation was the first report on the antibiofilm property of the v. pinnatifidum ethanol extract. in this research, we found that forming of bacterial biofilm could be potentially being managed using v. pinnatifidum plant extract, especially against gram (+) bacteria. detailed phytochemical investigations are required to determine the types of substances responsible for the biological activities of v. pinnatifidum plant species. we hope that our results will provide useful data for discovering new compounds with better activity than agents currently available. acknowledgments. this investigation is a part of master thesis of mehmet göse. this study was financially supported by the çanakkale onsekiz mart university scientific research projects coordination unit, turkey (fyl-2018-2693). this paper was presented at the symposium on the flora of southeastern serbia and neighboring regions, june, 20-23 2019, serbia. authors would like to thank assoc. prof. dr. ersin karabacak for defining plant species. references alekshun, m.n., levy, s.b. 2007: molecular mechanisms of antibacterial multidrug resistance. cell, 128(6): 1037-1050. amin, j.n, batool, m., abu-hadid, m.m. 2015: screening antibacterial and antifungal activities and evalution of exhaustive extractions yields for verbascum sinuatum l. international research ayurveda pharmacy, 6 : 105-110. anil, s., dosler, s., mericli, a. h. 2016: chemical composition and antimicrobial activity of verbascum caesareum. chemistry of natural compounds, 52(1): 125-126. antunes, l.c.m., ferreira, r.b., buckner, m.m., finlay, b.b. 2010: quorum sensing in bacterial virulence. microbiology, 156(8): 2271-2282. baytop, t. 1999: türkiye’de bitkiler ile tedavi. nobel tıp kitabevleri, i̇stanbul. 480 p. boğa, m., ertaş, a., yılmaz, m.a., kızıl, m., çeken, b., haşimi, n., özden t.y., demirci, s., yener, i̇., deveci, ö. 2016: uhplc-esi-ms/ ms and gc-ms analyses on phenolic, fatty acid and essential oil of verbascum pinetorum with antioxidant, anticholinesterase, antimicrobial and dna damage protection effects. iranian journal of pharmaceutical research, 15(3): 393-405. clsi, 2006. clinical and laboratory standarts 172 institute. methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard-seventh edition. m07a7, villanova, pa, usa. cox, s., mann, c., markham, l., bell, h., gustafson, j. 2000: the mode of action of the essential oil of melaleuca alternifolia (tea tree oil). journal of applied microbiology, 88(1): 170-175. das, m.p. 2014: effect of cell surface hydrophobicity in microbial biofilm formation. european journal of experimental biology, 4(2): 254-266. davis, p.h., mill, r.r., tan, k. 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vasudevan, r. 2014: biofilms: microbial cities of scientific significance. journal of microbiology & experimentation, 1(3): 1–16. vuong, c., kocianova, s., voyich, j.m., yao, y., fischer, e.r., deleo, f.r., otto, m. 2004: a crucial role for exopolysaccharide modification in bacterial biofilm formation, immune evasion and virulence. journal of biological chemistry, 279(52): 54881-54886. 173 biologica nyssana ● 10 (2) december 2019: 169-173 göse, hacıoğlu doğru ● antibiofilm activity of verbascum pinnatifidum vahl. ethanolic extract microsoft word bn-oa-0201-07 jovanovic_et_al biologica nyssana 2 (1) september 2011: 51-58 jovanović, b. et al. assessment of heavy metal load in chub liver… 51 original article ! assessment of heavy metal load in chub liver (cyprinidae – leuciscus cephalus) from the nišava river (serbia) boris jovanović*1, željko mihaljev2, stevan maletin3, dušan palić4 1interdepartmental toxicology program and fisheries biology program, departments of biomedical sciences and natural resource ecology and management, iowa state university, ia, usa 2scientific insitute „novi sad“ for veterinary medicine, novi sad, serbia 3faculty of agriculture, university of novi sad, serbia 4department of biomedical sciences, the college of veterinary medicine, iowa state university, ia, usa * e-mail: prcko@iastate.edu abstract: jovanović, b., mihaljev, ž., maletin, s., palić, d.: assessment of heavy metal load in chub liver (cyprinidae leuciscus cephalus) from the nišava river (serbia). biologica nyssana, 2 (1), september 2011: 51-58. the nišava river plays an important role as the source for both drinking water and agricultural irrigation due to its hydrological and geomorphological characteristics as the largest river in the region of southeast serbia. in this study we used the liver of the chub (leiciscus cephalus) as a tool for biomonitoring heavy metal accumulation along the river. chub specimens were sampled from two localities (one at the border with bulgaria and a second in the city of niš). concentrations were estimated for six heavy metals (iron, cadmium, copper, zinc, lead and manganese) in chub liver. low bioconcentration level was observed for most of the metals and the concentrations corresponded to the nominal concentration in livers of fish inhabiting metal unpolluted streams and rivers. however, cadmium concentration in the chub liver exceeded 0.5 mg kg-1, a several hundred folds increase from nominal concentration indicating a potential toxic exposure of the fish and of the stream ecosystem to this heavy metal. hepatosomatic indices were calculated and tested for the impact of metal concentrations on liver size. a decrease of the hepatic index was observed in fish with higher cadmium concentration, suggesting a possible impact on the health of the chub population in the nišava river. key words: biomonitoring, heavy metals, hepatosomatic index, leuciscus cephalus, nišava river introduction ! rivers represent the most complex aquatic systems in terms of transport and interactions of heavy metals with geochemical and biological processes. large surface to volume ratios typical of flowing waters support extensive interactions with the atmosphere, constant exchange of gases, and the introduction of material suspended in atmospheric precipitations. oxygen saturation in the surface layer of the water allows for sustained high redox potential, causing quick degradation of organic matter and maintenance of the oxidative state of iron and manganese as suspended colloid hydroxides (a l l a r d et al., 2004; wa r r e n & h a a c k 2001). chemical assays of water and sediments have traditionally been used as tools in ecotoxicological analysis, but the variable chemistry of freshwater streams limits their application in observing cumulative effects of contaminants on the stream biota. biomonitoring of hazardous substances in tissues of aquatic organisms have been successfully 2 (1) • september 2011: 51-58 biologica nyssana 2 (1) september 2011: 51-58 jovanović, b. et al. assessment of heavy metal load in chub liver… 52 applied in recent years as indicators of heavy metal pollution (e v a n s et al., 2000; l a m a s et al, 2007; t e o d o r o v i ć , 2001; v a n d e r o o s t et al., 2003; wa g n e r & b o m a n 2003). furthermore, the use of fish as bioindicators appears more timeand cost-effective when compared with the effort required for repetitive water chemistry sampling and the cost of assessing pollutants with standardized chemistry and toxicity assays (b a r b o u r , 1999). water use and consumption of fish contaminated with heavy metals have deleterious effects on human health, which was widely acknowledged after a series of events in the period from 1953 to 1960 when several thousand people died in minamata bay in japan as a result of poisoning caused by the consumption of mercurycontaminated fish (s m i t h & s m i t h 1975). observed toxic effects raised public safety and human health concerns repeatedly since minamata, prompting legislators to set limits on the lead, cadmium and mercury levels detected in the fish muscle, but other heavy metals and fish tissues were not included in the european union (eu) regulations (e u r o p e a n c o m m i s s i o n , 2001; e u r o p e a n c o m m i s s i o n , 2002). fish can absorb heavy metals through epithelial or mucosal surfaces of the skin, gills and gastrointestinal tract. metal ions can exert toxicity effects either directly at the gill surface (fast binding reactions), or passing through the gills and causing adverse effects in the internal organs (s i m k i s s & t a y l o r , 1989). however, the majority of heavy metal load is commonly ingested via water or feedstuff and transported to the bloodstream and liver via mucosal membranes and hepatic blood supply. therefore, heavy metals are accumulated in the liver and subjected to a detoxication process. being the primary target of metal deposition, the liver can be effectively used as a biomonitoring organ for heavy metal pollution of water ecosystems (i c e s , 1991; d e n n y et al., 1995; j ø r g e n s e n & p e d e r s e n , 1994; s w a i b u h l w a n g a et al.,, 2003; t e o d o r o v i ć , 2001). the presented study describes heavy metals loads in leuciscus cephalus l. 1758 cyprinidae livers in the nišava river, which is the major supply point for irrigation and drinking water to south-east serbia. research aims were to: a) determine the concentrations of six heavy metals (fe, cd, cu, zn, mn and pb) in l. cephalus liver; and b) assess the impact of the heavy metals on the size of l. cephalus liver as the major detoxification organ. using fish livers as bioindicators of water quality in streams appears to be a rapid and economical method for screening waterways for the presence of heavy metals. furthermore, collected information on water quality can be used to establish a broad categorization of the examined stream and has the potential to be used by environmental managers and policy makers to improve the management of the river, following the example of the nišava river canyon section, “sićevačka gorge nature park”, which is currently under strict state protection because of its biodiversity and for providing a habitat for a number of endemic and relict species. material and methods stream and sampling site description the nišava river with its tributary ginska is a 202 km long with a watershed area of 4068 km2. it is situated in south-east serbia and bulgaria (upper one fourth of the river) and it is the largest tributary of the južna figure 1. the geographic region of the nišava river, the two sampling stations (l1 and l2) are indicated with arrows biologica nyssana 2 (1) september 2011: 51-58 jovanović, b. et al. assessment of heavy metal load in chub liver… 53 morava river, part of the southern danube river watershed (g a v r i l o v i ć & d u k i ć , 2002). the nišava has two distinct parts: upstream of the town of bela palanka it is a narrow, rapid mountain stream ranging from 6 to 76 m in width and 0.6 to 2 m in depth with velocity from 1 to 2.5 m s-1 and flow of 0.3-270 m3 s-1 of water. after bela palanka and all the way to its mouth near the village of lalinac, the nišava becomes a meandering stream with increased width (15-100 m), depth (1-3 m), and flow that carries 3-700 m3 s-1 of water (b r a n k o v i ć , 1997). l. cephalus sampling was performed on two sites in may of 2005 (fig. 1). the nišava river at ivko’s watermill, 1 km upstream from the city of dimitrovgrad was selected as site l1. this is the first section of the river as it enters serbia and there are no larger settlements upstream to this sampling site. in this section the river has a width of 6 m, and a depth of no more than 1 m. the bottom is composed of gravel, and the banks are covered with thick vegetation. the majority of the benthofauna is composed of ephemeroptera and trichoptera larvae, while in the ichthyofauna the most dominant species are: alburnoides bipunctatus bloch, 1782 cyprinidae, barbus peloponnesius valenciennes, 1842 cyprinidae and l. cephalus. as site l2, the nišava river access point at the greek consulate in the city of niš was selected. the selected site is located on the last section of river, about 10 km before its mouth. the greek consulate site is located downstream of the niš city limits and other heavily populated areas (cities of dimitrovgrad, pirot, and bela palanka). the site was selected due to the high potential for heavy metal contamination from several upstream industrial areas. at this site, the nišava river is about 80 m wide, and between 0.2 and 1.2 m deep. the bottom is composed of gravel, and both sides of the river are paved with no vegetation. the icthyofauna is rich, with the predominant species being a. bipunctatus, barbus barbus l. 1758 cyprinidae, b. peloponnesius, l. cephalus, alburnus alburnus l. 1758 cyprinidae, chondrostoma nasus l. 1758 cyprinidae and rhodeus sericeus pallas, 1776 cyprinidae. fish sampling and tissue preparation l. cephalus is one of the few species that inhabit the nišava through the whole serbian section of the river, can be collected with low catch per unit effort using only basic sport fishing gear, and its trophic level (omnivore – insectivore) potentially allows for bioaccumulation of heavy metals in the organs due to both water and nutritional intake. chub sampling was performed by an angling technique using hook and line. upon capture, specimens were immediately anesthetized in chloroform, euthanized, and stored in a field fridge at 0-4 °c. wet weight and total length of each specimen was measured in the laboratory using digital scales (kern 440-33, d=0.01g). the age of specimens was determined using standard scale method (apha, 1995). to avoid contamination of samples, dissection was carried out using stainless steel instruments and latex gloves without talcum, and collected livers were weighed. analyses of heavy metals in l. cephalus livers l. cephalus liver samples were treated using standard nitric acid-perchloric acid digestion (apha, 1995). briefly, 10 ml of hno3 and hclo4 has been added to the sample and heated until the samples dissolved. dissolved sample was diluted to 100 ml with deionized water in volumetric flask. all digests were incubated for a minimum of five hours at room temperature, before being transferred to a conical flask, and then refrigerated for 48 h at 4 °c to allow complete settling before aspiration and analysis with a flame atomic absorption spectrometer (varian, spektr aa-10). a total of 10 samples were prepared for each locality. concentrations of fe, cd, cu, zn, mn, and pb were expressed as mg kg-1 of wet weight, as well as cumulative concentrations (the summary of fe, cd, cu, zn and mn). data analysis prior to any further analysis, all data were subjected to tests of normality (kolmogorovsmirnov) and homogeneity of variance (levene’s), and no deviations were detected. analysis of variance (anova; (s t a t s o f t , 1996)) was used to determine differences in metal loads between localities. to determine if the size of specimens was associated with the concentrations of metals in the liver, spearman’s rank correlation tests (s t a t s o f t , 1996) were conducted between metal concentrations from chub liver and total length of fish specimen (s w a i b u h l w a n g a , 2003). the hepatosomatic index was calculated according to the formula: hsi=m/m*100, where m is the mass of the liver; and m is the mass of the fish specimen. spearman’s rank correlation test (s t a t s o f t , 1996) was also used to explore the correlation level between the hepatosomatic index and concentrations of metals in the liver, as well as to test hypotheses as to the possible negative impacts to fish due to high metal concentrations. biologica nyssana 2 (1) september 2011: 51-58 jovanović, b. et al. assessment of heavy metal load in chub liver… 54 results total length of fish specimens ranged from 13.6 to 20.5 cm, with the age ranging from 2 years to 3 years. the smallest specimen weighed 24.6 g, while the largest one weighed 86.1 g. no correlations between fish total length and metal concentration in the liver were observed, neither with single metal concentrations nor with cumulative concentrations (spearman’s rank correlation, p > 0.05). mean concentrations of heavy metals in chub liver at the l1 site for fe, cd, cu, zn and mn are 76.73, 0.98, 2.98, 38.83 and 3.47 mg kg-1 wet weight; while at the l2 site, concentrations were of the same order of magnitude: 93.95, 0.57, 6.94, 31.81 and 2.82 mg kg-1 wet weight, respectively (fig. 2). in both sites, pb was below the detection limit of the instrument. the comparison of data from the two sites revealed a significantly increased concentration of cadmium and zinc (fig. 3) at site l1 (anova: f = 5.2, p < 0.05; f = 4.7, p < 0.05 respectively). there were no differences in fe, cu and mn concentrations. l1 fe cd cu zn mn -20 0 20 40 60 80 100 120 l2 fe cd cu zn mn -20 0 20 40 60 80 100 120 figure 2. heavy metal concentration in chub liver at site l1 and site l2. values on the y-axis represent mg kg-1 wet weight. boxes refer to standard errors and whiskers refer to standard deviations correlation tests between hepatosomatic index and metal concentration in liver can be used to infer potential deleterious effects of heavy metal bioaccumulation. the concentration of cadmium in fish collected at site l1 is strongly and negatively correlated with liver size in chub (spearman’s rank correlation, r = -0.87, p < 0.001), which suggests potential influence of a metal on fish metabolism (fig. 4). all other concentrations of metals, singular and cumulative, from either site did not show any significant correlation. hepatosomatic index was 1.36 + 0.11 (standard error of the mean) for site l1 and 1.57 + 0.12 for site l2. ±std. dev. ±std. err. mean 22 26 30 34 38 42 46 50 l1 zn l2 zn b* ±std. dev. ±std. err. mean 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 l1 cd l2 cd a* figure 3. differences in concentration of cadmium and zinc at localities l1 (3a) and l2 (3b). values on the y-axis represent mg kg-1 wet weight. boxes refer to standard errors and whiskers refer to standard deviations. * indicates that the effect is statistically significant (anova p<0.05) discussion progressive accumulation of metal residues during the aging process in fish has been discussed recently, and results presented in this study are in accordance with published data (s w a i b u h biologica nyssana 2 (1) september 2011: 51-58 jovanović, b. et al. assessment of heavy metal load in chub liver… 55 l w a n g a et al., 2003; vinagre et al., 2004). it has been demonstrated that in relatively young fish progressive accumulation is not significant (except for mercury), and it was proposed that only fish from 1+ to 4+ years old should be used for such analysis (t e o d o r o v i ć , 2001). most of the concentrations of heavy metals in l. cephalus liver were close to the nominal concentrations (t e o d o r o v i ć , 2001) that are present in livers of fish inhabiting metal unpolluted streams and rivers. however, concentrations of cd were several hundreds folds greater than the nominal concentrations (t e o d o r o v i ć , 2001). cd ls i 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 0.2 0.6 1.0 1.4 1.8 2.2 l1 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 cd 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 ls i l2 figure 4. linear regression (spearman’s rank correlation, r = 0.87, p < 0.001) of hepatosomatic index (y-axis) and concentration of cadmium in chub liver mg kg-1 wet weight from locality l1 (xaxis) and r= 0.07, p >> 0.05 from locality l2 a recent comparable study was conducted on the nišava river using b. peloponnesius as a model on four sites, including site l2 of the present study (b r a n k o v i ć , 2003), and a significantly lower concentration of lead (nil vs. 0.76 mg kg-1) and cadmium (0.57 vs 1.82 mg kg-1) were detected in the l. cephalus liver compared to b. peloponnesius. lower concentrations of pb and cd detected after two years could suggest that the water quality had improved, but also can reflect different ecotoxicological effects of heavy metals on two analyzed species. b. peloponnesius is a strict benthivore species and could be more likely to accumulate heavy metals when compared to l. cephalus, which also feed in the upper water column. cadmium concentration in chub liver was higher in locality l1, which was expected to be the less polluted site. it is unclear why this phenomenon occurred, as there are no known pollution sources upstream of the site on the serbian side of the border, unless cd is found in the waters flowing in from nearby bulgaria. cadmium at observed concentrations has the potential to affect the metabolism of the fish (fig. 4) and could account for the increase of zinc concentration in the livers on this locality (fig. 3). zinc is an antagonist to cadmium and adaptation has been observed that in the presence of toxic concentrations of cd, zinc can be more rapidly absorbed by receptor spots in the gills in order to lower cd toxicity (w i c k l u n d et al., 1988). although the concentration of the heavy metals in the liver can be used to estimate the overall heavy metal burden in the stream that the aquatic biota can uptake, it is unrelated to food safety of the fish used in human consumption. therefore, it is strongly advisable to compare all suspicious metal concentrations with the proposed maximal allowed concentration cited in the regulations before fish is used for this purpose. the cd concentration in the liver was converted to muscle concentration using the bioconcentration index for carp, in order to check if there are any possible threats for human health, (c i n i e r d e c o n t o et al., 1999). the maximal possible cd concentration was used as output corresponding to 53 µg l-1 exposure of cd for 127 days as described (c i n i e r d e c o n t o et al., 1999). a calculated estimate placed cd concentration in fish muscle at 0.07 mg kg-1 wet weight at site l1 and 0.04 mg kg-1 wet weight at l2. according to current eu legislation (european c o m m i s s i o n , 2002), the l1 values could represent potential threats to human health as the maximum allowed concentrations (mac) for cadmium in freshwater fish musculature are 0.05 mg kg-1 wet weight. however, serbian laws are much looser and the mac for cadmium is 0.1 mg kg-1 wet weight (a n o n , 1992). nevertheless, the observed concentrations of cd exceed european standards on locality l1 and suggest a pressing need for frequent monitoring and a stronger biologica nyssana 2 (1) september 2011: 51-58 jovanović, b. et al. assessment of heavy metal load in chub liver… 56 environmental policy in order to maintain the water quality in the nišava river. in conclusion, the concentrations of cadmium and zinc are increased in l. cephalus livers from the upper part of the river, and cd concentration is high enough to suggest a certain degree of effects on the metabolism of l. cephalus, also involving a possible buffer-like interaction with zinc. historical information on the presence of cadmium in the upper parts of the river system is not available, and we therefore propose that further investigations should also include the bulgarian river section. a potential risk to human health is observed due to increased cd concentration in fish tissues collected from the upper nišava river system. acknowledgments. we are very thankful to stefano mariani for the critical reading of the manuscript, and adolfo carrillo cabello for the help with english grammar. references allard, t., menguy, n., salomon, j., calligaro, t., weber, t., calas, g., benedetti, m.f., 2004: revealing forms of iron in river-borne material from major tropical rivers of the amazon basin (brazil). geochimica et cosmochimica acta, 68(14): 3079-3094. anon, 1992: pravilnik o količinama pesticida, 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behaviour in freshwater environments. earth-science reviews, 54(4): 261-320. wicklund, a., runn, p., norrgren, l., 1988: cadmium and zinc interactions in fish: effects of zinc on the uptake, organ distribution, and elimination of 109 cd in the zebrafish, brachydanio rerio. archives of environmental contamination and toxicology, 17(3): 345-354. biologica nyssana 2 (1) september 2011: 51-58 jovanović, b. et al. assessment of heavy metal load in chub liver… 58 anticancer compounds from medicinal plants biologica nyssana 5 (2)  december 2014: 83-90 dragović, r. et al.  accumulation of heavy metals in different parts … 83 original article received: 14 november 2014 revised: 8 december 2014 accepted: 20 december 2014 accumulation of heavy metals in different parts of russian thistle (salsola tragus, chenopodiaceae), a potential hyperaccumulator plant species ranko dragović1*, bojan zlatković1, snežana dragović2, jelena petrović3, ljiljana janković mandić2 1university of niš, faculty of sciences and mathematics, višegradska 33, 18000 niš, serbia 2university of belgrade, vinča institute of nuclear sciences, p.o. box 522, 11001 belgrade, serbia 3university of belgrade, institute for the application of nuclear energy, banatska 31b, 11080 belgrade * corresponding author: dragovicr@pmf.ni.ac.rs abstract: dragović, r., zlatković, b., dragović, s., petrović, j., janković mandić, lj.: accumulation of heavy metals in different parts of russian thistle (salsola tragus, chenopodiaceae), a potential hyperaccumulator plant species. biologica nyssana, 5 (2), december 2014: 83-90. distribution and accumulation of 10 heavy metals was observed in plant parts of species s. tragus, a native representative of serbian flora. according to high bioaccumulation values (baf), more than a half of the analyzed elements were bioaccumulated in roots, as well as in the aerial part of the plant. translocation factor (tf) value was calculated, showing the ratio between the concentration of metals in roots and aerial parts of the plant. the results indicate that s. tragus may accumulate significant amounts of cd, co, cr and pb. concentrations of some of the individual elements are shown to be statistically different depending on the sampled plant parts. potential capacity of s. tragus for hyperaccumulation of heavy metals and possible utilization of this plant for phytoremediation is discussed. key words: salsola tragus, heavy metals, plant parts, accumulation, serbia apstrakt: dragović, r., zlatković, b., dragović, s., petrović, j., janković mandić, lj.: akumulacija teških metala u različitim delovima sonjače (salsola tragus, chenopodiaceae), potencijalne hiperakumulatorske biljne vrste. biologica nyssana, 5 (2), decembar 2014: 83-90. istraživana je distribucija i akumulacija 10 teških metala u delovima biljke s. tragus, autohtonog predstavnika flore srbije. prema visokim vrednostima faktora bioakumulacije (baf), vise od polovine analiziranih elemenata je bioakumulirano u korenu, kao i u nadzemnim delovima biljke. izračunat je faktor translokacije (tf), koji predstavljaja odnos koncentracije datog elementa u korenu i one u nadzemnim delovima biljke. rezultati su pokazali da s. tragus može akumulirati značajne koncentracije cd, co, cr i pb. pokazano je da se koncentracije elemenata statistički razlikuju u zavisnosti od analiziranih delova biljke. diskutovan je potencijalni kapacitet hiperakumulacije teških metala ove biljke i mogućnost njene primene u fitoremedijaciji. ključne reči: salsola tragus, teški metali, delovi biljke, akumulacija, srbija 5 (2) • december 2014: 83-90 biologica nyssana 5 (2)  december 2014: 83-90 dragović, r. et al.  accumulation of heavy metals in different parts … 84 introduction anthropogenic activities have resulted in increased deposition and migration of potentially toxic heavy metals, posing a major risk for both human health and the environment. heavy metals may disrupt the physical, chemical and biological balance of the soil and therefore affect the growth of plants. however, many plant species have adapted to tolerate high concentrations of heavy metals without any detrimental effects on growth or survival (b r o o k s , 1998). a hyperaccumulator may be defined as a plant whose tissues and organs contain a metallic element at a concentration exceeding a specified threshold, when growing in nature (v a n d e r e n t et al., 2013). the threshold concentration should be two or three orders of magnitude higher than in leaves of most species on normal soils, and at least one order of magnitude greater than the usual range found in plants from metalliferous soils. based on these criteria, the proposed nominal threshold values (mg kg-1 of dry leaf tissue) are: 100 for cd, se and tl; 300 for co, cr and cu; 1000 for as, ni and pb; 3000 for zn and 10,000 for mn (v a n d e r e n t et al., 2013). salsola tragus l. (syn. s. kali subsp. ruthenica), commonly known as russian thistle or tumbleweed, is one of the most common species of tumbleweed belonging to s. kali aggregate. it is an annual, weedy plant species, which at maturation forms spherical bushes approximately 1 m in diameter, detaches from the root system and tumbles in the wind, spreading its seed (b a r g e r o n & s w e a r i n g e n , 2010). native distribution of the species overlaps large areas of europe and asia, but it is also naturalized in north, central and south america, africa and australia (m o s y a k i n , 2003). it has a comparatively wide distribution in serbia, especially in the northern and northeastern parts of the country (s l a v n i ć , 1972). it is generally distributed in sandy areas in coastal regions and semiarid and arid areas in lowlands, deserts, but it is also common at ruderal habitats and disturbed places in the settlements, across the roadsides, and cultivated fields. due to its wide distribution, adequate life form and other biological traits, this taxon is a good candidate for a biomonitoring species. it has been shown that tumbleweed is able to capture elements from the ambient air and that has capabilities for being used as biomonitor of environmental pollution by heavy metals (b e n i t e z , 2009). the tumbleweed is also able to grow in areas polluted by radionuclides (w a r r e n , 2001). the aim this study was to assess the hyperaccumulating capacity of aerial as well as underground parts of tumbleweed (s. tragus) collected in the urban and sub-urban area of the city of niš, southeast serbia. in this study we demonstrated distribution and accumulation of cadmium (cd), cobalt (co), chromium (cr), mercury (cu), iron (fe), manganese (mn), nickel (ni), lead (pb), zinc (zn) and vanadium (v) in the tissues of s. tragus. material and methods study area the study area covers the urban and suburban area of the city of niš, i.e. the peripheral parts of niš valley, a small part of the sićevo gorge and alluvial terraces of nišava river and the south morava river at their confluence. niš is a city in southern serbia, situated in the composite valley of the river nišava near its confluence with the south morava. the city of niš has an area of 597 km2 including niška banja and 68 suburban and rural settlements. with 393,357 inhabitants (census of 2011) it is the second largest city in serbia and one of the most important industrial centers in serbia (electronics, mechanical engineering, textile and tobacco industry). the nišava valley is located at the contact of young and accumulated layers of broken rocks belonging to the carpatho-balkanides and paleozoic formations belonging to rhodopian crystalline mass. in niš basin the following rock types are distributed: magmatites, metamorphites, carbonate rocks, clastites and non-consolidated or partly consolidated neogene sediments (k r s t i ć et al., 1980; r a k i ć et al., 1973). at the rims of niš basin, above these rocks the alluvial sediments are deposited. the alluvial soils dominated in the area. sićevo gorge is formed by vertical fluvial erosion in the pliocene limestones between the southern slopes of the svrljig mountains and the mountain suva planina. sampling locations in sićevo gorge are located next to the main road junction with high traffic frequency (v u j i s i ć et al., 1980). at the study sites, there are no significant deposits of heavy metals. sampling and sample preparation the samples of tumbleweed plants and underlying soils were collected in 2013 from 15 locations with different intensity and types of anthropogenic influence. the selection of sampling sites was influenced by spatial distribution of tumbleweed populations in the investigated area. underground (roots) and aerial parts (stems, leaves and inflorescences) of plants from the same location were separated and washed in a series of fresh and de-ionized water to remove any particles of soil. the biologica nyssana 5 (2)  december 2014: 83-90 dragović, r. et al.  accumulation of heavy metals in different parts … 85 material was chopped, dried at room temperature for two weeks and finally stored in paper bags, prior to chemical analysis. voucher specimens are deposited at the department of biology and ecology (hmn), faculty of science and mathematics, university of niš, under the acquisition numbers 7315-7324. the mineralization of soil samples was performed by slightly modified methods described by e d g e l l (1988). approximately 1-3 g (depending of expected total metal concentrations in soil) of air-dried, ground and sieved (< 0.2 mm) soil was weighed and transferred into cuvettes. each sample was weighed in two replications. the 10 ml of hno3 was added and the samples were heated on the heating plate gradually, until no brown fumes were emitted. the solution was allowed to evaporate approximately to 5 ml. after cooling, 2 ml of water and 3 ml of 30% h2o2 were added and heated until effervescence subsided and the solution cooled. additional h2o2 was added until effervescence ceased (but no more than 10 ml h2o2 was added). the heating was continuing, and the solution was allowed to evaporate to approximately 5 ml. dried and ground plant materials (root and aerial parts, separately) were weighed (approximately 0.5 g) and transferred into cuvettes for mineralization. each sample was weighed in two replications. the mineralization was performed by wet procedure, i.e. with the mixture of hno3 (65%) and h2so4 (min. 95%) (iso 6636/2: 1981). fig. 1. s. tragus at the collection site in suburban area of the city of niš both soil and plant extract were cooled, filtered through satorius stedim biotech filter paper (quantitative grade 391) and adjusted to the appropriate volume with hno3/h2o (3:1) solution. analytical methods soil and plant samples were analyzed by using an atomic absorption spectrometer shimadzu aa-7000. measurements was performed by flame technique with the detection limits (3σ) being approximately 0.1 ppm for v, 0.6 ppm for cr, 0.01 ppm for cu, zn and mn, 0.2 ppm for ni, 0.07 ppm for pb, 0.7 ppm for fe, 0.3 ppm for co and 0.03 for cd. the deuterium lamp (d2) method was used as background correction function. control software wizaard was used for shimadzu atomic absorption spectrophotometer. reference standard materials nist 1515 (apple leaves) and bipea quality control sample (soil 90-0115-0106) were used for the verification of the measurement. the bioaccumulation factor is calculated as follows (f e i j t e l et al., 1997; g o b a s & m o r r i s o n , 2000; m a c k a y & f r a s e r , 2000): bfi = ci/cs (1) where ci is the mean concentration of metal in roots (i=a) or aerial (i=b) parts of tumbleweed (mg kg-1) and cs is the mean metal concentration in underlying soil (mg kg-1). the plant could be considered as hyperaccumulator if the baf is higher than 1. translocation factor (tf) is calculated as ratio of the metal concentration in aerial parts of the plants to those in the roots (c u i et al., 2007; l i et al., 2007; s o u s a et al., 2008; m a l i k et al., 2010): tf = cb/ca (2) where cb is the mean metal concentration in aerial parts (mg kg-1) and ca is the mean metal concentration in roots of plant (mg kg-1). the tf˃1 indicates that the plant translocates metals effectively from the roots to its aerial parts (b a k e r & b r o o k s , 1989). statistical methods analysis of variance (anova): one-way anova was used in order to determine whether there are any differences in concentrations of heavy metals among different examined parts of the plant, and among the localities. the results of this analysis should also pinpoint the most important variables regarding concentrations of metal in tissues of two plant parts. correlation analysis: in order to affirm relationships between heavy metal concentrations in roots and aerial parts of tumbleweed, pearson’s correlation analysis was applied. statistical data processing was carried out by statistica 8 software. biologica nyssana 5 (2)  december 2014: 83-90 dragović, r. et al.  accumulation of heavy metals in different parts … 86 results and discussion the mean concentrations of heavy metals in different parts of tumbleweed and in underlying soils and values of bioaccumulation and transfer factors are presented in tab. 1. different plant species and genera grown on the same soil may display different metal uptake and movement in plant (m i l e s & p a r k e r , 1979). in addition to availability of metals in soil to plants, there are several factors that cause metal uptake and transport through the plant. the biological factors, including the capability to transport particular metals through transport systems, strongly affect the accumulation behavior of the plant. heavy metal absorption and accumulation varies according to plant taxa and presence of suitable metal forms for the plant uptake in the soil. the studies of de la r o s a et al. (2004) and g a r d e a -t o r r e s d a y et al. (2005) suggested that tumbleweeds could be considered as potential hyperaccumulating species for cadmium and chromium, respectively. one of the species, s. soda has also found to be hyperaccumulator for lead (l o r e s t a n i et al., 2011; 2013) and zinc (l o r e s t a n i et al., 2013) and therefore suitable candidate for phytoremediation of contaminated soils. the obtained baf values pointed out the tendency of s. tragus to accumulate cd, co, cr, ni, pb and v in both underground and aerial parts of the plant (tab. 1). the highest bioaccumulation was observed for cd, with baf values of 6.32 and 6.73 in underground and aerial parts of the plant, respectively. for most studied elements, the plants have shown higher bioaccumulation capacity in their upper parts, except for v, which was better represented in roots than in aerial parts. however, the number of analyzed cases with favorable transport of absorbed metals from root zone into the aerial parts of plant, shown as translocation factor (tf), was much lower. in this study, high values of translocation factor were recorded only for cd, co, cr and pb, in cases of realistic possibility of uniform accumulation of these elements in aerial parts of plant species s. tragus. while the translocation factor indicates the relative ratio of concentrations of a certain metal in root and aerial parts, the transport of metals through the plant is directly influenced by various ecological characteristics of the plant, primarily the type and level of development of transport tissues, xylem and phloem, or existence of special physiological mechanisms. the data collected on ten elements in both plant parts in all localities were used to determine the interaction among metal concentrations in different plant parts. in combination with the tf values this could provide additional information on possibility of their transport through plant tissues. the derived correlation coefficients, as a measure of linear strength of connection between a pair of observed factors, indicate strong connection between concentrations of pb, fe and ni in roots and in aerial parts of plant (table 2). there was also a strong and statistically highly significant (p < 0.001) positive correlation of concentrations of fe and pb in aerial and underground part of plant. at the same time other types of correlation between concentrations of other variables were also recorded. however, the values of the correlation coefficients were weak or medium-strength, indicating that in the species s. tragus there is a clear interaction between concentrations of adopted elements in aerial and underground parts of plant. existence of favorable transport through the tissues and successful overcoming of the barriers that might potentially disrupt transport of heavy metals through plants, speaks in favor of the possibility that s. tragus performs hyperaccumulation of pb, fe and ni in cases when these metals are present in the environment in greater concentrations. samples of roots and aerial parts of plant species s. tragus collected in the study area show variability in concentrations for all 10 analyzed elements. the original hypothesis assumed that, as table 1. mean concentrations of heavy metals in underground (ca) and aerial (cb) parts of tumbleweed and underlying soil cs (mg kg-1 d.w.) and mean values of bioaccumulation (baf) and transfer (tf) factors parameter cd co cr cu fe mn ni pb zn v ca 5.56 79.7 86.4 8.12 6120 19.9 75.2 185 14.9 140 cb 5.92 85.9 102 10.4 6470 43.5 72.2 188 28.7 87.7 cs 0.88 14.7 41.6 54.7 27310 320 41.7 101 63.0 43.4 bafa 6.32 5.42 2.08 0.15 0.22 0.06 1.80 1.82 0.24 3.18 bafb 6.73 5.85 2.46 0.19 0.24 0.14 1.73 1.85 0.46 2.02 tf 1.06 1.08 1.18 1.27 1.06 2.18 0.96 1.02 1.92 0.63 biologica nyssana 5 (2)  december 2014: 83-90 dragović, r. et al.  accumulation of heavy metals in different parts … 87 table 2. correlations between concentrations of heavy metals in roots and aerial parts of tumbleweed (high r values in bold) cdb cob crb cub feb mnb nib pbb znb vb cda 0.72 0.17 0.21 -0.08 -0.32 -0.09 0.05 -0.25 0.19 -0.11 coa 0.29 0.42 0.10 -0.32 -0.41 -0.15 -0.26 -0.48 0.25 0.03 cra -0.16 -0.10 0.49 0.52 0.17 0.20 0.21 0.11 -0.21 -0.37 cua -0.48 0.02 -0.06 0.30 0.40 0.32 0.01 0.43 -0.11 -0.47 fea -0.35 -0.15 -0.14 0.29 0.94 0.71 0.18 0.96 -0.03 -0.49 mna -0.43 0.05 0.03 0.33 0.67 0.65 0.24 0.70 0.03 -0.54 nia -0.05 -0.23 0.23 0.27 0.35 0.41 0.84 0.38 0.05 -0.06 pba -0.42 -0.25 -0.13 0.32 0.94 0.62 0.24 0.98 -0.02 -0.48 zna -0.15 -0.04 -0.00 0.10 -0.03 0.10 0.07 -0.04 0.38 -0.08 va -0.24 0.04 0.12 0.11 0.13 -0.21 -0.16 0.17 -0.56 0.10 a – roots, b-aerial parts different parts of plant differ in anatomicallymorphological and functional sense, the movement and potential accumulation of metals in tissues would also be different. therefore the differences in mean values of measured concentrations of metals in various parts of plants were tested, and characters with greatest impact in differentiation of samples from aerial and underground plant parts were determined. the results of unifactorial analysis have shown that there are statistically significant differences in concentrations of heavy metals depending on plant organ, but only for some metals (tab. 3). comparison of aerial and underground parts of plant s. tragus, at level of 15 collected samples, shows statistically highly significant concentrations of mn, zn and v. the possibility of differentiation is most influenced by mn, which is characterized by the highest f value among all statistically significant variables. the measured mean values of mn were greater (more than double) in tissues of aerial part than in the root of the analyzed plant species. the manganese is one of the most important elements, influencing the process of photosynthesis and some other metabolic processes, but it is believed to be easy to transport through transport tissues and accumulated primarily in green parts of plants (m a r s c h n e r , 1995). the manganese shows a general tendency of greater accumulation in shoots than in root of the same plant, as demonstrated in samples of a number of different plant species (m i l l a l e o et al., 2010). it was proven that mn is transported through xylem relatively easily, in contrast to transport in the opposite direction, through the phloem part of transport system toward the underground plant organs (p a g e & f e l l e r , 2005). the aerial parts of s. tragus have included concentration of zn almost double to that in the underground part. the zinc is similar to mn in being an important nutrient for plant organisms, playing a significant role in process of photosynthesis and shows tendency to accumulate in the aerial parts of plants. in experimental conditions it was proven that this metal accumulates in leaves, and in case of increased concentration in substrate there may be hyperaccumulation in leaves of a number of cultivated plant species (s e k a r a et al., 2005). table 3. analysis of variance of heavy metal concentrations between the root and the aerial parts of tumbleweed (significant values in bold) effect root/aerial part root/ locality aerial part/ locality variable f (df 1) f (df 10) f (df 10) cd 0.51 0.71 0.78 co 1.01 1.16 1.22 cr 0.30 2.14 1.04 cu 1.84 1.40 0.62 fe 0.11 3.22 3.20 mn 44.92 1.82 0.77 ni 0.09 3.22 6.95 pb 0.02 4.39 4.18 zn 22.13 0.69 1.32 v 12.68 0.49 1.14 athe significance levels: p˂0.05;  p˂0.01; p˂0.001 distribution of v in parts of plant body is different than distribution of other elements. depending on sampling locality, values of this element in roots of s. tragus were greater on average than the values for aerial parts (tab. 1). the mean values of all analyzed samples show concentrations of v in roots to be more than 1.5 times greater than in rest of the plant. in contrast to mn and zn which play important roles in various physiological processes in plants, the phenomenon of accumulation and transport of v in plants is still biologica nyssana 5 (2)  december 2014: 83-90 dragović, r. et al.  accumulation of heavy metals in different parts … 88 largely understudied. most plants absorb v in very small amounts in comparison to its availability in soil. results of several studies, including our research, indicate that this element is accumulated in the root and poorly transported toward the aerial organs of the plant (s z i k l a i et al., 1987; n o w a k o w s k y , 1993). possibility of stronger accumulation of v in the root than in stems, fruits and green leaves, was also proven experimentally (g i l et al., 1995; v a c h i r a p a t a m a et al., 2001). increased concentration of this element in environment and in plant and animal organisms may have a toxic effect on consumers. increase of level of v in water and soil is attributed to burning of coal and oil derivatives, as well as to use of phosphate fertilizers (r i n g e l b a n d & h e h l , 2000). according to literature data, increased level of v was primarily recorded in plants growing on alluvium soil (g i l et al., 1995). it must be noted that almost all samples of species s. tragus analyzed in this study were collected at agricultural land or recently abandoned cultivated land, formed on alluvial deposits of river valleys. although s. tragus is able to accumulate v, according to tf values for this element (0.63) it is similar to most other plants in not being able to hyperaccumulate it and transport it to aerial parts in favorable amounts. results of analyzing the concentrations of heavy metals in a single part of plant among 11 groups of localities defined according to the distance from the potential source of pollution (highway) paint a slightly different picture (tab. 3). it is assumed that determination of important variables is crucially affected by contamination of soil with heavy metals, unlike the previous case where the important factor was affinity for accumulation in plant organs. if roots and aerial parts of plants are compared according to spatial distribution of sampling sites, concentrations of fe, ni and pb become more important in differentiation of sample groups. comparison of mean values shows that approximately identical concentrations of ni and pb were recorded in roots and aerial parts of plants, while fe is characterized by a significantly higher concentration in the aerial part. the f value was most prominent for concentration of pb, which was shown to be more variable and statistically more significant than concentration of other metals regarding the concentration of metals in the root. the lead belongs to metals most commonly mentioned as primary contaminants in the environment. in this case, the role of this metal in differentiation of samples probably accentuates the level of contamination in sampling sites, which were situated in vicinity of traffic routes as contaminant sources. the significance of species s. tragus as an indicator organism for lead contamination of environment, as concentration of lead in plants is dependent on distance from traffic routes where lead gasses are emitted, was stressed by s i n e g a n i (2007). considering the aerial parts of plant, ni is more significant and represents a more significant variable than either pb or fe. the nickel does not have any great role as a plant nutrient, and increase of its concentration in plants is mostly caused by emission from industrial sources or by prolonged use of mineral fertilizers and pesticides containing that element (s e r e g i n & k o z h e v n i k o v a , 2006). conclusion according to its ability to accumulate several heavy metals, s. tragus may represent an interesting model of phytoremediation or biomonitoring. this plant can uptake cd, co, cr and pb from the soil and make significant deposits in its aerial parts. distribution and accumulation of heavy metals is not equal in all parts of a plant. differences in 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2001: effect of vanadium on plant growth and its accumulation in plant tissues. songklanakarin journal of science and technology, 33 (3): 255-261. warren, r.w., 2001: sorption and transport of radionuclides by tumbleweed from two plasticlined radioactive waste ponds. journal of environmental radioactivity, 54 (3): 361-376. čubrić & crnobrnja-isailović 2022, biologica nyssana 13(1) 13 (1) september 2022: 47-57 doi: 10.5281/zenodo.7375498 a view on human perception of snakes in serbia with special reference to nose-horned viper original article tijana čubrić department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, 18 000 niš, serbia tijana.cubric@pmf.edu.rs (corresponding author) jelka crnobrnja-isailović department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, 18 000 niš, serbia department of evolutionary biology, institute for biological research “siniša stanković”, university of belgrade, despota stefana 142, 11000 belgrade, serbia received: march 05, 2022 revised: september 13, 2022 accepted: september 21, 2022 abstract: snakes are frequently misunderstood and ill-perceived animals. having in mind the frequent killings of snakes near villages in serbia as their habitats coincide with agricultural land, we administered questionnaire to the local inhabitants at several localities to evaluate human perception of the snakes with a special reference to vipera ammodytes. we interviewed 87 individuals where the majority (68%) were men of age 65 and older. we classified responses to eight different topics: attitude, knowledge, awareness, folklore, knowledge about nose-horned vipers, possible threats to the vipers, direct killings and snakebite incidence. younger age groups of the respondents (18-24 and 25-34) had positive attitude and more knowledge about snakes while other age groups were ambivalent. further, gender groups were also ambivalent. almost half (45%) of respondents recognized v. ammodytes, but 36% stated that this viper “jumps and attacks” humans, and majority of respondents (61%) perceived this species as aggressive. regarding these results, we have proposed possible conservation measures. key words: ophiophobia, serbia, survey, snake awareness, folklore, vipera ammodytes apstrakt: pregled ljudskog stava o zmijama u srbiji sa posebnim osvrtom na poskoka zmije su često neshvaćene životinje. imajući u vidu česta ubijanja zmija na selima u srbiji obzirom na to da se staništa ovih životinja nekada nalaze u blizini ili na poljoprivrednim zemljištima, sprovedeno je istraživanje mišljenja o zmijama lokalnih meštana, putem dodele upitnika, na nekoliko lokaliteta kako bi bio procenjen stav prema zmijama sa posebnim osvrtom na vrstu vipera ammodytes. anketirano je 87 osoba od kojih su većina (68%) bili muškarci starosti 65 godina i više. odgovore smo klasifikovali u osam različitih tema: stav, znanje, svest, folklor, znanje o poskoku, moguće pretnje zmijama, direktna ubistva i učestalost zmijskih ujeda. mlađe starosne grupe ispitanika (18-24 i 25-34) imaju pozitivan stav i više znanja o zmijama, dok su ostale starosne grupe ambivalentne. dalje, rodne grupe su takođe bile ambivalentne. skoro polovina (45%) ispitanika prepoznala je vrstu v. ammodytes, ali je 36% izjavilo da ova zmija „skače i napada“ ljude, a većina ispitanika (61%) ovu vrstu doživljava kao agresivnu. u vezi sa ovim rezultatima, predložili smo potencijalne mere zaštite. ključne reči: ofiofobija, srbija, anketa, svest o zmijama, folklor, vipera ammodytes introduction territory of the republic of serbia is inhabited by ten snake species (tomović et al., 2015): seven are non-venomous and belong to family colubridae while three are from venomous snakes’ family viperidae (vipera ammodytes l. and v. berus l. are highly venomous, while v. ursinii b. is mildly venomous). in serbia, snake species are protected by the national law where all of them, except v. ammodytes, are in category “strictly protected” (v. ammodytes is in a lesser category “protected”) (tomović et al., 2015). vipera ammodytes (nosehorned viper) is widely distributed throughout the balkan peninsula (crnobrnja-isailović & haxhiu, 1997), where it have been tradionally persecuted and killed by local inhabitants, illegally traded as a pet, and over-collected for venom extraction (jelić et al., 2013). in serbia, where this viper inhabits area on the south of sava and danube rivers, during a 16 © 2022 čubrić & crnobrnja-isailović. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 47 year period (1993 2008), 9,800 adult nose-horned vipers were collected by local suppliers and sold to the national institute for immunology and virology for venom extraction, where they later died due to improper care (ajtić, 2009). the ban on harvesting nose-horned vipers in serbia was proclaimed in 2009. many of the snake species in serbia can be found near and/or in villages: e.g. dolichophis caspius g., natrix natrix l., zamenis longissimus l., coronella austriaca l. and v. ammodytes, among the other habitats, can be found on road banks, near or in the ruins and in abandoned houses – often in the basements (speybroeck et al., 2016). villagers are frequently encountering and killing snakes, and this persecution, besides the urban development, habitat fragmentation, road killings and pet trade, represents additional strong threat for the snake species (soulsbury & white, 2016; de souza et al., 2018). as snakes could be one of the apex predators in agricultural ecosystems, their eliminations by direct killings can severely damage trophic webs (mills et al., 1993). in regard to these anthropogenically caused threats to both non-venomous and venomous snake species in serbia, the primary goal of this study was to analize data obtained from questionnaires on human attitude, knowledge and awareness related to snakes in serbia and particularly to the nose-horned viper, and to help development of future conservation programs by suggesting conservation measures based on implementation of acquired knowledge. our secondary aim was to educate and possibly mitigate human-snake conflict by distributing leaflets and booklets among the respondents after the conducted survey. leaflets were providing general information about nose-horned viper, instruction for proper reaction in case of venomous snake bite, rules on how to behave in nature, and information about the importance of nose-horned vipers, while booklets were containing photos and cohesive information about non-venomous and venomous snake species in serbia. materials and methods data collection we conducted personal survey using structured anonymous questionnaire in several villages on the territories of svilajnac town (central serbia), protected area “sićevačka gorge” (southeastern serbia), “djerdap” national park (eastern serbia), bosilegrad town (southeasternmost part of serbia), and krupanj town (western serbia) in the year 2016. (fig. 1). we purposefully selected these villages because their inhabitants have been in direct contact with both agricultural land and forested areas which are suitable for snakes, their prey and predators. furthermore, the areas surrounding svilajnac and krupanj are known as localities where the nosehorned vipers were collected for venom extraction in the past (information obtained by previous personal communication and from the press). additionally, all these areas also represent habitats where we have been conducting monitoring of nose-horned viper’s populations. we have questioned any local inhabitant willing to help us and we have obtained consent from the participants for publication of this study and any accompanying photographs. we recorded age and gender of participants. the questionnaire consisted of 20 questions (fig. 2) defined in a simple and concise way as older inhabitants have little or no education. nineteen questions were close-ended with response options of “yes”, “no” or “i don’t know” and “i am not sure”, while one question was open-ended. 48 biologica nyssana ● 13 (1) september 2022: 47-57 čubrić & crnobrnja-isailović ● a view on human perception of snakes in serbia with special reference to nose-horned viper fig. 1. interviewing local villagers data analyses we classified respondents into two gender groups and separately, into following six age groups: 1) 1824, 2) 25-34, 3) 35-44, 4) 45-54, 5) 55-64 and 6) 65 and older. we classified questions into following topics: 1. attitude questions numbered 1, 4, 5, 10, 12 and 14 (fig. 2); positive attitude was defined if the respondents have chosen answer “no” in questions no. 1, 4, 5, 10, 12 and “b” or “v” in question no. 14; negative attitude was defined if they have chosen “yes” for the questions no. 1, 4, 5, 10, “yes” and “i don’t know” for question no. 12, and answer “a” for question no. 14. 2. knowledge questions no. 2, 3, 7, 9, 11 and 13 (fig. 2); demonstrates knowledge if the respondents have chosen answer “yes” in questions no. 2, 7, 9, 11, and “no” for questions 49 biologica nyssana ● 13 (1) september 2022: 47-57 čubrić & crnobrnja-isailović ● a view on human perception of snakes in serbia with special reference to nose-horned viper fig. 2. questionnaire 50 no. 3 and 13; demonstrates lack of knowledge if the respondents have chosen answer “no” in questions no. 2, 7, 9, 11, and “yes” for question no. 3 and “yes” and “i don’t know” for question no. 13. 3. awareness – questions no. 6, 15, 16 and 17; we classified respondents as “aware” if they answered “yes” on three or more questions and not aware otherwise. 4. folklore questions no. 13, 18 and 19. 5. knowledge about nose-horned viper in particular open-ended question no. 8. 6. possible threat to vipers question no. 20. 7. pressure on the snakes’ and in particular nosehorned viper’s local populations from direct killing with fear as possible source of conflict questions no. 1, 4 and 10. 8. snakebite incidence with lethal outcome question no. 11. for the attitude and knowledge, we analyzed answer scores using the non-parametric wilcoxon test with median scores as the depended variable (wilcoxon, 1992; rosner et al., 2006). we used one sample wilcoxon signed rank test to understand median scores for each demographic group (gender and age group) and wilcoxon sum rank test to compare differences in scores. we proposed zero hypotheses as follows: 1. attitude. respondents do not have positive attitude if h0=4 of 6 questions (as we had 6 questions, where median is 3.5; positive answers classified as the defined above), alternative hypothesis h>h0. 2. knowledge. respondents did not give correct biologica nyssana ● 13 (1) september 2022: 47-57 čubrić & crnobrnja-isailović ● a view on human perception of snakes in serbia with special reference to nose-horned viper table 1. positive and negative attitudes towards snakes in general, and nose-horned viper in particular (positive and negative answers for questions numbered 1, 4, 5, 10, 12,14) answers if h0=4 of 6 questions (as we had 6 questions, where median is 3.5; correct answers classified as the defined above), alternative hypothesis h>h0 we considered all results to be significant at p<0.05. all analyses where done by statistica 7.0. for the remaining six topics, we summarized the results of the questionnaire as the percentages of the total number of respondents who selected each response for each question. results demographics as the samples per village were not large enough, we analyzed them in total. we questioned 87 individuals where the majority were men (n=59; 68%) and 32% were women (n=28). most respondents were 65 and older 22% (n=19), followed by 55-64 -18% (n=16), 16% were of age groups 45-54 and 55-64 (n=14), and 14% were of age groups 25-34 and 18-24 (n=12). detailed percentages per every yes/no question per gender group are given in appendix 1, and per age group in appendix 2, while percentages of answers per gender on triple choice questions are given in appendix 3 and per age group in appendix 4. attitude men (n=59) gave 4 of 6 positive answers (median 35; tab. 1) and 2 of 6 negative answers (median 24) while women (n=28) gave 2 positive (median 20; tab. 1) and 4 negative answers (median=8) indicating that both groups have ambivalent attitude (tab. 2). regarding age groups, both group aged 18-24 (n=12) and 25-34 (n=12) had positive attitude positive attitude negative attitude number of respondents median range w p value median range w p value gender males 59 35 24-57 2.2 0.03* 24 2-35 2.2 0.03* females 28 20 5-27 2.2 0.03* 8 1-23 2.20 0.03* age group 18-24 12 8 5-12 1.99 0.04* 4 0-7 2.20 0.03* 25-34 12 9 3-12 1.85 0.06 3 0-9 2.20 0.03* 35-44 14 9.5 6-14 1.85 0.06 4.5 0-8 2.20 0.03* 45-54 14 8 6-14 2.02 0.04* 6 0-8 2.20 0.03* 55-64 16 8 7-14 2.20 0.03* 8 2-9 2.20 0.03* 65 and older 19 11 5-18 2.20 0.03* 8 1-14 2.20 0.03* * denotes p values<0.05 51 biologica nyssana ● 13 (1) september 2022: 47-57 čubrić & crnobrnja-isailović ● a view on human perception of snakes in serbia with special reference to nose-horned viper (median=11, median=8 respectively). knowledge men gave 4 of 6 correct answers (median 43.5; tab. 3) and 2 of 6 incorrect answers (median 15.5) while women gave 3 correct (median 12.5; tab. 3) and 3 incorrect answers (median=15.5) indicating that both groups had the same level of aptitude medium one (tab. 4). regarding age groups, both group aged 18-24 and 25-34 had correct 4 answers, indicating high level of aptitude (median=8.5; median=7.5 respectively, tab. 3, tab. 4) while other 3 groups had medium aptitude (tab. 3, tab. 4): 3544 gave 3 correct and two incorrect answers, and the equal number of correct and incorrect answers on one question (median=8; median=6 respectively; tab. 3); 45-54 gave the equal number of correct and incorrect answers on one question, 4 correct and 1 incorrect answer (median=10.5, median=3.5 respectively); 55-64 gave 4 correct and 2 incorrect answers (median=11, median=5 respectively); 65 and older gave 4 correct and 2 incorrect answers (median=10.5, median=8.5 respectively). awareness 70% of respondents answered that they know that snakes can be important part of ecosystem and useful in rodent populations control (appendix 1); 61% of respondents didn’t know that the nosehorned viper is lethargic snake as they considered it aggressive, while 10% of them didn’t believe in that statement; 57% of villagers didn’t know about the nose-horned viper’s important role in ecosystem, while 79% knew about the usage of v. ammodytes answering with 5 positive answers (median=8; median=9 respectively, tab. 1, tab. 2) while other 3 groups were ambivalent (tab. 1, tab. 2): 3544 gave four positive and two negative answers (median=9.5; median=4.5 respectively; tab. 1), 4554 gave the equal number of positive and negative answers on 2 questions, 3 positive and 1 negative answer (median=8, median=6 respectively), 5564 gave the equal number of positive and negative answers on 3 questions, 2 positive and 1 negative answer (median=8, median=8 respectively), 65 and older gave 3 positive and 3 negative answers table 2. scores for positive and negative attitudes toward snakes in general and nose-horned viper in particular, calculated separately for genders and age groups (questions numbered 1, 4, 5, 10, 12,14;h0=4, h>h0) positive attitude negative attitude w p value w p value gender males 0.31 0.75 1.36 0.17 females 5 0.24 0.10 0.91 age group 18-24 2.02 0.04* 2.20 0.03* 25-34 2.20 0.03* 1.21 0.22 35-44 0.73 0.46 1.82 0.07 45-54 0.07 0.50 2.20 0.3 55-64 1.57 0.11 2.02 0.04* 65 and older 1.75 0.07 1.61 0.10 correct answers incorrect answers number of respondents median range w p value median range w p value gender males 59 43.5 1-59 2.02 0.04* 15.5 0-58 2.20 0.03* females 28 12.5 1-26 2.20 0.02* 15.5 2-27 2.20 0.03* age group 18-24 12 8.5 0-12 2.02 0.04* 3.5 1-12 2.02 0.04* 25-34 12 7.5 0-12 2.02 0.04* 4.5 0-12 2.02 0.04* 35-44 14 8 0-13 2.20 0.03* 6 1-14 2.02 0.04* 45-54 14 10.5 0-14 2.02 0.04* 3.5 0-14 2.02 0.04* 55-64 16 11 0-16 2.02 0.04* 5 0-16 2.02 0.04* 65 and older 19 10.50 2-19 2.02 0.04* 8.5 0.17 2.20 0.03* table 3. correct and incorrect answers on questions related to the knowledge on snakes and nose-horned viper in particular (questions numbered 2,3,7,9,11,13) venom in anti-venom production. therefore, we classified them as not completely aware. folklore 39% of villagers did not think that nose-horned viper is used in human consumption, while 34% did not know the answer (appendix 3); 43% did not think that nose-horned viper’s meat can be used as a medicine, while 51% did not know the answer; 36% of respondents didn’t believe in the folklore premise that v. ammodytes “jumps and attacks” humans, while 30% did not know the answer. knowledge about nose-horned viper 45% of respondents gave correct answer when asked to describe the morphology of v. ammodytes, while 52 biologica nyssana ● 13 (1) september 2022: 47-57 čubrić & crnobrnja-isailović ● a view on human perception of snakes in serbia with special reference to nose-horned viper 36% gave incorrect answers and 20% did not know to describe the snake. in regarding to the animal’s behavior, most common mistakes were in describing this viper as an animal which is very aggressive and which “jumps”, while in regard to morphology of the snake most common mistake was that this species can be uniformly colored (often mentioned brown and red color). gender and age group differences in answers are given in fig. 3 and fig. 4, respectively. possible threat 32% of respondents answered they have heard or know person who had been (in case of svilajnac) or is (in case of krupanj) harvesting the nose-horned vipers for venom supply. direct killing and fear 53% of villagers were afraid of snakes, 51% of them never killed a snake while 84% never killed a nosehorned viper (appendix 1). interestingly, majority of the respondents who are afraid of snakes, never killed a snake (31% from 53%) while majority of the respondents who aren’t afraid of snakes did kill a snake in a past (31% from 47%) (fig. 5). snakebite occurrence only 2 respondents (2%) answered that they knew the person who died due to the snakebite. further observations in order to additionally evaluate human perception of snakes, here we report some further observations. in “sićevačka gorge” protected area, villagers have burned the vegetation subsequent day after we were seen performing morphometric measurements on one nose-horned viper. respondents from one locality (krupanj) stated that some neighbors captured nose-horned vipers, pulled out their fangs, skinned them and wore the skin as bracelets. in the year 2017, (year following our field activities and conducted survey), during our educational lecture in krupanj and field work in svilajnac, two of the inhabitants respectively stated that someone released nose-horned vipers in their village referring to us, which is of course false information. this was the main reason why we have suspended conducting survey further, as it draw’s attention on local snake table 4. scores for correct and incorrect answers on questions related to the knowledge on snakes and nose-horned viper in particular (questions numbered 2,3,7,9,11,13;h0=4, h>h0) correct answers incorrect answers w p value w p value gender males 0.73 0.46 0.31 0.75 females 0.94 0.34 1.15 0.25 age group 18-24 1.99 0.046* 0.20 0.83 25-34 2.20 0.03* 1.78 0.07 35-44 1.36 0.17 1.04 0.29 45-54 2.20 0.06 0.41 0.67 55-64 1.57 0.11 0.27 0.78 65 and older 0.73 0.46 1.78 0.07 fig. 3. knowledge about nose-horned viper among genders 53 biologica nyssana ● 13 (1) september 2022: 47-57 čubrić & crnobrnja-isailović ● a view on human perception of snakes in serbia with special reference to nose-horned viper populations in the areas where local inhabitants were not even aware of their close proximity. discussion in serbia, relatively ambivalent current attitude toward snakes is the result of intermixing tradition with contemporary citizen science and education. in fig. 4. knowledge about nose-horned viper among age groups fig. 5. fear and responses to snake encounters general, human perception of snakes can be positive or negative, ranging from fascination to fear and hatred (moura et al., 2010). reasons for fear or adoration can be various; from innate to learned fear with conjunction of folklore and spiritual beliefs. namely, south slavic believed in protectorancestor spirit which represented the male founder of the family who has been incarnated into a house54 biologica nyssana ● 13 (1) september 2022: 47-57 čubrić & crnobrnja-isailović ● a view on human perception of snakes in serbia with special reference to nose-horned viper snake, the white snake, which was called “house protector” (conrad, 2001). in serbia, in particular, one of traditional beliefs is that this white snake lives in the foundation wall of the house or nearby, where preys on mice, and must not be harmed as it was considered the source of good fortune (conrad, 2001). on the contrary, in some villages in serbia, on lazar’s saturday (christian calendar marking the transition from winter to spring and from death to life), villagers perform acts such as throwing stones, shouting, making loud noise to chase away snakes (moussakova, 2016). among the vlach people in eastern serbia, it is commonly believed that nosehorned viper can jump and follow a person for a long time; also, snake skulls and sheds are there the most often used in magical rituals (crnobrnja-isailović et al., 2015). the causes of ophiophobia are still up to debate; is ophiophobia innate, learned or both factors influence person’s cognitive response (isbell, 2006; van le et al., 2013). regardless the cause, the fear of snakes is most probably a silent stimulus and can easily be triggered and amplified by external causes such are secondary experiences i.e. a scary story (ohman & mineka, 2001). this could be the possible explanation for our finding that younger age groups (age 18-24 and 25-34) have positive attitude towards snakes as they were probably more educated and in our personal communication with them some stated that they learned about snakes more in the nature documentaries. nevertheless, in our sample more people were afraid of snakes than not and, interestingly, the ones who were afraid tend to kill them less and possibly due to their avoidance of being in the proximity of the animal. studies have shown that people with ophiophobia tend to avoid snakes (geer, 1965; lang, 1969). our results have shown that people who are not afraid of snakes tend to kill them more often, probably to protect themselves, or they display excitement seeking behavior (bradshaw et al., 2007), portrayed in movies, articles and documentaries with cheap sensationalism, including even charismatic storytellers whose reckless and attention seeking behavior lead to their death (bradshaw et al., 2007). direct killings and vigorous killings of snakes have also been frequently reported in other countries such are nepal (pandey et al.,2016), jordan (eid et al.,2021), etc. showing a consistency in ill-perceived attitude towards snakes. the same two age groups also gave most accurate answers about snakes. our findings imply that even though education about snakes is important, fear of snakes can be beneficial as causes people to avoid snakes altogether as similar observations in japan suggest (tanaka et al.,1999). even though 45% of respondents gave correct answer when asked to describe the morphology and behavior of v. ammodytes, 36% gave incorrect answers, frequently stating that this viper “jumps and attacks” humans. moreover, most respondents (61%) perceived this snake as aggressive, which is in concordance with previously mentioned folklore. despite this belief, only two respondents in our sample knew a person who died due to the snakebite, which further confirms this viper’s lethargic nature. this is in concordance with report in nikolić (2020) that 164 cases of venomous snakebites in serbia were recorded between 1893 and 2018, and that only four deaths were reported in a ten-year period, although serbia has no systematized information regarding this topic. having in mind unwillingness of some of the local villagers to participate in our study, it is important to reconsider that negative attitude could be more prevalent than described in this study. these inhabitants could decline to answer because they were aware of the potential law penalty for killing the wild animals although they would actually like to kill them; the other possible reason for declining to answer could be the feeling of animosity and distrust towards persons who study the species they are afraid of and/or do not like to see around. therefore, even though most respondents gave negative and neutral answer on question number 12, it is important to interpret the ambivalent attitude with caution. conservation implications despite limited sample size, which is the result of performing the study in small villages, unwillingnes of the inhabitants to participate in the survey and our further suspension of survey because of the possible negative influense on the local snake populations as described above and below, results of our study provided information about attitudes of people in the rural serbia toward local snake species, especially about the most common and persecuted venomous one. additionally, we revealed that, analysing answers on question number 20, even the ban has been proclaimed since 2009, some people have been illegaly harvesting nose-horned vipers (this information was reported to the institute for nature protection of serbia). all these findings encouraged us to recommend a few guidelines for future national snake conservation action plans, originally proposed by dodd on global level (2016) which we broadened and specified for the republic of serbia: a. set the goals. it is important to choose the adequate core audience for education activities (lectures, documentaries, etc). have in mind that people afraid of snakes mainly do not kill them, and that it is important to recognize and understand the reasons which motivate people unafraid of snakes 55 biologica nyssana ● 13 (1) september 2022: 47-57 čubrić & crnobrnja-isailović ● a view on human perception of snakes in serbia with special reference to nose-horned viper to kill them and therefore target the education activities towards them. b. choose the right education campaign, whether that be earned through media, digital media or direct community outreach. the education cannot be left to the income motivated zoos, serpentariums or sensationalized movies, documentaries and news reports. snakes must be documented exclusively in their natural habitats with minimum to no handling in order to not send potentially harmful message. c. it is important to be consistent and regularly engaging with members of local communities. although, if it is recognized that local villagers are not aware of snakes’ presence in their vicinity, it is best not to draw attention to them. our experience is that villagers have burned the vegetation one day after we were seen performing morphometric measurements on one nose-horned viper; additionally, two of the inhabitants respectively stated that someone released nose-horned vipers in their village. in this case, our survey and community outreach were contra productive and this must be taken into consideration. thankfully, answers on questions regarding folklore (questions no. 18. and no. 19.) were mostly negative compared to dangerous threat in form of overexploiting a wild reptile species for purpose of consuming its meat – the case of testudo hermanni in some parts of serbia (crnobrnja-isailović et al., 2015). nevertheless, as the one third of interviewed villagers did not know the answer on question no. 18, while one half did not know the answer on the question 19., it would be the best not to mention it anymore in order to do not suggest to particular people the idea for very dangerous actions. d. education of primary and secondary schoolchildren could be quite beneficial. ballouard et al. (2013) found that young children up to 14 years old, liked snakes and wanted them protected after they were given correct information and interacted with animals. furthermore, similar preliminary children response was also noted by us when giving lectures in the primary school in donji milanovac (near djerdap national park) and in the secondary school in svilajnac. acknowledgements. the fieldwork was kindly funded by the rufford foundation (grants no: 19578-1 and 233922) for tč. tč and jci were also supported by the ministry of education, science and technological development of republic of serbia – contracts no. 173025 (2016-2019), 451-03-68/2020-14/200124 (2020), 451-03-9/202114/200124 (2021) and 451-03-68/2022-14/200124 (2022). jci was additionally funded by the ministry of education, science and technological development of republic of serbia – contracts no. 451-03-68/202014/200007 (2020), 451-03-9/2021-14/200007 (2021)and 451-03-68/2022-14/200007 (2022). references ajtić, r. 2009: nose-horned viper (vipera ammodytes) conservation problems in serbia. protection of nature, 60: 319-326. ballouard, j. m., ajtic, r., balint, h., brito, j. c., crnobrnja-isailović, j., desmonts, d., elmouden, e.h., erdogan, m., feriche m., pleguezuelos, j.m., prokop, p., sanchez, a., santos, x., slimani, t., tomović,lj., usak,m., zuffi, m., bonnet, x. 2013: schoolchildren and one of the most unpopular animals: are they ready to protect snakes? anthrozoös, 26(1): 93-109. bradshaw, c., brook, b., mcmahon, c. 2007: dangers of sensationalizing conservation biology. conservation biology, 21(3): 570-571. crnobrnja-isailović, j., haxhiu, i. 1997: vipera ammodytes. in: gasc, j.p. et al. (ed.), atlas of amphibians and reptiles in europe: 384-385, societas europaea herpetologica and muséum national d’ histoire naturelle, paris. crnobrnja-isailović, j., milojković, d., macura, b. 2015: vodozemci i gmizavci djerdapa/ amphibians and reptiles of djerdap national park: 26-28. donji milanovac, serbia. conrad, j. l. 2001: male mythological beings among the south slavs. folklorica-journal of the slavic, east european, and eurasian folklore association, 6(1): 3-9. de souza, j. c., da silva, r. m., gonçalves, m. p. r., jardim, r. j. d., markwith, s. h. 2018: habitat use, ranching, and human-wildlife conflict within a fragmented landscape in the pantanal, brazil. biological conservation, 217: 349-357. dodd, c. k. (ed.) 2016: reptile ecology and conservation: a handbook of techniques. oxford university press. eid, e., al awaji, m., nasarat, h., alhiyasat, a. 2021: a perceptions and knowledge towards snakes: a study from jordan. herpetological conservation and biology, 16(2): 345-354. isbell, l.a. 2006: snakes as agents of evolutionary change in primate brains. journal of human evolution, 51(1): 1-35. jelić, d., ajtić, r., sterijovski, b., crnobrnjaisailović j., lelo s., tomović, lj. 2013: legal status and assessment of conservation threats to vipers 56 biologica nyssana ● 13 (1) september 2022: 47-57 čubrić & crnobrnja-isailović ● a view on human perception of snakes in serbia with special reference to nose-horned viper (reptilia: squamata: viperidae) of the western and central balkans. herpetological conservation and biology, 8 (3): 764-770. mills, l.s., soulé, m.e., doak, d.f. 1993: the keystone-species concept in ecology and conservation. bioscience, 43(4): 219-224. moura, m.r.d., costa, h.c., sao-pedro, v.d.a., fernandes, v.d., feio, r.n. 2010: the relationship between people and snakes in eastern minas gerais, southeastern brazil. biota neotropica, 10: 133-141. moussakova, e. 2016: the dragon/snake motif in the illuminated old glagolitic manuscripts. slovo, 66: 139-163. nikolić, s. 2020: venomous snakebites in serbia through 125 years: what we do (not) know in comparison with neighboring countries: a literature review. acta medica medianae, 59(4): 95-103. öhman, a., mineka, s. 2001: fears, phobias, and preparedness: toward an evolved module of fear and fear learning. psychological review, 108(3): 483. pandey, d.p., subedi pandey, g., devkota, k., goode, m. 2016: public perceptions of snakes and snakebite management: implications for conservation and human health in southern nepal. journal of ethnobiology and ethnomedicine, 12(1): 1-25. rosner, b., glynn, r.j., lee, m.l.t. 2006: the wilcoxon signed rank test for paired comparisons of clustered data. biometrics, 62(1): 185-192. soulsbury, c.d., white, p.c. 2015: human– wildlife interactions in urban areas: a review of conflicts, benefits and opportunities. wildlife research, 42(7): 541-553. speybroeck, j., beukema, w., bok, b., van der vort, j. 2016: field guide to the amphibians and reptiles of britain and europe. bloomsbury, london. tanaka, h., hayashi, y., nakamura, a. 1999: factors affecting annual incidence of habu bites, and how residents develop and transfer cognition of high risk sites. in: rodda,g.h., sawai, y., chiszar, d., tanaka, h. (ed.), problem snake management: thehabu and brown treesnake: 139–146, cornell university press, ithaka. tomović, lj., kalezić, m., džukić, g. 2015: crvena knjiga faune srbije. ii. gmizavci. univerzitet u beogradu, biološki fakultet & zzps. van le, q., isbell, l.a., matsumoto, j., nguyen, m., hori, e., maior, r.s., tomaz, c., tran, a.h., ono, t., nishijo, h. 2013: pulvinar neurons reveal neurobiological evidence of past selection for rapid detection of snakes. proceedings of the national academy of sciences, 110(47): 19000-19005. wilcoxon, f. 1992:. individual comparisons by ranking methods. in: kotz, s., johnson, n.l. (ed.), breakthroughs in statistics: 196-202, springer, new york. appendices appendix 1. percentage of answers per gender on yes/no questions total in percentage males females question number yes no yes no yes no 1 53 47 30 38 23 9 4 49 51 40 28 9 23 5 54 46 28 40 26 6 6 70 30 51 17 20 13 7 70 30 53 15 17 15 9 59 41 47 21 11 21 10 16 84 15 53 1 31 11 2 98 1 67 1 31 16 43 57 33 34 9 23 17 79 21 51 17 29 3 20 34 66 23 45 11 21 57 biologica nyssana ● 13 (1) september 2022: 47-57 čubrić & crnobrnja-isailović ● a view on human perception of snakes in serbia with special reference to nose-horned viper appendix 2. percentage of answers per age group on yes/no questions 18-24 25-34 35-44 45-54 55-64 65 and older question number yes no yes no yes no yes no yes no yes no 1 6 8 7 7 9 7 8 8 9 9 14 8 4 8 6 1 13 6 10 9 7 9 9 16 6 5 5 9 10 3 9 7 8 8 10 8 11 10 6 8 6 8 6 14 2 14 2 13 6 14 8 7 11 2 8 6 10 6 14 2 14 5 13 9 9 8 6 9 5 8 8 10 6 11 7 11 10 10 1 13 0 14 0 16 5 11 7 11 3 18 11 0 14 0 14 0 16 0 16 0 16 2 20 16 6 8 5 9 8 8 10 6 9 9 5 17 17 9 5 8 6 15 1 14 2 16 2 17 5 20 2 11 3 10 3 13 7 9 8 10 10 11 appendix 3. percentage of answers per gender on triple choice questions question number total male female 2 98 2 0 68 0 0 30 2 0 3 3 92 5 0 67 1 3 25 3 8 45 36 20 37 17 14 8 18 6 12 14 63 23 10 40 17 3 23 6 13 34 36 30 23 29 16 11 7 14 14 3 66 31 2 43 23 1 23 8 15 29 61 10 22 39 3 7 22 3 18 26 39 34 21 28 15 6 11 15 19 7 43 51 3 37 28 3 6 23 appendix 4. percentage of answers per age group on triple choice questions question number 18-24 25-34 35-44 45-54 55-64 65 and older 2 13 1 0 14 0 0 15 1 0 16 0 0 18 0 0 22 0 0 3 0 14 0 0 11 2 0 15 1 1 15 0 0 17 1 2 20 0 8 7 2 5 8 6 0 3 9 3 9 3 3 10 6 2 7 9 6 12 1 9 3 0 8 6 0 11 5 2 10 3 6 9 3 5 15 2 13 7 5 2 5 3 6 5 6 6 6 8 2 7 7 5 6 7 9 14 0 9 5 0 9 5 0 6 10 0 10 6 2 13 3 1 18 2 15 3 10 0 1 13 0 5 9 2 7 8 1 6 8 5 7 13 2 18 3 5 6 3 6 5 5 7 5 5 5 7 7 6 6 3 11 7 19 1 7 6 0 6 8 1 7 8 1 3 11 2 7 9 1 13 8 anticancer compounds from medicinal plants biologica nyssana 4 (1-2)  december 2013: 1-7 stešević, d. caković, d.  contribution to the alien flora of montenegro … 1 original article contribution to the alien flora of montenegro and supplementum to the preliminary list of plant invaders danijela stešević, danka caković faculty of natural sciences and mathematic, university of montenegro, džordža vašingtona bb, 81000 podgorica, montenegro * e-mail: danijela.stesevic@ac.me abstract: stešević, d., caković, d.: contribution to the alien flora of montenegro and supplementum to the preliminary list of plant invaders. biologica nyssana, 4 (1-2), december 2013: 1-7. this contribution is based on the field observations from 2011 to 2013. besides new data about distribution of some known plant invaders, one new alien species for the flora of montenegro is reportedsolidago gigantea. this plant was recorded in 2011, on two distinct localities near the road side in peri-urban area of nikšić and mojkovac, in the vicinity of gardens, were it has been grown as ornamental. in 2012 survey, species was again reported for mojkovac, but it disappeared from nikšić, due to environmental changes caused by road construction. remaining locality is placed near the tara river bank, so considering ecological preferences (roadsides, disturbed river banks and moist soils), this species might become more frequent in the area. it is included into the eppo list of invasive alien plants. in addition, alien plant tagetes minuta is added to the preliminary list of plant invaders in montenegro. key words: alien flora, invasive flora, montenegro, solidago gigantea, tagetes minuta introduction since our last publication from 2010 (s t e š e v i ć & p e t r o v i ć , 2010) situation with funding of the research and control of ias (invasive alien species) didn’t change much, but some progress in the legislative has been made. pursuant to the requirements of the convention on biological diversity, on 29 july 2010 th , the first national biodiversity strategy and action plan for 2010 2015th was adopted (www.gov.me/.../filedownload.aspx?rid=121033). this document defined the long-term goals and a number of measures for the protection of biodiversity and protected natural resources, as well as its implementation according to overall economic and social conditions in the country. the strategy has identified the main threats to biodiversity and ecosystems. introduction of alien and invasive species was considered as so far poorly understood threat, with comment that its greater significance among the threats to biodiversity can be expected soon. in the meantime, on 14 march 2013 th , the government of montenegro on the proposal agency for environmental protection adopted a regulation on the national list of indicators in environmental protection, by which alien and invasive species are an indicator (b05) of pressure on dpsir (driving forces pressures state-impact-response) model. therefore, in coming period they will be systematically monitored in annually dynamics, while summary data will be published each 10 years (official gazette no 29/2013). according to recommendations of l a m b d o n et al. (2008), national check list of alien and invasive flora need to be further assessed within respect to the invasion status and residence time of the species included. for future in-depth analyses it is crucial that especially the naturalized/casual status is evaluated. furthermore, 11 th sfses • 13-16 june 2013, vlasina lake 4 (1-2) • december 2013: 1-7 biologica nyssana 4 (1-2)  december 2013: 1-7 stešević, d. caković, d.  contribution to the alien flora of montenegro … 2 more rigorous approach is also needed at the national level in terms of recording of habitat (c h y t r ỳ et al., 2008) and abundance data, and according to recognized standards which are comparable across the continent. this paper is a contribution to the alien and invasive flora of montenegro and results given in it presents our field observation, gathered during different floristic and habitat investigations. material and methods data on invasive flora presented in this paper were gathered during different floristic and habitat investigations of central and coastal part of the country, undertaken in period late summer 2011 to spring 2013. each location where alien plants were recorded were geo-coded by using a gps device garmin e-trex vista c. field data about the habitats were also collected. collected specimens were deposited in the herbarium collection of the faculty of natural sciences in podgorica (tgu!), while identification of materials was conducted according to m c n e i l (1976) and p i g n a t t i (1982). results and discussion solidago gigantea aiton, giant goldenrod/late goldenrod (fam. asteraceae) during the floristic survey in augustseptember 2011,on two distinct localities near the road side in peri-urban area of nikšić (n42º 47’21'‘e 18º 55’42'') and mojkovac (n42º 57’28'‘e 19º 34’23''), several individuals of ornamental plant solidago gigantea aiton, were recorded. the plant belongs to the solidago canadensis l. complex within the subgenus triplinervae (w e b e r & j a c o b s , 2005). plant is known as a giant goldenrod/late goldenrod. it is up to 280 cm tall, erect rhizomatous perennial with annual aboveground shoots 5-11mm in diameter. shots are branched only in the inflorescence. they are mainly glabrous, or weakly hairy in the inflorescence. leaves are alternate, oblong to lanceolate, often acuminate, with margins mostly serrate, occasionally entire, triple-nerved, glabrous or sometimes pubescent beneath. they are largest in the middle of shoots and become smaller towards the apex and within inflorescence. inflorescences form broad pyramidal panicles with recurving branches and a central axis. bracts of the involucre are linear, obtuse or somewhat acute, 3.5-5mm. ray florets are golden yellow, female and fertile, disc florets are bisexual and fertile, capitula 3-5 mm long. achenes are pubescent, 1-2 mm long, with a pappus of 1 mm long (m c n e i l , 1976; w e b e r & j a c o b s , 2005). giant goldenrod originates from north america (m c n e i l , 1976). it was introduced into europe as an ornamental plant in the mid-1700s, with naturalized populations first observed in the mid-1800s (w a g e n i t z , 1979). continuous spread was noted between 1850 and 1950, by which time most of the present range was occupied (w e b e r , 1998). according to (euro+med, 2006-), plant is naturalized in west, central and part of south europe. it is included into the eppo list of invasive alien plants (http://www.eppo.int/invasive plants/ias_lists.htm#a1a2lists). it grows in a wide range of different soil conditions but is not shade tolerant (e l l e n b e r g et al., 1992). initially it occupied ruderal sites, such as roads, railway lines and rivers, it has also established in semi-natural and natural habitat since the late 19 th century (g u z i k o w a & m a y c o c k 1986; l o h m e y e r & s u k o p p , 1992). in the peri-urban area of nikšić and mojkovac, plant was reported near the roadside, in the vicinity of gardens, were it has been grown as ornamental. in 2012 survey, species was again fig. 1. solidago gigantea, plant habitus and detail of glabrous shoot (photo a. haines) http://www.eppo.int/invasive%20plants/ias_lists.htm#a1a2lists http://www.eppo.int/invasive%20plants/ias_lists.htm#a1a2lists biologica nyssana 4 (1-2)  december 2013: 1-7 stešević, d. caković, d.  contribution to the alien flora of montenegro … 3 reported for mojkovac, but it disappeared from nikšić, due to environmental changes caused by the road construction. the remaining locality in mojkovac is placed near the tara river bank, so considering habitat preferences: disturbed river banks and moist soils (j a c o b s et al., 2004; s t a n č i ć , 2010; k o u t i k a et al., 2011), this species might become more frequent in the area. besides giant goldenrod (solidago gigantea), canada goldenrod (s. canadensis l.) is even more frequently planted in central and northern part of montenegro. both species are known as honey bee plants. according to (w a l t e r , 1987) planting of goldenrods as feeding plant for bees, may also have facilitated its invasion into new habitats. these closely related species are distinguished by following features: s. gigantea have glabrous and glaucous shoots, while shoots of s. canadensis are at least weakly pubescent (fig. 1, 2). involucre in s. gigantea is 3.5-5mm, while in s.canadensis is up to 3mm. flowers of s. gigantean are bright yellow and the pappus is brownish-white, whereas the flowers of s. canadensis are somewhat lemon-yellow and the pappus silvery whitish (mcneil, 1976; pignatti, 1982). canada goldenrod is also on the eppo list of invasive alien plants (http://www.eppo.int/invasive_plants/ias_list s.htm#a1a2lists). considering bad habits of gardeners, who cut down the fruiting shoots or dump rhizome fragments on rubbish heaps outside the gardens, on riversides, brooks or waste places near the roadside, it is possible to generate subspontaneously growing individuals/populations of this species, too. in the field survey at the long ulcinj beach and buljarice bay, undertaken in late autumn 2012 to spring 2013, eleven natura 2000 habitats have been mapped: 1210 annual vegetation of drift lines, 1410 mediterranean salt meadows (juncetalia maritimi), 2110 embryonic shifting dune, 2120 shifting dunes along the shoreline with ammophila arenaria (white dunes), 2190 humid dune slack, 2220 dunes with euphorbia terracina, 2240 brachypodietalia dune grasslands with annuals, 2270 * wooded dunes with pinus pinea and/or pinus pinaster, 3170 * mediterranean temporary ponds, 92a0 salix alba and populus alba galleries sand 91aa eastern white-oak wood. almost each habitat type has an alien component of the flora (tab. 1). as it is shown in table 1, in the field survey of long ulcinj beach and buljarice bay, 28 alien species were recorded. the richest alien flora has the embryonic shifting dune and brachypodietalia dune grasslands with annuals (10 species each), while the poorest with alien species are mediterranean salty meadows (only one). number of aliens would definitely be much higher if the survey included whole hinterland of both localities and all floristic aspects. among them twenty are considered as invasive (stešević & petrović, 2010), while tagetes minuta is proposed to be included in the list. our own field investigation of the long ulcinj beach dates from the beginning of 2000, when species was noticed sporadically. in the meantime it occupied larger area in the first line of the beachs’hinterland, just near the sandy road that parallel goes with the sea-line, distant 250-600m from the sea. the most abundant population was recorded at ada bojana. as a new alien species for montenegro this plant has been recorded at the beginning of 1970es in boka kotroska bay: dobrota, kostanjica, kamenjari, igalo. actually, it has been reported as a noxious weed of whole mediterranean and submediterranean part of former yugoslavia, starting from sw istria to s macedonia (šilić, 1973). the plant is worldwide known as invasive south-american species that establishes readily in disturbed sites in the coastal areas and can form dense populations. the large size and dense growth makes it highly competitive to native plant species (weber 2003). considering the fact that plant is recorded at 3 distant localities: boka kotorska bay (šilić, 1973; obradović, 1980), ulcinj area and buljarice bay, therefore, it would be interesting to fig. 2. solidago canadensis, plant habitus (photo d. stešević) and detail of pubescent shoot (photo a. haines) http://www.eppo.int/invasive_plants/ias_lists.htm#a1a2lists http://www.eppo.int/invasive_plants/ias_lists.htm#a1a2lists biologica nyssana 4 (1-2)  december 2013: 1-7 stešević, d. caković, d.  contribution to the alien flora of montenegro … 4 make a survey and see if it is established at some “in between” places. general remarks of the survey are that the most aggressive and widespread aliens at surveyed sites at the long ulcinj beach and its hinterland are xanthium strumarium subsp. italicum, oenothera glazioviana, o. fallax, conyza canadensis, c. albida, aser squamatus, sporobolus poirettii, and tagetes minuta, while in buljarice bay are: paspalum dilatatum, sorghum halepense, conyza canadansis, c.albida and aster squamatus. alike other species, that can invade different kind of devastated habitats with small plant-cover value, dallis grasspaspalum dilatatum prefere the grassy one, on which it soon begins to dominate. it is often accompanied by sporobolus poiretti. conclusion i) alien flora of montenegro is richer for one species: giant goldenrodsolidago gigantea. besides it, another goldenrod species is frequently planted in central and northern part of montenegro canada goldenrod (s. canadensis). considering bad habits of gardeners, who cut down the fruiting shoots or dump rhizome fragments on rubbish heaps outside the gardens, on riversides, brooks or waste places near the roadside, it is possible to generate subspontaneously growing individuals/populations of this species, too. ii) the list of plant invaders in montenegro is enlarged for one species: tagetes minuta. the plant is recorded at 3 distant localities: boka kotorska bay, ulcinj area and buljarice bay, therefore it would be interesting to make a survey and see if it is established at some “in between” places. iii) the most aggressive and widespread aliens at surveyed sites at the long ulcinj beach and its hinterland are xanthium strumarium subsp. italicum, oenothera glazioviana, o. fallax, conyza canadensis, c. albida, aser squamatus, sporobolus poirettii, and tagetes minuta, while in buljarice bay are: paspalum dilatatum, sorghum halepense, conyza canadansis, c.albida and aster squamatus. alike other species, that can invade different kind of devastated habitats with small plant-cover value, dallis grasspaspalum dilatatum prefer the grassy one, on which it soon begins to dominate. it is often accompanied by sporobolus poiretti. table 1. alien and invasive species on different natura 2000 and anthropogenized habitats at the long ulcinj beach and buljarice bay species long ulcinj beach, habitat type/cooridnates buljarice bay, habitat type/cooridnates ailanthus altissima (mill.) swingle graveyard (n 42°11’18.1" e 18°59’25.7"). agave americana l. 2110 embryonic shifting dune (n 41°54'31.2" e 19°14'55.2"). amaranthus hybridus l. agricultural habitat (n 41°54'48.6" e 19°15'12.4", n 41°54'45.0" e 19°15'32.1"). amorpha fruticosa l. 92a0 salix alba and populus alba galleries (n 41°53’32.0" e 19°18’29.1"), 2270* wooded dunes with pinus pinea and/or pinus pinaster (n 41°54’37" e 19°15’02.0"), 2190 humide dune slack (n 41°54’19.4"e 19°16’00.8", n 41°54’09.8" e 19°16’42.3"), 2240 brachypodietalia dune grasslands with annuals (n 41°51’51.6" e 19°20’27"). aster squamatus (spreng.) hieron 1410 mediterranean salt meadows (juncetalia maritimi) (n 41°52’52.9 e 19°19’48.5), 2190 humide dune slack (n 41°54’19.4" e 19°16’00.8", n 41°53’19.0" e 19°18’42.6"), 2270* wooded dunes with pinus pinea and/or pinus pinaster (n 41°54’15.18" e 19°15’58"), ruderal habitat (n 41°53’14.9" e 19°18’29.2"). graveyard (n 42°11’18.1" e 18°59’25.7"). bidens subalternans dc. graveyard (n 42°11’18.1" e 18°59’25.7"). biologica nyssana 4 (1-2)  december 2013: 1-7 stešević, d. caković, d.  contribution to the alien flora of montenegro … 5 species long ulcinj beach, habitat type/cooridnates buljarice bay, habitat type/cooridnates conyza albida willd. 2110 embryonic shifting dune (n 41°54’39" e 19°14’34.7"), 2270* wooded dunes with pinus pinea and/or pinus pinaster (n 41°54’15.18" e 19°15’58"). agricultural habitat (n 41°54’45.0” e 19°15’32.1”), ruderal habitat (n 41°54’31.7“ e 19°16’21.1“,n 41°53’52.2“ e 19°17’58.2“, n 41°53’14.9“ e 19°18’29.2“). mesophilic meadows (n 42°11’02.7” e 18°58’05.5”), graveyard (n 42°11’18.1” e 18°59’25.7”). conyza canadensis (l.) cronq. 2110 embryonic shifting dune (n 41°54’ 31.2” e 19°14’ 55.2”), 2190 humide dune slack (n 41°54’19.4” e 19°16’00.8”, n 41°53’19.0” e 19°18’42.6”), 2240 brachypodietalia dune grasslands with annuals (n 41°54’08.1” e 19°16’59.8”, n 41°53’55.3” e 19°18’04.9”), 2270* wooded dunes with pinus pinea and/or pinus pinaster (n 41°54’33.0” e 19°15’23.3”, n 41°54’15.18” e 19°15’58”), agricultural habitat (n 41°54’48.6” e 19°15’12.4”), ruderal habitat (n 41°54’31.7” e 19°16’21.1”, n 41°53’52.2” e 19°17’58.2”n 41°53’14.9” e 19°18’29.2”, n 41°53’27.9” e 19°18’26.8”), agricultural land (n 41°54’02.7” e 19°186’00.2”). mesophilic meadows (n 42°11’02.7” e 18°58’05.5”), graveyard (n 42°11’18.1” e 18°59’25.7”). datura stramonium l. 2270* wooded dunes with pinus pinea and/or pinus pinaster (n 41°54’15.18” e 19°15’58”), agrucultural habitat (n 41°53’52.2” e 19°17’58.2”). chenopodium multiflorum moq. ruderal habitat (n 41°53’52.2“ e 19°17’58.2“). cuscuta caesattiana bertol. 2270* wooded dunes with pinus pinea and/or pinus pinaster (n 41°54’15.18” e 19°15’58”). datura stramonium l. agricultural habitat (n 41°54’45.0” e 19°15’32.1”), ruderal habitat (n 41°53’52.2” e 19°17’58.2”). eleusine indica (l.) gaertn. ruderal habitat (n 41°54’37.0" e 19°14’27.7", n 41°53’14.9" e 19°18’29.2"). euphorbia maculata l. ruderal habitat (n 41°53’14.9" e 19°18’29.2"). lepidium virginiacum l. graveyard (n 42°11’18.1" e 18°59’25.7"). melia azedarach l. 2110 embryonic shifting dune (n 41°54’ 31.2" e 19°14’ 55.2") roadside vegetation (n 42°11’32.0" e 18°58’20.6"). mentha × piperita ruderal habitat (n 41°53’52.2" e 19°17’58.2"). oenothera fallax renner & o. glazioviana micheli 2110 embryonic shifting dune (n 41°54’31" e 19°14’55.2", n 41°54’39" e 19°14’34.7", n 41°54’28.0" e 19°15’19.7", n 41°54’19.2" e 19°15’50.3"), 2120 shifting dunes along the shoreline with ammophila arenaria (white dunes) (n 41°53’47.4" e 19°17’21.0", n 41°53’32.0" e 19°18’01.0") ruderal habitat (n 41°53’27.9" e 19°18’26.8"). oenothera suaveolens desf. 2110 embryonic shifting dune (n 41°53’18.9" e 19°18’33.3"). biologica nyssana 4 (1-2)  december 2013: 1-7 stešević, d. caković, d.  contribution to the alien flora of montenegro … 6 species long ulcinj beach, habitat type/cooridnates buljarice bay, habitat type/cooridnates robinia pseudoacacia l. 2110 embryonic shifting dune (n 41°54’ 31.2" e 19°14’ 55.2"), 2270* wooded dunes with pinus pinea and/or pinus pinaster (n 41°54’37" e 19°15’02.0", n 41°54’15.18" e 19°15’58") ruderal habitat (n 41°54’40.2" e 19°14’10.4", n 41°54’40.2" e 19°14’10.4"), agricultural habitat (n 41°54’48.6" e 19°15’12.4"). parthenocissus quinquefolia (l.) planch. ruderal habitat (n 41°54’40.2" e 19°14’10.4"). paspalum dilatatum poir. 92a0 salix alba and populus alba galleries (n 41°54’31.5" e 19°15’53.9"). mesophilic meadows (n 42°11’42.2" e 18°58’10.3", n 42°11’36.1" e 18°58’17.1", n 42°11’02.7" e 18°58’05.5"). paspalum paspaloides (michx.) schribn. 2270* wooded dunes with pinus pinea and/or pinus pinaster (n 41°54’33.0" e 19°15’23.3") ruderal habitat (n 41°54’31.7" e 19°16’21.1", n 41°53’27.9" e 19°18’26.8"). phytolacca americana l. agricultural habitat (n 41°54’48.6" e 19°15’12.4"), ruderal habitat (n 41°54’40.2" e 19°14’10.4", n 41°54’31.7" e 19°16’21.1", n 41°53’52.2" e 19°17’58.2"). sorghum halepense (l.) pers. agricultural habitat (n 41°54’48.6" e 19°15’12.4", n 41°54’45.0" e 19°15’32.1"). mesophilic meadows (n 42°11’00.2" e 18°58’50.4", n 42°11’02.7" e 18°58’05.5"). sporobolus poirettii (r.et s.) hitche 2240 brachypodietalia dune grasslands with annuals (n 41°54’08.1" e 19°16’59.8", n 41°53’55.3" e 19°18’04.9"), 2270* wooded dunes with pinus pinea and/or pinus pinaster (n 41°54’15.18" e 19°15’58") 92a0 salix alba and populus alba galleries (n 41°54’31.5" e 19°15’53.9"9), agricultural habitat (n 41°54’48.6" e 19°15’12.4", n 41°54’45.0" e 19°15’32.1"), ruderal habitat (n 41°54’31.7" e 19°16’21.1", n 41°53’14.9" e 19°18’29.2", n 41°53’27.9" e 19°18’26.8"). mesophilic meadows (n 42°11’42.2" e 18°58’10.3"), graveyard (n 42°11’18.1" e 18°59’25.7"). tagetes minuta l. 2240 brachypodietalia dune grasslands with annuals, and ruderal habitat in line transect: (n 41°53’52.2" e 19°17’58.2" to n 41°51’51.6" e 19°20’27"). graveyard (n 42°11’18.1" e 18°59’25.7"). xanthium strumarium l. italicum (moretti) d.löve 2110 embryonic shifting dune (n 41°54’31" e 19°14’55.2", n 41°54’39" e 19°14’34.7", n 41°54’19.2" e 19°15’50.3", n 41°53’22.6" e 19°18’22.7"), 2270* wooded dunes with pinus pinea and/or pinus pinaster (n 41°54’15.18" e 19°15’58"), 2120 shifting dunes along the shoreline with ammophila arenaria (white dunes) (n 41°54’09.7" e 19°16’19.5", n 41°53’47.4" e 19°17’21.0", n 41°53’32.0" e 19°18’01.0", n 41°53’19.0" e 19°18’42.6"), ruderal habitat (n 41°54’40.2" e 19°14’10.4", n 41°54’31.7" e 19°16’21.1", n 41°53’14.9" e 19°18’29.2"), agricultural land (n 41°54’02.7" e 19°186’00.2", n 41°53’27.9" e 19°18’26.8"). 1210annual vegetation of drift lines (n 42°11’36.4" e 18°578’54.7"). yucca gloriosa l. 2110 embryonicshifting dune (n 41°54’ 31.2" e 19°14’ 55.2"). biologica nyssana 4 (1-2)  december 2013: 1-7 stešević, d. caković, d.  contribution to the alien flora of montenegro … 7 acknowledgements. authors would like to thank arthur haines for his valuable photo contribution. the field investigation was partly funded by undp, office in montenegro (project number mne 12-057) and by the montenegrin academy of sciences and arts. references chytrý, m., maskell, l., pino, j., pyšek, p., vilà, m., font, x., smart, s. 2008: habitat invasions by alien plants: a quantitative comparison between mediterranean, subcontinental and oceanic regions of europe. journal of applied ecology, 45: 448–458. ellenberg, h., weber, h.e., düll, r., wirth, v., werner, w., paulissen, d. 1992: zeigerwerte von pflanzen in mitteleuropa. scripta geobotanica, 18:1-258. guzikowa, m., maycock, p. f. 1986: the invasion and expansion of three north american species of goldenrod (solidago canadensis l. sensu lato, s. 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euro+med 2006-: euro+med plantbase the information resource for euro-mediterranean plant diversity. http://ww2.bgbm.org/europlusmed/ [accessed may 2013]. ministarstvo turizma i održivog razvoja 2013: drugi izvještaj o implementaciji nacionalne strategije biodiverziteta sa akcionim planom. http://www.gov.me/.../filedownload.aspx?rid=1210 33 [accessed semptember 2013]. http://www.eppo.int/invasive_plants/ias_lists. htm#a1a2lists, [accessed may 2103] yuvaraja et al. 2025, biologica nyssana 16(1) 233 16 (1) june 2025: 233-245 doi: 10.46793/biolnyss.16.1.18y isolation, characterization, in silico docking, and in vitro cytotoxicity study of isolated triterpene constituents from ehretia microphylla original article ramakrishnan yuvaraja department of pharmaceutical chemistry, ktn college of pharmacy, chalavara, kerala, india kullampalayam krishnasamy sivakumar department of pharmaceutical chemistry, sri venkateshwara college of pharmacy, ariyur, pondicherry, india sivakumarkk1174@gmail.com (corresponding author) santhiagu arockiasamy bioscience and engineering, nit calicut, kerala, india jayaraman rajangam department of pharmaceutical chemistry, sri venkateshwara college of pharmacy, ariyur, pondicherry, india arulkumaran govindarajan department of pharmaceutical chemistry, ktn college of pharmacy, chalavara, kerala, india received: december 16, 2024 revised: april 08, 2025 accepted: april 14, 2025 abstract: building on previous studies of ehretia microphylla, we quantified key phytochemicals rutin, gallic acid, and quercetin using high-performance thin-layer chromatography (hptlc), validated against standard references. from chloroform extract, three triterpenes were isolated and structurally characterized. these were identified as bauerenol (compound a), 11-oxo amyrin (compound b, a novel triterpene), and β-sitosterol (compound c). in silico docking studies were performed with the cancer-related protein (pdb: 1db1), yielding binding scores of −9.50, −9.44, and −7.44, and with the hepatoprotective target (pdb: 4fa6), with scores of −8.13, −8.42, and −8.54, respectively. compounds a and b exhibited significant anticancer potential, while compound c showed superior hepatoprotective activity. in vitro cytotoxicity studies on hepg2 cells revealed ic₅₀ values of 455, 538, and 556 μg/ml, and on nih 3t3 cells, ic₅₀ values were 1068, 1153, and 1310 μg/ml, indicating selective toxicity toward cancer cells. these findings highlight the therapeutic potential of triterpenes from e. microphylla, with compounds a and b for hepatocellular carcinoma treatment and compound c for hepatoprotection. key words: ehretia microphylla, triterpenes, hepatoprotective, cytotoxicity, docking studies apstrakt: izolacija, karakterizacija, in silico dokovanje i in vitro ispitivanje citotoksičnosti triterpenskih jedinjenja izolovanih iz vrste ehretia microphylla nadovezujući se na prethodna istraživanja vrste ehretia microphylla, kvantifikovali smo rutin, galnu kiselinu i kvercetin, primenom hptlc metodologije. iz hloroformskog ekstrakta izolovana su i identifikovana tri triterpenska jedinjenja: bauerenol (jedinjenje a), 11-okso-amirin (b, novi triterpen) i β-sitosterol (c). in silico dokovanja pokazala su afinitete ka ključnim proteinima povezanim sa karcinomom (pdb: 1db1), sa rezultatima dokovanja -9.50, -9.44 i -7.44, kao i sa hepatoprotektivnim ciljnim proteinom (pdb: 4fa6), sa skorovima -8.13, -8.42 i -8.54. jedinjenja a i b ispoljila su izražen antiproliferativni potencijal, dok je c pokazalo superiornu hepatoprotektivnu aktivnost. in vitro testiranja na hepg2 ćelijama pokazala su ic₅₀ vrednosti od 455, 538 i 556 μg/ml, a na nih 3t3 ćelijama ic₅₀ vrednosti od 1068, 1153 i 1310 μg/ml, ukazujući na selektivnu citotoksičnost prema ćelijama kancera. dobijeni rezultati potvrđuju terapijski potencijal triterpena vrste e. microphylla, sa jedinjenjima a i b perspektivnim za lečenje hepatocelularnog karcinoma i jedinjenjem c za hepatoprotektivno dejstvo. ključne reči: ehretia microphylla, triterpeni, hepatoprotektor, citotoksičnost, dokovanje introduction cancer is a general term for a large group of diseases that can affect any part of the body. under normal circumstances, cells grow and die in a regulated manner; however, cancer cells grow uncontrollably and evade programmed cell death. cancer is the second leading cause of mortality worldwide, following cardiovascular diseases (sharma et al., 2022). among the various types of cancer globally, © 2025 yuvaraja et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 234 the most common form of primary liver cancer is hepatocellular carcinoma (hcc), also known as hepatoma, which ranks fourth in the world in terms of cancer-related deaths (villanueva, 2019). according to annual projections, the world health organization estimates that more than one million patients will die from liver cancer by 2032 (world health organization, 2016). liver cancer cells are primarily hepatocytes and are often a complication of cirrhosis, which can be caused by hepatitis b or c virus infections or prolonged alcohol consumption. efforts to control liver cancer have significantly benefited from research into medicinal plants. traditional cancer treatment approaches include surgery, radiation therapy, targeted therapy, immunotherapy, hyperthermia, stem cell transplantation, photodynamic therapy, laser therapy, blood product donation, transfusion, and, most importantly, chemotherapy (maurya, 2014). however, despite the wide variety of synthetic drugs used in cancer chemotherapy and the therapeutic successes of various treatment regimens, the prevailing therapies have not always yielded the expected results. tumor relapse and metastasis frequently occur, necessitating further research into alternative and complementary therapies. there is a growing need for natural, selective active compounds with fewer side effects, lower costs, and more medicinal properties. compounds with minimal disease resistance are particularly crucial for cancer treatment, especially those derived from biological and natural sources (sharifi-rad et al., 2019). one such plant is ehretia microphylla lam. (em), which has been reported to contain numerous bioactive metabolites, including triterpenes, alkaloids, glycosides, tannins, α-amyrin, β-amyrin, saponins, flavonoids, and rosmarinic acid (aarthi et al., 2014; pooja et al., 2022; jiang et al., 2024). this plant, belonging to the boraginaceae family, is of significant medicinal value in the philippines and other asian countries. traditionally, em has been used in the form of a decoction to treat coughs, diarrhea with bloody discharge, and dysentery. scientific studies have also reported its antioxidant, antimicrobial, anti-hypercholesterolemic, and antiallergic activities (palaniappan et al., 2014; umali et al., 2014; cagampan et al., 2018). various phytochemical constituents, which possess a steroid nucleus, play a crucial role in hepatoprotective and anticancer activities. an example of such a compound is vitamin d. vitamin d also plays a crucial role in regulating the phosphatidylinositol 3-kinase alpha (pi3kα) pathway (yuan et al., 2021; shamsan et al., 2024), which is vital for cell growth, survival, and metabolism. dysregulation of the pi3kα pathway is implicated in various liver diseases (e.g., nonalcoholic fatty liver disease, fibrosis, and cirrhosis) as well as in cancer, making vitamin d a promising therapeutic agent for the prevention and management of these conditions. in liver disease, vitamin d's active form, calcitriol, modulates the pi3kα pathway (trump, 2018) to reduce inflammation, prevent fibrosis, and promote liver regeneration. it inhibits the activation of hepatic stellate cells (hscs), which are responsible for collagen production and liver scarring. by suppressing the pi3kα/akt signalling pathway, vitamin d reduces the activation of hscs, potentially slowing or reversing liver fibrosis progression (chen et al., 2022). moreover, vitamin d regulates inflammatory cytokines such as tnf-α and il-6, which contribute to liver injury, further supporting its hepatoprotective effects. in cancer, particularly liver cancer (hcc), vitamin d interferes with pi3kα signalling to inhibit tumor growth and metastasis. calcitriol can induce apoptosis in cancer cells and prevent their proliferation by modulating the pi3k/akt pathway (chiang & chen, 2011; zhang et al., 2020). it also reduces epithelial-to-mesenchymal transition (emt), a key step in cancer metastasis. additionally, vitamin d enhances immune surveillance by promoting the activity of immune cells like t cells and natural killer (nk) cells, which are involved in targeting and eliminating tumor cells. interestingly, the phytochemical constituents of e. microphylla extract have structures resembling steroids (such as the cyclopentanoperhydrophenanthrene ring structure), similar to vitamin d. overall, vitamin d’s modulation of pi3kα offers dual protective effects in both liver diseases and cancer, influencing inflammation, fibrosis, cell survival, and metastasis. in continuation of our research on e. microphylla (yuvaraja et al., 2020; yuvaraja et al., 2021), we report the quantification of major phytochemicals like rutin, gallic acid, and quercetin using highperformance thin-layer chromatography (hptlc). we also isolated three triterpene compounds from chloroform extracts of e. microphylla and characterized their structures. in silico docking, adme studies were conducted on these isolated triterpene constituents, focusing on key transcription factors involved in hepatocellular carcinoma (hcc). additionally, we explored the in vitro cytotoxicity of these compounds against hepg2 (human hepatoma) and nih 3t3 (mouse embryonic fibroblast) cell lines using the mtt assay. materials and methods collection of plant parts and instrumentation based on the literature review and traditional claims, biologica nyssana ● 16 (1) june 2025: 233-245 yuvaraja et al. ● isolation, characterization, in silico docking, and in vitro cytotoxicity study of isolated triterpene constituents from ehretia microphylla 235 biologica nyssana ● 16 (1) june 2025: 233-245 yuvaraja et al. ● isolation, characterization, in silico docking, and in vitro cytotoxicity study of isolated triterpene constituents from ehretia microphylla aerial parts of e. microphylla were collected locally in sattankulam at thoothukudi district in tamil nadu from june to august. the collected plants were authenticated by the survey of medicinal and aromatic plants unit siddha, central council for research in ayurvedic sciences (ccras), palayamkottai, tamil nadu, india. borosil glassware was used throughout the entire project. they were soaked in chromic acid for 3 days, washed with tap water, rinsed with distilled water, and dried in a hot air oven. analytical grade chemicals supplied by sd fine chemicals, srl, hi media, merck india, and sigma aldrich chemicals were used for this research. instruments like analytical weighing balance (shimadzu auw220d, shimadzu corporation, kyoto, japan), high-performance layer chromatography (hplc), (camag linomat 5 with tlc scanner 3, camag, muttenz, switzerland), ft-ir (jasco ft/ir-4100 jasco corporation, tokyo, japan), nmr (bruker avance iii 400 mhz, bruker biospin gmbh, rheinstetten, germany), gc-ms (perkinelmer clarus 500 gc-ms, perkinelmer inc., waltham, ma, usa) were used. the human hepatoma cell line (hepg2) and mouse embryonic fibroblasts cell line (nih 3t3) were obtained from the national centre for cell science (nccs), pune. cells were maintained at 370 °c, 5% co2, 95% air, and 100% relative humidity. maintenance cultures were passaged weekly, and the culture medium was changed twice a week. quantitative estimation of major phytochemicals by hptlc method the collection of plant material, extraction method, solvent system, percentage yield of extracts, identification, and quantitative phytochemicals were evaluated and reported. based on the earlier research, major phytochemicals present in the chloroform e. microphylla extract (cem) and ethyl acetate extract (eaem) were quantitatively estimated by the hptlc method (sajeeth et al., 2010). preparation of standard and sample solutions stock solutions of standard compounds were prepared by dissolving 100 mg of gallic acid, rutin, and quercetin in 100 ml of methanol (hptlc grade). 1000 mg of cem and eaem extract were dissolved in 100 ml of methanol, filtered using whatman no.1 filter paper, and stored in an ambercolored container at 4 °c. procedure for extraction of active constituents five microliters of gallic acid, rutin, quercetin, and extracts were spotted in the form of bands with a microliter syringe on pre-coated silica gel glass plate 60 f254 (10×10 cm with 0.2 mm thickness) using a camag linomat 5 applicator. the plates were prewashed with methanol and activated at 60 °c for 10 min before chromatography. after chamber saturation with the respective mobile phase, the sample-loaded plate was kept in a tlc twin trough developing chamber. the optimized chamber saturation time for the mobile phase was 5 min at room temperature. linear ascending development was carried out, and the plate was developed in the respective mobile phase up to 7 cm. the developed plate was then dried by hot air to evaporate solvents from the plate and develop bands. the dried plate was observed under uv light at 254 nm and 366 nm, and photo documentation was done. densitometric scanning was performed on camag tlc scanner 3 in the absorbance mode at 280 nm. the percentage of active constituents present in the chloroform and ethyl acetate extracts was compared with that of the standard (jain et al., 2006; jeganathan & kannan, 2008). isolation of triterpene constituents from cem by column chromatography based on the results obtained from phytochemical screening and tlc studies, the solvent system was selected for column chromatography using the isocratic elution technique (mahrath et al., 2015; mawa et al., 2016). the silica gel (60-120 mesh) was made into a suspension with the selected solvent system (chloroform:ethyl acetate). silica gel was pre-activated by heating in a hot air oven at 100 °c for 1 h. the bottom of the column was plugged with cotton, and then the silica gel slurry was poured into the column, which was filled with a solvent system to a height of 50 cm. after that, it was set aside for 10 min and allowed to settle. the cem was mixed with a small amount of silica gel and moistened with a solvent system, mixed well, and allowed to evaporate the solvent to get the dry residue. then, the dry residue was charged on the column with the help of a solvent system; after that, cotton was placed over it to avoid the disturbance of the top layer of the adsorbent, as a fresh mobile phase was added to the column. spectral data of isolated compounds compound a: (bauerenol): iupac name: 4, 4, 6b, 8a, 11, 12, 12b, 14b-octamethy l-1, 2, 3, 4, 4a, 5, 6b, 7, 8, 8a, 9, 10, 11, 12, 12a, 12b, 13, 14, 14a, 14bicosahydropicen-3-ol; mp: 278-283 °c; uv: 217; ir (kbr, cm-1): 3384 (oh stretch), 2962, 2917 (alicyclic -ch2 stretch), 2894 (alicyclic -chstretch), 1655 (c=c stretch); 1h nmr (dmso-d6) δ ppm: 0.74 to 1.39 (s, 24h, methyl -ch3, no resolved j 236 biologica nyssana ● 16 (1) june 2025: 233-245 yuvaraja et al. ● isolation, characterization, in silico docking, and in vitro cytotoxicity study of isolated triterpene constituents from ehretia microphylla value) 1.46 to 1.95 (s, 18h, -ch2-ch2ethylene), 1.48 to 1.59 (s, 6h, ch of methylene), 3.24 (d, 1h , =ch, j= 10.5 hz), 5.152 (s, 1h, hydroxyl -oh); 13c nmr: δ 145.35, 116.44, 79.06, 77.41, 56.22, 54.89, 50.41, 48.22, 41.23, 39.65, 38.77, 37.69, 36.88, 35.33, 35.20, 32.42, 32.04, 31.52, 29.70, 28.87, 27.68, 27.53, 25.63, 24.15, 23.66, 22.66, 22.55, 16.80,14.66, 12.98; exact mass: m/z: 426.72 [m]+; chemical formula: c30h50o; calculated elemental analysis: c84.44%; h11.81%; o3.75%. compound b: (11-oxo-aamyrin): iupac name: 10-hydroxy-1,2,4a,6a,9,9,12a-heptamethyl-1,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,1 4b-octadecahydropicen-13(2h)-on. mp: 280 – 285 °c; uv: 218; ir (kbr, cm-1): 3312 (-oh stretch), 2944 (alicyclic -ch2 stretch), 2857 (alicyclic -ch stretch), 1716 (c=o stretch).1h nmr (dmso-d6) δ ppm: 0.81 to 1.25 (s, 24h, methyl -ch3 no resolved j value), 1.27 to 1.55 (s, 16h, -ch2-ch2ethylene), 1.55to 1.64 (s, 6h, ch of methylene), 3.27 ( d 1h, =ch), 5.184 (s, 1h, hydroxyl -oh); 13c nmr: δ 200.13, 175.16, 129.26, 116.44, 79.27, 77.42, 71.81, 56.85, 55.94, 45.82, 42.34, 40.48, 39.67, 37.24, 36.14, 33.93, 31.89, 31.65, 28.91, 28.24, 26.06, 25.39, 24.29, 23.05, 20.12, 20.01, 19.81, 17.68, 16.43, 15.65; exact mass: m/z: 440.37 [m]+; chemical formula c30h48o2; calculated elemental analysis: c81.76%; h10.98%; o7.26.% compound c: (β-sitosterol): iupac name: 17-(5-ethyl-6-methylheptan-2-yl)-10, 13-dimethyl-2, 3, 4, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17tetradecahydro-1h cyclopenta[a]phenanthren-3-ol; mp: 135–136.5 °c; uv 220: ir (kbr, cm-1): 3100–3400 (-oh stretch), 2912 (alicyclic -ch2 stretch), 1379, 1465 (alicyclic -chstretch); 1h nmr (dmso-d6) δ ppm: 0.69 to 1.08 (s, 18h, methyl -ch3), 1.12 to 1.81 (s, 22h, -ch2-ch2ethylene), 1.53 to 1.99 (s, 8h, ch of methylene), 3.52 ( d 1h , =ch), 5.36 (s, 1h, hydroxyl -oh); 13c nmr: δ 140.75, 138.31, 121.71, 56.87, 56.77, 51.23, 45.84, 42.29, 40.48, 39.78, 37.25, 36.51, 36.14, 33.95, 33.71, 32.41, 31.90, 31.65, 29.16, 28.91, 28.24, 26.09, 25.40, 24.36, 23.07, 23.05, 21.20, 21.07, 12.28; exact mass: m/z: 397[m]+; chemical formula: c29h50o; calculated elemental analysis: c83.99%; h12.15%; o3.86%. cytotoxicity study by mtt assay cells were maintained at 37 ºc, 5% co2, 95% air, and 100% relative humidity. maintenance cultures were passaged weekly, and the culture medium was changed twice a week. a mitochondrial enzyme in living cells, succinate-dehydrogenase, cleaves the tetrazolium ring, converting the mtt to an insoluble purple formazan. therefore, the amount of formazan produced is directly proportional to the number of viable cells (mosmann, 1983; monks et al., 1991). after 48 h of incubation, 15 µl of mtt (5 mg/ml) in phosphate-buffered saline (pbs) was added to each well and incubated at 37 °c for 4 h. the medium with mtt was then flicked off, and the formed formazan crystals were solubilized in 100 µl of dmso and then the absorbance was measured at 570 nm using a microplate reader. the % cell inhibition was determined using the following formula: % cell inhibition=100 abs (sample)/abs (control)×100 a nonlinear regression graph was plotted between % cell inhibition and log10 concentration, and ic50 was determined using graphpad prism software. ligand docking and adme properties screening docking study is an online server for performing site-specific molecular docking. the target pdb: 1db1 & 4fa6 were uploaded, and the binding site for the ligand was specified by clicking on the "select binding center” button. the binding center was selected by opening the target visualizer, which will be considered as the isolated compounds a, b, and c, which were the geometric center of the binding center. the data, such as name, pdb id, uniprot name, uniprot accession id/ taxonomic id, organism, and resolution, were specified. the binding site in angstroms is set according to the size of the ligand, which typically covers a sufficiently large space for docking of the small molecule. the maximum number of binding modes and exhaustiveness were specified. this server uses autodock vina for docking calculations. ligands are ranked by using docking scores (the more negative, the better), which suggests how properly the ligand is anticipated to bind to the target. it is, however, also important to check the binding mode and analyze whether it is in agreement with active site residues (https://mcule.com/apps/1-click docking/). results and discussion isolation and structure characterization the hptlc study demonstrated that both extracts contain rutin, gallic acid, and quercetin. the rf value of e. microphylla extracts and standard specimens, rutin, gallic acid, and quercetin were presented in tab. 1. detection of bands at uv light 254 nm and the densitograms are offered in fig. 1 and fig. 2, respectively. the hptlc results proved that the rf values of specimen rutin, gallic acid, and quercetin are 0.18, 0.78, and 0.87, respectively. totally 9 spots were obtained in the chloroform extract. among the 9 spots of the chloroform extract, the rf value of spot 2 is 0.18, which matches the rf of standard rutin. 237 biologica nyssana ● 16 (1) june 2025: 233-245 yuvaraja et al. ● isolation, characterization, in silico docking, and in vitro cytotoxicity study of isolated triterpene constituents from ehretia microphylla table 1. rf values of cem and eaem, and standard markers peak name of the formulation/ volume (5.0 µl) track-1 (cem) track-2 (eaem) rf value of peak area of peak assigned substance rf value of peak area of peak assigned substance 1 0.01 3818.3 unknown 1.01 1271.9 unknown 2 0.18 15297.8 rutin 0.11 4695.9 unknown 3 0.26 609.5 unknown 0.18 17477.3 rutin 4 0.33 149.2 unknown 0.25 894.8 unknown 5 0.43 689.2 unknown 0.42 1880.4 unknown 6 0.49 322.2 unknown 0.58 1172.1 unknown 7 0.57 1072.4 unknown 0.63 12808.1 unknown 8 0.78 19300.4 gallic acid 0.78 22882.4 gallic acid 9 0.87 15130.9 quercetin 0.87 18758.3 quercetin standard/volume (5.0 µl) peak rf value of peak area of peak assigned substance track-3 (rutin) 1 0.18 58576.5 rutin track-4 (gallic acid) 1 0.78 54563.7 gallic acid track-5 (quercetin) 1 0.87 48339.1 quercetin fig. 1. detection of bands at uv light 254 nm track 1: cem, track 2: eaem, track 3: rutin, track 4: gallic acid, and track 5: quercetin the rf value of the 8th spot is 0.78, which exactly matches the rf value of standard gallic acid, and the rf value of spot 9 is 0.87, which is the rf value of standard quercetin. also, a total of 9 spots are obtained in the chromatogram of eaem. among the 9 spots of eaem, the rf values of 3, 8, and 9 spots are 0.18, 0.78, and 0.87, respectively, which indicate the presence of rutin, gallic acid, and quercetin, respectively. the quantification of the identified compounds (rutin, quercetin, and gallic acid) present in cem and eaem was calculated by using the peak area of the extracts cem and eaem and is given in tab. 2. the results revealed that eaem showed the presence of a higher amount of rutin, gallic acid, and quercetin compared with cem. the chloroform extract was subjected to silica gel column chromatography. the solvent system used for the chromatographic analysis of cem was presented in tab. 3. the column was eluted with the selected solvent system by the isocratic method and the fractions were collected in a clean 100 ml beaker up to 25 ml at a speed of 25 drops/min. each collected fraction was tested for the presence of various constituents by tlc for the number and types of constituents, and similar fractions were pooled together. the migration of spot-on chromatograms is indicated by the term rf value. the fractionated compounds a, b, and c were subjected to tlc analysis and characterized by various spectral analyses (uv, ir, mass, and nmr spectroscopy). the fractionated compounds a, b, and c were presented in fig. 3. the ir spectrum of isolated compound a displayed -oh stretch at 3384 cm-1, 238 biologica nyssana ● 16 (1) june 2025: 233-245 yuvaraja et al. ● isolation, characterization, in silico docking, and in vitro cytotoxicity study of isolated triterpene constituents from ehretia microphylla fig. 2. densitograms display of the standard and extracts table 2. quantification of identified compounds in cem and eaem using hptlc s. no track sample volume (µl) rutin (1 mg/ml) of extract (%) gallic acid (1 mg/ml) of extract (%) quercetin (1 mg/ml) of extract (%) 1 track-1 cem 5.0 0.4394 0.2774 0.2890 2 track-2 eaem 5.0 1.2550 0.4522 0.6613 table 3. solvent profile for column chromatography of fractionated compounds compounds solvent system solvent ratio a chloroform: methanol: acetic acid 15:8:3:2 b chloroform: methanol: water 15:3:1 c toluene: ethyl acetate: glacial acetic acid 8: 2: 0.2 -ch2 stretch at 2962 cm-1 and 2917 cm-1, alicyclic -chstretch at 2894 cm-1, and c=c stretch at 1655 cm-1, indicating the presence of these functional groups in compound a. the ir spectrum of fractionated compound b exhibited a stretch at 3312 cm-1, which indicates the presence of a hydrogenbonded oh group. the prominent absorption at 2944 cm-1 indicates ch2 stretching vibration due to the alicyclic ring. the band at 2857 cm-1 is considered for the alicyclic ch stretch. the wide band at 1716 cm-1 indicates c=o stretching. fractionated compound c showed an ir spectrum band in a wide range of 3100-3400 cm-1, indicating the oh stretch's presence in the compound. the stretch at 2912 cm-1 indicates the presence of the alicyclic ch2 group, and the bands at 1379, 1465 cm-1 are consistent with the alicyclic ch stretch. the interpretation of all the spectral studies gives the tentative possible structure of compounds a, b, and c. compound a is bauerenol (c30h50o) [triterpene alcohol], compound b is 11-oxo-amyrin (c30h48o2), and compound c was β-sitosterol (c29h50o). compounds a and c were previously reported in e. microphylla, and to the best of our knowledge, compound b is isolated for the first time from e. microphylla. compound a the 1h-nmr spectrum of compound a indicated the presence of 50 protons. multiple singlets observed 239 biologica nyssana ● 16 (1) june 2025: 233-245 yuvaraja et al. ● isolation, characterization, in silico docking, and in vitro cytotoxicity study of isolated triterpene constituents from ehretia microphylla fig. 3. compounds isolated from ehretia microphylla aerial parts between δ 0.74–1.39 ppm corresponded to 24 methyl protons, suggesting the presence of eight methyl groups, typical in highly substituted triterpenoid or steroidal structures. signals between δ 1.46–1.95 ppm (18h) and δ 1.48–1.59 ppm (6h) were assigned to methylene and methine protons, respectively. a doublet at δ 3.24 ppm (1h) suggested a vinylic proton, and a singlet at δ 5.152 ppm (1h) indicated a hydroxyl group. the 13c-nmr spectrum revealed 30 carbon atoms: 8 ch₃, 9 ch₂, 7 ch, and 6 quaternary carbons. notably, downfield signals at δ 145.35 and 116.44 ppm suggested the presence of an alkene moiety, while the resonance at δ 79.06 ppm was consistent with a carbon bearing a hydroxyl group. this spectral profile is consistent with a tetracyclic triterpenoid backbone bearing oxygenated functionalities. compound b compound b was found to contain 48 protons and 30 carbon atoms. its 1h-nmr spectrum exhibited singlets between δ 0.81–1.25 ppm (24h), indicating the presence of eight methyl groups. the singlets at δ 1.27–1.55 ppm (16h) and δ 1.55–1.64 ppm (6h) were attributed to methylene and methine protons, respectively. similar to compound a, a doublet at δ 3.27 ppm (1h) represented a vinylic proton, while the signal at δ 5.184 ppm (1h) corresponded to a hydroxyl group. the 13c-nmr spectrum also confirmed a 30-carbon framework comprising 8 ch₃, 8 ch₂, 7 ch, and 7 quaternary carbon atoms. the signal at δ 200.13 ppm indicated the presence of a carbonyl group, most likely a ketone, while δ 175.16 ppm suggested a carboxylic or ester carbon. the alkene carbons (δ 129.26 and 116.44 ppm), along with hydroxyl-bearing carbons (δ 79.27 and 71.81 ppm), highlighted the oxygenated and unsaturated nature of the compound. the presence of both ketone and carboxylic functionalities, along with olefinic and hydroxyl groups, suggests that compound b is more oxidized than compound a. the variations in quaternary carbon content also reflect a structural rearrangement, possibly due to oxidation or skeletal modifications. compound c compound c exhibited 50 protons and 29 carbon atoms, slightly fewer than compounds a and b. the 1h-nmr spectrum revealed δ 0.69–1.08 ppm (18h) from six methyl groups, δ 1.12–1.81 ppm (22h) attributed to methylene protons, δ 1.53–1.99 ppm (8h) corresponding to methine protons, δ 3.52 ppm (1h, doublet) as vinylic hydrogen, δ 5.36 ppm (1h, singlet) indicating a hydroxyl proton. the range δ 0.74–1.39 ppm in the 1h nmr of bauerenol corresponds to methyl protons (ch₃), most of which appear as singlets or pseudo-doublets in highly symmetrical triterpenes like bauerenol. here, an nmr signal was observed at δ 0.74–1.39 ppm as a singlet (s, 24h, ch₃), and no resolvable j-coupling was detected. methyl groups attached to quaternary or tertiary carbons usually appear as singlets because they do not couple significantly with neighboring protons. if attached to methylene (ch₂) or methine (ch), the coupling may occur, but in rigid triterpenoid structures, it's often too small to resolve. on the other hand, because it’s a multiplet signal 1.46-1.95 m (m, 18h, ch2), these protons likely experience overlapping couplings from neighboring protons in a rigid framework; no single j can define the whole group. the 13c-nmr data revealed 6 ch₃, 11 ch₂, 9 ch, and 3 quaternary carbons. the relatively high number of ch₂ and ch carbons, along with fewer quaternary centers, suggests a more linear or less fused-ring structure compared to a and b. the signals at δ 140.75, 138.31, and 121.71 ppm point to the presence of a double bond system, possibly part of a substituted olefinic chain. moreover, the lower number of quaternary carbons and the absence of carbonyl peaks in compound c suggest a less oxygenated framework, indicating that compound c may represent a precursor or a 240 reduced form relative to a and b. in silico screening and biological activities the biological potential of any legend is determined by a blend of factors, including in silico study of its binding affinity to target proteins pdb: 1db1 and pdb: 4fa6 with click docking, its absorption, distribution, metabolism, and excretion (adme) properties with star drop version-6.5, and its inhibition efficacy in cell-based mtt assay method. in this study, three isolated compounds a, b, and c, were evaluated for their anticancer properties in human hepatoma cancer cell lines (hepg2) and mouse embryonic fibroblasts normal cell lines (nih 3t3), with a focus on ic50 values and dosedependent inhibition. furthermore, in silico docking studies were employed to assess each compound’s binding affinity to key target proteins and adme properties, providing deeper insight into their biological profiles. this comprehensive comparison examines how each compound performs in terms of inhibition efficacy, binding affinity, and amino acid interactions to offer a complete evaluation of their potential for further drug development, especially for human hepatoma cancer cells (hepg2) and mouse embryonic fibroblast normal cells (nih 3t3). ultimately, the analysis will help determine which compound holds the most promise for therapeutic use and the reasons for this. in silico docking study in this study, we compare the docking results of three compounds (a, b, and c) against two targets: pdb: 1db1, a protein associated with cancer, and pdb: 4fa6, which is involved in hepatoprotection. we will also compare these compounds to standard drugs, such as vitamin d and pyrrolidinyl-pyridopyrimidinone derivatives (mehra et al., 2016; alwarhi et al., 2021), highlighting their roles as kinase inhibitors that target critical pathways, including pi3k/akt/mtor and cyclin-dependent kinases (cdks), both of which are associated with potent anticancer and hepatoprotective activities. the docking results are given in tab. 4. the docking scores of isolated compounds a and b (-9.50 and -9.44, respectively) are quite similar, indicating a strong binding affinity to pdb: 1db1, compared with isolated compound c which has a weaker docking score (-7.14). this suggests that both compounds a and b could potentially have a good therapeutic effect, particularly in terms of their anticancer activity. compounds a and b display interactions with leu-230, leu-233, val-234, ile271, trp-286, tyr-295, his-305, his-397 amino acid residues in the binding pockets. compound b has two more interactions with leu-313 and val-418 amino acid residues. compound c shows interactions with pro-145, tyr-147, asn-276, and glu-277 amino acid residues in the binding pockets. standard drug (vitamin d) demonstrated interactions with tyr143, leu-233, val-234, ser-237, arg-274, ser-275, ser-278, trp-286, his-305, ser-306, ser-314, his397, arg-391 amino acid residues in the binding pockets. the interactions between compounds a and b with the amino acid residues in the binding biologica nyssana ● 16 (1) june 2025: 233-245 yuvaraja et al. ● isolation, characterization, in silico docking, and in vitro cytotoxicity study of isolated triterpene constituents from ehretia microphylla table 4. docking study of isolated compounds against protein associated with cancer (pdb: 1db1) and hepatoprotection (pdb: 4fa6) s.no compound code docking score and amino acid residue interactions pdb: 1db1 pdb: 4fa6 1 a -9.50 leu-230, leu-233, val-234, lle-271, trp-286, tyr-295, his-305, his-397 -8.13 pro-563, ser-594, gln-629 2 b -9.44 leu-227, leu-230, val-234, lie-271, lie-268, trp-286, his-305, leu-313, val-418 -8.42 pro-563, lew-564, asp-632, ser-1032, ser-1044 3 c -7.14 pro-145, try-147, asn-276, glu-277, leu-320, leu-325, glu-327, his-330 -8.54 pro-563, ser-594, gln-629 4 standard -9.76 tyr-143, leu-233 val-234, ser-237 arg-274 ,ser-275 ser-278, trp-286 his-305, ser-306 ser-314, his-397 arg-391 -10.84 lys 802, asp 810, val 822, lys 833, val 851 and asp 933 241 biologica nyssana ● 16 (1) june 2025: 233-245 yuvaraja et al. ● isolation, characterization, in silico docking, and in vitro cytotoxicity study of isolated triterpene constituents from ehretia microphylla fig. 4. isolated compounds from the virtual screening (pdb: 1db1) and their 3d interaction diagram specifying the amino acid residue interaction of the compound with the binding pocket of the vitamin d receptor domain (vdr) pocket suggest a strong binding that likely involves hydrophobic and aromatic interactions, which are crucial for the stability of protein-ligand complexes. the presence of tyr-295 in both compounds a and b also highlights aromatic interactions, which are common in anticancer drugs due to their ability to stabilize interactions and interfere with key protein functions involved in tumor progression. compound b has additional interactions with leu-313 and val418, which may contribute to higher specificity and potentially fewer off-target effects, making it a more selective and effective compound. these residues are likely involved in more specific interactions, which could improve the overall pharmacokinetic profile, reducing systemic toxicity. compound c, in contrast, interacts with pro-145, tyr-147, asn-276, and glu-277, suggesting that its interactions are less stable and more flexible due to the presence of proline, a residue known for introducing flexibility in protein structures. the interactions involving proline could result in reduced specificity, leading to weaker binding compared to compounds a and b. for the hepatoprotective properties of isolated compounds, docking studies were carried out against pdb; 4fa6. compounds a, b, and c demonstrated -8.13, -8.42, and -8.54, respectively, revealing that compound c has a stronger docking score than the other two compounds. this suggests that compound c might have a stronger potential for hepatoprotective activity compared to the other two compounds. compounds a and c display interactions with pro-563, ser-594, and gln629 amino acid residues in the binding pockets. compound b shows interactions with pro-563, leu-564, asp-632, ser-1032, and ser-1044 amino acid residues in the binding pockets. standard drug (pyrrolidinyl pyrido pyrimidinone derivatives) demonstrated interactions with lys-802, asp-810, val-822, lys-833, val-851, and asp-933 amino acid residues in the binding pockets. compound c stands out due to its stronger docking score against pdb: 4fa6. this could make it a strong candidate for protecting liver cells from damage, although its weaker binding to pdb: 1db1 means it might not be as effective against cancer. the virtual screening of isolated compounds against vdr (pdb: 1db1) and pi3ka domain (pdb: 4fa6) and their 3d amino acid residue interaction are given in fig. 4 and fig. fig. 5. isolated compounds from the virtual screening (pdb: 4fa6) and their 3d interaction diagram specifying the amino acid residue interaction of the compound with the binding pocket of the pi3ka domain 242 biologica nyssana ● 16 (1) june 2025: 233-245 yuvaraja et al. ● isolation, characterization, in silico docking, and in vitro cytotoxicity study of isolated triterpene constituents from ehretia microphylla ta bl e 5. in s ili co a d m e pr op er tie s st ud y of is ol at ed c om po un ds compound id o ra l n on c n s sc or in g pr ofi le _ sc or e l og -s 2c 9 pk i he r g pi c 50 b b b lo g( [b ra in ]: [b lo od ]) h ia c at eg or y_ pr ob ab ili ty pgp ca te go ry _ pr ob ab ili ty l ip in sk i’s r ul e of f iv e pa ra m et er m .w t. (< 50 0) lo gp (< 5) h -b on d do no r (< 5) h -b on d ac ce pt or (< 10 ) v io la tio n a 0. 06 83 3 0. 74 58 5. 02 5. 31 4 0. 59 98 -" " = 0. 03 71 35 3; "" +" " = 0. 96 28 65 no "" = 0. 71 ;" "y es "" = 0. 29 42 6. 7 6. 06 1 1 1 b 0. 08 15 6 0. 88 72 5. 03 4. 91 4 0. 07 33 8 -" " = 0. 03 71 35 3; "" +" " = 0. 96 28 65 no "" = 0. 75 ;" "y es "" = 0. 25 44 0. 7 5. 37 6 1 2 1 c 0. 03 97 2 0. 24 06 4. 94 9 5. 89 4 0. 68 24 -" " = 0. 03 71 35 3; "" +" " = 0. 96 28 65 no "" = 0. 68 ;" "y es "" = 0. 32 41 4. 7 7. 11 7 1 1 1 5. in silico adme study the in silico study of absorption, distribution, metabolism, and excretion (adme) properties displays key parameters related to oral bioavailability, cns penetration, drug-likeness, and transport interaction characteristics. in silico adme properties study results of the isolated compound are given in tab. 5. the oral non-cns scoring profile evaluates the likelihood of the compound being orally bioavailable and not penetrating the central nervous system (cns). higher scores indicate better oral bioavailability and reduced cns penetration. compound b has the highest score (0.08156), suggesting it is more likely to be orally bioavailable and less likely to cross the blood-brain barrier (bbb). compound a has a moderate score (0.06833), implying good oral absorption and low cns penetration. compound c has the lowest score (0.03972), which might indicate a lower potential for oral bioavailability and possibly higher cns penetration compared to a and b. the logs value indicates water solubility, with higher values suggesting better solubility. water solubility plays a crucial role in absorption. compound b has the highest logs, indicating it is the most water-soluble and, therefore, likely to have better absorption characteristics. logp reflects the compound's lipophilicity, crucial for membrane permeability and distribution. compounds with high logp values tend to cross cellular membranes more easily but may have issues with solubility. compound c is the most lipophilic, which could indicate it has a higher tendency to accumulate in tissues but might also pose challenges for solubility and gastrointestinal absorption. compound b is less lipophilic and might have lower tissue accumulation, but its moderate lipophilicity also suggests a balanced absorption profile. the pki value represents the binding affinity to the cytochrome p450 enzyme (specifically cyp2c9). higher pki values indicate stronger inhibition, which can lead to drug-drug interactions. compounds a and b show similar, moderately strong inhibition of cyp2c9, which might indicate potential for drug-drug interactions, particularly in metabolism. the herg inhibition is a critical factor in assessing the potential for arrhythmia caused by drugs (qt interval prolongation). the pic50 value reflects the compound's affinity for the herg channel (higher is worse). compound c demonstrates the highest herg pic50, indicating it is the most likely to inhibit herg, which raises concerns about cardiovascular safety. compound a has a moderate herg pic50, which could indicate a lower risk of arrhythmia but still warrants caution. the bbb log 243 biologica nyssana ● 16 (1) june 2025: 233-245 yuvaraja et al. ● isolation, characterization, in silico docking, and in vitro cytotoxicity study of isolated triterpene constituents from ehretia microphylla concentration (µg/ml) % inhibition of compound a % inhibition of compound b % inhibition of compound c nih 3t3 hepg2 nih 3t3 hepg2 nih 3t3 hepg2 63 23.61 25.82 23.15 20.21 18.75 22.2 125 34.26 30.26 33.10 25.35 28.24 29.66 250 39.35 41.2 38.31 33.87 33.91 35.68 500 44.33 50.61 43.29 44.27 39.93 46.27 1000 47.34 67.54 46.99 60.98 46.06 55.49 ic50 values 1068 455 1153 538 1310 556 table 6. in vivo cytotoxicity screening of isolated compounds against hepg2 and nih 3t3 cell line ([brain]:[blood]) ratio indicates the potential for the compound to cross the blood-brain barrier. higher values suggest better penetration into the brain. compound c has the highest value, suggesting it is the most likely to penetrate the bbb and reach the cns, which might limit its suitability for cnssafe drug targets. compound a has a moderate bbb penetration potential, which is balanced for drugs that should remain outside the cns. compound b has the lowest bbb ratio, indicating it is the least likely to cross the blood-brain barrier, which may be beneficial for drugs intended for non-cns targets. all compounds have the same high probability (96.3%) of good human intestinal absorption. this suggests that all three compounds are likely to be well absorbed in the gastrointestinal tract, assuming other factors (e.g., solubility, permeability) are favorable. p-glycoprotein (p-gp) is a transporter that can efflux drugs out of cells, reducing bioavailability. the probabilities suggest that p-gp interaction is less likely for all three compounds, but compounds a and b have a higher likelihood of being substrates for p-gp (29% and 25% chance, respectively), which could reduce their bioavailability or complicate drug interactions. compound c is less likely to be a p-gp substrate (32% chance). all the isolated compounds were in violation of lipinski’s rule (i.e., the log p value is >5), indicating that compounds have more lipophilicity, which is crucial for membrane permeability and distribution. cell line study all three isolated compounds (a, b, and c) demonstrated dose-dependent inhibition in nih 3t3 cells. compound a displayed inhibition against the nih 3t3 cell line, with a percentage of 23.61% at 63 µg/ml increasing to 47.34% at 1000 µg/ ml, and the ic50 at 1068 µg/ml. the results of in vivo cytotoxicity screening of isolated compounds against hepg2 and nih 3t3 cell lines are given in tab. 6. compound b inhibition against the nih 3t3 cell line started at 23.15% at 63 µg/ml and increased to 46.99% at 1000 µg/ml, ic50 at 1153 µg/ml. compound c demonstrated inhibition against the nih 3t3 cells line from 18.75% at 63 µg/ml to 46.06% at 1000 µg/ml with an ic50 at 1310 µg/ml. on the other hand, inhibition against hepg2 cells line study, compound a shows a steeper increase in inhibition with increasing concentration in hepg2 cells at 25.82% at 63 µg/ml and rises to 67.54% at 1000 µg/ml, which ic50 of 455 µg/ml. compound b rises from 20.21% at 63 µg/ml to 60.98% at 1000 µg/ml with ic50 of 538 µg/ml. compound c displayed inhibition starting at 22.20% at 63 µg/ml and reached 55.49% at 1000 µg/ml, with an ic50 at 556 µg/ml. compound a exhibits greater inhibition against hepg2 liver cancer cells than against nih 3t3 fibroblasts, suggesting that it is more effective at lower doses for cancer cells and indicates selectivity for cancerous cells, which is a crucial characteristic for anticancer agents, as it helps reduce toxicity to normal tissues. compounds b and c are also more effective against hepg2 cells than nih 3t3 cells, with a steeper inhibition curve for hepg2. however, later, both compounds’ potency is slightly lower than compound a's in both cell lines. conclusion compound a demonstrates the strongest overall potency, particularly for hepg2 liver cancer cells, with -9.50 against cancer protein (pdb: 1db1), suggesting robust binding and pharmacological potential. however, its potential cardiovascular risks and cns penetration may require optimization before being widely used. compound a shows dual-target activity and favorable binding affinity to both anticancer activity (pdb: 1db1) and hepatoprotective properties (pdb: 4fa6). its more predictable pharmacokinetic profile and selective binding make it an excellent candidate for non-cns drug development, particularly in the context of liver cancer therapy. compound c has the weakest binding to pdb: 1db1, but its stronger binding to 244 biologica nyssana ● 16 (1) june 2025: 233-245 yuvaraja et al. ● isolation, characterization, in silico docking, and in vitro cytotoxicity study of isolated triterpene constituents from ehretia microphylla pdb: 4fa6 suggests potential for cns-targeted therapies. however, its overall potency is the lowest, and its solubility, lipophilicity, and cardiovascular concerns require significant optimization. compound b is the most promising candidate for further development, particularly for liver cancer therapies. compound a remains a strong candidate but requires optimization to address potential cns penetration and cardiovascular issues. compound c requires extensive modifications to improve its potency, selectivity, and safety profile before it can advance in drug development. acknowledgements. the authors would like to thank the school of biotechnology, national institute of technology, nit campus p.o. 673601, kozhikode, kerala, india; dr. a. santhiagu, professor, school of biotechnology, nitc, calicut, and kmch college of pharmacy, coimbatore for their kind support of my research studies. references aarthi, v., shakila, r., sasikala, e., & 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(2020). vitamin d signaling in inflammation and cancer: molecular mechanisms and therapeutic implications. current medicinal chemistry, 27(24), 4326–4339. https:// doi.org/10.2174/0929867326666191009152137 kravchun et al. 2025, biologica nyssana 16(1) 247 16 (1) june 2025: 247-257 doi: 10.46793/biolnyss.16.1.19k different types of crystalluria in patients with bone and joint tissue pathology: hematological and biochemical profiles original article pavlo grigorovich kravchun department of internal medicine no. 2, clinical immunology and allergology named after academician l.t. malaya inna pavlivna dunayeva department of internal medicine no. 2, clinical immunology and allergology named after academician l.t. malaya frida solomonivna leontyeva sytenko institute of spine and joint pathology, national academy of medical sciences of ukraine olena dmytrivna povelychenko sytenko institute of spine and joint pathology, national academy of medical sciences of ukraine valentyna yuriivna dielievska department of internal medicine no. 2, clinical immunology and allergology named after academician l.t. malaya valentinka_1987@ukr.net (corresponding author) received: december 26, 2024 revised: march 03, 2025 accepted: march 17, 2025 abstract: crystalluria, as a sign of dysmetabolic nephropathy, has been associated with bone and joint tissue pathology. the aim of the study was to reveal the structure of crystalluria in patients with bone and joint tissue pathology and to investigate the characteristics of biochemical parameters according to the type of dysmetabolic nephropathy. in this study, common blood analyses and biochemical parameters were investigated. individuals with bone and joint tissue pathology demonstrated prevalence of hyperoxaluria (59%) with less frequency of uraturia (25.7%) and phosphaturia (15.1%). hyperoxaluria was associated with high incidence of ligamentum ruptures and bone fractures (21.6%). the patients with hyperoxaluria showed presence of eosinophilia, increased levels of platelets, leukocytes, erythrocyte sedimentation rate, c-reactive protein and serum alkaline phosphatase, whereas the patients with uraturia demonstrated decreased level of platelets and increased level of serum glucose, uric acid and creatinine. patients with phosphaturia demonstrated increased serum alanine aminotransferase. the results presented in this paper revealed the differences in biochemical parameters of patients with different types of crystalluria, suggesting the necessity of its control to improve the prognosis of the treatment of patients with bone and joint tissue pathology. key words: calcium, joint, oxalates, urates, urea apstrakt: različiti tipovi kristalurije kod pacijenata sa patologijom koštanozglobnog tkiva: hematološki i biohemijski profili kristalurija, kao znak dismetaboličke nefropatije, povezuje se sa patologijom koštano-zglobnog tkiva. cilj ove studije bio je da se utvrdi struktura kristalurije kod osoba sa patologijom koštano-zglobnog tkiva i da se ispitaju karakteristike biohemijskih parametara u zavisnosti od tipa dismetaboličke nefropatije. u ovoj studiji analizirane su rutinske analize krvi i biohemijski parametri. kod osoba sa patologijom koštano-zglobnog tkiva preovlađivala je hiperoksalurija (59%), a ređe su zabeležene uraturija (25.7%) i fosfaturija (15.1%). hiperoksalurija je bila povezana sa visokim udelom ruptura ligamenata i preloma kostiju (21.6%). pacijenti sa hiperoksalurijom imali su eozinofiliju, povećane vrednosti trombocita, leukocita, brzine sedimentacije eritrocita, c-reaktivnog proteina i serumske alkalne fosfataze. s druge strane, pacijenti sa uraturijom pokazali su snižene vrednosti trombocita i povišene vrednosti glukoze u serumu, mokraćne kiseline i kreatinina. kod pacijenata sa fosfaturijom primećene su povišene vrednosti alanin-aminotransferaze u serumu. rezultati predstavljeni u ovom radu ukazuju na razlike u biohemijskim parametrima pacijenata sa različitim tipovima kristalurije, što sugeriše potrebu za njenom kontrolom radi poboljšanja prognoze lečenja osoba sa patologijom koštano-zglobnog tkiva. ključne reči: zglob, kalcijum, oksalati, urati, urea introduction dysmetabolic nephropathies are a group of diseases characterized by an interstitial process in the kidneys with crystalluria due to metabolic disorders (colaci et al., 2024). crystalluria may be transient (monotonous diet or enzyme disorders) and permanent (chronic diseases, metabolic disorders). the factors contributing to the formation of crystals include a high concentration of salts in urine, weak water regime, use of medications, urinary tract infection and digestive disorders (shastri et al., © 2025 kravchun et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 248 2023). the formation of crystals in body fluids in dysmetabolic nephropathies contributes to the development of bone and joint tissue pathology. the urinary syndrome in dysmetabolic nephropathy is characterized by the presence of urates, phosphates, oxalates, leukocyturia, erythrocyturia and proteinuria (timo et al., 2023). crystalluria and urolithiasis occupy one of the leading places in the structure of metabolic diseases. thus, 8–15% of the population of europe and north america suffer from urolithiasis (verdesca et al., 2011; qian et al., 2022). the enzymes involved in glyoxylate metabolism have been related to primary hyperoxalurias (moya et al., 2024). the oxalate metabolism is associated with coagulation system. thus, f1 fragment of prothrombin is excreted in urine and precipitates into calcium oxalate crystals, acting as inhibitor of calcium crystallization in urine (grover et al., 1999). people with hyperoxaluria have demonstrated increased serum level of glycoproteins and chondroitin sulfates (hesse et al., 1991). uraturia patients are characterized by accumulation of monosodium urate crystals in joint tissue, causing inflammation (han et al., 2024). high serum uric acid has been found to be associated with hypercoagulation, endothelial dysfunction and inflammation. the uric acid correlated with thromboembolism and left atrial thrombosis in systemic diseases, metabolic syndrome and arterial hypertension (chen et al., 2016; liu et al., 2022). in high concentrations, uric acid has been shown to predispose to type 2 diabetes mellitus due to the increased production of reactive oxygen species that reduce blood glucose uptake (wardhana et al., 2018; wang et al., 2022). the role of phosphorus in aminotransferase activity has been also discussed (zechner et al., 2021). meanwhile, the association of oxalates, uric acid and phosphates with bone and joint tissue pathology has been scarcely studied. the aim of the study was to analyze the incidence and the structure of crystalluria in patients with bone and joint tissue pathology and to investigate the serum biochemical parameters in every type of crystalluria. materials and methods the examined patients a total of 728 patients (382 female, 346 male), aged 32 to 81 years, with bone and joint tissue pathology (including coxarthrosis, gonarthrosis, ligament ruptures, and bone fractures) treated at the sytenko institute of spine and joint pathology were included in the study following institutional ethical approval. among them, 66 patients exhibited crystalluria and signs of dysmetabolic nephropathy and were assigned to the study group. this group was further divided into group i: patients with hyperoxaluria (n=39), group ii: patients with uraturia (n=17), and group iii: patients with phosphaturia (n=10). an additional 61 patients with bone and joint tissue pathology but without crystalluria were included as the control group. results were compared between the crystalluria subgroups and the control group. clinical examination of the patients included objective examination of organs and systems. ultrasound investigation of kidneys, complete urinalysis and serum biochemical analysis were performed, and revealed the presence of echopositive formations as a sign of crystals. microscopic examination of crystals the crystals were observed under micromed xs -3330 microscope at ×400 magnification. the salt excretion from the 24-hour urine collection was investigated (fig. 1). increased oxalates excretion (more than 1 mg/kg/day) was revealed in patients with hyperoxaluria. patients with uraturia showed increased urinary excretion of uric acid and calcium (uric acid level in urine: more than 4.0 mmol/l, and calcium: more than 7.5 mmol/l). high phosphorus excretion (more than 33 mmol/l) was detected in patients with phosphaturia. urea the content of urea (by color reaction with diacetylmonooxime, langenfeld et al., 2021) was performed with a spectrophotometer. the reaction between diacetyl monoxime and urea, in the presence of sulfuric acid, phosphoric acid, thiosemicarbazide, and ferric chloride, produces a chromophore whose absorbance was measured at a wavelength of 520 nm. creatinine concentration of creatinine was detected using commercial kit (felicit diagnostics) by color yaffe reaction (toora et al., 2002), where 1 ml of working solution was added to 0.25 ml of serum, afterwards the optical density of analyzed sample and standard sample was measured after 30 seconds (e2 and e2) and 90 seconds (e3 and e4) accordingly at wavelength of 505 nm. creatinine concentration was calculated using formula: creatinine concentration=concentration of calibrator sample×(e3-e1)/(e4-e2) total protein 4.0 ml of monoreagent (pyrrogalol red 50 mmol/l, sodium molybdate) were added to 0.08 ml of serum and incubated for 10 minutes at room temperature in dark conditions. the optical density of the test biologica nyssana ● 16 (1) june 2025: 247-257 kravchun et al. ● different types of crystalluria in patients with bone and joint tissue pathology: hematological and biochemical profiles 249 biologica nyssana ● 16 (1) june 2025: 247-257 kravchun et al. ● different types of crystalluria in patients with bone and joint tissue pathology: hematological and biochemical profiles sample (e test) and the calibration sample (e calibration) was measured against a blank sample. the concentration of total protein was calculated by formula: c=e test: e calibration × 1000 mg/l calcium, glycoproteins, and chondrotin sulfates total calcium (complexonometric method with murexide indicator; ritgen u, 2023), glycoproteins (according to the method of steinberg and docenko; shtenberg op et al., 1962), and chondroitin sulfates (measured by the nemeth-csok method in sluckiy's modification; de jong et al., 1989) were analyzed in patients with bone and joint tissue pathology using the respective procedures. b-lipoproteins b-lipoproteins were measured using a commercial kit (cormay ldl direct, poland) and analyzed spectrophotometrically at a wavelength of 630 nm, following the manufacturer’s instructions and the method described by alan (2006). a total of 900 µl of reagent 1 was mixed with 9 µl of serum and incubated for 5 minutes at 37 °c. subsequently, 300 µl of reagent 2 was added to the mixture, followed by an additional 5-minute incubation at 37 °c. uric acid a mixture of 2.4 ml of distilled water, 0.3 ml of serum, 0.15 ml of catalyst solution, and 0.15 ml of sodium tungstate was incubated for 10 minutes at room temperature, then centrifuged for 10 minutes at 2000 rpm. 1.0 ml of the centrifugate was added to 0.5 ml of sodium carbonate solution and 0.3 ml of phosphotungsten reagent, incubated for 30 minutes at room temperature. the optical density was measured spectrophotometrically at a wavelength of 650 nm. aspartate aminotransferase activity aspartate aminotransferase (ast) was detected spectrophotometrically on multiparametric photocolorimetric biochemical analyser alize (lisabio, france) by the dinitrophenylhydrazine method (huang, x.-j., et al., 2006). substrate-buffer solution (0.25 ml) was added to the blood serum (0.05 ml) and incubated for 60 minutes at 37 °c. 0.25 ml of 2,4 dinitrophenylhydrazine was added and incubated for 20 minutes at room temperature. 0.4 m sodium hydroxide solution (2.5 ml) was added and incubated for 10 minutes at room temperature. the optical density of the test sample for aspartate aminotransferase was measured against a control sample on a photometer at a wavelength of 500-560 nm. determination of enzyme activity was carried out according to the calibration schedule. the difference in optical densities of the test and control samples was noted on the ordinate axis, and the corresponding value of enzyme activity was found on the abscissa axis. alanine aminotransferase activity 1 ml of working solution (substrate solution and coenzyme-enzyme reagent) was mixed with 0.1 ml of serum. the optical density was measured at a wavelength of 340 nm after 1 (e1) and 3 (e2) minutes. the difference in extinction (e2-e1)/3 was calculated. urinalysis the urinalysis was carried out according to the methods listed in urinalysis: approved guideline (rabinovitch et al., 2009) and included estimation of ph (ph meter, omega engineering inc.), specific gravity (lab world urinometer, uk), leukocytes, presence of bacteria, erythrocytes (under micromed xs -3330 microscope), and glucose in the urine sample. fig. 1. microscopic appearance of oxalates (1), phosphates (2), and urates (3). 250 biologica nyssana ● 16 (1) june 2025: 247-257 kravchun et al. ● different types of crystalluria in patients with bone and joint tissue pathology: hematological and biochemical profiles the renal function was evaluated by calculation of glomerular filtration rate (gfr) (ckd-epi, 2021) using the following formula: egfr (ml/min/1.73 m2) = 142 x min (creatinine/k, 1) α × max (creatinine/k, 1) 1.200 × 0.9938age × 1.012 (if female) where k is 0.7 for women and 0.9 for men; α is 0.241 for women and 0.302 for men (kirsztajn et al., 2024). ethical consent the study was approved by the ethical committee of kharkiv national medical university (protocol n 21, 24.07.23). informed consent was obtained from all the participants. the study adhered to the ethical guidelines of the declaration of helsinki. statistical evaluation statistical analysis was performed using the software statistica 10.0 (stat soft inc., usa). all measurements were performed in triplicate and the data are presented as average ± standard deviation (sd) (for normally distributed data). the significance of the differences was determined using student’s t-test for parametric data. p values less than 5% were accepted as positive in the tests. correlation analysis was performed using spearman's correlation test. results and discussion crystalluria was detected in 9% (n=66) of patients with bone and joint tissue pathology (n=728). in accordance with the study objective, the study group (n=66) comprised patients with bone and joint tissue pathology who exhibited crystalluria. this group was further subdivided into three subgroups: patients with hyperoxaluria (group i, n=39), uraturia (group ii, n=17), and phosphaturia (group iii, n=10). patients with bone and joint tissue pathology but without crystalluria (n=61) were included as the control group. according to the international kdigo guidelines, group ii exhibited a lower glomerular filtration rate (gfr) (44.52±1.31 ml/min/1.73 m²) compared to the control group (86.34±2.30 ml/min/1.73 m²), group i (83.22±1.91 ml/min/1.73 m²), and group iii (85.10±2.01 ml/min/1.73 m²). patients with hyperoxaluria hyperoxaluria was the most frequently observed type of crystalluria among individuals with bone and joint tissue pathology, identified in 39 patients (59%). individuals with hyperoxaluria showed a high incidence of ligament ruptures and bone fractures (21.6%), a finding that has also been reported by other researchers (büscher et al., 2024). however, there is no clear evidence in the literature linking the severity of bone disease directly to oxalate levels (ben-shalom et al., 2021). hage et al. (2008) reported that dense and radiolucent metaphyseal bands, vertebral osteocondensations, and renal osteodystrophy are characteristic signs of oxalosis. although considerable progress has been made in understanding the pathophysiological mechanisms underlying hyperoxaluria, the diagnosis of oxalaterelated bone disease is often delayed, and the prognosis remains poor (demoulin et al., 2022). patients with hyperoxaluria demonstrated significantly elevated levels of leukocytes (by 34.2%), eosinophils (by 75%), erythrocyte sedimentation rate (esr) (by 90%), and platelets (by 21.8%) in the complete blood count compared to the control group (p<0.05) (tab. 1). the increased levels of leukocytes and esr suggest that disturbances in oxalate metabolism are accompanied by inflammatory processes, possibly due to bacterial involvement. the presence of pathogenic bacteria in calcium oxalate stones has been previously reported, and such bacteria are known to accelerate crystal growth (cooper et al., 2008). conversely, the beneficial bacterium oxalobacter formigenes has been shown to interact with the intestinal mucosa and enhance oxalate secretion (nazzal et al., 2021). the erythrocyte sedimentation rate (esr) is a known marker of inflammation severity, and some researchers have reported an association between hyperoxaluria and inflammatory responses (guo et al., 2020). in europe, a group of scientists formed the european hyperoxaluria consortium to advance research and collaboration in this field (bernd et al., 2009). the increased platelet count observed in patients with hyperoxaluria may be explained by a compensatory response of the bone marrow, as oxalic acid has been reported to exhibit anticoagulant properties (national center for biotechnology information, 2025). oxalate dysmetabolism, often resulting from low serum glycine levels, has been associated with disrupted redox homeostasis, inflammation, atherosclerosis, and cytopenia. furthermore, dysregulated oxalate metabolism has been implicated as a contributing factor in the development of thrombosis and atherosclerosis (liu et al., 2021). therefore, the elevated platelet levels detected in patients with hyperoxaluria may indicate a predisposition to thrombotic events. to date, no prior studies have reported an association between hyperoxaluria and elevated eosinophil counts. biochemical analysis revealed that individuals 251 biologica nyssana ● 16 (1) june 2025: 247-257 kravchun et al. ● different types of crystalluria in patients with bone and joint tissue pathology: hematological and biochemical profiles table 1. clinical blood parameters in patients with bone and joint tissue pathology presenting with hyperoxaluria, uraturia, and phosphaturia with hyperoxaluria had a 23.8% higher serum alkaline phosphatase level compared to the control group (tab. 2). elevated alkaline phosphatase, a known marker of osteomalacia, may help explain the high frequency of ligament ruptures and bone fractures observed in this study. the increased serum alkaline phosphatase levels in hyperoxaluric individuals are consistent with previous findings (bchir et al., 2022). b-lipoprotein levels did not differ significantly between groups, with mean values of 80.42±6.4 units in the hyperoxaluria group and 74.0±5.1 units in the control group. during periods of active oxalate excretion, individuals with hyperoxaluria demonstrated a 17.3% increase in serum alanine aminotransferase (alt) and a 77% increase in c-reactive protein (crp) levels (tab. 3). the elevated alt may reflect liver cell damage, potentially due to cholestasis and the concomitant rise in alkaline phosphatase. variables hyperoxaluria (n=39) uraturia (n=17) phosphaturia (n=10) control group (n=61) (mean±sd) erythrocytes (1012/l) 4.46±0.35 5.02±0.44 4.82±0.4 4.84±0.42 hemoglobin (g/l) 130.62±11.5 143.6±14.2 141.7±13.7 140.25±13.5 platelets (109/l) 334.67±21.4* 213.6±15.63* 270.63±17.8 274.22±19.1 leukocytes (109/l) 8.08±0.85* 5.79±0.6 6.8±0.74 5.96±0.63 esr (mm/h) 19.07±3.2* 10.66±1.8 11.24±2.1 10.1±1.7 eosinophils (%) 3.5±0.6* 2.2±0.43 2.25±0.43 2.0±0.41 segmented neutrophils (%) 58.7±7.2 59.34±7.56 57.08±6.54 53.33±6.1 lymphocytes (%) 26.85±3.74 27.5±3.85 32.18±4.2 28.33±4.0 monocytes (%) 8.21±0.78* 7.78±0.74 4.83±0.32 5.0±0.6 esr: erythrocyte sedimentation rate; *p<0.05 compared to control group table 2. biochemical parameters in patients with bone and joint tissue pathology presenting with hyperoxaluria, uraturia, and phosphaturia variables hyperoxaluria (n=39) uraturia (n=17) phosphaturia (n=10) bone fractures (n=17) control (n=61) (mean±sd) glucose (mmol/l) 5.3±0.24 6.4±0.3* 5.05±0.17 5.97±0.27* 5.1±0.2 total protein (g/l) 75.7±7.13 74.55±6.7 74.3±6.63 77.7±7.4 75.88±7.2 calcium (mmol/l) 2.4±0.15 2.42±0.17 2.3±0.11 2.39±0.13 2.43±0.18 alt (un/l) 24.96±1.6 29.83±1.7* 32.14±2.4* 23.32±1.56 22.97±2.2 ast (un/l) 29.35±1.77 34.66±2.54* 33.6±2.41* 26.82±1.64 27.28±1.7 alph(un/l) 394.7±26.2* 302.42±17.4 318.27±20.1 urea (mmol/l) 4.05±0.48 6.31±0.8* 4.55±0.7 4.41±0.6 4.21±0.57 creatinine (mcmol/l) 95.33±13.1 164.3±17.2* 89.33±12.4 88.22±11.5 91.32±12.73 thymol probe (units) 2.34±0.2 2.56±0.24* 2.06±0.15 2.64±0.27* 1,97±0.11 uric acid (mcmol/l) 361.4±20.6 442.0±25.2* 351.2±17.4 p<0.05 compared to control group. alt: alanine aminotransferase; ast: aspartate aminotransferase; alph: alkaline phosphatase 252 biologica nyssana ● 16 (1) june 2025: 247-257 kravchun et al. ● different types of crystalluria in patients with bone and joint tissue pathology: hematological and biochemical profiles table 3. dynamics of biochemical parameters during active oxalate excretion in patients with hyperoxaluria correlations correlation analysis in patients with hyperoxaluria revealed a strong positive association between serum glucose and b-lipoproteins (r=0.74). serum total protein levels were significantly correlated with chondroitin sulfates (r=0.68) and calcium (r=0.87) (tab. 4), consistent with previous studies reporting a relationship between total protein and serum calcium levels. calcium concentration was inversely correlated with thymol turbidity test results (r=-0.69) and positively correlated with chondroitin sulfates (r=0.55). (p<0.05). in patients with hyperoxaluria, serum calcium levels were inversely correlated with alt, ast, thymol turbidity test results, and crp. in contrast, the control group (without dysmetabolic nephropathies) showed a direct correlation between serum calcium and thymol probe (r=0.57), crp (r=0.36), alt (r=0.16), and ast (r=0.12)(p<0.05). these findings indicate that, in hyperoxaluria, reduced serum calcium levels are associated with systemic inflammation. patients with uraturia among individuals with bone and joint tissue pathology, uraturia was observed less frequently, in 17 patients (25.7%). the platelet count in patients with uraturia was 22.6% lower compared to the control group (p<0.05). this reduction may reflect a compensatory response of the bone marrow, possibly due to the stimulatory effect of uric acid on prothrombotic factors. our findings are consistent with previous studies reporting an association between elevated serum uric acid and reduced platelet count, as demonstrated by regression analyses in individuals with hypertension (tayefi et al., 2018; țăpoi et al., 2021; weng et al., 2023). serum creatinine levels in the uraturia group were significantly higher, by 81.3%, compared to the control group (165.5±27.4 µmol/l vs. 91.32±12.73 µmol/l; p<0.05), indicating impaired renal function. previous research has shown that uric acid exerts a detrimental effect on kidney function. this group also exhibited increased serum glucose levels (by 25.4%), and uric acid levels (by 25.9%) compared to controls (p<0.05). elevated glucose in individuals with uraturia suggests that uric acid may be involved in disorders of carbohydrate metabolism. hyperuricemia and liver dysfunction have been reported in association with diabetes, insulin resistance, obesity, and dyslipidemia. high serum uric acid levels have also been found in patients with diabetes mellitus, showing a positive correlation with glycated hemoglobin a1c. previous studies confirm that hyperuricemia, hyperuricosuria, and urate nephropathy are common in individuals with diabetes. furthermore, uric acid is structurally related to alloxan, a known diabetogenic compound. experimental studies have shown that alloxan administration can induce “uric acid diabetes” (wang et al., 2021; gapparova et al., 2023). therefore, the increased serum glucose observed in individuals variables present oxalate excretion (n=19) absent oxalate excretion (n=20) (mean±sd) calcium (mmol/l) 2.37±0.25 2.34±0.22 alt (un/l) 27.4±1.24* 23.42±1.1 ast (un/l) 29.21±1.4 26.87±1.2 crp (mg/l) 62.74±11.7* 35.7±7.2 alph (un/l) 345.67±26.8 334.56±22.1 p<0.05 table 4. correlation analysis of biochemical parameters in patients with hyperoxaluria variables calcium ast alt rbc hb esr glucose -0.6 -0.85 -0.6 0.57 total protein 0.87 0.2 0.38 -0.82 esr -0.55 -0.48 crp -0.38 -0.38 0.64 calcium -0.56 -0.3 ast: aspartate aminotransferase; alt: alanine aminotransferase; blp: b-lipoproteins; rbc: erythrocytes; hb: hemoglobin; crp: c-reactive protein; esr: erythrocyte sedimentation rate. only correlations with p<0.05 are shown urine specific gravity demonstrated strong positive correlations with serum glucose (r=0.77) and b-lipoproteins (r=0.94) (p<0.05). serum glucose also showed moderate correlations with total protein (r=0.30) and eosinophils (r=0.20) (p<0.05), while creatinine was negatively correlated with glucose (r= -0.65). a strong inverse correlation was observed between neutrophil count and total protein (r= -0.87) 253 biologica nyssana ● 16 (1) june 2025: 247-257 kravchun et al. ● different types of crystalluria in patients with bone and joint tissue pathology: hematological and biochemical profiles with uraturia in this study supports existing evidence suggesting that elevated uric acid contributes to carbohydrate dysmetabolism (yang et al., 2023). the levels of glycoproteins in individuals with hyperoxaluria (0.71±0.11 g/l) and uraturia (0.78±0.13 g/l) did not differ significantly from the control group (0.65±0.10 g/l). similarly, chondroitin sulfate levels were comparable across groups: 0.207±0.032 g/l in hyperoxaluria, 0.187±0.020 g/l in uraturia, and 0.190±0.020 g/l in controls. patients with phosphaturia phosphaturia was the least frequently observed type of crystalluria among individuals with bone and joint tissue pathology, detected in only 10 patients (15.1%). this group demonstrated a 22% increase in serum aspartate aminotransferase (ast) levels compared to the control group (p<0.05). correlation analysis in the phosphaturia group revealed only minor abnormalities. serum calcium levels correlated weakly with chondroitin sulfates (r=0.25), while both alanine aminotransferase (alt) and ast showed a moderate correlation with glycoproteins (r=0.66). chondroitin sulfate levels were also weakly associated with total protein (r=0.25) and glycoproteins (r=0.25) (p<0.05). patients with bone fractures exhibited increased serum glucose levels by 17% and thymol turbidity values by 34% (p<0.05). this study aimed to investigate differences in biochemical parameters among patients with bone and joint tissue pathology based on the type of crystalluria, and to compare these findings to a control group. additionally, the potential association between crystalluria type and bone or joint complications was explored. bone and joint tissue pathology remains a leading cause of morbidity and mortality, particularly in aging populations. identifying and managing risk factors is therefore essential for prevention and treatment. numerous risk factors have been implicated in the development of such pathologies, including elevated blood glucose, kidney disease, obesity, and dyslipidemia (i̇zki et al., 2024). kidney dysfunction has also been associated with vascular wall changes, leading to decreased arterial elasticity (sarısoy et al., 2024). however, the role of dysmetabolic nephropathy in bone and joint pathology remains poorly defined. crystalluria, characterized by increased urinary excretion of oxalates, urates, and phosphates, has been associated with disorders of connective tissue metabolism, which may contribute to degenerative and dystrophic changes in bones and joints (burlaka et al., 2021). the "calcium paradox," whereby endothelial cells adopt osteoblast-like characteristics and sequester calcium crystals, has been proposed as a contributing factor in bone fragility and calcinosis (seker et al., 2024). this study therefore sought to determine the incidence and structural distribution of crystalluria in patients with bone and joint tissue pathology and to analyze biochemical profiles according to the type of crystalluria (hyperoxaluria, uraturia, phosphaturia). crystalluria was present in 9% of the 728 patients with bone and joint tissue pathology. among these, hyperoxaluria was the most common form (59%), followed by uraturia (25.7%) and phosphaturia (15.1%). hyperoxaluria has been linked to gastrointestinal disorders such as chronic gastritis, biliary tract dysfunction, duodenal ulcers, chronic enterocolitis, and crohn’s disease (basoglu et al., 2023), as well as joint dysfunction and oxalate osteopathy (hortabaas et al., 2013; hassona et al., 2024). several studies have reported skeletal abnormalities in patients with hyperoxaluria (bacchetta et al., 2016). an inverse relationship between dietary calcium intake and the risk of stone formation has also been observed. notably, elevated serum alkaline phosphatase in individuals with hyperoxaluria has been linked to low-calcium diets. the ratio of ionized to bound calcium is influenced by serum albumin levels (lalayiannis et al., 2024), and our study confirmed a strong correlation between total protein and calcium levels (r=0.87, p<0.05). low dietary calcium intake contributes to bone demineralization, and individuals with kidney stones are at increased risk for osteoporotic fractures. other studies have confirmed a correlation between low calcium intake and reduced bone mineral density (joshi et al., 2015). supplementation with calcium and lactose has been shown to elevate serum calcium and alkaline phosphatase levels (yang et al., 2024). studies examining the association between serum uric acid and bone mineral density have produced conflicting results. some report a positive association between uric acid levels and bone density (ibrahim et al., 2021; kim et al., 2023; li et al., 2023), while others have found that increased uric acid may reduce the risk of osteoporosis (lin et al., 2019). at physiological levels, uric acid may offer protection against bone fractures. however, in cases of hyperuricemia and gout, uric acid is associated with increased bone resorption and fracture risk (yan et al., 2017). some researchers have identified a significant negative correlation between serum uric acid and trabecular bone score (kuwabara et al., 2023). uric acid has also been associated with altered liver enzyme profiles, including elevated alt levels. high uric acid concentrations may disrupt 254 antioxidant defenses and contribute to liver damage (anothaisintawee et al., 2017). increased levels of both alt and uric acid have been linked to a higher incidence of metabolic syndrome. importantly, this study revealed significant differences in platelet counts depending on the type of crystalluria. patients with hyperoxaluria had significantly higher platelet counts compared to those with uraturia (p<0.05). phosphaturia has been associated with skeletal abnormalities, osteopenia, and osteoporosis (benson et al., 2022). excessive phosphate excretion in urine may result in hypophosphatemia due to reduced intestinal absorption. renal phosphate waste may be secondary to elevated parathyroid hormone levels or the use of loop and thiazide diuretics. phosphaturia is linked to decreased bone strength and typically presents with a low urinary calcium-to-creatinine ratio and a serum calcium × phosphorus product below 30 (mitri et al., 2012). in summary, this study demonstrated distinct biochemical profiles associated with different types of crystalluria in patients with bone and joint tissue pathology. hyperoxaluria was associated with inflammation (leukocytosis, elevated esr), thrombocytosis (increased platelet count), and bile stagnation (elevated alkaline phosphatase). hyperglycemia was observed in patients with uraturia, underscoring the need for glucose monitoring in this population. elevated ast levels were found in individuals with phosphaturia. hyperoxaluria was also associated with an increased risk of bone fractures. while dysmetabolic changes are often difficult to reverse, dietary modifications and appropriate treatment of crystalluria may substantially improve clinical outcomes in patients with bone and joint tissue pathology. prior studies have shown that even in advanced stages of renal dysfunction, reducing dietary oxalate intake may normalize serum creatinine and urinary markers (sun et al., 2017). conclusion hyperoxaluria in individuals with bone and joint tissue pathology was associated with leukocytosis, eosinophilia, and elevated levels of both platelets and serum alkaline phosphatase. in contrast, individuals with uraturia demonstrated increased serum levels of glucose, uric acid, and creatinine, alongside a decreased platelet count. patients with phosphaturia exhibited elevated serum aspartate aminotransferase (ast) levels. these findings suggest that clinicians should closely monitor leukocyte count, eosinophils, platelet levels, and serum alkaline phosphatase in patients with hyperoxaluria; 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(2021). hypophosphatemia in acute liver failure of a broad range of etiologies is associated with phosphaturia without kidney damage or phosphatonin elevation. translational research, 238, 1–11. doi: 10.1016/j.trsl.2021.07.003 terzić, d., đekić, v., milivojević, j., branković, s., perišić, v., perišić, v., đokić, d.: yield components and yield of winter wheat in different years of research. biologica nyssana, 9 (2). december, 2018 biologica nyssana 9 (2) ⚫ december 2018: 119-131 terzić et al. ⚫ yield components and yield of winter wheat in... 119 original article received: 24 august 2018 revised: 23 november 2018 accepted: 10 december 2018 yield components and yield of winter wheat in different years of research dragan terzić1, vera đekić2, jelena milivojević2, snežana branković3, vesna perišić2, vladimir perišić2, dragoslav đokić1 1institute for forage crops, globoder bb, 37000 kruševac, serbia 2small grains research centre, save kovacevica 31, 34000 kragujevac, serbia 3university of kragujevac, faculty of science, institute of biology and ecology, radoje domanović 12, kragujevac, serbia * e-mail: verarajicic@yahoo.com abstract: terzić, d., đekić, v., milivojević, j., branković, s., perišić, v., perišić, v., đokić, d.: yield components and yield of winter wheat in different years of research. biologica nyssana, 9 (2). december, 2018: 119-131. field trial with wheat varieties perfekta, kg 56s, aleksandra and vizija was set on vertisol-type soil during the vegetation season 2010/11 and 2011/12. the aim of the research was to analyse the yield and grain yield components in four varieties of wheat cultivated on acid soil. the highest values of yield components and grain quality were established in the year with moderate temperatures and high precipitation in the vegetation year 2010/11. the kg 56s and vizija varieties had the highest yield of grain, the highest number of plants and spikes per m2. the perfekta variety showed the highest 1000 grain weight and grain weight per spike. the highest number of grains in the spike and the lowest average yield of grain during the research were recorded for the variety aleksandra. a significant positive correlation between grain yield and grain weight per spike, the number of grains in the spike and the number of plants per m2 were established, as well as between the 1000 grain weight and the number of grains in the spike and the grain weight per spike. key words: yield components, grain yield, wheat apstrakt: terzić, d., đekić, v., milivojević, j., branković, s., perišić, v., perišić, v., đokić, d.: komponente prinosa i prinos ozime pšenice u različitim godinama istraživanja. biologica nyssana, 9 (2). decembar, 2018: 119131. poljski ogled sa sortama pšenice perfekta, kg 56s, aleksandra i vizija postavljen je na zemljištu tipa vertisol tokom vegetacionih sezona 2010/11 i 2011/12 godine. cilj istraživanja je bio da se kod četiri sorte pšenice gajene na kiselom zemljištu analizira prinos i komponente prinosa zrna. najveće vrednosti komponenti prinosa i kvaliteta zrna ustanovljene su u godini sa umerenim temperaturama i velikom količinom padavina u vegetacionoj 2010/11 godini. sorte kg 56s i vizija imale su najveći prinos zrna, najveći broj biljaka i klasova po m2. sorta perfekta odlikovala se najvećom masom 1000 zrna i masom zrna po klasu. najveći broj zrna u 9 (2) • december 2018: 119-131 doi: 10.5281/zenodo.2538604 biologica nyssana 9 (2) ⚫ december 2018: 119-131 terzić et al. ⚫ yield components and yield of winter wheat in... 120 klasu i najmanji prosečan prinos zrna tokom istraživanja imala je sorta aleksandra. ustanovljena je značajno pozitivna korelacija između prinosa zrna i mase zrna po klasu, broja zrna po klasu i broja biljaka po m2, kao i između mase 1000 zrna i broja zrna po klasu i mase zrna po klasu. ključne reči: komponente prinosa, prinos zrna, pšenica introduction winter wheat (triticum aestivum l.) is one of the most important field crops in serbia, and it is cultivated on about 530,000 ha per year. average yields of wheat for the last 10 years in the main production areas of serbia have ranged from 4.5-8.0 t/ha. wheat production in serbia depends to a large extent on the environmental factors. the average grain yield of winter wheat and the potential of fertility of cultivated varieties are significantly different, especially in mountainous areas of serbia. hristov et al. (2011) and jocković et al. (2014) state that the yield and grain yield components of winter wheat vary considerably depending on the cultivation system, the applied doses of nitrogen, variety and conditions during the growing year, as well as their complex interactions. in addition to the genotype, the grain yield of winter wheat is largely influenced by the fertilization system, which is one of the key factors affecting the formed yield and its quality, but it must be harmonized with the climatic and soil conditions as well as the requirements of the variety (malešević et al., 2008; jaćimović et al., 2011; popović et al., 2011; đekić et al., 2014; jelic et al., 2015; terzić et al., 2018). research has shown that the yield of wheat depends on a number of components: the number of plants i.e. spikes per unit area, the number of grains in the spike, the weight of the grains in the spike and the absolute grain weight. there are complex interactions between these indicators, because in increasing the value of one parameter, the value of another is often reduced (borojević and williams, 1992; denčić et al., 2000; kandić et al., 2009; dimitrijević et al., 2011; laghari et al., 2011; perišić et al., 2011; đekić et al., 2012; hristov et al., 2012; milovanović et al., 2012; đurić et al., 2013; hagos and abay, 2013; mohamed et al., 2013). the variety as an autonomous genetic, biological and agronomic entity is one of the crucial factors both on quantitative and qualitative production levels (denčić and kobiljski, 2007). the increase in the yield of wheat depends primarily on cultivated variety, climatic conditions and applied cultivation technology (dodig et al., 2008; milovanović et al., 2011; đekić et al., 2012; hristov et al., 2014). the introduction into the production of varieties with increased genetic potential for yield and improved agronomic and technological traits represents the contribution of breeding to achieve higher production per unit area (mladenov et al., 2007; milovanović et al., 2008; luković et al., 2014; przulj et al., 2014; đurić et al., 2018). the genetic potential for yield can be increased in different ways: better use of genetic variability, better utilization of solar energy, increase in number and weight of grain, increase in total biomass of the plant, using heterosis, i.e. wheat hybrid (denčić et al., 2010). choosing the appropriate site/location, i.e. reonization of varieties, will contribute to a less variation in the yields achieved and to achieving better average results (biberdžić et al., 2005; williams et al., 2008; madić et al., 2010; dimitrijević et al., 2011; hristov et al., 2014; perišić, 2016; stevanović et al., 2018; đurić et al., 2018; djuric et al., 2018). bearing all this in mind, it is necessary that climatic conditions are in accordance with the biological requirements of the plants. in the last ten years, extreme temperatures and disturbances in the amount and distribution of precipitation have significantly affected the reduction in overall production of organic matter and yield reduction (hristov et al., 2013; popović et al., 2014). production of winter wheat with high grain yield and appropriate quality is possible only by choosing varieties of good quality with appropriate cultivation conditions and appropriate production technology. during the vegetation years (20102012), in the field trials, four winter wheat varieties were examined in order to determine the selection/breeding of the better varieties for the production conditions of central serbia. material and methods experimental design and soil conditions this study was conducted over a two-year period in the šumadija region, central serbia, on a vertisol soil, at kragujevac location, 173-220 m a. s. l. (44°00′51″ n and 20°54′42″ e), in a temperate continental climate having an average annual temperature of 11.5 °c, typical of šumadija districts in serbia and a rainfall amount of about 550 mm. the basic type of soil on which the trial was carried out in kragujevac was characterized as smonitsa in degradation. the physical properties of this soil are very unfavourable making it the type of heavy clay soil. according to the analysis, this soil was of a medium acid reaction (phkcl = 4.8) and poor in https://tools.wmflabs.org/geohack/geohack.php?pagename=%d0%9a%d1%80%d0%b0%d0%b3%d1%83%d1%98%d0%b5%d0%b2%d0%b0%d1%86&params=44.014167_n_20.911667_e_type:city https://tools.wmflabs.org/geohack/geohack.php?pagename=%d0%9a%d1%80%d0%b0%d0%b3%d1%83%d1%98%d0%b5%d0%b2%d0%b0%d1%86&params=44.014167_n_20.911667_e_type:city biologica nyssana 9 (2) ⚫ december 2018: 119-131 terzić et al. ⚫ yield components and yield of winter wheat in... 121 humus (2.65%). it was very poorly provided with accessible phosphorus (1-2 mg/100 g of soil) and provided with easily accessible potassium (20 mg/100 g of soil). during the 2010/11 and 2011/12 growing seasons, four varieties of winter wheat were investigated (perfekta, kg 56s, aleksandra and vizija) at the small grains research centre in kragujevac (in central serbia). the experiments were conducted in randomized block systems, with a plot size of 50 m2 (5 m x 10 m) in five replications. the sowing was carried out using a machine with row spacing of 12 cm. the soil on which the trial was conducted was uniform and well prepared. the amount of seed per square meter amounted to 400-450 viable seeds, depending on the traits of varieties. it was sown in the third decade of october, with 400 kg/ha of fertilizer npk 15:15:15, which was added in the fall, while during the spring fertilization soil was supplemented with 300 kg/ha (kan 27% n). during the trial, the influence of variety and year on productive yield elements was monitored (number of grains by spike, grain weight per spike, number of plants and spikes per m2 and yield of grain) and grain quality. determination of the number of spikes per m² was done by numbering them in the period of ripening from the surface of 1 m². the analysis of the fertility factors of the spikes (number of grains in the spike and the grain weight per spike) was done by measuring, counting and processing the obtained results. as a material, by random sample, 20 spikes in full maturity were taken from each variant and from all repetitions. after harvest, the yield from each elementary parcel was measured and expressed in kg/ha. the yield was corrected for grain moisture content of 14%. agroecologial conditions data in tab. 1 for the investigated period (20102012) clearly indicate that the years in which the research were conducted differed from the typical multi-annual average for kragujevac region, regarding the meteorological conditions. the average air temperature in 2010/11 was lower by 1.5 c and 2011/12 was lower by 2.1 c as compared to the long-term mean. the sum of rainfall precipitation in 2010/11 was lower by 86.2 mm, where the sum of rainfall in 2011/12 was by 12.5 mm higher than the average of many years and with a very uneven distribution of precipitation per months. compared to the long-term mean, total rainfall values, especially in the first year, second and third year, were considerably higher in february, april and may, whereas total rainfall in april 2010/11 decreased by 31.1 mm. given the high importance of sufficient rainfall amount during the spring months, particularly april and may, for wheat production, the distribution and amount of rainfall over the growing season 2010/11 were considerably more favourable, resulting in increased yields in this year. apart from the rainfall deficiency during the spring months and the non-uniform distribution of rainfall across months, an increase in average air temperatures was also observed. larger precipitation amounts during the spring months (march-may) caused rising groundwater levels and flooding of agricultural land. the excessive amount of moisture influenced the poorer heading and filling of grain, lodging of crops, the abundance of the weeds and the intense occurrence of the disease in the examined wheat and other winter grains. significant deviation of precipitation and temperature from the multi annual average is becoming more pronounced (dodig, 2010; đekić et al., 2015). it was found that newly-produced highyielding wheat varieties are less susceptible to temperature deviation (except for extreme) compared to precipitation (hodson and white, 2009; hristov et al., 2013). namely, the total amount of precipitation is reflected on the multi annual average, but the distribution, especially at critical stages of development, is significantly disturbed. it was found that winter precipitation significantly influences the table 1. mean monthly air temperature and precipitation amount (kragujevac) months interval x xi xii i ii iii iv v vi average mean monthly air temperature (oc) 2010/11 10.2 11.4 2.4 0.9 0.5 7.2 12.0 15.8 20.9 9.0 2011/12 10.4 3.1 4.6 0.7 -3.7 8.1 12.9 16.1 23.0 8.4 average 12.5 6.9 1.9 0.5 2.4 7.1 11.6 16.9 20.0 10.5 the amount of precipitation (mm) 2010/11 86.9 27.9 50.1 29.1 48.5 20.4 20.8 65.8 32.3 381.8 2011/12 33.3 1.3 43.3 117.2 60.1 5.7 74.5 87.3 57.8 480.5 average 45.4 48.9 56.6 58.2 46.6 32.4 51.9 57.6 70.4 468.0 biologica nyssana 9 (2) ⚫ december 2018: 119-131 terzić et al. ⚫ yield components and yield of winter wheat in... 122 realization of the production potential of wheat (malešević et al., 2011). in addition to the necessary reserve for the spring part of the vegetation, winter precipitation greatly influences the distribution of easily accessible nitrogen in the soil (đekić et al., 2014; jelic et al., 2015; terzić et al., 2018). statistical analysis on the basis of achieved research results the usual variation statistical indicators were calculated: average values. experimental data were analysed by descriptive and analytical statistics using the statistics module analyst program sas/stat (sas institute, 2000) for windows. all evaluations of significance were made on the basis of the anova test at 5% and 1% significance levels. relative dependence was defined through correlation analysis (pearson's correlation coefficient), and the coefficients that were obtained were tested at the 5% and 1% levels of significance. results and discussion productive elements of wheat grain yield and quality the average values of productive grain yield and quality traits of the investigated winter wheat varieties grown in the centre for small grains kragujevac are shown in tab. 2. in regard to grain yield, differences were established in the tested varieties. the average yield of wheat in the experiment was 5.091 t/ha, with a variation of 4.630 t/ha in the second year to 5.553 t/ha in the first year of the study. the highest grain yield in the vegetation year 2010/11 was established for the variety vizija (6.127 t/ha) and variety kg 56s (5.863 t/ha). in the second study year, cultivation, the highest average grain yield (5.159 t/ha) was recorded for the variety kg 56s. the highest average grain yield in the two-year period was recorded for kg 56s (5.511 t/ha) and vizija (5.485 t/ha), and the lowest for the variety aleksandra (4.628 t/ha). the weight of 1000 grains in the vegetation year 2010/11 year was significantly higher than in 2011/12 (tab. 2). the average weight of 1000 grains in the study was 43.13 g, with a variation of 42.05 g in the vegetation year 2011/12 to 44.21 g in the vegetation year 2010/11. the highest weight of 1000 grains in the first year was recorded for kg 56s (47.76 g) and perfekta (46.40 g) varieties. in the second year of the study, the highest weight of 1000 grains were achieved by the variety aleksandra (45.80 g). the variety perfekta had the highest average two-year value of the 1000 grain weight (45.00 g) and the lowest the variety vizija (41.98 g). the variety perfekta had the highest number of grains in the spike (45) in the vegetation year 2010/11 year, while in 2011/12 the highest value of the number of grains in the spike was recorded for the variety aleksandra (41). the average two-year value for number of grains in the spike in all wheat varieties examined was 39.65, with a variation of 37.50 in the vegetation year 2011/12 to 41.80 in the vegetation year 2010/11. the data on grain weight per spike, independent of the year, showed that there was a significant difference between the genotypes, with the variety perfekta (1.908 g) showing the highest average weight of grain per spike for the examined years. the average grain weight per spike in the study was 1.725 g, with a variation of 1.423 g to 2.026 g. the highest grain weight per spike in both years was recorded for the varieties perfekta and kg 56s. in regard to the number of plants per m2, significant differences were found in the studied wheat varieties (tab. 2). the average two-year plant count per m2 was 386.9, while the same ranged from 414.6 per m2 in the first year of research to 359.2 per m2 in the second vegetation season. the highest number of plants in vegetation year 2010/11 was established for the varieties vizija (437.2) and kg 56s (426.8). in the second year of testing, the highest number of plants per m2 was found in kg 56s (376.0). the average two-year value of number of spikes per m2 was 518.2, while the same ranged from 564.5 in the first year of research to 472.0 in the second vegetation season. the highest number of spikes in the vegetation year 2010/11 was recorded for the varieties vizija (604.0) and kg 56s (583.6). in the second year of the study, the highest number of spikes was recorded in the variety kg 56s (500.8). analysis of variance between observed traits of wheat the analysis of the variance of productive grain yield and quality traits in the examined kragujevac cultivars of winter wheat, grown in the centre for small grains in kragujevac during the two vegetation seasons, is shown in tab. 3. the estimation of the significance of the obtained results shows that there are statistically highly significant differences between the examined vegetation seasons and grain yield (fexp=14.5689), the number of grains in the spike (fexp=16.0342), the grain weight per spike (fexp=101.1208) and significant for the number of plants per m2 (fexp=8.9838). the statistically significant biologica nyssana 9 (2) ⚫ december 2018: 119-131 terzić et al. ⚫ yield components and yield of winter wheat in... 123 differences were established between the wheat varieties examined and grain yield (fexp=3.3398). the influence of the interaction of agro-climatic conditions and genotypes has had a significant influence on grain yield and grain weight per spike, as well as on the weight of 1000 grains between the tested winter wheat varieties. correlation dependence between tested wheat traits the average values of the pearson’s coefficient of correlation (r) of investigated winter wheat traits are shown in tab. 4. the established correlation coefficients between the yield of grain and the weight of 1000 grains, grain weight per spike and the number of table 2. average values of investigated triticale varieties traits varieties 2010/11 2011/12 average x s sx x s sx x s sx yield, (t/ha) perfekta 5.099 1.001 0.448 4.385 0.678 0.303 4.742 0.890 0.281 kg 56s 5.863 0.537 0.240 5.159 0.956 0.427 5.511 0.820 0.259 aleksandra 5.121 0.421 0.188 4.135 0.256 0.114 4.628 0.615 0.194 vizija 6.127 0.633 0.283 4.843 0.654 0.292 5.485 0.909 0.287 average 5.553 0.780 0.174 4.630 0.747 0.167 5.091 0.887 0.140 1000 grain weight, g perfekta 46.40 2.266 1.013 43.60 1.475 0.660 45.00 2.329 0.737 kg 56s 47.76 0.713 0.319 38.20 1.204 0.538 42.98 5.124 1.620 aleksandra 39.34 3.265 1.460 45.80 2.168 0.969 42.57 4.292 1.36 vizija 43.36 2.611 1.168 40.60 1.673 0.748 41.98 2.528 0.799 average 44.21 3.985 0.891 42.05 3.336 0.746 43.13 3.789 0.599 number of grains per spike perfekta 45.00 0.707 0.316 35.00 4.062 1.817 40.00 5.944 1.880 kg 56s 41.20 1.304 0.583 38.20 2.588 1.158 39.70 2.497 0.789 aleksandra 39.20 2.049 0.916 41.00 3.162 1.414 40.10 2.685 0.849 vizija 41.80 1.923 0.860 35.80 4.147 1.855 38.80 4.392 1.389 average 41.80 2.587 0.579 37.50 4.046 0.905 39.65 3.997 0.632 grain weight per spike, g perfekta 2.186 0.055 0.025 1.630 0.097 0.044 1.908 0.302 0.096 kg 56s 2.138 0.063 0.028 1.500 0.071 0.032 1.819 0.342 0.108 aleksandra 1.810 0.093 0.042 1.287 0.071 0.032 1.548 0.287 0.091 vizija 1.970 0.084 0.037 1.278 0.279 0.125 1.624 0.413 0.131 average 2.026 0.167 0.037 1.423 0.209 0.047 1.725 0.358 0.057 number of plants per m2 perfekta 390.8 44.623 19.956 368.0 88.136 39.416 379.4 66.946 21.170 kg 56s 426.8 65.629 29.350 376.0 49.639 22.199 401.4 61.043 19.303 aleksandra 403.6 81.761 36.564 331.2 39.233 17.545 367.4 71.492 22.608 vizija 437.2 46.981 21.010 361.6 48.711 21.784 399.4 60.193 19.035 average 414.6 59.602 13.327 359.2 57.273 12.807 386.9 64.154 10.144 number of spikes per m2 perfekta 522.0 46.217 20.669 460.8 18.199 8.139 491.4 46.227 14.618 kg 56s 583.6 65.106 29.116 500.8 41.415 18.521 542.2 67.458 21.332 aleksandra 548.4 85.725 38.337 456.0 14.491 6.481 502.2 75.704 23.940 vizija 604.0 22.935 10.257 470.4 32.354 14.469 537.2 75.213 23.785 average 564.5 63.640 14.230 472.0 31.855 7.123 518.2 68.274 10.795 biologica nyssana 9 (2) ⚫ december 2018: 119-131 terzić et al. ⚫ yield components and yield of winter wheat in... 124 plants and spikes per m2 in both vegetation seasons as well as during the two-year research were positive, except between the yield and the weight of 1000 grains in the second year of research in which significantly negative coefficient of correlation (r=0.480*) was established. highly significant and weak correlation was established between grain yield and grain weight per spike (r=0.479**) and medium with number of spikes per m2 (r=0.639*), while significant and weak correlations were established between grain yield and number (r=0.345*) of grains in the spike and number of plants per m2 (r=0.360*). the highest value of the positive and highly significant strong correlation, in the first year of the study, was established between the weight of 1000 grains and the grain weight per spike (r=0.754**), as well as between the number of grains in the spike and grain weight per spike (r=0.770**). the lowest value of the negative correlation in the same year of the study was established between the number of grains per spike and the number of plants per m2 (r=0.190). negative, weak and statistically significant correlation in the second year of the study was determined between grain yield and 1000 grain weight (r=-0.480*). positive and statistically highly significant correlation during the two-year investigation period was established between the grain weight per spike and the yield of grain, the weight of 1000 grains and the number of grains in the spike. positive, weak and significant correlations were found between the number of grains in the spike and the yield (r=0.345*) and the weight of 1000 grains (r=0.372*), as well as between the number of plants per m2 and yield (r=0.360*) and the grain weight per spike (r=0.390*) during the duration of the research. the weight of 1000 grains and the number of plants per m2 in both vegetation seasons, as well as during the duration of the experiment, had a negative coefficient of correlation. correlative dependence between traits showed a large variation depending on the test years which results from the interaction between the properties within each genotype and genotype interactions with environmental factors. this suggests that the selection of genotypes with longer spike (hence higher number of grains per spike) with good resistance to biotic and abiotic stresses contributed to table 3. analysis of variance of the tested parameters (anova) effect of year on the traits analysed traits mean sqr effect mean sqr error f (df 1, 38) p-level grain yield, t/ha 8.50 0.584 14.5689** 0.000484 1000 grain weight, g 46.87 13.503 3.4712 0.070187 number of grains per spike 184.90 11.532 16.0342** 0.000279 grain weight per spike, g 3.63 .036 101.1208** 0.000000 number of plants per m2 30691.60 3416.316 8.9838* 0.004780 number of spikes per m2 85562.50 2532.395 33.7872** 0.000001 effect of cultivar on the traits analysed traits mean sqr effect mean sqr error f (df 3, 36) p-level grain yield, t/ha 2.227 0.667 3.3398* 0.029847 1000 grain weight, g 17.185 14.123 1.2168 0.317626 number of grains per spike 3.500 17.017 0.2057 0.891792 grain weight per spike, g 0.280 0.115 2.4214 0.081864 number of plants per m2 2676.667 4235.600 0.6319 0.599209 number of spikes per m2 6370.767 4518.922 1.4098 0.25577 effect of the year x cultivar interaction traits mean sqr effect mean sqr error f (df 3, 32) p-level grain yield, t/ha 2.227 0.467 4.7713** 0.007358 1000 grain weight, g 17.185 4.280 4.0147* 0.015626 number of grains per spike 3.500 7.563 0.4628 0.710220 grain weight per spike, g 0.280 0.015 18.5725** 0.000000 number of plants per m2 2676.667 3666.800 0.7300 0.541718 number of spikes per m2 2302.500 2194.100 1.0494 0.384167 *statistically significant difference (p<0.05); **statistically high significant difference (p<0.01) biologica nyssana 9 (2) ⚫ december 2018: 119-131 terzić et al. ⚫ yield components and yield of winter wheat in... 125 the increase in production and yield stability of wheat in our agroecological conditions. discussion the highest average grain yield of 5.553 t/ha of studied wheat varieties was recorded in vegetation season 2010/11 and it was significantly higher than the yield in 2011/12 (4.630 t/ha), which can mostly be associated with higher precipitation during the first vegetation period. hassan et al. (2013) and perišić et al. (2016) have indicated similar results. after the formation of the number of spikes and the number of grains per spike during the vegetation stage, the yield becomes mainly determined by the weight of the grain (zafaranaderi et al., 2013). the yield of wheat grain, according to many authors, depends on several components: the number of spikes per unit area, the number of grains per spike, the weight of the grains per spike and the plant, as well as the weight of 1000 grains (hristov et al., 2008; petrović et al., 2010; perišić, 2016; djuric et al., 2018). varying of yield below 10% in exceptionally yielding years is not considered a serious problem in intensive wheat production. if these deviations are conditioned solely by the action of climatic factors (without loss during harvest or transport), using varietal agro-technology, it is possible to significantly reduce the disturbance of total grain production in one production area (malešević et al., 2011; jaćimović et al., 2012; đekić et al., 2014; jelić et al., 2015; terzić et al., 2018). in the production of wheat, the correct reonization (regional distribution) of varieties is very important, and it can contribute to a lesser variation in realized yields and achieving better average results (donmez et al., 2001; zečević et al., 2004; dencic and kobiljski, 2007; dodig et al., 2008; madić et al., 2010; dimitrijević et al., 2011; đekić et al., 2012, 2013). bearing all this in mind, it is necessary that climatic conditions are in accordance with the biological requirements of the plants. in the last few years, extreme temperatures and disturbances in the amount and distribution of precipitation have significantly affected the reduction in the total production of organic matter and yield reduction (hodson and white, 2009; đurić et al., 2013; hristov et al., 2013; popović et al., 2013; đekić et al., 2015; jelic et al., 2015). in the continental climate, winter wheat has long been exposed to the influence of weather conditions, and hence the climate extremes. according to the forecasts of the european commission for agriculture, global climate change will cause significant deviations from the average table 4. correlation coefficients by studied environments in winter wheat gy gw ngs gws np ns correlations between the traits analysed in the 2010/11 grain yield, gy 1.00 0.093 0.041 0.137 0.308 0.528* 1000 grain weight, gw 1.00 0.397 0.754** -0.118 -0.094 number of grains per spike, ngs 1.00 0.770** -0.190 -0.190 grain weight per spike, gws 1.00 -0.104 -0.178 number of plants per m2, np 1.00 0.675** number of spikes per m2, ns 1.00 correlations between the traits analysed in the 2011/12 grain yield, gy 1.00 -0.480* 0.113 0.011 0.020 0.350 1000 grain weight, gw 1.00 0.191 0.042 -0.288 -0.316 number of grains per spike, ngs 1.00 -0.356 -0.207 -0.037 grain weight per spike, gws 1.00 0.155 0.276 number of plants per m2, np 1.00 0.348 number of spikes per m2, ns 1.00 correlations between the traits analysed in the 2010-2012 grain yield, gy 1.00 0.022 0.345* 0.479** 0.360* 0.639** 1000 grain weight, gw 1.00 0.372* 0.433** -0.040 0.090 number of grains per spike, ngs 1.00 0.475** 0.091 0.309 grain weight per spike, gws 1.00 0.390* 0.584** number of plants per m2, np 1.00 0.655** number of spikes per m2, ns 1.00 *statistically significant (p<0.05); **statistically high significant (p<0.01) **grain yield, t/ha gy; 1000 grain weight, g gw; number of grains per spike -ngs; grain weight per spike, g gws; number of plants per m2 np; number of spikes per m2 ns. biologica nyssana 9 (2) ⚫ december 2018: 119-131 terzić et al. ⚫ yield components and yield of winter wheat in... 126 values of climatic factors in the field crop areas of serbia (vojvodina, mačva, stig), which is related to the increase in winter and reduction of summer precipitation, increased risk of drought and soil erosion, and to a certain extension of the vegetation period (which would lead to an increase in yield, but with higher variability). temperatures will increase slightly, which will impose changes in the sowing structure and a more careful selection of the variety, i.e. hybrid. according to this scenario, the biggest concern is the increase of winter precipitation (november-march), which will often cause flooding of lower terrains as well as greater possibility of soil erosion. in serbia, there are about 30 percent of the soil with a heavier mechanical composition that is susceptible to compaction and formation of the plow pan. frequent passing of machines and conventional tillage at low depths (due to weaker tractors) leads to the formation of a poorly permeable layer “plow pan” at a small depth and rapid saturation of the surface layer of the soil with water. the soil acidification process has been strengthened in recent times and tackling this problem is an important issue in order to reduce the impact from natural hazards in serbia. decrease of the acidity in acid soils contributes to the improvement of chemical, physical and biological properties of the soil. improvement of these soil properties contributes to better infiltration and water storage and reduction of damage and losses from natural hazards, including floods and droughts. these facts impose the need to change the system of crop fertilization and processing systems as well as land management in general. higher air temperatures in june and july of year 2012, compared to the first year of the study, caused a shortening of the period of filling of the grain, accelerated ripening and reduction of yield. the dependence of the yield on the conditions of the year was also emphasized by other authors (biberdžić et al., 2005; zecevic et al., 2010; perišić et al., 2016). a certain contribution to the continuous increase in grain yield is also interpreted by the increased use of nitrogen fertilizers, pesticide use, improved agro-technology (especially earlier sowing), as well as the positive interaction of these factors (jelić et al., 2012; đekić et al., 2014; terzić et al. 2018). the best genetic predispositions for yield were found in the kg 56s and vizija varieties, while the lowest yield in our study was recorded in the aleksandra variety and it was significantly lower in comparison to the other varieties tested. our results are in agreement with the results of milovanovic et al. (2008), who, based on their research, state the variety vizija as a variety of high genetic potential for yield with the possibility of its manifestation at different sites/locations. the differences in the yields that were observed in the tested varieties in our experiment are the result of varietal specificities, which are mostly genetically conditioned. thus, by analysing the obtained results we can conclude that there is a significant dependence of grain quality components on the genotype, which is in agreement with the results of laghari et al. (2011) and perišić (2016). the weight of 1000 grains in year 2010/11 was significantly higher compared to 2011/12 (44.21 g and 42.05 g). the highest value of the 1000 grain weight was established for the perfekta variety, the variety with the lowest number of grains per spike, and the lowest value for the variety vizija, which had the highest number of grains per spike. vegetation period in year 2011/12, at the time of grain filling, was marked by drought and high temperatures. climatic conditions are especially important during the filling of the grain, since the lack of moisture and high temperature during this period affects the decrease in the weight of 1000 grains (przulj et al., 2014), as confirmed by the results of these studies. perišić (2016) points to significant differences in grain weight between years, irrespective of the genotype. jaćimovic et al. (2012) find that the application of mineral fertilizers has a significant impact on the weight of 1000 grains, i.e. the grain weight is significantly higher in more intensively fertilized variants especially fertilized with nitrogen. the number of grains per spike is a very variable trait of the spike, because it depends on other components of the spike and factors of the external environment. the results of these investigations are in agreement with the previously performed studies by đurić et al. (2018) and established a high variability of the number of grains per spike and grain weight per spike djuric et al., (2018). these spike characteristics have a direct impact on the overall yield of wheat grain. the number of grains per spike is quite variable trait, which largely depends on the agroecological conditions of the year and the applied agro-technology (zecevic et al., 2004). the number of grains per spike is a yield component that is directly dependent on the number of spikelets per spike, the number of flowers per spikelet and the success of pollination and grain formation (borojević and williams, 1992). since these parameters significantly depend on the agroecological conditions of the year and the applied agro-technology, the number of grains per spike is quite variable trait. hristov et al. (2008) point out that the increase in the yield of grain per plant is directly determined by the number of grains per spike and the weight of 1000 grains, which is confirmed by the results of our research. milovanović et al. (2011), based on their research, suggest that better lines of triticale have 7595 grains per spike compared to 40-50 grains in biologica nyssana 9 (2) ⚫ december 2018: 119-131 terzić et al. ⚫ yield components and yield of winter wheat in... 127 wheat. the number of grains per spike for all investigated wheat genotypes ranged from 35 to 45 grains. in the first year of the research, which was more favourable, the number of grains per spike was 42, and in the second year of the research it was 37. the highest number of grains in the spike in the vegetation year 2010/11 was recorded for the perfekta variety (45), while in 2011/12 for the variety aleksandra (41). the average value of the number of grains per spike of 44.80 is established by perišić (2016). the same author states that the number of grains per spike has ranged from 38.61 to 47.76. the number of grains per spike or plant is the result of the number of spikelets and the number of flowers per spikelet, on the one hand, and the success of polination and formating of grains in these flowers, on the other hand, so that the number of grains depends to a large extent on the genetic basis of the variety, i.e. the gene with additive and non-additive effect (perišić et al., 2011; deletić et al., 2012; luković et al., 2014; đurić et al., 2018) and external factors (dodig, 2010; hassan et al., 2013). in the case of wheat genotypes, the increase in the number of grains in the spike was observed during the study in the case of a reduced number of spikes per m2, which is in agreement with the results of perišić (2016). according to garcia del moral et al. (2003), the yield stability under the influence of different environmental conditions is closely related to the number of grains per spike, because a large number of grains per spike allows the achievement of a large number of grains per unit area also in conditions of a smaller number of spikes. donmez et al. (2001) find that significant genetic progress is achieved by improving the yield components formed during the vegetative phase of wheat development (the number of spikes per surface area and the number of grains per spike), compared to the grain weight, a component formed at unfavourable temperature and humidity conditions. the number of spikes per unit area depends on the sowing density, the number of seedlings, the number of plants that managed to survive winter and the number of productive stems. the number of spikes is greatly affected by the conditions of cultivation, which is confirmed by our research. in our study, the highest number of spikes per m2 in the period 2010-2012 was realized by the varieties kg 56s and vizija. the highest number of spikes in the vegetation year 2010/11 was established for the varieties vizija (604.0) and kg 56s (583.6), while in the second year of the study, kg 56s (500.8) had the highest number of spikes. our results are in agreement with perišić (2016), who states that the number of spikes has ranged from 544.4 to 671.1 per m2 and that it was statistically significant among the wheat genotypes examined. biberdžic et al. (2005) pointed out that the number of spikes/m2, the number of grains per spike and the length of spike are important factors that influence grain yield. there are complex interactions between these indicators, as by increasing the value of one parameter, the value of another is often reduced (hristov et al., 2008), which is confirmed by the results of our research. also, it was noticed that in year 2010/11, due to favourable climatic conditions (at the time of emergence and tillering), in almost all genotypes, on average, statistically significantly higher number of spikes per m2 (564.5) was achieved compared to the second year (472.0). obviously, weather conditions (precipitation and temperature) were the least favourable in the second year of testing. it has been confirmed earlier by other authors that meteorological factors, and especially the moisture regime, can influence this property (joshi et al., 2002; janušauskaite and šidlauskas, 2004; erekul and köhn, 2006). in order to be able to perceive which traits have most influenced the formation of the grain yield, as well as the relationship between them, the correlation coefficients between the tested properties have been determined. the correlative dependence of the yield and the weight of the 1000 grains, depending on the investigated vegetation season in wheat, ranged from 0.480* (2011/12) to 0.093 (2010/11). grain yield depends directly on the number of grains per spike and the weight of 1000 grains (hristov, 2008). knowledge of correlation relations between yield components is of practical importance in the breeding process. correlation analysis is the most commonly used method for this purpose (hristov et al., 2011). since yield components directly or indirectly affect the yield, it is necessary to determine their correlation relations. these authors have established a highly significant correlation between the height of the plant and the length of the spike, the number of grains/ spike, the weight of the 1000 grains and the grain weight per plant, the highly significant correlation between the harvest index and plant height and the length of the spike. the weight of the grains per spike was in significant correlation with the yield (r=0.479**), the weight of 1000 grains (r=0.433**) and the number of grains in the spike (r=0.475**). many researchers are of the opinion that an increase in the number of grains per spike leads to an increase in the yield of wheat (zafarnaderi et al., 2013; đurić et al., 2016). many authors consider the number of grains per spike the most important component of the yield. in wheat, the coefficient of correlation showed values from non-significantly positive in 2010/11 (r=0.041) to significantly positive in the two-year investigation period (r=0.345*). many authors cite weak positive (mohammadi et al., 2013) to very positive biologica nyssana 9 (2) ⚫ december 2018: 119-131 terzić et al. ⚫ yield components and yield of winter wheat in... 128 correlations (djuric et al., 2018). according to the majority of researchers, the number of spikes per unit area is in significant positive correlation with the yield of grain (zecevic et al., 2004). iftikhar et al. (2012) find that the number of grains per spike and mass of 1000 grains are in a positive and statistically significant correlation with grain yield and point out that the number of grains per spike and weight of 1000 grains have a direct effect on yield and, therefore, can be used as direct selection criteria. new varieties are characterized by a higher number of spikes per unit area, but their relationship to yield is not always in a positive correlation (deletić et al., 2012). the same authors point out that in the determination of yield, the weight of the grain has the crucial role, and then the number of spikes and the number of grains per spike. perišić (2016) states that a higher number of productive trees cannot guarantee high yields. there are complex interdependent relations between all components of yield. the importance of these components in the formation of grain yield depends on the climatic conditions in critical phases of growth and development, applied agro-technology and various combinations and relationships of npk nutrients (popović et al., 2013; đekić et al., 2014; jelić et al., 2014; terzic et al., 2018). therefore, it is important to know the effect of these properties, i.e. yield components, as well as their interdependence on grain yield. it should be noted that the grain weight per plant or the yield per plant is a direct indicator of total yield, as well as all components of spikes that are in significant positive correlation with this parameter (hristov et al., 2008; đekić et al., 2013). the presence of phenotypic and genotypic correlations between fertility components and fertility itself were studied by kashif and khaliq (2004). they find that at the genotypic level there is a significant positive correlation of the height of the plant, the length of the spike, the number of spikelets per spike, the number of grains per spike, and the weight of 1000 grains with grain yield, while phenotypically there is a very significant association of these properties. due to this association of quantitative properties, it is necessary to select genotypes with a genetic basis in which a balance is achieved between the most important components (number of spikes per unit area, number of grains per spike and grain weight per spike). conclusion in the highest yield of grain, weight of 1000 grains, number of grains per spike, grain weight per spike and number of spikes per m2 in all wheat varieties were in the vegetation period with moderate temperatures at the time of grain filling and large amount of precipitation in the first vegetation period. grain yields in wheat cultivars ranged from 4.628 t/ha (aleksandra) to 5.511 t/ha (kg 56s). the average weight of 1000 grains in the study was 43.13 g, with a variation of 42.05 g in the vegetation year 2011/12 to 44.21 g in the vegetation year 2010/11. the number of grains per spike for all examined wheat genotypes varied from 35 to 45 grains. the coefficient of correlation between grain yield and grain weight per spike, number of grains per spike and number of plants per m2 showed significant positive values over the examined period, while the coefficient between 1000 grain weight and number of grains per spike and grain weight per spike had significant positive values. also, there were significant positive coefficients of correlation between the grain weight per spike with the number of grains per spike and the number of plants per m2. based on the results of the research it can be concluded that a greater number of traits have a decisive role in the formation of grain yield. the contribution of each individual trait may be different in various genotypes and in various environmental conditions so that the correlation between two quantitative traits is not fixed. this suggests that the selection of genotypes with longer spike with good resistance to biotic and abiotic stresses contributed to the increase in production and yield stability of wheat. acknowledgements. investigations necessary for this paper are part of the project tp 31054 and 31057 financed by the ministry of education, science and technology development of republic of serbia. references biberdžić, m., đorđević, m., barać, s., deletić, n., stojković, s. 2005: productivity of some winter wheat genotypes. genetika, 37(2): 131-136. borojević, s., williams, w. 1992: genotype x enviroment interactions for leaf area parametars and yield components and their effects on wheat yield. crop science 22(5): 1020-1025. deletić, n., stojković, s., gudžić, s., đurić, v., aksić, m. 2012: genotypic specificity of some winter wheat traits and their effect on grain yield. genetika, 44(2): 249-258. denčić, s., kastori, r., kobiljski, b., duggan, b. 2000: evaluation of grain yield and its components in wheat cultivars and landraces under near optimal and drought conditions. euphytica, 113: 43-52. dencic, s., kobiljski, b. 2007: organization of wheat genetic resources in collections. uvodno predavanje. 1st joint psu-uns international biologica nyssana 9 (2) ⚫ december 2018: 119-131 terzić et al. ⚫ yield components and yield of winter wheat in... 129 conference on bioscience: food, agriculture, and the environment. hat yai, thailand. ia 04: 6. denčić, s., kobiljski, b., mladenović, g., jestrović, z., štatkić, s., pavlović, m., orbović, b. 2010: sorta kao faktor proizvodnje pšenice. ratarstvo i povrtarstvo, 47(1): 317-324. dimitrijević, m., knežević, d., petrović, s., zečević, v., bošković, j., belić, m., pejić, b., banjac, b. 2011: stability of yield components in wheat (triticum aestivum l.). genetika, 43(1): 29-39. dodig, d., zorić, m., knežević, d., king, s., šurlanmomirović, g. 2008: genotype x environment interaction for wheat yield in different drought stress conditions and agronomic traits suitable for selection. australian journal of agricultural research, 59: 536-545. dodig, d. 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research, a total of 110 species belonging to 45 genera, 27 families were recorded in the temska village area. the examination of ecological-trophic structure showed that the most numerous were the mycorrhizal fungi. of all identified species 19 are protected by the national law and 16 are included in the preliminary national red list. key words: endangered, macrofungi , red list. introduction the village of temska (43° 15' 38" n, 22° 32' 39" e) is situated in the south-western foothill of the mountain stara planina, at an altitude of about 500 m (fig. 1). it is surrounded by the temska gorge, krušavica, temačko brdo, kulišta and ravna hills. the area has a temperate continental climate. mean annual temperature is 9°c, with the amountof precipitation ranging from 500mm to 1000mm per year (d u c i ć et al., 2005). alluvial deposits can be found in this area. rendzina soil type was developed on marlstone and limestone geological ground (a n đ e l k o v i ć 1958). although an overview of mycromycete fungi for stara planina mt. was given by i v a n c e v i c & b e r o n j a (2004), this is the first paper focusing on basidiomycetes of the area surrounding temska village. materials and methods fungal specimens were being collected in the period between 2010. and 2012. in spring, summer and autumn. endangered and protected species were not collected and were identified on the spot. a large proportion of fungal basidiocarps were collected at the altitudes between 500 and 1100 m in the oak forest belt (quercus cerris l., q. frainetto ten., q. petraea (mattuschka) liebl., q. pubescens willd.), also inhabited by some other wood species such as betula pendula roth, fraxinus excelsior l., f. ornus l., crataegus monogyna jacq., acer campestre l., a. pseudoplatanus l., a. tataricum l., etc. also a number of species was collected from a belt of coniferous forest, consisting mainly of planted pinus nigra j.f.arnold trees. the fungi were identified on the basis of macroscopic and microscopic morphological characteristics and specific chemical reactions of fruiting bodies according to specific identification keys: (b o ž a c 1978; f o h t 1986; b r e i t e n b a c h & k r ä n z l i n 1995; m a c a et al., 1995; m c k n i g h t et al., 1998; j o r d a n 2004; u z e l a c 2009). author citations for each taxon are abbreviated according to the index fungorum (2013). 3 (2) • decemer 2012: 91-96 biologica nyssana 2 (2)  december 2011: 91-96 sadiković d. et al.  basidiomycetes of temska village area... 92 table 1. list of mycromycete fungi with preferred habitat type and substrate taxa habitat life form agaricales agaricaceae agaricus bitorquis (quél.) sacc. edge of broadleaf forest saprophytic agaricus campestris var. campestris l. meadow saprophytic agaricus macrosporus (f.h. moller & jul. schaeff.) pilat meadow saprophytic agarius nivescens (f.h. møller) f.h. møller meadow saprophytic agaricus moelleri wasser. broadleaf forest saprophytic agaricus silvaticus schaeff. edge of broadleaf forest saprophytic lepiota castanea quél. mixed broadleaf forest saprophytic macrolepiota procera (scop.) singer meadow; edge of the forest saprophytic macrolepiota rhacodes (vittad.) singer meadow; edge of the forest saprophytic amanitaceae amanita caesarea (scop.) pers. edge of broadleaf forest mycorrhizal amanita fulva (schaeff.) fr. broadleaf forest mycorrhizal amanita lividopallescens (secr. ex boud.) kühner & romagn. mixed broadleaf forest mycorrhizal amanita pantherina (dc.) krombh. broadleaf forest mycorrhizal amanita phalloides (vaill. ex fr.) broadleaf forest mycorrhizal amanita rubescens pers. mixed broadleaf forest mycorrhizal amanita vaginata (bull.) fr. broadleaf forest mycorrhizal amanita vaginata var. alba (sacc.) romagn. broadleaf forest mycorrhizal amanita virosa (fr.) bertill mixed forest mycorrhizal coprinaceae coprinus niveus (pers.) meadow; tilled fields saprophytic coprinus comatus (o.f. müll.) pers. tilled fields saprophytic figure 1. geographical position of investigated area based on google earth satellite image biologica nyssana 3 (2)  december 2012: 91-96 sadiković d. et al.  basidiomycetes of temska village area.. 93 taxa habitat life form coprinus micaceus (bull.) fr. tilled fields saprophytic panaeolus acuminatus (schaeff.) edge of the forest saprophytic hygrophoraceae hygrocybe conica (schaeff.) quél. meadow; edge of the forest saprophytic marasmiaceae marasmius oreades (bolton) fr. meadow; edge of the forest saprophytic marasmius rotula (scop.) fr. meadow saprophytic marasmius torquescens quél. broadleaf forest saprophytic omphalotus olearius (dc.) singer broadleaf forest saprophytic mycenaceae mycena renati quél. mixed forest saprophytic mycena vitilis (fr.) quél. mixed forest saprophytic pleurotaceae pleurotus eryngii (dc.) quél. broadleaf forest parasitic pleurotus ostreatus (jacq.) p. kumm. broadleaf forest parasitic/ saprophytic pluteaceae pluteus cervinus (schaeff.) p. kumm. edge of broadleaf forest saprophytic pluteus salicinus (pers.) p. kumm. broadleaf forest saprophytic schizophyllaceae schizophyllum commune fr. mixed forest parasitic strophariaceae pholiota highlandensis quadr. & lunghini meadow saprophytic tricholomataceae clitocybe odora (bull.:fr.) kummer broadleaf forest saprophytic leucopaxillus giganteus (quél.) singer broadleaf forest saprophytic oudemansiella radicata (relhan) singer mixed broadleaf forest saprophytic rugosomyces ionides (bull.) bon broadleaf forest saprophytic tricholoma fulvum (fr.) bigeard & h. guill mixed broadleaf forest mycorrhizal auriculariales auriculariaceae auricularia auricula–judae (bull.) quél. mixed broadleaf forest saprophytic auricularia mesenterica (dicks.) pers. edge of the forest saprophytic boletales boletaceae boletus aestivalis (paulet) fr. broadleaf forest mycorrhizal boletus aereus secr. mixed broadleaf forest mycorrhizal boletus chrysenteron (bull.) edge of broadleaf forest mycorrhizal boletus calopus pers. broadleaf forest mycorrhizal boletus edulis (bull.) broadleaf forest mycorrhizal boletus luridus (schaeff.) meadow;edge of the forest; mycorrhizal boletus luridiformis (rostk.) meadow; edge of the forest; mycorrhizal boletus rubellus (krombh.) broadleaf forest mycorrhizal boletus satanas rostk. edge of broadleaf forest mycorrhizal boletus subtomentosus j.f. gmel. broadleaf forest mycorrhizal leccinum carpini (r. schulz) m.m. moser ex d.a. reid mixed broadleaf forest mycorrhizal leccinum scabrum (bull.) gray mixed broadleaf forest mycorrhizal leccinum variicolor watling mixed broadleaf forest mycorrhizal xerocomus porosporus (imler ex bon & g. moreno) contu mixed forest mycorrhizal biologica nyssana 3 (2)  december 2012: 91-96 sadiković d. et al.  basidiomycetes of temska village area... 94 taxa habitat life form xerocomus chrysenteron (bull.) quélet. mixed forest mycorrhizal suillaceae suillus granulatus (l.) roussel coniferous forest mycorrhizal suillus luteus (l.) roussel coniferous forest mycorrhizal cantharellales cantharellaceae craterellus cornucopioides (pers.) broadleaf forest saprophytic cantharellus cibarius fr. broadleaf forest saprophytic cantharellus cinereus (pers.) fr. broadleaf forest saprophytic dacrymycetales dacrymycetaceae ditiola peziziformis (lév.) d.a. reid broadleaf forest saprophytic lycoperdales lycoperdonaceae calvatia excipuliformis (scop.) perdeck mixed broadleaf forest saprophytic lycoperdon echinatum schumach. broadleaf forest saprophytic lycoperdon mammiforme pers. mixed broadleaf forest saprophytic lycoperdon perlatum pers. mixed broadleaf forest saprophytic geastrales geastraceae geastrum fornicatum (huds.) hook. mixed broadleaf forest saprophytic phallales gomphaceae ramaria eumorpha (p. karst.) corner mixed broadleaf forest mycorrhizal ramaria fennica (p. karst.) ricken mixed broadleaf forest mycorrhizal phallaceae phallus hadriani vent. edge of broadleaf forest saprophytic polyporales fomitopsidaceae postia stiptica (pers.) jülich mixed forest saprophytic ganodermataceae ganoderma lucidum (curtis) p. karst broadleaf forest parasitic/ saprophytic ganoderma resinaceum boud. broadleaf forest parasitic / saprophytic polyporaceae daedalea quercina (l.) pers. broadleaf forest saprophytic fomes fomentarius (l.) fr. broadleaf forest parasitic polyporus badius jungh. broadleaf forest saprophytic polyporus leptocephalus (jacq.) fr. broadleaf forest parasitic/ saprophytic polyporus squamosus (huds.) fr. broadleaf forest parasitic/ saprophytic trametes gibbosa (pers.) fr. mixed broadleaf forest saprophytic trametes hirsuta (wulfen) lloyd broadleaf forest saprophytic trametes versicolor (l.) lloyd broadleaf forest saprophytic flammulina velutipes (curtis) singer broadleaf forest saprophytic russulales auriscalpiaceae auriscalpium vulgare gray. coniferous forest saprophytic russulaceae lactarius aurantiofulvus j. blum ex bon meadow;edge of the forest mycorrhizal biologica nyssana 3 (2)  december 2012: 91-96 sadiković d. et al.  basidiomycetes of temska village area.. 95 results the total of 110 basidiomycetes were identified during the mycological investigation of the area surrounding temska village. these species of fungi belong to 45 genera, 27 families and 11 orders and are listed in the table 1 (table 1. list of mycromycete fungi with preferred habitat type and substrate). discussion taxonomic analysis mycological survey of the area surrounding the village temska has resulted in a total of 110 fungi species belonging to 45 genera, 27 families and 11 orders. the families with the highest species diversity are: russulaceae (24 species or 21.82 % of total species number), boletaceae (15 species or 16.64 %), agaricaceae (9 species or 8.19 %) and amanitaceae (9 species or 8.19 %). regarding the species diversity, the richest genera are: russula (16 species), boletus (10 species) and lactarius (8 species). myc 48% sap 43% par 4% par/sap 5% figure 2. the proportion of groups in fungal species (myc mycorrhizal fungi, sap saprophytic fungi, par/sap fungi with parasitic and/or saprophytic ecology, par – parasitic fungi) ecological-trophic structure four trophic groups of fungi were found in the plant communities of the temska village area. analysis of the data presented showed that taxa habitat life form lactarius deliciosus (l.) gray broadleaf forest mycorrhizal lactarius evosmus kuhner & romagnesi mixed broadleaf forest mycorrhizal lactarius fulvissimus (romagn.) broadleaf forest mycorrhizal lactarius piperatus (l.) pers. broadleaf forest mycorrhizal lactarius tabidus fr. edge of broadleaf forest mycorrhizal lactarius vellereus (fr.) fr. broadleaf forest mycorrhizal lactarius zonarius (bull.) meadow; edge of the forest mycorrhizal russula aeruginea lindbl. ex fr. edge of broadleaf forest mycorrhizal russula chloroides (krombholz) bresadola broadleaf forest mycorrhizal russula cyanoxantha (schaeff.) fr. broadleaf forest mycorrhizal russula emetica (schaeff.) pers. broadleaf forest mycorrhizal russula fellea (fr.) fr. broadleaf forest mycorrhizal russula fragilis var. fallax (schaeff.) massee edge of broadleaf forest mycorrhizal russula grisea fr. broadleaf forest mycorrhizal russula heterophylla (fr.) fr. broadleaf forest mycorrhizal russula lepida fr. broadleaf forest mycorrhizal russula mairei (singer) broadleaf forest mycorrhizal russula melliolens quél. broadleaf forest mycorrhizal russula nigricans fr. broadleaf forest mycorrhizal russula sanguinea fr. broadleaf forest mycorrhizal russula turci bres. broadleaf forest mycorrhizal russula vesca fr. broadleaf forest mycorrhizal russula virescens (schaeff.) fr. broadleaf forest mycorrhizal stereaceae stereum hirsutum (willd.) pers. mixed forest parasitic thelephorales bankeraceae sarcodon imbricatus (l.) p. karst. broadleaf forest mycorrhizal biologica nyssana 3 (2)  december 2012: 91-96 sadiković d. et al.  basidiomycetes of temska village area... 96 mycorrhizal fungi represent the highest number of species (48 % of total species number), followed by saprophytic fungi (43 %), fungi with parasitic and/or saprophytic ecology (5 %) and parasitic fungi (4 %) (fig. 2). protected and endangered fungal species 16 of the species recorded in this area are included in the preliminary national red list (ivancevic 1998) : agaricus macrosporous, amanita cesarea, hygrocybe conica, mycena renati, pluteus salicinus, leucopaxillus giganteus, boletus aereus, boletus satanas, xerocomus porosporus, cantharellus cinereus, geastrum fornicatum, phallus hadriani, ganoderma resinaceum, polyporus badius, lactarius zonarius, sarcodon imbracatus. by the nature conservation law (2009) and regulation on the proclamation and protection of strictly protected and protected wild species of plants, animals and fungi (2010), 4 of recorded species are marked as strictly protected: leucopaxillus giganteus, boletus satanas, geastrum fornicatum and phallus hadriani. the following 10 species are listed as protected: amanita cesarea, marasmius oreades, boletus areus, b. edulis, craterellus cornucopioides, cantharellus cibarius, c. cinereus, lactarius deliciosus, russula cyanoxanta, r. virescens (ivancevic et al., 2012). conclusion fungi are essential parts of ecosystems and should be conserved alongside their habitats (ing 1993). in order to establish the actual fungal diversity in the temska area, more extensive, purposeful and systematic mycological surveys are needed. in the course of these studies, numerous new records both for stara planina mountain and the temska area are expected. acknowledgments. the authors want to express their gratitude to branko jotić for the determination of forest plant species in habitats from which fungal specimens were collected. references anđelković, м. (1958). geološki sastav i tektonika jugozapadnih padina stare planine. beograd : naucno delo. božac, romano. 1978: gljive naših krajeva. grafički zavod hrvatske. breitenbach, j., kränzlin, f. 1995: pilze der schweiz. band 4. verlag mykologia, luzern. 371p. dučić, v., radovanović, m., milovanović, b. 2005: temperature variability on the area of stara planlna in the instrumental period. glasnik srpskog geografskog društva 85 (2): 23–28. focht, i. 1986. ključ za gljive: ilustrirani uvod u gljivarstvo. naprijed, zagreb. index fungorum. http://www.indexfungorum.org/names/names.asp [accessed january 2013] ing, b. 1993: towards a red list of endangered european macrofungi. in: pegler, d. n., l.boddy, b. ing & p. m. kirk (eds.) fungi of europe: investigation, recording and conservation. the royal botanic gardens, kew. 322p. ivančević, b. 1998: a preliminary red list of the macromycetes of yugoslavia. conservation of fungi in europe. università degli studi, siena. pp. 57-61. ivančević, b., beronja, j. 2004: first records of macromycetes from the serbian side of stara planina mts (balkan range). mycologia balcanica vol. 1(1): 15-19. ivančević, b., matavulj, m., vukojević, j., karaman, m. 2012: fungi in the legislation of the republic of serbia. zbornik matice srpske za prirodne nauke (123): 51–64. jordan, m. 2004: the encyclopedia of fungi: of britain and europe. frances fincoln ltd., london. josifović, m., (eds)., 1970-1976: flora sr srbije, iix. sanu. beograd. rikardo, m., ileš, m., unković, s. 1995: kako da raspoznate pečurke: jestive gljive i njihovi otrovni dvojnici. evro, beograd. mcknight, k., mcknight v. 1998: a field guide to mushrooms: north america. houghton mifflin harcourt, boston. milanović, a. 2010: klima stare planine. zbornik radova geografskog instituta “jovan cvijić”, sanu, 1-136. uzelac, b. 2009: gljive srbije i zapadnog balkana. bgv logik, beograd. microsoft word bn020201 ljupkovic bojic biologica nyssana 2 (2) december 2011: 00-00 ljupković r.b. et al.. removal cu(ii) ions from water... 85 original article ! removal cu(ii) ions from water using sulphuric acid treated lagenaria vulgaris shell (cucurbitaceae) radomir b. ljupković, jelena mitrović, miljana radović, miloš kostić, danijela bojić, dragana-linda mitić-stojanović, aleksandar lj. bojić department of chemistry, faculty of sciences and mathematics, university of niš * e-mail: bojica@pmf.ni.ac.rs abstract: ljupković, r.b., mitrović, j., radović, m., kostić, m., bojić, d., mitić-stojanović, d-l., bojić, a.lj.: removal cu(ii) ions from water using sulphuric acid treated lagenaria vulgaris shell (cucurbitaceae), biologica nyssana, 2 (2), december 2011: 85-89. removal of cu(ii) ions from water solutions by sulphuric acid treated lagenaria vulgaris shell (cclvb) was studied. batch experiments were done by shaking a fixed mass of biosorbent (1.0 g) with 250 cm3 of 50.0 mg dm–3 cu(ii) solutions, at ph ranged from 2 up to 6. metal concentration in the filtrates as well as in the initial solution was determined by flame atomic absorption spectrometry. results show that efficiency of cu(ii) ions uptake by sulphuric acid treated lagenaria vulgaris shell is significantly greater than raw lagenaria vulgaris biosorbent. in addition, there is no significant effect of initial ph of solution on cu(ii) ions uptake by cclvb and obtained biosorbent can be applied in a wide range of ph. key words: biosorption, lagenaria vulgaris, cu(ii), ph, kinetics introduction ! rapid industrialization throughout the world has generated huge volumes of wastes containing toxic materials, such as heavy metals, dyes, phenols and surfactants. the presence of heavy metals in wastewaters and surface waters is major concern of the public health and the environment. the increased use of metals and chemicals in the industrial processes have resulted in the generation of large quantities of aqueous effluents that contain high levels of metals, creating serious environmental disposal problems (y u et al., 2000). the main sources of copper pollution are metal cleaning and plating baths, pulp, paper board mills, wood pulp production, the fertilizer industry, etc. (d u n d a r et al., 2009). copper usually occurs in nature as oxides and sulfides. in acidic environments, a free aqueous cu2+ ion dominates. at ph 6–8, the predominant species are cu2+, cu(oh)2, cuhco3 +, cuco3 and cuoh+, while at ph > 10 the major species are cu(oh)3 − and cu(oh)4 2−. copper may be found as a contaminant in food, especially shellfish, liver, mushroom, nuts and chocolate. copper is essential to human life and health, but in large amount it is toxic as well. excess copper in the human body can cause stomach and intestinal distress such as nausea, vomiting, diarrhea and stomach cramps (n u h o g l u et al., 2002). various methods have been used to remove heavy metals from wastewater, such as reduction and precipitation, coagulation, flotation, adsorption on activated carbon, ion exchange, reverse osmosis and electrodialysis (d a n g et al., 2009). some of these processes are expensive, or have other disadvantages, such as incomplete removal of metals, limited tolerance to ph change, moderate or no metal selectivity, dependence of metal concentration in wastewater, and production of toxic 2 (2) • december 2011: 85-89 biologica nyssana 2 (2) december 2011: 85-89 ljupković r.b. et al. removal cu(ii) ions from water… 86 sludge or other waste products that also need disposal (a n t u n e s et al., 2003). in recent years, biosorption has been suggested as cheaper as and more effective than many chemical or physico-chemical technologies. (krishnani et al., 2008) biosorption can be defined as the ability of biological materials to accumulate heavy metals from wastewater through metabolically mediated or physico-chemical pathways of uptake (u c u n et al., 2002). various biomasses are used as potential sorbents, such as fungi, bacteria, algae, agricultural waste and waste from food industries, wood sawdust and bark, in their natural form or in chemically or physically modified form (b a s s o et al., 2002). lignocellulosic fiber is an unconventional low-cost sorbent that has been examined for potential use in removing heavy metals. however, the ion exchange or adsorption capacity of lignocellulosic fiber is lower than that of other sorbents. as a result, attempts have been made to modify various lignocellulosic fibers with different chemicals (m i n et al., 2004). in this study, lagenaria vulgaris was used as a potential biosorbent for removal of cu(ii) ions from water. biosorbent was prepared by sulphuric acid treatment of plant shell to improve sorption characteristics. materials and methods preparation of biosorbent lagenaria vulgaris (cucurbitaceae family) is a creeping, hardy plant. it is found growing mainly on alluvial sandy soil and red loam, on flat areas and moderate slopes, and in higher-lying areas, and needs light and warmth. the outer shell is hard and ligneous, covering the spongy white pith characterized by its bitter taste. the experiments in this study were carried out using the shell of lagenaria vulgaris grown in the southeastern area of serbia without irrigation and fertilization, at an altitude of 200 m. raw lagenaria vulgaris shell was air dried, washed with distilled water and grounded. grounded biomass was rinsed with 0.3 m hno3 for a 24 h. acid treated material was washed with a deionised water to remove excess of acid and stirred on a magnetic stirrer with 0.1 m naoh for a 30 min. neutralized biomass was washed with deionized water and dried in a oven on 55±50c. obtained biosorbent was fractionized using standard sieves (endecott, england) and particles with size from 0.8 up to 1.25 mm are used in this study. this material is designated as alvb (m i t i c s t o j a n o v i c et al., 2011). prepared biosorbent was soaked with concentrated sulphuric acid for a 2 h. temperature of mixture was held on 40oc. after treatment, obtained black powder was washed with deionised water, and 0.1 m naoh was added in small portions until ph reached 7-7.5. then, biomass was washed with deionized water, filtered and dried in an oven on 55±5oc. result of this process is the most similar to thermal carbonization, and we call it „cold carbonization“. obtained material is designated cclvb. batch experiments the batch experiments are conducted by stirring a fixed mass of biosorbent (1.0 g) with 250 cm3 of 50.0 mg dm–3 cu(ii) into glass beaker. the initial ph of solutions was adjusted using hno3 or naoh (0.01, 0.1m) in range from 2 up to 6. the mixture was agitated for 240 min to reach equilibrium state and filtered through filter paper (wathman no2). samples (5 cm3) were taken at the beginning (before adding the sorbent) and at predetermined time intervals (1, 2, 5, 10, 20, 40, 60, 90, 120 and 240 min) for the residual cu(ii) ion concentration in the solution. to preserve aliquots, 0.1 ml of concentrated hno3 was added. metal concentration in the filtrates as well as in the initial solution was determined by flame atomic absorption spectroscopy (model aanalyst 300, perkin elmer, usa). determine of phpzc point of zero charge was determined by ph drift method (y a n g et al., 2004; a z i z et al., 2009) with some modifications for lvb. as inert electrolyte was used 0.01m kno3. the ph of test solutions were adjusted in range between 2 and 10 using 0.01m hno3 and 0.01 m koh. a 0.2 g of biosorbents were added to 50 ml of test solutions into stopped glass tubes and equilibrated for 24 h. the final ph (phf) was measured after 24 h and plotted against the initial ph (phi). the ph at which the curve crosses line phi = phf was taken as phpzc. ph was measured by sension3 (hach, usa), which was calibrated before every measure. results and disscusion effect of contact time to determine the effect of contact time on copper biosorption, 250 ml of cu(ii) ions solution, concentration 50.0 mg dm–3, was treated with 1.0 g of l. vulgaris shell biomass at 25±0.5oc and initial biologica nyssana 2 (2) december 2011: 85-89 ljupković r.b. et al. removal cu(ii) ions from water… 87 ph 5.0. the residual concentration of cu(ii) ions was determined after 1, 2, 5, 10, 20, 40, 60, 90, 120 and 240 min. results are shown on figure 1. figure 1. effect of contact time on cu(ii) ions biosorption removal (ccu(ii) = 50 mg dm–3, phi 5.0, clvb = 4.0 g dm–3, t = 25±0.5oc) results show that sorption of copper ions onto the biosorbents was very fast, as the equilibrium state was reached in less than 10 min for cclvb. at the end of the treatment, after 240 min of contact time, cu(ii) ions removal efficiency is near 100% for cclvb. sulphuric acid treatment improves sorption characteristics of l. vulgaris shell biomass, because unmodified biosorbent reaches equilibrium within about 30 min with removal efficiency near 90% at the end of treatment (240 min). pseudo-second order model was used to describe the kinetics of adsorption of cu(ii) onto cclvb. the kinetic data were analyzed using linear form of pseudo-second order relation (h o and m c k a y , 1999): t qqkq t eet 11 2 2 += where qt and qe (mg g-1) are amounts of cu(ii) ions adsorbed on cclvb at time t and at equilibrium, and k2 (min g mg-1) is the rate constant of process. as can be seen from figure 2 and table 1, straight line with correlation coefficient 0.999 show that kinetics of adsorption of cu(ii) on cclvb can be good fitted by pseudo-second order kinetic model. kinetic data for alvb are also good fitted by pseudo-second order kinetic model. in accordance with the pseudosecond order mechanism, the rate of cu(ii) sorption process appear to be controlled by chemical process or chemisorption involving valence forces through sharing or exchange of electrons between sorbent and sorbate. figure 2. pseudo-second order kinetic model for cu(ii) ions sorption on cclvb (ccu(ii) = 50 mg dm–3, phi 5.0, clvb = 4.0 g dm–3, t = 25±0.5oc) point of zero charge the ph at which the sorbent surface charge takes a zero value is defined as point of zero charge (phpzc). at this ph, the charge of the positive surface sites is equal to that of the negative ones. at ph values of solution higher than phpzc, sorbent surface is negatively charged and could better interact with metal positive species while at ph values lower than phpzc, solid surface is positively charged and could interact with negative species. table 1. pseudo-second order kinetic model parameters for cu(ii) ions sorption process on cclvb material c0 (mg dm–3) k (min g mg-1) qe,exp (mg g-1) qe,cal (mg g-1) r2 cclvb 50 0.8102 10.90 11.11 0.999 alvb 50 0.0404 7.97 7.70 0.999 biologica nyssana 2 (2) december 2011: 85-89 ljupković r.b. et al. removal cu(ii) ions from water… 88 2 4 6 8 10 2 4 6 8 10 phf phi figure 3. determination of phpzc using dirft method results in figure 3, show that phpzc of sulphuric acid treated lvb is 4.95. it is much more acidic in comparison with starting material alvb, with phpzc value 6.10. heaving in mind applied chemical treatment, increased content of acid groups in lvb structure, probably sulfonic group, is expected. effect of initial ph to determine the effect of initial ph on cu(ii) ions removal, experiments were conducted at ph 2, 3, 4, 5 and 6, adjusted with hno3 or naoh. 1.0 g of cclvb was added to 250 ml of 50 mg dm–3 cu(ii) ions solutions. the mixture was agitated for 240 min and aliquots were taken at predefined time intervals. results are shown in figure 3. 2 3 4 5 6 4 6 8 10 12 q e (m g g-1 ) ph figure 4. effect of initial ph on cu(ii) ions removal by cclvb (ccu(ii) = 50 mg dm–3, phi 5.0, clvb = 4.0 g dm–3, t = 25±0.5oc) it is well known that ph has a great importance for cation sorption by biosorbents because it influences chemical speciation of the metal ions in solution and also on the ionization of chemically active sites on the sorbent (b a s c i et al., 2004; g o y a l et al., 2008; i f t i k h a r et al., 2009; d a n g et al., 2009). in contrast, results in figure 4. shown that there is a small effect of initial ph of metal solution on sorption of cu(ii) ions onto cclvb. in ph range from 3 up to 6 almost there is no effect of ph on cu(ii) removal efficiency. only at the initial ph 2 the efficiency of cu(ii) removal is slightly smaller, for about 10%. uptake of cu(ii) by cclvb was favored by ph increasing from 2 to 3 because of different reasons, as in the case of many other biosorbents. the lower sorption of divalent metals observed at low ph values has been attributed to following factors: (a) repulsion between positive charge of the sorbent and free metal cations; (b) competition between h+ and free metal cations for the sorbent active sites; (c) lower formation of complexes with metal ions due to protonation of surface functional groups, and (d) combination of several of these factors (f i o l and v i l l a e s c u s a , 2008; a z i z et al., 2009; i f t i k h a r et al., 2009). authors m i t i ć s t o j a n o v i ć et al. (2011) were shown that metal ions sorption by untreated l. vulgaris biomass is very affected by initial ph of metal solution. they found that sorption efficiency significantly rise with increasing of ph in range from 2 to 5, with maximum at ph 5, which is closely related with obtained phpzc value of 6.10 for alvb. however, for sulphuric acid threated lvb results are not completely consistent with observed phpzc, with value 4.95 (figure 3). this could mean that mechanism of cu(ii) ions sorption by cclvb is not strictly of ionexchange type. lewis base – lewis acid interactions between sorbent surface functional groups and metal ions may play a significant role in the copper ions removal by sulphuric acid treated l. vulgaris shell biomass. results can be, also, explained by presence of acidic –so3 – groups on surface of cclvb, which cannot be protonated on investigated ph values. in addition, sulphuric acid treatment changes the physical and chemical properties of biosorbent, which results in increase of efficiency for metal ions removal. it can be supposed that conc. h2so4 initiated bond cleavage, leading to dehydration and elimination reactions that release volatile products such as water, acetic acid, methanol and other chemical substances (g e r c e l et al., 2007). this is followed by partially aromaticity and recombination of species to form a stronger cross-linked solid (k h a l e d et al., 2008). o z e r et al. (2007) supposed that sulphuric acid reacts with hydroxyl groups in the lignin to form sulphuric esters as a biologica nyssana 2 (2) december 2011: 85-89 ljupković r.b. et al. removal cu(ii) ions from water… 89 non-ionic functional group, which may complex cations. obviously, mechanism of biosorption process is rather complex and involve many different processes: chemisorption, complexation, ion exchange, chelation, physical adsorption, which do not depend of ph on the same way (s u d et al., 2008). conclusion sulphuric acid treated lagenaria vulgaris shell is found to be very efficient biosorbent for cu(ii) ions removal from water. biosorption process was very fast, reaching maximum efficiency (almost 100% of cu(ii) ions was removed from solution) in the first 10 minutes. sorption kinetic was found to be best-fitted pseudo-second order model, which indicates that process is mostly based on chemisorption. biosorption on cclvb is not significantly dependent of initial ph and this material could be used for treatment of acidic wastewater loaded with copper. acknowledgements. authors would like to acknowledge for financial support to the ministry of education and science of the republic of serbia (project tr34008). references antunes, w.m., luna, a.s., henriques, c.a., da costa, a.c.a., 2003: an evaluation of copper biosorption by a brown seaweed under optimized conditiones, electronic journal of biotechnology, 6: 174-184. aziz, a., ouali, m.s., elandaloussi, e.h., de menorval, l.c. lindheimer, m., 2009: chemicaly modified olive stone: a low cost sorbent for heavy metals and basic dyes removal from aqueous solutions, journal of hazardous materials, 163: 441-447. basci, n., kocadagistan, e., kocadagistan, b., 2004: biosorptiion of copper (ii) from aqueous solutions by wheat shell, desalination, 164: 135-140. basso, m.c., gerrela, e.g., cukierman, a.l., 2002: lignocellulosic materials as a potential biosorbents of trace toxic metals from wastewater, industrial and 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1040-1045. yang, y., chun, y., sheng, g., huang, m., 2004: phdependence of pesticide adsorption by wheat-residuederived black carbon, langmuir, 20: 6736-6741. anticancer compounds from medicinal plants biologica nyssana 3 (2)  december 2012: 53-60 mitrović, t. et al.  bioindication of heavy metal pollution … 53 original article bioindication of heavy metal pollution in the area of southeastern serbia by using epiphytic lichen flavoparmelia caperata (l.) hale tatjana mitrović 1 , slaviša stamenković 1 , vladimir cvetković 1 *, tatjana đekić 2 , rada baošić 3 , jelena mutić 3 , tatjana anđelković 4 , aleksandar bojić 4 1 university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia 2 university of niš, faculty of sciences and mathematics, department of geography, višegradska 33, 18000 niš, serbia 3 university of belgrade, faculty of chemistry, studentski trg 12-16, 11158 belgrade, serbia 4 university of niš, faculty of sciences and mathematics, department of chemistry, višegradska 33, 18000 niš, serbia * e-mail: biovlada@yahoo.com abstract: mitrović, t., stamenković, s., cvetković, v., đekić, t., baošić, r., mutić, j., anđelković, t., bojić, a.: bioindication of heavy metal pollution in the area of southeastern serbia by using epiphytic lichen flavoparmelia caperata (l.) hale. biologica nyssana, 3 (2), december 2012: 53-60. the content and distribution of 21 metals in the central and peripheral parts of the foliose epiphytic lichen flavoparmelia caperata (l.) hale, collected in the area of southeastern serbia, were analysed in terms of biological monitoring. inductively coupled plasma atomic emission spectrometry revealed higher concentrations of as, b, ba, cd, ga, pb, se, cr, cu, fe, in, li and/or ni in peripheral, younger, parts and ba, k, tl, mg, na and/or zn in central, older parts of lichens. principal component analysis and hierarchical cluster analysis were used to identify the relationship among metals in samples and their possible sources. significant correlations were found among ni-cr, cd-ga-in-as-se, zn-ba, cu-pb-b, suggesting a common source of pollution. given the location of sampling, these findings probably reflect airborne metal pollution in relation to the main wind directions and vicinity of the roads and industrial complexes. the importance of this study is the evidence that the special nature reserve jelašnička gorge is influenced by pollution sources in the area. flavoparmelia caperata could be effective as an early indicator of environmental changes of the studied area. key words: air pollution, bioindication, flavoparmelia caperata, heavy metals, lichens introduction lichens have been the most widely accepted sensors of enviromental pollution since the 19th century (g r i n d o n , 1859). lack of waxy cuticle and associated stomatas, parenchymal nature, slow growth and long duration of lichens result in the absorption of pollutants (metals, nonmetals, radionuclides, etc) across entire thallus surface and thus allow long-term biomonitoring of persistent pollutants in the ecosystem. lichens depend on mineral nutrients in the form of soluble salts and particles from wet atmospheric deposition (precipitation and occult precipitation, like fog and dew) and dry atmospheric deposition (sedimentation, impaction and gaseous absorption). they are very efficient accumulators of heavy metals by: ion exchange at specific cation exchange 3 (2) • december 2012: 53-60 biologica nyssana 3 (2)  december 2012: 53-60 mitrović, t. et al.  bioindication of heavy metal pollution … 54 sites, intracellular uptake of metal solution, and entrapment of airborne, metal-rich particles (size 0.5 to 1.0 μm) (n a s h i i i , 2008). lichens are capable of accumulating heavy metals to a concentration that vastly exceeds their physiological requirements by sequestering them extracellulary as insoluble oxalates or lichen acid complexes (b e e b y , 2001). the presence of particulate materials on the lichen surface which are trapped between the hyphae of medulla is shown by electron microprobe studies (b a r g a g l i , 1998). metal accumulation is in correlation with the environmental levels of particulate materials (b a r i et al., 2001; b a r g a g l i et al., 2002 ; l o p p i et al., 2004) and spatialand/or temporaldeposition patterns of trace metals are demonstrated (z s c h a u et al., 2003). the concentration of sequestered trace metals in lichen really reflects ambient level of airborn trace metals in terms that after the withdrawal of the source of contamination from the ecosystem (closures of industries, iron-steel factories, mines, waste treatment plants, roads and railroads, etc) a dramatic decrease of trace metals concentration in lichen thalli is observed (w a l t h e r et al., 1990). n i m i s et al. (2001) discussed the importance of intraand interspecific variability in metal accumulation and relationship between metal content and the age of the thalli. in this study we investigated heavy metal pollution using flavoparmelia caperata (l.) hale as bioindicator and bioaccumulator. the study was performed at 3 different locations: northwest (near the village of cerje), southeast (near the village of vlase) and northeast (jelašnička gorge) of the biggest urban and industrial center of southeastern serbia niš (approximately 350 000 inhabitants). we would like to emphasize the importance of one location jelašnička gorge. this area, situated 15 km away from niš and 3 km away from niška spa, is a protected natural area having the status of a special nature reserve. one of the reasons to investigate this part of serbia is the fact that a similar study had never been undertaken previously, although the area is densely populated with large urban-industrial infrastructure, which thus has an important anthropogenic impact on surrounding nature and ecosystems. lichen samples were analyzed for metal content. composition data have been treated with figure 1. study area (jelašnica gorge, vlase and cerje in respect to the city of niš) withwind roses for the area of niš (data from republic hydrometeorological service of serbia) biologica nyssana 3 (2)  december 2012: 53-60 mitrović, t. et al.  bioindication of heavy metal pollution … 55 various statistical multivariate techniques, primarily principal component analysis and cluster analysis. material and methods study area the study was performed in the subrural and rural area of southeastern serbia. the collection sites were: jelašnička gorge (330 m altitude), vlase (350 m altitude) and cerje (600 m altitude) in the vicinity of roadside (2m, 500 m and 2000 m, respectively) (figure 1). the nearest urban and industrial area is the city of niš (approximately 350 000 inhabitants) (figure 1). the climate is moderate continental with mean annual rainfall ranging over 543.3 mm, a mean temperature of 11.5 °c, and a mean annual relative humidity of approximately 69 %. prevailing winds are northwesterly in winter and northeasterly and easterly in summer. wind roses are shown in figure 1. lichen material foliose lichen flavoparmelia caperata (l.) hale (syn. parmelia caperata (l.) ach.; common name: greenshield lichen) was collected in april 2009. lichen specimens were obtained from a height of 1.5-2 m above the ground, at the side of trunks of prunus domestica and salix sp. not affected by stemflow. the material from each location was sorted into two samples corresponding to peripheral and central parts of lichen. the samples were air-dried, grinded, homogenized and further analyzed. the determination of lichens was performed by using several standard keys (boqueras, 2000; dobson, 2005; wirth, 1995). lichen samples were deposited in the lichenological herbarium of the department of biology and ecology, faculty of sciences and mathematics, university of niš. reagents and chemicals all chemicals were of analytical grade and were supplied by merck (darmstadt, germany). all glassware was soaked in 4 mol/l hno3 for a minimum of 12 hours and rinsed well with distilled water. ultra-pure water was prepared by passing doubly de-ionized water through milli-q system (millipore simplicity 185 system incorporating dual uv filters (185 and 254 nm) to remove carbon contamination). multi-element stock solution containing 1.000 g/l of each elements was used to prepare intermediate multi-element standard solutions. sodium borhydride solutions were stabilized with sodium hydroxide (merck, germany) for hydride generation. table 1. instrument operating conditions for determination of heavy metals in lichen samples spectrometar icap 6500 (thermo scientific) nebulizer concentric spray chamber cyclonic radio frequency power (w) 1150 principal argon flow rate (l/min) 12 auxiliary argon flow rate (l/min) 0.5 nebulizer flow rate (l/min) 0.5 sample flow rate (ml/min) 1.0 detector cid86 table 2. selected emission lines element λ [nm] ag 328.0 as 193.7 b 249.6 ba 455.4 cd 228.8 co 228.6 cr 283.5 cu 324.7 fe 259.9 ga 294.3 in 230.6 k 766.4 li 670.7 mg 280.2 mn 257.8 na 588.9 ni 231.6 pb 220.3 sr 215.0 tl 276.7 zn 213.8 hg 253.6 se 196.0 instrumentation all the measurements were made with a inductively coupled atomic emission spectrometer model 6500 duo (thermo scientific, united kingdom) equipped with a cid86 chip detector. the system is equipped with an integrated unit for biologica nyssana 3 (2)  december 2012: 53-60 mitrović, t. et al.  bioindication of heavy metal pollution … 56 hydride generation. this instrument operates sequentially with both radial and axial torch configurations. the entire system is controlled by iteva software. instrument operating conditions for the determination of heavy metals in lichen samples and selected emission lines are shown in table 1 and table 2, respectively. microwave digestion was performed in a pressurized microwave (ethos 1, advanced microwave digestion system, milestone, italy) equipped with a rotor holding 10 polytetrafluoroethylene (ptfe) cuvettes. procedure for microwave digestion samples (0.5 g) were transfered into ptfe cuvette, 7 ml of concentrated hno3 and 1 ml 30% h2o2 were added. digestion was performed under the following programme: warmed up for 10 mins to 200° c and held for 10 mins at that temperature. after the cool off period samples were quantitatively transferred into a volumetric flask (25 ml). statistical analysis to identify the relationship among metals in samples and their possible sources, pearson’s correlation coefficient analysis, principal component analysis (pca) and cluster analysis (ca) were performed using pls toolbox version 5.2.2 (eigenvector research) for the matlab version 7.4.0.287 (r2007a) (mathworks, natick, ma, usa). the principal component analysis was performed using varimax normalized rotation. cluster analysis was performed on the data sets using the between-groups linkage based on correlation coefficients (pearson coefficient) by pair-wise deletion. normalized data set was analyzed by ward’s method using squared euclidean distances as a measure of similarity between metal concentrations. results and discussion the concentrations of 21 metals in lichen flavoparmelia caperata, collected at 3 different localities in southeastern serbia (jelašnička gorge, cerje and vlase), are given in table 3. the determination of metals was performed on both central and peripheral parts of lichens. central parts are inner, older parts of thalli and they have therefore been exposed to pollutants longer. peripheral parts comprise outmost 3-4 mm of the thalli, with highest physiological activity and the maximum age of 1 year (fisher & proctor, 1978; loppi et al., 1997). thus, peripheral parts represent recent changes of the environment. our data indicated differences in the metal content of central and peripheral parts of lichens. central parts tend to give higher concentrations of a number of metals as table 3. concentration of metals in central and peripheral parts of the thalli of flavoparmelia caperata ( g/g dry wt.) elements flavoparmelia caperata from jelašnička gorge flavoparmelia caperata from cerje flavoparmelia caperata from vlase center (sample 1) perifery (sample 2) center (sample 3) perifery (sample 4) center (sample 5) perifery (sample 6) as 0.0037 0.0038 0.0036 0.0033 0.0031 0.0027 b 19.4303 9.5051 6.1341 6.9682 5.8576 6.7637 ba 27.2333 21.5575 9.5615 24.5084 13.8145 15.2969 cd 0.2558 0.1942 0.1681 0.1864 0.1310 0.1546 co 0.1512 0.0822 1.0894 2.1865 0.0970 0.0856 cr 1.9119 1.4572 3.7140 1.4670 1.7550 1.4879 cu 8.3806 5.9573 15.3721 5.7307 6.1412 6.5179 fe 476.6209 374.5863 645.2168 397.9597 493.2096 399.2086 ga 0.1069 0.0000 0.0413 0.0000 0.0346 0.0177 in 0.0000 0.0000 0.0802 0.0341 0.0810 0.0000 k 2608.8040 2852.2720 2300.1670 2706.2540 2506.7160 3156.7050 li 0.6932 0.5035 0.7566 0.4607 0.5617 0.4582 mg 251.8673 315.6654 274.1127 313.7536 287.8818 373.2304 mn 14.1271 13.5242 13.6761 12.4049 13.9091 15.6287 na 71.1085 98.1346 89.5684 103.6953 78.8976 110.4994 ni 1.2703 1.0977 1.5682 1.1916 1.4227 1.3011 pb 22.3011 10.0742 12.0318 7.3955 9.8771 7.4223 se 0.0043 0.0045 0.0042 0.0039 0.0036 0.0031 sr 31.4610 23.8918 16.8445 11.3996 40.6104 32.6951 tl 0.0000 0.6093 0.0000 0.9164 0.0000 1.5415 zn 17.6053 17.0019 18.0832 19.5534 20.7641 24.2578 biologica nyssana 3 (2)  december 2012: 53-60 mitrović, t. et al.  bioindication of heavy metal pollution … 57 previously reported in the studies of nimis et al. (2001). pearson’s correlation coefficients for metals in lichen samples are shown in table 4. determined correlations between metals provided interesting information on the sources and pathways of the metals. the correlation matrix was created from the values of the variables of all 21 metals in 6 samples. the pair-wise method was employed for the missing values. the results showed that these metals were strongly interrelated (p<0.01), with correction coefficients ranging from -0.790 to 0.990 at the 99% confidence level. b, cd, ga and pb evidently displayed significant positive correlations with each other (see: table 3), which indicates their association in analyzed samples. other metals, like cr, cu, fe, li and ni, showed significant correlations, too. the exceptions were elemental pairs as-zn and se-zn with significant negative correlations and as, mn and zn without any correlations observed. in order to better describe the relationship among metals and/or samples, principal component analysis (pca) was performed. analytical data were represented in a multidimensional space with variables defining the axes, and projected into a few principal components (pcs) that were linear combinations of the original variables and described the maximum variation within the data (brereton, 2003). obtained results clearly showed that the co, mn and sr had no impact. this was in accordance with results of metals determination (table 3) due to small differences in theirs values in different samples. therefore, pca analysis was done without these metals resulting in a three-component model explaining 91.78% of the data variation. the first pc comprised 49.75% of the total data variability, and the cumulative variance explained by the first two components was 81.09%. the addition of more pcs did not significantly change the classification of the analytes described below. score values for the samples, i.e. their mutual projections, for the first two pcs are shown in figure 2. additionally, score values for the samples and metals, for the first two pcs are shown in figure 3. the first pc distinguished two separate groups of samples according to metals content and age of parts of the lichen thalli sampled. samples 1, 3 and 5 are central (older) parts of the thalli from different areas while 2, 4 and 6 are peripheral (younger) parts of the thalli. the first pc distinguished two separate groups with characteristic patterns of metals accumulation (samples 2, 4 and 6 with ba, k, mg, na, tl and zn figure 2. score values of the first and the second pcs for the samples additionally, score values for the samples and metals, for the first two pcs are shown in figure 3. figure 3. biplot of the first and the second pcs for the samples and metals and samples 1, 3 and 5 with as, b, cd, cr, cu, fe, ga, in, li, ni, pb and se). these results clearly defined the highest accumulated metals characteristic for every sample. the following metals were characteristic of sample 1: as, b, cd, ga, pb and se. higher concentration of ba, k and tl were noticed in samples 2 and 4. besides, ba was located on the border that defined a pc and it appeared characteristic of sample 1, too. the accumulation of cr, cu, fe, in, li and ni were characteristic for samples 3 and 5. increased concentrations of mg, na and zn were observed in sample 6. compared to location of sampling, it biologica nyssana 3 (2)  december 2012: 53-60 mitrović, t. et al.  bioindication of heavy metal pollution … 58 biologica nyssana 3 (2)  december 2012: 53-60 mitrović, t. et al.  bioindication of heavy metal pollution … 59 seems that the special nature reserve jelašnička gorge from which sample 1 and 2 originated, is becoming polluted due to vicinity of the road (2m) and towns (niš and niška spa, 15 km and 3 km, respectively). evidence for this could be found in the increased concentration of cd, pb and se as the levels of these elements are similar to literature values for high-density traffic areas (mendil et al., 2009). the other two locations of lichen sampling, cerje and vlase, with samples 3-6, showed richness in litophile elements and macronutrients such as: cr, cu, fe, in, k, li, mg and na. this could imply a specific composition of geological substrate, as well as the influence of vegetation, i.e. substrate from which lichen samples were collected. moreover, there is a certain amount of anthropogenic pressure and it is manifested by pollution originating from traffic origin. finally, metal concentration data were submitted to cluster analysis (ca) using correlation coefficient as similarity measure and weighted pair group as clustering algorithm. this method is the most appropriate for the validation of the correlation between variables and is often used in environmental studies. the obtained dendrogram is shown in figure 4. figure 4. dendrogram derived from the hierarchical cluster analysis of samples nevertheless the cluster analysis organized the metals (without macroconstituents) in such a way that within-group similarity was maximized and among-group similarity was minimized. the ca results for the heavy metals studied are shown in figure 5 as a dendrogram. figure 5 displays four clusters: (1) ni-cr; (2) cd-ga-in-as-se; (3) zn-ba; (4) cu-pb-b in agreement with the pca results. it is observed, however, that clusters 1 and 2 as well as clusters 3 and 4 join together at a relatively higher level, possibly implying a common source. fossil fuel emits ni, cd, cr, cu, pb, zn during combustion (aslan et al., 2011). besides, the wear of car tires, degradation of parts, peeling paints and greases and metals in catalysts could be sources of pollutants (pecheyran et al., 2000). although vehicles with unleaded petrol prevail, the high density traffic of old vehicles using leaded petrol and diesel oil is still present in serbian roads. zn, as well as cd, is well known as an atmophile element subject to longdistance transport (loppi & pirintsos, 2003). a part of zn could be obtained from supporting trees since higher plants are known to release 20 % of total zn coming from natural sources (nriagu, 1979). also, flavoparmelia caperata is known for its higher capacity for zn and cd uptake (nimis et al., 2001). figure 5. dendrogram derived from the hierarchical cluster analysis of metals content interestingly, cluster 4 indicates pollution caused by exhaust gases due to traffic, but having in mind the poor frequency of traffic at the roadsides in close range of the location of lichen sampling, one could wonder about the origin of cu, pb and b detected. this is probably due to the specific wind roses (see figure 1) which put this area under the indirect impact of metal industry, situated 10-15 km northwesterly (in the outskirt of the city of niš). further research and monitoring should be performed in order to obtain the right conclusions. conclusion our study confirms the metal accumulation capacity of lichen flavoparmelia caperata and its potential for biomonitoring. this lichen species could be effective as an early warning system for detecting changes in the environment. peripheral parts of lichen samples allow annual changes to be detected. detected accumulation of heavy metals in lichen samples is probably a result of the frequency of traffic and types of engines on the roads in this biologica nyssana 3 (2)  december 2012: 53-60 mitrović, t. et al.  bioindication of heavy metal pollution … 60 area, the activity of industrial complexes in and around the city of niš, soil and substrate compositions and predominant wind directions. further research and biomonitoring surveys of air pollution in the studied area should contribute to the amelioration of air quality and environment in southeastern serbia. acknowledgment. this research was supported by the ministry of science and education of the republic serbia during activities on the projects iii41018, oi 171025 and tr 34008. references aslan, a., çiçek, a., yazici, k., karagöz, y., turan, m., akku, f., yildirim, o.s. 2011: the assessment of lichens as bioindicator of heavy metal pollution from motor vehicles activites. african journal of agricultural research, 6 (7): 1698-1706. bargagli, r. 1998: trace elements in terrestrial plants: an ecophysiological approach to biomonitoring and biorecovery. springer verlag. berlin. bargagli, r., monaci, f., borghini, f., bravi, f., agnorelli, c. 2002: mosses and lichens as biomonitors of trace metals. a comparison study on hypnum cupressiforme and parmelia caperata in a former mining district in italy. environmental pollution, 116 (2): 279-287. bari, a., rosso, a., minciardi, m.r., troiani, f., piervittori, r. 2001: analysis of heavy metals in atmospheric particulates in relation to their bioaccumulation in explanted pseudevernia furfuracea thalli. environmental monitoring and assessment, 69 (3): 205-220. beeby, a. 2001: what do sentinels stand for? environmental pollution, 112 (2): 285-298. boqueras, m. 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(1): 27-32. walther, d.a., ramelow, g.j., beck, j.n., young, j.c., callahan, j.d., marcon, m.f. 1990: temporal changes in metal levels of the lichens parmatrema praesoredious and ramalina stenospora southwest louisiana. water, air and soil pollution, 53: 189-200. wirth, v. 1995: die flechten baden-württembergs, teil 1 & 2. eugen ulmer & co. gmbh. stuttgart. zschau, t., getty, s., gries, c., ameron, y., zambrano, a., nash, t. 2003: historical and current atmospheric deposition to the epilithic lichen xanthoparmelia in maricopa county, arizona. environmental pollution, 125 (1): 2130. rajicic et al. 2021, biologica nyssana 12(2) 12 (2) december 2021: 141-150 doi: 10.5281/zenodo.5759859 yield components and genetic potential of winter wheat on pseudogley soil of western serbia original article vera rajičić university of niš, faculty of agriculture, kosančićeva 4, 37000 kruševac, republic of serbia verarajicic@yahoo.com (corresponding author) dragan terzić university of niš, faculty of agriculture, kosančićeva 4, 37000 kruševac, republic of serbia violeta babić university of niš, faculty of agriculture, kosančićeva 4, 37000 kruševac, republic of serbia vesna perišić university of niš, faculty of agriculture, kosančićeva 4, 37000 kruševac, republic of serbia marijana dugalić university of niš, faculty of agriculture, kosančićeva 4, 37000 kruševac, republic of serbia dragoslav đokić university of niš, faculty of agriculture, kosančićeva 4, 37000 kruševac, republic of serbia snežana branković university of kragujevac, faculty of science, department of biology and ecology, radoja domanovića 12, kragujevac, republic of serbia received: july 09, 2021 revised: november 19, 2021 accepted: december 02, 2021 abstract: in order to determine the effect of genotype and vegetation season on the yield and components of the winter wheat yield an experiment was performed in the secondary agricultural-chemical school “dr đorđe radić” in kraljevo. five wheat varieties (kruna, renesansa, pobeda, ns 40s and takovčanka) were examined during two growing seasons in the agro-ecological conditions of western serbia. trials were arranged according to a randomized scheme in five repetitions. grain yield (gy), 1000 grain weight (tgw) and test weight (tw) in winter wheat grain were analyzed. the study was performed as an experiment based on extremely acidic pseudogley soil. on average, for all wheat varieties, during the two-year trial, the highest yield of winter wheat 4.673 t/ha was obtained in the variety renesansa. the renesansa variety showed the highest yield and 1000 grain weight. the highest 1000 grain weight had been established for wheat cultivar renesansa (43.72 g). the highest two-year average value of test weight was found in the varieties kruna and pobeda (77.52 kg/hl and 77.31 kg/hl). analysis of variance revealed a highly significant effect of the growing season on the yield and test weight of wheat. a highly significant effect of genotype on 1000 grain weight and test weight was determined. key words: yield components, grain yield, wheat apstrakt: komponente prinosa i genetski potencijal ozime pšenice na pseudoglejnom zemljištu zapadne srbije u cilju utvrđivanja uticaja genotipa i vegetacione sezone na prinos i komponente prinosa ozime pšenice izveden je ogled u srednjoj poljoprivrednohemijskoj školi „dr đorđe radić“ u kraljevu. ispitivano je pet sorti pšenice (kruna, renesansa, pobeda, ns 40s i takovčanka) tokom dve vegetacione sezone u agroekološkim uslovima zapadne srbije. ogledi su postavljeni po slučajnom blok sistemu u pet ponavljanja. analizirani su prinos zrna (pz), masa 1000 zrna (mhz) i hektolitarska masa (hm) u zrnu ozime pšenice. ogled je zasnovan na izuzetno kiselom zemljištu tipa pseudoglej. u proseku, za sve ispitivane sorte pšenice, tokom dvogodišnjeg ogleda, najveći prinos ozime pšenice od 4.673 t/ha dobijen je kod sorte renesansa. najveći prinos i masa 1000 zrna ustanovljena je kod sorte renesansa. najveća masa 1000 zrna ustanovljena je kod sorte pšenice renesansa (43.72 g). najveća dvogodišnja prosečna vrednost hektolitarske mase utvrđena je kod sorti kruna i pobeda (77.52 kg/hl i 77.31 kg/hl). analiza varijanse pokazala je veoma značajan uticaj vegetacije na prinos i hektolitarsku masu pšenice. utvrđen je visoko značajan uticaj genotipa na masu 1000 zrna i hektolitarsku masu. ključne reči: komponente prinosa, prinos zrna, pšenica introduction winter wheat (triticum aestivum l.) is one of the most important field crops in world and republic of serbia. world’s current average wheat production in tested period (2017-2018), was 752.841.037 tons and in republic of serbia 2.608.612 tons. the average area under wheat in the world in tested period was 216.141 mill. ha and in republic of serbia was 599.599 ha. the average wheat yield in the world was 3.483 t/ha and in republic of serbia was 4.333 t/ha (fao, 2021). the average yield of wheat in 2017 in the world was 3.538 t/ha and in the republic of serbia 4.092 t/ha. in 2018, the average yield of wheat in the world was 3.427 t/ha, and in the republic of serbia 4.574 t/ha (fao, 2021). © 2021 rajičić et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 141 wheat production in serbia depends to a large extent on the environmental factors. the grain yield and yield components of winter wheat vary considerably depending on the cultivation system, the applied doses of nitrogen, variety and conditions during the growing year, as well as their complex interactions (jaćimović et al., 2011; đekić et al., 2014; jelic et al., 2015; terzić et al., 2018; đekić et al., 2019a; popović et al., 2020; ljubičić et al., 2021). the interaction between the genotype and environment greatly limits the effectiveness of selection, if it is performed only on the basis of the average yield (dimitrijević et al., 2011; laghari et al., 2011; perišić et al., 2011; milovanović et al., 2012; đekić et al., 2013; hristov et al., 2014; jocković et al., 2014; đurić et al., 2016; terzić et al., 2018). varieties that have a smaller contribution to interaction are less sensitive to changing environmental conditions, such varieties are stable. genetic variation plays a significant role in the adaptability of wheat to stress caused by external environment (mladenov et al., 2007; milovanović et al., 2011; luković et al., 2014; đurić et al., 2018; rajičić et al., 2020). understanding the genetic basis of adaptation and its physiological causes is important for understanding the interactions, leading to more efficient choices of superior and stable genotypes (perišić, 2016; djuric et al., 2018; luković et al., 2020). the yield per unit area is the result of the action of factors of variety fertility in interaction with environmental factors. the main goal in breeding wheat is to create varieties with high yield potential and good grain quality suitable for animal feed or brewing industry (milovanović et al., 2011; 2012; hristov et al., 2014; perišić, 2016; đurić et al., 2018). improved production technology has contributed to the increase in yields in recent decades, because improved production technologies enable better realization of the potential for yield. high and stable yield of wheat is the main goal of breeding, especially in institutions where it is not possible to adequately test the breeding material on quality traits. yield increase is the result of improving germplasm and improvement of production technology (đurić et al., 2018). the need for a variety of short or long growing season depends on the environmental conditions of a particular area. in winter wheat often variations in yield can be detected, also between the years and between locations (đekić et al., 2015; đurić et al., 2018; jevtić and đekić, 2018; terzic et al., 2018; luković et al., 2020). the 1000 grain weight and test weight are direct components of grain yield and change under the influence of environmental factors, but primarily varietal characteristics. they indicate the grain size and are important criterions in breeding wheat. the 1000 grain weight in addition to the number of grains per spikes is the most important criterion in breeding wheat for yield increase (perišić, 2016). production of winter wheat with high grain yield and appropriate quality is possible only by choosing varieties of good quality with appropriate cultivation conditions and appropriate production technology. the aim of this study was the determination of the varieties and ecological environmental factors influence on differences in stability and adaptability of varieties regard the grain yield, 1000 grain weight and test weight of tested winter wheat varieties. during the vegetation years (2016-2018), in the field trials, five winter wheat varieties were examined in order to determine the selection of the better varieties for the production conditions in western serbia. materials and methods materials and field trials field experiments were performed on the experimental field of the secondary agriculturalchemical school “dr. đorđe radić” in kraljevo (43°43´00´´n, 20°40´60´´e, 198 m above sea level) during two growing seasons (2016/17 and 2017/18) in dry crop conditions, with the aim of analysing the yield and quality of winter wheat grains in western serbia. the experiments were conducted in randomized block systems, with a plot size of 50 m2 (5 m x 10 m) in five replications. wheat has been preceded by maize in all seasons. the basic tillage was done classically, up to 25 cm deep. the sowing was carried out using a machine with row spacing of 12 cm. the soil on which the trial was conducted was uniform and well prepared. the amount of seed per square meter amounted to 400-450 viable seeds, depending on the characteristics of varieties. it was sown in the third decade of october, with 300 kg/ha of fertilizer npk 15:15:15, which was added in the fall, while during the spring fertilization soil was carried out in 2-3 leaf stage was supplemented with 300 kg/ha (can-calcium-ammonium-nitrate, 27% n). the winter wheat varieties kruna and takovčanka originated from the centre for small grains in kragujevac and renesansa, pobeda and ns 40s originated from the institute of field and vegetable crops, novi sad were selected as the material for the experiment. the following properties were analyzed: grain yield (t/ha), 1000 grain weight (g) and test weight (kg/hl). grain yield was measured for each plot and converted to grain yield in t/ha based on 14% grain moisture, after which a sample was taken for analysis of 1000 grain weight and test weight of the wheat. 142 biologica nyssana ● 12 (2) december 2021: 141-150 rajičić et al. ● yield components and genetic potential of winter wheat on pseudogley soil of western serbia 143 soil conditions the trial at the secondary agricultural-chemical school “dr. đorđe radić” in kraljevo, was performed on soil that is characterized as pseudogley. the trial was performed on pseudogley soil, of unfavourable physical characteristics, of poor water-air regime with frequent water or air deficiencies. this soil has very bad physical properties (compacted, having a high content of silt and clay particles, with slow water percolation) and an extremely acid ph value (ph<4.5). its total humus content is relatively fair, but microbiological activity is low, because of its poor physical properties. it is characterized by low content of available phosphorus (6.70-6.90 mg/100g of soil) and potassium (7.80-9.80 mg/100g of soil). meteorological conditions this study was conducted over a two-year period in the kraljevo, located in the čačak-kraljevo valley that belongs to the western morava river region (tab. 1). it is surrounded by a large number of mountain ranges and their hills through which the wide river valleys of the west morava, the lower ibar and the lower gruža intersect. it is located at 20°40´60´´e longitude and 43°43´00´´n latitude. the soil of the studied area is in a zone of temperate continental climate, with uneven distribution of precipitation by months. based on the data of meteorological stations in kraljevo, in the years in which the researches were carried out, the conditions differed from the long-term average characteristic of the area. the data in tab. 1 for the investigated period (2016-2018) clearly indicate that the average air temperature was higher than the annual average by 0.8 ºc in 2017/18 and lower by 0.4 ºc in 2016/17 (tab. 1). average monthly air temperatures in 2016/17 were slightly lower, especially in january (-5.0 оc) than in 2017/18. however, in both years, temperatures were within optimal limits and had no negative effects on yield. the data presented in tab. 1 the wheat growing season analyzed (2016-2018) clearly suggest differences in weather conditions between the years of the study and the long-term mean for the region. the total amount of precipitation in the vegetation in 2016/17 was 524.4 mm and 758.7 mm in 2017/18. the total amounts of rainfall were above the perennial average in the surveyed growing seasons (2017/18), with a rather uneven monthly distribution. weather conditions in the growing period in 2017/18 were marked by high rainfall during march, while rainfall in april was significantly lower. variable and moderately warm weather, with less precipitation than average, marked may 2016/17 and 2017/18 (tab. 1). during the run of the experiment, the differences between the mean precipitation values and the perennial average in the second years of the study were the highest in march and june. higher precipitation and their better distribution in 2017/18, especially in october, march and june, contributed to better fertilizer decomposition and higher wheat grain yield. statistical analysis on the basis of achieved research results the usual variational statistical indicators were calculated: average values. experimental data were analyzed by descriptive and analytical statistics using the statistics module analyst program genstat (2013) for pc/windows 7. all evaluations of significance were made on the basis of the anova test at 5% and 1% significance levels. relative dependence was defined through correlation analysis (pearson’s correlation coefficient), and the coefficients that were obtained were tested at the 5% and 1% levels of significance. biologica nyssana ● 12 (2) december 2021: 141-150 rajičić et al. ● yield components and genetic potential of winter wheat on pseudogley soil of western serbia table 1. mean monthly air temperatures and precipitations in kraljevo, serbia (2016-2018), in relation to many years average months interval x xi xii i ii iii iv v vi average mean monthly air temperature (oc) 2016/17 7.6 6.0 5.8 -5.0 4.5 10.3 11.3 16.2 22.4 8.8 2017/18 11.9 6.8 4.0 2.7 2.0 6.5 16.6 19.1 20.9 10.0 average 11.6 7.7 2.0 0.6 3.3 7.9 12.5 16.3 20.9 9.2 the amount of precipitation (mm) 2016/17 84.1 77.6 9.4 22.1 35.3 57.7 82.1 99.9 56.2 524.4 2017/18 133.3 32.4 55.6 51.0 80.9 111.2 40.6 84.4 169.3 758.7 average 73.4 37.8 56.0 48.6 51.5 80.8 80.3 112.1 82.4 622.9 144 results grain yield and yield components the average values of productive grain yield and yield components of the investigated winter wheat varieties grown in the secondary agricultural-chemical school “dr. đorđe radić” in kraljevo are shown in tab. 2. grain yield: in regard to grain yield, differences were established in the tested varieties and growing seasons. the variety with the lowest average yield in all years was variety renesansa (4.673 t/ha). the highest average grain yield in the first year of testing was recorded for the variety renesansa (4.273 t/ha) and the lowest for the variety ns 40s (3.894 t/ha). the average grain yield of all varieties in 2016/17 was 4.077 t/ha. in the second year of testing, the highest yield of all varieties was achieved, also in this year, with the variety renesansa (5.073 t/ha). average yield of winter wheat for all varieties in 2017/18 was 4.639 t/ha and the average grain yield was 4.358 t/ha. the highest average grain yield in all years was the variety renesansa (4.673 t/ha) and the slightly lower yield the varieties takovčanka and pobeda (4.386 t/ha and 4.379 t/ha). 1000 grain weight: average values for 1000 grain weight of all studied varieties are presented in tab. 2. the average of 1000 grain weight of all tested varieties and years of winter wheat was 40.38 g. the variety ns 40s had the lowest 1000 grain weight for all years (34.68 g) and the highest 1000 grain weight the variety renesansa (43.72 g). in 2016/17, the average of 1000 grain weight of all varieties was 39.48 g and in 2017/18 was 41.28 g. the growing period in 2016/17 at the time of grain loading was marked by drought and high temperatures, which reduced the weight of 1000 grains. test weight: the average value for all varieties and years was 76.02 kg/hl (tab. 2). the lowest test weight grain of winter wheat at two years was recorded for the variety ns 40s (74.01 kg/hl), while the highest value for the varieties pobeda and kruna (77.31 kg/hl and 77.52 kg/hl). the average value of test weight of grain for all varieties was significantly higher in 2017/18 (77.26 kg/hl) compared to 2016/17 (74.78 kg/hl). regardless of the year, varibiologica nyssana ● 12 (2) december 2021: 141-150 rajičić et al. ● yield components and genetic potential of winter wheat on pseudogley soil of western serbia table 2. average values of investigated winter wheat varieties traits varieties 2016/17 2017/18 average x s sx x s sx x s sx yield, t/ha kruna 4.014 0.634 0.284 4.614 0.117 0.052 4.314 0.534 0.169 renesansa 4.273 0.443 0.198 5.073 0.475 0.213 4.673 0.604 0.191 pobeda 4.073 0.464 0.207 4.686 0.551 0.246 4.379 0.579 0.183 ns 40s 3.894 0.353 0.158 4.178 0.207 0.093 4.036 0.311 0.098 takovčanka 4.129 0.465 0.208 4.643 0.573 0.256 4.386 0.561 0.177 average 4.077 0.457 0.091 4.639 0.486 0.097 4.358 0.546 0.077 1000 grain weight, g kruna 39.49 0.905 0.405 40.94 1.781 0.797 40.21 1.536 0.486 renesansa 41.64 0.689 0.308 45.79 1.728 0.773 43.72 2.512 0.794 pobeda 40.98 0.655 0.293 43.84 1.427 0.638 42.41 1.838 0.581 ns 40s 34.45 0.636 0.284 34.91 0.578 0.259 34.68 0.622 0.197 takovčanka 40.82 1.192 0.533 40.94 1.781 0.797 40.88 1.430 0.452 average 39.48 2.772 0.554 41.28 4.008 0.801 40.38 3.530 0.499 test weight, kg/hl kruna 75.42 0.335 0.150 79.62 0.228 0.102 77.52 2.230 0.705 renesansa 74.70 0.400 0.179 78.03 0.480 0.215 76.36 1.804 0.570 pobeda 75.82 0.415 0.185 78.81 0.431 0.193 77.31 1.625 0.514 ns 40s 73.26 0.865 0.387 74.77 0.228 0.102 74.01 0.994 0.314 takovčanka 74.70 0.400 0.179 75.06 0.817 0.365 74.88 0.636 0.201 average 74.78 1.010 0.202 77.26 2.069 0.414 76.02 2.040 0.289 145 biologica nyssana ● 12 (2) december 2021: 141-150 rajičić et al. ● yield components and genetic potential of winter wheat on pseudogley soil of western serbia eties pobeda and kruna had the highest test weight. however, observed by years, there is considerable disagreement on their differences. the highest test weight in the first year of testing had variety pobeda (75.82 kg/hl) and the lowest variety kruna (75.42 kg/hl). in the second year of testing, variety kruna (79.62 kg/hl) had the highest test weight. analysis of variance between observed traits of wheat the analysis of yield variance, 1000 grain weight and test weight of winter wheat varieties during two growing seasons 2016/17 and 2017/18, are shown in tab. 3. analysis of variance in winter wheat genotypes tested showed statistically highly significant differences in yield and test weight in relation to the growing season (p<0.01). in regard to the 1000 grain weight and test weight, statistically highly significant differences were determined between the varieties of winter wheat. statistically highly significant difference in 1000 grain weight and test weight is determined under the influence of the interaction year x genotype. correlation dependence between tested wheat traits the average values of the pearson’s coefficient of correlation (r) of investigated winter wheat traits are shown in tab. 4. correlation coefficients based on all traits tested during 2016/17 and 2017/18 had positive. highly significant and positive correlation coefficients, during 2016/17, were found between 1000 grain weight and test weight (r=0.673**). during 2017/18, highly significant positive correlation coefficients were found between 1000 grain weight and test weight (r=0.596**) and significant and positive dependence was determined between yield and 1000 grain weight (r=0.496*). over a two-year study period (20162018), significant positive correlation coefficients were found between yields and 1000 grain weight (r=0.467*) and test weight (r=0.459*). also, highly significant positive correlations were found between 1000 grain weight and test weight (r=0.623**). discussion wheat grain yield depends on the type of soil and its fertility, weather conditions during the vegetation period, the genetic potential of the variety and the levels applied agrotechnical measures, especially mineral nutrition of plants with nitrogen (zafaranaderi et al., 2013; đekić et al., 2015; luković et al., 2016; perišić et al., 2016; đurić et al., 2018). small grains are very sensitive to soil acidity, although some genotypes show different tolerances. therefore, there is a need to grow wheat and other cereals on acid soils to isolate genotypes adapted to acid soils and different climatic conditions (jelić et al., 2016). numerous studies in our country and the world indicate that the combined application of organic and mineral fertilizers is the most efficient way to eliminate unfavorable production properties of acid table 3. analysis of variance of the tested parameters (anova) effect of year on the traits analyzed traits mean sqr effect mean sqr error f (1. 48)8 p-level grain yield (t/ha ) 3.948 0.223 17.733 0.000** 1000-grain weight (g) 40.825 11.874 3.438 0.070ns test weight (kg/hl) 76.756 2.651 28.951 0.000** effect of genotype on the traits analyzed traits mean sqr effect mean sqr error f (4. 45) p-level grain yield (t/ha ) 0.515 0.279 1.842 0.137 ns 1000-grain weight (g) 120.115 2.896 41.480 0.000** test weight (kg/hl) 23.414 2.452 9.547 0.000** effect of the year x genotype interaction traits mean sqr effect mean sqr error f (4. 40) p-level grain yield (t/ha ) 0.088 0.207 0.425 0.789 ns 1000-grain weight (g) 7.119 1.525 4.667 0.003** test weight (kg/hl) 5.860 0.254 23.069 0.000** *statistically significant (p<0.05); **statistically high significant (p<0.01) soils, which multiplies the yields of cultivated plants (đekić et al., 2013; jelić et al., 2014; biberdžić et al., 2014; rajičić et al., 2019). the results of our tests show that, on average for the investigated factors, the yield wheat grains amounted to 4.358 t/ha (tab. 2). the studied genotypes achieved the lowest yield values in 2016/17 years of research (4.077 t/ha) and the highest yield values in 2017/18 years (4.639 t/ha). živanović et al. (2017) gained similar wheat yields on the vertisol of šumadija (4.310-4.440 t/ha). the expressed differences in yield can be the result of the chemical properties of the given soil, since vertisol is of the acidic reaction and has lower nitrogen, phosphorus and potassium content compared to the alluvium. thus stanković (2009) pointed out that the yield of wheat grains depends on the type of soil and its fertility, weather conditions during the growing period, the genetic potential of the variety and the level of applied cultural practices measures. according to the authors đekić et al. (2019a), varieties of winter wheat tested to low soil ph values show very good results. based on the level of yield achieved, winter wheat varieties are known to be the most tolerant the kruna variety proved to be more adaptable. the same authors point out that grain yield of wheat cultivars ranged from 3.743 t/ha to 6.275 t/ha. newly created high-yielding wheat varieties have been found to be less responsive to temperature deviation than is the case with rainfall (đurić et al., 2013; hristov et al., 2013; đekić et al., 2015; jelic et al., 2015; luković et al., 2020; rajičić et al., 2020). đekić et al. (2019) pointed out that intensive preharvest rainfall in 2010 led to crop lodging in case of wheat cultivars (toplica and vizija), which reduced yield. jevtić & đekić (2018) pointed out that the yield of wheat varieties varied significantly by years of research and amounted from 3.125 to 3.518 t/ha in 2012/13, while a much higher yield of 4.395 to 4.729 t/ha was established in 2011/12. according to the authors terzić et al. (2018), average yield of wheat in the experiment was 5.091 t/ha, with a variation of 4.630 t/ha in the 2011/12 to 5.553 t/ha in the first year of the study (2010/11). biberdžić et al. (2020) investigated different wheat genotypes over a two-year study and reported that yields ranged from 3.090 to 3.745 t/ha in 2016/17 and from 3.839 to 4.770 t/ha in 2017/18. the same authors pointed out that grain yield of wheat cultivars ranged from 3.464 t/ha (the variety daria on vertisol) to 6.865 t/ha (the variety avenue on the alluvial). the 1000 grain weight is a very important qualitative characteristic of grain that has a strong impact on grain yield. the 1000 grain weight during the two-year study period was 40.38 g. the values obtained are slightly lower than the results obtained by biberdžić et al. (2020), jevtić & đekić (2018) and terzić et al. (2018). jevtić & đekić (2018), examined different wheat genotypes and stated that the 1000 grain weight ranged from 42.72 to 44.05 g. terzić et al. (2018) performed a two-year study and the results showed that the 1000 grain weight ranged from 41.98 g to 45.0 g. biberdžić et al. (2020) pointed out that the average weight of 1000 grains in the optimal sowing period was 41.8 g, while in the delayed time it was 40.08 g, which does not represent a statistically significant difference. test weight is an indicator of grain size and filling, and it is a very important parameter when buying 146 biologica nyssana ● 12 (2) december 2021: 141-150 rajičić et al. ● yield components and genetic potential of winter wheat on pseudogley soil of western serbia table 4. analysis of variance of the tested parameters (anova) grain yield, gy 1000 grain weight, gw test weight, tw correlations between the traits analysed in the 2016/17 grain yield, gy 1.00 0.272 0.183 1000 grain weight, gw 0.673** test weight, tw 1.00 correlations between the traits analysed in the 2017/18 grain yield, gy 1.00 0.496* 0.234 1000 grain weight, gw 0.596** test weight, tw 1.00 correlations between the traits analysed in the 2016-2018 grain yield, gy 1.00 0.467* 0.459* 1000 grain weight, gw 0.623** test weight, tw 1.00 *statistically significant (p<0.05); **statistically high significant (p<0.01) 147 biologica nyssana ● 12 (2) december 2021: 141-150 rajičić et al. ● yield components and genetic potential of winter wheat on pseudogley soil of western serbia wheat. the average two-year value of test weight was 76.02 kg/hl and the highest were obtained for the genotype kruna (77.52 kg/hl). the values obtained were slightly higher than the test weight obtained by jevtić & đekić (2018) and biberdžić et al. (2020). a significant influence of the growing season on test weight in wheat is also established by jevtić & đekić (2018). during a two-year study, jevtić & đekić (2018), have found that the test weight ranged from 69.5 to 71.03 kg/hl. according to the authors biberdžić et al. (2020), in the optimal sowing period, test weight was 71.42 kg/hl, while in the delayed time, it was 69.96 kg/hl, which does not represent a statistically significant difference. based on the analysis of variance, it can be concluded that the influence of growing season on grain yield was highly significant (fexp=17.733**). also, biberdžić et al. (2020), jevtić & đekić (2018) and terzić et al. (2018), state that differences in yields between years occur due to environmental conditions. the 1000 grain weight in the study showed highly significant dependence on the genotypes (fexp=41.480**) and very significant in the interaction of growing seasons and genotypes (fexp=4.667**). the present results confirm the opinion of many authors that the 1000 grain weight are strongly modified by the nutrient status of the environment and weather conditions (đekić et al. 2014; jevtić & đekić, 2018; terzić et al., 2018; rajičić et al., 2020). correlation analysis shows the intensity of dependence between studied traits. from the genetic aspect, correlation indicates links between genes, or the appearance of pleiotropic effects of genes. gene linkage pertains to their position on the same chromosome, while pleiotropy appears when one gene regulates the expression of several traits. a strong positive correlation between small grains yields and 1000 grain weight has been found by many researchers (terzic et al., 2018; rajičić et al., 2020) and medium positive dependence has been identified by jelic et al. (2013), đekić et al. (2014) and terzić et al. (2018). positive and weak dependence of yield and 1000 grain weight (r=0.31) was found by đekić et al. (2019b). iftikhar et al. (2012) have established that the 1000 grain weight have a positive and statistically significant correlation with grain yield. rajičić et al. (2020) found a highly significant and positive correlation between yield and 1000 grain weight in 2013/14 (r=0.683**) and highly significant and positive correlation coefficients in 2014/15 (r=0.733**). the duration of the grain filling period is significantly influenced by environmental factors, above all the high temperatures that shortened this period. selection of early-flowering varieties can influence the formation of more flowers and a longer grain filling period, i.e. the selection of higher yielding varieties (djuric et al., 2018). based on the results of the research it can be concluded that a greater number of traits have a decisive role in the formation of grain yield. the contribution of each individual trait may be different in various varieties and in various environmental conditions so that the correlation between two quantitative traits is not fixed. this results from the interaction between the traits of each genotype and the interaction of varieties with external factors. conclusions the highest yield of grain, 1000 grain weight and test weight in all wheat varieties were in the second vegetation period with moderate temperatures at the time of grain filling and a large amount of precipitation. grain yields in wheat varieties ranged from 4.036 t/ha (ns 40s) to 4.673 t/ha (renesansa). the average 1000 grain weight in the study was 40.38 g, with a variation of 39.48 g in the vegetation year 2016/17 to 41.28 g in the vegetation year 2017/18. the test weight for all examined wheat varieties in the study was 76.02 kg/hl and varied from 74.01 kg/ hl in variety ns 40s to 77.52 kg/hl in variety kruna. grain yield shows a tendency to grow in years with a larger amount and better distribution of precipitation during the critical phases of plant development. analysis of variance revealed a very significant influence of the growing season on grain yield and a highly significant influence of the year x variety interaction on grain yield. based on these results, it can be concluded that several traits have a decisive role in the formation of grain yield. the contribution of each individual feature can be different for different varieties and the various environmental conditions so that this results from the interaction between the features within each genotype and genotype interactions with environmental factors. acknowledgements. ph.d. vera rajičić and authors sincerely thanks to the secondary agricultural-chemical school “phd đorđe radić” in kraljevo in serbia, which has enabled the use and analysis of traits of the wheat. investigations necessary for this paper are part of the projects agreement number 451-03-09/2021-14/200383 financed by the ministry of education, science and technology development of republic of serbia. biologica nyssana ● 12 (2) december 2021: 141-150 rajičić et al. ● yield components and genetic potential of winter wheat on pseudogley soil of western serbia references biberdžić, m., jelić, m., knežević, b., 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research. biologica nyssana, 9(2): 119-131. rajičić et al. ● yield components and genetic potential of winter wheat on pseudogley soil of western serbia biologica nyssana ● 12 (2) december 2021: 141-150 149 zafaranaderi, n., aharizad, s., moha-mmadi, s.a. 2013: relationship between grain yield and related agronomic traits in bread wheat recombinant inbred lines under water deficit condition. annals of biological research, 4(4): 7-11. biologica nyssana ● 12 (2) december 2021: 141-150 rajičić et al. ● yield components and genetic potential of winter wheat on pseudogley soil of western serbia živanović, lj., popović v., ikanović j., kolarić, lj. 2017: uticaj količine i oblika azota na produktivnost ozime pšenice. “xxii savetovanje o biotehnologiji”, zbornik radova, knjiga 1. čačak. 150 raca, i., ljubisavljević, i., jušković, m., ranđelović, n., ranđelović, v.: comparative anatomical study of the taxa from series verni mathew (crocus l.) in serbia. biologica nyssana, 8 (1), september 2017 biologica nyssana 8 (1)  september 2017: 15-22 raca, i. et al.  comparative anatomical study of the taxa from… 15 original article received: 27 june 2017 revised: 25 july 2017 accepted: 12 august 2017 comparative anatomical study of the taxa from series verni mathew (crocus l.) in serbia irena raca*, irena ljubisavljević, marina jušković, novica ranđelović, vladimir ranđelović university of niš, faculty of science and mathematics, department of biology and ecology, višegradska 33, niš, serbia * e-mail: raca.irena@gmail.com abstract: raca, i., ljubisavljević, i., jušković, m., ranđelović, n., ranđelović, v.: comparative anatomical study of the taxa from series verni mathew (crocus l.) in serbia. biologica nyssana, 8 (1), september 2017: 15-22. the comparative leaf anatomy of three crocus l. taxa from series verni mathew (crocus heuffelianus herb., crocus tommasinianus herbert and crocus kosaninii pulević) in serbia is introduced. the general outlook of the cross sections of the leaves was defined with microphotographs. more precisely, leaf shape, leaf surface, mesophyll parenchyma and vascular bundle features were examined. the biggest differences were found at the level of vascular bundles (xylem area, phloem area, sclerenchyma area) and leaf blade features (section height, section length, arm length, white stripe width, lacuna area). in general, listed parameters had the lowest values in c. tommasinianus population, while the highest values could be found in population of c. heuffelianus. further investigations should be focused on discovering more localities followed by surveying the ecological factors of the habitats. key words: crocus, series verni, cross section, leaf apstrakt: raca, i., ljubisavljević, i., jušković, m., ranđelović, n., ranđelović, v.: uporedna anatomska studija taksona serije verni mathew (crocus l.) u srbiji. biologica nyssana, 8 (1), septembar 2017: 15-22. komparativna anatomska analiza listova tri taksona roda crocus l. a serije verni mathew (crocus heuffelianus herb., crocus tommasinianus herbert i crocus kosaninii pulević) iz srbije prikazana je na mikrofotografijama. konkretnije, razmatrani su oblik poprečnog preseka lista, površinske strukture listova, lisni mezofil i karakteristike provodnih snopića. najveće razlike zabeležene su na nivou provodnih snopića (površina ksilema, floema i sklerenhima) i parametara koji se odnose na oblik preseka (visina i dužina preseka, dužina ručice, širina bele pruge, površina centralnog parenhima). generalno, navedeni parametri imaju najniže izmerene vrednosti u populaciji vrste c. tommasinianus, dok su najviše izmerene vrednosti zabeležene u populaciji vrste c. heuffelianus. komparativna anatomska analiza obezbeđuje dodatne informacije o karakterima značajnim za diferencijaciju vrsta. kako god, buduća istraživanja bi trebalo da budu fokusirana na pronalaženje novih lokaliteta populacija od interesa, kao i na praćenje ekoloških uslova njihovih habitata. ključne reči: crocus, serija verni, poprečni presek, list 8 (1) • september 2017: 15-22 doi: 10.5281/zenodo.963053 biologica nyssana 8 (1)  september 2017: 15-22 raca, i. et al.  comparative anatomical study of the taxa from… 16 introduction saffrons are known as “golden spice” and ornamentals (c a n d a n , 2007). moreover crocus species have antitumor, antimutagenic, cytotoxic activities and inhibitory effect on nucleic acid synthesis in human malignant cells (f a t e h i et al., 2003). as a matter of fact, the genus crocus contains more than 200 taxa (r u k š ā n s , 2017) from the west mediterranean to central asia with poland as northern limit of distribution. in the latest detailed revision of the genus (m a t h e w et al., 2009), the genus was divided into: section crocus mathew and section nudiscapus mathew. the balkan peninsula is known as one center of diversity (m a t h e w , 1982). in serbia, there are 17 noticed species (r a n đ e l o v i ć et al., 1990). they belong to section crocus mathew (series verni mathew, scardici mathew, crocus mathew) and section nudiscapus mathew (series reticulati mathew, biflori mathew, flavi mathew) (r a n đ e l o v i ć et al., 1990). however, there are many contentious questions related to systematic positions of crocus taxa. one of them certainly refers to taxa from series verni. series verni is represented by three species in serbia: crocus heuffelianus herb.(fig. 1a), crocus kosaninii pulević (fig.1b) and crocus tommasinianus herb. (fig. 1c) (r a n đ e l o v i ć et al., 1990). since good differential markers can be found on the level of micromorphological aspects of the leaves, there have been several investigations about leaf anatomy of crocus taxa from different parts of its distribution range (r u d a l l & m a t h e w , 1990; a k a n , 2007; e r o l & k u ç u k e r , 2007; s a t i l , 2007; c a n d a n , 2007, 2015; k a n d e m i r , 2009, 2010, 2012; c o ş k u n , 2010; ö z d e m i r , 2010; y e t i ş e n , 2013). as can be seen in the literature, no detailed comparative study of series verni has been available, yet. therefore, the initial objective of this paper is to introduce the comparison of leaf anatomy in cross sections of series verni taxa in serbia and to identify their possible differences. material and methods plant samples were collected from natural populations in the flowering time. the sampling localities (fig. 2) with the associated information are given below (tab. 1). taxonomic description of the species was made according to r a n đ e l o v i ć et al. fig. 1. verni taxa in serbia: 1a. crocus heuffelianus, 1b. c. kosaninii, 1c. c. tommasinianus table 1. localities with associated information species crocus heuffelianus crocus kosaninii crocus tommasinianus locality velika ivanča strojkovce mađere n lat/e long 44º 42' 33" 20º 59' 20" 42º 54' 00" 21º 55' 00" 43º 41' 29" 21º 30' 34" altitude 173 m 305 m 207 m https://www.google.rs/url?sa=t&rct=j&q=&esrc=s&source=web&cd=7&cad=rja&uact=8&ved=0ahukewjtw_rjv_pmahviexokhauua0cqfghimay&url=https%3a%2f%2fwww.facebook.com%2foznur.kucuker&usg=afqjcngod6h_3lba3ebsio7vj928hxsvog&sig2=hl-gjbxdry4czpuo-bjrwg biologica nyssana 8 (1)  september 2017: 15-22 raca, i. et al.  comparative anatomical study of the taxa from… 17 (1990). specimens were deposited in herbarium moesiacum niš. five individuals per population were fixed in 50% alcohol for anatomical study, which was done in the laboratory for plant systematics and ecology, faculty of science and mathematics, university of niš. manual microtome (g l i g o r i j e v i ć & p e j č i n o v i ć , 1983) was utilized in order to make cross sections of the leaves. 30 transverse sections per population were stained with safranin alcian blue and examined by leica dm 1000 microscope afterwards. following parameters were considered in leaf cross sections: section height, section length, arm length, white stripe width, lacuna area, adaxial epidermis cells height and width, palisade cells height and width, palisade tissue height, spongy cells height and width, spongy tissue height, abaxial epidermis cells height and width, number of big vascular bundles, xylem area, phloem area and sclerenchyma area. listed anatomical features were measured with imagej software. anatomy characteristics were also described by microphotograps. results and discussion typical microphotographs of leaf cross sections for all of three verni species from serbia are presented in fig. 3. the first part of collage refers to the general outline of leaf cross sections. lateral arm details are given in the middle. the last part of collage represents the structure of big bundles positioned at the end of the arms. leaf blade: crocus leaves cross sections have a unique shape with a square and almost rectangular central keel and two lateral arms recurved towards the keel (r u d a l l & m a t h e w , 1990). however, there are some exceptions where the keel and arms are of the same length (c. scharojanii, c. carpetanus, c. scardicus). the leaf type was declared as morphologicaly and physiologicaly bifacial (e r o l , 2007). the keel is squared with wide base and acute corners in all verni cross sections. the length of arms and their curving degree differ amongst the species (fig. 3a) (tab. 2). the papillae are detected on the adaxial side at the end of the arms. also, the abaxial side of the corner of keel might have hair. the mentioned hair abundance pattern is characteristic of each of three species. leaf surface: adaxial epidermis cells are rectangular, while abaxial cells are elliptical in shape. the cuticle is thicker on the adaxial side of the leaf. stomata occurs on the abaxial surface in the region of arms and lateral sides of the keel. the stomata type is anomocytic (e r o l , 2007). mesophyll: large, almost round parenchyma cells are located in the central part of the keel. because of their thin walls, they might break down, forming an air space called lacuna. disintegration of parenchyma cell walls varies between species (fig. 3a, tab. 2). since parenchyma cells of the keel lack chloroplasts, lacuna area can be noticed as typical white stripe all along the central part of the crocus leaves (e r o l , 2007; y e t i ş e n , 2013). there are two types of parenchyma cells in the mesophyll of arms – palisade and spongy cells. palisade is oriented to the adaxial surface and it is made of two layers of polygonal cells. spongy parenchyma is three – four layered and consists elliptical irregular shaped cells sometimes with intercellular space within (fig. 4). two layers of palisade cells are noticed at the base of the keel as well. vascular bundles: collateral vascular bundles are located between the palisade and spongy cells in one row. there are four big vascular bundles (two of them are placed at the ends of the lateral arms, while two others are positioned in the base of the central keel) (fig. 3a). the number of small and the smallest vascular bundles depends on the leaf dimensions. additionally, they are located all along fig. 2. the geographical position of the investigated populations in serbia (1-crocus heuffelianus; 2-c. tommasinianus; 3-c. kosaninii) biologica nyssana 8 (1)  september 2017: 15-22 raca, i. et al.  comparative anatomical study of the taxa from… 18 the arms and lateral and basal side of the keel. the xylem is oriented towards the adaxial side with the phloem beneath. sclerenchyma “caps” below the phloem are well developed (fig. 5). the anatomy features measurements expressed through the average values and standard deviation are given in the table below (tab. 3). apparently, the highest values of leaf blade features (section height and length, arm length and lacuna area) are noticed in c. heuffelianus population. in the other two species values of those parameters are much lower. the white stripe is narrow in c. kosaninii cross sections (fig. 6). both adaxial and abaxial epidermis cells were found to be fig. 3. anatomic characters of leaves of crocus ser. verni species from serbia: a-leaf cross sections (5x), b-arm details (20x) and c-big bundles (40x) (a-c. heuffelianus; b-c. kosaninii; c-c. tommasinianus) table 2. qualitative anatomic characters of leaves of crocus ser. verni species from serbia crocus heuffelianus crocus kosaninii crocus tommasinianus leaf shape arms the longest with the highest curving degree shorter and slightly recurved keel rectangulared, with ± acute corners squared, with ± acute corners squared, with conspicuously acute corners epidermis cell shape adaxial rectangular, abaxial eliptical papillae finger-like, at the end of the arms and in the corner of the keel mesophyll parenchyma lacuna area large round cells disintegration of parenchyma cell walls is more frequent palisade 2 layers of polygonal cells spongy 3 4 layers of elliptical – irregular shaped cells big vascular bundles 2 of them at the ends of the lateral arms, 2 others at the base of the central keel sometimes one more pair in the middle of the arms biologica nyssana 8 (1)  september 2017: 15-22 raca, i. et al.  comparative anatomical study of the taxa from… 19 the largest in c. kosaninii population. palisade tissue characters are of a similar value. the thinnest spongy layer was observed in the investigated c. tommasinianus population. the most developed vascular tissue features (xylem, phloem and sclerenchyma cap area) were found in c. heuffelianus cross sections. crocus tommasinianus vascular bundles are the smallest, while c. kosaninii big bundles are middle-sized (fig. 6). the objective of this study was to introduce and compare leaf anatomy of the three crocus species from series verni present in serbia. leaf blade, epidermis, mesophyll and vascular bundle features were analyzed. as might be expected (r u d a l l & m a t h e w , 1990), those species tend to correspond closely in their leaf anatomy. however, major differencies can be found on the level of big vascular bundles and their examined parameters (xylem area, phloem area and sclerenchyma area). moreover, leaf blade features (section height and length, arm length, white stripe width and lacuna area) are good distinguishing characters as well. the table 3. quantitative anatomic characters of leaves of crocus ser. verni species from serbia species crocus heuffelianus crocus kosaninii crocus tommasinianus character mean ± stdev min max mean ± stdev min max mean ± stdev min max section height (µm) 881 ± 173 570 1210 654 ± 124 494 1017 630 ± 125 382 916 section length (µm) 7003 ± 1095 3611 9035 4527 ± 946 2810 6298 4152 ± 959 2391 5598 arm length (µm) 3139 ± 492 1684 4263 1908 ± 429 1109 2725 1701 ± 462 892 2467 white stripe width (µm) 752 ± 122 500 996 590 ± 93 434 792 740 ± 111 584 955 lacuna area (µm2) 371375 ± 105510 193667 648276 209992 ± 69767 111999 335380 248603 ± 74234 141373 395804 adaxial epidermis cell height (µm) 20 ± 3 16 28 22 ± 2 19 27 16 ± 2 11 20 adaxial epidermis cell width (µm) 18 ± 3 15 30 19 ± 2 16 25 17 ± 2 13 20 palisade cell height (µm) 37 ± 6 27 55 34 ± 4 27 42 34 ± 4 28 43 palisade cell width (µm) 15 ± 2 11 21 14 ± 1 12 17 16 ± 2 13 20 palisade tissue height (µm) 59 ± 13 35 88 59 ± 9 45 82 54 ± 7 40 66 spongy cell height (µm) 22 ± 3 16 29 22 ± 2 17 27 16 ± 2 12 20 spongy cell width (µm) 19 ± 3 15 26 20 ± 2 18 23 24 ± 3 17 30 spongy tissue height (µm) 68 ± 11 38 91 69 ± 16 38 107 47 ± 9 27 66 abaxial epidermis cell height (µm) 19 ± 2 14 24 22 ± 2 15 25 14 ± 2 11 18 abaxial epidermis cell width (µm) 18 ± 2 14 21 22 ± 2 17 27 16 ± 3 12 23 number of big bundles 4 ± 0 4 4 4 ± 0 4 4 4 ± 0 4 4 sclerenchyma cap area (µm2) 2614 ± 636 1323 3710 2219 ± 375 1596 2929 1459 ± 409 895 2303 phloem area (µm2) 1230 ± 245 669 1645 720 ± 337 338 1965 609 ± 87 442 768 xylem area (µm2) 2477 ± 586 1657 3946 1234 ± 491 621 2323 1059 ± 249 496 1509 biologica nyssana 8 (1)  september 2017: 15-22 raca, i. et al.  comparative anatomical study of the taxa from… 20 length of arms and their curving degree differ amongst the species (e r o l , 2007). crocus heuffelianus cross sections have the longest arms with conspicuously high curving degree. this population is also characterized by the most prominent vascular bundles. the features referred to leaf blade are of a higher value as well (section height and length, arm length, white stripe width, lacuna area). while c. tommasinianus can be distinguished from the other two species by the smallest vascular bundles, thinnest spongy layer and wide white stripe, c. kosaninii has middle sized vascular bundles and the biggest epidermal cells (both adaxial and abaxial). also, disintegration of parenchyma cell walls in lacuna area is more frequent in c. tommasinianus and c. kosaninii populations in comparision to c. heuffelianus. further researches should be carried out including more populations, other closely related species and surveying also the ecological conditions in each locality. this would allow us to evaluate which impact habitat factors have on the anatomy of leaves as well as the potential of leaf anatomy features as diagnostic characters. conclusion as could be seen from the literature, no detailed comparative study of series verni had been done before. therefore, the major aim of this paper was to introduce a leaf anatomy comparison of series verni taxa in serbia and to highlight the differences between them. this study is a basis for further researches within series verni. acknowledgements. the work was funded by the ministry of education, science and technological development of republic of serbia (project no. 173030). fig. 4. arm details on leaves cross sections of crocus ser. verni species from serbia (20x): a-c. heuffelianus; b-c. kosaninii; c-c. tommasinianus (ade-adaxial epidermis, p-palisade parenchyma, s-spongy parenchyma, abe-abaxial epidermis, st-stomata) fig. 5. big bundles on leaves cross sections of crocus ser. verni species from serbia (40x): a-c. heuffelianus; b-c. kosaninii; c-c. tommasinianus; (sc-sclerenchyma cap; ph-phloem; xy-xylem) biologica nyssana 8 (1)  september 2017: 15-22 raca, i. et al.  comparative anatomical study of the taxa from… 21 fig 6. box plots of quantitative anatomic characters of leaves of crocus ser. verni species from serbia: a-c. heuffelianus; b-c. kosaninii; c-c. tommasinianus (ad ec-adaxial epidermal cell; ab ec-abaxial epidermal cell) 200 700 1200 c b a section height (µm) 2000 4000 6000 8000 10000 c b a section length (µm) 500 1500 2500 3500 4500 c b a arm length (µm) 400 600 800 1000 c b a white stripe width (µm) 0,1 0,2 0,3 0,4 0,5 0,6 0,7 c b a lacuna area (mm2) 10 15 20 25 c b a ad ec height (µm) 12 17 22 27 32 c b a ad ec width (µm) 10 15 20 25 30 c b a ab ec height (µm) 10 15 20 25 30 c b a ab ec width (µm) 25 35 45 55 c b a palisade cell height (µm) 10 15 20 c b a palisade cell width (µm) 30 55 80 c b a palisade tissue height (µm) 10 20 30 c b a spongy cells height (µm) 10 20 30 c b a spongy cells width (µm) 20 45 70 95 c b a spongy tissue height (µm) 400 1400 2400 3400 c b a xylem area (µm2) 200 700 1200 1700 c b a phloem area (µm2) 500 2500 4500 c b a sclerenchyma cap area (µm2) biologica nyssana 8 (1)  september 2017: 15-22 raca, i. et al.  comparative anatomical study of the taxa from… 22 references akan, h., eker, i., satil, f. 2007: the morphological and anatomical properties of endemic crocus leichtlinii d. dewar bowles (iridaceae) in turkey. pakistan journal of 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(ed.), flora sr srbije viii: 2-12, srpska akademija nauka i umetnosti, beograd. yetişen, k., şen, u., yıldırım, t., özdemir, c. 2013: morphological and anatomical study on endemic crocus olivieri gay subsp. istanbulensis mathew subspecies (iridaceae). anadolu university journal of science and technology-c life sciences and biotechnology, 3 (1): 31-37. hacıoğlu doğru, acar, 2019, biologica nyssana 10(2) 10 (2) december 2019: 181-187 doi: 10.5281/zenodo.3600203 effects of salt stress factors on antimicrobial activity of two triticum aestivum l. varieties original article nurcihan hacıoğlu doğru department of biology, faculty of arts and sciences, çanakkale onsekiz mart university, çanakkale, turkey nurcihan.n@gmail.com (corresponding author) okan acar department of biology, faculty of arts and sciences, çanakkale onsekiz mart university, çanakkale, turkey oacar@comu.edu.tr received: july 10, 2019 revised: september 10, 2019 accepted: september 13, 2019 abstract: salinity is one of the most common environmental stress factors that adversely affect plant growth and crop production in cultivated areas worldwide. herbal or ‘alternative’ medicine is gaining popularity and scientific research about wheat grass as a “functional food” is becoming more available and popular. wheat grass, triticum aestivum l. has a long history and is widely used as a health food supplement. it is found to be used as a treatment for minor ailments, and also as a preventive dietary supplement and therapeutic drug. current study was aimed at evaluate antimicrobial properties of the two varieties of t. aestivum l. [cv. tosunbey (drought tolerant) and cv. sultan 95 (drought sensitive)], grown in three different conditions [(1) control; not treated with salt or acetyl salicylic acid; (2) treatment with sea water; (3) sea water and pre-treatment of seeds with acetyl salicylic acid]. the antimicrobial activity of the ethanol extracts of the two varieties of t. aestivum were assayed against escherichia coli nrrl b-3704, pseudomonas aeruginosa atcc 27853, proteus vulgaris atcc 13315, acinetobacter baumanii atcc 19606, bacillus subtilis atcc 6633, staphylococcus aureus atcc 25923, s. haemolyticus atcc 43252 and candida albicans atcc 10231 test microorganisms by agar disc diffusion method and broth microdilution method. the results showed that the ethanol extracts from the different treatments showed antimicrobial activities, with the diameters of the inhibition zone ranging from 8 to 13 mm and 2.5 to 20 µg/ ml, respectively. the highest antimicrobial activity was demonstrated against p. aeruginosa atcc 27853 by the extract of t. aestivum cv. sultan 95, which grown in sea water and whose seeds were pre-treated with acetyl salicylic acid. key words: antibacterial activity, t. aestivum cv. tosunbey, t. aestivum cv. sultan 95 apstract: efekat sonog stresa na antibakterijsku aktivnost dva varijeteta triticum aestivum l. salinitet je jedan od najčešćih sredinskih stresnih faktora koji nepovoljno utiču na rast biljaka i produkciju useva u kultivisanim područjima širom sveta. biljna ili “alternativna” medicina dobija na popularnosti i naučna istraživanja na temu pšenice kao “finkcionalne hrane” postaju sve dostupnija i popularnija. pšenica, triticum aestivum l., ima dugu istoriju i veoma je zastupljena kao dodatak ishrani. utvrđeno je da se koristi kao tretman za blaža oboljenja i kao preventivni dodatak ishrani i terapeutski agens. ovo istraživanje imalo je za cilj da utvrdi antimikrobni potencijal dva varijeteta t. aestivum l. [cv. tosunbey (tolerantna na sušu) i cv. sultan 95 (osetljiva na sušu)], uzgajanih u tri različita uslova [(1) kontrola; netretirana semena; (2) tretman morskom vodom; (3) tretman morskom vodom i pretretman semena acetil salicilnom kiselinom]. antimikrobna aktivnost etanolnih ekstrakata dva varijeteta t. aestivum testirana je u odnosu na escherichia coli nrrl b-3704, pseudomonas aeruginosa atcc 27853, proteus vulgaris atcc 13315, acinetobacter baumanii atcc 19606, bacillus subtilis atcc 6633, staphylococcus aureus atcc 25923, s. haemolyticus atcc 43252 i candida albicans atcc 10231, korišćenjem metoda disk difuzije na agaru i mikrodilucije. rezultati su pokazali da etanolni ekstrakti iz različitih uslova gajenja poseduju antimikrobnu aktivnost, sa dijametrima inhibicionih zona od 8 to 13 mm i minimalnim inhibitornim aktivnostima od 2.5 do 20 µg/ml. najveća antimikrobna aktivnost, utvrđena u odnosu na p. aeruginosa atcc 27853, pokazana je od strane ekstrakta t. aestivum cv. sultan 95, dobijenog od biljaka gajenih u morskoj vodi i čija su semena pretretirana acetil salicilnom kiselinom. ključne reči: antibakterijska aktivnost, t. aestivum cv. tosunbey, t. aestivum cv. sultan 95 © 2019 hacıoğlu doğru, acar. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work noncommercially under the same license as the original. 181 13th symposium on the flora of southeastern serbia and neighboring regions introduction antibiotic resistance has become a global concern (westh et al., 2004) and clinical efficiency of many existing antibiotics is being threatened by the emergence of multi-drug resistant pathogens as reported by bandow et al. (2003). so, there is an urgent need to develop new antimicrobial compounds which are more active against newer and re-emerging infectious diseases (rojas et al., 1992). medicinal plants have bioactive compounds which are used for curing of various human diseases and play an important role in healing. biosynthesis and accumulation of substances such as flavonoids, phenolic acid and anthocyanin enhances the biological activity (antimicrobial, antioxidant, etc.) of plant material (karakaş and türker, 2013). wheat grass, triticum aestivum l. has a long history and is widely used as a health food supplement. it is found to be utilized as a treatment for minor ailments and serious life-threatening diseases, and as a preventive dietary supplement and therapeutic drug. salinity is one of the most common environmental stress factors that affect plant growth and crop production in cultivated areas worldwide. primary saline conditions appear naturally in environment, while anthropogenic activities are responsible for secondary salinity. increased urbanization and deforestation are two important human-derived activities for salinity. a number of reports on soil salinization and their influences on crop productivity, land degradation and ecological disturbances are reported worldwide. excess amount of salts affected physical, chemical, as well as the biological properties of soils. plant health in saline soils considerably declines owing to poor nutrition, osmotic stress and reduced microbial diversity (paul and lade, 2014; vimal et al., 2018). the response of plants to physiological stress conditions (e.g. salinity) activates genes encoding pathogen-related proteins (prs) (sehgal and mohamad, 2018). therefore, prs are commonly synthesized in plants not only under biotic stress conditions, but also under abiotic stress conditions (such as osmotic shock, chemical injury, and aging). in fact, there are inducible defense proteins classified into 17 prs families in plants (van loon et al., 2006; sehgal and mohamad, 2018). the relationship between antifungal (schmidt at al., 2019) and antimicrobial resistance (sharma et al., 2018) has been shown in proteins induced by oxidative stress caused by environmental stresses. in addition, phytoalexins synthesized in response to stresses such as antimicrobial attack and physical injury in plants (ejike et al., 2013) have been shown to increase antimicrobial activity in the rice plant in relation to the physiology of chloroplast and mitochondria (li et al., 2011). accordingly, it is believed that increases in antimicrobial activity are a consequence of oxidative stress caused by environmental stresses. in this study, the effects of seawater induced salt stress (24-26% nacl, 24 h) and salicylic acid (sa) priming combinations were investigated on antimicrobial activity in 21-day-old seedlings of the two wheat varieties [cv. tosunbey (drought tolerant) and cv. sultan 95 (drought sensitive)]. material and methods the plants material: the seeds were sterilized by washing with 5% sodium hypochlorite solution for 5 min (1 time) and with sterile distilled water for 2.5 min (3 times). seawater is taken from the coast of kepez (çanakkale), 30 m from the coast, and filtered in the laboratory to remove the particles. salt concentration of clear seawater is 24-26% (türkoğlu, 2006) and it was mixed with distilled water to obtain 50% concentration (sw 50%), while full concentration (sw 100%) was also used in experiments. seedlings were planted in perlite:peat mixture (1:3) containing pots. plants were watered with hoagland nutrient solution (100%; steward, 1983) for 21 days. seedlings of t. aestivum l. cv. tosunbey (drought tolerant) and cv. sultan 95 (drought sensitive) were divided into three groups: (1) control; not treated with salt or acetyl salicylic acid; (2) treatment with 100 ml sea water (50% and 100 %); (3) sea water and pre-treatment of seeds with 1000 mg acetyl salicylic acid (during 2 hours in 2 x 500 mg aspirin tablets solution). leaf sampling was collected after 24 hours of salt treatment of 21d old plants. test microorganisms gram-negative bacteria (escherichia coli nrrl b-3704, pseudomonas aeruginosa atcc 27853, proteus vulgaris atcc 13315, acinetobacter baumanii atcc 19606), gram-positive bacteria (bacillus subtilis atcc 6633, staphylococcus aureus atcc 6538, staphylococcus haemolyticus atcc 43252) and yeast (candida albicans atcc 10231) were used for determining the antimicrobial activities of the two t. aestivum varieties. preparation of extracts for antimicrobial activity air-dried samples of two wheat varieties were grounded into a fine powder in a grinding mill. pulverized plant samples (1 g) were extracted with 10 ml of 80% ethanol, (1:10 w/v) using an orbital shaker for 8 h at room temperature. the extract was separated from the solids by filtration with whatman no. 1 filter paper. the remaining solids 182 biologica nyssana ● 10 (2) december 2019: 181-187 hacıoğlu doğru, acar ● effects of salt stress factors on antimicrobial activity of two triticum aestivum l. varieties 183 biologica nyssana ● 10 (2) december 2019: 181-187 ta bl e 1. a nt im ic ro bi al a ct iv ity re su lts o f t he tw o va rie tie s of t . a es tiv um e xt ra ct s te st m ic ro or ga ni sm s pl an t e xt ra ct s *d is c d iff us io na ( m m ) m ic (µ g/ m l) cv . t os un be y cv . s ul ta n 95 cv . t os un be y cv . s ul ta n 95 t1 t2 t3 t4 s1 s2 s3 s4 c on tr ol p1 0/ n y1 00 t1 t2 t3 t4 s1 s2 s3 s4 c on tr ol st / n y1 00 e . c ol i n r r l b37 04 8. 0 ± 0. 21 11 .0 ± 0. 2 8. 0 ± 0. 03 8. 0 ± 0. 03 12 .0 ± 0. 0 1 11 .0 ± 0. 03 7. 0 ± 0. 0 1 7. 0 ± 0. 0 1 16 .0 20 .0 2. 5 20 .0 20 .0 2. 5 2. 5 20 .0 20 .0 4. 0 p. a er ug in os a at c c 2 78 53 10 .0 ± 0. 11 9. 0 ± 0. 02 7. 0 ± 0. 2 10 .0 ± 0. 01 11 .0 ± 0. 12 7. 0 ± 0. 01 10 .0 ± 0. 02 13 .0 ± 0. 13 8. 0 5. 0 5. 0 20 .0 5. 0 2. 5 20 .0 10 .0 2. 5 1. 0 p. v ul ga ris at c c 1 33 15 13 .0 ± 0. 01 10 .0 ± 0. 0 1 8. 0 ± 0. 1 11 .0 ± 0. 14 12 .0 ± 0. 12 9. 0 ± 0. 03 9. 0 ± 0. 02 8. 0 ± 0. 01 13 .0 2. 5 5. 0 20 .0 5. 0 2. 5 10 .0 10 .0 20 .0 4. 0 a . b au m an ii at c c 1 96 06 10 .0 ± 0. 0 1 9. 0 ± 0. 12 8. 0 ± 0. 01 10 .0 ± 0. 12 10 .0 ± 0. 01 7. 0 ± 0. 03 10 .0 ± 0. 04 10 .0 ± 0. 12 12 .0 10 .0 20 .0 20 .0 10 .0 10 .0 20 .0 20 .0 20 .0 2. 0 b . s ub til is at c c 6 63 3 7. 0 ± 0. 14 8. 0 ± 0. 2 7. 0 ± 0. 12 9. 0 ± 0. 03 11 .0 ± 0. 12 9. 0 ± 0. 03 8. 0 ± 0. 12 10 .0 ± 0. 12 14 .0 20 .0 20 .0 20 .0 5. 0 2. 5 10 .0 20 .0 5. 0 4. 0 s . a ur eu s at c c 6 53 8p 11 .0 ± 0. 02 9. 0 ± 0. 12 8. 0 ± 0. 04 10 .0 ± 0. 1 10 .0 ± 0. 01 8. 0 ± 0. 01 12 .0 ± 0. 01 7. 0 ± 0. 0 1 15 .0 2. 5 5. 0 20 .0 5. 0 5. 0 20 .0 2. 5 20 .0 4. 0 s . h ae m ol yt ic us at c c 4 32 52 9. 0 ± 0. 03 10 .0 ± 0. 1 8. 0 ± 0. 12 10 .0 ± 0. 1 11 .0 ± 0. 1 10 .0 ± 0. 2 10 .0 ± 0. 0 1 8. 0 ± 0. 0 1 14 .0 5. 0 5. 0 20 .0 5. 0 5. 0 5. 0 5. 0 20 .0 5. 0 c an di da a lb ic an s at c c 1 02 31 7. 0 ± 0. 1 8 .0 ± 0. 1 10 ± 0. 1 11 .0 ± 0. 0 1 8. 0 ± 0. 1 7. 0 ± 0. 12 7. 0 ± 0. 03 7. 0 ± 0. 12 16 .0 20 .0 20 .0 10 .0 5. 0 20 .0 20 .0 20 .0 20 .0 2. 5 t: t . a es tiv um c v. t os un be y s: t . a es tiv um c v. s ul ta n 95 t1 : c on tro l; t2 : t re at m en t w ith a ce ty l s al ic yl ic a ci d; t 3: tr ea tm en t w ith s al in ity s tre ss ; t 4: s al in ity s tre ss a nd p re -tr ea tm en t o f s ee ds w ith a ce ty l s al ic yl ic a ci d s1 : c on tro l; s2 : t re at m en t w ith a ce ty l s al ic yl ic a ci d; s 3: tr ea tm en t w ith s al in ity s tre ss ; s 4: s al in ity s tre ss a nd p re -tr ea tm en t o f s ee ds w ith a ce ty l s al ic yl ic a ci d in hi bi tio n zo ne (m m ); a in cl ud es d ia m et er o f d is k (6 m m ); p1 0 = pe ni ci llin (1 0 ug /d is c) ; s t: s tre pt om yc in ; n y1 00 n ys ta tin 1 00 u g/ di sc hacıoğlu doğru, acar ● effects of salt stress factors on antimicrobial activity of two triticum aestivum l. varieties were extracted twice with the same solvent and the extracts were combined. extracts were stored in a refrigerator (4 °c) until analyzed (sultana et al., 2007). screening of antimicrobial activity disc diffusion assay empty sterilized antibiotic discs having a diameter of 6 mm (schleicher and schull no. 2668, dassel, germany) were each impregnated with 50 μl of the extract (10 mg/disc). all the bacteria mentioned above were incubated at 35 ± 0.1 °c for 24 h by inoculation into nutrient broth (difco laboratories, mi, usa) and the yeast culture studied was incubated in malt extract broth (difco laboratories, mi, usa) at 25 ± 0.1 °c for 48 h. an inoculum containing 106 bacterial cells or 108 yeast cells/ml was spread on mueller hinton agar (mha) (oxoid ltd., hampshire, uk) plates (1 ml inoculum/plate). the discs injected with extracts were placed on the inoculated agar by pressing slightly. petri dishes were placed at 4 °c for 2 h, afterwards the plate inoculated with the yeast culture was incubated at 25 ± 0.1 °c and bacteria were incubated at 35 ± 0.1 °c for 24 h (collins et al., 1989). at the end of the period, inhibition zones formed on the medium were measured in millimeters. studies were performed in triplicate. treatments with penicillin (p10), and nystatin (nys30) served as positive controls and treatments with ethanol, without bacterial or fungal materials served as negative controls. minimum inhibitory concentration assay minimum inhibitory concentration (mic) was investigated as recommended by instructions of the clinical and laboratory standards institute (clsi, 2006). the lowest concentration of extracts inhibiting visible growth of each test microorganisms was taken as the mic. the medium, 0.1% (w/v) streptomycin (st), nystatin (nys100) and 10% dmso were used as the non-treated, positive and negative controls, respectively (teanpaisan et al., 2017). statistical analysis the results of antimicrobial assay were mean ± sd of the three parallel assays. results and discussion a total of eight plant extracts were tested against eight test microorganisms. ethanol was selected as extraction solvent, because it is one of the best solvents used for the extraction of antimicrobial substances (jonathan and fasidi, 2003). the results showed that the ethanol extracts from the different treatments studied have antimicrobial activities, with the diameters of the inhibition zone 184 ranging from 8 to 13 mm and 2.5 to 20 µg/ml, respectively (tab. 1). the highest antimicrobial activity was demonstrated against p. aeruginosa atcc 27853 by the extract of t. aestivum cv. sultan 95 which grown in sea water and whose seeds were pre-treated with acetyl salicylic acid (s4). however, the disc diffusion method is a qualitative technique and is mainly used for selecting extracts with antimicrobial activity, mostly when diameter zones of inhibition are ≥ 10 mm (sánchez et al., 2016). so, s. haemolyticus atcc 43252 was the second most susceptible strain against the tested plants extracts. the same was found when observing the results of micro-dilution method. although the disc diffusion method is one of the inexpensive and less laborious antimicrobial testing methods currently available, this procedure suffers with the demerit of not always producing reproducible results for plant extracts (eloff, 1998; vambe et al., 2018). this is primarily because some constituents do not diffuse readily in polarized agarose gels (vambe et al., 2018). when the same extracts were screened using microdilution method, five plant extracts, namely t1, t2, s1, s2, s3 demonstrated higher antibacterial activity than the control antibiotic. this discordance observed in the results obtained from these two bioassays could be attributed to several factors (vambe et al., 2018). it is possible that antibacterial activity was masked in the disk diffusion assay by low diffusion potential. wheat is a grain containing protein, starch (vida et al., 2014), fiber, phytochemicals and antioxidant substances which have a very important place in human nutrition (andersson et al., 2013). while meeting the daily nutrition requirements with protein and starch, it facilitates digestion with fibers and thus reduces the risk of developing colon cancer (huang et al., 2015). this plant has been also shown to have anti-inflammatory, antioxidant, anti-carcinogenic, immuno-modulatory, laxative, astringent, diuretic, antibacterial and anti-aging properties and is a part of formulations as a highly effective antimicrobial agent (saha et al., 2018). in the literature there are many investigations about wheat grass antimicrobial activity. pallavi et al. (2011), tested wheat grass extracts against the gram-positive bacteria s. aureus, b. subtilis and gram-negative e. coli, using amoxicillin as standard. certain extracts exhibited considerable activity against b. subtilis and moderate activity against s. aureus and e. coli. ashok (2011) also reported antibacterial activity against e. coli, p. aeruginosa and s. aureus. antifungal activity was also reported against c. albicans. sundaresan et al. (2015), tested antimicrobial properties of 7th, 14th, biologica nyssana ● 10 (2) december 2019: 181-187 hacıoğlu doğru, acar ● effects of salt stress factors on antimicrobial activity of two triticum aestivum l. varieties and 21st day wheat grass extracts obtained with five different solvents (water, ethanol, methanol, ethyl acetate and hexane). all these extracts showed antibacterial activity against seven food borne pathogens. amongst them, hexane extracts from the seven-day-old wheat grass showed maximum antibacterial activity, especially against yersinia enterocolitica and listeria monocytogenes. das et al. (2012) found that acetone (80%) extract of wheatgrass was effective against five foodborne microorganisms, including the fungus aspergillus niger, a common contaminant of food. there appears to be a lack of published scientific data on the salt stress effect to antimicrobial properties of wheat grass, thereby highlighting the need for continued research. plants are frequently subjected to a variety of harsh environmental stresses such as scarcity of water, extreme temperatures, high soil salinity, herbivore attack, and pathogen infection diminishing their productivity (sewelam et al., 2016). different salt concentrations induce physiological and metabolic changes in plants affecting their seed germination, growth, development, yield and also decreases the rate of respiration and photosynthesis in plants. in addition to the growth and yield, the composition of bioactive compounds present in the aromatic and medicinal plants is affected by salinity (gil et al., 2002). the increased levels of plant secondary metabolites such as phenols, flavonoids, tannins, alkaloids and others under the influence of increased salt concentrations as a part of defense mechanism have been reported (kate, 2008). reactive oxygen species (ros) are continuously produced under normal environmental conditions in plant cells. bioactive compounds also trigger metabolic pathways in plant cells and result in a higher ros accumulation. the common response observed in salt-stressed plants is the generation of ros, highly reactive and responsible in damaging cell structures, nucleic acids, lipids and proteins. plants possess medicinal value with anti-inflammatory and antimicrobial activities; acquired resistance to stress induced by ros is due to the presence of several bio-active compounds (foyer et al., 1994). in the scientific literature, there are no data on the effect of salt stress on wheat grass antimicrobial activity. kotagiri et al. (2017) investigated the secondary metabolites and antimicrobial potential of leaf, stem and root ethanol and chloroform extracts of five different coleus species; c. aromaticus, c. amboinicus, c. barbatus, c. forskohlii and c. zeylanicus subjected to salinity stress. the up-regulation in the content of plant bioactive compounds, along with the antimicrobial activities of ethanol and chloroform extracts under the influence of salinity stress have been observed during the study in coleus. the leaf, stem and root extracts of all the five coleus species showed good antimicrobial activity against the tested pathogenic strains. the leaf extracts of coleus showed higher inhibitory activity compared to the stem and root extracts. ethanol extracts showed higher antimicrobial activity ranging from 1.5-100.0 mg/ml when compared with the chloroform extracts ranging from 0.97-250 mg/ ml respectively. the leaf, stem and root extracts of coleus showed effective antimicrobial activity against the pathogenic strains even under saline conditions is due to the up regulation of secondary metabolites which provides a scope of developing novel drugs to treat infectious diseases. our results have a correlation with the results of kotagiri et al (2017) about increasing antimicrobial activity under saline stress and pre-treatment of seeds with acetyl salicylic acid conditions, which is probably related to the up-regulation of secondary metabolites which provides a scope for developing novel drugs to treat p. aeruginosa atcc 27853. conclusion the screening of the plant extracts for antimicrobial activity has shown that higher plants represent a potential source of new antiinfective agents. herbal medicines are a valuable and rapidly available resource for primary health care and complementary health care systems. the results of the present study provide evidence that t. aestivum plant varieties grown in different condition represent an important asset to the health care in communities. 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serbia (coleoptera, cerambycidae) ivan gnjatović, vladimir žikić university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia * e-mail: ivangnjatovic81@gmail.com abstract: gnjatović, i., žikić, v.: cerambycids of southeast serbia (coleoptera, cerambycidae). biologica nyssana, 1 (1-2), december 2010: 111-115. the cerambycids (longhorn beetles) are known as very attractive insects, as well as pests, especially in forestry. our investigation and collecting the specimens on the territory of serbia has started in the middle of the nineteenth century. up today, 242 species were registered in the fauna of serbia (ilić, 2005). the first information about cerambycids in serbia was published by bobić (1871). significant contribution in researching of the family cerambycidae was given by košanin (1904). finally, there were several domestic authors occupied by longhorn beetles: adamović (1950), mikšić (1963), ćurčić et al. (2003) and ilić (2005). in this review forty-nine species reviewed from 34 genera which belong to five subfamilies: prioninae, lepturinae, spondylidinae, cerambycinae and lamiinae. examined material has been collected in the southeastern serbia. the most numerous were the subfamily cerambycinae, comprising 18 species from 15 genera. key words: coleoptera, checklist, southeast serbia introduction ! the cerambycidae comprises about 25,00030,000 species distributed in all zoogeographical regions worldwide. all known species are phytophagous; majority of their larvae are xylophagous (86%); some members feed on herbaceous plants (13%). they could cause serious damages on forest habitats, so they are marked as pests (bílý and mehl, 1989). there are about 2.500 species in the palaearctic fauna of which 625 occur in europe (b í l ý and m e h l , 1989; i l i ć , 2005). pančić was the first investigator who started collecting the specimens of coleoptera in serbia since 1846, but the first information about cerambycids in serbia has been published by b o b i ć (1891). significant contribution in researching of the family cerambycidae was given by k o š a n i n (1904). he has noted 72 species from various localities in serbia. long after that, a d a m o v i ć (1950) complements košanin’s coleopteran collection and reporting 25 new species. m i k š i ć (1963) published 278 species for the fauna of ex yugoslavia. for the territory of serbia he has noticed 141 species, based on following data of s v i r č e v (1938), k u t h y (1896), ž i v o j i n o v i ć (1950) and a d a m o v i ć (1950). in additional research m i k š i ć at al. (1971, 1973, and 1985) presented 283 species from 105 genera. in the latest publication ć u r č i ć et al. (2003), reported 5 subspecies, along with 49 species, belonging to 49 genera as well as two new species in serbia: leiopus femoratus faimaire and pachytodes cerambyciformis (schrank). up to year of 2005, it was described 242 species, 40 subspecies from 123 genera, 15 subgenera, 44 tribes and 6 subfamilies in serbia (i l i ć , 2005). most recent data shows there are 259 species of longhorn beetles in serbia (p i l & s t o j a n o v i ć , 2009). 10th sfses • 17-20 june 2010, vlasina lake1 (1-2) • december 2010: 111-115 biologica nyssana 1 (1-2) december 2010: 111-115 gnjatović, i.,žikić, v. cerambycids of southeast serbia… 112 material and methods greater part of samples comes from the student’s collections of insects. our analisys included the specimens from family cerambycidae, with except of the tribe of dorcadionini. investigated material was collected on 31 localities with different vegetation and habitat in southeastern serbia (details are given in fig. 1). samples were preserved in 70% ethyl-alcohol, and deployed in wooden boxes held at about 22 °c. all specimens are deposited in department of biology and ecology, faculty of sciences, university of niš, serbia. identification of the collected samples is performed by the keys: m i k š i ć and g e o r g i j e v i ć (1971, 1973); bense (1995). this investigation related to all representatives of the family cerambycidae, with the exception of the tribe dorcadionini. results and discussion a total number of 49 species were identified from 34 genera, from 20 tribes, belonging in 5 subfamilies: prioninae (2 species), lepturinae (15 species), spondylidinae (2 species), cerambycinae (18 species), lamiinae (12 species). new records for the fauna of investigated territory are marked by an asterisk (*) (tab. 1). subfamily prioninae tribe megopidini aegosoma scabricornis (scop. 1763), synonym megopis scabricornis – 3♀, serbia: bela palanka: čiflik, 26.06.2006, leg. i. gnjatović; 3♀, serbia: suva mt., 23.05.2007, leg. o. stojnev. prionus coriarus (l. 1758) – 1♂, serbia: niš 15.08.2005, leg. s. lukić; 2♂, serbia: stara mt., 07.07.2006, leg m. jovanović; 1♂, serbia: bela palanka: čiflik, 26.06.2006, leg. i. gnjatović; 1♂, serbia: vladičin han, 18.07.2008, leg. i. gnjatović. subfamily lepturinae tribe anaglyptini anastrangalia sanguinolenta l. 1761 – 1♂, serbia: čestin, 27.05.2007, leg. o.stojnev; 1♂, serbia: suva mt, 16.07.2007, leg. i. gnjatović; 1♂, serbia: vladičin han, 16.07.2008, leg. i. gnjatović. 1♀, serbia: pirot, 15.08.2006, leg. i. gnjatović. anastrangalia dubia (scop. 1763) – 1♀, serbia: vitkovac, 27.05.2008, leg. i. gnjatović. tribe asemini *brachyta balcanica (hampe 1870) – 1♂, serbia: niš, 16.07.2009, leg. i. gnjatović; 1♀, serbia: niš: bojanine vode, 16.07.2008, leg. i. gnjatović; 1♀, serbia: donji dušnik, 09.06.2007, leg. i. gnjatović. tribe lepturini cortodera flavimana (walt 1838) – 1♀, serbia: donji dušnik, 03.06.2006, leg. i. gnjatović; 1♂, serbia: donji dušnik, 01.06.2007, leg. i. gnjatović; 1♂, serbia: bosilegrad, 10.08.2008, leg. o. stojnev. corymbia scutellata (fab. 1781) – 1♀, serbia: niš, 16.07.2009, leg. i. gnjatović. leptura maculata poda, 1761, synonym rutpela maculata (poda 1761) – 1♀, 1♂, serbia: donji dušnik, 06.06.2006, leg. i. gnjatović; 1♂, serbia: novo selo, 19.07.2007, leg. j. mirković; 2♂, serbia: niš, 07.06.2009, leg. m. ristić; 1♀, serbia: vladičin han, 18.07.2008, leg. i. gnjatović; 1♂, serbia: vladičin han, 13.08.2008, leg. i. gnjatović; 1♀, 1♂, serbia: suva mt., 25.06.2007, leg. o.stojnev; 1♂, serbia: niš, 15.04.2009, m. stamenković; 1♂, serbia: babička gora, 05.07.2008, leg. i. gnjatović; 1♂, serbia: vrelo, 03.06.2006, leg. i. gnjatović; 1♂, serbia: stara mt., 11.07.2007, leg. i. gnjatović. leptura quadrifasciata l. 1758, -1♀, serbia: vladičin han, 10.08.2008, leg. i. gnjatović . pachytodes cerambyciformis (sch. 1787) -1♂, serbia: vladičin han, 23.08.2008, leg. i. gnjatović; 1♂, serbia: niš, 15.07.2007, leg. o. stojnev; 1♂, serbia: čestin, 27.06.2007, leg. i. gnjatović. pachytodes erraticus (dalman 1817) -1♂, serbia: vladičin han, 21.07.2008, leg. i. gnjatović. tribe rhagiini rhagium mordax deg. 1785 – 2♂, serbia: suva mt., 12.08.2008, leg. i. gnjatović; 1♀, serbia: vladičin han, 22.08.2008, leg. i. gnjatović. rhagium inquisitor l., 1758, – 1♀, serbia: suva mt., 12.08.2008, leg. i. gnjatović. rhagium bifasciatum fab. 1775 – 2♀, serbia: vladičin han, 22.08.2008, leg. i. gnjatović. rhagium sycophanta (sch. 1781) – 1♀, serbia: vladičin han, 22.08.2008, leg. i. gnjatović. tribe stenopterini stenurella bifasciata (mull. 1776) – 1♀, serbia: niš, 15.07.2008, leg. i. gnjatović. tribe tragosomatini vadonia unipunctata (fab. 1787) – 1♂, serbia: donji dušnik, 05.06.2006, leg. i. gnjatović. subfamily spondylidinae tribe asemini arhopalus ferus (mul. 1839) – 1♀, serbia: niška banja, 08.08.2007, leg. i. gnjatović. arhopalus rusticus (l. 1758) – 1♂, serbia: niška banja, 04.09.2007, leg. i. gnjatović; 1♀, serbia: niš: malča, 09.30.2007, leg. i. gnjatović. biologica nyssana 1 (1-2) december 2010: 111-115 gnjatović, i.,žikić, v. cerambycids of southeast serbia… 113 subfamily cerambycinae tribe callichromini aromia moschata (l. 1758) – 3♀, 4♂, serbia: suva mt., 07.07.2006, leg. m. jovanović; rosalia alpina (l.1758) – 1♂, serbia: leskovac, 17.07.2006, leg. i. gnjatović; 1♂, serbia: zlot, 10.08.2007, leg. d. gligorijević; 4♂, serbia: suva mt. 27.07.2007, leg. i. gnjatović; tribe callidini callidium violaceum (l. 1758) – 2♀, serbia: šišmanovac, 03.07.2007, leg. j. mirković; 1♂, serbia: vlasina lake, 22.06.2007, leg. i. gnjatović. hylotrupes bajulus (l. 1758) – 1♀, serbia: vranje, 15.07.2008, leg. i. gnjatović. phymatodes testaceus (l. 1758) – 1♂, serbia: vlasina lake, 22.06.2007, leg. i. gnjatović; 1♂, serbia: niš, 17.05.2005, leg. v. žikić; 1♀, serbia: izvor, 25.07.2006, leg. i. gnjatović; 2♂, 1♀, serbia: niš: novo selo, 22.08.2007, leg i. gnjatović. pyrrhidium sanguineum (l. 1758) – 1♂, serbia: bujanovac, 12.06.2006, leg. i. gnjatović; 2♂, serbia: niš, 12.08.2008, leg. a. savić. ropalopus clavipes (fab. 1775) – 1♀, serbia: vlasina lake, 07.06.2007, leg. i. gnjatović. tribe clytini chlorophorus figuratus (scop. 1763) – 1♂, serbia: vladičin han, 16.08.2008, leg. i. gnjatović. chlorophorus varius (mull. 1766) – 1♀, serbia: niš, 13.08.2009, leg. i. gnjatović. clytus arietis l. 1768, -1♀, serbia: vladičin han, 04.08.2008, leg. i. gnjatović. isotomus speciosus (schn. 1787) – 1♀, 2♀, serbia: niška banja, 23.09.2007, leg. i. gnjatović; 1♀, serbia: niš, 26.09.2009, leg. v. žikić. plagionotus arcuatus (l. 1758) – 1♀, 1♂, serbia: niš, 11.06.2007, leg. i. gnjatović; 2♂, serbia: novo selo, 22.08.2007, leg. i. gnjatović; 1♂, serbia: niš, 16.08.2008, leg. i. gnjatović; 1♂, serbia: zaječar, 16.08.2009, leg. i. gnjatović; 1♀, serbia: suva mt., 09.07.2007, leg. i. gnjatović; plagionotus floralis (pall. 1776) – 1♀, serbia: niš, 11.06.2007, leg. i. gnjatović; 1♂, serbia: niš, 04.07.2008, leg. i. gnjatović; 1♂, serbia: niš, 05.08.2008, leg. i. gnjatović; 1♂, serbia: niš, 15.04.2009, leg. m. stamenković; 1♀, serbia: kukavica mt., 07.06.2008, leg. i. gnjatović; 1♀, serbia: bujanovac, 04.08.2006, leg. i. gnjatović; 1♂, serbia: donji dušnik, 25.06.2006, leg. i. gnjatović; 1♀, serbia: bujanovac, 13.08.2006, leg. i. gnjatović; 1♂, serbia: petrova reka, 12.06.2006, leg. i. gnjatović. tribe cerambycini cerambyx cerdo (l. 1758) – 1♂, serbia: niš, 06.05.2008, leg. i. gnjatović; 4♀, 4♂, suva mt., 19.05.2008, leg. i. gnjatović. cerambyx scopolii fessly 1775 – 3♀, 2♂, serbia: niš, 06.05.2008, leg. i. gnjatović; 1♂, čestin, 15.06.2008, leg. i. gnjatović; 1♀, 3♂, stara mt., 29.05.2006, leg. i. gnjatović; 1♀, serbia: suva mt.: ličje 23.05.2007, i. gnjatović. tribe purpuriceini purpuricenus kaehleri (l., 1758) – 1♂, serbia: niš, 19.06.2009, leg. m. mladenović. tribe hesperophanini stromatium unicolor (oli., 1795) – 1♀, serbia: niš, 12.08.2007, m. jovanović; 1♂, serbia: niš: jelašnica, 26.08.2007 leg. i. gnjatović; 1♂, serbia: niš: gabrovac, 06.08.2006, leg i. gnjatović; 1♀, serbia: vlasina lake, 03.06.2008, leg. i. gnjatović; 1♂, serbia: niš: jelašnica, 01.10.2007, leg. i. gnjatović; serbia: 1♂, vladičin han 07.08.2008, leg. i. gnjatović; serbia: 1♀, stara mt., 13.07.2007, leg. i. gnjatović; 4♀, 3♂, serbia: niš, 15.07.2009, leg. a. vidojković. *trichoferus fasciculatus (fald.,1837) – 1♀, serbia: ličje, 21.05.2007, leg. i. gnjatović. subfamily lamiinae tribe agapanthini agapanthia cardui (l. 1767) – 1♀, 1♂, serbia: niš, 20.07.2006, leg. i. gnjatović. agapanthia dahli (rich. 1821) – 1♀, 1♂, serbia: niš, 11.07.2009, leg. a. vidojković; 1♂, serbia: niš: čegar, 12.06.2008, leg. i. gnjatović; 1♂, serbia: petrova reka, 12.06.2006, leg. i. gnjatović. agapanthia violaceae (fab. 1775) – 1♂, serbia: niš, 21.08.2008, leg. i. gnjatović; 1♂, serbia: niš, 20.07.2009, leg. i. gnjatović; 1♀, serbia: vlasina lake, 16.06.2007, leg. i. gnjatović; 1♀, serbia: niška banja, 17.07.2008, leg. i. gnjatović; 1♀, serbia: vranje, 10.06.2007, leg. i. gnjatović. agapanthia villosoviridescens (de geer 1775) – 1♀, 1♂, srebia: niška banja, 10.09.2007, leg. i. gnjatović; 1♀, 1♂,, serbia: niš; novo selo, 22.08.2007, leg. i. gnjatović; 2♀, serbia: zlot, 18.08.2007, leg. d. gligorijević; 1♀, serbia: batinac, 10.04.2007, leg. s. pištoljević. tribe mesosini mesosa nebulosa (fab. 1781) – 1♀, serbia: vlasina lake, 16.06.2007, leg. i. gnjatović; 1♀, serbia: niš, 04.07.2009, leg. i. gnjatović. tribe monochamini monochamus galloprovincialis (oli. 1795) – 1♂, serbia: niš, 16.06.2007, leg. i. gnjatović. tribe lamiini morimus funereus muls. 1863 – 1♂, serbia: niš: delijski vis, 13.04.2009, leg. m. stanković; 1♂, serbia: niš: delijski vis, 16.09.2009, leg. m. stanković; 4♀, 1♂, serbia: suva mt., 12.08.2008, leg. i. gnjatović. biologica nyssana 1 (1-2) december 2010: 111-115 gnjatović, i.,žikić, v. cerambycids of southeast serbia… 114 tribe phytoeciini *opsilia molybdaena (dal. 1817) – 1♂, serbia: niš, 06.07.2007, leg. i. gnjatović. phytoecia icterica (schall. 1783) – 1♂, serbia: niška banja, 25.05.2008, leg. i. gnjatović. phytoecia pustulata (sch., 1776) – 1♂, serbia: niš, 06.07.2007, leg. i. gnjatović. phytoecia virgula (char., 1825) – 1♂, serbia: niš, 08.08.2008, leg. i. gnjatović. tribe tetropini tetrops starkii chev. 1859 – 1♂, serbia: bovan lake, 01.05.2009, leg. v. žikić. table 1. the list of new species of investigated teritory. subfamily species locality prioninae brachyta balcanica (hampe, 1870) niš cerambycinae trichoferus fasciculatus (fald.,1837) ličje lamiinae opsilia molybdaena (dal., 1817) niš figure 1. map of serbia (utm 10x10), showing sites were investigated material was collected: en06 bujanovac; en17 vranje; en28 vladičin han, babička gora; en37 kukavica mt.; en53 šišmanovac; en77 leskovac; en79 donji dušnik, ličje; en89 suva mt., bojanine vode; en96 novo selo; en97 niš, gabrovac, delijski vis; en98 niška banja, jelašnica; ep07 čegar, vrelo; ep28 batinac; ep35 bovan lake; ep77 zlot; fn02 bosilegrad; fn20 vlasina lake; fn61 izvor; fn80 bela palanka – čiflik; fn81 petrova reka; fp02 vitkovac; vp30 stara mt.; vp50 zaječar; dp čestin conclusion we presented 49 species of the family cerambycidae from 34 genera. new records for the fauna of southeast serbia are: brachyta balcanica (prioninae), trichoferus fasciculatus (cerambycinae) and opsilia molybdaena (lamiinae). acknowledgment. the 1st author wish to express his thankful to dr p. jakšić (niš, serbia) for his help during research. the 2nd author wish to express his sincere thanks to the ministry of science and environment protection of the republic of serbia (grant no. 143006b). references adamović, ž. 1950: zbirka cerambycidae u prirodnjačkom muzeju srpske zemlje, 1. deo: prionini, cerambycini. glasnik prirodnjačkog muzeja srpske zemlje, beograd. b, (3-4), 343357. bense, u. 1995: longhorn beetles: illustrated key to the cerambycidae and vesperidae of europe. margraf verlag, weikersheim, 512 pp. bílý, s., mehl, o. 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mikšić, r., georgijević, e. 1973: cerambycidae jugoslavije, 2. deo. akademija nauka i umjetnosti bosne i hercegovine, sarajevo., 153 pp. mikšić, r., korpič, m. 1985: cerambycidae jugoslavije, 3. deo. akademija nauka i umjetnosti bosne i hercegovine, sarajevo, 145 pp. pil, n., stojanović, d. 2009: theophilea subcilindricollis hladil, 1988 a new longhorn beetle (coleoptera: cerambycidae) for serbian fauna. acta entomologica serbica, 14 (1): 125128. boskov et al. 2022, biologica nyssana 13(2) 13 (2) december 2022: 119-128 doi: 10.5281/zenodo.7437250 quality and stability of topical formulation based on the extract of black locust flower (robiniae pseudoacaciae flos) original article ivana a. boskov faculty of technology, university of niš, bulevar oslobođenja 124, 16000 leskovac, serbia ivannab.92@gmail.com (corresponding author) ivana m. savić gajić faculty of technology, university of niš, bulevar oslobođenja 124, 16000 leskovac, serbia ivan m. savić faculty of technology, university of niš, bulevar oslobođenja 124, 16000 leskovac, serbia received: june 30, 2022 revised: october 15, 2022 accepted: october 22, 2022 abstract: black locust flowers are the source of a significant amount of antioxidants that can be useful against free radicals formed on the skin after the environmental factors. having in mind these facts, the ultrasound-assisted extraction of antioxidants from plant materials was previously developed. the aim of this study was to prepare a topical formulation based on the extract of black locust flowers. the extract enriched with antioxidants was prepared using the ultrasound-assisted extraction using 60% (v/v) ethanol at 60 °c and a liquidto-solid ratio of 10 ml/g for 30 min. the two topical formulations in the form of o/w emulsion at different concentrations of the extract (1% and 2%) were prepared by a hot-hot emulsification process. the product quality was estimated and concluded that the emulsion is white in the semi-solid state. the formulations can have adequate application properties due to their present ingredients, which do not leave a greasy film on the skin. the ph value of the prepared formulations was according to the allowed range for the application to the skin. the proper choice of ingredients enabled the achievement of satisfactory formulation stability. the formulations can be microbiologically safe to use for human purposes. a better antimicrobial activity against the investigated microorganism strains showed the formulation with higher content of the extract so it can be selected as an adequate for further analysis. key words: black locust flowers, extract, topical formulation, quality control, stability apstrakt: kvalitet i stabilnost topikalne formulacije na bazi ekstrakata cvetova bagrema (robiniae pseudoacaciae flos) cvetovi bagrema su izvor značajnih količina antioksidanasa koji mogu biti korisni za hvatanje slobodnih radikala nastalih na koži nakon dejstva spoljašnjih faktora. imajući u vidu ove činjenice, ultrazvučna ekstrakcija antioksidanasa iz biljnog materijala je prethodno razvijena. cilj ove studije je bio da se pripremi topikalna formulacija na bazi ekstrakta cvetova bagrema. ekstrakt obogaćen antioksidansima je pripremljen primenom ultrazvučne ekstrakcije korišćenjem 60% (v/v) etanola na 60 °c i pri odnosu tečnosti i čvrste materije 10 ml/g u toku 30 min. dve topikalne formulacije u obliku u/v emulzije pri različitim koncentracijama ekstrakta (1% i 2%) pripremljene su toplo-toplo emulzifikacionim procesom. kvalitet proizvoda je procenjen i zaključeno je da su emulzije bele polučvrste konzistencije. formulacije mogu da imaju adekvatna aplikativna svojstva zbog prisutnih sastojaka, s obzirom da ne ostavljaju mastan film na koži. ph vrednost pripremljenih formulacija bio je u skladu sa dozvoljenim opsegom za primenu na koži. pravilan izbor sastojaka omogućile su postizanje zadovoljavajuće stabilnosti formulacije. formulacije mogu da budu mikrobiološki bezbedne za ljudsku upotrebu. bolju antimirkobnu aktivnost na analiziranim sojevima mikroorganizmima ispoljila je formulacija sa većim sadržajem ekstrakta, tako da za dalju analizu može biti pogodna. ključne reči: cvetovi bagrema, ekstrakt, topikalna formulacija, kontrola kvaliteta, stabilnost introduction the use of medicinal plants and their extracts in pharmaceutical and cosmetic industries increases due to the desire for a healthier lifestyle and youthful appearance. medicinal plants contain bioactive compounds, which have positive effects on human health (veiga et al., 2020). among the bioactive © 2022 boskov et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 119 compounds, polyphenols retaken a significant place. this group of compounds, also known as secondary metabolites of plants, shows antioxidant (de lima cherubim et al., 2020), antimicrobial, anticancer (dzah et al., 2020), anti-inflammatory, anticollagenase, and antielastase activities (madan & nanda, 2018). according to these properties and the ability to pass through the stratum corneum and penetrate the epidermis and dermis, they are suitable for topical application (zillich et al., 2015). they are topically applied for care (guimarães et al., 2021) and photoprotection of skin (farjadmand et al., 2021), as well as for the prevention of skin cancer (souto et al., 2019). to demonstrate their certain biological activities, polyphenolic compounds have to release from the formulation. the formulations should be chemically, physically, and microbiologically stable. to ensure good migration of polyphenolic compounds into the skin, their precipitation in the vehicle of the formulation should not occur. polyphenolic compounds precipitate rapidly because of their poor solubility in water (figueroa-robles et al., 2020). the solubilization of polyphenolic compounds can be improved and their precipitation can be prevented by the addition of surfactants, such as labrasol and tween (jee et al., 2019) or block copolymers (shin et al., 2018) in the formulation. emulsions are the most suitable type of vehicle for topical formulations since they have adequate solubilization capacity for lipophilic and hydrophilic ingredients (yang et al., 2020). the lower content of the oily phase in the emulsions makes it easier to release polyphenolic compounds and a higher rate of their penetration through the skin (zillich et al., 2013). this type of emulsion has a lower viscosity, which allows better diffusion of polyphenols within the emulsions. skin hyperhydration can be achieved using formulations with a high content of water (o/w emulsion) that cause the higher permeability of polyphenols through the skin. the addition of aqueous and oily plant extracts does not disturb the structure of the emulsion itself (smaoui et al., 2012), but it may affect its rheological properties. due to the dilution effect and the interaction of polyphenols with protein or polysaccharide emulsifiers (li et al., 2019), the viscosity is increased by the addition of the extract (leite et al., 2019). the interactions of polyphenols with nonionic or anionic surfactants, commonly used in emulsions, are rarely described. on the other hand, polyphenols have also surfactant properties (katsouli et al., 2017). the oxidative and storage stability of emulsions can be improved in the presence of polyphenols (choulitoudi et al., 2021). there are dermo-cosmetics formulations with polyphenolic compounds (quercetin, resveratrol, gallic acid, rutin, curcumin, apigenin, etc.) and 120 biologica nyssana ● 13 (2) december 2022: 119-128 boskov et al. ● quality and stability of topical formulation based on the extract of black locust flower (robiniae pseudoacaciae flos) extracts enriched with these compounds (grape extract, liquorice root extract, aloe vera extract, calendula extract, lilac extract, lavender extract, etc.) in the market. due to the growing market demand for dermo-cosmetic products recognized as safe, curative, and with synergistic or multifunctional effects, there is a continual need to find new sources of polyphenolic compounds. according to our previous studies, the ethanol extract of black locust flowers is an excellent source of polyphenolic compounds with expressed antioxidant activity (savic gajic et al., 2019). the aim of this study was to prepare the topical formulation with the extract of black locust flowers enriched with antioxidants. based on the properties of the topical formulation and its physico-chemical stability, the product quality was estimated. materials and methods in this study, sabowax sx and sabonal c1618 (cetearyl alcohol) (sabo s.p.a., italy), white beeswax (ceraalba, extra pure, centrohem, srbija), triethanolamine (care basf, germany), phenoxyethanol sa (schülke & mayr gmbh, germany), mineral oil (techno-chem d.o.o., serbia), glycerol (85%), carbomer, trichloroacetic acid (sigma-aldrich, serbia), and distilled water (aqua purificata) was used. ethanol extract of black locust flowers was obtained by ultrasound-assisted extraction (savic gajic et al., 2019). preparation of topical formulation the o/w topical formulations with and without (base) black locust flower extract were prepared according to tab. 1. the oily and water phases were prepared separately by heating in enameled paten via water bath at a temperature of 70±1 °c. the topical formulation was prepared by the hot-hot emulsification process. this process involves the addition of the oily phase into the water phase gradually with stirring using a laboratory propeller mixer at 800 rpm and constant temperature. after the emulsification process, a stirring of the phases was continued at 500 rpm until the temperature was below 40 °c. phenoxyethanol sa was added as a preservative after cooling the sample. triethanolamine was added to adjust the ph of the formulation. the prepared formulations were stored at room temperature (22±2 °c) in a plastic polypropylene container. the base was prepared in the same way but without the addition of the black locust flower extract. formulation quality assay organoleptic properties (appearance, color, homogeneity, and separation of phases) of the base biologica nyssana ● 13 (2) december 2022: 119-128 boskov et al. ● quality and stability of topical formulation based on the extract of black locust flower (robiniae pseudoacaciae flos) 121 and topical formulations with black locust flower extract (f1 and f2) were assessed by visual observation (ph. jug. iv, 1984). determination of emulsion type was achieved by electrical conductivity measuring (stojiljković et al., 2013). the measurement of electrical conductivity was performed by direct immersion of the conductometer electrode (cdm 230, radiometer, copenhagen, denmark) into the creams samples at room temperature (22±2 °c). before starting the work, the measuring electrode was calibrated with 0.01 mol/lkcl solution. to measure the ph value of the samples, a potentiometric method was used (stojiljković et al., 2013). a glass electrode of ph meter (hi 9321, hanna instruments, lisbon, portugal) was directly immersed in the samples at room temperature (22±2 °c). the device was calibrated with standard ph buffers of 4.0 and 7.0. the viscosity of prepared formulations was determined using a viscosimeter (visco basic plus, fungilab inc., usa) with an sp-r5 spindle, at a 12 rpm rotation speed (maia filho et al., 2012). physical stability of the formulations centrifugation assay was applied to assess the physical stability of the prepared samples on the gravity effect according to the previously described method with slight modification (juttulapa et al., 2017). precisely 4 g of the samples were weighed into a 10 ml plastic test cuvette and centrifuged twice at 3000 rpm for 15 min. a laboratory centrifuge (lc 320, tehtnica, železniki, slovenia) was used. the measurements were performed at room temperature (22±2 °c) 24 h after preparation. the samples were observed visually to notice eventual changes (phase separation). cyclic temperature stress test or accelerated aging test of formulations was realized through 6 cycles. each cycle included the following steps: 1) the samples were placed in the freezer at -10 °c for 24 h; 2) the samples were removed from the freezer and left at room temperature (22±2 °c) for 24 h; 3) the samples were placed in thermostat oven (sutjeska, yugoslavia) at the temperature of 40 °c for 24 h; 4) the samples were removed from the thermostat oven and left at room temperature (22±2 °c) for 24 h. at the same time, one sample was left under the isothermal condition at room temperature (22±2 °c), in a fridge at +4 °c, and in a thermostat oven at +40 °c. after the third and sixth cycles, the ph value and electrical conductivity were measured in each sample. if the product is stable, the appearance, color, and consistency should not change. a long-term aging test or shelf test was applied to estimate the physical stability of the prepared formulations based on the changes in organoleptic properties, ph values, and electrical conductivity. the samples were stored in primary packaging (plastic containers) at room temperature for 3 months, and the tests were performed 7, 30, 60, and 90 days after their preparation. the chemical stability of the extract in pure form and after its incorporation into the formulations (f1 and f2) was assessed based on the change in total antioxidant content (tac) over time. the tac in the black locust flower extract and formulations was determined by the folin-ciocalteu method (savic gajic et al., 2019). the analyzed samples ingredients base topical formulation f1 f2 oily phase white beeswax 1 1 1 mineral oil 6 6 6 sabowax sx 5 5 5 sabonal c1618 (cetearyl alcohol) 6.5 6.5 6.5 trichloroacetic acid 7 7 7 triethanolamine 0.7 0.7 0.7 phenoxyethanol sa 0.2 0.2 0.2 water phase water 68.1 67.1 66.1 carbomer 0.5 0.5 0.5 glycerin 5 5 5 black locust flower extract 1 2 table 1. composition of topical formulations expressed in the percentage (f1-topical formulation with 1% of the extract; f2–topical formulation with 2% of the extract) 122 biologica nyssana ● 13 (2) december 2022: 119-128 boskov et al. ● quality and stability of topical formulation based on the extract of black locust flower (robiniae pseudoacaciae flos) were exposed to the influence of lower and higher temperatures (-18 °c, +4 °c, and +22 °c), as well as daylight/darkness for 3 months. sampling was performed after 7, 30, 60, and 90 days, whereby the tac expressed as milligrams of gallic acid equivalent per 100 g of dry weight (mg gae/100 g) was determined for each sample. the absorbance of the samples was measured at 740 nm after incubation at room temperature (+22 °c) for 30 min. varian cary-100 uv-vis spectrophotometer (malgrave, victoria, australia) and 1×1 cm quartz cuvettes were applied for scanning the samples. microbiological safety of formulations the method is based on the isolation and identification of bacteria, yeasts, and molds present in the sample (https://www.paragraf.rs/ propisi/pravilnik-uslovima-pogledu-zdravstveneispravnosti-predmeta-opste-upotrebe.html).the samples were seeded on a nutrient medium and incubated under optimal conditions. antimicrobial activity antimicrobial activity of black locust flower extract, the base, and formulations was examined against gram-positive (staphylococcus aureus atcc 6538, streptococcus pneumoniae atcc 49619) and gram-negative bacteria (escherichia coli atcc 8739, proteus mirabilis atcc 25933, klebsiella pneumoniae atcc 10031), as well as on fungi (candida albicans atcc 10231) using disk diffusion method. the samples were dissolved in dimethyl sulfoxide (dmso) which was used as a negative control. the antibiotic medium-1 nutrient medium was used for the growth of bacteria, while sabouraud 4% dextrose agar nutrient medium was used for fungal growth. an adequate amount of nutrient medium was dissolved in purified water and then sterilized at 121 °c in an autoclave for 15 min. the sterilized medium was cooled to 40–45 °c and an appropriate concentration of microorganisms’ suspension was added to it. the nutrient medium (15 ml) was poured into petri dishes with a diameter of 90 mm. the paper discs with a diameter of 6 mm, soaked with 30 μl of extract (1 mg/ml) and formulations (0.1 g/ml) were arranged on a cooled and solid surface. gentamicin was used as a positive control. bacteria were incubated at 37 °c for 18–24 h and fungi at 25 °c for 24–48 h under anaerobic conditions. after the incubation, the inhibition zones were measured in millimeters. based on the inhibition zones, the antimicrobial activity of the samples was estimated. statistical analysis all obtained data were presented as mean value±standard deviation. all statistical analyzes were performed using the originpro9 software (originlab corporation, northampton, usa), with a significance level of p<0.05. results and discussion the o/w emulsion formulations were prepared by a technological procedure in the laboratory conditions. the oily phase consisted of white beeswax, mineral oil, sabowsax sx, cetearyl alcohol (sabonal c1618), trichloroacetic acid (tca), triethanolamine, and phenoxyethanol sa. white beeswax is a mixture of esters of higher fatty acid and higher fatty alcohols, free fatty acids and alcohols, carotenoids, aromatic and mineral substances. due to its regenerative, emollient, and soothing properties, white beeswax is suitable for dry skin and winter skincare routines. mineral oil is the odorless, colorless, and tasteless oily liquid of mineral origin. this oil is a mixture of saturated hydrocarbons, which due to their size cannot penetrate the skin and therefore do not close the skin pores. it is not carcinogenic and does not cause allergic reactions on the skin. it can form a protective layer on the skin that prevents transdermal water loss, making skin soft and smooth. white beeswax and mineral oil, in addition to achieving an occlusive effect, are also important for regulating the consistency of the developed formulation (baumann, 2011). sabowax sx is an anionic self-emulsifying o/w base and presents a mixture of cetearyl alcohol, fatty alcohols, and sodium alkyl sulfate. in topical formulations, it is used in concentrations of 4–7% and can improve the stability and consistency of the preparation. cetearyl alcohol (sabonal c1618) is a fatty alcohol used as an emulsifier in the formulations, improving the viscosity of the product. it has a soothing effect on the skin, making it smooth and soft. in the formulations, tca can be used in different concentrations depending on the purpose. it has a significant role in smoothing fine surface wrinkles, removing surface stains, and correcting pigment problems. in the developed formulations, triethanolamine was used as a ph adjusting agent. depending on the desired ph value, it can be used in the concentration of 0.1–1%. due to good antimicrobial properties and low toxicity, phenoxyethanol sa was used as a preservative for the developed formulation. if the formulation contains only one preservative, the maximum permissible concentration is 0.4%, while for a mixture of preservatives, the allowable value is 0.8%. unlike the oily phase, the water phase consisted of demineralized water, carbomer, and glycerin. prepared ethanol extract of black locust flower in the concentration of 1% and 2% was added to the water 123 biologica nyssana ● 13 (2) december 2022: 119-128 boskov et al. ● quality and stability of topical formulation based on the extract of black locust flower (robiniae pseudoacaciae flos) al., 2013). the electrical conductivity for the base, f1, and f2 of 78, 86, and 91 μs/cm, respectively, indicate that the prepared formulations are o/w emulsion. the physiological ph value of skin ranges from 4 to 7 (lambers et al., 2006), while the average value is 5.5. topical formulations should have a ph value close to this range. the ph values of the base, f1, and f2 were 6.41, 6.38, and 6.35, respectively. the addition of the extract to the base led to a slight decrease in the ph value (akhtar et al., 2011). the viscosity of the formulation is important for handling and storage. based on this parameter, the stability of the product is possible to evaluate. the measured values for the viscosity of base, f1, and f2 of 13.522, 13.261, and 12.789 mpa·s, respectively, indicated that the formulations were semi-solid consistent. the addition of extract to the base led to a decrease in viscosity. physical stability of formulations the prepared formulations were physically stable because there was no phase separation after the action of mechanical stress (centrifugation assay). shelflife assessment is a bottleneck during designing a new product. therefore, it is necessary to lead an „accelerated agingˮ test. applying this test in a short time interval can provide information about the physical stability of the product. in that case, it is necessary to observe parameters (ph value, electrical conductivity, stratification rate, flocculation, etc.) with rapid changes in values under the influence of some external factors. temperature is one of phase. the extract was obtained by ultrasoundassisted extraction and characterized in our previous study (savic gajic et al., 2019). the tac in the extract was 3.12 gae/100 g d.w. among the identified individual polyphenolic compounds, rutin was the most abundant compound (56.9 mg/100 g d.w.). the ic50 value of the extract was 120.5 µg/ ml, which makes it suitable for the development of dermo-cosmetic formulations. the water used in the formulations was toxins and microbes free. carbomer found wide application in the topical products as a safe substance with extremely low irritant and sensitizing potential. in the developed formulations, a carbomer was used to stabilize the emulsion. it should be in the concentration range of 0.1-0.5% to use as an emulsion stabilizer. glycerol as humectant was added to the aqueous phase of the emulsion. it is known that glycerol forms an invisible film on the skin which binds water molecules from the air and thus hydrates the skin. formulation quality assay the prepared formulations were a semi-solid consistency, homogeneous, white, and with a characteristic smell. the adequate selection of oily phase ingredients enabled the samples to be stable 24 h after preparation. determination of electrical conductivity is one of the most reliable methods for the determination of emulsion type. according to the literature data, the electrical conductivity higher than 50 μs/cm indicates o/w emulsion, while values less than 1 μs/cm indicates w/o emulsion (jiang et sample storage conditions parameters ph electrical conductivity (ms/cm) f1 cycle iii 6.39 85 cycle vi 6.34 86 f2 cycle iii 6.36 90 cycle vi 6.33 90 base cycle iii 6.41 77 cycle vi 6.39 78 comparative f1 +4 °c 6.38 86 room temperature 6.38 87 +40 °c 6.22 88 comparative f2 +4 °c 6.35 91 room temperature 6.36 92 +40 °c 6.19 91 comparative base +4 °c 6.39 76 room temperature 6.38 78 +40 °c 6.23 79 table 2. ph and electrical conductivity values of the formulations after the cyclic temperature stress test 124 biologica nyssana ● 13 (2) december 2022: 119-128 boskov et al. ● quality and stability of topical formulation based on the extract of black locust flower (robiniae pseudoacaciae flos) the most commonly used factors for accelerated testing of product stability. in the cyclic temperature stress test, the sample was alternately exposed to higher and lower temperatures, which can cause destabilizing phenomena (stratification, flocculation, and coalescence). in tab. 2, the measured values of ph and electrical conductivity for the samples after iii and iv cycles are presented. the comparative formulations were stored isothermally at +4 °c, room temperature (+22 °c), and +40 °c. the base, f1, and f2 had a slightly lower ph value after cycle vi compared to cycle iii. in comparative samples of the formulation was observed a slight decrease of ph value at +40 °c. the high temperature probably destabilized the formulation, but it did not affect the overall product quality because the ph value was still around 6.0. this value is acceptable since it is close to physiological ph and does not irritate the skin. the changes in electrical conductivity of samples were insignificant under all examined conditions. after using the cyclic temperature stress test, the prepared formulations were relatively stable, because the significant changes in the ph value and electrical conductivity were not noticed. also, the changes in the organoleptic properties were not observed. in addition to the accelerated aging tests (centrifugation assay and cyclic temperature stress test), a long-term aging test was also applied for assessing the physical stability of the prepared formulations. the samples were stored at room temperature after preparation for 3 months. the ph value and electrical conductivity were measured after 7, 30, 60, and 90 days of formulation preparation (tab. 3) the ph values of the formulations were decreased but in the range of acceptable limits. the decrease in the ph value was less than 10% for the formulations stored at room temperature for 90 days. these results indicated that the formulations are physically stable. unlike the ph value, the electrical conductivity was increased in all samples during the time. after the storage, the samples were without visible changes in the organoleptic properties. this behavior is the result of good physic-chemical properties and longterm stability of the formulations. table 3. the change of ph and electrical conductivity values of formulations during storage sample ph electrical conductivity (ms/cm) day 7 day 30 day 60 day 90 day 7 day 30 day 60 day 90 f1 6.34 6.19 6.02 5.90 87 88 89 90 f2 6.31 6.16 5.96 5.86 91 93 95 96 base 6.38 6.22 6.06 5.99 79 81 83 85 chemical stability of formulations polyphenols in their structure contain one or more benzene rings to which are attached at least two hydroxyl groups. these groups are highly reactive and susceptible to epimerization, autooxidation, esterification, alkylation, carboxymethylation and dealkylation reaction, chelate formation, and other reactions. these reactions result in a decrease in the stability or loss of the pharmacological activity of polyphenols (garcia et al., 2016). among the environmental factors, the effect of temperature and light were studied on the stability of polyphenols. to investigate the effect of temperature on stability, black locust flower extract was stored at -18 °c, +4 °c, and room temperature for 90 days. at -18 °c, a significant change in the tac (less than 2%) was not observed. this slight decrease is most likely due to the inhibition of phenoloxidase activity at lower temperatures (wei & zhang, 2008). at +4 °c and room temperature, the decrease in the tac was about 8% until day 90 which was in accordance with available results (tsali & goula, 2018). the effect of temperature on the stability of formulations was carried out in the same way as in the case of the extract. the smallest decrease in the tac was noticed for the samples stored at -18 °c (less than 1%), while this decrease was about 4% at higher temperatures (+4 °c and room temperature). the formulation can be considered stable since the decrease in the tac was less than 5% compared to the initial value (ich q1a (r2)). the satisfactory chemical stability of the formulations is the result of an adequate choice of ingredients, especially those in the oily phase. there is no available literature data about the stability of black locust flower extract. srivastava & gupta (2009) monitored the chemical stability of aqueous and methanol chamomile flower extract at different temperatures (-20, +4, and +25 °c) based on the change in the flavonoid content and obtained similar results. at +4 °c and room temperature, the flavonoid content decreased about 10% during 120 days. the storage of prepared chamomile extracts at -20 °c was not caused a significant reduction in the flavonoid content. certain polyphenolic compounds, such as quercetin (golonka et al., 2020), resveratrol (chen 125 biologica nyssana ● 13 (2) december 2022: 119-128 boskov et al. ● quality and stability of topical formulation based on the extract of black locust flower (robiniae pseudoacaciae flos) et al., 2020), and catechin (yuann et al., 2021), etc. are susceptible to photodegradation. isomerization and polymerization are the most common reactions that occur in the extract under the light effect (deng et al., 2018; latva-mäenpää et al., 2021). the chemical stability of extract and formulations stored in daylight and the dark for 90 days was estimated based on the changes in the tac. after 90 days of storage in daylight and the dark, the change in the tac was about 3% and 2%, respectively. also, the reduction in the tac was about 1.5% in daylight and 1.2% in the dark after 90 days. the formulation remained photostable after the addition of the black locust flower extract. also, it can be concluded that there were no interactions between the extract and other ingredients. microbiological safety of formulations microbiological analysis of the samples showed that the total number of aerobic mesophilic bacteria, yeasts, and mold spores in 1 g of the sample corresponds to the permissible number of microorganisms according to regulations (https:// www.paragraf.rs/propisi/pravilnik-uslovimapogledu-zdravstvene-ispravnosti-predmeta-opsteupotrebe.html). the presence of pathogenic bacteria was not confirmed in 0.1 g of the sample. therefore, it can be concluded that the prepared formulations were microbiologically safe. antimicrobial activity the antimicrobial activity of formulations with black locust flower extract (0.1 g/ml) was tested on appropriate strains of microorganisms. the base and extract were prepared at the concentration of 1 mg/ml and considered the positive controls. the results of antimicrobial activity are shown in tab. 4. both dmso solvent and the base did not show an inhibitory effect against the microorganism strains. gentamicin affected only bacteria, while it did not show an effect on the analyzed fungus. black locust flower extract showed the strongest activity against gram-positive bacteria (s. aureus atcc 6538, s. pneumoniae atcc 49619) and weaker against gram-negative (e. coli atcc 8739, p. mirabilis atcc 25933, k. pneumoniae atcc 10031). it did not show an inhibitory effect against the fungus c. albicans atcc 10231. s. aureus bacterium causes skin infections, such as ulcers, carbuncles, impetigo and cellulitis. this bacterium can also cause serious infections of postoperative wounds and burns. since the extract showed the strongest activity against these strains, it can be used in the treatment of skin infections. rosu et al. (2012) showed that ethanol extract of black locust flower has an effect on gram-positive bacteria at the concentration of 100 mg/ml. black locust flower essential oil has antimicrobial activity against selected food pathogens, such as s. aureus kctc 1621, bacillus subtilis kctc 3569, listeria monocytogenes kctc 3569, e. coli o157:h7, and salmonella enterica atcc 4731 with mic and mbc values of 250-1000 mg/ml (bhalla & bajpai, 2017). conclusions the topical formulations with black locust flower extract were developed in the laboratory conditions. the formulation was free of synthetic fragrances, colors, and aggressive emulsifiers. the prepared formulations were safe to use and their measured ph was about 6 corresponding to the ph value of healthy skin. the viscosity of 13.261 and 12.789 mpa·s for f1 and f2, respectively, indicated that the prepared formulations were semi-solid consistency. the formulations were stable since the significant changes in the ph value, electrical conductivity, organoleptic properties, and tac were not noticed during storage. satisfactory product stability was the result of an adequate choice of ingredients, especially for the oily phase. the optimal conditions microorganisms extract f1 f2 base dmso gentamicin s. aureus atcc 6538 +++ ++ +++ +++ s. pneumoniae atcc 49619 +++ ++ ++ +++ e. coli atcc 8739 ++ + ++ ++ p. mirabilis atcc 25933 ++ + + ++ k. pneumoniae atcc 10031 ++ + c. albicans atcc 10231 table 4. results of antimicrobial activity using disk diffusion method no antimicrobial activity (-), inhibition zone <15 mm. weak antimicrobial activity (+), inhibition zone of 15–16 mm. moderate antimicrobial activity (++), inhibition zone of 17–19 mm. high antimicrobial activity (+++), inhibition zone of 20–22 mm. strong antimicrobial activity (++++), inhibition zone of >23 mm. standard deviation±0,5 mm. biologica nyssana ● 13 (2) december 2022: 119-128 boskov et al. ● quality and stability of topical formulation based on the extract 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(scrophulariaceae) and implications for conservation biologica nyssana 7 (2)  december 2016: 101-106 petrova, g. et al.  genetic diversity of the critically endangered verbascum… 101 original article received: 08 october 2016 revised: 30 october 2016 accepted: 05 november 2016 genetic diversity of the critically endangered verbascum davidoffii murb. (scrophulariaceae) and implications for conservation galya petrova1*, stefan petrov2, svetlana bancheva3 1laboratory “photosynthesis activity and regulation”, institute of plant physiology and genetics, bulgarian academy of sciences, “acad. g. bonchev” str. 21, 1113 sofia, bulgaria 2gene regulation department, institute of molecular biology “roumen tsanev”, bulgarian academy of sciences, “acad. g. bonchev” str. 21, 1113 sofia, bulgaria 3department of plant and fungal diversity and resources, institute of biodiversity and ecosystem research, bulgarian academy of sciences, “acad. g. bonchev” str. 21, 1113 sofia, bulgaria * e-mail: galiaty@abv.bg abstract: petrova, g., petrov, s., bancheva, s.: genetic diversity of the critically endangered verbascum davidoffii murb. (scrophulariaceae) and implications for conservation. biologica nyssana, 7 (2), december 2016: 101-106. verbascum davidoffii murb. (scrophulariaceae), one of the rarest plant species in bulgarian flora, is a local endemic, protected by the national biodiversity act, included in the red list of vascular plants, as well as in the red data book of bulgaria with conservation status “critically endangered”. its distribution is limited due to anthropogenic pressure, specific ecological requirements and low reproductive capability. in this study, we aimed to measure the genetic diversity level in the unique single world population of verbascum davidoffii located in pirin national park, bulgaria. we found high genetic diversity in the excitant population of the species. the present study indicates that the primary objective in conservation of verbascum davidoffii is to preserve as much as possible of its evolutionary potential. key words: verbascum davidoffii, endemic species, genetic diversity, conservation apstrakt: petrova, g., petrov, s., bancheva, s.: genetički diverzitet kritično ugrožene vrste verbascum davidoffii murb. (scrophulariaceae i implikacije za konzervaciju. biologica nyssana, 7 (2), decembar 2016: 101-106. verbascum davidoffii murb. (scrophulariaceae), jedna je od najređih biljaka flore bugarske i lokalni endemit koji je zaštićen nacionalnim zakonom o biodiverzitetu, uključen u crvenu listu vaskularnih biljaka, kao i u crvenu knjigu bugarske sa konzervacionim statusom “kritično ugrožena”. njeno rasprostranjenje je ograničeno zbog antropogenog pritiska, specifičnih ekoloških prohteva i niske reproduktivne sposobnosti. u ovom istraživanju, naš cilj bio je da merimo nivo genetičkog diverziteta u jedinstvenoj populaciji verbascum davidoffii na svetu, lociranoj u nacionalnom parku pirin, bugarska, gde je utvrđen visok genetički diverzitet. 7 (2) • december 2016: 101-106 12th sfses • 16-19 june 2016, kopaonik mt doi: 10.5281/zenodo.200406 biologica nyssana 7 (2)  december 2016: 101-106 petrova, g. et al.  genetic diversity of the critically endangered verbascum… 102 ovo istraživanje je ukazalo na to da je primarni cilj u konzervaciji vrste verbascum davidoffii da se njen evolucioni potencijal sačuva u što većoj meri. ključne reči: verbascum davidoffii, endemična vrsta, genetički diverzitet, konzervacija introduction bulgarian vascular flora consists of more than 4000 species. this high level of plant diversity is determined by various factors and the geographical position of the country is a main factor, allowing the distribution of diverse floristic elements on a relatively restricted territory. the richest in endemic species are the genera centaurea (cornflower), verbascum (mullein), anthemis (dog fennel), silene (catchfly), etc. (b i s e r k o v et al., 2015). the genus verbascum comprises of about 360 species of flowering plants in the scrophulariaceae family (f i r a t , 2015). verbascum davidoffii murb. (scrophulariaceae), one of the rarest plant species in bulgarian flora, is a local endemic, protected by the national biodiversity act, included in the red list of vascular plants and in the red data book of bulgaria with conservation status “critically endangered”. the unique single world population of the species is located between the valleys of river bunderitsa and razlozhki suhodol, pirin national park, bulgaria (fig. 1). its distribution is limited due to anthropogenic pressure, specific ecological requirements and low reproductive capability (a s s y o v & d e n c h e v , 2015). genetic diversity is of fundamental importance in the continuity of a species as it provides the necessary adaptation to the prevailing biotic and abiotic factors, and enables changes in the genetic composition to cope with environmental changes (r a o & h o d g k i n , 2002). understanding of the genetic variation within and between populations is essential for the establishment of efficient conservation practices for rare plants. small populations are expected to demonstrate lower genetic diversities than larger populations and vice versa (w i l l i et al., 2006; l i et al., 2012). because of the small size of v. davidoffi population, we hypothesized that the genetic diversity of the species will be low. the loss of genetic diversity is one of the key aspects in many conservation programmes of threatened plant species (h a m r i c k & g o d t , 1996). thus, the present study was designed as a first attempt towards investigating the genetic diversity of v. davidoffii. the increasing availability of pcrbased molecular markers allows the detailed analyses and evaluation of genetic diversity in plants (i d r e e s & i r s h a d , 2014). currently, there is no an universal method for all applications and each technique has its own advantages and limitations. among the various types of available molecular fig. 1. geographic location of verbascum davidoffii population in bulgaria. biologica nyssana 7 (2)  december 2016: 101-106 petrova, g. et al.  genetic diversity of the critically endangered verbascum… 103 markers, inter-simple sequence repeats (issrs) do not need prior knowledge of the genome to be analyzed (z i e t k i e w i c z et al., 1994). the issr analysis requires comparatively low amount of dna, the utilization of long primers allows more stringent annealing temperatures and reveals more polymorphic fragments (f a n g & r o o s e , 1997; w o l f e & l i s t o n , 1998; c a m a c h o & l i s t o n , 2001). considering these advantages of issr primers, we attempted to use this marker system in order to analyze the pattern of genetic diversity of v. davidoffii, given that such knowledge will be important for more efficient planning of the strategies for conservation of this endangered plant species. material and methods species analyzed morphology and biology. biennial herbaceous plant. stem up to 80 cm high, usually unbranched. basal leaves obovate, densely hairy. flowers in groups of 2–4 in the upper part of the stem. calyx 5–merous, with black glandular hairs. corolla 5-merous, golden yellow, up to 3.5 cm in diameter. fruit spherical to ovate capsule. fl. vi–vii, fr. vii–ix. reproduction by seeds (a s s y o v & d e n c h e v , 2015). distribution in bulgaria and description of species’ habitat. the population of v. davidoffii consists of very low number of individuals distributed between the valleys of river bunderitsa and razlozhki suhodol, pirin national park. the park encompasses over 40 000 ha of unique nature, at an altitude between 1008 and 2914 m in the pirin mountains, southwest bulgaria. pirin national park comprises diverse limestone mountain landscapes with glacial lakes, waterfalls, caves and predominantly coniferous forests. v. davidoffii inhabits grassy openings and light, rocky places in forests of pinus heldreichii on calcareous soils (a s s y o v & d e n c h e v , 2015). plant material fourteen individuals were sampled during the flowering stage from the v. davidoffii population in pirin national park (latitude (n): 41°76'82''; longitude (e): 23°42'63''). dna extraction genomic dna was extracted from young leaves following the modified ctab procedure of d o y l e & d o y l e (1987). issr analysis issr primers (microsynth, balgach, switzerland) in the initial screening that had a high level of polymorphism, repeatability, and the best scorability were selected, after the screening of 35 primers on the subset of collected samples (tab. 1). polymerase chain reactions were performed in a volume of 25 µl, containing a final concentration of 1 × pcr buffer (fermentas, vilnius, lithuania), 1 u taq dna polymerase (fermentas, vilnius, lithuania), 100 µm of each dntp, 1 µm of each primer and 50 ng of extracted dna. pcr cycling conditions were as follows: 5 min. initial denaturation at 95 °c, 35 cycles of amplification [45 s at 94 °c, 1 min. at the annealing temperature (ta), 2 min. elongation at 72 °c] and a final elongation step of 5 min. at 72 °c. pcr experiments were performed with a tc-5000 gradient thermal cycler (techne, staffordshire, uk). to determine the optimal annealing temperature for each primer, an interval of 10 °c around the melting temperature (tm) was tested. the temperatures leading to clear patterns were then repeated until the optimal ta was selected for each primer for routine issr fingerprinting. the reproducibility of the table 1. sequences, annealing temperature, total number and number of polymorphic bands for selected issr primers in the present study of verbascum davidoffii. primer sequence (5'→3') total number of bands number of polymorphic bands percent of polymorphic loci (%) annealing temperature (ºc) (ga)8t 12 12 100 60 (ga)8a 13 13 100 60 (ca)8g 11 11 100 60 (ac)8t 17 17 100 60 (ac)8c 12 12 100 60 (ac)8g 9 9 100 60 (ag)8yt 9 9 100 55 (ag)8yc 5 5 100 55 (ga)8yg 5 5 100 55 (ac)8yt 10 10 100 55 103 103 100 biologica nyssana 7 (2)  december 2016: 101-106 petrova, g. et al.  genetic diversity of the critically endangered verbascum… 104 technique was tested by replicating each amplification reaction twice. to further ensure the quality, pcr reactions were performed with one positive and one negative control. the pcr products were analyzed on 2% agarose gels (fermentas, vilnius, lithuania) in 0.5 × tbe buffer. a 100-bp plus dna ladder size standard (fermentas, vilnius, lithuania) was used to estimate the length of pcr products. the gels were stained by incorporating 1.5 µl of ethidium bromide (0.5 mg/ml) in 100 ml agarose. electrophoresis was run for 1.5 h at 150 v, the issr profiles were visualized with a uv transilluminator (tfp-m/wl, vilber lourmat, eberhardzell, germany) and further analyzed with a video image analyzer. software data analysis the issr band profiles were treated as dominant markers and each locus was considered as a bi-allelic locus with one amplifiable and one null allele. the well resolved and consistently reproducible amplified dna fragments as bands were scored with regards to their presence (1) or absence (0). the assignment of issr bands to genetic loci was performed semi-automatically using the gelanalyzer 2010a image analysis software (http://www.gelanalyzer.com). the genetic diversity was measured using genalex v.6.5 (p e a k a l l & s m o u s e , 2012). results and discussion genetic diversity the low level of genetic diversity is considered to be a common feature of endemic plant species and it is generally attributable to the small size of their populations (h u a n g et al., 2009; z h u et al., 2009; p e t r o v a et al., 2014). however, in contrast to our expectations, the genetic pattern of v. davidoffii revealed in this study was characterized with a high level of genetic diversity. ten of 35 tested issr primers resulted in polymorphic issr profiles including 4 poly (ac), 3 poly (ga), 2 poly (ag) and 1 poly (ca) dinucleotide primers (tab. 1). the number of bands produced by the used primers ranged between 5 (primers (ag)8 yc and (ga)8yg) and 17 for the primer (ac)8t. the total number of alleles produced by the ten primers is 103. a 100% polymorphism was scored for all primers. examples of photographs illustrating the issr fingerprinting by selected primers are shown in fig. 2. these include issr fingerprinting revealed by primers (ca)8g (fig. 2a) and (ga)8yg (fig. 2b). the data from the issr-analysis were combined in a single matrix and the genetic coefficients (shannon’s information index, si; nei’s genetic diversity, h and unbiased diversity, hu) were calculated to describe the genetic pattern of the excitant population of v. davidoffii. the shannon’s information index (si) was estimated to be 0.406 and the issr derived unbiased diversity (hu = 0.266) from the present study was close to the average value of narrow and outcrossing plant species according to the nybom statistic, where estimates derived by the dominantly inherited markers are very similar and may be directly comparable (n y b o m , 2004). in summary, our study indicated that the critically endangered bulgarian endemic v. davidoffii is able to maintain high level of genetic diversity even its small population size. the observed maintenance of genetic diversity in v. davidoffii is surprising, considering the limited area of distribution of the species, as well as its "critically endangered" status. in recent years, a lot of studies revealed high levels of genetic variability in rare or narrow endemic plants (h e l e n u r m , 2001; z a w k o et al., 2001; x u e et al., 2004; j i a et al., 2016; t u r c h e t t o et al., 2016). among the various factors, the breeding system is an important factor that may be invoked to explain high levels of genetic diversity in rare plants (h a m r i c k & g o d t , 1996). previous studies on the mating system of v. davidoffii suggested that the species is predominantly outcrossing (s t e f a n o v a g a t e v a , 1995). the outcrossing and long-lived figure 2. examples of photographs illustrating issr fingerprints of verbascum davidoffii by issr primers (ca)8g (a) and (ga)8yg (b). lanes 1 ÷ 14 stand for individual samples from the population of the species. m: 100 bp plus dna ladder. biologica nyssana 7 (2)  december 2016: 101-106 petrova, g. et al.  genetic diversity of the critically endangered verbascum… 105 plants commonly have higher levels of genetic diversity than selfing plants (n y b o m , 2004)). therefore, we assume that the high level of genetic load maintained in v. davidoffii is probably due to its reproductive system and may be contributed to the outcrossing behavior of the species. generally, mountain plants are among the species most vulnerable to global warming, because of their isolation and narrow geographic distribution. altitudinal gradients in mountains modify environmental conditions and put populations under different selective pressures that can easily be linked to ongoing climate changes (w a l t h e r et al., 2005; g o n z a l o t u r p i n & h a z a r d , 2009). verbascum davidoffii is distributed in a single high altitude population (a s s y o v & d e n c h e v , 2015). in the present study, we did not observe a correlation between the issr derived genetic diversity of the species and the size of its population despite the theoretical prediction that small populations might lose genetic variation. thus, we hypothesize that the genetic diversity pattern of v. davidoffii may be a result from recently dispersed individuals from different population sources, and it may not respond immediately to the reduction in population size. conservation implications the high genetic diversity of v. davidoffii indicates that the major factors that threaten the persistence of its population are ecological factors rather than genetic. the main risk is that this species is found in only one population and no other natural populations were found. in order to protect it against extinction and loss of its available genetic resources, we recommend collection of seeds from as many individuals as possible and storage in seed banks and living collections. we suggest that conservation programs should be improved to protect its natural habitat. pirin national park was established in 1962 and it was added to the world heritage list in 1983. although, it has long been a subject to tourism pressure, largely caused by the development of ski facilities. such activities may affect the values and integrity of the property and therefore require rigorous control. the development of sustainable forms of tourism would guarantee preservation of ecosystems in the park. we recommend the establishment of variety of educational programs that directly present conservation and biodiversity issues. the key outcomes include enhanced visitor experience, administrative support, volunteer activities and ecological restoration. conclusion the high level of genetic diversity enables v. davidoffi to adapt to changing environments. the maintenance of genetic diversity in the excitant population of the species is an indicator that its current endangered status is not caused by genetic factors. the results reported herein suggest that the high genetic diversity of the species may derive from an ancestral population. however, the ecological requirements of the species, as well as its origin and relationship with other related species should be further elucidated. acknowledgements. the authors wish to thank chief assistant stoyan stoyanov (iber-bas) and dr. boryana sidjimova (iber-bas) for the samples of v. davidoffii used in this work. this work was supported by the bulgarian nsf under grant dfni-bo2/18. references assyov, b., denchev, c. 2015: verbascum davidoffii murb. in: biserkov, v. et al. 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doi: 10.5281/zenodo.7437290 spatial and temporal distribution of the macrozoobenthos community in ponds of southeastern serbia original article ana savić department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, niš, serbia ana.savic@pmf.edu.rs (corresponding author) nenad ilić department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, niš, serbia jelena grozdanović department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, niš, serbia miodrag đorđević department of mathematics, faculty of sciences and mathematics, university of niš, višegradska 33, niš, serbia mrđan đokić department of geography, faculty of sciences and mathematics, university of niš, višegradska 33, niš, serbia received: october 31, 2022 revised: november 09, 2022 accepted: november 15, 2022 abstract: ponds are a prevalent type of freshwater ecosystems worldwide, but most studies have been traditionally focused on deep stratified lakes. the region of southeastern serbia is showcasing the fact that research on this type of small water bodies is scarce. in present study was compared macroinvertebrates community in ponds in southeastern serbia, at different altitudes but with similar other parameters. the goals of this study include: determining the composition and structure of macrozoobenthos communities in ponds at different altitudes; determining the seasonal changes in macrozoobenthos communities in these ponds; and determining the microdistribution of macroinvertebrate taxa in these ponds along the gradient of depth. in studied ponds, structuring of communities was mostly contributed to by insect groups. in the lower-altitude pond these were chironomidae and coenagrionidae, while in the higher-altitude pond chironomidae and dytiscidae. our study has shown that in most cases the sample of macroinvertebrate community included a greater number of families in autumn than in spring season. the greatest number of families was recorded in samples collected in the shallowest part of the pond, both in the pond with a low and in the pond with a high altitude. key words: macroinvertebrates, ponds, southeastern serbia apstrakt: prostorna i vremenska distribucija makroinvertebratske zajednice u barama jugoistočne srbije iako bare predstavljaju široko rasprostranjene, česte ekosisteme u celom svetu, veliki deo istraživanja je koncentrisan na duboka, stratifikovana jezera. u oblasti jugoistočne srbije takodje postoji nedostatak istraživanja malih vodenih tela. u ovom istraživanju je uporedjivana makroinvertebratska zajednica u barama koje se karakterišu različitim nadmorskim visinama, dok su sa druge strane, one slične po drugim karakteristikama. ciljevi ovog istraživanja su bili da se odredi sastav i struktura zajednice makroinvertebrata u barama sa različitom nadmorskom visinom, da se proprate sezonske promene u ovim zajednicama i da se odredi mikrodistribucija predstavnika ove zajednice duž gradijenta dubine. sastavu zajednice najviše doprinose insekatske grupe. u bari sa nižom nadmorskom visinom najveći doprinos struktuiranju imaju chironomidae i coenagrionidae, dok u bari sa većom nadmorskom visinom chironomidae i dytiscidae. u istraživanju je konstatovano da u najvećem broju uzoraka zajednica ima u sastavu veći broj familija u jesenjem aspektu nego u prolećnom. najveći broj familija je konstatovan u najplićim delovima bara koje su bile predmet ovog istraživanja. ključne reči: makroinvertebrate, bare, jugoistočna srbija introduction over a long period of time, conservation activities have mostly been focused on extensive ecosystems, especially those that contain large portions of the earth’s biodiversity (brooks et al., 2006; hunter et al., 2017; savić et al., 2022a). on the other side of the spatial gradient, there are some conservation efforts focused on small natural features in contrast to the large-scale conservation (hunter et al., 2017). small natural features (snfs) are analogous to keystone species because they have an ecological im© 2022 savić et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 157 portance that is disproportionate to their size; sometimes because they provide resources that constrain key populations or processes that affect a much larger area; sometimes because they support unusually high diversity, abundance, or productivity (hunter et al., 2017; savić et al., 2022a). ponds can be defined as small (1 m2 to max 5 ha), natural or man-made shallow waterbodies (normally fresh water, but occasionally brackish), which holds water for at least three months of the year or more (céréghino et al., 2008). this is just one of the possible definitions found in scientific literature, and there are almost as many definitions as there are authors of papers on these ecosystems. ponds (and shallow lakes also) are a prevalent type of freshwater ecosystems worldwide, but most studies have been traditionally focused on deep stratified lakes (hilt et al., 2017). the region of southeastern serbia is also showcasing the fact that research on this type of small waterbodies is scarce. they attracted more attention by botanists (ranđelović et al., 2007), while there are only a few (more recent) papers on macroinvertebrate community of ponds (đorđević, 2017; petrović, 2017). knowledge of spatial and temporal distribution patterns reduces bias in the analysis of data and allows the elaboration of sampling strategies. this type of information is particularly needed for the communities of macrozoobenthos in ponds because they are poorly studied (oertli, 1995). space and time may be perceived as the two axes along which the changes in macroinvertebrate community come about. these changes may pertain to composition and structure of the community, its diversity, biomass and productivity, etc. the spatial distribution of macroinvertebrates depends on various factors, depending on which spatial scale is observed. regarding the micro-distribution, the key factors recognized in the previous studies included heterogeneity of substrate (heino, 2000), depth of water body and oxygen concentration (brinkhurst, 2002), interaction with other organisms and presence of aquatic plants (waters & san giovanni, 2002) etc. temporal variations in fauna can exist at scales ranging from minutes to years (oertli, 1995). in the present study we will investigate temporal variations in macroinvertebrate communities in terms of seasonal changes. considering mentioned, the goals of this study include: 1) determining the composition and structure of macrozoobenthos communities in ponds at different altitudes; 2) determining the seasonal changes in macrozoobenthos communities in these ponds; and 3) determining the microdistribution of macroinvertebrate taxa in these ponds along the gradient of depth. materials and methods the selected study sites included two ponds in southeastern serbia. one is situated in the close vicinity of city of niš (43°17′40.2’’n, 21°55′45.9’’e) at the altitude of 294 m above sea level, while the other is at vlasina plateau (42°40′42.2’’n, 22°21′17.2’’e) at the altitude of 1212 m (fig. 1). average annual temperature on localities are 11.65 oc and 5.72 oc, respectively. 158 biologica nyssana ● 13 (2) december 2022: 157-164 savić et al. ● spatial and temporal distribution of the macrozoobenthos community in ponds of southeastern serbia fig. 1. map of the study area samples were collected in two seasons: in late may and late september, according to the following procedure: five spots were selected at each pond and one sample was taken at each point. samples were taken during a transect, first sample at depth of 30 cm and each following sample at distance of 2 m toward the deepest part of the pond (fig. 2a and 2b). each sample of macrozoobenthos was collected with a net with square frame 30 x 30 cm, mesh size 300 μm and size of sampled surface 0.30 m x 0.30 m. during the sampling process, the frame was placed vertically at the bottom of the pond and pulled along the bottom in the same length as the side of the frame (30 cm). the collected material was transferred to plastic bags and then conserved biologica nyssana ● 13 (2) december 2022: 157-164 savić et al. ● spatial and temporal distribution of the macrozoobenthos community in ponds of southeastern serbia 159 in 70% ethyl alcohol. identification of material was performed by using following keys: belfiore (1983), nilsson (1997), vallenduuk & pillot (2007). simper analysis was used for analysing the year and seasonal structuring of the community, as well as for analysis and comparison of community structure along the gradient of depth. in other words, simper analysis was performed to test differences within faunal composition of groups, dissimilarities between and similarities within the above groups. this analysis was performed using statistical program primer 7.0 (clarke & gorley, 2015). results the samples at the pond in vicinity of niš included representatives of 14 families (550 individuals), with 9 families recorded during the spring sampling and 10 families in the autumn sampling. at the annual level, the best-represented family was coenagrionidae (fig. 3a). the spring aspect was dominated by family chironomidae and the autumn aspect by family coenagrionidae (fig. 3b and 3c). the comparison of localities along the gradient of depth at the annual level has shown that the greatest fig. 2. sampling transects in: a pond in the vicinity of niš; b – pond of the vlasina plateau fig. 3. percentages of macrozoobenthos groups (at family level) in pond in vicinity of niš: a annual; b – in spring period; c – in autumn period 160 biologica nyssana ● 13 (2) december 2022: 157-164 savić et al. ● spatial and temporal distribution of the macrozoobenthos community in ponds of southeastern serbia fig. 4. annual number of benthic macroinvertebrate families in pond in vicinity of niš, their seasonal dynamic and distribution along depth gradient (where loc 1a is the shallowest and loc 5a is the deepest) fig. 5. percentages of macrozoobenthos groups (at family level) in pond on vlasina plateau: a annual; b – in spring period; c – in autumn period number of families was recorded at the locality with shallowest water, while the smallest number was recorded at the locality with deepest water (fig. 4). in four out of five localities, the number of families was greater in autumn that during the spring period. the samples from the pond at the vlasina plateau included representatives of 8 families (241 1ndividuals) (fig. 5a), where both spring and autumn samples were represented by 5 families each. at the annual level, the bestrepresented family was chironomidae, fig. 6. annual number of benthic macroinvertebrate families in pond on vlasina plateau, their seasonal dynamic and distribution along depth gradient (where 1b is the shallowest and loc 5b is the deepest) both in spring and autumn periods (fig. 5b, fig. 5c), respectively. just as in the pond in vicinity of niš, at the annual level the sample from the shallowest locality included the greatest number of families and the sample from the deepest locality included the lowest number of families (fig. 6). in four of the five localities, the number of families was greater in autumn that during the spring period, just as in the first pond. the simper analysis at the annual level has shown that samples from the 161 biologica nyssana ● 13 (2) december 2022: 157-164 savić et al. ● spatial and temporal distribution of the macrozoobenthos community in ponds of southeastern serbia the two ponds have shown high percent of differences according to simper analysis (70.04). regarding the seasonal aspects of the community, by far the greatest impact on community structure at the vicinity of niš during the spring aspect was shown by chironomidae (89.96) with negligible presence of ceratopogonidae (4.41). the difference between samples from the two ponds was not particularly high (45.95%). during the autumn aspect, the community structure of the pond in vicinity of niš was mostly pond in vicinity of niš did not show high average similarity (41.46), while the samples from the pond at vlasina plateau have shown somewhat higher levels of similarity (58.24). the structuring of the community at the pond in vicinity of niš was mostly contributed to by families chironomidae (34.64%) and coenagrionidae (25.6%). the structuring of community in the pond at vlasina plateau was also mostly contributed to by family chironomidae (75.23%) while the next family was dytiscidae (14.27%). at the annual level, the communities of table 1. simper analysis of five groups of macroinvertebrates divided along the gradient of pond depth group i: average similarity=27.38 group ii: average similarity=20.00 group iii: average similarity=24.88 group iv: average similarity=37.91 group v: average similarity=31.58 group i and ii: average dissimilarity=65.08 group i and iii: average dissimilarity=52.47 group i and iv: average dissimilarity=54.29 group i and v: average dissimilarity=53.07 group ii and iii: average dissimilarity=77.01 group ii and iv: average dissimilarity=64.18 group ii and v: average dissimilarity=63.82 group iii and iv: average dissimilarity=53.87 group iii and v: average dissimilarity=60.49 group iv and v: average dissimilarity=54.64 family av. abund. contrib. % cum. % group i chironomidae 21 65.22 65.22 corixidae 4 17.39 82.61 dytiscidae 9.5 13.04 95.65 group ii dytiscidae 7.5 45.49 45.45 chironomidae 21 27.27 72.73 corixidae 4.5 27.27 100 group iii chironomidae 21 66.67 66.67 elmidae 6.5 18.52 85.19 ceratopogonidae 4 7.41 92.59 group iv chironomidae 32.5 86.21 86.21 dytiscidae 4 13.79 100 group v chironomidae 23 100 100 162 biologica nyssana ● 13 (2) december 2022: 157-164 savić et al. ● spatial and temporal distribution of the macrozoobenthos community in ponds of southeastern serbia determined by families coenagrionidae (42.43), tabanidae (20.71) and caenidae (16.79). on the other hand, the most dominant family in the community at vlasina plateau was chironomidae (50.9), but elmidae (39.6) were also significant. for the needs of analysing microdistribution of families, the samples were divided into five groups along the gradient of pond depth. the simper analysis has shown that family chironomidae is significantly contributing to structuring at all depths. the value was highest in the first sample from the shallowest water (65.22%). in the second group of samples, taken at slightly greater depth, the most dominant family was dytiscidae (45.45%) while chironomidae was only second. in the third group, chironomidae (66.6%) were joined by elmidae (18.52%), while in the fourth and fifth groups chironomidae became dominant again (tab. 1). discussion the variation of water temperature across spatiotemporal gradients is one of the key abiotic drivers that shape the distribution, ecology and biology of freshwater macroinvertebrates (vannote et al., 1980). latitudinal and altitudinal gradients are recognized as ecological analogues and drivers of environmental gradients, with both being considered proxies of temperature variation (dos santos et al., 2018; rendoll cárcamo et al., 2019). several studies have focused on temperature and elevation gradient effects on freshwater macroinvertebrates in central europe (novikmec et al., 2015), south america (nieto et al., 2016; shah et al., 2017; dos santos et al., 2018) etc. nevertheless, region of balkans, have received little attention in this regard. considering mentioned, here was compared macroinvertebrates community in ponds at different altitudes but with similar other parameters (similar pond size, way of water supply and hydrological regime). in our research greater number of familia was detected in the pond with lower altitude which is in concordance with the claim that different types of diversity are showing a similar pattern with values decreasing as elevation increased (rendoll cárcamo et al., 2019). the samples at the pond in vicinity of niš included representatives of 14 families, while samples from the pond at the vlasina plateau included representatives of 8 families. although inhabited by a smaller number of taxa, ponds at high elevations are a specific type of small waterbodies leading to high regional diversity (hamerlík et al., 2014; rendoll cárcamo et al., 2019). in the studied ponds, the total number of individuals (550 vs. 241) and abundance decreased with increase of altitude, matching the results by rendoll cárcamo et al. (2019). the simper analysis at the annual level has shown that samples from the pond in vicinity of niš did not show high average similarity (41.46), while the samples from the pond at vlasina plateau have shown somewhat higher levels of similarity (58.24). in other words, this fact indicates a more uniform community at high altitude, similar to some similar investigation in other geographic regions (rendoll cárcamo et al., 2019). in both studied ponds, structuring of communities was mostly contributed to by insect groups. in the lower-altitude pond these were chironomidae and coenagrionidae, while in the higher-altitude pond chironomidae and dytiscidae. the predominance of chironomids is a common pattern in european high altitude stagnant water bodies (fjellheim et al., 2009). role of representatives of order odonata in community structuring is somewhat less commonly observed in ponds (thornhill et al., 2017). our study has shown that in most cases the sample of macroinvertebrate community included a greater number of families in autumn than in spring season. there is still an ongoing debate in scientific circles on the best season to sample aquatic macroinvertebrates in ponds for biodiversity assessment, but our results match those from studies in northern europe where autumn is the most suitable period (hill et al., 2016). in both ponds, the greatest number of families was recorded in samples collected in the shallowest part of the pond. one possible reason is the fact that more different microhabitats are situated in this zone than in others. the second possible reason may pertain to development of riparian vegetation. therefore, conservation of riparian vegetation in both lotic and lentic ecosystems is seemingly crucial as it supports high values of macroinvertebrates diversity (savić et al., 2022b). conclusions our results have shown that ponds at higher altitudes were inhabited by a more uniform community of macroinvertebrates. although hosting a smaller diversity of macroinvertebrate communities than ponds at lower altitudes, the high-altitude ponds significantly contribute to regional diversity and therefore should be given more attention. this study also contributes to the debate on the best season to sample aquatic macroinvertebrates in ponds for biodiversity assessment: it is the autumn aspect. the future research will likely be directed toward studies of a larger number of small waterbodies of this and similar types. 163 biologica nyssana ● 13 (2) december 2022: 157-164 savić et al. ● spatial and temporal distribution of the macrozoobenthos community in ponds of southeastern serbia references belfiore, c. 1983: efemerotteri (ephemeroptera). in: ruffo s. 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adjacent lowlands: general ecology, tanypodinae. knnv publishing, zeist. 270 p. vannote, r.l., minshall, g.w., cummins, k.w, sedell, j.r., cushing, c.e. 1980: the river continuum concept. canadian journal of fisheries and aquatic sciences, 37(1): 130–137. waters, n.m., san giovanni, c.r. 2002: distribution and diversity of benthic macroinvertebrates associated with aquatic macrophytes. journal of freshwater ecology, 17(2): 223-232. karakuyu et al. 2025, biologica nyssana 16(1) 137 16 (1) june 2025: 137-144 doi: 10.46793/biolnyss.16.1.12k determination of fruit characteristics and biochemical contents of some almond cultivars (p. amygdalus l.) grown in the afyonkarahisar/ dinar region original article yavuz karakuyu ministry of agriculture and forestry, department of horticulture, afyon, türkiye adnan n. yildirim isparta university of applied sciences, agriculture faculty, department of horticulturae, isparta, türkiye adnanyildirim@isparta.edu.tr (corresponding author) ayşe vildan pepe isparta university of applied sciences, agriculture faculty, department of horticulturae, isparta, türkiye civan çelik isparta university of applied sciences, agriculture faculty, department of agricultural biotechnology, isparta, türkiye received: february11, 2025 revised: march 03, 2025 accepted: march 06, 2025 abstract: this study was conducted in a closed almond orchard in the dinar district, of afyonkarahisar province, from 2023-2024. the study aimed to determine the fruit characteristics and biochemical contents of ferragnes, ferraduel, and nonpareil almond cultivars. in the study, the fruit weight ranged from 2.01 g (nonpareil) to 5.18 g (ferragnes), the fruit width ranged from 19.87 mm (nonpareil) to 26.65 mm (ferragnes), the fruit length ranged from 32.66 mm (nonpareil) to 39.72 mm (ferragnes), and the shell thickness ranged from 1.75 mm (nonpareil) to 3.58 mm (ferraduel). the kernel weight varied between 1.07 g (nonpareil) and 1.69 g (ferragnes), the kernel width between 12.89 mm (nonpareil) and 16.20 mm (ferragnes), the kernel length between 22.54 mm (nonpareil) and 28.40 mm (ferragnes), the kernel thickness between 6.45 mm (nonpareil) and 7.65 mm (ferragnes), and the kernel ratio between 27.29% (ferraduel) and 53.40% (nonpareil). in terms of biochemical composition, total oil content ranged from 44.84% (ferraduel) to 49.02% (ferragnes), total phenolic content from 361.02 mg gae/100 g (nonpareil) to 429.2 mg gae/100 g (ferragnes), total flavonoid content from 30.49 mg cae/100 g (nonpareil) to 68.64 mg cae/100 g (ferragnes), total antioxidant content from 82.98% (nonpareil) to 87.00% (ferraduel), and total protein content from 16.64% (ferragnes) to 19.16% (ferraduel). key words: amygdalus communis l., kernel ratio, total phenolics, total oil apstrakt: određivanje karakteristika plodova i biohemijskog sastava nekih sorti badema (p. amygdalus l.) uzgajanih u regionu afjonkarahisar/ dinar ova studija je sprovedena u zatvorenom zasadu badema u okrugu dinar, u provinciji afjonkarahisar, tokom perioda od 2023 do 2024. godine. cilj istraživanja bio je da se odrede karakteristike plodova i biohemijski sastav sorti badema ferragnes, ferraduel i nonpareil. u okviru studije, masa ploda varirala je od 2,01 g (nonpareil) do 5,18 g (ferragnes), širina ploda od 19,87 mm (nonpareil) do 26,65 mm (ferragnes), dužina ploda od 32,66 mm (nonpareil) do 39,72 mm (ferragnes), dok se debljina ljuske kretala od 1,75 mm (nonpareil) do 3,58 mm (ferraduel). masa zrna varirala je između 1,07 g (nonpareil) i 1,69 g (ferragnes), širina zrna između 12,89 mm (nonpareil) i 16,20 mm (ferragnes), dužina zrna između 22,54 mm (nonpareil) i 28,40 mm (ferragnes), debljina zrna između 6,45 mm (nonpareil) i 7,65 mm (ferragnes), dok se udeo zrna kretao od 27,29% (ferraduel) do 53,40% (nonpareil). što se tiče biohemijskog sastava, ukupni sadržaj ulja varirao je od 44,84% (ferraduel) do 49,02% (ferragnes), ukupni sadržaj fenola od 361,02 mg gae/100 g (nonpareil) do 429,2 mg gae/100 g (ferragnes), ukupni sadržaj flavonoida od 30,49 mg cae/100 g (nonpareil) do 68,64 mg cae/100 g (ferragnes), ukupni antioksidativni kapacitet od 82,98% (nonpareil) do 87,00% (ferraduel), dok je ukupni sadržaj proteina varirao od 16,64% (ferragnes) do 19,16% (ferraduel). ključne reči: amygdalus communis l., udeo zrna, ukupan sadržaj fenola, ukupan sadržaj ulja © 2025 karakuyu et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 138 introduction almond [prunus dulcis (mill.) d.a. webb; sin. p. amygdalus batsch] is one of the oldest fruit species cultivated worldwide. türkiye's location within the near east and mediterranean basin, its favorable ecological conditions, and its position along migration routes have contributed to its rich genetic resources. for this reason, türkiye, which allows the natural growth of 9 out of nearly 40 almond species, is among the genetic centers of almonds (bayazit & küden, 2023). however, despite its high almond production potential, türkiye has not been able to fully utilize this capacity. the lack of standardization in almond production and producers' concerns about inconsistent yields are among the primary reasons for this situation. however, in recent years, increasing consumer awareness, the expansion of irrigated areas, and the development of clonal rootstocks and new varieties suitable for modern cultivation systems have contributed to the expansion of almond production areas, particularly in the mediterranean and southeastern anatolia regions, due to their ecological suitability. moreover, with the breeding and introduction of late-blooming and self-fertile new varieties, almond production has been steadily increasing both worldwide and in türkiye in recent years. in the last 10 years, the global almond production area has increased by 27.01%, expanding from 18.281.300 hectares to 23.220.670 hectares, while production has increased by 92.22%, rising from 1.828.130 tons to 3.513.970 tons. the united states ranks first in global almond production with 1.791.690 tons. it is followed by spain with 297.660 tons, australia with 260.000 tons, and türkiye with 170.000 tons (fao, 2023). in the last 10 years, türkiye's almond production area has increased significantly above the global average, expanding from 270.203 hectares to 686.966 hectares, an increase of 154.24%. meanwhile, production has risen from 73.230 tons by 132.15% to 170.000 tons. in türkiye, the highest almond production is recorded in adıyaman with 21.299 tons, followed by mersin with 19.052 tons and muğla with 9.785 tons (tüi̇k, 2023). nuts are highly nutritious fruits with a high oil content. it has been reported that meeting daily fat intake through nuts can significantly reduce cholesterol levels in humans (abbey et al., 1994). its high content of unsaturated fatty acids, protein, minerals, fiber, phytosterols, phenolics, and tocopherols, along with its diverse uses and consumption forms (raw, roasted, blanched, in the chocolate and creamy cake industry, as well as in the pharmaceutical and dye industries, and in animal feed production), is among the characteristics that distinguish almonds from other nuts (yada et al., 2011; yıldırım et al., 2016a; yıldırım et al., 2016b). additionally, the high antioxidant content of almond oil allows it to be used by aromatherapists today as a natural emollient and anti-aging agent (bolling, 2017; barreca et al., 2020). since most almond plantations have been propagated from seeds, there is a wide variation among types, leading to inconsistent yields and the inability to obtain standardized products. this situation results in low productivity despite the high number of almond trees in türkiye. therefore, it is necessary to identify suitable varieties for each region and establish new orchards using standardized rootstocks and cultivars to ensure high-quality products and increase production. this study aims to determine some fruit characteristics and the biochemical content of the almond cultivars 'ferragnes,' 'ferraduel,' and 'nonpareil' grown in the afyonkarahisar/dinar region. materials and methods material this study was conducted in yeşilçat village, dinar district, afyonkarahisar province, between 2023 and 2024, on three almond varieties (ferragnes, ferraduel, and nonpareil) grafted onto seedlings in a closed orchard planted with a 5 x 5 meter spacing. regular cultural practices such as pruning, fertilization, irrigation, and weed control have been carried out in the orchard. the orchard is located at an average altitude of 900 meters above sea level, and the trees' irrigation needs were met using a double-line drip irrigation system. fruit samples representing the tree from four different directions were collected from each almond variety and transported to a laboratory for measurement and analysis. the pomological analysis of the collected fruit samples was carried out in the laboratories of the department of horticultural sciences at isparta university of applied sciences, faculty of agriculture. method in the study, a total of 810 fruit samples were used, with 30 fruits per repetition for each variety, in order to determine their pomological characteristics. the collected fruits were transported to the pomology laboratory of the department of horticultural sciences at isparta university of applied sciences, faculty of agriculture, where the green hulls (mesocarp) were removed and the fruits were subjected to a drying process at 50-55 °c until their moisture content stabilized. the harvested fruits were measured for various parameters, including the fruit length, fruit width, biologica nyssana ● 16 (1) june 2025: 137-144 karakuyu et al. ● determination of fruit characteristics and biochemical contents of some almond cultivars (p. amygdalus l.) grown in the afyonkarahisar/ dinar region 139 biologica nyssana ● 16 (1) june 2025: 137-144 karakuyu et al. ● determination of fruit characteristics and biochemical contents of some almond cultivars (p. amygdalus l.) grown in the afyonkarahisar/ dinar region shell thickness, kernel length, kernel width, and almond thickness. these measurements were made using a digital caliper with an accuracy of 0.01 mm (yıldırım, 2007). the nut fruit weight and kernel weight were determined using a sensitive balance with an accuracy of 0.01 g (yıldırım, 2007). the total phenolic content was determined using the folin-ciocalteu method as described by singleton & rossi (1965). homogenized fruit pulp was subjected to extraction in tubes for 1 h using a solution of acetone, water, and acetic acid (70:29.5:0.5). accordingly, the samples were mixed in 0.1 ml of extract with de-ionized water and 0.1 ml (2n) folin-ciocalteu indicator and kept at room temperature for 6 min. then, 0.5 ml of 20% sodium carbonate solution was added and incubated at room temperature for 30 min for color development. after color development, the absorbance values of the samples were read in a spectrophotometer at 760 nm wavelength (model t60u, pg instruments usa). the total flavonoid content was determined according to the method described by kim et al. (2003). first, 1 ml of each sample was mixed with 0.3 ml of 5% sodium nitrite (nano2) solution and incubated for 5 min. 0.3 ml of 10% aluminium chloride (alcl3) solution was added for an additional 6 min incubation. afterwards, 2 ml of 1 m sodium hydroxide (naoh) was added, and the mixture was allowed to stand for 2 minutes. subsequently, 4 ml of distilled water was added thoroughly mixed, and the absorbance values of the samples were read at 510 nm wavelength. the total antioxidant capacity was determined using the method reported by kumaran & karunakaran (2006), employing 1,1-diphenyl-2picrylhydrazyl (dpph). first, 2 g of each sample were grounded and incubated in 20 ml of 80% methanol at -20 °c for 2 h. then, the mixture was centrifuged at 2.000 rpm for 5 min, and the supernatant was collected for analysis. following this step, 2 ml of 0.1 mm dpph dissolved in methanol was added to 100 µl of the supernatant. after 30 min of incubation, the absorbance was measured at 517 nm wavelength. in the study, the total oil content in the fruits was determined and calculated using the soxhlet method. the results were evaluated as percentages (james, 1995). the oils of the samples were extracted using the soxhlet method. hexane was used as a solvent. the oil samples obtained from seeds and fruits were converted to methyl esters using a mixture of sodium methoxide and methanol according to the method specified by knothe (2013). fatty acid analyses were performed using a shimadzu gc 2010 plus instrument with a cp-wax52cb 60 m column. helium gas was used as a carrier at a flow rate of 3 ml/min. the column temperature was 80 °c, the detector temperature was 265 °c, and the injection block temperature was 250 °c. the oven temperature programme. after holding the initial temperature of 80 °c for 4 minutes, the temperature was increased to 175 °c at a rate of 20 °c/min and held for 25 minutes. it was then increased to 215 °c at a rate of 4 °c/min and held for 2 minutes, and finally increased to 250 °c at a rate of 2 °c/min and held for 20 minutes. fatty acid methyl esters of fruit and seed samples were determined by comparison with the peak time of standards (37 fame mix, supelco). the phenolic compounds of the fruits were determined using a shimadzu hplc device according to the method of artik et al. (1998). for this purpose, 10 g of sample was taken; 0.1 g of bht (2,6-di-tert-butyl-4-methylphenol) and 20 ml of extraction solution were added, then crushed in a homogeniser. after being placed for 45 minutes in an ultrasonic bath and then 45 minutes on a shaker, it was filtered through whatman filter paper. the filtrate was passed through a 0.45 µm filter, and 20 µl of it was injected into the hplc apparatus. the results were expressed in µg/g (extraction solution: 80% methanol with 1% hcl). statistical analyses the research was designed according to a randomized block design with 3 replications and 3 trees per replication. the data obtained from the study were analyzed using the minitab statistical software. significant differences between the means were determined using the tukey multiple comparison test (p≤0.05) and are indicated by different letters. results and discussion in the study, the fruit and kernel characteristics of the almond varieties are presented in tab. 1. statistically significant differences were found between the almond cultivars in terms of both fruit and kernel characteristics (p≤0.05). in the study, the fruit weight ranged from 2.01 g (nonpareil) to 5.18 g (ferragnes); fruit width from 19.87 mm (nonpareil) to 26.65 mm (ferragnes); and fruit length from 32.66 mm (nonpareil) to 39.72 mm (ferragnes). the kernel length ranged from 1.75 mm (nonpareil) to 3.58 mm (ferraduel). the kernel attributes were recorded as follows: the kernel weight ranged from 1.07 g (nonpareil) to 1.69 g (ferragnes), kernel width from 12.89 mm (nonpareil) to 16.20 mm (ferragnes), kernel length from 22.54 mm (ferraduel) to 28.40 mm (ferragnes), and kernel thickness from 6.45 mm (nonpareil) to 7.65 mm (ferragnes). the kernel ratio ranged from 27.29% (ferraduel) to 53.40% 140 biologica nyssana ● 16 (1) june 2025: 137-144 karakuyu et al. ● determination of fruit characteristics and biochemical contents of some almond cultivars (p. amygdalus l.) grown in the afyonkarahisar/ dinar region ta bl e 1. f ru it an d ke rn el c ha ra ct er is tic s of a lm on d cu lti va rs c ul tiv ar s fw (g ) fw (m m ) fl (m m ) st (m m ) k w (g ) k w (m m ) k l (m m ) k t (m m ) k r (% ) fe rr ag ne s 5. 18 ±0 .0 3a 26 .6 5± 0. 03 a 39 .7 2± 0. 18 a 3. 32 ±0 .0 7a 1. 69 ±0 .0 2a 16 .2 0± 0. 13 a 28 .4 0± 0. 13 a 7. 65 ±0 .1 0a 32 .7 6± 0. 13 b fe rr ad ue l 4. 86 ±0 .0 9b 23 .0 3± 0. 11 b 32 .9 2± 0. 17 b 3. 58 ±0 .0 7a 1. 30 ±0 .0 1b 13 .8 4± 0. 20 b 22 .5 4± 0. 05 c 7. 06 ±0 .1 7b 27 .2 9± 0. 18 c n on pa re il 2. 01 ±0 .0 5c 19 .8 7± 1. 37 c 32 .6 6± 0. 49 b 1. 75 ±0 .3 0b 1. 07 ±0 .0 2c 12 .8 9± 0. 08 c 23 .1 5± 0. 09 b 6. 45 ±0 .0 5c 53 .4 0± 0. 53 a th e di ffe re nc es b et w ee n va lu es re pr es en te d by d iff er en t l et te rs in th e sa m e co lu m n ar e st at is tic al ly s ig ni fic an t ( p< 0. 05 ). (f w : f ru it w ei gn t, fw : f ru it w id th , f l: f ru it le ng th , s t: s he ll t hi ck ne ss , k w : k er ne l w ei gh t, kw : k er ne l w id th , k l: k er ne l l en gt h, k t: k er ne l t hi ck ne ss , kr :k er ne l r at io ) (nonpareil). the study reveals significant variations in the fruit characteristics between the cultivars. previous studies have also reported differences in fruit and kernel characteristics between cultivars and genotypes (viti & loreti, 1993; yıldırım, 2007; rapposelli et al., 2018; karaat, 2019; karahan, 2021; demirdaş, 2022; khojand et al., 2023; i̇sen, 2023). segura & kodad (2009) reported that the genetic traits of cultivars significantly influence the physical properties of almond fruits. in a high-quality almond variety, a thin shell and high kernel yield are desirable. the shell of almonds varies according to cultivars but constitutes 35-75% of the fruit (li et al., 2018). therefore, in hard-shelled almond varieties, shell thickness is one of the most important traits affecting fruit quality and kernel yield. while shell thickness is essentially a cultivar characteristic, it is also influenced by drought and cultural practices (goldhamer et al., 2006; romero et al., 2017; yıldırım et al., 2023). previous studies showed variations in fruit and kernel characteristics compared to our research results. in almond cultivation, ecological factors have a significant impact on quality traits (yıldız & perdahcı, 2019; bayazit & alaz, 2022), as well as cultural practices, especially plant nutrition, tree age, and yield conditions, also affect kernel fullness, size, and other characteristics (küden et al., 2000). similarly, environmental factors, cultural practices, and genetic traits have an impact on fruit quality, with cultivar, rootstock, and tree age having a stronger influence than genetic traits (atay et al., 2009; bayazit & alaz, 2022). the biochemical contents of almond varieties are presented in tab. 2. statistically significant differences were determined between almond cultivars in terms of total phenolic, flavonoid, and fat content (p≤0.05). the total phenolic content ranged from 361.02 mg gae/100 g (nonpareil) to 429.2 mg gae/100 g (ferragnes); the total flavonoid content ranged from 30.49 mg cae/100 g (nonpareil) to 68.64 mg cae/100 g (ferragnes); the total antioxidant content ranged from 82.98% (nonpareil) to 87.00% (ferraduel); the total protein content ranged from 16.64% (ferragnes) to 19.16% (ferraduel); and the total oil content ranged from 44.84% (ferraduel) to 49.02% (ferragnes). in previous studies, it has been reported ta bl e 2. b io ch em ic al c on te nt s of a lm on d cu lti va rs c ul tiv ar s t ot al p he no lic c on te nt (m g g a e /1 00 g ) t ot al f la va no id c on te nt (m g c a e /1 00 g ) t ot al a nt io xi da nt c ap ac ity (% in hi bi sy on ) t ot al pr ot ei n (% ) t ot al o il (% ) fe rr ag ne s 42 9. 2± 20 .0 a 68 .6 4± 2. 50 a 84 .8 6± 0. 83 a 16 .6 4± 1. 52 a 49 .0 0± 2. 24 a fe rr ad ue l 40 8. 77 ±7 .2 1a 33 .0 7± 1. 20 b 87 .0 0± 1. 35 a 19 .1 6± 1. 00 a 44 .8 2± 1. 01 a n on pa re il 36 1. 02 ±3 .3 4b 30 .4 9± 1. 39 b 82 .9 8± 4. 73 a 17 .4 8± 0. 25 a 46 .4 1± 2. 11 a th e di ffe re nc es b et w ee n va lu es re pr es en te d by d iff er en t l et te rs in th e sa m e co lu m n ar e st at is tic al ly s ig ni fic an t ( p< 0. 05 ) 141 biologica nyssana ● 16 (1) june 2025: 137-144 karakuyu et al. ● determination of fruit characteristics and biochemical contents of some almond cultivars (p. amygdalus l.) grown in the afyonkarahisar/ dinar region ta bl e 3. p he no lic c on te nt o f a lm on d cu lti va rs (m g/ kg ) c ul tiv ar s g al lic a ci d c at ec hi n phy dr ox yb en zo ic ac id c hl or og en ic ac id c aff ei c ac id e pi ca te ch in k ae m pf er ol l ut eo lin fe rr ag ne s 2. 00 ±0 .0 0c 50 .0 4± 0. 43 a 5. 13 ±0 .0 7a 11 .0 1± 0. 11 a 4. 14 ±0 .0 6a 20 .7 1± 0. 17 a 6. 10 ±0 .0 2a 0. 00 ±0 .0 0b fe rr ad ue l 4 .6 3± 0. 04 a 33 .8 2± 0. 25 b 4. 62 ±0 .0 5b 7. 85 ±0 .0 3b 2. 07 ±0 .0 2b 17 .5 2± 0. 14 b 2. 05 ±0 .0 7b 3, 52 ±0 .0 6a n on pa re il 2 .8 8± 0. 02 b 13 .5 6± 0. 06 c 1. 48 ±0 .0 3c 2. 50 ±0 .0 5c 0. 87 ±0 .0 2c 4. 20 ±0 .0 5c 1. 67 ±0 .0 0c 0. 00 ±0 .0 0b th e di ffe re nc es b et w ee n va lu es re pr es en te d by d iff er en t l et te rs in th e sa m e co lu m n ar e st at is tic al ly s ig ni fic an t ( p< 0. 05 ) that there are differences in total phenolic, flavonoid, antioxidant, protein, and oil content between cultivars and genotypes. it has been noted that the biochemical contents of almonds show considerable variation across cultivars, regions, and years, and these differences are influenced by factors such as cultural practices, ecology, ripeness, soil composition, post-harvest storage conditions, fungi, bacteria, and pests (yildız et al., 2014; maestri et al., 2015; rapposelli et al., 2018; oliveira et al., 2018; küçük, 2019; ossama et al., 2021; özcan, 2023; khojand et al., 2023; i̇sen, 2023; çakmak, 2023). furthermore, i̇sen (2023) stated that the chemical composition of the fruit in almond breeding could be considered a selection criterion; however, certain markers, such as total protein, is heavily influenced by environmental factors, making their use as a sole selection criterion misleading (beltrán sanahuja et al. 2021). in this study, the phenolic compounds of almond cultivars are presented in tab. 3. statistically significant differences were determined between almond cultivars in terms of phenolic compounds (p≤0.05). the amounts of gallic acid ranged from 2.00 mg/kg (ferragnes) to 4.63 mg/kg (ferraduel), catechin from 13.56 mg/kg (nonpareil) to 50.04 mg/ kg (ferragnes), p-hydroxybenzoic acid from 1.48 mg/kg (nonpareil) to 5.13 mg/kg (ferragnes), chlorogenic acid from 2.50 mg/kg (nonpareil) to 11.01 mg/kg (ferragnes), caffeic acid from 0.87 mg/kg (nonpareil) to 4.14 mg/kg (ferragnes), epicatechin from 4.20 mg/kg (nonpareil) to 20.71 mg/kg (ferragnes), kaempferol from 1.67 mg/kg (nonpareil) to 6.10 mg/kg (ferragnes), and luteolin from 0.00 mg/kg (nonpareil, ferragnes) to 3.52 mg/kg (ferraduel). bolling et al. (2010) stated that there were statistically significant differences between cultivars regarding phenolic content, with catechin having the highest value and naringenin the lowest. colic et al. (2017) reported that catechin, with an average of 46.3% of total phenols, was the dominant phenolic compound, followed by chlorogenic acid and rutin. in this study, lower values were obtained for some phenolic compounds and higher values for others. as mentioned in previous studies, this variation may be influenced by factors such as fungi, bacteria, pests, air, and light, and environmental factors in the growing region, cultivation techniques, fruit ripeness, soil properties of the region, and the genetic table 4. phenolic content of almond cultivars (mg/kg) cultivars ferragnes ferraduell nonpareil myristic acid 0.01±0.00b 0.00±0.00c 0.03±0.00a palmitic asit 5.99±0.03c 6.49±0.03b 6.65±0.01a palmitoleic asit 0.53±0.00c 0.59±0.00b 0.75±0.00a heptadecanoic acid 0.05±0.04a 0.10±0.00a 0.10±0.00a stearic acid 2.12±0.01b 2.03±0.00c 2.17±0.00a oleic acid 77.63±0.02a 70.53±0.01b 68.18±0.01c linoleic acid 13.41±0.01c 20.07±0.03b 21.89±0.01a arachidic acid 0.07±0.00a 0.08±0.00a 0.08±0.00a linolenic acid 0.06±0.00a 0.06±0.01a 0.07±0.00a behenic acid 0.02±0.00a 0.00±0.00b 0.02±0.00a arachidonic acid 0.01±0.00a 0.00±0.00b 0.01±0.00a arachidic acid 0.01±0.00a 0.01±0.00a 0.01±0.00a the differences between values represented by different letters in the same column are statistically significant (p<0.05) 142 from 16.64% (ferragnes) to 19.16% (ferraduel). this research is of great importance because it serves as a model for producers in a region where fruit cultivation is developing. this is especially significant since no previous studies have been conducted on almond cultivation using standard cultivars. the results indicate that almond cultivation is feasible in the dinar district of afyonkarahisar, a region characterised by severe winter cold and frequent late spring frosts. this is because the results of fruit weight and kernel ratio, the main quality attributes of almonds, are consistent with studies conducted in more suitable ecological conditions. in addition, the study highlights the need to conduct adaptation trials for extra-late almond cultivars, taking into account the regional ecology. the results of this research are expected to contribute to the fruit industry, pomology, and almond cultivation in the dinar district of afyonkarahisar. references abbey, m., noakes, m., belling, g.b., & nestel, p.j. (1994). partial replacement of saturated fatty acids with almonds or walnuts lowers total plasma cholesterol and low-density-lipoprotein cholesterol. the american journal of clinical nutrition, 59(5), 995-999. https://doi.org/10.1093/ajcn/59.5.995 alonso segura, j.m. & kodad, o. (2007). fruit quality in almond: physical aspects for breeding strategies. acta horticulturae, 814, 475-480. doi: 10.17660/actahortic.2009.814.80 artik, n., murakami, h., & mori, t. (1998). determination of phenolic compounds in pomegranate juice by using hplc. european journal of science and technology, 6(10), 32-37. atay, e., pırlak, l., & atay, a.n. (2009). factors affecting fruit size in apples. journal of agriculture faculty of ege university, 46(2), 137-144.19. banjanin, t., nikolic, d., uslu, n., gökmen, f., özcan, m.m., milatovic, d., & dursun, n. (2021). physicochemical properties, fatty acids, phenolic compounds, and mineral contents of 12 serbia regional and commercial almond cultivars. journal of food processing and preservation, 45(1), e15015. https://doi.org/10.1111/jfpp.15015 barreca, d., nabavi, s.m., sureda, a., rasekhian, m., raciti, r., silva, a.s., & mandalari, g. (2020). almonds (prunus dulcis mill. da webb): a source of nutrients and health-promoting compounds. nutrients, 12(3), 672. https://doi.org/10.3390/ nu12030672 bayazit, s. & alaz, m. (2022). yield and fruit characteristics of species and cultivars may also play a role (yıldırım et al., 2016; colic et al., 2017; banjanin et al., 2020). in this study, the fatty acid composition of almond cultivars is presented in tab. 4. statistically significant differences were determined between almond cultivars in terms of fatty acids (p≤0.05). the results showed that oleic acid, linoleic acid, palmitic acid, and stearic acid were the dominant fatty acids, making up 99% of the total oil content. among the unsaturated fatty acids, oleic acid ranged from 68.18% (nonpareil) to 77.63% (ferragnes), linoleic acid ranged from 13.41% (ferragnes) to 21.89% (nonpareil), and among the saturated fatty acids, palmitic acid ranged from 5.99% (ferragnes) to 6.65% (nonpareil), stearic acid ranged from 2.03% (ferraduel) to 2.17% (nonpareil). these results are similar to those found by özcan et al. (2011), kodad et al. (2014), and colic et al. (2017). however, banjanin et al. (2020) reported higher results. it has been reported that factors such as variety, location, harvest time, cultural practices, storage conditions, tree age, and ecology have a significant impact on characteristics such as fat, protein, mineral content, vitamins, and phytochemicals in almonds (yıldırım, 2016; özcan, 2023). conclusion this study aims to determine the fruit characteristics and some biochemical contents of ferragnes, ferraduel, and nonpareil almond varieties grown in an almond orchard located in yeşilçat village, dinar district, afyonkarahisar province. in the study, the fruit weight ranged from 2.01 g (nonpareil) to 5.18 g (ferragnes), fruit width from 19.87 mm (nonpareil) to 26.65 mm (ferragnes), fruit length from 32.66 mm (nonpareil) to 39.72 mm (ferragnes), and shell thickness from 1.75 mm (nonpareil) to 3.58 mm (ferraduel). the kernel weight varied between 1.07 g (nonpareil) and 1.69 g (ferragnes), kernel width between 12.89 mm (nonpareil) and 16.20 mm (ferragnes), kernel length between 22.54 mm (nonpareil) and 28.40 mm (ferragnes), kernel thickness between 6.45 mm (nonpareil) and 7.65 mm (ferragnes), and kernel ratio between 27.29% (ferraduel) and 53.40% (nonpareil). the study determined total oil content ranging from 44.84% (ferraduel) to 49.02% (ferragnes), total phenolic content from 361.02 mg gae/100 g (nonpareil) to 429.2 mg gae/100 g (ferragnes), total flavonoid content from 30.49 mg cae/100 g (nonpareil) to 68.64 mg cae/100 g (ferragnes), total antioxidant content from 82.98% (nonpareil) to 87.00% (ferraduel), and total protein content biologica nyssana ● 16 (1) june 2025: 137-144 karakuyu et al. ● determination of fruit characteristics and biochemical contents of some almond cultivars (p. amygdalus l.) grown in the afyonkarahisar/ dinar region 143 characteristics of some foreign almond varieties in the ecology of gaziantep. mustafa kemal university journal of agricultural sciences, 27(2), 374-383. https://doi.org/10.37908/mkutbd.1098934 bayazit, s. & küden, a. 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(2023). diversity of nut and kernel weight, oil content, and the main fatty acids of some almond cultivars and genotypes. journal of nuts, 14(1), 33-44. https://doi. org/10.22034/jon.2022.1945292.1145 kim, d.o., jeong, s.w., & lee, c.y. (2003). antioxidant capacity of phenolic phytochemicals from various cultivars of plums. food chemistry, 81(3), 321-326. knothe, g. (2013). avocado and olive oil methyl esters. biomass and bioenergy, 58, 143-148. https:// doi.org/10.1016/j.biombioe.2013.09.003 kodad, o., estopañán, g., juan, t., alonso, j.m., espiau, m.t., & i company, r.s. (2014). oil content, fatty acid composition and tocopherol concentration in the spanish almond genebank collection. scientia horticulturae, 177, 99-107. https://doi.org/10.1016/j.scienta.2014.07.045 kumaran, a. & joel karunakaran, r. (2006). antioxidant activities of the methanol extract of cardiospermum halicacabum. pharmaceutical biology, 44(2), 146-151. https://doi. org/10.1080/13880200600596302 küçük, e. 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(2015). variability in almond oil chemical traits from traditional cultivars and native genetic resources from argentina. food chemistry, 170, 55-61. https:// doi.org/10.1016/j.foodchem.2014.08.073 oliveira, i., meyer, a., afonso, s., ribeiro, c., & gonçalves, b. (2018). morphological, mechanical and antioxidant properties of portuguese almond cultivars. journal of food science and technology, 55, 467-478. ossama, k., khaoula, c., mina, e.b., said, e.n., josé, m.g.p., hassouna, g., & pedro, m.g. (2021). kernel quality evaluation of promising new almond germplasm grown in mountain and oasis agro-systems in morocco. agroforestry systems, 95, 625-640. özcan, m.m. (2023). a review on some properties of almond: ımpact of processing, fatty acids, polyphenols, nutrients, bioactive properties, and health aspects. journal of food science and technology, 60(5), 1493-1504. özcan, m.m., ünver, a., erkan, e., & arslan, d. (2011). characteristics of some almond kernel and oils. scientia horticulturae, 127(3), 330-333. https://doi.org/10.1016/j.scienta.2010.10.027 rapposelli, e., rigoldi, m.p., satta, d., delpiano, d., secci, s., & porceddu, a. (2018). genetic, phenotypic, and commercial characterization of an almond collection from sardinia. plants, 7(4), 86. https://doi.org/10.3390/plants7040086 romero, a., dicenta, f., miarnau, x., batlle, i., & chamakh, m. (2017). variability of almond shell mechanical strength. in vii international symposium on almonds and pistachios 1219. (pp. 45-50) https:// doi.org/10.17660/actahortic.2018.1219.8 singleton, v.l. & rossi, j.a. (1965). colorimetry of total phenolics with phosphomolybdicphosphotungstic acid reagents. american journal of enology and viticulture, 16(3), 144-158. https://doi. org/10.5344/ajev.1965.16.3.144 tüi̇k (türkiye i̇statistik kurumu). 2023. plant production statistics. retrieved from https://biruni. tuik.gov.tr/medas/?kn=92&locale=tr. erişim tarihi. 07.02.2025. viti, r. & loreti, f. (1993). research on the bioagronomic behaviour of 22 almond cultivars of various origins. in i international congress on almond 373 (pp. 49-56). https://doi.org/10.17660/ actahortic.1994.373.5 yada, s., lapsley, k., & huang, g. (2011). a review of composition studies of cultivated almonds: macronutrients and micronutrients. journal of food composition and analysis, 24(4-5), 469-480. https:// doi.org/10.1016/j.jfca.2011.01.007 yildiz, h., atli, h., tosun, m., ercişli, s., & pınar, h. (2014). antioxidant activity, total phenolic and flavonoid content of some local and cultivated almonds (prunus dulcis l.). oxidation communications, 37(3), 733-740. yıldız, e. & perdahcı, ç.e. (2019). adaptation of some almond varieties to the ecological conditions of uşak. erciyes journal of agriculture and animal sciences, 2(1), 11-19. yildirim, a.n., akinci-yildirim, f., san, b., & sesli, y. (2016a). total oil content and fatty acid profile of some almond (amygdalus communis l.) cultivars. polish journal of food and nutrition sciences, 66(3), 173-178. https://doi.org/10.1515/ pjfns-2015-0032 yıldırım, a.n., yildirim, f., şan, b., polat, m., & sesli, y. (2016b). variability of phenolic composition and tocopherol content of some commercial almond cultivars. journal of applied botany and food quality, 89, 163-170. yıldırım, a.n. (2007). selection of almonds (p. amygdalus l.) in the isparta region. (doctoral dissertation, adnan menderes university, institute of science, aydın). yıldırım, y., bilgin, n. a., & mısırlı, a. (2023). the effect of altitude on fruit characteristics of ferragnes and ferraduel almond varieties. bahçe, 52(2), 113-118. https://doi.org/10.53471/ bahce.1338213 biologica nyssana ● 16 (1) june 2025: 137-144 karakuyu et al. ● determination of fruit characteristics and biochemical contents of some almond cultivars (p. amygdalus l.) grown in the afyonkarahisar/ dinar region microsoft word bn020205 djurdjevic biologica nyssana 2 (2) december 2011: 129-130 đrđević a., đurić m. blepisanis vittipennis (reiche 1877) (coleoptera:… 129 short communication ! blepisanis vittipennis (reiche 1877) (coleoptera: cerambycidae), a new longhorn beetle for serbia aca đurđević1, milan đurić2 1university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia 2habiprot, belgrade, serbia * e-mail: djukiamphibia@gmail.com abstract: đurđević, a., đurić, m.: blepisanis vittipennis (reiche 1877) (coleoptera: cerambycidae), a new longhorn beetle for serbia, biologica nyssana, 2 (2), december 2011: 129-130. a longhorn beetle species, blepisanis vittipennis, known so far in europe but only from greece and bulgaria, was recorded for the first time in the fauna of serbia, increasing number of recorded longhorn beetle species to 238. key words: blepisanis vittipennis, cerambycidae, preševo, serbia. blepisanis vittipennis (r e i c h e , 1877) is one of two representatives of the genus blepisanis in europe. the other species, b. melanocephala (f a b r i c i u s , 1787) inhabits only island of sicily, while b. vittipennis is distributed in se balkan peninsula, caucasus, turkey and near east (h o s k o v e c & r e j z e k , 1991-2011). in the balkan peninsula, the species is reported as common in continental greece and bulgaria (s a m a , 2002), as well as in the near east turkey (ö z d i k m e n & t u r g u t , 2008). as a part of first systematic entomological research in serbia, on june 4 2011, a group of researchers visited poorly investigated region in southeast of serbia, miratovac village in vicinity of preševo. the most interesting habitats were found to the east of the village, at an altitude of 551 m, situated at 42°16΄20˝ n, 21°39΄1.6˝ e. the location is an area with a strong submediterranean influence, probably felt more than anywhere else in serbia. the biotope comprises mostly of fragmented quercus pubescens forest and dry, rocky pastures, despite the fact that wider area is predominantly used for crop planting. during that survey, one specimen of blepisanis vittipennis was found on dianthus sp. the series of photographs was taken, but since authors did not recognize species and significance of the finding, specimen was not collected for further analysis (fig. 1). good photographs enabled dr. nastas ilić to determine species and emphasize importance of this finding. first systematic review of longhorn beetles in serbia (i l i ć , 2005) listed a total of 245 taxa of this family, but some were marked with a question mark due to unreliable findings, some listed as species at the time were given status of subspecies. therefore, having in mind the most widely used taxonomy (fauna europaea, 2011), list of longhorn beetles recorded in serbia comprised 237 species (habiprot, 2005-2011), and with this finding comes up to 238. the finding of blepisanis vittipennis in serbia is significant because it increases the species areal towards the west, and vicinity of the location to macedonia implies that presence of the species in that country is probable. 2 (2) • december 2011: 129-130 biologica nyssana 2 (2) december 2011: 129-130 đrđević a., đurić m. blepisanis vittipennis (reiche 1877) (coleoptera:… 130 figure 1. blepisanis vittipennis near miratovac village. photo: milan đurić acknowledgements. the authors want to express their gratitude to dr. nastas ilić for unselfish support and sharing of his immense knowledge on longhorn beetles, and to bojan zlatković for plant determination, and also, to miloš popović and ivan simonović for their relentless support during field surveys. references fauna europaea, 2011. group coordinator audisio, p. fauna europaea version 2.4, web service available online at http://www.faunaeur.org habiprot, 2005-2011: alciphron – a database on insects of serbia. habiprot. belgrade. hoskovec, m. & rejzek, m. 1999-2011. cerambycidae, web service available online at http://www.cerambyx.uochb.cz/ ilić, n. 2005. strižibube srbije (coleoptera; cerambycidae) faunistički pregled, autorsko izdanje, beograd, 180pp. özdikmen, h. and turgut, s. 2008. new species and a new subspecies of the subgenus phytoecia (blepisanis) pascoe, 1866 from turkey (coleoptera: cerambycidae: lamiinae). mun. ent. zool. (3) 2: 568-581. sama, g.., 2002. atlas of the cerambycidae of europe and the mediterranean area vol. 1, nakladatelství kabourek, zlín (czech republic), 173 pp. milošević ilić et al., 2019, biologica nyssana 10(1) 10 (1) september 2019: 49-57 doi: 10.5281/zenodo.3464008 diversity of aphids (homoptera: aphididae) in southeastern serbia original article marijana ilić milošević department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, 18000 niš, serbia marijana183@gmail.com (corresponding author) vladimir žikić department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, 18000 niš, serbia vzikic@pmf.ni.ac.rs darija milenković department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, 18000 niš, serbia darija.velickovic@pmf.edu.rs saša s. stanković department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, 18000 niš, serbia ssasa@pmf.ni.ac.rs olivera petrović-obradović institute of phytomedicine, faculty of agriculture, university of belgrade, nemanjina 6, 11080 belgrade-zemun, serbia petrovic@agrif.bg.ac.rs received: august 2, 2019 revised: september 11, 2019 accepted: september 19, 2019 abstract: aphids represent one of the most significant pest species in agroecosystems. in this study we investigated aphid diversity in southeastern serbia. we recorded 132 species, mostly infesting host plants from the families asteraceae and rosaceae. the most diverse genus was aphis linnaeus with 34 species. within this genus, the most frequently sampled species was a. fabae scopoli. genera uroleucon mordvilko, brachycaudus van der goot and dysaphis börner were also represented by a large number of species. key words: aphids, southeastern serbia, species richness apstract: raznovrsnost biljnih vaši (homoptera: aphididae) u jugoistočnoj srbiji biljne vaši predstavljaju jednu od najznačajnijih grupa štetočina u agroekosistemima. u ovoj studiji istraživali smo raznovrsnost biljnih vaši u jugoistočnoj srbiji. zabeležili smo 132 vrste, koje uglavnom zaražavaju biljke domaćine iz porodice asteraceae i rosaceae. najraznovrsniji rod je aphis linnaeus sa 34 vrste. u ovom rodu, najčešće uzorkovana vrsta je a. fabae scopoli. rodovi uroleucon mordvilko, brachicaudus van der goot i disaphis borner takođe su predstavljeni velikim brojem vrsta. ključne reči: biljne vaši, jugoistočna srbija, bogatstvo vrsta introduction aphids (homoptera: aphididae) are one of the most economically important insect group in agriculture. they are significant plant pests, both in cultivated and self-seeding plants. apart from causing direct damage by sucking plant juices, they also transmit many plant viruses, such as the bean yellow mosaic virus, bean common mosaic virus, cucumber mosaic virus (gálvez & morales, 1989). aphids are distributed worldwide, but usually most common in temperate areas of the northern hemisphere. number of described species in world to this date is about 4700, of which about one third have been recorded from europe (coeur d’acier et al., 2010). within the territory of serbia, 374 species were found. the research on aphid fauna in serbia was most contributed by petrović (1998), petrovićobradović (2003), as well as poljaković-pajnik et al. (2002), vučetić et al. (2014) and petrović-obradović et al. (2007, 2010, 2018). the primary aim of this study was to explore the diversity of aphid fauna during the ten-year research period in the region of southeastern serbia. the secondary aim was to determine the number of trophic associations between aphid species and their host plants. materials and methods this study was based on material collected during the last ten years in different types of habitats © 2019 ilić-milošević et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 49 of southeastern serbia. aphids were collected from plant parts by using a fine thin brush and were transferred to plastic tubes filled with 90% ethanol. the specimens were identified according to keys prepared by blackman & eastop (1994, 2000, 2006). all analyzed material has been deposited at the faculty of agriculture, university of belgrade, serbia. plants were identified using josifović, ed. (19701976) and deposited in the herbarium of the faculty of sciences and mathematics, university of niš, serbia (hmn). results aphid-host plant associations species of aphididae are listed alphabetically, with the following order: host plant, host plant family in parenthesis, collection site, date of collection and legator (vž-vladimir žikić, dv-darija veličković, sss-saša s. stanković, mr-milica ristić, mimmarijana ilić milošević, mđ-maja đorđević, ml-maja lazarević, ns-nikola stanković, asaleksandra stojiljković, ms-milan stanisavljević, bz-bojan zlatković, nm-nikola mladenović, mmmajda mehanović, dn-danijela nikolić, zk-zorana kojičić, ljm-ljubomir mihajlović). acyrthosiphon caraganae (cholodkovsky, 1908) on: coronilla emerus (fabaceae), niš, niška banja, 19.5.2015, vž; pčinja canyon, prohor pčinjski, 10.6.2014, vž acyrthosiphon malvae (mosley, 1841) on: petunia sp. (solanaceae), prokuplje, 6.6.2017, dv; geranium dissectum (geraniaceae), niš, 26.4.2014, vž. acyrthosiphon pisum (harris, 1776) on: medicago sativa (fabaceae), lebane, konjino, 3.5.2014, sss; pisum sativum (fabaceae), prokuplje, gornja konjuša, 11.6.2017, mr; vicia angustifolia (fabaceae), niš, popovac, 27.4.2014, vž; vicia sp., vlasinsko jezero (lake), 6.8.2015, vž acyrthosiphon sp. on: coronilla varia, niš, 3.6.2016, vž anoecia corni (fabricius, 1775) on: cornus mas (cornaceae), niš, donji matejevac, 26.4.2018, mim; sićevačka klisura (gorge), ostrovica, 28.5.2013, mđ aphis acetosae linnaeus, 1761 on: rumex acetosella (polygonaceae), vlasinsko jezero (lake), 19.6.2018, vž aphis affinis del guercio, 1911 on: mentha aquatica (lamiaceae), bela palanka, divljana, 6.6.2014, vž; grdelička klisura (gorge), 5.6.2014, sss; m. longifolia, niš, niška banja, 23.5.2014, sss; pčinja canyon, gornji starac, 10.6.2014, sss; sićevačka klisura (gorge), sićevo, 24.5.2018, ml; vlasinsko jezero (lake), 6.8.2015, sss; vranje, 18.6.2016, ns; m. piperita, niš, bubanj, 5.6.2013, sss aphis balloticola szelegiewicz, 1968 on: ballota nigra (lamiaceae), niš, 31.5.2011, vž aphis confusa walker, 1849 on: knautia sp. (dipsacaceae), niš, niška banja, 31.5.2017, dv; sićevačka klisura (gorge), ostrovica, 28.5.2013, mđ aphis craccae linnaeus, 1758 on: vicia cracca, radan planina (mt.), 12.6.2016, vž; vlasinsko jezero (lake), 30.6.2016, sss aphis craccivora koch, 1854 on: cichorium intybus (asteraceae), leskovac, 30.5.2017, as; prokuplje, gornja konjuša, 11.6.2017, mr; coronilla varia, niš, 3.6.2015, vž; crepis biennis (asteraceae), niš, donji matejevac, 5.6.2013, vž; c. foetida, niš, 1.6.2013, vž; crepis sp., niš, 5.6.2013, vž; lactuca sp. (asteraceae), lebane, 1.6.2013, sss; medicago sativa, aleksinac, moravac, 18.6.2015, ms; lebane, 19.6.2011, sss; leskovac, 19.5.2017, as; melilotus alba (fabaceae), leskovac, 12.6.2017, as; robinia pseudoacacia (fabaceae), dukat (mt.), 7.8.2011, vž; leskovac, 25.5.2018, as; niš, 19.5.2013, vž; vranje, 17.6.2016, ns; vlasinsko jezero (lake), 7.8.2015, vž; salvia sp. (lamiaceae), niš, 5.6.2013, vž; sophora japonica (fabaceae), niš, niška banja, 6.6.2013, vž; trifolium lagopus (fabaceae), pčinja canyon, prohor pčinjski, 10.6.2014, bz; vicia cordata, niš, 21.5.2013, sss; pčinja canyon, gornji starac, 10.6.2014, sss; v. cracca, lebane, konjino, 31.5.2014, sss; prokuplje, beloljin, 14.5.2016, mr; prokuplje, bresničić, 15.6.2014, vž; vlasinsko jezero (lake), 8.8.2015, vž; vranje, 8.6.2016, ns; v. pannonica, radan planina (mt.), 12.6.2016, vž aphis crepidis (börner, 1940) on: crepis foetida, vlasinsko jezero (lake), 21.7.2013, sss aphis davletshinae hille ris lambers, 1966 on: malva sylvestris (malvaceae), prokuplje, gornja konjuša, 11.6.2017, mr aphis epilobii kaltenbach, 1843 on: epilobium montanum (onagraceae), grdelička klisura (gorge), 5.6.2014, sss aphis fabae scopoli, 1763 50 biologica nyssana ● 10 (1) september 2019: 49-57 ilić-milošević et al. ● diversity of aphids (homoptera: aphididae) in southeastern serbia 51 on: achillea millefolium asteraceae), prokuplje, gornja konjuša, 15.6.2016, mr; aegopodium podagraria (apiaceae), niš, 11.6.2014, vž; amaranthus retroflexus (amaranthaceae), niš, 1.7.2011, vž; prokuplje, gornja konjuša, 15.6.2016, mr; anthriscus sp. (apiaceae), niš, gornji matejevac, 25.5.2014, vž; prokuplje, 8.6.2017, dv; vlasinsko jezero (lake), 30.6.2016, sss; a. sylvaticus, prokuplje, gornja konjuša, 23.4.2016, mr; a. sylvestris, niš, niška banja, 14.5.2017, vž; radan planina (mt.), 6.6.2015, vž; arctium lappa (asteraceae), sićevačka klisura (gorge), ostrovica, 28.5.2013, mđ; vranje, 18.5.2018, nm; atriplex patula (chenopodiaceae), vranje, 17.7.2016, ns; ballota nigra, vranje, 4.5.2016, ns; beta vulgaris (chenopodiaceae), leskovac, 26.5.2017, as; bifora radians (apiaceae), niš, 22.5.2013, mđ; carduus acanthoides (asteraceae), leskovac, 25.5.2018, as; prokuplje, gornja konjuša, 29.5.2016, mr; centaurea cyanus (asteraceae), lebane, konjino, 31.5.2014, sss; chenopodium album (chenopodiaceae), lebane, 1.6.2013, sss; niš, 8.6.2015, sss; prokuplje, 6.6.2017, dv; prokuplje, gornja konjuša, 21.5.2017, mr; sićevačka klisura (gorge), 4.6.2011, vž; sićevačka klisura (gorge), sićevo, 17.7.2013, vž; vranje, 30.10.2016, ns; prokuplje, bresničić, 15.6.2014, sss; cichorium intybus, niš, 8.6.2015, sss; cirsium acanthoides (asteraceae), lebane, konjino, 31.5.2015, sss; c. arvense, leskovac, 18.6.2017, as; merošina, oblačinsko jezero (lake), 15.6.2014, sss; niš, 25.5.2014, sss; niš, kamenički vis, 4.6.2017, dv; sićevačka klisura (gorge), ravni do, 24.5.2015, vž; vlasinsko jezero (lake), 15.6.2013, sss; vranje, 20.6.2016, ns; c. vulgare, suva planina (mt.), bojanine vode, 7.7.2015, vž; vlasinsko jezero (lake), 6.8.2015, vž; coronilla varia, niš, 10.7.2014, vž; digitalis lanata (plantaginaceae), sićevačka klisura (gorge), sićevo, 24.5.2018, vž; euonymus japonicus (celastraceae), lebane, konjino, 31.5.2014, sss; fumaria officinalis (papaveraceae), niš, 22.5.2013, mđ; galium aparine (rubiaceae), lebane, konjino, 16.5.2015, sss; niš, 5.6.2013, mđ; niš, gornji matejevac, 25.5.2014, vž; niš, kamenički vis, 4.6.2017, dv; prokuplje, 8.6.2017, dv; sićevačka klisura (gorge), 24.5.2015, vž; sićevačka klisura (gorge), ravni do, 29.5.2014, vž; vlasinsko jezero (lake), 30.6.2016, sss; hedera helix (araliaceae), niš, 16.5.2015, vž; malva sylvestris, niš, 21.5.2018, nm; matricaria chamomilla (asteraceae), grdelička klisura (gorge), 5.6.2014, sss; lebane, 1.6.2013, sss; prokuplje, gornja konjuša, 11.6.2017, mr; sićevačka klisura (gorge), ostrovica, 28.5.2013, mđ; onopordum acanthium (asteraceae), niš, lalinac, 8.6.2013, sss; orlaya grandiflora (apiaceae), niš, gornji matejevac, 25.5.2014, vž; prokuplje, gornja konjuša, 13.6.2017, mr; papaver dubium (papaveraceae), niš, gornji matejevac, 25.5.2014, vž; p. rhoeas, niš, 5.6.2013, vž; niš, gornji matejevac, 25.5.2014, vž; sićevačka klisura (gorge), sićevo, 24.5.2018, ml; pastinaca hirsuta (apiaceae), vlasinsko jezero (lake), 6.8.2015, vž; philadelphus coronarius (hydrangeaceae), niš, 20.5.2014, sss; plantago major (plantaginaceae), niš, 6.6.2017, mr; ranunculus sp. (ranunculaceae), sićevačka klisura (gorge), ostrovic, 28.5.2013, mđ; rumex acetosella, leskovac, 6.5.2018, as; r. patientia, prokuplje, gornja konjuša, 23.4.2016, mr; vranje, 20.5.2018, nm; rumex sp., lebane, konjino, 31.5.2014, sss; niš, 22.5.2013, mđ; radan planina (mt.), 6.6.2015, vž; sićevačka klisura (gorge), sićevo, 24.5.2018, mim; suva planina (mt.), bojanine vode, 24.5.2018, ml; vranje, 7.6.2016, ns; solanum nigrum (solanaceae), niš, 8.10.2010, vž; vranje, 21.8.2016, ns; tamarix sp. (tamaricaceae), niš, niška banja, 29.5.2014, vž; tanacetum sp. (asteraceae), sićevačka klisura (gorge), sićevo, 24.5.2018, ml; tordylium maximum (apiaceae), niš, 20.5.2015, vž; torilis sp. (apiaceae), sićevačka klisura (gorge), sićevo, 7.7.2015, mim; valeriana officinalis (valerianaceae), vlasinsko jezero (lake), 21.7.2013, sss; viburnum lantana (viburnaceae), niš, 18.5.2013, mđ; xeranthemum annuum (asteraceae), prokuplje, gornja konjuša, 29.5.2016, mr; x. cylindraceum, sićevačka klisura (gorge), brljavski kamen, 7.7.2015, vž; yucca filamentosa (asparagaceae), niš, 2.7.2014, mm aphis galliscabri schrank, 1801 on: galium aparine, niš, 6.6.2017, mr; sićevačka klisura (gorge), sićevo, 24.5.2018, ml aphis gossypii glover, 1877 on: ballota nigra, niš, 18.5.2016, mr; prokuplje, gornja konjuša, 1.5.2016, mr; chrysanthemum indicum (asteraceae), lebane, konjino, 22.11.2014, sss; hibiscus syriacus (malvaceae), niš, 12.6.2011, vž; vranje, 9.5.2016, ns aphis grossulariae kaltenbach, 1843 on: lythrum salicaria (lythraceae), sićevačka klisura (gorge), sićevo, 7.7.2015, vž aphis hederae kaltenbach, 1843 on: hedera helix, niš, 1.6.2013, vž; prokuplje, gornja konjuša, 21.5.2017, mr aphis hieracii schrank, 1801 biologica nyssana ● 10 (1) september 2019: 49-57 ilić-milošević et al. ● diversity of aphids (homoptera: aphididae) in southeastern serbia 52 on: pilosella bauhini (asteraceae), sićevačka klisura (gorge), sićevo, 24.5.2018, vž aphis idaei van der goot, 1912 on: rubus ideus (rosaceae), suva planina (mt.), bojanine vode, 21.5.2014, vž aphis intybi koch, 1855 on: cichorium intybus, lebane, konjino, 31.5.2014, sss; leskovac, 28.5.2018, as; niš, 23.6.2016, mr; vranje, 28.5.2018, nm aphis nasturtii kaltenbach, 1843 on: jovibarba sp. (crassulaceae), niš, 20.6.2018, dn aphis nerii boyer de fonscolombe, 1841 on: nerium oleander (apocynaceae), leskovac, 5.6.2018, as aphis newtoni theobald, 1927 on: iris germanica (iridaceae), niš, 30.5.2013, vž aphis polygonata (nevsky, 1929) on: polygonum aviculare (polygonaceae), prokuplje, gornja konjuša, 29.5.2016, mr aphis pomi de geer, 1773 on: crataegus monogyna (rosaceae), vlasinsko jezero (lake), 15.6.2013, sss; cydonia oblonga (rosaceae), gadžin han, ovsinjinac, 20.5.2015, mr; malus pumila (rosaceae), niš, 30.5.2013, vž; niš, donji matejevac, 5.6.2013, vž; niš, pasi poljana, 27.5.2013, vž; prokuplje, gornja konjuša, 23.4.2016, mr; vranje, 28.5.2018, nm; vranje, golemo selo, 21.6.2017, nm; vranje, kopanjane, 1.7.2017, nm; prunus cerasifera (rosaceae), niš, 18.5.2016, mr; pyrus communis (rosaceae), prokuplje, 6.6.2017, dv; spiraea japonica (rosaceae), niš, niška banja, 3.5.2015, sss aphis ruborum (börner, 1932) on: rubus discolor, pčinja canyon, prohor pčinjski, 10.6.2014, vž; preševo, slavujevac, 10.6.2014, sss; r. fruticosus, niš, novo selo, 1.6.2015, mr; prokuplje, gornja konjuša, 23.5.2015, mr; rubus sp., lebane, konjino, 31.5.2014, sss; niš, 21.5.2013, vž; niš, niška banja, 23.5.2014, vž; sićevačka klisura (gorge), 4.6.2011, sss; sićevačka klisura (gorge), sićevo, 28.5.2013, vž; vranje, 4.5.2016, ns; r. ulmifolium, pčinja canyon, prohor pčinjski, 10.6.2014, sss aphis sambuci linnaeus, 1758 on: sambucus nigra (viburnaceae), merošina, oblačinsko jezero (lake), 19.4.2018, vž; niš, 15.5.2015, ml; prokuplje, 6.6.2017, dv; prokuplje, gornja konjuša, 21.5.2017, mr; sićevačka klisura (gorge), 12.5.2013, sss; suva planina (mt.), bojanine vode, 24.5.2018, ml aphis sanguisorbae (schrank, 1801) on: sanguisorba minor (rosaceae), niš, donji matejevac, 5.6.2013, vž; pčinja canyon, gornji starac, 10.6.2014, sss; prokuplje, gornja konjuša, 11.6.2017, mr aphis sedi kaltenbach, 1843 on: jovibarba heuffelii, niš, 13.6.2017, dn aphis spiraecola patch, 1914 on: chaenomeles japonica (rosaceae), niš, 30.5.2013, vž; malus pumila, leskovac, 7.5.2018, as; scabiosa argentea (dipsacaceae), pčinja canyon, prohor pčinjski, 10.6.2014, vž; campsis radicans (bignoniaceae), niš, 14.5.2013, sss aphis spiraephaga f.p. muller, 1961 on: spiraea japonica, niš, niška banja, 3.5.2015, sss aphis taraxacicola (börner, 1940) on: taraxacum officinale (asteraceae), prokuplje, gornja konjuša, 11.6.2017, mr aphis ulmariae schrank, 1801 on: filipendula ulmaria (rosaceae), vlasinsko jezero (lake), 27.5.2018, vž aphis umbrella (börner, 1950) on: malva sylvestris, sićevačka klisura (gorge), ostrovica, 28.5.2013, mđ; grdelička klisura (gorge), 5.6.2014, sss; niš, 17.6.2016, mr; vranje, 26.4.2016, ns aphis urticata j.f. gmelin, 1790 on: urtica dioica (urticaceae), niš, niška banja, 31.5.2017, dv; niš, 25.5.2014, sss; lebane, 1.6.2013, sss; niš, kamenički vis, 26.4.2018, mim; pčinja canyon, gornji starac, 10.6.2014, sss; prokuplje, gornja konjuša, 1.5.2016, mr; sićevačka klisura (gorge), 24.5.2015, vž; sićevačka klisura (gorge), ostrovica, 29.5.2014, vž; sićevačka klisura (gorge), sićevo, 28.5.2013, zk; vlasinsko jezero (lake), 6.6.2014, vž; vranje, 4.5.2016, ns aphis veratri walker, 1852 on: veratrum album (melanthiaceae), vlasinsko jezero (lake), 19.6.2018, vž aphis verbasci schrank, 1801 on: verbascum sp. (scrophulariaceae), niš, popovac, 8.6.2013, vž; vlasinsko jezero (lake), 6.6.2014, sss aulacorthum solani kaltenbach, 1843 on: lathyrus tuberosus (fabaceae), prokuplje, bresničić, 16.6.2014, sss; vinca minor (apocynaceae), niš, 1.5.2014, vž biologica nyssana ● 10 (1) september 2019: 49-57 ilić-milošević et al. ● diversity of aphids (homoptera: aphididae) in southeastern serbia 53 brachycaudus cardui (linnaeus, 1758) on: carduus acanthoides, leskovac, 25.5.2018, as; merošina, oblačinsko jezero (lake), 15.6.2014, sss; niš, 7.7.2013, vž; pčinja canyon, prohor pčinjski, 10.6.2014, vž; prokuplje, gornja konjuša, 11.6.2017, mr; carduus sp., pčinja canyon, prohor pčinjski, 10.6.2014, vž; cirsium acanthoides, lebane, konjino, 31.5.2014, sss; c. arvense, vlasinsko jezero (lake), 6.6.2014, sss; c. canddelabrum, sićevačka klisura (gorge), sićevo, 7.7.2015, mim; c. eriophorum, vlasinsko jezero (lake), 21.7.2013, sss; c. palustre, vlasinsko jezero (lake), 21.7.2013, sss; hibiscus syriacus, niš, 25.5.2013, vž; onopordum acanthium, niš, 3.6.2015, vž; niš, popovac, 8.6.2013, vž; preševo, slavujevac, 10.6.2014, sss; sićevačka klisura (gorge), ravni do, 24.5.2015, sss; prunus cerasifera, grdelička klisura (gorge), 5.6.2014, sss; niš, 22.5.2018, vž; prokuplje, gornja konjuša, 23.4.2016, mr; vlasinsko jezero (lake), 22.5.2016, vž; p. domestica, sićevačka klisura (gorge), 1.5.2013, vž; p. spinosa, niš, 23.5.2014, vž brachycaudus helichrisy (kaltenbach, 1843) on: matricaria chamomilla (asteraceae), prokuplje, gornja konjuša, 11.6.2017, mr; cephalaria transsilvanica (dipsacaceae), niš, 15.5.2013, vž; erigeron annuus (asteraceae), leskovac, 19.5.2017, as brachycaudus persicae passerini, 1860 on: prunus persica, niš, 20.5.2014, vž brachycaudus prunicola (kaltenbach, 1843) on: prunus cerasifera, niš, 3.6.2015, vž brachycaudus schwartzi (börner, 1931) on: prunus persica, niš, pasi poljana, 27.5.2013, vž brachycaudus sp. on: arctium lappa, merošina, oblačinsko jezero (lake), 15.6.2014, vž; prunus cerasifera, merošina, vlasinsko jezero (lake), 15.6.2013, vž brachycaudus tragopogonis (kaltenbach, 1843) on: scorzonera sp. (asteraceae), radan planina (mt.), 6.6.2015, vž; tragopogon dubius (asteraceae), niš, 24.5.2017, mr; prokuplje, gornja konjuša, 15.6.2016, mr; t. major, lebane, konjino, 31.5.2014, sss; niš, 23.5.2014, vž brevicoryne brassicae (linnaeus, 1758) on: alliaria petiolata (brassicaceae), gadžin han, ovsinjinac, 13.4.2016, mr calaphis flava mordvilko, 1928 on: betula pubescens (betulaceae), vlasinsko jezero (lake), 21.7.2013, sss capitophorus hippophaes walker, 1852 on: polygonum lapathifolium, niš, 13.10.2013, vž cavariella aegopodii (scopoli, 1763) on: aegopodium podagraria, niš, 2.6.2017, mim; chaerophyllum aureum (apiaceae), sićevačka klisura (gorge), ravni do, 24.5.2015, vž; foeniculum vulgare (apiaceae), sićevačka klisura (gorge), 6.6.2013, vž; sićevačka klisura (gorge), sićevo, 28.5.2013, zk cavariella theobaldi (gillette & bragg, 1918) on: pastinaca hirsuta, niš, 3.6.2015; vlasinsko jezero (lake), 15.6.2013, vž chaitophorus populeti (panzer, 1804) on: populus alba (salicaceae), leskovac, 19.5.2017, as chaitophorus sp. on: salix caprea (salicaceae), vlasinsko jezero (lake), 15.6.2013, sss; s. matsudana, niš, niška banja, 6.6.2013, vž chromaphis juglandicola (kaltenbach, 1843) on: juglans regia (juglandaceae), leskovac, 1.6.2013, vž; niš, 17.7.2011, vž; niš, popovac, 8.6.2013, vž; sićevačka klisura (gorge), ostrovica, 28.5.2013, mđ cinara cuneomaculata (del guercio, 1909) on: larix sp. (pinaceae), niš, 6.6.2017, mr cinara juniperi (de geer, 1773) on: juniperus oxicedrus (cupressaceae), pčinja canyon, gornji starac, 10.6.2014, sss cinara pilicornis (hartig, 1841) on: picea pungens (pinaceae), niš, 6.6.2017, mr cinara sp. on: pinus nigra (pinaceae), niš, gornji matejevac, 25.5.2014, vž; p. sylvestris, vlasinsko jezero (lake), 6.6.2014, sss corylobium avellanae (schrank, 1801) on: corylus avellana (corylaceae), niš, 6.6.2017, mr; niš, novo selo, 1.6.2015, mr ctenocallis dobrovljanskyi klodnitsky, 1924 on: chamaecytisus heuffelii (fabaceae), vlasinsko jezero (lake), 28.9.2017, vž drepanosiphum platanoidis (schrank, 1801) on: acer platanoides, vlasinsko jezero (lake), 6.8.2015, vž dysaphis crataegi (kaltenbach, 1843) on: orlaya grandiflora, radan planina (mt.), 12.6.2016, sss; vlasinsko jezero (lake), 15.6.2013, sss biologica nyssana ● 10 (1) september 2019: 49-57 ilić-milošević et al. ● diversity of aphids (homoptera: aphididae) in southeastern serbia 54 dysaphis devecta (walker, 1849) on: malus pumila, niš, novo selo, 17.4.2016, mr dysaphis plantaginea (passerini, 1860) on: malus pumila, niš, donji matejevac, 5.6.2013, vž; niš, pasi poljana, 27.5.2013, vž; vranje, 9.5.2016, ns; vranje, golemo selo, 3.5.2017, nm; vranje, katun, 8.5.2017, nm dysaphis pyri (boyer de fonscolombe, 1841) on: pyrus communis, lebane, konjino, 24.4.2016, sss; pyrus sp., vranje, 9.5.2016, ns dysaphis reaumuri (mordvilko, 1928) on: pyrus spinosa, preševo, slavujevac, 10.6.2014, sss dysaphis sp. on: foeniculum vulgare, sićevačka klisura (gorge), sićevo, 6.6.2013, vž; malus pumila, leskovac, 13.5.2018, as eucallipterus tiliae (linnaeus, 1758) on: tilia sp. (malvaceae), niš, niška banja, 6.6.2013, vž eulachnus sp. on: pinus nigra, vlasinsko jezero (lake), 7.8.2015, sss; p. sylvestris, vlasinsko jezero (lake), 6.8.2015, sss hyadaphis coriandri (b. das, 1918) on: foeniculum vulgare, niš, gornji matejevac, 25.5.2014, vž hyalopterus pruni (geoffroy, 1762) on: prunus cerasifera, niš, 26.4.2014, vž; vranje, 9.6.2016, ns; p. cerasus, vranje, 8.6.2016, ns; p. domestica, prokuplje, gornja konjuša, 13.6.2017, mr; vranje, 30.5.2017, ns; p. persica, niš, novo selo, 5.6.2013, vž; p. spinosa, niš, 7.5.2014, vž; vranje, 8.6.2016, ns hyperomyzus lactucae (linnaeus, 1758) on: sonchus sp. (asteraceae), grdelička klisura (gorge), 5.6.2014, sss; niš, 28.5.2014, sss; niš, gornji matejevac, 25.5.2014, vž liosomaphis berberidis (kaltenbach, 1843) on: berberis sp. (berberidaceae), vranje, 19.6.2016, ns macrosiphoniella artemisiae boyer de fonscolombe, 1841 on: artemisia absinthium (asteraceae), sićevačka klisura (gorge), 4.6.2011, vž; vladičin han, 2.6.2011, ljm; a. vulgaris, leskovac, 25.5.2018, as; niš, kamenički vis, 4.6.2017, nm macrosiphoniella sanborni (gillette, 1908) on: chrysanthemum sp., leskovac, 13.5.2018, as; niš, 3.11.2017, mr macrosiphoniella sp. on: achillea millefolium, vlasinsko jezero (lake), 8.8.2015, sss; centaurea rhenana, vlasinsko jezero (lake), 15.6.2013, vž macrosiphum euphorbiae (thomas, 1878) on: euphorbia esula (euphorbiaceae), niš, 26.4.2014, vž; malva sylvestris, vranje, 9.6.2016, ns macrosiphum funestum (macchiati, 1885) on: rubus sp., radan planina (mt.), 6.6.2015, vž macrosiphum rosae (linnaeus, 1758) on: dipsacus sp. (dipsacaceae), leskovac, 25.5.2018, as; rosa canina, niš, 30.5.2010, vž; prokuplje, gornja konjuša, 23.4.2016, mr; rosa sp., leskovac, 8.6.2018, as; niš, 31.5.2011, vž; niš, donji matejevac, 5.6.2013, vž; radan planina (mt.), 6.6.2015, vž; vranje, 7.6.2016, ns; scabiosa columbaria, vlasinsko jezero (lake), 19.6.2018, vž melanaphis pyraria (passerini, 1862) on: pyrus communis, leskovac, 1.6.2013, vž; prokuplje, gornja konjuša, 21.5.2017, mr metopeurum fuscoviride stroyan, 1950 on: tanacetum sp., sićevačka klisura (gorge), sićevo, 24.5.2018, ml; vranje, 20.6.2016, ns; t. vulgare, suva planina (mt.), bojanine vode, 7.7.2015, ml; svodje, 6.6.2014, vž; vlasinsko jezero (lake), 30.6.2016, sss metopolophium dirhodum (walker, 1849) on: triticum aestivum (poaceae), leskovac, 1.6.2013, vž microlophium carnosum (buckton, 1876) on: urtica dioica, niš, 18.5.2016, mr myzocallis carpini (koch, 1855) on: carpinus betulus (corylaceae), niš, niška banja, 29.5.2013, vž myzocallis coryli (goeze, 1778) on: corylus avellana, niš, novo selo, 1.6.2015, mr; c. colurna, niš, 21.5.2013, sss myzus cerasi (fabricius, 1775) on: prunus avium, leskovac, 1.6.2013, vž; niš, 30.5.2013, vž; niš, pasi poljana, 27.5.2013, vž; vranje, 4.5.2016, ns; p. cerasifera, niš, 18.5.2016, mr; vranje, 22.8.2016, ns; p. cerasus, leskovac, 18.5.2017, as; niš, 15.5.2013, sss; vranje, 9.5.2016, ns myzus linariae holman, 1965 on: linaria genistifolia (plantaginaceae), niš, gornji matejevac, 10.5.2015, sss; (plantaginaceae), niš, 2.7.2015, vž biologica nyssana ● 10 (1) september 2019: 49-57 ilić-milošević et al. ● diversity of aphids (homoptera: aphididae) in southeastern serbia myzus lythri (schrank, 1801) on: lythrum salicaria, niš, 2.7.2013, vž; sićevačka klisura (gorge), 28.5.2013, zk; sićevačka klisura (gorge), ostrovica, 29.5.2014, vž; vlasinsko jezero (lake), 15.6.2013, sss myzus persicae sulzer, 1776 on: capsicum annuum (solanaceae), lebane, 17.10.2011, sss; niš, 18.1.2019, vž; niš, popovac, 26.5.2013, vž; viola cornuta (violaceae), niš, 10.11.2011, vž; niš, popovac, 26.10.2011, vž myzus varians davidson, 1912 on: prunus persica, niš, 25.5.2014; vranje, 28.5.2018, nm nasonovia sp. on: pilosella officinarum (asteraceae), vlasinsko jezero (lake), 21.7.2013, sss ovatus insitus (walker, 1849) on: cydonia oblonga, gadžin han, ovsinjinac, 20.5.2015, mr panaphis juglandis (goeze, 1778) on: juglans regia, niš, 24.5.2017, mr; sićevačka klisura (gorge), 28.5.2013, mđ periphyllus aceris (linnaeus, 1761) on: acer campestre, niš, kamenički vis, 4.6.2014, sss; niš, gornji matejevac, 10.5.2015, vž; a. monspessulanum, niš, gornji matejevac, 26.4.2018, mim; niš, kamenički vis, 26.4.2018, mim periphyllus sp. on: acer campestre, niš, niška banja, 11.5.2014, sss; a. monspessulanum, niš, niška banja, 19.5.2015, vž; a. platanoides, niš, 22.5.2010, vž; a. pseudoplatanus, suva planina (mt.), bojanine vode, 21.5.2018, ml phorodon humuli (schrank, 1801) on: prunus cerasifera, lebane, konjino, 31.5.2014, sss; niš, niška banja, 23.5.2014, vž; prokuplje, gornja konjuša, 1.5.2016, mr; niš, 21.5.2013, vž; p. domestica, lebane, 1.6.2013, sss; niš, 28.5.2015, mr; niš, popovac, 20.5.2013, vž pterocomma pilosum buckton, 1879 on: salix alba, niš, donji matejevac, 3.6.2010, vž; s. caprea, vlasinsko jezero (lake), 18.6.2010, vž pterocomma populeum (kaltenbach, 1843) on: populus alba, sićevačka klisura (gorge), 29.5.2010, sss; p. nigra, lebane, konjino, 3.5.2014, sss; sićevačka klisura (gorge), 29.5.2010, sss pterocomma salicis (linnaeus, 1758) on: salix caprea, vlasinsko jezero (lake), 15.6.2013, vž pterocomma sp. on: populus tremula, niš, 25.5.2010, mim; salix caprea, stara planina (mt.), babin zub, 4.7.2010, vž rhopalosiphum maidis (fitch, 1856) on: zea mays (poaceae), sićevačka klisura (gorge), sićevo, 17.7.2013, vž rhopalosiphum nymphaeae (linnaeus, 1761) on: typha latifolia (typhaceae), bela palanka, divljana, 6.6.2014, vž; niš, niška banja, 23.7.2013, vž rhopalosiphum padi (linnaeus, 1758) on: hordeum murinum (poaceae), grdelička klisura (gorge), 5.6.2014, sss schizaphis graminum (rondani, 1852) on: zea mays, sićevačka klisura (gorge), sićevo, 17.7.2013, vž semiaphis pastinacae börner, 1950 on: pastinaca hirsuta, vlasinsko jezero (lake), 19.6.2018, vž sipha maydis passerini, 1860 on: elytrigia repens (poaceae), sićevačka klisura (gorge), 28.5.2013, zk; arrhenatherum elatius (poaceae), radan planina (mt.), 6.6.2015, vž; zea mays, niš, lalinac, 8.6.2013, sss sitobion avenae (fabricius, 1775) on: avena sativa (poaceae), lebane, 19.6.2011, sss; prokuplje, gornja konjuša, 11.6.2017, mr; triticum aestivum, leskovac, 1.6.2013, vž smiela sp. on: berteroa sp., leskovac, 21.5.2017, as tetraneura sp. on: ulmus campestris (ulmaceae), lebane, 1.6.2013, sss; ulmus sp. (ulmaceae), sićevačka klisura (gorge), 24.5.2018, vž thelaxes dryophilus (schrank, 1801) on: quercus robur (fagaceae), radan planina (mt.), 12.6.2016, sss tuberculatus sp. on: quercus cerris, niš, niška banja, 29.5.2013, sss uroleucon cichorii (koch, 1855) on: cichorium intybus, leskovac, 31.5.2017, as; crepis biennis, prokuplje, 6.6.2017, dv; c. foetida, leskovac, 19.5.2017, as; niš, kamenički vis, 4.6.2017, dv; prokuplje, gornja 55 biologica nyssana ● 10 (1) september 2019: 49-57 ilić-milošević et al. ● diversity of aphids (homoptera: aphididae) in southeastern serbia konjuša, 11.6.2017, mr; vlasinsko jezero (lake), 15.6.2013, sss; crepis sp., bela palanka, divljana, 6.6.2014, vž; vlasinsko jezero (lake), 19.6.2018, vž; lapsana communis (asteraceae), prokuplje, 6.6.2017, dv uroleucon jaceae (linnaeus, 1758) on: carthamus creticus (asteraceae), preševo, slavujevac, 10.6.2014, sss; c. lanatus, merošina, oblačinsko jezero (lake), 15.6.2014, vž; prokuplje, bresničić, 15.6.2014, sss; centaurea jacea, radan planina (mt.), 12.6.2016, sss; vlasinsko jezero (lake), 19.6.2018, vž; c. rhenana, radan planina (mt.), 12.6.2016, sss; vlasinsko jezero (lake), 15.6.2013, vž; c. salonitana, niš, 3.6.2015, vž; c. solstitialis, prokuplje, bresnčić, 15.6.2014, sss uroleucon picridis (fabricius, 1775) on: cichorium intybus, vlasinsko jezero (lake), 21.7.2013, sss; crepis paludosa, vlasinsko jezero (lake), 21.7.2013, sss uroleucon sonchi (linnaeus, 1767) on: sonchus oleraceus, prokuplje, gornja konjuša, 11.6.2017, mr uroleucon sp. on: chondrilla juncea (asteraceae), niš, 21.5.2013, vž; inula sp. (asteraceae), lebane, 1.6.2013, sss; lapsana communis, suva planina (mt.), bojanine vode, 24.5.2018, vž uroleucon tussilaginis (walker, 1850) on: tussilago farfara (asteraceae), banja topilo, 21.4.2018, vž discussion in this study we sampled 211 plant species, which were attacked by 118 aphid species within 46 genera and 9 subfamilies, formed 436 bi-trophic associations. most of the aphid species were collected on plants from the family asteraceae (105 associations) belonging to 27 genera, followed by the family rosaceae (88 associations) from 11 genera. among the aphid genera, aphis linnaeus was at the same time the most common (211 associations) and the most diverse aphid genus (34 species). within this genus, species a. fabae scopoli established the largest number of trophic associations (88), usually with plants from families asteraceae and rosaceae. aphis fabae has an almost cosmopolitan distribution and represents one of the most important agricultural pests (blackman & eastop, 2000, minks & harrewijn, 1989). also, a. craccivora koch formed 27 associations. as well as a. fabae, a. craccivora is a polyphagous species. however it prefers plants from family fabaceae (blackman & eastop, 2007). after the genus aphis, the next most diverse genera were brachycaudus van der goot (7 species), dysaphis börner (6 species) and uroleucon mordvilko (6 species). within the genus brachycaudus, the most frequently recorded species was b. cardui. this aphid altered the hosts between various fruit trees of genus prunus l. and usually carduus l. and cirsium mill. species (influentialpoints. 2018). within the genera dysaphis and uroleucon, d. plantaginea (linnaeus), u. cichorii (passerini) and u. jaceae (linnaeus) formed the largest number of associations. the primary hosts for d. plantaginea were species from genus malus mill (influentialpoints. 2018). in our study, we noticed that this aphid caused damage on species m. pumila mill. species u. cichorii mordvilko was usually collected on crepis l., while u. jaceae (linnaeus) on carthamus l. and centaurea l. species macrosiphum rosae (linnaeus) and myzus cerasi (fabricius) formed nine associations. all other recorded aphid species appeared in lower frequencies. this research contributes to a better knowledge about diversity of aphids in the territory of serbia. acknowledgements. this study was supported by the ministry of education, science and technological development of the republic of serbia (iii43001). references blackman, r.l., eastop, v.f. 1994: aphids on the world’s trees: an identification and information guide. cab international, wallingford, uk. 987p. blackman, r.l., eastop, v.f. 2000: aphids on the world’s crops: an identification and information guide. john wiley & sons, chichester, uk. 466p. blackman, r.l., eastop, v.f. 2006: aphids on the world’s herbaceous plants and shrubs, 1 volume set. john wiley & sons, chichester, uk. 1024p. blackman, r.l., eastop, v.f. 2007: taxonomic issues. in: van emden, h.f., harrington, r. 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(corresponding author) received: october 11, 2019 revised: october 13, 2019 accepted: november 24, 2019 abstract: bryological research in serbia can be divided into several periods and research fields, the first of which being floristic research. an important exploration of bryophytes in serbia started at the end of the 19th century, and with a few discontinuances it continued up today. during the last decade of the 20th century couple of researchers started their work on the exploration of bryophytes. they focused on bryologically important habitats like sources, riverbanks, peat bogs and mountains. a significant number of new species was reported for the country, but the revision of the old records has been done as well, hence today flora of serbia numbers 831 bryophyte taxa in total. progress of the botanical research has brought interdisciplinarity into bryology. as a result, bryology in serbia nowadays spread in different types of bryophyte research. for example, bryophytes are studied to investigate the biological, antimicrobial, antifungal and cytotoxic activity, biotransformation of molecules, phytochemical composition and as model organisms in stress physiology. special emphasis is also given on active conservation of the species. this represents only some directions of what is currently studying on bryophytes in serbia. key words: bryophytes, serbia, history, floristics, phytochemistry, biological activity apstract: kolekcija mahovina beou zanemareno nacionalno istraživačko blago istoriju briologije u srbiji možemo podeliti na nekoliko perioda i pravaca. prvi je pravac florističke (inventarizacione) briologije. naime značajnija briološka istraživanja u srbiji započeta su krajem devetnaestog veka i sa znatnim prekidima traju do danas. u poslednjoj dekadi dvadesetog veka pojavilo se nekoliko istraživača koji su se prevashodno posvetili istraživanju mahovina. istraživanja su usmerena na briološki zanimljiva staništa kao što su vrela, reke, tresetišta, planine. otkriven je značajan broj novih taksona ali izvršena je i revizija nekih ranijih podataka, tako da trenutno brioflora srbije broji 831 takson. kako je opšti način botaničkih istraživanja napredovao, i u briologiji se prešlo na multidisciplinarnost. grupa mlađih istraživača bavi se istraživanjima biološke aktivnosti mahovina, antimikrobne, antifungalne i citotoksične, fitohemijom mahovina, biotransformacijom molekula izolovanih iz mahovina ali i fiziologijom stresa. poseban akcenat je stavljen na aktivnu zaštitu i reintrodukciju. ovo je samo deo onoga što se danas iz briologije radi u srbiji. ključne reči: briofite, srbija, istorija, floristika, fitohemija, biološka aktivnost introduction bryological research in serbia could be divided into three periods of investigation, and several research fields. first period coincide to the onset of the botany in serbia, or with the work of josif pančić and his students. this period is characterized mainly by floristic or inventory studies, with relatively little bryophyte records made. after a brief break in investigation, research slightly intensified during the period from 1920s to 1960s with the work of group of phytocenologists. third, or the modern period, began in the end of 20th century, and is the most fruitful period so far (pantović & sabovljević, 2017). during this period, in addition to extensive floristic research, ecological investigations, the biological activity of moss extracts, their phytochemical analysis, as well as the transformation of molecules isolated from the extracts, were also initiated and successfully realized. at present, a lot of research is © 2019 veljić. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 87 13th symposium on the flora of southeastern serbia and neighboring regions conducting in the field of stress physiology in serbia, as well. a group of researchers is intensively working on the conservation of bryophytes, with an emphasis on active protection and reintroduction of the rare and threatened species. the institute of botany and botanical gardens “jevremovac” has valuable collection of over 260 species of living bryophytes from around the world, of which about 60% are regionally or globally endangered. floristic-ecological research of the bryophytes of serbia as a part of general botanical research in the territory of serbia, research of the bryophyte flora had started in the middle of the 19th century. the first bryophyte records were published in the works of the researchers of that time (grisebach, 1843; pančić, 1859, 1863). more extensive research of bryophyte flora has began at the end of the century when svetozar obradović, đura ilić and lujo adamović started researching bryophytes; they were sending most of the collected material for determination and verification by more experienced botanists (wettstein, 1890; simić, 1892; matouschek, 1899, 1901). simić (1897, 1898) published part of his research on the vranje surrounding in the reports of the vranje gymnasium. schiffner (1897) identified one species from the bornmuller’s herbarium. research results obtained during this period were summarized by jurišić (1900) and simić (1900). in these papers, they included part of their unpublished results as well jurišić cites 209 and simić 145 species. for the next ten years, bryological research was done mostly by katić (1900, 1903, 1906, 1907a, 1907b i 1909) and košanin (1909a, 1909b, 1910). after this period, bryological studies were done by podpera (1922), černjavski (1929, 1932, 1937, 1938), pichler (1931, 1939, 1940), grebenschikov (1943, 1949, 1950), soška (1949), knapp (1944), rudski (1949a, 1949b) and rajevski (1951). pavletić (1955) stated in his work “prodromus of the bryophyte flora of yugoslavia” 374 taxa (57 marchantiophyta, 317 bryophyta) to be present in serbia. since the 1950s, only sporadic bryophye records were published within the general floristic research (gigov & nikolić, 1954; gigov, 1956; slavnić, 1956; pavletić, 1956, 1968; janković & janković 1962; blečić & tatić, 1962; čolić et al., 1963; pavlović, 1951, 1964; mišić & popović, 1960; popović, 1966; filipović, 1966; guelmino, 1970, 1972; erdeši, 1971; babić, 1972; tešić et al., 1979; martinčič, 1980; janković & mišić 1980; jovanović 1980; janković & stevanović, 1981; mišić, 1982; grgić, 1983; gajić, 1983, 1986, 1988, 1989; gajić & karadžić, 1991; petković et al., 1988, 1990, 1991; mišić & panić, 1989). based on these studies, the list of bryophyte flora has been updated by gajić et al. (1991), who cited 415 bryophyte species in serbia (60 marchantiophyta, 355 bryophyta). the third period (or modern period) of bryophyte research began in the last decade of the 20th century when several researchers devoted their research to the bryophytes: ranđelović, 1994; stevanović et al., 1995; veljić et al,. 1996, 2001a, 2001b, 2004, 2006, 2008, 2013, 2016a, 2016b; veljić & marin, 1997; pavić et al., 1998; sabovljević & stevanović, 1999; petković et al., 2000; sabovljević, 1998, 1999, 2000, 2003a, 2003b, 2006; papp & sabovljević, 2001, 2002, 2010; grdović & blaženčić, 2001; sabovljević & cvetić, 2003; pócs et al., 2004; cvetić & sabovljević, 2005, papp & erzberger, 2005; grdović, 2005; grdović & stavretović, 2004, 2006; sabovljević & grdović, 2009; papp & erzberger, 2005, 2007a, 2007b, 2009; erzberger & papp, 2007; papp et al., 2004, 2006, 2012a, 2012b, 2013, 2014a, 2014b, 2014c, 2016a, 2016b, 2016c; ranđelović, zlatković, 2010; pantović & sabovljević, 2013, 2017; pantović et al., 2014; ilić et al., 2015. research was focused on biologically interesting and unexplored habitats such as springs, rivers, peatlands, mountains. this phase of intensive research of the flora and ecology of bryophytes is ongoing, and more recently two ph.d. dissertations focusing on bryophytes in serbia were done: “biogeographic and ecological study of the bryophyte flora of serbia” (pantović, 2018) and “diversity, distribution, differentiation of micro-habitats and the structure of moss communities of fruška gora mountain” (ilić, 2019). in this short period of time, 88 biologica nyssana ● 10 (2) december 2019: 87-98 veljić ● bryology in serbia: from floristics to chemosystematics fig. 1. collection of bryophytes in the beou herbarium. shown are digitized specimens included in the bryo database (photo j. pantović) a significant number of new taxa has been reported for the country, but also some previous data has been revised, so serbia currently numbers 830 bryophyte taxa (1 anthocerotophyta, 142 marchantiophyta, 687 bryophyta). historical data on the distribution and ecology of bryophytes has been collected and digitized into a unique database of bryophytes of serbia, the bryo database (fig. 1). updated lists of liverworts and mosses of serbia have been prepared and are currently in press. protection and conservation of bryophytes bryophytes, like all other living organisms, are exposed to various negative environmental factors. they are threatened similarily as vascular plants, but the attention and concern for their conservation is much lower. among bryophytes, there are taxa that are extinct, in different categories of threat, or rare, which has been confirmed by the increasing number of the regional red lists (sabovljević et al., 2004; marka et al., 2012; hodgetts et al., 2019). recognizing this global problem, a group of associates gathered around marko and aneta sabovljević (bbgb bryophyte biology group belgrade) has developed into the widely recognized group for bryophyte conservation (sabovljević et al., 2009; sabovljević et al., 2014; vujičić et al., 2012). the institute of botany and botanical garden “jevremovac” has an in vitro living collection of over 260 species of bryophytes from around the world, of which about 60% are regionally or globally endangered. nearly 52% biologica nyssana ● 10 (2) december 2019: 87-98 89 of the species in the collection are on the european and regional red lists. special attention is given to the active protection, like reintroduction and population strengthening of the rare and endangered taxa. in vitro propagated and grown plants are being prepared for reintroduction to the potentially suitable habitats. several species such as hendiella heimii and entostodon hungaricus (fig. 2) have been propagated this way and reintroduced to habitats in banat, serbia (sabovljević et al., 2012a, 2012b, 2016, 2018). ecophysiological research of bryophytes in serbia with progress of the botanical research, a multidisciplinary approach has been adopted in bryology as well. a group of young researchers has started working on bryophyte stress physiology (rajčić et al., 2015; vujičić, 2016; vujičić et al., 2016; ćosić et al., 2018). as a part of these studies, a doctoral dissertation entitled “biochemical and ecophysiological properties of selected moss species during salt stress in the axenical conditions” (vujičić, 2016) has been done. monitoring the parameters of morphogenesis revealed that bryophyes and vascular plants respond differently to the short-term and long-term salt stress, indicating different survival strategies with the increased salinity of the microenvironment. the effect of salt stress is also observed by the change in the concentration of tocopherols in mosses (physcomitrella patens, entosthodon hungaricus and henediella heimii) (fig. 3). namely, tocopherol as a non-enzymatic component plays an important role in the moss response to the stress caused by increased nacl concentration. the results of the study show that under long-term stress, the concentration of tocopherols increases on moderately saline media (50-100 mm nacl) in h. heimii. the salt stress affects concentration of proline and the ratio of proline and free amino acids in mosses (p. patens, e. hungaricus and h. heimii). proline as an essential amino acid plays a very important role in protecting the cell from osmotic stress, and it is involved in osmotic adaptation. it was discovered that proline concentration increases with the increase in salt concentration in all tested species during short-term stress, and that the ratio of proline and free amino acids increases with the increase in salt concentration in the medium. long-term salt stress induces a decrease of proline concentration in plants grown on a medium with the high concentration of fig. 2. reintroduction of henediella heimii (marked red) and enthostodon hungaricus (marked yellow), propagated axenically in vitro, acclimatized and finally released to nature on potentially suitable site in banat, serbia! (photo m. sabovljević) veljić ● bryology in serbia: from floristics to chemosystematics 90 salt. during long-term stress, the ratio of proline to free amino acids also increases with the increase of salt concentration in the medium. in addition to the non-enzymatic components, as a response to the salt stress enzyme activity (peroxidase, catalase, and superoxide dismutase) was monitored as well. phytochemical research of the bryophytes together with the ecophysiological studies, phytochemical studies of bryophytes have also been done. using the uhplc/-hesi-ms/ms methods, phenolic compounds were analyzed in physcomitrella patens (vujičić, 2016). in plants exposed to the short-term stress compared to plants exposed to the long-term salt stress, a significantly greater variety of compounds was observed. the predominantly detected compound was p-coumarin acid, which was detected in all treatments except in the control group. qualitative analysis of phenolic compounds in the plants exposed to the long-term stress showed that the number of detected phenolic compound decreases with the increase in salt concentration. in all tested species dominant phenolic compound was p-coumaric acid. the chemical composition has been investigated on selected moss species: rodobrium ontariense, atrichum undulatum, hypnum andoi (pejin et al., 2012a, 2012c). however, there are more studies of the chemical composition of liverworts. several liverwort taxa have been the subject of a doctoral dissertation: “chemical composition and antimicrobial activity of various liverwort extracts of scapania aspera m. & h. bernet, s. nemorea (l.) grolle, porella cordaeana (hub.) moore and p. arboris-vitae (with.) grolle)” (bukvički, 2014). analysis of the volatile compounds of different extracts identified 105 compounds in scapania aspera (bukvicki et al., 2013), 79 in scapania nemorea (bukvički et al., 2014), 84 in porella cordaeana (bukvički et al., 2012a)., and 66 in p. arboris-vitae (tyagi et al., 2013). chemical analysis of the volatile compounds of the extracts revealed the presence of large amounts of sesquiterpene and monoterpenes, and a much lower percentage of hydrocarbons, alcohols, aldehydes and ketones. gc-ms analysis of the hexane solution of methanolic extract of lophocolea bidentata showed two major compounds two sesquiterpene lactones. diplophyllolide (eudesmanic-type of sesquiterpene lactone), an important chemotaxonomic marker for marchantiophyta, and 5-hydroxydiplophyllolide have also been identified. bis-bibenzyls are a significant group of chemical compounds in liverworts. methanolic extract of marchantia polymorpha subsp. ruderalis contains bisbibenzyl marchantin a as a fig. 3. the appearance of the investigated species cultivated for three weeks in culture in vitro on a nutrient medium with the addition of various salt concentrations (photo m. vujičić) fig. 4. structures of seven new bisbibenzyls isolated from the methanol extract of the liverwort lunularia cruciata (novaković et al., 2019). biologica nyssana ● 10 (2) december 2019: 87-98 veljić ● bryology in serbia: from floristics to chemosystematics 91 major component (sabovljevic et al., 2017). seven new bis-bibenzyls were isolated from the methanol extract of the liverwort lunularia cruciata (fig. 4) along with one previously known bibenzyl and five known bisbibenzyls (novaković et al., 2019). these are acyclic bisbibenzyls, perrotetins and cyclic analogues, ricardines. recently, microbial transformation of molecules isolated from mosses has been the subject of the investigation. biotransformation represents an important strategy in the structural modification of natural compounds by microorganisms. biological activity of bryophytes secondary metabolites of bryophytes have important antibacterial and antifungal potential. this has been confirmed by a series of studies on both mosses and liverworts. among mosses, following species were studied: bryum argenteum (sabovljevic et al., 2006), pleurozium schreberi, palustriella commutata, homalothecium philippeanum, anomodon attenuatus, rhytidium rugosum, hylocomium splendens, dicranum scoparium, leucobryum glaucum (veljić et al., 2008), fontinalis antipyretica, hypnum cupressiforme, ctenidium molluscum (veljić et al., 2009), abietinella abietina, neckera crispa, platyhypnidium riparoides, cratoneuron filicinum, campylium protensum (bukvički et al., 2012b) and rhodobryum ontariense (pejin et al., 2012b). in general, all extracts showed stronger antifungal than antibacterial potential. however, significantly higher antimicrobial potential has been shown by secondary metabolites in liverworts. in addition to the species from scapania and porella genera that were already mentioned (bukvički, 2014), ptilidium pulcherrimum was investigated as well (veljić et al., 2010). tem analysis was performed to confirm antimicrobial activity and the mode of action. transmission electron micrographs of salmonella enteritidis (fig. 5) show the effect of the extracts: (a) control cells; (b) bacterial cell after the treatment with methanolic extract and (c) bacterial cell after the treatment of ethanol extract of the liverwort p. arborisvitae (fig. 5). more recently, the cytotoxic potential of various molecules (like terpenes, flavonoids, sterols, coumarins, and interesting macrocyclic compounds bibenzyls and bis-bibenzyls) present mainly in marchantiophyta, have also been investigated. the cytotoxic activity of the newly isolated compounds, as well as the already known ricardin g was investigated. the isolated compounds showed cytotoxic activity against a549 lung cancer cells with ic50 values of 5.0, 5.0 and 2.5 mm. pinguisane-type of compounds isolated from porella cordaeana showed imunomodulatory activity (radulović et al., 2016). this chronological overview gives a partial review of bryological researches in serbia, but also shows endless directions and possibilities of bryology. bryology has large potential and 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mathematics and natural science, universitas negeri medan, jl. william iskandar ps. v, deli serdang 20221, north sumatera, indonesia endanggultom@gmail.com (corresponding author) received: march 14, 2025 revised: may 16, 2025 accepted: may 16, 2025 abstract: trigona honey, produced by stingless bees, exhibits unique physicochemical characteristics influenced by environmental factors, nectar sources, and processing methods. this study investigates the relationship between honey color variation (yellow, red, and black) and its macronutrient composition and physicochemical properties, including moisture content, fat, protein, carbohydrate, and acidity levels, using standard laboratory methods. the findings reveal significant differences in composition among the samples, with black honey having the lowest moisture content (19.4%) and the highest acidity (134 ml naoh/kg), suggesting better stability and potential bioactive properties, while yellow honey had the highest moisture content (24.1%), making it more susceptible to fermentation. carbohydrate content was highest in red honey (37.3%), indicating its potential as an energy source. no protein was detected in any sample, and fat content ranged from 0.21% to 0.64%. the results suggest that darker-colored honey may contain higher levels of phenolic and flavonoid compounds, contributing to enhanced antioxidant and antimicrobial activity. this study underscores the importance of characterizing trigona honey for quality control, standardization, and potential applications in the food and pharmaceutical industries. further research is recommended to explore the functional properties of trigona honey, particularly its bioactive and therapeutic potential. key words: trigona honey, macronutrient composition, honey color, bioactive compounds apstrakt: makronutritivni sastav i fizičko-hemijske karakteristike obojenog meda trigona iz nacionalnog parka tesso nilo, riau trigona med, kojeg proizvode pčele bez bodlji, pokazuje jedinstvene fizičkohemijske karakteristike koje su pod uticajem faktora životne sredine, izvora nektara i metoda prerade. ova studija istražuje vezu između varijacija u boji meda (žuti, crveni i crni) i njegovog makronutritivnog sastava i fizičkohemijskih osobina, uključujući sadržaj vlage, masti, proteina, ugljenih hidrata i nivoa kiselosti, korišćenjem standardnih laboratorijskih metoda. rezultati ukazuju na značajne razlike među uzorcima u pogledu sastava: crni med je imao najniži sadržaj vlage (19,4%) i najviši nivo kiselosti (134 ml naoh/kg), što ukazuje na bolju stabilnost i potencijalna bioaktivna svojstva, dok je žuti med imao najviši sadržaj vlage (24,1%), što ga čini podložnijim fermentaciji. sadržaj ugljenih hidrata bio je najviši u crvenom medu (37,3%), što ukazuje na njegov potencijal kao izvor energije. u uzorcima nije potvrđeno prisustvo proteina, dok se sadržaj masti kretao od 0,21% do 0,64%. rezultati sugerišu da med tamnije boje može sadržati veće količine fenolnih i flavonoidnih jedinjenja, što doprinosi pojačanoj antioksidativnoj i antimikrobnoj aktivnosti. ova studija naglašava značaj karakterizacije trigona meda za potrebe kontrole kvaliteta, standardizacije i potencijalne primene u prehrambenoj i farmaceutskoj industriji. preporučuje se dalje istraživanje funkcionalnih svojstava meda trigona, posebno njegovog bioaktivnog i terapijskog potencijala. ključne reči: trigona med, makronutritivni sastav, boja meda, bioaktivna jedinjenja © 2025 simanullang et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 208 introduction honey is a natural organic substance produced from flower nectar by bees and is one of the most widely used natural sweeteners. chemically, honey is defined as a natural food composed primarily of sugars and water, along with minor components such as minerals, vitamins, amino acids, organic acids, flavonoids, other phenolic compounds, and aromatic substances. one type of honey that has gained increasing attention in recent years is trigona honey, produced by stingless bees of the genus trigona. this honey exhibits unique characteristics compared to honey produced by apis bees, particularly in terms of color, taste, viscosity, moisture content, sugar levels, and macronutrient composition, including protein, fat, and carbohydrates (gadge et al., 2024). additionally, trigona honey is known for its high acidity and complex bioactive compound composition, making it an intriguing subject for further research, particularly regarding its potential health benefits (zulkifli et al., 2023). several studies have shown that the chemical composition of honey is significantly influenced by nectar and pollen sources, which affect moisture, sugar profile, and acidity (fahrul et al., 2022; andrade velásquez et al., 2023). tesso nilo national park in riau province is a conservation area with high biodiversity that provides a natural habitat for trigona bees. the honey produced in this region is widely marketed, particularly in local markets. however, scientific studies on the macronutrient composition of this honey remain limited. characterizing the chemical composition of honey is essential for determining its quality and potential applications in the food and pharmaceutical industries (andrade velásquez et al., 2023). several studies have shown that the chemical composition of honey is significantly influenced by nectar sources, environmental conditions, and bee species. these variations directly impact honey's nutritional composition and physicochemical properties, including moisture content, carbohydrates, protein, fat, and acidity levels (gadge et al., 2024). honey contains more than 200 bioactive compounds, with sugars as its main component. fructose is the dominant sugar fraction (38–40%), followed by glucose (30–34%), maltose, and sucrose in smaller amounts. additionally, honey contains proteins, fats, organic acids, and minerals, with concentrations varying depending on its floral origin (alvarez-suarez et al., 2013). secondary metabolites such as polyphenols, flavonoids, enzymes, and carotenoid pigments are also found in honey and contribute to its biological activities, including antioxidant, antibacterial, and anti-inflammatory properties (cianciosi et al., 2018). various studies have demonstrated the potential of honey as a therapeutic agent in wound healing and infection treatment, as well as a nutritional supplement to enhance health (hadagali & chua, 2014). one of the primary distinguishing factors of trigona honey compared to other types of honey is its color variation, ranging from golden yellow to dark black. the color of honey is influenced by various factors, including the type of nectar collected by bees, the content of phenolic compounds and flavonoids, and oxidation processes during storage (sawarkar, 2024). studies have indicated that darkercolored honey generally contains higher antioxidant and mineral levels than lighter-colored honey. this is associated with the high polyphenol and organic acid content, which contributes to honey's chemical stability and biological effectiveness (luca et al., 2025). in addition to influencing antioxidant composition, honey color may also be related to its macronutrient content, including moisture, fat, protein, and carbohydrate levels. honey with high moisture content tends to have lower viscosity and a lighter color, whereas honey with lower moisture content and higher pigment concentration is typically darker and exhibits better stability during storage (vîjan et al., 2023). however, the direct correlation between honey color and macronutrient composition has not been extensively studied, particularly for trigona honey from indonesia. although previous studies have suggested that darker-colored honey generally contains higher levels of antioxidants and minerals attributed to polyphenol and organic acid content, such correlations have not been thoroughly examined in trigona honey from the tesso nilo region. this study aims to address that gap by exploring the relationship between color variation and the physicochemical composition specific to this biodiverse area. materials and methods collection of honey samples yellow, red, and black honey were obtained from tesso nilo national park, riau, using the drip method (fig. 1). moisture content test the moisture and sugar content tests were conducted using the refractometry method with a refractometer. the test was performed by placing a honey sample on the refractometer prism, and the refractive index value was then recorded. the moisture content standard is referred to as sni 01-3545-2013. biologica nyssana ● 16 (1) june 2025: 207-213 simanullang et al. ● a macronutrient composition and physicochemical characteristics of colored trigona honey from tesso nilo national park, riau 209 biologica nyssana ● 16 (1) june 2025: 207-213 simanullang et al. ● a macronutrient composition and physicochemical characteristics of colored trigona honey from tesso nilo national park, riau fat content test the fat content test was conducted using the soxhlet extraction method. a 2 g sample was placed in a filter paper and inserted into a fat extraction cartridge. the cartridge was then placed in a pre-weighed fat flask and connected to a soxhlet apparatus. the extraction cartridge was positioned in the soxhlet extraction chamber and rinsed with a fat-soluble solvent. the extraction tube was assembled onto the soxhlet distillation apparatus and heated at 40 °c using an electric heater for 6 hours. during distillation, the fat solvent evaporated and was collected in the extraction chamber. the solvent was then removed to prevent it from returning to the fat flask. afterward, the fat flask was dried in an oven at 105 °c and subsequently cooled in a desiccator until a constant weight was achieved. protein content test the protein content test was conducted using the kjeldahl method. in digestion, a 1 g sample was weighed and placed into a kjeldahl flask. then, half of a kjeldahl tablet (selenium) and 2 ml of concentrated h₂so₄ were added to the tube. the tube containing the solution was inserted into a digestion apparatus and heated at 400 °c for 1 hour until the solution turned transparent green. the solution was then allowed to cool. after digestion, the solution was transferred into a 100 ml volumetric flask and diluted with distilled water up to the marked line. a 5 ml aliquot of the diluted solution was then pipetted into a distillation apparatus. a solution of 5 ml naoh (30%) and a few drops of phenolphthalein (pp) indicator was prepared. additionally, 10 ml of 2% boric acid solution mixed with the pp indicator was used as the receiving solution. the condenser tip was rinsed with distilled water. finally, the solution was titrated with 0.01 n hcl until a color change was observed. carbohydrate content test a 25 ml sample solution was pipetted and mixed with 25 ml of luff-schoorl reagent. the mixture was then heated on a magnetic stirrer with thermal control under reflux conditions for 10 minutes. after heating, the solution was cooled, followed by the addition of 10 ml of 20% potassium iodide solution and 15 ml of 25% sulfuric acid (h₂so₄) added gradually. subsequently, a 0.5% starch indicator was added. the solution was then titrated with 0.1 n sodium thiosulfate. when foaming appeared, 1% starch solution was added and homogenized, and the titration continued until the solution turned milky white. acidity test the acidity test was conducted using an acid-base titration method based on sni 2018 standards. a 10 g honey sample was weighed and placed into a 250 ml erlenmeyer flask, then dissolved in 75 ml of distilled water. subsequently, 4–5 drops of phenolphthalein (pp) indicator were added. the honey solution was titrated with 0.1 n naoh solution until the endpoint was reached, indicated by a stable pink color that remained for 10 seconds. statistical analysis the physicochemical parameters of the three trigona honey color variants (yellow, red, and black) were compared using one-way analysis of variance (anova) and tukey’s post-hoc test to identify specific group differences. all statistical analyses were conducted using ibm spss statistics version 26, with a significance level set at p<0.05. however, due to the absence of replication, these analyses are exploratory and should be interpreted cautiously. results and discussion macronutrient testt the following are the test results (tab. 1) based on the macronutrient test conducted on red, yellow, and black trigona honey. moisture content moisture content is a crucial parameter in honey, influencing its stability, quality, and shelf life. the study results indicate that the moisture content of red, yellow, and black trigona honey is 22.4%, 24.1%, and 19.4%, respectively. according to ikhsan et al. (2022), higher moisture levels increase the risk of fermentation due to microbial activity, particularly yeasts, which can degrade honey quality. moisture content is influenced by environmental humidity, fig. 1. the appearance of trigona honey samples obtained from tesso nilo national park, riau: black, yellow and red (from left to right) 210 biologica nyssana ● 16 (1) june 2025: 207-213 zsimanullang et al. ● a macronutrient composition and physicochemical characteristics of colored trigona honey from tesso nilo national park, riau table 1. macronutrient test results of trigona honey no test parameters trigona honey red colour trigona honey yellow colour trigona honey black colour 1 taste characteristic sweet honey characteristic slightly sour honey characteristic slightly bitter honey 2 aroma characteristic honey characteristic honey characteristic honey 3 color reddish-brown yellow black 4 moisture content 22.4% 24.1% 19.4% 5 fat 0.21% 0.64% 0.25% 6 protein 0% 0% 0% 7 carbohydrates 37.3% 37.1% 33.6% 8 acidity 24.5 114% 134% temperature, hive position, and harvesting methods (kek et al., 2017). honey with high moisture content is more susceptible to fermentation, as yeast growth accelerates when moisture levels exceed 20% (da silva et al., 2024). the yellow trigona honey exhibited the highest moisture content (24.1%), making it more prone to fermentation than black trigona honey, which had the lowest moisture content (19.4%). harvest age is another critical factor affecting moisture content; honey that remains in the hive for an extended period has lower moisture levels due to continuous evaporation (zaldivar-ortega et al., 2024). environmental factors such as temperature, humidity, hive location, and harvesting techniques significantly influence honey’s moisture content. according to suhri & bahar (2023), moisture levels also depend on the ripening process within the hive, where prolonged hive storage allows for greater evaporation, reducing moisture content. furthermore, honey’s moisture content directly correlates with its viscosity—higher moisture content leads to lower viscosity, making it more fluid than honey with lower moisture content (pimentel et al., 2022). fat content the analysis showed that the fat content in red, yellow, and black trigona honey was 0.21%, 0.64%, and 0.25%, respectively. according to khalil et al. (2001), stingless bee honey generally contains very low fat levels, making it an insignificant fat source. hasan et al. (2023) reported that the lipid content in honey could also be influenced by the presence of wax or propolis that mixes with the honey during extraction. moreover, the harvesting method and filtration process affect the fat content in honey. although honey cannot be considered a primary dietary fat source, its minor lipid compounds contribute to its biological activity, including antimicrobial and antioxidant properties (olas, 2020). protein content proteins in honey are minor components derived from enzymes produced by bees and nectar and pollen collected during honey production. the study found that the protein content in red, yellow, and black trigona honey was 0.00%, significantly lower than the findings of kamal et al. (2022), who reported protein levels between 3.9% and 8.5%. according to mahani et al. (2025), protein content in honey is primarily influenced by the type of nectar consumed by bees and the environmental conditions in their habitat. several other factors may contribute to the low protein levels in trigona honey from riau, including filtration processes, harvesting methods, and storage duration, which can lead to protein degradation due to reduced enzymatic activity (hasan et al., 2023). additionally, braghini et al. (2020) found that honey protein content can be altered due to thermal processing during handling or prolonged storage. carbohydrates content carbohydrates are the primary components of honey, consisting mainly of monosaccharides (glucose and fructose), disaccharides (sucrose, maltose), and small amounts of oligosaccharides. the study results indicate that the carbohydrate content of red, yellow, and black trigona honey is 37.3%, 37.1%, and 33.6%, respectively. several factors, including moisture content, nectar source, and fermentation or storage processes, influence the variation in carbohydrate content. the sugar profile was predominantly composed of reducing sugars, including fructose and glucose, which this study did not quantify individually. nonetheless, higher 211 biologica nyssana ● 16 (1) june 2025: 207-213 simanullang et al. ● a macronutrient composition and physicochemical characteristics of colored trigona honey from tesso nilo national park, riau the lowest moisture content (19.4%) and highest acidity (134 ml naoh/kg), which are indicative of better chemical stability and possibly higher organic acid and phenolic content; red honey displayed the highest carbohydrate content (37.3%), reflecting its potential as an energy source; and yellow honey showed both the highest moisture content (24.1%) and fat content (0.64%), suggesting a greater susceptibility to fermentation. these trends are consistent with the literature indicating that honey color may correlate with specific physicochemical and bioactive properties. nevertheless, further studies with adequate biological replication are essential to validate these preliminary observations through statistically robust analysis. conclusion in conclusion, this study highlights that honey color may serve as an indicator of underlying physicochemical properties, potentially reflecting differences in botanical and environmental origin. these findings open avenues for using color variation for preliminary quality assessment in trigona honey. future studies should incorporate advanced analytical techniques to explore the specific bioactive compounds and their functional roles. references alvarez-suarez, j. m., giampieri, f., & battino, m. 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(2023). the potential neuroprotective effects of stingless bee honey [review]. aging neuroscience, 14. https://doi.org/10.3389/fnagi.2022.1048028 mahmutović-dizdarević et al. 2023, biologica nyssana 14(1) 14 (1) june 2023: 47-56 doi: 10.5281/zenodo.8027148 essential oils of selected citrus fruits and spice plants as potential antibacterial and antibiofilm agents original article irma mahmutović-dizdarević department of biology, faculty of science, university of sarajevo, zmaja od bosne 33-35, 71000 sarajevo, bosnia and herzegovina irma.m@pmf.unsa.ba (corresponding author) nejra bektaš department of genetics and bioengineering, international burch university, francuske revolucije bb, 71210 ilidža, bosnia and herzegovina šabanija gazić department of genetics and bioengineering, international burch university, francuske revolucije bb, 71210 ilidža, bosnia and herzegovina anesa jerković-mujkić department of biology, faculty of science, university of sarajevo, zmaja od bosne 33-35, 71000 sarajevo, bosnia and herzegovina mirsada hukić academy of sciences and arts of bosnia and herzegovina, center for disease control and geohealth studies, bistrik 7, 71000 sarajevo, bosnia and herzegovina institute for biomedical diagnostics and research nalaz, čekaluša 69, 71000 sarajevo, bosnia and herzegovina monia avdić department of genetics and bioengineering, international burch university, francuske revolucije bb, 71210 ilidža, bosnia and herzegovina academy of sciences and arts of bosnia and herzegovina, center for disease control and geohealth studies, bistrik 7, 71000 sarajevo, bosnia and herzegovina received: december 22, 2022 revised: april 07, 2023 accepted: april 17, 2023 abstract: this study evaluates the antibacterial and antibiofilm properties of essential oils (eos) from citrus lemon (l.) osbeck, lemon; citrus reticulata blanco, mandarine; nigella sativa l., black cumin, and foeniculum vulgare mill., fennel, using the disk-diffusion, broth microdilution, and tissue culture plate methods on 11 bacterial strains, including the multidrug-resistant (mdr). results showed that tested eos exhibit antibacterial effects, that are stronger in gram-positive bacteria. the widest inhibition zones were achieved with the black cumin eo against mdr staphylococcus aureus. values of the minimum inhibitory concentrations ranged from 250 to 750 µg/ml, while minimum bactericidal concentrations were 500-1000 µg/ml. black cumin eo performed strong antibiofilm features, with the total elimination of the biofilm in the case of different s. aureus strains (including methicillin-resistant, mrsa), and pseudomonas aeruginosa. all investigated eos exhibited strain-specific and dose-dependent antibacterial and antibiofilm activity. key words: bacterial biofilms, antibiofilm agents, essential oils, citrus lemon (l.) osbeck, citrus reticulata blanco, nigella sativa l., foeniculum vulgare mill. apstrakt: eterična ulja ploda odabranih citrusa i začinskih biljaka kao potencijalni antibakterijski i antibiofilm agensi ova studija evaluira antibakterijska i antibiofilm svojstva eteričnih ulja iz citrus lemon (l.) osbeck, limuna; citrus reticulata blanco, mandarine; nigella sativa l., crnog kima i foeniculum vulgare mill., komorača, korišćenjem disk-difuzione, mikrodilucijske i “tissue culture plate” metode na 11 sojeva bakterija, uključujući multirezistentne (mdr) sojeve. rezultati su pokazali da testirana eterična ulja ispoljavaju antibakterijski efekat, koji je jači kod gram-pozitivnih bakterija. najšire zone inhibicije su postignute delovanjem eteričnog ulja crnog kima na mdr staphylococcus aureus. vrednosti minimalne inhibitorne koncentracije su se kretale od 250 do 750 µg/ml, dok su minimalne baktericidne koncentracije bile 500-1000 µg/ml. eterično ulje crnog kima je ispoljilo jako antibiofilm dejstvo, sa totalnom eliminacijom biofilma u slučaju različitih sojeva s. aureus (uključujući meticilin-rezistentni, mrsa) i pseudomonas aeruginosa. sva ispitivana eterična ulja su ispoljila antibakterijsku i antibiofilm aktivnost koja je bila specifična za soj, te dozno-zavisna. ključne reči: bakterijski biofilmovi, antibiofilm agensi, eterična ulja, citrus lemon (l.) osbeck, citrus reticulata blanco, nigella sativa l., foeniculum vulgare mill. introduction antimicrobial resistance is considered one of the major global health challenges of the 21st century (hernando-amado et al., 2019). according to urbanchmiel et al. (2022), antibiotic resistance is acquired via several mechanisms such as active removal of the antibiotic from the cell, enzymatic modifications of the drug, modifications of cell components which are the target of the antibiotic, overexpression of an enzyme inactivated by the antibiotic, a change in the permeability of bacteria cell membranes, production of an alternative metabolic pathway, an increase in the concentration of a metabolite which is an antagonist of the antibiotic, a reduction in the amount or activity of an enzyme activating the precursor of the antibiotic, modifications in regulatory systems not associated with the direct mechanism © 2023 mahmutović-dizdarević et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 47 of action of the antibiotic, or a reduction in the demand for the product of the inhibited metabolic pathway. bacterial biofilm represents a structured consortium of microorganisms embedded in a self-produced matrix made from polysaccharides, proteins, and dna, characterized by increased resistance to antimicrobial agents (høiby et al., 2010). studies suggest that microbial cells within the biofilm have 10–1000 times more antibiotic resistance in comparison to planktonic cells (mah, 2012). furthermore, bacterial biofilms are involved in approximately 80% of chronic and recurrent microbial infections in humans (sharma et al., 2019). numerous investigations have shown that different plant products as well as plant active ingredients can inhibit the formation and development of bacterial biofilms, and eradicate mature biofilms (cheng et al., 2022). essential oils (eos) are the volatile secondary metabolites of plants, produced by more than 17.500 plant species from many angiosperm families, but only about 300 of them are commercialized (wińska et al., 2019). numerous biological activities such as antiseptic, antibacterial, antiviral, antioxidant, antiparasitic, antifungal, and insecticidal effects are previously reported for eos. also, eos are considered a powerful tool in the reduction of bacterial resistance (chouhan et al., 2017). essential oils as well as other bioactive products of medicinal plants have received excessive attention for their low toxicity, pharmacological activities, and economic viability (auddy et al., 2003). therefore, eos and their components are naturally occurring antimicrobial compounds with the potential to prevent the limitations of conventional antimicrobial agents (orhan-yanıkan et al., 2019). lemon, citrus limon (l.) osbeck (rutaceae), is a well-known and frequently used plant for different purposes, especially in cooking, but studies suggest its various biological activities such as anticancer, antioxidant, antimicrobial, etc. lemon is also proven for many beneficial effects on the nervous, cardiovascular, respiratory, and skeletal systems (klimek-szczykutowicz et al., 2020). bioactive properties such as antimicrobial, anticancer, antioxidant, antigenotoxic, hepatoprotective, etc. are also related to citrus reticulata blanco (rutaceae), the mandarin orange (musara et al., 2020). nigella sativa l. (ranunculaceae), known as black cumin, is a worldwide distributed plant with many curative effects on human health. the pharmacological significance of this species is mainly illustrated by its capacity to act as an antimicrobial and antioxidative agent (tabassum et al., 2018). fennel or foeniculum vulgare mill. (apiaceae), is a seasonal medicinal plant, grown by humans in nearly every region. due to its great aromaticity and spiciness, this is a popular cooking ingredient but nevertheless, pharmacological studies revealed that fennel possesses bioactive potential and could be used in the treatment of different diseases (tripathi et al., 2012). the main goal of this study was to evaluate the antibacterial and antibiofilm potential of essential oils made from well-known edible and spice plants: lemon, c. lemon (lemon), c. reticulata (mandarin orange), n. sativa (black cumin), and f. vulgare (fennel). materials and methods essential oils in this investigation pure eos derived from citrus lemon (l.) osbeck, citrus reticulata blanco, nigella sativa l., and foeniculum vulgare mill. (dea flores d.o.o., rijeka, croatia) were used. stock solutions of the test substances were prepared in 0.1% dimethyl sulfoxide ≥99% (dmso) (sigma-aldrich) and kept at room temperature in the dark. bacterial species investigation of antibacterial and antibiofilm properties of selected eos comprised a total of 11 bacteria, including the multidrug-resistant (mdr), strains: staphylococcus aureus atcc 6538 (sa1); s. aureus atcc 25923 (sa2); methicillin-resistant s. aureus (mrsa): s. aureus atcc 33591 (sa3), and s. aureus nctc 12493 (sa4); enterococcus faecalis atcc 29212 (ef); bacillus subtilis atcc 6633 (bs); escherichia coli 14169 (ec1); e. coli atcc 25922 (ec2); extended-spectrum beta-lactamase-producing (esbl) e. coli atcc 35218 (ec3); pseudomonas aeruginosa atcc 27853 (pa); salmonella enterica nctc 6017 (se). investigated bacterial strains were purchased from the american type culture collection (atcc) and national collection of type cultures (nctc) (microbiologics, st. cloud, minnesota, usa). inoculums were set in accordance with eucast (2017). overnight cultures were diluted in sterile saline solution to the final turbidity of 0.5 mcfarland standard, which corresponds to the bacterial concentration of 1.5 × 108 cfu/ml. determination of the inhibition zones the antibacterial activity of investigated essential oils was evaluated by using pure eo (100%), as well as different concentrations of eo (250, 500, and 750 µg/ml) aseptically dissolved in 0.1% dmso. for this part of the study, the disk diffusion method was applied (bauer et al., 1966). bacterial strains were cultured in mueller hinton (mh) medium (fluka biochemica; buchs, 48 biologica nyssana ● 14 (1) june 2023: 47-56 mahmutović-dizdarević et al. ● essential oils of selected citrus fruits and spice plants as potential antibacterial and antibiofilm agents biologica nyssana ● 14 (1) june 2023: 47-56 mahmutović-dizdarević et al. ● essential oils of selected citrus fruits and spice plants as potential antibacterial and antibiofilm agents with bacterial inoculum. inoculums were prepared as described above, i. e turbidity of 0.5 mcfarland standard and bacterial concentration of 1.5 × 108 cfu/ml. the adherence of bacteria in the presence of tsb was used for the determination of the biofilm formation. after the overnight incubation, the plates were emptied, washed in phosphate buffered saline, pbs (sigma-aldrich, usa), and stained with 0.1% crystal violet solution for 10 minutes. after washing, 96% ethanol was added to each well. results were analysed on the microplate reader (biochrom ez read 400) at 595 nm. this experiment was done in four replications, and results were given as the means ± stdev. the biofilm-forming category was determined according to stepanović et al. (2007) and by the biofilm classifier software ver 1.1. the optical density cut-off value (odc) was calculated as three standard deviations above the mean od of the negative control, while the biofilm categories were determined as follows: od ≤ odc: non-adherent (na), odc < od ≤ 2 x odc: weakly adherent (w), 2 x odc < od ≤ 4 x odc: moderately adherent (m), and 4 x odc < od: strongly adherent (s). statistical analysis for the calculation of the descriptive statistical parameters (mean values and standard deviation) and the percentage of biofilm inhibition microsoft office 2019 excel (microsoft corporation, usa) was used. data were further analyzed by one-way anova and post-hoc fisher’s lsd test (statistica 10; statsoft. inc.) at the significance level of p<0.05. results and discussion the antibacterial activity of essential oils is mainly associated with their hydrophobic properties, which ensure their distribution in the lipids of the cell membranes and mitochondria, which further affects the structural integrity and function of those structures and after all results in leakage of cell contents (tang et al., 2020). such events interfere with the atp balance and among others, have an impact on ph, protein synthesis, coagulation of the cytoplasmic material, dna disruption, and quorum sensing inhibition (el-tarabily et al., 2021). obtained results regarding the antibacterial activity of investigated eos through the measuring of inhibition zones are presented in table 1. overall observation showed that all investigated eos exhibited antibacterial potential against all tested bacteria except e. coli atcc 14169, e. coli atcc 35218 (esbl strain), and s. enterica nctc 6017. the most sensitive bacterial species was b. subtilis, with achieved inhibition zones with all tested eos, in all concentrations except the lowest dilution switzerland), and after adjusting the final density of inoculum (1.5 × 108 cfu/ml), spread over the growth medium plates. an amount of 10 µl of each concentration of eos was impregnated into the paper disk which was enough to achieve saturation. a total of six disks (four concentrations of eo, positive and negative control included) were placed on one 150 mm plate. after the application of eo, plates were incubated at 35±2 °c for 16-18 hours. as the positive control, antibiotics ampicillin (10µg), streptomycin (10µg), and colistin (10µg), all made by oxoid™ (great britain) were used, while 0.1% dmso served as the solvent control. the antibacterial effect was evaluated based on the diameter (mm) of inhibition zones. all tests were performed in triplicate, and the mean value ± standard deviation (stdev) was taken for further analysis. minimum inhibitory and minimum bactericidal concentration the minimum inhibitory concentration (mic) of eos was determined through the broth microdilution method (clsi, 2018). pure eos (100%) were dissolved in 0.1% dmso to achieve the stock concentration of 1000 µg/ml. the final volume of 100 µl of two-fold dilutions of eos ranging from 500 to 1.95 µg/ml, was applied in a 96-well microtiter plate containing the mueller hinton broth (sigma-aldrich). an amount of 10 µl of bacterial inoculum (concentration of 1.5 × 108 cfu/ml) was then added to each well. pure bacterial culture was taken as the growth control, and uninoculated media with the dmso was used as the negative control. after the overnight incubation, results were read on a microplate reader (biochrom ez read 400) at 595 nm. experiments were performed in quadruplets, and mic concentrations were determined based on the generated absorbance values. minimum bactericidal concentration (mbc) was evaluated by replating the inoculums from the wells without signs of growth on a sterile mueller hinton medium and observing the presence of growth after overnight incubation. evaluation of the antibiofilm activity in order to determine the biofilm formation in the presence of tested eos, the tissue culture plate method (tcp) in 96 well plates (merritt et al., 2005) was used, with the tryptic soy broth, tsb (sigmaaldrich) as the growing medium. the stock solution of eo in dmso was two-fold diluted in tsb up to the end concentration of 1.95 µg/ml. an amount of 100 µl of such dilution was added to each well, followed by the inoculation with 10 µl of the bacterial suspension. uninoculated media with dmso was used as the solvent control, while wells described as the untreated biofilms controls contained media 49 sa m pl e b ac te ri al st ra in s sa 1 sa 2 sa 3* sa 4* e f b s e c 1 e c 2 e c 3* pa se g ro w th in hi bi tio n (m m ) pure eo le m on n ia n ib n ic, d 19 .0 0± 0. 0e 11 .0 0± 0. 0f 20 .0 0± 0. 0 a ,b ,c ,e ,f, g, h, i,j n ig n ih, k, l n ic, k n ii n id, j,l m an da rin e 13 .3 0± 0. 6a n ib n ic, d n ie n if 12 .3 0± 1. 2 a ,b ,c ,e ,f, g, h, i,j n ig n ih, k, l n ic, k n ii n id, j,l b la ck c um in 19 .7 0± 1. 2a 21 .0 0± 0. 0b 38 .3 0± 2. 9c, d 32 .0 0± 1. 7e n if 30 .5 0± 0. 7 a ,b ,c ,e ,f, g, h, i,j n ig 30 .0 0± 0. 0h, k, l n ic, k 12 .0 0± 1. 0i n id, j,l fe nn el n ia n ib 8. 50 ±1 .4 c, d n ie n if 11 .3 0± 1. 5 a ,b ,c ,e ,f, g, h, i,j n ig n ih, k, l n ic, k n ii n id, j,l 250 µg/ml le m on n ia n ib n ic, d n ie n if n i a ,b ,c ,e ,f, g, h, i,j n ig n ih, k, l n ic, k n ii n id, j,l m an da rin e n ia n ib n ic, d n ie n if n i a ,b ,c ,e ,f, g, h, i,j n ig n ih, k, l n ic, k n ii n id, j,l b la ck c um in n ia n ib 17 .3 0± 2. 5c, d n ie n if n i a ,b ,c ,e ,f, g, h, i,j n ig n ih, k, l n ic, k n ii n id, j,l fe nn el n ia n ib n ic, d n ie n if n i a ,b ,c ,e ,f, g, h, i,j n ig n ih, k, l n ic, k n ii n id, j,l 500 µg/ml le m on n ia n ib n ic, d n ie 10 .0 0± 0. 0f 13 .0 0± 0. 0 a ,b ,c ,e ,f, g, h, i,j n ig n ih, k, l n ic, k n ii n id, j,l m an da rin e 10 .7 0± 0. 6a n ib n ic, d n ie n if 8. 30 ±0 .6 a ,b ,c ,e ,f, g, h, i,j n ig n ih, k, l n ic, k n ii n id, j,l b la ck c um in 7. 30 ±0 .6 a 9. 00 ±2 .6 b 30 .3 0± 2. 8c, d 20 .3 0± 2. 3e n if 27 .0 0± 1. 4 a ,b ,c ,e ,f, g, h, i,j n ig 21 .7 0± 2. 9h, k, l n ic, k 9. 30 ±0 .6 i n id, j,l fe nn el n ia n ib n ic, d n ie n if 7. 00 ±1 .7 a ,b ,c ,e ,f, g, h, i,j n ig n ih, k, l n ic, k n ii n id, j,l 750 µg/ml le m on n ia n ib n ic, d 16 .5 0± 0. 7e 9. 00 ±0 .0 f 14 .0 0± 0. 0 a ,b ,c ,e ,f, g, h, i,j n ig n ih, k, l n ic, k n ii n id, j,l m an da rin e 8. 00 ±1 .7 a n ib n ic, d n ie n if 8. 70 ±0 .6 a ,b ,c ,e ,f, g, h, i,j n ig n ih, k, l n ic, k n ii n id, j,l b la ck c um in 16 .7 0± 1. 2a 17 .3 0± 2. b 35 .2 0± 1. 7c, d 26 .3 0± 2. 5e n if 29 .0 0± 2. 6 a ,b ,c ,e ,f, g, h, i,j n ig 28 .3 0± 1. 5h, k, l n ic, k 10 .0 0± 0. 0i n id, j,l fe nn el n ia n ib 6. 60 ±1 .1 c, d n ie n if 8. 30 ±1 .1 a ,b ,c ,e ,f, g, h, i,j n ig n ih, k, l n ic, k n ii n id, j,l a m pi ci lli n 11 .0 0± 0. 0a 12 .0 0± 0. 0b n ic, d n ie 9. 00 ±0 .0 f 22 .0 0± 0. 0 a ,b ,c ,e ,f, g, h, i,j 14 .0 0± 0. 0g 16 .0 0± 0. 0h, k, l n ic, k n ii n id, j,l st re pt om yc in 9. 00 ±0 .0 a 10 .0 0± 0. 0b n ic, d n ie 15 .0 0± 0. 0f 20 .0 0± 0. 0 a ,b ,c ,e ,f, g, h, i,j 15 .0 0± 0. 0g 17 .0 0± 0. 0h, k, l 12 .0 0± 0. 0c, k 14 .0 0± 0. 0i 15 .0 0± 0. 0d, j,l c ol is tin n ia n ib n ic, d n ie n if 10 .0 0± 0. 0 a ,b ,c ,e ,f, g, h, i,j 16 .0 0± 0. 0g 15 .0 0± 0. 0h, k, l 10 .0 0± 0. 0c, k 17 .0 0± 0. 0i 11 .0 0± 0. 0d, j,l ta bl e 1. d ia m et er o f i nh ib iti on z on es o f i nv es tig at ed e ss en tia l o ils a nd s ta nd ar d an tib io tic s sa 1: s ta ph yl oc oc cu s au re us a tc c 6 53 8; s a2 : s . a ur eu s at c c 2 59 23 ; s a3 : s . a ur eu s at c c 3 35 91 (m r sa ); sa 4: s . a ur eu s n c tc 1 24 93 (m r sa ); ef : e nt er oc oc cu s fa ec al is a tc c 2 92 12 ; b s: b ac ill us s ub til is a tc c 6 63 3; e c 1: e sc he ric hi a co li at c c 1 41 69 ; e c 2: e . c ol i a tc c 2 59 22 ; e c 3: e . c ol i a tc c 35 21 8 (e sb l) ; p a: p se ud om on as a er ug in os a at c c 2 78 53 ; s e: s al m on el la e nt er ic a n c tc 6 01 7. * m d r s tra in s. le tte rs in s up er sc rip t i nd ic at e st at is tic al ly s ig ni fic an t d iff er en ce s af te r p er fo rm in g po st -h oc f is he r’s l sd te st . v al ue s w ith th e sa m e le tte r d iff er s ig ni fic an tly at p <0 .0 5. r es ul ts a re m ea n± sd ev . n i= n o in hi bi tio n. d m so =n i. mahmutović-dizdarević et al. ● essential oils of selected citrus fruits and spice plants as potential antibacterial and antibiofilm agents biologica nyssana ● 14 (1) june 2023: 47-56 50 mahmutović-dizdarević et al. ● essential oils of selected citrus fruits and spice plants as potential antibacterial and antibiofilm agents (250 µg/ml). in comparison to the antibacterial activity of standard antibiotics, the black cumin eo performed the greater growth inhibition of b. subtilis (tab.1). black cumin is recognized for its antimicrobial, immunomodulatory, antiinflammatory, and antioxidant features (ugur et al., 2016). dalli et al. (2021) investigated the chemical profile of the black cumin eo from different geographical areas. as the major components α-phellandrene, β-pinene, β-cymene, and 4-caranol were identified, and the antibacterial activity of this plant could be associated with them. according to previous findings (oussalah et al., 2006; saad et al., 2013), volatile compounds possess the capacity to inhibit the synthesis of structural macromolecules and growth regulators. in general, gram-positive bacteria included in this study were more susceptible to the activity of tested eos. growth inhibition zones observed in gram-positive strains ranged from 6.60±1.10 (s. aureus atcc 33591, mrsa strain; achieved by the activity of fennel eo at 750 µg/ml) to 38.30±2.90 (same strain; inhibited by the pure eo of black cumin). a possible explanation of such results could be related to the more complex and rigid outer membrane of gram-negative bacteria, where the lipopolysaccharide layer limits the diffusion of hydrophobic constituents, unlike the gram-positive bacteria, where peptidoglycan cell wall provides less resistance (patterson et al., 2019). suggested mechanisms of the antibacterial activity of eos are increased cell permeability and toxic effects on membrane structure (swamy et al., 2016; chouhan fig. 1. inhibited bacterial strains after the application of essential oils and antibiotics et al., 2017). furthermore, there should be mentioned that eos are composed of 20 to 60 different compounds (nazzaro et al., 2013), and specific ones could be responsible for the particular antibacterial activity. adequate diffusion of the active molecules through the agar medium requires optimization of medium concentration to achieve optimum activity of some chemical compounds. the results of uzair et al. (2017) suggest that essential oils exhibit a strong synergism with certain antibiotics, even in mdr bacterial strains. therefore, some essential oils that weren’t so effective alone, could be discussed as synergistic candidates. both mrsa strains included in this study were susceptible to eos, while the same strains were resistant to all standard antibiotics (tab. 1). the number of bacterial strains inhibited by the activity of investigated eos and antibiotics is presented in fig. 1. broth microdilution protocol was performed after all the results from the disk-diffusion method were gained. only those bacterial species where the inhibition with the investigated eo was achieved, were further analyzed in the sense of determining the minimum inhibitory concentration. minimum inhibitory concentrations of investigated eos against tested bacterial species ranged from 250 µg/ml, recorded in the case of the black cumin eo against s. aureus atcc 33591 (mrsa strain), while the highest mic values (750 µg/ml) are related to the fennel and lemon eos against s. aureus atcc 33591 and s. aureus nctc 12493, both mrsa strains. mic values of the fennel and lemon eos against mrsa strains (s. aureus atcc 33591 and s. aureus nctc 12493, respectively) were extrapolated from the disk-diffusion method since there was bacterial growth at 500 µg/ml, and no growth at 1000 µg/ml (pure oil in dmso), but a particular concentration of 750 µg/ml was not included in the decimal dilutions used in the broth microdilution method. obtained values are presented in tab. 2. in order to establish the mbc value, the results of the previously performed methods were taken into account. the mbc was determined for the eos with known mic. with the exception of the black cumin eo against the s. aureus atcc 33591 (mrsa), where mbc was determined at 500 µg/ml, other samples biologica nyssana ● 14 (1) june 2023: 47-56 51 52 biologica nyssana ● 14 (1) june 2023: 47-56 performed bactericidal activity as the pure eo, at 1000 µg/ml of dmso. for the evaluation of the antibiofilm potential of tested eos, the first step included the determination of the biofilm-forming category of investigated bacterial strains based on the results generated in positive controls. obtained results showed that strong biofilm-formers were: s. aureus atcc 6538 (sa1), s. aureus nctc 12493 (sa4; mrsa), e. coli atcc 35218 (ec3; esbl), and p. aeruginosa atcc 27853 (pa). moderately adherent biofilm was detected in s. aureus atcc 25923 (sa2), s. aureus atcc 33591 (sa3; mrsa), b. subtilis atcc 6633 (bs), and e. coli 14169 (ec1). weakly adherent biofilm was formed by e. faecalis atcc 29212 (ef), e. coli atcc 25922 (ec2), and s. enterica nctc 6017 (se). mean absorbance values with standard deviations are presented in fig. 2. mahmutović-dizdarević et al. ● essential oils of selected citrus fruits and spice plants as potential antibacterial and antibiofilm agents table 2. minimum inhibitory concentration (µg/ml) of tested essential oils and standard antibiotics essential oils bacterial strains sa1 sa2 sa3 sa4 ef bs ec1 ec2 ec3 pa se lemon 750* 500 500 mandarine 500 black cumin 500 500 250 500 500 500 500 fennel 750* 500 sa1: staphylococcus aureus atcc 6538; sa2: s. aureus atcc 25923; sa3: s. aureus atcc 33591 (mrsa); sa4: s. aureus nctc 12493 (mrsa); ef: enterococcus faecalis atcc 29212; bs: bacillus subtilis atcc 6633; ec1: escherichia coli atcc 14169; ec2: e. coli atcc 25922; ec3: e. coli atcc 35218 (esbl); pa: pseudomonas aeruginosa atcc 27853; se: salmonella enterica nctc 6017. *mic is extrapolated according to the results of the disk-diffusion method. fig. 2. biofilm-forming categories of investigated bacteria (red columns, strong biofilm-formers: sa1, staphylococcus aureus atcc; sa4, methicillin-resistant (mrsa) s. aureus nctc 12493; ec3, extendedspectrum beta-lactamase-producing (esbl) e. coli atcc 35218; pa, pseudomonas aeruginosa atcc 27853. yellow columns, moderate biofilm-formers: sa2, s. aureus atcc 25923; sa3, methicillin-resistant s. aureus (mrsa) atcc 33591; bs, bacillus subtilis atcc 6633; ec1, escherichia coli 14169. green columns, weak biofilm-formers: ef, enterococcus faecalis atcc 29212; ec2, e. coli atcc 25922; se, salmonella enterica nctc 6017) subinhibitory concentrations of tested eos were examined for antibiofilm properties. changes in the biofilm-forming capacity of investigated bacteria due to the activity of eos are presented in tab. 3. lemon eo decreased the biofilm-forming capacity of s. aureus nctc 12493 (mrsa), with full inhibition of the biofilm formation at a concentration of 62.5 µg/ml. the same sample removed the weakly adherent biofilm of e. faecalis atcc 29212 and the moderately adherent biofilm of b. subtilis atcc 6633 at 125 µg/ml. lemon eo is known for its antibacterial potential, but recently there are also studies regarding its antibiofilm effects (sun et al., 2018; luciardi et al., 2021; jamil et al., 2022). our investigation confirmed that lemon eo has the ability to inhibit bacterial growth, including the mdr strains. furthermore, antibiofilm activity is recorded against gram-positive bacteria. as with other eos, biological activity is typically associated with the most abundant compound. lawrence & palombo (2009) noted that particular constituents of eo don’t show the same level of antibacterial activity as the complete oils, which suggests the involvement of the minor components, that can act synergistically with major compounds. in the lemon eo the most abundant monoterpene hydrocarbons are d-limonene, γ-terpinene, and β-pinene, while among the oxygenated monoterpenes, the most abundant are α-terpineol, nerol, and geraniol (yazgan et al., 2019). mandarin orange eo leads to a reduction of biofilm formation of s. aureus atcc 6538 which is otherwise a good biofilm producer, and in b. subtilis atcc 6633 which has a moderate biofilm-forming capacity, to weakly adherent at 125 µg/ml. mandarin orange eo consists mainly of limonene and γ-terpinene, while other compounds include α-and β-pinene, β-myrcene, o-cymene, and b-thujene, with the limonene being considered the main constituent involved in bioactive and antimicrobial properties (song et al., 2020). song et al. (2021) noted that mandarin orange eo can inhibit biofilm formation and destroy mature biofilms, but these authors also emphasize the lower activity of eo against gram-negative bacteria. in accordance with other results of this study, black cumin eo showed great antibiofilm potential, and eradication of biofilm was recorded in the case of s. aureus atcc 6538, s. aureus atcc 33591 (mrsa), s. aureus nctc 12493 (mrsa), and p. aeruginosa atcc 27853. at the concentration of 125 µg/ ml of black cumin eo, there was no bacterial cell adherence in the abovementioned strains. bourgou et al. (2010) also noted differences in the chemical composition of the black cumin eo from various geographical areas, while chaieb et al. (2011) defined thymoquinone as the active principle of the black cumin with anti-biofilm potential. a review by forouzanfar et al. (2014) stands out the antimicrobial properties of black cumin, with an accent of wide medicinal use of this plant without reported side effects. fennel eo leads to the eradication of preformed biofilms of s. aureus atcc 33591 (mrsa) and b. subtilis atcc 6633 at the concentration of 125 µg/ml. antibacterial properties of fennel investigated earlier (diao et al., 2014; mutlu-ingok, 2021; alam et al., 2022) are recently supported by antibiofilm studies of this plant and its derivates (kwiatkowski et al., 2020). the major ingredient of the fennel eo is p-metoxypropenylobenzene, which is a by-product of terpene synthesis (kwiatkowski table 3. impact of the tested eos on the biofilm-forming capacity of investigated bacteria essential oil bacterial strains sa1 sa2 sa3 sa4 ef bs ec1 ec2 ec3 pa se lemon +++* +* ++* mandarine ++ + black cumin +++* + ++* +++* + + +++* fennel ++* ++* sa1: staphylococcus aureus atcc 6538; sa2: s. aureus atcc 25923; sa3: s. aureus atcc 33591 (mrsa); sa4: s. aureus nctc 12493 (mrsa); ef: enterococcus faecalis atcc 29212; bs: bacillus subtilis atcc 6633; ec1: escherichia coli atcc 14169; ec2: e. coli atcc 25922; ec3: e. coli atcc 35218 (esbl); pa: pseudomonas aeruginosa atcc 27853; se: salmonella enterica nctc 6017. changes in the biofilm-forming category: +++ decreasing up to three biofilm-forming categories ++ decreasing up to two biofilm-forming categories + decreasing in one biofilm-forming category * elimination of the biofilm in presence of a particular concentration of tested eo no antibiofilm activity mahmutović-dizdarević et al. ● essential oils of selected citrus fruits and spice plants as potential antibacterial and antibiofilm agents biologica nyssana ● 14 (1) june 2023: 47-56 53 biologica nyssana ● 14 (1) june 2023: 47-56 mahmutović-dizdarević et al. ● essential oils of selected citrus fruits and spice plants as potential antibacterial and antibiofilm agents et al., 2019). conclusion essential oils represent bioactive compounds of natural origin, where the proportion of active constituents vary due to geographical distribution and many environmental conditions. these slight differences in the chemical profile of the essential oil could be crucial in terms of the occurrence of bacterial resistance. the antibacterial potential of eos derived from four edible and spice plants: lemon, mandarin orange, black cumin, and fennel is emphasized through the results of this research. performed investigation showed that essential oils derived from c. lemon, c. reticulata, n. sativa, and f. vulgare exhibited antibacterial properties which are proved through different microbiological assays. gram-positive bacteria were more susceptible to the activity of eos. for instance, mdr s. aureus treated with black cumin eo had the greatest growth inhibition, which was not achieved by the activity of tested antibiotics. additionally, b. subtilis was successfully inhibited by all four eos. besides inhibition, investigated eo performed bactericidal properties. the antibiofilm activity was noted for all examined eos, with the most prominent activity of the black cumin eo, where complete inhibition of the biofilm formation was noted for strong biofilm-formers, including mdr strains. it could be concluded that all investigated essential oils displayed strain-specific and dose-dependent antibacterial activity. references alam, a., foudah, a. i., salkini, m. a., raish, m. & sawale, j. 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(2019). antimicrobial influence of nanoemulsified lemon essential oil and pure lemon essential oil on food-borne pathogens and fish spoilage bacteria. international journal of food microbiology, 306. mahmutović-dizdarević et al. ● essential oils of selected citrus fruits and spice plants as potential antibacterial and antibiofilm agents 56 microsoft word 0105_matovic_et_al biologica nyssana 1 (1-2) december 2010: 43-47 matović, m. et al. natural potentials of the medicinal plants… 43 original article ! natural potentials of the medicinal plants from the orchidaceae family with mucus as the main ingredients from zlatar mountain milić matović1, biljana nikolić2*, gorica đelić3, marija marković1 1 university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia 2 institute of forestry, kneza višeslava 3, 11030 belgrade, serbia 3faculty of sciences, university of kragujevac, radoja domanovića 12, 34000 kragujevac, serbia * e-mail: smikitis@gmail.com abstract: matović, m., nikolić, b., đelić, g., marković, m.: natural potentials of the medicinal plants from the orchidaceae family with mucus as the main ingredients from zlatar mountain. biologica nyssana, 1 (12), december 2010: 43-47. the spontaneous medicinal flora of zlatar mountain was studied in the aim of realizing the possibilities of its sustainable use for the needs of the pharmaceutical industry. the special attention was paid to genera orchis, ophrys, plathanthera, gimnadenia, etc. from the orchid family (orchidaceae) of which salep is made (tuber salep). salep is a typical mucous drug (contains over 50% of mucus), which is very beneficial and useful. the primary role of salep is to heal and strengthen the organism and urge the sexual and every other biological ability. orchids of which salep is made (orchis coriophora, orchis laxiflora, orchis morio, orchis mascula, orchis pallens, orchis purpurea, orchis simia, orchis tridentata and orchis ustulata) are to be found on numerous habitats of zlatar (in the bright forests, clearing areas and on forest meadows). key words: tuber salep, orchis, drug, pharmacopoeia, production of the orchids introduction ! species from orchidaceae family (order asparagales) are spread over the whole planet. there are about 22000 to 26000 species, classified in about 880 genera (s t e v e n s , 2001). the main development orchids reached in the tropic areas (particularly in the tropic forests), where they live as epiphytes or lianas. in the mild and cold zones they are quite sparse, and they usually grow on the lime base (p a v i ć e v i ć et al., 1968; s a r i ć , ed., 1986; o b r a t o v & m a t o v i ć 1993a; m a t o v i ć et al., 2005). because of the unusually beautiful, impressive and fragrant flowers, numerous species are grown as decorative plants in the form of the numerous strains and hybrids. as economically important plants, orchids has proportional small role. the most important of them is vanilla planifolia jacks., originating from mexico. some species from the orchid family (orchidaceae), are used in medicine because of the abundant content of mucus in the tuber (genus orchis, ophrys, plathantera, gimnadenia, etc). genus orchis consists of about 75 species which grow in europe, mild part of asia, in north africa, on the canary islands and in north america. endangerment of many plant species, their ecology as well as measures for their protection have already proposed (m a t o v i ć , 1985, 1986, 1993; m a t o v i ć et al., 1994, 2005; m a t o v i ć & t a t i ć , 1999; n i k o l i ć et al., 2006). 10th sfses • 17-20 june 2010, vlasina lake1 (1-2) • december 2010: 43-47 biologica nyssana 1 (1-2) december 2010: 43-47 matović, m. et al. natural potentials of the medicinal plants… 44 material and methods ! the greatest part of the data was collected by personal investigation in zlatar, starting from the very foot at the banks of lim, mileševka and uvac, to the greatest peaks of the mountain. this research has been performed since 2006. the spontaneous medicinal flora of zlatar as the natural healing factors in the forest ecosystems was studied in the aim of perceiving the possibilities of its sustainable use for the needs of the health tourism. the orchids of which salep is made are to be found on the numerous forest habitats of zlatar. the optimal conditions the orchids have in the bright forests, clearing areas and forest meadows. the plant species from the orchid family (orchidaceae) which are the subject of the research in this paper were determined by the keys of the modern flora: “pharmacopoea serbica“ (1881-1926), “lekovito bilje u srbiji“ (p e t r o v i ć , 1883), “parmacopoea jugoslavica“ (1933-1984), “flora europaea“ (t u t i n at al., 1964–1980), “flora sr srbije“ (j o s i f o v i ć et al., 1970–1977), “flora of serbia“ (s a r i ć 1986),“lekovite biljke srbije“ (s a r i ć , ed., 1989), o b r a t o v (1992). results and disscusion there are 13 species of orchids in flora of serbia (s a r i ć , 1989), and 9 of them from the genus orchis are present in the zlatar mountain: 1. orchis coriophora l. 1753, 2. orchis laxiflora lam. 1779, 3. orchis morio l. 1753, 4. orchis mascula l. 1755, orchis pallens l. 1771, 6. orchis purpurea hudson 1762, 7. orchis simia lam. 1779, 8. orchis tridentata scop. 1772, and 9. orchis ustulata l. 1753 (diklić in j o s i f o v i ć , 1976; o b r a t o v & m a t o v i ć 1992, 1993b,c; m a t o v i ć & m i h a j l o v , 1993; m a t o v i ć et al., 1993). orchids from zlatar mountain are extremely beautiful, colourful, small, but long-lasting herbaceous plants. their leaves are green, succulent, whole across the rib and have parallel nerves. flower stalk is straight and in the upper part it carries a bunch of unusually beautiful, irregular flowers, which vary in colours, and to the greater extent they vary in form and size. the orchids blossom from spring to the mid summer, which depends upon the altitude. the orchids sometimes have several rootlets and two tubers which are most often egg-shaped, after which the whole genus was named, which was derived from the greek word orchis = egg, because of the appearance of the tuberlets. the egg out of which the overground stem grows in the spring is bigger, but rugged, soft and dark. the egg which is smaller, but succulent, solid, firm, whole, young and bright is placed next to it. as summer goes by, the young egg becomes bigger, and the old one deteriorates and disappears. the healing power of the orchid is placed in the egg-shaped tubers of which salep is made (tuber salep). fig. 1. orchis morio , zlatar mt. (author: m. matović, 2010) the tubers are of different form and size. some of them are egg-shaped, whereas the others are paw and fork shaped. round tubers are of the smaller walnuts' size. the demand for them and their prices are higher than of the fork sized ones, although their healing value is the same. round tubers have the following orchid species from zlatar mountain: orchis mascula, orchis morio, orchis tridentata and orchis purpurea. they all have red or purple flowers. according to round tubers they are similar to some other orchids: orchis militaris and orchis mascula ssp. signifera. this group of the orchids is accompanied with the other similar species with the egg-shaped tubers from which salep is made. there are following species of such orchids: platanthera bifolia which have the white, fragrant flowers and oval leaves and ophrys sphegodes with the vertically eliptic to wide lancet-shaped leaves, the widest in the middle, and ponted on the top). finger-shaped tubers have the following orchids: orchis maculata, orchis latifolia, orchis palustris, orchis sambucina, as well as related species with the finger-shaped tubers (gymnadenia conopsea). while the orchids blossom only the young tuber is extracted. the trained collectors of medicinal plants also extract it after the blossom, as the yield is bigger at the time, and the young tuber is larger. however, one should be careful not to dig the tubers of the other non-medicinal or poisonous biologica nyssana 1 (1-2) december 2010: 43-47 matović, m. et al. natural potentials of the medicinal plants… 45 plants. the special attention should be paid to autumn saffron. extracted tubers are cleaned of soil, washed in cold water, stringed on the thread or place in the net, put in the boiling water, kept for a few minutes until the water boils again or scalded by hot water, then they are dried in the draft, in the sun or in the drier at temperature lower than 700c. by boiling water the tubers lose the unpleasant odour and bitterness which they have when they are fresh, they dry more quickly, starch grains are turned into the glue, and the dried tuber as a result becomes transparent and hornlike. the tubers which are not scalded dry slowly, darken and become moldy before they are completely dried. boling water destroy the enzymes and the salep obtained in such way can be preserve for a long time. the tubers prepared in such way are round, heart-shaped, whereas some resemble fist or are paw-fork-shaped, very hard, hornlike and very heavy (they look like pebble), little transparent on the surface, 1-4 cm long, and 0.5-3 cm wide. on the top they have a stamp of the stem bud. on the surface they are irregulary furrowlike, uneven, netlike rugged, whereas some are almost smooth or little rough. their colour is brownish or whiteyellow. their taste is very mucous and insipid. the name salep is derived from the persian sahlep i schalap= slimy, the name for the little tubers and for the hot drink which is made from them, either from the orchid species of the orchis genus or from the related genera gymnadenia, ophrys, platanthera, etc. the main ingredient of salep is mucus (about 50%), there are 25% of starch, about 5% of albumen, 1% of sugar. it also contains cellulose, fat substances, organic acid and other pharmacodynamic active substances. salep is a typical mucous drug, which is very beneficial and useful. by cooking in water the thick, sinewy, sticky, colloidal solutions are obtained. when used, it forms a noble membrane on the mucosa which alleviates pains and prevents inflammation. therefore, salep has been used in the scientific medicine for centuries as a mild, harmless, efficient healing remedy for treatment of the various diseases. the primary role of salep is to heal and strengthen the organism. salep is often used in the form of so-called "salep mucus". according to the regulations of our pharmacopoeia (pharmacopoeia jugoslavica) our pharmacies prepare this mucus when it is needed, with the doctor's prescription, in the following way: 1 g of salep in powder and 1 g of lactose (milky sugar) are mixed well and this mixture is put in the measured bottle. the mixture is drenched with 2 g of alcohol, mixed, filled up with 10-15 g of boiled destilled water the weight of which is up to 100g. the liquid is often shaked until it is completely cool and the even thich mucus is formed. the mucus prepared in this way is very useful and healing, but children do not like drinking it. therefore, prior to use it has to be mixed well in a glass with currant, orange, lemon, cherry juice, or some other refreshing drink. french and some other pharmacopoeias give this description for making the medicinesweet, pleasant for use, in contrast to a pure salep which is insipid and disagreeable, owing to which many persons, particularly children do not like drinking it: 30g of chocolate is dissolved in the mild heat, 1,000g of salep in the form of the noble powder is added, mixed well and poured in the forms which the children prefer. salep is drunk as a remedy for enjoyment, sweet, and nourishing drink. salep which is cooked in water or milk for a long time the makers sweetened with honey, spiced with aromatic herb powders and poured in order that it is drunk hot. salep is harmlees, nourishing and healing drink, and easily digested food, the use of which is yet to be affirmed. an important precondition for the preservation of the species diversity of orchids on their natural habitats is the large-scale orientation towards the plantation production (fig. 2 and 3). fig. 2. the production of the orchids from the culture, zlatar mt. (author: m. matović, 2010) conclusion relations between man and his search for the medicines in nature have a long tradition, about which different sources in the form of the written data, preserved monuments and original plant medicines testify. knowledge which these sources give to use is undoubtedly the result of a long human struggle with diseases, in which man learnt biologica nyssana 1 (1-2) december 2010: 43-47 matović, m. et al. natural potentials of the medicinal plants… 46 to find the medicines in plants, their bark, seedlings, fruits, leaves and other organs. for many medicines of plant origin, which were known in the oldest human civilizations as well and which have been used for millennia, the modern science confirmed the active effect and incorporated them in pharmacopoeia. fig. 3. experimental plots of orchids from the culture, zlatar mt. (author: m. matović, 2010) the orchids are widely spread in zlatar from the very foot to the top of the mountain. out of 75 species, which grow in europe, the mild part of asia, north africa, on the canary islands and in north america, 13 species are present in flora of serbia and some of them are rare and endangered, and we found 9 orchis species at zlatar mt. alongside with systematic and chemical diversity, zlatar orchids are very variable and morphological. this is particularly expressed in the variability of the shapes of the tubers ranging from round and egg-shaped to the paw-shaped with the numerous transitory forms. salep is obtained from little tubers is typical, mucous drug, which is very efficient and useful. by cooking in water the thick, sinewy, sticky and colloidal solutions are obtained. upon use, salep forms a noble membrane on the mucosa, which covers the mucus, alleviate and soothes the pains and inhibits the inflammation, because of which is has been used in medicine for centuries as a mild, harmless, efficient healing remedy for the treatment of the various diseases. thus, the primary role of salep is to heal and strengthen organism. a number of the medicinal plants which are in circulation have a limited spread in serbia, and owing to the use in folk and official medicine they become endangered, which points to the need of the limited collecting and possibility of the prohibition of the total use in serbia. primary protection of some representatives of orchid family (orchidaceae) comes from the 1973 cites (convention on international trade in endangered wild flora and fauna) and later, from iucn red list (orchid specialist group). for the individuals of these species, their parts and derivates, the trade is prohibited, except for seeds and pollen, seedlings or culture of tissues in vitro obtained individuals, as well as cut plants of flowers produced in the culture. alongside with the protection of these species, it is needed to protect the areas, such as protected natural properties, identify the centers of medicinal plants, as well as the parts of the network of the important plant areas. the protection is carried out in the habitat (in situ) and outside of it (ex situ). medicinal plants with mucous substances have a concrete ability as every other creatures or their groups to take part in the preservation of the accord and harmony in the biosphere. nature is perfectly rational and useful, so it left to a chance not even a particle of the biosphere which would endanger its sustainable development. at preservation and improvement of biodiversity of medicinal plants and other plant species the way of sustainable use of biological resources can be defined, if the economically important biological resources are concerned, or the revitalisation can be applied on the nature habitats, if we want to protect the economically important species. for both approaches it is needed to make the plan and programme of the protection, which contains the information about spread, ecology of the species, endangerment, measures for protection. biologica nyssana 1 (1-2) december 2010: 43-47 matović, m. et al. natural potentials of the medicinal plants… 47 references josifović, m., ed., 1970-1977: flora sr srbije. i-ix. srpska akademija nauka i umetnosti, beograd. matović, m. 1985: vegetacija kanjona mileševke. polimlje (prijepolje), 96 p. matović, m. 1986: biljni pokrivač okoline prijepolja. polimlje (prijepolje), 166 p. matović, m. 1993: reliktna vegetacija srednjeg polimlja. prirodno-matematički fakultet, kragujevac, 144 p. matović, m., mihajlov, m. 1993: contribution to the medical forests plants serbien pancicia and spruce (pancicio-piceetum abietis mat.) on mountain zlatar (serbia). archiv of farmacy, 56, belgrade, pp. 131-135. matović, m., mihajlov, m., svilkić, b., topuzović, m. 1993: the medical plants of the pastures and meadows in the valley of prijepolje (serbia), archiv of farmacy, 5-6, belgrade, pp. 141-144. matović, m., bukvić, s., jovičić, d. 1994: zaštita životne sredine i biljni svet, naučna knjiga, beograd. matović, m., tatić, b. 1999: endemične biljke. zavod za udžbenike i nastavna sredstva, beograd. matović, m., đelić, g., nikolić, b., braunović, s., brašanac, lj. 2005: istraživanje ugroženih retkih biljnih vrsta na području jugozapadne srbije. 8th symposium on flora of southeastern serbia and neighbouring regions. june 20 24, 2005, niš, abstracts, p. 45. nikolić, b., matović, m., rakonjac, lj. 2006: endemorelic tree species in southwest serbia. international scientific conference in occasion of 60 year of operation of institute of forestry, belgrade, serbia: sustainable use of forest ecosystems, the challenge of the 21th century, 8 10 th november 2006, donji milanovac, serbia, the book of abstracts, p. 145, proceeedings, pp. 575-582. obratov, d. 1992: flora i vegetacija planine zlatar. beograd: biološki fakultet, doktorska disertacija. obratov, d., matović, m. 1992: rare plant speces in the forest associations in the central polimlje. protection of nature, 45, belgrade, pp. 47-54. obratov, d., matović, m. 1993a: zastupljenost vrsta sa taninskim svojstvima u flori planine zlatar. iii simpozijum o flori jugoistočne srbije, leskovacpirot, zbornik radova, pp. 17-27. obratov, d., matović, m. 1993b: lekovite biljke smrčevih šuma planine zlatar. savetovanje o lekovitim i aromatičnim biljkama jugoslavije, zlatibor, 8-10. septembar 1993, izvodi radova, p. a-3. obratov, d., matović, m. 1993c: chorological ecomorphologie, taxonomie and phytocoenological characteristics of the species pancicia serbica vis., optima meeting, borovetz, bulgaria, pp. 8-9. pavićević, n. et al. 1968: zemljište starog vlaha i raške, institut za proučavanje zemljišta, beograd. petrović, s. 1883: lekovito bilje u srbiji. srpski arhiv za celokupno lekarstvo, 2, xvi. kraljevska srpska državna štamparija, beograd. ... ph. serb. i, 1881: pharmacopoea serbica, editio prima, typographia principetus serbe, belgradi. ... ph. serb. ii, 1908: srpska farmakopeja, drugo izdanje pharmakopoea serbica, edittio secunda, izdanje kralj. srb. ministarstva unutrašnjih dela, beograd. ... ph. serb. ii, 1926: srpska farmakopeja, drugo izdanje. pharmakopoea serbica, editio secunda, apotekarska komora kraljevine srba, hrvata i slovenaca, beograd. ... ph. jug. i, 1933: parmacopoea jugoslavica mcmxxxiii, jugoslovenska farmakopeja 1933, apotekarska komora, beograd. ... ph. jug. ii, 1951: farmakopeja fnrj, pharmacopoea jugoslavica, editio secunda, medicinska knjiga, beograd. ... ph. jug. iii, 1972: farmakopeja sfrj, pharmacopoea jugoslavica, editio quarta, vol. iii, savezni zavod za zdravstvenu zaštitu, beograd. ... ph. jug. iv, 1984: farmakopeja sfrj, pharmacopoea jugoslavica, edittio quarta, vol. iii, savezni zavod za zdravstvenu zaštitu, beograd. sarić, m. (ed.) 1989: lekovite biljke srbije, srpska akademija nauka i umetnosti, beograd. stevens, p.f. 2001: angiosperm phylogeny website, july 2010. http://www.mobot.org/mobot/research/apweb/ tutin, t.g., v.h. heywood, n.a. burges, d.m. moore, d.h. valentine, s.m. walters & d.a. webb (eds.), 1964-1980: flora europaea, i-v. cambridge university press. london anticancer compounds from medicinal plants biologica nyssana 3 (2)  december 2012: 61-67 jović j. et al..  influence of solvent on antimicrobial activity... 61 original article influence of solvent on antimicrobial activity of carlinae radix essential oil and decoct jovana jović * , tatjana mihajilov-krstev, andrea žabar, zorica stojanović-radić department of biology and ecology, faculty of science and mathematics, university of niš, višegradska 33, 18000 niš, serbia * e-mail: jovanajović85@gmail.com abstract: jović, j., mihajilov-krstev t., žabar a., stojanović-radić z.: influence of solvent on antimicrobial activity of carlinae radix essential oil and decoct. biologica nyssana, 3 (2), december 2012: 61-67. plants of the family asteraceae are known for their use in ethnopharmacology, available as commercial drugs. in this study, antimicrobial activity of carlinae radix commercial drug’s vinegar decoction and essential oil, dissolved in various solvents (etohethanol, dmsodimethyl sulfoxide and tween 80 polyoxyethylene sorbitan monolaurate) was tested to investigate the effect of solvents on activity and to compare the results with previous researches. the microdilution method was used to determine the minimum inhibitory concentration (mic) and minimum bactericidal concentration (mbc). the results showed that antimicrobial activity of the carlinae radix oil significantly depends on the solvent and that most efficient antimicrobial effect had the essential oil dissolved in 7% ethanol, which points to significant synergistic effect of the oil with this solvent. key words: antimicrobial activity, carlina acanthifolia l. root, synergistic activity, solvent, mic and mbc introduction plants of the family asteraceae are known for their use in ethnopharmacology. essential oil is present in high amounts in these plants, but in traditional medicine much more frequent way of utilisation is using of decoct. carlina acaulis l. (asteraceae) is widely spread herb in east asia and europe. commonly used part of this plant in ethnopharmacology is root, known as carlinae radix. in traditional medicine, well known use of this drug is in the form of tinctures and decoction against urinary tract infections, skin diseases and wound irrigation (t u c a k o v , 1971). it was confirmed that inulin and flavonoids from roots have the antitumor, antiviral, antibacterial, antidiabetic, antioxidant and neuroprotective activity (a l b u l e s c u et al., 2004; c h a n et al., 2010). previous investigations on this commercial drug’s composition, based on morphological and anatomical features of the dried root material, revealed that in serbia, carlinae radix mostly contains roots of c. acanthifolia, instead of c. acaulis (đ o r đ e v i ć et al., 2004; s t o j a n o v i ć r a d i ć , 2011). together with this, chemical analyses determined very similar chemical compositions of the oils from both species, suggesting that adulteration of the drugs would not affect biological activities of commercial drug material. studies of c. acanthifolia essential oil showed yield from 1-2%, while chemical analysis identified 11 compounds of the oil (đ o r đ e v i ć et al., 2005; s t o j a n o v i ć r a d i ć , 2011). carlina 3 (2) • december 2012: 61-67 biologica nyssana 3 (2)  december 2012: 61-67 jović j. et al..  influence of solvent on antimicrobial activity... 62 oxide was a major component (98.9±0.9%), which is known as potent antimicrobial compound (c h a l c h a t et al., 1996; w i c t h l et al., 2002). the essential oil showed inhibitory activity on reference strains of staphylococcus aureus, escherichia coli, pseudomonas aeruginosa, proteus vulgaris, klebsiella pneumoniae and candida albicans (đ o r đ e v i ć et al., 2007; s t o j a n o v i ć r a d i ć et al., 2012). together with essential oil’s antimicrobial activity, s t o j a n o v i ć r a d i ć et al. (2012) investigated antimicrobial properties of different root decocts, where vinegar decoct had the highest inhibitory effect on the bacterial strain s. aureus atcc 6538 in comparison to water and wine decocts (s t o j a n o v i ć r a d i ć et al., 2012). in this study, we tested the antimicrobial activity of vinegar decoct and essential oil isolated from commercial carlinae radix drug against total of eighteen microbial strains. among them, one set was made of atcc (american type culture collection) reference strains, while the second set was comprised of multiresistant clinical isolates from wounds. these strains were chosen in order to explore the effect on common wound pathogens, since this plant is commonly used for wound irrigation. together with this, the tested oil was dissolved in three different solvents (etoh ethanol, dmsodimethylsulfoxide and tween 80 polyoxyethylene sorbitan monolaurate) in order to investigate the influence of solvent to antimicrobial activity and compare the results with previous researches. materials and methods essential oil isolation the essential oil was obtained from 100 g of dried root material of the commercial herbal drug carlinae radix (“jeligor”, svrljig) by hydrodistillation method, using clevenger’ s apparatus (c l e v e n g e r , 1928). the essential oils (1.05–1.60 g per batch) were obtained in the mean yield of 1.05% (w/w). the obtained oils were separated by extraction with freshly distilled diethyl ether and dried over anhydrous magnesium sulphate. the solvent was evaporated under a gentle stream of nitrogen at room temperature in order to exclude any loss of the essential oil and immediately analyzed. when the oil yields were determined, after the bulk of ether was removed under a stream of n2, the residue was exposed to vacuum at room temperature for a short period to eliminate the solvent completely. the pure oil was then measured on an analytical balance and multiple gravimetric measurements were taken during 24 h to ensure that all of the solvent had evaporated. preparation of decoct. decoct was prepared from 2 g of the roots, which were cut into small pieces and then extracted with 100 ml of boiling apple vinegar for 10 min, as described previously (s t o j a n o v i ć r a d i ć et al., 2012). after the plant material was filtered off, the obtained extract (decoct) was used as such in the antimicrobial tests. microorganisms antimicrobial activity assays were performed against eight american type culture collection (atcc) strains: salmonella enteritidis 13076, pseudomonas aeruginosa 3554, enterococcus faecalis 19433, enterobacter aerogenes 13048, proteus mirabilis 12453, clostridium perfringens 19404, klebsiella pneumoniae 10031 and yeast candida albicans 10231. multiresistant bacterial strains were isolates from wounds (clinical isolatesci): enterobacter aerogenes, acinetobacter sp. (2 clinical isolates), proteus mirabilis, escherichia coli, staphylococcus aureus, pseudomonas aeruginosa (3 clinical isolates) and klebsiella oxytoca. bacterial strains were maintained on the nutrient agar and yeast on sabouraud dextrose agar (microbiological laboratory, department of biology and ecology, faculty of science and mathematics, niš). antimicrobial activity testing antimicrobial activity was evaluated by microdilution method as described previously (s t o j a n o v i ć r a d i ć et al., 2010). bacterial strains were transfered on new nutrient agar and candida albicans on sabouraud dextrose agar, and incubated for 18 h at 37°c. overnight strains were used to make suspensions in sterile saline solution (0.9% nacl). standard turbidity was adjusted to 0.5 mcfarland (density of bacterial cells 1.0-1.5 x 10 8 and yeast 1.0-1.5 x 10 7 cfu/ml) and this inoculum size was used to prepare a final colony number of 12 x 10 6 (1-2 x 10 5 for yeast) colony forming units (cfu/ml) in a plate with sterile mueller hinton broth (mhb). carlinae radix essential oil was dissolved in three different solvents: 70% ethanol, dmso and 0.05% tween 80. a serial doubling dilutions of the oil in mueller hinton broth (methodology 1) or in the same solvent (methodology 2) were prepared in 96 well microtiter plate and used for experimental work. biologica nyssana 3 (2)  december 2012: 61-67 jović j. et al..  influence of solvent on antimicrobial activity... 63 methodology 1. microtiter plate wells were filled with 100 µl mhb and 100 µl carlinae radix oil (dissolved in 70% ethanol) were added into the first wells. double dilutions were made by transferring 100 µl of the first dilution in subsequent wells (for each microorganism strain) in a concentration range from 0.03 mg ml -1 to 70 mg ml 1 . antimicrobial activity was tested in 96-well plates, prepared by dispensing 90 µl of mhb and 1 µl of the inoculum into each well. then, 10 µl of the appropriate oil dilutions were transferred to wells and initial concentration of the oil was 7 mg ml -1 . methodology 2. microtiter plates were filled with different solvents (100 µl in each well): 70% ethanol, dmso and 0.05% tween 80. double dilutions were made in solvent by adding carlinae radix oil (100 µl into the first wells), dissolved in the same solvent, and transferring 100 µl from the first in subsequent wells. antimicrobial activity was tested like in methodology 1, with oil concentration range from 0.003 mg ml -1 to 7 mg ml -1 . negative controls were solvents in inoculated broth: 7% ethanol, 10% dmso and 0.005% tween 80. in all tests, positive controls were tetracycline (bacteria) and nystatin (yeast), both in the concentration range from 0.25-512 µg ml -1 . decoct activity was tested in plates with mhb (100 µl). the first wells (for each microorganism strain) were filled with 100 µl of decoct for making a series of double dilutions and inoculated. concentration range of decoct was from 0.024% to 50% (v\v). negative control was commercial apple vinegar in initial concentration of 2% (v\v). after incubation period of 24 h at 37°c, bacterial growth was determined by adding 20 µl of 0.5% ttc (triphenyl tetrazolium chloride) aqueous solution. mic (minimum inhibitory concentration) was read as the lowest concentration of oil at which there was no visible growth and red color. the broth from wells without visible growth (100 µl) was transferred to mha (mueller hinton agar) for 24 h at 37°c. minimum bactericidal concentration (mbc) was defined as lowest oil concentration killing 99.9% of microorganism cells. control of microorganisms’ growth was inoculated broth (without oil), while non-inoculated wells were included to ensure broth sterility. the experiment was performed in triplicate and the mean values are presented. results and discussion the results of broth microdilution assay of carlinae radix essential oil are presented in table 1. in methodology 1, the oil showed mic/mbc activity in the range from 0.055-7.000/1.750-7.000 mg ml -1 . the highest microbistatic and microbicidal effect were manifested against reference strain pseudomonas aeruginosa 3554 (mic/mbc = 0.055/1.750 mg ml -1 ). also, significant activity was obtained against clinical isolates pseudomonas aeruginosa (ci 2), acinetobacter sp. (ci 2) (mic/mbc = 0.219/7.000 mg ml -1 ) and atcc strains enterobacter aerogenes 13048 (mic/mbc = 0.437/1.750 mg ml -1 ) and salmonella enteritidis 13076 (mic/mbc = 0.437-3.500 mg ml-1) (graph 1). clinical isolates klebsiella oxytoca, acinetobacter sp. (ci 1), escherichia coli, pseudomonas aeruginosa (ci 1) and proteus mirabilis were resistant to the highest tested concentration (7.000 mg ml -1 ). graph 1. minimum inhibitory concentrations of carlinae radix essential oil dissolved in ethanol (methodology 1). the bars above the concentration of 7.00 mg ml-1 are placed to represent strains resistant to the highest tested concentration of essential oil. microbial strain legend: 1enterobacter aerogenes (clinical isolate), 2acinetobacter sp. (clinical isolate 1), 3 proteus mirabilis (clinical isolate), 4escherichia coli (clinical isolate), 5staphylococcus aureus (clinical isolate), 6 pseudomonas aeruginosa (clinical isolate 1), 7pseudomonas aeruginosa (clinical isolate 2), 8klebsiella oxitoca (clinical isolate), 9pseudomonas aeruginosa (clinical isolate 3), 10 acinetobacter sp. (clinical isolate 2), 11: salmonella enteritidis 13076, 12pseudomonas aeruginosa 3554, 13 enterococcus faecalis 19433, 14enterobacter aerogenes 13048, 15proteus mirabilis 12453, 16clostridium perfringens 19404, 17klebsiella pneumoniae 10031 and 18 candida albicans 10231. biologica nyssana 3 (2)  december 2012: 61-67 jović j. et al..  influence of solvent on antimicrobial activity... 64 biologica nyssana 3 (2)  december 2012: 61-67 jović j. et al..  influence of solvent on antimicrobial activity... 65 generally, gram-negative bacterial strains possess higher resistance to external agents, which can be attributed to their characteristic membrane structure (beveridge, 1999). the results obtained in the present study for klebsiella pneumoniae 10031 and candida albicans 10231, showed higher resistance in comparison with the previous research (s t o j a n o v i ć r a d i ć et al., 2012). in previous investigation, solvent was absolute alcohol which could lead to synergism and, thus, higher activity of oil. results of microdilution method (methodology 2) where the essential oil was dissolved in ethanol showed inhibitory activity against all tested strains at surprisingly low concentrations, lower than the tested concentration range (mic<0.003 mg ml -1 ), but without bactericidal effect (mbc>7.000 mg ml -1 ) (table 1). the results could be explained by the synergism between the antimicrobial components of the oil and ethanol. this method used the same concentration of the solvent in all tested wells of the microtiter plate (7%) in combinations with different concentrations of the oil (0.003 mg ml -1 to 7 mg ml -1 ). in methodology 1, concentration of 7% ethanol was in the first well only and then subsequently two-fold diluted, so we can conclude that at this concentration, ethanol exhibits efficient synergistic action, probably by increasing permeability of the membrane for the active compounds of the essential oil. it is also very important to mention that this solvent (7% ethanol), tested alone against all model microorganisms did not exhibited any inhibitory effect. in this study, inhibitory effect of the oil dissolved in dmso ranged from 0.437 to 3.500 mg ml -1 (graph 2). h i l l i et al. (1997) tested thirteen oils dissolved in dmso and showed their antimicrobial activity. however, cinnamon oil, which was not dissolved in dmso had higher activity against the tested strains. the results confirmed that higher concentrations of dmso indicate antagonistic effect (h i l l i et al., 1997). 10% dmso and 0.005% tween 80 had no activity against our strains (except for candida albicans and pseudomonas aeruginosa clinical isolate 2), as in the previous research (p r a b u s e e n i v a s a n et al., 2006). increased resistance of microorganisms, compared to mic/mbc of the oil dissolved in ethanol, could be explained by antagonistic action of dmso with carlina oxide (the main component of the oil) or by the lack of membrane permeability alteration effect. graph 2. minimum inhibitory concentrations of carlinae radix essential oil dissolved in dmso (methodology 2) the oil dissolved in tween 80 had relatively low activity against all tested strains of microorganisms (mic = 3.500-7.000 mg ml -1 ) or the strains showed complete resistance. there was not bactericidal activity of oil in these solvents. b a u m a n n et al. (2011) confirmed the stimulatory effect of tween 80 on protein secretion in yeast (pichia pastoris). a source of oleic acid such as tween 80 enhanced subsequent acid survival of probiotic lactobacilli when added to the growth medium (c o r c o r a n et al., 2007). graph 3. minimum inhibitory concentrations of carlinae radix essential oil dissolved in tween 80 (methodology 2). the bars above the concentration of 7.00 are placed to represent strains resistant to the highest tested concentration of essential oil. biologica nyssana 3 (2)  december 2012: 61-67 jović j. et al..  influence of solvent on antimicrobial activity... 66 non-ionic detergents are characterized by their uncharged, hydrophilic head groups, so that could be the reason of low oil activity. antagonistic and synergistic effect is probably reflected by oil’s distribution between two phases, water (broth) and the solvent, which influences the effect of oil components on the microorganisms. bacterial strains also used in this study were multiresistant isolates from wounds. this set was chosen as model since, according to the literature, decoct of carlinae radix is used in traditional medicine for rinsing wounds on skin (k o j i ć et al., 1998). comparison of decoct activity with apple vinegar (control) confirmed that decoct exhibited antimicrobial activity. apple vinegar concentration was generally higher (or equal to) mic/mbc of the control when compared to mic/mbc of decoct. the results showed that decoct caused minimal inhibitory activity at a concentration of 0.7811.562% v\v and minimal bactericidal activity from 0.781-6.250% v\v. the highest resistance showed acinetobacter sp. (ci 1), pseudomonas aeruginosa (ci 3), enterobacter aerogenes 13048 and candida albicans 10231 (table 2). in previous studies, apple vinegar decoct showed higher activity against s. aureus (mic = 0.78%, mbc = 3.12%) when compared to water and wine decoct (s t o j a n o v i ć r a d i ć , 2011). activity can be attributed to the extracted compounds at low ph. graph 4. minimum inhibitory concentrations of carlinae radix decoct. the mic bars of decoct which are at the same values as the mic of vinegar present the lack of decoct activity (strains 1, 2, 3, 4, 5 and 9). table 2. antimicrobial activity of carlinae radix decoct bacterial/fungal strain strain type carlinae radix decoct apple vinegar mic mbc mic mbc (%) (%) (%) (%) enterobacter aerogenes clinical isolate 1.562 1.562 0.063 0.125 acinetobacter sp. clinical isolate 1 1.562 3.125 0.063 0.125 proteus mirabilis clinical isolate 1.562 1.562 0.063 0.125 escherichia coli clinical isolate 1.562 1.562 0.063 0.125 staphylococcus aureus clinical isolate 1.562 1.562 0.063 0.25 pseudomonas aeruginosa clinical isolate 1 0.781 1.562 0.063 0.125 pseudomonas aeruginosa clinical isolate 2 1.562 3.125 0.125 0.25 klebsiella oxitoca clinical isolate 1.562 3.125 0.125 0.25 pseudomonas aeruginosa clinical isolate 3 1.562 3.125 0.063 0.063 acinetobacter sp. clinical isolate 2 0.781 0.781 0.063 0.125 salmonella enteritidis atcc 13076 0.781 6.25 0.125 0.125 pseudomonas aeruginosa atcc 3554 0.781 3.125 0.063 0.063 enterococcus faecalis atcc 19433 1.562 3.125 0.125 0.125 enterobacter aerogenes atcc 13048 1.562 3.125 0.063 0.063 proteus mirabilis atcc 12453 1.562 1.562 0.125 0.125 clostridium perfringens atcc 19404 1.562 1.562 0.125 0.125 klebsiella pneumoniae atcc 10031 1.562 1.562 0.125 0.125 candida albicans atcc 10231 1.562 3.125 0.125 0.125 conclusion based on the obtained results it can be concluded that the antimicrobial activity of the oil carlinae radix depends on the solvent. the most efficient inhibitory effect had the oil dissolved in ethanol at 7% concentration, which can be explained by the effect of the ethanol in this concentration on membrane permeability, which leads to increased transfer of the oil’s active compounds into the target places on/inside the microbial cell. vinegar decoct of carlinae radix exhibited very significant antimicrobial activity, even against all tested multiresistant microbial strains, which justifies its ethnopharmacological utilization as an efficient biologica nyssana 3 (2)  december 2012: 61-67 jović j. et al..  influence of solvent on antimicrobial activity... 67 wound rinsing agent. as a final recommendation, the oil should be used in combination with ethanol (in the form of tinctures) at minimum 7% of the solvent concentration to achieve highly efficient antimicrobial effect. also, since the decoct of carlinae radix shows remarkable antimicrobial properties at very low percentage concentrations, it should be considered as more frequently used wound rinsing agent 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composition and antimicrobial properties. pharmaceutical biology, 50 (8): 933-40. tucakov j., 1971: lečenje biljem. fitoterapija. izdavačko preduzeće rad: beograd, jugoslavija, 402, 535-537. wichtl m., 2002: teedrogen und phytopharmaca. wissenschaftliche verlagsgesellschaft mbh, stuttgart, germany, pp. 114–115. willfort r., 1959: gesunheit durch heilkrauter. rudolf trauner verlag lintz, germany. http://www.ncbi.nlm.nih.gov/pubmed?term=stojanovi%c4%87-radi%c4%87%20z%5bauthor%5d&cauthor=true&cauthor_uid=22480199 http://www.ncbi.nlm.nih.gov/pubmed?term=%c4%8comi%c4%87%20l%5bauthor%5d&cauthor=true&cauthor_uid=22480199 http://www.ncbi.nlm.nih.gov/pubmed?term=radulovi%c4%87%20n%5bauthor%5d&cauthor=true&cauthor_uid=22480199 http://www.ncbi.nlm.nih.gov/pubmed?term=stojanovi%c4%87-radi%c4%87%20z%5bauthor%5d&cauthor=true&cauthor_uid=22480199 http://www.ncbi.nlm.nih.gov/pubmed?term=%c4%8comi%c4%87%20l%5bauthor%5d&cauthor=true&cauthor_uid=22480199 http://www.ncbi.nlm.nih.gov/pubmed?term=radulovi%c4%87%20n%5bauthor%5d&cauthor=true&cauthor_uid=22480199 http://www.ncbi.nlm.nih.gov/pubmed?term=blagojevi%c4%87%20p%5bauthor%5d&cauthor=true&cauthor_uid=22480199 http://www.ncbi.nlm.nih.gov/pubmed?term=blagojevi%c4%87%20p%5bauthor%5d&cauthor=true&cauthor_uid=22480199 http://www.ncbi.nlm.nih.gov/pubmed?term=mihajilov-krstev%20t%5bauthor%5d&cauthor=true&cauthor_uid=22480199 http://www.ncbi.nlm.nih.gov/pubmed?term=rajkovi%c4%87%20j%5bauthor%5d&cauthor=true&cauthor_uid=22480199 biologica nyssana 3 (2)  december 2012: 61-67 jović j. et al..  influence of solvent on antimicrobial activity... 68 anticancer compounds from medicinal plants biologica nyssana 4 (1-2)  december 2013: 75-79 žabar, a. et al.  larvicidal activity and in vitro … 75 original article larvicidal activity and in vitro effects of green tea (camellia sinensis l.) water infusion andrea žabar* 1 , vladimir cvetković 1 , jelena rajković 1 , jovana jović 1 , perica vasiljević 1 , tatjana mitrović 1 department of biology and ecology, faculty of science and mathematics,university of niš, višegradska 33,18000 niš * e-mail: andreazabar@yahoo.com abstract: žabar, a., cvetković, v., rajković, j., jović, j., vasiljević, p., mitrović, t.: larvicidal activity and in vitro effects of green tea (camellia sinensis l.) water infusion. biologica nyssana, 4 (1-2), december 2013: 7579. in this study green tea water infusion was tested on drosophila melanogaster wild-type larvae in vivo, also an in vitro antihemolytic and hemolytic tests were performed. three different concentrations were used 7.5 mg/ml, 37.5 mg/ml and 75 mg/ml, the lowest dose representing the recommended dose followed by five times and ten times higher doses. effect of these three concentrations was monitored and tested in vivo on drosophila melanogaster (meigen, 1830) wt (wild type) larval development and surviving. all three concentrations showed toxic effect for larvae, with toxicity being increased in dose – depended manner. the time needed for larvae to fully develop was delayed. this decrease of developmental time was in dose – dependent manner, too. amount of hemolysis caused by the lowest concentration was very small when compared with the percent of spontaneous hemolysis. other two higher concentrations, 37.5 mg/ml and 75 mg/ml, showed higher hemolytic effect. during the four hour incubation period percent of hemolysis grew in time – dependent manner. the highest hemolytic effect was recorded for the concentration of 37.5 mg/ml. antihemolytic test showed that the lowest concentration had the highest inhibitory effect to h2o2 induced hemolysis. the 37.5 mg/ml and 75 mg/ml concentrations had lower inhibitory effect when compared with the dose of 7.5 mg/ml. according to our study it can be concluded that the high concentrations of green tea water infusion exhibit larvicidal activity against d. melanogaster larvae, don't have protective effect to rbc membrane and cause greater hemolysis. key words: green tea; drosophila melanogaster wt; larvicidal activity; rbc; hemolytic test; antihemolytic test. introduction one of the most consumed beverages in the world is green tea. caffeine is one of the active compounds present in tea, and its content is around 3 to 4 % (w i l l s o n et al., 1992). caffeine may play an important role against some pathogens (van b r e d a et al., 2012). green tea is also a source of polyphenol antioxidants such as epicatechin, epigallocatechin, epicatechin gallate and epigallocatechin gallate (z h a n g et al., 1997, h o d g s o n , 2008). catechins have similar backbone but differ in number and location of hydroxyl groups. catechins are water – soluble, colorless compounds that make up to 30% of the dry leaf weight (g r a h a m , 1992). these natural antioxidants have been reported to have the ability of preventing cancer (l i et al., 2008), are effective scavengers of free radicals (n a n j o et al., 1996), inhibit hbv in vitro (x u et al., 2008). it has been 11 th sfses • 13-16 june 2013, vlasina lake 4 (1-2) • december 2013: 75-79 biologica nyssana 4 (1-2)  december 2013: 75-79 žabar, a. et al.  larvicidal activity and in vitro … 76 reported that one cup of tea can provide 150 – 200 mg of flavonoids. also, lower risk of heart disease and stroke are related to the flavonoids intake (h o d g s o n , 2008). isolated flavonoids at high physiological concentrations can reduce platelet aggregation and markers of platelet activation (r e i n et al., 2000). green tea extract can delay and inhibit oxidation of low-density lipoprotein ldl catalyzed by copper in vitro (y o k o z a w a et al., 1997). some authors report that drinking green tea may result in lower level of total triglyceride and cholesterol in serum together with deceased atherogenic index (i m a i et al., 1995). in view of variety of reports considering green tea, we have conducted a study to evaluate the effects of high concentrations of green tea water infusion. drosophila melanogaster is frequently used in larvacidal and insecticidal studies (m i y a z a w a et al., 1998; m i y a z a w a et al., 2000). our aim was to see whether these high doses have beneficial effects on rbc membrane and on drosophila melanogaster wt larvae development. to evaluate green tea infusion effect rbc membrane the hemolytic test with drabkin's reagent was used. also, to see whether high concentrations of green tea water infusion could inhibit lipid peroxidation caused by h2o2, the antihemolytic method was employed. materials and methods test organisms in this experiment, the drosophila melanogaster reference type 5 wt, originated from „bloomington drosophila stock centre”, indiana university, usa was used. fruit flies were cultivated on a standard drosophila medium (9% sugar, 10% corn meal, 2% agar, 2% yeast, with addition of fungicide nipagin ® ), in a standard cultivating 200 ml flasks on 25 o c, 60% humidity, with 12h/12h day/night cycle. adults were allowed to oviposit eggs during the eight hour period, after that they were removed from the flasks. then, after three days larvae which were 72±4h old, have been removed from the cultivating medium, washed with distilled water, and placed on the new medium containing green tea infusion. concentrations of green tea in medium were 7.5 mg/ml, 37.5 mg/ml and 75 mg/ml. experiment was done in triplicate. there were 15 larvae from the same hatch in each replicant flask, and there were 12 replicant flasks including the water control (negative control). replicants were incubated in standard way, already described. alive specimens were collected 10-16 days after the egg laying. rbc preparation a modified method from noudeh et al. (2009) was used. briefly, blood was collected in heparinized tubes from male wistar rats via cardiopuncture. whole blood was centrifuged at 2000 rpm for 10 minutes at 4 ºc. plasma and the buffy coat were removed and an equal volume of pbs (ph 7.4) was added. this was repeated three times, to obtain washed erythrocytes. at the end packed erythrocytes were diluted with pbs to obtain 4% suspension. rbc prepared in this way were used both for hemolytic and antihemolytic test. solutions of green tea infusion and pbs (0.9%, ph=7.4) were made to give the final concentrations of 7.5mg/ml, 37.5mg/ml and 75mg/ml. hemolytic test the following method was employed: 200 μl of green tea water infusions (concentrations 7.5 mg/ml, 37.5 mg/ml, 75 mg/ml) were incubated with 200 μl of rbc suspension at room temperature for one, two, three and four hours. after each hour of incubation samples were centrifuged at 1500 rpm for 10 minutes at 4 ºc. then, 200 μl of resulting supernatant was added to 3 ml of drabkin's reagent and the absorbance was measured at 540 nm on spectrophotometer (shimadzu uv-vis 1650, tokyo, japan). positive control consisted of 200 μl of distilled water and 200 μl rbc suspension and negative control were 200 μl of pbs and 200 μl rbc suspension. everything was done triplicate. percentage of hemolysis was calculated by the equation: % hemolysis = [(ab(sample) ab(negative control)) / ab(positive control)] * 100% (gould et al., 2000). antihemolytic test antihemolytic test was done by modified method previously described (ebrahimzadeh et al, 2010.) 2 ml of rbc suspension was pre-incubated with 0.5 ml of green tea water infusion. the final volume was made to be 5 ml with pbs, and the final concentrations of green tea infusions were 7.5 mg/ml, 37.5 mg/ml and 75 mg/ml. this mixture was incubated for 15 minutes at 37 ºc. then 0.5 ml of h2o2 dissolved in pbs was added to the mixture. concentration of h2o2 was made to give 90% of hemolysis after 4 hours. at the end of the incubation period of 4 hours, different concentration samples were spun at 1500 rpm for 10 min at 4 ºc. then 200 μl of supernatant was added to 3 ml of drabkin's reagent and the absorbance was measured at 540 nm. positive control consisted only of pbs and biologica nyssana 4 (1-2)  december 2013: 75-79 žabar, a. et al.  larvicidal activity and in vitro … 77 h2o2 without green tea infusion. experiments were done in triplicate. percentage of inhibition was calculated by equation: %inhibition = [(ab (h2o2) ab(sample) ) / ab (h2o2) ] *100% . results and discussion the absolute mortality was monitored number of hatched adult specimens that were scored and compared to total transferred larval specimens. it was observed that the mortality increases with the increase in green tea concentration in the medium (fig. 1). fig. 1. absolute mortality of d. melanogaster larvae in presence of green tea infusion development was also observed and number of pupae that were scored from ninth to sixteenth day of larval life (data not shown). in comparison with water control, developmental time from larvae to adults prolongs if the concentration of green tea infusion is higher. one of the active compounds of green tea is caffeine. according to the literature, content of the caffeine is around 3 to 4% (w i l l s o n et al., 1992). it is supposed that caffeine, localized in the vascular bundles of young camellia sinensis l. leaves plays a crucial role in the defense against various pathogens (v a n b r e d a et al., 2012.). itoyama and b i c u d o (1992 and 1997) showed harmful effects of caffeine on drosophila prosaltans d., related to fecundity, egg lying capacity and longevity which is consistent with our results. on the other hand, some studies show that camellia sinensis l., inhibits the accumulation of iron and thus extends the life span of drosophila melanogaster (m a s s i e et al., 1993). also, these results are consistent with previous studies of other authors who showed that polyphenols as antioxidants affect larval development in drosophila causing its time delay (r i c e -e v a n s et al., 1996; a r u o m a , 2003; k i m et al., 2004). results for both antihemolytic and hemolytic test are accordant. in both experiments the lowest doses of green tea infusion exibited slightly beneficial effect on rbc membrane (fig. 2 and 3). fig. 2. percentage of rbc hemolysis in presence of green tea water infusion http://www.ncbi.nlm.nih.gov/pubmed?term=massie%20hr%5bauthor%5d&cauthor=true&cauthor_uid=8326745 biologica nyssana 4 (1-2)  december 2013: 75-79 žabar, a. et al.  larvicidal activity and in vitro … 78 percentage of hemolysis was the lowest for the concentration of 7.5 mg/ml, and it grew as the time of incubation prolonged (after first hour it was 4.99% ± 3.28, second 4.56% ± 0.4, third 5.41% ± 2.22, fourth 5.8 % ± 1.43). other two higher doses showed an increase in rbc hemolysis also in time dependant manner. hemolysis was highest for the 37.5 mg/ml concentration. percent of hemolysis for the 37.5 mg/ml concentration was 15.16% ± 2.4 after first hour, and for the second, third and fourth hour of incubation it was 20.96 % ± 3.55, 20.86 % ± 1.27 and 20.52 % ± 3.3 respectively. percent of hemolysis for the concentration of 75 mg/ml was 8.45 % ± 3.79, 10.79 % ± 0.95, 11.63 % ± 1.33, 12.96 % ± 2.69 after first, second, third and fourth hour respectively. fig. 3. antihemolytic effect of green tea infusion according to the fick's law, diffusion flux from a membrane is proportional to concentration difference of both sides. so, an increase in concentration of green tea infusion in outer membrane leads to diffusion to internal membrane until it gets to specific concentration which can promote membrane disruption and induce hemolysis (k l e s z c z y n s k a et al., 2005.). lipid peroxidation of rat rbc induced by h2o2 creates membrane damage and hemolysis. the highest percentage of inhibition was recorded for the 7.5 mg/ml concentration (94.96 % ± 0.1). other two concentrations had lower inhibitory effect, and for 37.5 mg/ml dose it was 90.39 % ± 0.29, while for the 75 mg/ml it was 85.42 % ± 0.45. certain polyphenols may interact with membrane, leading to the decrease in its fluidity and the diffusion of free radical into the rbc (c o s t a et al., 2009.). in higher doses effects differ (fig. 3.), percentage of hemolysis inhibition is lower. high concentrations of green tea infusion don't have ameliorative effect against rbc free radical hemolysis. conclusion high concentrations of green tea water infusion exibit toxic effect to drosophila larvae. the time of development was prolonged. this is probably due to high concentration of active compounds present in green tea water infusion such as caffeine and catechins. also these results indicate that high doses of green tea infusion can cause a change in tonicity of the erythrocyte membrane. change in tonicity is probably due to the interactions of polyphenols with the 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characteristics of natural population of micromeria juliana (l.) benth. ex rchb. in bulgaria biologica nyssana 8 (1)  september 2017: 99-103 stojanović, g. et al.  first insight into chemical composition of… 99 original article received: 23 mart 2017 revised: 25 may 2017 accepted: 25 july 2017 first insight into the chemical composition of essential oils and head space volatiles obtained from fresh leaves and flowers of peucedanum longifolium waldst. & kit. gordana stojanović1*, olga jovanović1, bojan zlatković2, snežana jovanović1, ivana zrnzević1, novica ristić3 1university of niš, faculty of science and mathematics, department of chemistry, višegradska 33, niš, serbia 2university of niš, faculty of science and mathematics, department of biology and ecology, višegradska 33, niš, serbia 3university of priština, temporarily settled in kosovska mitrovica, faculty of natural science and mathematics, lole ribara 29, kosovska mitrovica, serbia * e-mail: stgocaus@yahoo.com abstract: stojanović, g., jovanović, o., zlatković, b., jovanović, s., zrnzević, i., ristić, n.: first insight into the chemical composition of essential oils and head space volatiles obtained from fresh leaves and flowers of peucedanum longifolium waldst. & kit.. biologica nyssana, 8 (1), september 2017: 99-103. for the first time, chemical composition of p. longifolium essential oils (eo) and head space (hs) volatiles obtained from the fresh leaves, harvested in different phases of the plant development, and inflorescences was compared. the major contributor of leaves essential oil in vegetative phase (ld) was β-elemene (44.1%). on the contrary, β-elemene (22.5%) was the second most abundant component, while (e)-β-ocimene (26.7%) was the first and cis-lanalool oxide (furanoid, 21.9%) was the third most represented compound in the leaves essential oils collected in blossoming phase (lf). (e)-β-ocimene was the major compound in both hs leaf samples but in very varying amounts (ld 81.8% and lf 27.4%). beside (e)-β-ocimene, p-cymene and limonene were represented more than 10% (13.6% and 15.2% respectively). the essential oil and hs volatiles of the inflorescences was characterized by a high content of β-phellandrene (16.4% and 20.4% respectively), α-phellandrene (22.5% and 13.9% respectively) and myrcene (23.1% and 22.1% respectively). the presented data showed that there was a significant difference in the composition of volatile components from different plant organs and also from the same organ in different stages of development. key words: peucedanum longifolium, essential oil composition, head space volatiles apstrakt: stojanović, g., jovanović, o., zlatković, b., jovanović, s., zrnzević, i., ristić, n.: prvi uvid u hemijski sastav etarskog ulja i "head space" (hs) isparljivih komponenti svežih listova u različitim fazama razvoja biljke i svežih cvetova peucedanum longifolium waldst. & kit.. biologica nyssana, 8 (1), septembar 2017: 99-103. po prvi put je određen i upoređen hemijski sastav etarskog ulja (eo) i "head space" (hs) isparljivih komponenti p. longifolium dobijenih iz svežih listova u različitim fazama razvoja biljke i svežih cvetova. najzastupljenija 8 (1) • september 2017: 99-103 doi: 10.5281/zenodo.964345 biologica nyssana 8 (1)  september 2017: 99-103 stojanović, g. et al.  first insight into chemical composition of… 100 komponenta etarskog ulja listova biljke u vegetativnoh fazi (ld) je bio β-elemen (44.1%). u etarskom ulju listova biljke u fazi cvetanja (lf) redosled zastupljenosti je bio: (e)-β-ocimen (26.7%), β-elemene (22.5%) i cis-lanalol-oksid (furanoid, 21.9%). (e)-β-ocimen je bio najzastupljeniji medju "head space" (hs) isparljivim komponentama oba hs uzorka listova (ld 81.8% i lf 27.4%). pored (e)-β-ocimena, p-cimene i limonene su bili zastupljeni preko 10% (13.6% i 15.2% respektivno). etarsko ulje i hs isparljive komponente cvetova je karakterisao visok sadržaj β-felandrena (16.4% i 20.4%, respektivno), α-felandrena (22.5% i 13.9%, respektivno) i mircena (23.1% and 22.1%, respektivno). prezentovani rezultati pokazuju da postoji razlika u sastavu isparljivih komponenti listova i cvetova p. longifolium kao i listova biljke u fazi razvića i cvetanja. ključne reči: peucedanum longifolium, sastav etarskog ulja, sastav "head space" isparljive komponente introduction about 120 peucedanum l. species are widespread in europe, asia and africa (s p a l i k et al., 2004). of this number, 29 species and subspecies grow in the territory of europe, of which 14 in serbia (n i k o l i ć , 1973). some of the members, e. g. p. longifolium waldst. & kit., p. cervaria (l.) lapeyr., p. officinale l., p. oreoselinum (l.) moench and p. arenarium waldst. & kit. are indicated in cardiovascular, respiratory, genitourinary and nervous system disorders, as used in traditional medicine of some balkan countries (p e t k o v & m a n o l o v , 1978; s a r i ć , 1989; м и т р е в , 1995). peucedanum longifolium (sect. peucedanum) is glabrous perennial plant with solid (60-200 cm high), branched steams and small, yellow flowers arranged in several compound inflorescences. the species inhabits dry grasslands and rocky, usually calcareous slopes of medium altitudes in the c, s & e parts of balkan peninsula, extending to the mountains of c romania (s p a l i k et al., 2004). however, this species is mainly reported for limestone habitats (n i k o l i ć , 1973), while we have surprisingly found it growing over silicate bedrock. previous phytochemical studies found coumarins and flavonoids in the roots, steams, flowers and fruits of p. longifolium (k u z m a n o v et al., 1980). evaluation of antioxidant and antimicrobial activity of essential oils (t e p e et al., 2011; i l i ć et al., 2015) and extracts (m a t e j i ć et al., 2013) was also reported. to the best of the authors’ knowledge, there are only few papers (t e p e et al., 2011; i l i ć et al., 2015; k a p e t a n o s et al., 2008) referring to the chemical composition of essential oil of air-dried aerial parts of p. longifolium in flowering phase and one report related to the volatiles of fresh aerial parts from initial stage of the plant development (j o v a n o v i ć et al., 2015). recently, chemical composition of the essential oil and head space volatiles obtained from the fresh root of p. longifolium was published (s t o j a n o v i ć et al., 2017). the present study reports, for the first time, chemical composition of the essential oil (eo) and head space (hs) volatiles obtained from the fresh leaves and flowers of the p. longifolium growing in unusual natural environments i.e. on silicious substrate. material and methods plant material plant material was collected from thermophilous grasslands at siliceous, rocky grounds of mt. stara planina. all specimens were collected from the same population, habitat and locality (topli do, e serbia, 43°17’59” n and 22°36’42” e). voucher specimens were deposited in the “herbarium moesiacum niš” (hmn), department of biology and ecology, faculty of science and mathematics, university of niš under the acquisition numbers 11780 (vegetative stage; 05.06.2015.) and 11781 (flowering stage; 05.07.2015.). isolation and analysis of volatiles isolation and analysis of volatiles was done as previously described (j o v a n o v i ć et al., 2015). the light yellow oil, which has pleasant odor, was obtained in a yield of 0.09%, 0.07%, and 0.38% from fresh leaves in the developing and flowering phase and from the fresh flowers (respectively). results and discussion oil yield of both leaf samples (0.09% ld and 0.07% lf) was many times lower than the oil yield obtained from the flower (0.38%). a total of 42 compounds, whose representation in at least one sample was greater than or equal 0.1, were identified in six examined samples: essential oils (eo) and head space (hs) volatiles of leaves in vegetative and flowering phase (ld and lf, respectively) as well as inflorescence samples (f) (tab. 1). monoterpenes were predominant in all samples except in sample eo ld which was dominated by sesquiterpene -elemene (44.1%). twenty compounds were identified in the hs ld, biologica nyssana 8 (1)  september 2017: 99-103 stojanović, g. et al.  first insight into chemical composition of… 101 accounting 98.9% of the hs volaties, of which almost four fifth was (e)--ocimene (81.8%). its presence was reduced to 27.4% in the hs lf sample (fig. 1). opposite to that, percentage representation of pinene (1.3% ld, 4.6% lf), sabinene (0.5% ld, 6.0% lf), -elemene (2.2% ld, 3.7% lf), β-pinene (0.6% ld, 8.0% lf), myrcene (1.0% ld, 5.9% l), p-cymene (0.6% ld, 13.6% l) and limonene (0.3% ld, 15.2% l) was increased in lf sample. regarding ld oil, it was characterized by elemene (44.1%) whose participation was approximately twice decreases in the lf sample (22.5%). in contrast to -elemene presence of the following components was greatly increased in the lf sample: (e)--ocimene (8.5% ld, 26.7% lf), cis-linalool oxide (8.2% ld, 21.9% lf), and translinalool oxide (4.5% ld, 11.2% lf). comparing mutual composition of hs and eo, it is evident that the cis-linalool oxide and translinalool oxide were not detected, even as a trace, in samples of hs. further, presence of -elemene was many times more in eo samples than in corresponding hs samples. it can be assumed that the oxides are formed from the acyclic monoterpenes during hydrodistillation (s c h o f i e l d et al., 2002) while lower temperature of preparation of hs samples in relation to the essential oils obtaining (80 °c vs. 100 °c) could be the reason for a lesser presence of -elemene in hs samples. composition of hs f and eo f was different from the corresponding leaves samples, particularly regarding to the -phellandrene. this p-menthane monoterpene was represented with 20.4% and 16.4% in hs f and eo f samples, respectively, while it was not detected in leaf samples at all. -phellandrene was identified in the both flower and leaf samples, but in different quantities (1.2% hs lf, 0.3 % eo lf, 13.9% hs f and 22.5% e of). myrcene was the most abundant compound in the hs f and eo f. comparing mutual composition hs f and eo f it could be noticed a difference in the composition of -pinene (3.8% and 1.8%, respectively) and -pinene (10.9% and 1.4%, respectively) as well as -elemene (0.8% and 5.0%, respectively). previous studies have been done with the entire dry above-ground part on the flowering stage (k a p e t a n o s et al., 2008; t e p e et al., 2011; i l i ć et al., 2015) or fresh above-ground part in the developing stage (j o v a n o v i ć et al., 2015). 8cedren-13-ol (33.7%), myrcene (15.9%), α-pinene (36.3%) and β-elemene (24.9%) (k a p e t a n o s et al., 2008; t e p e et al., 2011; i l i ć et al., 2015; j o v a n o v i ć et al., 2015), respectively, published as the most represented components of essential oils. oil yield of both leaf samples was approximate each other (0.09% ld and 0.07% lf) many times lower than the oil yield obtained from the flower (0.38%). chemical composition of the eo and hs volatiles obtained from the fresh root of p. longifolium growing on the same substrate as here examined sample (s t o j a n o v i ć et al., 2017) was totally different relative to composition of leaves and flowers. namely, α-pinene was the most abundant compound in root eo (60.3%) and root hs volatiles (76.3%) while β-elemene (eo 0.1%; hs bellow fig. 1. variation of the percentage content of the p. longifolium essential oil (eo) and head space volatile (hs) components (ld-leaf, developing phase; lf-leaf, flowering phase; f-flower) biologica nyssana 8 (1)  september 2017: 99-103 stojanović, g. et al.  first insight into chemical composition of… 102 0.1%), (e)-β-ocimene (eo and hs 1.2%) and myrcene (eo 2.3%; hs 2.1%) were manifold less represented comparing to their representation in eo and hs of here examined leaves and flowers. conclusion the results of this and previous studies show that p. longifolium, like most other species, has a different table 1. chemical composition (%) of p. longifolium essential oils (eo) and head space volatile (hs) constituents ri ai compound content (%) ld lf f hs eo hs eo hs eo 1. 802 801 hexanal 0.4 0.5 2. 840 831 (e)-2-hexenal 0.4 3. 854 850 (z)-3-hexenol tr 2.3 6.7 4. 867 863 hexanol 0.9 0.2 tr 5. 929 924 -thujene tr 0.1 3.7 0.1 2.6 1.3 6. 936 932 -pinene 1.3 0.2 4.6 tr 3.8 1.8 7. 951 946 camphene 0.1 0.3 0.9 0.3 8. 975 969 sabinene 0.5 0.3 6.0 0.4 4.5 9. 979 974 -pinene 0.6 0.3 8.0 0.7 10.9 1.4 10. 992 988 myrcene 1.0 0.4 5.9 0.1 22.1 23.1 11. 993 991 trans-dehydroxy-linalool oxide 0.1 12. 1007 1002 -phellandrene 0.2 1.2 0.3 13.9 22.5 13. 1008 1006 cis-dehydroxy-linalool oxide 0.1 14. 1020 1014 -terpinene tr 0.2 tr 15. 1027 1020 p-cymene 0.6 1.8 13.6 0.7 0.5 16. 1031 1024 limonene 0.3 0.9 15.2 2.2 13.2 8.2 17. 1039 1025 -phellandrene 20.4 16.4 18. 1041 1032 (z)-β-ocimene 5.5 0.8 3.2 1.2 1.3 0.8 19. 1050 1044 (e)-β-ocimene 81.8 8.5 27.4 26.7 7.0 7.1 20. 1061 1054 γ-terpinene 0.2 0.5 2.0 2.7 1.4 1.6 21. 1075 1067 cis-linalool oxide (furanoid) 8.2 tr 21.9 tr 1.7 22. 1090 1084 trans-linalool oxide(furanoid) 4.5 11.2 1.1 23. 1091 1086 terpinolene tr 0.4 24. 1101 1095 linalool 0.2 tr 0.1 25. 1180 1174 terpinen-4-ol 0.1 tr 0.1 26. 1389 1389 iso-longifolene 2.4 27. 1397 1389 -elemene 2.2 44.1 3.7 22.5 0.8 5.0 28. 1426 1417 (e)-caryophyllene 0.2 1.5 tr 0.3 0.1 29. 1461 1452 -humulene 0.2 7.9 tr tr 30. 1481 1476 -chamigrene 0.4 31. 1489 1478 γ-muurolene 0.3 32. 1488 1484 germacrene d 3.6 tr 0.4 0.3 0.6 33. 1494 1489 β-selinene 0.3 1.1 tr 0.3 0.7 34. 1502 1498 α-selinene 0.3 0.7 0.3 tr 35. 1503 1500 bicyclogermacrene 0.7 0.1 36. 1514 1508 germacrene a 2.7 5.3 0.6 0.8 0.2 37. 1529 1522 δ-cadinene 0.1 tr 38. 1585 1577 spathulenol 0.3 39. 1591 1582 caryophyllene oxide 0.2 40. 1618 1608 humulene epoxide ii 0.6 41. 1648 1640 epi--murrolol ( τ-muurolol) 0.1 42. 1663 1658 selin-11-en-4-α-ol 1.1 monoterpenoids 91.9 27.2 91.1 68.2 98.1 92.5 sesquiterpenoids 6.2 70.1 4.6 23.7 1.7 6.1 other 0.8 0 3.7 6.9 0 0 total (%) 98.9 97.3 99.4 98.8 99.8 99.2 hs – head space volatiles; eo – essential oil; ld–leaf, vegetative phase; lf – leaf, flowering phase; f – inflorescences; ri – experimental linear retention indices relative to c8-c40 alkanes on the hp-5ms; aiadam’s retention indices; tr– trace (< 0.05%) and not detected (-); the most represented components are in bold. biologica nyssana 8 (1)  september 2017: 99-103 stojanović, g. et al.  first insight into chemical composition of… 103 composition of the volatile components in various organs in the same vegetative phase and in the same organ in different stages of development. in accordance with that, depending on application, should be chosen in which stage of development and which plant organ should be collected. in the case of p. longifolium, if the intention is to use p. longifolium in perfumery, it is best to use its flower because of their pleasing aroma originating from high content of phellandrenes and ocimenes. if there is intention for utilization of p. longifolium in the pharmaceutical industry, it is the best to use leaf, especially in the development phase, because of the high content of βelemene for which benefits for cancer treatment have been previously reported (z h u et al., 2011). acknowledgements. the research was supported by the serbian ministry of education, science and technology development (grant no. 172047). references ilić, b., miladinović, d., kocić , b., miladinović, m., 2015: antibacterial profile of peucedanum longifolium essential oil. acta medica medianae, 54 (1): 20-26. jovanović, o. p., zlatković, b. k., jovanović, s. c., petrović, g., stojanović, g. s., 2015: composition of peucedanum longifolium waldst. & kit. essential oil and volatiles obtained by headspace. journal of essential oil research, 27 (3): 182-185. kapetanos, c., karioti, a., bojović, s., marin, p., veljić, m., skaltsa, h., 2008: chemical and principal-component analyses of the essential oils of apioideae taxa (apiaceae) from central balkan. chemistry and biodiversity, 5 (1): 101119. kuzmanov, b., andreev, n., kozovska, v., 1980: chemotaxonomic study on bulgarian species of peucedanum l. i. anales del jardín botánico de madrid, 37 (2): 779-788. matejić, j. s., džamić, a. m., ćirić, a. d., krivošej, z., ranđelović, v. n., marin, p. d., 2013: antioxidant and antimicrobial activities of extracts of four peucedanum l. species. digest journal of nanomaterials and biostructures, 8(2): 655-665. mitrev, a., 1995. peucedanum arenarium w. et k. in: bondev, i. 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(liliaceae) from serbia and montenegro original article jovana stojanović university of niš, faculty of sciences and mathematics, department of biology and ecology, niš, serbia jovanagreen6@gmail.com (corresponding author) irena raca university of niš, faculty of sciences and mathematics, department of biology and ecology, niš, serbia racairena@gmail.com jelena jevtić university of niš, faculty of sciences and mathematics, department of biology and ecology, niš, serbia j.jevtic93@gmail.com marina jušković university of niš, faculty of sciences and mathematics, department of biology and ecology, niš, serbia marinaju@pmf.ni.ac.rs vladimir ranđelović university of niš, faculty of sciences and mathematics, department of biology and ecology, niš, serbia vladar@pmf.ni.ac.rs received: november 12, 2019 revised: december 23, 2019 accepted: december 24, 2019 abstract: in this study, morphological and anatomical properties of species gagea pratensis (pers.) dumort. were investigated and described. the comparative analysis included six populations from serbia and montenegro. this study aimed to examine potential morpho-anatomical differentiation and the characters that contribute most to the differences between populations. statistical analyses were carried out for 19 morphological and 5 anatomical characters of 122 specimens using program statistica 7, including the analysis of variance (anova), principal component (pca) and discriminant analysis (cda). analysis of variance showed that almost all investigated characters are statistically significant for differentiation of analysed populations. according to the principal component analysis characters that contributed the most to the separation of populations were height of the whole plant, bulb width, width and length of basal leaf, width and indumentum type of peduncle, length of first cauline leaf, width and length of second cauline leaf, and width of outer and inner segments of perigone. the results of thr discriminant analysis showed the existence of morphoanatomical differentiation between analysed populations, separating the population from locality gamzigradska banja from all other populations. key words: gagea pratensis, morphology, anatomy, variability, differentiation apstract: komparativna morfo-anatomska analiza vrste gagea pratensis (pers.) dumort. (liliaceae) iz srbije i crne gore u ovoj studiji ispitivane su i opisivane morfološke i anatomske karakteristike vrste gagea pratensis (pers.) dumort. komparativnom analizom obuhvaćeno je šest populacija sa prostora srbije i crne gore. cilj istraživanja bio je određivanje potencijalne morfo-anatomske diferencijacije, kao i definisanje onih karaktera koji najviše doprinose postojanju razlika između populacija. sprovedene su statističke analize za 19 morfoloških i 5 anatomskih karaktera na 122 uzorka primenom programa statistica 7, uključujući analizu varijanse (anova), analizu glavnih komponenti (pca) i diskriminantnu analizu (cda). analiza varijanse je pokazala da gotovo svi istraživani karakteri imaju statistički značaj u diferencijaciji analiziranih populacija. na osnovu analize glavnih komponenti izdvojeni su karakteri koji su imali najveći uticaj na razdvajanje populacija i to su visina cele biljke, širina lukovice, širina i dužina bazalnog lista, širina i tip indumentuma stabla, dužina prvog pricvetnog lista, širina i dužina drugog pricvetnog lista i širina unutrašnjih i spoljašnjih segmenata perigona. rezultati diskriminantne analize ukazali su na postojanje morfo-anatomske diferencijacije, pri čemu je populacija sa gamzigradske banje bila jasno izdvojena od svih ostalih populacija. ključne reči: gagea pratensis, morfologija, anatomija, varijabilnost, diferencijacija introduction the genus gagea salisb. (liliaceae) is a taxonomically complicated group, comprising more than 320 species (levichev, 2013; peterson et al., 2016), distributed in temperate and subtropical regions of europe and asia (levichev, 1999a). the primary cause of the taxonomic problems in the genus gagea is the superficial similarity of most of the species (rix & woods, 1981). in the last few decades several new species of the genus gagea salisb. were described based on analyses of © 2019 stojanović et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 125 13th symposium on the flora of southeastern serbia and neighboring regions morphological and anatomical characters (zarrei and zarre, 2003; peruzzi et al., 2007; hamzaoğlu et al., 2008, zarrei et al., 2010a,b; levichev et al., 2019). according to levichev (in peterson et al., 2008) gagea pratensis (pers.) dumort. belongs to section gagea davlianidze, 1972, not. syst. geogr. inst. bot. thbilissiensis. 29: 73., genus gagea salisb., tribe tulipeae and family liliaceae (peruzzi, 2011). section gagea forms a well-defined monophyletic clade which is marked by four morphological synapomorphies angular peduncles and pedicels, second leaf concrescent with peduncle and seeds with distinct arillode (peruzzi et al., 2008a). gagea pratensis is distributed in most parts of europe and asia minor (diklić in josifović, 1975). it is characterized by the bulb and bulblet being exserted (peruzzi et al., 2007). basal leaves have nine or more vascular bundles, first cauline leaves have five barely pronounced keels, pedicels are triangular with six to eight vascular bundles and peduncles are polyedric with a ‘channel-like’ structure inside (peruzzi et al., 2007). gagea pratensis is a highly variable, complex and critical taxon, which is poorly investigated in the balkan peninsula. therefore, in this paper, the morphology and anatomy of g. pratensis from serbia and montenegro were studied in detail. the aim of the study was to examine potential morpho-anatomical differentiation of the studied populations, and to determine the characters that contribute most to the differences between populations. material and methods plant material plant samples were collected from 6 populations of g. pratensis from serbia and montenegro during the flowering time. the sampling localities with associated information are given in the tab. 1. one part of the collected plant material was deposited in herbarium moesiacum niš (hmn). the other part was fixed in 50% alcohol for anatomical study, which was done in the laboratory for plant systematics and ecology (department of biology and ecology, faculty of sciences and mathematics, university of niš, serbia). 126 biologica nyssana ● 10 (2) december 2019: 125-133 stojanović et al. ● comparative morpho-anatomical analysis of gagea pratensis (pers.) dumort. (liliaceae) from serbia and montenegro fig. 1. gagea pratensis: a – habitus, b – underground organs, c – floral details table 1. the sampling localities with associated information (v.r. v. ranđelović, i.r. i. raca, j.j. j. jevtić, d.h. d. harpke) locality n e altitude (m) habitat date collectors voucher gamzigradska banja 43° 92’38” 22° 17’ 03” 164 clearing in an oak forest 18.03.2017. v.r. hmn13289 the jelašnička gorge 43° 28’ 41” 22° 06’ 61” 460 arid subcontinental steppic grassland 23.03.2017. v.r, i.r., j.j. hmn13284 the hill rgotski kamen 44° 02’ 62” 22° 23’ 32” 225 arid subcontinental steppic grassland 18.03.2017. v.r. hmn13287 mt. seličevica 43° 18’ 38” 21° 83’ 09” 802 oak forest 23.03.2017. v.r, i.r., j.j. hmn13285 mt. lovćen 42° 24’ 02” 18º 49’ 33” 1749 subalpine pasture 07.04.2018. v.r, i.r., d.h. hmn13666 mt. vjetarnik 42° 36’ 30” 19º 25’ 49” 1075 10.04.2018. v.r, i.r., d.h. hmn13667 tissue was present below both adaxial and abaxial epidermis in all populations, except that from mt. vjetarnik, where some individuals had palisade tissue only below their abaxial epidermis. big and small vascular bundles were alternately lined in leaf mesophyll. every vascular bundle was surrounded by parenchymatous tissue organised in one or two layers. indumentum was absent from basal leaves. the anatomical characteristics of the first cauline leaf are presented in fig. 3. cross-sections of first cauline leaves of the species g. pratensis had a crescent shape. the anatomy of first cauline leaves is similar to the anatomy of basal leaves. the epidermis was made of square, rectangular to oval-shaped cells all arranged in one layer. leaves were amphystomatic. mesophyll was usually made of spongy parenchyma and one or two layers of palisade parenchyma. palisade tissue can be located only below abaxial (populations from mt. seličevica, mt. vjetarnik and mt. lovćen, and some individuals from the hill rgotski kamen and the jelašnička gorge), below both abaxial and adaxial epidermis (population from gamzigradska banja and some individuals from the hill rgotski kamen) or it can be absent (some individuals from the jelašnička gorge had mesophyll made only of spongy tissue). big and small vascular bundles were alternately lined in leaf morpho-anatomical analysis 19 morphological characters were defined from the fresh plant material. manual microtome (gligorijević & pejčinović, 1983) was utilized in order to make cross-sections of the basal leaves, first cauline leaves, peduncles and pedicels at their widest part. cross-sections were stained with safranin alcian blue, examined by leica dm 2500 microscope and photographed with camera leica dfc490. 5 anatomical characters were analysed. all investigated characters are represented in the tab. 3. statistical analysis basic descriptive statistics was performed for each quantitative character. the significance of differences between the populations studied was established by analysis of variance (anova). principal component analysis (pca) was conducted to determine which characters contributed most to the delimitation of populations, while discriminant analysis (cda) was used to determine the morpho-anatomical differentiation of investigated populations. all statistical analyses were performed using statistica 7.0 software (statsoft. inc., 2004). results the general habitus is represented in fig. 1a, including the details of the underground organs (fig. 1b) and flower (fig. 1c) (photo by j. stojanović). tunic’s colour in all flowering plants was either light or dark brown. in all investigated populations bulbils on peduncle and in the base of cauline leaves were absent. middle stem leaves and indumentum of basal leaves were lacking in all investigated populations. first cauline leaves were sparse ciliate, except some individuals from population from the hill rgotski kamen. peduncles were hairless in all populations, excluding some specimens from the locality of gamzigradska banja and mt. seličevica with sparse hairy peduncles. pedicels, outer perigone and inner perigone segments were glabrous in all populations. perigone segments were yellow on the inside (white colour was only present in the population from the hill rgotski kamen) and green on the outside. the anatomical characteristics of the basal leaf are presented in fig. 2. cross-sections of basal leaves of the species g. pratensis had recognisable v-shape in all investigated populations. both adaxial and abaxial epidermis were made of square, rectangular to oval-shaped cells all arranged in one layer. leaves were amphystomatic and stomata were at the same level as epidermal cells. some indications of mesophyll differentiation were noticed in all investigated populations. palisade biologica nyssana ● 10 (2) december 2019: 125-133 127 figure 2. a – v-shaped cross-section of basal leaf of g. pratensis; b – central part of basal leaf cross-section; c – central vacular bundle (ade – adaxial epidermis, abe – abaxial epidermis, ph – phloem, pp – palisade parenchyma, sp – spongy parenchyma, st – stomata, vb – vascular bundle, vs – vascular sheath, xy – xylem) stojanović et al. ● comparative morpho-anatomical analysis of gagea pratensis (pers.) dumort. (liliaceae) from serbia and montenegro mesophyll. every vascular bundle was surrounded by parenchymatous tissue organized in one or two layers. indumentum was present on first cauline leaves in all investigated populations. the anatomical characteristics of pedicels are presented in fig. 4. crosssections of pedicels of the species g. pratensis had recognizable triangular shape. the epidermis was made of square to oval cells organised in one layer. stomata were at the same level as epidermal cells. both cortex and pith, were made of parenchymatous cells with thin walls. the cortex was made of 3-5 layers of cells. vascular bundles were arranged in a circle, with xylem oriented toward the pith and surrounded by its cells. each vascular bundle was surrounded by a parenchymatous vascular sheath. sclerenchymatous 128 tissue was also present in the form of a ring, made of 2-3 layers of cells, located beneath the cortex and covering only the phloem. indumentum was absent from pedicels. microphotographs of peduncle cross-sections for all investigated populations are presented in fig. 5. this organ was very variable in shape. peduncles with square shape without channel-like structures were noticed in populations from the hill rgotski kamen, mt. lovćen and mt. vjetarnik; deltoid shape without channel-like structures from mt. lovćen and fig. 3. a – cross-section of first cauline leaf of g. pratensis; b – central part of cross-section; c – central vascular bundle; d – leaf edge (ade – adaxial epidermis, abe – abaxial epidermis, ph – phloem, pp – palisade parenchyma, sp – spongy parenchyma, st – stomata, t – trichome, vb – vascular bundle, vs – vascular sheath, xy – xylem) fig. 4. a – general outlook and b – anatomical features of triangular cross-section of pedicel of g. pratensis (cr – cortex, e epidermis, ph – phloem, pi – pith, sc – sclerenchyma, st – stomata, vb – vascular bundle, vs – vascular sheath, xy xylem) fig. 5. comparative overview of peduncle cross-sections of g. pratensis from localities: a – gamzigradska banja, b – seličevica, c – jelašnička klisura, d – rgotski kamen, e – lovćen, f – komovi. biologica nyssana ● 10 (2) december 2019: 125-133 stojanović et al. ● comparative morpho-anatomical analysis of gagea pratensis (pers.) dumort. (liliaceae) from serbia and montenegro 129 mt. komovi; deltoid shape with one channel-like structure from all populations except the populaton from locality gamzigradska banja. deltoid shape with an arm and two channel-like structures was present only in populations from gamzigradska banja and mt. seličevica. deltoid shape without arms and with 2 channel-like structures, as well as, irregular shape with two arms and 2 or 3 channellike structures was noticed only in population from gamzigradska banja. table 2. descriptive statistics of quantitative morphological and anatomical characters of analysed populations of the species g. pratensis. quantitative numerical values are expressed in mm, as mean±standard deviation locality gamzigradska banja jelašnička gorge rgotski kamen seličevica lovćen vjetarnik morphological characters mean±std.dev. height of the whole plant 162.16±26.63 77.85±15.12 113.19±24.54 124.44±14.79 95.98±21.22 85.38±23.95 number of bulbs in common tunic 3.00±0.00 1.00±0.00 1.00±0.00 3.00±1.00 1.00±0.00 1.00±0.00 width of bulb 20.17±4.41 7.03±1.17 9.55±1.83 15.95±3.59 4.74±1.46 7.07±1.55 width of basal leaf 4.51±0.74 2.30±0.63 2.71±0.53 3.54±0.48 2.19±0.63 2.97±0.74 length of basal leaf 217.61±40.80 113.99±30.33 147.28±34.12 159.38±19.89 140.36±38.16 162.93±31.39 width of peduncle 1.84±0.38 1.19±0.21 1.52±0.35 1.40±0.19 0.84±0.22 1.18±0.33 heigh of peduncle from bulb to first cauline leaf 91.23±20.35 46.38±17.10 65.48±22.72 67.62±16.12 56.53±18.27 38.70±18.29 width of first cauline leaf 6.79±1.30 4.73±6.19 5.68±1.20 5.07±0.91 5.70±1.34 6.48±1.72 length of first cauline leaf 60.63±16.64 23.73±6.25 42.59±14.51 42.65±9.24 40.02±9.22 48.03±15.73 width of second cauline leaf 5.07±1.12 1.64±0.57 2.60±0.61 2.88±0.63 2.67±0.59 2.84±0.85 length of second cauline leaf 41.90±9.90 13.13±4.15 24.48±7.90 25.96±5.56 21.69±5.04 26.17±10.41 width of pedicels 1.13±0.16 0.96±0.17 0.88±0.16 1.01±0.10 0.76±0.20 0.75±0.12 number of flowers 3.00±1.00 1.00±1.00 4.00±2.00 2.00±1.00 2.00±1.00 3.00±2.00 length of outer perigone segments 16.65±1.69 12.74±2.93 12.76±2.47 14.57±1.47 12.95±1.62 15.11±2.01 length of inner perigone segments 15.21±1.59 11.63±2.06 11.37±2.25 13.55±1.39 12.21±1.66 14.26±1.95 width of outer perigone segments 4.24±0.72 2.59±0.57 2.52±0.49 3.02±0.32 2.72±0.46 3.42±0.61 width of inner perigone segments 3.23±0.56 2.00±0.49 2.10±0.43 2.47±0.21 2.41±0.34 2.72±0.36 anatomical characters no. of vascular bundles in basal leaf 9.00±0.00 8.00±1.00 8.00±1.00 7.00±1.00 8.00±1.00 8.00±2.00 no. of vascular bundles in first cauline leaf 12.00±3.00 10.00±2.00 12.00±2.00 9.00±2.00 14.00±2.00 13.00±3.00 no. of vascular bundles in pedicel 6.00±1.00 6.00±0.00 6.00±0.00 6.00±0.00 6.00±0.00 6.00±0.00 no. of vascular bundles in peduncle 13.00±1.00 8.00±1.00 9.00±1.00 9.00±1.00 9.00±2.00 11.00±2.00 no. of channel-like structures in peduncle 2.00±0.00 1.00±0.00 0.00±1.00 1.00±1.00 0.00±1.00 1.00±0.00 biologica nyssana ● 10 (2) december 2019: 15-133 stojanović et al. ● comparative morpho-anatomical analysis of gagea pratensis (pers.) dumort. (liliaceae) from serbia and montenegro the anatomical structure was the same regardless the shape of peduncle (fig. 6). the epidermis was made of cubed to oval-shaped cells arranged in one layer. channel-like structures were limited by the cells which are similar to epidermal cells regarding their shape and size. stomata were present in epidermis, as well as in the layer of cells surrounding channel-like structures. cortex was made of 4-5 layers of oval-shaped parenchymal cells with thin walls. sclerenchymatous tissue was present in the form of a ring, made of 2-3 layers of cells, located beneath the cortex and covering only the phloem. vascular bundles were also arranged in the form of a ring. in the case of individuals with channel-like structures, there were additional vascular bundles (1-3) in the area surrounding this structure. it was common that two vascular bundles fuse in the region of xylem, while the phloem stays separated. pith was surrounding xylem, and it was made of parenchymal cells. indumentum was absent from the peduncle, except populations from localities gamzigradska banja and mt. seličevica, where some plants with hairy peduncles were recorded. descriptive statistics is represented in the tab. 2. the results of one-way analysis of variance (tab. 3) showed that all analysed characters had statistical 130 fig. 6. a – cross-section of peduncle of g. pratensis, b – channel-like structure, c – fusion of two vascular bundles in the region of xylem (ch – channel-like structure, cr – cortex, e epidermis, ph – phloem, pi – pith, sc – sclerenchyma, st – stomata, vb – vascular bundle, vs – vascular sheath, xy xylem) table 3. results of one-way analysis of variance (* p<0.05) and principal component analysis (pca) of morphological and anatomical characters of six populations of the species g. pratensis. the first three principal components accounted for 62.69% of the variance characters anova p-values pca 1 pca 2 pca 3 height of the whole plant 0.00* -0.81 -0.04 -0.31 number of bulbs in common tunic 0.00* -0.69 -0.43 0.15 width of bulb 0.00* -0.85 -0.35 -0.01 width of basal leaf 0.00* -0.89 -0.02 -0.06 length of basal leaf 0.00* -0.79 0.22 -0.18 width of peduncle 0.00* -0.72 -0.11 -0.39 height of peduncle from bulb to first cauline leaf 0.00* -0.61 -0.10 -0.36 indumentum of peduncle 0.00* -0.82 -0.18 0.11 first cauline leaf width 0.28 -0.31 0.36 -0.09 length of first cauline leaf 0.00* -0.78 0.41 -0.15 width of second cauline leaf 0.00* -0.90 0.21 -0.05 length of second cauline leaf 0.00* -0.88 0.26 -0.12 width of pedicel 0.00* -0.55 -0.42 -0.06 number of flowers 0.00* -0.36 0.45 -0.58 length of outer perigone segments 0.00* -0.64 0.13 0.35 length of inner perigone segments 0.00* -0.64 0.16 0.36 width of outer perigone segments 0.00* -0.80 0.18 0.30 width of inner perigone segments 0.00* -0.73 0.27 0.23 color of the inner side of perigone segments 0.00* -0.07 -0.01 0.53 no. of vascular bundles in basal leaf 0.00* -0.30 0.18 0.18 no. of vascular bundles in first cauline leaf 0.00* 0.00 0.68 0.16 no. of vascular bundles in pedicel 0.00* -0.27 -0.22 0.43 no. of vascular bundles in peduncle 0.00* -0.66 0.25 0.32 number of channel-like structures in peduncle 0.00* -0.68 -0.46 0.17 biologica nyssana ● 10 (2) december 2019: 125-133 stojanović et al. ● comparative morpho-anatomical analysis of gagea pratensis (pers.) dumort. (liliaceae) from serbia and montenegro significance for the formation of differences between analysed populations of species g. pratensis, except the width of the first cauline leaf. the results of principal component analysis (pca) showed that the three principal components contained 62.69% of the total variability. the most significant contribution to the formation of variability was provided by the first principal component with 44.77%, followed by the second one with 9.96% and the third one with 7.96% of variation. according to pca analysis (tab. 3) characters which contributed most to the differentiation of populations (factor loadings > 0.7) were height of the whole plant, width of bulb, width and length of basal leaf, width and type of indumentum of peduncle, length of first cauline leaf, width and length of second cauline leaf and width of outer and inner perigone segments. the results of discriminant analysis (cda) showed the existence of morpho-anatomical differentiation of analysed populations. populations from the jelašnička gorge, the hill rgotski kamen, mt. lovćen and mt. komovi are on the positive side of the first canonical root, while populations from mt. seličevica and gamzigradska banja are on the negative side. separation is also present on the second canonical root, where populations from the jelašnička gorge and mt. seličevica are on the positive, while populations from mt. lovćen and mt. vjetarnik are on the negative side (fig. 7). discussion in this study, the detailed morpho-anatomical comparison of six populations of g. pratensis from serbia and montenegro was conducted. the primary goal was to examine potential morpho-anatomical differentiation and to determine the characters that contribute most to the differences between populations. as previous studies showed, the morphological characters of significance for taxonomy within the genus gagea salisb. are number and dimensions of underground organs (levichev, 1999b; peruzzi & caparelli, 2007; zarrei et al., 2007; hamzaoğlu et al., 2008; peruzzi et al., 2008b; peruzzi et al., 2011), presence/absence of bulbils (levichev, 1999b; peruzzi et al., 2011; zarrei et al., 2011) and indumentum (hamzaoğlu et al., 2008; wörz et al., 2012), number and dimensions of basal and cauline leaves (rix & woods, 1981; peruzzi & caparelli, 2007; peruzzi et al., 2011; zarrei et al., 2011; wörz et al., 2012), and number, dimensions and colour of flowers (peruzzi & caparelli, 2007; zarrei et al., 2007; hamzaoğlu et al., 2008; peruzzi et al., 2008b; peruzzi et al., 2011; zarrei et al., 2011; levichev et al., 2019). our research confirmed the significance of previously mentioned morphological traits. gagea pratensis is highly variable taxon, therefore wide ranges of values were obtained for almost all investigated characteristics. maximal values of the morphological characters studied were mostly found in the population from gamzigradska banja, which makes it easily distinguishable from all the other populations. also, significant role in the taxonomy of this genus plays the anatomy of basal leaves (zarrei and zarre, 2003; peruzzi et al., 2007; peruzzi et al., 2008a; zarrei et al., 2010b; zarrei et al., 2010c), first cauline leaves (peruzzi et al., 2007), pedicels (peruzzi et al., 2007; peruzzi et al., 2008a, zarrei et al., 2010b; zarrei et al., 2010c) and peduncles (peruzzi et al., 2007; peruzzi et al., 2008a; ajani et al., 2010; zarrei et al., 2010b; zarrei et al., 2010c). relevant anatomical markers are the shape of cross-sections (grossheim, 1935; zarrei et al., 2010c) and the number of vascular bundles (heyn and dafni, 1977; zarrei et al., 2010c). investigated populations of g. pratensis had recognisable v-shaped basal leaves with inner part that was not fistulose, and rather, it was made up of mesophyll, which was in accordance to the study conducted by peruzzi et al. (2008a). mesophyll was not differentiated into palisade and spongy tissue, which was also confirmed by akyol et al. (2015), but some indications of differentiation were noticed in all investigated populations. according to grossheim (1935) presence/absence 131 fig. 7. canonical discriminant analysis of the six g. pratensis populations from serbia and montenegro biologica nyssana ● 10 (2) december 2019: 15-133 stojanović et al. ● comparative morpho-anatomical analysis of gagea pratensis (pers.) dumort. (liliaceae) from serbia and montenegro of central parenchyma in basal leaves is one of the notable characters in terms of taxonomy. this tissue was absent from basal leaves in all investigated populations. the number of vascular bundles in basal leaves was less than nine in all populations, except in population from gamzigradska banja. these results differ from the results obtained by peruzzi et al. (2007). first cauline leaves had similar anatomical structure as basal leaves and presence of keels was noticed, as in the study carried out by peruzzi et al. (2007). cross-sections of pedicels of g. pratensis are triangular (peruzzi et al., 2008a) and number of vascular bundles ranges from 6-8 (peruzzi et al., 2007), which is also confirmed by our study. of all the organs analysed, peduncles showed the highest variability in shape. according to levichev (in ajani et al., 2010) the form of a peduncle is particular, because the second basal leaf accretes with a peduncle from the basis up to an inflorescence and functions as the lower floral leaf. this accretion of basal leaf with a peduncle leads to a dissymmetric and the specific configuration of the transverse section of the peduncle. peduncles are often polyhedric with a ‘channel-like’ structure inside (peruzzi et al., 2007) and two circles of vascular bundles (levichev, 2001). in our research, number of channel-like structures was one in all investigated populations except the populations from seličevica (some plants had one, while others had two channel-like structures) and gamzigradska banja (some plants had two, while others had three channel-like structures). these two populations are also distinguished by the presence of arms and hairs on their peduncles, which has not been recorded in the previous studies. we assume that the largest channel-like structure often followed by the presence of arms is a result of an incomplete concrescence between the first cauline leaf and peduncle. conclusion populations of g. pratensis analysed in this study are morpho-anatomically differentiated. population from gamzigradska banja is separated from all the other populations based on height of the whole plant, width of bulb, width and length of basal leaf, width and indumentum type of peduncle, length of first cauline leaf, width and length of second cauline leaf, and width of outer and inner segments of perigone. the values of the characters analysed in the population from seličevica tend to be transit in between population from gamzigradska banja and all the others populations. in order to determine the basis of morphological and anatomical differences, it is necessary to carry out analysis of the influence of ecological factors on investigated characters, perform experiments with cultivated plants, and conduct a molecular analysis. acknowledgements. the work was funded by the ministry of education, science and technological development of republic of serbia (project no. 173030). references ajani, y., noroozi, j., levichev, i. g. 2010: gagea alexii (liliaceae), a new record from subnival zone of southern iran with key and notes on sect. incrustatae. pakistan journal of botany, 42: 67-77. akyol, y., kocabas, o., yetisen, k. 2015: morphological and anatomical investigations of some gagea salisb. species (liliaceae) in turkey. journal of biological research and development, 5(1): 1-10. diklić, n. 1975: gagea salisb. in: josifović, m. 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(liliaceae) from serbia and montenegro anticancer compounds from medicinal plants biologica nyssana 5 (1)  september 2014: 31-35 matejić, j. et al.  antimicrobial potential of essential oil from... 31 original article received: 10 july 2014 revised: 21 august 2014 accepted: 10 september 2014 antimicrobial potential of essential oil from pastinaca sativa l. jelena s. matejić 1 , ana m. džamić 2 , tatjana mihajilov-krstev 3 , vladimir n. ranđelović 3 , zoran đ. krivošej 4 , petar d. marin 2 1 university of niš, faculty of medicine, 18000 niš, serbia 2 university of belgrade, faculty of biology, institute of botany and botanical garden “jevremovac”, 11000 belgrade, serbia 3 university of niš, faculty of science and mathematics, 18000 niš, serbia 4 university of priština, department of biology, faculty of natural sciences, 38220 kosovska mitrovica, serbia e-mail: jekamatejic@gmail.com abstract: matejić, j., džamić, a., mihajlov-krstev, t., ranđelović, v., krivošej, z., marin, p.: antimicrobial potential of essential oil from pastinaca sativa l. biologica nyssana, 5 (1), septmeber 2014: 31-35. the aim of the current investigation was to evaluate the antimicrobial effect of pastinaca sativa l. (apiaceae) essential oil. the aerial parts of plants were collected at kopaonik mountain (serbia) and the essential oil has been isolated by hydrodistillation from this plant material. essential oil was dominated by (z)-β-ocimene (10.8%), hexyl butanoate (10.4%), (e)-β-farnesene (6.1%) and lavandulyl acetate (5.2%). the antimicrobial activity of the essential oil was investigated using a micro-well dilution assay against the most common human gastrointestinal pathogenic microbial strains: escherichia coli, pseudomonas aeruginosa, salmonella enteritidis, bacillus cereus, listeria monocytogenes, staphylococcus aureus and yeast candida albicans. the results showed that minimal inhibitory concentrations (mic) and minimal fungicidal concentrations of essential oil ranged from 0.72 μg/ml (for the most sensitive b. cereus) to above 92.5 mg/ml for s. enteritidis and l. monocytogenes. this finding suggests that p. sativa may be considered as a natural source of antimicrobial agents. key words: antimicrobial potential, essential oil, pastinaca sativa l. apstract: matejić, j., džamić, a., mihajlov-krstev, t., ranđelović, v., krivošej, z., marin, p.: antimikrobni potencijal etarskog ulja vreste pastinaca sativa l. biologica nyssana, 5 (1), septmeber 2014: 31-35. cilj ovog rada bio je utvrđivanje antimikrobne aktivnosti etarskog ulja vrste pastinaca sativa l. (apiaceae). nadzemni deo biljke sakupljen je na kopaoniku (srbija). etarsko ulje nadzemnog dela izolovano je metodom hidrodestilacije. kao glavne komponente etarskog ulja bile su: (z)-β-ocimen (10.8%), heksil butanoat (10.4%), (e)-β-farnezen (6.1%) i lavandulil acetat (5.2%). antimikrobna aktivnost etarskog ulja ispitivana je korišćenjem mikrodilucione metode na patogene gastrointestinalnog trakta kao što su: escherichia coli, pseudomonas aeruginosa, salmonella enteritidis, bacillus cereus, listeria monocytogenes, staphylococcus aureus i candida albicans. rezultati su pokazali da su se minimalne inhibitorne koncentracije (mic) kao i minimalne fungicidne koncentracije kretale u opsegu od 0.72 μg/ml (za najosetljiviju bakterijsku vrstu b. 5 (1) • september 2014: 31-35 biologica nyssana 5 (1)  september 2014: 31-35 matejić, j. et al.  antimicrobial potential of essential oil from... 32 cereus) do 92.5 mg/ml za s. enteritidis i l. monocytogenes. na osnovu dobijenih rezultata slobodno se može reći da p. sativa može služiti kao prirodni izvor antimikrobnih agenasa. ključne reči: antimikrobni potencijal, etarsko ulje, pastinaca sativa l. introduction essential oils are complex mixtures of compounds, synthesized in plant tissues as secondary metabolites. the role of essential oil in plant is multiple: defense against pathogens and herbivores, attraction of pollinators, inhibition of other plant's seed germination, formation of protective layer which reduces transpiration and formation of specific microclimate. due to their lipophylic character, essential oils can interact with microbial membranes and achieve significant antimicrobial effect (s t o j a n o v i ć r a d i ć , 2012). in serbian flora, family apiaceae consists of 138 species. from the genus pastinaca, two species are found in serbia p. sativa l. and p. hirsuta panč. (n i k o l i ć , 1973). pastinaca sativa l. is biennial, more or less pubescent plant native to europe except the arctic. stem is hollow or solid, angled or terete. basal leaves are usually simply pinnate. fruit is broadly elliptical and vittae on the commissural face not reaching the ends of the fruit (tutin, 1968). the seed of p. sativa species contains essential oils. the dominant constituents in this oil are myristicin (64.20%), β-ocimene (29.10%) and βfarnesene (24.50%). for this reason p. sativa is used in food industry in serbia (j a n č i ć e t a l ., 1995). in the oil obtained from crushed seeds of p. sativa subsp. urens from turkey, 18 components were characterized representing 95% of the oil with octyl butyrate (79.5%) and octyl hexanoate (5.3%) as the major constituents (k u r k c u o g l u e t a l ., 2006). a whole plant is used for strengthening the appetite, better digestion, and as a diuretic. the fruit is bitter aromatics that increases milk yield (t u c a k o v , 1986). the root is rich in starch and sugar and is used as food (parsnip), animal fodder, and for wine making. the sap is liable to cause skin irritation by sensitizing skin to uv radiation (z e h u i & w a t s o n , 2005). the root is delicious baked, and it can be used in soups, added to cakes, pies and puddings (f a c c i o l a , 1990). leaves and young shoots can be usedcooked with other greens as a vegetable or added to soups (l a u n e r t , 1981; f a c c i o l a , 1990). the seed is used as a condiment (l a u n e r t , 1981). it is similar in taste to dill (f a c c i o l a , 1990). native parts of plants have been applied in feeding as a spice, pharmaceutical industry and folk medicine (n i k o l i ć , 1973). material and methods plant material and isolation of the essential oils aerial parts of p. sativa were collected in july 2003 from kopaonik mountain. a voucher specimen (psa 60316), has been deposited at the herbarium of the institute of botany and botanical garden “jevremovac”, faculty of biology, university of belgrade (beou). the oil from the dried aerial parts (210 g) was separately obtained by hydro distillation for 3 h using a clevenger-type apparatus. yield of the essential oil from the aerial parts was 0.1%, ρ=925.5 g/cm 3 . antimicrobial activity microbial strains the antimicrobial activity of all tested samples was evaluated using laboratory control strains obtained from the american type culture collection: gram (-) bacteria escherichia coli atcc 25922, pseudomonas aeruginosa atcc 9027, salmonella enteritidis atcc 13076; gram (+) bacteria: bacillus cereus atcc 10876, listeria monocytogenes atcc 15313, staphylococcus aureus atcc 25923 and yeast candida albicans atcc 10231. micro-well dilution assay the inocula of the microbial strains were prepared from the overnight broth cultures and suspensions were adjusted to 0.5 mcfarland standard turbidity (corresponding to 10 7 -10 8 cfu/ml, depending on genera consensus standard by the nccls (2003)). serial doubling dilutions of the tested samples (tested oil in 5% dmso) were prepared in a 96/well microtiter plate over the range of 0.05–100 μl/ml (for oil) in inoculated muellerhinton broth. the final volume was 100 μl and the final microbial concentration was 10 6 cfu/ml in each well. the plate was incubated for 24 h at 37 ° c. all experiments were performed in triplicate. two controls were included medium with 30% ethanol (negative control) and medium with streptomycin, chloramphenicol and nystatin (positive control). microbial growth was determined by adding 20 μl of 0.5% triphenyl tetrazolium chloride (ttc) aqueous solution (s a r t o r a t t o et al., 2004). biologica nyssana 5 (1)  september 2014: 31-35 matejić, j. et al.  antimicrobial potential of essential oil from... 33 minimal inhibitory concentration (mic) was defined as the lowest concentration of the samples inhibiting visible growth (red colored pellet on the bottom of the wells after the addition of ttc). to determine mbc/mfc, the broth was taken from each well without visible growth and inoculated in mueller-hinton agar (mha) for 24 h at 37 ° c. minimal bactericidal/fungicidal concentration (mbc/mfc) was defined as the lowest samples concentration killing 99.9% of bacterial/fungal cells. results and discussion chemical analysis of p. sativa essential oil was already tested and the main components were: (z)-β-ocimene (10.8%), hexyl butanoate (10.4%), (e)-β-farnesene (6.1%) and lavandulyl acetate (5.2%), and p. hirsuta was dominated by butyl butyrate (2.5%). in both species, hexyl butanoate is dominant (10.4 and 55.4%, resp.) (k a p e t a n o s e t a l ., 2008). yield of the essential oil for 13 components in this essential oil varied in the range of 5-1% (germacrene d, (+)-γ-terpinene, arcurcumene, α-zingiberene, β-sesquiterpene, (+)-(e)β-caryophyllene, geranyl acetone, myrcene, βbourbonene, nonanyl hexanoate, nonanal, (+)-βpinene, ethyl 2-phenylbutanoate (k a p e t a n o s e t a l . , 2008). in k u b e c z k a & s t a h l (1977) study, oils from the wild parsnip p. sativa were characterized by the presence of octyl acetate. the composition of essential oils in the roots of p. sativa changes during the course of plant development: at the seedling stage (0-18 days), the primary oil ducts between the pericycle cells contain mainly the sesquiterpene hydrocarbon trans-β-farnesene (34.9%) with smaller amounts of myristicin (14.4%) and terpinolene (3.1%); as the roots thicken during the secondary stage (23-30 days) the sesquiterpene content decreases, whereas the myristicin and terpinolene contents increase; and finally, at the adult stage (38-160 days) when secondary oil ducts develop, the percentage of sesquiterpene falls to 1.5%, while that of myristicin and perpinolene increase to 62.6 and 25.5%, respectively (stahlbiskup & wichtmann, 1991). in the oil of p. hirsuta, twenty-nine compounds were identified in root oil and thirteen compounds in fruit oil (92.8% and 90.8% of the total oils, respectively) using gc-fid and gc-ms. the principal components of the root oil were apiole (56.0%) and myristicin (21.0%), followed by βbisabolene (7.2%). the main compounds in the fruits oil were hexyl hexanoate (59.8%) and hexyl butanoate (21.4%) (p e t r o v i ć e t a l . , 2008). our results of antimicrobial activity obtained for the examined essential oil are presented in table 2. we were using the antibiotics streptomycin, chloramphenicol and nystatin for comparison. the essential oil of p. sativa showed the greatest activity against b. cereus strains (mic=mbc=0.72 mg/ml). bacterial strains s. enteritidis and l. monocytogenes showed the lowest susceptibility to the tested essential oil. terpenes, in general, possess a wide spectrum of biological activity through which they appear to play a role in plant defense mechanisms (f r a g a , 2001, 2003). in addition, it was possible that components present in lower amounts in the oil might be involved in some type of synergism with the other active compounds (m a r i n o et al., 2001). due to their bioactivity, the great number of terpenes has been evaluated for antibacterial or antifungal activity. components of essential oil such as p-cymene, α-pinene, β-pinene, limonene, α-terpinene, α–terpinolene, caryophyllene oxide, camphene, 1,8-cineole and linalool have been reported for their antimicrobial activity (v i l j o e n , 2003; k n o b l o c h , 1989; s ö k m e n , 2004). the main components of p. sativa essential oil were not up to now evaluated for their antimicrobial activity. b r k o v i ć e t a l . (2006) tested antimicrobial activity of water, ethanol and ethyl acetate extracts from p. sativa and 11 other plants from the family of apiaceae. comparison with the other species showed that p. sativa had the least antimicrobial activity. the root oil of p. hirsuta pančić was active against all tested microorganisms and exhibited the best activity against b. subtilis (mic 6.25 µg/ml), s. aureus, m. luteus and c. albicans atcc 10259 (mic 12.5 µg/ml). the oil from the fruits of p. hirsuta was active only against e. coli and c. albicans (the most active against c. albicans atcc 10259: mic 12.5 µg/ml) (p e t r o v i ć e t a l . , 2008). when comparing the results, we can conclude that the results of antimicrobial activity points to higher potential of p. hirsuta than the herein tested oil. the reason for this can be different chemical composition of the root and the fruits as well as different solvent in which the oil is dissolved during the antimicrobial testing. biologica nyssana 5 (1)  september 2014: 31-35 matejić, j. et al.  antimicrobial potential of essential oil from... 34 table 2. antimicrobial activity of pastinaca sativa essential oil against pathogenic microbial strains using micro-well dilution assay pathogenic microbial strains p. sativa essential oil (mic/mbc(mfc) in mg/ml) antibiotic (mic/mbc(mfc) in μg/ml) gram (-) bacteria streptomycin e. coli atcc 25922 23.1/92.5 16.0/16.0 p. aeruginosa atcc 9027 46.2/46.2 8.0/8.0 s. enteritidis atcc 13076 92.5/92.5 4.0/4.0 gram (+) bacteria chloramphenicol b. cereus atcc 10876 0.72/0.72 4.0/16.0 l. monocytogenes atcc 15313 92.5/92.5 8.0/16.0 s. aureus atcc 25923 46.2/46.2 1.0/8.0 yeast nystatin c. albicans atcc 10231 46.2/46.2 16.0/16.0 conclusion the essential oil from pastinaca sativa inhibited the growth of tested microorganisms. these microorganisms cause food spoilage and are recognized as human and animal pathogens. thus, the oil from this species deserves further investigation in search for natural food preservatives. acknowledgements. the authors are grateful to the ministry of education and science of the republic of serbia for financial support (grant no. 173029). references brković, l.d., čomić, lj., solujić-sukdolak, s. 2006: antibacterial activity of some plants from family apiaceae in relation to selected phytopathogenic bacteria. kragujevac journal of science, 28: 65-72. facciola, s. 1990: cornucopia a source book of edible plants. kampong publications, california, usa. fraga, b.m. 2001: natural sesquiterpenoids. natural product reports, 18:650–673. fraga, b.m. 2003: natural sesquiterpenoids. natural product reports, 20:392–413. jаnčić, r., stošić, d., mimicа-dukić, n., lаkušić, b. 1995: aromаtične biljke srbije. nip dečije novine, beogrаd-gornji milаnovаc. kapetanos, c., karioti, a., bojović, s., marin, p., veljić, m., skaltsa, h. 2008: chemical and principal-component analyses of the essential oils of apioideae taxa (apiaceae) from central balkan. chemistry and biodiversity, 5:101-119. knobloch, k., iberl, a.p.b., weigand, h., weis, n. 1989: antibacterial and antifungal properties of essential oil components. journal of essential oil research, 1:119–128. kubeczka, k. h., stahl e. 1977: essential oils from the apiaceae (umbelliferae). ii. the essential oils rom the above ground parts of pastinaca sativa. planta medica, 31:173-184. kurkcuoglu, m., baser, k.h.c., vural, m. 2006: composition of the essential oil of pastinaca sativa l. subsp. urens (req. ex godron) celak. chemistry of natural compounds, 42(1):114115. launert, e., 1981: edible and medicinal plants. hamlyn, london. marino m, bersani c, comi g 2001: impedance measurements to study the antimicrobial activity of essential oils from lamiaceace and compositae. international journal of food microbiology, 67: 187–195. nccls – national committee for clinical laboratory standards, performance standards for anti-microbial susceptibility testing: eleventh informational supplement, document m100-s11, 2003: national committee for clinical laboratory standard, wayne, pa, usa. biologica nyssana 5 (1)  september 2014: 31-35 matejić, j. et al.  antimicrobial potential of essential oil from... 35 nikolić v. 1973: fam. apiaceae. in: josifivić m. 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(eds.), flora europaea 2. cambridge university press, london, uk, p. 364. viljoen, a., van vuuren, s., ernst, e., klepser, m., demirci, b., baser, h., et al. 2003: osmitopsis asteriscoides (asteraceae)-the antimicrobial activity and essential oil composition of a capedutch remedy. journal of ethnopharmacology, 88(2–3):137–143. zehui, p., watson, m.f. 2005: 94. pastinaca linnaeus, sp. pl. 1: 262. 1753. flora of china, 14, 193. biologica nyssana 5 (1)  september 2014: 31-35 matejić, j. et al.  antimicrobial potential of essential oil from... 36 i̇rez et al. 2021, biologica nyssana 12(1) 12 (1) september 2021: 55-62 doi: 10.5281/zenodo.5523017 fomes fomentarius (l.) fr. extracts as sources of an antioxidant, antimicrobial and antibiofilm agents original article eylül i̇rem i̇rez department of biology, faculty of arts and sciences, çanakkale onsekiz mart university, çanakkale, turkey eyluliremirez@gmail.com nurcihan hacioğlu doğru department of biology, faculty of arts and sciences, çanakkale onsekiz mart university, çanakkale, turkey nurcihan.n@gmail.com (corresponding author) neslihan demir department of biology, faculty of arts and sciences, çanakkale onsekiz mart university, çanakkale, turkey neslihandemir@comu.edu.tr received: mart 03, 2020 revised: december 22, 2020 accepted: february 08, 2021 abstract: this paper evaluated antioxidant, antimicrobial and antibiofilm activities of ethanol, methanol, acetone and chloroform extracts of lignicolous fungal species fomes fomentarius (l.) fr. (polypodiaceae). antiradical activity was evaluated by using dpph (2,2-diphenyl-1-picrylhydrazyl) assay. the antimicrobial screening was carried out via disc diffusion and microdilution methods in order to estimate minimum inhibitory (mic) and minimum bactericidal concentration (mbc) of analyzed extracts against seven standard bacteria and one yeast: escherichia coli nrrl b-3704, pseudomonas aeruginosa atcc 27853, proteus vulgaris atcc 13315, acinetobacter baumanii atcc 19606, bacillus subtilis atcc 6633, staphylococcus aureus atcc 25923, s. haemolyticus atcc 43252 and candida albicans atcc 10231. in vitro antibiofilm activities were investigated based on crystal violet binding assay. all the extracts showed higher antioxidant activity compared to bht (butylated hydroxytoluene) which was used as a standard. ethanol, methanol and acetone extracts of the tested macrofungus also showed higher antimicrobial effect against p. aeruginosa atcc 27853 in comparison to the antibiotic penicillin (p10). the lowest mic was recorded by ethanol extract against s. haemolyticus atcc 43252 (0.625 µg/ml). the highest antibiofilm activity was also noticed against biofilm formed by s. haemolyticus atcc 43252. the results indicated that the f. fomentarius tested represent potential source of natural bioactive compounds with respect to antimicrobial, antibiofilm and antioxidant activities. key words: antimicrobial, antibiofilm, antioxidant activity, fomes fomentarius apstract: fomes fomentarius (l.) fr. ekstrakti kao izvori antioksidanasa, antimikrobnih i antibiotskih sredstava ovaj rad se bavi procenom antioksidativne, antimikrobne i antibiofilm aktivnosti etanolnog, metanolnog, acetonskog i hloroformnog ekstrakta lignikolne gljive fomes fomentarius (l.) fr. (polyporaceae). antiradikalna aktivnost je utvrđena korišćenjem dpph (2,2-difenil-1-pikrilhidrazil) eseja. antimikrobni skrining je izvršen putem disk difuzione i mikrodilucione metode kako bi se utvrdile minimalna inhibitorna (mik) i minimalna baktericidna koncentracija (mbk) analiziranih ekstrakata u odnosu na sedam standardnih bakterija i jednu gljivu: escherichia coli nrrl b-3704, pseudomonas aeruginosa atcc 27853, proteus vulgarisatcc 13315, acinetobacter baumaniiatcc 19606, bacillus subtilis atcc 6633, staphylococcus aureus atcc 25923, s. haemolyticus atcc 43252 and candida albicans atcc 10231. in vitro antibiofilm aktivnost je ispitana korišćenjem kristal violet eseja. svi ekstraktu pokazali su veću antioksidativnu aktivnost u poređenju sa bht (butilovanim hidroksitoluenom) koji je korišćen kao standard. takođe, etanolni, metanolni i acetonski ekstrakti testirane makrogljive su u poređenju sa antibiotikom penicilinom (p10) pokazali veći antimikrobni efekat na p. aeruginosa atcc 27853. najniža mik vrednost je zabeležena zza etano0lni ekstrakt i to protiv s. haemolyticus atcc 43252 (0.625 µg/ml). najviša antibiofilm aktivnost uočena je kod biofilma formiranog od strane s. haemolyticus atcc 43252. rezultati su ukazalu da testirana vrsta f. fomentarius predstavlja potencijalni izvor prirodnih bioaktivnih jedinjenja u smislu antimikrobne, antibiofilm i antioksidativne aktivnosti. ključne reči: antimikrobna, antibiofilm, antioksidativna aktivnost, fomes fomentarius © 2021 i̇rez et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 55 introduction in recent years, the problem of antibiotic resistance, where bacterial and fungal pathogens developed numerous defense mechanisms against antimicrobial agents is increased, and scientific studies presented new and more powerful agents as an alternative to antibiotic therapy (heleno et al., 2013). the interest in natural compounds has been a common point for most biotechnology companies to be used in the production of new antimicrobial drugs (butler, 2004). microbial biofilms are communities of bacteria, embedded in a self-producing matrix, forming on living and nonliving solid surfaces (vasudevan, 2014). they are considered as an important virulence factor that causes persistent chronic and recurrent infections; they are highly resistant to antibiotics and host immune defenses. biofilm resistance is due to several reasons, like restricted diffusion of antibiotics into biofilm matrix, expression of multidrug efflux pumps, type iv secretion systems, decreased permeability, and the action of antibioticmodifying enzymes (alekshun and levy, 2007). the increased biofilm resistance to conventional treatments enhances the need to develop new control strategies (simões et al., 2007; sánchez et al., 2016). it is common to believe that it is difficult to prevent and treat biofilm infections. current findings regarding the fact that biofilms have a highly heterogeneous structure necessitates the development of new treatment strategies unlike the treatment strategies used in the destruction of microorganisms in the planktonic phase (clatworthy et al., 2007). besides its antimicrobial effects, some natural product extracts are known to prevent biofilm formation. due to the inadequate antimicrobial therapies in combating biofilms, researchers are directed towards the discovery and identification of new antimicrobial agents, especially natural (karaca et al., 2017). antioxidants have the ability to protect the body against damage caused by oxidative stress. the interest in natural antioxidants is increasing day by day. for example, polyphenols, found in medicinal plants and foods, can help prevent oxidative damage (silva et al., 2005). mushrooms are rich sources of antioxidant compounds such as phenolic compounds (phenolic acids and flavonoids) and tocopherols (cheung et al., 2003; ferreira et al., 2009; heleno et al., 2010; sun et al., 2011; dündar et al., 2016). fomes fomentarius (l.) fr., polyporaceae, tinder fungus is a woody, perennial fungus, large in size which develops as a parasite or saprophyte on the beech (fagus sylvatica l.) and other deciduous species. it is a white root fungus causing heart rot of the wood. in recent years, the active compounds of f. fomentarius have been studied extensively and most of its biological activities have been put forward. it is found that f. fomentarius have significant effects such as antioxidant (lee, 2005; vazirian et al., 2014; dündar et al., 2016; bal et al., 2017), antimicrobial (kolundžić et al., 2016), antifungal (dresch et al., 2015), anti-inflammatory (vazirian et al., 2014), cytotoxic (kolundžić et al., 2016), antitumor (chen et al., 2008), antiviral (aoki et al., 1993), dna protective (bal et al., 2017), fibrinolytic activity (sánchez-santillán et al., 2015). this extended study was to evaluate the antimicrobial, antibiofilm and antioxidant activities of ethanol, methanol, acetone and chlorofom extracts of f. fomentarius. although there have been extensive researches to reveal antioxidant and antimicrobial activity of f. fomentarius, studies on antibiofilm activity have not been found in the literature. we also aimed to determine antibiofilm potential of the four extracts obtained from f. fomentarius against important pathogen microorganisms for the first time. materials and methods the macrofungal material fomes fomentarius was collected from çanakkale, turkey on decaying stump in january, 2018 and identified by dr. ersin karabacak. preparation of extracts the sample of the dried macrofungus (15 g) was extracted with four solventsethanol, methanol, acetone and chloroform (150 ml) using the soxhlet apparatus (isolab). the extraction was continued until the final extract was colorless according to khan et al. (1988). the solvents were removed under reduced pressure and dried using a rotary evaporator (heidolp, laborota 4001, germany) at 55 °c. dry extract was taken into glass bottles with dark lid and stored at +4 °c. for the experiments, dissolution in dimethyl sulfoxide (dmso) (10%) and different concentrations of the prepared extract were sterilized by a membrane filter (0.2 µm) and prepared for screening. evaluation of antiradical activity dpph assay the antioxidant activity of the extracts was measured by using commercial free radical 2,2-diphenyl-1-picrylhydrazyl (dpph) following the procedure described by brand-williams et al. (1995), with slight modifications. the extracts were evaporated to dryness and re-suspended in dmso. butylated hydroxytoluene (bht) solution was used as a positive control. the absorbance 56 biologica nyssana ● 12 (1) september 2021: 55-62 i̇rez et al. ● fomes fomentarius (l.) fr. extracts as sources of an antioxidant, antimicrobial and antibiofilm agents 57 values of the samples were measured on a uv-vis spectrophotometer (pg instruments t+80) at 517 nm against a blank. the experiment was repeated three times and the arithmetic mean of the readings was taken. the radical scavenging activity of each sample was calculated using the following equation and the results were expressed as % inhibition. inhibition (%) = [(a0 a1) / a0] x 100 a0: extract or non-standard control absorbance, a1: extract or standard absorbance evaluation of antimicrobial activity sterilized antibiotic discs 6 mm in diameter (schleicher and schull no. 2668, dassel, germany) were impregnated with 50 μl of each extract (10 mg/disc) at concentration of 200 mg/ml. gram negative bacteria escherichia coli nrrl b-3704, pseudomonas aeruginosa atcc 27853, proteus vulgaris atcc 13315, acinetobacter baumanii atcc 19606, gram positive bacteria bacillus subtilis atcc 6633, staphylococcus aureus atcc 25923, s. haemolyticus atcc 43252 and yeast culture candida albicans atcc 10231 were used as the test microorganisms. all the bacteria were incubated at 35 ± 0.1 °c for 24 h by inoculation into nutrient broth (difco laboratories, mi, usa) and the yeast culture studied was incubated in malt extract broth (difco laboratories, mi, usa) at 25 ± 0.1 °c for 48 h. an inoculum containing 106 bacterial cells or 108 yeast cells/ml was spread on mueller hinton agar (mha) (oxoid ltd., hampshire, uk) plates (1 ml inoculum/plate). the discs injected with extracts were placed on the inoculated agar by pressing slightly. petri dishes were placed at ±4 °c for 2 h and incubated for 24 h and 48 h for bacteria and yeast, respectively (collins et al., 1989). at the end of the period, inhibition zones formed on the medium were evaluated in millimeters. studies were performed in triplicate. treatments with penicillin (p10), and nystatin (nys30) served as positive controls and treatments with ethanol, methanol, acetone and chloroform without fungal materials served as negative controls. for quantitative antimicrobial analyses, minimum inhibitory concentration (mic) values of all samples were determined. mic and mbc were investigated as recommended instruction of the clinical and laboratory standards institute (clsi, 2006). briefly, stock solution of each extract was diluted in the muller hinton broth (mhb) in two-fold serial dilutions to obtain final concentrations range of 200.156 mg/ml at a total volume of 100 μl per well in 96-well microtiter plates. the lowest concentration of extracts inhibiting visible growth of each test microorganisms was taken as the mic. the medium, 0.1% (w/v) streptomycin (st), nystatin (nys30) and 10% dmso were used as the non-treated, positive and negative controls, respectively (teanpasian et al., 2017). confirmation of mic and establishment of the minimum bactericidal concentration (mbc) and minimum fungicidal concentration (mfc), 10 µl of the following dilutions (40 to 2.5 µg/ml) were inoculated into plates with mha to evaluate microbial growth. after 24 h of incubations at 35 ± 0.1 °c for bacteria and 48 h at 25 ± 0.1 °c for the yeast culture, the plates with no apparent cfu of surviving microorganisms were determined. each experiment was repeated three times (pacheco-ordaz et al., 2018). biofilm inhibition assay microplate biofilm method (merrit et al., 2005) was used to evaluate the inhibition of biofilm formation by f. fomentarius extracts against tested microorganisms. cultures were incubated in 5 ml tryptic soy broth (tsb) medium containing 5% glucose. cultures were diluted 1:100 in tsb and loaded into each well in 4 sterile microplate. different concentrations of fungal extracts (mic and submic concentrations: 50, 25, 12.5% of mic) were prepared and transferred to each microplate well. after incubation at 37 ± 0.1 °c for 48 h planktonic bacteria were removed from the wells and wells were washed twice with distilled water. crystal violet solution (0.1%, 200 μl) was added to each well and incubated for 20 minutes. the crystal violet bounded extracts were poured and washed until the crystal violet was finally removed. the microtiter plates were inverted and the remaining liquid was drained and dried in room heat. finally, the adhered biofilm bounded crystal violet was eluted in ethanol (95%) and the absorbance was measured at 550 nm by using an automated elisa reader (biotek, uk). all experiments were repeated in triplicate. the calculation of the antibiofilm effect of the extract was made by the reduction formulation percentage. % inhibition = (acontrol – asample / acontrol) x 100 acontrol: absorbance of the control (containing 100 µl tbs instead of fungal extract) reaction, asample: absorbance of the tested compound results and discussion two polar (ethanol and methanol), one intermediate polar (acetone) and one nonpolar (chloroform) solvent were selected for comparison of biological activity of f. fomentarius. the choice of solvents depends on the type of macrofungi, part of the materials to be extracted, nature of the bioactive biologica nyssana ● 12 (1) september 2021: 55-62 i̇rez et al. ● fomes fomentarius (l.) fr. extracts as sources of an antioxidant, antimicrobial and antibiofilm agents 58 compounds, the availability of solvent and the literature. polar solvents such as ethanol, methanol and acetone were used in extraction of polar compounds, whereas nonpolar solvents such as chloroform were used in extraction of nonpolar compounds such as terpenoids, flavonoids, fats and oils (abubakar and hague, 2020). the findings showed that polar solvents, especially ethanol, are more successful in extraction of secondary metabolites with antioxidant, antimicrobial and antibiofilm activity. dpph assay is widely used in determination of the antioxidant activity of compounds and different fungal extracts. in this work, it was found that the inhibition percentage of ethanol extracts from f. fomentarius was slightly higher (89.95±0.21) than when acetone, methanol and chloroform solvents were applied (88.88±0.18, 87.34±0.11 and 80.93±0.07, respectively) (fig. 1). bht which was used as a standard showed lower antioxidant activity (75.81±0.03) compared with all other extracts, obtained by different solvents. studies have shown that the biological activities of the extracts can vary depending on the season, type of extract, dose and duration of application. depending on the extraction method, the level of bioactive substances can be observed (anlas et al., 2017). mircea et al. (2015) and kolundžić et al. (2016) reported that dpph radical scavenging activity of f. fomentarius methanol extract results correlated with the polyphenol content. in addition, f. fomentarius methanol and ethanol extracts have been reported to have the strongest dpph activity comparing to other solvents (bojin et al., 2020). the present study indicated that ethanol extract showed slightly higher antioxidant activities than the other solvents. the antimicrobial activities of the macrofungal extracts against different test strains were assessed according to inhibition zones diameter and mic and mbc or mfc values (tab. 1). all extracts showed higher antimicrobial effect against p. aeruginosa, p. vulgaris, s. haemolyticus, c. albicans except chloroform extract with inhibition zone of 10-13 mm. ethanol, methanol and acetone extracts of macrofungi also showed higher antimicrobial effect against p. aeruginosa in comparison to antibiotic p10. the lowest mic value was recorded by f. fomentarius ethanol extract against s. haemolyticus (0.625 μg/ml). also, ethanol extract showed higher mic against e.coli, b. subtilis, s. aureus and s. haemolyticus bacteria when compared to antibiotic streptomycin probably due to the polar component that the ethanol extract contain. there are many scientific reports about f. fomentarius antimicrobial activity (zhao et al., 2013; dündar et al., 2016; kolundžić et al., 2016; gedik et table 1. antimicrobial activity of f. fomentarius extracts fig. 1. dpph free scavenging activity of f. fomentarius extracts test microorganisms *disc diffusiona control antibiotics mic (µg/ml) control antibiotics mbc (mfc) mbc (mfc)/ mic e1 e2 e3 e4 p10 ny 100 e1 e2 e3 e4 st ny 100 e1 e2 e3 e4 e1 e2 e3 e4 e.coli 7.0 7.0 10.0 8.0 16.0 nt 2.5 2.5 10 20 4.0 nt 40 2.5 40 40 16 1 4 2 p. aeruginosa 10.0 11.0 10.0 7.0 8.0 nt 10 10 10 20 1.0 nt 40 40 40 40 4 4 4 2 p. vulgaris 13.0 10.0 10.0 7.0 13.0 nt 20 5 10 20 4.0 nt 40 40 40 40 2 8 4 2 a. baumanii 7.0 11.3 8.6 9.0 12.0 nt 5 5 10 20 2.0 nt 40 5 40 40 4 1 4 2 b. subtilis 13.0 9.0 8.3 8.0 14.0 nt 2.5 2.5 10 20 4.0 nt 40 2.5 40 40 16 1 4 2 s. aureus 13.0 10.6 8.3 9.0 15.0 nt 2.5 5 10 20 4.0 nt 40 5 40 40 16 1 4 2 s. haemolyticus 11.6 11.6 10.3 8.0 14.0 nt 0.6 5 10 20 5.0 nt 40 10 40 40 64 2 4 2 c. albicans 10.0 11.0 10.0 7.0 nt 16.0 5 5 5 20 nt 5.0 40 40 40 40 4 8 8 2 biologica nyssana ● 12 (1) september 2021: 55-62 i̇rez et al. ● fomes fomentarius (l.) fr. extracts as sources of an antioxidant, antimicrobial and antibiofilm agents al., 2019). similar to the present study, kolundžić et al. (2016) tested the antimicrobial activity of f. fomentarius extracts of different polarity, especially against gram-negative and gram-positive bacteria. they indicated that their f. fomentarius extracts (cyclohexane, dichloromethane, methanol and aqueous) displayed strong antimicrobial activity. gedik et al. (2019) also showed that ethanol and aqueous extracts of f. fomentarius have higher antibacterial activity against k. pneumoniae, a. baumanii, s. aureus, vancomycin-resistant enterococci (vre+), e. coli against standard antibiotics. in our study, a similar situation was obtained against p. vulgaris, b. subtilis, s. aureus bacteria. the results of zhao et al. (2013) demonstrated weak antimicrobial activity in isolated phenyl-ethanediols from the fruiting bodies of f. fomentarius. moreover, methanolic extracts of f. fomentarius have been found to be effective against m. luteus, but no activity was detected against s. aureus, e. coli, p. aeruginosa, b. subtilis, enterococcus faecalis (kolundžić et al., 2016). in the present study we obtained different results for antimicrobial activity of f. fomentarius against the tested microorganisms; that is possibly due to differences of fungal species habitat factors, ecological status, seasonal differences and variety of extraction methods. the results of potential inhibition of test microorganism’s biofilm formation by four extracts of f. fomentarius were shown in fig. 2. the mic values of f. fomentarius extracts were used to determine antibiofilm rates. it can be seen that antibiofilm rates of four extracts ranged from 5.03 to 95.2%. the results indicated that ethanol and methanol extracts of f. fomentarius could inhibit the biofilm formation of all the tested strains, significantly. the highest antibiofilm activity was noticed against biofilm formed by s. haemolyticus. both acetone and chloroform extracts showed very low antibiofilm effects compared to ethanol and methanol, except for c. albicans (fig. 2). although recent studies on the evaluation of medically important fomes species in this direction are few in the literature (bin et al., 2012; solmaz et al., 2013; alves et al., 2014; petrović et al., 2014; signoretto et al., 2014; carvalho et al., 2016; karaca et al., 2017), there is no study on the antibiofilm effects of the f. fomentarius on the related pathogen strains. alves et al. (2014) tested the antibiofilm capacity of different macrofungal species against e. coli, p. aeruginosa, a. baumannii and p. mirabilis strains. they found that all macrofungi methanol extracts showed strong antibiofilm activity against pathogens. petrović et al. (2014) also reported that hot water extract from agaricus blazei could influence on biofilm formation, twitching and swimming activity, pyocyanin production which are part of anti-quorum sensing activity. karaca et al. (2017) investigated antibiofilm potentials of methanolic and ethanolic extracts of the three medicinal macrofungi species and found that the highest antibiofilm activity was observed with ganoderma lucidum methanolic extract. therefore, the findings obtained from the present study are presenting the important contributions to the literature data on this subject. conclusions in the present study, it was determined that f. fomentarius demonstrated high antioxidant, 59 fig. 2. inhibition (%) of biofilms formation of f. fomentarius extracts (e1: ethanol, e2: methanol, e3: acetone, e4: chloroform) biologica nyssana ● 12 (1) september 2021: 55-62 i̇rez et al. ● fomes fomentarius (l.) fr. extracts as sources of an antioxidant, antimicrobial and antibiofilm agents antimicrobial and antibiofilm activities. in conclusion, it was determined that analyzed f. fomentarius extracts could be used after chemical characterization and toxicity testing as natural resources in the fields of medicine, pharmacology, nutrition and cosmetics due to their strong antioxidant and antimicrobial activities. acknowledgment. authors would like to thank assoc. prof. dr. ersin karaback for defining macrofungus species. this research is a part of mrs. eylül i̇rem i̇rez’s tübi̇tak-2209 a project which is supported with the frame of undergraduate students grant in biological science. references abubakar, a.r., hague, m. 2020: preparation of medicinal plants: basic extraction and fractionation procedures for experimental purposes, journal of pharmacy & bioallied sciences, 12(1): 1-10. alekshun, m.n., levy, s.b. 2007: molecular mechanisms of antibacterial multidrug resistance, cell, 128(6): 1037-1050. alves, m. j., ferreira, i. c., lourenço, i., costa, e., martins, a., pintado, m. 2014: wild mushroom extracts as inhibitors of bacterial biofilm formation. pathogens, 3(3): 667-679. anlas, c., ustuner, o., ustun alkan, f., bakirel, t., aydogan, m.n., baykan, erel, s. 2017: a comparative study on the antioxidant activities and phenolic contents of different extracts of achillea nobilis subsp. sipylea and alcea apterocarpa (fenzl) boiss, endemic plants in turkey. fresenius environmental bulletin, 26(2): 1423-1430. aoki, m., tan, m., fukushima, a., hieda, t., kubo, s., takabayashi m., ono, k., mikami, y. 1993: antiviral substances with systemic effects produced by basidiomycetes such as fomes fomentarius. bioscience, biotechnology, and biochemistry, 57(2): 278-82. bal, c., akgul, h., sevindik, m., akata, i., yumrutas, ö. 2017: determination of the antioxidative activities of six mushrooms. fresenius environmental bulletin, 26(10): 6246-6252. brand-williams, w., cuvelier, m.e., berset, c. 1995: use of a free radical method to evaluate antioxidant activity. lebensmittel-wissenschaft undtechnologie/food science and technology, 28:2530. bin, l., wei, l., xiaohong, c., mei, j., mingsheng, d. 2012: in vitro antibiofilm activity of the melanin from auricularia auricula, an edible jelly mushroom. annals of microbiology, 62(4): 1523-1530. bojin, l., serb, a., pascariu, m., moaca, a., kostici, r., purcarea, v.l., penescu, m. ivan, m.v., georgescu, m., sisu, e. 2020: assessment of antioxidant properties of different fomes fomentarius extracts. farmacia, 68(2): 322-328. butler, m.s. 2004: the role of natural product chemistry in drug discovery. journal of natural products, 67: 2141–2153. carvalho, m. p., gulotta, g., do amaral, m. w., lünsdorf, h., sasse, f., abraham, w. r. 2016: coprinus lactone protects the edible mushroom coprinus comatus against biofilm infections by blocking both quorum sensing and mura. environmental microbiology, 18(11): 4254-4264. chen, w., zhao, z., chen, s.f. 2008: optimization for the production of exopolysaccharide from fomes fomentarius in submerged culture and its antitumor effect in vitro. bioresource technology, 99(8): 3187–3194. cheung, l.m., cheung, p.c.k., ooi, v.e.c. 2003: antioxidant activity and total phenolics of edible mushroom extracts. food chemistry, 81: 249–255. clatworthy, a.e., pierson, e., hung, d.t. 2007: targeting virulence: a new paradigm for antimicrobial therapy. nature chemical biology, 3(9): 541-548. clsi, 2006. clinical and laboratory standarts institute. methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; 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(lepidoptera, bombycidae) original article aleksandra trajković university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia aleksandra.trajkovic2@pmf.edu.rs (corresponding author) marijana ilić milošević university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia saša s. stanković university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia maja lazarević university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia darija milenković university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia iva stojanović university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia marija krstić university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia vladimir žikić university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia received: april 17, 2024 revised: july 09, 2024 accepted: july 11, 2024 abstract: in this study, a total of 900 specimens of univoltine polyhybrid silkworm breed were reared across early, mid, and late breeding seasons. the objective of the presented work was to assess reported cases of rearing silkworm on alternative host plants and evaluate the feasibility of partially replacing the optimal food source, morus alba l., with the year-round available taraxacum officinale (l.) weber ex f.h.wigg., while assessing larval and pupal duration, l5 larval weight, cocoon weight, as well as larval mortality and larval–pupal intermediates (unsuccessful pupae). while the substitution showed a statistically significant effect only for the larval stage duration, different temperature ranges affected all investigated traits. the optimal values were achieved in groups bred during the mid-season (19–24.5 °c), where differences in success between the control and the treatment were minimal. further breeding will provide clearer insights into the limitations of this food replacement strategy within the system, depending on its research purpose. key words: diet, dandelion, silkworm rearing apstrakt: efekat parcijalne zamene duda maslačkom na pojedine biološke karakteristike svilene bube, bombyx mori l. (lepidoptera, bombycidae) za potrebe ovog istraživanja, kroz rani, srednji i kasni period sezone, odgojeno je 900 jedinki univoltnog polihibrida svilene bube. cilj predloženog istraživanja bio je testiranje objavljenih navoda o gajenju svilenih buba na alternativnim biljkama hraniteljkama, kao i preliminarna ocena izvodljivosti parcijalne zamene optimalnog izvora hrane, belog duda, morus alba l., lako dostupnim maslačkom, taraxacum officinale (l.) weber ex f.h.wigg. efekat zamene praćen je na nivou bioloških karakteristika, dužine trajanja stadijuma larve i lutke, težine l5 gusenica, težine svilenih kokona, mortaliteta gusenica i frekvencije javljanja neuspešnih međuformi larva-lutka. zamena je pokazala statistički značajan efekat na trajanje stadijuma larve, dok su različiti temperaturni opsezi uticali na sve ispitivane karakteristike. optimalne vrednosti postignute su u grupama gajenim u srednjem delu sezone (19– 24.5 °c), kada su razlike u uspehu gajenja između tretmana i kontrole bile minimalne. kontinuirani uzgoj će obezbediti bolji uvid u odnos prednosti i ograničenja ove izmene režima ishrane, u kontekstu krajnjeg cilja istraživanja. ključne reči: ishrana, maslačak, gajenje svilene bube introduction in addition to the honeybee and the common fruit fly, the silkworm, bombyx mori l. is among the most thoroughly studied insects in the world (cermeño et al., 2022). the silkworm’s economic importance extends beyond its well-known role in textile production. it also serves as a valuable model organism in scientific research, particularly in the fields of endocrinology, genetics, and immunology. additionally, silkworms are used as bioreactors and are of increasing interest in the fields of entomophagy (insect consumption as food) and nutraceutical development (development of food products with health benefits) (abdelli et al., 2018). domesticated for approximately five millennia, this species exhibits significant genetic diversity, comprising numerous geographical races, hybrids, and lines. different © 2024 trajković et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 105 breeds require specific environmental conditions (vimala et al., 2020), reflecting the agricultural trend towards optimizing voltinism, biomass yield, silk production, and resistance to pathogens. breeding programs, particularly advanced in china (xiang et al., 2018), india (sharma & kapoor, 2020), korea (kim et al., 2022), japan (onaga, 2012), as well as italy (cappellozza, 2015), bulgaria (tzenov et al. 2022), and romania (pop et al., 2018) for europe, continuously adapt to ensure optimal genetic performance. hybridization has further widened the diversity of biological and technological traits, leading to variations in larval and pupal duration, dietary preferences, artificial diet acceptance, temperature tolerance, cocoon characteristics, and silk filament properties (altman & farrell, 2022). native to china, the silkworm is a monophagous herbivore, requiring fresh leaves of the white mulberry, morus alba l., a woody species native to the himalayan region. due to the long-standing process of co-evolution and natural selection between silkworms and mulberry trees, moriculture and sericulture have grown in tandem (liu et al., 2023). understanding the mechanisms behind the mulberry-specific feeding preference has been a subject of extensive investigation, encompassing neuroregulatory, metabolomic, and genetic aspects. this feeding specialization results from an interplay of gustatory and olfactory cues, along with factors that either promote or inhibit feeding behavior (jia et al., 2020). mulberry leaves contain three groups of factors that promote food intake: attracting factors such as α, β-hexenal, β, γ-hexenol, citral, linalyl acetate, and linalool; a biting factor (primarily β-sitosterol) and several swallowing factors, predominantly cellulose (song et al., 2021). traditional sericulture, centered around feeding silkworms with fresh mulberry leaves, has historically served as a cornerstone for both moriculture and small farm establishments, offering numerous opportunities for economic growth in many regions. however, mulberry leaves are not readily available year-round, their quality decreases as the breeding season progresses, and they are susceptible to frost damage (urbanek krajnc et al., 2023). this is particularly important for bivoltine and polyvoltine breeds, but also in cases in which laboratories need to establish populations outside their usual breeding timeframe. additionally, mulberry leaves can harbor pathogens that pose a risk to silkworm colonies, even with proper disinfection protocols in place. these factors make mulberry leaves unsuitable for certain types of research and laboratory experiments that require consistent dietary conditions for the model organism (tao et al., 2022). consequently, extensive research has been conducted to evaluate alternative feeding options, including different varieties of mulberry, supplementation, or fortification with vitamins, but most importantly, development of artificial and semi-artificial diets that mimic mulberry leaves chemical composition for standard rearing protocols. although progress has been made in breeding silkworm varieties capable of efficiently utilizing artificial and semi-artificial diets, there are still limitations regarding the metabolic utilization of these diets compared to mulberry leaves (qin et al., 2020), leading to challenges such as weak larvae and low silk yields. even when the optimal nutritional ratio and efficient feeding and growth are achieved, metabolic utilization, convenience and exploring low-cost alternatives for artificial diets remain a critical focus for researchers in the field (dai et al., 2022). before interest rose in developing agar-based semi-artificial diets, the twentieth century was marked with attempts on breeding b. mori on alternative plants, especially in the late-summer or autumn season for bivoltine breeds or in regions unsuitable for moriculture. lambert (1917), samokhvalova (1958, 1959, 1971), golanski (1959), samokhvalova et al. (1961, 1972), arzone & marletto (1975), manchev (1981), nasreen et al. (1999) and more recently el-shewy & elgizawy (2017) reported experiments with several alternative host plants. underreporting of silkworm strain specifications, geographical differences, and nonstandardized environmental conditions make systematization in most publications on the subject impossible. this study aims to organize existing literature on the experimental use of alternative host plants based on the reported outcomes. given the promising results regarding t. officinale, the study further focuses on comparing the biological traits and rearing performance of two feeding modalities. the study will investigate the impact of breeding season (temperature conditions and host plant quality) on rearing performance not focused on silk yield. two diets will be tested: the preferred host plant, m. alba, and a combination of m. alba with t. officinale, which is readily available and easily cultivated in laboratory settings. materials and methods to systematize previous attempts at silkworm rearing on non-mulberry natural diets, existing reports on experimental trials designed as comparisons between mulberry diet and alternative host plants were retrieved via google scholar search engine without publication time limitations. the performed queries included the following phrases: “bombyx mori alternative host plants” “silkworm 106 biologica nyssana ● 15 (2) december 2024: trajković et al. ● effects of dandelion partial replacement on some biological traits of the silkworm, bombyx mori l. (lepidoptera, bombycidae) biologica nyssana ● 15 (2) december 2024: trajković et al. ● effects of dandelion partial replacement on some biological traits of the silkworm, bombyx mori l. (lepidoptera, bombycidae) 107 alternative host plants” “non-mulberry silkworm diets”, and “silkworm adaptability”. afterwards, the publications were treated as separate research cases based on the author or group of authors and scored by the overall reported success of the rearing program. for the experiment, the egg batches were obtained from crea – aa (padua, italy), specifically double-cross (126x125) x (129x127) nr.1a, produced in june 2022, and stored at 4° c until the beginning of the trials. to investigate the impact of fluctuating temperatures during early, mid, and late season (roughly april, may, and june for univoltine breeds), the rearing was conducted at room temperature, which was recorded daily, at ld 16:8 photoperiodic conditions. fresh mulberry leaves were collected from a local variety tree, grown without the use of pesticides, within the garden of the faculty of sciences and mathematics, university of niš, serbia. dandelion plants were sourced from pristine environments during the entire season. in the beginning of each experiment, 300 newly hatched larvae were carefully collected using a fine brush and transferred into perforated plastic petri dishes (10 larvae per dish). they remained in the petri dishes until the first molt was completed to secure higher humidity favorable for young larvae (lakshminarayana et al., 2002). ad libitum feeding regiment comprising mulberry (the control), and a combination of mulberry and dandelion in a 3:7 ratio (the treatment) was introduced immediately post-hatching. from the second instar onwards, the caterpillars were reared in perforated plastic containers. prior to use, the leaves of both plants were disinfected and dried as described in ayandokun & alamu (2020). mortality was monitored daily and calculated as cumulative rate (total number of dead larvae in all instars per group). all successful l5 larvae were weighed before pupation. container cleaning was performed as required. the number of unsuccessful pupae (larval-pupal intermediates) was recorded for each group. upon completion of feeding, the caterpillars were transferred to wooden frames covered with muslin cloth to facilitate cocoon spinning. pupal duration and cocoon weight were recorded accordingly. data analysis was performed in r studio environment for statistical computing. as all four recorded traits displayed non-normality, to reveal effects of factors (season and diet) on continuous variables (larval duration, larval weight, pupal duration, pupal weight) was subjected to the non-parametric alternative, kruskal–wallis analysis, with dunn’s post hoc test for pairwise comparison. the relatedness between the abovementioned factors and categorical dependent variables (occurrence of intermediates and cumulative larval mortality) was conducted through pearson’s chi-squared test, followed by the bonferroni post hoc test in r studio environment. results and discussion the literature survey identified 22 alternative host plants. among these, scorzonera hispanica l., lactuca sativa l., and taraxacum officinale (l.) weber ex f. h. wigg. stood out due to the significant research interest they have attracted. this conclusion is based on the total number of authors and rearing attempts associated with these plants (tab. 1). table 1. non-mulberry bombyx mori host plants tested under laboratory conditions alternative host plant rearing performance references* bougainvillea glabra choisy partially successful el-shewy & elgizawy (2017) carpinus betulus l. failed golanski (1959) cichorium intybus l. failed lambert (1917) citrus medica l. failed nasreen et al. (1999) cudrania triloba (carrière) bureau successful lambert (1917) ficus religiosa l. successful nasreen et al. (1999) ficus retusa l. partially successful el-shewy & elgizawy (2017) lactuca sativa l. failed lambert (1917), golanski (1959), samokhvalova (1976), nasreen et al. (1999), el-shewy & elgizawy (2017) lantana camara l. partially successful el-shewy & elgizawy (2017) maclura pomifera (raf.) c.k. schneid. failed lambert (1917) 108 biologica nyssana ● 15 (2) december 2024: trajković et al. ● effects of dandelion partial replacement on some biological traits of the silkworm, bombyx mori l. (lepidoptera, bombycidae) for the experimental part of the study, dandelion was chosen as the most convenient alternative host plant due to its availability and ease of cultivation. however, previous attempts highlighted limitations, as dandelion was found suitable only for the first three instars, with stunted growth and developmental issues reported. to address this, the experimental design incorporated a partial substitution strategy, supplementing dandelion with a small amount of white mulberry. all described modalities were successful in terms of reaching the adult stage. overall, mean values recorded for larval duration, larval weight, pupal duration, and pupal weight, as well as the mortality rate and the percentage of unsuccessful pupae (tab. 2), indicate effects of temperature variations on the development of silkworm. lower temperatures of the early season (ranging from 19 °c to 22 °c) resulted in prolonged larval development, while higher temperatures, typical of the late season, together malus domestica (borkh.) borkh. failed nasreen et al. (1999) prenanthes alba l. failed lambert (1917) pyrus communis l. failed nasreen et al. (1999) scorzonera hispanica l. successful lambert (1917), golanski (1959), samokhvalova et al. (1961), samokhvalova (1959, 1976), arzone & marletto (1975) taraxacum officinale weber ex f. h. wigg. partially successful lambert (1917), golanski (1959), samokhvalova et al. (1961), samokhvalova (1971), manchev (1981), samokhvalova (1976) tilia sp. unknown samokhvalova (1976) tragopogon porrifolius l. successful lambert (1917) tussilago farfara l. unknown lambert (1917) ulmus parvifolia jacq. partially successful khnournuti et al. (1965) ulmus pumila l. partially successful lambert (1917), golanski (1959), samokhvalova (1976) urtica dioica l. failed lambert (1917), samokhvalova (1976) vitis vinifera l. failed nasreen et al. (1999) * – and references cited therein; partially successful – to either a certain lx, with reduced economic value, high mortality, or successful if combined with other alternative host plants; successful – in multiple generations, selection advised. table 2. biological traits recorded during different parts of the silkworm breeding season (mean ± sd) season diet regiment temp. range (˚c) larval stage (days) cumulative larval mortality (%) l5 weight (g) larval-pupal intermediates (%) pupal duration (days) cocoon weight (g) early ma 19-22 36.50±1.68 0.06 3.61±0.32 10.64 9.90±1.60 1.77±.92 com 37.52±1.71 0.06 3.65±0.33 12.77 10.51±1.75 1.71±.07 midma 19-24.5 30.60±1.44 0.02 3.73±0.31 8.16 9.07±1.53 1.80±.07 com 28.08±1.43 0.04 3.71±0.30 6.25 8.89±1.37 1.79±.07 late ma 21.5-32 25.92±0.80 0.08 2.92±0.34 17.39 7.86±1.22 0.70±.11 com 25.84±2.56 0.12 1.94±0.47 20.55 7.64±0.93 0.68±.17 ma – mulberry leaves diet, com – dandelion-mulberry diet, l5 – fifth larval instar biologica nyssana ● 15 (2) december 2024: trajković et al. ● effects of dandelion partial replacement on some biological traits of the silkworm, bombyx mori l. (lepidoptera, bombycidae) 109 with the large temperature fluctuations (ranging from 21.5 °c to 32 °c) led to accelerated cocooning and thus, much smaller caterpillars and cocoons. these findings align with most of the previous studies, which suggest that consistent optimal or slightly fluctuating temperatures complement the larval development (zambrano-gonzález et al., 2023). additionally, these observations are consistent with expectations considering the temperate european climate (hăbeanu et al., 2023), and the breed used for the experiments. the mean number of days spent in the larval stage, as reported in table 1, appears consistent based on the mean values for all seasons and feeding modalities. regarding the investigated seasons, the kruskal-wallis test revealed significant differences for larval duration (h=256.97, p<0.01), larval weight (h=192.29, p<0.01), pupal duration (h=28.41, p<0.01), and cocoon weight (h=107.12, p=0.03). subsequent dunn’s pairwise comparison indicated significant differences among all three season groups for each of the four continuous variables (adjusted p<0.01). however, pearson’s chi-squared test found no statistically significant difference between season and larval mortality (p=0.07), although a significant association was observed between season and the occurrence of larval-pupal intermediates (x-squared=7.67, p=0.02). further analysis using the bonferroni post hoc test demonstrated that the mid-season group significantly differed from both the early season and late season groups (p = 0.04), which can potentially be explained by the lower occurrence of intermediates during the mid-season period. kruskal–wallis test showed significant differences among groups based on feeding modality (h=72.77, p<0.01), which also reflects in the higher standard deviation of values on combined diet. however, further testing did not show statistically significant differences for larval weight, pupal duration, nor cocoon weight. chi-sqaure test revealed no statistical significance for the larval mortality nor the occurrence of larval–pupal intermediates. based on this study, it can be concluded that the mid-season (may), characterized by slightly fluctuating temperatures within specific ranges, provides optimal conditions for silkworm growth and development. in terms of the inspected biological traits, the treatment yields results comparable to those of the control, the mulberry diet. while the early season (april, as soon as the buds open) also provides satisfactory results, it is associated with a considerably higher mortality rate compared to midseason rearing. in contrast, late-season rearing (june) is marked by a general decrease in biomass and a noticeable difference in progress among feeding modalities. caterpillars fed a combined diet lag by approximately 30% in terms of larval weight during late season rearing. this could be explained by the decline in dandelion quality, potential inhibitory effects on metabolism, and stress induced by high temperatures (filippo et al., 2022). with europe reembracing both traditional sericulture and b. mori research (marzoli et al., 2022), investigating partial replacement of mulberry with dandelion explored in this study can be of practical value, especially for laboratories trying to establish or maintain insect stocks. the standard mulberry diet can be challenging due to its seasonal availability, vulnerability to frost, and logistical constraints. the presented findings suggest that integrating dandelion into silkworm diets could offer a viable alternative, particularly during periods when mulberry is scarce, and when the breeding goal is not primarily silk yield. as suggested in the previous studies, acceptability of non-mulberry hosts can be increased through selective breeding (kim et al., 2022). the growing interest in entomophagy, where silkworm stands out as the most promising candidate within the lepidoptera order (van der fels, 2015), will require exploring the impact of non-mulberry hosts on the silkworm nutritional value. although our study revealed satisfactory larval growth and viability on the altered diet, in respect to the most optimal breeding season, in-depth understanding of this host-plant–herbivore interaction will allow utilization of silkworm in more diverse contexts. acknowledgements. authors would like to thank to dr. silvia cappellozza (crea-aa, padua, italy) for providing b. mori eggs as well as her continuous support, and dr. andreja urbanek krajnc (university of maribor, slovenia) for her logistical assistance that enabled this study. we are grateful to all undergraduate and master students from the faculty of sciences and mathematics, department of biology and ecology, university of niš, who helped with the experiments. this study is founded by the ministry of science, technological development and innovation of the republic of serbia (contract no. 45103-65/2024-03/200124, 451-03-66/2024-03/200124). 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(lepidoptera, bombycidae) 111 stanković, s. et al.  potential candidates for biological control of the black... biologica nyssana 6 (1)  september 2015: 49-54 stanković, s. et al.  potential candidates for biological control of the black... 49 original article received: 08 july 2015 revised: 13 july 2015 accepted: 01 august 2015 potential candidates for biological control of the black bean aphid aphis fabae in serbia saša s. stanković*, marijana ilić milošević, vladimir žikić university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia * e-mail: sasasta@gmail.com abstract: stanković, s.s., ilić milošević, m., žikić, v.: potential candidates for biological control of the black bean aphid aphis fabae in serbia. biologica nyssana, 6 (1), september 2015: 49-54. the black bean aphid is widely spread aphid species in the palaearctic, known to attack over 1150 plant species. because some of the host plants are of great agricultural interest, aphis fabae represent a very important pest. we assembled all data concerning the presence of this pest and connected it in tritrophic associations. in the period of 24 years investigation on the territory of serbia it has been recorded in 107 trophic associations. in total there are 145 findings of a. fabae parasitized by 19 taxa of aphidiinae (brackonidae) from seven genera. the most suitable biocontrol agents for the black bean aphid are lysiphlebus fabarum, binodoxys angelicae, lipolesis gracilis and the introduced species lysiphlebus testaceipes. key words: aphis fabae, parasitoids, aphidiinae, tritrophic associations apstrakt: stanković, s.s., ilić milošević, m., žikić, v.: potencijalni kandidati za biološku kontrolu crne repine vaši aphis fabae u srbiji. biologica nyssana, 6 (1), septembar 2015: 49-54. crna repina vaš je rasprostranjena širom palearktika. ova biljna vaš je zabeležena na preko 1150 vrsta biljaka. s obzirom na to da su mnoge biljke domaćini veoma bitne sa agrikulturnog stanovišta, aphis fabae predstavlja veoma vašnu štetočinu u zasadima gajenih biljaka. prikupljeni su podaci o prisustvu crne repine vaši u periodu od 24 godine na teritoriji srbije, a rezultati su predstavljeni u vidu tritrofičkih asocijacija. tokom istaraživanog perioda zabeleženo je 107 asocijacija. ukupno je registrovano 145 nalaza a. fabae koju je parazitiralo ukupno 19 taksona potfamilije aphidiinae (braconidae) iz sedam rodova. najpogodnije vrste za biološku kontrolu crne repine vaši su lysiphlebus fabarum, binodoxys angelicae, lipolesis gracilis, a takođe i introdukovana vrsta lysiphlebus testaceipes. key words: aphis fabae, parasitoidi, aphidiinae, tritrofičke asocijacije introduction the black bean aphid, aphis fabae scopoli 1763 (aphididae) is one of the most common aphid in europe. it is notorious for attacking many crops, especially broad bean vicia faba (fabaceae) and beet beta vulgaris (amaranthaceae), but also many other host plants which are of agricultural importance. according to h o l m a n (2008) a. fabae has been reported on over 1150 host plants. this species has almost global distribution; it is especially common in temperate regions (b l a c k m a n & e a s t o p , 2008). regarding its wide plant host range it is very difficult to comprehend biology and ecology of this particular group. therefore a. fabae (sensu lato) has long been 6 (1) • september 2015: 49-54 biologica nyssana 6 (1)  september 2015: 49-54 stanković, s. et al.  potential candidates for biological control of the black... 50 considered as a species complex and there were many attempts to resolve this problem (m ü l l e r & s t e i n e r , 1986; t h i e m e & d i x o n , 1996; r a y m o n d et al., 2001). today there are five recognized taxa of the a. fabae complex, three of them considered on a subspecies level: a. fabae fabae scopoli 1763, a. fabae cirsiiacanthoidis scopoli 1763, a. fabae mordvilkoi börner & janisch 1922; while a. evonymi fabricius 1775 and a. solanella theobald 1914 have the species status (v a n e m d e n & h a r r i n g t o n , 2007). beside direct damages inflicted by probing and sucking plant juices, a. fabae is an important vector of many plant viruses among other aphid species. one of the most important viruses is byv (beet yellows virus) which causes a yellowing disease in beta vulgaris and some other plants, but also bmv (brome mosaic virus) which primarily infects plants from poaceae family and cause damages to wheat plants. traditionally, aphids are treated chemically to prevent damages on agricultural plants; however, nowadays the biological control concept is of the highest importance. biologically, aphid number is controlled by predators, such are coccinellids, some hoverfly larvae; parasites, such are entomopathogenic nematodes, fungi and bacteria; and also by parasitoids, where the most important are wasps from the family braconidae, precisely the subfamily aphidiinae which are exclusive parasitoids of aphids. many aphidiinae species are economically very important as biological control agents in agroecosystems, therefore it is of great importance to know their taxonomy, biology and ecology for their successful usage against aphids (h å g v a r & h o f s v a n g , 1991; v ö l k l & m a c k a u e r , 2000). one of the most common parasitoid of a. fabae is an aphidiine wasp lysiphlebus fabarum (marshall 1896) which has been frequently reared from a. fabae feeding on crops and other plants (v ö l k l & s t e c h m a n n , 1998). beside l. fabarum, the black bean aphid is attacked by many other aphidiines such are ephedrus plagiator (nees 1811), binodoxys angelicae (haliday 1833), praon abjectum (haliday 1833) and others (s t a r ý , 1970). along native and common parasitoid wasps, a. fabae is very frequently parasitized by lysiphlebus testaceipes (cresson 1880) which is now rather invasive species previously introduced into the mediterranean area for biological control of citrus aphids (s t a r ý et al., 1988; ž i k i ć et al., 2015). research on a. fabae on the territory of serbia has been conducted since 1952 (m i l e t i ć , 1952a, 1952b), also aphid parasitoids were studied intensively in serbia (v u k a s o v i ć , 1928; t o m a n o v i ć & b r a j k o v i ć , 2001; t o m a n o v i ć et al., 1998; k a v a l l i e r a t o s et al., 2004, 2010; p e t r o v i ć et al., 2009). one of the recent surveys of aphidiine parasitoids in serbia was done three years ago where several parasitoids were recorded to attack a. fabae (ž i k i ć et al., 2012). having in mind the economic importance of a. fabae in crop productions, as well as its natural enemies such is wasps from the family aphidiinae, the authors strived to present tritrophic associations of the black bean aphid and its aphidiine parasitoids, but as well the host plant species on which aphids were found. also, we think this paper will serve as a good starting point toward practical usage of afidiinae parasitoids for biological control of a. fabae in serbia and beyond. material and methods the material was collected in a period of 24 years (1990-2014) research and collecting throughout the territory of serbia. most of the material was collected during 2000s, although some of the samples date from 1990s (table 1). infested plants were cut and placed into plastic cups along with aphids, a muslin cloth were put on the top of the cups to prevent the exit of insects but allowing ventilation. such prepared plastic cups were kept in laboratory condition with the constant temperature of 23 °c, relative humidity of 65%, and the photoperiod (l:d) 16:8h. each plant was prepared and photographed for later identification following j o s i f o v i ć (1972) and s a r i ć & d i k l i ć (1986). live aphids were preserved in 90% ethyl-alcohol and identified, following nomenclature r e m a u d i è r e & r e m a u d i è r e (1997). parasitoid wasps were preserved as dry material but most of them were put in plastic tubes with 90% ethyl-alcohol. morphological terminology of parasitoids follows s h a r k e y & w h a r t o n (1997). some of the wasp specimens were dissected and mounted onto microscopic slides in order to facilitate identification on the species level. results here we present 145 findings of a. fabae on 50 plant taxa. eight plant specimens were identified to the generic level, while for one plant specimen we have data only to the family level (tab. 1). in total, a. fabae was parasitized by 19 aphidiinae taxa from seven genera; aphidius, binodoxys, ephedrus, lipolexis, lysiphlebus, praon and trioxys. twelve taxa were identified to the species level, and the remaining seven taxa were identified to the generic biologica nyssana 6 (1)  september 2015: 49-54 stanković, s. et al.  potential candidates for biological control of the black... 51 table 1. the list of aphis fabae findings with its host plants and parasitoids. legend: ♂– male; ♀ – female; (+) – over the presented number; xx – no data about number of specimens. legator abbreviations: ap – anđeljko petrović, mđ – maja đorđević, mm – majda mehanović, ss – saša stanković, vž – vladimir žikić, zk – zorana kojičić, žt – željko tomanović, x – no data about legator. plant parasitoid no of spec. locality, date, legator initials aegopodium podagraria praon sp. 2♂ 3♀ niš, trošarina; 11.06.2014; vž lysiphlebus testaceipes 4♂ niš, trošarina; 11.06.2014; vž amaranthus retroflexus lipolexis gracilis 1♀ lebane, konjino; 06.07.2010; ss lysiphlebus fabarum 17♀ 20♂ lebane, konjino; 06.07.2010; ss anthriscus sylvestris lysiphlebus fabarum 50+♀ tara, perućac; 25.06.2014; ss apiaceae lipolexis gracilis 3♀ 2♂ brzeće; 29.07.2010; vž arctium lappa binodoxys angelicae 50+♀ sićevačka klisura, ostrovica; 28.05.2013; mđ lipolexis gracilis 2♀ sićevačka klisura, ostrovica; 28.05.2013; mđ lysiphlebus cardui 100+♀♂ sićevačka klisura, ostrovica; 28.05.2013; mđ lysiphlebus cardui 2♀ 6♂ sićevačka klisura; 23.06.2012; ss lysiphlebus fabarum 50+♀ 1♂ sićevačka klisura; 23.06.2012; ss lysiphlebus cardui 1♂ tara, mitrovac; 25.06.2013; vž beta vulgaris lysiphlebus fabarum 13♀ 7♂ zemun; 27.09.1990; x bifora radians lysiphlebus testaceipes 6♀ 5♂ niš, trošarina; 22.05.2013; mđ calendula officinalis binodoxys angelicae 1♀ 2♂ kruševac; 18.06.2013; zk capsella bursa pastoris lysiphlebus fabarum 3♂ vlasina, čemernik; 04.08.2011; vž carduus acanthoides lysiphlebus fabarum 13♀ slankamen; 24.06.2011; x carduus sp. lysiphlebus fabarum 1♀ petnica; 12.06.2011; ap lysiphlebus fabarum 22♀ kragujevac, ilićevo; 06.06.2011; x centaurea cyanus lysiphlebus fabarum 13♀ 7♂ lebane, konjino; 31.05.2014; ss chenopodium album aphidius matricariae 1♀ niš, popovac; 22.05.2010; vž binodoxys acalephae 23♀ 14♂ niš, popovac; 22.05.2010; vž ephedrus plagiator 1♂ niš, popovac; 22.05.2010; vž lipolexis gracilis 11♀ niš, popovac; 22.05.2010; vž lysiphlebus fabarum 1♀ niš, popovac; 22.05.2010; vž lysiphlebus fabarum 18♀ 4♂ sićevačka klisura; 04.06.2011; vž binodoxys angelicae 1♀ sićevačka klisura; 04.06.2011; vž lysiphlebus cardui 18♀ 3♂ sićevačka klisura, sićevo; 17.07.2013; ss binodoxys angelicae 1♂ sićevačka klisura, sićevo; 17.07.2013; ss binodoxys angelicae 11♀ niš, popovac; 25.06.2010; vž lipolexis gracilis 9♀ 3♂ niš, popovac; 24.07.2010; vž lysiphlebus cardui 23♀ 11♂ sićevačka klisura, sićevo; 23.06.2012; vž lipolexis gracilis 2♀ 5♂ sićevačka klisura, sićevo; 23.06.2012; vž lysiphlebus fabarum 2♀ 3♂ sićevačka klisura; 06.06.2013; vž binodoxys angelicae 2♀ 8♂ sićevačka klisura; 06.06.2013; vž lysiphlebus orientalis 4♀ slankamen; 24.06.2011; x praon volucre 1♀ 1♂ slankamen; 24.06.2011; x lysiphlebus fabarum 3♀ lebane; 01.06.2013; ss lysiphlebus fabarum 21♀ 1♂ sićevačka klisura, sićevo; 17.07.2013; ss binodoxys sp. 1♂ sićevačka klisura, sićevo; 17.07.2013; ss lysiphlebus fabarum 2♀ surčin; 15.06.2010; ap lysiphlebus fabarum 20♀ zemun; 17.10.1991; x lysiphlebus fabarum 14♀ zemun; 15.06.2011; ap lysiphlebus orientalis 28♀ zemun, galenika; 07.06.2011; ap binodoxys angelicae 1♀ zemun, galenika; 07.06.2011; ap lysiphlebus testaceipes 21♀ 26♂ niš; 03.06.2014; ss lysiphlebus fabarum 1♀ prokuplje, bresničić; 15.06.2014; ss cichorium sp. lipolexis gracilis 12♀ 2♂ sićevačka klisura; 04.06.2011; ss binodoxys angelicae 2♀ sićevačka klisura; 04.06.2011; ss cirsium arvense lysiphlebus fabarum 116♀ 37♂ vlasina; 03.08.2011; ss lysiphlebus fabarum 83♀ 85♂ vlasina; 03.08.2011; ss lysiphlebus fabarum 15♀ 4♂ vlasina, čemernik; 07.08.2010; ss lysiphlebus fabarum 1♀ 14♂ brzeće; 29.07.2010; ss lipolexis gracilis 6♀ 4♂ brzeće; 29.07.2010; ss lysiphlebus fabarum 1♀ 2♂ merošina, oblačinsko jezero; 15.06.2014; ss lysiphlebus fabarum 22♀ 1♂ ostružnica; 06.07.2011; x lysiphlebus testaceipes 8♂ 9♀ niš; 25.05.2014; ss lysiphlebus confusus 7♂ 2♀ niš; 25.05.2014; ss biologica nyssana 6 (1)  september 2015: 49-54 stanković, s. et al.  potential candidates for biological control of the black... 52 cirsium arvense lysiphlebus fabarum 8♀ 4♂ vlasina; 21.07.2013; ss lysiphlebus fabarum 15♀ 3♂ vlasina, čemernik; 07.08.2010; ss lysiphlebus fabarum 2♀ vlasina; 15.06.2013; ss lysiphlebus cardui 66♀ 16♂ suva planina, bojanine vode; 11.07.2010; vž lysiphlebus fabarum 7♀ 1♂ vlasina; 11.08.2006; vž lysiphlebus fabarum 62♂ vlasina; 28.06.2012; vž lysiphlebus fabarum 27♀ 15♂ vlasina; 06.08.2010; vž binodoxys acalephae 3♀ zlatibor; 26.06.2013; ss cirsium eriophorum lipolexis gracilis 4♀ 2♂ dukat; 07.08.2011; ss cirsium palustre lysiphlebus fabarum 9♀ 24♂ goč; 13.07.2011; x cirsium sp. lysiphlebus fabarum 12♀ 21♂ kopaonik; 17.07.2013; ss coreopsis verticillata lysiphlebus fabarum xx zemun; 17.10.1991; žt coronilla varia lysiphlebus testaceipes 2♀ niš, pantelej; 10.07.2014; vž digitalis ambigua lysiphlebus fabarum 12♀ tara, derventa; 03.07.2012; vž lysiphlebus cardui 8♀ tara, derventa; 03.07.2012; vž eryngium campestre lipolexis gracilis 7♀ 5♂ jerma canyon; 19.07.2013; vž euonymus japonicus lysiphlebus fabarum 11♀ 12♂ lebane, konjino; 31.05.2014; ss euonymus europaeus trioxys sp. xx beograd, radmilovac; 21.04.1995; x binodoxys angelicae 5♂ obedska bara; 01.04.2001; x galium aparine lysiphlebus testaceipes 1♂ 2♀ niš, gornji matejevac; 25.05.2014; vž lysiphlebus fabarum 37♀ 9♂ niš, tvrdjava; 05.06.2013; mđ ephedrus sp. 30♀ sićevačka klisura, ravni do; 29.05.2014; vž lipolexis sp. 7♀ 4♂ sićevačka klisura, ravni do; 29.05.2014; vž lysiphlebus cardui 10♂ vlasina; 05.08.2011; vž binodoxys sp. 2♀ 1♂ niš, bubanj; 28.05.2014; ss lactuca serriola lysiphlebus cardui 89♂ vlasina; 05.08.2011; ss levisticum officinale lysiphlebus fabarum 7♀ užice; 14.08.1999; žt matricaria chamomilla lipolexis sp. 2♀ 4♂ grdelička klisura; 05.06.2014; ss lysiphlebus fabarum 4♀ 1♂ grdelička klisura; 05.06.2014; ss lysiphlebus cardui 3♀ sićevačka klisura, ostrovica; 28.05.2013; mđ binodoxys angelicae 3♀ 1♂ lebane; 01.06.2013; ss praon volucre 3♀ 1♂ lebane; 01.06.2013; ss lysiphlebus fabarum 6♀ 2♂ lebane; 01.06.2013; ss lysiphlebus testaceipes 4♀ 10♂ lebane; 01.06.2013; ss lysiphlebus testacepies 50+♀ lebane, konjino; 31.05.2014; ss lysiphlebus fabarum 50+♀ lebane, konjino; 31.05.2014; ss lysiphlebus confusus 50+♀ lebane, konjino; 31.05.2014; ss matricaria perforata lysiphlebus fabarum 88♀ 60♂ vlasina; 05.08.2011; ss musa sapientum lysiphlebus fabarum 21♀ 4♂ kruševac; 21.06.2012; zk oenanthe stenoloba lysiphlebus fabarum 75♀ 31♂ vlasina; 05.08.2011; ss onopordum acanthium lysiphlebus fabarum 2♀ niš, lalinac; 08.06.2013; ss orlaya grandiflora lysiphlebus testaceipes 4♀ niš, gornji matejevac; 25.05.2014; vž papaver dubium praon sp. 1♀ niš, gornji matejevac; 25.05.2014; vž papaver rhoeas binodoxys angelicae 1♀ 1♂ niš, tvrdjava; 05.06.2013; mđ lysiphlebus fabarum 3♂ niš, tvrdjava; 05.06.2013; mđ lysiphlebus orientalis 1♀ 2♂ niš, tvrdjava; 05.06.2013; mđ praon volucre 1♀ niš, tvrdjava; 05.06.2013; mđ lysiphlebus testaceipes 2♀ 3♂ niš, tvrdjava; 05.06.2013; mđ lysiphlebus testaceipes 3♀ 1♂ niš, gornji matejevac; 25.05.2014; vž papaver sp. binodoxys angelicae 3♀ beograd, botanička bašta; 06.12.2013; x lysiphlebus orientalis 4♀ beograd, botanička bašta; 06.12.2013; x praon abjectum 3♀ 2♂ beograd, botanička bašta; 06.12.2013; x pastinaca sativa lysiphlebus fabarum 3♀ 1♂ beograd, radmilovac; 22.08.1996; x binodoxys angelicae 1♀ vlasina; 03.08.2011; ss pastinaca sp. binodoxys angelicae 1♀ nova varoš, suljova česma; 07.07.2013; x praon sp. 3♀ nova varoš, suljova česma; 07.07.2013; x philadelphus coronarius aphidius matricariae 1♂ niš; 05.05.2009; vž aphidius sp. 2♂ niš; 07.05.2014; ss praon sp. 2♂ 2♀ niš; 07.05.2014; ss binodoxys sp. 3♀ 5♂ niš; 07.05.2014; ss lysiphlebus testaceipes 1♀ 1♂ niš; 07.05.2014; ss lysiphlebus sp. 1♂ niš; 25.05.2014; ss aphidius sp. 1♂ niš; 25.05.2014; ss praon sp. 2♂ 2♀ niš; 25.05.2014; ss lysiphlebus testaceipes 1♂ 1♀ niš; 20.05.2014; ss biologica nyssana 6 (1)  september 2015: 49-54 stanković, s. et al.  potential candidates for biological control of the black... 53 philadelphus coronarius binodoxys sp. 1♀ srb, niš; 20.05.2014; ss plantago lanceolata lysiphlebus fabarum 9♀ 19♂ vlasina; 05.08.2011; ss polygonum aviculare lysiphlebus fabarum 42♀ 23♂ zemun; 06.10.2012; ss ranunculus sp. binodoxys angelicae 10♀ 5♂ sićevačka klisura, ostrovica; 28.05.2013; mđ lysiphlebus cardui 1♀ sićevačka klisura, ostrovica; 28.05.2013; mđ rumex sp. lysiphlebus fabarum 40♀ padinska skela; 06.04.2009; x lysiphlebus testaceipes 1♀ lebane, konjino; 31.05.2014; ss lysiphlebus confusus 2♂ lebane, konjino; 31.05.2014; ss lysiphlebus fabarum 4♂ niš, trošarina; 22.05.2013; mđ lysiphlebus fabarum 10♀ tara, derventa; 03.07.2012; ss lysiphlebus cardui 6♀ tara, derventa; 03.07.2012; ss solanum nigrum binodoxys angelicae 29♀ 16♂ niš; 08.10.2010; vž lysiphlebus fabarum 1♀ niš; 08.10.2010; vž lysiphlebus fabarum 2♀ obedska bara; 15.06.1996; žt tamarix sp. lysiphlebus testaceipes 30+♀ niš, niška banja; 29.05.2014; vž torilis microcarpa ephedrus plagiator 1♀ dukat; 07.08.2011; ss urtica dioica lysiphlebus fabarum 7♀ 2♂ smederevo; 27.05.2011; x valeriana officinalis lysiphlebus fabarum 2♀ 5♂ vlasina; 21.07.2013; ss lysiphlebus testaceipes 1♀ 5♂ vlasina; 21.07.2013; ss yucca filamentosa lysiphlebus testaceipes 7♀ 2♂ niš; 02.07.2014; mm zea mays lysiphlebus fabarum 2♀ 1♂ beljanica, resavska pećina; 30.07.1996; x lysiphlebus cardui 10♀ 10♂ lebane, konjino; 24.07.2012; ss level (tab. 1). in this survey we found 107 tritrophic associations; plant/aphid/parasitoid. the results of this research are given in a table form where all listed plants were infested by a. fabae, and the very same species was parasitized by the listed aphidiinae taxa (tab. 1). although the fauna of aphidiinae parasitoids has been relatively well investigated in serbia, we want to highlight the three new associations of a. fabae and its parasitoids since the last checklist of aphidiinae in serbia (ž i k i ć et al., 2012). aphidius matricariae haliday 1834 is now newly recorded to parasitize a. fabae in serbia, two specimens, one male and one female were found on chenopodium album (chenopodiaceae) and philadelphus coronarius (hydrangiaceae) (tab. 1). also, two invasive species from the genus lysiphlebus are reported to attack black bean aphid. lysiphlebus orientalis stary & rakhshani 2010, which was originally described from the northeast china as a specialized parasitoid of the soybean aphid (aphis glycines matsumura 1917) was recently recorded on the territory of serbia where as a host, among other aphid species, was a. fabae (p e t r o v i ć et al., 2013). also, lysiphlebus testaceipes, known to attack many aphids primarily in the mediterranean area, in some recent researches was recognized as a very competitive introduced species in the european mainland as well as in serbia (ž i k i ć et al., 2015). sublimating the results of this investigation we found that the most suitable biological control agent of a. fabae is lysiphlebus fabarum, but also binodoxys angelicae, lipolesis gracilis foerster 1862 and the previously mentioned introduced species l. testaceipes. acknowledgements. the authors wish to express great thankfulness to dr željko tomanović and dr anđeljko petrović from the faculty of biology, university of belgrade, also to maja đorđević, zorana kojičić and majda mehanović for sample collection. this investigation was supported by grant iii 43001 (the ministry of science, education and technological development of the republic of serbia). references blackman, r.l., eastop, v.f. 2008: aphids on the world's herbaceous plants and shrubs. john wiley & sons. 1460p. hågvar, e.b., hofsvang, t. 1991: aphid parasitoids (hymenoptera, aphidiidae): biology, host selection and use in biological control. biocontrol news and information, 12 (1): 13-42. holman, j. 2008: host plant catalog of aphids. springer, netherlands. 1216p. josifović, m. 1972: flora sr srbije. srpska akademija nauka i umetnosti. beograd. kavallieratos, n.g., tomanović, ž., starý, p., 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biologica nyssana 12(1) 12 (1) september 2021: 01-10 doi: 10.5281/zenodo.5522951 new floristic data in north macedonia with a first record of allium melanantherum pančić original article aco teofilovski public enterprise nacionalni šumi, pero nakov bb, 1000 skopje, north macedonia acoteofilovski@hotmail.com (corresponding author) received: december 08, 2020 revised: february 10, 2021 accepted: february 17, 2021 abstract: the author discloses new data referring to chorology and habitats of 12 vascular plants in north macedonia of which the balkan endemic allium melanantherum pančić, recorded in the subalpine belt of belasica mt., is reported for the first time in the flora of the country. new locality or localities are presented for the following rare or doubtfully known taxa: asplenium cuneifolium viv., athyrium distentifolium opiz, centaurea scabiosa subsp. spinulosa (spreng.) arcang., chaerophyllum bulbosum l., crepis baldaccii halácsy, dioscorea balcanica košanin, epilobium alpestre (jacq.) krock., eriophorum vaginatum l., euphorbia peplus l., orchis quadripunctata cirillo ex ten. and thelypteris palustris schott. literature data (if present) regarding the chorology of each of the treated taxa in the country are also provided and presented. key words: allium melanantherum, flora, species, distribution, north macedonia apstract: novi floristički podaci za severnu makedoniju sa prvim nalazom vrste allium melanantherum pančić autor otkriva nove podatke vezane za horologiju i staništa 12 vaskularnih biljaka severne makedonije, od kojih je balkanski endemit allium melanantherum pančić, pronađen u subalpskom pojasu planine belasica, po prvi put zabeležen u flori zemlje. nov lokalitet ili lokaliteti su predstavljeni za sledeće retke ili malo poznate taksone: asplenium cuneifolium viv., athyrium distentifolium opiz, centaurea scabiosa subsp. spinulosa (spreng.) arcang., chaerophyllum bulbosum l., crepis baldaccii halácsy, dioscorea balcanica košanin, epilobium alpestre (jacq.) krock., eriophorum vaginatum l., euphorbia peplus l., orchis quadripunctata cirillo ex ten. i thelypteris palustris schott. literaturni podaci (ukoliko postoje) vezani za horologiju svakog obrađenog taksona u zemlji su takođe obezbeđeni i predstavljeni. ključne reči: allium melanantherum, flora, vrste, rasprostranjenje, severna makedonija introduction with more than 3000 species, the vascular flora of north macedonia is among the richest in europe, particularly in regards to its relatively small area of 25713 km2. owing to the researches lasting more than 17 decades, a large amount of floristic data is already published making the flora of this country relatively well studied. however, the chorology of some taxa and taxonomic groups as well as the floristic composition of some parts of the country are still insufficiently known and numerous important data are continuously published. during the enthusiastic field work and performing some applicative projects in the recent years, the author had an opportunity to conduct certain floristic researches in many regions of north macedonia. thereby, an abundant herbarium material belonging to various taxonomic groups was collected and studied. in this work new data concerning some selected taxa are presented aiming to improve the knowledge for their chorology and ecology in the country and in their overall range of distribution. © 2021 teofilovski. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 1 materials and methods during the field work appropriate plant specimens were collected and herbarized. they are stored in the private herbarium of the author, provided with labels containing data regarding the locality, habitat, size and condition of the population. appropriate photographs of live plants are also taken. the plants were identified according to the flora of the republic of macedonia (micevski, 1985, 2001, 2005), flora europaea (tutin & al., 1964-1980) and some other regional floras and monographic works. relevant literature was checked in order to provide the existing data for each of the treated taxa. maps of distribution of most of the presented taxa are also given. results and discussion allium melanantherum pančić (amaryllidaceae) (fig. 1) belasica mt.: sečena skala, subalpine meadow, silicate, 1700 m, 41°20’29.28”n, 22°53’18.94”e, 18.8.2018, leg. a. teofilovski & d. mandzukovski, det. a. teofilovski & z. nikolov. this is a first record of this species in north macedonia. only a small population of no more than 30 individuals was found, spreading on an area of ca. 200 m2. the species grows on a dry subalpine meadow on siliceous geological substrate, accompanied with the following herbaceous plants and shrubs: allium flavum l., asperula aristata l. f., euphorbia barrelieri savi, festuca sp., genista carinalis griseb., hypericum olympicum l., rosa sp., scabiosa triniifolia friv., vaccinium vitis-idaea l., etc. allium melanantherum is a rare balkan endemic plant, which, beside in north macedonia is distributed also in: kosovo part of šar mountain (šutman, gora), several localities in e & se serbia, c & sw parts of bulgaria, and nc & ne floristic regions of greece (see anačkov, 2009; assyov et al., 2012; dimopoulos et al., 2013). having in consideration such distribution, its finding on the territory of north macedonia is not a surprise. this allium is considered generally a high-mountain species of open habitats, although in bulgaria it occurs also in the medium mountain belt, between 900 and 2400 m, on meadows, forest clearings and shrubby 2 fig. 1. allium melanantherum: a inflorescence, b – bulb, c habitat (belasica mt., sečena skala, photo a. teofilovski), d distribution in north macedonia biologica nyssana ● 12 (1) september 2021: 1-10 teofilovski ● new floristic data in north macedonia with a first record of allium melanantherum pančić 3 places (stojanov et al., 1966). similarly, in serbia it grows on high mountain xerophytic or mesophytic meadows and in greece between 1500 and 1900 m, in meadows and rocky places (andersson, 1991; anačkov, 2009). the cited authors do not provide data on the geological substrate of the species habitat. description: bulbs 1-1-5 cm in diameter; outer tunics membranous, with fine, close, parallel fibres separating at the top. stem 13-40 cm. leaves 1-3, up to 20 cm x 2 mm, filiform, sheathing the lower ¼-½ of the stem; margins scabrid. spathe 2-valved; valves unequal, one 0.5-2 cm, the other 1-6 cm, persistent, longer than the pedicels, narrowly ovate or lanceolate at base, abruptly contracted above into a filiform appendage. umbel fastigiate, 3to 25-flowered, with a few bulbils; pedicels 1-2.5 mm, unequal. perianth cylindrical; segments 8-10 x 1-2 mm, pink, with red mid-vein, very narrowly oblong, obtuse. stamens included; filaments c. 6 mm; anthers blackish; pollen yellow. capsule 4 mm (stearn, 1980). allium melanantherum belongs to allium sect. codonoprasum reichenb. (stearn 1981, anačkov 2009). it is easy distinguishable from the other representatives of this section occurring in the balkan peninsula (a. carinatum l., a. flavum l., a. oleraceum l., a. paniculatum s. lato, etc.) by the longer (7-10 mm) and obtuse perigon segments, stamens included in the perigon and dark blue (almost blackish) anthers. asplenium cuneifolium viv. (aspleniaceae) (fig. 2) kavadarci: majdan village, between the road to kruša and majdan river, serpentine, 1020 m, 41°9’10.53”n, 21°55’56.89”e, 5.9.2014, 8.10.2014, leg, det. a. teofilovski. a serpentinophytic species of a. adiantum-nigrum complex with insufficiently known distribution in north macedonia. micevski (1985) in the flora of s.r. macedonia reported this species only from the serpentine area nw of skopje, thereby questioning the previous reports from dudica mt. (konjsko) (bornmüller, 1928) and belasica mt. (mokrievo) (stojanov 1921). recently, it was reported also from nidže mt. (dobro pole) (teofilovski, 2011). on the locality near majdan village this species occurs rather sparse on an area of several hectares, in pinus nigra forests and forest openings, on serpentine geological substrate. athyrium distentifolium opiz (athyriaceae) (fig. 3) šar mountain: otunje village, ginov kamen, near silicate rocks, 1632 m, 42°4’35.68”n, 20°59’19.73”e, 15.11.2017, leg, det. a. teofilovski; belasica mt.: tromegje, 1760 m, between large siliceous stones, 41°20’26.48”n, 22°55’34.06”e, 18.8.2018, leg. a. teofilovski & d. mandzukovski, det. a. teofilovski. a widely distributed species in the north hemisphere but very rare in the balkan peninsula biologica nyssana ● 12 (1) september 2021: 1-10 teofilovski ● new floristic data in north macedonia with a first record of allium melanantherum pančić fig. 2. asplenium cuneifolium: a (kavadarci, majdan village, photo a. teofilovski); b distribution in north macedonia, solid circles herbarium data of the author, rings – data from literature fig. 3. distribution of athyrium distentifolium in north macedonia, solid circles herbarium data of the author, rings – data from literature 4 (croatia, montenegro, romania, bulgaria, north macedonia) (christenhusz et raab-straube, 2013). in north macedonia following two localities were previously known, both of them referring also to siliceous substrate: jablanica mt. (vevčani) and pelister (golemo ezero) (micevski, 1985). the recorded population on belasica mt. is only 200 m distant from the state border with greece and thus the presence of this species could be expected also on the territory of this country. centaurea scabiosa subsp. spinulosa (spreng.) arcang. (asteraceae) (fig. 4) delčevo: 2.6 km ne of zvegor village, meadow, 1015 m, 41°58’30.18”n, 22°50’12.49”e, 26.6.2020, leg. a. teofilovski & d. mandzukovski, det. a. teofilovski. this subspecies was previously known only from the following localities in the northern part of the country: vodno mt. (drenkovski, 1969) and suva gora mt. (lukovica, sedlarevo) (teofilovski, 2011). the only other report of c. scabiosa l. refers to the vicinity of strumica (rudski, 1943), but it is unclear to which infraspecific taxon might it refer as another two subspecies [subsp. scabiosa and subsp. fritschii (hayek) hayek] also occur in this part of balkan peninsula (greuter, 2006 +; dimopoulos et al., 2013). chaerophyllum bulbosum l. (apiaceae) (fig. 5) kičevo: prostranje village, meadow, 940 m, 41°21’30.42”n, 20°59’43.92”e, 22.6.2020, leg, det. a. teofilovski; makedonski brod: near the road to suvodol, 550 m, 41°30’40.43”n, 21°13’45.09”e, 23.6.2020, leg, det. a. teofilovski; kratovo: nw-n of grizilevci village, roadside, 1000-1100 m, 42°2’49.31”n, 22°8’36.54”e, 24.6.2017, leg, det. a. teofilovski; vinica: osojnica river, alder forest, 785 m, 41°47’49.09”n, 22°40’33.59”e, 18.6.2019, leg, det. a. teofilovski; selska river, alder forest, 820 m, 41°47’4.82”n, 22°40’12.95”e, 18.6.2019, observ. a. teofilovski; pehčevo: near the road to ramna reka, 1010 m, 41°43’40.37”n, 22°55’28.46”e, 12.6.2019, leg, det. a. teofilovski; berovo: 2.8 km e of the dam, waste place, 1260 biologica nyssana ● 12 (1) september 2021: 1-10 teofilovski ● new floristic data in north macedonia with a first record of allium melanantherum pančić fig. 4. centaurea scabiosa subsp. spinulosa: a capitulas (delčevo, zvegor village, photo. a. teofilovski); b distribution in north macedonia, solid circles herbarium data of the author, rings – data from literature fig. 5. chaerophyllum bulbosum: a (kratovo, grizilevci village, photo. a. teoflovski); b distribution in north macedonia, solid circles herbarium data of the author, rings – data from literature m, 41°40’5.06”n, 22°55’38.89”e, 28.6.2020, leg, det. a. teofilovski; toward prevedena, dump place, 41°37’13.35”n, 22°50’47.92”e, 1020 m, 2.8.2020, leg, det. a. teofilovski. the following few chorological data regarding this species are given in the flora of the republic of macedonia: kriva palanka (urumov 1923), between tetovo and gostivar, and few localities in the lower part of radika river basin (volkovija, jovan bigorski, bituše) (micevski 2005). the new localities indicate a more frequent occurrence of this species, though the abundance of almost all documented populations is rather low. crepis baldaccii halácsy (asteraceae) (fig. 6) bistra mt.: izvor village, near the road to gorni lopušnik, limestone rocks, 1340 m, 41°30’3.25”n, 20°45’5.24”e, 29.7 & 13.8.2020, leg, det. a. teofilovski; jablanica mt.: strižak, 1865 m, limestone rocks, 41°17’43”n, 20°32’14”e, 14.9.2016. leg, det. a. teofilovski; vevčanska lokva, limestone rocks, 1835 m, 41°14’32.08”n, 20°32’21.55”e, 23.8.2016, leg, det. a. teofilovski; čuma, 1700-1950 m, limestone rocks, 41°13’32.71”n, 20°31’50.09”e, 19.7.2017, observ. a. teofilovski (photo.); labuniški bačila, 41°16’5.53”n, 20°32’5.47”e, limestone rocks, 1900 m, 17.8.2017, observ. a. teofilovski (photo.); podgorečki bačila, 1780 m, 17.8.2017, observ. a. teofilovski (photo.). a balkan endemic species, which geographical range, beside north macedonia, include the mountains of n, c & s albania, e kosovo and nw greece (gajić, 1975; barina et al., 2017; dimopoulos et al., 2013). in north macedonia, previously it was known only from the peak of crn kamen on jablanica mt. (černjavski, 1943, sub c. baldaccii subsp. albanica jáv.), while the report from šar mountain (ljuboten) (c. baldaccii subsp. albanica) (teofilovski, 2015) is incorrect, based on specimens actually belonging to the closely related c. macedonica kitanov. according to the author’s field observation, c. baldaccii is a frequent species in the subalpine belt of jablanica mt. and seems to be very rare on bistra mt. it grows in cervices of limestone rocks while near podgorečki bačila probably also on silicate ones. curiously, despite its relatively wide occurrence on macedonian part of jablanica mt., so far it is not reported from the part of this mountain belonging to albania (kitanov 1948, barina & al. 2017). within c. baldaccii two subspecies (besides the typical one) are so far described subsp. albanica jáv. from n albania and subsp. carpini greuter from nw greece (jávorka et al., 1926; greuter, 1975). however, having in consideration the variations of the morphologic characteristic within the recorded populations on jablanica and bistra mts., both subspecies seem cannot be easily distinguished from the typical c. baldaccii. similar comments are also noted by kamari (1991) in the treatment of the genus crepis l. in the “mountain flora of greece”. dioscorea balcanica košanin (dioscoreaceae) (fig. 7) jablanica mt.: lukovo village, above the road to debar, 710 m, 41°22’0.29”n, 20°36’10.64”e, 11.7.2016, leg, det. a. teofilovski; modrič village, near the road to debar, 41°22’21.86”n, 20°35’45.68”e, 620 m, 15.6.2020, leg. a. teofilovski & z. nikolov, det. a. teofilovski; drenok river gorge, 41°23’43.08”n, 20°34’43.46”e, 694 m, 16.6.2020, leg. a. 5 biologica nyssana ● 12 (1) september 2021: 1-10 teofilovski ● new floristic data in north macedonia with a first record of allium melanantherum pančić fig. 6. crepis baldaccii: a plant with details of fruits and capitula (bistra mt., road to gorni lopušnik, photo. a. teofilovski); b distribution in north macedonia, solid circles herbarium data of the author, rings – data from literature teofilovski & z. nikolov, det. a. teofilovski; globočica hydropower plant, near the road to debar, 41°23’59.53”n, 20°35’0.93”e, 595 m, 15.6.2020, leg. a. teofilovski & z. nikolov, det. a. teofilovski. in the flora of north macedonia this w balkan endemic plant was reported only from a single locality on jablanica mt. (lukovo village) (rizovski, 1977), which represents the southernmost point of the species range. after the publication of this first record, no additional data regarding the chorology or ecology of this species in the country has been disclosed. the new localities extends the known finding site near lukovo village for 5 km toward north, to drenok river and globočica hydropower plant. the species thrives usually on shady places in thermo-mesophylous deciduous forests and their margins, on carbonate geological substrate. at the bottom of the drenok river gorge, it occurs within a relic forest community in which the following species participate: acer campestre l., acer obtusatum willd., acer pseudoplatanus l., aesculus hyppocastanum l., fraxinus excelsior l., fraxinus ornus l., hedera helix l., juglans regia l. (juv.), ostrya carpinifolia scop., sambucus nigra l., etc. epilobium alpestre (jacq.) krock. (onagraceae) (fig. 8) stogovo mt.: 3.9 km nw of ehloec village, wet places near mountain stream, 1670 m, 41°26’6.54”n, 20°43’56.71”e, 30.7.2020, leg, det. a. teofilovski. this epilobium has a wide distribution in c & s europe and part of sw asia, but becoming rare toward southernmost parts of europe and the balkan peninsula. among the countries neighboring north macedonia it is known only from bulgaria (balkan mts., rila mt., vitoša mt.) and serbia (kopaonik mt., golija mt., zlatibor mt., rakoš, kosovo), while doubtfully present in albania and missing from the flora of greece (diklić, 1973; dimopoulos et al., 2013; barina & al., 2018; vladimirov, 2019). the only previous record of this species in north macedonia originates from the upper mountain belt of bistra mt. (careva češma) (micevski, 2001). eriophorum vaginatum l. (cyperaceae) (fig. 9) maleševo mountains: čengino kale, wet place, 41°43’15.09”n, 23°1’43.81”e, 1690 m, leg. a. teofilovski & d. mandzukovski, det. a. teofilovski. a wetland species with a wide eurasian distribution but quiet rare in the flora of north macedonia. it was previously known from a few rather old reports, lacking any recent confirmation: jakupica mt. [salakovo ezero (košanin, 1911), pepelak (bornmüller, 1928)] and šar mountain (lukovo pole) (horvat, 1953). 6 biologica nyssana ● 12 (1) september 2021: 1-10 teofilovski ● new floristic data in north macedonia with a first record of allium melanantherum pančić fig. 7. dioscorea balcanica, male specimen (jablanica mt., modrič village, photo a. teofilovski) fig. 8. epilobium alpestre: a (stogovo mt., photo. a. teofilovski); b distribution oin north macedonia, solid circle herbarium data of the author, ring – data from literature euphorbia peplus l. (euphorbiaceae) (fig. 10) kičevo: 0.7 km sw of the city center, near a fence and on a concrete pavement, 41°30’27.20”n, 20°57’17.68”e, 22.6.2020, leg, det. a. teofilovski; tetovo: blagoja toska str., in concrete planters and between concrete blocks, 42°0’48.23”n, 20°58’48.74”e, 28.9.2020, leg, det. a. teofilovski. in the floristic literature this rare annual spurge is quoted only from two localities in the vicinity of dojran [dub mt. (cirimotić, 1958), nikolić (čarni et al., 2014)] and an unspecified locality between veles and gevgelija (nikolovski & cirimotić, 1958). its occurrence was not confirmed in the flora of the republic of macedonia (micevski, 1998). the observed populations in the urban areas of kičevo and tetovo are rather small, inhabiting ruderal habitats associated with various concrete constructions. orchis quadripunctata cirillo ex ten. (orchidaceae) (fig. 11) makedonski brod: zdunje village, near the bridge, thermophilous forest, limestone, 595 m, 41°46’31.68”n, 21°9’17.08”e, 12.5.2016, leg, det. a. teofilovski. the recorded population which consists of a few tenths of individuals, grows on shallow soil in a sparse thermophilous forest of quercus trojana webb. this north-mediterranean species was known only from a single old report referring to the hilly area near radobil village (prilep) (bornmüller 1928). the new record confirms the poorly known presence of this orchid in the country flora, thereby, extending its general range of distribution more deeply toward the continental part of the balkan peninsula. among the countries neighboring north macedonia it occurs only in albania (frequent in the southern part and scattered elsewhere) and greece (in all floristic regions) (dimopulos et al., 2013; barina et al., 2017). thelypteris palustris schott (thelypteridaceae) (fig. 12) tetovo: jančiste village, alder forest, 400 m, 42°3’23.36”n, 21°7’12.13”e, 3.8.2018, leg, det. a. teofilovski; ohrid: belčišta village, sini viroj, alder forest, 41°18’51.65”n, 20°48’57.68”e, 23.6.2020, leg, det. a. teofilovski. a wetland fern distributed throughout much of the north hemisphere but only with a rare occurrence in the country. in the flora of the republic of 7 biologica nyssana ● 12 (1) september 2021: 1-10 teofilovski ● new floristic data in north macedonia with a first record of allium melanantherum pančić fig. 9. eriophorum vaginatum: a habitat with a detail of a basal part of inflorescence (maleševo mountains, čengino kale, photo. a. teofilovski); b distribution in north macedonia, solid circles herbarium data of the author, rings – data from literature fig. 10. distribution of euphorbia peplus in north macedonia, solid circles herbarium data of the author, rings – data from literature, ring in brackets – insufficiently specified locality from literature macedonia this species is reported from alder forests or wet habitats on three localities: strumica (bansko), gostivar (vranovci) and kožuf mt. (visoka čuka) (micevski, 1985). its actual presence on the cited localities needs to be checked as such habitats are frequently being disturbed by the adverse human activities. conclusions during the recent floristic researches new data regarding the chorology and habitats of 11 rare or poorly known plants and one species new for the country flora are recognized and presented in this work. the balkan endemic allium melanantherum is recorded for the first time in the flora of north macedonia, in the subalpine belt of belasica mt. (sečena skala). considering its occurrence in e & se serbia, c & sw parts of bulgaria, n greece and kosovo part of šar mountain, the finding on the territory of north macedonia was expected in some extend. another one balkan endemic crepis baldaccii, previously known only from an old literature report from jablanica mt. (crn vrv), is confirmed on several new localities of the same mountain with addition of a first record on bistra mt. a previous report from šar mountain (ljuboten) is revised and dismissed as an error. orchis quadripunctata, previously poorly known only from an almost one-century old literature data referring to radobil village (kavadarci), is confirmed for the country flora with its finding near zdunje village (makedonski brod). new locality or localities accompanied with data regarding the habitats, are added for eight rare plants which were previously known only from one or few localities: asplenium cuneifolium kavadarci (majdan village), athyrium distentifolium – šar mountain (otunje village), belasica mt. (tromegje), centaurea scabiosa subsp. spinulosa – delčevo (zvegor village), dioscorea balcanica jablanica mt. (modrič village, drenok river, globočica), epilobium alpestre stogovo mt. (ehloec village), eriophorum vaginatum – maleševo mountains (čengino kale), euphorbia peplus tetovo, kičevo, thelypteris palustris – tetovo (jančište village), ohrid (belčišta village). the new localities of chaerophyllum bulbosum from the vicinity of kratovo, pehčevo, berovo, kičevo and makedonski brod, indicate its wider distribution in north macedonia then it was previously considered. references anačkov, g. 2009: taksonomija i horologija roda allium l. 1754 (amaryllidales, alliaceae) u srbiji. phd thesis, pp. 1-253. univerzitet u novom sadu, prirodno-matematički fakultet. biologica nyssana ● 12 (1) september 2021: 1-10 teofilovski ● new floristic data in north macedonia with a first record of allium melanantherum pančić fig. 11. orchis quadripunctata: a flowers (makedonski brod, zdunje village, photo. a. teofilovski); b distribution in north macedonia, solid circle herbarium data of the author, ring – data from literature fig. 12. distribution of thelypteris palustris in north macedonia, solid circles herbarium data of the author, rings – data from literature 8 andersson, i.a. 1991: allium l. in: strid. a, tan, k. 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(eds.), new floristic records in the balkans: 40. phytologia balcanica, 25(3): 295–335. biologica nyssana ● 12 (1) september 2021: 1-10 teofilovski ● new floristic data in north macedonia with a first record of allium melanantherum pančić 10 atsatan and diler 2025, biologica nyssana 16(2) 16 (2) december 2025: doi: 10.46793/biolnyss.16.2.4a determination of regional changes in morphological characteristics of perch (perca fluviatilis) caught in different lakes of türkiye original article kamil atsatan isparta university of applied sciences, faculty of aquaculture, department of fisheries basic sciences, isparta, türkiye kamilatsatan@isparta.edu.tr (corresponding author) i̇brahim diler bursa technical university, faculty of engineering and natural sciences, department of bioengineering, bursa, türkiye received: july 15, 2025 revised: august 18, 2025 accepted: august 19, 2025 abstract: this study investigated the morphological traits, growth performance, and organosomatic parameters of perch (perca fluviatilis) populations collected from twelve lakes and reservoirs in türkiye. the morphometric measurements, growth indicators, and condition factors of the specimens were analysed in detail. the results revealed significant differences in morphometric and growth characteristics among populations. these variations are closely linked to environmental factors, including water temperature, habitat structure, food availability, and trophic interactions. populations from konya (altınapa), afyon (şeyitler), and i̇zmir-tahtalı exhibited superior growth performance and higher morphometric values, while individuals from adana (seyhan), ankara (çamlıdere), and kırşehir (hirfanlı) exhibited lower values. these results suggest that freshwater perch populations exhibit significant morphological and physiological plasticity in response to varying habitat conditions. these differences highlight the importance of considering regional environmental factors when developing stock management and conservation strategies for this species. key words: pearch, morphometric variation, growth performance, environmental factors, population differences apstrakt: određivanje regionalnih promena morfoloških karakteristika smuđa (perca fluviatilis) ulovljenog u različitim jezerima turske ova studija je ispitivala morfološke osobine, performanse rasta i organosomatske parametre populacija štuke (perca fluviatilis) prikupljenih iz dvanaest jezera i akumulacija u turskoj. morfometrijska merenja, pokazatelji rasta i faktori kondicije ispitanih jedinki detaljno su analizirani. rezultati su pokazali značajne razlike u morfometrijskim i karakteristikama rasta između populacija. ove varijacije su usko povezane sa faktorima životne sredine, uključujući temperaturu vode, strukturu staništa, dostupnost hrane i trofičke interakcije. populacije iz konje (altınapa), afjona (şeyitler) i izmira-tahtalı pokazale su bolje performanse rasta i veće morfometrijske vrednosti, dok su jedinke iz adane (seyhan), ankare (çamlıdere) i kırşehira (hirfanlı) imale niže vrednosti. ovi rezultati sugerišu da populacije slatkovodne štuke pokazuju značajnu morfološku i fiziološku plastičnost kao odgovor na različite uslove staništa. ove razlike naglašavaju značaj uzimanja u obzir regionalnih faktora životne sredine pri razvoju strategija upravljanja populacijom i zaštite ove vrste. ključne reči: štuka, morfometrijske varijacije, performanse rasta, faktori životne sredine, razlike između populacija introduction the perch (perca fluviatilis l.) is a medium-sized fish that belongs to the percidae family (mcdowall, 1996). it was first described from swedish lakes in 1730 by peter artedi, and later formally classified by carl linnaeus in 1758, based on artedi’s studies (thorpe, 1977; pimakhin et al., 2015). the percidae family comprises 10 genera and 195 species (berra, 2001). according to fossil records, the genus perca is estimated to have emerged approximately 19.8 million years ago, during the early miocene epoch (stepien et al., 2015). this genus includes three recognized species: © 2025 atsatan and diler. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. • perca fluviatilis (linnaeus, 1758), • perca flavescens (mitchill, 1814; commonly known as the yellow perch, widely distributed in north america), • perca schrenckii (kessler, 1874; commonly known as the balkhash perch, with a restricted distribution in lakes of central asia). the body of the perch is laterally compressed and covered with oval-shaped ctenoid scales. the lateral line arches upward, terminating before reaching the caudal fin. the anterior dorsal region appears hump-backed. the preoperculum exhibits a series of serrations, while the postoperculum terminates in a pronounced spine-like projection. the mouth is large, bearing sharp teeth on both the palatine and vomer bones. the head length is approximately one-fourth of the total body length. although the interorbital region of the skull is scaleless, the cheeks are covered with ctenoid scales. the dorsal fins are located close together. the eyes are relatively large, positioned near the top of the head, and their diameter is about one-fifth to one-sixth of the head length. the first dorsal fin gradually decreases in height from anterior to posterior. the anal fin originates slightly behind the second dorsal fin. the caudal fin is slightly emarginate, with rounded lobes. the general body coloration is brownish-green, with 6–8 dark green vertical bars along the flanks. a distinct black blotch is present on the posterior part of the first dorsal fin. the ventral, anal, and caudal fins are typically orange-red, while the other fins are colorless. the body length may reach up to 50 cm. local and regional environmental conditions influence both the body size and morphology of perch populations (persson, 1987; olsson et al., 2007; kerstin & petersson, 2023). temperature, in particular, is a significant factor affecting the size of the perch. in warmer lakes, where perch populations tend to be denser, the average individual size is usually smaller due to increased competition for limited resources (kerstin & petersson, 2023). in this study, the morphological characteristics, growth performance, and organosomatic parameters of perch individuals captured from natural water bodies in different regions of türkiye were thoroughly investigated. the primary aim of the research was to identify the morphological variations of perch populations inhabiting distinct geographical regions, compare their growth performance, and evaluate their physiological status based on organosomatic indices. materials and methods materials the fish specimens used in this study were collected from the following water bodies: darıderesi dam (isparta), yedikır dam lake (amasya), ürkmez dam (i̇zmir), tahtalı dam (i̇zmir), seyhan dam (adana), şeyitler dam (afyon), denizli pond (kocaeli), karaağaç pond (uşak), hirfanlı dam (kırşehir), çamlıdere dam (ankara), altınapa dam (konya), and ladik lake (samsun). the fish were captured using gillnets with a mesh size of 4 mm. a total of 300 specimens were analyzed, with 25 fish collected from each sampling location. all specimens were obtained utilizing gillnet fishing. methods determination of morphological characteristics the morphological characteristics of the perch were measured according to the method described by pravdin et al. (1966) (fig. 1). biologica nyssana ● 16 (2) december 2025: atsatan and diler ● determination of regional changes in morphological characteristics of perch (perca fluviatilis) caught in different lakes of türkiye fig. 1. measurement diagram of the perch: ab–total body length, an–snout length, np–horizontal eye diameter, po–postorbital head length, ao–head length, lm–head height, gh–maximum body height, ik– minimum body height, aq–distance to the first dorsal fin, fd–caudal peduncle length, tu–first dorsal fin height, t1u1–second dorsal fin height, yy1–anal fin base length, ej–anal fin height biologica nyssana ● 16 (2) december 2025: atsatan and diler ● determination of regional changes in morphological characteristics of perch (perca fluviatilis) caught in different lakes of türkiye growth performance and organosomatic parameters the perch specimens used in this study were captured during their natural spawning season, from december to february. it was determined that approximately 95% of the captured individuals were females, and all biometric and biochemical parameters analyzed in the study were evaluated based on the females. this approach minimized the gender-related variations, especially when assessing reproductive-related biometric data. hepatosomatic index (hsi): the hepatosomatic index (hsi) value was calculated as the percentage of the fish’s liver weight (g) to its body weight (g), using the following formula (hoşsu et al., 2001): hsi=(liver weight (g)/total body weight (g))×100 renosomatic index (rsi): the renosomatic index (rsi) value was calculated as the percentage ratio of the fish’s kidney weight (g) to its body weight (g), using the following formula (silva & anderson, 1995; hoşsu et al., 2001): rsi=(kidney weight (g)/total body weight (g))×100 viscerosomatic index (vsi): the viscerosomatic index (vsi) value was calculated as the percentage ratio of the fish’s internal organ weight (g) to its body weight (g), using the following formula (hoşsu et al., 2001): vsi=(internal organ weight (g)/total body weight (g))×100 carcass yield: carcass yield was calculated as the weight of the fish after removing its internal organs divided by its total body weight, expressed as a percentage: carcass yield=(weight of fish after removing internal organs/total body weight)×100 gonadosomatic index (gsi): the gonadosomatic index (gsi) value was calculated as the percentage ratio of the gonad weight (g) to the total body weight (g), using the following formula: gsi=(gonad weight (g)/total body weight (g))×100 statistical analyses a total of 300 fish were used to determine the morphological and growth parameters, with five repetitions and five fish per repetition. the obtained data were subjected to analysis of variance using the minitab software. significant differences among the means were determined using tukey’s multiple comparison test (p≤0.05) and are presented with different letters. results and discussion morphological characteristics the morphological characteristics of perch specimens collected from various dams and lakes located in different geographical regions of türkiye are presented in tab. 1. statistical evaluations revealed that all morphometric characters differed significantly among the studied populations (p<0.05). these findings highlight the influence of environmental conditions and habitat differences on the morphological variations of the species. regarding total length (ab), the highest mean value was recorded in specimens collected from tahtalı dam (i̇zmir) with 263.92 mm, while the lowest mean total length was observed in specimens from darıderesi dam (isparta) with 113.32 mm. for head morphology measurements, including head length (ao), postorbital length (po), and anterior snout length (an), the specimens collected from i̇zmir and konya exhibited the highest values, whereas those from isparta and uşak had notably lower measurements. in terms of maximum body depth (gh), a significant similarity was observed among the specimens from i̇zmir tahtalı dam (83.96 mm), konya altınapa dam (81.52 mm), and afyon şeyitler dam (80.64 mm). the lowest value was found in the specimens from isparta darıderesi dam (35.44 mm). measurements related to fin morphology such as first dorsal fin height (tu), second dorsal fin height (t₁u₁), anal fin base length (yy₁), and anal fin height (ej) also showed distinct variations among different localities. specimens from i̇zmir and konya had longer dorsal and anal fins, while those from isparta and uşak displayed the lowest values. for horizontal eye diameter (np), the maximum value of 11.00 mm was recorded in specimens from afyon, i̇zmir, and konya. in contrast, the lowest measurement of 4.76 mm was observed in the specimens from isparta. growth performance and organosomatic parameters the growth parameters of the perch specimens collected from various dams and lakes across different geographical regions of türkiye are presented in tab. 2. significant differences were observed among individuals in terms of live body weight. the highest mean body weight was recorded in the konya population (264.20 g), followed by afyon (220.20 g), i̇zmir-tahtalı (212.10 g), and samsun (197.20 g) populations. the lowest mean body weights were biologica nyssana ● 16 (2) december 2025: atsatan and diler ● determination of regional changes in morphological characteristics of perch (perca fluviatilis) caught in different lakes of türkiye ta bl e 1. m or ph ol og ic al c ha ra ct er is tic s of p er ch c ol le ct ed fr om d iff er en t l oc at io ns d am s a nd l ak es ab an np po ao lm gh a d a n a (s ey ha n d am ) 14 7. 12 ±6 .5 5e 9. 96 ±1 .0 2f 6. 00 ±0 .0 0e 18 .6 8± 0. 69 e 30 .4 0± 2. 76 e 32 .8 4± 0. 37 g 46 .6 0± 3. 75 e a fy o n (ş ey itl er d am ) 25 4. 44 ±1 1. 03 a 17 .3 6± 0. 70 b 11 .0 0± 0. 00 a 31 .9 6± 1. 02 a 52 .2 0± 3. 90 a 54 .8 4± 0. 37 b 80 .6 4± 6. 36 a a m a sy a (y ed ik ır d am l ak e) 21 5. 40 ±9 .7 0c 14 .4 8± 0. 51 d 9. 00 ±0 .0 0c 26 .9 6± 1. 02 c 44 .4 8± 3. 29 bc 46 .8 4± 0. 37 d 68 .4 8± 5. 38 b c a n k a r a (ç am lıd er e d am ) 14 0. 04 ±6 .4 1ef 9. 48 ±0 .5 1fg 6. 00 ±0 0e 17 .6 8± 0. 69 ef 29 .0 0± 2. 32 e 30 .8 4± 0. 37 h 44 .4 8± 3. 52 e is pa r t a (d ar ıd er es i d am ) 11 3. 32 ±5 .9 8g 7. 40 ±0 .5 0h 4. 76 ±0 .5 2f 13 .7 6± 1. 30 g 22 .8 8± 2. 60 f 24 .0 4± 1. 64 j 35 .4 4± 4. 06 f i̇z m i̇r (t ah ta lı d am ) 26 3. 92 ±1 1. 75 a 18 .3 6± 0. 70 a 11 .0 0± 0. 00 a 32 .9 6± 1. 02 a 54 .2 4± 4. 00 a 56 .8 4± 0. 37 a 83 .9 6± 6. 75 a i̇z m i̇r (ü rk m ez d am ) 22 8. 52 ±1 0. 21 b 15 .4 4± 0. 82 c 10 .0 0± 0. 00 b 28 .8 0± 1. 08 b 46 .9 6± 3. 48 b 49 .3 6± 0. 75 c 72 .8 0± 5. 94 b k ir şe h i̇r (h ir fa nl ı d am ) 14 6± 15 .1 6e 9. 52 ±1 .0 8fg 6. 00 ±0 .5 7e 17 .9 2± 1. 90 ef 29 .3 2± 3. 81 e 30 .8 8± 3. 16 h 45 .7 2± 6. 37 e k o c a e l i̇ (d en iz li po nd ) 16 8. 16 ±1 7. 34 d 11 .1 2± 1. 09 e 6. 84 ±0 .6 8d 20 .7 2± 2. 28 d 34 .0 0± 4. 31 d 35 .8 0± 3. 61 f 52 .5 6± 7. 45 d k o n y a (a ltı na pa d am ) 26 0. 68 ±1 3. 93 a 17 .3 2± 0. 90 b 10 .7 2± 0. 67 a 32 .0 4± 2. 82 a 52 .6 0± 6. 04 a 55 .4 0± 4. 01 ab 81 .5 2± 9. 18 a sa m su n (l ad ik p on d) 20 7. 76 ±9 .3 c 14 .3 6± 0. 70 d 9. 00 ±0 .0 0c 25 .9 2± 0. 90 c 42 .6 8± 3. 11 c 44 .8 4± 0. 37 e 66 .2 0± 5. 29 c u şa k (k ar aa ğa ç po nd ) 13 5. 16 ±1 3. 97 f 9. 08 ±0 .9 0g 5. 84 ±0 .6 8e 16 .5 6± 1. 80 f 27 .3 2± 3. 55 e 28 .8 0± 2. 88 ı 42 .0 8± 5. 82 e d am s a nd l ak es ik aq fd tu t₁u ₁ yy ₁ ej a d a n a (s ey ha n d am ) 15 .4 4± 0. 82 e 32 .2 4± 2. 04 e 18 .1 6± 0. 98 e 26 .3 6± 2. 48 de 14 .4 4± 1. 41 cd 21 .7 6± 1. 20 e 13 .5 2± 1. 29 de a fy o n (ş ey itl er d am ) 26 .3 2± 1. 06 a 54 .7 2± 2. 92 a 30 .9 2± 1. 68 a 45 .6 0± 4. 10 a 24 .4 8± 2. 38 a 37 .6 8± 2. 21 a 22 .7 6± 2. 58 a observed in the ankara (109.20 g), adana (121.50 g), and uşak (119.39 g) populations. regarding liver weight, the highest values were found in konya specimens (3.36 g), followed by afyon specimens (2.83 g) and i̇zmir-tahtalı specimens (2.61 g). the lowest liver weights were recorded in the populations of ankara (1.47 g), adana (1.63 g), and isparta (1.50 g). these c on tin ua tio n of th e ta bl e 1 biologica nyssana ● 16 (2) december 2025: atsatan and diler ● determination of regional changes in morphological characteristics of perch (perca fluviatilis) caught in different lakes of türkiye *d iff er en ce s be tw ee n m ea ns in di ca te d by s ep ar at e le tte rs a re s ig ni fic an t ( p≤ 0. 05 ) ab –t ot al b od y le ng th , an –s no ut le ng th , np –h or iz on ta l e ye d ia m et er , po –p os to rb ita l h ea d le ng th , ao –h ea d le ng th , lm –h ea d he ig ht , gh – m ax im um b od y he ig ht , i k– m in im um b od y he ig ht , a q– di st an ce to th e fir st d or sa l fi n, fd –c au da l p ed un cl e le ng th , t u– fir st d or sa l fi n he ig ht , t 1u 1– se co nd d or sa l fi n he ig ht , y y 1– an al fi n ba se le ng th , e j– an al fi n he ig ht a m a sy a (y ed ik ır d am l ak e) 22 .3 2± 1. 06 bc 46 .6 8± 2. 44 bc 26 .2 0± 1. 32 bc 38 .8 0± 3. 52 bc 20 .8 4± 1. 99 b 32 .0 8± 1. 82 bc 19 .3 6± 2. 19 bc a n k a r a (ç am lıd er e d am ) 14 .4 4± 0. 82 ef 30 .4 8± 1. 66 ef 17 .1 6± 0. 98 ef 25 .3 2± 2. 37 6e 13 .4 4± 1. 41 d 20 .7 6± 1. 20 ef 12 .5 2± 1. 29 e is pa r t a (d ar ıd er es i d am ) 11 .2 8± 1. 02 g 24 .3 2± 2. 35 g 13 .4 4± 1. 29 g 19 .8 0± 2. 88 f 10 .8 4± 1. 40 e 16 .8 8± 1. 42 g 10 .2 8± 1. 17 f i̇z m i̇r (t ah ta lı d am ) 27 .3 2± 1. 06 a 56 .7 2± 2. 92 a 31 .9 2± 1. 68 a 47 .6 0± 4. 10 a 25 .4 8± 2. 38 a 39 .3 2± 2. 61 a 23 .7 6± 2. 58 a i̇z m i̇r (ü rk m ez d am ) 23 .4 4± 0. 82 b 49 .2 4± 2. 77 b 27 .7 2± 1. 62 b 41 .1 6± 3. 61 b 22 .0 4± 1. 85 b 33 .9 2± 2. 08 b 20 .6 4± 2. 03 b k ir şe h i̇r (h ir fa nl ı d am ) 14 .4 8± 1. 91 ef 31 .3 2± 3. 95 ef 17 .5 6± 1. 75 ef 25 .6 0± 3. 90 e 13 .9 6± 2. 40 d 21 .6 0± 2. 19 ef 13 .2 4± 1. 60 e k o c a e l i̇ (d en iz li po nd ) 17 .2 4± 2. 20 d 36 .0 0± 4. 48 d 20 .1 2± 2. 18 d 29 .4 0± 4. 44 d 16 .2 0± 2. 73 c 24 .9 6± 2. 45 d 15 .3 6± 2. 03 d k o n y a (a ltı na pa d am ) 26 .3 2± 2. 28 a 55 .7 6± 5. 33 a 31 .2 4± 2. 77 a 45 .6 ±6 .5 6a 25 .0 0± 3. 24 a 38 .6 0± 3. 16 a 23 .6 4± 2. 70 a sa m su n (l ad ik p on d) 21 .4 4± 0. 82 c 44 .7 2± 2. 39 c 25 .1 6± 1. 21 c 37 .4 4± 3. 15 c 20 .0 4± 1. 85 b 30 .9 2± 2. 08 c 18 .6 4± 2. 03 c u şa k (k ar aa ğa ç po nd ) 13 .4 4± 1. 75 f 29 ±3 .6 1f 16 .0 8± 1. 70 f 23 .6 0± 3. 70 e 12 .9 6± 2. 20 d 19 .8 0± 1. 97 f 12 .2 8± 1. 51 e differences reflect variations in energy reserves and metabolic conditions among the populations. the gonad weight, an important indicator of reproductive capacity, was highest in konya (30.78 g), i̇zmir-tahtalı (26.45 g), and afyon (26.41 g) specimens. the lowest values were recorded in the populations of adana (3.51 g), ankara (3.66 g), and kırşehir (3.76 g). these findings suggest that reproductive timing and maturation stages may vary among populations. for kidney weight, the highest averages were noted in konya (1.07 g), followed by afyon (0.96 g) and i̇zmir-tahtalı (0.87 g), while the lowest value was observed in the kırşehir population (0.42 g). the highest visceral weight was recorded in the konya population (35.70 g), followed by the afyon population (30.43 g) and the i̇zmir-tahtalı population (29.45 g). the lowest visceral weight was found in specimens from ankara (17.10 g). in terms of carcass weight, the highest value was observed in konya (228.50 g), with afyon (189.80 g) and i̇zmir-tahtalı (182.60 g) populations also showing relatively high carcass weights. the lowest carcass weights were recorded in ankara (92.09 g) and adana (102.90 g) specimens. the hepatosomatic index (hsi) showed limited variation among populations. the highest hsi values were found in amasya (1.47%), followed by adana and ankara (both 1.35%), while the lowest values were recorded in the i̇zmir-tahtalı and ürkmez populations (1.26%). the renosomatic index (rsi) showed relatively low intraspecific variation. the highest rsi was biologica nyssana ● 16 (2) december 2025: atsatan and diler ● determination of regional changes in morphological characteristics of perch (perca fluviatilis) caught in different lakes of türkiye ta bl e 2. g ro w th p er fo rm an ce a nd o rg an os om at ic p ar am et er s of p er ch s co lle ct ed fr om d iff er en t l oc at io ns d am s a nd l ak es w e ig h t (g ) l iv e r (g ) g o n a d w e ig h t (g ) k id n e y w e ig h t (g ) w e ig h t o f in t e r n a l o r g a n s (g ) c a r c a se w e ig h t (g ) h si̇ (% ) a d a n a (s ey ha n d am ) 12 1. 50 ±4 0. 20 de 1. 63 ±0 .4 9cd e 3. 51 ±0 .7 7c 0. 50 ±0 .0 55 de 18 .5 8± 4. 82 de 10 2. 90 ±3 5. 40 de 1. 35 ±0 .0 3 a fy o n (ş ey itl er d am ) 22 0. 20 ±2 3. 40 ab 2. 83 ±0 .2 8ab 26 .4 1± 2. 76 ab 0. 96 ±0 .0 2ab 30 .4 3± 2. 81 ab 18 9. 80 ±2 0. 60 ab 1. 28 ±0 .0 0 a m a sy a (y ed ik ır d am l ak e) 16 6. 70 ±2 6. 90 bc de 2. 42 ±0 .2 0ab cd e 20 .1 0± 3. 13 b 0. 72 ±0 .0 9bc d 24 .0 1± 3. 22 bc de 14 2. 70 ±2 3. 60 bc de 1. 47 ±0 .2 1 a n k a r a (ç am lıd er e d am ) 10 9. 20 ±1 2. 94 e 1. 47 ±0 .1 5e 3. 66 ±0 .6 0c 0. 47 ±0 .0 5de 17 .1 0± 1. 55 e 92 .0 9± 11 .3 9e 1. 35 ±0 .0 1 is pa r t a (d ar ıd er es i d am ) 11 7. 76 ±5 .4 0de 1. 50 ±0 .2 2de 5. 84 ±0 .9 6c 0. 61 ±0 .1 8cd e 18 .1 3± 0. 64 de 99 .6 3± 4. 75 de 1. 27 ±0 .1 3 i̇z m i̇r (t ah ta lı d am ) 21 2. 10 ±4 2. 50 ab 2. 61 ±0 .1 3ab c 26 .4 5± 4. 48 ab 0. 87 ±0 .0 4ab c 29 .4 5± 5. 10 ab 18 2. 60 ±3 7. 40 ab 1. 26 ±0 .2 i̇z m i̇r (ü rk m ez d am ) 17 6. 87 ±1 6. 43 bc d 2. 27 8± 0. 79 bc de 18 .8 4± 5. 36 b 0. 72 ±0 .0 7bc d 25 .2 2± 1. 97 bc d 15 1. 64 ±1 4. 46 bc d 1. 26 ±0 .3 6 k ir şe h i̇r (h ir fa nl ı d am ) 13 5. 51 ±7 .4 7cd e 1. 8± 0. 09 bc de 3. 76 ±0 .5 48 c 0. 42 ±0 .0 5e 20 .2 6± 0. 89 cd e 11 5. 25 ±6 .5 7cd e 1. 32 ±0 .0 0 k o c a e l i̇ (d en iz li l ak e) 18 0. 69 ±9 .9 5bc d 2. 35 ±0 .1 2ab cd e 21 .7 5± 1. 17 b 0. 70 ±0 .1 8bc de 25 .6 8± 1. 19 bc d 15 5± 8. 76 bc d 1. 30 ±0 .0 0 k o n y a (a ltı na pa d am ) 26 4. 20 ±2 4. 30 a 3. 36 ±0 .2 9a 30 .7 8± 30 a 1. 07 ±0 .1 2a 35 .7 0± 2. 92 a 22 8. 50 ±2 1. 40 a 1. 27 ±0 .0 0 sa m su n (l ad ik l ak e) 19 7. 20 ±1 7. 60 ab c 2. 54 8± 0. 21 ab cd 22 .3 8± 2. 26 b 0. 76 ±0 .0 4bc d 27 .6 6± 2. 12 ab c 16 9. 51 ±1 5. 51 ab c 1. 29 ±0 .0 0 u şa k (k ar aa ğa ç l ak e) 11 9. 39 ±1 1. 0de 1. 58 ±0 .5 3cd e 4. 40 ±0 .3 4c 0. 51 ±0 .1 1de 18 .3 2± 1. 33 de 10 1. 06 ±9 .7 6de 1. 31 ±0 .3 5 and reservoirs located in various geographical regions of türkiye were compared. the morphometric features of freshwater perch are closely related to the environmental conditions of their habitats. poulet et al. (2004) investigated the morphometric and genetic variations among zander (sander lucioperca) populations inhabiting different subbasins of the rhône delta. they reported significant morphometric differences between populations collected from distinct deltaic regions, particularly in traits such as body depth, head length, and caudal peduncle measurements. the authors suggested that such morphological variations, shaped by environmental adaptations, may lead to adaptive divergence among populations in conjunction with genetic differentiation. in the present study, our findings confirm that the morphometric characteristics of freshwater perch individuals are directly influenced by the environmental conditions of their habitats, consistent with previous literature. specifically, individuals biologica nyssana ● 16 (2) december 2025: atsatan and diler ● determination of regional changes in morphological characteristics of perch (perca fluviatilis) caught in different lakes of türkiye d am s a nd l ak es r si̇ (% ) v si̇ (% ) c a r c a ss y ie l d (% ) g si (% ) a d a n a (s ey ha n d am ) 0. 43 ±0 .1 2 15 .5 1± 1. 00 ab 84 .4 8± 1. 00 ef 3. 02 ±0 .8 8b a fy o n (ş ey itl er d am ) 0. 44 ±0 .0 3 13 .8 3± 0. 20 ef 86 .1 6± 0. 20 ab 11 .9 9± 0. 02 a a m a sy a (y ed ik ır d am l ak e) 0. 44 ±0 .1 2 14 .4 4± 0. 42 bc de f 85 .5 5± 0. 42 ab cd e 12 .0 9± 1. 01 a a n k a r a (ç am lıd er e d am ) 0. 43 ±0 .0 5 15 .6 9± 0. 41 a 84 .3 0± 0. 41 f 3. 36 ±0 .5 5b is pa r t a (d ar ıd er es i d am ) 0. 52 ±0 .1 7 15 .4 0± 0. 16 ab c 84 .5 9± 0. 16 de f 4. 98 ±0 .9 8b i̇z m i̇r (t ah ta lı d am ) 0. 42 ±0 .1 0 13 .9 4± 0. 44 ef 86 .0 5± 0. 44 ab 12 .5 7± 1. 18 a i̇z m i̇r (ü rk m ez d am ) 0. 40 ±0 .0 2 14 .2 7± 0. 22 cd ef 85 .7 2± 0. 22 ab cd 10 .5 2± 2. 16 0a k ir şe h i̇r (h ir fa nl ı d am ) 0. 31 ±0 .0 3 14 .9 57 9± 0. 16 ab cd e 85 .0 4± 0. 16 bc de f 2. 77 ±0 .3 2b k o c a e l i̇ (d en iz li l ak e) 0. 39 ±0 .1 1 14 .2 1± 0. 12 de f 85 .7 8± 0. 12 ab c 12 .0 4± 0. 01 a k o n y a (a ltı na pa d am ) 0. 40 ±0 .0 3 13 .5 2± 0. 13 8f 86 .4 7± 0. 13 a 11 .7 7± 2. 10 a sa m su n (l ad ik l ak e) 0. 38 ±0 .0 2 14 .0 4± 0. 19 ef 85 .9 6± 0. 19 ab 11 .3 8± 1. 19 a u şa k (k ar aa ğa ç l ak e) 0. 42 ±0 .0 5 15 .3 7± 0. 32 ab cd 84 .6 2± 0. 32 cd ef 3. 69 ±0 .2 2b c on tin ua tio n of th e ta bl e 2 *d iff er en ce s be tw ee n m ea ns in di ca te d by s ep ar at e le tte rs a re s ig ni fic an t ( p≤ 0. 05 ). h ep at oso m at ic in de x (h si ), r en os om at ic in de x (r si ), vi sc er os om at ic in de x (v si ), g on ad os om at ic in de x (g si ) observed in isparta (0.52%), and the lowest in kırşehir (0.31%). the highest viscerosomatic index (vsi) was recorded in the ankara population (15.69%), followed by adana (15.51%) and isparta (15.40%), while the lowest vsi was observed in konya (13.52%). regarding carcass yield, the highest values were recorded in the konya population (86.47%), followed by the afyon (86.16%) and i̇zmir-tahtalı (86.05%) populations. the lowest carcass yield was observed in adana (84.48%) specimens. for the gonadosomatic index (gsi), the highest values were observed in i̇zmir-tahtalı (12.57%), amasya (12.09%), afyon (11.99%), and konya (11.77%) populations, while the lowest values were recorded in kırşehir (2.77%), adana (3.02%), and ankara (3.36%) populations. in this study, the morphological, genetic, and physiological characteristics of freshwater perch (perca fluviatilis) populations collected from lakes collected from konya, afyon, and i̇zmir-tahtalı exhibited superior growth indicators, including body weight, liver and gonad weights, and carcass yield, whereas those from adana, ankara, and kırşehir displayed lower values. these differences offer significant insights into how environmental factors, including food availability, water quality, temperature, and pollution, impact growth performance. couture and rajotte (2003) examined parameters like length-weight relationships, liver and gonadosomatic indices (lsi and gsi), and condition factors (k) in perca flavescens collected from three lakes in the sudbury region with varying levels of metal contamination. they observed that individuals from highly polluted lakes exhibited notably lower condition factors, higher liver somatic indices, lower gonadosomatic indices, and deviations in length-weight relationships. the authors reported growth retardation in some individuals from polluted environments, which they attributed to environmental stress. mairesse et al. (2005) noted that cultured fish tend to have shorter and deeper bodies compared to wild individuals, which display longer and slimmer body shapes. overton et al. (2008) emphasized the direct influence of environmental factors on fish growth. in this context, the statistically significant differences observed in head morphology parameters such as head length, postorbital length, and anterior snout length among individuals from different locations in our study further highlight the role of habitat heterogeneity in shaping growth performance. our findings are also supported by kocovsky et al. (2013), who examined morphometric traits such as standard length, total length, head length, body depth, and caudal peduncle thickness in p. flavescens populations from the great lakes region of north america and found that these characteristics varied significantly according to geographic origin. they suggested that these differences could be linked to adaptations to environmental factors such as food sources, water flow, and predation pressure. these observations align with the variations detected among freshwater perch populations from different regions of türkiye in our study. rowinski et al. (2015) investigated the potential effects of climate change on fish morphology by examining the impact of elevated water temperatures on the body shape of freshwater perch. they compared populations from long-term thermal discharge areas (nuclear power plant effluents) with those from natural thermal conditions in sweden. their findings indicated that individuals from warmer environments had shorter bodies, larger heads, and shorter caudal peduncles compared to those from natural habitats. these results offer important evidence on how temperature regimes and thus metabolic activity can influence morphological traits, especially in ecologically diverse countries like türkiye, supporting the differences observed in our study. baranov (2021) explored the effects of different habitats (river vs. lake) on the morphology of freshwater perch in the loz’va river and surrounding mountainous and semi-mountainous regions in russia. he reported distinct morphometric profiles among individuals from flowing river environments characterized by slimmer and longer bodies compared to those from stagnant lake environments, which exhibited shorter body shapes. terentjev and berezina (2022) conducted a comparative study on the ecological characteristics, morphological structure, and feeding habits of freshwater perch in dystrophic and oligotrophic lakes in northern karelia during the autumn and winter periods. they found that individuals from dystrophic lakes exhibited smaller body sizes and slower growth rates, whereas those from oligotrophic lakes had larger body morphologies and higher condition factors. faulks et al. (2015) examined the genetic and morphological differentiation of freshwater perch and rutilus rutilus populations inhabiting littoral (shoreline) and pelagic (open-water) habitats. they observed that pelagic individuals had longer, narrower bodies and larger eyes, features advantageous for swimming speed and prey detection in open water. in contrast, littoral individuals exhibited deeper body shapes and shorter head-to-body ratios. the authors emphasized that these morphological differences could result from ecological factors, phenotypic plasticity, and possible adaptive evolutionary processes. in the present study, disparities in parameters such as total length, head length, and body proportions among individuals further substantiate the impact of environmental factors, including water temperature, trophic structure, and oxygen levels, on morphological traits. these findings corroborate previously reported evidence of habitat-driven morphological plasticity. the highest morphometric values were generally recorded in individuals from reservoirs in western and central anatolia, particularly i̇zmir-tahtalı, konya-altınapa, and afyon-şeyitler. these results clearly indicate that regional environmental factors and habitat characteristics significantly influence the morphological traits of freshwater perch. evaluation of growth parameters revealed distinct biometric and physiological differences among populations. konya (altınapa), afyon (şeyitler), and i̇zmir-tahtalı populations stood out biologica nyssana ● 16 (2) december 2025: atsatan and diler ● determination of regional changes in morphological characteristics of perch (perca fluviatilis) caught in different lakes of türkiye biologica nyssana ● 16 (2) december 2025: atsatan and diler ● determination of regional changes in morphological characteristics of perch (perca fluviatilis) caught in different lakes of türkiye with higher live and carcass weights, well-developed gonads, and high carcass yields, suggesting superior growth performance and reproductive potential. in contrast, individuals from adana (seyhan) and ankara (çamlıdere) exhibited lower growth and reproductive parameters, further highlighting the influence of environmental conditions on physiological development. overall, the findings demonstrate significant differences in biological productivity among freshwater perch populations from different regions, emphasizing the need to consider these variations in management and conservation strategies. conclusion in this study, detailed analyses were conducted on the morphological measurements, growth parameters, and condition factors of perca fluviatilis specimens collected from twelve different lakes and reservoirs located in various geographical regions of türkiye. the evaluation revealed distinct morphometric differences among populations, indicating that these variations are shaped by the environmental conditions of their respective habitats. specifically, factors such as water temperature, food availability and quality, habitat structure, trophic interactions, and competition intensity were found to exert dominant and directional effects on morphology and growth performance. specimens obtained from the altınapa dam (konya), şeyitler dam (afyon), and tahtalı dam (i̇zmir) displayed superior performance in both morphometric traits and growth parameters, exhibiting higher body weight, liver and gonad weights, and increased live and carcass yield. conversely, individuals from locations such as the seyhan dam (adana), çamlıdere dam (ankara), and seyfe lake (kırşehir) demonstrated lower morphometric values and growth performance. these findings suggest that environmental stressors such as temperature fluctuations, poor water quality, pollutants, or limited food resources may adversely affect the physiological development and biological productivity of populations. furthermore, head morphology traits (head length, postorbital length, and anterior snout length) and body ratios varied significantly in relation to habitat heterogeneity, with differences likely driven by factors such as trophic level, water temperature, and oxygen regimes. overall, the results demonstrated that freshwater perch populations in türkiye exhibit a high degree of morphological and physiological plasticity in response to environmental conditions. this plasticity represents a critical factor for understanding natural population dynamics and should be carefully considered in the development of management and conservation strategies. in conclusion, this study revealed significant differences in growth performance, morphological structure, and condition factors among freshwater perch populations from different habitats, highlighting the complex and multifaceted influence of environmental factors on these traits. references baranov, v.y. (2021). variability in body shape of the perch perca fluviatilis linnaeus, 1758 in mountain and semimountain reaches of the loz’va river and adjacent lakes (northern urals). contemporary problems of ecology, 14, 27–36. https://doi.org/10.1134/s1995425521010029 berra, t.m. (2001). freshwater fish distribution. san diego: academic press. couture, p., & rajotte, j.w. (2003). morphometric and metabolic indicators of metal stress in wild yellow perch (perca flavescens) from sudbury, ontario: a review. journal of environmental monitoring, 5(2), 216–221. faulks, l., svanbäck, r., eklöv, p., & östman, ö. (2015). genetic and morphological divergence along the littoral–pelagic axis in two common and sympatric fishes: perch, perca fluviatilis (percidae) and roach, rutilus rutilus (cyprinidae). biological journal of the linnean society, 114(4), 929–940. https://doi.org/10.1111/bij.12452 hoşsu, b., korkut, a. y., & firat, a. (2001). fish nutrition and feed technology i. ege university faculty of fisheries publications, 50, 295. kerstin, h., & petersson, e. (2023). are perch (perca fluviatilis l.) getting larger or smaller in swedish lakes? ecology of freshwater fish, 32(4), 735–749. https://doi.org/10.1111/eff.12719 kocovsky, p.m., sullivan, t.j., knight, c.t., & stepien, c.a. (2013). genetic and morphometric differences demonstrate fine‐scale population substructure of the yellow perch perca flavescens: need for redefined management units. journal of fish biology, 82(6), 2015–2030. https://doi. org/10.1111/jfb.12129 mairesse, g., thomas, m., gardeur, j.n., & brun-bellut, j. (2005). appearance and technological characteristics in wild and reared eurasian perch, perca fluviatilis (l.). aquaculture, 246(1–4), 295–311. https://doi.org/10.1016/j. aquaculture.2004.09.023 biologica nyssana ● 16 (2) december 2025: atsatan and diler ● determination of regional changes in morphological characteristics of perch (perca fluviatilis) caught in different lakes of türkiye mcdowall, r.m. (ed.). (1996). freshwater fishes of south-eastern australia (2nd ed.). sydney: reed books. olsson, j., svanbäck, r., & eklöv, p. (2007). effects of resource level and habitat type on behavioral and morphological plasticity in eurasian perch. oecologia, 152, 48–56. https://doi.org/10.1007/ s00442-006-0588-8 overton, j.l., bayley, m., paulsen, h., & wang, t. (2008). salinity tolerance of cultured eurasian perch, perca fluviatilis l.: effects on growth and on survival as a function of temperature. aquaculture, 277(3–4), 282–286. https://doi.org/10.1016/j. aquaculture.2008.02.029 persson, l. (1987). the effects of resource availability and distribution on size class interactions in perch, perca fluviatilis. oikos, 148–160. pimakhin, a., kouřil, j., stejskal, v., & žák, j. (2015). the effect of geographical origin of perch (perca fluviatilis l. 1758) populations on growth rates under natural and aquaculture conditions: a review. journal of applied ichthyology, 31, 56–63. https://doi.org/10.1111/jai.12901 poulet, n., berrebi, p., crivelli, a.j., lek, s., & argillier, c. (2004). genetic and morphometric variations in the pikeperch (sander lucioperca l.) of a fragmented delta. archiv für hydrobiologie, 159(4), 531–554. https://doi.org/10.1127/00039136/2004/0159-0531 pravdin, i.f. (1966). guide to the study of fish. moscow: food industry publishing house. rowinski, p. k., mateos‐gonzalez, f., sandblom, e., jutfelt, f., ekström, a., & sundström, l. f. (2015). warming alters the body shape of european perch perca fluviatilis. journal of fish biology, 87(5), 1234–1247. https://doi.org/10.1111/jfb.12785 de silva, s.s., & anderson, t.a. (1994). fish nutrition in aquaculture. springer netherlands. https://doi.org/10.1007/978-94-011-0623-5 stepien, c.a., behrmann-godel, j., & bernatchez, l. (2015). evolutionary relationships, population genetics, and ecological and genomic adaptations of perch (perca). in biology of perch (pp. 7–46). terentjev, p.m., & berezina, n.a. (2022). ecological and morphological characteristics and feeding of perch (perca fluviatilis) in the autumn– winter period in dystrophic and oligotrophic lakes of northern karelia (russia). inland water biology, 15(6), 915–928. https://doi.org/10.1134/ s1995082922060177 thorpe, j.e. (1977). morphology, physiology, behavior, and ecology of perca fluviatilis l. and p. flavescens mitchill. journal of the fisheries board of canada, 34(10), 1504–1514. https://doi. org/10.1139/f77-215 plants and insects in interactions: multitrophic associations 75 reviev article received: 31 october 2016 revised: 12 november 2016 accepted: 24 november 2016 plants and insects in interactions: multitrophic associations vladimir žikić, marijana ilić milošević, maja lazarević*, saša s. stanković university of niš, faculty of science and mathematics, department of biology and ecology, niš, serbia * e-mail: majalazarevic9@gmail.com abstract: žikić, v., ilić milošević, m., lazarević, m., stanković, s.s.: plants and insects in interactions: multitrophic associations. biologica nyssana, 7 (2), december 2016: 75-82. studying a biological species, we are very often obliged to examine many other species which are directly or indirectly related to the particular species in order to have broader perspective of its being. interactions between organisms can be perceived on various levels concerning biological study areas, such as ecology, physiology, morphology etc. in this place, the authors have given some peculiar examples concerning parasitism. this specific interaction is very common in the animal kingdom, having in mind that about a half of all known organisms are parasitic. the importance of this phenomenon is reflected on many aspects of life, propelling evolution and speciation of each member in the relation. key words: parasitism, parasitoids, hosts, defending strategies apstrakt: žikić, v., ilić milošević, m., lazarević, m., stanković, s.s.: interakcije biljaka i insekata: multitrofičke asocijacije. biologica nyssana, 7 (2), decembar 2016: 75-82. proučavajući određene biološke vrste često smo primorani da izučavamo i mnoge druge koje su direktno ili indirektno povezane sa njom u cilju šireg sagledavanja njene celokupne biologije. interakcije između organizama mogu biti proučavane na različitim biološkim nivoima, kao što su ekologija, fiziologija, morfologija itd. u ovom slučaju, autori su prezentovali neobične primere parazitizma. ova specifična interakcija je veoma česta u životinjskom carstvu, imajući u vidu da skoro polovina poznatih organizama pripada parazitima. značaj ovog fenomena se ogleda u mnogim aspektima života, podstičući evoluciju i specijaciju svakog člana posmatrane interakcije. ključne reči: parazitizam, parazitoidi, domaćini, odbrambene strategije introduction the vast majority of plants have evolved along with insects. a lot of insect species directly depend on plants which represent a resource for the whole, or at least a part of insects’ life cycle. in this trophic association plants can offer roots, bulbs, trunks, branches, leaves, flowers, fruits and seed. even dead plants or their parts can be of use for decomposing insects. other connections between plants and insects imply symbiosis or mutualism. a specific relationship between plants and most insects is pollination; this particular act can be performed by hymenopterans, butterflies and moths, 7 (2) • december 2016: 75-82 12th sfses • 16-19 june 2016, kopaonik mt doi: 10.5281/zenodo.200402 biologica nyssana 7 (2)  december 2016: 75-82 žikić, v. et al.  plants and insects in interactions… 76 horned beetles, many flies etc., of which a great number represent very specialized forms as far as their morphology or behaviour is concerned (b r o n s t e i n et al., 2006). for example, moths from the family sphingidae are associated with plants whose flowers have deep calyx forming tubes (f a e g r i & v a n d e r p i j l , 1979; h a b e r & f r a n k i e , 1989). hovering around the plants these moths suck nectar through a very elongated proboscis. this type of feeding is not unique in the animal world; convergent evolution has appeared four times in non-related nectar feeders. in mammals, bats are a very good example, while among birds there are hummingbirds, and also two groups of insects: hoverflies (diptera: syrphidae) and hawk moths (k i t c h i n g , 2002). the list of attended plants is huge, ranging from those with small flowers with short calyx such as verbena l., lantana l., lobelia l. and buddleja l., over petunia juss. or nicotiana l., and furthermore to plants with very large flowers with deep calyx such as various species of datura l. or darwin orchid, angraecum sesquipedale thouars with a calyx which is 30 cm deep. otherwise, the group of solitary bees (megachilidae), e.g., eucera longicornis (l.), some digger wasps (crabronidae) and some mining bees (andrenidae) specialize in the pollination of various orchids imitating the females; in the first place from the genus ophrys l. (w i l l m e r , 2011). for many snapdragon species the only solution of being pollinated was to establish an intimate relation with some bumblebees which are capable to open the “lips” of the flowers using their strong hind legs (e.g., o d e l l et al., 1999). myrmecochory is a special kind of ant-plant interaction where ants are engaged in seed dispersal. for this purpose myrmecochorous plants shaped their seeds to carry elaiosomes, various external appendages rich of nutrients such as lipids, amino acid, or other attractive substances to ants (g i l a d i , 2006). some stick insects (phasmida) have taken advantage of this ant activity by producing seed-like eggs which are carried by ants into their nests where the conditions are favourable for their development (c o m p t o n & w a r e , 1991; s t a n t o n et al., 2015). in central america there is an example of true symbiosis between ants and plants. the acacia ant – pseudomyrmex ferruginea f. smith, lives in small colonies in the base of the horns of the bullhorn acacia – vachellia cornigera (l.) finding a shelter there, but at the same time offering protection to its host (j a n z e n , 1967; p i p e r , 2007) as ants attack all approaching animals and plants by stinging them. a specific way of insect parasitism takes place in galls. galls or cecidia are transformations of plant parts (external tissues) such as leaves, branches, roots, petals or flowers, caused by insect or mite activities. plant tissues show abnormal outgrowths like tumours in animals. the most important gallinducing insects are gall wasps, cynipidae (hymenoptera), gall midges cecidomyiidae (diptera), gall aphids aphididae (hemiptera) and some species from the group of leaf-miner flies, agromyzidae (diptera) (e.g., s p e n c e r , 1973; s t o n e et al., 2002; y u k a w a & r o h f r i t s c h , 2005; f l o a t e , 2010). galls and cecidia arise after females inject cecidogenous fluid, along with egg, in the plant tissue, e.g. tenthredinidae (hymenoptera) (k o p e l k e , 1998). afterwards, parasite larvae force the neighbouring plant cells into gall to start the division process of meristems. parasites use the galls as their habitat and shelter; also exploiting the sugars accumulated in it, they also use the galls as a food source (l a r s o n & w h i t h a m , 1991). a very bizarre relationship between plants and insects is found in carnivorous plants such as drosera l., nepenthes l., sarracenia l. or dionaea muscipula ellis that “consume” insects. leaves of those mainly bog inhabiting plants form traps, usually pitfalls, filled with water and enzymes (phytotelmata). all kind of traps in carnivorous plants are supplied with attractive colour, odour and specialized nectary glands. those glands produce sweet smelling nectar to attract and catch insects later digesting them, using externally excreted enzymes. in this way, plants obtain minerals from insect’s body, since the boggy soil is poor. once those plants have to be pollinated, they need insects again. then, carnivorous plants suspend all the attractants in their traps, focusing on their flower equipment in order to attract pollinators. this convergent behaviour evolved independently in both dicotyle and monocotyle plant lineages, terrestrial and underwater plants (a l b e r t et al., 1992). plants, hosts and parasitoids predators, parasites and parasitoids have an indirect connection with plants via their herbivorous pray or host which is essential for their life cycles (p r i c e et al., 1980; s t o t z et al., 1999). in the case of insect parasitoids, the host is usually required during the larval stage. the plant role in this play is to unintentionally help the host to become less attractive or even poisonous for its enemies. the six-spot burnet, zygaena filipendulae l. is capable of sequestering alkaloids from the food plant lotus corniculatus l. in this cases the cyanogenic glucoside linamarin, also lotaustralin are biosynthesized in poison thus decreasing predation of larvae and adults (d a v i s & n a h r s t e d t , 1979; z a g r o b e l n y et al., 2007). this poison protects a biologica nyssana 7 (2)  december 2016: 75-82 žikić, v. et al.  plants and insects in interactions… 77 very high percentage of adult moths while larvae still remain exposed for various parasitoids such as various hymenopterans and tachinids (tachinidae: diptera) (ž i k i ć et al., 2013). the great majority of parasitoid insects belong to the hymenoptera insect order. with some exceptions, they come from the division parasitica. parasitoid members of the other division aculeata, are in minority and their kind of parasitism is a little different (e.g., chrysididae, sphecidae, etc.) (w c i s l o et al., 1985; w i n t e r h a g e n , 2015). the second important group of parasitoids are tachinid flies (feener & brown, 1997); also, there are some sporadic cases in coleoptera, lepidoptera, trichoptera, neuroptera and strepsiptera (p i e r c e , 1995; h e r a t y , 2009). a simple association between a plant (primary food producer), an herbivorous host (primary food consumer) and a parasitoid (secondary food consumer) is being considered through trophic chains; this threesome is usually referred to as a tritrophic association. higher trophic levels with more members include more than one parasitoid species and also hyperparasitoids primary food consumer forming a multitrophic association. parasitoid larvae are carnivorous, feeding outside or inside the host body. conversely, adults are free living organisms feeding on flowers (nectar) or honeydew produced by green aphids. the final effect of host-parasitoid interaction is death of the host, which makes these organisms very powerful biological weapon. nowadays, several companies have developed technologies for commercial production of a few dozen parasitoid species on artificial nutrient medium. those species are applicable both in open fields as well as in greenhouses (o l k o w s k i et al., 2003; h a l e & h e n s l e y , 2010). there are various classifications of parasitoids based on different criteria concerning their behaviour, life cycle or number of eggs laid per single host. on the basis of oviposition location parasitoids can be ectoor endoparasitoids; whether or not parasitoid larvae alter hosts behaviour they could be idiobionts or koinobionts accordingly; according to the number of eggs laid per host there are two cases, solitary (a single egg per host) or gregarious (usually few to more than a hundred eggs laid per host) (g o d f r a y , 1994). ectoparasitoids lay their eggs on the host body surface. if the female causes a permanent paralysis of the host and parasitoid larvae feed outside the host body, those species represent idiobionts (many ichneumonidae and some braconidae). on the contrary, if the host continues its life after the larvae have been hatched on its body, those parasitoids are considered as koinobionts. those are parasitoids which only temporarily paralyse the host, and they are capable of regulating its behaviour and life cycle until they reach maturity (d e s n e u x et al., 2009). usually, parasitoid larva kills the host at the end of its larval stage. an example of ectoparasitic koinobionts is an ichneumonid wasp acrodactyla quadrisculpta (gravenhorst) which parasitizes a spider, tetragnatha montana simon (m i l l e r et al., 2013). the majority of koinobionts represent endoparasitoids, which develop inside the host body. in some cases, such as the species macrocentrus curtis (braconidae), the female lays only one egg in the host body which undertakes the process of polyembryony and ultimately gives dozens of individuals which are genetically the same (clones) and thus increasing its parasitic capacity (h o w a r d , 1906; c u s h m a n , 1913; p a r k e r , 1931; q u i c k e , 2014). another strategy of increasing parasitic capacity can be seen in gregarious parasitoids (e.g., eulophidae, encyrtidae, braconidae) where females lay a couple of eggs per host, but their offspring is not genetically the same and both sexes are developed (q u i c k e , 2014). multitrophic associations usually include hyperparasitoids, the species that parasitize primary parasitoids. however, the hyperparasitism does not end here; on the highest level of trophic chains we are talking about hyperparasitoids of the second and the third level. clearly, in the trophic chain with a maximum number of members in a raw is consisted of: 1) primary food producer (plant), 2) primary food consumer (host), 3) secondary food consumer (primary parasitoid), 4) tertiary food consumer (secondary parasitoid = primary hyperparasitoid), 5) quaternary food consumer (tertiary parasitoid = secondary hyperparasitoid) and 6) quinary food consumer (quaternary parasitoid = tertiary hyperparasitoid). h a r v e y et al. (2009) explained this phenomenon working on the wasp cotesia glomerata (l.) (braconidae) as a primary parasitoid which is hyperparasitized by lysibia nana gravenhorst (ichneumonidae) which is a secondary parasitoid. furthermore, the species l. nana was hyperparasitized by another ichneumonid wasp gelis agilis (fabricius), appearing as a tertiary parasitoid. finally, there are some other species from the same genus, gelis attack g. agilis. interspecific parasitism is considered in more details in s u l l i v a n (1987). a kind of parasitism which is known as kleptoparasitism is the interaction where one animal takes food from another. in parasitoids kleptoparasitism occurs in cuckoo wasps (chrysididae). the cuckoo wasp omalus panzer is the parasitoid of the larvae of another hymenopteran family, crabronidae (w i n t e r h a g e n , 2015). this species does not oviposit directly in crabronid larva, but in aphids, even though an aphid is many times biologica nyssana 7 (2)  december 2016: 75-82 žikić, v. et al.  plants and insects in interactions… 78 smaller than the cuckoo wasp larva. crabronid females collect the aphids and carry them in a previously made nest to serve as food for their larvae. accidentally, a crabronid female picks up a parasitized aphid with the egg of a cuckoo wasp which has been laid inside its body. in this way a crabronid wasp brings the parasitoid in its own nest. ultimately, when the cuckoo wasp larva emerges from its egg and then from the aphid body, it starts looking for food devouring the crabronid larva, and also the collected aphids. exceptions exist in almost every group of sibling organisms. well know example of exception is panda – being herbivore among the mammal’s order carnivora. similarly, among the large groups of parasitica, there are some exceptions as well; many species of the family agaonidae (fig wasps) have developed a unique mutualistic association with various ficus l. trees, and more specifically with their flowers. the odour released from the fig flowers (syconium) attracts a fertilized female wasp to enter the syconium through a tiny opening in the centre. in this way, the wasp is carrying pollen from the flower from which it emerged. however, the main reason of its presence is to lay eggs in fig’s female flowers turning a syconium to a fig fruit (k j e l l b e r g et al., 2005). plant defending strategies and plant communications there is a secret language in which plants communicate with each other in many ways (e.g., allelopathy) (b a l u š k a & n i n k o v i ć , 2010). modern definition of allelopathy is “chemical interactions that involve toxic allelochemicals and should be distinguished from plant chemical communication” (s c h e n k et al., 1999; s c h e n k & s e a b l o o m , 2010). a way of emanating evaporative chemical signals via air is the one that is best investigated (v e t & d i c k e , 1992; b r u c e et al., 2005). f o w l e r & l a w t o n (1985) found that the chemicals are a blend of organic molecules such as alcohols, aldehydes, ketones and esters which are known as volatile organic compounds (vocs) also called kairomones (more recent, semiochemicals). recent studies reveal that plants as the lowest trophic level, when attacked by herbivores such as aphids, scale insects or caterpillars, emit any substance produced by an individual of one species that benefits both the producer and the recipient which is of a different species synomones which attract natural enemies i.e. parasitoids and predators in order to defend themselves (v e t & d i c k e , 1992; t u r l i n g s et al., 1995). this group of authors also revealed that the signalling role probably evolved secondarily as a result of plant responses that produce toxins and repellents against pests and also antibiotics against pathogens. studying olfactory physiology and behaviour of a predaceous beetle thanasimus formicarius (l.) (coleoptera: cleridae), the authors z h a n g & s c h l y t e r (2010) showed that the specific chemical substances from other nontarget hosts and plants, which are not even a part of the particular trophic association, can inhibit the attraction of chemical signals of attacked host/prey. in other words, neighbouring target plants resort to mimicry, thus stopping to smell out, becoming less attractive for pests. the other way of plant communication is through soil. they can do that directly by roots, or most often using an “internet” made of fungi. by linking to the fungal network, plants exchange information, nutrients and toxic chemicals with their neighbours (s o n g et al., 2010). mutually-beneficial relationships between plants and fungi, known as mycorrhiza can be found in a great majority of plants. host-parasitoid coevolution under the pressure of parasitoids, hosts develop various defending strategies (k r a a i j e v e l d et al., 1998). a simple type of protection that females use for their offspring is laying eggs in hidden places. for example, some siricidae (hymenoptera: symphyta) have very long and strong ovipositors with which they drill wood and lay their eggs deep inside it, thus minimizing the chance of their larvae to be parasitized. some other species, such as sawflies (hymenoptera: argidae and tenthredinidae) saw plant stems where they will oviposit. in symphyta there are also the species causing leaf mines or galls as it was previously explained (h a w k i n s , 1988; h e r i n g , 2013). the immune system can protect the host against parasitoids to varying degrees (k r a a i j e v e l d & g o d f r a y , 1999; c a r t o n et al., 2008). in a process of encapsulation hosts can eliminate parasitoids, forming a multi-layered cyst made of hemocytes. the hemocytes found in insect’s hemolymph are cells with the phagocyte function (g o d f r a y , 1994). endosybionts are present in many living beings, irrespective of whether they are algae, plants, fungi or animals. these are organisms that live within the body or cells of other organisms. the most famous examples of endosymbiosis are nitrogenfixing bacteria associated with legume roots (franche et al., 2009), single-cell algae living in reef-building corals (r o w a n , 1998) and in some turbellaria (b a r n e a h et al., 2007), or some protists and prokaryotes which inhabit the intestine of termites biologica nyssana 7 (2)  december 2016: 75-82 žikić, v. et al.  plants and insects in interactions… 79 (o h k u m a , 2008) and other wood-consuming insects. there are also bacterial endosymbionts that protect host insects, e.g., aphids against parasitoids. bacteria from the genus buchnera overproduce tryptophan (plasmid borne enzyme) and other amino acids that minimize the exposure of aphids to parasitoids (r o u h b a k h s h et al., 1997). hamiltonella defensa is a species of bacteria, that lives as an endosymbiont of aphids (most studied is the pea aphid – acyrthosiphon pisum harris) providing its host protection against parasitoids. mechanism of protection implies that h. defensa is infected with temperate bacteriophages called acyrthosiphon pisum secondary endosymbionts (apses) (v a n d e r w i l k et al., 1999; v o r b u r g e r , 2014). those bacteriophages encode different toxin genes killing the parasitoid eggs or larvae thus protecting the aphid host (d e g n a n & m o r a n , 2008). endosymbiotic male-killing bacteria from the genera wolbachia and rickettsia eliminate only the male sex in parasitoid embryos defined by genetics. it is not yet clear why males are more sensitive to wolbachia; it might be due to the fact that they have only one x chromosome or because they lack the whole garniture of chromosomes (haploids) (e.g., h u r s t & j i g g i n s , 2000; k o n d o et al., 2002; b e l s h a w & q u i c k e , 2003; w e r r e n at al., 2008). parasitoids also have evolved a variety of strategies to overcome host immune responses, but on the other hand they developed strategies to defend themselves against hyperparasitoids. there are several strategies to accomplish host defending mechanisms: by avoiding oviposition in body parts accessible to host hemocytes or by disrupting the host immune system injecting specific chemicals into the host at the time of oviposition; for example: apanteles (hymenoptera: braconidae) injects baculovirus-like nucleocapsids in the caterpillar body (s t o l t z & v i n s o n , 1977). also, remnants of polydnaviruses found in larvae of some parasitoid taxa disrupt the capsule around the parasitoid which is made of host hemocytes (b é z i e r et al., 2009). polydnaviruses destroy those hemocytes or alter their ability to aggregate and form a strong layer around parasitoid larvae (v i n s o n , 1990). these endosymbionts played one of the main roles in evolution of host range and in defining parasitoidhost compatibility (s c h l e n k e et al., 2007). conclusion although this overview represents only a glimpse of a fantastic array of interactions among insects themselves and plants, it gives an interesting insight and perspective of these phenomena. beside the fact that many details are concealed or maybe beyond our reach, the accumulated knowledge and permanent investigation of this topic has provided us with many benefits. the most useful is pest management and using biocontrol agents to combat pest insects. this has some serious repercussions in healthier food production and sustainable agriculture. “let it be borne in mind how infinitely complex and close-fitting are the mutual relations of all organic beings to each other and to their physical conditions of life” (d a r w i n , 1872). acknowledgements. we would like to thank to the organisation board of the 12th symposium on the flora of south-eastern serbia and neighbouring regions, kopaonik mt. review of english language was done by antonis mylonopoulos, komotiní, greece. we also thank to dr. andjeljko petrović for the selection of references to write this manuscript. the authors participate in the project of the ministry of education, science and technological development of the republic of serbia, grant no. iii43001.this study is partly supported by eu project biocomes (fp7-kbbe-2013-7). references albert, v.a., williams, s.e., chase, m.w. 1992: carnivorous plants: phylogeny and structural evolution. american association for the advancement of science, 257 (5076): 1491-1495. baluška, f., ninković, v. 2010: plant communication from an ecological perspective. springer science & business media. 252pp barneah, o., brickner, i., hooge, m., weis, v.m., lajeunesse, t.c., benayahu, y. 2007: three party symbiosis: acoelomorph worms, corals and unicellular algal symbionts in eilat (red sea). marine biology, 151 (4): 1215-1223. belshaw, r., quicke, d.l. 2003: the cytogenetics of thelytoky in a predominantly asexual parasitoid wasp with covert sex. genome, 46 (1): 170-173. bézier, a., annaheim, m., herbinière, j., wetterwald, c., gyapay, g., bernard-samain, s., pfister-wilhem, r. 2009: polydnaviruses of braconid wasps derive from an ancestral nudivirus. science, 323 (5916): 926-930. bronstein, j.l., alarcón, r., geber, m. 2006: the evolution of plant–insect mutualisms. new phytologist, 172 (3): 412-428. bruce, t.j., wadhams, l.j., woodcock, c.m. 2005: insect host location: a volatile situation. trends in plant science, 10 (6): 269-274. carton, y., poirié, m., nappi, a.j. 2008: insect immune resistance to parasitoids. insect science, 15 (1): 67-87. biologica nyssana 7 (2)  december 2016: 75-82 žikić, v. et al.  plants and insects in interactions… 80 compton, s., ware, a.b. 1991: ants disperse the elaisosome-bearing eggs of an african stick insect. psyche, 98 (2-3): 207-214. cushman, r.a. 1913: biological notes on a few rare or little known parasitic hymenoptera. proceedings of the entomological society of washington, 15: 153-155. darwin, c. 1872: on the origin of species by means of natural selection, or the preservation of favoured races in the struggle for life. john murray. london. 502 p. lulu. com. davis, r.h., nahrstedt, a. 1979: linamarin and lotaustralin as the source of cyanide in zygaena filipendulae. comparative biochemistry and physiology, 64b: 395-397. degnan, p.h., moran, n.a. 2008: diverse phageencoded toxins in a protective insect endosymbiont. applied and environmental microbiology, 74 (21): 6782-6791. desneux, n., barta, r.j., delebecque, c.j., heimpel, g.e. 2009: transient host paralysis as a means of reducing self-superparasitism in koinobiont endoparasitoids. journal of insect physiology, 55 (4): 321-327. faegri, k., van der pijl, l. 1979: principles of pollination ecology. pergamon press, oxford. 244p feener jr, d.h., brown, b.v. 1997: diptera as parasitoids. annual review of entomology, 42 (1): 73-97. floate, k.d. 2010. gall-inducing aphids and mites associated with the hybrid complex of cottonwoods, populus spp. (salicaceae), on canada’s grasslands. in shorthouse, j.d. and floate, k.d. 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(lepidoptera: zygaenidae). archives of biological sciences, 65 (3): 1027-1035. kuna et al. 2024, biologica nyssana 15(2) 15 (2) december 2024: 113-121 doi: 10.5281/zenodo.13847338 assessment of the cytostatic, cytotoxic and antibacterial potential of aluminium (iii) chloride hexahydrate original article džana kuna university of sarajevo, faculty of science, department of biology, zmaja od bosne 33-35, 71000 sarajevo, bosnia and herzegovina irma mahmutović-dizdarević university of sarajevo, faculty of science, department of biology, zmaja od bosne 33-35, 71000 sarajevo, bosnia and herzegovina irma.m@pmf.unsa.ba (corresponding author) renata bešta-gajević university of sarajevo, faculty of science, department of biology, zmaja od bosne 33-35, 71000 sarajevo, bosnia and herzegovina aner mesić university of sarajevo, faculty of science, department of biology, zmaja od bosne 33-35, 71000 sarajevo, bosnia and herzegovina received: may 09, 2024 revised: september 16, 2024 accepted: september 19, 2024 abstract: even though aluminium does not participate in any known biochemical process in living organisms, it is omnipresent in various products intended for human consumption. this study aimed to evaluate the cytostatic and cytotoxic effects of aluminium (iii) chloride hexahydrate (alcl3) in cultured human peripheral blood lymphocytes, as well as its antimicrobial potential using agar diffusion method. our results showed a significant deviation in the mitotic index (mi) values between 2.20, 2.40 and 2.50% of alcl3 and control group. mi value decreased with an increase in the concentration of alcl3. an increased frequency of apoptotic cells at all concentrations of the alcl3 was detected, whereby the significantly higher frequency of apoptotic cells was detected between 2.20 and 2.30% alcl3 and the control group. staphylococcus aureus and escherichia coli were resistant to the tested compound. inhibition zones detected in bacillus subtilis and pseudomonas aeruginosa were 13.67±0.58 mm at 1 mg/ml, 12.00±0.00 mm at 10 mg/ml; and 13.00±0.00 mm at 1 mg/ ml, and 14.67±0.58 mm et 10 mg/ml, respectively. our results indicate that alcl3 possesses antiproliferative and apoptosis-inducing potential. discrete antibacterial effects were demonstrated. further studies are needed to strengthen these findings. key words: aluminium (iii) chloride hexahydrate, human lymphocytes, toxicological effects, antibacterial activity, agar diffusion method apstrakt: procena citostatskog, citotoksičnog i antibakterijskog potencijala aluminijum (iii) hlorid heksahidrata iako aluminijum ne učestvuje ni u jednom poznatom biohemijskom procesu u živim organizmima, on je sveprisutan u raznim proizvodima namenjenim ljudskoj upotrebi. ova studija je imala za cilj da proceni citostatske i citotoksične efekte aluminijum (iii) hlorid heksahidrata (alcl3) u kultivisanim limfocitima periferne krvi čoveka, kao i njegov antimikrobni potencijal metodom agar difuzije. naši rezultati su pokazali značajno odstupanje vrednosti mitotičkog indeksa (mi) između 2.20, 2.40 i 2.50% alcl3 i kontrolne grupe. vrednost mi se smanjivala sa povećanjem koncentracije alcl3. utvrđena je povećana učestalost apoptotičkih ćelija pri svim koncentracijama alcl3, pri čemu je značajno veća učestalost apoptotičkih ćelija između 2.20 i 2.30% alcl3 i kontrolne grupe. staphylococcus aureus i escherichia coli su bili otporni na testirano jedinjenje. zone inhibicije uočene kod bacillus subtilis i pseudomonas aeruginosa bile su 13.67±0.58 mm pri 1 mg/ml, 12.00±0.00 mm pri 10 mg/ml; i 13.00±0.00 mm pri 1 mg/ml, i 14.67±0.58 mm pri 10 mg/ ml, respektivno. naši rezultati pokazuju da alcl3 poseduje antiproliferativni potencijal i potencijal indukcije apoptoze. pokazani su diskretni antibakterijski efekti. potrebne su dalje studije kako bi se ovi nalazi ojačali. ključne reči: aluminijum (iii) hlorid heksahidrat, humani limfociti, toksikološki efekti, antibakterijska aktivnost, metod agar difuzije introduction metal ions are present in more than one-third of all cellular proteins, and are pivotal for the activity of these proteins (martinez-finley, 2012). aluminium represents the most abundant metal, which does not participate in any known biochemical process in living organisms (tenan et al., 2021). despite this, © 2024 kuna et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 113 aluminium is nowadays a ubiquitous component of human life, since it is added to water, food, medicines, vaccines, cosmetic and personal care products intended for human use (tenan et al., 2021; rahimzadeh et al., 2022). extraneous metals can disturb the delicate equilibrium that allows the correct course of cellular activities, and thus exhibit toxic and/or carcinogenic effects (beyersmann & hartwig, 2008; jan et al., 2015). aluminium chloride represents an inorganic compound with the formula alcl3. it forms a hexahydrate containing six water molecules of hydration. the anhydrous form as well as the hexahydrate are colourless crystals. the anhydrous form is commercially important since it possesses a low melting and boiling point (helmboldt et al., 2007). prolo et al. (2007) demonstrated that aluminium reduced the proliferative response of normal human peripheral blood mononuclear cells to phytohemagglutinin stimulation. furthermore, it has been shown that aluminium inhibited the proliferation of the ht-29 cells (yu et al., 2019), as well as that aluminium trichloride inhibited the proliferation of osteoblasts (huang et al., 2017). considering its large presence in products that people consume daily, as a result of continuous exposure aluminium accumulates in various organs including bone, brain, kidney, liver, lung, and mammary gland (exley, 2013; darbre, 2016). high levels of aluminium are associated with dialysis, encephalopathy, osteomalacia, and parkinson’s disease (alfrey et al., 1976; ellis et al., 1979; hirsch et al., 1991). aluminium has also been described as a potential human carcinogen involved in the etiology of breast cancer, based on the experimental evidence that daily application of antiperspirants, especially those rich in aluminium salts, contribute to an increased incidence of breast cancers (darbre, 2001; mcgrath, 2003). in support of this, linhart et al. (2017) demonstrated that frequent use of underarm cosmetic products may lead to an accumulation of aluminium in breast tissue, suggesting a possible association between breast cancer risk and frequent use of these products. earlier investigations discussed the antiseptic and antibacterial activity of some aluminium salts (cromwell & leffler, 1942; shelley et al., 1953; aguilar et al., 1956), as well as the validity of using the aluminium salts in cosmetic preparations for their antibacterial potential (blank & dawes, 1960). the antimicrobial activity of aluminium is investigated by londono et al. (2017) and the study suggests a potential synergism with other metals to achieve antibacterial effects. through evolution, microorganisms found ways to avoid environmental stress, and one of such mechanisms is illustrated via the production of siderophores. siderophores could be defined as low-molecularweight molecules with a high and specific affinity to chelate metals, such as iron (hider & kong, 2010). timofeeva et al. (2022) investigated siderophores involved in iron uptake and mentioned about 20 different bacterial genera that are known for siderophore production. the research of farh et al. (2017) suggests that the production of siderophores is related to bacterial resistance to aluminium toxicity. therefore, the present study aimed to assess the cytostatic, cytotoxic, and antibacterial potential of aluminium (iii) chloride hexahydrate (alcl3). materials and methods peripheral blood lymphocyte culture for this research, whole blood from two healthy female donors, aged 24, who were non-smokers was used. blood is collected into sterile vials with 68 iu of sodium heparin per milliliter of blood (bd vacutainer vr, becton, dickinson and co., nj, usa). lymphocyte cultures were prepared according to moorhead et al. (1960) with some modifications. in that sense, 0.5 ml of whole blood was added to 5 ml rpmi-based complete medium containing fetal bovine serum (fbs), l-glutamine and phytohemagglutinin (pha) (gibcotm pbmax™ karyotyping medium, invitrogen, ca, usa). lymphocyte cultures were incubated in total for 72 hours at 37 °c. after 48 hours, cultures were treated with alcl3 in four test concentrations (2.20, 2.30, 2.40 and 2.50%) determined according to sccs (2020), and incubated for another 24 hours. in addition to cultures with four concentrations of the test substance (alcl3), cultures containing untreated lymphocytes (control group), were incubated. two replicates for each test concentration and control group were made. two hours before the end of the incubation period, colcemidtm (sigma-aldrich, st. louis, mo, usa) was added to the cultures at a final concentration of 0.2 μg/ml to block cell division in metaphase. after the end of the incubation period, the cultures were treated with fresh hypotonic solution (0.075 m kcl) to lyse the cells. afterward, cultures were fixed with methanol/glacial acetic acid (3:1 v/v) fixative. the lymphocytes were then spread onto clean, cooled glass slides that were left to airdry, and then stained with 10% giemsa solution. microscopic analysis the cytogenetic analysis included the examination of two microscope slides (two replicates) for each of the tested concentrations of alcl3 and the control group. the analysis of the mitotic index (mi) value, as well as the frequency of apoptotic cells (cytotoxic biomarkers), was performed in 1000 cells per slide 114 biologica nyssana ● 15 (2) december 2024: kuna et al. ● assessment of the cytostatic, cytotoxic and antibacterial potential of aluminium (iii) chloride hexahydrate biologica nyssana ● 15 (2) december 2024: kuna et al. ● assessment of the cytostatic, cytotoxic and antibacterial potential of aluminium (iii) chloride hexahydrate 115 (a total of 2000 cells per concentration of the test substance and the control group). the mi was calculated as the ratio between the number of cells in the division and the total number of cells analyzed neglecting broken cells, clumped cells and cellular debris. microbiological analysis to test the antimicrobial activity of alcl3, a solid substance was dissolved in dimethyl sulfoxide, dmso (sigma aldrich, st. louis, mo, usa) to the final concentrations of 1 mg/ml and 10 mg/ml. as the positive control, 10 µg/disk of ampicillin (oxoid tm, watham, ma, usa) was used. the antibacterial activity of alcl3 was evaluated using a total of four bacteria: bacillus subtilis atcc 6633, staphylococcus aureus atcc 6538, pseudomonas aeruginosa atcc 27853, and escherichia coli atcc 8739. tested bacteria were obtained from the american type culture collection (atcc) and inoculums were prepared according to eucast (2017). overnight cultures were dissolved in sterile saline solution to achieve the final turbidity of 0.5 mcfarland standard, which represents the bacterial cell concentration of 1.5 × 108 cfu/ml. agar diffusion method the agar diffusion method is performed according to balouiri et al. (2016). inoculation of the agar plates was performed using the sterile swabs soaked in a suspension of test bacteria, and the seeded plates were left at room temperature for 15 minutes to allow the better absorption of the applied inoculum into the substrate. plates were then drilled to make wells with 8 mm in diameter. the amount of 50 µl of alcl3 was transferred into the wells and plates were incubated for 16 to 18 hours at 37 °c. both concentrations of alcl3 were triple tested. the antibacterial activity was evaluated based on the diameter of inhibition zones, after the alcl3 diffusion in the medium. statistical analysis all descriptive statistical computations were conducted by use of microsoft excel 2019 (microsoft corporation, redmond, usa). one-way anova with the post hoc multiple comparison tests (lsd) in terms of determining significant differences between the test compound (alcl3) and the control for all analyzed parameters was performed using ibm spss statistics for windows, version 20.0 (armonk, ny, usa) for the results of cytogenetic analysis, and statistica 10 (statsoft.inc., tulsa, ok, usa) for the results of microbiological assay. differences were considered statistically significant at the p<0.05 level. results and discussion cytostatic and cytotoxic effects of alcl3 the results of alcl3 effects on cell proliferation are summarized in tab. 1. mitotic index (mi) values varied depending on the applied concentration of the test substance. in that sense, a significant deviation of the mi values in mutual comparisons between the groups treated with alcl3 at concentrations of 2.20, 2.40 and 2.50% alcl3 and the control group was detected. the highest mi value was observed at a concentration 2.20% of alcl3, with a tendency for mi value to decrease with an increase in the concentration of the test substance. as for the cytotoxic effects (tab. 1), the results revealed that the frequency of apoptosis was elevated at all four tested concentrations of alcl3 as compared to the control group. however, a significantly higher frequency of apoptotic cells was observed between 2.20% and 2.30% of alcl3 and the control group. table 1. cytostatic and cytotoxic effects of alcl3 in human lymphocytes alcl3 concentration (%) mitotic index apoptotic cells 2.50 0.25±0.07* 6.50±0.07 2.40 0.87±0.40* 7.50±2.12 2.30 3.00±0.56 17.00±2.82* 2.20 5.10±1.27* 15.50±3.53* control group 2.90±0.07 3.50±2.12 results are presented as mean ± sd; the mitotic index is presented as a percentage; *statistically significant differences as compared to the control group (p<0.05) it is known that aluminium, even though is the most abundant metal, is not involved in any known biochemical process in living organisms. however, due to its mobilization from minerals which is encouraged by various human activities, aluminium is nowadays an omnipresent component of human life. aluminium is an active ingredient present in many products for human use such as vaccines, drugs, antiperspirants, cosmetics, agricultural pesticides and foods (sakhawoth et al., 2021; tenan et al., 2021). this study aimed to evaluate the possible cytostatic and cytotoxic effects of alcl3 in human peripheral lymphocytes. by analyzing the mitotic index, we observed a significant dose-dependent reduction of the mitotic index values, which provides evidence that aluminium is directly affecting the inhibition of cell proliferation. these findings confirm those obtained 116 biologica nyssana ● 15 (2) december 2024: kuna et al. ● assessment of the cytostatic, cytotoxic and antibacterial potential of aluminium (iii) chloride hexahydrate by prolo et al. (2007) whose study showed that aluminium diminished the proliferative response of normal human peripheral blood mononuclear cells to phytohemagglutinin stimulation. furthermore, our results are in concordance with those which demonstrated that aluminium inhibited the proliferation of the ht-29 cells (yu et al., 2019), as well as those which showed that aluminium trichloride inhibited osteoblastic proliferation (huang et al., 2017). the analysis of cytotoxicity markers showed a significantly higher frequency of apoptosis between alcl3 (2.20% and 2.30%) and control. our results are supportive of those obtained by yu et al. (2019) who showed that aluminium induced cellular apoptosis in ht-29, a human colon cancer cell line. similarly, prolo et al. (2007) demonstrated that aluminium increases apoptosis of cultured human cells (saos2). tenan et al. (2021) demonstrated that aluminium at low doses did not significantly affect cell viability, but did significantly increase chromosome doublestrand breaks. this could be if not the most dangerous situation, then certainly one of the most dangerous, since these damaged cells have the potential to survive and disseminate karyotypic abnormalities. this suggests that low chronic doses of aluminium have the potential to be more damaging than higher doses, making them more likely to cause cell elimination by cytotoxicity. cytotoxicity or cellular transformation might be dependent on several factors such as dose and duration of exposure to the foreign metal, with chronic low-dose exposure possibly favoring carcinogenesis (zhao et al., 2014), which is by the reality of human exposure to low doses absorbed from various sources daily. dna double-strand breaks represent the most cytotoxic lesions of those occurring in the dna, which can lead to cell death, and/or to genome mutagenesis and chromosomal instability. even though most of these rearrangements exhibit harmful effects on cellular survival, single events can ensure clonal advantage and result in abnormal cellular proliferation and cancer (gómez-herreros, 2019). apoptosis and the genes involved in its control have a significant impact on the malignant phenotype (katavic et al., 2023). disruption of apoptosis can promote tumor formation by promoting cell proliferation through the expansion of selected clones (labi & erlacher, 2015). the link between the increase in the rate of apoptosis and the development of tumors was demonstrated during the study of the development of breast cancer in mice, highlighting that the apoptosis inducer cd95 promoted cancer growth (wang et al., 2013). experimental studies indicate that the long-term use of aluminium may correlate with breast cancer development and progression, due to its possible estrogen-like activities (gorgogietas et al., 2018). in another study, alcl3 was used, and loss of contact inhibition, as well as anchorage-independent growth in mcf-10a human mammary epithelial cells, were observed. these processes were preceded by increased dna synthesis, the occurrence of doublestrand breaks, and accelerated aging in proliferating cultures, as well as in proliferating primary human mammary epithelial cells (sappino et al., 2012). the foregoing undoubtedly suggests that alcl3, by causing an increased frequency of apoptosis, could affect the malignant transformation of cells in humans. antibacterial activity the obtained results of the antibacterial activity of investigated aluminium salt showed susceptibility in the case of bacillus subtilis and pseudomonas aeruginosa, while staphylococcus aureus and escherichia coli were resistant to the investigated compound in both tested concentrations of 1 mg/ ml and 10 mg/ml (tab. 2). the concentration of 1 mg/ml of alcl3 caused an inhibition zone of 13.67±0.58 mm in b. subtilis, and 13.00±0.00 mm in p. aeruginosa, while the concentration of 10 mg/ml of tested substance generated inhibition zones of 12.00±0.00 mm and 14.67±0.58 mm in b. subtilis and p. aeruginosa respectively. antibiotic ampicillin performed strong antibacterial activity, with the more extensive growth inhibition of grampositive bacteria in comparison to gram-negative strains (tab. 2). solvent control showed that dmso did not perform antibacterial action. our investigation regarding the antibacterial properties of tested alcl3 provided interesting results, bearing in mind that a particular compound is used in cosmetic products due to its antiseptic potential. staphylococcus aureus and escherichia coli used in this study were completely resistant to both concentrations of the alcl3, while in bacillus subtilis and pseudomonas aeruginosa discrete inhibition zones have been detected. impact of the aluminium on the bacterial cell could be discussed at several levels. aluminium can bind with dna, bacterial cell membranes, and the cell wall. also, the presence of the aluminium ion in the cell causes competition of iron and magnesium uptake which changes enzymatic reactions (piña & cervantes, 1996). this could be responsible for the decline in the overall growth rate of bacteria (maier, 2009). the research of londono et al. (2017) noted that aluminium alone is not responsible for bactericidal effects, but rather is the combination with other metals. that kind of chemical interaction could be related to the formation of a toxic environment for the bacteria biologica nyssana ● 15 (2) december 2024: kuna et al. ● assessment of the cytostatic, cytotoxic and antibacterial potential of aluminium (iii) chloride hexahydrate 117 (preston et al., 2000). according to schoonen & lanzirotti (2006), the ph strongly influences metal toxicity. this study used dmso as a solvent, which is an alkaline reagent, and this might be connected to the observed subtle bactericidal effects. besides the already mentioned, microorganisms have developed numerous mechanisms to cope with a variety of toxic metals to survive (mittal et al., 2003). aluminiumresistant bacteria from different genera have been previously reported, and according to farh et al. (2017), the resistance to aluminium toxicity is mainly correlated to the production of siderophores. timofeeva et al. (2022) gave a summarized list of the siderophore-producing bacteria, that could be classified into the genera: azotobacter, azospirillum, bacillus, dickeya, enterobacter, klebsiella, kosakonia, methylobacterium, nocardia, pantoea, paenibacillus, pseudomonas, rhodococcus, serratia, streptomyces, etc. the study of schalk et al. (2011) highlighted that other metals besides iron can activate the production of siderophores, as well as that some siderophores except for their preference for iron can chelate other elements and, in that way, maintain the homeostasis of metal in the bacterial cell. aluminium easily forms complexes with siderophores, based on the similarity in size and charge between the aluminium and iron ions, and besides the passive aluminium uptake, siderophoremediated uptake of this metal is recognized among the microorganisms (bojić et al., 2002). according to their chemical nature, siderophores are classified into several groups, where the hydroxamate siderophores characteristically form a bidentate ligand with fe ions (ustiatik et al., 2021; timofeeva et al., 2022). it is shown that bacillus megaterium produces two hydroxamate siderophores (byers et al., 1967), that are capable of chelating aluminium. the siderophore transport receptor absorbs the aluminium which provides an additional pathway for the aluminium accumulation. this process is closely related to the overall implications of metal toxicity (hu & boyer, 1996). bacillus cereus also stays viable in the increased concentration of aluminium (ji et al., 2016). furthermore, the study of hazarika et al. (2023) confirmed that mitigation of al-toxicity through the activity of b. subtilis is connected to siderophore production. cornelis (2008) noted that pyoverdine found in p. aeruginosa can complex aluminium with high affinity, which implies that is involved in the sequestration of toxic metals. some other species of the genus pseudomonas are also able to tolerate aluminium-induced stress (appanna et al., 1994; singh et al., 2005; purwanti et al., 2019; mozumder et al., 2022). the literature is generally scarce regarding the tolerance of aluminium toxicity in s. aureus. ghssein & ezzedine (2022) emphasize the presence of high-affinity metal import pathways, including metal ions acquisition, recruitment, and metal-chelate complex import. therefore, siderophores together with metallophores increase the infectious properties of s. aureus. these findings are in accordance with the investigation of aras et al. (2023) where the impact of aluminium chlorohydrate on the development of antibiotic resistance in staphylococcus epidermidis is highlighted. guida et al. (1991) discuss the aluminium toxicity to e. coli and suggest ph as one of the crucial factors in that term. also, the same study observed that aluminium binding is happening intracellularly as well as at the bacterial cell surface, while intracellular binding sites involve iron transport pathways. bojić et al. (2002) observed that aluminium toxicity to e. coli depends on the type of growing medium, mainly because of the different uptake of the metal in different media. al-khikani et al. (2023) investigated the effects of aluminium potassium sulphate on e. coli and the results were concentration-dependent, where the higher concentrations of the substance were effective, while lower concentrations inhibited bacterial growth only in combination with antibiotics, probably due to the synergistic activity. the strain of e. coli used in our experiment performed low susceptibility to standtable 2. observed zones of inhibition (mm) due to the activity of alcl3 results are presented as mean ± sd; all values differ significantly at p<0.05 after performing the post hoc fisher’s lsd test tested bacteria investigated substance alcl3 ampicillin (10 µg) dmso (≥99.7%)1 mg/ml 10 mg/ml bacillus subtilis atcc 6633 13.67±0.58 12.00±0.00 48.13±0.32 ni staphylococcus aureus atcc 6538 ni ni 32.97±0.25 ni pseudomonas aeruginosa atcc 27853 13.00±0.00 14.67±0.58 13.10±0.21 ni escherichia coli atcc 8739 ni ni 8.93±0.31 ni beyersmann, d., & hartwig, a. 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(lamiaceae, nepetoideae) is widely distributed in the mediterranean region on rocky habitats. in bulgarian flora the genus is represented by four species that have relatively limited distribution but they are used widely as herbs and spices. micromeria species have been applied for the treatment of diseases of the cardiovascular system, digestive tract, respiratory system (asthma), skin inflammations (s a i d et al., 2002). the species are reported to have antimicrobial, antioxidant, gastroprotective, hepatoprotective, cytotoxic, antiinflammatory, anticholinesterase and analgesic activity (ö z t ü r k et al., 2011, v l a d i m i r k n e ž e v i ć et al., 2011, h e r k e n et al., 2012, s h e h a b & a b u -g h a r b i e h , 2012, a b u g h a r b i e h et al., 2013, b u k v i c k i et al., 2015). micromeria dalmatica benth. is balkan endemic distributed in bulgaria, greece, crit, crna gora (p e t r o v a & v l a d i m i r o v , 2010). populations of the species of bulgarian, serbian (montenegro) and greek origin have been well studied for essential oil composition (s l a v k o v s k a et al., 2005, k o s t a d i n o v a et al., 2007, k a r o u s o u et al., 2012). for the essential oil of the species has been reported to possess high antimicrobial efficacy against food spoilage microorganisms (b u k v i c k i et al., 2015). flavonoid profile of m. dalmadca is characterized by the presence of thymonin (5,6,4'-trihydroxy-7,8,3'trimethoxyflavone) as a major exudate flavonoid aglycone and acacetin (4'-methoxy-5,7dihydroxyflavone) derivatives as the most abundant flavonoid glycosides (t o m a s -b a r b e r a n et al., 1991, m a r i n et al., 2001). metabolite profiling is an analytical method for relative quantitation of a mixture of compounds from biological samples using chromatography and universal detection technologies (gc-ms, lcms). these techniques allow rapid identification of huge number of metabolites. metabolite profiling have been used in plant and fungi chemotaxonomy, for screening purposes, quality control and standardization purposes as well as for search of new natural products (f r i s v a d et al., 2008, f i s c h e d i c k et al., 2010, hill & r o e s s n e r , 2013, v r a n c h e v a et al., 2014, r o h l o f f , 2015). as a part of comprehensive survey of chemical composition and biological activity of bulgarian species of genus micromeria in the present study the metabolite profile and free radical scavenging activity of micromeria dalmatica benth were examined. material and methods plant material m. dalmatica was collected from vlahina mt, a part of west bulgarian frontier mts, in august, 2015. samples, upper flowering part of the plants were collected at full flowering phase without traces of soil, dust or parts of other plants. after that, samples were air-dried in place with good ventilation, away from direct sunlight. acetone exudate air-dried, but not ground (3g) plant material of aerial parts of m. dalmatica was briefly (2-3 min) rinsed with acetone at room temperature to dissolve the lipophilic components accumulated on the surface. the obtained acetone filtrate was then dried using a rotary-evaporator to give a crude extract. methanol extract 100 µg of plant material as well as internal standards of 50 µg of nonadecanoic acid, 50 µg of ribitol and 50 µg of 3,4 dichloro-4-hidroxy benzoic acid were placed in 2 ml ependorf tubes and extracted with 1 ml of meoh for 2 h at room temperature assisted by an ultrasonic bath for 15 min at 70 ˚c every 30 min, after that the sample was centrifuged. aliquot of 800 ul was transferred in other ependorf tubes and was added of 500 µl h2o and 500 µl of chcl3, vortexing for 2 min, and the mixture was centrifuged. the chloroform fraction was separated, evaporated and transmethylated with 2% of h2so4 in meoh at 60˚c for 18 h, than lipids were extracted with nhexane (2x500 µl) which was dried with anhydrous na2so4 and evaporated to obtain lipid fraction. an aliqote of 100 µl from the aqueous fraction was placed in glass vial and evaporated in a speed-vac to obtain polar fraction. the rest of aqueous fraction was hydrolyzed with 0.5 ml of 1n naoh for 18 h at 60˚c. after acidification to ph 1-2 with conc. hcl, the phenolic compounds were extracted with etoac (2x500 µl) which was dried with anhydrous na2so4 and evaporated to obtain phenolic fraction. the fractions of the methanolic extract as well as 20 mg of the acetone exudate were silylated with 50 μl of n,o-bis-(trimethylsilyl)trifluoro-acetamide (bstfa) in 50 μl of pyridine for 2 h at 50°c. metabolite analysis the gc–ms spectra were recorded on a termo scientific focus gc coupled with termo scientific dsq mass detector operating in ei mode at 70 ev. adb-5ms column (30 m x 0.25 mm x 0.25 m) was used. the temperature program was: 100-180 oc at 15 oc x min-1, 180-300 20 at 5 oc x min-1 and 10 min hold at 300 oc. the injector temperature was 250 oc. biologica nyssana 7 (2)  december 2016: 159-165 nikolova, m. et al.  gc-ms metabolic profiling and free radical… 161 table 1. identified compounds in the acetone exudate and methanolic extract of micromeria dalmatica by gc/ms compounds ri acetone exudate* methanol extract* lipid fraction polar fraction phenolic fraction alkanes (hydrocarbones) tridecane 1300 0,13 tetradecane 1400 3,42 pentadecane 1500 0,31 hexadecane 1600 2,84 heptadecane 1700 1,20 octadecane 1800 1,94 eicosane 2000 0,92 docosane 2200 0,37 tricosane 2300 0,11 pentacosane 2500 1,18 heptacosane 2700 4,5 2,10 octacosane 2800 0,89 3,45 nonacosane 2900 0,72 triacontane 3000 1,53 4,30 hentriacontane 3100 13,13 dotriacontane 3200 1,23 fatty alkohols 1-dodecanol 1560 39,96 4,23 1-tetradecanol 1757 0,64 0,69 1,52 1-hexadecanol 1955 3,55 10,61 1-octadec-9z-enol trimethylsilyl ether 2125 4,43 8,91 1-octadecanol 2152 0,13 1,72 5,72 1-eicosanol 2350 0,09 1-docosanol 2546 0,21 1-tetracosanol 2741 0,56 1-hexacosanol 2938 0,03 1-octacosanol 3133 0,04 0,17 fatty acids octanoic acid (caprylic acid, 8:0) 1575 0,17 tetradecanoic acid, methyl ester** (myristic acid, 14:0) 1721 0,62 hexadecanoic acid, methyl ether (palmitic acid, 16:0)** 1922 15,95 hexadecanoic acid (palmitic acid, 16:0) 2041 11,23 2,20 9,71 octadecadienoic acid 9,12(z,z),methyl ester (linoleic acid, 18:2)** 2090 4,16 octadecatrienoic acid 9,12,15-(z,z,z), methyl ether (α-linolenic acid 18:3) 2098 8,54 octadecanoic acid, methyl ester ** (stearic acid,18:0) 2124 3,52 octadecadienoic acid 9,12-(z,z) (linoleic acid, 18:2) 2204 2,42 octadecatrienoic acid 9,12,15-(z,z,z), methyl ether (α-linolenic acid 18:3) 2211 5,67 octadecanoic acid (stearic acid,18:0) 2238 0,10 octadecanoic acid, 2,3bis[(trimethylsilyl)oxy]propyl ester 2775 4,14 organic acids malic acid 1473 1,02 erythronic acid 1526 0,12 biologica nyssana 7 (2)  december 2016: 159-165 nikolova, m. et al.  gc-ms metabolic profiling and free radical… 162 table 1. continued glycerides glycerol 1258 2,88 1,03 15,25 hexadecanoylglycerol 2582 4,06 monooctadecanoylglycerol 2775 4,14 tocoferols α-tocopherol 3122 0,351 phytosterols β-sitosterol 3335 2,57 2,17 triternes amyrin 3415 13,58 polyoles erythritol 2,21 arabitol 1706 0,34 meso-erythritol 1711 5,87 7,35 myo-inositol 2080 1,22 13,18 mannose 1919 0,44 galactose 1926 0,14 2,3,4,5-tetrahydroxypentanoic acid1,4-lactone (arabinonic acid, 1,4lactone) 1624 1,53 monosaccharides arabinose 1683 2,39 monosaccharide 1 1792 7,78 fructose i 1800 1,58 6,69 fructose derivative 1805 0,83 3,99 fructose ii 1837 0,12 5,66 fructose iii 1855 0,04 6,47 glucose 1882 2,94 6,02 mannose 1918 7,16 monosaccharide 2 1969 2,95 12,40 monoaccharide 3 2539 0,25 disaccarides disacharide 1 2498 0,18 sucrose 2628 4,80 56,96 trisaccharides rafinnose 3420 0,68 amino acids 0,17 glycine 1121 0,21 flavonoid aglycones 2628 apigenin-4’-methyl ether 3040 0,17 quercetagein 3,6,7-trimethyl ether 3400 4,78 hydroxycinnamic acids quinic acid 1846 29,74 caffeic acid trans 2131 1,66 benzoates 3,5-bis(trimethylsiloxy)benzoic acid 1999 12,75 legend: *-data are expressed as percentage of the total peak area [%] **-due to the way of processing of the lipid fraction the fatty acid is detected as methyl ester the flow rate of carrier gas (helium) was 0.8 ml x min-1. the split ratio was 1:10 1 µl of the solution was injected. the metabolites were identified as tmsi derivatives comparing their mass spectra and kovats indexes (ri) with those of an on-line available plant specific database (the golm metabolome database; http://csbdb.mpimp-golm.mpg.de/csbdb/gmd/home/ gmd_sm.html), the nist 05 database and mass spectra available in the on-line lipid library (http://www. lipidlibrary.co.uk/ms/ms01/index.htm), nist 05 database and literature data as indicated in tab. 1. the measured mass spectra were deconvoluted by the automated mass spectral deconvolution and identification system (amdis), before comparison with the databases. then, the spectra of individual components were transferred to the nist mass spectral search program ms search http://www/ biologica nyssana 7 (2)  december 2016: 159-165 nikolova, m. et al.  gc-ms metabolic profiling and free radical… 163 2.0 where they were matched against reference compounds of the nist mass spectral library 2005 and the golm metabolome database. ri of the compounds were recorded with standard nhydrocarbon calibration mixture (c9-c36) (restek, cat no. 31614, supplied by teknokroma, spain) using amdis 3.6 software. free radical scavenging activity the stable 2,2-diphenyl-1-picryl hydrazyl radical (dpph) was used for determination of free radical scavenging activity of studied samples (stanojević et al., 2009). different concentrations (10, 20, 50 100 and 200 µg/ml in methanol) of m. dalmatica extract were added at an equal volume (2.5 ml) to methanol solution of dpph (0.3 mm, 1 ml). after 30 min at room temperature, the ab values were measured at 517 nm on a spectrophotometer (jenway 6320d) and converted into the percentage antioxidant activity using the following equation: dpph antiradical scavenging capacity (%) = [1–(absample–abblank)/abcontrol] × 100 methanol (1.0 ml) plus plant extract solution (2.5 ml) was used as a blank, while dpph solution plus methanol was used as a control.the ic50 values were calculated by software prizm 3.00. all of the experiments were carried out in triplicate. results and discussion metabolite analysis acetone exudate and methanolic extract from aerial parts of micromeria dalmatica were analyzed by gc/ms. in the acetone exudate of the sample 174 chemical peak signals were detected. a part of them was identified as representatives of aklanes, fatty alcohols, fatty acids, triterpenes, flavonoid aglycones, and other (tab. 1). eight n-aklanes were found and they ranged from 23 to 32 carbon numbers. among them the hentriacontane c31h64 was the most abundant (13,13%) that in accordance with previously reported data for micromeria cristata and m. juliana (r e d d y et al., 2000). seven fatty alcohols were identified as minor components in the acetone exudate. palmitic and linoleic acids were presented in the most significant relative amount. amyrin was detected as major triterpene in the acetone exudate. quercetagetin 3,6,7-trimethyl ether and apigenin 4'-methyl ether were identified as representatives of flavonoid aglycones. methyl derivatives of quercetagetin are rarely reported to lamiaceae family (g r a y e r et al., 2010), however this is the first time that quercetagetin derivative has been found in the genus micromeria. in the methanolic extract (lipid, polar and phenolic fractions) of m. dalmatica more than 100 chromatographic peaks were detected, including organic, fatty and phenolic acids, saccharides, polyoles, phytosterols, alkanes and other (tab. 1). unlike the acetone exudate in the lipid fraction of the methanolic extract alkanes with carbon number from 13 to 22 were found. alkanes with carbon number 30, 28 and 16 are dominant. six fatty alcohols were identified among them dodecanol was the most abundant (39,96%). seven fatty acids are found, the main being methyl ether of palmitic acid (16:0) followed by linolenic acid (18:3) and linoleic acid (18:2). this fatty acid composition confirms data previously reported for micromeria thymifolia and micromeria albanica (r i s t i ć et al., 1997). in the polar fraction the main carbohydrate was sucrose, approximately 50%. many monosaccharides and their derivatives were detected. among identified phenolic compounds quinic acid was the most abundant followed by 3,5-bis(trimethylsiloxy) benzoic acid. free radical scavenging activity the dpph assay has been widely used to evaluate the free radical scavenging effectiveness of various antioxidant substances and plant extracts (m a r i n o v a & b a t c h v a r o v , 2011). hydromethanolic extract of m. dalmatica was studied for in vitro antioxidant 2,2-diphenyl-1picrylhydrazyl (dpph) free radical-scavenging activity. the inhibitory concentration (ic50) of extract needed to inhibit 50% of the dpph radicals was calculated to be 21.36 µg/ml. the received value is similar to that obtained for micromeria croatica, m. juliana and m. thymifolia extracts and demonstrate considerable activity to scavenge dpph radicals (v l a d i m i r -k n e ž e v i ć et al., 2011). the commercial antioxidant butylated hydroxytoluene (bht) was used as positive control and its ic50 value was 12.6 µg/ml. conclusion the present study is first report on gc/ms based metabolite profiling of micromeria dalmatica. most of the compounds were reported here for the first time for the species. results obtained on the lipid composition appropriate complements the available literature data on other micromeria species and can be used in comparative chemotaxonomic analyzes. this is the first report on the occurrence of quercetagetin derivatives in genus micromeria. dpph assay shows high antioxidant potential of micromeria dalmatica extract. biologica 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abstract: insects, nematodes, and aquatic animals face several biotic and abiotic stressors that can significantly affect their fitness – specifically damaging their cellular protein function. as a result, they have evolved sophisticated stress-responsive mechanisms. certain endogenous proteins, the small heat shock proteins (shsps), are proposed to maintain the stability and function of proteins under stress. since the identification of the first shsps, an increasing number of shsps, mainly due to the new robust sequencing tools, continue to be identified and reported to play a critical role in the response of organisms to stress. this review explores and summarizes the contributions of the shsps implicated in the stress response of different animal species in unique environments. understanding their function is crucial for advancing our knowledge of how different animal species adapt to harsh environments while maintaining cellular homeostasis. key words: abiotic stress, biotic stress, aquatic animals, cell damage, insects, nematodes, resistance apstrakt: uvidi u male proteine toplotnog šoka: pokretači tolerancije na sredinski stres kod odabranih životinjskih vrsta insekti, nematode i vodeni organizmi izloženi su brojnim biotičkim i abiotičkim stresorima koji mogu značajno uticati na njihovu prilagođenost, posebno narušavajući funkciju ćelijskih proteina. kao odgovor na to, razvilio su sofisticirane mehanizme adaptacije. među ključnim endogenim proteinima, mali proteini toplotnog šoka (shsps) igraju ključnu ulogu u očuvanju stabilnosti i funkcije proteina pod stresom. od identifikacije prvog shsp-a, sve veći broj biva otkriven zahvaljujući naprednim sekvencirajućim alatima, a njihov značaj u stresnom odgovoru organizama postaje sve jasniji. ovaj rad istražuje i sumira ulogu shsp-a u prilagođavanju različitih životinjskih vrsta specifičnim ekstremnim uslovima. razumevanje njihove funkcije ključno je za dublji uvid u mehanizme kojima se organizmi prilagođavaju nepovoljnim sredinskim faktorima, istovremeno održavajući ćelijski balans. ključne reči: abiotički stres, biotički stres, insekti, nematode, otpornost, oštećenje ćelija, vodeni organizmi introduction insects, nematodes, and aquatic animals are regularly exposed to biotic and abiotic stressors such as increasing temperature, salinity, and heavy metal accumulation that can significantly affect their physiology, growth and development, behavior, and survival (häder et al., 2020; mugwanya et al., 2022; viitasalo & bonsdorff, 2022). changes in stressors above an organism’s threshold can disrupt its intracellular proteins and consequently induce physiological stress, cell damage, and death (basha et al., 2012; mugwanya et al., 2022). for example, in the fruit fly drosophila virilis, exposure of pupae to heat stress delayed their sexual maturity, whereas in adults, it caused sterility within seven days post-exposure (walsh et al., 2021). in the face of climate change, stressors will likely become more acute or chronic and, therefore, have more impact on organisms (guillén et al., 2022). in a simulation experiment, increased heatwaves (frequency and severity) reduced the chemosensitivity of bumblebees to sense and locate plants as their food source (nooten et al., 2024). this can, consequently, © 2025 bwambale et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 1 2 lead to the decline of the bumblebee population/ colony and food security (nooten et al., 2024). while stressors pose significant challenges to organisms, they also drive evolutionary adaptations. organisms have evolved and can employ either behavioral adaptations to avoid or escape the stressor or physiological adaptations to endure the stressor better (colinet et al., 2015; king & macrae, 2015). as a physiological response, cells synthesize small heat shock proteins (shsps) that function as chaperons by binding to the partially folded or denatured substrate proteins, preventing them from irreversible folding/denaturation or enhancing their correct functional foldings (gething & sambrook, 1992; feder & hofmann, 1999; sørensen et al., 2003; basha et al., 2012; gonzález‐tokman et al., 2020). these proteins have both constitutive and stress-inducible members (colinet et al., 2010b), with a low molecular weight (typically 12 – 42 kda) and a highly conserved α-crystallin domain (acd) consisting of 80 – 100 amino acids (morris & aquilina, 2010). under stress conditions, some shsps can accumulate and reach up to 1% of the total cellular proteins (sun et al., 2002). unlike other heat shock proteins (hsp) families, shsps do not require atp to perform their chaperone activity (miernyk, 1999). thus, they can act rapidly in response to stress, and their function is less costly energy-wise. the chaperone role of shsps is not only during stress exposure but also during normal development. shsps have been implicated in various biological processes such as cell growth, differentiation, lipid membrane polymorphism (tsvetkova et al., 2002; sun & macrae, 2005; eisenhardt, 2013), diapause (gkouvitsas et al., 2008), and lifespan (morrow et al., 2004). for instance, shsp p26 in brine shrimp (artemia sp.) has been reported to confer resistance to artemia embryos exposed to stressors, e.g., extreme temperatures, prolonged anoxia, and desiccation during encystment and diapause (qiu et al., 2006). it is noteworthy that in all developmental stages, p26 is expressed exclusively in the embryo stage (willsie & clegg, 2001) and constitutes 10 – 15 % of the total yolk protein. while the chaperone function/mechanism of p26 is reported to be by the formation of ~500kda oligomers (willsie & clegg, 2002; hibshman et al., 2023), other in vitro studies conducted with transfected mammalian cells cultured under stress show that it could be through apoptosis inhibition (villeneuve et al., 2006). transfected mammalian cells have exhibited enhanced thermotolerance (villeneuve et al., 2006), and resistance to oxidative damage induced by hydrogen peroxide (willsie & clegg, 2001). the high evolutionary conservation observed in shsps across nematodes, insects, fish, and other organisms underscores their importance in fundamental cellular processes. their amino acid sequence alignment of shsps reveals how these proteins are conserved across these taxa. in nematodes, particularly in caenorhabditis elegans, the conservation of shsps is evident. for instance, multiple amino acid sequence alignment of cehsp12 using mafft v7.490 algorithm (katoh et al., n.d.; katoh & standley, 2013) in geneious prime software 2024.0.7 showed over 86% mean pairwise similarity across various species within the caenorhabditis genus (fig. 1), similarly, in fish, such as carassius sp., cyprinus sp., sinocyclochellus sp., ctenopharyngodon sp., and culter sp., the shsp family is well-conserved, with hsp20 showing over 95% pairwise similarity in sequence alignment (fig. 2). moreover, in insects, amyelois transitella, 2 biologica nyssana ● 16 (1) june 2025: 1-22 bwambale et al. ● insights into small heat shock proteins: drivers of environmental stress tolerance in selected animal species fig. 1. amino acid sequence alignment of cehsp12 among different nematode species. the alignment was performed using the mafft algorithm in geneious prime software 2024.0.7. the mean pairwise identity over all pairs in the column is depicted-green: 30% to 100% identity; red: <30% identity 3 biologica nyssana ● 16 (1) june 2025: 1-22 bwambale et al. ● insights into small heat shock proteins: drivers of environmental stress tolerance in selected animal species 3 manduca sexta, leptosia nina, zerene cesonia, colias sp., bombyx mori, maniola sp., ostrinia nubialist, parnassius sp., chilo sp. and loxostege sticticalis, all exhibit more than 90% pairwise similarity of shsp19 (fig. 3). other hsp families, mainly classified based on molecular weight, are excellently reviewed (tissières et al., 1974; sørensen et al., 2003). in insects, nematodes, and aquatic animals, the first shsps were recognized and extracted from d. melanogaster in 1974 (tissières et al., 1974), c. elegans in 1983 (russnak et al., 1983), and brine shrimp (artemia sp.) in 1999 (liang & macrae, 1999), respectively. since then, because of genomics and transcriptomics, an increasing number of shsps has continued to be reported in several of these organisms such as the diamondback moth (plutella xylostella), c. elegans, meloidogyne hapla, penaeus fig. 2. amino acid sequence alignment of shsp19 among different insect species. the alignment was performed using the mafft algorithm in geneious prime software 2024.0.7. the mean pairwise identity over all pairs in the column is depicted-green: 30% to 100% identity; red: <30% identity 44 biologica nyssana ● 16 (1) june 2025: 1-22 bwambale et al. ● insights into small heat shock proteins: drivers of environmental stress tolerance in selected animal species fig. 3. amino acid sequence alignment of shsp20 among different fish species. the alignment was performed using the mafft algorithm in geneious prime software 2024.0.7. the mean pairwise identity over all pairs in the column is depicted—green: 30% to 100% identity; red: <30% identity monodon, v. philippinarum and the goldfish (carassius auratus) (chen & zhang, 2015; chen et al., 2022; flis et al., 2024). excellent reviews of shsp’s role and association in physiological responses to stress have been made particularly; in insect responses to temperature extremes, crowding, starvation, and hypoxia/ anoxia (denlinger et al., 2001; king & macrae, 2015; wang et al., 2016; gonzález‐tokman et al., 2020), however, no comprehensive reviews on the role of shsps in nematodes and aquatic animals have been made in response to abiotic and biotic stress. as such, this review summarizes what is known, to date, about several shsps involved in the response of insects, nematodes, and aquatic animals to abiotic and biotic stressors. understanding their function is crucial for advancing our knowledge in agricultural pest management, climate change adaptation, and biotechnology. we explore how shsps contribute to the resilience of these organisms under various environmental stressors, highlighting their relevance in mitigating environmental stress and maintaining cellular homeostasis. 5 biologica nyssana ● 16 (1) june 2025: 1-22 bwambale et al. ● insights into small heat shock proteins: drivers of environmental stress tolerance in selected animal species small heat shock proteins temperature extremes temperature is one of the most important determining factors of insect abundance and distribution (bale et al., 2002; king & macrae, 2015). exposure to high temperatures, even for a short time, can negatively affect insect growth and development (abdullah, 1961; gilbert & raworth, 1996). for instance, exposure of the fruit fly zeugodacus tau to high temperatures (34°c and 38°c) for 12 hours exhibited changes in their mating behavior, antioxidant defense, and detoxifying enzymes in a sex-specific manner (li et al., 2023). li et al. (2023) further noted that high-temperature exposure promoted mating, a behavioral adaptation strategy for survival under environmental stress. insects produce shsps as one of the various physiological responses to exposure to temperature extremes (feder & hofmann, 1999; gonzález‐tokman et al., 2020). several shsps have been associated with affording heat tolerance, heat acclimation, and hardening (dahlgaard et al., 1998; huang et al., 2007). for example, in a study tracking the level of shsps in outdoor larvae of the gall fly, eurosta solidaginis, the shsp αbcrystallin increased in expression levels prior to and during the cold winter weather (approx. at -10°c to 10°c from december to february) suggesting that it contributes to protection from cold (zhang et al., 2011). within certain limits of temperature stress, insects can recover from stress-induced coma when the stress is removed. in drosophila melanogaster, two genes (hsp22 and hsp23) were upregulated during recovery (at 25°c) but not during the cold exposure itself (at 0°c for 12 h) (colinet et al., 2010a). besides, observations of upregulation and involvement of shsps in thermal tolerance have also been studied through gene knockdown by rnai. for instance, the thermal tolerance of the soil-dwelling beetle gastrophysa atrocyanea to daily exposure to high temperatures reaching 50°c for 12 minutes, as indicated by its viability, lowered to 9% when two shsps genes (i.e., shsps 21 and 23) were silenced compared to the > 67% viability in the control (atungulu et al., 2006). desiccation stress maintaining water balance is a critical requirement for insects (hadley, 1994), as dehydration can lead to significant physiological injuries such as protein denaturation, reactive oxygen species (ros) production, nucleic acid damage, and membrane damage through lipid peroxidation (hansen et al., 2006) and, consequently, death (lopez-martinez et al., 2008). due to the severity of the cellular damage resulting from dehydration, it is better prevented than repaired. thus, it is advantageous for the insect to initiate an early response to dehydration. several shsps have been identified to offset or respond to dehydration/rehydration changes – suggesting their importance in preventing possible protein denaturation. for example, the shsp, smhsp, in the larvae of belgica antarctica, an insect that lives in antarctica, reacted to varying levels of dehydration and rehydration (lopez-martinez et al., 2008). this antarctic midge larva spends most of its time frozen in ice, but during summer, the ice melts, exposing it to potential dehydrating conditions such as intense sunlight and wind. under fast dehydration (75% rh for 36 h) – quick loss of water, shsp mrna was increased, and levels continued to be high during rapid rehydration (done by submerging larvae in water for two hours), indicating involvement in preparation for stress exposure. several other cases of shsps involvement in desiccation are presented in tab. 1. pesticides and heavy metals stress exposure to pesticides is a significant contributor to terrestrial insect population decline (sánchez-bayo & wyckhuys, 2019; van klink et al., 2020). pesticide exposure is linked to increased ros production in cells, which, if not neutralized or regulated to low levels, targets proteins whose amino acid residues are oxidation-sensitive and consequently can change protein functional structure and/or cause aggregation (kannan & jain, 2000; reichmann et al., 2018; martelli et al., 2020; li et al., 2022). besides exposure to targeted insecticides, insects can also be impacted by exposure to non-insect targeted pesticides, such as herbicides, due to sharing a common habitat with the target pests (cheng et al., 2018; d’ávila et al., 2018). for instance, glyphosate, the most used herbicide worldwide, negatively affects insect physiology, delays the growth and development of larvae, and disrupts the foraging behavior of worker honeybees apis mellifera (farina et al., 2019). also, glyphosate impaired the habituation of mosquito larvae (aedes aegypti) under field conditions (baglan et al., 2018). insects respond to pesticide exposure by overproducing shsps. ros activates heat shock factor (hsf1) to stimulate the expression of these shsps (rajak & roy, 2018). shsps contribute to repairing damage from pesticide-induced oxidative stress (otaka et al., 2006), stimulate the activity of antioxidant-related genes and antioxidant enzymes, or hinder the activation of ros transcription factors (somensi et al., 2017; yu et al., 2019; shan et al., 2020). in apis cerana, a knockdown of the shsp gene, accshsp21.7, decreased its resistance to oxidative stress resulting from exposure to glyphosate, resulting in a significantly increased 6 biologica nyssana ● 16 (1) june 2025: 1-22 bwambale et al. ● insights into small heat shock proteins: drivers of environmental stress tolerance in selected animal species ta bl e 1. e xp re ss io n of s h sp s in in se ct s in re sp on se to s ev er al s tre ss or s in se ct sp ec ie s sm al l h ea t s ho ck p ro te in /g en e e xp re ss io n pa tt er n r ef er en ce s te m pe ra tu re st re ss c or n st al k bo re r (s es am ia n on ag ri oi de s) sn oh sp 19 .5 a nd s no h sp 20 .8 m r n a le ve ls fo r b ot h ge ne s w er e up re gu la te d (w ith in 1 5 m in ) b y he at sh oc k at 4 0 °c a nd w he n la rv ae re co ve re d (a t 2 5° c ) a fte r c ol d sh oc k tre at m en t ( 5 °c to 1 7 °c ). g ko uv its as e t a l. (2 00 8) le af b ee tle (g as tro ph ys a at ro cy an ea ) sh sp s 2 1 an d sh sp s 2 3 r n a i k no ck do w n of m r n a o f b ot h ge ne s de cr ea se d vi ab ili ty fr om > 6 7% to 9 % w he n ex po se d to d ai ly h ea t s ho ck o f 5 0 °c fo r 1 2 m in ut es . a tu ng ul u et a l. (2 00 6) li ri om yz a sa tiv ae ls -h sp 19 .5 , l shs p2 0. 8 an d ls -h sp 21 .7 m r n a le ve ls o f a ll w er e si gn ifi ca nt ly in du ce d by c ol d tre at m en t ( 0 °c to 15 °c fo r 1 h r) w ith ls -h sp 20 .8 m r n a d is pl ay in g th e gr ea te st se ns iti vi ty . t hi s s ug ge st s t ha t d iff er en t s h sp s m ay re sp on d to v ar io us st re ss in te ns iti es . h ua ng e t a l. (2 00 9) eu ro st a so lid ag in is α c ry st al lin s a nd β -c ry st al lin s th e sh sp s α b -c ry st al lin in cr ea se d in p ro te in le ve ls ju st p rio r t o an d du rin g a 3m on th w in te r se as on a t a pp ro x. 10 °c to 1 0 °c . p ro te in le ve ls o f b ot h αa a nd α b c ry st al lin w er e hi gh ly in du ce d in re sp on se to th aw c on di tio ns (3 °c ). zh an g et a l. (2 01 1) si lk w or m (b om by x m or i) sh sp 19 .9 , s hs p2 0. 1, sh sp 20 .4 , s hs p2 0. 8, sh sp 21 .4 , sh sp 23 .7 a nd sh sp 21 .4 . m r n a le ve ls o f a ll ge ne s w er e up re gu la te d by h ea t s tre ss (4 5° c fo r 3 5 m in ut es ) e xc ep t sh sp 21 .4 , w hi ch w as d ow nr eg ul at ed . sa ka no e t a l. (2 00 6) , l i e t a l. (2 01 2) r ed fl ou r b ee tle (t ri bo liu m c as ta ne um ) tc hs p1 8. 3 m r n a le ve ls o f t he g en e w er e up re gu la te d in re sp on se to h ea t s tre ss (4 5° c fo r 1 – 1 2 h) b ut no t t o co ld st re ss (4 °c fo r 1 – 1 2 h) . x ie e t a l. (2 01 9) fr ui tfl y (d ro so ph ila m el an og as te r) h sp 22 a nd h sp 23 th e re m ov al o f t he g en e’ s m r n a b y r n a i in te rr up te d re co ve ry (t im e to re co ve r a nd m ob ili ty fo llo w in g re co ve ry ) f ro m c hi ll in ju ry th us sh ow in g th at u pr eg ul at io n of th e ge ne s i s re qu ire d fo r r ec ov er y, b ut n ot d ur in g th e co ld st re ss it se lf. c ol in et e t a l. (2 01 0a ) 7 biologica nyssana ● 16 (1) june 2025: 1-22 bwambale et al. ● insights into small heat shock proteins: drivers of environmental stress tolerance in selected animal species fl es h fly (s ar co ph ag a cr as -s ip al pi s) h sp 23 d el et io n of g en es ’ m r n a re du ce d co ld ha rd in es s. r in eh ar t e t a l. (2 00 7) w es te rn fl ow er th rip (f ra nk lin ie lla o cc id en ta lis ) fo h sp 11 .6 a nd f oh sp 28 .0 m r n a le ve ls o f b ot h ge ne s w er e in du ce d by b ot h lo w (10 °c to 0 °c fo r 1 h ) a nd h ig h (3 9° c a nd 4 1° c fo r 1 h ) t em pe ra tu re s w ith m ax im um e xp re ss io n le ve ls a tta in ed a fte r 0. 5 – 1 h of te m pe ra tu re st re ss e xp os ur e. a ls o, th er m ot ol er an ce w as re du ce d w he n bo th g en es w er e si le nc ed b y r n a i. y ua n et a l. (2 02 2) c hi lo su pp re ss al is c sh sp 19 .0 m r n a le ve l o f t he g en e w as u pr eg ul at ed a s a re sp on se to h ea t a nd c ol d st re ss e xt re m es in a ra ng e of – 11 °c to 4 3 °c (w ith 2 7 °c a s a co nt ro l) fo r e xp os ur e du ra tio n of 2 h . d on g et a l. (2 02 1) sp od op te ra fr ug ip er da sf sh sp 21 .3 , s fs h sp 20 , s fs h sp 20 .1 , s fs h sp 19 .3 , an d sf sh sp 29 . a ll m r n a s o f t he g en es w er e si gn ifi ca nt ly up re gu la te d at b ot h te m pe ra tu re e xt re m es (4 2° c a nd 4 °c fo r a d ur at io n be tw ee n 0 – 15 0 m in ut es ) e xc ep t t w o ge ne s (s fs h sp 20 .1 a nd s fs h sp 19 .3 ) i n th e ad ul t m al es th at d id n ot re sp on d to th e 4° c tre at m en t. y an g et a l. (2 02 1) pe st ic id e an d he av y m et al st re ss ap is c er an a ce ra na ac cs h sp 21 .7 a k no ck do w n of th e sh sp g en e’ s m r n a de cr ea se d th e re si st an ce o f t he in se ct to a co m m er ci al h er bi ci de g ly ph os at e, re su lti ng in si gn ifi ca nt m or ta lit y. h ua ng e t a l. (2 02 3) fa ll ar m yw or m (s po do pt er a fr ug ip er da ) sh sp 19 .0 7, sh sp 20 .7 a nd sh sp 19 .7 4. m r n a le ve ls o f a ll ge ne s w er e up re gu la te d fo llo w in g ex po su re to th e c hl or an tra ni lip ro le pe st ic id e. t ho ug h sh sp 19 .7 4 re ac he d m ax im um m r n a e xp re ss io n le ve ls fa st er (8 h af te r e xp os ur e) th an th e re st (1 2h ), its le ve ls pl um m et ed a t 1 2 h af te r e xp os ur e, su gg es tin g a m om en ta ry re sp on si ve ne ss o f s h sp s to pe st ic id e tre at m en t. sa m an ta e t a l. (2 02 1) 8 biologica nyssana ● 16 (1) june 2025: 1-22 bwambale et al. ● insights into small heat shock proteins: drivers of environmental stress tolerance in selected animal species d ia m on db ac k m ot h (p lu te lla x yl os te lla ) fo ur te en sh sp s ( sh sp 27 .5 , s h sp 28 .9 , s h sp 21 .6 , sh sp 18 .8 , s h sp 19 .2 2, sh sp 21 .8 , s h sp 21 .9 , sh sp 22 .1 , s h sp 23 .4 , s h sp 19 .5 , s h sp 20 .0 6, sh sp 20 .0 9, sh sp 19 .2 3, sh sp 20 .1 ) fo ur th in st ar la rv ae w er e ex po se d to v ar io us pe st ic id es a nd h ea vy m et al s f or 2 4 hr . s h sp m r n a le ve l r es po ns es w er e as b el ow : b et acy pe rm et hr in p es tic id e si gn ifi ca nt ly up re gu la te d al l e xc ep t s h sp 20 .0 9, w he re as ch lo rf en ap yr p es tic id e do w nr eg ul at ed a ll ex ce pt sh sp 28 .9 . e xp re ss io n re sp on se s t o in do xa ca rb a nd c an th ar id in w er e irr eg ul ar . ex po su re to h 2o 2 fo r 2 4 h do w nr eg ul at ed fi ve sh sp s ( sh sp 19 .2 2, sh sp 19 .2 3, sh sp 21 .6 , sh sp 22 .1 , a nd sh sp 23 .4 )c op pe r ( c u2 +) do w nr eg ul at ed th re e sh sp s ( sh sp 20 .1 sh sp 22 .1 , s h sp 28 .9 ) a nd u pr eg ul at ed se ve n sh sp s ( sh sp 19 .2 2, sh sp 19 .2 3, sh sp 20 .0 6, sh sp 20 .0 9, sh sp 21 .8 , s h sp 21 .9 , s h sp 27 .5 ). m an ga ne se (m n2 +) u pr eg ul at ed fo ur sh sp s (s h sp 20 .1 , s h sp 21 .6 , s h sp 22 .1 , s h sp 28 .9 ) an d up re gu la te d al l t he re st . n ic ke l ( n i2 +) up re gu la te d (s h sp 19 .2 2, sh sp 19 .5 , sh sp 20 .0 6, sh sp 20 .0 9) , n ot in du ce d (s h sp 20 .1 , s h sp 21 .8 , s h sp 21 .9 ), an d th e re st w er e do w nre gu la te d. g en e ex pr es si on re sp on se to l ea d (p b2 +) w as ir re gu la r. c he n & z ha ng (2 01 5) d ap hn ia m ag na el ev en sh sp g en es (t er m ed d m sh sp 1 d m sh sp 11 ) in se ct e xp os ur e to h ea vy m et al s ( c d2+ , c u2+ , a nd z n2+ ) u pr eg ul at ed m r n a le ve ls of d m sh sp 1 an d d m sh sp 5. r n a i kn oc kd ow n of g en es d m sh sp 1– 21 , e xc ep t d m sh sp 11 –1 2. 8, in cr ea se d su sc ep tib ili ty to he av y m et al st re ss e xp os ur e. li e t a l. (2 02 2) c hi ro no m us ri pa ri us hs p1 7, h sp 21 , h sp 22 , h sp 23 , h sp 24 , h sp 27 , an d hs p3 4 fo llo w in g ac ut e ex po su re to c ad m iu m (c d) , m r n a le ve ls o f hs p2 3, h sp 24 , h sp 27 , a nd h sp 34 w er e up re gu la te d, w he re as le ve ls o f hs p1 7 an d hs p2 1 re m ai ne d un al te re d. t hi s in di ca te s t ha t s h sp s h av e di ve rs e ro le s d ur in g re sp on se to c d. m ar tín -f ol ga r & m ar tín ez g ui ta rte (2 01 7) 9 biologica nyssana ● 16 (1) june 2025: 1-22 bwambale et al. ● insights into small heat shock proteins: drivers of environmental stress tolerance in selected animal species c hi ne se ri ce gr as sh op pe r ( o xy a ch in en si s) o cg rp 78 , o ch sp 70 , o ch sp 90 , a nd o ch sp 40 fo llo w in g ex po su re to c ad m iu m (c d) , m r n a ex pr es si on le ve ls o f a ll ge ne s i nc re as ed , re ac hi ng a m ax im um w ith in a sh or t p er io d (6 h) , a lb ei t d ec re as in g si gn ifi ca nt ly a fte r 1 2 h. zh an g et a l. (2 01 5) c ro w di ng s tr es s m ig ra to ry lo cu st s (l oc us ta m ig ra to ri a l) h sp 20 .5 , h sp 20 .6 , a nd h sp 20 .7 m r n a s o f a ll sh ps w er e m or e ex pr es se d in g re ga rio us p ha se s ( re pr es en tin g hi gh po pu la tio n de ns ity ) c om pa re d to so lit ar y ph as es (r ep re se nt in g lo w p op ul at io n de ns ity ) w an g et a l. (2 00 7) a us tra lia n pl ag ue lo cu st (c ho rt oi ce te s t er m in ife ra ) h sp 20 .5 a nd h sp 20 .7 c ro w di ng (d ur in g th e gr eg ar io us p ha se ) re su lte d in a 2 – 3 fo ld si gn ifi ca nt u pr eg ul at io n of m r n a le ve ls o f b ot h ge ne s. c ha pu is e t a l. (2 01 1) st ar va tio n st re ss m ul be rr y py ra lid c at er pi lla r (g ly ph od es p yl oa lis ) g ph sp 19 .5 , 2 0, 2 0. 2, a nd 2 1. 6, g ph sp 21 .8 a nd g ph sp 21 .4 g en es w er e up re gu la te d tim ede pe nd en tly , re ac hi ng m ax im um le ve ls o n th e si xt h da y of fo od d ep riv at io n. o n th e co nt ra ry , m r n a e xp re ss io n le ve ls o f t w o g ps h sp s (g ph sp 21 .8 a nd g ph sp 21 .4 ) d em on st ra te d in te rm itt en t d ow nr eg ul at io n in c om pa ris on to th e co nt ro l a t 2 o r 4 d ay s f ol lo w in g th e st ar va tio n pe rio d. c hu e t a l. (2 02 0) fr ui tfl y (d ro so ph ila m el an og as te r) h sp 27 sh sp w as k no ck ed o ut , a nd fl ie s s ho w ed a si gn ifi ca nt d ec re as e in re si st an ce to st ar va tio n. h ao e t a l. (2 00 7) h ou se fly (m us ca d om es tic a) m do m h sp 27 , m do m h sp 10 , m do m h sp 27 .1 , a nd m do m h sp 27 .2 th e m r n a e xp re ss io n of m do m h sp 27 w as si gn ifi ca nt ly d ow nr eg ul at ed a fte r a 6h st ar va tio n pe rio d, w he re as m r n a ex pr es si on o f o th er 3 m do m h sp s (m do m h sp 10 , m do m h sp 27 .1 an d m do m h sp 27 .2 ) w er e no t s ig ni fic an tly aff ec te d ti an e t a l. (2 01 8) r ed fl ou r b ee tle (t ri bo liu m c as ta ne um ) tc hs p1 8. 3 w he n m r n a o f s h sp g en e w as k no ck ed do w n, th e lif es pa n of a du lt be et le s w as re du ce d by 1 5. 8% (t he y di ed w ith in 1 8 da ys a fte r st ar va tio n) c om pa re d to th e co nt ro l g ro up . x ie e t a l. (2 01 9) 10 biologica nyssana ● 16 (1) june 2025: 1-22 bwambale et al. ● insights into small heat shock proteins: drivers of environmental stress tolerance in selected animal species pt er om al us p up ar um pp h sp 20 m r n a le ve ls o f t he sh sp in cr ea se d si gn ifi ca nt ly a fte r 6 h o f s ta rv at io n bu t de cl in ed a fte r 2 4 h w an g et a l. (2 01 2) d ia m on db ac k m ot h (p lu te lla x yl os te lla ) sh sp 20 .1 , s h sp 21 .6 , s h sp 22 .1 , a nd sh sp 28 .9 m r n a e xp re ss io n le ve ls o f a ll sh sp s w er e si gn ifi ca nt ly d ow nr eg ul at ed fo llo w in g fo od st ar va tio n fo r 2 1 h. c he n & z ha ng (2 01 5) mortality rate (huang et al., 2023). the promoter region of the accshsp21.7 gene was rich in binding sites for oxidative stress transcription factors creb (camp response element-binding protein) and hsf (heat shock factor) (huang et al., 2023). creb can help the insect save energy by transitioning to the diapause stage (king & macrae, 2015) e.g., under hypoxia conditions (wang et al., 2020), whereas hsf turns on heat shock genes that subsequently drive more shsp synthesis (brunquell et al., 2016). the buildup of heavy metals (hms) beyond their required range is toxic and has been linked to oxidative stress, as the buildup enables the formation of free radicals (jomova & valko, 2011; jaishankar et al., 2014). also, metals such as cadmium (cd) can disrupt the proteins involved in dna repair and dna damage signaling (hartwig et al., 2002). in addition, cd has a high affinity for and interacts with the thiol groups in cysteine residues, disrupting/denaturing protein structure and consequently stimulating the expression of chaperone proteins such as shsps (martín-folgar & martínez-guitarte, 2017). in daphnia magna, a model insect for investigating hm toxicity, shsps like dmshsp1 and dmshsp5 were upregulated when the insect was exposed to hms (cadmium ions (cd2+), copper ions (cu2+), and zinc ions (zn2+), perhaps preventing potential protein agglutination and denaturation (li et al., 2022a). rnai knockdown of these genes (i.e. dmshsp1 and dmshsp5) increased susceptibility to hm stress exposure (li et al., 2022). it is noteworthy that besides metallothionein (mt), shsps are being explored as possible bioindicators of metal contamination because of their sensitivity to small changes in cell homeostasis and their conserved nature in many species (denlinger et al., 2001; morales et al., 2011; martín-folgar & martínez-guitarte, 2017). hypoxia/anoxia stress several adult insects or during certain stages of their life cycle live in habitats with consistent or bursts of oxygen levels below the normal 20.94%, such as ground burrows, decaying organic matter, aquatic ecosystems, flood-prone soils, and high altitudes (hoback & stanley, 2001). under hypoxic conditions, organisms employ shortand long-term mechanisms to increase their access to oxygen or decrease their oxygen demand, and failure can result in tissue damage or death (liu et al., 2006; michaud et al., 2011). therefore, the ability to survive in hypoxic or anoxic conditions is necessary for the survival of these insects. michaud et al. (2011) and zhang et al. (2011) reported an association of shsp upregulation with insect exposure to hypoxia/anoxia conditions. in s. crassipalpis, which typically burrows underground (basson & terblanche, 2010), mrna levels of three shsps (hsp25, hsp23, and hsp18) were upregulated during hypoxia (3% oxygen) treatment application, and declining 2 h post-treatment application (michaud et al., 2011). in larvae of gall fly, eurosta solidaginis, the protein levels of both αa and αb crystallin increased in response to anoxia (exposure period of 24 h under nitrogen gas at 15°c) (zhang et al., 2011). these studies suggest that the respective shsps are involved in anoxic/hypoxic stress response with a potential role in maintaining protein homeostasis during and shortly after stress exposure. crowding and starvation stresses generally, when organisms are crowded, they are at risk of being stressed (wang et al., 2007), and consequently, their growth and development processes are affected. for instance, in the migratory locust species locusta migratoria l, the fecundity of solitary locusts was higher compared to that of gregarious locusts (representative of crowding) (albrecht et al., 1959). as a stress factor, population density has been shown to affect the physiology of organisms, such as inducing the production of shsps, as shown in tab. 1. in l. migratoria l, mrnas of three shsps (hsp20.5, hsp20.6, and hsp20.7) were more expressed in gregarious phases compared to solitary phases (wang et al., 2007). it is worth noting that insects can experience multiple stressors in crowded conditions, including starvation, pathogen infection and desiccation. therefore, given 11 biologica nyssana ● 16 (1) june 2025: 1-22 bwambale et al. ● insights into small heat shock proteins: drivers of environmental stress tolerance in selected animal species 11 that shsp induction is in response to any stressor that causes protein unfolding (parsell & lindquist, 1993), it is difficult to decipher the contributions of a single stressor to shsp induction (wang et al., 2007). within the context of starvation, when an insect is deprived of food, its energy homeostasis can be damaged (chu et al., 2020). shsps have been implicated in responding to this stress sensitively (tab. 1). for instance, in the 4-day-old larvae of the housefly musca domestica, the expression of mdomhsp27 was significantly down-regulated after a six-hour starvation period (tian et al., 2018). under conditions of starvation, silencing of tchsp18.3 in tribolium castaneum (xie et al., 2019), and hsp27 gene in drosophila melanogaster (hao et al., 2007) reduced the lifespan of the insect by 15.8% and 39% compared to the control. these findings suggested that shsps can be sensitively responsive to starvation in insects. parasitism stress parasitism can induce shsps in host organisms (rinehart et al., 2002), for instance eliciting shsps in response to the venom injection. envenomation of pieris rapae by the endoparasitic wasp pteromalus puparum stimulates the expression of hsp20 in the host (zhu et al., 2013). also, envenomation of the larvae of plodia interpunctella by the ectoparasitoid bracon hebetor stimulated upregulation of shsp (shsp) (shim et al., 2008). these results suggest that shsps could be involved in immune responses to parasite attack. uv radiation stress some studies have indicated a correlation between uv-induced stress and the expression of shsps in insects. uv radiation, such as uv-a, photo-oxidizes photosensitizers inside insect cells resulting in the production of large quantities of ros that ultimately impair the function of proteins (cadet et al., 2005; meng et al., 2009; meyer-rochow et al., 2002). for instance, uv-a irradiation on spodoptera frugiperda adults resulted in increased expression levels of five shsps genes (sfshsp21.3, sfshsp20, sfshsp20.1, sfshsp19.3, and sfshsp29) indicating that they play a role in molecular response mechanisms (yang et al., 2021). similarly, expression levels of hsp22.6 and hsp27.6 in the honeybee apis cerana cerana (liu et al., 2012; zhang et al., 2014), as well as those of hsp27 and hsp21.8b in the beetle tribolium castaneum, were upregulated upon exposure to uv radiation (sang et al., 2012; xie et al., 2018). small heat shock proteins in nematodes heat stress heat stress is a major environmental challenge for nematodes, particularly with the increasing impact of global climate change. increasing temperatures can cause protein denaturation and malfunction, which in turn activates the heat shock response (kayastha et al., 2024). during this process, shsps are upregulated to aid in the refolding of damaged proteins, thus boosting the thermotolerance of nematodes (kyriakou et al., 2022). research has demonstrated that c. elegans relies on shsps for survival under high-temperature conditions, highlighting their critical role in protecting against heat-induced stress (zevian & yanowitz, 2014). for instance, cehsp16.1 and cehsp16.2 are among the first proteins to be upregulated in response to heat shock at 35°c. their deletion significantly reduces survival under prolonged heat stress, specifically at 35°c over 2 hours (strayer et al., 2003). other nematodes also exhibit a robust heat shock response. for example, in meloidogyne hapla, shsps mhhsp12.2, mhhsp6, mhhsp1, and mhhsp43, are induced by heat stress with higher expression levels at 35-40°c (flis et al., 2024). in bursaphelenchus xylophilus, bx-shsp16a, bx-shsp16b, bxshsp21, and bxshsp25 are upregulated in response to both heat and cold stress, playing a dual role in temperature adaptation (wang et al., 2016). in strongyloides ratti, the expression of srhsp17 is significantly elevated during heat stress and when exposed to pathogens, particularly in the infective larval stage whereas shsp18 and shsp22 are upregulated at 37°c in third-stage larvae of the filarial nematode brugia pahangi (jecock & devaney, 1992; younis et al., 2011). although these findings strongly suggest a protective role, they primarily indicate correlation rather than definitive causation. cold stress although often less discussed, cold stress can also impact nematodes, particularly in environments with seasonal temperature fluctuations. cold exposure can affect the fluidity of cellular membranes and the stability of proteins (fonseca et al., 2019). shsps are involved in the cold shock response of organisms, helping to preserve protein stability under low temperatures (sadura & janeczko, 2024). evidence suggests that shsps are upregulated in nematodes exposed to cold environments, aiding in their ability to maintain cellular functions and protect proteins from cold-induced damage (peter & candido, 2002). for example, in c. elegans, heat shock factor 1 (hsf-1) triggered inducing the nematode into an arrested state of development (diapause) at 4°c, and this is essential for maintaining cellular stability under cold temperatures (horikawa et al., 2024). similarly, in m. hapla, mhhsp12.2 plays a dual role, being upregulated both during heat and cold shock. 12 biologica nyssana ● 16 (1) june 2025: 1-22 bwambale et al. ● insights into small heat shock proteins: drivers of environmental stress tolerance in selected animal species the expression of this gene at low temperatures (-20°c) enhances cold tolerance, allowing the nematode to survive periods of cold exposure during the winter season (flis et al., 2024). additionally, in panagrolaimus davidi, a cold-adapted nematode from antarctic environments, shsps are crucial in ensuring survival at subzero temperatures, by maintaining protein stability and membrane fluidity under freezing conditions (thorne et al., 2014). desiccation stress desiccation, or dehydration, also poses a serious threat to nematode survival. during dehydration, cellular structures, including proteins, are at risk of damage. shsps play a vital role in enhancing desiccation tolerance by stabilizing proteins and cellular structures and preventing irreversible damage during water loss (hibshman et al., 2023). nematodes can survive extreme desiccation, which helps them maintain cellular integrity during periods of water scarcity (adhikari et al., 2010). in c. elegans, the dauer larvae stage is particularly adapted to survive harsh desiccation conditions and exhibits high levels of shsp p16 (erkut et al., 2013). thus, shsps enable nematodes to thrive in environments where water availability is unpredictable, making them essential for desiccation tolerance. similarly, in panagrolaimus superbus, a model species for desiccation research, both pshsp17.1, and pshsp20 are upregulated during desiccation and dehydration (tyson et al., 2012). anhydrobiosis and hypoxia stress in extreme cases, such as anhydrobiosis (a state of suspended animation in response to complete desiccation) and hypoxia (low oxygen conditions), shsps have been found to play a protective role. during anhydrobiosis, where nematodes experience extreme dehydration, shsps help stabilize cellular components, preventing damage during the transition to and from the dried and hypoxic state (lee & lee, 2013). in heterorhabditis indica, dnj-13, which is a class b j-protein belonging to hsp40 family, is upregulated when subjected to anhydrobiotic conditions (balakumaran et al., 2022) allowing the nematode to survive complete dehydration for extended periods. similarly, under hypoxic conditions, shsps contribute to cellular protection by maintaining protein homeostasis. for example, in c. elegans, the shsp hsp-16.1 and hsp-16.2 were upregulated following a one-hour exposure to hypoxic conditions (hong et al., 2004). while shsps likely play a significant role in stabilizing cellular components during these stresses, it is important to note that anhydrobiosis is a complex process involving the coordinated action of multiple genes in nematodes (evangelista et al., 2017). chemical stress nematodes, like many organisms, are frequently exposed to a variety of chemical stressors in their environments, including hms, pesticides, and other toxic compounds (kim et al., 2014). in c. elegans, shsps are often co-expressed with other antioxidant enzymes, such as superoxide dismutase (sod) and catalase (cat), providing a coordinated defense against oxidative damage caused by hm stress (sampayo et al., 2003). ezemaduka et al. (2017b), showed that the expression of cehsp17 increases when c. elegans are exposed to toxic hms like cd and zn. as earlier mentioned in the previous section, these metals disrupt protein folding and lead to the formation of misfolded protein aggregates. shsps such as cehsp17 play a critical role in preventing irreversible aggregation and facilitating the disaggregation of misfolded proteins. pesticides and other xenobiotic compounds are widely present in agricultural soils, posing a significant chemical stress challenge to nematodes. shsps have been implicated in the defense against pesticide-induced stress. for example, exposure to the synthetic pyrethroid insecticide cypermethrin leads to oxidative damage and stress responses in c. elegans. shashikumar & rajini, 2010, demonstrated that cypermethrin exposure at non-lethal concentrations induces the expression of hsp-16 in a concentrationand timedependent manner. marked upregulation of hsp-16 was observed throughout the nematode’s body after 24 hours of exposure, suggesting its involvement in mitigating cypermethrin-induced stress. while these findings highlight the potential role of hsp16 in responding to pesticide stress, the study did not provide direct evidence of reduced protein aggregation or enhanced cellular recovery in the presence versus the absence of hsp-16. further research is required to clarify the mechanistic role of shsps in protecting against pesticide-induced damage. small heat shock proteins in aquatic animals heavy metal, temperature, and salinity stress poor disposal of industrial waste often leads to contamination and accumulation of heavy metals (hms) in aquatic ecosystems (gaur et al., 2020; sonone et al., 2021). cadmium, considered the most dangerous hm to aquatic animals, is the most common contaminant from industrial waste (khan et al., 2022). even at low concentrations, this hm can accumulate in the vital organs of aquatic animals, leading to their death (moiseenko & 13 biologica nyssana ● 16 (1) june 2025: 1-22 bwambale et al. ● insights into small heat shock proteins: drivers of environmental stress tolerance in selected animal species gashkina, 2020; das et al., 2023;). as mentioned in previous sections, hm stress leads to oxidative stress in exposed tissues due to the accumulation of ros (tamás et al., 2017; georgiadou et al., 2018). moreover, hms and metalloids negatively impact protein homeostasis and the viability of cells by interfering with the protein folding processes in cells (tamás et al., 2014; balali-mood et al., 2021). shsps have been reported to play a vital role in alleviating the stress effects of hms in certain aquatic animals. mnhsp28.6 was found to predominately accumulate in the muscles of the oriental river prawn (macrobrachium nipponense) upon exposure to cd and cu ions and hence thought to be involved in animal response to hm (yuan et al., 2019). moreover, in vitro expression of this shsp in escherichia coli conferred host cells impressive protection against hydrogen peroxide (h₂o₂) compared to control e. coli suggesting its responsibility in alleviating oxidative stress induced by h₂o₂ (yuan et al., 2019). in another study, zhang et al. (2013) demonstrated that exposing razor clam (sinonovacula constricta) to hm stress (cd and lead (pb)) led to an increased accumulation of scshsp in haemocytes, gonads, and mantle hence confirming shsp’s linkage to tolerance responses to hm toxicity stress in s. constricta. likewise, yang et al. (2012) observed increased expression of shsp lchsp27 in the liver and brain of yellow croaker (larimichthys crocea) exposed to cd ions. moreover, lchsp27 expression was enhanced in the liver under high and low temperatures thus indicating its role in protecting the liver from multiple stress factors. in another study, li et al. (2010) reported improvement in the level of expression of vpshsp-2 in manila clam (venerupis philippinarum) exposed to cd ion stress. a previous study on hard clam (meretrix meretrix) showed that exposing the animal to cd stress led to increased expression of mrna levels of mm-hsp 20 in the digestive gland and hemocytes hence indicating its role in hm detoxification and protection against oxidative stress (li et al., 2013). li et al. (2022) exposed daphnia magna to different levels of heavy metals (cd, cu, and zn), and the authors observed the expression of eleven shsps (dmshsp1 – dmshsp11) under hm stress. exposing the animals to cu2+ strongly induced the expression of dmshsp1 while it was moderately induced under cd2+ and zn2+ exposure. the authors also observed that dmshsp5 was induced in all the hm stress. temperature stress is one of the major abiotic stress factors affecting the survival of aquatic animals. changes in the environmental temperatures below or above the organism’s thermal threshold cause oxidative stress that could hinder the survival and general well-being of the organism (mugwanya et al., 2022). the expression of shsps in tissues of stressed animals could aid in the alleviation of temperature stress effects in the organisms. for instance, bildik et al. (2019) investigated changes in the expression of hsps (shsp 30) in gilt-head bream (sparus aurata) exposed to high and low temperatures and observed a two-fold increase in the expression of hsp30 under higher temperatures (27°c), which they correlated to improved thermal tolerance in fish. in another study, chen et al. (2022) observed an increase in the relative mrna expression of shsp20 in the spleen of goldfish (carassius auratus) exposed to high temperatures (32°c), which correlated with fish thermal tolerance. currie (2000) demonstrated that acclimatization of rainbow trout (oncorhynchus mykiss) to higher temperatures (25°c) led to increased expression of shsp30 in all tissues except blood. likewise, liu et al. (2019) reported increased expression of shsp30 mrna levels in the gills of o. mykiss exposed to temperature stress (25°c), indicating its association with heat stress tolerance in fish. a previous study on pool barb (puntius sophore) showed increased expression of hsp47 when exposed to temperature stress (41°c) (mahanty et al., 2017). salinity stress in aquatic animals leads to the accumulation of ros in tissues, an overaccumulation of which leads to cell damage and apoptosis. expression of certain shsps in aquatic animals exposed to high or low salinity stress has been shown to enhance the stress tolerance of the organism. shekhar et al. (2013) investigated stress responses of p. monodon exposed to low salinity stress and observed increased expression of hsp21 in tissues thus suggesting its role in salinity stress adaptive mechanism in shrimp. a recent study by zarei et al. (2024) has shown that an increase in mrna expression levels of hsp27 was associated with immune responses and cell survival in sterlet sturgeon (acipenser ruthenus) under salinity stress. pathogenic infection shsps induced by biotic stress play a significant role in protein folding, enhancement of immune responses, and protection against diseases caused by various pathogenic organisms and viruses (muthusamy et al., 2017). although several studies have previously reported on the induction of shsps by abiotic stress factors, little information is known about the induction of shsps by abiotic stress factors. certain studies have suggested that members of the hsp20, and hsp37, among others, occur in crustaceans and several fish species. chen et al. (2022) conducted a study on goldfish (c. auratus) to understand how pathogen infection can 14 biologica nyssana ● 16 (1) june 2025: 1-22 bwambale et al. ● insights into small heat shock proteins: drivers of environmental stress tolerance in selected animal species induce the expression of shsps in animal tissues. the authors observed that intraperitoneal injection of lipopolysaccharide (lps) or polyinosinicpolycytidylic acid (poly i:c) induced the expression of hsp20 in the spleen, thus improving the tolerance of the fish to stress. arockiaraj et al. (2012) reported that the expression of mrhsp37 in freshwater prawns (macrobrachium rosenbergii) triggered an immune response upon exposure to pathogen infection (hypodermal and hematopoietic necrosis virus). li et al. (2010) demonstrated that exposing manila clam (v. philippinarum) to a pathogen challenge (vibrio anguillarum) induced the upregulation of vpshp-1in haemocytes and increased 1.5-fold and 9.9-fold 6 h and 96 h post-infection, respectively. likewise, the mrna expression level of vpshp-2 increased by 8.7-fold compared to the control group 24 h post-infection. certain studies have, however, shown a downregulation of shsps in tissues of aquatic animals exposed to biotic stress factors. for instance, huang et al. (2008) demonstrated that when challenged with the white spot syndrome virus, penaeus monodon has a significantly decreased expression of hsp21 thus indicating that the gene regulation of shsp 21 was negatively affected by the virus. conclusions environmental stressors pose significant challenges to the survival, fitness, and distribution of insects, nematodes, and aquatic organisms, particularly in the context of climate change and increased anthropogenic pressures. small heat shock proteins (shsps) play an essential role in the resilience of these organisms. this review highlights the broad and highly conserved role of shsps across species in responding to various biotic and abiotic stressors discussed above. future research should aim to unravel further the molecular mechanisms and pathways regulated by shsps, particularly in the context of multiple simultaneous stressors. integrating genomics, proteomics, and transcriptomics approaches will be key to uncovering the full spectrum of shsp functions. moreover, exploring the evolutionary conservation of shsps across taxa will provide deeper insights into their potential as universal stress-resilience mechanisms. ultimately, enhancing our understanding of shsps will not only advance fundamental biological knowledge, but also inform practical applications in fields such as agriculture, conservation biology, and environmental monitoring, helping to safeguard biodiversity and ecosystem stability in an era of unprecedented environmental change. acknowledgements. the authors wish to thank mr. fahad kimera, manager of agriculture cares research, for his professional support during the writing of this manuscript. references abdullah, m. 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tamilnadu, india https://orcid.org/0000-0002-5480-5782 received: september 06, 2024 revised: september 28, 2024 accepted: september 30, 2024 abstract: pleurotus mushrooms, commonly known as oyster mushrooms, are celebrated for their edibility and emerging health benefits. this study investigates the bioactive components and functional groups in pleurotus florida mushrooms, focusing on polysaccharides, peptides, fatty acids, and phenolic compounds. comprehensive analysis identified functional groups and diverse range of 37 bioactive compounds, each exhibiting distinct biological activities. these include treatment for phobic disorders, antiseborrheic effects, sclerosant properties, cholesterol antagonism, antieczematic effects, regulation of lipid metabolism, antihypercholesterolemic properties, antisecretoric activity, and cardiovascular analeptic effects. additionally, the study highlights the potential synergistic effects of these compounds, which may contribute to their overall therapeutic efficacy. the findings underscore the promise of pleurotus florida as a valuable source of functional compounds with diverse and significant therapeutic applications, warranting further research to fully explore their potential benefits in clinical settings. key words: bioactive compounds, pleurotus florida, ft-ir, gcms, oyster mushroom apstrakt: bioaktivne komponente i terapeutski potencijal pleurotus florida: sveobuhvatna analiza pečurke roda pleurotus, poznate kao bukovače, poznate su po svojoj jestivosti i sve većim zdravstvenim koristima. ova studija istražuje bioaktivne komponente i funkcionalne grupe u pečurkama pleurotus florida, sa fokusom na polisaharide, peptide, masne kiseline i fenolna jedinjenja. detaljna analiza je identifikovala funkcionalne grupe i širok spektar od 37 bioaktivnih jedinjenja, od kojih svako ispoljava različite biološke aktivnosti. ove aktivnosti uključuju lečenje fobičnih poremećaja, antiseboroične efekte, sklerozantna svojstva, antagonizam holesterola, antiekzematske efekte, regulaciju metabolizma lipida, antihiperholesterolemična svojstva, antisekretornu aktivnost i kardiovaskularne analeptičke efekte. pored toga, studija ističe potencijalne sinergističke efekte ovih jedinjenja, koji mogu doprineti njihovoj ukupnoj terapijskoj efikasnosti. rezultati naglašavaju obećavajući potencijal pleurotus florida kao vrednog izvora funkcionalnih jedinjenja sa različitim i značajnim terapeutskim primenama, što opravdava dalje istraživanje kako bi se u potpunosti istražile njihove moguće koristi u kliničkim uslovima. ključne reči: bioaktivna jedinjenja, pleurotus florida, ft-ir, gcms, bukovača introduction mushrooms are the spore-bearing fruiting bodies of matured mycelium typically formed above the soil or on its substrate. greeks and romans have taken mushrooms as food since ancient times. chinese considered mushroom as an elixir of life and romans regarded it as food of god. mushroom belongs to the class basidomycota of kingdom fungi because of unique fungal characteristics and development (song, 2004). mushrooms have drawn attention as a therapeutic food, as a source of drugs and nutritional supplements since they are hyped for their antioxidant, antitumor and antimicrobial properties. in addition to their pharmacological properties, mushrooms have become much more significant in our diet due to its high nutritional value, high protein content and low fat / energy content (khatun © 2024 selvaananthi and beulah jerlin. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 135 et al., 2012). mushrooms have predominantly been employed in conventional medicinal practices across china and various other asian nations. these fungi exhibit a plethora of beneficial properties, including antineoplastic, antibacterial, antiviral, hypoglycaemic, hypocholesterolemic, antiinflammatory, and antioxidant effects (guillamón et al., 2010; wasser, 2014). the present study reveals the various types of bioactive compounds, potential health benefits, and the exciting possibilities they hold for future applications in medicine and functional foods. materials and methods ft-ir mushroom sample was lyophilized and mixed with kbr pellets and then subjected to ft-ir spectral analysis. the dried pellets were subjected to ft-ir spectroscopy measurement in the spectral range of 4000-400 cm with resolution of 4 cm. the results were compared with standard values and the functional groups were identified. gc-ms analysis gc-ms analysis of the mushroom sample was performed using a gc clarus 500 perkin-elmer system comprising an aoc 20i autosampler and gas chromatograph interfaced to a mass spectrometer (gc-ms) equipped with an elite-1, fused silica capillary column (330 mm×0.25 mm id×1μm df, composed of 100% dimethyl polysiloxane). for gc-ms detection, an electron ionization system with ionizing energy of 70 ev was used. helium gas (99.999%) was used as the carrier gas at constant flow rate of 1ml/minute and an injection volume of 0.5 μl was employed (split ratio of 10:1); injector temperature 250 °c; ion-source temperature 280 °c. the oven temperature was programmed from 110 °c (isothermal for 2 minutes), with an increase of 10 °c/ minute, to 200 °c, then 5 °c/minute to 280 °c, ending with a 9 minutes isothermal at 280 °c. mass spectra were taken at 70 ev; a scan interval of 0.5 seconds and fragments from 40 to 550 da. total gc running time was 36 minutes. the relative percentage of each component was calculated by comparing its average peak area to the total areas, software adopted to handle mass spectra and chromatograms was a turbo mass. interpretation on mass spectrum of gc-ms was conducted using the database of national institute of standard and technology (nist) having more than 62,000 patterns. the spectrum of the unknown components was compared with the spectrum of the known components stored in the nist library. the name, molecular weight and structure of the components of the test materials were ascertained. results fi-ir analysis ft-ir analysis of the pleurotus florida mushroom powder provides the information about the functional groups present in it (fig. 1, tab. 1). the peak at 3387.25 is associated with n-h stretching, which is characteristic of aliphatic primary amines, and it shows medium intensity. a weak peak at 2081.93 136 biologica nyssana ● 15 (2) december 2024: selvaananthi and beulah jerlin ● bioactive components and therapeutic potential of pleurotus florida: a comprehensive analysis fig. 1. ft-ir spectrum of pleurotus florida biologica nyssana ● 15 (2) december 2024: selvaananthi and beulah jerlin ● bioactive components and therapeutic potential of pleurotus florida: a comprehensive analysis 137 corresponds to c≡c stretching, indicating the presence of an alkyne group. aromatic compounds are represented by a weak c-h bending peak at 1627.13, while a medium-intensity c-h bending peak at 1459.97 suggests the presence of alkanes. the 1380.70 peak is associated with c-h bending in aldehydes and is of medium intensity. a strong peak at 1138.83 corresponds to c-o stretching in aliphatic ethers. alkenes are identified by two peaks: a strong c=c bending peak at 959.82 and a medium-intensity c=c bending peak at 800.45. halo compounds are indicated by strong peaks at 608.48 and 510.66, corresponding to c-br stretching and c-i stretching, respectively. these peaks help to elucidate the chemical structure and functional groups present in the sample. gc-ms analysis from the gc-ms analysis of the mushroom extract totally 37 compounds were identified (fig. 2). with the help of the online tool pass online its biological activity was identified. tab. 2 presents a list of compounds along with their molecular weight, peak area, molecular structure, bioactivity, and probability of activity. the bioactive compounds are shown to have a wide range of therapeutic properties, such as antineoplastic, anti-inflammatory, lipid metabolism regulation, cardiovascular benefits, etc. the probability of bioactivity for each compound highlights its potential efficacy in various medical treatments. antineoplastic treatments antineoplastic agents play a critical role in inhibiting the growth and spread of tumors. several compounds are identified with antineoplastic properties in the table 1. ft-ir analysis of pleurotus florida s. no. peak value group class peak details 1 3387.3 n-h stretching aliphatic primary amine medium 2 2081.9 c≡c stretching alkyne weak 3 1627.1 c-h bending aromatic compound weak 4 1460 c-h bending alkane medium 5 1380.7 c-h bending aldehyde medium 6 1138.8 c-o stretching aliphatic ether strong 7 959.82 c=c bending alkene strong 8 800.45 c=c bending alkene medium 9 608.48 c-br stretching halo compound strong 10 510.66 c-i stretching halo compound strong fig. 2. gc-ms spectrum of pleurotus florida 138 biologica nyssana ● 15 (2) december 2024: selvaananthi and beulah jerlin ● bioactive components and therapeutic potential of pleurotus florida: a comprehensive analysis ta bl e 2. b io ac tiv e co m po un ds id en tifi ed b y g c -m s an al ys is o f p le ur ot us fl or id a s. n o. r /t n am e of th e c om po un d m ol ec ul ar w ei gh t pe ak a re a % m ol ec ul ar s tr uc tu re b io ac tiv ity pr ob ab ili ty of a ct iv ity 1 5. 49 5 tr ie th yl si la no l 13 2 1. 41 c c [s i]( c c )( c c )o a nt in eo pl as tic 0. 99 5 2 5. 49 5 fo rm ic a ci d 11 8 1. 41 c (= o )o 3 5. 49 5 m et ho xy ac et al de hy de di et hy l a ce ta l 14 8 1. 41 c c o c (c o c )( o c (c )c )c a nt io st eo po ro tic , a nt io be si ty , b on e di se as es tre at m en t 0. 95 4, 0. 95 8, 0. 92 8 4 7. 67 5 b en ze ne , 1, 2, 3tri m et hy l 12 0 23 .9 6 c c 1= c (c (= c c =c 1) c )c a nt is eb or rh ei c 0. 85 5 7. 67 5 b en ze ne , 1, 2, 4tri m et hy l 12 0 23 .9 6 c c 1= c c (= c (c =c 1) c )c a nt is eb or rh ei c, c ar m in at iv e 0. 86 , 0 .8 28 6 8. 64 1h ex an ol , 2 -e th yl 13 0 2. 06 c c c c c (c o )c c sc le ro sa nt , p ho bi c di so rd er s t re at m en t 0. 89 7, 0 .8 96 7 8. 64 2pr op yl -1 -p en ta no l 13 0 2. 06 c c c c (c c c )c o ph ob ic d is or de rs tre at m en t, sc le ro sa nt 0. 90 0, 0 .8 6 8 12 .4 1 1, 4: 3, 6d ia nh yd ro -. al ph a. -d gl uc op yr an os e 14 4 10 .2 1 c 1[ +f 15 c @ @ h ]2 [c @ @ h ]3 [c @ h ]( o 1) [c @ h ]( [c @ @ h ]( o 2) o 3) o c al ci um re gu la to r, a nt ie no pl as tic , a nt iin fla m m at or y, r es te no si s t re at m en t(n on h od gk in 's ly m ph om a) 0. 92 5, 0 .9 12 , 0. 87 8 9 12 .4 1 4, 5o ct an ed io l, 2, 7di m et hy l17 4 10 .2 1 c c (c )c c (c (c c (c )c )o )o ph ob ic d is or de rs tre at m en t, sc le ro sa nt , a nt is eb or rh ei c 0. 92 2, 0 .8 54 , 0. 82 2 10 12 .4 1 m et ha cr yl ic a ci d, et hy l e st er 11 4 10 .2 1 c c o c (= o )c (= c )c sk in ir rit at io n, a nt ie cz em at ic , ph ob ic d is or de rs tre at m en t, pe di cu lic id e, c ho le st er ol a nt ag on is t, le uk op oi es is st im ul an t 0. 95 7, 0 .8 74 , 0. 82 6, 0 .7 9, 0. 72 7, 0 .7 23 11 12 .4 1 5d ec an on e, 6hy dr ox y17 2 10 .2 1 c c c c c (c (= o )c c c c )o le uk op oi es is st im ul an t 0. 77 7 biologica nyssana ● 15 (2) december 2024: selvaananthi and beulah jerlin ● bioactive components and therapeutic potential of pleurotus florida: a comprehensive analysis 139 12 12 .4 1 4o ct an on e, 5hy dr ox y2, 7di m et hy l17 2 10 .2 1 c c (c )c c (c (= o )c c (c )c )o ph ob ic d is or de rs tr ea tm en t 0. 87 2 13 14 .2 5 m al ic a ci d 13 4 3. 46 c (c (c (= o )o )o )c (= o )o a nt is eb or rh ei c 0. 79 3 14 14 .2 5 pr op an oi c ac id , 2m et hy l-, p ro py l es te r 13 0 3. 46 c c c o c (= o )c (c )c ph ob ic d is or de rs tre at m en t, a nt is eb or rh ei c, sc le ro sa nt , a nt ie cz em at ic 0. 93 3, 0 .8 81 , 0. 79 2, 0 .7 56 15 14 .2 5 pr op an oi c ac id , 2m et hy l-, 2 -e th yl -3 hy dr ox yh ex yl e st er 21 6 3. 46 c c c c (c (c c )c o c (= o )c (c )c )o sc le ro sa nt , l eu ko po ie si s st im ul an t 0. 84 0, 0 .7 25 16 14 .2 5 3h yd ro xy de ca no ic ac id 18 8 3. 46 c c c c c c c c (c c (= o )o )o li pi d m et ab ol is m re gu la to r 0. 88 8 17 15 .1 7 te tra de ca ne 19 8 2. 59 c c c c c c c c c c c c c c a nt ie cz em at ic , a nt is eb or rh ei c 0. 89 5, 0 .8 57 18 15 .1 7 h ex ad ec an e 22 6 2. 59 c c c c c c c c c c c c c c c c a nt ie cz em at ic , a nt is eb or rh ei c 0. 89 5, 8 .8 57 19 15 .1 7 pe nt ad ec an e 21 2 2. 59 c c c c c c c c c c c c c c c a nt ie cz em at ic 0. 89 5 20 17 .2 8 d ie th yl p ht ha la te 22 2 3. 15 c c o c (= o )c 1= c c =c c =c 1c (= o )o c c c ar di ov as cu la r a na le pt ic 0. 86 8 21 20 .2 6 n on ad ec an e 26 8 1. 32 c c c c c c c c c c c c c c c c c c c c ar di ov as cu la r a na le pt ic 0. 86 8 22 20 .2 6 o ct ad ec an e 25 4 1. 32 c c c c c c c c c c c c c c c c c c a nt ie cz em at ic , a nt is eb or rh ei c, c ar di ov as cu la r a na le pt ic 0. 89 5, 0 .8 57 , 0. 86 8 23 21 .2 9 pe nt ad ec an oi c ac id , et hy l e st er 27 0 0. 74 c c c c c c c c c c c c c c c (= o )o c c m uc os iti s t re at m en t 0. 81 5 24 21 .2 9 et hy l 1 3m et hy lte tra de ca no at e 27 0 0. 74 c c o c (= o )c c c c c c c c c c c c (c )c sc le ro sa nt , l ip id m et ab ol is m re gu la to r, c ho le st er ol a nt ag on is t 0. 83 2, 0 .8 07 , 0. 79 3 25 21 .2 9 h ex ad ec an oi c ac id , et hy l e st er 28 4 9. 6 c c c c c c c c c c c c c c c c (= o )o c c fi br in ol yt ic , m uc os iti s t re at m en t 0. 76 8, 0 .7 71 26 22 .3 5 h ep ta de ca no ic a ci d, et hy l e st er 29 8 9. 6 c c c c c c c c c c c c c c c c c (= o )o c c a nt ie cz em at ic , le uk op oi es is st im ul an t 0. 91 9, 0 .8 50 biologica nyssana ● 15 (2) december 2024: selvaananthi and beulah jerlin ● bioactive components and therapeutic potential of pleurotus florida: a comprehensive analysis 27 24 .0 4 9, 12 -o ct ad ec ad ie no ic ac id (z ,z )28 0 38 .9 8 c c c c c /c =c \c /c =c \c c c c c c c c (= o )o li pi d m et ab ol is m re gu la to r, m uc om em br an ou s pr ot ec to r, c ho le st er ol an ta go ni st , a nt ih yp er ch ol es te ro le m ic , a nt is ec re to ric 0. 94 3, 0 .9 04 , 0. 87 6, 0 .8 31 , 0. 82 8 28 24 .0 4 li no le ic a ci d et hy l es te r 30 8 38 .9 8 c c c c c /c =c \c /c =c \c c c c c c c c (= o )o c c a nt ie cz em at ic , l ip id m et ab ol is m re gu la to r 0. 96 3, 0 .9 43 29 24 .0 4 9, 12 -o ct ad ec ad ie no ic ac id , e th yl e st er 30 8 38 .9 8 c c c c c /c =c /c /c =c /c c c c c c c c (= o )o c c a nt ie cz em at ic , l ip id m et ab ol is m re gu la to r, m uc om em br an ou s pr ot ec to r, c ho le st er ol an ta go ni st 0. 96 3, 0 .9 43 , 0. 90 4, 0 .8 31 30 24 .0 4 et hy l 9 .c is .,1 1. tra ns .oc ta de ca di en oa te 30 8 38 .9 8 c c c c c c /c =c /c =c \c c c c c c c c (= o )o c c a nt ie cz em at ic , l ip id m et ab ol is m re gu la to r, m uc om em br an ou s pr ot ec to r 0. 94 0, 0 .9 38 , 0. 90 4 31 27 .3 8 b is (2 -e th yl he xy l) ph th al at e 39 0 1. 54 c c c c c (c c )c o c (= o )c 1= c c =c c =c 1c (= o ) o c c (c c )c c c c c ho le st er ol a nt ag on is t, pl at el et a gg re ga tio n st im ul an t 0. 81 7, 0 .8 11 32 27 .3 8 d iis oo ct yl p ht ha la te 39 0 1. 54 c c (c )c c c c c o c (= o )c 1= c c =c c =c 1c (= o ) o c c c c c c (c )c ph ob ic d is or de rs tre at m en t, a nt is eb or rh ei c, sc le ro sa nt , f ib rin ol yt ic 0. 94 8, 0 .8 44 , 0. 79 6, 0 .7 44 33 27 .3 8 h ex ac os an e 36 6 0. 97 c c c c c c c c c c c c c c c c c c c c c c c c c c ph ob ic d is or de rs tre at m en t, a nt ie cz em at ic , c ar di ov as cu la r a na le pt ic 0. 92 4, 0 .8 95 , 0. 86 8 34 27 .3 8 h en ei co sa ne 29 6 0. 97 c c c c c c c c c c c c c c c c c c c c c ph ob ic d is or de rs tre at m en t, a nt ie cz em at ic , c ar di ov as cu la r a na le pt ic , le uk op oi es is st im ul an t 0. 92 4, 0 .8 95 , 0. 86 8, 0 .8 23 35 27 .3 8 pe nt ac os an e 35 2 0. 97 c c c c c c c c c c c c c c c c c c c c c c c c c ph ob ic d is or de rs tre at m en t, a nt ie cz em at ic , c ar di ov as cu la r a na le pt ic 0. 92 4, 0 .8 95 , 0. 86 8 140 biologica nyssana ● 15 (2) december 2024: selvaananthi and beulah jerlin ● bioactive components and therapeutic potential of pleurotus florida: a comprehensive analysis 141 36 27 .3 8 o ct ac os an e 39 4 0. 97 c c c c c c c c c c c c c c c c c c c c c c c c c c c c ph ob ic d is or de rs tre at m en t, a nt ie cz em at ic , c ar di ov as cu la r a na le pt ic 0. 92 4, 0 .8 95 , 0. 86 8 37 27 .3 8 te tra te tra co nt an e 61 8 0. 97 c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c ph ob ic d is or de rs tre at m en t, a nt ie cz em at ic , c ar di ov as cu la r a na le pt ic 0. 92 4, 0 .8 95 , 0. 86 8 study. triethylsilanol and 1,4:3,6-dianhydro-alpha-dglucopyranose are among the bioactive compounds known for their potential in cancer treatment. these compounds could inhibit cancer cell proliferation and possibly serve in therapeutic applications for various cancers, including nonhodgkin’s lymphoma. antiosteoporotic treatments bone health is crucial for preventing osteoporosis, a condition where bones become weak and brittle. methoxyacetaldehyde diethyl acetal has demonstrated antiosteoporotic potential, indicating that it could play a role in bone disease prevention or treatment. by improving bone density and preventing bone loss, this compound shows promise in contributing to treatments aimed at strengthening skeletal health. antiobesity treatments obesity is a significant health challenge, and the need for antiobesity agents is evergrowing. methoxyacetaldehyde diethyl acetal has been highlighted for its potential antiobesity effects. this compound could help regulate body fat and metabolism, providing support in weight management therapies, potentially reducing the risk of obesity-related diseases. antiseborrheic treatments seborrhea is a skin condition characterized by excessive oil production, leading to scaly patches and dandruff. several compounds, including benzene (1,2,3-trimethyl and 1,2,4-trimethyl), propanoic acid, 2-methyl-, propyl ester, malic acid, 4,5-octanediol, 2,7-dimethyl-, ocatadecane and hexadecane have been identified for their antiseborrheic properties. these compounds could aid in reducing excessive oil production and inflammation, thus contributing to the treatment of seborrheic dermatitis and related skin conditions. sclerosant treatments sclerosants are used to treat varicose veins by causing the veins to shrink. compounds such as ethyl 13-methyl-tetradecanoate, 1-hexanol, 2-ethyl, 2-propyl-1-pentanol, propanoic acid, 2-methyl-, propyl ester, propanoic acid, 2-methyl-, 2-ethyl-3hydroxyhexyl ester and 4,5-octanediol, 2,7-dimethyl exhibit sclerosant properties. these compounds could contribute to therapies designed to reduce the appearance of varicose veins and improve vascular health by inducing the closure of the affected veins. phobic disorders treatments phobic disorders, such as anxiety and irrational fears, can be debilitating. several compounds, including 1-hexanol, 2-ethyl, 2-propyl-1-pentanol, 4,5-octanediol, 2,7-dimethyl-, methacrylic acid, ethyl ester, propanoic acid, 2-methyl-, propyl ester, heneicosane, hexacosane, pentacosane, octacosane, tetratetracontane and diisooctyl phthalate are linked to the treatment of phobic disorders. these compounds may help regulate the nervous system, offering potential in managing symptoms of anxiety, stress, and related disorders, contributing to mental health therapies. lipid metabolism regulation proper lipid metabolism is vital for maintaining healthy cholesterol levels and preventing cardiovascular diseases. compounds like bis(2-ethylhexyl) phthalate, ethyl 13-methyltetradecanoate, 3-hydroxydecanoic acid, linoleic acid ethyl ester, 9,12-octadecadienoic acid (z,z), 9,12-octadecadienoic acid, ethyl ester, methacrylic acid, ethyl ester and ethyl 9.cis.,11. trans.-octadecadienoate are known to regulate lipid metabolism. these bioactive molecules could aid in breaking down fats and maintaining healthy lipid profiles, potentially benefiting individuals with metabolic disorders. cholesterol antagonism high cholesterol levels are a significant risk factor for cardiovascular diseases. several compounds, including henicosane, linoleic acid ethyl ester, 9,12-octadecadienoic acid (z,z), and ethyl 9.cis., 11.trans.-octadecadienoate, demonstrate cholesterol antagonist properties. these compounds could help reduce cholesterol levels, supporting heart health and preventing conditions such as atherosclerosis mycelium of p. ostreatus effectively inhibited the proliferation of neoplastic cells associated with ehrlich tumor and sarcoma 180. protein extract derived from p. ostreatus demonstrated a significant therapeutic impact on the colorectal cancer cell line sw 480 as well as on the monocytic leukaemia cell line thp-1 by promoting their apoptosis (wu et al., 2011). furthermore, protein extracts from p. eryngii and p. ostreatus (eryngin and pleurostrin) exhibit notable antifungal and antibacterial activities (erjavec et al., 2012). the species p. cornucopiae has yielded two oligopeptides that display antihypertensive effects (jang et al., 2011). additionally, the mycelium and fruiting bodies of p. citrinopileatus are characterized by high concentrations of ergothioneine, a watersoluble amino acid recognized for its exceptional antioxidant properties (lin et al., 2016). ribonuclease derived from p. djamor demonstrates an inhibitory effect on the proliferation of hepatic and breast cancer cells (wu et al., 2010). investigations have established that the polysaccharide-peptide complex obtained from p. abalonus significantly lowers blood glucose levels in murine models (chen et al., 2015). lectins isolated from pleurotus citrinopileatus exhibit both antineoplastic and antiviral properties (li et al., 2008). terpenoids extracted from p. cornucopiae showed cytotoxic properties towards hela and hepg2 cancer cell lines (wang et al., 2013). menikpurage et al. (2009) and alam et al. (2009) performed an investigation involving animal subjects, demonstrating that lovastatin present in the powdered fruiting bodies of p. osteratus and p. sajor-caju favorably influenced lipid profiles as well as hepatic and renal functions. the concentrations of total cholesterol and triglycerides in the bloodstream of the rats were found to be reduced. the application of oyster mushrooms within the realms of cosmetology and dermatology represents another pertinent subject. owing to their rich content of antioxidants, along with anti-aging, anti-wrinkle, whitening, and moisturizing agents, extracts from oyster mushrooms are suitable for the formulation of various cosmetics and cosmeceuticals. certain countries offer soap featuring p. ostreatus extract, known for its skin regeneration properties. the utilization of a pleuran-based cream as an adjunctive therapy for patients suffering from atopic dermatitis has showed promising outcomes. recent scientific literature substantiates the efficacy of various oyster mushroom species as viable raw materials for the cosmetics sector (taofiq et al., 2016; wu et al., 2016; morris et al., 2017). cholesterol is a crucial lipophilic molecule essential for human survival, playing numerous roles in maintaining cellular function. it serves and coronary artery disease. leukopoiesis stimulation leukopoiesis, the production of white blood cells, is essential for immune system function. compounds such as heptadecanoic acid, ethyl ester, 5-decanone, 6-hydroxy-, henicosane, methacrylic acid, ethyl ester, propanoic acid, 2-methyl-, 2-ethyl3-hydroxyhexyl ester and hexadecanoic acid, ethyl ester have shown the ability to stimulate leukopoiesis. these compounds could help boost the immune system by increasing white blood cell counts, making them potentially useful in treating conditions like neutropenia and enhancing immune response. cardiovascular analeptic treatments cardiovascular analeptics are agents that stimulate heart function and improve circulatory health. diethyl phthalate, nonadecane, ocatadecane, hexacosane, pentacosane, octacosane, tetratetracontane, and heneicosane have been found to exhibit cardiovascular analeptic properties. these compounds could help enhance heart function and overall cardiovascular health, making them potential candidates for managing heart conditions and improving circulation. discussion the pleurotus genus comprises approximately 40 distinct species, among which several hold considerable economic importance. the fruiting bodies of oyster mushrooms possess significant nutritional value and confer health-promoting benefits. furthermore, numerous species within the pleurotus genus have been recognized as sources of compounds with established medicinal attributes, including polysaccharides, peptides, and proteins, terpenoids, fatty acid esters, and polyphenols. the bioactive compounds present in pleurotus species demonstrate a variety of beneficial properties, including immunostimulation, anti-neoplastic effects, anti-diabetic activity, anti-atherosclerotic effects, anti-inflammatory responses, antibacterial properties, and antioxidative capabilities. the multifaceted positive effects on the human body can be attributed to the synergistic interactions of these bioactive compounds (golak-siwulska et al., 2018). the fruiting bodies of various species of oyster mushrooms have yielded an extensive array of polysaccharides exhibiting several biological activities. zhang et al. (2012) successfully isolated two distinct fractions of polysaccharides from p. ostreatus, demonstrating their significant antioxidative capabilities. facchini et al. (2014) reported that polysaccharides derived from the biologica nyssana ● 15 (2) december 2024: selvaananthi and beulah jerlin ● bioactive components and therapeutic potential of pleurotus florida: a comprehensive analysis 142 biologica nyssana ● 15 (2) december 2024: selvaananthi and beulah jerlin ● bioactive components and therapeutic potential of pleurotus florida: a comprehensive analysis 143 as a precursor in the synthesis of vitamin d, steroid hormones and sex hormones. additionally, cholesterol is a key component of bile salts, which aid in the digestion and absorption of fat-soluble vitamins a, d, e, and k (ciaula et al., 2017). the impact of diet on cholesterol levels varies among individuals (stone & lloyd-jones, 2015). certain compounds in mushrooms help maintain healthy blood cholesterol levels and eliminate unwanted fats from the body. psilocybin, a classic hallucinogen produced by over 100 species of mushrooms worldwide, binds with high affinity to several serotonin receptors, including 5-ht1a, 5-ht2a, and 5-ht2c, which are located in various brain regions such as the cerebral cortex and thalamus. daniel & haberman (2017) provide an in-depth review of psilocybin’s potential against various mental health conditions, including depression, anxiety disorders, obsessivecompulsive disorder, alcohol use disorder, and tobacco use disorder. various substances extracted from macro fungi, particularly mushrooms, such as ceramides, lentinan, schizophyllan, omega-3, 6, and 9 fatty acids, carotenoids, and resveratrol, are increasingly being incorporated into cosmetics (hyde et al., 2010; camassola, 2013). conclusion the study has revealed a rich array of bioactive components, including polysaccharides, peptides, fatty acids, and phenolic compounds, each associated with various therapeutic activities. the study identified 37 distinct bioactive compounds, demonstrating a wide spectrum of biological effects such as treatment for phobic disorders, antiseborrheic and antieczematic properties, cholesterol antagonism, and regulation of lipid metabolism. the results suggest that pleurotus florida holds considerable promise as a source of functional compounds with diverse health benefits. these findings support the potential of these mushrooms for use in developing novel therapeutic interventions and highlight the need for further research to elucidate the mechanisms underlying their efficacy and to explore their clinical applications more comprehensively. references alam, n., amin, r., khan, a., ara, i., shim, m.j., lee, m.w., & lee1, t.s. 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(2016). mushroom extracts and compounds in cosmetics, cosmeceuticals and nutricosmetics – a review. industrial crops and products, 90, 38-48. biologica nyssana ● 15 (2) december 2024: selvaananthi and beulah jerlin ● bioactive components and therapeutic potential of pleurotus florida: a comprehensive analysis 144 stanojević et al 2020, biologica nyssana 11(2) 11 (2) december 2020: 109-114 doi: 10.5281/zenodo.4393961 copper(ii) compounds as effective agents against colletotrichum acutatum causing anthracnose of strawberry original article ivana stanojević faculty of agriculture, university of niš, kosančićeva 4, 37000 kruševac, serbia stanojevic.ivana85@gmail.com biljana glišić faculty of science, department of chemistry, university of kragujevac, r. domanovića 12, 34000 kragujevac, serbia bglisic@kg.ac.rs sanja živković faculty of agriculture, university of niš, kosančićeva 4, 37000 kruševac, serbia gajicsanja43@gmail.com sonja filipović faculty of agriculture, university of niš, kosančićeva 4, 37000 kruševac, serbia sonjafilipovic86@yahoo.com snežana andjelković institue for forage crops, kruševac, 37251, globoder, serbia snezana.andjelkovic@ikbks.com tanja vasić faculty of agriculture, university of niš, kosančićeva 4, 37000 kruševac, serbia tanjavasic82@gmail.com (corresponding author) received: may 21, 2020 revised: september 18, 2020 accepted: november 30, 2020 abstract: the aim of this study was to evaluate the in vitro antifungal effects of copper(ii) compounds on the growth of the fungus colletotrichum acutatum isolated from strawberry. the growth inhibition activity of na2[cu(1,3pddadp)].6h2o complex (1,3-pddadp is 1,3-propanediamine-n,n’-diacetaten,n’-di-3-propionate anion) against this fungus was compared to those of cuso4 .5h2o and ba2(1,3-pddadp).8h2o. additionally, the effects of these compounds on sporulation level and the number of conidia per 1 mm2 of colony were investigated. the obtained results showed that the highest percentage of inhibition for mycelium growth was achieved at a concentration of 100 and 200 µg/ml for all applied compounds. the biological activities of the investigated compounds were compared with those for the commercial formulation of fungicide metod 480 sc (captan). key words: antifugal activity, colletotrichum acutatum, copper(ii) compounds, strawberry apstract: jedinjenja bakra(ii) kao efikasni agensi prema gljivi colletotrichum acutatum, uzročniku antraknoze jagoda cilj ovog rada je ispitivanje in vitro antifungalne aktivnosti jedinjenja bakra(ii) prema gljivi colletotrichum acutatum koja je izolovana iz jagode. ispitivana je aktivnost na2[cu(1,3-pddadp)].6h2o kompleksa (1,3-pddadp je 1,3-propandiamin-n,n’-diacetat-n,n’-di-3-propionat), pri čemu su dobijeni rezultati poređeni sa aktivnošću cuso4 .5h2o i ba2(1,3-pddadp).8h2o. dodatno je analiziran je uticaj različitih koncentracija ovih jedinjenja na intenzitet sporulacije i broj konidija po 1 mm2 kulture c. acutatum. dobijeni rezultati su pokazali da sva ispitivana jedinjenja imaju najveću aktivnost pri koncentraciji od 100 i 200 µg/ml. biološka aktivnost ispitivanih jedinjenja je poređena sa odgovarajućom aktivnošću komercijalnog fungicida metod 480 sc kaptan. ključne reči: antifungalna aktivnost, colletotrichum acutatum, jedinjenja bakra(ii), jagoda introduction strawberry (fragaria ananassa) is commercially the most important fruit, which is grown worldwide and is predominant species cultivated for production globally (fao, 2000). one of the species that has been reported to cause wide spread disease in strawberry, namely anthracnose, is the fungus colletotrichum acutatum (sutton, 1992; maas and palm, 1997; freeman et al., 1998; wedge et al., 1999). this fungus can attack the plant at all stages of development, including flowers, leaves, stems and roots. however, it causes the greatest damage to fruits, causing significant losses in strawberry production (mertely et al., 2018). there are only a few available fungicides which are effective against c. acutatum, nevertheless a long and frequent use of these fungicides led to the © 2020 stanojević et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 109 resistance development (maas, 2004). considering the heavy burden for the strawberry production sector worldwide that anthracnose is causing, development of advanced and efficient therapeutic options became of great importance. with the increasing emergence of drug-resistant c. acutatum species and due to a lack of potent agent against anthracnose, a special attention has been devoted to the evaluation of antimicrobial potential of metal compounds (schatzschneider, 2019). among them, copper(ii) compounds have received a particular interest, due to their wellknown application in agriculture as fungicides, nutritionals and algicides (richardson, 1997). the importance of copper(ii)-based compounds as fungicides in viticulture has been recognized after discovery of the bordeaux mixture, which consists of copper(ii) sulfate, lime and water. nowadays, the pharmaceutical companies have been produced different copper(ii)-based fungicides in soluble forms of sulfates, oxychlorides, acetates, carbonates, oleates, silicates and hydroxides. copper(ii) oxychloride has been widely used due to its good crop tolerance and effectiveness against different phytopathogenic fungi such as rhizoctonia solani, r. bataticola, botrytis cinerea, fusarium semitectum, f. culmorum, f. moniliforme, f. solani, f. oxysporum, stemphylium radicinum, sclerotinia sclerotiorum and colletotrichum gloeosporioides (gharieb et al., 2004). copper(ii)-based fungicides have a broad spectrum of activity against different fungal and bacterial microorganisms as a consequence of their ability to be attached on the plant surface. at the same time, they are economically acceptable and do not lead to resistance development (müller et al., 2000). with the aim to selectively deliver cu(ii) ion to the diseased tissues, different copper(ii) complexes with various ligands have been synthesized and reported to possess significant antibacterial and antifungal activities (singh et al., 2008; singh et al., 2009; patel et al., 2010; ng et al., 2016). beside copper(ii) compounds, diaminopolycarboxylate ligands which belong to edta family (ethylenediamine-n,n,n’,n’-tetraacetate anion) and their metal complexes have shown a remarkable antimicrobial activity (anderegg, 1987; bulman, 1987; douglas and radanović, 1993). for instance, edta has shown activity against different gramnegative and gram-positive bacteria, yeasts, amoeba and fungi (brown and richards, 1965; kite and hatton, 2014). considering the importance of copper(ii) compounds and diaminopolycarboxylate ligands in design of antifungal agents, in the present study, we have synthesized and biologically evaluated na2[cu(1,3-pddadp)].6h2o complex (1,3-pddadp is 1,3-propanediamine-n,n’-diacetate-n,n’-di-3propionate anion) against the fungus c. acutatum. the obtained biological results for this complex was compared to those for cuso4 .5h2o and ba2(1,3pddadp).8h2o. material and methods materials barium salt of diaminopolycarboxylate ligand from edta-family, ba2(1,3-pddadp).8h2o, and copper(ii) complex na2[cu(1,3-pddadp)].6h2o were prepared according to a procedure published in the literature (radanović et al., 1992; radanović et al., 1997). other reagents were obtained commercially and used without further purification. test organism in this experiment, the isolate of c. acutatum was used. it was isolated from strawberries grown in serbia. the isolate was determined on the basis of morphological, pathogenic and molecular characteristics and maintained on a potato-dextrose agar (pda) at 25 °c. antifungal activity antifungal activity of the three compounds cuso4 .5h2o, ba2(1,3-pddadp).8h2o and na2[cu(1,3-pddadp)].6h2o was tested on pda in petri dishes of 90 mm diameter. substrates were inoculated by mycelial fragments of c. acutatum isolates taken from the edge of 7-days old culture. tested compounds were dissolved in distilled water at three different concentrations: 100, 200 and 300 µg/ml. the solutions were incorporated into the autoclaved pda, which was cooled to 55 °c. cultures prepared in this way were grown for 7 days in a thermostat at 25 °c. the commercial formulation of fungicide metod 480 sc (captan, galenika fitofarmacija) was used as a positive control and applied at a concentration recommended for practical application. the negative control variant, fungal isolates grown on pda, were used under identical conditions without described treatments (zang et al., 2012). after seven days, the linear increase in the studied cultures was measured. the inhibition of the fungal growth expressed in percentage terms was determined from the growth in the test plate relative to the respective control plate as given below: inhibition (%) = (c-t) 100 / c where, c = diameter of fungal growth in the control plate and t = diameter of fungal growth in the test plate. sporulation level ten days after treatment, the effects of cuso4 .5h2o, ba2(1,3-pddadp).8h2o and na2[cu(1,3-pddadp)].6h2o 110 biologica nyssana ● 11 (2) december 2020: 109-114 stanojevic et al. ● copper(ii) compounds as effective agents against colletotrichum acutatum causing anthracnose of strawberry 111 on c. acutatum sporulation levels were determined. determination of sporulation levels was performed using a tahoma haemocytometer. for this purpose, a spore suspension was prepared by adding 5 ml of distilled water to the petri dish with a culture of isolate c. acutatum. the sporulation level was expressed according to the scale by quesada and lopez (1980), where: + = poor sporulation (<5.000 spores/ml), ++ = medium sporulation (5.000-10.000 spores/ml) and +++ = abundant sporulation (>10.000 spores/ml). the experiment was set in three repetitions. the number of conidia per 1 mm2 of colony was determined based on the number of conidia/ ml and the area of the colony, which was calculated from the proportion of paper weight on which the contours of colonies were drawn and paper weight of 1 dm2 (stojanović, 1997). the results obtained during the research were processed by anova analysis with the statistical program statistica 8.0 (statsoft inc., usa). duncan’s test was conducted to analyze the difference between various pre-treatments. a value of p = 0.05 was considered statistically significant. results and discussion biological activity all tested compounds showed a remarkable growth inhibition of c. acutatum mycelia. the inhibition of mycelial growth was observed at concentrations of 100 μg/ml and higher, for all tested compounds, na2[cu(1,3-pddadp)].6h2o, cuso4 .5h2o and ba2(1,3-pddadp).8h2o, and was dependent on the applied concentration of the compounds. moreover, a significant percentage inhibition of c. acutatum mycelium compared to the commercial captan formulation (metod 480 sc, galenika fitofarmacija) was achieved at all applied concentrations (100, 200 and 300 µg/ml) of the tested compounds (tab. 1 and figs. 1 and 2). the linear growth of colonies of the tested c. acutatum isolate was statistically significantly influenced by the concentrations of the applied compounds. during the study, it was found that the fig. 1. influence of two copper(ii) compounds, na2[cu(1,3pddadp)].6h2o and cuso4.5h2o on the growth inhibition of c. acutatum mycelium biologica nyssana ● 11 (2) december 2020: 109-114 stanojevic et al. ● copper(ii) compounds as effective agents against colletotrichum acutatum causing anthracnose of strawberry table 1. the percentage of growth inhibition of c. acutatum by na2[cu(1,3-pddadp)].6h2o, cuso4.5h2o and ba2(1,3-pddadp).8h2o compounds compound 100 µg/ml 200 µg/ml 300 µg/ml na2[cu(1,3-pddadp)].6h2o 36.3b,c 38.7 b 31.6 c cuso4 .5h2o 35.3b,c 34.7 c 39.3 b ba2(1,3-pddadp).8h2o 39.7 b 34.3 b 40.0 b control c. acutatum 53.3a 53.3 a 53.3 a metod 480 sc 100d 100d 100d * the data in rows marked by the same letter are not statistically significantly different based on duncan test (p = 0.05) 112 highest linear growth of the colony of c. acutatum was achieved after treatment with cuso4 .5h2o and ba2(1,3-pddadp).8h2o at a concentration of 300 µg/ ml relative to the negative control (pure culture of c. acutatum) (tab. 1, fig. 2b and 2c). contrary to this, the smallest increase in mycelium of the fungus c. acutatum was after treatment with na2[cu(1,3pddadp)].6h2o and cuso4 .5h2o at concentrations of 100 and 200 µg/ml (fig. 2a and 2b). considering this, it can be concluded that the minimum inhibitory concentrations (mics) against the c. acutatum are at 100 and 200 µg/ml for the tested compounds (tab. 1 and fig. 2). oziengbe and osazee (2012) evaluated the inhibitory effect of copper(ii) sulfate on the linear growth of c. gloeosporioides mycelium. their results showed that this copper(ii) salt reduced the linear growth of this fungus in all applied concentrations (0.2, 0.4, 0.6 and 0.8 mg/l). on the other hand, everett and timudotorrevilla (2007) tested four commercial copperbased preparations (champ (copper-hydroxide, 37.5% copper), kocide (copperhydroxide, 35% copper), kocide (copper-hydroxide, 46.1% copper) and cuprofix disperss (copper-hydroxosulphate, 20% copper)) against the most important pathogens on avocado, colletotrichum acutatum, c. gloeosporioides, botryosphaeria parva, b. dothidea and phomopsis spp. in new zealand. the results showed that four copper formulations inhibited the germination of spores of all tested fungi, but at high concentrations (i.e. 40.9 µg/ml for copper hydroxide (46.1% copper) against c. gloeosporioides). the copper formulation that was effective at the lowest concentrations against the germination of botryosphaeria parva spores was copper hydroxide (37.5% copper), the most effective against both species of colletotrichum was copper hydroxosulfate (20% copper), while the most table 2. influence of the investigated compounds on the number of conidia per mm2 of c. acutatum compound 100 µg/ml 200 µg/ml 300 µg/ml na2[cu(1,3-pddadp)].6h2o + ++ + cuso4 .5h2o +++ ++ ++ ba2(1,3-pddadp).8h2o ++ + +++ control c. acutatum +++ +++ +++ metod 480 sc * sporulation: + = poor sporulation, ++ = medium sporulation, +++ = abundant sporulation fig. 2. the effect of na2[cu(1,3-pddadp)].6h2o complex (a), cuso4.5h2o (b) and ba2(1,3-pddadp).8h2o (c) on the growth of c. acutatum isolate in vitro after seven days. biologica nyssana ● 11 (2) december 2020: 109-114 stanojevic et al. ● copper(ii) compounds as effective agents against colletotrichum acutatum causing anthracnose of strawberry effective against b. dothidea and phomopsis sp. was copper-hydroxide (46.1% copper) (everett and timudo-torrevilla, 2007). sporulation level the number of conidia formed per mm2 of colony was statistically significantly conditioned by the applied concentrations of the tested compounds in c. acutatum culture. the lowest number of conidia per mm2 was found for na2[cu(1,3-pddadp)].6h2o at 100 and 300 µg/ml, for cuso4 .5h2o at 200 and 300 µg/ml, and for ba2(1,3-pddadp).8h2o at 100 and 200 µg/ml. the highest number of spores per mm2 was observed for cuso4 .5h2o and ba2(1,3-pddadp).8h2o at concentrations of 100 and 300 µg/ml, respectively (tab. 2). as can be seen from tab. 3, the ability of the fungus c. acutatum to form conidia is affected by different concentrations of the applied compounds, na2[cu(1,3-pddadp)].6h2o, cuso4 .5h2o and ba2(1,3-pddadp).8h2o. in the treatment with captan which was used as a positive control, no sporulation of c. acutatum occurred. in the case of ba2(1,3pddadp).8h2o at 200 µg/ml and for na2[cu(1,3pddadp)].6h2o complex at 100 and 300 µg/ml, a lower and medium level of sporulation was noticed. contrary to this, the abundant sporulation was observed for the cuso4 .5h2o at 100 µg/ml, as well as for ba2(1,3-pddadp).8h2o at 300 µg/ml (tab. 3). spores are very important for the spread of the disease on strawberry plantations. thus, the number of spores and the intensity of sporulation play a significant role in the spread of pathogens and the progress of infection. spores of the pathogen are passively dispersed by spraying rain or irrigation water which causes infection and pathogen develop on immature fruits and young tissues, and these spores germinate and penetrate the cuticle and epidermis to multiply through the tissues (babović, 2003). symptoms include small black spots and/ or large black lesions on the tissue surface, which coalesce and penetrate deep into the fruit, resulting in fruit rot. borkow and gabbay (2009) found that different copper formulations have inhibited a spore germination of colletotrichum gloeosporioides at different concentrations (0.2, 0.4, 0.6 and 0.8 mg/l). similar conclusions were drawn by oziengbe and osazee (2012), who tested different concentrations of copper compounds against the isolates of colletotrichum gloeosporioides originated from mango. conclusion in conclusion, the safety of copper for humans and its powerful biocidal properties allow the use of copper and its compounds for different applications. this study showed that copper(ii) compounds in the form of complex or simple inorganic salt could inhibit the growth of colletotrichum acutatum mycelium, by affecting the intensity of sporulation and the number of spores per mm2 in vitro, while strongly causing defensive reactions on the strawberry fruit. references anderegg, g., 1987: complexones. in: wilkinson g, editor. comprehensive coordination chemistry vol. 2, pergamon press, oxford. babović m. 2003: osnovi patоlogije biljaka. poljoprivredni fakultet, univerzitet u beogradu, beograd. 651 p. borkow g. and gabbay j. 2009: copper, an ancient remedy returning to fight microbial. fungal and viral 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(eds.): colletotrichum: biology, pathology and control. cab international, wallingford, oxon, uk, 1-26 wedge, d. e., riley, m. b., tainter, f. h. 1999: phytotoxicity of discula destructiva culture filtrates to cornus spp. and the relationship to disease symptomology. plant disease, 83(4): 377-380. zhang, c.q., liu, y.h., wu, h.m., xu, b.c. sun, p.l., and xu, z.h. 2012: baseline sensitivity of pestalotiopsis microspora, which causes black spot disease on chinese hickory (carya cathayensis), to pyraclostrobin. crop protection 42: 256-259. 114 biologica nyssana ● 11 (2) december 2020: 109-114 stanojevic et al. ● copper(ii) compounds as effective agents against colletotrichum acutatum causing anthracnose of strawberry microsoft word 0305_zivanovic_et_al biologica nyssana 1 (1-2) december 2010: 71-75 mitrović, b. et al. chenopodium rubrum l. as a model plant… 31 original article ! chenopodium murale l., a long-day plant as a model for physiological and biochemical research branka živanović1, aleksandra mitrović1*, jelena bogdanović pristov1, ksenija radotić hadži-manić1, ljubinka ćulafić2 1 institute for multidisciplinary research, university of belgrade, kneza višeslava 1, 11030 belgrade, serbia 2 institute of botany and botanical garden "jevremovac", takovska 43, faculty of biology, university of belgrade, 11000 belgrade, serbia * e-mail: mita@imsi.rs abstract: živanović, b., mitrović, a., bogdanović-pristov, j., radotić hadži-manić, k., ćulafić, lj.: chenopodium murale l., a long-day plant as a model for physiological and biochemical research. biologica nyssana, 1 (1-2), december 2010: 71-75. chenopodium murale l. genus chenopodium family chenopodiaceae is a weedy annual widely distributed in serbia. this is a long-day plant and an early flowering species. we used culture of intact plants in vitro and antioxidative enzymes detection in order to examine the effect of gibberellic acid (ga3) on two key processes during ontogenesis – germination and flowering. our results showed a sequential expression of antioxidative enzymes during germination. in dry seeds and during early imbibition catalase (cat) and superoxide dismutase (sod) activities showed no changes, while peroxidase (pod) activity was under the level of delectability. during radicule protrusion cat and sod activity increased. early seedling development correlates with decrease in sod, increase in cat and appearance of pod activity. ga3 delayed and synchronized germination. c. murale photoperiodic sensitivity for flowering shows age-dependant oscillatory changes. glucose and ga3 have cumulative stimulatory effect on its flowering in vitro. the exposure of 2 months old vegetative plants to continuous darkness, in the presence of ga3 in culture media, resulted in flowering. therefore transferring to darkness canceled photoperiodic control in c.murale and flowering occurred under autonomous mechanism. we suggest c.murale as a suitable model for investigation of physiological and biochemical mechanisms of growth and developmental processes. key words: catalase, chenopodium murale l., germination, gibberelic acid, flowering, peroxidase, superoxide dismutase introduction ! chenopodium murale l. belongs to the family chenopodiaceae, genus chenopodium. ecotype 197 is characterized by c u m m i n g (1967) as a facultative long-day annual and an early flowering species. it is sensitive to photoperiodic flowering induction as early as at the phase of the 1st pair of leaves (p a v l o v á et al., 1989). 10 cycles of continuous light (24 h light) are necessary for photoperiodic flowering induction, regardless of plant age (p a v l o v á et al., 1989). intact c. murale plants, cultured in vitro on optimal media composition and under adequate photoperiodic regime, produces seeds in 18 weeks. gibberellins (gas) are very potent plant growth regulators. gas plays an important role in stimulation of seed germination (b e w l e y , 1997). they are efficient in breaking seed dormancy, by their ability to overcome the requirements for environmental factors, and in accelerating germination in non-dormant seeds (b e w l e y & 10th sfses • 17-20 june 2010, vlasina lake1 (1-2) • december 2010: 71-75 biologica nyssana 1 (1-2) december 2010: 71-75 mitrović, b. et al. chenopodium rubrum l. as a model plant… 72 b l a c k , 1982). ga3 also induces or promotes flowering, being particularly effective in long-day species (e v a n s et al., 1999; ž i v a n o v i ć et al., 1995; m i t r o v i ć et al., 2000; m i t r o v i ć et al., 2000; m i t r o v i ć et al., 2003). continuous production and removal of reactive oxygen species (ros) is, besides as a phenomenon with negative consequences (damage of cell membranes and organelles), linked with a signaling role in plant developmental processes. antioxidative enzymes, catalase (cat), superoxide dismutase (sod) and peroxidase (pod) are engaged in the scavenging of ros (dat et al., 2000) and therefore participate in regulation of plant growth and development (m i t r o v i ć , 2007) as well as in the protection against pathogens or abiotic stress (h e n d r y & c r a w f o r d , 1994). this is a review of our previously reported data that describe the effects of gibberellic acid in regulation of c. murale seed germination and flowering, as two key processes in plant ontogenesis. results and disscusion seed germination seed germination starts with imbibition, and ends with radicule protrusion. it can be divided in three phases: imbibition (rapid initial water uptake – physical process characteristic also for dead seeds), the plato phase (small change of water content but high metabolic activity) and further water uptake coinciding with radicule protrusion and growth (g i b a et al., 2004). plato phase, as the most important in regulation of germination, involves the activation of specific enzymes at the appropriate time and regulation of their activity (r i l e y , 1987). it was supposed that accumulation of reactive oxygen species (ros), during seed imbibition, leads to germination (b a i l l y , 2004). therefore, antioxidant enzymes have been considered to be of particular importance for the completion of germination. sequential expression of antioxidative enzymes and their importance during germination in different plant species was shown (d u č i ć et al., 2003/4; b a i l l y et al., 2000; p u n t a r u l o et al., 1991; b o g d a n o v i ć et al., 2008). gibberellic acid delayed and synchronized c. murale germination and showed similar effect on protein content and activities and isoenzyme pattern of antioxidative enzymes (fig. 1, fig. 2) (b o g d a n o v i ć et al., 2008). this suggests that ga3 and ros, thorough activities of antioxidative enzymes, participate in the same signaling pathway in germination. protein concentration increased at the time of c.murale ridicule protrusion (b o g d a n o v i ć et al., 2008), connected with release from storage and de novo synthesis of building and regulatory proteins in the emerging seedlings (r o b e r t s , 1972). fig. 1. c. murale germination (%) on distilled water or ga3 (160 μm) solution. data points are averages of four replicates of 50 seeds each. modified from bogdanović et al., 2008 in dry seeds and during early imbibition catalase (cat) and superoxide dismutase (sod) activities showed no changes, while peroxidase (pod) activity was under the level of delectability (fig. 2). cat activity increased during radicule protrusion and early seedling development, by appearance of a new cat isoform. pick in sod activity coincided with radicule protrusion and early seedling development. based on the specific inhibition of sod activity by kcn and h2o2, we showed that c.murale seeds/seedlings contain only mn sod form. pod activity appeared prior to or simultaneously with radicule protrusion and increased during early seedling growth, presented with two basic isoforms (b o g d a n o v i ć et al., 2008). specific role of pods in regulation of final phases of germination and early seedling growth was suggested in many species (s c h o p f e r et al., 2001; d u č i ć et al., 2003/4; p r o d a n o v i ć et al., 2007; b o g d a n o v i ć et al., 2008). pods have a very important role in physiological processes in plants (g a s p a r et al. 1991), but their exact relationship to developmental events is often obscure by their extensive polymorphism in a single plants species. changes in cat, sod and pod activities could be “the markers” of different phases of germination. decrease in sod activity (h2o2 producing enzyme), increase in cat and appearance of pod activity (h2o2 consuming enzymes) coincide with early seedling development in c. murale. these data, accompanied with similar findings concerning seed germination in different plant species, suggest that decrease in h2o2 level biologica nyssana 1 (1-2) december 2010: 71-75 mitrović, b. et al. chenopodium rubrum l. as a model plant… 73 might be involved in regulation of this process (bogdanović et al., 2008). fig. 2. catalase, superoxide-dismutase and peroxidase activities [mg g-1 (d.w.)] during c. murale seed germination on distilled water or ga3 (160 μm) solution. modified from bogdanović et al., 2008 flowering transition from vegetative to reproductive phase of development is controlled by genetical (autonomous) and ecological (photoperiod and/or temperature) factors. autonomous control that depends on plant age is the basic control level. induced or photoperiodic control, alone or in combination with temperature, is the second level of flowering control that stimulates or inhibits genetically determined flowering. in c.murale plants photoperiodic sensitivity is age-dependant. it shows oscillatory changes with aging (expressed by number of leaves) (p a v l o v á et al., 1989; m i t r o v i ć et al., 2000). 10 cycles of continuous light (24 h light) are necessary for photoperiodic flower induction, regardless of plant age (p a v l o v á et al., 1989; m i t r o v i ć et al., 2000). glucose and ga3 have cumulative stimulatory effect on c.murale flowering in vitro under inductive photoperiodic conditions, probably affecting flower development, rather than flower initiation (tab. 1). under photoperiod noninductive for flowering neither glucose nor ga3 were able to compensate for c.murale photoperiodic requirements for flowering (mitrović et al., 2000). the photoperiodic control is loosened with aging in some plants (perilla nankensis, rudbeckia bicolor) (b e r n i e r et al., 1981; c h a i l a k h y a n , 1988). exposure to darkness cancels photoperiodic control, so the flowering is controlled only by the autonomous mechanism (c h a i l a k h y a n , 1988; m i t r o v i ć et al., 2003). some long-day and shortday plants produces flowers under continuous darkness (f i f e and price, 1953; sugino, 1957; inouye et al., 1964; t a k i m o t o , 1960; m i t r o v i ć et al., 2003; c v e t i ć et al., 2004). in 5 – 7 month old long-day plant rudbeckia bicolor flowering occurred after exposure to darkness (c h a i l a k h y a n , 1988). in 2 months old c. murale plants, exposed to continuous darkness for 10 days, the addition of ga3 in culture medium was necessary for transition to flowering (tab. 1) (mitrović et al., 2003). ga3 alone was not able to compensate for c. murale photoperiodic demands for flowering. so we suggest that ga3 affected flower development, rather than flower initiation. c.murale plants partly loose demands for specific day length with aging, so transferring to darkness cancels photoperiodic control and flowering occurs under autonomous mechanism (m i t r o v i ć et al., 2003). conclusion this is a review of our work on model plant chenopodium murale. our results improved the knowledge of the role of gibberellic acid in two key processes during ontogenesis germination and flowering. ga3 delay and synchronize germination, while it stimulates flowering. changes in activities and isoforms of antioxidative enzymes could be “the markers” of different phases of seed germination. intact c.murale plants have the ability to be induced for flowering in total darkness in vitro. acknowledgments. this work was supported by a grant (№. 143043) from the ministry of science and technological development, republic of serbia. biologica nyssana 1 (1-2) december 2010: 71-75 mitrović, b. et al. chenopodium rubrum l. as a model plant… 74 references bailly, c. 2004: active oxygen species and antioxidants in seed biology. seed science research, 14: 93-107. bailly, c., benamar, a., corbineau, f., côme, d. 2000: antioxidant systems in sunflower (helianthus annuus l.) seeds as affected by priming. seed science research, 10: 35-42,. bernier, g., kinet, j-m., sachs, r.m. 1981: age and flower initiation. in: the physiology of flowering i: 106-115, crc press., boca raton. bewley, j.d., black, m. 1994: seeds. physiology of development and germination. 2nd ed. plenum press, new york. bewley, j.d. 1997: seed germination and dormancy. plant cell, 9: 1055-1066. bogdanović, j., radotić, k., mitrović, a. 2008: changes in activities of antioxidant enzymes during chenopodium murale seed germination. biologia plantarum, 52: 396-400. chailakhyan, m.ch. 1988: regulyatsiya tsveteniya u vysshikh rastenii. 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(ed): seed ecology: 189-218, butterworts, london. schopfer, p., plachy, c., frahry, g. 2001: release of reactive oxygen intermediates (superoxide radicals, hydrogen peroxide, and hydroxyl radicals) and peroxidase in germinating radish seeds controlled by light, gibberellin, and abscisic acid. plant physiology, 125: 1591-1602. takimoto, a. 1960: effect of sucrose on flower initiation of pharbitis nil in aseptic culture. plant cell physiology, 1: 241-246. živanović, b., ćulafić, lj., filipović, a. 1995: the effects of hormones and saccharides on growth and flowering of green and herbicides-treated chenopodium rubrum l. plants. biologia plantarum, 37: 257-264. sarajlić, n., jogan, n.: alien flora of the city of sarajevo (bosnia and herzegovina). biologica nyssana, 8 (2). biologica nyssana 8 (2)  december 2017: 129-136 sarajlić, n., jogan, n.  alien flora of the city of sarajevo… 129 original article received: 09 november 2017 revised: 24 december 2017 accepted: 25 december 2017 alien flora of the city of sarajevo (bosnia and herzegovina) nermina sarajlić1, nejc jogan2 1ornithological society „naše ptice“, semira frašte 6, 71000 sarajevo, bosnia and herzegovina 2university of ljubljana, biotechnical faculty, department of biology, večna pot 111, 1000 ljubljana, slovenia * e-mail: nermina_sarajlic@yahoo.com abstract: sarajlić, n., jogan, n.: alien flora of the city of sarajevo (bosnia and herzegovina). biologica nyssana, 8 (2). december, 2017: 129-136. this paper presents the inventory and brief analysis of 82 alien vascular plant taxa of the urban and suburban area of the city of sarajevo. the checklist is based on field work that has been carried out from summer 2015 to autumn 2017. each taxon is given with information concerning its life-form, time and mode of introduction, geographic origin and previously reported occurrence in the area. most alien plants were the members of the asteraceae (incl. cichoriaceae), therophytes, neophytes, originated from north and south america and deliberately introduced. the values of indicators of anthropogenic changes showed the considerable anthropogenic influence on the total flora. key words: alien plants, urban flora, sarajevo apstrakt: sarajlić, n., jogan, n.: alohtona flora grada sarajeva (bosna i hercegovina). biologica nyssana, 8 (2), decembar, 2017: 129-136. u radu je dat spisak i kratka analiza 82 alohtona taksona vaskularnih biljaka zabeleženih u urbanom i suburbanom delu grada sarajeva od leta 2015. do jeseni 2017. godine. za svaki takson navedeni su podaci o životnoj formi, vremenu i načinu introdukcije, poreklu i najraniji objavljeni podatak o njegovom prisustvu na istraživanom području. izračunati su indikatori antropogene promene flore. najbrojnije alohtone vrste zabeležene tokom ovog istraživanja su pripadnici porodice asteraceae (uključujući cichoriaceae), terofite, neofite poreklom iz severne i južne amerike, koje su u evropu unesene namerno. vrednosti indeksa antropogene promene pokazuju značajan antropogeni uticaj na ukupnu floru grada sarajeva. ključne reči: alohtone biljke, urbana flora, sarajevo 8 (2) • december 2017: 129-136 doi: 10.5281/zenodo.1135953 biologica nyssana 8 (2)  december 2017: 129-136 sarajlić, n., jogan, n.  alien flora of the city of sarajevo… 130 introduction urban habitats are generally characterized by high levels of disturbance, high fertility and heterogeneity of habitats, suitable for development of numerous plant species, including many alien plants. alien (exotic, adventive, introduced, allochthonous, nonindigenous, non-native) plants are those whose presence in a certain area is due to intentional or accidental introduction as a result of human activities (p y š e k , 1995; r i c h a r d s o n et al., 2000; p y š e k et al., 2004). according to their spreading ecology in a certain territory, alien plants can be divided in three groups: casual, naturalized and invasive. according to the time when they were introduced into europe, alien plants can be classified as archaeophytes (introduced before the year 1500) and neophytes (introduced after the year 1500) (p y š e k , 1995, k o w a r i k , 1995; p y š e k et al., 2002; p r e s t o n et al., 2004; l a s o r t e et al., 2007). some of them were introduced deliberately, as ornamental, edible or medicinal plants, and others came to europe accidentally, without the deliberate actions of humans. the city of sarajevo was founded in the 15th century, but expanded, mostly towards the west, only during the post-world war ii era. intense exchange of goods since ancient times and further urbanization, construction of roads and railway enabled the introduction of new plants, some of which slowly become naturalized part of the flora. literature on alien flora of the city of sarajevo is scarce, and the presence of some alien plants is either mentioned in general floristic studies (k o m š a , 1928; p a v l o v i ć , 1987; t o m o v i ć -h a d ž i a v d i ć & š o l j a n , 2006), or studies focused on particular species (s l a v n i ć , 1960; a b a d ž i ć , 1986/87; š o l j a n & m u r a t o v i ć , 2000; š o l j a n , 2011; m e m i š e v i ć -h o d ž i ć et al., 2015; s a r a j l i ć et al., 2016; s u l j i ć et al., 2016). this paper presents a list of hitherto recorded alien species and a brief analysis of the alien flora of the city of sarajevo. the inventory of 82 taxa is provided based on the field work performed in 2015, 2016 and 2017, updating previous floristic knowledge for the investigated area. material and methods study area the city of sarajevo is located in the sarajevo valley, on the banks of miljacka river, at an average altitude between 520 and 750 m a. s. l. (fig. 1). the central parts of the city are situated in the lower parts of fig. 1. geographical position of investigated area (the yellow line limits the area in which the survey was performed) biologica nyssana 8 (2)  december 2017: 129-136 sarajlić, n., jogan, n.  alien flora of the city of sarajevo… 131 sarajevo polje, and the suburbs are scattered on the slopes of surrounding hills and mountains. the area of sarajevo is under influence of mid-european continental climate from the north, and the mediterranean from the south. field investigation the field survey was performed in the urban part of four municipalities (novi grad, novo sarajevo, centar, stari grad) of the city of sarajevo (fig. 1), in an area of 32 km2, from the summer of 2015 to the autumn of 2017. only taxa that had not been deliberately planted were recorded. data analysis the nomenclature follows euro+med plantbase and the international organization for plant information database. asteraceae family was considered in wider sense (incl. cichoriaceae). the taxa are listed in alphabetic order, followed by designations for life form (phanerophyte ph, chamaephyte ch, hemicryptophyte h, geophyte g, hydrophyte hy and therophyte t), period of introduction (archaeophytes arc and neophytes – neo), estimated mode of introduction into the region (deliberate, by planting – del, accidental – acc or in both modes d-a) geographic origin and reported first record for the sarajevo area. life forms were given according to r a u n k i a e r (1934), and time and mode of introduction and geographic origin according to p y š e k et al. (2002), m o s y a k i n & y a v o r s k a (2003) and i g n a t i e v a & k o n e c h n a y a (2004). first records for the area of sarajevo were given according to available literature sources (see references and appendix 1). the indicators of anthropogenic changes in the flora of sarajevo were calculated after j a c k o w i a k (1990, 2006), according to w i t o s ł a w s k i & b o m a n o w s k a (2009) as in tab. 1. results during this research, a total of 82 alien plant taxa from 71 genera and 38 families were recorded. the families with the highest number of alien taxa were asteraceae (21%), poaceae (10%), brassicaceae (7%), fabaceae (6%), amaranthaceae (5%) and solanaceae (5%) (tab. 2). the other families were represented only with one or two taxa. table 2. the most abundant families in the alien flora of the city of sarajevo family no. of taxa % of total alien flora asteraceae 17 20.73 poaceae 8 9.75 brassicaceae 6 7.31 fabaceae 5 6.09 amaranthaceae 4 4.88 solanaceae 4 4.88 analysis of life forms showed the domination of therophytes with 47 taxa (57%), followed by phanerophytes with 14 taxa (17%) geophytes (10 taxa, 12%), hemicryphophytes (10 taxa, 12%), and chamaephytes (1 or 1%). the alien flora of the city of sarajevo consisted of 28 (34%) archaeophytes and 54 (66%) neophytes. analysis of the geographical origin of the alien flora of sarajevo (tab. 3) showed that the majority of species originated from n. and s. america (37 taxa, 45%), followed by those originating from asia (24 taxa, 24%), mediterranean (13 taxa, 16%), eurasia (10 taxa, 12%) and africa (2 taxa, 2%). table 1. indicators of anthropogenic changes (an – total number of alien species, sp – number of native species, mt – number of permanently established alien species (ar + kn df), ar – number of archaeophytes, kn – number of kenophytes (neophytes), df – number of diaphytes (casual aliens)). indicator formula for calculation indicator of total anthropophytization iant = an/(sp+an) ×100% indicator of permanent anthropophytization ianp = mt/(sp+mt) ×100% indicator of total archaeophytization iart = ar/(sp+an) ×100% indicator of permanent archaeophytization iarp = ar/(sp+mt) ×100% indicator of total kenophytization iknt = kn/(sp+an) ×100% indicator of permanent kenophytization iknp = kn/(sp+mt) ×100% indicator of modernization im = kn/mt ×100% indicator of fluctuation changes if = df/(sp+an) ×100% biologica nyssana 8 (2)  december 2017: 129-136 sarajlić, n., jogan, n.  alien flora of the city of sarajevo… 132 table 3. an analysis of the geographical origin of the alien flora of the city of sarajevo region subregion no. of taxa % of total alien flora africa 2 2.44 east (af-e) 1 1.22 north (af-n) 1 1.22 america 37 45.12 south (am-s) 7 8.54 north (am-n) 30 36.58 asia 24 29.27 asia (as) 4 4.88 central (as-c) 1 1.22 east (as-e) 8 9.76 south-west (as-sw) 4 4.88 west (as-w) 3 3.66 eurasia (eu-as) 10 12.19 mediterranean (m) 13 15.85 total 82 100.00 indicators of anthropogenic changes values in the flora of sarajevo are presented in tab. 4. table 4. indicators of anthropogenic changes in the city of sarajevo indicator % iant 12.09 ianp 8.17 iart 4.13 iarp 4.31 iknt 7.96 iknp 8.32 im 65.85 if 4.45 according to our research, which has been taking place since summer 2015, the urban flora of sarajevo consists of 678 taxa. the indicators of anthropophytization (iant = 12.09%; ianp = 8.17%) showed the considerable anthropogenic influence on the total flora of sarajevo, and higher indicator values of kenophytization (iknt = 7.96%; iknp = 8.32%) showed that the flora of sarajevo is more influenced by neophytes than by archaeophytes (iart = 4.13%; iarp = 4.31%). in both archaeophytes and neophytes, the values of total and permanent indicators were similar, showing that alien flora is well established. this was confirmed by the low value of the indicator of fluctuating changes (if = 4.45%). discussion cities usually contain large proportion of alien plants (w i t t i g , 2004), due to developed transport network and diversity of habitats that can be easily colonized by alien species. during this research, we found that the families with the highest number of taxa were asteraceae and poaceae. those two families were also dominant in alien flora of zagreb (h u d i n a et al., 2012), sisak (p r u š a et al., 2013), podgorica (s t e š e v i ć et al., 2014) and mostar (m a s l o , 2015), probably due to high reproduction rate and specialised dispersion structures on the seeds (p y š e k , 1997). in alien flora of sarajevo, therophytes were the dominant life-form. the same was recorded in sisak (croatia) by p r u š a et al. (2013) and in mostar by m a s l o (2015). the domination of therophytes is due to the warm and dry microclimate (the “urban heat-island effect”) that characterize urban environments, providing conditions that allow many alien species with higher temperature requirements and tolerance for arid environments to become established. all mentioned cities have the significant share of phanerophytes in their alien flora, mostly due to woody ornamental aliens escaping from cultivation. during this research, neophytes were more numerous than archaeophytes. more neophytes (55%) were also recorded in alien flora of mostar by m a s l o (2015). according to d e l t r e d i c i (2010), the ratio of neophytes to archaeophytes rises in direct relation to the intensity of human disturbance. this is due to the fact that archaeophytes are typically associated with traditional rural environments or intermediate levels of anthropogenic activities (p r e s t o n et al., 2004), and neophytes are more common in highly disturbed modern anthropogenous habitats covering huge areas with little vegetation coverage and with developed transport facilities and industrial infrastructure contributing to the ‘urban heat-island effect’, which provides distinctive environmental conditions that favor the establishment of species from warmer and drier areas (p y š e k , 1998; p y š e k et al., 2002; g o d e f r o i d & k o e d a m , 2007; l a s o r t e et al., 2007). also, the cultivation of numerous foreign species along roads, in parks and gardens, presents the source for neophyte colonization (w i t t i g , 2004). the analysis of the geographical origin of the alien flora of sarajevo showed that the most plants originated from n. and s. america and asia. the same was found in the city of podgorica (s t e š e v i ć et al., 2014) and mostar (m a s l o , 2015). the alien plants originating from n. and s. america and asia biologica nyssana 8 (2)  december 2017: 129-136 sarajlić, n., jogan, n.  alien flora of the city of sarajevo… 133 also dominate in urban flora of italian cities (c e l e s t i g r a p o w & b l a s i , 1998). this is in a relation with the increased number of neophytes in urban areas, due to the fact that archaeophytes originate primarily from southern europe and the near east, and neophytes from north america and eastern asia (p y š e k et al., 2002; l a s o r t e et al., 2007). the indicators of anthropophytization (iant = 12.09%; ianp = 8.17%) showed considerable anthropogenic influence on the total flora of the city of sarajevo, but were not as high as in savica park area near zagreb (iant = 28.78%; ianp = 26.76%) calculated by a l e g r o et al. (2013) or rzeszów foothills in poland (iant = 21.3%) by jaźwa & s t a d n i c k a -f u t o m a (2015). this is probably due to the fact that some habitats in the city of sarajevo (forests and natural grasslands in the suburban parts of city) had very small number of aliens in comparison to the native plants. according to available literature sources, the first alien plants reported from the area of sarajevo were panicum miliaceum, linum usitatissimum, aesculus hippocastanum, capsella bursa-pastoris, conyza canadensis, helianthus annuus, h. tuberosus, juglans regia, phalaris canariensis, pyrus communis, sinapis arvensis and syringa vulgaris (h o f m a n n , 1882). on the other hand, species recorded only recently before our study were acer negundo, ailanthus altissima, antirrhinum majus, oenothera biennis (t o m o v i ć h a d ž i a v d i ć & š o l j a n , 2006), sedum sarmentosum (š o l j a n , 2011) and reynoutria japonica (s a r a j l i ć et al., 2016). during our research, 37 taxa for which there was no earlier data for the area of sarajevo were found (appendix 1). conclusion the results of 3-years research showed that the alien flora of the city of sarajevo consisted of 82 species from 71 genera and 38 families, of which the asteraceae (21%) were the most numerous, followed by poaceae (10%) and brassicaceae (7%). the analysis of life forms showed the domination of therophytes (57%), followed by phanerophytes (17%). neophytes (66%) were more numerous than archaeophytes (34%). analysis of the mode of introduction showed that most aliens (49%) were introduced to europe deliberately, as horticultural or agricultural plants. most alien plants recorded in the area of the city of sarajevo originated from n. and s. americas (45%) and asia (24%). during this research, 37 taxa for which there was no earlier data for the area of sarajevo were found. the values of indicators of anthropogenic changes showed the considerable anthropogenic influence on the total flora of sarajevo. references abadžić, s. 1986/87: prilog poznavanju horologije i ekologije dviju adventivnih vrsta – echinocystis lobata (michx) torrey et gray i bidens bipinnata l. u flori bosne i hercegovine. glasnik zemaljskog muzeja bosne i hercegovine (prirodne nauke), nova serija, 25-26: 71-77. alegro, a., bogdanović, s., rešetnik, i., boršić, i., cigić, p., nikolić, t. 2013: flora of the seminatural marshland savica, part of the (sub)urban flora of the city of zagreb (croatia). natura croatica, 22 (1): 111–134. beck, g. 1887a: flora von südbosnien und der angrenzenden hercegovina. ii theil. annalen des naturhistorischen museums in wien, 2 (1): 35-76. beck, g. 1887b: flora von südbosnien und der angrenzenden hercegovina. iii theil. annalen des naturhistorischen museums in wien, 2 (2): 81-184. beck, g. 1891: flora von südbosnien und der angrenzenden hercegovina. annalen des naturhistorischen museums in wien, 6: 307-344. beck, g. 1896: flora von südbosnien und der angrenzenden hercegovina viii theil (fortsetzung). annalen des naturhistorischen museums in wien, 11 (1): 39-80. beck, g. 1906: flora bosne, hercegovine i novopazarskog sandžaka ii (3.) dio. glasnik zemaljskog muzeja u bosni i hercegovini, 18 (4): 468-495. beck, g. 1916: flora bosne, hercegovine i novopazarskog sandžaka ii (7.) dio. nastavak. glasnik zemaljskog muzeja u bosni i hercegovini, 28 (1): 41-168. celesti grapow, l., blasi, c. 1998: a comparison of the urban flora of different phytoclimatic regions in italy. global ecology and biogeography letters, 7 (5): 367-378. del tredici, p. 2010: spontaneous urban vegetation reflections of change in a globalized world. nature and culture, 5 (3): 299-315. euro+med (2006-): euro+med plantbase the information resource for euro-mediterranean plant diversity. published on the internet http://ww2.bgbm.org/europlusmed/ [accessed december 2017] formanek, e. 1888: beitrag zur flora von bosnien und der hercegovina. österreichische botanische zeitschrift 38: 271-279. formanek, e. 1889: beiträge zur flora von bosnien und der hercegovina. österreichische botanische zeitschrift, 39: 22-28. biologica nyssana 8 (2)  december 2017: 129-136 sarajlić, n., jogan, n.  alien flora of the city of sarajevo… 134 godefroid, s., koedam, n. 2007: urban plants species patterns are highly driven by density and function of built– up areas. landscape ecology, 22: 1227–1239. hofmann, f. 1882: beitrag zur kenntnis der flora von bosnien. österreichische botanische zeitschrift, 32: 73-81; 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allochthonous species in the flora of bosnia and herzegovina. herbologia, 12 (3): 15-21. šoljan, d., muratović, e., jukić, lj., merdan, a., ravlić, v. 2006: caryophyllaceae, fabaceae, lamiaceae, ranunculaceae i scrophulariaceae u flori kanjona rijeke miljacke od bentbaše do kozije ćuprije. hrvatska misao, 6: 7-23 šoljan, d., muratović, e. 2000: rasprostranjenost vrste ambrosia artemisiifolia l. na području grada sarajeva. herbologia, 1 (1): 41-47. tomović-hadžiavdić v., šoljan, d. 2006: urbana flora sarajeva. glasnik zemaljskog muzeja (prirodne nauke), nova sveska, 32, 121–135. witosławski, p., bomanowska, a. 2009: southern european species in the flora of towns in the central poland. botanica serbica, 33 (2): 115-129. wittig, r. 2004: the origin and development of the urban flora of central europe. urban ecosystems, 7: 323–333. appendix 1. alien flora of the city of sarajevo (lf-life form, pi-period of introduction, mi-mode of introduction, oorigin) species family lf pi mi o 1st record 1. abutilon theophrasti medik. malvaceae t arc acc as-e 2. acer negundo l. aceraceae ph neo del am-n tomović-hadžiavdić & šoljan (2006) 3. aesculus hippocastanum l. hippocastanaceae ph neo del m hofmann (1882) 4. ailanthus altissima (mill.) swingle simaroubaceae ph neo del as-e tomović-hadžiavdić & šoljan (2006) 5. alcea rosea l. malvaceae h neo del m 6. amaranthus albus l. amaranthaceae t neo acc am-n maly (1919) 7. amaranthus caudatus l. amaranthaceae t neo del am-s 8. amaranthus graecizans l. amaranthaceae t neo acc af-e 9. amaranthus retroflexus l. amaranthaceae t arc acc am-n beck (1887a) 10. ambrosia artemisiifolia l. asteraceae t neo acc am-n maksimović (1990) 11. anagallis arvensis l. primulaceae t arc acc m beck (1887b) 12. angelica archangelica l. apiaceae h arc d-a as-e 13. antirrhinum majus l. scrophulariaceae t arc d-a m tomović-hadžiavdić & šoljan (2006) 14. armoracia rusticana gaertn, may & scherb. brassicaceae g arc d-a eu-as beck (1916) 15. artemisia annua l. asteraceae t neo acc as-e beck (1887b) 16. bassia scoparia (l.) a. j. scott chenopodiaceae t neo del as-c maly (1910) 17. bidens frondosus l. asteraceae t neo acc am-n 18. brassica rapa (l.) l. brassicaceae t arc del m maly (1904) 19. calendula officinalis l. asteraceae t arc del m 20. capsella bursa-pastoris (l.) medik. brassicaceae h arc acc eu-as hofmann (1882) 21. catalpa bignonioides walter bignoniaceae ph neo del am-n 22. commelina communis l. commelinaceae g neo del as-e 23. cuscuta campestris yuncker cuscutaceae t neo acc am-n 24. datura stramonium l. solanaceae t neo acc am-n beck (1887b) 25. eleusine indica (l.) gaertn. poaceae t neo acc as 26. erigeron annuus (l.) desf. asteraceae t neo acc am-n maly (1910) 27. erigeron canadensis l. asteraceae t neo acc am-n hofmann (1882) 28. euphorbia prostrata aiton euphorbiaceae t neo acc am-n 29. galinsoga parviflora cav. asteraceae t neo acc am-n maly (1933) 30. galinsoga quadriradiata ruiz & pav. asteraceae t neo acc am-n 31. gleditsia triacanthos l. fabaceae ph neo del am-n šoljan et al. (2006) 32. helianthus annuus l. asteraceae t neo del am-n hofmann (1882) 33. helianthus tuberosus l. asteraceae g neo del am-n hofmann (1882) biologica nyssana 8 (2)  december 2017: 129-136 sarajlić, n., jogan, n.  alien flora of the city of sarajevo… 136 34. hemerocallis fulva (l.) l. xanthorrhoeaceae g neo del as-e maly (1928) 35. hesperis matronalis l. brassicaceae h arc acc eu-as 36. impatiens balfourii hook. f. balsaminaceae t neo del as 37. impatiens glandulifera royle balsaminaceae t neo del as 38. iris germanica l. iridaceae g arc del eu-as 39. juglans regia l. juglandaceae ph arc del as-sw hofmann (1882) 40. juncus tenuis willd. juncaceae h neo acc am-n 41. lepidium virginicum l. brassicaceae t neo acc am-n 42. linum usitatissimum l. linaceae t arc d-a as-w formanek (1889) 43. lonicera nitida e.h. wilson caprifoliaceae ph neo del az-sw 44. lycopersicon esculentum mill. solanaceae t neo del am-s 45. matricaria discoidea dc asteraceae t neo acc am-n maly (1912) 46. medicago arabica (l.) huds. fabaceae t arc acc m beck (1887b) 47. medicago sativa l. fabaceae h arc acc eu-as beck (1896) 48. morus nigra l. moraceae ph arc del as-sw 49. oenothera biennis l. onagraceae h neo del am-n tomović-hadžiavdić & šoljan (2006) 50. oenothera erythrosepala borbas rosaceae h neo del am-n 51. oxalis dillenii jacq. oxalidaceae h neo acc am-n maly (1928) 52. oxalis fontana bunge oxalidaceae h neo acc am-n 53. panicum capillare l. poaceae t neo acc am-n 54. panicum miliaceum l. poaceae t arc d-a as-e formanek (1888) 55. papaver rhoeas l. papaveraceae t arc acc m beck (1916) 56. parthenocissus quinquefolia (l.) planch. vitaceae ph neo del am-n 57. paspalum dilatatum poir. poaceae t neo acc am-s 58. petunia × hybrida (hook.) vilm. solanaceae t neo del am-s 59. persicaria maculosa gray polygonaceae t arc acc eu-as komša (1928) 60. phalaris canariensis l. poaceae t neo acc m hofmann (1882) 61. platycladus orientalis (l.) franco cupressaceae ph arc del eu-as 62. portulaca oleracea l. portulacaceae t arc acc m beck (1906) 63. pyrus communis l. rosaceae ph arc del eu-as hofmann (1882) 64. ranunculus arvensis l. ranunculaceae t arc acc m beck (1891) 65. reynoutria japonica houtt. polygonaceae g neo del as-e sarajlić et al. (2016) 66. rhus typhina l. anacardiaceae ph neo del am-n 67. robinia pseudoacacia l. fabaceae ph neo del am-n maly (1928) 68. sedum sarmentosum bunge crassulaceae ch neo del as šoljan (2011) 69. senecio inaequidens dc. asteraceae t neo acc af-s 70. sinapis arvensis l. brassicaceae t arc d-a m hofmann (1882) 71. solanum tuberosum l. solanaceae g neo del am-s 72. solidago canadensis l. asteraceae g neo del am-n 73. solidago gigantea aiton asteraceae g neo del am-n 74. sorghum halepense (l.) pers. poaceae g arc acc m 75. syringa vulgaris l. oleaceae ph arc del eu-as hofmann (1882) 76. tagetes patula l. asteraceae t neo del am-s 77. tanacetum parthenium (l.) sch.bip. asteraceae t neo del as-w maly (1904) 78. trifolium arvense l. fabaceae t arc del eu-as maly (1904) 79. triticum aestivum l. poaceae t arc del as-sw 80. veronica persica poir. scrophulariaceae t neo acc as-w beck (1887b) 81. xanthium strumarium l. asteraceae t neo acc am-n maly (1912) 82. zea mays l. poaceae t neo del am-s beck (1887a) abbreviations: life forms: ph phanerophyte, ch chamaephyte, h hemicryptophyte, g geophyte, t therophyte; period of introduction: arc archaeophyte, neo – neophyte; mode of introduction: acc accidental, del deliberate, d-a in both ways. aladejana, oluduro 2021, biologica nyssana 12(1) 12 (1) september 2021: 79-86 doi: 10.5281/zenodo.5523057 molecular characterization of extended spectrum β-lactamaseproducingenterobacteriaceae from bats (eidolon helvum) faeces in osun state, nigeria original article oluwatoyin modupe aladejana department of biological sciences, microbiology unit, kings university, odeomu, osun state nigeria om.aladejana@kingsuniversity.edu.ng (corresponding author) anthonia olufunke oluduro department of microbiology, faculty of science, obafemi awolowo university, ile-ife, osun state, nigeria aoluduro2003@yahoo.co.uk received: october 23, 2020 revised: april 01, 2021 accepted: april 04, 2021 abstract: enterobacteriaceae from faecal samples of eidolon helvum were isolated and identified biochemically then further confirmed using analytical profile index 20e kit. antibiotic susceptibility testing and extended spectrum b-lactamase (esbl) production using mast disc were carried out. detection of ctx-m gene was done using polymerase chain reaction (pcr) employing appropriate primers. a total of 337 bacterial isolates was recovered from the studied locations consisting of forty-one species belonging to fifteen genera. species of citrobacter, enterobacter, salmonella, klebsiella spp, and escherichia coli were the most abundant and common to the three studied locations, with salmonella spp being the most predominant. a total of 35.9% of the selected isolates was produced elbs and 14.28% of the isolates harbours ctx-m gene. the study concludes that e. helvum in the study areas are reservoirs of enterobacteriaceae and some harboured ctx-m gene which confirmed their pathogenicity with public health consequences. key words: eidolon helvum, enterobacteriaceae, extended spectrum b-lactamase apstract: molekularna karakterizacija enterobacteriaceae iz fecesa slepog miša (eidolon helvum) u osunu, nigerija koje produkuju β-laktamaze proširenog spektra enterobacteriaceae su izolovane iz fekalnih uzoraka eidolon helvum i biohemijski identifikovane korišćenjem api (analytical profile index) 20e kita. korišćenjem mast diskova urađeno je i testiranje osetljivosti na antibiotike i testiranje produkcije β-laktamaze proširenog spektra (esbl). detekcija gena ctx-m je urađena je polimeraznom lančanom reakcijom (pcr) korišćenjem odgovarajućih prajmera. na ispitivanim lokacijama utvrđdeno je ukupno 337 izolata koji pripadaju 41 vrsti iz 15 rodova. vrste citrobacter, enterobacter, salmonella, klebsiella spp, i escherichia coli bile su najzastupljenije i česte za tri ispitivane lokacije, sa najčešćom salmonella spp. ukupno 35.9% odabranih izolata produkovalo je esbl i 14.28% izolata nosilo je gen ctx-m. istraživanje je zaključilo da su e. helvum rezervoari enterobacteriaceae u istraživnim oblastima i da neki nose ctx-m gen koji potvrđuje njihovu patogenost i posledice po javno zdravlje. ključne reči: eidolon helvum, enterobacteriaceae, β-laktamaze proširenog spektra introduction enterobacteriaceae consist of a large number of closely related bacterial species that inhabit the large bowel of man and animals, soil, water, and decaying matter (hassan et al., 2011). these organisms are responsible for various infections including nosocomial or hospital-acquired infections that cause urinary tract and wound infections, pneumonia, meningitis and septicaemia, among others (ruiz et al., 2002). wild animals can serve as sources of various infectious diseases, including the majority of nosocomial or hospital-acquired and illnesses resulting from environmental contamination. emerging infectious diseases are increasingly originating from wildlife. different activities result © 2021 aladejana, oluduro. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 79 into emerging infectious diseases of wildlife and they include:alteration of an ecosystem, transfer of pathogens and vectors by human or other agents as well as advice in microbial genomic studies which reveals more about the infectious agents. the transmission of pathogens from wildlife to other animals and humans could be through direct or indirect contact (rabinowitz et al., 2009). wildlife although not directly exposed to antimicrobial agents can acquire resistance to clinically used antimicrobial agents through contact with the polluted environment, domestic animals and humans. bat is a natural reservoir of deadly viruses, such as rabies, ebola viruses and zoonotic bacterial pathogens, such as escherichia coli and staphylococcus aureus among others. (oluduro, 2012; akobi et al., 2012). bat can easily spread multiple antibiotic resistant enterobacteriaceae due to their versatile feeding and migratory capability. antibiotics are used in the treatment of infections caused by enterobacteriaceae. the active antimicrobial agents available for use against these organisms include; fluoroquinolones, β -lactams and aminoglycosides. resistance to betalactams in enterobacteriaceae is mainly conferred by β-lactamases. these enzymes inactivate betalactam antibiotics by hydrolysis. the most common esbls are shv-, tem-, ctx-m. the ctx-ms are increasingly detected worldwide (paterson & bonomo, 2005; pierre et al., 2020). materials and methods study area the study area were selected cities where bats roost over a human-populated place in osun state, nigeria. these included: ile ife lies between 7°31’14’’ n and 7°31’14’’.7612 n and longitudes 4°32’3.161’’e and 4°32’2.591’’ e coordinates, osogbo (machine tools area, km 8, osogboikirun road) located at 7°50’9.0168’’ n and 4°36’30.0708’’ e and ilesa (oba’s palace area, ilesa) located at 7°37’0’’ n and 4°43’0’’ e (fig. 1). sample collection faecal droppings of bats were collected randomly from various roosting places in osun state, in the locations stated above. the samples were collected between 6.00 a.m and 7.00 a.m according to the method of akobi et al. (2012) and transported to the laboratory immediately in an ice bag for bacteriological analysis. isolation of bacteria the faecal samples collected were immediately streaked on prepared macconkey agar plates and incubated at 37 °c for 24 hours for isolation of enterobacteriaceae. distinct colonies were picked and sub-cultured by successive streaking on freshly prepared macconkey agar plates, incubated at 37 °c for 24 hours and a pure isolates were obtained. these were stored on nutrient agar slant and kept in the refrigerator at 4 °c for further use. all the isolates were identified using cultural, morphological, microscopic examination and biochemical tests following standard procedures and interpreted according to bergey’s manual of systematic bacteriology. isolates were further confirmed using analytical profile index (api) 20e test kit (biomérieux, inc., france). 80 biologica nyssana ● 12 (1) september 2021: 79-86 aladejana, oluduro ● molecular characterisation of extended spectrum β-lactamase-producingenterobacteriaceae from bats faeces... fig. 1. map of osun state and the study areas 81 phenotypic detection of extended spectrum b-lactamase (esbl) production selected multiple antibiotic-resistant enterobacteriaceae isolates from faecal samples of bats were tested for esbl production. this was carried out by using mask disc comprising six antibiotics of ceftazidime (30 µg), ceftazidime plus clavulanic acid (30 µg), cefotaxime (30 µg), cefotaxime plus clavulanic acid (30 µg), cefpodoxime (30 µg), and cefpodoxime plus clavulanic acid (30 µg). the standardised inoculum of the isolates were inoculated aseptically on müeller-hinton agar plates. these discs were firmly placed on the surface of the culture plates aseptically using a sterile forceps and incubated in an inverted position at 37 °c for 24 hours. the diameters of inhibition zones of each antibiotic on the bacterial isolates were measured with a transparent ruler to the nearest millimetre. an increase in zone diameter greater than or equal to 5 mm in the presence of clavulanic acid from any or all the sets of mast id esbl detection disc was considered to indicate the presence of esbl in the tested organisms. detection of resistance gene (ctm gene) in selected esbl-producing enterobacteriaceae isolates by polymerase chain reaction (pcr) for the extraction of dna, randomly selected esbl-producing enterobacteriaceae isolates grown overnight were transferred to eppendorf tube and spun down at 14,000 rpm for 2 minutes. the supernatant was discarded and 600 µl of prewarmed extraction buffer cetyl trimethyl ammonium bromide (ctab) was added to the pellet and incubated at 65 °c for 30 minutes. the sample was removed from the incubator and allowed to cool to room temperature, chloroform was added and mixed by inverting tubes for 15 minutes. after that, the sample was spun at 14,000 rpm for 15 minutes and the supernatant was transferred into a new eppendorf tube, afterwards equal volume of cold isopropanol was added to precipitate the dna. the sample was kept in the freezer for 1hour and later spun at 14,000 rpm for 10 minutes. the supernatant was discarded and the pellet was washed with 70% ethanol. the sample was then air dried for 30 minutes on the bench. the pellet was resuspended in 100 µl of sterile distilled water. the dna concentration of the samples was measured on spectrophotometer at 260 nm and 280 nm and the genomic purity was determined. the genomic purity was between 1.8 –2.0 µl for all the dna samples. ctx-m genes were profiled in the isolates by pcr using the primer ctx-m-f atgtgcagyaccagtaargtkatggc, ctx-m-r-tgggtraartargtsaccagaa ysagcgg (8). pcr was performed in a total volume of 25 μl containing 2.5 μl of both the forward and the reverse of the primers, 12.5 μl master mix, 2.5 μl nuclease free water and 5 μl of the extracted dna (as dna template), then dna amplification was carried out with the thermal cycler. the reaction mixtures were amplified by denaturation for 4 minutes at 95 °c, 30 cycles at 95 °c for 30 seconds, annealing temperature at 55 °c for 60 seconds. the pcr products were electrophoresed using 1.5% agarose gel. it was stained with 1% ethidium bromide and run at 80 v for 2 hours, the electrophoretic products were scanned with uvtransilluminator. data analysis data were analysed using spss software (version 20). proportions and the actual number of esblproducing enterobacteriaceae isolates was used to describe frequency outputs for categorical variables. mean and standard deviation were used to describe continuous variables. results in all, 337 bacterial isolates were recovered from the three locations which includes 142 (42.12%) isolates from obafemi awolowo university, ile ife, 84 (24.93 %) from nigeria machine tools area, osogbo and 111 (32.94 %) from oba’s palace area, ilesa all in osun state, southwest, nigeria. the identified enterobacteriaceae belong to 15 genera comprising 41 different species. the bacterial species identified based on their various biochemical reactions to the api kits include the following: citrobacter freundi, c. koseri, c. diversus, c. werkmanii, c. rodentium; enterobacter aerogenes, e. intermedius, e. cloacae, e. aquatilis e. asburiae, e. sakazakii; proteus vulgaris, p. mirabilis, salmonella arizonae, s. bongori, s. typhi, s. enterica, s. choleraesuis; klebsiella pneumoniae, k. oxytoca, k. ornithinolytica, k. planticola; shigella sonnei, sh. dysentery, sh. flexineris; serratia. liquefaciens, s. marcescens, s. plymutica, s. odorifera; raoultella terrigena, r. ornithinolytica; yersinia frederiksenii, y. aleksiciae; y. mollaretii; escherichia coli, erwinia amylovora, edwardsiella ictaluri, kluyvery ascorbate, providenica rettgeri, and rahnella aquatilis. table 1 shows the distribution of the bacterial isolates in the bats faeces across the three studied locations. for ile-ife location, s. arizonae has the highest frequency of 24 (16.90%) followed by e. coli 17 (11.97%) and the least frequency of 1 (0.70%) were recorded in e. sakazaki, e. hormaecei, k. planticola, k. ornitinolytica, r. aquatilis, r. terribiologica nyssana ● 12 (1) september 2021: 79-86 aladejana, oluduro ● molecular characterisation of extended spectrum β-lactamase-producingenterobacteriaceae from bats faeces... 82 table 1. the percentage of enterobacteriaceae isolates in faeces of eidolon helvum from the three locations bacterial isolates ile-ife (n=142) (%) osogbo (n=84) (%) ilesa (n=111) (%) number of isolates (n=337) (%) citrobacter freundi 3(2.11) 2(2.38) 5(4.51) 10 (2.97) c. koseri 3(2.11) 2(2.38) 3 (2.70) 8 (2.37) c. werkmanii 1(0.70) 1 (0.30) c. rodentium 6(4.23) 3 (0.89) c. farmer 2(1.41) 2 (0.59) c. diversus 4 (3.60) 4(1.19) escherichia coli 17 (11.97) 11(13.10) 8 (7.20) 36 (10.68) enterobacter aerogenes 2 (1.41) 5(5.59) 6 (5.40) 13 (3.86) e. aquatilis 1(1.19) 1 (0.30) e. intermidius 3 (2.11) 2(2.38) 5 (1.48) e. asburiae 12(8.45) 4(2.80) 5 (4.50) 21 (0.21) e. cloacae 2 ( 1.41) 1(1.19) 6 (5.40) 9 (2.67) e. sakazaki 1 (0.70) 2(2.38) 3 (0.89) e. hormaecei 1 (0.70) 1 (0.30) edwardsiella ictaluri 3 (2.11) 3 (0.89) erwinia amylovora 1 (0.90) 1 (0.30) klebsiella pneumonia 10 (7.04) 10 (11.91) 11 (9.91) 31 (9.20) k. planticola 1 (0.70) 3 (2.70) 4(1.19) k. oxytoca 13 (9.16) 9 (10.71) 5 (4.51) 27 (8.01) k. ornitinolytica 1 (0.70) 1 (0.30) kluyvery ascorbate 2(1.41) 1 (1.19) 1 (0.90) 4 (1.19) proteus mirabilis 5(3.04) 1 (1.19) 6 (5.40) 12 (3.56) p. vulgaris 2(1.41) 1(1.19) 3 (2.70) 6 (1.78) providenica rettgeri 2(1.41) 2 (0.60) rahnella aquatilis 1(0.70) 1 (0.30) raoultella terrigena 1 (0.70) 1 (0.30) r. ornithinolytica 3 (2.11) 3 (0.89) serratia plymutica 5 (3.04) 3 (2.70) 8 (2.37) s. odorifera 2 (1.41) 2 (1.80) 4 (1.19) s. liquefaciens 1 (0.70) 1 (1.19) 2 (0.60) s. marcescens 3 (2.11) 2 (1.80) 5 (1.48) salmonella typhi 1 (1.19) 4 (3.60) 5 (1.48) s. enterica 7 (8.33) 5 (4.51) 12 (3.56) s. bongori 2 (1.41) 2 (2.38) 4 (1.19) s. arizonae 24 (16.90) 14(16.67) 12 (10.80) 50 (41.56) s. choleraesuis 1 (1.19) 1 (0.30) shigella flexineris 3 (2.11) 3(3.57) 3 (2.70) 9 (2.67) s. sonnei 4(4.76) 4 (3.60) 8 (2.37) yersinia aleksiciae 1 (0.70) 1(1.19) 2 (0.60) y. mollaretii 1 (0.70) 2 (1.80) 3 (0.89) y. fredrikseni 3 (2.11) 3 (0.89) biologica nyssana ● 12 (1) september 2021: 79-86 aladejana, oluduro ● molecular characterisation of extended spectrum β-lactamase-producingenterobacteriaceae from bats faeces... gena, s. liquefaciens, y. aleksiciae and y. mollaretii. for osogbo location, salmonella arizonae has the highest frequency of 14 (16.67%) followed by escherichia coli 11 (13.10%) and the leat frequency of 1 (1.19%) were recorded in enterobacter aquatilis, e. cloacae, kluyvery ascorbate, proteus mirabilis, p. vulgaris, s. liquefaciens, s. typhi, s. choleraesuis and y. aleksiciae. also for ilesa location. s. arizonae has the highest frequency of 12 (10.80%) followed by k. pneumoniae 11 (9.91%) and the leat frequency of 1 (0.9%) were recorded in erwinia amylovora and kluyvery ascorbate. in total, s. arizonae has the hig-hest frequency of 50 (41.56%), followed by e. coli 36 (10.68 %), k. pneumoniae 31 (9.20 %), k. oxytoca 27 (8.01%). salmonella spp., citrobacter spp., enterobacter spp., klebsiella spp., yersinia spp. and e. coli were most abundant and common to the three locations as depicted in fig. 2. table 2 shows the occurrence of the esbl-producing isolates in selected multiple antibiotic resistant bacterial isolates from bat faecal samples. the esblproduction by selected multiple antibiotic-resistant isolates using mast disc showed that 14 (35.90%) of the 39 selected antibiotic-resistant isolates were positive to esbls production. 83 fig. 2. distribution of bacterial isolates common to the three locations (ile-ife, osogbo and ilesa) fig 3: agarose gel electrophoresis of ctx –m (529 bp) amplicons of the selected multiple antibiotics resistant e coli spp. isolated from faecal samples of eidolon helvum from three locations key lane m –dna ladder (100 bp). lanes 1 to 3 were amplicons from e. coli lanes 4 to 6 were amplicons from enterobacter sakazakii lanes 7 and 10 were amplicons from salmonella arizonae lanes 11 and 12 were amplicons from serretia liquefacience lanes 13 and 14 were amplicons from citrobacter koseri biologica nyssana ● 12 (1) september 2021: 79-86 aladejana, oluduro ● molecular characterisation of extended spectrum β-lactamase-producingenterobacteriaceae from bats faeces... the ctx –m (529 bp) gene was detected in e. coli and e. sakazakii as shown in fig. 3. discussion fifteen different genera comprising 41 different species of enterobacteriaceae were recovered from the faecal samples of bats from the three study locations (ile-ife, osogbo and ilesa) in osun state, nigeria. the high diversity of isolates could be as a result of the ability of bats to travel far distance, which may result with feeding from different habitats. the enterobacteriaceae recovered from the faecal samples of bats in this study were bacterial isolates that are significant in faecal samples of animals (both domestic and wild) across the world (sader et al., 2018). salmonella arizonae has the highest frequency of 50 (41.56%), followed by escherichia coli 36 (10.68 ), k. pneumoniae 31 (9.20%) and k. oxytoca 27 (8.01%). klebsiella pneumoniae had the highest occurrence of 10 (7.04%), 10 (11.91%) and 11 (9.91%) in ileife, osogbo and ilesa, respectively. meanwhile, k. oxytoca had the next high percentage occurrence of 13 (9.16%), 9 (10.71%) and 5 (4.91%) in ileife, osogbo and ilesa location. these percentage occurrences were relatively low for k. pneumoniae (35.8 %) but relatively high for k. oxytoca (0.9%) when compared to the occurrence from wild birds (10). escherichia coli had a prevalence of 17 (11.97%), 11 (13.10%) and 8 (7.20%) in ile-ife, osogbo and ilesa locations, respectively. this percentage occurrence was relatively low compared to 50.5 % occurrence reported from wild birds (carlos et al., 2016). the result of this study can be compared to the record of the presence of e. coli in the faecal samples of bats in ile-ife, nigeria (oluduro, 2012). salmonella spp. and escherichia coli have also been isolated from bat faeces in trinidad according to adesiyun (2009). ten (10) genera were common to the three studied locations while 13 species were common to the three studied locations, 13 species were common to two of the locations and 15 species were present in only one studied location. ile-ife location has the highest diversity of enterobacteriaceae with a total of 34 different bacteria species identified, osogbo has a total of 21 different species while ilesa has a total of 25 different species identified. the results show great diversity of enterobacteriacea in the studied area, although statistical data indicate that there is no significant difference in the occurrence of isolates across the three locations. bats are unique with their ability to fly long distance (calisher et al., 2006). therefore they can migrate from one geographical location to another. this capability makes it easy for them to acquire new microorganism either from the environment, contact with one another or other animals which aid the resistance of their microbial commensals to antimicrobial agents. the antimicrobial resistance among these bacteria leads to ineffective treatment of the infections caused by these organisms. various mechanisms of drug resistance have been attributed with the production of betalactamases which leads to resistance. the increase in the prevalence of esbl-producing isolates globally increases the burden of treating infectious disease especially in developing countries with poor resources and weak health management system. in the recent decade, esbl-producing enterobacteriaceae has resulted in an increase of resistance to β-lactam antibiotics leading to challenges in the management of infections caused by these bacteria (cantòn & coque, 2006). in this study, 35.9% of the selected multiple resistant isolates were positive to (esbls) produc84 s/n isolates name specific isolate frequency 1 escherichia coli a84, b83, c42 3 2 enterobacter sakazakii a3, b29, a120 3 3 salmonella arizonae a5, b6, c15, b32, b44 4 4 serretia liquefacience a140, a69 2 5 citrobacter koseri a105, b3 2 * isolate code isolates from ile-ife: a84, a3, a120, a5, a140, a69 and a105 isolates from osogbo: b83, b29, b6, b32, b44, and b3 isolates from ilesa: c42 and c25 table 2. occurrence of extended-spectrum β-lactamase (esbl) -producing isolates from faecal samples of eidolon helvum from the three locations using mast disc biologica nyssana ● 12 (1) september 2021: 79-86 aladejana, oluduro ● molecular characterisation of extended spectrum β-lactamase-producingenterobacteriaceae from bats faeces... tion. it is an indication that substantial amount of the multiple resistant isolates were esbls producers. the reason may be that the third-generation cephalosporins are commonly in use over a long period in the studied areas and might have been abused, therefore leading to the acquisition of resistance mechanisms by the organisms. the decreased susceptibility of these antimicrobial could also be as a result of the production of esbl and ampc b-lactamase. two of the isolates profiled harboured ctx-m gene (e. coli (b83 from osogbo) and enterobacter sakazakii (a120 from ife)). although the ctx-m family of esbls has been reported in germany since 1989 (doi et al., 2017) it has now spread to different parts of the world. the ctx-m enzyme has been found in e. coli and klebsiella spp., more frequently then in other enterobacteriaceae species (bush, 1989). wildlife are not directly exposed to clinically used antibiotics, but they could have contacted it through environment (sewages and animal manure). resistance could also be gained through feeding on some materials that contain antimicrobial substances in nature, like plants, but this still remain unclear. due to the ability of migratory birds to travel far distance, they can carry antibiotic-resistant bacteria from one continent to another leading to global transmission of bacterial resistance among bats. direct contact with faeces of wildlife and indirect contact through food, water and air is an important risk factor for the dissemination and transmission of pathogens or antimicrobial resistance from animals to both other animals and humans. conclusions currently, no studies have described the prevalence of esbl-producing enterobacteriaceae in faecal samples of eidolon helvum in nigeria, hence the study is novel. although much has not been done on the probable source of contamination, however the result of this study indicates that bat is a source of esbl-producing enterobacteriaceae and hence prevention need to be taken since they roost over human-populated area and release their faeces indiscriminately. acknowledgements. i acknowledge professor oluduro’s laboratory, department of microbiology, 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in men. journal of clinical microbiological, 40: 4445–4449. sader, h.s., castanheira, m., duncan, l.r., flamm, r.k. 2018. antimicrobial susceptibility of enterobacteriaceae and pseudomonas aeruginosa isolates from united states medical centers stratified by infection type: results from the international network for optimal resistance monitoring (inform) surveillance program, 20152016. diagnostic microbiology infectious diseases, 92(1): 69-74. 86 biologica nyssana ● 12 (1) september 2021: 79-86 aladejana, oluduro ● molecular characterisation of extended spectrum β-lactamase-producingenterobacteriaceae from bats faeces... aćimović et al., 2019, biologica nyssana 10(1) 10 (1) september 2019: 23-28 doi: 10.5281/zenodo.3463994 the chemical composition of the essential oil of dracocephalum moldavica l. from vojvodina province (serbia) original article milica aćimović institute of field and vegetable crops novi sad, maksima gorkog 30, 21000 novi sad, serbia acimovicbabicmilica@gmail.com (corresponding author) jovana stanković institute of chemistry, technology and metallurgy, njegoševa 12, 11000 belgrade, serbia jovana_stankovic@hotmail.com mirjana cvetković institute of chemistry, technology and metallurgy, njegoševa 12, 11000 belgrade, serbia miracvet13@gmail.com marina todosijević university of belgrade, faculty of chemistry, studentski trg 12-16, 11000 belgrade, serbia marinab@chem.bg.ac.rs milica rat university of novi sad, faculty of science, trg dositeja obradovića 3, 21000 novi sad, serbia milica.rat@dbe.uns.ac.rs received: mart 7, 2019 revised: may 21, 2019 accepted: august 26, 2019 abstract: dracocephalum moldavica l., also called moldavian balm or moldavian dragonhead, is native to temperate climate of asia, but it was naturalized in eastern and central europe, north africa, china and north-eastern united states. this is an annual plant, with numerous stems (up to 6), 22-45 cm high, and blue flowers arranged in pseudo-whorls growing in leaf axils. essential oil accumulates in exogenous oil-containing cells at the dorsal sides of the leaves, and in the inflorescence. because of this, the entire plant has a citrus-like flavor, resembling that of lemon balm and catnip. this plant is extensively used as a spice and for composition of tea blends, in food aromatization (canned fish, jams, candies, syrups), perfumery, alcohol industry, soaps and detergents. dracocephalum moldavica from vojvodina province, serbia contains geranial (29.6%), geranyl acetate (27.2%) and neral (19.4%) as the most abundant compounds. further investigations will be focused on the influence of weather conditions on essential oil composition, as well as on bioactive potential of this essential oil. key words: moldavian balm, moldavian dragonhead, gc-ms analysis, geranial, geranyl acetate, neral apstract: hemijski sastav etarskog ulja dragocephalum moldavica l. iz vojvodine (srbija) biljka dracocephalum moldavica l., poznata je kao moldavska melisa ili moldavska zmajeglavka. poreklom je iz umerenog klimata azije, ali je naturalizovana u istočnoj i centralnoj evropi, severnoj africi, kini i severo-istočnom delu sad. iz korena ove jednogodišnje biljke izbija veći broj stabljika (do 6), koje su 22-45 cm visine, sa plavim cvetovima raspoređenim u lažnim pršljenastim cvastima u pazusima listova. etarsko ulje se akumulira u egzogenim uljanim ćelijama sa donje strane listova i u cvetovima. zbog etarskog ulja koje ima citrusnu notu ova biljka podseća na matičnjak i macinu travu. biljka se intenzivno koristi kao začin i kao dodatak čajnim mešavinama, za aromatizaciju hrane (konzervirana riba, džemovi, slatkiši, sirupi), parfimeriji, alkoholnoj industriji, proizvodnji sapuna i deterdženata. etarsko ulje d. moldavica iz ap vojvodine, srbije sadrži geranial (29,6%), geranil-acetat (27,2%) i neral (19,4%) kao najzastupljenije komponente. dalja istraživanja biće usmerena na uticaj vremenskih uslova na sastav etarskog ulja, kao i na njegov biološki potencijal. ključne reči: moldavska melisa, moldavska zmajeglavka, gc-ms analiza, geranial, geranil acetat, neral introduction dracocephalum moldavica l., also called moldavian balm or moldavian dragonhead, is native to temperate climate of asia, but it was naturalized in eastern and central europe, north africa, china and north-eastern united states. this species belongs to the subtribe nepetinae, tribe menthae of lamiaceae family. the genus contains 71 species, widespread across northern hemisphere regions (naderifar et al., 2015; amirnia et al., 2017). this annual plant is very beautiful, with numerous stems (up to 6), 22-45 cm high, and blue flowers arranged in pseudo-whorls growing in leaf axils. © 2019 aćimović et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 23 essential oil accumulates in exogenous oil-containing cells at the dorsal sides of the leaves, and in the inflorescence. because of this, the entire plant has a citrus-like flavor, resembling that of lemon balm (melissa officinalis l.) and catnip (nepeta cataria l.). apart from essential oil, the plant produces nectar, due to which it is grown as a honey-bearing plant and cultivated in gardens and parks as an ornamental plant (aćimović et al., 2019). this plant with its citrus like flavor is extensively used as a spice and for composition of tea blends, because of neral and geranial as major constituents of essential oil. dracocephalum moldavica is used in food aromatization (canned fish, jams, candies, syrups), perfumery, alcohol industry, soaps and detergents. apart from this, dried leaves have the potential of being used as a functional additive for extruded crisps with high nutritional value, especially because of the dietary fiber and rosmarinic acid content, strong antioxidant potential and acceptable sensory properties (wojtowicz et al., 2017). seed is a good source of fatty oil with spicy taste and aromatic odor, rich in unsaturated fatty acids, principally the linolenic and linoleic acids. this categorizes d. moldavica seed into the group of raw materials suitable for nutraceuticals, food supplements, and functional food applications (aćimović et al., 2019). moreover, the application of d. moldavica residues as bagasse waste (oilcake) collected after pressing and added to corn crisps could be an effective way of limiting the oil waste after pressing and increasing the sustainability of waste management. a new range of nutritionally valuable snacks could be introduced to the market (oniszczuk et al., 2017). furthermore, numerous investigations show that this plant possesses good antioxidative (aprotosoaie et al., 2016; aslanipour et al., 2017; weremczukjeżynaet al., 2017; ehsani et al., 2017; fallah et al., 2018), antimicrobial (pak et al., 2016; ehsani et al., 2017; keikhaie et al., 2018) and insecticidal activity (chu et al., 2011; ding et al., 2015). it is also used as antinociceptive (maham et al., 2013), sedative (martínez-vázquez et al., 2012), neuroprotective (sun et al., 2014), as well as cardiotonic agent (najafi et al., 2009; zeng et al., 2018), and for treating chronic mountain sickness (maimaitiyiming et al., 2014). this species is not well known in serbia. it was introduced in serbia in the collection garden of institute of field and vegetable crops, from romania (kišgeci et al., 1982). the aim of this paper is to analyze the essential oil obtained from d. moldavica grown in agroecological conditions of vojvodina province, serbia. materials and methods plant material the plant (fig. 1) grown at institute of field and vegetable crops novi sad, during 2018, were confirmed by m. rat and deposited at the herbarium of biology and ecology (buns herbarium), faculty of natural sciences, university of novi sad, as d. moldavica, voucher specimens 2-1468. during the flowering stage (june), the aboveground parts were cut, dried and used for essential oil extraction. essential oil extraction the dried aboveground parts of d. moldavica were subjected to hydro-distillation using an all glass clevenger-type apparatus to extract essential oils. the samples were ground, homogenized and made into a fine powder. in order to extract the essential oils, 100 g of the powder was placed in 1 l conical flask and connected to the clevenger apparatus. 500 ml of distilled water was added to the flask and heated to the boiling point. the steam in combination with the essential oils was distilled into a graduated cylinder for 4 h and then separated from aqueous layer. the yield essential oil was very low, so it is extracted with n-hexane, dried over anhydrous sodium sulfate and evaporated. the obtained oil was kept refrigerated at +4 °c until required for further analysis. 24 biologica nyssana ● 10 (1) september 2019: 23-28 aćimović et al. ● the chemical composition of the essential oil of dracocephalum moldavica l. from vojvodina province (serbia) fig. 1. dracocephalum moldavica gc/ms analysis gas chromatographic-mass spectrometric analysis was performed using an agilent 6890 gas chromatograph coupled with an agilent 5973 network mass selective detector (msd) (both agilent, santa clara, usa), in positive ion electron impact (ei) mode. the separation was effected using agilent 19091s-433 hp-5ms fused silica capillary column with 30 m × 0.25 mm i.d., 0.25 μm film thickness. the gc oven temperature was programmed from 60 °c to 285 °c at a rate of 3 °c/min. helium was used as carrier gas; inlet pressure was 20.3 kpa; linear velocity was 1 ml/ min at 210 °c. injector temperature: 250 °c; injection mode: splitless. ms scan conditions: ms source temperature, 230 °c; ms quad temperature, 150 °c; energy, 70 ev; mass scan range, 40–550 amu. 25 identification of volatile compounds the identification of components was carried out on the basis of kovats retention index and by comparison with reference mass spectra (wiley and nist databases). results and discussion in d. moldavica essential oil 88 compounds were detected, among which geranial (29.6%), geranyl acetate (27.2%) and neral (19.4%) were the most abundant, comprising 76.2%. other significant compounds were: geraniol (5.4%), neryl acetate (3.0%), piperitone (1.8%) and vulgarone b (1.6%). other compounds were present in the amount less than 1.0%, among which 31 unidentified compounds table 1. chemical composition of d. moldavica no compound ri rt % no compound ri rt % 1 1-octen-3-ol 982 7.14 0.2 46 trans-α-farnesene 1514 29.23 tr 2 3-octanone 990 7.38 0.2 47 italicene ether 1540 30.28 tr 3 dehydro-1,8-cineole 997 7.59 tr 48 spathulenol 1582 32.032 0.2 4 3-octanol 1000 7.65 0.1 49 caryophyllene oxide 1587 32.25 0.4 5 bergamal 1057 9.70 0.1 50 viridiflorol 1596 32.60 tr 6 cis-linalool oxide 1076 10.42 tr 51 salvial-4(14)-en-1-one 1598 32.67 tr 7 trans-linalool oxide 1093 11.04 tr 52 ni-12 1602 32.88 0.1 8 linalool 1104 11.47 0.4 53 ni-13 1610 33.17 0.1 9 ni-1 1109 11.65 0.1 54 humulene epoxide ii 1613 33.28 0.1 10 1-octen-3-yl acetate 1116 11.98 tr 55 1,10-di-epi-cubenol 1619 33.51 tr 11 ni-2 1142 13.10 0.1 56 ni-14 1630 33.93 0.1 12 exo-isocitral 1148 13.34 0.1 57 ni-15 1634 34.09 0.1 13 trans-chrysanthemal 1152 13.54 0.3 58 ni-16 1648 34.63 0.1 14 nerol oxide 1156 13.76 0.1 59 vulgarone b 1654 34.87 1.6 15 cis-isocitral 1166 14.18 0.6 60 ni-17 1659 35.02 0.1 16 ni-3 1175 14.59 0.2 61 cis-calamenen-10-ol 1663 35.20 0.1 17 rosefuran epoxide 1177 14.62 0.1 62 ni-18 1668 35.37 0.1 18 trans-isocitral 1184 14.96 0.9 63 ni-19 1675 35.67 0.2 19 α-terpineol 1193 15.34 tr 64 germacra-4(15),5,10(14)-trien-1-α-ol 1690 36.25 0.1 20 ni-4 1196 15.50 0.2 65 cyclocolorenone 1764 38.92 0.1 21 ni-5 1209 16.01 0.2 66 ni-20 1829 41.28 0.1 22 ni-6 1222 16.57 0.1 67 6,10,14-trimethyl-2-pentadecanone 1851 42.02 0.3 23 ni-7 1226 16.77 0.1 68 ni-21 1926 44.59 0.1 24 nerol 1232 17.04 0.4 69 ni-22 2011 47.47 0.1 25 ni-8 1236 17.22 0.2 70 ni-23 2038 48.29 0.1 26 neral 1248 17.76 19.4 71 manool 2068 49.18 0.1 27 piperitone 1258 18.21 1.8 72 ni-24 2082 49.60 0.1 28 geraniol 1262 18.38 5.4 73 ni-25 1997 50.02 0.1 29 ni-9 1264 18.48 0.2 74 ni-26 2116 50.62 0.1 30 geranial 1280 19.18 29.6 75 ni-27 2126 50.91 0.3 31 trans-anethole 1291 19.65 tr 76 ni-28 2156 51.80 0.2 32 thymol 1298 19.96 tr 77 ni-29 2164 52.05 0.2 33 carvacrol 1307 20.35 0.7 78 ni-30 2218 53.65 0.1 34 methyl geranate 1328 21.26 0.2 79 ni-31 2238 54.26 0.1 35 neryl acetate 1369 23.08 3.0 80 tricosane 2307 56.54 0.1 36 α-copaene 1380 23.58 0.1 81 pentacosane 2503 62.04 0.2 37 geranyl acetate 1393 24.15 27.2 82 hexacosane 2603 64.65 tr 38 decyl acetate 1412 25.07 0.1 83 heptacosane 2706 67.19 0.3 39 trans-caryophyllene 1425 25.49 0.2 84 2-methyloctacosane 2780 68.96 tr 40 α-humulene 1459 26.91 0.1 85 octacosane 2806 69.60 tr 41 ni-10 1464 27.11 0.1 86 nonacosane 2906 71.96 0.3 42 ni-11 1469 27.35 0.1 87 triacontane 3056 74.23 tr 43 germacrene d 1488 28.10 0.1 88 untriacontane 3105 76.45 0.1 44 ar-curcumene 1489 28.15 0.1 total identified 95.5 45 trans-β-ionone 1491 28.24 tr total ni 4.1 ri – retention index, rt – retention time, ni – not identified compounds (mass spectrum of these compounds, m/z (intensity) are shown at figures 2-32), tr – compound presented in traces (less than 0.1%) biologica nyssana ● 10 (1) september 2019: 23-28 aćimović et al. ● the chemical composition of the essential oil of dracocephalum moldavica l. from vojvodina province (serbia) 26 comprising 4.1% (tab. 1). a gc-fid chromatogram of d. moldavica essential oil is shown at fig 2. chemical composition of d. moldavica essential oil was previously studied in egypt (hussein et al., 2006; el-baky and el-bsroty, 2008; aziz et al., 2013; ahl et al., 2015; hegazy et al., 2016), iran (omidbaigi et al., 2010; maham et al., 2013, golparvar et al., 2016, ehsani et al., 2017; janmohammadi et al., 2017; fallah et al., 2018), turkey (eshan et al., 2014), ukraine (kotyuk and rakhmetov, 2017) and china (chu et al., 2011). it is established that chemical composition of essential oil from aerial parts of d. moldavica depends on many factors, among which origin, cropping system, fertilization, salt stress, weed management, etc. (aziz et al., 2013; janmohammadi et al., 2017; fallah et al., 2018). the principal compounds in almost all essential oils are neral, geranial, geranyl acetate and geraniol. however, the content of neral in essential oil varied between 10.25 and 43.49%, while geranial ranged between 9.10 and 42.45%. furthermore, geranyl acetate and geraniol content varied in larger scale, between 0.20%-40.40%, and 0.50 and 28.14%, respectively (aćimović et al., 2019). investigations show that the geranyl acetate, geranial and geraniol in essential oil reach their maximum levels during the flowering, while the content of neral, decreases during flowering. these observations indicate that the biosynthesis of geranyl acetate is dominant at the beginning of the vegetative period, but is superseded by biosynthesis of geranial and geraniol, from the early stage of flowering. it indicated that the optimal harvest time proved to be during the flowering stage, when the oil content is the highest and thus also the amount of the main terpenes is the highest (holm et al., 1988). conclusion dracocephalum moldavica from serbia contains geranial (29.6%), geranyl acetate (27.2%) and neral (19.4%) as the most abundant compounds. further investigations will be focused on the influence of weather conditions on essential oil composition, as well as on bioactive potential of this essential oil. acknowledgements. this investigation is supported by the ministry of education, science and technological development of the republic of serbia grant number tr31025. references aćimović, m., sikora, v., brdar-jokanović, m., kiprovski, b., popović, v., koren, a., puvača, n. 2019: dracocephalum 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kocira, s., wojtunik, k., mitrus m., kocira a., widelski, j., skalicka-wozniak, k. 2017: application of moldavian dragonhead (dracocephalum moldavica l.) leaves addition as a functional component of nutritionally valuable corn snacks. journal of food science and technology, 54(10):3218-3229. zeng, c., jiang, w., yang, x., he, c., wang, w., xing, j. 2018: pretreatment with total flavonoid extract from dracocephalum moldavica l. attenuates ischemia reperfusion-induced apoptosis. scientific reports, 8:17491. stanojević et al. 2022, biologica nyssana 13(2) 13 (2) december 2022: 205-216 doi: 10.5281/zenodo.7476279 comparative analysis of chemical composition and antioxidant activity of essential oil isolated from orange and red marigold (tagetes patula l.) flower petals original article jelena stanojević faculty of technology, university of niš, bulevar oslobođenja 124, 16000 leskovac, serbia jstanojevic@tf.ni.ac.rs (corresponding author) nataša simonović faculty of technology, university of niš, bulevar oslobođenja 124, 16000 leskovac, serbia ljiljana stanojević faculty of technology, university of niš, bulevar oslobođenja 124, 16000 leskovac, serbia zoran ilić faculty of agriculture, university of priština, kopaonička bb, 38219 lešak, serbia aleksandra milenković faculty of technology, university of niš, bulevar oslobođenja 124, 16000 leskovac, serbia jelena zvezdanović faculty of technology, university of niš, bulevar oslobođenja 124, 16000 leskovac, serbia dragan cvetković faculty of technology, university of niš, bulevar oslobođenja 124, 16000 leskovac, serbia received: november 20, 2022 revised: december 12, 2022 accepted: december 15, 2022 abstract: the present study aimed to determine and compare chemical composition and antioxidant activity of essential oils (eos) isolated from marigolds (tagetes patula l.) cultivated in the garden in southeast serbia. the eos were isolated from dry orange and red flower petals by clevenger type hydrodistillation during 2 h by using 1:15 m/v hydromodule. their qualitative composition was determined by gc/ms and quantitative by gc/fid method. the antioxidant activity was determined by using the dpph assay. the most abundant components in the essential oil isolated from orange flower petals were geranyl acetate (36.7%) and (e)-caryophyllene (31.6%) while the one isolated from the red flower petals contained (e)-caryophyllene (69.4%) in the highest percentage. since phototoxic thiophenes were identified in both eos, they should not be used as components in cosmetic products for applications on areas of skin exposed to sunshine. essential oils showed similar antioxidant activity, with orange flower eo being somewhat better. key words: marigold, tagetes patula l. flower petals, thiophenes, gc/ms, antioxidant activity apstrakt: uporedna analiza hemijskog sastava i antioksidativne aktivnosti etarskog ulja izolovanog iz narandžastih i crvenih cvetnih latica baštenske kadife (tagetes patula l.) cilj ovog rada bio je određivanje i poređenje hemijskog sastava i antioksidativne aktivnosti etarskih ulja izolovanih iz baštenske kadife (tagetes patula l.) gajene u jugostočnoj srbiji. etarska ulja su izolovana iz suvih narandžastih i crvenih cvetnih latica clevenger hidrodestilacijom u toku 2 h pri hidromodulu 1:15 m/v. kvalitativni sastav izolovanih etarskih ulja određen je gc/ms metodom a kvantitivni sastav gc/fid metodom. antioksidativna aktivnost određena je dpph testom. najzastupljeniji sastojci u etarskom ulju izolovanom iz narandžastih latica bili su geranil acetat (36.7%) i (e)-kariofilen (31.6%) dok je u etarskom ulju izolovanom iz crvenih latica (e)-kariofilen bio zastupljen u najvećem procentu. s obzirom na to da su fototoksični tiofeni identifikovani u oba etarska ulja, ova etarska ulja ne bi trebalo koristiti kao komponente u kozmetičkim proizvodima namenjenim nezi delova tela izloženih sunčevim zracima. etarska ulja su pokazala slična antioksidativna svojstva, pri čemu je etarsko ulje izolovano iz narandžastih cvetnih latica nešto bolje. ključne reči: baštenska kadifa, tagetes patula l. cvetne latice, tiofeni, gc/ms, antioksidativna aktivnost introduction the life of people and animals on the earth, as members of delicate world ecosystem, is possible due to the most important link of the chain with which they live in symbiosis – plants. people use plants more than 10,000 years. they cultivate them, consume (as the main source of vitamin c), and use as animal feed, as well as for fuel production (parisi et al., 2010). the “dark side” of ethnobotany is reflected in phytodermatosis (dermatitis caused by plants) occurrence. it could be caused by the direct contact with plants or by association with sunlight. skin reactions caused by contact with plants could © 2022 stanojević et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 205 14th symposium on the flora of southeastern serbia and neighboring regions be divided as follows: dermatitis by physical trauma, dermatitis by pharmacological action, ige-mediated dermatitis, dermatitis by irritation, sunlight-induced dermatitis and dermatitis by sensitization. the letter occurs when sensitizing substances are present. among other plant families, these substances are present in ornamental plants such as chrysantemum, daisies, and marigolds from sunflower (asteraceae) family (dos reis, 2010). the genus tagetes consists of annual and perennial herbaceous plants, commonly called marigolds. it is native to the area from south-western america to argentina, with maximum variations occurring in mexico, but also naturalized all over the globe (singh et al., 2015). phytochemical studies of different plant parts revealed the presence of phenylpropanoids, carotenoids (such as lutein, zeaxanthin, neoxanthin plus violaxanthin, β-carotene, lycopene, α-cryptoxanthin, phytoene and phytofluene), flavonoids, thiophenes, and triterpeniods (priyanka et al., 2013; salehi et al., 2018; singh et al., 2020). there are at least fifty-six tagetes species (salehi et al., 2018) and the most widely known are t. erecta (also known as mexican marigold), t. patula (french marigold, dwarf marigold) and t. minuta (black mint or stinking roger). they are mainly studied in the field of agriculture, because of fungicidal, bactericidal, and insecticidal activities (singh et al., 2015; salehi et al., 2018). t. minuta is mainly cultivated for its essential oil, while t. patula and t. erecta have floricultural use. the flowers of african marigolds are yellow to orange and do not include red coloured marigolds, while french marigolds have red, orange and yellow as well as red and orange bicolour patterns flowers (fig. 1). the marigold species are traditionally used as analgesics, antiseptics, carminatives, diuretics, stimulants etc. in mexico; in food seasoning and for insect repellence in usa; and in ceremonial purposes in india, mexico and guatemala. recently, they have been recognised as commercial resources of essential oils and biologically active compounds for potential use as agrochemicals, colorants and nutritional supplements as well as in cosmetics (singh et al., 2015). the aerial parts of marigolds are rich sources of strongly aromatic eo (tagetes oil), mainly used for perfumes production (singh et al., 2020). generally, tagetes essential oils are rich in monoterpene hydrocarbons (such as ocimenes, limonene, etc.) and in acyclic monoterpene ketones (such as tagetone, dihydrotagetone, and tagetenone) which are the primary odorants – fig. 2. there are also sesquiterpene hydrocarbons and oxygenated compounds but in much lower amounts. however, the chemical diversity is quite high (tomova et al., 2005; singh et al., 2015). marigolds are very popular as garden plants in southeastern serbia. however, there is scarce information on the chemical composition and antioxidant properties of their essential oil. therefore, the aim of the present study was to determine the chemical composition of essential oils isolated from red and orange tagetes patula l. flower petals grown in southeastern serbia in order to detect potentially present phototoxic compounds as well as to determine and compare their antioxidant activities. materials and methods plant material the seeds of tagetes patula l. (tagetes patula mix 5436) were purchased in local agricultural pharmacy (producer: semenarna ljubljana, slovenia). plant material was cultivated in the garden in village manastirište (municipality of vlasotince, southeastern serbia; 42.9612° n, 22.1590° e). the orange and red flowers in their full flowering state (july and august 2022) were collected and dried in a shadowy place, at room temperature. before 206 biologica nyssana ● 13 (2) december 2022: 205-216 stanojević et al. ● comparative analysis of chemical composition and antioxidant activity of essential oil isolated from orange and red marigold (tagetes patula l.) flower petals fig. 1. the aerial parts of tagetes patula l. (a, c) with dry orange (b) and red (d) flower petals used in this study biologica nyssana ● 13 (2) december 2022: 205-216 stanojević et al. ● comparative analysis of chemical composition and antioxidant activity of essential oil isolated from orange and red marigold (tagetes patula l.) flower petals 207 the analysis, the dried flower petals were separated from the capitula and ground in an electric grinder (moulinex multi moulinette 3 in 1, 500 w). chemicals and reagents the following chemicals and reagents were used in this study: ethanol, 96% (centrochem, zemun, serbia); 1,1-diphenyl-2-picrylhydrazyl (dpph radical), butylated hydroxytoluene (bht); alkane standard solution c8-c20 (~40 mg/l each, in hexane), alkane standard solution c21-c40; (e)-caryophyllene (≥98.0%, sum of enantiomers); β-pinene (≥99%); γ-terpinene (97%); linalool (97%) (sigma chemical company, st. louis, mo, usa); terpinen-4-ol (97%, j&k scientific ltd., beijing, china); hplc grade hexane (≥95%, fisher scientific, uk), and redistilled water. isolation of essential oil the essential oils (eos) were isolated by clevenger type hydrodistillation from dry flower petals during 2 h by using 1:15 m/v hydromodule. the volatile compounds were collected in hexane (1 ml) added fig. 2. chemical structures of the primary odorants present in tagetes eos in a graduated tube of the apparatus. the hexane solution was separated from the water phase and evaporated at room temperature. the residual essential oil was measured gravimetrically and stored at 4 °c until analysed. the yield of the essential oils was determined in g per 100 grams of the extracted plant material (g/100 g p.m.). gas chromatography-mass spectrometry (gc/ ms) and gas chromatography-flame ionization detection (gc/fid) analyses gc/ms analysis was performed on agilent technologies 7890b gas chromatograph, equipped with nonpolar, silica capillary column, hp-5ms (5% diphenyland 95% dimethyl-polysiloxane, 30 m × 0.25 mm, 0.25 μm film thickness; agilent technologies, santa clara, ca, usa) and coupled with inert, selective 5977a mass detector of the same company. the essential oils were dissolved in diethyl ether in concentrations of ~19 mg/ml (ofeo) and ~33 mg/ml (rfeo). one μl of the solution prepared was injected to the gc column through a split/splitless inlet set at 220 °c in 20:1 208 biologica nyssana ● 13 (2) december 2022: 205-216 stanojević et al. ● comparative analysis of chemical composition and antioxidant activity of essential oil isolated from orange and red marigold (tagetes patula l.) flower petals split mode. helium was used as the carrier gas, at a constant flow rate of 1 ml/min. the oven temperature increased from 60 °c to 246 °c at the rate of 3 °c/ min. temperatures of the msd transfer line, ion source and quadrupole mass analyzer were set at 300 °c, 230 °c and 150 °c, respectively. the ionization voltage was 70 ev and mass range m/z 41-415. gc/fid analysis was carried out under identical experimental conditions as gc/ms. the flows of the carrier gas (he), make up gas (n2), fuel gas (h2), and oxidizing gas (air) were 1, 25, 30, and 400 ml/ min, respectively. the temperature of the flameionization detector (fid) was set at 300 °c. data processing was performed using msd chemstation, masshunter qualitative analysis and amdis_32 softwares (agilent technologies, usa). retention indices of the components from the analyzed samples were experimentally determined using a homologous series of n-alkanes from c8-c20 and c21-c40 as standards. essential oil constituents identification was based on the comparison of their retention indices (riexp) with those available in literature (adams, 2007) (rilit); their mass spectra with those of authentic standard as well as with those from willey 6, nist2011 and rtlpest3 libraries (ms) and wherever possible, by co-injection with an authentic standard (co-i). quantification was done by an external standard method according to the procedure described by sparkman et al. (2011). the standards used were in the concentration ranges as follows: β-pinene (0.1252 mg/ml), linalool (1.67-15 mg/ml), γ-terpinene (0.75-5 mg/ml), terpinen-4-ol (0.0625-1 mg/ml) and (e)-caryophyllene (0.0625-1 mg/ml). the response factor (rf) for each standard used was calculated as follows: rf=areastd/ssd where areastd is the peak area of the analyte standard and cstd is the concentration of the standard used. the values of the mean response factors for the 5-points calibration curves of each standard used were: 8.39×106; 2.45×107; 3.02×107; 8.36×106; and 6.83×106 for β-pinene, γ-terpinene, linalool, terpinen-4-ol, and (e)-caryophyllene, respectively. then, the mean value of response factors (rfmean=1.56×107 for rfeo and 1.51×107 for ofeo) was calculated and used for quantification according to the following formula: cx=areax/rfmean where cx is the concentration of an analyte in the sample (in mg/ml of eo), areax is the peak area of the analyte in the sample and rfmean is the mean response factor of the standards used (sparkman et al., 2011). the calculated concentrations of the β-pinene, and γ-terpinene in rfeo were: 0.02 mg/ml and 0.03 mg/ ml respectively, while the concentrations of linalool, terpinen-4-ol, and (e)-caryophyllene were: 0.16 mg/ ml; 0.15 mg/ml; and 45.79 mg/ml in rfeo and 0.20 mg/ml; 0.11 mg/ml, and 12.12 mg/ml in ofeo, respectively. finally, the content of each component in the sample expressed in % was normalized to get percents according to the formula: c (%)=(cx/σcx)x100 where cx is the concentration of each component in the sample and ʃcx is the total concentration of all components in the sample. dpph assay the ability of the essential oils to scavenge free dpph radicals was determined using the dpph assay. essential oils were dissolved in ethanol and a series of different concentrations (0.4-13.5 mg/ml and 0.3-10.3 mg/ml for eo isolated from red (rfeo) and orange flower petals (ofeo, respectively)) were prepared. ethanol solution of dpph radical (0.3 ml, 300 μmol solution (3×10-4 mol/l)) was added to 0.75 ml of the prepared essential oil solutions („sample”) and the absorbance was measured at 517 nm after 20 min, 40 min, 60 min, 90 min, and 120 minutes incubation with radical (as). absorbance at 517 nm was determined for ethanolic solution of dpph radical („control” ac), diluted in the aforementioned ratio (0.3 ml of the dpph radical of the given concentration with 0.75 ml ethanol added) as well as for the ethanolic solution of the essential oil which is not treated with dpph radical solution (“blank” ab). ethanol was used as a blank. free radical scavenging activity was calculated according to the formula: dpph radical scavenging capacity (%)=100-[(as-ab)x(100/ac)] all absorbances were measured on uv-vis varian-cary 100 conc. spectrophotometer. bht (dissolved in ethanol; concentration range 0.04-1.3 mg/ml) was used as a positive control. results and discussion qualitative and quantitative composition of essential oils the yields of pale yellow coloured essential oils were 0.03 g/100 g p.m and 0.01 g/100 g p.m for rfeo and ofeo, respectively. total ion chromatograms (tics) of eos studied are given in fig. 3, while their qualitative and quantitative composition is given in tab. 1. the chemical structures of the most abundant compounds are shown in fig. 4. 209 biologica nyssana ● 13 (2) december 2022: 205-216 stanojević et al. ● comparative analysis of chemical composition and antioxidant activity of essential oil isolated from orange and red marigold (tagetes patula l.) flower petals fig. 3. tic chromatogram of (a) ofeo, and (b) rfeo fig. 4. chemical structures of the most abundant compounds present in isolated eos 210 biologica nyssana ● 13 (2) december 2022: 205-216 stanojević et al. ● comparative analysis of chemical composition and antioxidant activity of essential oil isolated from orange and red marigold (tagetes patula l.) flower petals table 1. chemical composition of essential oils isolated from orange and red flower petals of t. patula l. no. tret, min compound riexp rilit method of identification content (%) c (mg/ml of eo) ofeo rfeo ofeo rfeo 1 7.92 sabinene 965 969a ri, ms tr tr 2 8.03 β-pinene 968 974a ri, ms, co-i tr tr 3 8.46 2-pentyl furan 983 984a ri, ms tr tr 4 10.11 (z)-β-ocimene 1,030 1,032a ri, ms 0.4 2.7 0.1 0.9 5 10.49 (e)-β-ocimene 1,040 1,044a ri, ms 0.2 0.1 6 10.92 γ-terpinene 1,051 1,054a ri, ms, co-i tr tr 7 12.06 terpinolene 1,081 1,086a ri, ms tr tr 8 12.25 p-cymenene 1,086 1,089a ri, ms tr tr 9 12.72 linalool 1,098 1,095a ri, ms, co-i 0.4 tr 0.1 tr 10 14.44 (e)-tagetone 1,132 1,139a ri, ms tr tr 11 15.98 terpinen-4-ol 1,169 1,174a ri, ms, co-i tr tr tr tr 12 16.58 p-cymen-8-ol 1,184 1,179a ri, ms tr tr 13 17.95 coahuilensol, methyl ether 1,216 1,219a ri, ms tr tr 14 18.19 (z)-ocimenone 1,222 1,226a ri, ms tr 0.3 tr 0.1 15 18.60 (e)-ocimenone 1,232 1,235a ri, ms tr tr 16 19.15 piperitone 1,245 1,249a ri, ms 0.3 0.1 17 20.30 isobornyl acetate 1,276 1,283a ri, ms tr tr 18 21.97 silphiperfol-5-ene 1,324 1,326a ri, ms tr tr 19 22.25 presilphiperfol-7-ene 1,330 1,334a ri, ms tr tr 20 22.89 piperitenone 1,337 1,340a ri, ms 0.5 1.5 0.1 0.5 21 23.04 α-terpinyl acetate 1,340 1,346a ri, ms tr tr 22 23.73 eugenol 1,357 1,356a ri, ms, co-i tr tr tr tr 23 23.93 piperitenone oxide 1,361 1,366a ri, ms tr tr 24 24.09 α-copaene 1,365 1,374a ri, ms tr tr tr tr 25 24.59 geranyl acetate 1,377 1,379a ri, ms 37.6 7.2 26 26.21 (e)-caryophyllene 1,416 1,417a ri, ms, co-i 31.6 69.4 6.0 22.8 27 26.51 α-trans-bergamotene 1,423 1,432a ri, ms tr tr 28 27.30 geranyl acetone 1,443 1,453a ri, ms tr tr tr tr 29 27.38 α-humulene 1,444 1,452a ri, ms 1.0 1.7 0.2 0.6 30 28.53 germacrene d 1,488 1,484a ri, ms 2.8 8.2 0.5 2.7 31 29.08 bicyclogermacrene 1,490 1,500a ri, ms 1.1 3.6 0.2 1.2 32 29.39 (e)-methyl isoeugenol 1,493 1,491a ri, ms tr tr 33 29.45 (e,e)-α-farnesene 1,495 1,505a ri, ms 0.6 0.2 34 29.56 β-bisabolene 1,498 1,505a ri, ms 12.5 2.4 35 30.10 γ-cadinene 1,511 1,513a ri, ms tr tr tr tr 36 30.78 (e)-γ-bisabolene 1,529 1,529a ri, ms 0.9 0.4 0.2 0.1 37 32.51 caryophyllene oxide 1,574 1,582a ri, ms 5.7 4.5 1.1 1.5 38 34.65 caryophylla4(12),8(13)-dien-5α-ol 1,631 1,639a ri, ms tr tr tr tr 211 biologica nyssana ● 13 (2) december 2022: 205-216 stanojević et al. ● comparative analysis of chemical composition and antioxidant activity of essential oil isolated from orange and red marigold (tagetes patula l.) flower petals 39 35.32 α-cadinol 1,648 1,652a ri, ms tr tr tr tr 40 36.63 shyobunol 1,684 1,688a ri, ms tr tr tr tr 41 36.81 eudesm-7(1l)-en-4-ol 1,688 1,698a ri, ms 0.9 tr 0.2 tr 42 37.18 (2e)-tridecenol acetate 1,698 1,703a ri, ms 0.8 tr 0.2 tr 43 41.65 hexahydrofarnesyl acetone 1,850 1,846b ri, ms 0.6 0.3 0.1 0.1 44 44.05 (5z,9e)-farnesyl acetone 1,897 1,889a ri, ms tr tr 45 44.27 5-(3-buten-1-ynyl)2,2'-bithiophene (bbt) 1,902 1,892c ri, ms 0.4 0.1 46 46.12 palmitic acid 1,960 1,959b ri, ms 1.2 1.9 0.2 0.6 47 49.29 5-(3-penten-1-ynyl)2,2-bithiophene (pbt) 2,053 2,043c ri, ms 0.7 1.5 0.1 0.5 48 53.28 2,2':5',2''-terthiophene (α-terthiophene) 2,181 2,171c ri, ms 0.9 0.3 49 55.35 tricosane 2,296 2,300a ri, ms, co-i 0.5 0.7 0.1 0.2 total identified 99.2 99.1 19.0 32.6 grouped components (%) (mg/ml of eo) monoterpene hydrocarbons (1, 2, 4-8) 0.4 2.9 0.1 1.0 oxygenated monoterpenes (9-21, 23, 25, 28) 38.5 2.1 7.4 0.7 sesquiterpene hydrocarbons (24, 26, 27, 29-31, 33-36) 49.9 83.9 9.5 27.6 oxygenated sesquiterpenes (37-41, 43, 44) 7.2 4.8 1.4 1.6 phenylpropanoids (22, 32) tr tr tr tr thiophenes (45, 47, 48) 0.7 2.8 0.1 0.9 others (3, 42, 46, 49) 2.5 2.6 0.5 0.8 tret.: retention time; rilit retention indices from literature (aadams (2007); bbalogun et al. (2017); cszarka et al. (2007)); riexp: experimentally determined retention indices using a homologous series of n-alkanes (c8-c20 and c21-c40) on the hp-5ms column. ms: constituent identified by mass-spectra comparison; ri: constituent identified by retention index matching; co-i: constituent identity confirmed by gc co-injection of an authentic sample; tr = trace amount (<0.05%; <0.1 mg/ml); ofeo essential oil isolated from orange flower petals, rfeo essential oil isolated from red flower petals according to the results of gc/ms analysis, 30 and 42 compounds were identified in ofeo and rfeo, comprising 99.2%, and 99.1% of total eo composition, respectively. the most abundant groups of compounds in ofeo were sesquiterpene hydrocarbons (49.9%) and oxygenated monoterpenes (38.5%). on the other side, sesquiterpene hydrocarbons with 83.9% in the total essential oil composition were the dominant group in rfeo (tab. 1). among sesquiterpene hydrocarbons (e)-caryophyllene was the dominant compound in both eos, with 31.6% and 69.4%, in total ofeo and rfeo composition, respectively while β-bisabolene, with 12.5% was second most dominant compound in ofeo. β-bisabolene was not detected in rfeo. among oxygenated monoterpenes, the dominant one was geranyl acetate with 37.6% in total ofeo composition (structures given in fig. 3). it was not detected in rfeo. the results obtained are in agreement with the study by szarka et al. (2007). the authors hydrodistilled eo from t. patula flowers that had been air-dried, and the most abundant compound found there was (e)-caryophyllene, which accounted for 50.2% (compared to 69.4% in herein study). geographical origin plays an important role in the chemical diversity not just among species but even in the same species. for example, krishna et al. (2002) analysed eos of the capitula, leaves and shoots of tagetes patula l. raised in the cimap experimental biologica nyssana ● 13 (2) december 2022: 205-216 stanojević et al. ● comparative analysis of chemical composition and antioxidant activity of essential oil isolated from orange and red marigold (tagetes patula l.) flower petals 212 farm at lucknow (india). the main constituents of eo isolated from capitula were (z)-β-ocimene (19.9%), (z)-tagetenone (12.4%), (e)-tagetenone (10.4%), piperitenone (5.8%) and (e)-caryophyllene (15.1%) (krishna et al., 2002). on the other hand, the eo isolated from cultivated t. patula flowers grown in mandal (uttarakhand, india) contained β-ocimene (22.11%), α-terpinolene (14.59%), and (e)-caryophyllene (12.69%) as the most abundant compounds (negi et al., 2013). the most abundant components of the eo isolated from fresh flowers of t. patula grown in new delhi (india) were (e)caryophyllene (3.92-42.76%), germacrene-d (1.486.72%), (z)-tagetone (1.29-4.38), caryophyllene oxide (0.68-24.3%), and piperitone oxide (0.111.23) (tamut et al., 2019). in the eo isolated from t. patula flowers sampled during august in areas of erbil province (iraq), (e)-caryophyllene (20.59%), and (e)-ocimenone (12.08%) were the most abundant components while geranyl acetate was not identified (safar et al., 2020). zarate-escobedo et al. (2018) reported the presence of geranyl acetate for genus tagetes for the first time. they determined the chemical composition of the essential oil hydrodistilled from floral stems of 14 t. lucida populations from north and south of the state of mexico, where six types of soils and six climatic conditions were detected. the plant material was collected from september to october 2014 and in september 2015. in southern populations with a warm climate, the major compounds were monoterpenes: geranyl acetate (ranging from 12% to 40%) and β-ocimene (14% to 24%) depending on the location (zarate-escobedo et al., 2018). it is postulated that terpenoid compounds play a role in an ecological interaction of plants with biotic and abiotic factors of their environment by defending plants from herbivores and pathogens (toxins or repellents) or being the signals and rewards to pollinators. the constant evolution of new terpenoids structures is enabled by the evolution of new genes encoding new enzymes capable of making such new metabolites (pichersky & raguso, 2016). what is more, according to the available literature data single compounds such as bisabolene, (e)caryophyllene, camphor, (e)-β-farnesene, pinene, and linalol have been recognized as good repellents towards aphids and various pests (hori, 1998; isman, 2000; halbert et al., 2009; pascual-villalobos et al., 2017; dardouri et al., 2019). taking into account that (e)-caryophyllene and β-bisabolene were present in considerable amounts in the eos isolated in this study (tab. 1), they could be considered as a potential source of natural pesticides. it is well known that acyclic monoterpenes (ocimenones) including (z)-βand (e)-β-ocimene, (z)and (e)-tagetone and (z)and (e)-tagetenone are formed by chemical modification (such as hydrolysis, dehydration, oxidation and reduction which are catalyzed by specific enzymes) of either gpp or neryl pyrophosphate (npp). on the other hand, the biosynthesis of sesquiterpenes proceeds through the precursor farnesyl pyrophosphate (fpp) which is formed by condensation of gpp with one molecule of ipp (singh et al., 2015). given that terpenoids’ biosynthesis is genetically determined, finding the reason for the difference in chemical composition between ofeo and rfeo, regarding the most abundant components, goes beyond the scope of this paper. geranyl acetate is an acyclic monoterpenes ester with great economic value. it is widely used in cosmetic industry due to its „rose-like” odor. in cymbopogon spp. geranyl acetate is biosynthesed by acetylation of geraniol catalyzed by geraniol acetyl transferase (gat) (ganjewala & luthra, 2010). phosphatase (gppase) mediates the formation of geraniol while geranyl acetate esterase (gae) catalizes deacetylation of geranyl acetate into geraniol. thus, the content of geraniol depends on the relative activities of these three (gppase, gat, and gae) enzymes. geraniol was not identified in herein studied eo. the possible reason could be that the plant material was collected in the phase when gae (catalyzing transformation of geranyl acetate to geraniol) had little or no activity. having in mind that essential oils isolated from „geranylacetate rich” plants like palmarosa (cymbopogon martini (roxb.) wats. var. motia burk.) contained 4.3-14.8% of geranyl acetate (rajeswara rao et al., 2009); wild carrot (daucus carota l. ssp. carota) mature umbels 16.5% (staniszewska et al., 2005); lemongrass (cymbopogon flexuosus (steud) wats.) 25.9% (kulkarni et al., 1997); pastinocello fruits (daucus carota ssp. major) 34.2% (flamini et al., 2014); coriander (coriandrum sativum l.) fruits 46.27% (msaada et al., 2007); and the ofeo isolated in this paper (containing 37.6%) could be considered as a potential source of geranyl acetate. the thiophenes, with bbt, pbt, and α-terthienyl as representatives, were also identified, with 0.7% and 2.8% in ofeo and rfeo, respectively (tab. 1). although the thiophenes are the main secondary metabolites of tagetes roots in this paper they were identified in flower petals, which is in agreement with the study of szarka et al. (2007). namely, szarka et al. (2007) studied the composition of eos isolated from hairy roots, normal roots and flowers and the major thiophene of flower eo was pbt with 6.0% in the total eo composition (szarka et al., 2007). the identity of thiophenes identified in this paper was confirmed by comparing mass spectrum from the hit 213 biologica nyssana ● 13 (2) december 2022: 205-216 stanojević et al. ● comparative analysis of chemical composition and antioxidant activity of essential oil isolated from orange and red marigold (tagetes patula l.) flower petals list in mass spectra libraries but also with the mass spectra given in the study of szarka et al. (2006) as well as by comparing experimentally obtained retention indices with the retention indices given in the study of szarka et al. (2007). on the other side, in the study of arciniegas et al. (2020), the authors determined the antioxidant and photosensitizer activities of extracts and isolates of genus dyssodia (asteraceae, tageteae). the antioxidant activity was determined by the interactions with copper ion (cu2+) observed in epr, as well as by the dpph and the thiobarbituric reactive substances (tbars) methods. their photosensitizer activities were observed as the the abilities to produce 1o 2 by electron paramagnetic resonance (epr). they isolated seven thiophene derivatives, among others 2,2′:5′,2′′‐terthiophene, and 5‐(3‐buten‐1‐ynyl)‐2,2′‐bithiophene, which were identified in this study as well. both thiophenes had no antioxidant activity determined by the dpph assay, so they surely not contribute to the antioxidant activity of the eos studied in this paper. on the other side, α‐terthiophene and related compounds are known as photosensitizers by their activity to generate singlet oxygen (1o2) under uv irradiation regime (arciniegas et al., 2020). what is more, these compounds, and especially α-terthienyl show enhanced nematocidal activity in the presence of sunlight (uv-a), but also antibiotic, ovicidal, algicidal, larvicidal, and antifeedant activities (d’auria et al., 1987). antioxidant activity the antioxidant activity of isolated eos was determined by the dpph assay and compared both mutually, and with bht as a positive control. their dpph radical scavenging activity determined after 20 min, 40 min, 60 min, 90 min, and 120 min incubation with the dpph radical is shown in fig. 5. the ec50 values obtained from the graphs given in fig. 5 are presented in the tab. 2. according to the results obtained, antioxidant activity of both eos and bht was dependant on both concentration and incubation time with the dpph radical, as already stated (stanojević et al., 2016). generally, the duration of incubation time of dpph radical with samples depends on the type of sample, taking longer time for interaction with the weak antioxidants (bal et al., 2021). the effect of incubation time studied in this paper indicated that dpph radical scavenging activity of both eos and bht reached the maximum after 120 minutes of incubation (tab. 2). however bht, as a phenolic representative of synthetic antioxidants used in this paper, reduced the 50% of the initial dpph radical concentration (the ec50 value) after 20 minutes of incubation and showed ~12 and ~14 times stronger fig. 5. antioxidant activity of (a) ofeo, (b) rfeo, and (c) bht antioxidant activity in comparison to ofeo and rfeo after 120 minutes of incubation, respectively. therefore, the isolated eos had similar antioxidant biologica nyssana ● 13 (2) december 2022: 205-216 stanojević et al. ● comparative analysis of chemical composition and antioxidant activity of essential oil isolated from orange and red marigold (tagetes patula l.) flower petals 214 activity and both are weaker antioxidants than bht. comparing the chemical composition of isolated eos, the main groups were sesquiterpenes hydrocarbons (83.9% vs. 55.6%) and oxygenated monoterpenes (2.1% vs. 38.5%) in rfeo and ofeo, respectively. according to the study by ruberto & barrata (2000), oxygenated monoterpenes have better antioxidant activity in comparison to monoterpenes hydrocarbons, sesquiterpenes hydrocarbons, and oxygenated sesquiterpenes. the best antioxidant activity in the mentioned study showed phenolic compounds (such as thymol and carvacrol) due to their redox properties, and the ability to neutralize free radicals (ruberto & barrata, 2000). considering that ofeo contained considerable amount of oxygenated monoterpenes (38.5%) in comparison to rfeo (2.1%), its slightly better antioxidant activity could be ascribed to their presence, especially to the presence of geranyl acetate due to its capacity to reduce free radical stability via electron or hydrogen donating mechanisms (seema farhath et al., 2013). on the other side, phenolic compounds (particularly eugenol) were identified in traces in both isolated eos, so their weak antioxidant activity is not surprising. conclusions the essential oils hydrodistilled from dry red and orange tagetes patula l. flower petals cultivated in south-eastern serbia are rich sources of sesquiterpene hydrocarbons ((e)-caryophyllene in rfeo comprising 69.4% of total eo composition) and oxygenated monoterpenes (geranyl acetate in ofeo comprising 37.6% of total eo composition). they have also contained sulphurated tiophenes (bbt, pbt and ttp) with 2.8% and 0.7% in total rfeo and ofeo composition, respectively. having in mind that α-terthienyl, also called terthiophene (ttp) and 5-(3-penten-1-ynyl)-2,2-bithienyl (pbt) are phototoxic compounds, they should not be used as components in cosmetic products for applications on areas of skin exposed to sunshine. being weaker antioxidants in comparison to the synthetic antioxidant bht, they could not be used as its natural alternative. on the other side, the overuse of synthetic pesticides causes pest resistance and makes them one of the major pollutants in soil and water, as well as toxic substances for humans and animals. taking into account that (e)-caryophyllene and β-bisabolene present in considerable amounts in the eos isolated in this study are recognized as good repellents (besides thiophenes). the authors would like to draw attention to marigolds, not just as garden ornamental flowers but also as a potential source and appropriate basis for future development of naturally based herbicides. acknowledgements. this work was supported by the ministry of education, science and technological development of the republic of serbia under the program of financing scientific research work, number 451-0368/2022-14/ 200133. nataša simonović and aleksandra milenković are scholars of the ministry of education, science and technological development of the republic of serbia. references adams, r.p. 2007: identification of essential oil components by gas chromatography /mass 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nizhny novgorod state pedagogical university, nizhny novgorod, russia maria a. krivonogova minin nizhny novgorod state pedagogical university, nizhny novgorod, russia anna a. shestakova lobachevsky state university of nizhny novgorod, nizhny novgorod, russia received: january 29, 2025 revised: march 05, 2025 accepted: march 06, 2025 abstract: the paper aimed to evaluate the current state of the breeding population of the house martin delichon urbicum in the nizhny novgorod oblast based on the analysis of the results of our field surveys and data collected by citizen science. based on repeated counts of nests in 10 colonies, we discovered the number of house martins decreased by an average of 7.16% per year from 2012 to 2024. based on the average annual population decline, the probable number of nests in 49 colonies from 786 to 916 nests is calculated in 2024. the calculated regional number of house martins ranges from 2,116 to 2,488 pairs. the prospect of further research is the organization of a regional census of the house martins, the development of the database of colonies and further monitoring of the species conditions in the oblast, and the implementation of european experience in artificial nest erecting. key words: western house martin, delichon urbicum, red data book, nizhny novgorod oblast, population trends apstrakt: rasprostranjenost i brojnost vrste delichon urbicum u nižnjenovgorodskoj oblasti rad je imao za cilj da proceni trenutno stanje gnezdilišne populacije gradske laste (delichon urbicum) u nižnjenovgorodskoj oblasti na osnovu analize rezultata terenskih istraživanja i podataka prikupljenih kroz građansku nauku. na osnovu ponovljenih prebrojavanja gnezda u 10 kolonija, otkrili smo da se brojnost gradskih lasta smanjivala u proseku za 7,16% godišnje u periodu od 2012. do 2024. godine. na osnovu prosečnog godišnjeg opadanja brojnosti populacije, procenjeni broj gnezda u 49 kolonija u 2024. godini iznosi između 786 i 916. izračunata regionalna brojnost gradskih lasta kreće se od 2.116 do 2.488 parova. perspektive daljih istraživanja uključuju organizaciju regionalnog popisa gradskih lasta, razvoj baze podataka o kolonijama i kontinuirani monitoring stanja vrste u oblasti, kao i implementaciju evropskih iskustava u postavljanju veštačkih gnezda. ključne reči: zapadna gradska lasta, delichon urbicum, crvena knjiga, nižnjenovgorodska oblast, populacioni trendovi introduction the western house martin delichon urbicum is a widespread synanthropic bird, a vivid example of an urbanized species (von blotzheim et al., 2012), which is, in the middle zone of european russia, associated with settlements and is not found in natural habitats. until recently, the status of the species was of least concern everywhere. however, estimates of the species numbers are very different in recent publications. for example, the global species population ranges from 10.5 to 500 million adults, and in europe – from 22.4 to 47.2 million adults (dvořáková et al., 2024). according to the iucn red list of threatened species (2024) data, the global species population is estimated at 38.3 – 80.2 million adults, and the european population is 23-48.1 million adults. in some european cities, the breeding density of house martin is very high (jerzy, 2001; kopij, 2016). many specialists note the negative population trend for the species and discuss the reasons for such decline (evans et al., 2012; schmolz, 2020; kette et al., 2021; birdlife international, 2023; dvořáková et al., 2024). © 2025 bakka et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 318 the nizhny novgorod oblast (s=76,600 km2) is located in the center of the russian plain. western house martin is listed in the second edition of the regional red data book (bakka & kayumov, 2014). during the designing of the third edition of the regional red book, specialists were discussing the need to preserve the conservation status of this synanthropic species, which inspired the authors to conduct this study aimed at identifying the regional numbers of the species and its population trends. in the first half of the xx century, the western house martin was a common breeding species in the nizhny novgorod region and spread almost as widely as the barn swallow. however, there were no specific data on the localities of colonies and the species (serebrovkiy, 1918; puzanov et al., 1955; vorontsov, 1967). there is only information about eight localities for 10 specimens from the collection of the zoological museum of the nizhny novgorod state university, of which nine were found in nizhny novgorod and its surroundings, and one near the mikhaylovka village on the modern territory of vyksa (fig. 1). the species was probably of least concern until the 1990s. based on data obtained in the 1980s and 1990s, we made an expert assessment of its regional population of 5,000-10,000 pairs. this number was already significantly lower than the number of other swallows. by 2012, only five small colonies of house martin had remained in nizhny novgorod, where a total of 25-35 pairs breed. a colony in the avtozavodsky district of the city, numbering dozens of pairs, has disappeared. the breeding population of house martin in arzamas has been reduced to 4-5 microcolonies, which consist of two or three breeding pairs. besides nizhny novgorod and arzamas, 8085 pairs of house martin were known to nest in other seven settlements. these data became the reason to list the house martin in the red data book of the nizhny novgorod oblast (bakka & kayumov, 2014), as well as the basis for estimating the regional population number of the species in 200-400 pairs (bakka & kiseleva, 2007, 2017). during the longterm monitoring of the avifauna of the nizhny novgorod airport, the house martin was observed only twice: in 2013 and 2018 (noskova et al., 2020). approving the house martin as a protected biologica nyssana ● 16 (1) june 2025: 317-331 bakka et al. ● distribution and numbers of delichon urbicum in the nizhny novgorod oblast fig. 1. records of western house martin in the nizhny novgorod oblast in the first half of the xx century (made by the authors based on the materials of the collection of the zoological museum of the lobachevsky state university of nizhny novgorod) leaves species in the oblast has provided scientific interest to study the distribution and abundance of the species, including by scientific volunteers (citizen science). after 2014, a significant number of previously unknown habitats of the species were discovered in villages and small towns of the nizhny novgorod oblast. it became clear that the species number estimated as 200-400 pairs (bakka & kiseleva, 2017) is significantly underestimated. it was important to determine the reasons for such differences in population estimates: is it the species recovery, its relocation from large cities to rural areas or the insufficiency of census data for the late xx – early xxi centuries; and to estimate the modern species number in the region. materials and methods this paper aims to estimate the current state of the 319 biologica nyssana ● 16 (1) june 2025: 317-331 bakka et al. ● distribution and numbers of delichon urbicum in the nizhny novgorod oblast breeding population of the western house martin in a typical region of the central part of the russian plain on the example of the nizhny novgorod oblast. to achieve this goal, the tasks are as follows: • to analyze all data on the numbers and distribution of house martin in the nizhny novgorod oblast obtained by authors and scientific volunteers in the xxi century; • to estimate its current population numbers and trends; • to reveal the species’ habits in its distribution in the oblast; • to provide reasons for preserving the conservation status of the species; • to develop proposals for the organization of further study, monitoring of the species populations and their protection. generally, the direct counting of active swallow nests seems to be a reliable method for estimating the size of breeding populations and their trends on large spatial and temporal scales (tella et al., 2024). researchers also note the important role of civic science in studying the distribution and numbers of swallows (dickinson et al., 2012; kettel et al., 2021). since direct counts were carried out only in a part of the known colonies of the house martin in the oblast, we had to extrapolate the results of our surveys using data of citizen science from the site inaturalist.com, as well as an expert assessment of probable underestimation. all our data on records of house martins in the nizhny novgorod oblast in 2007-2024 were summarized, including the results of the nest census in colonies (76 units of information: data on 66 colonies, nine records of adults during the breeding season and one registration of a flock of house martins during the autumn migration). the data of citizen science collected in 2007-2024 and mainly presented on inaturalistwebsite were also processed (100 units of information: 48 registrations of colonies and 52 records of adults during the breeding season). databases on all the records have been compiled, including 184 units of information, including eight localities of specimens from the collection of the zoological museum of nizhny novgorod state university, which were shot in the first half of the xx century. in addition, information about two colonies that became extinct in the 2000s were taken into account (bakka & kayumov, 2014). the localities of the house martin records in the nizhny novgorod oblast in 2007-2024 are shown in fig. 2. all the analyzed data on the habitats of the house martin in the nizhny novgorod oblast and the general logic of the study are presented in fig. 3. a gis-theme has been created in order to identify the overlaps of our data and the information of citizen science, data obtained by different authors at different times, but related to the same localities of the species. the distribution of western house martin nesting sites on the territory of the nizhny novgorod oblast was analyzed: significant differences in the species density in four natural regions in the oblast fig. 2. records of the western house martin in the n. novgorod oblast in 2007–2024 (designed based on the results of our surveys and data of citizen science from inaturalist-website) 320 biologica nyssana ● 16 (1) june 2025: 317-331 bakka et al. ● distribution and numbers of delichon urbicum in the nizhny novgorod oblast have been shown. we found 10 colonies of the western house martin being surveyed repeatedly from 2012 to 2024: nine colonies were surveyed twice and one colony – four times. based on these data, the average population trend for a year was calculated. the obtained value was used in further calculations. the census of active nests during the period of surveys was carried out in 49 colonies of western house martins, but in different years, including the census in 16 colonies in 2024. using the calculated value of the average annual population trend, the active nest numbers in colonies counted in earlier years allow us to project the status in 2024. the average colony size in 2024 was calculated. this value was used to estimate the number of western house martins in 52 localities, where the number of nests was not counted. as a result, we obtained the total number of western house martins in all habitats identified in the nizhny novgorod oblast. a histogram for the distribution of colonies by the number of nests has been designed. the low share of overlaps identified by the authors and scientific volunteers was accepted as evidence of a significant underestimation. to evaluate the probable underestimation, the number of records and the number of breeding pairs were determined for 57 municipalities of the nizhny novgorod oblast in four natural regions. if the borders of the natural regions divided the municipal district, then its parts were considered separately. for each natural region, the average number of known and projected colonies in a municipality was calculated. in districts, where the known number of records was less than the average, the data was considered incomplete (underestimated) and, during further evaluation, replaced with average values. the underestimation in the colony numbers was calculated, and the number of nests at those localities was calculated based on the average size of western house martin colonies in the nizhny novgorod oblast. based on these extrapolations, we estimated the data insufficiency, which we consider to be minimal. taking into account this value, the probable species number in the region was determined. all calculations were carried out using the microsoft excel software. results as a result of the analysis of data obtained, we discovered 10 colonies of the western house martin being surveyed repeatedly from 2012 to 2024: nine fig. 3. all the analyzed data on the habitats of the house martin in the nizhny novgorod oblast and the general logic of the study (designed by authors) 321 biologica nyssana ● 16 (1) june 2025: 317-331 bakka et al. ● distribution and numbers of delichon urbicum in the nizhny novgorod oblast colonies were surveyed twice and one colony – four times. empty cells in the columns indicate the absence of census data for a particular year, but not table 1. number of active nests in colonies of the western house martin, which were surveyed repeatedly from 2012 to 2024 (designed by authors) colony localities number of active nests in the colony 2012 2014 2015 2019 2021 2022 2023 2024 vad (vadskiy district) 51 41 lopatino (vadskiy district) 44 31 kuz’miyar (vorotynskiy district) 20 22 bokovaya (semenovskiy district) 10 14 9 4 knyaginino (knyagininskiy district) 25 22 pechi (lukoyanovskiy district) 11 12 pokrovka (lukoyanovskiy district) 7 5 sergach (segachskiy district) 12 5 ardatov (ardatovskiy district) 12 5 lyskovo (lyskovskiy district) 32 28 the absence of nests (tab. 1). the change in the number of active nests in the western house martin colonies during the surveyed period is presented in tab. 2. the negative trend was noted for most colonies. considering these data, the average population in the period from 2012 to 2024 was 7.16% per year (stddev=9.37). the confidence interval for the selected probability of 90% ranges table 2. the change in the number of active nests in the western house martin colonies from 2012 to 2024 (designed by authors) colony localities the period between censuses, years the change in the number of active nests in a colony the absolute value for the period between censuses % of previous census value average per year, % vad 4 -10 -19.6 -4.9 lopatino 2 -13 -29.5 -14.75 kuz’miyar 2 2 10 5 bokovaya 7 4 40 5.71 3 -5 -35.7 -11.9 2 -5 -55.6 -27.8 knyaginino 2 -3 -12 -6 pechi 3 1 9.1 3.03 pokrovka 3 -2 -28.6 -9.53 sergach 9 -7 -58.3 -6.48 ardatov 10 -7 -58.3 -5.83 lyskovo 1 -4 -12.5 -12.5 total: -7.16 from -2.71% to -11.61%. the census of nests during the period of surveys was carried out in 49 colonies of western house martins, but in different years, including the census in 16 colonies in 2024 (tab. 3). a total of 956 nests were counted in those 49 colonies. using the calculated value of the average annual decrease in the number of western house martins (min – 2.71%, max – 11.61%), we projected the nest numbers in colonies in 2024. the total number of western house martins in 49 colonies ranged from 786 to 916 nests, 322 biologica nyssana ● 16 (1) june 2025: 317-331 bakka et al. ● distribution and numbers of delichon urbicum in the nizhny novgorod oblast table 3. the results of nest census in the western house martin colonies in different years and projected nest numbers in 2024 (designed by authors) colony locality year counted active nests period since the census to 2024, years population trend to 2024, % of counted nest number projected nest number in 2024 min max min max potrokhovo village (krasnobakovsky district) 2015 5 9 -105 -24 0 4 norkovka village (vyksa) 2023 1 1 -12 -3 1 1 novaya village (chkalovsk) 2022 35 2 -23 -5 27 33 bokovaya village (semenovsky district) 2024 4 0 0 0 4 4 sechenovo (sechenovsky district) 2013 5 11 -128 -3 0 4 perevoz (perevozsky district) 2013 39 11 -128 -3 0 27 bolshoe boldino (bolsheboldinsky district) 2013 5 11 -128 -3 1 4 pochinki (pochinkovsky district) 2014 18 10 -116 -27 0 13 ichalki (perevozsky district) 2015 1 9 -105 -24 0 1 knyaginino (knyagininsky district) 2019 5 5 -58 -14 2 4 kommunar village (pochinkovsky district) 2019 6 5 -58 -14 3 5 neley village (pervomaisk) 2019 15 5 -58 -14 6 13 village (pervomaisk) 2019 18 5 -58 -14 8 16 vladimirskoye (voskresensky district) 2019 1 5 -58 -14 0 1 knyaginino (knyagininsky district) 2022 2 2 -23 -5 2 2 bol’shoe murashkino (bolshemurashkinsky district) 2022 4 2 -23 -5 3 4 sergach (sergachsky district) 2022 9 2 -23 -5 7 9 pechi village (lukoyanovsky district) 2022 13 2 -23 -5 10 12 pokrovka village (lukoyanovsky district) 2022 5 2 -23 -5 4 5 shutilovo (pervomaisk) 2022 112 2 -23 -5 86 106 glukhovo (diveevsky district) 2022 5 2 -23 -5 4 5 kuzhendeevo (ardatovsky district) 2022 5 2 -23 -5 4 5 rozhentsovo (sharangsky district) 2022 6 2 -23 -5 5 6 uren (urensky district) 2022 2 2 -23 -5 2 2 vad (vadsky district) 2023 41 1 -12 -3 36 40 lopatino (vadsky district) 2023 31 1 -12 -3 27 30 s. valgusy (buturlinsky district) 2023 3 1 -12 -3 3 3 bridge over the ezhat river at panovoosanovo (gaginsky district) 2023 48 1 -12 -3 42 47 kuzmiyar (g.o. vorotynsky district) 2023 22 1 -12 -3 19 21 turbanka village (spassky district) 2023 6 1 -12 -3 5 6 lukoyanov (lukoyanovsky district) 2023 5 1 -12 -3 4 5 shatki (shatkovsky district) 2023 4 1 -12 -3 4 4 vyesdnoe (arzamas) 2023 54 1 -12 -3 48 53 arzamas (arzamas) 2023 15 1 -12 -3 13 15 323 biologica nyssana ● 16 (1) june 2025: 317-331 bakka et al. ● distribution and numbers of delichon urbicum in the nizhny novgorod oblast sosnovskoye (sosnovsky district) 2024 1 0 0 0 1 1 lyskovo (lyskovsky district) 2024 28 0 0 0 28 28 knyaginino (knyagininsky district) 2024 22 0 0 0 22 22 sergach (sergachsky district) 2024 2 0 0 0 2 2 sergach (sergachsky district) 2024 19 0 0 0 19 19 sergach (sergachsky district) 2024 5 0 0 0 5 5 bolshaya pokrovskaya str., 29 (nizhny novgorod) 2024 45 0 0 0 45 45 sergach (sergachsky district) 2024 32 0 0 0 32 32 building near the highway 1.5 km southwest of kislovka village (lyskovsky district) 2024 6 0 0 0 6 6 building near the highway n. novgorod vyksa (pavlovsky district) 2024 5 0 0 0 5 5 blagodatovka near the highway (voznesensky district) 2024 139 0 0 0 139 139 ardatov (ardatovsky district) 2024 5 0 0 0 5 5 krasnoe (arzamas) 2024 19 0 0 0 19 19 razino (lukoyanovsky district) 2024 36 0 0 0 36 36 l’vovo (vorotynsky district) 2024 42 0 0 0 42 42 total 786 916 with an average of 851 nests in 2024 (tab. 3). the distribution of western house martin colonies in the nizhny novgorod oblast (n=49) by the nest numbers in 2024 is shown in fig. 4, and the distribution of the total nest numbers by colonies of fig. 4. the distribution of western house martin colonies (n=49) in the nizhny novgorod oblast by the nest numbers in 2024 (designed by authors) different sizes is shown in fig. 5. following data from tab. 3, the average probable colony size ranged from 16 to 19 nests in 2024. these values were used to estimate the number of western house martins in 52 localities where the 324 number of active nests was not counted. the total estimated number of nests there ranged from 832 to 988. as a result, we obtained the total number of western house martins in all habitats discovered in the nizhny novgorod oblast: from 1,617 to 1,904 pairs. the distribution of the discovered western house martin habitats across the territory of the nizhny novgorod region is shown in fig. 6, which clearly illustrates the unevenness of this distribution. there are differences in the occurrence of the species in different natural regions. the western house martin breeds with the highest density in the south-east of the region and with the lowest density in the north. the results of the estimation of the probable underestimation are presented in tab. 4. in general, for the nizhny novgorod oblast, the colony numbers were underestimated by 31, and the number of breeding pairs was underestimated by a range from 499 to 584. according to our estimate, the share of the missed part of the regional population is 30.7%. the number of colonies and nests of the western house martin in different natural regions of the nizhny novgorod oblast is shown in tab. 5, and the results of the breeding density calculation are shown in tab. 6. discussion the census of occupied and empty nests is proposed as one of the methods for assessing the long-term biologica nyssana ● 16 (1) june 2025: 317-331 bakka et al. ● distribution and numbers of delichon urbicum in the nizhny novgorod oblast fig. 5. the distribution of the total active nest numbers by colonies of different sizes (n=49) (designed by authors) population trend of the western house martin population in colonies (tella et al., 2024). this method is practically not applicable in the nizhny novgorod oblast. our observations indicate that western house martin nests rarely existed for more than one year. despite the status of a protected species, building owners clean the walls of old swallow buildings almost every year. it has been shown that the breeding success of western house martins in nests from the previous year is higher than in newly built ones (kettel et al., 2021). therefore, the regular destruction of nests, even after the fledglings leave, can be a serious limiting factor for the species. on the other hand, this is a reason why we have never found swallow nests occupied by passer domesticus. several authors note that the impact of sparrows reduces the breeding success of swallows (maréchal, 1986; jerzy, 2001; ieziekel & yosef, 2020; tella et al., 2024). another limiting factor for the western house martin can be the high number of swifts apus wapus in the towns of the nizhny novgorod oblast. according to some researchers, when the numbers of flying insects are relatively low in towns, the swift is a trophic competitor to the western house martin (gashek & krasutsky, 2021), although researchers note more sophisticated mechanisms associated with the isotopic range of resources consumed by the western house martin in wintering grounds (evans et al., 2012). many authors declare the negative effects of 325 biologica nyssana ● 16 (1) june 2025: 317-331 bakka et al. ● distribution and numbers of delichon urbicum in the nizhny novgorod oblast fig. 6. the distribution of the western house martin colonies in natural regions of the nizhny novgorod oblast (designed by authors) climate change on western house martins, both directly and indirectly, through the impact on insect accessibility (imlay & leonard, 2019; brahmia et al., 2021; dvořáková et al., 2024). at the same time, the western house martin can enter torpor during cold spells and safely experience negative weather conditions (prinzinger & siedle, 1988). this adaptation reduces the impact of negative climate during the breeding season. our results indicate that in the nizhny novgorod oblast, the western house martin forms mainly small colonies (less than 9 nests). the share of such small colonies is 57% (28 out of 49 surveyed colonies) of the total number. at the same time, only 12.5% of breeding pairs inhabit such a large number of small colonies. only 3 large colonies of more than 50 nests have been found in the oblast (6% of the number of discovered colonies), 33% of the registered nests are in these localities. a significant number of small colonies and single nests are evidence of the species’ disadvantage. the breeding success in small colonies is known to be less, since one pair spends much more time on nest building (up to two days) than in a large colony, where swallows together build a nest in 2 h (ieziekel & yosef, 2020). the increasing use of plastic in the construction and facing of houses reduces the breeding success of swallows (kettel et al., 2021). the erecting of artificial nests as an effective method of maintaining swallow populations is discussed in several papers (mcneil & clark, 1977; willi et al., 2011; kettel et al., 2021; elle & lanfer, 2023; allera et al., 2024). an essential factor in the artificial nest occupancy by birds is their erecting near natural colonies of western house martins (meister & ehrengruber, 2015). we believe the experience of artificial nest erecting for western house martins should be adopted in the nizhny novgorod oblast. despite its synanthropy and longterm adaptation to living next to humans, the western house martin turned out to be a vulnerable species. the problem is that a species related to the human being becomes hostage to changing human activity without direct impact on nature: changes of materials and construction of houses (repairs, cleaning, etc.). thus, synanthropic species depends on this situation which may cause its instability. a species being adapted and living next to a human does not mean prosperous, and the western house martin demonstrates this well. in addition, the vulnerability of the species increases due to its coloniality. breeding pairs are concentrated in a few suitable habitats. the locality of colonies in northern eurasia is determined by the need to combine man–made structures suitable for nesting and the presence of numerous objects of its diet – flying insects (mel'nikov, 2016; møller, 2019; gashek & krasutsky, 2021). according to our estimate, the modern number of western house martins in the nizhny novgorod 326 biologica nyssana ● 16 (1) june 2025: 317-331 bakka et al. ● distribution and numbers of delichon urbicum in the nizhny novgorod oblast ta bl e 4. t ot al n um be rs o f c ol on ie s an d ac tiv e ne st s of th e w es te rn h ou se m ar tin in th e na tu ra l r eg io ns o f n . n ov go ro d o bl as t ( de si gn ed b y au th or s) d is tr ic t n um be r of lo ca lit ie s ( co lo ni es ) n um be r of n es ts (p ai rs ) in k no w n lo ca lit ie s pr ob ab le un de re st im at io n to ta l kn ow n pr oj ec te d m in u nd er -e st im at io n m in m ax m in m ax m in m ax th e n or th er n tr an sv ol ga re gi on v ar na vi ns ky d is tr ic t 0 0. 5 0. 5 0 0 8 10 8 10 v et lu zh sk y di st ri ct 0 0. 5 0. 5 0 0 8 10 8 10 v os kr es en sk y di st ri ct 0 0. 5 0. 5 0 0 8 10 8 10 sh ak hu ns ky d is tr ic t 0 0. 5 0. 5 0 0 8 10 8 10 se m en ov sk y di st ri ct 0 0. 5 0. 5 0 0 8 10 8 10 so ko ls ky d is tr ic t 0 0. 5 0. 5 0 0 8 10 8 10 k ov er ni ns ky d is tr ic t 0 0. 5 0. 5 0 0 8 10 8 10 k ra sn ob ak ov sk y di st ri ct 3 3 0 32 42 0 0 32 42 t on ki ns ky d is tr ic t 0 0. 5 0. 5 0 0 8 10 8 10 t on sh ae vs ky d is tr ic t 1 1 0 16 19 0 0 16 19 u re ns ky d is tr ic t 1 1 0 2 2 0 0 2 2 sh ar an gs ky d is tr ic t 1 1 0 5 6 0 0 5 6 a ve ra ge fo r t he re gi on 0. 5 0. 8 0. 3 4. 6 5. 8 5. 3 6. 7 9. 9 12 .4 n or th er n tr an sv ol ga re gi on , t ot al 6 10 4 55 69 64 80 11 9 14 9 th e so ut he rn t ra ns -v ol ga re gi on a nd v ol ga -o ka in te rfl uv es v os kr es en sk y di st ri ct 3 3 0 32 39 0 0 32 39 v or ot yn sk y di st ri ct 1 1 0 19 21 0 0 19 21 b or sk y di st ri ct 2 2 0 32 38 0 0 32 38 se m en ov sk y di st ri ct 1 1 0 4 4 0 0 4 4 g or od et sk y di st ri ct 0 1 1 0 0 16 19 16 19 l ys ko vs ky d is tr ic t 0 1 1 0 0 16 19 16 19 b al ak hn in sk y di st ri ct 0 1 1 0 0 16 19 16 19 v ol od ar sk y di st ri ct 2 2 0 32 38 0 0 32 38 d ze rz hi ns k 0 1 1 0 0 16 19 16 19 327 biologica nyssana ● 16 (1) june 2025: 317-331 bakka et al. ● distribution and numbers of delichon urbicum in the nizhny novgorod oblast c hk al ov sk y di st ri ct 3 3 0 59 71 0 0 59 71 pa vl ov sk y di st ri ct 0 1 1 0 0 16 19 16 19 a ve ra ge fo r t he re gi on 1. 1 1. 5 0. 5 16 .2 19 .2 7. 3 8. 6 23 .5 27 .8 so ut he rn t ra ns -v ol ga re gi on a nd v ol ga o ka in te rfl uv es , t ot al 12 17 5 17 8 21 1 80 95 25 8 30 6 th e w es te rn c is -v ol ga re gi on b og or od sk y di st ri ct 4 4 0 64 76 0 0 64 76 v ac hs ky d is tr ic t 0 2 2 0 0 32 38 32 38 v oz ne se ns ky d is tr ic t 1 2 1 13 9 13 9 16 19 15 5 15 8 a rz am as 2 2 0 32 38 0 0 32 38 v yk su ns ky d is tr ic t 2 2 0 17 20 0 0 17 20 k ul eb ak sk y di st ri ct 1 2 1 16 19 16 19 32 38 n iz hn y n ov go ro d 4 4 0 93 10 2 0 0 93 10 2 n av as hi ns ky d is tr ic t 0 2 2 0 0 32 38 32 38 sa ro v 4 4 0 64 76 0 0 64 76 pa vl ov sk y di st ri ct 3 3 0 37 43 0 0 37 43 so sn ov sk y di st ri ct 2 2 0 17 20 0 0 17 20 a ve ra ge fo r t he re gi on 2. 1 2. 6 0. 5 43 .5 48 .5 8. 7 10 .4 52 .3 58 .8 w es te rn c is -v ol ga re gi on , t ot al 23 29 6 47 9 53 3 96 11 4 57 5 64 7 th e e as te rn c is -v ol ga re gi on a rd at ov sk y di st ri ct 2 2. 6 0. 6 9 10 10 11 19 21 b ol sh eb ol di ns ky d is tr ic t 1 2. 6 1. 6 1 4 26 30 27 34 b ol sh em ur as hk in sk y di st ri ct 4 4 0 51 61 0 0 51 61 b ut ur lin sk y di st ri ct 2 2. 6 0. 6 19 22 10 11 29 33 v ad sk y di st ri ct 2 2. 6 0. 6 63 70 10 11 73 81 v or ot yn sk y di st ri ct 1 2. 6 1. 6 42 42 26 30 68 72 a rz am as 4 4 0 96 10 6 0 0 96 10 6 pe rv om ai sk y di st ri ct 5 5 0 13 2 17 3 0 0 13 2 17 3 pe re vo zs ky d is tr ic t 3 3 0 16 47 0 0 16 47 g ag in sk y di st ri ct 2 2. 6 0. 6 58 66 10 11 68 77 328 bakka et al. ● distribution and numbers of delichon urbicum in the nizhny novgorod oblast biologica nyssana ● 16 (1) june 2025: 317-331 d al ne ko ns ta nt in ov sk y di st ri ct 3 3 0 48 57 0 0 48 57 d iv ee vs ky d is tr ic t 2 2. 6 0. 6 20 24 10 11 30 35 k ny ag in in sk y di st ri ct 3 3 0 26 28 0 0 26 28 k ra sn oo kt ya br sk y di st ri ct 1 2. 6 1. 6 16 19 26 30 42 49 k st ov sk y di st ri ct 5 5 0 80 95 0 0 80 95 l uk oy an ov sk y di st ri ct 5 5 0 70 77 0 0 70 77 l ys ko vs ky d is tr ic t 2 2. 6 0. 6 34 34 10 11 44 45 pi ln in sk y di st ri ct 0 2. 6 2. 6 0 0 42 49 42 49 po ch in ko vs ky d is tr ic t 5 5 0 51 75 0 0 51 75 se rg ac hs ky d is tr ic t 5 5 0 65 67 0 0 65 67 se ch en ov sk y di st ri ct 1 2. 6 1. 6 0 4 26 30 26 34 sp as sk y di st ri ct 1 2. 6 1. 6 5 6 26 30 31 36 sh at ko vs ky d is tr ic t 1 2. 6 1. 6 4 4 26 30 30 34 a ve ra ge fo r t he re gi on 2. 6 3. 3 0. 7 39 .4 47 .4 11 .2 12 .8 50 .6 60 .3 e as te rn c is -v ol ga re gi on , t ot al 60 75 .8 15 .8 90 6 10 91 25 8 29 5 11 64 13 86 n iz hn y n ov go ro d ob la st , t ot al 10 1 13 2 31 16 18 19 04 49 8 58 4 21 16 24 88 ta bl e 5. t he to ta l n um be r o f c ol on ie s an d ne st s of th e w es te rn h ou se m ar tin in n at ur al re gi on s of th e n iz hn y n ov go ro d o bl as t ( de si gn ed by a ut ho rs ) n at ur al r eg io ns n um be r of lo ca lit ie s ( co lo ni es ) n um be r of n es ts (p ai rs ) kn ow n co ns id er in g th e pr oj ec te d on es kn ow n co ns id er in g th e pr oj ec te d on es t he n or th er n t ra ns -v ol ga r eg io n 6 10 61 12 9 t he s ou th er n t ra ns -v ol ga r eg io n an d th e v ol ga -o ka in te rfl uv es 12 17 20 4 29 4 w es te rn c is -v ol ga r eg io n 23 29 51 5 62 3 e as te rn c is -v ol ga r eg io n 60 76 10 51 13 31 n iz hn y n ov go ro d ob la st , t ot al 10 1 13 2 18 31 23 77 329 bakka et al. ● distribution and numbers of delichon urbicum in the nizhny novgorod oblast biologica nyssana ● 16 (1) june 2025: 317-331 table 6. the breeding density of the western house martin in natural regions of the nizhny novgorod oblast (designed by authors) natural regions density of localities, colonies/1000 sq.km density, nests (pairs)/1000 sq.km known considering the projected ones known considering the projected ones the northern trans-volga region 0.24 0.41 2.48 5.24 the southern trans-volga region and the volga-oka interfluves 0.78 1.11 13.29 19.16 western cis-volga region 1.77 2.23 39.68 48 eastern cis-volga region 2.52 3.19 44.07 55.81 nizhny novgorod oblast, total 1.32 1.72 23.85 30.96 oblast ranges from 2,116 to 2,488 pairs. this value turned out to be higher than previous estimates, which caused the species to be included in the regional red data book (bakka & kiseleva, 2007). however, for a small bird from passeriformes, the average breeding density of about 30 pairs per 1,000 km2 is extremely low. according to zoologists in the xx century, the western house martin number was not much lower than the number of the barn swallow hirundo rustica (serebrovsky, 1918; puzanov et al., 1955; vorontsov, 1967), at present its number is ten times lower (bakka & kiseleva, 2017). the analysis of the monitoring results of separate colonies (tabs. 1, 2) showed a negative trend in the funnel population in the nizhny novgorod region: the average annual decrease is 7.16% (the confidence interval with a probability of 90% is from 2.71% to 11.61%). the confirmed population decline and the presence of the threats mentioned above indicate the need to keep the conservation status of the western house martin in the oblast. this status is the only legal instrument to protect colonies from destruction during the repair and reconstruction of buildings. the distribution of western house martin colonies in the nizhny novgorod oblast is very uneven. several researchers associate similar unevenness in other regions either with landscape features (and determine the presence of building material for nests, for example) or with the humidity of the territory (mel'nikov, 2016; jokimäki et al., 2018; gashek & krasutsky, 2021). in the nizhny novgorod oblast, we observe an obvious decrease in the density of the western house martin from the forest-steppe (eastern cis-volga region) through the subzone of subtaiga forests to the southern taiga (northern trans-volga region). at the same time, the central part of the oblast, where pine forests end wetlands predominate on ancient alluvial and fluvioglacial sands, is more inhabited than the north of the region, where soils are often clayish. the small number of overlaps of western house martin registrations made by us and the scientific volunteers indicates the insufficiency of the data. estimating the regional population of the western house martin, we had to use a complex of calculations and extrapolations. the evaluation of the probable underestimation was 30.7%. we believe the effective protection of the species should be provided with more comprehensive data on its abundance and distribution. the regional census of the western house martin with the involvement of citizen science should be organized. as a result of the census, the regional database of the western house martin colonies should be developed. based on such a database, a representative sample of western house martin colonies should be identified to monitor and control the species number. conclusion we summarized and analyzed all information about the species registrations in the territory of the oblast and the census carried out in separate colonies by us and by use of citizen science methods. a database has been developed containing 184 units of information: 76 units of information were obtained by us, 100 – by scientific volunteers, and 8 – data from museum collections. according to our estimation the total number of the species ranges from 2,116 to 2,488 pairs in the nizhny novgorod oblast. the average decrease in the western house martin population in the oblast was 7.16% per year. we have identified the uneven distribution of the western house martin in the nizhny novgorod oblast. the breeding density of western house martin significantly decreases from the forest steppe through the subzone of the subtaiga forests to the southern taiga. the low species number along with a negative population trend and the presence of serious threats 330 to the species provides the basis for keeping the conservation status of the western house martin in the nizhny novgorod oblast. we have demonstrated the necessity of organizing the regional census of the western house martin, developing the regional database and continuing the monitoring. as a measure of species protection, it is important to prohibit the regularly destroying of western house martin nests after the fledglings leave during the repair and reconstruction of buildings. it is necessary to implement the positive experience in artificial nest erecting in the nizhny novgorod oblast, including this activity in the regional program of conservation measures for rare wildlife species. thus, the results obtained allow us to outline the prospects for further study of the revealed problem, identify the limiting factors for the species and make it clear in the upcoming third edition of the red data book of the nizhny 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(2011). barn swallows hirundo rustica need livestock, common house martins delichon urbicum artificial nests. ornithologische beobachter, 108(3), 215-224. bakka et al. ● distribution and numbers of delichon urbicum in the nizhny novgorod oblast biologica nyssana ● 16 (1) june 2025: 317-331 sarajli' et al., 2019, biologica nyssana 10(2) 10 (2) december 2019: 135-142 doi: 10.5281/zenodo.3600191 spontaneous flora of the vraca memorial park (sarajevo, bosnia and herzegovina) original article nermina sarajlić ornithological society „naše ptice“, semira frašte 6, 71000 sarajevo, bosnia and herzegovina nermina.sarajlic@ptice.ba (corresponding author) nejc jogan university of ljubljana, biotechnical faculty, department of biology, večna pot 111, 1000 ljubljana, slovenia jernej.jogan@bf.uni-lj.si senad murtić department of plant production, faculty of agricultural and food sciences, university of sarajevo, zmaja od bosne 8, 71000 sarajevo, bosnia and herzegovina murticsenad@hotmail.com vladimir ranđelović university of niš, faculty of sciences and mathematics, department of biology and ecology, niš, serbia vladar@pmf.ni.ac.rs received: october 12, 2019 revised: november 19, 2019 accepted: november 24, 2019 abstract: the vraca memorial park was built in 1980-1981 around the old austrohungarian fortress located above the city of sarajevo, on northwestern slopes of trebević mountain slightly above 600 m asl. it covers an area of approximately 8 ha. the southwestern part of the park is mostly covered by semi-natural forest, the central part is mostly paved, whereas the northeastern part is partly covered by ornamental forest and some grassland patches. despite being declared a national monument of bosnia and herzegovina in 2005, the park is neglected and ruined, which allowed diverse subspontaneous vegetation to develop. the paper presents the results of systematic research and analysis of the spontaneous vascular flora of the vraca memorial park. a total of 280 species of 182 genera and 67 families were recorded. with 37 species, poaceae are the most abundant, followed by compositae with 29, and fabaceae and rosaceae (23 species each). numerous seedlings of shrubs and trees planted for ornamental purposes were observed, as well as the presence of two protected orchidaceae species. key words: sarajevo, urban flora, urban habitats, alien flora, indicators of anthropogenic changes apstract: spontana flora spomen-parka vraca (sarajevo, bosna i hercegovina) spomen-park vraca izgrađen je u periodu 1980-1981. godine oko stare austrougarske tvrđave koja se nalazi iznad grada sarajeva, na severozapadnim padinama planine trebević, malo iznad 600 m nadmorske visine. prostire se na površini od oko 8 ha. jugozapadni deo parka obrastao je poluprirodnom šumom, centralni deo je popločan, a severoistočni prekriven travnatum površinama i ukrasnim drvećem i grmljem. iako je 2005. proglašen nacionalnim spomenikom bosne i hercegovine, park je u velikoj meri zapušten, što je omogućilo razvoj raznovrsne subspontane vegetacije. u radu su prikazani rezultati sistematskog istraživanja i analize spontane vaskularne flore spomenparka vraca, gde je zabeleženo ukupno 280 vrsta iz 182 roda i 67 porodica, od kojih su najbrojnije poaceae sa 37 vrsta, a slede compositae sa 29 i fabaceae i rosaceae (svaka sa 23 vrste). u parku je zabeležen veliki broj klijanaca ukrasnog drveća i grmlja, kao i dve zaštićene vrste iz porodice orchidaceae. ključne reči: sarajevo, urbana flora, urbana staništa, strana flora, indikatori antropogenih promena introduction parks are open urban spaces generally reserved for public use. they are often larger than other urban green spaces and tend to consist of different habitats with a number of native and introduced plant species, combined to satisfy recreational and other requirements of urban green areas. nielsen et al. (2014) reviewed 62 papers on biodiversity of parks, and concluded that the findings consistently showed that parks are among the most species rich types of urban green spaces. they also noted that most papers indicated large share of non-native species in parks. the same was noted for park of lužnica manor © 2019 sarajlić et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 135 13th symposium on the flora of southeastern serbia and neighboring regions near zaprešić (hršak et al., 2015) and large public park in tirana (mesiti & dinga, 2016). changes in management of urban parks were found to have significant effect on changes in composition of flora (bianco et al., 2003). despite being declared a national monument of bosnia and herzegovina in 2005 (komisija za očuvanje nacionalnih spomenika, 2005), the vraca memorial park, which was built around the old austro-hungarian fortress located above the city of sarajevo, and open to the public on 25. november 1981 (sulejmanagić, 2017), is neglected and ruined, which allowed diverse subspontaneous vegetation to develop. the paper presents the results of systematic research and an analysis of the spontaneous vascular flora of the vraca memorial park. materials and methods study area the vraca memorial park is located above the city of sarajevo, on the top of a small range of northwestern slopes of trebević mountain reaching slightly above 600 m asl. it covers an area of approximately 8 ha. the southwestern part is mostly covered by semi-natural forest, central part is mostly paved by various types of stones, and northeastern part is covered by partly ornamental forest and with some grassland patches. the area is under influence of mid-european continental climate from the north, and the mediterranean from the south. field investigation the field survey was performed from the summer of 2015 to the spring of 2019. a complete list of taxa developed in three “sub-habitat” types (walls/ pavements, grassland and forest) of the vraca memorial park was made. ornamental species were listed only if specimens developed in a site where there were obviously not deliberately planted (e.g. in cracks of the wall or pavement) were observed. data analysis the nomenclature of plants follows euro+med plantbase and the international organization for plant information database. the analyses of plant life forms (phanerophyte ph, chamaephyte ch, hemicryptophyte h, geophyte g, hydrophyte hy and therophyte t), as well as origin of plants are given. life forms were given according to raunkiaer (1934), and degree of naturalization according to pyšek et al. (2002), mosyakin & yavorska (2003) and ignatieva & konechnaya (2004). the indicators of anthropogenic changes in the flora of vraca memorial park were calculated according to jackowiak (1990, 2006), as stated by witosławski & bomanowska (2009) (tab. 1). results and discussion during this research, a total of 280 species of 182 genera and 67 families were recorded. the most abundant families were poaceae (37 taxa, 13.21%), compositae (29 taxa, 10.36%), fabaceae and rosaceae (23 taxa, 8.21% each) (tab. 2). poaceae and compositae also were the dominant families in the “grand park of tirana” in albania (mesiti & dinga, 2016) and park of lužnica manor in croatia (hršak et al., 2015). 136 table 1. indicators of anthropogenic changes indicator of total anthropophytization iant = an/(sp+an) ×100% indicator of permanent anthropophytization ianp = mt/(sp+mt) ×100% indicator of total archaeophytization iart = ar/(sp+an) ×100% indicator of permanent archaeophytization iarp = ar/(sp+mt) ×100% indicator of total kenophytization iknt = kn/(sp+an) ×100% indicator of permanent kenophytization iknp = kn/(sp+mt) ×100% indicator of modernization im = kn/mt ×100% indicator of fluctuation changes if = df/(sp+an) ×100% an – total number of alien species, sp – number of native species, mt – number of permanently established alien species (ar + kn df), ar – number of archaeophytes, kn – number of kenophytes (neophytes), df – number of diaphytes (casual aliens). table 2. the most abundant families in the flora of vraca memorial park family no. of taxa % of total park flora (280) poaceae 37 13.21 compositae 29 10.36 rosaceae 23 8.21 fabaceae 23 8.21 lamiaceae 14 5 caryophyllaceae 11 3.93 brassicaceae 10 3.57 table 3. number of taxa in different habitats of vraca memorial park habitat no. of taxa % of total park flora (280) walls/pavement 174 62.14 forest 196 70 grassland 116 41.43 biologica nyssana ● 10 (2) december 2019: 135-142 sarajlić et al. ● spontaneous flora of the vraca memorial park (sarajevo, bosnia and herzegovina) least numerous group (2.87%), in grassland it was the phanerophytes (2.59%) and in forested parts of the park, chamaephytes (4.59%) (tab. 4). the alien flora of the vraca memorial park consists of 24 taxa (8.57%), 7 of which (29.17%) are archaeophytes, and 17 (70.83%) neophytes. according to del tredici (2010), the ratio of neophytes to archaeophytes rises in direct relation to the intensity of human disturbance. this is due to the fact that archaeophytes are typically associated with traditional rural environments or intermediate levels of anthropogenic activities (preston et al., 2004), and neophytes are more common in highly disturbed anthropogenous habitats, which provide distinctive environmental conditions that favor the establishment of species from warmer and drier areas (pyšek, 1998; pyšek et al., 2002; godefroid & koedam, 2007; la sorte et al., 2007). of three habitats in the vraca memorial park, most alien plant species were registered in cracks of walls and pavements, followed by forested parts of the park and only 6 alien species were recorded in grasslands (tab. 5). indicators of anthropogenic changes values in the flora of vraca memorial park are presented in tab. 6. the indicators of anthropophytization (iant = 8.57%; ianp = 7.25%) showed the considerable anthropogenic influence on the total flora of vraca memorial park, but were not as high as in savica park area near zagreb (iant = 28.78%; ianp = 26.76%) calculatmost taxa (196, or 70% of the total number of taxa of the park) were recorded in forested part of the vraca memorial park, and the least (116, or 41.43%) in the grassland patches in the northeast (tab. 3). the analysis of life forms in the entire park showed the domination of hemicryptophytes with 130 taxa (46.43%), followed by therophytes (68 taxa, 24.28%), phanerophytes (47 taxa, 16.78%) geophytes (20 taxa, 7.14%), and chamaephytes (15 taxa, 5.36%). hemicryptophytes also represented the major part of taxa in all three habitats of the park (44.83% in walls/pavements, 46.94% in forested part of the park and 65.52% in grassland). therophytes were the second most numerous group in walls/ pavements (27.01%) and grasslands (20.69%). the domination of therophytes and hemicryptopytes in the wall flora was also observed by altay et al. (2010). in forested part of the park, phanerophytes were the second most numerous group (21.43%). in walls/pavement habitats, the geophytes were the 137 table 4. distribution of life forms in vraca memorial park, per habitat types life form habitat ch g h ph t no. % no. % no. % no. % no. % walls/pavement 10 5.75 5 2.87 78 44.83 31 17.82 47 27.01 forest 9 4.59 15 7.65 92 46.94 42 21.43 38 19.39 grassland 4 3.45 9 7.76 76 65.52 3 2.59 24 20.69 entire park 15 5.36 20 7.14 130 46.43 47 16.78 68 24.28 table 5. alien flora of vraca memorial park, per habitat types alien (total) naturalized invasive archaeophytes neophytes walls/pavement 17 13 10 4 13 forest 15 7 4 7 8 grassland 6 4 3 3 3 entire park 24 16 11 7 17 table 6. indicators of anthropogenic changes in the vraca memorial park indicator (%) walls/ pavement forest grassland entire park iant 9.77 7.65 5.17 8.57 ianp 8.72 6.22 5.17 7.25 iart 2.3 3.06 2.59 2.14 iarp 2.33 3.11 2.59 2.17 iknt 6.32 3.06 2.59 5 iknp 6.39 3.11 2.59 5.07 im 73.33 50 50 70 if 1.15 1.53 0 1.43 biologica nyssana ● 10 (2) december 2019: 135-142 sarajlić et al. ● spontaneous flora of the vraca memorial park (sarajevo, bosnia and herzegovina) ed by alegro et al. (2013) or rzeszów foothills in poland (iant = 21.3%) by jaźwa & stadnicka-futoma (2015). this is probably due to the fact that very small number of aliens in comparison to the native plants was registered in forested area and grassland. higher indicator values of kenophytization (iknt = 5%; iknp = 5.07%) showed that the flora of the park is more influenced by neophytes than by archaeophytes (iart = 2.14%; iarp = 2.17%). in both archaeophytes and neophytes, the values of total and permanent indicators were similar, showing that alien flora is well established, which was confirmed by the low value of the indicator of fluctuating changes (if = 4.45%). the forested part of the vraca memorial park is characterized by numerous seedlings of shrubs and trees planted for ornamental purposes, notably aesculus hippocastanum l., catalpa bignonioides walter, quercus rubra l., parthenocissus quinquefolia (l.) planchon, and mahonia aquifolium (pursh.) nutt. were observed, as well as the presence of two orchidaceae species included in the red list of flora of federation of bosnia and herzegovina: cephalanthera damasonium (mill.) druce (nt) and cephalanthera longifolia (l.) fritsch (vu) (đug et al., 2013). the flora developing in the vertical parts of walls was characterized by the presence of widespread and ruderal plants typical for rocky places and uncultivated lands, heliophilous, droughtresistant taxa with shallow root system, capable of growing on a thin soil layer. in pavements, tops and bottoms of the walls, where the soil layer is thicker, shade-tolerant species, some of which require higher humidity are developed. the same was noted by pavlova & tonkov (2005) and nedelcheva (2011). numerous seedlings of spiraea japonica l. were observed in cracks of the pavement in vraca memorial park. the vegetation of the park is similar to the vegetation of trampled and paved habitat types in sarajevo, and developed partly as a result of early naturalization of planted ornamental species. conclusion current neglected state of the vraca memorial park led to the development of diverse subspontaneous vegetation, similar to the vegetation of trampled and paved habitat types in sarajevo. the field survey performed in an area of 8 km2, in three “sub-habitat” types (walls/pavements, grassland and forest) of the vraca memorial park. a total of 280 species of 182 genera and 67 families were recorded. most taxa were recorded in forested part of the memorial park, and the least in the grassland patches in the northeast. the park’s flora is dominated by hemicryptophytes and native plants are more numerous in relation to 138 alien taxa, but the indicators of anthropophytization still showed the considerable anthropogenic influence on the total flora of vraca memorial park. the presence of two orchidaceae species included in the red list of flora of federation of bosnia and herzegovina indicates the potential importance of this area for conservation of native species. 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international organization for plant information 1996-2007 http://ww2.bgbm.org/iopi/gpc/default. asp [accessed september 2019] witosławski, p., bomanowska, a. 2009: southern european species in the flora of towns in the central poland. botanica serbica 33 (2): 115-129. biologica nyssana ● 10 (2) december 2019: 135-142 sarajlić et al. ● spontaneous flora of the vraca memorial park (sarajevo, bosnia and herzegovina) n o. taxon w al ls /p av em en t g ra ss la nd fo re st 1 acer campestre l. + + 2 acer negundo l. + 3 acer platanoides l. + + 4 acer pseudoplatanus l. + + 5 acer tataricum l. + + 6 achillea millefolium l. + + + 7 aegopodium podagraria l. + 8 aesculus hippocastanum l. + + 9 agrimonia eupatoria l. + 10 agrostis gigantea roth + 11 agrostis stolonifera l. + + + 12 ailanthus altissima (mill.) swingle + 13 ajuga genevensis l. + + + 14 ajuga reptans l. + + + 15 alliaria petiolata (m. bieb.) cavara et grande + + + 16 allium scorodoprasum l. + 17 ambrosia artemisiifolia l. + 18 amelanchier ovalis medik. + 19 anisantha sterilis (l.) nevski + + + 20 anthriscus sylvestris (l.) hoffm. + + 21 apera spica-venti (l.) p.beauv. + + 22 arabis hirsuta (l.) scop. + 23 arctium lappa l. + + 24 arctium tomentosum mill. + 25 aremonia agrimonoides (l.) dc. + + 26 arenaria serpyllifolia l. + + 27 arrhenatherum elatius (l.) p.beauv. ex j.presl et c.presl + + + 28 artemisia vulgaris l. + + + 29 arum maculatum l. + 30 asplenium ruta-muraria l. + 31 asplenium trichomanes l. + 32 atriplex patula l. + annex 1. overview of flora of vraca monumental park 140 33 ballota nigra l. + 34 barbarea vulgaris r. br. + 35 bellis perennis l. + + + 36 betula pendula roth + + 37 brachypodium sylvaticum (huds.) p.beauv. + + 38 bromopsis benekenii (lange) holub + 39 bromopsis erecta (huds.) fourr. + + 40 bromopsis inermis (leyss.) holub + 41 bromus commutatus schrad. + + + 42 bromus hordeaceus l. + + + 43 bromus squarrosus l. + 44 calystegia sepium (l.) r. br. + + + 45 calystegia silvatica (kit.) griseb. + 46 campanula patula l. + + 47 campanula rapunculus l. + 48 capsella bursa-pastoris (l.) medik. + + + 49 cardamine hirsuta l. + + 50 carex hirta l. + + 51 carex spicata huds. + + 52 carex sylvatica huds. + 53 carpinus betulus l. + 54 catalpa bignonioides walter + + 55 centaurea jacea l. + 56 centaurea scabiosa l. + 57 centaurea stoebe l. + 58 centaurium pulchellum (sw.) druce + 59 cephalanthera damasonium (mill.) druce + 60 cephalanthera longifolia (l.) fritsch + 61 cerastium arvense l. + 62 cerastium fontanum baumg. + + + 63 cerastium glomeratum thuill. + + + 64 chelidonium majus l. + + 65 cichorium intybus l. + + + 66 cirsium arvense (l.) scop. + 67 cirsium vulgare (savi) ten. + + 68 clematis vitalba l. + + + 69 clinopodium acinos (l.) kuntze + + 70 clinopodium vulgare l. + + + 71 convolvulus arvensis l. + + + 72 cornus sanguinea l. + + 73 corylus avellana l. + 74 cotoneaster horizontalis decne. + 75 crataegus monogyna jacq. + 76 crepis biennis l. + 77 cruciata laevipes opiz + + 78 cymbalaria muralis p.gaertn., b.mey. et scherb. + 79 cynosurus cristatus l. + + 80 dactylis glomerata l. + + + 81 daucus carota l. + + + 82 dianthus barbatus l. + 83 dioscorea communis (l.) caddick & wilkin + 84 diplotaxis muralis (l.) dc + 85 dipsacus fullonum l. + 86 dipsacus laciniatus l. + + + 87 echinochloa crus-galli (l.) p.beauv. + 88 echium vulgare l. + 89 elytrigia repens (l.) nevski + + + 90 epilobium roseum schreber + 91 equisetum arvense l. + 92 equisetum telmateia ehrh. + 93 eragrostis minor host + 94 erigeron annuus (l.) pers. + + + 95 erigeron canadensis l. + 96 erodium cicutarium (l.) ľ hér. + + 97 euphorbia amygdaloides l. + 98 euphorbia cyparissias l. + + 99 euphorbia esula l. + 100 euphorbia helioscopia l. + 101 euphorbia platyphyllos l. + 102 festuca heterophylla lam. + 103 festuca rubra l. + + 104 festulolium loliaceum (huds.) p.fourn + 105 ficaria verna huds. + 106 filipendula ulmaria (l.) maxim. + 107 filipendula vulgaris moench + + 108 fragaria vesca l. + + 109 fraxinus excelsior l. + + 110 fraxinus ornus l. + + + 111 galium aparine l. + + + 112 galium mollugo l. + + + 113 galium verum l. + + + 114 geranium columbinum l. + 115 geranium molle l. + + 116 geranium pusillum burm. f. + + 117 geranium robertianum l. + + + 118 geum urbanum l. + + + 119 glechoma hederacea l. + + + 120 hedera helix l. + + 121 heracleum sphondylium l. + + 122 holcus lanatus l. + + + 123 hordeum murinum l. + + + 124 hypericum perforatum l. + + + 125 juglans regia l. + + 126 juncus articulatus l. + 127 juncus bufonius l. + 128 juncus compressus jacq. + 129 juncus tenuis willd. + 130 knautia arvensis (l.) dc. + 131 lactuca muralis (l.) gaertn. + + 132 lactuca serriola l. + + biologica nyssana ● 10 (2) december 2019: 135-142 sarajlić et al. ● spontaneous flora of the vraca memorial park (sarajevo, bosnia and herzegovina) 141 133 lamium maculatum l. + 134 lamium purpureum l. + 135 lapsana communis l. + + 136 lathyrus latifolius l. + + 137 lathyrus nissolia l. + + 138 lathyrus pratensis l. + 139 lathyrus tuberosus l. + + 140 leontodon hispidus l. + 141 lepidium campestre (l.) r. br. + + 142 lepidium draba l. + 143 leucanthemum vulgare lam. + 144 ligustrum vulgare l. + 145 linaria vulgaris mill. + + 146 lolium perenne l. + + + 147 lonicera caprifolium l. + + 148 lonicera nitida wilson + 149 lotus corniculatus l. + + + 150 lysimachia nummularia l. + + 151 lythrum salicaria l. + + + 152 mahonia aquifolium (pursh.) nutt. + 153 malva moschata l. + 154 matricaria discoidea dc. + 155 medicago falcata l. + + + 156 medicago lupulina l. + + + 157 medicago sativa l. + + 158 melica ciliata l. + + 159 melilotus albus medik. + 160 melilotus officinalis (l.) lam. + + 161 mentha longifolia (l.) l. + + + 162 microrrhinum minus (l.) fourr. + 163 myosotis arvensis (l.) hill + + + 164 myosotis ramosissima rochel + 165 myosotis sylvatica hoffm. + 166 ochlopoa annua (l.) h. scholz + + + 167 origanum vulgare l. + 168 ornithogalum pyrenaicum l. + 169 oxalis corniculata l. + + 170 oxalis fontana bunge + 171 parthenocissus quinquefolia (l.) planchon + + 172 pastinaca sativa l. + 173 persicaria lapathifolia (l.) delarbre + 174 petrorhagia saxifraga (l.) link + 175 phleum pratense l. + 176 physalis alkekengi l. + + 177 picea abies (l.) h. karst. + 178 picris hieracioides l. + + 179 pimpinella saxifraga l. + 180 plantago lanceolata l. + + + 181 plantago major l. + + + 182 plantago media l. + + + 183 poa angustifolia l. + 184 poa bulbosa l. + 185 poa compressa l. + + 186 poa nemoralis l. + + 187 poa pratensis l. + 188 poa trivialis l. + + + 189 polygonum aviculare l. + 190 populus nigra l. + 191 potentilla argentea l. + 192 potentilla micrantha ramond ex dc. + 193 potentilla reptans l. + + + 194 primula acaulis (l.) l. + 195 prunella vulgaris l. + + + 196 prunus avium l. + 197 prunus cerasifera ehrh. + + + 198 prunus spinosa l. + 199 pteridium aquilinum (l.) kuhn + 200 quercus robur l. + + 201 quercus rubra l. + 202 ranunculus acris l. + + 203 reseda lutea l. + 204 rhinanthus rumelicus velen. + 205 robinia pseudoacacia l. + + 206 rorippa sylvestris (l.) besser + + 207 rosa arvensis huds. + + 208 rosa canina l. + + 209 rubus caesius l. + + 210 rubus plicatus weihe et nees + + 211 rumex acetosa l. + 212 rumex crispus l. + + + 213 rumex obtusifolius l. + 214 rumex patientia l. + + 215 rumex sanguineus l. + 216 sagina apetala ard. + 217 sagina procumbens l. + 218 salix alba l. + 219 salix caprea l. + 220 sambucus ebulus l. + + 221 sambucus nigra l. + + 222 sanguisorba minor scop. + + + 223 sanguisorba minor subsp. balearica (nyman) muñoz garm. & c. navarro + 224 schedonorus arundinaceus (schreb.) dumort. + + + 225 schedonorus pratensis (huds.) p. beauv. + + 226 sedum album l. + 227 sedum rupestre l. + 228 sedum sexangulare l. + 229 senecio vulgaris l. + 230 setaria viridis (l.) p.beauv. + 231 silene vulgaris (moench) garcke + 232 sisymbrium officinale (l.) scop. + 233 smyrnium perfoliatum l. + biologica nyssana ● 10 (2) december 2019: 135-142 sarajlić et al. ● spontaneous flora of the vraca memorial park (sarajevo, bosnia and herzegovina) 142 234 solanum nigrum l. + 235 solidago gigantea aiton + + 236 sonchus asper (l.) hill + 237 sonchus oleraceus l. + + 238 sorbus aucuparia l. + 239 spiraea japonica l. f. + 240 stachys sylvatica l. + 241 stellaria graminea l. 242 stellaria media (l.) vill. + 243 syringa vulgaris l. + 244 taraxacum sect. taraxacum f. h. wigg. + + + 245 taxus baccata l. + 246 teucrium chamaedrys l. + 247 thymus pulegioides l. + + + 248 tilia cordata mill. + 249 tilia platyphyllos scop. + + 250 torilis arvensis (huds.) link + 251 torilis japonica (houtt.) dc. + + 252 trifolium aureum pollich + + 253 trifolium campestre schreber + + 254 trifolium dubium sibth. + 255 trifolium hybridum l. + 256 trifolium medium l. + 257 trifolium pratense l. + + + 258 trifolium repens l. + + + 259 tripleurospermum inodorum (l.) sch. bip. + + 260 trisetum flavescens (l.) p.beauv. + + + 261 tropaeolum majus l. + 262 tussilago farfara l. + + 263 ulmus minor mill. + + 264 urtica dioica l. + + + 265 verbascum thapsus l. + 266 verbena officinalis l. + + + 267 veronica arvensis l. + + + 268 veronica chamaedrys l. + 269 veronica hederifolia l. + 270 veronica persica poir. + + 271 viburnum lantana l. + 272 viburnum opulus l. + 273 vicia cracca l. + + 274 vicia hirsuta (l.) gray + 275 vicia sativa l. + + 276 vicia tetrasperma (l.) schreber + + 277 vicia villosa roth ssp. varia (host) corb. + 278 vinca minor l. + 279 viscum album l. + 280 vulpia myuros (l.) c.c.gmel. + + biologica nyssana ● 10 (2) december 2019: 135-142 sarajlić et al. ● spontaneous flora of the vraca memorial park (sarajevo, bosnia and herzegovina) nikolić, d. et al.  the influence of orographical and bioclimatic factors … biologica nyssana 6 (1)  september 2015: 1-9 nikolić, d. et al.  the influence of orographical and bioclimatic factors … 1 original article received: 15 july 2015 revised: 18 august 2015 accepted: 01 september 2015 the influence of orographical and bioclimatic factors on morphological variability of analyzed characters of jovibarba heuffelii (schott) a. löve & d. löve (crassulaceae) danijela nikolić1*, jasmina šinžar-sekulić2, vladimir ranđelović1, dmitar lakušić2 1university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia 2university of belgrade, faculty of biology, institute of botany and botanical garden „jevremovac”, takovska 43, 11000 belgrade, serbia * e-mail: danid@pmf.ni.ac.rs abstract: nikolić, d., šinžar-sekulić, j., ranđelović, v., lakušić, d.: the influence of orographical and bioclimatic factors on morphological variability of analyzed characters of jovibarba heuffelii (schott) a. löve & d. löve (crassulaceae). biologica nyssana, 6 (1), september 2015: 1-9. the goal of this paper was to determine the extent of morphological variability in species j. heuffelii caused by orographic and bioclimatic factors. samples were collected from 14 populations of species j. heuffelii, from the territories of serbia, macedonia, bulgaria and romania. for this purpose cluster analysis (upgma) on bioclimatic parameters and regression analysis were performed. the cluster analysis of bioclimatic factors has shown that the study area was influenced by semiarid temperate-continental or subcontinental climate, continental mountain climate and humid mountain climate. among the orographic factors, the greatest influence on morphological characters of species j. heuffelii was determined for altitude, exposition and slope of the terrain. the mean temperature of the wettest quartile (bio8) and temperature seasonality (bio4) have shown the greatest influence on the morphological characters of j. heuffelii. key words: jovibarba heuffelii, morphological variability, environmental factors apstrakt: nikolić, d., šinžar-sekulić, j., ranđelović, v., lakušić, d.: uticaj orografskih i bioklimatskih faktora na morfološku varijabilnost analiziranih karaktera jovibarba heuffelii (schott) a. löve & d. löve (crassulaceae). biologica nyssana, 6 (1), septembar 2015: 1-9. cilj ovog rada je bio da se utvrdi u kolikoj meri je velika morfološka varijabilnost kod vrste j. heuffelii uslovljena uticajem orografskih i bioklimatskih faktora. prikupljeni su uzorci 14 populacija vrste j. heuffelii sa područja srbije, makedonije, bugarske i rumunije. urađena je klaster analiza (upgma) sa bioklimatskim parametrima i regresiona analiza. klaster analiza bioklimatskih faktora je pokazala da je istraživano područje pod uticajem tri tipa klime: semiaridne umereno kontinentalne ili subkontinentalne klime, kontinentalne planinske klime i humidne planinske klime. od orografskih faktora pored nadmorske visine, ekspozicija i nagib terena imaju najveći uticaj na morfološke karaktere vrste j. heuffelii. srednja temperatura najvlažnijeg kvartala (bio8) i temperaturna sezonalnost (bio4) najviše utiču na morfološke karaktere j. heuffelii. key words: jovibarba heuffelii, morfološka varijabilnost, ekološki faktori 6 (1) • september 2015: 1-9 biologica nyssana 6 (1)  september 2015: 1-9 nikolić, d. et al.  the influence of orographical and bioclimatic factors … 2 introduction j. heuffelii (crassulaceae) is a succulent xerophyte primarily inhabiting rocky habitats at various substrates (limestone, serpentinite, silicate) in a range of altitudes from the coastline to 2500 m above sea level (b a r c a & n i c u l a e , 2005; l a k u š i ć et al., 2005; d i m i t r i j e v i ć et al., 2011). distribution of this species in balkan peninsula and the southern carpathians indicates that this is a european endemic species (m e u s e l , 1965; j a l a s et al., 1999). according to the climatic division of southeastern europe (h o r v a t et al., 1974) and division into main climate and biome types by w a l t e r and l e i t h (1964), there are two main climate types within the range of j. heuffelii complex: temperate-continental climate (type vi) and mountain climate (type x). the temperatecontinental climate is represented by subtype vi 3 semiarid temperate-continental climate (moesiancarpathian variant). within the mountain climate there are two subtypes: x 2 humid mountain climate of alpine type (illyrian variant) and x 3 – continental mountain climate (moesian-carpathian variant). this species is characterized by a very high morphological variability, as evidenced by recent studies on variability of morphological characteristics of vegetative and reproductive organs (d i m i t r i j e v i ć et al., 2011) and analysis of morphological characteristics of nectaries (n i k o l i ć et al., 2015a). the impact of orographic and bioclimatic habitat factors on morphological variability was analyzed in paper by n i k o l i ć et al. (2015b), while this paper pertains only to populations from identical mountain habitats, while gorge and canyon populations were omitted from the analysis. regression analysis and cluster analysis (upgma) based on matrix of bioclimatic parameters were performed in order to determine the extent of impact of orographic and bioclimatic factors on variability of morphological characteristics and differentiation of all analyzed populations. table 1. the list of population of j. heuffelii used in this study. vouchers are deposited in the herbarium of the institute of botany, faculty of biology, university of belgrade (beou) population coordinate substrate altitude voucher individuals 1. ro-domogled 44°52'41.70"n 22°25'54.11"e limestone 1300 beou16510 20 2. sr-gradac 44°15'17.98"n 19°53'23.00"e limestone 490 beou-16458 21 3. sr-suvaja 44°10'54.98"n 19°52'11.08"e limestone 417 beou-16457 20 4. sr-studenica 43°29'20.56"n 20°32'7.74"e serpentinite 486 beou-16461 20 5. srnebeske stolice 43°15'34.14"n 20°49'33.59"e serpentinite 1907 beou-16468 22 6. sr-treska 43°15'36.31"n 20°47'6.40"e serpentinite 1628 beou-16462 20 7. sr-basarski kamik 43° 9'37.29"n 22°42'9.57"e limestone 1350 beou16460 25 8. sr-radan 42°55'4.99"n 21°33'25.74"e silicate 802 beou-16456 20 9. sr-pljačkovica 42°34'47.20"n, 21°53'31.09"e silicate 674 beou-16465 21 10. srbesna kobila 42°31'45.08"n 22°13'51.10"e silicate 1900 beou16463 30 11. sr-stara planina 43°23'23.41"n 22°38'1.98"e silicate 1840 beou-16459 20 12. bu-trojanski prolaz 42°46'1.62"n 24°37'2.30"e silicate 1400 beou-16509 20 13. ma-treskavec 41°24'14.73"n 21°32'14.44"e silicate 1250 beou16511 20 14. mamavrovo 41°38'14.33"n 20°42'29.90"e limestone 1300 beou16512 20 biologica nyssana 6 (1)  september 2015: 1-9 nikolić, d. et al.  the influence of orographical and bioclimatic factors … 3 table 2. morphological characters investigated using the populations of j. heuffelii described in table 1. morphological characters abbreviations 1 diameter of rosette (mm) ros_d 2 number of leaves in rosette leros_n 3 length of biggest leaf (mm) leros_l max 4 width of biggest leaf (mm) leros_w max 5 distance of widest part of leaf from the top of the leaf (mm) apex_d_ros 6 length of spike (mm) leros_sp_l 7 length of cilia (mm) leros_ci_l 8 width of cartilaginous leaf edge (mm) leros_ed_w 9 height of stem to lowest flower branch (mm) ste_h 10 number of leaves at stem leste_n 11 length of middle leaf on stem (mm) midleste_l 12 width of middle leaf on stem (mm) midleste_w 13 distance of widest part of leaf from the top of the leaf (mm) apex_d_ste 14 number of floral branches flobra_n 15 number of flowers at stage of ripening fruit flo_n 16 length of longest branch in fruit (mm) flobra_l 17 length of sepal (mm) sep_l 18 width of sepal (mm) sep_w 19 length of petal (mm) pet_l 20 width of petal (mm) pet_w 21 length of longest filament (mm) fil_l max 22 height of ovary (mm) ova_h 23 height of stylus (mm) sty_h 24 height of central tooth on petal (mm) centoo_h 25 height of lateral tooth on petal (mm) lattoo_h 26 height of fruit (mm) fru_h 27 width of fruit (mm) fru_w 28 length of rostrum (mm) rost_l 29 total seed length (mm) see_l 30 total seed width (mm) see_w 31 width of central longitudinal fold (costa) (mm) cos_w 32 width of nectary (mm) nect_w 33 height of nectary (mm) nect_h 34 the angle between carpels and nectaries (degree) nect-ang material and methods plant material fourteen populations (299 individuals) of j. heuffelii were collected for morphological analysis. the samples were taken from serbia, macedonia bulgaria and romania (during two growing seasons 2010, 2011). the voucher specimens are deposited in the herbarium of the institute of botany and botanical garden “jevremovac", faculty of biology, university of belgrade beou (tab. 1). morphometric analyses morphometric analyses were performed on dissected plant organs (leaves, stems, flowers, fruits and seeds). leaves and inflorescences were stored in a glycerol: 96% ethanol solution (50:50). for measuring of flowers, the microscope slides were used, where all flower parts (sepals, petals, stamens, carpels) were separated and individually placed on the microscope slide. slides were first scanned (scanexpress a3 usb, mustek) and then measured by using the leicaqwin image analyzing program (leica image software). for purposes of measuring biologica nyssana 6 (1)  september 2015: 1-9 nikolić, d. et al.  the influence of orographical and bioclimatic factors … 4 the smallest details on leaves, flowers and nectaries, these organs were photographed with the leica dm 1000 microscope while seeds were photographed with the leica mz16 a stereomicroscope. thirty four morphological characters were used in these analyses (tab. 2). statistical analysis cluster analysis (upgma) was performed in order to evaluate the bioclimatic differentiation between the habitats of the 14 investigated populations. each location was characterized using 19 bioclimatic parameters, extracted from the worldclim set of global climate layers (h i j m a n s et al., 2005). the extraction of bioclimatic parameters was done with diva-gis 7.5 software (h i j m a n s et al., 2012). regression analysis (linear regression) was performed in order to estimate the correlations between the variation of morphological characters of j. heuffelii and basic orographic, geological, and bioclimatic habitat characteristics, as well as the geographic position of each population. the geographic positions were recorded using a handheld global positioning system (gps garmin etrex vista® c). orographic characteristics including altitude, slope and aspect were calculated from the shuttle radar topography mission (reuter et al., 2007) at an approximate 90 m pixel resolution using arcgis 10 spatial analyst. prior to the regression analysis, habitat characteristics were tested for multicollinearity. bioclimatic predictors that have shown significant correlations with other predictors were not used in regression analysis of morphological characters. all statistical analyses were performed using the package statistica 5.1 (statsoft, 1996). results and discussion cluster analysis of bioclimatic data cluster analysis based on bioclimatic factors showed presence of 3 clusters (fig. 1). the first cluster (c1) included localities with semiarid, temperate-continental climate or subcontinental climates. these habitats appear in gorges of rivers gradac, suvaja and studenica as well as at domogled in romania. this cluster also included populations from bulgaria (bu-trojanski prolaz central part of stara planina) and the population from mt. stara planina in the e part of serbia. the second cluster (c2) contained localities with continental mountain climates (sr-besna kobila, sr-radan, sr-basarski kamik, sr-pljačkovica and ma-treskavec). the third cluster (c3) includes localities with humid mountain climate (sr-treska, sr-nebeske stolice and ma-mavrovo). fig. 1. results of cluster analysis for populations of j. heuffelii based on habitat climatic characteristics (c1, c2, c3, climate type, for details see tab. 2.) biologica nyssana 6 (1)  september 2015: 1-9 nikolić, d. et al.  the influence of orographical and bioclimatic factors … 5 t a b le 3 . m in im al , av er ag e an d m ax im al v al u e o f 1 9 b io cl im at ic p ar am et er s o f lo ca li ti es s tu d ie d . c 1 , c 2 a n d c 3 r ep re se n t cl im at e ty p es c o rr es p o n d t o t h e g ro u p in g s o b ta in ed f ro m t h e cl u st er a n al y si s o f b io cl im at ic p ar am et er s (s ee f ig . 1 ). t h e b io cl im at ic v ar ia b le s th at w er e u se d a s p re d ic to rs i n r eg re ss io n a n al y si s ar e m ar k ed w it h a st er is k ( * ). b io cl im a ti c p a ra m et er s c 1 c 2 c 3 se m i -a ri d m o d er at el y co n ti n en ta l cl im at e co n ti n en ta l m o u n ta in cl im at e h u m id m o u n ta in c li m at e m in a v g m ax m in a v g m ax m in a v g m ax a n n u al m ea n t em p er at u re ( 1 ) 3 .3 7 7 .7 1 1 1 .3 3 3 .3 0 7 .7 4 9 .7 2 2 .8 7 4 .2 3 4 .9 8 m ea n m o n th ly t em p er at u re r an g e (2 ) * 7 .9 8 9 .1 1 0 .1 8 .7 3 9 .1 1 0 .3 6 7 .4 8 8 .1 6 9 .0 4 is o th er m al it y ( 2 /7 ) (* 1 0 0 ) (3 ) * 2 9 .4 9 3 1 .7 4 3 3 .6 7 3 1 .6 4 3 3 .3 0 3 4 .6 4 2 9 .3 4 3 0 .8 8 3 3 .4 9 t em p er at u re s ea so n al it y ( s t d * 1 0 0 ) (4 ) * 6 7 6 .6 0 7 2 4 .8 4 7 5 5 .9 2 6 6 1 .0 8 7 2 1 .5 2 7 5 0 .3 9 6 4 5 .8 4 6 6 8 .4 9 6 9 1 .6 4 m ax . te m p er at u re o f th e w ar m es t m o n th ( 5 ) 1 7 .2 2 2 .6 5 2 6 .8 1 7 .6 2 3 .4 8 2 4 .1 1 6 .4 1 8 .3 1 9 .6 m in . te m p er at u re o f th e co ld es t m o n th ( 6 ) -3 .2 -5 .9 7 -8 .9 -4 .4 -5 .9 3 -9 -7 .4 -8 .1 -9 .1 a n n u al t em p er at u re r an g e (5 – 6 ) (7 ) * 2 6 .1 2 8 .6 2 3 0 .2 2 6 .6 2 9 .4 3 0 .4 2 5 .5 2 6 .4 2 7 m ea n t em p er at u re o f th e w et te st q u ar te r (8 ) 9 .6 1 4 .6 1 1 8 .4 5 3 .9 3 1 0 .2 9 1 3 .5 5 -1 .3 6 .3 6 1 1 .3 2 m ea n t em p er at u re o f th e d ri es t q u ar te r (9 ) * -0 .9 2 3 .4 5 9 .5 8 1 .8 1 1 .2 7 1 6 .5 2 -2 .7 5 6 .0 6 1 2 .6 3 m ea n t em p er at u re o f th e w ar m es t q u ar te r (1 0 ) 1 1 .4 8 1 6 .3 6 2 0 .1 7 1 1 .2 5 1 6 .3 5 1 8 .5 8 1 0 .8 5 1 2 .2 6 1 3 .1 2 m ea n t em p er at u re o f th e co ld es t q u ar te r (1 1 ) -4 .9 5 -1 .4 2 2 .0 1 -3 .3 -1 .3 4 0 .1 7 -5 .3 8 -4 .0 2 -2 .9 a n n u al p re ci p it at io n ( 1 2 ) * 7 7 8 8 1 5 .1 7 8 4 9 6 0 6 6 6 5 .7 5 7 4 2 9 5 0 1 0 0 1 .6 7 1 0 4 5 p re ci p it at io n o f th e w et te st m o n th ( 1 3 ) 8 7 1 0 0 .3 3 1 2 1 6 8 7 6 .7 5 8 4 1 0 4 1 1 3 .6 7 1 2 7 p re ci p it at io n o f th e d ri es t m o n th ( 1 4 ) * 4 2 5 0 5 5 4 0 4 2 .7 5 4 6 5 6 6 2 .6 7 6 9 p re ci p it at io n s ea so n al it y ( c v ) (1 5 ) * 1 7 .5 7 2 3 .1 3 3 4 .4 0 1 6 .6 7 1 9 .1 0 2 1 .2 9 1 5 .1 3 1 8 .9 2 6 .2 4 p re ci p it at io n o f th e w et te st q u ar te r (1 6 ) 2 5 5 2 6 9 .1 3 3 1 0 1 7 2 2 0 0 .7 5 2 3 1 2 8 5 3 1 2 .3 3 3 4 8 p re ci p it at io n o f th e d ri es t q u ar te r (1 7 ) 1 4 7 1 6 2 .1 7 1 7 5 1 2 7 1 3 6 .2 5 1 4 7 1 7 8 2 0 2 2 2 2 p re ci p it at io n o f th e w ar m es t q u ar te r (1 8 ) * 2 2 0 2 4 5 .1 7 2 9 1 1 3 6 1 6 1 .7 5 2 0 4 1 7 9 2 3 5 2 7 2 p re ci p it at io n o f th e co ld es t q u ar te r (1 9 ) * 1 5 8 1 7 7 .5 1 8 7 1 4 7 1 6 0 1 9 4 2 1 8 2 5 5 3 1 0 biologica nyssana 6 (1)  september 2015: 1-9 nikolić, d. et al.  the influence of orographical and bioclimatic factors … 6 t a b le 4 . s u m ar y s ta ti st ic s o f re g re ss io n ( r 2 ) fo r in d ep en d en t b io cl im at ic , g eo g ra p h ic al a n d g eo lo g ic al v ar ia b le s. a cr o n y m a l t a s p s l o b io 2 b io 3 b io 4 b io 7 b io 8 b io 9 b io 1 2 b io 1 3 b io 1 4 b io 1 5 b io 1 8 b io 1 9 r o s_ d 0 .0 7 * * 0 .0 2 0 .0 1 0 .0 2 0 .0 7 * * 0 .0 5 0 .0 0 0 .2 6 0 .1 1 0 .0 1 0 .0 0 0 .0 5 0 .0 1 0 .1 6 0 .0 2 l er o s_ n 0 .2 6 * 0 .1 3 * 0 .0 2 0 .2 5 * 0 .1 3 * 0 .1 9 * 0 .2 8 * 0 .0 8 * * 0 .0 1 0 .0 2 0 .0 0 0 .0 4 0 .0 2 0 .0 1 0 .0 2 l er o s_ l m ax 0 .0 9 * 0 .0 2 0 .0 1 0 .0 0 0 .0 5 0 .1 3 * 0 .0 2 0 .2 2 * 0 .0 1 * 0 .0 0 0 .0 3 0 .0 0 0 .0 9 * 0 .1 6 * 0 .0 9 * l er o s_ w m ax 0 .1 2 * 0 .0 2 0 .0 0 0 .0 0 0 .0 4 0 .1 6 * 0 .0 4 0 .2 0 * 0 .1 9 * 0 .0 1 0 .0 5 0 .0 2 0 .0 3 0 .1 4 * 0 .0 2 a p ex _ d 1 0 .0 6 0 .0 2 0 .0 0 0 .0 0 0 .0 0 0 .0 7 0 .0 2 0 .1 0 * 0 .0 4 0 .0 0 0 .0 0 0 .0 0 0 .0 2 0 .0 4 0 .0 4 l er o s_ s p _ l 0 .0 0 0 .0 0 0 .0 3 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .0 1 0 .0 2 0 .0 2 0 .0 1 0 .0 0 0 .0 1 0 .0 2 l er o s_ c i_ l 0 .0 0 0 .0 1 0 .0 7 0 .0 0 0 .0 1 0 .0 4 0 .0 1 0 .0 0 0 .0 1 0 .0 0 0 .0 2 0 .0 2 0 .1 0 * 0 .0 0 0 .0 2 l er o s_ e d _ w 0 .0 3 0 .0 4 0 .0 4 0 .0 0 0 .0 0 0 .0 1 0 .0 1 0 .0 3 0 .0 1 0 .0 0 0 .0 3 0 .0 7 * * 0 .1 4 * 0 .0 1 0 .0 1 s te _ h 0 .1 7 * 0 .0 4 0 .0 1 0 .0 0 0 .0 4 0 .1 7 * 0 .0 5 0 .3 0 * 0 .1 2 * 0 .0 0 0 .0 0 0 .0 4 0 .0 1 0 .0 8 * * 0 .0 5 l es te _ n 0 .0 0 0 .0 1 0 .0 0 0 .0 6 0 .1 2 * 0 .0 0 0 .0 1 0 .0 5 0 .0 7 * * 0 .0 3 0 .0 0 0 .1 6 * 0 .1 3 * 0 .0 3 0 .0 0 m id l es te _ l 0 .0 0 0 .0 1 0 .0 0 0 .0 3 0 .0 5 0 .0 0 0 .0 1 0 .0 4 0 .0 3 0 .0 0 0 .0 2 0 .0 0 0 .0 4 0 .1 0 * 0 .0 2 m id l es te _ w 0 .0 0 0 .0 0 0 .0 1 0 .0 9 * 0 .1 9 * 0 .0 2 0 .0 2 0 .1 0 * 0 .2 0 * 0 .0 6 0 .0 7 * * 0 .0 9 * 0 .0 0 0 .2 2 * 0 .0 0 a p ex _ d 2 0 .0 5 0 .0 0 0 .0 1 0 .0 0 0 .0 5 0 .0 7 * * 0 .0 1 0 .2 3 * 0 .0 8 * * 0 .0 1 0 .0 2 0 .0 1 0 .0 4 0 .0 7 * * 0 .1 0 * s ep _ l 0 .0 3 0 .0 0 0 .0 9 * 0 .0 0 0 .0 0 0 .0 1 0 .0 0 0 .0 8 * * 0 .0 2 0 .0 6 0 .1 0 * 0 .0 0 0 .0 6 0 .0 0 0 .0 6 s ep _ w 0 .0 2 0 .0 0 0 .0 7 0 .0 0 0 .0 1 0 .0 1 0 .0 0 0 .0 7 * * 0 .0 0 0 .0 1 0 .0 0 0 .0 0 0 .0 0 0 .0 1 0 .0 3 p et _ l 0 .2 2 * 0 .0 6 0 .0 4 0 .0 2 0 .0 1 0 .1 9 * 0 .0 9 * 0 .3 1 * 0 .0 5 0 .0 1 0 .0 1 0 .0 2 0 .0 3 0 .0 3 0 .0 6 p et _ w 0 .0 1 0 .0 1 0 .0 4 0 .0 3 0 .0 8 * * 0 .0 1 0 .0 0 0 .0 4 0 .0 4 0 .0 1 0 .0 0 0 .1 1 * 0 .0 9 * 0 .0 1 0 .0 0 f il _ l m ax 0 .2 3 * 0 .1 1 * 0 .0 0 0 .0 2 0 .0 0 0 .1 7 * 0 .0 8 * * 0 .3 4 * 0 .0 8 * * 0 .0 0 0 .0 1 0 .0 5 0 .0 5 0 .0 6 0 .0 3 o v a_ h 0 .2 3 * 0 .1 3 * 0 .0 4 0 .0 1 0 .0 1 0 .1 5 * 0 .0 7 * * 0 .3 1 * 0 .0 4 0 .0 0 0 .0 0 0 .0 3 0 .0 2 0 .0 4 0 .0 3 s ty _ h 0 .2 9 * 0 .1 5 * 0 .0 0 0 .0 4 0 .0 0 0 .2 9 * 0 .1 6 * 0 .3 6 * 0 .1 0 * 0 .0 0 0 .0 0 0 .0 2 0 .0 1 0 .0 9 * 0 .0 7 * * c en t o o _ h 0 .0 2 0 .0 0 0 .0 5 0 .0 1 0 .0 0 0 .0 0 0 .0 1 0 .0 2 0 .0 1 0 .0 2 0 .0 9 * 0 .0 0 0 .1 2 * 0 .0 3 0 .0 1 l at t o o _ h 0 .0 1 0 .0 4 0 .0 1 0 .0 0 0 .0 0 0 .0 1 0 .0 0 0 .0 1 0 .0 1 0 .0 1 0 .0 9 * 0 .0 0 0 .1 7 * 0 .0 8 * * 0 .0 0 f ru _ h 0 .3 1 * 0 .1 4 * 0 .0 2 0 .0 3 0 .0 0 0 .2 2 * 0 .1 2 * 0 .4 2 * 0 .0 6 0 .0 0 0 .0 2 0 .0 5 0 .0 7 0 .0 4 0 .0 5 f ru _ w 0 .3 1 * 0 .1 4 * 0 .0 4 0 .0 3 0 .0 0 0 .2 1 * 0 .1 2 * 0 .4 0 * 0 .0 5 0 .0 0 0 .0 2 0 .0 4 0 .0 7 0 .0 3 0 .0 5 r o st _ l 0 .2 5 * 0 .3 7 * 0 .0 8 * * 0 .0 4 0 .0 0 0 .2 6 * 0 .1 3 * 0 .1 9 * 0 .0 0 0 .0 0 0 .0 7 * * 0 .0 0 0 .2 1 * 0 .1 1 * 0 .0 6 f lo b ra _ n 0 .0 3 0 .0 2 0 .0 0 0 .0 1 0 .0 5 0 .0 3 0 .0 0 0 .1 1 * 0 .0 7 * * 0 .0 1 0 .0 1 0 .0 3 0 .0 0 0 .0 9 * 0 .0 0 f lo _ n 0 .0 9 0 .0 5 0 .0 0 0 .0 1 0 .0 0 0 .0 6 0 .0 3 0 .1 0 * 0 .0 4 0 .0 0 0 .0 1 0 .0 0 0 .0 1 0 .0 4 0 .0 1 f il _ l m ax 0 .1 1 0 .0 0 0 .0 0 0 .0 0 0 .0 6 0 .1 3 * 0 .0 2 0 .3 8 * 0 .2 0 * 0 .0 2 0 .0 7 0 .0 2 0 .1 1 * 0 .0 6 0 .1 0 * s ee _ l 0 .0 7 * * 0 .0 8 * * 0 .0 2 0 .0 0 0 .0 0 0 .0 2 0 .0 1 0 .0 7 * * 0 .0 6 0 .0 2 0 .0 6 0 .0 0 0 .0 6 0 .0 9 * 0 .0 0 s ee _ w 0 .1 3 * 0 .0 3 0 .0 0 0 .0 5 0 .0 2 0 .0 5 0 .0 6 0 .0 8 * * 0 .0 4 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 c o s_ w 0 .0 2 0 .0 0 0 .0 2 0 .0 1 0 .0 0 0 .0 1 0 .0 1 0 .0 7 0 .0 1 0 .0 1 0 .1 7 * 0 .0 3 0 .4 1 * 0 .0 1 0 .0 0 n ec t_ w 0 .0 8 * * 0 .1 0 * 0 .0 3 0 .1 7 * 0 .1 3 * 0 .0 5 0 .1 4 * 0 .0 0 0 .0 6 0 .0 6 0 .1 1 * 0 .0 2 0 .0 4 0 .1 2 * 0 .0 2 n ec t_ h 0 .0 4 0 .0 4 0 .0 0 0 .0 0 0 .0 1 0 .0 7 * * 0 .0 3 0 .0 4 0 .0 5 0 .0 3 0 .0 3 0 .0 3 0 .0 0 0 .0 4 0 .0 0 n ec ta n g 0 .0 4 0 .0 1 0 .0 3 0 .0 8 * * 0 .0 8 * * 0 .0 1 0 .0 5 0 .0 0 0 .0 1 0 .0 5 0 .0 4 0 .0 4 0 .0 0 0 .0 5 0 .0 1 a b b re v ia ti o n s: a l t -a lt it u d e, a s p as p ec t, s l o -s lo p e, b io b io cl im at ic c h ar ac te ri st ic s (s ee t ab le 3 ) * p < 0 .0 5 , * * p < 0 .0 1 biologica nyssana 6 (1)  september 2015: 1-9 nikolić, d. et al.  the influence of orographical and bioclimatic factors … 7 the analysis of the three clusters regarding the values of bioclimatic parameters (tab. 3) shows that type c1 is characterized by the highest values of bio1 bioclimatic parameter, the mean annual temperature within the range 3.37-11.33 °c. c2 type has shown variation in this character in range of 3.30-9.72 °c, while c3 climate type has shown the lowest values of this parameters, 2.87 4.98 °c (tab. 3). a similar trend was noticed in bioclimatic parameters bio5, bio6, bio8, bio10 and bio11, while in the bioclimatic parameter bio9 (mean temperature of the driest quarter) the highest values were recorded for the second type of climate c2 (1.8-16.52 °c), followed by the third type c3 (–2.75 -12.63 °c) while the lowest values were recorded in the first climate type c1 (–0.92-9.58 °c). the analysis of these three clusters according to bioclimatic factors (tab. 3) indicated that the greatest differences between clusters were present in the amount of precipitation, as measured by the factors annual precipitation (bio12), precipitation of the driest month (bio14) and precipitation of the wettest quarter (bio16). the largest amount of precipitation was present in the third cluster, the second had the lowest amount of precipitation, while the first cluster was intermediate. considering the temperature, the highest values were recorded in canyons and river gorges (c1) and the lowest values in the third cluster (c3). there is an important question: to which extent do bioclimatic factors in analyzed habitats cause the differentiation in populations? the answer to this question may be provided by cluster analysis including both the bioclimatic characters and the morphological characteristics of individuals from the analyzed populations. if the position of populations in the cluster analysis of bioclimatic factors matches the position obtained from morphometric characters, it may be concluded that such grouping is a result of impact by various bioclimatic factors and vice versa (k u z m a n o v i ć et al., 2011). as cluster analysis of bioclimatic factors have shown that the study area is strongly differentiated into three main clusters: one with semiarid temperate continental or subcontinental climate, another with continental mountain climate and the third cluster with humid mountain climate, it might be expected that the cluster analysis of morphological characters would also produce three main clusters. however, the analysis has shown that within the cluster analysis of morphological characters all populations were divided into four clusters (n i k o l i ć et al., 2015b), while the distribution of individual clades (populations) was not matched to the distribution of populations obtained in the cluster analysis of bioclimatic parameters. this type of distribution indicates that bioclimatic factors are not the single reason for such morphological differentiation of populations. results of the cluster analysis performed solely on mountain populations were similar. the effect of microclimatic differences caused by orography on morphological differentiation was avoided in this analysis, as all mountain populations inhabit open grassland habitats from classes festuco-brometea and elyno-seslerietea. although both cluster analyses, of morphological characters and bioclimatic parameters, have shown separation into two groups, the placement of individual populations into these groups is different, indicating that bioclimatic parameters are not crucial in differentiation of populations (n i k o l i ć et al., 2015b). regression analysis (linear regression) regression analysis showed that orographic factors (altitude, aspect and slope) influenced the morphological characters of the species j. heuffelii. the geological substrate was not analyzed, as this factor showed a high level of co-linearity with other factors (tab. 4). altitude had the greatest influence on the following morphological characters: length of rostrum, height of fruit, height of stylus, width of fruit, number of leaves in rosette, length of the longest branch in floral maturation stage, length of the longest filament. exposition aspect is also one of the key abiotic factors, with the greatest impact on morphological characters of reproductive organs (ovary stylus, fruit and nectary). the morphological differentiation of the analyzed populations is also highly influenced by the bioclimatic factors precipitation and temperature. the greatest correlation with the analyzed characters was shown by mean temperature of driest quarter (bio8) and temperature seasonality (bio4). these two bioclimatic factors have the highest influence on the same characters that are highly influenced by altitude. there is a somewhat smaller but still significant impact of precipitation seasonality (bio15), with the greatest impact on following characters: length of petal, height of ovary, height of the stem to the lowest flower branch, width of the nectary, width of the middle leaf on the stem, width of the largest leaf, length of the largest leaf, width of the central longitudinal fold (costa). there is a somewhat smaller but still significant impact of precipitation of warmest quarter (bio18) and mean temperature of driest quarter (bio9). biologica nyssana 6 (1)  september 2015: 1-9 nikolić, d. et al.  the influence of orographical and bioclimatic factors … 8 the geographic variability in plant morphology is the result of phenotypic changes expressed as an answer to local ecological conditions, genetic variability and evolution among the populations, as well as the biogeographic history of species (e l l i s o n et al., 2004). some of the morphological characteristics have genetic origins, for example leaf shape, but they may also be strongly influenced by local conditions in which the plants develop (t h o m p s o n , 1991; s c h l i c h t i n g & p i g l i u c c i , 1998). altitude is a factor with significant impact on variability of morphological characteristics of analyzed populations, as shown by regression analysis. change in altitude causes changes in other factors, including temperature, air humidity, precipitation and partial pressure of gasses in the atmosphere, causing the adaptive response in plants and therefore also changes in morphology and physiology (k ö r n e r , 1999). altitude may have a strong impact on both leaf morphology and physiology in individuals from various populations within the same species (h o v e n d e n & v a n d e r s c h o o r , 2004). with increase of altitude the length and width of leaves generally decrease (k ö r n e r et al., 1986), while thickness of leaves increases (k ö r n e r et al., 1989; r o d e r i c k et al., 2000). regression analysis has supported the hypothesis that altitude has the greatest impact on variability of morphological characters in j. heuffelii populations. conclusion the regression analysis has shown that morphological characteristics are under stronger influence by seasonal dynamics in temperature and precipitation than by the total amount of precipitation or mean annual temperature, which are the most commonly analyzed climatic factors for any geographic area. in addition to altitude, the orographic factors with the strongest impact on morphological characters of species j. heuffelii are exposition and slope of terrain. the bioclimatic parameters may also influence the variability in morphological characters. the greatest impact was recorded in mean temperature of wettest quartile (bio8) and temperature seasonality (bio4). they are mostly influencing the following characters: height and width of fruit, height of stylus, length of rostrum, number of leaves in the rosette, length of the longest flowering branch and length of longest filament of the stamen. the seasonal character of precipitation (bio15) was shown to influence size of leaves, height of stem and reproductive characters: length of petal, height of ovary and width of nectary. these results suggest that temperature conditions and precipitation may contribute to plant phenotype in various habitat types. this trend was also recorded in the populations from canyons and river gorges with c1 type of climate. populations with the highest value of bio15 parameter (seasonality of precipitation) show greater dimensions of leaves than the populations from localities with c2 and c3 types of climate, where values of bio15 parameter are lower. it remains to be determined if the large morphological variability of j. heuffelii populations in the territory of central balkans 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qosja et al., 1992; qosja et al., 1996; vangjeli etal., 2000) from the institute of biological research, academy of sciences, albania. the material for “flora of albania” is taken mainly by the national herbarium and includes over 150,000 specimens. many scientific publications of foreign and albanian botanists, who have carried out expeditions throughout albania, have been used. the territory of albania includes a wide range of coastal habitats from maritime limestone and sandstone cliffs to sand dunes, saline grasslands and brackish waters to mountains above 2000 m (barina et al., 2017). from year 2000 when the publication of the 4th and last volume of “flora of albania” was completed and onwards, a considerable number of taxa have been reported by many national and foreign authors. from the general analysis of all these studies it results that the albanian flora already consists of about 3,629 species which belong to 960 genera and 170 families (meço & mullaj, 2017). © 2022 saliaj et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 11 lamiaceae (mint family) has a cosmopolitan distribution (heywood et al., 2007). the enlarged lamiaceae contain about 236 genera (raymond et al., 2004). the largest genera are salvia (900), scutellaria (360), stachys (300), plectranthus (300), hyptis (280), teucrium (250), vitex (250), thymus (220), and nepeta (200) (raymond et al., 2004) (https://en.wikipedia.org/wiki/lamiaceae). the stems usually, but not exclusively, have a square cross-sectional. its flowers are bilaterally symmetrical with 5 petals and 5 sepals. lamiaceae are commonly bisexual and they appear to have one whorl of flowers even though they are comprised of two clusters. the leaves are either decussated (crossing one another) or whorled. these plants are most frequently shrubs or trees but could also be vines. this family includes the characteristically aromatic herbs used in cooking such as rosemary, oregano, thyme, basil, mint, marjoram, sage, and lavender (patwardhan & mashelkar, 2009). in the 3rd published volume of “flora of albania” lamiaceae family is recognized as labiatae family and includes 32 genera. the aim of this study is to bring an update of data on one of the large families of “flora of albania”, lamiaceae family, obtained from the use of modern methods of molecular analysis, research expeditions for the recognition and discovery of new species by albanian botanists or foreigners, by completing the lists of families or genera with newly discovered species, establishing current names if they have changed starting from family, genus and species, etc. materials and methods most of the data for the critical revision of lamiaceae family in albanian flora has been extracted from literature, such as the four volumes of “flora of albania” (paparisto et al., 1988; qosja et al., 1992; qosja et al., 1996; vangjeli et al., 2000), the five volumes of the “flora europaea” (tutin et al., 19641980), the medchecklist (greuter et al., 1984-1989) and the euro+med plantbase (http://ww2.bgbm. org/europlusmed/query.asp). euro+med plantbase has been selected as a reference for the taxa names. we have also referred, as an important study on the flora of albania, to the monograph “checklist of vascular plants of albania” (barina et al., 2018). also, to complete the study with the discoveries of recent years, all new species found in the territory of albania (year 2018-2022) and published in international scientific journals have been taken into consideration. additionally, the rest of the information is gathered through visiting the national herbarium of albania at the research center of flora and fauna. results and discussion after revising the family lamiaceae using the above methodology, it results that in albania from this family already exist 32 genera, but most of them with lot of changes that needs to be updated for the albanian flora. all the genera that have undergone major changes have been analyzed one by one. genus ajuga l. ajuga genus is represented by 11 taxa (tab. 1), of which 3 (2 types and 1 species) are not published in any of the volumes of “flora of albania”: 1. ajuga chamaepitys (l.) schreb. subsp. chamaepitys (barina et al., 2018) 2. ajuga pyramidalis l. subsp. pyramidalis (the euro+med plantbase, 2010) 3. ajuga laxmannii (l.) benth. (barina et al., 2018) genus ballota l. ballota genus is represented by 7 taxa (tab. 1), of which 2 have new accepted names by “the euro+med plantbase”. 1. ballota hispanica (l.) benth. in the 3rd volume of “flora of albania” this species is known as ballota rupestris (biv.) vis. 2. ballota nigra subsp. ruderalis (sw.) briq. this subspecies in the same volume of “flora of albania” is known as subspecies unicata. genus clinopodium l. this genus for albania brings many changes. first, according to the euro+med plantbase (http://ww2. bgbm.org/europlusmed/query.asp), the genera calamintha and acinos, which are present in the 3rd published volume of “flora of albania”, have already changed the name of genus and are part of the genus clinopodium. from the genus clinopodium in “flora of albania”, only two taxa are mentioned: clinopodium vulgare l., and clinopodium vulgare l. subsp. vulgare. meanwhile, with all the analysis done, already, 23 taxa exist for the genus clinopodium (tab. 1). 1. clinopodium acinos (l.) kuntze. known as acinos arvensis (lam.) dandyin “flora of albania” 3rd edition. 2. clinopodium alpinum (l.) kuntze. known as acinos alpinus (l.) moench in “flora of albania” 3rd edition. 3. clinopodium alpinum subsp. albanicum (kümmerle & jáv.) govaerts. new subspecies according to the euro+med plantbase 2010, not published in any of the volumes of “flora of albania”. 12 biologica nyssana ● 13 (1) september 2022: 11-25 saliaj et al. ● critical revision of lamiaceae family in albanian flora table 1. lamiaceae family in albanian flora genera species ajuga l. ajuga chamaepitys (l.) schreb. ajuga chamaepitys subsp. chia (schreb.) ajuga chamaepitys (l.) schreb. subsp. chamaepitys ajuga genevensis l. ajuga orientalis l. ajuga piskoi degen & bald. ajuga pyramidalis l. ajuga pyramidalis l. subsp. pyramidalis ajuga reptans l. ajuga laxmannii (l.) benth. ajuga iva (l.) schreb. ballota l. ballota hispanica (l.) benth. ballota macedonica vandas ballota nigra l. ballota nigra subsp. foetida hayek ballota nigra l. subsp. nigra. ballota nigra subsp. sericea (vandas) patzak. ballota nigra subsp. ruderalis (sw.) briq. clinopodium l. clinopodium acinos (l.) kuntze clinopodium alpinum (l.) kuntze clinopodium alpinum subsp. alpinum clinopodium alpinum subsp. albanicum (kümmerle & jáv.) govaerts clinopodium alpinum subsp. hungaricum (simonk.) govaerts clinopodium alpinum subsp. meridionale (nyman) govaerts clinopodium grandiflorum (l.) kuntze clinopodium grandiflorum (l.) kuntze subsp. grandiflorum clinopodium menthifolium (host) stace clinopodium menthifolium subsp. ascendens (jord.) govaerts clinopodium menthifolium (host) stace subsp. menthifolium clinopodium nepeta (l.) kuntze clinopodium nepeta (l.) savi subsp. nepeta clinopodium nepeta subsp. glandulosum (req.) govaerts clinopodium suaveolens (sm.) kuntze clinopodium serpyllifolium (m. bieb.) kuntze clinopodium thymifolium (scop.) kuntze clinopodium vardarense (šilic) govaerts clinopodium vulgare l. clinopodium vulgare subsp. orientale bothmer clinopodium vulgare l. subsp. vulgare biologica nyssana ● 13 (1) september 2022: 11-25 saliaj et al. ● critical revision of lamiaceae family in albanian flora 13 14 biologica nyssana ● 13 (1) september 2022: 11-25 saliaj et al. ● critical revision of lamiaceae family in albanian flora genera species clinopodium l. clinopodium graveolens subsp. rotundifolium (pers.) govaerts clinopodium suaveolens (sibth. & sm.) g. don. galeopsis l. galeopsis bifida boenn. galeopsis ladanum l. galeopsis pubescens besser galeopsis speciosa mill. galeopsis tetrahit l. glechoma l. glechoma hederacea l. glechoma hirsuta waldst. & kit. hyssopus l. hyssopus officinalis l. hyssopus officinalis subsp. aristatus (godr.) nyman. hyssopus officinalis l. subsp. officinalis lamium l. lamium amplexicaule l. lamium amplexicaule l. var. amplexicaule lamium galeobdolon (l.) crantz. lamium galeobdolon (l.) crantz subsp. galeobdolon lamium galeobdolon subsp. montanum (pers.) hayek lamium garganicum l. lamium garganicum l. subsp. garganicum lamium garganicum subsp. striatum (sm.) hayek lamium maculatum (l.) l. lamium purpureum l. lamium purpureum l. var. purpureum lamium bifidum cirillo lamium flexuosum ten. lamium orvala l. leonurus l. leonurus cardiaca l. lavandula l. lavandula angustifolia mill. lycopus l. lycopus europaeus l. lycopus exaltatus l. f. marrubium l. marrubium anisodon k. koch. marrubium cylleneum boiss. & heldr. marrubium incanum desr. marrubium peregrinum l. marrubium thessalum boiss. & heldr. marrubium vulgare l. marrubium × paniculatum desr. melissa l. melissa officinalis l. melissa officinalis subsp. altissima (sm.) arcang. melissa officinalis subsp. officinalis 15 biologica nyssana ● 13 (1) september 2022: 11-25 saliaj et al. ● critical revision of lamiaceae family in albanian flora genera species melittis l. melittis melissophyllum l. melittis melissophyllum subsp. albida (guss.) p. w. ball mentha l. mentha aquatica l. mentha longifolia (l.) l. mentha longifolia (l.) l. subsp. longifolia mentha pulegium l. mentha spicata l. mentha spicata subsp. condensata (briq.) greuter & burdet. mentha spicata l. subsp. spicata mentha × dumetorum schult. mentha × piperita l. mentha × rotundifolia (l.) huds. mentha suaveolens ehrh. micromeria benth. micromeria albanica (k. malý) šilic micromeria cremnophila boiss. & heldr. micromeria cremnophila boiss. & heldr. subsp. cremnophila micromeria cristata (hampe) griseb. micromeria cristata (hampe) griseb. subsp. cristata micromeria cristata subsp. kosaninii (šilic) ined. micromeria croatica (pers.) schott. micromeria graeca (l.) benth. micromeria graeca subsp. fruticulosa (bertol.) guinea micromeria juliana (l.) benth. ex rchb. micromeria parviflora rchb. micromeria microphylla (d'urv.) benth. micromeria myrtifolia boiss. & hohen. micromeria nervosa (desf.) benth. nepeta l. nepeta cataria l. nepeta nuda l. nepeta nuda l. subsp. nuda nepeta parnassica heldr. & sart. nepeta spruneri boiss. nepeta argolica baden. nepeta argolica baden subsp. malacotrichos (baden) a.strid & kit. tan. ocimum l. ocimum basilicum l. origanum l. origanum vulgare l. origanum vulgare subsp. hirtum (link) ietsw. origanum vulgare l. subsp. vulgare origanum vulgare l. subsp. viridulum (martrindonos) nyman. origanum majorana l. 16 biologica nyssana ● 13 (1) september 2022: 11-25 saliaj et al. ● critical revision of lamiaceae family in albanian flora genera species phlomis l. phlomis fruticosa l. phlomis herba-venti l. phlomis herba-venti l. subsp. herba-venti phlomis tuberosa l. prasium l. prasium majus l. prunella l. prunella albanica pénzes. prunella grandiflora (l.) scholler. prunella grandiflora (l.) scholler subsp. grandiflora prunella laciniata (l.) l. prunella vulgaris l. prunella vulgaris l. subsp. vulgaris prunella × intermedia link. prunella × surrecta dumort. rosmarinus l. rosmarinus officinalis l. rosmarinus officinalis l. subsp. officinalis salvia l. salvia amplexicaulis lam. salvia argentea l. salvia candidissima vahl. salvia fruticosa mill. salvia glutinosa l. salvia nemorosa l. salvia nemorosa l. subsp. nemorosa salvia officinalis l. salvia officinalis l. subsp. officinalis salvia pratensis l. salvia pratensis l. subsp. pratensis salvia ringens sm. salvia sclarea l. salvia tomentosa mill. salvia verbenaca l. salvia verticillata l. salvia verticillata l. subsp. verticillata salvia virgata jacq. salvia viridis l. salvia aethiopis l. salvia microphylla kunth. salvia splendens sellow ex schult. satureja l. satureja cuneifolia ten. satureja hortensis l. 17 biologica nyssana ● 13 (1) september 2022: 11-25 saliaj et al. ● critical revision of lamiaceae family in albanian flora genera species scutellaria l. satureja montana l. satureja montana l. subsp. montana satureja montana l. subsp. variegata (host) p.e.ball. satureja subspicata bartl. ex vis. satureja subspicata bartl. ex vis. subsp. subspicata satureja parnassica heldr. & sart. ex boiss. satureja kitaibelii wierzb. ex heuff. scutellaria alpina l. scutellaria altissima l. scutellaria columnae all. scutellaria columnae all. subsp. columnae scutellaria galericulata l. scutellaria orientalis l. scutellaria orientalis subsp. pinnatifida j. r. edm. scutellaria rupestris boiss. & heldr. scutellaria rupestris subsp. adenotricha (boiss. & heldr.) greuter & burdet. scutellaria hastifolia l. scutellaria rubicunda hornem. scutellaria rubicunda hornem. subsp. rubicunda sideritis l. sideritis montana l. sideritis montana l. subsp. montana sideritis raeseri boiss. & heldr. sideritis raeseri boiss. & heldr. subsp. raeseri sideritis romana l. sideritis romana l. subsp. romana sideritis romana l. subsp. purpurea (talbot ex benth.) sideritis scardica griseb. stachys l. stachys albanica markgr. stachys alopecuros (l.) benth. stachys alopecuros (l.) benth. subsp. alopecuros stachys alpina l. stachys alpina l. subsp. alpina stachys alpina l. subsp. dinarica murb. stachys anisochila vis. & pancic. stachys annua (l.) l. stachys annua (l.) l. subsp. annua stachys arvensis (l.) l. stachys atherocalyx k. koch. stachys beckeana dörfl. & hayek stachys cretica l. genera species stachys l. stachys cretica subsp. cassia (boiss.) rech. f. stachys cretica l. subsp. cretica stachys cretica subsp. salviifolia (ten.) rech. f. stachys cretica subsp. bulgarica rech. f. stachys germanica l. stachys germanica l. subsp. germanica stachys germanica subsp. heldreichii (boiss.) hayek stachys germanica subsp. penicillata (heldr. & sart. ex boiss.) nyman stachys maritima gouan. stachys menthifolia vis. stachys mollissima willd. stachys obliqua waldst. & kit. stachys officinalis (l.) trevis. stachys officinalis (l.) trevis. subsp. officinalis stachys officinalis subsp. skipetarum jáv. stachys palustris l. stachys recta l. stachys recta subsp. baldaccii (k. malý) hayek. stachys recta subsp. doerfleri (hayek) hayek. stachys recta l. subsp. recta stachys recta subsp. subcrenata (vis.) briq. stachys recta subsp. labiosa (bertol.) briq. stachys scardica (griseb.) hayek. stachys serbica pancic. stachys sericophylla halácsy. stachys spinulosa sm. stachys sylvatica l. stachys tymphaea hausskn. stachys canescens bory & chaub. stachys parolinii vis. stachys plumosa griseb. teucrium l. teucrium arduinii l. teucrium capitatum l. teucrium capitatum l. subsp. capitatum teucrium chamaedrys l. teucrium chamaedrys l. subsp. chamaedrys teucrium flavum l. teucrium flavum l. subsp. flavum teucrium montanum l. teucrium montanum l. subsp. montanum 18 biologica nyssana ● 13 (1) september 2022: 11-25 saliaj et al. ● critical revision of lamiaceae family in albanian flora 19 biologica nyssana ● 13 (1) september 2022: 11-25 saliaj et al. ● critical revision of lamiaceae family in albanian flora genera species thymbra l. teucrium scordium l. teucrium scordium subsp. scordioides (schreb.) arcang. teucrium polium l. teucrium fruticans l. teucrium divaricatum sieber ex heldr. teucrium botrys l. thymbra capitata (l.) cav. thymus l. thymus boissieri halácsy. thymus doerfleri ronniger. thymus longicaulis c. presl. thymus longicaulis subsp. chaubardii (rchb. f.) jalas. thymus longicaulis c. presl subsp. longicaulis thymus parnassicus halácsy. thymus praecox opiz. thymus praecox subsp. jankae (čelak.) jalas. thymus praecox subsp. polytrichus (a. kern. ex borbás) jalas. thymus praecox subsp. zygiformis (heinr. braun ex wettst.) jalas. thymus pulegioides l. thymus pulegioides subsp. montanus (benth.) ronniger. thymus pulegioides l. subsp. pulegioides thymus sibthorpii benth. thymus striatus vahl. thymus teucrioides boiss. & spruner. thymus teucrioides boiss. & spruner subsp. teucrioides thymus thracicus velen. thymus × oblongifolius opiz. thymus ciliatopubescens (halácsy) halácsy. thymus dacicus borb. thymus degenii heinr. braun. thymus odoratissimus mill. thymus heterotrichus griseb. thymus roegneri k. koch thymus leucotrichus halácsy. thymus serpyllum l. thymus stojanovii degen. vitex l. vitex agnus-castus l. ziziphora l. ziziphora capitata l. ziziphora capitata l. subsp. capitata 4. clinopodium alpinum subsp. hungaricum (simonk.) govaerts. known as acinos alpinus subsp. majoranifolius (mill.) p.w.ball in “flora of albania”. biologica nyssana ● 13 (1) september 2022: 11-25 20 saliaj et al. ● critical revision of lamiaceae family in albanian flora 5. clinopodium alpinum subsp. meridionale (nyman) govaerts. in “flora of albania” 3rd edition it’s known as acinos alpinus subsp. meridionalis (nyman) p.w.ball. 6. clinopodium alpinum subsp. alpinum (not published in any of the volumes of “flora of albania”), according to barina et al., (2018), it’s present in albania under the genus acinos). 7. clinopodium grandiflorum (l.) kuntze. known as calamintha grandiflora (l.) moench. in “flora of albania” 3rd edition. 8. clinopodium grandiflorum (l.) kuntze subsp. grandiflorum. new subspecies according to the euro+med plantbase 2010, not published in any of the volumes of “flora of albania”. 9. clinopodium menthifolium (host) stace. known as calamintha sylvatica bromf. in “flora of albania”. 10. clinopodium menthifolium subsp. ascendens (jord.) govaerts. in “flora of albania” 3rd edition it’s known as calamintha sylvatica bromf. subsp. ascendens (jord.) p.w.ball. 11. clinopodium menthifolium (host) stace subsp. menthifolium. new subspecies according to the euro+med plantbase 2010, not published in any of the volumes of “flora of albania”. 12. clinopodium nepeta (l.) kuntze. known as calamintha nepeta (l.) savi (l.) moench in “flora of albania”. 13. clinopodium nepeta subsp. glandulosum (req.) govaerts. in “flora of albania” it’s known as calamintha nepeta (l.) savi subsp. glandulosa (req.) p.w.ball. 14. clinopodium nepeta (l.) savi subsp. nepeta. in “flora of albania” it’s known as calamintha nepeta (l.) savi subsp. nepeta. 15. clinopodium suaveolens (sm.) kuntze. new species according to the euro+med plantbase 2010, not published in any of the volumes of “flora of albania”. 16. clinopodium thymifolium (scop.) kuntze. in “flora of albania” it is known as micromeria thymifolia (scop.) fritsch. 17. clinopodium vardarense (icilic) govaerts. known as satureja vardarensis (šilić) greuter & burdet in “flora of albania” 3rd volume. 18. clinopodium vulgare subsp. orientale bothmer. known as clinopodium vulgare subsp. arundanum boiss. in “flora of albania”. 19. clinopodium serpyllifolium (m. bieb.) kuntze. it’s not published in any of the volumes of “flora of albania”, but according to barina et al. (2018), it’s known as micromeria fruticosa (l.) druce subsp. serpyllifolia (m.bieb.) p.h.davis. 20. clinopodium graveolens subsp. rotundifolium (pers.) govaerts. it’s not published in any of the volumes of “flora of albania”. according to barina et al. (2018), it is present in albania under the name acinos rotundifolius pers. 21. clinopodium suaveolens (sibth. & sm.) g. don. it’s not published in any of the volumes of “flora of albania”. according to barina et al. (2018), it’s present in albania under the genus acinos. genus hyssopus l. hyssopus genus consists in 1 species and 2 subspecies (tab. 1), of which in “flora of albania” only the species hyssopus officinalis l. is found, without its type and the other subspecies. 1. hyssopus officinalis subsp. aristatus (godr.) nyman (the euro+med plantbase, 2010). 2. hyssopus officinalis l. subsp. officinalis (the euro+med plantbase, 2010). genus lamium l. even the genus lamium has undergone many taxonomic changes over the last few years. it is now accepted that this genus includes 14 taxa (tab. 1), of which 8 taxa are not found published in any of the volumes of “flora of albania”. 1. lamium amplexicaule l. var. amplexicaule (the euro+med plantbase, 2010). 2. lamium galeobdolon (l.) crantz. in “flora of albania” 3rd edition it’s known as lamiastrum galeobdolon (l.), so the name of the genus has changed from lamiastrum to lamium. 3. lamium galeobdolon (l.) crantz subsp. galeobdolon. in “flora of albania” 3rd edition it is known as lamiastrum galeobdolon (l.) subsp. galeobdolon, so the name of the genus has changed from lamiastrum to lamium. 4. lamium galeobdolon subsp. montanum (pers.) hayek. in “flora of albania” 3rd edition it is known as lamiastrum galeobdolon (l.) subsp. montanum, so the name of the genus has changed from lamiastrum to lamium. 5. lamium garganicum subsp. striatum (sm.) saliaj et al. ● critical revision of lamiaceae family in albanian florabiologica nyssana ● 13 (1) september 2022: 11-25 21 hayek (the euro+med plantbase, 2010, barina et al., 2018). 6. lamium purpureum l. var. purpureum (the euro+med plantbase, 2010). 7. lamium flexuosum ten. (barina et al., 2018). 8. lamium orvala l. this species is most likely missing in albania and its presence needs further confirmation. according to barina et al. (2018), the presence of this species is reported only once (hayek, 1917). genus marrubium l. this genus in albania is already represented by 7 species (tab. 1), of which 3 are not found in “flora of albania” published editions. 1. marrubium anisodon k. koch. in the 3rd volume of “flora of albania”, it is known as marrubium alternidens rech. 2. marrubium thessalum boiss. & heldr. (barina et al., 2018). 3. marrubium × paniculatum desr. this hybrid species is most likely missing in albania and its presence needs further confirmation. according to barina et al. (2018), the presence of this species is reported only once (schütt, ~1939). genus mentha l. mentha genus has also undergone many taxonomic changes. it is now accepted that 11 taxa belong to this genus (tab. 1), of which 5 (2 types, 1 subspecies and 2 hybrid species) are not found in published editions of “flora of albania”. 1. mentha longifolia (l.) l. subsp. longifolia (the euro+med plantbase, 2010) 2. mentha spicata subsp. condensata (briq.) greuter & burdet. new name for albania according to the euro+med plantbase, in “flora of albania” 3rd edition, it’s known as mentha microphylla k. koch. 3. mentha spicata l. subsp. spicata (the euro+med plantbase, 2010) 4. mentha × dumetorum schult. the presence of this hybrid species in albania needs further confirmation, because according to barina et al. (2018), its presence is reported only once (schütt ,~1939). 5. mentha × rotundifolia (l.) huds. (barina et al., 2018) genus micromeria benth. the genus micromeria is already represented by 14 taxa (tab. 1), of which 8 taxa are not found in the “flora of albania”. 1. micromeria albanica (k. malý) šilic. (barina et al., 2018) 2. micromeria cremnophila boiss. & heldr. subsp. cremnophila (the euro+med plantbase, 2010) 3. micromeria cristata (hampe) griseb. subsp. cristata. according to barina et al. (2018), the presence of this subspecies in albania needs further confirmation, because it is reported only once (greuter et al., 1986). 4. micromeria cristata subsp. kosaninii (šilic) ined. (barina et al., 2018) 5. micromeria croatica (pers.) schott. (barina et al., 2018) 6. micromeria graeca subsp. fruticulosa (bertol.) guinea. new subspecies according to the euro+med plantbase 2010. 7. micromeria microphylla (d’urv.) benth. (barina et al., 2018) 8. micromeria nervosa (desf.) benth. (barina et al., 2018) genus nepeta l. nepeta genus is represented by 7 taxa (tab. 1), of which only 3 (1 type, 1 species and 1 subspecies) are not found in the volumes of “flora of albania”. 1. nepeta nuda l. subsp. nuda (the euro+med plantbase, 2010) 2. nepeta argolica baden (barina et al., 2018) 3. nepeta argolica baden subsp. malacotrichos (baden) a. strid & kit. tan. (barina et al., 2018) genus origanum l. in albania this genus is represented by 5 taxa (tab. 1), of which only 3 taxa (1 type and 2 subspecies) are not found in the “flora of albania” published editions. 1. origanum vulgare subsp. hirtum (link) ietsw. (barina et al., 2018) 2. origanum vulgare l. subsp. vulgare (barina et al., 2018) 3. origanum vulgare l. subsp. viridulum (martrindonos) nyman. (barina et al., 2018) genus phlomis l. in albania, nowadays, this genus is represented by 4 taxa (tab. 1), of which 2 (1 type and 1 species) are not published in “flora of albania”. 1. phlomis herba-venti l. subsp. herba-venti (the euro+med plantbase, 2010) 2. phlomis tuberosa l. (barina et al., 2018) biologica nyssana ● 13 (1) september 2022: 11-25 saliaj et al. ● critical revision of lamiaceae family in albanian flora genus prunella l. the genus prunella is already represented by 8 taxa (tab. 1), of which 5 (1 species, 2 types and 2 hybrid species) are not found in “flora of albania” published volumes. 1. prunella albanica pénzes (the euro+med plantbase, 2010) 2. prunella grandiflora (l.) scholler subsp. grandiflora (the euro+med plantbase, 2010) 3. prunella vulgaris l. subsp. vulgaris (the euro+med plantbase, 2010) 4. prunella × intermedia link. (barina et al., 2018) 5. prunella × surrecta dumort. according to barina et al. (2018), for this hybrid species there is lack of information (no reported location), so it may need further confirmation. genus rosmarinus l. in albania this genus already has as representative 1 species (rosmarinus officinalis l.) also found in “flora of albania”, but its type rosmarinus officinalis l. subsp. officinalis is missing (the euro+med plantbase, 2010). genus salvia l. salvia genus has 22 representative taxa found in albania (tab. 1), of which 8 (4 types and 4 species) are not published in the volumes of “flora of albania”. 1. salvia fruticosa mill. in “flora of albania” 3rd edition, it’s known as salvia triloba l.f. 2. salvia nemorosa l. subsp. nemorosa (the euro+med plantbase, 2010) 3. salvia officinalis l. subsp. officinalis (the euro+med plantbase, 2010) 4. salvia pratensis l. subsp. pratensis (the euro+med plantbase, 2010) 5. salvia tomentosa mill. new name for albania (the euro+med plantbase), in “flora of albania”, it’s known as salvia grandiflora etl. 6. salvia verticillata l. subsp. verticillata (the euro+med plantbase, 2010) 7. salvia aethiopis l. (barina et al., 2018) 8. salvia microphylla kunth. according to barina et al. (2018), this species is only found cultivated in albania. genus satureja l. the genus satureja in albania is represented by 9 taxa (tab. 1), of which 3 (2 types and 1 species) are not published in “flora of albania” and 2 species come with new accepted names by “the euro +med plantbase”. 1. satureja kitaibelii wierzb. ex heuff. new name for albania, in “flora of albania” 3rd volume, this species is known as satureja montana l. subsp. kitaibelii (wierzb.) p.w.ball. 2. satureja montana l. subsp. variegata (host) p.w.ball. (barina et al., 2018) 3. satureja subspicata bartl. ex vis. new name for albania (the euro+med plantbase), in “flora of albania” 3rd volume, this species is known as satureja montana l. subsp. illyrica. 4. satureja subspicata bartl. ex vis. subsp. subspicata (the euro+med plantbase, 2010) 5. satureja parnassica heldr. & sart. ex boiss. according to barina et al., 2018, the presence of this species in albania is mentioned only once (kárpáti & kárpáti, 1961), so it may need further confirmation. genus scutellaria l. this genus in albania is represented by 12 taxa (tab. 1), of which 3 (1 type and 2 subspecies) are not published in the “flora of albania” books and one has a new updated name according to “the euro+med plantbase”. 1. scutellaria columnae all. subsp. columnae (the euro+med plantbase, 2010) 2. scutellaria orientalis subsp. pinnatifida j. r. edm. (the euro+med plantbase, 2010, barina et al., 2018) 3. scutellaria rupestris boiss. & heldr. new name for albania, in “flora of albania” 3rd edition, this species is known as scutellaria rubicunda hornem. subsp. rupestris (boiss. & heldr.). 4. scutellaria rupestris subsp. adenotricha (boiss. & heldr.) greuter & burdet. (the euro+med plantbase, 2010) genus sideritis l. sideritis genus in albania is represented by 8 taxa (tab. 1), of which only 2 types are not published in “flora of albania”. 1. sideritis montana l. subsp. montana (the euro+med plantbase, 2010) 2. sideritis raeseri boiss. & heldr. subsp. raeseri (the euro+med plantbase, 2010) genus stachys l. the genus stachys has the largest number of species in the lamiaceae family. this genus has a total of 44 22 biologica nyssana ● 13 (1) september 2022: 11-25 saliaj et al. ● critical revision of lamiaceae family in albanian flora taxa (tab. 1), of which 16 (5 types, 8 subspecies and 3 species) are not found in “flora of albania” books and one has changed its name. 1. stachys alopecuros (l.) benth. subsp. alopecuros (the euro+med plantbase, 2010) 2. stachys alpina l. subsp. alpina (barina et al., 2018) 3. stachys alpina l. subsp. dinarica murb. (barina et al., 2018) 4. stachys annua (l.) l. subsp. annua (the euro+med plantbase, 2010) 5. stachys cretica subsp. cassia (boiss.) rech. f. (barina et al., 2018) 6. stachys cretica l. subsp. cretica. the presence of this subspecies in albania needs further confirmation due to lack of records (barina et al., 2018) 7. stachys cretica subsp. salviifolia (ten.) rech. f. (barina et al., 2018) 8. stachys cretica subsp. bulgarica rech.f. (barina et al., 2018) 9. stachys germanica subsp. penicillata (heldr. & sart. ex boiss.) nyman. (the euro+med plantbase, 2010) 10. stachys mollissima willd. new name for albania, in “flora of albania” 3rd book, this species is known as stachys decumbens pers. 11. stachys officinalis (l.) trevis. subsp. officinalis (the euro+med plantbase, 2010) 12. stachys officinalis subsp. skipetarum jáv. (barina et al., 2018) 13. stachys recta subsp. baldaccii (k. malý) hayek. (barina et al., 2018) 14. stachys recta subsp. doerfleri (hayek) hayek. (barina et al., 2018) 15. stachys canescens bory & chaub. (barina et al., 2018) 16. stachys parolinii vis. (barina et al., 2018) 17. stachys plumosa griseb. (barina et al., 2018) genus teucrium l. this genus is represented by 15 taxa (tab. 1), of which 6 (4 types and 2 species) are new to albanian flora and one has a new updated name according to “the euro+med plantbase”. 1. teucrium capitatum l. new name for albania, in “flora of albania” 3rd book, this species is known as subspecies: teucrium polium l. subsp. capitatum (l.) arcang. so it has passed from subspecies to species. 2. teucrium capitatum l. subsp. capitatum (the euro+med plantbase, 2010) 3. teucrium chamaedrys l. subsp. chamaedrys (the euro+med plantbase, 2010) 4. teucrium flavum l. subsp. flavum (the euro+med plantbase, 2010) 5. teucrium montanum l. subsp. montanum (the euro+med plantbase, 2010) 6. teucrium divaricatum sieber ex heldr. (the euro+med plantbase, 2010) 7. teucrium botrys l. according to barina et al. (2018), the presence of this species in albania is mentioned only once (demiri, 1983), so it may need further confirmation. genus thymbra l. this genus is mentioned only by the euro+med plantbase for the distribution of one species in albania. this genus is not found in any of the 4 published books of “flora of albania” thymbra capitata (l.) cav. in “flora of albania” the species is known as thymus capitata l. genus thymus l. this is also a genus that consists in a large number of taxa, a total of 28 (tab. 1), of which 19 taxa are new for albanian flora and 2 of the species come with new accepted names according to “the euro+med plantbase”. 1. thymus boissieri halácsy. in “flora of albania” 3rd volume, this species is known as thymus cherlerioides. 2. thymus doerfleri ronniger. (barina et al., 2018) 3. thymus longicaulis subsp. chaubardii (rchb. f.) jalas. (the euro+med plantbase, 2010) 4. thymus longicaulis c. presl subsp. longicaulis (the euro+med plantbase, 2010) 5. thymus parnassicus halácsy (the euro+med plantbase, 2010) 6. thymus praecox subsp. jankae (.elak.) jalas. in “flora of albania” 3rd volume, this species is known as thymus praecox subsp. scorpilii (vele.) jalas. 7. thymus pulegioides subsp. montanus (benth.) ronniger (the euro+med plantbase, 2010) 8. thymus pulegioides l. subsp. pulegioides (the euro+med plantbase, 2010) 9. thymus sibthorpii benth. (barina et al., 2018) 10. thymus teucrioides boiss. & spruner subsp. teucrioides (the euro+med plantbase, 23 biologica nyssana ● 13 (1) september 2022: 11-25 saliaj et al. ● critical revision of lamiaceae family in albanian flora 2010) 11. thymus thracicus velen. (barina et al., 2018) 12. thymus × oblongifolius opiz. according to barina et al. (2018), the presence of this hybrid species in albania is reported only once (jávorka, 1922), so it may need further confirmation. 13. thymus ciliatopubescens (halácsy) halácsy (barina et al., 2018) 14. thymus dacicus borb. according to barina et al. (2018), the presence of this species in albania is reported only once (hayek, 1924), so it may need further confirmation. 15. thymus degenii heinr. brown. according to barina et al. (2018), the presence of this species in albania is reported only once (jávorka, 1926), so it may need further confirmation. 16. thymus odoratissimus mill. according to barina et al. (2018), the presence of this species in albania is reported only once (greuter et al., 1986), so it may need further confirmation. 17. thymus heterotrichus griseb. (barina et al., 2018) 18. thymus roegneri k. koch. according to the euro+med plantbase 2010, this is the updated and accepted name for thymus hirsutus m. bieb. 19. thymus leucotrichus halácsy. according to barina et al. (2018), the presence of this species in albania is confirmed since 1933: (hayek (1933), rechinger (1939), mitrushi (1966), meyer (2011), rakaj et al. (2013), vangjeli (2015). 20. thymus serpyllum l. according to barina et al. (2018), the presence of this species in albania is reported only once (heldreich, 1879), so it may need further confirmation. 21. thymus stojanovii degen. the presence of this species in albania needs further confirmation. it is reported by barina et al. (2018) referred to other references: grimus (1871), bornmüller (1937), mitrushi (1966), tutin et al. (1972), qosja (1973), demiri (1983), greuter et al. (1986) and ball (2011) . some of the genera with a small number of species/subspecies have not undergone any taxonomic change over the years and are found the same in “flora of albania”, such as galeopsis l., glechoma l., leonurus l., lavandula l., lycopus l., melissa l., melittis l., ocimum l., and prasium l. vitex l. genus in the 3rd published edition “flora of albania” is not part of labiatae family, but is found under verbenaceae family (qosja et al., 1996). ziziphora l. genus has 2 representative taxa in albania, 1 species and 1 subspecies/the type, of which only the species (ziziphora capitata l.) is published in “flora of albania”, not its type (ziziphora capitata l. subsp. capitata). after finishing the revision of lamiaceae family for the flora of albania, referring all the time to the only documented books (4 volumes of “flora of albania”), a lot of changes should be highlighted. lamiaceae family is found in the 3rd volume of “flora of albania” with the name “labiatae” and has a total of 32 genera (qosja et al., 1996). lamiaceae family in albania (following the above methodology) is represented by a total of 32 genera and 276 taxa (species/subspecies/varieties) (barina et al., 2018). two genera: acinos miller and calamintha miller do not exist so far, this two genera are part now of clinopodium l genus. also lamiastrum heister ex fabr., genus does not exist anymore, it is now placed under the lamium l. genus. two new genera belong now to this family, thymbra and vitex, both with one representative species in albania. (http://ww2.bgbm.org/europlusmed/ query.asp). from 276 taxa of this family only 143 are also found unchanged like in the 3rd edition of “flora of albania”, whereas 133 taxa are new for the flora (with changed species/subspecies name or not even found in any of “flora of albania” published volumes). the total composition of lamiaceae family in albanian flora is found in tab. 1. references barina z., somogyi g., pifko d., rakaj m. 2018: checklist of vascular plants of albania. phytotaxa, 378(1): 1-339. barina, z., mullaj, a., pifkó, d., somogyi, g., meco, m., rakaj, m. 2017: distribution maps. – in: barina, z. (ed.): distribution atlas of vascular plants in albania. hungarian natural history museum. budapest. pp. 47–445. greuter, w., von raab-straube, e. (eds) 2008: med-checklist: a critical inventory of vascular plants of the circum-mediterranean countries. dicotyledones. optima secretariat. palermo. harley, r. m., atkins, s., budantsev, a. l., cantino, p. d., conn, b. j., grayer, r., harley, m. m., de kok, r., krestovskaja, t., morales, r., paton, a. j., ryding, o., upson, t. 2004: labiatae. in: kubitzki, k. (ed.): the families and genera of 24 biologica nyssana ● 13 (1) september 2022: 11-25 saliaj et al. ● critical revision of lamiaceae family in albanian flora vascular plants, vii. springer. germany. heywood, v. h., brummitt, r. k., culham, a., seberg, o. 2007: flowering plant families of the world. firefly books. canada. ontario. meço, m., mullaj, a. 2015: phenological aspects of albanian flora. proceedings of international conference on soil. agricultural university of tirana. paparisto, k., demiri, m., mitrushi, i., qosja, xh. 1988: flora e shqipërisë, 1. academy of sciences, center of biological research. albania. patwardhan, b., mashelkar, r. a. 2009: traditional medicine-inspired approaches to drug discovery: can ayurveda show the way forward? drug discovery today, 14(15-16): 804-811. qosja, xh., paparisto, k., demiri, i., vangjeli, j., balza, e. 1992: flora e shqipërisë, 2. academy of sciences, center of biological research. albania. qosja, xh., paparisto, k., vangjeli, j., ruci, b., mullaj, a. 1996: flora e shqipërisë, 3. academy of sciences, center of biological research. albania. the euro+med plantbase (https://ww2.bgbm.org/ europlusmed/ptaxondetail.asp?nameid=111505& ptreffk=8000000) vangjeli, j., ruci, b., mullaj, a., qosja, xh., paparisto, k. 2000: flora e shqipërisë, 4. academy of sciences, center of biological research. albania. 25 mohammadi et al. 2020, biologica nyssana 11(1) 11 (1) september 2020: 45-54 doi: 10.5281/zenodo.4060294 a real-time pcr assay for evaluation of drought tolerance in a new tea clone original article narjes mohammadi department of biology, faculty of science, university of guilan, rasht, iran mohammadi_narges32@yahoo.com akbar norastehnia department of biology, faculty of science, university of guilan, rasht, iran norasteh@guilan.ac.ir (corresponding author) mohannad m. sohani department of biology, faculty of science, university of guilan, rasht, iran msohani@guilan.ac.ir korosh falakroo tea research institute, lahijan, iran k.falakro@areo.ir received: september 14, 2019 revised: february 24, 2020 accepted: march 23, 2020 abstract: drought stress induces oxidative stress with subsequent increases in reactive oxygen species ros in plants. to evaluate oxidative stress intensity with re-irrigation, changes in superoxide dismutase (sod) expression were investigated by real-time pcr in two tea clones, dn and 100. results showed that sod expression and other measured factors, except for carotenoids, increased under drought stress in clone dn, but the changes were not significant in clone 100, compared to control. all of the meaningful increases in evaluated enzymatic and non-enzymatic antioxidant factors were eliminated with re-irrigation. the observed changes indicate that clone dn activated its antioxidant defense system in response to drought. in contrast, the lack of response in clone 100 indicates higher levels of tolerance of this new clone against drought. key words: drought, oxidative stress, proline, superoxide dismutase, tea apstract: ukupan sadržaj fenola i antioksidativni potencijal različitih varijeteta brassica oleracea stres izazvan sušom indukuje oksidativni stres sa naknadnim povećanjem ros-a u biljkama. da bi se procenio intenzitet oksidativnog stresa ponovnim navodnjavanjem, ispitivane su promene ekspresije superoksid dismutaze (sod) pomoću pcr-a u realnom vremenu u 2 čajna klona dn i 100. rezultati su pokazali da ekspresija sod-a i drugih merenih faktora, osim karotenoida, povećava stres izazvan sušom u klonu dn, dok promene nisu bile značajne u klonu 100, u poređenju sa kontrolom. sva značajna povećanja procenjenih enzimskih i neenzimskih antioksidativnih faktora eliminisana su ponovnim navodnjavanjem. uočene promene pokazuju da je klon dn aktivirao svoj antioksidativni odbrambeni sistem kao odgovor na sušu. suprotno tome, nedostatak odgovora u klonu 100 ukazuje na viši nivo tolerancije ovog novog klona na sušu. ključne reči: suša, oksidativni stres, prolin, superoksid dizmutaza, čaj introduction plants are often exposed to many stresses, such as drought, high temperatures, and high salt levels, heavy metals and finally oxidative stress. drought is one of the environmental stresses which has harmful effects on all stages of growth and triggers the production of ros in plants (farooq et al., 2009). the amount of damage caused by water shortage depends on the plant species, genotype, duration and intensity of water deficit, soil characteristics, age and developmental stage of the plant (sánchez et al., 2001). in addition to the physiological changes that occur in plants due to water shortages, stress also inhibits photochemical activity and reduces calvin cycle activity (monakhova & chernyad’ev, 2004) accumulation of ros is one of the many biochemical changes which occurs in plants under stress, as an inevitable and normal product of cell metabolism and oxidative damage caused by ros is the major limiting factor for plant growth (allen & ort, 2001). while there are several sources of ros production, and whereas some of the most common sources of ros are the electron transport chains in mitochondria and chloroplasts, chloroplasts are the main source of ros production in plants (noctor et al., © 2020 mahammadi et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 45 2014). plants respond to increased ros amounts with activation of their enzymatic and non-enzymatic defense systems (zhu, 2001; mukhopadhyay ,2014). among the most important crops subjected to stress in iran is tea. the total area under tea cultivation in the north of iran is over 40 thousand hectares. 4-4,5 percent of the total tea in the world is produced in iran. it is essential to elevate the amount of product produced per unit to match the increasing consumption of tea. one important strategy for achieving this goal is reducing the environmental damage caused by stresses such as dehydration. although most tea gardens in iran are located in areas with high rainfall, reduced crop yields are nevertheless encountered due to the unequal distribution of rainfall in different seasons, especially in the growing season. research has shown that in areas where the annual rainfall is less than 1150 mm, tea plant growth is reduced due to stress. to reduce losses due to water deficits, varieties should be carefully selected to resist this stress, because damage to the plant is dependent greatly on the plants’ stress tolerance. the first intracellular line of defense is sod. phospholipid membranes are non-permeable to o2 (asada, 2006). it is therefore essential that sod be present to remove them in places where these compounds are formed. based on required metal co-factors, sod enzymes are classified into 4 groups: fesod, mn-sod, cu/zn-sod, and ni-sod. these isoforms are located in different cellular compartments: fe-sod is in chloroplasts as is cu/zn-sod, the latter of which is also present in cytosol and extracellular spaces. mn-sod is found in mitochondria and peroxisomes (jamal et al., 2006), and the fourth isoform of sod, ni-sod, which contains nickel in its active site has been found in several types of soil bacteria (bannister et al., 1987; bowler et al., 1992). many attempts have been made to produce transgenic plants, variously using all 3 isoforms of sod enzymes. most of these reports have noted that sod over-expression led to increased tolerance towards oxidative stress (faize et al., 2011). however, only in transgenic plants containing mn-sod protection was induced against, for example, reduced biomass and leaf damage (van breusegem et al., 1999; samis et al., 2002). many studies have been consistent with this data and mn-sod has been purified from pea (sevilla et al., 1980; palma et al., 1998), corn (baum & scandalios, 1981), pine (streller et al., 1994) and tea under cold stress (vyas & kumar, 2005). however, research has not been carried out on the role of this isoform in the improvement of drought tolerance in tea. in light of the results in other plants, mn-sod is expected to play a crucial role in the improvement of drought tolerance in tea. hence, in this study, the role of this isoform in resistance to drought was examined. complex responses of plants to abiotic stresses are manifested in the expression of multiple genes and various biochemical and molecular pathways. understanding these differences will lead to the identification of genes and molecular processes that play roles in a variety of conditions within biological systems (moody, 2001). in this study, the expression levels of sod transcripts were measured by real-time pcr. to evaluate the adaptation capacity of the two clones investigated 100 and dn against oxidative stress caused by drought, some indicators of oxidative stress, such as malondialdehyde (mda), carotenoid and proline content, chlorophyll a and total chlorophyll were also investigated. material and methods sampling two selected genotypes belonging to the tea research center, namely dn and 100, were used in this study. farm sampling was done from 8th july till 25th july for 17 days at the fashalam research station, iran. the experiment was arranged with a randomized complete block design with 3 replicates. 10-year-old vegetatively propagated tea plants (70-80 cm, height) were used under farm conditions (at ~24-33°c day/night and relative humidity of 75-80%) at longitude and latitude 49°55ʹ10ʺ and 37°8ʹ20ʺ, respectively. soil was sandy-loam with ph 5.8 and electric conductivity = 270. based on data obtained previously, a 10-day drought stress was imposed on the plants. this was followed by a one week irrigation period (twice a week, according to the usual practice of the research station) to exit from the stress condition. soil moisture content (smc) was measured on days 0, 5, 10 and 15th after withholding water began (black, 1965). sampling from the third terminal leaf was carried out in 2 steps, at day 10 and day 17. these samples provided data showing the effects of drought stress and exit from drought conditions, respectively. leaves were moved in liquid nitrogen and kept at -70 °c until needed. total rna extraction fresh leaf tissue, 0.1 g, was used for rna extraction according to kit instructions (cinnagen co.), with slight modification. because high levels of secondary metabolites in tea prevent high-quality rna extraction, isoamyl chloroform (1:24) was used to minimize the deposition of these compounds in rna isolates, and sodium acetate (2 m) was applied to optimize rna precipitation. after extraction, the resulting pellet was dissolved in 50 μl depc-treated water and maintained at -70 °c. to determine the 46 biologica nyssana ● 11 (1) september 2020: 45-54 mohammadi et al. ● a real-time pcr assay for evaluation of drought tolerance in a new tea clone 47 biologica nyssana ● 11 (1) september 2020: 45-54 mohammadi et al. ● a real-time pcr assay for evaluation of drought tolerance in a new tea clone quality of the extracted rna, 1% agarose gel electrophoresis was used. to determine the quantity and the degree of contamination of the rna, absorbance was read at a260/280 and a260/230 nm. rnas concentration in µg/µl was calculated as: cdna synthesis by the protocol included with the accupower rt premix kit (bioneer), one microgram of rna was used for cdna synthesis. first, 1 µg of rna was mixed with 0.5 µg of oligo dt primer and the mixture was incubated at 70 °c for 5 min to complete annealing. the product was then transferred to accupower rt premix microtube and the volume was brought to 20 µl with depc-treated water. this was vortexed for a few seconds and placed for 60 min at 42 °c. finally, the mixture was placed at 94 °c for 5 min and the synthesized cdna was maintained at -20 °c. primer design primers used for mn-sod gene and the 18s rrna reference gene were designed using oligo7 software (molecular biology insights, inc) (tab. 1). amplifications were performed in a total reaction volume of 25 µl containing 0.5 mm of dntp mix, 1 µl of each primer, 2.5 µl of 10x pcr buffer, 0.75 mm mgcl2 25mm, 0.3 unit taq dna polymerase and 4µl of template cdna and 14.95 µl sterile distilled water with an initial denaturing step of 95 °c for 5 min, followed by 40 amplification cycles of 95 °c for 45 s, 60.5±5 °c for 45 s, and 72 °c for 90 s and a final extension step of 72 °c for 5 min. finally 60.5 °c was selected for annealing tempreture. rtpcr was used to determine the appropriate annealing temperature of the primers. pcr products were electrophoresed on a 2% agarose gel. real-time pcr various approaches can be used for understanding gene expression in different circumstances. using of real-time pcr allows precise quantization of gene expression with a minimum amount of nucleic acid sample. measurement of mn-sod gene expression was performed on treated and control, untreated samples. the reaction mixture contained 12.5 μl real-time master mix, 2 μl each of forward and reverse primers, 6 μl template cdna and 2.5 μl sterile distilled water, with a final volume of 25 μl by 35 amplification cycles. 3 replicates were prepared from each sample and, for standardization, the housekeeping gene (18s rrna) was used. finally, after standardizing the data using the expression level of genes in the samples, the actual amount of target gene expression (mn-sod) was determined using model 2ctδδ (livak & schmittgen, 2001) at the level of mrna synthesis. statistical analysis using one way anova and duncan test were performed in spss 18 and charts exhibiting the changes in gene expression were plotted in excel. measurement of leaf relative water content relative water content (rwc) was measured on days 0 (control), 5, 10 and 17 based on barrs and weatherley (1962). after calculating the wet (or fresh) weight (fw), dry weight (dw) and saturated weight (sw), the percentage of leaf rwc in the two clones was obtained in triplicate and finally, rwc percentage was calculated by following formula: rwc = (fw dw/sw dw) × 100 measurement of proline proline measurement was carried out with ninhydrin reagent according to bates et al. (1973). for this purpose, 0.5 g of fresh leaf tissue, glacial acetic acid and toluene were used. after preparation of the extracts, absorbance of the upper layer (containing toluene and proline) was read at 520 nm. by using the standard curve in terms of micrograms per gram fresh weight of leaves, proline content was determined. measurement of lipid peroxidation to determine the extent of lipid peroxidation, mda concentration was measured using the method of heath & packer (1968). for this purpose, 0.5 g of table 1. sequence of designed primers for the rt-pcr analysis accession number putative function tm (°c) primer sequence (5´-3´) size of amplicon ay641734.2 manganese superoxide dismutase 60.5 f: cggagggcatatcaaccact r: cacccatccagagcctcgt 121 bp ay563528.1 rrna 18 57.7 f: caacacggggaaacttaccag r: taaccagacaaatcgctccac 178 bp fig. 1. a) total rna extracted from tea leaves and electrophoresed on 1% agarose gel with bands of the 5/8s, 18s, and 28s rrna, indicating the absence of rna degradation and lack of protein and dna contamination in the samples; b) 2% agarose gel of mn-sod gene rt-pcr product in clones 100 and dn. bands 1-4 and 5-8 are related to the 121 bp fragment of mn-sod gene in clones dn and 100, respectively. the annealing temperature 48 fresh leaf tissue was ground with 5 ml of 5% trichloroacetic acid. the extract was centrifuged at 14000 rpm for 15 minutes at room temperature and the supernatant was separated using a micropipette. an equal volume of 20% tca containing 0.5% tba was then added to the supernatant. the mixture was placed in a boiling water bath at 100°c for 30 min. and immediately cooled on ice. then, 2 ml of the supernatant was transferred to a microtube and centrifuged for 5 min at 7500 rpm. mda + tba complex absorbance was read at 532 nm and the non-specific absorption of the pigment was determined at 600 nm and subtracted from absorption at 532 nm. the extinction coefficient of 155 mm-1 cm-1 was used to calculate the concentration of mda. finally, the reaction product of lipid peroxidation (malondialdehyde) was calculated as nm per gram of fresh weight. measurement of chlorophyll and carotenoids pigment measurement was performed based on lichtenthaler (1987). chlorophyll and carotenoid concentrations were obtained in terms of µg/ cm2 of leaf area. results extraction of rna from tea leaves the extracted rna bands can be seen in fig. 1a. 3 bands of rrna, corresponding to 5.8s 18s and 28s, are present in the rna extract, with the width and intensity of the 28s band higher than the other bands. the extracted rna sample was used for cdna synthesis in rt-pcr (fig. 1a). fig. 2. melting curves of rt-pcr product (mn-sod gene) in clones 100 and dn. dna was detected with cyber green. a: melting curve of mn-sod gene related to clone dn in control treatments. b: melting curve of mn-sod gene related to clone dn in 10 days drought treatment. c: melting curve of mn-sod gene related to clone 100 in control treatments. d: melting curve of mn-sod gene related to clone 100 in 10 days drought treatment. e: melting curve of mn-sod gene related to clone dn in 17-day treatment (retrieved by re-watering). f: melting curve of mn-sod gene related to clone 100 in 17-day treatment (retrieved by re-watering). biologica nyssana ● 11 (1) september 2020: 45-54 mohammadi et al. ● a real-time pcr assay for evaluation of drought tolerance in a new tea clone 49 electrophoresis and melting curve of rt-pcr products of mn-sod gene as is shown in fig. 1b and 2, the melting temperature for the 121 bp fragment of mn-sod gene rtpcr product and of the rrna 18s gene are 73.5 °c and 77.5 °c, respectively (fig. 1b, fig. 2). as shown in fig. 3 measurement of mn-sod gene expression was carried out in leaf tissue from 2 clones 100 and dn at 0, 10 and 17 days after treatment. standardization of gene expression data was carried out using 18s rrna. examining gene expression during drought stress and re-irrigation yielded different results. results indicate that the greatest amount of mn-sod expression was observed in clones of dn after undergoing drought conditions for 10 days. significant increases in mrna synthesis of mn-sod gene occurred during this period and expression level decreased significantly by day 17 day, i.e., after the 7 day alleviation of the drought conditions, compared to expression levels after the 10 day exposure to drought conditions in the same clone. in contrast, mn-sod gene expression did not significantly change in clone 100 in any of the 2 treatments (fig. 3). measurement of relative water content and soil moisture content measurement of smc showed that soil moisture was decreased meaningfully in all treatments which were inducted by imposition of drought conditions (fig. 4). the effect of drought on rwc changes in the 100 and dn clones showed that responses were similar in both clones and rwc was reduced meaningfully after 10 days of drought stress. in the recovery treatment, rwc levels increased in both clones. this increase was similar in the 2 clones (fig. 4). proline measurement a comparison of proline content in clones dn and 100 indicates that the responses of the two clones differed in samples under drought and recovery conditions. proline levels increased under drought stress in clone dn, whereas significant increases were not observed in clone 100 compared to control. proline content of clone dn was also reduced significantly after the recovery treatment; however, changes of proline content were again not statistically significant in clone 100 at this point (fig. 5). fig. 3. changes in mrna level of the mn-sod gene in clones 100 and dn in treatments of control (0), 10 days without watering (10) and treatments recovered with reirrigation for 7 days (17). data is average of three replicates ± standard error (se) respectively. different letters indicate significant differences between treatments according to duncan’s test with p <0.05. fig. 4. changes in rwc of clones 100 and dn in treatments of control (0), 10 days without watering (10) and treatments recovered with re-irrigation for 7 days (17). data is average of three replicates ± standard error (se) respectively. different letters indicate significant differences between treatments according to duncan’s test with p <0.05. fig. 5. changes of proline content of clones 100 and dn in treatments of control (0), 10 days without watering (10) and treatments recovered with re-irrigation for 7 days (17). data is average of three replicates ± standard error (se) respectively. different letters indicate significant differences between treatments according to duncan’s test with p <0.05. biologica nyssana ● 11 (1) september 2020: 45-54 mohammadi et al. ● a real-time pcr assay for evaluation of drought tolerance in a new tea clone 50 measurement of lipid peroxidation as seen in fig. 6 changes in malondialdehyde levels in clone 100 are different from those in clone dn under drought stress. mda content does not increase in clone 100 under drought stress, mda levels are very substantially increased in clone dn after 10 days of drought treatment. mda levels in clone dn dropped dramatically in the 7 day recovery period, as assayed on day 17 day of the overall experiment (fig. 6). measurement of photosynthetic pigments measurement of carotenoids data shows that the highest concentrations of carotenoids occurred in clone 100 in the 10 day treatment. changes in carotenoid level in clone dn are substantially lower than in clone 100 (fig. 7). measurement of chlorophyll a measurement of chlorophyll a in the 2 clones showed that in clone dn levels of chlorophyll a were increased after 10 days of drought stress, whereas significant changes were not observed in clone 100. the amount of chlorophyll a in clone dn was significantly greater than in clone 100 after the 10 day drought treatment. by day 17, i.e., after 7 days recovery, the amount of chlorophyll a in clone dn had dropped dramatically and the levels in it and dn were not significantly different (fig. 8). measurement of total chlorophyll measurement of total chlorophyll content from the 2 clones indicated that chlorophyll content in clone 100 did not vary throughout the experiment. although total chlorophyll content appeared higher at day 10 compared to day 0, these differences were not statistically significant. however, there were significant differences in clone dn after the recovery period, i.e. at day 17 compared to day 10 days and the control (fig. 9). discussion many studies have shown that in photosynthetic plants there is a strong relationship between tolerance to oxidative stress induced by environmental stresses and increasing concentrations of antioxidant enzymes (sairam & saxena, 2000; sairam & srivastava, 2001). researchers have shown that the concentrations of antioxidant enzymes double under stress, resulting, in particular, in increased resistance fig. 6. changes of mda content of clones 100 and dn in treatments of control (0), 10 days without watering (10) and treatments recovered with re-irrigation for 7 days (17). data is average of three replicates ± standard error (se) respectively. different letters indicate significant differences between treatments according to duncan’s test and with p <0.05. fig. 7. changes in carotenoid content in clones 100 and dn in treatments of control (0), 10 days without watering (10) and treatments recovered with re-irrigation for 7 days (17). data is average of three replicates ± standard error (se) respectively. different letters indicate significant differences between treatments according to duncan’s test with p <0.05. fig. 8. changes of chlorophyll a content in clones 100 and dn in treatments of control (0), 10 days without watering (10) and treatments recovered with re-irrigation for 7 days (17). data is average of three replicates ± standard error (se) respectively. different letters indicate significant differences between treatments according to duncan’s test with p <0.05. biologica nyssana ● 11 (1) september 2020: 45-54 mohammadi et al. ● a real-time pcr assay for evaluation of drought tolerance in a new tea clone 51 to oxidative stress. drought stress increases the activity of glutathione reductase and sod especially (gamble & burke, 1984; lascano et al., 2001). this is not to say that all cultivars of a particular species are stressed to the same degree by drought conditions. in the present study, a significant increase was observed in mn-sod gene expression in clone dn during 10 days of drought treatment, while no significant increase in the expression of this gene was found in clone 100. this correlated well with the other physiological parameters assayed and reflected clone 100’s greater resistance to drought in the first place. clone dn activates resistant mechanisms against oxidative stress caused by drought by increasing the expression of mn-sod because it was more greatly stressed by the drought conditions. malondialdehyde content rose, also, in only clone dn and not in clone 100. increases in this product represent an increase in lipid peroxidation due to ros production and there is a positive correlation (r2=0.66) between the amount of malondialdehyde and sod gene expression. it can be said, therefore, that sod gene expression in clone dn was increased to mitigate the effects of generated ros. again, neither mda content nor sod gene expression were significantly increased in clone 100, indicating that a lower level of cellular stress was induced by drought conditions in this cultivar. on the other hand, appropriate conditions are provided for the growth of clones with re-watering, which is accompanied by reduced expression of the sod gene in clone dn, indicating a reduction in oxidative stress in this clone. ten days of drought applied to clone 100 did not result in considerable stress responses involving a significant stimulation of the antioxidant defense system. many reports are indicating mn-sod expression and activity increases during stress (shah & nahakpam, 2012). for example, increased resistance to cold stress was correlated with a simultaneous increase in the expression of mn-sod in chlorella ellipsoidea (clare et al., 1984). similarly, transformation of wheat plants with the mn-sod gene from brassica species and its overexpression increased resistance to oxidative stress and aluminum toxicity (gachon et al., 2004) in the transgenic wheat. this method to increase sod activity has also been reported in a study by bowler et al. (1992). they found that transformation of maize chloroplasts with the tobacco mn-sod gene enhanced cold tolerance. also, increased expression of mn-sod in tobacco considerably enhances resistance against oxidative stress. in studies conducted by upadhyaya et al. (2008), performed on 4 tea clones, a decrease in sod activity occurred during 10 days drought stress in clone tv 1 that is resistant to water stress, while an increase in activity was seen in the sensitive clone tv 20. rwc is a very important index of plant water status and dehydration tolerance, and consequently reflects the activity of metabolites in the tissue. rwc is at its highest level in the early stages of leaf development and decreases with increasing leaf dry matter and maturation. this physiological parameter has a direct relationship with water uptake of root and loss of water through respiration (anjum et al., 2011). different cultivars show different levels of rwc, depending on duration of stress and ability of the genotypes to maintain osmotic potential in drought conditions at different stages of development (siddique et al., 2000; allen & ort, 2001). interestingly, a comparison of rwc in clones dn and 100 revealed that rwc was the same in both and was similarly reduced after 10 days of drought stress; whilst re-watering caused an essentially identical increase in rwc in the leaves of both clones. that the decrease in leaf water content happens equally in both clones but causes a different response in these clones strongly indicates that lowered rwc does not necessarily result in oxidative stress. although not receiving adequate moisture is observed in both examined clones, similar water deficits will not necessarily stimulate defense responses in the two clones equally. each clone can activate its defense system tailored to its needs and ability. it will be very interesting to learn what the bases of this difference are. perhaps cytoplasmic proline levels are important here. clone 100 showed much higher basal proline than did clone dn. relationships between the production of free radicals and proline accumulation in plants indicate fig. 9. changes of total chlorophyll content in clones 100 and dn in treatments of control (0), 10 days without watering (10) and treatments recovered with re-irrigation for 7 days (17). data is average of three replicates ± standard error (se) respectively. different letters indicate significant differences between treatments according to duncan’s test with p <0.05. biologica nyssana ● 11 (1) september 2020: 45-54 mohammadi et al. ● a real-time pcr assay for evaluation of drought tolerance in a new tea clone a role for proline in reducing toxicity, a non-enzymatic defense mechanism for removing free radicals (matysik et al., 2002). on the other hand, increases in proline, which reduces the acidity of the cytosol, can be due to various stresses (kurkdjian & guern, 1989). removing excess h+ as a positive effect of proline synthesis results in a reduction of stress. the current study suggests that a change in proline content in tea plant is regulating the level of osmotic potential that ultimately controls the water absorption. the same phenomenon has been previously observed in cotton (parida et al., 2008), rice (pirdashti et al., 2009), maize (valentovic et al., 2006) and in tea (upadhyaya & panda, 2013). our investigation shows that higher drought resistance of clone 100 compared to clone dn is due to its higher proline level. this capability of clone 100 might be more effective in weak or moderate drought and probably less effective in extreme drought conditions. clones dn and 100 differed also in their chlorophyll a profiles throughout the experiment. chlorophyll a levels rose under drought conditions in clone dn but were unchanged in clone 100. similarly, upon relief of drought, chlorophyll a levels (and, in this case, total chlorophyll as well) dropped in clone dn and, again, did not significantly change in clone 100. changes in chlorophyll levels during stress are related to the duration and intensity of the stress (zhang & kirkham, 1996). chapman & barreto (1997) believe that the growth stage, cultivar and environmental conditions may be important factors influencing the amount of chlorophyll pigments under stress. sairam et al. (2000 and 2001) have also stipulated that higher levels of carotenoids and chlorophylls in plants under drought stress conditions can be associated with different resistance genotypes. machado & paulsen (2001) found that rapid physiological changes such as tubular leaves, reduced leaf area, and increased stomatal resistance may be factors in the apparent increase in chlorophylls content, as part of an avoidance mechanism to drought stress. during stressful conditions, plants reduce transpiration levels to prevent water loss through lower leaf surfaces. consequently, the chlorophyll content increases per unit leaf area, despite the decrease in total chlorophyll content. clone dn is a drought resistant cultivar and is considered as a reference strain for evaluating new iranian clones used at tea research institute of iran. perhaps at least a part of the changes in chlorophylls in this cultivar was related to the higher oxidative stress intensity experienced by this clone during the drought conditions. after the 7 day re-watering period the morphological changes mentioned above were reversed but chlorophyll levels nevertheless fell in clone dn to values lower than in control. it is possible that enhanced chlorophyll synthesis could not be upgraded along with these morphological changes and/or not enough time was available for chlorophyll synthesis to reach levels present in control samples. clone 100 displayed better tolerance to drought and changes in chlorophyll content in these clones were minimal. again, it will be very interesting to learn the basis for the lack of apparent oxidative stress in clone 100 as contrasted to the clear oxidative stress experienced by clone dn under conditions of drought. conclusion 10-day drought stress was imposed on two clones of tea plants. this was followed by one week irrigation period (twice a week, according to the usual practice of the research station) to exit from the stress condition. the results showed that 10-day stress caused significant changes in metabolism and activity of antioxidant system in dn clone, while these changes were not observed in clones 100. measurement of membrane lipid peroxidation, as an important physiological indicator to detection oxidative stress intensity and check out the mn-sod gene expression as the first defense enzyme against oxidative stress, showed that during the 10 days of water stress, the clone 100 is not affected, whereas dn clone showed increase in proline, mda and also increased expression of superoxide dismutase. with the re-irrigation of the samples, the expression of superoxide dismutase and the levels of malondialdehyde are also decreased in dn clone similar to clone 100, indicating that this clone has been out of stress. therefore, in terms of some responses to drought stress, clone 100 can be introduced as a more compatible clone for planting in the north region of iran. acknowledgements. the authors acknowledge for the plant material provided by 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nyssana 11(1) 11 (1) september 2020: 1-7 doi: 10.5281/zenodo.4060278 affordable chironomid housing: proposed modifications of standard oecd substrate for testing of chemicals on aquatic midges original article dimitrija savić-zdravković department of biology and ecology, faculty of sciences and mathematics, university of niš, serbia dimitrija.savic@pmf.edu.rs (corresponding author) aca đurđević department of biology and ecology, faculty of sciences and mathematics, university of niš, serbia djukiamphibia@gmail.com zorana lazarević department of biology and ecology, faculty of sciences and mathematics, university of niš, serbia zorana.lazarevic92@gmail.com nastasja manić department of biology and ecology, faculty of sciences and mathematics, university of niš, serbia nastasja.mitic@gmail.com djuradj milošević department of biology and ecology, faculty of sciences and mathematics, university of niš, serbia djuradj@pmf.ni.ac.rs received: february 19, 2020 revised: march 23, 2020 accepted: march 26, 2020 abstract: this study was conducted in order to formulate a modification of standard substrate for laboratory bioassays (oecd protocol number 218: ”sediment water chironomid toxicity test using spiked sediment“), with as few constituents as possible, yet enabling the highest larval survival of model organism: chironomus tentans (chironomidae, diptera). laboratory experiment consisted of two bioassays: first with substrate mixture of two and more ingredients tested: standard oecd substrate, standard substrate with medical clay, peat + clay, peat + sand, clay + sand; second with every ingredient individually tested: coarse sand, fine sand, peat, clay and no substrate. highest larval survival was observed in sand + peat (≈85%) and coarse sand substrate (≈82%), whilst clay + peat and peat substrate caused the highest larval mortality (≈65% and 46% respectively). no larvae survived in treatment without any substrata, indicating the absolute necessity and importance of substrate presence for larval survival. key words: chironomus tentans, bioassay, life traits, oecd guidelines, sediments, substrate, toxicity tests apstract: pristupačno stanovanje za hironomide: predlog izmene standardne podloge oecd-a za ispitivanje hemijskih agenasa na vodenim komarcima ovo istraživanje sprovedeno je radi formulisanja modifikovane standardne podloge namenjene laboratorijskim biološkim testovima (protokol oecd-a broj 218: “test toksičnosti na hironomidama u sistemu sediment-voda, sa supstancom u sedimentu”), sa što manje sastojaka koja omogućava visoku stopu preživljavanja larvi model organizama: chironomus tentans (chironomidae, diptera). laboratorijski eksperiment sastojao se od dva biološka testa: u prvom je testirana mešavina dva ili više sastojaka: standardna oecd podloga, standardna podloga sa medicinskom glinom, treset + glina, treset + pesak i glina + pesak; u drugom je testiran svaki sastojak podloge individualno: krupan pesak, sitan pesak, treset, glina i tretman bez podloge. najveće preživljavanje uočeno je u podlozi sa sastavom pesak + treset (≈85%) i podlozi sa krupnim peskom (≈82%), dok su podloge sastavljene iz mešavine gline i treseta ili samo treseta izazvale najveći mortalitet larvi (≈65% i 46% respektivno). ni jedna larva nije preživela u tretmanu bez ikakve podloge, ukazujući na potpunu neophodnost i značaj prisustva podloge za preživljavanje larvi. ključne reči: chironomus tentans, biološki test, životni parametri, oecd smernice, sedimenti, podloga, testovi toksičnosti introduction in aquatic ecosystems, sediments represent a vital and specific habitat for many organisms providing feeding, spawning, hiding, and rearing areas. on the other hand, sediments in aquatic habitats can accumulate anthropogenic chemicals and waste materials, which can be directly toxic to sediment-dwelling organisms or bioaccumulate in the food chain contributing to a variety of environmental problems (den besten and munawar, 2016; savić-zdravković et al. 2020). considering this enormous environmental problem, ecotoxicological testing of water and sediment based on information from chemical analyses, but also on effect measurements, i.e. bioassays or toxicity tests, has been developed over the years (den besten and munawar, 2016). these tests have been standardized and numerous regulations and guidelines have been developed, providing direct, quantifiable evidence of biological con© 2020 savić-zdravković et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work noncommercially under the same license as the original. 1 sequences of sediment contamination (taylor and scroggins, 2013). great efforts has been invested to harmonize testing and to facilitate the mutual recognition of data by american public health association (apha), american society for testing and materials international (astm), environment canada (ec), european committee for standardization or comite´ europe´en de normalisation (cen), international organization for standardization (iso), organisation for economic co-operation and development (oecd) and united states environmental protection agency (us epa) (taylor and scroggins, 2013). in europe, toxicity tests are conducted by the guidelines developed by oecd (oecd, 2020). the results from oecd test guidelines are intended to be accepted by all eu member states by the mad principle (oecd agreement on mutual acceptance of data in the assessment of chemicals, http://www. oecd.org/env/ehs/mutualacceptanceofdatamad.htm). a wide range of toxicity tests and test organisms exist for both freshwater and marine organisms, but there is a need for adjusting the existing protocols in a sense of new chemical substances and new possible tested endpoints (hund-rinke et al. 2016) and there is allowance for case-specific final modifications of the testing methodology. very important part of the macrozoobenthos in the freshwater ecosystems is the group of chironomids, known as non-biting midges (dickman and rygiel, 1996). chironomidae are a family of the order diptera, representing world-wide distributed midge flies with exceptional species richness and abundance (ferrington, 2008). the family is divided into 11 subfamilies and 22 nominal tribes (ferrington, 2008). chironomids could be found in all sorts of habitats, from aquatic to terrestrial ones, but a total of 339 genera and 4,147 species are strictly aquatic in the larval stage (ferrington, 2008). occupying almost every type and conditions of aquatic habitats and having extraordinary ecological range and sensitivity makes them very good environmental indicators (dickman and rygiel, 1996). chironomids usually exhibit specific phenotypes as a response to the presence of different stressors (wiederholm, 1984). in polluted areas midges with deformed phenotypes were found, which lead to conclusion that aquatic pollutants cause deformities in chironomids, thus making them widely used as indicators of environmental stress in studies assessing the ecological impact of human activities (lenat, 1993). hamilton and saether (1971) were the first to propose the usage of chironomid deformities as biological indicators of freshwater quality, and a lot of research has been done ever since. in fact recent meta-analysis found 41 studies on chemical effects on chironomid deformities in laboratory experiments, especially larval mouthpart deformities (gagliardi et al. 2016). this study, by gagliardi et al. 2016, also emphasizes the inconsistency in the published data due to several factors, amongst which are mortality effects and background stressors (referring to all stressors apart from the one that is tested in the particular study, including inbreeding, parental effects, inadequate substrates and unknown stressors in substrates and food), that influence the “reliability” of conducted studies. the design of the appropriate bioassay depends on the intended application of the test. when examining chemicals’ effects on structures in chironomid larvae, especially mouthparts, it is of great importance to have minimal wear of the structures and minimize additional deformities caused by sediment they are reared in. in few papers, substrate consisting only on quartz sand has been proposed as suitable for this kind of studies (bird, 1997; langer-jaesrich et al. 2010), but no detailed studies have been conducted. in this study, modifications of the standard oecd protocol number 218, entitled ”sediment water chironomid toxicity test using spiked sediment“, designed to assess the effects of prolonged exposure of chemicals to the sediment-dwelling larvae, have been made. this was done in order to formulate a substrate with as few constituents as possible for bioassays where morphological changes on the larvae are examined as an endpoint. the aim of this study was to compare the effect of different standard substrate modifications on the survival on c. tentans larvae and to propose a simplified substrate formulation (lowering the possibility of damage or deformation of the larval mouth apparatus) that will enable favorable larval survival. the proposed modifications of sediment standards are based upon the laboratory experiments on chironomus tentans species. material and methods experimental organisms specimens of chironomus tentans, fabricius, 1805, (diptera, chironomidae), used in the experiments were from the stock cultures reared in the laboratory of the faculty of science and mathematics, university of niš (niš, serbia). stock cultures were reared under the oecd guidelines (oecd; 2004). specimens were reared in glass tanks with water temperature 21.0±0.5 °c, a light-dark cycle of 16:8h artificial daylight and under constant aeration. the artificial sediment used for rearing the laboratory cultures was shredded paper sediment. larvae were fed with tetramin® fish food flake mixture. about a week before the start of the experiment, fresh egg cases have been isolated from the stock aquaria into 2 biologica nyssana ● 11 (1) september 2020: 1-7 savić-zdravković et al. ● affordable chrionomid housing: proposed modifications of standard oecd substrate for testing of chemicals on aquatic midges petri dishes filled with water from the stock culture aquaria for hatching. experimental design sediment bioassays were performed following oecd guidelines for the testing of chemicals, section 2, specifically the test number 218: sedimentwater chironomid toxicity using spiked sediment (oecd, 2004), further referred to as oecd test 218. larvae were exposed to sediment in the test vessels (glass 600 ml beakers measuring 8 cm in diameter) which were composed of 2 cm sediment layer surrounded by a mixture of deionized and tap water (ph 6.5±0.4; hardness 7.3 °dh; electrical conductivity 325 μs/cm3). twenty freshly hatched 1st instar larvae were placed in each test vessel (larvae were 2 days old), at 21 ± 1 °c, with gentle aeration and 16:8 light-dark cycle. three replicas were made for each treatment, i.e. tested substrate type. on termination of the experiment (each bioassay was terminated after 12 days when larvae reached 4th stadium), the number of larvae recovered was recorded from each substrate. survival (s) was calculated by the formula: s=ne/na, where ne is the sum of midge larvae survived at the end of the experiment per vessel and na is number of larvae introduced per vessel. in order for test to be valid, the average survival of c. tentans must be greater than or equal to 70% in the control at the end of the test, according to the protocol. modification of the formulated substrate the following formulated substrate, based on the artificial soil used in oecd test 218 (oecd, 2004), is recommended: 1. 4-5% (dry weight) peat: as close to ph 5.5 to 6.0 as possible; it is important to use peat in powder form, finely ground (particle size less than 1 mm) and only air dried. 2. 20% (dry weight) kaolin clay (kaolinite content preferably above 30%). 3. 75-76% (dry weight) quartz sand (fine sand should predominate with more than 50 percent of the particles between 50 and 200 μm). deionised water is added to obtain a moisture content of the final mixture in a range of 30 to 50 % and calcium carbonate of chemically pure quality (caco3) is added to adjust the ph of the final mixture of the sediment to 7.0 ± 0.5. organic carbon content of the final mixture should be 2% (±0.5%) and is to be adjusted by the use of appropriate amounts of peat and sand, according to the proposed proportions. the experiment consisted of the two identically structured bioassays with different modifications of thhe recommended standard oecd substrate. 3 the modifications consisted of simplification, i.e. excluding one or more of the requested constituents. in the first bioassay we tested the mixture of two and more ingredients, whilst in the second we tested every ingredient individually (for sterilization each ingredient was thoroughly rinsed with water, than incubated 3 h at 500 °c, except peat, which was air dried as suggested by the protocol). each tested substrate (treatment) had three replicas and in each replica the ratio of the depth of the substrate layer to the depth of the overlying water was 1:4 (overlaying water consisted of mixture of dechlorinated tap and deionized water in 1:1 ratio). first bioassay was conducted with following five treatments, i.e. substrate mixtures: 1. standard oecd substrate (sand + peat + clay) (further referred to as standard) 2. standard substrate with medicinal clay (sand + peat + medicinal clay) (further referred to as s+p+mc) 3. standard substrate without sand (peat + clay) (further referred to as p+c) 4. standard substrate without clay (peat + sand) (further referred to as p+s) 5. standard substrate without peat (clay + sand) (further referred to as c+s) second bioassay was conducted with following five treatments, i.e. individual ingredient substrates: 1. coarse quartz sand (canary grain sand which is available from pet stores with >50% of the particles were in the range of 400–1000 μm) (further referred to as cs) 2. fine sand (commercially available sandbox sand with >50% of the particles were in the range of 50–200 μm) (further referred to as fs) 3. peat (further referred to as p) 4. clay (further referred to as c) 5. without substrate (vessels with only water) (further referred to as 0) data analysis the datasets from both bioassays were of normal distribution (kolmogorov-smirnov with lilliefors significance correction and shapiro-wilk test of normality, significance <0.05), therefore the data was analyzed with parametric tests (one way anova). in order to test the significant differences in mortality rates between all tested combinations of substrates, the datasets from both experiments were analyzed separately, than were pooled together for the analysis. for post hoc comparison of larval mortality between groups, i.e. tested substrates, tukey’s honestly significant difference (tukey hsd) test was used, with significance set at p<0.05. all statistical analyses were conducted in ibm spss statistics software version 19 © spss inc. 1989, 2010. biologica nyssana ● 11 (1) september 2020: 1-7 savić-zdravković et al. ● affordable chrionomid housing: proposed modifications of standard oecd substrate for testing of chemicals on aquatic midges 4 results bioassay with ingredient mixture substrate types the 1st bioassay was terminated after 12 days. according to protocol propositions, the average survival of c. tentans larve must be greater than or equal to 70% in the control at the end of the test, which was the case for only two tested substrates: standard and p+s substrate. one way anova showed statistically significant differences in mean survival values among the groups, i.e. tested substrates (p=0.049, f=3.518). post hoc test showed that survival of chironomid larvae in substrate p+c was the lowest and significantly different (p<0.05, mean difference = – 0.316) than in the substrate p+s, where the highest survival was recorded (tab. 1). bioassay with individual ingredient substrate types the 2nd bioassay was terminated after 12 days. according to protocol propositions, the average survival of c. tentans larve must be greater than or equal to 70% in the control at the end of the test, which was the case for only two tested substrates: cs substrate and fs substrate. one way anova showed statistically significant differences among the groups, i.e. tested substrates (p=0.001, f=11.019). post hoc test showed statistically significant differences between the following groups (tukey hsd p<0.05): none of the larvae survived in the substrate 0, which was significantly lower survival than in all other substrates, accept in substrate p. survival of chironomid larvae in substrate p was the second lowest and significantly different than in substrate cs, where the highest survival was recorded (tab. 2). all substrate types when combining the results from both bioassays (fig. 1), and excluding the treatment 0 (in which no larvae survived), one way anova showed statistically significant differences among the groups, i.e. tested substrates (p=0.025, f=2.2998). larval survival in the substrate cs and p+s was the highest among all substrates and significantly higher only than survival in substrate p, which was the lowest (tukey hsd p<0.05). discussion when conducting eco-toxicological tests on chironomids it is essential to propose a simple and effective bioassay design that will enable detecting the influence of examined stressor with desired specific endpoints. if we consider sublethal effects of the tested stressor as specific endpoints we desire to investigate (i.e. larval deformities, stress enzyme activity, dna damage etc.), the bioassay that ensures survival of at least 70% of the population in the control, with proper substrate formulation that reduces the morphological alteration, variability of important population parameters and other background stressors, is essential. to propose optimal simplified substrate formulation for laboratory bioassays designed to assess the effects of prolonged exposure of chemicals to the sediment-dwelling freshwater chironomid biologica nyssana ● 11 (1) september 2020: 1-7 savić-zdravković et al. ● affordable chrionomid housing: proposed modifications of standard oecd substrate for testing of chemicals on aquatic midges table 1. survival mean values ± sd in % of c. tentans larvae in different ingredient mixture substrate types. calculated as the mean values of three replicas for each tested substrate. code substrate type mean survival ± sd% standard standard oecd substrate (sand + peat + clay) 76.7 ± 7.6 s+p+mc standard substrate with medicinal clay (sand + peat + medicinal clay) 61.7 ± 15.3 p+c standard substrate without sand (peat + clay) 53.3 ± 12.6 b p+s standard substrate without clay (peat + sand) 85 ± 15. a c+s standard substrate without peat (clay + sand) 63.3 ± 2.9 treatment a is statistically significantly different from treatment b (tukey hsd p<0.05) table 2. survival and mortality mean values ± sd in % of c. tentans larvae in different individual ingredient substrate types. calculated as the mean values of three replicas for each tested substrate. code substrate type mean survival ± sd % cs coarse sand 81.67 ± 17.55 a fs fine sand 73.3 ± 12.5 p peat 35 ± 31.2 b c clay 61.6 ± 7.6 0 without substrate 0.0 ± 0.0 c the treatment a is statistically significantly different from treatments b and c, whilst treatment c is statistically significantly different from all other treatments accept b (tukey hsd p<0.05) 5 larvae, we compared the effect of different standard oecd substrate modifications on the survival on c. tentans larvae. a total of 10 substrate types, i.e. treatments, were tested: 9 combinations of substrate constituents and one treatment with no substrate at all. acceptable survival (70% and above) was recorded in only following four substrates (listed by average survival in descending order): p+s > cs > standard > fs. in all other substrates the recorded survival was lower than 70%, whilst the survival in treatment with no substrate was 0%. most of the existing internationally harmonized ecotoxicological bioassays were developed through research and development, validation and peer review before they were officially published as documents with standard conditions, procedures, and guidance (davis, 1977). typically, several years of development precedes establishment of bioassay protocols and new additions to existing rules and regulations are regularly made in line with contemporary trends and market demand. therefore, these protocols can be modified with respect to their function depending on the specific requirements of the tested species and the tested hypothesis (iso, 2003). sediment dwelling larvae of chironomids represent ideal test organisms for sediment ecotoxicity tests, especially since they usually exhibit specific phenotypes as a response to the presence of different stressors (wiederholm, 1984) and are most diverse and abundant group out of all macroinvertebrates (milošević, 2013). the standard oecd test 218, recommends using formulated substrate because of several advantages: reduced experimental variability, reduced costs in comparison to using field sediments as substrate, replicability and comparison of results between studies (davis, 1977; oecd, 2004). however, modifications of the recommended substrate are approved, as long as clean and uncontaminated substrate which provides satisfactory environment for larval development and 70% or higher survival (oecd, 2004). providing more economical and simpler design which leads to same results in a more cost-effective way is always encouraged. this is why in this study we investigated the possibility of creating a simplified substrate that provides sufficient survival rate, with as few ingredients as possible to reduce possible influence on larval mouth apparatus morphology, reduce contamination, cost and time of substrate preparation and therefore increase the efficiency of laboratory bioassays. to simplify the existing oecd formulated substrate, we firstly excluded only one of the standard three recommended substrate constituents, formulating the substrate out of two mixed ingredients. thereunto we tested the standard oecd substrate and standard substrate with medicinal clay (commercially available and less expensive clay than proposed kaolin clay). we recorded the highest survival in the s + p substrate (≈85%) and in the standard (≈77%), whilst the lowest survival was in c + p sediment (≈53%) (tab. 1). we then excluded two out of three oecd substrate constituents, formulating the substrate out of only one ingredient. thereunto we tested the larval survival in vessels containing only water and no substrate at all. we recorded the highest survival in cs substrate (≈82%), following by the high survival in the fs substrate (≈73%). no larvae survived the end of the experiment in treatment 0, the jars without any substrata (tab. 2), indicating the absolute necessity fig. 1. larval survival in all tested combinations of substrate types, i.e. treatments, presented together. treatments from the 1st bioassay are colored blue and treatments form the 2nd bioassay are colored green. a – boxplots of survival rates with sd values; b – average survival rates sorted from highest to lowest. treatments a are statistically significantly different from treatments b (tukey hsd p<0.05). biologica nyssana ● 11 (1) september 2020: 1-7 savić-zdravković et al. ● affordable chrionomid housing: proposed modifications of standard oecd substrate for testing of chemicals on aquatic midges 6 of substrate presence for larval survival. just few previous studies conducted the usage of different substrate formulations in the experiments with chironomids as model organisms: the study of bird, (1997), where c. tentans larvae were cultured on four different substrates (shredded paper, coarse sand, fine sand and silty clay), showed the highest survival in paper sediment. this study of bird suggests that substrates with sand are less suitable for chironomids because some of the food is lost between the sand grains and is not accessible to the larvae. however, this study encourages the use of coarse sand if the bioassay is designed to examine larval mentum deformities, since larvae reared in coarse sand had less worn (p<0.05) teeth than those reared in the other substrates. chironomus tentans larvae belong to gatherers/collectors functional feeding group (ffg), meaning that they collect small food particles (<1mm) that get lodged between rocks or in deep pools (brabec et al., 2017). given the size of the particles they can eat, it is possible for the larvae to ingest the substrate particles and mistake them for food, if they are less than 1 mm in size, therefore lowering the amount of food eaten and increasing mortality rates. the substrate composed of coarse sand (with particles around 1 mm) is too large to be ingested by the larvae and therefore does not cause higher mortality, deformation and damage of the oral apparatus. in our study, the survival recorded on both coarse and fine sand substrate was within the allowed survival rate, and in the coarse sand it was even greater than in a standard substrate. when combining the results from both bioassays and comparing all possible substrate combinations (fig. 1), the descending order of suitable substrates (from highest to the lowest larval survival recorded) is the following: c+s > standard > f+s > c+s > c > s+p+mc > p+c > p > 0. significant variation in larval survival on different substrates was observed, but the only significant difference was between p substrate (where the lowest survival of ≈35% is recorded) and cs and p+s substrate (where the highest survival is recorded: ≈82% and ≈85% respectively). the average larval survival in the standard substrate was among the highest of all treatments (≈77%), but when excluding clay form the standard mixture or excluding clay and peat (leaving only coarse sand sediment) the larval survival increased, justifying our aim to simplify the substrate formulation. considering that acceptable survival of above 70% was recorded in only four substrates: c+s, cs, standard and fs, only these combinations of ingredients could be recommended for further usage as substrata in further laboratory bioassays. those four tested substrates were not mutually statistically significantly different in terms of larval survival in the present experiment. using the substrate composed of peat and sand sediment has shown to be the most convenient in the terms of survival of the larvae, but sediments that consist of only peat, or peat mixed with other ingredients, such as clay, have shown to cause higher mortality. high survival is evident in the sediments containing sand, which is in accordance with research of langer-jaesrich et al. (2010) which suggested the use of quartz sand substrate in bioassays (particle size 0.1–0.3 mm) as free of possible organic contaminations, and proposed by several other authors (bird, 1997, meregalli and ollevier, 2001; meregalli et al., 2001). there has never been a systematic proposal for modification of oecd test 218 (oecd, 2004; oecd, 2005). this could be due to the fact that research and development of a test method is typically the most time-consuming aspect of the standardization process, as it requires one to become familiar with all aspects of the test organism and experimental design. the importance of uniformity and consistency in ecotoxicological studies is indisputable, but in order to have solid comparative framework there are still a lot of problems that need to be overcome in the future. on the basis of this study, conducted under controlled laboratory conditions, we could propose further usage of substrate consisting only of quartz sand (especially coarse canary grain sand which is available from pet stores with >50% of the particles in the range of 400–1000 μm) as substrate in laboratory bioassays on c. tentans species. the substrate consisting only of one ingredient could be lowering possible negative effects and “background noise” in bioassays and increasing the reproducibility and ease of performing experiments: sand could be obtained by cheaper prices in the pet stores, could be easily sterilized (thoroughly rinsed with water, than burned 3 h at 500 °c) and chironomid larvae could be better visible and more easily separated from the sediment at the end of the experiment. further study on the influence of substrate to more specific, sublethal endpoints, such as mentum and mandible ware, deformity rates, body weight, larval shape and size has to be conducted on all four proposed suitable substrates in order to confirm the preposition of their usage in bioassays where sublethal effects on the larvae are examined. acknowledgements. this work was financed by the ministry of education, science and technological development, republic of serbia, grant no. iii43002. references brabec, k., janecek, b.f.u., rossaro, b., spies m., bitusik, p., syrovatka, v., schmidt-kloiber, a. 2017: chironomidae indicator database. biologica nyssana ● 11 (1) september 2020: 1-7 savić-zdravković et al. ● affordable chrionomid housing: proposed modifications of standard oecd substrate for testing of chemicals on aquatic midges 7 euro-limpacs project (contract no. gocect-2003-505540), dataset “chironomidae”, workpackage, 7. https://www.freshwaterecology. info/ bird, g. a. 1997: deformities in cultured chironomus tentans larvae and the influence of substrate on growth, survival and mentum wear. environmental monitoring and assessment, 45(3): 273-283. den besten, p.j., & munawar, m. 2016: ecotoxicological testing of marine and freshwater ecosystems: emerging techniques, trends and strategies. crc press, boca raton. 297 p. davis, j.c. 1977: standardization and protocols of bioassaystheir role and significance for monitoring, research and regulatory usage. in proceedings of the 3 rd aquatic toxicity workshop, halifax, nova scotia nov. 2-3, 1976, environment canada, tech. report. dickman, m., & rygiel, g. 1996: chironomid larval deformity frequencies, mortality, and diversity in heavy-metal contaminated sediments of a canadian riverine wetland. environment international, 22(6): 693-703. ferrington, l. c. 2008: global diversity of nonbiting midges (chironomidae; insecta-diptera) in freshwater. hydrobiologia, 595(1): 447. gagliardi, b. s., pettigrove, v. j., long, s. m., & hoffmann, a. a. 2016: a meta-analysis evaluating the relationship between aquatic contaminants and chironomid larval deformities in laboratory studies. environmental science & technology, 50(23): 12903-12911. hamilton, a.l. & saether, o.a. 1971: the occurrence of characteristic deformities in the chironomid larvae of several canadian lakes. the canadian entomologist, 103(3): 363-368. hund-rinke, k., baun, a., cupi, d., fernandes, t.f., handy, r., kinross, j.h., navas, j.m., peijnenburg, w., schlich, k., shaw, b.j., scottfordsmand, j.j. 2016: regulatory ecotoxicity testing of nanomaterials–proposed modifications of oecd test guidelines based on laboratory experience with silver and titanium dioxide nanoparticles. nanotoxicology, 10(10): 1442-1447. international organization for standardization, 2003: soil quality-guidance on the ecotoxicological characterization of soils and soil materials. iso. https://www.iso.org/obp/ui/#iso:std:iso:15799:ed2:v1:en langer-jaesrich, m., köhler, h. r., & gerhardt, a. 2010: can mouth part deformities of chironomus riparius serve as indicators for water and sediment pollution? a laboratory approach. journal of soils and sediments, 10(3): 414-422. lenat, d. r. 1993: using mentum deformities of chironomus larvae to evaluate the effects of toxicity and organic loading in streams. journal of the north american benthological society, 12(3): 265-269. meregalli, g., & ollevier, f. 2001: exposure of chironomus riparius larvae to 17α-ethynylestradiol: effects on survival and mouthpart deformities. science of the total environment, 269(1): 157-161. milošević, d., simić, v., stojković, m., čerba, d., mančev, d., petrović, a., paunović, m. 2013: spatio-temporal pattern of the chironomidae community: toward the use of non-biting midges in bioassessment programs. aquatic ecology, 47(1): 37-55. oecd 2020: oecd guidelines for the testing of chemicals, section 2. effects on biotic systems. https://doi.org/10.1787/20745761 oecd 2005: guidance document on the validation and international acceptance of new or updated test methods for hazard assessment, (env/jm/ mono(2005)14). http://www.oecd.org/officialdocuments/publicdisplaydocumentpdf/?doclanguage= en&cote=env/jm/mono(2005)14 oecd 2004: test no. 218: sediment-water chironomid toxicity using spiked sediment, oecd guidelines for the testing of chemicals, section 2. oecd publishing, paris, https://doi. org/10.1787/9789264070264-en. savić‐zdravković, d., milošević, d., uluer, e., duran, h., matić, s., stanić, s., vidmar, j., ščančar, j., dikic, d., jovanović, b. 2020: a multiparametric approach to cerium oxide nanoparticle toxicity assessment in non‐biting midges. environmental toxicology and chemistry, 39(1):131-140. taylor, l.n., & scroggins, r.p. 2013: standardization of ecotoxicological tests: the process. in: férard, j.f., blaise, c. (ed): encyclopedia of aquatic ecotoxicology. springer, dordrecht, 1221 p. wiederholm, t. 1984: incidence of deformed chironomid larvae (diptera: chironomidae) in swedish lakes. hydrobiologia, 109(3): 243-249. biologica nyssana ● 11 (1) september 2020: 1-7 savić-zdravković et al. ● affordable chrionomid housing: proposed modifications of standard oecd substrate for testing of chemicals on aquatic midges anticancer compounds from medicinal plants biologica nyssana 5 (2)  december 2014: 91-102 zlatković, i. et al.  taxonomical, phztogeographical and ecological … 91 original article received: 17 jul 2014 revised: 21 september 2014 accepted: 10 november 2014 taxonomical, phytogeographical and ecological analysis of the salt marsh flora of central and southern serbia ivana zlatković1*, bojan zlatković2, vladimir ranđelović2, dragana jenačković2, lidija amidžić3 1 college of agriculture and food technology, ćirila and metodija 1, 18400 prokuplje, serbia 2university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia 3 university singidunum, faculty of applied ecology, belgrade, serbia *corresponding autor: gajevicivana@yahoo.com abstract: zlatković, i., zlatković, b., ranđelović, v., jenačković, d., amidžić, l.: taxonomical, phytogeographical and ecological analysis of the salt marsh flora of central and southern serbia. biologica nyssana, 5 (2), december 2014: 91-102. the floristic studies of salt marshes of central and southern serbia in period 2000-2014 have shown presence of 333 taxa within 176 genera and 46 families. the phytogeographical structure is dominated by taxa with wide distribution (291 or 87.39%). the best represented chorological types are: eurasian, holarctic, mediterranean-submediterranean and cosmopolitan. the analysis of representation of life forms has shown that salt marsh flora in this part of serbia has therophytic character, with a significant participation of hemicryptophytes. presence of a high number of threatened taxa, including some listed in “red book of flora of serbia, 1” as critically endangered (cr) plant taxa, indicates pronounced importance of these habitats for biodiversity conservation. key words: flora, chorological type, halophyte, life form, salt marsh area apstrakt: zlatković, i., zlatković, b., ranđelović, v., jenačković, d., amidžić, l.: taksonomska, fitogeografska i ekološka naliza flore slatina centralne i južne srbije. biologica nyssana, 5 (2), decembar 2014: 91-102. florističkim istraživanjima slatina centralne i južne srbije u periodu od 2000-2014. godine, utvrđeno je prisustvo 333 taksona svrstanih u 176 rodova i 46 familija. u fitogeografskom smislu dominiraju taksoni širokog rasprostranjenja (291, 87,39%). najzastupljeniji horološki tipovi su: evroazijski, holarktički, mediteransko-submediteranski i kosmopolitski. analiza zastupljenosti pojedinih životnih formi pokazala je da flora slatina ovog dela srbije ima terofitski karakter, sa značajnim učešćem hemikriptofita. prisustvo velikog broja ugroženih taksona, od kojih su neki našli svoje mesto i u „crvenoj knjizi flore srbije, 1“ kao krajnje ugroženi (cr) biljni taksoni, ukazuje na veliki značaj ovih staništa u smislu očuvanja biodiverziteta. key words: flora, horološki tip, halofite, životna forma, slatinska područja 5 (2) • december 2014: 91-102 biologica nyssana 5 (2)  december 2014: 91-102 zlatković, i. et al.  taxonomical, phytogeographical and ecological analysis … 92 introduction the continental salt marshes belong to fragile, highly threatened habitats. they occupy large areas in pannonian and walachian plains and neighboring lowland regions. however, existence of halomorphic soil and salt marsh habitats is extremely rare in serbia south of vojvodina. in the area from the southern edge of pannonian basin to ovče polje in macedonia salt marshes include only a few small localities in river valleys of južna morava and toplica. in this area the salt marshes represent the relict habitats, where characteristic flora and vegetation have developed under the conditions of isolation and high anthropogenous pressure. vegetation of continental salt marshes is welldeveloped in the pannonian and walachian plains and neighboring lowland regions. the salt marsh areas of central and southern serbia are different from salt marshes of pannonian and walachian basins in the phytogeographical sense, as shown by studies of their flora. salt marsh flora in the study area was a subject of interest for many botanists, starting with p a n č i ć (1884). there are some scarce and non-systematized literature data on salt marsh flora recorded in vicinity of bujanovac, oslare and levosoje (s l a v n i ć , 1940), aleksandrovac (s l a v n i ć , 1940; z l a t k o v i ć et al., 2003), lalinac and oblačina salt marshes (p a n č i ć , 1884; n i k e t i ć , 1993; m i l o s a v l j e v i ć et al., 2002). halophyte vegetation of vicinity of prokuplje was also studied (r a n đ e l o v i ć et al., 2000). the goal of this paper is to present in a systematic manner all available literature and herbarium data, as well as data gathered in our own research and to perform a floristic analysis of these interesting habitats. investigated area the study area includes localities in southern and central serbia (fig. 1, tab. 1), divided into three groups: aleksandrovac salt marsh area, lalinac salt marsh area and bresničić salt marsh area. fig 1. geographical position of the investigated localities biologica nyssana 5 (2)  december 2014: 91-102 zlatković, i. et al.  taxonomical, phytogeographical and ecological analysis … 93 tabela 1. list of sites with geographical and climatic data (a – altitude, t mean annual temperature, m – mean annual precipitation) (temperature and precipitation data taken from diva-gis, 2002) locality n latitude e longitude a (m) t (co) m (mm) 1. levosoje 42°25'50'' 21°44'50'' 400 11,2 553 2. oslare 42°25'39'' 21°43'21'' 405 11,2 553 3. bujanovac 42°27' 21°45' 395 11,2 550 4. aleksandrovac 42°29'20'' 21°54'12'' 401 11,2 540 5. neradovac 42°31'17'' 21°52'56'' 403 11,0 550 6. lalinac 43°20'42'' 21°44'45'' 200 11,2 633 7. oblačina 43°18'26'' 21°40'54'' 285 11,1 636 8. lepaja 43°17'35'' 21°39'50'' 285 11,1 636 9. suva česma 43°13'58'' 21°30'43'' 257 10,9 655 10. bresničić 43°14'50'' 21°27'10'' 293 10,8 670 the aleksandrovac salt marsh area includes several smaller localities (aleksandrovac, oslare, levosoje, bujanovac and neradovac) with developed halomorphic soil and very interesting flora and vegetation. the greatest surface area of salt marshes in this area is in vicinity of aleksandrovac. the lalinac salt marsh area is situated at the foothills of mt. mali jastrebac. this area includes lalinačka, oblačinska and lepajska salt marshes. in the valley of river toplica, in the area between prokuplje and kuršumlija (bresničić salt marsh area), there are several salt marsh fragments. this paper includes data on the better-preserved salt marsh fragments suva česma and salt marshes near bresničić. material and methods the floristic list was composed according to literature data (s l a v n i ć , 1940; n i k e t i ć , 1995; m i l o s a v l j e v i ć et al., 2002; z l a t k o v i ć et al., 2003), herbarium collections (hmn) and our own studies in period 2000-2014. the list of localities is included in tab. 1. the collected material was herbarized and herbarium specimens stored in the herbarium collection “herbarium moesiacum” at the faculty of science and mathematics in niš (hmn). identification of plant material was performed by using keys in “flora europaea” (t u t i n et al., 1964-1980), “flora of serbia” (j o s i f o v i ć (ed.), 1970-1978; s a r i ć (ed.), 1986, 1992) and “flora of nr bulgaria” (j o r d a n o v (ed.) 1963-1979; v e l č e v 1979-1995). the nomenclature was correlated with med-checklist (g r e u t e r et al., 1984-1989), flora europaea (t u t i n et al., 19641980), and euro+med plantbase (euro+med 2006). the chorological types of plant taxa were defined by m e u s e l et al. (1965, 1978), m e u s e l and j ä g e r (1992) and s t e v a n o v i ć (1992). the life forms were presented according to raunkier’s principles, further developed by ellenberg and mueller-dombois (e l l e n b e r g & m u e l l e r -d o m b o i s , 1967 in m u e l l e r d o m b o i s & e l l e n b e r g , 1974) and modified according to s t e v a n o v i ć (1992) in “flora of serbia”. the abbreviations used for life forms follow the chart in “flora of serbia” (s t e v a n o v i ć , 1992b). the list of taxa follows this scheme: name of taxon – life form – chorological type (number of locality according to literature data) author (year); (number of locality in our study) results list of vascular plant taxa magnoliophyta magnoliopsida boraginaceae asperugo procumbens l. t rept ea (6) buglossoides arvensis (l.) i. m. johnston t scap ea (6, 10) heliotropium europaeum l. t scap – e(w)a (6) h. supinum l. t scap – e(w)a (2, 3, 4, 5) slavnić (1940) – (7) myosotis arvensis (l.) hill t scap-ros ea (6, 9) m. stricta link ex roemer & schultes t scap-ros ea (4) nonea pallens petrović t scap – pont/balk (6) campanulaceae legousia speculum-veneris (l.) chaix t scap – cemed-pont (6) caryophyllaceae cerastium brachypetalum pers. t scap – e(w)a (6) c. dubium (bast.) o. schwartz t caesp ce (4, 6, 9) c. glomeratum thuill. t caesp cosm (4, 6) c. pumilum curtis t scap-ros – ce-med-pont (4, 5, 6) c. semidecandrum l. t scap – e(w)a (4, 6) dianthus monadelphus vent. subsp. pallens (sibth. & sm.) w. greuter & burdet ch caesp – medsmed/balk (6) niketić (1995); biologica nyssana 5 (2)  december 2014: 91-102 zlatković, i. et al.  taxonomical, phytogeographical and ecological analysis … 94 d. pinifolius sibth. & sm. ch caesp – med-smed/balk (6) niketić (1995); d. viscidus bory & chaub t scap – med-smed/balk (2, 3, 4, 5) slavnić (1940); (4) gypsophila muralis l. t caesp ea (4, 7) holosteum umbellatum l. t scap – ea (4) minuartia viscosa (schreber) schinz & thell. t caesp – e(w)a (4) moenchia graeca boiss. & heldr. t caesp – medsmed/balk (4) m. mantica (l.) bartl. t scap – e(w)a (4, 6) petrorhagia prolifera (l.) p. w. ball & heywood t scap – e(w)a (6, 7, 10) scleranthus annuus l. t caesp ce-med (6) s. polycarpos l. t caesp ce-med (4) spergula arvensis l. t scap cosm (4) spergularia rubra (l.) j. & c. presl t caesp cosm (2, 3, 4, 5) slavnić (1940); (1, 4) vaccaria pyramidata medicus t scap cosm (6) chenopodiaceae atriplex prostrata boucher ex dc. t scap hol (5) slavinić (1940); (6, 9) a. rosea l. t scap – ea (2) slavinić (1940) a. tatarica l. t scap ea (2) slavinić (1940); (4, 10) camphorosma monspeliaca l. ch suffr caesp medpont (6) niketić (1995); (6) milosavljević & al. (2002); (6) chenopodium glaucum l. t scap ea (2, 3, 4, 5) slavnić (1940); (7, 9) c. hybridum l. t scap hol (4) c. rubrum l. t scap hol (1, 4, 10) c. urbicum l. t scap еа (10) polycnemum arvense l. t rept еа (6) salsola kali l. t scap еа (6) compositae achillea collina j. becker ex reichenb. h scap eа (6) a. crithmifolia waldst. & kit. h scap – med-pont (9) aster oleifolius (lam.) wagenitz h scap pont (6) niketić (1995); (6) a. sedifolius l. subsp. canus (waldst.& kit.) merxm. h scap – med-pont/balk (6) niketić (1995); (6) bellis perennis l. h ros ce-med (2, 3, 4, 5) slavinić (1940); (4) bidens tripartita l. t scap ea (4,5,2,3) slavinić (1940) carduus crispus l. t/h scap bienn ea (6) carthamus lanatus l. h scap ce-med-pont (6) centaurea pannonica (heuffel) simonkai h scap ce (6) niketić (1995); (4) c. salonitana vis. h scap pont (6) c. solsticialis l. t scap med-pont (6, 10) chamomilla recutita (l.) rauschert t scap ea (4, 6, 9) chondrilla juncea l. h scap – e(w)a (2, 3, 4, 5) slavnić (1940) cichorium intybus l. h scap cosm (4) cirsium arvense (l.) scop. h scap ea (6) crepis setosa haller fil. t scap med-smed (9) filaginella uliginosa (l.) opiz t scap ea (4,5,2,3) slavnić (1940); hypochoeris radicata l. h ros – e(w)a (4) inula britannica l. h scap ea (1, 6, 7) i. oculus-christi l. h scap-semiros – med-pont (6) lactuca saligna l. t/h scap bienn ce-med-pont (4) leucanthemum vulgare lam. h scap ea (6) onopordum acanthium l. t scap – e(w)a (6) picris echioides l. h scap med-smed (6) niketić (1995); (6,10) p. hieracioides l. t/h scap bienn e(w)a (6) pulicaria dysenterica (l.) bernh. h scap e(w)a (4) scorzonera cana (c. a. meyer) o. hoffm. h scap – ce-med-pont (6) niketić (1995); (6) milosavljević & al (2002); (4, 2, 1, 6, 9, 10) senecio aquaticus hill subsp. barbareifolius (wimmern & grab) h scap ce-med (1, 6) s. erucifolius l.h scap ce-med (6) s. vernalis waldst. & kit. t scap ce-med (6, 10) s. vulgaris l. t scap ea (6) sonchus asper (l.) hill t/h scap bienn ea (8) s. oleraceus l. t/h scap bienn ea (10) taraxacum erythrospermum andraz. ex besser h ros med-pont (4) t. palustre (lyons) symons h ros ce (4, 5) t. serotinum (waldst. & kit. ) poiret h ros med-pont (4) tragopogon pratensis l. h scap ce-med-pont (4) xanthium strumarium l. t scap antrop (2, 3, 4, 5) slavnić (1940) xeranthemum annuum l. t scap med-smed (6) x. cylindraceum sibth. & sm. t scap med-pont (6) crassulaceae sedum caespitosum (cav.) dc. t succ ce-med (4, 6, 10) cruciferae berteroa incana (l.) dc. t/h scap bienn ea (2, 3, 4, 5) slavnić (1940) bunias erucago l. t scap med-pont (6, 9) calepina irregularis (asso) thell. h scap med-pont (9) camelina sativa (l.) crantz t scap ce-med-pont (6) capsella bursa-pastoris (l.) medicus t scap-ros cosm (6) cardamine hirsuta l. t scap-ros ea (4, 6) cardaria draba (l.) desv. h scap ce-med (9) coronopus didymus (l.) sm. h scap ce-med (6) erophila verna (l.) chevall. t ros e(w)a (4, 6) erysimum repandum l. t scap e(w)a (6) lepidium perfoliatum l. t scap-ros ce-med-pont (6) milosavljević & al (2002); (4) zlatković & al (2003); (4, 6, 9, 10) l. ruderale l. t scap-ros e(w)a (1, 4, 6 ,7, 8, 10) myagrum perfoliatum l. t scap med-pont (6) neslia paniculata (l.) desv. subsp. thracica (velen.) bornm. t scap ea (6) rorippa sylvestris (l.) besser h scap-semiros ea (9) teesdalia coronopifolia (j. p. berg.) thell. t scap-ros ce-med-pont (4) thlaspi arvense l. t scap ea (10) dipsacaceae dipsacus fullonum l. t/h scap bienn e(w)a (6) knautia arvensis (l.) coulter t/h scap bienn e(w)a (6) euphorbiaceae euphorbia chamaesyce l. t scap – ea (6) biologica nyssana 5 (2)  december 2014: 91-102 zlatković, i. et al.  taxonomical, phztogeographical and ecological … 95 e. falcata l. t scap – ea (6) e. helioscopia l. t scap – ea (4, 6) e. salicifolia host h scap ce (10) gentianaceae centaurium erythraea rafin. t scap – med-pont (6, 7) c. littorale (d. turner) gilmour subsp. uliginosum (waldst. & kit.) rothm. t scap -ce-pont (6, 8) c. pulchellum (swartz) druce t scap ce-pont (4, 8) geraniaceae erodium cicutarium (l) l'her. t scap-ros cosm (4, 6) geranium dissectum l. t scap e(w)a (10) g. molle l. t scap e(w)a (10) guttiferae hypericum elegans stephan ex willd. h scap ea (6) haloragaceae myriophyllum spicatum l. hydt rad hol (4) labiatae lamium amplexicaule l. t scap – e(w)a (6) mentha aquatica l. h scap -cosm – (4) m. pulegium l. h scap ea (4, 6) m. spicata l. h scap antrop (6) phlomis tuberosa l. g tub/h scap – med-pont (6) niketić (1995); (6) salvia aethiopis l. t/h scap bienn ea (6, 7, 10) s. pratensis l. h scap – ce (6, 9, 10) stachys cassia (boiss.) boiss. h scap – med-smed (6) niketić (1995); s. germanica l. h scap – ce-med-pont (6) s. milanii petrović t scap – med-smed/balk (6) niketić (1995); (6) milosavljević & al (2002.); (6) teucrium chamaedrys l. ch suffr caesp e(w)a (6) t. polium l. ch suffr caesp – med-pont (6) t. scordium l. subsp. scordioides (schreber) maire & petitmengin h scap – med-smed (4, 6, 7,8) thymus pannonicus all. ch suffr rept ce (10) galega officinalis l. h scap e(w)a (6) lathyrus aphaca l t scap hol (6) l. hirsutus l. t scap ce-med (4, 6, 10) l. tuberosus l. g tub rept ea (6) lotus angustissimus l. t scap – med-pont (4, 6, 10) l. corniculatus l. h scap ce-med (6) l. tenuis waldst. & kit. ex willd. h scap – ce-med (2, 3, 4, 5) slavnić (1940) medicago arabica (l.) hudson t/h scap – med-pont (9) m. lupulina l. t scap ea (6) m. minima (l.) bartal. t scap ea (4, 6) m. rigidula (l.) all. t scap ce-med-pont (4, 6) m. officinalis (l.) pallas t/h scap bienn ea (6) ononis arvensis l. ch suffr caesp ea (4) o. spinosa l. ch suffr caesp ea (6) pisum sativum l. subsp. arvense (l.) asch. & graebn. t scap cemed-pont (6) trifolium dubium sibth. t scap ce-med-pont (10) t. echinatum bieb. h scap med-pont (6, 7, 8, 10) t. fragiferum l. h rept ea (4) t. incarnatum l. t scap med-smed (9) t. lappaceum l. t scap med-smed (6, 7, 10) t. nigrescens viv. t scap ce-med (4, 10) t. patens schreber t scap ce-med (6) t. pratense l. h scap ea (6) t. repens l. h rept hol (6) t. resupinatum l. h scap med-pont (4, 6, 9, 10) t. striatum l. h scap ce-med-pon (4) t. strictum l. h scap ea (6) t. subterraneum l. t rept med-pont (4) vicia grandiflora scop. t scap ce-med-pont (9) v. hirsuta (l.) s. f. gray t scap ce-med (9) v. narborensis l. t scap ce-med-pont (6) v. serratifolia jacq. t scap ce-med-pont (6, 10) linaceae linum corymbulosum reichenb. t scap med-smed (6) lythraceae lythrum hyssopifolia l. t scap cosm (1, 4) l. salicaria l. h scap ea (6) malvaceae althaea hirsuta l. t scap med-pont (1, 6) a. officinalis l. h scap ea (1, 6) onagraceae epilobium tetragonum l. h scap hol (10) papaveraceae fumaria officinalis l. t scap ea (6, 9) f. parviflora lam. t scap – phol-ptrop (6) f. vaillantii loisel. t scap ea (9) papaver dubium l. t scap – med-pont (2, 3, 4, 5) slavnić (1940) p. rhoeas l. t scap – med-pont (2, 3, 4, 5) slavnić (1940) plantaginaceae plantago coronopus l. t ros ce-med (2, 3, 4, 5) slavnić (1940); (4) zlatković & al (2003); (4) p. lanceolata l. h ros ea (4, 6, 9, 10) p. major l. subsp. intermedia (dc.) arcangeli h ros hol (3) slavnić (1940); (4) p. media l. h ros ea (8, 9) plumbaginaceae limonium gmelinii (willd.) o. kuntze h scap-ros/ch herb succ ea (6) niketić (1995); (6) milosavljević & al (2002.); (6, 7, 10) polygonaceae polygonum amphibium l. hydg rhiz hol (3) slavnić (1940) p. lapathifolium l. t scap hol (4) slavnić (1940) p. patulum bieb. t scap hol (2, 3, 4, 5) slavnić (1940); (4) rumex conglomeratus murray h scap ea (4, 6, 7) r. palustris sm. t scap ea (1, 7, 9) r. sanguineus l. h scap – med-pont (10) r. stenophyllus ledeb. h scap ea (7) portulacaceae montia fontana l. subsp. chondrosperma (fenzl) walters t rept cosm (4) ranunculaceae adonis aestivalis l t scap med-pont (9) a. flammea jacq. t scap med-pont (6) biologica nyssana 5 (2)  december 2014: 91-102 zlatković, i. et al.  taxonomical, phytogeographical and ecological analysis … 96 a. vernalis l. g rhiz caesp med-pont (6) niketić (1995); (10) consolida orientalis (gay) schodinger t scap e-medsmed-pon (2, 3, 4, 5) slavnić (1940); (6) c regalis s. f. gray t scap med-pont (2, 3, 4, 5) slavnić (1940); (6) myosurus minimus l. t ros med-pont – (4) zlatković & al (2003); (4, 6, 7) ranunculus acris l. h scap-semiros hol (4, 6) r. circinatus sibth. – hydt ea (4) r. ficaria l. subsp. calthifolius (reichenb.) arcangeli g tub rept ea (4) r. fluitans lam. – hydt ea (4) slavnić (1940); r. marginatus d'urv. t scap – ce-med-pont (4) r. ophioglossifolius vill. t/h scap bienn – med-smed (4) slavnić (1940) r. pedatus waldst. & kit. g tub/h scap ea (4, 6, 7, 9, 10) r. repens l. h rept ea (4, 6) r. sardous crantz t scap med-pon (9, 10) r. sceleratus l. t scap ea (7, 10) r. serbicus vis. h /g rhiz scap – med-smed/apembalk (6) r. trichophyllus chaix hydt cosm (4) slavnić (1940); (6) r. velutinus ten. h scap med-smed (4) thalictrum lucidum l. h scap ce (6) t. minus l. h scap ea (6) rosaceae potentilla reptans l. h rept ea (6) rubiaceae asperula purpurea (l.) ehrend. ch suffr caesp medsmed (6) galium parisiense l. t scap ce-med (4) g. tenuissimum bieb. t scap med-pont (6, 10) g. tricornutum dandy t scap ea (6) g. verum l. h scap ea (6, 10) sherardia arvensis l. t rept ea (4, 6) santalaceae comandra elegans (rochel ex reichenb.) reichenb. fil. ch suffr caesp – pont/balk (6) niketić (1995) scrophulariaceae kickxia elatine (l.) dumort. t rept e(w)a (2, 3, 4, 5) slavnić (1940); (4, 7, 10) k. spuria (l.) dumort. t rept e(w)a (2, 3, 4, 5) slavnić (1940); (4, 10) melampyrum arvense l. t scap ce-med (6) parentucellia latifolia (l.) caruel t scap ce-medpont (4) verbascum blattaria l. t/h scap bienn ea (6, 10) veronica acinifolia l. t scap ea (4, 6) v. anagallis-aquatica l. h scap ea (1, 7, 8) v. arvensis l. t scap ea (6) v. beccabunga l. h rept ea (4) v. hederifolia l. t caesp med-smed (6) v. polita fries t caesp ea (6) v. verna l. t caesp ea (10) umbelliferae anthriscus sylvestris (l.) hoffm. h scap ce (9) bifora radians bieb. t scap ce-med-pont (6) bupleurum rotundifolium l. t scap ea (6) b. tenuissimum l. t scap ce-med-pont (2, 3, 4, 5) slavnić (1940); (4, 6, 7, 10) oenanthe fistulosa l. h scap ce-med (1) o. silaifolia bieb h scap ce-med (4, 6, 10) pastinaca sativa l. t/h scap bienn ea (6) torilis nodosa (l.) gaertner -t scap ce-med-pont (7) turgenia latifolia (l.) hoffm. t scap ce-med-pont (6) valerianaceae valerianella carinata loisel. t scap ce-med (9) v. dentata (l.) pollich t scap ce-med (6) v. locusta (l.) laterrade t scap ce-med (4, 6) violaceae viola kitaibeliana schultes t caesp ce-med (6) monocotyledones alismataceae alisma plantago-aquatica l. hydg rhiz hol (4) slavnić (1940); (4, 8) amaryllidaceae leucojum aestivum l. g bulb – e(w)a (9) araceae arum orientale bieb. g rhiz caesp e(w)a (6) butomaceae butomus umbellatus l. hydg rhiz ea (1, 4) cyperaceae carex distans l. h caesp ea (1, 4, 5, 6, 9, 10) c. divisa hudson g rhiz caesp e(w)a (4, 3) slavnić (1940); (1, 4, 6, 7, 8, 9, 10) c. elata all. h caesp ce (4) slavinić (1940) c. hirta l. g rhiz caesp hol (9) c. hordeistichos vill. g rhiz caesp hol (7) c. otrubae podp. h/g rhiz caesp – e(w)a (4, 6, 9) c. tomentosa l. h/g rhiz caesp e(w)a (4, 6) c. vulpina l. h/g rhiz caesp e(w)a (1, 4, 6, 9, 10) cyperus flavescens l. t caesp cosm (2, 3, 4, 5) slavnić (1940); (8) c. fuscus l. t caesp hol (2, 3, 4, 5) slavnić (1940); (4, 7, 8, 9) c. longus l. g rhiz caesp ea (4) slavinić (1940); (1, 2) c. pannonicus jacq. t caesp ce-pont (4, 3) slavinić (1940); (4, 6, 9, 10) eleocharis palustris (l.) roemer & schultes hydg rhyz cosm (2, 3, 4, 5) slavnić (1940); (4, 6, 7) fimbristylis bisumbellata (forskål) bubani t caesp cosm (4) slavnić (1940); (4) zlatković & al (2003) scirpoides holoschoenus (l.) soják – h caesp/g rhiz ea (1) scirpus lacustris l. subsp. lacustris g rhiz caesp ea (4) slavnić (1940); s. lacustris l. subsp. tabenaemontani (c. c. gmelin) syme ea (4, 2, 6, 7, 8, 10) bolboschoenus maritimus (l.) palla g rhiz ceasp cosm (2, 3, 4, 5) slavnić (1940); (6) niketić (1995); (4) zlatković (2003); (1, 4, 6, 7, 9, 10) b. glaucus (lam.) s.g. sm. g rhiz ceasp cosm (6). biologica nyssana 5 (2)  december 2014: 91-102 zlatković, i. et al.  taxonomical, phztogeographical and ecological … 97 gramineae agropyron cristatum (l.) gaertner g rhiz caesp cemed-pont (6) niketić (1995); (6) agrostis capillaris l. h caesp hol (4) a. stolonifera l. h caesp hol (2, 3, 4, 5) slavnić (1940); alopecurus aequalis sobol. t rept ea (6) a. myosuroides hudson t rept e(w)a (6) a. pratensis l. h caesp ea (10) a. rendlei eig. t rept hol (4, 6, 9) a. odoratum l. h caesp ea (4) arrhenatherum elatius (l.) beauv. ex j. & c. presl h caesp – e(w)a (9) beckmannia eruciformis (l.) host h caesp hol (1) bromus arvensis l. t scap ea (4) b. commutatus schrader t scap ce-med (4) b. hordeaceus l. t scap hol (4, 9, 10) b. racemosus l. t scap ce (4) b. scoparius l. t scap med-pont-s sib (6) b. squarrosus l. t/h caesp bienn med-smed (6) b. tectorum l. t scap ce (9) calamagrostis arundinacea (l.) roth g rhiz caesp ce-pont (1) catabrosa aquatica (l.) beauv. g rhiz caesp hol (6, 8) crypsis aculeata (l.) aiton t caesp med-pont (2, 3) slavinić (1940); (4) zlatković & al (2003); (6, 10) c. alopecuroides (piller & mitt.) schrader t caesp ce-med-pont (4, 2, 3) slavinić (1940); (4, 8) c. schoenoides (l.) lam. t caesp med-pont (2, 3, 4, 5) slavinić (1940); (4, 6, 10) cynodon dactylon (l.) pers. g rhiz rept cosm (4, 6, 10) echinochloa crus-galli (l.) beauv. t caesp cosm (4, 3) slavinić (1940); eragrostis cilianensis (all.) f. t. hubbard t caesp ea (6) e. minor host t caesp hol (4) e. pilosa (l.) beauv. t caesp cosm (4, 10) festuca valesiaca schleicher ex gaudin h caesp cemed (7, 10) hordeum hystrix roth h caesp ce-med-pont (2, 3, 4, 5) slavinić (1940); (6) milosavljević & al (2002.); (4, 6, 8, 9, 10) h. murinum l. h caesp ce-med-pont (9) h. secalinum schreber h caesp hol (6, 7, 8, 10) lolium perenne l. h caesp ea (9) l. rigidum gaudin t caesp – med-smed (4, 10) melica transsilvanica schu h caesp ce (6) molineriella minuta (l.) rouy t scap medsmed/balk (4) phleum pratense l. subsp. bertolonii (dc) bornm h caesp ce-med (4) pholiurus pannonicus (host) trin. t caesp ce-medpont (4) zlatković & al (2003); (4, 6, 7, 8, 10) phragmites australis (cav.) trin. ex steudel hydg rhiz cosm (4) slavnić (1940); (4, 6, 8, 9) poa annua l. t caesp cosm (4, 6) p. bulbosa l. h caesp ea (4, 9) p. palustris l. h caesp hol (9) puccinellia distans (l.) parl. h caesp hol (2, 3, 4, 5) slavinić (1940); (6) niketić (1995); (6) milosavljević & al (2002.); (4, 6, 7, 8, 9, 10) p. festuciformis (host) parl. subsp. convoluta (hoernm.) w.e. hughes h caesp med-pont (4) zlatković & al (2003); (2, 1) sclerochloa dura (l.) beauv. t caesp ea(9) taeniatherum caput-medusae (l.) nevski t scap med-pont (10) tragus racemosus (l.) all. t caesp cosm (4) vulpia bromoides (l.) s. f. gray t scap med-pont (4) v. myuros (l.) c. c. gmelin t scap ce-med-pont (4, 6, 10) iridaceae crocus chrysanthus (herbert) herbert g bulb – medsmed/balk-anat (6) niketić (1995); juncaceae juncus articulatus l. g rhiz caesp hol (4, 3) slavinić (1940); (1, 4) j. bufonius l. t caesp hol (2, 3, 4, 5) slavinić (1940); (1, 4) j. compressus jacq. g rhiz caesp ea (6, 7, 9, 10) j. effusus l. g rhiz caesp cosm (6) j. gerardii loisel. g rhiz caesp hol (3, 4) slavnić (1940); (4, 6, 8, 10) j. inflexus l. g rhiz caesp hol (4) slavnić (1940); (6) j. ranarius song. & perr. t caesp ce-med-pont (2, 3, 4, 5) slavinić (1940); (7, 8) j. tenageia l. fil. t caesp e(w)a (1) lemnaceae lemna gibba l. hyd t cosm (7, 10) l. minor l. hyd t cosm (4) slavnić (1940); liliaceae allium cupani rafin. g bulb – med-smed/apen-balk (6) niketić (1995); a. guttatum steven subsp. dalmaticum (a. kerner ex janchen) g bulb med-smed/balk (6) niketić (1995); (4, 6, 10) a. scorodoprasum l. g bulb ce-med-pont (6) niketić (1995); (6) a. spaerocephalon l. g bulb ce-med-pont (6) a. vineale l. g bulb hol (6) muscari tenuiflorum tausch g bulb med-pont (6) ornithogalum divergens boreau g bulb ce-med-pont (5) o. pyramidale l. g bulb med-pont (6, 10) o. refractum kit. ex schlecht. g bulb med-pont (6) scilla autumnalis l. g bulb med-pont (4, 6) sternbergia colchiciflora waldst. & kit. g bulb cemed-pont (6) niketić (1995); orchidaceae orchis morio l. g tub scap ce-med-pont (4) spiranthes spiralis (l.) chevall. g tub scap ce-medpont (4) potamogetonaceae potamogeton pectinatus l. hydt cosm (4) slavnić (1940); p. polygonifolius pourret hydt cosm (4) slavnić (1940); biologica nyssana 5 (2)  december 2014: 91-102 zlatković, i. et al.  taxonomical, phytogeographical and ecological analysis … 98 sparganiaceae sparganium erectum l. hydg rhiz ea (4) slavnić (1940); typhaceae typha angustifolia l. hydg rhiz cosm (4) slavnić (1940); t. latifolia l. hydg rhiz cosm (4) slavnić (1940); (7, 10) zannichelliaceae zannichellia palustris l. hydt cosm (10) discussion the results of original studies performed in salt marsh fragments of central and southern serbia have shown presence of 334 taxa at species and subspecies level, belonging to 177 genera and 46 families. all taxa belong to division magnoliophyta. class magnoliopsida includes 232 species or 69.46% of total flora, while class liliopsida includes 102 species or 30.54% of flora in the study area. detailed taxonomic analysis was used to determine number of species and subspecies, genera and families in each salt marsh area (tab. 2). as expected, most species were recorded at lalinac salt marsh area (207 species) which includes the bestpreserved and so far best-studied fragments of salt marsh areas in serbia south of the pannonian basin. table 2. total taxa (nt), genera (ng) and families (nf) in salt marsh areas of central and southern serbia salt marsh area nt ng nf aleksandrovac 168 102 34 lalinac 207 126 39 bresničić 114 76 30 the analysis of representation of genera (tab. 3) and species in certain families (fig. 2) has shown that family gramineae has the highest number of taxa at aleksandrovac (19 genera, 27 species) and bresničić salt marsh areas (16 genera, 23 species), while family compositae had the highest number of taxa at lalinac salt marsh area (15 genera, 25 species). the pronounced diversity of family gramineae is attributed to strong steppe influence on formation of salt marsh flora. the high number of taxa from family compositae was expected as this family is richest in taxa both in balkan peninsula and in europe (t u r i l l 1929). analysis of representation of species in certain genera (fig. 3) has shown that the largest genera were ranunculus, trifolium, carex and juncus. table 3. comparative overview of families with the highest number of species in a salt marsh areas of central and southern serbia (a – aleksandrovac, l – lalinac, b – bresničić) a l b gramineae 30 21 23 compositae 18 24 9 cyperaceae 16 13 9 caryophyllaceae 13 12 3 ranunculaceae 13 13 5 legumonosae 11 21 12 cruciferae 6 11 7 liliaceae 3 10 5 labiatae 3 12 3 one important characteristic of flora in all three salt marsh areas is complete absence of highly halophilic genera such as suaeda and salicornia, in contrast to salt marshes of macedonia and pannonian plain. the phytogeographical analysis (tab. 4) has shown that floras of all three salt marsh areas are dominated by species with wide distribution, as a consequence of floristic uniformity of this habitat type as well as the pronounced anthropogenous influences on the broader regions. the dominance of taxa with eurasian range types indicates continental character of these areas. although they are few in number, the species of mediterranean-submediterranean distribution type play an important role in structure of the flora of aleksandrovac salt marsh area. this group also includes two endemic (allium guttatum subsp. dalmaticum and dianthus viscidus) and one subendemic taxon (moenchia graeca). dianthus viscidus and moenchia graeca are differential species in comparison to salt marshes of jastrebac foothills (bresničić and lalinac salt marsh area). group of differential species also includes the mediterraneanmacedonianthracian species molineriella minuta, which was first recorded in serbia during the studies of salt marshes of aleksandrovac area (z l a t k o v i ć et al., 2005). species puccinellia festuciformis also has a significant differential role, appearing as a dominant and characteristic species of association montiopuccinellietum festuciformis at the salt marshes in vicinity of vranje and bujanovac. flora of lalinac salt marsh area is predominantly characterized by species of pontiansouth siberian range type, with a differential character in comparison to aleksandrovac and bresničić salt marsh area. analysis of flora of lalinac salt marsh area has shown presence of the greatest number of endemics (stachys milanii and biologica nyssana 5 (2)  december 2014: 91-102 zlatković, i. et al.  taxonomical, phztogeographical and ecological … 99 allium guttatum subsp. dalmaticum) and subendemics (dianthus pinifolius, allium cupanii, ranunculus serbicus and achillea crithmifolia) in comparison to other salt marshes of central and southern serbia. this group may also include the species with ranges including part of balkan peninsula, pannonian plain and the western part of pontian-south siberian floristic region (comandra elegans, dianthus monadelphus, aster sedifolius, aster oleifolius, centaurea salonitana and nonea pallens). the greatest phytogeographical significance is certainly that of the endemic species stachys milanii. in addition, the lalinac salt marsh area also hosts another critically endangered species of serbian flora, camphorosma monspeliaca, creating characteristic chamaephyte associations. the bresničić salt marsh area was characterized by absence of any taxa of pontiansouth siberian range types, although the steppe influence is reflected in presence of 31 taxa with ranges mostly occupying the pontian-south siberian region. at the salt marshes of this area there was 30 18 16 13 13 11 21 24 13 13 12 21 23 9 9 5 3 12 0 5 10 15 20 25 30 35 gra m in eae com posit ae cyp era ce ae ran uncu la ce ae car yo phyll ac eae le gum in osa e aleksandrovac area lalinac area bresničić area fig. 2. comparative overview of families with the highest number of species in a salt marsh areas of central and southern serbia 10 6 6 5 4 4 3 1 6 6 5 7 2 5 5 5 3 5 2 6 2 1 1 1 0 2 4 6 8 10 12 ranuncu lu s car ex ju ncu s trif oliu m bro m us cera st iu m vero nica alliu m aleksandrovac area lalinac area bresničić area fig. 3. comparative overview of genera with the highest number of species in a salt marsh areas of central and southern serbia biologica nyssana 5 (2)  december 2014: 91-102 zlatković, i. et al.  taxonomical, phytogeographical and ecological analysis … 100 only one endemic (allium guttatum subsp. dalmaticum) and one subendemic taxon (achillea crithmifolia). flora of salt marshes of central and southern serbia includes 6 main (fig. 4) and two transitional life forms of plants. characteristic feature is significant participation of therophytes (about 50% of total flora in all three salt marsh areas) and complete absence of phanerophytes. the therophyte character of flora in these habitats is explained by presence of relatively arid and unsuitable habitats for plant life. additional factor favoring presence of therophytes and reducing the other ecological groups is substrate salinity. high levels of presence of therophytes within the spectrum of life forms of salt marsh flora are also contributed to by strong influence of the anthropogenous factor. in all salt marsh areas the hemicryptophytes table 4. comparative overview of presence of chorological types in the flora of salt marshes of central and southern serbia (n – number of species) s a l t m a r c h a r e a aleksandrovac lalinac bresničić chorological type n % n % n % cosmopolitan (cosm) 24 14.29 15 7.25 10 8.77 antropohoric (antrop) 1 0.60 1 0.48 0 0.00 holarctic (hol) 22 13.10 14 6.76 11 9.65 paleoholarctic-paleothropic (ph-pt) 0 0.00 1 0.48 0 0.00 eurasian (ea) 41 24.40 59 28.50 30 26.32 euro-west asian (e(w)a) 16 9.52 22 10.63 11 9.65 central european-mediterranean-pontic (ce-med-pont) 19 11.31 25 12.08 13 11.40 central european-pontic (ce-pont) 2 1.19 3 1.45 1 0.88 mediterranean-submediterranean-pontic (med-pont) 17 10.12 27 13.04 17 14.91 pontic-south siberian (pont) 0 0.00 4 1.93 0 0.00 central european (ce) 5 2.98 5 2.42 6 5.26 central european-mediterranean (ce-med) 13 7.74 15 7.25 9 7.89 mediterranean-submediterranean (med-smed) 8 4.76 16 7.73 6 5.26 fig. 4. comparative overview of the percentage representation of life forms in the flora of the investigated salt march areas biologica nyssana 5 (2)  december 2014: 91-102 zlatković, i. et al.  taxonomical, phztogeographical and ecological … 101 occupy the second place by number of species. the third place is occupied by the life form of geophytes. the pronounced presence of geophytes in salt marsh habitats is explained by their adaptation to unfavorable habitat and climate conditions, primarily during the arid summer period. presence of deeper water level in spring period and deeper temporary and permanent ponds enables appearance of hydrotherophytes and hydrogeophytes. the real hydrophytes were best represented at the aleksandrovac salt marsh area, due to proximity of accumulation lake. the water from the accumulation floods the coastal part of salt marsh in spring months, remaining for several months and enabling development of marsh vegetation of saline terrain. salt marshes are characterized by specific water regime. during the vegetation period they are flooded for a certain period of time. these conditions are not suitable for development of most chamaephytes. the percentage of chamaephytes is somewhat greater at lalinac salt marsh area than in other habitats, as a consequence of presence of welldeveloped steppe habitats. furthermore, the salt marshes of lalinac area are the only site where exclusively chamaephyte phytocoenoses of halophyte camhorosma mospelliaca were recorded. regarding the taxonomic diversity and endemism of flora, the salt marsh areas of central and southern serbia may be considered to be one of the more important centers of diversity for salt marsh flora and vegetation in the region south of the pannonian plain. as all three salt marsh areas are under strong anthropogenous influence, it is necessary to implement appropriate measure for conservation and protection of their flora and vegetation. acknowledgements. the ministry of education, science and technological development of the republic of serbia (grant 173030) supported this research. one part of the results was collected during the research activities in period 2003-2005, within the project “flora and vegetation of salt marshes of central and southern serbia and their valorization from the aspect of conservation”, financially supported by the institute for nature conservation of serbia. references euro+med, 2006-: euro+med plantbase the information resource for euro-mediterranean plant diversity. published on the internet http://ww2.bgbm.org/europlusmed/ greuter, w., burdet, h.m., long, g. (ed.), 19841989: med-checklist, 1, 3, 4. gèneve. йорданов, д., (ед.), 1963-1986: флора на нр българия, i-viii. издателство на бан. софия. josifović, m. (ed.), 1970-1977: flora sr srbije, iix. sanu. beograd. meussel, h., jager, e., weinert, e., 1965: vergleinchende chorologie der zentraleuropaischen flora. veb. gustav fischer verlag, 1. jena. meussel, h., jager, e., raischert, s., weinert, e., 1978: vergleinchende chorologie der zentraleuropaischen flora. veb. gustav fischer verlag, 2. jena. meusel, h., jager, e., 1992: vergleichende chorologie der zentraleuropaischen flora, karten, literatur, register. gustav fischer, jena, stuttgart, new york. milosavljević, v., ranđelović, v., zlatković, b., 2002: vegetacija lalinačke slatine kod niša. 7. simpozijum o flori srbije i susednih područja. zbornik rezimea, 47. dimitrovgrad. mueller-dombois, d., ellenberg, h., 1974: aims and methods of vegetation ecology. john wiley and sons. new york. niketić, m., 1995: pregled flore šireg područja lalinačke slatine kod niša. ii simpozijum o flori srbije (iv simpozijum o flori jugoistočne srbije). – zbornik rezimea, 34, vranje. pančić, j., 1884: dodatak flori kneževine srbije. kraljevska srpska državna štamparija. beograd. ranđelović, v., amidžić, l., ilić, n., 2000: halofitska vegetacija okoline prokuplja. 6. simpozijum o flori jugoistočne srbije i susednih područja. zbornik rezimea, 39. sokobanja. raunkiaer, c. 1934: the life forms of plants and statistical plant geography; being the collected papers of c. raunkiaer, translated into english by h. g. carter, a. g. transley and miss fausboll. london. sarić, m. (ed.), 1986: flora sr srbije, x. sanu. beograd. sarić, m. (ed.), 1992: flora srbije, i (2 izd.). sanu. beograd. slavnić, ž., 1940: prilog halofitskoj flori i vegetaciji jugoistočne srbije. glasnik skopskog naučnog društva, 22: 65-77, skoplje. stevanović, v. 1992: floristička podela teritorije srbije sa pregledom viših horiona i odgovarajućih flornih elemenata. in: sarić, m.r. (ed.), flora srbije, 1, 47-56. sanu, beograd. turrill, w.b., 1929: the plant-life of the balkan peninsula. a phytogeographical study. clarendon, oxford. 490 p. tutin, t. g., heywood, v. h., burges, n. a., valentine, d. h., walters, s. m. & webb, d. a. (eds.), 1964: flora europaea, 1. cambridge. biologica nyssana 5 (2)  december 2014: 91-102 zlatković, i. et al.  taxonomical, phytogeographical and ecological analysis … 102 велчев, в. (ед.), 1989: флора на нр българия, ix. издателство на бан. софия. zlatković, b, randjelović, v., amidzić, l., 2003: flora, vegetation and conservation of aleksandrovac's salth marsh. third international balkan botanical congress. abstracts, 134, sarajevo. zlatković, b, randjelović, v., amidzić, l., 2005: novi podaci o flori slatina centralne i južne srbije. 8. simpozijum o flori jugoistočne srbije i susednih područja. selvaananthi and beulah jerlin 2024, biologica nyssana 15(2) 15 (2) december 2024: 91-96 doi: 10.5281/zenodo.13944272 effect of agricultural substrates mixture on yield and growth performance of two different species of pleurotus original article selvaananthi a. research scholar, register number: 21212212262009, department of botany, st. mary’s college, (autonomous), thoothukudi-1, affiliated to manonmaniam sundaranar university, abishekapatti, tirunelveli 627012, tamilnadu, india selvaananthia1997@gmail.com (corresponding author) https://orcid.org/0000-0001-5498-7582 beulah jerlin s. assistant professor, department of botany, st. mary’s college (autonomous), thoothukudi-1, affiliated to manonmaniam sundaranar university, abishekapatti, tirunelveli 627012, tamilnadu, india https://orcid.org/0000-0002-5480-5782 received: august 06, 2024 revised: september 30, 2024 accepted: october 03, 2024 abstract: mushroom cultivation provides a sustainable way to manage agricultural waste by utilizing organic residues as substrates. this approach not only promotes recycling but also improves agricultural productivity by reusing available resources. this study examines the growth and yield performance of pleurotus florida and p. ostreatus on three substrates such as paddy straw (a), banana leaves (b), and a 1:1 mix of both (c). pleurotus florida colonizes the substrate faster, while p. ostreatus outperforms in yield, biological efficiency, and organic mass loss. both species produced higher yields on substrates b and c than on a. moreover, both species reduced substrate ph effectively. pleurotus ostreatus acidified the substrate more efficiently than p. florida. the findings emphasize the importance of selecting appropriate substrates to optimize oyster mushroom cultivation, improving both yield and substrate utilization efficiency. key words: oyster mushroom, agriculture waste, paddy straw, banana leaves apstrakt: efekat mešavine poljoprivrednih supstrata na prinos i performanse rasta dve različite vrste roda pleurotus gajenje pečuraka predstavlja održiv način upravljanja poljoprivrednim otpadom korišćenjem organskih ostataka kao supstrata. ovaj pristup ne samo da podstiče reciklažu, već i poboljšava poljoprivrednu produktivnost ponovnim korišćenjem dostupnih resursa. ova studija ispituje rast i performanse prinosa vrsta pleurotus florida i p. ostreatus na tri supstrata: pirinčana slama (a), listovi banane (b) i mešavina ova dva supstrata u odnosu 1:1 (c). pleurotus florida brže kolonizuje supstrat, dok p. ostreatus daje bolje rezultate u pogledu prinosa, biološke efikasnosti i gubitka organske mase. obe vrste su produkovale veće prinose na supstratima b i c u poređenju sa supstratom a. pored toga, obe vrste su efikasno smanjile ph supstrata, s tim što je vrsta p. ostreatus to postigla efikasnije od vrste p. florida. rezultati naglašavaju važnost odabira odgovarajućih supstrata za optimizaciju uzgoja bukovača, poboljšanje prinosa i efikasnost korišćenja supstrata. ključne reči: bukovača, poljoprivredni otpad, pirinčana slama, listovi banane introduction mushroom cultivation has gained significant attention in recent years due to its potential for sustainable food production, waste recycling, and health benefits. among the various species cultivated, pleurotus florida and p. ostreatus are particularly popular due to their rapid growth, high nutritional value, and adaptability to a wide range of substrates. these oyster mushrooms are known for their unique flavors, medicinal properties, and economic importance, making them valuable in both culinary and medicinal markets. oyster mushrooms are esteemed as a food source today not only for their unique flavor and texture (mowsurni & chowdhury, 2010). individuals are increasingly prioritizing their dietary choices and physical activity in contemporary society (tagde et al., 2021). beneficial chemical, nutritional, and therapeutic attributes (sifat et al., 2020) as well as their associated health benefits add more interest towards oyster mushrooms (roy et al., 2015). pleurotus has emerged as the most widely consumed and favored mushroom due to its appealing taste, © 2024 selvaananthi and beulah jerlin. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 91 distinctive flavor, substantial nutritional profile, and therapeutic benefits. these fungi provide a healthy alternative that contains minimal calories, fats, and sugars, yet offers a wealth of fiber, protein, chitin, and vital minerals. agricultural waste, consisting of residues left after crop harvest and processing, represents a significant and often underutilized resource in many farming systems. this waste includes straw, husks, leaves, stalks and other plant materials that are typically discarded or burned, contributing to environmental pollution and greenhouse gas emissions (iscia & demirer, 2007). however, these agricultural wastes hold considerable potential as substrates for mushroom cultivation. by converting agricultural waste into a growth medium for mushrooms, farmers can enhance the sustainability of their operations, reduce waste disposal costs and generate an additional source of income (rachna & sodhi, 2013). understanding the growth performance, morphological characteristics, and substrate interactions of these species is crucial for optimizing cultivation practices and maximizing yield. this study focuses on comparing p. florida and p. ostreatus across two substrates such as paddy straw and banana leaves. the study also delves into the morphological aspects of the mushrooms, examining pileus and stipe dimensions, which are important for marketability and consumer preference. additionally, the changes in substrate ph provide insights into the metabolic activities of the fungi and their ability to utilize and degrade the substrate efficiently. materials and methods collection of agricultural waste material agro-waste materials such as paddy straw and banana leaves were used for the present study. paddy straw and banana leaves were collected from the farmers. these substrates were dried and stored for further use. purchase of spawn sorghum grain-based spawns of p. florida and p. ostreatus were procured from the indian institute of horticulture research, bangalore and used for the experiment. experimental design selected species were cultivated by bag method using two different agro-waste as substrates. substrate a: paddy straw substrate b: banana leaves substrate c: 1:1 ratio of paddy straw and banana leaf cultivation of mushrooms in the present study, selected edible oyster mushrooms were cultivated by following the standard procedure of tamil nadu agricultural university. the selected agro wastes were cut into small pieces (6-10 cm) and soaked in water for 12–14 h. the soaked substrates were sterilized at 121 °c for 20-30 min by using an autoclave and excess amount of water content was removed. before starting the packing of mushroom growing bags, hands were washed thoroughly with the help of antiseptic lotion. a polypropylene bag with a size of 60×30 cm and with a thickness of 80 gauge was used for the cultivation. the bottom end of the bag was tied with the help of thread and turned toward the inside. the sterilized substrate was filled in the bag to a height of 3 inches. a handful of grainbased spawn was sprinkled over the layer. likewise, a few layers were placed on the bag. finally, the bag was pressed gently and tied with a thread. a few holes were made in the bags to facilitate ventilation. the spawned bags were kept in a dark room for 1 week to facilitate the spawn running and colonization. then the bags were transferred to the cropping room. the optimum temperature of 22 °c to 25 °c and required humidity of 85% was maintained by spraying water on the walls of the mushroom unit four to five times a day. after colonization, the mycelium starts to produce its reproductive structure called fruiting bodies. initially, it looked like a pinhead, and it was transformed into a fully matured fruiting body within two days. after maturity, the pileus edges start to shrink towards the inside. at this stage, the fruiting bodies were collected manually and used for further experiments. growth parameters colonizing period (spawn run): colonizing period, also known as pawn running or mycelium running, is a crucial phase in mushroom cultivation. the spawn run period is the time span from spawning to the first appearance of pinheads, indicating that the mycelium has fully colonized the substrate (pandey et al., 2023). the number of days required for the colonization of fungal mycelium is counted from the day of packing. pinheads and fruitbodies developed: pinheads and fruiting bodies developed on the substrate were counted manually. yield: after the maturation, the fruitbodies were handpicked and immediately weighed. biological efficiency (carvalho et al., 2012): biological efficiency is a term frequently used in the mushroom industry to describe the potential of the macro fungus to yield a fruiting body (mushroom) from a known dry weight of substrate. 92 biologica nyssana ● 15 (2) december 2024: selvaananthi and beulah jerlin ● effect of agricultural substrates mixture on yield and growth performance of two different species of pleurotus biologica nyssana ● 15 (2) december 2024: selvaananthi and beulah jerlin ● effect of agricultural substrates mixture on yield and growth performance of two different species of pleurotus 93 biological efficiency (%) = (fresh weight of mushroom/dry weight of the substrate) × 100 organic mass loss (carvalho et al., 2012): the organic mass loss of the substrate was calculated by using the following formula. organic mass loss (%) = (initial substrate dry mass – residual/initial substrate dry mass) × 100 morphological parameters: length and width of stipe and pileus were measured immediately after harvesting with the help of thread and measuring scale. ph analysis of the substrate (jackson, 1965): the ph of the substrates was determined using a digital ph meter. ten grams of sample were taken in 100 ml distilled water and stirred continuously for 30 min with a glass rod. the ph of the suspension was recorded after half an hour of settling by the ph meter pre-calibrated using standard buffers of ph 4.0, 7.0 and 9.0. results and discussion table 1 presents the comparative data on the growth and yield of two species of mushrooms, p. florida and p. ostreatus, grown on three different substrates labelled a, b, and c. the key metrics provided include the colonizing period, yield, number of pinheads, percentage of pinheads that develop into fruiting bodies, biological efficiency and organic mass loss. colonizing period (spawn run) and pinhead initiation during this phase, the mushroom mycelium, which is the vegetative part of the fungus, colonizes the substrate. the process begins with the inoculation of the substrate with mushroom spawn. the spawn contains mycelium, which acts similarly to seeds in plants. for pleurotus species spawn run duration differs depending on the species type and substrate used. pleurotus florida consistently shows a shorter spawn run period compared to p. ostreatus across all substrates, with the shortest time on substrate a (18 days) and the longest on substrates b and c (21 days). pleurotus ostreatus has a longer spawn run period, ranging from 24 days on substrate a to 27 days on substrate b. pinhead initiation is the stage where the first mushroom primordia or „pinheads” emerge. it is a critical phase in mushroom cultivation, signifying the transition from vegetative growth to the fruiting stage. this phase is pivotal because it directly influences the subsequent development and yield of the mushrooms. in the current study, p. florida demonstrated faster pinhead initiation across all substrates compared to p. ostreatus. for p. florida, pinheads appeared after 23 days on substrate a, 26 days on substrate b, and 24 days on substrate c. in contrast, p. ostreatus exhibited a longer time to pinhead initiation, with 29 days on substrate a, and 31 days on both substrates b and c. these differences highlighted the quicker adaptation and readiness of p. florida to fruit across various substrates, which could be advantageous for growers seeking shorter production cycles. understanding the factors that influence pinhead initiation, such as substrate composition, environmental conditions, and species-specific growth characteristics, is essential for optimizing cultivation practices to maximize yield and efficiency. our results were found to be similar to the results of pandey et al. (2023) who reported colonization and pin head formation around 20 to 28 days in which banana leaves take maximum days. the faster mycelium growth rate in rice straw can be linked to its balanced composition of alpha cellulose, hemicellulose, and lignin (mondal et al., 2010). the time required for pinhead formation varies based on the substrate and is affected by factors like moisture content and nutrient availability (iqbal et al., 2016; muswati et al., table 1. effect of different substrate on the growth, morphology and yield performance of pleurotus florida and pleurotus ostreatus sp ec ie s su bs tr at es sp aw n ru n pe ri od (d ay s) pi nh ea d in iti at io n (d ay s) y ie ld (g ra m ) n o. o f p in h ea ds fr ui tin g bo dy d ev el op ed fr om p in he ad s ( % ) b io lo gi ca l e ffi ci en cy (% ) o rg an ic m as s l os s ( % ) p. florida a 18 23 425 70 49.5 85 30 b 21 26 460 55 64.2 92 35 c 21 24 445 59 71.2 89 32 p. ostreatus a 24 29 510 51 55.2 102 38 b 27 31 565 42 53. 9 113 43 c 26 31 571 47 63.8 114.2 42 94 biologica nyssana ● 15 (2) december 2024: selvaananthi and beulah jerlin ● effect of agricultural substrates mixture on yield and growth performance of two different species of pleurotus 2021), a trend reflected in our study as well. the differences in pinhead formation times across various substrates can be explained by the variations in nutrient and moisture availability. number of pinheads and percentage of fruitbodies developed from pinheads in terms of the number of pinheads, p. florida outperforms p. ostreatus across all substrates. the higher number of pinheads for p. florida suggests a greater initial fruiting potential, which could lead to higher overall yields if a significant proportion of these pinheads develop into mature fruiting bodies. the conversion of pinheads into fruiting bodies, however, presents a more nuanced picture. on substrate a, p. ostreatus has a higher conversion rate of 55.2%, indicating that a larger proportion of its initial pinheads develop into mature fruiting bodies compared to p. florida, which has a conversion rate of 49.5%. conversely, on substrate b, p. florida demonstrates a superior conversion rate of 64.2%, significantly higher than the 53.9% observed for p. ostreatus. this trend is further accentuated in substrate c, where p. florida achieves a conversion rate of 71.2%, outperforming p. ostreatus, which has a conversion rate of 63.8%. yield, biological efficiency and organic mass loss when comparing the yield, p. ostreatus performs better than p. florida across all substrates. for substrate a, p. ostreatus yields 510 grams, whereas p. florida produces 425 grams. this trend continues with substrate b and c. this data highlights p. ostreatus as the more productive species in terms of yield across all tested substrates. biological efficiency, which measures the ratio of the weight of the harvested mushrooms to the weight of the dry substrate, expressed as a percentage, also shows a clear advantage for p. ostreatus. on substrate a, b and c p. ostreatus achieves a greater biological efficiency than p. florida. these results indicate that p. ostreatus is more efficient in converting substrate into mushroom biomass. organic mass loss, indicating the percentage of organic matter in the substrate that is consumed during the cultivation process, varies between the two species. p. ostreatus tends to have higher organic mass loss across all substrates, which suggests a more extensive utilization of the substrate. the higher organic mass loss for p. ostreatus could correlate with its higher yield and biological efficiency, reflecting a more complete breakdown and utilization of the substrate components. pleurotus ostreatus demonstrates superior performance in terms of yield, biological efficiency, and organic mass loss compared to p. florida across all substrates. pleurotus ostreatus yields more mushrooms, exhibits higher biological efficiency, and incurs greater organic mass loss, indicating a more effective conversion of substrate into biomass. thomas et al. (2022) determined the biological efficiency of p. ostreatus which falls between 54.5% and 130.9%. the findings of neupane et al. (2018) also reported highest yield on banana leaf substrates than rice straw, wheat straw, rice straw + wheat straw and sawdust. our results didn’t agree with the results of mahat et al. (2022) who reported lowest yield in banana leaf substrates and the reason might be the use of semi-dried leaves in their experiment which contain low lignocellulose to hydrolyze into sugar. tirkey et al. (2017) reported highest yield with a biological efficiency of 91% in the banana leaf substrates. length and width of pileus and stipe pleurotus florida and p. ostreatus exhibit different trends in pileus dimensions across the substrates (tab. 2). on substrate a, p. florida shows a slightly larger pileus length (6.9 cm) and width (7.7 cm) compared to p. ostreatus (6.3 cm length, 7.2 cm width). substrate b results in p. ostreatus having a slightly longer pileus (6.5 cm) than p. florida (6.2 cm), but p. florida maintains a broader pileus width (7.4 cm vs. 7.9 cm for p. ostreatus). on substrate c, both species show increased pileus dimensions, with p. ostreatus having a slightly longer pileus (6.9 cm) compared to p. florida (6.6 cm), while p. florida has a wider pileus (9.2 cm) compared to p. ostreatus table 2. length and width of the pileus and stipe t re at m en t pleurotus florida pleurotus ostreatus pileus stipe pileus stipe length (cm) width (cm) length (cm) width (cm) length (cm) width (cm) length (cm) width (cm) a 6.9±0.3 7.7±0.4 3.4±0.4 1±0.4 6.3±0.4 7.2±0.3 2.8±1.1 1.7±0.4 b 6.2±0.5 7.4±0.8 3.1±0.2 1.1±0.3 6.5±0.6 7.9±0.5 1.9±0.9 1.5±0.5 c 6.6.0±0.9 9.2±1.2 2.7±0.2 1.5±0.3 6.9±0.4 8.1±0.4 2.2±0.5 1.8±0.2 biologica nyssana ● 15 (2) december 2024: selvaananthi and beulah jerlin ● effect of agricultural substrates mixture on yield and growth performance of two different species of pleurotus 95 (8.1 cm). regarding the stipe dimensions, p. florida shows longer stipe lengths across all substrates compared to p. ostreatus. in terms of stipe width, p. ostreatus generally has a broader stipe across all substrates. pleurotus florida generally shows longer pileus lengths and widths on substrates a and c compared to p. ostreatus, while p. ostreatus demonstrates slightly longer pileus lengths on substrate b and consistently broader pileus widths across all substrates. for stipe dimensions, p. florida consistently exhibits longer stipe lengths, whereas p. ostreatus shows greater stipe widths across all substrates. these morphological differences can influence the visual appeal and marketability of the mushrooms, as well as their suitability for different culinary applications. our report corroborated with the findings of dubey et al. (2019) who reported highest stipe length in rice straw substrates. ph changes in substrates during the mushroom cultivation process, significant changes in substrate ph were observed for both species (tab. 3). on substrate a, the initial ph of 6.3 was reduced to 5.2 by p. florida and to 5.5 by p. ostreatus, indicating that p. florida was more effective in acidifying the substrate. for substrate b, which had an initial ph of 5.2, both species lowered the ph, with p. florida achieving a final ph of 4.6 and p. ostreatus reaching 4.5, showing that p. ostreatus had a slightly greater capacity to reduce the ph. on substrate c, starting with an initial ph of 5.6, p. florida decreased the ph to 5.1, while p. ostreatus reduced it further to 4.9, highlighting the superior ability of p. ostreatus to acidify the substrate. these results demonstrate the impact of both mushroom species on substrate ph, with p. ostreatus generally achieving lower ph levels compared to p. florida. the ph of the substrate plays a crucial role in oyster mushroom cultivation. the reduction in ph may be due to the metabolic activity and decomposition process of the substrate by the fungus. research conducted by zubairi et al. (2022) found that the ph values of the substrate before spawning were between 8 and 9, which dropped to between 4 and 6 after spawning, demonstrating a significant reduction and acidification of the substrate. conclusion this study highlights the effectiveness of paddy straw, banana leaves and their combinations in optimizing the growth, yield, and biological efficiency of p. florida and p. ostreatus. pleurotus ostreatus demonstrated higher yield and biological efficiency, while p. florida exhibited faster growth rates, making both species viable for different cultivation goals. the results underscore the potential of using readily available agricultural waste to reduce costs and promote sustainable practices. future research should focus on refining substrate combinations and investigating more agricultural residues to improve yield, growth efficiency and improved nutrient content. additionally, understanding the genetic mechanisms behind substrate utilization could lead to even more effective cultivation techniques. farmers are encouraged to explore local agricultural waste as substrates, particularly rice straw and banana leaves which showed promising results in this study. adopting these sustainable cultivation methods can lower production costs, increase yields, and contribute to environmental conservation. farmers should also monitor moisture and nutrient levels in substrates to optimize mushroom growth and maximize their economic returns. references carvalho, c.s.m., aguiar, l.v.b., & andrade, m.c.n. 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(2007). biogas production potential from cotton wastes. renewable energy, 32(5), 750-757. table 3. ph changes in the substrate used for pleurotus florida and pleurotus ostreatus su bs tr at e in iti al ph final ph p. florida p. ostreatus a 6.3 5.2 5.5 b 5.2 4.6 4.5 c 5.6 5.1 4.9 journal of krishi vigyan, 2(1), 26-29. roy, d.n., azad, a.k., sultana, f., anisuzzaman, a.s.m., & khondkar, p. (2015). nutritional profile and mineral composition of two edible mushroom varieties consumed and cultivated in bangladesh. the journal of phytopharmacology, 4(4), 217220. sifat, n., lovely, f., zihad, s.n.k., hossain, m.g., shilpi, j.a., grice, i.d. & uddin, s.j. (2020). investigation of the nutritional value and antioxidant activities of common bangladeshi edible mushrooms. clinical phytoscience, 6, 1-10. tagde, p., tagde, p., islam, f., tagde, s., shah, m., hussain, z.d., & abdel-daim, m.m. (2021). the multifaceted role of curcumin in advanced nanocurcumin form in the treatment and management of chronic disorders. molecules, 26(23), 7109. thomas, a.s.s., arivu, s., shanmugam, v., gundupalli, m.p., & amornraksa, s. (2022). paddy straw: an economical substrate for oyster mushroom (pleurotus ostreatus) cultivation. e3s web of conferences, 355, 02019. tirkey, v.j., simon, s., & lal, a.a. (2017). efficacy of different substrates on the growth, yield and nutritional composition of oyster mushroom pleurotus florida (mont.) singer. journal of pharmacognosy and phytochemistry, 6(4), 10971100. zubairi, s.i., zabidi, n.a.s.m., azman, z.z., kamaruddin, s.n.d.m., kasim, z.m., lazim, a.m., nurzahim, z., & jamil, m.s.m. (2022). pleurotus ostreatus cultivation: physico-chemical characteristics of a robust pre-blocks oyster mushroom substrate with absorptive starch binders. sains malaysiana, 51(2), 329-343. https://doi. org/10.17576/jsm-2022-5102-01 jackson, m.l. (1965). soil chemical analysisadvanced course. usa, wisckonsin: university of wisconsin, department of soils. mahat, j., dhami, d., bhatt, d., & bhandari, k. (2022). effect of different substrates on growth and yield performance of oyster (pleurotus ostreatus) mushroom at gokuleshwor, baitadi. tropical agroecosystems, 3(1), 07-11. mondal, s., rehana, j., noman, m., & adhikary, s. (2010). comparative study on growth and yield performance of oyster mushroom (pleurotus florida) on different substrates. journal of the bangladesh agricultural university, 8(2), 213–220. mowsurni, f.r. & chowdhury, m.b.k. (2010). oyster mushroom: biochemical and medicinal prospects. bangladesh journal of medical biochemistry, 3(1), 23-28. muswati, c., simango, k., tapfumaneyi, l., mutetwa, m., & ngezimana, w. (2021). the effects of different substrate combinations on growth and yield of oyster mushroom (pleurotus ostreatus). international journal of agronomy, 1–10. neupane, s., thakur, v., bhatta, b., pathak, p., gautam, b.b., & aryal, l. (2018). performance of different substrates on the production of oyster mushroom (pleurotus florida) at gokuleshwor, darchula. international journal of scientific and research publications, 8(1), 231-240. pandey, k.r., joshi, y.r., bhattarai, s., joshi, d., subedi, s., pant, p.k., & khatri, s. (2023). evaluation of local substrates as rice straw alternatives for oyster mushroom (pleurotus ostreatus) cultivation in resource-constrained darchula, nepal. archives of agriculture and environmental science, 8(4), 535-544. rachna, g.r. & sodhi, g.p.s. (2013). evaluation of vocational training programmes organized on mushroom farming by krishi vigyan kendra patiala. biologica nyssana ● 15 (2) december 2024: selvaananthi and beulah jerlin ● effect of agricultural substrates mixture on yield and growth performance of two different species of pleurotus 96 stojičić, d., tošić, s., pavlović, j., golubović, a., simonović, j., zlatković, b.: factors influencing axillary bud induction on nodal segments of micromeria pulegium (rochel) benth. biologica nyssana, 8 (1), september 2017 biologica nyssana 8 (1)  september 2017: 93-98 stojičić, d. et al.  factors influencing axillary bud induction on nodal… 93 original article received: 16 june 2017 revised: 29 june 2017 accepted: 15 july 2017 factors influencing axillary bud induction on nodal segments of micromeria pulegium (rochel) benth. dragana stojičić*, svetlana tošić, jovana pavlović, aleksandra golubović, jelena simonović, bojan zlatković university of niš, faculty of science and mathematics, department of biology and ecology, višegradska 33, niš, serbia * e-mail: draganadstojicic@gmail.com abstract: stojičić, d., tošić, s., pavlović, j., golubović, a., simonović, j., zlatković, b.: factors influencing axillary bud induction on nodal segments of micromeria pulegium (rochel) benth. biologica nyssana, 8 (1), september 2017: 93-98. micromeria pulegium (rochel) benth. is an endemic species from family lamiaceae. plants from this family are characterized by presence of secondary metabolites and antioxidant components. micromeria pulegium contains pulegone which is a potential bio-insecticide and a bio-pesticide. natural populations of this species are so small that there is a need for an alternative way of propagate and proliferation of individuals. method of micropropagation was used with the goal of mass production of plants with the chemical composition of essential oils as similar as possible to that in wild-harvested plants. this paper presents the study on influence of concentration of mineral salts, carbon sources (sucrose and maltose) and nitrogen source (casein hydrolysate) on process of in vitro regeneration of plants through induction of axillary buds on the nodal segments of micromeria pulegium. the greatest number of axillary buds was formed in explants grown on ms culture medium with 3% sucrose and 500 mg/l casein hydrolysate. key words: axillary bud induction, shoot culture, biomass production apstrakt: stojičić, d., tošić, s., pavlović, j., golubović, a., simonović, j., zlatković, b.: faktori koji utiču na indukciju aksilarnih pupoljka na nodalnim segmentima vrste micromeria pulegium (rochel) benth.. biologica nyssana, 8 (1), septembar 2017: 93-98. micromeria pulegium (rochel) benth. je endemična vrsta iz familije lamiaceae. ovu familiju karakteriše prisustvo sekundarnih metabolita i antioksidativnih komponenti. micromeria pulegium sadrži pulegon koji je potencijalni bio-insekticid i biopesticid. prirodne populacije ove vrste imaju mali broj jedinki pa se nameće potreba za pronalaženjem načina njihovog gajenja i umnožavanja. u cilju masovne produkcije biljaka čiji će hemijski sastav etarskih ulja biti najsličniji onome koji sadrže biljke iz prirode primenjena je metoda mikropropagacije. u ovom radu ispitan je uticaj jačine mineralnih soli, izvora ugljenika (saharoze i maltoze) i izvora azota (kazein hidrolizata) na proces regeneracije biljaka in vitro putem indukcije aksilarnih pupoljaka na nodalnim segmentima micromeria pulegium. najveći broj aksilarnih pupoljaka je formiran na eksplantatima gajenim na ms hranljivoj podlozi sa 3% saharoze i 500 mg/l kazein hidrolizata. ključne reči: indukcija aksilarnih pupoljaka, kultura izdanaka, produkcija biomase 8 (1) • september 2017: 93-98 doi: 10.5281/zenodo.1003217 biologica nyssana 8 (1)  september 2017: 93-98 stojičić, d. et al.  factors influencing axillary bud induction on nodal… 94 introduction micromeria pulegium (syn. clinopodium pulegium) belongs to section pseudomelissa within the genus micromeria benth. (family lamiaceae). this genus includes four sections: pseudomelissa, micromeria, cymularia and pineolentia (h a r l e y et al., 2004). according to molecular evidence, emphasizing similarity of selected morphological traits, b r ä u c h l e r et al. (2006) have included species of genus micromeria, sect. pseudomelissa, into clinopodium l. m. pulegium is an endemic species of southern carpathians. its range includes romania, serbia and federation of bosnia and herzegovina. in serbia it was recorded in the east, in the area of svrljiški timok gorge, while previously it used to be present at mountain tara (š i l i ć , 1979). gorge of svrljiški timok is situated at low altitude, so summers are very warm and dry, while winters are characterized by strong winds and snow. habitats are rocky and steep, mostly gorges at 1000-1200 m above sea level. micromeria pulegium is a perennial, medium-sized, erect plant (fig. 1), and its leaves are densely covered in small glands, rendering a pleasant aroma to the plant. species of genus micromeria are characterized by presence of secondary metabolites, serving as the foundation of their diverse biological activity (v l a d i m i r k n e ž e v i ć et al., 2000). these species have antimicrobial properties (s a r a c & u g u r , 2007), mostly based on phenol compounds, flavones and flavonoids, terpenoids and alkaloids (c o w a n et al., 1999; c o s e n t i n o et al., 1999). these aromatic plants have been traditionally used as spices and in alternative medicine (a l h a m w i et al., 2011; d u d a i et al., 2001; t e l c i et al., 2007). antioxidant and antimicrobial activities were recorded in both micropropagated plants of m. pulegium and those collected from natural habitats (t o š i ć et al., 2015). chemical composition of m. pulegium essential oils from native and micropropagated plants was also studied (s t o j i č i ć et al., 2016). endemic species with small populations such as those of m. pulegium, situated in vicinity of urban environment, are often under negative anthropogenic influence. in order to preserve biodiversity, various biotechnological methods have been developed, and among them micropropagation of plants in vitro is highly important (p a u n e s c u , 2009; r e e d et al., 2011; s h a r m a & s h a r m a , 2013). through micropropagation it is possible to multiply selected genotypes and chemotypes of different plants, avoiding collection from their natural habitat. in vitro propagation from field-grown plants through multiplication of nodal segments (axillary shoot formation) is a good method of producing a large number of plants without changing the chemical composition (s a n t o s -g o m e s & f e r n a n d e s f e r r e i r a 2003; a f f o n s o et al. 2007). this study was initiated in order to improve the reliable protocol for rapid propagation of m. pulegium through axillary bud induction from nodal explants. material and methods plant material and source of explants aerial parts of m. pulegium plants, at the vegetative stage of development, were collected from natural populations in svrljiški timok gorge, in august 2012 (fig. 1). voucher specimen (nº 6912) was deposited in the herbarium collection of the faculty of science and mathematics, university of niš (hmn). nodal segments (one-node stem segments, 1 cm long and bearing two axillary buds) were surface-sterilized for 30 min with 25% solution of sodium hypochlorite (6% active chlorine) containing two drops of liquid detergent. after three rinsing in sterile distilled water, the explants were treated with 5% solution of nystatin for 24 hours in order to eliminate possible fungal infections. after that nodal segments were rinsed three times with sterile distilled water, and they were placed in different variations of culture medium. every jar closed with polycarbonate cover. fig. 1. wild-growing plant m. pulegium, svrljiški timok george biologica nyssana 8 (1)  september 2017: 93-98 stojičić, d. et al.  factors influencing axillary bud induction on nodal… 95 culture medium and culture conditions isolated nodal segments were placed horizontally on basal murashige and skoog (ms) medium (1962) supplemented with 3% sucrose (w/v) and 0.7% (w/v) agar (torlak, belgrade) in 250-ml glass jars containing 25 ml of the medium, if not stated otherwise. ten explants were placed in each jar. the ph of the media was adjusted to 5.8 prior to autoclaving at 114 °c for 25 min. cultures were maintained at 25 ± 2°c under conditions of a 16 h/8 h photoperiod, with a photon flux density 45 µe m-2 s-1 provided by cool white fluorescent lamps, at 25 ± 2 °c. variations of media medium strength – the effects of five different medium strengths were tested: 0; 0.25; 0.5; 1.0; or 2.0 times those of ms. the other components in all five media were the same as in ms medium. effects of carbohydrates to determine the influence of different carbon sources, ms medium was supplemented with one of two carbohydrates (sucrose or maltose), each at five different concentrations (0; 0.5; 1; 3; or 5%). effect of enzymatic casein hydrolyzate (ch) as a source of organic nitrogen, ch was tested at the following concentrations 0; 125; 250; 375; or 500 mg/l. measured parameters after 4 weeks in culture, the explants formed axillary buds. the explants reacting positively to treatment were recorded and following parameters were measured. the number of explants producing shoots and number of shoots per explant, as well as explant fresh and dry weight were recorded in order to evaluate the effect of nutritive factors on shoot multiplication. the dry weight of shoots was recorded after drying in separate paper containers for 24 h. buds shorter than 1 mm were disregarded. the table 1. effect of medium strength on micromeria pulegium shoot proliferation, number of shoots per explant, shoot length and shoot fresh and dry weight after 28 days of culture ms explants producing shoots (%) number of shoots per explant shoot length (mm) explant fresh weight (g) explant dry weight (g) 0ms 0 0 0 0 0 0.25ms 80.0 ± 0.1a 8.15 ± 0.32a 6.25 ± 0.40a 0.09 ± 0.01a 0.009 ± 0.001a 0.5ms 85.0 ± 0.1b 8.52 ± 0.51a 8.48 ± 0.46b 0.11 ± 0.01b 0.010 ± 0.001a 1ms 93.3 ± 0.2c 12.32 ± 0.71b 9.31 ± 0.80c 0.21 ± 0.02c 0.017 ± 0.002b 2ms 81.7 ± 0.2a 12.12 ± 0.81b 9.88 ± 1.11d 0.22 ± 0.05c 0.019 ± 0.003b values are mean ± se, n = 60. means in the column followed by different letters are different according to lsd multiple range test (p ≤ 0.05) table 2. effect of carbon source on micromeria pulegium shoot proliferation, number of shoots per explant, shoot length and shoot fresh and dry weight after 28 days of culture carbon source (%) explants producing shoots (%) number of shoots per explant shoot length (mm) explant fresh weight (g) explant dry weight (g) 0 51.7 ± 0.1a 2.33 ± 0.93a 4.93 ± 0.33a 0.06 ± 0.02a 0.002 ± 0.001a maltose 0.5 70.0 ± 0.1b 4.84 ± 0.46b 6.41 ± 0.35ab 0.10 ± 0.02ab 0.005 ± 0.001b 1.0 75.0 ± 0.1b 4.94 ± 0.66b 6.43 ± 0.48ab 0.15 ± 0.03bc 0.005 ± 0.001b 3.0 76.7 ± 0.2bc 4.60 ± 0.66b 6.05 ± 0.48ab 0.11 ± 0.03ab 0.006 ± 0.001b 5.0 68.3 ± 0.2b 6.09 ± 0.21c 6.07 ± 0.44ab 0.24 ± 0.01e 0.020 ± 0.001d sucrose 0.5 80.0 ± 0.1c 9.09 ± 0.21d 7.43 ± 0.44c 0.21 ± 0.01d 0.017 ± 0.001c 1.0 85.0 ± 0.1c 12.52 ± 0.21e 7.00 ± 0.44c 0.18 ± 0.01c 0.016 ± 0.001c 3.0 93.3 ± 0.2d 12.32 ± 0.71e 9.31 ± 0.80d 0.21 ± 0.02d 0.017 ± 0.002c 5.0 81.7 ± 0.2c 10.09 ± 0.21de 6.33 ± 0.44ab 0.21 ± 0.01d 0.019 ± 0.001c values are mean ± se, n = 60. means in the column followed by different letters are different according to lsd multiple range test (p ≤ 0.05) biologica nyssana 8 (1)  september 2017: 93-98 stojičić, d. et al.  factors influencing axillary bud induction on nodal… 96 efficiency of different treatments on growth rate was determined by comparing biomass increase and in vitro proliferation rates, using different methods. biomass increase was calculated on both fresh and dry weight basis. proliferation rate was assessed by counting the number of shoots at subculture and following 4-week treatment under previously defined conditions. statistical methods for each treatment, a total of 60 nodal segments (ten explants per jar) were used, divided into two replicates. data collected from experiments were calculated and statistically analyzed, and differences were tested for significance using anova multiple range test at the significance level of p ≤ 0.05. results and discussion micropropagation is a good method for achieving uniform plant material, and at the same time use of nodal segments for plant regeneration with the goal of mass production is considered a reliable method for many lamiaceae species (d o d e et al., 2003). in most species of lamiaceae family, shoot proliferation demands presence of cytokinin in the nutrient medium, with or without auxin (s a h a et al., 2012; b a k h t i a r et al., 2014). however, the nodal explants of m. pulegium cultured on ms medium without plant growth regulators have produced shoots (s t o j i č i ć et al., 2016). these results were used as a foundation for determining the influence of nutritive factors of nutrient substrate on production of axillary buds on nodal segments of m. pulegium. research on the effects of five different medium strengths has shown that medium without salts and vitamins was not sufficient for development of axillary buds, and necrosis of whole explants happened already in the second week (tab. 1). on medium supplemented with salts and vitamins, the percentage of explants developing shoots was greater when concentration of salts increased. the maximum axillary bud proliferation was obtained at 1ms and 2ms, in contrast to results by f a d e l et al. (2010) with mentha spicata l. and m i š i ć et al. (2006) with salvia brachyodon. they achieved the best results by using medium with ms salts reduced to one half (0.5ms). supplementation of the medium with salts and vitamins promoted elongation of the shoot. the greatest average length of axillary buds was developed on explants grown at medium with 2ms. the explants with the greatest average fresh and dry weight were grown on 1ms and 2ms medium. fig. 2. in vitro plantlets cultured on medium with 750 mg/l caseine hydrolysate (left), flowering in vitro plantlets on same medium (right) explants of m. pulegium grown on culture medium ms without a source of carbon were light green, with sporadically developed axillary buds in leaf axils. the percentage of explants with developing axillary buds was the smallest in the whole study (52%). the values of number of buds, length of buds, fresh and dry mass were also significantly smaller than in axillary buds grown in medium supplemented with sugar (tab. 2). sucrose is often the best source of carbon in micropropagation of various species from family lamiaceae (s u j a n a & n a i d u , 2011). after adding maltose or sucrose table 3. effect of casein hydrolyzate on micromeria pulegium shoot proliferation, number of shoots per explant, shoot length and shoot fresh and dry weight after 28 days of culture ch (mg/l) explants producing shoots (%) number of shoots per explant shoot length (mm) explant fresh weight (g) explant dry weight (g) 0 93.3 ± 0.2b 12.32 ± 0.71a 9.31 ± 0.80a 0.21 ± 0.02b 0.017 ± 0.002a 125 80.0 ± 0.1a 12.11 ± 0.45a 9.90 ± 0.70a 0.19 ± 0.02a 0.018 ± 0.002a 250 90.0 ± 0.1b 12.27 ± 0.24a 9.51 ± 0.51a 0.19 ± 0.02a 0.017 ± 0.002a 500 93.3 ± 0.2b 16.93 ± 0.38c 15.36 ± 0.57b 0.25 ± 0.04c 0.023 ± 0.002b 750 81.7 ± 0.2a 15.06 ± 0.42b 15.01 ± 1.10b 0.25 ± 0.02c 0.024 ± 0.002b values are mean ± se, n = 60. means in the column followed by different letters are different according to lsd multiple range test (p ≤ 0.05) biologica nyssana 8 (1)  september 2017: 93-98 stojičić, d. et al.  factors influencing axillary bud induction on nodal… 97 sugars to the nutrient medium, the percentage of m. pulegium explants with axillary buds has significantly increased (68-93%). the explants grown on medium with sucrose were healthy, with good branching, green, with elongated internodes and a large number of developed axillary buds. the results of our study show that sucrose was a better source of carbon for m. pulegium than maltose. in all studied concentrations sucrose caused a greater number of axillary buds than when the medium contained maltose. the maximum number of buds developed on medium with 1% and 3% sucrose, while the greatest length was recorded in axillary buds growing on medium with 3% sucrose. the explants grown on medium with maltose had bushlike form with shorter internodes. therefore the greatest values of average dry and fresh biomass were recorded in explants grown on medium with the greatest concentration of maltose. casein hydrolysate is commonly used in micropropagation as a source of nitrogen. most of the recorded effect was stimulative (s t o j i č i ć et al., 2008). explants of m. pulegium grown on ms medium with or without casein hydrolysate showed no morphological differences. they were elongated, branching explants, dark green in color, with a large number of developed axillary buds (fig. 2). the stimulative effect of casein hydrolysate was manifested in increase of length of axillary buds and their fresh and dry biomass (tab. 3). on medium with the lowest concentrations of casein hydrolysate this increase was not statistically significant. however, use of casein hydrolysate in concentrations of 500 and 750 mg/l is justified as it leads to statistically significant increase in bud length and biomass. the maximum length of axillary buds was recorded in explants grown on medium with 500 and 750 mg/l of casein hydrolysate. some of the explants grown on this medium produced flowers (fig. 2). in some of the explants grown on medium with the greatest concentration of casein hydrolysate adventive roots have formed spontaneously without addition of auxin to the medium. root formation without auxin was observed in a number of lamiaceae species (z u z a r t e et al., 2010; bassolino et al., 2015), where roots were induced on plant growth regulator free medium. however, in m. pulegium shoots rooted spontaneously on the plant growth regulator free medium; hence, the auxin treatment was used to promote rooting (s t o j i č i ć et al., 2016). conclusion micromeria pulegium (rochel) benth. is characterized by relatively high amounts of the essential oils rich in pulegone that potentially may be used as bio-insecticide and bio-pesticide (k o u l et al. 2008). specimens collected in the wild and grown in vitro both produced essential oil of relatively stable composition (s t o j i č i ć et al., 2016). careful selection of the culture conditions may increase accumulation of biomass and production of secondary metabolites, which may be employed to obtain essential oils for commercial use. results indicate that micropropagation of m. pulegium is influenced by composition of nutritive medium. selection of proper mineral salt concentration, carbon source and especially presence of nitrogen source were shown to be important for stimulation of development and growth of axillary buds in this species. the presented protocol may be used as a foundation in further research in order to produce m. pulegium plants with desired characteristics, especially for production of secondary metabolites. acknowledgements. this research was supported by the ministry of education, science and technological development of the republic of serbia (grant 173015, 173030). references affonso, v. r., bizzo, h. r., salguiero lage, c. l., sato, a. 2009: influence of growth regulators in biomass production and volatile profile of in vitro plantlets of thymus vulgaris l. journal of agricultural and food chemistry, 57 (14): 6392– 6395. al-hamwi, m., bakkour, y., abou-ela, m., ellakany, a., tabcheh, m., el-omar, f. 2011: chemical composition and seasonal variation of the essential oil of micromeria fruticosa. journal of natural products, 4: 147-149. bakhtiar, z., mirjalili, m. h., sonboli, a., moridi farimani, m., ayyari, m. 2014: in vitro propagation, genetic and phytochemical assessment of thymus persicus—a medicinally important source of pentacyclic triterpenoids. biologia, 69: 594–603. bassolino, l., giacomelli, e., giovanelli, s., pistelli, l., casetti, a., damonte, g., bisio, a., ruffoni, b. 2015: tissue culture and aromatic profile in salvia dolomitica codd. plant cell, tissue and organ culture (pctoc), 121 (1): 83–95. brauchler, c., meimberg, h., heubl, g. 2006: new names in old world clinopodium—the transfer of the species of micromeria sect. pseudomelissa to clinopodium. taxon, 55: 977–981. cosentino, s., tuberoso, cig., pisano, b., satta, m., mascia, v., arzedi, e. 1999: in vitro antimicrobial activity and chemical composition of sardinian biologica nyssana 8 (1)  september 2017: 93-98 stojičić, d. et al.  factors influencing axillary bud induction on nodal… 98 thymus essential oils. letters in applied microbiology, 29: 130–135. cowan, m. m. 1999: plant products as antimicrobial agents. clinical microbiology reviews, 12 (4): 564-582. dudai, n., larkov, o., ravid, u., putievsky, e., lewinsohn, e. 2001: development control of monoterpene content and composition in micromeria fruticosa (l.) druce. annals of botany, 88 (3): 349-354. fadel, d. kintzios, s., economou, a., moschopoulou, g., constantinidou, h.i. 2010: effect of different strength of medium on organogenesis, phenolic accumulation and antioxidant activity of spearmint (mentha spicata l.). the open horticulture journal, 3: 3135. harley, r. m., atkins, s., budantsev, a. l., cantino, p. d., conn, b. j., grayer, r., harley, m. m., de kok, r., krestovskaja, t., morales, r., paton, a. j., ryding, o., upson, t. 2004: labiatae. in: kadereit, j. w., kubitzki, k. 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three selected micromeria species from croatia. molecules, 16: 1454-1470. zuzarte, m. r., dinis, a. m., cavaleiro, c., salgueiro, l. r., canhoto, j. m. 2010: trichomes, essential oils and in vitro propagation of lavandula pedunculata (lamiaceae). industrial crops and products, 32:580–587. alfatlawi 2025, biologica nyssana 16(2) 16 (2) december 2025: doi: 10.46793/biolnyss.16.2.16a osteoprotegerin and dna repair gene (ogg1 and xrcc1) in females with breast cancer original article wafaa raji alfatlawi applied chemistry department, college of applied science, university of technology, iraq orcid: 0000-0002-7266-1233 wafaa.r.mohammed@uotechnology.edu.iq (corresponding author) received: july 28, 2025 revised: november 07, 2025 accepted: november 10, 2025 abstract: breast cancer is one of the most common cancers globally and is the second leading cause of death among women. osteoprotegerin (opg) is a member of the tumor necrosis factor receptor superfamily. the ogg1 gene encodes an 8-oxoguanine dna glycosylase, a crucial enzyme in the base excision repair pathway that removes oxidative dna damage to prevent mutations. the xrcc1 gene encodes a scaffold protein essential for the repair of dna damage. the aim of this study was to investigate the associations between breast cancer risk and opg as a diagnostic marker in iraqi females. additionally, we investigated whether breast cancer risk is associated with four amino acid substitution variants in dna repair genes, including xrcc1 arg194trp and ogg1 ser326cys, which are involved in nucleotide excision repair. one hundred thirty-two samples were collected from the baghdad teaching hospital in baghdad, iraq. results show significant differences in opg levels with a p-value of ≤ 0.05. in the ogg1, the allele c/c showed a high odds ratio (or=3.5) in newly diagnosed females with breast cancer. in the chemotherapy patient group, the allele g/g shows a high or of 1.8. the xrcc1 gene analysis revealed that allele a/g shows a high or of 15.2 in newly diagnosed females with breast cancer, while in chemotherapy patients, the allele a/g shows a high or of 2.79. gene polymorphism ser326cys (rs1052133) is linked to increased risk of breast cancer. the xrcc1 arg399gln polymorphism has been related in a significant way to increased breast cancer risk, particularly in newly diagnosed and chemotherapy-treated females. key words: breast cancer, dna repair, osteoprotegerin, xrcc1, ogg1 apstrakt: osteoprotegerin i geni za popravku dnk (ogg1 i xrcc1) kod žena sa karcinomom dojke karcinom dojke je jedan od najčešćih kancera globalno i predstavlja drugi vodeći uzrok smrti kod žena. osteoprotegrin (opg) je član superfamilije receptora tumor nekroznih faktora. gen ogg1 kodira 8-oksoguanin dnk glikozilazu, ključni enzim u procesu popravke putem isecanja baza, kojim se uklanjaju oksidativna oštećenja na dnk. gen xrcc1 kodira uvijen protein, koji je takođe ključan za popravku dnk oštećenja. cilj ove studije bio je da se istraži povezanost između rizika za karcinom dojke i opg-a kao dijagnostičkog markera kod iračanki. pored ovoga, istraživali smo i da li je rizik za karcinom dojke povezan sa četiri varijanti substitucije aminokiselina kod gena za popravku dnk, uključujući xrcc1 arg194trp i ogg1 ser326cys, koji imaju ulogu u popravci dnk isecanjem nukleotida. sto trideset i dva uzorka sakupljena su u edukativnoj bolnici u bagdadu, irak. rezultati su pokazali značajne razlike u nivoima opg sa p-vrednostima ≤ 0,05. kod ogg1, alel c/c pokazao je veliki odnos šansi (or=3,5) kod novodijagnostikovanih žena sa karcinomom dojke. u grupi pacijentkinja koje su primale hemoterapiju, alel g/g pokazao je visok or od 1,8. analiza gena xrcc1 pokazala je da alel a/g pokazuje visok or od 15,2 kod novodijagnostikovanih žena sa karcinomom dojke, dok je alel a/g imao od od 2,79. genski polimorfizam ser326cys (rs1052133) se povezuje sa povećanim rizikom od karcinoma dojke. polimorfizam arg399gln xrcc1 gena je značajno povezan sa povećanim rizikom od kancera dojke, posebno kod novodijagnostikovanih pacijentkinja i onih tretiranih hemoterapijom. ključne reči: karcinom dojke, popravka dnk, osteoprotegerin, xrcc1, ogg1 © 2025 alfatlawi et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. introduction breast cancer is one of the most common cancers in women worldwide, accounting for approximately 570,000 deaths in 2015. over 1.5 million women (25% of all women with cancer) are diagnosed with breast cancer every year throughout the world. the cancer rates have increased in iraq in recent years. this might be explained by increasing life expectancy coupled with changing patterns of behavioral risk factors associated with higher noncommunicable disease risk, such as tobacco and alcohol use, obesity, physical inactivity, and an unhealthy diet. this ratio is expected to increase across all cancer types (salih et al., 2025). breast cancer is a metastatic cancer and can commonly transfer to distant organs such as the liver, bones, lungs, and brain, which mainly accounts for its incurability. early diagnosis of the disease can lead to a good prognosis and a high survival rate (siegel et al., 2018). osteoprotegerin (opg) is a glycoprotein that belongs to the tumor necrosis factor receptor superfamily (tnfrsf11b) and forms homodimers to function as a secreted decoy receptor for ligands like rankl and trail. its primary role is to regulate bone turnover by inhibiting osteoclast genesis and other biological processes, including immune responses, vascular health, and cancer progression (wang et al., 2022). elevated opg levels have been linked to poorer outcomes in some cancers, such as pancreatic cancer and hepatocellular carcinoma, suggesting its value as a prognostic marker. therapeutically, opg can reduce tumor burden in bone metastasis models by inhibiting osteoclast activity, and its ability to block trail offers a potential avenue for enhancing apoptosis in tumor cells (geerts et al., 2020). elfar et al. (2017) tested blood opg and rankl levels and found that serum opg had a sensitivity of 59% and a specificity of 92% in identifying women with breast cancer, while the corresponding values for the rankl/ opg ratio were 73% and 72%, respectively. opg modulates mammary epithelial cell proliferation. thus, the development of breast cancer is affected by abnormalities in the rank–rankl pathway, and the progression of breast cancer is influenced by rank–rankl expression, which is directly linked to the survival and proliferation of mammary epithelial cells (wu et al., 2020). chemotherapy drugs are designed to create free radicals within tumor cells, causing lethal damage to dna and other crucial cellular components, leading to the death of the cell and cancer growth inhibition. the resulting oxidative stress can also lead to serious side effects related to chemotherapy (tas et al., 2005). numerous studies have investigated the role of vitamin d in reducing the risk of breast cancer (nsaif, 2019; nechuta et al., 2011). vitamin d and calcium are metabolically highly correlated dietary factors that exhibit anticarcinogenic effects by regulating cell proliferation, differentiation, and apoptosis in both normal and malignant breast cells (momivand et al., 2024). brca1/2 mutations significantly increase breast cancer risk by impairing dna repair, while variants in xrcc1 and ogg1, which are also involved in dna repair, can modify this risk. xrcc1 acts as a scaffold protein in base excision repair (ber), while ogg1 repairs oxidative dna damage and coordinates other repair enzymes like dna polymerase β and dna ligase iii. mutations in ogg1 and xrcc1 do not cause hereditary breast cancer like brca1/2, but polymorphisms within these genes may affect cancer susceptibility (barili et al., 2024). vitamin d 1,25(oh)2d, the active form of vitamin d, exerts its effects mainly through binding to the nuclear vitamin d receptor (vdr) and then binding to specific dna sequences and vitamin d response elements. through this genomic pathway, 1,25(oh)2d regulates the expression of specific genes in a tissue-specific manner. many studies have shown that 1,25(oh) 2d can inhibit cellular proliferation and angiogenesis, while inducing differentiation and apoptosis in both normal and malignant breast cells (dubey & sharma, 2025). the aim of this study was to investigate the associations between breast cancer risk and the biochemical parameter opg as a diagnostic marker in iraqi females with breast cancer and other parameters. patients were classified into subgroups based on prediagnosis and chemotherapy status, and the results were compared with those of the control group. also, we tested whether breast cancer risk is associated with four amino acid substitution variants in dna repair genes, including xrcc1 arg194trp and ogg1 ser326cys, which are involved in nucleotide excision repair. materials and methods study design the current case-control study includes a total of 132 females with an age range of 25-45 years. the samples were classified into three groups: g1 as the control group, g2 as newly diagnosed with breast cancer, and g3 as women who were given the first and/or second doses of chemotherapy, as shown in fig. 1. flow diagram. all females received medical care at medical city, oncology teaching hospital, department of early diagnosis of breast cancer, baghdad, iraq. the sample collection period was from january to april 2024. biologica nyssana ● 16 (2) december 2025: alfatlawi ● osteoprotegerin and dna repair gene (ogg1 and xrcc1) in females with breast cancer biologica nyssana ● 16 (2) december 2025: alfatlawi ● osteoprotegerin and dna repair gene (ogg1 and xrcc1) in females with breast cancer inclusion criteria: the inclusion criteria for patients were newly diagnosed females with breast cancer, and females who received the first or second dose of chemotherapy, aged between 25 and 45 years old, and before menopause. exclusion criteria: the exclusion criteria for patients were females at menopause and any cases with chronic inflammatory diseases. sample collection: after collecting 10 ml of whole blood, 5 ml of the blood were allowed to clot by leaving the sample at room temperature. this procedure usually takes 10-20 minutes. after removing the clot by centrifuging at 2,000-3,000 rpm for 20 minutes, the biochemical assay was performed. the second part (5 ml) was placed into an edta tube for molecular testing; the resulting sera and blood were stored at -20 °c until the assay. methods biochemical analyses: osteoprotegerin was measured using the opg kit (catalogue number: sl1314hu). the eliza method involves pre-coating the microplate with an opg-specific antibody, adding serum samples to the appropriate microplate wells, and combining them with the specific antibody. this is followed by the addition of horseradish peroxidase-conjugated antibody and, finally, the addition of tmb substrate to stop the reaction. vitamins: the kits used to measure vitamins a, e, and d were from human sunlong/china cat. no.: sl1833hu, sunlong/china cat. no.: sl2328hu, and sunlong/china cat. no.: sl1834hu, respectively. the enzymatic method was used to evaluate calcium, pth, alt, albumin, urea, creatinine, and mda. these methods for analyzing these parameters offer improved specificity and accuracy compared to traditional chemical methods. molecular analyses: pcr-rflp genotyping assays: genomic dna was isolated from 200 µl of whole blood, using the wizprep™ gdna mini kit (blood) (republic of korea). a qualitative pcr protocol was used to detect the presence of ogg1 and xrcc1 genes. pcr-rflp involves digesting pcr products with a restriction enzyme, with the conditions depending on the specific enzyme used and requiring a specific number of enzyme units per amount of dna. a typical protocol includes incubating the pcr product with the enzyme and its corresponding buffer at the enzyme's optimal temperature for a specified time, often between 30 minutes and 3 hours. the amount of enzyme used is critical and is expressed in units, where one unit is the amount of enzyme required to digest a specific amount of dna. preparation of primers: the primers used to identify and amplify the ogg1 (ser326cys) and xrcc1 (arg399gln) polymorphisms by the pcrrflp method, as described by berg et al. (2012), were prepared by integrated dna technologies (idt) in canada. using primers from alpha dna usa, a 200 bp fragment of the hogg1 gene and a 248 bp fragment of the xrcc1 gene were amplified using a specific primer. the pcr materials for the hogg1 and xrcc1 genes are shown in tab.1. fig. 1. flow diagram of the study target gene sequence of primers allele and size of pcr products (bp) gc (%) tm (ᵒc) ogg1 ser326 cys f1: 5'-gga agg tgc ttg ggg aat-3' for the c allele (200 bp) 55 62 r1: 5'-act gtc act agt ctc acc ag-3' 55 62 xrcc1 arg399 gln f1: 5'-caa gta cag cca ggt cct ag-3' for the g allele (248bp) 55 56 r1: 5'-cct tcc ctc atc tgg agt ac-3' 50 60 table 1. sequences of primers used in this study biologica nyssana ● 16 (2) december 2025: alfatlawi ● osteoprotegerin and dna repair gene (ogg1 and xrcc1) in females with breast cancer reverse: 5'-cct tcc ctc atc tcg agt ac3: consequently, the arg/arg (homozygote), arg/gln (heterozygote), and gln/gln genotypes result in 89 and 159 bp; 89, 159, and 248 bp; and 248 bp digestion, respectively. rflp cuts a dna sequence into pieces by using restriction enzymes ncil; the action of this enzyme is specific. statistical analysis: version 26 of spss was utilized for data analysis. mean ± standard deviation (sd) was used to illustrate the results. the data were compared using independent-sample t-tests. correlation was tested using the spearman rank correlation test. significance was set at p<0.05. the demographic characteristics of the cases and controls were compared using fisher's exact tests, student's t-tests, and x² tests. after adjusting for subject characteristics, ors and 95% cis were computed using logistic regression. each two-way interaction between genotypes was taken into consideration. the model was eliminated of non-significant interactions by applying a backward-stepping algorithm. the pcr-rflp and genotyping: a) rs1052133/ogg1 gene: simple pcr-rflp was used to detect the ser326cys variant. the primers used for amplification of the ogg1 gene exon 7 are a-ogg1 forward: 5'-gga agg tgc ttg ggg aat-3 and b-ogg1 reverse: 5'-act gtc act agt ctc acc ag-3'. consequently, the ser/ser (homozygote), ser/cys (heterozygote), and cys/cys genotypes result in 200 bp, 200 and 100 bp, and 100 bp digestion fragments, respectively. restriction fragment length polymorphism (rflp) is a technique that cuts a dna sequence using restriction enzymes (such as fnu4h1) into specific pieces. fig. (2-6) represent the electrophoresis patterns in the studied groups of the ogg1 and xrcc1 genes. b) xrcc1 gene: the distribution of genotyping of codon 399 of xrcc1 following primers is designed to encompass the arg399gln polymorphism site. axrcc1 forward: 5'-caa gta cag cca ggt cctag-3 bxrcc1 fig. 2. electrophoresis pattern of 200 bp pcr product of healthy females, digested with fnu4hi restriction enzyme (2.5% agarose gel). lanes 1, 3, 4 homozygous: gg genotype (100 bp); lanes 6, 7, 8, 11, 12, 13, 18, 21, 28 homozygous: cc genotype (200 bp). lanes 2, 5, 9, 10, 14, 15, 16, 17, 19, 20, 22, 23, 24, 25, 26, 27 heterozygous: gc genotype (100/200 bp). m: dna molecular marker 100 bp in size. bands in the gel were stained with red stain. fig. 3. electrophoresis pattern of 200 bp pcr product of newly diagnosed females, digested with fnu4hi restriction enzyme (2.5% agarose gel). lanes 7, 8, 21, 24, 27 homozygous: gg genotype (100 bp); lanes 1, 2, 5, 6, 11, 14, 16, 17, 18, 20, 22, 23, 25, 26, 28 homozygous: cc genotype (200 bp). lanes 3, 4, 9, 10, 12, 13, 19 heterozygous: gc genotype (100/200 bp). m: dna molecular marker 100 bp in size. bands in the gel were stained with red stain. biologica nyssana ● 16 (2) december 2025: alfatlawi ● osteoprotegerin and dna repair gene (ogg1 and xrcc1) in females with breast cancer fig. 4. electrophoresis pattern of 200 bp pcr product of chemotherapy-treated females, digested with fnu4hi restriction enzyme (2.5% agarose gel): lanes 1, 2, 3, 4, 5, 6, 7, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 homozygous: gg genotype (100 bp); lanes 8 and 9 homozygous: cc genotype (200 bp). m: dna molecular marker 100 bp in size. bands in the gel were stained with red stain. fig. 5. electrophoresis pattern of 248 bp pcr products of newly diagnosed females digested with ncil restriction enzyme (2.5% agarose gel). lanes 1-3, 7-16, 18, 20-22, 25, 27 heterozygous: ag genotype (248/159/89 bp); lanes 19 and 26 homozygous: gg genotype (159/89 bp). lanes 4, 5, 6, 17, 23, 24, 28 homozygous: aa genotype (248 bp). m: dna molecular marker 100 bp in size. bands in the gel were stained with red stain. fig. 6. electrophoresis pattern of 248 bp pcr product (chemotherapy) digested with ncil restriction enzyme (2.5% agarose gel). lanes 2, 3, 7, 12, 13, 14 heterozygous: ag genotype (248/159/89 bp); lane zero homozygous: gg genotype (159/89 bp). lanes 1, 4, 5, 6, 8, 9, 10, 11, 15, 16, 17, 18, 19, 20 homozygous: aa genotype (248 bp). m: dna molecular marker 100 bp in size. bands in the gel were stained with red stain. online tool https://www.easycalculation.com/ health/hardy-weinberg-equilibrium-calculator. php was used to determine the variance of various genotypes in accordance with the hardy-weinberg equilibrium (hwe) principle. biologica nyssana ● 16 (2) december 2025: alfatlawi ● osteoprotegerin and dna repair gene (ogg1 and xrcc1) in females with breast cancer results the body mass index and age in tab. 2 show that there was no significant difference between the patient groups and the control group (p>0.05). tab. 3 shows that all parameters except calcium, opg, alp, estrogen and albumin had significant differences between the groups, especially for vitamin e and d. ogg1 polymorphisms in newly diagnosed patients: tab. 4 presents the frequency distribution of ogg1 gene polymorphisms in the newly diagnosed group compared to the healthy control. the allele c/c (homozygous) genotype was more likely to have a risk of breast cancer (p<0.05), showing an odds ratio (or) of 3.5-fold higher risk (95% ci = 1.22–10.01) when compared with the control group. the allele c/g was associated with a low level of breast cancer risk, with a p<0.05, an odds ratio (or) of 0.2, indicating a 0.2-fold higher risk compared to the control group. in contrast, the allele g/g had an or of 1.8 with a non-significant p-value (p>0.05). the genotype distribution in the ogg1 gene in the healthy group (a) and the newly diagnosed group (b) is shown in fig. 7. ogg1 polymorphisms in the chemotherapy group: the frequency distribution of ogg1 gene polymorphism of the chemotherapy group compared with the healthy control is represented in tab. 5. the allele c/c was more common among chemotherapy patients, with a significantly higher risk (or=3.5, p<0.05) compared with the control parameters group mean ± sd p-value (g1) n=34 (g2) n=34 (g3) n=34 g1 & g2 g1 & g3 g2 & g3 age, years 38.0 ± 4.38 44.04 ± 3.18 37.04 ± 3.38 0.20 0.36 0.12 bmi, kg/m2 26.04 ± 3.38 26.80 ± 7.68 28.14 ± 6.43 0.86 0.33 0.63 table 2. age and bmi in the studied groups p significant at p≤0.05, highly significant at p<0.001, non-significant at p>0.05 g1: control group, g2: newly diagnosed, g3: chemotherapy group parameters group mean ± sd p-value (g1) n=34 (g2) n=34 (g3) n=34 g1 & g2 g1 & g3 g2 & g3 vitamin a (μg\dl) 57.9±16.5 62.6 ±15.8 58.2 ±18.1 0.044 0.33 0.045 vitamin e (μg\dl) 484.1 ±190.2 385.9 ±195.6 587.3 ±256.6 0.016 0.019 0.01 vitamin d (ng\dl) 20.04±2.80 4.98 ± 1.67 9.38 ± 1.43 0.001 0.001 0.001 calcium (mg\dl) 9.28 ± 0.43 9.23 ± 0.38 9.21 ± 0.45 0.866 0.780 0.986 opg (pg\ml) 324.11 ± 104.73 315.0 ± 123.98 313.38 ± 109.02 0.941 0.919 0.998 urea (mg\dl) 27.5 ±8.4 38.3 ±9.6 27.7 ±6.6 0.011 0.32 0.02 creatinine (mg\dl) 0.7 ±0.2 1.0 ±0.2 0.7 ±0.2 0.04 0.04 0.04 alp (u\l) 100.39 ± 20.76 107.70 ± 31.09 105.09 ± 22.97 0.462 0.725 0.906 cortisol (nmol\l) 265.94 ± 81.82 294.89 ± 99.06 345.85 ± 97.66 0.409 0.002 0.067 estrogen (pg\ml) 87.93 ± 37.01 75.60 ± 24.99 76.74 ± 21.87 0.185 0.247 0.985 pth (pg\ml) 68.52 ± 20.44 167.79 ± 35.21 136.52 ± 58.56 0.001 0.001 0.779 uric acid (mg\dl) 3.9± 0.8 4.8 ±1.3 5.0 ±1.1 0.19 0.013 0.18 albumin (g\dl) 4.8±0.25 4.7 ±1.2 4.5 ±0.95 0.13 0.09 0.17 mda (mg\dl) 6.4 ±4.3 4.5 ±3.2 3.0 ±2.3 0.1 0.01 0.15 table 3. biochemical parameter levels in the studied groups bolded: significant at p≤0.05, highly significant at p<0.001, non-significant at p>0.05 g1: control group, g2: newly diagnosed, g3: chemotherapy group biologica nyssana ● 16 (2) december 2025: alfatlawi ● osteoprotegerin and dna repair gene (ogg1 and xrcc1) in females with breast cancer group. on the other hand, a significant difference was also observed in allele c/g (p<0.05), but with a low or of 0.2. a significant association was also observed for the c/g genotype (p<0.05), but with a low odds ratio (or=0.2), indicating a reduced risk of breast cancer. the g/g genotype showed a higher risk (or=1.8); however, this association was not statistically significant (p>0.05). the distribution of ogg1 gene genotypes in the control group (a) and the chemotherapy-treated group (b) is presented in fig. 8. amplification of the xrcc1 gene and polymorphisms: a specific region of the genome was amplified by using pcr. the gel electrophoresis technique is used to visualize the amplified fragment, which is 248 bp in size. a 100 bp dna ladder was used as a molecular weight marker, and xrcc1 polymorphisms were genotyped using a pcr method and primers. the forward primer 5'caa gta cag cca ggt cct ag-3', and the reverse primer 5'-cct tcc ctc atc tgg agt ac-3', were used to amplify the genomic dna, and this process was followed by restriction enzyme digestion. a single band of 248 bp indicated the aa (homozygote), two bands of 89/159 indicated the gg (homozygote), and three bands of 248/159/89 polymorphism in ogg1 groups p-value % x2 or ci 95%g1 n=34 g2 n=34 observed expected observed expected cc 13 10.8 18 15.4 p<0.05 51.3 5.5 3.5 1.22-10.01 cg 18 14.4 11 12.2 p<0.001 40.7 8.3 0.2 0.07-0.61 gg 3 4.8 5 2.4 p>0.05 8 0.5 1.8 0.4-8.11 table 4. genotype distribution of the ogg1 gene c/c, c/g, and g/g polymorphism in newly diagnosed and healthy control groups g1: control group, g2: newly diagnosed patients group, or: odds ratio, p-value: probability considered significant at level p<0.05, ci: confidence interval fig. 7. genotype distribution in the ogg1 gene in the healthy group (a) and the newly diagnosed group (b) polymorphism in ogg1 groups p-value % x2 or ci 95%g2 n=34 g1 n=34 observed expected observed expected cc 9 10.8 11 0.2 p<0.05 1 2.8 3.5 1.22-10.01 cg 18 14.4 5 3.6 p<0.001 18 18.8 0.2 0.07-0.61 gg 7 4.8 18 16.2 p>0.05 81 31.5 1.8 0.4-8.11 table 5. genotype distribution of the ogg1 gene c/c, c/g, and g/g polymorphism in healthy controls and chemotherapy group g1: control group, g2: chemotherapy patient group, or: odds ratio, p-value: probability considered significant at level p<0.05, ci: confidence interval biologica nyssana ● 16 (2) december 2025: alfatlawi ● osteoprotegerin and dna repair gene (ogg1 and xrcc1) in females with breast cancer bp indicated the ag genotype (heterozygote). the frequency distribution of xrcc1 polymorphisms in newly diagnosed female patients compared to a healthy control is represented in tab. 6. a higher risk for breast cancer was observed in the a/g allele, with a highly significant p≤0.01 when compared with the control group. the a/a genotype showed a significant difference (p≤0.01) but was linked to a lower risk (or = 0.11), while the g/g genotype showed no significant association (p≥0.05, or = 0.5, 95% ci). the genotype distribution in the xrcc1 gene for the control (a) and newly diagnosed patient groups (b) is shown in fig. 9. xrcc1 polymorphisms in the chemotherapy group: tab. 7 presents the frequency distribution of xrcc1 gene polymorphisms in the chemotherapy group compared to the healthy control group. the heterozygous genotype a/g showed a higher breast cancer risk with an or of 2.79, while for a/a and g/g, they were non-significant with 0.85 and 0.14, respectively. when compared with the control group, the p≥0.05 in all three genotypes, indicating no statistically significant difference. the genotype distribution in the xrcc1 gene in the control group (a) and chemotherapy group (b) is shown in fig. 10. discussion biochemical parameters: the potential role of vitamins a, e, and d in preventing and treating tumors is a subject of growing interest, supported by a combination of preclinical and clinical studies, as well as safety considerations. vitamins play complex roles in cancer prevention, antioxidant resistance, inflammatory modulation, and epigenetic regulation, which can enhance outcomes by affecting cell differentiation, apoptosis, and proliferation, as well as combating oxidative stress and dna damage (talib et al., 2024). patients who had breast cancer are more likely to have uremia than the control group. increased migration and invasion of breast carcinoma cells are key events in the development of metastasis to lymph nodes and distant organs, which can lead to an elevated level of urea in the blood (yang et al., 2025). increased mda concentration has been reported in breast cancer, gastric and lung cancer, and several studies present evidence that ros are involved in the progression and etiology of this type of cancer (gubaljevic et al., 2018). opg in breast cancer: according to the study in the european prospective investigation for cancer and nutrition (epic)(sarink et al., 2017), elevated serum opg is linked to higher overall and specific mortality rates for breast cancer. its action promoted cell fig. 8. genotype distribution in the ogg1 gene in the control group (a) and in the chemotherapy patient group (b) polymorphism in xrcc1 groups p-value % x2 or ci 95%g1 n=34 g2 n=34 observed expected observed expected aa 22 20.8 7 10.2 p<0.001 34 15.0 0.11 0.04-0.35 ag 4 8.3 21 14.6 p<0.001 48.7 19.8 15.2 4.18-55.02 gg 8 0.9 6 5.2 p>0.05 17.3 0.7 0.5 0.08-2.67 table 6. xrcc1 gene a/a, a/g, and g/g polymorhism genotype distribution in the newly diagnosed brest cancer patients and healthy controls g1: control group, g2: newly diagnosed patients group, or: odds ratio, p-value: probability considered significant at level p<0.05, ci: confidence interval biologica nyssana ● 16 (2) december 2025: alfatlawi ● osteoprotegerin and dna repair gene (ogg1 and xrcc1) in females with breast cancer apoptosis and extended the survival of inflammatory cells by interacting with the endoplasmic reticulum (er) chaperone and master regulator of the unfolded protein response, glucose-regulated protein/binding immunoglobulin protein (grp78/bip). in orthotopic breast cancer xenografts, recombinant opg (ropg) inhibited tumor development and metastasis by reducing the activity of the β-catenin pathway, thereby limiting breast cancer stemness (liu et al., 2015). additionally, studies have shown that opg may have indirect tumor-promoting effects on the development of breast cancer in addition to its direct proliferative effects on breast cancer cells, and in breast cancer tumorigenesis, one hypothesis implies that opg's pro-angiogenic activities encourage the growth of tumors. another hypothesis suggests that opg causes proliferation and aneuploidy in normal mammary epithelial cells, thereby reprogramming them into a tumorigenic state and accelerating cancer development (infante et al., 2019). the mechanism behind the association between bmi and breast cancer risk is controversial; the positive association between bmi and breast cancer risk was speculated to result from the higher level of estrogen derived from the aromatization of androstenedione within the larger fat reserves of women of higher bmi. the negative relationship between higher bmi and breast cancer risk may come from a protective effect of increased weight in the early premenopausal years, which is a predictor of longer anovulatory cycles and a lower level of progesterone and estrogen (radhi et al., 2025) . ogg1 polymorphisms in the newly diagnosed patients group: according to studies done by fig. 9. genotype distribution in the xrcc1 gene in control group (a) and newly diagnosed patient group (b) polymorphism in xrcc1 groups p-value % x2 or ci 95%g1 n=34 g2 n=34 observed expected observed expected aa 22 20.8 14 14.5 p>0.05 72.5 0.07 0.85 0.25-2.89 ag 8 8.3 12 5 p>0.05 25 2.1 2.79 0.69-11.22 gg 4 0.9 8 0.5 p>0.05 2.5 2.9 0.14 0.01-2.63 table 7. genotype distribution of the xrcc1 gene a/a, a/g, and g/g polymorphism in healthy controls and female breast cancer chemotherapy groups g1: control group, g2: chemotherapy patients group, or: odds ratio, p-value: probability considered significant at level p<0.05, ci: confidence interval fig. 10. genotype distribution in the xrcc1 gene in the control group (a) and chemotherapy group (b) biologica nyssana ● 16 (2) december 2025: alfatlawi ● osteoprotegerin and dna repair gene (ogg1 and xrcc1) in females with breast cancer sharma et al. (2020) and huang & zhou (2021), many dna repair genes have different alleles, some of which may alter dna repairing capacity; clarifying the etiology of cancer may be facilitated by the characterization of these genotypic differences in dna repair functions and their association with cancer. our results imply that variations in amino acid substitution of dna repair genes in two repair pathways, especially when combined, may increase the risk of breast cancer and also revealed that female patients with an allele g/g (homozygous) genotype with a risk factor of 4% were more likely to have a disease compared to patients who carry allele a/a (homozygous) genotype and allele a/g (heterozygous) genotype. the arg399gln mutation is located in a hydrophobic region of the xrcc1 gene, which resides between the domains that interact with adp-ribosyltransferase and dna polymerase. the functional significance of the arg194trp region remains unclear, and further research is needed to elucidate its role in dna strand break repair and common oxidative damage. current data indicate that genetic variants in xrcc1 arg399gln correlate with breast cancer risk, aligning with the findings of ruan et al. (2025). nonetheless, the xrcc1 arg194trp variant was not associated with breast cancer risk in an alternative investigation by zoubi et al. (2015). in this study, the or of 81.0 could be explained by genetic instability and impaired dna repair, which significantly increase an individual's susceptibility to cancer due to the accumulation of mutations in critical genes. association between the risk of breast cancer and rs1052133 single-nucleotide polymorphisms: three genetic variants in ogg1 (ser326cys, 7143a/g, and 11657a/g) were linked to the incidence of breast cancer, as shown by rossner and colleagues (rossner et al., 2006). it is yet unknown how oxidative stress affects the risk of breast cancer. ogg1, which catalyzes the removal of dna's 8-oxodeoxyguanosine, the most prevalent lesion resulting from oxidative stress, 8-oxodeoxyguanosine, is highly mutagenic. our aim was to study the association between the polymorphism in the ogg1 gene (snp: rs1052133 (ser326cys)) on exon 7 and genetic variability in ogg1. a variety of human malignancies commonly lose the ogg1 genetic locus (3p26.2); ogg1 mutations, on the other hand, indicate that the observed disease development may be due to factors other than the gene's role in carcinogenesis in the 3p26 chromosome region (ali et al., 2015). different gene sequence mutations can have either no effect or limit the activity of the gene, and this disorder may arise from the majority of these changes that impact gene activity. the mechanism of protecting the genome's integrity, resulting in minimizing the quantity of mutations caused by carcinogens, involves the cellular dna repair process, which stabilizes the genome (clarke & mostoslavsky, 2022). our findings concurred with the results of yuan et al. (2010), who found that the hogg1 ser326cys polymorphism is associated with a high risk of breast cancer in a meta-analysis study. according to a study of wang et al. (2012), women who carry a variant allele of hogg1 ser326cys are likely to be at a higher risk of developing breast cancer. other research has shown that the genotype distribution of the hogg1 ser326cys polymorphism may not have a significant impact on breast cancer incidence (cai et al., 2006). association between the risk of breast cancer and xrcc1 single nucleotide polymorphisms: xrcc1 is believed to be involved in the multistep base excision repair pathway, which eliminates "non-bulky" base adducts created by methylation, oxidation, reduction, or fragmentation of bases by ionizing radiation or oxidative damage, according to several studies on the gene's association with breast cancer risk (hindi et al., 2021; datkhile et al., 2023). the distributions of xrcc1 genotypes may affect an individual's vulnerability to breast cancer, as demonstrated by the study of filip et al. (2025), who investigated an association between dna repair and breast cancer histopathological subtypes. other research has demonstrated that the xrcc1 gene polymorphism's allele frequency distribution may not significantly affect american white women's chance of developing breast cancer (falagan-lotsch et al., 2009). conclusion osteoprotegerin has important roles in breast cancer by promoting tumor growth and spread, but also in the prevention of bone breakdown from metastases. low concentrations of vitamin d are associated with an enhanced risk of breast cancer by affecting cell growth and estrogen release. the ogg1 gene plays a crucial role in repairing dna damage, and its polymorphism, ser326cys (rs1052133), is associated with an increased risk of breast cancer due to the decreased efficiency of the ogg1 protein and impaired dna repair activity. the xrcc1 arg399gln polymorphism has been related in a significant way with increased breast cancer risk, particularly in newly diagnosed and chemotherapy-treated females, as it affects dna repair function, leading to genomic instability and cancer development. in our study, bmi did not show a significant difference between the patient and control groups, although it affected insulin levels and estrogen production. ethical approval: the applied chemistry department, college of applied sciences, university of technology, iraq, approved this study. references ali, k., mahjabeen, i., sabir, m., mehmood, h., & kayani, m. a. 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(2015). x-ray repair crosscomplementing protein 1 and 3 polymorphisms and susceptibility of breast cancer in a jordanian population. saudi medical journal, 36(10), 1163– 1167. https://doi.org/10.15537/smj.2015.10.12659 alfatlawi ● osteoprotegerin and dna repair gene (ogg1 and xrcc1) in females with breast cancer biologica nyssana ● 16 (2) december 2025: dimitrijević et al. 2025, biologica nyssana 16(1) 177 16 (1) june 2025: 177-187 doi: 10.46793/biolnyss.16.1.6d chemical composition and antifungal effect of the essential oil of helichrysum italicum (roth) g. don against clinical isolates of candida spp. original article marina dimitrijević university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18 000 niš, serbia marina.dimitrijevic@pmf.edu.rs (corresponding author) zorica stojanović-radić university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18 000 niš, serbia niko radulović university of niš, faculty of sciences and mathematics, department of chemistry, višegradska 33, 18 000 niš, serbia milica nešić university of niš, faculty of sciences and mathematics, department of chemistry, višegradska 33, 18 000 niš, serbia milica pejčić pejić university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18 000 niš, serbia received: october 29, 2024 revised: january 30, 2025 accepted: january 31, 2025 abstract: this study investigated the chemical composition and antifungal properties of the essential oil derived from helichrysum italicum (roth) g. don, a plant belonging to asteraceae family. a total of 52 compounds were identified using the gc/ms analysis with the main components being neryl acetate (13.4%) and γ-curcumene (10.3%). we examined the antifungal activity of h. italicum oil against human candida isolates and its interaction with the conventional antifungal agent, nystatin. additionally, the effect of oil on biofilm production by candida isolates was investigated. the results revealed a notably strong anti-candida effect, with mic values ranging from 0.312 to 5.00 mg/ml. the oil exhibited a synergistic effect with the tested antifungal drug and notable antibiofilm activity. these findings support further research into plant secondary metabolites as potential new antifungal agents. key words: candidosis, immortelle essential oil, antifungal activity apstrakt: hemijski sastav i antifungalni efekat etarskog ulja helichrysum italicum (roth) g. don na kliničke izolate candida spp. u studiji je predstavljen hemijski sastav i antifungalna svojstva etarskog ulja smilja (helichrysum italicum (roth) g. don) iz porodice asteraceae. ukupno je identifikovano 52 jedinjenja koristeći gc/ms tehnike a među najzastupljenijim komponentama bili su neryl acetat (13.4%) i γ-kurkumen (10.3%). ispitana je antifungalna aktivnost ulja h. italicum na humane izolate kandida, kao i njegova interakcija sa konvencionalnim antifungalnim agensom, nistatinom. pored toga, ispitan je uticaj etarskog ulja na proces formiranja biofilma kod vrsta c. albicans i c. krusei. rezultati su pokazali značajan anti-candida efekat sa mik vrednostima u rasponu od 0,312 do 5,00 mg/ml. ulje smilja pokazalo je sinergistički efekat u kombinaciji sa testiranim antifungalnim agensom, kao i statistički značajnu aktivnost u smanjenju biofilm formi. dobijeni podaci mogu doprineti daljim istraživanjima o biljnim sekundarnim metabolitima kao potencijalno novim antifungalnim agensima. ključne reči: kandidoze, etarsko ulje smilja, antifungalna aktivnost introduction the genus helichrysum mill. comprises over a thousand taxa, known for their prolific production of various secondary metabolites, including flavonoids, pyrones, triterpenoids, and sesquiterpenes. these compounds are likely responsible for the remarkable efficacy of the essential oils derived from plants within this genus. the name helichrysum is derived from the greek words „helios” (sun) and „chrysos” (gold), reflecting the bright yellow flowers characteristic for these plants (sala et al., 2003; rosa et al., 2007). the most extensively studied species within this genus is helichrysum italicum (roth) g.don, a mediterranean aromatic shrub known for its distinctive yellow flowers. it belongs to the order asterales and the family asteraceae. immortelle, a common name for h. italicum, is frequently found along the eastern coastline and the islands of the adriatic sea. in traditional medicine, it is commonly used as a diuretic, choleretic, and expectorant (chinou et al., 1996). ethnopharmacological studies across various regions of europe have shown that © 2025 dimitrijević et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 178 h. italicum is traditionally used to treat numerous ailments, including those requiring antimicrobial, antioxidant, antiviral, and antiproliferative effects (sala et al., 2003; rosa et al., 2007; antunes viegas et al., 2014; ninčević et al., 2019; mollova et al., 2020; aćimović et al., 2021; genčić et al., 2021; šovljanski et al., 2024). the genus candida comprises over 200 species, though only a few are pathogenic to humans, all are opportunistic pathogens. typically, candida species are part of the normal microbiota of the human body, classifying them as commensal microorganisms. in healthy individuals, they are considered a harmless component of the microbiome and do not cause infection. however, candida albicans is the most prevalent cause of human fungal infections and is a leading cause of nosocomial infections, particularly in immunocompromised patients (mayer et al., 2013; sawant & khan, 2017; dadar et al., 2018; talapko et al., 2021). in recent years, research on natural substances as potential antifungal agents has intensified due to the rising resistance of candida isolates to antifungal drugs and the occurrence of recurrent infections. given the prolonged antifungal therapy often required for individuals with confirmed candidosis, alternative treatments could significantly improve treatment efficacy (cid-chevecich et al., 2022; blanc et al., 2023; karpiński et al., 2023). our study aimed to investigate the chemical composition and inhibitory effect of immortelle essential oil against clinical human candida isolates, as well as a reference strain of c. albicans atcc 24433. the study also examined the potential synergistic interaction between this essential oil and the commercial antifungal agent nystatin. additionally, it evaluated the oil’s effectiveness in reducing biofilm production when treated with varying concentrations of the tested substances. materials and methods microorganisms clinical strains of candida were isolated from various sites of patients with confirmed candidosis. the isolates were identified as c. krusei and c. albicans using chromogenic candida agar (hicrome™ candida differential agar; himedia) based on their growth characteristics, as well as the observation of their morphological and biochemical properties. the isolates were cultured on sabouraud dextrose agar (sda) (himedia, mumbai, india) in a thermostat before being used for the experiment. following the acquisition of overnight cultures, yeast suspensions were prepared, and their density was adjusted to 10⁷ cfu/ml by using a densitometer (den-1, biosan). these cultures were then used to conduct experiments outlined in our research. essential oils the essential oil (eo) used in the study was commercially available and purchased from a local store in niš, serbia. this oil was obtained through the distillation of species h. italicum (siempre viva oils, niš district, serbia). the oil was dissolved in dimethyl sulfoxide (dmso) solvent for use in the experiments. gc and gc/ms analyses gc/ms analyses (performed in triplicate) were conducted using a hewlett-packard 6890n gas chromatograph (agilent technologies, santa clara, california, usa) equipped with a db5ms fused silica capillary column ((5%-phenyl)methylpolysiloxane, 30 m × 0.25 mm, film thickness 0.25 µm) and coupled to a 5975b mass selective detector. the injector and interface temperatures were set at 250 °c and 300 °c, respectively. the oven temperature was programmed to increase from 70 °c to 290 °c at a rate of 5 °c/min, followed by an isothermal period of 10 min. helium was used as the carrier gas at a flow rate of 1.0 ml/min. samples (1.0 μl of essential oil solutions, prepared by dissolving 10.0 mg of essential oil in 1.0 ml of diethyl ether) were injected in pulsed split mode (with an initial flow rate of 1.5 ml/min for 0.5 min, then reduced to 1.0 ml/min for the remainder of the analysis; split ratio 40:1). the ms conditions included an ionization voltage of 70 ev, an acquisition mass range of m/z 35-650, and a scan time of 0.32 s. the percentage composition of the essential oils was calculated based on the gc peak areas without any corrections. constituents were identified by comparing their linear retention indices (relative to c8-c33 n-alkanes on the db-5ms column) with literature values (adams, 2007) and mass spectra with those of authentic standards, as well as databases such as wiley 6, nist11, and massfinder 2.3, and a custom ms library containing spectra of known oils and pure substances. wherever possible, identification was further confirmed through coinjection with authentic samples. microdilution method the microdilution method was used to examine the potential activity of the tested essential oil against candida isolates (stojanović-radić et al., 2020; cid-chevecich et al., 2022). the method involves preparing serial dilutions of the substances used in the experiment to determine the minimum concentrations required to inhibit the growth of candida cells. the inhibitory activity test was conducted in sabouraud dextrose broth (sdb) (himedia, mumbai, india), biologica nyssana ● 16 (1) june 2025: 177-187 dimitrijević et al. ● chemical composition and antifungal effect of the essential oil of helichrysum italicum (roth) g. don against clinical isolates of candida spp. 179 biologica nyssana ● 16 (1) june 2025: 177-187 dimitrijević et al. ● chemical composition and antifungal effect of the essential oil of helichrysum italicum (roth) g. don against clinical isolates of candida spp. which was used to inoculate the microtiter plates. the initial concentration of the tested essential oil was 10 mg/ml, with the antifungal agent nystatin serving as a positive control. prepared cell suspensions were applied to microtiter plates, where a series of dilutions of the immortelle oil had already been transferred. the plates were then incubated at 35 °c for 48 h, after which each substance’s mic (minimum inhibitory concentration) was determined. the experiment was conducted in triplicate. checkerboard assay the checkerboard method involves using various dilutions of two substances, applied in a cross-pattern on microtiter plates to create different combinations of agents at different doses (silva et al., 2011; stringaro et al., 2014; de castro et al., 2015). this method is used to examine the type of interaction between potential antifungal agents, determining whether their effects are synergistic, additive, or antagonistic. in our research, the immortelle oil was tested in combination with a commercial antimycotic drug (nystatin). the prepared plates were incubated at 35 °c for 48 h, after which fic (fractional inhibitory concentration) values were recorded. the data were then used to calculate the fic index, which determined the outcome of the experiment. the fici was interpreted as a synergistic effect when it is ≤0.5, as additive or indifferent when it is >0.5 and ≤2, and as antagonistic when its value is >2. anti-biofilm assay the ability to produce biofilms and the efficacy of essential oil in reducing biofilm formation were assessed using the crystal violet (cv) method (stepanović et al., 2007; stojanović-radić et al., 2016, 2020). this method was carried out in 96-well microtiter plates, with yeast suspensions adjusted to a final concentration of 5×105 cfu/ml. the test was conducted in a sterile rpmi-1640 medium (sigmaaldrich) containing 0.8% glucose, while a sample of immortelle essential oil was dissolved in dmso solvent for the experiment. two concentrations of the test agent, mic and half-mic, were used, with nystatin as the positive control. the prepared plates were incubated at an optimal temperature of 35 °c for 48 h. after the incubation period, the contents of the plates were aspirated, and the wells were washed twice with sterile phosphatebuffered saline (pbs, ph 7.4). the plates were then dried, and a 0.8% crystal violet solution was added for 20 min. after staining, the plates were washed, and 250 µl of 96% ethanol (v/v) was added to each well for decolorization, which lasted 45 min. the resulting contents were then transferred to new sterile plates for absorbance reading. the absorbance was measured at 595 nm using an elisa reader (multiscan™ fc microplate reader, thermo scientific). statistical analysis the data from our study were statistically analyzed using prism software (graphpad software, version 5.01). the statistical analysis employed was anova, followed by a tukey post hoc test. all experiments were conducted in triplicate, with a 95% confidence level. statistical significance was defined as p<0.05. results and discussion chemical composition a total of 52 compounds were identified using the gc/ms analysis (tab. 1), with the most abundant components being neryl acetate (13.4%), γ-curcumene (10.3%), β-selinene (9.3%) and α-pinene (8.3%). all other isolated components were present in significantly lower concentrations in the composition of the commercial immortelle essential oil sample. these components (neryl acetate, γ-curcumene, β-selinene, and α-pinene) were also the most abundant in the h. italicum essential oils examined by other authors (staver et al., 2018; tzanova et al., 2018; aćimović et al., 2021). in their study, tzanova et al. (2018) investigated the chemical composition of essential oils from h. italicum, which was introduced from corsica (france) and cultivated in south bulgaria. using the gc/ms method, they identified a total of 41 components. the main compounds in the essential oil were αand γ-curcumene (12.46–25.76%), neryl acetate (12.02– 20.6%), and α-pinene (5.59–19.52%). staver et al. (2018) used fresh aerial parts of h. italicum from central dalmatia, croatia, which were hydrodistilled to identify its chemical composition. a total of 38 compounds were identified, with α-pinene (21.6%) and neryl acetate (7.9%) being the most abundant components of the essential oil. in contrast, the results of the study by ćavar zeljković et al. (2015) revealed a distinct chemical composition of h. italicum essential oil from various locations along the adriatic coast in croatia. the oils were extremely rich in sesquiterpene hydrocarbons, with isomers of curcumene being the dominant compounds (ćavar zeljković et al., 2015). the chemical composition of h. italicum essential oil in the study conducted by acimović et al. (2021) was analyzed using gc/fid and gc/ms methods. a total of 70 compounds were detected, with 17 unidentified components making up 8.5% of the total composition. the dominant 180 biologica nyssana ● 16 (1) june 2025: 177-187 dimitrijević et al. ● chemical composition and antifungal effect of the essential oil of helichrysum italicum (roth) g. don against clinical isolates of candida spp. table 1. chemical composition of h. italicum essential oil tested for anti-candida activity ria rib constituentc content (%)d 932 932 α-pinenee 8.3 946 945 α-fenchene 0.5 948 946 camphenee tr 951 953 thuja-2,4(10)-diene tr 976 974 β-pinenee 0.3 995 1000 isobutyl 2-methylbutanoatee tr 1022 1020 p-cymenee tr 1026 1024 limonenee 0.7 1028 1026 1.8-cineolee 0.7 1042 1045 isobutyl angelatee tr 1066 1067 cis-linalool oxide (furanoid)e tr 1083 1084 trans-linalool oxide (furanoid)e 0.1 1095 1095 linaloole 1.0 1118 1118 exo-fenchole tr 1122 1119 trans-p-mentha-2.8-dien-1-ol tr 1125 1122 α-campholenal 0.8 1142 1135 trans-pinocarveole 0.5 1145 1143 isoamyl angelatee 2.5 1164 1160 pinocarvonee tr 1173 1165 borneole tr 1182 1174 terpinen-4-ole tr 1197 1186 α-terpineole tr 1199 1195 myrtenale 0.6 1211 1204 verbenonee 0.7 1224 1226 cis-carveol tr 1250 1239 carvonee tr 1281 1280 (z)-3-hexenyl angelatee 0.4 1288 1291 trans-verbenyl acetatee tr 1350 1343 limonene glycol tr 1355 1359 neryl acetatee 13.4 1371 1376 α-ylangene 0.4 1378 1379 isoitalicene tr 1409 1405 italicene 3.5 1413 1411 α-cisbergamotene 1.1 1434 1432 α-transbergamotene 0.7 1441 1438 4,6,9-trimethyldec-8-ene-3,5-dione (diketo tautomer)e tr 1444 1452 neryl propionate 3.4 1468 1464 α-acoradiene 0.4 1472 1469 β-acoradiene 0.4 1475 1471 4,5-di-epi-aristolochene tr 1477 1485 selina-4,11-diene 181 biologica nyssana ● 16 (1) june 2025: 177-187 dimitrijević et al. ● chemical composition and antifungal effect of the essential oil of helichrysum italicum (roth) g. don against clinical isolates of candida spp. compounds included γ-curcumene (13.6%), β-selinene (12.2%), and α-pinene (11.8%), followed by β-caryophyllene (6.7%) and arcurcumene (5.0%). it is important to emphasize that the efficacy of immortelle essential oil is highly dependent on the proportions of its chemical components, which can vary based on the chemotype (aćimović, 2023). anticandidal activity the results of our investigation into the antifungal potential of the tested essential oil are presented in tab. 2. the table shows the minimum concentrations of the natural agent required to inhibit the growth of clinical candida isolates and the reference strain c. albicans atcc 24433. the data obtained through the microdilution method demonstrated a strong antifungal potential of the tested essential oil, with mic values ranging from 0.312 to 5 mg/ml. the c. krusei species were particularly sensitive to the oil, with cell growth inhibition occurring at 0.312 mg/ml in isolates c3 and c10. candidosis poses a significant challenge due to increasing resistance to conventional drugs, primarily as a result of the prolonged therapy required for such infections. comparing our obtained results with those previously reported in the literature, the essential oil of h. italicum demonstrates a remarkable inhibitory effect against candida strains (mastelic et al., 2005; djihane et al., 2017; staver et al., 2018; bacic et al., 2021; zheljazkov et al., 2022). the study by mastelic et al. (2005) investigated the chemical composition and antimicrobial potential of h. italicum essential 1481 1481 γ-curcumene 10.4 1485 1491 2,4,6,9-tetramethyldec-8-ene-3,5-dione, diastereomer i (diketo tautomer) tr 1494 1491 β-selinene 9.3 1501 1498 α-selinene 1.5 1517 1513 γ-cadinene 0.6 1527 1528 cis-calamenene tr 1555 1547 italicene epoxide 0.8 1572 1578 3,5,7,10-tetramethylundec-9-ene-4,6-dione, diastereomer i (diketo tautomer) 1.7 1581 1582 ar-tumerol 0.8 1589 1582 caryophyllene oxidee 6.3 1616 1608 humulene epoxide ii 0.8 aretention indices determined experimentally on a db-5ms column relative to a series of c7-c40 n-alkanes. bliterature values of retention indices taken from adams (adams, 2007) or nist (nist mass spectrometry data center, william e. wallace, n.d.) collection, if not stated otherwise. ccompound identified based on mass spectra and retention indices matching with literature data, if not stated otherwise. dvalues are means of three individualanalyses. ecompound identity confirmed by a co-injection experiment with a standard. ftr, trace amounts (<0.05%) table 2. antifungal activity of h. italicum essential oil against clinical isolates of candida spp. and reference strain c. albicans atcc 24433 tested yeasts h. italicum oil nystatin mic (mg/ml) mic (mg/ml) c1 candida albicans 5.00 0.009 c2 candida albicans 1.25 0.004 c3 candida krusei 0.31 0.009 c4 candida albicans 2.50 0.004 c5 candida albicans 1.25 0.004 c6 candida albicans 0.62 0.004 c7 candida krusei 0.62 0.004 c8 candida albicans 2.50 0.009 c9 candida albicans 1.25 0.004 c10 candida krusei 0.31 0.002 c11 candida albicans 0.62 0.004 mic – minimum inhibitory concentration. oil from croatia. the experiment tested selected strains of staphylococcus aureus, escherichia coli, pseudomonas aeruginosa, and c. albicans. the findings revealed that the essential oil significantly inhibited the growth of the yeast c. albicans and the gram-positive bacteria s. aureus. research by djihane et al. (2017) found that c. albicans atcc 10231 exhibited the highest sensitivity (6.32 µg/ ml) to the action of immortelle essential oil. in their study, the aerial parts of h. italicum were collected in june 2013 during the flowering period 182 activity against c. krusei and c. tropicalis, with the highest activity observed against the gram-positive bacteria s. aureus. the previously reported data on the antifungal properties of immortelle essential oil in the literature prompted us to investigate its synergistic potential and antibiofilm effect against various clinical isolates of candida species. synergistic potential in our study, we investigated the interaction between the essential oil of h. italicum and the commercial antifungal agent (tab. 3). in this test, both the reference strain and two representatives of human clinical candida isolates were evaluated. a synergistic effect was observed between immortelle oil and nystatin against the standard strain of c. albicans atcc 24433, with an fic index of 0.187. this type of interaction was also observed against isolate c7 (c. krusei), with an fic index of 0.25, as well as against isolate c9 (c. albicans). notably, no antagonistic interactions were detected between the tested substances. it is important to highlight that the mic values indicating growth inhibition showed a significant decrease. this suggests that similar antifungal effects can be achieved with lower doses of antifungal agents, demonstrating the cooperative interaction between the tested substances. according to the existing literature, several studies highlight the synergistic potential of essential oils, both as standalone treatments and in combination with established antifungal agents (orchard et al., 2019; soulaimani et al., 2021; blanc et al., 2023). the study conducted by stojanović-radić et al. (2020) has also demonstrated a synergistic effect of inula helenium l. oil (asteraceae) with nystatin against clinical isolates of candida. this may from bejaia, northern algeria. the essential oil from this collection was characterized by a high content of oxygen-containing sesquiterpenes (76.7%) and monoterpenes (61.42%). major constituents included α-cedrene (13.61%), α-curcumene (11.41%), ceranyl acetate (10.05%), limonene (6.07%), nerol (5.04%), neryl acetate (4.91%), and α-pinene (3.78%). staver et al. (2018) described the effects of h. italicum essential oil, sourced from central dalmatia (croatia), against various microorganisms. notably, at a concentration of 6.4 mg/ml, the oil exhibited an antimicrobial effect on clinical isolates of mrsa, s. epidermidis, and c. albicans. in contrast, our research demonstrated even more significant results: the growth of the reference strain c. albicans atcc 24433 was inhibited at a concentration of 0.625 mg/ml, while all clinical isolates of candida were inhibited at concentrations ≤5 mg/ml. the study by bacic et al. (2021) examined the chemical composition and antimicrobial effects of h. italicum essential oil from bosnia and herzegovina. a total of 41 components were identified, with the main constituents being α-pinene, neryl acetate, γ-curcumene, and β-selinene. when tested against the reference strain of c. albicans atcc 10231 using the disc diffusion method, the essential oil exhibited weak anti-candida activity. zheljazkov et al. (2022) presented the chemical composition and antimicrobial potential of essential oils from the species h. arenarium (l.) moench and h. italicum. they tested h. italicum essential oils derived from plants introduced from bosnia, corsica, and france. the main constituents identified were neryl acetate (4.04–14.87%) and β-himachalene (9.9–10.99%). the results obtained using the disk diffusion method demonstrated moderate antifungal biologica nyssana ● 16 (1) june 2025: 177-187 dimitrijević et al. ● chemical composition and antifungal effect of the essential oil of helichrysum italicum (roth) g. don against clinical isolates of candida spp. table 3. interaction between the essential oil and nystatin against clinical isolates of candida spp. and the reference strain of c. albicans atcc 24433 combinations candida albicans atcc 24433 candida krusei c7 candida albicans c9 mic alone mic combined fic mic alone mic combined fic mic alone mic combined fic eo+n immortelle 0.625 0.039 0.062 0.625 0.078 0.125 1.25 0.312 0.25 nystatin 0.004 0.0005 0.125 0.004 0.0005 0.125 0.004 0.001 0.25 *fici interaction 0.187 syn 0.25 syn 0.5 syn mic – minimum inhibitory concentration (mg/ml); fic – fractional inhibitory concentration; fici – fractional inhibitory concentration index; syn – synergism 183 be a consequence of i. helenium oil’s chemical composition, where sesquiterpenes dominate. however, we did not find any available studies made to directly compare our results on the interaction between a commercial sample of immortelle eo and the antifungal agent nystatin against human candida isolates. the results of our study demonstrated that immortelle oil significantly inhibited the growth of planktonic candida cells, both as a standalone treatment and in combination with nystatin. these findings underscore the importance of exploring the potential synergism of essential oils, as such research could contribute to developing new strategies for treating fungal infections. effect of the immortelle oil on biofilm production we investigated the biofilm-forming ability of candida species and evaluated the potential of h. italicum essential oil to inhibit biofilm formation. the results are presented in figs. 1 and 2. a significant reduction in biofilm formation was observed after treatment with h. italicum essential oil at both mic and half-mic concentrations for isolates c1, c4, c5, and c11. the inhibition percentage was approximately 60% or lower, demonstrating the strong efficacy of immortelle oil against clinical human candida isolates. the reference strain c. albicans atcc 24433 also exhibited high sensitivity to the tested essential oil, significantly reducing biofilm formation by over 60%. moderate reduction efficiency was observed with isolates c2 and c8, where the inhibition percentage was around 40% or slightly lower, depending on the agent concentration and the specific candida isolate. the literature emphasizes the notable ability of natural products from various plant species to reduce biofilm formation (butassi et al., 2021; karpiński et al., 2021). for instance, butassi et al. (2021) provided a comprehensive literature review (covering the period from 2017 to may 2021) on essential oils, propolis, plant extracts, algae, lichens, microbial compounds, and nanosystems containing natural products. the review discusses their in vitro and in vivo potential to modulate fungal biofilms. these findings demonstrated that various plant secondary metabolites and their compounds exhibit significant activity inhibiting biofilm formation and reducing mature biofilm structures against candida isolates. in the review study by karpiński et al. (2021) 69 plant-derived compounds from plants belonging to various families were shown activity against candida biofilms. among these compounds, monoterpenes were the most prevalent, followed by sesquiterpene lactones and sesquiterpenes. since our commercial sample of h. italicum contains the main compounds that fall into the sesquiterpene biologica nyssana ● 16 (1) june 2025: 177-187 dimitrijević et al. ● chemical composition and antifungal effect of the essential oil of helichrysum italicum (roth) g. don against clinical isolates of candida spp. fig. 1. effects of the immortelle oil on candida biofilm production. helichrysum italicum essential oil (hieo); nystatin (n). statistical analysis used by anova with tuckey post hoc test ****p<0.0001; **p<0.001; **p<0.01; *p<0.05 vs. control 184 and monoterpenes category, these components likely contribute to the significant antibiofilm activity observed in our research (figs. 1 and 2). in their study, manoharan et al. (2017) screened 83 essential oils for their effectiveness against c. albicans biofilm formation, including the essential oil of h. coriaceum (dc.) harv. the results showed the biofilm reduction by immortelle essential oil, which contains β-caryophyllene a sesquiterpene compound known for its bioactive properties (manoharan et al., 2017). to date, we have not found relevant literature to directly compare our findings on the antibiofilm effects of immortelle essential oil against clinical isolates of candida, the causative agent of candidosis. given the limited relevant literature on the antifungal potential of immortelle oil (h. italicum) on planktonic and biofilm forms of the genus candida, our study provides a valuable contribution to ongoing and future research in this field. conclusion our research demonstrated a potent anticandida effect of the tested essential oil from the helichrysum genus. in this study, we analyzed the chemical composition of h. italicum essential oil, identifying a total of 52 compounds. the most abundant constituents were neryl acetate, α-pinene, γ-curcumen, and β-selinene. immortelle essential oil, evaluated using the microdilution method, exhibited a significant inhibitory effect on tested strains of the genus candida. additionally, the oil was examined for potential synergistic interactions with nystatin on selected clinical candida isolates, with the results confirming a notable synergistic potential. the potential to reduce candida biofilm formation after treatment with immortelle oil at mic and half-mic concentrations was also investigated. significant antibiofilm activity was observed in most treated strains when compared to the control group. the data obtained suggest that immortelle oil, which effectively inhibits both planktonic and biofilm-forming candida cells, holds promise as a potential alternative agent treatment for candidosis. our research provides a strong foundation for further testing on clinical strains responsible for candidosis, particularly given the rising incidence of recurrent infections and growing resistance to current antifungal treatments. acknowledgements. the work was supported by the ministry of science, technological development and innovations of the republic of serbia (contract number: 451-03-136/2025-03/ 200124 and 451-03-137/2025-03/ 200124). biologica nyssana ● 16 (1) june 2025: 177-187 dimitrijević et al. ● chemical composition and antifungal effect of the essential oil of helichrysum italicum (roth) g. don against clinical isolates of candida spp. fig. 2. effects of the immortelle oil on candida biofilm production. helichrysum italicum essential oil (hieo); nystatin (n). statistical analysis used by anova with tuckey post hoc test ****p<0.0001; **p<0.001; **p<0.01; *p<0.05 vs. control 185 biologica nyssana ● 16 (1) june 2025: 177-187 dimitrijević et al. ● chemical composition and antifungal effect of the essential oil of helichrysum italicum (roth) g. don against clinical isolates of candida spp. references aćimović, m. 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(2022). chemical profile and antimicrobial activity of the essential oils of helichrysum arenarium (l.) moench. and helichrysum italicum (roth.) g. don. plants, 11(7), 951. https://doi.org/10.3390/plants11070951/s1 jenačković, d. et al.  contribution to the knowledge of distribution… biologica nyssana 6 (2)  december 2015: 59-65 jenačković, d. et al.  contribution to the knowledge of distribution… 59 original article received: 01 august 2015 revised: 12 october 2015 accepted: 01 december 2015 contribution to the knowledge of distribution of certain macrophytes, invasive and threatened species in serbia dragana jenačković*, milica miljković, dragana mitrović, vladimir ranđelović university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia * e-mail: draganaj@pmf.ni.ac.rs abstract: jenačković, d., miljković, m., mitrović, d., ranđelović, v.: contribution to the knowledge of distribution of certain macrophytes, invasive and threatened species in serbia. biologica nyssana, 6 (2), december 2015: 59-65. this paper contains a short report about new and unpublished chorological data for the following species: hydrocharis morsus-ranae l., bidens cernuus l., najas marina l., potamogeton lucens l., schoenoplectus triqueter (l.) palla, cyperus rotundus l., polypogon monspeliensis (l.) desf. and paspalum distichum l.. despite the fact that this paper provides insight into the distribution of analyzed species, it could be used as a base for invasive species’ paspalum distichum l. expanding patterns research, and as a base for reevaluation of the threatened status of schoenoplectus triqueter (l.) palla and cyperus rotundus l. in serbia. key words: macrophytes, invasive and threatened species, serbia, distribution apstrakt: jenačković, d., miljković, m., mitrović, d., ranđelović, v.: prilog poznavanju rasprostranjenja pojednih makrofitskih, invazivnih i ugroženih vrsta u srbiji. biologica nyssana, 6 (2), december 2015: 59-65. u ovom radu dat je pregled novih i nepublikovanih podataka o rasprostranjenu sledećih vrsta: hydrocharis morsus-ranae l., bidens cernuus l., najas marina l., potamogeton lucens l., schoenoplectus triqueter (l.) palla, cyperus rotundus l., polypogon monspeliensis (l.) desf. i paspalum distichum l.. prikazani horološki podaci pružaju kompletniji uvid u rasprostranjenje analiziranih makrofita, predstavljaju osnovu za detaljnija proučavanja puteva introdukcije i širenja invazivne vrste paspalum distichum l. i od velikog su značaja za procenu statusa ugroženosti vrsta schoenoplectus triqueter (l.) palla i cyperus rotundus l. u srbiji. key words: makrofite, invazivne i ugrožene vrste, srbija, rasprostranjenje introduction in the period of 2011-2014 years, floristic and phytocoenological researching of aquatic and swamp vegetation was performed in the region of the southern serbia. on this occasion, new points of distribution for the following species: hydrocharis morsus-ranae l., bidens cernuus l., najas marina l., potamogeton lucens l., schoenoplectus triqueter (l.) palla, cyperus rotundus l., polypogon monspeliensis (l.) desf. and paspalum distichum l. are recorded (fig. 1). unfortunately, in serbia macrophytes are extensively studied in terms of distribution, ecology and coenotic affiliation on almost entire territory of vojvodina province, while in the other parts of country they are sporadically 6 (2) • december 2015: 59-65 biologica nyssana 6 (2)  december 2015: 59-65 jenačković, d. et al.  contribution to the knowledge of distribution… 60 investigated. significant contribution to the collection of the chorological data for the mentioned plants group on the southern area of the sava and danube rivers are given by j o v a n o v i ć (1965), t o p u z o v i ć et al. (2003), r a n đ e l o v i ć et al. (2007), s t a n k o v i ć et al. (2009), r a n đ e l o v i ć & zlatković (2010). material and methods the data about distribution of analyzed plant species in serbia were collected by reviewing relevant literature sources. further, herbarium specimens deposited in the herbarium of the institute of botany and botanical garden “jevremovac”, university of belgrade (beou) and the herbarium of the faculty of sciences and mathematics, department of biology and ecology, university of niš (hmn) were used as a source of distribution data. the new chorological data were obtained during floristic researching which was carried out at the territory of the se serbia. the distribution of studied species is presented on 10 x 10 km2 utm grid. determination of the collected plant materials was performed using dichotomous identification keys (t u t i n et al. (eds.), 1964-1980; j o s i f o v i ć (ed.), 1970-1980). nomenclature of the studied species is consistent with euro+med plantbase (euro+med, 2006-). the threatened status for all researched species is considered in terms of iucn (2000) criteria and categories. results and discussion 1. bidens cernuus l. (compositae) general distribution: europe, siberia, central asia, southeastern asia, japan, central africa, north america and galapagos. distribution in serbia: published literature data points that this species is presented in the following areas: bačka region (babić, 1971; lazić, 2006), banat region (obradović et al., 1977), šumadija region (topuzović et al., 2003), se serbia (ranđelović & stamenković, 1983; ranđelović & zlatković, 2010) and s serbia (jovanović, 2004) (fig. 2). new and unpublished distribution data: smilovci lake (fn57), caricetum ripariae, 715 m, 07.07.2013., coll./det. d. jenačković. habitat description: this plant prefers wet, muddy habitats which are characterized by significant water level fluctuation during growing season. iucn threatened status in the world: least concern (lc). iucn threatened status in serbia: not threatened. 2. najas marina l. (najadaceae) general distribution: most of europe, most of asia (except west asia), indonesia, australia, mauritius, north africa, south and southeastern africa, most of north america (except alaska and canada), middle america, south america (venezuela, brazil and argentina) and hawaii. accordingly, it could be said that najas marina l. belongs to the group of cosmopolitan species. this species is native to europe, asia and africa. in the last century it was recorded in america where it has the status of alien species. distribution in serbia: the distribution pattern of najas marina l. in serbia could be met according to literature and herbarium data (fig. 2). this species is recorded in banat region (slavnić, 1956; butorac, 1995; polić et al., 2004; radulović, 2005; knežević et al., 2008; polić et al., 2008; ljevnaić-mašić, 2010), bačka region (slavnić, 1956; čanak & dokić 1969; obradović et al., 1977; vučković et al., 1998; lazić et al., 2004; lazić, 2006; stojanović et al., 2007), srem region (obradović et al., 1977; vukov et al., 2012), šumadija region (topuzović et al., 2003), e serbia (stanković et al., 2009), w serbia and vicinity of belgrade (blaženčić, 1997). new and unpublished distribution data: debelica village (fp03), potamo-najadetum, 175 m, 20.07.2011., coll./det. d. jenačković; smilovci lake (fn57), potamo-najadetum, 712 m, 05.08.2012., coll./det. d. jenačković. habitat description: najas marina l. shows preferences to fresh water and brackish water. iucn threatened status in the world: least concern (lc). iucn threatened status in serbia: not threatened. 3. potamogeton lucens l. (potamogetonaceae) general distribution: most of europe, most of asia (except central asia), australia, north africa (morocco), tanzania, madagascar, south america (brazil and argentina), bahamas islands and north america (except alaska and canada). distribution in serbia: potamogeton lucens l. is recorded in banat region (polić et al., 2004; radulović, 2005; knežević et al., 2008; polić et al., 2008; ljevnaić-mašić, 2010), bačka region (slavnić, 1956; stojanović et al., 1994; radulović, biologica nyssana 6 (2)  december 2015: 59-65 jenačković, d. et al.  contribution to the knowledge of distribution… 61 2000; lazić et al., 2004; panjković, 2005; lazić, 2006; stojanović et al., 2007), srem region (panjković, 2005; vukov et al., 2012), e serbia (stanković et al., 2009), se serbia (petković, 1983) and vicinity of belgrade (blaženčić, 1997). the first data about distribution of potamogeton lucens l. in the vicinity of niš is given by sava petrović (petrović, s., 1879, beou) (fig. 2). new and unpublished distribution data: vrtište village (ep60), scirpo-phragmitetum subass. typhetosum angustifoliae, 181 m, 02.06.2013., coll./det. d. jenačković; smilovci lake (fn57), potamo-najadetum, 712 m, 05.08.2012., coll./det. d. jenačković. habitat description: potamogeton lucens l. inhabits relatively depth, clear, alkaline and nutrient poor waters. iucn threatened status in the world: least concern (lc). iucn threatened status in serbia: not threatened. fig. 1. the photographs of studied species 4. schoenoplectus triqueter (l.) palla (cyperaceae) general distribution: central and west europe, most of asia (except north asia), africa (sudan and south africa), north america (the united states). schoenoplectus triqueter (l.) palla is considered an autochthonous species to europe and asia while in the other parts of areal it is introduced. distribution in serbia: there is a little data about distribution of schoenoplectus triqueter (l.) palla in serbia. it has been recorded in bačka region (babić, 1971; panjković et al., 2010) and srem region (slavnić, 1956; stanković, 2011) (fig. 2). only one herbarium data about distribution of this species exists for region positioned outside the boundaries of the vojvodina province. it refers to west serbia (zlatibor mountain). new and unpublished distribution data: se serbia: pukovac village (en67), 190 m, 11.09.2014., coll./det. m. miljković. habitat description: this plant grows on wet and muddy habitats which occur at the banks of rivers, marches and the other types of aquatic habitats. iucn threatened status in the world: least concern (lc). iucn threatened status in serbia: critically endangered vulnerable (data deficient) ((cr vu (dd)). biologica nyssana 6 (2)  december 2015: 59-65 jenačković, d. et al.  contribution to the knowledge of distribution… 62 5. cyperus rotundus l. (cyperaceae) general distribution: europe (except north, central and east europe), asia minor, south, central and southeastern asia, indonesia, australia, africa, north america (except alaska and canada), south america and islands in atlantic, indian and pacific ocean. distribution in serbia: the only literature data about distribution of cyperus rotundus l. in serbia are published in “the red data book of flora of serbia 1” (stevanović (ed.), 1999). according to this literature source, cyperus rotundus l. is recorded on the following locations: the velika morava river near ćuprija (ep26), miljkovo (ep19) and the južna morava river near vrtište (ep60). in hmn is stored exemplar of the mentioned species from the village klisura (en68) while beou contains specimens from the sands around the village vrtište (ep60) (fig. 2). new and unpublished distribution data: se serbia: pukovac village (en67), 190 m, 20.08.2013., coll./det. d. jenačković & d. mitrović. habitat description: cyperus rotundus l. grows at sandy habitats which are positioned on the bank of aquatic bodies. iucn threatened status in the world: least concern (lc). iucn threatened status in serbia: endangered – vulnerable (en b2c vu a1c; b1; e). 6. polypogon monspeliensis (l.) desf. (gramineae) general distribution: europe (except central, north and east europe), asia minor, south, central and east asia, australia, new zealand, africa, north america, middle america, west part of south america, islands in atlantic and pacific ocean. thus, polypogon monspeliensis (l.) desf. could be defined as cosmopolitan species which is native to the area of europe, asia and africa while to the other continent it is introduced. distribution in serbia: distribution of polypogon monspeliensis (l.) desf. in serbia is not known (fig. 2). new and unpublished distribution data: se serbia: pukovac village (en67), 190 m, 20.08.2013., coll./det. d. jenačković & d. mitrović. habitat description: polypogon monspeliensis (l.) desf. shows preference to the humid habitats which are placed around swamps, backwaters and depressions. the specificity of this plant is ability to tolerate the higher level of salinity, so that it often occurs in salt marshes. iucn threatened status in the world: least concern (lc). iucn threatened status in serbia: not threatened. 7. paspalum distichum l. (gramineae) general distribution: europe (except north, central and east europe), asia minor, south asia, east asia, australia, africa (except central and east africa), north america (except alaska and canada), middle america, south america, islands in atlantic and pacific ocean. this plant has a status of alien species for the european area. distribution in serbia: paspalum distichum l. is an invasive, emergent plant. according to the published data (stevanović et al., 2004; polić, 2006), this species massively occurred near the danube river between belgrade and đerdap dam (fig. 2). in the southern part of serbia, it is recorded in the vicinity of niš (ranđelović et al., 2007). the other data about distribution are unpublished and they confirm spreading of paspalum distichum l. in the vicinity of belgrade and banat region. ability to displace the autochthonous vegetation makes it a threat to native biological diversity. new and unpublished distribution data: se serbia: klisura village (en68), 11.10.2014., coll./det. m. miljković. habitat description: paspalum distichum l. grows on humid habitats which are often flooded at the beginning of growing season. iucn threatened status in the world: least concern (lc). iucn threatened status in serbia: not threatened. 8. hydrocharis morsus-ranae l. (hydrocharitaceae) general distribution: most of europe, siberia, caucasus, asia minor, middle asia, north africa (morocco and algeria) and madagascar, north america. hydrocharis morsus-ranae l. is native species to europe, asia and africa while it has been introduced to the territory of north america (catling et al., 2003). distribution in serbia: hydrocharis morsus-ranae l. is common species in aquatic and semi-aquatic vegetation of vojvodina province. according to the literature data, this plant is distributed in the banat biologica nyssana 6 (2)  december 2015: 59-65 jenačković, d. et al.  contribution to the knowledge of distribution… 63 fig.2. the utm grid maps of distribution of the researched species region (slavnić, 1956; butorac, 1995; stojanović & lazić, 1998; polić et al., 2004; radulović, 2005; knežević et al., 2008; ljevnaić-mašić, 2010), bačka region (slavnić, 1956; stojanović et al., 1994; stojanović & lazić, 1998; vučković et al., 1998; radulović, 2000; borišev, 2002; lazić, 2003; lazić et al., 2004; nikolić, 2004; panjković, 2005; lazić, 2006; stojanović et al., 2007), srem region (slavnić, 1956; obradović et al., 1977; igić et al., 2004; stanković, 2011; vukov et al., 2012) and the vicinity of belgrade (jovanović, 2011). in beou a few exemplars of the mentioned species are stored and they refer to localities (mala krsna and obrenovac) which are placed in the south of the sava and danube rivers (fig. 2). the first literature data about presence of this species in central serbia is given by pančić (1874). new and unpublished distribution data: vrtište village (ep60), scirpo-phragmitetum subass. phragmitetosum, 185 m, 15.07.2012., coll./det. d. jenačković. habitat description: hydrocharis morsus-ranae l. grows in shallow, mesotrophic, calcareous waters which are protected from direct wave and wind action. iucn threatened status in the world: least concern (lc). iucn threatened status in serbia: not threatened. conclusion the presented results are important to prepare the new edition of flora serbia and red book of flora serbia. particularly significant are data about distribution of species polypogon monspeliensis (l.) desf. which has been not recorded in serbia until to now. further, presence of species paspalum distichum l. on the banks of the južna morava river is significant for tracking the expanding pattern of this invasive species in our country. information about distribution of threatened species: schoenoplectus triqueter (l.) palla and cyperus rotundus l. are also very important. their significance is reflected in reevaluation of the threatened status and implementation of conservation process. biologica nyssana 6 (2)  december 2015: 59-65 jenačković, d. et al.  contribution to the knowledge of distribution… 64 acknowledgements. this study was supported by the project of ministry of science and technological development of republic of serbia (project no. 173030). references babić, n. 1971: močvarna i livadska vegetacija koviljskog rita. zbornik matice srpske za prirodne nauke, 41: 19-87. blaženčić, j. 1997: florističke karakteristike makrofitske vegetacije savskog jezera kod beograda (srbija, jugoslavija). glasnik instituta za botaniku i botaničke bašte univerziteta u beogradu, 29: 167-173. borišev, m. 2002: akvatične makrofite jegričke (temerin-gospođinci). diplomski rad, prirodnomatematički fakultet, univerzitet u novom sadu. novi sad. butorac, b. 1995: review of aquatic vegetation of the regional park "stari begej". tiszcia, 29: 27-32. čanak, m., dokić, m. 1969: naseljavanje osnovne kanalske mreže hidrosistema dunav-tisa-dunav vodenim makrofitama. letopis naučnih radova poljoprivrednog fakulteta, novi sad, 13: 121132. catling, p. m., mitrow, g., haber, e., posluszny, u., charlton, w. a. 2003: the biology of canadian weeds. 124. hydrocharis morsus-ranae l.. canadian journal of plant science, 83 (4): 10011016. euro+med, 2006(continuously updated): euro+med plantbase the information resource for euro-mediterranean plant diversity. published on http://www.emplantbase.org/home.html igić, r., polić, d., borišev, m., janauer, g. a. 2004: aquatic macrophytes of “krstonošića okno” (obedska bara swamp). limnological reports, proceedings of the 35th iad conference, novi sad, 35: 463-468. iucn world commission on protected areas and europark federation 2000. guidelines for protected area management categories. grafenau, brussels. http://www.iucnredlist.org josifović, m. 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(eds.), 1964-1980. flora europaea, i-v. cambridge university press, london. vučković, m., stojanović, s., stanković, ž., žderić, m., radulović, s. 1998: floristical and phytocoenological characteristics of canal network of danube-tisza-danube hydrosystem in bačka. ekologija, 33: 71-76. vukov, d., igić, r., rućando, m., radulović, s. 2012: diversity of vascular hydrophytes in the zasavica river (serbia): changes after thirteen years. archives of biological sciences, 64 (4): 1607-1617. biologica nyssana 6 (2)  december 2015: 59-65 jenačković, d. et al.  contribution to the knowledge of distribution… 66 liana et al. 2025, biologica nyssana 16(2) 16 (2) december 2025: doi: 10.46793/biolnyss.16.2.8l cell deaths and their associated mechanisms original article phey liana department of clinical pathology, faculty of medicine, universitas sriwijaya-rsup dr. mohammad hoesin, palembang, indonesia pheyliana@fk.unsri.ac.id (corresponding author) masagus irsan saleh department of pharmacology, faculty of medicine, universitas sriwijaya, palembang, indonesia desi oktariana department of physiology, faculty of medicine, universitas sriwijaya, palembang, indonesia tungki pratama umar division of surgery and interventional science, faculty of medicine, university college london, london, uk received: june 29, 2025 revised: september 04, 2025 accepted: september 04, 2025 abstract: cell death is a crucial process in every organism, maintaining a balance between the number of new cells generated through mitosis and the number of damaged or unnecessary cells removed from the body. it happens from the embryonic process throughout every life stage of a human being. regulated cell death, known as apoptosis, is the most common form, while accidental or uncontrolled cell death, known as necrosis, is associated with pathological processes. in addition to these types of cell death, there are numerous other types, including necroptosis (a controlled cell death with an inflammatory process) and autophagy (mainly a part of the immune process). it is important to note that inhibition of regulated cell death can impact the normal cell cycle and can lead to drug resistance. a classic example of dysfunctional cell death regulation is cancer development. this review summarizes cell death types and their associated mechanisms. key words: accidental cell death, apoptosis, necrosis, regulated cell death apstrakt: ćelijske smrti mehanizmi udruženi sa njima ćelijska smrt je ključni proces u svakom organizmu, jer održava ravnotežu između broja novih ćelija nastalih mitozom i broja oštećenih ili nepotrebnih ćelija uklonjenih iz organizma. ona se odvija od embrionalnog razvoja pa tokom svih životnih stadijuma čoveka. regulisana ćelijska smrt, poznata kao apoptoza, predstavlja najčešći oblik, dok je slučajna ili nekontrolisana ćelijska smrt, poznata kao nekroza, povezana sa patološkim procesima. pored ovih oblika ćelijske smrti, postoji i niz drugih tipova, uključujući nekroptozu (regulisanu ćelijsku smrt sa inflamatornim procesom) i autofagiju (uglavnom deo imunskog procesa). važno je napomenuti da inhibicija regulisane ćelijske smrti može uticati na normalan ćelijski ciklus i dovesti do rezistencije na lekove. klasičan primer disfunkcionalne regulacije ćelijske smrti jeste razvoj kancera. ovaj pregled sumira tipove ćelijske smrti i njihove povezane mehanizme. ključne reči: slučajna ćelijska smrt, apoptoza, nekroza, regulisana ćelijska smrt introduction every organism, either multicellular and unicellular, has a specific mechanism to preserve a homeostatic state between the amount of new cells that emerged through mitosis and the number of damaged or unfavorable cells that need to be eliminated from the organism’s body (spiesser et al., 2012; alvarado et al., 2018). continuous cell turnover is essential to produce physical features that consist of fingers and toes, which emerge after developing from the webbed limbs first noticed in the human fetus, observed around week 10 of gestation (hurle & ganan, 1986). meanwhile, uncontrolled cellular proliferation and growth may cause the development of several debilitating diseases, with cancer as the main example (feitelson et al., 2015). in contrast, an extremely high cell death rate can lead to many disorders, including rheumatoid arthritis, alzheimer's disease (ad), and parkinson's disease (gorman, 2008; d’arcy, 2019). cell death can occur through several mechanisms. it can happen in a programmed (controlled) way, encompassing a multitude of biochemical, physical, and molecular operations, as well as uncontrolled ones, resulting in the leakage of cell contents into neighboring tissues. these processes may trigger further inflammatory response (particularly in unprogrammed cell death) or not (fink & cookson, 2005). apoptosis, a type of regulated cell death, is considered as the most prevalent type of cell death with its importance © 2025 liana et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. throughout the body (d’arcy, 2019). meanwhile, the uncontrolled or accidental form of cell death is called necrosis, which is particularly associated with pathological processes (including hypoxia, microbial infection, physical stress, and chemical agents) (fathima et al., 2023) . the mechanisms associated with mitosis regulation (stimulation or inhibition), cellular abnormality detection, and programmed cell death (pcd) initiation (apoptosis, pyroptosis or autophagy) in animals involve a long list of regulatory genes (d’arcy, 2019). while the cell cycle and cell death require a complicated interaction of gene products, certain receptors, enzymes, and regulatory proteins have been discovered to play key roles in those events. mutations or aberrant expression of these regulatory proteins may affect the normal cell cycle (whitfield et al., 2002; abreu velez & howard, 2015). for example, enzymes of the b-cell lymphoma-2 (bcl2) family and transglutaminase-2 either trigger or inhibit apoptosis based on their expression (upregulation or downregulation), localization or conformation (budillon et al., 2013; murphy et al., 2013). regulated cell death inhibition may directly affect cancer cells' susceptibility to chemotherapeutic drugs, resulting in drug resistance (mansoori et al., 2017). this is illustrated in the case of increased transglutaminase-2 expression, which is associated with poor prognosis and relapse of acute myelocytic leukemia (aml) (pierce et al., 2013; jacamo et al., 2016). this review will briefly overview cell death types and their associated mechanisms. cell death: classification and explanation the classification of cell death was initially based on the morphology and structure of tissues and cells. one of the earliest classifications was made by schweichel and merker in 1973, when they published a system based on morphology to classify three types of cell death, namely type i, type ii, and type iii (schweichel & merker, 1973). type i cell death, also known as apoptosis, is defined by cellular shrinkage (pyknosis), membrane prominence (blebbing), apoptotic body emergence, deoxyribonucleic acid (dna) disintegration (karyorrhexis), and chromatin condensation (portt et al., 2011). this type of cell death is additionally recognized as shrinkage necrosis, considered a type of non-pathologic cell death first described by a team of investigators led by john kerr (1971). next, type ii cell death is defined as autophagy-dependent cell death that results in the formation of autophagic vacuoles consisting of cytoplasmic substances and organelles to create autophagic bodies. the process is mediated through lysosomal involvement and may be observed in several cases, broadly classified as microand macroautophagy (todde et al., 2009; denton & kumar, 2019). meanwhile, type iii cell death, frequently designated as necrosis, is marked by membrane damage and expansion of subcellular organelles. necrosis is widely defined as uncontrollable cell death and is mainly a pathological process (tang et al., 2019). for several decades, apoptosis was regarded as the only plausible kind of programmed cell death (suzanne & steller, 2013). necrosis, conversely, was perceived as an unregulated, inadvertent consequence of excessive physical or chemical injury. however, this dichotomy has been superseded by a more nuanced, function-oriented framework determined by the nomenclature committee on cell death (nccd) (galluzzi et al., 2018). currently, cell death has been classified as either accidental cell death (acd) or regulated cell death (rcd), depending on functionality. acd is prompted by a sudden assault or damage that disables all conceivable control systems. rcd, on the other hand, has a distinct signaling cascade carried out by a specialized set of effector molecules, as well as distinctive activities, molecular structures, and immunological consequences. it can also be pharmacologically or genetically modulated (galluzzi et al., 2018; van der meeren et al., 2023). rcd is referred to as pcd when it develops under physiological conditions, such as during embryonic development (díazmendoza et al., 2013; tang et al., 2019). rcd can be divided into at least eleven types depending on its molecular features, including apoptosis, necroptosis, pyroptosis, ferroptosis, entotic cell death, netotic cell death (netosis), parthanatos, lysosomedependent cell death (lcd), autophagy-dependent cell death, alkaliptosis, and oxeiptosis (galluzzi et al., 2018). this paradigm shift was prompted by the realization that necrosis, equivalent to apoptosis, can occur in a regulated fashion, a phenomenon now termed necroptosis, which serves as a host protective mechanism, exhibiting antipathogenic and anticancer properties (khoury et al., 2020; ye et al., 2023). accidental cell death: necrosis necrosis (fig. 1), unlike apoptosis, is an unregulated type of cell death that occurs due to sudden external insults such as hypoxia, radiation, extreme heat, and toxins that cause severe damage and render cells unable to function properly. this process frequently entails a surge in the expression of diverse proinflammatory factors, such as nuclear factor-κb (li et al., 2001). these scenarios result in cell swelling (oncosis) followed by rupture of the cell membrane, leading to a release of cell contents to the surrounding region, which triggers an inflammatory biologica nyssana ● 16 (2) december 2025: liana et al. ● cell deaths and their associated mechanisms biologica nyssana ● 16 (2) december 2025: liana et al. ● cell deaths and their associated mechanisms fig. 1. necrosis process chain reaction and leads to tissue breakdown (helgoe et al., 2024). overall, the necrosis process does not require energy. the notion of necrosis as a contrast to apoptosis is valuable. however, in cell culture, what is frequently referred to as the necrosis process is basically the remains of cells in the late-stage apoptotic events, where there is a compromised structural integrity of apoptotic bodies. this typically appears as the presence of cellular chunks in the media, which is mistakenly attributed to necrosis (berghe et al., 2010). within a living organism, phagocytosis typically occurs, where circulating white blood cells devour and remove these apoptotic bodies. however, in a monoculture, this phagocytosis process does not occur (liang et al., 2014). this can complicate identifying the precise process by which cell death occurs. regulated cell death apoptosis apoptosis (fig. 2) is considered the archetypal form of rcd, a meticulously orchestrated process of cellular self-dismantling directed by a family of cysteine-aspartic proteases known as caspases (brentnall et al., 2013). caspases may be classified into two groups: initiator and executor caspases. initiator caspases (caspases 8 and 9) are activated from dormant procaspase forms upon detection of cell injury, and they subsequently trigger executor caspases (caspases 3, 6, and 7) (aral et al., 2019). these enzymes initiate a series of processes that lead to dna fragmentation via endonuclease activation, nuclear protein disintegration, cytoskeleton disruption, protein crosslinking, production of ligands for phagocytic cells, and apoptotic body formation. an important feature to note is that the cell membrane remains intact as the cell is packaged into smaller, membrane-bound vesicles called apoptotic bodies during the process (zhao et al., 2021). macrophages then swiftly phagocytose the apoptotic bodies that have formed, leading to the non-inflammatory removal of malignant and precancerous cells and a decrease in tissue damage from erroneous immune reactions (hirayama et al., 2017). apoptosis is estimated to occur at a rate of 1×109 cells daily in the human body (elliott & ravichandran, 2010). this process is characterized by an interruption of cell division and expansion, followed by a sequence of steps to prepare the cell for controlled death so that its materials do not leak into the vicinity (d’arcy, 2019). apoptosis may occur through two major pathways (nosalova et al., 2023): the intrinsic (mitochondrial) pathway, responsive to intracellular stressors such as dna damage and regulated by the b-cell lymphoma-2 (bcl-2) protein family, and the extrinsic (death receptor/dr) pathway, initiated by extracellular ligands such as tumor necrosis factor (tnf) or fas ligand (fasl). the complementary actions of extrinsic and intrinsic apoptotic pathways guarantee that damaged cells are eliminated from the body and multicellular organisms continue to function normally (zhong et al., 2020; nosalova biologica nyssana ● 16 (2) december 2025: liana et al. ● cell deaths and their associated mechanisms et al., 2023). apoptosis regulation issues can result in pathological conditions in several diseases. for instance, in degenerative illnesses like ad, where caspase overactivation—a crucial class of enzymes implicated as an apoptosis driver—appears to be the cause of neuronal death (sheffield et al., 2006). this is also a necessary process to prevent malignancy (abastado, 2015). the intrinsic pathway of apoptosis can be initiated from a positive or negative process and depends on chemicals generated from the mitochondria. the lack of growth factors, hormones, and cytokines in the cell's surroundings produces negative signals (orzaez et al., 2009). in addition, several cellular pro-apoptotic molecules, such as puma, noxa, and bax, which are typically suppressed in a physiological state, become active and are able to start apoptosis after reaching the activation threshold and with a deficiency of the previously mentioned pro-survival signals (zhang et al., 2016). positive situations, including hypoxia, poisons, radiation, reactive oxygen species overproduction, viruses, and different hazardous agents, are additional variables that trigger apoptosis (guhl et al., 2003; sun & mei, 2024). two subsets of bcl-2 proteins known as antiapoptotic proteins, including bcl-2, bcl-xl, bcl-w, bfl-1, and mcl-1, control the intrinsic pathway (zhu et al., 2007). these substances control apoptosis by preventing the release of cytochrome c (cytc) from mitochondria. on the other hand, apoptosis is regulated by pro-apoptotic proteins like hrk, bak, bad, bcl-xs, bid, bik, and bim, which release cytc from mitochondria (liu et al., 2013). caspases 8 and 9 are activated, and apoptosomes are formed due to the release of cytc from mitochondria. cell death is caused by the activation of caspase-3 by these activated caspases (aral et al., 2019). the extrinsic mechanism of apoptosis involves specific receptors binding to ligands, forming a disc complex, and activating caspases. this pathway is initiated when nk cells or macrophages produce death ligands that bind to the dr on the target cell membrane (schulze-osthoff et al., 1998; park et al., 2012). the role of these death receptors in the extrinsic pathway is significant, as they trigger the pathway by activating procaspase 8 to caspase 8 (schulze-osthoff et al., 1998). there are several dr types, such as cd95 (also known as fas and apo-1, tumor necrosis factor/tnf receptor 1 (tnfr1), tnf receptor superfamily member 25 (tnfrsf25)/dr3, tnf-related apoptosis-inducing ligand receptor 1 (trail-r1)/dr4, and tnf-related apoptosisinducing ligand receptor 2 (trail-r2)/dr5 (fossati et al., 2012; bittner et al., 2016). however, the first two receptors are the most well-known ones. these receptors contain a death domain, which requires adaptor proteins to operate properly, such as the fas-associated death domain (fadd) and tnf receptor-associated death domain (tradd). these proteins bind to the death receptor, strengthening the ligand-receptor-adaptor protein complex known as the death-inducing signaling complex (disc) (fossati et al., 2012; cavalcante et al., 2019). disc then activates procaspase 8, which later activates fig. 2. apoptosis process biologica nyssana ● 16 (2) december 2025: liana et al. ● cell deaths and their associated mechanisms following phosphorylation, mlkl oligomerizes and shifts from the cytoplasm to the cell membrane. this causes membrane permeabilization, probably owing to mlkl attachment to phosphatidylinositol lipids and cardiolipin, which results in cell death (loftus et al., 2022). ferroptosis ferroptosis is a unique type of rcd defined by the accumulation of lipid peroxides to fatal amounts based on an iron-dependent process (miao et al., 2023). it is distinguished biochemically and structurally from other forms of cell death and is typified by compression of the mitochondria and greater cellular membrane density with sparing of the nucleus. ferroptosis is caused when the action of proferroptotic agents (such as iron and polyunsaturated fatty acids [pufas]) and anti-ferroptotic protection mechanisms are unmatched. glutathione peroxidase 4 (gpx4) is the key enzyme governing protection against ferroptosis. it utilizes glutathione (gsh) to prevent the accumulation of lipid peroxides, thereby interfering with the system xc⁻ or incapacitating gpx4, which leads to unregulated lipoperoxidation and cell death (stockwell et al., 2020). it is reliant substantially on the availability of labile iron that participates in fenton reactions to generate reactive oxygen species (ros) causing lipoperoxidation (xu et al., 2024). pyroptosis classical apoptosis can be recognized by the partitioning of internal materials inside the cell and the removal of cellular debris without causing collateral injury to adjacent tissues (fink & cookson, 2005). zychlinsky et al. (1992) discovered pyroptosis for the first time, a type of cell death caused by proinflammatory impulses and linked to an inflammatory process. pyroptosis was first detected as a suicide mechanism of the gram-negative bacterial pathogen shigella flexneri-infected macrophages, and later in salmonella-infected cells (zychlinsky et al., 1992; hilbi et al., 1998; hersh et al., 1999). this is a highly inflammatory form of rcd mediated by the gasdermin family of poreforming proteins, considered as a key component of the innate immune response to pathogen infection and other danger signals (he et al., 2015). pyroptosis is primarily instigated by the activation of inflammasomes the intracellular multimeric sensing complexes of pathogen-associated molecular patterns (pamps) or damage-associated molecular patterns damps (zhang et al., 2021). activated inflammasomes (particularly the nlrp3 inflammasome) result in cleavage and activation of inflammatory caspases like caspase-1 within caspase 8 and triggers apoptosis (kober et al., 2011). necroptosis in 2005, a new cell death variant was discovered which resembled necrosis but had distinct characteristics. it is an alternate form of necrosis; however, in this circumstance, cells may undergo necrosis in a controlled manner (conrad et al., 2016), implying that apoptosis is not necessarily the favored mode of cell death. this sort of cell death is known as necroptosis (degterev et al., 2005). necroptosis is a pro-inflammatory, lytic rcd type that serves as an established viral defense process that allows cells to commit „cellular suicide” in a caspase-independent fashion in the presence of viral caspase inhibitors (associated with apoptosis blockage) to limit virus reproduction (orzalli & kagan, 2017). furthermore, necroptosis has been linked to inflammatory disorders occurring in several organ systems, including the pulmonary, gastrointestinal, neurological, and cardiovascular systems (khoury et al., 2020). in an environment-deprived scenario, necroptosis is modulated by the receptor-interacting proteins 1 and 3 (rip1 and rip3) (moriwaki et al., 2015). the dr is the most recognized activation mechanism for necroptosis, which is most typically driven by tnfr1. simultaneously, other factors, including tumor necrosis factor receptor-related apoptosisinducing ligand (trail) and the fas receptor, may act to trigger necroptosis (kim & li, 2013). ligand attachment to tnfr1 can initiate prosurvival complex i activation, including tnfrassociated death domain (tradd), rip1, and some e3 ubiquitin ligases. rip1 is polyubiquitinated within the complex i; meanwhile, deubiquitination of rip1 causes the formation of complex iia or iib. complex iia drives caspase 8 activation and induces apoptosis, while its inhibition results in the formation of complex iib, which stimulates necroptosis (chen et al., 2019). to initiate necroptosis, complex iib requires rip1 to partner with rip3 and cause autoand transphosphorylation, which leads to the oligomerization of phosphorylated rip3. this results in the formation of necrosomes, which are amyloid-like multiprotein complexes that distinguish them from uncontrolled necrosis. morphologically, necroptosis mimics unregulated necrosis, marked by oncosis, the eventual burst of the plasma membrane, and the release of pro-inflammatory intracellular components. mixed lineage kinase domain-like pseudokinase (mlkl), in addition to rip1 and rip3, has a role in necroptosis (cho et al., 2009; berghe et al., 2014). rip3 recruits mlkl and phosphorylates it at threonine 357 and serine 358. biologica nyssana ● 16 (2) december 2025: liana et al. ● cell deaths and their associated mechanisms the canonical pathway or caspase-4/5 (human) and caspase-11 (mouse) within the non-canonical signaling pathway (zhang et al., 2024). activated caspases (for example procaspase 1 to caspase 1) cleave a gasdermin family protein, mostly gasdermin d (gsdmd), into an n-terminal poreforming domain and a c-terminal inhibitor domain (he et al., 2015). there is also a secretion of the pro-inflammatory cytokines interleukin 1β (il-1β) and il-18 in their active forms, causing cell death and the release of inflammatory cytokines into the surrounding milieu. the n-terminal cleavage product oligomerizes and inserts into the cell membrane and forms small pores which permit ions to move through, resulting to a discrepancy of internal and extracellular ionic slopes (after potassium efflux); water then permeates the cell, causing cell expansion and lysis, in addition to a collapse of cell membrane integrity (wagatsuma & nakase, 2020). autophagy autophagy is when cellular constituents, such as macroproteins or organelles, are engulfed in the lysosome (intracellularly) to undergo enzymatic breakdown. the lysosome subsequently digests the substrate, and all elements that cannot be reconstituted to form novel cell components or organelles are further broken down and utilized as an energy source (ballabio & bonifacino, 2019). autophagy may be induced by several stressors, including nutritional shortages (calorie deprivation), signals occurring during cellular differentiation and embryogenesis, and compromised organellar interfaces. autophagy has also been implicated in adaptive and innate immune reactions, such as defense against viral infection (he et al., 2018). following the breakdown of intracellular pathogens, the antigen is presented to the major histocompatibility complex (mhc) class ii, and the nucleic acid from the pathogenic organism is then transferred to the toll-like receptor. although autophagy is commonly employed in recycling cellular parts, it may also cause cell demise. it has been attributed to the clearance of senescent cells from old tissues and eradication of neoplastic tumors. disruption of autophagy may contribute to malignant development. it is also linked (particularly in older species) to the formation of protein clumps in neurons and the aftermath of neurodegenerative disorders such as ad (barmaki et al., 2023). autophagy can be classified into macro-, micro-, and selective autophagy. the most well-discussed type is macroautophagy. in macroautophagy, double-membrane vesicles known as autophagosomes envelop the whole cell surface. these autophagosomes subsequently merge with lysosomes to form autophagolysosomes, where the proteases eliminate their contents (kroemer & levine, 2008). microautophagy occurs when organelles or cytosolic areas engage immediately with lysosomes. microautophagy is considerably more targeted compared to macroautophagy and can be initiated by signaling compounds present on the outer layers of defective organelles, such as peroxisomes and mitochondria, causing lysosomes to fuse with these organelles (settembre et al., 2013). selective autophagy, also referred to as chaperonemediated autophagy, occurs when a cytosolic chaperone targets proteins in the cytoplasm for union with lysosomes via communications between the chaperone and pentapeptides found in the substrate's amino acid sequence (choi et al., 2023). the substrate protein attaches to the lysosomal receptor lamp-2a and is transported to the lysosome for breakdown (bandyopadhyay et al., 2008). the autophagy process starts with the action of the ulk1 complex. a phosphoinositide-kinase (p13k) complex is needed to produce a phagophore, mainly the class iii complex. pi3k itself can be classified into four classes (class ia, ib, ii, and iii) with two main subunit types: a regulatory and a catalytic subunit. class ia, the most studied type of pi3k complex, has five subunits (p50α, p55α, p55γ, p85β, and p85α). meanwhile, the one specific for the autophagy process has vps15 as the regulatory and vps34 as the catalytic subunit to trigger the conversion of phosphatidylinositol (pi) to phosphatidylinositol 3-phosphate (pi(3) p) (engelman et al., 2006). the atg16l, atg5, and atg12 complexes, together with the lipidated microtubule-associated protein light chain 3 (lc3ii), facilitate phagophore elongation and serve as prerequisites for autophagosome construction (park et al., 2016; li et al., 2020). the p60 protein interacts with other proteins and is annihilated. p60 interacts with lc3ii, resulting in the formation of autophagosomes that engulf target proteins and organelles. autophagosomes then combine with lysosomes, whose contents are digested (dice, 2007). comparison of different major rcd types is available in tab. 1. other rcd classifications in addition to the previously described rcd types, several other kinds will be discussed below. this includes parthanatos, entosis, neutrophil extracellular traps (net)osis – suicidal pathway, lysosome-dependent cell death (lcd), alkaliptosis, oxeiptosis, and anoikis (galluzzi et al., 2018; khan et al., 2022). parthanatos is an rcd that depends on poly(adpribose) polymerase 1 (parp1) (conrad et al., 2016). biologica nyssana ● 16 (2) december 2025: liana et al. ● cell deaths and their associated mechanisms ta bl e 1. c om pa ris on o f m aj or re gu la te d ce ll de at h pa th w ay s ac sl 4: a cy l-c oa s yn th et as e lo ng c ha in f am ily m em be r 4 , a pa f1: a po pt ot ic p ep tid as e ac tiv at in g fa ct or 1 , a tg : a ut op ha gy -re la te d pr ot ei ns , b c l2: bce ll ly m ph om a 2, d a m ps : d am ag eas so ci at ed m ol ec ul ar p at te rn s, d n a : d eo xy rib on uc le ic a ci d, f as l: f as l ig an d, g px 4: g lu ta th io ne p er ox id as e 4, g sd m d : g as de rm in d , g sd m e: g as de rm in e , i l1β : i nt er le uk in -1 b et a, il -1 8: in te rle uk in -1 8, l ps : l ip op ol ys ac ch ar id e, m lk l: m ix ed l in ea ge k in as e d om ai nli ke p se ud ok in as e, m to r : m am m al ia n ta rg et o f r ap am yc in , p a m ps : p at ho ge nas so ci at ed m ol ec ul ar p at te rn s, p u fa s: p ol yu ns at ur at ed f at ty ac id s, r ip k 1: r ec ep to r-i nt er ac tin g se rin e/ th re on in epr ot ei n ki na se 1 , r ip k 3: r ec ep to r-i nt er ac tin g se rin e/ th re on in epr ot ei n ki na se 3 , r sl 3: r as se le ct iv e le th al 3 , t n f: t um or n ec ro si s fa ct or fe at ur e a po pt os is n ec ro pt os is fe rr op to si s a ut op ha gy py ro pt os is k ey m ed ia to rs c as pa se s ( in iti at or : 8 , 9; e xe cu tio ne r: 3, 6 , 7 ), b c l2 fa m ily , a pa f1 r ip k 1, r ip k 3, m lk l ir on , a c sl 4, g px 4, pu fa s a tg p ro te in s: a tg 5, a tg 7, a tg 12 , a nd a tg 8 g as de rm in s ( g sd m d , g sd m e) , c as pa se s ( 1, 4 , 5, 1 1) , i nfl am m as om es m or ph ol og y c el l s hr in ka ge , ch ro m at in c on de ns at io n, m em br an e bl eb bi ng , ap op to tic b od ie s c el l s w el lin g (o nc os is ), m em br an e ru pt ur e, or ga ne lle sw el lin g m ito ch on dr ia l sh rin ka ge , i nc re as ed m em br an e de ns ity , in ta ct n uc le us fo rm at io n of a d ou bl em em br an e ve si cl e (a ut op ha go so m e) c el l s w el lin g, m em br an e ru pt ur e, p or e fo rm at io n, „p yr op to tic b od ie s” pr im ar y t ri gg er s d n a d am ag e, g ro w th fa ct or w ith dr aw al , d ea th re ce pt or li ga tio n (t n f, fa sl ) d ea th re ce pt or lig at io n+ ca sp as e in hi bi tio n, p a m ps / d a m ps , v ira l i nf ec tio n g lu ta m at e, g px 4 in hi bi tio n, ir on ov er lo ad , o xi da tiv e st re ss c el lu la r s ta rv at io n, nu tri en t a nd g ro w th fa ct or d ep riv at io n, ce llu la r s tre ss a nd or ga ne lle d am ag e pa m ps /d a m ps (e .g ., lp s) , p at ho ge n in fe ct io n, ch em ot he ra py im m un ol og ic al c on se qu en ce g en er al ly n on in fla m m at or y („ si le nt ”) h ig hl y in fla m m at or y (d a m p re le as e) c on te xt -d ep en de nt , c an be im m un og en ic c on te xt -d ep en de nt h ig hl y in fla m m at or y (i l1β /il -1 8 re le as e) k ey p ha rm ac ol og ic al m od ul at or s in du ce rs : b h 3 m im et ic s ( e. g. , v en et oc la x) . in hi bi to rs : p an -c as pa se in hi bi to rs (e .g ., zv a d fm k) in hi bi to rs : r ip k 1 in hi bi to rs (e .g ., n ec ro st at in -1 , g sk 29 82 77 2) , m lk l in hi bi to rs (e .g ., n ec ro su lfo na m id e) in du ce rs : e ra st in , r sl 3. in hi bi to rs : fe rr os ta tin -1 , li pr ox st at in -1 , i ro n ch el at or s in du ce rs : m to r in hi bi to r r ap am yc in an d its d er iv at iv es . in hi bi to rs : c hl or oq ui ne a nd h yd ro xy ch lo ro qu in e in hi bi to rs : c as pa se -1 in hi bi to rs (e .g ., v x -7 65 ), g sd m d in hi bi to rs the process is triggered by oxidative stress, resulting in dna injury and chromatinolysis. parthanotic cell death does not involve the generation of apoptotic bodies or the breakdown of dna. furthermore, there is no cell swelling, although there is a rupture of the plasma membrane (andrabi et al., 2008). parp1 is a chromatin-associated nuclear protein essential for singleand double-strand dna repair. parp1 detects dna damage and uses nicotinamide adenine dinucleotide (nad+) and atp to initiate poly(adp-ribose)-ylation (vekariya et al., 2024). the parthanatos cycle necessitates the involvement of the apoptotic dnase endonuclease g (endog) and the apoptosis-inducing factor mitochondriaassociated 1 (aifm1, commonly referred to as aif). the binding of parp1 to aifm1 induces the translocation of aifm1 through the mitochondria to the cell nucleus, leading to parthanatotic chromatinolysis. the pathway can be obstructed by reducing parp1 expression through the action of the poly(adp-ribose)-degrading protein adpribosylhydrolase-like 2 (adprhl2, often referred to as arh3) (wang et al., 2011; fernández-lázaro et al., 2024). recently, it was discovered that macrophage migration inhibitory factor (mif) is an aifm1-binding protein with nuclease activity that produces large dna fragments during parthanatos induction (weiß et al., 2023). entosis is a form of cellular cannibalism wherein one cell swallows and annihilates another. entosis and entotic cell death mostly transpire in epithelial tumor cells under several situations, including abnormal proliferation, glucose deficiency, matrixinduced mortality, and mitotic pressure (durgan et al., 2017). entosis is demonstrated by the appearance of cell-in-cell structures in cancer patients' urine and ascitic fluid, with a frequency proportionate to tumor stage. cell adhesion and cytoskeletal rearrangement pathways are crucial in controlling entosis induction, even if the underlying mechanisms are unclear. entotic cell death entails the digestion of ingested cells by host cells, involving lc3-associated phagocytosis (lap) and the cathepsin b-mediated lysosomal degradation cascade (martins et al., 2017). entosis, however, does not include effector caspases and unregulated by bcl2 family proteins, important apoptotic characteristics (li et al., 2015). netotic cell death (netosis) is a form of rcd characterized by the expulsion of neutrophil extracellular traps (nets) (liana et al., 2022a). nets are protein structures resembling dna generated by cells reacting to viral or non-infectious stimuli. elevated netosis not only inhibits the advancement of pathogenic infiltration by sequestering harmful organisms but also induces the release of damps. this process may result in autoimmune disorders (liana et al., 2022b). netosis is a dynamic process that responds to several stimuli, including ros production by nadph oxidase, autophagy, and the secretion and transference of granule enzymes such as elastase, matrix metalloproteinase (mmp), and myeloperoxidase. subsequently, histone citrullination occurs, characterized by chromatin decondensation, cellular membrane disintegration, and the release of chromatin fibers (jorch & kubes, 2017). lcd, frequently referred to as lysosomal cell death, is an instance of rcd caused by the ejection of hydrolytic enzymes into the cytoplasm as a consequence of lysosomal barrier perforation. this process happens when cells are exposed to lysosomotropic detergents (sphingosine or phosphatidic acid) and ros (fogde et al., 2022). cathepsins, a class of lysosomal hydrolase enzymes, are essential in lysosomal degradation. inhibition of cathepsin expression or action can avert lcd. nonetheless, cathepsin is not the sole contributor to lcd, as lysosomes also sequester excess iron (lin et al., 2010). this implies that lysosomal membrane permeation could contribute to the expulsion of this hazardous metal into the cytosol, thus triggering ferroptosis, which was discussed in a previous section (min & kwon, 2020). alkaliptosis is a newly identified type of rcd that initiates through intracellular alkalinization (song et al., 2018). disruption of the nuclear factor kappa b kinase subunit beta (ikbkb, or ikkβ)nf;κβ pathway, which leads to the downregulation of carbonic anhydrase 9 (ca9), an enzyme critical for ph regulation, is an essential alkaliptosis mediator (liu et al., 2019). it was determined following jtc801 administration, an opioid analgesic, which effectively eradicates cell lines of human malignancies, causing cellular demise that are not characteristic of with apoptosis, necroptosis, autophagy, or ferroptosis (song et al., 2018). anoikis is a specific type of rcd that prohibits detached cells from surviving and populating in the wrong places. this is a vital process for preserving tissue homeostasis, making cells die after their detachment from the extracellular matrix (ecm) (taddei et al., 2012). this phenomenon is especially significant for epithelial and endothelial cells, which greatly depend on interactions with the ecm for survival and appropriate function (giannoni et al., 2008). moreover, anoikis is essential to prevent metastasis of cancer cells. however, in some cases, cancer cells develop an anoikis resistance, which allows them to enter the metastatic cascade, such as by the involvement of the muc1 extracellular domain. it enables cancer cells to withstand detachment, infiltrate the lymphatic or blood biologica nyssana ● 16 (2) december 2025: liana et al. ● cell deaths and their associated mechanisms systems, and eventually colonize far-off locations to develop secondary tumors (zhao et al., 2014). like apoptosis, anoikis is a process marked by cellular and molecular alterations, including membrane blebbing, chromatin condensation, dna fragmentation, and caspase activation. these processes result in the methodical breakdown and elimination of the cell without causing an inflammatory reaction (park et al., 2023). oxeiptosis is a form of rcd induced by oxygen radicals. it results from the activation of the keap-1-pgam5-aifm1 pathway. ozone or h2o2-induced oxeiptosis does not engage apoptotic or pyroptotic caspases, necroptosis, autophagy, or ferroptosis (holze et al., 2018). the keap1nfe2l2 pathway is recognized for facilitating cytoprotective responses to oxidative stress. active keap1 facilitates h2o2-induced oxeiptosis through a mechanism involving pgam5, a mitochondrial serine-threonine phosphatase that dephosphorylates aifm1 at ser116 (holze et al., 2018; tang et al., 2019). therapeutic implications the unraveling of the molecular processes governing rcd has reshaped medication development, shifting from broad cytotoxic drugs to personalized therapies designed to modulate certain cell death pathways. this approach could effectively eliminate malignant cells, such as cancer cells, or prevent cell death when it is inappropriate, as shown in neurodegenerative and inflammatory illnesses. the evasion of apoptosis is a hallmark of cancer, enabling tumor cells to endure stress associated with the disease and resist therapeutic interventions (mirzayans, 2024). a common method for this evasion is the overexpression of anti-apoptotic proteins from the bcl-2 group. this understanding led to the development of a class of medications known as „bh3 mimetics”, which are small compounds designed to mimic the bh3-only pro-apoptotic proteins that naturally counteract their anti-apoptotic counterparts (hassig et al., 2014; fulda, 2015). a drug, venetoclax, the inaugural bcl-2 inhibitor of its type, represents a significant advancement in this domain, particularly for treating numerous hematologic malignancies. in chronic lymphocytic leukemia (cll), particularly in individuals with the highrisk 17p deletion who often demonstrate resistance to conventional chemoimmunotherapy, venetoclax has exhibited substantial and enduring results (stilgenbauer et al., 2016). similarly, in acute myeloid leukemia (aml), the combination of venetoclax with hypomethylating drugs (e.g., azacytidine and decitabine) has become a new standard of care (ucciero et al., 2023). inhibiting cell death is the primary approach to treating numerous chronic inflammatory and neurodegenerative disorders. halting necroptosis is thought to mitigate tissue damage and disease progression in this context. ripk1, the primary kinase in the system, has been the principal focus of pharmacological development. the discovery of necrostatin-1, a small-molecule inhibitor of ripk1, represented the first pharmacological instrument to demonstrate the therapeutic effectiveness of necroptosis inhibition in many preclinical illness settings (vandenabeele et al., 2013). subsequently, more potent and specific next-generation ripk1 inhibitors with enhanced pharmacological characteristics were developed and evaluated in clinical studies. several drugs, including gsk2982772, sar443060 (dnl747), and sar443820 (dnl788), have been evaluated in phase i and ii trials for various neurodegenerative and inflammatory illnesses (vissers et al., 2022; hincelin-mery et al., 2024; gonen et al., 2025). although these drugs demonstrated a favorable safety profile, its clinical application has been challenging due to the lack of significant medical benefit. inducing ferroptosis in cancer highlights a unique therapeutic paradox: the same procedure that kills cancer cells can also be inhibited to preserve neurons, highlighting its context-dependent role in disease. as a result, multiple preclinical studies have demonstrated the significant efficacy of tool compounds like erastin and rsl3 in diverse cancer types (ghoochani et al., 2021). meanwhile, several potent ferroptosis inhibitors, such as the radical-trapping antioxidants ferrostatin-1 and liproxstatin-1, have consistently shown advantageous neuroprotective effects in animal models of neurodegenerative diseases (zilka et al., 2017). the therapeutic challenge is to develop ferroptosis inhibitors capable of traversing the blood-brain barrier, exhibiting high specificity, and possessing an extensive therapeutic window. the summary regarding cell death targeting to treat numerous diseases is available in fig. 3. immunogenic cell death and anti-tumor immune response immunogenic cell death (icd) has revolutionized the fields of cell death and immunology, illustrating that particular forms of rcd can convert dying tumor cells into a potent in situ vaccine capable of eliciting a robust and enduring anti-tumor immune response. this concept is now integral to contemporary cancer immunotherapy. biologica nyssana ● 16 (2) december 2025: liana et al. ● cell deaths and their associated mechanisms an icd is a variant of rcd that possesses sufficient strength to initiate an adaptive immune response against antigens derived from necrotic cells. the prompt release or exposure of a particular collection of molecules known as damps confers this immunogenicity (galluzzi et al., 2020). an identifiable „immunogenic triad” of damps is considered the hallmark of genuine icd. these damps initiate a complex immunological response that transforms a tumor microenvironment (tme) that is unresponsive to the immune system, or „cold”, into an inflamed or „hot” state, facilitating the infiltration of activated antigen-presenting cells (apcs) and effector t cells (galluzzi et al., 2020; solari et al., 2020; thiruppathi et al., 2024). this mechanism initiates a tumor-specific immune cycle. synergistic strategies have shown improved tumor control and survival compared to singular modalities. the interaction between cell death modulators and immune checkpoint blockade (icb) transcends drugs that induce traditional icd within the tumor cells themselves (arimoto et al., 2024). research on the bcl-2 inhibitor venetoclax in solid tumor models has revealed a significant alternative mechanism. venetoclax, a selective bcl-2 inhibitor, eliminates naive and memory t cells reliant on bcl-2, while sparing effector t cells that depend on bcl-xl and exhibit resistance to venetoclax. this mechanism induces an „immune-sculpting” impact in the tme, which selectively eliminates less active t-cell subsets, hence enhancing conditions for pd1-expressing effector t cells, the primary target of immune checkpoint blockade (icb) (ludwig et al., 2021; nagasaki et al., 2024). a major challenge in the clinical application of icd-based combination medicines is the selection of individuals who are most likely to benefit. this has prompted the quest for reliable biomarkers capable of predicting or monitoring the onset of an immunogenic response in patients. potential biomarkers can be broadly categorized into two types: those that quantify the damps directly and those that assess the ensuing immune responses (jiang et al., 2021). nonetheless, the results have been inconsistent across different cancer types and treatment modalities due to multiple challenges, including the technical difficulties in standardizing assays, the short half-life of these markers, and the release of damps like hmgb1 during non-icd death or sterile inflammation, which limits their specificity as icd biomarkers (ahmed & tait, 2020; thiruppathi et al., 2024). conclusion cell death is a natural process that maintains bodily homeostasis. the body's primary strategy for maintaining homeostasis is the selective elimination of 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(1992). shigella flexneri induces apoptosis in infected macrophages. nature, 358(6382), 167–169. https://doi.org/10.1038/358167a0 mirić 2022, biologica nyssana 13(1) 13 (1) september 2022: 27-31 doi: 10.5281/zenodo.7117534 population size of the bluethroat (cyanecula svecica) in the lower course of great bačka canal original article radislav mirić mir.radislav@gmail.com (corresponding author) received: september 01, 2021 revised: december 10, 2021 accepted: may 24, 2022 abstract: a census of the territorial males of the bluethroat (cyanecula svecica) was conducted in the year 2014 on the great bačka canal and surrounding wetlands. a total of 32 territorial males were recorded on the total transect length of 67.9 km. bluethroat population in the study area presents 12-20% of the total estimated population of this species in serbia. a significant short-term threat recorded was surface water regime maintenance since the removal of vegetation during this activity led to the destruction of habitat in several potential bluethroat territories. key words: breeding birds, habitat destruction, population density, wetland apstrakt: veličina populacije modrovoljke (cyanecula svecica) u donjem toku velikog bačkog kanala tokom 2014. godine sproveden je popis teritorijalnih mužjaka modrovoljke (cyanecula svecica) na velikom bačkom kanalu i okolnim vlažnim područjima. ukupno je zabeleženo 34 teritorijalna mužjaka na pređenih 67.9 km transekata. populacija modrovoljke zabeležena na ovom području predstavlja 11-19% celokupne procenjene populacije ove vrste u srbiji. kao značajan kratkoročni ugrožavajući činilac zabeleženo je održavanje kanalske mreže, pošto je uklanjanjem vegetacije tokom ove aktivnosti uništeno stanište na nekoliko mogućih teritorija modrovoljke. ključne reči: reproduktivno zrele ptice, uništavanje staništa, gustina populacije, vlažno introduction the bluethroat (cyanecula svecica) is a species of passerine bird belonging to the flycatcher family (muscicapidae). its breeding range covers a vast area from western europe across northern asia to the western coast of alaska (cramp, 1988). the species prefers ecotone habitats such as foresttundra, mountain steps, subalpine bush, coasts of lakes, ponds, and rivers with willows, reed beds, and other emergent vegetation (collar, 2019). it nests under the dense cover of mid-height vegetation with patches of bare ground (collar, 2019). there are 11 subspecies recognized in the world of which two are recorded in serbia. cyanecula svecica is a sporadic migrant, while c. cyanecula is a regular breeder in the northern part of the country. the population breeding in central europe prefers habitats covered with reeds natural and man-made ditches, canals, sewage farms, and fishponds (krüger, 1997; orłowski & sęk, 2005; petersen et al., 2005; van turnhout et al., 2010). the breeding period lasts from april to july, after which it remains in the vicinity until august and even mid-september (tomik, 2011). wintering range covers the mediterranean, sub-saharan africa, and south asia (kováts, 2009; collar, 2019). the breeding population in serbia is estimated at 170-280 breeding pairs with a stable population trend (puzović et al., 2015). although the current population is considered stable, historical data from the 19th century suggest a severe reduction of the breeding range (šćiban et al., 2015). in addition, we assume that bluethroat suffered a severe decline in serbia as a result of wetland drainage and vegetation succession as it happened in neighbouring countries (grüll, 2001). because threatening factors are still abundant, the species is considered near threatened in the red book of the fauna of serbia (mirić et al., 2018). the main aim of this paper is to quantify the population size and density of bluethroat in the lower course of the great bačka canal with surrounding wetlands and to evaluate it’s significance in comparison with a total country population. © 2022 mirić. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 27 materials and methods the study area is located within the vojvodina province (n 45.54°, e 19.90°) and includes a section of the great bačka canal that runs from srbobran to bačko gradište, lower courses of krivaja and beljanska bara rivers which are medium-sized rivers with the slow flow (in the further text slow-flowing rivers). adjacent 3-5 m wide and 1.5-3 m deep drainage ditches and surrounding ponds, that are in some cases completely overgrown with reed (ponds smaller than 10 ha) were part of the study area. listed wetlands are predominantly surrounded by arable lands and have suffered significant changes after reclamation activity that has been undertaken from 19th to the mid-20th century (tomić, 1974; stojšić, 1977). the water level is maintained artificially with little fluctuation throughout the year. the width of the floodplain is 50 m on average. the majority of the banks are covered with reeds which form a belt from 1 to 130 m in width. the water surface is at 78 m a.s.l., whilst surrounding land rises to 85 m a.s.l. (tomić, 1990). the dominant plant species is the common reed (phragmites australis) which creates many communities with the other species such as cattail (typha sp.), grey willow (salix cinerea), crack willow (salix fragilis), and white willow (salix alba) (lakušić et al., 2005; dobretić & stojšić, 2011). the census of bluethroats took place from the 10th to the 23rd of april 2014 on sunny days, without wind and rain, from 4 a.m. to 1 p.m. according to merilä & sorjonen (1994) the census should be ended before 10 a.m. because of a significant decrease in territorial male activity. in this study, since the level of bluethroat activity remained stable well after 8 a.m., census lasted until 1 p.m (fig. 1, fig. 2). a total transect length of 67.9 km was covered, of which: 19.8 km along great bačka canal, 16.3 km along beljanska bara river, 9.6 km along krivaja river, 13.3 km along ponds and 8.9 km along drainage ditches. sampling was undertaken based on the line transects with the stop points and the use of male call tape lure. along each transect, every 300 m, bluethroat song was played two times for 1 minute followed by 1 minute pause for listening after every song playback. distance between count points in this census was increased compared to 200 m that sorjonen & merilä (2000) and peris & mendes (2010) propose in order to reduce the chance of double counting. also, the terrain is deforested and flat, thus there are no obstacles for sound to spread. every male recorded singing or in display flight, which stands for possible and probable breeding in 28 biologica nyssana ● 13 (1) september 2022: 27-31 mirić ● population size of the bluethroat (cyanecula svecica) in the lower course of great bačka canal fig. 1. time of song/song flight recorded without song playback fig. 2. time of every song/song flight recorded european breeding bird atlas methodology (ebcc 2015), is considered territorial. area of suitable habitat, used to calculate bluethroat density, includes reed bed surface around surveyed water bodies, within a distance belt of 60 m from transects. the detection rate of bluethroats diminishes dramatically when they are more than 60 meters away from the observer (musseau & beslic, 2018), hence this distance is chosen. habitat surface was calculated using google earth pro and arcmap 10.8 software and in total it amounts to 171.88 ha, of which: 111.04 ha around slowflowing rivers, 44.34 ha on ponds, and 16.5 ha along drainage ditches. bluethroat density was estimated by dividing the number of territorial males by the area of suitable habitat. google earth pro was also used to calculate reed bed width and to digitalize the location of recorded birds. arcmap 10.8 was used for map production. results during the study period 32 territorial males were recorded (fig. 3). out of this number 18 males were counted around slow-flowing rivers, 9 were situated on ponds and 5 of them were in drainage ditches. the average bluethroat density is 1.86/10 ha. separately for each habitat type, bluethroat 29 densities are the following: 1.62/10 ha for slowflowing rivers, 2.03/10 ha for ponds and 3.03/10 ha for drainage ditches. discussion if we compare the number of territorial males registered in this study (n = 32) with puzović et al. (2015) population estimate for bluethroat in serbia, we can say it represents 11-19% of the total population of the country. number of males registered in study area is minimal, as it is known that bluethroat males significantly reduce their response to a tape lure after acquiring a mate (sorjonen & merilä, 2000) and since they have an evident habit of discretion if observer is too close to it (musseau & beslic, 2018). biologica nyssana ● 13 (1) september 2022: 27-31 mirić ● population size of the bluethroat (cyanecula svecica) in the lower course of great bačka canal however, the estimate given by puzović et al. (2015) was based on sporadic findings from large wetlands (e.g. kormanjoš, 1989; gergelj et al., 2000; žuljević & đapić, 2002; vig et al., 2009; radišić & tucakov, 2010; tucakov, 2011; vig et al., 2012) without any systematic research. this study indicates that small wetlands such as drainage ditches and ponds are significant for this species. this is also shown in the research performed in lower saxony (krüger, 2002), in which around 50% of territories are found on fig. 3. study area with potential bluethroat territories drainage ditches. when we consider that the province of vojvodina has over 20,000 km of drainage ditches (vranešević, 2015), and we multiply it by the 5 males found on 8.9 km of the same habitat in this study, we get an estimate of over 11,000 males just on drainage ditches. unfortunately, there is no available data from other parts of the country, so this estimate should be taken with reserve. a few conspicuous but under-researched habitatrelated occurrences were also noticed. on territories of 18 bluethroat males some amount of burnt reed was registered. some reed beds were reduced to only a few m2 which males used for perch and shelter. a similar case was recorded in poland where the fire occurred during the breeding season, but it did not chase birds away from their territories (orłowski & sęk, 2005). also, 24 bluethroat males were recorded in habitats with the one-year-old reed, which is not in line with the findings of vadász and csörgő (2009) who assumed that such habitat is deprived of species diversity and abundance. it is worth to mention that in the years following this study several cases of bluethroat habitat destruction were recorded in study area. on seven locations where bluethroats were recorded, habitats were destroyed, in some cases even during the breed30 ing season. habitat destruction is the consequence of surface water regime maintenance, such as drainage ditch maintenance and reed bed removal. this problem was recorded earlier in the vicinity of the study area, where two males lost their territories because of drainage ditch maintenance (šćiban, 2004). with this in mind, drainage ditches can be considered ecological traps for bluethroat. besides the aforementioned problem, additional threats for bluethroat in the entirety of its range in serbia and in central europe are e.g., long-term water level decrease, vegetation succession, reduction of wetlands, and possible predation pressure (grüll, 2001; vadász et al., 2011; čížková et al., 2013). these threats have the greatest impact on the birds that live in the habitats surveyed in this study, such as ponds, so developing national conservation plans for them could be important for bluethroat conservation in serbia. acknowledgements. i would like to thank draženko rajković and marko šćiban for advice given during paper writing,vukašin kartalović for helping with the graphic presentation, and milica krmar for english proofreading. references čížková, h., květ, j., comín, f.a., laiho, r., pokorný, j., pithart, d. 2013: actual state of european wetlands and their possible future in the context of global climate change. aquatic sciences, 75: 3-26. collar, n. 2019: bluethroat (cyanecula svecica). in: del hoyo, j., elliott, a., sargatal, j., christie, d.a., de juana (eds.), handbook of the birds of the world alive. lynx edicions, barcelona. cramp, s. 1988: the birds of the western palearctic, 5. oxford university press, oxford. dobretić, v., stojšić, v. 2011: park prirode “beljanska bara” predlog za stavljanje pod zaštitu zaštićeno područje iii kategorije. pokrajinski zavod za zaštitu prirode, novi sad. european bird census council (ebcc) (2015): ebba2 methodology. https://www.ebba2.info/wpcontent/uploads/2015/01/ebba2_methodology_ final.pdf (accessed on august 1st, 2021). gergelj, j., tot, l., frank, z. 2000: ptice potisja od kanjiže do novog bečeja. ciconia, 9: 121-154. grüll, a. 2001: investigations on the population of the white-spotted bluethroat (luscinia svecica cyanecula) in the area of lake neusiedl (burgenland, austria). egretta 44: 1-44. kováts, l. 2009: bluethroat luscinia svecica (linnaeus, 1758). in: csörgő, t., karcza, z., halmos, g., magyar, g., gyurácz, t., szép, t., bankovics, a., schmidt, s., schmidt, e. 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(eds), crvena knjiga faune srbije iii – ptice. beograd: zavod za zaštitu prirode srbije, univerzitet u novom sadu, prirodno-matematički fakultet, departman za biologiju i ekologiju i društvo za zaštitu i proučavanje ptica srbije. 442-444 p. musseau, r., beslic, s. 2018: high densities of the french coastal endemic bluethroat (cyanecula svecica namentum) revealed in intertidal reed beds and conservation perspectives towards sea level rise. revue d’ecologie (terre et vie), 73(2): 115-121. orłowski, g., sęk, m. 2005: semi-natural reedbeds as breeding habitat of bluethroat (luscinia svecica l.) on a sewage farm inwrocław city (south-western poland). polish journal of ecology, 53:133-140. peris, j., mendes, s. 2010: comparación de dos métodos de censo para estimar la población reproductora del pechiazul luscinia svecica azuricollis. ardeola, 57 (especial):117-122. biologica nyssana ● 13 (1) september 2022: 27-31 mirić ● population size of the bluethroat (cyanecula svecica) in the lower course of great bačka canal 31 biologica nyssana ● 13 (1) september 2022: 27-31 mirić ● population size of the bluethroat (cyanecula svecica) in the lower course of great bačka canal petersen, b., krüger, t., zang, h. 2005: blaukehlchen – luscinia svecica (l., 1758). in: zang, h., heckenroth, h., südbeck, p. (eds): die vögel niedersachsens und des landes bremen – drosseln, grasmücken, fliegenschnäpper, naturschutz und landschaftspflege in niedersachsen, sonderreihe b heft 2.9. hannover. 50-62 p. puzović, s., radišić, d., ružić, m., rajković, d., radaković, m., pantović, u., janković, m., stojnić, n., šćiban, m., tucakov, m., gergelj, j., sekulić, g., agošton, a., raković, m. 2015: ptice srbije: procena veličina populacija i trendova gnezdarica 2008 – 2013. društvo za zaštitu i proučavanje ptica srbije i prirodno-matematički fakultet, departman za biologiju i ekologiju, univerzitet u novom sadu, novi sad. radišić, d., tucakov, m. 2010: sastav i status faune ptica ribnjaka kod bača u periodu 2000-2010. ciconia, 19: 33-47. sorjonen, j., merilä, j. 2000: response of male bluethroats luscinia svecica to song playback: evidence of territorial function of song and song flights. ornis fennica, 77: 43-47. stojšić, m. 1977: odvodnjavanje ravničarskog dela vojvodine. vodoprivreda, 45-46: 19-23. šćiban, m. 2000: podaci o nekim zanimljivim vrstama ptica uz veliki bački kanal od kule do čuruga. ciconia, 9:185-186. šćiban, m. 2004: bluethroath luscinia svecica. acrocephalus, 25: 234. šćiban, m., rajković, d., radišić, d., vasić, v., pantović, u. 2015: ptice srbije – kritički spisak vrsta. pokrajinski zavod za zaštitu prirode i društvo za zaštitu i proučavanje ptica srbije, novi sad. tomić, p. 1974: turija, fizičko – geografske odlike okoline. zemlja i ljudi, 24: 139-144. tomić, p. 1990: geografska monografija turije. prirodno-matematički fakultet, institut za geografiju novi sad, novi sad. tomik, a. 2011: inventarizacija gnijezdeće populacije modrovoljke erithacus svecicus i žutog voljića hippolais icterina, konačno izvješće. hrvatsko društvo za zaštitu ptica i prirode, osijek. 49 p. tucakov, m. 2011: potamišje – dinamično poplavno područje. iucn, beograd. vadász, c., csörgő, t. 2009: evaluation of regulations of the agro-environmental program focusing on reedbed management, based on their effects on the breeding passerine assemblage. természetvédelmi közlemények, 15: 235-245. vadász, c., mogyorósi, s., pellinger, a., aleksza, r., biró, c. 2011: results of the breeding passerine census carried out at the hungarian part of lake fertő in 2008. ornis hungarica, 19: 11-20. van turnhout, c.a.m., hagemeijer, e.j.m., foppen, r.p.b. 2010: long-term population developments in typical marshland birds in the netherlands. ardea, 98: 283-299. vig, l., balog, i., pete, g. 2009: ptice jegričke. društvo ljubitelja prirode “falco”, temerin. vig, l., đureković-tešić, o., marić, b., stojanović, t., puzović, s., stojnić, n., knežev, m., lazić, l., stojanović, v. 2012: slano kopovo. edicija “ramsarska područja vojvodine” knjiga iii. pokrajinski sekretarijat za urbanizam, graditeljstvo i zaštitu životne sredine, novi sad. vranešević, m. 2015: biotehničke mere kao mogućnost za povećanje efikasnosti sistema za odvodnjavanje. phd thesis. poljoprivredni fakultet, novi sad. žuljević, a., đapić, d. 2002: podaci o fauni ptica bare “jezero” kod stanišića u severozapadnoj bačkoj. ciconia, 11: 123-126. stojanović 2025, biologica nyssana 16(2) 16 (2) december 2025: doi: 10.46793/biolnyss.16.2.5s influence of milk fat-to-protein ratio on the energetic status of holstein-friesian cows in early lactation original article goran stojanović toplica academy of applied studies, department of agriculture and food technology studies, prokuplje, serbia gstojanovic2002@yahoo.com (corresponding author) received: july 18, 2025 revised: august 12, 2025 accepted: august 12, 2025 abstract: this study presents the results of an investigation into the organic components of milk in holstein-friesian cows during early lactation, with a particular emphasis on analyzing key biochemical parameters—specifically protein and fat—and their interrelationship. special attention was given to evaluating the correlation between these components in order to identify cows experiencing a pronounced negative energy balance during early lactation, while simultaneously aiming to prevent metabolic disorders and reproductive complications. the research was conducted on 90 holstein-friesian dairy cows at an early stage of lactation, on a farm located in the municipality of dimitrovgrad. routine sampling for milk biochemical analysis was used for this purpose, focusing on fat and protein content and their ratio. the analysis was performed using a milkoscan device. the results indicate that the energy status of high-yielding dairy cows has a significant influence on their production performance, reproductive efficiency, and overall health. key words: early lactation, nutritional status, organic components, metabolism, energy balance apstrakt: uticaj odnosa masti i proteina u mleku holštajn-frizijskih krava na energetski balans u ranoj laktaciji ovaj rad prikazuje rezultate istraživanja organskih sastojaka mleka kod krava holštajn-frizijske rase tokom rane laktacije, s posebnim fokusom na analizu ključnih biohemijskih parametara (proteina i masti) i njihov međusobni odnos. posebna pažnja posvećena je oceni korelacije ovih komponenti kako bi se identifikovale krave sa izraženim negativnim energetskim bilansom u fazi rane laktacije, uz istovremeno sprečavanje metaboličkih i reproduktivnih poremećaja. istraživanje je sprovedeno na 90 mlečnih krava holštajn-frizijske rase u ranoj fazi laktacije, na farmi krava u opštini dimitrovgrad. za potrebe istraživanja iskorišćeno je rutinsko uzorkovanje za određivanje biohemijskog sastava mleka, a u ovom radu je analiziran sadržaj masti i proteina, kao i njihov međusobni odnos. sadržaj masti i proteina određeni su pomoću uređaja milkoskan. rezultati ukazuju da energetski status visokoproduktivnih mlečnih krava značajno utiče na njihove proizvodne rezultate, reproduktivne performanse i zdravstveno stanje. ključne reči: rana laktacija, nutritivni status, organski sastojci, metabolizam, energetski balans introduction the most significant health disorders in highyielding dairy cows arise as a result of disturbances in energy metabolism. it is well established that the most critical phase for the metabolic stability of high-producing cows is the transition from the dry period to early lactation. during this phase, the animal’s energy and nutrient demands change drastically (šamanc et al., 2006, 2011; lin, 2024). while energy balance is positive during late pregnancy, it becomes negative immediately after calving, when lactation begins and the organism’s energy demands sharply increase. in conditions of negative energy balance, the body utilizes its internal energy reserves first glycogen, then fat, and finally protein stores (nogalski et al., 2012; roche et al., 2013). successful adaptation of the metabolic and endocrine systems during this period enables the cow to transition from a state of late gestation to one © 2025 stojanović. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. of intensive lactation (kirovski et al., 2011, 2012; cabezas-garcia et al., 2021; barłowska et al., 2024). the consequences of energy imbalance manifest as negative energy balance, during which cows may develop hepatic lipidosis (“fatty liver”) and other metabolic disorders (šamanc et al., 2006, 2011, 2013). the quality of milk intended for human consumption is directly dependent on the maintenance of metabolic stability in dairy cows (toscano et al., 2023). increased lipomobilization, commonly observed during energy deficits, can elevate milk fat content while simultaneously reducing protein levels changes that may adversely affect the technological properties of milk during processing (van knegsel et al., 2005; kirovski et al., 2011, 2012; knob et al., 2021). it is evident that intensive cattle farming practices, aimed at maximizing the genetic potential for milk production and reproductive performance, may contribute to disturbances in energy metabolism. as previously noted, energy reserves in high-yielding cows are stored in adipose tissue during the late lactation phase and the dry period. since cows in early lactation are typically unable to ingest sufficient quantities of food to meet their elevated energy requirements, the energy deficit is compensated by mobilizing body fat stores. therefore, assessing energy reserves during the periparturient period is a crucial procedure for determining the animal’s energetic condition (šamanc et al., 2006, 2011, 2013; kirovski et al., 2011, 2012, 2013; đoković et al., 2016; bojković-kovačević, 2016). given these considerations, maintaining metabolic stability in cows during the periparturient and early lactation periods is essential, not only to achieve high milk yields but also to ensure milk that meets the nutritional and hygienic standards of the modern market. only through proper nutritional management, veterinary care, and rigorous control of raw milk quality can high-quality dairy products be consistently produced for human consumption, thereby supporting a sustainable and competitive dairy industry (kirovski et al., 2011, 2012; bojkovićkovačević, 2016; đoković et al., 2016; barłowska et al., 2024). in the past decade, the determination of organic component concentrations in milk has been increasingly used as a method for assessing the energy status of dairy cows (van knegsel et al., 2005; kampl, 2005; šamanc et al., 2006; kirovski et al., 2011, 2012; knob et al., 2021). such an assessment is highly reliable and easily applicable, as sample collection is non-stressful for the animal. moreover, it is highly cost-effective when integrated into the routine analysis of raw milk components conducted for milk quality evaluation (kampl, 2005; šamanc et al., 2006). the energy supply status of animals can also be determined based on the ratio of fat to protein content in milk (šamanc et al., 2006; kirovski et al., 2011, 2012, 2013; trifkovic et al., 2015). specifically, during intensive lipomobilization, the concentration of free fatty acids in the blood increases, leading to enhanced synthesis of milk fat and its concentration in milk (nogalski et al., 2012). if the milk protein content exceeds 3.2% and the milk fat content remains below 4.5%, the energy supply is considered adequate. conversely, if the fat content in milk increases while the protein content decreases simultaneously, this indicates an energy deficit within the herd (kirovski et al., 2011, 2012; lakić et al., 2018; knob et al., 2021). materials and methods the study was conducted during july and august 2023 on a holstein-friesian dairy farm located in the municipality of dimitrovgrad. the investigation included a total of 90 cows in early lactation (the first 60 days postpartum). the cows were divided into three groups (a, b, and c): group a consisted of cows within the first 15 days of lactation, group b included those within the first 30 days, and group c included cows at 60 days of lactation. the cows were in their first to sixth lactation, with an average daily milk yield of 27.2 liters. milking was performed twice daily, in the morning and evening. for the purpose of this study, milk samples were collected during morning milking as part of the routine analysis of biochemical composition. the samples were transported to the laboratory in portable coolers at 8 °c and then stored at -20 °c until analysis. the data used in this study pertained to the fat and protein content of milk. these parameters were analyzed using a milkoscan device (series 130, model 10900, a/s, n. foss electric) at the eko-lab d.o.o. laboratory in belgrade (idf, 2015; lefier et al., 1996). the fat-to-protein ratio was evaluated in order to reliably diagnose the energy status of the cows under investigation (kirovski et al., 2011, 2013; đoković et al., 2016). the results were statistically processed and presented in tables using descriptive statistical parameters, including arithmetic means (x), standard deviations (sd), standard errors (se), coefficients of variation (cv), and ranges (iv). additionally, pearson’s correlation coefficient was calculated for selected biochemical components using microsoft excel (office 365). biologica nyssana ● 16 (2) december 2025: stojanović ● influence of milk fat-to-protein ratio on the energetic status of holstein-friesian cows in early lactation biologica nyssana ● 16 (2) december 2025: stojanović ● influence of milk fat-to-protein ratio on the energetic status of holstein-friesian cows in early lactation results and discussion the protein and fat content of milk samples collected during morning milking were thoroughly analyzed and are presented in tab. 1. these biochemical parameters in milk serve as key indicators of proper nutrition and metabolic function in cows and are sufficient for the rapid and efficient assessment of the energy status of the examined animals. optimal range, while the other two farms, particularly farm a, exhibited lower values. the decrease in milk fat content observed on two farms is most often attributed to changes in cow nutrition and energy status. several hypotheses exist regarding the mechanisms responsible for the onset of milk fat depression syndrome. most agree that it results from alterations in ruminal digestion processes. as a consequence, the supply of acetate decreases, along with the rate of milk fat synthesis, since acetate is known to be the primary precursor for milk fat synthesis in mammary epithelial cells (grummer, 1993). the fat content in the milk of cows in groups a, b, and c was individually measured at 2.76±0.83%, 3.15±1.03%, and 3.41±1.12%, respectively. the fat content in the milk of cows in group a was significantly lower than that of group c (p< 0.05). the overall average fat content across all cows was 3.11±1.02%. the investigation of milk organic components conducted by šamanc et al. (2006) found that the fat content in milk from cows on farms b and d was 1.66±0.75% and 1.07±0.26%, respectively. based on these results, it can be concluded that these farms likely experienced milk fat depression syndrome. this diagnosis is made when milk fat content is at least 50% below the optimal level, typically in the range of 1.2% to 1.7%. on the other two farms (farms a and c), the milk fat content was 3.89±0.77% and 3.38±0.93%, respectively values considered normal for this breed (šamanc et al., 2006). the findings of this study are consistent with those reported by other authors, including kirovski et al. (2012) and đoković et al. (2016). in comparison with the study by đoković et al. (2016), which reported a milk fat content of 3.42±0.48% in holstein cows, our results fall within a similar range, confirming a general trend in milk production across farms. however, compared to their findings on protein concentration, holstein cows exhibited significantly higher levels at the beginning of lactation (4.67±2.72%), while our results indicate a lower protein content in the milk of the cows examined in this study. during intensive fat mobilization, the concentration of free fatty acids in the blood increases. this is reflected in the extent of fat synthesis within the mammary gland, leading to elevated levels of milk fat in such cases (kirovski et al., 2011, 2012, 2013). based on the obtained results, dietary adjustments and improvements in the health status of the cows can be recommended (kirovski et al., 2012). the examination of milk biochemical components urea, protein, and fat can contribute to a better table 1. milk protein (%) and fat (%) content in holstein-friesian cows sampled during morning milking milk fat (%) milk protein (%) group a (n=30) x 2.76 3.03 sd 0.83 0.15 se 0.15 0.03 cv 30.07 0.49 iv 1.26-4.62 2.71-3.38 group b (n=30) x 3.15 3.16 sd 1.03 0.25 se 0.18 0.05 cv 32.69 0.79 iv 0.95-4.88 2.71-3.87 group c (n=30) x 3.41 3.15 sd 1.12 0.21 se 0.20 0.04 cv 32.84 0.66 iv 0.96-6.56 2.89-3.72 a, b and c combined (n=90) x 3.11 3.11 sd 1.02 0.21 se 0.11 0.02 cv 32.79 0.67 iv 0.95-6.56 2.71-3.87 šamanc et al. (2006) found that the greatest deviations from physiological values in milk occur in fat content and urea concentration, while lactose and protein levels vary to a lesser extent. in their study of four farms, they reported that milk fat content ranged from 1.07% to 3.89%. the results of our research showed that the average milk fat content on the studied farms was 2.76±0.83%, 3.15±1.03%, and 3.41±1.12%, respectively. the fat content in milk from cows on farm a was significantly lower compared to that of farm c (p<0.05). compared to the data reported by those authors, only farm c had fat content within the biologica nyssana ● 16 (2) december 2025: stojanović ● influence of milk fat-to-protein ratio on the energetic status of holstein-friesian cows in early lactation understanding of the energy status of high-yielding dairy cows and allow for the timely implementation of appropriate measures to prevent numerous health disorders that result from negative energy balance (šamanc et al., 2006; kirovski et al., 2011, 2012; đoković et al., 2016). based on statistical analysis, pearson’s correlation coefficient between milk fat and protein content was determined to be approximately r ≈ 0.730, indicating a moderately strong positive correlation between these variables. this correlation suggests that an increase in milk protein content is significantly associated with an increase in milk fat content, reflecting the interrelated synthesis processes of these components within the mammary gland. given that the levels of fat and protein in milk reflect not only the genetic potential of the cows but also the quality of their diet and metabolic status, these findings confirm that a balanced intake of protein and energy in the diet contributes to optimal milk quality (kirovski et al., 2011; toscano et al., 2023). the testing of the null hypothesis (h₀), which posited that there is no statistically significant relationship between these parameters, resulted in its rejection, as the correlation was found to be statistically significant (p<0.05). this finding is consistent with the results reported in the literature. these data further emphasize the importance of properly formulated nutrition in achieving optimal nutritional status and health in dairy cows, particularly during phases of high production demands (roy et al., 2011). from fig. 1, it is observed that the majority of data points fall within the lower-left quadrant, indicating a significant energy deficit based on the ratio of milk fat content (below 4.5%) and protein content (below 3.2%) in the examined cows. a smaller proportion of cows, located in the lower-right quadrant, did not exhibit a negative energy balance, meaning they were fed an optimal amount of energy appropriate for the demands of early lactation (the first 60 days postpartum). based on the fat-to-protein ratio, šamanc et al. (2006) determined that a significant energy supply deficit was present in the majority of cows. this observation is also confirmed by the results of our study. a large portion of the cows examined in the study by kampl (2005) were likewise positioned in the lower-left quadrant, indicating a pronounced energy deficit, which aligns with the findings of our research. from the overall analysis of the fat-toprotein ratio, šamanc et al. (2006) also concluded that the majority of cows across the four examined farms exhibited an energy deficit an observation that closely aligns with the results of our investigation. the findings of this study are valuable for further research and for improving feeding management strategies to prevent metabolic and reproductive disorders, as observed in cow populations experiencing negative energy balance. these observations are consistent with the research outcomes of other authors as well (roy et al., 2011; schwab et al., 2017). conclusion based on the presented findings, it can be concluded that the analysis of biochemical milk components (urea, total protein and fat) can significantly contribute to a better understanding of the energy status of high-yielding dairy cows, as well as to the timely implementation of appropriate measures to prevent various health disorders arising from negative energy balance. the use of milk organic components as indicators of cows’ energy status has gained considerable interest among veterinary professionals due to its practical applicability in field conditions. this approach simplifies both the sampling of biological material and the required laboratory analyses. compared to the long-standing metabolic profiling method, which involves testing a large number of parameters, this method represents a rational and financially feasible diagnostic approach for any producer, regardless of whether they operate a small-scale or large-scale cattle farm. our results support the widely accepted view that fig. 1. correlation between milk fat and protein content in morning milking, aggregated across largescale farms a, b, and c, in holstein-friesian dairy cows. biologica nyssana ● 16 (2) december 2025: stojanović ● influence of milk fat-to-protein ratio on the energetic status of holstein-friesian cows in early lactation mlečnih krava u mleku i njihovo korišćenje u programu zdravstvene preventive i intenziviranja proizvodnje i reprodukcije. in zbornik radova, 4. simpozijum “ishrana, reprodukcija i zaštita zdravlja goveda”, subotica, 2005. kirovski, d., šamanc, h., prodanović, r., sladojević, ž., & vujanac, i. (2011). evaluation of dairy cows energy status by biochemical analysis of organic components of milk. in proceedings of days of veterinary medicine, 9–11 september 2011, ohrid, macedonia. kirovski, d., šamanc, h., prodanović, r., sladojević, ž., & vujanac, i. (2012). nutritivni status visoko mlečnih krava: procena na osnovu sastava mleka. veterinarski žurnal republike srpske, xiii(2), 109–116. https://doi.org/10.7251/ vjrs1302109k kirovski, d., šamanc, h., vujanac, i., prodanović, r., sladojević, ž., & savić, đ. (2013). prediction of energy balance of early lactation dairy cows from milk composition measures at individual cow level. in ii international symposium and xviii scientific conference of agronomists of republic of srpska, 26–29 march, trebinje, bosnia and herzegovina, book of abstracts. knob, d.a., thaler neto, a., schweizer, h., weigand, a.c., kappes, r., & scholz, a.m. (2021). energy balance indicators during the transition period and early lactation of purebred holstein and simmental cows and their crosses. animals, 11(2), 309. https://doi.org/10.3390/ani11020309 lakić, i., cincović, m.r., belić, b., đoković, r., majkić, m., petrović, m.ž., & nikolić, s. (2018). lipoliza i ketogeneza kod krava u ranoj laktaciji. acta agriculturae serbica, 23(46), 265–276. https:// doi.org/10.5937/aaser1846265l lefier, d., grappin, r., & pochet, s. (1996). determination of fat, protein, and lactose in raw milk by fourier transform infrared spectroscopy and by analysis with a conventional filter-based milk analyzer. journal of aoac international, 79(3), 711–717. https://doi.org/10.1093/jaoac/79.3.711 lin, x.f. (2024). optimizing animal energy metabolism: nutritional strategies in agricultural production. international journal of molecular zoology, 14(5), 255–264. https://doi.org/10.5376/ ijmz.2024.14.0023 nogalski, z., wroński, m., sobczuk-szul, m., mochol, m., & pogorzelska, p. (2012). the effect of body energy reserve mobilization on the fatty acid profile of milk in high-yielding cows. asian-australasian journal of animal sciences, the health condition and production performance of high-yielding dairy cows in our country are most often a reflection of inadequately applied modern feeding technologies. the consequences of such practices include prolonged energy deficits and numerous metabolic disorders, accompanied by consistently reduced milk production and impaired reproductive performance. this, in turn, leads to significant financial losses and inefficient utilization of the cows’ genetic potential for milk production. this issue was common in all former state-owned farms and is now increasingly evident on newly established small-scale farms with genetically superior holstein and simmental cattle. references barłowska, j., wójcik-saganek, a., litwińczuk, z., brodziak, a., teter, a., & kędzierskamatysek, m. (2024). nutritional value and technological parameters of milk from organic and conventional farms in poland. annals of animal science, 24(3), 949–964. https://doi.org/10.2478/ aoas-2024-0008 bojković-kovačević, s.s. (2016). metabolički status krava holštajn rase u peripartalnom periodu kao prognostički faktor proizvodnih rezultata u ranoj laktaciji [metabolic status of peripartal holstein cows, as a prognostic factor of production results in early lactation] (doctoral dissertation, university of belgrade, faculty of veterinary medicine). https:// phaidrabg.bg.ac.rs/view/o:14511 cabezas-garcia, e.h., gordon, a.w., mulligan, f.j., & ferris, c.p. (2021). revisiting the relationships between fat-to-protein ratio in milk and energy balance in dairy cows of different parities, and at different stages of lactation. animals, 11(11), 3256. https://doi.org/10.3390/ani11113256 đoković, r., ilić, z., kurćubić, v., petrović, m. p., caro-petrović, v., milošević, b., & omerović, i. (2016). određivanje metaboličkog i hranidbenog statusa kod mlečnih krava tokom početka i sredine laktacije. biotechnology in animal husbandry, 32(1), 1–8. https://doi.org/10.2298/bah1601001d grummer, r.r. (1993). etiology of lipid-related metabolic disorders in periparturient dairy cows. journal of dairy science, 76(12), 3882–3896. https://doi.org/10.3168/jds.s0022-0302(93)77729-2 idf (2015). milk and milk products – guidance on the application of mid-infrared spectrometry for the determination of fat, protein, and other components (idf standard 141c:2015). kampl, b. (2005). pokazatelji energetskog deficita šamanc, h., kirovski, d., nada, l., celeska, i., bojković-kovačević, s., sladojević, ž., & ivanov, i. (2013). a comparison of energy balance-related variable concentrations in jugular and mammary vein blood of dairy cows with different milk yield. acta veterinaria hungarica, 61(1), 52–63. https:// doi.org/10.1556/avet.2013.055 toscano, a., giannuzzi, d., pegolo, s., vanzin, a., bisutti, v., gallo, l., trevisi, e., cecchinato, a., & schiavon, s. (2023). associations between the detailed milk mineral profile, milk composition, and metabolic status in holstein cows. journal of dairy science, 106(9), 6577–6591. https://doi. org/10.3168/jds.2022-23161 trifkovic, j., savic, d., jotanovic, s., & vekic, m. (2015). organic milk ingredients as an indicator of energy status of cows with different milk production during the first 100 days of lactation. the journal of agriculture and forestry, 61(1), 251–259. https:// doi.org/10.17707/agricultforest.61.1.32 van knegsel, a.t.m., van den brand, h., dijkstra, j., tamminga, s., & kemp, b. (2005). effect of dietary energy source on energy balance, production, metabolic disorders and reproduction in lactating dairy cattle. reproduction nutrition development, 45(6), 665–688. https://doi. org/10.1051/rnd:2005059 25(12), 1712–1720. https://doi.org/10.5713/ ajas.2012.12279 roche, j.r., bell, a.w., overton, t.r., & loor, j.j. (2013). nutritional management of the transition cow in the 21st century – a paradigm shift in thinking. animal production science, 53(9), 1000–1023. https://doi.org/10.1071/an12293 roy, b., brahma, b., ghosh, s., pankaj, p.k., & mandal, g. (2011). evaluation of milk urea concentration as a useful indicator for dairy herd management: a review. asian journal of animal and veterinary advances, 1(1), 1–19. https://doi. org/10.3923/ajava.2011.1.19 schwab, c.g., & broderick, g.a. (2017). a 100-year review: protein and amino acid nutrition in dairy cows. journal of dairy science, 100(12), 10094–10112. https://doi.org/10.3168/jds.201713320 šamanc, h., kirovski, d., dimitrijević, b., vujanac, i., damjanović, z., & polovina, m. (2006). procena energetskog statusa krava u laktaciji određivanjem koncentracije organskih sastojaka mleka. veterinarski glasnik, 60(5-6), 283–297. https://doi.org/10.2298/vetgl0606283s šamanc, h., kirovski, d., stojić, v., stojanović, d., vujanac, i., prodanović, r., & bojkovićkovačević, s. (2011). application of metabolic profile test in the prediction and diagnosis of fatty liver in holstein cows. acta veterinaria beograd, 61(6), 543–553. https://doi.org/10.2298/ avb1106543s biologica nyssana ● 16 (2) december 2025: stojanović ● influence of milk fat-to-protein ratio on the energetic status of holstein-friesian cows in early lactation vitkova, a., balabanova, v.: trials on the introduction and cultivation of arnica montana l. (asteraceae) in bulgaria. biologica nyssana, 9 (1). september, 2018: 21-29. biologica nyssana 9 (1) ⚫ september 2018: 21-29 vitkova, balabanova ⚫ trials on the introduction and cultivation of arnica… 21 original article received: 15 mart 2018 revised: 29 june 2018 accepted: 22 august 2018 trials on the introduction and cultivation of arnica montana l. (asteraceae) in bulgaria antonina vitkova1, vessela balabanova2 1institute of biodiversity and ecosystem research, department of plant and fungal diversity and resources, bulgarian academy of science, acad. g. bonchev str., bl. 23, 1113 sofia, bulgaria 2department of pharmacognosy, faculty of pharmacy, medical university sofia, dunav str. 2, 1000 sofia, bulgaria * e-mail: vitkova17@abv.bg abstract: vitkova, a., balabanova, v.: trials on the introduction and cultivation of arnica montana l. (asteraceae) in bulgaria. biologica nyssana, 9 (1). september, 2018: 21-29. the aim of the present study was to consider the possibilities for introduction and sustainable cultivation of arnica montana in bulgaria. the research was conducted on two experimental fields in different floristic regions of the country at 1 400-1 500 m altitude. the experiment was conducted with seeds of two origins: a natural population from the carpathians (ukraine) and the variety "arbo" (germany). comparison between in vivo and in vitro propagated plants under field conditions was carried out. during four vegetation seasons the growth and development of the plants were monitored and the survival rate was evaluated. there were no significant morphological differences between the plants of both studied origins while in vivo and in vitro propagated plants had high parameters variation. the obtained results proved the possibility of sustainable cultivation of a. montana in the high-mountain regions of bulgaria. key words: arnica montana, bulgaria, field cultivation, medicinal plant apstrakt: vitkova, a., balabanova, v.: ispitivanja introdukcije i kultivacije vrste arnica montana l. (asteraceae) u bugarskoj. biologica nyssana, 9 (1). septembar, 2018: 21-29. cilj ovog istraživanja bio je da se razmotre mogućnosti introdukcije i održive kultivacije vrste arnica montana u bugarskoj. istraživanje je bilo izvedeno na dva ogledna polja koja su pripadala različitim florističkim regionima na nadmorskoj visini od 1400-1500 m. eksperiment je izveden sa semenjem čije je poreklo bilo iz dva različita izvora: prirodna populacija sa karpata (ukrajina) i varijetet „arbo“ (nemačka). izvršeno je poređenje in vivo i in vitro propagiranih biljaka u terenskim uslovima. tokom četiri vegetacione sezone, praćeni su rast i razvoj biljaka i procenjena je stopa preživljavanja. nije bilo značajnih morfoloških razlika između biljaka različitog porekla, dok su in vivo i in vitro propagirane biljke imale visoke varijacije parametara. 9 (1) • september 2018: 21-29 doi: 10.5281/zenodo.1470844 biologica nyssana 9 (1) ⚫ september 2018: 21-29 vitkova, balabanova ⚫ trials on the introduction and cultivation of arnica… 22 dobijeni rezultati dokazali su mogućnost održive kultivacije a. montana u visoko planinskim regionima bugarske. ključne reči: arnica montana, bugarska, kultivacija na oglednim poljima, lekovita biljka introduction arnica montana l. (asteraceae) is a valuable medicinal plant widely used in the phytotherapy in the form of numerous preparations (merfort, 2002). the pharmaceutical industry uses the herbal drug arnicae flos (arnica flower heads) (european pharmacopoeia, 2011) that contains sesquiterpene lactones, flavonoids and phenolic acids (lyss et al., 1998; nikolova et al., 2013; todorova et al., 2016) and it is applicable in the production of creams and lotions for muscles and bone injuries as well as in the treatment of hematomas (klaas et al., 2002). the studied species is native to europe. it is distributed from southern norway and latvia, southward to portugal, east across europe to the north apennines in italy and south carpathians in romania (ferguson, 1976; smallfield & douglas, 2008). investigations have been carried out on the influence of environmental factors on plant material quality from natural populations of a. montana (ucenic & mastorakis, 2007), the effect of altitude on the phenolic content in arnica flower heads (spitaler et al., 2008) and also on the presence of mycorrhiza under field conditions (ryszka et al., 2010). the results of the attempts to cultivate mountain arnica in some european countries could help to find appropriate environmental conditions for its sustainable cultivation. such studies have already been made in germany (weyel, 1989; bomme et al., 1995), poland (jurkiewicz et al., 2010; sugier et al., 2013), switzerland (delabays & mangel, 1991), italy (aiello et al., 2012), finland (galambosi, 2004) and serbia (plevljakušić et al., 2014). in bulgaria, experiences on in vivo and in vitro propagation of a. montana have been conducted by balabanova & vitkova (2010), evstatieva et al. (2012) and balabanova et al. (2013). a. montana is not native in the bulgarian flora. however, more than 100 years ago, this species has been found in the subalpine zone in the rila mt. at 1 800 m a.s.l. and a specimen was deposited in the herbarium of sofia university "st. kliment ohridski" (so 86331). the main purposes of the present study were: to establish an approach for cultivation of arnica montana in bulgaria; to find regions in the country with appropriate environmental conditions for its sustainable cultivation; to make a comparison of in vivo and in vitro propagated plants grown in different regions in bulgaria on the basis of their biological features. material and methods our preliminary studies have been conducted on fields in the region sofia (544 m a.s.l.) and the town of pirdop (696 m a.s.l.). the obtained results showed that the climatic conditions at these altitudes are not suitable for successful cultivation of a. montana in bulgaria (tab. 1). after this conclusion we started to look for mountain areas with appropriate table 1. experimental field environmental conditions experimental fields ecological characteristic vitosha mt. sofia city western rhodopes mts. pirdop city gps coordinates n 42o36´ e 23o14´ n 42o41´ e 23o20´ n 41o50´ e 24o07´ n 42°42´ e 24°11´ altitude [m] 1 404 544 1 500 696 exposition sw sw nw s slope [o] 8 1 3 1 soil type brown mountain-forest alluvial-diluvial meadow brown mountain-forest cinnamon forest area [m2] 30 10 30 20 n [%] 0.459 0.147 p2o [mg/100 g] 4.10 19.83 k2o [ mg/100 g] 7.8 16.6 ph 6.1 6.5 humus % 7.02 2.84 biologica nyssana 9 (1) ⚫ september 2018: 21-29 vitkova, balabanova ⚫ trials on the introduction and cultivation of arnica… 23 environmental conditions where the study could be realized. the field experiments were carried out during the period 2009-2013 at two experimental fields of the institute of biodiversity and ecosystem research, bulgarian academy of science: “beglika” locality (western rhodope mts.) at 1500 m a.s.l. and “zlatni mostove” locality (vitosha mt.) at 1400 m a.s.l. the experimental fields are located in the european continental climatic region of bulgaria (subev & stanev, 1963). the experimental fields' environmental characteristics are presented in tab. 1. the soil analysis was carried out at the laboratory of soil analysis at the university of forestry, sofia, bulgaria. the soil type is determined according to ninov (2002). the data for the average temperature and precipitation for the experimental fields were obtained by national hydrometeorological institute, bulgarian academy of science, sofia, bulgaria. the experiment was carried out with seeds (achenes) of two origins: a natural population from the carpathians (ukraine) and a variety "arbo" (germany). comparative study on in vivo and in vitro propagated plants under field conditions was carried out. the field observation on 1-, 2and 3-year old plants was conducted through 2011-2013 growing seasons. in vivo seedlings were produced from seeds of both origins under greenhouse conditions following the smallfield and douglas (2008) methodology. in december 2010, dark and mature achenes were sown in plastic plates with 50 jacks, each 5 cm in diameter, in commercial peat substrate "tera mix" and pine bark (1:1) (fig. 1). thereafter, the soil was slightly pressed in order to improve its close contact with the seeds. the seeds germinated after 10-12 days at 1820 °c. when the plants formed six leaves, they were put into individual pots (d =12 cm) with the above mentioned substrate. we received the in vitro propagated plants bred by assoc. prof. ely zayova from the institute of plant physiology and genetics, bulgarian academy of sciences, sofia, bulgaria. the seedlings were planted in coconut sawdust (noramix group, sri lanka) and their adaptation was conducted under greenhouse conditions. two months later, the seedlings were planted into individual pots (d=12 cm) in peat substrate with natural zeolite (tera mix, bulgaria) and pine bark (1:1). the in vitro plants were included in the study to determine the possibility of plants growing associated with a future selection based on biotechnological breeding approaches. in may 2011, six-month-old in vivo and in vitro plants were transferred to the experimental fields of “beglika” and “zlatni mostove” localities. the seedlings were transplanted in 30 x 40 cm distance, thus ensuring a plant density of 10-12 plant/m2 according to a previous research (sugier et al., 2013). further, the cultivation model should be adjusted for field plantation and the seedlings should be planted in 70 x 30 cm pattern due to the mechanical maintenance. the experimental replication was made up of 46 plants. experimental complete plot consisted of two and three replications for in vitro and in vivo propagated plants, respectively (each replication of 3.8 m2). the areas were kept clean by hand hoeing and a regular irrigation was made. direct seed sowing on the field was conducted in the beginning of april and mid-september. the seeds were sown in shallow furrows in the soil which were at a distance of 30 cm in depth of 4-5 cm. the furrows were filled with a mixture of peat substrate with natural zeolite (tera mix, bulgaria) and pine bark (1:1). after sowing, the soil was treated with a wooden roller and the areas were regularly moistened. the phenological observations were carried out following beydeman (1974). the plant development was monitored throughout 5 phenophases and the duration of each phase was recorded (tab. 2). table 2. duration of phenophases in a. montana development under field conditions experimental fields duration of phenophase (days) vitosha mt. rhodopes mts. vegetative 18 20 flower buds formation 20 16 full flower 30 24 ripe fruit 19 28 end of vegetation 60 55 total duration of vegetation 147 143 in order to establish the plant morphometric variability due to the seed origin, in vivo and in vitro seedlings production and the environmental conditions in the regions of plants cultivation, 8 parameters were examined on 3-year-old plants, as follows: rosette diameter stem height; affiliated rosettes number; leaves number per a rosette; length and width of rosette leaf; flower head diameter; flower heads number per plant. morphometrical measurements were performed on 3-year-old plants during the full flowering stage. the obtained data were statistically calculated by medcalc program, biologica nyssana 9 (1) ⚫ september 2018: 21-29 vitkova, balabanova ⚫ trials on the introduction and cultivation of arnica… 24 expressed by average value (m) and standard deviation (sd), and the differences of the parameters variation were shown (p<0.1%) (tab. 3). results and discussion plants development in greenhouse and adaption to field conditions in vivo propagated plants the present study proved that the seeds from both origins natural population (ukraine) and variety ”arbo” (germany) germinated jointly 10 12 days after sowing under greenhouse conditions and the germination reached a rate up to 70 80%. during the first two months, the seedlings formed roots (5-6 cm) and 4 small leaves (fig.1). six-month-old plants had rosettes with diameter of 10.00 – 12.00 cm consisting of 8-10 leaves. kating & seidel (1967) reported that the nursery stage is critical for arnica cultivation, which prompted us to be careful during this period of plant growing. in vitro propagated plants two-month-old in vitro plants were transferred from the coconut sawdust into a mixture of commercial (tera mix, bulgaria) and pine bark (1:1) substrate. after the transplanting, some of the plants failed to adapt and 15-20% of them died. the surviving plants had a successful adaptation and developed well in the greenhouse conditions during the next 4 months. the 6-month-old plants with well-formed roots and a rosette of leaves with diameter of 10.00-12.00 cm were transferred to the fields. direct seed sowing on the fields the seeds from the two investigated origins did not germinate on the experimental fields of "beglika" and "zlatni mostove" localities. the literary data on cultivated a. montana by direct seed sowing on the field indicated that the seeds germinated jointly, but the juvenile plants were vulnerable to the climate conditions (sugier et al., 2013). our results proved that this approach is not a reliable method for cultivation of mountain arnica in bulgaria. plant adaptation after transferring to the fields in the middle of may 2011, in vivo and in vitro plants were transferred from greenhouse to the mountain experimental fields. plants have successfully adapted to the new conditions. at the end of the studied period table 3. morphometric characteristic of the plants at vitosha mt. and rhodopes mts. parameter [m±sd] sample rosette diameter plant height rosette number leaves number per rosette leaf length leaf width flower head diameter flower heads number амvu1 28.11±4.19 46.33±5.51 3.56±0.92 7.78±1.67 11.80±1.56 3.60±0.76 6.77±0.59 7.94±4.29 амvu2 24.25±6.27 42.94±7.09 5.75±3.20 8.25±1.28 9.60±2.16 3.44±1.10 5.41±0.48 16.63±12.32 амvg1 25.36±5.50 40.90±4.53 3.64±1.69 8.18±1.89 10.62±1.98 3.57±0.79 6.28±0.64 8.09±5.57 амvg2 22.30±2.05 38.50±4.21 4.60±0.89 8.00±1.41 9.20±1.15 3.45±0.72 5.36±0.31 12.40±8.36 амru1 31.87±5.64 33.91±4.12 8.83±2.23 7.65±0.98 11.72±2.51 2.82±0.51 6.57±0.79 23.00±11.87 амru2 29.50±4.04 30.33±3.62 7.67±2.42 7.67±0.82 11.68±1.93 3.77±0.59 6.12±0.80 20.60±10.53 амrg1 33.41±4.10 32.23±5.95 9.71±2.46 8.23±0.89 12.77±2.70 2.61±0.41 6.56±0.80 24.02±17.93 амrg2 25.80±1.92 28.40±6.54 6.00±1.58 7.60±0.89 10.70±1.35 3.10±0.55 5.88±0.25 13.80±3.49 legend: amvu1 a. montana, ukraine, in vivo plants, vitosha mt.; amvu2 a. montana, ukraine, in vitro plants, vitosha mt.; amvg1 a. montana, germany, in vivo plants, vitosha mt.; amvg2 a. montana, germany, in vitro plants, vitosha mt.; amru1 a. montana, ukraine, in vivo plants, rhodopes mts.; amru2 a. montana, ukraine, in vitro plants, rhodopes mts.; amrg1 a. montana, germany, in vivo plants, rhodopes mts.; amrg2 a. montana, germany, in vitro plants, rhodopes mts. fig.1. two-month-old in vivo seedlings bred under greenhouse conditions biologica nyssana 9 (1) ⚫ september 2018: 21-29 vitkova, balabanova ⚫ trials on the introduction and cultivation of arnica… 25 (2013) in the rhodope mts. (“beglika” locality), 80% in vivo and 60% in vitro plants (variety "arbo") and 65% in vivo and 60% in vitro plants (ukraine) have adapted and completed their full development. regarding vitosha mt. (“zlatni mostove” locality), 75% in vivo and 33% in vitro plants (variety "arbo") and 41% in vivo, 25% in vitro plants (ukraine) have been adapted. these results showed that the percentage of successfully adapted in vivo plants at field conditions was higher than in vitro plants and the best results were established in “beglika” locality. our research found a chlorosis in 5% of the plants at “beglika” locality which had resulted on plant death. this is the evidence that the chlorosis correlates with high phosphorus content in the soil (delabays & mange, 1991). the soil analysis has proven that in the locality “beglika” p2o5 content was 19.83 mg/100 g while on the locality “zlatni mostove” it was significantly lower (4.10 mg/100 g). it was found that the plants are very sensitive to drought stress which was also reported by weyel (1989). this conclusion shows that a regular irrigation of the experimental fields is needed, especially during the driest and warmest months july and august. a drought stress was observed in the plants grown on vitosha mt. this is mainly due to the southwest exposure of the experimental area, which leads to intense and prolonged sunshine during the summer months. to the causes of the drought stress on the plants, the soil drying due to the slope of the terrain (8o) can also be added as well as the inability to keep the precipitations falling. due to the meteorological survey, the average monthly air temperatures at “zlatni mostove” locality during april 2009 october 2013 were as follow: at the beginning of the period the temperatures were slightly above the monthly norms (perennial average). during the years 2010, 2011, 2012 and 2013, the temperatures were close to the monthly rates. according to the precipitation values for the above-mentioned period, a significant fluctuation in the amount was recorded. in the spring and late autumn, the rainfalls were generally in the norm, while in summer and early autumn, the received data were close to the minimum. regarding “beglika” experimental field, the average monthly air temperatures during january 2010 october 2013 were generally close to the above-mentioned perennial averages and the temperatures were considerably high in august september. concerning the precipitation values, the data were more variable compared to the perennial average. in the spring and early autumn, rainfalls were close to the average monthly rate, but were below the norm during the period august october. the data obtained in the study showed that the most suitable period for planting and establishing a mountain arnica field plantation in bulgaria is at the end of may or in early june. in this period, the plants assimilate the spring humidity, temperature is appropriate and the plants adapt to the field conditions. if the planting is done in late autumn (november/december), some plants will fail to accommodate to a sharp drop in temperature, freezing of the soil and a large number of them will not survive. if the planting is done at the early october, the plants adaptation will be successful. vegetative propagation it was found that at the beginning of their development, the plants of mountain arnica grown under greenhouse conditions formed offspring of rosettes. at the end of the first vegetation season, 100% of the plants produced up to 2-3 rosettes. two, three and four-year-old arnica plants had about 3-5, 6 10 and up to 11-25 offspring rosettes per plant, respectively (fig. 2). through a division of offspring rosettes, seedlings which can be planted in the field were obtained. the observations showed that 100% of the plants survived and 40% of them blossomed in the same vegetation season. fig. 2. affiliated rosettes of mountain arnica biologica nyssana 9 (1) ⚫ september 2018: 21-29 vitkova, balabanova ⚫ trials on the introduction and cultivation of arnica… 26 phenological observation of plants grown in different floristic regions the phenological observation is important in the process of plant introduction and cultivation into a new geographical area. this study provides information on the duration of the vegetation season and different phenophases. in the presented research it was found that the growing season on “beglika” and “zlatni mostove” experimental fields lasted 143 and 147 days, respectively and that the plant development passed throughout five phenophases (tab. 2). vegetative phase. the growing season of 2-, 3 and 4-year-old plants began in the second half of april in the two experimental fields. regarding vitosha mt., the plants began to form new leaves in the middle of may and the vegetative phase lasted 18 days, while at the rhodopes mts. that happened 5 days later and the vegetative phase lasted 20-22 days. flower buds. this phase started at the end of may in the “zlatni mostove”locality and in the first half of june in “beglika” locality. this phase ended in the second half of june for the plants on both fields. mountain arnica plants began to bloom in the second growing season as 70% of them formed flower buds. in the third growing season, all plants have flower buds and entered into a generative period. it was found that the plants formed single flower buds until the middle of august. full flowering. the plants in “beglika” locality began to bloom two weeks after the flower buds formation and the phase lasted 14 days (fig. 3b). during the period 1-5 july, all the plants were blossoming and unripe seeds in the flower heads were observed. on the same plant, single flower buds, heads in full flowering stage and blooming heads with unripe achenes were present. the full flowering of the plants at “zlatni mostove” locality started in middle of june and ended in middle of july (fig. 3a). these results showed a slightly longer flowering phase for the plants from this locality compared to “beglika” locality. this is probably mainly due to the climatic features of these two plots. the number of flower stems and flower heads increased during the plant growth and development. concerning 4-yearold plants, the flower stem and flower heads number for one plant was 12-20 (25) and 35-50, respectively (fig. 4). ripe fruits. it was found that after the blossoming of the first flower heads, in the middle of july, the seed formation began at both experimental fields. in the beginning of this period, there were a small number of mature seeds in the flower heads. ripe fruit formation was observed in the first half of august, when 80-90% of the seeds were mature. end of vegetation. this phase began in early september when the stems were dying. leaves that have appeared in the same year usually survived the winter. the dried stems fell off at the end of october. variation of plant morphometric characteristics the data on the morphometrical research showed the following results: the rosette diameter was characterized by a very low to medium variability and a large scattering of the sign; stem height was fig. 3. three-year-old plants in flowering phase at “zlatni mostove“ (a) and “beglika“ (b) experimental fields biologica nyssana 9 (1) ⚫ september 2018: 21-29 vitkova, balabanova ⚫ trials on the introduction and cultivation of arnica… 27 characterized by low to medium volatility and a very large sign scattering; rosette number and inflorescence number per plant were characterized by high to very high variability and a large scattering of the parameters; leaves number per rosette was from low to high variability and relatively large sign dissipation; length/width of rosette leaf was characterized by medium to high volatility and a wide scattering of the signs; flower head diameter was characterized by low volatility and low parameter dissipation. plant morphometric variability according to their geographic origin our study found that statistically proven differences exist on plant height, leaves number in the rosette and flower head diameter (p<0.1%) for the plants of both studied origins (tab. 3). the plants from ukraine, grown on both experimental fields, had higher values of the morphometrical characteristics compared to those of variety “arbo” (germany). morphometrical variability of in vitro and in vivo propagated plants statistically proven differences exist on all studied parameters, except stem height and leaves number per rosette (p<0.1%) (tab. 3). morphometrical variability of the plants depending on the region of cultivation significant differences for the plants grown at both experimental fields for the most parameters were proven except leaves number per rosette and leaf length (p<0.1%) (tab. 3). it was found that plants of the two origins grown at “zlatni mostove” locality had higher stems and larger leaves number per a rosette. higher values for number of the flower heads and flower head diameter were established for the plants at “beglika” locality. the least changing parameter was flower head diameter indicating that it is a conservative feature. the flower heads number per plant is an important characteristic for mountain arnica cultivation and its commercial use. the data proved that number of flower heads was significantly larger for the plants cultivated on “beglika” experimental field. arnicae flos yield regarding our previous study, a higher yield was obtained for the plants originating in ukraine. the highest yield was registered for in vivo propagated plants up to 268 kg/ha (balabanova & vitkova, 2016). the harvest of the two studied origins was relatively similar in the experimental plot on vitosha mt. unlike those grown in rhodope mts. the highest quantity of herbal drug arnicae flos was reported for 5-year-old mountain arnica. conclusion a comparative study on the cultivation of a. montana in two floristic regions in bulgaria was carried out for the first time. the obtained results proved good acclimatization of the seedlings to the experimental fields at altitude of 1 400 m (vitosha mt.) and 1 500 m (rhodopes mts.). the in vivo plants were better adapted than those propagated by in vitro methods. the most important factors for a. montana sustainable cultivation were the climate and soil conditions. for the establishment of field plantation should be preferred flat terrain with northern exposure or a northern component. the soil should be well structured and slightly acidic (ph 6.1-6.5). the plants propagated by seeds passed into generative phase during the second year, while those fig.4. four-year-old plants in flowering phase at “beglika” experimental field biologica nyssana 9 (1) ⚫ september 2018: 21-29 vitkova, balabanova ⚫ trials on the introduction and cultivation of arnica… 28 obtained by dividing of offspring rosettes during the first year. the total duration of mountain arnica growing season was 143-147 days and the plants passed throughout five phenological phases. there were no significant morphological differences between the plants of both studied origins while in vivo and in vitro propagated plants had high parameters variation. the most variable was the parameter flower heads number per plant. the present study proves that a. montana could be cultivated successfully in the high-mountainous regions of bulgaria. acknowledgements. the authors are grateful to prof. alexander tashev from the university of forestry in sofia, bulgaria, who collected the mountain arnica seeds from the natural population in the carpathians, ukraine. special thanks also to assoc. prof. ely zayova from the institute of plant physiology and genetics, bulgarian academy of science, sofia, bulgaria, for breeding and providing the in vitro plants. references aiello, n., scartezzini, f., vender, c. 2012: cultivation trial of arnica montana wild accessions – results of second year. acta horticulturae, 955: 253-257. balabanova, v., vitkova, a. 2010: pecularities in ontogenesis of arnica montana l. in bulgaria. comptes rendus de l,academie bulgare des sciences, 2 (63): 1301-1306. balabanova, v., vitkova, a., 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(eds.). flora europaea, vol. 4. cambridge univ. press, london, 189-190. galambosi, b. 2004: introduction of arnica montana l. in finland. arzneiund gewürzpflanzen, 4: 174-179. jurkiewicz, a., ryszka, p., anielska, t., waligorski, p., bialonska, d., goralska, k., tsimilli-michael, m., turnau, k. 2010: optimization of culture conditions of arnica montana l.: effects of mycorrhizal fungi and competing plant. mycorrhiza, 20: 293-306. kating, h., seidel, f. 1967: cultivation experiments with arnica species. planta medica, 15: 148-258 klaas, ch. a., wagner, g., laufer, st., sosa, s., loggia, r. d., bomme, u., pahl, h. l., merfort, i. 2002: studies on the anti-inflammatory activity of phytopharmaceuticals prepared from arnica flowers. planta medica, 68: 385-391. lyss g., schmidt t., merfoft i., pahl., h.1997: helenalin, an anti-inflammatory sesquiterpene lactone from arnica, selectively inhibits transcription factor nf-kb. biological chemistry, 378: 951-61. nikolova, m., petrova, m., zayova, e., vitkova, a., evstatieva, l. 2013: comparative study of in vitro, ex vitro and in vivo grown plants of arnica montana polyphenols and free radical scavenging activity. acta botanica croatica, 72 (1): 13-22. ninov, n. 2002: soils. in: kopralev, i (ed.). geography of bulgaria. sofia, publ. house “farcom”. pljevljakušič, d., janković, t., jelačić, s., novaković, m., menković, n., beatović, d., dajić-stevanović, z. 2014: morphological and chemical characterization of arnica montana l. under different cultivation models. industrial crops and products, 52: 233-244. ryszka, p., blaszkowski, jurkiewicz, a., turnau, k. 2010: arbuscular mycorrhiza of arnica montana under field condition conventional and molecular studies. mycrorrhiza, 20: 551-557. smallfield, b., douglas, m. 2008: arnica montana: a grower,s guide for commercial production in new zealand, christchurch, new zealand. 15 p. spitaler, r., winkler, a., lins, i., yanar, s., stuppner, h., zidorn, ch. 2008: altitudinal biologica nyssana 9 (1) ⚫ september 2018: 21-29 vitkova, balabanova ⚫ trials on the introduction and cultivation of arnica… 29 variation of phenolic contents in flowering heads of arnica montana cv. arbo: 3-year comparison. journal of chemical ecology, 34: 369-375. subev, l., stanev, s. 1963: climatic regions of bulgaria and their climate. state publishing house for agricultural literature. sofia. 178 p. sugier, d., sugier, p., gawlik-dziki, u. 2013: propagation and introduction of arnica montana l. into cultivation: a step to reduce the pressure on endangered and high -valued medicinal plant species. hindawi publishing corporation. the scientific journal, article id 414363, http://dx.doi.org/10.1155/2013/414363 todorova, m., trendafilova, a., vitkova, a., petrova, m., zayova, e., antonova, d. 2016: developmental and environmental effects of sesquiterpene lactones in cultivated arnica montana l. chemistry and biodiversity, 13 (8): 976-981. ucenic, c., mastorakis, n. 2007: the impact to environmental issues in the supply chain for a natural resource: the case study of arnica montana from romania. proceedings of the 2nd iasme/wseas international conference on energy & enviroment (ee'07), portoroz, slovenia, may 15-17. weyel, a. 1989: improvement of the domestication properties of wild plant populations and selected genotype of arnica montana l. phd thesis, univ. giessen, germany. 135 p. anticancer compounds from medicinal plants biologica nyssana 4 (1-2)  december 2013: 41-48 atanacković v. et al.  morphological and anatomical observatoris on … 41 original article morphological and anatomical observations on ranunculus auricomus l. var. biformis l. in vlasina lake, serbia valentina atanacković 1 , vladimir ranđelović 2 , rashid ismael hag ibrahim 3 1 university of lleida, etsea, department hbj, av. alcalde rovira roure 191, 25198 lleida, spain; 2 university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia 3 department of horticulture, gyeongnam national university of science and technology, jinju 660-758, republic of korea * e-mail: valentina.atanackovic@gmail.com abstract: atanacković, v., ranđelović, v., ibrahim, r.i.h.: morphological and anatomical observatoris on raunculus auricomus l. var. biformis l. in vlasina lake, serbia . biologica nyssana, 4 (1-2), december 2013: 41-48. r. auricomus var. biformis l. (r. binatus kit.) grows mainly in the suburbs of vlasina plateau beside a variety of other species and forms one part of the peat-land association. specimens were collected from the northeast coastal flooded area of vlasina lake (latitude: 42° 41' 46 n, longitude: 22° 22' 14 e). in this study, some morphological and anatomical characteristics were investigated. samples for anatomical analysis were prepared according to the standard methods described by ruzin (1999) though, slightly modified (atanackovic et al., 2012). morphological features such as leaf, petiole, stem, and root have been described. the anatomy presented in this work form the first of its kind available in the literature about this variety found in the vlasina lake. transverse sections of ground-leaf, upper-leaf, petiole, stem and root have been examined and photographed. the observed morphological and anatomical feauters showed that the species grows in these flooding conditions. key words: anatomy, leaf, morphology, petiole, ranunculus, root, stem introduction ranunculus l. or buttercup is the largest genus of ranunculaceae that contains around 600 species and described as cosmopolitan (t a m u r a , 1993) that mostly distributed in all continents throughout temperate, arctic, sub-antarctic and rarely in high elevated areas of the tropic zones excluding the antarctic (e r s t a n d s u k h o r u k o v , 2011). ranunculus grows in a wide range of habitats such as rivers, lakes, wet soils, damp/dry meadows and forests, and shows several morphological adaptations to different habitats (p a u n et al., 2006). ranunculus is well established in central europe where the yellow buttercups are found in meadows, pastures, or ruderal places during spring and summer (s t e i n b a c h a n d g o t t s b e r g e r , 1994). r. auricomus complex (aggregate) represents one of the larger clades within ranunculus (h ö r a n d l et al., 2005) that is distributed throughout europe to western siberia, from the arctic zone to the mediterranean region, greenland and alaska (j a l a s a n d s u o m i n e n , 1989). r. auricomus complex is highly diverse in morphotypes, cytotypes and ecotypes, which were the reasons to be considered as a separate section (l o o s , 1997) and often subdivided into four morphological groups; auricomus, cassubicus, fallax and monophyllus sensu (e r i c s s o n , 1992; 2001). 11 th sfses • 13-16 june 2013, vlasina lake 4 (1-2) • december 2013: 41-48 biologica nyssana 4 (1-2)  december 2013: 41-48 atanacković v. et al.  morphological and anatomical observatoris on … 42 h ö r a n d l et al. (2005) reported that various morphological adaptations and reproductive strategies such as vegetative reproduction (stolons), self compatibility (in water-buttercups), and likely agamospermy (r. auricomus complex) could play important role in the ranunculus ability to colonize different habitats, altitudes, and latitudes. despite the morphological adaptations, r. auricomus var. biformis is in an extremely high risk of extinction at the regional scale and it is recognized as a critically endangered variety (r a n đ e l o v i ć et al., 2010). temporary or continuous flooding of soil with fresh water occurs as a result of overflowing of the lake (r a n đ e l o v i ć , z l a t k o v i ć , 2010). the mechanisms by which flood-tolerant plants survive water-logging are complex and involve interactions of morphological, anatomical, and physiological adaptations (k o z l o w s k i , 1997; s t e v e n s et al., 2002). generally, adverse effects of flooding include changes in plant distribution and composition as well as changes in shoot growth of many plants due to suppressed leaf formation and expansion of leaves and internodes. soil inundation leads to increased length of the first internode and furthermore, flooding may also increases intercellular spaces in the tissues of flood-tolerant plants (a t a n a c k o v i c et al., 2012). certain terrestrial plant species respond to poor aeration in flooded areas and produce aerenchyma mainly in the cortical tissues of the stem and root (j a c k s o n a n d c o l m e r , 2005; s t e v e n s et al., 2002; jung et al., 2008). thus, flooded habitats may change the anatomical structure such as the cuticle layer and phloem in leaves, pith cavity area and vessel lumen in stem (t a o et al., 2009). different plants express different morphological or anatomical adjustments in response to environmental changes, which act as mechanisms of survival and reproduction under a wide range of conditions (b e n z et al., 2007). we assumed that r. auricomus var. biformis adapted and survived in temporal flooded peat bog habitat due to morphological and/or anatomical adjustments. hence, its morphological and anatomical features of leaf, petiole and stem and anatomical adaptations of root were investigated. material and methods sampling of plant material plant material were collected from the northeast coastal flooded area of vlasina lake at latitude 42° 41' 46 n and longitude 22° 22' 14 e (fig. 1). the variety was identified by professor vladimir stevanovic. plant samples were fixed in 50% ethanol and samples were also deposited and recorded at the herbarium and wet collection of the department of ecology and geography of plants, institute of botany and botanical garden “jevremovac”, faculty of biology, university of belgrade, serbia. fig. 2. r. auricomus var. biformis plant growing in a peat bog habitat fig. 1. geographical position of investigated area biologica nyssana 4 (1-2)  december 2013: 41-48 atanacković v. et al.  morphological and anatomical observatoris on … 43 preparation of plant material for anatomical analysis anatomical analysis of leaf, petiole, stem and root were performed. samples were prepared according to the standard methods described by r u z i n (1999), though slightly modified (a t a n a c k o v i ć et al., 2012). morphological observations and anatomical analysis under light microscope certain quantitative measures were carried out for leaf, stem and root of r. auricomus var. biformis as well as description of petioles. morphometric measures were taken for leaves that included; leaf length, leaf length from base to the widest part, leaf width, leaf perimeter, and leaf area, while those for the plant included; length of each of the first three internodes, stem length and total number of internodes. measurements performed on the cross section of leaf included; leaf thickness, width and thickness of epidermal cells and surface, total thickness of mesophyll including thickness of spongy and palisade tissues and number of layers for each tissue. the diameter of primary cortex and central cylinder at the radius of root cross section were also measured. statistical analysis to increase the accuracy of data and statistical analysis, each character was measured on 25 samples of leaf, stem and root of individuals from the plant population. all measurements of quantitative characters were performed on an “image analyzer” software package (qwin, leica microsystems imaging solutions ltd, cambridge, uk), and then the measurement results were processed by the software spss 15 for windows (spss 15.0. 2006). for each character, basic parameters were statistically analyzed such as mean, minimum value, maximum value and standard errors. results and discussion in serbia, r. auricomus var. biformis grows in light and moist deciduous forests, bushes, thickets, damp meadows, fields from lowland regions to alps belt, but grows mainly in the suburbs of vlasina plateau beside a variety of other species. r. auricomus var. biformis became part of the peatland association that includes caricetum goodenowii and caricion cannescentis-nigrae, which belong to the class caricetalia fusca and scheuchzerio-caricetea fusca (r a n đ e l o v i ć et al., 2010). r. auricomus var. biformis is a perennial plant that grows during spring-summer in a halfrosette and hemicryptophyte (fig. 2). ground leaves are rounded, kidney-shaped and partially divided into lobes. the stem is erect, in the range of 9.5 cm high and rarely covered with trichomes. the root is short and vertical with numerous hair roots. morphological characteristics leaf r. auricomus var. biformis has two heterophyllous leaves here named as upper and ground leaves, which differ in shape and size. upper leaves are sessile and cut into 4-8 linear-lanceolate lobes. these leaves are fan-like-shaped, soft, shiny, smooth and dark green, while the reverse side is light green and the length of leaf is nearly six times its width (fig. 3a). there are two types of ground leaves namely ground leaves that are not lobed (fig. 3b) and ground leaves that are divided into lobes (fig. 3c). deep ground leaves have larger lobes with rounded tips, sharply-toothed secondary parts and sometimes appear jagged with lateral lobes. ground leaves are kidney-shaped, shallow, soft, smooth, and glossy green with long petioles. the length of leaf is slightly less than or equal to its width (table1). fig. 3. morphological features of upper (a) and ground leaves; not lobed (b) and lobed (c) from r. auricomus var. biformis. a b c biologica nyssana 4 (1-2)  december 2013: 41-48 atanacković v. et al.  morphological and anatomical observatoris on … 44 stem r. auricomus var. biformis is an erect plant, branched and can reach 9.5 cm tall (fig. 2). two or three internodes of various lengths were observed and while the first internode is the longest, the second and third were slightly shorter (table 2). anatomical characteristics upper leaf the upper leaf of r. auricomus var. biformis is thinner compared with the ground leaf (table 1). the adaxial layer of the epidermis consists of larger cells and a few stomata were observed (fig. 4a). the mesophyll composed of the palisade tissue of 2-3 layers and the spongy tissue of 4 layers of cells and large intercellular spaces, but cells are smaller than in the ground leaves. the palisade tissue is slightly thicker than the spongy tissue (fig. 4b). the vascular bundle is collateral and surrounded by sclerenchyma tissue (fig. 4c). ground leaf the two types of ground leaves are not different in structure as seen from the cross sections. the epidermis was observed as one layer of large cells on the adaxial side of leaf and the exterior walls are thicker and slightly prominent. stomata were seen on both adaxial and abaxial surfaces or leaves are amfistomatic. similar to the upper leaves, the mesophyll is divided into the palisade tissue that consists of 2-3 layers of extremely large cells, but the first layer of cells is slightly longer than other layers and smaller intercellular spaces were observed. the spongy tissue of 4-6 layers of cells that are irregular in shape, thick-walled and separated by large intercellular spaces (fig. 5a). the vascular bundles are collateral, enclosed in sclerenchyma tissue (fig. 5b) and the number of vascular bundles is between 28 and 30 (table 1). petiole the cross section of the petiole of ground leaf; lobed or not, is kidney-shaped and showed clearly distinct epidermis, primary cortex, central cylinder and pith cavity on one side towards the centre. the epidermis of the petiole consisted mostly of one layer, but two layers in some parts, covered with a well-developed cuticle and stomata were observed. the primary cortex consists of small parenchyma cells that are densely packed in groups of 6-7 layers separated by large intercellular spaces and sometimes there might be one or two large voids. five vascular bundles were seen and the central bundle is the largest, while the marginal were the least developed. all bundles are partially enveloped in sclerenchyma that was observed on both sides of the phloem and xylem and the cambium was visible in the central bundle. the pith cavity is irregular in shape (saddle-shaped) and broken in some areas and remains of the pith could be seen (fig. 6). stem the cross section of stem clearly showed distinct epidermis, primary cortex, vascular tissue and pith cavity located at the centre. the epidermis of stem consists of mostly two layers covered with a well-developed cuticle and stomata were also observable in some areas (fig. 7a). the primary cortex consists of slightly thicker-walled small parenchyma cells and densely packed but in some areas large intercellular spaces were visible. the chloroplasts and starch reserves were observable in parenchyma cells (fig. 7b). the vascular tissue consists of 9-10 vascular bundles partially enveloped in sclerenchyma tissue, which is found only on the phloem side of the vascular bundle. in addition to the basic parenchyma tissue, the vascular tissue contains xylem vessels and phloem in a collateral arrangement and remains of the cambium could be seen (fig. 7c). the central cavity forms the pith cavity and remains of the pith were observed in some areas (fig. 7a). the thickness of stem cortex is ¾ of the radius of pith cavity (table 2). root the cross section of root showed the primary cortex and central cylinder (fig. 8a). the parenchyma of primary cortex consists of thinwalled, densely packed cells filled with reserve materials and intercellular spaces between cells scarcely observable. the endodermis forms the last layer of the primary cortex. the vascular tissue consists of the pericycle of 1 or 2 layers of cells, the xylem and phloem that were arranged in radials (fig. 8b). the xylem consists of 3-4 bundles each contains 4-10 vessels and typical to the normal root with a central stele. the plates of phloem are radial in the vascular bundles. the protophloem mesh tubes were seen larger and located on the periphery, while the metaphloem is near the centre (fig. 8c). the primary cortex is five times larger than the central cylinder (table 2). biologica nyssana 4 (1-2)  december 2013: 41-48 atanacković v. et al.  morphological and anatomical observatoris on … 45 fig. 4. a cross section through the upper leaf of r. auricomus var. biformis (a) shows the upper and lower epidermis, mesophyll tissue; palisade and spongy tissue, vascular bundle of mid-rip, and stomata (b) and close up of the vascular bundle of mid-rip (c). ep; epidermis, p; palisade tissue, s; spongy tissue, vb; vascular bundle of mid-rip, st; stoma, x; xylem vessel. table 1. morphological and anatomical features of the upper and ground leaf from r. auricomus var. biformis 1 mean ± standard error fig. 5. a cross section through a ground leaf of r. auricomus var. biformis (a) shows a vascular bundle (b). ep; epidermis, st; stoma, p; palisade tissue, s; spongy tissue, x; xylem vessel, ph; phloem, sc; sclerenchyma. upper leaf ground leaf (not lobed) 1 ground leaf (lobed) morphology leaf length (mm) 06.20 ± 0.20 / / length from the base to widest part (mm) 03.07 ± 0.10 05.8 ± 0.30 05.5 ± 0.2 leaf width (mm) 01.40 ± 0.40 15.6 ± 0.60 18.2 ± 0.3 leaf perimeter (mm) 71.20 ± 0.20 51.7 ± 0.30 31.4 ± 0.1 leaf blade area (cm²) 09.80 ± 0.01 14.3 ± 0.01 03.1 ± 0.1 anatomy leaf thickness (µm) 106.813 ± 0.4 120.80 ± 0.2 114.0 ± 0.2 width of upper epidermis (µm) 014.300 ± 0.6 012.40 ± 0.4 012.4 ± 0.3 thickness of upper epidermis (µm) 010.900 ± 0.5 010.40 ± 0.3 010.6 ± 0.4 mesophyll tissue thickness (µm) 086.700 ± 0.4 099.90 ± 0.2 098.9 ± 0.3 palisade tissue thickness (µm) 056.800 ± 0.2 056.50 ± 0.2 045.7 ± 0.2 spongy tissue thickness (µm) 046.900 ± 0.3 055.00 ± 0.1 055.1 ± 0.1 the number of layers of palisade tissue 002.000 ± 0.0 003.00 ± 0.0 003.0 ± 0.0 the number of layers of spongy tissue 004.000 ± 0.0 006.00 ± 0.2 005.0 ± 0.2 width of lower epidermis (µm) 010.900 ± 0.5 009.20 ± 0.3 008.8 ± 0.3 thickness of lower epidermis (µm) 009.200 ± 0.4 008.92 ± 0.3 008.5 ± 0.2 ep p s vb a b c st x st ep sc x ph p s a b biologica nyssana 4 (1-2)  december 2013: 41-48 atanacković v. et al.  morphological and anatomical observatoris on … 46 fig. 6. a cross section of the leaf petiole of r. auricomus var. biformis. ep; epidermis, vb; central vascular bundle, pc; pith cavity. fig. 7. a cross section through the stem of r. auricomus var. biformis shows a distinct epidermis, primary cortex, vascular tissue and pith cavity located at the centre (a) a vascular bundle in a parenchyma tissue (b) and details of a vascular bundle (c). ep; epidermis, co; cortex, vp; vascular bundle, pc; pith cavity, sc; sclerenchyma, pa; parenchyma, x; xylem vessel, ca; cambium residues, ph; phloem. fig. 8. a cross section through the root of r. auricomus var. biformis (a) shows the central cylinder and cortex (b) and details of the central cylinder (c). co; cortex, cc; central cylinder, en; endodermis, x; xylem vessel, ph; phloem. vb pc ep a b a b c sc pa ca ph x pc ep co vb a b c cc co x en ph biologica nyssana 4 (1-2)  december 2013: 41-48 atanacković v. et al.  morphological and anatomical observatoris on … 47 table 2. morphological and anatomical features of the stem and root of r. auricomus var. biformis feature mean ± se 1 stem morphology stem length (cm) 9.40 ± 0.005 length of first internode 1(cm) 5.72 ± 0.400 length of second internode (cm) 1.96 ± 0.400 length of third internode 3(cm) 3.07 ± 0.400 total number of internodes 2.30 ± 0.900 stem anatomy stem cortex thickness (µm) 303.1 ± 0.1 radius of pith cavity of stem (µm) 399.9 ± 0.9 root anatomy cortex diameter of root (µm) 527.9 ± 0.1 radius of central cylinder (µm) 126.5 ± 0.5 1 mean ± standard error discussion r. auricomus var. biformis is characterized by a terrestrial life form. despite this terrestrial mode of life, r. auricomus var. biformis managed to adapt and survive during periods of drought in mostly wet meadows. the durability of this modified form of life varies and depends on microclimatic changes in a given habitat (personal observation during this study). morpho-anatomical analysis indicated that r. auricomus var. biformis considers specific structure and typical terrestrial/aquatic forms. r. auricomus var. biformis belongs to the ecological group of hygromesophytes and grows as a terrestrial plant in temperate and moist areas on the slopes around vlasina lake that is characterized by slight changes of humidity. on this terrestrial type and according to the position on plant, two types of ground kidney-shaped leaves are formed. these leaves are characterized by one layer of epidermis on the adaxial surface, large cells, elongated palisade tissue cells and large intercellular spaces, which can be correlated with significant surface moisture due the wet habitat. the plant is characterized by a small diameter of the primary cortex of stem. morphological characteristics of leaves and stem two kinds of heterophyllous leaves were observed, namely; upper linear-lanceolated-lobed and sessile leaves and ground leaves that were lobed or not. the length of the internodes varied and the first internode was the longest that could be a consequence of the wet habitat (table 2), which is in contrary to the shortest first internode of r. lingua from the same area (atanackovic et al., 2012). anatomical characteristics of leaf, petiole, stem and root the upper leaf of r. auricomus var. biformis was thinner compared with the ground leaf (table 1). the three types of leaves contained one layer of epidermis and the stomata were rarely observed, which could be an adaptation to the habitat. smaller intercellular spaces were seen in the mesophyll of the upper leaves compared with the ground leaves. large intercellular spaces between cells in the spongy tissue and long palisade cells are characteristics influenced by water availability on leaves. the plant stem showed structural features of the genus ranunculus that consists of primary cortex, vascular tissue and pith cavity located at the centre (fig. 7). one layer of epidermal cells contains only few stomata, primary cortex composed of small cells that are separated by large intercellular spaces, which could be a characteristic anatomical feature of a plant living in a wet habitat. the central cylinder consists of 9-10 vascular bundles. the petiole of ground leaves showed similar structure as the stem, but less number of vascular bundles, i.e. around 5. the root consisted of primary cortex and central cylinder, though the structure was not different from other ranunculus species. conclusion the goal of this study was to observe the morphological characteristics of leaves and stem and anatomical characteristics of leaves, petiole, stem and root of r. auricomus var. biformis in peat bog habitat, in se serbia. the wet habitat partially influenced the anatomical structure of leaf, stem and root, while the morphological characteristics did not show any influence by the habitat except for the length of first internode of the stem. nevertheless, the constraints in a term of temporal flooding could not affect the species due the stable structure of plant organs. acknowledgements. this study was supported by the project of ministry of education, science and technological development of republic of serbia (project no. 173030). we wish to thank vladimir stevanovic for the identification of the species and variety in the laboratory at the department of ecology and geography of plants, university of belgrade. we are also thankful to branka stevanovic for her useful comments on the anatomy part of this study. biologica nyssana 4 (1-2)  december 2013: 41-48 atanacković v. et al.  morphological and anatomical observatoris on … 48 references atanacković, v., ranđelović, v., ibrahim, r.i.h. 2012: morphological and anatomical observations on ranunculus lingua l. under flooding conditions in vlasina lake (se serbia), biologica nyssana, 3 (2): 97-103. benz b.r., rhode j.m., cruzan m.b. 2007: aerenchyma development and elevated alcohol dehydrogenase activity as alternative responses to hypoxic soils in the piriqueta caroliniana complex. amer j of bot 94(4): 542-550. debell d.s., hook d.d., mckee jr w.h. 1984: growth and physiology of loblolly pine roots under various water table level and phosphorous treatments. for sci 30:705-714. ericsson s. 1992: the microspecies of the ranunculus auricomus complex treated at the species level. ann bot fenn 29: 123-158. ericsson s. 2001: ranunculus auricomus complex. in: jonsell b. and karlsson t. 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gabes, tunisia, university of carthage, tunisia faculty of sciences of gabes, university of gabes, tunisia national engineering school of gabes, laboratory of energy, water, environment and processes, university of gabes, tunisia bouabidiamen@gmail.com (corresponding author) mehrez romdhane national engineering school of gabes, laboratory of energy, water, environment and processes, university of gabes, tunisia ezzeddine saadaoui agricultural experimentation unit of gabes, tunisia, university of carthage, tunisia received: december 31, 2024 revised: february 01, 2025 accepted: february 05, 2025 abstract: the apocynaceae family includes the evergreen ornamental shrub known as oleander (nerium oleander). numerous cultivars were listed in this species, and these differ essentially by the color of the flower. the level of diversity within this species is linked to several factors, such as the geographical region where it grows naturally, environmental conditions, and genetic diversity. numerous studies of this plant have highlighted bioactive ingredients and several pharmacological effects. nerium oleander is an extremely toxic and even fatal plant because cardiac glycosides such as oleandrin and neriin are constituents present in different parts of the plant. however, a few common oleander pests occasionally feed on the bush. despite the danger, oleander is of great medicinal importance. compounds such as terpenes, steroids, polyphenols, and flavonoids have been identified in extracts of different plant parts. the essential oil also includes compounds such as digitoxigenin. its compounds are involved in several biological activities such as antiinflammatory, antioxidant, anticancer, hepatoprotective, antimicrobial, larvicidal, and antidiabetic activities which can be used to produce natural drugs. key words: nerium oleander, biological activities, chemical composition, diversity, toxicity apstrakt: nerium oleander l.: pregled diverziteta, toksičnosti, hemijskog sastava i bioloških aktivnosti porodica apocynaceae obuhvata zimzeleni ukrasni žbun poznat kao oleander (nerium oleander). ova vrsta obuhvata brojne kultivare koji se uglavnom razlikuju po boji cveta. nivo diverziteta unutar vrste zavisi od više faktora, uključujući geografsko područje prirodnog rasprostranjenja, ekološke uslove i genetičku raznovrsnost. brojna istraživanja ove biljke istakla su prisustvo bioaktivnih sastojaka i različite farmakološke efekte. nerium oleander je izrazito toksična, pa čak i smrtonosna biljka, jer različiti delovi sadrže srčane glikozide, poput oleandrina i neriina. ipak, pojedine štetočine povremeno se hrane ovom biljkom. uprkos svojoj toksičnosti, oleander ima značajnu medicinsku primenu. u ekstraktima različitih delova biljke identifikovana su jedinjenja poput terpena, steroida, polifenola i flavonoida. etarsko ulje sadrži i jedinjenja kao što je digitoksigenin. njegovi bioaktivni sastojci učestvuju u brojnim biološkim aktivnostima, uključujući antiinflamatorno, antioksidativno, antikancerogeno, hepatoprotektivno, antimikrobno, larvicidno i antidijabetičko dejstvo, te se mogu koristiti u proizvodnji prirodnih lekova. ključne reči: nerium oleander, biološke aktivnosti, hemijski sastav, diverzitet, toksičnost introduction nerium oleander l. or oleander is a mediterranean species grown worldwide as an ornamental plant. it is a 2–5 m tall evergreen shrub with 5–20 cm long leaves. flowers display a range of hues, including pink, red, white, peach, and yellow (dey et al., 2015). it has been introduced to several regions of africa, such as tunisia and algeria. this plant is widely distributed in the mediterranean region and subtropical asia (bandara et al., 2010), but it has now expanded worldwide, including the united states, australia, china, and middle eastern countries. the presence of oleanders around the © 2025 bouabidi et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 36 mediterranean basin has been referenced since the miocene (palamarev, 1989). oleander has the potential to be lethal for people, animals, and even insects (langford & boor, 1996) due to the presence of cardenolides, which include oleandrin, neriin, and digitoxigenin. although it is toxic in all its parts, this species is widely cultivated, making it one of the most toxic cultivated plants (saadaoui et al., 2023). despite the fact that it is the only species of the genus nerium, it presents various forms of variability (color, leaf shape, seed shape, etc.) (pagen, 1987). this diversity is linked to genetic factors, mainly the mode of reproduction. oleander is a hermaphrodite, but new genotypes might arise because of its highly heterozygous and allogamous nature. several chemical screening applied to medicinal plants confirmed that n. oleander produces many bioactive metabolites grouped as pregnanes (bai et al., 2007), cardenolides (bai et al., 2010), phenolics, cardiac glycosides (aiazzi-mancini, 1962; bauer et al., 1984), alkaloids, tannins, flavonoids and terpenoids (siddiqui et al., 1995) that are integrated and assembled in all parts or specific parts of the plant, and that some of them have significant pharmacological interest. nerium oleander has proved its efficiency in medicine by treating different pathologies. identifying the metabolites present in the plant is based on various approaches, some of which could be enzymatic, ultrasonic, or fluid methods, giving different content of chemical compounds (zaid et al., 2022). the richness of n. oleander with diverse chemical compounds, mainly secondary metabolites, gives it various biological activities such as hepatoprotective (singhal, 2012), anticancer (montano, 2013), antidiarrheal, larvicidal (raveen, 2014), antihelmintic (native, 2014), anti-ulcer (sabira, 1998) and cytotoxic (hassan, 2011). different improvements are reported in common cancer treatments by finding secondary compounds of natural products and medicinal herbs (ayouaz et al., 2023). not only extracts, but also n. oleander essential oil have been the object of numerous studies. it was analyzed and reported to possess high antimicrobial (derwich et al., 2010), antioxidant, and antitumor activities (ali et al., 2010). this study's purpose is to compile information concerning n. oleander in terms of genetic diversity, toxicity, and the responsible causative agents. this review is also designed to highlight this plant's phytochemical composition and different biological activities. description and distribution of n. oleander nerium oleander is an evergreen shrub regularly grown as an attractive plant in gardens and public city areas. at present, it is the only species listed in the genus nerium. it has linear and leathery leaves in various colors, from dark green to grey-green, with separate light yellowish veins. the flowers of n. oleander are funnel-shaped and fragrant, with single or double flowers, white, yellow, pink, or red. its fruit is a narrow sheath holding many silkyhaired seeds. oleander has flexible branches with smooth pale green to light gray bark that releases a milky juice when cut. each stem node has two or three narrow elliptical leaves with entire margins on short petioles (garima & amla, 2010). the plant has extensive root systems and is often used to stabilize soil in warmer areas (garima & amla, 2010; sinha & biswas, 2016). nerium oleander is a species with hermaphrodite flowers, theoretically fully selfcompatible, although the spatial separation of pollen and stigma prevents self-fertilization, leading to high heterozygosity when the plants are reproduced by seeds (lazzaro et al., 2018), and it shows great variability in seedling populations (garima & amla, 2010; sinha & biswas, 2016). nerium oleander is introduced to many parts of the world with mediterranean or subtropical climates (california, australia, and others) (bañon et al., 2006). taxonomic ambiguity morphological traits were utilized in the early stages of taxonomic categorization and systematic classification to classify forms. however, their weaknesses and disadvantages were increasingly evident as they were used. the environment can strongly influence them, and they are therefore not directly heritable, which explains the great confusion regarding n. oleander and its different varieties. for a long time, nerium oleander has been assigned to the apocynaceae family (ramade, 2008). oleander variants are still difficult to name and identify, mostly because of materials sold under dubious labels. many cultivars have been selected within this species, but they differ only in certain morphological characteristics. pages (1987) listed more than 400 cultivars based mainly on morphological characters (fig. 1) such as shape, flower color, and leaves (often variegated). several studies (pagen, 1987; ebrahimi et al., 2018; al-snafi, 2020) confirmed that n. oleander is the only species of the genus nerium, and they listed the rest of the varieties as n. oleander synonyms. however, other studies revealed that n. indicum, originally from asia, is a second species in the genus nerium (bi et al., 2016). this taxonomic ambiguity confirms that the classification of this species is only based on the morphological aspect; thus, it remains a subject of research and discussion in the scientific community. according to the the plant list (page, 2025), synonyms include: nerium indicum, nerium indicum subsp. kotschyi, nerium indicum var. leubiologica nyssana ● 16 (1) june 2025: 35-52 bouabidi et al. ● nerium oleander l.: a review of diversity, toxicity, chemical compositions and biological activities 37 biologica nyssana ● 16 (1) june 2025: 35-52 bouabidi et al. ● nerium oleander l.: a review of diversity, toxicity, chemical compositions and biological activities canthum, nerion oleandrum, nerium carneum, nerium flavescens, nerium floridum, nerium grandiflorum, nerium indicum f.leucanthum, nerium indicum var. lutescens, nerium odoratissimum, nerium odoratum, nerium odorum, nerium oleander var. indicum, nerium indicum f. lutescens, nerium indicum var. plenum, nerium japonicum, nerium kotschyi, nerium latifolium, nerium lauriforme, nerium luteum, nerium mascatense, , nerium oleander subsp. kurdicum, nerium splendens, nerium thyrsiflorum, nerium verecundum, oleander indica, and oleander vulgaris (http://www.theplantlist.org/tpl/record/ kew-135196). nerium oleander pests oleander blooms profusely in the summer and fall with large, fragrant blossoms. oleander is a resilient evergreen plant that blooms in intense heat and drought. despite its defensive and toxic secondary metabolites, n. oleander is attacked by many phytophagous pests, essentially aphids such as aphis nerii (homoptera: aphididae) and the striped mealybug, ferrisia virgata (homoptera: pseudococcidae) (el-shazly, 2002). aphids can be found throughout the growing season, although they are often most prevalent in the spring. several pests, including oleander caterpillars like syntomeida epilais, can harm any plant part. the xylella fastidiosa strain causes oleander leaf scorch, and aspidiotus nerii, whitish insects found on leaves' top or bottom surfaces, causes malformation, shriveling, and plant death. these pests have a distinct appearance at every stage of development, and eggs colonize the undersides of leaves (popenoe et al., 2019). genetic diversity of nerium oleander genetic diversity of n. oleander can be investigated by using agro-morphological (alizadeh et al., 2017; roughani et al., 2018), biochemical (keshavarzi et al., 2015), cytogenetic (salmasi et al., n.d.); and molecular markers (portis et al., 2004; ibrahim et al., 2014). morphological analysis of oleander grown spontaneously or cultivated in tunisia and belonging to different bioclimates, has shown high intra-specific diversity (saadaoui et al., 2023). sdspage (sodium dodecyl sulfate polyacrylamide gel electrophoresis) is one of numerous biochemical procedures that have been effectively utilized to address taxonomy and evolutionary issues with several plants (khan, 1992; rabbani et al., 2001). in this context, de britto & sebastian (n.d.) analysed the protein variability among five varieties of n. oleander with flowers in various colors through sds page electrophoresis, and the results clearly showed that there was a high degree of diversity among these varieties of plant species. portis et al. (2004) reported genetic relatedness among 71 accessions of n. oleander using an amplified fragment length polymorphism (aflp) marker. their results demonstrated that, compared to the few morphological features typically employed for variety differentiation, the aflp technique provides far more information about the genetic relationships and origins of accessions. also, they highlighted a high variability related to the fact that oleander is substantially allogamous and highly heterozygous. furthermore, ibrahim et al. (2014) studied the cytogenetic diversity of several apocynaceae species populations, such as n. oleander. they found that all populations are diploid and that there is variation in karyotypic traits within and between species. oleander grows in the mild, temperate climate of the mediterranean basin in gullies, rivers, and fig. 1. six n. oleander cultivars: a) n. oleander with double dark pink flowers and variegated leaves; b) n. oleander with red flowers; c) n. oleander with simple light pink flowers; d) n. oleander with double white flowers; e) n. oleander with pink flowers; f) n. oleander with double pink flowers and simple leaves 38 biologica nyssana ● 16 (1) june 2025: 35-52 bouabidi et al. ● nerium oleander l.: a review of diversity, toxicity, chemical compositions and biological activities streambanks. there is a dearth of information on the phylogeography of mediterranean riparian tree and shrub species. a study by mateu-andrés et al. (2015) performed on mediterranean natural populations of oleander which used plastid dna showed a high homogeneity at both intraand interpopulation levels in this species. a low mutation rate and/or recent recolonization of the mediterranean basin could explain the absence of plastid variability in oleander. according to fussi et al. (2010; 2012), the latest post-glacial period's quick and recent recolonization may cause this low variability. lorenzo et al. (2018) tried to comprehend the origin of oleander populations in montecristo and pistoia (italy) using issr (inter-simple sequence repeat) molecular markers. because of the accuracy and repeatability of the results, plant population genetics uses this genome-scanning technology extensively (rakoczy-trojanowska & bolibok, 2004). the results highlight medium levels of genetic diversity, hardly higher than those reported by hamrick & godt (1996) for long-lived outcrossing taxa (0.180) with a widespread distribution (0.183), which seems to support an anthropic origin of such population. this hypothesis is also suggested by the general relativeness between montecristo and pistoia, which may arise from a possible common anthropic origin of these two populations (lazzaro et al., 2018). actually, no recent study has focused on the genetic diversity of n. oleander and the origin of different cultivars. thus, an accurate method for their identification and characterization is necessary, mainly to confirm the presence of a single species and determine the genetic relationship between different varieties and cultivars. toxicity nerium oleander is indeed a highly toxic plant, recognized for its dangerous cardiac glycosides (turan et al., 2006). these toxins include compounds like oleandrin, neriin, and digitoxigenin, all of which impact the heart by inhibiting na⁺/k⁺-atpase pump activity, leading to a toxic buildup of intracellular na⁺ and ca²⁺. this effect, which resembles the cardiac impact of digitoxin (from digitalis or foxglove) (bandara et al., 2010), can lead to severe cardiovascular effects, including increased contraction force and potential heart failure (blum & rieders, 1987). oleander's toxicity varies by plant part and flower color, with red-flowered plants typically producing higher glycoside concentrations than white-flowered ones (karawya et al., 1973). roots and seeds contain the highest glycoside levels (bandara et al., 2010). the main cardiac glycoside of n. oleander is oleandrin (hameed et al., 2015). oleandrin (c32h48o9) is a white crystalline powder with a melting point of 250 °c and a molecular weight of 576.727 da, and it is insoluble in water and soluble in methanol, ethanol, and chloroform (zhai et al., 2022). the roots contain the highest concentration of oleandrin, followed by the leaves, stems, and flowers. the concentrations of oleandrin in the various plant sections were as follows: 0.18 to 0.31 mg/g dry weight in the leaves, 0.12 to 0.23 mg/g dry weight in the stem, and 0.34 to 0.64 mg/g dry weight in the roots (tayoub et al., 2014). several other cardenolides are added to oleandrin: nerizoside, neritaloside, odoroside, and nerioside (rashan et al., 2011). neriin is also known as a potent cardiac glycoside, present in all parts of the plant (tab. 1). digitoxigenin, with a molecular formula of c23h34o4 inhibits the heart's functioning by hindering the action of na⁺/k⁺-atpases. the two cardiac glycosides, oleandrin and neriin, are the most noteworthy of these toxins. these poisons may be found in many plant sections, but they are more prevalent in the sap. the bark of oleanders contains rosagenin, which is recognized for having effects similar to those of strychnine, along with many other unidentified chemicals that may have potentially harmful effects (dardona & shahabuddin, 2022). nerium oleander is highly toxic to humans, animals, and certain insects, with all parts of the plant (fresh, dried, or boiled) capable of causing fatal poisoning. even minimal ingestion can be deadly due to its cardiac effects (al-snafi, 2020). clinical and toxicological studies report that in humans, n. oleander ingestion causes severe gastrointestinal symptoms like nausea, vomiting, colic, and bloody diarrhea, as well as cardiac issues such as decreased pulse rate, irregular heartbeat, and respiratory paralysis, which can lead to death. secondary neurological symptoms like tremors, drowsiness, ataxia, and hypotension have also been noted, with some cases including seizures (gopalakrishnan et al., 2017). in animals, oleander poisoning has been documented across species (barbosa et al., 2008). horses, for instance, often die within 8 to 10 hours after ingesting lethal amounts, displaying symptoms like cold extremities, ataxia, colic, and convulsions (turner & torres, 2017). cattle show signs such as polydipsia, bloody diarrhea, muscle tremors, fever, dehydration, tachypnea, and tachycardia, with post-mortem examinations revealing extensive hemorrhaging in the lungs and heart (souza, 2018). accidental and/or experimental cases of oleander poisoning have been described in several other species, including monkeys (schwartz et al., 1974), rabbits (al-farwachi et al., 2008), and goats (barbosa et al., 2008). animals' lungs, liver, and heart are affected negatively by the toxicity of n. oleander leaves or flowers. nerium oleander leaf extract causes mononuclear infiltration in the lung, especially around the blood vessels (abbasi et al., 39 biologica nyssana ● 16 (1) june 2025: 35-52 bouabidi et al. ● nerium oleander l.: a review of diversity, toxicity, chemical compositions and biological activities 2018), histopathological changes in the lung tissue (abbasi et al., 2014), as well as interstitial hemorrhage in the lung one hour after receiving the oleander, resulting in congestion and edema (aslani et al., 2004; aslani et al., 2007). majeed (2012) demonstrated that n. oleander flower extract caused severe blood vessel congestion and edema around the esophagus, particularly at high doses. the liver can also be affected by the toxicity of n. oleander components, particularly from the leaves, which can cause different levels of bleeding, hepatocyte degeneration and localized necrosis, hepatocyte necrosis, fatty degeneration, and infiltration of mononuclear inflammatory cells (ozmaie et al., 2013). depending on the extract dose, the administration of n. oleander revealed various characteristics of cardiac toxicity. in cardiac cells, n. oleander leaf extract causes pathomorphological alterations and intrasarcoplasmic vacuole myocytolysis (taheri et al., 2013). an earlier study conducted by aslani et al. (2004) on the cardiotoxicity impact of n. oleander (110 mg/ kg, orally, single dose) in male sheep indicated that sinus bradycardia was seen as the first symptom in electrocardiogram (ecg) 0.5 h after receiving this plant (aslani et al., 2004). nerium oleander toxicity can also affect insects. the presence of lipophilic components explains the insecticidal effect of n. oleander extracts. zaid et al. (2022) assessed n. oleander's insecticidal efficacy against chaitophorus leucomelas viviparous females. four days following the treatment, at a concentration of 5.15 g/m2, a 100% mortality rate was noted. phytochemical screening nerium oleander contain a high number of chemical compounds, the bulk of which are listed below, according to the results of the phytochemical screening: adenerine, neriin, digitoxigenin, cardenolides, bufadienolides, ouabain, proscillaridin, kanersoide, neriumoside, cis and trans karenin, oleandrin, folinrin, 4-oxooctyl-2-hydroxy-undecanoate, heptacosane-3-enyl-5-hydroxyhexanoate, betulin, betulinic acid, stigmasterol, quercetin5-o-[α-l-rhamnopyranosyl(1→6)]-β-d-glucopyranoside and kaempferol-5-o-[α-lrhamnopyranosyl-(1→6)]-βd-glucopyranoside (hase et al., 2016). the plant's leaves contain a variety of physiologically significant metabolites, including proteins, carbohydrates, alkaloids, flavonoids, terpenoids, cardiac glycosides, tannins, and saponins (suganya, 2012). nawaz et al. (2023) recently isolated a new bioactive steroid 3β-acetoxy-5, 25(26) diene, 24β-hydroxy lanostane from this plant. extract composition many approaches are employed to extract the chemical components of n. oleander, with varying outcomes. plant sections displayed a variety of phytochemical substances. essential oils were extracted using polar and non-polar solvents, including ethanol, diethyl ether, hexane, cyclohexane, and ethyl acetate. certain metabolites are only synthesized under specific environments, or their contents significantly increase under these conditions (ebrahimi et al., 2018). every part of n. oleander revealed a specific compound. the preliminary phytochemical screening showed that the leaves of this plant contain carbohydrates, flavonoids, alkaloids, steroids, cardiac glycosides, and tannins (sinha & biswas, 2016). oleandrin, neriin, cardenolides, gentiobiosyl, and odoroside are the primary glycosides (farooqui & tyagi, 2018). the seeds possess glucosides (oleandrine, odorosidesandadigoside). the bark also component molecular formula structure molecular weight (g/ mol) oleandrin c32h48o9 576.72 neriin c24h40n2 356.6 digitoxigenin c23h34o4 374.5 table 1. molecular formula, structure, and molecular weight of n. oleander cardiac glycosides (oleandrin, neriin, digitoxigenin) 40 a pectic polysaccharide made up primarily of galacturonic acid, in addition to rhamnose, arabinose, and galactose, made up the majority of the fraction (67%). the hydro-ethanolic extract of the n. oleander plant collected in morocco underwent phytochemical screening, which identified the presence of coumarins, terpenes, triterpenes, flavonoids, and contains glucosides (rosaginoside, nerioside, and corteneroside), and the roots contain steroids (garima & amla, 2010). fifteen chemicals were detected in the n. oleander alkaloid leaf extract, and eight alkaloid components were found in the methanolic extract (hameed et al., 2015) and are presented in tab. 2. the maximum concentration of phenobiologica nyssana ● 16 (1) june 2025: 35-52 bouabidi et al. ● nerium oleander l.: a review of diversity, toxicity, chemical compositions and biological activities table 2. chemical composition of n. oleander extracts plant part extract type composition references leaves ethanol extraxt siloxane compounds: cycloheptasiloxane, tetradecamethyl, cyclooctasiloxane, hexadecamethyl, cyclononasiloxane, octadecamethyl, cyclodecasiloxane, eicosamethyl hameed et al., 2015 methanolic extraxt cyclopentenes: 2-cyclopenten-1-one, 2-cyclohexen-1-one, 5-hydroxy-methylfurfural, β-d-allopyranoside fatty acids: 9,12,15-octadecatrienoic acid, octadecane phenolic compounds: rutin, catechin, epicatechin, quercetin, quenonic acid flavonoids: isoquercetin, luteolin-7-o-glucoside triterpenoids: betulinic acid, oleanolic acid, hederagenin cardenolides: oleandrin, neriin, digitoxigenin hameed et al., 2015; saranya et al., 2017; ling et al., 2025 ethyl acetate extract triterpenes: taraxasterin-type (e.g., 20β,28-epoxy-28αmethoxy-taraxasteran-3β-ol) and ursane-type (e.g., 3β-hydroxyurs-12-en-28-aldehyde) zhao et al., 2006 aqueous extract pectic polysaccharides: primarily galacturonic acid, with rhamnose, arabinose, and galactose sinha & biswas, 2016 flowers methanolic extract phenolic compounds: rutin, catechin, epicatechin, quercetin and quenonic acid; other compounds: maltol, oleic acid, cis-vaccenic acid, benzene, 4h-pyran-4-one saranya et al., 2017; saeed et al., 2023 ethanol extraxt flavonoids: kaempferol 3-o-β-glucopyranoside and chlorogenic acid atay balkan et al., 2018 roots methanolic extract phenolic compounds gunes et al., 2017 twigs phenolic derivatives: caffeic acid, ferulic acid, p-coumaric acid ling et al., 2025 lic compounds, which are found in both leaves and flowers of n. oleander, such as rutin, epicatechin, quercetin, and quenonic acid, were found in the methanolic extract of the plant in vitro (saranya et al., 2017). the ethyl acetate extract of n. oleander leaves yielded the taraxasterane-type triterpenes (20 β,28-epoxy-28α-methoxytaraxasteran-3β-ol and 20β,28-epoxytaraxaster-21-en-3β-ol) and ursanetype triterpenes (28-nor-urs-12-ene-3β,17β-diol and 3β-hydroxyurs-12-en-28-aldehyde) (zhao et al., 2006). a 2.3% crude polysaccharide was obtained from a water extract of crushed n. oleander leaves. sterols. however, no mucilage, tannins, or leucoanthocyanins were found (el-akhal et al., 2015). this result is consistent with previous research demonstrating that flavonoids, coumarins, and triterpenes are produced by apocynaceae family plants, including nerium (sedaghat et al., 2011; roni et al., 2013). according to the study of zaid et al. (2022), 38 chemical components, mostly made up of the terpenoid and fatty acid group (oleic, linolenic, palmitic, and stearic), were identified in the methanolic extract. the majority of terpene compounds that are associated with oxygenated derivatives include 41 p-cresol, isopulegol, guaiol, phenylethyl alcohol, durene, beta-myrcene, phytol, gamma-sitosterol, and 8-quinolinol. verbenone, squalene, 2-(4h)benzofuranone, 4-methyl, and caryophyllene oxide. apart from these findings, additional compounds were extracted from the ethyl acetate sub-extract of the ethanolic extract of n. oleander flowers (atay balkan et al., 2018). the recent study by ling et al. (2025) identified 50 compounds in the leaves of n. oleander and 25 compounds in the twigs, including cardenolides, flavonoids, phenolic derivatives, and triterpenoids, with a greater diversity of compounds found in the leaves compared to the twigs. essential oil of n. oleander the essential oils of n. oleander flowers from china, morocco, and saudi arabia exhibit distinct chemical profiles, reflecting the influence of geographic origin on their composition and properties. morocco stands out with a higher essential oil yield (1.76%) (derwich et al., 2010), as well as the presence of bioactive compounds such as neriin (22.56%) and digitoxigenin (11.25%). while neriin remains poorly studied, it may possess interesting biological properties, whereas digitoxigenin is a well-known cardiotonic compound used to treat heart conditions (patel, 2016). limonene (5.01%), known for its aromatic properties and applications in the perfume and food flavoring industries, is also present in oleander essential oil (sun, 2007). in oil from saudi arabia, camphor (12.76%), a compound widely studied for its analgesic, anti-inflammatory, and decongestant properties (dos santos et al., 2021) dominates in the chemical profile (ali et al., 2010). moreover, the presence of thymol (8.43%) in saudi oil is notable, as this compound is recognized for its antiseptic, antifungal, and antioxidant properties (marchese et al., 2016). in china, bi et al. (2016) identified twenty-nine components from n. indicum flowers, corresponding to 83.1% of the total composition. the data show the presence of compounds such as tricosane (7.4%) and pentanal (4.4%), although no major compound clearly dominates the chemical profile (bi et al., 2016). other minor compounds have been identified in this study with a percentage higher than 1% and are detailed in tab. 3. several studies (derwich et al., 2010; ali et al., 2010; bi et al., 2016) confirmed that geographic origin is a key factor contributing to the chemical composition of essential oils (szakiel et al., 2011). however, further research is needed to explore the impact of extraction methods, cultivation conditions, and seasonal variations. biologica nyssana ● 16 (1) june 2025: 35-52 bouabidi et al. ● nerium oleander l.: a review of diversity, toxicity, chemical compositions and biological activities table 3. essential oil yields in n. oleander in flowers of china, marocco and saudi arabia and the main compounds with a percentage higher than 1% origin china morocco saudi arabia plant part flowers chemical compounds (%) tricosane (7.4) isocaryophyllene (1.1) myristicin (1.04) 1-nonanal (2.7) terpinene-4-ol (3.98) cuparene (1.76) phenylacetaldehyde (2.9) myrtenal (1.25) β-bisabolene (1.01) docosane (3.2) neriin (22.56) β-cubenene (1.87) hexyl alcohol (2.4) digitoxigenin (11.25) camphene (2.75) 2,3,7 trimethyldecane (1.2) α-terpinene (1.52) β-elemene (1.08) pentalan (4.4) amorphane (8.11) β-funebrene (2.77) butyl isobutyl phtalate (1.3) 1,8-cineole (6.58) α-phellandrene (1.43) hexanal (1.2) α-pinene (5.54) β-ocimene (1.05) furfural (1.6) clarence (5.12) β-sesquiphellandrene (1.98) heneicosane (8.7) limonene (5.01) ocimene (1.79) 2-hexenal (3.2) β-phellandrene (4.84) caryophyllene (3.43) linoleic acid (1.8) sabinene (3.22) guaiol (1.88) leaf alcohol (5.9) globulol (1.1) α-cubebene (3.43) linolenic acid (3.4) 3-carene (2.56) α-humulene (2.43) 42 biological activities of nerium oleander nerium oleander components are becoming more and more interesting because of their significance, widespread consumer acceptance, and potential for multipurpose functional use despite their toxicity (sawamura, 2000; ormancey, 2001; sacchetti et al., 2005). in prior pharmacological investigations, this plant has exhibited anti-inflammatory, analgesic, hypolipidemic, anticancer, antibacterial, antiparasitic, anti-inflammatory, dermatological, and cardiovascular properties (al-snafi, 2020). other studies revealed antifungal, molluscicidal, cytotoxic, larvicidal, and insecticidal activities (hussain & gorsi, 2004; hadizadeh et al., 2009; elakhal et al., 2015). antimicrobial activities of nerium oleander according to several studies (cao et al., 2009; havlik et al., 2009), oleander essential oil has considerable antibacterial properties due to various components, including phenolic and terpene compounds (tab. 4). in saudi arabia, essential oil extracted from oleander flowers was used to evaluate antimicrobial activity, and excellent results were obtained (ali et al., 2010). in this same study, two gram-positive bacteria (bacillus subtilis and staphylococcus aureus), two gram-negative bacteria (escherichia coli and salmonella typhimurium), and three fungal species (fusarium oxysporum, rhizoctonia solani, and macrophoma mangiferiae) were tested. on the other hand, the antibacterial activity of the n. oleander essential oil extracted by hydro-distillation inhibited the pseudomonas aeruginosa biofilm at very low concentration (almanaa et al., 2021). the antibacterial properties of essential oils derived from n. oleander were investigated in morocco by applying minimum inhibitory concentration (mic) and disk diffusion testing methods. as test bacterial strains, escherichia coli, pseudomonas aeruginosa, and staphylococcus aureus were employed. according to the results, the strain of e. coli that showed the highest susceptibility to this oil had an inhibition zone of 28.89 mm. with an inhibition zone of 18.22 mm, p. aeruginosa was more sensitive than the other microorganisms, while inhibitory zones of 6.32 mm were found against s. aureus, indicating modest activity (derwich et al., 2010). mouhcine et al. (2019) also investigated the biologica nyssana ● 16 (1) june 2025: 35-52 bouabidi et al. ● nerium oleander l.: a review of diversity, toxicity, chemical compositions and biological activities chemical compounds (%) heptacosane (4.4) isoledene (2.94) β-charnigrene (1.08) 2,6-di-tert-butyl-p-cresol (2.7) verbenol (1.24); humulene (2.29) trans-calarnenene (0.82); germacrene d (2.76) pentacosane (4.5) β-pinene (2.01) β-selinene (1.98) lauric acid (1.6) seychellene (1.09) γ-curcumene (1.09) hexacosane (1.4) cymen-8-ol (1.67) camphore (12.76) tetradecanal (1.7) ylangene (1.2) myrcene (1.31) 1-heptacosanol (1.7) germacrene-d (1.01) thymol (8.43) myristic acid (1.8); diisobutylphtalate (1.4); 4-octadecanolide (2.1); 1,19-eicosadiene (1.6); 1,6-cyclodecadiene (2.3); 6,10,14-trimethyl-2-pentadecanone (2.7) patchoulene (1.02) α-campholenal (5.05); eugenol (10.45); α-copaene (1.5); δ-cadinene (1.27) essential oils yield (%) 0.11 1.76 0.1 total oil (%) 83.1 93.21 94.69 references bi et al., 2016 derwich et al., 2010 ali et al., 2010 43 biologica nyssana ● 16 (1) june 2025: 35-52 bouabidi et al. ● nerium oleander l.: a review of diversity, toxicity, chemical compositions and biological activities antibacterial activity of n. oleander extracts. six bacterial strains were used, including 3 grampositive bacteria: enterococcus faecalis, listeria monocytogenes, and staphylococcus aureus, and three gram-negative bacteria: escherichia coli, pseudomonas aeruginosa, and salmonella typhimurium. the outcomes showed that the e. faecalis strain was susceptible to the effects of both ethanolic and aqueous extracts, with the inhibition zone diameter reaching 5.3 mm and 10 mm, respectively. in contrast, l. monocytogenes was not sensitive to the action of the aqueous extract. the other organisms that were tested, including p. aeruginosa, s. aureus cect 476, e. coli, and s. typhimurium, were resistant to both extracts. numerous other studies have focused on the organs of n. oleander (flowers, leaves, roots, roots bark) to study the antibacterial activity against a high number and types of bacteria. the antibacterial activity of the leaf and flower extracts was investigated against b.cereus, b. pumilus, bacillus epidermidis, and erwinia carotovora. strong antibacterial action against both gram-negative and gram-positive bacteria was demonstrated by dichloromethane and methanol extracts of leaves and flowers (excluding staphylococcus epidermidis) (namian et al., 2013). jeyachandran et al. (2010) observed that the methanolic extract of n. oleander showed maximum zone of inhibition (28 mm) against s. typhi. hussain & gorsi (2004) focused on the antibacterial activity of n. oleander root, bark, and leaf extracts against aspergillus niger, bacillus pumilus, staphylococcus aureus, and escherichia coli. after an incubation period of 24 hours, the chloroform, ethanol, and methanol extracts of n. oleander exhibited strong action against every tested bacterium (growth inhibition zone: 20–23 mm). none of the crude extracts chloroform, ethanol, or methanol exhibited any antifungal action against a. niger. on the other hand, studies of antifungal and antiviral activities of n. oleander remain rare compared to other activities; very few studies focused on this plant species. siddiqui et al. (2016) examined the antifungal activities of n. oleander using aqueous, methanol, ethanol, chloroform, and acetone extracts from its leaves, stem, and root against three fungal species: macrophomina phaseolina, sclerotium rolfsii, and fusarium oxysporum. similarly, el sawi et al. (2010) investigated the antifungal activity of n. oleander extracts against six fungal species: aspergillus flavus, a. fumigatus, a. niger, fusarium moniliforme, penicillium expansum, and rhizopus oryzae. the crude extract exhibited the highest antifungal activity against a. flavus, with inhibitory zones of 15 mm and 20 mm. among the tested species, a. flavus had the lowest minimum inhibitory concentration (mic) of the active crude extract (25 μg/ml), while the mic values for the remaining strains were as follows: r. oryzae >100 μg/ml, p. expansum 50 μg/ml, f. moniliforme >100 μg/ml, and a. fumigatus >100 μg/ml. the primary studies focusing on antibacterial activity are summarized in tab. 5. cytotoxic activity of n. oleander essential oils and their components are used in medicine as constituents of different medical products and have stronger biological activities, such as cytotoxic activity. nerium oleander oil was tested against several carcinoma cell lines, revealing a gradual increase in antitumor activity. ali et al. (2010) tested n. oleander oil against the growth of ehrlich ascites carcinoma cell line. the results demonstrated that oleander essential oil significantly improves anti-tumor activity, which reaches 100% with 8 µl/ml of oleander essential oil. several researchers have studied the cytotoxic effect of extracts and components isolated from n. oleander. mouhcine et al. (2019) have investigated the cytotoxic capabilities using the wst-1 bioassay on two human cancer cell lines: mda-mb-231 for breast cancer and ht-29 for colon adenocarcinoma. table 4. antimicrobial activity of n. oleander essential oil activity plant part microorganism result reference antibacterial flowers g+: bacillus subtilis and staphyllococcus aureus; g-: escherichia coli, salmonella typhimirium, pseudomonas aeruginosa + ali et al., 2010; derwich et al., 2010; almanaa et al., 2021 leaves g-: escherichia coli and salmonella typhimirium + almanaa et al., 2021 antifugal flowers fusarium oxysporum, rhizoctonia solani, macrophoma mangiferae + ali et al., 2010 *(+): effect / (-): no effect; *g+: gram positive bacteria/g-: gram negative bacteria 44 biologica nyssana ● 16 (1) june 2025: 35-52 bouabidi et al. ● nerium oleander l.: a review of diversity, toxicity, chemical compositions and biological activities table 5. antimicrobial activities of n. oleander extracts (antiviral, antibacterial and antifungal) activity extract plant part strains tested result references antibacterial ethanolic and aqueous crude extracts leaves g+: listeria monocytogenes and enterococcus faecalis + mouhcine et al., 2019 g+: staphylococcus aureus; g-: escherichia coli, pseudomonas aeruginosa and salmonella typhimurium dichloromethane and methanol extracts flowers and leaves g-: escherichia coli, erwinia carotovora; g+ : staphylococcus aureus, bacillus cereus, bacillus pumilus + namian et al., 2013 g+: staphylococcus epidermidis chloroform, ethanol and methanol extracts root, bark and leaves g+: bacillus pumilus, bacillus subtilis, staphylococcus aureus; g: escherichia coli + hussain & gorsi, 2004 aqueous and ethanol extracts leaves shigella dysenteriae, aeromonas hydrophila, escherichia coli, enterobacter spp., klebsiella spp., pseudomonas aeruginosa and staphylococcus aureus + aboud, 2015 aqueous extract leaves g+: bacillus subtilis, staphylococcus aureus; g: pseudomonasaeruginosa, escherichia coli and proteus mirabilis + minnat, 2016 ethanolic, aqueous and chloroform extracts flowers g+: bacillus subtilis, staphylococcus aureus; g: escherichia coli, salmonella typhi and pseudomonas aeruginosa + saranya et al., 2017 ethanolic extract leaves g+: staphylococcus aureus; g-: escherichia coli and pseudomonas aeruginosa + malik et al., 2015 crude and pure extracts flowers g-: escherichia coli, pseudomonas aeruginosa and salmonella enteritidis; g+: bacillus subtilis, listeria monocytogenes and staphylococcus aureus + el sawi et al., 2010 methanolic extract white flowers g+: listeria monocytogenes, staphylococcus caprae; g-: shigella dysenteriae and salmonella sp. + saeed et al., 2023 methanolic extract white, red and pink flowers g-: escherichia coli; g+: streptococcus group a cardona & shahabuddin, 2022 45 biologica nyssana ● 16 (1) june 2025: 35-52 bouabidi et al. ● nerium oleander l.: a review of diversity, toxicity, chemical compositions and biological activities based on cell viability indicators, the wst-1 test showed that both ethanolic and aqueous extracts decreased cell viability in both cell lines. according to their findings, the aqueous extract exhibited higher activity than the ethanolic extract. for mdamb-231 cells, the ic50 values were 1.67 μg/ml and 2.36 μg/ml, respectively, while for ht29 cells, they were 2.89 μg/ml and 5.09 μg/ml. barai et al. (2018) used the phytochemicals included in n. oleander's stem bark extract to synthesize stable, gold-conjugated nanoparticles that preferentially induce the apoptosis of cancer cells, particularly the mcf-7 breast cancer cell line. at 74 μg/ml, it is destroying the cancer cells. according to other studies, such as those conducted by calderón-montaño et al. (2013), n. oleander extract exhibited a strong effect on the a549 lung cancer cell line. its cytotoxicity was found to be significantly higher than that of nonmalignant cell lines, and its potency and selectivity were comparable to those of the anticancer drug cisplatin. furthermore, using an orthotopic human pancreatic cancer model, researchers investigated the anticancer effectiveness of pbi-05204, a supercritical co₂ extract of n. oleander containing oleandrin. the results showed that pbi-05204 inhibited the proliferation of panc-1 tumor cells. additionally, pbi-05204 reduced the expression of pakt, ps6, and p4ebp1 in panc-1 tumor tissues and human pancreatic cancer cell lines in a concentrationdependent manner (pan et al., 2015; newman & yang, 2015). the primary cause of the intriguing cytotoxicity of n. oleander extracts on cell lines is the presence of cardiac glycosides, which have been antibacterial white, red and pink flowers g+: staphylococcus aureus + (except white) ethanolic extract g-: escherichia coli g+: streptococcus group a (except red) g+: staphylococcus aureus + antifungal chloroform, ethanol, and methanol extracts roots, bark, and leaves aspergillus niger hussain & gorsi, 2004 ethanolic, aqueous and chloroform extracts flowers aspergillus niger, a. flavus, a. fumigatus, rhizopus spp. + saranya et al., 2017 crude and pure extracts aspergillus flavus, a. fumigatus, a. niger, fusarium moniliforme, penicilium expansum, rhizopus oryzae + el sawi et al., 2010 aqueous, methanol, ethanol, chloroform, and acetone extracts roots, leaves, stems macrophomina phaseolina, slerotium rolfsii, fusarium oxysporum + siddiqui et al., 2016 antiviral aqueous extract n/a hiv + singh et al., 2013 cold extract, hot extract n/a poliovirus type 1 (sb-1) + sanna et al., 2021 herpes simplex virus type 1 (hsv-1), vesicular stomatitis virus (vsv), reovirus type-1 (reo-1), human immunodeficiency virus type1 (hiv-1), yellow fever virus (yfv) *(+): effect / (-): no effect; *g+: gram positive bacteria/g-: gram negative bacteria 46 extensively studied for their anticancer properties. in human pancreatic tumor cells (panc-1), the main glycoside extracted from n. oleander inhibited cell proliferation and arrested cells at the g2/m stage of the cell cycle (newman et al., 2007). oleandrin, the main compound with cytotoxic activity, has been extensively studied without overlooking the effects of other glycosides such as odorside, neritaloside, and the aglycone oleandrigenin (wang et al., 2000). using various cancer cell lines, the anvirzel™ supplement, derived from n. oleander and containing oleandrin, odorside, neritaloside, and the aglycone oleandrigenin, has demonstrated notable anticancer activity. additionally, fractions containing cardenolides from the cold aqueous extract of n. oleander leaves exhibited anticancer properties against 36 human tumor cell lines, with an ic₅₀ value of 0.85 μg/ml (rashan et al., 2011). insecticidal activity of n. oleander certain plant extracts are employed as phytopesticides or bioinsecticides to counteract the overuse of pesticides. these substitute methods have less of an adverse effect on the environment and are more considerate of the health of people and animals (regnault-roger et al., 1993; kellouche, 2005). in this context, n. oleander has been the subject of several studies on its toxicity against some insects. this plant is toxic to larvae of culex pipiens (barbouche et al., 2001), rhizotrogini and lymantra dispar (madaci et al., 2008; kerris et al., 2008) and to schistocerca gregaria in morocco (bagari et al., 2013). harizia and doumandji (2014) reported that the insecticidal efficacy of n. oleander essential oil resulted in total mortality of schistocerca gregaria larvae (5th larval stage) by the 7th day after treatment. the essential oil exhibited insecticidal effects by reducing body weight and deterring consumption. similarly, bagari et al. (2013) observed complete mortality after 12 days when larvae were fed exclusively on fresh leaves of the same plant during the fourth stage of development. oleander extracts have demonstrated larvicidal effects against various insect species. according to rao et al. (2012), the aqueous leaf extract exhibited insecticidal activity against culex tritaeniorhynchus and c. gelidus. several studies have also reported that ethanol extracts from n. oleander leaves possess larvicidal effects against aedes aegypti mosquitoes (komalamisra et al., 2005), the third and fourth larval stages of culex pipiens (el-akhal et al., 2015), as well as the second instar larvae of the medically important false stable fly, muscina stabulans (elshazly et al., 1996). in another study, c. pipiens was used as a test subject to evaluate the larvicidal properties of water, chloroform, acetone, and diethyl ether extracts of n. oleander leaves. based on lc₅₀ values, the toxicity of these four extracts was higher at 10 °c than at 35 °c (el-sayed & el-bassiony, 2016). using enzymatic, ultrasonography, and supercritical fluid extraction techniques, researchers identified 38 bioactive compounds, including d-limonene. this compound exhibited insecticidal activity by reducing certain reproductive parameters in viviparous female chaitophorus leucomelas (zaid et al., 2022). furthermore, natural populations of c. leucomelas exposed to a 5.15 g/m² methanol leaf extract under semi-controlled field conditions exhibited 100% adjusted mortality after four days. moreover, a recent study by al-ansi et al. (2024) suggested that n. oleander leaf extracts could serve as a natural alternative to synthetic insecticides against pachycondyla sennaarensis ants, while emphasizing the need for strict precautions in their application. the phytochemical components, including flavonoids, sterols, terpenes, triterpenes, and coumarins, may contribute to the larvicidal activity observed in n. oleander extracts (el-akhal et al., 2015). the larvicidal activity of n. oleander flowers against culex quinquefasciatus, the filarial vector, was studied. mortality was observed during both 24 and 48 hours. the larvicidal activity of hexane flower extract was maximal after 24 and 48 hours, with lc50 values of 102.54 ppm and 61.11 ppm, respectively (raveen et al., 2014). therefore, the effect of the plant (toxic, repellent, or antifeedant) and the type of extract utilized (aqueous extract, essential oil, fresh leaves), as stated by these authors, are mostly responsible for the variability of larval mortality (harizia & doumandji, 2014). antioxidant activity nerium oleander has strong antioxidant properties; extracts from its leaves, stems, and roots effectively scavenge free radicals and can be utilized as a natural source of powerful antioxidants (farooqui & tyagi, 2018). the compounds extracted from the leaves demonstrated excellent hydroxyl radical, peroxynitrite, hypochlorous acid scavenging, and iron chelation activity. the stem had the most potent capacity to scavenge dpph radicals and nitric oxide, whereas the root demonstrated lipid peroxidation, superoxide anion, hydrogen peroxide, and singlet oxygen scavenging activities (dey & chaudhuri, 2014). it has also been found that n. oleander flowers can serve as an excellent source of natural antioxidants (mohadjerani, 2012). both leaves and flowers have been shown to contain phenolic compounds, such as rutin, catechine, epicatechin, quercitin, and quenonique acid (saranya et al., 2017), that display the highest levels of antioxidant activity (garima, 2011). bouabidi et al. ● nerium oleander l.: a review of diversity, toxicity, chemical compositions and biological activities biologica nyssana ● 16 (1) june 2025: 35-52 47 table 6. pharmacological effect of n. oleander extracts effect plant extract experimental model result references anti-inflammatory flower ethanolic extract mice inhibition erk phosphorylation (by 20.53% at 200 μg/ml); concentration-dependent inhibition of protein (albumin) denaturation at concentrations ranging from 100 to 500 μg/ml; significant antinociceptive activity against p-benzoquinone-induced abdominal contractions erdemoglu et al., 2003; mary et al., 2017; atay balkan et al., 2018 dermatological aqueous leaf extract rabbit complete healing at 6-7 days minnat, 2016 anti-hyperlipidemic hydroethanolic extracts of n. oleander flowers rats a dose-dependent, considerable ameliorative effect on increased lipids and lipoproteins in comparison to standard (test conducted in hyperlipidemic rats); beneficial effects on cholesterol metabolism-related gene gayathri et al., 2013; demirel kars et al., 2014 nervous effects hydroalcoholic, methanol, chloroform flowers extracts mice significant reduction in spontaneous locomotor activity, anxiolytic activity singhal & gupta, 2011; shashikala et al., 2018 antidiabetic hydromethanolic leaf extract mice antihyperlipidemic activity, percentage decrease in different livel marker enzymes, decrease in triglyceride and cholesterol levels (test applied in diabetic mice induced by alloxan) dey et al., 2015 cardiovascular methanolic leaf extracts rats the elevation of marker enzymes in plasma, including lactate dehydrogenase, γ-glutamyl transferase, creatine kinase (ck-mb and creatine phosphokinase), aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase, was prevented in rats by pretreatment with the extract (10, 30, and 100 mg/kg) and propranolol for two weeks after isoproterenol challenge (test against isoproterenolinduced myocardial toxicity in rats compared to propranolol) gayathri et al., 2011 bouabidi et al. ● nerium oleander l.: a review of diversity, toxicity, chemical compositions and biological activities biologica nyssana ● 16 (1) june 2025: 35-52 48 nerium oleander essential oil possessed significant antioxidant activity, which was studied by three methods (dpph assay, β-carotene/linoleic acid bleaching assay, and ferric reducing power assay)(al-snafi, 2020). when compared to synthetic antioxidants such as trolox and bht, oleander essential oil exhibited much higher antioxidant activity (ali et al., 2010). additional research, such as that conducted by bi et al. (2016), found that the essential oil of n. indicum (also known as n. oleander syn) exhibited notable scavenging properties against free radicals such as dpph, atbs, and superoxide anion, with ic50 values of 45.29, 32.47, and 67.31 g/ml. the antioxidant properties of the leaves and flower extract were assessed by namian et al. (2013) using dpph at doses of 0.5, 0.25, 0.125, 0.0625, 0.0312, 0.0156, 0.0078, 0.0039 and 0.0019 mg/ml. the leaves and flowers methanol extracts demonstrated strong antioxidant activity, with ic50 values of 0.27 and 0.2 mg/ml, respectively. the antioxidant activity of water, methanol, water: methanol and acetone extracts of n. oleander grown in the north of iran was studied by employing various in-vitro assays (dpph free radical scavenging, reducing power and total antioxidant capacity). the extracts with the highest antioxidant potency were the methanolic and aqueous methanolic extracts. four extracts of n. oleander leaves (water, methanol, water: methanol and acetone) showed total antioxidant activity of 1.280, 1.246, 0.982, and 0.912, while for flowers, there are 2.330, 1.386, 1.596, 2.930 mg ascorbic acid equivalents/mg extract (al-snai et al., 2019). oleander flower extract's antioxidant capacity was assessed utilizing the dpph free radical scavenging assay and the reducing power assay. according to the results of applied assays, the ethanolic extract demonstrated a significant scavenging capacity and reducing power activity (saranya et al., 2017). ethanol and aqueous extracts of n. oleander were also investigated for their antioxidant activities with dpph scavenging assay and the β-carotene bleaching test. mouhcine et al. (2019) reported the high antioxidant capacity of n. oleander extracts. while all the tested concentrations of the aqueous extract had comparable results to bht, with an impact above 50%, the ethanol extract's ic50 was 2.2 mg/l. furthermore, the β-carotene bleaching assay demonstrated that both n. oleander extracts significantly reduced the oxidation of β-carotene. oleander various extracts were investigated for pharmacological activities and were found to possess anti-inflammatory, skin-healing, antihyperlipidemic, anxiolytic, positive cardiovascular and hepatoprotective effects (tab. 6). conclusion nerium oleander is a unique species characterized by high intraspecific diversity, many varieties, and significant toxicity. extracts from this species contain various chemical compounds, the most important of which include carbohydrates, proteins, alkaloids, flavonoids, terpenoids, cardiac glycosides, tannins, and saponins. neriin, oleandrin, cardenolides, gentiobiosyl, and odoroside are the primary toxic compounds of this plant. these toxic chemicals have been utilized as effective pharmaceutical agents for treating various infectious and chronic diseases. additionally, n. oleander extracts are known for their diverse biological activities, including antimicrobial, antioxidant, and insecticidal properties. furthermore, n. oleander exhibits potent cytotoxic activity, making it a promising candidate for future cancer treatments due to the presence of active glycosides such as oleandrin and is also being explored as a potential new treatment for hiv. references aboud, a. s. 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(2006). taraxasterane-and ursane-type triterpenes from nerium oleander and their biological activities. journal of natural products, 69(8), 1164-1167. bouabidi et al. ● nerium oleander l.: a review of diversity, toxicity, chemical compositions and biological activities biologica nyssana ● 16 (1) june 2025: 35-52 vasić et al 2020, biologica nyssana 11(2) 11 (2) december 2020: 115-119 doi: 10.5281/zenodo.4393965 resistance to anthracnose (colletotrichum linicola) on different cultivars of red clover (trifolium pratense) in serbia original article tanja vasić faculty of agriculture, university of niš, kosančićeva 4, 37000 kruševac, serbia tanjavasic82@gmail.com (corresponding author) sanja živković faculty of agriculture, university of niš, kosančićeva 4, 37000 kruševac, serbia gajicsanja43@gmail.com mitra debasis division of soil science and agricultural chemistry, icar indian institute of horticultural research, karnataka 560 089, india debasismitra3@gmail.com ivana stanojević faculty of agriculture, university of niš, kosančićeva 4, 37000 kruševac, serbia stanojevic.ivana85@gmail.com sonja filipović faculty of agriculture, university of niš, kosančićeva 4, 37000 kruševac, serbia sonjafilipovic86@yahoo.com biljana anđelić faculty of agriculture, university of niš, kosančićeva 4, 37000 kruševac, serbia andjelic.biljana@ni.ac.rs jordan marković institute for forage crops, kruševac, 37251, globoder, serbia jordan.markovic@ikbks.com received: february 05, 2020 revised: november 23, 2020 accepted: november 30, 2020 abstract: in addition to alfalfa, red clover (trifolium pratense l.) is the most important perennial forage legume grown in serbia. it is rich in proteins that are important for the nutrition of livestock. red clover, like many legumes, is parasitized by fungi of the genus colletotrichum, and the consequence is significant economic damage in the form of reduced yields and reduced hay quality. in this study, sixteen cultivars of red clover of different geographical origin were tested for the resistance to the isolate coll-44 (c. linicola). the susceptibility of different cultivars of red clover to the tested c. linicola isolate was determined using the scale of 1-5. six cultivars (wilo, repio, viglana, kolubara, k-39 and k-17) showed high levels of resistance to the tested isolate of c. linicola. based on the severity index, the most resistant to the isolate of c. linicola was the american variety wilo with a severity index of 1.6 (50%), while the lowest severity index of the serbian variety k-39 was 2.6 (20%). the purpose of this research was to evaluate the susceptibility and tolerance level of 16 red clover cultivars to anthracnose causative agents, in this case, c. linicola. key words: colletotrichum linicola, cultivar, resistance, trifolium pratense apstract: otpornost na antraknozu (colletotrichum linicola) kod različitih sorti crvene deteline (trifolium pratense) u srbiji pored lucerke, crvena detelina (trifolium pratense l.) je najvažnija višegodišnja krmna leguminoza koja se gaji u srbiji. bogata je proteinima koji su važni za ishranu domaćih životinja. crvenu detelinu kao i mnoge leguminoze parazitiraju gljive iz roda colletotrichum, nanoseći značajne ekonomske štete u vidi smanjenja prinosa i kvaliteta sena. u ovom radu testirano je šesnaest sorti crvene deteline različitog geografskog porekla na otpornost prema izolatu coll-44 (c. linicola). osetljivost različitih sorti crvene deteline prema ispitivanom izolatu c. linicola određena je prema skali od 1-5. šest sorti wilo, repio, viglana, kolubara, k-39 and k-17 pokazalo je visok nivo otpornosti na testirani izolat c. linicola. na osnovu indeksa otpornosti najotpornijom prema izolatu c. linicola se pokazala američka sorta wilo sa indeksom otpornosti 1.6, dok je najniži indeks otpornosti kod srpske sorte k-39 iznosio 2.6. cilj ovog istraživanja bio je da se oceni stepen osetljivosti i tolerantnosti kod 16 sorti crvene deteline prema prouzrokovačima antraknoze, u ovom slučaju c. linicola. ključne reči: colletotrichum linicola, sorta, otpornost, trifolium pratense introduction red clover (trifolium pratense l.) is one of the most important forage crops in serbia. as such, it represents an essential element in the crop rotations. in recent years, it has been observed that the life span and quality of red clover hay decreased significantly, and, as a reason for this occurrence, fungal species of the genus colletotrichum (in particular, c. trifolii bain et essary, c. destructivum o’gara, c. coccodes (wallr.) hughes, c. truncatum (schw.) andrus and moore, c. dematium (pers.) grove and c. linicola pethybr. and laff) were cited (schubiger et al., 2004; jacob et al., 2010; vasić et al., 2014). anthracnose © 2020 vasić et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 115 decreases the vigour of the individual plants and thins out the plant population. during the summer and autumn, diseased plants start to appear in the red clover and alfalfa fields. these diseased plants have typical appearance. plants, in the advanced stages of the disease, develop deformations of the upper part of the stem. stem infection results in wilting and death of the upper portion of the stem and thus making the characteristic “shepherd’s crook” symptom (fig. 1). this is considered as the typical symptom of the disease. as the disease progresses, the infection spreads and passes from the stem to the root neck and the root area. crown and root infection is characterized by dry rot with a change in the color of the affected tissue to blue-black (vasić et al., 2016). damage caused by presence and development of pathogen agent cause the reduction of quantity and quality of fresh mass of 10% up to 30% depending on cultivar of red clover and alfalfa, species of pathogen, climatic and edaphic factors (stuteville and erwin, 1990; vasić, 2010b). the diseased plants can, thus, decay during one growing season or be severely weakened, often succumbing to frost in winter conditions (schubiger et al., 2003). since the growing importance of red clover crops in serbia, the aim of this experiment was to determine the susceptibility and tolerance level of different red clovers cultivars to anthracnose agents, in this case, c. linicola, and to find the right sources of resistance. material and methods in june-july, 2018, sixteen commercial cultivars of red clover were tested for resistance to the anthracnose causative agent c. linicola. the experiment was done in the greenhouse of the institute for forage crops in kruševac. commercial red clover cultivars of different origin were used for the experiment. cultivars were as following: k-9, k-17, k-27, k-32, k-38, k-39 (institute for forage crops, kruševac, serbia), una and kolubara (institute of field and vegetable crops, novi sad, serbia), diplomat (deutsche saatveredelung ag – dsv, lippstadt, germany), nigula (estonian crop research institute, estonia), longevo (istituto sperimentale per le colture foraggere, lodi, italy), viglana, manuela (research institute of plant production in piestany, slovakia) and wilo, repio and diana (us dairy forage research dairy farm, wisconsin, usa). one isolate from serbia, coll44, was used in this study and was identified as c. linicola originated from alfalfa from serbia, based on morphological, pathological and molecular traits. colonies of isolate c. linicola were light to dark olive green. in cultures on pda medium, acervuli were formed. conidia from acervuli were released in mucous masses that were orange to cream-pink in color (vasić et al., 2014). the sowing of the red clover seeds was done using plastic containers at a depth of 2 cm in sterile substrate in a greenhouse. the conidial suspension was prepared from cultures of the tested isolate. it was grown for ten days on pda medium at 25 °c in the dark. the concentration of spores was determined by hemocytometer by thoma and it was 4-6 x 104 spores/ml. inoculated red clover plants were 7 weeks old and they were scarred prior to inoculation by cutting and sprayed with a spore suspension. the experiment was conducted in five repetitions, 5 plants per genotype and red clover plants inoculated with sterile water used as a negative control. plants were categorized in 5 score classes, based on the anthracnose reaction using the 1 to 5 scale (1 = no stem lesions or only a few small water-soaked or black spots; 2 = stems with elongated black lesions but without acervuli; 3 = stems with long, wide, but non-gridling lesions, with acervuli present; 4 = large, coalescing and sporulating lesions which kill 116 fig. 1. “shepherd’s crook” symptom on red clover biologica nyssana ● 11 (2) december 2020: 115-119 vasić et al. ● resistance to anthracnose (colletotrichum linicola) on different cultivars of red clover (trifolium pratense) in serbia 117 upper part of seedling; 5 = seedling dead) (ivanović and ivanović, 2005). based on the results, the severity index was calculated according to the following formula: i severity index ntotal number of seedling n number of seedlings in class k number of categories k number of individual categories s sum of products according to ostazeski et al. (1969), where 1 and 2 are assigned to resistant plants, plants were scored individually, four weeks after inoculation. disease intensity or severity of infected plants was calculated using the severity index. when the severity index is used as a criterion, all cultivars are classified with severity index from 2.72 to 2.25 can be classified as resistant cultivars (ostazeski et al., 1979). results the studied cultivars showed a clear difference in sensitivity to the tested isolate of c. linicola. the number of surviving plants varied over a wide range. four weeks after the inoculation of the tested plants, primarily necrotic lesions occurred on the stem and there was slight twist of the top of the upper third of the stem. necrotic lesions did spread further throughout the plant and it led to the complete drying and decay of some plants. the intensity of disease development was evaluated using the scale of 1-5. the response of the tested red clover cultivars after inoculation with the tested c. linicola isolate as well as the severity index score are shown in tab. 1. in the conditions of this experiment, the most resistant was the american cultivar of red clover wilo, which had the severity index of 1.6 and 50% of resistant plants. furthermore, vinglana cultivar, with the severity index 2.4, and repio, with the severity index 2.6, showed high levels of resistance. also, serbian varieties k-39, k-17 and kolubara showed significant resistance, and their severity indices ranged from 2.6 to 2.8, while the percentage of resistant plants for all tested cultivars was 20. the most sensitive was cultivar diana, with the severity index 3.6 and 10% of the healthy plants, while cultivars k-27 and diplomat had severity indices 3.4 without healthy plants. discussion according to results of ostazeski et al. (1969), sensitive cultivars include cultivars with 10% of healthy plants, while resistant cultivars include cultivars with over 65% of healthy plants. vasić et al. (2015) tested thirteen (k-27, k-39, biologica nyssana ● 11 (2) december 2020: 115-119 vasić et al. ● resistance to anthracnose (colletotrichum linicola) on different cultivars of red clover (trifolium pratense) in serbia table 1. evaluation of sensitivity in commercial cultivars of red clovers (trifolium pratense l.) to isolate c. linicola (coll-44) cultivars plants categorised by ostazeski scale (%) severity index resistant plants (1+2%)1 2 3 4 5 k-9 0 20 10 20 0 3.0 20 k-17 0 20 20 10 0 2.8 20 k-27 0 0 30 20 0 3.4 0 k-32 0 10 30 10 0 3.0 10 k-38 0 0 30 20 0 3.4 0 k-39 0 20 30 0 0 2.6 20 diplomat 0 0 30 20 0 3.4 0 nigula 0 10 30 20 0 3.0 10 viglana 10 10 30 0 0 2.4 20 manuela 0 10 20 10 20 3.4 10 longevo 0 10 20 10 0 3.4 10 kolubara 0 20 20 10 0 2.8 20 una 0 10 30 10 0 3.0 10 wilo 20 30 0 0 0 1.6 50 repio 0 20 30 0 0 2.6 20 diana 0 10 10 20 0 3.6 10 control 118 k-32, k-38, una, kolubara, čiroku, lea, virgiana, valentine, wilo, repio and diana) commercial cultivars of red clover for resistance to the anthracnose causative agent c. destructivum. the reactions of the tested cultivars were different. the most resistant to c. destructivum was the cultivar wilo with severity index 1.9, while cultivar valentina showed the highest sensitivity to the tested isolate. despite the large number of studies, which indicated that resistance to anthracnose is controlled by major dominant or recessive genes and other resistance mechanisms, resistance to c. trifolii is not clear enough (schubiger et al., 2004). schubiger et al. (2003) found that the degree of resistance to colletotrichum trifolii was different in different cultivars of red clover. the anthracnose resistance of the red clover plants ranged from 3% to 52% (schubiger et al., 2004). according to boller et al. (2004), merula and pavo were red clover cultivars of the mattenklee type resistant to the causative agents of anthracnose, c. trifolii. according to jacob et al. (2010), the german cultivars of red clover showed a wide range of resistance to colletotrichum trifolii with plant survival from 29% to 87%. schubiger et al. (2004) examined twelve swiss cultivars of red clover (leisi, renova, ruettinova, milvus, formica, corvus, merula, pavo, temara, vanessa, larus, astur) considering the resistance to c. destructivum. in their research, they stated that none of the red clover cultivars were severely affected by c. destructivum. also, jacob et al. (2015) tested thirtyeight red clover cultivars listed in germany, for the resistance to c. trifolii and found that resistance of red clover to anthracnose was highly heritable. vasić et al. (2010b) reported that different cultivars of red clover (manuela, margot, k-39, k-32 and breed population l-50) show different levels of resistance depending on different c. trifolii isolates. the degree of resistance of the red clover cultivar depended on the virulence of the c. trifolii isolates. vasić et al. (2010a) showed that alfalfa clones had different resistance to c. trifolii. by comparing american and serbian cultivars, it can be noted that the american alfalfa cultivars had the highest resistance level (64.5% of plant survival). the most susceptible tested cultivar had the plant survival rate of 56.4%. conclusion it can be concluded, from the presented assessment, that red clover cultivars showed variations in their resistance response to c. linicola. six cultivars of wilo, repio, viglana, kolubara, k-39 and k-17 showed resistance to the tested isolate, and could be listed for cultivation under integrated production systems and used in the development of new resistant red clover cultivars. references boller, b., tanner, p., schubiger, f.x. 2004: merula und pavo: neue, ausdauernde mattenkleesorten. agrarforschung, 11(5): 162-167. ivanović, m., ivanović, d. 2005: bolesti voćaka i vinove loze i njihovo suzbijanje. poljoprivredni fakultet. beograd. jacob i., hartmann s., schubiger f.x., struck c. 2010: genetic diversity of red clover varieties listed in germany concerning the resistance to southern anthracnose. grassland science in europe, 15: 344346. jacob, i., hartmann, s., schubiger, f. x., struck, c. 2015: resistance screening of red clover cultivars to colletotrichum trifolii and improving the resistance level through recurrent selection. euphytica, 204: 303-310. ostazeski, s.a., barnes, d.k., hanson, c.h. 1969: laboratory selection of alfalfa for resistance to anthracnose, colletotrichum trifolii. crop science, 9: 351-354. ostazeski, s.a., elgin, j.h., jr., mcmurtray, j.e. 1979: occurrence of anthracnoses on formerly anthracnose-resistant “arc” alfalfa. plant disease report, 63: 734-736. stuteville d.l., erwin d.c. 1990: compendium of alfalfa diseases, second edition. the american phytopathological society st. paul, minnesota. schubiger f.x., streckeisen p., boller b. 2003: resistance to southern anthracnose (colletotrichum trifolii) in cultivars of red clover (trifolium pratense). czech journal of genetics and plant breeding, 39 (special issue): 309-312. schubiger, f.x., alconz, e., streckeisen, p., boller, b. 2004: resistance to southen anthracnoses (colletotrichum trifolii) in red clover (trifolium pratense). agrarforschung, 11(5): 168-173. vasić, t., radović, j., lugić, z., marković, j., jevtić, g., gajić, s. 2010a: occurrence of colletotrichum trifolii (bain et essary), the inducer of alfalfa anthracnose in serbia. in: c. huyghe (ed.): sustainable use of genetic diversity in forage and turf breeding. springer, 53: 369-374. vasić, t., lugić, z., anđelković, s., štrbanović, r., marković, j., gajić, s, anđelković, b. 2010b: the impact of colletotrichum trifolii isolates on resistance in different red clover cultivars. proceedings of the xii international symposium on forage crops of republic of serbia, 26-28. may, kruševac, serbia, biotechnology in animal husbandry, 26 (spec.issue), book 2: 51-56. biologica nyssana ● 11 (2) december 2020: 115-119 vasić et al. ● resistance to anthracnose (colletotrichum linicola) on different cultivars of red clover (trifolium pratense) in serbia vasić, t., bulajić, a., krnjaja, v., jevremović, d., živković, s., andjelković, b. 2014: first report of anthracnose on alfalfa caused by colletotrichum linicola in serbia. plant disease, 98, (9): 1276. vasić, t., lugić, z., terzić, d., milenković, j., marković, j., živković, s. 2015: the impact of colletotrichum destructivum on resistance in different red clover cultivars. book of proceedings/ sixth international scientific agricultural symposium “agrosym 2015”, 15th-18th october; mountain jahorina, bosnia and herzegovina, 972976. vasić, t., jevremović, d., andjelković, s., marković, j., zornić, v., babić, s., leposavić, a. 2016: morphological and molecular identification of a new alfalfa parasite-colletotrichum linicola in serbia. book of proceedings vii international scientific agriculture symposium “agrosym 2016” jahorina, october 06 09, pp. 1480-1485. 119 biologica nyssana ● 11 (2) december 2020: 115-119 vasić et al. ● resistance to anthracnose (colletotrichum linicola) on different cultivars of red clover (trifolium pratense) in serbia volatiles of quince fruit and leaf (cydonia oblonga mill.) from serbia biologica nyssana 7 (2)  december 2016: 145-149 veličković, d.t. et al.  volatiles of quince fruit and leaf… 145 original article received: 08 october 2016 revised: 30 october 2016 accepted: 24 november 2016 volatiles of quince fruit and leaf (cydonia oblonga mill.) from serbia dragan t. veličković1*, mihailo s. ristić2, nebojša p. milosavljević1, dejan n. davidović1, dragan m. milenović3, ana s. veličković4 1college of agriculture and food technology, prokuplje, serbia, 2institute for medicinal plant research "dr. josif pančić", belgrade, serebia 3"zdravlje-actavis" company, leskovac, serbia 4out-patient clinic leskovac health center vučje, koste stamenkovića bb, vučje, serbia * e-mail: dvelickovic7@ptt.rs abstract: veličković, d.t., ristić, m.s., milosavljević, n.p., davidović, d.n., milenović, d.m. veličković, a.s.: volatiles of quince fruit and leaf (cydonia oblonga mill.) from serbia. biologica nyssana, 7 (2), december 2016: 145-149. the subject of this study is the chemical composition of volatile fractions from the fruit and leaf of quince (cydonia oblonga mill.). dominant components were ethyl 2-methylbutanoate, (e,e)--farnesene, ethyl(2e,4z)-decadienoate, pentadecanol, -acoradienol, ethyl decanoate, ethyl octanoate, (e)-nerolidol, ethyl dodecanoate, 14-hydroxy-9-epi-(e)-caryophyllene, (2z,6e)-farnesol, -cedrene. volatile fraction of the fruit was also characterized by ethyl-(4z)-decenoate, ethyl 9-dodecenoate, ethyl hexanoate, ethyl nonanoate and cyclocitral, where in the leaf n-octanal, n-hexanol, n-nonanal and benzaldehyde were present. key words: quince fruit, quince leaf, volatiles, 2-methylbutanoate, ethyl-(2e,4z)-decadienoate apstrakt: veličković, d.t., ristić, m.s., milosavljević, n.p., davidović, d.n., milenović, d.m. veličković, a.s.: isparljive supstancije ploda i lista dunje (cydonia oblonga mill.) iz srbije. biologica nyssana, 7 (2), decembar 2016: 145-149. predmet ove studije je hemijski sastav isparljivih frakcija ploda i lista dunje (cydonia oblonga mill.). dominantne komponente su bile etil 2-metilbutanoat, (e,e)--farnezen, etil-(2e,4z)-dekadienoat, pentadekanol, -akoradienol, etil dekanoat, etil oktanoat, (e)-nerolidol, etil dodekanoat, 14-hidroksi-9-epi-(e)kariofilen, (2z,6e)-farnezol, -cedren. isparljivu frakciju ploda karakterišu etil-(4z)-decenoat, etil 9dodecenoat, etil heksanoat, etil nonanoat i -ciklocitral, dok su u listu bili prisutni n-oktanal, n-heksanol, nnonanal i benzaldehid. key words: plod dunje, list dunje, isparljive supstancije, 2-metilbutanoat, etil-(2e,4z)-dekadienoat 7 (2) • december 2016: 145-149 12th sfses • 16-19 june 2016, kopaonik mt doi: 10.5281/zenodo.200413 biologica nyssana 7 (2)  december 2016: 145-149 veličković, d.t. et al.  volatiles of quince fruit and leaf… 146 introduction quince fruit and its products (jam, marmalade, compote, juice, quince-brandy) are highly appreciated in the market. some studies have shown that the quince is an excellent natural source of polyphenols and flavonoids (o l i v e i r a et al., 2007; s i l v a et al., 2008), giving it a very important role and position in traditional medicine. the fruit is used in the treatment of dysentery, the leaf can be used as a sedative and seed as an emulsifying agent in hair-fixing lotions (e l a n d s e n & m a g n e y , 1992; b e r g m a n et al., 1996). in addition to fructose, glucose and sorbitol, malic, tartaric, citric, phytic and quinic acids, and crude fiber (o l i v e i r a et al., 2008; r o d r i g u e z g u i s a d o et al., 2009; s z y c h o w s k i et al., 2014), as main phenolic components in the extract of quince leaf were found 5-o-caffeoylquinic acid and 3-o-caffeoylquinic acid (c o s t a et al., 2009). latest studies show that c. oblonga is a rich source of chlorogenic acids, proanthocyanidins and flavonol c and o-glycosides (k a r a r et al., 2014), as well as the volatile components (s c h m a r r & b e r n h a r d t , 2010). in the seeds of quince linoleic, palmitic, oleic, stearic, eicosanoic, palmitoleic and arachidonic acids were identified (d a n e s h v a n d et al., 2012; s z y c h o w s k i et al., 2014). among a large number of metabolites isolated from the quince fruit skin, a few have been identified and tested on radicalscavenging and antioxidant activities (a l e s i a n i et al., 2010). a l s n a f i (2016) states antioxidant potential, immunological, antiallergic, cardiovascular, reproductive, dermatological, anticancer, antiinflammatory, antidiabetic, protective effects, as well as effect on respiratory smooth muscle contraction. the main goal of this study was to determine quince volatiles of serbian origin, and compare our results with accessible publications. material and methods plant material for this study, it was used the quince cydonia oblonga mill. var. leskovačka, from locality džigolj, municipality prokuplje, central serbia (j o v a n o v i ć , 1972). the authors are very grateful to prof. jugoslav trajković (college of agriculture and food technology) for botanical identification of the plant. ripe fruits were collected in october 2013. isolation of volatiles (vs) volatiles were isolated from small pieces of quince fruit and cut fresh leaves. the materials were put into a round-bottom flask of 2 l, and hydrodistillation process was performed using a clevenger-type apparatus (ph. eur. 4, 2002), with hydromodule 1 : 10. the vs were taken up in cyclohexane (1 ml), which was inserted in a graduated tube of the apparatus. solutions were stored for a few hours at 4 °c in the dark until being analyzed by gc. identification of volatiles (vs) analytical gas chromatography (gc-fid) and combination of gas chromatography and mass spectrometry (gc-ms) were used for the characterization of volatiles trapped in graduated tube of the clevenger type apparatus used. gc-fid analysis was carried out on an agilent technologies, model 7890a gas chromatograph, equipped with split-splitless injector and automatic liquid sampler (als), attached to hp-5ms column (30 m × 320 m, 0.25 m film thickness) and fitted to flame ionization detector (fid). carrier gas flow rate (h2) was 1 ml/min, split ratio 1:30, injector temperature was 250 °c, detector temperature 300 °c, while column temperature was linearly programmed from 40 °c to 260 °c (at a rate of 4 °c/min), and then kept isothermally at 260 °c for 15 min. sample solutions were prepared in cyclohexane (in conc. of approximately 10 g/l), and injected by als (2 l). the same analytical conditions as those mentioned for gc-fid were employed for gc-ms analysis, along with column hp-5ms (30 m × 250 m, 0.25 m film thickness), using hp g 1800c series ii gcd system [hewlett-packard, palo alto, ca (usa)]. helium was used as a carrier gas. transfer line was heated at 260 °c. mass spectra were acquired in ei mode (70 ev) in m/z range 40450. the amounts of 0.2 l of sample solutions were injected. constituents were identified by comparison of their mass spectra with those stored in ms libraries (wiley 275, nist05 and adams 2007), using different search engines (pbm, nist 2.0), as well as using calibrated amdis (ver. 2.64) for determination and comparison of retention indices (a d a m s , 2007). similarly, quantification of present constituents was achieved by normalization method, based upon the area percent report obtained by gc-fid. statistics has been covered by fid specification (results with a range of deviation for the level 1%). biologica nyssana 7 (2)  december 2016: 145-149 veličković, d.t. et al.  volatiles of quince fruit and leaf… 147 table 1. volatiles of different parts of quince (%, w/w) constituents kil fruit leaf ethyl 2-methylbutanoate 864 1.4 23.4 n-hexanol 863 0.3 1.3 2-methylbutyl acetate 875 tr. 2-heptanol 896 0.7 0.7 n.i. 0.4 n.i. 0.3 ethyl pentanoate (ethyl valerate) 901 tr. cumene 924 tr. 0.7 ethyl tiglate 929 0.5 0.2 benzaldehyde 952 1.0 6-methyl-5-hepten-2-one 981 0.2 tr. ethyl hexanoate (ethyl caproate) 997 2.0 tr. n-octanal 998 tr. 2.0 (2e,4e)-heptadienal 1005 tr. hexyl acetate 1007 tr. -phellandrene 1025 tr. 1,8-cineole 1026 tr. ethyl 2-hexenoate 1038 0.1 -terpinene 1054 tr. n-octanol 1063 0.1 cis-linalool oxide 1067 tr. fenchone 1083 tr. linalool 1095 tr. ethyl heptanoate (ethyl enanthate) 1097 0.8 tr. 4-ethyl-2-hexynal n.a. 0.6 n-nonanal 1100 tr. 1.1 (z)-isocitral 1160 0.9 0.6 ethyl 3-(methylthio)-(z)2-propenoate n.a. 0.3 camphor 1141 tr. ethyl-(4e)-octenoate 1184 0.2 -terpineol 1186 tr. ethyl octanoate (ethyl caprilate) 1196 4.3 1.9 n-decanal 1201 0.3 -cyclocitral 1217 1.2 0.6 ethyl-(2e)-octenoate 1245 0.5 ionene 1258 0.6 0.3 vitispirane 1272 1.0 0.6 ethyl nonanoate (ethyl pelargonate) 1294 1.4 0.7 (2e,4e)-decadienal 1315 0.3 n.i. 0.5 n.i. 0.1 ethyl-(4z)-decenoate 1380 2.7 1.9 ethyl-(4e)-decenoate 1388 0.5 ethyl decanoate (ethyl caprinate) 1395 6.3 3.9 (e)--damascone 1413 0.9 0.6 -cedrene 1421 2.5 1.5 constituents kil fruit leaf (e)--farnesene 1454 tr. ethyl-(2e,4z)decadienoate 1467 8.3 4.2 (e)--ionone 1487 tr. tr. ethyl undecanoate 1498 0.7 0.7 (e,e)--farnesene 1505 18.4 16.1 -dehydro-arhimachalene 1516 0.6 megastigmatrienone ** n.a. 0.2 (e)-nerolidol 1561 4.1 2.7 ethyl 9-dodecenoate n.a. 2.2 ethyl dodecanoate (ethyl laurate) 1594 3.2 3.8 -atlantol 1608 tr. n.i. 3.0 1.7 n.i. 0.4 himachalol 1652 0.5 0.3 lyral 1665 tr. 14-hydroxy-9-epi-(e)caryophyllene 1670 3.8 2.3 n.i. 0.4 n.i. 0.2 (2z,6z)-farnesol 1698 0.5 (2e,6z)-farnesal 1713 0.6 0.5 (2z,6e)-farnesol 1722 3.2 1.7 ethyl 3hydroxydodecanoate n.a. 0.2 -acoradienol 1762 7.6 5.1 pentadecanol 1773 7.8 5.2 n.i. 0.1 ethyl tetradecanoate (ethyl myristate) 1795 tr. hexahydrofarnesyl acetone 1838 0.5 n-hexadecanol 1874 0.3 nonadecane 1900 0.3 methyl hexadecanoate (methyl palmitate) 1922 0.5 n.i. 0.2 ambrettolide n.a. 0.2 ethyl hexadecanoate (ethyl palmitate) 1992 tr. eicosane 2000 tr. 0.5 octadecanol 2077 0.1 heneicosane 2100 0.3 1.0 ethyl oleate (ethyl (z)-9octadecenoate) 2179 0.7 0.7 docosane 2200 0.2 n.i. 0.8 tricosane 2300 0.2 0.9 tetracosane 2400 0.2 1.1 n.i. 0.6 pentacosane 2500 0.3 1.4 hexacosane 2600 0.2 1.2 heptacosane 2700 0.3 2.4 % w/w mass percent defined by peak area percent determined by integration (gc-fid) biologica nyssana 7 (2)  december 2016: 145-149 veličković, d.t. et al.  volatiles of quince fruit and leaf… 148 kil kovats (retention) index (adams, 2007) n.a. not available n.i. not identified tr. traces (< 0.1%) ** tentative identification results and discussion table 1. shows a comparative overview of volatiles in the quince fruit and leaf, where 94.8% and 96.2% of the components were identified from the total mass, respectively. components which dominate in the quince fruit and leaf are (e,e)--farnesene (18.4% and 16.1%), ethyl-(2e,4z)-decadienoate (8.3% and 4.2%), pentadecanol (7.8% and 5.2%), -acoradienol (7.6% and 5.1%), ethyl decanoate (6.3% and 3.9%), ethyl octanoate (4.3% and 1.9%), (e)-nerolidol (4.1% and 2.7%) and ethyl dodecanoate (3.2% and 3.8%). the above components are more present in the fruit, with the exception of ethyl dodecanoate. one of the essential components of the leaf is ethyl 2methylbutanoate with 23.4%, which is present in the fruit with only 1.4%. some more components that characterize the volatile fraction of the fruit are 14-hydroxy-9-epi(e)-caryophyllene (3.8%), (2z,6e)-farnesol (3.2%), ethyl-(4z)-decenoate (2.7%), -cedrene (2.5%), ethyl 9-dodecenoate (2.2%), ethyl hexanoate (2.0%), ethyl nonanoate (1.4%) and -cyclocitral (1.2%). the leaf is characterized by 14-hydroxy-9-epi-(e)caryophyllene (2.3%), n-octanal (2.0%), (2z,6e)farnesol (1.7%), -cedrene (1.5%), n-hexanol (1.3%), n-nonanal (1.1%), benzaldehyde (1.0%), as well as the compounds of the cosane group. the presence of fatty acid esters is obvious, especially in the quince fruit: ethyl decanoate (6.3%) > ethyl octanoate (4.3%) > ethyl dodecanoate (3.2%) > ethyl hexanoate (2.0%) > ethyl nonanoate (1.4%) > ethyl heptanoate (0.8%) > ethyl undecanoate (0.7%) > ethyl oleate (0.7%). ethyl pentanoate, ethyl tetradecanoate and ethyl hexadecanoate occur only in traces. s c h m a r r & b e r n h a r d t (2010) identified esters in the quince fruit (ethyl butanoate, ethyl 2methylbutanoate, 2-methylbutyl acetate, butyl acetate, hexyl acetate), alcohols (butanol, 2methylbutanol, hexanol, (e)-2-hexenol), carbonyls (hexanal, (z)-3-hexenal, (e)-2-octenal, (e/z)-2nonenal, 1-octen-3-one), and terpenes ((e)-damascenone, limonene, farnesene, linalool, eugenol). the authors assume that (e,z)and (e,e)ethyl 2,4-decadienoate could be characteristic esters responsible for flavor. s o u s a et al. (2007) identified trans-9-amino-8-hydroxy-2,7-dimethylnona-2,4-dienoic acid glucopyranosyl ester, homomonoterpenic compound which could be chemical marker. also, main volatiles of quince fruit were trans--farnesene, furfural, megastigma-4,6,8-trien3-one, trans-marmelo lactone and cis-marmelo lactone (t s u n e y a et al., 1983). the results of u m a n o et al. (1986) showed that main volatile constituents of quince fruit peel were ethyl propionate, ethyl acetate, ethyl dodecanoate, ethyl octanoate, ethanol, 2-methyl-1-propanol. the newest chemical analysis of c. oblonga showed that it contained phenolics, pectin, essential and volatile oils. the analysis of the leaves essential oils (a l s n a f i , 2016) showed that it contained aromatic aldehyde, fatty acid, oxygenated monoterpene, and sesquiterpene hydrocarbon (benzaldehyde, hexadecanoic acid, linalool, (e)-β-ionone, germacrene d). we have also identified some of those substances, especially regarding the ethyl 2methylbutanoate, ethyl-(2e,4z)-decadienoate, (e,e)-farnesene, ethyl octanoate, and ethyl dodecanoate. conclusion the presented results show that the quince fruit and leaf are an extraordinary source of substances, which from the chemical standpoint, belong to different groups of compounds. quince fruit was rich in fatty acid esters. among the volatiles, ethyl 2methylbutanoate and ethyl-(2e,4z)-decadienoate were the most abundant which could be responsible for flavor. acknowledgements. the ministry of education and science of the republic of serbia supported this study through the projects no. 172047 and 173021. references adams, r.p. 2007: identification of essential oil components by gas chromatography / mass spectrometry. 4th ed. allured publishing corp., carol stream, il 60188 usa. alesiani, d., canini, a., d’abrosca, b., dellagreca, m., fiorentino, a., mastellone, c., monaco, p., pacifico, s. 2010: antioxidant and antiproliferative activities of phytochemicals from quince (cydonia vulgaris) peels. food chemistry, 118: 199-207. al-snafi, a. e. 2016: the medical importance of cydonia oblonga a review. iosr journal of pharmacy, 6 (6): 87-99. bergman, r., afifi, a.k., heidgerip, p.m. 1996: text of histology. 9th ed. saunders wb, montréal, 159 p. biologica nyssana 7 (2)  december 2016: 145-149 veličković, d.t. et al.  volatiles of quince fruit and leaf… 149 costa, r.m., magalhães, a.s., pereira, j.a., andrade, p.b., valentão, p., carvalho, m., silva, b.m. 2009: evaluation of free radical-scavenging and antihemolytic activities of quince (cydonia oblonga) leaf: a comparative study with green tea (camellia sinensis). food and chemical toxicology, 47: 860-865. daneshvand, b., ara, k.m., raofie, f. 2012: comparison of supercritical fluid extraction and ultrasound-assisted extraction of fatty acids from quince (cydonia oblonga miller) seed using response surface methodology and central composite design. journal of chromatography a, 1252: 1-7. elandsen, s., magney, j. 1992: color atlas of histology. 6th ed. mosby, toronto, 104 p. european pharmacopoeia, 2002. 4th edn., strasbourg. jovanović, b. 1972. genus cydonia mill. in: josifović, m. (ed.), flora sr srbije. iv: 130-131, sanu, beograd. karar, e.g.m., pletzer, d., jaiswal, r., weingart, h., kuhnert, n. 2014: identification, characterization, isolation and activity against escherichia coli of quince (cydonia oblonga) fruit polyphenols. food research international, 65: 121-129. oliveira, a.p., pereira, j.a., andrade, p.b., valentão, p., seabra, r.m., silva, b.m. 2007: phenolic profile of cydonia oblonga miller leaf. journal of agricultural and food chemistry, 55: 7926-7930. oliveira, a.p., pereira, j.a., andrade, p.b., valentão, p., seabra, r.m., silva, b.m. 2008: organic acids composition of cydonia oblonga miller leaf. food chemistry, 111: 393-399. rodriguez-guisado, i., hernández, f., melgarejo, p., legua, p., martínez, r., martínez, j.j. 2009: chemical, morphological and organoleptical characterisation of five spanish quince tree clones (cydonia oblonga miller). scientia horticulturae, 122: 491-496. schmarr, h.g., bernhardt, j. 2010: profiling analysis of volatile compounds from fruits using comprehensive two-dimensional gas chromatography and image processing techniques. journal of chromatography a, 1217: 565-574. silva, b.m., valentão, p., seabra, r.m., andrade, p.b. 2008. quince (cydonia oblonga miller): an interesting dietary source of bioactive compounds. in: papadopoulos, k.n. (ed.), food chemistry research developments: 243-266, nova science publishers, inc., new york. sousa, c., silva, m.b., andrade, b.p., valentão, p., silva, a., ferreres, f., seabra, m.r., ferreira, a.m. 2007: homo-monoterpenic compounds as chemical markers for cydonia oblonga miller. food chemistry, 100: 331-338. szychowski, j.p., munera-picazo, s., szumny, a., carbonell-barrachina, a.á., hernández, f. 2014: quality parameters, bio-compounds, antioxidant activity and sensory attributes of spanish quinces (cydonia oblonga miller). scientia horticulturae, 165: 163-170. tsuneya, t., ishihara, m., shiota, h., shiga, m. 1983: volatile components of quince fruit (cydonia oblonga mill.). agricultural and biological chemistry, 47 (11): 2495-2502. umano, k., shoji, a., hagi, y., shibamoto, t. 1986: volatile constituents of peel of quince fruit, cydonia oblonga miller. journal of agricultural and food chemistry, 34 (4): 593-596. novica ranđelović: diary of a collaboration. biologica nyssana, 8 (1) 5 essay received: 17 january 2017 novica ranđelović: diary of a collaboration david a. hill budapest, hungary * e-mail: futured@hu.inter.net in summer 1980 i was appointed as the british council lektor at the primary teacher training college (viša pedagoška škola) in prizren, kosovo. i had specifically chosen that particular lektor post out of various that were offered me (priština, niš, skopje, belgrade) because of what i already knew about the natural history of the area and the serbian monasteries. i had started studying botany at the age of 12, written my first article and given my first public lecture aged 16, and had articles published in watsonia and the alpine garden society journal by the age of 27. and as well as 25 years in the uk, i has also spent 2 years in northern italy, honing my alpine and mediterranean botanical skills. thus when i arrived in prizren i was ready for a new experience with the balkan flora. of course, i was fairly badly equipped for this in terms of botanical field guides. i had polunin’s flowers of europe, polunin & everard’s trees & bushes of europe, and various uk, northern european and mediterranean guides, though this was fortunately soon augmented by polunin’s flowers of greece & the balkans, which came out in 1980, and i bought when home in the uk in summer 1981. so i did the best i could. i should say that as well as flowers, i was also studying any other wildlife that came my way, especially birds and butterflies, for which i had adequate books for the region. my first finds of local crocuses came on 17.03.81, when i discovered a lilac-flowered species with a netted corm, which i misidentified as crocus reticulatus. polunin (1980) told me that crocus dalmaticus, which it later turned out to be, was in the velebit mountains, so i didn’t consider it. this turned out to be an important find, extending the known range of the species considerably. i also found it at nearby landovica. and so i trawled the prizren area, kosovo, the mountains around finding many, many new and interesting flowers, besides the first crocus. i also found crocus chrysanthus on dulje between prizren and priština in spring 1982. but then i left prizren and moved to niš university to be british council lektor there, starting in september 1982. i was by this time living with a serbian lektor in english, and it was she who found novica ranđelovic for me. she was travelling on a bus between priština and niš in february 1983, when she overheard two gentlemen talking about flowers. she interrupted them and explained my situation – british amateur botanist, no contacts with local botanists, could they help? and novica – one of the gentlemen! – did. and so on 25.02.83, i drove down to his home in doljevac to meet novica, show him some of my botanical work, and discuss flowers with him, not least crocuses, in which we soon found we were both very interested. he told me about the jelašnica and sičevo gorges west of niš, and i visited both on the next two days, finding crocus adami in flower as he’d suggested. then on 11.03.83, novica and i went out into the field for the first time together. he took me to seličevica to see crocus alexandrii, amongst other things. it was the start of a long, happy and fruitful period of collaborative botanical work. i was very pleased when novica spoke to my partner 8 (1) • september 2017: 05-06 doi: 10.5281/zenodo.962791 biologica nyssana 8 (1)  september 2017: 05-06 hill, d.a.  novica ranđelović: diary of a collaboration 6 after that first meeting and said ‘i thought you said he was an amateur – he really knows a lot!’ and so the idea gradually took shape that we would work to revise the information on the genus crocus in serbia, but also do lots of other botany together outside the spring and autumn crocusflowering seasons. this list will give you an idea of the intensity of our field work: 1983: 11.03: seličevica; 13.03: rača; 24.04: rujan planina / pohor pčinjski; 15.05 stolovi; 29.05: ostrozub; 05.06 suva planina; 15.10: rujan planina; 1984: 03.03: vučje; 18.03: bujanovacpreševo; 24.03: trgovište; 01.04: bujanovacpreševo; 15.04: vlasinsko jezero; 03.06: preševo; 10.06: basara; 04.07: vlasinsko jezero; 20.10: trgovište / radovnica; 1985: 17.03: velika kopašnica / velika grabovnica / vučje; 24.03: prosek / sićevo / staničenje; 03.04: rujan planina; 06.04: trnava; 27.04: subotinac; 03.05: radovnica / novo selo; 19.05: vidlič / basara; 08.06: svrljig; 14.06: subotinac; 22.09: subotinac; 1986: 08.03: konjuh; 15.03: preševo / rujan planina / bujanovac / kopašnica; 22.03: preševo / katlanovo / prilep / pletvar / belavodna / prisad; 30.03: skopje: kitka / skopska crna gora; 02.04: staničenje; 05.04: vrska čuka; 09.04: subotinac; 12.04: vlasotinsko jezero; 23.04: šar planina (stojkova kuća / prevalac) / prizren / landovica / beli drim gorge / pojata / štimlje. my involvement in the fieldwork stopped then, because in august 1986 my six year limit of time in yugoslavia was up, and i moved to milan, italy to work at the british council there. i returned to serbia each holiday up until the wars began in 1990, and so met novica from time to time. the last time we met face-to-face was on 26.08.89. however, we kept in close touch, because from december 1988 we were working on our book the genus crocus l. in serbia which was published by sanu, belgrade which i finally got a copy of in october 1990. the fieldwork listed above was often done in conjunction with other botanists, most frequently spas sotirov and vlastimir stamenković. we drove everywhere either in novica’s yellow citroen dyane, or my red renault 4tl; in fact, we often used my car because in those days petrol was issued using coupons, and locals could only have so many per month, while foreigners could buy as many as they wanted (at a higher rate, of course!)… so i always had plenty. many of the places listed above were visited primarily to look for crocuses, and repeat visits were to check on species we knew about. others were general botanizing expeditions aimed at deepening knowledge about the flora of southern parts of serbia. the various visits to subotinac were for a project on the flora of oil shale, which i presented at a symposium in belgrade in june 1986. a number of articles were published in different places describing our finds. of course, i have not listed the numerous meetings and phone calls that novica and i had to talk over our finds, examine material, check the books, especially with reference to the new species we found for science, crocus rujanensis randjelović et hill, first seen on 18.03.84. so who was this novica randjelović that i shared so much time, so many car journeys, so much foot-slogging over rough terrain, so many cups of coffee, and so much discussion with over a period of three years? first of all, he was a kind and generous man, someone who was able to accept this younger, enthusiastic foreigner into his home and make him feel welcome, and that in a time when foreigners were still widely treated with suspicion. secondly, he was someone who wore his knowledge lightly and with humility – he never wanted to push what he knew at you to show it off; rather, it was there as a constant resource. thirdly, he was endlessly curious and enthusiastic, always wanting to do new projects, discover new plants in different areas. i appreciated all of these characteristics greatly. we had a lot of fun on all those trips as well as doing a lot of serious work, too. i regret that things ended as they did. the wars came, and i never got back to niš. however, the bond i feel to novica has remained a deep and important part of me. i salute him and thank him on his 80th birthday. may there be many more. janmohammadi et al. 2025, biologica nyssana 16(2) 16 (2) december 2025: doi: 10.46793/biolnyss.16.2.7j morphophysiological response of sunflower (helianthus annuus) to various farmyard manure and irrigation frequency original article mohsen janmohammadi university of maragheh, faculty of agriculture, department of plant production and genetics, maragheh, iran mjanmohammadi@maragheh.ac.ir (corresponding author) asghar ebadi-segherloo moghan college of agriculture and natural resources, university of mohaghegh ardabili, ardabil, 1136756199, iran naser sabaghnia university of maragheh, faculty of agriculture, department of plant production and genetics, maragheh, iran mehdi mohebodini department of horticultural science, faculty of agricultural science and natural resources, university of mohaghegh ardabili, ardabil, iran received: june 14, 2025 revised: july 03, 2025 accepted: july 24, 2025 abstract: a field trial was set up to evaluate the effects of different irrigation levels (i1: rainfed and no irrigation, i2: 2 times supplementary irrigation at head development and seed setting stages, i3: 3 times irrigation at stalking, heading, and seed filling stages, i4: full irrigation) and the use of various types of farmyard manure (m0: no manure application, m1: cow manure, m2: sheep manure, m4: poultry manure) on growth, yield, and oil content of sunflower seeds. the largest heads were produced using poultry and sheep manure under full irrigation and i3 conditions. the highest lateral canopy growth was achieved with the use of i4+m4. the highest percentage of oil was obtained under conditions i4 and i3, using m1, m4 and m2, respectively. reducing the frequency of irrigation to three times, along with the use of farmyard manure, resulted in acceptable economic performance while reducing water consumption. key words: head diameter, empty achenes, oilseed yield, vegetative growth, water saving, water supply apstrakt: morfo-fiziološki odgovor suncokreta (helianthus annuus) na različito stajsko đubrivo i učestalost navodnjavanja eksperiment je postavljen na terenu, sa ciljem da se ispitaju efekti različitih nivoa navodnjavanja (i1: bez navodnjavanja, samo kiša; i2: dodatno navodnjavanje dva puta, u fazama formiranja glavice i zametanja semena; i3: navodnjavanje tri puta, u fazama porasta stabljike, formiranja glavice i semena; i4: potpuno navodnjavanje) i upotrebe različitih vrsta stajnjaka (m0: bez stajnjaka, m1: kravlji stajnjak, m2: ovčiji stajnjak, m4: živinski stajnjak) na rast, prinos i sadržaj ulja u semenu suncokreta. najveće glavice dobijene su primenom živinskog i ovčijeg stajnjaka, pri potpunom navodnjavanju i uslovima u tretmanu i3. najveći bočni porast lisne mase postignut je kombinacijom i4+m4. najveći procenat ulja dobijen je pri uslovima i4 i i3, korišćenjem m1, m4 i m2, redom. smanjenje učestalosti navodnjavanja na tri puta, uz primenu stajnjaka, dovelo je do ekonomski prihvatljivih rezultata, uz istovremeno smanjenje potrošnje vode. ključne reči: prečnik glavice, prazne ahenije, prinos uljarica, vegetativni rast, ušteda vode, snabdevanje vodom introduction the population of the world is growing rapidly, and the low amount of vegetable oil produced in most developing countries will pose a serious challenge to food security (afzal et al., 2022). therefore, the cultivation of oilseed crops is of great importance. sunflower (helianthus annuus l.) is one of the most important oilseed crops and is relatively adaptable to semi-arid conditions. it has been cultivated in eastern north america by indigenous people since about 4000 bc (adeleke & babalola, 2020). sunflower achene typically contains about 20% protein and 50% fat. the major fatty acids are linoleic acid (65%) and oleic acid (25%). in recent decades, breeding and selection processes for this plant have intensified, and relatively drought-tolerant cultivars adapted to the semi-arid mediterranean regions have been released. sunflower seeds have high nutritional value and contain about 45% oil, more than 30% protein, and some important elements in the lipid structure, which creates a valuable nutritional © 2025 janmohammadi et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. composition (petraru et al., 2021). although sunflowers can tolerate water shortage stress to some extent, the impact of global warming on the intensification of climate change in arid and semi-arid regions has caused the distribution of precipitation to change drastically and the total annual precipitation to decrease. the most important sunflower-producing countries are russia with 9.9×106 ha of cultivated area and 18.1×106 t of achenes production, ukraine with 5.2×106 ha of cultivated area and 12.7×106 t of achenes production, argentina with 2.4×106 ha of cultivated area and about 5 million t of achenes production, china with 1×106 ha of cultivated area and 3×106 t of achenes production, and romania with 1.1×106 ha of cultivated area and about 2×106 t of harvested achenes (fao, 2023). the area under sunflower cultivation in iran is 62,000 ha, and the amount of harvested sunflower seeds is 65,000 t (fao, 2023). the amount of sunflower seed imports in iran has exceeded 99,000 t, worth 126,000 $, which places it in sixteenth place in the list of global sunflower seed importers. considering this amount of import, assuming a yield of at least 2 t per ha, 50,000 ha of sunflower land are needed to compensate for the quantity of imports. therefore, it is necessary to increase production through improving agronomic methods, modifying soil conditions, and applying climate-smart methods. the low rainfall in semi-arid regions has caused the soils in these areas to have relatively little development and evolution. the soils in these areas have low vegetation cover, poor structure, very little residue return, quick degradation of organic matter, and high ph (naorem et al., 2023). likewise, low soil moisture and low soil retention capacity are among the most important factors limiting production. however, it seems that the use of some climatefriendly and eco-friendly management practices, such as the use of farmyard manure, can improve soil conditions for sunflower production (derpsch et al., 2024). given the development of the livestock industry alongside agriculture and the production of a significant volume of farmyard manure, this cheap and accessible input can play a role in improving soil conditions and mitigating the effects of global warming (nogués et al., 2023). farmyard manure application may increase the soil organic matter (som) and improve structural properties and water permeability. the use of organic fertilizers can reduce soil compaction and increase cation exchange capacity. the use of farmyard manures adjusts the soil ph, which can accelerate the release of nutrients in the rhizosphere (ferreira et al., 2022). adjusting irrigation regularity in h. annus is critical for improving the water use efficiency and achene yield. while irrigation during critical stages of plant development can lead to higher yields, it may also increase water use efficiency due to decreased water loss through evaporation and drainage. although some researchers have investigated the effect of applying livestock manures to sunflower fields in semi-arid climates (maia filho et al., 2013; vilvert et al., 2023; mokgolo et al., 2024), there is still no comprehensive information on how these fertilizers affect different moisture regimes and the differences between livestock manures. this is important because the diet of small livestock (like sheep and goats), large livestock (like cattle), and poultry (like chickens and turkeys) differs significantly due to their digestive systems, nutritional needs, and production goals. furthermore, it seems that the differences in the feeding habits of small, large livestock, and poultry, as well as their different digestive systems, result in the production of different qualities of manure. under conditions where rainfall distribution is symmetrical and normal during the season, sunflowers can produce economically without irrigation and in a rainfed manner. however, considering the changes in rainfall patterns caused by global warming, the use of supplementary irrigation techniques seems necessary. supplementary irrigation is a method of water management that, under rainfed conditions, reduces the amount of rainfall by providing multiple irrigations during key growth stages, reducing severe yield loss and improving water efficiency compared to full irrigation methods. garcíalópez et al. (2016) reported that the use of supplementary irrigation in semi-arid regions significantly increased sunflower yield and also increased nitrogen fertilizer use efficiency. due to the decrease in rainfall at the end of the season and coinciding with the reproductive stages of sunflower in semi-arid mediterranean regions, supplementary irrigation before anthesis up to flowering significantly increased yield compared to rainfed conditions (giannini et al., 2022). this experiment aimed to evaluate the effect of different supplementary irrigation regimes and the application of different farmyard manures on sunflower performance in northwest iran. materials and methods site description this field experiment was conducted during the 2023-2024 agricultural year in a private research farm in the kharajoo region, northwest iran, east azerbaijan. the site was located at longitude 46° 55’ e and latitude 37° 33’ n, at 1716 m a.s.l. the total annual precipitation in the region was 392 mm, and the mean annual minimum (tmin) biologica nyssana ● 16 (2) december 2025: janmohammadi et al. ● morphophysiological response of sunflower (helianthus annuus) to various farmyard manure and irrigation frequency biologica nyssana ● 16 (2) december 2025: janmohammadi et al. ● morphophysiological response of sunflower (helianthus annuus) to various farmyard manure and irrigation frequency and maximum temperatures (tmax) were 5 °c and 26 °c, respectively, making it a cold semiarid climate, often designated bsk in the köppen climate classification. monthly precipitation and air temperature conditions are shown in fig. 1. the soil texture was silty loam, and soil analysis is shown in tab. 1. experimental treatments a field trial was designed to estimate the effect of applying different livestock manures under different irrigation regimes. the arrangement and allocation of treatments to plots was based on a threereplicated split plot with based on rcbd. the main plots with dimensions of 19×4 m were allocated to 4 irrigation regimes. irrigation levels were: 1 rainfed (no irrigation) and rainfall-dependent growth, 2 two supplementary irrigations during advanced reproductive stages including the stage of pod development and seed setting (200 mm), 3 three supplementary irrigations during the vegetative (stem elongation) and advanced reproductive stages (270 mm), 4 full irrigation (530 mm). subplots with an area of 4×4 were allocated to different types of livestock manure. subfactors at four levels included 1 no manure application, 2 the use of 10 t ha-1 of cow manure collected from a beef calf farm before planting, 3 the use of 10 t ha-1 of fig. 1. precipitation and air temperature trends in the studied area during the experimental year table 1. the status of soil available elements and some chemical components of livestock manures ph ec oc% tn% p k c/n ca fe zn mn soil 7.74 2.51 0.71 0.09 10.217 215 1.69 37.29 1.242 3.45 0.97 cow fym 6.61 4.62 28.37 1.87 1.53 192 14.32 193.7 142.62 128 210.3 sheep fym 6.52 5.37 31.92 2.69 1.31 342 24.60 268.1 664 91.7 301.5 poultry manure 6.43 3.67 39.41 3.42 2.74 436 31.28 318 410.2 194.6 261.4 the element concentration is mg kg-1. ec: electrical conductivity (ds m-1), oc: organic carbon, tn: total nitrogen, soil ph in h2o refers to the measurement of soil acidity or alkalinity in a water solution, fym: farm yard mannure biologica nyssana ● 16 (2) december 2025: janmohammadi et al. ● morphophysiological response of sunflower (helianthus annuus) to various farmyard manure and irrigation frequency sheep manure collected from a sheep overnight stable, 4 application of 10 t ha-1 of poultry manure collected from a broiler farm. a one-meter nonplanting margin was considered between the main and sub-plots to prevent moisture leakage and nutrient leaching. the nutritional value of manures and some of their natural characteristics are briefly summarised in tab. 1. seed planting the field was under rainfed wheat cultivation in the previous crop year. after the wheat harvest in july, the soil was plowed with 4-bottom share moldboard plows. in november, after determining the boundaries of the main and subplots, different types of manure were applied to the soil surface according to the area of the subplots and mixed with the topsoil to a depth of 15 cm using a shovel. the single-cross hybrid golsa was obtained from the seed and plant improvement institute, iran. this variety has been produced by crossing the restorer rgk2 with the cytoplasmic male sterile line agk2. the good characteristics of this variety include being single-branched, uniform maturity, full fertility, early maturity, and high yield. the growth period of this plant is 100 to 105 days, and it is classified as an early variety. it is resistant to rust, tolerant to charcoal rot, and relatively susceptible to downy mildew. this oil variety can be used as a first crop in temperate and cold regions, and as a summer crop in temperate and central regions of iran. the weight of a thousand seeds in the golsa variety is 50 g, and the grain yield is about 2.7 kg ha-1. the height of the plant in this variety is approximately 180 cm. after secondary tillage and creating relatively soft, lump-free soil, the surface shape was transformed into a ridge-furrow archaeological pattern through a furrower. seeds were sown manually at the top of the ridges at a depth of 4 cm on march 20. through multiple seed sowing per hole, the chances of seeding emergence increased. however, after seedling establishment, excess seedlings were removed. a density of 9 plants per m2 was obtained with a rectangular planting pattern with 60 cm distance between rows and 15 cm intra-row spacing. during seed planting, npk fertilizer was applied as a band utilization 2 cm below the seeds. the recommended rates of nitrogen, phosphorous, and potassium for the studied site were 120 kg ha-1, 80 kg ha-1, and 40 kg ha-1, respectively. n, p, and k n, p, and k were provided by the urea, triple superphosphate, and potassium sulfate fertilizers. irrigation was carried out through polyethylene pipes equipped with volumetric meters and in the form of a surface furrow system. due to the presence of precipitation during the vegetative stages, supplemental irrigation was performed at the stem elongation stage with a depth of 60 mm, and supplemental irrigation was performed at the reproductive stage with a depth of 100 mm. the amount of water consumed through irrigation under full irrigation conditions was 510 mm. weed control was done through hand weeding and spraying sethoxydim herbicide at 1.5 l ha-1. measurements of the traits at the head emergence stage, canopy extent was obtained by measuring the distance between the tip of the leftmost leaf to the rightmost leaves in the middle of the canopy using a meter. during the achenes setting stage (bbch-scale: 71), photosynthetic pigments were measured using a handheld leaf chlorophyll meter (spad-tys-a, china). at the end of the reproductive period, when the backs of the heads turned brown, the characteristics of the crop, such as head diameter, number of filled and empty achenes in the head, were measured or counted. using a 1 m2 randomly in the plots, all the heads in the area inside the quadrat were harvested, and after drying the heads at 60 °c for 36 h, the seeds were separated and weighed. the weight of one hundred seeds was obtained using a seed counter and a digital scale. the measurement of the seed oil content was done using a soxhlet apparatus and n-hexane as the extraction solution. relative leaf water content (rwc) was measured at the achene setting stage. the rwc was obtained through the method of soltys-kalina et al. (2016). leaf disc samples were prepared from the upper leaves early in the morning using a punch (fresh weight: fw), after that they were placed in distilled water for 8 h (turgid weight: tw) and then leaf samples were dried in an electric oven at 65 °c; their dry weight (dw) was calculated. rwc=[(fw−dw)/(tw−dw)]×100. data analysis before the analysis of variance, the normality of the data distribution was examined by minitab software. anova was carried out by sas. the mean comparison was performed by the lsd test. boxplots were drawn by spss, and principal component analysis plots were done by the minitab package. results the effect of different types of animal farmyard manure (m) and irrigation regimes (i) on canopy spread and plant height was significant (p<0.01). canopy width in plants grown under fully irrigated conditions (i4) was 51% greater than those grown under rainfed conditions (i1). the mean comparison of stem length between moisture regimes showed that no significant difference was observed between rainbiologica nyssana ● 16 (2) december 2025: janmohammadi et al. ● morphophysiological response of sunflower (helianthus annuus) to various farmyard manure and irrigation frequency nfa increased by 32%. the most effective manures for nfa were sheep and poultry manure, which increased this trait by 18% and 15%, respectively, compared to the m1. the plants grown under i4m3 produced the highest number of achenes compared to other conditions. under i3 circumstances, the highest nfa was obtained with the use of poultry manure. the ratio of empty achenes (rea) was also affected by i×m. the highest rea was observed under full irrigation and no fertilizer application (4.98%). plants grown under i2m1 and i3m1 conditions were in second and third place in terms of rea (fig. 3). the lowest rea was observed in plants grown under i1m4 (2.95%) and i3m2 (3.09%). achen weight responded strongly to moisture regimes, and increasing irrigation frequency increased this trait. the use of farmyard manure, especially sheep manure and poultry manure, also increased achene weight compared to the control. the heaviest achenes were obtained under i4+m3 and i4+m4 conditions. irrigation levels and manure applications also affected the oil content of achene (p<0.01). although no significant difference was observed between the manures (m2, m3, m4) in terms of achene oil content, the use of manures increased the oil content by about 2% compared to the control. full irrigation conditions increased the oil content of achen by 5% compared to rainfed conditions. the highest oil content was obtained under i4m4 (41.39%) and i4m2 (41.69%). the evaluation of achene yield indicated that the interaction effects of irrigation and manure were significant for this trait. the best achene yield was obtained with the application of farmyard manures (poultry, sheep, and cow) under full irrigation conditions with values of 1806 kg ha-1, 1799 kg ha-1, and 1762 kg ha-1. under i3 conditions, the application of poultry and sheep manure showed a greater improvement effect than cow manure. the achene yield in the latter conditions was 1680 kg ha1, 1659 kg ha-1, and 1659 kg ha-1. the lowest achene yield was recorded under i1m1 conditions. under rainfed circumstances (i1), the application of sheep manure resulted in the highest achene yield (1007 kg ha-1). the results showed that under supplemental irrigation conditions, the application of poultry manure significantly increased the achene yield (fig. 4). monograph pca showed that pc1 separated the optimal moisture conditions of i4 and i3 from other irrigation levels. the highest values for the evaluated agronomic traits were recorded under full irrigation conditions and three times supplemental irrigation. pc2 could distinguish the low-effective levels of fertilizer treatments from the poultry and sheep fed and two times of supplementary irrigation (i2), but i4 and i3 (three times of supplementary irrigation) increased this component by 52% and 40%. among farmyard manures, the use of cow manure (m2) resulted in the highest lateral canopy growth (20% vs. control). however, the tallest plants were obtained with poultry manure and sheep manure, which increased longitudinal growth by 27% and 15%, respectively, compared to the control (tab. 2). the i4+m3 condition resulted in the highest lateral canopy growth, and the smallest canopies were observed under rainfed conditions without manure application (41.33 cm). although increasing irrigation frequency increased longitudinal growth, the effectiveness of farm yard manures became more pronounced with improved moisture compared to the control. under i2 conditions, the difference between fertilizers in terms of plant height was quite obvious, while under i1 conditions, no difference was observed between fertilizers in terms of longitudinal growth. the application of (sheep and poultry) m3 and m4 had a greater effect on vegetative growth components compared to the control. stem diameter was affected by soil moisture supply, and the thickest stems were produced by plants under i4 and i3 conditions with the application of m4 and m3, respectively. regardless of the type of fertilizer, the use of all farmyard manures increased the leaf number (ln) compared to m1, and on the other hand, increasing the frequency of irrigation also improved this trait. the highest ln was recorded in plants grown under i3m3, i4m2, i4m3, and i4m4. the head diameter was affected by i×m at the 5% level. under rainfed conditions, the use of farmyard manures did not have a significant effect on this trait. with increasing irrigation frequency, the effectiveness of farmyard manures on head diameter increased, and the highest diameter was obtained under i4+m3 conditions (19.19 cm). under supplementary irrigation conditions, i3, the use of poultry manure resulted in the highest head diameter. the relative leaf water content is shown in fig. 2. the interaction effects of i×m on rwc were significant (p<0.01). under rainfed conditions, poultry manure application increased rwc. application of cow and sheep manure under moisturelimited conditions decreased rwc compared to the m1 condition. this could be due to the presence of nitrogen and higher water consumption of plants with the applications of m2 and m3. under i3 conditions, sheep manure application increased rwc. the results showed that the effect of fertilizers on rwc is largely influenced by irrigation regimes. the number of filled achenes (nfa) was also affected by manure and irrigation. as irrigation frequency increased from i2 to i3, the proportion of biologica nyssana ● 16 (2) december 2025: janmohammadi et al. ● morphophysiological response of sunflower (helianthus annuus) to various farmyard manure and irrigation frequency table 2. the effect of different livestock fertilizers in different irrigation regimes on sunflower performance in the drought-prone region of northwestern iran effects cc cs ph sd ln hd nfa wha oc irrigations (i) i1 40.38d 47.75c 107.00d 1.25b 15.24c 13.21d 498.50d 3.89d 35.73d i2 53.49c 46.83c 124.25c 1.28b 18.73b 14.73c 559.87c 4.16c 38.73c i3 55.59b 65.92b 150.08b 1.43a 24.75a 15.85b 675.97b 4.39b 39.86b i4 60.16a 72.25a 162.75a 1.40a 25.41a 18.09a 892.92a 4.96a 40.60a manure (m) m1 48.55c 52.75c 125.75c 1.32ab 19.54b 14.85b 590.22c 4.16c 37.55b m2 51.69b 63.333a 136.17b 1.31b 21.33a 15.55a 654.72b 4.25b 39.12a m3 54.12a 58.33b 140.33ab 1.39a 21.90a 15.77a 699.45a 4.49a 38.92a m4 55.28a 58.33b 141.83a 1.33ab 21.35a 15.73a 682.87ab 4.50a 39.32a interactions i1m1 36.13h 41.33i 99.33h 1.16f 14.11i 12.89h 422.47h 3.76i 35.87g i1 m2 39.73g 54.33f 110.67g 1.21ef 15.67hi 13.07h 479.33gh 3.87hi 35.97g i1 m3 42.03fg 46.67gh 105.00gh 1.36cd 16.84hg 13.10h 535.33fg 3.86hi 35.97g i1 m4 43.63f 48.67gh 113.00g 1.25f 14.33i 13.80gh 556.87f 4.07efg 35.13g i2m1 49.30e 44.33hi 123.33f 1.30def 17.53fg 14.67g 561.27f 4.06fg 37.65f i2 m2 50.13de 49.67g 122.67f 1.30def 18.80ef 15.03def 591.53f 4.01gh 39.00def i2 m3 58.37bc 45.00hi 125.67f 1.26f 18.20fg 14.83efg 557.27f 4.37d 38.70fe i2m4 56.17c 48.33gh 125.33f 1.24def 20.40e 14.40fg 529.4fg 4.20ef 39.57cde i3m1 52.47d 61.00e 143.00de 1.49abc 23.07d 14.43fg 568.47f 4.07efg 37.63f i3 m2 57.47bc 65.00de 160.00b 1.36cd 24.47cd 16.07cd 675e 4.23de 39.83bcde i3m3 56.23c 71.33b 145.00cde 1.56a 26.43a 15.93cde 695.87de 4.64c 40.77abc i3 m4 56.20c 66.33cd 152.33bc 1.32de 25.03ac 16.97bc 764.53cd 4.61c 41.20ab i4m1 56.30c 64.33de 137.33e 1.33de 23.47cd 17.42b 808.67bc 4.75bc 39.06def i4 m2 59.41b 84.33a 151.33bcd 1.37bcd 26.40a 18.02b 873b 4.90b 41.69a i4 m3 59.83b 70.33bc 185.67a 1.38bcd 26.13ab 19.19a 1009.33a 5.08a 40.25abcd i4 m4 65.10a 70.00bc 176.67a 1.50ab 25.63ab 17.73b 880.67b 5.11a 41.39a significance level i <0.0001 <0.0001 <0.0001 0.002 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 f <0.0001 <0.0001 <0.0001 0.1100 <0.0001 0.0100 <0.0001 <0.0001 <0.0001 i×f 0.0011 <0.0001 <0.0001 0.0149 0.0369 0.0272 0.0017 0.0046 0.0155 cv% 3.18 4.47 4.05 6.26 6.87 4.37 6.75 2.24 2.21 i1: rainfed or no-irrigated condition, i2: two-time supplemental irrigation, i3: three-time supplementary irrigation, i4: full irrigation, m1: control or non-use of farm yard manure, m2: the application of 10 t ha-1 cow manure, m3: the application of 10 t ha-1 sheep manure, m4: use of 10 t ha-1 poultry manure. cv: the coefficient of variation (%), cc: leaf chlorophyll (spad unit), cs: canopy spread (cm), ph: height of stem (cm), sd: diameter of the stem (cm), ln: leaf number per plant, hd: head diameter (cm), nfa: number of the filled achene per head, wha: weight of hundred achenes (g), oc: achene oil content (%). for each trait, the means with dissimilar letters show that they have been differently affected by treatments at p<0.05 when evaluated by the lsd test. significance test (p values) is shown for determining the significance level; the values lower than 0.05 and 0.01 are significant at 5% and 1% statistical level, respectively. biologica nyssana ● 16 (2) december 2025: janmohammadi et al. ● morphophysiological response of sunflower (helianthus annuus) to various farmyard manure and irrigation frequency fig. 2. the mean comparison of the relative water content (rwc) in sunflower leaves under different manure application conditions and soil moisture regimes in northwest iran. i1: rainfed, i2: two-time supplemental irrigation, i3: three-time supplemental irrigation, i4: full irrigation. m1: control or no manure application, m2: application of cow manure, m3: sheep manure, m4: poultry manure fig. 3. the proportion of empty achenes in sunflowers was affected by irrigation regimes and farmyard manures. i1: rainfed, i2: two-time supplemental irrigation, i3: three-time supplemental irrigation, i4: full irrigation. m1: control or no manure application, m2: application of cow manure, m3: sheep manure, m4: poultry manure manures. the best appearance of sunflower and the highest vegetative growth were obtained under m3 and m4 conditions (fig. 5). discussion soil condition assessment showed that the soil in the study area was severely limited due to low organic matter and nutrients. in addition, meteorological data showed that the end season of the sunflower growth period is characterized by dry spells. the total annual rainfall in the region was 390 mm, while the estimated evapotranspiration rate for the region was 594 mm. these data indicate that precipitation is low compared to evapotranspiration unless soil moisture reserves are significant at the beginning of the growing season. the results indicated that vegetative traits such as stem height, canopy spread, stem diameter, and leaf number showed their lowest amounts under rainfed conditions and increased significantly with increasing irrigation frequency. only the stem diameter did not show a significant difference between supplementary and full irrigation levels. according to the hypothesis of acidic cell growth, water accumulation in vacuoles is the main driving factor in plant growth (jiang et al., 2021), and it seems natural that reducing irrigation frequency would severely affect growth. chlorophyll, as the most important photosynthetic pigment, also showed a significant decrease with reducing irrigation frequency. it seems that water scarcity increases the decomposition or inhibition of the biosynthesis of this pigment. these findings are consistent with the results of kheshtpaz et al. (2024), who reported that water deficit stress reduced leaf chlorophyll content. a relatively similar trend was observed for the number of leaves per plant. in this connection, it can be stated that water deficit stress affects both the size of photosynthetic sources and the power of the sources (zhao et al., 2020). in addition to the effects of water deficit stress on growth, reduced photosensitizer levels and reduced chlorophyll content can also limit growth by reducing the amount of current photo-assimilates (yusuf & hamed, 2021). on the other hand, the application of farmyard manure improved the vegetative traits compared to the control conditions (m1). this increase can be attributed to overcoming physical limitations of the soil by supplying a significant volume of organic matter from farmyard manure (nouraein et al., 2019). also, the use of farmyard manure can facilitate and expedite the supply and availability of nutrients by adjusting the soil ph and optimizing the rhizosphere condition (liu et al., biologica nyssana ● 16 (2) december 2025: janmohammadi et al. ● morphophysiological response of sunflower (helianthus annuus) to various farmyard manure and irrigation frequency fig. 4. evaluation of sunflower seed yield grown under different levels of supplementary irrigation and application of various types of farmyard fertilizers in the northwestern region of iran. i1: rainfed, i2: twotime supplemental irrigation, i3: three-time supplemental irrigation, i4: full irrigation. m1: control or manure application, m2: application of cow manure, m3: sheep manure, m4: poultry manure 2024). among the livestock fertilizers studied, the use of poultry and sheep manure increased chlorophyll content the most. given the key role of nitrogen in the structure of this pigment, it seems that poultry and sheep manure provided greater amounts of nitrogen to the root system (tagar et al., 2016). in a brief comparison of the applied manures with the control conditions, it was found that all manures led to increased growth. the soil of this region, like other semi-arid regions of northwest iran, had very low organic matter, and it appears that the application of manures, by improving the organic matter content of the soil, improved some key soil properties such as water retention capacity, the formation of micropores, and the cation exchange capacity of the soil (zhu et al., 2025). the results showed that the effect of water supply under different conditions of manure application was different. the application of manure increased the effect of supplementary irrigation on both vegetative growth components and achene yield components. in the present experiment, the highest yield of achene was obtained under full irrigation conditions, using either poultry or sheep manure. considering six irrigation events carried out under full irrigation conditions, 530 mm of water was used. however, water resources in the region are facing serious limitations, and providing water during sunflower growth due to its simultaneous irrigation with other crops is one of the key challenges facing local farmers (kouchakkhani et al., 2024). therefore, it appears that the use of three-time supplemental irrigation (i3) in conjunction with farmyard manure can significantly reduce water consumption by producing achene relatively reasonably and economically, thereby conserving water resources for other crops. although increasing the frequency of irrigation resulted in a higher achene number in the head, it also increased the proportion of unfilled achenes. this could be due to the limitation of photoassimilative resources to fill the empty cup of the produced achenes. the intensification of climate change has also complicated the situation regarding available water resources in the region (janmohammadi & sabaghnia, 2023), which increases the acceptability of using supplementary irrigation (i3) instead of full irrigation among farmers. eliminating three irrigations saves 260 mm of water, which can be used to produce another crop. our previous findings also indicated significant water savings by using supplemental irrigation techniques in chickpea fields (janmohammadi et al., 2024). the long-term application of manure can improve soil properties and enhance the rate of root development, allowing roots to spread deeper into the soil, which can further reduce the need for irrigation (liu et al., 2013). the qualitative characteristics of the oil, such biologica nyssana ● 16 (2) december 2025: janmohammadi et al. ● morphophysiological response of sunflower (helianthus annuus) to various farmyard manure and irrigation frequency fig. 5. principal component analysis plot to evaluate the similarity of the effects of supplementary irrigation treatments and different farmyard fertilizers on growth traits and yield of sunflower. i1: rainfed, i2: twotime supplemental irrigation, i3: three-time supplemental irrigation, i4: full irrigation. m1: control or manure application, m2: application of cow manure, m3: sheep manure, m4: poultry manure as oil content, also showed that under i3 conditions, where this component showed an acceptable increase with the use of poultry manure, and it can be stated that the oil yield under the mentioned conditions can be economical and acceptable. the findings showed that supplementary irrigation should not only focus on the reproductive stages, but also that applying supplementary irrigation once during vegetative growth can improve vegetative growth and the sufficient production of photo-assimilates, allowing the plant to enter the reproductive stage with sufficient photo-assimilate reserves. this will compensate for the lack of photo-assimilates during the formation of achene components through the utilization of photo-assimilate reserves and the remobilization process (saadatmand et al., 2024). conclusion the annual precipitation and rainfall distribution in the studied location were insufficient for rainfed sunflower production, and the crop’s economic performance was not encouraging. therefore, supplemental irrigation for sunflower production is a climate-smart solution to prevent the negative impact of water shortage stress, especially at the end of the season. although the application of all farmyard fertilizers improved the growth and achene yield compared to the control, some differences in the effect of fertilizers were evident. under rainfed conditions, sheep manure application increased achene yield. with increasing soil moisture through irrigation, the effectiveness of livestock fertilizers also showed a significant increase. however, the scarcity of water resources in the region is a serious limiting factor, and the use of three supplementary irrigations, as well as the use of poultry and sheep manures, had the best effect, and the water efficiency was also significantly increased. the use of livestock manures alone is not sufficient to improve growth and increase achene yield under rainfed conditions, and supplementary irrigations must be included in sunflower field management and crop production systems. references adeleke, b.s. & babalola, o.o. (2020). oilseed crop sunflower (helianthus annuus) as a source of food: nutritional and health benefits. food science & nutrition, 8(9), 4666–4684. https://doi.org/10.1002/ fsn3.1783 afzal, m.f., khalid, w., khalid, m.a., zubair, m., akram, s., kauser, s., noreen, s., jamal, a., khan, m.k., & al-farga, a. (2022). recent industrials extraction of plant seeds oil used in the development of functional food products: a review. international journal of food properties, 25(1), 2530–2550. https://doi.org/10.1080/10942912.2022 .2144882 derpsch, r., kassam, a., reicosky, d., friedrich, t., calegari, a., basch, g., & dos santos, d.r. (2024). nature’s laws of declining soil productivity and conservation agriculture. soil security, 14, 100127. https://doi.org/10.1016/j. soisec.2024.100127 fao (food and agriculture organization of the united nations). 2023. crop production and trade data. retrieved from http://www.fao.org/faostat/ en/#home ferreira, p.a.a., ceretta, c.a., lourenzi, c.r., de conti, l., marchezan, c., girotto, e., tiecher, t.l., palermo n.m., parent, l.-é., & brunetto, g. (2022). long-term effects of animal manures on nutrient recovery and soil quality in acid typic hapludalf under no-till conditions. agronomy, 12(2), 243. https://doi.org/10.3390/agronomy12020243 garcía-lópez, j., lorite, i.j., garcía-ruiz, r., ordoñez, r., & dominguez, j. (2016). yield response of sunflower to irrigation and fertilization under semi-arid conditions. agricultural water management, 176, 151–162. https://doi. org/10.1016/j.agwat.2016.05.020 giannini, v., mula, l., carta, m., patteri, g., & roggero, p.p. (2022). interplay of irrigation strategies and sowing dates on sunflower yield in semi-arid mediterranean areas. agricultural water management, 260, 107287. https://doi.org/10.1016/j. agwat.2021.107287 janmohammadi, m., kouchakkhani, h., & sabaghnia, n. (2024). the effect of supplemental irrigation and exogenous application of glycine betaine on chickpea performance in the semiarid region. journal of agriculture faculty of ege university, 61(2), 189–199. https://doi. org/10.20289/zfdergi.1402726 janmohammadi, m. & sabaghnia, n. (2023). strategies to alleviate the unusual effects of climate change on crop production: a thirsty and warm future, low crop quality. a review. biologia, 69(2), 121– 133. https://doi.org/10.6001/biologija.2023.69.2.1 jiang, y.t., yang, l.h., ferjani, a., & lin, w.h. (2021). multiple functions of the vacuole in plant growth and fruit quality. molecular horticulture, 1, 1–13. https://doi.org/10.1186/s43897-021-00008-7 kheshtpaz, n., janmohammadi, m., & sabaghnia, n. (2024). foliar spray can improve rapeseed yield components under continuous irrigation. pesquisa agropecuária tropical, 54, e80539. https://doi. biologica nyssana ● 16 (2) december 2025: janmohammadi et al. ● morphophysiological response of sunflower (helianthus annuus) to various farmyard manure and irrigation frequency org/10.1590/1983-40632024v5480539 kouchakkhani, h., janmohammadi, m., & sabaghnia, n. (2024). the effects of water stress and plant density on vegetative and reproductive characteristics of safflower in the semi-arid region. studia universitatis babeş-bolyai, biologia, 69(2). https://doi.org/10.24193/subbbiol.2024.2.05 liu, y., lan, x., hou, h., ji, j., liu, x., & lv, z. (2024). multifaceted ability of organic fertilizers to improve crop productivity and abiotic stress tolerance: review and perspectives. agronomy, 14(6), 1141. https://doi.org/10.3390/agronomy14061141 liu, c.-a., li, f.-r., zhou, l.-m., zhang, r.-h., jia, y., lin, s.l., wang, l.j., siddique, k.h.m., & li, f.m. (2013). effect of organic manure and fertilizer on soil water and crop yields in newly-built terraces with loess soils in a semi-arid environment. agricultural water management, 117, 123–132. https://doi.org/10.1016/j.agwat.2012.11.002 maia filho, f.d.c.f., mesquita, e.f.d., guerra, h.o.c., moura, m.f., & chaves, l.h.g. (2013). effect of cattle manure on sunflower production and water use in two types of soil. revista ceres, 60, 397–405. https://doi.org/10.1590/s0034737x2013000300013 mokgolo, m.j., zerizghy, m.g., & mzezewa, j. 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(2019). short-term effects of tillage intensity and fertilization on sunflower yield, achene quality, and soil physicochemical properties under semi-arid conditions. applied sciences, 9(24), 5482. https://doi.org/10.3390/app9245482 petraru, a., ursachi, f., & amariei, s. (2021). nutritional characteristics assessment of sunflower seeds, oil and cake. perspective of using sunflower oilcakes as a functional ingredient. plants, 10(11), 2487. https://doi.org/10.3390/plants10112487 saadatmand, m., soltani najafabadi, m., & mirfakhraei, s.r. (2024). exploring sourcesink relationship for the formation of grain yield in sunflower. journal of agricultural science and technology, 26(6), 1373–1388. https://doi. org/10.22034/jast.26.6.1373 soltys-kalina, d., plich, j., strzelczyk-żyta, d., śliwka, j., & marczewski, w. (2016). the effect of drought stress on the leaf relative water content and tuber yield of a half-sib family of ’katahdin’derived potato cultivars. breeding science, 66(2), 328–331. https://doi.org/10.1270/jsbbs.66.328 tagar, a.a., tagar, s.a., leghari, n., shah, a.r., shah, a.m., shah, s.b., & memon, a. (2016). nitrogen content in cattle, sheep and poultry manures: its escalation and shortfall over time. science international, 28, 4943–4945. vilvert, e., zander, p., mgeni, c.p., sieber, s., & lana, m.a. (2023). assessment of nitrogen management on sunflower yield and its economic response in smallholder farms in a semi-arid region. international journal of plant production, 17(1), 109–119. https://doi.org/10.1007/s42106-022-00224-1 yusuf, n. & hamed, n.f.i. (2021). effects of water deficit on the growth and chlorophyll content of capsicum frutescens. journal of sustainability science and management, 16, 148–158. https://doi. org/10.46754/jssm.2021.08.013 zhao, w., liu, l., shen, q., yang, j., han, x., tian, f., & wu, j. (2020). effects of water stress on photosynthesis, yield, and water use efficiency in winter wheat. water, 12(8), 2127. https://doi. org/10.3390/w12082127 zhu, z., zhang, y., tao, w., zhang, x., xu, z., & xu, c. (2025). the biological effects of biochar on soil’s physical and chemical characteristics: a review. sustainability, 17(5), 2214. https://doi. org/10.3390/su17052214 biologica nyssana ● 16 (2) december 2025: janmohammadi et al. ● morphophysiological response of sunflower (helianthus annuus) to various farmyard manure and irrigation frequency microsoft word bn-oa-0201-04 stancic et al biologica nyssana 2 (1) september 2011: 35-38 stančev, i. et al. yield and morphological characteristics of some types… 35 original article ! yield and morphological characteristics of some types of cms hybrids of tobacco prilep ivica stančić*, saša petrović, jelica živić college of agriculture and food technology, prokuplje * e-mail: istancic@medianis.net abstract: stančić, i., petrović, s., živić, j.: yield and morphological characteristics of some types of cms hybrids of tobacco prilep. biologica nyssana, 2 (1), september 2011: 35-38. this paper presents characteristics of the four newly created hybrid varieties of tobacco prilep. the survey was conducted in period from year 2005. to 2006, and was performed at the experimental field of ad selekcija in aleksinac. the aim was to perform a comparative analysis of the newly created cms hybrid varieties of tobacco type prilep through testing the yield and morphological characteristics. relating morphological traits, all tested hybrids showed the characteristic of the oriental tobacco type prilep. the hybrid p-3 had a slightly greater plant height, whereas the hybrid p-2 achieved the greatest number of leaves per plant and the hybrid p-4 some greater size of the largest leaf as well as the length of internodes. the hybrid p-3 exhibited a much greater yield of dry leaves than hybrids p-2 and p-4. the same hybrid achieved significantly better features in proportional representation of high classes compared to the hybrid p-2. key words: tobacco, prilep, hybrid, dry leaf yield, morphological characteristics introduction ! production area of tobacco (nicotiana tabacum l.) has decreased in the recent period in serbia. according to the report of the serbian chamber of commerce, the production was only 320.000 kilograms in 2009. production of oriental tobacco is mainly conducted in the regions of south morava and kosovo and metohija. one of the reasons for production decrease is the present assortment which does not satisfy either the yield or the quality (s t a n č i ć et al., 1998). for this reasons, breeders are trying to create hybrids based on cytoplasmic male sterility (cms). in this way, the illegal seed production will be prevented, and conditions for achieving varietal uniformity and good yield and quality will be also created in the entire production area. knowledge in gene pool of tobacco could assist in selection of materials for breeding or genetic investigations (m o o n et al., 2009). on the basis of the needs for oriental tobacco that is present 8-10% in the manufacture of cigarettes, the goal of the work was to perform a comparative analysis of the newly created cms hybrid varieties of tobacco type prilep through testing the yield and morphological characteristics. materials and methods the survey comprised four hybrids based on cms of type prilep: p-1, p-2, p-3 and p-4. the process of plant breeding was carried out through a series of crossbreeding, where sterile analogues were conducted at first. the subsequent procedure comprised the research of combining potentials of the achieved material in order to find the best parental genotypes of f1 hybrids. the survey was conducted from year 2005. to 2006., 2 (1) • september 2011: 35-38 biologica nyssana 2 (1) september 2011: 35-38 stančev, i. et al. yield and morphological characteristics of some types… 36 table 1. morphological characteristics of tested tobacco hybrids hybrids plant height including flower (cm) number of leaves per plant internodes length (cm) length of leaf (cm) width of leaf (cm) ratio leaf length / width p-1 49 36 1,6 13,1 7,9 1,66 p-2 50 39 1,8 13,2 8,1 1,63 p-3 55 38 2,2 13,7 8,1 1,69 p-4 49 35 2,5 15,2 8,9 1,71 table 2. yield of leaves (kg/ha) and percentage of high classes (i and ii) hybrids yield of leaves (kg/ha) rank % representation (i and class ii) rank p-1 1410 2 26,56 2 p-2 1356 4 22,31 4 p-3 1494 1 28,76 1 p-4 1361 3 23,38 3 lsd 0,05 0,01 93,72 130,23 6,12 8,32 cv 5,62 3,21 at the experimental field of ad selekcija in aleksinac. the experiment was set up according to a completely randomized block design with five replications. during the experiments the optimal agricultural practices for oriental tobacco cultivation of were applied. yield of dry leaves (kg/ha) and calculation of the proportional representation of high classes (i and ii) (j o v a n o v i ć , d., 2001) were obtained after harvesting, drying and grading. investigation of the morphological characteristics was carried out during the vegetation period, and ten plants from each trial were used as a representative sample. the measurement was done on the insertion of the middle leaf and it included the height of the plants with a flower, the number of leaves per plant, the length of internodes in the middle of the stem, the size of leaves and the ratio of leaf length and width. results and discussion morphological traits of the tested hybrids were obtained during the vegetation period (tab. 2). the hybrid p-3 achieved the highest height (55 cm), while hybrids p-1 and p-4 expressed some lower values. regarding the number of leaves per plant, the hybrid p-2 had the greatest value (39), whereas the hybrid p-4 the lowest (35). investigation of the genetic variability of plant height and number of leaves per plant in some varieties of oriental tobacco and their f1 hybrids is a significant precondition for high and stable yield (k o r u b i n -a l e k s o s k a , a., 2010). the highest (2.5 cm) and lowest (1.6) length of internodes were found for hybrids p-4 and p1respectively. the greatest leaf size was found in the hybrid p-4 with an average length of 15.2 cm and an average width of 8.9 cm. the hybrid p-1 had the smallest leaf with an average length of 13.1 cm and an average width of 7.9 cm. m i c e s k a et al. 2003 obtained higher values for the examined morphological characteristics of tobacco type prilep. regarding the ratio of leaf length and width, the hybrid p-4 showed the greatest value (1.71), while the lowest value (1.66) was found for the hybrid p1. bearing in mind the correlation between leaf size and its chemical composition, relation of length and width of the leaf is an important parameter for the quality of oriental tobacco. the parameters of morphological characteristics of tested hybrids were typical for the oriental tobacco type prilep. the leaf yield is a trait that is influenced by many interrelated factors. genetic properties of tobacco hybrids and agro-ecological conditions of production are the decisive factors in formation of organic and mineral complex of substances, affecting the corresponding leaf yield. the hybrid p3 achieved the greatest dry leaf yield (1494 kg / ha), while the hybrids p-2 and p-4 exhibited lower values (tab.1). statistically significant differences were found among the hybrids. the hybrid p-3 achieved significantly greater yield comparing to hybrids p-2 and p-4. no significant differences were obtained among other tested hybrids. tobacco breeders focus their program on those quantitative traits that are highly associated with leaf yield (d r a z i c et al., 2010). the quality of tobacco was expressed through the percentage of high classes of the leaves (i and ii, tab. 1). the hybrid p-3 exhibited the highest percentage of high class representation (28.76%), biologica nyssana 2 (1) september 2011: 35-38 stančev, i. et al. yield and morphological characteristics of some types… 37 while the hybrid p-2 had the lowest participation of high classes in dry leaf (22.31%). the hybrid p-3 showed some better characteristics compared to the hybrid p-2 in term of the proportional distribution of high classes. there was no statistically significant difference among other tested hybrids. the obtained yield values and the percentage of high classes of the leaves are in accordance with the results obtained by m i c e s k a & d i m i t r i e s k i (2000). conclusion the obtained mean values and variability parameters show that the tested hybrids exhibited a high variability in yield and morphological traits. the genotypes usually varied in leaf yield, the percentage of high classes, plant height and internodes length. the hybrid p-3 achieved the highest yield of dry leaves. it also had the best proportional distribution of high classes, whereas morphological characteristics of this hybrid were typical for the oriental tobacco type prilep. genetic potential and morphological characteristics of the hybrid p-3 could be included in breeding program of oriental tobacco. references dražić, s., prodanović, s., živanović, t. 2010: identification of donor lines containing favorable alleles for quantitative traits in burley tobacco (n. tabacum l.). genetika, vol 42, no. 2, 287 297. jovanović, d. 2001: priručnik za proizvodnju duvana.digp “prosveta”, niš korubin-aleksoska, a., nikova, v., aleksoski, j. 2010: regression analysis of the interitance of leaf size in f1 and f2 propgenies in various tobacco genotypes. biotechnology & biotechnological equipment. eq. volume 24, number 2. p. 401-406. miceska, g., dimitreski, m. 2000: some characteristics tobacco variety p 10-3/2 (in vitro), transplanted in field conditions. tutun/tobacco science and profession.vol. 50, no 4-6, p. 61-69. miceska, g., dimitreski, m., sspasenoski, m. 2003: the effect of common mosaic virus (tmv) on morphological characteristic of plants from tobcco type prilep. tutun/tobacco science and profession. vol. 53, no 9-10, p. 268-277. moon, h. s., j. m. nifong, j. s. nicholson, a. heineman, k. lion, r. van der hoeven, a. j. hayes & r. s.lewis 2009: microsatellite-based analysis of tobacco (nicotiana tabacum l.). crop sci, 49,2149-2159. stančić, i., popović, r., pešić, v. 1998: analiza fizičkih i hemijskih osobina lista kod nekih cms hibrida orijentalnih duvana. tutun/tobacco. vol. 48 no. 1-6. p.58-64. biologica nyssana 2 (1) june 2011: x-x čavlović d. et al. allochthonous woody taxa in zasavica ecosystem 38 džamić & alimpić aradski 2022, biologica nyssana 13(2) 13 (2) december 2022: 83-87 doi: 10.5281/zenodo.7437204 in memoriam professor sonja duletić-laušević (1962-2021) original article ana m. džamić university of belgrade, faculty of biology, institute of botany and botanical garden “jevremovac”, studenski trg 16, 11000 belgrade, serbia simicana@bio.bg.ac.rs (corresponding author) ana z. alimpić aradski university of belgrade, faculty of biology, institute of botany and botanical garden “jevremovac”, studenski trg 16, 11000 belgrade, serbia professor sonja duletić-laušević suddenly passed away on december 18, 2021, leaving a large void behind. her passing left us in disbelief and sadness. as a full professor at the university of belgrade, faculty of biology, she made great contributions both as a teacher and scientist. sonja n. duletić-laušević was born on february 14, 1962, in belgrade where she finished primary school and gymnasium. she enrolled in the department of biology of the faculty of science and mathematics, university of belgrade, and graduated in 1986 in the group of general biology. she received a master’s degree at the university of belgrade faculty of biology in 1990 defending the thesis entitled: “investigation of the production of t-2 toxin and its effect on rec mutants of escherichia coli”. in 1995, she defended her doctoral thesis with the title “comparative anatomical analysis of the vascular system of the genus helianthus l. genotypes” at the university of belgrade faculty of biology. her first job was at the department of plant physiology (mycology laboratory) of the institute for biological research „siniša stanković” in belgrade (1987-1988) where she was a junior assistant. in 1989 she was employed as a teaching assistant at the chair of plant morphology and systematics, institute of botany, university of belgrade faculty of biology. she was promoted to assistant professor in 1997, associate professor in 2005, and full professor in 2019 at the chair of plant morphology and systematics, at the faculty of biology. she participated in the preparation and realization of practical and theoretical courses organized by the chair of plant morphology and systematics at all study levels. she taught the morphology of plants and general botany course to numerous generations. in recent years, she has been teaching anatomy and morphology of plants and botany at the undergraduate level. she is a co-author of numerous textbooks and practicums. she is the coauthor of the digital content visual dictionary of plant anatomy and morphology. she was the mentor of three doctoral dissertations and a member of nine defense comissions, as well as numerous master’s, and diploma theses. her scientific opus includes over 230 bibliographic units, © 2022 džamić & alimpić aradski. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 83 14th symposium on the flora of southeastern serbia and neighboring regions 84 biologica nyssana ● 13 (2) december 2022: 83-87 džamić & alimpić aradski ● in memoriam professor sonja duletić-laušević (1962-2021) over 730 citations, and an h-index of 16. she worked in a specific scientific field of morphology, phytochemistry, and systematics of plants and extensively carried out anatomical, histochemical, and micromorphological analyses of plants. she placed special emphasis on the analysis of plant organs of representatives of the lamiaceae family. special focus of her research was glandular trichomes as the main secretory structures synthesizing diverse bioactive compounds. she was also involved in phytochemical and biological activity research on plants. at the same time, she worked in the field of mycology and collaborates with a group coordinated by professor jelena vukujević. professor solomon wasser. apart from the scientific research work that takes place at the faculty of biology, she continued work with colleagues from mycology and collaborated with many colleagues from our or other institutions. she was a member of several scientific associartions: serbian biological society and serbian society for microscopy; serbian chemical society; international sunflower association; optima; amapseec. also, she was a member of the editorial board of the international scientific journal botanica serbica. she served as a reviewer of many scientific journals and student books and was a commitee member for many teaching and scientific promotion. professor sonja with the members of mycology research group (december 2009, institute of botany and botanical garden „jevremovac” since the beginning of her career, she has been engaged in numerous national and international projects. as part of the international project “higher education reform in biological sciences”, tempus jep 40094 (2006-2009), dedicated to the reform of study programs at the faculty of biology, she stayed at the universities of turin and nice in december 2006. in 2004, she stayed at the university of haifa (israel) in cooperation with in addition, she was working on the popularization of botany and was co-author of botany for kids which is translated into spanish, chinese, and turkish languages, and after that as a longtime editor of textbook editions of the publishing house creative center. we will remember her as an enthusiastic scientist and teacher, a careful person who enjoyed in work with young scientists and students. we worked 85 biologica nyssana ● 13 (2) december 2022: 83-87 džamić & alimpić aradski ● in memoriam professor sonja duletić-laušević (1962-2021) with her, discussed, made and achieved goals. she suggested to the future generation that „the challenge is to be a botanist and know that you belong to the few who feel at home among plants”. this sentence represents sonja’s thinking and we will keep her in our memory for a long time. bibliography books and practicums vukojević, j., duletić-laušević, s. 2004: patogene gljive povrća i voća u srbiji. nnk international, beograd. isbn 86-83635-30-9. petković, b., merkulov, lj., duletić-laušević, s. 2005: anatomija biljaka sa praktikumom. nnk international, beograd. isbn 86-907471-1-7. petković, b., merkulov, lj., duletić-laušević, s. 2005: morfologija biljaka sa praktikumom. nnk international, beograd. isbn 86-907471-2-5. duletić-laušević, s., janošević, d. 2008: botanika, edicija saznaj i probaj, kreativni centar, belgrade. petković, b., duletić-laušević, s. (2001): praktikum iz anatomije i morfologije biljaka. nnk international, beograd. isbn 86-83635-066. (14) duletić-laušević, s., janaćković, p., džamić, a., veljić, m., grujić, s., rajčević, n. 2019: praktikum iz botanike. biološki fakultet univerziteta u beogradu, beograd. isbn: 978-86-7078-153-5. petković, b., merkulov, lj., duletić-laušević, s. 2012: anatomija i morfologija biljaka sa praktikumom. biološki fakultet univerziteta u beogradu. isbn 978-86-7078-084-2. duletić-laušević, s., janaćković, p., grujićjovanović, s., marin, m., džamić, a., rajčević, n., gradojević, j. (2009): vizuelni rečnik anatomije i morfologije biljaka. biološki fakultet univerziteta u beogradu. digitalni sadržaj. chapters in monographs vukojević, j., muntañola-cvetković, m., duletićlaušević, s. 2002: diaporthe/phomopsis helianthi, the sunflower pathogen: twenty years of research. in: environment and crop production (dris, r., jain, s. m., khan, i. a. (eds.), crc press, taylor & francis group. pp. 99-110. isbn: 9781578082575. professor sonja with the closest collaborators (september 2014, university of belgrade) 86 biologica nyssana ● 13 (2) december 2022: 83-87 džamić & alimpić aradski ● in memoriam professor sonja duletić-laušević (1962-2021) duletić-laušević, s, vukojević, j., soković, m. 2005: fungi as causative agents of diseases of vegetables and fruits. in: crops, growth, quality & biotechnology. dris, r. (ed.) wfl publisher science and technology. pp. 405-424. isbn: 95291-8601-0. vukojević, j., duletić-laušević, s., glamočlija, j. 2005: pathogenic fungi of vegetables and rosaceae fruits. in: crops, growth, quality & biotechnology. dris, r. (ed.) wfl publisher science and technology. pp. 425-448. isbn: 95291-8601-0. alimpić aradski, a., oalđe, m., duletić-laušević, s. 2021: salvia officinalis: traditional medicinal plant with novel therapeutical perspectives. in: salvia officinalis: production, cultivation and uses, cameron ashton (ed.). nova science publishers, new york, usa. pp. 1-72. isbn: 978-1-53619-531-6. selected papers marin, p., sajdl, v., kapor, s., tatić, b., petković, b., duletić, s. 1992: fatty acids of the stachyoideae. biochemical systematics and ecology, 20(4): 389392. marin, p.d., petković, b., duletić, s. 1994: nutlet sculpturing of selected teucrium species (lamiaceae): a character of taxonomic significance. plant systematics and evolution, 192: 199-214. marin, m., koko, v., duletić-laušević, s., marin, p.d., rančić, d., dajić stevanović z. 2006: glandular trichomes on the leaves of rosmarinus officinalis: morphology, stereology and histochemistry. south african journal of botany, 72: 378-382. stajić, m., persky, l., hadar, y., friesem, d., duletić-laušević, s., wasser, s., nevo, e. 2006: effect of copper and manganese ions on activities of laccase and peroxidases in three pleurotus species grown on agricultural wastes. applied biochemistry and biotechnology 128(1): 87-96. marin m., koko, v., duletić-laušević, s., marin, p.d. 2008: micromorphology of trichomes of thymus malyi (lamiaceae). journal of microscopy, 232(3): 406-409. stajić, m., vukojević, j., duletić-laušević, s. 2009: biology of pleurotus eryngii and role in biotechnological processes: a review. critical reviews in biotechnology, 29(1): 55-66. stajić, m., kukavica, b., vukojević, j. simonić, j., veljović-jovanović, s., duletić-laušević, s. 2010: wheat straw conversion by enzymatic system of ganoderma lucidum. bioresources 5(4): 23622373. ćilerdžić, j., stajić, m., vukojević, j., duletićlaušević, s., knežević a. 2011: potential of trametes hirsuta to produce ligninolytic enzymes during degradation of agricultural residues. bioresources, 6(3): 2885-2895. stajić, m., vukojević, j., knežević, a., duletićlaušević, s., milovanović, i. 2013: antioxidant protective effects of mushroom metabolites. current topics in medicinal chemistry, 13(21): 2660-2676. al sheef, n.b., duletić-laušević, s., janošević, d., budimir, s., marin, m., alimpić, a., giweli, a.a.m., marin, p.d. 2013: micromorphology аnd ultrastructure of trichomes of libyan salvia fruticosa mill. archives of biological sciences, 65(1): 239-248. alimpić, a., pljevljakušić, d., šavikin, k., knežević, a., ćurčić, m., veličković, d., stević, t., petrović, g., matevski, v., vukojević, j., marković, s., marin, p. d., duletić-laušević, s. 2015: composition and biological effects of salvia ringens (lamiaceae) essential oil and extracts. industrial crops and products. 76: 702-709. alimpić, a., knežević, a., milutinović, m., stević, t., šavikin, k., stajić, m., marković, s., marin, p.d., matevski, v., duletić-laušević, s. 2017: biological activities and chemical composition of salvia amplexicaulis lam. extracts. industrial crops and products. 105: 1-9. alimpić, a., knežević, a., šavikin, k., ćurčić, m., veličković, d., stević, t., matevski, v., stajić, m., marković, s., marin, p.d., duletić-laušević, s. 2017: composition and biological activities of different extracts of salvia jurisicii, a rare and endemic macedonian species. plant biosystems, 151(6): 1002-1011. živković, j. č., barreira, j. c. m., šavikin, k., alimpić, a., stojković, d., dias, m. i., santosbuelga, c., duletić-laušević, s., ferreira, i. c. f. r. 2017: chemical profiling and assessment of antineurodegenerative and antioxidant properties of veronica teucrium l. and veronica jacquinii baumg. chemistry and biodiversity, 14(8): 1-11. šavikin, k., živković j., alimpić, a., zdunić, g., janković, t., duletić-laušević, s., menković, n. 2018: activity guided fractionation of pomegranate extract and its antioxidant, antidiabetic and antineurodegenerative properties. industrial crops and products. 113: 142-149. ćilerdžić, j. l., sofrenić, i.v., tešević, v. v., biologica nyssana ● 13 (2) december 2022: 83-87 džamić & alimpić aradski ● in memoriam professor sonja duletić-laušević (1962-2021) brčeski, i. d., duletić-laušević, s. n., vukojević. j. b., stajić, m. m. 2018: neuroprotective potential and chemical profile of alternatively cultivated ganoderma lucidum basidiocarps. chemistry and biodiversity, 15(5): e1800036. duletić-laušević, s. alimpić aradski, a., šavikin, k., knežević, a. milutinović, m., stević, t., vukojević, j., marković, s., marin, p. d. 2018: composition and biological activities of libyan salvia fruticosa mill. and s. lanigera poir. extracts. south african journal of botany, 117: 101-109. savić, a., jarić, s., dajić stevanović, z., duletićlaušević, s. 2019: ethnobotanical survey and traditional use of local pear varieties (pyrus communis l.) in polimlje, southwest serbia. genetic resources and crop evolution, 66(3): 589-609. alimpić aradski, a., janošević, d., pećinar, i., budimir, s., dajić stevanović, z., matevski, v., marin, p.d., duletić-laušević, s. 2021: micromorphological and anatomical characteristics of salvia amplexicaulis lam., s. jurisicii košanin and s. ringens sibth. & sm. (lamiaceae). plant biosystems, 155(1): 92-108. oalđe, m., kolarević, s., živković, j., alimpić aradski, a., marić, j. j., kolarević, m. k., đorđević, j., marin, p. d., šavikin, k., vuković-gačić, b., duletić-laušević, s. 2021: a comprehensive assessment of the chemical composition, antioxidant, genoprotective and antigenotoxic activities of lamiaceae species using different experimental models in vitro. food and function, 12: 3233-3245. oalđe pavlović, m., kolarević, s., đorđević, j., jovanović marić, j., lunić, t., mandić, m., kračun kolarević, m., živković, j., alimpić aradski, a., marin, p. d., šavikin, k., vuković-gačić, b., božić nedeljković, b., duletić-laušević, s. 2021: a study of phytochemistry, genoprotective activity, and antitumor effects of extracts of the selected lamiaceae species. plants, 10(11): 2306. 87 terzić et al. 2022, biologica nyssana 13(2) 13 (2) december 2022: 129-139 doi: 10.5281/zenodo.7437258 genetic potential of novi sad winter wheat varieties on smonica-type soil original article dragan terzić faculty of agriculture, university of niš, kosančićeva 4, 37000 kruševac, republic of serbia vera rajičić faculty of agriculture, university of niš, kosančićeva 4, 37000 kruševac, republic of serbia verarajicic@yahoo.com (corresponding author) milan biberdžić faculty of agriculture, university of priština-kosovska mitrovica, kopaonička bb, 38228 lešak, serbia vesna perišić faculty of agriculture, university of niš, kosančićeva 4, 37000 kruševac, republic of serbia marijana dugalić faculty of agriculture, university of niš, kosančićeva 4, 37000 kruševac, republic of serbia dragoslav đokić faculty of agriculture, university of niš, kosančićeva 4, 37000 kruševac, republic of serbia snežana branković university of kragujevac, faculty of science, department of biology and ecology, radoja domanovića 12, kragujevac, republic of serbia received: september 01, 2022 revised: october 27, 2022 accepted: november 04, 2022 abstract: the field experiment with the wheat varieties simonida, ns 40 s, zvezdana, ns ilina, ns futura, ns mila and ns obala was set up on the smonica-type soil during the 2016/17 and 2017/18 growing seasons. the aim of the research was to analyse the yield, weight of 1000 grains and hectolitre weight of seven novi sad wheat varieties grown on slightly acidic soil. the highest values of the observed properties were established in the 2017/18 vegetation year with moderate temperatures and a higher amount of precipitation. varieties ns futura, ns obala and ns 40 s had the highest grain yield during the two-year study. the simonida variety was characterized by the highest hectolitre weight. the highest values of hectolitre weight were found in the zvezdana variety in both study years. the analysis of variance established a highly significant influence of the interaction of agro climatic conditions and genotype on the weight of 1000 grains and the hectolitre weight of the tested varieties of winter wheat. key words: grain yield, hectolitre weight, variety, wheat apstrakt: genetski potencijal novosadskih sorti ozime pšenice na zemljištu tipa smonice poljski ogled sa sortama pšenice simonida, ns 40 s, zvezdana, ilina, futura, mila i obala postavljen je na zemljištu tipa smonica tokom vegetacionih sezona 2016/17 i 2017/18 godine. cilj istraživanja je bio da se kod sedam novosadskih sorti pšenice gajene na slabo kiselom zemljištu analizira prinos, masa 1000 zrna i hektolitarska masa. najveće vrednosti ispitivanih osobina ustanovljene su u godini sa umerenim temperaturama i većom količinom padavina u vegetacionoj 2017/18 godini. sorte ns futura, ns obala i ns 40 s imale su najveći prinos zrna tokom dvogodišnjeg istraživanja. sorta simonida odlikovala se najvećom hektolitarskom masom. najveće vrednosti hektolitarske mase ustanovljene su kod sorte zvezdana u obe ispitivane godine. analizom varijanse ustanovljen je visoko značajan uticaj interakcije agroklimatskih uslova i genotipa na masu 1000 zrna i hektolitarsku masu kod ispitivanih sorti ozime pšenice. ključne reči: hektolitarska masa, prinos zrna, pšenica, sorta introduction winter wheat (triticum aestivum l.) is one of the most important agricultural crops in serbia. according to fao data, in year 2017 in the republic of serbia, wheat was harvested from 556,115 hectares with an average yield of 4.092 t/ha, while in 2018 it was harvested from 643,083 hectares with an average yield of 4.574 t/ha. statistical data show that in the past two-year period, wheat in the world has been grown on over 216,120,193 hectares with an average yield of 3,480 t/ha, while in the republic of serbia in the same period, the area under wheat has amounted to 599,599 ha with an average yield of 4,333 t/ha (fao, 2021). the average yields of wheat for the last 10 years in the main production areas of serbia have ranged from 4.5-8.0 t/ha. wheat production in serbia largely depends on external environmental factors. the average grain yields of winter wheat in our country and the fertility potential of cultivated varieties differ significantly, especially in the mountainous regions of serbia (jelić et al., 2015, 2016; jevtić & đekić, 2018). the yield and yield components of © 2022 terzić et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 129 winter wheat vary significantly depending on the cultivation system, applied nitrogen doses, variety and conditions present during growing season, as well as their complex interactions (đekić et al., 2012; tmušić et al., 2021; rajičić et al., 2021). in addition to the genotype, the grain yield of winter wheat is greatly influenced by the fertilization system, as one of the key factors that affects the formed yield and its quality, but it should be harmonized with climatic and soil conditions, as well as the requirements of the variety (đekić et al., 2014; terzić et al., 2018; rajičić et al., 2019; biberdžić et al., 2020b; đurić et al., 2020; luković et al., 2020). in the production of wheat, proper zoning of varieties is very important, and it can contribute to less variation in realized yields and achieving better average results (đurić et al., 2018; terzić et al., 2018; rajičić et al., 2020; grčak et al., 2020). with all that in mind, it is necessary that the climatic conditions are in accordance with the biological requirements of the plants. in the last few years, extreme temperatures and disturbances in the amount and distribution of precipitation have significantly influenced the reduction of the total production of organic matter and the reduction of yields (đurić et al., 2016; luković et al., 2017; grčak et al., 2018; đekić et al., 2019; popović et al., 2020; biberdžić et al., 2021b). the aim of the study was to analyse grain yield and some yield traits and examine the influence of varieties and ecological and environmental factors on the differences in stability and adaptability of the tested winter wheat genotypes. during two growing seasons, in field trials, seven winter wheat genotypes were examined in order to determine the selection of better varieties for production conditions in western serbia. materials and methods materials and field trials the trial was carried out on the experimental field of the secondary agricultural school with the student home “ljubo mićić” in požega (43°50´41´´n latitude, 20°02´11´´e longitude, 310 m above sea level), town and municipality in the zlatibor district of western serbia. during two growing seasons (2016/17 and 2017/18), in conditions of dry crop production, tests were carried out with the aim to analyse the yield and quality of winter wheat grains and determine and choose better varieties for production conditions in western serbia. the experiment was set up according to the random block system in four repetitions, with the size of the elementary plot 100 m2 (10 x 10 m). the soil on which the experiment was set up was the smonica-type soil. the main crop (preceding crop) in both years was corn. sowing was done with a small mechanical seeder in both years in the middle of the third decade of october, at an inter-row distance of 12.5 cm and 3 cm in a row. the amount of seeds per square meter was 400-450 germinated seeds, depending on the characteristics of the variety. the total amount of fertilizer 300 kg/ ha npk 15:15:15, where one third of nitrogen was distributed by hand on the ploughing surface before the pre-sowing soil preparation. at the beginning of intensive plant growth, in the 2-3 leaf phase, at the beginning of march, 200 kg/ha of kan (cancalcium-ammonium-nitrate, 27% n) was applied. wheat varieties simonida, ns 40 s, zvezdana, ns ilina, ns futura, ns mila and ns obala of the institute of field and vegetable crops, novi sad, were selected as material for the trial. the following properties were analysed: grain yield (t/ha), weight of 1000 grains (g) and hectolitre weight (kg/hl). at the stage of full maturity, a sample of 30 plants from five plots was taken to determine the weight of 1000 grains. after harvesting, the grain yield from each plot was measured and converted to yield in t/ha based on 14% grain moisture, after which a sample was taken for analysis of the hectolitre weight of wheat. soil conditions the trial was carried out on the experimental field of the secondary agricultural school with the student home “ljubo mićić” in požega, on a plot of smonicatype soil. the soil on which the research was carried out has unfavourable physical properties, poor water-air regime with frequent water deficiency. the soil is slightly acidic (ph<6.5), compact, with a high content of silt and clay particles and slow percolation of water. the soil is well supplied with humus (3.1%) and has good microbiological activity, due to the introduction of manure. it is characterized by a low content of available phosphorus (10.7 mg/100 g of soil) and an optimal content of potassium (20 mg/100 g of soil). meteorological conditions the požega area is characterized by a moderately continental climate, with an uneven monthly distribution of precipitation. the latitude of the meteorological station in požega is 43°50´41´´n, the longitude is 20°02´11´´e, and the altitude is 310 m above sea level. on the basis of the data provided by the meteorological station, the years in which the surveys were conducted differed from the multiannual average for the given area. the average air temperature in 2016/17 was lower by 0.33 ºc, while in 2017/18 it was higher by 0.91 ºc compared to the 130 biologica nyssana ● 13 (2) december 2022: 129-139 terzić et al. ● genetic potential of novi sad winter wheat varieties on smonica-type soil 131 multi-annual average (tab. 1). the data presented in tab. 1 for the studied vegetation period (2016-2018) clearly indicate that the years in which the tests were performed differed in terms of meteorological conditions from the multiannual average characteristic of this area. the total amount of precipitation during the trial was below the multi-annual average in the 2016/17 vegetation season, with a rather uneven distribution by month (501.1 mm), while in the same period during the second vegetation season years 2017/18, the amount of precipitation was higher and amounted to 605.9 mm, with an even monthly distribution (tab. 1). weather conditions in the growing season in 2016 and 2017 were marked by a high amount of precipitation in october, while in may 2016 and 2017 there was less precipitation compared to the multi-annual average. may 2017 and 2018 and june 2017 were characterized by changeable and moderately warm weather with less precipitation than average. the vegetation period 2016/17 was rainy, especially from the beginning of heading until harvest (may–june). in the same year, precipitation was lower than the multi-annual average from december to march. during the tillering period, which took place during march, the applied amounts of nitrogen fertilizers in the soil were not sufficiently used due to insufficient moisture, as the nitrogen fertilizer did not change into easily accessible forms for the plant and was not used by the plants. nitrogen, due to insufficient moisture, did not play the role of yield builder, which resulted in a decrease in yield in year 2017. in april, precipitation was above the multi-annual average. the total amount of precipitation was 39.4 mm less than the multiannual average (tab. 1). conditions for germination and initial growth of plants in the first year (2017/18) were significantly more favourable. the period from november to february was favourable for germination, sprouting and tillering of wheat. the continuation of tillering and the beginning of stem elongation during the month of march was with a slightly higher amount of precipitation compared to the multi-annual average. in the month of march, the precipitation was above the multi-annual average, which had a favourable effect on the period of fertilization and filling of grains (tab. 1). changeable and moderately warm weather with more precipitation than average marked june 2018. in the growing season 2017/18, the sum of precipitation was higher than the multiannual average by 65.4 mm. based on the fact that the amount of precipitation and temperature in the period march–june are very important for the development of wheat, the second year can be characterized as more favourable in terms of the distribution and amount of precipitation. temperature variations on average were greater in the first compared to the second growing season. vegetation period of wheat during testing in 2017/18 mostly took place in conditions of higher temperatures compared to the multi-annual average (tab. 1). relatively warm weather during the entire vegetation period with a relatively sufficient amount of precipitation enabled intensive germination, sprouting/shooting and normal development of all phases of winter wheat plant development. in the final stages of growth and development, during the month of june, high temperatures contributed to the early drying of leaves and accelerated ripening. statistical analysis experimental data were analysed by descriptive and analytical statistics using the statistics module analyst program genstat (2013) for pc/windows 7. the usual variational statistical indicator was calculated, average value (x), as well as standard deviation (s) and standard error (sx). evaluation of significance was made on the basis of the anova test at 5% and 1% significance levels. relative biologica nyssana ● 13 (2) december 2022: 129-139 terzić et al. ● genetic potential of novi sad winter wheat varieties on smonica-type soil table 1. mean monthly air temperatures and precipitations in požega, serbia months interval x xi xii i ii iii iv v vi average mean monthly air temperature (oc) 2016/17 9.7 4.6 -1.5 -6.6 2.9 8.3 9.4 15.3 20.4 6.94 2017/18 10.1 4.5 2.1 0.4 0.8 4.4 14.5 17.5 19.3 8.18 average 10.4 4.9 -0.1 -1.3 1.0 5.7 10.6 15.2 19.0 7.27 the amount of precipitation (mm) 2016/17 81.0 85.9 9.1 20.6 30.9 35.9 76.7 76.8 84.2 501.1 2017/18 108.1 29.8 68.9 48.5 76.7 109.6 20.4 48.5 95.4 605.9 average 60.6 54.3 54.3 41.1 46.1 52.7 60.1 82.2 89.1 540.5 132 dependence was defined through correlation analysis (pearson’s coefficient), and the values of coefficient that were obtained were tested at the 5% and 1% levels of significance. results grain yield and yield components the average values of the yield, weight of 1000 grains and hectolitre weight of the tested wheat varieties of the institute of field and vegetable crops from novi sad are shown in tab. 2. grain yield: in regard to grain yield, differences were observed between the tested varieties. the average yield of wheat for all tested genotypes in the experiment was 5.188 t/ha. the higher average grain yield of 5.585 t/ha of the tested wheat varieties was achieved in the 2017/18 growing season and was higher by 793 kg/ha compared to the yield in vegetation season 2016/17 (4.792 t/ha), which can mainly be related to the adequate distribution of precipitation during the second vegetation period (tab. 2). the highest grain yield in the 2016/17 vegetation season was realized with the ns futura variety (5.175 t/ha), but a good yield was also achieved with the ns obala variety (5.025 t/ha). in the second study year, the ns futura variety had the highest average grain yield (6.087 t/ha), but a good yield was also achieved with the ns obala (5.802 t/ha) and ns 40 s (5.697 t/ha) varieties. the variety ns fubiologica nyssana ● 13 (2) december 2022: 129-139 terzić et al. ● genetic potential of novi sad winter wheat varieties on smonica-type soil table 2. average values of investigated winter wheat varieties traits (x-average value, s-standard deviation and sx-standard error) varieties 2016/17 2017/18 average x s sx x s sx x s sx yield, t/ha simonida 4.253 0.251 0.126 5.447 0.329 0.165 4.850 0.693 0.245 ns 40 s 4.876 0.517 0.259 5.697 0.517 0.259 5.286 0.650 0.230 zvezdana 4.523 0.468 0.234 5.556 0.444 0.222 5.040 0.695 0.246 ns ilina 4.892 0.450 0.225 5.326 0.540 0.270 5.109 0.515 0.182 ns futura 5.175 0.192 0.096 6.087 0.580 0.290 5.631 0.631 0.223 ns mila 4.799 0.502 0.251 5.178 0.319 0.160 4.989 0.439 0.155 ns obala 5.025 0.126 0.063 5.802 0.580 0.290 5.414 0.569 0.201 average 4.792 0.451 0.085 5.585 0.516 0.097 5.188 0.625 0.083 1000 grain weight, g simonida 43.45 1.072 0.536 50.73 0.348 0.174 47.09 3.965 1.402 ns 40 s 42.72 0.644 0.322 44.96 1.173 0.586 43.84 1.485 0.525 zvezdana 41.03 1.757 0.879 43.52 1.683 0.841 42.28 2.076 0.734 ns ilina 36.72 0.769 0.384 43.22 1.408 0.704 39.97 3.632 1.284 ns futura 42.54 0.853 0.426 44.36 0.844 0.422 43.45 1.251 0.442 ns mila 42.03 0.755 0.378 41.44 0.779 0.390 41.73 0.778 0.275 ns obala 41.64 1.134 0.567 44.77 0.505 0.253 43.20 1.860 0.658 average 41.44 2.296 0.434 44.71 2.899 0.548 43.08 3.072 0.410 hectolitre weight, kg/hl simonida 75.05 0.816 0.408 77.00 0.252 0.126 76.03 1.183 0.418 ns 40 s 74.50 0.443 0.222 78.16 0.278 0.139 76.33 1.987 0.703 zvezdana 77.35 0.476 0.238 79.00 0.503 0.252 78.18 0.992 0.351 ns ilina 71.90 2.253 1.127 78.18 0.708 0.354 75.04 3.693 1.306 ns futura 74.60 0.755 0.378 77.55 0.258 0.129 76.08 1.661 0.587 ns mila 70.90 1.969 0.984 76.70 1.112 0.556 73.80 3.435 1.215 ns obala 73.65 0.231 0.115 73.80 0.252 0.126 73.73 0.237 0.084 average 73.99 2.280 0.431 77.20 1.668 0.315 75.60 2.556 0.341 133 biologica nyssana ● 13 (2) december 2022: 129-139 terzić et al. ● genetic potential of novi sad winter wheat varieties on smonica-type soil tura had the highest average grain yield during the two-year study (5.631 t/ha), and varieties simonida (4.850 t/ha) and ns mila (4.989 t/ha) the lowest. 1000 grain weight: the grain of the tested wheat varieties had good physical properties, especially the 1000 grain weight. the weight of 1000 grains in 2017/18 was significantly higher than in 2016/17 (tab. 2). the vegetation period in year 2017, at the time of grain filling, was marked by drought and high temperatures, which affected the reduction of 1000 grain weight. the highest 1000 grain weight during the two-year research period was recorded for the simonida variety (47.09 g), and the lowest for the ns ilina variety (39.97 g). the simonida variety had the highest 1000 grain weight in the 2017 vegetation year (43.45 g), and the ns ilina variety had the lowest (36.72 g). in the second year of research, the simonida variety had the highest 1000 grain weight (50.73 g), and the lowest was recorded for the ns mila variety (41.44 g). test weight: hectolitre weight is an indicator of grain quality, especially its monetary value. the obtained data on the hectolitre weight, regardless of the year, showed that there was a significant difference between the genotypes, with the zvezdana variety having the highest hectolitre weight (78.18 kg/hl) on average for the trial years. the average value of hectolitre weight during the two-year research was 75.60 kg/hl. the higher average value of hectolitre weight was achieved in the 2017/18 growing season (77.20 kg/hl) compared to the in-vegetation season 2016/17 (73.99 kg/hl), (tab. 2). in the first research year, the zvezdana variety had the highest hectolitre weight (77.35 kg/hl), while in the second year, the highest hectolitre weight was observed for the zvezdana variety (79.00 kg/hl) and with a slightly lower value for the varieties ns ilina (78.18 kg/hl) and ns 40 s (78.16 kg/hl). analysis of variance between observed traits of wheat the analysis of yield variance, weight of 1000 grains and hectolitre weight of the tested winter wheat varieties from novi sad during two vegetation seasons are shown in tab. 3. the influence of year and variety and their interactions on the tested properties of winter wheat are shown in tab 3. based on the analysis of variance, it can be concluded that the interaction of variety and year in tested wheat varieties did not significantly affect the grain yield, while it had a very significant effect on 1000 grain weight (fexp=13.456**) and hectolitre weight (fexp=10.742**) in tested varieties of winter wheat. the influence of year on the yield, 1000 grain weight and hectolitre weight of tested winter wheat varieties was highly significant. no significant influence of the variety on grain yield was found, among the tested wheat genotypes, while highly significant differences were found in the weight of 1000 grains and hectolitre weight. correlation dependence between tested wheat traits the average values of pearson’s correlation coeftable 3. analysis of variance of the tested parameters (anova) *statistically significant (p<0.05); **statistically high significant (p<0.01) effect of year on the traits analyzed traits mean sqr effect mean sqr error f (1.54) p-level grain yield (t/ha) 8.801 0.234 37.524 0.000** 1000 grain weight (g) 149.635 6.839 21.879 0.000** hectolitre weight (kg/hl) 143.840 3.990 36.052 0.000** effect of genotype on the traits analyzed traits mean sqr effect mean sqr error f (6.49) p-level grain yield (t/ha) 0.585 0.366 1.597 0.168 ns 1000 grain weight (g) 38.595 5.865 6.581 0.000** hectolitre weight (kg/hl) 19.525 4.942 3.951 0.003** effect of the year x genotype interaction traits mean sqr effect mean sqr error f (6.42) p-level grain yield (t/ha) 0.178 0.192 0.924 0.488 ns 1000 grain weight (g) 15.101 1.122 13.456 0.000** hectolitre weight (kg/hl) 9.920 0.923 10.742 0.000** ficient (r) of the tested traits of winter wheat are shown in tab. 4. correlations between grain yield and hectolitre weight of the tested genotypes of winter wheat at the požega location show a negative value in both investigated vegetation seasons. correlations between grain yield and 1000 grain weight show a negative value in the first year of research (2016/17), while correlations between 1000 grain weight and hectolitre weight show a negative value in the vegetation year 2017/18. correlations between grain yield and 1000 grain weight and between the 1000 grain weight and hectolitre weight of winter wheat genotypes tested at the požega location during the two-year study show a positive and highly significant value. correlations between grain yield and hectolitreweight, during the two-year study, show a positive and significant value. discussion adverse weather conditions such as frost and temperature stress, and short periods with high temperatures, have a strong negative impact on plant production and represent a great risk (rajičić et al., 2020; biberdžić et al., 2021a; ljubičić et al., 2021). according to lalić et al. (2014), the negative impact of high temperatures on winter wheat is reflected in a reduced number of grains, an increase in the intensity of grain filling (up to a certain limit), as well as a shortening of the duration of the filling period, which results in a decrease in yield when temperatures exceed the critical level above 35 oc. in numerous studies, soil drying or shortening of phenophases during the growing season are reported as the cause of winter wheat yield reduction with increasing temperatures (mohamed et al., 2013). in their research, ottman et al. (2012), report that wheat yield decreased by 6.9% with each degree increase in temperature during the season when it exceeded 16.3 °c. a 2.5 °c increase in night-time temperatures in northern china resulted in a 27% drop in wheat yields (fang et al., 2010). the significant deviation of precipitation and temperature from the multi-annual average is becoming more pronounced (dimitrijević et al., 2011; jocković et al., 2014). it has been established that the newly created high-yielding varieties of wheat react less to temperature deviations (except for extremes), compared to precipitation (milovanović et al., 2012; đekić et al., 2015, 2017). namely, the total amount of precipitation is maintained at the multi-annual average, but the distribution, especially in critical stages of development, is significantly disturbed (đekić et al., 2013; zafaranaderi et al., 2013). it has been established that winter precipitation significantly affects the realization of the production potential of wheat (laghari et al., 2011; luković et al., 2016). in addition to the necessary reserve for the spring part of the vegetation, winter precipitation greatly affects the distribution of easily accessible nitrogen in the soil (biberdžić et al., 2014; terzić et al., 2018; rajičić et al., 2019). the variety, as an autonomous genetic, biological, and agronomic entity, is one of the decisive factors on both the quantitative and qualitative level of production (đurić et al., 2020). the increase in 134 biologica nyssana ● 13 (2) december 2022: 129-139 terzić et al. ● genetic potential of novi sad winter wheat varieties on smonica-type soil table 4. correlation coefficients by studied environments in winter wheat *statistically significant (p<0.05); **statistically high significant (p<0.01) grain yield, gy 1000 grain weight, gw hectolitre weight, hw correlations between the traits analysed in the 2016/17 grain yield, gy 1.00 -0.037 -0.232 1000 grain weight, gw 0.295 hectolitre weight, hw 1.00 correlations between the traits analysed in the 2017/18 grain yield, gy 1.00 0.045 -0.129 1000 grain weight, gw -0.066 hectolitre weight, hw 1.00 correlations between the traits analysed in the 2016-2018 grain yield, gy 1.00 0.351** 0.297* 1000 grain weight, gw 0.416** hectolitre weight, hw 1.00 135 biologica nyssana ● 13 (2) december 2022: 129-139 terzić et al. ● genetic potential of novi sad winter wheat varieties on smonica-type soil wheat yield primarily depends on the cultivated variety, climatic conditions and applied cultivation technology (jelić et al., 2015; đekić et al., 2018). the introduction into production of varieties with increased genetic potential for yield and improved agronomic and technological properties represents the contribution of breeding to achieving higher production per unit area (perišić et al., 2016; branković et al., 2018; đurić et al., 2018). the genetic potential for yield can be increased in different ways: better use of genetic variability, better use of solar energy, increasing the number and weight of grains, increasing the total biomass of the plant, using heterosis, i.e., wheat hybrid (đurić et al., 2016). grain yield: grain yield is a complex property because it depends on several factors, primarily: genotype, external environment, and their interaction. a high and stable yield, first of all, must be economically justified, which can be achieved, by respecting varietal agrotechnics and with favourable agro climatic conditions. the results of our two-year study show that the grain yield of the tested wheat varieties was 5.188 t/ha (tab. 2). the 2018 production year had a higher average grain yield compared to the 2017 production year, which was mostly contributed by favorable weather conditions. the yield of winter wheat in 2018 was 5.585 t/ha and varied in the range from 5.178 t/ha to 6.087 t/ha. the lower quality and quantity of the yield in the 2017 production year was influenced by unfavourable temperature conditions and uneven distribution of precipitation in periods of the year that coincided with the sensitive stages of wheat development, which affected the yield, which was 4.792 t/ha (tab. 2). temperature, precipitation, and sufficient amount of water in the soil are the three most important reasons for yield instability in our area. in the ecological conditions of serbia, high temperatures and water deficit during the may-june period led to a reduction in yield and a deterioration in the technological properties of grain, which is why by extending the total vegetation period the period of grain filling for the purpose of yield increase cannot be extended. živanović et al. (2017), show slightly lower wheat yields on vertisol-type land in šumadija (from 4.31 t/ha to 4.44 t/ha), obtained during year 2017. đurić et al. (2013), state that the yields in the trial were very high and ranged from 8.00 t/ha of grain in case of the variety esperia to 9.60 t/ha in pobeda and talas varieties. however, in the first three varieties talas, victory and ns 40 s, difference in yield was about 400 kg and that is negligible considering the total yield. hristov et al. (2013) state that in a threeyear period, all varieties, except pobeda, achieved a higher yield at a higher density. the lower yield of the pobeda variety was primarily the result of longstanding water on the plot. also, with the exception of the problematic year 2010, varieties pobeda (10.76 t/ha) and renesansa (11.05 t/ha) achieved higher yields in 2011, indicating that in order to make correct conclusions, it is necessary to analyse a larger number of localities over a longer period of time. đekić et al. (2013), examined five winter wheat varieties (takovčanka, kg 100, kg 56s, ana morava and lazarica). the average grain yield of the tested wheat varieties ranged from 3.011 t/ha to 3.774 t/ha. in regard to the physical characteristics of the grain, the ana morava variety achieved the highest average yield in both growing seasons (4.499 t/ha and 3.049 t/ha). terzić et al. (2018) point out that the average yield of wheat in the trial was 5.091 t/ha, ranging from 4.630 t/ha in the second year to 5.553 t/ha in the first year of the study. the highest grain yield in the 2010/11 vegetation year was established with the vizija variety (6.127 t/ha), but a good yield was also achieved with the kg 56s variety (5.863 t/ha). in the second year of research, the variety kg 56s had the highest average grain yield (5.159 t/ha). the highest average grain yield in the two-year period was observed for the varieties kg 56s (5.511 t/ha) and vizija (5.485 t/ha), and the lowest for the variety aleksandra (4.628 t/ha). đurić et al. (2016) point out that the pkb vizantija variety achieved an average grain yield of 8.356 t/ ha. rajičić et al. (2021) have found that the highest winter wheat yield of 4.673 t/ha was obtained with the renesansa variety. analysis of variance showed a very significant influence of vegetation on wheat yield. 1000 grain weight: the 1000 grain weight is a direct component of yield and at the same time an important indicator of grain quality. it represents a highly variable property which depends to a high degree on the conditions of the external environment. numerous authors (milovanović et al., 2011; đekić et al., 2012; đurić et al., 2013) have pointed out that the 1000 grain weight is a varietal characteristic and that there is a significantly greater variation between different genotypes than between applied treatments or external environmental factors. during the two-year trial, the grain of the tested wheat varieties was characterized by good physical properties, especially the 1000 grain weight. the 1000 grain weight during the two-year research period was 43.08 g, ranging from 39.97 g to 47.09 g. the obtained values are close to the values obtained by jevtić and đekić (2018) and terzić et al. (2018), and slightly higher than the results obtained by biberdžić et al. (2020a). đurić et al. (2013) state that the weight of 1000 grains ranged from 38.80 biologica nyssana ● 13 (2) december 2022: 129-139 terzić et al. ● genetic potential of novi sad winter wheat varieties on smonica-type soil g in the variety ns 40 s to 46.50 g in the variety pobeda. đekić et al. (2013) point out that the average value of the 1000 grain weight in the tested wheat varieties ranged from 36.23 to 42.70 g. examining different variants of fertilization in the wheat variety takovčanka, đekić et al. (2014) have found that the weight of 1000 grains was the highest with balanced fertilization with all three nutrients in the variant n80p60k60 (44.46 g) and variant n80p100k60 (43.38 g). đekić et al. (2015a), point out that in the first year of research, the vizija variety had the highest weight of 1000 grains (42.65 g), while the kruna variety had a slightly lower value (41.63 g). in the second year of research, the weight of 1000 grains of the vizija variety was by 1.036 g higher than the kruna variety. đekić et al. (2015b), have examined six winter wheat varieties from kragujevac, with the aim of determining and choosing the best varieties for serbia’s production conditions. based on a twoyear study, they concluded that the highest two-year average value of the weight of 1000 grains was observed in the variety planeta (44.22 g), while the lowest was reported for the variety vizija (37.53 g). the results of the research by jelic et al. (2015), show that the highest 1000 grain weight of 47.06 g was achieved with the complete application of mineral npk, lime and manure with a higher dose of caco3 of 5 t/ha and phosphorus of 100 kg/ha p2o5. terzić et al. (2018) point out that the average weight of 1000 grains was 43.13 g, ranging from 42.05 g to 44.21 g. in the study of five varieties of wheat (kruna, renesansa, pobeda, ns 40 s and takovčanka) during two growing seasons in the agro ecological conditions of western serbia, rajičić et al. (2021) have found that the highest weight of 1000 grains was obtained in case of the variety renesansa (43.72 g). by analysis of variance, the same authors have found a highly significant influence of genotype on the weight of 1000 grains. hectolitre weight: hectolitre weight is a complex quantitative trait that is determined by the action of a large number of genes under the strong influence of the external environment (đurić et al., 2013). the average value of hectolitre weight during the twoyear research was 75.60 kg/hl, ranging from 73.73 kg/hl to 78.18 kg/hl. the highest value of hectolitre weight in both examined years was achieved by the variety zvezdana (77.35 kg/hl and 79.00 kg/hl). the obtained hectolitre weight values were slightly higher than the values obtained by jelic et al. (2015), jevtić and đekić (2018) and biberdžić et al. (2020a), and lower than the results obtained by đurić et al. (2013). it is generally considered that grain with a higher hectolitre weight is of better quality compared to that with lower values (đurić et al., 2013; đekić et al., 2015b; rajičić et al., 2021). đurić et al. (2013) state that the obtained hectolitre weight values are relatively high, which indicates a good marketable quality, ranging from 78.9 kg/hl for the ns 40 s variety to 84.4 kg/hl for the merkur variety. đekić et al. (2014) have found the highest value of hectolitre weight in the variant with individual fertilization with 60 kg/ha of phosphorus (71.57 kg/hl) and in the variant that was fertilized with 80 kg/ha of nitrogen, 60 kg/ha of phosphorus and 60 kg/ha of potassium (71.49 kg/hl). by evaluating the significance, they have found statistically very significant differences for the hectolitre weight between the tested fertilization variants. đekić et al. (2015b) by using the analysis of variance, have established highly significant differences for the hectolitre weight values between the tested wheat varieties. according to the results of jelić et al. (2015), observed by fertilization variants, the highest hectolitre weight of grain was recorded for variant f5 (70.91 kg/hl), which was fertilized with 120 kg/ha n, 100 kg/ha p2o5, 60 kg/ha k2o, 5.0 t/ha of caco3 and with 20 t/ha of manure. jevtić and đekić (2018) point out that the hectolitre grain weight of the tested wheat varieties, during the two-year study, ranged from 69.5 to 71.03 kg/hl. regarding the optimal wheat sowing date, biberdžić et al. (2020a) have found that the hectolitre weight of grain was 71.42 kg/hl, while in the case of later sowing it was 69.96 kg/hl, which does not represent a statistically significant difference. rajičić et al. (2021) have found that the highest two-year average value of hectolitre weight was observed for varieties kruna and pobeda (77.52 kg/hl and 77.31 kg/hl, respectively). they established a very significant influence of vegetation and genotype on the hectolitre weight of wheat. conclusions based on the obtained results, it can be concluded that the winter wheat variety ns futura achieved the best results in terms of grain yield, while the simonida variety had the highest of 1000 grain weight, and the variety zvezdana had the highest hectolitre weight in the observed two-year period. the highest values for yield of wheat grains, 1000 grain weight and hectolitre weight, were observed in the 2017/18 vegetation season with moderate temperatures at the time of grain filling and a large amount of precipitation evenly distributed in the second part of the vegetation season. the grain yield of the tested wheat varieties in the two-year period ranged from 4.850 t/ha (simonida) to 5.631 t/ha (ns futura). the average hectolitre weight during the two-year research was 75.60 kg/hl, with 73.99 kg/hl in the 2016/17 growing season and 77.20 kg/hl in the 136 2017/18 vegetation season. grain yield showed a tendency to increase in years with higher sum of precipitation and better distribution of rainfall during critical stages of plant development. the analysis of variance established a very significant influence of the interaction of year x variety on the weight of 1000 grains and the hectolitre weight of the examined wheat varieties, while the influence of the vegetation season on all the examined traits was statistically justified. many traits play a decisive role in the formation of grain yield. the contribution of each individual trait can be different in different genotypes and in different environmental conditions. based on the obtained results, it can be concluded that the sowing structure should be based on more than one variety, in order to reduce the risk of the unpredictability of each individual vegetation, regardless of the reliability of the criteria for selection of variety for sowing in a particular research year. acknowledgements. ph.d. dragan terzić and authors sincerely thanks to the secondary agricultural school with the student dormitory “ljubo mićić” in požega in serbia, which has enabled the use and analysis of traits of the wheat. investigations necessary for this paper are part of the projects agreement number 451-03-09/202114/200383 financed by the ministry of education, science and technology development of republic of serbia. references biberdžić, m., jelić, m., knežević, b., barać, s., lalević, d. 2014: yield of wheat in pseudogley in dependence of fertilisation with mineral fertilisers, lime fertiliser and manure. agriculture & forestry, 60(4): 7-13. biberdžić, m., barać, s., deletić, n., stojković, s., madić, m., lalević, d., đekić, v., stojiljković, j. 2020a: the effect of sowing time on the yield of some wheat varieties. 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december 2018: 63-75 bogosavljević, zlatković ⚫ report on the new floristic data from serbia ii 63 original article received: 15 november 2018 revised: 29 november 2018 accepted: 12 december 2018 report on the new floristic data from serbia ii stefan bogosavljević1, bojan zlatković2 1lilly drogerie, health institution, trg oslobođenja 14, 19350 knjaževac, serbia 2 university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia * e-mail: stefan.bogosavljevic@gmail.com abstract: bogosavljević, s., zlatković, b.: report on the new floristic data from serbia ii. biologica nyssana, 9 (2). december, 2018: 63-75. this paper presents new chorological data for 16 taxa of vascular flora of serbia (records no. 1-16). these taxa belong to following families: amaryllidaceae (1-2), apiaceae (3), aristolochiaceae (4), aspleniaceae (5), asteraceae (6-9), iridaceae (11), juncaceae (12), ophioglossaceae (13), ranunculaceae (14), rutaceae (15) and urticaceae (16), also including the possibly first record of opuntia humifusa (raf.) raf., cactaceae (10) in serbia. key words: distribution, chorological data ii, serbia, vascular flora apstrakt: bogosavljević, s., zlatković, b.: izveštaj o novim florističkim podacima iz srbije ii. biologica nyssana, 9 (2). decembar, 2018: 63-75. prikazani su novi horološki podaci za 16 taksona vaskularne flore srbije (izveštaji br. 1-16). taksoni pripadaju sledećim familijama: amaryllidaceae (1-2), apiaceae (3), aristolochiaceae (4), aspleniaceae (5), asteraceae (6-9), iridaceae (11), juncaceae (12), ophioglossaceae (13), ranunculaceae (14), rutaceae (15) i urticaceae (16), uključujući i najverovatnije prvi podatak o prisustvu vrste opuntia humifusa (raf.) raf., cactaceae (10) u srbiji. ključne reči: distribucija, horološki podaci ii, srbija, vaskularna flora introduction this study represents a continuation of chorological studies of vascular flora of serbia. new distribution data are presented for 14 plant species and 2 subspecies, including representatives of rare, threatened and protected taxa as well as some invasive species in process of range increase. these data may provide additional information on distribution of taxa included in the edition “flora of 9 (2) • december 2018: 63-75 doi: 10.5281/zenodo.2538596 biologica nyssana 9 (2) ⚫ december 2018: 63-75 bogosavljević, zlatković ⚫ report on the new floristic data from serbia ii 64 sr serbia (i-x)” (josifović ed., 1970-1977; diklić, 1977; nikolić et al., 1986; sarić & diklić, 1986), as well as the taxa that will be included in the new edition “the flora of serbia” (stevanović ed., 1992, 2012). in addition, survey of existing literature yielded no previous data on appearance of the invasive species opuntia humifusa (raf.) raf. (cactaceae) in serbia, so we believe that this was probably the first scientific set of data on its presence in this country. material and methods identification of collected plant material was performed according to josifović ed. (1970-1977), stevanović, ed. (1992, 2012), tutin et al. (1964), nardi (1984), trinajstić (1990), majure et al. (2017) and pardo & alonso (2017). in addition, some of collected specimens (aristolochia lutea desf.) were compared to virtual collections at herbarium of institute of botany, university of vienna (wu) and herbarium of botanic garden and botanical museum berlin-dahlem (b) (http://herbarium.univie.ac.at/ database/search.php). the description of species opuntia humifusa was based on literature sources (petrova et al., 2013; majure et al., 2017) and combined with observations of collected material. plant material was preserved and stored at the herbarium of the faculty of sciences and mathematics, department of biology and ecology, university of niš (hmn), with acquisition numbers given in the brackets. invasiveness status was determined using the terminology according to lambdon et al. (2008). distribution of the studied taxa within the territory of serbia was determined and mapped in utm grid system (10 x 10 sq. km., utm zone 34t) (lampinen, 2001). the nomenclature and classification of taxa were matched with the plant list database (http://www.theplantlist.org/). in order to identify regions of the data occurrence in serbia we used a geographic regionalization according to marković (1970), modified by stevanović ed. (1999). data on population (subpopulation) vulnerability, number of individuals and habitats of the species were based on personal observations. results and discussion amaryllidaceae 1. allium guttatum subsp. dalmaticum (a. kern. ex janch.) stearn (east): surroundings of dimitrovgrad, gulenovci village, odorovačko polje, steppelike habitats in karst field, limestone, 690 m, fn47, 10.06.2016, b. zlatković & n. stanković (hmn 13818). after new literature data on topic of distribution of this critically endangered taxon in flora of serbia were published pertaining to central and southeastern serbia (tomović et al., 2009), the species was also recorded in eastern serbia. all previously known sites for this species in serbia were steppe-like, slightly salt-affected habitats of saltpans. this locality is the first in serbia where this species was recorded on limestone as a substrate lacking salt deposits, and it appears in a small population of 2030 individuals just as in all other localities. 2. galanthus elwesii hook.f. (east): surroundings of knjaževac, mt. tupižnica peak, shrubs and fragments of beech forests, limestone, 1140 m, ep93, 29.03.2017, m. ranđelović & s. bogosavljević (hmn 13819). according to jovanović et al. (2016), this species was present in serbia only in vicinity of niš and pirot in eastern serbia. the new locality is situated on a limestone plateau of mt. tupižnica, in the zone of beech forests, where individuals of this species appear in scrub vegetation, sometimes between stone blocks, as well as within various derivatives of disrupted forest vegetation, primarily in fragments of beech forests. the only species previously recorded in literature for the area of mt. tupižnica was g. nivalis l. (fritsch, 1909). apiaceae 3. trinia ramosissima (fisch. ex trevir.) w.d.j. koch (east): surroundings of knjaževac, podvis village, dry grasslands, 385 m, ep92, 09.06.2016, coll. s. bogosavljević & b. zlatković (hmn 13820). (east): surroundings of niš, kunovica village, ploče plateau, steppe-like grassland, limestone, 659 m, en99, 15.06.2016, b. zlatković (hmn 13822). (east): surroundings of niš, vlase village, mt. seličevica, silicate, 308 m, en78, 29.06.2005, b. zlatković (hmn 13821). in serbia, trinia ramosissima is relatively rare, with a status of protected species (anonymous, 2010). according to butorac et al. (1991) it was recorded in serbia in a small number of localities in vojvodina and central serbia. new research has shown that this species also appears in southwestern serbia (papović et al., 2014). in the localities http://herbarium.univie.ac.at/ biologica nyssana 9 (2) ⚫ december 2018: 63-75 bogosavljević, zlatković ⚫ report on the new floristic data from serbia ii 65 included in this study it was recorded in several small populations in eastern serbia, appearing together with other mostly steppe or steppe-mediterranean elements (chrysopogon gryllus, erysimum diffusum, galatella linosyris, linum tenuifolium, scabiosa micrantha, vinca herbacea). its habitats include dry pastures, edges of forests and forest glades within the thermophilic oak forests. aristolochiaceae 4. aristolochia lutea desf. (fig. 1a, b). (central): ibar river valley, surroundings of ušće, loose, rocky material in juniperus oxycedrus stand, ultramafite, 410 m, dp61, 22.05.2003, b. zlatković (hmn 13823). (central): brezna, brezanska river gorge, rocky places in oak forest, ultramafite, 350 m, dp72, 06. 2001, b. zlatković (hmn 13826); mt. stolovi, meljanica gorge, rocky places and thermophilous oak forest, ultramafite, 400 m, dp72, 05.2001, b. zlatković (hmn 13824). (central): mt. stolovi, čukar, screes and rocky places, serpentinit, 930 m, dp62, 06.2001, b. zlatković (hmn 13827). (western): mt. tara, brusnica gorge, ostryopinetum nigrae, limestone, cp66, 07.2001, b. zlatković (hmn 13825). (south): mt. rujan, orljak, thermophilous oak forest, silicate, em67, 06. 06. 2009, b. zlatković (hmn 13828). this species belongs to aggregate aristolochia pallida which includes following species: a. pallida, a. tyrrhena, a. lutea, a. elongata, a. macedonica, a. croatica and a. merxmuelleri (tatić, 1999). within the taxonomic revision of genus aristolochia l. by nardi (1984) it was determined that the name “aristolochia pallida willd.” should only apply to the taxon appearing in the western part of alps massif, with only a few localities outside of this region, while a. lutea is widely distributed in the regions to the east of the alps, the apennines and balkan peninsula, pannonia, the carpathian mountains and in the most of the asia minor. as a. pallida and a. lutea are morphologically very similar, it should be noted that their most important differential character is ratio between the length of perigone tube and the length of perigone limb. in a. pallida the perigone tube is of equal length or slightly shorter than the limb (which is also wider than the tube), while in a. lutea the perigone tube is significantly longer than the limb, which is of equal width or slightly narrower than the perigone tube (nardi, 1984; trinajstić, 1990) (fig. 1b). considering these facts we believe that hypothesis by tatić et al. (1995), that a. lutea is present in serbia but misidentified as a. pallida in floras, scientific papers and herbarium collections, is justified. this species was first recognized as a. pallida in flora of serbia by pančić (1874), citing its presence at two localities in šumadija. gajić (1970) considers it widespread in serbia but without citing precise localities. however, in the new edition of flora of serbia (gajić, 1992) it is stated that a. pallida does not live in serbia, while data by mayer & greuter (1985), stating that a. lutea is present in serbia, were not taken in consideration. according to our research and the literature references on distribution of a. pallida, which within the study area with high probability pertains to the present species (pančić, 1874; ilić, 1900; ranđelović & stamenković, 1979; ranđelović, 1979/1980; ranđelović & stamenković, 1984; stamenković, 1985; tatić et al., 1995; jotić et al., 2013), it may be concluded that a. lutea appears in several regions of serbia (fig. 1a), where it is represented by small populations isolated from each other. aspleniaceae 5. asplenium lepidum c. presl (east): surroundings of knjaževac, stogazovac village, ždrelo gorge, crevices in sheltered parts of rocks, 390 m, limestone ep93, 08.05.2016, coll. m. ranđelović & s. bogosavljević (hmn 13829). this species of fern inhabits specific protected shaded habitats in crevices of limestone rocks, often appearing in gorges and canyons of hill region (stevanović et al., 1991). due to its specific ecological demands it is relatively rare and localized in serbia, mostly in eastern and western part of state (stevanović et al., 1991). asteraceae 6. bidens connata muhl. ex willd. (northeast): the town of prahovo, prahovo port, banks of the danube, 40 m, fq20, 22.09.2016, coll. s. bogosavljević, m. ranđelović & b. zlatković (hmn 13830). (northeast): mihajlovac village, banks of the danube, 40 m, fq11, 22.09.2016, coll. s. bogosavljević, m. ranđelović & b. zlatković (hmn 13831). (northeast): the town of kladovo, banks of the danube, ruderal, 40 m, fq24, 29.10.2016, coll. m. ranđelović & s. bogosavljević (hmn 13832). bidens connata is characteristic for north america as an autochthonous species, however today biologica nyssana 9 (2) ⚫ december 2018: 63-75 bogosavljević, zlatković ⚫ report on the new floristic data from serbia ii 66 its presence is also recorded in 14 european countries, including serbia where it is considered an invasive naturalized species (bogosavljević & zlatković, 2015). new data on distribution of this species in serbia pertain to localities in north-eastern serbia along river danube, at the romanian border. in romania this species was recorded only in the southeastern part of country (anastasiu et al., 2007), but the results of our study indicate that it should be expected in southwestern romania as well as in northwestern parts of bulgaria. fig. 1. a distribution of aristolochia lutea in serbia; b a. lutea flower; c ophioglossum vulgatum general appearance; d distribution of o. vulgatum in serbia (legend: black circles literature data, red circles new data, white circles imprecise literature data). biologica nyssana 9 (2) ⚫ december 2018: 63-75 bogosavljević, zlatković ⚫ report on the new floristic data from serbia ii 67 7. bidens vulgata greene (fig. 2a). (northeast): surroundings of negotin, srbovo village, jasenička reka channel, nitrophilous, damp places along the forest roads, 50 m, fp39, 22.09.2016, coll. s. bogosavljević, m. ranđelović & b. zlatković (hmn 13833). the only known record of this species is from a single locality in northwestern serbia (tatić & žukovski, 1973). it originated in north america and was first introduced to europe in mid-20th century, to france and romania, while today it is present in a number of european countries (petrova & vladimirov, 2009). however, due to high morphological and ecological similarity to the widespread species b. frondosa, b. vulgata probably remained unnoticed in appropriate habitats in most of its artificial range. 8. erigeron sumatrensis retz. (syn. conyza sumatrensis (retz.) e. walker, c. albida spreng.) (northeast): the town of kladovo, banks of the danube, ruderal, 50 m, fq24, 29.10.2016, coll. m. ranđelović & s. bogosavljević (hmn 13835). (east): surroundings of knjaževac, rgošte village, ruderal, 250 m, ep92, 25.08.2016, coll. m. ranđelović & s. bogosavljević (hmn 13834). (šumadija): the town of ripanj, ruderal, 255 m, dq64, 15.09.2017, m. ranđelović & s. bogosavljević (field obs.). new data on distribution of e. sumatrensis in serbia indicate that the invasive character of this species is becoming more pronounced. although until recently it was known from central, south and eastern serbia (zlatković & bogosavljević, 2014) and belgrade (niketić & jovanović, 2002), the new data also indicate its presence in northeastern serbia. the locality in northeastern serbia is situated immediately next to the romanian border, so this species should be also expected in southwestern romania, in addition to known records from the southeastern part of that country (anastasiu & memedemin, 2012). this species reaches high abundance of individuals in all new localities in serbia, inhabiting various ruderal habitats within human settlements. 9. symphyotrichum novae-angliae (l.) g.l.nesom (syn. aster novae-angliae l.) (east): surroundings of knjaževac, rgošte village, banjica pool, arable fields, 235 m, ep92, 13.10.2013, coll. b. zlatković & s. bogosavljević (hmn 13836). new data on distribution of this invasive species indicate that it continues to spread through serbia, as expected due to its invasive potential. so far it has been recorded in eastern and western serbia as naturalized, while in many localities it was recorded as a cultivated species (lakušić & jovanović, 2012). the population recorded near knjaževac includes self-propagated individuals in wetlands along the riverbank and the peripheral parts of cultivated plots. monitoring of this species in the last several years (until 2017) has shown no significant increase in number of individuals or tendencies of range spread. cactaceae 10. opuntia humifusa (raf.) raf., med. fl. 2: 247. (1830) (fig. 2b, c, d) (southeast): pčinja valley, jablanica village, dry sand and rocky places, 475 m, em78, 09.07.2016, coll. b. zlatković & s. bogosavljević (hmn 13837). family cactaceae includes about 1810 species, with natural range in tropical and subtropical areas of america (essl & kobler, 2008). however, several representatives of this family are considered to be significant allochthonous elements distributed in other warmer parts of the world, while in europe their invasiveness is mostly connected to the mediterranean region (essl & kobler, 2008). there are 26-29 invasive species of cacti recorded in europe (lambdon et al., 2008; novoa et al., 2015). according to essl & kobler (2008) and novoa et al. (2015), most invasive species from family cactaceae, at both global (27) and european (20) level, belong to genus opuntia (l.) mill., which at the same time is also the richest genus of this family (essl & kobler, 2008). as a number of species of this genus is used in horticulture, in suitable conditions individuals often show subspontaneous, either reproductive or vegetative dispersal into natural ecosystems (tashev, 2012). presence of this species in serbia was presented in a study by tashev (2012), where it is mentioned as a naturalized species, and that author cites erre et al. (2009). however, the overview of original data (erre et al. 2009) indicates that this was an erroneous interpretation. the other literature data from serbia do not show presence of o. humifusa. opuntia humifusa is a succulent plant with strongly branching, often creeping and rooting stems. the vegetative shoots in this cactus include tightly connected cladodes. they form links of 1-4 segments, often with lateral branches at the top and at the base. the cladodes are elliptical to oval, dark green, fleshy, sometimes with ridged stripes, 3-17 cm long and 4-12 cm wide, with 4-5 diagonal areoles at the widest part. the glochids are numerous, biologica nyssana 9 (2) ⚫ december 2018: 63-75 bogosavljević, zlatković ⚫ report on the new floristic data from serbia ii 68 inconspicuous. spines are absent, which is one of the main characteristics setting this species apart from the other species of complex opuntia humifusa. the flowers are 5-9 cm in diameter, hermaphroditic, while outer layers of perianth are dark green to slightly greyish-green in color. there are 8-9 inner segments and they are bright yellow. the stamens are numerous, with yellow or yellowish-green filaments. the stigma is white, with 6-7 lobes. the fruit is a berry, 2.5-5 cm long, at first green but becoming red to orange-red as it matures, with areoles and glochids. there are numerous seeds, 4-5 mm wide and with thick coating. the number of chromosomes is 2n=44. fig. 2. a bidens vulgata infructescence; b opuntia humifusa flower, c general appearance, d mowed plants; e iris sibirica flower; f haplophyllum suaveolens general appearance; g distribution of h. suaveolens in serbia (legend as in fig.1). biologica nyssana 9 (2) ⚫ december 2018: 63-75 bogosavljević, zlatković ⚫ report on the new floristic data from serbia ii 69 genus opuntia shows high taxonomic complexity and most species are characterized by high infraspecific variability (fateryga & bagrikova, 2017). some of the species may also be represented by atypical forms within their secondary range, so there are objective problems in species identification (fateryga & bagrikova, 2017). during the last taxonomic revision there were 8 recognized species within the complex o. humifusa s.l. (majure et al., 2017). opuntia humifusa is morphologically most similar to species o. mesacantha raf., which is characterized by presence of spines at least in the terminal part of cladodes. parts of stem in o. mesacantha are more or less easily separable from each other, while there are just 3-4 areoles in the diagonal of the widest part of cladodes. population of o. humifusa in pčinja valley is present in dry grassland and stony areas, along the local road, most probably representing a garden escape. other plants recorded in the same habitat include: centaurea stoebe, chondrilla juncea, clinopodium alpinum subsp. hungaricum, cynodon dactylon, erodium cicutarium, filago arvensis, herniaria glabra, petrorhagia saxifraga and scabiosa argentea. the habitats described above are in direct contact with stands of association scabiosotrifolion dalmatici, as evidenced by the combination of weed and pontian-submediterranean elements developing under similar ecological conditions. in this phase of spread, the species is successfully reproducing by vegetative means. although certain individuals produce abundant flowers and fruits, it is not known whether other means of dispersion are present at this locality. population of o. humifusa in pčinja valley presently occupies space of several ares and is observed to a hundred individuals. this population is observed for several years, and we believe that introduction of this species took place a few decades ago. mowing was recorded at the locality, leading to partial hindrance to further spread of this species. analysis of existing data on distribution of this species in europe (fateryga & bagrikova, 2017) indicates that it is present in 10 countries including serbia. in half of these countries it is considered an invasive species, and in other half a naturalized species. taking into account the total abundance of population, the types of habitats where this species grows and the period of time elapsed since the first record in serbia, we conclude that o. humifusa should be considered to have the status of a casual alien species in our country. at this moment, the species does not pose any significant threat to autochthonous ecosystems and habitats in areas where it has been recorded. iridaceae 11. iris sibirica l. (fig. 2e). (east): surroundings of knjaževac, podvis village, reed and sedge beds, 385 m, ep92, 26.05.2016, coll. s. bogosavljević (hmn 13838). in serbia, iris sibirica is one of the strictly protected species (anonymous, 2010). according to kostrakiewicz (2007), it belongs to the euro-siberian element. its natural area includes central and eastern europe, from northeastern turkey, european part of russia and western siberia to northern italy, but presently it is rare throughout the range (kostrakiewicz-gierałt, 2013). in balkan peninsula this species is rare with a disjunctive range in central bosnia, bulgaria, montenegro and serbia. in serbia it was recorded at several localities in vojvodina, central and western part of country (stevanović et al., 1993). at the new locality its population includes a small number of individuals thriving in an area of only several square meters. the other species at the site include: anacamptis palustris, iris pseudacorus, ophioglossum vulgatum, salix caprea, scirpus silvaticus, typha latifolia etc. it is necessary to implement special protection measures, while the main negative factors threatening the remaining individuals of i. sibirica in this habitat include: drying and turning the natural habitats into cultivated land, succession of wet meadows and reed beds into shrub and forest vegetation, plantation farming of poplar trees etc. juncaceae 12. juncus capitatus weigel (east): surroundings of pirot, temska village, temštica river, wet, eroded sandy and rocky places, 500 m, fn39, 28.05.2016, coll. s. bogosavljević, m. ranđelović & b. zlatković (hmn 13839). this threatened taxon was until recently believed to have gone extinct from the territory of serbia (ranđelović, 1999), but this study has shown presence of a third known site (tomović et al., 2009; zlatković & bogosavljević, 2014). this species was first recorded in eastern serbia, on eroded wet areas along roads, on silicate substrate. ophioglossaceae 13. ophioglossum vulgatum l. (fig. 1c, d) (east): surroundings of knjaževac, podvis village, wet meadows, 385 m, ep92, biologica nyssana 9 (2) ⚫ december 2018: 63-75 bogosavljević, zlatković ⚫ report on the new floristic data from serbia ii 70 26.05.2016, coll. s. bogosavljević (hmn 13840). (east): surroundings of svrljig, lalinac village, lalinac river, wet meadows, 385 m, ep80, 29.04.2016, coll. s. bogosavljević, m. ranđelović & b. zlatković (hmn 13841). (east): surroundings of pirot, barje čiflik village, čeltaš, wet meadows, 390 m, fn27, 28.05.2016, coll. s. bogosavljević, m. ranđelović & b. zlatković (hmn 13842). (east): surroundings of knjaževac, gornja kamenica village, papratska river, wet meadows, 360 m, fp11, 02.06.2017, coll. s. bogosavljević, m. ranđelović & b. zlatković (hmn 13843). (east): surroundings of dimitrovgrad, mazgoš village, alluvium, 661 m, salicetum albofragilis, fn56, 22.06.2013, coll. b. zlatković & n. stanković (hmn 13844); protopopinci village, alluvium, 666 m, wet meadows, molinio-arrhenatheretea, fn56, 22.06.2013, coll. b. zlatković & n. stanković (hmn 13845). (east): sićevo gorge, dolac village, alluvium, 265 m, wet meadows, en98, 28.05.2016, coll. b. zlatković, s. bogosavljević & m. ranđelović (hmn 13846). according to krivošej et al. (2013), ophioglosum vulgatum is distributed from the northernmost areas in serbia, in bačka, across central, west, northeastern and eastern serbia, as well as kosovo and metohija. the new points of distribution indicate presence of a number of sites for this species in eastern and southeastern serbia. this species inhabits fragile habitat types such as wetland and marsh meadows and reed beds, but recorded abundance in new localities was never particularly high. the map of present distribution in serbia (stevanović ed., 1992; zlatković & ranđelović, 1993/4; mihajlović, 1995; ranđelović et al., 2000; zlatković, 2011; krivošej et al., 2013), including new data on range, is presented in fig. 2d. in serbia this species is included in the group of plant species protected by law (anonymous, 2010). ranunculaceae 14. eranthis hyemalis (l.) salisb. (fig. 3a, b, c, d, e) (east): surroundings of knjaževac, podvis village, thermophilous oak forests and edges, 325-395 m, n-ne, ep92, 02.03.2017, coll. m. ranđelović & s. bogosavljević, 31.03.2017, coll. m. ranđelović & s. bogosavljević and 11.04.2017 coll. m. ranđelović & s. bogosavljević (hmn 13848, 13847, 13858); orešac village, golemi kamen, thermophilous oak forests, 365 m, ep92, 05.02.2018, coll. m. ranđelović & s. bogosavljević (hmn 13851). (east): surroundings of knjaževac, orešac village, ploča, carpino orientalis-quercetum mixtum, 600 m, ep91, 13.03.2018, coll. m. ranđelović & s. bogosavljević (hmn 13852). (east): surroundings of zaječar, vratarnica village, zmijanac (provalina), oak forests, 235 m, fp04, 24.04.2017, coll. m. ranđelović & s. bogosavljević (hmn 13849). (east): the town of niš, gorica hill (tri lipe), forest fragments and lime plantations in suburban area of the city, 265 m, en79, 12.02.2017, coll. b. zlatković (hmn 13850). in serbia eranthis hyemalis is included in the category of strictly protected species (anonymous, 2010), and as a critically endangered taxon it was included in the red book of flora of serbia (budak, 1999). at the locality in eastern serbia (podvis), this species lives at the edges but also inside thermophilic oak forests developed on higher, warmer slopes of the gorge of svrljiški timok. at this locality, the population developed on the surface area of about 10 ha in form of seven smaller spatial fragments, each with 50-100 individuals. as part of this area is mostly inaccessible, we assume that this population includes a much greater number of individuals. our estimate of ratio of flowering/fruit-bearing vs. juvenile individuals in this population is 30:70. in addition to e. hyemalis, the habitat at podvis site also includes following herbaceous species: anemone apeninna, arabis procurrens, asplenium ceterach, a. trichomanes, cardamine graeca, corydalis solida, ficaria verna, fragaria vesca, glechoma hederacea, hedera helix, helleborus odorus, lamium purpureum, orchis purpurea, polypodium vulgare, potentilla micrantha, ruscus aculeatus, smirnium perfoliatum etc. approximation of population (subpopulation) size at a different locality within the same gorge (golemi kamen) has shown that number of e. hyemalis was even greater than in the first mentioned locality, considering more than a thousand individuals. as both localities are situated within the same utm square, the total number of individuals in the utm square is probably more than 1500. at the locality ploča there were only a few dozen recorded individuals within the stand of oriental hornbeam shrub. another group (100-150 individuals) of e. hyemalis was also recorded in anthropogenously altered forest stands and linden plantations at hill gorica (tri lipe) in immediate vicinity of niš. however, this population develops in an immediate vicinity of a human settlement and almost certainly is subspontaneous in origin. biologica nyssana 9 (2) ⚫ december 2018: 63-75 bogosavljević, zlatković ⚫ report on the new floristic data from serbia ii 71 the observe of number of individuals in other populations in eastern serbia, at localities (vratarnica, provalina) known from literature sources (petrović & lakusić, 2017), is at about several hundred individuals, but a surprisingly small number of reproductively mature individuals was recorded in field studies. in all known localities a high percentage of individuals were infected by a type of parasitic fungus. fig. 3. a eranthis hyemalis flower, b fruit, c general appearance, native habitat, d subspontaneous specimens; e distribution of e. hyemalis in serbia and observed number of individuals (legend: circles with black dots in center literature data, circles with white dots in center new data): white circle not observed, green circle above 10000 individuals, purple circle between 1500 and 10000 individuals, yellow circle between 100 and 1500 individuals, red circle under 100 individuals; black circle probably extinct population. biologica nyssana 9 (2) ⚫ december 2018: 63-75 bogosavljević, zlatković ⚫ report on the new floristic data from serbia ii 72 in light of new literature data (petrović & lakusić, 2017), and results of our own fieldwork, e. hyemalis should remain within the group of critically endangered (cr) taxa of flora in serbia, as it is represented by a small number of spatially distant natural populations with a relatively small number of individuals. however, it is important to note that the region of eastern serbia, according to observed population sizes in all known localities, contains a significant part of population of this species in serbia. localities in vicinity of knjaževac and zaječar are, after the locality near bačka palanka (panjković et al., 2015; kiš et al., 2016), certainly the richest habitats in serbia. therefore we believe that it is necessary to take conservation measures so this species would survive in these localities in eastern serbia. the locality near vratarnica is already enclosed within the nature park stara planina (anonymous, 2008), but the locality near knjaževac is not included in any form of spatial protection. the modified distribution map of e. hyemalis in serbia, with addition of new distribution points and data on reevaluated number of individuals in comparison to budak (1999) and petrović & lakusić (2017), is presented in fig. 3e. rutaceae 15. haplophyllum suaveolens ledeb. (fig. 2f, g) (east): surroundings of svrljig, plužina village, visoka stena, dry grasslands, 750 m, ep81, 18.05.2018, coll. m. ranđelović & s. bogosavljević (hmn 13853). (east): surroundings of dimitrovgrad, dry steppe-like habitats, 610 m, fn46, 15.05.2018, coll. s. bogosavljević, m. ranđelović & b. zlatković (hmn 13854). (east): surroundings of niš, sićevo village, rocky places, 360 m, en89, 20.05.2014, coll. b. zlatković (hmn 13855). (east): surroundings of niš, kamenica village, dry grasslands, 440 m, ep70, 23.05.2014, coll. b. zlatković (hmn 13856). the range of this steppe species reaches from southern siberia to asia minor on one side, and romania and the eastern part of balkan peninsula on the other side (mayer & wraber, 1974). within the balkan peninsula the species is present in bulgaria, macedonia, greece, albania and serbia (hayek, 1924; tutin et al., 1968). it inhabits dry, grassy, partially rocky terrain on limestone substrate. haplophyllum suaveolens is a strictly protected species in serbia (anonymous, 2010). population size is observed to a few hundred in vicinity of svrljig and only a handful of individuals in new localities in vicinity of niš and dimitrovgrad. the present distribution of this species in serbia is presented in fig. 2g, according to literature data (diklić, 1973; lakušić & niketić, 1988; ranđelović et al., 2000; ranđelović et al., 2007; pavlović et al., 2018) and results of our current research. urticaceae 16. parietaria lusitanica l. subsp. serbica (pančić) p.w.ball (east): surroundings of knjaževac, stogazovac village, ždrelo gorge, fissures of sheltered rocks, 390 m, limestone ep93, 08.05.2016, coll. m. ranđelović & s. bogosavljević (hmn 13857). this subendemic species inhabits limestone rock shelters, cave entrances and other similar habitats of southern carpathians, dobrogea and eastern part of balkan peninsula (stevanović et al., 2014). the new locality fits within the southwestern part of global range of this rare species. in serbia, parietaria lusitanica subsp. serbica is protected by law (anonymous, 2010). acknowledgements. the authors would like to thank milica ranđelović for help during the field work. the ministry of education, science and technological development of the republic of serbia (grant 173030) supported this research. references anastasiu, p., negrean, g., basnou, c., sîrbu, c., oprea, a. 2007: a preliminary study on the neophytes of wetlands in romania. in: rabitsch, w., essl f. & klingenstein, f. 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(eds.) 1968: flora europaea, 2. university press. cambridge. zlatković, b. 2011: flora i fitogeografska pripadnost doline reke pčinje u jugoistočnoj srbiji. doktorska disertacija. univerzitet u beogradu, biološki fakultet. beograd. zlatković, b., bogosavljević, s. 2014: report on the new floristic data from serbia. biologica nyssana, 5 (2): 123-129. zlatković, b., ranđelović, v. 1993-1994: ugroženost i zaštita flore sićevačke klisure. zaštita prirode, 46-47: 191-199. http://www.theplantlist.org/ antioxidant activity of oregano essential oil (origanum vulgare l.) biologica nyssana 7 (2)  december 2016: 131-139 stanojević, lj.p. et al.  antioxidant activity of oregano essential oil… 131 original article received: 30 june 2016 revised: 31 october 2016 accepted: 24 november 2016 antioxidant activity of oregano essential oil (origanum vulgare l.) ljiljana p. stanojević*, jelena s. stanojević, dragan j. cvetković, dušica p. ilić faculty of technology, university of niš, bulevar oslobodenja 124, 16000 leskovac, serbia * e-mail: stanojevic@tf.ni.ac.rs abstract: stanojević, lj.p., stanojević, j.s., cvetković, d.j., ilić, d.p.: antioxidant activity of oregano essential oil (origanum vulgare l.). biologica nyssana, 7 (2), december 2016: 131-139. essential oil obtained from oregano (origanum vulgare l.) by clevenger-type hydrodistillation and hydromodulus 1:10 m/v during 180 minutes, has been investigated in this work. qualitative and quantitative composition of the oil was determined by gc-ms and gc-fid spectrometry. antioxidant activity of the obtained oil was examined spectrophotometrically by dpph test (after 20, 30, 45 and 60 minutes of incubation) and tba-mda assay. the yield of essential oil was 4.1 ml/100 g of plant material. seven components were identified: α-thujene, myrcene, α-terpinene, o-cymene, γ-terpinene, thymol and carvacrol. the major components were thymol (45%) and carvacrol (37.4%). oil incubated for 60 minutes has shown the best antioxidant activity according to dpph test. the concentrations of essential oil, required for neutralization of 50% of initial dpph radical concentration (ec50), were 0.761, 0.590, 0.360 and 0.326 mg/ml, after 20, 30, 45 and 60 minutes of incubation, respectively. lipid peroxidation inhibition of 92.3% was achieved by 1.35 mg/ml essential oil concentration. the results obtained indicate that oregano essential oil is a good source of natural antioxidants with potential application in food and pharmaceutical industries, as a safer alternative to the synthetic antioxidants. key words: origanum vulgare l., essential oil, antioxidant activity, gc-ms analysis apstrakt: stanojević, lj.p., stanojević, j.s., cvetković, d.j., ilić, d.p.: antioksidativna aktivnost etarskog ulja vranilove trave (origanum vulgare l.). biologica nyssana, 7 (2), december 2016: 131-139. u ovom radu je ispitivano etarsko ulje vranilove trave (origanum vulgare l.) dobijeno clevenger hidrodestilacijom pri hidromodulu 1:10 m/v, u toku 180 minuta. kvalitativni i kvantitativni sastav dobijenog etarskog ulja određen je gc-ms i gc-fid spektrometrijom. antioksidativna aktivnost ulja je određena spektrofotometrijski, dpph testom (posle 20,30, 45 i 60 minuta inkubacije) i tba-mda testom. prinos etarskog ulja bio je 4.1 ml/100 g biljnog materijala. identifikovano je sedam komponenti: α-tujen, mircen, αterpinen, o-cimen, γ-terpinen, timol i karvakrol. kao glavne komponente identifikovane su timol (45%) i karvakrol (37,4%). najbolju dpph-antioksidativnu aktivnost pokazalo je ulje nakon 60 min inkubacije. koncentracije etarskog ulja neophodne za neutralizaciju 50% od početne koncentracije dpph radikala (ec50 vrednosti) bile su 0,761; 0,590; 0,360 i 0,326 mg/ml, nakon 20, 30, 45 i 60 min inkubacije, respektivno. inhibicija lipidne peroksidacije od 92,3% je postignuta sa 1,35 mg/ml eterskog ulja. dobijeni rezultati sugerišu da dobijeno etarsko ulje vranilove trave predstavlja dobar izvor prirodnih antioksidanasa sa potencijalnom primenom u prehrambenoj i farmaceutskoj industriji kao bezbednija alternartiva sintetskim antioksidansima. ključne reči: origanum vulgare l., etarsko ulje, antioksidativna aktivnost, gc-ms analiza 7 (2) • december 2016: 131-139 12th sfses • 16-19 june 2016, kopaonik mt doi: 10.5281/zenodo.200410 biologica nyssana 7 (2)  december 2016: 131-139 stanojević, lj.p. et al.  antioxidant activity of oregano essential oil… 132 introduction the oxidation process is one of the major causes of food spoilage, which results in rancidity and deterioration of the nutritional quality, color, flavor, texture, and safety of foods (b e t a i e b et al., 2010). a significant number of herbs is used as natural preservatives in food industry. besides being used to achieve the proper flavor and to intensify the flavors some spices and herbs exhibit antioxidant effects which is of great importance for food industry (s t a n k o v i ć & s t a n o j e v i ć , 2014; t o n g n u a n c h a n et al., 2014). plants rich in antioxidant compounds are present in food industry (b e t a i e b et al., 2010). there are evident requirements for increasing application of natural antioxidants obtained from plant material. undesirable side effects of synthetic antioxidants, such as butyl hydroxy anisole (bha) and butylated hydroxytoluene (bht), which are associated with their toxic and carcinogenic effects (m a e s t r i et al., 2006), are the reason of such requirements. essential oils are very heterogeneous group of complex mixtures of secondary plant metabolites. composition of essential oil may be different between different species or varieties, related to different cultivation, origin, vegetative stage and growing seasons of the plant (v a z i r i a n et al., 2015). beside aroma, odor and fragrance of many of the oils, some of them have also been confirmed to possess antioxidant activities (b e t a i e b et al., 2010; v a z i r i a n et al., 2015). essential oils are also widely used in perfumes, cosmetics, aromatherapy and nutrition (b u r t , 2004; b a k k a l i et al., 2008). earlier studies have been shown that, among the herbs and spices which are extensively studied, the plants from the lamiaceae (labiatae) family possess a significant antioxidant activity (t s i m i d o u & b o s k o u , 1994; l a g o u r i et al., 1993). within this family oregano (origanum vulgare l.) is probably one of most widely used aromatic plant which essential oils are particularly rich in monoand sesquiterpenes (d e f a l c o et al., 2013). the genus origanum includes around 38 species, most of which are indigenous to the mediterranean, euro-siberian and irano-siberian regions (s a h i n et al., 2004). origanum vulgare l. is one of the most widely among all the species within the genus which distributed all over the europe, west and central asia up to taiwan (s a h i n et al., 2004; r a d u š i e n e et al, 2008). it is one of the most important culinary herbs in the world. leaves and flowers of oregano are traditionally used to cure cough and sore throats and for relieve of gastrointestinal disorders. origanum vulgare is a source of essential oils and phenolic metabolites (r a d u š i e n e et al, 2008). the main bioactive components of oregano are phenolic components (s e g e i t k u j a w a et al., 1990; k u l e v a n o v a et al., 2001; l e u n g & f o s t e r , 2003; r a d u š i e n ė et al., 2008) and essential oil (l e u n g & f o s t e r , 2003). origanum vulgare contains 0.1-1.0% of essential oil composed of thymol, carvacrol, -bisabolene, caryophyllene, pcimene, borneol, linalool, linalyl acetate, geranyl acetate, -pinene, -pinene, -terpinene, with highly variable relative proportion, depending on source (l e u n g & f o s t e r , 2003). oregano essential oils have been shown to possess antioxidant, antibacterial, antifungal, diaphoretic, carminative, antispasmodic and analgesic activities (d e f a l c o et al., 2013). thymol and carvacrol, usually the major phenols present in oregano, have strong fungicidal, anthelmintic, irritant, and other properties (l e u n g & f o s t e r , 2003). based on numerous studies it was established that oregano essential oil, rich in thymol and carvacrol, has a significant antioxidant activity in the process of the lard oxidation (l a g o u r i et al., 1993; t s i m i d o u & b o s k o u ,1994). y a n i s h l i e v a and m a r i n o v a (1995) examined the antioxidant activity of hexane extracts from oregano grown in bulgaria, and the mechanism of action of pure thymol and carvacrol (y a n i s h l i e v a et al.,1999), while kulišić and coworker presented different methods for antioxidant activity of oregano essential oil testing (k u l i š i ć et al., 2004). the aim of the present study was to examine the antioxidant properties of oregano essential oil, originated from serbia, by using two different methods, namely, dpph test, and tba-mda assay. material and methods plant material the commercial sample of aerial parts of origanum vulgare l. (origani herba) was purchased from the local health food store in leskovac, serbia. according to the declaration, the material used for investigations originates from serbia (packed by: malina-impex d.o.o. popučke b.b., valjevo) chemicals and reagent ethanol, 96% (centrochem, zemun, serbia), 1,1diphenyl-2-picrylhydrazyl (dpph radical), butylated hydroxy toluene (bht), thiobarbituric acid (tba), 2,2'-azobis (2-methylpropionamidine) dihydrochloride (aaph) (sigma chemical company, st. louis, usa), trichloroacetic acid (tca) (j.t. baker, biologica nyssana 7 (2)  december 2016: 131-139 stanojević, lj.p. et al.  antioxidant activity of oregano essential oil… 133 va deventer, netherlands). phospholipids (phospholipon® 90; pl90) were a gift from phospholipid gmbh (cologne, germany). according to the declaration pl90 mixture consisting of phosphatidylcholine 94.6%, lyso-phosphatidylcholine 1.3%; fatty acid: palmitic acid 12 ± 2%, stearic acid 3 ± 1%, oleic acid 10 ± 3%, linoleic acid 66 ± 5%, linoleic acid 5% ± 2; peroxide number 1.4. isolation of essential oil essential oil from aerial parts of o. vulgare was isolated by classic clevenger-type hydrodistillation according to ph. jug. v (2000). plant material (50 g) was immersed in 500 ml of water in round bottom flask, and the oil was isolated using a clevenger-type apparatus for 180 min. the resulting essential oil was dried over anhydrous sodium sulfate, filtered and stored at +4 °c in a well-filled, airtight container, protected from light, until the analysis. gc-ms and gc-fid analysis gc-ms analysis of the oregano essential oil was performed on agilent technologies 7890b gas chromatograph, equipped with weakly polar, silica capillary column, hp-5ms (5% diphenyland 95% dimethyl-polysiloxane, 30 m x 0.25 mm, 0.25 μm film thickness; agilent technologies, usa) and coupled with inert, selective 5977a mass detector of the same company. one μl of the sample dissolved in diethyl ether in the concentration of 1000 ppm was injected in 20:1 split mode. helium was used as the carrier gas, at a constant flow rate of 1 ml/min. the oven temperature was programmed from 50 °c for 2.25 minutes and then increased to 290 °c at the rate of 4 °c/min. temperatures of the msd transfer line, ion source and quadruple mass analyzer were set at 300 °c, 230 °c and 150 °c, respectively. the ionization voltage was 70 ev and mass range m/z 35650. gc-fid analysis was carried out under identical experimental conditions as gc-ms. the temperature of the flame-ionization detector (fid) was set at 300°c. data processing was performed using msd chemstation, masshunter qualitative analysis and amdis 32 softwares (agilent technologies, usa). retention indices of the components from the analyzed samples were experimentally determined using a homologous series of n-alkanes from c8-c20 as standards. compounds identification was based on the comparison of their retention indices (riexp – tab. 1) with those available in literature (adams, 2007) (rilit – tab. 1), as well as their mass spectra with those from willey, nist and rtlpest libraries. the percentage composition of particular components in the essential oil was determined on the basis of automatically integrated peak areas of the gc-fid signal. antioxidant activity dpph assay antioxidant activity of oregano essential oil was determined by dpph test (a q u i n o et al., 2002; c h o i et al., 2002; s a n c h e z m o r e n o , 2002). the essential oil was dissolved in ethanol (96%) and a series of different concentration solutions were prepared (0.098 to 12.5 mg/ml). the ethanol solution of dpph radical (1 ml, 3×10-4 mol/l) was added to 2.5 ml of each essential oil solutions. absorbance of one sample was immediately measured at 517 nm, while the other samples were incubated at room temperature in the dark, for 20, 30, 45 and 60 minutes, and the absorbance was also measured at 517 nm (as). the absorbance at 517 nm was measured for pure ethanol solution of dpph radical prepared as described above – 1 ml of the dpph radical (3×10-4 mol/l) diluted with 2.5 ml of ethanol, ac), as well as for the essential oil before treatment with dpph radical (2.5 ml of essential oil diluted with 1 ml of ethanol, ab). free radical scavenging capacity was calculated by the eq. 1 (s t a n o j e v i ć et al., 2015): dpph rsc (%) =          c bs a aa 100 100 ......... (1) rscradicals scavenging capacity ec50 value was defined as essential oil concentration needed for the neutralization of 50% of the initial dpph radical concentration. this value was determined by interpolation from the linear regression analysis in the concentration range between 0.098 and 0.391 mg/ml of essential oil added to the reaction mixture. bht was used as the reference compound. tba-mda assay thiobarbituric acid – malondialdehyde (tba-mda) test is one of the most commonly used tests for lipid peroxidation process monitoring in vitro. it’s based on heating of sample with tba in acidic environment, i.e. on tba and mda reaction. mda is end-product of lipid peroxidation formed during hydroperoxide degradation which build pink chromogen ([tba]2-malondialdehyde adduct) with absorption maximum at 532 nm. colored complex is formed by condensation of 2 moles of tba and 1 mole of mda only from the fatty acid chains that contain at least three double bonds (h a l l i w e l l & c h i r i c o , 1993; l a g u e r r e et al., 2007). the antioxidant activity of oregano essential oil was determined by the method developed for caroteinoid, biologica nyssana 7 (2)  december 2016: 131-139 stanojević, lj.p. et al.  antioxidant activity of oregano essential oil… 134 flavonoids, extracts and some potential antioxidants (c v e t k o v i c & m a r k o v i c , 2008; c v e t k o v i c et al., 2011; z v e z d a n o v i ć et al., 2014; s t a n o j e v i ć et al., 2015a), with some modifications. "sample" contains 0.3 cm3 of pl90 methanolic solution (1·10-2 mol/l), and essential oil ethanolic solution (0.042-5.375 mg/ml) in 2:1 (v/v) ratio. lipid peroxidation was initiated by addition of 0.2 cm3 of aqueous solution of thermal azo-initiator aaph (2.2·10-2 mol/l) during 3 h at a temperature of 40 °c, protected from light. after incubation, 1 ml of aqueous solution of tca (5.5%), 0.5 ml of methanolic solution of bht (1∙10-3 mol/l) and 0.5 ml of tba (4.2∙10-2 mol/l in 5∙10-2 mol/l naoh) were added to the reaction mixture. the mixture was incubated for 10 min at 65 °c and then centrifuged for 5 min at 13800 rpm. the increase in absorbance of the supernatant at 532 nm represents an absorption maximum of generated tba-mda complex. the absorbance of pl90 solution, where lipid peroxidation is initiated with aaph, treated with tba (”control“), as well as of pl90 solution, without lipid peroxidation initiation, but also treated with tba (”blank“). lipid peroxidation inhibition was calculated by the eq. 2 (stanojević et al., 2015a):   100 bc sc aa ) a(a lpi (%) ............................. (2) lpiinhibitionxidationlipid pero where ac – represents the absorbance of ”control“; as – absorbance of ”sample“; ab – absorbance of ”blank“. bht (0.0125-0.05 mg/ml) was used as the reference compound. all experiments were carried out in three replications. data were expressed as mean ± standard deviation. the obtained data were analyzed by microsoft excel 2007 and origin 7 trial. results and discussion hydrodistillation kinetics and oregano essential oil composition figure 1 shows the influence of hydrodistillation time on the essential oil yield. maximal essential oil yield of 4.1 ml/100g of plant material was achieved after 180 minutes. the hydrodistillation curve shows that there are two different periods of hydrodistillation (fig. 1). the essential oil was evaporated out from the surface from the cells of plant material in the first period (the fast oil hydrodistillation). in the second, slow oil hydrodistillation period, a slow molecular diffusion of the essential oil from internal part from cells of plants material occurred. fig. 1. hydrodistillation kinetics of oregano essential oil in the tab. 1 and on fig. 2 the results of gc– ms analysis of oregano essential oil are presented. seven components were identified in essential oil: αthujene, myrcene, α-terpinene, o-cymene, γterpinene, thymol and carvacrol. the major components were thymol (45%) and carvacrol (37.4%). the content of thymol and carvacrol in the oregano essential oil from various regions was different: 35% and 32% of thymol and carvacrol, respectively, from dalmacia (k u l i š i ć et al., 2004), 0.8 and 0.6% respectively, from turkey (s a h i n et al., 2004), 1.1 and 14.3% respectively, from southern italy (d e f a l c o et al., 2013), 37.1 and 9.6 respectively, from iran (v a z i r i a n et al., 2015), 31.8 and 0.2%, respectively from portugal (g a l e g o et al, 2008). other investigators reported a lower content of carvacrol in oregano essential oil (less than 35%) compared to the oregano from serbia. these changes of thymol and carvacrol content in the oregano essential oil, and changes in the compositions of the oil might arise from several environmental reasons (climatic, seasonal, geographical), used drying method of plant material and genetic differences of the plant material (f a b e r et al., 1997; c a l l a n et al., 2007; g h a s s e m i g o l e z a n i et al., 2008; f i g i e l et al, 2010; s t a n o j e v i ć et al., 2011; d e f a l c o et al, 2013). significantly higher content of this two bioactive components with an array of different biological activities is especially important (u l t e e et al., 2002; n o s t r o et al., 2007; w a n g et al., 2009; m a t h e l a et al., 2010; m e h d i et al., 2011). the major components are believed to be mainly responsible for the biological activity of this b a k k a l i , 2008). based on the results obtained in biologica nyssana 7 (2)  december 2016: 131-139 stanojević, lj.p. et al.  antioxidant activity of oregano essential oil… 135 particular essential oil (b a i l e r et al., 2001; our investigation, the oregano essential oil from serbia could be a potential natural source of thymol and carvacrol. antioxidant activity dpph test is most commonly in vitro method for antioxidant activities determination of plant extracts and essential oils. it is very suitable and useful for the antioxidant activity determination since it’s fast and sufficiently sensitive (m o l y n e u x , 2004). this method is based on hydrogen atoms or electrons exchange between antioxidant molecules and dpph radicals in the solution (s a n c h e z m o r e n o et al., 2002). dpph radical scavenging activity of isolated oregano essential oil was shown on fig. 3, while the ec50 values are listed in tab. 2. degree of dpph radical neutralization depends on oil concentration as well as the incubation time it increases with the concentration increase which is the lowest for non incubated samples (fig. 3). the highest antioxidant activity (about 95%) has been measured after 60 minutes of incubation. the ec50 (dpph) value of fig. 2. gc-fid chromatogram of oregano essential oil table 1. chemical composition of origanum vulgare essential oil no. tret., min compound riexp rilit method of identification content, % monoterpene hydrocarbons (14.4%) 1 9.78 α-thujene 925 924 ri, ms 0.4 2 11.99 myrcene 987 988 ri, ms 1.0 3 12.93 α-terpinene 1014 1014 ri, ms 1.1 4 13.22 o-cymene 1022 1022 ri, ms 4.4 5 14.46 γ-terpinene 1057 1054 ri, ms 7.5 phenols (82.4%) 6 22.59 thymol 1292 1289 ri, ms 45.0 7 22.92 carvacrol 1300 1298 ri, ms 37.4 total 96.8% tret.: retention time; rilit a,b-retention indices from literature (adams, 2007), respectively; riexp: experimentally determined retention indices using a homologous series of n-alkanes (c8-c20) on the hp-5ms column. ms: constituent identified by mass-spectra comparison; ri: constituent identified by retention index matching. biologica nyssana 7 (2)  december 2016: 131-139 stanojević, lj.p. et al.  antioxidant activity of oregano essential oil… 136 synthetic antioxidant bht was 0.021 mg/ml, indicating better antioxidant activity compared to the tested essential oil. mentioned antioxidant is the most commonly used synthetic antioxidant, but with harmful effects in human body (i t o et al., 1986). so, presented results suggest that oregano essential oil can be potentially used as a safer alternative to synthetic antioxidants in pharmaceutical and food industries. fig. 3. dpph antioxidant activity of oregano essential oil k u l i š i ć et al. (2004) and s a h i n et al. (s a h i n et al., 2004) have come to similar results in their investigations. in earlier reports, thymol and carvacrol, in particular, were found to be main antioxidant components of essential oil from different origanum species (b a r r a t a et al., 1998; r u b e r t o et al., 2002; p u e r t e s -m e j i a et al., 2002). because the main components of the oregano essential oil in our investigations are phenolics thymol and carvacrol, they are probably mostly responsible for the high degree of dpph radical neutralization (y a n i s h l i e v a & m a r i n o v a , 1995; k u l i š i ć et al., 2004; s a h i n et al., 2004). the tba method is sensitive and achieves reproducible results. this method is preferable in order to obtain useful data in an environment similar to the real-life situation (k u l i š i ć et al., 2004). fig. 4 shows degree of lipid peroxidation inhibition by investigated oregano essential oil, while tab. 2 shows the ec50 value (tba-mda) of the oil. fig. 4. inhibition of lipid peroxidation by oregano essential oil (tba-mda test) based on the presented results it can be concluded that isolated essential oil from oregano shows good concentration dependent antioxidant activity. the highest degree of lipid peroxidation inhibition was achieved by essential oil in concentration of 3.0 mg/ml. the ec50 (tba-mda) value of synthetic antioxidant bht was 0.0185 mg/ml. k u l i š i ć et al. (2004) reported antioxidant activity of oregano essential oil from dalmacia and its fractions, measured by tba method. in addition, c a p e c k a et al. (2005) found that o. vulgare showed the strongest inhibition of linolenic acid peroxidation. our results of antioxidant activity are in accordance with s a h i n et al. (2004) who reported that essential oil of origanum vulgare ssp. vulgare possesses antioxidant compounds, and therefore can be used as a natural preservative in food and/or pharmaceutical industry. presented results confirm that oregano essential oil possesses remarkable antioxidant activities as assessed by two different methods. this biological effect is probably due to the presence of thymol and carvacrol, but possible synergistic effect table 2. ec50 values of oregano essential oil ec50 (dpph), mg/ml 20 min 30 min 45 min 60 min 0.761  0.003 0.590  0.005 0.360  0.004 0.326  0.002 ec50 (tba-mda), mg/ml 0.455  0.004 biologica nyssana 7 (2)  december 2016: 131-139 stanojević, lj.p. et al.  antioxidant activity of oregano essential oil… 137 among other compounds in the oil can be also suggested. conclusion the presented results indicate that the oregano essential oil could be used as potential source of natural antioxidants for the food, pharmaceutical and chemical industry. 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(salisb.) reichb. original article anđela v. dragićević university of niš, faculty of medicine, department of pharmacy, serbia dragicevic.andjela@gmail.com (corresponding author) jelena s. matejić university of niš, faculty of medicine, department of pharmacy, serbia nada kovačević university of belgrade, faculty of pharmacy, serbia silva dobrić university of defence in belgrade, medical faculty of the military medical academy, serbia dragana r. pavlović university of niš, faculty of medicine, department of pharmacy, serbia received: january 16, 2024 revised: april 03, 2024 accepted: april 05, 2024 abstract: bruckenthalia spiculifolia belongs to the ericaceae family. the aim of this study was to quantify the total content of phenols, tannins, and flavonoids spectrophotometrically and to evaluate the in vivo and in vitro antioxidant and anti-inflammatory activity of the ethanolic extract of the root of b. spiculifolia. the antioxidant activity with ic50 2.59±0.10 μg/ml and 9.10±0.81 μg/ml in dpph and β-carotene/linoleic acid assay, respectively, was measured. significant anti-inflammatory activity was observed by inhibiting the denaturation of bovine serum albumin by 87±0.004% and reducing the inflammatory response induced by carrageenan in rats in a dose-dependent manner. in view of the demonstrated effects, the ethanolic extract from the root of b. spiculifolia can potentially be used as a new natural source for the pharmaceutical industry. key words: bsa, carrageenan, erica spiculifolia, phytochemical compounds apstrakt: antioksidativna i anti-inflamatorna studija etanolnog ekstrakta korena bruckenthalia spiculifolia (salisb.) reichb. bruckenthalia spiculifolia pripada familiji ericaceae. cilj ovog rada je spektrofotometrijska kvantifikacija ukupnih fenola, tanina i flavonoida, kao i in vitro i in vivo procena antioksidativne i antiinflamatorne aktivnosti etanolnog ekstrakta korena b. spiculifolia. etanolni ekstrakt korena je pokazao antioksidativnu aktivnost u dpph i β-karoten/linoleinska kiselina testu (sa ic50 2,59±0,10 μg/ml i 9,10±0,81 μg/ml). značajna antiinflamatorna aktivnost je zabeležena uz denaturaciju goveđeg serumskog albumina od 87±0,004% i dozno zavisno smanjenje inflamatornog odgovora izazvanog karagenanom kod pacova. s obzirom na pokazanu antioksidativnu i antiinflamatornu aktivnost, etanolni ekstrakt korena b. spiculifolia se može potencijalno koristiti kao novi prirodni resurs u farmaceutskoj industriji. ključne reči: bsa, karagenin, erica spiculifolia, fitohemijska jedinjenja introduction bruckentalia spiculifolia salisb (syn. erica spiculifolia salisb., brukentalia spiculifolia drude) is the monospecific genus of cosmopolitan ericaceae family (jovanović, 1972). today, the high mountain regions of greece, romania, bulgaria, turkey, macedonia, and serbia are the only places where b. spiculifolia can be found (turrill, 1929; horvat et al., 1974; polunin & walters, 1985). the absence of bracteoles, the partially fused sepals, and a unique type of caryology are features of this genus (oliver 1989, 2000). the discovery of bruckenthalia seeds in a deposit at gort in county galway, ireland, in 1959 provided the first evidence of the plant’s earlier occurrence at sites in western europe (jessen et al., 1959). however, their true parentage was not discovered until phillips & sparks (1976) refuted their initial claim that they were descended from erica scoparia var. macrosperma. bruckenthalia spiculifolia is an evergreen shrub with tiny flowers (corolla 2.5 – 3 mm long) grouped in terminal inflorescences. a low-growing, bushy shrub or subshrub has a pistillate apex that is the same diameter as the rest of the pistil, but is often mistakenly called a „stigma”. this plant species © 2024 dragićević et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 37 grows on acidic soils in mountainous shrublands between 1,400 and 2,500 meters (mitić et al., 2018). the soils of the ecosystems where this species lives are volcanic rocks with mostly acidic character (between 4 and 6) (fagúndez & izco, 2008). folk uses of erica species include antirheumatic, diuretic, and astringent effects, as well as treatment of urinary tract infections, wounds, snakebites, and inflammation-related diseases, including draining edema (mitić et al., 2018). as far as we know, the available literature data on b. spiculifolia are scarce, especially for the ethanolic extract. there are several studies whose aim was to investigate the primary phytochemical composition and antioxidant activity of the ethanolic extract of b. spiculifolia. according to pavlović et al. (2009) significant amounts of polyphenolic compounds, including tannins and flavonoids, were found in the ethanolic extract of aboveground parts of the b. spiculifolia. the aforementioned authors observed the remarkable antioxidant activity of the ethanolic herbal extract as determined by three assay systems, namely total antioxidant capacity (frap), free radical scavenging (dpph), and inhibition of lipid peroxidation (pavlović et al., 2009). qualitative and quantitative hplc analysis of phenolic compounds, total phenylpropanoids content, and inhibition of β-carotene bleaching of the ethanolic leaf extract of b. spiculifolia was evaluated (pavlović et al., 2013). yet, there is no data in the literature on phytochemical compounds, and antioxidant and anti-inflammatory activity of the root extract of b. spiculifolia. the aim of this study was to determine the content of the total phenolic, total tannin, and total flavonoid compounds of ethanolic extract of the root of b. spiculifolia. furthermore, the antioxidant activity of the ethanolic root extract is investigated with two different complementary in vitro tests: dpph radical scavenging activity and β-carotene/ linolenic acid assay. the anti-inflammatory activity of the ethanolic root extract was determined in vitro by a protein denaturation assay using an aqueous solution of bovine serum albumin and in vivo in a carrageenan-induced rat paw oedema assay. materials and methods plant material, extraction, and chemical analysis the plant species was collected in serbia (vlasina lake) and identified by professor bojan zlatković, from the department of biology and ecology, faculty of sciences and mathematics, university of niš. the b. spiculifolia (voucher no. 3364) has been deposited in the herbarium of the faculty of pharmacy, university of belgrade. the root was percolated with 70%, v/v ethanol according to ph. eur. 10.0 (2020) and then evaporated to dryness under vacuum at 40 °c; the extraction yield was 11.5%. all chemicals used were obtained from sigma aldrich (usa), or zorka pharma (šabac, serbia). all solvents and chemicals were of analytical grade. determination of total phenolic contents the total phenolic content of ethanolic extract of the root of b. spiculifolia was determined using the folin‐ciocalteu method (makkar et al., 2000; maksimović et al., 2005). the properly diluted test samples are filled into test tubes with ethanol (70% v/v) as solvent. then the folin‐ciocalteu reagent, previously diluted 1:1 v/v with distilled water, and 20% na2co3 solution were added to test tubes. the tubes were then shaken vigorously for 40 min to develop the blue color and the absorbance was measured spectrophotometrically at 725 nm in comparison to a blank containing extraction solvent instead of the sample. the total phenolic content of the root extract was calculated and expressed as mg catechin equivalents (ce) per g of dry extract using a (+)-catechin calibration curve (1-5 μg/ml range). the total phenolic assay was done in triplicate. determination of total tannin content the total tannin content of the ethanolic extract of the root of b. spiculifolia was determined using the same folin‐ciocalteu method (makkar et al., 2000; maksimović et al., 2005). polyvinylpolypyrrolidone was added to the test tubes containing the sample and then shaken vigorously. the supernatant contains all phenolic compounds except the tannins. the test was performed on the clear supernatant and the results were expressed in mg catechin equivalents (ce) per g dry extract. the experiment was done in triplicate. the content of non-tannin polyphenols is also expressed as mg catechin equivalents per g of dry extract and total tannin content is obtained from the difference. determination of total flavonoid content the total flavonoid content of the ethanolic extract of the root of b. spiculifolia was estimated according to lamaison & carnat (1990). aluminum trichloride (alcl3) in ethanol was mixed with the same volume of the extract solution. the blank sample consisted of alcl3 with ethanol without extract solution. after incubation for 1 h, the absorbance was measured spectrophotometrically at 430 nm. the total flavonoid content of the root extract was calculated and expressed as mg rutin (ru)/g dry extract using a rutin calibration curve (0.5-5 μg/ml range). the total flavonoid assay was done in triplicate. 38 biologica nyssana ● 15 (1) june 2024: 37-45 dragićević et al. ● antioxidative and anti-inflammatory study on the ethanolic extract of the root of bruckenthalia spiculifolia (salisb.) reichb. biologica nyssana ● 15 (1) june 2024: 37-45 dragićević et al. ● antioxidative and anti-inflammatory study on the ethanolic extract of the root of bruckenthalia spiculifolia (salisb.) reichb. 39 antioxidant activity examination dpph assay the dpph (1,1-diphenyl-2 picrylhydrazyl) free radical scavenging assay was used to measure the antioxidant activity of the ethanolic extract of the root of b. spiculifolia according to pavlović et al. (2017). increasing volumes (0.1 ml, 0.2 ml, 0.4 ml 0.6 ml, 0.8 ml) of the ethanolic extract with dpph in 96% (v/v) ethanol were added to a 96-well microtitration plate. the plate was stored in the dark for 30 min at room temperature, then the absorbance was measured on an elisa reader at 540 nm against 96% (v/v) ethanol as a blank. the synthetic antioxidants bht and bha were used as reference substances, while dpph with 96% (v/v) ethanol was used as a control. inhibition of dpph free radical in percent was calculated according to: % dpph = (ac – as) / ac × 100 where ac is the absorbance of the control, and as is the absorbance of the sample. the ability to neutralize a free radical can be determined by the ic50 value, i.e. the concentration of the extract that removes (neutralizes) 50% of the stable dpph• radicals from the solution (cuendet et al., 1997). β-carotene bleaching assay the β-carotene bleaching assay was used to evaluate the ability of the ethanolic extract of the root of b. spiculifolia to inhibit lipid peroxidation according to koleva et al. (2002). to perform the assay, tween-20 and linoleic acid were combined with β-carotene solution in chloroform, and the mixture was evaporated under vacuum at a temperature of 40 °c. after adding distilled water, an emulsion was formed by shaking the mixture. a freshly prepared β-carotene-linoleic acid emulsion was added to the sample in a 96-well microtitration plate. the control contains solvent (70% v/v) instead of the extract solution. the absorbance was read on an elisa reader at 450 nm after the plate had been shaken on a microplate shaker. after reading the initial absorbance, the plate is placed in an incubator at 55 °c for 120 min, after which the absorbance is read again. the percentage inhibition of β-carotene bleaching by the samples was calculated according to the formula (barros et al., 2007): % inhibition = 100 (a120 / a0) × 100 where a120 is the absorbance of the sample at t=120 min and a0 is the absorbance of the sample at t=0 min. the synthetic antioxidants bht and bha were used as reference substances. anti-inflammatory activity examination animals and housing adult male wistar rats (200–250 g) from the animal farm of the medical military academy in belgrade, were used in this study. the experiment was conducted at the medical faculty of the military medical academy, university of defence in belgrade in accordance with the internationally accepted principles for laboratory animal use and care in the european community (eec directive of 1986; 86/609/eec) adopted by ethical committee of military medical academy, belgrade (no. 32307-06862/2016-05/2). evaluation of in vitro anti-inflammatory activity the in vitro anti-inflammatory activity of the ethanolic extract of the root of b. spiculifolia was investigated using the protein denaturation assay according to lavanya et al. (2010). the test solution consists of an ethanolic extract of the root and a 5% w/v aqueous solution of bovine serum albumin (bsa). a distilled water and a 5% w/v aqueous solution of bovine serum albumin served as a control. the results were compared with the standard solution, which consisted of diclofenac sodium and a 5% w/v aqueous solution of bovine serum albumin. the absorbance was measured at 340 nm using an elisa microplate reader. the inhibition (%) of protein denaturation was calculated by applying the following equation: protein denaturation (%) = 100 ((optical density of test solution optical density of product)/optical density of test control) × 100) the control represents 100% protein denaturation. the results were compared with diclofenac (100 µg/ ml). evaluation of in vivo anti-inflammatory activity the in vivo anti-inflammatory activity of the ethanolic extract of the root of b. spiculifolia was investigated in carrageenan-induced paw oedema assay according to dobrić et al. (2017). the tested root extract was dissolved in dimethyl sulfoxide (dmso) and administered per os by gastric tube at a dose of 50, 100, and 200 mg/kg body weight, 60 min before the onset of inflammation. groups of 6 animals were formed for each dose. oedema was induced by injection of 0.5% carrageenan solution into the right hind paw of rats. the same volume of saline solution was injected into the left hind paw of the rats. the control animals were administered a vehicle (dmso) per os at 1 ml/kg. indomethacin, which was also dissolved in dmso and administered per os, served as the reference drug at a dose of 4 mg/kg. three hours after injection of carrageenan, i.e. saline solution, and 4 h after administration of the extracts, the difference in mass between the right and left paw, indicated the size of the oedema, i.e. the degree of inflammation was measured (dobrić et al. 2017). statistical analysis data for both the in vitro and the in vivo assay were expressed as the mean ± sd value. a one-way analysis of variance (anova) followed by tukey’s post-hoc analysis (for in vitro tests) or mannwhitney u analysis (for in vivo tests) was used to determine whether there were statistically significant differences between treatments (graphpad prism version 5.03, san diego, ca, usa). results and discussion numerous biological effects of phytochemical compounds from plant extracts have been documented in the scientific literature (sharma et al., 2021). the most important phytochemical compounds include phenols, tannins, flavonoids, alkaloids, glycosides, steroids, and others. most of the intended beneficial properties are due to these chemical compounds (alzamel, 2022). phenolic compounds have been extensively studied for their biological effects, such as antioxidant, anti-inflammatory, antimicrobial, antihypertensive, anticarcinogenic, and antimutagenic effects, which may be beneficial to human health (shahidi & ambigaipalan, 2015; cosme et al., 2020). pharmaceutical applications of tannins include astringents, diuretics, gastric and duodenal antitumor agents, antioxidants, antiseptics, anti-inflammatory agents, and hemostatics (ribeiro et al., 2022). various positive effects are attributed to flavonoids, including cytotoxic, anti-cancer, antibacterial, anti-inflammatory, anti-allergic, antioxidant, and vascular effects (mutha et al., 2021). the folin-ciocalteu method was used to determine the total phenolic content of the ethanolic extract of the root of b. spiculifolia, which was 194.41±5.74 ce/g (tab. 1). the tannin compounds were also examined using the same method. we found that the extract contained 150.17±7.52 ce/g tannins (tab. 1). the total flavonoid content in the ethanolic extract, expressed as mg rutin per g dry extract, was also analyzed. the results showed that the ethanolic extract of the root contains 3.51±0.06 mg ru/g flavonoids (tab. 1). as the obtained results indicate, the studied ethanolic root extract of b. spiculifolia contains considerable amounts of phenolic, tannic and flavonoid compounds. these phytochemical compounds may be responsible for many of the health benefits of this extract. in previous study significant amounts of phenolic compounds were found in plant materials and dry leaves extracts of b. spiculifolia: 3.71±0.05 and 11.79±0.25% flavonoids, 4.67±0.08 and 19.22±0.48% total polyphenols, 0.86±0.06 and 3.99±0.07% non-tannins, 3.80±0.06 and 15.23±0.07% tannins, respectively (pavlović et al., 2009). in the study conducted by pavlović et al. (2013) seven phenolic compounds were identified in the dry ethanolic leaf extracts. the main phenolic compounds were quercitrin (70.91 mg/g), quercetin 3-o-glucoside (32.5 mg/g), and quercetin (3.77 mg/g) (pavlović et al., 2013). the antioxidant activity of ethanolic extract of the root of b. spiculifolia was examined by two complementary tests: dpph free radical scavenging activity and β-carotene bleaching assay. the investigated ethanolic extract of root showed antioxidant activity with ic50 2.59±0.10 μg/ml and 9.10±0.81 μg/ml in dpph and β-carotene bleaching assay, respectively (tab. 1). according to pavlović et al. (2009), the ethanolic herbal extract of b. spiculifolia showed anti-radical activity with ic50 10.22±0.74 µg/ml. based on the results of our study and the study conducted by pavlović et al. biologica nyssana ● 15 (1) june 2024: 37-45 dragićević et al. ● antioxidative and anti-inflammatory study on the ethanolic extract of the root of bruckenthalia spiculifolia (salisb.) reichb. 40 table 1. phenolic compounds contents and ic50 values in in vitro antioxidant assays of ethanolic extract of the root of b. spiculifolia (salisb.) reichb. phenolic content (mg ce/g) total flavonoid content (mg ru/g) radical scavenging assay ic50 (μg/ml) β-carotene linoleic assay ic50 (μg/ml) total phenolic content (mg ce/g) total nontannin content (mg ce/g) total tannin content (mg ce/g) 194.41±5.74 44.23±12.29 150.17±7.52 3.5±0.06 2.59±0.10 9.10±0.81 bht 22.82±2.07 0.03±0.00 bha 2.44±0.09 0.04±0.01 data are given as mean ± sd (n=3) biologica nyssana ● 15 (1) june 2024: 37-45 dragićević et al. ● antioxidative and anti-inflammatory study on the ethanolic extract of the root of bruckenthalia spiculifolia (salisb.) reichb. (2009) the ethanolic extract of the root has a higher dpph radical scavenging activity. the plant species were collected from different locations, which could be one reason for the different results. further, the different antioxidant activity could be due to the fact that different plant parts were used to prepare the ethanolic extract and the phytoconstituents are different (pavlović et al., 2009). the dry ethanolic herbal extract in concentration of 62.5 µg/ml reduced the bleaching of β-carotene by more than 95% (pavlović et al., 2013). these results are similar to those found for the same extracts tested to inhibit lipid peroxidation in liposomes (pavlović et al., 2009). in a recent study by pavlović et al. (2024), the ethanolic extract of the leaves, flowers, and aboveground parts as well as different fractions of the extract, except for the petroleum ether fraction, showed excellent antioxidant activity in the two complementary assay methods: dpph radical scavenging activity and inhibition of lipid peroxidation in the β-carotene-linolenic acid assay. for comparison, the abilities of the commercially available synthetic antioxidants bht and bha were investigated. under the same conditions under which the extract was tested, the ic50 values for bht (22.82±2.07 μg/ml; 0.03±0.00 μg/ml) and bha (2.44±0.09 μg/ml; 0.04±0.01 μg/ml;) were determined in dpph and β-carotene bleaching assay, respectively (tab. 1). by comparing the concentrations obtained, it can be concluded that the ethanolic root extract showed a better ability of free radicals removal from the commercial antioxidant bht. the reaction mechanism between antioxidants and dpph• radicals depends on the structural conformations of antioxidants, a large number of compounds react slower, and more complex mechanisms are involved (prior et al., 2005). if the radical formed by the antioxidant is not stable enough, many other reactions are also possible. due to the complex reactions involving three different reaction pathways between bht and dpph• radicals, the obtained results can be explained (bondet et al., 1997). on the other hand, the synthetic antioxidants showed significantly greater activity in stopping the chain reaction of lipid peroxidation. a significant correlation between antioxidant capacity and total phenolic content suggests that phenolic substances are the most responsible for the antioxidant capacity. the antioxidant activity of phenols comes from their high reactivity as electron or hydrogen donors, from properties of radicals formed from phenols to stabilize and delocalize the unpaired electron and to chelate transition metal ions (gebicki & nauser, 2021; andrés et al., 2023). according to cui et al. (2013) and dharmadeva et al. (2018), one of the causes of inflammation is the production of autoantigens through protein denaturation. changes in hydrophobic, disulfide, hydrogen, and electrostatic bonds may be part of the mechanism of protein denaturation (cui et al., 2013; dharmadeva et al., 2018). consequently, the inhibition of protein denaturation can reduce inflammatory activity. the standard drug in this study was diclofenac, a non-steroidal antiinflammatory drug (nsaid). nsaids have an antiinflammatory effect by preventing the function of the enzyme cyclooxygenase. however, these drugs can also cause constipation, bleeding, ulcers, and perforations (sohail et al., 2023). in the current study, ethanolic extract of the root of b. spiculifolia showed a significant anti-inflammatory effect with a percentage inhibition of bsa denaturation of 87±0.004%, which is comparable although lower than the standard value for diclofenac (95.6±0.001%). the secondary metabolites like phenolic and tannin compounds which were found in preliminary phytochemical screening might be responsible for this activity (fürst & zündorf, 2014; ghosh, 2015). therefore, it is necessary to explore plant-based anti-inflammatory drugs that are natural, safe, and have few side effects. pavlović et al. (2024) discovered that the ethanolic extract of leaves, flowers, and aboveground part of b. spiculifolia and the various fractions, with the exception of petroleum ether, exhibited significant and similar inhibition of bsa denaturation as diclofenac. in addition, the anti-inflammatory effect of the ethanolic extract of the root of b. spiculifolia was investigated in an in vivo assay of carrageenan-induced paw oedema. based on the difference between rats treated with the active substance and rats treated with the vehicle (control), the anti-inflammatory effect of the tested extract and reference drug was found in a carrageenan-induced paw oedema assay. oral administration of the ethanolic extract of the root of b. spiculifolia statistically significantly inhibited the carrageenan-induced inflammatory response in rats in a dose-dependent manner, but differences in the effect between individual doses only in some cases reach the level of statistical significance (far right column of tab. 2). the strongest inhibition 52.37±7.03% was observed at a dose of 200 mg/kg ethanolic extract of root, which was higher than that of the reference drug at a dose of 4 mg/kg, while the vehicle (dmso) at a dose of 1 ml/kg showed no effect. the mechanism of action of carrageenaninduced inflammation is biphasic, with the first phase characterized by the release of histamine, serotonin, and bradykinin, and to a lesser extent prostaglandins produced by cyclooxygenase enzymes (cox) in the first hour, while the second phase is attributed to neutrophil infiltration, and the continuing of 41 biologica nyssana ● 15 (1) june 2024: 37-45 dragićević et al. ● antioxidative and anti-inflammatory study on the ethanolic extract of the root of bruckenthalia spiculifolia (salisb.) reichb. the prostaglandin generation (brooks & day, 1991; gilligan et al., 1994). the delayed phase of carrageenan-induced acute inflammation involves the release of pro-inflammatory cytokines such as tumor necrosis factor (tnf-α) and interleukin-1 β (il-1 β) as well as neutrophil-derived free radicals and nitric oxide (no) (halici et al., 2007). the results of the current study show that the extract reduces the acute inflammation caused by carrageenan. this effect can be explained by the inhibition of the release or synthesis of cyclooxygenase and/ or lipoxygenase products since the production of arachidonic acid metabolites is an important factor involved in both phases of carrageenan-induced inflammation (guay et al., 2004). in addition, the anti-inflammatory effect of the ethanolic extract of the root of b. spiculifolia could be related to the inhibition of neutrophil infiltration and the elimination of free radicals, which are characteristics of the second phase of inflammation (halici et al., 2007). a statistically significant anti-inflammatory effect, ranging from 17.8% to 63%, was obtained from ethanol extracts of the leaves, flowers, and aboveground parts as well as from petroleum ether, ethyl acetate, and water fractionations of the ethanol extract of b. spiculifolia (pavlović et al., 2024). in the in vivo study, it was found that the studied gel containing 2% dry leaf extract of b. spiculifolia significantly reduced the degree of skin irritation and reversed the ph of the skin disturbed by the preirritation with sodium lauryl sulfate (pavlović et al., 2013). further, ethanolic extracts of leaves, flowers, and herbs of b. spiculifolia significantly inhibited no production by macrophages stimulated with lipopolysaccharide (pavlović et al., 2022). our study of the ethanolic extract of the root of b. spiculifolia has shown that the investigated extract contains significant amounts of phenolic, tannin, and flavonoid compounds, as well as considerable in vitro antioxidant and in vitro/in vivo anti-inflammatory activity, providing a basis for future investigations. identification of the molecules responsible for the pharmacological activity requires phytochemical studies to separate the compounds. additional spectral evaluation of the isolated chemicals will also yield a potential drug for later use. conclusion the studied ethanolic root extract of b. spiculifolia contains considerable amounts of phenolic, tannic, and flavonoid compounds. these phytochemical compounds may be responsible for many of the health benefits of this extract. the studied ethanolic extract of the root showed significant antioxidant and anti-inflammatory activity in both in vitro and in vivo studies. however, further studies are required to isolate the active constituents responsible for the observed effect and find potential applications of b. spiculifolia root in light of these and future findings. acknowledgements. this research was supported by the ministry of science, technological development and innovation of the republic of serbia (grant no. 45103-47/2023-01/200113) and the faculty of medicine university of niš internal scientific project (no. 15). references alzamel, n.m. 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(solanaceae), a new alien species … 47 original article received: 25 july 2017 revised: 12 august 2017 accepted: 30 august 2017 datura inoxia mill. (solanaceae), a new alien species in serbia dmitar lakušić1, milica rat2, goran anačkov2, slobodan jovanović1 1university of belgrade, faculty of biology, institute of botany and botanical garden “jevremovac”, takovska 43, 11 000 belgrade, serbia 2university of novi sad, faculty of science, department of biology and ecology, trg dositeja obradovića 2, 21 000 novi sad, serbia * e-mail: dlakusic@bio.bg.ac.rs abstract: lakušić, d., rat, m., anačkov, g., jovanović, s.: datura inoxia mill. (solanaceae), a new alien species in serbia. biologica nyssana, 8 (1), september 2017: 47-51. as ornamental plant originated from central america, datura inoxia mill. has been introduced to many areas beyond its natural range. however, in many places, mainly in the mediterranean region d. inoxia has escaped from cultivation and is being established as an alien. in relevant botanical literature its occurrence on the balkan peninsula has not been reported for albania, bosnia & herzegovina, macedonia (fyrom) and serbia. based on our field and herbarium studies, we have found that d. inoxia is also present in serbia, both as cultivated ornamental plant, but as well as an escape, with established populations. we have registered this species in 23 localities in srem, bačka, banat, šumadija and ne serbia regions, which are situated in 16 utm 10 km x 10 km squares. most of the findings (16 localities) refer to a small group of individuals which are cultivated, while the remaining seven relate to plants that have escaped from cultivation and have established small wild populations in the surrounding ruderal habitats, edges of natural or planted forest, waste and arable fields. although we have registered only seven small groups of individuals that have escaped from cultivation, due to its capacity to invade natural habitats d. inoxia can be considered as potential threat for natural biodiversity in serbia. key words: datura inoxia, alien species, distribution, cultivation, invasion apstrakt: lakušić, d., rat, m., anačkov, g., jovanović, s.: datura inoxia mill. (solanaceae), nova strana vrsta u srbiji. biologica nyssana, 8 (1), septembar 2017: 47-51. kao ukrasna biljka koja potiče iz centralne amerike, datura inoxia mill. je uvedena u mnoge oblasti izvan njenog prirodnog areala. međutim, na mnogim mestima, uglavnom u mediteranu, ona je pobegla iz kultivacije i postala strana vrsta. u relevantnoj botaničkoj literaturi njeno prirustvo na balkanskom poluostrvu nije registrovano za albaniju, bosnu i hercegovinu, makedoniju i srbiju. na osnovu našeg istraživanja utvrđeno je da je d. inoxia prisutan i u srbiji, i to kao kultivisana ornamentalna biljka, ali i kao strana vrsta sa uspostavljenim populacijama i na staništima izvan kultivacije. ova vrsta je registrovana na 23 lokaliteta u regionima srem, bačka, banat, šumadija i ne srbija. registrovani lokaliteti se nalaze u 16 utm kvadrata 10 x 10 km. većina nalaza (16 lokaliteta) odnosi se na male grupe gajenih individual koje, dok se preostalih sedam nalaza odnosi na biljke koje su uspostavile male divlje populacije u okolnim ruderalnim staništima, ivicama prirodnih ili zasađenih šuma, deponijama ili obradivim površinama. iako smo registrovali samo sedam malih 8 (1) • september 2017: 47-51 doi: 10.5281/zenodo.963583 biologica nyssana 8 (1)  september 2017: 47-51 lakušić, d. et al.  datura inoxia mill. (solanaceae), a new alien species … 48 grupa pojedinaca koji su pobegli iz kultivacije, zbog svoje sposobnosti za invaziju prirodnih staništa d. inoxia se može smatrati potencijalnom pretnjom za autohtoni biodiverzitet u srbiji. ključne reči: datura inoxia, strane vrste, rasprostranjenje, gajenje, invazija introduction datura inoxia mill. is an annual, ornamental but extremely toxic plant. it originates from central america. due to its attractive flowers and increased growing in the gardens for decorative purposes, it has been introduced to many areas beyond its natural range (africa, asia, australia and europe). however, in many places, mainly in the mediterranean region (spain, portugal, france, italy, turkey), d. inoxia has escaped from cultivation and is being established as an alien species while in other european countries its alien status is unknown (daisie, 2017). there is the evidence that in vojvodina province this species is cultivated as medicinal plant and that it occasionally escapes from cultivation (o b r a d o v i ć & j a n j a t o v i ć , 1982). in relevant botanical literature its occurrence on the balkan peninsula has not been reported for albania, bosnia & herzegovina, macedonia (fyrom) and serbia (daisie, 2017, c a k o v i ć et al., 2014). according to g r e u t e r and r a u s (2005) datura inoxia has been known from bulgaria (sofia district) since 1933. in romania it is a casual alien, distributed along black sea coastal area, mainly in habitats close to harbors (a n a s t a s i u et al., 2011). the first data for greece refer to plants collected by a. yannitsaros in october 1966, on a rubbish-tip on waste ground on the south peloponnesian island of kithira (y a n n i t s a r o s , 1998). later, the plant was recorded in several localities in the coastal and mainland greece (c a k o v i ć et al., 2014). in croatia the species was reported approximately 30 years ago (h e ć i m o v i ć , 1981). like in greece, in croatia datura inoxia has spread along roads, in courtyards, gardens and in ruderal vegetation, not only to a number of locations in the littoral region, but also within the continental parts (h e ć i m o v i ć , 1981, 1982, f r a n j i ć , 1993, p a n d ž a & s t a n č i ć , 1990, t r i n a j s t i ć et al., 1993, f r a n j i ć et al., 1998, p a n d ž a et al., 2001). recently, in 2012, species was reported for the first time in montenegro, where it was recorded on waste ground at long ulcinj beach (c a k o v i ć et al., 2014). material and methods the study was carried out on recent field studies, analysis of herbarium material deposited at beou and buns (herbarium acronyms according to t h i e r s , 2017+), as well as literature data. the locations for occurrences of the species in the field were recorded with gps (garmin etrex legend hcx and garmin etrex vista c). all other data on the distribution were georeferenced in oziexplorer 3.95 4s program. the chorological data are presented according to the grid map with squares of c. 10 km × 10 km, based on the universal transverse mercator (utm) projection (l a m p i n e n , 2001), greed zone 34t. latitudes and longitudes are given according to the world geodetic system 84 (wgs84). results and discussion based on our field and herbarium studies, we have found that d. inoxia is also present in serbia, both as cultivated ornamental plant, but as well as an escape, with established populations. we have registered this species in 23 localities in srem, bačka, banat, šumadija and ne serbia regions, which are situated in 16 utm 10 km x 10 km squares (fig. 1). most of the findings (16 localities) refer to a small group of individuals which are cultivated, while the remaining seven relate to plants that have escaped from cultivation and have established small wild populations in the surrounding ruderal habitats, edges of natural or planted forest, waste and arable fields (fig. 2). monitoring of few populations (stepojevac, ritopek and golubac ponikva) from 2013 to 2016 did not show any significant spreading into the surrounding hinterland. chorological data bačka: cr45  apatin, in surroundings, cultivated (perić, r. summer 1998, buns) cr75  rumenka, cemetery (ujhelji, s. 23.10.2003, buns) cs90  subotička peščara kelebija-radanovac, planted pinus-robinia forest, 46.134546 n, 19.642232 e, 124 m (radak, b., bokić, b., rat, m. field obs., 12.08.2011) banat: dr57  kikinda 45.83 n, 20.45 e, 80 m, cultivated (fodulović, g. field obs., 09. 2013) srem: dq25  kupinovo, forest edge, waste and the arable fields in vicinity of the settlement, 44.711295 biologica nyssana 8 (1)  september 2017: 47-51 lakušić, d. et al.  datura inoxia mill. (solanaceae), a new alien species … 49 n, 20.045730 e, 74m (knežević, j., kovački, m., rat, m. field obs., 14.10.2016) šumadija: dq42 stepojevac, 44.511594 n, 20.294598 e, 130 m, escaped from cultivation (lakušić, d. field obs., 10.09.2015, ibid 09.10.2016) dq55 beograd braće jerković, 44.772 n, 20.490 e, 150 m, cultivated (fodulović, g. field obs., 09. 2013) dq55 beograd braće jerković, 44.769621 n, 20.498191 e, 175 m, cultivated (fodulović, g. field obs., 08.09. 2016) dq55 beograd železnik, 44.738512 n, 20.386443 e, 90 m, cultivated (lakušić, d. field obs., 10.09.2015) dq56 beograd dorćol, visokog stevana 21, 44.825409 n, 20.458966 e, 80 m, cultivated (dnevne novine blic 12.7.2013) dq56 beograd zvezdara, marka oreškovića, 44.803009 n, 20.487444 e, 120 m, cultivated (lakušić, d. field obs., 10.2015) dq56 beograd zvezdara, vojvode brane, 44.804743 n, 20.483909 e, 120 m, escaped from cultivation (lakušić, d. field obs., 10.09.2015) dq56 beograd zvezdara, vojvode savatija, 44.804695 n, 20.483720 e, 125 m, cultivated (lakušić, d. field obs., 09. 2013) dq65 beograd ritopek, 44.764100 n, 20.564839 e, 200 m, escaped from cultivation (lakušić, d. field obs. 12.02.2016, ibid. 09.10.2016) dq65 beograd selo rakovica ispod avale, 44.720943 n, 20.499923 e, 200 m, cultivated (lakušić, d. field obs., 10.09.2015) dq66 beograd karaburma, marijane gregoran, 44.813257 n, 20.508549 e, 115 m, cultivated (lakušić, d. field obs., 09. 2013) dq72 mladenovac, 44.468310 n, 20.666330 e, 150 m, cultivated (lakušić, d. field obs. 11.08.2015) dq74 grocka 44.682760 n, 20.706863 e, 110 m, cultivated (lakušić, d. field obs., 06.09. 2016) ne serbia: eq24 veliko gradište, sirakovo 44.679089 n, 21.338209 e, 160 m, escaped from cultivation (lakušić, d. field obs., 25.09. 2013) eq35 veliko gradište, kumane 44.730360 n, 21.466415 e, 75 m, cultivated (lakušić, d. field obs., 25.09. 2013) eq35 veliko gradište, topolovnik 44.720534 n, 21.443139 e, 75 m, cultivated (lakušić, d. field obs., 25.09. 2013) eq44 golubac, ponikva 44.689409 n, 21.570992 e, 120 m, escaped from cultivation (lakušić, d. 37683, 25.09. 2013, beou) eq92 donji milanovac, centar 44.465961 n, 22.153158 e, 70 m, cultivated (lakušić, d. field obs., 25.09. 2013) conclusion in conclusion, although we have registered only seven small groups of individuals that have escaped from cultivation, due to its capacity to invade natural – fig. 1. distribution of datura innoxia mill. in serbia. (utm grid zone 34t; basic square 10 x 10 km, according to l a m p i n e n , 2001). locality symbols:  cultivated plants  established populations biologica nyssana 8 (1)  september 2017: 47-51 lakušić, d. et al.  datura inoxia mill. (solanaceae), a new alien species … 50 habitats d. inoxia can be considered as potential threat for natural biodiversity in serbia. according to field records, its status in serbia is established alien species, with low invasiveness impact, i.e. it is casual species. nevertheless, on a local level it can be considered an important invader. the most vulnerable habitats are sandy areas along danube and continental sands, that are close to human settlements. in the same time, the wide range of adaptations on different artificial habitats and our records altogether with already published data show that datura inoxia is spreading in the continental and pannonian part of the se europe and thus can be expected in albania, bosnia & herzegovina and macedonia (fyrom) as well. acknowledgements. research was supported by the ministry of education and science, the republic of serbia, grant 173030, and financial mechanism of the european economic area 2009-2014, programme bg03 biodiversity and ecosystem services д-33-51/30.06.2015 (esenias-tools). references anastasiu, p., negrean, g., samoila, c., memedemin, d., cogalniceanu, d. 2011: a comparative analysis of alien plant species along the romanian black sea coastal area. the role of harbours. journal of coastal conservation, 15: 595-606. caković, d., stešević, d., vuksanović, s., tan, k. 2014: colchicum cupanii guss. subsp. glossophyllum (heldr.) rouy, datura innoxia mill. and eclipta prostrata (l.) l., new floristic records in montenegro and western balkan. acta botanica croatica, 73(1): 255–265. daisie european invasive alien species gateway, 2017. datura innoxia. available from: http://www.europealiens.org/speciesfactsheet.do?speciesid=20353# [accessed 30th june 2017]. franjić, j., trinajstić, i., škvorc, ž. 1998: a contribution to the knowledge of the spreading of some neophytes in croatia. fragmenta phytomedica et herbologica, 26: 5–17. fig. 2. naturalized plants of datura innoxia mill. golubac, ponikva – eq44, 44.689409 n, 21.570992 e, 120 m (lakušić, d. field obs., 25.09. 2013, photo d. lakušić). biologica nyssana 8 (1)  september 2017: 47-51 lakušić, d. et al.  datura inoxia mill. 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(solanaceae) in croatia. natura croatica, 8: 117–124. thiers, b. 2013 (continuously updated). index herbariorum: a global directory of public herbaria and associated staff. new york botanical garden's virtual herbarium. available from: http://sweetgum.nybg.org/ih/ (accessed 2017). trinajstić, i., pavletić, z., franić, j., liber, z. 1993: contribution to the knowledge of the neophytic flora of makarska littoral (dalmacija, hrvatska) . fragmenta phytomedica et herbologica, 21: 57– 62. yannitsaros, a. 1998: additions to the flora of kithira (greece) i. willdenowia, 28: 77–94. anticancer compounds from medicinal plants biologica nyssana 4 (1-2)  december 2013: 49-55 mitic, v. et al.  chemometric analysis of chlorophyll… … 49 original article chemometric analysis of chlorophyll a, b and carotenoid content in green leafy vegetables violeta mitić, vesna stankov jovanović, marija dimitrijević, jelena cvetković, , goran petrović, gordana stojanović university of nis, faculty of science and mathematics, department of chemistry, nis, serbia e-mail: violetamitic@yahoo.com abstract: mitić, v., stankov jovanović, v., dimitrijević, m., cvetković, j., petrović, g., stojanović, g.: chemometric analysis of chlorophyll a, b and carotenoid content in green leafy vegetables. biologica nyssana, 4 (1-2), december 2013: 49-55. effects of cooking on chlorophyll a and b and total carotenoid content in seven leafy green vegetables (brussels sprout, white cabbage, kale, chard, garden patience, broccoli and spinach) were evaluated. pigment content varied between species. chlorophyll a content was higher in all analyzed vegetables, compared to chlorophyll b and carotenoid content. boiling caused significant loss of chlorophyll a, chlorophyll b and carotenoids in kale (90.30%, 96.32% and 98.09%, respectively). cluster analysis was applied on pigment content in fresh and boiled vegetables and two statistically significant clusters were obtained, with difference in spinach position. key words: carotenoid content, chlorophyll content, chemometrics, cluster analysis, leafy green vegetables introduction leafy green vegetables are widely consumed in serbia. most of the vegetables are cooked by boiling in water or consumed fresh. fresh leafy vegetables contain valuable food components, such as chlorophylls, carotenoids, vitamins and phenolic compounds. these compounds are associated with dietary activities (k i m u r a & r o d r i g u e z a m a y a , 2003; g u t i e r r e z e t a l ., 2008). leafy green vegetables are well known for their characteristic flavor, color, and therapeutic value (f a l l e r & f i a l h o , 2009). these vegetables also contain chlorophylls and carotenoids (k i m u r a & r o d r i g u e z a m a y a , 2002), which are responsible for visual quality attributes (x u e & y a n g , 2009). chlorophylls are the earth’s most important organic molecules as they are necessary for photosynthesis (m a r k o v i ć e t a l ., 2012). chlorophyll is green pigment, which contain magnesium ion at the center of the porphyrin ring. there are various types of chlorophyll structure (chlorophyll a, chlorophyll b, chlorophyll c1, chlorophyll c2, chlorophyll d), but plants contain chlorophyll a and b. chlorophyll b differs from chlorophyll a only in aldehyde group bonded to the porphyrin, compared to the methyl group for chlorophyll a. chlorophyll a is photosynthetic pigment and absorbs blue, red and violet wavelengths in the visible spectrum, while chlorophyll b absorbs blue light and is used to complete the absorption spectrum of chlorophyll a. carotenoids are an important group of pigments in bacteria, algae and higher plants, where they function as accessory photosynthetic pigments covering regions of the visible spectrum not utilized by chlorophylls. carotenoids belong to tetraterpenoids, and they are split into two classes: 11 th sfses • 13-16 june 2013, vlasina lake 4 (1-2) • december 2013: 49-55 http://en.wikipedia.org/wiki/magnesium http://en.wikipedia.org/wiki/ion biologica nyssana 4 (1-2)  december 2013: 49-55 mitic, v. et al.  chemometric analysis of chlorophyll… … 50 xanthophylls (which contain oxygen) and carotenes (hydrocarbons, contain no oxygen). chlorophylls and carotenoids have an important role in the prevention of various diseases such as cancer, cardiovascular diseases and other chronic diseases (s a n g e e t h a & b a s k a r a n , 2010). cooking process can cause changes in physical characteristics and chemical composition of vegetables (z h a n g & h a m a u z u , 2004). chlorophylls are known to be easily degraded by acids, heat and light (t o n u c c i & v o n e l b e , 1992). food color plays important role in product acceptability, so it is important to prevent chlorophyll loss. the reason for the green color loss during processing is attributed to conversion of chlorophylls to pheophytins. this study was undertaken to evaluate pigment content in fresh and boiled broccoli (brassica oleracea var. silvestris), brussels sprouts (brassica oleracea var. gemmifera), white cabbage (brassica oleracea var. capitata), kale (brassica oleracea var. sabauda), chard (beta vulgaris), spinach (spinacia oleracea) and garden patience (rumex patientia). cluster analysis was applied to group vegetables based on their pigment content. material and methods fresh broccoli, brussels sprouts, white cabbage, kale, chard, spinach and garden patience were purchased from local market in niš – serbia. the contents of chlorophyll and carotenoids were determined spectrophotometrically. pigment content was determined in fresh and boiled vegetables. vegetable (10 g) was added to 50 ml of water and cooked for 5 minutes. samples were drained off and cooled. vegetables were cut into small pieces and weighed to 0.5000 g. measured vegetable material was mixed with acetone and homogenised in a mortar. mgco3 was added before vegetable homogenization to prevent chlorophyll pheophytinization. mixtures were filtered, mortar and pestle were washed several times with acetone, and the content was quantitatively transferred to the filter. the filter was washed with acetone so that the rest of the filter was completely white. filtrate was diluted with acetone to a total volume of 25 ml. apsorbance of prepared mixtures was recorded at 662, 644 and 440 nm using acetone as blank and pigment content was calculated using the formula of holm and wetsttein: chlorophyll а = 9.784·a662 – 0.990·a644 chlorophyll b = 21.426·a644 – 4.650·a662 chlorophyll a + b = 5.134·a662 + 20.436·a644 carotenoids = 4.695·a440 – 0.268·(a + b); а = absorbency at corresponding wave length, values 9.784, 0.990, 21.426, 4.650 and 0.288 is the molar absorptivity coefficient according to holm (1954) and wetsttein (1957) for acetone (absorption of 1 cm). after calculating the concentrations, the amounts of pigment per g of fresh matter were calculated applying the formula: c = content of pigment (mg/g) of fresh matter; c1 = the concentration of pigment calculated by the previous formula (mg/l); v = the starting volume of extract (ml); r = dilution; m = the weighed fresh plant (g). statistical analysis all results are expressed as mean of three determinations. arithmetic mean, the standard deviation and the coefficient of variation and correlations were calculated applying software statistica 7 (statsoft, inc., tulsa, ok, usa). cluster analysis (ca) is a multivariate technique, with the purpose of classifying the objects of the system into categories or clusters based on their similarities (richard & dean, 2002). ca is reported in several publications for the analysis of foodstuff (cam et al., 2009, tomsone et al., 2012). results obtained by cluster analysis are typically illustrated by a dendrogram. cluster analysis was done by means of ward’s method using euclidean distances as a measure of similarity. ward’s method attempts to minimize the sum of squares of any two (hypothetical) clusters that can be formed at each step. euclidean distance is most common way to measure distance between objects. the distances can be affected by differences in scale among the dimensions from which the distances are computed, so the variables must be standardized. the linkage distance is reported as dlink/dmax, which represents the quotient between the linkage distances for a particular case divided by the maximal linkage distance. the data derived from this study were originally centered by logarithmic transformation with all variables standardized. statistica 7 software was used to compile the dendrogram. biologica nyssana 4 (1-2)  december 2013: 49-55 mitic, v. et al.  chemometric analysis of chlorophyll… … 51 results and discussion analyses performed on selected vegetables showed a large variety of chlorophyll a, b and carotenoid content. chlorophyll and carotenoid content in green leafy vegetables was measured in fresh and boiled vegetables and the results were presented in figure1, 2 and 3. chlorophyll a content varied between species, from 0.047 mg/g fresh vegetables (f.v.) in brussels sprout, to 0.837 mg/g f.v. in garden patience (figure 1). amount of chlorophyll a was higher than chlorophyll b and carotenoids in all selected vegetables, which is in agreement with literature (lopez-ayerra et al., 1998). in fresh vegetables chlorophyll b ranking was: spinach kale garden patience chard broccoli cabbage brussels sprout (figure 2). these results showed that the chlorophyll concentration in the leafy green vegetables is dependent on species. the highest carotenoid content (figure 3) in spinach (0.191 mg/g f.v.) was seven times higher than than the lowest content in brussels sprout (0.024 mg/g f.v.). fig. 1. chlorophyll a content in fresh and boiled vegetables fig. 2 chlorophyll b content in fresh and boiled vegetables biologica nyssana 4 (1-2)  december 2013: 49-55 mitic, v. et al.  chemometric analysis of chlorophyll… … 52 fig. 3. carotenoid content in fresh and boiled vegetables most vegetables are cooked by boiling before consumed. cooking process has influence on chemical composition of vegetables (ismail et al., 2004; z h a n g and h a m a u z u ; 2004). s a h l i n et al. (2004) concluded that boiling, baking and frying had effect on the ascorbic acid, total phenolic, lycopene and antioxidant activity of the tomatoes. few data on the chlorophyll and carotenoid content of these vegetables are found in the literature, and disagreement with literature data may be caused by differences in varieties used and analytical methods applied. t u r k m e n et al. (2006) reported that chlorophyll a+b content in boiled spinach and broccoli was lower than in fresh vegetables, which is in agreement with our study. boiling caused significant loss of chlorophyll a, chlorophyll b and carotenoids in kale (90.30%, 96.32% and 98.09%, respectively). reduction of chlorophyll a and b content is attributed to the degradation of chlorophylls in their main derivatives pheophytin a and b, respectively (b o e k e l , 1999). pheophytin formation is result of mg 2+ eliminiation of chlorophyll. pheophytin can be produced by an acidic cooking environment or by prolonged cooking. in boiled vegetables, chlorophyll a content was lower than in fresh ones, except in brussels sprout. boiled brussels sprout contained 0,534 mg/g f.v. chlorophyll a, which is almost twice higher than chlorophyll a content in fresh vegetables. chlorophyll b content was lower in boiled than in fresh vegetables. chlorophyll a and b content decreased the most in kale (96.32% and 90.30%, respectively). carotenoid content was higher in boiled brussels sprout and chard than in fresh ones. in other vegetables tested, carotenoid content decreased, ranged from 28.07% in broccoli to 98.09% in kale. chemometrics is the branch of chemistry dealing with the analysis of chemical data (extracting information from data) and ensuring that experimental data contain maximum information (the design of experiments) (wold, 1995). chemometrics is used to classify of food products based on their main compounds (woodcock et al., 2007). in cluster analysis (ca), samples are grouped based on similarities. in this study ca was performed to the standardised data, on chlorophyll a, chlorophyll b and carotenoid content in fresh and boiled vegetables. two clusters were formed after applying cluster analysis to pigment content in fresh vegetables. brussels sprout, cabbage, broccoili and kale formed one cluster, while spinach, garden patience and chard grouped in second cluster. brussels sprout and cabbage showed lowest pigment content, so it was expected that they are „nearest neighbors“. euclidean distance between this two species was lowest (0.09). largest distance was observed between brussels sprout and garden patience (0.84), and accordingly differences between their pigment content was highest. biologica nyssana 4 (1-2)  december 2013: 49-55 mitic, v. et al.  chemometric analysis of chlorophyll… … 53 tree diagram for 7 cases ward`s method euclidean distances 0 20 40 60 80 100 120 (dlink/dmax)*100 chard garden patience spinach kale broccoli cabbage brussels sprouts fig. 4. dendrogram obtained by cluster analysis using means of pigment content in fresh vegetables table 1. euclidean distances among analyzed fresh vegetables euclidean distances (fresh vegetables) brussels sprouts broccoli cabbage kale spinach garden patience chard brussels sprouts 0.00 0.24 0.09 0.36 0.80 0.84 0.58 broccoli 0.24 0.00 0.18 0.17 0.57 0.63 0.37 cabbage 0.09 0.18 0.00 0.27 0.75 0.80 0.55 kale 0.36 0.17 0.27 0.00 0.57 0.65 0.42 spinach 0.80 0.57 0.75 0.57 0.00 0.11 0.23 garden patience 0.84 0.63 0.80 0.65 0.11 0.00 0.28 chard 0.58 0.37 0.55 0.42 0.23 0.28 0.00 ca was performed on pigment content in boiled vegetables (figure 5). according to ca, boiled leafy green vegetables can be grouped as follows: brussels sprout, cabbage, kale, broccoli and spinach (cluster 1) and garden patience and chard (cluster 2). clustering according boiled vegetables pigment content shows different grouping of vegetables. compared to dendrogram compiled using fresh vegetables pigment content, this one differs in spinach position. boiled spinach have similar pigment content as brussels sprout, cabbage, kale and broccoli, while fresh spinach is more similar to garden patience and chard. nearest neighbors are boiled cabbage and kale, with euclidean distance of 0.01. biologica nyssana 4 (1-2)  december 2013: 49-55 mitic, v. et al.  chemometric analysis of chlorophyll… … 54 tree diagram for 7 cases ward`s method euclidean distances 0 20 40 60 80 100 120 (dlink/dmax)*100 chard garden patience spinach broccoli kale cabbage brussels sprouts fig. 5. dendrogram obtained by cluster analysis using means of pigment content in boiled vegetables table 2. euclidean distances among analyzed boiled vegetables euclidean distances (fresh vegetables) brussels sprouts broccoli cabbage kale spinach garden patience chard brussels sprouts 0.00 0.10 0.08 0.08 0.18 0.55 0.43 broccoli 0.10 0.00 0.18 0.18 0.08 0.46 0.33 cabbage 0.08 0.18 0.00 0.01 0.26 0.63 0.51 kale 0.08 0.18 0.01 0.00 0.26 0.62 0.51 spinach 0.18 0.08 0.26 0.26 0.00 0.39 0.26 garden patience 0.55 0.46 0.63 0.62 0.39 0.00 0.27 chard 0.43 0.33 0.51 0.51 0.26 0.27 0.00 conclusion chlorophyll a, b and carotenoid content varied between species. chlorophylls and carotenoids have significant role in human diet, so it is important to determine their loss during cooking process. largest decrease of all analysed pigments was recorded in kale. cluster analysis can be used to classify vegetables according to certain properties. according to pigment content in fresh vegetables, two statistical significant clusters were obtained. pigment content in boiled vegetables also yelds dendrogram with two clusterts, but with different grouping. biologica nyssana 4 (1-2)  december 2013: 49-55 mitic, v. et al.  chemometric analysis of chlorophyll… … 55 acknowledgements: the research was supported by ministry of education, science and technological development of the republic of serbia [oi 172047; oi 172051]. references boekel, m.a.j.s. 1999: testing of kinetic models: usefulness of the multiresponse approach as applied to chlorophyll degradation in foods. food research international, 32 (4): 261–269. cam m., hısıl y., durmaz g. 2009: classification of eight pomegranate juices based on antioxidant capacity measured by four methods, food chemistry 112 (): 721– 726 faller, a.l.k., fialho, e. 2009: the antioxidant capacity and polyphenol 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acid, carotenoids and antioxidant activity of broccoli and their changes during conventional and microwave cooking. food chemistry, 88 (4): 503–509. stojanović et al., 2019, biologica nyssana 10(2) 10 (2) december 2019: 189-197 doi: 10.5281/zenodo.3600205 evaluation of the reference genes in human adipose tissue and lipoma samples original article sanja stojanović university of niš, faculty of medicine, department of biology and human genetics and department for cell and tissue engineering, 18000 niš, serbia s.sanja88@gmail.com (corresponding author) stevo najman university of niš, faculty of medicine, department of biology and human genetics and department for cell and tissue engineering, 18000 niš, serbia stevo.najman@medfak.ni.ac.rs vladimir cvetković university of niš, faculty of sciences and mathematics, department of biology and ecology, 18000 niš, serbia biovlada@yahoo.com aleksandra korać university of belgrade, faculty of biology, 11000 belgrade, serbia aleksandra.korac@bio.bg.ac.rs received: november 1, 2019 revised: december 12, 2019 accepted: december 21, 2019 abstract: housekeeping genes, by definition and function, should be constitutively and stably expressed in all cells of the organism, regardless of the cell type and function they accomplish. however, it was observed that expression of housekeeping genes, used as reference genes in gene expression analysis, varies greatly in different cells and tissue types and is also dependent on the experimental conditions and treatments. the aim of our study was to examine the expression patterns of the most frequently used reference genes (gapdh, actb and rrn18s) in the samples of human subcutaneous adipose tissue (scwat) and benign adipose tissue tumors (lipomas). the obtained results have shown that the expression of all three examined housekeeping genes is slightly lower in lipoma samples compared to scwat samples and that gapdh is the most stable housekeeping gene in examined samples so it can be recommended as an optimal reference gene for gene expression analysis in human scwat and lipoma samples. key words: reference genes, gene expression, adipose tissue, lipoma, scwat apstract: evaluacija referentnih gena u uzorcima humanog masnog tkiva i lipoma housekeeping geni bi, po definiciji i funkciji, trebalo da se konstitutivno i stabilno eksprimiraju u svim ćelijama organizma, nezavisno od tipa ćelija i funkcije koju one obavljaju. primećeno je međutim da ekspresija housekeeping gena, koji se koriste kao referentni geni u analizi ekspresije gena, varira u velikoj meri zavisno od tipa ćelija i tkiva i da zavisi takođe od eksperimentalnih uslova i tretmana. cilj našeg istraživanja je bio ispitati obrazac ekspresije najčešće korišćenih referentnih gena (gapdh, actb i rrn18s) u uzorcima humanog potkožnog masnog tkiva (scwat) i benignih tumora masnog tkiva (lipoma). dobijeni rezultati su pokazali da je ekspresija sva tri ispitivana gena nešto niža u uzorcima lipoma u odnosu na uzorke scwat, da je gapdh najstabilniji housekeeping gen u ispitivanim uzorcima i da se može preporučiti kao optimalan referentni gen za analizu ekspresije gena u uzorcima humanog scwat i lipoma. ključne reči: referentni geni, genska ekspresija, masno tkivo, lipomi, scwat introduction housekeeping genes, by definition and function, should be constitutively and stably expressed in all cells of the organism, regardless of the cell type function and they accomplish, and are considered to be the minimal set of genes necessary for the life of an organism (eisenberg and levanon, 2013). since they are essential for maintaining the basic cellular structures and functions, they are expressed at all stages of the development and growth of an organism, as well as in physiological and pathophysiological states (eisenberg and levanon, 2013). in biomedical research, housekeeping genes are widely used as reference genes, i.e. internal controls. the largest application in this field is for the purposes of gene expression analysis using qrt-pcr (quantitative reverse-transcription polymerase chain reaction) method, where housekeeping genes play a role of endogenous control for normalizing the expres© 2019 stojanović et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 189 sion of a gene of interest (goi) (bustin and nolan, 2004; eisenberg and levanon, 2013; caracausi et al., 2017). many studies have shown that the results obtained using this method are largely influenced by various factors, such as experimental conditions, sample size and rna isolation techniques, but also rna integrity, and the efficiency of reverse transcription reaction (rna reversely transcribed into cdna) and the quality of the cdna obtained (bustin, 2002; bustin and nolan, 2004). the influence of these factors is due to the multistage manipulations of the samples prior to the qpcr reaction itself (rna isolation, reverse transcription etc.), as well as the use of different reagents as obligatory components of the kits that are necessary during all the above steps. all of these can significantly affect the efficacy and specificity of the qpcr reaction itself, so certain internal controls need to be included in the analysis to increase efficiency and to make the final result as realistic and highly specific as possible for the studied goi. housekeeping (reference) genes are used to correct, i.e. to normalize variations in the expression of the goi from sample to sample, thereby avoiding errors and increasing the efficiency of the rt-pcr method when quantifying the expression of the goi. in order for a housekeeping gene to be considered as the most suitable reference gene used for normalization in gene expression analysis it should meet the following criteria: to be expressed at a sufficient level so it can be easily detected, to have stable/constant expression in different cell types in an examined organism and within the same cell type undergoing different treatments or experimental conditions and to be ubiquitously expressed (caracausi et al., 2017). thus, the proper selection of the reference gene for studying gene expression by qrt-pcr is extremely important and can be very challenging (andersen et al., 2004; derveaux et al., 2010; zhang et al., 2016). although many different reference genes have been used in the research to date, no “ideal” reference gene has been found yet (radonić et al., 2004; andersen et al., 2004). the stability of the commonly used reference genes has been shown to be relative, since it was observed that their expression varies greatly in different cell and tissue types and also depending on the experimental conditions and different treatments (thellin et al., 1999; schmittgen and zakrajsek, 2000; suzuki et al., 2000; warrington et al., 2000; tricarico et al., 2002; andersen et al., 2004; dheda et al., 2004). in the literature, among the reference genes used in the studies of gene expression in biomedical research, various genes can be found (eisenberg and levanon, 2013; taube et al., 2015; zhang et al., 2016; almeida-oliveira et al., 2017), however among the most frequently used, regardless of the sample type and organism, are genes for: glyceraldehyde-3-phosphate dehydrogenase (gapdh), beta actin (actb) and 18s ribosomal rna (rrn18s) (gabrielsson et al., 2005; almeida-oliveira et al., 2017), while in the studies that analyzed gene expression in different adipose tissue samples, in addition to these three the most common, genes for ldl receptor related protein 10 (lrp10), beta-2-microglobulin (b2m) and beta-glucuronidase (gusb) were also used. lipomas represent one of the most common soft tissue neoplasms of mesenchymal origin (mohammed et al., 2014; omonte et al., 2016) with still unclear etiology and pathogenesis. although lipomas are very common soft tissue tumors, there is insufficient information on their molecular characteristics and the potential mechanisms of their formation. to understand the mechanisms of lipoma formation and to find potential ways for their adequate treatment, detailed characterization of these tumors at molecular level needs to be performed in addition to already performed studies on the implications of mesenchymal stem cells in the pathogenesis of lipoma (stojanović et al., 2018; stojanović and najman, 2019). gene expression analysis gives us insight into the changes that have occurred in adipose tissue that may have led to its dysfunction and tumor development, so it is necessary to have reliable reference genes to perceive the real state of transcriptome and to find target places for eventual therapy. considering the very different data on reference genes used in gene expression analysis and the extent to which their expression varies, both in different tissue types and in different samples of the same tissue type, the aim of this study was to examine the expression patterns of the most frequently used reference genes (gapdh, actb and rrn18s) in the samples of human subcutaneous adipose tissue (scwat) and benign adipose tissue tumors (lipomas). materials and methods tissue samples lipoma tissue samples were obtained after surgical removal of the lipomas, previously clinically diagnosed as benign adipose tissue tumors, while scwat samples were taken during other non-cancerous surgeries patients underwent. all samples were obtained at clinical center niš, serbia, and all patients included in the study gave their informed written consent. the study was approved by the local ethics committee of the faculty of medicine, university of niš, serbia (approvals 01-6481-15 and 12-6316-2/4). a total of 37 tissue samples were analyzed, of which 24 lipoma and 13 scwat samples, patients of both sexes aged 21 to 70 years. tissues 190 biologica nyssana ● 10 (2) december 2019: 189-197 stojanović et al. ● evaluation of the reference genes in human adipose tissue and lipoma samples resis gel. the expression pattern of the examined housekeeping (reference) genes, as well as the variation of their expression among the samples of both study groups, was analyzed based on the obtained ct (cycle threshold) values. statistical analysis results in the form of obtained ct values were statistically processed. as indicators of central tendency mean and median values were calculated, while standard deviation (sd) and coefficient of variation (cv) were calculated as indicators of the variability. the results are presented as boxplots, made in graphpad prism 5, with median values and the range of minimum and maximum values. statistically significant differences in the expression of the examined reference genes between lipoma and scwat samples were analyzed by anova and mann – whitney u tests. values of p < 0.05 were considered as statistically significant. results the specificity of qpcr reactions was confirmed by melting curve analysis and the results are presented in fig. 1. based on the obtained melting curves, it can be seen that in all qpcr reactions the specific primer binding products were obtained. only one peak for rrn18s (a), actb (b) and gapdh (c) for each qpcr sample reaction that can be observed in the graphs indicates the presence of only one specific reaction product per sample. the results of the gene expression pattern analysis of gapdh, actb and rrn18s housekeeping genes are presented as ct values in fig. 2. it can be noticed that rrn18s is highly expressed in all samples, as shown by low ct values (fig. 2a). the expression of rrn18s is slightly lower in lipoma than in scwat samples, although the resulting difference is not statistically significant (p = 0.86). the expression of rrn18s is highly variable in both examined groups of samples, which can be noticed based on the distribution of ct values (fig. 2a). the expression of actb was statistically significantly lower in lipoma compared to scwat samples (p < 0.05) (fig 2b). broad range and uneven distribution of the values can be noticed in both lipoma and scwat group, indicating a relatively large variability in actb expression in different samples within both study groups (fig. 2b). the results of gapdh expression (fig. 2c) indicate statistically significantly lower level of expression of this gene in lipoma compared to scwat samples (p < 0.05), which can be seen from the higher ct values in lipoma samples. on the other hand, narrower range and more uniform distribution of ct values can be observed in the case of gapdh expression compared to the expression were sampled from several body localizations such as: head, oral cavity, neck, arm, leg, abdomen, back, hip, knee and groin. all patients had a body mass index less than 30, which excluded pathologically obese patients. gene expression analysis rna isolation and reverse transcription immediately after sampling, parts of the lipoma and scwat were stored in rnalater® at -80 °c until rna isolation. total rna was isolated using rneasy lipid tissue mini kit (qiagen, venlo, netherlands) according to the manufacturer’s instructions, after tissue homogenization in trizol reagent (qiagen), using tissue homogenizer (th220, omni international, sad). on column removal of residual genomic dna was performed during rna isolation using dnase i rnase-free kit (qiagen). rna concentration was determined immediately after isolation on the qubit® fluorimeter (thermo scientific, waltham, ma, usa) using qubit™ rna br assay kit (thermo scientific), and then isolated rna was stored at -80 °c until further use. total rna was subsequently transcribed into a single-stranded cdna using high-capacity cdna reverse transcription kit (applied biosystems, foster city, ca, usa), according to the manufacturer’s instructions. the reverse transcription reaction was carried out in surecycler8800 (agilent technologies, santa clara, ca, usa), with 400 ng of total rna per reaction per sample. the reverse transcription reaction was performed according to the following program: 10 min at 25 °c, 120 min at 37 °c, 5 min at 85 °c and cooling at 4 °c. the synthesized cdna was stored at -80 °c and used later to determine relative gene expression. real-time pcr analysis of the relative gene expression in tissue samples, by sybr green based real-time qpcr method, was performed in mic qpcr cycler (bio molecular systems, australia). the reactions were prepared using luna® universal qpcr master mix (new england biolabs, ipswich, ma, usa) according to the manufacturer’s instructions. universal rox dye was used as a reference dye. predesigned primer sets (quantitect primer assay kits, qiagen) were used for the following genes: gapdh (qt00079247), rrn18s (qt00199367) and actb (qt00095431). the protocol conditions were as follows: (1) enzyme activation: 60 s at 95 oc (1 cycle); (2) denaturation: 15 s at 95 oc and annealing/extension (with data acquisition): 30 s at 60 oc (40 cycles). the specific binding of primers was confirmed by melting curve analysis for each reaction and by visualizing specific length products on electropho191 biologica nyssana ● 10 (2) december 2019: 189-197 stojanović et al. ● evaluation of the reference genes in human adipose tissue and lipoma samples 192 fig. 1. melting curves for all qpcr products; reference genes: rrn18s (a), actb (b) and gapdh (c) fig. 2. expression pattern of rrn18s (a), actb (b) and gapdh (c) housekeeping genes in lipoma and scwat samples; the results are presented as ct values; boxplot charts with median and the range of minimum and maximum values biologica nyssana ● 10 (2) december 2019: 189-197 stojanović et al. ● evaluation of the reference genes in human adipose tissue and lipoma samples of previously analyzed rrn18s and actb genes. the cv, as a measure of deviation from the mean, is the lowest in the case of gapdh gene in both lipoma and scwat group of samples (tab. 1) which confirms the findings that gapdh is the most stable reference gene in our study in lipoma and scwat samples. for all tested genes, mean and median ct values were higher in lipoma compared to scwat group of samples, indicating that the expression of all three examined reference genes was slightly lower in lipoma samples compared to scwat samples. statistically significant difference (p < 0.05) in gene expression between lipoma and scwat samples was noticed for actb and gapdh, but not for rrn18s. similar values of the mean and median can be observed for all tested samples in the case of all examined genes, indicating a relatively symmetric distribution of values in both groups. discussion to adequately analyze the expression of goi in examined samples and to increase the efficiency of qpcr reactions, it is very important to choose the optimal housekeeping gene. gapdh, actb and rrn18s are the most commonly used reference genes that can be found in published studies that dealt with gene expression analyses wherein gapdh was recommended as the reference gene with the most stable expression. however, numerous literature data indicates that there is a great variability in the expression of these genes both in samples of different tissues and samples of the same tissue and cell type as well as under different experimental conditions, treatments and metabolic states. there are various computer programs, statistical methods and software tools such as normfinder, genorm, bestkeeper and global pattern recognition for identification and selection of an optimal reference gene (de spiegelaere et al., 2015; perez et al., 2017) that can predict the expression pattern of housekeeping genes in the desired samples, but they cannot confidently predict the experimental conditions and how it will affect the expression of reference genes. when adipose tissue is analyzed good prediction of this type cannot be done since adipose tissue is an endocrine and paracrine organ that is greatly influenced by various factors such as nutrition, hormonal status, metabolic status, physical activity, temperature, etc. and also its characteristics vary from organism to organism and within different anatomical localizations of the same organism. therefore, the aim of this study was to examine the expression pattern of three the most commonly used housekeeping genes in the scwat and lipoma samples, and to determine which housekeeping gene is the most optimal to be used as a reference gene in gene expression analysis by real-time pcr. due to interand intraindividual differences in housekeeping gene expression in human tissue samples, some authors consider that, in the case of comparison, more than one housekeeping gene should be used to normalize the level of the rna of the target gene (tricarico et al., 2002). in the study that analyzed the expression patterns of housekeeping genes in colon and bladder cancer samples by rt-pcr, housekeeping genes ubc, gapdh and tpt1 were shown to be suitable for normalization in colon cancer samples, while hspcb, tegt and atp5b were suitable for normalization in bladder cancer samples (andersen et al., 2004). in the study in which the expression of several housekeeping genes was analyzed in 16 different human tissue samples by real-time pcr, the rpii gene has been shown to be suitable as a reference gene for a large number of different tissues (radonić et al., 2004), but authors have noticed that the expression level of examined genes was signifi193 table 1. statistical parameters of reference genes’ expression analysis in lipoma and scwat samples rrn18s actb gapdh lipomas scwat lipomas scwat lipomas scwat mean 6.89 6.82 16.13 15.58 17.03 16.61 median 6.94 6.79 16.11 15.33 17.05 16.65 minimum 6.05 5.33 15.06 14.83 15.86 15.63 maximum 7.63 7.67 17.52 17.30 18.05 17.32 standard deviation 0.53 0.68 0.64 0.79 0.58 0.44 coefficient of variation (%) 7.69 9.97 3.97 5.10 3.41 2.65 statistical significance p = 0.86 p < 0.05 p < 0.05 biologica nyssana ● 10 (2) december 2019: 189-197 stojanović et al. ● evaluation of the reference genes in human adipose tissue and lipoma samples 194 cantly different in different tissue types. in the same study, in performed in vitro experiments on cell cultures, it has been shown that different treatments have the least effect on rpii expression (radonić et al., 2004). when expression level of 10 commonly used reference genes was analyzed (gadph, ppia, actb, ywhaz, rrn18s, b2m, ubc, tbp, rplp and hprt) in human heart tissue samples, ppia, rplp and gadph genes have been shown to be the most stable as estimated by genorm and normfinder and were recommended for the analysis of this tissue type (pérez et al., 2007). no statistically significant difference was found between the rats on the lipid-rich diet and the control group when the expression levels of the reference genes rrn18s, actb, gapdh and 36b4 were examined in three different types of rat adipose tissue of both groups of rats, but it was noticed that rrn18s was the most expressed gene in all samples (zhang et al., 2016) which correlates with the results obtained in our study. the stability of reference genes’ expression was examined by genorm, normfinder and bestkeeper softwares and the results showed that gapdh was the most stable reference gene in ewat (epididymal white adipose tissue) samples, but in ibeat (inguinal beige adipose tissue) and bat (brown adipose tissue) samples its expression was the most variable (zhang et al., 2016). the results obtained with genorm and normfinder differed from those obtained by other analyzes, so the authors proposed 36b4 and gapdh as the best combination of reference genes for ewat samples, while for ibeat and bat samples they suggested 36b4 and actb (zhang et al., 2016). changes in the expression of housekeeping genes were monitored during adipogenic differentiation of primary preadipocytes as well as under various experimental conditions and treatments of human primary mature adipocytes and its has been shown that the expression of gapdh and tfr housekeeping genes was the most stable in different experimental conditions (gorzelniak et al., 2001). the effect of adipogenic differentiation, cryopreservation and medium supplementation on the stability of 11 reference genes was examined in adscs in vitro and estimated by genorm software (dessels and pepper, 2019). it has been shown in this study that different experimental conditions significantly influence the expression stability of reference genes while the most stable expression in all experimental conditions was observed for ywhaz, hprt, tbp and actb (dessels and pepper, 2019). expression of 12 commonly used reference genes was analyzed in different murine adipose tissue samples of animals subjected to different treatments and the results showed that the tbp gene has stable expression in examined adipose tissue samples of the control group and is proved to be suitable for the analysis of gene expression in bat of the control and obese mice (almeida-oliveira et al., 2017). the atpf1 gene was stably expressed in the subcutaneous and perigonadal adipose tissue samples of control and obese mice and was proved to be the best for normalizing gene expression in adipose tissue samples of streptozotocin-treated animals (almeida-oliveira et al., 2017). authors of this study conclude that there is no ideally stable reference gene under all experimental conditions, but on the basis of the results obtained, they propose tbp and atpf1 as suitable reference genes for gene expression normalization in mouse adipose tissue samples in various metabolic states and disorders (almeida-oliveira et al., 2017). when several commonly used reference genes as well as some new candidates were analyzed by qpcr and microchip analysis in perirenal bat and subcutaneous adipose tissue, among commonly used, rplp0, lrp10, ywhaz and gapdh showed the lowest variability in examined samples in this study as evaluated by normfinder and genorm software (taube et al., 2015). the authors of this study conclude that all frequently used reference genes, except rrn18s, show acceptably low variability (taube et al., 2015). also, authors identified potentially new reference genes such as: gnb1, gnb2 and psmb2, which proved to be even more stable than the commonly used reference genes (taube et al., 2015). our results have shown that the expression of gapdh was the most stable in both groups of examined samples (lipoma and scwat), and varied the least between different patients within examined groups. expression analysis of a large number of reference genes in subcutaneous and omental adipose tissue samples has shown that rplp0 is the most suitable reference gene for adipose tissue. however, in one study it was shown that the expression of rplp0, when analyzed in relation to the lrp10, was significantly influenced by the diet-induced weight loss (gabrielsson et al., 2005). in examined adipose tissue samples in that study, it was shown that gapdh expression varied in greater extent in all examined groups compared to rrn18s and actb, which is contrary to what was observed in cultures of primary human adipocytes in vitro for the same genes, where expression of gapdh was stable in all samples and experimentally conditions (gorzelniak et al., 2001). this finding in tissue samples is contrary to what we obtained in our study. gabrielsson et al. (2005) propose lrp10, as the most suitable reference gene for gene expression analysis in human adipose tissue samples. expression of ipo8 and fbxl10, as potential reference genes, was examined by real-time pcr in omental and subcutaneous adipose tissue samples as biologica nyssana ● 10 (2) december 2019: 189-197 stojanović et al. ● evaluation of the reference genes in human adipose tissue and lipoma samples 195 well as in cultured primary preadipocytes and it has been shown that ipo8 expression was more stable in tissue samples of both adipose tissue depots as well as in preadipocytes isolated from subcutaneous adipose tissue, compared to fbxl10 expression that was more stable in preadipocytes from omental adipose tissue (hurtado del pozo et al., 2010). as the expression level of these two genes did not change during adipogenesis, the authors of this study concluded that both ipo8 and fbxl10 are good candidates for reference genes for gene expression analysis in human omental and subcutaneous adipose tissue samples as well as in differentiation of preadipocytes isolated from these tissues (hurtado del pozo et al., 2010). perez et al. (2017) analyzed the expression stability of a large number of reference genes by δct method in genorm, normfinder and bestkeeper software, using different adipocyte and muscle cell cultures in vitro, under different treatments and conditions, in animal muscle and adipose tissue samples of different metabolic states as well as in human muscle and adipose tissue samples. different results were obtained for different tissue types of different origin and under different conditions but general observation was that rrn18s was highly expressed in human samples and that gene for β-actin, although often used alone as a reference gene in rt-qpcr, is one of the most unstable genes in all muscle and adipose tissue samples, and is not recommended for this type of research (perez et al., 2017). the selection of an optimal reference gene for gene expression analysis is of great importance, because its stability in different samples and conditions will determine the final results obtained for the expression of goi and their reliability. zhang et al. (2016) demonstrated how the reference genes influence the expression of lep and ucp-1 gene in different adipose tissue samples of rats on a high-fat diet and control normal-fed rats. the expression of both examined goi was normalized by the expression of the most stable and the least stable reference genes. different gene expression patterns were obtained for lep and ucp-1 gene in different adipose tissue samples and different groups of animals when expression was normalized with the most and the least stable reference genes previously determined for each type of examined adipose tissue (zhang et al., 2016). based on the literature data, we can conclude that it is difficult to find an “ideal” reference gene that will have stable expression in different tissue types and samples of the same tissue type but under different experimental conditions and treatments. it is a common occurrence that one reference gene is suitable for one tissue type or treatment but not for the others, so it is difficult to compare the expression of goi between different samples. in our study, gapdh was shown to be the least variable and the most stable examined housekeeping gene in both lipoma and scwat group which is of great importance for the gene expression analysis and comparison between lipoma and scwat samples that could reveal the molecular mechanisms involved in the formation of lipoma. it can be noted that the expression of all examined housekeeping genes is slightly lower in lipoma samples compared to scwat, which obviously represents a characteristic of lipomas and reflects their basal metabolic status. conclusion based on the obtained results we can conclude that the expression of all three examined housekeeping genes (rrn18s, actb and gapdh) is slightly lower in lipoma samples compared to scwat samples and that expression of gapdh is the least variable and the most uniform in both examined groups of samples. we can consider gapdh as the most stable housekeeping gene in our study and recommend it as an optimal reference gene for gene expression analysis in human scwat and lipoma samples and its use for normalization of gene expression in realtime pcr method. acknowledgments. this study was supported by the ministry of education, science and technological development, of the republic of serbia (grant no. iii 41017). references almeida-oliveira, f., leandro, j.g.b., ausina, p., sola-penna, m., majerowicz, d. 2017: reference genes for quantitative pcr in the adipose tissue of mice with metabolic disease. biomedicine & pharmacotherapy, 88: 948-955. andersen, c.l., jensen, j.l., ørntoft, t.f. 2004: normalization of real-time quantitative reverse transcription-pcr data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. cancer research, 64(15): 5245-50. bustin, s.a. 2002: quantification of mrna using real-time reverse transcription pcr (rtpcr): trends and problems. journal of 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biologica nyssana ● 10 (2) december 2019: 189-197 stojanović et al. ● evaluation of the reference genes in human adipose tissue and lipoma samples 197 analytical biochemistry, 309(2): 293-300. warrington, j.a., nair, a., mahadevappa, m., tsyganskaya, m. 2000: comparison of human adult and fetal expression and identification of 535 housekeeping/maintenance genes. physiological genomics, 2: 143-7. zhang, w.x., fan, j., ma, j., rao, y.s., zhang, l., yan, y.e. 2016: selection of suitable reference genes for quantitative real-time pcr normalization in three types of rat adipose tissue. international journal of molecular sciences, 17(6): e968. biologica nyssana ● 10 (2) december 2019: 189-197 stojanović et al. ● evaluation of the reference genes in human adipose tissue and lipoma samples orchid flora of the muntele mic (caraş – severin country, romania) biologica nyssana 7 (2) ⚫ december 2016: 107-112 milanovici, s. ⚫ the orchid flora of the muntele mic… 107 original article received: 25 june 2018 revised: 28 sptember 2018 accepted: 18 november 2018* the orchid flora of the muntele mic (caraş – severin county, romania) sretco milanovici natural science section, banat national museum, huniade square no. 1, timișoara city, timiș county, romania * e-mail: milanovici.sretco@wildcarpathiangarden.com abstract: milanovici, s.: the orchid flora of the muntele mic (caraş – severin county, romania). biologica nyssana, 7 (2), december 2016: 107-112. muntele mic mountain is located in the southwestern part of romania and belongs to the southern carpathians. although relatively small, muntele mic contains most of typical mountain and high-mountain habitats. the field research regarding the orchid’s family in the muntele mic area, have started in the summer of 2009. owing to easy access (asphalt road that goes to the tourist center of muntele mic), although it is classified as part of the european natura 2000 network (rosci0126 munţii ţarcu), the area is influenced by negative anthropogenic factors. although considered to be a very anthropized area, the field research concluded that there are 10 species of orchids growing in this location, of which three: gymnadenia frivaldii hampe ex griseb., dactylorhiza fuchsii (druce) soó and dactylorhiza saccifera (brongn.) soó), were not mentioned in the literature data. key words: orchids, conservation, threats, muntele mic, romania apstrakt: milanovici, s.: flora orhideja planine muntele mic (caraş – severin county, romania). biologica nyssana, 7 (2), decembar 2016: 107-112. planina muntele mic nalazi se u jugozapadnom delu rumunije i pripada južnim karpatima. iako relativno male površine, muntele mic sadrži većinu karakterističnih planinskih i visokoplaninskih staništa. sistematska terenska istraživanja porodice orhideja planine muntele mic, započeta su u proleće 2009 godine. zahvaljujući lakom pristupu (asfaltni put koji ide do turističkog centra muntele mic), iako svrstan kao deo mreže natura 2000 rosci0126 munţii ţarcu, pod snažnim je udarom negativnih faktora antropogenog porekla. iako se smatra veoma antropizovanim, terenskim istraživanjem konstatovano je da ovde raste 10 vrsta orhideja, od čega se tri: gymnadenia frivaldii hampe ex griseb., dactylorhiza fuchsii (druce) soó i dactylorhiza saccifera (brongn.) soó), ne pominju u stručnoj literaturi. ključne reči: orhideje, problemi zaštite, muntele mic, rumunija introduction according to “flora româniei” (vol. vii, p a u c a et al., in s a v u l e s c u , 1972) there are 56 orchid species growing in romania. in the last decades, some taxa, formerly classified as subspecies, became species per se (a typical example is several species of the epipactis genus). in 2009 ciocârlan mentioned 7 (2) • december 2016: 107-112 12th sfses • 16-19 june 2016, kopaonik mt doi: 10.5281/zenodo.2528264 biologica nyssana 7 (2) ⚫ december 2016: 107-112 milanovici, s. ⚫ the orchid flora of the muntele mic… 108 58 orchid species recorded for the romanian flora (ciocârlan, 2009), while according to sârbu this number reaches 60 (s â r b u et al., 2013). three more species are to be added to the last figure mentioned: epipactis guegelii robatsch (r o b a t s c h , 1996), epipactis persica (soó) hausskn. ex nannf. (w u c h e r p f e n n i g , 2008) and epipactis greuteri h. baumann et künkele (a r d e l e a n , 2011). so far, the total amount of orchid species recorded in romania is 62. we do not regard this figure as final. in his reference work b o ş c a i u (1971) mentions 16 orchid species for the muntele mic area. the geographical unity of ţarcu mountains, whose part is the muntele mic, cover the northwestern region of meridional carpathian mountains. material and methods the field studies on the orchidaceae l. family in the muntele mic mountain area, regarding the species richness, distribution, size and dynamics of populations as well as the acknowledgment of the threats with (direct and indirect) impact upon the orchid species and populations have started in 2009 and ended in 2015 (fig. 1, 2b). the main bibliography related to the muntele mic mountain, started with the basic studies: b o ș c a i u (1971) and p a u c a et al., in s a v u l e s c u – “flora r. s. românia”, vol. xii, (1972). to understand the biology and ecology of terrestrial orchid species, literature data of d r e s s l e r (1981, 1983) and r a s m u s s e n (1995) were used. the determination of taxa up to the species level has been done based on the data provided by “flora of the s. r. romania” vol. xii (p a u c a et al., in s a v u l e s c u , 1972) and the nomenclature of used taxa has been harmonized to world checklist of monocotyledons database (2003). also, other relevant literary sources used (s o ó , 1973; m o o r e , 1980; d e l f o r g e , 2006; c i o c â r l a n , 2009) and related websites have been used. the area where the field studies were conducted covers only the zone of the alpine meadows, spruce and beech forests, along the mixed ones, mesophilic mountain meadows (especially those located in the upper part of the craiu valley) to the lowest altitude of 600 m. results and discussion the herein presented studies on the orchid species comprised the area of muntele mic, from an altitude of 600 m to the top of the mountain. although in his reference work boşcaiu (1971) mentioned 16 orchid species for the muntele mic zone, we have no fig. 1. geographical position of the muntele mic investigated area. biologica nyssana 7 (2) ⚫ december 2016: 107-112 milanovici, s. ⚫ the orchid flora of the muntele mic… 109 field confirmation (in the researched zone above 600 m) for seven species, namely: listera ovata (l.) r.br., platanthera chlorantha (custer) rchb., anacamptis palustris subsp. elegans (heuff.) r.m.bateman, pridgeon & m.w.chase, anacamptis coriophora (l.) r.m.bateman, pridgeon & m.w.chase, orchis mascula subsp. signifera (vest) soó and anacamptis morio (l.) r.m.bateman, pridgeon & m.w.chase, neotinea ustulata (l.) r.m.bateman, pridgeon & m.w.chase. instead, in the lower meadows area (borlova zone) and beech forests, below 600 m, one can notice the presence of most of the species mentioned above. the fieldwork recorded the presence of three orchid species, not mentioned in the relevant literature (esp boşcaiu, 1971) for the muntele mic zone, specifically leucorchis friwaldskiana (hampe) fuss, dactylorhiza fuchsii (druce) soó and dactylorhiza saccifera (brongn.) soó. in the following text, a description is given for each species confirmed in the fieldwork of the present study (muntele mic zone, above 600 m elevation): the genus dactyloriza neck. ex nevski. dactylorhiza cordigera (fr.) soó habitat: it sprouts individually or in groups of up to dozens of specimen on the eastern slope of the mountain, on wet meadows, on the banks of the torrents coming down from the mountain or in bogs, at an altitude between 1450 and 1600 m (fig. 2a); status of population: population is relatively large and stabile. following the multiannual countering, one can find at least 500 blooming specimens (mostly on the eastern slope of muntele mic); threats: over pasturing, leveling of the meadows for skiing tracks, off road motorcycling. dactylorhiza fuchsii (druce) soó habitat: sporadic and very rare; it had been found in a small number of specimens on the upper part of valea craiu, at the confluence of craiu and cuntu streams, at the edge of the forest, 610 m in altitude; status of population: population comprises around 20 specimens; threats: deforestation along removal of timber logs and their storage along the road; observation: newly found species for the muntele mic zone. dactylorhiza maculata (l.) soó habitat: sporadic and very rare; it had been found in a small number of specimens in the mesophilic meadow at the confluence of craiu and cuntu streams, 610 m in altitude (fig. 2c); status of population: population comprises around 20 specimens; threats: deforestation along removal of timber logs and their storage along the road. dactylorhiza viridis (l.) r.m.bateman, pridgeon & m.w.chase (syn. coeloglossum viride (l.) hartm.) habitat: very rare; a number of 5 specimens had been found, on shadowy rocks (northern exposure), on the eastern slope of the mountain; altitude: 1600 m; status of population: population comprises maximum 10 specimens; threats: intensive pasture. dactylorhiza saccifera (brongn.) soó habitat: found individually or in groups of up to ten specimens at the forest edge (near the paved road that climbs toward muntele mic resort), from valea sebeşului (600 m) along wet meadows from climbing road and the bottom of meadows on the eastern slope of the mountain, on an altitude between 600 and 1.400 m (fig. 2g); status of population: a fairly numerous population, comprising around 200 specimens; threats: intensive pasture, deforestation along removal of timber logs and their storage along the road, off road motorcycling; observation: newly found species for the muntele mic zone. the genus neottia guett. neottia nidus-avis (l.) rich. habitat: found individually or in groups of up to ten specimens in beech or mixed beech – spruce forests, preponderantly below 600 m altitude. as the altitude grows, the specimen became increasingly rare. some rare specimen have been detected on the eastern slope of muntele mic, about 800 m altitude , in the mixed beech – spruce forest; status of population: a very numerous population, comprising around 300 specimens (above 600 m); threats: deforestation along removal of timber logs, pasture on forest ground. the genus gymnadenia rich. gymnadenia conopsea (l.) r.br. habitat: very rare; a few specimens were found on the alpine meadows on the eastern slope, at an altitude between 1.300 and 1.500 m; status of population: given the reduced number of found specimen, one cannot correctly assess the size biologica nyssana 7 (2) ⚫ december 2016: 107-112 milanovici, s. ⚫ the orchid flora of the muntele mic… 110 fig. 2: a) dactylorhiza cordigera (fr.) soó (photo: 18.06.2009); b) muntele mic – general view (photo: 05.06.2015); c) dactylorhiza maculata (l.) soó (photo: 18.06.2009); d) gymnadenia frivaldii hampe ex griseb. (photo: 18.06.2009); e) pseudorchis albida (l.) á. löve & d. löve (photo: 18.06.2009); f) epipactis helleborine (l.) crantz (photo: 20.08.2015); g) dactylorhiza saccifera (brongn.) soó (photo: 18.06.2009). biologica nyssana 7 (2) ⚫ december 2016: 107-112 milanovici, s. ⚫ the orchid flora of the muntele mic… 111 of the population (taking also into account the dormancy, frequently met with the terrestrial orchid species); threats: over pasturing, leveling of meadows for skiing tracks, off road motorcycling. gymnadenia frivaldii hampe ex griseb. habitat: a relatively large population was identified in a single area on muntele mic (on a surface of about 50 sq m), on the eastern slope, in a mesophyllic alpine meadow, 1.460 m altitude (fig. 2d); status of population: the population is relatively numerous, concentrated on a very small surface and comprises some 200 bloomy specimens; threats: extremely threatened by over pasture; not far from the habitat of this species, a farm was built two years ago; the species is also threatened by off road motorcycling. observation: newly found species for the muntele mic zone. the genus pseudorchis ség. pseudorchis albida (l.) á. löve & d. löve habitat: a very small number of specimens were found on the eastern slope, 1400 m altitude (fig. 2e); status of population: very rare; a total of six bloomy specimens, in different locations and different years; threats: extremely threatened by over pasture, the more so as closely to its habitat a sheepfold was installed 2 years ago; the species is also threatened by off road motorcycling. the genus epipactis zinn epipactis helleborine (l.) crantz habitat: a relatively large population of some 200 specimens was found in a small spruce forest, near a torrent, on the border of the alpine meadows. it is interesting that in the lower areas, although the habitat is friendlier to this species, not a single specimen had been identified (fig. 2f); status of population: relative large population, comprising some 200 specimens (around 1200 m altitude); the entire population is concentrated on some 100 sq m; threats: pasturing on forest ground (closely to its habitat a sheepfold was installed 2 years ago) and technical improvements for a skiing track. conclusion although relatively isolated from tarcu mt range (to which it belongs to), muntele mic mt contains most of the typical high-mountain habitats. field studies on the orchid species and their populations started in 2009 and ended in 2015. since the investigated area comprised area above 600 m a.s.l. up to the highest peak of the muntele mic mt (1802 m a.s.l.), the number of recorded species was 10, which was lower than number recorded by boșcaiu (1971). however, the herein presented results revealed the existance of three new species for muntele mic mt, namely gymnadenia frivaldii, dactylorhiza fuchsii and dactylorhiza saccifera. the dactylorhiza cordigera species, according to multiannual monitoring, presents the largest population (at least 500 bloomy specimens, most of them on the east slope of muntele mic). the rarest species are: pseudorchis albida, gymnadenia conopsea (!?), dactylorhiza viridis, dactylorhiza fuchsii and dactylorhiza maculata, as the number of specimens for each of these species is up to 20. considering the easy access up the mountain (asphalted road to muntele mic resort), the existing habitats are subject to an extremely severe negative anthropogenic impact. the greatest threats to the species and their habitats come from leveling of the meadows for skiing tracks and adjacent infrastructure, unfortunately highly aggressive and irreversible (including the south western part). it is followed by the over pasturing, due to existence of at least three sheepfolds on muntele mic. another threat comes from the illegal off-road motorcycling races. references ardelean, corina, 2011: epipactis greuteri (orchidaceae) a new species for romanian flora. journal europäischer orchideen, 43 (3): 527534. boşcaiu, n. 1971: flora şi vegetaţia munţilor ţarcu, godeanu şi cernei. edit. academiei republicii socialiste românia, bucureşti. 494 p.. chase, m. w., cameron, k. m., barrett, r. l., freudenstein, j. v. 2003: dna data and orchidaceae systematics: a new phylogenic classification. in: dixon k.w., kell, s.p., barrett, r.l., cribb, p.j., eds. orchid conservation. kota kinabalu, sabah: natural history publications, 69–90. ciocârlan, v. 2009: flora ilustrată a româniei. pteridophyta et spermatophyte, ed. ceres, bucureşti. 1141 p.. cribb, p.j., kell, s.p., dixon, k.w., barrett, r.l. 2003: orchid conservation: a global perspective. in: dixon k. w., kell, s. p., barrett, r. l., cribb, p.j., eds. orchid conservation. kota kinabalu, sabah, natural history publications, 1–24. biologica nyssana 7 (2) ⚫ december 2016: 107-112 milanovici, s. ⚫ the orchid flora of the muntele mic… 112 delforge, p. 2006: orchids of europe, north africa and the middle east. a&c black, london. 640 p.. dressler, r. l. 1981: the orchids, natural history and classification. cambridge, ma: harvard university press. 332 p.. dressler, r. l. 1993: phylogeny and classification of the orchid family, dioscorides press. 314 p.. govaerts, r. 2003: world checklist of monocotyledons database in access: 1-71827. the board of trustees of the royal botanic gardens, kew. nyárády, e. i. 1958: flora şi vegetaţia munţilor retezat. edit. acad. r.p.r. bucurești. 195 p.. oltean, m., negrean, g., popescu, a., roman, n., dihoru, g., sanda, v., mihăilescu, s. 1994: lista roşie a plantelor superioare din românia, st. sin. doc. ec., 1/1994, bucureşti. pócs, t. 1957: contributions á la flore des carpathes orientaux et meridionaux. annales historiconaturales musei nationalis hungarici, 8: 205-217. robatsch, k. 1996: epipactis guegelii k. robatsch spec. nov., eine neue epipactis-art aus rumänien. journal europäischer orchideen, 28 (4):765-772. rogobete, gh., grozav, adia, beutură, d., nemeş i. 2007: soil acidification by ferrolysis in a pedological sequence of muntele mic, caraşseverin county. factori şi procese pedogenetice din zona temperată 6 s. nouă, 67-74. sanda, v., öllerer, kinga, burescu, p. 2008: fitocenozele din românia. sintaxonomie, structură, dinamică şi evoluţie. ed. ars docendi, universitatea din bucureşti. 570 p.. sârbu, i., ştefan, n., oprea, a. 2013: plante vasculare din românia. determinator ilustrat de teren. ed. victor b victor, bucureşti. 1320 p.. savulescu, t., (ed) 1972: flora republicii socialiste românia, vol. xii. editura academiei republicii socialiste românia, bucureşti. soó, r. 1973: a magyar flóra és vegetáció rendszertani – növényföldrajzi kézikonyve v. akadémiai kiadó, budapest. 723 p.. tutin, t.g., heywood, v.h., burges, n.a., valentine, d.h., eds. 1980: flora europaea, vol. v. cambridge university press, cambridge. urdea, p., törok-oance, m., ardelean, m., vuia, f., voiculescu, m. 2009: geomorphological aspects of the human impact in the alpine area of southern carpathians (romania). hrvatski geografski glasnik, 71 (1): 19-32. wucherpfennig, w. 2008: epipactis persica and andere orchideen des donaudeltas (rumänien). – ber. arbeitskrs. heimische orchideen, 25 (1):85110. http://www.theplantlist.org http://apps.kew.org/wcsp/home.do http://rbg-web2.rbge.org.uk/fe/fe.html  note: this article has been retracted due to technical and grammar errors and plagiarisms in introduction, which were not influenced by the originality and importance of research results in 23 aprile 2018. in agreement with the reviewers, the editor decided to approve resubmission of the manuscript and publication in the same issue of the journal. urosevic et al. 2021, biologica nyssana 12(2) 12 (2) december 2021: 151-157 doi: 10.5281/zenodo.5763846 new findings of kotschy’s gecko, mediodactylus kotschyi (steindachner, 1870) in serbia, with a particular focus on recently recorded populations in niš and sremska mitrovica original article aleksandar urošević institute for biological research “siniša stanković”, national institute of republic of serbia, university of belgrade, bulevar despota stefana 142, belgrade, serbia aurosevic@ibiss.bg.ac.rs (corresponding author) marko maričić institute of zoology, faculty of biology, university of belgrade, studentski trg 16, belgrade, serbia tijana vučić institute of zoology, faculty of biology, university of belgrade, studentski trg 16, belgrade, serbia vladimir žikić faculty of sciences and mathematics, university of niš, višegradska 33, 18000 niš, serbia saša s. stanković faculty of sciences and mathematics, university of niš, višegradska 33, 18000 niš, serbia mirko šević institute of zoology, faculty of biology, university of belgrade, studentski trg 16, belgrade, serbia marko anđelković institute for biological research “siniša stanković”, national institute of republic of serbia, university of belgrade, bulevar despota stefana 142, belgrade, serbia received: october 26, 2021 revised: november 24, 2021 accepted: december 03, 2021 abstract: kotschy’s gecko (mediodactylus kotschyi) is native to the eastern mediterranean basin but well known as a successful colonizer. the most probable way of its spreading is cargo and passenger traffic. in serbia, m. kotschyi is assumed native only in prizren. anthropogenic introductions were confirmed in belgrade, novi sad, pančevo and smederevo. this study aimed to summarize data of m. kotschyi findings with reference to recently confirmed population in niš and newly recorded population in sremska mitrovica. these two populations are well-established with multiple pieces of evidence of reproduction. pholidosis data were collected for several individuals to confirm their specific status. new and previously recorded introduced populations of kotchy’s gecko in serbia belong to m. kotschyi bibroni based on morphology. the introduction was most likely related to railways and further spread in urban zones is expected. it seems that introduced populations do not threaten native ecosystems as these populations are appeared to be highly localized in urban habitats. key words: anthropogenic introduction, colonizing species, distribution, m. kotschyi bibroni apstrakt: novi nalazi kočijevog gekona, mediodactylus kotschyi (steindachner, 1870) u srbiji, sa posebnim fokusom na nedavno zabeležene populacije u nišu i sremskoj mitrovici kočijev gekon (mediodactylus kotschyi) se autohtono javlja u basenu istočnog sredozemlja ali je dobro poznat i kao uspešan kolonizator. najverovatniji načini njegovog širenja su teretni i putnički saobraćaj. u srbiji, kočijev gekon se smatra autohtonim samo na području prizrena. antropogene introdukcije potvrđene su u beogradu, novom sadu, pančevu i smederevu. cilj ove studije je da sumira podatke o nalazima m. kotschyi sa posebnom pažnjom na nedavno potvrđeno prisustvo populacije u nišu i novozabeleženu populaciju u sremskoj mitrovici. ove dve populacije su dobro uspostavljene, sa višestrukim dokazima reprodukcije. podaci o folidozi prikupljeni su za veći broj jedinki da bi se potvrdio njihov status vrste. nove i prethodno zabeležene populacije kočijevog gekona u srbiji prema morfologiji pripadaju m. kotschyi bibroni podvrsti. introdukcije su najverovatnije vezane za železnički saobraćaj i očekivano je dalje širenje ove vrste u urbanim zonama. pretpostavlja se da introdukovane populacije ne ugrožavaju autohtone ekosisteme jer su po svoj prilici veoma lokalizovane na urbana staništa. ključne reči: antropogena introdukcija, kolonizujuća vrsta, distribucija, m. kotschyi bibroni introduction lizards, and particularly geckos, are well known as adept colonizers that significantly spread their range via human activity (flower, 1933; davis, 1974; conant & collins, 1998; jesus et al., 2002). it was even assumed that many populations of the hemidactylus (gekkonidae, squamata) and tarentola (phyllodactylidae, squamata) geckos in the mediterranean basin are of anthropogenic origin (harris et al., 2004; kasapidis et al., 2005). kotschy’s gecko (mediodactylus kotschyi (steindachner, 1870), gekkonidae, squamata) is a small, crepuscular, or nocturnal gecko species © 2021 urošević et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 151 native to the eastern mediterranean basin (böhme et al., 2009). recently, based on nuclear and mitochondrial dna analysis, it was discovered that it is a species complex consisting of five species – m. kotschyi (mainland of balkan peninsula, most of aegean islands, italy), m. orientalis (levant, cyprus, southern anatolia, south-eastern aegean islands), m. danilewskii (black sea region, southwestern anatolia and gavdos island, greece), m. bartoni (crete and surrounding islets) and m. oertzeni (southern dodecanese islands) (kotsakiozi et al., 2018). there are known cases of individuals of m. kotschyi species complex introduction outside the native range in hungary (farkas et al., 1999), serbia (ajtić, 2009; tomović et al., 2014; balej & jablonski, 2015; urošević, 2016; urošević et al., 2016, 2019), romania, bulgaria (koynova et al., 2020) and italy (mares & novarni, 2020). in serbia, it is assumed native only in prizren, metohija (ajtić & tomović, 2001), according to the distribution of mediterranean fauna in serbia and its neighbouring countries (haxhiu, 1998; ajtić & тomović, 2001; ajtić, 2004). other populations that were discovered outside of metohija are presumed to be introduced during the historical times or, more likely, recently (ajtić, 2009; tomović et al., 2014; balej & jablonski, 2015; urošević, 2016; urošević et al., 2016, 2019). the introductions have been confirmed in novi sad (ajtić, 2009; balej & jablonski, 2015;), belgrade [vračar (balej & jablonski, 2015), stari grad – kneginje zorke st., zemun (urošević et al., 2016)], smederevo [smederevo fortress (urošević, 2016; urošević et al., 2016)] and pančevo (urošević et al., 2019). finding from niš has been mentioned in the literature (ajtić, 2009; urošević et al., 2016), but it was based only on one reported sighting in a local coffee warehouse, and it has lacked an expert confirmation. with the recent taxonomic revision of the m. kotschyi group (kotsakiozi et al., 2018), the need to correctly assess the specific status of the introduced populations emerged. although the molecular analyses are not congruent with most of the described subspecies, the newly described species are very close to the main groups defined by beutler (1981). also, ajtić (2014) showed that discrimination between the main groups (for instance, the mainland kotschyi and danilewskii) is feasible via morphological characters. our main assumption was that the introductions occurred by land, from the continental parts of the balkan peninsula. since the geckos could have originated from the black sea coast region and therefore belong to the m. danilewskii group (d. jablonski, personal communication) we wanted to assess meristic characters through which we could identify the approximate origin of the colonized geckos, before we conduct the dna analyses. the main aim of this study is to provide a comprehensive list of the published and unpublished known findings of the kotschy’s gecko in serbia, and morphologically assess specific/subspecific status of the populations in niš (confirmed for the first time) and sremska mitrovica (newly discovered), since they are well-established, numerous and with multiple proofs of successful reproduction observed by the authors. materials and methods the presence of kotschy’s geckos at the studied sites was detected by visual observation or by listening to 152 biologica nyssana ● 12 (2) december 2021: 151-157 urošević et al. ● new findings of kotschy’s gecko, mediodactylus kotschyi (steindachner, 1870) in serbia, with a particular focus on recently recorded populations in niš and sremska mitrovica fig. 1. meristic data. a) rdt – number of rows of dorsal tubercles; b) supl. – number of supralabial tubercles; infl. – number of infralabial tubercles; otic tubercles – arrangement, c) pap – number of preanal pores; d) lct – number of lateral caudal tubercles 153 the characteristic “vocalization”, usually during the late afternoon or in the evenings, after sunset, during the summer and early autumn of 2020 and 2021. for confirmation, voucher photos of the animals were taken with a digital camera, and the precise locations were georeferenced (via application gps measurer – area, perimeter, distance, poi; v. 1.6.6 for android, edsa). when possible, the animals were collected by hand to examine pholidosis and take a tail tip as a sample for the subsequent dna analyses. the detailed pholidosis data was collected for several individuals in niš and sremska mitrovica. activities such as mating and feeding, as well as presence of juvenile animals or gravid females were also noted if observed. the pholidosis was compared to the diagnostic characters of the m. kotschyi former subspecies (biserkov et al., 2007; ajtić, 2009 and references there in) with an emphasis on the former subspecies native to the continental part of the balkans – m. k. bibroni (beutler & gruber, 1977), m. k. skopjensis biologica nyssana ● 12 (2) december 2021: 151-157 urošević et al. ● new findings of kotschy’s gecko, mediodactylus kotschyi (steindachner, 1870) in serbia, with a particular focus on recently recorded populations in niš and sremska mitrovica (karaman, 1965), m. k. rumelicus (müller, 1940) and m. k. danilewskii (strauch, 1887), since we assumed that the multiple introductions were most likely via the continental transportation routes. specific traits that we examined were: 1) layout of the tubercles around the ear opening, 2) shape of the dorsal tubercles, 3) number of rows of the dorsal tubercles (rdt), 4) number of supralabials (supl.) and infralabials (infl.), 5) number of the preanal pores (pap), and 6) number of lateral caudal tubercles (lct) (fig. 1). results the intensive research re-confirmed the presence of kotschy’s geckos at most of the known locations from the literature (fig. 2, supplementary table 1). the new findings were reported from the town of sremska mitrovica and a few localities in belgrade, as well as the city of niš, where the expert confirmation of the introduction was provided for the first time. in novi sad, belgrade, smederevo and niš, the findings were often localized to specific walls, buildings, or streets. in those places, the species seems to prefer the walls that are oriented towards the south and/or west, and that receive a lot of insolation during the day. in sremska mitrovica, the geckos seem to be frequent throughout the old town centre, probably due to the abundance of old, mutually connected buildings which provide suitable habitats. the pholidosis characters of the observed individuals in niš and sremska mitrovica are given in tab. 1. all animals had between 11 and 13 rows of dorsal tubercles (most had 12), between 8 and 10 supralabial scales (most having 9), 6 or 7 infralabial scales (most having 7) and 2 or 3 lateral caudal tubercles. all males in the sample had 4 preanal pores. all animals had large, keeled dorsal tubercles and almost all individuals had otic tubercles above and in front of ear opening (only one had above, in front and behind). in niš, mating was observed and one gravid female with two visible eggs was caught (fig. 3). in sremska mitrovica, three caught animals were subadults. we also observed feeding on arachnids in niš. many animals in both populations had regenerated tails, which is a strong indication that they are subject to predation (by wall lizards, feral cats, etc.) or aggressive intraspecific behaviour. discussion mediterranean geckos are known as successful colonizers and their spread to the north is constrained only by their association with anthropogenic habitats fig. 2. map of m. kotschyi distribution in serbia. empty blue dots represent literature reports, blue dots with black centre represent literature records reconfirmed in the field during the last survey, and red dots denote new distribution records. population in prizren, that is considered as native, is presented as a large blue dot 154 biologica nyssana ● 12 (2) december 2021: 151-157 urošević et al. ● new findings of kotschy’s gecko, mediodactylus kotschyi (steindachner, 1870) in serbia, with a particular focus on recently recorded populations in niš and sremska mitrovica and climate (meshaka et al., 2005). in serbia, the establishment of kotschy’s geckos in the urban areas is well known and documented (ajtić, 2009; tomović et al., 2014; balej & jablonski, 2015; urošević, 2016; urošević et al., 2016, 2019). by now, only the population in prizren is considered to be autochthonous (ajtić & tomović, 2001). due to its isolation from the rest of the native range of m. kotschyi, it is hypothesized that the prizren population could also be a result of the historical introduction (ajtić & tomović, 2001; ajtić, 2004, 2009). however, the isolation could be explained by the lack of distribution data from the north albania and metohija regions (haxhiu, 1998), since these areas generally lack systematic faunistic research (ajtić & tomović, 2001; ajtić, 2009; tomović et al., 2014). the rest of the populations in serbia are considered to be anthropogenic introductions (ajtić, 2009; urošević et al., 2016). since the discovered population in novi sad appeared stable, it was supposed that the introduction took place long ago (ajtić, 2009). the other populations that were discovered afterwards also seemed well established, albeit localized, and could be detected throughout consecutive years, with most being reconfirmed during the last field surveys. hypotheses of historical introduction could not explain how well-established populations of partially diurnal geckos were not noticed earlier. the deliberate introductions in hungary showed that these geckos can establish breeding colonies quickly (farkas et al., 1999). urošević et al. (2016) proposed a scenario of table 1. meristic data for the collected individuals. abbreviations: ind. – individual; rdt – number of rows of dorsal tubercles; supl. – number of supralabial tubercles; infl. – number of infralabial tubercles; pap – number of preanal pores; lct – number of lateral caudal tubercles; sdt – shape of dorsal tubercles; subad. – subadult. locality ind. sex rdt supl. infl. pap lct sdt otic tubercules note niš ni001 m 12 9 7 4 3 large, keeled above and in front niš ni002 f 12 10 6 / 2 large, keeled above and in front gravid niš ni003 m 12 9 7 4 / large, keeled above and in front niš ni004 f 11 9 7 / 3 large, keeled above and in front sremska mitrovica sm001 f 13 9 7 / 3 large, keeled above and in front subad. sremska mitrovica sm002 f 11 8 6 / 3 large, keeled above and in front sremska mitrovica sm003 m 13 9 7 4 3 large, keeled above, in front and behind sremska mitrovica sm004 f 12 10 7 / 3 large, keeled above and in front sremska mitrovica sm005 f 13 9 7 / 2 large, keeled above and in front sremska mitrovica sm006 f 11 8 7 / 2 large, keeled above and in front subad. sremska mitrovica sm007 f 13 9 7 / 2 large, keeled above and in front subad. sremska mitrovica sm008 f 12 8 6 / 2 large, keeled above and in front sremska mitrovica sm009 m 12 9 7 4 2 large, keeled above and in front 155 biologica nyssana ● 12 (2) december 2021: 151-157 urošević et al. ● new findings of kotschy’s gecko, mediodactylus kotschyi (steindachner, 1870) in serbia, with a particular focus on recently recorded populations in niš and sremska mitrovica fig. 3. evidence of m. kotschyi reproduction in the niš population. a) mating pair; b) gravid female with visible eggs relatively recent (15–20 years ago), simultaneous introduction into the large urban centres. ajtić (2009) assumed that geckos were introduced accidentally throughout the known localities in serbia, via cargo shipments from south or east. through the centuries, balkan peninsula harboured an important network of trading routes. for instance, via militaris (later known as “tsarigrad road”) had been traced in the 1st century ad. some of the places where gecko populations were confirmed (prizren, belgrade, sremska mitrovica, pančevo, smederevo, and niš) were the important trading hubs and caravan stations during the roman and ottoman empires (simonović, 2003; ajtić, 2009; urošević, 2016; urošević et al., 2016). today, these places are well connected by road and railroad networks. railroads are suggested as an especially probable means of introductions, since most of the known introductions in serbia are situated up to 1.5 km far from the railroad stations and junctions, and railroad beds can also provide good migratory corridors for reptiles (gherghel et al., 2009; urošević et al., 2019). this could also have facilitated the spread of geckos via cargo or passenger traffic (urošević, 2016). according to the meristic characters interpretable as diagnostic for the subspecies (biserkov et al., 2007; ajtić, 2009), geckos from the populations in niš and sremska mitrovica clearly belonged to the former m. kotschyi bibroni subspecies, characteristic for the central and southern balkans – males had four preanal pores, all examined individuals had two or three lateral caudal tubercles, most animals had otic tubercles only above and in front of the ear opening (which rules out m. kotschyi rumelicus) and dorsal tubercles were large and strongly keeled. animals observed in belgrade, novi sad and smederevo that were photographed also had strongly keeled dorsal tubercles and otic tubercles only above and in front of the ear opening. the number of rows of the dorsal tubercles ruled out the geographically closest subspecies, m. kotschyi skopjensis, since it usually has only 10 rows of tubercles, while the animals we observed had between 11 and 13 (most 12) rows, which corresponds with m. kotschyi bibroni group (ajtić, 2009). according to the molecular data, the group m. kotschyi bibroni, which inhabits the mainland balkans, belongs to the m. kotschyi species (kotsakiozi et al., 2018). although kotschy’s gecko seems to be spreading in serbia, there are no known negative effects which it can have on native ecosystems (urošević et al., 2016). all known introduced populations are localized in the urban zones, sometimes only on specific buildings. in the north of its native range, the species is also almost exclusively associated with human dwellings, in urban or suburban zones (ajtić & tomović, 2001; arnold & ovenden, 2002; ajtić, 2009). we expect further spread of kotschy’s gecko predominantly into urban areas, towns and cities that, due to the “heat island effect“, provide 156 biologica nyssana ● 12 (2) december 2021: 151-157 urošević et al. ● new findings of kotschy’s gecko, mediodactylus kotschyi (steindachner, 1870) in serbia, with a particular focus on recently recorded populations in niš and sremska mitrovica adequate thermal conditions for the predominantly mediterranean species. acknowledgements. we would like to thank ljiljana tomović and ilias strachinis for their constructive reviews that greatly improved our manuscript, ana miletić, jelena pavlović and sara krunić for the help in the field, and dunja čolak and ksenia prądzyńska for sharing their field data. the study was partially financed by the serbian ministry of the education, science and technological development, grant nos. 451-03-9/2021-14/ 200007 and 451-03-9/2021-14/ 200178. animals and dna samples were collected with permissions from the serbian ministry of environmental protection, permit nos. 353-012876/2019-04 and 353-01-2716/2020-04. references ajtić, r., tomović, l. 2001: first record of kotschy’s gecko cyrtodactylus kotschyi (steindachner, 1870) (gekkonidae, lacertilia) in fr yugoslavia. archives of biological sciences, 53: 23–24. ajtić, r. 2004: morphological and ecological characteristics of population of kotchy’s gecko (cyrtodactylus kotchyi, steindachner, 1870) (gekkonidae, lacertilia) from part of the balkan peninsula. protection of nature, 56: 69–78. ajtić, r. 2009: morfološke, biogeografske i ekološke odlike kočijevog gekona (cyrtodactylus kotschyi steindachner, 1870) sa kopnenog dela areala. master thesis. odsek za biologiju i ekologiju, prirodno-matematički fakultet, univerzitet u nišu. niš. ajtić, r. 2014: morphological, biogeographical and ecological characteristics of kotschy’s gecko (cyrtodactylus kotschyi steindachner, 1870 gekkonidae) from the mainland portion of its distribution range. fauna balkana, 3: 1–70. arnold, n., ovenden, d. 2002: a field guide to the reptiles and amphibians of britain and europe. harper collins publishers, london. balej, p., jablonski, d. 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antimicrobial activity of satureja l. essential oils against phytopathogenic bacteria erwinia amylovora tatjana mihajilov-krstev1, dragan radnović2, dušanka kitić3 1 university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia 2 faculty of sciences and mathematics, university of novi sad, serbia 3 faculty of medicine, university of niš, serbia * e-mail: nis_mikrobi@yhoo.com abstract: mihajilov-krstev, t., radnović, d., kitić, d.: antimicrobial activity of satureja l. essential oils against phytopathogenic bacteria erwinia amylovora. biologica nyssana, 1 (1-2), december 2010: 95-98. in this paper, antimicrobial activity of s. kitaibelii wierzb. ex heuff., s. montana ssp. montana l., s. adamovicii šilić and s. fukarekii šilić has been investigated. during the testing, two methods were used: disc-diffusion and broth micro-dilution. the results showed high sensitivity of this bacteria to all four essential oils. the essential oil of s. montana ssp. montana exhibited the largest inhibition zone (25 mm), while s. adamovicii showed the highest inhibitory and bactericidal activity (mic=mbc=0.09 μlml-1). all investigated oils showed the same values for mic and mbc, which means that the oils posses bactericidal effect at very low concentrations and they could be used as non-harmful source of bactericides. key words: genus satureja l., essential oils, antimicrobial activity, erwinia amylovora introduction ! erwinia is a genus of enterobacteriaceae bacteria containing mostly plant pathogenic species which was named for the first phytobacteriologist, erwin smith. a well-known member of this genus is the species e. amylovara, which causes fireblight on apple, pear, and other rosaceous crops. this species produce enzymes that hydrolyze pectin between individual plant cells. this causes the cells to separate, a disease plant pathologists term plant rot. fireblight is a systemic disease. the term "fireblight" describes the appearance of the disease, which can make affected areas appear blackened, shrunken and cracked, as though scorched by fire. sprays of the antibiotics, streptomycin or terramycin can prevent new infections (f a l k e n s t e i n et al., 1998). during the last several decades, natural products with antimicrobial effect are investigated in order to eliminate the use of synthetic antibiotics which cause the resistance of microorganisms and can exhibit side effects to human health. aromatic plants are known for a very long time and they are used in phytotherapy and food preservation (b u r t , 2004). among the aromatic plant species, genus satureja l. occupies a special position. essential oils of satureja l. species showed very significant antimicrobial activity against various species of bacteria and fungi (c i a n i et al., 2000; a z a z et al., 2002; s a h i n et al., 2003; c h o r i a n o p o u l o s et al., 2004; b e ž i ć et al., 2005; s k o č i b u š i ć & b e ž i ć , 2004, 2006; a d i g u z e l et al., 2007; r a z z a g h i -a b y a n e h et al., 2008). in this paper, disc-diffusion and broth microdilution method were used to investigate antibacterial effect of essential oils of four species of satureja l. genus against phytopathogenic bacteria e. amylovora. according to our knowledge, these are the first investigations of essential oil’s effect against this bacterium. considering these 10th sfses • 17-20 june 2010, vlasina lake1 (1-2) • december 2010: 95-98 biologica nyssana 1 (1-2) december 2010: 95-98 mihajilov-krstev et al. antimicrobial activity of satureja l. essential oils… 96 facts, the results of the present study can find application in the pomology as harmless and natural bactericidal agents. material and methods ! plant material the aerial parts of wild growing plant materials of eight satureja species were collected during the beginning of the flowering stage in serbia (s. kitaibelii wierzb. ex heuff.), montenegro (s. monatana l.) and f.y.r.o.m. (s. fukarekii šilić and s. adamovicii šilić). voucher specimens were deposited in the herbarium at the faculty of natural sceince and mathematics of the university of novi sad. extraction of the essential oils air-drying of the plant was performed in a shady place at room temperature for 10 days. dried aerial parts (100 g) of plants, were cut and subjected to the hydro-distillation for 3 h, using clevengertype apparatus. the resulting essential oils were dried over anhydrous sodium sulfate and stored at 4°c. antimicrobial screening the antimicrobial activities of the tested essential oils were evaluated using disc-diffusion and broth micro-well dilution method. all tests were performed in triplicate with two growth controls: ethanol (negative control) and streptomycin (positive control). microbial strain. laboratory control strain erwinia amylovora ncppb 595. disc-diffusion assay. antimicrobial tests were carried out by disc-diffusion method using 100 μl of suspension (containing 2.0 x 108 cfu/ml of bacteria) spread on mueller-hinton agar (mha, torlak) in sterilized petri dishes (90 mm in diameter). the discs (6 mm in diameter, himedia laboratories pvt. limited) were impregnated with 15 μl of oil dilution (2 %, 5 % and 10 %) and placed on the inoculated agar. the inoculated plates were kept at 4 0c for 2 h and incubated at 37 0c for 24h. antimicrobial activity was evaluated by measuring the zone of inhibition against the test bacterial strain. micro-well dilution assay. the inocula of the bacterial strain was prepared from overnight broth culture and suspension was adjusted to 0.5 mcfarland standard turbidity (corresponding to 107108 cfu ml-1) (consensus standard by the nccls). ethanol was used to dissolve the essential oils and then diluted to the highest concentration (500 μl ml-1). a serial doubling dilutions of the oils were prepared in a 96/well microtiter plate over the range of 50.000.02μl ml-1 in inoculated nutrient broth (the final concentration in each well adjusted to 2.0 x 106 cfu ml-1). the plates were incubated for 24 h at 370c. the microbial growth was determined by absorbance at 620nm using the universal microplate reader (thermolabsystems, multiskan ex, software for multiscan ver.2.6.). the highest dilution without growth is the minimum inhibitory concentration – mic. to determine mbc, broth was taken from each well and inoculated in mueller hinton agar (mha) for 24 h at 37 0c. the mbc is defined as the lowest concentration of the essential oil at which inoculated microorganism was 99.9 % killed. statistical analysis of data. analysis of variance (anova) was used to determine the significance (p≤0.05) of the data obtained in all experiments. results and discussion the dominant components of satureja l. essential oils were monoterpenes carvacrol and thymol, p-cymene, limonene and also monoterpenoid alcohols borneol and linallole. considering sesquiterpenes, the most ambudant were β-caryophyllene, caryophyllene–oxide and spathulenol (p a v l o v i ć et al., 1987; s l a v k o v s k a et al., 2001; a z a z et al., 2002; s a h i n et al., 2003; c h o r i a n o p o u l o s et al., 2004; b e ž i ć et al., 2005; s k o č i b u š i ć & b e ž i ć , 2004; a d i g u z e l et al., 2007; r a z z a g h i -a b y a n e h et al., 2008).according to the results of cited authors rich in carvacrol was essential oil of the species satureja montana ssp. montana l. (16.1 – 52.4 %) (p a v l o v i ć et al., 1987; s l a v k o v s k a et al., 2001; s k o č i b u š i ć et al., 2004). monoterpenoidal alcohol borneol was present in high content in the satureja fukarekii šilić (55.0 %) essential oil (p a v l o v i ć et al., 1987). significantly smaller content of borneol was present in the satureja kitaibelii wierzb. ex heuff. (9.8 %) oil, where dominant compounds were the precursors of carvacrol p–cymene (20.9 %) and limonene (16.0 %) (s l a v k o v s k a et al., 2001). in the oil of the species satureja adamovicii, p-cymol was present (40.0 %) together with 1,8-cineole and limonene (35.0 %) (p a v l o v i ć et al., 1987). the results of disc-diffusion testing showed that all tested oils (0.75 μl/disc) ehibited very significant antimicrobial activity against e. amylovora. the oils showed higher activity than the biologica nyssana 1 (1-2) december 2010: 95-98 mihajilov-krstev et al. antimicrobial activity of satureja l. essential oils… 97 tested reference antibiotic streptomycin (38 mm, 30 μl of the active substance/disc). especially high activity showed the oil isolated from s. montana ssp. montana with inhibition zone of 25 mm (fig. 1). fig.1. mean diameter of inhibition zone for satureja essential oils and streptomycin (30 μg/disc) against erwinia amylovora, obtained by disc-diffusion method the results of broth micro-well dilution method confirmed high essential oil activity of four satureja l. species (fig. 2). their mic/mbc values were in the range from 0.09 – 0.18 μl ml-1, which is very good considering the values of the reference antibiotic, streptomycin (mic/mbc = 8.0/16.0 μl ml-1). fig. 2. the mic/mbc values (μl ml-1) of the four satureja essential oils and streptomycin against erwinia amylovora by the broth microdilution method phenolic compound carvacrol, monoterpene limonene and alcoholes linalool and borneol are mostly responsible for the obtained high antimicrobial activity. the most possible mechanism is sinergistic effect of sesquiterpenes like β-caryophyllene and caryophyllene–oxide with other active compounds. beside this, it is wellknown that some compounds do not possess antimicrobial activity, but can enhance the activity of some other antimicrobial compounds by incorporating into the membrane bilayer and enabling the passage of the active compounds. one of these components is p-cymene, which is constituent of allmost all investigated species of the genus satureja l. (lamber et al., 2001; arfa ben et al., 2006). conclusion essential oils of four species from the genus satureja l. showed high antibacterial activity against e. amylowora. that can be explained by high content of monoterpenes and sesquiterpenes, which are well known and many times proved as antimicrobials. these results demonstrated that essential oils of the investigated species could be used as a natural potential antimicrobial agents in prevention and treatment of "fireblight". references adiguzel, a., ozer, h., kilic, h. and cetin, b. 2007: screening of antimicrobial activity of essential oil and methanol extract of satureja hortensis on foodborne bacteria and fungi. chez j food sci., 25: 81-89. arfa ben, a., combes, s., preziosi-belloy, l., gontard, n. and chalier, p. 2006: antimicrobial activity of carvacrol related to its chemical structure. letters in apllied microbiology, 43: 149-154. azaz, d., demirci, f., satil, f., kurcouglu, m. and baser, k.h. 2002: antibacterial activity of some satureja. z naturforsch, 57: 817-821. baser, k.h.c., ozek, t., kirimer, n. and tumen, g. 2004: a comparative study of the essential oils of wild and cultivated satureja hortensis l. jeor, 16: 584-589. bežić, n., skočibušić, m. and dunkić, v. 2005: phytochemical composition and antimicrobial activity of satureja montana l. and satureja cuneifolia ten. essential oils. acta bot croat., 64: 313-322. burt, s. 2004: essential oils: their antibacterial properties and potential applications in food – a 0 2 4 6 8 10 12 14 16 satureja montana satureja kitaibelii satureja fukarekii satureja adamovicii streptomycin μlm 1 0 5 10 15 20 25 30 35 40 satureja montana satureja kitaibelii satureja fukarekii satureja adamovicii streptomycin zone of inhibition in mm biologica nyssana 1 (1-2) december 2010: 95-98 mihajilov-krstev et al. antimicrobial activity of satureja l. essential oils… 98 review. international journal of food microbiology, 94: 223-253. chorianopoulos, n., kalpoutzakis, e., aligiannis, n., mitaku, s., nychas, g.j. and haroutounian, s.a. 2004: essential oils of satureja, origanum, and thymus species: chemical composition and antibacterial activities against foodborne pathogens. j agric food chem., 52: 8261-8267. ciani, m., menghini, l., mariani, f., pagiotii, r., menghini, a. and fatichenti, f. 2000: antimicrobial properties of essential oil of satureja montana l., on pathogenic and spoilage yeasts. biotechnology letters, 22: 1007-1010. dorman, h.j.d. and deans, s.g. 2000: antimicrobial agents from plants: antibacterial activity of plant volatile oils. journal of applied microbiology, 88: 308-316. falkenstein, h., bellemann, p., walter, s. zeller, w., geider, k. 1988: identification of erwinia amylovora, the fireblight pathogen, by colony hybridization with dna from plasmid pea29. applied and environmental microbiology, 54(11): 2798-2802. lamber, r.j.w., skandamis, p.j., coote, g. and nychas, j.e. 2001: a stady of the minimum inhibitory concentration and mode of action of oregano essential oil, thymol and carvacrol. journal of applied microbiology, 91: 453-462. nccls – national committee for clinical laboratory standards. 2000: performance standards for anti-microbial susceptibility testing: eleventh informational supplement. document m100-s11. national committee for clinical laboratory standard, wayne, pa, usa. pavlović, s., živanović, p., jančić, r., todorović, b., ševarda, a.l., kuznjecova, g.a. 1987: the qualitative composition of the essential oil in species of genus satureja l. (lamiaceae) distributed in yugoslavia. biosistematika, 13: 19-24. razzaghi-abyaneh, m., shams-ghahfarokhi, m., yoshinari, t., rezaee, m.b., jaimand, k., nagasawa, h., sakuda, s. 2008: inhibitory effects of satureja hortensis l. essential oil on growth and aflatoxin production by aspergillus parasiticus. international journal of food microbiology, 123: 228–233. sahin, f., karaman, i., gulluce, m., oguta, h., sengul, m., adiguzel, a., ozturk, s. and kotan, r. 2003: evaluation of antimicrobial activities of satureja hortensis l. j ethnopharmacol, 87: 6165. skočibušić, m. and bežić, n. 2004: chemical composition and antimicrobial variability of satureja montana l. essential oils produced during ontogenesis. jeor, 16: 387-391. skočibušić, m. and bežić, n. 2006: phytochemical composition and antimicrobial activities of the essential oils from satureja subspicata vis. growing in croatia. food chemistry, 96: 20-28. slavkovska, v., jančić, r., bojović, s., milosavljević, s. and djoković, d. 2001: variability of essential oil of satureja montana l. and satureja kitaibelii wierzb. ex heuff. from the central part of balkan peninsula. phytochemistry, 57: 71-76. anticancer compounds from medicinal plants biologica nyssana 5 (2)  december 2014: 103-112 papović, o. et al.  phytogeographical analysis and endemism of the flora … 103 original article received: 21 september 2014 revised: 06 december 2014 accepted: 20 december 2014 phytogeographical characteristics and endemism of the flora of rogozna mt. (sw serbia) olivera papović1, milica miljković2, novica ranđelović2, vladimir ranđelović2 1university of pristine, faculty of sciences and mathematics, department of biology, ive lole ribara 29, 38220 kosovska mitrovica, serbia 2university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia * corresponding autor: olja.bio@open.telekom.rs abstract: papović, o., miljković, m., ranđelović, n., ranđelović, v.: phytogeographical characteristics and endemism of the flora of rogozna mt. (sw serbia). biologica nyssana, 5 (2), december 2014: 103-112. based on presence of area types and area groups, the phytogeographical analysis show that rogozna mt. is area with eurasian-submediterranean-centraleuropean-pontic characteristics. eurasian area type is dominant in the flora of rogozna mt. detailed analysis of the eurasian area type showed a numerous presence of the species with central european-mediterranean (73) and central european-mediterranean-pontic (73) types of distribution. the influence of the mediterranean region is especially pronounced. there are 117 species with mediterranean-submediterranean type of distribution, many of which are endemics or subendemics. typical pontic elements of flora are present in very low percent (1.27%), but taxa with mediterranean-pontic (14.7%) and central-european-mediterranean-pontic (9.38%) are numerous. the presence of an imposing number of endemic taxa (51) has a great significance from the aspect of biodiversity and conservation of the area. key words: area-types and groups, endemites, flora apstrakt: papović, o., miljković, m., ranđelović, n., ranđelović, v.: fitogeografske karakteristike i endemizam flore planine rogozne (jz srbija). biologica nyssana, 5 (2), decembar 2014: 103-112. na osnovu zastupljenosti areal-tipova i areal-grupa, fitogeografskom analizom je utvrđeno da planina rogozna predstavlja područje sa evroazijsko-submediteransko-srednjeevropsko-pontskim karakteristikama. evroazijski areal-tip je dominantan u flori rogozne. detaljna analiza evroazijskog areal-tipa pokazuje brojno učešće taksona srednjeevropsko-mediteranskog (73) i srednjeevropsko-mediteransko-pontskog (73 taksona) tipa distribucije. uticaj mediteranskog regiona je naročito izražen. prisutno je 117 taksona mediteranskosubmediteranskog tipa distribucije, od kojih su mnoge endemiti i subendemiti. tipični pontski elementi flore je prisutan sa malim procentom (1.27%), ali su taksoni sa mediteransko-pontskim (14.7%) i srednjeevropskomediteransko-pontskim (9.38%) brojni. prisustvo impozantnog broja endemičnih taksona (51) ima veliki značaj sa aspekta biodiverziteta i očuvanje područja. ključne reči: areal-tipovi i grupe, endemiti, flora 5 (2) • december 2014: 103-112 biologica nyssana 5 (2)  december 2014: 103-112 papović, o. et al.  phytogeographical analysis and endemism of the flora … 104 introduction rogozna is the mountain located in southwestern serbia (p a p o v i ć et al., 2014). according to phytogeographical division of the southeastern europe (h o r v a t et al., 1974) the rogozna mt. is situated near the border of the illyrian, west mesian and scardo-pindhian provinces of the balkan floristic subregions (fig. 1). such position of the mountain reflects to a large extent on the genesis of its flora. in addition, geological composition of the terrain has a significant influence on the phytogeographical and ecological characteristics of the flora. it is represented by upper cretaceous flysch of kosovska mitrovica which seals the immediate contact between the ophiolite mélange of the vardar zone and drina–ivanjica metamorphic unit in the east and west, respectively (b o r o j e v i ć š o š t a r i ć et al., 2012). in the central part of mountain, serpentine rocks (ophiolite) are brought to the surface. east and west of them widespread dacite-andesite and quartz latite (m i l o v a n o v i ć & k a r a m a t a , 1960; u r o š e v i ć et al., 1966, 1970). on the surface of serpentine rocks, very fragile and fine-grained, poor soil is developed, whose water and mineral regime are unfavourable (p r o d a n o v i ć et al., 2010). areas of the soil-rock systems, which may be grouped together as serpentines occur in many parts of the world and, wherever they occur, are known by their remarkable plant life (w h i t t a k e r , 1954). the plants which develop on serpentines are called serpentinophytes (p a v l o v i ć , 1962; v a s i ć & d i k l i ć , 2001). according to preference for the serpentine supstrate, serpentinophyte can be obligate (plants which are mainly growing on serpentine) and facultative (plants which often grow on serpentine, but also inhabit other kinds of supstrate) (s t e v a n o v i ć et al., 2003). the aims of this study are to determine phytogeographical characteristics of the flora of rogozna mt. based on the presence of area types and groups of plant taxa. special attention was paid to the obligate serpentinophytes and endemics. material and methods the analysis is based on the floristic list shown in the article “analysis of the flora of rogozna mountain in southwestern serbia“ (p a p o v i ć et al., 2014) supplemented (see tab. 1.) by data from the literature (r e x h e p i , 1979; k r i v o š e j et al., 2013; p r o d a n o v i ć et al., 2013) and herbarium collection (hmn). fig 1. position of rogozna mt. on phytogeographical map of the southeastern europe based on h o r v a t et al. (1974) biologica nyssana 5 (2)  december 2014: 103-112 papović, o. et al.  phytogeographical analysis and endemism of the flora … 105 nomenclature was correlated with medchecklist (g r e u t e r et al., 1984-1989), flora europaea (t u t i n et al., 1964-1980), and euro+med plantbase (euro+med, 2006). area types, groups and floristic elements of the plant species were defined by m e u s e l et al. (1965, 1978), m e u s e l and j ä g e r (1992) and s t e v a n o v i ć (1992). results revision of the flora of rogozna mt. list of the species shown in previous article (p a p o v i ć et al., 2014) is supplemented with 17 species (tab. 1). after herbarium revision, it was established that misidentification occurred in the case of 6 species, where data related to other taxa (tab. 2). some of the species do not grow on the rogozna mt., such as andromeda polyfolia l., potentilla leucopolitana p.j. mueller, angelica verticilaris l., hacquetia epipactis (scop.) dc., tordilium apulum l., cerastium transsilvanicum schur., dianthus ciliatus guss., dianthus serotinus waldst. & kit. and melampyrum fimbriatum vandas. phytogeographical analysis for the phytogeographical analysis, all plant taxa were classified into 12 area types and 20 area groups (tab. 3). as expected, the largest number of the species has eurasian type of distribution (360 taxa), but also numerous taxa that have a mediterranean (118) and meridional-submeridional (121) type of distribution. the other area types and groups contribute with small percentages to the chorological composition of the rogozna mt. flora. table 1. the list of supplemented plant species according to the literature and herbarium data plant species data asparagus tenuifolius lam., asparagaceae rexhepi, 1979 bupleurum tenuissimum l., apiaceae prodanović et al., 2013 laburnum alpinum (mill.) bercht. et j. presl., fabaceae prodanović et al., 2013 ophioglossum vulgatum l., ophioglossaceae krivošej et al., 2013 hedera helix l., araliaceae hmn sedum album l., crassulaceae hmn carex caryophyllea latour., cyperaceae hmn cheilanthes persica (bory) kuhn, adiantaceae hmn euphorbia amygdaloides l., euphorbiaceae hmn robinia pseudoacacia l., fabaceae hmn quercus frainetto ten., fagaceae hmn erodium cicutarium (l.) l'herit, geraniaceae hmn scilla bifolia l., liliaceae hmn corydalis solida (l.) clairv., plantaginaceae hmn plantago lanceolata l., plantaginaceae hmn p. major l., plantaginaceae hmn anthoxanthum odoratum l., poaceae hmn table 2. revision of the list of flora of rogozna mt. from p a p o v i ć et al. (2014) incorrectly listed taxa correct name alnus viridis (chaix) lam. et d.c. a. glutinosa (l.) gaertn. cerastium grandiflorum waldst. & kit. c. decalvans schlosser & vuk. cerastium sylvaticum waldst. & kit. c. fontanum baumg. dianthus pinifolius sibth. & sm. ssp. pinifolius d. pinifolius sibth. & sm. ssp. serbicus wettst. silene bupleuroides l. ssp. bupleuroides s. bupleuroides l. ssp. staticifolia (sibth. & sm.) chowd. sempervivum heuffelii schott jovibarba heuffelii (schott) á. löve & d. löve tulipa scardica bornm. t. serbica tatić et krivošej biologica nyssana 5 (2)  december 2014: 103-112 papović, o. et al.  phytogeographical analysis and endemism of the flora … 106 table 3. phytogeographical spectrum of the rogozna mt. (n – number of taxa, % percent of the total flora) area type/area group/area subgroup n % cosmopolitan (cosm) 23 2.92 holarctic (hol) 38 4.82 palaeoholarctic-palaeotropic (ph-pt) 6 0.76 boreal (bor) 7 0.89 arctic-alpine (a-a) 2 0.25 antropohoric (ant) 7 0.89 eurasian (ea) 358 45.43 eurasian (ea) 213 27.03 eurasian (ea) 117 14.83 west eurasian (eaw) 96 12.17 central european-mediterranean-pontic (ce-m-p) 73 9.26 c. european-mediterranean-pontic-oriental-turanian (ce-med-pont-or-tur) 3 0.38 c. european-mediterranean-pontic-oriental (ce-med-pont-or) 22 2.79 c. european-mediterranean-pontic (ce-med-pont) 45 5.70 c. european-pontic (ce-pont) 3 0.38 central european-mediterranean (ce-med) 73 9.26 central european-mediterranean (ce-med) 66 8.37 central european-submediterranean (ce-smed) 7 0.89 eurasian mountain (eam) 50 6.34 eurasian mountain (eam) 6 0.76 eurasian mountain (eam) 2 0.25 west eurasian mountain (eamw) 4 0.51 south european mountain (sem) 18 2.28 s. european mountain (sem) 12 1.39 apenninic-balkanic-anatolian (ap-balk-an) 1 0.13 balkanic (balk) 5 0.63 central s. european mountain (csem) 26 3.30 csem-caucasian (csem-cau) 1 0.13 central s. european mountain (csem) 18 2.28 (illyrian)-balkanic ((illyr)-balk) 5 0.64 balkanic-anatolian (balk-an) 1 0.13 alpine-balkanic-carpathian (alp-balk-carp) 1 0.13 central european (ce) 51 6.46 centraleuropean (ce) 41 5.20 alpine-apenninic-balkanic (alp-apen-balk) 1 0.13 alpine-balkanic-carpathian (alp-balk-carp) 1 0.13 balkanic-carpathian (balk-carp) 3 0.38 pannonian-balkanic (pan-balk) 1 0.13 balkanic (balk) 4 0.51 pontic-south siberian (pont-ss) 10 1.27 balkanic-pontic (balk-pont) 1 0.13 pontic (pont) 9 1.14 meridional-submeridional (msm) 118 14.96 mediterranean-pontic-oriental (med-pont-or) 4 0.51 mediterranean-submediterranean-oriental-turanian (med-smed-or-turan) 1 0.13 mediterranean-submediterranean-oriental (med-smed-or) 3 0.38 medeterranean-pontic (med-pont) 114 14.45 atlantic-medeterranean-pontic (atl-med-pont) 2 0.25 medeterranean-pontic (med-pont) 35 4.44 submediterranean-pontic (smed-pont) 77 9.76 mediterranean-submediterranean (med-smed) 118 14.95 mediterranean-submediterranean (med-smed) 51 6.46 biologica nyssana 5 (2)  december 2014: 103-112 papović, o. et al.  phytogeographical analysis and endemism of the flora … 107 area type/area group/area subgroup n % mediterranean-submediterranean (med-smed) 28 3.55 mediterranean-e. submediterranean (med-esmed) 3 0.38 submediterranean (smed) 20 2.53 e. mediterranean-e. submediterranean (emed-esmed) 67 8.19 e. mediterranean 12 1.52 alpine-(illyrian)-balkanic (alp-(illyr)-balk) 5 0.64 alpine-balcanic-carpathian (alp-balk-carp) 1 0.13 apenninic-(dacian-illyrian)-balkanic (apen-(dac-illyr)-balk) 4 0.51 apenninic-balkanic-anatolian (apen-balk-anatol) 1 0.13 (illyrian)-balkanic-anatolian-(crimean) ((illyr)-balk-anat-(crim)) 5 0.64 balkanic-dacian-(danubian) (balk-dac-(danub) 2 0.25 illyrian-balkanic-anatolian-caucasian (illyr-balk-anat-cau) 1 0.13 balkanic-caucasian (balk-cau) 2 0.25 illyrian-balkanic-crimean (illyr-balk-crim) 1 0.13 (illyrian)-balkanic-carpathian (balk-carp) 4 0.51 (illyrian)-balkanic ((illyr)-balk) 29 3.66 total 789 100.00 table 4. list of the obligate serpentinophytes distributed on rogozna mt. plant taxa area type floristic element stachys recta l. subsp. baldaccii (k. malý) haye sem dinaric-scardo-pindhian bromus pannonicus kummer & sendtner msm balkan-pontic-pannonian asplenium cuneifolium viv. ce central european silene paradoxa l. med-smed mediterranean-submediterranean paragymnopteris marantae (l.) k. h. shing med-smed mediterranean-submediterranean hypochaeris illyrica k. malý med-smed e illyrian-moesian halacsya sendtneri (boiss.) dörfler med-smed moesian-scardo-pindhian scrophularia tristis (k. malý) šilić med-smed illyrian-moesian alyssum bertolonii desv. med-smed moesian-scardo-pindhian alyssum markgrafii o. e. schulz ex markgraf med-smed moesian-scardo-pindhian fumana bonapartei maire & petitmengin med-smed illyrian-scardo-pindhian sedum serpentini janchen med-smed moesian-scardo-pindhian scabiosa fumarioides vis. & pančić med-smed moesian-scardo-pindhian thymus lykae degen med-smed w moesian-scardo-pindhian tulipa serbica tatić & krivošej med-smed w moesian orobanche nowackiana markgraf med-smed dinaric-scardo-pindhian helleborus serbicus adamović med-smed dinaric-w moesian galium rubrum l. med-smed c mediterranean-submediterranean haplophyllum boissieranum vis. & pančić med-smed moesian-scardo-pindhian obligate serpentinophytes and its phytogeographical characteristics on the rogozna mt. 19 plant taxa from the group of obligate serpentinophytes based on s t e v a n o v i ć et al. (2003) were recorded (tab. 4). one of these tulipa serbica, is the local endemic for rogozna mt. (t a t i ć & k r i v o š e j , 1997). most of the other obligate serpentinophytes are the balkan endemics (12 taxa) or subendemics (2 taxa), while 4 taxa have a wider distribution – central european or mediterraneansubmediterranean. endemism of the flora the endemic plants are very important plant group for understanding the florogenetic tendencies and phytogeographic characteristics of each region (r a n đ e l o v i ć et al., 2008). based on the complete list of plant taxa inhabiting the rogozna mt., a list of 51 balkan endemic taxa was prepared (tab. 5). the largest number of the endemic taxa has a mediteranean-submediteranean type of distribution (fig. 2). biologica nyssana 5 (2)  december 2014: 103-112 papović, o. et al.  phytogeographical analysis and endemism of the flora … 108 table 5. list of the balkan endemics distributed on rogozna mt. plant taxa area type floristic element campanula sparsa friv. subsp. sphaerothrix (griseb.) hayek ce adriatic-egean-macedonic-thracianmoesian-dacian trifolium medium l. subsp. balcanicum velen. ce dinaric-moesian-scardo-pindhian acer hyrcanum fischer & c. a. meyer subsp. intermedium (pančić) bornm. ce dinaric-moesian-scardo-pindhianpeloponnesian acer heldreichii orph. ex boiss. subsp. visianii k. malý ce moesian-scardo-pindhian trifolium velenovskyi vandas ce moesian-scardo-pindhian knautia dinarica (murb.) borbás subsp. dinarica eam/csem dinaric-moesian stachys alpina l. subsp. dinarica murb. eam/csem dinaric-scardo-pindhian viola aetolica boiss. & heldr. subsp. kopaonikensis pančić ex tomović & niketić, ined. eam/csem dinaric-w moesian pastinaca hirsuta pančić eam/csem e moesian tephroseris crassifolia (schultes) griseb. & schenk eam/csem illyrian-moesian silene sendtneri boiss. subsp. sendtneri eam/csem illyrian-moesian-scardo-pindhian campanula moesiaca velen. eam/csem moesian-scardo-pindhian melampyrum scardicum wettst. eam/csem moesian-scardo-pindhian trifolium trichopterum pančić eam/csem scardo-pindhian-macedonic-thracian cerastium decalvans schlosser & vuk. subsp. leontopodium (stoj. et stef.) niketic eam/sem dinaric-moesian-scardo-pindhianhellenic-peloponnesian dianthus cruentus griseb. subsp. cruentus eam/sem dinaric-moesian-scardo-pindhianpeloponnesian stachys scardica (griseb.) hayek eam/sem dinaric-moesian-scardo-pindhianthessaly acinos alpinus (l.) moench subsp. albanicus (kümmerle & jáv.) niketić eam/sem dinaric-scardo-pindhian stachys recta l. subsp. baldaccii (k. malý) haye eam/sem dinaric-scardo-pindhian pimpinella serbica (vis.) bentham & hooker fil. ex drude eam/sem moesian-scardo-pindhian erysimum kuemmerlei jáv. eam/sem moesian-scardo-pindhianmacedonic-thracian dianthus pinifolius sibth. & sm. subsp. serbicus wettst. med-smed dacian-moesian-scardo-pindhian linum hologynum reichenb. med-smed dacian-moesian-scardo-pindhian galatella albanica degen med-smed dinaric-adriatic-w moesian-scardopindhian eryngium palmatum pančić & vis. med-smed dinaric-moesian-scardo-pindhian trifolium pignantii fauché & chaub. med-smed dinaric-moesian-scardo-pindhianthessaly alyssum markgrafii o. e. schulz ex markgraf med-smed dinaric-scardo-pindhian orobanche nowackiana markgraf med-smed dinaric-scardo-pindhian melampyrum heracleoticum boiss. & orph. med-smed dinaric-scardo-pindhian-thessaly helleborus serbicus adamović med-smed dinaric-w moesian linaria rubioides vis. & pančić subsp. rubioides med-smed dinaric-w moesian melampyrum hoermannianum k. malý med-smed e illyrian-dinaric-moesian hypochaeris illyrica k. malý med-smed illyrian-moesian scrophularia tristis (k. malý) šilić med-smed illyrian onosma echioides l. dalmatica (scheele) peruzzi & n. g. passal. med-smed illyrian-adratic-dinaric hieracium tommasinianum k. malý med-smed illyrian-moesian-scardo-pindhian scabiosa fumarioides vis. & pančić med-smed illyrian-moesian-scardo-pindhian acanthus hungaricus (borbás) baenitz med-smed illyrian-moesian-scardo-pindhianmacedonic-thracian thymus praecox opiz subsp. jankae (čelak.) jalas med-smed illyrian-moesian-scardo-pindhianmacedonic-thracian fumana bonapartei maire & petitmengin med-smed illyrian-scardo-pindhian biologica nyssana 5 (2)  december 2014: 103-112 papović, o. et al.  phytogeographical analysis and endemism of the flora … 109 plant taxa area type floristic element halacsya sendtneri (boiss.) dörfler med-smed illyrian-w moesian-scardo-pindhian sedum serpentini janchen med-smed illyrian-w moesian-scardo-pindhian potentilla visianii pančić med-smed illyrian-w moesian-scardo-pindhian haplophyllum boissieranum vis. & pančić med-smed illyrian-w moesian-scardo-pindhian eryngium serbicum pančić med-smed moesian tragopogon pterodes pančić ex petrović med-smed moesian-macedonic-thracian alyssum montanum l. subsp. serbicum novák med-smed moesian-scardo-pindhian bupleurum apiculatum friv. med-smed moesian-thessaly-macedonicthracian-aegean tulipa serbica tatić & krivošej med-smed w moesian linum tauricum willd. subsp. serbicum (podp.) petrova med-smed w moesian thymus lykae degen med-smed w moesian-scardo-pindhian fig 2. representation of area types in the endemic flora of the rogozna mt. discussion based on presence of area types and area groups, the phytogeographical analysis show that rogozna mt. is area with eurasian-submediterraneancentraleuropeanpontic characteristics. eurasian area type is dominant in the flora of rogozna mt. the dominance of eurasian floristic elements is decisive for the genesis of the flora on this mountain. however, detailed analysis of the eurasian area type showed a numerous presence of the species with central european-mediterranean (73) and central european-mediterranean-pontic (73) types of distribution, which indicates a significant influence of these floristic chorions on the genesis of the flora. however, in comparison to the ibar river valley, where the pontic flora and related area groups (central europeanmediterranean-pontic and mediterranean-pontic) are numerous (p r o d a n o v i ć , 2007; p r o d a n o v i ć et al. 2012), pontian floristic region made less influence on the genesis of the rogozna mt. flora (fig. 3). the influence of the mediterranean region is especially pronounced. there are 117 species with mediterranean-submediterranean type of distribution, many of which are endemics or subendemics. out of these, 51 species (6.46%) are widespread in the mediterranean. that is more than the percent of these floristic elements in the flora of kosovo (5.23%) (r e x h e p i , 1997). the mediterranean exhibits influence through the valley of the river ibar. as confirmation of this, there is more pronounced presence of mediterranean species in this river valley (72 taxa, 8.79%) (fig. 3) (based on the floristic list from p r o d a n o v i ć , 2007). most of the obligate serpentinophytes has a mediterranean type of distribution (tab. 4). serpentine massifs are characterized by significant presence of the pontic taxa. according to s t e v a n o v i ć et al. (2003) the spread of pontic flora throughout the central and east balkans was especially pronounced during ice age. serpentine habitats were very suitable for these plants (j a k o v l j e v i ć et al., 2011). on the rogozna mt. typical pontic elements of flora are present in very low percent (10 taxa or 1.27%), but taxa with mediterranean-pontic (116 or 14.7%) and centraleuropean-mediterranean-pontic (74 or 9.38%) are numerous. small presence of the plant taxa with pontic type of distribution is a consequence of large distance from the pontic region. additionally, the high massif of kopaonik mt. is a significant barrier which prevents the spread of pontic elements of flora from the east. floristic endemism of the serpentine areas in the balkans is very pronounced (s t e v a n o v i ć et al., 2003; m i l l a k u et al. 2008; p a v l o v a , 2007; t o m o v i ć , 2007; t o m o v i ć et al., 2014). the presence of an imposing number of endemic taxa has biologica nyssana 5 (2)  december 2014: 103-112 papović, o. et al.  phytogeographical analysis and endemism of the flora … 110 fig 3. comparative spectrum of the basic area groups in the flora of the rogozna mt. (tab. 3) and the ibar river valley (according to the data of p r o d a n o v i ć , 2007) fig 4. spectrum of the floristic elements corresponding to the floristic provinces (total) and area type (different colour) of the endemic flora of rogozna mt. biologica nyssana 5 (2)  december 2014: 103-112 papović, o. et al.  phytogeographical analysis and endemism of the flora … 111 a great significance from the aspect of biodiversity and conservation of the area, emphasizing the fact that the rogozna mt. represents an important center of the endemic balkan flora in serbia. the total list of the endemic flora of the rogozna mt. can be divided into four area-groups (fig. 2). the strong influence of the mediterraneansubmediterranean floristic chorion, which advances through the ibar valley, is observable in the areaspectrum of the endemic flora. the largest number of endemics (30 taxa) belongs to the mediterraneansubmediterranean area group. half of them belong to a group of obligate serpentinophytes (tab. 4). they are distributed into 20 floristic elements (tab. 5), where illyrian-w moesian-scardo-pindhian element is represented by 4 taxa, while others are represented by one or two taxa. the geomorphological characteristics of the area, primarily altitude (1479 m) (p a p o v i ć et al., 2014), have enabled a strong influence of the eurasian mountainous chorion on florogenesis. the consequence of this influence is the presence of much endemics belonging to the south european mountainous (7 taxa) and central south european mountainous (9 taxa) area group (tab. 5). except for the prevailing illyrian-moesianscardo-pindhian endemic taxa (including illyrian-w moesian-scardo-pindhian, illyrian-moesian and illyrian-scardo-pindhian) from different area groups (10 taxa), it should be noted that the moesianscardo-pindhian endemics (including w moesianscardo-pindhian) (7 taxa), regardless of the area group, represent one of the dominant group in the endemic flora of the rogozna mt. additionally, all other endemics at least partially by its own areal spread into one of these floristic provinces (tab. 5, fig 4). this is confirmation of the fact that this region is located at the border of illyrian, west moesian and scardo-pinhian provinces of the balkan floristic subregion. acknowledgements. the ministry of education, science and technological development of the republic of serbia (grant 173030) supported this research. references borojević-šoštarić, s., cvetković, v., neubauer, f., palinkaš, l. a., bernroider, m., & genser, j., 2012: oligocene shoshonitic rocks of the rogozna mts. 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(ed.), flora srbije, 1, 47-56. sanu, beograd. stevanović, v., tan, k., iatrou, g., 2003: distribution of the endemic balkan flora on serpentine. plant systematic and evolution, 242: 149-170. tatić, b., krivošej, z., 1997: tulipa serbica (liliaceae), a new species from serbia. bocconea, 5: 733-736. tomović, g., 2007: phytogeographycal reference, distribution and diversity centres of the balkan endemic flora in serbia. phd thesis, university of belgrade. (in serbian, with english abstract). tomović, g., niketić, m., lakušić, d., ranđelović, v., stevanović, v., 2014: balkan endemic plants in central serbia and kosovo regions: distribution patterns, ecological characteristics, and centres of diversity. botanical journal of the linnean society, 176 (2): 173-202. tutin, t.g., heywood, v.h., burges, n.a., moore, d.m., valentine, d.h., walters, s.m., webb, d.a. (ed.), 1964-1980: flora europaea, i-v. cambridge, university press. london. urošević, m., pavlović, z, klisić, m., karamata, s., malešević, m., stefanović, m., marković, o., trifunović, s., 1966: tumač za list ogk novi pazar, 77. savezni geološki zavod, beograd. urošević, m., pavlović, z, klisić, m., brković, t., malešević, m., trifunović, s., 1970: osnovna geološka karta 1:100000, list novi pazar, k 3430. savezni geološki zavod, beograd. vasić, o., diklić, n., 2001: the flora and vegetation on serpentinites in serbia a review. bocconea, 13: 151-164. whittaker, r.h., 1954: the ecology of serpentine soils. ecology, 35 (2): 258-288. stanković et al., 2019, biologica nyssana 10(1) 10 (1) september 2019: 59-61 doi: 10.5281/zenodo.3464010 contribution to the knowledge of tachinid fauna in serbia short communication saša s. stanković university of niš, department of biology and ecology, faculty of sciences and mathematics, višegradska 33, 18000 niš, serbia sasasta@gmail.com (corresponding author) marijana ilić milošević department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, 18000 niš, serbia marijana183@gmail.com vladimir žikić department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, 18000 niš, serbia vzikic@pmf.ni.ac.rs hans-peter tschorsnig staatliches museum für naturkunde, rosenstein 1, 70191 stuttgart, germany hanspeter.tschorsnig@smns-bw.de received: august 5, 2019 revised: september 7, 2019 accepted: september 9, 2019 abstract: tachinid flies were reared from several species of lepidoptera larvae during the year 2018. eight species were recorded from two subfamilies, exoristinae and tachininae. the species ceromya bicolor is new for the serbian fauna. also, for the same territory, the genus ceromya is recorded for the first time. key words: tachinidae, ceromya bicolor, parasitoids, serbia apstract: doprinos poznavanju faune muva guseničarki u srbiji muve guseničarke su odgajene iz nekoliko vrsta larvi lepidoptera tokom 2018. godine. registrovano je osam vrsta iz dve podfamilije, exoristinae i tachininae. vrsta ceromya bicolor je nova za faunu srbije. takođe, za istu teritoriju je prvi put registrovan rod ceromya. ključne reči: tachinidae, ceromya bicolor, parazitoidi, srbija tachinid flies are well known parasitoids of other arthropods. they parasitize almost exclusively insects, especially larval lepidoptera, but very rarely spiders, scorpions and centipedes (vincent, 1985; williams et al., 1990; stireman et al., 2006). besides various parasitic hymenoptera, tachinids are the second most important natural enemies of many pest insects in both natural and agroecosystems. many tachinid species are being used in pest managements in forestry and crop production systems with notable success (greathead, 1986; grenier, 1988). the family tachinidae is one of the most diverse among diptera order, so far over 10,000 species are described worldwide from about 1,520 genera (irwin et al., 2003). the family is divided in four subfamilies: exoristinae, dexiinae, phasiinae and tachininae (herting & dely draskovits, 1993). although, the group is the most diverse in tropical regions, in europe there are registered about 877 species (according to fauna europaea, internet database). this database listed 242 species for the territory of serbia and montenegro. however, some recent faunistic papers reported several more species for the territory of serbia. for example, hubenov (2008) reported 288 species for the serbian fauna, stanković et al. (2014) reported two more species and later stanković et al. (2018) published an updated list of 295 species from which five are newly recorded and the new genus buquetia for the investigated territory. tachinids were reared from lepidoptera larvae during the year 2018 mostly from the southern part of serbia. the host larvae were grown in special plastic boxes in the laboratory until parasitoids emergence. after rearing the adults of tachinid flies, they were put in small plastic vials and preserved in 96 % ethanol. the identification of the species was done by the last author of the paper. the rearing of parasitoids and all the material is stored at the department of biology and ecology, faculty of science and mathematics, university of niš, serbia. the remarks are based on a catalogue on palaearctic tachinid-host records (tschorsnig, 2017). new species and genus is newly reported for the investigated territory and marked by an asterisk (*). © 2019 stanković, s.s. et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 59 biologica nyssana ● 10 (1) september 2019: 59-61 stanković et al. ● contribution to the knowledge of tachinid fauna in serbia species list subfamily exoristinae erycia fatua (meigen, 1824) 3 ♂, 1 ♀, serbia, vlasina lake, 27.v 2018. ex melitaea didyma (esper, 1778) (nymphalidae), on centaurea jacea l., leg. v. žikić. remark: a parasitoid of melitaea, m. didyma was already known (see tschorsnig, 2017: 181). erycia festinans (meigen, 1824) 2 ♂, 4 ♀, serbia, vlasina lake, 27.v 2018. ex melitaea phoebe (denis & schiffermüller, 1775) (nymphalidae), on centaurea jacea l., leg. s. stanković. remark: a parasitoid of melitaea, m. phoebe was already known. erycia festinans is morphologically very similar to erycia fatua (see herting, 1973; tschorsnig & herting, 1994). having the same hosts, there remains some doubt if both are really separate species. (see tschorsnig, 2017: 182). eumea mitis (meigen, 1824) 1 ♂, serbia, niš, brzi brod, 3.v 2018. unknown host, on sambucus ebulus l., leg. v. žikić. remark: a parasitoid of noctuidae and several microlepidoteran families (see tschorsnig, 2017: 187188). eurysthaea scutellaris (robineau-desvoidy, 1848) 1 ♂, serbia, sićevačka klisura (gorge), ostrovica, 1.v 2018. ex yponomeuta sp. (yponomeutidae) on euonymus japonicus thunb, leg. v. žikić. remark: common parasitoid of yponomeuta spp. (see tschorsnig, 2017: 215-216). masicera sphingivora (robineau-desvoidy, 1830) 2 ♀, serbia, vlasina lake, 27.v 2018. ex melitaea didyma (esper, 1778) (nymphalidae), on centaurea jacea l., leg. s. stanković. remark: a parasitoid with many hosts, also m. didyma was already known (see tschorsnig, 2017: 232). phryxe hirta (bigot, 1880) 1 ♂, serbia, vlasina lake, 27.v 2018. ex heterogynis zikici de freina, 2018 (heterogynidae) on chamaecytisus heuffelii (wierzb. ex griseb. and schenk) rothm, leg v. žikić. remark: typical parasitoid of heterogynis spp. (see tschorsnig, 2017: 117). phryxe vulgaris (fallén, 1810) 1 ♀, serbia, sićevačka klisura (gorge), 7.v 2018. ex brenthis daphne (bergsträsser, 1780) (nymphalidae), on rubus sp. leg v. žikić. remark: a common species which has many hosts; also b. daphne is among it (see tschorsnig, 2017: 133). subfamily tachininae *ceromya bicolor (meigen, 1824) 2 ♂, 2 ♀, serbia, paraćin, bukovik, 5.v 2018. unknown host, on unknown plant, leg n. veljković. remark: a small species which occasionally develops in numbers in large lasiocampidae (see tschorsnig, 2017: 272). here we present eight tachinid species collected in serbia from two subfamilies, seven from exoristinae and one from tachininae. the species erycia festinans is still not listed in fauna europaea for the territory of serbia although it is reported recently as a new species for this country in stanković et al. (2018). the same is with the species phryxe hirta which is reported as a new species for serbia in stanković et al. (2014). the species ceromya bicolor is the new species for the serbian fauna, as well as the genus ceromya. these observations of l. senator represent a contribution to the distribution and population census of this species, and also the need of protection of its habitat. acknowledgements. this study was supported by the ministry of education, science and technological development of the republic of serbia (iii43001). we thank to kjeld brem sørensen and boženka hric for the identification of caterpillars. references greathead, d.j. 1986: parasitoids in classical biological control. in: insect parasitoids. 13th symposium of the royal entomological society of london, 18-19 september 1985 at the department of physics lecture theatre, imperial college, london (pp. 289-318). academic press. grenier, s. 1988: applied biological control with tachinid flies (diptera, tachinidae): a review. anzeiger für schädlingskunde pflanzenschutz umweltschutz, 51: 49-56. herting, b. 1973: beiträge zur kenntnis der europäischen raupenfliegen (dipt. tachinidae) xiii. stuttgarter beiträge zur naturkunde, serie a (biologie) 254: 18 pp. herting, b., dely-draskovits, a. 1993: family tachinidae. in: soós, a., papp, l. (ed.), catalogue of palaearctic diptera 13: 118-624, hungarian natural history museum, budapest. hubenov, z. 2008: composition and zoogeographical characteristics of the family tachinidae (diptera: insecta) in serbia and bulgaria. advances in arachnology and developmental biology, 12: 375394. 60 irwin, m.e., schlinger, e.i., thompson, f.c. 2003: diptera, trueflies. in: goodman, s. m., benstead, j. p. (ed.), the natural history of madagascar, 692702, university of chicago press. chicago/london 1728 p. stanković, s.s., žikić, v., hric, b., tschorsnig, h.p. 2014: several records of tachinidae (diptera) reared from their hosts in serbia and montenegro. biologica nyssana, 5 (1): 71-73. stanković, s.s., žikić, v., ilić milošević, m., ritt, r., tschorsnig, h.p. 2018: tachinid fauna of serbia and montenegro updated with new findings (diptera: tachinidae). journal of the entomological research society, 20 (3): 53-66. stireman iii, j.o., o’hara, j.e., wood, d.m. 2006: tachinidae: evolution, behavior, and ecology. annual review of entomology, 51: 525-555. tschorsnig, h.p., herting, b. 1994: die raupenfliegen (diptera: tachinidae) mitteleuropas: bestimmungstabellen und angaben zur verbreitung und ökologie der einzelnen arten. stuttgarter beiträge zur naturkunde, serie a (biologie), 506: 170 pp. tschorsnig, h. p. 2017: preliminary host catalogue of palaearctic tachinidae (diptera). web page: www. nadsdiptera. org/tach/worldtachs/catpalhosts/ home. html) (date accessed: 15.08. 2017). vincent, l.s. 1985: the first record of a tachinid fly as an internal parasitoid of a spider (diptera: tachinidae; araneae: antrodiaetidae). panpacific entomologist, 61: 224-235. williams, s.c., arnaud, p.h., lowe, g. 1990: parasitism of anuroctonus phaiodactylus (wood) and vaejovis spinigerus (wood) (scorpiones: vaejovidae) by spilochaetosoma californicum smith (diptera: tachinidae), and a review of parasitism in scorpions. myia, 5: 11-27. 61 biologica nyssana ● 10 (1) september 2019: 59-61 stanković et al. ● contribution to the knowledge of tachinid fauna in serbia jevtić, a., đekić, v.: influence of growing season on some agronomic characteristics of winter wheat cultivars. biologica nyssana, 9 (2). december, 2018 biologica nyssana 9 (2) ⚫ december 2018: 119-131 jevtić, a., đekić, v. ⚫ influence of growing season on some agronomic... 133 original article received: 24 january 2018 revised: 22 august 2018 accepted: 6 november 2018 influence of growing season on some agronomic characteristics of winter wheat cultivars aleksandra jevtić1, vera đekić2 1high agricultural food school of professional studies, ćirila and metodija 1, 18400 prokuplje, serbia 2small grains research centre, save kovačevića 31, 34000 kragujevac, serbia * e-mail: verarajicic@yahoo.com abstract: jevtić, a., đekić, v.: influence of growing season on some agronomic characteristics of winter wheat cultivars. biologica nyssana, 9 (2). december, 2018: 133-139. the experiment was established in the experimental field of the small grains research centre in kragujevac during the two years. the winter wheat varieties used in the experiment was takovčanka, kruna, toplica and planeta. the following characteristics were analysed: grain yield, plant height, number of plants per m2, 1000 grain weight and test weight. the highest yield of all tested varieties of winter wheat was achieved by takovčanka (4.124 t/ha). the highest plant height for all years was recorded for the cultivar toplica (81.90 cm). the highest 1000 grain weight of investigation on winter wheat takovčanka cultivar (44.05 g). the highest two-year average value of test weight was found in the cultivars kruna and planeta (71.03 kg/hl). analysis of variance was found to have the highly significant effect of years in grain yield, plant height, 1000-grain weight and test weight and significant differences in number of plants per m2 at investigated wheat were found in relation to the year. key words: cultivar, grain yield, winter wheat apstrakt: jevtić, a., đekić, v.: uticaj vegetacije na neke agronomske osobine kod ozime pšenice. biologica nyssana, 9 (2). decembar, 2018: 133-139. eksperiment je izveden na oglednom polju centra za strna žita u kragujevcu tokom dve vegetacione godine. u eksperimentu su korišćene sorte ozime pšenice takovčanka, kruna, toplica i planeta. analizirane su sledeće osobine: prinos zrna, visina biljke, broj biljaka po m2, masa 1000 zrna i hektolitarska masa. najveći prinos kod svih ispitanih sorti ozime pšenice ostvarila je sorta takovčanka (4.124 t/ha). najveću visinu biljaka tokom eksperimenta ostvarila je sorta toplica (81,90 cm). najveću masu 1000 zrna kod ispitivanih sorti ozime pšenice ostvarila je sorta takovčanka (44,05 g). najveću prosečnu dvogodišnju vrednost hektolitarske mase ustanovljena je kod sorti kruna i planeta (71,03 kg/hl). analizom varijanse utvrđen je veoma značajan efekat godine na prinos zrna, visinu biljaka, masu 1000 zrna i hektolitarsku masu, a značajne razlike ustanovljene su kod broja biljaka po m2. ključne reči: ozima pšenica, prinos zrna, sorta 9 (2) • december 2018: 133-139 doi: 10.5281/zenodo.2538606 biologica nyssana 9 (2) ⚫ december 2018: 119-131 jevtić, a., đekić, v. ⚫ influence of growing season on some agronomic... 134 introduction new perspective wheat lines and varieties has more and better filled grain, higher yield, grain mass and farinaceous content, while proteins and lysine were smaller compared with older varieties (đekić et al., 2012, jelić et al., 2013). wheat cultivars that were in production until the end of the eighties were characterized by the lower yields, good technological quality and higher stem sensitive on lodging (zečević et al., 2007). due to lower resistance on lodging, that cultivars were grown at modest soils and therefore they had lower yields (đekić et al., 2014; jelić et al., 2012). agronomic cultivar value depends not only on its genetic potential for yield, but also on its ability to achieve genetic potential under different conditions of production (malešević, 2008). new varieties are characterized by good technological quality, better resistance to lodging and diseases, shorter stem and more efficient assimilates usage (browne et al., 2006; jovanović et al., 2006; malešević et al., 2008; hristov et al., 2011; đekić et al., 2016). several factors are decisive in increasing wheat yields: the cultivar, cultural practices, agroecological conditions, local climatic and soil characteristics, mineral nutrition and adequate protection from plant diseases, pests and weeds. in regard to the elements of mineral nutrition, nitrogen plays a major role in increasing the yield of wheat (kastori et al., 2005; jovanović et al., 2006; malešević, 2008; đekić et al., 2014; jelić et al., 2014). the aim of this study was the determination of the cultivars and ecological environmental factors influence on differences in stability and adaptability of cultivars regard the grain yield, plant height, number of plants per m2, 1000 grain weight and test weight of tested winter wheat cultivars, as well as specificity cultivars exploring in relation to growing seasons conditions. material and methods experimental design and statistical analysis during the 2011/12 and 2012/13 growing seasons, four cultivars of winter wheat (takovčanka, kruna, toplica and planeta), cultivated at the small grains research centre in kragujevac were investigated. experiments have been conducted in randomized block systems, with a plot size of 10 m2 (2 m x 5 m) in five replicates. the usual techniques for wheat production were applied, and it was done in the optimum sowing time in late october. 400 kg/ha of fertilizer npk 15:15:15 was added in the fall on the investigated plots, while during the spring fertilization, 300 kg/ha (kan) was supplemented. the following properties were analyzed: grain yield (t/ha), plant height (cm), number of plants per m2, 1000 grain weight (g) and test weight (kg/hl). on the basis of achieved research results the usual variational statistical indicators were calculated: average values, standard error and standard deviation. statistical analysis was made in the module analyst program sas/stat (sas institute, 2000). meteorological conditions this study was conducted over a three-year period in the šumadija region, central serbia, on a vertisol soil, at kragujevac location, 173-220 m a. s. l. (44° 22′ n, 20°56′ e), in a temperate continental climate having an average annual temperature of 11.5 °c typical of šumadija districts in serbia and a rainfall amount of about 550 mm. kragujevac area is characterized by a moderate continental climate, which general feature is uneven distribution of rainfall by month. the data in tab. 1 for the investigated period (2011-2013) clearly indicate that the years in which the researches were conducted differed from the typical multi-year average of kragujevac region regard the meteorological conditions. table 1. middle monthly air temperature and precipitation amount (kragujevac) year months average x xi xii i ii iii iv v vi mean monthly air temperature (oc) 2011/12 10.4 3.1 4.6 0.7 -3.7 8.1 12.9 16.1 23.0 8.35 2012/13 13.5 9.5 1.7 2.9 4.0 6.5 13.4 18.2 19.9 9.96 average 12.5 6.9 1.9 0.5 2.4 7.1 11.6 16.9 20.0 8.87 the amount of rainfall (mm) 2011/12 33.3 1.3 43.3 117.2 60.1 5.7 74.5 87.3 57.8 480.5 2012/13 56.2 17.7 16.4 62.4 84.3 102.0 41.2 70.8 30.3 481.3 average 57.6 70.4 71.5 58.5 62.7 45.4 48.9 56.6 58.2 529.8 biologica nyssana 9 (2) ⚫ december 2018: 119-131 jevtić, a., đekić, v. ⚫ influence of growing season on some agronomic... 135 the average air temperature in 2011/12 was lower by 0.52 c and 2012/13 was higher by 1.09 c. the sum of rainfall precipitation in 2011/12 was lower by 49.3 mm, where the sum of rainfall in 2012/13 was 48.5 mm lower than the average of many years and with a very uneven distribution of precipitation per months. spring months march and april in 2011/12 were the surplus of precipitation, what affected unfavorable on the crops. during the march in 2012/13 it was 102.0 mm of rainfall, what was 56.6 mm more compared with the perennial average. regarding the high importance of sufficient rainfall amounts during the spring months, particularly may for small grains production, the distribution and amount of rainfall over the growing season 2011/12 were considerably more favorable, resulting with the increment of yields during that year. based on the fact that sufficient amounts of rainfall in these months are very important for the successful production of cereal crops it can be concluded that the years in which the researches were conducted were favorable for the wheat growing. soil and weather conditions before the commencement of the experiment, soil samples were taken from the sample surface and the chemical analysis of soil was performed. on the basis of obtained results, it was revealed that the soil belongs to the smonitza type, with relatively high clay content, and unfavorable physical properties. the humus content in the surface layer of soil was low (2.38-2.64%), and a substitution and total hydrolytic acidity were quite high (ph h2o=5.99, kcl=4.56). the soil was medium provided with total nitrogen (0,11-0,13% n) and easily accessible potassium (10-14 mg/100 g soil k2o), while the available phosphorus content was low (under 10 mg/100 g of soil p2o5). results and discussion average values of grain yield (t/ha), plant height (cm), number of plants per m2, 1000-grain weight (g) and test weight (kg/hl) at investigated kragujevac’s winter wheat cultivars grown at the small grains research centre in kragujevac during two growing seasons, 2011/12 and 2012/13, are presented in tab. 2. during the first year of investigations, cultivar takovčanka achieved the highest grains yield (4.729 t/ha), followed by kruna (4.642 t/ha) and toplica (4.473 t/ha), while the lowest yield was at planeta cultivar (4.395 t/ha). during the second year of investigations (2012/13), the yield of takovčanka cultivar was the highest with 3.518 t/ha, while the slightly lower yield was realized by kruna cultivar (3.276 t/ha). average grains yield observed in the two-year period was the highest at takovčanka variety (4.124 t/ha), while the lowest yield was obtained by kruna cultivar (3.959 t/ha). during the first and second years of investigation, toplica cultivar achieved the highest average plant height (85.80 cm and 78.00 cm) compared with other tested wheat cultivars. in the two-year period, the highest average plant height of winter wheat cultivar investigated was achieved in the toplica cultivar (81.90 cm) and planeta cultivar (78.60 cm). number of plants per m2 of wheat significantly varied across years, from 374 to 454 in 2011/12, from 342 to 368 plants per m2 in 2012/13. during the first year of investigations, the highest average value of number of plants per m2 of wheat achieved the kruna cultivar (454) and planeta (437). during the second year of investigations, the highest average value of number of plants per m2 of wheat achieved the kruna cultivar (368). in the two-year period, the highest average number of plants per m2 of winter wheat cultivar investigated was achieved in the kruna cultivar (411 plants per m2). during the first years of investigation, planeta cultivar achieved the highest average 1000 grain weight (44.90 g) and takovčanka cultivar (44.72 g). during the second year of investigations (2009/10), the highest average value of 1000 grain weight achieved the takovčanka cultivar (43.38 g) compared with other tested wheat cultivars. in the two-year period, the highest average thousand grain weight of winter wheat cultivar investigated was achieved in the takovčanka and planeta cultivars (44.05 g and 43.31 g). the wheat cultivar kruna has achieved the highest test weight in the first years of investigation compared to other tested wheat cultivars (73.05 kg/hl). during the second year of investigations (2012/13), the test weight of planeta cultivar was the highest with 69.81 kg/hl, while the slightly lower test weight was realized by kruna cultivar (69.01 kg/hl). the average two-year value of test weight at kruna and planeta cultivars was 71.03 kg/hl, while the lowest average was at takovčanka and toplica cultivars (70.00 kg/hl and 69.50 kg/hl). table 3 shows the impact of the year, cultivar and interaction of year x cultivar on yield, plant height, number of plants per m2, 1000-grain weight and test weight. analysis of variance was found highly significant effect of year on the grain yield (f=48.911**), plant height (f=26.863**), 1000-grain weight (f=2.616**) and test weight (f=3.634**), and biologica nyssana 9 (2) ⚫ december 2018: 119-131 jevtić, a., đekić, v. ⚫ influence of growing season on some agronomic... 136 significant effect for the number of plants per m2 (f=5.910*). discussion in regard to the tested genotypes winter wheat, variety takovčanka achieved the highest yield of grain (4.124 t/ha). of the tested years, particularly in 2011/12 year, the highest yields were achieved. considerable variation in yield depending on years of research have established biberdžić et al. (2005), jovanović et al. (2006), malešević et al. (2008), hristov et al. (2011), jelić et al. (2013), đekić et al. (2016). perišić et al. (2009), pointing out that among the kg cultivars, very high grain yield demonstrated winter wheat cultivars kruna and vizija. the takovčanka cultivar had stable and high grain yields, while cv. toplica demonstrated good yield followed by high quality. wheat productivity and grain quality in central serbia are governed by a range of factors, notably climate, soil, genetics and crop nutrition. soil acidity in wheat fields in central serbia has become a severe problem that leads to a significant decline in grain yield and quality of wheat (đekić et al., 2013, jelić et al., 2015). the yield per unit area is the result of the action of factors of variety in interaction with environmental factors. therefore, the yield a relative term and is determined by the variety, environmental conditions and the level of applied technology. yield is largely dependent on the genetic potential, which could be defined the yield of variety which was grown in conditions on which it had been adapted, with adequately amounts of water and nutrients and efficient control of pests, diseases, weeds and other stresses (đekić et al., 2016). yields considerably vary primarily as a result of agro-ecological conditions during the growing season (zečević et al., 2010; hristov et al., 2011; đekić et al., 2015). analysis of variance showed highly significant effect of year on the grain yield (p<0.01). considerable variation in yield depending on years of research has been established by glamočlija et al. (2010), milovanovic et al. (2011), đekić et al. (2012; 2014) and jelic et al. (2015). table 2. average values of investigated wheat cultivars characteristics varieties 2011/12 2012/13 average x s sx x s sx x s sx grain yield, t/ha takovčanka 4.729 0.567 0.253 3.518 0.566 0.253 4.124 0.832 0.263 kruna 4.642 1.014 0.454 3.276 0.278 0.124 3.959 1.005 0.318 toplica 4.473 0.258 0.115 3.125 0.692 0.309 3.799 0.864 0.273 planeta 4.395 0.757 0.338 3.160 0.402 0.180 3.778 0.866 0.274 plant height, cm takovčanka 83.60 9.317 4.166 71.20 2.588 1.158 77.40 9.180 2.903 kruna 83.40 4.722 2.112 72.20 3.365 1.505 77.80 7.056 2.231 toplica 85.80 9.418 4.212 78.00 3.021 1.351 81.90 7.770 2.457 planeta 81.80 6.140 2.746 75.40 2.631 1.177 78.60 5.587 1.767 number of plants per m2 takovčanka 374 73.245 32.756 362 38.220 17.093 368 55.44 17.532 kruna 454 66.969 29.949 368 88.136 39.416 411 86.493 27.351 toplica 375 71.688 32.060 342 46.785 20.923 359 59.630 18.857 planeta 437 82.384 36.843 362 48.711 21.784 399 75.113 23.753 1000 grain weight, g takovčanka 44.72 1.994 0.892 43.38 1.240 0.554 44.05 1.717 0.543 kruna 43.86 2.317 1.036 41.82 1.215 0.543 42.84 2.049 0.648 toplica 44.24 1.212 0.542 41.20 2.051 0.917 42.72 2.256 0.713 planeta 44.90 1.044 0.467 41.92 1.305 0.583 43.41 1.925 0.609 test weight, kg/hl takovčanka 72.92 0.784 0.351 67.08 1.994 0.892 70.00 3.393 1.073 kruna 73.05 1.789 0.800 69.01 2.128 0.952 71.03 2.823 0.893 toplica 71.37 1.647 0.736 67.63 2.706 1.210 69.50 2.889 0.913 planeta 72.25 1.661 0.743 69.81 1.152 0.515 71.03 1.863 0.589 biologica nyssana 9 (2) ⚫ december 2018: 119-131 jevtić, a., đekić, v. ⚫ influence of growing season on some agronomic... 137 milovanovic et al. (2011), states that in serbia higher yields are achieved by varieties with shorter growing season because they manage to form the largest part of the yield before the occurence of high temperatures. in this study, in both years, the wheat was not exposed to extremely high temperatures so early growth did not come into its own. the 1000-grain weight of winter wheat significantly varied across cultivars, from 42.72 g in cultivar toplica to 44.05 g in cultivar takovčanka. highly significant influence of the year on 1000-grain weight was established at investigated winter wheat cultivars by variance analysis. a number of authors (jelić et al. 2013; đekić et al. 2014, 2016), underline that 1000-grain weight is a cultivar-specific trait, with considerably higher variations being observed among genotypes than among treatments or environmental factors. jelic et al. (2015) have presented the 1000grain weight of three winter wheat cultivars (pobeda, planeta and nora). the 1000-grain weight of wheat variety was 42.62 g in control to 47.06 g while wheat treatment in the combined treatment with lime, manure and mineral npk fertilizer at the rates of caco3 of 5 t/ha and phosphorus of 100 kg/ha. a number of authors underlined that 1000-grain weight is a cultivar-specific trait, with considerably higher variations being observed among genotypes than among treatments or environmental factors (malešević et al., 2008; hristov et al., 2011; đekić et al., 2012, 2013; jelic et al., 2015). the genotype kruna had an extreme value (73.05 kg/hl) in the first years (2011/12) were recorded. the average two-year value of test weight at kruna and planeta cultivars was 71.03 kg/hl. particularly in the second year, genotypes had significantly lower test weight compared to first year. based on the analysis of variance, it can be concluded that there are significant differences in test weight in regard to the year of investigation, which is in accordance with the results obtained by đekić et al. (2015). the test weight of winter wheat ranged from 80 (takovčanka) to 82 kg/hl in cultivar toplica (protić et al., 2013). jelic et al. (2015) have examined the average test weight of winter wheat, which ranged from 68.66 kg/hl to 70.91 kg/hl. a number of authors (browne et al., 2006; đekić et al., 2013, 2016; jelić et al., 2013, 2014) underline that grain of investigated wheat cultivars was characterized by good physical characteristics; especially regarding the test weight and 1000 grain weight. determined average values of these characteristics in the study were slightly higher than the values obtained by jelić et al. (2013) and đekić et al. (2015). table 3. analysis of variance of the tested parameters (anova) effect of year on the traits analyzed traits mean sqr effect mean sqr error f(df1,2) 1, 38 p-level grain yield (t/ha) 16.64 0.340 48.91138 0.000000 plant height (cm) 893.03 33.243 26.86321 0.000007 number of plants per m2 26419.60 4470.631 5.90959 0.019886 1000-grain weight (g) 55.22 2.616 21.10920 0.000047 test weight (kg/hl) 161.20 3.634 44.35568 0.000000 effect of cultivar on the traits analyzed traits mean sqr effect mean sqr error f(df1,2) 3, 36 p-level grain yield (t/ha) 0.259 0.800 0.324121 0.807880 plant height (cm) 41.825 56.411 0.741432 0.534361 number of plants per m2 6177.467 4938.089 1.250983 0.305709 1000-grain weight (g) 3.715 3.986 0.932025 0.435230 test weight (kg/hl) 5.878 7.824 0.751257 0.528824 effect of the year x cultivar interaction traits mean sqr effect mean sqr error f(df1,2) 3, 32 p-level grain yield (t/ha) 0.015 0.378 0.040450 0.988943 plant height (cm) 19.825 33.697 0.588333 0.627103 number of plants per m2 3028.667 4445.800 0.681242 0.570020 1000-grain weight (g) 1.658 2.603 0.636829 0.596804 test weight (kg/hl) 4.906 3.305 1.484619 0.237321 *statisticaly significant difference (p<0.05) **statisticaly high significant difference (p<0.01) biologica nyssana 9 (2) ⚫ december 2018: 119-131 jevtić, a., đekić, v. ⚫ influence of growing season on some agronomic... 138 in the growing season 2011/12 which were more favourable for wheat production in terms of agro-meteorological conditions, a significantly higher grain yield was obtained in all winter wheat cultivars. highly significant impact of the year on grain yield, plant height, 1000-grain weight and test weight were determined in investigated winter wheat cultivars by using the variance analysis, while the influence of year on number of plants per m2 was statistically significant. there are significant phenotypic differences among wheat cultivars with regard to grain quality. these differences result from the action of different genes that control characters for wheat quality (perišić et al., 2009). nonetheless, environmental factors play a major role in the expression of genotype characteristics (misic & mladenov, 1998; glamočlija et al., 2010; milovanović et al., 2011; đekić et al., 2013; jelić et al., 2013). their impact, however, is rarely optimal; one or more of them will always limit the yield and quality of the product. for this reason, it is very important to determine the variation of environmental factors and their effects on the processes that determine wheat quality. the present results confirm the opinion of many authors that the traits analyzed are genetically determined but are strongly modified by the nutrient status of the environment and weather conditions (jelić et al., 2014; đekić et al., 2016). due to the global change of climate, drought is a frequent occurrence both in southern parts of central europe and in the balkan. owing to such climatic conditions, southern parts of central europe and southern europe are considered suitable for the production of bread wheat (misic and mladenov, 1998; glamočlija et al., 2010; milovanović et al., 2011). conclusion based on the results during the two-year investigation on four kragujevac’s winter wheat cultivars, it can be concluded that the grain yield of wheat ranged from 3.778 t/ha (planeta) to 4.124 t/ha (takovčanka). the highest average plant height of winter wheat cultivar investigated was achieved in the toplica cultivar (81.90 cm). during the two-year the 1000 grain weight of investigation on winter wheat cultivars ranged from 42.72 g (toplica) to 44.05 g (takovčanka). winter wheat cultivars had test weight greater than 70 kg/hl, except of cultivar toplica (69.50 kg/hl). during 2011/12, statistically significantly higher grain yield per area unit, as well as 1000 grain weight was achieved, compared with 2012/13 year. highly significant differences in yield, plant height, 1000-grain weight and test weight and significant differences in number of plants per m2 at investigated wheat were found in relation to the year. the interaction of grain yield, plant height, number of plants per m2, 1000-grain weight and test weight and year x cultivar had no significant differences at investigated wheat cultivars. rainy weather in the spring month during 2013 caused water lodging, the occurrence of plant lodging and plant diseases, which influenced poorer quality and lower average yield of wheat in the 2013/14 season compared to the 2011/12 season. acknowledgements. investigations necessary for this paper are part of the project tp 31054 "development of new cereals cultivation technologies on acid soils by usage of modern biotechnology", financed by the ministry of education, science and technology development of republic of serbia. references biberdžić, m., đorđević, m., barać, s., deletić, n., & stojković, s. 2005: productivity of some winter wheat genotypes. genetika, 37(2), 131-136. browne, r.a., 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interaction on grain yield of winter wheat varieties. bulgarian journal of agricultural science, 19(4), 697-700. sas/stat (2000). user's guide, version 9.1.3. sas institute inc. zečević, v., knežević, d., mićanović, d. 2007: variability of technological quality components in winter wheat. genetics, belgrade, 39: 365-374. zečević, v., bošković, j., knežević, d., mićanović, d., madić, m. 2010: ecological and genetic variability of wheat quality components. kragujevac journal science, 32: 89-94. microsoft word bn020206 marko lazic biologica nyssana 2 (2) december 2011: 00-00 nahirnić a. supplements of butterfly fauna… 131 short communication ! accessory femoral pores in podarcis muralis from southern serbia marko m. lazić1, jelka crnobrnja-isailović1, 2 1university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia 2institute for biological research „siniša stanković“, university of belgrade, serbia * e-mail: markol@pmf.ni.ac.rs abstract: lazić, m.l., crnobrnja-isailović, j.: accessory femoral pores in podarcis muralis from southern serbia, biologica nyssana, 2 (2), december 2011: 131-133. abnormalities in scalation are common in lizards. bilateral asymmetries are frequent, as are supernumerary scales. factors possibly responsible for such developmental anomalies are inbreeding and environmental stress. here, we report two cases of accessory femoral pores in a podarcis muralis from southern serbia. key words: abnormal scalation, podarcis muralis, femoral pores, developmental instability, environmental stress developmental stability is the ability of individuals to undergo stable development of their phenotype under a range of environmental conditions (m ø l l e r , 1997). the origin of the disturbances is assumed to be genetic, environmental, or the product of a genotypeenvironment interaction (m a r k o w , 1995). deviations from developmental stability arise when organism fails to buffer such disturbances. developmental anomalies and asymmetries in bilateral traits are common in lizards (b r a ñ a & j i , 2000; c m o b m j a -i s a i l o v i c et al., 2005). there is generally negative correlation between developmental instability and fitness components such as growth, fecundity and longevity and these relations can be either direct and indirect (m ø l l e r , 1997). here, we report two cases of supernumerary femoral pores in common wall lizard (podarcis muralis) from southern serbia. podarcis muralis is small lacertid lizard species, distributed in central and southern europe, but its range extends to northwestern asia minor (g a s c et al., 1997; a r n o l d & o v e n d e n , 2002). it is one of the most widespread lizard species in the central balkans (r a d o v a n o v i ć , 1951), and, consequently, susceptible to various kinds of negative anthropogenic impacts, as pesticide use (g u i l l a u m e , 1997). in spring 2011, during collection of adult p. muralis in southern serbia, adult female (63.0 mm svl) and adult male (52.3 mm svl) with supernumerary femoral pores were sampled. female was sampled in the niš fortress (n 43° 19.477’ e 021° 53.864’) in the center of the city of niš. the habitat consisted of tall stone walls up to 10 meters with open grass. accessory rows of femoral pores were noted in both hind limbs (figure 1).the accessory pores (left n =14, right n =8) were smaller and located parallel. on left hind limb they were located along the entire length of normal series and on the right they were in a central position and posterior to the normal series (left n = 20, right n = 20). this was the only case of supernumerary femoral pores in 155 individuals examined from this population (appr. 0.64% of entire sample). male was 2 (2) • december 2011: 131-133 biologica nyssana 2 (2) december 2011: 131-133 lazić m.l., crnobrnja-isailović j. accessory femoral pores in podarcis… 132 collected in sićevo gorge, 15 km from the city of niš (n 43° 20.307’ e 022° 05.000’). habitat consisted of stone walls up to 4 meters high, large and small piles of stones and knocked down trees. accessory rows of femoral pores (n=4) were noted on left hind limb (figure 2) but above the normal series, in central position and anterior of the normal series (left n=16, right n=17). this was the only one with this type of anomaly in 126 examined individuals (appr. 0.79% of entire sample). after photographing and measuring, animals were released to the site of capture. there are few records of accessory femoral pores in lizards. in portugal, supernumerary femoral pores were found in adult female podarcis bocagei (k a l i o n t z o p o u l o u & c a r r e t e r o , 2006). as in this study, frequency of occurrence was quite low, one individual from 37 sampled (2.70%). moreover, they didn’t find any such case in 380 lizards of the same species sampled from seven localities. accessory femoral pores were recorded in the collared lizard, crotaphytus collaris (w a l k e r , 1980) with 63.7% of males and 36.3% of females displaying the anomaly, which was attributed to inbreeding since the population was small and isolated. these anomalies were also recorded among iguanians (t a n n e r & a v e r y , 1964). figure 1. female specimen of podarcis muralis from niš fortress, southern serbia. the white arrows on each side delimit the additional row of femoral pores figure 2. male specimen of podarcis muralis from sićevo gorge, southern serbia. the white arrows on each side delimit the additional row of femoral pores biologica nyssana 2 (2) december 2011: 131-133 lazić m.l., crnobrnja-isailović j. accessory femoral pores in podarcis… 133 two populations of common wall lizard from serbia analysed here are not completely isolated from the nearby ones. both human settlements and natural habitats (rocks, gorges) are more or less connected by some sort of corridors (railways, stone piles, edges of magistral roads, traditional houses) that provide opportunities for migration. therefore, abnormal scalation found in studied individuals is more likely to be caused by environmental stress from environmental polution (as pesticide use), than by inbreeding. however, it is important to mention that another two population samples, collected from additional two localities (one urban and one rural), did not contain individuals with accessory femoral pores. our preliminary analises showed that environmental stress impact occur in another urban locality, at least (l a z i ć et al., submitted), but this type of anomaly was not detected. acknowledgements. this work was supported by grant no. 173025 of ministry of education and science of republic of serbia. references arnold, e.n., ovenden, d.w. 2002: a field guide to the reptiles and amphibians of britain and europe. harper collins publishers ltd., london, 288 pp. braña, f., ji, x. 2000: influence of incubation temperature on morphology, locomotor performance, and early growth of hatchling wall lizards (podarcis muralis). the journal of experimental zoology, 286 (4): 422-433. crnobrnja-isailović j., aleksić i., bejaković, d. 2005: fluctuating asymmetry in podarcis muralis populations from southern montenegro: detection of environmental stress in insular populations. amphibiareptilia, 26 (2): 149-158. gasc, j-p., cabela, a., crnobrnja-isailović, j., dolmen, d., grossenbacher, k., haffner, p., lescure, j., martens, h., martinez-rica, j.p., maurin, h., oliveira, m.l., sofianidou, t.s., veith, m. and zuiderwijk, a. (eds), 1997: atlas of amphibians and reptiles in europe. societas europaea herpetologica and museum national d' histoire naturelle (iegb/spn), paris. guillaume, cl.p. 1997: podarcis muralis (laurenti, 1768). in: casc, j.p., cabela, a., crnobrnjaisailovic, j., dolmen, d., grossenbacher, k., haffner, p., lescure, j., martens, h., martinezrica, j.p., maurin, h., oliveira, m.e., sofianidou, t.s., veith, m., zuiderwijk, a. (eds), atlas of amphibians and reptiles in europe, societas europaea herpetologica and museum national d’ histoire naturelle (iegp/spn), paris, p. 286-287. kaliontzopoulou, a., carretero, m.a. 2006: podarcis bocagei (bocage's wali lizard) abnormal scalation. herpetological review, 37(4): 470-471. markow, a.t. 1995: evolutionary ecology and developmental instability. annu. rev. entomol, 40: 105-120 møller, p.a. 1997: developmental stability and fitness: a review. the american naturalist, 149 (5): 916-932. radovanović, m. 1951: vodozemci i gmizavci naše zemlje. beograd (srpsko biološko društvo). tanner, w.w., avery, f.d. 1964: a new sauromalus obesus from the upper colorado basin of utah. herpetologica, 20 (1): 38-42. walker, j.m. 1980: accessory femoral pores in a colony of the collared lizard, crotaphytus collaris in texas. journal of herpetology, 14 (4): 417-418. anticancer compounds from medicinal plants biologica nyssana 2 (2)  december 2011: 00-00 ljupković r.b. et al..  removal cu(ii) ions from water... 37 original article contribution to the knowledge of jumping spiders (araneae: salticidae) from vicinity of jagodina, central serbia boban stanković 1* 1 city goverment of jagodina, department of environmental protection, kralja petra i, no 6, 35000 jagodina, serbia * e-mail: boban.stankovic035@gmail.com abstract: stanković, b.: contribution to the knowledge of jumping spiders (araneae: salticidae) from vicinity of jagodina, central serbia, biologica nyssana, 3 (1), september 2012: 37-42. during last 10 years, based on personal collectings, 21 species from 14 genera of salticidae (araneae) are recorded from vicinity of jagodina: ballus chalybeius, carrhotus xanthogramma, evarcha arcuata, evarcha falcata, heliophanus auratus, heliophanus cupreus, heliophanus flavipes, heliophanus kochii, icius hamatus, icius subinermis, leptorchestes berolinensis, macaroeris nidicolens, marpissa muscosa, marpissa nivoyi, mendoza canestrinii, pellenes tripunctatus, phintella castriesiana, phlegra fasciata, pseudeuophrys erratica, pseudeuophrys lanigera, salticus scenicus. all those species are provided with habitat notes and global distribution. new records for the spider fauna of serbia are heliophanus kochii (simon 1868), icius subinermis (simon, 1937), marpissa nivoyi (lucas, 1846) and mendoza canestrinii (ninni, 1868). key words: salticidae, jumping spiders, jagodina, serbia. introduction the salticidae blackwall, 1841 or jumping spiders, are the most diverse globally distributed spider family mostly tropical, with 5337 species placed in 573 genera (p l a t n i c k , 2011). in the spider fauna of europe, this family is represented with 396 species (h e l s d i n g e n , 2010). the salticid fauna of serbia is not yet fully known. according to d e l t s h e v et al. (2003), only 49 species of jumping spiders have been recorded in serbia. s t a n k o v i ć (2010) reported two species of salticidae as new for the fauna of serbia: icius hamatus (c. l. koch, 1846) and pseudeuophrys lanigera (simon, 1871). in this paper, original faunistic records and habitat notes of 21 species from 14 genera of jumping spiders from vicinity of jagodina with information of globally distribution and zoogeographic analysis are presented. new species records for the spider fauna of serbia are: heliophanus kochii (simon, 1868), icius subinermis (simon, 1937), marpissa nivoyi (lucas, 1846) and mendoza canestrinii (ninni, 1868). materials and methods study area. all specimens of the jumping spiders species were collected in wide vicinity of jagodina (the central part of serbia) (utm ep16,17,26,27; 21°11‘-21°21‘, 43°56‘-44°03‘; altitude range 109-300m). the study area included parts of middle morava valley and southeastern šumadija. it belongs to the peripannonic region of serbia. there are three landscapes types (biomes) in the study area: biome of submediterranean oak woodlands, biome of south european deciduous montane woodlands and biome of south european 3 (1) • september 2012: 37-42 http://www.faunaeur.org/full_results.php?id=182682 http://www.faunaeur.org/full_results.php?id=182682 biologica nyssana 3 (1)  september 2012: 37-42 stanković, b.  contribution to the knowledge… 38 deciduous woods in lowland and inundated areas (m a t v e j e v & p u n c e r , 1989). the climate is moderately continental. according to the relevant meteorological station in ćuprija, the average annual air temperatures are between 11.2 and 11.7°c approximately, and the average annual rainfall is 619 mm. during the eight month period of march-november, the average monthly air temperatures were higher to 10°c. collection, preservation and identification of specimens. the material has been collected mainly during last 10 years by hand, sweep net and pitfall trapping by the author. material was preserved in 70% ethyl alcohol with added glycerin (9:1 by volume) and kept in the author’s collection. determinations, photographs and drawings were made using stereomicroscope (btc stm-1 x20) and light microscope (meopta) from preserved specimens. for new records, photos or original drawings of the male palp or female epigyne are presented. determinations were made mainly according to p r ó s z y ń s k i (1976, 1979, 1997, 2003, 2005), h e i m e r & n e n t w i g (1991), r a k o v & l o g u n o v (1996a), r a k o v (1997), l o g u n o v (1996, 1998b, 1999b), ż a b k a (1997) and m e t z n e r (1999, 2011). the nomenclature follows p l a t n i c k (2011). results and discussion ballus chalybeius (walckenaer, 1802) material examined: 1♂, đurđevo brdo near jagodina, 17.06.2007; 1♂, đurđevo brdo – ćelijan, 2.07.2007; 1♂, đurđevo brdo, 23.04.2010; 1♂, jagodina, 13.05.2011. distribution: europe, north africa to central asia (p l a t n i c k , 2011). habitat: high density shrubs of ligustrum vulgare, prunus spinosa, rosa sp., crataegus monogyna, also urban habitats, gardens (present data). carrhotus xanthogramma (latreille, 1819) material examined: 1♂, panjevački rit, on young leaves of verbascum nigrum, 11.05.2005; 1♀, đurđevo brdo, 4.06.2006; 1♂, road for vill. gornje štiplje, in oak wood, 9.07.2008; 1♂, rit vill.bukovče, 28.05.2009. distribution: amphi-eurasian subborealsubtropical species (l o g u n o v & g u s e i n o v , 2001). habitat: low vegetation, meadows, on oak trunk, shrubs and bushes (present data). evarcha arcuata (clerck, 1757) material examined: 1♂, crni vrh, near vill.gornje štiplje, 14.07.2005; 2♀♀, đurđevo brdo, 5.20.04.2007; 2♀♀, đurđevo brdo, 10.-20.07.2007; 1♂, panjevački rit, 14.05.2008; 1♂, jagodina, graveyard, 5.06.2010. distribution: trans-euroasian temperate species (l o g u n o v & g u s e i n o v , 2001). habitat: low vegetation, bushes, grassy habitat, meadows (present data). evarcha falcata (clerck, 1757) material examined: 1♂, rit-vill.ribnik, 28.05.2009; 1♀, lipar, 4.07.2010. distribution: euro-siberian temperate species (l o g u n o v & g u s e i n o v , 2001). habitat: meadows, grass and bushes in oak woods (quercetum confertae-cerris rudski) (present data). heliophanus auratus (c.l.koch, 1835) material examined: 1♀, jagodina, near r.belica, 1.06.2007; 1♂, rit-vill.ribnik, 28.05.2009; 1♂, glogovačke bare, 17.07.2010. distribution: euro-siberio-central asian temperate species (l o g u n o v & g u s e i n o v , 2001). habitat: wet meadows-grass vegetation, agricultural fields (present data). heliophanus cupreus (walckenaer, 1802) material examined: 1♂, vicinity of jagodina, 3.08.2005; 1♂, đurđevo brdo, 4.06.2006; 1 juv., jagodina, 19.06.2006; 2♂♂, đurđevo brdo, 17.06.2007; 2♂♂, vill.ribare, bank of velika morava, 10.08.2008; 1♀, rit vill. ribnik, r.belica valley, 28.05.2009; 1♂, lipar, 25.04.2010. distribution: euro-caucasian (r a k o v & l o g u n o v , 1996a). habitat: meadows, shrubs, gardens, vineyards, agricultural fields (present data). heliophanus flavipes (hahn, 1832) material examined: 1♀, jagodina – vill. rakitovo, along bank of river lugomir, 19.04.2006; 1♂, vill.bukovče, 28.05.2009; 2♀♀, panjevački rit, 31.05.2009; 1♂ juvenile, vill.končarevo, near r.velika morava, 23.07.2010. distribution: trans palearctic temperate species (l o g u n o v & g u s e i n o v , 2001). habitat: high grass meadows, wet grassy habitat, in the litter of populus woods (present data). biologica nyssana 3 (1)  september 2012: 37-42 stanković, b.  contribution to the knowledge… 39 a b c figure 1. heliophanus kochii, male, dorsal view (a), male palp, ventral (b), lateral view (c). heliophanus kochii (simon 1868), fig. 1. material examined: 1♂, vill.ribare, 30.06.2003; 1♂, jagodina – vill.rakitovo, along bank of river lugomir, 1.05.2005; 1♀, jagodina, near river belica, 2.08.2007; 1♂, vill.končarevo, lake predor, 29.05.2009. distribution: palearctic (p l a t n i c k , 2011). new for serbia. habitat: grassy meadows (present data). icius hamatus (c. l. koch, 1846) material examined: only previous records (stanković, 2010): 1♂, jagodina, 21.07.1998; 1♀, đurđevo brdo near jagodina in dry grassy habitat, 12.06.2005. distribution: widely distributed in mediterranean regions (southern europe, mediterranean islands and north africa) (a l i c a t a & c a n t a r e l l a 1993). habitat: urban habitat, in grass (present data). icius subinermis (simon, 1937), fig. 2. material examined: 1♀, jagodina, 19.06.2004. distribution: presumably western mediterranean range (a l i c a t a & c a n t a r e l l a , 1993), although the species has been found in germany (p l a t n i c k , 2011), slovenia (k o s t a n j š e k & f i š e r , 2005) and switzerland (b l i c k e t a l ., 2004). new for serbia. habitat: i.subinermis favors moist habitats, for example near streams or on moist meadows. it builds a silken retreat in infructescences of rushes or under rocks near rivers or creeks. it will retreat there when the weather is unfavorable (b e l l m a n n , 1997); urban habitat (present data); remarks: the records of mediterranean species i.subinermis, indicates the possibility of the introduction with imported fruits and spread into synanthropic habitats, like second mediterranean species from this genus icius hamatus, recorded in central and west europe (t o m a s i e w i c z & w e s o ł o w s k a , 2006). however, another possibility, may be indicates spread of distribution from macedonia. figure 2. icius subinermis, epigyne. a b figure 3. leptorchestes berolinensis, male palp, ventral (a), lateral view (b). leptorchestes berolinensis (c. l. koch, 1846), fig. 3 material examined: the suburb of jagodina, road for vill.štiplje: (2♂♂, 1♀), 16.05.2002; (1♀, 1 juvenile), 22.06.2002; (1♂, 1♀), 2.07.2003; 1♂, 9.06.2004. biologica nyssana 3 (1)  september 2012: 37-42 stanković, b.  contribution to the knowledge… 40 distribution: europe to turkmenistan (p l a t n i c k , 2011). this is a second records of l.berolinensis in serbia. habitat: sunny walls, tree barks, wood, in grass and fences (present data). macaroeris nidicolens (walckenaer, 1802) material examined: 1♂ juvenile, đurđevo brdo, 23.06.2004; 1♂, bank of velika morava, vill.ribare, 14.06.2005; 1♂ juvenile, jagodina, 13.07.2005; 1♂ juvenile, jagodina – rusko groblje, 5.07.2008; 1♀ juvenile, đurđevo brdo, 28.06.2009. distribution: west palearctic subboreal species (l o g u n o v & r a k o v , 1998), occuring from the canary islands and madeira (w u n d e r h i c h , 1991) to turkmenistan (f e t , 1983). habitat: grassy vegetation, woods (present data). marpissa muscosa (clerck, 1757) material examined: jagodina: 1♀, 12.05.2002; 1♀, 25.04.2004; 2♀♀, 17.08.2006; 1♀, ?.07.2008. distribution: european temperate species (l o g u n o v & g u s e i n o v , 2001). habitat: this species was collected only in urban habitat at shadow and dry places around the houses, external walls of the houses (present data). a b figure 4. marpissa nivoyi, female dorsal view adult (a) and juvenile (b). marpissa nivoyi (lucas, 1846), fig.4. material examined: jagodina: 1♀ juvenile, 11.05.2011; 1♀, 22.05.2011. distribution: south european – central asian subboreal species (l o g u n o v & g u s e i n o v , 2001). new for serbia. habitat: urban habitat: gardens, tree-line, on leave of tilia sp. (present data). mendoza canestrinii (ninni, 1868), fig. 5. material examined: 1♂, rit-vill. ribnik (river belica valley), 3.05.2009. distribution: trans-eurasian subboreal-subtropical species (l o g u n o v & g u s e i n o v , 2001), with the easternmost localities in china (s c h e n k e l , 1963; w e s o ł o w s k a , 1981). new for serbia. habitat: swamp, on leave of iris sp. (present data). figure 5. mendoza canestrinii, male lateral view pellenes tripunctatus (walckenaer, 1802) material examined: 1♀, lipar, 28.05.2003. distribution: euro-siberian temperate species (l o g u n o v & g u s e i n o v , 2001). habitat: in grass of edge of oak woods (present data). phintella castriesiana (grube, 1861) material examined: 1♀, vicinity of jagodina, weeds near road, 11.06.2006; 1♀, jagodina, near r.belica, 26.05.2008; 1♂, vicinity of jagodina, on leaves of dock, 19.04.2011. distribution: amphi-eurasian subboreal species (l o g u n o v & g u s e i n o v , 2001). habitat: in broad-leaved vegetation, weeds (present data). phlegra fasciata (hahn, 1926) material examined: 1♀, đurđevo brdo – ćelijan, in grass, 2.07.2007. distribution: trans-eurasian temperate-subtropical species (l o g u n o v & g u s e i n o v , 2001). habitat: at sunny and dry places, in grass, on stones and on banks of waters (n e n t w i g e t a l ., 2010). pseudeuophrys erratica (walckenaer, 1826) material examined: 1♀, jagodina, 29.04.2002; 1♀, jagodina, near r.belica, 3.06.2004; 1♂, panjevački rit, 17.06.2008; 1♀, đurđevo brdo, 1.07.2008; 1 biologica nyssana 3 (1)  september 2012: 37-42 stanković, b.  contribution to the knowledge… 41 juvenile, jagodina, 23.04.2009; 1♂, rit vill.bukovče, 15.05.2011; 1♂, jagodina, 24.08.2011. distribution: trans-eurasian temperate species (l o g u n o v & g u s e i n o v , 2001). in nearctic found in new jersey (l o g u n o v , 1998b). habitat: lowland grassy vegetation, meadows in the river valley, also urban habitat (present data). pseudeuophrys lanigera (simon, 1871) material examined: jagodina, indoors in my apartment on the 8 th floor: (4 ♀♀ juvenile, 1♀) unknown data; 1♀, ?.09.2002; (1♂, 1♀ juvenile), 04.2003; 1♂, ?.08.2004; (1♀, 1♂), 11.2004; 1♀, 3.10.2009; 1♀ juvenile, 11.03.2010; 1♀, 5.10.2010; (1♂, 1♀), 12.04.2011; 1♂, 7.08.2011. distribution: western, central and southern europe, eastward to the caucasus mts (l o g u n o v 1998b, l o g u n o v & g u s e i n o v , 2001). habitat: synanthropic species, walls of buildings (present data). salticus scenicus (clerck, 1757) material examined: jagodina: 1♀, 12.05.2002; 1♂, 4.08.2003; 1♀, 24.07.2006; 1♂, 7.07.2009; 1♂, vill.bukovče, on wall of school, 21.07.2005; 1♀, đurđevo brdo, 13.05.2007. distribution: holarctic temperate species (l o g u n o v & g u s e i n o v , 2001). habitat: on sunny walls and barks (present data). conclusion total of 21 species from 14 genera of family salticidae are presented. new records for the spider fauna of serbia are: heliophanus kochii (simon 1868), icius subinermis (simon, 1937), marpissa nivoyi (lucas, 1846) and mendoza canestrinii (ninni, 1868). from the results presented in this study, a total of 55 species of salticidae registered in serbia. in the area and vicinity of jagodina registered 38.2% of total. zoogeographical analysis of species found in the region jagodina (fig. 6), it can be concluded that the widespread species (holarctic, palearctic) most represented with 18 species or 86%. the largest number of species with transpalearctic distribution, 8 species or 38,1%, followed by 7 westpalearctic species (33.3%), 3 mediterranean species (14,3%), 2 amphipalearctic species (9.5%), and the 1 holarctic species (4.8%). jumping spiders are grouped into zoogeographical categories according to p r ó s z y ń s k i (1976), l o g u n o v (1992), s z u t s e t a l . (2003). 38,1 33,3 14,3 9,5 4,8 0 5 10 15 20 25 30 35 40 tp al wp al m ed ap al ho l figure 6. – zoogeografical composition of jumping spider fauna (araneae: salticidae) from vicinity of jagodina. (tpal – transpalearctic, wpal – westpalearctic, med – mediterranean, apal – amphipalearctic, hol – holarctic). acknowledgements. i wish to express my warmest thanks to dr. dmitri v. logunov (curator of arthropods, the manchester museum) for reviewing the manuscript and confirmed determinations of the new species records. references alicata p., cantarella t. 1993: the euromediterranean species of icius (araneae, salticidae): a critical revision and description of two new species. animalia, 20(1/3): 111-131. bellmann, h. 1997: spinnentiere europas. kosmosatlas, stuttgart, franckh-kosmos verlags-gmbh & co. blick, t., bosmans, r., buchar, j., gajdos, p., hänggi, a., helsdingen, p., van, ruzicka,v., starega, w., thaler, k. 2004: checkliste der spinnen mitteleuropas (arachnida: araneae). version 1. dezember 2004. http://www.arages.de/checklist.html#2004_aran eae deltshev, c., ćurčić, b. p. m., blagoev, g. a. 2003: spiders of serbia. institute of zoology, belgrade, monographs 7., 832pp. fet, v., ya 1983: the fauna of aranei of the southwestern kapetdagh//ent. obozr. 62(4): 835-845. heimer, s., nentwig, w. 1991: spinnen mitteleuropas. verlag paul parey, berlin & hamburg. helsdingen, p.j. van, 2010: araneae, in: fauna europaea database european spiders and their distribution distribution version 2010.1, online at www.european-arachnology.org. kostanjšek, r., fišer, c. 2005: new records of jumping spiders (araneae: salticidae) for slovenia. natura sloveniae, 7(1): 5-11. http://www.faunaeur.org/full_results.php?id=182682 http://www.faunaeur.org/full_results.php?id=182682 biologica nyssana 3 (1)  september 2012: 37-42 stanković, b.  contribution to the knowledge… 42 logunov, d. v. 1992: preliminary report on the euro-siberian faunal connections of jumping spiders (araneae, salticidae). annales zoologici fennici, 201: 71-76. logunov, d. v. 1996: notes on a jumping spider collection from israel (aranei salticidae). arthropoda selecta, 5(1/2): 55-61. logunov, d. v. 1998b: pseudeuophrys is a valid genus of the jumping spiders (araneae, salticidae). revue arachnologique, 12(11): 109128. logunov d.v. 1999b: redefinition of the genera marpissa c. l. koch, 1946 and mendoza peckham & peckham, 1894 in the scope of the hoalarctic fauna (araneae, salticidae). revue arachnologique, 13(3). 25-60. logonuv, d. v., guseinov, e. f. 2001: faunistic review of the jumping spiders of azerbaijan (aranei: salticidae), with additional faunistic records from neighboring caucasian countries. arthropoda selecta, 10: 243-260. logunov, d. v., rakov, s. y. 1998: miscellaneous notes on middle asian jumping spiders (aranei: salticidae). arthropoda selecta, 7(2): 117-144. matvejev, s., puncer, i. 1989: karta bioma – predeli jugoslavije. prirodnjački muzej, beograd. metzner, h. 1999: die springspinnen (araneae, salticidae) griechenlands. andrias, 14: 1-279. metzner, h. 2011: worldwide database of jumping spiders (arachnida, araneae, salticidae). http://www.jumping-spiders. com/site/ impressum. html nentwig, w., blick, t., gloor, d., hänggi, a., kropf, c. 2010: spiders of europe. www.araneae.unibe.ch. version of access date.araneae spinnen europas identification key to spiders of europe http://www.araneae.unibe.ch/ platnick, n.i. 2011: the world spider catalog, version 11.5. american museum of natural history, online at http://research.amnh.org/ entomology/spiders/ catalog. doi: 10.5531/db.iz. 0001. proszynski, j. 1976: studium systematyczno zoogeograficzne nad rdzina salticidae regionow palearktycznego i nearktycznego. wysza skola pedagogiczna w siedlcach, 6: 260 pp. proszynski, j. (1979): systematic studies on east palearctic salticidae iii. remarks on salticidae of the ussr. annales zoologici warszava, 34(11): 299-369. proszynski, j. 1997: salticidae (araneae) of the world, part i: diagnostic drawings libraryby. http://www.salticidae.org. proszynski, j. 2003: salticidae (araneae) of the levant. annales zoologici warszava, 53(1): 1180. prószyński, j. 2005: catalogue of salticidae (araneae). [http://salticidae.org/salticid/catalog/ thorell.htm]. rakov s.y, 1997: a review of the spider genus evarcha simon, 1902 in middle asia (aranei, salticidae). arthropoda selecta, 6(1/2): 105-112. rakov, s., yu., logunov, d.v. 1996a: a critical review of the genus heliophanus c.l.koch, 1833 from middle asian and the caucasus (aranei, salticidae). arthropoda selecta, 5(3-4): 67-104. schenkel, e. 1963: ostasiatische spinnen aus dem museum d'histoire naturelle de paris. memoires memoirs du museum d'histoire naturelle, 25(1): 1-481. stanković, b. 2010: two new species of salticidae (aranea) for the fauna of serbia. bulletin of the natural history museum, 3: 133-136. szuts, t., szinetar, c., samu, f., szita, e. 2003: check list of the hungarian salticidae with biogeographical notes. arachnologische mitteilungen basel, 25:45-61. tomasiewicz, b., wesolowska, w. 2006: icius hamatus (salticidae, araneae) in poland? polish journal of entomology, 75: 339-342. wesołowska, w. 1981: salticidae (aranei) from north korea, china and mongolia. annales zoologici warszava, 36: 45-83. wunderlich, j. 1991: höhlentiere der kanarischen inseln. mitt. dt. höhl.-karstf, 37(2): 33. żabka m. 1997: salticidae pajaki skaczace (arachnida: araneae). fauna polski 19. muzeum i instytut zoologii pan, warszawa. anticancer compounds from medicinal plants biologica nyssana 2 (2)  december 2011: 00-00 ljupković r.b. et al..  removal cu(ii) ions from water... 1 original article floristic diversity of plants spontaneously spreading in the botanical garden of the university of łódź (poland) anna bomanowska 1 , maria kurzac 1 , agnieszka stefaniak* 1 1 department of geobotany and plant ecology, university of łódź, banacha 12/16, pl-90237 łódź; * e-mail: stefa@biol.uni.lodz.pl abstract: bomanowska, a., kurzac, m., stefaniak, a.: floristic diversity of plants spontaneously spreading in the botanical garden of the university of łódź (poland), biologica nyssana, 3 (1), september 2012: 01-10. the aim of the paper is to characterize plants spontaneously spreading outside the flower beds within the territory of the botanical garden of the university of łódź. floristic studies rivealed that many cultivated species also appear outside their original planting sites. total of 124 taxa of vascular plants were found to be subject of spontaneous spreading. various directions of spontaneous migration of species from sites of their planting were observed within the garden: the “oak-hornbeam lawn”, shaded lawns, sunny lawns, shaded paths, sunny paths, limestone rock garden on hillside. most species occurred exclusively in one of the abovelisted sites. the analysed group of vascular plants includes a predominant representation of native species – 103 species (83.1 %) and predominance of herbal perennial plants – hemicryptophytes (80 species; 67.7 %). key words: botanical garden, migration patterns, plant collection, spontaneous spread introduction there are many reasons for growing plants in modern botanic gardens, including species conservation, scientific research, cultivar development, education, public amenity and pleasure, and cultural heritage (w y s e j a c k s o n & s u t h e r l a n d , 2000, m a u n d e r et al., 2001). historically, their main activities were horticultural and taxonomic research, but the urgent need for biodiversity conservation, biotechnological outreach and education have become an increasingly important focus (k o j s & k o j s , 2004, r y b c z y ń s k i et al., 2004, h a v e n s et al., 2006, c h a n g et al., 2008). for this reason, botanical gardens are areas of intensive cultivation of numerous plants and a key pathway for the introduction of new species. most plant species introduced to a new country or region has originated from ornamental horticulture (d e h n e n s c h m u t z & t o u z a , 2008, v i r t u e et al., 2008, g a l e r a & s u d n i k w ó j c i k o w s k a , 2010). botanical gardens, as well as private flower gardens are often in service to alien taxa as bridgeheads in the process of successful colonisation of new territories (r e i c h a r d & w h i t e , 2001, s m i t h et al., 2006, d e h n e n -s c h m u t z et al., 2007, d a w s o n et al., 2008, v i r t u e et al., 2008, g a l e r a & s u d n i k -w ó j c i k o w s k a , 2010). due to this phenomenon, botanical gardens might influence the plant species inventory that contributes to enrichment of country or regional flora with new, mainly exotic taxa. floristic studies carried out in the teaching and experimental botanic garden of the university of łódź, led to the observation that many cultivated species also appear outside of their original planting 3 (1) • september 2012: 01-10 biologica nyssana 3 (1)  september 2012: 01-10 bomanowska, a. et al.  floristic diversity of plants spontaneously… 2 sites. these observations supplemented by the analysis of data contained in the documentation of garden collections, formed the basis for the preparation of the present work, aiming to characterize those plants that spontaneously spread outside the flower beds within the territory of the university botanical garden. materials and methods the botanical garden of the university of lodz is situated in the centre of lodz (19° 29’ 05” e and 51° 46’ 40” n) and occupies total area of 1.02 ha. the garden does not constitute a separate legal entity, being internally managed by the faculty of biology and environmental protection. the object was established in 1985, and has status of a teaching and experimental garden, i.e. the collections are mainly used for teaching purposes and for scientific research. management duties include the selection of plant material, creation of necessary collections to be used for the education of biology and environmental protection students, and care of aesthetic qualities and the external image of the garden. the garden features herbaceous and ligneous plants, represented by species of both native and foreign origin (s t e f a n i a k & b o m a n o w s k a , 2011). collection of the garden includes 793 taxa: 295 species and varieties of trees and shrubs as well as 498 taxa of herbaceous plants (s t e f a n i a k & b o m a n o w s k a , 2011). the herbaceous collection is spread over 189 cultivation plots, each having an area of 3.75 m 2 . garden collections are a reflection of habitat and taxonomic diversity of the vascular flora of poland. numerous plants of alien origin (mainly mediterranean, american) are also cultivated in the garden. detailed floristic studies were carried out in the garden in the period of 2007-2008. observations were performed both on the cultivation plots and on the dividing paths and in the internal buffer zone of the garden. the present study includes selected data from these floristic observations, with some additional data from the years 2006 and 2009. only those cultivated species, for which individuals where observed to have spontaneously spread outside their original planting sites, were included in the analysis. the relevant individuals occurred outside the actual area of cultivation of the given plant species, on ground with a different land management profile, e.g. on a path, a lawn, in the garden buffer zone etc. in the case of vegetatively reproducing plants, only independent separated rametes were considered as spontaneously occurring individuals (f a l i ń s k a , 2002). biological classification of species was based on the system of raunkiaer’s life forms, with the life form for each species taken from z a r z y c k i et al. (2002) and from the „biolflor” database (http://www.ufz.de/biolflor). the remaining data on the biology of a species such as manner of reproduction, dispersal of diasporas and life strategies were compiled from available sources (v a n d e r p j l , 1972, h a n d e l & b e a t i e , 1990, l i n d a c h e r , 1995, t o k a r s k a -g u z i k , 2005) as well as from the on-line databases (biolflor, floraweb, l i u et al., 2008) and personal observations. the geographical and historical classification of the flora as proposed by k o r n a ś (1981) and modified by j a c k o w i a k (1990). status of species within this classification was determined using the studies of z a j ą c (1979), m i r e k et al. (2002), t o k a r s k a -g u z i k (2005), but it was related to local conditions. due to the history of ground usage and the group of analysed plants (exclusively species escaping from cultivation), all analysed species were treated as synanthropic ones. native species were ascribed the status of oekiophytic apophytes (oekiophytes), i.e. native species escaping from cultivation (s u d n i k -w ó j c i k o w s k a & k o ź n i e w s k a , 1988). species of alien origin, on the other hand, were classified as metaphytes (archaeophytes and kenophytes) if they are permanently naturalised in the polish flora and occur spontaneously in plant communities, or as diaphytes (ergasiophygophytes) if they are exclusively cultivated plants which appear in the spontaneous flora of poland transiently as a result of temporary “escape” from cultivation. the nomenclature of herbaceous vascular plants follows m i r e k et al. (2002) and g a w r y ś (2009), whereas names of woody species are from s e n e t a & d o l a t o w s k i (2000). results in the course of field research in the garden, 124 taxa of vascular plants were found to be subjects of spontaneous spreading, belonging to 47 families and 107 genera (table 1). in distinguished group the most numerously represented families are: asteraceae (12 species) and lamiaceae (11), as well as poaceae (8) and ranunculaceae (8) and rosaceae and apiaceae represented by seven species each (table 1). the majority of families (35) have only one or two species. the analysed group of vascular plants includes a predominant representation of native species, i.e. oekiophytes – 103 species (83.1%). among alien species, ergasiophygophytes (14 species; 11.3%) biologica nyssana 3 (1)  september 2012: 01-10 bomanowska, a. et al.  floristic diversity of plants spontaneously… 3 predominate over metaphytes (7; 5.6%). the comparision of the life form spectrum showed a predominance of herbal perennial plants – hemicryptophytes (80 species; 64.5%) over other life forms. 11 species (8.9%) are chamaephytes. the proportion of woody species (megafanerophytes, nanofanerophytes) and species which are both geophytes and hemicryptophytes is 7.3% for each of these groups. table 1. cultivated taxa spontaneously spreading in the experimental and teaching botanical garden of the university of łódź. abbreviations and symbols: status (affiliation to geographical-historical groups): oe – oekiophyte, me – metaphyte, ar – archaeophyte, ke – kenophyte, er – ergasiophygophyte; life forms: ch – chamaephyte, g – geophyte, h – hemicrytophyte, m – megaphanerophyte, n – nanophanerophyte, li – liana; life strategy: c – competitor, s – stres tolerator , r – ruderal; types of reproduction: s – seed, sv – seed and vegetatively, ssv – mosty by seed, rarely vegetatively, vvs – mosty vegetatively, rarely by seed, v – vegetatively; dispersal mode: ane – anemochory, aut – autochory, anthr – anthropochory, bar – barochory, dys – dyszoochory, end – endozoochory, epi – epizoochory, hyd – hydrochory, myr – myrmecochory; directions of spontaneous migration: ohl – oak-hornbeam lawn, shl – shaded lawns, sul – sunny lawns, shp – shaded paths, sup – sunny paths, lim – limestone rock, hill – hillside. no. species family status l ife fo rm l ife strateg y r ep ro d u ctio n d isp ersal m o d e directions of spontaneous migration o h l s h l s u l s h p s u p l im h il l 1. abies alba l. pinaceae oe m c s ane + + + + + + + 2. acer platanoides l. aceraceae oe m c s ane + + + + + + + 3. acer pseudoplatanus l. aceraceae oe m c s ane + + + + + + + 4. aegopodium podagraria l. apiaceae oe h c vvs aut + 5. agrimonia eupatoria l. rosaceae oe h c s epi + 6. agrimonia procera wallr. rosaceae oe h cs sv epi + 7. ajuga reptans l. lamiaceae oe h csr sv myr, aut + 8. allium ursinum l. liliaceae oe g csr ssv end + 9. angelica archangelica l. subsp. archangelica apiaceae oe h cs s ane, hyd + 10. anthemis tinctoria l. asteraceae me [ar] h cs s aut, epi + 11. anthriscus sylvestris (l.) hoffm. apiaceae oe h c ssv epi, dys + 12. aquilegia x hybrida hort. ranunculaceae er h ? s ? + 13. arrhenatherum elatius (l.) p. beauv. ex j. presl et c. presl poaceae oe h c ssv ane, anthr + 14. asclepias syriaca l. asclepiadaceae me [ke] g, h c sv ane, anthr + 15. astragalus glycyphyllos l. fabaceae oe h c s epi, dys + 16. betonica officinalis l. lamiaceae oe h c sv ? + 17. betula pendula roth betulaceae oe m c s ane, epi + + + + + + + 18. borago officinalis l. boraginaceae er h cr s epi + 19. brachypodium sylvaticum (huds.) p. beauv. poaceae oe h cs sv epi + 20. campanula rapunculoides l. campanulaceae oe h csr sv aut + + + + + + + 21. cardamine pratensis l. brassicaceae oe h csr vvs aut + 22. carex brizoides l. cyperaceae oe g,h csr sv ane, aut + 23. carum carvi l. apiaceae oe h c s dys + 24. centaurea jacea l. asteraceae oe h c s dys, ane, aut + 25. centaurea stoebe l. asteraceae oe h csr s ane, epi, anthr + 26. cerastium tomentosum l. caryophyllaceae er ch c sv ane + 27. cerinthe minor l. boraginaceae me [ar] h csr s ? + 28. chaerophyllum hirsutum l. apiaceae oe h c sv epi, ane + 29. clematis vitalba l. ranunculaceae me [ke] n c s ane, epi, aut + + + + + + + 30. clinopodium vulgare l. lamiaceae oe h cs sv ane, epi, aut + biologica nyssana 3 (1)  september 2012: 01-10 bomanowska, a. et al.  floristic diversity of plants spontaneously… 4 31. convallaria majalis l. liliaceae oe g, h cs sv end, aut, anthr + 32. coronilla varia l. fabaceae oe h c ? epi, ane, aut + 33. corydalis cava (l.) schweigg. et körte fumariaceae oe g csr s aut, myr + 34. corynephorus canescens (l.) p. beauv. poaceae oe h cs s ane + 35. cynoglossum officinale l. boraginaceae oe h cs s epi + + + + + + + 36. deschampsia cespitosa (l.) p.beauv. poaceae oe h c s ane, anthr + + + + + + + 37. dianthus barbatus l. caryophyllaceae er ch ? s ane, anthr + 38. digitalis grandiflora mill. scrophulariaceae oe g, h c sv aut, ane, epi + 39. epiliobium hirsutum l. oenotheraceae oe h c sv ane + 40. epilobium roseum schreb. oenotheraceae oe h cs sv ane + 41. eranthis hyemalis (l.) salisb. ranunculaceae er g csr s ? + + + + + + + 42. euonymus europea l. celastraceae oe n c s end + 43. euphorbia cyparissias l. euphorbiaceae oe g,h csr sv aut, myr + 44. festuca ovina l. poaceae oe h csr s epi, dys, aut, anthr + + 45. ficaria verna huds. ranunculaceae oe g, h csr vvs aut, myr + 46. fragaria vesca l. rosaceae oe h csr sv end, epi, aut + 47. galium mollugo l. rubiaceae oe h c s dys + 48. galium verum l. rubiaceae oe h cs sv epi + + 49. genista tinctoria l. fabaceae oe ch cs s aut + + 50. geranium pratense l. geraniaceae oe h c sv aut, epi + 51. geum urbanum l. rosaceae oe h csr ssv epi + 52. hedera helix l. araliaceae oe n,ch sc sv end + 53. helianthemum nummularium (l.) mill. cistaceae oe ch cs ssv ? + 54. hepatica nobilis schreb. ranunculaceae oe h csr sv aut, myr + 55. heracleum sphondylium l. apiaceae oe h c ssv dys, epi, ane + 56. hieracium pilosella l. asteraceae oe h csr sv ane, aut, epi + 57. hieracium sabaudum l. asteraceae oe h c sv ane + 58. hippophaë rhamnoides l. eleagnaceae oe n c sv end, anthr + + + + + + + 59. holcus lanatus l. poaceae oe h c sv ane, end, anthr + + + + + + + 60. holcus mollis l. poaceae oe g, h csr vvs ane, anthr + + + + + + + 61. humulus lupulus l. cannabaceae oe h, li c sv ane + 62. inula helenium l. asteraceae me [ke] h c sv ane, epi, myr + + 63. junglans regia l. junglandaceae er m c s bar, ane, end + + + + + + + 64. knautia arvensis (l.) j.m.coult. dipsacaceae oe h c s end, epi + 65. lathyrus pratensis l. fabaceae oe h c ssv aut, ane, dys + 66. lathyrus vernus (l.) bernh. fabaceae oe g csr sv aut + 67. leontodon hispidus l. asteraceae oe h csr sv ane + + + + + + + 68. leucanthemum vulgare lam. asteraceae oe h c sv ane, epi, dys + 69. ligustrum vulgare l. oleaceae oe n c s end, anthr, aut + + + + + + + 70. linum perenne l. linaceae me [ke] h cs sv aut, epi, anthr + + 71. lychnis flos cuculi l. caryophyllaceae oe h csr sv ane, epi + 72. lysimachia vulgaris l. primulaceae oe h cs sv ane, epi, hyd, aut + 73. lythrum salicaria l. lythraceae oe h cs ssv ane, epi + + + + + + + 74. mahonia aquifolium (pursh) nutt. berberidaceae er n c sv anthr, end + + + + + + + 75. melica nutans l. poaceae oe g,h cs sv aut, myr + 76. melissa officinalis l. lamiaceae er h c sv ? + 77. mercurialis perennis l. euphorbiaceae oe g,h cs sv aut, myr + 78. origanum vulgare l. lamiaceae oe c,h csr sv ane, aut + + biologica nyssana 3 (1)  september 2012: 01-10 bomanowska, a. et al.  floristic diversity of plants spontaneously… 5 79. ornithogalum umbellatum l. liliaceae oe g csr vvs bar, myr, aut + + + + + + + 80. picea abies (l.) h.karst. pinaceae oe m c s ane, end + + + + + + + 81. pimpinella saxifraga l. apiaceae oe h cs s anthr, epi + 82. plantago media l. plantaginaceae oe h csr sv aut + 83. potentilla micrantha ramond ex dc. rosaceae oe h csr sv ane, aut + 84. primula veris huds. primulaceae oe h csr sv ane + 85. prunella vulgaris l. lamiaceae oe h csr sv hyd, epi, dys, aut + 86. pulmonaria obscura dumort. boraginaceae oe h csr sv myr + 87. pulmonaria officinalis l. boraginaceae oe h csr sv myr + 88. pyracantha coccinea m. roem. rosaceae er n ? ? end + + + + + + + 89. quercus robur l. fagaceae oe m c s ane, end, bar + + + + + + + 90. ranunculus cassubicus l. ranunculaceae oe h ? sv epi + + 91. ranunculus lanunginosus l. ranunculaceae oe h cs sv epi + 92. rudbeckia laciniata l. 'gold kugel' asteraceae me [ke] h c sv ane, aut, epi, myr + 93. rumex acetosa l. polygonaceae oe h c sv ane, epi, dys, hyd + + + + + + + 94. salvia sclarea l. lamiaceae er h ? ? aut + + + + + + + 95. salvia verticillata l. lamiaceae oe h csr ssv aut + + 96. sanguisorba minor scop. rosaceae oe h csr ssv ane, hyd, aut + 97. scabiosa ochroleuca l. dipsacaceae oe h csr s ane + + 98. scilla sibirica haw. liliaceae er g csr sv aut, myr, anthr + 99. scorzonera hispanica l. asteraceae er h csr s ? + 100. scropularia nodosa l. scrophulariaceae oe h cs sv ane, epi, aut + + 101. sedum acre l. crassulaceae oe ch s sv hyd, myr, aut + 102. sedum reflexum l. crassulaceae oe ch s sv hyd, myr, aut + 103. sedum sexangulare l. crassulaceae oe ch s sv hyd, myr, aut + 104. solanum dulcamara l. solanaceae oe n, li c sv end, epi, aut + 105. stachys recta l. lamiaceae oe h csr s ? + 106. stachys sylvatica l. lamiaceae oe h cs sv epi, ane, hyd, aut + 107. tanacetum balsamita l. asteraceae er h c sv ane, epi, anthr + 108. taxus x media rehder taxaceae er n ? ? end + + + + + + + 109. thalictrum minus l. ranunculaceae oe h cs sv ane, epi, hyd + + + + + + + 110. thymus kosteleckyanus opiz lamiaceae oe ch csr s aut + + 111. thymus pulegioides l. lamiaceae oe ch csr ssv aut, myr + + 112. tilia cordata mill. tiliaceae oe m c ssv ane + + + + + + + 113. tragopogon orientalis l. asteraceae oe h csr s ane, epi + 114. trifolium fragiferum l. fabaceae oe h csr ssv ane, epi + 115. ulmus levis pall. ulmaceae oe m c ssv ane + + + + + + + 116. valeriana officinalis l. valerianaeae oe h c sv ane, epi, hyd, aut + + + + + + + 117. verbascum densiflorum bertol. scrophulariaceae oe h c s ane, epi + 118. verbascum nigrum l. scrophulariaceae oe h c s ane, epi + 119. verbascum phlomoides l. scrophulariaceae oe h c s ane + 120. veronica teucrium l. scrophulariaceae oe t c s ane, aut, hyd, anthr + + 121. vinca minor l. apocynaceae oe ch cs sv aut, myr, anthr + 122. vincetoxicum hirundinaria medik. asclepiadaceae oe h cs sv ane, aut + 123. viola reihenbachiana jord. ex boreau violaceae oe h csr sv aut, myr + 124. viola riviniana rchb. violaceae oe h csr sv myr + biologica nyssana 3 (1)  september 2012: 01-10 bomanowska, a. et al.  floristic diversity of plants spontaneously… 6 as many as 49 escaped species are plants with competitive strategy. quite a large group (25 species) are species whose competitive abilities are limited by the stress (cs strategy). as many as 40 are species with the mixed, csr strategy. only one species (borago officinalis) represents the cr strategy. the distinguished group of escaped plants the most numerous are species that reproduce by seeds (40 species; 32.3%) or both by seeds and during vegetatively (75; 60.5%). only 5 species prefer vegetative reproduction: aegopodium podagraria, cardamine pratensis, ficaria verna, holcus mollis, ornithogallum umbellatum (table 1). the group of spontaneously spreading species in the garden is very diverse with respect to different ways of spreading. anemochorous plants prevail over others – 54 species (43.5%). the share of myrmeko-, baroand autochorous species is also significant. most species use several ways of dispersal. the migration of species appears to be multidirectional (fig.1, table 1). various directions of spontaneous migration of species from sites of their planting were observed within the garden: the “oakhornbeam lawn”, shaded lawns, sunny lawns, shaded paths, sunny paths, limestone rock garden on the hillside. most species (93; 75%) occurred exclusively in one of the above-listed sites. among abovementioned species, most have chosen shaded places, such as lawns, paths and “oak-hornbeam lawn” (43 species; 34.7%). escaped species appeared also quite numerously on the sunny paths and lawns (37 species; 29.8%; fig.1). only 13 species (10.5%) occurred spontaneously in limestone rock garden and hillside. there was also a relatively numerous group of plants (28 species; 22.6%) which spread all over the territory of the garden discussion the phenomenon of spontaneous spread of cultivated species, observed in the botanical garden of the university of łódź, confirms the tendencies observed in other botanical gardens (r e i c h a r d & w h i t e , 2001, g a l e r a , 2003, g a l e r a & s u d n i k -w ó j c i k o w s k a , 2004, 2010, d a w s o n et al., 2008, v i r t u e et al,. 2008). even though the number of plants escaping from cultivation is much smaller there (124) than in other gardens in poland (g a l e r a , 2003), this stems probably from the smaller size and less rich collection of the garden in łódź. still, on the local scale the “escaped species” flora is relatively rich, since it constitutes 15.5% of the whole plant collection in the garden. characteristic features of the described group are determined by the specific range of plants cultivated in the particular garden as well as biological propensities of some species for spreading. the influence of these local factors is figure 1. directions of spontaneous migrations of cultivated plants in the university botanical garden. abbreviations: see table 1. biologica nyssana 3 (1)  september 2012: 01-10 bomanowska, a. et al.  floristic diversity of plants spontaneously… 7 reflected both in the taxonomic structure and in the ecological characteristics of the investigated group of species. the high frequency of escaping plants from poaceae and asteraceae families is related to the fact that the majority of the herbaceous collection in the garden belongs to these families, like the majority of species in the polish flora (s t e f a n i a k & b o m a n o w s k a , 2011). moreover, biological characteristics of many species from these families (i.a. the ability to use various dispersal routes) predisposes them to spread and to occupy new diverse habitats (m i z i a n t y , 1995; p y š e k , 1997). the biological spectrum with its preponderance of hemicryptophytes is also a reflection of the species composition of polish flora and selection of species cultivated in the garden (z a r z y c k i et al., 2002, s t e f a n i a k & b o m a n o w s k a , 2011). the collection of herbaceous plants is composed mainly of perennials and a small number of biannual species. the lack of therophytes among spontaneously spreading plants may be explained by the fact that few plants from this group are cultivated in the university garden, with most of them being cultivars which produce no seeds. in other investigated polish botanical gardens this is numerous group of runaway species (g a l e r a & s u d n i k -w ó j c i k o w s k a , 2004). the most numerous group of the described flora is oekiophytes (native escaped species). the cultivated flora of university garden is also represented mainly by this group, while in the remaining botanical gardens in poland there is similar share of two groups: native species and ergasiophygophytes (g a l e r a & s u d n i k w ó j c i k o w s k a , 2004). it is probably because the garden collection is composed mainly of native species (s t e f a n i a k & b o m a n o w s k a , 2011). distribution of grime’s life strategies showing a similar share of species representing ctype and csr-type strategies in the described flora reflects the habitat conditions in the garden which is an anthropogenically transformed area, but with stable ecological conditions (with homogeneous manners of exploitation). this conclusion may be supported by the preference for generative reproduction shown by spontaneously spreading species, even though it requires significantly more ecological effort than vegetative reproduction (f a l i ń s k a , 2002). most plants that spread spontaneously in the university botanical garden uses several different ways of dispersion of propagules. the diversity of strategies helps species to spreadp0’’’, migrate and settle on new habitats (v a n d e r p i j l , 1972, k o l l m a n n , 2000, s o o n s & o z i n g a , 2000, v i t t o z & e n g l e r , 2007). composition of the described group is clearly dominated by anemochorous plants, including species from asteraceae and poaceae families, as well as some trees (e.g. maples). these results are in agreement with those obtained for warsaw botanical gardens (s u d n i k -w ó j c i k o w s k a & g a l e r a , 2005). species disseminated by the wind spread in different directions: the lawns and paths, both sunny and shady, and on limestone hills. in case of many plant species the main vector which spreads propagules are animals, including birds (a d a m o w s k i & k n o p i k , 1996, g o s p e r et al., 2005 and literature cited therein). garden of the university is a food source and resting site for many bird species, which eat the fleshy fruits of many species (e.g. taxus x media, euonymus europaea, mahonia aquifolia, pyracantha coccinea), thereby contributing to their spread. plants with seeds that have prehensile trichomes or hooks (e.g. agrimonia officinalis, borago officinalis) are spread around within the garden by mammals, especially by rodents, but also by house cats (own observations). seeds of species such as corydalis cava, scilla sibirica, viola reichenbachiana, v. riviniana, as well as some plants from asteraceae and lamiaceae families, which have elaiosome bodies, are spread by ants (v a n d e r p i j l , 1972, h a n d e l , 1990, g o r b et al., 2000). anthropochory is also of significant importance in spread of plants (f a l i ń s k i , 1972, v i t t o z & e n g l e r , 2007). the unintentional transport of diaspores of in the garden takes place both during gardening procedures and while teaching field classes with groups of students. in this manner, the following species may have spread: arrhenatherum elatius, centaurea stoebe, cynoglossum officinale. species spreading in zoochoric or anthropochoric way, most often spread on paths – places where most visitors walk, as well as living and penetrating it occasionally animals. among native species which spontaneously spread in the university garden particular speciallz significant is very rare species, e.g. potentilla micrantha, a vulnerable species (vu) included in the polish red data book of plants (k a ź m i e r c z a k o w a & z a r z y c k i , 2002). it is a characteristic species of broadleaf forests, and for this reason it has been planted in the most shaded site, the so-called “oak-hornbeam lawn”. secondary localities of this plant appeared spontaneously on lawns in other parts of the garden. this is another biologica nyssana 3 (1)  september 2012: 01-10 bomanowska, a. et al.  floristic diversity of plants spontaneously… 8 case of the positive function of botanical gardens in aiding the spread of plants. this escape of species from flower beds in the garden can be dangerous to the native flora if it contributes to expansion of invasive species (r e i c h a r d & w h i t e , 2001, d e h n e n s c h m u t z et al., 2007, d a w s o n et al., 2008, k ř i v á n e k & p y š e k , 2008, g a l e r a & s u d n i k -w ó j c i k o w s k a , 2010). it seems that this is not a real and present danger in the described case, since among the plants which appear spontaneously in the garden, few show any tendencies towards expansion or invasiveness. potentially, this description may fit only to asclepias syriaca and carex brizoides, two species which have a high reproductive potential, which grow quickly and reproduce both via seeds and vegetatively (l i u et al., 2008; biolflor database). the common milkweed is a new arrival in our flora (1872 – first record in polish territory; t o k a r s k a g u z i k , 2005), introduced as an ornamental plant, which has in recent times spread rather quickly in anthropogenic habitats (t o k a r s k a -g u z i k , 2005). in the university garden, individuals of the milkweed have also been observed to quickly occupy new habitats. the quaking grass sedge, although native to our flora, is considered an invasive species and an indicator of degeneration of oak-hornbeam forest phytocoenoses (c h m u r a & s i e r k a , 2007). the potential threat to the local flora may be derived from spontaneously spreading cultivars that are able to hybridise with native species (j a c k o w i a k , 1999). invasive species can show a number of patterns of hybridization with native species, from hybridization without introgression to complete admixture (r h y m e r & s i m b e r l o f f , 1996, a l l e n d o r f et al., 2001). among the species described here, this type of danger may be expected from the hybrid taxon aquilegia x hybrida. this plant undergoes intense generative reproduction and has a tendency to spread quickly, so that cultivating it in the vicinity of natural localities of the original native species aquilegia vulgaris carries the risk of introgressive hybridisation (g a l e r a , 2003). even though a realistic evaluation of the potential of these two species for further expansion is very difficult since the colonisation success of an alien species depends on numerous factors that are often difficult to predict (h e g e r & t r e p l , 2003, h i e r r o et al., 2005, s h a r m a et al., 2005), their spontaneous spread in the garden should not be treated too lightly. it is difficult to predict whether the spontaneous escape of cultivated plants from sites of cultivation within the łódź university botanical garden may be the beginning of their further spread outside its boundaries. the time course of observations was relatively short and it is unknown how long the escaped plants may persevere in the new sites where they grow, especially since most taxa were characterised by low frequency of occurrence. conclusion taking into account the composition of group of escaped plants (predominance of native species, high number of ergasiophygophytes among the alien species) and location of the garden in the centre of a major city, further spontaneous spread of described species seems rather 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(anacardiaceae) in north africa original article zahra robã bouabdelli centre de recherche en agropastoralisme (crapast), djelfa, algeria bzahraroba@gmail.com (corresponding author) abdelghafour doghbage centre de recherche en agropastoralisme (crapast), djelfa, algeria safia belhadj ziane achour university, faculty of science of nature and life, djelfa 17007, algeria hafidh zemour ista, university of larbi ben m’hidi, oum el bouaghi, algeria fathi abdellatif belhouadjeb centre de recherche en agropastoralisme (crapast), djelfa, algeria received: may 19, 2025 revised: october 16, 2025 accepted: october 21, 2025 abstract: arbuscular mycorrhizal fungi (amf) have a vital role in natural ecosystems, intensifying nutrient cycling, plant competition, and species interactions. these microbes establish symbiotic relationships with a multitude of plant species, promoting optimal nutrient absorption and plant development. this study allowed the characterization of the mycorrhizal status of pistacia terebinthus in algeria, where we chose two forests located in djelfa to carry out the sampling, senelba and guetia. four parameters were estimated: infection frequency (f%), colonization intensity (m%), arbuscular content (a%), and vesicular content (v%) to calculate the colonization rate of mycorrhizal fungi in the fine roots of this species. in addition, the physical and chemical soil parameters were analyzed: ph, electrical conductivity, granulometric, calcium carbonate, and organic matter percentage. results showed that the roots of the p. terebinthus trees are colonized by arbuscular mycorrhizal fungi (amf). mycorrhization parameters revealed a frequency of colonization (f%) of up to 92%, mycorrhizal intensity (m%) reaching 19.7%, abundance of arbusculars (a%) and vesicles (v%) in the mycorrhizal parts of root fragments (respectively: 15.5% and 11%). physical and chemical analysis of its ground notes that these soils have an alkaline ph, unsalted, rich in organic matter, and low in total calcium carbonate. a total of 7 species belonging to five genera, three families, and two orders of glomeromycota were detected in the soil samples. these results indicate that there is a significant relation between this species and mycorrhizal fungi. for future research, isolating and using high-performing strains of arbuscular mycorrhizal fungi (amf) identified in the semi-arid forests of algeria, it would be possible to develop effective mycorrhizal inoculants. such an approach would not only enhance the native p. terebinthus-amf associations but would also contribute to the sustainable cultivation of pistachios in arid and semi-arid regions. key words: arbuscular mycorrhizal fungi, north africa, pistacia terebinthus, seasons, soil apstrakt: prostorno-vremenska varijacija endomikorize između dve populacije pistacia terebinthus l. (anacardiaceae) u severnoj africi arbuskularne mikorizne gljive (amf) imaju ključnu ulogu u prirodnim ekosistemima, jer intenziviraju kruženje hranljivih materija, konkurenciju među biljkama i međusobne interakcije vrsta. ovi mikroorganizmi uspostavljaju simbiotske odnose sa brojnim biljnim vrstama, čime podstiču optimalnu apsorpciju hranljivih materija i rast biljaka. ovim istraživanjem izvršena je karakterizacija mikoriznog statusa vrste pistacia terebinthus u alžiru, pri čemu su za uzorkovanje odabrane dve šume u oblasti dželfa – senelba i guetia. procijenjena su četiri parametra: učestalost infekcije (f%), intenzitet kolonizacije (m%), sadržaj arbuskula (a%) i sadržaj vezikula (v%), kako bi se izračunala stopa kolonizacije mikoriznih gljiva u finim korenovima ove vrste. pored toga, analizirani su fizičko-hemijski parametri zemljišta: ph, električna provodljivost, granulometrijski sastav, sadržaj kalcijum-karbonata i procenat organske materije. rezultati su pokazali da su korenovi stabla p. terebinthus kolonizovani arbuskularnim mikoriznim gljivama (amf). parametri mikorizacije ukazuju na učestalost kolonizacije (f%) do 92%, intenzitet mikorize (m%) od 19,7%, te prisustvo arbuskula (a%) i vezikula (v%) u mikoriznim delovima korenovih fragmenata (odnosno 15,5% i 11%). fizičko-hemijska analiza zemljišta pokazala je da ova tla imaju alkalan ph, nisu zasoljena, bogata su organskom materijom i sadrže mali procenat ukupnog kalcijum-karbonata. u uzorcima zemljišta identifikovano je ukupno sedam vrsta koje pripadaju pet rodova, tri porodice i dva reda iz koljena glomeromycota. ovi rezultati ukazuju na značajnu povezanost između ove biljne vrste i mikoriznih gljiva. u budućim istraživanjima, izolovanjem i korišćenjem visokoperformansnih sojeva arbuskularnih mikoriznih gljiva (amf) identifikovanih u polusušnim šumama alžira, moguće je razviti efikasne mikorizne inokulante. takav pristup ne bi samo unapredio prirodne asocijacije p. terebinthus–amf, već bi doprineo i održivoj kultivaciji pistacija u sušnim i polusušnim regionima. ključne reči: arbuskularne mikorizne gljive, severna afrika, pistacia terebinthus, sezone, zemljište © 2025 bouabdelli et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original introduction the cultivation of pistachio (pistacia vera l.) has gained increasing attention due to the fruit’s proven antioxidant and anti-inflammatory properties, making it valuable both nutritionally and economically (ağlar et al., 2020). pistachio fruits are rich in fibers, minerals, and unsaturated fats. however, its annual yield often fluctuates, influenced by the selection of appropriate rootstocks and varying weather conditions (aznarte-mellado et al., 2014). rootstocks play a critical role in the growth, development, and quality of pistachio trees, affecting yield, precocity, and nutrient uptake (ozturk & ozturk, 2014; yıldız et al., 2018). pistachios are economically propagated by grafting onto pistacia terebinthus l. trees, whose adaptability to diverse ecological conditions enhances species resilience (moriana et al., 2018). a crucial factor in pistachio cultivation is the symbiotic relationship between plants and arbuscular mycorrhizal fungi (amf). mycorrhizae improve nutrient uptake, particularly nitrogen and phosphorus, which are otherwise inaccessible to plants (genre et al., 2023; ioanna & stavropoulos, 2023). amf also play a protective role by reducing disease incidence in host plants through mechanisms such as competition for carbon sources, pathogen inhibition, and the activation of plant biochemical and molecular defenses (tian et al., 2021; balestrini et al., 2015). this partnership not only enhances nutrient acquisition but also increases the pistachio tree’s resilience in various climates and soils, ultimately contributing to sustainable agriculture practices. in exchange, it receives the plant’s reduced carbon (luginbuehl, 2017; oldroyd & downie, 2017). both partners benefit from this mutual exchange, which helps to boost plant productivity. these responses enhance plant performance, photosynthetic efficiency, and biomass production under abiotic stress. amf helps maintain resilient ecosystems by improving soil structure, nutrient cycling, and carbon sequestration (menge, 2023). biotic and abiotic constraints in semi-arid mediterranean ecosystems have significant impacts on crop yields and agricultural sustainability. understanding interactions between amf and plant is crucial for effective conservation strategies aimed at preserving biodiversity and ecosystem services provided by natural habitats. the field of arbuscular mycorrhizal fungi (amf) and their ecological role in agricultural ecosystems has experienced notable advancements in recent years. nevertheless, despite these advancements, numerous unanswered questions persist, compelling the need for additional research to delve further into the intricacies of amfmediated interactions. it is widely recognized that amf can have an impact on plant competition and promote the coexistence of various species (wang et al., 2022). the study reported here aimed to conduct a detailed examination of the seasonal variation of amf root colonization of pistacia terebinthus, the relationship between edaphic factors, and amf spore morphological identification. two natural forest study sites in djelfa, algeria, were selected for this study. this is the first comprehensive study of the mycorrhization of p. terebinthus in these regions. the results of this study could give us an idea of the behavior of p. vera rootstock for the purpose of growing them in semi-arid regions. materials and methods study area the present study was conducted in the province of djelfa, located in north-central algeria. within this region, two forest sites were selected: guetia (34°33′n, 2°48′e) and senelba (34°53′n, 3°03′e). root samples were of pistacia terebinthus were collected in 2014. both sites represent typical forest ecosystems of the semi-arid zone. meteorological data for the studied period were obtained from the national office of meteorology (onm, djelfa). to define the bioclimatic stage of these forests, climatic parameters such as maximum temperature (mt), minimum temperature (mt), and rainfall were analyzed over a 14-year period (1990–2014). the main geographical and ecological characteristics of these forests are summarized in tab. 1. roots material, collection and preparation of roots roots were collected concurrently with soil samples at a depth of 20 cm. for each station, five distinct pistacia terebinthus individuals were randomly selected. surface litter was removed, and samples were taken by digging in three locations around each tree, arranged in a triangular pattern with angles at least one meter apart to ensure a representative sampling of the root system. the collected samples biologica nyssana ● 16 (2) december 2025: bouabdelli et al. ● spatiotemporal variation of endomycorrhizae among two populations of pistacia terebinthus l. (anacardiaceae) in north africa latitude longitude altitude (m) mt (°c) mt (°c) p (mm/an) q3 bioclimatic levels guetia 34°33’n 2°48’e 1320 33.21 -0.44 336.1 33.05 semi-arid (cold winter) senelba 34°53’n 3°03’e 1363 32.91 -0.63 344.7 35.25 semi-arid (cold winter) table 1. determination of the bioclimatic levels of the study sites mt: maximum temperature, mt: minimum temperature, p: pluviometry, q: pluviothermic quotient biologica nyssana ● 16 (2) december 2025: bouabdelli et al. ● spatiotemporal variation of endomycorrhizae among two populations of pistacia terebinthus l. (anacardiaceae) in north africa were placed in plastic bags and transported to the laboratory. sampling was conducted during the spring, summer, and autumn seasons. endomycorrhizal coloration the harvested roots are gently washed in a beaker on a shaker to remove adhering soil particles. the modified method of philips & hayman (1970) is used to highlight the light microscope mycorrhizal infection. the roots are cut into pieces about 1 cm long, placed in a 10% potassium hydroxide (koh) solution, and then oven-dried at 90 °c for 1 hour. to remove the rest of the pigments, the roots were transferred to a 10% solution of h2o2 at 90 °c for 20 minutes until the roots turned white. the roots are rinsed several times with water, passed through a 10% lactic acid bath, and then transferred to a trypan blue solution, which is placed in the oven at 90 °c for 1 hour. after removing the roots from the dye solution, they were rinsed with tap water and placed in a petri dish containing glycerol. estimation of mycorrhization colonization root fragments are carefully crushed and observed by light microscopy at magnifications of 100 and 400. several scoring systems are used to assess the importance of mycorrhizal infection. the system proposed by trouvelot et al. (1986) was adopted in this study. using this method of colonization, five key parameters were calculated as follows: frequency of mycorrhizal colonization (f%): this represents the percentage of root fragments colonized by endomycorrhizae, indicating the extent of root system colonization. intensity of mycorrhizal colonization in the root system (m%): this measures the degree of colonization within the root system, providing insight into the depth of the symbiotic relationship. abundance of arbuscules in the root system (a%): this parameter reflects the proportion of the root system containing arbuscules, which are specialized structures formed by mycorrhizal fungi for nutrient exchange. abundance of vesicles in the root system (v%): this measures the presence of vesicles, storage structures formed by the fungi, within the root system. these parameters help assess the overall extent and functional efficiency of mycorrhizal colonization in the sampled roots. calculation of these parameters was performed using the software program mycocalc available at www.dijon.inra.fr (invam). physico-chemical analyses of soils we collected approximately 1 to 2 kg of soil around p. terebinthus roots in 3 different places (from a depth of 20 cm). soils were taken to the laboratory for physico-chemical analysis. we used the methods described by aubert (1978) in his book “method of soil analysis.” we analyzed: ph and electrical conductivity (ec) on a soil-water suspension (1/5), granulometric composition, calcium carbonate content (tcaco3), and organic matter percentage. statistical analysis statistica version 10 software was used to conduct descriptive statistics. subsequently, the assessed parameters were analyzed for significant differences using analyses of variance (anovas) based on the specified factors and their interactions. all anovas were conducted with a significance level set at p=0.05. this was followed by principal component analysis (pca). results and discussion mycorrhizal fungi observation of the roots through the binocular magnifying glass reveals the absence of a fungal mantle. the roots are slightly transformed, slightly swollen at the end, and have absorbent hairs on their surface. nevertheless, at the guetia station (spring season), we noticed the presence of some structures origin of the deformation of the rootlets in some trees (3 trees), this can be due to a colonization of other fungi forming ecto-endomycorrhiza (double colonization) or to rhizo-endophytes (mycoendophytes) which can generate ecto-mycorrhizoidal structures (fig. 1). two forms were recorded during our observations: the simple form characterized by a swelling of the lateral rootlets at the end (fig. 1, a and b) and the coralloid or dichotomous form characterized by the branching of the lateral rootlets (fig. 1, c and d), which are surrounded by an extra-matrix network of mycelial cords (branched aggregates of hyphae) (fig. 1, d and e). microscopic observations of root fragments of p. terebinthus, collected during the three seasons (summer, autumn, and spring) and crushed and stained with trypan blue, allowed us to highlight the presence of arbuscular mycorrhizae at the studied stations. this type of mycorrhiza is characterized by the presence of three different fungal structures: hyphae, vesicles, and arbuscules (fig. 2, a and b). the different characteristics of the three structures are presented in tab. 2. tab. 3 presents the mycorrhization parameters of pistacia terebinthus at two forest stations, senelba and guetia, across three sampling seasons. overall, biologica nyssana ● 16 (2) december 2025: bouabdelli et al. ● spatiotemporal variation of endomycorrhizae among two populations of pistacia terebinthus l. (anacardiaceae) in north africa p. terebinthus exhibited a very high infection frequency (f%), averaging 92%, which indicates that arbuscular mycorrhizal fungi (amf) are well established in its rhizosphere. the frequency reached 100% at senelba for all seasons, while guetia showed slightly lower and more variable values (80–92%). this confirms a strong spatial influence, with senelba providing more favorable edaphic or climatic conditions for amf colonization. the intensity of colonization (m%) was moderate overall (mean=19.7%) and significantly higher at senelba (26%) than at guetia (13%), suggesting denser intraradical fungal development in senelba soils. seasonally, fig. 1. stereomicroscope observation of roots (r) morphology of pistacia terebinthus (goutia forest spring): a-b: simple mycorrhiza sm, with root hairs rh, c: coralloid or dichotomous mycorrhiza dm, d-e: fungal coat fc. vesicules arbuscules hyphae two shapes oval (+++) round (++) irregular two locations intracellular (++) extracellular varied size large (+) small arum type «senelba and guetia» (+++) paris type «guetia» (+/-) thick siphoned intercellular hyphae forming a skeleton and much branched (+++) coiled thick intracellular hyphae (pelotons) (+) hyphae with arbuscules or vesicles at their ends (+) table 2. different amf structures observed in p. terebinthus in the two stations colonization intensity was greatest in summer (≈30%) and lowest in autumn (≈7-18%), reflecting the positive effect of warmer, drier conditions on fungal activity and plant dependence on the symbiosis. the arbuscular content (a%), representing functional exchange structures, followed a similar trend—higher at senelba (up to 25%) than at guetia (≈15%)—with maximum development during spring and summer, indicating periods of active nutrient transfer between the host and the fungus. the vesicle content (v%), which reflects storage structures within roots, was also more pronounced at senelba (up to 18%) than at guetia (≈10%), particularly in summer and autumn. this suggests enhanced fungal storage activity under seasonal water stress. statistical analysis revealed that both station and season factors influenced mycorrhizal parameters, with senelba consistently showing higher values. these results demonstrate that p. terebinthus maintains a strong and functionally diverse amf association, modulated by environmental conditions, especially temperature and soil moisture. identification of amf of p. terebinthus the morphological identification of the spores allowed us to observe various morphotypes that differ among themselves in size, color, and shape (rounded or oblong) (fig. 3). a total of 7 species belonging to five genera, three families, and two orders of glomeromycota were detected in the soil samples. two species for each of the genera glomus and gigaspora, one species for each of the genera septoglomus, funneliformis, and acaulospora. two major families have been listed, glomeraceae (glomerales) and gigasporaceae (diversisporales) (tab. 4). shannon’s diversity index varies between 1.04 and 1.61 and follows the guetia ˃ senelba gradient. despite the climatic homogeneity of the two stations (cool semi-arid). the guetia forest is richer in amf species. biologica nyssana ● 16 (2) december 2025: bouabdelli et al. ● spatiotemporal variation of endomycorrhizae among two populations of pistacia terebinthus l. (anacardiaceae) in north africa fig. 2a. microscopic observations of stained root of pistacia terebinthus showing different mycorrhizal structures. a–b: the hartig network hn formed between cortical cells’ parenchyma illustrates the intercellular colonization typical of ectoendomycorrhizal associations (guetia forest, spring), c: fungal hyphae h, extending through the root tissues (guetia forest, spring). d: coiled structure c observed within cortical cells parenchyma, indicating an intracellular fungal interaction (senelba forest, autumn). e–f: developed arbuscules a inside the cortical cells of the parenchyma, characteristic of arbuscular mycorrhizal symbiosis. magnification (x 40). fig. 2b. microscopic observations of the stained root of pistacia terebinthus showing vesicular structures formed during arbuscular mycorrhizal colonization: a–d: intracellular vesicles iv, localized within the cortical cell parenchyma ccp, indicating lipid or nutrient storage structures characteristic of mature arbuscular mycorrhizal symbioses. e–f: extracellular vesicles ev, located between cortical cells. a and b: guetia forest, summer, c and e: senelba forest, summer, d: senelba forest, autumn, f: senelba forest, spring. magnification (x 40). parameters season frequency of infection f (%) intensity of colonization m (%) arbuscular content of infection a (%) vesicle content of infection v (%) senelba (s) spring 100±0 28.8±20.5 10.0±4.2 6.1±2.9 summer 100±0 30.9±12.1 25.5±12.9 18.7±15.5 autumn 100±0 18.7±10.4 13.5±9 10.3±7.6 mea. s±sd 100ns±0 26.1ns±14.9 16.3ns±11.1 11.2ns±10.2 guetia (t) spring 80±44.7 16±9.6 30.4±36.7 22.8±33.4 summer 92.0±17.9 16.4±14.5 11.6±14 7.9±10.3 autumn 80±44.7 7.4±4.8 2±1.5 1.4±0.9 mea. s±sd 84ns±35.6 13.3*±10.6 14.6ns±24.3 10.7ns±20.9 season factor mea. e±sd 92ns±26.1 19.7ns±14.3 15.5ns±18.6 11ns±16 station factor mea. e±sd 92ns±26.1 19.7*±14.3 15.5ns±18.6 11ns±16 season-station interaction mea. e±sd 92ns±26.1 19.7ns±14.3 15.5ns±18.6 11ns±16 table 3. mycorhization parameters of p. terebinthus (senelba and guetia) mea. s±sd: mean station, mea. e±sd: mean species, mea. g±sd: mean genus, sd: standard deviation, statistical significance level at p=0.05, * p<0.05, ** p<0.01, *** p<0.001, ns: no significant biologica nyssana ● 16 (2) december 2025: bouabdelli et al. ● spatiotemporal variation of endomycorrhizae among two populations of pistacia terebinthus l. (anacardiaceae) in north africa physico-chemical analysis of the soils the average ph of the two stations is 8.2 (alkaline soil). a higher value (8.4) is recorded at senelba (with 8.0 for guetia). the three seasons, spring, autumn, and summer, have ph-water values between 8.4, 8.2, and 8.1, respectively. for electrical conductivity, the average rate is 0.2 ms/cm. the soils are non-saline, with electrical conductivity values of 0.23 ms/cm in senelba and 0.15 ms/cm in guetia. ecs vary little between seasons (0.22, 0.21, and 0.15 ms/cm for spring, summer, and autumn, respectively). for organic matter, the two stations are rich (7.9% for guetia and 4.8% for senelba, with an average of 6.3%). spring and summer have high rates of 8.4% and 7.8% respectively, while autumn is the season when this rate is low (2.8%). the average total limestone value is 10.6% for both stations, representing a low amount of total caco3 (12% and 9.2% for guetia and senelba, respectively). it is noted that the highest values are marked in spring (12.5%), and decrease in summer (12.0%), then in autumn (7.2%) (tab. 5). the soil at guetia is characterized by a loam texture, with a relatively balanced composition of sand (44.2%), silt (42.8%), and clay (13%). in contrast, the senelba site exhibits a sandy-loam texture, dominated by a high proportion of silt (81.33%) and a lower content of sand (13.77%) and clay (4.9%) (tab. 6). c la ss o rd er fa m ily g en us id en tifi ed sp ec ie s u ni de nt ifi ed sp ec ie s st at io ns se ne ba (s ) g ue tia (t ) g lo m er om yc èt es g lo m er al es g lo m er ac ea e g lo m us g lo m us sp .1 * g lo m er om yc èt es g lo m er al es g lo m er ac ea e g lo m us g lo m us sp .2 * g lo m er om yc èt es g lo m er al es g lo m er ac ea e se pt og lo m us s. co ns tri ct um * * g lo m er om yc èt es g lo m er al es g lo m er ac ea e fu nn el ifo rm is f. m os se ae * g lo m er om yc èt es d iv er sis po ra le s g ig as po ra ce ae g ig as po ra g ig as po ra sp .1 * g lo m er om yc èt es d iv er sis po ra le s g ig as po ra ce ae g ig as po ra g ig as po ra sp .4 * g lo m er om yc èt es d iv er sis po ra le s a ca ul os po ra ce ae a ca ul os po ra a ca ul os po ra sp .1 * * to ta l 2 3 5 2 5 4 5 ta bl e 4. a m f sp ec ie s is ol at ed fr om th e rh iz os ph er e of p . t er eb in th us fig. 3. spores of different amf species isolated from the rhizosphere under p. terebinthus: a-d: spores isolated from guetia forest. e-i: spores isolated from the senelba forest. magnification (x 40). bar 30 μm. biologica nyssana ● 16 (2) december 2025: bouabdelli et al. ● spatiotemporal variation of endomycorrhizae among two populations of pistacia terebinthus l. (anacardiaceae) in north africa data analysis the correlation matrix between the mycorrhization soil and climatic parameters shows several significant correlations (tab. 7): between arbuscular (a) and vesicular (v) content (r=0.98); between conductivity (ec) and ph (r=0.57), and between colonization intensity (m) and electrical conductivity (ec) (r=0.63). the correlation circle shows that the majority of the variables (except f, om, and tl) are correlated with the two axes, explaining 69.53% of the variance. the two soil variables (ph and ec) and all the climatic variables are correlated with axis 1; the two mycorrhization variables (a and v) are correlated with axis 2, while the m variable is correlated with two axes (fig. 4a). consequently, this interaction made it possible to group all the trees of the two populations into two distinct groups (fig. 4b). axis 1 seems to reflect an increasing aridity index (q3), where the different stations are discriminated according to their bioclimates into two distinct groups. parameters season ph-water electrical conductivity (ms/cm) organic matter (%) total caco3 (%) senelba (s) spring 2.4±1.3 5.6±1.4 summer 8.7±0.1 0.3±0.1 7.9±2.2 11.2±6.7 autumn 8.2±0.1 0.3±0.1 4.2±1 10.8±6.3 mea. s±sd 8.4***±0.3 0.2*±0.1 4.8***±2.8 9.2ns±5.6 guetia (t) spring 8.1±0.0 0.1±0.01 3.2±0.7 8.8±6.5 summer 8.1±0.1 0.2±0.02 9±1.2 13.8±6.6 autumn 7.9±0.2 0.2±0.02 11.4±2.2 13.3±6.6 mea. s±sd 8.0ns±0.1 0.2*±0.02 7.9***±3.8 12ns±6.5 season factor mea. e±sd 8.2*±0.3 0.2*±0.1 6.3***±3.6 10.6ns±6.1 station factor mea. e±sd 8.2***±0.3 0.2***±0.1 6.3*±3.6 10.6ns±6.1 season-station interaction mea. e±sd 8.2***±0.3 0.2***±0.1 6.3***±3.6 10.6ns±6.1 table 5. physico-chemical analysis of soils (senelba and guetia) mea. s±sd: mean station, mea. e±sd: mean species, mea. g±sd: mean genus, sd: standard deviation, statistical significance level at p=0.05, * p<0.05, ** p<0.01, *** p<0.001, ns: no significant element guetia (t) senelba(s) sand (%) 44.2 13.77 silt (%) 42.8 81.33 clay (%) 13.0 4.90 texture loam sandy-loam table 6. particle size analysis of soils in the studied populations of p. terebinthus fig. 4. principal component analysis, a: circle of correlation of variables for p. terebinthus; f frequency of mycorrhization. m intensity of colonization of the root cortex. a arbuscular content. v vesicular content. ph ph-water. ec electrical conductivity. mo organic matter. ct total limestone. alt altitude. q3 rainfall quotient. tm mean maximum temperature of the hottest month in degrees celsius. tm mean minimum temperature of the coldest month in degrees celsius. b: scatterplot of samples, s senelba, t guetia, g1 group 1, g2 group 2. biologica nyssana ● 16 (2) december 2025: bouabdelli et al. ● spatiotemporal variation of endomycorrhizae among two populations of pistacia terebinthus l. (anacardiaceae) in north africa f% m % a % v % ph ec (m s/ cm ) o m % tl % p (m m /y ) m t (° c ) m t (° c ) a t ( m ) q 3 f% 1 0. 42 0. 26 0. 21 0. 2 0. 22 -0 .0 5 -0 .1 6 -0 .3 1 0. 31 -0 .3 1 0. 31 -0 .3 1 m % 1 0. 36 0. 33 0. 34 0. 63 0. 12 0. 07 -0 .4 6 0. 46 -0 .4 6 0. 46 -0 .4 6 a % 1 0. 98 -0 .0 5 0. 1 0. 16 0. 29 -0 .0 5 0. 05 -0 .0 5 0. 05 -0 .0 5 v % 1 -0 .0 7 0. 07 0. 13 0. 3 -0 .0 2 0. 02 -0 .0 2 0. 02 -0 .0 2 ph 1 0. 57 -0 .0 8 -0 .1 4 -0 .7 1 0. 71 -0 .7 1 0. 71 -0 .7 1 ec (m s/ cm ) 1 0. 18 -0 .0 2 -0 .5 7 0. 57 -0 .5 7 0. 57 -0 .5 7 o m % 1 0. 28 0. 43 -0 .4 3 0. 43 -0 .4 3 0. 43 tl % 1 0. 23 -0 .2 3 0. 23 -0 .2 3 0. 23 p (m m /y ) 1 -1 1 -1 1 m t (° c ) 1 -1 1 -1 m t (° c ) 1 -1 1 a t ( m ) 1 -1 q 3 1 ta bl e 7. p ea rs on c or re la tio n m at rix (p ˂0 .0 5) fo r m yc or rh iz at io nn , s oi l a nd c lim at ic v ar ia bl es s tu di ed o f p . t er eb in th us f% : f re qu en cy o f m yc or rh iz al c ol on iz at io n, m % : i nt en si ty o f m yc or rh iz al c ol on iz at io n in th e r oo t s ys te m , a % : a bu nd an ce o f a rb us cu le s in th e r oo t s ys te m , v % : ab un da nc e of v es ic le s in th e r oo t s ys te m , p h: p h w at er , e c: e le ct ric al c on du ct iv ity (m illi si em en s pe r c en tim et er ), o m : o rg an ic m at te r, tl : t ot al l im es to ne , m t: m ax im um te m pe ra tu re , m t: m in im um te m pe ra tu re , p : p lu vi om et ry (m m p er y ea r), q 3: p lu vi ot he rm ic q uo tie nt , a t: al tit ud e (m et er ) group 1 is made up of the station of senelba (s), characterized by high values of ph, ec, tm, and at. group 2 includes the station of guetia (t), which has high values of p, mt, and q3. for the p. terebinthus species, the matrix of correlations between the parameters of mycorrhization and those of the soils shows significant correlations between all the parameters of mycorrhization: between the ph and the total limestone (r=0.73), and between the ph and colonization intensity (r=-0.57) (tab. 7). discussion there has been no research on mycorrhizae assessment in pistacia terebinthus rhizospheres’ soil; however, many other studies on other species, such as pistacia atlantica, have been conducted (hamada et al., 2013; alguacil et al., 2016). in our study, rhizospheric roots of p. terebinthus were colonized by both arbuscular mycorrhizal fungi (amf) and ectoendomycorrhizae (ecm) types. according to brundrett (2004), the same types are found in the quercus suber species sampled in mediterranean forests. the root deformation observed in some trees of p. terebinthus is of particular ecological significance, as it indicates active interactions between roots and diverse fungal partners. such morphological modifications reflect the establishment of complex symbiotic structures that can enhance nutrient exchange, improve root surface area, and contribute to the functional diversity and adaptability of the mycorrhizal association. previous studies have demonstrated that single colonization by either endophyte fungi (dse) or ecto-endomycorrhizal fungi (ecm) can negatively affect plant growth, whereas dual colonization tends to mitigate or neutralize this inhibitory effect (jumpponen & trappe, 1998; püschel et al., 2013). this synergistic effect is attributed to the complementary ecological functions of both fungal groups: dse contribute to the mineralization of organic matter and nutrient mobilization, while ecm fungi enhance nutrient uptake efficiency and regulate endophytic colonization (mandyam & jumpponen, 2005; smith & read, 2008; kariman et al., 2018). we found high total amf colonization rates in the roots. this rate reached up to 100%. our results are similar to those of ortas et al. (2013) on four forest trees, where the colonization rate ranged from 62.07% to 100%. our results suggest that p. terebinthus is well adapted to these two forest conditions and promotes arbuscular mycorrhizal symbiosis. data for the winter season were not collected due to limited accessibility to the study area during this period. harsh climatic conditions and restricted access to the forest made sampling difficult, preventing the collection of root and soil samples. consequently, only data from spring, summer, and autumn were included in this study arbuscular mycorrhizal (am) fungi play crucial functional roles in ecosystems, including facilitating nutrient uptake, modifying the physical properties of soil, and altering plant interactions with other biota (smith & read, 2008). however, the composition, specificity, and abundance of these fungal communities in semiarid conditions, particularly in tropical regions, are poorly understood (gai et al., 2006). the soils in this study area are alkaline and calcareous, which, according to ortas et al. (2013), leads to phosphorus precipitation, making it less available to plants. by interacting with the abiotic environment, mycelium can directly impact ecosystem functioning (leifheit et al., 2014). furthermore, hoeksema et al. (2010) discovered that the presence of am fungi reduced variation in community productivity across strong gradients of phosphorus and nitrogen limitations while also increasing plant community-level stoichiometric homeostasis. the particle size analysis revealed clear differences in soil texture between the two studied sites. a loam texture characterizes the soil at guetia, and this composition suggests a medium-textured soil that favors good aeration and moderate water retention, suitable for root development. in contrast, the senelba site exhibits a sandy-loam texture, dominated by a high proportion of silt (81.33%) and a lower content of sand (13.77%). this texture indicates lighter soil with better drainage but lower nutrient-holding capacity. such variation in soil texture between sites may influence water availability, nutrient dynamics, and consequently, the mycorrhizal colonization of pistacia terebinthus roots. notably, amf colonization intensity was significantly and negatively correlated with electrical conductivity. previous studies have also demonstrated this correlation (koide & kabir, 2000; lekberg & koide, 2005; munkvold et al., 2004). however, no significant correlation was observed between amf colonization rate and other soil parameters in our study. our results differ from those of ortas et al. (2013), who found that amf colonization intensity was significantly and negatively correlated with soil total carbon and organic matter content. selecting the rootstock in pistachio orchards is one of the most critical decisions, mainly in conditions of water scarcity. however, there are only a few studies that report the response of pistachio to water stress according to rootstock. according to trotta et al. (1996), mycorrhiza-treated plants achieved approximately 80% positive grafts using p. terebinthus as a rootstock for p. vera buds. our results suggest a great affinity between these fungi and p. terebinthus, making it a suitable rootstock for p. vera buds in semi-arid forests for two reasons: 1. the rehabilitation and restoration of degraded forests in these regions. 2. increasing almond production in our country. symbiotic arbuscular mycorrhizal fungi (amf) represent a cost-effective and non-destructive strategy for enhancing crop yield and promoting a sustainable agricultural system with low inputs. these fungi efficiently absorb low-level soil nutrients, significantly impacting ecosystem functioning and stability (giovannetti & avio, 2002). as vital components of soil microbiology, amfs can be harnessed to improve crop development, potentially reducing or eliminating the need for chemical fertilizers and pesticides in organic agriculture (gianinazzi et al., 2010; veresoglou et al., 2012). previous research by hartnett & wilson (1999) indicated that the application of benomyl in dry grassland plots suppressed am fungi, leading to reduced temporal stability in community productivity. amf-colonized plants exhibit superior soil utilization, enhanced nutrient absorption, and greater resilience to drought, salinity, and heavy metals. additionally, they offer benefits as plant growth promoters and biological control agents against phytopathogens (pozo & azcónaguilar, 2007). conclusion given the key role of mycorrhizal fungi in the poor soils of fragile ecosystems in arid and semi-arid areas, identifying some of these fungi has enabled us to understand their ecology across different soil types. the use of molecular techniques for the identification of amf species as a complement to the morphological tool is desired in order to proceed with the isolation of the most efficient strains which plays a key role in the ecosystem productivity and stability with the aim of their use on a large scale for mycorrhization of young pistachio trees to be planted in favorable areas for the reforestation of the green dam. funding and acknowledgments: this research was funded by le ministère de l’enseignement supérieur et de la recherche scientifique, algeria. references ağlar, m., bostan, a., & ünal, t. 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(2020). arbuscular mycorrhizal fungi as biological control agents in agricultural ecosystems. biological control, 139, 104092. https://doi.org/10.1016/j.biocontrol.2019.104092 anoop and vengatachalam 2025, biologica nyssana 16(1) 53 16 (1) june 2025: 53-57 doi: 10.46793/biolnyss.16.1.22a vicoa indica: unveiling the therapeutic properties and phytochemical profiling of a lesser-known medicinal plant review article t.v. anoop department of pharmacology, vinayaka mission’s college of pharmacy, vinayaka mission's research foundation, ariyanoor, salem, india gomathi vengatachalam department of pharmacology, vinayaka mission’s college of pharmacy, vinayaka mission's research foundation, ariyanoor, salem, india gomicology@gmail.com (corresponding author) received: january 10, 2025 revised: february 06, 2025 accepted: march 06, 2025 abstract: vicoa indica (l.) dc., a member of the asteraceae (compositae) family, is a well-known traditional medicinal plant widely available in tropical regions, including india. various parts of this plant have been evaluated for their pharmacological properties. traditionally, tribes in northern india have used v. indica as a contraceptive. the plant’s biological activity is attributed to its diverse phytochemical composition, including sesquiterpenoid lactones, triterpenoids, and flavonoids. several studies have reported its antiinflammatory, contraceptive, antioxidant, and antimicrobial properties. this review compiles the existing scientific literature on the traditional medicinal uses of v. indica. however, limited research has been conducted on this species, highlighting the need for further studies to explore its therapeutic potential comprehensively. key words: antioxidant, anti-inflammatory, antimicrobial, asteraceae, banjauri, contraceptive, sesquiterpenoid lactones, vicoa indica, vicolides apstrakt: vicoa indica: otkrivanje terapeutskih svojstava i fitohemijski profil manje poznate lekovite biljke vicoa indica (l.) dc., pripadnik porodice asteraceae (compositae), je dobro poznata tradicionalna lekovita biljka široko rasprostranjena u tropskim područjima, uključujući i indiju. različiti delovi ove biljke su proučavani zbog svojih farmakoloških svojstava. tradicionalno, plemena severne indije su koristila v. indica kao kontraceptiv. biološka aktivnost biljke pripisuje se njenom raznovrsnom fitohemijskom sastavu, koji uključuje seskviterpenoidne laktonе, triterpenoide i flavonoide. nekoliko istraživanja je izveštavalo o njenim protivupalnim, kontraceptivnim, antioksidativnim i antimikrobnim svojstvima. ovaj pregled objedinjuje postojeću naučnu literaturu o tradicionalnoj upotrebi v. indica kao lekovite biljke. ipak, sprovedena su ograničena istraživanja na ovoj vrsti, što naglašava potrebu za daljim proučavanjima kako bi se u potpunosti ispitali njeni terapeutski potencijali. ključne reči: oksidansi, antiinflamatorno, antiseptičko, asteraceae, banjauri, kontraceptiv, seskviterpenoidni laktoni, vicoa indica, vikolidi introduction the field of traditional medicine continues to play a significant role in global healthcare, with increasing interest in their integration into modern medicine. the growing interest in these practices, combined with ongoing research and regulatory efforts, is shaping the future of traditional medicine (rizvi et al., 2022). there is increasing public interest in natural and holistic approaches to health, leading to greater acceptance and use of traditional medicines (wyk et al., 2020). a desire for alternative treatments and a growing awareness of the potential benefits of traditional practices drives this interest. many organizations promote research, develop regulatory frameworks, and integrate traditional medicine into broader health strategies. ethno-pharmacological research on vicoa indica has predominantly focused on asian countries, where the plant is primarily used in traditional medicine (misra et al., 2011). the plant, belonging to the family asteraceae, is a perennial herb distributed throughout central asia and northeast tropical africa (tiwari et. al., 2018). so far, only limited research has been conducted on this plant, which is summarized below. © 2025 anoop and vengatachalam. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 54 botanical description and distribution the plant is found in west tropical africa, india, pakistan, myanmar, china, sri lanka, and thailand. in india, it occurs in the himalayas and the western ghats (misra et al., 2011). this annual herb is widely cultivated in india, especially in the states of kerala, tamil nadu, andhra pradesh, karnataka, and odisha. vicoa indica, commonly known as golden daisy, is an erect annual herb reaching a height of 1–3 feet. the upper portion bears lush, cylindrical branches along the stems. the sessile leaves are oblong-lanceolate, acute, entire or serrulate, coarse or scabrid, with short appressed hairs on both sides. they vary in size, are dilated, and have rounded auricles at the base, typically measuring 3 to 8 cm in length. yellow flower heads, 1–2 cm wide, appear singly on thin stems. the ray flowers number 12–24, each with three teeth at the apex; they are thin and accompanied by yellow disc florets. the flowers are bright yellow. flowering and fruiting occur from september to march (pullaiah et al., 2009; tiwari et al., 2018). it is considered a weed of cultivation and a colonizer, commonly growing in plains and paddy fields up to 1300 m altitude. phytochemistry qualitative tests on various parts of vicoa indica have been conducted to detect secondary metabolites. the plant is rich in phytocompounds such as flavonoids, sesquiterpene lactones, alkaloids, phenolic compounds, triterpenes, and others. several sesquiterpenoid lactones were isolated from different extracts of v. indica and named vicolides a, b, c, d, and e (purushothaman et al., 1981; vasanth et al., 1995). the structures of these compounds were elucidated using x-ray crystallography, chemical, and spectroscopic methods. studies also revealed the presence of other phytochemicals, including triacontanol, campesterol, a rare flavonoid—3′,5,6trihydroxy-4′,7-dimethoxyflavone—and β-sitosterol (purushothaman et al., 1986). sixteen n-alkanes and nine n-alkanoic acid ethers were isolated from aerial parts using the urea adduct method (balakrishna et al., 1993). chloroform and methanolic extracts contained compounds such as vicogenin and viscose b (vasanth et al., 1992). extracts of the aerial parts also showed the presence of germacranolide (sawaikar et al., 1998). additionally, mossa et al. (1997) reported four new sesquiterpene lactones: 10α-hydroxy-14h-inuviscolide, 4α,5αepoxy-10α,11β,13h,14h-1-epi-inuviscolide 3-β-dglucoside, 2α-o-acetyl-3β-hydroxyalantolactone, and 2α,3α-dihydroxyalloalantolactone, along with several known compounds and thymol esters. despite the isolation of many bioactive compounds, their pharmacokinetic profiles, bioavailability, and specific mechanisms of action remain largely unexplored. most research has been conducted in vitro, with limited in vivo validation. uses in folk and traditional medicine traditionally, women use a decoction of v. indica to induce sterility. the plant is well known in northern indian states as banjauri, meaning a female infertility agent (dhall et al., 1988; basak et al., 2016). banjauri is believed to prevent pregnancy for five to seven years, and sometimes it is also taken during menstrual cycles, although the results are not consistently reliable. in an ethnopharmacological study of the “baiga” tribe in madhya pradesh, an aqueous decoction of vicoa indica was used to treat rheumatism and arthritis (tiwari et al., 2018). tribes in the nandurbar district of maharashtra used an infusion of the whole plant as an abortifacient agent (tayade et al., 2005). additionally, the roots of the plant are traditionally used to treat cough and jaundice (oudhia et al., 2001–2003). despite its widespread use in traditional medicine, v. indica has not been extensively studied in clinical settings. this reliance on traditional knowledge underscores the need for controlled pharmacological studies to validate its safety and efficacy. biological activities antibacterial activity several reports suggest the antibacterial potential of vicoa indica. the methanol extract of leaves proved effective against bacterial strains such as vibrio parahaemolyticus, klebsiella pneumoniae, and escherichia coli, indicating the plant's potential for treating numerous infectious illnesses (kesavan et al., 2007). however, aqueous and ethanolic extracts of vicoa indica leaves did not show promising antibacterial activity when tested against k. pneumoniae and pseudomonas aeruginosa (harish et al., 2010). gold nanoparticles (aunps) synthesized from the leaf extract demonstrated significant inhibitory ability against human pathogenic bacteria (hpb) (deivanathan et al., 2023). gopalakrishnan et al. (2013) compared extracts of various parts of different plants for antibacterial activity. chloroform and acetone extracts of roots, flowers, leaves, and stem parts of v. indica showed inhibitory activity against e. coli. among the various parts investigated, the leaf extract showed the maximum inhibition against the bacterial strain (gopalakrishnan et al., 2013). gopal et al. (1992) isolated vicolides a, b, c, and d, previously reported from the chloroform extract of the whole plant and subjected them to antibacterial studies against six strains. all vicolides showed moderate activity against bacillus subtilis. vicolide b also biologica nyssana ● 16 (1) june 2025: 53-57 anoop and vengatachalam ● vicoa indica: unveiling the therapeutic properties and phytochemical profiling of a lesser-known medicinal plant 55 biologica nyssana ● 16 (1) june 2025: 53-57 anoop and vengatachalam ● vicoa indica: unveiling the therapeutic properties and phytochemical profiling of a lesser-known medicinal plant showed reasonable efficacy against staphylococcus aureus, while vicolide d was active against staphylococcus citreus (gopal et al., 1992). antifertility activity in india, many plants have traditionally been used for birth control by tribal communities, and vicoa indica is one such plant, commonly known as banjauri. ancient people widely used it as an abortifacient agent and to induce infertility in females. female albino rats treated with v. indica demonstrated the antifertility activity of vicolide b (vasanth et al., 1990), which was identified as a natural antiestrogenic compound. it also exhibited abortifacient effects despite neither being progestational nor antiprogestational. animal studies on bonnet monkeys (macaca radiata) further validated its contraceptive potential (rao et al., 1996). in this study, female monkeys were treated on days 1–14 and 9–14 of their menstrual cycle or 2–5 days postpartum, and fertility was assessed in the subsequent cycle. the results showed that treatment from day 1 to day 14 of the cycle protected the monkeys from pregnancy; however, contraception failed during the postpartum period. treated animals did not conceive even after 13 ovulatory cycles with fertile males, while all vehicle-fed monkeys became pregnant within just three cycles. the safety and contraceptive efficacy of v. indica (banjauri) have also been evaluated in female human subjects using modern clinical trial methods. when administered at a dose of 15 g once daily for 3 days across three cycles, the drug was found to be free from side effects and exhibited doserelated antifertility activity, which could potentially be enhanced by modifying the drug delivery system (dall et al., 1988). antiviral activity the antiviral properties of the plant extracts were screened for their inhibitory activity against herpes simplex virus (hsv) replication using in vitro methods. the study revealed that the aqueous root extract showed maximum inhibition against hsv1 replication, comparable to the standard drug acyclovir (rani et al., 2013). epilupeol, an antiviral compound, was isolated from the plant’s hexane extract. its antiviral activity was demonstrated using a chicken embryo model, establishing the plant’s effectiveness in treating ranikhet disease virus (rdv) (chowdhury et al., 1990). anti-inflammatory and analgesic activity the flavone isolated from the aerial parts of vicoa indica was evaluated for analgesic and antiinflammatory properties using in vivo models (krishnaveni et al., 1997). the isolate was found to effectively inhibit responses in both antiinflammatory and analgesic models. the analgesic effect is attributed to its action on opioid receptors. additionally, the compound showed consistent antiedema effects across three experimental models: carrageenan-, histamine-, and turpentine-induced edema. sesquiterpene lactones, namely vicolides a, b, c, and d, exhibited anti-inflammatory effects on cotton pellet granulomas in rats treated with 10 mg/kg body weight via the subcutaneous route. vicolides c and d reduced serum and liver protein levels, as well as the activities of sgot, sgpt, and acid and alkaline phosphatases. the potent anti-inflammatory properties of vicolide c, which contains an ester moiety and a 3,4-epoxy group, and vicolide d, characterized by an epoxy angeloyl group, are linked to their molecular structures. the antipyretic action of vicolide d was observed at a dosage of 250 mg/kg of body weight (alam et al., 1992). antioxidant activity natural antioxidants play an important role in maintaining and protecting human health, often preferred over synthetic antioxidants. gold nanoparticles synthesized from the leaf extract demonstrated free radical scavenging activity (deivanathan et al., 2023). the total phenolic content in the plant extract was measured at 135 gae per microgram (chethan et al., 2013). hepatoprotective activity methanolic extracts of v. indica leaves demonstrated hepatoprotective effects in ccl₄induced hepatic toxicity rat models (prasanth et al., 2020). however, the underlying mechanisms of hepatoprotection—such as cyp450 modulation and antioxidant pathways—remain speculative. further research should incorporate targeted proteomic and metabolomic analyses, along with long-term toxicity assessments. conclusion in summary, vicoa indica emerges as a plant of significant therapeutic potential, highlighted by its diverse phytochemical composition and a wide range of pharmacological activities, including antibacterial, analgesic, anti-inflammatory, antioxidant, antifertility, and hepatoprotective effects. the comprehensive data presented here provide compelling evidence supporting its potential applications in various therapeutic contexts. to fully harness the medicinal value of v. indica, further clinical and pharmacological studies are needed to explore its extracts and isolated compounds. 56 biologica nyssana ● 16 (1) june 2025: 53-57 anoop and vengatachalam ● vicoa indica: unveiling the therapeutic properties and phytochemical profiling of a lesser-known medicinal plant kesavan, s., devarajan, n., chokkalingam, m., chinthambi, v., & nandakumar, n. 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(2018). validity of ethnopharmacological uses of vicoa indica (l.) dc., family-asal-khaykanee et al. 2025, biologica nyssana 16(1) 287 16 (1) june 2025: 287-296 doi: 10.46793/biolnyss.16.1.15a role of 11β-hydroxysteroid dehydrogenase gene polymorphisms rs846910 and rs12086634 in the etiology of hepatocellular carcinoma original article ahmed mudher al-khaykanee general directorate of education of babylon governorate, iraq ahmedmudher14@gmail.com (corresponding author) soura alaa hussein department of technical nursing, technical institute-baghdad, middle technical university, 10074, iraq z muhsen m ali department of technical anesthesia technical institute-baghdad, middle technical university, 10074, iraq received: december 06, 2024 revised: february 17, 2025 accepted: march 17, 2025 abstract: in asia, the most frequent cause of hepatocellular carcinoma (hcc) is chronic hepatitis b. the hepatitis b virus's (hbv) genomic integration is probably a precursor to carcinogenesis. the integrated hbv genome may directly activate nearby cellular genes to give the liver cells a selective growth advantage. our study's goal was to determine if genetic variants in 11β-hsd1, rs846910 and rs12086634 could be linked to chronic hepatitis b virus (hbv) infection and hepatocellular carcinoma (hcc). 150 samples were collected from people suffering from hepatitis b with hcc, hcc, and healthy people. the samples were genotyped for 11β hsd1 (rs846910 and rs12086634) using allele-specific real-time pcr. after performing genotyping of the 11β-hsd1 gene using real-time pcr, both alleles g and a of rs846910 were found to be more prevalent in the two groups of hepatocellular carcinoma and hepatitis b. this gives a clear indication about the possibility of contracting hepatocellular carcinoma and hepatitis b. a different result was found than previously, as the tt genotype of rs12086634 showed a protective effect against hepatocellular carcinoma and hepatitis b virus infection, while the ag genotype of rs846910 increased the risk of hepatocellular carcinoma. these genetic variations are clearly significant biomarkers for hepatocellular carcinoma. key words: single nucleotide polymorphism, 11β hsd1, hepatocellular carcinoma (hcc) apstrakt: uloga polimorfizama gena 11β-hidroksisteroid dehidrogenaze rs846910 i rs12086634 u etiologiji hepatocelularnog carcinoma u aziji, najčešći uzročnik hepatocelularnog karcinoma (hcc) je hronični hepatitis b. genomska integracija hepatitis b virusa (hbv) je verovatno prekursor karcinogeneze. integrisani hbv genom može direktno aktivirati obližnje ćelijske gene, omogućavajući ćelijama jetre selektivnu prednost u rastu. cilj naše studije bio je da ispita da li genetske varijante u 11β-hsd1, rs846910 i rs12086634 mogu biti povezane sa hroničnom infekcijom hepatitis b virusa (hbv) i hepatocelularnim karcinomom (hcc). ispitano je 150 uzoraka sakupljenih od osoba sa hepatitisom b i hcc, osoba sa hcc, kao i zdravih ispitanika. genotipizacija uzoraka na 11β-hsd1 (rs846910 i rs12086634) izvršena je uz pomoć alel-specifične real-time pcr metode. nakon genotipizacije 11β-hsd1 gena uz pomoć real-time pcr metode, utvrđeno je da su oba alela, g i a, polimorfizma rs846910 zastupljenija u grupama pacijenata sa hepatocelularnim karcinomom i hepatitisom b, što ukazuje na povećan rizik od obolevanja od ovih bolesti. nasuprot ranijim rezultatima, genotip tt polimorfizma rs12086634 pokazao je zaštitni efekat protiv hepatocelularnog karcinoma i infekcija izazvanih hepatitis b virusom, dok je ag genotip rs846910 bio povezan sa povećanim rizikom od hepatocelularnog karcinoma. ove genetske varijacije predstavljaju značajne biomarkere za hepatocelularni karcinom. ključne reči: jednonukleotidni polimorfizam (snp), 11β hsd1, hepatocelularni karcinom (hcc) © 2025 al-khaykanee et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 288 introduction liver cancer ranks sixth in terms of prevalence and number of cases in the world, and fourth in terms of the number of deaths resulting from it. it is a global health problem and one of its causes is nonalcoholic fatty liver disease, a risk factor for this type of cancer (brown et al., 2023). hepatocellular carcinoma is currently the most common in north africa, southeast asia, and east asia (rumgay et al., 2022). the risk of developing hepatocellular carcinoma increases when there is cirrhosis of the liver, and it rarely occurs in a healthy liver. one of the main causes of infection is the hepatitis b virus, which is responsible for about 50% of cases of this type of cancer (akinyemiju et al., 2017). the 5-year survival rate for hepatocellular carcinoma is approximately 18.4% (leowattana et al., 2023). hepatitis b virus (hbv) is a member of the hepadnaviridae family and consists of only 3,200 nucleotides and is the smallest virus that infects humans. the absence of a proofreading mechanism in hbv reverse transcriptase facilitates the emergence of a varied range of genomic variants throughout different stages of infection. analysis of hbv genomic sequence divergence has identified several sub genotypes and ten hbv genotypes (trépo et al., 2014; razavi-shearer et al., 2018). the lack of a proofreading mechanism in hbv reverse transcriptase facilitates a diverse array of genomic variations for selective proliferation throughout various stages of infection. analysis of hbv genomic sequence divergence has identified multiple sub genotypes and eleven hbv genotypes (assefa et al., 2024). hepatitis b virus (hbv) belongs to the hepadnaviridae family. it is an epidemic liver disease. people who carry this virus are more susceptible to liver cancer. it has the ability to multiply within liver cells and interacts with many proteins. it also leads to gradual liver fibrosis and is considered a risk factor for hcc or hepatocellular carcinoma, adversely affecting the survival of people with hepatitis b virus, while concomitant hepatitis d virus infection exacerbates the condition. evidence indicates that hbv infection correlates with the likelihood of developing hcc, irrespective of the presence of underlying liver cirrhosis, through multiple direct and indirect pathways that facilitate hepatocarcinogenesis (rizzo et al., 2022). the human chromosome 1 has the hsd11b1 gene. in humans, the gene located on chromosome 16 encodes 11-hsd2. the two types of 11-hydroxysteroid dehydrogenases are microsomal enzymes associated with the cellular membrane of the endoplasmic reticulum. 11-hsd1 is expressed in the brain, hepatic adipose tissue, and arteries. its function is to promote the enactment of the glucocorticoid receptor by facilitating the centralization of the dynamic type of glucocorticoids in the tissue. as a result, 11-hsd2 is present in tissues connected to mineralocorticoid activity, the kidney, the placenta, and the salivary organs (studzińska et al., 2018; de kloet et al., 2019). increased cortisol levels are observed during the bipolar episode, suggesting activation of the hpa (watson et al., 2012). various analyses have suggested that 11β-hsd1 plays a major and helpful role in the metabolic cycles that underlie various diseases. in animals lacking 11β-hsd1, the lack of glucocorticoid recovery in the liver and adipose tissue provided a protective effect against insulin resistance and hyperglycemia (kotelevtsev et al., 1997; morgan & tomlinson, 2010). one single nucleotide polymorphism (snp) of the hsd11β1 quality that has been studied is rs846910, which is located in the 5' promoter region of the hsd11β1 gene revealed a conflicting association between metabolic disorders and type 2 diabetes in different populations (nair et al., 2004). we speculate that 11β-hsd1 gene polymorphisms, particularly rs846910 and rs12086634, may be linked to the onset of hcc based on the findings of this investigation. materials and methods this study included 150 samples divided into three groups. the first group included 50 samples from individuals with hepatocellular carcinoma and hepatitis b virus carriers. the second group included 50 samples from individuals carrying only hepatitis b virus. the third group included 50 samples from healthy individuals. samples were collected from marjan teaching hospital in the babylon governorate, department of oncology and gastroenterology, between 2022 and 2024. ethical approval was obtained from the relevant committees in compliance with the helsinki principles. all participants provided informed consent. the control group comprised 50 healthy adults devoid of any history of liver disease, hypertension, renal illness, diabetes, or other chronic conditions. they were also free of hepatitis b or c virus infections, or hiv. there were 25 males and 25 females among the control group. sample collection blood samples of 8 ml were collected from each participant and processed as follows: 2 ml were placed in edta tubes for dna extraction, 2 ml were used for a complete blood count (cbc) analysis, and the remaining 4 ml were placed in plain biologica nyssana ● 16 (1) june 2025: 287-296 al-khaykanee et al. ● role of 11β-hydroxysteroid dehydrogenase gene polymorphisms rs846910 and rs12086634 in the etiology of hepatocellular carcinoma 289 biologica nyssana ● 16 (1) june 2025: 287-296 al-khaykanee et al. ● role of 11β-hydroxysteroid dehydrogenase gene polymorphisms rs846910 and rs12086634 in the etiology of hepatocellular carcinoma tubes, allowed to clot for 15 minutes, centrifuged at 3000 rpm for 10 minutes, and then divided into aliquots for liver function testing. hepatitis markers and alpha-fetoprotein (afp) were analyzed using an electrochemiluminescence assay (ecla), and hbv-rna quantification was performed using the cobas taqman hbv quantitative assay, version 2.0. hbv genotypes were determined using the linear probe assay with the inno-lipa hbv 2 kit from innogenetics (uk). snp assay of 11β hsd1 rs846910 and rs12086634 and dna extraction genomic dna was extracted using purelink genomic dna extraction kits (usa) according to the manufacturer's instructions (qiagen). single nucleotide polymorphism (snp) assays for 11β-hsd1 rs846910 and rs12086634 were conducted using taqman genotyping test kits with the applied biosystems 7500 real-time pcr instrument (foster city, ca, usa). the primer and probe sequences for 11β-hsd1 rs846910 were as follows: forward primer 5′-ctctgttgcttgcttgcttgattc-3′, reverse primer 5 ′ -agagcaggctttcagcaga ta-3′ , and probe 5 ′ -ctggtgggaat[vic]/ [fam]tcaaatcagagaga-3′ (genbank nc_000001.11:209701908:a:g). for 11β-hsd1 rs12086634, the forward primer was 5 ′ -ggaggagaatgggaaaggtat caac-3′ , the reverse primer was 5 ′ -tcctcctgcaagagatggctatatt-3′ , and the probe was 5 ′ -cccagaggatttct[vic]/ [ f a m ] c a g a t g a t t t c t 3 ′ ( g e n b a n k nc_000001.11:209706913:t:g). the pcr reaction (25 µl) mixture contained 12.5 µl of taqman master mix, 1.25 µl of the snp assay, 6.25 µl of nuclease-free water, and 5 µl (10 ng/µl) of genomic dna template. negative controls included 5 µl of dnase-free water. the mixture was thoroughly mixed, centrifuged briefly, and loaded onto the pcr cycler (applied bio systems ® 7500 real-time pcr). genotypes were determined using the taqman allelic discrimination test, and unidentified samples were categorized as homozygous or heterozygous based on allelic differentiation analysis. laboratory analyses liver function tests included the measurement of alkaline phosphatase (alp), albumin, total protein, aspartate aminotransferase (ast), and alanine aminotransferase (alt) using the monarch-240 clinical chemistry analyzer. hepatitis b surface antigen (hbsag) was assessed using a chemiluminescence assay kit from siemens healthcare diagnostics. cbc was performed with the sysmex xp-300 fully automated 3-part cell counter (japan). routine assessments for hepatic and renal functions, hepatitis markers, afp measurement, and hbv-rna detection were conducted for all groups. statical analysis data were analyzed using spss version 20. numerical variables were expressed as mean ± standard deviation (sd), while categorical variables were presented as frequencies and percentages. the mann-whitney u test was employed to compare nonparametric variables between two groups, and the chi-square test was used to compare categorical variables. correlations between numerical variables were analyzed using spearman's correlation coefficient (rs). ors were calculated using logistic regression with ci 95%. a p-value≤0.05 was considered statistically significant, with p<0.01 indicating a highly significant result. results and discussion this study included 150 samples, consisting of 50 patients with hepatocellular carcinoma (hcc) and hepatitis b virus (hbv), 50 patients with hbv infection, and 50 healthy controls. the demographic and laboratory data for the patient and control groups are summarized in tab. 1. no statistically significant differences were observed for age and sex between the groups, indicating adequate matching. however, statistically significant differences were detected between the hbv and hcc groups compared to the controls regarding the laboratory tests (tab. 1). the results revealed significant differences in the distribution of genotypes and alleles for the rs12086634 and rs846910 polymorphisms among the hbv, hcc, and control groups, suggesting a potential association between these polymorphisms and the development of hbv and hcc (tab. 2). in the case of 11β-hsd1 rs12086634, the gg and tg genotypes were more prevalent in the hbv (18% and 72%, respectively) and hcc patients (16% and 80%, respectively), whereas the control group exhibited the highest frequency of the tt genotype (82%, p<0.001). allelic distribution comparisons revealed that the g allele was significantly more frequent in the hbv (54%) and hcc (56%) groups compared to the control group (13%, p<0.001). this suggests that the g allele may contribute to an increased risk of both diseases. in contrast, the t allele was the most common in the control group (87%) compared to hbv (46%) and hcc (44%). this allele may play a protective role against disease 290 biologica nyssana ● 16 (1) june 2025: 287-296 al-khaykanee et al. ● role of 11β-hydroxysteroid dehydrogenase gene polymorphisms rs846910 and rs12086634 in the etiology of hepatocellular carcinoma table 1. demographic and laboratory data for the hbv, hcc, and control groups variable mean ± sd. hbv n=50 hcc n=50 control n=50 p-value gender: male n. (%) 22 (46) 18 (42) 25 (64) 0.325 female n. (%) 28 (54) 32 (58) 25 (36) 0.325 age (years) 56.55±4.45 60.7±1.3 58.55±6.18 0.121 wbcs (x 103/µl) 6.11±2.87a 5.12±1.88a 7.22±0.11 <0.001* hb (gm/dl) 9.45±1.43a 8.87±0.11a 11.78±1.22 <0.001* platelets (x 103/µl) 119.6±40.31a 98.8±20.37a 271.6±20.41 <0.001* p.c.v (%) 70.80±9.53a 69.21±5.55a 89.45±5.65 <0.001* ast (iu/l) 43.86±12.15a 103.7±18.31a 17.4±4.66 <0.001* alt(iu/l) 58.64±12.50a 189.6±264.22a,b 13.11±6.58 <0.001* t. bilirubin (mg/dl) 2.88±1.86a 0.68±0.05a 2.96±0.1 <0.001* albumin (gm/dl) 2.96±0.22a 2.7±0.65a,b 4.56±0.19 <0.001* t. protein (gm/dl) 7.33±1.02a 6.44±0.96a 7.88±0.65 <0.001* alkaline phosphatase (iu/l) 68.06±44.82b 130.5±57.32a 67.14±11.40 <0.001* afp (ng/ml) 11.24±2.60 28.27±20.64a <0.001* viral load (iu) 698.96.9±1067.4 7435.21±1153.6a <0.001* * statistically significant at p≤0.05, a significantly differs from control group, b significantly differs from hbv group table 2. the distribution of genotypes and alleles for the 11β-hsd1 rs846910 and rs12086634 gene polymorphisms among the hbv, hcc, and control groups hbv (n=50) hcc (n=50) control (n=50) χ2 p no. % no. % no. % rs12086634 gg 9 18% 8 (16%) 4 (8%) 50.47* <0.001*tg 36 72% 40 (80%) 5 (10%) tt 5 (10% 2 (4%) 41 (82%) allele g 54 (54%) 56 (56%) 13 (13%) 46.83* <0.001* t 46 (46%) 44 (44%) 87 (87%) rs846910 aa 24 (48%) 22 (44%) 46 (92%) 71.51* <0.001* ag 26 (52%) 28 (56%) 4 (8%) allele g 26 (26%) 28 (28%) 96 (96%) 65.264* <0.001* a 74 (74%) 72 (72%) 4 (4%) * statistically significant at p≤0.05 291 biologica nyssana ● 16 (1) june 2025: 287-296 al-khaykanee et al. ● role of 11β-hydroxysteroid dehydrogenase gene polymorphisms rs846910 and rs12086634 in the etiology of hepatocellular carcinoma progression. the genotype distribution of the 11β-hsd1 rs846910 polymorphism also showed a clear difference between the three studied groups, with the aa genotype being the most common in the control group (92%), hbv (48%), and hcc (44%). the ag genotype was the most common in the hcc group (56%), less common in the hbv (52%), and very rare in the control group (8%). the results indicate that the distribution of the a allele may be associated with the risk of infection with hepatitis b virus and hepatocellular carcinoma, and the exact opposite for the g allele, as it was more widespread and distributed in the control group (96%) compared to its prevalence in the liver cancer group (28%) and in the hepatitis b virus group (26%), so it is considered a protective factor. these distributions of the genetic patterns in both rs12086634r and rs846910 indicate that they may have an important role in the genetic predisposition to infection with the hepatitis b virus and its development into hepatocellular carcinoma. the overrepresentation of specific alleles in the disease groups highlights their potential as genetic markers for susceptibility or protection. the findings provide evidence of significant associations between the rs12086634 and rs846910 polymorphisms and hbv and hcc. the g allele in rs12086634 and the a allele in rs846910 appear to be risk factors, while the t allele in rs12086634 and the g allele in rs846910 might have protective effects. additionally, we analyzed the association of 11β-hsd1 rs846910 and rs12086634 polymorphisms across the three groups by comparison of their genotypic frequencies. this provided a valuable insight into the potential influence of these polymorphisms on the risk of hcc and hbv infection. regarding the rs12086634 polymorphism, the analysis revealed significant differences in the genotypic distributions between the groups. by comparing hepatitis b virus (hbv) with hepatocellular carcinoma (hcc), the tg genotype showed a strong association (or=25.11, p<0.001), indicating an increased risk of hcc in individuals carrying the tg genotype for rs12086634. additionally, the tt genotype also showed a significant association with both the hcc subset compared to the control group (or=0.009, p=0.0133) and hbv versus the control group (or=0.024, p<0.001). this suggests that the tt genotype is a protective factor against hbv and hcc for rs12086634. significant results emerged, as the t allele consistently showed a lower odds ratio compared to the g allele in all groups studied. therefore, the t allele for rs12086634 is considered to reduce the risk of hepatocellular carcinoma and hepatitis b virus infection. the gg genotype of the rs846910 polymorphism showed a significant association with a low odds ratio (or=0.045, p<0.001) when comparing the hepatocellular carcinoma group with the hepatitis b virus group. this indicates that it is a protective factor against hepatocellular carcinoma in individuals carrying this genotype. in contrast, the ga genotype was a risk factor for hepatocellular carcinoma compared to the hepatitis b virus (or=21.77, p<0.001) and control groups (or=19.11, p<0.001), representing a risk factor for hepatocellular carcinoma. in the comparison between hbv and control, the ga genotype was associated with a very high odds ratio (or=40.77, p<0.001), suggesting a strong correlation with hbv infection risk. the a allele was consistently associated with higher odds of both hbv and hcc, with g allele showing a protective role. the p-values for the majority of comparisons between the groups were statistically significant, particularly for the tg and ga genotypes, indicating a strong association between these genotypes and the presence of liver disease. the distribution of observed and expected genotypic frequencies for rs12086634 and rs846910 polymorphisms was also analyzed across the three groups (tab. 4). the chi-square test (χ2) and p-values were used to assess deviations from hardy-weinberg equilibrium (hwe) within these groups. in the caase of rs12086634, the observed frequencies of the gg, tg, and tt genotypes in the hcc group showed a significant deviation from the expected frequencies (χ2=0.271, p=0.018), indicating disequilibrium, reinforcing the hypothesis that the rs12086634 polymorphism may play a role in liver cancer susceptibility. conversely, the hbv and control groups showed no significant deviations (χ2=1.69, p=0.184; χ2=3.87, p=0.325 for the hbv and control groups, respectively), indicating that the distribution of rs12086634 genotypes in both healthy individuals and the hbv group follows the expected patterns (tab. 4). for rs846910, the hcc group showed a significant deviation from hwe (χ2=0.214, p=0.015), mainly due to an overrepresentation of the ga genotype compared to the expected frequencies (28 vs. 9.4, tab. 4). this suggests a potential association between the ga genotype and increased hcc susceptibility. in contrast, the hbv and control groups did not show a significant deviation (χ2=4.22, p=0.251, χ2=6.32, p=0.536 for the hbv and control groups, respectively), suggesting that rs846910 may not play a major role in hbv susceptibility in the control group. the significant deviations from hwe observed in the hcc group for both polymorphisms suggest that rs12086634 and rs846910 may influence susceptibility to hcc, potentially through their 292 biologica nyssana ● 16 (1) june 2025: 287-296 al-khaykanee et al. ● role of 11β-hydroxysteroid dehydrogenase gene polymorphisms rs846910 and rs12086634 in the etiology of hepatocellular carcinoma table 3. genotypic frequencies and associations of 11β-hsd1 rs846910 and rs12086634 in hbv, hcc, and control groups p1 or1 (ci. 95%) p2 or2 (ci. 95%) p3 or3 (ci. 95%) rs12086634 genotype gg 0.42 0.69 (0.26-1.83) 0.036 3.63 (1.082-12.18) 0.14 2.52 (0.722-8.81) tg <0.001 25.11 (8.36-71.22) <0.001* 23.14 (7.61-70.30) <0.001* 23.14 (7.61-70.30) tt <0.001* 0.03 (0.002-0.087) 0.0133* 0.009 (0.002-0.044) <0.001* 0.024 (0.0076-0.078) allele g r t <0.001* 0.12 (0.08-0.74) <0.001* 0.01 (0.05-0.20) <0.001* 0.12 (0.063-0.52) rs846910 genotype gg <0.001* 0.045 (0.016-0.130) <0.001* 0.53 (0.018-0.03) <0.001* 0.024 (0.007-0.083) ga <0.001* 21.77 (7.68-61.74) <0.001* 19.11 (0.51-0.64) <0.001* 40.77 (12.02-138.29) allele g r a <0.001* 0.84 (0.48-1.48) <0.001* 18.10 (6.17-53.08) <0.001* 15.86 (5.39-46.66) p1 hcc vs hbv, p2 hcc vs control, p3 – hbv vs control, or1 hcc vs hbv, or2 hcc vs control, or3 hbv vs control, ci confidence interval, * statistically significant at p≤0.05 roles in regulating biological processes associated with liver disease progression. the defect in the hepatitis b virus 11β-hsd1 gene rs12086634 demonstrates its importance in the pathogenesis of this virus and highlights the importance of the 11β-hsd1 rs846910 and rs12086634 gene polymorphisms in understanding the genetic basis of hepatocellular carcinoma and hepatitis b virus. future studies require increasing the sample size and further functional analyses to confirm the results and to elucidate the mechanisms underlying this association. hepatocellular carcinoma (hcc) constitutes a significant public health issue, with an expected 905,677 new cases and 830,180 fatalities reported globally in 2020. consequently, hcc is projected to be the third most fatal cancer form, mostly due to diagnostic delays and insufficiently efficient treatment techniques (sung et al., 2021). nearly 90% of all hcc cases are associated with identifiable risk factors, namely chronic viral hepatitis and alcohol misuse. chronic infection with hepatitis b virus (hbv) is the primary cause of hcc globally, succeeded by chronic infection with hepatitis c virus (hcv) (galle et al., 2018; alkhaykanee et al., 2021). consequently, the world health organization has designated hbv and hcv as oncoviruses (mesri et al., 2014). hepatitis b contributes directly or indirectly to liver cancer by destabilizing the cell genome and producing genetic modifications in the host dna, leading to chromosomal reconfiguration and aberrant 293 activation of oncogenes and tumor suppressor genes through integration or mutation of host genes. it may also induce malignant transformation of hepatocytes by activating various cancer-related signaling pathways (jiang et al., 2021). 11beta-hydroxysteroid dehydrogenase type 1 (11β-hsd1) catalyzes the conversion of glucocorticoids from the inactive cortisone to the active cortisol, serving a vital role in glycemic regulation. growing evidence suggests that heightened glycolytic activity is significantly associated with postoperative recurrence and prognosis of hepatocellular carcinoma (hcc) (liu et al., 2015). in this study, the polymorphism of the 11β-hsd1 gen g/t was evaluated in the three studied groups (hcc, hbv and control group). analyses revealed significant measurable variations in genotype and allele frequency of the 11β-hsd1 rs12086634 gene. the increase was with the prevalence of the g/t genotype in hcc patients and its presence was higher than in patients infected with hbv only. the tt genotype was the most common in the control group, as the t allele is associated with resistance to hbv infection and is considered defensive against the development of hcc. in this study, we observed the distribution of the 11β-hsd1 rs846910 genotype among the three studied groups, where the ga genotype was slightly more prevalent in hcc patients than in kidney infection only, while the ga genotype was more normal in the control group. in general, the distribution of the aa genotype was less prevalent. in this study, we investigated 11β-hsd1 gene association with certain diseases, explored the relationship of the polymorphism of these gene with hbv risk and hcc contaminated patients. 11β-hsd1 is present in the liver, adipose tissue, and kidneys, among other organs, and performs multiple physiological functions, including intracellular and oxysterol metabolism; its dysregulated expression is linked to insulin resistance, visceral adiposity and hypotension (seckl et al., 2004). 11β-hsd1 is a microsomal compound that catalyzes the change of the active steroid cortisol to the inactive metabolite cortisone (michalaki et al., 2012). the 11β-hsd1 gene is considered as hereditary risk factors for polycystic ovary condition and could potentially be used to evaluate for early detection of pcos (soliman et al., 2020). furthermore, the expression level of 11β-hsd1 in subcutaneous adipose tissue was associated with fasting plasma glucose levels and correlated with lipids. the gene expression of 11β-hsd1 significantly increases the risk of diabetes mellitus and hypertension in obese individuals (aziz et al., 2023). 11β-hsd1 might be a significant protein in the pathogenesis of fatty liver and obesity (lutz et al., 2016). a study conducted by shareef & al-attar (2019) confirmed that snp rs2086634 t/g of the 11β-hsd1 gene is significantly associated with polycystic ovary syndrome. in addition, the g allele of this snp showed a positive association with obesity (moon et al., 2013). there is a relationship between elevated liver enzymes alt, liver cells damage, and hsd11b1 polymorphisms (rs12086634) in patients suffering from nafld (devang et al., 2017). also, there is an association between the t/g genetic polymorphism biologica nyssana ● 16 (1) june 2025: 287-296 al-khaykanee et al. ● role of 11β-hydroxysteroid dehydrogenase gene polymorphisms rs846910 and rs12086634 in the etiology of hepatocellular carcinoma table 4. observed vs. expected genotypic frequencies and hardy-weinberg equilibrium analysis for 11β-hsd1 rs846910 and rs12086634 polymorphisms observed expected χ2 p rs12086634 hcc (n=50) gg 8 12 0.271 0.018*tg 40 83 tt 2 5 hbv (n=50) gg 9 10 1.69 0.184tg 36 34 tt 5 6 control (n=50) gg 4 5 3.87 0.325tg 5 7 tt 41 38 rs846910 hcc (n=50) aa 22 21 0.214 0.015 ga 28 29 hbv (n=50) aa 24 23 4.22 0.251 ga 26 27 control (n=50) aa 46 44 6.32 0.536 ga 4 6 * statistically significant at p≤0.05 294 in hsd11b1 rs12086634 and increased visceral obesity as a result of increased hsd11b1 activity (gambineri et al., 2011). 11β-hsd1, containing the two snps rs846910 (a) and rs12086634 (t), results in enhanced 11β-hsd1 expression and activity, which supports the development of metabolic disorders (gambineri et al., 2011). studies on the association of snps rs846910 and rs12086634 with liver disorders have not yet been conducted, despite the fact that there are several studies and discoveries on these snps. to confirm the correlations found in this study and clarify the biological processes behind these genetic predispositions, more research is required. conclusion in conclusion, the study provides clear evidence about the relationship between genetic polymorphisms in rs12086634 and rs846910 within the 11β-hsd1 gene and the possibility of developing hepatocellular carcinoma. in the case of people infected with the hepatitis b virus, as the g allele of rs12086634 was found to be more prevalent in the hepatocellular carcinoma group and the hepatitis b virus group, it is considered a risk factor for cancer, while the t allele was found to be protective. for rs846910, ga was the most common genotype in both the liver cancer group and 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(2012). a randomized trial to examine the effect of mifepristone on neuropsychological performance and mood in patients with bipolar depression. biological psychiatry, 72(11), 943-949.‏ doi: 10.1016/j.biopsych.2012.05.029. georgiev 2023, biologica nyssana 14(2) 14 (2) december 2023: 125-127 doi: 10.5281/zenodo.10209820 a new species of tapinella enderlein, 1908 (insecta: psocoptera), tapinella zirobwensis n. sp., from uganda original article dilian georgiev department of ecology and environmental conservation, university of plovdiv, 24 tsar assen street, 4000 plovdiv, bulgaria diliangeorgiev@gmail.com (corresponding author) received: january 06, 2023 revised: november 08, 2023 accepted: november 09, 2023 abstract: a new species of tapinella enderlein, 1908 from uganda was described and named tapinella zirobwensis n. sp. it was collected near zirobwe town, in agricultural lands with eucalypt plantations and bushes. the type material is deposited at the national museum of natural history, sofia, bulgaria. only a female specimen was collected and described. key words: africa, biodiversity, equatorial, insects, research apstrakt: nova vrsta tapinella enderlein, 1908 (insecta: psocoptera), tapinella zirobwensis n. sp., iz ugande u radu je opisana novootkrivena vrsta iz roda tapinella enderlein, 1908, nazvana tapinella zirobwensis n. sp., koja je pronađena u ugandi. jedinka je prikupljena u blizini grada zirobwe, na poljoprivrednom zemljištu sa zasadom eukaliptusa i žbunastih biljaka. tokom istraživanja pronađena je samo jedna jedinka (ženka) i deponovana u nacionalnom muzeju prirodne istorije u sofiji, bugarska. ključne reči: afrika, biodiverzitet, ekvator, insekti, istraživanje introduction faunistic and taxonomic works on the psocid fauna of uganda are very scarce. information about this insect group was published in the papers of karny (1924), pearman (1934), smithers (1960), and mockford (1993). only 14 species of psocoptera were known from this region till now (lienhard, 2016). in this paper a new species of tapinella enderlein, 1908 from uganda is described. this genus contains mainly tropical species and is not studied in detail in the area of equatorial africa. materials and methods psocoptera were collected from uganda by beating the vegetation on 28.07.2022., in the area of zirobwe town (about 40 km north of kampala city). the specimens were stored in 96% ethanol. the photos (specimens were placed in glycerin, because of its greater transparency) were taken by the canon powershot sx500is camera through the eyepiece of a light microscope optika. measurements were taken by the eyepiece micrometer. the type material was deposited at the national museum of natural history, sofia, bulgaria (nmnh). the species discussed in the paper were evaluated according to the original descriptions, redescriptions, and published identification keys. measurements followed lienhard (1998) methodology. measurements abbreviations (all in mm in the text): lc=body length; a=antenna length, p4: fourth segment of maxillary palp, f+tr=hind femur and trochanter length; t=hind tibia length; t1, t2, t3=tarsomeres of hindtarsus (lengths measured from condyle to condyle), fw=forewing, hw=hindwing, d=anteroposterior diameter of the compound eye, io=shortest distance between compound eyes. results family: pachytroctidae enderlein, 1904 tapinella zirobwensis n. sp. material examined: holotype: 1 ♀, 28.07.2022, uganda, about 40 km n of kampala city, ne of zirobwe town, near dulo school, agricultural lands (fields and eucalypt plantations with bushes and single trees), n00 42 11.8 e32 42 58.2, 1061 m a.s.l., nmnh – sofia, bulgaria. © 2023 georgiev. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 125 type locality: uganda, about 40 km n of kampala city, ne of zirobwe town, near dulo school, agricultural lands (fields and eucalypt plantations with bushes and single trees), n00 42 11.8 e32 42 58.2, 1061 m a. s. l. description (after four months in 96% ethanol): female: coloration: head creamy with dark brown lateral stripe at each side from the postclypeus to the antennal socket, continuing to the eye and the thorax. vertex and frons with two symmetrical elongate darker spots at each side as shown on fig. 1a, fig. b. antennae and palpi blackish brown. compound eyes gray with a brown spot in the middle at the level of the longitudinal stripe of the head. ocelli pale, partly surrownded by dark red-brown pigment at their inner side. thorax and abdomen unregularly brown with lighter gaps, dirty whitish ventrally. abdominal apex paler dorsally. forewings blackish brown, the pigment is a little bit denser near veins, cell cu2 paler (but not white). hind wings are the same color, but paler. morphology: macropterous. three ocelli present. lacinia with three cusps almost equal in length (fig. 1g). fore and hind wings slender with venation typical for the genus (fig. 1c, d). subgenital plate with t-sclerite as typical for the genus, having long slender curved arms (fig. 1f). epiproct rounded, paraprocts triangular. both with dense setae of two size types: very long ones and such of medium size, about twice shorter (fig. 1e). measurements (in mm): holotype (female): lc = 1.12; f+tr = 0.40; t = 0.54; t1 = 0.18, t2 = 0.05, t3 = 0.06, fw = 1.0, hw = 0.82, d = 0.06, io = 0.26, io/d = 4.33. male: unknown. diagnosis: t. zirobwensis n. sp. has a specific combination of coloration of its body parts. all known species from the genus which have brown abdomens are with brown head too (pearman, 1932; badonnel, 1977; thornton, 1984 and other) but in the new species the head is paler with a dark pattern. its head coloration and markings are same with those of t. maculata (known from belize, guadeloupe, guatemala, jamaica, mexico, usa, venezuela) (mockford & gurney, 1956) but differs by its abdomen coloration and lacinia. by its brown abdomen and slightly paler middle area of forewing cell cu2, the new species resembles t. fusca badonnel, 1977 (angola) (badonnel, 1977) but differs in its head and eye coloration, as well as the lack of pale areas at cells r and a of the forewing. etymology: named after the area of zirobwe town, uganda, where the species was found. habitat: the species was collected from an agricultural area that consists of open fields and eucalypt forest plantations. the exact microhabitat is not known but presumably dry leaves of bushes or palms. discussion psocoptera is an order of insects that is poorly studied worldwide. many species are of unclear origin, distribution, ecology and taxonomic status. many species are described from only one known specimen (like t. zirobwensis n. sp.), especially from the tropical areas, sometimes even in poor condition and with missing body parts (badonnel, 1949, 1977; broadhead & richards, 1980). the paucity of genetic studies in general for the order also makes comparing the genotypes of newly described species impossible, even if they are sequenced. sequencing for any species of the genus tapinella is lacking. concerning all these facts, the biogeographical implications of the discovery, and the conservation status of the species are quite unclear and many 126 biologica nyssana ● 14 (2) december 2023: 125-127 georgiev ● a new species of tapinella enderlein, 1908 (insecta: psocoptera), tapinella zirobwensis n. sp., from uganda fig. 1. tapinella zirobwensis n. sp., female, holotype: a – lateral view, b – dorsal view, c – forewing, d – hindwing, e – paraproct, f – subgenital plate, g – lacinia (only a and b to scale). photographs and drawings made by the author. biologica nyssana ● 14 (2) december 2023: 125-127 georgiev ● a new species of tapinella enderlein, 1908 (insecta: psocoptera), tapinella zirobwensis n. sp., from uganda 127 future studies across the tropics are needed to reach the point of making any conclusions. the wing venation is probably a not so good taxonomic feature for tapinella because of its variability and was not considered in this paper. author’s observations showed that in some genera that have both wingless and winged forms (like tapinella), there are also some intermediate transitional specimens with different development and abnormalities of wing venation (for example the rhyopsocus species). badonnel (1949) and lienhard (2008) showed different venation of the forewing of t. curvata badonnel, 1949. the main characters accepted to delimit the species of tapinella are body coloration and morphology of subgenital plate, paraprocts, epiproct and lacinia. conclusion it is evident that many new country records and new species to science could be expected from the rich and diverse equatorial area of uganda. taking into consideration this publication and the previous works of georgiev (2023a, b, c), a total of 54 species of psocoptera are now known from this country. further research activities on the psocoptera are recommended for this region, as new records and species can be expected. references badonnel, a. (1949). psocopteres du congo belge (3e note). bulletin de l’institut royal des sciences naturelles de belgique, 25(11), 1-64. badonnel, a. (1977). psocoptères de l’angola. v. publicacões culturais da companhia de diamantes de angola, 89, 103-152. broadhead, e., & richards, a. (1980). the peripsocidae and psocidae (psocoptera) of east africa. systematic entomology, 5, 357-397. georgiev, d. (2023a). new records of psocoptera (insecta) from uganda. zoonotes, 214, 1-4. georgiev, d. (2023b). on the rhyopsocus hagen, 1876 (insecta: psocoptera) of east africa with a description of two new species. historia naturalis bulgarica, 45(2), 31-37. georgiev, d. (2023c) a new species of liposcelis motschulsky, 1852 from uganda. spixiana, 45(2), 257-260. lienhard, c. (2008). order psocoptera. in: harten van a. (ed.), arthropod fauna of the united arab emirates, 1st ed., pp. 104-132, dar al ummah printing, abu dhabi, uae. mockford, e., & gurney, a. (1956). a review of the psocids, or book-lice and bark-lice, of texas (psocoptera). journal of the washington academy of sciences, 46(11), 353-368. pearman, j. (1932). a new species of tapinella (psocoptera). stylops, 1(11), 240-242. thornton, i. (1984). psocoptera and wallace’s line: collections from the islands of bali and lombok. treubia, 29(2), 83-177. anticancer compounds from medicinal plants biologica nyssana 5 (1)  september 2014: 1-10 kamberović, j. et al.  marshland vegetation of the order phragmitetalia … 1 original article received: 26 june 2014 revised: 28 august 2014 accepted: 10 september 2014 marshland vegetation of the order phragmitetalia on shores of mine pit lakes in north-eastern bosnia and herzegovina jasmina kamberović 1* , senka barudanović 2 , ermin mašić 2 , anita dedić 3 1 university of tuzla, faculty of natural sciences and mathematics, department of biology, univerzitetska 4, 75 000 tuzla, bosnia and herzegovina 2 university of sarajevo, faculty of natural sciences and mathematics, department of biology, ulica zmaja od bosne 33-35, 71 000 sarajevo, bosnia and herzegovina 3 university of mostar, faculty of science and education, department of biology, matice hrvatske bb, 88 000 mostar, bosnia and herzegovina * e-mail: jasmina.kamberovic@untz.ba abstract: kamberović, j., barudanović, s., mašić, e., dedić, a.: marshland vegetation of the order phragmitetalia on shores of mine pit lakes in north-eastern bosnia and herzegovina. biologica nyssana, 5 (1), septemeber 2014: 1-10. marshland vegetation of the order phragmitetalia w. koch 1926 on the four mine pit lakes in the wider area of tuzla was investigated during 2008. in total, two plant communities were noted. on the lakes suhodanj, mušićko and šićki brod association typhetum latifoliae g. lang 1973 were recorded, within which was noted 33 plant species. on the shores of lakes ramićko and šićki brod the dominant association was phragmitetum australis schmale 1939, within which were determined 32 plant species. this paper analyzes the life forms and bioindicator values of plant species. the research results indicate a successful process of colonization by macrophyte species on absolutely degraded habitats such as mine pit lakes. key words: bioindicators, biodiversity, colonization, mine pit lakes, vegetation, wetlands apstrakt: kamberović, j., barudanović, s., mašić, e., dedić, a.: močvarna vegetacija reda phragmitetalia na obalama kopovskih jezera u severoistočnoj bosni i hercegovini.. biologica nyssana, 5 (1), septemebar 2014: 1-10. močvarna vegetacija reda phragmitetalia w. koch 26 istraživana je u toku 2008. godine na obalama četiri kopovska jezera šireg područja tuzle. na istraživanom području određene su dve biljne zajednice. na jezerima suhodanj, mušićko i šićki brod prisutna je zajednica typhetum latifoliae g. lang 1973, u sklopu koje je zabeleženo prisustvo 33 biljne vrste. na obalama jezera ramićko i šićki brod dominantna je zajednica phragmitetum australis schmale 1939, gde su određene 32 biljne vrste. u radu su analizirane životne forme, bioindikatorske vrednosti i socijabilnost biljnih vrsta. rezultati istraživanja upućuju na uspešan proces kolonizacije močvarnih biljnih vrsta na apsolutno degradiranim staništima, kao što su kopovska jezera. key words: bioindikatori, biodiverzitet, kolonizacija, kopovska jezera, vegetacija, vlažna staništa 5 (1) • september 2014: 1-10 biologica nyssana 5 (1)  september 2014: 1-10 kamberović, j. et al.  marshland vegetation of the order phragmitetalia … 2 introduction plant communities of the order phragmitetalia represent clearly defined zone of emersal vegetation on shores of lakes, ponds and river valleys (r a n đ e l o v i ć et al., 2007; p o l i ć , 2006). they develop on wetlands, gley soils, often peaty soils, with intensive reduction processes (r e d ž i ć et al., 2009). in bosnia and herzegovina they are spread on the area of hutovo blato, bardača, ždralovac, velika and mala tišina near bosanski šamac, some parts of the lake modrac and are fragmented around many small lakes (r e d ž i ć et al., eds., 2008). studies of plant communities of the class phragmitetea in bosnia and herzegovina have been known for a sub-mediterranean area (j a s p r i c a et al., 2003), where in hutovo blato 11 plant communities from three vegetation alliances were determined. in addition to the above mentioned natural habitats, emergent hydrophytes in progradation processes also inhabit shores of anthropogenicallyformed lakes. examples are numerous pit lakes, created in the process of surface coal mining. pit lakes have the potential to conserve wetland biodiversity (b a r u d a n o v i ć & k a m b e r o v i ć , 2008), being overgrown by vegetation of submerged hydrophytes (k a m b e r o v i ć & b a r u d a n o v i ć , 2012) and weed vegetation (b a r u d a n o v i ć & k a m b e r o v i ć , 2011). since the community structure of emergent hydrophytes on the shores of pit lakes in tuzla region is insufficiently known, this paper aims to (i) determine the structure of communities from the order phragmitetalia at artificially formed habitats, (ii) compare these communities with the structure of communities at natural habitats and (iii) assess the ecological characteristics of the habitat on the basis of ecological indexes. study area the research on marshland vegetation was carried out in 2008 in northeastern bosnia and herzegovina on the shores of four pit lakes: suhodanj lake (su, fig. 1), mušićko lake (mj, fig. 2), ramićko lake (rj, fig. 3) and šićki brod lake (šb, fig. 4). lakes were formed during the period from 1982-1987 and each have their own water regime. the lakes suhodanj and ramićko were created by damming the flow of surface water with the tailings material on a partly degraded terrain. the lakes mušićko and šićki brod were formed in the final crater after the cessation of surface coal mining. basic characteristics of the figure 1. lake suhodanj (su) figure 2. lake mušićko (mj) figure 3. lake ramićko (rj) figure 4. lake šićki brod (šb) biologica nyssana 5 (1)  september 2014: 1-10 kamberović, j. et al.  marshland vegetation of the order phragmitetalia … 3 table 1. the basic characteristics of the researched lakes (k a m b e r o v i ć , 2010) table 2. the physical and chemical parameters of water quality of the studied lakes in august 2008 indicators su šb mj rj water temperature ( 0 c) 26 25.5 24 26 turbidity (ntu) 1.5 1.3 2.5 3.5 ph 8.1 8.2 7.8 8.3 total alkalinity (mg caco3l -1 ) 157 147 287 172 carbonate hardness (°dh) 8.8 8.3 16.1 9.7 noncarbonated hardness (°dh) 11.2 8.3 40.3 0.1 total hardness (°dh) 20.2 26.5 56.4 9.8 dissolved oxygen (mgl -1 ) 10 8.7 10.2 8.5 oxygen saturation (%) 116 109 127 104 kmno4 consumption (mgl -1 ) 10.5 14.2 12.6 17.2 total nitrogen (mgl -1 ) 0.18 0.02 1.46 0.18 ammonium (mgl -1 ) 0.16 0.02 0.59 0.16 nitrite (mgl -1 ) 0.018 0.004 0.424 0.004 nitrate (mgl -1 ) 0 0 0.45 0.02 calcium (mgl -1 ) 44.1 122 222.4 31.1 magnesium (mgl -1 ) 60.2 40.7 110 23.7 electrical conductivity (µs/cm) 535 682 1562 280 studied lakes are given in tab. 1. the research area belongs to the temperate continental climate region. the average temperature varies from 9 0 c to 10.6 0 c, and the annual precipitation ranges from 830 l/m 2 to 1.150 l/m 2 (s m a j i ć , 2007). material and methods phytocenological relevés of coastal vegetation were carried out using the zűrich–montpellier school in three vegetation seasons (b r a u n b l a n q u e t , 1964). phytocenological relevés from the spring season with the designation of the lake are additionally marked with the letter "a", the summer season with the letter "b" and the autumn season with the letter "c". determination of plants is done on the basis of herbarium materials according to the relevant floras (t u t i n e t a l ., eds., 19641993, d o m a c , 2002, javorka & c s a p o d y , 1979). the cover of taxa was determined using the numerical scale according to westhoff van der maarel (k o j i ć e t a l ., 1997), and syntaxonomic affiliation of associations according to l a k u š i ć e t a l . (1977). life forms of plant species are taken from o b e r d o r f e r (1979). sociability of plant species and environmental factors (temperature, humidity, soil acidity, nitrification, light, continentality and salinity) are given according to ellenberg (b o r h i d i , 1993). the research of physical/chemical properties of water was done on the field and in the laboratory. the water temperature and the ph of water were lakes suhodanj (su šićki brod (šb) mušići (mj) ramići (rj) year of origin 1985 1987 1982 1983 elevation (m) 293 207 357 378 geographic coordinates 44°23`37``n 44°31`42``n 44°23`45``n 44°25`06``n 18°38`41``e 18°34`44``e 18°30`24``e 18°25`29``e lake's surface (ha) 2.5 20.8 5.53 6.2 max. lake's depth (m) 18 33 6 18 geological basis marl sandy marl marl and serpentine biologica nyssana 5 (1)  september 2014: 1-10 kamberović, j. et al.  marshland vegetation of the order phragmitetalia … 4 measured on the field, while other physical/chemical parameters (turbidity, total alkalinity, carbonate hardness, noncarbonated hardness, total hardness, dissolved oxygen, oxygen saturation, kmno4 consumption, total nitrogen, ammonium, nitrite, nitrate, calcium, magnesium, electrical conductivity) were determined in the laboratory of the institute for chemical engineering in tuzla, by the standard procedure of the american health organization (apha, awwa & wef, 1995). in the statistical analysis we used complete linkage clustering method in primer 6 (c l a r k e & g o r l e y , 2006). the diversity of assemblages was quantified with the shannon-wiener diversity index by the use of the biodiversity pro version 2 software packages (m c a l e e c e et al., 1997). results and discussion physico-chemical analysis of water the water of all four researched lakes has a weak alkaline reaction (ph 7.8-8.3). most pit lakes in the world have an acid reaction of water (n i x d o r f et al., 2005). v e l a g i ć h a b u l et al. (2005) concluded that neutralisation and acidification in mining lakes of the tuzla area are to date producing a cirumneutral water quality. investigated lakes except lake mušićko have sufficient oxygen regime and generally low concentrations of nutrients. lake mušićko has the highest level of water hardness, high calcium and magnesium concentrations, high value of electrical conductivity and the highest value of total nitrogen (tab. 2). previous studies (k a m b e r o v i c & b a r u d a n o v i ć , 2012) found that the lakes suhodanj and šićki brod are characterized by oligo/ß-mesosaprobic status of water with submerged vegetation of charophyta. lakes mušićko and ramićko have ß-mesosaprobic water status and are partially overgrown with vegetation of the alliance magnopotamion. marshland vegetation marshland vegetation of the order phragmitetalia w. koch 1926 in the study area is represented with the alliance phragmition australis w. koch 1926 and two plant association: typhetum latifoliae g. lang 1973 and phragmitetum australis schmale 1939. the community typhetum latifoliae g. lang 1973 occurs in habitats with shallow water (up to 0.5 meters deep) at the lakes suhodanj, mušićko and šićki brod (tab. 3). it is most developed at the lake mušićko and has a dominant role in the appearance of the lake scenery. besides broadleaf cattail (typha latifolia), in this community also occur other semiaquatic plants: mentha aquatica, lycopus europaeus, alisma plantago-aquatica, typha angustifolia and juncus effusus. this community is a very widespread community of shallow parts of water pools with a quiet eutrophic water (s t a n č i ć , 2010). mostly it represents the transition between the water vegetation and the vegetation of reed beds. communities of cattail are resistant for a longer period of drying, salinization and water pollution (p o l i ć , 2006). the community phragmitetum australis schmale 1939 occurs on some more drained terrain compared to the previous community and it was identified at two lakes. on the lake šićki brod it grows over the northwestern lake shore and has a significant role in progradation processes, while at ramićko lake occurs in the form of a narrow zone on the west shore. the floristic composition is dominated by common reed phragmites australis, while all other species are represented with clearly low covering degree (tab. 4). determination of plant communities was confirmed by cluster analysis. clearly separated two clusters indicate differentiation in two plant communities, while the subgroups are formed in relation to the research sites (fig. 5). the floristic composition of the association changes over the year. relevés in the spring season have lower number of species compared to the summer and autumn relevés when terrestrial plant species occure. in cluster analysis, in addition to the original, for comparison purposes we included also results of other similar research. from the dendrogram is evident that the community of reed beds on the lake šićki brod is most similar to stands in serbia and croatia (l a z i ć et al., 2005, p o l i ć , 2006, s t a n č i ć , 2010) with which it shares only three common characteristic plant species of the order phragmitetalia (phragmites australis, mentha aquatica and lycopus europaeus). the community of reed beds on ramićko lake is most similar to the stands in the sub-mediterranean part of bosnia and herzegovina (j a s p r i c a et al., 2003) with which it has four plant species in common (phragmites australis, scirpus sylvaticus, potentilla reptans and calystegia sepium). the communities of cattail at pit lakes in comparison with other studies do not show a great similarity, except for those on the lake šićki brod. differences in the structure of the examined communities are pronounced because of the participation of some characteristic plant species at the localities in serbia, croatia and herzegovina (e.g. glyceria maxima, acorus biologica nyssana 5 (1)  september 2014: 1-10 kamberović, j. et al.  marshland vegetation of the order phragmitetalia … 5 calamus, scirpus lacustris) that are absent at pit lakes. table 3. association typhetum latifoliae g. lang 1973 in littoral of studied pit lakes (the cover of taxa is given according to westhoff van der maarel) association typhetum latifoliae g. lang 1973 relevé number 1 2 3 4 5 6 7 8 9 the locality mja mjb mjc sua sub suc šba šbb šbc d eg re e o f p re se n ce area of relevé (m 2 ) 50 50 50 50 50 50 30 30 30 density (%) 60 85 85 80 95 90 90 95 95 char. ass. typha latifolia l. 8 9 9 8 8 8 8 8 8 v typha angustifolia l. 3 3 3 7 7 7 iv phragmites australis (cav.) trin. ex steud. 5 5 5 ii char. alliance phragmition, char. order phragmitetalia & char. class mentha aquatica l. 3 3 3 2 2 3 iv lycopus europaeus l. 3 3 3 2 3 3 iv alisma plantago-aquatica l. 3 3 3 2 3 3 iv sparganium erectum l. 3 3 3 ii carex vulpina l. 3 3 2 ii lythrum salicaria l. 2 2 2 ii phalaris arundinacea l. 2 3 3 ii epilobium palustre l. 2 3 ii companions cyperus fuscus l. 2 3 3 2 2 2 2 2 v ranunculus repens l. 2 2 3 2 iii juncus effusus l. 3 3 3 ii lysimachia vulgaris l. 3 2 2 ii inula britannica l. 2 2 3 ii eupatorium cannabinum l. 2 3 3 ii elymus repens (l.) gould 2 3 3 ii potentilla reptans l. 2 2 2 ii plantago major l. 2 2 2 ii equisetum telmateia ehrh. 2 3 3 ii erigeron annuus (l.) pers. 2 2 2 ii tussilago farfara l. 2 2 2 ii artemisia vulgaris l. 2 2 2 ii polygonum persicaria l. 2 2 2 ii rumex sanguineus l. 2 3 ii poa pratensis l. 2 2 ii prunella vulgaris l. 2 2 ii bidens tripartita l. 2 2 ii polygonum lapathifolium l. 3 3 ii carex hirta l. 3 i mentha pulegium l. 2 i lycopus exaltatus l. 2 i biologica nyssana 5 (1)  september 2014: 1-10 kamberović, j. et al.  marshland vegetation of the order phragmitetalia … 6 table 4. association phragmitetum australis schmale 1939 in littoral of studied pit lakes (the cover of taxa is given according to westhoff van der maarel) association phragmitetum australis schmale 1939 relevé number 1 2 3 4 5 6 the locality šba šbb šbc rja rjb rjc d eg re e o f p re se n ce area of relevé (m 2 ) 30 30 30 50 50 50 density (%) 80 90 90 100 100 100 char. ass. phragmites australis (cav.) trin. ex steud. 9 9 9 9 9 9 v typha latifolia l. 3 3 3 iii char. alliance phragmition, char. order phragmitetalia & char. class lycopus europeus l. 2 2 2 2 2 2 v alisma plantago-aquatica l. 2 2 2 2 2 2 v epilobium palustre l. 2 2 2 3 iv mentha aquatica l. 2 2 2 iii sparganium erectum l. 2 2 2 iii scirpus sylvaticus l. 2 2 2 iii lythrum salicaria l. 3 3 ii companions ranunculus repens l. 3 2 2 3 3 3 v poa pratensis l. 2 2 3 iii salix purpurea l. 2 2 2 iii equisetum palustre l. 2 2 2 2 iv elymus repens (l.) gould 5 5 5 iii potentilla reptans l. 3 3 3 iii agrostis stolonifera l. 3 3 3 iii calystegia sepium /l./r.br. 5 5 5 iii plantago altissima l. 2 2 2 iii tanacetum vulgare l. 2 2 ii conyza canadensis (l.) cronquist 2 2 ii cyperus fuscus l. 2 2 ii eupatorium cannabinum l. 3 3 ii mentha pulegium l. 3 2 ii bidens tripartita l. 3 3 ii polygonum mite schrank 3 3 ii polygonum lapathifolium l. 2 2 ii echinocystis lobata (michx.) torr. & a.gray 5 5 ii atriplex patula srn. 3 3 ii trifolium repens l. 3 3 ii sonchus arvensis l. 2 3 ii pastinaca sativa l. 2 2 ii verbena officinalis l. 2 2 ii biologica nyssana 5 (1)  september 2014: 1-10 kamberović, j. et al.  marshland vegetation of the order phragmitetalia … 7 figure 5. dendrogram of the original (mj, su, šb and rj) and campared relevés (s t a n č i ć , 2010, p o l i ć , 2006, j a s p r i c a et al., 2003, l a z i ć 2006), complete linkage cluster analysis. p.a. phragmitetum australis; t.l. typhetum latifoliae. biodiversity of associations as part of the order phragmitetalia at the researched sites, a total of 50 plant species were determined. the value of shannon -wiener diversity index ranges from 2.10 to 3.15 (fig. 6). within the community typhetum latifoliae were recorded 33 plant species, and the largest number of species was observed on the lake mušićko (19). in the community phragmitetum australis were determined 32 plant species. here we observed largest deviations in biodiversity with a small number of species at the lake šićki brod (13), which is probably caused by unfavorable pedological conditions (sandy soil and poor water regime). shannon index results mja typh.lat. mjb typh.lat. m.j. typh.lat. su.a typh.lat. su.b typh.lat. su.c typh.lat. š.b.a-typh.lat. š.b.b typh.lat. š.b.c-typh.lat. š.b.a -phr. aus. š.b.b phr. aus.. š.b.c phr. aus. r.j.a phr. aus. r.j.b phr. aus. r.j.c phr. aus. v a lu e sample 3,1 3,2 3,3 2,4 2,5 2,6 2,7 2,8 2,9 2,10 2,11 2,12 2,13 3,14 3,15 0 1 2 3 4 0 5 10 15 figure 6. the values of shannon wiener diversity index biologica nyssana 5 (1)  september 2014: 1-10 kamberović, j. et al.  marshland vegetation of the order phragmitetalia … 8 figure 7. ecological analysis of the studied association (t-temperature, w-humidity, r-soil acidity, n-nitrification, l-light, k-continentality, s-salinity) ecological analysis of associations ecological indexes of plant species from the analyzed communities indicate that stands appear on alkaline (r=6.3-6.8), non-salinated (s=0.5-0.7), well exposed to sunlight or shaded occasionally (l=7.17.6), briefly flooded (w=7.5-8.7) and mesotrophic soils (n=5-6.7). habitat requirements about heat are moderate (t=5.1-5.8), and are characterized by a large proportion of sub-oceanic species which find their main areal in the central europe (k=4-4.6) (fig. 7). comparing ecological indexes of the studied communities with the stands in the "labudovo okno", all values are in close ranges, except for the humidity index, which was lower for the pit lakes habitats (w = 7.5 8.7), indicating a less favorable hydro regime. biological spectrum and sociability of plant species in the studied communities at the localities of the lakes suhodanj and šićki brod the most dominant life form are hydrophytes (up to 46.15%), while hemicryptophytes are dominant at other sites (up to 44.8%) (fig. 8). compared to the stands of the order phragmitetalia in serbia (p o l i ć , 2006) and croatia (s t a n č i ć , 2010), where the dominance in the stands is achieved by hydrophytes, hemicryptophytes and geophytes, in the communities on pit lake shores the share therophytes is not negligible (7.69 25.92%). this phenomenon makes the community's response to frequent changes in water levels and to periods of drought during the summer months. figure 8. the biological spectrum of the studied association (t therophytes, g geophytes, ch chamaephytes, h hemicriptophytes, w hydrophytes, pn phanerophytes) biologica nyssana 5 (1)  september 2014: 1-10 kamberović, j. et al.  marshland vegetation of the order phragmitetalia … 9 figure 9. the social behavior types spectrum of the studied association (ac invasive species, rc ruderal competitors, w weed species, dc pioneer species of secondary successions, g associated plant species, c competitors, s sensitive species) the spectrum of sociability of the plant species indicates the state of the natural vegetation progradation. analysis of the spectrum of plant species sociability indicates that the most frequent are pioneering elements of secondary successions (dt = 23.07 55%) and associated plant species (g=18,18-30,76%). a lower frequency have the competitor plant species (c = 7.69 27.23) and they are most numerous in the communities on the lake šićki brod (fig. 9). weed plant species are also present (plantago major, bidens tripartita, artemisia vulgaris, atriplex patula, verbena officinalis and sonchus arvensis), and from invasive species there were identified three plant species (echinocystis lobata, conyza canadensis and erigeron annuus). r a n d j e l o v i c et al. (2007) emphasize the eurivalence of the community of reed beds for many environmental factors, therefore it is very often penetrated by elements of meadow and weed vegetation. on the shores of pit lakes this phenomenon is very evident, due to close contact of these communities with trampled and ruderal habitats in landfills of tailings, leading to the appearance of invasive plant species. progradation process in natural habitats the communities of reed beds and cattail play a crucial role for the stability and the climax of wetland ecosystems, while in the degraded habitats such as pit lakes they are crucial for the restoration and progradation. vegetation of cattail often grow over anthropogenic habitats of eutrophic character that had been under water for a long period of time (s t a n č i ć , 2010). among the researched localities it was most developed at the lake mušićko, which has a minimum depth, a small slope of the southern shore and water of lower quality. at the lakes suhodanj and šićki brod this community grows over a very small area (up to 50 m 2 ). the factors that prevent its spread are probably variations of water levels and the dominance of communities of the class bidentetea tripartitae (b a r u d a n o v i ć & k a m b e r o v i ć , 2011) on the lake suhodanj and a large slope of the littoral zone on the lake šićki brod. the vegetation of reed beds is more eurivalent for the moisture factor. therefore, at the lakes with large slope of the littoral zone (šićki brod and ramićko lakes), where there are not achieved conditions for communities of cattail, reed beds grow over the coastal region and tolerate longer periods of dryness. frequent fluctuations of water levels throughout the year and steep banks of pit lakes allow colonization of emersed hydrophytes in fragments only on the flat shores. therefore the progradation processes in degraded habitats in terms of establishing wetland ecosystems are possible only in lakes with lower slopes of the shore and more or less stable water levels. conclusion on the shores of four pit lakes within the order phragmitetalia there were determined 50 plant species within two plant communities. in phytocenological releves thirteen species characteristic of the association, alliance, order or class were identified. sociability spectrum of plant species, along with the domination by species of pioneering elements of secondary succession, indicates the state of natural progradation processes. bearing in mind that pit lakes in the initial stages of biologica nyssana 5 (1)  september 2014: 1-10 kamberović, j. et al.  marshland vegetation of the order phragmitetalia … 10 formation are absolutely degraded surfaces, the results of research point to the successful colonization of wetland plant species, whose natural progradation is ongoing. references apha, awwa & wef, 1995: standard methods for the examination of water and wastewater. 19 th edition, washington. barudanović, s., kamberović, j. 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(ed.) 2008: bosna i hercegovina – zemlja raznolikosti, pregled i stanje biološke i pejzažne raznolikosti bosne i hercegovine, prvi izvještaj bih za cbd, bemust, sarajevo. redžić, s., barudanović, s. trakić, s., kulijer, d. 2009. reedbeds, tall sedges and vegetation of phragmito-magnocaricetea in: biodiversity working group 2009: habitat interpretation sheets natura 2000 habitat types occurring along the sava river. final technical document, no. bwg-2008-01:44. stančić, z. 2010: marshland vegetation of the class phragmito-magnocaricetea in northwest croatia (krapina river valley). biologia, 65 (1): 39-53. tutin, t. g., heywood, v. h., burges, n. a., valentine d. h., walters, s.m. & webb, d.a. /ed./ 1964-1993: flora europaea 1-5, cambridge university press, cambridge. velagić-habul, e., bašagić, m. & omanović, e. 2005: uticaj jalovišta površinskih kopova uglja na kvalitet vode tekućica. radovi poljoprivrednog fakulteta univerziteta u sarajevu, l. 56. biologica nyssana 5 (1)  september 2014: 1-10 kamberović, j. et al.  marshland vegetation of the order phragmitetalia … 11 nikolic et al. 2021, biologica nyssana 12(2) 12 (2) december 2021: 159-165 doi: 10.5281/zenodo.5759870 evaluation of ecological awareness and superstition on hermann’s tortoise in eastern and southern serbia original article marko nikolić faculty of sciences and mathematics, university of niš, višegradska 33, 18000 niš, serbia biological society “dr. sava petrović”, višegradska 33, 18000 niš, serbia marko@bddsp.org.rs (corresponding author) dimitrija savić-zdravković faculty of sciences and mathematics, university of niš, višegradska 33, 18000 niš, serbia biological society “dr. sava petrović”, višegradska 33, 18000 niš, serbia jelka crnobrnja-isailović faculty of sciences and mathematics, university of niš, višegradska 33, 18000 niš, serbia institute for biological research “siniša stanković” – institute of national importance for republic of serbia, university of belgrade, despota stefana 142, 11000 belgrade, serbia received: november 01, 2021 revised: november 26, 2021 accepted: december 02, 2021 abstract: folk beliefs, religion and mythology, created through various cultural influences in serbia, shaped today’s attitudes toward wildlife. in this study we evaluated different levels of ecological awareness and superstition extent related to hermann’s tortoise (testudo hermanni) through a systematized face-to-face questionnaire. the study was conducted in two protected and two unprotected areas of southern and eastern serbia. the results confirmed the presence of myths and superstitions amongst locals and existing conflict between the humans and t. hermanni. these results also showed the lack of knowledge about national regulations related to protection of nature and t. hermanni, even in protected areas, with statistically signifficant differences among localities. to reduce this human/wildlife conflict, future conservation measures would have to take into account the views of the local population. key words: testudo hermanni, serbia, ethnozoology, survey, questionnaire, protected areas, human-wildlife conflict apstrakt: procena nivoa ekološke svesti i sujeverja o šumskoj kornjači u istočnoj i južnoj srbiji narodna verovanja, religija i mitologija, nastali pod dejstvom različitih kulturoloških uticaja u srbiji, oblikovali su današnji odnos čoveka prema prirodi. u ovoj studiji procenjivali smo različite nivoe ekološke svesti i sujeverja lokalnog stanovništva o šumskoj kornjači (testudo hermanni) putem anketiranja. studija je sprovedena na dva zaštićena i dva nezaštićena područja južne i istočne srbije. rezultati su potvrdili prisustvo mitova i sujeverja među lokalnim stanovništvom i postojanje sukoba između ljudi i šumske kornjače. ovi rezultati, takođe, pokazuju i nedostatak znanja o nacionalnim propisima koji se odnose na zaštitu prirode i zaštitu šumske kornjače, čak i kod stanovništva u zaštićenim područjima, sa statistički značajnim razlikama među lokalitetima. da bi se smanjio ovaj sukob između ljudi i divljih životinja, buduće mere zaštite bi morale da uzmu u obzir stavove lokalnog stanovništva. ključne reči: testudo hermanni, srbija, etnozoologija, anketa, upitnik, zaštićena područja, sukob između ljudi i divljih životinja introduction as it has been shown in many places worldwide, attitude of the local community regarding importance and conservation of wildlife plays a major role in restricting direct and indirect human impact on habitats and populations of endangered species. local resource users can affect the implementation of conservation measures positively or negatively, therefore being a powerful conservation factor (ramstad et al., 2007; røskaft, et al., 2007; ebua et al., 2011; talukdar & gupta, 2018). local folk beliefs, religion and mythology contribute vastly to the formation of attitudes and opinions of the native local human population (further referred to as locals) towards nature and wildlife. therefore, in recent decades, interdisciplinary fields of study, such as ethnozoology, nature conservation marketing, or social psychology, have made significant contributions to biodiversity conservation (dickman, 2010; alves & souto, 2015; wright et al., 2015). in serbia, various cultural influences shaped today’s attitudes toward wildlife. one of the most serious wildlife threats in this country is illegal collecting for a variety of reasons, major ones being: for pet shops, use in food, folk medicine, or due to superstition and magic rituals (durst & mikuška, 2017; nikolić & golubović, 2017; jovanović et al., © 2021 nikolić et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 159 2020). tolstoy & radenkovic (2001) describe the role of many animal species in local mythology, and eastern hermann’s tortoise (testudo hermanni boettgeri) is one of them. in addition to already known threatening factors such as natural catastrophes, road casualties, habitat fragmentation and loss (including urbanization), a direct human-wildlife conflict is also present (van dijk et al., 2004; bertolero et al., 2011; fernández–chacón et al., 2011; celse et al., 2014; nikolić & crnobrnja-isailović, 2017). nowadays, one of the most common myths about hermann’s tortoise is the belief in the healing properties of tortoise blood, which is believed to help treat serious illnesses (nikolić & crnobrnja-isailović, 2017). according to the red book of reptiles of serbia (tomović et al., 2015), the hermann’s tortoise belongs to national category nt (djordjević & ljubisavljević, 2015) and its’ global iucn red list status is also nt (van dijk et al., 2004). besides, hermann’s tortoise is listed in annexes ii and iv of the habitats directive, in annex ii of the bern convention, and in annex ii of the cites convention. although hermann’s tortoise is protected by both international and national acts, the problem of illegal hunting and trading is still unresolved. tortoises are being collected and transported mainly to european union countries. thus, in 2005, 504 hermann’s tortoises were seized on the border between serbia and croatia, and a similar situation occurred in 2006 with 22 hermann’s and greek tortoises (testudo graeca) (jovanović & ajtić, 2011). in order to solve this problem, it is necessary to understand it. success in reducing negative anthropogenic impact and maintaining long-term sustainable management of natural resources depends a lot on the support of the local community. also, understanding the attitudes of the locals, taking into account their needs, and respecting their opinions should become a priority in creating effective protection and conservation measures (macura et al., 2011). the main aim of this research was to determine and evaluate different levels of ecological awareness and superstition extent occurring among local inhabitants in protected and unprotected areas situated in the southern and eastern regions of serbia. to achieve this goal, we conducted a survey about the awareness of the locals on the existence of protected areas and knowledge of national regulations related to protection of both nature and hermann’s tortoise. finally, we examined the presence of myths and superstitions amongst locals, as well as their perceptions of the costs and benefits of hermann’s tortoise protection. materials and methods study area surveying was conducted in a total of four locations in the municipalities of serbia two in the east and two in the south of the country (fig. 1). two of the four sites were located in protected areas: donji milanovac, on the territory of the “djerdap” national park in eastern serbia, and kunovica, 15 km from the city of niš, on the territory of “sićevačka klisura” nature park in southern serbia (niška banja municipality). the other two sites were not located within the protected areas: kladovo in eastern serbia and leskovac in southern serbia. the presence of hermann’s tortoise populations was confirmed on all 4 localities (golubović et al., 2019; nikolić et al., 2020). sampling fieldwork was carried out from may to october 2016. the survey was conducted for 5 days per 160 biologica nyssana ● 12 (2) december 2021: 159-165 nikolić et al. ● evaluation of ecological awareness and superstition on hermann’s tortoise in eastern and southern serbia fig. 1. map of serbia with the research localities marked as following: 1 – donji milanovac, 2 – kladovo, 3 – niš, 4 – leskovac; 1 44°27’50.48”n, 22° 9’2.63”e; 2 44°36’24.96”n, 22°36’49.19”e; 3 43°19’5.73”n, 21°53’47.89”e; 4 42°59’48.00”n, 21°56’39.03”e 161 locality, and residents were interviewed between 9:00 and 17:00. the research team toured the defined area on foot and, according to the principle of chance, the residents were interviewed face-toface. the conversation with one respondent lasted 15-20 minutes. during one day, 20-25 respondents were interviewed. our total sample included 389 inhabitants (tab. 1). questionnaire the face-to-face questionnaire was designed based on a study by veličković et al. (2015). the questions were divided into three sections. the first section contained questions about the respondents’ awareness of the existence and role of protected areas, and the presence of such areas in their close surroundings. the second section contained questions about superstition on one hand; as well as questions about the presence of myths related to the healing properties of hermann’s tortoise and whether the respondents believed in them, on the other hand. respondents were also able to state whether they were aware of what was believed to be used for these purposes (blood, eggs, legs or something else that was not listed), which diseases are believed to be curable in this way, have they heard of cures, and have they had similar experiences. finally, respondents answered questions about the existence of legal protection for hermann’s tortoise and whether conservation actions should be done to biologica nyssana ● 12 (2) december 2021: 159-165 nikolić et al. ● evaluation of ecological awareness and superstition on hermann’s tortoise in eastern and southern serbia table 1. basic data about respondents locality region protected area number of inhabitants number of respondents average age (%) male respondents (%) donji milanovac east serbia yes 2410 97 49.1 49.5 kladovo east serbia no 8869 93 44.5 55.9 niš (niška banja municipality) south serbia yes 14098 100 48.6 40 leskovac south serbia no 60288 99 49.8 51.5 preserve this species. the questions were formulated in a non-suggestive way to enable to the respondents to give as honest and complete answers as possible. statistical analysis respondents’ answers to superstition questions were presented in the form of categorical variables, and the chi-square (with yates correction for continuity for 2x2 tables) test of the ibm spss statistics ver. 25. software package was used to assess the statistical significance of differences in respondents’ answers. the awareness of the locals about the existence of protected areas in their surroundings and the presence of myths about the healing properties of tortoises was marked with appropriate codes in relation to the attitude towards their claims (1–yes, 2–no, 3–not familiar with the topic). the data was organized using microsoft office 365 excel apps, where graphic figures were also created. results the survey conducted at four localities in serbia included a total of 389 respondents (tab. 1). by analyzing the survey results and applying the chi-square test (tab. 2) we found that there were mild statistically significant differences between the number of superstitious residents of protected areas compared to other respondents: x2(1, n=389) = 4.07, p=0.044. the results have shown that many respondents believe in the myth that blood, eggs or meat of hermann’s tortoise have healing properties (tab. 3). as many as 72.6% of respondents in eastern serbia answered that these myths are present in their environment and that they knew someone who used tortoises for healing purposes. in relation to 55.3% of respondents in southern serbia, significant statistical differences were found: x2(1, n=389)=11.93, p=0.001. statistically significant differences were also found when it comes to the percentage distribution by localities x2(3, n=389) =16.24, p=0.001. most respondents who believed table 2. superstitious respondents in protected and unprotected areas (in percentages) % of respondents df syg. protected area 34.2 1 0.044 non-protected area 24.4 1 162 in the myth of the healing properties of hermann’s tortoise were recorded in donji milanovac (76.3%), and the least in leskovac (50%) (tab. 4). out of a total of 197 respondents living in selected localities within protected areas in southern and eastern serbia, 55.3% of respondents were informed about the existence of protected areas and of the fact that they are located in their immediate vicinity (fig. 2). additionally, 44.7% of respondents outside protected areas were informed according to the survey. the most informed inhabitants were found in donji milanovac (68%, p=0.000), and the least in leskovac (19%, p=0.000). attitudes of the locals that the hermann’s tortoise should be protected and preserved were statistically significantly differentamong localities x2 (3, n =389)=8.71, p=0.033. out of the total number of respondents who agree that hermann’s tortoise should be protected, the smallest share was donji milanovac with 23% (fig. 3), which is located in both eastern serbia and in the protected area. discussion the interaction of the local, indigenous human population with nature is conditioned by various direct or indirect reasons. the direct reasons are the purchase of food, the purchase of medicinal raw materials, or the quest for new natural sites (alves & souto, 2015). as humans have become the dominant species on the planet (ehrlich & ehrlich, 2008), throughout history human civilization has influenced the living world and biodiversity in many ways. in general, the interaction of the locals with nature has always taken place in different ways, and they vary depending on the cultural influence and the environment itself that we observe (alves & souto, 2015). understanding the ecological context for resolving human-wildlife conflicts is not enough; therefore, different socio-economic aspects, cultural influences, attitudes of the actors involved and interactions between them must be considered (salom et al., 2021). our research has shown that 34.2% of the population in protected areas was superstitious (tab. 2), in comparison to 24.4% (p=0.044) of biologica nyssana ● 12 (2) december 2021: 159-165 nikolić et al. ● evaluation of ecological awareness and superstition on hermann’s tortoise in eastern and southern serbia table 3. respondents per region who believe in the healing properties of hermann’s tortoise (in percentages) region % of respondents df syg. east serbia 72.6 1 0.001 south serbia 55.3 1 fig. 2. awareness of the respondents about the existence of protected areas in their environment in relation to whether they live in the territory of the protected area or not fig. 3. respondents who are aware that the hermann’s tortoise should be protected by law (percentage representation of respondents in relation to the total sample) locality ht df syg. pa df syg. donji milanovac 76.3 3 0.001 68.0 3 0.001 kladovo 68.8 3 65.6 3 niš 60.6 3 33.3 3 leskovac 50.0 3 19.0 3 table 4. respondents per locality who believe in the healing properties of hermann’s tortoise (in percentages) (column “ht”) and difference inawareness of the respondents from different localities about the existence of protected areas in their surroundings (column “pa”) 163 biologica nyssana ● 12 (2) december 2021: 159-165 nikolić et al. ● evaluation of ecological awareness and superstition on hermann’s tortoise in eastern and southern serbia superstitious people who live in non-protected areas. the population that inhabits protected areas lives in more rural environment (economically underdeveloped areas far from large settlements), so it can be assumed that this is another reason why myths and folk beliefs are more present there. in the results of the study by nikolić et al. (2019) it was stated that superstition is more present in eastern than in southern serbia. the presence of superstitions related to wild animals also means a greater threat to wildlife (kulišić et al., 1998). our previous research confirmed the assumption that local beliefs and myths can be a powerful cause of animal endangerment in both eastern and southern serbia (nikolić & crnobrnja-isailović, 2017; nikolić et al., 2018). a superstitious human population also could mean a greater impact of myths and folk beliefs on increase of hunting, killing and expelling animals from their natural habitat. in tab. 3, 72.6% (p=0.001) of the respondents who inhabit the protected areas believed that hermann’s tortoise has healing properties. myths about the healing properties of hermann’s tortoise blood, eggs or meat are most present in donji milanovac (76.3%, p=0.001), followed by kladovo (68.8%, p=0.001). also, in southern serbia, the presence of these myths was higher in protected areas, but in a smaller percentage than in the east of the country (tab. 3). one of the warning results of the research is the uninformedness of local inhabitants about the legal regulations on the protection of nature and, particularly, on the protection of the hermann’s tortoise. according to the collected data, 55.3% of the population in protected areas knows about the existence of protection regimes in their environment. although no statistically significant difference was found in relation to the population living outside the protected areas (fig. 2), the worrying fact is that only slightly more than half of the population was informed on this issue. the results from tab. 4 revealed that 68% of the inhabitants of donji milanovac knew about the existence of the national park (x2(3, n=389) = 69.080, p<0.001). thus, almost a third of the respondents did not know that they live within the territory of the largest protected area in serbia, which covers 63,608.45 ha and was placed under protection in 1974 (stanisavljević et al., 2012). the lowest level of awareness in the population from southern serbia was in leskovac–only 19% (p<0.001). there, the inhabitants live far from protected areas, so only a fifth of the total surveyed population was aware of their existence. we recorded large populations of hermann’s tortoise near all four investigated localities (nikolić et al., 2020), so we also know that the residents are in frequent contact with them. but, this study shows more warning results, and that is the attitude of the respondents on the topic of whether hermann’s tortoise should be protected. our research showed that 80.2% of the respondents are aware that hermann’s tortoise should be protected (p=0.033), but a negligible number of respondents knew that this species isalready protected by law (n=7 in total) (službeni glasnik rs, 5/2010, 47/2011, 32/2016 i 98/2016). however, among the localities, the smallest share (23%) belonged again to donji milanovac (fig. 3). as 25.8% (p=0.033) of the inhabitants of donji milanovac were aware that hermann’s tortoise should not be protected (tab. 5), and, at the same time, the presence of folk beliefs and myths was greatest in that locality, this lack of informedness plus superstition suppose to be a direct threat to the populations of this species in the national park. what is also warning are different motives of the respondents when it comes to “protection” of the hermann’s tortoise. an indirect threat to this species can behidden by the motives for protection (ballouard et al., 2020): the motive of caring for hermann’s tortoise should be investigated further, because his “care” for tortoises can be a concern to have enough tortoises for healing, illegal hunting, or for various magical rituals. hermann’s tortoises live near humans, they are harmless, they are slow and relatively small, so people can easily take them from nature to keep as pets (williams, 1999). also, many people find that collecting hermann’s tortoises and carrying them home is safer for the tortoise, than if they are in their natural habitat, because they look vulnerable. this parallel between caring for and endangering tortoises is widespread in children (ballouard et al., 2020). the adequate tool for reducing this type of negative anthropogenic impact in creating successful conservation strategies is education (howe, 2009). education of young people (students and other locals), should become a priority in the sphere of nature conservation, as table 5. the attitude of the respondents per locality that the hermann’s tortoise should be protected and preserved locality % of respondents df syg. donji milanovac 74.2 3 0.033 kladovo 79.6 3 niš 89.9 3 leskovac 77.0 3 well as continued education of the locals as a part of conservation activities in protected areas. the presence of myths and superstitions, as well as residents’ perceptions of the benefits and costs of hermann’s tortoise protection was, according to our knowledge, examined for the first time in the recent scientific literature in serbia. it revealed that the conflict between the humans and this species still cannot be neglected and that, to reduce this conflict, future conservation measures would have to take into account the views of the local population, and even to involve the locals in the planning and implementation of conservation actions. acknowledgements. we are grateful to the rufford small grants foundation for financial support (grants no. 18761-1, 22238-2, 26448-b and 34161-d). we are also indebted to many volunteers – members of biological society “dr. sava petrovic” for participating in the suveying. thanks to phd biljana macura for her help in creating the questionnaire and thanks to phd miloš popović for help in creating maps for the manuscript. jci was also supported the ministry of education, science and technological development of the republic of serbia (contracts nos. 451-03-68/2020-14/200007 and 451-0368/2020-14/200124). 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(brahma kamal) original article debasis mitra department of biotechnology, graphic era (deemed to be university), dehradun, uttarakhand, india debasismitra3@gmail.com (corresponding author) navendra uniyal department of biotechnology, graphic era (deemed to be university), dehradun, uttarakhand, india navendraunniyal121@gmail.com komal sharma department of biotechnology, graphic era (deemed to be university), dehradun, uttarakhand, india komal.sharma638@gmail.com anju rani department of life sciences, graphic era (deemed to be university), dehradun, uttarakhand, india teotia_anju29@rediffmail.com lok man singh palni vice chancellor, graphic era (deemed to be university), dehradun, uttarakhand, india lmspalni@rediffmail.com akansha chauhan department of biotechnology, graphic era (deemed to be university), dehradun, uttarakhand, india akansha0911chauhan@gmail.com prabhakar semwal uttarakhand state council for science and technology (ucost), vigyan dhaam, dehradun, uttarakhand, india. semwal.prabhakar@gmail.com poonam arya department of biotechnology, graphic era (deemed to be university), dehradun, uttarakhand, india aryapoonam73@gmail.com received: november 06, 2019 revised: january 21, 2020 accepted: january 26, 2020 abstract: the rhizospheric association of bacteria in terrestrial plants across the hilly region has tremendous potential for plant growth-promotion (pgp) and protection. this is the first study which aims to isolate some rhizo-bacteria from the different rhizospheric soil samples of s. obvallata in the himalayan region. genotypic, biochemical and pgp traits shows genetically similar bacteria species to have diverse pgp potential as shown by iaa production of 87.567 μg ml-1 by bacillus sp. d5 and phosphate solubilization potential 30 mm halo zones are shown by bacillus sp. d9. growth of isolates was in the ranges of temperature (0-60 °c), salt (0-20%), which shows their tendencies towards surviving harsh environmental conditions. the isolates were further evaluated on amaranthus cruentus, which shows a significant improvement in growth compared to control. the novel study finds the bacterial community at high altitudes to survive extreme climatic variability through association with plants by developing a commensalistic relationship with host. key words: s. obvallata, indigenous rhizobacteria, plant growth promotion, amaranth apstract: izolacija i uticaj rizobakterija iz saussurea obvallata (dc.) edgew (brahma kamal) bakterijska rizosferna asocijacija na terestričnim biljkama širom brdskog regiona ima ogroman potencijal za stimulaciju biljnog rasta (pgp) i zaštitu. ovo je prva studija koja ima za cilj izolovanje nekih rizobakterija iz različitih uzoraka zemljišta rizosfere vrste saussurea obvallata u regionu himalaja. genotipska, biohemijska i pgp svojstva pokazuju da genetski slične vrste bakterija imaju raznovrstan pgp potencijal, kao što pokazuje iaa produkcija od 87.567 µg/ml od strane bacillus sp. d5 i potencijali za solubilizaciju fosfata od 30 mm halo zone od strane bacillus sp. d9. rast izolata je bio u rasponu temperature (0-60°c) i soli (0-20%), što pokazuje njihove tendencije prema preživljavanju surovih uslova životne sredine. izolati su dalje procenjeni na vrsti amaranthus cruentus, što je pokazalo značajno poboljšanje rasta u odnosu na kontrolu. nova studija otkriva da bakterijska zajednica na velikim nadmorskim visinama preživljava ekstremne klimatske varijacije povezivanjem sa biljkama i razvijanjem komensalističke veze sa domaćinom. ključne reči: s. obvallata, autohtone rizobakterije, stimulacija biljnog rasta, amaranthus introduction saussurea obvallata (dc.) edgew. (brahma kamal), the state flower of uttarakhand, india is an endemic herb of the himalayan region (encompassing the indian himalayan region, northern burma and southwest china). the plant is distributed at an altitudinal range of 3000–4800 m (semwal and pant, 2013). brahma kamal is used for the preparation of traditional medicines by the local peoples in tibet and other places including garhwal himalayas. it is well known that indigenous rhizobacteria exert beneficial effects on plants productivity and sustenance of soil health through their capacity for phosphate solubilization (sarikhani et al., 2019), indole acetic acid (iaa) (selvakumar et al., 2008), ammonia, hcn and cell wall degrading enzyme production (tsegaye et al., 2019) etc. pgpr are heterogeneous groups of microbes associated with the plant’s root in diverse ways (nath et al., 2015). in the rhizobac© 2020 mitra et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 35 terial population, 2-5% of total the beneficial rhizobacteria are involved in plant growth promotion (bakker and schippers, 1987; antoun and kloepper 2001; ahemad and kibret, 2014). over 95% of the rhizobacteria existing in the roots and plants obtain many nutrients through the soil bacteria (ji et al., 2014). different pgpr, including associative and symbiotic bacteria such as pseudomonas sp., azospirillum sp., azotobacter sp., rhizobium sp., klebsiella sp., enterobacter sp., alcaligenes sp., arthrobacter sp., burkholderia sp., bacillus sp., and serratia sp. groups have been used for their beneficial effects on plant growth and improvement (kloepper and beauchamp, 1992; höflich et al., 1994). rhizospheric microbial population mainly promote plant health, they stimulate plant growth directly by producing or changing the concentration of plant growth regulators (kloepper et al., 1980; ramette et al., 2003; sparzak-stefanowska et al., 2019) like gibberellic acid, iaa, cytokinins (glick, 1995; vessey, 2003), asymbiotic n2 fixation (ardakani et al., 2010; bhattacharyya and jha, 2012). these populations also achieve antagonism against phytopathogenic microorganisms by producing antibiotics, fluorescent pigment, enzyme, cyanide, phenolics, signal compounds (niranjan et al., 2005), as well as solubilization of mineral phosphates and other nutrients (johri et al., 1999; sharma and johri 2003). inoculums have been used to increase plant yields in several countries, which is reported in research articles (dursun et al., 2019) and commercial products are currently available (backer et al., 2018). we are addressing for the first time isolation of high altitude rhizobacteria from s. obvallata (brahma kamal) rhizospheric soil in the himalayan region and their beneficial effects, harsh environment survival strategy and plant growth promotion in amaranthus cruentus. we proposed that these isolates may also have the ability to express activity towards promoting sustainability in harsh environments. material and methods the four rhizospheric soil samples of s. obvallata were collected from different place of kedarnath valley, uttarakhand, india and samples collection sites were described by semwal et al. (2018) and mitra et al. (2019). the soil samples were serially diluted and 100 μl of dilution sample was plated onto nutrient agar medium (nam) (himedia, mv002, india) for the isolation of potential pgp. all isolated strains were stored at -20 °c in nutrient broth containing 15% (v/v) glycerol. morphological and biochemical characterization tests viz. imvic test, citrate utilization, starch hydrolysis, phenylalanine deaminase, urease test, casein hydrolysis, h2s production, peroxidase test, nitrate utilization, and growth in different media macconkey’s agar, esculine hydrolysis, mannitol salt agar tests were carried out followed by macfaddin, 1985; harley and prescott, 2002; anija, 2003; dubey and maheshwari, 2007; cappuccino and sherman, 2008; brown, 2009 respectively. to identify the morphological characteristic of all isolates, gram staining, negative staining, shape, margin, elevation, form, motility and pigment were recorded. a loopful of bacteria was inoculated and incubated into pre-sterilized peptone broth containing 1% of tryptone for 48 h at 28 °c. after 48 h, 1 ml of kovac’s reagent (himedia, india) was added to all tubes including control, gently shaked for few second and left to react for 5-10 minutes. the appearance of red/ pink ring at the top is the clear indication of iaa production (loper and schroth, 1987; dubey and maheshwari, 2007). as described in gordon and weber (1951), the standard graph of iaa was prepared for the quantitative estimate of iaa production. different iaa concentrations were prepared as aqueous solution of iaa ranging from 5-150 μg ml-1. to each 1 ml of the standard, 2 ml of 2% fecl3 (0.5 m) in 35% perchloric acid i.e. salkowaski reagent was added and readings were taken after 30 minutes at 535 nm using uv-visible spectrophotometer (thermo scientific™ genesys-10s-uv). the standard graph was finally prepared by plotting concentration of iaa in ppm vs optical density at 535 nm. bacterial cultures were grown for 48 h in nutrient broth. fully grown cultures were centrifuged at 4000 rpm for 20 minutes at 4 °c. the supernatant (1 ml) was mixed with 4 ml of salkowaski reagent. samples were left at 28 °c for 30 minutes. development of pink color indicates the production of iaa. optical density was measured at 535 nm with a spectrophotometer. the concentration of iaa produced by bacterial cultures was measured with the help of standard graph of iaa (loper and schroth, 1987). phosphate solubilization ability of the isolates was evaluated in pikovskaya’s (pkv) agar medium (himedia, india) incorporated with tri-calcium phosphate (tcp) [ca3 (po4)5] as insoluble phosphate. pkv media plate was prepared and point inoculation was transferred in each plate at 28 °c for 5 days. after incubation, positive phosphate solubilizing bacteria which give clear zone around the colony were measured (fiske and subbarow, 1925). isolated strains were grown overnight in 10% trypone soy agar supplemented with glycine (4.4 gly l-1) (himedia, india) (bakker et al., 2003). a whatman no. 1 filter paper soaked in 2% sodium carbonate and 0.4% picric acid solution was placed to the underside of the petri dish lids (macfaddin, 1980; results explained by cook, 1993) and kept at 28 °c for 4 days. for the ammonia production, all isolated 36 biologica nyssana ● 11 (1) september 2020: 35-44 mitra et al. ● isolation and impacts of rhizobacteria from saussurea obvallata (dc.) edgew. (brahma kamal) bacterial strain was grown in 4% peptone broth for 48 h at 28 °c. detection of ammonia production was done by adding dropwise of 1.5 ml nessler’s reagent (himedia, india) (bakker et al., 2003). cellulase enzyme production was determined from clear zone test in cmc (carboxyl methyl cellulose) agar (cmc 100.00 g, peptone 5.0, agar: 15.00 g, distilled water 1000 ml, ph: 7.2) and czapekmineral salt medium (nano3 2.2 g, k2hpo4 1.0 g, mgso4.7h2o 0.7 g, kcl 0.5 g, cmc 5.0 g, peptone 2.5 g, agar 18.0 g, distilled water 1000 ml, ph 6.0) (bakthavatchalu et al., 2012). two days after incubation, 2 ml of congo red solution (0.1% congo red in 1 m nacl) was added in each plate. all the plates were then gently shaken. a positive result was indicated when a clear zone and light develops (qadri et al., 1988). protease activity (casein degradation) was determined from the clear zone in skim milk agar (skim milk powder 100.0 g, peptone 5.0 g, agar 15.0 g, distilled water 1000 ml and ph 7.2) (vijayaraghavan and vincent, 2013). the biofilm experiment was performed in 10 ml glass bottles. three milliliters of nutrient broth (without nacl) was dispensed and inoculated with an overnight experiment culture. the bottles were incubated under the static condition for 5 days, the wells were washed thrice with distilled water and the biofilm was stained with 1% crystal violet dye solution for 15 min, and again the wells were rinsed thrice with distilled water to wash off the unbound dye. quantification of the biofilm biomass was done by absolute alcohol and recording the absorbance at 600 nm according to srinandan et al. (2010). antibiotic resistance testing was determined by disc diffusion method (penicillin: sd028-1vl, erythromycin: sd083-1vl, streptomycin: sd091-1vl, chloramphenicol: sd153-1vl and tetracycline: sd1331vl, himedia, india) using nutrient agar plates. the bacterial suspension was inoculated on the nutrient agar plate by swabbing to give a smooth lawn and antibiotic discs were properly placed on the plate for overnight. the plates were observed and recorded the zone of inhibition around the antibiotic disc. all isolates antibiotic profile diversity was analyzed by ntsys 2.02e software, where 0 denotes susceptible and 1 denotes resistance to a particular antibiotic source. the data were analyzed using the similarity for qualitative data subroutine of ntsys-pc2 package. the isolates were grouped as per jaccard’s similarity index by the unweighted paired group method using arithmetic means (upgma) and depicted as dendrogram (ansari et al., 2014). antibiotics test result were graphically determined by the r-heatmap plot analysis where 0 denotes susceptible and 1 denotes resistance to a particular antibiotic source. 37 to find out the optimal conditions for the growth of bacterial isolate, the effect of different temperature levels on the development of isolates was examined. 0 °c, 4 °c, 28 °c, 37 °c and 60 °c were mainly used and bacterial suspension (100 μl) was placed in a tube containing 10 ml nutrient broth. the tube was incubated at the predetermined temperatures for 48 hours. the effect of different salt concentration on bacterial growth was studied with nutrient broth containing salt concentrations ranging from 0-20%. the effect on different salt concentration (0% and 8% nacl) for indole-3-acetic acid production in tryptone broth. this was investigated by using tryptone broth containing 0% and 8% nacl and following 48 h of incubation, the effects on growth rate were measured by spectrophotometric measurements at 660 nm. the pot experiment was conducted (two times) in the net-house, graphic era (deemed to be university), dehradun (30°19’n, 78°04’e, 650 m) with two times sterile soil. amaranthus cruentus seeds were surface sterilized, the seeds were dried for 2 hours in sterile conditions and subsequently planted into the pots prepared for treatments. there were five replicates per treatment with 15 seeds planted per pot. two bacterial isolates viz. bacillus sp. d5 and bacillus sp. d9 were selected based on pgp activity. soil treatments consisted of 0.5 kg lignite (carrier material) and 100 ml of inoculum (d5 and d9; separately) solution and sterile distilled water was the control. after 30 days, the length and mass of shoots and roots were taken to determine the effect of treatments on the growth of a. cruentus plants. diversity and statistical analysis were done by using ntsys 2.02e software. experimental data were analyzed using standard analysis of variance (anova) and hau, hisar online statistical analysis software results ten pgpr isolates (pseudomonas sp. d1, bacillus sp. d2, bacillus sp. d3, bacillus sp. d4, bacillus sp. d5, bacillus sp. d6, bacillus sp. d7, bacillus sp. d8, bacillus sp. d9 and bacillus sp. d10) were chosen from a total of eighty-five isolates using the serial dilution technique of rhizospheric soil samples of s. obvallata. all isolates were screened and selected on the basis of growth rate, morphological (fig. 1) and biochemical characteristics. this is the first research report that the d1, d2 and d3 isolated from hathi parwat (hp), d4 and d5 from maha panth (mp), d6 and d7 from madhu ganga (mg) and d8, d9 and d10 from cheer ganga (cg) of kedarnath valley. similarly, mitra et al. (2019) reported that some dominant fungi viz. phanerochaete chrysosporium, aspergillus fumigatus and trichoderma longibrachiatum was found and identified through 16s rdna sequencing (its4 and its5) from the same biologica nyssana ● 11 (1) september 2020: 35-44 mitra et al. ● isolation and impacts of rhizobacteria from saussurea obvallata (dc.) edgew. (brahma kamal) 38 rhizospheric soil samples of s. obvallata. biological communities in the indian himalayas include one of the biggest altitudinal encompass in the world and a portion of the more extravagant gatherings of wild and medicinal plants are found in this area (chauhan et al., 2015). presentation and exploitation of pgpr in agro-biological systems improve plant-microbes interactions that may influence biological communities supportability, farming efficiency, and ecological quality (zahid et al., 2015). the rhizosphere is a special specialty for different kinds of microorganisms in the soil (agrawal and johri, 2014). semwal et al. (2018) reported that s. obvallata rhizospheric and non-rhizospheric soils macro micro nutrients analysis and range of different nutrients availability viz. nitrogen, phosphorus, potassium, zinc, copper, iron, manganese in normal and suggested that they are helpful for development and growth for this endangered plant. so, in our present study, ten pgpr bacterial isolates were isolated and screened for pgp abilities (kloepper et al., 1988). this study was conducted to isolate the native low temperature growing potential pgpr from the himalayan region rhizospheric soil of s. obvallata plant and showed that some common pgprs like bacillus sp. and pseudomonas sp. were present in this rhizospheric area. similarly, majeed et al. (2015) reported that they are isolated some pgpr for the root endosphere and rhizosphere of wheat from the himalayan region and isolates showed pgp ability like iaa production, n2 fixation and p solubilization. agrawal and johri (2014) reported that fiftyfive rhizobacteria were isolated, characterized and screened pgp attribute from the central himalayan region, forest sites; altitude 3500, 12000 and 3100 ft. biochemical test results of all bacterial isolates were analyzed by heatmap plot and represented the results where black colour denotes negative and gray colour denotes positive activity (fig. 2). heatmap plot colour analysis showed that most (approx. <50%) isolates has positive biochemical activity. iaa production is a natural property of rhizobacteria that enhance and facilitate plant growth and development. quantitative assay of iaa production of rhizobacteria was done by the spectrophotometfig 1. the figure shows the percentage abundances of bacteria as determined under microscope fig 2. biochemical test results represented by heatmap plot (mr and vp: mr/vp test, c: citrate utilization, sh: starch hydrolysis, pd: phenylalanine deaminase, ut: urease test, ch: casein hydrolysis, h2s: h2s production, pt: peroxidase test, nr: nitrate utilization, ma: macconkey’s agar, eh: esculine hydrolysis and msa: mannitol salt agar) biologica nyssana ● 11 (1) september 2020: 35-44 mitra et al. ● isolation and impacts of rhizobacteria from saussurea obvallata (dc.) edgew. (brahma kamal) 39 ric analysis. the concentration of iaa produced by bacterial cultures was measured with the help of the standard graph of iaa. in this study, six qualitative screened positive bacterial isolates were taken for the quantitative assay and d5 isolate iaa production is 87.567 μg ml-1. phosphate solubilizing bacteria used as inoculants simultaneously increases phosphate uptake by the plant and crop yield (rodríguez and fraga, 1999). phosphate solubilization activity was determined by the development of the clear zone around the bacterial colony. isolate d9 is a high phosphate solubilizer bacterial strain which is used for the treatments. d9 showed the 30 mm zone (diameter) on pkv agar media (fig. 3a) after 5 days of incubation. four phosphate solubilizer bacteria strains were grown in pkv medium (ph 6.35) for 3 and 5 days at 28 °c. after the given time, ph of the culture medium was checked by ph meter and decreased ph rate can be observed in fig. 3b. in the similar study, panday et al. (2002) reported and isolated p. corrugata from the low-temperature location of sikkim and reported that all isolates could grow and solubilize phosphate in the range of 4 to 35 °c. all isolates were grown in 10% tryptone soy agar with glycine (4.4 gly l-1) on the plates sealed with parafilm®. the production of hcn was determined by the change in colour of filter paper from yellow to red-brown. after the incubation of all bacterial culture in medium, the nessler’s reagent was added. ammonia produced by bacterial isolates formed the yellowish-brown colour in the medium. cell wall degrading enzyme production was determined by congo red method and protease activity was determined by clear zone formation on the test plate. by this, the production of the most important plant growth hormones was recorded (fig. 4). fig 3. a bacillus sp. d9 isolate phosphate solubilization zone on pkv media after 5 days; b ph level in pkv broth fig 4. isolates plant growth-promoting test results are evaluated graphically by heatmap plot where gray color: (+) pgpr and black colour: (-) pgpr. (iaa: iaa production, ps: phosphate solubilization, hcn: hcn production, ap: ammonia production, c: cellulase test and p: protease test) fig 5. biofilm formation by isolates biologica nyssana ● 11 (1) september 2020: 35-44 mitra et al. ● isolation and impacts of rhizobacteria from saussurea obvallata (dc.) edgew. (brahma kamal) 40 quantification of the biofilm biomass results by the bacterial isolates are presented in fig. 5. isolates d2, d5, d7 and d9 showed high biofilm formation after 5 days of incubation. the antibiotic sensitivity was determined by disc diffusion method, where five antibiotics were used and the results of resistance and susceptibility of the isolates are presented in fig. 6a. antibiotic diversity of the isolates was analyzed by ntsys and dendrogram, where d2 and d5 showed sensitivity to all tested antibiotics (fig. 6b). heatmap plot showed that d6, d7, d8, d9 and d10 are resistant to penicillin and d1 showed resistance to erythromycin and tetracycline (fig. 6c). bacterial growth under different temperature and nacl concentration were determined by measuring the optical density (od) at 660 nm by spectrophotometer (fig. 7a, b). in this study d2, d5 and d9 bacterial isolates demonstrated the highest growth rate at 0 c, while 28 and 37 were optimal growth temperatures for most other isolated strains. all isolates showed low growth rate at 60 °c. our study suggested that the most suitable salt concentrations differed among the strains. the result showed that optimal growth and survival were at 0% for d8, 0.5% for most of the strains (d1, d2, d3, d7, d9 and d10), while three strains (d4 d5 d6) had the best growth at 5% nacl concentration (fig. 7b). iaa is a common product of l-tryptophan metabolism by numerous microorganisms but in the medium salt concentrations affects the growth rate of microorganism. all isolates were grown in tryptone broth with different salt concentration. d6 and d7 growth rate were higher at 8% nacl and d9 growth rate was constant at 0% and 8% nacl (fig. 8). the results of the soil treatments with potential pgpr isolates viz. d5 and d9 on the growth of a. cruentus showed significant effect in enhancing the plant’s shoot root height and weight of inoculated plants as compared to uninoculated control (fig. 9). in experiment 1, d5 and d9 showed a positive result in the development of shoot but in experiment 2, d5 showed high effect in enhancing shoots and roots as compared to control. discussion pgpr are originally free-living soil bacteria that colonize the rhizosphere or the tissues of living plants. once the bacteria are associated with the plant by colonizing the rhizosphere around the roots, the rhizoplane (root surface) or the root itself (rodriguez-navarro et al., 2007), the bacteria can increase the development of the plant through indirect or direct mechanisms. in our study, pgpr isolates viz. d4, d5, d6 and d9 showed positive for phosphate solubilization and ph ranged from 4.00 – 5.00. d9 bacterial isolates showed the highest zone in pkv plate. highest biofilm formation showed by rhizobacterial isolates d1, d2, d5, d7 and d9 was very evident at 600 nm readings. from the compatibility results, d1, d5 and d9 isolates were selected for the pot experiment to assess its performance in terms of growth, nutrient uptake and stress conditions survival ability. pot experiment results showed fig 6. a effect of antibiotics to different isolates; b antibiotics diversity of isolates analyzed by ntsys; c antibiotics test results analyzed by heatmap plot where gray color: resistance and black color: susceptibility (p10: penicillin, e10: erythromycin, s25: streptomycin, c25: chloramphenicol, t10: tetracycline biologica nyssana ● 11 (1) september 2020: 35-44 mitra et al. ● isolation and impacts of rhizobacteria from saussurea obvallata (dc.) edgew. (brahma kamal) 41 fig 7. a isolates growth under the different temperature; b isolates growth under the different salt concentration fig 8. growth rate in tryptone broth with different salt concentration biologica nyssana ● 11 (1) september 2020: 35-44 mitra et al. ● isolation and impacts of rhizobacteria from saussurea obvallata (dc.) edgew. (brahma kamal) that soil treatments with these isolates significantly enhanced the plant growth of inoculated amaranth plants as compared to uninoculated control. conclusion in our study, we used bacillus sp. d5 and bacillus sp. d9 as a bio-inoculum and phosphate solubilizer from the rhizospheric soil samples of s. obvallata (brahma kamal) in the himalayan region. the present study has clearly demonstrated the pgp trails and application on amaranths with bacillus sp. d5 and bacillus sp. d9 having a positive influence in growth parameter in comparison with the control. the study suggested that low-temperature bacterial community growth has multifunctional characteristics in growth promotion and develops a green biofertilizer for hill farming. acknowledgements. we are grateful to the founder and president, prof. (dr.) kamal ghanshala, graphic era (deemed to be university), dehradun and giving student research grants for this research work. we are also grateful to prof. ashish thapliyal, professor and head, department of biotechnology, graphic era (deemed to be university), dehradun. first author thanks to devvrat, ph.d. scholar, graphic era (deemed to be university), dehradun, india and ansuman senapati, srf, icarnrri, cuttack, india for his help in data analysis references agrawal, p.k., johri, b.n. 2014: characterization of plant growth promoting rhizobacteria from rhizospheric soil of himalayan region. octa journal of biosciences, 2(2): 69-75. ahemad, m., kibret, m. 2014: mechanisms and applications of plant growth promoting rhizobacteria: current perspective, journal of king saud university – science, 26(1): 1-20. biologica nyssana ● 11 (1) september 2020: 35-44 mitra et al. ● isolation and impacts of rhizobacteria from saussurea obvallata (dc.) edgew. 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(figure 1. wild cherry seed). all statistical analyses of the data were made using the spss 15.0 package for windows. fig. 1. wild cherry seed results and discussion the results obtained show the presence of a high level of intrapopulational, as well as interpopulational, morphological variability in the natural populations of the wild cherry which have been investigated. analyses of population differentiation have not confirmed our expectations. a discriminant analysis by groups formed according to classes of altitude (fig. 1) and ecological and vegetation zoning (fig. 3) has shown that there are no separations within the studied populations, which implies the possibility to use seed and planting material from different altitudes and ecological and vegetation zones within the studied area. conclusion the analyses of 6 morphological characteristics in 12 natural populations of the wild cherry in bosnia and herzegovina showed statistically significant differences between investigated populations. differentiation in natural populations of the wild cherry was very low and identified only in fruit dimension characteristics. fig. 2. discriminant analysis by groups according to classes of altitude fig. 3. discriminant analysis by groups according to ecological and vegetation zoning biologica nyssana 4 (1-2)  december 2013: 15-17 ballian, d., mujagić-pašić, a..  morphological variability of the fruit… 17 references ballian, d. 2000: početna istraživanja varijabilnosti morfoloških svojstava sjemena divlje trešnje (prunus avium l.). šumarski list, 124 (5– 6): 271–278. ballian, d. 2002: variability of characteristics of the wild cherry blossom (prunus avium l.) in the region of central bosnia. ann sci for, 25(2): 19. herman, j. 1971: šumarska dendrologija. stanbiro. zagreb, 466 p. ninkovski, i. 1998: trešnja. nauka i praksa, beograd, 151 p. vavilov, n. i. 1935: teoretičerskie osnovi selekcii rastenij. gos izdat kolhoznoj i sovhoznoj literaturi, moskva-leningrad. žukovsky, p. m. 1965: main gene centers of cultivated plants and their wild relation within the territory of the ussr. euphytica, p 14. microsoft word 0603_jaksic_momirovic biologica nyssana 1 (1-2) december 2010: 123-130 jakšić, p., momirović, m. contribution to understanding the origin… 123 original article ! contribution to understanding the origin and the genesis of the nišava riverside valley fauna predrag jakšić*, milan momirović university of niš, faculty of science and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia *e-mail: jaksic@pmf.ni.ac.rs abstract: jakšić, p., momirović, m.: contribution to understanding the origin and the genesis of the nišava riverside valley fauna. biologica nyssana, 1 (1-2), december 2010: 123-130. the fact that very early biologists registered an extraordinary faunistic richnes in the greater area of niš (nišava’s riverside) is pointed out. the conditions that contributed to this variety are analyzed. the importance of geological history of this region is explained, from the ancient lake phase trough the glacial epochs including the present state. the dilemma about lake terraces and the water level of the neogene lake system is discussed. a list of gorges, speleological objects, springs, mountain tops as habitats of endemic and relict species is given. all the endemic and relict species in these habitats are listed. key words: fauna origin, fauna genesis, nišava riverside, serbia introduction ! the area of the nišava river valley attracted the attention of geographers and biologists very early. as early as 1891 đ. j o v a n o v i ć wrote about the caves in the gorge and in 1909 p. j a n k o v i ć devoted a comprehensive monograph to the development nišavska valley. among botanists s. p e t r o v i ć researched the flora of this region on two occasions (in 1882 and 1885, respectively). at the very early stages of research of the ponišavlje fauna, both botanists and zoologists noticed the diversity and large numbers of endemic and relic species. thus, for example, p a v l o v i ć (1912) in his monograph on the land snails in serbia pointed out the presence of numerous endemic species, and based on this data he produced one of the first zoogeographic maps in serbia, where the nišava valley was tagged as an area in which tertiary relics existed. subsequent studies of many botanists and zoologists had this view only updated and confirmed. the reason for this abundance is explained using geological and tectonic history of the area, its central position in the balkan peninsula and the topographic disposition of the terrain. from the valley of niš, through the surrounding hills and cliffs of numerous rivers, the suva planina mt., the vidlič mt. and other mountains of the wider area and geological complexity orographical areas offer good macroclimatic and microclimate conditions, and the significant complexity of habitat necessary for the existence of various elements of the flora and the fauna. besides the gorges, which serve as an excellent refuge, there are also wells and springs, numerous caves, forest litter (bedding) and xerothermal habitats on the tops of the surrounding mountains. the central position of this area in the balkan peninsula, in turn, allowed the presence of various elements of the fauna: central-european, pannonian, dacian, pontic, east asian, etc. 10th sfses • 17-20 june 2010, vlasina lake1 (1-2) • december 2010: 123-130 biologica nyssana 1 (1-2) december 2010: 123-130 jakšić, p., momirović, m. contribution to understanding the origin… 124 material and methods the area considered includes the headwaters of the svrljig timok, the reka river valley, the nišava valley from bela palanka to niš (gorge), the corresponding slopes of the svrljiška and suva planina mt., the jelašnica and prvokutinska gorges, caves and springs in the area. in the basin of svrljig timok, taken into consideration are okapina 1, okapina 2, tor, bezimena okapina, great okapina, small okapina, okapina at ostenjak, the church ruins okapina 1, the church ruins okapina 2, the church ruins okapina 3, the ripaljka cave, tunnel cave; in nišava basin, the objects considered are the okapina on the jecava, the eagle gap, mečija duvka, celenkina duvka, golema duvka, ladarova duvka, goveda duvka, great okapina 1, great okapina 2, okapina near the tunnel , small okapina, popova duvka (savnjak) paramanska okapina, ogorelička cave, the cave at zavrte, jovina duvka, great balanica, small balanica, popova duvka (brljevski stone), svinjarska duvka, radonjino konopljište, tunnel cave. wells and springs taken into consideration include bojanine vode and vrelo on the suva planina mt. finally, mountain peaks of xerothermic character taken into consideration are ramnište 951m, crni vrh 1164 m, mečji vrh 1184 m and golemi vrh 1535 m, all on the suva planina mt. on this geographic terrain the presence of tertiary relict species and postglacial xerothermic steppe species is analyzed. in this work the method of causal induction method was used (js mill) in which the conclusion is based on the search for causes. this method has many variations, and we applied the mode of agreement and difference, a method of reasoning where cause and effect correlate. geological-tectonic history and the history of climate change in the area are observed as a cause and the abundance of the species as a result. results representatives of nišava valley flora and fauna of preglacial age and xerothermic relics a) representatives of the flora. among tertiary relics we can cite the following species: juglans regia, syringa vulgaris, corylus colurna, acer intermedium, staphylea pinnata, daphne laureola, dictamnus albus, edraianthus serbicus and, especially, endemorelict species micromeria cristata, parietaria serbica, ramonda serbica and ramonda nathaliae. from the period of the relatively warm and dry interglacial (xerothermal) remained the steppe relics: adonis vernalis, prunus fruticosa, prunus tenela, asparagus tenuifolius, sternbergia colchiciflora, hyacinthella leucophea, ranunculus illyricus, cachrys alpina et al. (l a z a r e v i ć et al., 2007). b) representatives of the fauna. a survey of the fauna is limited to two groups of invertebrates: mollusca and arthropoda. significant contribution to the knowledge of mollusca in this area was given by p a v l o v i c (1912). the author cites the following relict and endemic species in the gorge: cabinet annularis stud, hyaline nitens michaud., was also found near the village prekonoge, zonitoides nitidus müll., patula solaria mke., eulota fruticum müll., vallonia pulchella müll., fruticicola ( monache) incarnata müll., campylaea trizon zgl., carthusian carthusian müll., buliminus (zebra) detritus müll., buliminus (ena) obscura müll., chondrula tridens müll., chondrula (mastus) venerabilis ziege., caecilianella acicula müll., orcula doliolum brug, pupa frumentum drp., modicella avenacea brug., pupilla muscorum l., isthmia minutissima hartm., isthmia salurensis reinhardt, clausiliastra laminate mont., alinda plicata drp., alinda biplicata mont., herilla frauenfeldi zelebor, carinigera eximia mollendo . the last one listed is an endemic species growing on the bare and sun-exposed limestone and found in bela palanka at vrelo in the gorge on the road village mokro, above the modro spring, near the village of krupac and also in pleš on nišavska side. finally, there are also types of carychium minimum müll., in both sićevo and jelašnička gorge acme serbica clessin was found, and ericia costulata (zgl.) rossmore. species uncinaria petrovici pavlović was found on vidlič above visočke ržane. species kuzmićia pygmaea mollendo. is distributed only in southern and southeastern serbia, in the analyzed area it was found on a suva planina mt., in the jelašnička and sićevo gorges, on pleš mt. and the village of prekonoge. different groups of arthropod fauna have also been the subject of intensive research by many authors. thus p l j a k i ć (1977) in this area identified the presence of many primitive species of lower crustaceans from oniscoidea: hyloniscus riparius (cl koch, 1838) in the sićevo gorge, hyloniscus transsylvanicus verhoeff, 1901 in the spring at ostrovica, hyloniscus kopaonicensis buturović, 1960 in the upper and lower dušnik, trichoniscus naissensis pljakić, 1977 in the three biologica nyssana 1 (1-2) december 2010: 123-130 jakšić, p., momirović, m. contribution to understanding the origin… 125 caves (donja cave in refuge, great belanica and kojina cave) and trichoniscus bononensis sotirova pljakić, 1977 in the cave kravljansko pester. the summation of known diplopoda in this area was given by makarov et al. (2004). the authors cite several cavernlike species: dorypetalum degenerans (latzel, 1884) and megaphyllum austriacum (latzel, 1884) from a cave in the barski reed, svarogosoma bozidarcurcici makarov, 2003 from a cave in sava’s fall (trem), nopoiulius kochii (gervais, 1847) from the great belanica and typhloiulus (typhloiulus) nevo makarov, mitić & curcio, 2002 from the great cave. the opilionida identified are paranemastoma (buresiolla) bureschi (roewer, 1926) and cyphophthalmus serbicus (hadži, 1973) in cerjanska caves. in the pseudoscorpiones group ć u r č i ć et al. (2004) found the species roncus remesianensis ćurčić, 1981, roncus timacensis ćurčić, 1981 and roncus jelasnicae ćurčić and dimitrijevic, 2009. the recognized species of spiders (araneae) include pholcomma gibbum (westring, 1851), steatoda triangulus (walckenaer, 1802) and lepthyphantes leprosus (ohlert, 1865) in košuća cave (kravlje), lepthyphantes trnovensis (drensky, 1931) in the cave near niš and mettelina merianae (scopoli, 1763) in jelašnička cave. among ticks (ixodidae), the species found include eschatocephalus vespertilionis cl koch, 1844 at ogorelačkoj cave. the presence of numerous representatives of insecta has been determined. the representatives of the species staphylinidae are bisnius cephalotes (gravenhorst, 1802) and aleochara (xenochara) funebris wollaston, 1864 from the ogorelačka cave, and the species quedius (microsaurus) mesomelinus skoraszewskyi korge, 1961 and atheta (alaobia) caving erichson, 1839 from both belanica and the ogorelačka cave. the representatives of the beetles (carabidae) are the species duvalius (paraduvalius) winkler (jeannel, 1923) from the ogorelačka cave, and straight prekonoga oven and laemostenus (pristonychus) terricola punctatus (dejean, 1828) from the ogorelačka cave, and the species trechus (trechus) irenis csik, 1912 from the cerjanska cave. finally, the orthoptera (raphidophoridae) species observed are troglophilus neglectus vlasinensis maran, 1958 in the belanica and ogorelačka cave. the ogorelačka cave has long been known for existence of the cave bear (d j o r d j e v i ć , 1891). j o v a n o v i ć (1891) established its presence in the prekonoška cave. herak (1947) proved that the cave bear in the balkan peninsula is not indigenous but has in fact come from the region of the alps at a later date diluvium. discussion from the description it is evident that the presence of the glacial elements in the analyzed region is reduced to a minimum. in association with the pine (sorbet-mughetum jn.) on the steep, cold northern slopes of the highest part of the suva planina mt. at about 1400 m above sea level, all the way to the highest peaks, some of the glacial relics can be found, such as eg. dryas octopetala. this means that the glaciation in this region has significantly disrupted the starting wildlife. if the climatic factor was not crucial, then it can be assumed that the proper interpretation of the geological history is the key to understanding the origin and genesis of the recent flora and fauna in the nišava riverside valley. in the geological-tectonic history of the area the most important event was the lake stage. from the point of view of modern science, it is surprising that as early as 1909, 100 years ago, j a n k o v i ć published an extensive study of the history of the development of the nišavska valley. the author of this work claims: "the niška valley is a drained pool of a large neogen lake, which existed probably until the end of the pliocene." in the same paragraph, the author presents another observation: "the niška valley with its configuration and its main plastic lines independent of the nišava valley and its actions, because the river formed only after the runoff neogen lake"; also extremely important is that we note that the author of this work gives the original drawings of the land and sea during the three phases of the neogen. the suva planina mountain, the pleš, the vidlič mt. and other ranges are presented in these maps as islands. j a n k o v i ć in summarizing his results highlighted the existence of two systems of terraces: the high abrasion (lake) and a system of low (fluvial, river) terraces and that by them we can see that the great lakes declined successively and in breaks. the assessed absolute levels and relative levels of lake shores in stages are also determined. c v i j i ć (1913) in his study of the rtanj mountain pays attention to rtanj lake plasticity. cvijić found the presence of lake terraces at 940-830 m, then at 680-670 m, and at the level of 600 m, he also links these terraces to pont. writing about the prvokutinska gorge, which was cut by kutina, a left tributary of nišava, m a r t i n o v i ć (1976) linked its genesis with the lake stage, the end of the miocene and stated that the area of today's gorge was the lake narrow of the niš lake basin. by analyzing the formation of dolomite in neogen lake sediments in the niš area of the morava riverside, n i k o l i ć & j a n j i ć (1986) came to the conclusion that in certain phases of the existence of biologica nyssana 1 (1-2) december 2010: 123-130 jakšić, p., momirović, m. contribution to understanding the origin… 126 the lake there occurred the salinisation of the water environment, an increased salinity, higher temperatures and evaporation, causing a deposit of dolomite. by analyzing the neogen series of the litostratigraphic column, the authors established the first cycle of sedimentation in the lower miocene, the second cycle of sedimentation taking place during the middle miocene, the third cycle of sedimentation proceeding during the mio-pliocene, and after that the last phase was implemented in the genesis of neogen sedimentation. a similar view was proposed by a n d j e l k o v i ć et al. (1991), who pointed out that during the pontian the marine environment had been reduced to remnants of paratethys in the pannonian and dacian areas, while in other parts of the former sea came to a loss of salinity and the formation of wetlands covered by dense vegetation taxodium forests. during the dacian era, the complete withdrawal of paratethys from serbia occurred, this territory, therefore, existing as a land mass from 12 (12.5) million years ago until the present day. m a r t i n o v i ć (1968) compiled a very detailed survey of the genesis of the sićevo gorge. he singled out six lake levels, i.e. six lake terrace: the highest abrasive track at the level of 1020 1000 m; the second terrace is at 860 m, the third terrace at 830-820 m; the next level of 760 750 m has been developed in the sićevo gorge and on the outskirts of the niš valley; the fifth level is a distinct riparian terraces in the gorge at a height of 720-700; the last level was at an altitude of 680-660 m; this level is also present along the perimeter of the niš valley (where it is represented at two levels due to intense tectonics of the terrain). martinović says that up to a height of 720 m terrace is of pannonian age (upper miocene, about 12-13 million years), lower than 460 m of pontus (pliocene, about 11 to 11.5 million years) and below this level are the diluvial ones; the last lowest terrace is recent. the pontic era climate in serbia is characterized by relatively high temperature and high humidity, as evidenced by the sediments and deposits of tacsodium. during the dacian era, the climate did not significantly change, which is proven by the process of paleocrustification (a n d j e l k o v i ć et al., 1991). in the debate about the level of neogen lakes in the south of serbia, m i l i ć (1982) showed that there was no communication between pannonian lake and aegean lake through grdelica gorge or other possible locations. but the author also pointed out that such communication was possible in the upper miocene and that the link extended west of the mount kukavica to the preševo-kumanovo basin. the author specifieds that this connection had existed during the pannonian. however, the views put forward by j a n k o v i ć (1909), c v i j i ć (1913) and others are opposed by the contemporary authors. thus, p e t r o v i ć d. (1988) critically examined cvijić’s understanding that during the upper and lower pliocene there existed a unique aegean lake that cut high terraces of 760-740 and 670 m. the shallow character of pont sediments, as encountered in the nišava riverside valley, and their low height exclude the possibility that the pontic water in general could reach the height of the coastal line of 940 m and above, and cut the surface at a height of 850 m. the existence of epigenetic gorges excludes the possibility that under the height of their upper edges surface abrasions could be carved, because the central lake level had to be above them. if we take the contour line of 857 m as a reference point selected as the middle of the three phases that janković presented on his maps [which is approximately equal to the highest correlated level terrace of 873 meters of the current sea levels, according to n i k o l i ć (1991)], then we come to the conclusion that the islands could not be used to maintain tertiary elements of flora and fauna (fig. 1). fig. 1. a reconstruction of the land and sea on the contour line of 857 m, as understood by janković (1909). the legend: 1 sea, 2 land, 3 river network, 4 caves analyzed in this paper, svrp the svrljiške planine mt., sp the suva planina mt.. such an image completely excludes the existence of gorges, caves and springs, all modern caves (represented by circles in the drawing) would in that time have been submerged. the abrasion and fluvial terraces seen in the field are real; their biologica nyssana 1 (1-2) december 2010: 123-130 jakšić, p., momirović, m. contribution to understanding the origin… 127 existence is not doubted by anyone. on the given maps of land and sea during the sarmat, pannonian and pontian, a n d j e l k o v i ć et al. (1991) specified the lines of the lake in that period, showing its successive reduction. these maps match well with the maps of jovanović for the period of pannon. however, substantial differences appear in the level andjelkovic et al. emphasize that this is a shallow lake, unlike jovanović’s view. finally, andjelkovic et al. pointed out that during the pontian there occurred a positive epirogenesis, an upheaval of the land that led to the withdrawal of the sea and the transition of plain lands to hill lands. now, we can set a theory that in this way the terraces were successively raised to the level seen today or that the level of the sea was more or less constant, and that the marking of terraces went successively, and that, due to positive epirogenesis after forming, they would be raised to this level. n i k o l i ć (2007), in a study of abrasive terraces, definitely confirmed this view. the author provided a precise series of successive levels of abrasion terrace for serbia (especially in the catchment area of south morava); according to the data, the altitude of the abrasion terrace showed these values: 60, 75, 100, 120, 140, 175, 200, 220, 240, 260 and 280 m. in the same article the author presented the data on a curve of eustatic fluctuations of the mediterranean sea in the pleistocene, based on which it is shown that the sea level used to be between + 280 in the gűnz 1 period gűnz 2 (560 thousand years ago), over 120 in wűrm 3 (about 30 thousand years), to the present level of 0 m. caves and species found in them can be the key to solving the dilemma presented here. according to the hypothesis of j a n k o v i ć (1909), as shown in figure 1, all existing caves during the neogen and until the end of the pliocene were submerged; it is unlikely that they existed at all. therefore, it follows that they were developed only after the runoff of the neogen lake, their maximum age being 11.5 million years. in speleology it is a common knowledge that in the dinarides and carpatho-balkanides there are old cave systems of preglacial age. most caves of the alpine arc are of glacial age, some of them are only 40,000 to 50,000 years old. there is little data about caves in eastern serbia. g a v r i l o v i ć (1967) described the genesis of the vlaška cave in the resava spring. the cave was formed by water, its formation probably began in early pleistocene and the formation of main channels in late pleistocene. a similar view was expressed by p e t r o v i ć (1977) in discussion about the genesis of the bogovinska cave. the author concluded that the formation of the bogovinska cave is related to the first half of the pleistocene, it being the oldest stage in the formation of this cave. we also have reliable data for the zlotska cave. the underground stream had built the channels of the zlotska cave during the late pleistocene and the formation was completed in holocene (brown, 1957/58). in the relative proximity of the area that was analyzed in this paper is the seselačka pećura in soko banja. p e t r o v i ć (1984) concluded that it was a younger object, formed at the turn of pleistocene to holocene. bearing in mind the relative closeness of these caves with other speleological objects in the nišava valley, as well as an exact geological-tectonic history and identical climatic conditions, we can conclude that the caves in the nišava valley are also of pleistocene age. this viewpoint can serve as an explanation for significant differences in cavern-like fauna of cave systems in eastern serbia (which are relatively poor in the fauna) and cave systems of dinarides (who hold one of the richest fauna in the world). cave systems in eastern serbia were formed at the end of the glaciation, when most termophile species of the tertiary age had already extinct. outside the context of this paper we present an interesting observation. specifically, the caves of eastern serbia have developed as part of two carbonate platforms: “kučajsko-tupižnička carbonate platform” and “miročka carbonate platform”. both platforms were developed in the mesozoic, in the upper jurassic and lower cretaceous (about 150 million years ago). "overlaying" the map of distribution of carbonate rocks in serbia with the map of neogen basins in serbia shows that limestone areas were not flooded by neogen waters they existed as dry land (figure 2). so the question is: why are there no old cave systems to them? one possible explanation is that at the time the position of these masses was much closer to the equator, and in such circumstances, in the absence of cold waters the forming of caverns was difficult. r i b e r a et al. (2010) analyzed the age of 57 species of cave-dwelling beetles from leptodirini (leiodidae, cholevinae). the authors concluded that the colonization of cavern like habitat in the pyrenees had begun shortly after their formation in the early oligocene. so, today's abundance of species from this group is understandable having in mind that their evolution took 35 million years. the authors also concluded autochthony of this group; it has evolved in the same area where its ancestors used to be. the pyrenees are represented by another tertiary relic ramonda myconi (fam. biologica nyssana 1 (1-2) december 2010: 123-130 jakšić, p., momirović, m. contribution to understanding the origin … 128 fig. 2. mutual spatial relationship – right, between the neogene basins of serbia (j a n k o v i c , 1990) left, and the range of karst (g a v r i l o v i c , 1976) in the middle. gesneriaeceae), this species makes it possible to make a complete parallel with the area that we have analyzed in this paper. as the species mentioned in the pyrenees, our ramonda serbica and ramonda nathaliae are tertiary relicts. but while in the caves of the pyrenees there are 57 analyzed species from leptodirini, to this time there are no species of this group in analyzed caves of nišava valley. by this means, the opinions of g a v r i l o v i ć (1967) and p e t r o v i ć (1977) are confirmed about the pleistocenic age of caves in eastern serbia. the known leptodirini are leiodidae from eastern serbia, the representatives of the endogean and not of the cavern-like fauna. (c u r č i ć et al., 2009). why did this group fail at adaptive radiation of cavern-like habitats? probably because the continuous temperature stability of the region and the presence of optimum moisture content in litter and endogean conditions allowed a comfortable existence. the area of deciduous forests, which dominate the carpatho-balkan mountains of eastern serbia, is characterized by the presence of a powerful layer of organo-mineral complexes; that is why the representatives of the endogean fauna did not have an ecophysiological need to retreat to cavern-like habitats. the areas affected by the influence of the pleistocene climate, but which were not covered in ice, have a well-preserved terrestrial fauna. we conclude that the process of formation of cavern-like fauna in the carpatho-balkanides started, but has not yet been completed. but, on the other hand, cavern-like leptodirini of the pyrenees have their analogues in eastern serbia. an analogy is found in the group pseudoscorpiones according to c u r č i ć et al. (2004) out of 25 species from this group distributed in eastern serbia, 20 of them live in caves, while only 5 species have been found outside the cave. conclusion the fauna of the nišava riverside valley is represented by species from different geochronologycal epochs: the species of tertiary age as the oldest (represented by thermophile species), the species of the pleistocene age (represented by glacial species) and species of the holocene age, of postglacial character, as the youngest (represented by xerothermic relicts). the opinion of j a n k o v i ć (1909) on the relationship between the land and the sea during the neogen must be taken with caution. the opinion of p e t r o v i ć (1988) is more likely, that in this area a shallow sea existed and the surrounding terrain upheaved successively. the current lake terraces explain the stages of ground upheaval, rather than the stages of the sea outflow. based on cavern-like fauna composition, and based on analogy of genesis of some caves in eastern serbia, it is certain that the cavern-like objects in the nišava riverside valley are of pleistocene age. the forming process of the cavern-like fauna has begun; in some groups it has advanced far (pseudoscorpiones), in some groups it is still in its infancy (leptodirini, leiodidae), but it certainly has not yet reached the scale of what we encounter in the dinarides. on the other hand, the species of tertiary age are abundantly represented in the gorges, springs and wells, especially in forest litter, in damp and shady habitats. also, they can be found in high mountain habitats of oromediteranic thermophilic character. biologica nyssana 1 (1-2) december 2010: 123-130 jakšić, p., momirović, m. contribution to understanding the origin… 129 references cvijić, j., 1913. rtanj. glasnik geografskog društva, 2: 279-298, beograd. ćurčić, b.p.m. dimitrijević, n.r., legakis, a., 2004. the pseudoscorpions of serbia, montenegro and the republic of macedonia. belgrade-athens. ćurčić, s.b., brajković, m.m., ćurčić, b.p.m., 2007. the carabids of serbia. belgrade – vienna. ćurčić, s.b., makarov, s.e. and ćurčić, b.p.m., 2009. a revision of magdelainella jeannel, with a description of m. studenicae sp. n. 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(the flora of sićevo gorge). diplomski rad, pp. 1127. univerzitet u novom sadu, pmf, institut za biologiju. novi sad. (manuscript). zlatković, b. i ranđelović, v., 1993-94. ugroženost i zaštita flore sićevačke klisure. [endangerment and protection of flora of sićevačka klisura (the sicevo gorge)]. zaštita prirode, 46-47: 191-199, beograd. microsoft word 0306_vujicic_et_al konacno biologica nyssana 1 (1-2) december 2010: 77-82 vujičić, m. et al. axenically culturing the bryophytes … 77 original article ! axenically culturing the bryophytes: a case study of the moss herzogiella seligeri (brid.) z. iwats. (plagiotheciaceae) milorad vujičić*, aneta sabovljević, marko sabovljević institute of botany and garden jevremovac, faculty of biology, university of belgrade, takovska 43, 11000 belgrade, serbia *e-mail: milorad@bio.bg.ac.rs abstract: vujičić, m., sabovljević, a., sabovljević, m.: axenically culturing the bryophytes: a case study of the moss herzogiella seligeri (brid.) z. iwats. (plagiotheciaceae). biologica nyssana, 1 (1-2), december 2010: 77-82. a moss genus herzogiella, from the pleurocarpous family plagiotheciaceae contains only seven species world wide. it occurs in north, central and south america, europe and asia. in europe, only three species occurred, namely h. seligeri, h. striatella and h. turfacea of which, the last one is threatened. with aim to develop the methodology for protection, conservation and active propagation of h. turfacea, more commonly distributed counterpart, h. seligeri, were taken from the national park fruška gora and axenically culture were established. the study gives overview into the problems of sterilization, in vitro establishing, development, propagation and biology of species, as well as indices applicable to threatened counterpart. key words: herzogiella seligeri, in vitro introduction ! bryophytes (comprising mosses, liverworts, hornworts and alies) are the second largest group of higher plants after flowering plants, with estimated 15,000 species worldwide (h a l l i n g b ä c k and h o d g e t t s , 2000). bryophytes, although the second largest group of terrestrial plants, received much less attention in conservation and protection and in comparison to vascular plants and higher animals much less are known on their biology. they comprise very diverse plant groups (e.g. peatmosses, latern-mosses, leafy liverworts) with quite diverse biological characteristics (i.e. structure, size, ecology etc). although culturing plant tissues and organs under axenic conditions was firstly established and profitably employed in bryophytes, especially mosses (s e r v e t t a z , 1913), bryophytes did not retain for long their rightful place as a highly favored research object; therefore most studies of plant morphogenesis are now being done on vascular plants. besides the problems with bryophyte establishment in axenic culture, it is often problem of material availability, genetic variability of material, disposal of axenic organisms leaving on bryophytes and low level of species biology knowledge (e.g. d u c k e t t et al., 2004). apart from economic considerations of experimental work with bryophytes, many fundamental and applicative physiological, genetical, morphogenetic, ecological and evolutionary, as well as other problems could be studied more easily in bryophytes rather than in vascular plants (s a b o v l j e v i ć et al., 2003). bryophytes are useful objects for the elucidation of comlex biological processes such as apogamy, apospory, stress-induced cellular responses in plants, and the fusion and growth of protoplast, etc (l a l , 1984; c o v e et al., 1997; o l i v e r and w o o d , 1997; s h u m a k e r and d i e t r i c h , 1998; r e s k i , 1998; w o o d et al., 2000; c v e t i ć et al., 2005, 2009; b o g d a n o v i ć et al., 2009; v u j i č i ć et al. 2010). 10th sfses • 17-20 june 2010, vlasina lake1 (1-2) • december 2010: 77-82 biologica nyssana 1 (1-2) december 2010: 77-82 vujičić, m. et al. axenically culturing the bryophytes… 78 besides, axenical cultivation of bryophytes as well as developing of methodology in propagation of bryophytes are significant in rare species conservation both for ex situ and reintroduction (e.g. b a t r a et al., 2003; b i j e l o v i ć et al., 2004; s a b o v l j e v i ć et al., 2005; r o w n t r e e and r a m s a y , 2 0 0 5 ; 2 0 0 9 ; g o n z a l e z et al., 2 0 0 6 ; m a l l o n et al., 2007; r o w n t r e e , 2006; c v e t i ć et al., 2007; b r e z e a n u et al, 2008; c h e n et al., 2009; v u j i č i ć et al., 2009, r o w n t r e e et al., in press). this is especially valuable for the species like bryophytes many of which are dioecious and possibly long-lastingly in sterile condition naturally. axenic culturing of bryophytes seems to be so complicated that many investigators gave up the attempt. however, due to possible interaction with other organisms in non axenic conditions, sterile culturing is necessary for certain experimental procedures. progress in bryophyte tissue culture has not gone as fast as in culture of the cells of vascular plants, and the number of cases achieved still does not satisfy sufficiently the demands of various research fields (f e l i x , 1994). like other members of the bryophyta, the mosses are diverse haploid-dominant plants. mosses did not received a lot attention in chemistry research as a source of newly and/or bioactive compounds (s a b o v l j e v i ć and s a b o v l j e v i ć , 2008). however, the problem for analyzing and/or certain substance production in larger amount is often inadequate axenical material, i.e. impossibility to have clean material in enough amount neither to establish bryophyte monoculture fields. one of solution, even it seems problematic one is to establish in vitro culture, to find the proper developmental conditions and to propagate it for the wanted purpose (s a b o v l j e v i ć et al., 2010) in this study, we have focused to pleurocarpous moss herzogiella seligeri. the aim of the present study was to establish stable in vitro culture of this species and examine its development under axenic conditions. since up to date any plagiothecioid moss were not cultivated axenically, the true challenge was to establish the axenic culture of this moss having in mind its tiny and tender morphology and anatomy. material and methods a moss h. seligeri (bridel) z. iwats. (syn. leskea seligeri bridel, musc. rec. 2(2): 47. 1801; dolichotheca seligeri (bridel) loeske; isopterygium seligeri (bridel) c. jensen; plagiothecium silesianum (weber & mohr) schimper; plagiothecium seligeri (bridel) lindberg; sharpiella seligeri (bridel) z. iwatsuki) has been studied. it is wide spread in contrast to its rare and endangered counterpart h. turfacea (i r e l a n d , 1992). h. seligeri is one of the first invading species on the rotten logs and tree bases of all kind of trees. the capsules are mature at the beginning of summer. this is a species distinctive by its widespreading leaves, appearing in several rows, and its long (2-3.5 mm), arcuate capsules. plants in thin mats, lightto yellowish green, glossy. stems to 30 × 1.5-3 mm, prostrate to ascending, pseudoparaphyllia lacking. leaves widespreading, ovate to ovate-lanceolate, smooth, nondecurrent or 1-3 cells indistinctly decurrent, 12.5 × 0.5-0.9 mm, margins serrulate to serrate; cell walls pitted at leaf base, indistinctly pitted distally, sometimes pits lacking; median cells 30-70 × 5-7 μm; alar cells quadrate to short-rectangular, sometimes rounded to oval and inflated, 17-48 × 1226 μm. sexual condition autoicous. seta light brown to red, 1.5-2.5 cm. capsule light brown to reddish brown, inclined, 2-3.5 × 0.5-0.8 mm, cylindric, strongly arcuate, when dry contracted below mouth; operculum conic, 0.4-0.6 mm. spores 12-22 μm. the materials for axenic culture were collected in fruška gora mt in march 2007 on rotten log and the voucher specimen was deposited in the bryophyte collection of the belgrade university herbarium (beou 4421). after collection, the plants were stored in plastic bags at +4°c, till the begging of the experimental work. in the laboratory conditions, the material were cleaned under dissecting microscope from the visible mechanical impurity. the sporophyte with mature almost ripen but unopened capsules were separated, and the gametophyte tips consisted of the leafy stems of ca. 10 mm longitude were separated carefully, placed in glasses, covered with cheese cloth, and rinsed with tap water for 30 minutes. sporophytes and gametophyte parts were then disinfected for 5 minutes with 3, 5, 7, 10, 13% or 15% solution of sodium hypochlorite (commercial bleach, naocl). finally, they were rinsed three times in sterile deionised water. as a basal medium for establishment of in vitro culture, we used murashige and skoog (1962) (ms) medium containing murashige and skoog mineral salts and vitamins, 100 mg/l inositol, 0.70% (w/v) agar (torlak purified, belgrade), and 3% sucrose and bcd medium (see s a b o v l j e v i ć et al. 2009 for the media details). once, the establishment was done, and the plants produced, the in vitro developed plant segments (tips and protonema pieces) were used for further developmental experiments. biologica nyssana 1 (1-2) december 2010: 77-82 vujičić, m. et al. axenically culturing the bryophytes … 79 in order to observe the influence of sucrose and/or mineral salts on the morphogenesis of this species, the following medium composition combination were tested: ms1: half strength of ms mineral salts, sugar free; ms2: half strength of ms mineral salts, 1.5% sucrose; ms3: half strength of ms mineral salts, 3% sucrose; ms4: ms mineral salts, sugar free; ms5: ms mineral salts, 1.5% sucrose; ms6: ms mineral salts, 3% sucrose; bcd1: bcd mineral salts, 1.5% sucrose; bcd2: bcd mineral salts, 3% sucrose; bcd3: bcd mineral salts, sugar free; the ph of the media was adjusted to 5.8 before autoclaving at 114°c for 25 minutes. the temperature and light duration varied in combined with sets of media: combination c1: 16/8 hours of light to darkness, at 25 ± 2°c. combination c2: 8/16 hours of light to darkness, at 20 ± 2°c. combination c3: 16/8 hours of light to darkness, at 20 ± 2°c. combination c4: 16/8 hours of light to darkness, at 18 ± 2°c. light was supplied by cool-white fluorescent tubes at a photon fluency rate of 47 μmol/m2s. cultures were subcultured for a period of 4-6 weeks. for analysis of condition set influence to development 10mm long apical segments (gametophyte), spores or protonema were transferred to various nutrient media. for each medium composition combined with light conditions, 40 transplants of h. seligeri were cultivated. the influence of tested environmental condition was quantified after 30 days by visually estimation of the plant habitat marked as the best those of appearance like in nature. results and discussion the attempts to establish the axenic culture from gametophytes i.e. 10mm plant tips failed since the concentration for surface sterilizations killed the plant material or was not effective enough to kill the xenic organisms on the plants and not to harm the plants at the same time. so, even there where the plants survive the bleach surface sterilization and transferred to the mineral salts, it was overgrown quickly with fungi, algae and bacterias. the try outs to leave it until transferred plantlets overgrow the xenic organisms, for the purpose of the use of newly grown tips, remained useless (note that the survival does not mean axenic as well). there is rather low probability percentage of good bleach surface sterilization of the moss tips, without harming plants, for establishment in vitro culture (e.g. s a b o v l j e v i ć et al., 2003) surface sterilization of the sporophytes was more successful since we choose the almost mature but unopened capsules and did the sterilization in various concentration of bleech for 5 minutes like for the gametophytes. the advantage of this process was that we did not need the capsules material itself (so we could harmed it lethally) but the spores from inside that should remain viable. once, the surface of sporophytes was sterilized, the capsules were opened in sterile conditions and the spores were taken out with sterile needle to the mineral salt containing media. the success of this way starting culture concerning sterilization of start plant material was achieved with 100% at 10% bleach for 5 minutes. in higher concentration the sterilization percentage remain high but the bleach started to harm the spores quantified by spore germination slightly decrease. 0 2 4 6 8 10 12 14 16 -10 0 10 20 30 40 50 60 70 80 90 100 110 su rv iv al (% ) bleach concentration (%) gametophyte sporophyte fig 1. percentage of surviving of gametophyte and sporophyte parts after bleach treatment. the best bleach concentration for surface sterilizing of h. seligeri was 10%. the percentage of tested propagules, both of gametophyte tips and sporophytes survival, decreased with concentration increase (fig.1). however, the bleach could not offer proper sterilization since propagules contained hardly disposal contamination afterwards with fungi, algae or bacteria, or the high concentration is lethal in high percentage (above 10%). the bleach concentration under 10% are functional since the propagules survived in high percentage, but not 100% of used material was axenical. bleach concentrations above 7% for five minutes exposure biologica nyssana 1 (1-2) december 2010: 77-82 vujičić, m. et al. axenically culturing the bryophytes… 80 kill most of the propagules and are not appropriate for this moss gametophytes in vitro culture establishment. figs. 2. and 3. a detail of herzogiella seligeri gemetophyte developed in in vitro condition spores were germinated on ms medium enriched with sucrose (ms3). after releasing from the capsules, spores germinated in relatively high percentage (up to 100%). however, on the ms medium enriched with sucrose they remain in the phase of primary protonema. the subculturing to fresh medium with sucrose was not a signal for plantlets to pass to the next developmental stage. the protonema is not spreading far from spore in any media tested. the variation of light-length and temperature condition in h. seligeri cultures did not show significantly different behavior (figs. 2 and 3). a set of various combination of light length, temperature and mineral salts were tested to achieve the bud induction and gametophyte development. it can be concluded that in the condition when medium contain the sugar (ms2, ms3, ms5, ms6, bcd1, bcd2) the spore germination is stimulated, but the gametophyte development stopped at protonemal eventually caulonemal stage. schoefield (1981) stated that in most bryophytes spores germinate 7-30 days after exposure of spores to good conditions. in our case, it was quicker when media contained sucrose (7-10 days) than the spore germination on sucrose free media. interestingly, difference in gametophyte development was achieved when sucrose was put by (ms1, ms4). on bcd sugar free (bcd3), the bud formation was noticed after a month. bopp (1952) explained that in native conditions protonema have to achieved the certain size which then produce enough amount of kinetin-like growth regulators released in substrate. this is a trigger for bud induction or passing from filamentous to meristemal growth. buds developed rapidly into a stem which again branched and continue growing achieving full size and normal leaf shapes of natural plants but not the plant shape (figs. 4 and 5). a rather very humid air condition of the growth-dishes favors elongation and growth. figs. 4. and 5. herzogiella seligeri gemetophyte developed in in vitro condition the 10mm shoot and branch tips were used further for subculturing into new media combined with four combination of controlled conditions of day length and temperature. biologica nyssana 1 (1-2) december 2010: 77-82 vujičić, m. et al. axenically culturing the bryophytes … 81 the best developed and the most similar to the plants developed in nature were grown on ms1 and bcd3 at temperature of 18°c or 20±2°c, at both day length. in the temperature of 25±2°c the plants produced slightly smaller, shorter, tinny, fragile and unbranched shoots, often developing contamination of 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(dicranaceae, bryophyta). botanica serbica 33(2): 137-140. vujičić, m., cvetić, t., sabovljević, a., sabovljević, m. 2010. axenically culturing the bryophytes: a case study of the liverwort marchantia polymorpha l. ssp. ruderalis bischl. & boisselier (marchantiophyta, marchantiaceae). kragujevac journal of science 32: 73-81. wood, a. j., oliver, m. j., cove, d. j. 2000 new frontiers in bryology and lichenology. bryophytes as model systems. bryologist 103: 128–133. đokić et al. 2023, biologica nyssana 14(1) 14 (1) june 2023: 57-63 doi: 10.5281/zenodo.8027182 impact of cuscuta spp. seed size variability on machine operation during seed finishing of natural alfalfa seeds original article dragoslav đokić faculty of agriculture, university of niš, kosančićeva 4, kruševac, serbi djdragosla01@gmail.com (corresponding author) violeta oro institute for plant protection and environment, 11000 belgrade, teodora drajzera 9, serbia ratibor štrbanović institute for plant protection and environment, 11000 belgrade, teodora drajzera 9, serbia dobrivoj poštić institute for plant protection and environment, 11000 belgrade, teodora drajzera 9, serbia jasmina milenković institute for forage crops, 37251 globoder-kruševac, serbia jasmina knežević faculty of agriculture, university of priština kosovska mitrovica, lešak 38219, kopaonička bb, serbia rade stanisavljević institute for plant protection and environment, 11000 belgrade, teodora drajzera 9, serbia stanisavljevirade@gmail.com (corresponding author) received: december 05, 2022 revised: march 02, 2023 accepted: march 07, 2023 abstract: the alfalfa seeds intended for marketing must not contain any seeds of the dodder (cuscuta spp.), because it is a quarantine weed in serbia and the world. cuscuta spp. are frequently found in natural alfalfa seeds; nevertheless and because of this, the seed must be processed, i.e. cleaned of weeds. the paper presents a study of seed size variability of cuscuta spp. of four seed lots of natural alfalfa seeds originating from banat, serbia. from each of the four examined seed lots of natural alfalfa seeds, a difference in seed size was determined for 30 seeds of cuscuta spp. per lot. the difference in seed length of cuscuta spp. was 371 µm, and the difference in seed width was 330 µm. the coefficient of variation varied from cv=8.356% to 12.00% for seed width; and from cv=9.383% to 10.69% for seed length. seed size of cuscuta spp. did not significantly affect the efficiency of magnetic separator work in extracting cuscuta spp. seeds from natural alfalfa seeds. key words: cuscuta spp., variability, seed width and seed length, seed processing apstrakt: uticaj varijabilnosti veličine semena cuscuta spp. na rad mašina pri doradi naturalnog semena lucerke seme viline kosice (cuscuta spp.) je karantinski korov u srbiji i svetu i zato se u semenu lucerke koje je namenjeno prodaji, ne sme naći seme korova cuscuta spp. međutim, seme cuscuta spp. se relativno često može naći u naturalnom semenu lucerke i zbog toga se seme mora doraditi, odnosno očistiti od korova. u radu je prikazano ispitivanje varijabilnosti veličine semena cuscuta spp. koja je izdvojena iz naturalnog semena četiri partije semena lucerke poreklom iz banata, srbija. na po 30 semena cuscuta spp., koja su izdvojena iz svake od četiri ispitivane partije naturalnog semena lucerke, uvrđena je varijabilnost za veličini semena. razlika po dužini semena cuscuta spp. je bila 371 µm, a po širini 330 µm. izraženo kroz varijabilnost, koeficijent varijacije se kretao od cv=8.356 do 12.0% za širinu semena i od cv=9.383 do 10.69% za dužinu semena. veličina semena cuscuta spp. nije uticala značajno na efikasnost rada magnetnog separatora prilikom izdvajanja iz naturalnog semena lucerke ključne reči: cuscuta spp., varijabilnost, širina i dužina semena, proces dorade introduction dodder (cuscuta spp.) belongs to the genus convolvulaceae which includes about 200 mostly parasitic species (costea et al., 2015 a,b). it is a parasitic plant that causes significant economic damage around the world (dawson et al., 1994; costea & tardif, 2006; sarić-krsmanović et al., 2020). it is especially harmful in alfalfa seed crops (dawson et al., 1994; sarić-krsmanović et al., 2020). dodder seed causes damage by extracting nutrients from the parent plant; thus, the parent plant weakens, becomes exhausted and lags behind in development. about ten species of cuscuta spp. were detected in serbia in the 1960s and 1970s, and they can cause significant damage to agricultural crops (stojanović et al., 1973), with a particularly large problem in alfalfa (mijatović & stojanović, 1968). in agronomic practice, it is considered that it is best to control cuscuta spp. in the seed crop before harvesting. nevertheless, it happens that alfalfa seed crops often contain cuscuta spp. according to janjic et al. (2005), in serbia, protection from cuscuta spp. began with weed control in crops that had © 2023 đokić et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 57 already been infected. however, a better approach is to apply preventive measures that should be the basic way of weed control. the properties of the dodder seed, and above all, its vitality in the soil, enables the weeded parcels to become a source of further infection during the next ten or more years. therefore, the basic strategy in the fight must be to prevent infestation of parcels (janjić et al., 2005). regardless of the numerous ways of testing and experience in biology and agronomic practice to study its characteristics and control to this day, it is widespread and causes significant damage in the world (abdel-khalik, 2006; córdoba et al., 2021) and serbia (sarić-krsmanović et al., 2022). due to the close resemblance in size between cuscuta spp. and alfalfa seeds, the presence of cuscuta spp. in the seed crop not only directly affects seed production but also presents substantial challenges for seed finishing. the dispersal of cuscuta spp. in the alfalfa seed-growing region is another major issue. also, a significant issue is the ability of cuscuta spp. seeds to retain germination in the soil for decades and germinate again after alfalfa resowing (jayasuriya et al., 2008; olszewski et al., 2020). especially this was a problem for seed production in the eighties of the last century (čuturilo & nikolić, 1986). it is necessary to study the advantages of extracting the seeds of cuscuta sp. from natural alfalfa seeds on a laboratory machine laboratory magnetic separator by german manufacturer emceka gompper and compare it with the efficiency of extracting cuscuta sp. seeds in a conventional way. further studies of weeds cuscuta spp. in serbia have given some solutions in some aspects, but the question of cuscuta spp. is not close to being fully solved (sarić-krsmanović et al., 2020; 2022). it should be added that the seeds of cuscuta spp. in alfalfa seed are considered quarantine weeds which means that such seed cannot be placed on the market gazette of sfry, 1987 (47/87), gazette of the republic of serbia, 45 2005, and supplement to 2021 which is in line with ista rules (ista 2021), which mainly deals with quality and seed trade in europe. the aim of this research was to extract cuscuta spp. seeds from four seed lots of natural alfalfa seed, and examine its variability in seed size. then, to determine whether the size of cuscuta spp seeds affects the efficiency of seed finishing. additionally, to contrast the effectiveness of cuscuta spp. seed separation from natural alfalfa seeds on a laboratory magnetic separator made by german company emceka gompper with the effectiveness of cuscuta spp. seed separation on equipment used in the industrial-standard process of processing natural alfalfa seeds. materials and methods seeds of four seed lots of alfalfa were collected from the banat region (the part of serbia where alfalfa for seed production is grown the most). the seed lots were selected on the basis of a test sample, according to which the seed lots (i to iv) of natural alfalfa seed had approximately the same amount of quarantine weed cuscuta spp. the analyzes were performed in an accredited laboratory for seed quality control, institute of plant protection and environment belgrade. a precise electronic scale was used to measure the samples. the seed samples for analysis weighed 5 g (working) samples in accordance with the regulations of the law on seeds and planting material. all conditions related to the method of production, processing, use, circulation, import and testing are prescribed seeds of agricultural plants (gazette of the sfry no. 47, 1987 and official gazette of the republic of serbia no. 45, 2005). the quality of alfalfa seeds should correspond to the regulation on the quality of seeds of agricultural plants, which is harmonized with the international regulation on the seed quality ista 2021. subsample i: a sample of 5 grams of natural alfalfa seed was obtained from each seed lot. the length and width of the dodder seeds from this weed were measured using the random selection method on a sample of 30 seeds. the length and width of the seeds were examined with an olympus bh-2 microscope. for each seed lot, the average, lowest and highest value (min. and max.), standard deviation (stdev), coefficient of variation (cv%), and standard error of mean were determined to determine the variation in seed size of cuscuta spp. (sem). for each seed lot, the correlation coefficients (r) between cuscuta spp. seeds’ length and width were computed. subsample iia: from each seed lot, 1 kg of seeds was divided into four parts (repetitions) and cleaned on a laboratory magnetic separator. subsample iib: in the seed processing facility of the company „herba” d.o.o. belgrade, the seed lots are processed in the traditional industrial manner, also in four repetitions. all seed batches of natural alfalfa were processed using a selector from the cimbria brand, model delta super 102. this selector features three shaker shoes with two 625 mm x 800 mm sieves each, for a total of six 3 m2 sieves. i shaker shoe has two sieves with round holes of 2.25 mm and 2.25 mm, ii shaker shoe has two sieves with round openings of 1.4 mm and 1.4 mm, and iii shaker shoe has two sieves with rectangular openings of 0.7 mm and 0.5 mm (#-wire). sieves with the following 58 biologica nyssana ● 14 (1) june 2023: 57-63 đokić et al. ● impact of cuscuta spp. seed size variability on machine operation during seed finishing of natural alfalfa seeds biologica nyssana ● 14 (1) june 2023: 57-63 đokić et al. ● impact of cuscuta spp. seed size variability on machine operation during seed finishing of natural alfalfa seeds is the passage through the magnetic separator, where the seeds of the dodder are separated, as well as other harmful weeds and impurities that are undesirable in alfalfa seeds. separation of weeds, especially weeds with wrinkled and unsmooth seeds and other impurities, on this machine is done by electromagnetic force. a certain quantity of steel powder with water is used for the technological process of weed separation on a magnetic separator. between the results of the separation of cuscuta spp. seeds, obtained by industrial and laboratory processing, the correlation coefficient (r) was calculated for each seed lot of natural alfalfa seeds. statistical analysis was done via the freeware software package minitab 16. results and discussion in our experiments, dodder seed, isolated from natural seed lots, showed variability in terms of average seed size (tab. 1). the highest average length and width of seeds of cuscuta spp. was in the third seed lot (1,361 µm and 1,197 µm), which is 371 µm more in length, and 330 µm more in width, compared to the seed lot with the smallest seed (lot iv: length 990 µm and width 867 µm). perforations are used in practice to remove cuscuta spp. quarantine weeds from alfalfa: 1.85 mm, 2.00 mm, and 2.25 mm. other seeds (like lolium sp.) are typically sieved using sieves with openings smaller than 4.5 mm. the sieves are arranged in size order from top to bottom, with larger opening sieves at the top and smaller opening sieves at the bottom. seeds are divided into length and width using rectangular and round sieves, respectively. based on the results of the purity test of the natural seed sampled (determination of the type of inert substances, the presence of weeds and quarantine weeds cuscuta sp., rumex sp., ambrosia sp. and other impurities). depending on the level of purity of the natural seed achieved after the primary seed finishing on the cimbria selector, a subsequent step of seed processing on the cimbria trier model hsr 4020 rl may be required. a total of two segments, one for round seed and the other for long seed, make up the mantle, which is about 600 mm in diameter and 2,000 mm long. a mantle with alveoli with a diameter of less than 2 millimetres was employed in this instance of completing alfalfa seeds. a cimbria gravity table can be used in the seed processing procedure if necessary. the last, final phase of alfalfa seed processing 59 table 1. the seed size variability of cuscuta spp. detected in four seed lots of natural alfalfa seed cuscuta spp. seeds (serial number) seed dimensions of cuscuta spp. detected in a seed lots of natural alfalfa seeds (μm) i ii iii iv length width length width length width length width 1 1053 951 1275 879 1256 1156 1189 1025 2 1062 988 1283 1157 1325 1265 956 845 3 971 1109 1495 1530 1256 1253 899 802 4 1254 1086 1210 1169 1152 989 1075 895 5 1225 1114 1340 1156 1155 1156 1172 956 6 1102 978 1380 1155 1456 1356 856 802 7 1221 1121 1434 1423 1352 1302 848 832 8 1489 1178 1340 1049 1299 1255 1025 1002 9 1164 1014 1183 1127 1158 1199 975 887 10 1206 1066 1196 1073 1522 1325 887 799 11 1381 1114 1298 1181 1632 1025 926 802 12 1094 1026 1564 1346 1452 1152 1175 1011 13 1145 1128 1090 971 1289 1136 956 789 14 1178 1078 1116 1098 1325 1256 875 803 15 1301 1082 1278 1151 1256 1302 1125 1033 16 1236 1027 1198 1140 1488 1235 963 805 17 1341 1341 1313 1051 1502 1156 1023 921 18 1131 1131 1309 1155 1558 1235 985 875 đokić et al. ● impact of cuscuta spp. seed size variability on machine operation during seed finishing of natural alfalfa seeds biologica nyssana ● 14 (1) june 2023: 57-63 60 cuscuta spp. seeds (serial number) seed dimensions of cuscuta spp. detected in a seed lots of natural alfalfa seeds (μm) i ii iii iv length width length width length width length width 19 1143 1143 1211 1068 1365 1099 885 756 20 1208 1208 1619 1398 1236 1156 1025 901 21 1078 1078 1201 1030 1320 1299 1125 1023 22 1347 1347 1330 1272 1398 1005 982 789 23 1214 1214 1193 971 1299 1125 976 821 24 1379 1379 1488 1084 1365 1233 1087 902 25 1242 1242 1211 1063 1455 1302 965 842 26 1314 1314 1269 1163 1266 1235 875 702 27 1347 1347 1448 1172 1566 1099 1023 825 28 1231 1231 1373 1196 1488 1263 986 756 29 1306 1021 1188 1051 1259 1198 831 799 30 1221 956 1626 1221 1368 1022 1025 1002 average 1219 1134 1315 1150 1361 1197 990 867 min 971 951 1090 879 1152 989 831 702 max 1489 1379 1626 1530 1632 1356 1189 1033 stdev 115.2 123.1 140.6 138.0 127.7 99.99 100.7 92.59 cv (%) 9.447 10.86 10.69 12.00 9.383 8.356 10.17 10.68 sem 13.97 14.93 17.05 16.74 15.48 12.13 12.21 11.23 for the dodder seed width, the highest variability was in the second seed lot (cv=12.00%), and the lowest was in the third seed lot (cv=8.356%). the variability of seed size that occurred in different seed lots can be statistically considered low (hadživuković, 1991). ho & costea (2018) stated significantly higher variability for the seed size of cuscuta spp. however, this study was done at different geographical distances and was different from the region where our experiment had been placed. within the genus convolvulaceae, the seed size of cuscuta spp. is most affected by the species (costea et al., 2015a,b). the following physical properties are considered to be the most important for seed finishing: humidity, shape, dimensions, sphericity, mass of 1,000 seeds, volume and porosity, volume-hectolitre mass, density, static and dynamic angle of internal friction (free fall angle), and external condition seed surfaces (copeland & mcdonald, 2004; black et al., 2006; babić & babić, 2012; baskakov et al., 2018). in our trials, all seed lots showed a positive correlation between seed length and width, whereas three seed lots (i, ii and iv) had a very high correlation between these features (p≤0.001). in seed lot iii, seed length and width did not correlate significantly (p≥0.05). also, positive correlative interdependence was high in all seed lots (p≤0.001) (tab. 2). healthy alfalfa seeds have a smooth surface, and after mixing the seeds with steel powder and water in a certain proportion, the steel powder does not stay on that surface. in contrast, the dodder seed has a grain that is porous, and steel powder remains on it, which allows it to separate from alfalfa seeds (fig. 1, fig. 2). table 2. simple correlations (r) between length and width of cuscuta spp. seeds (n=30) seed dimensions of cuscuta spp. originating from lots of natural alfalfa seed (μm) i ii iii iv length width length width length width length width r=0.579*** r=0.672*** r=0.046 ns r=0.818*** * p≤0.05, ** p≤0.01, *** p≤0.001, ns – not significant p≥0.05 đokić et al. ● impact of cuscuta spp. seed size variability on machine operation during seed finishing of natural alfalfa seeds the electromagnetic cleaning machine is used for the final cleaning of alfalfa seeds from the seed of weeds. the quality of weed cleaning on this machine depends on the correct ratio of water and magnetic seed cleaning powder and the amount of seed passed through the magnetized cleaning rollers (đokić & stanisavljević, 2012; uhlarik et al., 2018; đokić et al., 2020). in our experiment, a simple correlation coefficient between the length and width of cuscuta spp. seeds fig. 1. laboratory magnetic separator german manufacturer emceka gompper was very significant (r=0.784; tab. 3). biologica nyssana ● 14 (1) june 2023: 57-63 61 fig. 2. seed of cuscuta spp. table 3. simple correlations (r) between length and width of cuscuta spp. seeds separated from all seed lots of natural seed (n=120) length width r=0.784*** ***, p≤0.001 the principle of operation of electromagnetic separators (trifolin machine-detector) is based on electromagnetic action. they are intended for separating weed seeds with wrinkled and unsmooth surfaces and other impurities. the success of removing the dodder from alfalfa seeds also depends on the method of applying the appropriate metal powder (milošević et al., 1996). electromagnetic seed cleaning machines are very important due to their high-quality cleaning performance. such a high quality of cleanliness cannot be achieved by pneumatic cleaning, triers or selector screens (kozlov, 2013.). the seed of cuscuta spp. was completely isolated from all seed lots of alfalfa seeds in our laboratory tests. also, the seed size did not affect the separation efficiency (tab. 4). table 4. efficiency (%) of separating cuscuta spp. seeds on a laboratory magnetic separator seed size cuscuta spp. (length d μm and width w μm) separated from seed lots of natural seed of alfalfa i ii iii iv l w l w l w l w 100a 100a 100a 100a 100a 100a 100a 100a a tukey’s test (p≤0.05) for length, a tukey’s test (p≤0.0) for width in seed finishing practice, the assumption is that laboratory and industrial processing of alfalfa seeds should be equally or similarly effective in removing cuscuta spp. from natural alfalfa seeds. table 5. simple correlations (r) between the isolation of cuscuta spp. from four seed lots of natural alfalfa seeds in laboratory conditions and in conventional alfalfa seed processing (n=4). seed lots of natural alfalfa seed i ii iii iv r=0.984** r=0.931* r=0.999*** r=0.966** * p≤0.05, ** p≤0.01, *** p≤0.001, ns – not significant (p≥0.05) 62 biologica nyssana ● 14 (1) june 2023: 57-63 in our study, there was a positive and highly correlated relationship (p≤0.05 to p≤0.001) between the efficiency of magnetic separators used in the laboratory and those used in industry (tab. 5), which is in agreement with the results of lin et al. (2022). conclusion an average sample of thirty seeds of cuscuta spp. taken from four seed lots, differed by 371 μm in length and by 330 μm in width. dodder seed isolated from all four seed lots showed variability for width from cv=8.356 (seed from the third lot) to cv=12.00% (seed from the second lot). between the length of the seeds of cuscuta spp. and width was a positive correlation, but at a different level of statistical significance (p≥0.05 to p≤0.001). the influence of cuscuta spp. seed size in the process of its separating from natural alfalfa seeds was not significant. there was a positive and significant correlation (p≤0.05 to p≤0.001) between the separation of cuscuta spp. seeds by laboratory and industrial magnetic separator. according to the law on seed material, processed alfalfa seeds must not contain any seeds of quarantined weed cuscuta spp, regardless of the type and dimensions of cuscuta spp. with electromagnetic cleaning machines, the seeds of the cuscuta spp. must be completely removed from the alfalfa seeds that are being processed. acknowledgements. research was financed by the ministry of education, science and technological development, republic of serbia (contract reg. no. 45103-68/2022-14/200383, 451-03-68/2022-14/ 200010, 451-03-68/2022-14, 451-03-9/2022-14/200133) references abdel-khalik, k.n. 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(1991). statistički metodi s primenom u poljoprivrednim i biološkim istraživanjima (2ed edition). srbija, novi sad: poljoprivredni fakultet. đokić et al. ● impact of cuscuta spp. seed size variability on machine operation during seed finishing of natural alfalfa seeds ho, a. & costea, m. (2018). diversity, evolution and taxonomic significance of fruit in cuscuta (dodder, convolvulaceae); the evolutionary advantages of indehiscence. perspectives in plant ecology, evolution and systematics, 32, 1–17. ista (international seed testing association). 2021. rules for testing seeds. switzerland, zurich. janjić, v., marisavljević, d., & pavlović, d. (2005). vilina kosica i mogućnost suzbijanja. biljni lekar, 5, 590-595. jayasuriya, k.m., baskin, j.m., geneve, r.l., baskin, c.c., & chien, c.t. 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(2022). variability in early seed development of 26 populations of cuscuta campestris yunck.: the significance of host, seed age, morphological trait, light, temperature, and genetic variance. agronomy, 12, 559. stojanović, d., mijatović, k., & borić, b. (1973). identifikacija vrsta rasprostranjenih na teritoriji sr srbije. biljni lekar, 18(3-4), 95-100. uhlarik, a., popov, s., karagić, đ., ponjičan, o., & turan, j. (2018). alfalfa seed cleaning using a magnetic separator. journal on processing and energy in agriculture, 22(4), 192-195. đokić et al. ● impact of cuscuta spp. seed size variability on machine operation during seed finishing of natural alfalfa seeds biologica nyssana ● 14 (1) june 2023: 57-63 63 anticancer compounds from medicinal plants biologica nyssana 2 (2)  december 2011: 00-00 ljupković r.b. et al..  removal cu(ii) ions from water... 69 original article antimicrobial activity of the three commercial drug’s essential oils: chamomillae flos, calendulae flos and millefolii herba zorica stojanović-radić university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia e-mail: zstojanovicradic@yahoo.com abstract: stojanović-radić, z.: antimicrobial activity of the three commercial drug’s essential oils: chamomillae flos, calendulae flos and millefolii herba. biologica nyssana, 3 (2), december 2012: 69-76. commercial herbal drugs of the three plants belonging to family asteraceae were used for the extraction of the essential oils (hydrodistillation method). since various factors during the processing of the plant material can affect several characteristics of essential oils such as yield, chemical composition and, thus, biological activities of essential oils, comparison of the antimicrobial activity of the herein isolated oils was done with avaiable literature sources dealing with this subject. in order to evaluate antimicrobial potential of the isolated essential oils, a broth microdilution method was employed. the results pointed to very high activity of the tested oils, in all cases much stronger in comparison to the previous results. as a conclusion, the adverse effects of plant material processing did not affect its antimicrobial potential, while the obtained high antimicrobial activity can be explained by sinergistic action of the herein used solvent with essential oils. key words: achillea millefolium, antimicrobial activity, calendula officinalis, chamomilla recutita, herbal drug introduction essential oils are complex mixtures of compounds, synthetized in plant tissues as secondary metabolites. the role of essential oil in plant is multiple: defence of pathogens and herbovores, attraction of insects, inhibition of other plant's seed germination, formation of protective layer which reduces transpiration and formation of specific microclimate. due to their lipophylic character, essential oils can interact with microbial membranes and achieve significant antimicrobial effect. for this reason, during the last several decades, much attention has been devoted to plant essential oils, especially to their antimicrobial activity against various pathogenic and nonpathogenic species. during the research of some plant's medicinal properties, very important indicator of antimicrobial potential is ethnopharmacological use for the treatment of infective diseases. owing to this, many studies have been focused to ethnopharmacological plants and their antimicrobial activity, as a novel source of antimicrobial compounds. in serbia, plants belonging to family asteraceae are very significant medicinal plants and are used extensively in both traditional medicine and current phytotherapy. among them, very commonly used ones are chamomile, yarrow and calendule. yarrow (achillea millefolium) is an aromatic, perennial herbaceous plant, widely distributed in serbia. healing properties of yarrow are well known from the ancient times and it is used in both official and alternative medicine. according to ph. eur. iv, biological source of the yarrow herbal drug is strictly the species achillea millefolium. however, some authors (b o ž i n et al., 2008) stated that achillea millefolium, in wider sence, represents a cytogenetic and chemically polymorphous aggregate 3 (2) • december 2012: 69-76 biologica nyssana 3 (2)  december 2012: 69-76 stojanović-radić z.  antimicrobial activity of.. 70 of 12 species, characterized by a well defined morphological, anatomical and caryological features, while the existence of the naturally occurring hybrids increases the number of the representatives in the aggregate. herbal drug used in medicinal purposes is the above-ground part of the plant in bloom (millefolii herba). rarely, some parts can be used as individual drugs – inflorescence (millefolii flos) or leaves (millefolii folium) (s a r i ć , 1989). the main utilization is connected to the treatment of the various gastrointestinal disorders owing to its carminative, holagogue and spasmolytic effects. also, it can be used to treat hemorrhoids, hypertension, thrombosis, inflammations and respiratory infections (s a r i ć , 1989; k o j i ć et al., 1998; t a j i k et al., 2008; s a n t ’ a n n a et al., 2009). applied externally, yarrow can heal inflammations of skin and mucosal surfaces; it promotes epitelization, heals the wounds and stops the bleeding process by constricting the blood vessels. essential oil of this plant yields from 0.2-1% and can contain up to 50% of chamazulenes (r o s s , 2003). investigations of its chemical composition showed that it is very variable, depending on factors such as geographical origin, duration of inflorescence and level of polyploidy – the oil has characteristic blue color only in the case of its isolation from tetraploid specimens (k u b e l k a et al., 1999; r o h l o f f et al., 2000; b o š k o v i ć et al., 2005), and it is connected to the presence of the sesquiterpene chamazulene. literature data on antimicrobial activity of achillea millefolium are relatively poor, since the most number of investigations studied antimicrobial effects of its various extracts (k o j i ć et al., 1998; n a s c i m e n t o et al., 2000; h o l e t z et al., 2002; c a n d a n et al., 2003; s t o j a n o v i ć et al., 2005; t a j i k et al., 2008). only one of these studies investigated antimicrobial potential of the yarrow essential oil (c a n d a n et al., 2003), where moderate antimicrobial activity was detected. calendula officinalis l. is used extensively in ethnopharmacology. the most frequent application of this plant is in the form of tincture or creme for curing the wounds, abscesses and decubitus. it has beneficial effect to gastrointestinal and urogenital disorders, and it is also used for sedative purposes (w i l l f o r t , 1959). the famous wound healing properties of this plant prompted investigations which proved its proliferative effects (c h a n d r a n & k u t t o n , 2008; l e a c h , 2008). marigold contains small amounts of essential oil: 0.06-0.3% (n a g u i b et al., 2005), but due to its significant pharmacological activities, it has been studied well. in the most papers, its main component is α-cadinol (c h a l c h a t et al., 1991, o k o h et al., 2007, g a z i m et al., 2008a, p e t r o v i ć et al., 2010). investigations of marigold’s essential oil antimicrobial activity inhibited growth of bacillus subtilis, escherichia coli, staphylococcus aureus, pseudomonas aeruginosa and candida albicans (j a n s s e n et al., 1986). also, it has been shown that it possess potent activity against candida albicans and staphylococcus aureus clinical isolates (g a z i m et al., 2008b; f i t et al., 2009). chamomilla recutita is an annual, perennial plant, native in southeastern europe, but today widely distributed and can be found in north africa, asia, america and new zealand (p i r z a d et al., 2006). chamomille is used in therapy since an ancient times (о w l i a et al., 2007). owing to its medicinal properties, it is included as a drug in pharmacopeia of 26 countries (p i r z a d et al., 2006; о w l i a et al., 2007; s h i k o v et al., 2008). this plant possesses anti-inflammatory, anesthetic, antiseptic, sedative, carminative, antidiarrhoeic and vulnerary properties (p i r z a d et al., 2006; m i l l e r , 2009). scientifically investigated and confirmed pharmacological properties of chamomile are multiple antioxidant (a s g a r y et al., 2002; o w l i a et al., 2007; r o m e i l a h , 2009; a y o u g h i et al., 2011), аntiulcer (k a r b a l a y d o s t & n o o r a f s h a n , 2009), antiinflammatory (p r e s i b e l l a et al., 2006; m c k a y & b l u m b e r g , 2006), vulnerary (m c k a y & b l u m b e r g , 2006; m a r t i n s et al., 2009), anticoagulant, antitumor, antigenotoxic, hypocholesterolemic, antispasmolitic and sedative (m c k a y & b l u m b e r g , 2006). the most pharmacologically active compounds are those found in chamomile essential oil – chamazulene, αbisabolol, bisabolol oxides, farnesene and cyclic esters (o r a v et al., 2001; m a r t i n s et al., 2009), but flavonoids, coumarins and pyrones are also significantly active compounds, found in chamomille (g r g e š i n a et al., 1995; p i r z a d et al., 2006). antimicrobial activity of chamomile is confirmed against 25 different gram positive and gram negative bacterial strains, as well as against 20 lysteria monocytogenes strains (l i s b a l h i n et al., 1998). the same study showed antifungal activity against aspergillus niger, a. ochraceus and fusarium moniliforme. study of o w l i a et al. (2007) showed antistreptococcal activity of chamomile essential oil, which was also active against mycobacterium tuberculosis, salmonella typhymurium and staphylococcus aureus (p i r z a d et al., 2006). beside antibacterial activity, it was confirmed that chamomile essential oil possess antiviral activity against polio and herpes viruses (mcka y & b l u m b e r g , 2006). biologica nyssana 3 (2)  december 2012: 69-76 stojanović-radić z.  antimicrobial activity of.. 71 since most of the literature data about antimicrobial activity of these plants is based on unprecise method (disc diffusion) or the data are relatively old, the main goal of this paper was to investigate antimicrobial properties of these three plants, but using more precise microdilution method and to compare the obtained results with the previous ones on this subject. owing to the fact that commercial plant material (for medicinal purposes) can be found at the markets nowdays, there is high possibility that it possess different properties than the same material collected by botanical experts. the processing after the collection such as drying, time of grounding and storage period before the isolation are very important since they can significantly affect chemical composition of essential oil and, thus, antimicrobial and other biological activities. in the light of these facts, this investigation used commercial plant material which had a lot of potential to differe when compared to personally collected material by relevant experts. the paper provides updated and more precise data on these essential oil's antimicrobial activity, as well as information whether usage of commercial material can significantly affect expected beneficial properties. materials and methods plant material plant material used for isolation of the essential oils was puchased in the form of monocomponent herbal drug from the following commercial sources: jeligor, svrljig (yarrow), montes, leskovac (chamomille) and „josif pančić” institute, belgrade (marigold). isolation of the essential oil air-dried, to constant weight, the commercial sample of carlinae radix drug (three batches of 100-150 g) were subjected to hydrodistillation with ca. 1 l of distilled water for 3.5 h using the original clevenger-type apparatus (c l e v e n g e r , 1928). the obtained oils were separated by extraction with freshly distilled diethyl ether and dried over anhydrous magnesium sulphate. the solvent was evaporated under a gentle stream of nitrogen at room temperature in order to exclude any loss of the essential oil and immediately analyzed. when the oil yields were determined, after the bulk of ether was removed under a stream of n2, the residue was exposed to vacuum at room temperature for a short period to eliminate the solvent completely. the pure oil was then measured on an analytical balance and multiple gravimetric measurements were taken during 24 h to ensure that all of the solvent had evaporated. microorganisms and culture conditions antimicrobial activity assays were performed against seven american type culture collection (atcc) strains: gram positive staphylococcus aureus 6538, gram negative escherichia coli 8739, e. coli 25922, proteus vulgaris atcc 8427, pseudomonas aeruginosa atcc 9027, klebsiella pneumoniae 10031 and the yeast candida albicans atcc 10231. bacterial strains were maintained on nutrient agar (na), while c. albicans was maintained on sabouraud dextrose agar (sda) in the culture collection of the microbiology laboratory, department of biology and ecology, faculty of science and mathematics, university of niš. determination of mic and mbc/mfc (minimal inhibitory/microbicidal) concentrations antimicrobial activity determination was performed by a microdilution method as described previously (s t o j a n o v i ć r a d i ć et al., 2012). briefly, overnight cultures of microorganisms were used for the preparation of suspension. final size of the bacterial inoculum was 5 x 10 5 cfu ml -1 . stock solutions of the essential oil were prepared in 50% aqueous ethanol, whose final concentration never exceeded 5% (v/v) in a well. dilution series were prepared in 96-well microtitre plates in the concentration range from 0.001-50 µl ml -1 (essential oil). after attaining the right dilutions, the inoculums were added to all wells. the plates were incubated for 24 h at 37 ºc (bacteria) and 48 h at 30 ºc (yeast). bacterial growth was determined by adding 20 μl of 0.5% triphenyl tetrazolium chloride (ttc) aqueous solution. one inoculated well was included to allow control of the broth suitability for organism growth. one non-inoculated well, free of antimicrobial agents, was also included to ensure medium sterility. positive controls were tetracycline and nystatine, while the solvent for the oil dilution (ethanol) was used as negative control. minimal inhibitory concentration (mic) was defined as the lowest concentration of the oil inhibiting visible biologica nyssana 3 (2)  december 2012: 69-76 stojanović-radić z.  antimicrobial activity of.. 72 growth (red colored pellet on the bottom of wells after the addition of ttc), while the minimal microbicidal (bactericidal and fungicidal) concentration (mbc/mfc) was defined as the lowest oil concentration killing 99.9% of bacterial/fungal cells. to determine mbc/mfc, the broth was taken from each well without visible growth and inoculated in mueller hinton agar (mha) for 24 h at 37 ºc and in sabouraud dextrose agar (sda) for 48 h at 30 ºc in the case of the tested yeast. experiments were done in quintuplicate. results and discussion essential oils were isolated and yields, together with features are presented in the table 1. the highest yield of essential oil was in the case of yarrow, which classifies this plant into the category of aromatic plants. considering literature data about content of essential oils in the investigated plants, the only one which had different amount is chamomile. in the present work, it yielded only 0.1%, which is lower than the up to now determined range from 0.2-1.9%. this can be explained by material processing before purchasing and distillation, where evaporation of the oil during long storage could contribute to the herein obtained lower yield. the results of antimicrobial activity determination showed activity of the three essential oils in the range from 0.09-50.00 µl ml -1 . the most active essential oil was the one isolated from milefolii herba drug, while the essential oil from calendulae flos exhibited the lowest activity among the tested ones. negative control (solvent = ethanol) showed no activity against any of the tested microbial strains. considering activity in comparison to the reference antibiotics (positive controls), all tested oils showed lower activity than the antibiotic (figures 1-3). considering essential oil of chamomilla recutita, the results of microdilution method revealed very significant activity, which was higher than those reported in previous studies on this subject. the range of the active concentrations was from 0.10-1.73 µl ml -1 (table 2, figure 1). among the tested microbial species, candida albicans showed the highest sensitivity, with inhibited growth at only 0.10 µl ml -1 , while concentration of 0.32 µl ml -1 showed fungicidal effect. on the other hand, the most resistant strain was escherichia coli (atcc 8739), where active concentrations were mic=1.73 µl ml -1 and mbc=2.60 µl ml -1 . table 1. yield and features of the essential oils isolated from commercial material of herbal drugs belonging to three species from family asteraceae herbal drug plant species stated as the only one component of the herbal drug features of isolated essential oil yield of isolated essential oil range of yields according to literature data chamomilae flos chamomila recutita liquid, specific blue color, intensive pleasant aroma 0. 100% 0.20-1.90% milefolii herba achillea millefolium liquid, specific blue color, intensive pleasant aroma 0. 430% 0.20-1.00% calendulae flos calendula officinalis liquid, colorless, intensive pleasant aroma 0. 063% 0.06-0.30% biologica nyssana 3 (2)  december 2012: 69-76 stojanović-radić z.  antimicrobial activity of.. 73 previous investigations on chamomile antimicrobial activity were numerous (l i s b a l h i n et al., 1998; a l i s m a i l & t a l a l , 2003; p i r z a d et al., 2006; o w l i a et al., 2007; s h i k o v et al., 2008; p e r e i r a et al., 2008) and it is considered as a plant with moderate antimicrobial activity. it possesses activity against both gram positive and gram negative strains, as well as against fungi (r o m e i l a h , 2009). essential oil isolated from this plant species showed antibacterial activity at concentration of 25 mg ml -1 against bacillus subtilis, staphylococcus aureus, streptococcus mutans and streptococcus salivarius as well as against candida albicans (a g g a g & y o u s e f , 1972; b e r r y , 1995). in the study of l i s b a l c h i n et al. (1998), it has been reported that this oil has effect against 25 different bacterial species and 20 strains of lysteria monocytogenes. the same study revealed significant antifungal activity against aspergillus niger, a. ochraceus and fusarium culmorum. different streptococcal species streptococcus pyogenes, s. mutans, s. salivarius, s. faecalis and s. sanguis were inhibited by chamomile essential oil in the concentration range from 0.10 – 4.0 µl ml -1 (o w l i a et al., 2007), which is similar to the active concentration range in the present work. the mechanism of chamomile essential oil’s activity was investigated against fungal species, where it was reported that it increases permeability of cytoplasmic membrane, which leads to osmotic pressure disturbance and death of the cell as a consequence (t o l o u e e et al., 2010). however, due to different methods used in the mentioned investigations (disc diffusion in most cases), as well as different solvents used (dimethyl sulfoxide), data from the present paper cannot be compared properly to the previously obtained results (a g g a g & y o u s e f , 1972; b e r r y , 1995; l i s b a l h i n et al., 1998). in some of the existing papers, α-bisabolol is considered as the main carrier of the activity (o w l i a et al., 2007; r o m e i l a h , 2009), which is mentioned in some studies as antifungal compound (p a u l i , 2006; t o l o u e e et al., 2010). it inhibits the synthesis of ergosterol, which would explain the highest activity against fungal organism obtained in the present investigation. results of calendulae flos essential oil’s antimicrobial activity testing showed very high effect against all tested microbial strains. obtained active concentration were in the range from 0.0918.7 µl ml -1 (table 2, figure 2). this essential oil exhibited the highest activity against klebsiella pneumoniae and pseudomonas aeruginosa, while escherichia coli 8739 showed the highest resistance, where higher concentration of 18.7 µl ml 1 was necessary for inhibition of its growth. yeast candida albicans showed very significant sensitivity to this oil, which points to the potential of this plant in the treatment of fungal infections. figure 1. antimicrobial activity of chamomillae flos essential oil compared to the reference antibiotic (µg ml-1) тable 2. minimal inhibitory and bactericidal concentrations of essential oils from commercial milefolii herba, calendulae flos and chamomillae flos herbal drugs (µl ml -1 ) bacterial/fungal species атcc number milefolii herba calendulae flos chamomillae flos ab mic mbc mic mbc mic mbc mic staphylococcus aureus 6538 0.29 0.78 0.14 0.39 0.16 0.16 0.39 escherichia coli 25922 0.78 3.12 3.12 6.25 0.12 0.32 1.56 escherichia coli 8739 0.65 1.56 18.7 50.0 1.73 2.60 1.56 klebsiella pneumoniae 10031 0.32 12.50 0.09 0.19 0.13 2.60 0.78 pseudomonas aeruginosa 9027 0.09 0.39 0.09 0.19 0.13 0.32 1.56 proteus vulgaris 8427 0.32 3.12 0.10 0.78 0.10 1.30 1.56 candida albicans 10231 0.12 0.39 0.19 0.19 0.10 0.32 6.25 abreference antibiotic (tetracycline and nystatin), given in µg ml-1 biologica nyssana 3 (2)  december 2012: 69-76 stojanović-radić z.  antimicrobial activity of.. 74 marigold is mentioned in the literature data as a plant with high antimicrobial potential with reported activity against gram positive, gram negative and fungal species (j a n s s e n et al., 1986; i a u k et al., 2003). studies dealing with investigation of antimicrobial effect of marigold’s essential oil isolated from flowers (j a n s s e n et al. 1986; g a z i m et al., 2008b; f i t et al., 2009) reported its activity against bacillus subtilis, escherichia coli, staphylococcus aureus, pseudomonas aeruginosa and candida albicans (j a n s s e n et al. 1986). also, this essential oil showed extraordinary effect against 35 different isolates from skin lesions, with average zone of inhibition of 21.3 mm (at concentration of 30 µl/disc), which was the second strongest activity among ten tested essential oils (aloe vera, rattle, fir, savory, marigold, coconut, eucalyptus, lavender, mint and pine), isolated from medicinal plants (f i t et al., 2009). study on antimicrobial activity of marigold’s essential oil against 23 clinical isolates of candida albicans showed intensive antifungal activity, comparable or sometimes even several times higher than nystatin (g a z i m et al., 2008b). all reported studies pointed to very high antimicrobial potential of marigold, where we must highlight that the lack of results obtained by microdilution method makes the comparison with the present ones very difficult. certainly, according to the previous results, the herein obtained activity was expected. previous studies on chemical composition of this essential oil reported the presence of many compounds already reported as antimicrobial such as monoterpenes αthujone, α-pinene, limonene or 1,8-cineole. therefore, we can suppose that the obtained activity might be a result of their synergistic effect. figure 2. antimicrobial activity of calendulae flos essential oil compared to the reference antibiotic (µg ml-1) essential oil isolated from commercial drug of yarrow showed unexpected, very high activity, showing antimicrobial effect against all tested strains. inhibitory concentrations were in the range from 0.09-0.78 µl ml -1 , while concentrations from 0.39-12.50 µl ml -1 exhibited microbicidal effect (table 1, figure 3). this oil showed the highest activity against bacterial species pseudomonas aeruginosa (mic=0.09 µl ml -1 , mbc=0.39 µl ml -1 ), followed by candida albicans with mic at 0.12 µl ml -1 and mbc at 0.39 µl ml -1 . the highest tolerance to the essential oil presence showed both species of escherichia coli, where e. coli 25923 was more resistant and showed sensitivity at 0.78 µl ml -1 (mic) and 3.12 µl ml -1 (mbc). previous investigation of this plant essential oil’s antimicrobial activity (c a n d a n et al., 2003; b o ž i n et al., 2008) pointed to moderate effect, while different extracts in most cases showed no activity. comparison of the present results with the previous one obtained by the same antimicrobial testing method (c a n d a n et al., 2003) showed extremely higher activity of the herein tested essential oil. the oil from the mentioned investigation showed antimicrobial activity in the range from 4.50-72.00 mg ml -1 , while the oil in the present study was active at concentrations from 0.08-0.60 mg ml -1 , which can be explained by eventual qualitative differences of the compared essential oils, as well as by different solvents used. c a n d a n et al. (2003) used 0.5% tween 80 for solution and dispersion of the essential oil, while the present study used ethanol as solvent. beside this, the authors of the previous study added solvent directly into the medium and then performed the dilution of essential oil. in the present study, due to figure 3. antimicrobial activity of milefolii herba essential oil compared to the reference antibiotic (µg ml-1) biologica nyssana 3 (2)  december 2012: 69-76 stojanović-radić z.  antimicrobial activity of.. 75 very weak solubility, the oil was first dissolved in 50% ethanol. after that, dilutions were made in the same solvent, which was followed by adding of these solutions into the wells of the microtiter plate in order to achieve appropriate concentrations. owing to the low solubility of the yarrow essential oil, it is possible that the previous study did not have completely dissolved oil, which affected its activity due to lower final concentration of the oil in the wells. also, better activity obtained in this study might be connected to the synergistic activity with ethanol, which does not affect the growth of bacteria in the final concentration, but might increase the permeability of the bacterial membrane and facilitate the passage of the active compounds to its target place. conclusion as a final conclusion, we can state that commercial plant material does not have significantly altered biological properties as a consequence of the plant material processing. the only difference was yield of chamomile essential oil, which can be related to evaporation of oil during a longer period of storage. antimicrobial activity of the tested essential oils was, in all three cases very significant and in the range or even lower than the previously investigated essential oil. higher activity obtained in the present investigation is probably due to more precise and quantitative method used herein (microdilution method), while used solvent might contribute by synergistic action with essential oils. synergism of these mixtures might be very promising and future studies should be focused on this object of investigation. references aggag, m.e., yousef, r.t. 1972. study on antimicrobial activity of chamomile oil. planta medica, 22(2): 140-151. al-ismail, k.m., talal, a. 2003. a study of the effect of water and alcohol extracts of some plants as antioxidants and antimicrobial on longterm storage of anhydrous butter fat. dirasat: agricultural sciences, 30(3): 330–337. asgary, s., naderi, g., bashardoost, n., etminan, z. 2002. antioxidant effect of the essential oil and extract of matricaria chamomilla l. on isolated rat hepatocytes. journal of medicinal plants, 1(1): 69-76. ayoughi, f., barzegar, m., sahari, m.a., naghdibadi, h. 2011. chemical compositions of essential oils of artemisia dracunculus l. and endemic matricaria chamomilla l. and an evaluation of their antioxidative effects. journal of agriculture, science and technology, 13(1): 79-88. berry, m. 1995. the chamomiles. pharmaceutical journal, 254: 191-193. bošković, z., radulović, n., stojanović, g. 2005. essential oil composition of four achillea species from the balkans and its chemotaxonomic significance. chemistry of natural compounds, 41(6): 674–678. božin, b. mimica-dukić, n., bogavac, m., suvajdžić, lj., simin, n., samojlik, i., couladis, m. 2008. chemical composition, antioxidant and antibacterial properties of achillea collina becker ex heimerl s.l. and a. pannonica scheele essential oils. molecules, 13(13): 2058-2068. candan, f., unlu, m., tepe, b., daferera, d., polissiou, m., sökmen, a., akpulat, h.a. 2003. antioxidant and antimicrobial activity of the essential oil and methanol extracts of achillea millefolium subsp. millefolium afan. 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heilkrauter. rudolf trauner verlag lintz, germany. vukotić et al. 2022, biologica nyssana 13(1) 13 (1) september 2022: 59-81 doi: 10.5281/zenodo.7117592 aquatic and wetland plant communities of the pusta river (southern serbia): floristic characteristics and syntaxonomic affiliation original article danica vukotić department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, 18 000 niš, serbia faculty of electronic engineering, university of niš, aleksandra medvedeva 14, 18106 niš, serbia danica.vukotic@elfak.ni.ac.rs (corresponding author) dragana jenačković gocić department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, 18 000 niš, serbia dušica p. ilić faculty of electronic engineering, university of niš, aleksandra medvedeva 14, 18106 niš, serbia danijela nikolić department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, 18 000 niš, serbia vladimir ranđelović department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, 18 000 niš, serbia received: july 19, 2022 revised: september 20, 2022 accepted: september 28, 2022 abstract: at the end of summer 2021, phytocenological research was conducted on aquatic and wetland vegetation of the pusta river left tributary of the south morava river. using upgma classification method and the bray-curtis distance on a set of 97 relevés and 44 species, degree of floristic differentiation between studied vegetation plots was quantified. it was established that 7 associations take part in composing vegetation of the pusta river – myriophyllo-potametum soó 1934, potametum nodosi soó (1928) 1960, segal 1964, phragmitetum australis savič 1926, typhetum latifoliae nowiński 1930, glycerio-sparganietum neglecti koch 1926, phalaridetum arundinaceae libbert 1931 and polygono-bidentetum tripartitae (w. koch 1926) lohm. 1950. the plant associations of vegetation class potamogetonetea klika in klika et novák 1941potametum nodosi and myriophyllo-potametum, are floristically the poorest and entirely consist of native plant species. the associations of vegetation class phragmito-magnocaricetea klika in klika et novák 1941 typhetum latifoliae (34 taxa), glycerio-sparganietum neglecti (33 taxa), phragmitetum australis (24 taxa) and phalaridetum arundinaceae (20 taxa) consist of higher number of species, including invasive plants. vegetation class bidentetea tx. et al. ex von rochow 1951 is represented by only one association polygono-bidentetum tripartitae, which consists of 24 taxa, with a share of 16.67% of allochthonous species. collected phytocenological data are going to be good basis for the future research of the vegetation of lotic ecosystems in the southern serbia. key words: aquatic and wetland vegetation, pusta river, upgma classification apstrakt: vodene i močvarne biljne zajednice puste reke (južna srbija): sintaksonomska pripadnost i florističke karakteristike krajem leta 2021. godine, sprovedena su fitocenološka istraživanja vodene i močvarne vegetacije puste reke – leve pritoke južne morave. korišćenjem upgma klasifikacione metode i bray-curtis-ove distance nad setom podataka od 97 fitocenoloških snimaka i 44 vrsta kvantifikovan je stepen florističke diferencijacije između proučavanih sastojina. ustanovljeno je da u izgradnji biljnog pokrivača puste reke učestvuje 7 biljnih zajednica myriophyllopotametum soó 1934, potametum nodosi soó (1928) 1960, segal 1964, phragmitetum australis savič 1926, typhetum latifoliae nowiński 1930, glycerio-sparganietum neglecti koch 1926, phalaridetum arundinaceae libbert 1931 i polygono-bidentetum tripartitae (w. koch 1926) lohm. 1950. asocijacije vegetacijske klase potamogetonetea klika in klika et novák 1941 potametum nodosi i myriophyllo-potametum, su floristički najsiromašnije i u potpunosti izgrađene od autohnotnih biljnih vrsta. u sastav zajednica vegetacijske klase phragmito-magnocaricetea klika in klika et novák 1941 typhetum latifoliae (34 vrste), glycerio-sparganietum neglecti (33 vrste), phragmitetum australis (24 vrste) i phalaridetum arundinaceae (20 vrste) ulazi veći broj vrsta, uključujući i invazivne biljne vrste. vegetacija klase bidentetea tx. et al. ex von rochow 1951 predstavljena je zajednicom polygono-bidentetum tripartitae izgrađenom od 24 vrsta, sa udelom alohtonih vrsta od 16.67%. prikupljeni fitocenološki podaci predstavljaju svojevrsnu osnovu za buduća, intenzivnija istraživanja biljnog pokrivača lotičkih ekosistema južne srbije. ključne reči: vodena i močvarna vegetacija, pusta reka, upgma klasifikacija © 2022 vukotić et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 59 14th symposium on the flora of southeastern serbia and neighboring regions introduction macrophytes are a complex group of plants adapted to the life in water. they include all aquatic vascular plants and aquatic mosses, and some authors also include macroscopic algae (stevanović & janković, 2001). they form aquatic plant communities that, compared to terrestrial communities, consist of a smaller number of species and, in most cases, are dominated by one species. macrophytes can be both submerged and floating, and can be further divided into morphoecological types of plants that are rooted and those that are not rooted, but whose roots float in water, or these types of plants don’t develop roots at all (stevanović & janković, 2001). communities of rooted, floating plants as well as submerged macrophytes belong to the vegetation class potamogetonetea klika in klika et novák 1941, while floating, non-rooted macrophytes of relatively still and nutrient-rich waters, belong syntaxonomically to the vegetation class lemnetea o. de bolòs et masclans 1955 (mucina et al., 2016). aquatic vegetation in serbia has been previously studied in lentic systems (vukov et al., 2003; topuzović et al., 2009; stanković et al., 2009, ranđelović & zlatković, 2010; marković et al., 2015), while in the lotic systems it has been studied only in a few rivers and channels (stojanović et al., 2007; vukov et al., 2008; jenačković et al., 2010; ljevnaić-mašić, 2010; džigurski et al., 2016). wetland vegetation belongs to the class phragmito-magnocaricetea klika in klika et novák 1941 (mucina et al., 2016). in contrast to aquatic vegetation, the structure and physiognomy of phytocenoses in wetlands is determined by emergent macrophytes. this type of vegetation develops in places where water dries out during the summer months but remains close to the ground surface, as well as in habitats that are flooded throughout the year (ranđelović et al., 2007a). wetland vegetation provides refuge for many species, often rare and endangered, that inhabit only habitats where wetland vegetation is present. they are considered very sensitive and one of the most endangered habitats in the world, and their degradation is continuous and uncontrolled (svitok et al., 2011). wetland vegetation in serbia has been studied mainly in the plains of vojvodina (slavnić, 1956; vukićević et al., 1966; babić, 1971; stojanović et al., 1987; butorac & crnčević, 1987; parabućski & butorac, 1994; polić, 2006; stojanović et al., 2007; ljevnaić-mašić, 2010; džigurski et al., 2010; džigurski et al., 2011), while it has been less researched in the southern parts of serbia (katić, 1910; košanin, 1910; mišić et al., 1987; ranđelović, 1978; ranđelović & zlatković, 2010; jenačković et al., 2010; jenačković, 2017; jenačković et al., 2019). aquatic and wetland vegetation show high floristic and ecological similarity, which is why they are often studied together. these types of vegetation on the south morava river and its tributaries have not been sufficiently investigated in terms of their floristic composition and ecological differentiation. wetland vegetation south of the autonomous province of vojvodina was examined near vlaško polje (jovanović, 1958), in the valley of the velika morava river (jovanović, 1965), near kragujevac (veljović, 1967), in the valley of the južna morava river (ranđelović, 1988), in the moravica river (milenović & ranđelović, 2005), in the batušinačke swamps located to the left of the south morava river (ranđelović et al., 2007a), on the banks of the murina river, brestovac (ranđelović et al., 2007b), also on the banks of vlasina lake, cvetkova river, dedina dolina (ranđelović & zlatković, 2010), in the svrljiški and beli timok rivers (jenačković et al., 2010), smilovsko lake, krupac lake, oblačina lake, wetland habitats in surroundings of the cities of bela palanka and prokuplje, as well as near the villages of žitkovac, vrtište, medoševac, lepaja, bresničić, lalinac and levosoje which have been investigated by jenačković (2017). aquatic vegetation was also investigated in the beli and svrljiški timok rivers (jenačković et al., 2010). the classification of aquatic and semi-aquatic vegetation of lakes in serbia was also carried out by laketić (2013), as well as the review of complete aquatic vegetation studied so far in serbia (cvijanović et al., 2018). the main objectives of this paper are to determine: • the diversity of aquatic and wetland phytocenoses of the pusta river, • the degree of floristic differentiation among the communities, • the syntaxonomic affiliation of the registered communities. materials and methods research area phytocenological research was conducted on the aquatic and wetland vegetation of the pusta river along its course from the municipality of bojnik to the municipality of doljevac (fig. 1). the pusta river is located in the southern part of serbia and is a left tributary of the south morava river. the river’s spring is located near the village of donji statovac. it is 71 km long and its catchment is 569 km2. it flows into the south morava river near the village of pukovac (gavrilović & dukić, 2002). phytocenological research of aquatic and wetland communities was conducted during september 2021, 60 biologica nyssana ● 13 (1) september 2022: 59-81 vukotić et al. ● aquatic and wetland plant communities of the pusta river (southern serbia): floristic characteristics and syntaxonomic affiliation 61 biologica nyssana ● 13 (1) september 2022: 59-81 vukotić et al. ● aquatic and wetland plant communities of the pusta river (southern serbia): floristic characteristics and syntaxonomic affiliation as this part of the vegetation season is considered optimal for their sampling (jenačković et al., 2019). phytocenological relevés were recorded in the lower course of the pusta river, from its confluence with the south morava river to the municipality of bojnik, along a river length of about 35 km. the largest number of phytocenological relevés were recorded in the part of the river stream near the villages of pukovac and kočane, as in these localities the phytocenoses of aquatic and wetlands were fully developed. in the part of the river course between the village of pukovac and the municipality of bojnik, the areas with wetland vegetation were drastically reduced. the wetland phytocenoses were represented only by a few smaller patches of vegetation plots that were several kilometers apart. the cleaning of the riverbed significantly destroyed the wetland vegetation in bojnik, which is believed to have developed in the riparian zone. the phytocenological surveys were carried out according to the principle of the swiss-french braun-blanquet school (braun-blanquet, 1964). the areas of the sampled stands had a standard size of 4 m2 for aquatic vegetation and 16 m2 for wetland vegetation. sampling points were georeferenced using the gps device (global positioning system). the plant material collected in the sampled stands was deposited in the herbarium collection “herbarium moesiacum” of the faculty of natural sciences and mathematics (hmn) of the university of niš. the determination was performed with the help of dichotomous identification keys: “flora sr srbije i-x” (josifović, 1970-1980, sarić, 1986, 1992), “флора на нр българияˮ (йорданов, 19631986, велчeв, 1989), and “flora europaea” (tutin et al., 1964-1980). the nomenclature and taxonomy of plant species were standardized according to the euro+medplantbase database (https://www. emplantbase.org). nomenclature of syntaxa, at the level of vegetation classes, orders and alliances was standardized according to mucina et al. (2016). the names of associations, and their syntaxonomic positions, have been adjusted with tzonev (2009), cvijanović (2018) and landucci et al. (2020). statistical analyses the turboveg software package was used to digitize 97 phytocenological relevés recorded during field research (hennekens & schaminée, 2001). classification analysis was performed using the upgma (unweighted pair group method with arithmetic mean) classification method and the bray-curtis similarity index. the value of the “crispness of classification” (botta-dukát et al., 2005) was used to determine the optimal number of clusters. groups of phytocenological relevés belonging to one or more associations, identified by the hierarchical classification analysis as a separate group (cluster), are characterized by diagnostic species. diagnostic species were determined based fig. 1. map of the investigated area on the pusta river, start point (bojnik) and end point (doljevac) 62 on the phi-coefficient, which expresses the degree of taxon fidelity to a particular cluster (chytrý et al., 2002). in addition, virtual standardization of all vegetation groups was performed to eliminate the dependence of the phi-coefficient on the size of the vegetation groups – clusters (tichý & chytrý, 2006). fisher’s accuracy test was used to exclude all phi-coefficient values that were not statistically significant at the significance level p<0.05. species with a phi-coefficient greater than 0.20 were considered as diagnostic species, while species with a phi-coefficient greater than 0.50 were considered as highly diagnostic species. in addition to the classification analysis, where the optimal number of clusters was determined, one more cluster analysis was performed to better understand the floristic similarity between individual phytocenological relevés. second cluster analysis was performed without determining the optimal number of clusters. the dominant and constant species were determined for the vegetation groups identified using the results of the classification analysis. dominant species were determined by calculating the average cover values in relevés within the groups using the cover index (ic) (lausi et al., 1982) and the share in total cover (d%) (surina, 2005). dominant species were determined to be those that were present in at least 10% of the relevés with a cover of at least 25%. constant species were considered those that occurred in at least 40% of the relevés within the clusters. the determination of diagnostic, constant and dominant species within the clusters, as well as the determination of the optimal number of clusters during classification analysis, were performed using the software package juice 7.0 (tichý, 2002, http://www.sci.muni.cz/botany/juice). the software package juice 7.0 was also used to create the biologica nyssana ● 13 (1) september 2022: 59-81 vukotić et al. ● aquatic and wetland plant communities of the pusta river (southern serbia): floristic characteristics and syntaxonomic affiliation combined synoptic table. the combined synoptic table contains information about the floristic composition of the studied communities and the quantitative proportion of species in each of them. results and discussion based on the results of the classification analysis, with determined optimal number of clusters (fig. 2) and the composition of the diagnostic species within the clusters defined (tab. 1), four main vegetation groups (clusters) were distinguished. the results of classification analysis, performed without determining optimal number of clusters, are presented in fig. 3, and illustrate the floristic similarities between individual relevés more clearly. based on the results of these analyses, it can be noticed that cluster ii (fig. 2) is clearly divided into 3 subclusters (fig. 3), and cluster iv (fig. 2) into 2 subclusters (fig. 3), resulting in 7 vegetation groups that participate in the construction of the plant cover of the investigated part of the pusta river. the first cluster (fig. 2) corresponds with relevés of typhetum latifoliae nowiński 1930 (fig. 3), second one (fig. 2), divided into 3 subclusters (fig. 3), unifies relevés of phalaridetum arundinaceae libbert 1931, polygono-bidentetum tripartitae (w. koch 1926) lohm. 1950 and glycerio-sparganietum neglecti koch 1926, third cluster corresponds with relevés of phragmitetum australis savič 1926, and fourth, divided into two subclusters, unifies relevés of potametum nodosi soó (1928) 1960, segal 1964 and myriophyllo-potametum soó 1934. classification analysis showed that the greatest floristic similarity exists between the clusters i and ii, i.e., the communities typhetum latifoliae nowiński 1930 (cluster i) and phalaridetum arundinaceae libbert 1931, polygono-bidentetum tripartitae (w. koch 1926) lohm. 1950 and glycerio-sparganietum fig. 2. dendrogram obtained as a result of the hierarchical upgma classification on bray-curtis resemblance matrix along with determining optimal number of clusters. 63 biologica nyssana ● 13 (1) september 2022: 59-81 vukotić et al. ● aquatic and wetland plant communities of the pusta river (southern serbia): floristic characteristics and syntaxonomic affiliation table 1. the combined synoptic table with the information on frequency and phi-coefficient values (x100) for species occurring in four optimal cluster groups defined by upgma classification analysis. the values for the frequency are represented by the base, while the values for fidelity are given in the form of superscripts species clusters i ii iii iv typha latifolia l. 100 85.6 14 11 . salix alba l. 50 31.3 33 22 . sparganium erectum l. 16 91 69 22 8 lythrum salicaria l. 62 77 42.8 22 . mentha aquatica l. 16 47 42.1 11 . polygonum mite schrank 22 37 29.8 11 . phalaris arundinacea l. 6 28 40 . . tanacetum vulgare l. 3 21 24.7 11 . phragmites communis trin. . . 100100 . urtica dioica l. 25 26 56 37.9 . echinocystis lobata (michx.) torr. & a. gray 3 2 33 46.1 . robinia pseudoacacia l. 6 9 33 37.2 . myriophyllum spicatum l. 6 19 . 92 79.9 potamogeton nodosus poir. 16 28 11 85 60.4 bidens tripartitus l. 47 47 56 . polygonum lapathifolium l. 28 25 22 . salix euxina i. v. belyaeva 31 21 33 . xanthium strumarium l. 28 35 11 . populus sp. 16 9 11 . epilobium hirsutum l. 12 12 11 .. lycopus europaeus l. 9 21 22 rubus caesius l. 6 12 22 . galium aparine l. 6 9 11 . potamogeton crispus l. 3 2 . 15 lemna minor l. 3 7 . 8 calystegia sepium (l.) r. br. 19 23 . . leersia oryzoides (l.) sw. 12 19 . . solanum dulcamara l. 12 19 . . amorpha fruticosa l. 9 9 . . echinochloa crus-galli (l.) p. beauv. 3 9 . . humulus lupulus l. 9 . 11 . rumex conglomeratus murray 3 2 . . lysimachia vulgaris l. 3 2 . . erigeron canadensis l. . 5 11 . scutellaria galericulata l. . 5 . . aristolochia clematitis . 2 . . chenopodium album l. . 2 . . lathyrus latifolius l. . 2 . . neglecti koch 1926 (cluster ii). cluster iv differs the most as this cluster consists of associations potametum nodosi soó (1928) 1960, segal 1964 and myriophyllo-potametum soó 1934 presenting aquatic vegetation (fig. 2). association typhetum latifoliae is characterized by presence of highly diagnostic species typha latifolia (tab. 1). it is floristically rich, includes 34 taxa, and its constant species are lythrum salicaria, salix alba and bidens tripartitus (appendix 1). the stands of this community have been developed in the coastal zone, where t. latifolia has been partly in the water and borders with the stands of myriophyllopotametum and potametum nodosi (fig. 4). on the banks of the river, it forms a dense, impassable cover, and is intersected in some places by the glycerio-sparganietum neglecti. in southern part of serbia, stands of this association were recorded in the batušinačke swamps (ranđelović et al., 2007a), on the banks of the grlište hydro-accumulation (stanković et al., 2009), vlasina lake (ranđelović & zlatković, 2010). in ribinac, the stands in which typha latifolia is the dominant species together with phragmites communis were recorded (ranđelović et al., 2007b). the spectrum of accompanying species is diverse and depends on the habitat. according 64 biologica nyssana ● 13 (1) september 2022: 59-81 vukotić et al. ● aquatic and wetland plant communities of the pusta river (southern serbia): floristic characteristics and syntaxonomic affiliation to šumberová (2011), short-term floodplains are usually much richer in the number of taxa and include not only common wetland plants (e.g. lycopus europaeus l. and lythrum salicaria l.) but also tall sedges (e.g. carex riparia curtis) and species typical for meadow vegetation (e.g. equisetum palustre l., galium palustre l. and symphytum officinale l.). in long-term floodplains, especially in highly eutrophic wetlands, the macrophyte lemna minor l. is common (šumberová, 2011), which is also recorded in the studied stands of the pusta river. it is necessary to stress that high values of phicoefficient for species salix alba (tab. 1) is related with including one relevé characterized by high percentage value of cover of salix alba in database. on the territory investigated, salix alba occurs frequently within stands of the typhetum latifoliae, at its upper border or at the elevated parts of the bank where it occurs mainly with one or two individuals. in some places, one to two individuals of salix alba can be found due to the lack of wetland vegetation. salix alba represents the remnants of once-present floodplain forests that developed near the pusta river. today, the remains of flooded forests in the lower course of the river, are represented only by certain species of the genus salix and populus, some fig. 3. dendrogram obtained as a result of the hierarchical upgma classification on bray-curtis resemblance matrix without identifying optimal number of clusters species clusters i ii iii iv scrophularia nodosa l. . 2 . . berula erecta (huds.) coville . 2 . . stellaria neglecta (lej.) weihe . 2 . . eleocharis palustris (l.) r. br. . 2 . . stachys palustris l. 3 . . . althaea officinalis l. 3 . . . 65 biologica nyssana ● 13 (1) september 2022: 59-81 vukotić et al. ● aquatic and wetland plant communities of the pusta river (southern serbia): floristic characteristics and syntaxonomic affiliation of which extend into the wetland vegetation. the stands of phalaridetum arundinaceae (fig. 4) have been developed on the raised banks of the riverbed, exposed only to occasional floods, after which the surface water stagnates for a short time. the diagnostic species of this association is phalaris arundinacea, while the constant species are mentha aquatica, lythrum salicaria, sparganium erectum, salix alba and xanthium strumarium. it is floristically rich and consists of 20 taxa (appendix 2). this plant community fig. 4. the stands of associations typhetum latifoliae nowiński 1930 (1,2), phalaridetum arundinaceae libbert 1931 (3), phragmitetum australis savič 1926 (4), glycerio-sparganietum neglecti koch 1926 (5) and potametum nodosi soó (1928) 1960, segal 1964 (6) developed in riverbed and on the banks of pusta river, as well. occurs in habitats with pronounced fluctuations of the flood level. the development is conditioned by the hydrological regime of groundwater along the river course (ranđelović & zlatković, 2010). it is present in nutrient rich habitats, on sandy-gravelly to clay substrates. with its strong organic production, this community contributes to the overgrowth of ponds and shallow depressions (panjković, 2005). sometimes it occurs in deeper depressions where shallow (<0.3 m) surface water is retained during most of the growing season (jenačković, 2017). 66 biologica nyssana ● 13 (1) september 2022: 59-81 vukotić et al. ● aquatic and wetland plant communities of the pusta river (southern serbia): floristic characteristics and syntaxonomic affiliation phalarietum arundinaceae is only fragmentarily developed in several localities in southern serbia. according to ranđelović et al. (2007b), the best developed stands are along the šaranica stream near the villages of brestovac, pukovac and bubanj, in the valleys of the vlasina river and murina river. also, stands of this association were recorded on seasonally flooded habitats near the villages of gornje međurovo, levosoje, podvis, vrtište and žitkovac (jenačković et al., 2017). association polygono-bidentetum tripartitae consists of 24 taxa (appendix 3). its dominant species are polygonum lapathifolium and bidens tripartitus (appendix 3). since there are only three vegetation plots, a large number of constant species is noticeable, with a proportion of 16.67% of allochthonous species. this community develops on the banks of the river, where the water occasionally floods the banks, and retreats during the summer months. it is widespread in places along the banks of the pusta river especially where wetland vegetation is absent. also, this community was phytosociological described in svrljiški timok river and beli timok river, where it had a similar species composition (jenačković et al., 2010). similar floristic composition was recorded in nitrophilous stands developed on the shores of artificial lakes placed near the city of tuzla (barudanović & kamberović, 2011). glycerio-sparganietum neglecti is floristically rich association (33 taxa) (appendix 4) and develops on the banks of the river, in the form of narrow belts, some of which extend into the riverbed, and some on slightly elevated banks (fig. 4). its diagnostic and, at the same time, dominant species is sparganium erectum, while the constant species of association are lythrum salicaria and bidens tripartitus. native species have the largest share in the composition of this phytocenosis, but invasive, non-native species such as echinocystis lobata, echinochloa crus-galli, amorpha fruticosa and robinia pseudacacia (lazarević et al., 2012) were also recorded. a few years ago, in the part of the pusta river which flow through the village of pukovac and suffering strong anthropogenic influence via discharge of waste water, the stands of this association were phytocoenological described (jenačković, 2017). stands recorded in svrljiški timok river and beli timok river (jenačković et al., 2010), whose the most common species are sparganium erectum and mentha aquatica, show a remarkable floristical similarity with the stands developed in the pusta river. so far, communities with domination of sparganium erectum have been recorded in the batušinačke swamps (ranđelović et al., 2007a), on suitable habitats near the village of bubanj (ranđelović, 1988), žitkovac, vrtište, prokuplje, gornje međurovo, podvis, rgošte, levosoje (jenačković et al., 2019) and on the banks of vlasina lake (ranđelović & zlatković, 2010) and smilovsko lake (jenačković et al., 2019). the stands of the association glycerio-sparganietum neglecti koch 1926 are frequently involved in the establishment of the plant cover of lowland rivers, not only in our country, but also in the other european countries (gecheva et al., 2013). the dominant species of this community – sparganium erectum, is considered a reliable bioindicator of eutrophic waters (schneider & melzer, 2003), which explains the frequent occurrence of its monodominant communities in the lower reaches of the river, where high concentrations of nutrients, especially nitrogen, accumulate and are maintained as a result of anthropogenic influence. sparganium erectum shows exceptional frequency in the studied rivers of bulgaria, where it occurs in no less than 80 of 223 studied sites, with a frequency of 35% (gecheva et al., 2013). the taxa sparganium erectum and typha latifolia show ecological preferences for wetlands with similar physicochemical properties. they find optimal growing conditions in habitats characterized by relatively deep, slightly acidic water with low electrical conductivity. they prefer habitats where the substrate has the following characteristics: low ph, low values of conductivity values and concentration of carbonates, bicarbonates and potassium (jenačković, 2017). on the area investigated, stands of phragmitetum australis (fig. 4) develops on the highest parts of the riverbanks, often along the road. they have been recorded in only 9 localities in the villages of kočane and pukovac. this community is floristically rich and consists of 24 taxa (appendix 5), and its highly diagnostic species is phragmites communis (tab. 1). in addition to the species phragmites communis, high values of phi-coefficient were established for the species urtica dioica, echinocystis lobata and robinia pseudoacacia. these values probably reflect reduced substrate moisture and increased concentrations of nutrients, primarily nitrogen. abundant presence of individuals of echinocystis lobata and robinia pseudacacia can be explained by their high invasive capacity (lazarević et al., 2012). often, in nature, stands of phragmitetum australis are bordered to the stands of typhetum latifoliae. they usually prefer mineral sand, mineral-organic, as well as organic substrate (pełechaty, 1999). in terms of water depth and degree of soil moisture, they successfully develop on diverse habitat types completely flooded, occasionally flooded and non-flooded. according to jenačković (2017), dominant species of association phragmitetum 67 biologica nyssana ● 13 (1) september 2022: 59-81 vukotić et al. ● aquatic and wetland plant communities of the pusta river (southern serbia): floristic characteristics and syntaxonomic affiliation australis shows affinities to the habitats with high values of substrate ph. stands of phragmitetum australis are widespread in the central balkans. in serbia, they have been recorded in the velika morava river (jovanović, 1958; veljović, 1967), on the slopes of stara planina (mišić et al., 1987), in the south morava basin (ranđelović, 1988), in the toplica river (perišić et al., 2003), on the shores of vlasina lake (ranđelović & zlatković, 2010), smilovsko lake, oblačina lake, near the villages of žitkovac, lalinac, vrtište, rgošte and levosoje, on habitats placed in suburb of the city of bela palanka (jenačković et al., 2019). otherwise, the communities phragmitetum australis, typhetum latifoliae and glycerio-sparganietum neglecti belong to the phytocenoses that most frequently overgrow the wetland habitats of the central balkans (jenačković, 2017). the stands of the myriophyllo-potametum develop in the riverbed itself and form a relatively stable submerged community, consisting mainly of rooted hydrophytes (myriophyllum spicatum and potamogeton crispus) and floating hydrophytes (lemna minor). the association develops on a muddy waterbed at depths of up to 2 m, forming a dense cover. in some parts of the pusta river, the stands of this association form small, separated groups which look like islands composed of plants, while in other parts of the stream they cover the entire water surface from one bank to the another. they prefer slow to moderately slow water flow, but they also tolerate strong water strikes, and are indicators of mesotrophic and eutrophic waters. the association is floristically poor and consists of 3 taxa (appendix 6). myriophyllum spicatum is the highly diagnostic species (tab. 1), while the status of constant species have potamogeton nodosus and potamogeton crispus (appendix 6). study conducted on the aquatic vegetation of rivers in slovenia (germ et al., 2021) have shown that myriophyllum spicatum and potamogeton nodosus are the most common aquatic species in undisturbed aquatic ecosystems, while investigation of slovakian rivers have confirmed that the highest relative mass in rivers has myriophyllum spicatum (hrivnak et al., 2006). the species myriophyllum spicatum, potamogeton crispus and lemna minor have taken part in composing aquatic vegetation of the svrljiški and beli timok rivers (jenačković et al., 2010), river systems and channels in the northern part of serbia (radulović et al., 2010; vukov et al., 2012; džigurski et al., 2016; vukov et al., 2017) as well. otherwise, myriophyllum spicatum and potamogeton nodosus are indicators of eutrophic waters (schneider & melzer, 2003), which explains their occurrence in the lower parts of the river, where high nutrient concentrations accumulate and remain. potametum nodosi develops in the riverbed itself, mainly along the coast, and partly invades coastal vegetation where water flow is slower. in deeper parts of the riverbed, however, the community borders with myriophyllo-potametum. the highly diagnostic species of this community is potamogeton nodosus (tab. 1), and the constant species is myriophyllum spicatum (appendix 7). the community is relatively species-poor and is composed of only 4 taxa (appendix 7). a similar floristic composition have had stands developed in the svrljiški and beli timok rivers (jenačković et al., 2010). floristically, this community is also described in the moravica river, highlighting rare and endangered species (ljevnaić-mašić et al., 2016). the stands noted in the danube-tisa-danube canal in vojvodina, in the northern part of serbia, have had a slightly richer floristic composition compared to the stands recorded in pusta river (džigurski et al., 2016). it is known that both, wetlands and aquatic vegetation, have developed over the years on the once disturbed river. since the vegetation along the entire course of the river has never been described completely so far, but only sporadically, we do not have the possibility to compare the data. what is certain is that this area has been disturbed several times and has changed due to the needs of agriculture. nowadays, the river is surrounded on all sides by fields and arable land, and thus exposed to constant anthropogenic influences. the previously described associations belong to the following vegetation classes, orders and alliances: potamogetonetea klika in klika et novák 1941 potamogetonetalia koch 1926 potamogetonion libbert 1931 myriophyllo-potametum soó 1934 potametum nodosi soó (1928) 1960, segal 1964 phragmito-magnocaricetea klika in klika et novák 1941 pragmitetalia communis koch 1926 phragmition communis koch 1926 phragmitetum australis savič 1926 typhetum latifoliae nowiński 1930 glycerio-sparganietum neglecti koch 1926 phalaridetum arundinaceae libbert 1931 bidentetea tx. et al. ex von rochow 1951 bidentetalia br.-bl. et tx. ex klika et hadač 1944 bidention tripartitae nordhagen ex klika et hadač 1944 polygono-bidentetum tripartitae (w. koch 1926) lohm. 1950 68 biologica nyssana ● 13 (1) september 2022: 59-81 vukotić et al. ● aquatic and wetland plant communities of the pusta river (southern serbia): floristic characteristics and syntaxonomic affiliation conclusions based on the results of the classification analyses and composition of diagnostic species per clusters, 7 associations typhetum latifoliae nowiński 1930, phalaridetum arundinaceae libbert 1931, polygono-bidentetum tripartitae (w. koch 1926) lohm. 1950, glycerio-sparganietum neglecti koch 1926, phragmitetum australis savič 1926, potametum nodosi soó (1928) 1960, segal 1964 and myriophyllo-potametum soó 1934, are described on the territory researched. the stands of typhetum latifoliae nowiński 1930 are abundantly developed in the part of the flow from the confluence of the pusta river to south morava river all the way upstream to the village of pukovac, where they are the most developed and occupies the largest recorded area on the pusta river. the stands of glycerio-sparganietum neglecti koch 1926 were developed in some places along the whole studied part of the river, and they were mainly developed 2-3 km upstream from the village of pukovac. the stands of phalaridetum arundinaceae libbert 1931, polygono-bidentetum tripartitae (w. koch 1926) lohm. 1950, phragmitetum australis savič 1926, diverse in size, occur sporadically. in the village of pukovac and a few kilometers upstream, the aquatic vegetation is more abundantly developed and in some places forms a dense cover that covers the entire water surface. this is one of the first complete research of this kind conducted on the pusta river, and it represents a good basis for future research on the vegetation of lotic ecosystems in southern serbia. acknowledgements. this work has been supported by the ministry of education, science and technological development of republic of serbia (grant no. 451-0368/2022-14/200102 and no. 451-03-68/2022-14/200124). references babić, n. 1971: močvarna i livadska vegetacija koviljskog rita. zbornik matice srpske za prirodne nauke, 41: 19-87. barudanović, s., kamberović, j. 2011: weed vegetation on the shores of artificial reservoirs of surface mining pits in the area of tuzla. herbologia, 12(3): 1-14. botta-dukát, z., chytrý, m., hájková, p., havlová, m. 2005: vegetation of lowland wet meadows along a climatic continentality gradient in central europe. preslia, 77: 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(1 98 2) ; d % s ha re in to ta l co ve ra ge a cc or di ng to s ur in a (2 00 5) ; s % sp ec ie s pe rc en ta ge fr eq ue nc y w ith in th e cl us te r a pp en di ce s 72 73 biologica nyssana ● 13 (1) september 2022: 59-81 vukotić et al. ● aquatic and wetland plant communities of the pusta river (southern serbia): floristic characteristics and syntaxonomic affiliation n um be r of re le vé s 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 ic d % s % le er si a or yz oi de s . + . . + . . . . + . . . . . . . . . . . . . . . . . . . r . . 2. 4 1. 3 12 .5 so la nu m du lc am ar a . + . . . + . . . + . . . . . . . . . . . . . . . . r . . . . . 2. 4 1. 3 12 .5 po ta m og et on no do su s + . . . . . . . . . . r . . . . . . . . . . . . r r . r . . . . 2. 1 1. 1 15 .6 ep ilo bi um hi rs ut um . . + . + r . . . . . . . . . . . . . . . . . . . r . . . . . . 2. 1 1. 1 12 .5 am or ph a fr ut ic os a . . . . . . + + . . . . . . . . . . . r . . . . . . . . . . . . 1. 7 0. 9 9. 4 h um ul us lu pu lu s . . . . . r . . . . . . . . . . . . . . . . . . . . + . . . r . 1. 4 0. 7 9. 4 ph al ar is ar un di na ce a + . . . . . . . . . . . . . . + . . . . . . . . . . . . . . . . 1. 4 0. 7 6. 3 ly co pu s eu ro pa eu s . . r r . . . . . . . . . . . . . . . . . . . . . r . . . . . . 1 0. 5 9. 4 st ac hy s pa lu st ri s . . . . . 1 . . . . . . . . . . . . . . . . . . . . . . . . . . 1 0. 5 3. 1 ro bi ni a ps eu do ac ac ia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . r + . 1 0. 5 6. 3 ru m ex co ng lo m er at us + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0. 7 0. 4 3. 1 ly si m ac hi a vu lg ar is . . . . + . . . . . . . . . . . . . . . . . . . . . . . . . . . 0. 7 0. 4 3. 1 ta na ce tu m vu lg ar e . . . . . . . . . . . . + . . . . . . . . . . . . . . . . . . . 0. 7 0. 4 3. 1 m yr io ph yl lu m sp ic at um . . . . . . . . . . . . . . . . . . . . r . . . r . . . . . . . 0. 7 0. 4 6. 3 ru bu s c ae si us . . . . . . . . . . . . . . . . . . . . . . . . . . r r . . . . 0. 7 0. 4 6. 3 g al iu m ap ar in e . . . . . r . . . . . . . . . . . . . . . . . . . . r . . . . . 0. 7 0. 4 6. 3 al th ae a offi ci na lis . . . . . . . . . . . . r . . . . . . . . . . . . . . . . . . . 0. 3 0. 2 3. 1 po ta m og et on cr is pu s . . . . . . . . . . . . . . . . . . . . . r . . . . . . . . . . 0. 3 0. 2 3. 1 ec hi no ch lo a cr us -g al li . . . . . . . . . . . . . . . . . . . . . . . . . . . . r . . . 0. 3 0. 2 3. 1 biologica nyssana ● 13 (1) september 2022: 59-81 vukotić et al. ● aquatic and wetland plant communities of the pusta river (southern serbia): floristic characteristics and syntaxonomic affiliation n um be r of re le vé s 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 ic d % s % le m na m in or . . . . . . . . . . . . . . . . . . . . . . . . . . . . r . . . 0. 3 0. 2 3. 1 ec hi no cy st is lo ba ta . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . r . 0. 3 0. 2 3. 1 74 75 biologica nyssana ● 13 (1) september 2022: 59-81 vukotić et al. ● aquatic and wetland plant communities of the pusta river (southern serbia): floristic characteristics and syntaxonomic affiliation appendix 2. phytocoenological table of phalaridetum arundinaceae libbert 1931. ic coverage index according to lausi et al. (1982); d% share in total coverage according to surina (2005); s% species percentage frequency within the cluster number of relevés 1 2 3 4 5 6 7 8 ic d% s% locality kočane kosančić recording date 8 sep 12 sep 25 sep elevation (m) 194 194 194 195 195 195 195 222 latitude 43 .1 14 3 43 .1 15 3 43 .1 13 43 .1 12 4 43 .1 89 3 43 .1 88 5 43 .1 87 4 43 .0 78 8 longitude 21 .5 03 21 .5 03 5 21 .5 03 1 21 .5 03 5 21 .8 43 5 21 .8 43 7 21 .8 44 3 21 .7 86 4 phalaris arundinacea 5 5 5 5 5 5 4 5 99 41 100 mentha aquatica r + + 1 + . 2 . 21 8.6 75 lythrum salicaria r + + + + . + . 15 6.3 75 sparganium erectum + + + + . 1 . . 15 6.3 63 salix alba + + + + . . . + 14 5.7 63 xanthium strumarium . . + + + . + . 11 4.6 50 populus sp. + r . . 1 . . . 8.3 3.4 38 leersia oryzoides + . + + . . . . 8.3 3.4 38 typha latifolia + . . . + . + . 8.3 3.4 38 urtica dioica r . + . r . . . 8.3 3.4 38 epilobium hirsutum r . . . + . . . 4.2 1.7 25 polygonum mite + . . . r . . . 4.2 1.7 25 calystegia sepium . . + . . r . . 4.2 1.7 25 tanacetum vulgare . . 1 . . . . . 4.2 1.7 13 bidens tripartitus . . . . . . r + 4.2 1.7 25 salix euxina + . . . . . . . 2.8 1.1 13 amorpha fruticosa . + . . . . . . 2.8 1.1 13 lycopus europaeus . . . . + . . . 2.8 1.1 13 polygonum lapathifolium . . . . + . . . 2.8 1.1 13 myriophyllum spicatum . . . . . . . + 2.8 1.1 13 biologica nyssana ● 13 (1) september 2022: 59-81 vukotić et al. ● aquatic and wetland plant communities of the pusta river (southern serbia): floristic characteristics and syntaxonomic affiliation appendix 3. phytocoenological table of polygono-bidentetum tripartitae (w. koch 1926) lohm. 1950. ic coverage index according to lausi et al. (1982); d% share in total coverage according to surina (2005); s% species percentage frequency within the cluster number of relevés 1 2 3 ic d% s% locality kočane recording date 08 sep elevation (m) 195 latitude 43 .1 14 1 43 .1 14 3 43 .1 12 9 longitude 21 .5 02 8 21 .5 03 21 .5 03 1 polygonum lapathifolium 4 5 4 93 23 100 bidens tripartitus 1 1 4 52 13 100 polygonum mite 2 + + 33 8.4 100 lythrum salicaria 1 + . 19 4.7 67 xanthium strumarium + 1 . 19 4.7 67 leersia oryzoides . 2 . 19 4.7 33 sparganium erectum + + . 15 3.7 67 calystegia sepium + + 15 3.7 67 echinochloa crus-galli r + . 11 2.8 67 erigeron canadensis . r + 11 2.8 67 tanacetum vulgare . r + 11 2.8 67 lysimachia vulgaris 1 . . 11 2.8 33 mentha aquatica + . . 7.4 1.9 33 lycopus europaeus + . . 7.4 1.9 33 aristolochia clematitis + . . 7.4 1.9 33 urtica dioica + . . 7.4 1.9 33 phalaris arundinacea + . . 7.4 1.9 33 solanum dulcamara + . . 7.4 1.9 33 rubus caesius + . . 7.4 1.9 33 amorpha fruticosa . + . 7.4 1.9 33 salix euxina . + . 7.4 1.9 33 chenopodium album . + . 7.4 1.9 33 robinia pseudoacacia . . + 7.4 1.9 33 rumex conglomeratus . . + 7.4 1.9 33 76 77 biologica nyssana ● 13 (1) september 2022: 59-81 vukotić et al. ● aquatic and wetland plant communities of the pusta river (southern serbia): floristic characteristics and syntaxonomic affiliation a pp en di x 4. p hy to co en ol og ic al ta bl e of g ly ce rio -s pa rg an ie tu m n eg le ct i k oc h 19 26 . i c c ov er ag e in de x ac co rd in g to l au si e t a l. (1 98 2) ; d % sh ar e in to ta l c ov er ag e ac co rd in g to s ur in a (2 00 5) ; s % sp ec ie s pe rc en ta ge fr eq ue nc y w ith in th e cl us te r n um be r of re le vé s 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 ic d % s % l oc al ity k oč an e pu ko va c donje brijanje bojnik r ec or di ng da te 08 s ep 12 s ep 16 s ep 25 s ep e le va tio n (m ) 19 5 19 6 20 0 21 8 25 1 l at itu de 43.11356 43.11404 43.11429 43.11311 43.11255 43.11239 43.11222 43.18922 43.18825 43.18822 43.18779 43.18643 43.18616 43.18478 43.16677 43.16702 43.16714 43.16611 43.16495 43.16474 43.16388 43.16378 43.16343 43.16296 43.16218 43.16152 43.16073 43.16055 43.09847 43.0984 43.01257 l on gi tu de 21.50288 21.50282 21.50307 21.50306 21.50336 21.5034 21.50352 21.84363 21.84367 21.84347 21.84394 21.84498 21.84496 21.84558 21.85568 21.85548 21.85567 21.85677 21.86029 21.86074 21.86166 21.86159 21.86172 21.86183 21.86182 21.8619 21.8626 21.86237 21.81782 21.81832 21.72439 sp ar ga ni um er ec tu m 5 5 5 5 5 5 5 5 5 5 5 5 4 5 3 5 5 5 5 5 5 5 5 5 5 5 5 5 5 4 5 98 .6 48 .8 10 0 ly th ru m sa lic ar ia 1 1 + + + + + + . 1 + r + + 2 + r + . r . + r + r r . r . . . 16 .8 8. 3 77 .4 po ly go nu m m ite . + + 1 . . . . . . . . . + . . + . r . . . + . . r . . + 4 + 9. 7 4. 8 35 .5 bi de ns tr ip ar tit us . + r . + + . . . . . . . + . . + . + r r . + r r . . . + + + 9 4. 4 48 .4 po ly go nu m la pa th ifo liu m . 1 + + . . + . . . . . . + . . 1 . r r . . + . . . . . + . 1 8. 2 4. 1 35 .5 m en th a aq ua tic a . + . + 1 . . . + + . + + . r r . r . . r . . . . r . . . . . 7. 2 3. 6 38 .7 sa lix a lb a . . + r + . + . . . + . 1 . . . . . . . . . . . . . . + + . . 5. 7 2. 8 25 .8 po ta m og et on no do su s . . . . . + r + . . . + r . . . . r r r r . . . r . r r . . . 5. 4 2. 7 38 .7 sa lix e ux in a . r . . . . . . . . . . 1 . . + . . . . r . . . . + . . + + . 4. 7 2. 3 22 .6 xa nt hi um st ru m ar iu m . . . . . r . . + . . . . + . . r . r . . . . r . r . . . r + 4. 3 2. 1 29 biologica nyssana ● 13 (1) september 2022: 59-81 vukotić et al. ● aquatic and wetland plant communities of the pusta river (southern serbia): floristic characteristics and syntaxonomic affiliation n um be r of re le vé s 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 ic d % s % ly co pu s eu ro pa eu s . + . . . . . . . . . . . . r . . r r . . . . . . r . r . r . 2. 9 1. 4 22 .6 ph al ar is ar un di na ce a 1 . . . . . . . . + . . + . . . . . . . . . . . . . . . . . . 2. 5 1. 2 9. 7 ty ph a la tif ol ia + . . + . . . . . . . . . . 1 . . . . . . . . . . . . . . . . 2. 5 1. 2 9. 7 u rt ic a di oi ca . . . r . . . r . . . . r . r . . r . . . . . . . r . . . . r 2. 5 1. 2 22 .6 m yr io ph yl lu m sp ic at um . . . . . . . . . . . . . . . . . r . r r . . r r . r r . . . 2. 5 1. 2 22 .6 ta na ce tu m vu lg ar e . . . . . + . . . . . . . . . . . r . . . . r . . r . r . . . 2. 2 1. 1 16 .1 c al ys te gi a se pi um . . . . . . . . . . . r . . r . . r . . . . . . . r . . . r r 2. 2 1. 1 19 .4 ro bi ni a ps eu do ac ac ia . . . . . . . . . + . . . . . . . . . r . . . . . . . + . . . 1. 8 0. 9 9. 7 so la nu m du lc am ar a . r . . . . . . . . . . . . + r . . . . . . . . . . . . . . . 1. 4 0. 7 9. 7 am or ph a fr ut ic os a . . + . + . . . . . . . . . . . . . . . . . . . . . . . . . . 1. 4 0. 7 6. 5 le er si a or yz oi de s . . . + . . . . . . . . . . . . . r . r . . . . . . . . . . . 1. 4 0. 7 9. 7 g al iu m ap ar in e . . . . . . . r . . . . . . . . . r . . . . r . . r . . . . . 1. 4 0. 7 12 .9 ru bu s c ae si us . . . . . . . . . . . r . . . . . . r r . r . . . . . . . . . 1. 4 0. 7 12 .9 ep ilo bi um hi rs ut um . . . . . . . . . . . . . . . . . . . r . r . . . r . . . . . 1. 1 0. 5 9. 7 le m na m in or . . . . . . . . . . . . . . . . . . . r . . . r . . . . . r . 1. 1 0. 5 9. 7 ec hi no ch lo a cr us -g al li . . r . . . . . . . . . . . . . . . . . . . . . . . r . . . . 0. 7 0. 4 6. 5 po ta m og et on cr is pu s . . . . . . . + . . . . . . . . . . . . . . . . . . . . . . . 0. 7 0. 4 3. 2 sc ut el la ri a ga le ri cu la ta . . . . . . . . . . . . . . . . . r . . . . . r . . . . . . . 0. 7 0. 4 6. 5 sc ro ph ul ar ia no do sa . . . . . . . . . . . . . . . . . r . . . . . . . . . . . . . 0. 4 0. 2 3. 2 78 79 biologica nyssana ● 13 (1) september 2022: 59-81 vukotić et al. ● aquatic and wetland plant communities of the pusta river (southern serbia): floristic characteristics and syntaxonomic affiliationr n um be r of re le vé s 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 ic d % s % be ru la e re ct a . . . . . . . . . . . . . . . . . . . . r . . . . . . . . . . 0. 4 0. 2 3. 2 st el la ri a ne gl ec ta . . . . . . . . . . . . . . . . . . . . . . . . . r . . . . . 0. 4 0. 2 3. 2 ec hi no cy st is lo ba ta . . . . . . . . . . . . . . . . . . . . . . . . . r . . . . . 0. 4 0. 2 3. 2 el eo ch ar is pa lu st ri s . . . . . . . . . . . . . . . . . . . . . . . . . . r . . . . 0. 4 0. 2 3. 2 biologica nyssana ● 13 (1) september 2022: 59-81 vukotić et al. ● aquatic and wetland plant communities of the pusta river (southern serbia): floristic characteristics and syntaxonomic affiliation appendix 5. phytocoenological table of phragmitetum australis savič 1926. ic coverage index according to lausi et al. (1982); d% share in total coverage according to surina (2005); s% species percentage frequency within the cluster number of relevés 1 2 3 4 5 6 7 8 9 ic d% s% locality kočane pukovac recording date 08 sep 12 sep 16 sep elevation (m) 195 200 latitude 43 .1 12 7 43 .1 88 43 .1 85 3 43 .1 84 8 43 .1 64 5 43 .1 63 6 43 .1 63 5 43 .1 63 1 43 .1 63 longitude 21 .5 03 3 21 .8 43 8 21 .8 45 3 21 .8 45 5 21 .8 61 2 21 .8 61 6 21 .8 61 7 21 .8 61 8 21 .8 62 1 phragmites communis 5 5 5 5 5 5 5 5 5 100 56 100 bidens tripartitus + . . . + r r r . 9 5 56 urtica dioica r . r . + r r . . 7 4 56 salix.euxina . . . . + + + . . 7 4 33 robinia pseudoacacia . r + . . . . . + 6 4 33 salix alba + + . . . . . . . 5 3 22 echinocystis lobata . . . . + . . r r 5 3 33 typha latifolia 1 . . . . . . . . 4 2 11 lythrum salicaria + . . . . r . . . 4 2 22 polygonum lapathifolium + . . . . . . r . 4 2 22 sparganium erectum . . . r + . . . . 4 2 22 humulus lupulus + . . . . . . . . 3 1 11 mentha aquatica + . . . . . . . . 3 1 11 polygonum mite . . . . + . . . . 3 1 11 epilobium hirsutum . . . . + . . . . 3 1 11 galium aparine . . . . + . . . . 3 1 11 rubus caesius . . . . . r r . . 3 1 22 lycopus europaeus . . . . . r r . . 3 1 22 populus sp. . r . . . . . . . 1 1 11 potamogeton nodosus . . . r . . . . . 1 1 11 erigeron canadensis . . . . . r . . . 1 1 11 tanacetum vulgare . . . . . r . . . 1 1 11 xanthium strumarium . . . . . . r . . 1 1 11 80 81 biologica nyssana ● 13 (1) september 2022: 59-81 vukotić et al. ● aquatic and wetland plant communities of the pusta river (southern serbia): floristic characteristics and syntaxonomic affiliation appendix 6. phytocoenological table of myriophyllo-potametum soó 1934. ic coverage index according to lausi et al. (1982); d% share in total coverage according to surina (2005); s% species percentage frequency within the cluster number of relevés 1 2 3 4 5 ic d% s% locality kočane pukovac recording date 08 sep 16 sep elevation (m) 195 200 latitude 43 .1 14 6 43 .1 64 5 43 .1 64 43 .1 62 3 43 .1 66 8 longitude 21 .5 03 2 21 .8 61 2 21 .8 61 6 21 .8 61 7 21 .8 55 8 myriophyllum spicatum 5 5 4 5 5 98 69 100 potamogeton crispus 1 . . . 4 24 17 40 potamogeton nodosus + . 2 + . 20 14 60 appendix 7. phytocoenological table of potametum nodosi soó (1928) 1960, segal 1964. ic coverage index according to lausi et al. (1982); d% share in total coverage according to surina (2005); s% species percentage frequency within the cluster number of relevés 1 2 3 4 5 6 7 8 ic d% s% locality pukovac recording date 16 sep elevation (m) 200 latitude 43 .1 66 8 43 .1 66 3 43 .1 65 3 43 .1 64 9 43 .1 64 7 43 .1 61 7 43 .1 60 8 43 .1 60 6 longitude 21 .8 55 8 21 .8 56 7 21 .8 59 1 21 .8 59 8 21 .8 60 9 21 .8 62 2 21 .8 62 6 21 .8 62 7 potamogeton nodosus 5 5 5 4 4 5 5 5 97 63 100 myriophyllum spicatum . 4 1 + 1 1 2 4 44 29 87.5 sparganium erectum . . . 3 . . . . 10 6 12.5 lemna minor . . . . + . . . 3 2 12.5 grdovć' et al., 2019, biologica nyssana 10(2) 10 (2) december 2019: 99-103 doi: 10.5281/zenodo.3600181 bryophyte collection beou – a neglected national research treasure original article svetlana grdović faculty of veterinary medicine, university of belgrade, 11000 belgrade, serbia cecag@vet.bg.ac.rs (corresponding author) jovana pantović university of belgrade, faculty of biology, institute of botany and botanical garden, takovska 43, 11000 belgrade, serbia jpantovic@bio.bg.ac.rs milan veljić university of belgrade, faculty of biology, institute of botany and botanical garden, takovska 43, 11000 belgrade, serbia veljicm@bio.bg.ac.rs marko sabovljević university of belgrade, faculty of biology, institute of botany and botanical garden, takovska 43, 11000 belgrade, serbia marko@bio.bg.ac.rs received: october 13, 2019 revised: november 07, 2019 accepted: december 19, 2019 abstract: considering recent progress of bryology in serbia digitisation of bryophyte collection within beou (bryo beou) was initiated. it is the most important and the largest collection of the bryophytes in the country, and represent a significant basis for all further research and analysis of bryophyte flora of serbia. this collection also keeps relevant material from neighbouring countries that are considered poorly investigated (e.g. n. macedonia, albania, bosnia and herzegovina). here an overview of the specimens kept in the collection, data-based up to 2019, is given. however, work on the organisation and systematisation of this growing collection is ongoing. key words: bryophytes, herbarium, collection, digitisation, serbia apstract: kolekcija briofita beou zanemareno nacionalno istraživačko blago sa razvojem briologije u srbiji, koja je decenijama bila zapostavljena, započeta je digitalizacija kolekcije briofita u okviru beou herbarijuma (bryo beou). ova kolekcija je najznačajnija i najveća kolekcija briofita u zemlji, i predstavlja važnu osnovu za sva buduća istraživanja i analize flore briofita u srbiji. zbirka sadrži i značajan materijal iz drugih zemalja koje se smatraju slabo ili nedovoljno briološki istraženim (npr. severna makedonija, albanija, bosna i hercegovina). u radu je dat trenutni pregled briološke zbirke, sa presekom digitalizacije do 2019. godine. rad na organizaciji i sistematizaciji ove kolekcije je i dalje u toku. ključne reči: briofite, herbarijum, zbirka, digitalizacija, srbija the value of herbarium collections herbaria document the world’s flora and provide a record of botanical diversity. historically, the primary importance of herbarium specimens was related to the questions of taxonomy, which includes identification, nomenclature, and classification of taxa (e. g. janković et al., 2014; niketić, 2014; di pietro et al., 2017). beyond this, herbarium collections have become crucial for a wide array of studies. primarily, they ensure a continuous record of species distributions (buzurović et al., 2013), and even its predictions (loiselle et al., 2008; wollan et al., 2008; feeley & silman, 2011). hence, historical plant collections are of great value for studying changes in time of flora and vegetation due to the global climate change or degradation of habitats. herbarium collections can also be used to monitor the spread of invasive taxa (delisle et al., 2003; crawford & hoagland, 2009; jenačković et al., 2015), population trends and traits of rare and endangered taxa (christenson, 1994), and to identify priority sites for conservation. historical herbarium specimens are also an important source of information for the conservation of rare or even extinct taxa (kricsfalusy & trevisan, 2014), but also for (phylo)genetic and evolutionary studies. the collections represent an important educational resource for researchers and students. herbarium beou one of the most important and largest herbarium collections in southeastern europe is located at the institute of botany and botanical garden “jevremovac” of the faculty of biology, university of belgrade. it was founded almost 160 years ago, in 1860, when the famous serbian botanist josif pančić gave his collection of pressed plants to the great school, cur© 2019 grdović et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 99 13th symposium on the flora of southeastern serbia and neighboring regions rent university of belgrade (vukojičić et al., 2011). at that time, the collection numbered 20.000 specimens of 6.000 different species that he was gathering during his field trips, but also as an exchange with the european herbaria such as germany, france, italy, spain and russia. pančić’s collection (herbarium pancicianum) is of exceptional scientific, historical and cultural value and importance. in addition to pančić, many other serbian botanists of the 19th and 20th centuries have contributed to the enrichment of the herbarium, such as s. petrović, p. jurišić, đ. ilić, đ. ničić, s. pelivanović, n. košanin, t. soška, l. adamović, v. blečić, i. rudski, p. černjavski, b. tatić, m.m. janković, j. blaženčić and many others. over the past 30 years, researchers of the recent generations (v. stevanović, d. lakušić, s. jovanović, g. tomović, s. vukojičić, and others) have contributed significantly to the herbarium collection, with over 60.000 herbarium specimens collected mostly from the balkan peninsula. herbarium of the university of belgrade today numbers more than 180.000 specimens of vascular plants, bryophytes (bryo/beou) and algae. it is included in the global directory of registered herbaria (index herbariorum), under the herbarium code beou. bryophyte collections beou bryology in serbia started in the middle of the 19th century. during the so-called early period of exploration (pantović & sabovljević, 2017), many serbian botanists and teachers from pančić, katić, jurišić, simić and soška, to košanin, rudski, černjavski and grebenščikov in addition to the vascular flora also collected bryophytes. bryophyte material collected by the first researchers is kept at the natural history museum in belgrade (pavletić, 1955), which is also included in the index herbariorum under code beo. material from this collection is from localities from all over former yugoslavia and the balkan peninsula. although historically important, this collection was forgotten and remained neglected with unnamed specimens for a long time. according to the catalogue, it numbers over 800 specimens. a far greater collection of bryophytes has been kept at the beou herbarium, which has been intensively increasing over the last twenty years. it is estimated to have over 18.000 specimens (vukojičić at al., 2011), of which 8.000 are identified and data-based, and about 10.000 undetermined specimens. the collection was established in the 1990s, within the two chairs of the institute of botany. at the department of plant ecology and phytogeography, professor vladimir stevanović and his associates collected bryophytes while exploring the flora and vegetation of the durmitor mountain in montenegro. then svetlana grdović enriched the collection with bryophytes from belgrade and its surroundings (e. g. grdović, 2005; grdović & stevanović, 2006;). from 2000 until today, professor marko sabovljević had the highest importance for the enrichment of the herbarium collection. together with his foreign and local coworkers (beata papp, erszebet szurdoki and jovana pantović), or by himself, he has collected an impressive number of herbarium specimens of mosses and liverworts from serbia and balkan countries (e. g. 100 biologica nyssana ● 10 (2) december 2019: 99-103 grdović et al. ● bryophyte collection beou – a neglected national research treasure fig. 1. labelled and digitised moss specimen in the bryophyte collection beou fig. 2. stored boxes of bryophyte collection in the beou herbarium all original data on taxa, locality, collector, date, locality, habitat and ecology and identifier, were entered into excel spreadsheet. specimens have been identified to at least genus level, although the majority was identified to a species or lower level. specimens are kept in paper envelops, labelled (fig. 1) and stored in card boxes (fig. 2) in controlled conditions. the collection of bryophytes at the beou herbarium is of great scientific and cultural value today. bryophyte specimens originate from 6 different continents: europe, asia, africa, north america, australia and antarctica, from 46 different countries. however, the majority of the specimens are from europe (fig. 3). the largest number was collected on the territory of serbia, altogether 4696 bryophyte specimens, followed by montenegro 544, slovenia 327, bosnia and herzegovina 295, italy 241 and so on (fig 4). however, these numbers refer only to identified specimens. until the 1990s mosses were sporadically collected, hence only 613 records in the bryo beou collection were collected before 1990. from 1990 onwards, bryophyte flora has been intensively studied and all other records originate from this so-called modern period (pantović & sabovljević, 2017) of exploration. by years (fig. 5), largest number of 4696 544 327 295 241 236 236 204 130 127 116 105 102 sabovljević & cvetić, 2003; papp & sabovljević, 2010; sabovljević et al., 2010), but also from other european countries (e. g. papp & sabovljević, 2009; papp et al., 2016). at the same time in the 1990s, at the department of plant morphology and systematics, professor milan veljić has started his collection of bryophytes, which is now part of the bryo beou. he collected material from various sites in serbia (e. g. veljić et al., 2001, 2008). part of this bryo beou collection is valuable material from nature reserves and protected areas in serbia, collected and published afterwards by mića popović in the middle of the last century (popović, 1966). the modern period of the bryo beou collection considering recent progress of bryology in serbia a comprehensive data summarisation on the distribution of bryophytes in serbia was necessary to be able to critically assess certain taxa and to update the country list of the species. for that purpose, bryo database was established (pantović & sabovljević, 2017), and besides aggregation of literature data, digitisation of bryophyte herbarium specimens within beou was initiated. 8084 specimens have been digitised so far, but a large part of the collection has not been identified nor digitised yet. biologica nyssana ● 10 (2) december 2019: 99-103 101 fig. 2. map indicating the number of specimens in bryo beou collected from different european countries fig. 4. countries with more than 100 specimens in the bryophyte collection grdović et al. ● bryophyte collection beou – a neglected national research treasure identified herbarium specimens was collected in 2000 and 2001 (951 and 676), followed by over 500 specimens in 2013, 2016 and 2018, and more than 400 specimens collected in 1997, 1998, 2002, 2014 and 2015. more than half (66%) of the specimens, a total of 5112, was collected by marko sabovljević and his coworkers. analysis of the period of collecting showed that the majority of the field trips were done in the spring and summer months (fig. 6). largest number of bryophyte specimens was collected in may (1334) and july (1240) while the least records were collected in january (79) and december (166). all digitised bryophytes can be grouped into 797 different taxa and 294 genera. bryo beou collection and its digitised data rep0 100 200 300 400 500 600 700 800 19 91 19 93 19 94 19 95 19 96 19 97 19 98 19 99 20 00 20 03 20 04 20 05 20 06 20 07 20 08 20 09 20 10 20 11 20 12 20 13 20 14 20 15 20 16 20 17 20 18 102 resent an important basis for all further research and analysis of bryophyte flora of serbia. this collection also keeps important material, although not yet completely identified and digitised, from neighbouring countries that are considered poorly investigated (e.g. macedonia, albania, bosnia and herzegovina). work on the organisation and systematisation of this growing collection is ongoing. references buzurović, u., stevanović, v., niketić, m., jakov-ljević, k., tomović, g. 2013: on the distribution of goniolimon tataricum (plumbaginaceae) in serbia. botanica serbica, 37(2): 167-172. christenson, e. a. 1994: significant collections of orchidaceae conserved in herbarium hamburgense (hbg). brittonia, 46(4): 344-354. crawford, p. h., hoagland, b. w. 2009: can herbarium records be used to map alien species invasion and native species expansion over the past 100 years?. journal of biogeography, 36(4): 651-661. delisle, f., lavoie, c., jean, m., lachance, d. 2003: reconstructing the spread of invasive plants: taking into account biases associated with herbarium specimens. journal of biogeography, 30(7): 1033-1042. di pietro, r., kuzmanović, n., iamonico, d., lakušić, d. 2017: nomenclatural and taxonomic notes on sesleria sect. argenteae (poaceae). phytotaxa, 309(2): 101-117. feeley, k. j., silman, m. r. 2011: keep collecting: accurate species distribution modelling requires more collections than previously thought. diversity and distributions, 17(6): 1132-1140. grdović, s. 2005: mahovine šireg područja beograda i njihov bioindikatorski značaj, zadužbina andrejević, beograd. 130 p. grdović, s., stevanović, v. 2006: the moss flora in the central urban area of belgrade. archives of biological sciences 58(1): 55-59. janković, i., kuzmanović, n., clementi, m., lakušić, d. 2014: lectotypification of campanula secundiflora vis. & pančić (campanulaceae), a species of european concern. botanica serbica, 38(2): 269-271. biologica nyssana ● 10 (2) december 2019: 99-103 fig. 5. number of collected specimens per year collected only during the modern period of investigation (since 1990 onwards) fig. 6. number of collected specimens per each calendar month grdović et al. ● bryophyte collection beou – a neglected national research treasure 79 546 858 748 1334 924 1240 820 330 301 479 166 103 jenačković, d., miljković, m., mitrović, d., ranđelović, v. 2015: contribution to the knowledge of distribution of certain macrophytes, invasive and threatened species in serbia. biologica nyssana, 6(2): 59-65. kricsfalusy, v. v., trevisan, n. 2014: prioritizing regionally rare plant species for conservation using herbarium data. biodiversity and conservation, 23(1): 39-61. loiselle, b. a., jørgensen, p. m., consiglio, t., jiménez, i., blake, j. g., lohmann, l. g., montiel, o. m. 2008: predicting species distributions from herbarium collections: does climate bias in collection sampling influence model outcomes?. journal of biogeography, 35(1): 105-116. niketić, m. 2014: nomenclature review of the plants published by josif pančić (nomenclator pancicianus novus). botanica serbica, 38(2): 209-236. pantović, j., sabovljević, m. 2017: overview of bryophyte flora research in serbia with presentation of the serbian bryo database. botanica serbica, 41(2): 153-162. papp, b., pantović, j., szurdoki, e., sabovljević, m. s. 2016: new bryophyte records for the republic of macedonia. journal of bryology, 38(2): 168-171. papp, b., sabovljević, m. 2009: notes on some new and interesting bryophyte records from croatia. journal of bryology, 31(4): 272-275. papp, b., sabovljević, m. 2010: contribution to the bryophyte flora of the vršačke planine mts., serbia. botanica serbica, 34(2): 107-110. pavletić, z. 1955: prodromus flore briofita jugoslavije. jugoslavenska akademija znanosti i umetnosti. 578 p. popović, m. 1966: prilog poznavanju mahovina u rezervatima i zaštićenim područjima u srbiji. zaštita prirode, 33: 219-228. sabovljević, m., cvetić, t. 2003: bryophyte flora of avala mt. (c. serbia, yugoslavia). lindbergia, 28: 90-96. sabovljević, m., papp, b., szurdoki, e. 2010: new bryophyte records to some countries of the southeastern europe. cryptogamie, bryologie, 31(3): 289-292. veljić, m., marin, p., boža, p., petković, b. 2001: bryoflora of some well-springs of the dinaric alps and carpathian karst in serbia. bocconea, 13: 343351. vukojičić, s., lakušić, d., jovanović, s., marin, p.d., tomović, g., sabovljević, m., šinžarsekulić, j., veljić, m., cvijan, m., blaženčić, j., stevanović, v. 2011: university of belgrade herbarium treasury of data and challenges for future research. on the occasion of the 150th anniversary of university of belgrade herbarium (18602010). botanica serbica, 35:163-178. wollan, a. k., bakkestuen, v., kauserud, h., gulden, g., halvorsen, r. 2008: modelling and predicting fungal distribution patterns using herbarium data. journal of biogeography, 35(12): 2298-2310. biologica nyssana ● 10 (2) december 2019: 99-103 grdović et al. ● bryophyte collection beou – a neglected national research treasure anticancer compounds from medicinal plants biologica nyssana 5 (1)  september 2014: 1-10 stojković, m. et al.  antioxidant potential of tanacetum vulgare l. … 47 original article received: 3 september 2014 revised: 5 september 2014 accepted: 10 september 2014 antioxidant potential of tanacetum vulgare l. extracts milan b. stojković 1* , snežana s. mitić, jovana lj. pavlović, branka t. stojanović, dušan đ. paunović 1 department of chemistry, faculty of science and mathematics, university of niš, 33 višegradska street, 18 000 niš, serbia * e-mail: milan@svrljig.net abstract: stojković, m., mitić, s., pavlović, j., stojanović, b., paunović, d.: antioxidant potential of tanacetum vulgare l. extract. biologica nyssana, 5 (1), septmeber 2014: 47-51. antioxidant activity of tanacetum vulgare l. extracts was determined. areal plant parts (leaves and flowers) were dried, grinded and extracted with five different solvents: methanol, ethanol, acetone, water and isopropanol. total phenols and total flavonoids were determined and four in vitro antioxidant assays were applied. the best extraction medium (considering only phenols and flavonoids) was methanol and 62.7 mg galic acid equivalents per gram of dry weight were found (for leaves extract). comparing amounts of phenolic compounds found in the extracts and its antioxidant potential with other herbal teas and extracts, it may be concluded that t. vulgare is a plant species destitute with phenolic compounds. obtained results suggest that phenolic compounds, present in the plant tissues, are carriers of antioxidant properties. key words: antioxidant, flavonoid, extrac, phenol, tanacetum vulgare apstract: stojković, m., mitić, s., pavlović, j., stojanović, b., paunović, d.: antioksidantni potencijal ekstrakta biljne vrste tanacetum vulgare l. biologica nyssana, 5 (1), septmeber 2014: 47-51. određivana je antioksidantna aktivnost ekstrakta biljne vrste tanacetum vulgare l. nadzemni delovi ove biljne vrste (listovi i cvetovi) su sušeni, samleveni i ekstrakovani sa pet različitih rastvarača (metanol, etanol, aceton, voda i izopropanol). određen je sadržaj totalnih fenola i totalnih flavonoida kao i antioksidantna aktivnost pomoću četiri in vitro metoda. kao najbolje ekstrakciono sredstvo (za fenole i flavonoide) pokazao se metanol i nađeno je 62.7 mg ekvivalenata galne kiseline po gramu suve biljne mase (u ekstraktu lista). ako nađeni sadrđaj fenola i flavonoida uporedimo sa sličnim ekstraktima ili biljnim čajevima, možemo zaključiti da je biljna vrsta t. vulgare relativno siromašna fenolnim jedinjenjima. dobijeni rezultati sugeriču da su fenolna jedinjenja, prisutna u biljnim tkivima, nosioci antioksidantnih osobina. ključne reči: antioksidant, flavonoid, ekstrakt, fenol, tanacetum vulgare introduction tanacetum is a genus of about 160 species of flowering plants in the asteraceae family, native to many regions of the northern hemisphere. they are known commonly as tansies in english and povratič in serbian. tanacetum vulgare l. (common tansy) is perennial, herbaceous plant species native to temperate europe and asia but invasive in other parts of world. 5 (1) • september 2014: 47-51 biologica nyssana 5 (1)  september 2014: 47-51 stojković, m. et al.  antioxidant potential of tanacetum vulgare l. … 48 tansy is a well-known herbal plant widely used in traditional medicine in south-eastern serbia (k o j i ć et al., 1998). in recent times, preparing teas is contraindicated because of the toxic monoterpene, α-thujone, present in the plant tissue (b a r a n a u s k i e n e et al., 2014). tea prepared from t. vulgare is used as an antihelminthic, carminative, antispasmodic, stimulant to abdominal viscera, tonic, emmenagogue, antidiabetic, diuretic and antihypertensive (l a h l o u et al., 2008). various authors showed that the water and lipophilic extracts from t. vulgare possess antitumor, antiinflammatory, antioxidant and antimicrobial activity (l a h l o u et al., 2008). to explore potential health benefits, dried parts of t. vulgare (flowers and leaves) were extracted with five different solvents (methanol, ethanol, acetone, water and isopropanol) and total phenols and flavonoids were determined. series of in vitro antioxidant assays were performed (scavenging dpph radical, scavenging abts radical, iron(iii) to iron(ii) reduction assay and cupric ion reducing antioxidant capacity assay). material and methods plant material and extract preparation areal parts of tanacetum vulgare l. (asteraceae) were collected in mai 2012 from the mountain peak babin zub (latitude: 43.363 n; longitude: 22.585 e), south-east serbia. the plant material was air dried about two weeks, out of direct sunlight. flowers and leaves were separated from the stems and were grinded into fine powder. exact masses (0.25 g) of powdered flowers and leaves were extracted with five different solvents (methanol, ethanol, acetone, water and isopropanol) on ultrasonic bath. every batch was extracted three times for 30 minutes, with 20+20+10 ml of appropriate solvent. reagents 1,1-diphenyl-2-picrylhydrazyl radical (dpph), 2,2’azino-bis(3-ethylbenzothiazoline-6sulfonic acid) (abts), potassium-persulfate and catechin were obtained from sigma-aldrich (steinheim, germany), methanol, ethanol and acetone were purchased from j. t. baker (deventer, holland). gallic acid was obtained from carl roth (karlsruhe, germany). trolox (6-hydroxy-2,5,7,8tetramethylchroman-2-carboxylic acid) were obtained from acros organics (geel, belgium). folin-ciocalteu reagent, na2hpo4, nah2po4, na2co3, nano2, naoh, feso4 ∙ 7h2o, k3[fe(cn)6], trichloroacetic acid (tca) and alcl3 were purchased from merck (darmstadt, germany). all other chemicals were of analytical grade, and were used as received except that the solvents were distilled prior to use. determination of total phenolic (tp) content total phenolic content of the extracts were determined using folin-ciocalteau assay (m i t i ć e t a l ., 2011). briefly, 0.4 ml of extract were mixed with 2.0 ml of (20% w/v) na2co3 solution and 0.5 ml of fc reagent and made up to 10 ml with deionized water. the solution was mixed and, after aging for 120 min at 25˚c, absorbance was measured at 760 nm, using agilent 8453 uv-visible spectrophotometer (agilent technologies, usa). results were expressed as mg of gallic acid equivalents (gae) per g of the dry sample. determination of total flavonoid (tf) content the total flavonoid content of t. vulgare extracts was determined by a colorimetric method according to m i t i ć e t a l . (2012). a known volume of the samples was mixed with 2 ml of distilled water and subsequently with 0.3 ml of a nano2 solution (5%, w/w). after 5 min, 3 ml of alcl3 solution (1%, w/w) was added and the solution left for 5 min at room temperature. then, 2 ml of naoh solution (1 mol/l) was added to the mixture diluted with deionized water to the final volume of 10 ml. the mixture was thoroughly mixed and absorbance was immediately measured at 510 nm. results were expressed as mg catechin equivalent (ce) per g of dry weight (d.w.). dpph free radical-scavenging assay the antioxidant capacity of t. vulgare solvent extracts was studied through the evaluation of the extracts’ free radical-scavenging effect on 1,1diphenyl-2-picrylhydrazyl (dpph) radical (s t o j k o v i ć e t a l ., 2014). an aliquot (0.1 ml) of the different extracts was mixed with 2.5 ml of 100 µmol/l dpph methanol solution. the mixture was thoroughly vortex-mixed, kept out of light for 30 min and absorption was measured at 515 nm. the absorption of the blank containing the same amount of methanol and dpph solution was prepared and measured daily. the radical scavenging activity was calculated using the following formula: scavenging effect (%) = [1-(absorbance of the sample/absorbance of the blank)] ∙ 100 biologica nyssana 5 (1)  september 2014: 47-51 stojković, m. et al.  antioxidant potential of tanacetum vulgare l. … 49 the results were expressed as mg of trolox equivalents (te) per 1 g of sample. abts radical-scavenging capacity assay the abts radical cation (abts •+ ) solution was prepared by the reaction of solutions of 7 µmol/l abts and 2.45 µmol/l of potassium persufate, at 23°c in the dark for 16 h. the abts •+ solution was then diluted with 80% (v/v) aqueous ethanol to obtain a solution with the absorbance of 0.700 ± 0.020 at 734 nm (v e l j k o v i ć e t a l ., 2013). the abts •+ solution (3.9 ml) was added to 0.1 ml of the test sample and mixed thoroughly. the reaction mixture was left to stand at 23°c for 6 min and then the absorbance was measured at 734 nm. the total antioxidant activity of t. vulgare extracts was expressed as mg of te per g of dry weight. radical scavenging activity was calculated using the following formula: scavenging effect (%) = [1-(absorbance of sample/absorbance of blank)] ∙100 iron(iii) to iron(ii) reduction assay (ira) iron(iii) to iron(ii) reduction assay was performed according to s t o j k o v i ć et al. (2014). different dilutions of the extracts (0.5 ml) were added to the mixture of 1.25 ml of the phosphate buffer (0.2 mol/l, ph 6.6) and 1.25 ml of potassium ferricyanide (1%, w/w). the resultant solution was incubated at 50°c for 20 min. after that, trichloroacetic acid solution (1.25 ml, 10%, w/w) was added, diluted with 4.25 ml of water and 0.85 ml of ferric chloride solution (0.1%, w/w) was added. after 30 min, the absorbance was measured at 700 nm. ira of the extracts was expressed as mg gallic acid equivalents per g of dry weight of the sample. cupric ion reducing antioxidant capacity (cuprac) the cuprac method was applied as described by s t o j k o v i ć et al. (2014). a mixture comprised of 1 ml of 10 mmol/l copper(ii) chloride, 1 ml of 1mol/l ammonium acetate buffer at ph 7.0, and 1 ml of 7.5 mmol/l neocuproine solution was prepared, x ml sample solution and (1−x) ml distilled water were added, and well mixed (total volume: 4.0 ml). this final mixture in a stoppered test tube was left to stand at room temperature for 30 min. after that, the absorbance at 450 nm was measured against a blank. the total antioxidant activity of t. vulgare extracts was expressed as µmol of te per g of dry weight. statistical analysis all results were expressed as the mean ± standard deviation. statistically significant differences were determined by one-way analysis of variance (anova) followed by tukey´s post hoc test for multiple comparison (graph pad prism version 5.03, san diego, ca, usa). probability values (p) less than 0.05 were considered to be statistically significant. results and discussion phenolic compounds (phenolic acids and flavonoids) are the natural constituents of plants. they are secondary metabolites and produced by plants as a response to different environmental factors (light, chilling, pollution, etc.) and to defend injured plants. phenolic compounds are crucial for plant growth and reproduction. also, they have various roles in plants (protective, signal molecules, gives colours of flowers, fruits, and leaves, etc.) but they are not understood completely (g h a s e m z a d e h & g h a s e m z a d e h , 2011). polyphenols are well known for their antioxidant activity as radical scavengers and possible beneficial roles in human health, such as reducing the risk of cancer, cardiovascular and other diseases (m i t i ć et al., 2012). the main carriers of antioxidant properties in plant tissues are phenolic compounds. thus, total phenols and total flavonoids of t. vulgare’s flowers and leaves extracts were determined. the antioxidant capacity of the extracts were studied through four in vitro antioxidant assays: scavenging dpph radical, scavenging abts radical, iron(iii) to iron(ii) reduction assay (ira) and cupric ion reducing antioxidant capacity assay (cuprac). obtained results are shown in tab. 1. total phenols (tp) in various extracts are in the range from 62.7 to 12.2 mg of galic acid equivalents (gae) per g of dried weight (dw) for leaf extracts and from 42.1 to 7.2 mg gae/g dw for flower extracts. methanol was observed as the best extraction medium for total phenolic compounds among the others solvents used (ethanol, water, isopropanol and acetone). the lowest amounts of phenolic compounds were extracted with acetone. total flavonoids found in mentioned extracts are in the range from 18.2 to 4.7 mg of catechin equivalents (ce)/g dw for leaf extracts and from 10.5 to 4.0 mg ce/g dw for flower extracts. the best solvent for flavonoid extraction was water, dissimilar to total phenols where methanol was the best one, but the observed difference was small biologica nyssana 5 (1)  september 2014: 47-51 stojković, m. et al.  antioxidant potential of tanacetum vulgare l. … 50 table 1: total phenols, total flavonoids and in vitro antioxidant tests tp tf abts dpph ira cuprac mg gae/g dw mg ce/g dw mg te/g dw mg te/g dw mg gae/g dw mg te/g dw l ea v es ethanol 31.6 ± 0.5 9.3 ± 0.3 25.5 ± 0.5 12.3 ± 0.5 18.7 ± 0.5 115 ± 3 methanol 62.7 ± 0.5 15.3 ± 0.5 41.2 ± 0.5 28.1 ± 0.5 34.2 ± 0.5 214 ± 5 acetone 12.2 ± 0.5 4.7 ± 0.1 7.6 ± 0.2 4.9 ± 0.1 10.2 ± 0.5 53 ± 1 water 47.8 ± 0.5 18.2 ± 0.5 22.2 ± 0.5 25.9 ± 0.5 35.2 ± 0.5 129 ± 3 isopropanol 12.3 ± 0.5 6.3 ± 0.1 5.5 ± 0.2 4.2 ± 0.1 9.9 ± 0.5 34 ± 1 f lo w er s ethanol 19.0 ± 0.5 6.0 ± 0.1 12.2 ± 0.3 7.6 ± 0.3 13.9 ± 0.5 47 ± 1 methanol 42.1 ± 0.5 10.1 ± 0.3 27.6 ± 0.5 15.8 ± 0.5 27.0 ± 0.5 164 ± 3 acetone 7.2 ± 0.3 4.0 ± 0.1 4.5 ± 0.1 3.5 ± 0.1 9.2 ± 0.5 18.1 ± 0.5 water 42.3 ± 0.5 10.5 ± 0.3 19.9 ± 0.5 21.7 ± 0.5 25.8 ± 0.5 121 ± 3 isopropanol 10.3 ± 0.5 5.6 ± 0.1 4.8 ± 0.1 5.5 ± 0.2 10.0 ± 0.5 33 ± 1 table 2: correlation coefficient between total phenols, total flavonoids and different in vitro antioxidant assays tp tf abts dpph ira cuprac tp 1 0.9094 0.9474 0.9735 0.9737 0.9677 tf 1 0.7871 0.9470 0.9601 0.8158 abts 1 0.8613 0.8739 0.9790 dpph 1 0.9802 0.8943 ira 1 0.9118 cuprac 1 (leaf) or insignificant compared to the standard deviation (flower). phenol and flavonoid contents in various herbal teas (v e l j k o v i ć et al., 2013) are in the range from 26 to 241 mg gae/g dw and from 12 to 85 mg cae/g dw for phenols and flavonoids, respectively. according to this data, t. vulgare is a plant with small contents of phenolic compounds. 1,1-diphenyl-2-picrylhydrazyl radical (dpph) is a stable nitrogen-centered free radical whose color changes from violet to yellow upon reduction either by the process of hydrogen or electron-donation. antioxidant capacity were in the range from 28.1 mg trolox equivalents (te)/g dw (methanol extract of leaf) to 3.5 mg te/g dw (ethanol extract of flower). the abts assay method is based on the ability of antioxidant molecules to quench the longlived abts cation radicals (which ethanol solution is blue-green with characteristic absorption at 734 nm). the addition of antioxidants to the solution of prepared radical cation reduces it to abts, causing decolorizing. results were expressed as mg trolox equivalent per g of dw. antioxidant capacity, determined with this assay, was in the range from 41.2 mg trolox equivalents (te)/g dw (methanol extract of leaf) to 4.5 mg te/g dw (ethanol extract of flower). ira is based on reduction of ferricyanide ions to ferrocyanide in the presence of reductants (antioxidants) in the samples by donating an electron. the ferrocyanide ions then react with fe 3+ ions forming prussian blue which strongly absorbs on 700 nm. increasing absorbance at 700 nm indicates an increase in antioxidant ability of a sample. leaf extracts of both water and methanol showed the highest values for reduction capacity (35.2 and 34.2 mg gae/g dw, respectively). acetone and isopropanol extracts of leaf and flower showed the lowest antioxidant capacity and there is no significant difference between the values according to anova. another antioxidant assay used was cuprac. it is based on reduction of cu 2+ ion to cu + ion which reacts with neocuproine giving the complex with absorption maximum at 450 nm. the biologica nyssana 5 (1)  september 2014: 47-51 stojković, m. et al.  antioxidant potential of tanacetum vulgare l. … 51 best antioxidant capacity showed methanol extract of leaf (214 mg te/g dw) while isopropanol extracts of both leaf and flower had the lowest capacity (34 and 33 mg te/g dw, respectively). correlation coefficient between total phenols, total flavonoids and different in vitro antioxidant assays are calculated and showed in the tab. 2. the high values for the coefficients between tp and various in vitro tests suggest that phenolic compounds are the main carriers of antioxidant power in the plant tissues. also, the high values for the coefficients among various in vitro tests proposed that the experimental errors were minor. conclusion methanol extracts showed the best antioxidant potential among the other solvents used (ethanol, water, acetone and isopropanol). the best extraction medium considering only phenols and flavonoids was also methanol (62.7 mg galic acid equivalents per gram of dry weight were found for leaves extract). methanol is an extremely toxic solvent and it is not applicable for human usage, especially methanol extracts. on the other hand, water extracts are suitable for human intake and. obtained results suggest that antioxidant potential of water extracts were as high as the appropriate methanol ones. in some cases water extracts showed higher antioxidant potential. it can be concluded that the water extraction is a good way to obtain phenolic compounds from t. vulgare. comparing amounts of phenolic compounds found in the extracts and its antioxidant potential with other herbal teas and extracts, it may be concluded that t. vulgare is a plant species destitute with phenolic compounds. obtained results suggest that phenolic compounds, presents in the plant tissues, are carriers of antioxidant properties. acknowledgements. this work was supported by the ministry of education and science of serbia (project no 172061 and project no 31060). the authors are grateful for the financial support provided by this ministry. references baranauskiene, r., kazernaviciute, r., pukalskiene, m., mazdzieriene, r., venskutonis, p., 2014: agrorefinery of tanacetum vulgare l. into valuable products andevaluation of their antioxidant properties and phytochemicalcomposition. industrial crops and products, 60: 113–122. ghasemzadeh, a., ghasemzadeh, n., 2011: flavonoids and phenolic acids: role and biochemical activity in plants and human. journal of medicinal plants research, 5: 6697– 6708. kojić, m., stamenković, v., jovanović, d. 1998: lekovite biljke jugoistočne srbije. zavod za udžbenike i nastavna sredstva, beograd. lahlou, s., tangi, k., lyoussi, b., morel, n., 2008: vascular effects of tanacetum vulgare l. leaf extract: in vitro pharmacological study. journal of ethnopharmacology, 120: 98–102. mitic, s., stojkovic, m., pavlovic, j., mitic, m., stojanovic, b., 2012: antioxidant activity, phenolic and mineral content of stachys germanica l. (lamiaceae). oxidation communications, 35, (4): 1011–1020. stojkovic, n., stojkovic, m., marinkovic, m., chopra, g., kostic, d., zarubica, a., 2014: polyphenol content and antioxidant activity of anthemis cretica l. (asteraceae). oxidation communications, 37, (1): 237–246. veljković, j,. pavlović, a., mitić, s., tošić, s., stojanović, g., kaličanin, b., stanković, d., stojković, m., mitić, m., brcanović, j., 2013: evaluation of individual phenolic compounds and antioxidant properties of black, green, herbal and fruit tea infusions consumed in serbia: spectrophotometrical and electrochemical approaches. journal of food and nutrition research, 52: 12–24. biologica nyssana 5 (1)  september 2014: 47-51 stojković, m. et al.  antioxidant potential of tanacetum vulgare l. … 52 a note on scavenging behaviour of adult hermann’s tortoise biologica nyssana 7 (1)  september 2016: 53-55 nikolić, m. et al.  a note on scavenging behaviour of adult hermann's tortoise... 53 short communication received: 26 december 2015 revised: 15 february 2016 accepted: 01 june 2016 a note on scavenging behaviour of adult hermann’s tortoise (testudo hermanni) marko nikolić1,2*, dimitrija savić1,2, maja ilić1,2, dragana stojadinović1, jelka crnobrnjaisailović1,3 1faculty of sciences and mathematics, university of niš, višegradska 33, 18000 niš, serbia 2biological society “dr. sava petrović”, visegradska 33, 18000 niš, serbia 3institute for biological research “siniša stanković”, university of belgrade, despota stefana 142, 11000 belgrade, serbia * e-mail: zerocool.axl@gmail.com abstract: nikolić, m., savić, d., ilić, m., stojadinović, d., crnobrnja-isailović, j.: a note on scavenging behaviour of adult hermann’s tortoise (testudo hermanni). biologica nyssana, 7 (1), september 2016: 53-55. report of the first observation of scavenging behaviour in the population of testudo hermanni boettgeri that has been monitored for six years in the village kunovica near the city of niš in serbia. on 31 may 2015 at 10:18 a.m., the adult tortoise was observed while eating a dead european green lizard (lacerta viridis). key words: testudo hermanni, diet, scavenging behavior, serbia apstrakt: nikolić, m., savić, d., ilić, m., stojadinović, d., crnobrnja-isailović, j.: beleška o strvinarskom ponašanju kod adulta šumske kornjače (testudo hermanni). biologica nyssana, 7 (1), septembar 2016: 5355. beleška o prvom zapažanju strvinarskog ponašanja u populaciji šumske kornjače testudo hermanni boettgeri, čiji monitoring se sprovodi već šest godina u selu kunovica, u blizini grada niša u srbiji. adultna kornjača je uočena prilikom hranjenja lešinom zelembaća (lacerta viridis) 31. maja 2015. u 10:18. key words: testudo hermanni, ishrana, strvinarsko ponašanje, srbija introduction testudinidae is the family of terrestrial chelonians (i.e. tortoises) which are, apart from a few species of lizards, the only terrestrial ectothermic vertebrates with generalized herbivorous or omnivorous feeding habits (d e l v e c c h i o et al., 2011). l u i s e l l i (2006) reviewed general dietary habits of 50 species from the family testudinidae and 15 species from the families geoemydidae and emydidae. of those, about 66% of terrestrial chelonians were exclusively herbivorous, 33% were omnivorous, and only one species (terrapene carolina) was predominantly carnivorous. herbivory in tortoises is not obligatory, as many species also feed on different food that 7 (1) • september 2016: 53-55 doi: 10.5281/zenodo.159104 biologica nyssana 7 (1)  september 2016: 53-55 nikolić, m. et al.  a note on scavenging behaviour of adult hermann's tortoise... 54 includes mushrooms, soil, sand, pebbles, and animal matter (m o s k o v i t s & b j o r n d a l , 1990; c e l s e et al., 2014). testudo hermanni is a southern european species (b e r t o l e r o et al., 2011). several studies about its diet, done in croatia, italy, france and spain, revealed the total of 134 plant species (46 families) on the menu, where the most frequently used plants were from the families asteraceae and fabaceae, and a bit less used species were from the families ranunculaceae and poaceae (for more detailed list see in v e t t e r , 2006). according to data from corsica, the hermann's tortoise is almost exclusively vegetarian (97.4% plants in 997 feedings observed in the wild), and its highly diverse food spectrum includes at least 250 species (c e l s e et al., 2014). although hermann’s tortoises avoid ligneous, aromatic, resinous, milky or hairy plant species, it has been shown that they consume some plants that are toxic to other animal species, such as tammus communis, arum sp. and ranunculus sp. (v e t t e r , 2006; b e r t o l e r o et al., 2011), perhaps to neutralize intestinal parasites (l o n g e p i e r r e & g r e n o t , 1999). in addition to various vascular plants, hermann’s tortoises occasionally feed on mushrooms, colonies of cyanobacteria (nostoc sp.), different species of invertebrates, and feces from various mammal species (human, dog, rabbit, goat, and pig) which seem to be appreciated for the hair and bone fragments or moisture that they contain, as well as on carrion (v e t t e r , 2006). heterospecific coprophagy was also reported elsewhere (v e t t e r , 2006). the ingestion of soil and stones can be a common albeit rarely observed behavior of desert tortoises (i.e. gopherus sp.). the deficiency of a mineral other than calcium or a ratio of minerals may be responsible for the ingestion of bones, stones, and soils by tortoises (e s q u e & p e t e r s , 1994). also, it is known that hermann’s tortoises occasionally ingest soil (geophagy) to acquire minerals (đ o r đ e v i ć & g o l u b o v i ć , 2014). here we report on the first observation of scavenging behaviour in the population of testudo hermanni boettgeri that has been monitored for six years in the village kunovica near the city of niš, serbia (43° 18’ 00’’ n; 22° 05’ 29’’ e). feeding behavior was previously observed in 27 of 1398 records collected during the fieldwork from 2010 to 2015. in all those 27 cases tortoises were feeding on plants. however, on 31 may 2015, at 10:18 a.m., the adult tortoise was observed while eating the remains of a dead european green lizard (lacerta viridis) (fig. 1). kunovica is a typical highland village area, situated at 621 m a.s.l., with the surface of 13.41 km2 and agricultural part covering about 4.44 km2. it is characterized by diverse vegetation and habitat composition, as about 58% of the land is covered with forests, 19% with fields and gardens, pastures constitute 6%, and there are also abandoned and active vineyards, and orchards (t u r n š e k , 2006). all these facts suggest optimal plant resources for local hermann’s tortoises. fig. 1. the adult hermann’s tortoise (testudo hermanni boettgeri) eating the remains of dead european green lizard (lacerta viridis) biologica nyssana 7 (1)  september 2016: 53-55 nikolić, m. et al.  a note on scavenging behaviour of adult hermann's tortoise... 55 b u d ó et al. (2009) recorded scavenging behaviour in t. h. hermanni in albera, (catalonia, spain) (one tortoise was observed while eating the remains of a dead bird). to our knowledge, this kind of feeding behaviour was reported in c e l s e et al. (2014) as sporadically detected in tortoise populations in serbia, but no details were provided. on the basis of relations between habitat preferences and dietary habits in tortoises, their sporadical carnivorous diet is considered as relic (n a t c h e v et al., 2015, but see j a c k s o n et al., 1999). therefore, further investigation about variation in its feeding habits is necessary for building more comprehensive knowledge on hermann’s tortoise ecological requirements. acknowledgements. this study was supported by grant no173025 ministry of education, science and technological development of republic of serbia. references bertolero, a., cheylan, m., hailey, a., livoreil, b., & willemsen, r.e. 2011: testudo hermanni (gmelin 1789): hermann's tortoise. in: rhodin, a.g.j., pritchard, p.c.h., van dijk, p.p., saumure, r.a., buhlmann, k.a., iverson, j.b., mittermeier, r.a. (eds.), conservation biology of freshwater turtles and tortoises: a compilation project of the iucn/ssc tortoise and freshwater turtle specialist group. chelonian research monographs, 5: 059.1–059.20. budó, j., capalleras, x., fèlix, j., font, j. 2009: aportacions sobrel’estudi de l’alimentació de la tortuga mediterrània (testudo hermanni hermanni) (gmelin, 1789) a la serra de l’albera (catalunya). butlletí de la societat catalana d’herpetologia, 18: 109–115. celse, j., catard, a., caron, s., ballouard, j.-m., gagno, s., jardé, n., cheylan, m., astruc, g., croquet, v., bosc, m., petenian, f. 2014: management guide of populations and habitats of the hermann's tortoise. life, 8. del vecchio, s., burke, r.l., rugiero, l., capula, m., luiselli, l. 2011: seasonal changes in the diet of testudo hermanni hermanni in central italy. herpetologica, 67: 236–249. đorđević, s., golubović, a. 2013: geophagy in the hermann’s tortoise, testudo hermanni. photo note hyla herpetological bulletin, 2013 (1): 4647. esque, t. c., peters, e. l. 1994: ingestion of bones, stones, and soil by desert tortoises. fish and wildlife research, 13: 73-84. jackson, d. r., ostertag, t. e. 1999: gopherus polyphemus (gopher tortoise). scavenging. herpetological review, 30 (1): 40. longepierre, s., grenot, c. 1999: some effects of intestinal nematodes on plant foraging behaviour of testudo hermanni hermanni in the south of france. in: miaud, c., guyetant, g. (eds.), current studies in herpetology, 277-284, le bourget du lac (seh). luiselli, l. 2006: resource partitioning in the communities of terrestrial turtles: a review of the evidences. revue d ecologie la terre et la vie, 61: 353–365. moskovits, d.k., bjorndal, k.a. 1990: diet and food preferences of the tortoises geochelone carbonaria and g. denticulata in northwestern brazil. herpetologica, 46(2): 207–218. natchev, n., tzankov, n., werneburg, i., heiss, e. 2015: feeding behavior in a “basal” tortoise provides insights on the transitional feeding mode at the dawn of modern land turtle evolution. peerj, 3: e1172. turnšek, b.a. 2006: the village of kunovica in the sustainable development context. facta universitatis – series: architecture and civil engineering, 4 (1): 25–39. vetter, h. 2006: chelonian library 2. hermann's tortoise. testudo hermanni, t. boettgeri and t. hercegovinensis. – frankfurt/main (edition chimaira), 325 pp. petrović, g., stamenković, j., stojanović, g., zlatković, b., jovanović, o.: essential oil analysis of different plant parts of geocaryum cynapioides (guss.) l. engstrand. biologica nyssana, 9 (1). september, 2018: 31-35. biologica nyssana 9 (1) ⚫ september 2018: 31-35 petrović et al. ⚫ essential oil analysis of different plant parts of geocaryum… 31 original article received: 29 january 2018 revised: 29 mart 2018 accepted: 27 june 2018 essential oil analysis of different plant parts of geocaryum cynapioides (guss.) l. engstrand goran petrović1, jelena stamenković1, gordana stojanović1, bojan zlatković2, olga jovanović1 1university of niš, faculty of science and mathematics, department of chemistry, višegradska 33, 18000 niš, serbia 2university of niš, faculty of science and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia * e-mail: jelena.stamenkovic@pmf.edu.rs abstract: petrović, g., stamenković, j., stojanović, g., zlatković, b., jovanović, o.: essential oil analysis of different plant parts of geocaryum cynapioides (guss.) l. engstrand. biologica nyssana, 9 (1). september, 2018: 31-35. phytochemical analysis by gc and gc/ms of the essential oil samples, obtained from fresh aerial parts, shoots and inflorescences of geocaryum cynapioides (guss.) l. engstrand, allowed the identification of 55 components in total, comprising 99.5%, 99.7% and 99.0% of the oils compositions, respectively. regarding the aerial parts essential oil, the major of 52 identified volatile compounds were (e)-β-farnesene (66.6%), 7epi-cis-sesquisabinene hydrate (17.4%) and (e,e)-α-farnesene (3.7%). the same compounds were dominant among the 48 components detected in the shoots and 42 volatiles in inflorescences oil, but in different proportions. hydrocarbon sesquiterpenes had the highest contribution in all investigated geocaryum cynapioides essential oils, with a share of about three quarters of all, followed by one quarter of oxygenated sesquiterpenes, while the monoterpenoid and nonterpenoid compounds were detected only in trace amounts. key words: geocaryum cynapioides, essential oil, phytochemical analysis, gc/ms apstrakt: petrović, g., stamenković, j., stojanović, g., zlatković, b., jovanović, o.: analiza etarskih ulja iz različitih biljnih delova vrste geocaryum cynapioides (guss.) l. engstrand. biologica nyssana, 9 (1). septembar, 2018: 31-35. fitohemijskom analizom uzoraka etarskih ulja izolovanih iz svežeg biljnog materijala različitih delova (nadzemnog dela, stabljike i cvasti) biljne vrste geocaryum cynapioides (guss.) l. engstrand, pomoću gc i gc/ms metode, identifikovano je ukupno 55 komponenti, što predstavlja 99,5% nadzemnog dela, 99,7% stabljike i 99,0% cvasti. u etarskom ulju izolovanom iz svežeg nadzemnog dela biljke identifikovane su ukupno 52 komponente među kojima su (e)-β-farnezen (66,6%), 7-epi-cis-seskvisabinen hidrat (17,4%) i (e,e)-α-farnezen (3,7%) bile najzastupljenije. iste te komponente su bile dominantne i u etarskim uljima 9 (1) • september 2018: 31-35 doi: 10.5281/zenodo.1470846 biologica nyssana 9 (1) ⚫ september 2018: 31-35 petrović et al. ⚫ essential oil analysis of different plant parts of geocaryum… 32 izolovanim iz stabljike i cvasti ali u različitim procentnim udelima. u svim ispitivanim uzorcima tri četvrtine ukupnog sastava etarskog ulja činili su ugljovodonični seskviterpeni, zatim oksigenovani seskviterpeni koji su činili jednu četvrtinu, dok su monoterpenoidi i neterpenoidne komponente detektovani samo u tragovima. ključne reči: geocaryum cynapioides, etarsko ulje, fitohemijska analiza, gc/ms introduction the genus geocaryum l. (apiaceae) is small genus, comprised of only 8 accepted species (the plant list, 2012). it belongs to the tribe scandiceae, subtribe scandicinae (spalik & downie, 2001) with 11 another larger genera like chaerophyllum, anthriscus, osmorhiza etc. geocaryum cynapioides (guss.) l. engstrand is endemic species which inhabits pastures and meadows of the high mountains in southern parts of the balkan peninsula and central and southern parts of italy (ball, 1968). it is a perennial herb, 45 cm tall, with flexuous or curved internodes and undivided uppermost cauline leaves, linear or filiform lamina up to 20 mm, flowering from may to july. according to the plant list database, g. cynapioides has as many as 18 synonymous names classifying it in other genera such as bunium, carum or even chaerophyllum. downie (downie et al., 2010) finally hemotaxonomically defined its position within the tribe scandiceae on the basis of his investigation of nuclear ribosomal dna. but, it is very unusual that there are no any previous data about the chemical composition of essential oils, as well as about the extracts of species belonging to this genus, and their biological activities. as far as we know, there is only one report on chemical composition of g. cynapioides essential oil (radulović et al., 2008) which relates to the plant as a whole, including underground tubers, for which it is literally known that they might be completely different in chemical composition. the aim of this study was to perform a detailed phytochemical analysis of geocaryum cynapioides (guss.) l. engstrand essential oils obtained from different plant parts. material and methods plant material the plant material (flowering stage) was collected at vlasina lake plateau, serbia, in june 2017 and was identified by one of the authors bojan zlatković. the voucher specimen was deposited in the herbarium moesiacum niš (hmn), department of biology and ecology, faculty of science and mathematics, university of niš under the acquisition number 13317. sample preparation the essential oils samples were prepared by hydrodistillation of fresh chopped whole aerial plant parts (194 g), shoots (158 g) and inflorescences (54 g), for 2.5 hours using clevenger type apparatus, according to the method recommended in british pharmacopoeia (british pharmacopoeia, 1988). the essential oils were extracted with n-hexane, dried over anhydrous sodium sulfate and evaporated. the yields of the oils were 0.06%, 0.05% and 0.08%, based on the weight of fresh plant, respectively. samples were re-dissolved in n-hexane to obtain the desired optimal concentrations and stored at -20 °c in the dark until analyzed. hexane analytical reagent grade, fisher chemical, uk; na2so4 pro analysis grade, sigma-aldrich, germany. gc and gc/ms analysis the sample of essential oil (20 mg/ml) was analyzed by a 7890/7000b gc/ms/ms triple quadrupole system in ms1 scan mode (agilent technologies, usa) equipped with a combi pal sampler. the fused silica capillary column hp-5 ms (5% phenylmethylsiloxane, 30 m x 0.25 mm, film thickness 0.25 μm) was used. the injector and interface operated at 250 and 300 °c, respectively. temperature program: from 50 to 290 °c at a heating rate of 4 °c min-1. the carrier gas was helium with a flow of 1.0 ml min-1. one microliter of the oil solution in hexane was injected (1:100, split ratio 40:1). post run: back flash for 1.89 minutes, at 280 °c, with helium pressure of 50 psi. ms conditions were as follows: ionization voltage of 70 ev, acquisition mass range 40-440, scan time 0.32 seconds. gc-fid analysis was carried out under the same experimental conditions using the same column as described for the gc/ms. the percentage composition of the sample was computed as an average of the gc peak areas obtained in triplicate, without any corrections. identification of volatile compounds eo constituents were identified by comparison of their linear retention indices relative to c8-c32 alkanes on the hp-5 ms column (van den dool & kratz, 1963), with literature values and their ms with biologica nyssana 9 (1) ⚫ september 2018: 31-35 petrović et al. ⚫ essential oil analysis of different plant parts of geocaryum… 33 table 1. chemical compositions of the g. cynapioides essential oils obtained by gc/ms relative amount % riexp riref compound so io ao class 998 988 myrcene tr tr tr m 1034 1024 limonene tr tr tr m 1042 1032 (z)-β-ocimene 1.5 0.2 1.0 m 1052 1044 (e)-β-ocimene 0.6 0.1 0.4 m 1083 1088* 1-nonen-3-ol 0.1 tr o 1091 1086 terpinolene 0.2 0.1 m 1101 1095 linalool tr tr mo 1100 1100 undecane tr o 1106 1100 n-nonanal tr 0.1 tr o 1147 1144 2(z)-nonen-1-al 0.1 tr o 1161 1157 2(e)-nonen-1-al tr tr o 1287 1283 isobornyl acetate tr tr mo 1291 1288 lavandulyl acetate tr tr mo 1299 1300 tridecane tr tr o 1278 1374 α-copaene tr tr tr s 1383 1379 geranyl acetate 0.1 tr mo 1388 1387 β-bourbonene 0.1 tr s 1394 1389 β-elemene 0.1 tr s 1406 1405 sesquithujene 0.1 0.1 tr s 1416 1410 α-cedrene tr tr tr s 1423 1417 (e)-caryophyllene 0.8 1.6 1.0 s 1429 1429* himachala-2,4-diene 0.2 0.1 0.1 s 1432 1430 β-copaene tr . tr s 1437 1432 α-trans-bergamotene tr tr tr s 1444 1440 (z)-β-farnesene tr tr tr s 1461 1454 (e)-β-farnesene 61.0 72.0 66.6 s 1481 1481 γ-curcumene 0.1 0.1 s 1485 1484 germacrene d 2.7 3.0 2.8 s 1494 1491* (z,e)-α-farnesene 1.8 1.2 1.5 s 1500 1500 bicyclogermacrene 0.6 0.2 0.4 s 1503 1506 (z)-α-bisabolene 0.1 tr 0.1 s 1508 1505 (e,e)-α-farnesene 4.4 2.6 3.7 s 1513 1514 β-curcumene 0.5 0.2 0.4 s 1525 1521 β-sesquiphellandrene tr tr tr s 1525 1522 δ-cadinene 0.2 tr 0.1 s 1533 1529 (e)-γ-bisabolene tr tr tr s 1555 1542 cis-sesquisabinene hydrate 2.1 1.4 1.7 so 1564 1561 (e)-nerolidol 0.4 0.3 0.3 so 1579 1574 germacrene d-4-ol 0.1 tr so 1583 1577 trans-sesquisabinene hydrate 0.4 0.1 0.1 so 1593 1586* 7-epi-cis-sesquisabinene hydrate 19.3 13.1 17.4 so 1611 1611 tetradecanal tr tr o 1634 1632 α-acorenol 0.6 0.3 0.4 so 1651 1636 β-acorenol 0.2 0.1 0.1 so 1672 1670 epi-β-bisabolol 0.7 0.4 0.5 so 1685 1683 epi-α-bisabolol 0.2 0.1 so 1698 1699 β-sinensal tr tr tr so 1900 1900 nonadecane 0.1 tr o 2000 2000 eicosane tr tr o 2100 2100 heneicosane 0.1 tr o 2200 2200 docosane tr o biologica nyssana 9 (1) ⚫ september 2018: 31-35 petrović et al. ⚫ essential oil analysis of different plant parts of geocaryum… 34 those of authentic standards, as well as those from adams (adams, 2007), wiley 6, nist11, agilent mass hunter workstation b.06.00 software and a homemade ms library with the spectra corresponding to pure substances and components of known eos by the application of the amdis software (automated mass spectral deconvolution and identification system, ver. 2.1, dtra/nist, 2011). results and discussion chemical compositions of the aerial plant parts, shoots and inflorescences essential oils of geocaryum cynapioides, obtained by gc and gc/ms analyses, are presented in tab. 1. number of identified compounds in aerial parts (ao), inflorescences (io) and shoots (so) samples were 52, 42 and 48 respectively, representing 99.5%, 99.0% and 99.7% of total volatiles. the composition of the essential oils of the shoots and the oil obtained from inflorescences was found to be quite comparable. hydrocarbon sesquiterpenes had the highest contribution in all investigated g. cynapioides essential oils, with a share of about three quarters of all, followed by one quarter of oxygenated sesquiterpenes. the shoot essential oil had a little bit lower content of hydrocarbon sesquiterpenes compared to the inflorescences oil, 72.6% and 81.1%, respectively. in both essential oils this class of compounds predominated over the oxygenated sesquiterpenes. a reverse distribution was observed for the oxygenated sesquiterpenes which are prevalent in shoots essential oil over the inflorescences oil (24.0% vs. 15.7%, respectively). hydrocarbon monoterpenes were present in negligible amounts in both oils, while the oxygenated monterpenes were not detected at all. obtained results indicate that the composition of the g. cynapioides aerial parts essential oil represents the average value of the contents of essential oils derived from inflorescences and shoots essential oils. phytochemical analysis by gc and gc/ms of the essential oil samples, obtained from fresh aerial parts, shoots and inflorescences of g. cynapioides, allowed the identification of 55 components in total. regarding the aerial parts essential oil, the major of 52 identified volatile compounds were (e)-βfamesene (66.6%), 7-epi-cis-sesquisabinene hydrate (17.4%) and (e,e)-α-farnesene (3.7%). the same compounds were dominant among the 48 components detected in the shoots and 42 volatiles in inflorescences oil, but in different proportions. as we already shown, chemical composition of inflorescences could be pretty similar (kostevski et al., 2016) or essentially various (petrović et al., 2018; stamenković et al., 2015) in comparison to the composition of the shoots essential oil. on the other hand, the composition of the volatile components obtained from all the above-ground parts of the plant and root is always completely different (petrović et al., 2017; stamenković et al. 2015). compared with the previously published results of g. cynapioides essential oil (radulović et al., 2008), certain differences in chemical compositions of aerial parts essential oils can be observed. that can be explained by the fact that sample a consisted of fresh aerial parts (inflorescences, stems and leaves) and underground tubers, while sample b was composed of stems, leaves and umbels full of ripe fruit (fruiting stage). sample b differs significantly both by the number of identified components and by the relative content of compound classes, especially in relation to monoterpenoids content (hydrocarbon monoterpenes relative amount % riexp riref compound so io ao class 2300 2300 tricosane 0.1 0.6 0.2 o 2500 2500 pentacosane tr 0.4 0.1 o 2700 2700 heptacosane 0.1 0.2 0.1 o 2900 2900 nonacosane 0.3 0.4 0.2 o total 99.7 99.0 99.5 monoterpene hydrocarbons m 2.3 0.3 1.5 monoterpene oxygenated mo 0.1 sesquiterpene hydrocarbons s 72.6 81.1 76.8 sesquiterpene oxygenated so 24.0 15.7 20.6 others o 0.7 1.9 0.6 compounds are listed in order of elution from a hp-5 ms column; riref: literature retention indices; riexp: experimental retention indices relative to c8-c32 n-alkanes; (*): identified by nist chemistry webbook retention indices; tr: traces (<0.1%); (-): not detected. essential oils: so shoots; io inflorescences; ao aerial plant parts. biologica nyssana 9 (1) ⚫ september 2018: 31-35 petrović et al. ⚫ essential oil analysis of different plant parts of geocaryum… 35 6.5% and oxygenated monterpenes 4.9%). however, the main difference that can be noticed is that one of the main components identified in our investigation, 7-epi-cis-sesquisabinene hydrate, was not found at all. the probable reason is the similarity of the mass spectra and the proximity of the retention indices of this compound and trans-sesquisabinene hydrate, which was found in a significant amount, which led to a misidentification. according to the results of this study, the essential oil of g. cynapioides consisted mainly of sesquiterpene compounds, implying that there are great differences in composition of investigated essential oil compared to other reports about the scandicinae species` essential oils. considering the fact that geocaryum genus is not investigated at all, more studies are needed regarding essential oil constituents of its species. obtained data could be helpful in future research to clarify the chemotaxonomic relationships between the genera within the tribe scandiceae and subtribe scandicinae. acknowledgements. the authors are grateful to the ministry of education, science and technological development for financial support through the grant within frame of basic research, no 172047. references adams, r. p. 2007: identification of essential oil components by gas chromatography and mass spectrometry (4th ed.). carol stream: allured publishing corporation. ball, p. w. 1968: huetia p. w. boiss. in: t. g. tutin, v. h. heywood, n. a. burges, d. m. moore, d. h., valentine, s. m. walters, d. a. webb (eds.), flora europaea 2 (pp. 330). cambridge: cambridge university press. downie, s. r., katz-downie, d. s., spalik, k. 2000: a phylogeny of apiaceae tribe scandiceae: evidence from nuclear ribosomal dna internal transcribed spacer sequences. american journal of botany, 87: 76-95. kostevski, i., petrović, g., stojanović, g., stamenković, j., zlatković, b. 2016: chemical composition of achillea coarctata essential oil and headspace volatiles. natural product communications, 11 (4): 543-545. petrović, g., ilić, m., stankov-jovanović, v., stojanović, g., jovanović, s. 2018: phytochemical analysis of saponaria officinalis l. shoots and flowers essential oils. natural product research, 32 (3): 331-334. petrović, g., stamenković, j., stojanović, g., mitić, v., zlatković, b. 2017: chemical profile of essential oils and headspace volatiles of chaerophyllum hirsutum from serbia. natural product communications, 12 (9): 1513-1515. radulović, n., đorđević, n., zlatković, b., palić, r. 2008: composition of the essential oil of geocaryum cynapioides (guss.) l. engstrand. chemical papers, 62 (6): 603-607. spalik, k. & downie, s. 2001: the utility of morphological characters for inferring phylogeny in scandiceae subtribe scandicinae (apiaceae). annals of the missouri botanical garden, 88 (2): 270-301. stamenković, j., stojanović, g., radojković, i., petrović, g., zlatković, b. 2015: chemical composition of the essential oil from chaerophyllum temulum (apiaceae). natural product communications, 10 (8): 439-441. the plant list [internet]. royal botanic gardens, kew and missouri botanical garden; [version 1.1, september 2013]. available from: http:// http://www.theplantlist.org/ van den dool, h. & kratz, p. d. 1963: a generalization of the retention index system including linear temperature programmed gasliquid partition chromatography. journal of chromatography, 11, 463-471. anticancer compounds from medicinal plants biologica nyssana 5 (1)  september 2014: 37-46 joković, n. et al.  screening of lactic acid bacteria isolated … 37 original article received: 18 june 2014 revised: 25 august 2014 accepted: 10 september 2014 screening of lactic acid bacteria isolated from serbian kajmak for use in starter cultures nataša joković 1 *, jelena rajković 1 , katarina veljović 2 , maja tolinački 2 , ljubiša topisirović 2 1 department of biology and ecology, faculty of science and mathematics, university of nis, visegradska 33, 18000 nis, serbia. 2 institute of molecular genetics and genetic engineering, university of belgrade, vojvode stepe 444a, p.o. box 23, 11010 belgrade, serbia. * e-mail: jole35@open.telekom.rs abstract: joković, n., rajković, j., veljović, k., tolinački, m., topisirović, lj.: screening of lactic acid bacteria isolated from serbian kajmak for use in starter cultures. biologica nyssana, 5 (1), septmeber 2014: 37-46. one hundred and seventy eight isolates of lactic acid bacteria (lab) were isolated by pour plate and enrichment techniques from a sample of milk used for kajmak production and three samples of kajmak from one month ripening period. the identification of isolates was performed by phenotypic characterization followed by molecular identification using (gtg)5-pcr and sequence analysis of 16s rrna gene. isolates belonged to lactococcus lactis and enterococcus faecium were found in milk and kajmak samples while leuconostoc mesenteroides and enterococcus durans were the most frequently isolated species from kajmak samples. streptococcus thermophilus were isolated from milk sample only with enrichment technique. further characterization of lab isolates was done for technological properties which are important for industrial application of lab. strains of lc. lactis and s. thermophilus that showed very good acidification and proteolityc activities and l. mesenteroides strains that metabolized citrate can be used in development of starter cultures for eventual industrial production of kajmak. additionally, producers of antimicrobial compounds belonged to lc. lactis subsp. lactis biovar. diacetylactis can be used for control of undesirable microflora in kajmak production. key words: kajmak, lactic acid bacteria, technological characterization apstract: joković, n., rajković, j., veljović, k., tolinački, m., topisirović, lj.: ispitivanje bakterija mlečne kiseline izolovanih iz srpskog kajmaka za upotrebu u starter kulturama. biologica nyssana, 5 (1), septmeber 2014: 37-46. sto i sedamdeset osam izolata bakterija mlečne kiseline (bmk) izolovano je metodom izlivanja ploča i tehnikom obogaćivanja iz uzorka mleka korišćenog za proizvodnju kajmaka i tri uzorka kajmaka starosti do jednog meseca. preliminarna identifikacija izolata rađena je fenotipskim metodama dok su molekulrne metode identifikacije, pcr sa (gtg)5 prajmrom i sekvenciranje 16s rrnk gena, korišćene za identifikaciju izolata do nivoa vrste. izolati identifikovani kao lactococcus lactis i enterococcus faecium izolovani su iz uzoraka mleka i kajmaka dok su vrste leuconostoc mesenteroides i enterococcus durans najčešće izolovane vrste iz uzoraka kajmaka. streptococcus thermophilus izolati dobijeni su iz uzorka mleka samo tehnikom 5 (1) • september 2014: 37-46 biologica nyssana 5 (1)  september 2014: 37-46 joković, n. et al.  screening of lactic acid bacteria isolated … 38 obogaćivanja. dalja karakterizacija bmk izolata rađena je na osnovu tehnoloških svojstava koja su značajna za industrijsku primenu bmk. sojevi lc. lactis i s. thermophilus koji su imali veoma dobru sposobnost acidifikacije i proteolitičku aktivnost kao i l. mesenteroides sojevi sa sposobnošću metabolizma citrata mogu se upotrebiti za razvoj starter kultura za industrijsku proizvodnju kajmaka. osim toga, izolati identifikovani kao lc. lactis subsp. lactis biovar. diacetilactis koji sintetišu antimikrobna jedinjenja mogu se koristiti za kontrolu nepoželjne mikroflore u proizvodnji kajmaka. ključne reči: kajmak, bakterije mlečne kiseline, tehnološka karakterizacija izolata introduction kajmak is a delicious artisanal dairy product of serbia and neighboring balkan countries. it is made by fermentation of milk fat that rises on the top of cooked milk after slowly cooling. thin layer of aggregated milk fats and proteins is then salted and placed layer by layer in special wooden vessels where ripening takes place. kajmak can be consumed as a fresh up to 7 days of fermentation, or mature kajmak that can be aged for several months. in the most of the dairy products, lactic acid bacteria (lab) are the dominant microbial population which metabolic products play crucial role in formation of the taste, smell, texture and quality of final product (w o u t e r s et al., 2002). in a previous work, we have shown that the most ubiquitous lab isolated from kajmak samples belong to genera leuconostoc, enterococcus, lactobacillus and lactococcus (j o k o v i ć et al., 2008). in serbia, kajmak is made at farmhouse level in central and west part of the country and the sale is mainly limited to the open markets. production of kajmak is always based on non-standardized traditional techniques without addition of starter cultures. therefore, the fermentation process depends entirely on the natural microbial flora that originates from the milk and from the environment. consequently, final products are sensitive to contamination and spoilage and have variable quality, poor hygiene and doubtful safety. on the other hand, some attempts for industrial production of kajmak did not succeed because sensory characteristics of final products were not the same as in original products from households (p u d j a e t a l . , 2008). in modern dairy industry, the main approach to obtain traditional dairy products with improved and reproducible qualities is usage of defined commercial starter cultures made with strains isolated from original products (c h a m m a s et al., 2006). these strains are best adapted on food substrate and their metabolic activities contribute to evolution of typical and unique organoleptic characteristics of traditional products. the searching for strains that can be used in construction of starter cultures includes screening of a large number of isolates selected from naturally occurring processes in term of their technological capabilities that are relevant for obtaining more quality product (a s t e r i et al., 2009). the aim of the present study was screening of lab isolates that were predominant in the early phases of kajmak ripening. for that purpose, isolation and identification of lab from the milk used for manufacturing of kajmak and samples of resulting kajmaks in the first period of ripening were performed. additionally, the technological properties of obtained isolates were characterized. this information could make possible selection of strains that could be eventually used in development of starter cultures for the industrial production of kajmak. material and methods bacterial strains, media, and growth conditions bacterial strains used in this study are presented in table 1. mrs broth (ph 5.7) (merck, gmbh, darmstadt, germany) was used for growth of leuconostoc and lactobacillus strains whereas lactococcus, streptococcus and enterococcus strains were cultured in m17 broth (ph 7.2) (merck, gmbh, darmstadt, germany) supplemented with glucose (0.5%, w/v; gm17 broth). a solid medium was prepared by adding agar (2% w/v; torlak, belgrade, serbia) to each medium. the inoculated media were incubated overnight at appropriate temperatures depending on the strain. manufacture and sampling of kajmak one batch of kajmak was manufactured from cows’ milk in a household in the western part of serbia according to traditional procedures without addition of starter cultures. the household was selected as their kajmak had high quality properties. raw milk was cooked to boiling, with occasional stirring to prevent burnt. then, hot milk was poured into the open wooden shallow vessels that were placed on the shelves in the special room for producing kajmak called “creamery”. the next biologica nyssana 5 (1)  september 2014: 37-46 kamberović, j. et al.  marshland vegetation of the order phragmitetalia … 39 table 1. list of reference strains used bacterial strains lactococcus lactis np45 a  lactococcus lactis ssp. cremoris ns1 a lactococcus lactis subsp. lactis bgmn1-596 a lactococcus lactis subsp. lactis biovar. diacetylactis s50 a enterococcus faecium bggj8-3* enterococcus durans bgzls20-35b* streptococcus thermophilus bgdk1-4a lactobacillus paracasei subsp. paracasei bgbuk2-16/k4 a lactobacillus plantarum a112 a leuconostoc mesenteroides ssp. mesenteroides nrrl b-512 leuconostoc mesenteroides ssp. mesenteroides nrrl b-3470 a -strains used in antimicrobial activity assay *strains identified by molecular methods in the laboratorium voor microbiologie, universitet gent, gent, belgium nrrl – agricultural research service culture collection, peoria, il, usa 12 h milk in vessels was cooling at room temperature with a gradual separation of cream on the surface of the milk. afterwards, the formed layer of fats and proteins was carefully skimmed with perforated spoon, drained and placed in the wooden tub. salt was added between the layers from different vessels. the tub was placed in the creamery where ripening took place for one month. sample of milk immediately after boiling, designated as bgnk1, and the samples of kajmak after 5, 15 and 30 days of ripening (designated as bgnk5, bgnk15 and bgnk30, respectively) were collected for microbiological analyses. the collected samples of milk and kajmak were kept in sterile bags at 4°c until the analyses which were performed within the following 12 h. isolation of lactic acid bacteria (lab) the isolation of lab was performed by pour plate and enrichment techniques. in the pour plate technique, the samples (10 g or 10 ml) were homogenized in 90 ml of sterile 2% (w/v) sodium citrate solution (ph 7.5) preheated to 45°c. decimal dilutions of the homogenates were prepared with 0.85% (w/v) sterile saline and were plated on suitable media for isolation of lab: m17 agar (merck, gmbh, darmstadt, germany) supplemented with glucose (gm17), mrs agar (merck, gmbh, darmstadt, germany) and mayeux, sandine, elliker (mse) agar (mayeux et al., 1962). the plates were incubated at 30°c and 45°c for 3 days. after the incubation period, five single colonies were randomly picked from agar plates and streaked on new agar plates for purification. in the enrichment technique, 0.1 ml of the first dilution was transferred into 10 ml of mrs or gm17 broth as well as into 10 ml of 10% skimmed milk. tubes were incubated at 30 o c and 45 o c for 48 h. after the incubation, loopful of grown culture was streaked on mrs and gm17 agar plates and incubated for additional 48 h at appropriate temperatures and under anaerobic conditions (anaerocult a, gaspakmerck, germany). from each plate, few colonies showing different appearance were randomly picked and streaked on new plates in order to obtain pure colonies. phenotypic identification of isolates gram-positive, catalase-negative isolates were grouped and preliminary identified according to the following phenotypic tests: morphology under microscopic examination; the production of gas from glucose in mrs broth lacking beef extract and containing inverted durham tubes; hydrolysis of arginine; hydrolysis of esculin in esculin broth (torlak, belgrade, serbia); the growth in gm17 or mrs broth with 4% and 6.5% (w/v) nacl for 5 days and the growth in 10% skimmed-milk medium. the growth at different temperatures was determined in mrs broth for bacilli and cocobacilli at 15°c and 45°c or in gm17 broth for cocci at 10°c and 45 for 5 days. bile esculin agar (himedia, mubai, india) was used for the presumptive identification of enterococci. biologica nyssana 5 (1)  september 2014: 37-46 joković, n. et al.  screening of lactic acid bacteria isolated … 40 molecular identification of isolates molecular identification of selected isolates was done by rep-pcr method with (gtg)5-primer and by sequencing of 16s rrna gene. the total dna extraction from pure cultures, pcr amplification with (gtg)5-primer, electrophoresis and statistical similarity among fingerprints of the isolates and the reference strains were performed as previously described (j o k o v i ć et al., 2008). sequencing of the 16s rrna gene was done by using total dna as a template for pcr amplifications with u968 (5΄aacgcgaagaaccttac-3΄) and l1401 (5΄gcgtgtgtacaagaccc-3΄) primers (z o e t e n d a l et al., 1998; r a n d a z z o et al., 2002). the amplified fragments were sequenced by macrogen (sequencing service macrogen, seoul, south korea). the sequences were analysed in the ncbi database using blast, the standard nucleotide-nucleotide homology search (http://www.ncbi.nlm.nih.gov/blast). technological characterization of isolates the acidifying activity of isolates was evaluated by measuring the ph of inoculating 10% (w/v) reconstructed skim milk (rsm). overnight lab cultures grown at appropriate temperatures were centrifuged at 5000 rpm for 10 min, washed with peptone water and inoculated (1% v/v) in 10 ml rsm. ph was measured after 6 and 24 h of incubation at appropriate temperature and values were expressed as ph decrease (ph), in relation to the ph values of non-inoculated control milk. the proteolytic activity of the isolates was detected after growing of isolates for 24 h in rsm as described for acidifying activity. after incubation, the proteins from 0.5 ml of the culture were precipitated with trichloroacetic acid. the proteolytic activity of isolates was determined by the o-pa (o-phthaldialdehyde) method of church and associates (1983). the values were calculated from calibration curve with glycin and expressed in mmglyl -1 . citrate utilization was detected on kempler and mckay agar plates (k e m p l e r & m c k a y , 1981). citrate-positive colonies were blue after 48 h of incubation at appropriate temperature. production of diacetyl in milk was determined after a growing of isolates in rsm for 24 h as described above. qualitative determination of diacetyl was done by addition of 0.5 ml of naphthol (1% w/v) and koh (16% w/v) solution in 1 ml of milk culture. after incubation at 30°c for 10 min results were read. appearance of red ring was considered as positive result (k i n g , 1948). lipolytic activity of lab was identified on tributyrin agar plates (merck, gmbh, darmstadt, germany). the plates were incubated for 7 days at appropriate temperatures and observed daily for clear zones around the colonies. production of exopolysaccharides was detected on reconstructed mrs medium containing 100 g l -1 of sucrose, glucose, lactose, maltose or fructose. the plates were incubated anaerobically for 48 h at appropriate temperature and isolates were tested for slime formation using the inoculated loop method. the lab isolates were screened for antimicrobial activity by the agar-well diffusion method (t a g g & m c g i v e n , 1971). different indicator strains used in this study are listed in table 1. the protein nature of antimicrobial compounds was confirmed by adding a crystal of pronase e (sigma chemie gmbh, deisenhofen, germany) near to the edge of the well containing the strains with a potential production of bacteriocins. the plates were incubated overnight at 30°c. the appearance of a clear zone of inhibition around the well, but not near of pronase e crystal, was considered as positive result for possible bacteriocin production results and discussion identification of isolates sensory properties of dairy products manufactured without addition of starter cultures, as kajmak is, entirely depend on the microflora found in milk and the contaminants from the environment (p o z n a n s k i e t a l ., 2004). in order to determine which part of the lab populations derived directly from the milk, 178 gram-positive and catalase-negative isolates were collected from milk used for kajmak production (bgnk1) and kajmak samples bgnk5, bgnk15 and bgnk30. on the basis of physiological tests that were used for the preliminary identification of isolates to the genus level, all isolates were grouped into eight physiological groups (tab. 2). representatives of various physiological groups, a total of 76, were further selected for molecular identification by (gtg)5-pcr fingerprinting. a dendrogram derived by comparing the similarity of digitally reproduced (gtg)5 fingerprints of isolates and reference strains is presented in fig. 1. biologica nyssana 5 (1)  september 2014: 37-46 kamberović, j. et al.  marshland vegetation of the order phragmitetalia … 41 figure 1. dendrogram based on statistical analysis of the (gtg)5–pcr fingerprints. *indicates the isolates that were subjected to sequencing of 16s rdna the results of physiological tests showed that three groups of coccal isolates can be distinguished (tab. 2). based on a dendrogram derived from (gtg)5-pcr fingerprints these isolates were identified as lactococcus lactis, streptococcus thermophilus, enterococcus faecium and enterococcus durans. the (gtg)5-pcr identification was confirmed by sequencing of 16s rdna for two s. thermophilus, three e. durans and one e. faecium isolates. heterofermentative coccoid isolates showing arginine negative phenotype were supposed to belong to genus leuconostoc (tab. 2). these isolates were identified to species level as leuconostoc mesenteroides (fig. 1) as they had similar (gtg)5-pcr fingerprints with leuconostoc mesenteroides reference strains nrrl b-512 and nrrl b-347. three isolates shaped as very short rods were also heterofermentative with the identical (gtg)5 pcr fingerprints (fig. 1). but these biologica nyssana 5 (1)  september 2014: 37-46 joković, n. et al.  screening of lactic acid bacteria isolated … 42 table 2. differentiation of lab isolated from milk and kajmaks samples based on morphological and physiological characteristics, and the identification of representative strains from each group by (gtg)5-pcr and sequencing of 16s rdna cell morphology and genus1 p ro d u ct io n o f c o 2 growth on growth in broth with nacl m il k cl o tt in g hydrolysis of b s a 2 n o i so la te s identified by 45oc 15 oc 10 oc 4% 6.5% arginine esculin (gtg)5-pcr as sequencing of 16s rdna as cocci lactococcus sp. i nd + + + + + 14 lc. lactis (14)3 nd enterococcus sp. + nd + + + + + + + 53 e. faecium (5) e. durans (14) e. faecium (1) e. durans (3) streptococcus sp. + nd + + 7 s. thermophilus (4) s. thermophilus (2) coccoid leuconostoc sp. i + + nd + + +v + 53 l. mesenteroides (15) nd leuconostoc sp. ii + + nd + +v + 28 l. mesenteroides (13) nd leuconostoc sp. iii + + nd + + +v 13 l. mesenteroides (5) nd leuconostoc sp. iv + + nd + + 7 l. mesenteroides (3) nd rods (very short) weisella sp. + + nd + + + 3 nd (3) w. cibaria (1) + positive; negative; +v reaction positive for some isolates; nd – not determined 1 -classification based on morphological and physiological characteristics 2 -black colonies on bsa (bile esculin agar) 3 -numbers in parenthesis represent the number of isolates that underwent genetic identification 0 10 20 30 40 50 60 70 80 90 100 bgnk1 bgnk5 bgnk15 bgnk30 samples fr e q u e n c y o f is o la ti o n weissella cibaria leuconostoc mesenteroides enterococcus durans enterococcus faecium lactococcus lactis figure 2. distribution of lab species in milk and kajmak samples based on a pour-plate technique isolates were not identified with rep-pcr method because (gtg)5-pcr fingerprints of these isolates were not related to any band pattern of reference strains from our collection. the 16s rdna sequencing result of one representative isolate from this group showed that it belonged to the species weissella cibaria (table 2). the identification of isolates allowed us to describe the distribution of lab in milk (bgnk1) and kajmak samples (bgnk5, bgnk15 and bgnk30). lab species isolated from milk and kajmak samples were different (fig. 2). e. faecium and lc. lactis were only species isolated from milk sample by a pour plate technique, biologica nyssana 5 (1)  september 2014: 37-46 joković, n. et al.  screening of lactic acid bacteria isolated … 43 while s. thermophilus was isolated from the milk by enrichment technique. lc. lactis, e. faecium, and s. thermophilus are commonly isolated species from milk, while the lactobacilli and leuconostocs are always isolated in lower percentage (f r a n c i o s i et al., 2009; g i a n n i n o et al., 2009). on the other hand, l. mesenteroides was the most dominant species in all analyzed kajmak samples. this species was found in high percentage (56%) in 5-day-old sample of kajmak (bgnk5) and its frequency of isolation increased throughout of the ripening (fig. 2). leuconostocs are ubiquitous in nature due to their ability to survive longer on the surface of different materials or during pasteurization (m a r t l e y & c r o w , 1993; o g i e r et al., 2008). since leuconostocs were not isolated from the milk, they probably got into the kajmak from the environment or they were present in the milk in such small numbers that they could not be detected by used isolation techniques. lc. lactis isolates were isolated by pour plate technique from 5-day-old (bgnk5) and 15-day-old samples of kajmaks (bgnk15) although its frequency of isolation was very low and decreased during fermentation (13% and 9%, respectively) (fig. 2). in the 30-day-old kajmak sample (bgnk30) lactococci were isolated only with enrichment technique. the reduction in the number of lactococci is probably the result of a slight salt content and dry matter increase and ph values decrease during the ripening of kajmak (j o k o v i ć et al., 2008). these conditions inhibited the growth of lactococci, which are much more sensitive to adverse conditions prevailing during the ripening of dairy products compared to leuconostocs and enterococci (l o p e z d i a z et al., 2000). two species of enterococcal isolates were detected in kajmak samples. e. faecium isolates detected in milk, were also isolated from 5-day-old kajmak sample (bgnk5) while e. durans isolates were detected in all samples of kajmak and their number increased slightly during ripening (fig. 2). e. durans isolates were not previously detected in the analyzed samples of kajmak (j o k o v i ć et al., 2008) but they were found commonly in milk and different types of cheeses (o g i e r & s e r r o r , 2008). enterococci have a high tolerance to adverse conditions such as salt and acidity during the ripening of dairy products which explains their dominance in many dairy products (m a r i n o et al., 2003). isolates identified as w. cibaria were found in 5-day-old (bgnk5) and 15-day-old samples of kajmak (bgnk15) in low percentage (7% and 3%, respectively). w. cibaria isolates have been detected in foods of various origins, as well as among some clinical isolates of human and animal origin (b j o r k r o t h et al., 2002; o u a d g h i r i et al., 2009). technological characterization of isolates all isolates were tested for technological characteristics important for use of lab in dairy industry. assays included acidifying, proteolityc and lipolytic activity, ability to metabolize citrate and produce diacetyl and production of eps and inhibitory substances. acidifying and proteolityc activity in all dairy products rapid production of lactic acid by lab at the beginning of fermentation is a crucial step for getting products with good sensory and hygienic properties. fast production of lactic acid by lab strains depends on their proteolytic system, ability to metabolize lactose and bacterial resistance to acid stress (g a l i a et al., 2009). therefore, acidifying and proteolityc activities of the isolates from dairy products are very important for their selection in starter cultures. in our study, streptococci showed the highest acidifying activity in milk (fig. 3) and all strains tested clotted skimmed milk after 24 h of incubation. four of them decreased ph of milk for 6 h of incubation to the value around 5.30. lactococcal strains had lower initial values of ph than streptococci but in latter stage of growing in milk some of them enhanced their acidifying activity reaching ph around 4.40 after 24 h incubation period. lc. lactis and s. thermophilus strains isolated from different dairy products, most often, have better activity in milk than other lab isolates (a y a d et al., 2004; b a d i s et al., 2004). enterococcal strains were weak acid producers and no of the isolate could decrease ph of milk under 5.00 after 24 h of growth in the milk. all strains of l. mesenteroides showed low acidifying ability and 30% could not reduce ph of milk at all while strains of w. cibaria practically could not grow in milk (fig. 3). a limited number of lab strains with good activity in milk was detected by analyzing lab isolates from other artisanal dairy products (a s t e r i et al., 2009; m o h a m m e d et al., 2009). the assay for proteolytic activity using the ophthaldialdehyde method showed that lactococci released the highest amount of amino groups in milk with average value of 1.97 mmgly l -1 (fig. 3). s. thermophilus isolates had lower proteolytic activity followed by e. durans and e. faecium isolates. biologica nyssana 5 (1)  september 2014: 37-46 kamberović, j. et al.  marshland vegetation of the order phragmitetalia … 44 figure 3. acidifying and proteolytic activities of isolates the error bars represent the means of standard deviation isolates identified as leuconostocs and weisella showed the lowest level of proteolytic activity (fig. 3). these results were similar to that described by other authors who showed that lactococci had the best proteolytic activity among lab (g o n z a l e z et al., 2010). five of the fourteen strains of lc. lactis with the highest level of proteolytic activity (2.08-2.28 mmglyl -1 ) were the most rapidly acidifying lactoccocal strains. these strains are good candidates for construction of starter cultures for kajmak production. utilization of citrate and production of diacetyl possibility of diacetyl synthesis from citrate is characteristic of certain lab strains included in the composition of starter cultures. diacetyl is one of the most important compounds that affect the aroma of dairy products giving them flavor similar to that of butter (m a d e r a et al., 2003). in this study, six strains of lc. lactis had blue appearance on kempler and mckey agar plates and produced diacetyl in milk after 24 h of incubation. therefore, these isolates were identified as lactococcus lactis subsp. lactis biovar. diacetylactis. the most of enterococcal isolates had ability of diacetyl production (five e. faecium and twenty-five e. durans isolates) but none of them could use citrate on kempler and mckey agar. these results indicate that enterococci might use other substrates for the synthesis of diacetyl, as have been shown for some lactococci (b a r s & y v o n , 2008). on the other hand, thirty four leuconostoc isolates were able to metabolize citrate on kempler and mckey agar plates but diacetyl synthesis in milk was not detected. since leuconostocs produce diacetyl only in medium with lower ph values (g a r a b a l et al., 2008) that had not been detected in this study due to their poor growth in milk, more detailed investigations on this matter are required. lipolytic activity five e. durans and one e. faecium strains showed lipolytic activity when assayed on tributirin agar. these strains were weakly lipolytic as halos around colonies were very small. in general, lab have poor lypolitic activity, but for certain strains it has been shown to hydrolyze mainly triglycerides containing fatty acids with medium length chains (k a t z et al., 2002). lipolytic strains of the genus enterococcus have increased lipase activity compared to the strains of the genera lactococcus and leuconostoc (a s t e r i et al., 2009). production of eps in the dairy industry, lab strains that synthesize eps have effects on the organoleptic properties of the product and use of these strains in functional starter cultures may be significant (l e r o y & d e v u y s t, 2004). among the tested strains, only sixty-seven l. mesenteroides and three w. cibaria isolates produced eps on the mrs medium with the addition of sucrose. antimicrobial activity the most important antimicrobial substances that are produced by lab are bacteriocins due to their possible application in food biopreservation (s i m o v a et al., 2009). results showed that four lc. lactis subsp. lactis biovar. diacetylactis strains biologica nyssana 5 (1)  september 2014: 37-46 joković, n. et al.  screening of lactic acid bacteria isolated … 45 produced bacteriocins with inhibitory effect on the growth of lactococcal indicator strains lc. lactis ssp. cremoris ns1 and lc. lactis subsp. lactis bgmn1596 used in the experiment. lab strains that produce bacteriocins are a part of starter cultures for improving the microbiological quality and safety of dairy products, while the adjunct starters composed of sensitive strains and bacteriocin producer strains lead to increasing in the degree of autolysis and accelerate the ripening (a y a d et al., 2004). conclusion kajmak is a delicatessen dairy product of balkan countries that is not present in the world market because of not standardized production in households that leads to the variability and poor safety of final products. these problems can be overcome by the industrial production of kajmak with the addition of starter cultures composed of the appropriate strains isolated from kajmak samples and well characterized. the results of the present research indicate that among isolated lab a few interesting strains could be used in construction of starter cultures for industrial production of kajmak. four lactococcal strains that showed fast acidification rate and good proteolityc activity as well as rapid acidifying s. thermophilus strains could be used in starter cultures for rapid production of lactic acid at the beginning of kajmak fermentation. strains of lactococcus lactis subsp. lactis biovar. diacetylactis that produce diacetyl and protein antimicrobial substances may have double role, in getting the specific flavor and control of undesirable microflora. among l. mesenteroides isolates, strains that metabolized citrate, also may have important role in the formation of sensory properties of kajmak. acknowledgments. this work was supported by the ministry of education and science of the republic of serbia grant no.: 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communities of active bacteria. applied and environment microbiology, 64:3854-3859. acar, hacıoğlu doğru, 2019, biologica nyssana 10(2) 10 (2) december 2019: 175-179 doi: 10.5281/zenodo.3600199 effects of drought stress factors on antibacterial activity of two triticum aestivum l. varieties original article okan acar department of biology, faculty of arts and sciences, çanakkale onsekiz mart university, çanakkale, turkey oacar@comu.edu.tr nurcihan hacıoğlu doğru department of biology, faculty of arts and sciences, çanakkale onsekiz mart university, çanakkale, turkey nurcihan.n@gmail.com (corresponding author) received: july 10, 2019 revised: september 10, 2019 accepted: september 13, 2019 abstract: triticum aestivum l. (wheat grass), one of the members of poaceae family, has been considered to be a very efficient therapeutic drug. in the present study, we propose to evaluate antibacterial effects of the two varieties of t. aestivum l. [cv. tosunbey (drought tolerant) and cv. sultan 95 (drought-sensitive)] which grown in three different stress conditions [(1) drought stress; (2) pre-treatment of seeds with acetyl salicylic acid; (3) drought stress and pre-treatment of seeds with acetyl salicylic acid]. the antibacterial activity of the ethanol extracts was assayed against five pathogens (pseudomonas aeruginosa atcc 27853, proteus vulgaris atcc 13315, escherichia coli nrrl b-3704, staphylococcus aureus atcc 25923 and bacillus subtilis atcc 6633) by agar disc diffusion and micro broth dilution methods. the results showed that the ethanol extracts from the different studied treatments showed antibacterial activities, with the diameters of the inhibition zone ranging from 8 to 15 mm and minimum inhibitory concentration ranging from 2.5 to 20.0 µg/ml, respectively. the highest antibacterial activity, against b. subtilis atcc 6633, was demonstrated by the extract of t. aestivum cv. sultan 95, which grown in conditions where drought stress and pre-treatment of seeds with acetyl salicylic acid were combined. key words: antibacterial activity, t. aestivum cv. tosunbey, t. aestivum cv. sultan 95 apstract: antibiofilm aktivnost etanolnog ekstrakta verbascum pinnatifidum vahl. triticum aestivum l. (pšenica), jedan od članova familije poaceae, smatra se za veoma efikasnu terapeutsku drogu. u ovom istraživanju, utvrđivan je antibakterijski efekat dva varijeteta t. aestivum l. [cv. tosunbey (tolerantna na sušu) i cv. sultan 95 (osetljiva na sušu)] koji su uzgajani u tri različita uslova stresa [(1) sres suše; (2) semena pretretirana acetil salicilnom kiselinom; (3) sres suše i pretretman semena acetil salicilnom kiselinom]. antibakterijska aktivnost etanolnih ekstrakata ispitivana je u odnosu na pet patogenih vrsta (pseudomonas aeruginosa atcc 27853, proteus vulgaris atcc 13315, escherichia coli nrrl b-3704, staphylococcus aureus atcc 25923 i bacillus subtilis atcc 6633) korišćenjem disk difuzije na agaru i mikrodilucione metode. rezultati su pokazali da etanolni ekstrakti dobijeni iz ispitivanih različitih uslova gajenja poseduju antimikrobnu aktivnost, sa dijametrima inhibicionih zona koji su varirali od 8 to 15 mm i minimalnim inhibitornim koncentracijama od 2,5 do 20,0 µg/ml. najveća antibakterijska aktivnost, utvrđena u odnosu na b. subtilis atcc 6633, pokazana je od strane ekstrakta t. aestivum cv. sultan 95, koji je rastao u uslovima kombinacije sušnog stresa i pretretmana semena acetil salicilnom kiselinom. ključne reči: antibakterijska aktivnost, t. aestivum cv. tosunbey, t. aestivum cv. sultan 95 introduction plants contain numerous of constituents and are valuable sources of new and biologically active molecules having antimicrobial properties that are important for drug designing against diseases (das et al., 2010; bhattacharjee and islam, 2015; saha et al., 2018). wheat as one of the members of gramineae family, has been known for very efficient medical values and several therapeutic drugs that include bran and germ. it is used as a protection against various diseases such as constipation, heart diseases, appendicitis, obesity, diabetes and condition of the colon called diverticulum (hadjivassiliou et al., © 2019 acar, hacıoğlu doğru. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work noncommercially under the same license as the original. 175 13th symposium on the flora of southeastern serbia and neighboring regions 2003; saha et al., 2018). triticum aestivum has also been shown to be a rich source of vitamins a, c, e and b complex, including b12 (ashok, 2011; sundaresan et al., 2015). it contains a multitude of minerals like calcium, phosphorus, magnesium, alkaline earth metals, potassium, zinc, boron, and molybdenum. other compounds that are making this grass therapeutically effective are the indole compounds, choline and laetrile (amygdalin) (padalia et al., 2011; sundaresan et al., 2015). antibiotic resistance has become a global concern (westh et al., 2004; saha et al., 2018) and clinical efficiency of many existing antibiotics is being threatened by the emergence of multidrug-resistant pathogens as reported by saha et al. (2018). so, there is an urgent need to develop new antimicrobial compounds which are more active against novel and re-emerging infectious diseases (rojas et al., 1992; saha et al., 2018). duke and ayensu (1985) reported that wheat is an easily grown plant in the world and that the young stems had proven effective for the treatment of biliousness, intoxication and removing skin blemishes, antipyretic, antihydrotic and sedative. triticum aestivum has also been used against cough, sore throat, malaise, spasmic pain, abdominal coldness and constipation (ashok, 2011). wheat is also known to have anticancer and antimicrobial properties (gregorova et al., 2015). drought is the most critical limiting factor for crop production and is becoming an increasingly severe problem in many regions of the world. abiotic stress conditions such as drought, salinity, cold stress can induce the synthesis of pathogen-associated proteins (prs) in plants (van loon et al., 2006; sehgal and mohamad, 2018). in severe drought conditions, prs such as chitinase and glucanase have been activated in wheat (gregorova et al., 2015). in addition, tdltp4, a pr protein, was found to be higher in wheat variety with salt and drought tolerance (safi et al., 2015). on the other hand, prs induced by the effect of salicylic acid (sa) has been shown to have antimicrobial activity in vitro (van loon et al., 2006). also, sa applications have been reported to increase antimicrobial activity in turnip (thiruvengadam et al., 2016) and sesame plants (hossein et al., 2016). it is known that antifungal and antimicrobial activity increases due to oxidative stress induced by environmental stresses (sharma et al., 2018; schmidt et al., 2019). the relationship between antimicrobial activity and prs, produced by plants under environmental stress conditions such as defensins (schmidt et al., 2019) and phytoalexins (ejike et al., 2013), has been demonstrated earlier (li et al., 2011). in this study, the effects of drought and salicylic acid (sa) priming combinations were investigated on antibacterial activity in 21-day-old seedlings of two t. aestivum varieties. material and methods plant materials the seeds were sterilized by washing with 5% sodium hypochlorite solution for 5 min (1 time) and with sterile distilled water for 2.5 min (3 times). two tablets of 500 mg aspirin were thoroughly dissolved in 500 ml of distilled water and sterile seeds were kept in this solution for 2 hours for ascorbic acid (asa) priming. at the end of the period, the seeds were placed on moist filter paper and germinated for three days. seedlings were planted in perlite:peat mixture (1:3) containing pots. plants were watered with hoagland nutrient solution (100%; steward, 1983) during 21 days in growth chamber (22-24 oc, photoperiod 16/8 day/night). drought stress application was carried out with water scarcity for 5 days starting from the 21st day. seedlings of t. aestivum l. cv. tosunbey (drought tolerant) and cv. sultan 95 (drought-sensitive) were divided into four groups: (1) control (2) drought stress; (3) pretreatment of seeds with acetyl salicylic acid; (4) drought stress and pre-treatment of seeds with acetyl salicylic acid. leaf sampling was done on 21-day old plants. preparation of plant extracts air-dried samples of two wheat varieties were grounded into a fine powder in a grinding mill. pulverized plant samples (1 g) were extracted with 10 ml of 80% ethanol, (1:10 w/v) using an orbital shaker for 8 h at room temperature. the extract was separated from the solids by filtration with whatman no. 1 filter paper. the remaining solids were extracted twice with the same solvent and extracts combined. extracts were stored in a refrigerator (4 oc) until analyzed (sultana et al., 2007). test microorganisms gram-negative bacteria (pseudomonas aeruginosa atcc 27853, proteus vulgaris atcc 13315, escherichia coli nrrlb-3704), gram-positive bacteria (staphylococcus aureus atcc 6538p, bacillus subtilis atcc 6633) were used for determining the antibacterial activities of two t. aestivum varieties. screening for antimicrobial activities disc diffusion method was used for qualitative analyses of two t. aestivum species extracts (collins et al., 1989). studies were performed in triplicate. treatments with penicillin (p10) served as positive controls, respectively and treatments with ethanol without plant extracts served as negative controls. 176 biologica nyssana ● 10 (2) december 2019: 175-179 acar, hacıoğlu doğru ● effects of drought stress factors on antibacterial activity of two triticum aestivum l. varieties minimum inhibitory concentration assay minimum inhibitory concentration (mic) was investigated as recommended by the clinical and laboratory standards institute (clsi, 2006). the lowest concentration of extracts inhibiting visible growth of each test microorganisms was taken as the mic. the medium, 0.1% (w/v) streptomycin (st), nystatin (nys100) and 10% dmso were used as the non-treated, positive and negative controls, respectively (teanpasian et al., 2017). results and discussion tab. 1 indicated antimicrobial activity of two varieties of t. aestivum plant extracts and the inhibition zones formed by standard antibiotic disks and mic ratios. the results of antimicrobial testing showed that the ethanol extracts from the different treatments studied showed variable antibacterial activities, with the diameters of the inhibition zone ranging from 8 to 15 mm and 2.5 to 20.0 µg/ml, respectively. the highest antibacterial activity was demonstrated by the extract of t. aestivum cv. sultan 95, grown in drought conditions (s3 in tab.1), against b. subtilis atcc 6633. triticum aestivum plant varieties used in this study showed higher antibacterial activity against gram-positive bacteria (b. subtilis atcc 6633) than gram-negative test bacteria. the results in present study are parallel to those reported in the previous investigations. in general, the distinctive feature of gram-negative bacteria is the presence of a double membrane surrounding each bacterial cell. although all bacteria have an inner cell membrane, gramnegative bacteria have a unique outer envelope. this outer membrane excludes certain drugs and antibiotics from penetrating the cell, partially accounting for why gram-negative bacteria are generally more resistant to antibiotics than grampositive bacteria (dülger and dülger, 2018). in the literature there are many investigations about wheat grass antimicrobial activity (pallavi et al., 2011; das et al., 2012; sundaresan et al., 2015). however, there is no data effect of drought stress on wheat grass antibacterial activity in the literature. drought and salinity conditions can cause oxidative stress due to osmotic stress. this may induce the emergence of prs such as phytoalexin and defensin (van loon et al., 2006; sehgal and mohamad, 2018). our results showed the highest antibacterial activity in drought-sensitive sultan 95 with sa and drought treatments together. accordingly, our results are consistent with studies in which severe drought stress activates prs in wheat (gregorova et al., 2015), and increases a pr which causes higher fungal resistance in drought-tolerant wheat (safi et al., 2015). in addition, the increases in 177 biologica nyssana ● 10 (2) december 2019: 175-179 acar, hacıoğlu doğru ● effects of drought stress factors on antibacterial activity of two triticum aestivum l. varieties ta bl e 1. d is c d iff us io n, m ic ra tio s of th e tw o va rie tie s of t . a es tiv um e xt ra ct s te st b ac te ria pl an t e xt ra ct s *d is c d iff us io na (m m ) m ic (µ g/ m l) cv . t os un be y cv . s ul ta n 95 cv . t os un be y cv . s ul ta n 95 t1 t2 t3 t4 s1 s2 s3 s4 t1 t2 t3 t4 s1 s2 s3 s4 e .c ol i n r r lb -3 70 4 8. 0 11 .0 10 .0 7. 0 12 .0 11 .0 7. 0 7. 0 16 .0 20 .0 2. 5 10 .0 20 .0 2. 5 2. 5 20 .0 20 .0 4. 0 p. a er ug in os a at c c 2 78 53 10 .0 9. 0 7. 0 7. 0 11 .0 7. 0 9. 0 9. 0 8. 0 5. 0 5. 0 20 .0 20 .0 2. 5 20 .0 10 .0 2. 5 1. 0 p. v ul ga ris a tc c 1 33 15 13 .0 10 .0 13 .0 8. 0 12 .0 9. 0 11 .0 10 .0 13 .0 2. 5 5. 0 2. 5 20 .0 2. 5 10 .0 10 .0 20 .0 4. 0 s . a ur eu s at c c 6 53 8p 7. 0 8. 0 7. 0 9. 0 11 .0 9. 0 10 .0 12 .0 14 .0 20 .0 20 .0 20 .0 5. 0 2. 5 10 .0 5. 0 5. 0 4. 0 b . s ub til is a tc c 6 63 3 11 .0 9. 0 10 .0 11 .0 10 .0 8. 0 15 .0 13 .0 15 .0 2. 5 5. 0 10 .0 5. 0 5. 0 20 .0 2. 5 20 .0 4. 0 t: t . a es tiv um c v. t os un be y s: t . a es tiv um c v. s ul ta n 95 t1 : c on tro l; t2 : t re at m en t w ith a ce ty l s al ic yl ic a ci d; t 3: tr ea tm en t w ith d ro ug ht s tre ss ; t 4: d ro ug ht s tre ss a nd p re -tr ea tm en t o f s ee ds w ith a ce ty l s al ic yl ic a ci d s1 : c on tro l; s2 : t re at m en t w ith a ce ty l s al ic yl ic a ci d; s 3: tr ea tm en t w ith d ro ug ht s tre ss ; s 4: d ro ug ht s tre ss a nd p re -tr ea tm en t o f s ee ds w ith a ce ty l s al ic yl ic a ci d in hi bi tio n zo ne (m m ); a in cl ud es d ia m et er o f d is k (6 m m ); p1 0 = pe ni ci llin (1 0 ug /d is c) ; s t: st re pt om yc in antimicrobial activity by sa priming were similarly shown to increase antimicrobial activity in sa applications in sesame (hossein et al., 2016) and turnip (thiruvengadam et al., 2016). as a result, sa and drought application showed high antibacterial activity in drought-sensitive variety. this may indicate different prs synthesized in this variety compared to the tolerant one. the occurrence of lower antimicrobial activity in the tolerant variety compared to the sensitive variety suggests that some other stress factor is needed due to its tolerance to drought. conclusion this study was conducted to evaluate the antibacterial activity of two t. aestivum varieties under three different stress conditions. natural products are important source of new drugs which are having importance in modern medicine. the results pointed that the ethanol extracts from plants treated with combined stress conditions had significant antibacterial activities against p. vulgaris atcc 13315 (t3) and b. subtilis atcc 6633 (s3). the need to increase food production and exhaustion of wheat genetic resources has increased interest in alternative approaches for wheat improvement, including the use of different stress conditions. it is well known that abiotic stress leads to a series of morphological, physiological, biochemical, and molecular changes that adversely affect plant growth, productivity and antagonistic activity (hayat et al., 2013; ripa et al., 2019). therefore, selection, screening, and application of the stress-tolerant t. aestivum plant varieties for better farming would considerably facilitate the farming community by overcoming such extreme climate changes. this is the first report on the bioactivities of two varieties of t. aestivum grown in different drought conditions and pre-treatment of seeds with acetyl salicylic acid. this approach may also allow new kind of research in medicinal usage or development of drug research. acknowledgments. this paper was presented at the symposium on the flora of southeastern serbia and neighboring regions, june, 20-23 2019, serbia. references ashok, s.a. 2011: phytochemical and pharmacological screening of wheat grass juice (triticum aestivum l.). international journal of pharmaceutical sciences review and research, 9: 159-164. bhattacharjee, b., islam, s.m.s. 2015: assessment of antibacterial and antifungal activities of the extracts of rhynchostylis retusa. blumea medici178 nal orchid. world journal of pharmacy and pharmaceutical sciences, 4: 74-87. clsi, 2006: methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; 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(ed.) plant viruses vol. ii: pathology: 65-84. crc press, boca raton. sharma, a., sharma, d., verma, s.k. 2018: in silico study of iron, zinc and copper binding proteins of pseudomonas syringae pv. lapsa: emphasis on secreted metalloproteins. frontiers in microbiology, 9: 1838. sultana, b., anwar, f., przybylski, r. 2007: antioxidant activity of phenolic components present in barks of barks of azadirachta indica, terminalia arjuna, acacia nilotica, and eugenia jambolana lam. trees. food chemistry, 104: 1106-1114. sundaresan, a., selvi, a., manonmani, h.k. 2015: the anti-microbial properties of triticum aestivum (wheat grass). international journal of biotechnology for wellness industries, 4:(3): 84-91. van loon, l.c., rep, m.p. 2006: significance of inducible defense-related proteins in infected plants. annual review of plant pathology, 44: 135-162. thiruvengadam, m., baskar, v., kim, s.h., chung, i.m. 2016: effects of abscisic acid, jasmonic acid and salicylic acid on the content of phytochemicals and their gene expression profiles and biological activity in turnip (brassica rapa ssp. rapa). plant growth regulation, 80(3): 377-390. westh, h., zinn, c.s., rosdahl v.t. 2004: an international multicenter study of antimicrobial consumption and resistance in staphylococcus aureus isolates from 15 hospitals in 14 countries. microbial drug resistance, 10: 169-76. 179 biologica nyssana ● 10 (2) december 2019: 175-179 acar, hacıoğlu doğru ● effects of drought stress factors on antibacterial activity of two triticum aestivum l. varieties anticancer compounds from medicinal plants biologica nyssana 2 (2)  december 2011: 00-00 ljupković r.b. et al..  removal cu(ii) ions from water... 31 original article a contribution to the agromyzid leaf miners (diptera: agromyzidae) of iran zahra shahreki 1 , ehsan rakhshani* 1 , mitsohiru sasakawa 2 1 department of entomology, college of agriculture, university of zabol, 98615-538, zabol, i.r. iran 2 korigaoka, hirakata city, osaka pref., 573 japan * e-mail: rakhshani@uoz.ac.ir abstract: shahreki, z., rakshani, e., sasakawa, m.: a contribution to the agromyzid leaf miners (diptera: agromyzidae) of iran, biologica nyssana, 3 (1), september 2012: 31-36. a study was carried out during 2009 and 2010 in sistan region located in the eastern part of iran, for elucidating the agromyzid fauna. the specimens were collected by rearing of the mine-infested leaves of different cultivated and non-cultivated host plants. the leafminers of two subfamilies, agromyzinae and phytomyzinae, were studied in this paper. eight species belonging to five genera of the agromyzidae were collected. among them, three species, including chromatomyia nigra (hardy, 1849), melangromyza cunctans (meigen, 1830) and phytoliriomyza dorsata (siebke, 1863), are recorded for the first time for the territory of iran. the last species is also representative of the new genus record for iran. comments on general distribution of each species are presented here. key words: agromyzinae, eastern iran, host association, new record, phytomyzinae introduction the agromyzidae (leaf mining flies) is one of the largest dipteran families, with more than 3000 species belonging to 30 genera worldwide. about 1165 species have been recorded from the palaearctic region (civelek, 2003, 2008; çikman & sasakawa, 2008; dursun et al., 2010), one of the largest genera, the genus liriomyza mik, with over 300 species worldwide (spencer, 1973) appear common in the palaearctic region, but poorly represented in the oriental region (sasakawa, 1972). the agromyzid leaf miners including many species of economic importance, limiting production in several agroecosystems (spencer, 1973). most of species are cosmopolitan, polyphagous pests of agricultural crops and ornamental plants with an economic importance (spencer, 1973; dempewolf, 2006). they undermine the internal tissues of leaves, stems, fruits and even roots. leafminers can cause direct damage to the photosynthetic tissue of host plants caused by larval leaf mining (johnson et al., 1983) and esthetic damage caused by oviposition. adult males and females suck the exuding sap from the parenchyma cells on the punctures produced by adult females just after oviposition (spencer, 1973). extensive mining can result in premature leaf drop and leading to lack of shading (capinera, 2001). apart from direct damage caused by larval feeding on leaf tissues, the females may even act as vectors for diseases during oviposition (matteoni & broadbent, 1988; zitter & tsai, 1977). taxonomically, the agromyzids are considered one of the most difficult groups among the diptera order. this is because of high degree of uniformity between species and the small size of specimens. identification of species is an important 3 (1) • september 2012: 31-36 biologica nyssana 3 (1)  september 2012: 31-36 shahreki, z. et al.  a contribution to the agromyzid… 32 step prerequisite for establishment of effective pest control program and the quarantine measures, especially in the case of biological control (parrellaa & keil, 1984). a few attempts have been made for investigation of faunal diversity (dousti, 2010), biology (haghani et al., 2007; farrokhi et al., 2004) and the natural enemies (asadi et al., 2006; fathi, 2011) of the leafminer flies in iran. the scanty information were summarized by dousti (2010), in which the record of 26 species from iran, indicating the necessity of further investigations to extend the knowledge about the diversity and taxonomy of this poorly known group of insects. in spite of substantial efforts that have been made on systematic of agromyzidae species, there is a significant lack of knowledge in iran. furthermore, there is no record from sistan region which is located in the eastern part of the country. here, we present the data of the faunistic survey on agromyzidae from sistan region, including host associations and comments on their general distributions. materials and methods the leaves, stems and other parts of the host plants infested by the mining agromyzid larvae were collected in the various cultivated and noncultivated areas in sistan region during the period of 2009–2010. the infested plant materials were cut, preferably with representative part of the plants and then directly placed in plastic boxes (10 × 12 cm) covered with mesh on the upper side due to ventilation. the rearing boxes were then transferred to laboratory, inside the climatic chamber, under the constant conditions with the temperature of 25±1ºc and 65±5% rh. samples were kept for about 1–2 weeks expecting of emergence of the adults. adults were caught by an aspirator and gathered in the test tubes containing of 75% ethanol. illustrations were made using the nikon smz645 stereomicroscope and nikon eclips e200 microscope equipped with the canon ixus 100 is digital camera. all the samples are preserved in the collection of second author. results and disscusion eight species of agromyzidae belong to five genera and two subfamilies were identified in association with 11 different species of host plants. one genus and three species listed below are considered to be new records for iran. the newly recorded taxa are indicated by an asterisk ( ). subfamily: agromyzinae melanagromyza cunctans (meigen, 1830)* (figures 7, 15) distribution: mediterranean and western european countries. material examined: 2♀ 2♂, reared from lycopersicon esculentum – solanaceae, zabol, 11iii-2010; leg.: z.s. subfamily: phytomyzinae calycomyza humeralis (von roser, 1840) (figures 1, 9) distribution: nearctic and palaearctic region. material examined: 1♂, reared from triticum aestivum – poaceae, hamoon plain, 28-v-2009; leg.: z.s. chromatomyia horticola (gourea, 1851) (figures 2, 10) distribution: holarctic, oriental and afrotropical. expanded to central asia and europe. material examined: 30♀ 8♂, reared from gaillardia grandiflora – asteraceae, sistan dam, 20-i-2009; 2♀ 20♂, reared from helianthus annus – asteraceae, zahak, 24-iii-2009; 10♀ 8♂, reared from silybum marianum – asteraceae, zabol, 11xi-2009; 8♀ 12♂, reared from brassica rapa – brassicaceae, zahak, 06-iv-2010; 5♀ 3♂, cucumis sativus – cucurbitaceae, zahak, 02-v-2009; leg.: z.s. chromatomyia nigra (meigen, 1830)* (figures 3, 11) distribution: europe, northern asia, northern america. new record from iran. material examined: 5♀ & 3♂, reared from triticum aestivum – poaceae, hirmand, 28-v-2009; leg.: z.s. liriomyza congesta (becker, 1903) (figs. 4, 12) distribution: widespread in europe and north africa. also recorded from the eastern palaearctic and india. material examined: material: 1♀, reared from trigonella foenum-graecum – fabaceae, hamoon plain, 05-iv-2009; 2♀, reared from medicago sativa – fabaceae, zabol, 21-iv-2010; 2♂, hamoon plain, 05-xi-2009; leg.: z.s. biologica nyssana 3 (1)  september 2012: 31-36 shahreki, z. et al.  a contribution to the agromyzid… 33 figures 1-8. lateral hibatate of the adult leafminers. 1. calycomyza humeralis, 2chromatomyia horticola, 3. chromatomyia nigra, 4. liriomyza congesta, 5. liriomyza sativae, 6. liriomyza trifolii, 7. melanagromyza cunctans, 8. phytoliriomyza dorsata. biologica nyssana 3 (1)  september 2012: 31-36 shahreki, z. et al.  a contribution to the agromyzid… 34 liriomyza sativae (blanchard, 1938) (figure 5, 13) distribution: cosmopolitan. material examined: 1♀ 22♂, reared from malva sylvestris – malvaceae, zabol, 04-v-2009; leg.: z.s. liriomyza trifolii (burgess, 1880) (figure 6, 14) distribution: nearctic in origin, but a cosmopolitan species. material examined: material: 5♀ 10♂, reared from cucumis sativus – cucrubitaceae, zahak, 05-iv2009; 25♀ 6♂, reared from lactuca serriola – asteraceae, zahak, 19-iv-2009; leg.: z.s. phytoliriomyza dorsata (siebke, 1864)* (figures 8, 16) distribution: western palaearactic, north america. new record of the genus phytoliriomyza hendel from iran. material examined: 1♀, gaillardia grandiflora – asteraceae, zabol, 8-x-2009; leg.: z.s. figures 9-16. wing of agromyzid leafminers, 9. calycomyza humeralis, 10chromatomyia horticola, 11. chromatomyia nigra, 12. liriomyza congesta, 13. liriomyza sativae, 14. liriomyza trifolii, 15. melanagromyza cunctans, 16. phytoliriomyza dorsata. biologica nyssana 3 (1)  september 2012: 31-36 shahreki, z. et al.  a contribution to the agromyzid… 35 eight species belonging to five genera are known to occur in sistan region. all of them are newly recorded from eastern part of iran, too. mining larvae of different host plants are observed in cultivated or non-cultivated fields attacking crops in sistan region. most of these species have been found in warm or temperate and humid regions in the world (spencer, 1990). two species, l. trifolii and l. sativae are polyphagous leafminers known as important agricultural pests (darvas et al., 2000). among the studied agromyzids, three species, melanagromyza cunctans, phytoliriomyza dorsata and chromatomyia nigra were recorded for the first time for iran. the genus chromatomyia includes the polyphagous leafminer species, which are common agricultural pests on cosmopolitan plant species of grasses: poaceae (gramineae) (griffiths 1974, 1980). chromatomyia nigra was another newly recorded species from iran, which is an unusual species in having the densely hairy eyes and a pair of ventral processes on the distiphallus. this is an almost cosmopolitan species occurs on leaves of grasses, poaceae (gramineae) and asteraceae (compositae), aster and bellis. next species chromatomyia horticola considered also as a polyphagous species feeding on more than 30 plant families (griffiths 1974, 1980). melanagromyza hendel is the second large genus (spencer, 1966) and one of the most homogeneous taxa within the family agromyzidae (spencer, 1990). it is characterized by having a wide range of hosts undermining 23 plant families (benavent-corai et al., 2005). melanagromyza genus represents mainly internal stem-borers, but the species which has been described as melanagromyza symphyti griffiths, 1963 undermining thick-leaf stalks. the species such as melanagromyza cunctans that is newly recorded from iran, it is known as a gall-causer on lotus spp. in europe (spencer, 1976). it was collected in late winter from stems of tomato. another species of this genus, melanagromyza sativae spencer has already been recorded from the northern part of iran (spencer, 1990). it was also reared from the stems of the plants belong to the family apiaceae in fars province (dousti, 2010). the genus phytoliriomyza hendel is reported here for the first time from iran. less than 20 species of the genus phytoliriomyza have been recorded in the palaearctic region (zlobin, 2005). as a new host record, phytoliriomyza dorsata has been reared from the leaves of gaillardia grandiflora in spring. conclusion the present contribution increased our knowledge about the occurrence and the host associations of agromyzid leaf miner at a small area of eastern iran. the identified species were mostly the common and frequently encountered agricultural pests, as well as the new records, which have also economic importance in some other countries. on the other hand, further investigations are necessary to expand the data about the occurrence of other species and their host plants as an ecological refugia for the natural enemies especially the parasitoids. acknowledgements. this study was supported by the grant no. 89-9198, university of zabol, iran. references asadi, r., talebi, a.a., fathipour, y., moharramipour, s. & rakhshani, e. 2006: identification of parasitoids and seasonal parasitism of the agromyzid leafminers genus liriomyza (dip.: agromyzidae) in varamin, iran. journal of agricultural science and technology, 8: 293-303. benavent-corai, j., m. martinez & jiménez-peydró, r. 2005: catalogue of the host-plants of the world agromyzidae (diptera). bolletino di zoologia agraria e di bachicoltura. serie ii, 37: 1-97. capinera, j.l. 2001: handbook of vegetable pests. academic press, new york. 729 pp. çikman, e. & sasakawa, m. 2008: the turkish agromyzidae (diptera), with descriptions of four new species. entomological science, 11 (1): 81 86. civelek, h.s. 2003: checklist of agromyzidae (diptera) of turkey, with a new record. phytoparasitica, 31 (2): 132-138. darvas, b., skuhrav, m., & andersen, a. 2000: agricultural dipteran pests of the palaearctic region. in: papp, l. & darvas, b. 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specialization in the world agromyzidae (diptera). bulletin of the entomological society of america, 30: 22-25. spencer, k.a. 1972: diptera, agromyzidae. handbooks for the identification of british insects, 10: 1-136. zitter, t.a. & tsai, j.h. 1977: transmission of three potyviruses by the leafminer liriomyza sativae (diptera: agromyzidae). plant disease report, 61: 1025-1029. zlobin, v.v. 2005: studies on european species of the genus phytoliriomyza hendel (diptera: agromyzidae). russian entomological journal, 14(2): 119-123. microsoft word 0404_cavar_et_al biologica nyssana 1 (1-2) december 2010: 99-103 čavar, s. et al. comparison of essential oil composition of stachys … 31 original article ! comparison of essential oil composition of stachys menthifolia vis. from two natural habitats in croatia sanja ćavar1,2*, milka maksimović1, marija edita šolić3 1university of sarajevo, faculty of science, department of chemistry, zmaja od bosne 33-35, 71000 sarajevo, bosnia and herzegovina 2university of ljubljana, faculty of chemistry and chemical technology, aškerčeva 5, 1000 ljubljana, slovenia 3institute „mountain and sea“, franjevački put 1, 21300 makarska, croatia e-mail: sanja.cavar@pmf.unsa.ba abstract: ćavar, s., maksimović, m., šolić, m. e.: comparison of essential oil composition of stachys menthifolia vis. from two natural habitats in croatia. biologica nyssana, 1 (1-2), december 2010: 99-103. stachys menthifolia vis. is an endemic species from the balkan peninsula. aerial parts of the plant were collected from its natural habitat near dubrovnik. hydrodistilled volatile oil obtained from the plant material of s. menthifolia was subjected to gas chromatographic analysis coupled to mass spectrometry. more than 70 compounds were identified, representing 94.5% of the total oil. the major constituents of the oil were diterpenoid abietatriene (11.7%), and sesquiterpene hydrocarbons α-bisabolene (8.4%), and β-caryophyllene (7.4%). presented results are comparable to our previous findings on essential oil composition of the same species from biokovo mountain, with small differences in quantitative and qualitative constitution of the oil. although plants belonging to the stachys genus show significant variability in their chemical compositions depending on the location and stage of plant development, this work indicates that chemical polymorphism of endemic s. menthifolia does not manifest in the region of croatian mediterranean area. key words: abietatriene, essential oil, gc-ms, stachys menthifolia vis. introduction ! the genus stachys is one of the largest representative genera of the lamiaceae family, and includes about 300 species in the subtropical and tropical regions of both hemispheres (m a b b e r l e y , 1997). like most of species belonging to the family lamiaceae, stachys species produce essential oils, but the composition of volatile compounds is known only in a small number of species (m a l y , 1985; c a k i r et al., 1997; m a r i o t t i et al., 1997; p é l i s s i e r at al., 1999; c h a l c h a t et al., 2001; k u k i ć et al., 2006; p a l i ć et al., 2006; r a d u l o v i c et al., 2007). available literature data indicate the existence of a chemical polymorphism of essential oil composition among stachys taxa. stachys menthifolia vis. is an endemic species of the west balkan region. š i l i ć & š o l i ć (2001) discovered its natural habitat in the region of biokovo mountain in croatia. the first location in croatia was near dubrovnik, discovered by roberto visiani, who was the first who described this species. continuing our phytochemical research on s. menthifolia, this paper presents the essential oil composition of the plant collected from its natural habitat near dubrovnik. 10th sfses • 17-20 june 2010, vlasina lake1 (1-2) • december 2010: 99-103 biologica nyssana 1 (1-2) december 2010: 99-103 ćavar, s. et al. comparison of essential oil composition of stachys … 32 material and methods plant material was collected in may 2008 from the natural habitat near dubrovnik. voucher specimens have been deposited at the department of chemistry, faculty of science, university of sarajevo, bosnia and herzegovina. all applied reagents were of the highest purity available and purchased from the sigmaaldrich chemical company. essential oil from the air-dried parts of s. menthifolia was isolated by hydrodistillation according to european pharmacopoeia for 2 h. the oil was extracted with dichloromethane and dried over anhydrous sodium sulphate and stored at 4°c in the dark until the analysis. gas chromatography-mass spectrometry analysis was carried out on a hewlett-packard 6890 series ii gas chromatograph fitted with a fused silica hp-5 (5% phenyl methyl siloxane) capillary column (30 m × 0.252 mm, 0.25 μm film thickness), coupled to a hp 6890 series ii mass selective detector (msd). column temperature was programmed from 60°c to 240°c at 3°c min-1, and helium was used as carrier gas. other operating conditions were as follows: inlet pressure 9.43 psi, injector temperature 250°c, detector temperature 280°c, split ratio 1:25, injection volume 1 μl. ionization of the sample components was performed in the ei mode, (70 ev), with scan range 20-555 amu, and scan time 1.60 s. the linear retention indices, ri, for all compounds were determined by injection of the hexane solution containing the homologous series of c8-c26 n-alkanes (v a n d e n d o o l & k r a t z , 1963). the identification of the essential oil constituents was accomplished by the visual interpretation, comparing their retention indices and mass spectra with literature data (a d a m s , 2007) by computer library search (hp chemstation computer library nbs75k.l, nist/epa/nih mass spectral library 2.0 and mass finder 4 computer software and terpenoids library), and in the laboratory own database. semi-quantitative analysis was carried out directly from peak areas in the gc profile. results and discussion the yield of s. menthifolia hydrodistilled oil was 0.09% based on the dry weight of the plant material. exactly seventy-six compounds were identified in the essential oil, representing 94.5% of the total oil. the components identified in investigated sample of s. menthifolia, their retention indices, and percentage composition is summarized in table 1. the major constituents of the oil were diterpenoid abietatriene (11.7%), and sesquiterpene hydrocarbons α-bisabolene (8.4%), and βcaryophyllene (7.4%). in general, sesquiterpenoid compounds predominate in the oil with 59.1%, where 35.1% are sesquiterpene hydrocarbons. presented results can be compared to essential oil composition of s. menthifolia from biokovo mountain (ć a v a r et al., 2010), with some differences in quantitative and qualitative constitution of the oil (figure 1). oxygenated sesquiterpenes were the most abundant volatiles in s. menthifolia from biokovo (table 1), followed by diterpene hydrocarbons. this sample was characterized as the oil of abietatriene– 8−α−acetoxyelemol chemotype. significant differences between these samples were in the content of aromatic compounds, particularly 4’methoxyacetophenone, which was not found in the population from dubrovnik. moreover, the content of sesquiterpene hydrocarbons was much higher in the sample from dubrovnik. the observed differences might be explained by the fact that this plant material was harvested one month earlier than the sample from biokovo. figure 1. gc chromatograms of essential oil of s. menthifolia from dubrovnik (1) and biokovo (2) literature survey on studies related to the essential oils obtained from the plants belonging to stachys genus from the balkan peninsula, showed significant variability in their chemical composition depending on location and stages of plant development. essential oil obtained from s. milanii petrović (p a l i ć et al., 2006) had borneol and terpinen-4-ol as the most abundant compounds, while the major constituents of essential oil from bosnian s. alpina l. spp. dinarica murb. were βbiologica nyssana 1 (1-2) december 2010: 99-103 čavar, s. et al. comparison of essential oil composition of stachys … 33 caryophyllene and germacrene d (k u k i ć et al., 2006). dehydroabietane is the predominant compound in the essential oil of s. plumosa griseb. (p e t r o v i ć et al., 2006). sesquiterpenoids were the main class of the constituents in croatian stachys species as follows: (e)-nerolidol in s. alpina; germacrene d in s. officinalis, s. recta subsp. subcrenata, s. salviifolia, and s. sylvatica, caryophyllene oxide, along with 1-octen-3-ol, in s. palustris, while s. recta subsp. recta contained βionone as the dominant volatile constituent (b i l u š i ć v u n d a c et al., 2006). table 1. comparison of essential oil composition of s. menthifolia from croatia # ri compound dubrovnik ra† (%) biokovo* ra (%) 1. 801 hexanal 0.3 tr‡-0.2 2. 847 (2e)-hexenal 0.6 tr-0.2 3. 861 n-hexanol 0.3 4. 874 2-methyl butyl acetate 0.2 5. 930 α-pinene 0.3 tr-1.0 6. 975 1-octen-3-ol 0.9 tr-0.5 7. 1026 limonene 1.0 0.1-3.4 8. 1041 benzene acetaldehyde 0.1 tr 9. 1056 γ-terpinene 0.2 tr 10. 1099 linalool 3.6 0.7-1.7 11. 1103 nonanal 0.2 tr 12. 1107 2-methyl butyl isovalerate 0.5 13. 1111 1-octen-3-yl acetate 1.9 14. 1122 3-octanol acetate 0.4 15. 1189 α-terpineol 0.3 tr-0.3 16. 1190 methyl salycilate tr tr 17. 1255 linalool acetate 0.3 18. 1286 trans-linalool oxide acetate (pyranoid) 0.2 19. 1373 α-copaene 0.5 tr-0.3 20. 1381 β-bourbonene 0.3 tr 21. 1416 β-caryophyllene 7.4 0.5-2.8 22. 1422 β-maaliene 0.2 23. 1434 β-guaiene 0.1 tr 24. 1450 α-humulene 3.4 tr-0.2 25. 1456 (e)-β-farnesene 1.4 26. 1478 γ-curcumene 0.3 27. 1481 ar-curcumene 0.3 tr-0.2 28. 1492 α-muurolene 0.6 tr-0.2 29. 1499 (e,e)-α-farnesene 1.3 30. 1503 α-zingiberene 3.8 31. 1508 α-bisabolene 8.4 tr-0.1 32. 1512 γ-cadinene 1.7 tr-0.4 33. 1522 δ-cadinene 3.3 34. 1530 trans-cadina-1,4-diene 0.3 35. 1535 α-cadinene 0.9 tr-0.2 36. 1547 elemol 1.4 2.2-3.6 37. 1564 2-epi-(e)-β-caryophyllene 0.3 38. 1574 spathulenol 0.7 0.7-1.7 39. 1579 caryophyllene oxide tr 2.4-5.2 40. 1581 clovenol 1.1 41. 1588 globulol 1.9 1.5-2.4 42. 1600 guaiol tr tr-0.3 43. 1606 β-oplopenone 0.3 tr 44. 1627 trans-isolongifolanone 0.9 1.2-1.6 45. 1629 γ-eudesmol 0.9 1.1-1.9 biologica nyssana 1 (1-2) december 2010: 99-103 ćavar, s. et al. comparison of essential oil composition of stachys … 34 r a d u l o v i c et al., (2007) suggested that high degree of variation in the main volatiles of the species of stachys genus, especially of germacrene d and its congeners, was correlated with possible rearrangements under hydrodistillation conditions. according to the presence and quantity of dominant compounds, the essential oil of investigated populations of s. menthifolia significantly differs from the previously published data concerning this species (s k a l t s a et al., 2003). greek s. menthifolia essential oil had abietatriene (13.7%), kaurene (9.0%) and 13-epimanoyl oxide (7.5%) as the most abundant compounds. the observed differences in qualitative and quantitative composition of the essential oils between these two geographically isolated populations of s. menthifolia, confirm the influence of the environmental conditions on the volatiles # ri compound dubrovnik ra† (%) biokovo* ra (%) 46. 1633 caryophylla-4(15),8(13)-dien-5-α-ol 0.4 tr-0.4 47. 1640 epi-α-murrolol 1.7 1.8-2.6 48. 1647 β-eudesmol 2.3 2.4-5.0 49. 1650 α-eudesmol 1.5 1.4-3.3 50. 1652 α-cadinol 1.5 2.0-3.1 51. 1655 allo-aromadendrene epoxide 1.3 52. 1669 valeranone 4.5 53. 1676 n-tetradecanol 0.5 0.5-0.6 54. 1682 α-bisabolol 0.4 55. 1695 isolongifolol 2.7 56. 1761 benzyl benzoate tr 57. 1788 8-α-acetoxyelemol 2.1 6.9-21.3 58. 1807 cryptomeridiol 0.3 0.5-6.7 59. 1844 6,10,14-trimethylpentadecan-2-one 0.2 60. 1903 isopimara-9(11),15-diene 0.6 0-5-1.1 61. 1954 pimaradiene 0.5 0.2-0.8 62. 1963 n-hexadecanoic acid 0.7 63. 1983 manool oxide 1.9 0.7-2.3 64. 2002 (e)-labda-7,12,14-triene 0.3 0.5-2.7 65. 2006 13-epi-dolabradien tr 0.3-0.6 66. 2050 abietatriene 11.7 3.5-21.1 67. 2073 abietadiene 0.6 tr-0.8 68. 2141 abienol 0.4 0.7-0.9 69. 2206 2-keto manool oxide 0.7 0.7-1.2 70. 2226 7-α-hydroxy manool 0.4 0.2-0.3 71. 2258 larixol 0.5 0.4-1.1 72. 2288 dehydroabietal 1.9 1.5 73. 2298 n-tricosane tr tr 74. 2499 n-pentacosane 0.3 tr-0.2 75. 2599 n-hexacosane tr tr-0.2 76. 2698 n-heptacosane 0.6 aliphatic compounds 7.4 0.5-1.4 aromatic compounds 2.2 4.5-17.0 monoterpene hydrocarbons 1.5 0.1-4.6 oxygenated monoterpenes 4.4 1.7-2.9 sesquiterpene hydrocarbons 35.1 1.4-6.4 oxygenated sesquiterpenes 24.0 48.4-58.9 diterpene hydrocarbons 13.1 3.5-25.2 oxygenated diterpenes 6.8 1.5-5.8 total identified 94.5 86.8-90.8 *data taken from ć a v a r et al., (2010); †ra-relative area; ‡tr-trace (<0.1%). biologica nyssana 1 (1-2) december 2010: 99-103 čavar, s. et al. comparison of essential oil composition of stachys … 35 found in plants. it is essential to notify that these prominent variations were not retrieved in the populations from croatia. conclusion in conclusion, gc-ms analysis on the essential oil of endemic s. menthifolia populations from croatian mediterranean area indicated similarities in qualitative, but small differences in quantitative composition of their essential oils. although plants belonging to stachys genus show significant variability in their chemical compositions depending on the location and stage of plant development, this work indicates that chemical polymorphism of endemic s. menthifolia does not significantly manifest in the region of croatian mediterranean area. references adams, r. p. 2007: identification of essential oil components by gas chromatography/mass spectrometry, 4th ed. allured publ., carol stream, il. bilušić vundac,v., pfeifhofer, h. w., brantner, a. h., males, z., plazibat, m. 2006: essential oil of seven stachys taxa from croatia. biochemical systematics and ecology, 34, 875-881. cakir, a., duru, m. e., harmandar, m., izumi, s., hirata, t. 1997: the volatile constituents of stachys recta l. and stachys balansae l. from turkey. flavour and fragrance journal, 12, 215-218. ćavar, s, maksimović, m., vidic, d., šolić, m. e. 2010: chemical composition of the essential oil of stachys menthifolia vis. pharmaceutical biology, 48, 170-176. chalchat, j. c. petrović, s. d., maksimović z. a., gorunović, m. s. 2001: essential oil of stachys officinalis l. (trevis), lamiaceae, from montenegro. journal of essential oil research, 13, 286-287. kukić, j. petrović, s., pavlović, m., couladis, m., tzakou, o., niketić, m. 2006: composition of the stachys alpina l. ssp. dinarica murb. flavour and fragrance journal, 21, 539-542. mabberley, d. j. 1997: the plant-book, 2nd ed. cambridge university press, cambridge, new york, melbourne. maly, e. 1985: paper chromatography of the essential oils occurring in the genus stachys. journal of chromatography, 333, 288-289. mariotti, j. p., costa, j., bianchini, a., bernardini, a.f., casanova, j.  1997: composition and variability of the essential oil of stachys glutinosa l. from corsica (france). flavour and fragrance journal, 12, 205-209. palić, r. m., lazarević, j. s., stojanović, g. s., ranđelović, v. n.  2006: chemical composition and antimicrobial activity of the essential oil of stachys milanii petrovic. journal of essential oil research, 18, 290-292. pélissier, y., marion, s., rapior, s. 1999: volatile constituents of stachys corsica (pers.) (lamiaceae). journal of essential oil research, 11, 63-64. petrovic, s., ristic, m., milenkovic, m., kukic, j., antic-stankovic, j., niketic, m. 2006: composition and antimicrobial activity of essential oil of stachys plumosa griseb. flavour and fragrance journal, 21, 250-252. radulović, n., lazarević, j., ristić, n., palić, r. 2007: chemotaxonomic significance of the volatiles in the genus stachys (lamiaceae): essential oil composition of four balkan stachys species. biochemical systematics and ecology, 35, 196-208. šilić, č., šolić, m. e. 2002: the taxonomy, chorology and ecology of stachys menthifolia vis. (lamiaceae) in the north-west part of its distribution area. acta botanica croatica, 61, 5156. skaltsa, h. d. demetzos, c., lazari, d., soković, m. 2003: essential oil analysis and antimicrobial activity of eight stachys species from greece. phytochemistry, 64, 743-752. van den dool, h., kratz, p. d. 1963: a generalization of the retention index system including linear temperature programmed gasliquid partition chromatography. journal of chromatography, 11, 463-471. anticancer compounds from medicinal plants biologica nyssana 2 (2)  december 2011: 00-00 ljupković r.b. et al..  removal cu(ii) ions from water... 11 original article preliminary investigation of free radical scavenging activity and total phenolic content of three cultivated thymus chemotypes from bulgaria milena nikolova 1* genka zhekova 2 anatoli dzhurmanski 2 1 institute of biodiversity and ecosystem research, 23, acad.g. bonchev st., 1113 sofia, bulgaria 2 institute of roses, essential and medicinal cultures, bul. osvobozhdenie 49, 6100 kazanlak, bulgaria * e-mail: milena_n@bio.bas.bg abstract: nikolova, m., jekova, g., dzhurmanski, a.: preliminary investigation of free radical scavenging activity and total phenolic content of three cultivated thymus chemotypes from bulgaria , biologica nyssana, 3 (1), september 2012: 11-15. methanol extracts of three varieties of thymus “slava”, “german winter” and “pagane”, representatives respectively of citral, thymol and geraniol chemotype were evaluated for their antioxidant potential and phenolic content. total content of phenols was determined by spectrophotometric method using folinciocalteu reagent. the antioxidant potential of methanol extracts was estimated by 1,1-diphenyl-2picrylhydrazyl radical (dpph) free radical assay. all of the examined extracts exhibited significant free radical scavenging activity and their ic50 values were below 50 μg/ml. the highest antioxidant activity was determined for the extract of thymol chemotype “german winter” with ic50 value – 19.34 µg/ml. the extract of this variety had the highest amount of phenols 75.61 mg/g extract. the other two varieties “slava” and “pagane” have a lower content of phenols and antiradical activity. influence of three stages of flower development on antiradical activity and total phenolic content was examined for variety “pagane”. the highest antioxidant activity was determined on the extract prepared from plant material collected at the stage “end of flowering”. for the first time, provides data on the phenolic content and antiradical activity of varieties “slava” and “pagane”. these results are the basis for selection of varieties with high antioxidant potential for the preparation of cosmetic and herbal products. key words: thymus vulgaris, thymus marschalianus, dpph, citral, thymol, geraniol introduction the genus thymus l. (lamiaceae) comprises about 300 species of perennial aromatic, herbaceous plants with many subspecies, varieties, subvarieties and forms. the plants are extensively used in phytotherapy, cosmetic and food industries (s t a n e v , 1974; n i k o l o v , 2006). essential oils, flavonoids (highly methylated flavonoids, luteolin derivatives), biphenils and phenolic compounds have been determined as main constituents of the thyme with antioxidant, antiseptic, antiinflamatory and antimicrobial properties (j o v a n o v i ć et al., 1995; h a r a g u c h i et al., 1996; f e c k a & t u r e k , 2008; b o r o s a et al., 2010). the thyme shows a great species diversity which reflects to the composition of the essential oil content. based on the dominant monoterpene in the essential oils of different thymus chemotypes, compounds such as geraniol, thymol, α-terpineol, linalool, carvacrol can be distinguished (t h o m p s o n et al., 2003; c h i z z o l a et al., 3 (1) • september 2012: 11-15 biologica nyssana 3 (1)  september 2012: 11-15 nikolova, m. et al.  preliminary investigation of free radical… 12 2008). in recent years, several reports have been published concerning the antioxidant activity on the essential oils on different thyme chemotype (j u k i et al., 2005; b o u n a t i r o u et al., 2007; m i g u e l et al., 2007; s t o i l o v a et al., 2008; g r i g o r e et al., 2010; t e p e et al., 2011) while the studies on antioxidant activity of methanol extracts from different chemotypes and varieties are insufficient (j o v a n o v i ć et al., 1995; k u l i š i ć et al., 2006). a positive correlation between antioxidant activity and phenolic content in plant extracts has been well documented (m i l i a u s k a s a et al., 2004; k i s e l o v a et al., 2006). widely used methods for assessing the total content of phenols and antioxidant properties of plant extracts are based on folin-ciocalteu reagent and 1-diphenyl-2picrylhydrazyl (dpph) radicals (k i s e l o v a et al., 2006, g i o r g i et al., 2009; n i ć i f o r o v i ć et al., 2010; m a r i n o v a & b a t c h v a r o v , 2011). the purpose of the present work is to examine antioxidant activity and total phenolic content varieties created and cultivated in bulgaria: thyme “slava” (thymus vulgaris, citral chemotype) and “pagane” (thymus marschalianus wild., geraniol chemotype) as well as of the introduced variety “german winter” (thymus vulgaris, thymol chemotype). this study contributes to the knowledge of the antioxidant properties of the thymus chemotypes. materials and methods plant material the plants used for the present study were cultivated in the experimental field of institute of roses, essential and medicinal cultures, kazanlak. aerial parts of two varieties “slava” and “german winter” were collected at full flowering stage. aerial parts of the variety “pagane”, collected in three stages of flowering – beginning of flowering, full flowering and end of flowering were analyzed. preparations of extracts air-dried, powdered plant material (1 g) was extracted with 80% methanol in an ultrasonic bath. after evaporation of the solvent the crude extract was subjected to subsequent analyses. determination of total phenolic content total phenolic content of the methanol extracts was determined by employing the methods given in the literature including folin–ciocalteu reagent and gallic acid as standard (g i o r g i et al., 2009; n i ć i f o r o v i ć et al., 2010). plant extracts were diluted to the concentration of 1 mg/ml, and aliquots of 0.25 ml were mixed with 2.5 ml of folin–ciocalteu reagent (previously diluted 10-fold with distilled water) and 2 ml of na2co3 (6%). after 1 h of staying at the room temperature, the absorbances of the samples were measured at 765 nm on spectrophotometer versus blank sample. total phenols were determined as gallic acid equivalents (mg ga/g extract) by the following formula: c = c x v / m where c total content of phenolic compounds, mg/g plant extract, in gae; c the concentration of gallic acid established from the calibration curve, mg/ml; v the volume of extract, ml; m the weight of pure plant methanolic extract, g. free radical scavenging activity free radical scavenging activity of plant extracts was evaluated using a 1,1-diphenyl-2picrylhydrazyl (dpph) assay (c h o i et al., 2002; s t a n o j e v i ć et al., 2009). different concentrations of plant extract (10, 20, 50, 100 and 200 μg/ml, in methanol) were added at an equal volume (2.5 ml) to methanol solution of dpph (0.3 mm, 1 ml). after 30 min at room temperature, the ab (absorbance) values were measured at 517 nm on a spectrophotometer (jenway 6320d) and converted into the percentage antioxidant activity using the following equation: dpph antiradical scavenging capacity (%) = [ 1( absample – abblank ) / abcontrol ] x 100 plant extract solution (2.5 ml) in methanol (1.0 ml) was used as a blank, while dpph solution in methanol was used as a control. the ic50 values were calculated by sigmoid non-linear regression model using plots, where the abscissa represented the concentration of tested plant extracts and the ordinate the average percent of scavenging capacity (software prizm 3.00). ic50 values denote the concentration of sample required to scavenge 50% of dpph radical. statistical analysis statistical analysis was carried out using excel. all experiments were performed in triplicate. results are presented as a value ± standard deviation (sd). significant levels were defined at p<0.05. biologica nyssana 3 (1)  september 2012: 11-15 nikolova, m. et al.  preliminary investigation of free radical… 13 results and discussion methanol extracts of three chemotypes of thymus (citral, thymol and geraniol) were examined for their antiradical potential using a dpph assay and expressed as ic50 valueextract concentration providing 50% inhibition of the dpph solution (table 1). the all examined extracts exhibited considerable free radical scavenging activity and their ic50 values were below 50 μg/ml. the extract of variety “german winter” (thymol chemotype) showed the strongest antioxidant activity with ic50 value 19.34 µg/ml, followed by citral chemotype “slava” (33.18µg/ml) and geraniol chemotype “pagane” (47.72 µg/ml). the established differences in the antioxidant potential of studied varieties can be explained by different chemotype – different composition of essential oils. the thyme with the highest content of thymol (“german winter”) has the strongest antioxidant activity. this is in accordance with results reported by chizzola et al. (2008) which prove that the essential oil with high content of thymol and/or carvacrol have the highest antioxidant activity. these data allow us to assume antioxidant potential of thyme based on the essential oil composition and individual components (geraniol, linalool, thymol, etc.). a total phenolic content of studied varieties of thymus “slava”, “german winter” and “pagane” was determined (table 1). the extracts of variety “german winter” contain the highest amount of phenols (75.61 mg/g), followed by “slava” (56.6 mg/g) and “pagane” (45.42 mg/g). the greater amount of phenolic compounds positively correlated to more potent radical scavenging effect. carried out correlation analyses shows strong positive mutual dependence between total phenolic content and antioxidant (dpph scavenging) activity of investigated extracts (r=0, 9812). this is in accordance of several reports for such positive correlation between phenols and antioxidant activity of plant extracts (m i l i a u s k a s a et al., 2004; c a i et al., 2004; s h a n et al., 2005; k i s e l o v a et al., 2006; v i l l a ñ o et al., 2007). the antioxidant potential and total phenolic content of extracts obtained from material collected at differing stages of flower development of the variety “pagane” was examined. it was found that extracts of plant material gathered at stage “end of flowering” had the highest antioxidant activity with ic50 value of 34.28 µg/ml (table 2). the extracts prepared from plant material collected at the stages “beginning of flowering” and “full flowering” showed ic50 values 47.72 and 67.00 µg/ml, respectively. thyme is recommended to be picked up at the “end of flowering” stage, when their antioxidant activity is 40% higher than in a “full flowering” stage and 95% higher than in stage “beginning of flowering”. opposite to the results on antioxidant activity, it was found that there are no statistical significant differences (p>0,05) in the phenolic content of the examined extracts during flowering period. total phenols ranges from 44.08 to 46.82 mg/g. this fact suggests that radical scavenging activity of the studied extracts is not only due to phenol compounds. table 1. total phenolic content and free radical scavenging activity of methanol extracts of three thymus varieties thymus varieties total phenols mg gae/g extract* free radical scavenging activity ic50 (µg/ml)* pagane 46.82±1.22 47.72±5.41 slava 56.6±1.25 33.18±1.89 german winter 75.61±2.71 19.34±1.76 * values represent mean ±sd, n=3 table 2. total phenolic and antiradical activity of extracts of variety “pagane” collected at three stage of flowering stage of flowering total phenols mg gae/g extract* free radical scavenging activity ic50 (µg/ml)* beginning of flowering 44.08±1,80 67.00±7.63 full flowering 46.82±1.22 47.72±5.41 end of flowering 45.42±2.62 34.28±1.98 * values represent mean ±sd, n=3 biologica nyssana 3 (1)  september 2012: 11-15 nikolova, m. et al.  preliminary investigation of free radical… 14 conclusion in the present study, the total phenolic content and antioxidant capacity of three varieties of thymus “slava”, “german winter” and “pagane” were determined. high positive correlation was found between investigated indexes. bulgarian varieties thyme – “slava” and “pagane” was studied for the first time for their total phenolic content and the antioxidant activity. the methanol extracts of all examined samples showed high radical scavenging activity (ic50 <50 µg/ml). extracts of thymol chemotype “german winter” exhibited the strongest antiradical activity and highest content of phenols among studied varieties. therefore, thymol chemotype can be priority for utilization in phytoterapy and cosmetics. the recommended concentration of thyme solution for lipid stabilizing in cosmetics and food products is 50 µg/ml, when it provides 95% free radical inhibition whilst for the other types of thyme, it is twice higher. thyme is recommended to be picked up at the “end of flowering” stage, because then the content of antioxidants is the highest. obtained data will be employed as a basis for selection of varieties with high antioxidant potential for the preparation of cosmetic and herbal products, as well as for the optimization of harvesting time. references borosa, b., jakabová, s., dörnyei, a., horváth, g., pluhár z., kilár, f., felingera, a. 2010: determination of polyphenolic compounds by liquid chromatography–mass spectrometry in thymus species. journal of chromatography a, 1217: 7972–7980. bounatirou, s., smiti, s., miguel, m.g., faleiro, l., rejeb, m.n., neffati, m., costa, m.m., figueiredo, a.c., barroso, j.g., pedro, l.g. 2007: chemical composition, antioxidant and antibacterial activities of the essential oils isolated from tunisian thymus capitatus hoff. et link. food chemistry, 105: 146–155 cai, y., luo, q., sun, m., corke, h. 2004: antioxidant activity and phenolic compounds of 112 chinese medicinal plants associated with anticancer. life science, 74: 2157–2184. chizzola, r., michitsch, h., franz, c. 2008: antioxidative properties of thymus vulgaris leaves: comparison of different extracts and essential oil chemotypes, journal of agricultural and food chemistry, 56: 6897–6904. choi, c.w., kim, s.c., hwang, s.s., choi, b.k., ahn, h.j., lee, m.y. 2002: antioxidant activity and free radical scavenging capacity between korean medicinal plants and flavonoids by assay – guided comparsion. plant science, 163: 11611168. nikolov, s. (ed.), 2006: specialized encyclopedia of medicinal plants. trud publishing house, sofia. 566 p. (in bulgarian) fecka, i., turek, s. 2008: determination of polyphenolic compounds in commercial herbal drugs and spices from lamiaceae: thyme, wild thyme and sweet marjoram by chromatographic techniques. food chemistry, 108: 1039-1053. giorgi, a., mingozzi, m., madeo, m., speranza, g., cocucci, m. 2009: effect of nitrogen starvation on the phenolic metabolism and antioxidant properties of yarrow (achillea collina becker ex rchb.). food chemistry, 114: 204-211. grigore, a., ina, p., colceru-mihul, s., bubueanu, c., draghici e., ichim, m. 2010: chemical composition and antioxidant chemical composition and antioxidant activity of thymus vulgaris l. volatile oil obtained by two different methods. romanian biotechnological letters, 15: 5436–5443. haraguchi, h., saito, t., ishikawa, h., date, h., kataoka, s., tamura, y., mizutani, k. 1996: antiperoxidative components in thymus vulgaris. planta medica, 62: 217-221. juki, m., milo, m. 2005: catalytic oxidation and antioxidant properties of thyme essential oils (thymus vulgarae l.). croatica chemica acta, 78: 105-110. kiselova, y., ivanova, d., chervenkov, t., gerova, d., galunska, b., yankova, t. 2006: correlation between the in vitro antioxidant activity and polyphenol content of aqueous extracts from bulgarian herbs. phytotherapy research, 20: 961-965. kulišić, t., dragović-uzelac v., miloš, m. 2006: antioxidant activity of aqueous tea infusions prepared from oregano, thyme and wild thyme. food technology and biotechnology, 44: 485– 492. marinova, g., batchvarov, v. 2011: evaluation of the methods for determination of the free radical scavenging activity by dpph. bulgarian journal of agricultural science, 17: 11-24. miguel, m.g., costa, l.a., figueiredo, a.c., barroso, j.g., pedro, l.g. 2007: assessment of the antioxidant ability of thymus albicans, th. mastichina, th. camphoratus and th. carnosus essential oils by tbars and micellar model systems, natural product communications, 2: 399-406. miliauskasa, g., venskutonisa, p.r., van beek, t.a. 2004: screening of radical scavenging activity of biologica nyssana 3 (1)  september 2012: 11-15 nikolova, m. et al.  preliminary investigation of free radical… 15 some medicinal and aromatic plant extracts. food chemistry, 85: 231–237. nićiforović, n., mihailović, v., masković, p., solujić, s., stojković, a., muratspahić, d.p. 2010: antioxidant activity of selected plant species; potential new sources of natural antioxidants. food and chemical toxicology, 48: 3125-3130. picuric jovanovic, k., milovanovic, m., vrbaski, z. 1995: thymus vulgaris as a sourse of natural lipid antioxidant. review of research work at the faculty of agriculture, 40: 141-146. thompson, j.d., chalchat, j.c., michet, a., linhart, y.b., ehlers, b. 2003: qualitative and quantitative variation in monoterpene cooccurrence and composition in the essential oil of thymus vulgaris chemotype. journal of chemical ecology, 29: 859-880. tepe, b., sarikurkcu, c., berk, s., alim, a., akpulat, h.a. 2011: chemical composition, radical scavenging and antimicrobial activity of the essential oils of thymus boveii and thymus hyemalis. records of natural products, 5: 208220. shan, b., cai, y.z., sun, m., corke, h. 2005: antioxidant capacity of 26 spice extracts and characterization of their phenolic constituents. journal of agricultural and food chemistry, 53: 7749–7759. stanev, d. 1974: study of thymus vulgaris in bulgaria. plant science, 2: 29-33. stanojević, l., stanković, m., nikolić, v., nikolić, l., ristić, d., čanadanovic-brunet, j., tumbas, v. 2009: antioxidant activity and total phenolic and flavonoid contents of hieracium pilosella l. extracts. sensors, 9: 5702-5714. stoilova, i., bail, s., buchbauer, g., krastanov, a., stoyanova, a., schmidt, e., jirovetz, l. 2008: chemical composition, olfactory evaluation and antioxidant effects of an essential oil of thymus vulgaris l. from germany. natural product communications, 3: 1047-1050. villaño, d., fernández-pachón, m.s., moyá, m.l., troncoso, a.m., garcía-parrilla, m.c. 2007: radical scavenging ability of polyphenolic compounds towards dpph free radical. talanta, 71: 230–235. 16 xhulaj, 2019, biologica nyssana 10(2) 10 (2) december 2019: 155-158 doi: 10.5281/zenodo.3600193 preliminary data on lichens from albanian alps (razëm locality, northern albania) original article skerdilaid xhulaj research center of flora and fauna, faculty of natural sciences, university of tirana, bulevardi zog i, nr. 25/1, tirana, albania skerdilaid.xhulaj@fshn.edu.al (corresponding author) received: october 7, 2019 revised: december 3, 2019 accepted: december 6, 2019 abstract: a list of 82 taxa of lichens collected in the albanian alps in two nearby localities is presented here. more than half of them (58 taxa) represent new record for the investigation area. key words: albanian alps, biodiversity, lichens, new records apstract: preliminarni podaci o lišajevima sa albanskih alpa (razëm locality, northern albania) prikazan je spisak od 82 vrste lišajeva prikupljenih u albanskim alpima na dva obližnja lokaliteta. više od polovine njih (58 vrsta) predstavlja novi nalaz za područje istraživanja. ključne reči: albanski alpi, biodiverzitet lišajevi, novi nalazi introduction studies on albanian lichen flora have been and remained low in number. most of these studies have been conducted by foreign authors, increasingly highlighting the need for local specialists in this field. based on historical data, the earliest works belong to körber (1867) and zahlbruckner (1897), who provided information on areas now outside the borders of albania and belonging to montenegro, kosovo and northern macedonia (hafellner & kashta, 2003). data on some specimens of lichens, collected by j. b. kümmerle in the northeastern part of the country, are published by szatala & timko (1926). during his visit in 1924 and 1928, markgraf, while studying albanian vegetation, also collected lichens, most of which were identified by j. hillmann and mentioned by markgraf (1927, 1931) as part of his herbarium. the first important work on albanian lichen flora comes from hafellner & kashta (2003), which provides about 137 taxa of lichens, collected mainly in the north of the country. hafellner (2007), summarizes a list of species reported up to that time for albania by various authors. his list included about 191 taxa of lichens. this list has occasionally been enriched with scarce data from various authors such as: llop et al. (2007), mayrhofer & sheard (2007), hafellner (2009), obermayer (2009), kukwa (2011) and hafellner & zimmermann (2012) (svoboda et al., 2012). the most complete information so far on albanian lichen flora appears to be that of svoboda et al. (2012). of the 43 areas visited within this study, a list of 333 lichen taxa was compiled, 205 of which reported for the first time for albania (svoboda et al., 2012). despite studies and reports to date, the lichen flora of our country, based on climatic and geological conditions, is represented by an even greater number of species. in this aspect, the paper is a contribution to the recognition and enrichment of albanian lichen flora. materials and methods specimens were collected during field trips carried out in kastrat, razëm (malësi e madhe district). the collected specimens were initially divided by growth form and within the growth form were further grouped by the type of their fruiting bodies. lichens were determined by standard microscopic methods (stereo and microscope) and relevant literature: poelt (1969), poelt & vězda (1977, 1981), clauzade & roux (1985), wirth (1995), nimis & © 2019 xhulaj. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 155 13th symposium on the flora of southeastern serbia and neighboring regions martellos (2004), nimis & martellos (2008), smith et al. (2009) and wirth et al. (2013). the collected specimens are deposited in the author’s personal herbarium. the following provides more detailed data on two of six localities where the study was conducted. specimens from other localities, specifically 1, 2, 3 and 6 are still being analyzed in the laboratory and identification is ongoing, so therefore they have not been included here. data about localities locality 4: kastrat, northern albania, malësia e madhe district, c. 3 km from razëm village, beech forest, 42°20’45”n/19°32’52”e, alt. c. 1080 m, 23.04.2018. locality 5: kastrat, northern albania, malësia e madhe district, c. 1.5 km from vila alpini, beech forest, 42°20’24.47’’n/ 19°32’43.56’’e, alt. c. 1112m, 23.04.2018. list of substrates and their abbreviations cor: on bark (used in the case of phorophyte not identified) cal: on limestone ter-cal: on calcareous soil bry: on bryophytes fag: on fagus sylvatica results preliminary results concluded in 82 taxa which have been determined so far. 61 taxa were recorded on trees. a few stones, comprising 14 saxicolous lichens, were picked up together with common terricolous species, 21 in all. species are arranged alphabetically. numbers 4 and 5 correspond to the localities listed above. the abbreviations following this number correspond to the substrates listed also above. author’s initials and numbers in brackets show individual consecutive numbering systems used by the author to identify and locate individual specimens. an asterisk (*) indicates a new record for the investigation area. list of species anaptychia ciliaris (l.) körb. 4, 5 cor (sx 4021, 5001) *arthonia lapidicola (taylor) branth & rostr. 5 cal (sx 5003) *arthonia radiata (pers.) ach. 4, 5 cor (sx 4002, 5004) *aspicilia calcarea (l.) mudd 5 cal (sx 5005) *athallia holocarpa (hoffmann) arup, frödén & søchting 4 cor (sx 4003) *bagliettoa marmorea (scop.) gueidan & cl. roux 4, 5 cal (sx 4004, 5006) *buellia poeltii schauer (turner & borrer ex sm.) almb. 4, 5 fag (sx 4005, 5007) *caloplaca aurantia (pers.) hellb. 5 cal (sx 5008) caloplaca cerina (ehrh. ex hedw.) th.fr. var. cerina 4, 5, cor (sx 4005, 5008) *caloplaca cerinelloides (erichsen) poelt 4, 5 cor (sx 4006, 5010) caloplaca ferruginea (huds.) th. fr. 4 cor (sx 4007) *caloplaca flavorubescens (huds.) j.r. laundon 5 cor (sx 5012) *caloplaca haematites (chaub. ex st.amans) zwackh 5 cor (sx 5013) candelariella vitellina (hoffm.) müll. arg. 4, 5 cor (sx 4007, 5018) candelariella xanthostigma (ach.) lettau 4, 5 cor (sx 4009, 5020) candelariella sp. 4 cor (sx 4010) *catillaria nigroclavata (nyl.) schuler 4 cor (sx 4014) *catinaria atropurpurea (schaer.) vězda & poelt 4 fag (sx 4015) *circinaria contorta (hoffm.) a. nordin, s. savic & tibell 5 cal (sx 5022) *cladonia convoluta (lam.) p. caut. 5 ter-cal (sx 5023) cladonia furcata (huds.) schrad. 5 ter-cal (sx 5024) cladonia rangiformis hoffm. 4 ter-cal (sx 4020) collema subflaccidum degel. 4 cor (sx 4025) *collema subnigrescens degel. 4, 5 cor (sx 4025, 5031) *collema tenax (sw.) ach. 4, 5 cal (sx 4026, 5032) *haematomma ochroleucum var. ochroleucum (neck) j.r. laundon 4, 5 fag (sx 4027, 5034) *hypogymnia tubulosa (schaer.) hav. 4 cor (sx 4028) lathagrium cristatum (l.) otálora, p.m. jørg. & wedin 4, 5 cal (sx 4029, 5035) *lecania cyrtella (ach.) th. fr. 4, 5 cor (sx 4041, 5038) *lecania koerberiana j. lahm 4 cor (sx 4042) *lecanora allophana nyl. 4 cor (sx 4043) 156 biologica nyssana ● 10 (2) december 2019: 155-158 xhulaj ● preliminary data on lichens from albanian alps (razëm locality, northern albania) *phaeophyscia endophoenicea (harm.) moberg 4, 5 cor (sx 4071, 5061) *phaeophyscia orbicularis (neck.) moberg 4, 5, cor (sx 4072, 5062) *phaeophyscia poeltii (frey) clauzade & cl. roux 5 cor (sx 5063) *phlyctis argena (sprengel) flotow 4, 5 cor (sx 4082, 5065) *physcia adscendens (fr.) h.olivier 4, 5, cor (sx 4083, 5066) physcia aipolia (ehrh. ex humb.) fürnr. 4 cor (sx 4084) *physcia biziana (a.massal.) zahlbr. 4 cor (sx 4085) *physcia dubia (hoffm.) lettau 5 cor (sx 5067) *physcia semipinnata (j.f.gmel.) moberg. 4 cor (sx 4086) *physma omphalarioides (anzi) arnold 4, 5 cor (sx 4087, 5068) pseudevernia furfuracea (l.) zopf 5 cor (sx 5081) *ramalina farinacea (l.) ach. 5 cor (sx 5085) *ramonia luteola vězda 4, 5 fag (sx 4092, 5087) *rinodina albana (a. massal.) a. massal. 5 cor (sx 5088) *rinodina pyrina (ach.) arnold 4, 5 cor (sx 4094, 5090) *rinodina sophodes (ach.) massal. 4, 5 cor (sx 4095, 5091) *scoliciosporum umbrinum (ach.) arnold 4, 5 fag (sx 4096, 5092) squamarina cartilaginea (with.) p. james 5 ter-cal (sx 5100) *staurothele caesia (arnold) arnold 5 cal (sx 5101) *variospora velana (a. massal.) arup, søchting & frödén 4, 5 cal (sx 4100, 5102) verrucaria muralis ach. 5 cal (sx 5103) verrucaria nigrescens pers. 4, 5 cal (sx 4101, 5104) xanthoria parietina (l.) th. fr. 4, 5 cor (sx 4105, 5108) *xanthoria polycarpa (hoffm.) rieber 4, 5 cor (sx 4106, 5109) discussion the findings show many aspects. pectenia atlantica, peltigera collina, nephroma laevigatum, nephroma parile, leptogium saturnium, lecanora intumescens, ramonia luteola, catinaria artropurpurea show that the forest is primeval. the two last mentioned lecanora carpinea (l.) vain. 4 cor (sx 4044) *lecanora chlarotera nyl. 5 cor (sx 5040) *lecanora coiliocarpa (ach.) nyl. 4, 5 cor (sx 4045, 5041) *lecanora hagenii (ach.) ach. 5 cor (sx 5042) *lecanora intumescens (rebent.) rabenh. 4 fag (sx 4046) *lecanora leptyrodes (nyl.) degel. 5 cor (sx 5043) *lecanora muralis (schreb.) rabenh. 5 cal (sx 5044) *lecanora subfuscata f. variolosa (körb.) grummann 4, 5 cor (sx 4047, 5045) *lecidella elaeochroma (ach.) m. choisy 5 cor (sx 5046) lecidella euphorea (flörke) hertel 4 cor (sx 4048) *leptogium massiliense nyl. 4 cal (sx 4049) leptogium saturninum (dickson) nyl. 4, 5 cor (sx 4050, 5047) *lobothallia radiosa (hoffm.) hafellner 5 cal (sx 5048) melanelixia glabra (schaer.) o.blanco et al. 4 cor (sx 4051) *nephroma laevigatum ach. 4, 5 bry (sx 4061, 5050) nephroma parile (ach.) ach. 5 cor (sx 5051) *ochrolechia alboflavescens (wulfen) zahlbr. 4, 5 cor (sx 4062, 5052) *parmelia atlantica ach. 4 cor (sx 4063) *parmelia exasperata de not. 5 cor (sx 5053) parmelina pastillifera (harm.) hale 5 cor (sx 5054) *parvoplaca cf. suspiciosa (nylander) arup, søchting & frödén 4, 5 cor (sx 4064, 5055) pectenia atlantica (degel.) p.m. jørg. 4, 5 cor (sx 4065, 5056) *peltigera collina (ach.) schrad. 4, 5 fag (4066, 5057) *pertusaria albescens var. corallina (zahlbr.) j.r. laundon 4, 5 cor (sx 4067, 5058) *pertusaria hymenea (ach.) schaer. 4 cor (sx 4068) pertusaria pertusa (l.) tuck. 4, 5 cor (sx 4069, 5059) *phaeophyscia ciliata (hoffm.) moberg 4, 5 cor (sx 4070, 5060) 157 biologica nyssana ● 10 (2) december 2019: 155-158 xhulaj ● preliminary data on lichens from albanian alps (razëm locality, northern albania) species also show an alpine feature together with lecanora leptyrodes, buellia poeltii, phaeophyscia poeltii, and lecanora coiliocarpa. an oceanic feature is disclosed by physma omphalarioides, collema subnigrescens and parmelia atlantica. especially the habitation, but also a road through the area bring about the presence of physcia, xanthoria and rinodina species, and lecanora allophana. everywhere in the mountain area a rather common species on deciduous trees was a parvoplaca cf. suspicosa which is a scandinavian species (hitherto found only in the north of sweden). presumably the specimens found in razëm represent some mediterranean alpine species. some taxa have not been finally determined. by defining all the taxa found, a more complete interpretation of the lichen flora of this area can be made. references clauzade, g., roux, c. 1985: likenoj de okcidenta eŭropo. ilustrita determinlibro. bulletin de la société botanique du centre-ouest. nouvelle série. numéro spécial, 7: 1-893. hafellner, j., kashta, l. 2003: miscellaneous records of lichens and lichenicolous fungi from albania. herzogia, 16: 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hauck, m., schultz, m. 2013: die flechten deutschlands. 2 bände. stuttgart: eugen ulmer: 1244 pp zahlbruckner, a. 1897: lichenes albanici a cl. j. dörfler anno 1893 lecti. rep. krypt. lit. beibl. hedwigia 36: 1-4. biologica nyssana ● 10 (2) december 2019: 155-158 xhulaj ● preliminary data on lichens from albanian alps (razëm locality, northern albania) lošić et al. 2020, biologica nyssana 11(1) 11 (1) september 2020: 55-58 doi: 10.5281/zenodo.4060300 total phenol content and antioxidant potential of different brassica oleracea varieties original article ajna lošić laboratory for plant physiology, faculty of science, university of sarajevo, sarajevo, bosnia and herzegovina ajna.losic@gmail.com adisa parić laboratory for plant physiology, faculty of science, university of sarajevo, sarajevo, bosnia and herzegovina adisacausevic@hotmail.com arnela demir laboratory for plant physiology, faculty of science, university of sarajevo, sarajevo, bosnia and herzegovina arnela_s1@yahoo.com erna karalija laboratory for plant physiology, faculty of science, university of sarajevo, sarajevo, bosnia and herzegovina erna.karalija@gmail.com (corresponding author) received: february 20, 2020 revised: march 24, 2020 accepted: march 28, 2020 abstract: varieties of brassica oleracea are commonly used in human nutrition. beside nutritive role it is suggested that diets reach in cabbage play important role in disease prevention. the aim of this study was to determine total phenol content and the antioxidant potential of different brassica oleracea varieties (broccoli, brussel sprouts, cauliflower, green cabbage and red cabbage). the highest content of total phenols was found in red cabbage and the lowest in broccoli. red cabbage had the highest ability to remove free radicals, while the lowest radical scavenging activity was recorded for broccoli. strong correlation between phenolic content and antioxidant activity suggests the importance of phenolics in health benefits of cabbages. the study identified differences between different varieties suggesting different nutritional advantages, providing useful information in selection of right cabbage variety as diet supplement in disease prevention. key words: brussel sprouts, broccoli, cauliflower, dpph radical scavenging, green cabbage, phenols, red cabbage apstract: ukupan sadržaj fenola i antioksidativni potencijal različitih varijeteta brassica oleracea različiti varijeteti vrste brassica oleracea često se koriste u ishrani ljudi. pored hranljive uloge, smatra se da ishrana bogata kupusom ima važnu ulogu u prevenciji bolesti. cilj ovog istraživanja bio je da se utvrdi ukupan sadržaj fenola i antioksidativni potencijal različitih varijeteta vrste brassica oleracea (brokoli, kelj pupčar, karfiol, zeleni kupus i crveni kupus). najveći sadržaj ukupnih fenola utvrđen je u crvenom kupusu, a najmanji u brokoliju. crveni kupus je imao najveću sposobnost uklanjanja slobodnih radikala, dok je najmanja aktivnost uklanjanja radikala zabeležena kod brokolija. izražena korelacija između sadržaja fenola i antioksidativne aktivnosti ukazuje na značaj fenola u zdravstvenim prednostima kupusa. studija je identifikovala razlike između različitih varijeteta koje sugerišu različite nutritivne prednosti, pružajući korisne informacije u izboru prave sorte kupusa kao dodatak ishrani u prevenciji bolesti. ključne reči: kelj pupčar, brokoli, karfiol, dpph uklanjanje radikala, zeleni kupus, fenoli, crveni kupus introduction brassicaceae (cruciferae) or the cabbage family includes many economically important, edible and industrial types of oilseeds, vegetables, spices and fodder. this family is also known to comprise more than 120 wild species, some of which are very important for agriculture e.g. sinapsis arvensis and raphanus sativus (warwick, 2011). the long history of plant selection has resulted that this crop group is highly variable in use, appearance and phytochemistry. brassica oleracea is a good example with several important varieties: broccoli, cauliflower, brussel sprouts, cabbage, kale (bjorkman et al., 2011). oxidative stress has been identified as a major cause of the development and progression of several diseases. supplementation with exogenous antioxidant or enhancement of endogenous antioxidant defence of the body is a promising way of counteracting the reactive oxygen species (ros) side effects that cause oxidative damage. plants have long been a source of exogenous antioxidants. two-thirds of © 2020 lošić et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 55 the worlds’ plant species are thought to be medically significant, and almost all of them have excellent antioxidant potential. plants have effective, complex enzymatic and non-enzymatic antioxidant defence systems to avoid the toxic effects of free radicals and non-enzymatic antioxidant system including secondary metabolites as antioxidants (kasote et al., 2015). phenolic compounds from plants represent the largest group of secondary plant metabolites found in fruits, vegetables and teas. they are characterized by antioxidant, anti-inflammatory, anticancerogenic and other biological properties, and can protect plants from oxidative stress and some diseases (ahmed et al., 2017). over the last few decades, vegetables from the brassicaceae family have come under scientific attention because of numerous epidemiological studies that provide evidence that diets rich in these vegetables are associated with a reduces risk of several types of cancer and other chronic diseases. because of that, these vegetables are considered promising sources of metabolites for cancer prevention, and mechanism of anticancerogenic activity is mostly associated with glucosinolate degradation products (šamec et al., 2019). the aim of this study was to determine the content of total phenols and antioxidant potential of selected brassica oleracea varieties (broccoli, cauliflower, brussel sprouts, green cabbage, and red cabbage). material and methods plant material five mostly consumed brassica oleracea varieties were selected for the analysis: b. oleracea var. capitata (green cabbage) – fresh leaves; b. oleracea var. capitata f. rubra (red cabbage, bosnia and herzegovina) – fresh leaves; b. oleracea var. italica (broccoli) – broccoli flowers (frozen by rapid freezing technique); b. oleracea var. botrytis (cauliflower) – fresh flowers of cauliflower; b. oleracea var. gemmifera (brussel sprouts) – axillary buds were used (frozen by rapid freezing technique). extract preparation all samples were air-dried and extraction was performed by maceration with 80% ethanol. prepared macerate was incubated in the dark at 4 °c for 24 hours. after incubation, macerate was centrifuged at 5000 rpm for 30 minutes, and supernatant was collected for the analysis. extract yield was determined for all samples. analysis of total phenol content analysis of phenol content in ethanol extracts of different b. oleracea varieties was performed using folin ciocalteu reagent (fcr), by diluting 20 ml of extract with 1580 ml water and adding 100 ml of fcr, followed by addition of 300 ml 7.5% na2co3 after 3 min of incubation (wolfe et al., 2003). total phenols were quantified based on the direction of the calibration curve of gallic acid. the obtained values are expressed as mg of gallic acid equivalents per gram of dry material (mg ga/gdw). analysis of antioxidant capacity the dpph (2,2 – diphenyl – 1 – picrylhydrazyl) method was used to determine the antioxidant activity of the tested extracts (mishra et al., 2012). a stock solution of dpph was prepared by weighing 50 mg of dpph and dissolving in 100 ml of 96% ethanol. a working dpph was prepared by diluting 5 ml of stock solution with 96% ethanol to the 50 ml mark and by adjusting dpph absorbance reading to 1.123 at 517 nm (lambda 25; perkin elmer). extract testing was performed by mixing 100 ml of extract with diluted dpph and final absorbance reading was recorded after 30 minutes against 96% ethanol (blank). free radical scavenging activity was calculated by the formula: aa% antioxidant capacity; a(t0) – absorbance at 0 seconds; a(60) – absorbance after 60 seconds. naringenin was used as a standard in concentration of 1 mg/ml. statistical analysis the data were expressed as means of three independent replicates ± standard deviation. all data were statistically evaluated by analysis of variance with the anova test (p< 0.05 significance level), and the means were analyzed by regression analysis by newman keuls test. pearson’s correlation was used to assess relationships between the variables. all statistical testing was performed using statistica 10 software (copyright© statsoft, inc. 19842011). results and discussion differences in extract yield for different b. oleracea cultivars were recorded. the highest yield was noted for green cabbage (21.5%), followed by red cabbage, cauliflower and sprouts, and the lowest yield was registered for broccoli (2.6%) (tab. 1). the differences in extract yield for the analyzed b. oleracea varieties can be attributed to the availability of the components that are extracted from the sample, which in itself depends on the analyzed variety (hsu et al., 2006). also, the effectiveness of the solvent 56 biologica nyssana ● 11 (1) september 2020: 55-58 lošić et al. ● total phenol content and antioxidant potential of different brassica oleracea varieties 57 biologica nyssana ● 11 (1) september 2020: 55-58 lošić et al. ● total phenol content and antioxidant potential of different brassica oleracea varieties used for extraction to dissolve endogenous components has a significant effect on the yield of the extracts (siddhuraju & becker, 2003). other factors that can affect yield of extract solvent polarity, extraction time and temperature as well as the chemical nature of the sample itself (sun & ho, 2005). the choice of extraction solvent was selected according to targeted components (phenolics). the analysis of total phenolic content showed high concentrations in red cabbage, while broccoli contained only small amounts of phenolics (tab. 1). compared to other extraction solvents, 80% ethanol is less effective and methanol extracts are richer in phenolics as previously recorded by jaiswal et al. (2011). high phenolic content in red cabbage is probably result of high sinapic acid content as previously recorded in other studies (mattila & hellström, 2007). analysis of dpph radical scavenging activity by ethanol extracts of different varieties of b. oleracea are shown in tab. 1. the antioxidant capacity of the tested extracts ranged from 86.63% to 7.14% for red cabbage and broccoli, respectively. total phenolic content strongly correlated with dpph scavenging activity (r2 0,9755; p<0,05). high antioxidant capacity of red cabbage has been previously recorded by several authors (podsędek et al., 2006; isabelle et al., 2010; rokayya et al., 2013; liang et al., 2019). miller et al. (2000) studied the antioxidant effects of different vegetables and noticed a striking difference between red and green cabbage, suggesting a strong role of red cabbage pigment in antioxidant activity. oxidative stress is one of major causes for pathophysiology of several cardiovascular diseases (lahera et al., 2007). dietary phenolic antioxidants play significant role in counteracting cardiovascular diseases (chiu et al., 2018) and intake of red cabbage is beneficial for human health, as suggested by our study as well. conclusion presented study evaluated different brassica oleracea varieties for their phenolic content and antioxidative capacity. among the tested varieties, red cabbage had the highest phenolic content and significant antioxidant capacity, suggesting the importance of red cabbage in human nutrition. references ahmed, e., arshad, m., khan, m.z., amjad, h.s., sadaf, h.m., riaz, i., sabir, s., ahmad, n., 2017: secondary metabolites and their multidimensional prospective in plant life. journal of pharmacognosy and phytochemistry, 6 (2): 205-214. björkman, m., klingen, i., birch, a.n., bones, a.m., bruce, t.j., johansen, t.j., meadow r., mølmann, j., seljåsen, r., smart, l.e., stewart, d. 2011: phytochemicals of brassicaceae in plant protection and human health-influences of climate, environment and agronomic practice. phytochemistry, 72 (7): 538-556. hsu, b., coupar, i.m., ng, k. 2006: antioxidant activity of hot water extract from the fruit of the doum palm, hyphaene thebaica. food chemistry, 98 (2): 317-328. table 1. extract yield, total phenol content and antioxidant capacity of different brassica oleracea varieties sample extract yield (%) total phenol content (mg ga/g dw) antioxidant capacity (%) red cabbage b. oleracea var. capitata f. rubra 12.00b ± 0.10 27.97a ± 3.23 86.63a ± 1.97 green cabbage b. oleracea var. capitata 21.50a ± 0.12 7.89bc ± 0.88 19.88c ± 0.60 broccoli b. oleracea var. italica 2.60d ± 0.09 3.41d ± 0.73 7.14d ± 0.30 brussel sprouts b. oleracea var. gemmifera 9.30c ± 0.09 6.78c ± 0.67 25.76b ± 0.26 cauliflower b. oleracea var. botrytis 11.20bc ± 0.11 8.78b ± 0.90 23.49b ± 1.28 standard (naringenin) 90.00a ± 3.89 data expressed as means ± standard deviation. means of samples not sharing the same letter in one column are significantly different (p < 0.05); ga equivalents of gallic acid; dw dry weight. 58 isabelle, m., lee, b.l., lim, m.t., koh, w.p., huang, d., ong, c.n. 2010: antioxidant activity and profiles of common vegetables in singapore. food chemistry, 120 (4): 993-1003. jaiswal, a.k., rajauria, g., abu-ghannam, n., gupta, s. 2011: phenolic composition, antioxidant capacity and antibacterial activity of selected irish brassica vegetables. natural product communications, 6 (9): 1299-1304. kasote, d.m., katyare, s.s., hegde, m.v., bae, h. 2015: significance of antioxidant potential of plants and its relevance to therapeutic applications. international journal of biological sciences, 11 (8): 982–991. lahera, v., goicoechea, m., garcia de vinuesa, s., miana, m., heras, n.d.l., cachofeiro, v., luno, j. 2007: endothelial dysfunction, oxidative stress and inflammation in atherosclerosis: beneficial effects of statins. current medicinal chemistry, 14 (2): 243-248. liang, y., li, y., zhang, l., liu, x. 2019: phytochemicals and antioxidant activity in four varieties of head cabbages commonly consumed in china. food production, processing and nutrition, 1 (1): 3. mattila, p., hellstrōm, j. 2007: phenolic acids in potatoes, vegetables, and some of their products. journal of food composition and analysis, 20 (3): 152-160. miller, h.e., rigelhof, f., marquart, l., prakash, a., kanter, m. 2000: antioxidant content of whole grain breakfast cereals, fruits and vegetables. journal of the american college of nutrition, 19 (3): 312-319. mishra, k., ojha, h., chaudhury, n.k. 2012: estimation of antiradical properties of antioxidans using dpph assay: a critical review and results. food chemistry, 130 (4): 1036-1043. podsȩdek, a., sosnowska, d., redzynia, m., anders, b. 2006: antioxidant capacity and content of brassica oleracea dietary antioxidants. international journal of food science and technology, 41 (1): 4958. rokayya, s., li, c.j., zhao, y., li, y., sun, c.h. 2013: cabbage (brassica oleracea l. var. capitata) phytochemicals with antioxidant and antiinflammatory potential. asian pacific journal of cancer prevention, 14 (11): 6657-6662. siddhuraju, p., becker, k. 2003: antioxidant properties of various solvent extracts of total phenolic constituents from three different agroclimatic origins of drumstick tree (moringa oleifera lam.) leaves. journal of agricultural and food chemistry, 51 (8): 2144-2155. sun, t., ho, c.t. 2005: antioxidant activities of buckwheat extracts. food chemistry, 90 (4): 743749. šamec, d., urlić, b., salopek-sondi, b. 2019: kale (brassica oleracea var. acephala) as a superfood: review of the scientific evidence behind the statement. critical reviews in food science and nutrition, 59 (15): 2411-2422. warwick, s.i. 2011: brassicaceae in agriculture. in: schmidt, r., bancroft, i. (eds.), genetics and genomics of the brassicaceae, 33-65, springer, new york, usa. wolfe, k., wu, x., liu, r.h. 2003: antioxidant activity of apple peels. journal of agricultural and food chemistry, 51 (3): 609-614. biologica nyssana ● 11 (1) september 2020: 55-58 lošić et al. ● total phenol content and antioxidant potential of different brassica oleracea varieties savić, a., đorđević, m., jušković, m., pešić, v.: ecological analysis of macroinvertebrate communities based on functional feeding types: a case study in southeastern serbia. biologica nyssana, 8 (2). biologica nyssana 8 (2)  december 2017: 159-166 savić, a. et al.  ecological analysis of macroinvertebrate communities… 159 original article received: 22 november 2017 revised: 30 november 2017 accepted: 17 december 2017 ecological analysis of macroinvertebrate communities based on functional feeding groups: a case study in southeastern serbia savić ana1*, đorđević miodrag2 , jušković marina1, vladimir pešić3 1department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, 18000 nis, serbia 2department of mathematics, faculty of sciences and mathematics, university of niš, višegradska 33, 18000 nis, serbia 3department of biology, university of montenegro, podgorica, montenegro * e-mail: anka@pmf.ni.ac.rs abstract: savić, a., đorđević, m., jušković, m., pešić, v.: ecological analysis of macroinvertebrate communities based on functional feeding groups: a case study in southeastern serbia. biologica nyssana, 8 (2). december, 2017: 159-166. to examine if the macroinvertebrate community corresponds to the rcc (river continuum concept), which describes longitudinal patterns in allochthonous and autochthonous energy input and the associated feeding categories of macroinvertebrates along the lotic continuum, it was necessary to determine the functional feeding groups – ffgs. the goals of the research were: to examine whether a macroinvertebrate community matches the rcc; to use the functional feeding groups to determine the attributes of the ecosystem; to test if the itc index is appropriate for use in this part of the world and to determine water quality along the river course of the nišava river. macroinvertebrate samples and physicochemical data were analyzed for 10 localities along the 151 km long stretch of the nišava river in southeastern serbia, over a one-year period. out of all the collected specimens (9837 individuals) 49.6% belong to shredders, 31.77% to scrapers, 12.25% to collectors and 6.4% to predators. on the annual level only locality nine belongs to autotrophic type; relation p/r=1.44. locality four has the most heterotrophic character, the lowest channel stability and the most disturbed predator-prey relationships. the results of the research reveal that the trends found in relative functional group abundance do not correspond with the tendencies predicted by the rcc. the ffg ratios surrogate are consistent with the observations of the properties of the ecosystem at the sampling localities. according to the index of trophic completeness, most of the localities included in the research belong to good water quality (seven out of ten), while other localities belong to moderate water quality (three out of ten). key words: macroinvertebrates, functional feeding groups, index of trophic completeness apstrakt: savić, a., đorđević, m., jušković, m., pešić, v.: ekološka analiza zajednice makroinvertebrata na osnovu funkcionalnih grupa zasnovanih na tipu ishrane: primer iz jugoistočne srbije. biologica nyssana, 8 (2), decembar, 2017: 159-166. rcc (river continuum concept) opisuje longitudinalne šablone u inputu autohtone i alohtone energije, i njihovu povezanost sa kategorijama ishrane makroinvertebrata duž rečnog kontinuuma. da bi se utvrdilo da li 8 (2) • december 2017: 159-166 doi: 10.5281/zenodo.1135973 biologica nyssana 8 (2)  december 2017: 159-166 savić, a. et al.  ecological analysis of macroinvertebrate communities… 160 zajednica makroinvertebrata prati rcc neophodno je odrediti funkcionalne grupe na osnovu tipa ishrane (ffg). tokom jedne godine makroinvertebratska zajednica i fizičko hemijski podaci su analizirani na 12 lokaliteta duž 151 km rečnog toka nišave, koja se nalazi na jugoistoku srbije. od ukupnog broja individua (10519) 48.99% pripada sekačima, 31.49% grebačima, 13.02% sakupljačima i 6.5% predatorima. na osnovu ffg odredjeni su sledeći ekosistemski atributi: autotrofnost/heterotrofnost lokaliteta, stabilnost rečnog kanala, odnos predator-plen i odnos cpom/fpom (krupne čestice organske materije/sitne čestice organske materije). na godišnjem nivou, samo lokalitet 9 ima autotrofnu prirodu (p/r=1.44). lokalitet 4 je sa najizraženijim heterotrofnim karakterom, sa najmanjom stabilnošću rečnog kanala i sa najnarušenijim odnosom predator-plen. odnos cpom/fpom pokazuje da reka nišava ima regularnu asambleju sekača (na svim lokalitetima odnos je >0.25) što je povezano sa uslovima u riparijalnoj zoni. ključne reči: makroinvertebrate, funkcionalne grupe na osnovu tipa ishrane, indeks of trophic completeness introduction of all the freshwater organisms that have been considered for use in biological monitoring, benthic macroinvertebrates are recommended most often (c a r t e r et al., 2007). macroinvertebrates have been used to evaluate the effects of anthropogenic stressors at all levels of biological organization, from the molecular to the ecosystem (r o s e n b e r g & r e s h , 1993). functional feeding group (ffg) approach, described more than 40 years ago (c u m m i n s , 1973), has been modified in some details since then (e.g., c u m m i n s & k l u g , 1979, w a l l a c e & m e r r i t t , 1980, c u m m i n s & w i l z b a c h , 1985, m e r r i t t & c u m m i n s 1996, m e r r i t t et al., 1999, 2002), but the basis of ffg relationships remains quite simple. ffgs are based on a direct correspondence between the categories of nutritional resources present in the environment and the populations of freshwater invertebrates that are adapted to efficiently harvest a given food resource (m e r r i t t & c u m m i n s , 2007). the analysis of the trophic structure of benthic macroinvertebrate communities can be used in biological assessments of the condition of river ecosystems. using the trophic or functional approach, the index of trophic completeness (itc) was developed (p a v l u k et al., 2000). also, ffg ratios can be used as surrogates for these aquatic ecosystem attributes and serve as a useful assessment of the ecological condition freshwater ecosystems (m e r r i t t & c u m m i n s , 2007). in this study, we wanted to examine whether the macroinvertebrate community matched the rcc (river continuum concept) (v a n o t e et al., 1980) in the river, or its trophic structure (distribution pattern of ffg) was more influenced by pollution. the goal was to use functional feeding groups to determine the ecosystem attributes: indication of autotrophic/heterotrophic type; ratio between fpom fig. 1. the map of the studied area with localities biologica nyssana 8 (2)  december 2017: 159-166 savić, a. et al.  ecological analysis of macroinvertebrate communities… 161 in transport and fpom in sediment; stability of channels (river channel resistance capacity to the detachment of bed and bank materials); and predator control. another goal of the research was to test is itc index appropriate for use in this part of the world, because there was no evidence about its use in the region of the balkan peninsula. in addition, the study set out to determine water quality along the river course of the nišava river based on these two types of analysis. material and methods the nišava river (fig. 1) belongs to the black sea drainage basin. it originates in western bulgaria, on the stara planina mt., and flows in a southeastnorthwest direction. it is 218 km long, of which 67 km flows through bulgaria, and 151 km through serbia. ten localities were chosen along the entire course of the nišava river in serbia. the odd numbered localities are positioned upstream of the settlements, and even numbered downstream of the settlements. sampling was performed each month, from may 2006 to april 2007. all localities were sampled on a single day during each field trip. biochemical oxygen demand (bod5) was estimated using the standard methodology recommended by apha (1999). dissolved oxygen, ph and conductivity were measured using a wtw®multi 340i probe. the concentration of total nitrogen (tn) and phosphorus (tp) were determined in the field, using a photometer – system pc multi direct lovibond ® meter. the percentage of substrate was observed visually; the classification of mineral substrates by particle sizes according to w e n t w o r t h (1922) and v e r d o n s c h o t (1999) was used. since larger substrates require greater stream power for movement, they are physically more stable (g u r t z & w a l l a c e 1984); thus, pebbles, cobbles and boulders consolidated a stable substrate. water turbidity was measured with a lovibond® checkit device. stream order was determined according to s t r a h l e r (1952). macrozoobenthos were sampled at each locality over a 50 m river stretch with a square frame kick net (35 × 35 cm, mesh size 300 μm). three 3minute samples were taken during each visit to include different substrates (boulder, cobble, pebble, sand, silt, and detritus) and flow regime zones at each location. the net was held perpendicular to the flow and the substrate was vigorously disturbed in front of the net. as the substrate was disturbed, sampling moved progressively upstream. the three samples were then pooled, representing a single monthly sample for each site. this sampling procedure was previously evaluated by preliminary test sampling, and three replicates proved to be sufficient to capture the maximum number of taxa. all samples were elutriated in the field and the organisms were fixed in 4% formaldehyde solution and returned to the laboratory for sorting. the material was identified using identification keys: for oligochaeta b r i n k h u r s t & j a m i e s o n (1971) and h r a b e (1981); for hirudinea m a n n & w a t s o n (1964); for freshwater snails m a c a n & c o o p e r (1994) and p f l e g e r (1990, 2000); for odonata n i l s o o n (1997) and b e š o v s k i (1994); for ephemeroptera b e l f i o r e (1983) and e l l i o t et al. (1988), for trichoptera w a l a c e et al. (1990), e d i n g t o n & h i l d r e w (1995) and p e s c a d o r et al. (1995), for plecoptera h y n e s (1967) and z w i c k (2004); for diptera n i l s s o n (1997), v a l l e n d u u k & p i l l o t (2007) and p i l l o t (2009). for each taxon the functional feeding group was determined based on: m o o g (1995), g r a f et al. (2006) and p a v l u k et al. (2000). the ecosystem attributes where calculated according to m e r r i t t & c u m m i n s (2007) and m e r r i t t et al. (2002). itc index and water quality class are determined by de v a a t e & p a v l u k (2004). quality class score is calculated by the formula:    n i itot cc 1 , where ctot is the total score, n is the number of trophic guilds present in the data-set, and ci is the ln transformed indication value of trophic guild i. the relation between ctot and the quality classes is given in de v a a t e & p a v l u k (2004). results the community of macroinvertebrates was collected at ten localities along the entire course of the nišava river. the river is a 4th to 7th stream order (according to s t r a h l e r , 1952) (tab. 1). table 1 shows that the concentrations of total nitrogen and total phosphorus are the highest at localities 4 and 10. these localities are under the highest anthropogenic pressure. the 4th locality is under the impact of industrial wastewater from tire factory ‘tigar’. the 10th locality is downstream from the biggest city along the entire length of the river – niš. during one-year research 9837 individuals of macroinvertebrates were collected. out of the total number, shredders were the most present 4877 individuals. they were followed by scrapers (3125 individuals), collectors (1205 individuals) and finally predators (630 individuals) (fig. 2). biologica nyssana 8 (2)  december 2017: 159-166 savić, a. et al.  ecological analysis of macroinvertebrate communities… 162 table 2 shows that the highest number of predators were detected at localities 4 and 10, which have already been mentioned as localities with the highest concentrations of nutrients. the greatest percentage of predators are present at locality 4 (fig. 3). fig. 2. percentage of ffg on annual level according to the ratio p/r (tab. 3) at annual level, nine localities on the nišava river were characterized as heterotrophic (the exception is locality 9). according to the ratio tfpom/bfpom, fpom in transport is greater than normal particulate loading in suspension on locality 7. at other localities, there is more fpom in sediments (benthos) than in suspension. the stability of the river channel is the lowest at locality 4. also, it is low at locality 10. the ratio cpom/fpom indicates that the river nišava has normal shedder association linked to functioning riparian system along the whole river stretch. the predator-prey relationship is the most disturbed at locality 4. according to d e v a a t e & p a v l u k (2004), localities 2, 3, 5 and 7 belong to class ii (good quality), subclass i; locality 8 belongs to class ii (good quality), subclass ii; localities 6 and 9 belong to class ii (good quality), subclass iii. on the other hand, localities 1, 4 and 10 belong to class iii (moderate), subclass ii (tab. 4). discussion the trends found in relative functional group abundance in the research do not correspond with the tendencies predicted by the rcc. similar results were obtained in the research conducted by s k a f f (2010). the rcc assumes that the highest proportion of shredders will be present in lowstreams order (v a n n o t e et al. 1980). the data obtained in this study illustrate that this is not the case more shredders (in %) were found at locality 10 (the 7th stream order) than at locality 1 (the 4th stream order). one possible explanation for the disparity between the observed and the expected feeding group proportions may be relatively small ranges of physical stream characteristics found between stream orders. the 6th order (locality 3) has average depth 89.5 cm, while the 7th order (the locality 10) has average depth 96.5 cm (s a v i ć , 2012). similar results were obtained by s k a f f (2010). the use of stream order to characterize a stream is sometimes deemed to be a misrepresentation of the true size. some researchers consider drainage area and discharge to be better indicators (a l l e n & h o e k s t a , 1992). another explanation could be simply a general lack of riparian cover to supply litter inputs (c u m m i n s et al., 2005) in low stream order parts of the river stretch. according to the p/r ratio, nine sites (out of ten) were characterized as heterotrophic (tab. 3). it means that the dominant base food chain for the invertebrate communities, at most localities, was judged to be allochthonous detritus, largely from the riparian zone. locality 4 was the most heterotrophic. 12% 6% 32% 50% collectors predators scrapers shredders table 1. average annual values of environmental parameters at each locality (loc) studied along the nišava river. so-stream order; tp-total phosphorus in mg/l; tn-total nitrogen in mg/l; o-oxygen in mg/l; bod5biochemical oxygen demand in mg/l; tu-turbidity in ntu; con-conductivity in s/cm; ss-stable substrate in %. loc 1 loc 2 loc3 loc 4 loc 5 loc 6 loc 7 loc 8 loc 9 loc 10 so 4 5 6 6 7 7 7 7 7 7 tp 0.02 0.08 0.04 0.10 0.07 0.07 0.07 0.06 0.07 0.11 tn 0.27 0.28 0.08 0.34 0.10 0.12 0.12 0.08 0.09 0.21 o 6.72 7.28 8.04 7.09 7.85 8.34 8.37 7.98 7.56 6.50 bod5 1.68 3.04 2.74 4.16 3.08 3.37 3.61 3.08 3.17 2.99 ph 7.56 6.54 6.17 6.59 6.98 6.56 6.30 7.34 6.15 6.54 tu 26.60 35.92 12.33 18.20 21.98 20.72 5.28 12.90 6.94 3.75 con 496.00 536.42 460.08 459.42 395.75 403.83 414.17 411.08 413.58 575.67 ss 67.50 79.67 78.33 46.50 76.67 52.58 83.75 89.17 66.67 70.42 biologica nyssana 8 (2)  december 2017: 159-166 savić, a. et al.  ecological analysis of macroinvertebrate communities… 163 this is in accordance with the fact that the highest value of bod5 and the lowest value of oxygen concentration were found there (tab. 1). the tfpom/bfpom ratio revealed significant amounts of fpom in transport at locality 7 (tab. 3) which was an indicator of high quality of water (c u m m i n s et al., 2005). this is in accordance with the fact that the highest concentration of oxygen was detected at locality 7 (tab. 1). the stability of the stream channel is the key attribute of a stream/river ecosystem (m e r r i t t & c u m m i n s , 2007) as reflected in the relative permanence (stability) of various bottom materials. if the bottom is stable, invertebrates that cling to surfaces of stones or large wood, while feeding on attached algae will be more abundant (c u m m i n s et al., 2005). the ffg surrogate ratio for channel stability revealed that localities 1, 2, 4 and 10 were below the threshold of 0.50. localities 3 and 5 were on the borderline. the lowest values were at locality 4. this is also in accordance with the fact that the lowest percentage of stable substrates were found at locality 4 (tab. 1). according to m e r r i t t et al. (2002) for the ffg surrogate ratio for predator-prey balance the locality 4 is (tab. 3) the locality with the most deviation from typical values of this ratio (<0.15). in other words, only localities 4 and 10 are not with ‘normal’ predator-prey balance, or top-down control. table 2. number of specimens of ffg at each locality localities 1 2 3 4 5 6 7 8 9 10 collectors 34 228 301 16 115 91 187 124 68 41 predators 24 60 62 170 45 34 80 27 29 99 scrapers 38 197 186 3 834 329 407 371 731 29 shredders 99 416 247 90 1687 573 563 379 432 391 fig. 3. percentage of ffg at ten localities table 3. ecosystem attributes calculated using ffg according to m e r r i t t and c u m m i n s (2007). localities p/r tfpom/bfpom stability of the river channel top-down control 1 0.29 0.12 0.32 0.14 2 0.31 0.37 0.43 0.07 3 0.34 0.18 0.47 0.09 4 0.02 0 0.03 1.56 5 0.45 0.07 0.47 0.02 6 0.49 0.38 0.55 0.02 7 0.54 0.62 0.71 0.07 8 0.74 0.1 0.79 0.03 9 1.44 0.23 1.51 0.01 10 0.07 0.02 0.07 0.2 biologica nyssana 8 (2)  december 2017: 159-166 savić, a. et al.  ecological analysis of macroinvertebrate communities… 164 this is in accordance with the fact that proportion of some ffg could be changed under some pollutants (p a v l u k et al., 2000). the index of trophic completeness (itc) at locality 3 was with high value (itc=27.4), at locality 4 (downstream of water discharge point from tire factory ‘tigar’) it dropped to itc=17.7, then after a distance of 34.9 km, at locality 5, it increased again to itc=26.5; at locality 6 it dropped to itc=22.3 and 8.1 km further downstream, at locality 7, it increased to itc=26.9 (s a v i ć , 2012). on the other hand, the situation was different between localities 8 and 9, where instead of the expected increase of itc, its value decreased from itc=24.1 to itc=21.9. the distance between these two localities is only 4.8 km, which suggest that the minimal distance for the river self-purification processes to become effective is at least 8 km (s a v i ć et al., 2013). the odd-numbered localities are positioned upstream, and evennumbered downstream of the settlements. a clear table 4. values for itc on investigated localities. n number of species in each guild; a – relative number of species in each trophic guild; c=100/a. guild loc 1 2 3 4 5 6 7 8 9 10 11 12 ctot 1 n 6 3 3 5 0 9 11 3 1 0 0 0 a 14.6 7.32 7.32 12.2 0 21.9 26.8 7.32 2.44 0 0 0 c 6.83 13.7 13.7 8.19 0 4.57 3.73 13.7 41 0 0 0 ln c 1.92 2.61 2.61 2.1 0 1.52 1.32 2.61 3.71 0 0 0 18.4 2 n 5 4 7 3 0 16 17 5 1 2 0 2 a 8.05 6.45 11.3 4.84 0 25.7 27.4 8.05 1.61 3.22 0 3.22 c 12.4 15.5 8.86 20.7 0 3.88 3.65 12.4 62.1 31.1 0 31.1 ln c 2.52 2.74 2.17 3.03 0 1.35 1.28 2.52 4.13 3.43 0 3.43 26.6 3 n 7 6 8 5 0 15 22 3 1 2 0 1 a 10 8.56 11.4 7.13 0 21.4 31.4 4.28 1.43 2.86 0 1.43 c 10 11.7 8.75 14 0 4.66 3.17 23.4 69.9 35 0 69.9 ln c 2.3 2.46 2.17 2.63 0 1.54 1.14 3.15 4.25 3.55 0 4.25 27.4 4 n 2 1 0 2 0 4 5 1 3 3 0 0 a 9.52 4.75 0 9.52 0 19.1 23.8 4.75 14.3 14.3 0 0 c 10.5 21.1 0 10.5 0 5.25 4.19 21.1 7 7 0 0 ln c 2.34 3.04 0 2.34 0 1.65 1.42 3.04 1.94 1.94 0 0 17.7 5 n 4 2 3 3 1 12 12 2 5 1 0 0 a 8.88 4.44 6.66 6.66 2.22 26.7 26.7 4.44 11.1 2.22 0 0 c 11.3 22.5 15 15 45 3.75 3.75 22.5 9 45 0 0 ln c 2.42 3.11 2.71 2.71 3.81 1.32 1.32 3.11 2.19 3.81 0 0 26.5 6 n 6 3 4 3 0 12 9 0 4 1 0 1 a 13.9 6.97 9.3 6.97 0 27.9 20.9 0 9.3 2.32 0 2.32 c 7.17 14.3 10.8 14.3 0 3.57 4.77 0 10.8 43.1 0 43.1 ln c 1.96 2.66 2.36 2.66 0 1.26 1.55 0 2.36 3.75 0 3.75 22.3 7 n 7 4 7 6 0 23 14 2 4 2 0 1 a 10 5.71 10 8.56 0 32.9 20 2.86 5.71 2.86 0 1.43 c 10 17.5 10 11.7 0 3.04 5 35 17.5 35 0 69.9 ln c 2.3 2.86 2.3 2.46 0 1.11 1.61 3.55 2.86 3.55 0 4.25 26.9 8 n 5 3 3 3 0 20 15 1 3 1 0 0 a 9.25 5.55 5.55 5.55 0 37 27.8 1.85 5.55 1.85 0 0 c 10.8 18 18 18 0 2.7 3.6 54.1 18 54.1 0 0 ln c 2.37 2.88 2.88 2.88 0 0.98 1.27 3.98 2.88 3.98 0 0 24.1 9 n 4 4 4 4 0 12 14 2 3 2 0 0 a 8.15 8.15 8.15 8.15 0 24.5 28.6 4.07 6.12 4.07 0 0 c 12.3 12.3 12.3 12.3 0 4.07 3.5 24.6 16.3 24.6 0 0 ln c 2.51 2.51 2.51 2.51 0 1.4 1.25 3.2 2.79 3.2 0 0 21.9 10 n 1 2 0 3 0 12 10 0 1 3 0 0 a 3.12 6.25 0 9.37 0 37.5 31.3 0 3.12 9.37 0 0 c 32.1 16 0 10.7 0 2.66 3.2 0 32.1 10.7 0 0 ln c 3.46 2.77 0 2.36 0 0.98 1.15 0 3.46 2.36 0 0 16.5 biologica nyssana 8 (2)  december 2017: 159-166 savić, a. et al.  ecological analysis of macroinvertebrate communities… 165 pattern can be seen: values of ctot are higher at oddnumbered localities, except when it comes to localities 1 and 2. this could be explained by facts that all other pairs of localities (3 and 4; 5 and 6; 7 and 8; 9 and 10) belong to the same stream order (tab. 1). locality 1 belongs to the 4th stream order, while locality 2 belongs to the 5th stream order. according to that, the index of trophic completeness could be sensitive on river section which is opposite to conclusion to some investigation in different parts of the world. conclusion the trends found in relative functional group abundance in this study do not correspond with the tendencies predicted by the rcc. a possible explanation for the disparity between the observed and expected feeding group proportions may be the relatively small ranges of physical stream characteristics found between stream orders. another explanation could be disturbance of riparian vegetation. surrogate measures for ecosystem attributes were used for the very first time in this region. the ffg ratios surrogate are consistent with the observations of the properties of the ecosystem at the sampling localities. the results show that the use of surrogates of ecosystem attributes is quite adequate for this purpose since the results obtained in this way match the results obtained by direct measurements of the attributes. however, direct measurements are often more difficult and require additional time and money. therefore, this principle should be used more often. the index of trophic completeness is considered to be a promising tool for determination of water quality in our region. according to this index, most of the localities included in the research belong to good water quality (seven out of ten) while other localities belong to moderate water quality (three out of ten). references allen, t.f.h., hoeksta, t.w. 1992: toward a unified ecology. columbia university press, new york, 384 p. apha, 1999. standard methods for the examination of water, wastewater. port city press, baltimore, maryland. belfiore, c. 1983: efemerotteri (ephemeroptera). in: ruffo s. 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paleartic genera of stoneflies (plecoptera) in larval stage. limnologica, 34: 315-348. бешовски, в. л. 1994. фауна на българия, 23: insecta, odonata. издателствона бан. софия, 372 p. spas krumov sotirov (1929-2016) naturalist and biologist biologica nyssana 7 (2)  december 2016: 57-60 ranđelović, n. et al.  in memoriam spas krumov sotirov… 57 in memoriam spas krumov sotirov (1929-2016) naturalist and biologist novica ranđelović1, vlastimir stamenković2, tatjana mihajilov-krstev1 1department of biology and ecology, faculty of sciences and mathematics, university of niš, serbia 2dd “zdravlje” leskovac, serbia * e-mail: tatjanamk@pmf.ni.ac.rs prof. dr spas sotirov was born on 9th january, 1929 in dimitrovgrad, where he had finished both elementary and high school. dr sotirov finished his bachelor biology studies in 1954. at the faculty of biology in belgrade, after which he had finished and defended his master (1962) and phd (1970) theses at the same institution. his professional carrier started in gymnasium in dimitrovgrad where he worked as a professor. during the period from 1958 to 1966, he worked as a teaching assistant at the faculty of veterinary medicine in belgrade and faculty of technology in novi sad. over the following years, during the period 1966-1982, he worked in institute 7 (2) • december 2016: 57-60 12th sfses • 16-19 june 2016, kopaonik mt doi: 10.5281/zenodo.200398 biologica nyssana 7 (2)  december 2016: 57-60 ranđelović, n. et al.  in memoriam spas krumov sotirov… 58 for development of education in niš. his academic carrier started with his promotion into assistant professor in 1982 at the faculty of technology in leskovac, where he was promoted into associate professor (1987) and finally to full professor in 1990. he was involved as an organizer of many symposiums, scientific and specialized workshops. among his organizing activities, one of the most significant place takes symposium entitled one hundred years of “flora of niš surrounding area”, where he was plenary speaker on the subject on dr sava petrović. he gave high contribution to education of the new generations of faculty and other students. as a member of the national education council of the republic of serbia he was working on didactic problems in teaching and as a reviewer on many textbooks written for elementary education. dr sotirov was coauthor of the capital monography “encyclopedia of niš” and worked as a reviewer of special issues of the journals “politika” and “savremena biologija”. also, he was a collaborator and a member of editorial board in the journals “bratstvo”, “most” and “drugarče”. his scientific opus includes working and contribution on many scientific projects on the subject on fundamental and also fieldwork researches on flora and vegetation, as well as resolving of significant problems such as possibilities of using autochthonous plant material in alimentary industry, technology of fruits and vegetables, development of food technology concepts and creation of the modern managing, control and production mechanisms in food industry. he was author of over 50 scientific publications and books. bibliography sotirov, s. 1968: ribe okoline pirota. pirotski zbornik, 1, pirot. sotirov, s. 1973: u dolini jerme susret tradicije prirode sa tradicijom kulture. narodne novine, niš. sotirov, s. 1974: procvat jestvastveništva u starom nišu. gradina, 1-2, niš. sotirov, s. 1977: po dolinata na erma (2. izdanje). bratstvo, niš. ranđelović, n. jovanović, v. sotirov, s. stamenković, v. ružić, m. 1980: campanula versicolor andrews nova vrsta u flori srbije. glasnik prirodnjačkog muzeja u beogradu, 35 (b): 53-56. ranđelović, n., matinović, ž., sotirov, s., jovanović, v., ružić, m., stamenković, v., hill, d.a., krivošej, z., randjelović, v. 1983: prunus laurocerasus l. na ostrozubu cveta. glasnik prirodnjačkog muzeja, b, 38. beograd. fig. 1. the last lecture at the university of niš biologica nyssana 7 (2)  december 2016: 57-60 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(izveštaj u okviru projekta). leskovac. sotirov, s. 1985: biološko-ekološka edukacija studenata prehrambenih tehnologija. iii kongres ekologa jugoslavije, sarajevo. sotirov, s. 1985: dr sava petrović život i delo, stogodišnjica "flore okoline niša", niš. sotirov, s., ranđelović, n. 1985: prošlost, sadašnjost i budućnost ekosistema na području regiona niš. niški zbornik, 15: 189-197, niš. ranđelović, n., sotirov, s. 1985: rezultati vegetacijskih i florističkih istraživanja jugoistočne srbije i njihov značaj za regionalno planiranje. naučno-istraživački rad i njegova uloga u daljem razvoju regiona niš. niš. ranđelović, n., hill, d.a., sotirov, s. 1985: specifičnosti flore uljanih škriljaca kod aleksinca. stogodišnjica "flore okoline niša", niš. martinović, ž., sotirov, s., randjelović, n. 1985: biljno-geografske karakteristike miljkovačke klisure. zbornik radova stogodišnjica "flore okoline niša”, 89-93, niš. palić, r., anđelković, s., sotirov, s. 1985: prilog taksonomiji satureja montana i satureja kitaibelii w. et k. stogodišnjica "flore okoline niša", niš. sotirov, s., komadinić, v. 1986: gajenje, pomološka i tehnološka svojstva niške "džinke”. (izvod). vii kongres biologa jugoslavije, budva. sotirov, s. 1986: biološko-ekološka zasnovanost industrijske prerade voća i povrća (izvod). vii kongres biologa jugoslavije, budva. sotirov, s. 1986: aktuelni problemi prerade kafe u južnomoravskom regionu. most, 98, niš. sotirov, s. 1986: iskorišćavanje autohtonih biljnih sirovina. most, 99, niš. sotirov, s. 1986: osavremenjivanje upravljačkih mehanizama prehrambene industrije. most, 100, niš. palić, r., sotirov, s., mihajlović, p. 1986: promena količine i sastava etarskog ulja lavandula angustifolia mill. (izvod). glasnik hemičara i tehnologa kosova, 6 (1), priština. sotirov, s., palić, r. 1986: kvalitet lavandinog ulja iz okoline sićeva i mogućnost upotrebe za aromatizaciju prehrambenih proizvoda. (izvod, rukopis). i simpozijum o flori i vegetaciji sr srbije, beograd. ranđelović, n., stamenković, v., sotirov, s. 1986: vegetacija vlasine i krajišta. most, 101-102: 6578, niš. ranđelović, n., hill, d.a., sotirov, s. 1986: specifičnost flore uljnih škriljaca kod aleksinca. i simpozijum o flori i vegetaciji srbije, 27, beograd. ranđelović, n., sotirov, s., jovanović, v., ružić, m., stamenković, v. 1986: rezultati florističkih, vegetacijskih i fitohemijskih istraživanja na području jugoistočne srbije za period 1945-1985 godine. i simpozijum o flori i vegetaciji srbije, 34, beograd. sotirov, s., jakšić, m. 1987: problemi za sotirov, s., jakšić, m. 1987: problemi zaštite i unapređivanja radne i životne sredine u svetlosti sistemskostrukturne koncepcije. zbornik tehnološkog fakulteta, 3-4: 175-180, leskovac. sotirov, s., tasić, s. 1987: korišćenje semena grožđa u proizvodnji kavovina. prethodno saopštenje. zbornik radova tehnološkog fakulteta, 3-4: 201202, leskovac. tasić, s., sotirov, s., ristić, m., palić, r., stanković, m. 1987: ispitivanje sastava semena grožđa. i. sastav frakcije masnih kiselina. xxix savetovanje hemičara sr srbije, beograd. sotirov, s., stojiljković, s., davidović, i. 1988: blanširanje krompira surutkom. prethodno saopštenje. zbornik radova tehnološkog fakulteta, 5-6; 253-256, leskovac. randjelović, n., stamenković, v., sotirov, s. 1988: flora v okolnostitena bosilegrad. most, 112: 4754, niš. stamenković, v., ranđelović, n., sotirov, s. 1989: lekovite biljke užeg područja vlasine. leskovački zbornik, 29: 367-375. ranđelović, n., jovanović, v., ružić, m., sotirov, s., satamenković, v., hill, d.a., ranđelović, v. 1989: lekovite biljke toplice. leskovački zbornik, 29: 375-381. sotirov, s., randjelović, n., stamenković, v. 1990: prilog metodologiji kartiranja i eksploatacije samoniklih resursa lekovitog i začinskog bilja. 2. simpozijum o flori jugoistočne srbije i susednih područja, zbornik radova tehnološkog fakulteta, 7-8: 7-11, leskovac. biologica nyssana 7 (2)  december 2016: 57-60 ranđelović, n. et al.  in memoriam spas krumov sotirov… 60 ranđelović, v., sotirov, s., stamenković, v., redžepi, f., ranđelović, v., zlatković, b. 1990: lekovite biljke subregiona pirot. 2. simpozijum o flori jugoistočne srbije i susednih područja, zbornik radova tehnološkog fakulteta, 7: 29-35, leskovac. ranđelović, n., stamenković, v., sotirov, s. jovanović, v., ranđelović, v. 1990: prirodni resursi divljeg lekovitog bilja niškog subregiona. 2. simpozijum o flori jugoistočne srbije i susednih područja, zbornik radova tehnološkog fakulteta, 7-8: 35-40. leskovac. martinović, ž., ranđelović, n., sotirov, s. 1990: o nalazištu ramondije u dolini toponičke reke. 2. simpozijum o flori jugoistočne srbije i susednih područja, zbornik radova tehnološkog fakulteta, 7: 99-111, leskovac. sotirov, s., popović, r., ranđelović, n., stamenković, v., stojanović, v., anđelković, s., simić, n. 1990: prilog proučavanju ruta graveolens l. iz okoline sićeva. 2. simpozijum o flori jugoistočne srbije i susednih područja, zbornik radova tehnološkog fakulteta, 7-8: 195199, leskovac. đorđević, s., đorđević, i. sotirov, s., gudžić, b. 1990: ispitivanje hemijskog sastava ostrozubske šljive sa posebnim osvrtom na sadržaj minerala. 2. simpozijum o flori jugoistočne srbije i susednih područja, zbornik radova tehnološkog fakulteta, 7-8: 199-204. leskovac. sotirov, s., đorđević, s., tomić, v. zdravković, b. 1990: mineralni sastav droge i infuzuma začinsko-aromatičnih biljaka iz okoline sićeva. 2. simpozijum o flori jugoistočne srbije i susednih područja, zbornik radova tehnološkog fakulteta, 7: 205-209, leskovac. sotirov, s., stamenković, v., ranđelović, n., jančić, n. 1990: dobijanje kavovine od ploda castanea sativa mill. 2. simpozijum o flori jugoistočne srbije i susednih područja, zbornik radova tehnološkog fakulteta, 7-8: 209-213, leskovac. martinović, m.ž., sotirov, s., ranđelović, n., stojanović, d.: uticaj radioaktivnosti na održanje endemoreliktne vrste ramondia serbica. zbornik radova sa 3. simpozijuma o flori jugoistočne srbije i susednih područja, 207-220, pirot. sotirov, s., ranđelović, n., stamenković, v. 1993: prirodnjačke vrednosti pirotskog ponišavlja s posebnim osvrtom na floru i vegetaciju (pregled dosadašnjih istraživanja). plenarni referat. zbornik rezimea, iii simpozijum o flori jugoistočne srbije, 2-10, pirot. ranđelović, n., stamenković, v., sotirov, s, ranđelović, v., zlatković, b. 1993: prilog flori klisure reke jerme. izvodi radova. iii simpozijum o flori jugoistone srbije, 34, pirot. stamenković v., sotirov s., cvetković d., đorđević s., ranđelović n. 1993: dobijanje zelenih začina od svežih plodova paprike i nadzemnih listova crnog luka. izvodi radova. savetovanje o lekovitim i aromatičnim biljkama jugoslavije, 41-42, zlatibor. stamenković v., sotirov s., ranđelović n., cvetanović b., nikolić m. 1993: korišćenje listova vinove loze u prehrambenoj industriji. izvodi radova. savetovanje o lekovitim i aromatičnim biljkama jugoslavije, 39-40, zlatibor. sotirov, s. 1995: poganovskijot manastir i negovotoprirodnoto okruženija. most, niš. sotirov. s. 1995: 600-godišnine na poganovskija manastir (serijal). bratstvo, niš. sotirov, s. 1999: prilog poznavanja života i dela dr save petrovića. acta medica, 3, niš; sotirov, s., zlatković, b., ranđelović, v. 2002: antropogene fitocenoze sa mahonijom (mahonia aquifolium (purch.) nutt.) u okolini dimitrovgrada. vii simpozijum o flori jugoistočne srbije i susednih područja, zbornik rezimea, dimitrovgrad. sotirov, s. 2002: poganovo zveno meždu zemjata i neboto. narodna biblioteka, dimitrovgrad. sotirov, s. 2005: zelene spirale roda ramonda. književna kolonija sićevo, str. 49-77, niš. sotirov, s. 2006: sićevski kolopleti. zograf. sotirov, s. 2010: dr sava petrović život, lik i delo. unus mundus, 35, niš. milosavljević, v., avramović, d., sotirov, s. 2010: treća južnosrpska floristička-fitocenološka škola. 10. simpozijum o flori jugoistočne srbije i susednih regiona, zbornik rezimea, niš. sotirov, s., stanojević, v. 2012: treći alen kamen cerjanska pećina i topilo. academia homo turisticus, niš. sotirov, s. 2012: prodor darvinizma u srbiji. unus mundus, 42, niš. sotirov, s. 2012: u potrazi za panacejom. unus mundus, 42. niš. sotirov, s. 2014: miljenice profesora pančića. univerzitetska biblioteka nikola tesla, niš. karadžić, b.: chasmophytic forests of ostrya carpinifolia in west-serbian canyons. biologica nyssana, 8 (1), september 2017 biologica nyssana 8 (1)  september 2017: 73-81 karadžić, b. et al.  chasmophytic forests of ostrya carpinifolia… 73 original article received: 22 july 2017 revised: 17 august 2017 accepted: 30 august 2017 chasmophytic forests of ostrya carpinifolia in west-serbian canyons branko karadžić institute for biological research of belgrade university, bul. despota stefana 142, 11000 belgrade, serbia * e-mail: branko@ibiss.bg.ac.yu abstract: karadžić, b.: chasmophytic forests of ostrya carpinifolia in west-serbian canyons. biologica nyssana, 8 (1), september 2017: 73-81. variability patterns and biodiversity components of chasmophytic forests of ostrya carpinifolia in westserbian ravine habitats were analyzed in this article. investigations were performed in four gorges along gornja trešnjica, gradac, ljutina and lim rivers. chasmophytic hop hornbeam forests occur on steep slopes (25° to 50°) at elevations ranging from 260 m to 1200 m, on all aspects. these forests grow on screes, rocky clifs, colluvial gravel, on shallow soils on dolomites, limestone and serpentine. canonical correspondence analysis indicates that moisture, light and temperature gradients are the main factors affecting diversification of investigated forests. the greatest alpha diversity was detected in brodarevo and gostun gorges, along the lim river. key words: alpha diversity, beta diversity, ostrya carpinifolia, chasmophytes, canonical correspondence analysis apstrakt: karadžić, b.: hazmofitske šume sa ostrya carpinifolia u zapadnoj srbiji. biologica nyssana, 8 (1), septembar 2017: 73-81. u ovom radu ispitivani su obrasci varijabilnosti i komponente biodiverziteta hazmofitskih šuma ostrya carpinifolia u klisurskim habitatima zapadne srbije. istraživanja su sprovedena u četiri klisure duž reka gornja trešnjica, gradac, ljutina i lim. hazmofitske šume crnog graba se javljaju na strmim padinama (25° do 50°) i visinama od 260 m do 1200 m. ove šume rastu na siparima, kamenitim liticama, koluvijalnom šljunku, plitkim zemljištima na dolomitu, krečnjaku i serpentinima. kanonska korespodentna analiza ukazala je da gradijenti vlage, svetlosti i temperature predstavljaju glavne faktore koji utiču na diverzifikaciju ispitivanih šuma. najveći alfa diverzitet uočen je u klisurama brodareva i gostuna duž reke lim. ključne reči: alfa diverzitet, beta diverzitet, ostrya carpinifolia, hazmofite, kanonijska korespodentna analiza 8 (1) • september 2017: 73-81 doi: 10.5281/zenodo.964117 biologica nyssana 8 (1)  september 2017: 73-81 karadžić, b. et al.  chasmophytic forests of ostrya carpinifolia… 74 introduction the chasmophitic vegetation is very complex, since it involves communities on calcareous, serpentine and silicate rocks, at different altitudes, from colline (usually ravine) habitats to nival regions (l a k u š i ć & k a r a d ž i ć , 2010). these communities are intrazonal, since they occur in all vegetation zones. chasmophytes in ravine habitats usually belong to the group of (paleo) endemic rare species. in ravine habitats, these species found a refuge from unfavourable climatic conditions, stronger competitors from neighbouring non-ravine communities, grazing and human-induced disturbances. this article is focused on the chasmophytic forests, dominated by european hop hornbeam (ostrya carpinifolia scop.). the forests are located in ravine habitats along the gradac, gornja trešnjica, ljutina and lim rivers. ostrya carpinifolia scop. is a species adapted to warm to moderate climate. this species has a wide tolerance limits with respect to both, the light and moisture gradients (popović, et al., 1996). therefore it (co)dominates in a wide spectrum of communities that belong to ecologically different alliances (t r i n a j s t i ć & c e r o v e č k i , 1978, p u n c e r & z u p a n č i č , 1982, t o m i ć , 1994, 2000). distribution of ostrya carpinifolia comprises apennines, tyrol, western parts of the balkan peninsula, asia minor and lebanon (m e u s e l et al., 1965). the closest aliens of this species occur in east asia and north as well as central america (t r i n a j s t i ć & c e r o v e č k i , 1978). such distribution clearly indicates a tertiary disjunction of the genus ostrya scop. european hop hornbeam codominates with other (sub)mediterranean xeric trees (carpinus orientalis mill., quercus pubescens willd., paliurus spina-christi mill.) in xeric communities of the alliance carpinion orientalis horvat 1954, along the adriatic sea and in regions where the maritime influence extends deep into the continent, along the rivers una, neretva, drim and vardar. in more humid habitats, these forests are replaced by the amphiadriatic mesic calcareous climte-zonal alliance fraxino orni-ostryion carpinifoliae tomažič 1940 = ostryo-carpinion orientalis horvat 1959 (h o r v a t et al., 1974, č a r n i et al., 2009, m u c i n a et al., 2016). hop hornbeam also occurs on relict pinus nigra forests on dolomite and ultramafic substrates of the dinarides (alliance erico carneae-fraxinion orni horvat 1959). due to a broad ecological amplitude with respect to moisture, ostrya carpinifolia forms mesic intrazonal forests in inland ravine habitats. these forests are included into the alliance tilio platyphylli acerion pseudoplatani klika 1955 (b o r h i d i et al., 1999, k o š i r et al., 2008, d a k s k o b l e r et al., 2013). broad-leaved ravine tilio-acerion forests specify an eu priority type of ecosystems (r o d w e l l et al., 1998). compared with tilio platyphylli acerion pseudoplatani forests, the hop hornbeam forests in ravine habitats of inland balkan aea are more diverse (k a r a d ž i ć et al., 2001, 2015). the aim of present study is to describe structure of the mixed black hornbeam forests that are located in west-serbian canyons, to relate their floristic composition with environmental factors and to detect patterns of and -diversity. material and methods the study area is located in western parts of the central serbia. investigations were performed in four ravine habitats along the gradac, gornja trešnjica, ljutina and lim rivers. the altitudinal range and geographic coordinates of investigated localities are presented in table 1. the eastermost and northernmost locality is the gorge of the gradac river. this gorge is exposed to the influence of panonnian plane. the vegetation samples (relevés) were collected seasonally, from early spring to late autumn. combined cover-abundance values of b r a u n b l a n q u e t ’s (1965) alphanumeric scale were replaced by a nine-degree numeric values (w e s t h o f f & v a n d e r m a a r e l , 1973). the taxonomic nomenclature was harmonized with the plant list database that involves accepted latin name for vascular plants and bryophytes (http://www.theplantlist.org). differentiation of communities with respect to environmental variables was assessed using the table 1. geographic position and altitudinal range of investigated localities rivers locality altitudinal range angular coordinates gornja trešnjica trešnjica 315-855 44.08447 n 19.32165 e lim brodarevo, gostun 500-1080 43.10567 n 19.46642 e ljutina ljutina 619-1200 43.28807 n 19.24945 e gradac (sušaja) lastva 261-819 44.11722 n 19.51642 e biologica nyssana 8 (1)  september 2017: 73-81 karadžić, b. et al.  chasmophytic forests of ostrya carpinifolia… 75 canonical correspondence analysis (t e r b r a a k , 1986). environmental variables within analyzed communities were assessed using the weighted average of ecological indicator values (k o j i ć et al., 1997). alpha diversity was calculated using the shanon function    n i ii pph 1 log , where pi is the proportion for species within community. according to h i l l (1973) and p i e l o u (1974), the shanon’s function is a special case of renyi's general entropy function. beta diversity was detected according to the r o u t l e d g e (1977) method: 1 2 2    sr s  , where s is the total number of species, and r is the number of species pairs whose distributions overlap. above equation specifies the changing rates of compositional turnover along an environmental gradient (w i l s o n & s h m i d a , 1984). all statistical analyses were performed using “flora” software package (k a r a d ž i ć , 2013). results and discussion floristic differences among analyzed canyons ravine forests in serbian gorges and canyons occur on screes, rocky clifs, colluvial gravel and also on developed soils, mainly shallow rankers, rendzinas and brown calcareous soils (p a v l o v i ć et al., 2017). screes were recorded in all investigated ravines. they are covered by sparsely distributed individuals of ostrya carpinifolia scop., fraxinus ornus l., frangula rupestris (scop.) schur. and cotinus coggygria scop. on more favourable soils, the hop hornbeam occurs in combination with carpinus orientalis l., rhamnus saxatilis jacq., r. cathartica l., r. alpina subsp. fallax (boiss.) maire & petitm., tilia platyphyllos scop., acer campestre l., juglans regia l., carpinus betulus l., viburnum lantana l., quercus cerris l., q. petraea (matt.) liebl., q. pubescens willd., fagus sylvatica l. etc, forming complex polydominant communities. in order to detect patterns of floristic variability of analyzed forests a set of multivariate statistical methods was performed. correspondence analysis (ca) and principal components analysis (pca) with their canonical forms are the most frequently used ordination methods in ecology (j o n g m a n et al., 1987; l e g e n d r e & l e g e n d r e , 2003; g r e e n a c r e , 2010). principal axes obtained by either ca or pca are mutually uncorrelated, but not necessarily independent. a fig 1. correspondence analysis (a) and discriminant correspondence analysis (b) of analyzed forests. analyses were performed on a hypercorrelated matrix, in order to avoid undesirable guttman effect biologica nyssana 8 (1)  september 2017: 73-81 karadžić, b. et al.  chasmophytic forests of ostrya carpinifolia… 76 spurious polynomial relation between ordination axes (the arch effect or guttman effect) is a wellknown drawback of ca and pca. compared to ca, pca is more sensitive to the arch effect, especially in the case when beta diversity (species turnover) along spatial or environmental gradients is high (j o n g m a n et al., 1987, k a r a d ž i ć & p o p o v i ć , 1994, k a r a d ž i ć et al., 1999). k a r a d ž i ć et al. (2014) revealed that the hypercorrelated matrices significantly reduce the guttman effect. therefore, all multivariate analyses were performed on hypercorrelated matrices. the results of correspondence analysis (fig. 1) clearly indicate that the forests continuously intergrade. this is a consequence of polydominant structure and a great number of species with overlapping distribution. contrary to ordinary correspondence analysis, the discriminant correspondence analysis (g r e e n a c r e , 2000), clearly separates chasmophytic forests of ostrya carpinifolia in different gorges. first principal axis separates analyzed forests according to the altitudinal gradient (from the lowermost gorges along the gradac and gornja trešnjica to mountainous communities in ljutina and brodarevo gorges). environmental conditions specific topography of gorges and canyons significantly modifies microclimate conditions. solar rays hit the opposite slopes of canyons at different angles. amount of solar energy per unit surface is much greater on southern than on northern slopes. moreover, the evaporation is much greater on southern than on northern slopes. such conditions form strong environmental gradients. effects of environmental variables on floristic differentiation of analyzed forests were detected using canonical correspondence analysis (cca). the canonical ordination axes maximize floristic variability of a data set that can be explained by a set of environmental variables. the results of cca (fig. 2) clearly indicate that the forests continuously intergrade. this is a consequence of polydominant structure and a great number of species with overlapping distribution. the main factors that affected floristic differentiation of analyzed forests are moisture, light and temperature. thermophylous communities covering the gorge of the gradac river are clearly separated from other communities. fig. 3. components of alpha diversity in chasmophytic hop hornbeam communities fig 2. canonical correspondence analysis of chasmophytic hop hornbeam forests in investigated gorges biologica nyssana 8 (1)  september 2017: 73-81 karadžić, b. et al.  chasmophytic forests of ostrya carpinifolia… 77 diversity components of analyzed forests biological diversity is the term, which in its broadest sense means variability of the living world. such general definition needs further elaboration, since biological variability is a multi-component, complex parameter that involves intraspecies (population) and interspecies (cenotic) variety (w h i t t a k e r , 1972; k a r a d ž i ć & m a r i n k o v i ć , 2009). intraspecies or population variability is the result of genetic variability, the variability induced by influence of environmental factors and the variability which arises from the interaction of genetic background and fig 4. beta diversity of hop hornbeam forests along the gradients of moisture (a), temperature (b), light (c), soil acidity (d) and soil nitrogen (e) biologica nyssana 8 (1)  september 2017: 73-81 karadžić, b. et al.  chasmophytic forests of ostrya carpinifolia… 78 environmental conditions. interspecies or coenotic variability can be divided into within-community variability (alpha diversity), variability between communities (beta diversity) and the total variability in a given region (gamma diversity). alpha diversity (within-community diversity) depends on species richness and equitability of species abundances in a community. both, species richness and entropy are the greatest in brodarevo (and nearby gostun) gorges along the lim river. a high species richness and shanon’s entropy were recorded also in communities that inhabit the ljutina river gorge. the lowermost values of alpha diversity components were recorded in communities covering the gorge of the gradac river (fig. 3). a n d e r s o n et al. (2015) distinguish two types of beta diversity: directional turnover along a gradient and nondirectional between-community variation. directional beta-diversity (or extent of species replacement along environmental gradients) indicates the degree to which habitats have been partitioned by species. replacement of species (species turnover) is a function of the habitat heterogeneity and ecological tolerance of species (w h i t t a k e r , 1972; k a r a d ž i ć et al., 2003). beta diversity was calculated for each adjacent pairs of sites that are ordered along an environmental gradient (fig. 4). irrespective on environmental gradient, the greatest values of beta diversity were observed in communities that inhabit gorges along the lim river. endemism of hasmophytic taxa according to v e l č e v et al. (1992), the balkan endemics may be divided into two main categories: paleo-endemics and neo-endemics. the first group involves tertiary relics, with low-genetic variability, diploid or low-ploid chromosome number and fig 5. important endemic chasmophytes in analyzed forests: onosma stellulatum wald. et kit (a), lathyrus binatus pančić (b), aquilegia grata zimmeter (c), achillea ageratifolia (sibth. & sm.) boiss. (d), daphne malyana blečić (e), athamanta turbith (l.) brot. subsp. haynaldii (borbas & uechtr.) tutin. (f), campanula secundiflora vis. & pančić (g), corydalis ochroleuca koch. (h) and hieracium waldsteinii tausch (i). biologica nyssana 8 (1)  september 2017: 73-81 karadžić, b. et al.  chasmophytic forests of ostrya carpinifolia… 79 apparent disjunct distribution with its closest relatives. due to long time scale isolation and absence of genetic contacts with their relatives, these species formed stable genotypes under particular conditions. neo-endemics are species that appeared during quartenary climatic stresses. these species are characterized by polyploidy (tetraploid or high ploid chromosome numbers). tertiary relics comprise species that dominated in europe at the beginning of the so-called quaternary period. repeated swings of glacial and interglacial periods affected significant reduction of floristic diversity in europe. periodic latitudinal (northward and southward) migrations of flora were obstructed by the mountain barriers. the east–west orientation of european main mountain massifs prevented latitudinal migration of tertiary relics. therefore, the remnants of tertiary flora persisted in three south-european peninsulas (iberia, apennines and the balkans), mostly in ravine habitats, where specific topography attenuated climate oscillations. the most important endemorelics in analyzed communities are onosma stellulatum wald. et kit, lathyrus binatus pančić, aquilegia grata zimmeter, achillea ageratifolia (sibth. & sm.) boiss., daphne malyana blečić, athamanta turbith (l.) brot. subsp. haynaldii (borbas & uechtr.) tutin., campanula secundiflora vis. & pančić, corydalis ochroleuca koch., hieracium waldsteinii tausch, cardamine glauca sprengel, dianthus petraeus waldst. & kit. seseli rigidum waldst. & kit., edraianthus graminifolius (l.) a. subsp. jugoslavicus lakušić. some (steno)endemic taxa that inhabit investigated forests are presented in fig. 5. most of endemic species in serbia belong to the group of paleo-endemics and high-mountain endemics. the third group of the endemics belongs to the serpentinophytes. the serpentine soils are characterized by low levels of the essential plant elements (nitrogen, phosphorus, potassium and calcium), as well as high levels of iron, magnesium, and manganese, and toxic levels of chromium, cobalt and nickel. due to the “serpentine stress” (toxic effects of heavy metals, nutrient shortages and droughts), most plant species avoid serpentine soils. a small percent of serpentine-tolerant taxa has evolved morpho-anatomical and physiological adaptations that allow them to survive in extremely unfavourable conditions. strong selective pressures of serpentine soil and spatial isolation of serpentine regions resulted with high percent of endemic serpentinophyte taxa in the balkans (j a k o v l j e v i ć et al., 2011). the most important serpentinophytes that inhabit analyzed communities are euphorbia glabriflora vis., scrophularia tristis maly and asplenium cuneifolium viv. these species occur on patches of serpentinous soil in gorges along the lim river. the chasmophitic vegetation is distributed on rocky habitats of paleo-temperate region (p i g n a t t i et al., 1995). despite the wide distribution, chasmophitic vegetation is fragmented and isolated. gene flow between population in fragmented and geographically isolated habitats is obstructed. even within the same saxatile region the effective gene flow between chasmophytic populations may be considerably inhibited through the frequent failure of seed to establish new individuals (d a v i s , 1951). the first step on the colonization of bare-rock must be the establishment of seedlings. this occurs in microsites with finely divided material, especially crevices, in which rootsystems can find anchorage. most of the holes suitable for ecesis are already occupied in saxatile habitats, so that it is extremely difficult for the individual to produce progeny. due to a low effective population size, the effects of genetic drift and inbreeding depression may lead to the accident elimination of alleles and increased proportion of homozygous (often nonadaptive) alleles. ecological plasticity of rare chasmophytes is narrowed because of reduced genetic variability. therefore, most of rare chasmophytes belong to the group of critically endangered taxa. reduced gene flow, in combination with both, the strong natural selection in adverse saxatile habitats, together with genetic drift due to reduced effective population size, resulted in amazing diversification of chasmophytic flora. most chasmophytic species are endemics. strong selective pressures and reduced gene flow due to both, geographical isolation of saxatile areas and ecessis problems resulted in fast speciation of chasmophytic taxa with relatively narrow distribution. conclusion polydominant forest vegetation in west-serbian canyons represents a valuable pool of species diversity. a great heterogeneity of environmental conditions and specific history of biota in the canyons resulted with complex communities that represent 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natural sciences and mathematics, skopje, north macedonia trajce talevski department of cyprinid fauna, hydrobiological institute, ohrid, north macedonia received: may 16, 2025 revised: august 12, 2025 accepted: august 13, 2025 abstract: this study investigates the use of the fish embryo toxicity test on early life stages of pachychilon pictum, obtained from wild populations in lake ohrid. various chemical compounds were assessed for mortality, and median lethal concentrations at 96h post fertilization were determined. the findings demonstrated the successful application of the test requirements, confirming that embryos of this species are suitable for toxicity testing. notably, this represents the first ever toxicological investigation conducted on a fish species from lake ohrid, highlighting the importance of the resulting data. key words: fish embryo, lake ohrid, pachychilon pictum, toxicity apstrakt: akutna toksičnost na ranim razvojnim stadijumima vrste pachychilon pictum (heckel & knerr, 1878) iz ohridskog jezera u ovom istraživanju ispitivana je primena testa embrionalne toksičnosti riba na ranim razvojnim stadijumima vrste pachychilon pictum, dobijenim iz divljih populacija u ohridskom jezeru. procenjivani su različiti hemijski spojevi u pogledu smrtnosti, a određene su srednje letalne koncentracije nakon 96 sati od oplođenja. rezultati su pokazali uspešnu primenu metodoloških zahteva testa, potvrđujući da su embrioni ove vrste pogodni za ispitivanje toksičnosti. važno je istaći da ovo predstavlja prvo toksiološko istraživanje sprovedeno na jednoj ribljoj vrsti iz ohridskog jezera, što naglašava značaj dobijenih podataka. ključne reči: embrion ribe, ohridsko jezero, pachychilon pictum, toksičnost introduction many aquatic bodies are currently subjected to a variety of environmental stressors, which have the potential to negatively alter the conditions of water ecosystems and biodiversity. the growing awareness of the possible environmental effects of human activity has led to the development of numerous methodologies that attempt to predict how stress could impact fish populations (power, 1997). these methodologies are based on determination of physiological, biochemical, genotoxic changes in fish (van der oost et al., 2003), and occurrence of mortality (wedekind et al., 2007). high amounts of anthropogenic contaminants entering the environment have caused the necessity for reliable and accurate toxicity tests (fent, 2004). acute toxicity tests are significant in environmental risk assessment as they provide an evaluation of the relative toxicity of substances in a variety of species (wedekind et al., 2007). fish, as the most widespread aquatic vertebrate class, have been considered an essential part of toxicity assessment testing (braunbeck & lammer, 2005). acute fish toxicity data are regarded as an essential requirement in the hazard and environmental risk evaluation of different types of chemicals (cooney, 1995). primarily, most of the acute toxicity tests are applied using adult fish, which requires the sacrifice of lots of specimens. because the targeted endpoint of acute toxicity testing is mortality, fish are most likely to experience pain and suffering (chandroo et al., 2004), which conflicts with current animal welfare legislations. the developing fish embryo and larvae are regarded as the most sensitive stages in the life cycle of a fish, with expressed sensitivity to environmental changes (von westernhagen, 1988). with that in considera© 2025 trajchevski et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. tion, and the fact that the early life stages of fish are not protected by the legislations like the european union directive 2010/63/eu on the protection of animals used for scientific purposes, the early life stages of fish proved to be excellent candidates for an alternative test subject. the acute fish embryo toxicity test was designed as an alternative method and validated by the organisation for economic cooperation and development (oecd) (busquet et al., 2014) as test guideline oecd 236 (oecd, 2013). it has been demonstrated that fish embryo tests can be used to measure and rank different types of toxic substances, confirming the embryo toxicity tests as a useful method for assessing environmental dangers and threats to the ecological status of waterbodies (rundeva, 2014). studies have confirmed that acute toxicity tests with fish embryos are good predictors of the results obtained with acute tests on adult fish, and therefore they are a good testing alternative (belanger et al., 2013). besides their sensitivity, these tests have practical advantages over tests with adult fish, in the sense that they require less test volume and space, which enables the use of greater numbers of test organisms and replicates (wedekind et al., 2007). although the design of this test is based on the early life stages of zebrafish (danio rerio) as test organism, to encompass possible different species sensitivities, it was proposed to apply and adapt the protocol to other fish species (braunbeck et al., 2005; lammer et al., 2009; belanger et al., 2013). different fish species may vary in their sensitivity to environmental contaminants (nagel, 2002), and according to wedekind et al. (2007), in acute toxicity testing, fish species should be representative of the region where they occur. lake ohrid is an oligotrophic water body located on the balkan peninsula, and as the oldest natural lake in europe, it has a high level of endemism and, as a part of that endemism, a unique fish fauna (albrecht & wilke, 2008). as for now, there are no ecotoxicological studies incorporating toxicity tests on any of the fish species from lake ohrid. with the increasing anthropogenic pressures on the lake, present from decades ago (matzinger et al., 2006; kostoski et al., 2010), and the industrialization and urbanization of the area in modern times, toxicity data could be of great importance for environmental and risk assessment. therefore, the aim of this study was to apply and adapt the embryo toxicity test to the early life stages of pachychilon pictum (heckel & knerr, 1878), a representative of the cyprinid fish fauna from lake ohrid. this is the first study of this kind, applied to fish from lake ohrid, and for the fish species native to the freshwater bodies of north macedonia in general. materials and methods the embryos for this study were provided through artificial reproduction of wild specimens of pachychilon pictum. it is a cyprinid fish species, endemic to the freshwater bodies of the southwestern balkan (lakes ohrid and skadar, river black drin) (kottelat & freyhof, 2007; talevski et al., 2025). male and female specimens were sampled during the spawning period in early summer. the sampling took place in the littoral area of lake ohrid, using gill nets with various mesh sizes. reproductive material (eggs and milt) was collected by stripping, after which the fish were released back into the lake. the artificial reproduction was carried out by using the “dry method” (billard et al., 1996), and the acquired fertilized embryos were used for the acute toxicity test. for the implementation of the acute toxicity test, eight chemical compounds were selected. the selected chemicals were of different types and had different levels of toxicity. they are commonly used in various industries as raw materials, precursors, active substances, and agricultural biocides. according to the globally harmonized system of classification and labelling of chemicals (ghs) (un, 2023), these chemicals have different toxicity to fish, ranging from non-toxic to very toxic (oecd, 2011; oecd, 2012). the ranges of the test concentrations used for the toxicity test were selected based on data obtained from literature from zebrafish based toxicity tests. this was done to compare the sensitivity of p. pictum embryos to zebrafish as the standard model for toxicity testing. the test concentrations were obtained from the oecd validation study for the acute fish embryo toxicity test (oecd, 2011; oecd, 2012; busquet et al., 2014). the chemicals used for preparation of the solutions had a minimum purity grade of ≥ 98%, and all test solutions were freshly prepared using standardized dilution water (oecd, 2013). the tested chemical compounds, along with the test concentrations, are presented in tab. 1. the acute toxicity test was performed according to the oecd 236 test guidelines (oecd, 2013). freshly fertilized embryos were inspected for fertilization success, and only embryos without any irregularities were used for the test. after selection, the embryos were transferred to 24-well plates filled with 2 ml of the test solution, with one embryo placed in each well. on one plate, 20 embryos were placed in the test solution, and 4 embryos in dilution water, which served as an internal control for the plate. the test uses positive control (4 mg/l biologica nyssana ● 16 (2) december 2025: trajchevski et al. ● acute toxicity on early life stages of pachychilon pictum (heckel & knerr, 1878) from lake ohrid biologica nyssana ● 16 (2) december 2025: trajchevski et al. ● acute toxicity on early life stages of pachychilon pictum (heckel & knerr, 1878) from lake ohrid 3,4 – dichloroaniline) and negative control (dilution water), and is considered valid if the mortality rate in the positive control is minimum 30% and the maximum mortality rate in the negative control is 10%. the well plates were placed in an incubator at 26±1 °c, under semi-static exposure, and the tests were performed in duplicate. embryos were observed daily (24, 48, 72, 96 hpf hours post fertilization) for the occurrence of mortality, determined by the endpoints according to oecd 236 (oecd, 2013). the endpoints specified in the guidelines are: embryo coagulation, lack of somite formation, lack of tail detachment, and lack of heart beat (oecd, 2013). the embryos were observed and analyzed under an inverted microscope zeiss primovert (carl zeiss gmbh, oberkochen, germany), and pictures were taken using the zeiss zen software. lethal concentrations corresponding to effect levels of 10% (lc10) and 50% (lc50) were estimated at 96 hours post fertilization. these values, along with the 95% confidence limits, were calculated using probit analysis performed with statistical software (spss 23.0). results and discussion the mortality of pachychilon pictum embryos was recorded according to the endpoints of the test guideline oecd 236. the values for lc10 and lc50 at 96 hpf for the tested chemical compounds are shown in tab. 2. the highest lc50 value was observed for triethylene glycol, and the lowest for 4,6-dinitro-o-cresol. there was no observed mortality in the control group or in the internal plate controls. the embryos of p. pictum have transparent chorion, allowing direct observation of the embryonic development (fig. 1). during the test, we documented the occurrence of two of the required endpoints: coagulation of the embryo and lack of heartbeat (fig. 1). in the control group, all embryos were hatched at 72 hpf which is slightly faster compared to zebrafish, with a hatching period at 72 to 96 hpf (braunbeck & lammer, 2005; braunbeck et al., 2005) in the same water temperature. although the fish embryo acute toxicity test is designed on the basis of zebrafish embryos, this study showed that it can be successfully applied to different fish species. in that regard, p. pictum joins the group of fish species subjected to this test protocol (lammer et al., 2009; fiorino et al., 2018). the test validity was verified by confirming that the mortality observed in both the negative and positive control plates fell within the table 1. list of the chemical compounds and the test concentrations chemical cas number test concentrations (mg/l) chemical type copper (ii) sulfate pentahydrate 7758-99-8 0.15, 0.3, 0.6, 1.2, 2.4 biocide dibutyl maleate 105-76-0 0.25, 0.5, 1, 2, 4 industrial organic 3,4-dichloroaniline 95-76-1 0.5, 1, 2, 3.7, 4, 8 industrial organic 4,6-dinitro-o-cresol 534-52-1 0.18, 0.32, 0.58, 1.05, 1.89 biocide ethanol 64-17-5 5300, 8000, 12000, 18000, 27000 industrial organic 1-octanol 111-87-5 2.5, 5, 10, 20, 40 industrial organic sodium chloride 7647-14-5 1000, 2000, 4000, 8000, 16000 industrial inorganic triethylene glycol 112-27-6 20, 30, 45, 67.5, 101.25 industrial organic table 2. acute toxicity data from the fish embryo test with p. pictum embryos chemical lc10 95% confidence limits lc50 95% confidence limits copper (ii) sulfate pentahydrate 0.708 0.468–0.891 1.342 1.095–1.69 dibutyl maleate 0.792 0.512–1.02 1.657 1.337–2.094 3,4-dichloroaniline 1.056 0.413– 1.532 2.242 1.519–3.174 4,6-dinitro-o-cresol 0.217 0.152–0.267 0.383 0.320–0.459 ethanol 6214 4903–7081 8706 7763–9779 1-octanol 7.142 4.450–9.432 17.038 13.506–22.270 sodium chloride 2712 1795–3451 5380 4369–6699 triethylene glycol 34852 28124–38917 44597 39798–49124 biologica nyssana ● 16 (2) december 2025: trajchevski et al. ● acute toxicity on early life stages of pachychilon pictum (heckel & knerr, 1878) from lake ohrid placed in the category ≤ 1 mg/l, which according to the ghs system of classification of chemicals (un, 2023) is very toxic to fish. this means that p. pictum embryos are less sensitive regarding these two chemicals than zebrafish embryos (tab. 1). ethanol, sodium chloride, and triethylene glycol, according to the lc50 values, can be designated as non-toxic to p. pictum embryos, which is confirmed by other research based on zebrafish (busquet et al., 2014; von hellfeld et al., 2020). while lc50 values for most of the tested chemicals from the p. pictum test were generally comparable in their toxicity with those observed using zebrafish embryos as test subjects (busquet et al., 2014), variations were observed, possibly reflecting species specific sensitivity to certain chemicals. these findings support the recommendations from previous studies that a greater variety of fish species should be involved and adapted to embryo toxicity testing (nagel, 2002; braunbeck et al., 2005; wedekind et al., 2007). this could be particularly important when conducting toxicity tests on wild populations. the resulting data would be valuable for assessing environmental risks and potential negative effects on the fish populations and the ecosystems in general. according to wedekind et al. (2007), a broader range of species should be tested, with embryos collected from natural populations of various species, including most fish that occur in the habitats for which the test substance is potentially relevant. range specified by the test guideline. the rest of the validity requirements were also met, such as: high fertilization rate for the batch of eggs, maintained temperature at 26±1 °c for the duration of the test, and hatching rate of more than 80% in the negative control (oecd, 2013). the notion that we only detected two out of four mortality endpoints could be due to species specific differences in embryo development. all designated endpoints in p. pictum embryos were visible even at 24 hpf, which is not the case for zebrafish embryos (oecd, 2013). according to braunbeck & lammer (2005), in the zebrafish based test, all of the endpoints are observed until 48h post fertilization. based on our observations, p. pictum embryos had a slightly faster development rate than zebrafish embryos in the same water temperature. this could possibly be adjusted by changing the temperature during the test. as expected, the embryos of p. pictum manifested different sensitivity to different types of chemical compounds (tab. 2). 4,6-dinitro-o-cresol exhibited the highest level of toxicity, which correlates with other studies (busquet et al., 2014; von hellfeld et al., 2020). this compound is part of a group of herbicides that have a unique mechanism of toxicity, by uncoupling the oxidative phosphorylation and interfering with the production of atp. this leads to energy not being available for essential functions in the cells (solomon et al., 2014). the results for copper (ii) sulfate and dibutyl maleate gave different values for lc50 compared to zebrafish values from the validation study (oecd 2011, oecd 2012, busquet et al., 2014). in that study, these compounds were fig. 1. normal embryo development in the control plate (a); coagulated embryo and mortality observed by lack of heart beat in deformed hatched larva (b) biologica nyssana ● 16 (2) december 2025: trajchevski et al. ● acute toxicity on early life stages of pachychilon pictum (heckel & knerr, 1878) from lake ohrid php/altex/article/view/911 braunbeck, t., & lammer e. (2005). draft detailed review paper on fish embryo toxicity assays. german federal environment agency. chandroo, k.p., duncan, i.j.h., & moccia r.d. (2004). can fish suffer?: perspectives on sentience, pain, fear and stress. applied animal behaviour science, 86(3-4), 225-250. https://doi.org/10.1016/j. applanim.2004.02.004 cooney, j.d. (1995). freshwater tests. in: rand, g.m. (ed), fundamentals of aquatic toxicology (2nd ed). boca raton, us: taylor & francis group, crc press. https://doi.org/10.1201/9781003075363 european union (2010). directive 2010/63/eu of the european parliament and of the council of 22 september 2010 on the protection of animals used for scientific purposes. http://data.europa.eu/eli/ dir/2010/63/oj fent, k. (2004). ecotoxicological problems associated with contaminated sites. toxicology letters, 205(3), 223-240. https://doi.org/10.1016/j. tox.2004.06.060 fiorino, e., sehonova, p., plhalova, l., blahoba, j., svobodova, z., faggio, c. (2018). effects of glyphosate on early life stages: comparison between cyprinus carpio and danio rerio. environmental science and pollution research, 25, 8542-8549. https://doi.org /10.1007/s11356-017-1141-5 jezierska, b., ługowska, k., & witeska, m. (2008). the effects of heavy metals on embryonic development of fish (a review). fish phisiology and biochemistry, 35, 625-640. https://doi.org/10.1007/ s10695-008-9284-4 kostoski, g., albrecht, c., trajanovski, s., & wilke, t. (2010). a freshwater biodiversity hotspot under pressure assessing threats and identifying conservation needs for ancient lake ohrid. biogeosciences, 7, 3999-4015. https://doi. org/10.5194/bg-7-3999-2010 kottelat. m. & freyhof, j. (2007). handbook of europen freshwater fishes. switzerland, cornol: publications kottelat. lammer, e., carr, g.j., wendler, k., rawlings, j.m., belanger, s.e., & braunbeck, t. (2009). is the fish embryo toxicity test (fet) with the zebrafish (danio rerio) a potential alternative for the fish acute toxicity test? comparative biochemistry and physiology part c, 149, 196–209 https://doi. org/10.1016/j.cbpc.2008.11.006 matzinger, a., spirkovki, z., patceva, s., & wuest, a. (2006). sensitivity of ancient lake ohrid conclusion in conclusion, our results showed that embryos from pachychilon pictum are suitable to be tested using the acute embryo toxicity test, and the guidelines of the oecd 236 protocol were successfully applied. given that this is the first ecotoxicological study conducted on fish from lake ohrid, the generated toxicological data hold significant value. with the increasing anthropological pressure on the ecosystems, this data could be used for future evaluations of environmental threats and potential negative impacts. our study contributes to the embryo toxicity research by adding another fish species to the increasing group of tested species and gives an insight into the applicability of this test to wild fish populations, and the possibility of their use in ecotoxicological evaluations. references albrecht, c., wilke, t. (2008). ancient lake ohrid: biodiversity and evolution. hydrobiologia, 615, 103-140. https://doi.org/10.1007/s10750-0089558-y busquet, f., strecker, r., rawlings, j.m., belanger, s.e., braunbeck, t., carr, g.j., cenjin, p., fochtman, p., gourmelon, a., hubler, n., kleensang, a., knobel, m., kussatz, c., legler, j., lillicrap, a., martinez-jeronimo, f., polleichter, c., rzodezcko, h., salinas, e., shneider, k.e., scholz, s., van den brandhof, e., van der ven, l.t.m., walter-rhode, s., weight s., witters, h., & halder, m. (2014). oecd validation study to assess intra and inter laboratory reproducibility of the zebrafish embryo toxicity test for acute aquatic toxicity testing. regulatory toxicology and pharmacology, 69(3), 496-511. https://doi. org/0.1016/j.yrtph.2014.05.018 belanger, s.e., rawlings, j.m., & carr, g.j. (2013). use of fish embryo toxicity for the prediction of acute fish toxicity to chemicals. environmental toxicology and chemistry, 32(8), 1768-1783. https://doi.org/10.1002/etc.2244 billard, r., cosson, j., perchec, g., & linhart, o. (1996). biology of sperm and artificial reproduction in carp. aquaculture, 129, 95-112. https://doi. org/10.1016/0044-8486(94)00231-c braunbeck, t., boettcher, m., hollert, h., kosmehl, t., lammer, e., leist, e., rudolf, m., & seitz, n. (2005). towards an alternative for the acute fish lc(50) test in chemical assessment: the fish embryo toxicity test goes multi-species—an update. altex, 22(2), 87–102. https://altex.org/index. j. (ed): endemic species from around the world: teaching for sustainability. intechopen. doi: 10.5772/intechopen.1003342 united nations (2023). globally harmonized system of classification and labelling of chemicals (ghs) (10th ed.). usa, new york: united nations publications. van der oost, r., beyer, j., & vermulen, n.p. (2003). fish bioaccumulation and biomarkers in environmental risk assessment: a review. enironmental toxicology and pharmacology, 13(2), 57-149. https://doi.org/10.1016/s13826689(02)00126-6 von hellfeld, r., brotzmann, k., baumann, l., strecker, r., & braunbeck, t. (2020). adverse effects in the fish embryo acute toxicity (fet) test: a catalogue of unspecific morphological changes versus more specific effects in zebrafish (danio rerio) embryos. environmental sciences europe, 32, 122. https://doi.org/10.1186/s12302-020-00398-3 von westernhagen, h. (1988). sublethal effects of pollutants on fish eggs and larvae. in: noar, w.s., & randall, d.j. (ed). physiology of developing eggs, part a: eggs and larvae. usa: academic press. https://doi.org/10.1016/s1546-5098(08)60201-0 wedekind, c., von siebnethal, b., & gingold, r. (2007). the weaker points of fish acute toxicity test and how test on embryos can solve some issues. environmental pollution, 148, 385-389. https://doi. org/10.1016/j.envpol.2006.11.022 to local anthropogenic impacts and global warming. journal of great lakes research, 32(1), 158-179. https://doi.org/10.3394/0380-1330(2006)32[158:so alot]2.0.co;2 nagel, r. (2002). dart: the embryo test with the zebrafish danio rerio – a general model in ecotoxicology and toxicology. altex, 19, 38–48. https://altex.org/index.php/altex/article/view/2125 oecd (2011). validation report (phase 1) for the zebrafish embryo toxicity test part 2. no. 157. oecd (2012). annexes to the validation report (phase 2) for the zebrafish embryo toxicity test. no. 179. oecd (2013). guideline for testing of chemicals, test no. 236: fish embryo acute toxicity (fet) test. oecd publishing. https://doi. org/10.1787/9789264203709-en power, m. (1997). assesing the effects of environmental stressors on fish populations. aquatic toxicology, 39, 151-169 https://doi.org/10.1016/ s0166-445x(97)00020-9 rundeva, i. (2014). biomarkers for stress in fish embryos and larvae. boca raton, us: taylor & francis group, crc press. https://doi.org/10.1201/ b15378 solomon, k.r., dalhoff, k., volz, d., & van der kraak, g. (2014). effects of herbicides on fish, in: tierney, k.b., farrell, a.p., & brauner, c.j. (ed). organic chemical toxicology of fishes. usa: academic press. https://doi.org/10.1016/b978-012-398254-4.00007-8 talevski, t., talevska, m., & talevska-leshoska, a. (2025). endemic fish species in the macedonian fresh water. in: cano ortiz, a., & pena-martinez biologica nyssana ● 16 (2) december 2025: trajchevski et al. ● acute toxicity on early life stages of pachychilon pictum (heckel & knerr, 1878) from lake ohrid novica ranđelović: tireless botanist explores on the verge of his ninth decade 1 novica ranđelović: tireless botanist explores on the verge of his ninth decade fig. 1. novica ranđelović and his crocuses: a crocus rujanensis n. randj. et d.a. hill; b c. jablanicensis n. randj. et v. randj.; c – c. danubensis h. kernd., e. pasche, n. randj. & v. randj. exactly ten years ago, 9th symposium on the flora of southeastern serbia and neighboring regions has been organized in honor of 70th birthday of novica randjelović. he was a founder of non-formal south serbian botanical school, symposium on the flora of southeastern serbia and neighboring regions, the first professor and founder of the department for biology and ecology at the university of niš (s t a m e n k o v i ć , 2007). even though he was in his eight decade, professor randjelović tirelessly continues his research activities. he published many scientific papers. especially important are those describing a new species from his favorite genus crocus c. jablanicensis (r a n đ e l o v i ć et al., 2011) i c. danubensis (h a r p k e et al., 2014). two species belonging to this genus were named in his honor – crocus novicii (m i l j k o v i ć et al., 2016) and c. randjeloviciorum (h a r p k e et al., 2017). he published 13 papers (6 in international journals) in the period from 2007 to 2017. besides that, he is still a regular participant of all botanical scientific symposiums held on the territory of entire balkan peninsula. 8 (1) • september 2017: 01-03 doi: 10.5281/zenodo.962581 biologica nyssana 8 (1)  september 2017: 01-03  novica ranđelović: tireless botanist explores on the verge... 2 bibliography of n. ranđelović in period from 2007 to 2017 ranđelović, n., sekovski, ž., dimeska, g. 2007: systematic, chorological and genetic research into the genus crocus l. macedonia collection of papers devoted to academician kiril micevski, 97-131. ranđelović, v., zlatković, b., milosavljević, v., ranđelović, n. 2008: the endemic flora of bosilegrad surroundings (krajište region) in southeastern serbia. phytologia balcanica, 14 (3): 367-375. milosavljević, v., ranđelović, v., zlatković, b., ranđelović, n. 2008: phytocenologic diversity of krajište in southeastern serbia. natura montenegrina, 7 (3): 193-204. stanisavljević, d. m., đorđević, s. m., ristić, m. s., veličković, d.t., ranđelović, n.v. 2010: effects of different drying methods on the yield and the composition of essential oil from herb mentha longifolia (l.) huds. biologica nyssana, 1 (1-2): 89-93. zlatković, b., nikolić, lj., ranđelović, v., ranđelović, n., stevanović, v. 2011: comparative analyses of the vascular flora of the pcinja river gorges in serbia and macedonia. archives of biological sciences, 63 (4): 11571166. ranđelović, n., ranđelović, v., hristovski, n. 2012: crocus jablanicensis (iridaceae), a new species from the republic of macedonia, balkan peninsula. annales botanici fennici, 49 (1-2): 99102. velickovic, d.t., ristic, m.s., bjelakovic, l.l., karabegovic, i.t., stojicevic, s.s., lazic, m.l., randjelovic, n.v. 2012: chemical composition of dictamnus albus l. essential oil from serbia. agro food industry hi tech, 23, 3. miljković, m., ranđelović, n., ranđelović, v. 2012: phytogeographical analysis of the flora of miljkovačka gorge in eastern serbia. biologica nyssana 3 (2): 77-90. harpke, d., peruzzi, l., kerndorff, h., karamplianis, th., constantinidis, th., ranđelović, v., ranđelović, n., jušković, m., pasche, e., blattner, f.r., 2014: phylogeny, geographic distribution and new taxonomic circumscription of the crocus reticulatus species group (iridaceae). turkish journal of botany, 38 (6): 1182-1198. stanisavljevic, d., dordevic, s., milenkovic, m., lazic, m., velickovic, d., randelovic, n., zlatkovic, b. 2014: antimicrobial and antioxidant activity of the essential oils obtained from mentha longifolia (l.) hudson, dried by three different techniques. records of natural products, 8 (1), 61. papović, o., miljković, m., ranđelović, n., ranđelović, v., 2014: analysis of the flora of rogozna mountain in southwestern serbia. biologica nyssana, 5 (1): 17-30. papović, o., miljković, m., ranđelović, n., ranđelović, v. 2014: phytogeographical characteristics and endemism of the flora of rogozna mt. (sw serbia). biologica nyssana, 5 (2): 103-112. fig. 2. novica ranđelović with associates from serbia and germany on the field biologica nyssana 8 (1)  september 2017: 01-03  novica ranđelović: tireless botanist explores on the verge... 3 harpke, d., carta, a., tomović, g., ranđelović, v., ranđelović, n., blattner, f. r., peruzzi, l. 2015: phylogeny, karyotype evolution and taxonomy of crocus series verni (iridaceae). plant systematics and evolution, 301(1): 309-325. references harpke, d., peruzzi, l., kerndorff, h., karamplianis, th., constantinidis, th., ranđelović, v., ranđelović, n., jušković, m., pasche, e., blattner, f.r. 2014: phylogeny, geographic distribution and new taxonomic circumscription of the crocus reticulatus species group (iridaceae). turkish journal of botany, 38(6): 1182-1198. harpke, d., kerndorff, h., raca, i., pasche, e. 2017: a new serbian endemic species of the genus crocus (iridaceae). biologica nyssana, 8 (1): 0713. miljković, m., ranđelović, v., harpke, d. 2016: a new species of crocus (iridaceae) from southern albania (sw balkan peninsula). phytotaxa, 265 (1), 39-49. ranđelović, n., ranđelović, v., hristovski, n. 2012: crocus jablanicensis (iridaceae), a new species from the republic of macedonia, balkan peninsula. annales botanici fennici, 49 (1-2): 99102. stamenković, v. 2007: 70 godina života prof. dr novice ranđelovića i 45 godina obrazovnovaspitnog i naučno-istraživačkog rada. proceedings of 9th symposium on flora of southeastern serbia and neighboring regions, niš, 1-7. microsoft word bn-oa-0201-09 zivkovic & jovanovic biologica nyssana 2 (1) september 2011: 51-61 živković, d., jovanović, b. spatial morphometric plasticitz of spirlin… 67 original article ! spatial morphometric plasticity of spirlin alburnoides bipunctatus (bloch, 1782) phenotype from the nišava river, serbia, danube basin dušan živković1, boris jovanović*2 1 universityof niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, niš, serbia 2 interdepartmental toxicology program and fisheries biology program, departments of biomedical sciences and natural resource ecology and management, iowa state university, ames, ia, usa * e-mail: prcko@iastate.edu abstract: živković, d., jovanović, b.: spatial morphometric plasticity of spirlin alburnoides bipunctatus (bloch, 1782) phenotype from the nišava river, serbia, danube basin. biologica nyssana, 2 (1), september 2011: 67-77. variation of 22 morphometric and 4 meristic characters of the species alburnoides bipuuctatus from nišava river were analyzed. specimens were collected from four sampling stations along the putative mesohabitat gradient of river influence and were grouped into the categories with similar total length. morphometric characters varied immensely between different mesohabitats outlining morphometric plasticity. among several morphometry features that were influenced by different mesohabitats the most observable differences were noted for: postocular distance, anal fin height, minimal body height and preanal length. regarding the meristic features, number of soft rays in the anal fin did varied significantly among sampling stations while fourth hard ray in the anal fin for this species was reported for the first time ever. pharyngeal teeth formula of spirlin also showed variability across different mesohabitats. in conclusion, it appears that spirlin express a big morphological plasticity in relation to spatial and mesohabitat distribution and there is a possibility that spirlin from nišava river is a complex of neospecies in formation/morphotypes/ecotypes rather than a single population. key words: alburnoides bipunctatus, cyprinidae, mesohabitat, phenotype plasticity introduction ! spirlin alburnoides bipunctatus (bloch, 1782) – cyprinidae is a freshwater species reaching a maximal length of 13-14 cm (l e l e k , 1987). it is a sub-montane species typically present in upper and middle rithronic communities. it is widely distributed in europe and in north and west asia including the turkey and iran (b o g u t s k a y a , 1997, l e l e k , 1987). due to spirlin population abundance in majority of its areal, it is an important prey item of many predatory fish species, while its main food items are diatoms and green algae (t r e e r et al., 2006). spirlin is listed in iucn as a species of least concern, but locally threatened, and its status in serbia is lr (lc) (s i m o n o v i ć , 2001). several subspecies have been described for european region: a. bipunctatus rossicus (berg) from volga basin; a. bipunctatus strymonicus (chichkoff) from marica drainage; a. bipunctatus ohridanus (karaman) and a. bipunctatus fasciatus (nordmann) from ohrid lake and drim basin and a.bipunctatus prespensis (karaman) from lake prespa (l e l e k , 1987). at least six subspecies are present in asia (l e l e k , 1987). nominate subspecies inhabit both europe and asia. its areal is 2 (1) • september 2011: 67-77 biologica nyssana 2 (1) september 2011: 51-61 živković, d., jovanović, b. spatial morphometric plasticitz of spirlin… 68 from loire drainage in france eastward, in nearly all rivers draining to southern baltic, north, black and azov seas; caspian basin, in upper volga and from kura drainage southward to iranian tributaries of caspian (f r o e s e & p a u l y , 2011). nominate subspecies is also the one inhabiting nišava river (s i m o n o v i ć , 2001). recently, a relationship between morphometry and microhabitat utilization by a. bipunctatus has been established (copp et al., 2010; k o v a č et al., 2006). the differences observed were mainly due to different preference for water velocity, depth and substratum, within the same sampling station, as the fish get older (c o p p et al., 2010). such, segregation is likely connected with very specific abrupt isometric growth of a. bipunctatus two periods of isometric growth with different proportional values interrupted with short interval of allometric growth (k o v a č et al., 2006). such adaptation is increasing swimming performance of spirlin and provides better microhabitat utilization. while microhabitat analyses are based on identifying the segregation and preferences toward specific ecological factors within a habitat, mesohabitat modelling is based on a coarser scale covering longer sections of the river with mixture of ecological factors and adds the spatial component. the degree of spatial influence can be immense when analysing species with high morphometric plasticity potential. spatial component as a function of mesohabitat was not evaluated in the past regarding variation in morphometry for this species. therefore the aim of this study was to investigate whether the phenotypes of the spirilin will be significantly different along the same river. materials and methods stream and sampling site description the nišava river with its tributary ginska is a 202 km long with a watershed area of 4068 km2. it is situated in southeast serbia and bulgaria (upper one fourth of the river) and it is the largest tributary of the južna morava river, part of the southern danube river watershed (g a v r i l o v i ć & d u k i ć , 2002). the nišava has two distinct parts: upstream of the town of bela palanka it is a narrow, rapid mountain stream ranging from 6 to 76 m in width and 0.6 to 2 m in depth with velocity from 1 to 2.5 m s-1 and discharge of 0.3-270 m3 s-1 of water. after bela palanka and all the way to južna morava river, the nišava becomes a meandering stream with increased width and depth, and flow that carries up to 700 m3 s-1 of water (b r a n k o v i ć , 1997). sampling of spirlin was performed on four sites (fig. 1). the nišava river at ivko’s watermill, 1 km upstream from the city of dimitrovgrad was selected as site l1. this is the first section of the river as it enters serbia and there are no larger settlements upstream to this sampling site. in this section the river has a width of 3-6 m, and a depth of no more than 1 m. the bottom is composed of gravel, and the banks are covered with thick vegetation. the section is composed of light riffles intersperse with glide sections. the majority of the benthofauna is composed of ephemeroptera and trichoptera taxons, while in the ichthyofauna the most dominant species are: spirlin, brook barbel barbus balcanicus (kotlik, tsigenopoulos, rab & b e r r e b i 2002) cyprinidae and chub leuciscus cephalus (l. 1758) cyprinidae. figure 1. geographic region of nišava river. the four sampling stations (l1-l4) are indicated with dotted arrows biologica nyssana 2 (1) september 2011: 51-61 živković, d., jovanović, b. spatial morphometric plasticitz of spirlin… 69 site l2 – tributary of temska stream to nišava river. temska stream is the typical salmonid stream and the biggest right tributary of nišava river. the tributary section is enveloped in thick jungle-like vegetation, and river bottom is rocky with cobble size stones. the average water discharge is 10.5 m3 s-1 (ž i v i ć et al., 2005). mouth of the tributary is about 4 m wide with depth of 0.5 m and the whole section is with light riffles. benthofauna is almost exclusively composed of over dominant order trichoptera with occasional representatives of nematomorpha, hirudinea, odonata and megaloptera (ž i v i ć et al., 2005). ichthyofauna is similar to site l1 with addition of bleak alburnus alburnus (l. 1758) cyprinidae, barbel barbus barbus (l. 1758) – cyprinidae and nase chondrostoma nasus (l. 1758) cyprinidae. site l3 nišava river after town of bela palanka and before sićevo gorge. this section of river has biggest width, and it is essentially one long glide with mixture of sandy and muddy bottom with dept often over 1.5 m. as site l4, the nišava river before its entry of the major city of niš was selected (suburbia donja vrežina). the selected site is located on the last section of river, about 12-15 km before its mouth. in this section the nišava river is between 0.2 and 1.2 m deep and is a mixture of small riffle areas and stretches of slow moving water. the bottom is composed of gravel with copious cladophora sp. algae clinging on the bottom. benthofauna is composed predominantly of classes gastropoda, hirudinea, clitellata and malacostraca (gammaridae). icthyofauna is rich, with the predominant species such as: spirlin, barbel, brook barbel, chub, bleak, nase, perch perca fluviatilis (l. 1758) – percidae, bitterling rhodeus sericeus (p a l l a s , 1776) cyprinidae, carassius gibelio (b l o c h , 1783) cyprinidae, carp cyprinus carpio (l. 1758) cyprinidae, roach rutilus rutilus (l. 1758) cyprinidae, gudgeon gobio spp., as well as other typical lowland cyprinid species. morphometry several hundreds of spirlin specimens were sampled at each site ignoring the differences in microhabitats and sampling along the whole profile section. after the sampling, 40 specimens > 60 mm were chosen at random from each of the sampling sites and 22 morphometric and 4 meristic characters were measured or counted. list of measured morphometric characteristics is provided in fig. 2. all measurements were taken with 0.1 mm precision using vernier. list of meristic features counted is outlined in fig. 3. in adition mass of specimens were recorded using digital scale (kern 440’33, readability d = 0.01 g). figure 2. morphometry of a. bipunctatus a-b2: total length (longitudo totalis) a-b1: fork length (longitudo caudalis) a-v: standard length (longitudo corporis) c-g: proposal length (longitudo praedorsalis) g-d: base length of dorsal fin (longitudo dorsalis pinnae) hd: dorsal fin height (altitudo dorsalis pinnae) dc: postdorsal length (longitudo corporis postdorsalis pinnae) i-l: head length (longitudo capitis) i-j: preocular distance (spatium preorbitale) j-e: horisontal diameter of eye (diameter oculi horizonialis) k-l: postocular distance (spatium postorbitale) h-i-l: maximal head height (altitudo capitis) m-n: prepectoral length (longitudo praepectoralis) o-p: preventral length (longitudo praeventralis) o-a: preanal length (longitudo praeanalis) g-r: base length of anal fin (longitudo analis pinnae) hp: pectoral fin height (altitudo pectoralis pinnae) hv: ventral fin height (altitudo ventralis pinnae) ha: anal fin height (altitudo analis pinnae) t-v: caudal trunk length (longitudo pedunculi caudalis) h: maximal body height (altitudo corporis maxima) h: minimal body height (altitudo corporis minima) biologica nyssana 2 (1) september 2011: 51-61 živković, d., jovanović, b. spatial morphometric plasticitz of spirlin… 70 statistics for morphometry data sample from each sampling site was divided into two size categories based on the total length of the individuals: category a 60 80 mm; category b 80 100 mm. similar approach was previously used with success to assess difference in morphometry characteristics in mixed age sample for spirlin (k o v a č et al., 2006; c o p p et al., 2010). there were only five specimens in total longer than 100 mm (the longest was 122 mm) and these specimens were discarded from any further analysis. analysis of variance (anova) was used to depict any differences in characters between different sampling sites for each category separately followed by tukey – hsd post hoc comparison of means test. only values with p < 0.05 were considered statistically significant. results morphometrics morphometric results for each category are presented in tab. 1. total length of specimens in category a did not varied significantly between sampling sites (anova; df total = 63; f = 2.50; p > 0.05); however there was a significant difference in morphometry of 9 out of 22 characters (tab. 2). standard length (a-v) of specimens from site l1 was significantly smaller than the ones from l2 and l4 indicating a longer tail fin. specimens from l1 had shorter base of the dorsal fin (g-d) comparing to l4 specimens; l1 specimens also had shorter postdorsal length (dc) comparing to the l2 and l4 specimens. there was a small difference in preventral length (o-p) and preanal length (o-a) with o-p being shorter in l1 compared to l2 specimens and o-a being shorter in l1 as opposed to l4. caudal trunk length (t-v) was also shorter in l1 vs. l2 specimens. the most observable differences were for postocular distance (k-l); anal fin height (ha); and minimal body height (h) (fig. 4). postocular distance from l4 is significantly smaller comparing to specimens from any other sampling site. anal fin height was considerably bigger in l2 specimens comparing to any other sampling location while minimal body height of l1 was significantly smaller comparing to other samples. mass of the specimens varied slightly between localities for category a (anova; df total = 63; f = 4.59; p < 0.01). average mass with standard deviation of the specimens from l1-l4 were: 3.31 + 0.74; 4.64 + 0.53; 4.24 + 1.26; and 3.95 + 1.02 g respectively. category b significantly varied in total length between sampling locations (anova; f = 13.04; df total = 82; p << 0.001) (fig. 5). specimens from l1 and l4 sites had the same mean total length and were significantly different from l2 and l3 specimens. sample l2 and l3 had the approximately the same mean total length. since there was obvious difference in total length the rest of 21 morphometric features were compared only between the samples with the same mean. l1 vs. l4 and l2 vs. l3. results are presented in fig. 6. mass of the specimens varied significantly between localities for category b (anova; df total = 82; f = 16.55; p < 0.001). average mass with standard deviation of the specimens from l1-l4 were 6.34 + 1.23; 8.19 + 1.65; 9.32 + 1.46; and 6.42 + 1.20 g respectively. meristics number of scales in lateral line did not varied significantly between sampling sites (anova; df total = 159; f=0.41; p >> 0.05) and the average scale number formula for the whole river profile is 43-50 8-10/3-5. there was no difference in average number of hard or soft rays in the dorsal fin between sampling sites (anova; df total = 159; figure 3. meristic characters of a. bipunctatus. d: number of rays in dorsal fin separately for hard and soft (numerus radiorum pinnae dorsalis) a: number of rays in anal fin separately for hard and soft (numerus radiorum pinnae analis) ll: number of sacles in the lateral line (squamae linea lateralis) shown in red; number of scales above and below the lateral line (squamae linea trasversalis superior et squamae linea trasversalis inferior) shown in ocher. df: number of pharyngeal teeth (dentes pharyngeales) biologica nyssana 2 (1) september 2011: 67-77 živković, d., jovanović, b. spatial morphometric plasticitz of spirlin… 71 l1 l2 l3 l4 78 80 82 84 86 88 90 92 94 96 98 ab2 l1 l2 l3 l4 5.0 5.2 5.4 5.6 5.8 6.0 6.2 6.4 6.6 6.8 7.0 7.2 7.4 7.6 7.8 8.0 kl l1 l2 l3 l4 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 12.0 ha l1 l2 l3 l4 4.8 5.0 5.2 5.4 5.6 5.8 6.0 6.2 6.4 6.6 6.8 7.0 h figure 4. difference in a. bipunctatus category a morphometry characteristics between four sampling locations. y axis is in mm corresponding to the proper measurement (figure 1). l1 – l4 are different sampling locations. boxes refer to standard error and whiskers to standard deviation figure 5. distribution of a. bipunctatus category a total length morphometry across four sampling locations. y axis is in mm of total length. l1 – l4 are different sampling location. boxes refer to standard error and whiskers to standard deviation biologica nyssana 2 (1) september 2011: 51-61 živković, d., jovanović, b. spatial morphometric plasticitz of spirlin… 72 table 1. morpfometric analysis of a. bipunctatus. all data except column % are in mm. % refers to the percent of given character compared to total length of the specimen. std refers to standard deviation. category a category b mean % std min max mean % std min max a-b2 70.9 100.0 5.3 60.7 79.0 89.1 100.0 5.3 80.0 100.0 a-b1 64.5 91.0 4.9 54.5 72.4 81.4 91.4 5.1 72.1 96.0 a-v 58.0 81.8 4.5 50.0 65.7 73.3 82.3 4.6 64.2 83.0 c-g 30.6 43.2 2.7 25.5 37.3 38.6 43.3 2.8 33.0 44.5 g-d 7.29 10.3 0.9 5.2 9.7 9.4 10.5 0.8 7.7 11.5 hd 11.7 16.5 1.2 8.2 14.2 15.2 17.1 1.4 12.0 19.0 d-c 19.7 27.8 1.8 16.0 23.6 24.6 27.6 1.9 20.0 29.0 i-l 14.5 20.5 1.2 12.0 17.0 18.0 20.2 1.2 14.0 20.6 i-j 3.9 5.5 0.6 2.6 5.2 4.9 5.5 0.4 4.0 6.0 j-e 4.2 5.9 0.5 3.3 6.5 5.0 5.6 0.3 4.0 5.7 k-l 6.3 8.9 0.7 4.9 8.0 8.0 9.0 0.9 4.9 10.1 h-i-l 11.1 15.7 0.9 9.5 13.0 13.9 15.6 1.0 12.2 16.7 m-n 14.5 20.5 1.3 11.8 17.4 18.1 20.3 1.4 14.0 21.3 o-p 27.1 38.2 2.8 20.0 32.6 34.3 38.5 2.3 28.6 40.0 o-a 37.2 52.5 3.0 30.5 43.0 47.5 53.3 3.6 40.0 57.2 g-r 9.6 13.5 1.2 7.0 12.0 12.6 14.1 1.3 10.0 17.0 hp 11.2 15.8 1.1 8.6 13.2 14.2 15.9 1.2 12.0 17.4 hv 8.8 12.4 1.0 7.0 11.4 11.0 12.3 1.1 9.0 13.0 ha 9.5 13.4 1.3 6.0 12.0 11.8 13.2 1.3 9.0 15.2 t-v 11.9 16.8 1.3 9.7 15.2 15.1 16.9 1.3 12.7 18.0 h 15.7 22.1 1.6 12.6 20.0 21.0 23.6 2.1 16.4 25.0 h 5.9 8.3 0.67 4.4 7.0 7.5 8.4 0.7 5.8 9.0 table 2. analysis of variance and post hoc comparison of means of a. bipunctatus category a morphometry. l1 – l4 refers to the sampling site. +; ++; +++ means that the effect is statistically significant with p value being < 0.05; < 0.01; and < 0.001 respectively. ns means that the effect is not statistically significant. a-v g-d dc k-l o-p o-a ha t-v h p value + + + +++ + + ++ + ++ anova p – value tukey hsd l1 vs. l2 + ns + ns + ns ++ + + l1 vs. l3 ns ns ns ns ns ns ns ns + l1 vs. l4 + + + +++ ns + ns ns + l2 vs. l3 ns ns ns ns ns ns + ns ns l2 vs. l4 ns ns ns + ns ns ++ ns ns l3 vs. l4 ns ns ns +++ ns ns ns ns ns biologica nyssana 2 (1) september 2011: 51-61 živković, d., jovanović, b. spatial morphometric plasticitz of spirlin… 73 table 3. distribution of hard and soft rays in the anal fin of a. bipunctatus from nišava river. l1 – l4 refers to sampling locations. roman numerals refer to hard rays while arabic refers to soft rays. data are presented as % of sample. table 4. distribution of pharyngeal teeth of a. bipunctatus from nišava river. l1 – l4 refers to sampling locations. data are presented as % of the sample. f = 0.06; p >> 0.05). the overall number of rays in dorsal fin was d, ii-iii + (7-8) 9 (10). number of hard rays in anal fin did not varied statistically among the samples (anova; df total = 159; f=0.84; p >> 0.05), however for the first time in science a fourth hard ray in anal fin was detected for this species which has never been documented elsewhere. the fourth hard ray was present only in the specimens from sampling station l2 and l3. on the other hand, number of soft rays in the anal fin did varied significantly among sampling stations (anova; df total = 159; f = 5.61; p < 0.01). there was significantly higher number of soft rays in the specimens from l2. distribution of hard and soft rays from anal fin across sampling stations is presented in tab. 3. formula of anal fin rays is: a, (ii) iii (iv) + (10) (12) 13-15 (16). pharyngeal teeth of spirlin are in two rows. first row have two teeth, while the number of teeth in second raw is variable. specimens from l3 had more pharyngeal teeth on the right branchial arch than specimens from any other sample (anova; df total = 159; f = 9.08; p < 0.001). commonly these specimens had 7 teeth on the right branchial arch while specimens from other samples had 5 or 6 (tab. 4). discussion it is important to note that for the overall sample (category a + b) individuals from l2 and l3 on average have the biggest total length and mass. also, the biggest total length of any individual was 120 mm for l2 and 104.6 for l3 while the same parameter was only 90 mm for l1 and 91 mm for l4. one of the possible reasons for this might be better feeding conditions on sites l2 and l3. the studies performed in the fair vicinities of these localities demonstrated abundance of diatoms (t r a j k o v i c et al., 2008) which are spirlin’s primary food item (t r e e r et al., 2006) as well as caddis flies (ž i v i ć et al., 2005) which are secondary food item (t r e e r et al., 2006). better feeding can lead to faster growth rate which for this species is particularly characterized with two periods of isometric growth with different proportional values interrupted with short interval of allometric growth (k o v a č et al., 2006). thus, a difference in a rate of allometric growth can result in drastic changes of morphometry. in general, l2 and l3 spirlin had biggest anal fin height, as well as the biggest maximal body height and maximal observed total length, indicating better growth than spirlin ii iii iv 10 11 12 13 14 15 16 l1 12.5 87.5 0 0 0 5 45 37.5 12.5 0 l2 12.5 82.5 5 0 0 2.5 12.5 42.5 37.5 5 l3 5 92.5 2.5 0 0 10 30 50 10 0 l4 5 95 0 2.5 0 15 17.5 50 12.5 2.5 average 8.75 89.38 1.875 0.625 0 8.13 26.3 45 18.125 1.875 left branchial arch right branchial arch 3 4 5 6 7 3 4 5 6 7 l1 12.5 22.5 30 27.5 7.5 5 7.5 20 17.5 50 l2 2.5 17.5 35 35 10 5 12.5 20 25 37.5 l3 5 10 35 45 5 0 2.5 5 12.5 80 l4 2.5 17.5 37.5 37.5 5 5 5 35 37.5 17.5 average 5.625 16.875 34.375 36.25 6.875 3.75 6.875 20 23.125 46.25 biologica nyssana 2 (1) september 2011: 67-77 živković, d., jovanović, b. spatial morphometric plasticitz of spirlin… 74 l1 l4 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 kl c l1 l4 15.0 15.5 16.0 16.5 17.0 17.5 18.0 18.5 19.0 i-l a l1 l4 15.0 15.5 16.0 16.5 17.0 17.5 18.0 18.5 19.0 m -n a l2 l3 12.5 13.0 13.5 14.0 14.5 15.0 15.5 16.0 hi-l b l2 l3 32 33 34 35 36 37 38 op b l2 l3 42 44 46 48 50 52 54 oa c l2 l3 13.5 14.0 14.5 15.0 15.5 16.0 16.5 17.0 17.5 t-v b l2 l3 18 19 20 21 22 23 24 25 h c l2 l3 6.5 7.0 7.5 8.0 8.5 h b figure 6. difference in a. bipunctatus category b morphometry characteristics. y axis is in mm corresponding to the proper measurement (figure 1). l1 – l4 are different sampling locations. boxes refer to standard error and whiskers to standard deviation from other localities. spirlin of both categories from the l1 locality compared to spirlin from other samples had shorter base of the dorsal fin, shorter postdorsal, preventral, preanal, caudal trunk and minimal body length while having longer head, post ocular and prepoctaral length and theoretically longer tail fin. essentially this gives them a streamlined, torpedo-like shape with powerful head to pectoral region and slender, narrow streamline body for propelling themselves through the faster water current. fish with more streamline body have less drag (by minimizing viscous force) in a faster current, spending less energy by utilizing continuous, rather than burst-and-coast swimming mode (m ü l l e r et al., 2000; p e t r e l l & j o n e s , 2000). results of this investigation suggest that morphometry of spirlin can vary significantly for some characters, while others are not affected. comparison of this study with previous investigation of nominate subspecies features inhabiting different mesohabitat type (river gradac) identified head length (i-l); maximal head height (hi-l); base length of dorsal fin (g-d); caudal trunk length (t-v); maximal body height; and minimal body height (h) as characters with biggest variability (s t a n i s a v l j e v i ć et al., 1999). in conclusion, it appears that spirlin express a big morphological plasticity in relation to spatial and mesohabitat distribution. whether the plasticity was based on the influence of environmental factors, or it resulted from the expression of an organism's genes as well as the interactions between the two so far is not known. however, it is known that the karyotype of spirlin varies significantly on a spatial scale (k i l i c -d e m i r o k & ü n l ü , 2004). biologica nyssana 2 (1) september 2011: 51-61 živković, d., jovanović, b. spatial morphometric plasticitz of spirlin… 75 coupled with the number of known subspecies (l e l e k , 1987); the number of newly discovered sister species which were until recently classified as nominate subspecies (b o g u t s k a y a et al., 2010; c o a d & b o g u t s k a y a , 2009); and the new meristic characteristics presented in this manuscript it is reasonable to conclude that taxon a. bipunctatus bipunctatus is most likely complex of cryptic species/subspecies/neospecies in formation/morphotypes/ecotypes rather than a single nominate subspecies. it is possible (especially for species in formation) that species complex has the same genetic material (genome) which can be accounted for successful reproduction among different members, but completely different gene expression (transcriptome). it is hard to carry out such experiments for the species for which genome is not yet sequenced and most of the time pieces of information can be obtained through expressed sequence tag (est) sequencing coupled with genomics which is a laborious process based on pure luck of proper est extraction (j o v a n o v i ć et al., in press). however, the recent advances in "next generation" sequencing platforms and technologies, for quantitative comparative transcriptomics allowed for successful differentiation in the hepatic transcriptomes between two lake trout ecotypes, and demonstrated that this approach could be used to simultaneously discover and quantify differentially expressed genes in a nonmodel organism lacking a sequenced genome (g o e t z et al., 2010). this would be the right approach to discern whether spirlin from nišava river is a complex of different transcriptomic populations/ecotypes or not, and determine the level of crypticism within a complex if any. acknowledgements. we are thankful to dr stevan maletin for the useful comments regarding morphometry techniques. references bogutskaya, n.g., (1997). contribution to the knowledge of leuciscine fishes of asia minor. part 2. an annotated check-list of leuciscine fishes (leuciscinae, cyprinidae) of turkey with descriptions of a new species and two new subspecies. mitteilungen hamburgisches zoologisches museum und institut 94, 161-186. bogutskaya, n.g., zupančič, p. & naseka, a.m. (2010). two new 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(2006). diet and growth of spirlin, alburnoides bipunctatus in the barbel zone of the sava river. folia zoologica, 55(1): 97–106. živić, i., marković, z. & ilić, j. (2005). composition, structure, and seasonal dynamics of macrozoobenthos in the temska and visočica rivers (serbia). archive of biological sciences, 57 (2): 107-118. brahim et al. 2025, biologica nyssana 16(2) 16 (2) december 2025: doi: 10.46793/biolnyss.16.2.18b entomological diversity associated with apple trees in the aurès region (northeastern algeria) original article imene brahim university of batna 1, institute of veterinary and agronomic sciences, department of agronomy, 05000 batna, algeria; lapapeza laboratory, department of agricultural sciences, institute of veterinary and agricultural sciences, university of batna 1, 05000 batna, algeria. smail chafaa university of batna 2, department of ecology and environment, batna, algeria; laboratory of cellular and molecular physiotoxicology-biomolecules, faculty of science of nature and life, 05000 batna, algeria. s.chafaa@univ-batna2.dz. (corresponding author) fateh mimeche university of m’sila, department of agricultural sciences, 28000 m’sila, algeria. mohamed kebieche university of batna 2, department of ecology and environment, batna, algeria; laboratory of cellular and molecular physiotoxicology-biomolecules, faculty of science of nature and life, 05000 batna, algeria received: july 16, 2025 revised: november 20, 2025 accepted: november 28, 2025 abstract: pomiculture is a social and economic activity that holds considerable importance in the aurès region. to learn more about the diversity of insects that coexist with apples in this particular region, especially around tazoult, four types of sampling have been carried out: classical visual harvesting, yellow traps, white traps, and barber pots from february until april. the results recorded 156 individuals belonging to 17 species, arranged into 6 orders and 16 families, further grouped into four trophic categories: phytophages, predators, coprophagous, and saprophagous. among them, phytophages were the most dominant during our inventory. the shannon diversity index, h', showed high values, indicating high specific diversity. it could be explained by the diversification of environments at this period, which provided more favorable conditions for the establishment of larger numbers of individuals per species. in addition, close-to-unity equity values recorded suggest a high balance among the populations of the different sampled insect species at our study station. this study reveals the strong insect diversity of aurès apple orchards and highlights their ecological importance for regional conservation. it encourages knowledge of agricultural ecosystem interactions and identifies key issues for sustainable pomiculture management. key words: inventory, malus domestica borkh., insect, arthropoda, orchard apstrakt: entomološka raznovrsnost stabala jabuke u području aures (severoistočni alžir) pomikultura je društvena i ekonomska delatnost koja ima veliki značaj u aures oblasti. kako bi se bolje upoznala raznovrsnost insekata koji koegzistiraju sa jabukama u ovoj specifičnoj regiji, posebno u okolini tazoulta, sprovedene su četiri vrste uzorkovanja: klasično vizuelno sakupljanje, žute klopke, bele klopke i barberove klopke, u periodu od februara do aprila. rezultati su zabeležili 156 jedinki koje pripadaju 17 vrsta, raspoređenih u 6 redova i 16 familija, dalje grupisanih u četiri trofičke kategorije: fitofagi, predatori, koprofagi i saprofagi. među njima, fitofagi su bili najdominantniji tokom našeg inventarisanja. indeks diverziteta po shannonu, h', pokazao je visoke vrednosti, što ukazuje na veliku specifičnu raznovrsnost. ovo se može objasniti diverzifikacijom staništa u ovom periodu, što je obezbedilo povoljnije uslove za uspostavljanje većeg broja jedinki po vrsti. pored toga, zabeležene vrednosti jednakosti bliske jedinstvu ukazuju na visok balans među populacijama različitih vrsta insekata prikupljenih na našoj istraživačkoj stanici. ova studija otkriva veliku raznovrsnost insekata u voćnjacima jabuke u oblasti aures i naglašava njihov ekološki značaj za regionalnu konzervaciju. ona podstiče razumevanje interakcija u poljoprivrednim ekosistemima i identifikuje ključne izazove za održivo upravljanje pomikulturom. ključne reči: inventarisanje, malus domestica borkh., insekt, arthropoda, voćnjak © 2025 brahim et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. introduction the apple tree (malus domestica borkh.) is one of the most economically important fruit crops globally, with significant production and consumption levels (fao, 2021). in algeria, apple cultivation plays a crucial role in agricultural development, particularly in the northeastern region, including batna, where orchards contribute substantially to local economies and rural livelihoods. despite its agronomic importance, apple production faces numerous challenges, including pest infestations that can severely impact yield and fruit quality (dsa, 2020). this trend reflects not only the growing enthusiasm for apple cultivation in this region but also the strategic value of this crop to the local economy. the increased apple production in algeria is expected to achieve food security, improve farmers’ incomes by meeting the ever-increasing demand in the domestic market, and export the surplus. the result has been that the cultivation of apple trees has been one of the main issues in the country’s agrarian and economic development. however, despite increasing areas of the plantation, apple production remains scarce. it can be explained by several factors, including cultivation method, orchard age, fertilization, soil care, and phytosanitary treatments. however, the apple tree, like all trees, offers the best conditions necessary for the development of diseases, which can be fungal, bacterial, or viral, and for the appearance of animal pests, especially insects (brahim, 2022). the apple tree is particularly susceptible to a variety of pests. notable among these are the codling moth (cydia pomonella l.), various types of aphids such as the grain aphid (dysaphis plantaginea (passerini, 1860)) and the apple aphid (aphis pomi de geer, 1773), as well as the san josé scale (diaspidiotus perniciosus (comstock, 1881) ). the most important mites, amongst others, include the two-spotted spider mite (tetranychus urticae koch, 1836) and european red mite (panonychus ulmi koch (1836)) (gautier, 2001; chafaa, 2008; brahim, 2022). arthropods also play a fundamental role in pest biological control in agroecosystems (desneux et al., 2007) and in the food webs of many animal species. intensive management practices are used in apple orchards, including high application rates of pesticides, herbicides, and fertilizers, which can be very harmful to environmental and local biodiversity within the orchard. such methods affect the arthropod community, which is usually crucial to ecological balance and a key component of biodiversity in agroecosystems (chakravarthy et al., 2016; herz et al., 2019). many researchers have assessed habitat quality and measured habitat differences using arthropods (ali-shtayeh et al., 2010). in algeria, some remarkable works on apple tree parasites include those of frah et al. (2009), mahdjane (2013), medjkane & laguel (2015), and guermah et al. (2019; 2022). at the international level, the works of hulle et al. (1998) and cross et al. (2006) are among the most important ones published. these will add to the overall understanding of the phytosanitary problems the apple tree faces, with integrated management as a means of increasing its fruit production. the objective of the present study is to contribute to the analysis of insect diversity in an apple orchard in the tazoult region. through various capture techniques, this work will contribute to knowledge of the species found in this environment and explain their distribution within their biotope. materials and methods study area the station where this study was conducted is located on the left side of the national road (n° 31), 4.7 km north of the tazoult area and 12.8 km southeast of the chief town of the batna department in northeast algeria. the climate of the study area during the period 2010–2019 was characterized by a total average precipitation of 294.54 mm and an average temperature ranging from 0.09 °c in january to 36.36 °c in july. the study area is in a semi-arid bioclimatic stage with cool winters. this orchard was established in 2020 at 35°30’31” north latitude, 6°16’00” east longitude, and 1200 m of altitude. it has a rectangular shape and covers an area of 1.6 hectares. it features a total of 750 apple trees, divided into two varieties: red delicious is the most dominant, followed by red gala. the orchard consists of 9 rows, with a row spacing of 4 m and a tree spacing of 1.2 m. the irrigation system employed is drip irrigation, and fertilization is carried out using organic manure and mineral fertilizers (n, p, k). sampling different sampling methods were used during the winter-spring seasons, specifically from february to april 2023, when most insects were active. in this perspective, a qualitative inventory of this fauna was made by the classical methods of sampling with visual observation, following to colas (1974), and a quantitative one using different techniques: yellow traps, barber pots, and white traps, were used to realize a complete insect biodiversity inventory (chafaa et al., 2019; mimeche et al., 2024). insects were sampled every two weeks, and the collected species were taken to the laboratory and preserved in tubes containing 70% alcohol, each labeled with the date of collection, the type of trap used, and the site of collection. in the laboratory, the captured insects were sorted, counted, and identified to species and developmental stage using taxonomic keys from colas (1974), dierl & ring (1992), and chouinard et al. (2000). the voucher specimens were deposited at the department of ecology and environment (university of batna 2, batna, algeria). statistical analysis the relative abundance (fa) of each taxonomic order was calculated as its percentage contribution to the total specimen count. diversity assessment incorporated: (i) species richness (s), (ii) the shanbiologica nyssana ● 16 (2) december 2025: brahim et al. ● entomological diversity associated with apple trees in the aurès region (northeastern algeria) biologica nyssana ● 16 (2) december 2025: brahim et al. ● entomological diversity associated with apple trees in the aurès region (northeastern algeria) non-wiener index (h’ = -σ(pi×lnpi), where pi = ni/n), and (iii) pielou’s evenness (e = h’/ln(s)). β-diversity was evaluated using the sørensen dissimilarity index (βsør). nonparametric comparisons were performed using the kruskal-wallis test, complemented by permanova with bray-curtis dissimilarities. all analyses were conducted in past 4.16 (2024), with nmds ordination and permutation tests (n = 999) used for validation. diagnostic checks included calculating stress values for nmds and testing normality assumptions. results and discussion systematic inventory the sampling techniques employed at an apple orchard in tazoult enabled us to compile a taxonomic list of the recorded species. this study identified 17 species, based on the identification of 156 individuals belonging to the class insecta, across six orders and 16 families. the species were categorized into four trophic groups: phytophages, predators, coprophages, and saprophages (tab. 1). as noted by dajoz (2002), the class insecta represents one of the largest classes in the animal kingdom; it is eudoxic, free-living, and embraces a very wide range of ecological niches. moreover, beetles are the most abundant and species-rich insects. recent studies in regions like khenchela (tayaa, 2024) and bouira (maandi & sadoudi, 2020) report similar coleopteran dominance in apple agroecosystems. the obtained results are very close to those of frah et al. (2009) in the bouhmama c la ss o rd er s fa m ili es sp ec ie s d ye llo w tr ap s w hi te tr ap s b ar be r po ts v is ua l h un tin g to ta l in se ct a c ol eo pt er a c oc ci ne lli da e c oc ci ne lla se pt em pu m et at a pr 0 3 0 2 5 g eo tru pi da e g eo tr up es sp , sa p 0 8 6 1 15 sc ar ab ae id ae ta m ar us g ib bo su s c op 0 0 0 2 2 tr op in ot a hi rt a ph y 0 38 0 0 38 c oc ci ne lli da e th ea v ig iti du op un ct at a ph y 0 0 0 1 1 c hr ys om el id ae pl ag io de ra v er si co lo ra ph y 0 1 0 0 1 te ne br io ni da e te ne br io sp . ph y 0 37 0 0 37 d ip te ra b ib io ni da e bi bi oa l b ip en ni s ph y 0 3 0 0 3 c hl or op id ae m us ca d om es tic a po l 0 4 0 0 4 c ul ic id ae c ul ex sp . po l 2 8 0 0 10 h em ip te ra ly ga ei da e lg ae us m el ita ri s ph y 0 0 1 0 1 h ym en op te ra a nd re ni de a an dr en a ha em or rh oa ph y 0 0 2 0 2 a pi da e ap is m el lif er a ph y 25 6 0 0 31 m yr m ic id ae m on ic a ru bi da ph y 0 2 0 0 2 le pi do pt er a h al ic tid ae h al ic tu s s ca bi os ae ph y 1 1 0 0 2 to rtr ic id ae c yd ia p om on el la ph y 1 0 0 0 1 o rth op te ra g ry lli da e g ry llu s b im ac ul at us ph y 0 0 1 0 1 to ta l 6 16 17 29 11 1 10 6 15 6 ta bl e 1. t ax on om ic in ve nt or y of s pe ci es re co rd ed in t az ou lt ap pl e or ch ar d biologica nyssana ● 16 (2) december 2025: brahim et al. ● entomological diversity associated with apple trees in the aurès region (northeastern algeria) ecological flexibility of coleoptera noted by guermah et al. (2022) helps explain their prevalence across these studies. medjkane & laguel (2015), in a study dedicated to the entomofauna of apple cultivation, reported that insects accounted for 81.47%, followed by arachnids at 16.19% and myriapods at 2.34%. mahdjane (2013) noted that insects formed 77.35%, arachnids 11.62%, myriapods 8.9%, and crustaceans 2.11% in his inventory of plum trees in the tadmait region. this consistent arthropod distribution across crops suggests stable ecosystem patterns, though benameur-saggou et al. (2021) note significant pesticide-induced variations in ouargla ladybird populations. medjkane & laguel (2015) also reported that, with regard to barber traps, hymenopterans accounted for 31% of the catch, followed by beetles at 23% and dipterans at 11%. the same authors, using colored traps, showed that hymenopterans accounted for 40% and dipterans for 30% of the recorded specimens. these trap-specific responses mirror the findings of masquin et al. (2020), who reported that yellow traps showed 71.4% selectivity for diptera in agroforestry systems. in a study of insects in broad bean crops in the tizi-ouzou region, mezani et al. (2016) found that the orders coleoptera and hymenoptera were the most abundant in barber traps, comprising 23.80% and 23.38% of the catch, respectively. in contrast, coleoptera was the dominant order in both colored traps (28.62%) and sweep nets (57.3%). maandi & sadoudi (2020) confirmed similar coleopteran trapping efficiencies in bouira orchards, suggesting method-dependent sampling biases. in an inventory on entomofauna associated with olive in the region of tlemcen, a frequency of 51.51% hemiptera, 21.21% coleoptera, and 18.18% hymenoptera was found in a treated station (zenaidi & hamour, 2015). on the other hand, this order is followed respectively by that of the hymenoptera (03 species, 17.65%) and of the coleoptera (07 species, 41.18%). this insect order is quantitatively ranked region (east of algeria). indeed, this order is represented by 52 species, one-third of the 157 identified species. comparative data from ouargla (benameur-saggou et al., 2021) and lakhdaria (gacem & mezerdi, 2022) show parallel patterns in the representation of hymenopterans and dipterans. it is worth noting that coleoptera is followed by hymenoptera and diptera, each represented by three species. lepidoptera, orthoptera, and hemiptera, which are ranked fourth, fifth, and sixth, respectively, are under-represented in our work, possibly due to microhabitat specificity (guermah et al., 2022) or sampling bias (maandi & sadoudi, 2020). the total of 156 individuals reflects both the orchard’s edge habitats, which support diverse entomofauna (benameur-saggou et al., 2021), and vegetation structure that favors arthropod diversity (masquin et al., 2020). the kruskal-wallis test showed that trap-specific species preferences (χ²=9.10, p<0.01) were significant. several apple pests have been reported, including tropinota hirta poda, which causes damage to floral structures (çakmak & şahin, 2018), and cydia pomonella l., an internationally important pest. insect predators such as coccinella septempunctata l. (3.2% of captures) are at lower densities than those reported in agroforestry ecosystems (masquin et al., 2020) and may be affected by pesticides (benameursaggou et al., 2021). this provides evidence for the need for integrated pest management strategies that incorporate habitat diversification (guermah et al., 2022) and reduced use of broad-spectrum pesticides (guermah et al., 2019). distribution of species among insect orders analysis of species diversity by order (fig. 1) shows that beetles lead with seven species, followed by dipterans and hymenopterans with three species each. lepidopterans are represented by two species, and hemipterans and orthopterans by one species each. our results confirm that the falling belongs to six different orders, and that beetles are the most abundant, with an astounding 41.18%; dipterans and hymenopterans each contributed 17.65% to the total; lepidopterans constitute 11.76%, while orthopterans and hemipterans were poorly represented in our site of study. these patterns align closely with recent findings by tayaa (2024) in khenchela apple orchards and bayan et al. (2023) in bouira fruit ecosystems, confirming the consistent dominance of coleoptera in algerian pomology systems. in algeria, our results corroborate those of frah et al. (2009), who conducted a survey of the entomofauna of apple trees in the aurès region, indicating that beetles constituted an important proportion of 38% of the overall inventory. the fig. 1. species importance by order biologica nyssana ● 16 (2) december 2025: brahim et al. ● entomological diversity associated with apple trees in the aurès region (northeastern algeria) second after coleoptera. this treatment-dependent shift parallels the documentation of pesticide impacts on trophic structures in algerian orchards by guermah et al. (2019). trophic status of recorded species it can be seen from the inventory of the species that most of them are phytophagous, accounting for 70.59% of the total inventoried species. then, the sequence of polyphagous species accounts for 11.76%, while the remaining trophic statuses each account for 5.88%. this trophic structure is consistent with the findings of the bouhmama apple orchards, which reported 68-72% phytophagy (tayaa, 2024), and it represents mediterranean agroecosystem structures (bayan et al., 2023). regarding trophic status, the results are quite close to those obtained by marniche (2001) in ichkeul (tunisia), who captured 95 species of phytophagous insects, representing half of the 195 species recorded for the site. similar hierarchies were documented in bouira orchards, where hymenoptera consistently represented 15-20% of catches across trap types (maandi & sadoudi, 2020). however, being few in number compared to other insect categories, predators play a very important ecological role as regulators of other insect populations. recent studies in ouargla (benameur-saggou et al., 2021) demonstrate that even at low abundances (5-8%), predatory coccinellids can suppress 4060% of aphid infestations in untreated orchards. other than that, saprophagous and coprophagous organisms participate in nutrient recycling, thereby contributing to the overall health of the ecosystem. guermah et al. (2022) quantified this contribution, showing these groups accelerate organic matter decomposition by 30-50% in algerian fruit orchards. among the ecological factors determining the dynamics and stabilization of possible pest populations, attention shall be given to community density, the presence of effective predators and parasites, the physiological condition of plants, and their spatial arrangement. maandi & sadoudi (2020) emphasize that orchards maintaining predator: prey ratios above 1:10 show 75% fewer pest outbreaks, while masquin et al. (2020) note that intercropping can enhance this effect by 25-40%. many insects appear to be important pollinators of apples. apis mellifera l. is the most important pollinator of apples in algeria and elsewhere worldwide. in addition to apis, other bee pollinators of apples include the genera andrena (grab et al., 2019) and halictus (kapkoti et al., 2016). these wild bees complement honeybee pollination, contributing 15-30% of flower visits in diversified orchards (guermah et al., 2019), though their populations remain vulnerable to pesticide use (gacem & mezerdi, 2022). monthly population diversity the diversity parameters, calculated month by month for insect populations on apple trees in the tazoult region, are presented in tab. 2 below. as reported by blondel (1979), a community is considered more diverse when the diversity index (h’) is higher. this aligns with contemporary studies of algerian orchards (maandi & sadoudi, 2020; tayaa, 2024), in which h’ >2.0 consistently indicated high biodiversity zones. the shannon index, calculated for may and march, indicates that these periods exhibit the greatest diversity and even distribution (n’zala et al., 1997). similar seasonal peaks were observed in bouhmama apple orchards, where march-april showed 25-30% higher diversity than winter months (bayan et al., 2023). the equity value remains above 0.5 over the three months under study, indicating that insect populations in the apple orchard are well balanced. values >0.5 confirm stable ecosystem functioning according to masquin et al. (2020), though optimal orchard management maintains e>0.7 (maandi & sadoudi, 2020). moreover, shannon index testing for barber pots and colored traps shows that these methods have the highest diversity. comparative research in ouargla (benameur-saggou et al., 2021) revealed 40% more species trapped by colored traps than by barber pots in spring seasons, as demonstrated by the current research. the variations in shannon index values are explained by n’zala et al. (1997), who reported that in favorable environmental conditions, species diversity is high, with each species composed of a few individuals. recent research by guermah et al. (2022) puts this at 15-20% species increase for every 10% increase in floral resource availability. conversely, under unfavorable conditions, only a limited number of species are found, whereas in favorable conditions, each species has a larger population. blondel (1979) notes that the abundance of available plant species plays a significant role in month n s h’ h’max e february 11 3 1.1 1.58 0.69 march 16 6 2.22 2.58 0.86 april 129 13 2.67 3.7 0.72 total 156 22 2.99 4.46 0.67 table 2. total richness (s), shannon index (h'), maximum diversity (h'max) and evenness (e) of the populations recorded on the study site biologica nyssana ● 16 (2) december 2025: brahim et al. ● entomological diversity associated with apple trees in the aurès region (northeastern algeria) determining the richness of the animal community. in tizi-ouzou orchards, guermah et al. (2019) demonstrated that every additional plant species increased arthropod richness by 2.3 species on average. consequently, the insect community is closely linked to the structural complexity, the abundance of vegetation, and the diversity of ecological niches in the environment. trophic network structure revealed by trapping the diversity parameters calculated for the study site and the various trap types employed are presented in tab. 3. the shannon diversity index (h’) values are as follows: for barber pots, the diversity is h’ = 1.57 bits (maximum diversity, h’max = 2 bits); for yellow traps, h’ = 0.79 (h’max = 2 bits); and for white traps, h’ = 2.47 (h’max = 3.46 bits). complementing these findings, bray-curtis dissimilarity analysis revealed strong methodological partitioning (tab. 4). it confirmed strong partitioning among trap types, with maximal divergence between yellow traps and barber pots (bc=1.00), indicating nearly exclusive species composition. this was reinforced by β-diversity decomposition, which showed dominant species turnover (βsør=0.75), with replacement processes (βsim=0.58) accounting for 78% of total dissimilarity, far exceeding nestedness effects (βnes=0.17). the ecological basis for these patterns emerged clearly: yellow traps selectively captured airborne taxa, such as apis mellifera l. (25 individuals) and culex sp. (2 individuals), while barber pots specialized on soildwelling geotrupes sp. (6 individuals). white traps showed intermediate selectivity, with moderate overlap with visual hunting methods (bc=0.73) for flower-visiting species. anosim validation (r=0.52, p=0.001) confirmed that approximately half of the community variation was attributable to trap-type selection. together, these results demonstrate that methodological differences drive substantial turnover in sampled communities, with each technique preferentially accessing particular ecological guilds within the orchard ecosystem. while tayaa (2024) reported similar trap preferences, our permanova analysis confirms the significant impact of sampling methods on community composition, with trap type explaining 62% of the observed variance (f=18.3; p<0.001), far exceeding the marginal effect of month, which accounts for only 16% of the variance (f=3.1; p=0.02). this hierarchy of factors supports the previously identified trophic specializations, where each technique targets distinct ecological guilds. the interaction network analysis reveals three fundamental ecological patterns in the orchard system. first, phytophagous insects dominate trophic interactions (70% of links), with tropinota hirta l. emerging as a keystone species due to its extensive plant associations. second, predators account for less than 5% of interactions, indicating constrained biological control potential. third, trap types show distinct guild specialization: barber pots selectively capture soil-dwelling decomposers (geotrupes sp.), yellow traps target airborne taxa, while white traps demonstrate intermediate versatility for flower-visiting species. these functional patterns correlate strongly with our diversity metrics. the predator deficit aligns with their low representation in yellow trap catches, and the apparent isolation of saprophagous species confirms barber pots’ specificity for soil-dwelling taxa (βsim=0.58). together, these findings demonstrate that different trap types sample discrete ecological modules within the orchard ecosystem, with each method accessing particular functional groups. these results concur with trends logged in similar algerian agroecosystems: white traps’ higher efficiency (h’=2.47) mirrors findings in ouargla, where they outperformed yellow traps in capturing coleoptera due to phototaxis (benameur-saggou et al., 2021). meanwhile, the moderate diversity of barber pots (h’=1.57) reflects their documented efficacy for ground-dwelling taxa, as observed in yellow trap white trap barber pot visual hunting yellow trap 0.00 0.83 1.00 0.89 white trap 0.83 0.00 0.78 0.73 barber pot 1.00 0.78 0.00 0.86 visual hunting 0.89 0.73 0.86 0 table 4. bray-curtis dissimilarity matrix between sampling methods methods n s h’ h’max e yellow traps 29 4 0.79 2 0.4 white traps 111 11 2.47 3.46 0.71 barber pots 10 4 1.57 2 0.79 visual hunting 6 4 1.92 2 0.96 total 156 23 2.99 4.52 0.66 table 3. number of individuals (n), total richness (s), diversity index (h’), maximum diversity (h’max), and evenness (e) of the populations recorded according to trap types at the study site biologica nyssana ● 16 (2) december 2025: brahim et al. ● entomological diversity associated with apple trees in the aurès region (northeastern algeria) khenchela apple orchards (tayaa, 2024), although they may underrepresent canopy-associated species. lower h’ values here (1.57–2.47) compared to polycultures (e.g., mezani et al., 2016: h’=4.95; yasri et al., 2006: h’=4.63) likely reflect simpler orchard composition (masquin et al., 2020). evenness for each trap type is closer to 1, as indicated by e=0.97 for barber pots, 0.71 for white traps, and 0.4 for yellow traps, consistent with tayaa (2024), who found that e increased with trap complexity (yellow < white < barber). djetti et al. (2015) estimated evenness at the following rates: e=0.77 in the sub-humid bioclimatic zone (el harrach) and e=0.88 in the semi-arid bioclimatic zone by using barber pots and colored traps. frah et al. (2015) reported a diversity index of 0.77 in a study of the arthropod fauna of an olive grove in sefiane (batna). the diversity value e=0.9 indicated by chikhi & doumandji (2007) at maâmria is consistent with this result. metaanalyses guermah et al. (2022) suggest that e>0.7 indicates stable agroecosystems, while e<0.5 (e.g., yellow traps here) may reflect sampling bias or habitat simplification. the preference of coleoptera for white traps (varandi et al., 2018) was confirmed, with our results showing a 3.1× difference in captures (111 vs. 29 individuals), consistent with maandi & sadoudi (2020) in bouira (3.2× difference). trophic structure and community dynamics the analysis of species interactions revealed distinct ecological patterns within the orchard ecosystem. phytophagous insects dominated the trophic network, accounting for 70% of all observed interactions, with the scarab beetle tropinota hirta l. emerging as a particularly influential species due to its numerous plant associations. in contrast, predatory taxa such as coccinella septempunctata l. represented less than 5% of interactions, suggesting limited natural pest control potential in the studied system. the saprophagous group, including geotrupes species captured exclusively in barber pots, formed an ecologically distinct module, reinforcing this trap type’s specificity for soil-dwelling decomposers. these functional patterns correlate strongly with the trap-based diversity metrics, where low predator representation aligns with their scarcity in yellow trap catches, while the clear separation of decomposers supports the β-diversity findings (βsim=0.58), demonstrating strong species turnover between trap types. conclusion this study examines the trophic structure of mediterranean agroecosystems, highlighting the dominance of phytophagous species, particularly tropinota hirta poda. the low number of predators indicates a potential imbalance in natural pest control. different trap types—barber traps for soil decomposers such as geotrupes sp., yellow traps for airborne species, and white traps for flower visitors— each play a unique role in sampling different ecological groups. the findings emphasize the need for integrated management strategies, including increasing predator populations, optimizing farming practices such as agroforestry, and ongoing multimethod monitoring. additionally, conserving pollinators like apis mellifera l. is vital for balancing productivity and sustainability in algerian orchards. references ali-shtayeh, m. s., ali, w. m. d., & jamous, r. m. 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(2015). inventaire qualitatif et quantitatif des invertébrés inféodés à la culture de pommier… (master’s dissertation). mouloud mammeri university. anticancer compounds from medicinal plants biologica nyssana 7 (1)  september 2016: 41-46 čubrić, t.  species distribution modeling techniques as a tool in… 41 original article received: 30 mart 2016 revised: 27 april 2016 accepted: 01 june 2016 species distribution modeling techniques as a tool in preliminary assessment of special nature reserve “goč-gvozdac” tijana čubrić* university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia * e-mail: tijanacubric@hotmail.com abstract: čubrić, t.: species distribution modeling techniques as a tool in preliminary assessment of special nature reserve “goč-gvozdac”. biologica nyssana, 7 (1), september 2016: 41-46. effective conservation actions such as defining new nature reserve require accurate estimates of the spatial distributions of the target species. species distribution models provide habitat suitability maps for studied species. in this paper we used maxent software to estimate the distribution and extent of potential suitable habitat of five amphibian and reptilian species (mesotriton alpestris, bombina variegata, testudo hermanni, lacerta viridis and vipera ammodytes) in the special nature reserve “goč-gvozdac” (central serbia) in order to assess how much of the potential habitats is included in this reserve. comparing produced suitable habitat maps of the species with a map of the special nature reserve “goč – gvozdac” we concluded that the reserve boundaries do not coincide with the proposed distribution of suitable habitats for m. alpestris, t. hermanni, l. viridis and v. ammodytes, and therefore this reserve does not contribute much to the protection of local populations of these species. key words: species distribution modeling, maxent, nature reserve, reptiles, amphibians apstrakt: čubrić, t.: modelovanje prostornog rasprostranjenja vrsta kao tehnika u preliminarnom ocenjivanju specijalnog rezervata prirode „goč-gvozdac“. biologica nyssana, 7 (1), septembar 2016: 41-46. efektivne konzervacione mere kao što je definisanje novog zaštićenog područja zahtevaju precizne procene prostornog rasprostranjenja vrsta koja se štite. uz pomoć modelovanja prostornog rasprostranjenja vrsta, dobijaju se mape pogodnog staništa za istraživane vrste. u ovom radu korišćen je programski paket maxent sa ciljem procene rasprostranjenja pogodnih staništa za pet vrsta vodozemaca i gmizavaca (mesotriton alpestris, bombina variegata, testudo hermanni, lacerta viridis i vipera ammodytes) u specijalnom rezervatu prirode „goč-gvozdac“ (centralna srbija), kako bi se procenilo koliko se potencijalnih pogodnih staništa za ove vrste nalazi unutar teritorije ovog rezervata. poređenjem dobijenih mapa pogodnih staništa sa mapom specijalnog rezervata prirode „goč-gvozdac“ zaključili smo da se granice ovog rezervata ne poklapaju sa predloženim rasprostranjenjem pogodnih staništa za vrste m. alpestris, t. hermanni, l. viridis i v. ammodytes, te stoga ovaj rezervat ne doprinosi zaštiti lokalnih populacija ovih vrsta. key words: modelovanje prostornog rasprostranjenja vrsta, maxent, rezervati prirode, gmizavci, vodozemci 7 (1) • september 2016: 41-46 doi: 10.5281/zenodo.159102 biologica nyssana 7 (1)  september 2016: 41-46 čubrić, t.  species distribution modeling techniques as a tool in… 42 introduction the most effective way to preserve species diversity is conservation of viable populations in their natural habitats through establishment of network of protected areas (r o d r i g u e z -r e y et al., 2013). knowing where species are likely to occur is a basic part of natural resource management (r u s t o n et al., 2004). effective conservation actions such as defining new nature reserve require accurate estimates of the spatial distribution of the target species (h e r n a n d e z et al., 2006). with this information in hand conservationists can also predict species’ response in form of change in it’s local distribution due to landscape alteration and environmental change (h e r n a n d e z et al., 2006). species distribution models (sdms) are empirical models that relate field observations to environmental predictor variables based on statistically or theoretically derived response surfaces (g u i s a n & z i m m e r m a n n , 2000). species distribution modeling can provide a measure of a species occupancy potential in areas which are not covered by biological surveys and therefore these techniques are becoming an indispensable tool in conservation planning (g u i s a n & z i m m e r m a n n , 2000; c o r s i , 2000; l o i s e l l e et al., 2003), especially when funding and time are limited. also, predicted species distribution data from sdms are commonly used for conservation planning because the alternatives (e.g. survey data) are often incomplete or spatially biased (a n d e l m a n & w i l l i n g , 2002). one of the sdm software is maxent (p h i l l i p s et al., 2006). maxent is a generalpurpose method for characterizing probability distributions from incomplete information (p h i l l i p s et al., 2006). maxent combines distribution data with environmental factors and assesses the probability of presence of one species in a given cell on the basis of environmental features in that cell. maxent calculates the range of species distribution in order to find the species distribution of maximum entropy – closest to the uniform (p h i l l i p s et al., 2006). distribution based on maximum entropy means that the species are evenly distributed through environmental variables. mean task in this modeling program is to predict suitable habitat in relation to environmental variables. it requires only presence data. in this paper maxent was used in predicting suitable habitats for two amphibian and three reptilian species: mesotriton alpestris (amphibia: urodela: salamandridae), bombina variegata (amphibia: anura: bombinatoridae), testudo hermanni (reptilia: chelonia: testudines: testudinidae), lacerta viridis (reptilia: squamata: sauria: lacertidae) and vipera ammodytes (reptilia: squamata: serpentes: viperidae). nine amphibian and twelve reptilian species inhabits special nature reserve "goč gvozdac". the five chosen species are the most easy to spot while performing transects. in addition, all five species are protected under national law. also, they are listed under the iucn red list (www.iucnredlist.org) and under the annexes of bern convention (www.coe.int). m. alpestris, b. variegata, l. viridis and v. ammodytes are listed in the last concern iucn red list category and t. hermanni is listed in the near threatened category (www.iucnredlist.org). also, except m. alpestris, all the other selected species are considered of community interest in need of strict protection (annex iv) in habitat directive (a n o n y m o u s , 1992); additionally, b. variegata and t. hermanni are considered also a species which conservation requires the designation of special areas of conservation (annex ii in habitat directive). the objectives of this study are: 1) to use habitat suitability models to determine potential current distribution of chosen species throughout the study area 2) to estimate the distribution and extent of species potential habitat in the special nature reserve “goč-gvozdac” and 3) to assess how much of the potential habitats is included in this reserve. the basic question was whether the border of the reserve is set up in a way to contribute to the protection of the most important local habitats of these species. material and methods study area as the aim of this study was to evaluate special nature reserve “goč-gvozdac”, study area was wider than the reserve itself (fig. 1) and included the nature reserve (positioned in the middle of study area), the valleys of the ibar and west morava rivers and slopes of kopaonik and golija mountains. special nature reserve “goč – gvozdac” is located in central serbia, between 43° 31' and 43° 34' north latitude and 20° 37' and 20° 47' east longitude. it represents a forested mountainous area with its highest peak of 1484 m. and covers 3957 ha in total. the field research lasted from april to september 2014. the survey was carried out through 49 transects of the average length of 5 km set in randomly selected locations. the geographic coordinates of the localities where the species were found were collected and they included 10 records of m. alpestris, 22 records of b. variegata, 11 records of t. hermanni, 25 records of l. viridis and 10 records of v. ammodytes. biologica nyssana 7 (1)  september 2016: 41-46 čubrić, t.  species distribution modeling techniques as a tool in… 43 fig. 1. study area (red polygon with dimensions 50x50km). in the middle of the study area is located special nature reserve “goč-gvozdac” table 1. climatic variables used in models bio1 annual mean temperature bio2 mean diurnal range (mean of monthly (max temp min temp)) bio3 isothermality (bio2/bio7) (* 100) bio4 temperature seasonality (standard deviation *100) bio5 max temperature of warmest month bio6 min temperature of coldest month bio7 temperature annual range (bio5bio6) bio8 mean temperature of wettest quarter bio9 mean temperature of driest quarter bio10 mean temperature of warmest quarter bio11 mean temperature of coldest quarter bio12 annual precipitation bio13 precipitation of wettest month bio14 precipitation of driest month bio15 precipitation seasonality (coefficient of variation) bio16 precipitation of wettest quarter bio17 precipitation of driest quarter bio18 precipitation of warmest quarter bio19 precipitation of coldest quarter environmental variables climate variables were collected from worldclim databank while corine map was used for land cover data. worldclim base is a collection of maps having medium, minimum and maximum monthly temperature and precipitation for the entire land area of the planet. the maps are of high resolution (30 arc sec) which corresponds to a resolution of approximately 1 km2. the worldclim databank consists of 19 climatic variables (tab. 1). corine digital map is created for 29 european countries with a scale 1: 100 000 or 25 ha minimum size of the unit. resolution of all variables was set to a resolution of corine land cover maps (about 100x100 m). the variables were prepared using esri arcmap 9.3. species distribution modeling following basic parameters of the model maxent were used: calibration (training) data were generated by random selection of 75% occurrence records and 25% for testing. the chosen maxent default settings were: maximum number of iterations = 500, convergence threshold = 10-5, maximum number of background points = 10000. we used a regularization multiplier = 2. in order to validate the obtained models, the predicted values of habitat suitability assigned to presence and pseudo-absence of the data in the test subset were compared by producing the ‘receiver operating characteristic’ (roc) plots (fielding and bell, 1997) and deriving the relative ‘area under curve’ (auc) value (faraggi and reiser, 2002) through a jack-knife procedure. the main positive feature of this method consists of being a single threshold-independent measure for model performance. maps of suitable habitat (raster maps) were made in maxent. the maps of suitable habitats were then overlapped with the map of the special nature reserve using diva-gis 7.5. maps were compared in order to determine which parts of the reserve are suitable. we analyzed the binary maps with suitable and unsuitable habitats for the species. this allowed the calculation of the suitable habitat area. then, we analyzed the size of suitable habitat area of each species in the reserve and the relation between the area of suitable habitat in the reserve and the total area of suitable habitat in the study area. results model performance the models got relatively good performance. training auc was 0.924 for m. alpestris, 0.887 for b. variegata, 0.870 for t. hermanni, 0.863 for l. viridis and 0.754 for v. ammodytes, comparing binary maps of suitable habitats with the map of special nature reserve “goč-gvozdac” comparing the distribution of suitable habitats with a map of the reserve revealed that the suitable habitats for m. alpestris (fig. 2), t. hermanni (fig. 4), l. viridis (fig. 5) and v. ammodytes (fig. 6) are mostly located outside the borders of the reserve and that biologica nyssana 7 (1)  september 2016: 41-46 čubrić, t.  species distribution modeling techniques as a tool in… 44 very small part of the suitable habitat occurs on the northern border of the reserve. the large part of the reserve (75%) has suitable habitats for b. variegata (fig. 3), and the surface of suitable habitat in the study area is greater than the surface of the reserve. consequently, the largest portion of the surface of a suitable habitat in the reserve in relation to the total area of suitable habitat had b. variegata (tab. 2), and the lowest m. alpestris. discussion one of the most important aspects of amphibian and reptile biology that should be clarified for the efficient planning of conservation measures is represented by the patterns of species distribution, as well as by the environmental factors that influence such patterns (b o m b i , 2010). comparing the distribution of suitable habitat maps of the studied species with a map of the special nature reserve "goč gvozdac" we can conclude that the reserve boundaries do not coincide with the distribution of suitable habitats for m. alpestris, t. hermanni, l. viridis and v. ammodytes. therefore we can conclude that this reserve does not contribute much to the protection of local populations of these species. on the contrary, the area of suitable habitat for b. variegata occupies 75% of the reserve and the proposed reserve boundaries contribute to its protection. for m. alpestris it would be good to expand the northern boundaries of the reserve in a way to protect also potentially important habitats north of the reserve. given the fact that the area of suitable habitat for other analyzed species exceeds the area of the reserve, one of the potential measures to protect their local populations would be the establishment of additional new protected areas. areas for protection are often selected to cover the maximum number of species (c h u r c h et al. 1996; k i e s t e r et al., 1996). various authors (p r e s s e y & n i c h o l l s , 1989) recommend maximizing biodiversity in a protected area in order to be able to protect them with limited financial resources. therefore, it would be more economical to establish one large protected area by expanding the boundaries of the existing one, or to design a connected network of small protected areas. fig. 2. comparison of binary map of suitable habitat with a map of the reserve. the dark polygon represents the territory of the special nature reserve “goč-gvozdac” and hatched part represents the suitable habitat for species mesotriton alpestris. fig. 3. comparison of binary map of suitable habitat with a map of the reserve. the green polygon represents the territory of the special nature reserve “goč-gvozdac” and hatched part represents the suitable habitat for species bombina variegata. fig. 4. comparison of binary map of suitable habitat with a map of the reserve. the green polygon represents the territory of the special nature reserve “goč-gvozdac” and hatched part represents the suitable habitat for species testudo hermanni. biologica nyssana 7 (1)  september 2016: 41-46 čubrić, t.  species distribution modeling techniques as a tool in… 45 protected parts of nature that have been identified on the basis of their range and predictions of species distribution are likely to be more comprehensive and more representative than those that are identified solely on the basis of available presence data of the species. this is because the species presence is limited to the area where field research was conducted while species distribution models predict species distribution in and out of the researched area (r o n d i n i n i at al., 2006). also, simple probability-based approaches may be considered an alternative to more complex population viability analysis when conservation decisions involve large number of species and there is little time and few resources available (a r a ú j o et al. 2002). in this study it is achieved to evaluate the suitability of the total special reserve for selected species in regard to the wider area, although field work has not been conducted in all spatial segments. obviously, this study could be improved. first, vegetation map with better resolution would be more appropriate because finer resolution usually provides better predictions for fixed or very locally mobile organisms (g u i s a n & t h u i l l e r , 2005) such as species in this study. also, researched area was relatively small for resolution of variables. further, identifying small habitat features (such as cave openings, vegetation patches) is also important as v. ammodytes and l. viridis use their environments at a relatively fine spatial scale. new types of remotely sensed data such as laser altimetry or light detection and ranging (lidar) (l e f s k y et al., 2002) should prove useful for generating detailed habitat maps. various authors (r o n d i n i n i at al., 2005) concluded that there are no available maps showing presence of small, temporary water bodies that are important for certain species (e.g. m. alpestris and b. variegata) and that represents an obstacle in sdm. it may also happen that the resolution of variables does not match the scale at which species use environment. also, the relatively small number of samples should be taken into account in interpreting the results of this study, although maxent represents the best of the available programs for modeling with quantity of samples up to 5,10, 15 and 20 (h e r n a n d e z et al., 2006). also, fig. 5. comparison of binary map of suitable habitat with a map of the reserve. the green polygon represents the territory of the special nature reserve “goč-gvozdac” and hatched part represents the suitable habitat for species lacerta viridis. fig. 6. comparison of binary map of suitable habitat with a map of the reserve. the green polygon represents the territory of the special nature reserve “goč-gvozdac” and hatched part represents the suitable habitat for species vipera ammodytes. table 2. area of suitable habitats for analyzed species (snr-special nature reserve) species size of suitable habitat within the study area size of suitable habitat within the snr % of the size of suitable habitat within the snr in relation to the size of suitable habitat within the study area % of the size of suitable habitat within the snr in relation to the size of the reserve (3957 ha) mesotriton alpestris 2916,8171 ha 1,8224 ha 0,062 0,046 bombina variegata 70980,2766 ha 2998,6221 ha 4,22 75,78 testudo hermanni 57018,9638 ha 130,9016 ha 0,23 3,30 lacerta viridis 57559,7044 ha 224,1334 ha 0,39 5,66 vipera ammodytes 120566,0616 ha 1086,9871 ha 0,9 27,46 biologica nyssana 7 (1)  september 2016: 41-46 čubrić, t.  species distribution modeling techniques as a tool in… 46 the model is more accurate for species with a small distribution range and narrow ecological valence (h e r n a n d e z et al., 2006). conclusion because contemporary research in the field of conservation planning has focused on the development of theories and tools to design reserve networks that protect biodiversity in an efficient and representative manner (a r a u j o & w i l l i a m s , 2000; a r a u j o et al., 2002; c a b e z a et al., 2004), sdms (like models in this study) can be used as a foundation for the development of finer scale models, usable in a) definition of suitable habitat, b) identification of boundaries for new conservation areas and c) assessment of existing area to secure the protection of amphibian and reptilian species. acknowledgements. i am grateful to dimitrije radišić for support and help for realizing my ideas and to nikola vuletić for a company in the field. references andelman, s.j., willing, m.r. 2002: alternative configurations of conservation reserves for paraguayan bats: considerations of spatial scale. conservation biology, 16: 1352-1363. anonymous, 1992: council directive on the conservation of natural habitats and of wild fauna and flora 92/43/eec. araujo, m.b., williams, p.h., fuller, r.j. 2002: dynamics of extinction and the selection of nature reserves. proceedings of the royal society of london series b-biological sciences 269: 1971– 1980. araújo, m. b., williams, p. h. 2000: selecting areas for species persistence using occurrence data. biological conservation, 96 (3): 331-345. bombi p. 2010: toward a new instrument for identifying the italian hotspots of biodiversity: a case study of the amphibians and reptiles of sicily. italian journal of zoology, 77: 453-459. church, r. l., stoms, d. m., davis, f. w. 1996: reserve selection as a maximal covering location problem. biological conservation, 76 (2): 105-112. cabeza, m., araújo, m. b., wilson, r. j., thomas, c. d., cowley, m. j., moilanen, a. 2004: combining probabilities of occurrence with spatial reserve design. journal of applied ecology, 41 (2): 252-262. corsi, f. 2000: modelling species distribution with gis. in: boitani, l. and fuller, t. k. (eds), research techniques in animal ecology; controversies and consequences. columbia univ. press, pp. 389-434. faraggi d, reiser b. 2002: estimation of the area under the roc curve. statistics in medicine, 21: 3093–3106. fielding ah, bell jf. 1997: a review of methods for the assessment of prediction errors in conservation presence/absence models. environmental conservation, 24: 38–49. guisan, a., thuiller, w. 2005: predicting species distribution: offering more than simple habitat models. ecology letters, 8 (9): 993-1009. guisan, a., zimmermann, n. e. 2000: predictive habitat distribution models in ecology. ecological modelling, 135: 147-186. hernandez, p. a., graham, c. h., master, l. l., albert, d. l. 2006: the effect of sample size and species characteristics on performance of different species distribution modeling methods. ecography, 29 (5): 773-785. kiester, a. r., scott, j. m., csuti, b., noss, r. f., butterfield, b., sahr, k.,white, d. 1996: conservation prioritization using gap data. conservation biology, 10 (5): 1332-1342. lefsky, m.a., cohen w.b., parker g.g., harding d.j. 2002: lidar remote sensing for ecosystem studies. bioscience, 52: 19-30. loiselle, b. a., howell, c. a., graham, c. h., goerck, j. m., brooks, t., smith, k. g., williams, p. h. 2003: avoiding pitfalls of using species distribution models in conservation planning. conservation biology, 17 (6): 1591-1600. phillips, s.j., anderson, r.p., schapire, r.e. 2006: maximum entropy modelling of species geographic distributions. ecological modelling, 190: 231–259. pressey, r. l., nicholls, a. o. 1989: efficiency in conservation evaluation: scoring versus iterative approaches. biological conservation, 50 (1): 199-218. rodríguez-rey, m., jiménez-valverde, a., acevedo, p. 2013: species distribution models predict range expansion better than chance but not better than a simple dispersal model. ecological modelling, 256: 1-5. ruston, s.p., ormerod s.j., kerby g. 2004: new paradigms for modeling species distributions? journal of applied ecology, 41: 193 –200. rondinini, c., stuart, s., boitani, l. 2005: habitat suitability models and the shortfall in conservation planning for african vertebrates. conservation biology, 19 (5): 1488-1497. rondinini, c., wilson, k. a., boitani, l., grantham, h., possingham, h. p. 2006: tradeoffs of different types of species occurrence data for use in systematic conservation planning. ecology letters, 9 (10): 1136-1145 anticancer compounds from medicinal plants biologica nyssana 4 (1-2)  december 2013: 81-85 stamenković, j. et al.  optimization of hplc method for the… 81 original article optimization of hplc method for the isolation of hypericum perforatum l. methanol extract jelena stamenković, ivana radojković, aleksandra đorđević, olga jovanović, goran petrović, gordana stojanović department of chemistry, faculty of science and mathematics, university of niš, serbia * e-mail: jelena.stamenkovic@pmf.edu.rs abstract: stamenković, j., radojković, i., đorđević, a., jovanović, o., petrović, g., stojanović, g.: optimization of hplc method for the isolation of hypericum perforatum l. methanol extract. biologica nyssana, 4 (12), december 2013: 81-85. st. john's wort (hypericum perforatum l.) is one of the most studied plant species in the family hypericaceae. the aim of this study was the identification of the constituents of methanol extract of h. perforatum and optimization of conditions for their isolation. the main components of the methanol extract were isolated on preparative zorbax eclipse xdb c18 column with solvent system consisting of methanol and 1x10 -2 m ammonium acetate in water. constituents of the extract were identified by comparing their retention times with the retention times of the standards, with the literature data and the uv spectra. by varying the conditions of chromatography, the optimal conditions for isolation of the methanol extract constituents were determined: mobile phase consisting of methanol and 1x10 -2 m ammonium acetate in water in ratio 1 : 1, sample concentration 100 mg/ml, sample volume 30 µl, flow 2 ml/min. under these conditions 7 components of the methanol extract were isolated. key words: hypericum perforatum l., methanol extract, isolation, preparative hplc . introduction st. john’s wort (hypericum perforatum) is a herbaceous perennial plant of the hypericaceae family, which is distributed in europe, asia, and northern africa, and naturalized in the us. the plant grows approximately one metre high with opposite and paired branches. the leaves are opposite and sessile, up to 2 cm long, oblong and contain numerous translucent glandular dots, which are visible against light. the yellow flowers contain 5 petals with many stamens protruding. these flowers contain a group of reddish fluorescent dianthrone pigments with biological activity (v a t t i k u t i & c i d d i , 2005). hypericum species are medicinal plants known as healing herbs due to their various medicinal properties for the last two hundred years. oily hypericum preparations may be applied externally to treat minor burns, wounds, inflammation of the skin, and nerve pain (b l u m e n t h a l et al., 1998). internally, the herbal preparation is indicated for the treatment of anxiety and depression. st john’s wort (hypericum perforatum l.) has a chemical composition well studied: anthraquinone derivatives naphthodianthrones – hypericin, pseudohypericin and isohypericin, protohypericin and protopseudohypericin (biosynthetic precursors of hypericin and pseudohypericin, respectively); flavonoids – flavonols (kaempferol, quercetin), flavones (luteolin) and glycosides (hyperoside, isoquercitrin, quercitrin, and rutin), biflavonoids including biapigenin (a flavone) and amentoflavone (a biapigenin derivative) and catechins (flavonoids 11 th sfses • 13-16 june 2013, vlasina lake 4 (1-2) • december 2013: 81-85 biologica nyssana 4 (1-2)  december 2013: 81-85 stamenković, j. et al.  optimization of hplc method for the… 82 often associated with condensed tannins); prenylated phloroglucinols – hyperforin and adhyperforin; tannins; procyanidins (condensed type); other phenols – caffeic, chlorogenic, pcoumaric, ferulic, p-hydroxybenzoic and vanillic acids; volatile oils, carotenoids, choline, β-sitosterol etc. (c r e t u et al., 2011). naphthodianthrones, namely hypericin and pseudohypericin, are found in the flowering portions of the plant. until recently, these two compounds were considered responsible for the purported antidepressant effect of hypericum. numerous flavonoid compounds, including hyperoside, quercitrin, isoquercitrin, rutin, quercetin, campferol, luteolin, and myricetin, as well as biflavonoids i3,ii8-biapigenin and i3′,ii8-biapigenin (amentoflavone) are found in the aboveground portions of the plant, including the leaves, stalk, flowers, and buds. phloroglucinol compounds, including hyperforin and adhyperforin, are present in the flowers and buds (g r e e s o n et al., 2001). several methods for the analysis of h. perforatum by tlc, photometry, hplc, hplc-ms and hplc-nmr have been reported, but most of these focus on the determination of naphthodianthrones and/or hyperforin only (gray et al., 2000; liu et al., 2000; mulinacci et al., 1999). the aim of this study was to develop the new simple and efficient method for isolation and identification of the hypericum perforatum l. methanol extract main constituents. for this purpose, the following methods were used: maceration of dried plant material with methanol to obtain the extract; high-pressure chromatography (hplc) method for the isolation, identification and determination of purity of isolated compounds. materials and methods plant material was collected in the vicinity of leskovac in july 2011. voucher specimen is deposited at the herbarium of the department of biology and ecology, faculty of sciences and mathematics, university of niš. naphthodianthrones and hyperforin are known to be thermolabile and especially photosensitive; thus, the extraction process was performed at room temperature and protected from the light (w i r z et al., 2001; o r t h et al., 1999). dry aerial part of the plant was chopped and powdered. 60 g of dry material was transferred in a dark bottle and submeged in 600 ml (1:10, v/v) methanol (sigma aldrich, p.a.), and left in the dark for 48 hours. the methanol extract was filtered through filter paper (white bar) into the premeasured flask. methanol was evaporated under reduced pressure in a rotary evaporator and 12.10 g of dry extract was obtained. re-extraction of the previously treated material was performed with another 500 ml of methanol. after 5 days, the methanol extract was again filtered through filter paper (white bar) into the same flask. the extract was evaporated and 16.28 g of dry extract was obtained in whole after repeated extraction. the dry extract was extracted with 100 ml of hexane (sigma-aldrich, p.a.) in the ultrasonic bath for 30 min. the crude hexane extract was evaporated to dryness at reduced pressure by rotoevaporation and 0.28 g of dry material was obtained. the respective yields were calculated as percentage using the formula: (crude extract weight/plant material weight) x 100 and were: 20.16%, 27.13% and 1.72% respectively. for hplc analysis a weighed amount of extract was dissolved in hplc grade methanol. all samples were filtered through a 0.45 μm filter before undertaking hplc analysis. each sample solution was injected in triplicate and standard deviations were below 2.2 % for all experiments, indicating that the method is precise and reproducible. hplc analysis was performed on hplc chromatography with dad detector (hplc system 1200 series, agilent technologies, usa). separation and purification of the extract components was performed on zorbax eclipse xdb c18 column with dimensions (5 μm, 150 mm × 4.6 mm, analytical and 5 μm, 250 mm x 9.4 mm, semipreparative). solvent system consisting of methanol and 1x10 -2 m ammonium acetate in water was used as the mobile phase in isocratic and gradient mode. the ratio of the solvents was varied in order to determine the optimum conditions for separation. all experiments were carried out at room temperature (25±2°c). chromatograms were recorded at different wavelengths. the constituents of the extract were identified by comparing their retention times with the retention times of standards, based on literature data and comparing the uv spectra. fractions were isolated and purified using the fraction collector. results and discussion there are numerous literature data on the hplc analysis of different extracts h. perforatum l. (b r o l i s et al., 1998; r o e m p p et al., 2004). the fact common to all of the researched methods is that thay were developed primarily for analytical biologica nyssana 4 (1-2)  december 2013: 81-85 stamenković, j. et al.  optimization of hplc method for the… 83 purposes in order to achieve a better separation of the constituents of the extract (l i & f i t z l o f f , 2001). described methods are based on the usage of the system with different solvents in gradient and isocratic mode. therefore they are not suitable to be applied in preparative purposes primarily due to long duration of analysis. in order to determine the optimal conditions for isolating components, preliminary investigations were carried out on the analytical column, varying the amount of injected sample, eluent polarity and flow rate of mobile phase. work on the analytical column was carried out in isocratic and gradient mode and chromatograms were recorded at different wavelengths. the methanol extract first was eluated on the analytical column under isocratic conditions. the mobile phase was run at flow rate of 0.5 ml/min and consisted of methanol : water (1 % hcooh) = 80 : 20.5 µl of sample (concentration 5 mg/ml) was injected in all experiments performed on analytical column. since the separation in isocratic mode was not obtained in satisfactory manner, gradient elution was performed by using the following solvent gradient: 0-5 min – 50 % meoh; 5-10 min – 70 % meoh; 10-30 min – 90 % meoh. the flow-rate was kept at 0.5 ml/min. as gradient conditions did not provide good results, the possibility of using isocratic method with the changed polarity of the mobile phase was tested. a mixture of methanol : water (1x10 -2 m ch3coonh4) = 50 : 50 was used as mobile phase. the resulting chromatogram is shown in fig. 1. fig. 1. hplc chromatogram obtained in isocratic conditions, methanol : water (1x10 -2 m ch3coonh4) = 50 : 50. fig. 2. hplc chromatogram with the sample volume of 20 µl, at a flow rate of 2 ml/min. this system was the basis for the transition from the analytical to semipreparative column, by varying the flow rate and injected sample volume, in order to obtain optimal results. all samples that were injected to semipreparative column were significantly more concentrated (100 mg/ml), aimed to increase method efficiency. fig. 2 shows the chromatogram of hplc analysis that was performed on semipreparative column with the optimal composition of mobile biologica nyssana 4 (1-2)  december 2013: 81-85 stamenković, j. et al.  optimization of hplc method for the… 84 phase (methanol and 1x10 -2 m ammonium acetate in water, ratio (1:1). this system was chosen because of good separation and short runtime. injection volume of the sample was 20 µl, with a flow rate of 2 ml/min. the peaks in the hplc chromatogram were identified based on literature data (hansen et al.,1999), by comparing the retention time and uv spectra (hillwing et al., 2008) with reference standards. fig. 3. hplc chromatogram with the sample volume of 30 µl, at a flow rate of 2 ml/min. table 1. retention time of identified and isolated compounds, mass of isolated compounds and their purity. no. compound rt (min) isolated mass (mg) purity % 1. chlorogenic acid 4.033 / / 2. gallic acid 4.213 / / 3. transp-coumaric acid 4.535 / / 4. unidentified compound 1 5.600 1.4 87.65 5. rutin 7.425 / / 6. hyperoside 7.425 / / 7. isoquercitrin 7.631 16.8 79.84 8. quercitrin 8.467 / / 9. quercetin 9.444 5.5 92.71 10. pseudohypericin 10.634 0.8 96.66 11. hypericin 12.860 1.0 97.12 12. unidentified compound 2 16.464 0.9 97.43 13. hyperforin 20.974 1.4 98.26 table 1 shows the retention times of identified and isolated components, mass of isolated components and their purity, based on the integral of the peak areas. the purity was determined by the integration of hplc spectra of the isolated compounds and it was directly proportional to the achieved resolution. in order to increase method efficiency, 40 μl of sample was injected, but this led to the reduced resolution. as a compromise solution, 30 μl of sample volume was injected at the same flow rate of 2 ml/min (fig. 3). based on obtained chromatograms, it was concluded that the best results for the separation and isolation of constituents of h. perforatum l. methanol extract on the semipreparative column, were obteined by injecting the volume of 30 µl of the sample (concentration 100 mg/ml) at a flow rate of 2 ml/min. biologica nyssana 4 (1-2)  december 2013: 81-85 stamenković, j. et al.  optimization of hplc method for the… 85 conclusion the results showed that the optimal conditions for isolation of the constituents of hypericum perforatum l. methanol extract on the semipreparative column were: mobile phase methanol : water (1x10 -2 m ch3coonh4) = 50 : 50, sample concentration 100 mg/ml, injection sample volume 30 µl and the flow rate of 2 ml/min. seven compounds were isolated from the extract with purity : unidentified compound (peak 4) 87.65 %, isoquercitrin (peak 7) 79.84 %, quercetin (peak 9) 92.71 %, pseudohypericin (peak 10) 96.66 %, hypericin (peak 11), 97.12 %, unidentified compound (peak 12) 97.43 %, hyperforin (peak 13) 98.26 %. according to obtained results a new simple isocratic hplc method was developed, allowing the baseline separation of thirteen main hypericum perforatum standard compounds in less than 25 min, which means an improved separation 25-50 min less than other published systems. acknowledgements. the results obtained in this study are part of the research project 172047, funded by ministry of education, science and technological development of serbia. references blumenthal, m., busse, w.r., goldberg, a., grüenwald, j., hall, t., riggins, c.w., rister, r.s.(eds), 1998: the complete german commission e monographs: therapeutic guide to herbal medicines. american botanical council, boston. brolis, m., gabetta, b., fuzzati, n., pace, r., panzeri, f., peterlongo, f. 1998: identification by 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toxicological, and clinical literature. psychopharmacology, 153 (4): 402-414. hansen, s.h.,. jensen, a., cornett, c., bjørnsdottir, i., taylor, s., wright, b., wilson, i.d. 1999: high-performance liquid chromatography online coupled to high-field nmr and mass spectrometry for structure elucidation of constituents of hypericum perforatum l. analytical chemistry, 71 (22): 5235–5241. hillwig, m.l., hammer, k.d.p., birt, d.f., wurtele, e.s. 2008: characterizing the metabolic fingerprint and anti-inflammatory activity of hypericum gentianoides. journal of agricultural and food chemistry, 56 (12): 4359–4366. li, w., fitzloff, j.f. 2001: high performance liquid chromatographic analysis of st. john’s wort with photodiode array detection. journal of chromatography, 765 (1): 99–105. liu, f.f., ang, c.y., heinze, t.m., rankin, j.d., beger, r.d., freeman, j.p., lay, j.o. 2000: evaluation of major active components in st. john’s wort dietary supplements by highperformance liquid chromatography with photodiode array detection and electrospray mass spectrometric confirmation. journal of chromatography a, 888 (1–2): 85–92. mulinacci, n., bardazzi, c., romani, a., pinelli, p., vincieri, f.f., costantini, a. 1999: hplc-dad and tlc-densitometry for quantification of hypericin in hypericum perforatum l. extracts. chromatographia, 49 (3-4): 197-201. orth, h.c.j., rentel, c., schmidt, p.c. 1999: isolation, purity analysis and stability of hyperforin as a standard material from hypericum perforatum l. journal of pharmacy and phamacology, 51: 193-200. römpp h., seger c., kaiser c.s., haslinger e., schmidt p.c. 2004: enrichment of hyperforin from st. john’s wort (hypericum perforatum) by pilot-scale supercritical carbon dioxide extraction. european journal of pharmaceutical sciences, 21: 443-451. vattikuti u.m.r., ciddi, v. 2005: an overview on hypericum perforatum linn. natural product radiance, 4 (5): 368-381. http://www.ncbi.nlm.nih.gov/pubmed?term=gray%20de%5bauthor%5d&cauthor=true&cauthor_uid=10995119 http://www.ncbi.nlm.nih.gov/pubmed?term=rottinghaus%20ge%5bauthor%5d&cauthor=true&cauthor_uid=10995119 http://www.ncbi.nlm.nih.gov/pubmed?term=garrett%20he%5bauthor%5d&cauthor=true&cauthor_uid=10995119 http://www.ncbi.nlm.nih.gov/pubmed?term=pallardy%20sg%5bauthor%5d&cauthor=true&cauthor_uid=10995119 vitorovic et al. 2021, biologica nyssana 12(2) 12 (2) december 2021: 113-122 doi: 10.5281/zenodo.5759855 antioxidant potential of commercial hemp seed oils and cbd oil original article jelena vitorović faculty of sciences and mathematics, university of niš, višegradska 33, 18000 niš, serbia jelena.rajkovic@pmf.edu.rs (corresponding author) nataša joković faculty of sciences and mathematics, university of niš, višegradska 33, 18000 niš, serbia andrea žabar popović faculty of sciences and mathematics, university of niš, višegradska 33, 18000 niš, serbia received: october 12, 2021 revised: november 24, 2021 accepted: november 25, 2021 abstract: the present study investigates the antioxidant capacity of commercial hemp seed oils, 10% cbd (cannabidiol) enriched hemp seed oil and extra virgin olive oil used for comparison. results indicate a difference in antioxidant activities among tested oils. cbd oil with ic50 of 0.86±0.06 mg/ml was shown to be a better scavenger of hydroxyl radical and dpph (1,1-diphenyl-2-picrylhydrazyl) radical (ic50 value of 5.99±0.34 mg/ml) in comparison with other oils. cbd oil also had the best ability to reduce fe3+ from ferricyanide at a concentration of 20 mg/ml (0.73) and the highest total antioxidant capacity (245±35.16 gae). two commercial fresh hemp seed oils that showed small differences in antioxidant activity had better antioxidant properties than olive oil in three performed assays, while olive oil had the highest total antioxidant capacity. hemp seed oil showed a lower antioxidant potential after a year of storage. key words: cannabis sativa, hemp, cbd, seed oil, antioxidant apstrakt: antioksidativni potencijal komercijalnih ulja iz semena konoplje i cbd ulja cilj studije je ispitivanje antioksidativnog kapaciteta komercijalnih ulja dobijenih iz semena konoplje, ulja iz semena konoplje obogaćenog 10% cbdom (kanabidiol) i ekstra devičanskog maslinovog ulja koje je korišćeno za poređenje. dobijeni rezultati ukazuju na razliku u antioksidativnoj aktivnosti među testiranim uljima. cbd ulje koje ima ic50 vrednost 0.86±0.06 mg/ml se pokazalo kao bolji “hvatač” hidroksilnog radikala i dpph (1,1-difenil2-pikrilhidrazil) radikala (ic50 vrednost 5.99±0.34 mg/ml) u poređenju sa drugim uljima. cbd ulje je takođe imalo najveću sposobnost da redukuje fe3+ u koncentraciji od 20 mg/ml (0.73) i najveći ukupni antioksidativni kapacitet (245±35.16 gae). sveža komercijalna ulja iz semena konoplje koja su pokazala male razlike u antioksidativnoj aktivnosti su imala bolje antioksidativne karakteristike od maslinovog ulja u tri korišćena testa dok je maslinovo ulje imalo najveći ukupni antioksidativni kapacitet. ulje iz semena konoplje je pokazalo slabiji antioksidativni potencijal godinu dana nakon otvaranja. ključne reči: cannabis sativa, konoplja, cbd, ulje semena, antioksidans introduction reactive oxygen species (ros) are highly reactive molecules generated normally in cells during the metabolism of oxygen. they are mainly derived from aerobic respiration during the mitochondrial electron transport but other pathways including the activity of oxidoreductase enzymes and metal catalyzed oxidation can also be involved in their production (birben et al., 2012; ozcan & ogun, 2015). ros include free radicals and radical precursors such as superoxide anion, hydroxyl radical, singlet oxygen and hydrogen peroxide. free radicals are unstable highly reactive species because of possessing unpaired electrons that tend to pair with other molecules. at physiological concentrations, they participate in cell signaling for normal biological processes including cell proliferation, apoptosis, gene expression but when the redox balance is disturbed they can cause tissue damage by the oxidation of cellular components such as membrane lipids, proteins and dnas (salganik, 2001). there is a lot of evidence that ros underly many chronic diseases including cancer, cardiovascular diseases, neurodegenerative diseases, inflammatory diseases etc. (halliwell, 2006; halliwell, 2007; ferguson, 2010). the organism defends itself using endogenous cell antioxidant protection system consisted of non© 2021 vitorović et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 113 enzymatic and enzymatic components (kurutas, 2015). it maintains balance between ros production and removal and thus prevents organism from oxidative stress and the diseases associated with it. antioxidants can also be exogenous, ingested via antioxidant-rich food and supplements and in this way, they can help the antioxidative defense of the cell to cope with free radicals directly by scavenging them (breaking the chain reaction) or indirectly by upregulating the antioxidant defense or inhibiting the ros production. they act as free radicals scavengers, reducing agents, metal chelators and singlet oxygen quenchers (lobo et al., 2010). in this way, they prevent cell damage and consequent diseases (cox et al., 2000; eastwood, 1999). the use of synthetic antioxidants as food preservatives or supplements has been shown to be harmful to health (witschi, 1986). on the other hand, plants are rich source of natural antioxidants such as phenolics, flavonoids, tannins, carotenoids and vitamins with excellent antioxidant properties that have been shown in many studies. therefore, consuming food rich in natural antioxidants is crucial for health and the development of natural products with antioxidants is of great interest to the food industry. the plant seed oils occupy a special place in the food industry due to their nutritional value and high content of unsaturated fatty acids, as well as due to antioxidant compounds which, in addition to contributing to the stability of the oil, also have various pharmacological properties (kriese et al., 2004). various seed oils that have good antioxidant capacity are in use (ramadan & moersel, 2006; smeriglio et al., 2016). among them, special attention is paid to hemp seed oil (hso), the product of the cannabis sativa l. (cannabaceae) plant, due to the specific ratio of fatty acids in its composition. the oil obtained from hemp seeds has high nutritional value and a number of health effects (leizer et al., 2000; callaway, 2004). it contains >80% polyunsaturated fatty acids (pufas) with linoleic acid as dominant, present in concentration of 52-62%, followed with α-linolenic acid in a concentrations ranging from 12-23% (leizer et al., 2000). the linoleic acid (18:2 omega-6) and αlinolenic acid (18:3 omega-3) are present in a favorable ratio of 3:1 found to contribute to the better lipid profiles and have a cardioprotective effect (callaway, 2004; simopoulos, 2008; yang et al., 2016). hso contained also γ-linolenic acid that showed significant anti-inflammatory and immunoregulatory activity (kapoor & huang, 2006). the chemical analysis of the hemp seed oil also showed the presence of tocopherols, phenols, polyphenols and lignanamides which contribute to the stability of the oil and at the same time they are natural antioxidants desirable in nutrition (kriese et al., 2004; yan et al., 2015; andre et al., 2016; smeriglio et al., 2016). tocopherols are significant in the prevention of the oxidation of pufa in oils and have many beneficial health effects including prevention of cardiovascular diseases, cancer, and age-related macular degeneration (kriese et al., 2004). the chemical composition of the hemp seed oil indicates its function in maintaining redox balance and lowering oxidative stress and associated diseases. in vivo studies have shown a beneficial effect of hemp seed oil on oxidative status and reduction of oxidative stress (vitorović et al., 2021). cannabis sativa l. plant is known for the presence of compounds called cannabinoids such as delta-9 tetrahydrocannabinol (thc) and cannabidiol (cbd). unlike thc, the most famous psychoactive cannabinoid, cannabidiol is a non-psychoactive phytocannabinoid obtained from leaves and flowers of the hemp plant that showed many pharmacological activities and therefore it is in the center of interests of the pharmacological industry. however, although preclinical and pilot studies suggest the use of cbd in the treatment of different conditions such as epilepsy, migraine, alzheimer’s disease, chronic pain, inflammatory conditions etc. (friedman & devinsky, 2015; watt and karl, 2017; irving et al., 2018; van dolah et al., 2019), it is not allowed yet as a therapeutic drugs. it is already known that cbd has anti-inflammatory and antioxidant activities (costa et al., 2007; irving et al., 2018; pellati et al., 2018; atalay et al., 2020) which make it a promising agent in the prevention and treatment of illness associated with oxidative stress. cbd showed both, direct antioxidant effects through the impact on the ros generation and components of antioxidative defense and indirect antioxidant effects, reacting with other molecules that are important for redox balance (atalay et al., 2020). the hemp industry become increasingly popular worldwide and many different commercial hemp products can be found in the market. the aim of the present study was to examine the possible differences in the antioxidant potential of commercial hemp seed oils from different manufacturers and to compare the effectiveness with the antioxidant capacity of cbd oil. the antioxidant activity was also compared with the well-known extra virgin olive oil, known to have oleic acid (mufa) as a basic fatty acid in the oil (5583%) and a lot of minor compounds (about 2% of the olive oil weight) including polyphenols, carotenoids, squalene and tocopherols (cicerale et al., 2008; kabaran, 2018). since the hemp seed oil is highly rich in unsaturated fatty acids that are susceptible to oxidation which impairs their stability and function, another goal of the study was to examine how one year of storage affects the antioxidant ability of 114 biologica nyssana ● 12 (2) december 2021: 113-122 vitorović et al. ● antioxidant potential of commercial hemp seed oils and cbd oil 115 the oil. in this case, the same commercial oil was used for the tests after the opening (hso2) and a year later (hso1) and the obtained results were compared. materials and methods oil samples in our study, we used two commercial hemp seed oils (world of hemp, kisac and gramina, novi sad), commercial cbd oil (gramina, novi sad) and extra virgin olive oil (latzimas s.a., crete). hemp seed oils were obtained by cold pressing while cbd oil (cbd enriched hemp seed oil) represents 10% ethanolic extract of cannabidiol dissolved in the cold pressed hemp seed oil. oil samples used in study are listed in the tab. 1. old and new hemp seed oils represent the samples of the same oil analyzed immediately after opening (new hemp seed oil, hso2) and one year after opening (old hemp seed oil, hso1). different oil concentrations in antioxidant assays were prepared in ethanol. bht was used as a positive control in all tests. in vitro antioxidant activities of oils dpph radical scavenging assay dpph assay is using to assess the reduction capacity of the antioxidants toward dpph radical. the method is based on the scavenging of the 1,1-diphenyl-2-picryl-hydrazyl (dpph) radical by the antioxidants which change the initial purple color of dpph solution to pale yellow due to reduction reaction. the assay was performed according to the previously described method with some modifications (minioti & georgiou, 2010). hemp seed oils and olive oil in concentrations of 20, 60, 80, and 120 mg/ml and cbd oil in concentrations of 1, 5, 10, 15 and 20 mg/ml were mixed with 4 ml of dpph solution in ethanol (0.1 mm). the mixtures were placed in the dark for 1 hour at room biologica nyssana ● 12 (2) december 2021: 113-122 vitorović et al. ● antioxidant potential of commercial hemp seed oils and cbd oil temperature. discoloration of the initial purple color of dpph solution indicates the antioxidant potential of oils, which was measured at 517 nm against ethanol blank on uv-vis spectrophotometer (shimadzu, 1650 pc). dpph scavenging activity was calculated from the following formula: dpph scavenging (%)=((abcontrol–absample)/ abcontrol)*100 ic50 which represents the concentration of the oil required to scavenge 50% of dpph was calculated and utilized for easier comparations. the lower ic50 indicate the better antioxidant capacity of oils. oh radical scavenging assay hydroxyl radical scavenging assay was conducted according to the previous study with some modifications (yang et al., 2017). hydroxyl radical was generated in vitro through the fenton reaction model system. the addition of the salicylic acid (trapping agents) served to trap hydroxyl radicals because of its very short half-life leading to produce dihydroxybenzoic acids with absorption at 510 nm. samples were dissolved in ethanol in concentrations of 0.5, 1, 2, 4 and 6 mg/ml for hsos and olive oil and 0.01, 0.05, 0.1, 0.5, 1 and 2 mg/ml for cbd oil. 2 ml of each oil concentration was mixed with 0.6 ml 8 mm aqueous solution of feso4 and 2 ml 3 mm ethanol solution of salicylic acid. the reaction was activated by adding 0.5 ml of 0.6% h2o2, and the mixture was incubated at 37 °c for 30 min. after cooling, mixtures were centrifuged at 2000 rpm for 10 min and absorbances of supernatants were measured at 510 nm. the hydroxyl radical scavenging activity (%) was calculated from the formula: [1 – (aa – ac) /ab] × 100% where: aa represents the absorbance of the whole sample, ab represents mixture without hydrogen peroxide, with 0.5 ml dh20 , ac represents blank without oil with 2 ml of ethanol. ic50 was determined from the plot where % scavenging activity is presented in the function of concentration and utilized for easier comparations. ferric reducing power assay the method was used according to previous studies (baba & malik, 2015; bhatti et al., 2015). 0.2 ml of each oil (concentrations 5, 10, 20, 40 mg/ml for hsos and olive oil and 0.1, 0.5, 1, 10 and 20 mg/ ml for cbd oil) was added to 0.5 ml of 0.2 mm, ph 6.6 phosphate buffer and 0.5 ml of 1% potassium ferricyanide. this mixture was incubated for 20 table 1. oil samples used in antioxidant tests samples abv. manufacturer old hemp seed oil hso1 world of hemp, kisac new hemp seed oil hso2 world of hemp, kisac gramina hemp seed oil hso3 gramina, novi sad extra virgin olive oil evoo latzimas s.a., crete cannabidiol oil cbd gramina, novi sad 116 minutes at 50 °c. then, 0.5 ml of 10% tca was added to the mixture followed by centrifugation at 3000 rpm for 10 minutes. 0.5 ml of supernatant was mixed with 0.5 ml of distilled water and 0.1 ml of ferric chloride (0.1%). after incubation at room temperature for 10 minutes, the absorbances were measured at 700 nm. the reducing power of the oil is represented as a function of concentration. phosphomolybdenum assay for total antioxidant capacity to determine the total antioxidant capacity of the oils, the test with phosphomolybdenum was performed and the gallic acid was used as a standard. the test is based on the reduction of mo (vi) to mo (v) by the antioxidants and formation of a green phosphate/mo (v) complex at acid ph. hemp seed oils and olive oil were tested in different concentrations prepared in ethanol (5, 20, 60 and 120 mg/ml). cbd oil was tested in concentrations: 0.1, 0.5, 1, 5 and 10 mg/ml. in the test, 0.1 ml of each concentration was mixed with a previously prepared reagent composed of 0.6 m sulfuric acid, 28 mm sodium phosphate and 4 mm ammonium molybdate as described previously (chahar et al., 2012). incubation was performed at 95 °c for 90 min. the mixtures were cooled and the absorbances were measured at 695 nm. standard curve of gallic acid prepared in concentrations of 50, 100, 150, 200 and 250 µg/ml in ethanol was used to express the total antioxidant capacity of oil as a µg equivalent of gallic acid (gae). statistical analysis all the data were expressed as means ± standard deviation (sd). the one-way analysis of variance (anova) and tukey’s post hoc multiple comparison test were used for analysis. for p<0.05 differences were considered statistically significant. results and discussion free radical scavenging activity free radicals are very dangerous reactive species capable of damaging biomolecules in cells such as dna, proteins and lipids (salganik, 2001). therefore, the capability to scavenge free radicals is a very important feature of antioxidants. in our study, the capability of oils to scavenge free radicals were tested in dpph and oh scavenging assays (tab. 2, fig. 1 and 2). dpph scavenging activity dpph assay was conducted to estimate the antiradical activity of different oils toward dpph radical. results are presented in tab. 2 and fig. 1. cbd oil showed the best free radical scavenging activity in dpph assay with ic50 of 5.99±0.34 mg/ ml. the percentage of scavenging activity ranged from 32.34% to 99.69 % in the used concentrations of cbd oil (1 to 20 mg/ml). to reach 50% efficiency in scavenging dpph radicals, it is required about ten times higher concentration of evoo and hso1 and about seven times higher concentration of hso2 and hso3 compared to cbd oil. maximum inhibitory activity was noticed in the concentration of 120 mg/ ml for all these oils and fig. 1a shows that hso2 and hso3 showed a similar correlation between the concentration and the scavenging activity. the other two oils were less efficient scavengers until the concentration of 120 mg/ml where all the oils achieved a high degree of inhibition. this can also be seen from the calculated ic50value (tab. 2) which was lower for the hso2 and hso3 oils (39.96±1.01 and 43.09±1.21 respectively) than for hso1 and evoo (61.42±0.81 and 64.03±0.81, respectively). results also indicate a lowering in the antioxidant capacity of the oil after one year of storage. the capacity of scavenging was reduced by 35% in biologica nyssana ● 12 (2) december 2021: 113-122 vitorović et al. ● antioxidant potential of commercial hemp seed oils and cbd oil table 2. antioxidant activities of hemp seed, cbd and extra virgin olive oil antioxidant assays samples hso1 hso2 hso3 evoo cbd bht dpph assay (ic50, mg/ml) 61.42±0.81b 39.96±1.01d 43.09±1.2c 64.03±0.8a 5.99±0.34e 0.06±0.01f phosphomolybdenum assay (gae) 13±1.04b 28.6±2.07b 35.9±0.99b 40.3±3.97b 245±35.2a / hydroxyl radical scavenging assay (ic50, mg/ml) 6.45±0.35b 4.61±0.21c 3.56±0.15d 7.15±0.24a 0.86±0.06e 1.36±0.07e data are presented as mean ± sd of triplicate determinations. values in the same row with different superscript letters are significantly different (p<0.05). bht was used as a positive control 117 biologica nyssana ● 12 (2) december 2021: 113-122 vitorović et al. ● antioxidant potential of commercial hemp seed oils and cbd oil one year and, according to results this reduced oil activity was a little lower than the activity of olive oil to scavenge dpph radical (tab. 2). the most commonly used antioxidant bht had much lower ic50 than all oils (0.06±0.01 mg/ml). however, it is shown that synthetic antioxidants such as bht can cause organ damage and act as tumor promoting compounds in laboratory animals (vuolo & schuessler, 1985; witschi, 1986) and that is the most important reason for using natural antioxidants, safe and effective in reducing oxidative stress. dpph assay as a widely used test for measuring the antiradical ability of bioactive compounds belongs to a group of tests based on both, single electron transfer (sem) and hydrogen atom transfer (hat) mechanisms. studies that investigated in vitro antioxidant activity of seed oils showed their good scavenging activity toward dpph radical indicating that the unsaturated fatty acids, phospholipids, phytosterols and tocopherols are carriers of this activity (luzia et al., 2013; cherbi et al., 2017). in the paper of smeriglio et al. (2016), finola hso also showed antiradical activity and the percentage of inhibition of free radicals was the highest in the case of whole oil followed by lipophilic and hydrophilic fractions. the authors explained this activity by means of the presence of high content of flavonoids in oil that are effective in free radical scavenging and metal chelating activity fig. 1. dpph scavenging activity a) hemp seed oils and extra virgin olive oil, b) cbd oil, c) bht. data are presented as mean ± sd of triplicate determinations of the percentage scavenging of free radicals. bht was used as a positive control fig. 2. hydroxyl free radical scavenging activity presented via ic50 values (mg/ml) for different oils. data are presented as mean ± sd of triplicate determinations. values marked with different letters are significantly different (p<0.05). bht was used as a positive control (kandaswami & middleton, 1994). the weaker dpph radical scavenging activity of olive oil in comparison with hso noticed in our study was also showed in the study of ramadan et al. (2006). they found a negative correlation between radical scavenging activity and amounts of phenolics and tocopherols and a positive correlation with levels of unsaponifiable and phytosterols among oils with the high radical scavenging activity level (coriander seed oil and black cumin seed oil) and concluded that scavenging of dpph was combined action of different compounds in oils. it is most likely that the composition and proportion of the fatty acids and ancillary components from seed oils such are lipophilic flavonoids, tocopherol, or some other bioactive substances, are responsible for the dpph scavenging by hso. in our study, cbd enriched hemp seed oil was also tested, and it was shown that cbd oil is a strong scavenger of dpph radical which is consistent with other studies (hacke et al., 2019; kitamura et al., 2020; petrovici et al., 2021). cbd is a compound with two phenolic groups in the structure and an antioxidant mechanism characteristic of phenol derivates that make it a strong antioxidant. phenol compounds are strong dpph scavengers by hydrogen transfer mechanism (leopoldini et al., 2004; leopoldini et al., 2011). like polyphenols, cbd can eliminate free radicals via electron or hydrogen transfer from the phenol group (borges & da silva, 2017). hydroxyl free radical (oh) scavenging activity hydroxyl radical is the most biologically active and dangerous free radical which can cause serious damage to biomolecules and lead to the development of disease (lipinski, 2011; birben et al., 2012; ozcan & ogun, 2015). therefore, hydroxyl radical scavengers are very important for human’s health. generation of hydroxyl radical in fenton reaction in the presence of hydrogen peroxide and iron ions is a model used in vitro to estimate hydroxyl radical scavenging by the antioxidants. for easier comparison, the ic50was calculated for all tested oils and presented in tab. 2 and fig. 2. all the ic50 values are significantly different except ic50 for cbd and bht indicating their similar hydroxyl radical scavenging ability. ic50 for the cbd oil (0.86±0.06 mg/ml) was the lowest among oils (fig. 2). moreover, all analyzed hemp seed oils had lower ic50 than olive oil indicating that they, together with cbd that was also dissolved in hso, are better oh radical scavengers. good oh radical scavenging activity of cbd oil was previously explained by the presence of two phenolic groups in cannabinoids (borges & da silva, 2017; atalay et al., 2020). additionally, cbd is dissolved in hso rich in pufa that are known as good oh radical scavengers. the high affinity of hydroxyl radical to double bonds of pufa that turn into single bounds is thought to be responsible for the scavenging activity of pufa (lipinski, 2011). hso is rich in pufa (70 to 90% of hso are pufa) and their content is much higher than in olive oil (kabaran, 2018) that can explain its better oh radical scavenging activity. the presence of polyphenolic natural substances such as flavonoids, also contributes to the antioxidant capacity of oil. polyphenols, in addition to their ability to be oxidized, have also the ability to scavenge hydroxyl radicals reducing the double bonds to single ones and thus creating hydroxyl derivatives. this is only the case of polyphenols with available ortho position in the ring. thus, phenol compounds that can be found in olive oil and in hso are also responsible for their ability to scavenge oh radicals (tuck et al., 2001). hso2 and hso3 were shown as excellent scavengers of oh radical especially hso3 with the lowest ic50value (3.56±0.15). the scavenging ability of hso decreased after one year of storage which can be seen through a comparison of ic50 of old and new oil (6.45±0.35 for old hso and 4.61±0.21 for the new hso). this is probably because of the oxidation of the fatty acids in oil which impairs their anti-oh radical function. metal reducing ability of oils reduction of metals by natural compounds is another method used to evaluate the antioxidant ability (capacity) of natural compounds. for that purpose, we performed ferric reducing power assay and phosphomolybdenum assay. ferric reducing power the ability of oils to reduce fe3+ from ferricyanide into fe2+ (ferrous) is mainly characteristic of antioxidants such are phenol derivatives. the reducing power of oils is presented in fig. 3. absorbance obtained at 700 nm is directly proportional to the reducing capacity of the oils. the results showed an increase in reducing potential with increasing concentration of oils. the highest reducing activity showed cbd oil at a concentration of 20 mg/ml (0.73±0.01). hso2 and hso3 showed maximal reducing activity of around 0.5 under the concentration of 40 mg/ml (0.51±0.01 and 0.50±0.01, respectively). concentration of cbd oil of 0.1 mg/ml showed similar reducing power (0.24±0.004) as all other hsos (hso1, hso2 and hso3) in the concentration of 5 mg/ml (0.25±0.01, 0.24±0.003 and 0.22±0.006, respectively) as can be seen in fig. 3a and b. cbd can reduce compounds by donating electrons and also can donate h atom to neutralize free radicals (borges & da silva, 118 biologica nyssana ● 12 (2) december 2021: 113-122 vitorović et al. ● antioxidant potential of commercial hemp seed oils and cbd oil 119 biologica nyssana ● 12 (2) december 2021: 113-122 vitorović et al. ● antioxidant potential of commercial hemp seed oils and cbd oil 2017; atalay et al., 2020). petrovici et al. (2021) suggested that cbd-enriched hemp oil showed high antioxidant activity having the ability to reduce iron, scavenge free radicals and inhibit lipid peroxidation. reducing power of bht was higher compared to all oils and the nearest to cbd oil especially in lower concentrations (fig. 3b). in comparison with olive oil, all three samples of hso in all tested concentrations showed better reducing power (fig. 3a). the results from the reducing power assay showed that the old oil (hso1) had a very similar or even slightly higher reducing power than the new (hso2) in lower concentrations than 40 mg/ml, which can be seen on fig. 3a. a slight decrease in the reducing ability of the hso after one year was noticed in concentration of 40 mg/ml (0.51±0.01 for the new hso and 0.44±0.01 for the old hso). results that showed reducing power of the hsos are of great importance indicating oil potential to limit the generation of free radicals via fenton reaction. the reducing power of hemp seed extracts determined by frap assay was evaluated in the study of irakli et al. (2019). it was also shown that essential oil from the aerial parts of c. sativa showed good reducing power (zengin et al., 2018). cold pressed hso from finola cultivar of cannabis sativa reduced ferric ions, with the reducing power being better in the lipophilic fraction than in the hydrophilic fraction of oil (smeriglio et al., 2016). authors suggested that probably the polyphenols or tocopherol present in different amounts in hso were mainly responsible for these reducing abilities. in the work of petrovici et al. (2021), it was shown that reducing ability of cbd oil was in the line with the ability of phenolic derivatives. total antioxidant capacity the total antioxidant capacity (tac) of oils was estimated by phosphomolybdenum assay. the molybdenum assay serves to predict the antioxidant activity of the sample by measuring the reduction degree of mo (vi) to mo (v) by the oils and formation of green phosphate/mo (v) complex with absorption at 695 nm. tac of each oil was calculated as µg of gallic acid equivalent per mg of oils (gae) using a standard gallic acid curve. in accordance with other antioxidant tests, cbd oil showed the highest total antioxidant capacity (245.00±35.16) with the statistically significant difference in comparison with all other oils (tab. 2). fig. 3. ferric reducing potential of oils a) hemp seed oils (hso) and extra virgin olive oil (evoo), b) cbd oil and bht. data are presented as mean ± sd of triplicate determinations. bht was used as a positive control fig. 4. total antioxidant capacity of hemp seed oils and extra virgin olive oil. data are presented as mean ± sd of triplicate determinations. total antioxidant capacity is expressed as µg of gallic acid equivalent per mg of oil (gae). values marked with different letters are significantly different (p<0.05) the results obtained from phosphomolybdenum assay for other oils than cbd (fig. 4) showed that all the oil samples reduce mo (vi) to mo (v) with higher total antioxidant capacity obtain for evoo (44.3±3.97 gae) in comparison with all three samples of hso (35.9±0.99 gae, 28.6±2.07 gae and 13±1.04 gae for the hso3, hso2 and hso1, respectively). this could be probably explained by the presence of the antioxidants such are phenolic biologica nyssana ● 12 (2) december 2021: 113-122 120 vitorović et al. ● antioxidant potential of commercial hemp seed oils and cbd oil molecules and other minor compounds in olive oil since olive oil in addition to oleic acid, contains polyphenols that are known to be responsible for such antioxidant activities (bendini et al., 2007; lee et al., 2008). total antioxidant activity for old hso was 13±1.04 gae which was more than twice less compared to fresh oil (28.6±2.07 gae) indicating a loss of antioxidant power with storage time. conclusions oils rich in pufa such as hemp seed oil have been the subject of various discussions regarding the impact on diseases associated with oxidative stress. in vivo studies have shown a beneficial effect of hemp oil on oxidative status and reduction of oxidative stress. a part of the antioxidant activities is probably due to the presence of various metabolites and antioxidants such as polyphenols or tocopherols present in the oil. the high amount of unsaturated fatty acids also indicates antioxidant activity because of the reactions that occur at the double bonds. in vitro antioxidant tests showed good antiradical and reduction capabilities of hsos. therefore, the use of hemp seed oil may be a significant contribution to the protection of diseases caused by oxidative stress but there is still a need for more in vivo evidence. two commercial fresh hemp seed oils showed small differences in activity with little advantage for hso3 noticed in the ability to scavenge oh radical and in total antioxidant capacity compared to hso2 which showed better dpph activity. differences are probably due to the small variations in their composition. more in vivo studies are needed to examine their possible differences in the effect. hemp seed oil didn’t completely lose the antioxidant ability a year after opening although the antioxidant capacity was lower in most tests. although the difference between cbd oil and other hemp products is often not clearly known on the market, these results indicate a difference in activity and much better antioxidant power of cbd oil which is the result of its chemical composition. in the ability to neutralize oh radicals, cbd oil has been shown to be a better antiradical scavenger even than the synthetic bht. acknowledgements. this study was supported by the ministry of education, science, and technological development of the republic of serbia (contract number: 451-03-9/2021-14/200124). references andre, c.m., hausman, j-f., guerriero, g. 2016: cannabis sativa: the plant of the thousand and one molecules. frontiers in plant science, 7: 19. atalay, s., jarocka-karpowicz, i., skrzydlewska, e. 2020: antioxidative and anti-inflammatory properties of cannabidiol. antioxidants, 9(1): 21. baba, s.a., malik, s.a. 2015: determination of total phenolic and flavonoid content, antimicrobial and antioxidant activity of a root extract of arisaema jacquemontii blume. journal of taibah university for science, 9(4): 449-454. bendini, a., cerretani, l., carrasco-pancorbo, a., gómez-caravaca, a.m., segura-carretero, a., fernández-gutiérrez, a., lercker, g. 2007: phenolic molecules in virgin olive oils: a survey of their sensory properties, health effects, antioxidant activity and analytical methods. an overview of the last decade alessandra. molecules, 12(8): 16791719. bhatti, m.z., ali, a., ahmad, a., saeed, a., malik, s.a. 2015: antioxidant and phytochemical analysis of ranunculus arvensis l. extracts. bmc research notes, 8(1): 1-8. birben, e., sahiner, u.m., sackesen, c., erzurum, s., kalayci, o. 2012: oxidative stress and antioxidant defense. world allergy organization journal, 5(1): 9-19. borges, r., da silva, a. 2017: cannabidiol as an antioxidant. in: preedy, v. 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medicine and cellular longevity, 2017. zengin, g., menghini, l., di sotto, a., mancinelli, r., sisto, f., carradori, s., cesa, s., fraschetti, c., filippi, a.,angiolella, l. 2018: chromatographic analyses, in vitro biological activities, and cytotoxicity of cannabis sativa l. essential oil: a multidisciplinary study. molecules, 23(12): 3266. vitorović et al. ● antioxidant potential of commercial hemp seed oils and cbd oil biologica nyssana ● 12 (2) december 2021: 113-122 122 xhulaj 2022, biologica nyssana 13(2) 13 (2) december 2022: 173-178 doi: 10.5281/zenodo.7437326 various records of lichens from southeastern albania original article skerdilaid xhulaj research center of flora and fauna, faculty of natural sciences, university of tirana, bulevardi zog i, nr. 25/1, tirana, albania skerdilaid.xhulaj@fshn.edu.al (corresponding author) received: october 10, 2022 revised: november 16, 2022 accepted: november 23, 2022 abstract: 117 taxa of lichens from two neighboring districts (pogradec and korçë) in the southeast of albania are presented. 62 taxa are new for the study area and 27 taxa are new to albania. key words: biodiversity, lichens, flora of albania, new records apstrakt: različiti zapisi o lišajima iz jugoistočne albanije predstavljeno je 117 taksona lišaja iz dva susedna okruga (pogradec i korçë) na jugoistoku albanije. 62 taksona su nova za područje istraživanja i 27 taksona su nova za albaniju. ključne reči: biodiverzitet, lišaji, flora albanije, novi zapisi introduction hitherto, no thorough investigation about the lichens of this area has been carried out. as a result, publications are quite sporadic or lacking. markgraf (1927, 1931) has produced a few records from korçë district as part of his expedition focused on the flora and vegetation of albania. hafellner (2007) provides a checklist of lichens based on historical records by several authors. this list does not include any species of lichen from the area specifically covered in this investigation, except those reported by markgraf (1927, 1931). svoboda et al. (2012) have reported a greater number of lichens from 5 localities visited in this area (1 in the district of pogradec and 4 in the district of korça) compared to the above-mentioned authors. it should be noted that their localities in the district of korça were positioned within the bredhi i drenovës national park, leaving other areas or habitats of this district uncovered. the results presented below are based on fieldwork undertaken by author encouraged by the need to increase and improve knowledge on the diversity of lichens in albania. materials and methods specimens were collected by the author during field trips carried out in both districts. the sampling localities were chosen according to diversity of the habitats paying attention to the substrates. specimens were determined using lichenological routine light microscopy methods and based on relevant literature according to poelt (1969), poelt & vězda (1977, 1981), clauzade & roux (1985), wirth (1995), nimis & martellos (2004), smith et al. (2009), wirth et al. (2013). the nomenclature follows nimis & martellos (2008) and reflects a more modern concept of genus. the specimens are deposited in the author’s personal herbarium. list of sampling localities 1: pogradec district, calcareous hills w of ohrid lake c. 20 km n of pogradec, 41°03’40”n/20°36’36.2”e, alt. c. 700 m, 10.06.2020. 2: pogradec district, calcareous rocks on carpinus scrub, near the village rëmenj, c. 16 km s of pogradec, an area characterized by drought but influenced by human activity, 40°51’05.6”n/20°36’49.4”e, alt. c. 830 m, 10.06.2020. 3: pogradec district, near the village peshkëpi, c. 20 km e of pogradec, 40°53’45.9”n/20°46’45.7”e, alt. c. 940 m, 11.06.2020. 4: pogradec district, near the village guri i bardhë, c. 22 km w of pogradec, hedges between pastures, 40°52’53.8”n/20°33’03.6”e, alt. c. 1050 m, 11.06.2020. 5: korçë district, on roadside trees (populus), c. 5 km nw of korça, 40°39’13.8”n/20°44’48”e, alt. c. 850 m, 12.06.2020. 6: korçë district, on robinia behind the memorial of martyrs in korçë, 40°37’10.3”n/20°47’22.4”e, alt. 950 m, 12.06.2020. © 2022 xhulaj. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 173 14th symposium on the flora of southeastern serbia and neighboring regions 174 biologica nyssana ● 13 (2) december 2022: 173-178 xhulaj ● various records of lichens from southeastern albania 7: korçë district, bozdovec, bredhi i drenovës national park, c. 7 km e of the village drenovë, on hill wooded by middle-aged mixed forest with dominance of abies borisii-regis in the northern part of the park, 40°34’36.8”n/20°50’34.5”e, alt. 1360 m, 13.06.2020. 8: korçë district, korçë, bredhi i drenovës national park, tourist route in the southern part of the park, 40°32’33.3”n/20°49’18.2”e, alt. 1240-1300 m, 14.06.2020. 9: korçë district, near the village vithkuq, c. 32 km sw of korçë, limestone outcrops, mixed deciduous forest, 40°31’05.3”n/20°35’09.4”e, alt. c. 1230 m, 15.06.2020. 10: korçë district, near the village dardhë, c. 18 km se of korçë, mixed forest, 40°30’54”n/20°49’25”e, alt. c. 1250 m, 16.06.2020. list of substrates and their abbreviations: cor: on bark of trees cal: on limestone ter-cal: on calcareous soil sil: on siliceous rocks bry: on bryophytes abb: abies borisii-regis aps: acer pseudoplatanus car: carpinus sp. fag: fagus sylvatica pop: populus sp. qce: quercus cerris rps: robinia pseudoacacia sal: salix sp. results a total of 117 taxa of lichens were recorded, including 62 lichens new to the investigation area and 27 new to the country. species are arranged alphabetically. numbers from 1 to 10 correspond to the localities listed above; the abbreviations following this number correspond to the substrates listed above. author’s initials and numbers in brackets show individual consecutive numbering systems used by the author to identify and locate individual specimens. new records for the investigation area are indicated with an asterisk (*), and new records for albania with a ring (o). acarospora cervina a. massal. 1, 2, 5, 9 cal (sx 7108, 7211, 8521, 8964) *acarospora glaucocarpa (ach.) körb. 1 cal (sx 7133) anaptychia ciliaris (l.) flot. 1 car, 6 rps (sx 7113, 8656) *oarthonia apatetica (a. massal.) th. fr. 3, 4, 5, cor (sx 7314, 7404, 8593) arthonia didyma körb. 7, 8, 10 abb (sx 8772, 8811, 8123) athallia alnetorum (giralt, nimis & poelt) arup, frödén & søchting 4 qce, 5 pop (sx 7415, 8542) *oathallia cerinella (nyl.) arup, frödén & søchting 6 cor (sx 8619) athallia holocarpa (hoffm.) arup, frödén & søchting 5 cor (sx 8565) *oathallia pyracea (ach.) arup, frödén & søchting 4, 7, 8, 10 aps (sx 7423, 8735, 8809, 8176) *bagliettoa marmorea (scop.) gueidan & cl. roux 1 cal (sx 7186) *obagliettoa parmigera (j. steiner) vězda & poelt 1 cal (sx 7153) *obagliettoa parmigerella (zahlbr.) vězda & poelt 1 cal (sx 7104) blastenia crenularia (with.) arup, søchting & frödén 1, 2, 4 cal (sx 7111, 7270, 7441) blastenia ferruginea (huds.) a. massal. 3, 4, 6, 9 cor (sx 7315, 7476, 8655, 8911) calicium salicinum pers. 7, 8 abb (sx 8768, 8832) caloplaca cerina (hedw.) th. fr. 1 cor, 7 fag, 8 fag, 9 sal (sx 7174, 8761, 8844, 8924) *caloplaca conversa (kremp.) jatta 1 cal (sx 7168) *candelariella aurella (hoffm.) zahlbr. 1 cal (sx 7156) *candelariella vitellina (hoffm.) müll. arg 6, 10 cor (sx 8660, 8111) candelariella xanthostigma (ach.) lettau 3, 4, 6 cor, 7, 8 abb (sx 7310, 7437, 8692, 8750, 8812) *circinaria calcarea (l.) a. nordin, savić & tibell 1, 2, 3, 6, 9, 10 cal (sx 7137, 7261, 7303, 8605, 8922, 8108) *circinaria contorta (hoffm.) a. nordin, savić & tibell 1, 2, 3, 6, 9 cal (sx 7157, 7224, 7302, 8613, 8957) *ocircinaria hoffmanniana (s. ekman & fröberg ex r. sant.) a. nordin 1, 2 cal (sx 7107, 7223) 175 biologica nyssana ● 13 (2) december 2022: 173-178 xhulaj ● various records of lichens from southeastern albania *cladonia pyxidata (l.) hoffm. f. pocillum (ach.) nyl. 1, 2, ter-cal (sx 7105, 7219) *cladonia pyxidata (l.) hoffm. f. pyxidata 1, 2, 3, 4 ter-cal, 7, 9, 10 bry (sx 7152, 7261, 7308, 7430, 8708, 8964, 8152) collema flaccidum (ach.) ach. 1, 2, 3, 4 ter-cal (sx 7170, 7252, 7344, 7408) *oenchylium polycarpon (hoffm.) otálora, p.m. jørg. & wedin subsp. polycarpon 1, 2, 4 cal (sx 7114, 7216, 7426) enchylium tenax (sw.) gray 1, 2, 3, 4 ter-cal (sx 7175, 7246, 7322, 7467) *fuscopannaria praetermissa (nyl.) p.m. jørg. 1 ter-cal, 9 bry (sx 7169, 8913) *ogyalolechia flavorubescens (huds.) søchting, frödén & arup var. flavorubescens 2 car, 5 pop (sx 7266, 8590) hypogymnia farinacea zopf 7, 8 abb (sx 8718, 8875) *lathagrium auriforme (with.) otálora, p.m. jørg. & wedin 1 cal (sx 7103) *lathagrium cristatum (l.) otálora, p.m. jørg. & wedin 1, 2, 9 cal (sx 7161, 7230, 8959) lecanora allophana (ach.) nyl. f. allophana 3, 5, 6 pop, 7, 8, 10 fag (sx 7317, 8546, 8643, 8715, 8808, 8151) lecanora allophana f. sorediata vain. 3, 5 pop (sx 7347, 8513) *lecanora argentata (ach.) malme 7, 8, 9 fag (sx 8780, 8867, 8934) *lecanora carpinea (l.) vain. 3, 4, 10 cor (sx 7319, 7440, 8105) lecanora chlarotera nyl. subsp. chlarotera 3, 4 cor, 8 fag (sx 7324, 7449, 8833) lecanora intumescens (rebent.) rabenh. 7, 8 fag (sx 8762, 8843) lecanora leptyrodes (nyl.) degel. 1, 3, 4, 8, 9, 10 cor (sx 7102, 7366, 7465, 8812, 8918, 8116) lecanora salicicola h. magn. 4, 9 cor (sx 7409, 8912) lecanora subcarpinea szatala 7, 8, 10 abb (sx 8709, 8822, 8160) lecidea atrobrunnea (dc.) schaer. subsp. atrobrunnea 3, 10 sil (sx 7313, 8149) *lecidella elaeochroma (ach.) m. choisy var. elaeochroma 1, 3, 5, 6, 10 cor (sx 7127, 7329, 8597, 8617, 8121) *lecidella euphorea (flörke) hertel 1, 2, 7, 9 cor (sx 7106, 7255, 8731, 8961) lecidella flavosorediata (vězda) hertel & leuckert 5, 6, 9 cor (sx 8536, 8671, 8953) lepra albescens (huds.) hafellner 9, 10 cor (sx 8926, 8172) leptogium saturninum (dicks.) nyl. 4 cor, 8 fag (sx 7413, 8841) *lobothallia radiosa (hoffm.) hafellner 1, 2, 9 cal (sx 7119, 7279, 8948) melanohalea exasperata (de not.) o. blanco, a. crespo, divakar, essl., d. hawksw. & lumbsch 9, 10 cor (sx 8931, 8124) melanohalea laciniatula (h. olivier) o. blanco, a. crespo, divakar, essl., d. hawksw. & lumbsch 8 abb (sx 8847) *omicarea globulosella (nyl.) coppins 10 abb (sx 8131) *myriolecis dispersa (pers.) sliwa, zhao xin & lumbsch 2, 9 cal (sx 7253, 8938) myriolecis hagenii (ach.) sliwa, zhao xin & lumbsch 3, 5, 6 cor (sx 7316, 8594, 8612) nephroma resupinatum (l.) ach. 4, 9, 10 cor (sx 7432, 8925, 8137) ochrolechia pallescens (l.) a. massal. 7 aps, 8 abb (sx 8710, 8859) ochrolechia szatalaensis verseghy 10 abb (sx 8128) parmelia submontana hale 7 fag, 10 abb (sx 8724, 8162) phaeophyscia nigricans (flörke) moberg 2 cor, 5 pop (sx 7247, 8567) phaeophyscia orbicularis (neck.) moberg 1, 2, 5, 6 cor (sx 7158, 7271, 8525, 8644) *physcia adscendens h. olivier 2, 6, 9 cor (sx 7281, 8672, 8903) physcia aipolia (humb.) fürnr. 7 abb, 9 cor (sx 8704, 8962) *physcia biziana (a. massal.) zahlbr. var. biziana 176 biologica nyssana ● 13 (2) december 2022: 173-178 xhulaj ● various records of lichens from southeastern albania 4, 6, 9, 10 cor (sx 7405, 8640, 8958, 8136) *physcia dubia (hoffm.) lettau 2 cal, 6 cor (sx 7267, 8673) *physcia leptalea (ach.) dc. 9 cor (sx 8919) *physcia stellaris (l.) nyl. 5, 9 cor (sx 8553, 8914) *physciella chloantha (ach.) essl. 9 cor (sx 8933) physconia distorta (with.) j.r. laundon 7 fag, 8 aps, 9 abb (sx 8759, 8848, 8927) physconia perisidiosa (erichsen) moberg 8 abb (sx 8814) *oplacidium lachneum (ach.) b. de lesd. 1, 2 ter-cal, 9 bry (sx 7139, 7222, 8924) *oplacocarpus schaereri (fr.) breuss 1, 2 cal (sx 7199, 7286) *oplacolecis opaca (dufour) hafellner 2 cal (sx 7280) *placynthium nigrum (huds.) gray 2 cal (sx 7278) pleurosticta acetabulum (neck.) elix & lumbsch 7, 8 abb (sx 8725, 8846) *opolycauliona polycarpa (hoffm.) frödén, arup & søchting 5 cor (sx 8518) protoblastenia incrustans (dc.) j. steiner var. incrustans 1, 2, 9 cal (sx 7116, 7265, 8952) *oprotoparmeliopsis graeca (j.steiner) sipman & cl. roux 2 cal (sx 7251) *protoparmeliopsis muralis (schreb.) m. choisy var. muralis 1, 9 cal (sx 7177, 8906) psora rubiformis (ach.) hook. 7 sil, 10 bry (sx 8757, 8125) *opyrenodesmia chalybaea (fr.) a. massal. 1, 2, 4 cal (sx 7163, 7249, 7463) *pyrenodesmia variabilis (pers.) a. massal. 2, 4, 9 cal (sx 7245, 7433, 8923) ramalina calicaris (l.) fr. 3, 4 cor, 8, 10 abb (sx 7345, 7434, 8823, 8142) ramalina canariensis j. steiner 3, 4, 9 cor (sx 7328, 7481, 8907) ramalina fraxinea (l.) ach. 7, 8, 9 cor (sx 8792, 8836, 8951) *rinodina albana (a. massal.) a. massal. 3, 6 cor (sx 7320, 8606) *orinodina anomala (zahlbr.) h. mayrhofer & giralt 2 cor (sx 7204) *orinodina bischoffii (hepp) a. massal. 1, 2 cal (sx 7162, 7222) *rinodina exigua (ach.) gray 3, 9 cor (sx 7338, 8929) rinodina immersa (körb.) j. steiner 1, 2, 4, 9 cal (sx 7190, 7283, 7444, 8902) *orinodina pyrina (ach.) arnold 2 cor (sx 7286) *orinodinella dubyanoides (hepp) h. mayrhofer & poelt 1, 2 cal (sx 7193, 7232) sanguineodiscus haematites (chaub. ex st.-amans) i.v. frolov & vondrák 1, 2, 4, 8 cor (sx 7130, 7243, 7461, 8834) *sarcogyne regularis körb. var. regularis 2 cal (sx 7273) scoliciosporum umbrinum (ach.) arnold 7, 8, 10 abb (sx 8736, 8882, 8153) *oscytinium fragile (taylor) otálora, p.m. jørg. & wedin 1, 2 cal (sx 7144, 7213) scytinium lichenoides (l.) otálora, p.m. jørg. & wedin 1, 2 ter-cal, 6, 10 bry (sx 7164, 7248, 8639, 8117) scytinium pulvinatum (hoffm.) otálora, p.m. jørg. & wedin 1, 2, 7 ter-cal (sx 7165, 7250, 8732) scytinium tenuissimum (hoffm.) otálora, p.m. jørg. & wedin 1, 2 cor (sx 7166, 7254) squamarina cartilaginea (with.) p. james 1, 2 ter-cal (sx 7124, 7257) staurothele areolata (ach.) lettau 7, 8, 10 sil (sx 8734, 8845, 8143) *osynalissa ramulosa (bernh.) fr. 2 cal (sx 7264) *thalloidima candidum (weber) a.massal. 1, 2 cal (sx 7112, 7238) *othalloidima diffractum (a. massal.) a. massal. 1 cal (sx 7129) biologica nyssana ● 13 (2) december 2022: 173-178 xhulaj ● various records of lichens from southeastern albania thalloidima opuntioides (vill.) kistenich, timdal, bendiksby & s.ekman 2 cal, 9 bry (sx 7229, 8963) usnea barbata (l.) f.h. wigg. 8 abb (sx 8821) usnea perplexans stirt. 10 abb (sx 8104) *variospora aurantia (pers.) arup, frödén & søchting 1, 9 cal (sx 7146, 8928) *ovariospora dalmatica (a. massal.) 1, 2 cal (sx 7142, 7233) *ovariospora flavescens (huds.) arup, frödén & søchting 2 cal (sx 7236) *overrucaria caerulea dc. 1, 2 cal (sx 7191, 7260) *verrucaria nigrescens pers. 1, 2 cal (sx 7192, 7258) *xanthocarpia ochracea (schaer.) a. massal. & de not. 1, 2 cal (sx 7115, 7242) *oxanthomendoza fallax (hepp) søchting, kärnefelt & s.y. kondr. 5 cor (sx 8508) xanthomendoza fulva (hoffm.) søchting, kärnefelt & s.y. kondr. 8, 10 cor (sx 8863, 8133) *oxanthoparmelia glabrans (nyl.) o. blanco, a. crespo, elix, d. hawksw. & lumbsch 7 sil (sx 8755) xanthoparmelia pulla (ach.) o. blanco, a. crespo, elix, d. hawksw. & lumbsch 7, 10 sil (sx 8758, 8122) *xanthoria parietina (l.) th. fr. 1, 2 cal, 4, 5, 6, 9 cor (sx 7109, 7263, 7435, 8541, 8654, 8921) discussion thus far, 55 taxa of lichens were known from both districts (markgraf, 1927, 1931; svoboda et al., 2012). this study is able to add a further 62 taxa. more than 60% of the lichen taxa added by this contribution are crustose and many of them are widely distributed in the mediterranean region. regarding the substrate ecology of the newly recorded lichen taxa, 58% of the added taxa are saxicolous, 35% are corticolous and only 6% are terricolous or muscicolous. new lichen species for the albania are arthonia apatetica, athallia cerinella, a. pyracea, bagliettoa parmigera, b. parmigerella, circinaria hoffmanniana, enchylium polycarpon subsp. polycarpon, gyalolechia flavorubescens var. flavorubescens, micarea globulosella, placidium lachneum, placocarpus schaereri, placolecis opaca, polycauliona polycarpa, protoparmeliopsis graeca, pyrenodesmia chalybaea, rinodina anomala, r. bischoffii, r. pyrina, rinodinella dubyanoides, scytinium fragile, synalissa ramulosa, thalloidima diffractum, variospora dalmatica, v. flavescens, verrucaria caerulea, xanthomendoza fallax and xanthoparmelia glabrans. it should be noted: in korça town itself were only found three species, miserably developed (physcia biziana, physciella chloantha and phaeophyscia orbicularis). compared to the coastal areas visited so far this is poor, presumably because of the dry climate far from the coast. calcareous rocks on carpinus scrub south of pogradec, an area characterized by drought but influenced by human activity. to be noticed on carpinus: anaptychia ciliaris, gyalolechia flavorubescens, physcia leptalea, rinodina pyrina. on limestone: placocarpus schaereri, placolecis opaca, scytinium fragile, synalissa ramulosa and thalloidima diffractum. conclusions despite the considerable number of taxa found and localities visited, it is expected that the lichen flora should be richer due to the fact that some areas are difficult to reach and the large extent of both districts. although many factors influence the species composition of the investigated localities, it may be concluded that the general climate and the potential natural vegetation/vegetation zones adjust the general pattern of the lichenic flora, while the stand parameters and the conditions of the instant surroundings, especially the topography-influenced local climatic conditions and the present vegetation surrounding the habitats, may affect the species composition to a considerable extent. references clauzade, g., roux, c. 1985: likenoj de okcidenta eŭropo. ilustrita determinlibro. bulletin de la société botanique du centre-ouest. nouvelle série. numéro spécial, 7: 1-893. hafellner, j. 2007: checklist and bibliography of lichenized and lichenicolous fungi so far reported from albania (version 05-2007). fritschiana (graz), 59: 1-18 177 biologica nyssana ● 13 (2) december 2022: 173-178 xhulaj ● various records of lichens from southeastern albania poelt, j., vězda, a. 1981: bestimmungsschlüssel europäischer flechten. ergänzungsband ii. bibliotheca lichenologica, 16: 1-390. smith, c.w., aptroot, a., coppins, b.j., fletcher, a., gilbert, o.l., james, p.w., wolse-ley, p.a. 2009: the lichens of great britain and ireland. the british lichen society & the natural history museum. london. 1046 p. svoboda, d., bouda, f., malíček, j., hafellner, j. 2012: a contribution to the knowledge of lichenized and lichenicolous fungi in albania. herzogia, 25: 149-165. wirth, v. 1995: flechtenflora. bestimmung und ökologische kennzeichnung der flechten südwestdeutschlands und angrenzender gebiete. 2. auflage. stuttgart: eugen ulmer. 661 p. wirth, v., hauck, m., schultz, m. 2013: die flechten deutschlands. 2 bände. stuttgart: eugen ulmer. 1244 p. markgraf, f. 1927: an den grenzen des mittelmeergebietes. pflanzengeographie von mittelalbanien. feddes repertorium beiheft, 45: 1-217, vegetationskarte. markgraf, f. 1931: pflanzen aus albanien 1928. denkschriften der kaiserlichen akademie der wissenschaften/mathematischnaturwissenschaftliche classe, 102: 317-360, tab. nimis, p.l., martellos, s. 2004: keys to the lichens of italy. vol. 1: terricolous species. edizioni goliardiche. trieste. 346 p. nimis, p.l., martellos, s. 2008: italic the information system on italian lichens. version 4.0. university of trieste. deparrtment of biology, in4.0/1 (http://dbiodbs.univ.trieste.it/). poelt, j. 1969: bestimmungsschlüssel europäischer flechten. lehre: cramer. 757 p. poelt, j., vězda, a. 1977: bestimmungsschlüssel europäischer flechten. ergänzungsband i. bibliotheca lichenologica, 9: 1-258. 178 nikolić et al. 2024, biologica nyssana 15(2) 15 (2) december 2024: 75-81 doi: 10.5281/zenodo.13847305 effect of different extraction solvents on total phenolic and flavonoid contents and antioxidant activity of the bolboschoenus laticarpus rhizome extracts original article danijela nikolić university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia danid@pmf.ni.ac.rs (corresponding author) andrea žabar popović university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia milica vidanović university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia dragana jenačković gocić university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia perica vasiljević university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia marina jušković university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia received: june 21, 2024 revised: september 06, 2024 accepted: september 10, 2024 abstract: the aim of this study was to determine and compare antioxidant activity and total phenol and flavonoid content in various rhizome extracts of bolboschoenus laticarpus. methanolic, ethanolic, chloroform, and ethyl acetate extracts were tested at 250, 500, 625, 750, 825, and 1,000 µg/ ml of dry extract concentrations. the antioxidant activity of the extracts was evaluated using the dpph method, the total flavonoid content by the aluminium chloride (alcl3) method and the total phenolic content by the folin-ciocalteau method. the results revealed that ethanolic extract had the highest antioxidant capacity (ic50, 0.981 mg/ml) while the chloroform extract showed the lowest activity (ic50, 11.78 mg/ml). analysis of total phenolic content in different extract types showed that ethyl acetate extract had the highest concentration of phenols while chloroform extracts had the lowest values. the highest concentration of flavonoids was noticed in ethyl acetate extract while the lowest concentration was determined in methanolic extract. this study confirmed that rhizome of b. laticarpus has significant antioxidant capacity and is a good source of phenols and flavonoids. key words: bolboschoenus laticarpus, different solvents, antioxidant activity, phenols, flavonoids apstrakt: efekat različitih rastvarača za ekstrakciju na ukupnu količinu fenola i flavonoida kao i na antioksidativnu aktivnost ekstrakata rizoma vrste bolboschoenus laticarpus cilj ovog rada je bio da se utvrdi prisustvo ukupnuh fenola i flavonoida kao i antioksidativni potencijal različitih tipova ekstrakata iz rizoma vrste bolboschoenus laticarpus. testirani su metanolni, etanolni, hloroformski i etil acetatni ekstrakti pri koncentracijama od 250, 500, 625, 750, 825 i 1,000 µg/ml. antioksidativna aktivnost ekstrakta je utvrđena korišćenjem dpph metode, ukupna količina flavonoida je određena aluminijum-hlorid (alcl3) metodom dok je ukupna količina fenola određena metodom po folin-ciocalteou. rezultati su pokazali da je etanolni ekstrakt imao najvišu antioksidativnu aktivnost (ic50, 0.981 mg/ml), dok je hloroformski ekstrakt pokazao najnižu aktivnost (ic50, 11.78 mg/ml). analiza ukupne količine fenola je pokazala da etil acetatni ekstrakt ima najveću količinu ukupnih fenola, dok je hloroformski ekstrakt imao najmanju. najveća koncentracija flavonoida je zabeležena u etil acetatnom ekstraktu, dok je najmanja koncentracija flavonoida određena u metanolnom ekstraktu. ovi rezultati pokazuju da rizom b. laticarpus vrste pokazuje značajan antioksidativni kapacitet i dobar je izvor fenola i flavonoida. ključne reči: bolboschoenus laticarpus, različiti rastvarači, antioksidativna aktivnost, fenoli, flavonoidi introduction for a long time bolboschoenus laticarpus marhold, hroudová, duchácek & zákravsky, was considered a hybrid between the species b. yagara (ohwi) y. c. yang & m. zhan and b. maritimus (l.) palla (browning et al., 1996). however, hroudová et al. (1999) proposed a different parentage for b. © 2024 nikolić et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 75 laticarpus b. planiculmis × b. yagara which is more likely according to ecological requirements and chromosome numbers (jarolimova & hroudová, 1998). in 2004 marhold et al. compared morphological characteristics of populations belonging to hybrid b. maritimus × b. yagara with other related taxa native to central europe and described b. laticarpus as a new species, undoubtedly of hybrid origin but represents a wellestablished taxon occurring independently of the area of putative parents. bolboschoenus laticarpus is a perennial herbaceous plant with a branched underground rhizome, which bears a 2-3 cm elliptical to spherical tuber. stems are erect, trigonous, about 0.71.1 m tall, with leaves spirally arranged in three lines (hroudová et al., 2007). according to the identification key for european species of the genus bolboschoenus (hroudová et al., 2007), the main morphological characteristics for distinguishing b. laticarpus from other european bolboschoenus species are: branched inflorescence, consisting of a central group of 2-7 clustered, sessile spikelets and of 2-5 rays bearing fascicles of 2-4 spikelets, the rays more than two times longer than sessile spikelets, total number of spikelets on rays higher than or same as number of sessile spikelets, broad achenes (2.02.4 mm wide), compressed obtusely-trigonous in cross-section, thin exocarp but visibly developed, formed of slightly elongated cells. according to hroudová et al. (2007), b. laticarpus is a frequent species mostly distributed in central europe while it is rare in southern and southeastern europe and inhabits river floodplains, next to canals and occasionally flooded areas (hroudová et al., 1999). it can form large littoral zones in aquatic habitats, but it also thrives in terrestrial habitats that were temporarily flooded (mikulka et al., 1999). in serbia b. laticarpus occupies different habitat types: river floodplains, places near water channels, and the shores of lakes and ponds. it is recorded along the danube and tisa rivers in vojvodina and in a few localities in the south-eastern and southern parts of serbia (nikolić et al., 2019). it is known that some representatives of the genus bolboschoenus are used for practical purposes and in medicine (ewing, 1983; powell et al., 1987; simpson & inglis, 2001). the stems of b. maritimus are woven into baskets and made into blankets in ethiopia, while in india the seeds are used in food or made into flour (simpson & inglis, 2001). bolboschoenus maritimus is also used in medicine as an agent against tumors and leukaemia (powell, 1987). powell (1987) examined the alcoholic extract from the seeds of b. maritimus and analysed the bioactive components as well as their effect on leukaemia. in china, the root of b. maritimus is used as an astringent and diuretic (ewing, 1983). grigore & oprica (2015) analysed the possibility of antioxidant potential in some halophytes, including b. maritimus. numerous methods exist for extracting antioxidants from plants, including soxhlet extraction, maceration, supercritical fluid extraction, subcritical water extraction, and ultrasound-assisted extraction (do et al., 2014). the extraction yield and antioxidant activity are influenced not only by the chosen extraction technique but also by the solvent utilized (dapkevicius et al., 1998). the solubility of different antioxidant compounds, which vary in their chemical characteristics and polarities, may vary depending on the solvent employed (turkmen et al., 2006). polar solvents are commonly employed to extract polyphenols from plant matrices (anokwuru et al., 2011). among them, aqueous mixtures containing ethanol, methanol, acetone, and ethyl acetate are the most suitable (sultana et al., 2009). ethanol is recognized as an effective solvent for polyphenol extraction and is considered safe for human consumption. methanol is often preferred for extracting lower molecular weight polyphenols, while aqueous acetone is known for its efficacy in extracting higher molecular weight flavanols (dai & mumper, 2010). the species b. laticarpus has not been investigated in terms of antioxidant activity and total phenolic and flavonoid content so far. accordingly, the following goals were established: 1) determination of the antioxidant activity of different extracts of the rhizome of b. laticarpus. 2) determination of the total amount of phenol and flavonoids in the analysed different extracts of the rhizome of b. laticarpus. materials and methods plant material for the purposes of this research, rhizomes of the species b. laticarpus were collected from may to september 2018 at the oblačina location (se serbia). the material was identified based on the key for european and serbian species (hroudová et al., 2007; nikolić et al., 2019) and deposited in the herbarium moesiacum niš (hmn) of the faculty of sciences and mathematics (13511 hmn). the plant material was air-dried and stored in a dry room at room temperature until further processing. obtaining extracts dried and crushed plant material (10 g) was extracted using different organic solvents: ethanol, methanol, ethyl acetate, and chloroform (100 ml). 76 biologica nyssana ● 15 (2) december 2024: nikolić et al. ● effect of different extraction solvents on total phenolic and flavonoid contents and antioxidant activity of the bolboschoenus laticarpus rhizome extracts biologica nyssana ● 15 (2) december 2024: nikolić et al. ● effect of different extraction solvents on total phenolic and flavonoid contents and antioxidant activity of the bolboschoenus laticarpus rhizome extracts 77 the extraction was done within 24 h in a dark place at room temperature. extraction in the first and the last hour of the scheduled time took place in an ultrasonic bath. after filtering, the extracts are evaporated to dryness using a vacuum evaporator. all extracts were prepared to the concentration of 1 mg/ml. the extracts were packed in bottles and stored in a dark and cool place. chemicals and reagents chemicals and reagents used for antioxidant analysis were butylated hydroxytoluene (bht) („zorka pharma” šabac) and 2,2-diphenyl-1picrylhydrazyl (dpph) (sigma chemicals co. (st. louis, mo, usa)). the folin-ciocalteau phenolic reagent (merck (darmstadt, germany) and gallic acid (numex chemical products (india)) were used to determine the total phenolic content. na2co3, alcl3 and naoh („centrochem doo” stara pazova, serbia) and quercetin (med chem expres usa) were used. all solvents and substances were p.a. purity. determination of total phenolic content the total phenolic content of the extracts was determined spectrophotometrically according to folin-ciocalteau (singleton et al., 1999) with some modifications. the 300 µl extract solution and 1,500 µl 1:10 of folin-ciocalteau reagent were mixed and after 6 min in the dark 1,200 µl (7.5%) of na2co3 was added. after standing in the dark for 2 h at room temperature, the absorbance was measured at 740 nm wavelength (uv-1650pc, shimadzu 1650, europe). the total phenolic content was calculated based on the calibration curve of gallic acid (ga) in concentrations of 0.02-0.1 mg/ml. data are expressed as mg gallic acid equivalents per gram of extract (gae/g). determination of total flavonoid content the alcl3 (aluminium chloride) method (chang et al., 2002) was used to determine the total flavonoid content. aliquots of extracts and standards were dissolved in methanol. the 1 ml was added to a standard 10 ml dish containing 4 ml of distilled water (dh2o). then 0.3 ml of 5% nano2 was added. after 5 min, 0.3 ml of alcl3 (10%) was added. after 6 min, 2 ml of naoh (1m) was added and the total volume of the reaction mixture was adjusted with 2.4 ml of dh2o. the mixture was shaken well and allowed to incubate at room temperature for 30 min. absorbance was measured at 510 nm. the total flavonoid content is expressed as mg equivalent of quercetin per gram of extract (mg que/g extract). a standard curve was created for quercetin in the concentration range of 0.02, 0.04, 0.06, 0.08, and 0.1 mg/ml. measurements were made in relation to a blank sample (all substances used in the same ratio without extracts or standards). determination of antioxidant activity dpph method the antioxidant activity of the extracts was determined using the dpph method (blois, 1958). immediately before the experiment, a fresh dpph working solution (concentration 0.004%) was prepared. the initial stock of the extract had a concentration of 1 mg/ml. dilutions were made in concentrations of 1,000 µg/ml, 825 µg/ml, 750 µg/ml, 625 µg/ml, 500 µg/ml, and 250 µg/ml. the following procedure was used for each tested concentration and for each type of extract: 300 µl of extract of a certain concentration was mixed with 2,700 µl of a methanol solution of dpph, and the mixture was left in the dark for 30 min at a room temperature and after the reaction time, the absorbance was measured at 517 nm at spectrophotometer (a1). each sample as well as bht (positive control) was measured in 3 replicates. results are presented as mean ± standard deviation. the percentage of dpph radical inhibition (it can also be indicated as the percentage of antioxidant activity, %aa) is calculated using the formula: % dpph radical inhibition (at 517 nm) = [(a0 – a1) / a0] × 100 a0 – absorbance of the blank sample (dpph dissolved in methanol); a1 – sample absorbance. concentrations that inhibit 50% of dpph radicals (ic50) were obtained from the curve (microsoft excel 2007) where the dependence between dpph radical inhibition and concentration is presented for each type of extract as well as for the control. to obtain the ic50 value, an xy-scatter graph is used, on the basis of which the equation of the curve is determined. solving the curve equation for x gives the ic50 value. the r2 coefficient of correlation represents the probability of the accuracy of the constructed curve. a shimadzu uv-vis 1650pc spectrophotometer was used for the determination of antioxidant activity (dpph), as well as for the determination of total phenols and flavonoids. all measurements were repeated 3 times and presented as the mean value obtained from 3 repetitions ± standard deviation. calculations and construction of curves were performed using the ms office excel 2007 software package. 78 biologica nyssana ● 15 (2) december 2024: nikolić et al. ● effect of different extraction solvents on total phenolic and flavonoid contents and antioxidant activity of the bolboschoenus laticarpus rhizome extracts results and discussion total phenolic and flavonoid content of different extracts of bolboschoenus laticarpus rhizome the ethanolic extract had the highest yield, while the lowest yield was noticed in chloroform extract (tab. 1). the total phenolic content determined within the investigated extracts ranges from 7 to 80 mg gae/g of extracts (tab. 1). these values are higher than value 4.28 mg gae/g obtained for species b. maritimus (grigore & oprica, 2015). although the ethanolic extracts had the highest yield, the highest phenolic content was present in the ethyl acetate extract. the ethanolic extract had a higher amount of phenolic content compared to the methanolic and chloroform extract, and a lower amount compared to the ethyl acetate extract. the advantage of the ethanolic extracts compared to the methanolic extracts is also confirmed by yu et al. (2005) who investigated the effects of extraction solvents on concentration and antioxidant activity of peanut skin. the methanolic extract shows a lower phenolic content compared to the ethanolic and ethyl acetate extracts, and a higher content compared to the chloroform extract. the smallest amount of phenolic content is present in the chloroform extract (tab. 1). antioxidant activity of different extracts of bolboschoenus laticarpus rhizome analysis of the antioxidant activity showed that the ethanolic extract showed the highest antioxidant activity, while the lowest activity was recorded in the chloroform extract (tab. 2). similar results were reported by popescu et al. (2016) for scirpus holoschoenus l. rhizome extracts, namely between different solvents such as water, acetone, ethyl acetate and ethanol, where the highest antioxidant activity was noticed with ethanolic extract. the positive control had a low ic50 value, which is explained by its high antioxidant activity. ethyl-acetate extract showed higher antioxidant activity compared to methanol extract (tab. 2, fig. 1). depending on the different concentrations, the antioxidant activity of the examined extracts was calculated. the highest value of antioxidant potential was noticed in the ethanolic extract (50.13±0.78) at a concentration of 1,000 µg/ml, while the chloroform extract had the lowest value (5.30±2.33) at the same concentrations. it was observed that the ethyl acetate extract at lower concentrations (750, 625 and 500 µg/ml) had a higher antioxidant activity compared to the ethanol extract (tab. 2, fig. 1). table 1. yield, total phenolic content (tpc) and total flavonoid content (tfc) of tested extracts extract extraction yield (g) tpc (mg of ga/g of extracts) tfc (mg of que/g of extracts) methanolic 1.47 29 103 ethanolic 2.87 69 209 ethyl acetate 0.24 80 539 chloroform 0.22 7 417 flavonoids have the ability to scavenge reactive oxygen species due to their phenolic hydroxyl groups, which gives them good antioxidant potential (cao et al., 1997). the presence of a high content of flavonoids (yellow complex) in the extracts directly contributes to a strong antioxidant activity whose role is to remove free radicals. the amount of total flavonoids in different extracts of the analyzed plant species ranges from 103 to 539 mg que/g extract (tab. 1). the highest concentration of flavonoids was found in the ethyl acetate extract. the chloroform extract showed a lower concentration of flavonoids compared to the ethyl acetate extract, and a higher concentration compared to the methanolic and ethanolic extracts. in the case of the ethanolic extract, a lower content of flavonoids was determined compared to the ethyl acetate and chloroform extracts. the lowest concentration was found in the methanolic extract (tab. 1). it is considered that the antioxidant capacity of the analysed plant species depends on the type of solvent and concentration which is also confirmed in many studies (zhou & yu, 2004; yu et al., 2005; turkmen et al., 2006). the positive correlation between the concentration of phenolic content and antioxidant activity is already reported in many studies (yu et al., 2005; ghasemzadeh et al., 2010; anokwuru et al., 2011) which is partially in agreement with our results. in our study ethyl acetate extract which had the highest phenolic and flavonoid content showed the highest antioxidant activities at concentrations of 750, 625, and 500 µg/ml, while ethanol extracts at higher concentrations (1,000 and 825 µg/ml) showed the highest antioxidant activities. it was expected that ethyl acetate extract would have higher antioxidant activity at all concentrations but surprisingly the ethanol extract has the higher antioxidant activity which is similar to study by mahboubi et al. (2013) where the aqueous extract of scrophularia striata biologica nyssana ● 15 (2) december 2024: nikolić et al. ● effect of different extraction solvents on total phenolic and flavonoid contents and antioxidant activity of the bolboschoenus laticarpus rhizome extracts 79 boiss. had higher antioxidant activity than ethyl acetate extracts. the observed ic₅₀ values indicated that the ethanol extract (ic₅₀=0.981 mg/ml) exhibited the highest antioxidant activity, followed by the ethyl acetate extract (ic₅₀=0.988 mg/ml) and the methanolic extract (ic₅₀=2.897 mg/ml). the chloroform extract (ic₅₀=11.78 mg/ml) showed the lowest antioxidant activity (tab. 2). our study confirmed that extract yields, total phenolic and flavonoid contents, and resulting antioxidant activities of the plant materials are strongly dependent on the nature of the extraction solvent, due to the presence of different antioxidant compounds with various chemical characteristics and polarities that may or may not be soluble in a particular solvent which is also confirmed by jakopic et al. (2009) and sultana et al. (2009). conclusion bolboschoenus laticarpus rhizome represents a good source of phenolics and flavonoids and showed significant antioxidant activity. ethyl acetate and ethanol were found to be the most effective solvents for extracting these compounds from b. laticarpus, yielding the highest antioxidant activity and the greatest amounts of total phenolics and flavonoids. further research is necessary to identify its chemical composition and bioactive compounds responsible for the antioxidant properties. acknowledgements. the research was supported by the ministry of science, technological development, and innovation of the republic of serbia [evidence numbers: 451-03-65/2024-03/200124 and 451-03-66/202403/200124]. fig. 1. comparative overview of dpph radical inhibition percentage (%aa) in different b. laticarpus rhizome extracts and bht table 2. dpph radical scavenging activity and ic50 values by different extracts of b. laticarpus rhizomes dpph % scavenging activity concentration (µg/ml) methanolic extract ethanolic extract ethyl acetate extract chloroform extract bht 1,000 19.91±0.93* 50.13±0.78 49.53±4.98 5.30±2.33 68.39±1.30 825 16.20±0.41 44.35±1.45 42.16±3.71 5.72±0.50 58.62±1.37 750 14.98±0.63 39.65±0.58 40.96±0.10 5.05±0.59 55.41±1.96 625 12.28±0.63 32.99±0.36 35.28±0.82 4.18±0.47 48.84±0.77 500 13.47±1.43 26.47±4.58 28.72±0.61 3.66±0.82 33.01±4.35 250 6.78±0.40 16.91±2.11 16.62±0.29 2.63±0.67 21.25±1.11 ic50 2,897.97 981.02 988.13 11,782.22 695.05 * values are expressed in mean ± sd of triplicate measurement hroudová, z., zákravský, p., duchaček, m., & marhold, k. 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(2004). effects of extraction solvent on wheat bran antioxidant activity estimation. lebennsmittel wissenschaft und technologie, 37, 717–721. biologica nyssana ● 15 (2) december 2024: nikolić et al. ● effect of different extraction solvents on total phenolic and flavonoid contents and antioxidant activity of the bolboschoenus laticarpus rhizome extracts 81 kajevska, i., kuštera, m., cvijetan, i.: a contribution to the knowledge of ascomycetes in eastern serbia. biologica nyssana, 9 (2). december, 2018 biologica nyssana 9 (2) ⚫ december 2018: 77-88 kajevska et al. ⚫ a contribution to the knowledge of ascomycetes... 77 original article received: 7 jene 2018 revised: 28 august 2018 accepted: 24 september 2018 a contribution to the knowledge of ascomycetes in eastern serbia iskra kajevska1, marjan kuštera2, ivana cvijetan3 1faculty of sciences and mathematics, university of niš, višegradska 33, 18000 niš, serbia 2mycological society naissus, dvanaeste srpske brigade 7, 18000 niš, republic of serbia 3mycological society of niš, bul. svetog cara konstantina 37/34, 18000 niš, republic of serbia * e-mail: iskra.kajevska@pmf.edu.rs abstract: kajevska, i., kuštera, m., cvijetan, i.: a contribution to the knowledge of ascomycetes in eastern serbia. biologica nyssana, 9 (2). december, 2018: 77-88. mycological field research conducted from 2015 2018 in the eastern part of serbia resulted with a registration of a new data on 9 insufficiently known or new ascomycetes for the country: cordyceps militaris, ophiocordyceps gracilis, peziza phyllogena, neottiella vivida, scutellinia legalie, s. trechispora, plectania melastoma, pseudoplectania nigrella and urnula mediterranea. all species are presented with color photos of fresh specimens, microscopic morphology and notes on their taxonomy, ecology and distribution in the country. key words: ascomycetes, serbia apstrakt: kajevska, i., kuštera, m., cvijetan, i.: prilog poznavanju askomiceta istočne srbije. biologica nyssana, 9 (2). decembar, 2018: 77-88. mikološko terensko istraživanje obavljeno u periodu od 2015. do 2018. godine u istočnom delu srbije rezultiralo je registracijom novih podataka o 9 nedovoljno poznatih ili novih askomiceta za područje ove države: cordiceps militaris, ophiocordiceps gracilis, peziza phillogena, neottiella vivida, scutellinia legalie, s. trechispora, plectania melastoma, pseudoplectania nigrella i urnula mediterranea. sve vrste su predstavljene fotografijama svežih uzoraka, mikroskopskom morfologijom i podacima o njihovoj taksonomiji, ekologiji i distribuciji u srbiji. ključne reči: askomicete, srbija introduction although the interest in mycology, especially regarding fungal diversity is steadily increasing in serbia, the knowledge of the distribution of ascomycetes in serbia is scarce and limited. the eastern part of serbia is characterized with biologically rich land ecosystems but in terms of 9 (2) • december 2018: 77-88 doi: 10.5281/zenodo.2538598 biologica nyssana 9 (2) ⚫ december 2018: 77-88 kajevska et al. ⚫ a contribution to the knowledge of ascomycetes... 78 fungi, especially in ascomycota diversity, so far is the least explored area. therefore, the main aim of this study is to provide a contribution to the mycological knowledge of some ascomycetes collected from the eastern part of serbia which are insufficiently known or represent new species for the country. in scientific literature, a small number of mycological papers is dealing with species diversity of ascomycota in serbia. significant contribution to the research on ascomycota was made on np fruška gora, situated in the northern part of serbia, representing a checklist and a database of fungi (savić, 2016; http://www.naturefg.com/pages/fuascomycota.htm). some valuable published data regarding on distribution of this fungal phylum are as follows: ranojević (1910, 1914), ivančević & beronja (2004), marjanović et al. (2010), karaman et al. (2012); sadiković & kuštera (2013); ivančević (2016), milosavljević (2016), savić et al. (2016), savić & karaman (2016), vukojević et al. (2016). material and methods sources of information for this paper were available as unpublished data of ascomycetes registered during field surveys conducted in various habitats of selected locations in the eastern part of serbia. informations of research notes, revised exsiccates fig. 1. map of vicinity of the city of niš in serbia with marked sites where species have been registered biologica nyssana 9 (2) ⚫ december 2018: 77-88 kajevska et al. ⚫ a contribution to the knowledge of ascomycetes... 79 and references of those previously reported taxa from the country are also provided. collection of fungi was made from 2015 2017 by members of the mycological society of niš and mycological society naissus, niš. laboratory analyses were obtained with the use of optika n 400m and leica dm 1000 microscopes, and by examination and measuring of spores and other elements of fresh and dry material in water, 5% koh, lugol, cotton blue and melzer’s reagents. the descriptions of macroand micro characters are based exclusively on the collected material. all specimens are preserved in the private fungal collection of the society (m.s.n.). the location of the sites was determined using a gps receiver and all examined localities are provided with geographical coordinates. the sites where the taxa were registered are marked on a map (fig. 1). specimens were identified according to the following taxonomical books and keys: dennis (1960), breitenbach & kränzlin (1981), moser (1983), table 1. taxonomic review of investigated species of ascomycetes in eastern serbia class order family species sordariomycetes hypocreales clavicipitaceae cordyceps militaris ophiocordycipitaceae ophiocordyceps gracilis pezizomycetes pezizales pezizaceae peziza phyllogena pyrenomataceae neottiella vivida scutellinia legaliae scutellinia trechispora sarcosomataceae plectania melastoma pseudoplectania nigrella urnula mediterraneaea fig. 2. in situ macro-photos of the examined species. a) scutellinia legalie, ph.: i. kajevska; b) peziza phyllogena, ph.: s. lazarević; c) s.trechispora, ph.:i. kajevska; d) ophiocordyceps gracilis; ph.: s. lazarević; e) pseudoplectania nigrella, ph.: i. kajevska; f) plectania melastoma, ph.: s. lazarević, v. lilić; g) cordyceps militaris, ph.: g.taskov, s. lazarević; h) urnula mediterranea, ph.: i. kajevska; i) neottiella vivida, ph.: s.lazarević. biologica nyssana 9 (2) ⚫ december 2018: 77-88 kajevska et al. ⚫ a contribution to the knowledge of ascomycetes... 80 medardi (2006), hansen & knudsen (2000). names of the species have been modified according to index fungorum (http://www.indexfungorum.org) and mycobank (http://www.mycobank.org). the following abbreviations are used: mt. mountain, r. river, v. village, * new species for serbia. results and discussion the biodiversity of ascomycota in serbia is unknown for the most part. field research on mycodiversity conducted from 2015 2018 in the eastern part of serbia resulted with a registration of the following new and noteworthy taxa for the country: cordyceps militaris, ophiocordyceps gracilis, peziza phyllogena, neottiella vivida, scutellinia legalie, s. trechispora, plectania melastoma, pseudoplectania nigrella and urnula mediterranea (tab. 1, fig. 2). macroand microscopic descriptions, notes on ecology and diversity in the country are provided exclusively on the collected material. data on presence in the country of cordyceps militaris, peziza phyllogena, scutellinia trechispora and plectania melastoma hasn’t been reported in scientific literature up to now and scattered information were known only from web, macroscopic photos and personal communications. neottiella vivida, ophiocordyceps gracilis, scutellinia legalie and pseudoplectania nigrella are reported for serbia for the first time. a second country record is confirmed for the rare european species urnula mediterranea. fam.: clavicipitaceae (lindau) earle cordyceps militaris (l.) link description: stroma 10 70 x 2 10 mm wide, solitary or in small groups, club-shaped, cylindrical, bright orange to orange-red with abundant perithecia. stem 10 25 x 3 5 mm, smooth, wavy, paler and whitish at base (fig. 2.g). perithecia 500 720 x 300 480 μm, ovoid, usually 2/3 submersed in the stroma, paler in colour. asci 300 510 x 3.5 5 μm, narrow, cylindrical, not staining blue in iodine. ascospores elongate, filiform, multiseptate, breaking into bacilliform cell fragments with dimension of 2 4.5 x 1 1.5 μm (fig. 3). specimen examined: gabrovac v., near st. trojica monastery (niš), seličevica mt., deciduous forest, on a lepidopteran pupae half buried in soil, among moss, 316 m, n 43°15’55”, e 21°55’28”, 25/12/2015, m. kuštera, m.s.n. 25/12/15 104; gabrovac v., above st. trojica monastery (niš), seličevica mt., quercus cerris and carpinus orientalis forest, on a lepidopteran pupae, 400 m, n 43°16’13”, e 21°55’12”, 09/5/2017, n. pavković, m.s.n. 09/05/17 – 195; gadžin han town, above, suva planina mt. (niš), quercus cerris forest with carpinus orientalis, on larvae, 631 m, n 43°12’25”, e 22°03’05”, 01/06/2017/18, i. kajevska, m.s.n. 01/06/17 190. claviceps militaris is a type species of the genus with a worldwide distribution. this entomopathogenic species parasites insect larvae or pupae of several genera of lepidoptera and hymenoptera order (webster & weber, 2007). there are several species of the genus recorded in the northern part of serbia (more on this in savić et al., 2016). however, the cosmopolitan species c. militaris has not been reported in scientific literature for the mycobiota of serbia thus far. unpublished data with exsiccate and in situ photographs from the eastern part of serbia, were also kindly provided from grozdan taskov (pers.comm.): four well developed fruiting bodies found on lepidopteran pupae in pinus nigra plantings, near the city of pirot (n 43°10’18” e 22°36’56”; 11/2016). our material was collected in deciduous forests, all found on a lepidopteran pupae half buried in soil, except for one collection with small fruiting bodies emerging from larvae. as indicated above, we do not consider it as a rare species. fig. 3. microscopic photographs of cordyceps militaris, a) perithecia in melzer’s reagent (mlz); b) perithecium in lugol’s solution (iki); c) asci in iki; d) asci in mlz; e) asci tips in iki; f) spores in mlz (x400); g) spores in iki (x1000). photo – i. kajevska. biologica nyssana 9 (2) ⚫ december 2018: 77-88 kajevska et al. ⚫ a contribution to the knowledge of ascomycetes... 81 fam.: ophiocordycipitaceae g. h. sung, j.m. sung, howel-jones & spatafora *ophiocordyceps gracilis (grev.) g. h. sung, j.m. sung, howel-jones & spatafora (syn: cordyceps gracilis (grev.) durieu & mont.) description: stroma 8 x 4 mm wide, solitary, capitate, dark yellow to orange with abundant dark orange perithecial ostioles; flesh thick and paler than surface. stem 35 x 2.5 mm unfertile part of the stroma, cylindric, faintly floccose, somewhat wavy, sulphur yellow at base, paler and whitish near the fertile part of the stroma (fig. 2.d). perithecia (550)630 750 x 210 270 μm, ovoid, completelly immersed in the stroma, paler in colour. asci 370 500 x 3 5 μm, narrow, cylindrical, not staining blue in iodine, swollen at the apex. ascospores elongate, filiform, multiseptate, breaking into bacilliform cell fragments with dimension of (3)5 8 x 1 1.5 μm (fig. 4). specimen examined: niška banja city municipality (niš), suva planina mt., deciduous forest (populus tremula, p. nigra, salix alba), on lepidopteran larva, humid place, 200 m, n 43°18’14”, e 21°59’52”, 13/04/2018, s. lazarević (leg. d. vučić), m.s.n. 13/04/18 228. ophiocordyceps gracilis is a cosmopolitan but rarely reported species throughout the world. no occurrence for serbia is cited for this species, commonly known as cordyceps gracilis and this record is possibly the first for the country. a single stroma of o. gracilis has been found on lepidopteran larva emerging from the host completely covered by sulphur yellow mycelial strands in deciduous forest, predominantly polars. macro and microscopically the examined material is in accordance with the description given by mains (1951, 1958), eckblad (1967), allard (1998), breitenbach & kränzlin (1984), hansen and knudsen (eds.) (2000). fam.: pezizaceae dumort. peziza phyllogena cooke description: apothecia 5 45 mm in diameter, cup-shaped to disk-shaped, often irregularly shaped when mature; hymenium smooth, wrinkled in the center, brown with violet tinge; the outer surface is granular, brown to lilac, purplish at base; margin with purplish granules, slightly involute, and undulate; flesh thick and pale violet (fig. 2.b). asci 220 300 x 12 16 μm, cylindrical, amyloid, operculate, 8 spored, apex obtuse or truncate, ascus base with croziers. paraphyses 310 370 x 3 4 μm, cylindrical, septate, slightly enlarged at apex 4 6 μm. ascospores (13)17 19(21) x 6 11 μm, ellipsoid to narrowly ellipsoid, hyaline, structure with fine warts sometimes forming small ridges. medullarly excipulum of textura intricata, with fig. 4. microscopic photographs of ophiocordyceps gracilis, a) perithecia in (iki); b) asci in congo red (cr); c) asci tips (x1000) in cr; d) ascus and spores (x400) in cr; e) spores (x1000) in cr. photo – i. kajevska. fig. 5. microscopic photographs of peziza phyllogena. a) ascus in mlz and lpcb; b) ascus apex in lpcb; c) spores (x400) in lpcb; d) excipulum in mlz; e) ascus base in lpcb; f) hymenium (asci and spores) in lpcb. photo – i. kajevska. biologica nyssana 9 (2) ⚫ december 2018: 77-88 kajevska et al. ⚫ a contribution to the knowledge of ascomycetes... 82 hyaline cylindrical cells. ectal excipulum of textura globulosa-angularis with cells of 20 40 μm wide (fig. 5). specimen examined: popova glava hill, between hum v. and rujnik v. (niš), kalafat mt., quercus sp. and carpinus orientalis forest, on soil and wood debris, 700 m, n 43°23’25”, e 21°53’44”, 09/05/2016/7/8, i. kajevska (leg. s. lazarević), m.s.n. 09/05/17 116. no published record is reported from serbia for this species and so far, we managed to register only one locality of p. phyllogena (popova glava hill) in this part of the country. on this locality, in 2016 large quantity of this species was observed, but during the next two years these populations were with scattered number of fruiting bodies. this humus saprotroph may be confused with p. badia but its occurrence in late autumn and its reticulate spores removes any suspicion (pfister, 1987; elliot & kaufert, 1974). fam.: pyronemataceae corda *neottiella vivida (nyl.) dennis description: apothecia 2 13 x 3 8 mm in diameter, disk – shaped to cup-shaped; hymenium smooth or wrinkled with maturity, bright orange to dark orange-red when dry; excipulum concolorous with the hymenium or paler, villose with white soft and tender (delicate) hairs (up to 3.5 mm) which are mostly concentrate at the margin; margin entire or lobed, somewhat eroded; flesh thick and white. stem central if present, villose and thick (fig. 2.i). asci 150 270 x 15 18 μm, cylindrical, 8 spored, uniseriate, apex obtuse or truncate, not staining blue in iodine, ascus base with croziers. paraphyses 170 300 x 2 3 μm, narrowly cylindrical, swollen at the apex (5 6 μm) and often wry, filled with orange granules vivid in cotton blue or becoming greenish in melzer’s reagent. ascospores 21 25(-27) x 14 15(-17) μm, hyaline, thick-walled, ellipsoid, usually contains 1 2 large globose oil drops (7 10(18) μm); ornamentation verrucose on oil immersion, with small isolated warts but some are connected and form also isolated ridges. hairs 170 300 x 4 8 μm, hyaline or greenish in melzer’s reagent, usually with 2 3 septa, thickened walls, base of hair with somewhat clavate widening but mostly wry and widened (4 10 μm) with median constriction, tips obtuse. ectal excipulum of textura angularisintricata, with cells of 20 40 μm wide. medullarly excipulum of textura intricata, with hyphae 3 7 μm wide, hyaline, with thin walls (fig. 6). specimen examined: grbavče v., svrljiške planine mt. (svrljig), deciduous forest (carpinus orientalis, quercus spp., on moss rhizoids of polytrichum spp., 550 m, n 43°23’54”, e 22°00’36”, 29/09/2016, i. kajevska (leg. n. pavković), m.s.n. 29/09/16 101; gradac resort, above knez selo v. (niš), kalafat mt., fagus forest, on moss rhizoids, 450 m, n 43°23’21”, e 21°00’06”, 18/10/2016, i. kajevska (leg. n. pavković and s. lazarević), m.s.n. 18/10/16 102. neottiela vivida is bryoparasitic species which occurs with stipe base attached to moss rhizoids predominantly on polytrichum spp. important microscopic character of n. vivida which distinguishes it from other neottiella species is its warted spores (senn irlet, 1989; yao & spooner, 1996a; eckstein et al., 2014). distinguishable feature can also be the hair bases with its widened base and median constriction. its occurrence in europe is widespread but it is rarely reported. also, available literature search indicates no published data of this species from serbia. during field studies held in september and october 2016 in the eastern part of serbia, two localities of this species have been recorded (svrljiške planine mt. and gradac resort) and are reported here as new for the country. all findings have been observed at altitude bellow 700 m in fig. 6. microscopic photographs of neottiella vivida, a) ascus with spores and paraphysis in iki; b) spores in iki (x400); c) hymenium (asci, ascospores, and paraphyses) in lactophenol cotton blue solution (lpcb); d) spores in lpcb (x1000); e) ascus base in iki; f) medullary excipulum in lpcb; g) hair in lpcb; h) ectal excipulum in lpcb. photo i. kajevska. biologica nyssana 9 (2) ⚫ december 2018: 77-88 kajevska et al. ⚫ a contribution to the knowledge of ascomycetes... 83 xeric to mesic deciduous forest associations, predominantly quercus spp. *scutellinia legaliae lohmeyer & häffner description: apothecia 2 8 mm in diameter, disc shaped, hymenium dark orange and red, smooth; the outer surface covered with short dark brown thick hairs; margin distinct, covered with thick pointed or tapered hairs; flesh thick and yellowish (fig. 2.a). asci 200 310 x 18 23 μm, inamyloid, cylindrical, operculate, 8 spored, apex obtuse, ascus base with croziers. paraphyses 270 330 x 3 4 μm, cylindrical, septate, filled with orange carotenoid crystals, enlarged tips 6 12 μm. hairs: 350 650 μm, septate, mostly simple and bifurkate at base. ascospores 15.5 17.5 μm, globose, hyaline, surface covered with acuminate spines 2 5 μm long. medullarly excipulum of textura intricata, with hyaline hyphae of 5 15 μm wide. ectal excipulum of textura globulosa with cells of 20 50 μm wide (fig. 7). specimen examined: above gabrovac v., near st. trojica monastery (niš), seličevica mt., quercus cerris forest with carpinus orientalis, on wet clay soil, 450 m, n 43°15’40.6”, e 21°55’25.6”, 04/06/2017, i. kajevska (leg. s. lazarević), m.s.n. 04/06/17 199. according to schumacher (1990), s. legalie is a rarely recorded species in europe. for serbia it is reported for the first time. material examined was found on wet soil along forest pathway in quercus cerris forest on humid and open place unlike s. trechispora which obviously prefers humid but shaded places. although it is referred as more south european species preferring warm climates (schumacher 1990; matočec et al. 1995), hitherto our field studies from the eastern part characterized by warmer climate have resulted in only one data of this species consisting of four small fruiting bodies (two of them in its initial phase). scutellinia trechispora (berk. & broome) lambotte description: apothecia 3 10 mm in diameter, discshaped, hymenium dark orange and red, smooth; the outer surface covered with short dark brown thick hairs; margin distinct, covered with densely thick and sharply pointed hairs; flesh thick and white (fig. 2.c). asci 270 320 x 20 25 μm, inamyloid, cylindrical, operculate, 8 spored, apex obtuse, ascus base with croziers. paraphyses 250 330 x 2 4 μm, cylindrical, few septas, filled with orange carotenoid crystals well vivid in congo red, enlarged tips 6 9 μm. hairs: 500 1700 μm, septate, sharply pointed, mostly bifurkate at base. ascospores 15 18 μm, globose, hyaline, surface covered with truncate spines 2 4 μm high and 1 2 μm wide. medullarly excipulum of textura intricata, with hyaline hyphae of 5 15 μm wide. ectal excipulum of textura globulosa and angularis with cells of 20 50 μm wide (fig. 8). specimen examined: above gadžin han town (niš), suva planina mt., quercus cerris forest, on wet soil, under leaf litter and wood debris, by a stream, 600 m, n 43°12’25”, e 22°03’04”, 01/06/2017, i. kajevska, m.s.n. 01/06/17 213. no published data of this common european species have been reported for serbia up till now. it can be easily delimited from other taxa of the genus by its truncate spores (lohmeyer & häffner, 1983; schumacher, 1990; matočec et al., 1995; yao & spooner 1996b). macro and microscopically, our collection was consistent with published descriptions, except for the length of the hairs which is somewhat shorter that those noted by schumacher (1990). ecologically is also in accordance with the indications given by the aforementioned authors. our specimens consisting of five well-developed fruiting bodies have been found on wet soil in oak forest, barely visible among the humid leaf litter. fig. 7. microscopic photographs of scutellinia legalie, a) hairs (x100) in iki; b) hair tip in lpcb; c) hair base in lpcb; d) ascus apex in lpcb; e) ascus with spores in lpcb; f) spores within ascus (x1000) in lpcb; g) spores (x1000) in lpcb; h) spores (x1000) in iki. photo – i. kajevska. biologica nyssana 9 (2) ⚫ december 2018: 77-88 kajevska et al. ⚫ a contribution to the knowledge of ascomycetes... 84 fam.: sarcosomataceae kobayasi plectania melastoma (sowerby) fuckel description: apothecia 0.3 17 x 0.5 15 mm diam., at first roughly spherical with hollow opening, becoming cup-shaped, concave; hymenium smooth, shining, dark brown or black; excipulum wrinkled, concolorous with the hymenium, covered with orange granules when young especially in the upper part but seldom or missing when mature; flesh tough, gelatinous when wet, gray; margin entire, incurved and covered abundantly with orange granules especially when young. stem 0.2 0.5 mm, if present than central, thick, at base covered with black mycelial threads (fig. 2.f). asci 270 430 x 9 13 μm, cylindrical, long attenuated, 8 spored, uniseriate, thick walled, apex obtuse or truncate but with somewhat eccentric operculum, not staining blue in iodine, ascus base flexuous and tapered. paraphyses 270 400 x 2 μm, filiform, often forked or branched, flexuous, septate. hymenial hairs 260 410 x 2 3 μm, cylindrical, with obtuse apex and one septum at the base. ascospores (20) 21 24(27) x 9 10(12) μm, hyaline, elliptic, fusiform, slightly thick-walled, multiguttulate with small oil drops; smooth or barely visible ornamentation on oil immersion. medullarly excipulum of textura intricata, with hyphae 2 5 μm wide, smooth, hyaline, cylindrical, immersed in a gel. ectal excipulum of textura globulosa-angularis, with cells of 10 13 μm wide, thick walled, brownish. tomentum (basal hyphae) 6 8 μm wide, flexuous, with scarce septa, encrusting pigments, thick walled, dark brown (fig. 9). specimen examined: gabrovac v., near st. trojica monastery (niš), seličevica mt., quercus pubescens and q. cerris forest, on wet fallen oak branches of small diameter, 330 m, n 43°15’16”, e 21°44’75”, 28/02/2015, m. kuštera & i. cvijetan (leg. v. lilić), m.s.n. 28/02/15 – 106; bojanine vode resort, above gornja studena v., suva planina mt., fagus forest, on fallen branches and small sized decaying organic matter, 700 m, n 43°13’18”, e 22°07’20”, 24/04/2015, m. kuštera & i. cvijetan (leg. n. pavković), m.s.n. 24/05/15 107. plectania melastoma is well distinguishable from other species of the genus by the presence of the orange granules on the excipulum surface of the fruiting bodies which may last for centuries as noted by agnelo & carbone (2011). same authors also point out that spores are not quite smooth but has exclusively fine ornamentation (agnelo & carbone, 2011). p. melastoma is widespread species but also reported as rare in many fig. 8. microscopic photographs of scutellinia trechispora. a) ascus with paraphyses in iki; b) hairs (x200) in lpcb; c) hair base in iki; d) spores (x1000) in iki; e) spores (x1000) in lpcb. photo – i. kajevska. fig. 9. microscopic photographs of plectania melastoma, a) hymenium (asci, ascospores, hymenial hairs/seta and paraphyses) in lpcb; b) ascus apex in lpcb; c) ascus base in lpcb; d) paraphysis (branching) in lpcb; e) spores in mlz (x400); f) medullary excipulum in lpcb; g) ectal excipulum in lpcb; h) basal hyphae in mlz; i) spores in iki (x400). photo – i. kajevska. biologica nyssana 9 (2) ⚫ december 2018: 77-88 kajevska et al. ⚫ a contribution to the knowledge of ascomycetes... 85 european countries and listed in many national redlists of fungi (gierczyk et al., 2010). this saprotrophic fungus had not been reported before in scientific literature for serbia. data on p. melastoma published in this paper comes from two localities (gabrovac vill. and bojanine vode resort). investigated specimens were found in the base of a slope where lot of organic matter is accumulated in mixed deciduous forests (carpinus orientalis, quercus cerris, acer campestre, fagus sylvatica, robinia pseudoacacia), humid for a long time. the species was found for the first time in 2015. and all records occur from the southern mountain hills at altitude bellow 700 m. glejdura et al. (2011) states that in slovakia p. melastoma occurs at altitude of 280 – 750 m and its occurrence is confined on small diameter branches, humid places and on localities with no or low sign of human activity and these parameters are in accordance with our findings too. *pseudoplectania nigrella (pers.) fuckel description: apothecia 10 25 x 5 15 mm in diameter, cup shaped to disk-shaped; hymenium shiny black or dark brown, smooth or wrinkled with maturity; the outer surface dark brown to blakish, tomentose with dark brown to blackish, convolute and thick hairs, margin entire or undulate, also with hairs which are light brown when young; flesh thick, white and grayish (fig. 2.e). asci 280 320 x 10 14 μm, inamyloid, cylindrical, operculate, 8 spored, thick walled, apex obtuse or truncate, base attenuated. paraphyses 270 330 x 2 3 μm, filiform, septate, irregularly wry tips, some branched. hymenial hairs: 280 350 x 2 4 μm, narrowly cylindrical, straight, with one septum at base. ascospores (9-)10 12 μm, spherical, smooth, hyaline, thick walled, with gelatinous outer layer, inside contains many small globose oil drops. hairs (outher surface): 4 9 μm in diameter, cylindrical and curved, dark brown, thick-walled, septate. ectal excipulum of textura angularis, with dark brown cells of 18 25 μm wide. medullarly excipulum of textura intricata, with hyaline hyphae of 4 8 μm wide. tomentum (basal hyphae): 4 9 μm wide, cylindrical, curved, dark brown, thick walled, few septas (fig. 10). specimen examined: between gabrovac v. and gornje vlase v. (niš), seličevica mt., pinus sylvestris plantings, on soil, among moss, 398 m, n 43°16’6.34”, e 21°55’7.04”, 24/02/2017, i. kajevska, m.s.n. 24/02/17 158. pseudoplectania nigrella has not been previously reported from serbia and these are new data for the country. it has been found at four different localities, all from the eastern part of the country but exsiccate has been preserved only from the vicinity of gabrovac vill., with two welldeveloped fruiting bodies. macroand microscopically specimens are in accordance with the descriptions given by saver (1913), le gal (1953), paden (1983), breitenbach & kränzlin (1984), korf & zhuang (1991), hansen and knudsen (eds.) (2000), medardi (2006), carbone et al. (2012). data of this species in serbia comes from coniferous forests: 2 collections from fir forest and 2 from pine plantations. ascomata from all collections have been observed at the edge of the forests, about 1-2 m near the conifer trees, on an open area and amongst moss and fallen needles. urnula mediterraneaea (m. carbone, agnello & baglivo) m. carbone, agnello & p. alvarado (syn.: plectania mediterranea m. carbone, agnello & baglivo) description of studied specimen: apothecia 10 50 x 7 30 mm in diameter, cup shaped, hymenium brownish, medium reddish shade of brown, smooth fig. 10. microscopic photographs of pseudoplectania nigrella, a) hymenium (asci, ascospores, hymenial hairs/seta and paraphyses) in iki; b) hymenial hair (in middle) and paraphyses, and ascus with spores in iki; c) ascus apex in cr; d) spores (x1000) in cr; e) hymenial hairs with paraphyses in cr; f) medullary excipulum in iki; g) ectal excipulum in iki; h) basal hyphae in iki. photo – i. kajevska. biologica nyssana 9 (2) ⚫ december 2018: 77-88 kajevska et al. ⚫ a contribution to the knowledge of ascomycetes... 86 at first and wrinkled later; the outer surface is dark brown, rough in touch, covered with densely dark brown and thick hairs; margin crenate; flesh gelatinous, grayish (fig. 2.h). stem 5 20 mm, central, concolorous with the apothecia, with densely dark hairs. asci 450 550 x 12 15 μm, inamyloid, cylindrical, operculate, 8 spored, apex obtuse or truncate, ascus base without croziers. paraphyses 400 570 x 2 4 μm, cylindrical, septate, many with curved tips. hymenial hairs 450 600 x 3 4 μm, cylindrical, straight, apex obtuse. ascospores 22 27(29) x 10 14 μm, globose, hyaline, surface covered with truncate spines 2 4 μm high and 1 2 μm wide. medullarly excipulum of textura intricata, with hyaline hyphae of 3 7 μm wide. ectal excipulum of textura globulosa to angularis with brownish cells of 8 15 μm wide. tomentum (basal hyphae): 4 8 μm wide, cylindrical, curved, dark-brown, thick walled, septate (fig. 11). specimen examined: above gadžn han town (niš), suva planina mt., quercus cerris forest, on wet soil, under leaf litter and wood debris, by a stream, 600 m, n 43°12’25”, e 22°03’04”, 01/06/2017, i. kajevska, m.s.n. 01/06/17 213. urnula mediterranea is a recently described species (carbone et al., 2009) and it is an extremely rare european species. so far it is registered only from: italy, spain, france, serbia and greece (kaounas et al., 2015; milosavljević, 2017). the first record of this mediterranean species in serbia has been surprisingly reported from the central region of the country, a discovery which also represents the first record from the continental europe, and the species was found precisely in 2014, a year followed by heaviest rainfall, floods and temperatures above the average since 1951 (milosavljević, 2017). here we report the second record of u. mediterranea for serbia, located in the eastern part of the country (above gadžin han town, suva planina mt.). specimens on this locality have been registered in 2017 which was normal and dry in a relation of precipitation unlike 2014, but apothecia were found on a very humid place in quercus cerris forest under carpinus orientalis. our collection conforms well to the description given in carbone et al. (2009), although the length of spores has been quite smaller and did not exceed 30 μm but carlo agnello indicates that the dimension of spores is quite variable in this species (pers. comm.). acknowledgements. we are grateful to all members of the current mycological societies from niš for providing the fungal material in further examination. we also want to express our appreciation to prof. mitko karadelev for his valuable comments and advices in the preparation of this manuscript. references agnello, c., carbone, m., 2011: appunti di studio su plectania melastoma. rivista di micologia, 54 (4), 315-337. allard, c., 1998: recontres avec cordyceps gracilis mont. et durieu. le mystére des rendez-vous mycologiques. bulletin trimestriel de la société mycologique de france, 114: 29-33. breitenbach, j. kränzlin, f., 1981: pilze der schweiz band 1. ascomyceten. verlag micologia, luzern, switzerland. 303 p. carbone, μ., agnello, c., baglivo, a., 2009: plectania mediterranea una nuova specie 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scutellinia. mycological research, 100 (7), 859-865. microsoft word bn020201a jotic et al biologica nyssana 2 (2) december 2011: 00-00 nahirnić a. supplements of butterfly fauna… 91 original article ! the vascular flora of the vučje hill near pirot city (eastern serbia) branko jotić1, marija marković1, bojana petrović2, ivana fusijanović1, dragana pavlović3, vladimir ranđelović1 1university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia 2 institute for nature conservation of serbia, dr. ivana ribara 91, 11070 novi beograd, serbia 3university of kragujevac, faculty of sciences and mathematics, institute of biology and ecology, radoja domanovića 14, 34000 kragujevac, serbia * e-mail: branko_jotic@live.com abstract: jotić, b., marković, m., petrović, b., fusijanović, i., pavlović, d., ranđelović, v.: the vascular flora of the vučje hill near pirot city (eastern serbia). biologica nyssana, 2 (2), december 2011: 91-106. as a result of the floristic investigations of the vučje hill carried out betwenn 2008 and 2010, 534 plant taxa belonging to 277 genera and 67 plant families were recorded. phytogeographical analysis showed that the species of eurasian area type are the most abundant ones. there are 10 endemic taxa in flora of investigated area. the hemicriptophytic character of flora with significant contribution of therophytes and geophytes, is established by analyzing the presence of plant life forms. 31 species are listed as protected or strictly protected in national legislature, while some of them are of international importance for conservation. key words: endemic, flora, pirot city, vučje hill introduction ! the vučje hill (1188 m) is situated in eastern serbia, 8 km se of pirot city, above the krupac village (fig. 1). this is the part of mt. vidlič, westernmost part of the mountain system stara planina. the hill is elongated in the southeasternnorthwestern direction. in contrast to other regions of mountain system stara planina, mt. vidlič are represented by unique geological structure and climate conditions. the geological structure of the most part of mt. vidlič is represented by limestone, while other serbian parts of stara planina are built of silicate (v i d a n o v i ć , ed., 1960). the pedological substrate is consisted of reddish soil tiller and mountain soil. the climate of this area is transitional humid continental, between dry climate of pirot region and wet climate on mt. stara planina (v i d a n o v i ć , ed., 1960). although the flora of this part of mt. stara planina was investigated by p a n č i ć (1884) and a d a m o v i ć (1911), it has not been well known. the vučje hill flora is not investigated up to now. materials and methods the flora of vučje hill were studied in the period between 2008 and 2010. herbarium specimens are deposited at the herbarium moesiacum (hmn). determination of plant material was carried out using the keys from the regional flora reference (j o s i f o v i ć , ed. 1970-1977; v e l č e v , ed. 19821989). the nomenclature follows med-checklist (greuter et al., 1984-1989) and flora europaea (t u t i n et al., eds., 1964-1980). 2 (2) • december 2011: 91-106 biologica nyssana 2 (2) december 2011: 91-106 jotić b. et al. the vascular flora of the vučje hill… 92 for the area types classification m e u s e l et al. (1965, 1978), m e u s e l and j ä g e r (1992) and s t e v a n o v i ć (1992) were used. life forms were determined according to m u e l l e r d o m b o i s and e l l e n b e r g (1974) and s t e v a n o v i ć (1992a). results as a result of the floristic investigations of vučje hill in eastern serbia, 534 plant taxa belonging to 277 genera and 67 families were recorded (tab. 1). figure 1. geographical position of investigated area table 1. list of vascular flora with life forms and area-types taxa life form area type polypodiopsida aspleniaceae asplenium ceterach l. semp ch herb caesp me asplenium ruta-muraria l semp ch herb caesp hol asplenium trichomanes l. semp ch herb semiros csm dryopteridaceae dryopteris filix-mas (l.) schoot a meg g rhiz hol hypolepidaceae pteridium aquilinum (l.) kuhn a meg-alt g rhiz csm polypodiaceae polypodium vulgare l. mac fo semp g rhiz hol woodsiaceae cystopteris fragilis (l.) bernh. fo dec ch herb semiros csm biologica nyssana 2 (2) december 2011: 91-106 jotić b. et al. the vascular flora of the vučje hill… 93 taxa life form area type pinopsida cupressaceae juniperus communis l. a semp np scap hol magnoliopsida acanthaceae acanthus balcanicus heywood & i.b.k.richardson a meg-alt h scap semiros eas aceraceae acer campestre l. fo dec mes p scap eas acer hyrcanum fischer & c. a. meyer subsp. intermedium (pančić) bornm. fo dec mes p scap me acer monspessulanum l. fo dec mi-mes p scap med-smed apiaceae aegopodium podagraria l. v-a mes-meg h scap/g rhiz eas angelica sylvestris l. a mac-alt h scap bor bupleurum flavicans boiss. & heldr. a mi-mes t scap med-smed bupleurum praealtum l. v-a mi-mes t scap med-smed bupleurum veronense turra a mes t scap eas caucalis platycarpos l. v-a mes t scap eas chaerophyllum aureum l. a mes-alt h scap eas daucus carota l. a meg t/h scap hol eryngium campestre l. v-a mes-meg h semiros eas eryngium palmatum pančić et vis. a mes-meg h scap semiros med-smed freyera cynapoides (guss.) griseb. v-a mes-mac g bulb eamt orlaya grandiflora (l.) hoffm. a mes t scap eas scandix pectin-veneris l. v-a mes t scap med-smed smyrnium perfoliatum l. a mes-meg h scap med-smed torilis arvensis (hudson) link a meg t scap csm torilis heterophylla guss. a mes t scap med-smed torilis leptophylla (l.) reichenb. a mes t scap eas apocynaceae vinca herbacea waldst. et kit. v-a mi-mes ch suff rept p araliaceae hedera helix l. aut fo semp alt s lig me aristolochiaceae aristolochia clematitis l. v-a mes-meg g rad eas asarum europaeum l. fo semp ch herb rept eas asclepiadaceae vincetoxicum hirudinaria medicus a mes-alt h scap eas compositae (asteraceae) achillea clypeolata sibth. & sm. a mes-meg h scap med-smed achillea crithmifolia waldst. et kit. a mes-meg h scap msm achillea millefolium l. a meg h scap hol achillea pannonica scheele a meg h scap hol ambrosia artemisiifolia l. a-aut mes t scap adv anthemis arvensis l. a mes-mac t scap med-smed anthemis ruthenica bieb. a mes-meg t scap medsmed artemisia alba turra aut meg fo dec ch suffr caesp eamt artemisia scoparia waldst. et kit. a-aut meg t scap bienn eas artemisia vulgaris l. aut meg-alt h scap hol bellis perennis l. v-a mi-mes h ros eas biologica nyssana 2 (2) december 2011: 91-106 jotić b. et al. the vascular flora of the vučje hill… 94 taxa life form area type carduus acanthoides l. a mac h scap me carduus candicans waldst. et kit. subsp. globifer (velen.) kazmi a mes-meg h scap msm carduus nutans l. a mes-meg h scap me carlina vulgaris l. a mes-meg t/h scap bienn eas centaurea biebersteinii dc. a mes-meg h scap eas centaurea calcitrapa l. a mes-meg h caesp eas centaurea cyanus l. v-a mes-mac t scap csm centaurea scabiosa l. a meg h scap eas centaurea stenolepis a. kerner. a mes-meg h scap med-smed centaurea triumfetti all. a mes-meg h scap eamt chamomilla recutita (l.) rauschert a mes-meg t scap csm chondrilla juncea l. a mes-meg t scap bienn med-smed cichorium intybus l. a mac h scap csm cirsium acaule scop. a mes h ros eas cirsium vulgare (savi) ten. a mes-alt t/h scap bienn eas conyza canadensis (l.) cronquist a-aut mes-alt t scap adv crepis biennis l. v-a mac h scap bienn me crepis foetida l. a mes-meg t ros eas crepis sancta (l.) babcock v mi-mes t scap eas crepis setosa haller a mes-alt t scap med-smed crupina vulgaris cass v-a mes t scap eas doronicum columnae ten. v mes-meg h scap/g rhiz eamt erigeron acer l. a mes-meg h scap bienn hol hieracium cymosum l. a mes-meg h scap ros eamt hieracium pilosella l. a mi-mes h ros eas hieracium praealtum vill. ex gochnat subsp. bauhinii (besser) petunnikov a meg-alt h scap me inula britannica l. a mes-meg h scap eas inula conyza dc. a mes-meg h scap eas inula oculus-christi l. a mes-meg h scap p lactuca saligna l. a meg-alt t/h scap bienn eas lactuca serriola l. a-aut mes-mac t scap bienn eas lactuca viminea (l.) presl. a mes-mac h scap bienn eas lapsana communis l. a meg-alt t scap eas leontodon crispus l. a mi-mes h ros med-smed leontodon hispidus l. a mi-mac h ros eas leucanthemum vulgare lam. a mes-meg h scap eas logfia minima (sm.) dumort. a mi-mes t scap eas matricaria perforata mérat a-aut mes-meg t/h scap bienn eas mycelis muralis (l.) dumort. a mes-alt h scap eas onopordum acanthium l. a meg-alt h scap bienn eas picris hieracioides l. a-aut meg-alt h scap bienn eas ptilostemon afer (jacq.) w. greuter a mac-meg h scap bienn med-smed scorzonera cana (c. a. meyer) o.hoffm. a mes-mac h scap eas scorzonera hispanica l. a mac-meg h scap p senecio vernalis waldst. et kit. v mes t scap eas sonchus asper (l.) hill a-aut meg-alt t scap bienn eas sonchus oleraceus l. a meg-alt t/h scap bienn csm taraxacum officinale weber v-aut mes h ros eas tragopogon balcanus velen. a mes-meg h scap bienn med-smed tragopogon pratensis l. a mes-meg h scap eas xanthium spinosum l. a mes-meg t scap adv xeranthemum annuum l. a mes-meg t scap med-smed biologica nyssana 2 (2) december 2011: 91-106 jotić b. et al. the vascular flora of the vučje hill… 95 taxa life form area type boraginaceae anchusa barrelieri (all.) vitman a mac-alt h scap bienn eas anchusa officinalis l. a alt h scap bienn eas asperugo procumbens l. v mi-mes t scap eas buglossoides arvensis (l.) i. m. johnston v-a mes t scap eas buglossoides purpurocaerulea (l.) i. m. johnston v-a mac h scap eas echium italicum l. v-a mac-meg h scap bienn med-smed echium vulgare l. a mac h scap eas myosotis arvensis (l.) hill v-a mi-mes t scap hol myosotis caespitosa f.c.schultz. a mes h caesp bienn hol myosotis sylvatica hoffm. v mes t/h scap ros eas nonea pulla (l.) dc. v-a mes-mac h scap bien eas pulmonaria officinalis l. v mes-mac h scap me symphytum tuberosum l. v mes-mac h scap/g tub msm criciferae (brassicaceae) aethionema saxatile (l.) r. br. a mi-mes t scap med-smed alliaria petiolata (bieb.) cav. & grande v-a mes-meg t/h scap eas alyssum alyssoides (l.) l. v mi-mes t scap eas alyssum montanum l. v mi-mes fo dec ch suffr rept eas alyssum petraeum ard. a mes h scap ros bienn med-smed alyssum repens baumg. v mi-mes fo dec ch suffr p arabidopsis thaliana (l.) heuff. v mi-mes t scap ros eas arabis glabra (l.) bernh. a meg-alt h scap bienn hol arabis hirsuta (l.) scop. a mes-meg h ros bienn csm arabis recta vill. v meg h scap bienn eas arabis sagittata (bertol.) dc. a mes-meg h ros bienn eas arabis turrita l. v-a meg h scap semiros eas berteroa incana (l.) dc. a mes-mac t/h scap eas camelina rumelica velen. a mes t scap-ros med-smed capsella bursa – pastoris (l.) medicus v-aut mi-meg t/h ros bienn csm cardamine bulbifera (l.) crantz v-a mes-mac g rhiz eas cardamine hirsuta l. a mi-mes t scap ros eas cardaria draba (l.) desv. v-a mes-meg h scap eas draba muralis l. v mi-meg t scap csm erophila verna (l.) besser v mi-mes t ros eas erysimum cuspidatum (bieb.) dc. a mac t/h ros bienn p erysimum diffusum ehrh. a mes-meg t/h scap eas erysimum hieracifolium l. a mes h scap bienn me erysimum odoratum ehrh. a meg t scap bienn me lepidium campestre (l.) r. br. a meg t/h scap bienn eas sinapis arvensis l. v-a mes-mac t scap eas sysimbrium orientale l. a meg t scap bienn eas thlaspi arvense l. v mes-mac t scap eas thlaspi kovatsii heuff. a mes-alt h scap p thlaspi perfoliatum l. v mi-mes t scap semiros eas campanulaceae asyneuma canescens (w. et k.) gris. et sch. a mes-meg h scap eas campanula bononiensis l. a mes-meg h scap eas campanula glomerata l. a mes-mac h scap eas campanula persicifolia l. a mes-meg h scap me campanula sparsa friv. a mes-mac t scap me caprifoliaceae lonicera xylosteum l. fo dec mi p caesp eas biologica nyssana 2 (2) december 2011: 91-106 jotić b. et al. the vascular flora of the vučje hill… 96 taxa life form area type sambucus ebulus l. a alt g rad/h scap eas sambucus nigra l. fo dec mi p scap eas viburnum lantana l. fo dec n-mi p caesp eas caryophyllaceae agrostemma githago l. a meg t scap eas arenaria serpyllifolia l. v-a mi-mes t scap eas cerastium brachypetalum desp. a mi-mes t scap eas cerastium glomeratum thuill. v-a mi-mes t scap csm cerastium pumilum curt. a mes h scap eas cerastium semidecandrum l. v mi-mes t scap eas dianthus carthusianorum l. a mes-mac h scap caesp eas dianthus cruentus griseb. a mes-meg h scap caesp eamt herniaria hirsuta l. a mi-mes t/h rept eas herniaria incana lam. a mi h caesp rept eas holosteum umbellatum l. a mi t scap eas lychnis coronaria (l.) desr. a mes-meg h scap eas minuartia verna (l.) hiern. a mi-mes h caesp hol petrorhagia illyrica p.w.ball et heywood a-aut mes h/ch herb caesp med-smed petrorhagia prolifera (l.) p. w. ball & heywood a mes t scap eas petrorhagia saxifraga (l.) link a mes-mac ch caesp eas scleranthus neglectus rochel ex baumg. a mes t caesp eas scleranthus perennis l. subsp. dichotomus schur a mes-mac h scap rept eas silene bupleuroides chater et walters a mes-mac h scap eas silene otites (l.) wiebl a mes-meg h scap eas silene vulgaris (moench.) garcke a mes-meg h scap/g rad eas stellaria holostea l. a mes-mac h scap eas stellaria media (l.) vill. a mes-meg h scap rept csm celastraceae evonymus europaeus l. fo dec n-mi p caesp eas evonymus latifolius (l.) miller fo dec n-mi p caesp eas evonymus verrucosus scop. fo dec n-mi p caesp eas cistaceae fumana procumbens (dunal) gren. & gordon v-a mi-mes ch frut eas helianthemum ledifolium (l.) miller a mi t scap med-smed helianthemum nummularium (l.) miller a mes-meg fo dec ch suffr caesp eas helianthemum salicifolium (l.) miller v mi-mes t scap med-smed convolvulaceae convolvulus arvensis l. a mes-alt s herb eas convolvulus canthabrica l. a mes-meg h scap eas cuscuta approximata bab. t/s par hol cuscuta epithymum (l.) l. t/s par hol cuscuta europaea l. t/s par eas cornaceae cornus mas l. fo dec mi p caesp eas cornus sanguinea l. fo dec mi-mes p caesp me corylaceae carpinus betulus l. fo dec meg-alt p caesp scap eas carpinus orientalis miller fo dec mi-mes p caesp med-smed corylus avellana l. fo dec meg-alt p caesp eas corylus colurna l. fo dec mes p scap med-smed crassulaceae jovibarba heuffelii a. et d. löve a mes fo semp ch herb ros succ p biologica nyssana 2 (2) december 2011: 91-106 jotić b. et al. the vascular flora of the vučje hill… 97 taxa life form area type sedum acre l. a mi-mes ch herb scap succ me sedum album l. a mi-mes ch herb scap succ med-smed sedum annuum l. a n-mi ch herb scap succ aa sedum hispanicum l. a mi t/h succ eas sedum ochroleucum chaix. a mes ch herb succ med-smed sedum sexangulare l. a mi-mes ch herb succ me sedum telephium l. subsp. maximum (l.) krock. a mac h scap succ eas dipsacaceae cephalaria laevigata (waldst. & kit.) schrader a meg-alt ch suffrut med-smed knautia arvensis (l.) coult. v-a mes-alt h scap eas knautia integrifolia (l.) bertol a mes-meg t scap med-smed scabiosa argentea l. a mes-meg h scap p euphorbiaceae euphorbia amygdaloides l. a mes-mac t scap eas euphorbia cyparissias l. v-a mes-mac h scap eas euphorbia esula l. a meg-alt h scap me euphorbia falcata l. a-aut mi-mes t scap eas euphorbia helioscopia l. a mi-meg t scap eas euphorbia nicaeensis all. subsp. glareosa (pallas ex bieb.) a. r. sm. v-a mes h scap eas euphorbia taurinensis all. v-a mes t scap med-smed euphorbia seguierana necker subsp. niciciana (borbás ex novák) rech. fil. a mes-meg h scap eas euphorbia platyphyllos l. a-aut mes-mac t scap eas euphorbia salicifolia host. v-a mes h scap eas leguminosae (fabaceae) anthyllis vulneraria l. a mes-meg h scap eas astragalus glycyphyllos l. a mes-meg h scap rept eas astragalus onobrychis l. a mes fo ch suffrut rept eas chamaecytisus austriacus (l.) link v-a mes-mac fo dec ch suffrut caesp msm chamaecytisus ciliatus (wahlenb.) rothm. v-a mes-mac fo dec ch suffrut caesp msm chamaecytisus glaber (l. fil.) rothm. v-a mes-meg fo dec ch frut caesp eamt chamaecytisus rochelii (wierzb.) rothm. v-a mes-meg fo dec ch frut caesp msm chamaespartium sagittale (l.) p. gibbs a mes fo dec ch suffrut caesp eas coronilla coronata l. v-a mes fo dec ch suffrut caesp eas coronilla emerus l. a fo dec np caesp med-smed coronilla scorpioides (l.) koch v mi-mes t scap med-smed coronilla varia l. a mes-meg h scap eas cytisus procumbens (waldst. & kit. ex willd.) sprengel a mes fo dec ch suffrut caesp med-smed dorycnium pentaphyllum scop. subsp. germanicum (gremli) gams a mes fo dec ch suffrut caesp msm genista ovata waldst. et kit. a mes-mac fo dec ch suffrut caesp eas lathyrus aphaca l. a mes t/s herb eas lathyrus cicera l. a mes-mac t scap med-smed lathyrus inconspicuus l. a mes t scap med-smed lathyrus nissolia l. a mes-meg t scap eas lathyrus pallescens (bieb.) c. koch. a mes-mac t scap p lathyrus pratensis l. a mes-meg h scap eas lathyrus setifolius l. a mes-mac t rept med-smed lathyrus venetus (mill.) wohlf. a mes-meg h scap p lotus corniculatus l. a mes h scap hol medicago lupulina l. a mes t/h scap hol biologica nyssana 2 (2) december 2011: 91-106 jotić b. et al. the vascular flora of the vučje hill… 98 taxa life form area type medicago minima (l.) bartal v mi-mes t scap eas medicago rigidula (l.) all. v mi-mes t scap eas medicago sativa l. a mes-meg h scap adv medicago sativa l. subsp. falcata (l.) arcangeli a mes-meg h scap eas melilotus officinalis (l.) pallas a meg t/h scap bienn eas onobrychis alba (waldst. et kit.) desv. a mes-meg h scap med-smed onobrychis arenaria (kit.) dc. v-a mes-meg h scap eas onobrychis ocellata g. beck. a mes h scap med-smed onobrychis viciifolia scop. a mes-meg h scap med-smed onоnis pusilla l. a-aut mi-mes fo dec ch suffrut caesp eas ononis spinosa l. a mes-meg fo dec ch suffrut caesp eas oxytropis pilosa (l.) dc. a mes h scap eas trifolium alpestre l. a mes-mac h scap eas trifolium arvense l. a mes t/h scap bienn eas trifolium badium schreber a mes-meg h scap eamt trifolium campestre schreber a mes-mac t scap eas trifolium incarnatum l. a mes-meg t scap eas trifolium medium l. v-a mes-mac h scap eas trifolium montanum l. a mes h scap eas trifolium pratense l. a mes h scap eas trifolium repens l. a mi h rept hol trifolium scabrum l. a mi-mes t scap med-smed trifolium striatum l. a mes t scap eas vicia cracca l. a meg-alt h/s scap herb eas vicia hirsuta (l.) s. f. gray a mes-meg t/s scap herb eas vicia lathyroides l. v-a mes-meg t scap eas vicia pannonica crantz v-a mes t/s scap herb p vicia sativa l. subsp. nigra (l.) ehrh. v-a mes-meg t scap bienn eas vicia tenuifolia roth. a mac-alt h/s scap herb eas vicia tetrasperma (l.) schreber v-a mes t/s herb eas fagaceae fagus moesiaca (k. maly) czecz. fo dec meg-alt p scap me quercus cerris l. fo dec mes p scap med-smed quercus petraea (mattuschka) liebl. fo dec meg-alt p scap me quercus pubescens willd. fo dec mes p scap med-smed fumariaceae corydalis solida l. v mes g tub me fumaria officinalis l. a mes t scap eas fumaria vaillantii loisel. a mi-mes t scap eas gentianaceae gentiana cruciata l. a mes-meg g rad scap eas geraniaceae erodium ciconium (l.) l’her. v-a mi-mes t scap msm erodium cicutarium (l.) ľ herit. v-a mi-mes t ros csm geranium columbinum l. a mes-meg t scap eas geranium dissectum l. a mi-meg t scap eas geranium molle l. v mes t scap eas geranium rotundifolium l. a mi-meg t caesp eas geranium sanguineum l. v-a mes h semiros eas globulariaceae globularia punctata lapeyr. v-a mes h scap eas biologica nyssana 2 (2) december 2011: 91-106 jotić b. et al. the vascular flora of the vučje hill… 99 taxa life form area type grossulariaceae ribes alpinum l. fo dec mi p caesp scap eas hypericaceae hypericum barbatum jacq. a mes-mac h scap eas hypericum montanum l. a mes-meg h scap me hypericum perforatum l. a mes-meg h scap eas hypericum rumelicum boiss. a mi-mes h scap med-smed labiatae (lamiaceae) acinos alpinus (l.) moench a mes t scap/g rhiz eas acinos arvensis (lam.) dandy a mi-mes t scap/g rhiz eas ajuga chamaepytus (l.) shreber subsp. chia (schreber) arcangeli a mi-mes t scap med-smed ajuga genevensis l. v-a mes-mac h scap ros eas ajuga laxmannii (l.) bentham v-a mes h scap/g rhiz p ajuga reptans l. a mes h rept eas ballota nigra l. a meg h scap eas clinopodium vulgare l. a mes-meg h scap hol glechoma hirsuta waldst. et kit. a mes h rept eas hyssopus officinalis l. aut mes-meg ch frut eas lamiastrum galeobdolon (l.) ehrend. & polatschek v-a mes-mac h scap me lamium amplexicaule l. v mi-mes t scap hol lamium bifidum cyr. v-a mi-mes t scap med-smed lamium maculatum l. v mes-mac h scap eas lamium purpureum l. v mi-mes t scap eas marrubium peregrinum l. a mes-meg h scap eas marrubium vulgare l. a meg h scap eas mentha longifolia (l.) hudson a mes-meg h scap hol nepeta nuda l. a mac-meg h scap eas origanum vulgare l. a mes-meg h scap eas prunella laciniata (l.) l. a mes-mac h scap eas prunella vulgaris l. a mi-mes h scap semiros eas salvia aethiopis l. a mes-mac h scap p salvia austriaca jacq. a mes-mac h scap p salvia nemorosa l. a mes-meg h scap eas satureja montana l. subsp. kitaibelii (wierzb.) p.w. ball a mes ch suffrut caesp eamt sideritis montana l. a mi-mes t scap eas stachys germanica l. a meg h scap msm stachys recta l. a mes-meg h scap eas teucrium chamaedrys l. a mes ch suffrut caesp eas teucrium montanum l. a mes ch suffrut caesp eamt thymus glabrescens willd. a mi-mac ch herb rept eas thymus pannonicus all. a mi-mac ch rept me thymus praecox opiz subsp. jankae (čelak.) jalas a mi ch herb rept pulv med-smed thymus pulegioides l. a mi-mes ch herb rept me ziziphora capitata l. a mi-mes t scap med-smed linaceae linum hologynum reichenb. a mes-mac h caesp med-smed linum tenuifolium l. a mesmac h scap eas malvaceae althaea hirsuta l. a mes t scap eas althaea officinalis l. a meg h scap bienn eas biologica nyssana 2 (2) december 2011: 91-106 jotić b. et al. the vascular flora of the vučje hill… 100 taxa life form area type malva neglecta wallr. v-a mes-mac t/h scap eas malva sylvestris l. v-a mes-alt h scap eas oleaceae fraxinus ornus l. fo dec mi-mes p scap eas ligustrum vulgare l. fo dec mi p caesp eas syringa vulgaris l. fo dec mi p caesp med-smed orobanchaceae orobanche gracilis sm. a mes-mac ep par g eas orobanche nana (reuter) noë ex g. beck a mi-mes ep par g p papaveraceae papaver dubium l. a meg t scap eas papaver rhoeas l. a mes-meg t scap eas plantaginaceae plantago argentea chaix a mi-meg h ros eamt plantago lanceolata l. a mi-meg h ros eas plantago maior l. a mes-meg h ros csm plantago media l. a mes-meg h ros eas polygalaceae polygala comosa schkuhr a mi-mes h scap eas polygala vulgaris l. a mi-mes h scap eas polygonaceae fallopia convolvulus (l.) á. löve a-aut mes t/s rept-herb eas rumex patientia l. a mes-alt h scap semiros eas rumex sanguineus l. a meg h scap hol primulaceae anagallis arvensis l. v-a mi-mes t scap csm primula veris l. v mi-mes h ros eas ranunculaceae adonis flammea jacq. a mes t scap p adonis vernalis l. v mes-meg g rhiz p anemone nemorosa l. v mi-mes g rhiz hol anemone ranunculoides l. v mi-mes g rhiz me clematis vitalba l. a dec s lig eas consolida regalis s. f. gray a mes-meg t scap eas delphinium fissum waldst. et kit. a meg-alt g tub med-smed helleborus odorus waldts. & kit. v meg g rhiz me isopyrum thalictroides l. v mes g rhiz eas nigella arvensis l. a-aut mes t scap eas nigella damascena l. a mes t scap med-smed pulsatilla montana (hoppe) reichenb. subsp. bulgarica rummelspecher v mes h scap semiros eas ranunculus bulbosus l. a mes-meg h scap eas ranunculus ficaria l. v mi-mes g tub me ranunculus millefoliatus wahl. v-a mes h scap semiros med-smed ranunculus polyanthemos l. a meg h scap semiros eas ranunculus sardous crantz a mes-meg t scap eas thalictrum aquilegifolium l. a meg-alt h scap me resedaceae reseda lutea l. a mes-meg t/h scap bienn eas biologica nyssana 2 (2) december 2011: 91-106 jotić b. et al. the vascular flora of the vučje hill… 101 taxa life form area type rosaceae agrimonia eupatoria ledeb. a meg h scap eas aremonia agrimonioides (l.) neck. v mi-mes h scap ros eamt cotoneaster integerrimus medicus a mac ch frut rept me crataegus monogyna jacq. fo dec mi p caesp eas filipendula vulgaris moench a mes-meg h scap eas fragaria moschata duchesne a mes h rept ros eas fragaria vesca l. a mes h rept csm fragaria viridis duchesne a mes h rept eas geum urbanum l. a mes-meg h scap hol malus pumila miller fo dec mi-mes p scap p malus sylvestris miller fo dec mi-mes p scap eas potentilla argentea l. a mes-meg h scap me potentilla cinerea chaix ex vill. v mi-mes h caesp rept eas potentilla micrantha ramond ex d.c. v mi-mes h scap med-smed potentilla recta l. a mes-meg h scap eas prunus spinosa l. fo dec n p caesp eas prunus tenella batsch fo dec n p caesp eas pyrus amygdaliformis vill. fo dec n p caesp med-smed pyrus pyraster burgsd. fo dec mes p scap eas rosa canina l. fo dec n p caesp eas rosa micrantha borrer ex sm. fo dec n p caesp eas rosa pimpinellifolia l. fo dec n p caesp eas sanguisorba minor scop. a mes-meg h scap eas sorbus torminalis (l.) crantz fo dec mes p scap eas rubiaceae asperula aristata l. subsp. scabra (j. & c. presl) nyman a mes-meg h scap me asperula cynanchica l. v-a mes-mac h scap eas crucianella angustifolia l. a mi-mes t scap med-smed cruciata glabra (l.) ehrend. v-a mi-mes h scap eas cruciata laevipes opiz v-a mes-mac h scap eas cruciata pedemontana (bellardi) ehrend. v-a mes-meg h scap med-smed galium album miller a meg-alt h/s scap herb me galium aparine l. v-a mes-meg t/s herb csm galium verum l. a mes-meg h scap eas sherardia arvensis l. v-a mi-mes t scap med-smed rutaceae dictamnus albus l. v-a mes-alt ch suffrut caesp eas salicaceae salix purpurea l. fo dec mi p caesp eas santalaceae thesium alpinum l. a mes-mac h scap eamt thesium arvense horvatovszky a mi-mes h scap eas thesium divaricatum jan ex mert. & koch a mes h scap med-smed scrophullariaceae chaenorhinum minus (l.) lange a-aut mi-mes t scap eas digitalis lanata ehrh. a mac-alt h scap me euphrasia pectinata ten. a mi-mes t scap/ep semipar eas kickxia spuria (l.) dumort a-aut mes t rept eas lathraea squamarria l. a mes-meg g par eas linaria angustissima (lois.) borb. a mes h/g scap rhiz med-smed biologica nyssana 2 (2) december 2011: 91-106 jotić b. et al. the vascular flora of the vučje hill… 102 taxa life form area type linaria genistifolia (l.) mill. a mes-mac h scap eas linaria genistifolia (l.) miller subsp. dalmatica (l.) maire & petitmengin a meg-alt h scap eas linaria rubioides vis. & pančić subsp. nissana (petrović) niketić & tomović a mes h scap med-smed linaria vulgaris l. a mes-mac h scap eas rhinanthus rumelicus velen. a mi-mac t scap/ep semipar eas scrophularia canina l. a mes-mac h scap med-smed verbascum lychnitis l. a mac-alt t/h scap ros bienn eas verbascum phlomoides l. v-a mes-alt h ros eas verbascum phoeniceum l. v-a mac-meg h ros eas veronica austriaca l. a mes-mac h scap eas veronica chamaedrys l. v-a mi-mes h scap eas veronica hederifolia l. v mi-mes t scap eas veronica persica poiret v-aut mi-mes t scap adv veronica praecox all. v mi-mes t scap bienn eas veronica verna l. v-a mi-mes t scap bienn me solanaceae hyascyamus niger l. v-a mes-meg t scap bienn eas tiliaceae tilia platyphyllos scop. fo dec mes p scap me ulmaceae ulmus glabra hudson fo dec mes p scap me ulmus minor mill. fo dec mes p scap med-smed ulmus procera salisb. fo dec mes p scap me urticaceae urtica dioica l. a mes-meg t/h scap hol valerianaceae valeriana officinalis l. a meg-alt h scap eas valerianella coronata (l.) dc. v mi-mes t scap med-smed valerianella dentata (l.) pollich v mi-mes t scap eas valerianella locusta (l.) laterrade v mes t scap csm valerianella rimosa bast. v mes t scap eas verbenaceae verbena officinalis l. a mes-meg h scap eas violaceae viola alba besser v mi-mes h rept semiros eas viola arvensis murray v-aut mi-mac t scap csm viola canina l. v mi-mes h scap semiros hol viola hirta murray v mi-mes h scap me viola jordanii hanry v mes h scap ros p viola kitaibeliana schultes v n-mi t scap eas viola odorata l. v mi-mes h rept ros eas viola tricolor l. subsp. macedonica (boiss. & heldr.) a. schmidt v mi-mes t/h scap med-smed liliopsida araceae arum maculatum l. v mes-meg g rhiz me cyperaceae carex caryophyllea latourr. a mes-meg h caesp eas biologica nyssana 2 (2) december 2011: 91-106 jotić b. et al. the vascular flora of the vučje hill… 103 taxa life form area type carex divulsa stokes v-a mes-mac h caesp hol carex halleriana asso v mes-mac h caesp med-smed carex humilis leysser a mi-mes h caesp eas carex pilosa scop. v-a mes-meg h caesp eas dioscoreaceae tamus communis l. v alt s/g herb rhiz med-smed iridaceae crocus adami gay v mi g bulb msm liliaceae allium albidum fischer ex bieb. a mes g bulb p allium cupani rafin a mes g bulb med-smed allium flavum l. a mes-mac g bulb msm allium moschatum l. a mes g bulb eas allium scorodoprasum (l.) subsp. rotundum (l.) stearn a mes-meg g bulb eas allium sphaerocephalon l a mes-meg g bulb eas anthericum ramosum l. a mes-meg g rhiz eas asparagus officinalis l. a meg-alt g rhiz p asparagus tenuifolius lam. a meg-alt g rhiz eas colchicum autumnale l. aut mes g bulb eas erythronium dens – canis l. v mes g bulb eas gagea lutea (l.) ker-gawler v mes g bulb eas gagea arvensis (pers.) dumort. v mi-mes g bulb eas gagea minima (l.) ker-gawler v mi g bulb eas gagea pratensis (pers.) dumort. a mi-mes g bulb eas gagea pusilla (f. w. schmidt) schultes & schultes fil v mi g bulb eas hyacinthella leucophaea (c. koch) schur a mi-mes g bulb eas muscari comosum (l.) miller v mes g bulb eas muscari neglectum guss. ex ten. v mes g bulb eas muscari tenuiflorum tausch v mes g bulb p ornithogalum collinum guss. v-a mi-mes g bulb eas ornithogalum ortophyllum ten. subsp. kochii (parl.) zahar. v mi-mes g bulb eas ornithogalum pyramidale l. v mi-mes g bulb eas ornithogalum umbellatum l. v-a mes g bulb eas polygonatum odoratum (miller) druce a mes-meg g rhiz eas scilla bifolia l. v mes g bulb eas veratrum nigrum l. a meg-alt g rhiz eas orchidaceae listera ovata (l.) r. br. a mes g tub scap eas ophrys apifera hudson a mes g tub scap eas ophrys cornuta steven a mes g tub scap eas orchis morio l. v-a mes g tub scap eas orchis pallens l v mes g tub scap eas poaceae aegilops geniculata roth. a mi-mes t caesp med-smed agropyron cristatum (l.) gaertne a mes-meg g rhiz caesp eas agrostis capillaris l a mes-meg h caesp hol alopecurus pratensis l. a mes-meg h caesp eas antoxanthum odoratum l. a mes-meg h caesp eas avenula pubescens (hudson) dumort. a mes-alt h caesp eas biologica nyssana 2 (2) december 2011: 91-106 jotić b. et al. the vascular flora of the vučje hill… 104 taxa life form area type brachypodium pinnatum (l.) beauv. a mac-alt h caesp eas brachypodium sylvaticum (hudson) beauv. a mac-alt h caesp eas briza media l. a mes-meg h caesp me bromus commutatus schrader a meg t scap msm bromus hordeaceus l. a mes-meg t caesp eas bromus ramosus hudson a meg-alt h caesp eas bromus squarosus l. a mes-mac t caesp bienn med-smed chrysopogon gryllus (l.) trin. a alt h caesp eas cleistogenes serotina (l.) keng a mes-mac h caesp med-smed dactylis glomerata l. a meg h caesp eas dasypyrum villosumr. br. ex fresen. a mes-meg t caesp med-smed dichanthium ischaemum (l.)roberty a mes-meg h caesp eas elymus repens (l.) gould a mes-meg g rhiz caesp csm festuca pratensis huds. a mac-meg h caesp eas festuca rubra l. a meg h caesp hol festuca valesiaca schleicher ex gaudin a mes-mac h caesp eas holcus mollis l. a mes-mac h caesp eas hordeum murinum l. subsp. leporinum (link) arcangeli a mes t caesp med-smed koeleria eryostachya pančić a meg h caesp me koeleria glauca (schreder) dc. a mes-meg h caesp eas koeleria glaucovirens domin a mes-meg h caesp med-smed koeleria macrantha (ledeb.) schultes a mes-mac h caesp hol koeleria nitidula velen. a mes-meg h caesp med-smed lolium perenne l. a mes-mac h caesp eas melica ciliata l. a mes-meg h caesp eas melica transsilvanica schur a mes-meg h caesp p melica uniflora retz. a mes-mac h caesp me phleum pratense l. a mes-alt h caesp me poa annua l. a-aut mi-mes t/h caesp csm poa bulbosa l. a mes-meg h caesp eas poa compressa l a mes-mac h caesp csm poa nemoralis l. a mes-meg h caesp hol poa pratensis l. a mes-meg h scap eas sesleria latifolia (adamović) degen a mes-meg h caesp eamt sesleria rigida heuffel ex reichenb. a mes-meg h caesp eamt setaria viridis (l.) beauv. a mes-mac h caesp eas sorghum halepense (l.) pers. a-aut meg-alt g rhiz caesp adv stipa capillata l. a mac-meg h caesp eas stipa pulcherrima c. koch. a mac-alt h caesp eas tragus racemosus (l.) all. a mi-mes t caesp-rept csm vulpia myuros (l.) c. c. gmelin a mes-meg t caesp csm disscusion taxonomic analysis divisio pteridophyta was represented by single class polypodiopsida with only 7 species. pinophyta was represented with single class pinopsida and just one species. divisio magnoliophyta was represented by 526 species, from which 439 are classified as dicotyledons (magnoliopsida) and 87 as monocotyledons (liliopsida). in taxonomic spectrum of the flora of investigated area the most numerous in genera were compositae (36), gramineae (29), labiatae (20), criciferae (17) and leguminosae (17). families with the most number of species were compositae (63), leguminosae (55), gremineae (47) and labiatae (36). the analysis of the particular genera occurrence in flora of the vučje hill has shown biologica nyssana 2 (2) december 2011: 91-106 jotić b. et al. the vascular flora of the vučje hill… 105 domination of representatives of genera trifolium (11), euphorbia (10), lathyrus (8), viola (8), vicia (7) sedum (7), following by the genuses centaurea, veronica and allium with 6 species in each genus. phytogeographical analisys for the phytogeographical analysis, all plant taxa were classified into 10 area types (fig. 2) according to the classification by s t e v a n o v i ć (1992). eurasian area type (eas) was the most dominant in the flora of the vučje hill with 303 taxa (56.7%). the second one was mediterraneansubmediterranean area type (med) with 75 species (7.9%). me 8% med 14% p 4% hol 6% csm 5% adv 1% eam 3% eas 57% msm 2% ab 0% figure 2. area spectrum of the flora of vučje hill: csm – cosmopolitan, hol – holarctic, ab – arctoalpian + boreal, me – middle-european, eas – euroasian, eamt – euroasian mountain, p – pontic, msm – merridional-submerridional, med – mediterranean-submediterranean, adv adventitious there were 10 balcan endemic taxa in the flora of the vučje hill. these represents 3,48% of the total number of balkan endemics in serbian flora, which includes 287 taxa in rank of species and subspecies (s t e v a n o v i ć et al., 1999). small number of endemic floral elements are expected due to uniformity of habitats in the wider area of vučje. the recorded balkan endemics are: acanthus balcanicus, acer hyrcanum subsp. intermedium, bupleurum flavicans, eryngium palmatum, hypericum rumeliacum, thymus praecox subsp. jankae, pulsatilla montana subsp. bulgarica, linaria rubioides subsp. nissana, viola tricolor subsp. macedonica and sesleria latifolia. biological spectrum the analysis of plant life forms (fig. 3) showed that flora of investigated area was of hemicryptophyta type, as well as the flora of serbia (d i k l i ć , 1984) and balkan peninsula (turill, 1929). the hemicryptophytes were represented with 45%; theropytes followed with 26,9% and geophytes with 11,1%. the climatics characteristics of this area caused the high percentage of hemicryptophyta. h 45% g 11% t 27% s 3% p 7% ch 7% figure 3. biological spectrum of the flora of vučje hill: p – phanerophyta, ch – chamaephyta, h – hemycryptophyta, g – geophyta, t – terophyta, s scandetophyta threatened status even the flora of the vučje hill is diverse and rich, consisting of 526 plant taxa, 63 representatives are considered to be threatened in flora of serbia. in the flora of the vučje hill were registered 9 strictly protected species: acer hyrcanum subsp. intermedium, vinca herbacea, sideritis montana, adonis vernalis, pulsatilla montana subsp. bulgarica, gagea minima, ophrys apifera, orchis pallens and stipa pulcherrima (anonimous, 2010). also, in the flora of the vučje hill were registered 22 protected species, such as: achillea clypeolata, corylus colurna, allium albidum, lilium martagon, listera ovata, prunus tenella, ajuga laxmanii, salvia austriaca and other (anonimous, 2010). in the flora of the study area registered 33 species, that are covered by regulation on collecting, use and sale, such as: hedera helix, asarum europaeum, achillea millefolium, pulmonaria officinalis, primula veris, rosa canina, teucrium chamaedrys and other (anonimous, 2010a). references adamović, l., 1911: flora jugoistočne srbije. jazu. zagreb. anonimous, 2010: službeni glаsnik republike srbije br. 5/10, prаvilnik o proglаšenju i zаštiti biologica nyssana 2 (2) december 2011: 91-106 jotić b. et al. the vascular flora of the vučje hill… 106 strogo zаštićenih i zаštićenih divljih vrstа biljаkа, životinjа i gljivа anonimous, 2010a: službeni glаsnik republike srbije br. 31/05, 45/05-isprаvkа, 22/07, 38/08 i 9/10, uredbа o stаvljаnju pod kontrolu korišćenjа i prometа divlje flore i fаune ćirić, j., živković, v., 1974: krupac. geografija, istorija, društveni život, narodnooslobodilački rat. izdanje muzeja ponišavlja – pirot i balkanološkog instituta univerziteta – niš. diklić, n., 1984: životne forme biljnih vrsta i biološki spektar flore sr srbije. in: sarić, m. (ed.): vegetacija sr srbije, i: 291-316. sanu, posebna izdanja. beograd. greuter, w., burdet, h.m., long, g., eds., 19841989: med-checklist, 1, 3, 4. gèneve. javorka, s., csapody, v., 1991: iconographia florae partis austro-orientalis europe centralis. akademia kiado. budapest. josifović, m., ed., 1970-1976: flora sr srbije, i-ix. sanu. beograd. marinkov, j., 1999: zabrđe. prirodno – privredni potencijali. narodna biblioteka dimitrovgrad. dimitrovgrad. meussel, h., jäger, e., weinert, e., 1965: vergleinchende chorologie der zentraleuropaischen flora. veb. gustav fischer verlag, 1. jena. meussel, h., jäger, e., raischert, s., weinert, e., 1978: vergleinchende chorologie der zentraleuropaischen flora. veb. gustav fischer verlag, 2. jena. meusel, h., jäger, e., 1992: vergleinchende chorologie der zentraleuropaischen flora. veb. gustav fischer verlag, 3. jena. mueller – dombois, d., ellenberg, h., 1974: aims and methods of vegetation ecology. john wiley and sons. new york. pančić, j., 1884: dodatak flori kneževine srbije. kraljevska srpska državna štamparija, beograd. stevanović, v., ed., 1999: crvena knjiga flore srbije. 1, iščezli i krajnje ugroženi taksoni. ministarstvo za životnu sredinu republike srbije, zavod za zaštitu prirode srbije. beograd. stevanović, v., 1992: floristička podela teritorije srbije sa pregledom viših horiona i odgovarajućih flornih elemenata. in sarić, m. (ed.): flora srbije, i. (drugo izdanje). sanu. beograd. 49 – 65. stevanović, v., 1992a: klasifikacija životnih formi flore srbije. in sarić, m. (ed.): flora srbije i. (drugo izdanje). sanu. beograd. 39 – 46. turill, w.b., 1929: the plant life of the balkan peninsula. a phytogeographical study. oxford. tutin, t.g., heywood, v.h., burges, n.a., moore, d.m., valentine, d.h., walters, s.m., webb, d.a., eds., 1964-1980: flora europaea, i-v. cambridge university press. london. vidanović, g., 1960: vidlič – zabrđe, prilog poznavanju privrednog tipa, ekonomsko – geografska studija, srpska akademija nauka, posebna izdanja geografskog instituta, knjiga 15, beograd. frequency of isolation and antibiotic resistance patterns of bacterial isolates from wound infections biologica nyssana 7 (2)  december 2016: 151-158 stojanović-radić, z. et al.  frequency of isolation and antibiotic… 151 original article received: 03 october 2016 revised: 20 november 2016 accepted: 29 november 2016 frequency of isolation and antibiotic resistance patterns of bacterial isolates from wound infections zorica stojanović-radić*, marina dimitrijević, nikola stanković, ana aleksić, milica pejčić department of biology and ecology, faculty of science and mathematics, university of niš * e-mail: zstojanovic@pmf.ni.ac.rs abstract: stojanović-radić, z., dimitrijević, m., stanković, n., aleksić, a., pejčić, m.: frequency of isolation and antibiotic resistance patterns of bacterial isolates from wound infections. biologica nyssana, 7 (2), december 2016: 151-158. six hundred and thirteen bacterial strains were isolated from wound swabs and the isolates were identified on the basis of growth on differential and selective media. in order to test the sensitivity of isolated strains to different antibiotics, the disc diffusion method, according to eucast protocol v 5.0 was used. the most common species isolated from wound swabs was staphylococcus epidermidis (18.4%), followed by staphylococcus aureus, pseudomonas aeruginosa and enterococcus faecalis (16.8%, 12.7% and 10.4%, respectively). the maximum resistance of gram-positive cocci was observed to penicillin and the lowest to linezolid. gram-negative bacteria showed the highest resistance to tetracyclines, while the same strains demonstrated the highest sensitivity to polypeptide antibiotics. comparison of the resistance patterns of gramnegative and gram-positive bacterial strains showed significant difference in the tetracycline efficiency. key words: skin infections, wound swab, antibiotic resistance, s. epidermidis, s. aureus, p. aeruginosa apstrakt: stojanović-radić, z., dimitrijević, m., stanković, n., aleksić, a., pejčić, m.: učestalost i rezistencija bakterijskih izolata iz infekcija rana. biologica nyssana, 7 (2), decembar 2016: 151-158. mikrobiološkom analizom uzoraka, izolovano je 613 bakterijskih sojeva iz briseva rana. izolati su identifikovani na osnovu rasta na diferencijalnim i selektivnim podlogama. ispitivanje osetljivosti izolovanih sojeva na različite antibiotike rađeno je metodom disk difuzije po eucast v 5.0 protokolu. najčešće izolovana bakterija iz briseva rana bila je staphylococcus epidermidis (18.4%), nakon koje su najučestalije izolovane bile staphylococcus aureus, pseudomonas aeruginosa i enterococcus faecalis. najveća rezistencija gram-pozitivnih koka primećena je na peniciline a najmanja na linezolid. gram-negativne bakterije su pokazale najveću rezistenciju na tetracikline, dok su isti sojevi ispoljili najveću osetljivost na antibiotike iz klase polipeptidnih antibiotika. poređenjem obrazaca rezistencije gram-negativnih i gram-pozitivnih bakterijskih sojeva utvrđena je značajna razlika u efikasnosti primene tetraciklina. key words: infekcije kože, bris rane, antibiotska rezistencija, s. epidermidis, s. aureus, p. aeruginosa 7 (2) • december 2016: 151-158 12th sfses • 16-19 june 2016, kopaonik mt doi: 10.5281/zenodo.200414 biologica nyssana 7 (2)  december 2016: 151-158 stojanović-radić, z. et al.  frequency of isolation and antibiotic… 152 introduction loss of skin integrity (trauma) caused by mechanical, biological or chemical agent provides a suitable environment for infectious agent entrance, colonization and consequenting acute/chronic infection (giacometti et al., 2000; macedo & santos, 2005). wound infections are very frequent in outpatients and clinical patients, where they constitute a major healing barrier, which affects patient's quality of life. such infected wounds are the cause of large patient's physical and mental discomfort owing to associated pain, hypersensitivity and accompanying unpleasant odour (kotz et al., 2009). the most prevalent organisms that have been associated with acute and chronic wound infections are staphylococcus aureus (including mrsa – methycillin resistant staphylococcus aureus), pseudomonas aeruginosa and members of family enterobacteriaceae, such as escherichia coli, proteus vulgaris, klebsiella sp., yeast candida albicans and others (akinkunmi et al., 2014; bessa et al., 2015). emerging occurence and spread of (multi)drug resistant microbial pathogens present a significant challenge in providing an effective health care and, therefore, a worldwide medical issue. extensive use of antimicrobial agents in the treatment of commonly isolated pathogens results with emergence of their increased resistance to these drugs. also, prescribing of broad spectrum antibiotics for uncomplicated infections presents one of the most responsible approaches for emerging of resistance (mcquiston haslund et al., 2013). among resistant microbes, those showing multiresistance (mdr – multiple drug resistance) are the main concern in antimicrobial treatment today, owing to accompanying morbidity, mortality and costs (veličković -radovanović et al., 2009). among isolates from wound infections, many of them represent multiresistant microorganisms. literature on this subject showed alarming data, with reported multiresistance of many bacterial species, such as p. aeruginosa, s. aureus (mrsa), as well as species from genera enterococcus and acinetobacter (cetinkaya et al., 2000; rice, 2001; sader et al., 2001; guzmanblanco et al., 2009; moniri et al., 2009). due to the fact that there are differences in recommendations considering the empiric first-choice antibiotic treatment among european countries (mcquiston haslund et al., 2013), diverse reports on isolation frequency and resistance patterns for the same species, but isolated in different countries, are not a surprising fact (sader et al., 2001; shit tu et al., 2002; hiransuthikul et al., 2004; oguntibeju & nwobu, 2004; macedo & santos , 2005; anguzu & olila , 2007; pondei et al., 2013). in serbia, study in aleksinac (stanković nedeljković et al., 2012) investigated frequency of p. aeruginosa isolates from wound infection swabs and reported its high incidence rate of 36.6%. jovanović et al. (2014) investigated isolation frequencies of surgical and traumatic wounds causative agents and confirmed that there are differences in incidences among different clinics (abdominal surgery clinic and orthopedy and traumatology clinic in zemun and belgrade). among isolated bacteria, the most prevalent ones were e. coli (48%), pseudomonas spp. (12%), klebsiella spp. (11%), acinetobacter spp. (7%) in abdominal wounds, while s. aureus (36.4%), pseudomonas spp. (20.1%) and acinetobacter spp. (7.1%) were the most common causative agents of traumatic wound infections. according to the presented facts, periodical surveillance of predominant pathogens and also reporting of their antimicrobial resistance patterns are neccessary actions in each country and its distant regions, whose results will be applied in adjusting therapies to the application of proper and effective antibiotics. therefore, the goal of this study was to investigate local (territory of niš) predominant pathogens, recovered from wound infections during a one year period (january to december, 2015). also, the study included determination of their resistance patterns in order to, based on these results, recommend the best antimicrobial therapy approach for the treatment of wound infections in niš region. material and methods specimen collection the present study has been conducted by analyzing 526 wound swab samples, collected from wound infections of polyclinic human (niš) outpatients. the wound samples were collected using a sterile cotton swab, which were used for gentle swabbing of the inner surface of the infected area, afterwards the swabs were aseptically transported to the laboratory. bacterial samples were collected during a one year period between january and december 2015. a total of 613 bacterial isolates were covered by analysis in order to determine the frequency and resistance patterns of bacterial strains. identification of the pathogenic strains isolation of bacteria from wound swabs was done by inoculating the samples on blood agar. the inoculated media were incubated at 35-37 ºc for 16 to 24 hours. bacterial colonies on the agar plates were transported to a panel of selective and differential biologica nyssana 7 (2)  december 2016: 151-158 stojanović-radić, z. et al.  frequency of isolation and antibiotic… 153 media (himedia, india) in order to complete their identification to the genus or species level, afterwards bacterial isolates were further identified using a battery of biochemical tests. endo and macconkey agar were used for identification of the strains belonging to the family enterobacteriaceae. morphological and cultural characteristics were observed on kligler's double sugar medium and peptone water to demonstrate the indole production. other biochemical tests included citrate and urea utilization, and also fermentation of mannitol according to bergey’s manual of determinative microbiology (2000). susceptibility of isolates to antibiotics susceptibility testing was performed according to the standardized eucast protocol v 5.0. briefly, overnight cultures of isolates on mueller hinton agar incubated at 37 °c for 24 h were used for making bacterial suspensions in 0.85% nacl (w/w) and adjusted to mcfarland 0.5 turbidity standards. prepared suspensions were used for inoculation of the mueller hinton agar plate’s surface, afterwards the antibiotic discs were placed on the inoculated agar surface. after the incubation period of 24 h at 37 °c, the inhibition zones around the discs were measured and interpretation of the inhibition zone values (s-sensitive / r resistant) was based on the eucast v 5.0 criteria. results and discussion the six hundred and thirteen strains were obtained from 526 samples of wound swabs collected during 2015. the most frequently isolated species was s. epidermidis (18.4%), which belongs to the grampositive cocci. two species of gram positive cocci, s. aureus and e. faecalis were also isolated with high frequency, 16.8% and 10.4% respectively. the most common gram-negative bacteria in samples were p. aeruginosa (12.7%) and e. coli (9.0%). the other bacteria were isolated in a relatively low percentage, and citrobacter sp. was the least detected isolate in the swabs (fig. 1). the results of our research indicate no correlation with previous studies on this subject, conducted in other countries, where s. epidermidis, reported here as the most dominant isolate showed relatively low isolation frequency (sader et al., 2001; shit tu et al., 2002; hiransuthikul et al., 2004; oguntibeju & nwobu, 2004; macedo & santos, 2005; anguzu & olila , 2007; pondei et al., 2013). two different studies on isolation frequency of wound isolates in brasil (sader et al., 2001; macedo & santos , 2005) showed s. aureus and p. aeruginosa to be the dominant ones. investigation in thailand after tzunami (hiransuthikal et al., 2004) reported aeromonas sp. to be the most common wound pathogen, followed by e. coli and s. aureus. similar research in uganda (anguzu & olila , 2007) showed that p. mirabilis ranks as the second one by its isolation frequency, right after s. aureus, which was the most frequent wound pathogen. in nigeria, investigation of shit tu et al. (2002) reported s. aureus (25.3%), e. coli (12.3%) and p. aeruginosa (9.3%) to be the most prevalent isolates from wounds. two more recent studies (oguntibeju & nwobu, 2004; fig. 1. the frequency of bacteria isolated from wound swab 18.4% 16.8% 12.7% 10.4% 9.0% 6.2% 4.6% 4.2% 4.1% 3.6% 2.8% 2.8% 1.6% 1.0% 0.8% 0.3% 0.3% 0.2% 0.2% s. epidermidis s. aureus p. aeruginosa e. faecalis e. coli staphylococcus sp. p. mirabilis klebsiella sp. enterobacter sp. pseudomonas spp. acinetobacter spp. s. alfa haemolyticus p. vulgaris m. morganii serratia sp. s. beta haemolyticus p. rettgeri citrobacter sp. gbacilli biologica nyssana 7 (2)  december 2016: 151-158 stojanović-radić, z. et al.  frequency of isolation and antibiotic… 154 pondei et al., 2013) in the same country, showed significant changes in these frequencies, where p. aeruginosa strains were the most dominant (33.3% and 32.6%, respectively). considering studies performed in serbia (stanković -nedeljković et al., 2012; jovanović et al., 2014), which reported gram negative bacteria p. aeruginosa and e. coli to be the most prevalent ones, there is also high disagreement with the obtained results in this study, where two gram positive strains presented those with the highest isolation frequency. the isolates of s. epidermidis were absolutely resistant (100.0%) to the penicillin antibiotics (penicillin g, ampicillin and amoxicillin), while a higher percentage of isolates showed sensitivity to amoxicillin/clavulanic acid and piperacillin. the carbapenems were significantly effective against the tested strains of s. epidermidis. the absolute fig. 2. frequency of antibiotic resistance of s. epidermidis pen g-penicillin, amp-ampicillin, amo-amoxicillin, amc-amoxicillin/clavulanic acid, taz-tazobactam, pippiperacillin/tazobactam, mer-meropenem, imi-imipenem, cef-cefalexin, cfc-cefaclor, cfx-ceftriaxone, cfrcefuroxime, cfm-cefotaxime, cld-clindamicin, g-gentamicin, cip-ciprofloxacin, t/s -trimethoprim/sulfametoxazol, fuc-fusidic acid, rif-rifampicin, tei-teicoplanin, chl-chloramphenicol, lin-linezolid, erm-erythromicin fig. 3. frequency of antibiotic resistance of s. aureus pen g-penicillin, amp-ampicillin, amo-amoxicillin, amc-amoxicillin/clavulanic acid, taz-tazobactam, pippiperacillin/tazobactam, mer-meropenem, imi-imipenem, lin-linezolid, cef-cefalexin, cfc-cefaclor, cfrcefuroxime, dox-doxycicline, chl-chlomphenicol, erm-erythromicin, cld-clindamicin, g-gentamicin, cipciprofloxacin, t/s-trimethoprim/sulfametoxazol, fuc-fusidic acid, rif-rifampicin, tei-teicoplanin 0,0% 10,0% 20,0% 30,0% 40,0% 50,0% 60,0% 70,0% 80,0% 90,0% 100,0% 0,00% 10,00% 20,00% 30,00% 40,00% 50,00% 60,00% 70,00% 80,00% 90,00% 100,00% biologica nyssana 7 (2)  december 2016: 151-158 stojanović-radić, z. et al.  frequency of isolation and antibiotic… 155 sensitivity was found in the cases of glycopeptide antibiotics (teicoplanin) and linezolid (fig. 2). the results of research conducted in mexico do not indicate similarity with the results of our study (castro-alarcon et al., 2011). in 2014, study conducted on s. epidermidis isolates obtained from intensive care unit patients showed the highest resistance to erythromycin (59.4%), tetracycline (57.8%) and trimethoprim-sulfamethoxazole (53.1%) (najar-peerayeh et al., 2014). in the present study, percentage of the strains resistant to erythromycin and trimethoprim-sulfamethoxazole were also very high (74.1% and 63.2%, respectively) and had the similar relation (trimethoprimsulfamethoxazole demonstrated higher efficacy against isolates). considering s. aureus isolates, resistance to ampicillin, penicillin g and amoxicillin, which all belong to penicillins, amounted 98.7% for all three antibiotics. study of isolates obtained from the patients of the instutute for public health in ćuprija (serbia) reported similar data, showing high resistance of these strains to penicillin group of antibiotics (petrović -jeremić et al., 2008). all tested strains of s. aureus were sensitive to carbapenems (imipenem and meropenem), linezolid, doxycycline, fusidic acid and teicoplanin (fig. 3). investigation in nigeria (fadeyi et al., 2008) found the highest resistance of s. aureus isolates to fluoroquinolones, which was not the case in our study. contrary to this, sader et al. (2001) reported almost identical results with ours for the glycopeptide antibiotics (vancomycin and teicoplanin), which were the most active against all s. aureus isolates (100.0% susceptibility) in both studies. pseudomonas aeruginosa showed high rates of resistance to several classes of antibiotics (fig. 4). in this study, we established the maximum resistance (100.0%) to ampicillin, amoxicillin/clavulanic acid, cefuroxime, and trimethoprim/sulfometaxazol, while slightly lower number of isolates (92.9%) showed resistance against ceftriaxone. out of 78 p. fig. 4. frequency of antibiotic resistance of p. aeruginosa amp-ampicillin, amc-amoxicillin/clavulanic acid, taz-tazobactam, pip-piperacillin/tazobactam, imi-imipenem, mer-meropenem, cfx-ceftriaxone, cfd-ceftazidime, cfp-cefepime, cfr-cefuroxime, g-gentamicin, amkamikacin, tbr-tobramycin, nor-norfloxacin, cip-ciprofloxacin, lev-levofloxacin, t/strimethoprim/sulfametoxazol, col-colistin 0,00% 10,00% 20,00% 30,00% 40,00% 50,00% 60,00% 70,00% 80,00% 90,00% 100,00% fig. 5. frequency of antibiotic resistance of e. faecalis amp-ampicillin, lin-linezolid, dox-doxycicline, chlchloramphenicol, erm-erythromicin, g-gentamicin, g fgentamicin forte, cip-ciprofloxacin, lev-levofloxacin, rif-rifampicin, van-vancomycin, tei-teicoplanin 0,00% 10,00% 20,00% 30,00% 40,00% 50,00% 60,00% 70,00% 80,00% biologica nyssana 7 (2)  december 2016: 151-158 stojanović-radić, z. et al.  frequency of isolation and antibiotic… 156 aeruginosa isolates, 7.9% were resistant to imipenem and even lower percentage to carbapenems (3.5%). carmeli et al. (1999) got similar results about the susceptibility of p. aeruginosa to different classes of antibiotics. absolute sensitivity of the p. aeruginosa isolates has been observed toward tazobactam (penicillin group of antibiotics) and to polypeptide antibiotic colistin. similar data were obtained by the research of nosocomial infection in boston (troil let et al., 1997). the strains of e. faecalis were in the highest percentage resistant to erythromycin (73.3%), which belongs to the macrolide group of antibiotics (fig. 5). the greatest sensitivity of the pathogen was determined to the glycopeptide antibiotics, vancomycin and teicoplanin, while all tested isolates of e. faecalis showed sensitivity to linezolid. all tested isolates were sensitive to vancomycin which is highly important, bearing in mind the fact that occurrence of multi-resistant enterococci, in particular vancomycin resistant enterococci (vre) is continuously reported (rice, 2001). in comparison to the survey conducted in kuwait (udo et al., 2002), there was a great similarity in the results. examination of individual resistance of all other isolated bacteria wasn’t the part of the reported results of this research, because of their low isolation frequency from wound swabs and, therefore, low number for analysis performance. a small number of isolates wouldn’t give a true picture of the resistance/susceptibility of bacteria to different drugs. these isolates were taken into account in determining the total resistance of gram-positive and gram-negative bacteria to various classes of antibiotics (fig. 6). significant difference in the tetracycline efficiency was found when the resistance patterns of gram-negative and gram-positive bacterial strains were compared. gram-negative bacilli were resistant to tetracycline in 60.8% of samples, while grampositive cocci showed lower resistance (15.1%). the carbapenems were the most active against most of the gram-positive and gram-negative bacteria (>80.0% susceptible). these results are similar with research in uganda (anguzu & olila , 2007). conclusion a total of 613 bacterial isolates were isolated and identified from 526 samples of wound swabs. the most common causative agents of wound infection were the members of the genus staphylococcus. staphylococcus epidermidis was the predominant species (18.4%), followed by s. aureus (16.8%), p. aeruginosa (12.7%) and e. faecalis (10.4%). the gram-positive cocci showed the highest resistance rates to penicillin, while gram-negative bacteria were mostly resistant to tetracycline. significant difference in the tetracycline efficiency was found when comparing the resistance patterns of gramnegative and gram-positive bacterial strains. according to the obtained results, antibiotic therapy of wound infections in niš and surrounding area should always include previous isolation and antibiogram of the pathogen, but when it is not possible, the treatment of choice should include carbapenems or their combination with tetracyclines in order to cover both possible groups of causative organisms. at the same time, this approach leaves a battery of the last-resort antibiotics (e.g. linezolid, fig. 6. comparison of the resistance patterns of gram-negative and gram-positive bacterial wound isolates 0,0% 10,0% 20,0% 30,0% 40,0% 50,0% 60,0% 70,0% 80,0% g+ gbiologica nyssana 7 (2)  december 2016: 151-158 stojanović-radić, z. et al.  frequency of isolation and antibiotic… 157 teicoplanin) for each group of bacteria, which are found to be very efficient against obtained isolates. acknowledgements. this work was funded by the ministry of education, science and technological development of the republic of serbia (project 172061). references akinkumi, e.o., adesunkanmi, a.r., & lamikanra, a. 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correlation between antibiotic consumption and bacterial resistance as quality indicator of proper use of these drugs i inpatients. vojnosanitetski pregled, 66(4): 307-312. saliaj et al. 2024, biologica nyssana 15(1) 15 (1) june 2024: 1-11 doi: 10.5281/zenodo.11517530 spontaneous flora of the urban areas of tirana, three new species recorded for the first time in albania original article oresta saliaj research center of flora and fauna, faculty of natural sciences, university of tirana, petro nini luarasi street, no. 80, albania oresta.saliaj@fshn.edu.al (corresponding author) ermelinda gjeta research center of flora and fauna, faculty of natural sciences, university of tirana, albania julian shehu research center of flora and fauna, faculty of natural sciences, university of tirana, albania alfred mullaj research center of flora and fauna, faculty of natural sciences, university of tirana, albania received: april 12, 2024 revised: may 02, 2024 accepted: may 21, 2024 abstract: this study investigates the diversity and characteristics of spontaneous flora in urban areas of tirana, capital of albania. field surveys, based on a defined methodology, conducted during the spring-summer period of 2023, revealed a total of 262 plant species belonging to 53 families. the taxonomic distribution highlights poaceae and compositae as the most species-rich families. three new plant species were documented for the first time in albania, increasing the list of flora diversity of the country. biogeographical analysis indicates a dominance of euro-mediterranean species, reflecting the region’s geographical influences. furthermore, an analysis of flowering periods shows seasonal variations, with summer hosting the highest number of flowering species. these findings contribute to our understanding of urban plant diversity, adaptation strategies, and biogeographical patterns in tirana. nevertheless, the surveyed area is rather small, only 125 ha, and further research in the future is needed and might reveal new findings. key words: spontaneous flora, tirana, new records, urban areas apstrakt: spontana flora urbanih područja tirane, tri nove vrste zabeležene u albaniji ova studija istražuje diverzitet i karakteristike spontane flore u urbanim područjima tirane, albanija. terenska istraživanja, zasnovana na definisanoj metodologiji, sprovedena tokom prolećno-letnjeg perioda 2023. godine, otkrila su ukupno 262 biljne vrste koje se mogu svrstati u 53 porodice. taksonomska distribucija ističe porodice poaceae i compositae kao najbogatije vrstama. tri nove biljne vrste su dokumentovane po prvi put u albaniji, čime je povećan floristički spisak zemlje. biogeografska analiza ukazuje na dominaciju evromediteranskih vrsta, što odražava geografske uticaje regiona. analiza perioda cvetanja pokazuje sezonske varijacije, pri čemu je leto period sa najvećim brojem cvetajućih vrsta. ova saznanja doprinose razumevanju urbane biljne raznovrsnosti, adaptacionih strategija i biogeografskih obrazaca u tirani. ipak, istraživano područje je relativno malo, samo 125 ha, te su potrebna dalja istraživanja u budućnosti koja bi mogla otkriti nove nalaze. ključne reči: spontana flora, tirana, novi nalazi, urbana područja introduction the city of tirana, as the capital of albania, stands out as the most urbanized area in the country, boasting a population exceeding 1 million inhabitants as reported by the institute of statistics in year 2023. over the past three decades, rapid urban expansion has been marked by the spread of tall buildings and an increased population density per unit of land. the trend towards urbanization has been associated with a decline in biodiversity within urban landscapes (malkinson et al., 2018). within tirana’s urban areas, the flora predominantly comprises cultivated plants, although spontaneous species also boom in locations such as sidewalks, parks, and building walls. spontaneous vegetation, characterized by plants not deliberately planted by humans and not native to natural habitats, is a common feature of urban environments (cervelli, 2013). these spontaneous plants are vital biological assets, harboring a rich diversity of species with varied floral, foliar, and fruit characteristics, as well as biomass. importantly, spontaneous vegetation exhibits a notably higher proportion of native species © 2024 saliaj et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 1 compared to cultivated varieties (li et al., 2019). despite the significance of urban ecosystems, research on such environments remains limited in albania. therefore, the objective of this study is to investigate the diversity of spontaneous flora within tirana’s urban areas, focusing on various biological aspects of these species. materials and methods the field work for data collection was carried out during the spring-summer period of 2023. the field trips were carried out in 20 sampling areas defined and selected randomly, according to the selected methodology. the statue of scanderbeg in scanderbeg square is taken as the central point. a circular area with a radius of 2 km has been selected from this statue (fig. 1). grids of 250 m × 250 m squares were drawn on this layer and within these grids, 20 were selected and sampled using the random selection function in the qgis 3.4 madeira program. one of the selected grids is located in the largest park of tirana city (tirana lake park). this area is different from the 19 other grids which have a typical flora of the urban areas, because in this park are still present some semi-natural and natural habitats. field data recording was conducted using the “teneriffa topo maps” application, which facilitates efficient data entry, and point georeferencing with coordinates (fig. 2). additionally, this application enables the extraction of collected data in formats compatible with gis programs, which can then be further processed in excel for the generation of final lists. during the fieldwork, plant identification was primarily conducted on-site in most cases. for taxa that posed identification challenges, plant specimens were collected and further analyzed in the laboratory. the identification process in the laboratory involved consulting relevant literature sources such as the “flora of albania” volumes 1-4 published by the academy of sciences between 1988 and 2000 and “flora europaea” by tutin et al. (1964-1980). the euro+med plantbase database was utilized to determine the scientific names of the identified species. for each of the 20 sampling squares, a list is provided of the plants found, including their species name, family, chorology, phenology (flowering period), and biological form.. these elements were thoroughly analyzed as part of the study. 2 biologica nyssana ● 15 (1) june 2024: 1-11 saliaj et al. ● spontaneous flora of the urban areas of tirana, three new species recorded for the first time in albania fig. 1. map of the sampling stations biologica nyssana ● 15 (1) june 2024: 1-11 saliaj et al. ● spontaneous flora of the urban areas of tirana, three new species recorded for the first time in albania 3 results and discussion the survey conducted to investigate the spontaneous flora in the urban areas of tirana revealed a total of 262 species (tab. 1), belonging to 53 families (tab. 2). in tirana lake park, apart from the typical ruderal flora, species not common for the urban areas such as: potentilla micrantha dc., phillyrea latifolia l., quercus pubescens willd., dorycnium pentaphyllum scop., carpinus orientalis, rosa sempervirens l., etc. (tab. 1) were also found, due to the presence of their natural habitats in the park. fig. 2. pictures from tenerife topo maps application table 1. list of species found in the surveyed area no. species no. species 1 amaranthus blitum l. 12 arum maculatum l. 2 apium graveolens l. 13 arum italicum mill. 3 orlaya grandiflora (l.) hoffm. 14 hedera helix l. 4 daucus carota l. 15 aristolochia clematitis l. 5 eryngium amethystinum l. 16 asparagus acutifolius l. 6 eryngium campestre l. 17 ruscus aculeatus l. 7 scandix pecten-veneris l. 18 asplenium adiantum-nigrum l. 8 tordylium apulum l. 19 asplenium ceterach l. 9 torilis arvensis (huds.) link 20 asplenium trichomanes l. 10 torilis leptophylla (l.) rchb. f. 21 cynoglossum officinale l. 11 vinca major l. 22 echium italicum l. 4 biologica nyssana ● 15 (1) june 2024: 1-11 saliaj et al. ● spontaneous flora of the urban areas of tirana, three new species recorded for the first time in albania no. species no. species 23 echium plantagineum l. 65 filago germanica (l.) huds. 24 echium vulgare l. 66 helminthotheca echioides (l.) holub 25 heliotropium europaeum l. 67 hypochaeris achyrophorus l. 26 calepina irregularis (asso) thell. 68 hypochoeris glabra l. 27 sinapis alba l. 69 lactuca muralis (l.) gaertn. 28 capsella bursa-pastoris (l.) medik. 70 lactuca saligna l. 29 diplotaxis muralis (l.) dc. 71 lactuca serriola l. 30 rapistrum rugosum (l.) all. 72 lactuca viminea (l.) j. presl & c. presl 31 rorippa amphibia (l.) besser 73 matricaria chamomilla l. 32 humulus lupulus l. 74 picris hieracioides l. 33 arenaria leptoclados (rchb.) guss. 75 scolymus hispanicus l. 34 sagina apetala ard. 76 senecio vulgaris l. 35 saponaria officinalis l. 77 sonchus arvensis l. 36 arenaria serpyllifolia l. 78 sonchus asper (l.) hill 37 cerastium brachypetalum pers. 79 sonchus oleraceus l. 38 cerastium glomeratum thuill. 80 taraxacum sect. taraxacum f. h. wigg. 39 herniaria glabra l. 81 tussilago farfara l. 40 polycarpon tetraphyllum (l.) l. 82 urospermum picroides (l.) f. w. schmidt 41 stellaria media (l.) cirillo 83 pilosella officinarum f. w. schultz & sch. bip. 42 chenopodiastrum murale (l.) s. fuentes & al. 84 lactuca virosa l. 43 chenopodium album l. 85 calystegia sepium (l.) r. br. 44 hypericum perforatum l. 86 convolvulus arvensis l. 45 carduus acanthoides l. 87 cornus mas l. 46 cirsium italicum dc. 88 cornus sanguinea l. 47 crepis setosa haller f. 89 carpinus orientalis mill. 48 artemisia absinthium l. 90 ostrya carpinifolia scop. 49 crepis sancta (l.) bornm. 91 sedum album l. 50 picris hieracioides l. 92 sedum dasyphyllum l. 51 artemisia annua l. 93 bryonia alba l. 52 artemisia vulgaris l. 94 carex divulsa stokes 53 bellis perennis l. 95 carex flacca schreb. 54 bidens tripartitus l. 96 carex hirta l. 55 centaurea calcitrapa l. 97 cyperus rotundus l. 56 centaurea solstitialis l. 98 scirpoides holoschoenus (l.) soják 57 cichorium intybus l. 99 cystopteris fragilis (l.) bernh. 58 cirsium arvense (l.) scop. 100 pteridium aquilinum (l.) kuhn 59 cirsium vulgare (savi) ten. 101 dioscorea communis (l.) caddick & wilkin 60 coleostephus myconis (l.) rchb. f. 102 equisetum arvense l. 61 crepis foetida l. 103 euphorbia terracina l. 62 crepis neglecta l. 104 chrozophora tinctoria (l.) a. juss. 63 crepis pulchra l. 105 euphorbia chamaesyce l. 64 dittrichia viscosa (l.) greuter 106 euphorbia helioscopia l. biologica nyssana ● 15 (1) june 2024: 1-11 saliaj et al. ● spontaneous flora of the urban areas of tirana, three new species recorded for the first time in albania no. species no. species 107 euphorbia maculata l. 147 mentha pulegium l. 108 euphorbia peplus l. 148 mentha spicata l. 109 dorycnium pentaphyllum scop. 149 mentha x piperita l. 110 vicia hybrida l. 150 origanum vulgare l. 111 trifolium fragiferum l. 151 prunella vulgaris l. 112 trifolium resupinatum l. 152 stachys germanica l. 113 colutea arborescens l. 153 linum trigynum l. 114 dorycnium hirsutum (l.) ser. 154 lythrum salicaria l. 115 lathyrus annuus l. 155 malva parviflora l. 116 lotus angustissimus l. 156 alcea biennis winterl 117 lotus corniculatus l. 157 malva neglecta wallr. 118 medicago arabica (l.) huds. 158 malva sylvestris l. 119 medicago lupulina l. 159 phillyrea latifolia l. 120 medicago orbicularis (l.) bartal. 160 epilobium dodonaei vill. 121 medicago polymorpha l. 161 epilobium hirsutum l. 122 trifolium campestre schreb 162 epilobium lanceolatum sebast. & mauri 123 trifolium hybridum l. 163 oxalis corniculata l. 124 trifolium nigrescens viv. 164 veronica polita fr. 125 trifolium repens l. 165 cymbalaria muralis p. gaertn. , b. mey. & scherb. 126 vicia cracca l. 166 kickxia elatine (l.) dumort. 127 vicia grandiflora scop. 167 linaria vulgaris mill. 128 vicia hirsuta (l.) gray 168 plantago lanceolata l. 129 vicia sativa l. 169 plantago major l. 130 quercus cerris l. 170 plantago media l. 131 quercus pubescens willd. 171 veronica anagallis-aquatica l. 132 blackstonia perfoliata (l.) huds. 172 veronica arvensis l. 133 centaurium erythraea rafn 173 veronica chamaedrys l. 134 geranium dissectum l. 174 veronica prostrata l. 135 erodium cicutarium (l.) l'hér. 175 veronica serpyllifolia l. 136 geranium molle l. 176 veronica beccabunga l. 137 geranium purpureum vill. 177 lolium rigidum subsp. lepturoides sennen & mauricio 138 geranium sanguineum l. 178 alopecurus pratensis l. 139 ballota nigra l. 179 brachypodium pinnatum (l.) p. beauv. 140 prunella laciniata (l.) l. 180 achnatherum bromoides (l.) p. beauv. 141 clinopodium nepeta (l.) kuntze 181 aegilops geniculata roth 142 clinopodium vulgare l. 182 aegilops neglecta bertol. 143 marrubium vulgare l. 183 agrostis capillaris l. 144 melissa officinalis l. 184 agrostis stolonifera l. 145 mentha arvensis l. 185 anisantha rigida (roth) hyl. 146 mentha longifolia (l.) l. 186 anisantha sterilis (l.) nevski 5 biologica nyssana ● 15 (1) june 2024: 1-11 saliaj et al. ● spontaneous flora of the urban areas of tirana, three new species recorded for the first time in albania no. species no. species 187 anisantha tectorum (l.) nevski 225 polygonum aviculare l. 188 avena barbata link 226 rumex conglomeratus murray 189 avena fatua l. 227 rumex crispus l. 190 avena sterilis l. 228 rumex patientia l. 191 bromus hordeaceus l. 229 rumex pulcher l. 192 catapodium rigidum (l.) c. e. hubb. 230 portulaca oleracea l. 193 cynodon dactylon (l.) pers. 231 lysimachia nummularia l. 194 cynosurus echinatus l. 232 anagallis arvensis l. 195 dactylis glomerata l. 233 anagallis foemina mill. 196 digitaria sanguinalis (l.) scop. 234 adiantum capillus-veneris l. 197 echinochloa crus-galli (l.) p. beauv. 235 clematis vitalba l. 198 holcus lanatus l. 236 ranunculus acris l. 199 hordeum murinum l. 237 ranunculus muricatus l. 200 lagurus ovatus l. 238 ranunculus sardous crantz 201 lolium multiflorum lam. 239 aremonia agrimonoides (l.) dc. 202 lolium perenne l. 240 geum urbanum l. 203 ochlopoa annua (l.) h. scholz 241 potentilla micrantha dc. 204 phalaris brachystachys link 242 potentilla reptans l. 205 phleum echinatum host 243 potentilla supina l. 206 phragmites australis (cav.) steud. 244 rosa canina l. 207 piptatherum miliaceum (l.) coss. 245 rosa sempervirens l. 208 poa pratensis l. 246 rubus ulmifolius schott 209 poa trivialis l. 247 sanguisorba minor scop. 210 polypogon viridis (gouan) breistr. 248 galium aparine l. 211 rostraria cristata (l.) tzvelev 249 galium tricornutum dandy 212 setaria pumila (poir.) roem. & schult. 250 sherardia arvensis l. 213 setaria verticillata (l.) p. beauv. 251 verbascum blattaria l. 214 setaria viridis (l.) p. beauv. 252 verbascum sinuatum l. 215 sorghum halepense (l.) pers. 253 verbascum thapsus l. 216 trisetaria panicea (lam.) paunero 254 solanum nigrum l. 217 triticum aestivum l. 255 solanum villosum mill. 218 triticum aestivum l. subsp. aestivum 256 urtica dioica l. 219 vulpia myuros (l.) c. c. gmel. 257 parietaria judaica l. 220 zea mays l. 258 verbena officinalis l. 221 schedonorus arundinaceus (schreb.) dumort. 259 sambucus ebulus l. 222 schedonorus pratensis (huds.) p. beauv. 260 viola odorata l. 223 persicaria maculosa gray 261 tribulus terrestris l. 224 fallopia convolvulus (l.) á. löve 262 cenraurea benedicta (l.) l. the results of this study give new data of the distribution area of some species, not mentioned in previous publications. epilobium lanceolatum sebast. & mauri, a typical mountain species (beech woods, shady rocks) (barina et al., 2017), is found only once in one of the selected stations of the urban areas of tirana (fig. 3). malva parviflora l., is another finding of this study, which according to barina et al. (2017, 2018), is the species erroneously 6 biologica nyssana ● 15 (1) june 2024: 1-11 saliaj et al. ● spontaneous flora of the urban areas of tirana, three new species recorded for the first time in albania 7 table 2. list of families to which the plant species belong and the number of species per family family species family species amaranthaceae juss. 1 gentianaceae juss. 2 apiaceae lindl. 9 geraniaceae juss. 5 apocynaceae juss. 1 lamiaceae lindl. 14 araceae 2 linaceae perleb. 1 araliaceae juss. 1 lythraceae j. st.-hil. 1 aristolochiaceae juss. 1 malvaceae juss. 4 asparagaceae 2 oleaceae hoffmanns. & link 1 aspleniaceae newman 3 onagraceae juss. 3 boraginaceae juss. 5 oxalidaceae r. br. 1 brassicaceae burnett 6 plantaginaceae juss. 13 cannabaceae martinov 1 poaceae barnhart 46 caryophyllaceae juss. 9 polygonaceae juss. 7 chenopodiaceae vent. 2 portulacaceae juss. 1 clusiaceae lindl. 1 primulaceae vent. 3 compositae giseke 41 pteridaceae e. d. m. kirchn. 1 convolvulaceae juss. 2 ranunculaceae juss. 4 cornaceae (dumort.) dumort. 2 rosaceae 9 corylaceae mirb. 2 rubiaceae juss. 3 crassulaceae dc. 2 scrophulariaceae juss. 3 cucurbitaceae juss. 1 solanaceae 2 cyperaceae juss. 5 urticaceae juss. 2 cystopteridaceae schmakov 1 verbenaceae 1 dennstaedtiaceae lotsy 1 viburnaceae raf. 1 dioscoreaceae 1 violaceae batsch 1 equisetaceae dc. 1 zygophyllaceae r. br. 1 euphorbiaceae juss. 6 fabaceae lindl. 21 fagaceae dumort. 2 total 262 reported for the country (fig. 3). the casual finding of hypochaeris achyrophorus l. during the filedwork, represents a new distribution area of this species, previously reported only in the southern part of the country (delvinë, gjirokastër, kolonjë, saranda) (vangjeli, 2018). the findings indicate that the two most wellrepresented families are poaceae, with 46 species, and compositae, with 41 species. interestingly, these two families also rank among the most speciesrich families in the “flora of albania” 4th edition (vangjeli et al., 2000). additionally, approximately 21 families, namely amaranthaceae, apocynaceae, araliaceae, aristolochiaceae, cannabaceae, clusiaceae, cucurbitaceae, cystopteridaceae, dennstaedtiaceae, dioscoreaceae, equisetaceae, linaceae, lythraceae, oleaceae, oxalidaceae, portulacaceae, pteridaceae, verbenaceae, viburnaceae, violaceae and zygophyllaceae, are each represented by only one species in the surveyed area. three new species have been documented for the first time in albania as a result of this study (fig. 4): 1. compositae family lactuca virosa l. (coordinates: 19.826632, 41.322492) 2. lamiaceae family mentha arvensis l. (coordinates: 19.82493639, 41.32649646) 3. poaceae family trisetaria panicea (lam.) paunero (coordinates: 19.8130488, 41.340192) the distribution map of mentha arvensis l. biologica nyssana ● 15 (1) june 2024: 1-11 saliaj et al. ● spontaneous flora of the urban areas of tirana, three new species recorded for the first time in albania fig. 3. photographs of epilobium lanceolatum sebast. & mauri (left) and malva parviflora l. (right) fig. 4. photographs of trisetaria panicea (lam.) paunero (left) and lactuca virosa l. (right) 8 biologica nyssana ● 15 (1) june 2024: 1-11 saliaj et al. ● spontaneous flora of the urban areas of tirana, three new species recorded for the first time in albania 9 indicates that this species is native to all european countries and western asia, except for albania. lactuca virosa l. is distributed across most of europe, being native to countries such as greece, north macedonia, and italy (as per euro plus med data base). trisetaria panicea (lam.) paunero is native to the western and central parts of europe, including italy. with these new findings, the european distribution maps for these species are now completed with new data. based on raunkiaer’s (1934) classification of plant adaptation strategies, the spontaneous flora of urban areas in tirana is predominantly composed of therophytes and hemicryptophytes, with 117 and 101 species, respectively. geophytes represent the next most abundant group with 22 species, followed by phanerophytes with 11 species. nanophanerophytes and chamaephytes exhibit the lowest species count, with only 5 and 6 species respectively (fig. 5). fig. 5. classification of life forms/species fig. 6. chorological types of spontaneous species recorded in urban tirana biologica nyssana ● 15 (1) june 2024: 1-11 saliaj et al. ● spontaneous flora of the urban areas of tirana, three new species recorded for the first time in albania fig. 7. the analyses of flowering species per months in terms of chorological types, the spontaneous flora of tirana is characterized by a dominance of euro-mediterranean element, comprising 66 species, followed by eurasian element with 51 species, european element with 38 species, mediterranean element with 42 species, paleotemperate element with 26 species (fig. 6). these findings align with albania’s geographical location, explaining the prevalence of these biogeographical types. other chorological types exhibit fewer than 15 representative species each. we examined the flowering patterns of plant species over the course of a year, based on literature review for autumn and winter season (“flora of albania”, vol i-iv, 1988-2000) and from the field observation during spring-summer season. in january, 6 species from our survey are in flower, followed by an increase to 12 species in february. march marked a substantial rise, with 54 species flowering. april and may exhibited a significant surge in flowering, with 110 and 184 species respectively, showcasing the peak of floral diversity and activity during the spring season. as the summer months progressed, the number of flowering species remained relatively high, with june, july, and august recording 218, 192, and 156 species, respectively. the transition to autumn saw a gradual decline in flowering, yet september and october still boasted 94 and 50 species respectively. by november and december, the number of flowering species decreased to 13 and 7, marking the end of the flowering season for many plants in our study area (fig. 7). conclusion this study highlights the diverse flora found within urban areas of tirana, albania, showing a total of 262 species from 53 different families, with poaceae and compositae emerging as the most speciesrich families. three plant species lactuca virosa l., mentha arvensis l., trisetaria panicea (lam.) paunero are reported for the first time in albania, adding to the botanical diversity of the country. the dominance of euro-mediterranean species in tirana’s urban flora features the influence of the region’s geographical position on plant distribution. the analysis of flowering periods reveals seasonal variations, with summer hosting the highest number of flowering species, followed by spring, autumn, and winter. raunkiaer’s classification highlights the prevalence of therophytes and hemicryptophytes in tirana’s urban flora, indicating specific adaptation strategies to urban environments. references barina, z., mullaj, a., pifkó, d., somogyi, g., meco, m., & rakaj, m. (2017). distribution atlas of vascular plants in albania. hungarian natural history museum, hungary. isbn 978-963-987729-0. cervelli, e.w., lundholm, j.t., & du, x. (2013). spontaneous urban vegetation and habitat heterogeneity in xi’an, china. landscape and urban planning. https://doi.org/10.1016/j. landurbplan.2013.08.001 10 biologica nyssana ● 15 (1) june 2024: 1-11 saliaj et al. ● spontaneous flora of the urban areas of tirana, three new species recorded for the first time in albania euro+med. (2006+). euro+med plantbase the information resource for euro-mediterranean plant diversity. published at http://www.europlusmed.org [accessed 29/05/2024]. instat (institute of statistics). (2023). population of albania on 1st january 2023, tirana. available from http://www.instat.gov.al/al/temat/treguesitdemografik%c3%ab-dhe li, x.p., fan, sh.x., hao, p.y., & dong, l. (2019). temporal variations of spontaneous plants colonizing in different types of planted vegetation a case of beijing olympic forest park. urban forestry & urban greening, 46, 126459. malkinson, d., kopel, d., & wittenberg, l. (2018). from rural-urban gradients to patch – matrix frameworks: plant diversity patterns in urban landscapes. landscape and urban planning, 169, 260–268. https://doi.org/10.1016/j. landurbplan.2017.09.021 paparisto, k., demiri, m., mitrushi, i., & qosja, xh. (1988). flora of albania. vol. 1. tirana: science academy of albania republic, center of biological research. pils, g. (2016). illustrated flora of albania (private edition). isbn 978-3-200-04853-9. qosja, xh., paparisto, k., demiri, i., vangjeli, j., & balza, e. (1992). flora e shqipërisë. vol. 2. tirana: science academy of albania republic, center of biological research. qosja, xh., paparisto, k., vangjeli, j., ruci, b., & mullaj, a. (1996). flora e shqipërisë. vol. 3. tirana: science academy of albania republic, center of biological research. raunkiaer, c. (1934). the life forms of plants and statistical plant geography. oxford: clarendon press. tutin, t.g., heywood, v.h., burges, n.a., valentine, d.h., walters, s.m., & webb, d.a. (1972). flora europaea, volume 3: diapensiaceae to myoporaceae. cambridge: university press. vangjeli, j., ruci, b., mullaj, a., qosja, xh., & paparisto, k. (2000). flora e shqipërisë. vol. 4. tirana: science academy of albania republic, center of biological research. vangjeli, j. (2018). flora of albania atlas. isbn 978-9928-237-31-6. 11 microsoft word 0301_calic-dragosavac_radojevic biologica nyssana 1 (1-2) december 2010: 49-55 ćalić-dragosavac, d., radojević, lj. improvement of maturation… 49 original article ! improvement of maturation and conversion of horse chestnut androgenic embryos dušica ćalić-dragosavac*, ljiljana radojević department of plant physiology, institute for biological research, "siniša stanković'', bulevar despota stefana 142, belgrade, serbia * e-mail: calic@ibiss.bg.ac.rs abstract: ćalić-dragosavac, d., radojević, lj.: improvement of maturation and conversion of horse chestnut androgenic embryos. biologica nyssana, 1 (1-2), december 2010: 49-55. horse chestnut (aesculus hippocastanum l., hippocastanaceae) is a relict species of the tertiary flora and endemit of balkan peninsula. it has enormous horticultular and medical important. horse chestnut trees are native to the balkan peninsula, but grow as ornamental trees in parks and avenues throughout the northern hemisphere. because of the slow and difficult reproduction of great importance to be fast and cheap in vitro multiplication. possible solution is regenerated by androgenesis. microspore culture has been used in recent years as a tool for producing haploid plants in a varyety of higher plants, but the low frequencies of microspore-derived plants restrict the use of the technique in plant breeding. key words: aesculus hippocastanum, anther culture, maturation, ploidy level, suspension culture introduction ! horse chestnut showed slow and difficult reproduction in nature conditions. possible solution of this problem is regeneration by androgenesis in vitro. androgenesis in vitro has been used in recent years as a tool for producing haploid plants in a varyety of higher plants, but the low frequencies of microspore-derived plants restrict the use of the technique in plant breeding. regeneration in the genus aesculus via androgenesis has been demonstrated for a. hippocastanum (r a d o j e v i ć , 1978; ć a l i ć et al., 2003), a. carnea (r a d o j e v i ć et al., 1989; m a r i n k o v i ć & r a d o j e v i ć , 1992) and a. flava (ćalić et al., 2005; ćalić-dragosavac et al., 2010). androgenesis provides a large number of embryos at defined stages of development, and allows alterations of the embryonic environment through manipulations of culture conditions. regeneration of this species via androgenesis provides a means of propagation and a model system for conducting physiological and biochemical studies. although such embryos may appear “morphologically” mature, they do not perform well during postembryogenic growth without the imposition of a drying period. improvement of embryo quality can be achieved through the application of osmotic stress, which is an important factor for directing embryo development and maturation both in vivo and in vitro (c a p u a n a & d e b e r g , 1997; t r o c h et al., 2009). generation of horse chestnut somatic embryos is commonly achieved by transferring embryogenic tissue onto an activated charcoal (ac), abscisic acid (aba), polyethylene glycol (peg) and manitol-containing maturation media (c a p u a n a & d e b e r g , 1997). 10th sfses • 17-20 june 2010, vlasina lake1 (1-2) • december 2010: 49-55 biologica nyssana 1 (1-2) december 2010: 49-55 ćalić-dragosavac, d., radojević, lj. improvement of maturation… 50 activated charcoal is commonly used in tissue culture media to darken the immediate media surroundings and to absorb inhibitory or toxic substances and plant growth regulators (m o s h k o v et al., 2008). charcoal has been used in all stages of somatic embryogenesis to increase initiation frequencies of pinus taeda l. (p u l l m a n & j o n s o n , 2002), induce embryogenic tissue on vegetative shoot apices of mature trees of pinus patula (m a l a b a d i & v a n s t a d e n , 2005), improve yield and quality of somatic embryos during maturation (c a p u a n a & d e b e r g , 1997; c a r a w a y & m e r k l e , 1997; l i et al., 1997, 1998; g r o l l et al., 2002; p u l l m a n et al., 2005; l e l u w a l t e r et al., 2006), and most frequently during germination (v o o k o v á & k o r m u t ’ á k , 2001; s a l a j et al., 2004; a n d r a d e & m e r k l e , 2005). aba is commonly used at various concentrations to improve somatic embryo development of abies species prior to plantlet regeneration (s a l a j o v á et al., 1996; v o o k o v á & k o r m u t ’ á k , 2001). abscisic acid has been found to stimulate the production of cotyledonary embryos and regulate the course of embryo maturation (b e c w a r et al., 1987; b o u l a y et al., 1988; d u n s t a n et al., 1988; v o n a r n o l d & h a k m a n 1988; r o b e r t s et al., 1990a). the ability of aba to inhibit precocious germination has facilitated the development of procedures for mass propagation of spuce emblings (r o b e r t s et al., 1990b; w e b s t e r et al., 1990). osmotic treatments have been used to influence embryo maturation in many gymnosperms and angiosperms (c a p u a n a & d e b e r g h , 1997). aba induction of desiccation tolerance may be linked to accumulation of late embryogenesis – abundant proteins that may protect tissues (d u r e et al., 1989). the effect of peg mimics the naturally occurring water stress on seeds during the late stages of maturation. water stress caused by peg and increased concentrations of aba are essential for somatic embryo development to accumulation of storage compounds and inhibit precocious germination (s t r a s o l l a & y e u n g , 2003). peg enhanced somatic embryo maturation in several species, including picea glauca (a t t r e e et al., 1990, 1991, 1992, 1995), abies numidica (v o o k o v á & k o r m u t ’ á k , 2002) and abies hybrids (s a l a j & s a l a j , 2003; s a l a j et al., 2004). the combined application of aba and peg, a non-plasmolyzing osmoticum, has become a routine method for stimulating embryo maturation (a t t r e e & f o w k e , 1993). the aim of this research was to study influence of activated charcoal (ac), abscisic acid (aba) and polyethylene glycol (peg) on the maturation and conversion of horse chestnut microspore-derived embryo for the diversity protection and conservation. material and methods ! closed flower buds (4-5 mm long) used in the experiments were obtained from 100 years old aesculus hippocastanum l. tree growing in the botanical garden "jevremovac" of the belgrade university. the buds were surface sterilized with 95 % and 70 % ethanol. basal medium (bm) contained murashige and skoog (1962; ms) mineral salts, 2 % sucrose and was supplemented with the following (mg l-1): panthotehenic acid (10), nicotinic acid (5), vitamin b1 (2), adenine sulphate (2), myo-inositol (100) and casein-hydrolysate (200). uninuclear microspores culture was established on induction ms liquid (l) medium. induction l medium contained bm with 2,4-d dichlorphenoxyacetic acid (2,4-d) and kinetin (kin), 1.0 mg l-1 of each. about 100 anthers with uninucleate microspores per erlenmeyer flask with filter (50 μm) and 100 cm3 l medium for androgenic induction were cultivated. isolation and culture of microspores the anthers were macerated in a glass petri dish with scalpel through a 50-μm metal sieve in l induction medium. the microspores collected on the surface of the 50-μm sieve were carefully washed with same medium. the microspore suspension were subcultured every 30 days and refreshed with liquid l medium. after 2 months, a microspore suspension was plated by bergmann technique (1960) on a solid medium with reduced concentration of 2,4-d (0.01 mg l-1) and same concentration of kin. after medium for multiplication embryos were cultured on media for embryo maturation supplemented with ac, aba and peg 4000. various concentrations (0.1; 0.5 and 1%) of activated charcoal (ac) were tested. aba (2.5; 10.0 and 25.0 mg l-1) alone, as well as in combination with 1 % ac was investigated. also, influence of peg (5, 25 and 50 g l-1) and combinations peg (5 and 50 g l-1) with ac (1 g l-1) on maturation of androgenic embryos were studied. we used thirteen different maturation media which are presented in figures 1-4. subculturing was done every one month and the maturation phase took 3 months. cultures were monitored at the time of sub-culturing and the mature embryos were biologica nyssana 1 (1-2) december 2010: 49-55 ćalić-dragosavac, d., radojević, lj. improvement of maturation… 51 transferred to hormone and supplement free germination medium. all media were sterilized by autoclaving at 0.9 x 105 kpa and 1140c for 25 min. androgenic embryos were grown at 25 ± 1 0c and a 16-h photoperiod with irradiance of 33 45 μmol m-2s-1 produced by cool white fluorescent tubes. embryos on maturation media were investigated after 3 months. three characteristics were evaluated: 1) shoot elongation; 2) radicle development and 3) conversion into the plants. determination of ploidy level nuclear suspension from androgenic embryos in the cotyledonary stage of development were prepared. young leaf material of horse chestnut was used as control. plant material was macerated with a sharp razor blade in a ice-cold neutral buffer, and placed in plastic petri dishes. neutral dna buffer (ph 7) with 15 mm hepes, 1 mm edta, 80 mm kcl, 20 mm nacl, 0,5 mm spermine, 300 mm sucrose, 0.2 % triton x-100, 15 mm dte (dithiothreitol) and 2 mg l-1 dapi was used (modified by de laat and blaas, 1984). after maceration, the buffered mixture (ca. 2 ml), was passed through a nylon filter of 30 μm mesh size, stained with dapi, and analysed in a flow cytometer. fluorescence levels were determined by a photomultiplier and coverted in voltage pulses that were processed with pc. ploidy level of androgenic embryos was evaluated by flow citometry, using a pas ii cytometer (partec gmbh), equipped with a high pressure mercury lamp (osram hbo 100 w/2) and using the excitation filters ug-1, bg-31, kg-1 and tk-420 and emission filters tk560 and gg435. statistics and repetition sixty androgenic embryos per medium were analysed. three repetitions were performed per each medium. the total number of pollen grains analyzed for each studied medium was 180. the results were assessed using the variation analysis. results were tested according snk (student newman keuls) test (significance level α= 0.05) for determination statistical significant differencesis among treatments. results androgenesis of horse chestnut micospores was induced in liquid and solid ms (murashige and skoog, 1962) medium with 2,4-d acid and kin (1.0 mg l-1 of each). light and scanning microscopy confirmed that androgenic embryos of horse chestnut grown in suspension formed by direct division of microspores. after transfer to basal medium (bm), androgenic embryos showed asynchronous development. great numbers of these embryos were irregular, showed hypertrophy and had abnormal cotyledons or lacked a hypocotyl. percentage germination of androgenic embryos was followed after 3 months of growing on different maturation media. the best results of germination of horse chestnut androgenic embryos which maturated on media supplemented with ac (99%), alone, and in combination with peg (100%) were observed in microspore culture (fig. 1 and 4). with increasing concentration of ac increased the percentage of embryo germination from 92 to 99% after 3 months in suspension culture. root shoot w hole plants control ac 1 g/l ac 5g/l ac 10g/l 0 20 40 60 80 100 % control ac 1 g/l ac 5g/l ac 10g/l fig. 1. effect of ac on maturation microsporederived embryos, after three months root shoot w hole plants control aba 2.5 g/l aba 10 g/l aba 20g/l 0 10 20 30 40 50 60 70 % control aba 2.5 g/l aba 10 g/l aba 20g/l figure 2. effect of aba on microspore-derived embryos, after three months also, the greatest number of androgenic plants originating from microspore (18 %) culture was formed on the medium with supplemented with biologica nyssana 1 (1-2) december 2010: 49-55 ćalić-dragosavac, d., radojević, lj. improvement of maturation… 52 1 % ac (fig. 1). also, cultures on media containing 1 % ac showed better maturation, germination; shoot elongation and conversion into the plants (fig. 1). however, the number of germinated embryos with increasing concentration of aba (from 2.5 to 20 mg l-1) in media was decreased (fig. 2). lowest germination percentages, 37 % and 39 % in microspore culture were obtained on maturation media with aba 20 mg l-1 alone and in combination with ac 1g l-1. root shoot w hole plants control peg 5 g/l peg 25 g/l peg 50 g/l 0 10 20 30 40 50 60 70 80 90% control peg 5 g/l peg 25 g/l peg 50 g/l figure 3. effect of peg on microspore-derived embryos, after three months root shoot w hole plants control peg 5 g/l +ac 1g/l peg 50 g/ + ac 1g/l 0 20 40 60 80 100 % control peg 5 g/l +ac 1g/l peg 50 g/ + ac 1g/l figure 4. effect of peg and ac on maturation microspore-derived embryos, after three months aba in concentration of 2.5 mg l-1 had a positive effect on shoot (23 %) formation. peg in maturation media improved androgenic embryo germination and conversion into the plants. androgenic embryos on media containing peg (50 g l-1), in combination with ac (1 g l-1) showed rapid development of cotyledonary stage embryos and lowered percentage of abnormal structures. increasing peg from 5 to 50 g l-1 in the maturation media improved germination of androgenic embryos originating from microspore culture from 62 to 87 %. concentration of ac 1 g l-1 in maturation medium with 5 and 50 g l-1 of peg had a further beneficial effect on germination of embryos derived from microspore (79-100 %) culture. the addition of ac and peg to the maturation medium significantly increased the percentage of androgenic embryos forming shoots and increased the conversion frequencies in microspore (5-13 %; fig. 4) culture. cytogenetic analysis of androgenic embryos originating from anther and microspore culture was done after a first generation of regenerants (after 3 months) and after 3 years of subculturing. all androgenic embryos after 3 months of maturation treatments from microspore culture were haploid. however, only 10 % androgenic microspore embryos retained haploidity, while 10.5 % were diploid, 73.5 % tetraploid and 6 % octaploid (fig. 5) after 3 years of subculturing. discussion some of the horse chestnut androgenic embryos were irregular, hypertrophic and had abnormal cotyledons or lacked hypocotyls. this phenomenon is frequently observed in somatic embryos of trees (perez et al., 1986; capuana and deberg, 1997). therefore, addition of different concentrations of ac, aba or peg in media with horse chestnut androgenic embryos has overcome this problem. our results about influence of activated charcoal on the in vitro androgenesis in correlated with the results of özkum çiner and tipirdamaz (2002). the effect of activated charcoal has been attributed to the absorption of inhibitory substances (abscisic acid, phenolics) from the medium (thomas, 2008). in the current study, the best results of germination, maturation and conversion androgenic embryos into the plants were achieved on medium with 1 % activated charcoal which is in accordance with the results pullman et al. (2005). also, aba and water stress may keep an embryo in a maturation state by encouraging development and preventing germination. abscisic acid has been found to stimulate the production of cotyledonary embryos with normal morphology and to regulate the course of horse chestnut somatic embryos (capuana and deberg, 1997; troch et al., 2009) as well as american chestnut (robichaud et al., 2004). biologica nyssana 1 (1-2) december 2010: 49-55 ćalić-dragosavac, d., radojević, lj. improvement of maturation… 53 significant accumulation of biomass and storage products in the cotyledons of chestnut embryos may not be critical for germination under in vitro conditions. in fact, capuana and deberg (1997) and troch et al. (2009) noted that the highest conversion frequencies were obtained with embryos possessing thin cotyledons, while embryos bearing large, thick cotyledons, resembling those of mature zygotic embryos, did not regenerate plants all. aba also promotes the development of globular embryos in embryogenic cultures of horse chestnut as well as in spruce (von arnold and hakman, 1988). peg-derived horse chestnut embryos were also less aberrant than control embryos as well as somatic horse chestnut embryos (troch et al., 2009) and picea glauca embryos (attree et al., 1991). our results that peg increased germination of horse chestnut androgenic embryos are in correlation with results attree et al. (1992) and capuana and deberg (1997). attree et al. (1995) published that drying was essential for subsequent normal growth of picea glauca somatic embryos following maturation on peg. the ploidy level of regenerated embryos and plantlets at different growth stages was determined using flow cytometry and chromosome counts. diploid, tetraploid and octaploid plants were present among the horse chestnut regenerants. this could be a result of spontaneous chromosome doubling occurring during androgenesis. these results suggested that ploidy level significantly increased during long-term cultures (geier, 1991). in contrary of androgenic embryos, somatic embryos of horse chestnut did not show major genetic changes (troch et al., 2009). however, further experiments by this author are necessary to determine whether horse chestnut somatic embryos are genetically stable during long-term cultures. conclusion to the best of our knowledge, this is the first report about improvement of maturation and conversion of horse chestnut androgenic embryo for 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canadian journal of forest research, 20: 1759-1765. matejić et al. 2021, biologica nyssana 12(2) 12 (2) december 2021: 87-111 doi: 10.5281/zenodo.5759846 frequency of herbal medicinal products use in southeastern serbia original article matejić jelena university of niš, faculty of medicine, department of pharmacy, bulevar dr zorana đinđića 81, 18000 niš, serbia jekamatejic@gmail.com (corresponding author) pavlović dragana university of niš, faculty of medicine, department of pharmacy, bulevar dr zorana đinđića 81, 18000 niš, serbia stefanović nikola university of niš, faculty of medicine, department of pharmacy, bulevar dr zorana đinđića 81, 18000 niš, serbia stojanović milica university of niš, faculty of medicine, department of pharmacy, bulevar dr zorana đinđića 81, 18000 niš, serbia žarković lazar university of belgrade faculty of biology, institute of botany and botanical garden “jevremovac”, studentski trg 16, 11000 belgrade, serbia marin petar university of belgrade faculty of biology, institute of botany and botanical garden “jevremovac”, studentski trg 16, 11000 belgrade, serbia džamić ana university of belgrade faculty of biology, institute of botany and botanical garden “jevremovac”, studentski trg 16, 11000 belgrade, serbia received: july 18, 2021 revised: october 03, 2021 accepted: november 01, 2021 abstract: the trend of herbal medicine use continues even today, despite the growing number of different synthetic, conventional drugs and the huge progress of the pharmaceutical industry. in recent decades, there has been an increasing use of different herbal medicinal products (hmp) in the prevention of diseases, treatment of chronic and recurrent conditions and diseases, and for maintaining good health. the aim of our paper was to determine the use of hmp in the area of southeastern serbia. the survey method was applied through a pre-compiled questionnaire in the zaječar, sokobanja and aleksinac cities. the results of the research showed that there were 70 different hmp, which were most often used preventively (32.00%), while the rest were used in hypertension (23.50%), prostate hyperplasia (8.50%), diabetes (6.53%), depression (4.60%) and glaucoma (3.90%). the composition of the used products included 84 different herbal species, the most common of which were citrus x aurantium l. (5.90%) and cucurbita moschata duchesne (3.60%). when it comes to prevention and/or treatment of diseases, statistical analysis showed that the respondents living in the aleksinac and surrounding used more plant species compared to people from sokobanja and zaječar, while there was no difference between the respondents in the number of hmp used. respondents who did not have prescribed synthetic drugs, used hmp for the purpose of prevention in a higher percentage. it can be concluded that the respondents mostly use hmp as preventive means, but they also use them as an accompanying therapy. key words: herbal medicinal products, treatment, frequency, prevention, southeastern serbia apstrakt: učestalost korišćenja lekovitih biljnih produkata u jugoistočnoj srbiji trend korišćenja biljnih lekova nastavlja se i danas, bez obzira na postojanje sve većeg broja različitih sintetičkih, konvencionalnih lekova i ogroman napredak farmaceutske industrije. poslednjih decenija je konstatovana sve veća primena biljnih lekova u prevenciji bolesti, lečenju hroničnih i rekurentnih stanja i oboljenja, kao i za održavanje zdravlja. cilj našeg rada bio je da se utvrdi upotreba lekovitih biljnih produkata (lbp) na teritoriji jugoistočne srbije. primenjivana je metoda anketiranja putem unapred sastavljenog upitnika u sledećim gradovima: zaječar, sokobanja i aleksinac. rezultati istraživanja pokazali su da je u upotrebi bilo 70 različitih lbp, koji su najčešće korišćeni preventivno (32,00%), dok su ostali upotrebljavani kod hipertenzije (23,50%), hiperplazije prostate (8,50%), šećerne bolesti (6,53%), depresije (4,60%) i glaukoma (3,90%). sastav korišćenih produkata sadržao je 84 različitih biljnih vrsta, a najčeće su bile zastupljene citrus x aurantium l. (5,90%) i cucurbita moschata duchesne (3,60%). statistička analiza je pokazala da su ispitanici koji žive na teritoriji aleksinca koristili više biljnih vrsta kada je reč o prevenciji i/ili lečenju oboljenja u poređenju sa stanovnicima sokobanje i zaječara, dok nije bilo razlike među ispitanicima kada je reč o broju samih lbp. ispitanici koji nisu imali propisane sintetičke lekove u terapiji u većem procentu su koristili lbp u svrhu prevencije. može se zaključiti da ispitanici u većoj meri koriste lbp kao preventivna sredstva, ali ih u velikom broju koriste i kao prateću terapiju. ključne reči: lekoviti biljni produkti, lečenje, učestalost, prevencija, jugoistočna srbija © 2021 matejić et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 87 introduction herbs have been used as the first drugs in the treatment of diseases, and in recent decades their use has been increasingly important in medicine (barnes, 2012; heinrich et al., 2018; zhang, 2018). herbal therapy is used by 75% of the world’s population. this is due to better acceptability of herbal medicines, fewer side effects that can occur during therapy as well as the fact that they are cheaper (sam, 2019). in addition, patients are more likely to opt for different herbal products as they believe that they are completely safe because they are of natural origin, and consequentlly that their concomitant application with conventional medicines is not dangerous at all. for these reasons, patients usually do not report the use of herbal medicine to their doctors and pharmacists (dimitrijević et al., 2014). yet, in some cases, herbal medicinal products could have a harmful effect on health if they are not used in the prescribed manner and under the supervision of a professional or could lead to harmful interactions with conventional drugs (petrović et al., 2012). herbal medicines, traditional herbal medicines and herbal teas are used for maintaining good health, prevention of diseases, treatment of chronic and recurrent conditions, diseases treatment on the recommendation of a doctor or pharmacist, and more often on the basis of the patient’s decision (petrović et al., 2012; luketina-šunjka et al., 2020). the use of medicinal plants in serbia has a long tradition, and our previous work (matejić et al. 2020) showed the exceptional presence of medicinal plant species in eastern and southeastern serbia that are used for a large number of different indications. the aim of this study was to investigate the frequency of herbal medicinal products (hmp): herbal medicines, traditional herbal medicines, herbal supplements, and different other herbal products that could be found in public pharmacy in southeastern serbia and to determine how much and in what way herbs are used in the treatment of patients in southeastern serbia. in this regard, it was necessary to answer the following questions: (1) what are the most commonly used hmp, (2) for what therapeutic purposes are hmp used, (3) which conventional drug therapy is used in combination with hmp, (4) how does age affect the frequency of hmp use, (5) are there differences in the frequency of herbal use among men and women? materials and methods research area the research was conducted in southeastern serbia, more precisely in three municipalities: zaječar, sokobanja and aleksinac. the municipalities of zaječar and sokobanja belong to the zaječar district, while the municipality of aleksinac belongs to the nišava district. the municipality of zaječar covers an area of 1069 km2 and has 53,509 inhabitants in mid-2019 (statistical office of the republic of serbia, 2020). according to the latest research of the public health institute “timok” zaječar (2020), the total number of residents of this municipality who were ill in 2019 was 39,240. residents mostly suffered from respiratory diseases, i.e., 25.1% of the total number of patients. then they suffered from cardiovascular diseases, about 16.0% of them. musculoskeletal system and connective tissue diseases are in the third place with a share in the total morbidity of 11.0%. they were followed by urogenital system disorders (8.2%) and diseases from the group of symptoms, which includes pathological clinical and laboratory findings with 5.9% of the total morbidity. digestive system diseases are in the sixth place, accounting for 5.0% of the total number of patients. after that, there are diseases related to the endocrine system, nutrition and metabolism, with a share in the total morbidity of 4.9%. in the seventh place are injuries, poisonings and the consequences of external factors (4.7%). skin and subcutaneous tissue diseases account for 3.8%, while mental and behavioral disorders include 3.7% of the total number of patients. there are also diseases from other groups that do not individually contribute much to the number of patients and together make up the remaining 11.7%. in 2019, the most common diagnosis was high blood pressure making 12.5% of the total number of diseases. other common diagnoses were: acute pharyngitis and tonsillitis (11.0%); acute bronchitis and bronchiolitis (6.3%); other back diseases (5.7%); other symptoms, signs and pathological clinical and laboratory findings (4.0%); diabetes (3.8%); cystitis (3.2%); rhinitis, sinusitis and laryngitis (2.9%); degenerative joint disease and skin and subcutaneous tissue diseases (2.6%). the remaining 45% are other diseases. in 2019, the department for women’s health service of the municipality of zaječar reported 1,063 patients with various diseases. most of them suffered from diseases of the urogenital system, as many as 75%. they are followed by diseases from the group of tumors with 13.9%, and then infectious and parasitic diseases with 10.9% of the total number of patients. the diagnoses made in the patients were: menstrual disorders (16.5%), cervicitis (16.3%), menopausal diseases (15.2%), salpingitis and oophoritis (9.0%) and female infertility (8.6%). one third of the total number are other diagnoses that were encountered in 2019 in women. 88 biologica nyssana ● 12 (2) december 2021: 87-111 matejić et al. ● frequency of herbal medicinal products use in southeastern serbia 89 the municipality of sokobanja covers an area of 525 km2 with a total population of 14,755 in mid2016 (statistical office of the republic of serbia, 2017). based on the analysis of the public health institute niš (2017), a total of 23,700 patients were registered in the municipality of sokobanja in 2016. most of them suffered from cardiovascular diseases, namely 31.6% of the total morbidity. respiratory diseases accounted about 24.7%. in the third place are genitourinary diseases with 7.5%, and in the fourth place are musculoskeletal and connective tissue diseases with 7.1% of the total number of patients. these are followed by mental and behavioral disorders with a share in the total morbidity of 4.7%. digestive diseases are in the sixth place, accounting for 3.3%, followed by diseases from the group of symptoms, which includes pathological clinical and laboratory findings with 2.9%. they are followed by diseases from the group of factors that affect the health condition and contact with the health service, about 0.9%. other groups of diseases together make up 17.3% of the total number of patients. the most common diagnosis in 2016 was high blood pressure with 22.0% of the total number of diagnoses. it is followed by: acute pharyngitis and tonsillitis (10.0%), other back diseases (5.0%), tracheitis, emphysema and other obstructive lung diseases (4.5%) and cystitis (3.6%). there are various other diagnoses that together make up the remaining 54.5% of the total number of diseases. during 2016, the department for women’s health service of the municipality of sokobanja registered 934 patients. in as many as 80.5% of cases, the patients suffered from genitourinary diseases. in the second place are diseases from the group of tumors with 7.2%, and in the third are diseases from the group of factors that affect health and contact with the health service with 5.2% of the total number of patients. they are followed by skin and subcutaneous tissue diseases, as well as diseases from the group of symptoms, which includes pathological clinical and laboratory findings, which account for 5.1% of the total morbidity. after that, there are infectious and parasitic diseases, with about 0.6%. conditions belonging to the group of pregnancies, births and postpartum period make up 0.5% of the total number of patients. the five most common diagnoses in women were: pelvic inflammatory diseases (19.9%), menstrual disorders (17.8%), breast disease (13.4%), menopausal symptoms and cystitis (9.9%). these five diagnoses make up as much as 71.6% of all registered diseases. according to data from 2015, the municipality of aleksinac has 49,290 inhabitants on an area of 707 km2 (statistical office of the republic of serbia, 2016). based on the latest research by the public health institute niš (2017), there were a total of 57,242 patients in 2015. cardiovascular diseases took the first place, accounting for 24.3% of the total morbidity. next were respiratory diseases, with about 19.0%. in the third place are musculoskeletal and connective tissue diseases with 8.3%, and in the fourth place are genitourinary diseases with 7.0% of the total number of patients. endocrine diseases make up 5.9%, while mental and behavioral disorders make up 4.4%. there are also diseases from other groups that together contribute to the total morbidity with 31.0%. the most common diagnosis was high blood pressure with 19.9% of the total number of all diseases. other diseases are: acute pharyngitis and tonsillitis (6.6%), back diseases (5.7%), diabetes (4.4%), cystitis (3.7%). other types of diagnoses make up the remaining 59.8%. in the department for women’s health service of the municipality of aleksinac, there were 3,464 registered patients during 2015. women mostly suffered from genitourinary diseases, as many as 78.7%. in the second place are the factors that affect the health condition and contact with the health service with a share in the total morbidity of 9.5%. they are followed by diseases from the group of tumors with 7.8%, then conditions belonging to the group of pregnancies, births and postpartum period with 2.2% and diseases from the group of symptoms, which includes pathological clinical and laboratory findings with 1.0%. blood and blood cell diseases as well as endocrine diseases make up 0.3% of the total number of patients. other registered diseases account for 0.4%. frequent diagnoses in patients were: other pelvic inflammatory diseases (25.5%), menstrual disorders (16.2%), other genitourinary diseases, cystitis and menopause (8.4%) and menopausal diseases (8.2%). the remaining third of total diseases are other types of diagnoses. ethnopharmacological survey the survey has been conducted in a period of three months, more precisely from november 15, 2020 to january 15, 2021. we used a pre-compiled questionnaire, with the help of adonis pharmacy (https://www.adonisapoteka.rs). the questionnaires were completed thanks to employed masters of pharmacy, with the consent of the respondents, in accordance with all codes of ethics, in pharmacies located in the territory of southeastern serbia, in the three surveyed municipalities zaječar, sokobanja and aleksinac. a total of 100 surveys were collected: 13 in zaječar, 66 in sokobanja, and 21 in aleksinac. a total of 46 men and 54 women aged 1 to 88 were surveyed. the questionnaire consisted 6 questions biologica nyssana ● 12 (2) december 2021: 87-111 matejić et al. ● frequency of herbal medicinal products use in southeastern serbia 90 (one close and five open), and gives the following data on the respondents: • gender • age • chronic diseases yes or no • chronic diseases they suffer from, if yes • treatment of chronic disease • name of the hmp they use the obtained data were then analyzed and compared with the literature. chronic disease treatment was classified based on groups of drugs as prescribed by the agency for drugs and medical devices of serbia (2021). the hmp was studied in detail, starting from its pharmaceutical form, through the plants that are part of it and contribute to its action. for each plant species, we specified its folk and latin name according to the plant list (2000) (http://www.theplantlist.org), the medicinal part used and the form in which it is contained in the medicine. then, each hmp was associated with an appropriate chronic disease, the therapy of which it could contribute to. in the end, we did a systematization according to plant species (tab.1). statistical analysis the characteristics of the study group were defined as frequency (with or without percentage). kruskal-wallis test and mann-whitney u test (when data normality was not satisfied) were used to compare continuous data regarding the use of plants preparations and plants species between defined groups of informants. the chi-square (χ2) test was used to compare the distribution of plants preparations and plants species use between defined groups of informants. statistical analyses were performed using spss statistical software (version 20.0) at the significance level set at p<0.05. results and discussion quantitative analyses the results of the research showed that there were 70 different herbal medicines in use. the highest percentage was used preventively (32.00%), while the others were used in hypertension (23.50%), prostate hyperplasia (8.50%), diabetes (6.53%), depression (4.60%), glaucoma (3.90%) and angina pectoris and neurasthenia (2.60%). in their regular therapy, patients used drugs from the following groups: beta blockers (12.30%); ace inhibitors, calcium antagonists and prostate hyperplasia therapy (6.60%); oral antidiabetics (6.20%); antihypertensive (5.30%); benzodiazepines (4.90%) and glaucoma therapy (4.10%). the most commonly used drugs were: betamsal (0.4 mg) and concor cor (1.25 mg) (2.90%); cornelin (10 mg) and tamsol (0.4 mg) (2.50%); bromazepam (3 mg), diazepam (5 mg), tensec (5 mg) and xalatan sol (2.10%); cosopt sol, glucophage (1000 mg) and spironolakton (25 mg) (1.60%) and amaryl (2 mg), binevol (5 mg), cornelin (20 mg), diclofenac duo (75 mg), enalapril (20 mg), prilinda (5 mg) and tritace comp (5 mg + 25 mg) (1.20%). the largest number of hmp was in the form of: capsules (25.50%); tablets (23.60%); herbal tea (17.30%); solution (6.40%) and syrup (5.50%). the most commonly used were: urasan forte (7.30%); comprosta forte (6.40%); phlebodia 600 (4.50%); senna tea (3,60%); bilobil intense, peppermint tea, palisept and venodia plus (2.70%), alpenkräuter cream, gingival gel, hipervag intim, lagosa 150, prospan syrup, prospan effervescent tablets, prostamol uno, prostenal perfect, relax, rtanj tea (winter savory) and venalek (1.8%). herbs whose medicinal ingredients are part of herbal medicines include 84 different species. all species originate from 45 different families, of which the largest number of species originate from the following families: rutaceae (6.30%); rosaceae (3.60%); myrtaceae and urticaceae (2.70%). the most common were the following species: citrus x aurantium l. (5.90%); cucurbita moschata duchesne (3.60%); brassica napus l., foeniculum vulgare mill. and valeriana officinalis l. (3.20%); ginkgo biloba l., melissa officinalis l., senna alexandrina mill. and urtica dioica l. (2.70%); hedera helix l., hypericum perforatum l., mentha x piperita l., rosmarinus officinalis l. and origanum vulgare l. (2.30%); betula pubescens ehrh., calendula officinalis l., eucalyptus globulus labill., humulus lupulus l., matricaria chamomilla l., rosa canina l., vaccinium myrtillus l. and zea mays l. (1.80%); achillea millefolium l., aesculus hippocastanum l., equisetum arvense l., glycyrrhiza glabra l., mentha arvensis l., oenothera biennis l., petroselinum crispum (mill.) fuss, silybum marianum (l.) gaertn. and vitis vinifera l. (1.40%). the parts of the plant that were used as the source of ingredients in the largest percentage were as follows: folium (36.20%); flos (12.70%); herba and radix (10.40%); fructus (10.00%); semen (7.70%) and pericarpium (6.30%). herbal ingredients were most often in the form of an extract (49.30%); then in the form of crushed herbal drugs (17.60%); essential oil (15.80%); isolated substances (7.20%) and in the form of powdered herbal drugs (5.40%). statistical analysis a detailed statistical data processing was used to determine the frequency of use of hmp and plants in southeastern serbia. biologica nyssana ● 12 (2) december 2021: 87-111 matejić et al. ● frequency of herbal medicinal products use in southeastern serbia 91 biologica nyssana ● 12 (2) december 2021: 87-111 matejić et al. ● frequency of herbal medicinal products use in southeastern serbia ta bl e 1. f re qu en cy o f h m p us e in s ou th ea st er n se rb ia l at in n am e, s er bi an na m e an d fa m ily pa rt o f a pl an t fo rm ph ar m . f or m n am e of h m p a ll m ed ic in es us ed w ith h m p t he ra py gr ou ps pr es en t d is ea se s w he n h m p w as u se d ab ie s s ib ir ic a le de b. , si bi rs ka je la , p in ac ea e fo liu m es se nt ia l o il cr ea m a lp sk a kr em a / / / ab ie s a lb a m ill ., je la , pi na ce ae fo liu m es se nt ia l o il oi nt m en t pa vl e ba lz am / / / ac hi lle a m ill ef ol iu m l. , h aj du čk a tra va , a st er ac ea e he rb a ex tra ct oi nt m en t, liq ui d a sa m c rv en i ko nj sk i ba lz am , fe m is an a ka pi , f em is an b k ap i b ro m az ep am (3 m g) , si da ta (5 0 m g) , v iv ac e (5 m g) , v iv ac e pl us a c e in hi bi to rs , an tid ep re ss an t dr ug , an tih yp er te ns iv e dr ug , be nz od ia ze pi ne de pr es si on , h yp er te ns io n ae sc ul us hi pp oc as ta nu m l. , d iv lji k es te n, sa pi nd ac ea e se m en ex tra ct , es se nt ia l o il ge ll, o in tm en t, ta bl et a sa m c rv en i ko nj sk i ba lz am , er ba ve n re ta rd , k es te n ge l b ro m az ep am (3 m g) , si da ta (5 0 m g) , v iv ac e (5 m g) , v iv ac e pl us a c e in hi bi to rs , an tid ep re ss an t dr ug , an tih yp er te ns iv e dr ug , be nz od ia ze pi ne de pr es si on , h yp er te ns io n al ch em ill a xa nt ho ch lo ra r ot hm ., vi ra k, r os ac ea e he rb a ex tra ct liq ui d fe m is an a ka pi / / / al oe fe ro x m ill ., al oj a, x an th or rh oe ac ea e su cc us oi l, po w de re d he rb al su bs ta nc e cr ea m , t ab le t te n he rb s, v ita lis sp or ts cr ea m / / ch ro ni c ob st ip at io n al th ae a offi ci na lis l ., be li sl ez , m al va ce ae fo liu m , r ad ix ex tra ct , fr ag m en te d he rb al su bs ta nc e he rb al te a, pa st ill es , s yr up b eb a c si ru p be lo g sl ez a, č aj k or en a be lo g sl ez a, č aj p ro tiv br on hi tis a, h er bi ko si ru p be lo g sl ez a, pr ot ec t b el i sl ez b ip re z (2 .5 m g) , c or ne lin (1 0 m g) , en al ap ril (1 0 m g) , k lo m et ol (1 0 m g) , m ac ro pe n (4 00 m g) , m ol ic or (2 m g) a c e in hi bi to rs , ca lc iu m an ta go ni st s, an tia ng in al d ru g, an tie m et ic d ru g, be ta b lo ck er , m ac ro lid e an tib io tic an gi na p ec to ris , hy pe rte ns io n biologica nyssana ● 12 (2) december 2021: 87-111 92 matejić et al. ● frequency of herbal medicinal products use in southeastern serbia an no na m ur ic at a l. , gr av io la , a nn on ac ea e fo liu m po w de re d he rb al su bs ta nc e ca ps ul e a m az on sk a gr av io la b is op ro lo l (5 m g) , i ru zi d (2 0 m g+ 1 2. 5 m g) an tih yp er te ns iv e dr ug , b et a bl oc ke r hy pe rte ns io n ar ct os ta ph yl os u va ur si (l .) sp re ng ., uv a, er ic ac ea e fo liu m fr ag m en te d he rb al su bs ta nc e he rb al te a č aj li st a uv e, k irk ol in a lu x ča j z a m rš av lje nj e b ip re z (2 .5 m g) , c or ne lin (1 0 m g) , m ol ic or (2 m g) ca lc iu m an ta go ni st s, an tia ng in al dr ug , b et a bl oc ke r an gi na p ec to ris , hy pe rte ns io n ar te m is ia a bs in th iu m l. , p el en , a st er ac ea e he rb a tin ct ur e sy ru p a m ar ap ha rm si ru p / / / be tu la p ub es ce ns eh rh ., br ez a, b et ul ac ea e fo liu m ex tra ct , tin ct ur e, fr ag m en te d he rb al su bs ta nc e he rb al te a, po w de r, liq ui d k ap i z a m rš av lje nj e, u ro bi tra t in st an t č aj , u ro pr ot ek t č aj in ko nt an (1 5 m g) , m ar oc en (5 00 m g) , pa nr az ol (2 0 m g) pr ot on p um p in hi bi to r, qu in ol on e an tib io tic , in co nt in en ce tre at m en t in co nt in en ce br as si ca n ap us l. , u lja na re pi ca , b ra ss ic ac ea e flo s ex tra ct ta bl et c on pr os ta fo rte a m ar yl (2 m g) , a m ar yl (4 m g) , a m lo di pi n (5 m g) , b en ep ro st (5 m g) , b et am sa l (0 .4 m g) , b in ev ol (5 m g) , b ro m az ep am (3 m g) , c or ne lin (1 0 m g) , c or ne lin (2 0 m g) , c os op t so l, d at us t (0 .5 m g) , d ila co r (0 .2 5 m g) , g lu co ph ag e (1 00 0 m g) , a c e in hi bi to rs , ca lc iu m an ta go ni st s, an tih yp er te ns iv e dr ug , be nz od ia ze pi ne , be ta b lo ck er , in su lin , ca rd io to ni c ag en t, or al an tid ia be tic dr ug , g la uc om a tre at m en t, be ni gn p ro st at ic hy pe rp la si a tre at m en t, vi ta m in at ria l fi br ill at io n an d at ria l fl ut te r, de pr es si on , gl au co m a, b en ig n pr os ta tic h yp er pl as ia , hy pe rte ns io n, d ia be te s ty pe 2 , d ia be te s t yp e 1 l at in n am e, s er bi an na m e an d fa m ily pa rt o f a pl an t fo rm ph ar m . f or m n am e of h m p a ll m ed ic in es us ed w ith h m p t he ra py gr ou ps pr es en t d is ea se s w he n h m p w as u se d biologica nyssana ● 12 (2) december 2021: 87-111 matejić et al. ● frequency of herbal medicinal products use in southeastern serbia 93 l at in n am e, s er bi an na m e an d fa m ily pa rt o f a pl an t fo rm ph ar m . f or m n am e of h m p a ll m ed ic in es us ed w ith h m p t he ra py gr ou ps pr es en t d is ea se s w he n h m p w as u se d in su m an b as al so lo st ar (1 00 ij/ m l), m on op ril pl us (2 0 m g + 12 .5 m g) , p ril in da (5 m g) , p rin or m (1 00 m g) , t am so l (0 .4 m g) , te lm ic or (4 0 m g) , t en se c (5 m g) , t rit ac e co m p (5 m g + 25 m g) , v ig an to l s ol , x al at an so l c al en du la o ffi ci na lis l. , n ev en , a st er ac ea e flo s ex tra ct , es se nt ia l o il liq ui d, v ag in al ta bl et fe m is an a ka pi , f em is an b k ap i, h ip er va g in tim / / / c am el lia si ne ns is (l .) k un tz e, z el en i č aj , th ea ce ae fo liu m fr ag m en te d he rb al su bs ta nc e he rb al te a k irk ol in a lu x ča j z a m rš av lje nj e / / / c ar um c ar vi l ., ki m , a pi ac ea e fr uc tu s ex tra ct po w de r h ip p ča j z a do jil je / / / c en ta ur iu m e ry th ra ea r af n, k ič ic a, g en tia na ce ae he rb a tin ct ur e sy ru p a m ar ap ha rm si ru p / / / c en te lla a si at ic a (l .) u rb ., ce nt el a, a pi ac ea e he rb a ex tra ct ta bl et er ba ve n re ta rd / / / c he lid on iu m m aj us l ., liš ei vi ca , p ap av er ac ea e he rb a fr ag m en te d he rb al su bs ta nc e he rb al te a č aj h er be liš ei vi ce / / / c itr us li m on (l .) o sb ec k, li m un , pe ric ar pi um es se nt ia l o il ca ps ul e g el om yt ro l fo rte b ru fe n (4 00 m g) no ns te ro id al si nu si tis biologica nyssana ● 12 (2) december 2021: 87-111 94 matejić et al. ● frequency of herbal medicinal products use in southeastern serbia l at in n am e, s er bi an na m e an d fa m ily pa rt o f a pl an t fo rm ph ar m . f or m n am e of h m p a ll m ed ic in es us ed w ith h m p t he ra py gr ou ps pr es en t d is ea se s w he n h m p w as u se d r ut ac ea e an tiin fla m m at ory d ru g c itr us x a ur an tiu m l. , g or ka n ar an dž a, r ut ac ea e pe ric ar pi um is ol at ed a ct iv e co m po ne nt s, es se nt ia l o il ca ps ul e, ta bl et d et ra le x, fl ux iv , g el om yt ro l fo rte , ph le bo di a (6 00 m g) , v en al ek , v en od ia p lu s a m lo di pi n (1 0 m g) , a zo pt so l, b et am sa l (0 .4 m g) , b en ep ro st (5 m g) , b is op ro lo l (2 .5 m g) , b ro m az ep am (3 m g) , b ru fe n (4 00 m g) , c ar di op iri n (1 00 m g) , c on co r co r ( 1. 25 m g) , c or as p (1 00 m g) , c or ne lin (2 0 m g) , d ia ze pa m (5 m g) , en al ap ril (1 0 m g) , en al ap ril (2 0 m g) , f ar in (5 m g) , f le ka ni d (1 00 m g) , h em op re s (5 0 m g + 5 m g) , h yp ol ip (2 0 m g) , la si x (4 0 m g) , m on iz ol (2 0 m g) , n ev ot en s ( 5 m g) , pr es ol ol (5 0 m g) , sp iro no la kt on (2 5 m g) , t en se c (5 m g) , tr im et ac or (3 5 m g) , a c e in hi bi to rs , ca lc iu m an ta go ni st s, an tia ng in al d ru g, an tia rr hy th m ic dr ug , be nz od ia ze pi ne , be ta b lo ck er , di ur et ic , hy po lip id em ic dr ug , a nt ip la te le t ag en t, no ns te ro id al an tiin fla m m at or y dr ug , o ra l an tic oa gu la nt , gl au co m a tre at m en t, be ni gn p ro st at ic hy pe rp la si a tre at m en t an gi na p ec to ris , de pr es si on , g la uc om a, hy pe rli pi da em ia , b en ig n pr os ta tic h yp er pl as ia , hy pe rte ns io n, ne ur as th en ia , s in us iti s, ca rd ia c in su ffi ci en cy , ve in th ro m bo si s biologica nyssana ● 12 (2) december 2021: 87-111 matejić et al. ● frequency of herbal medicinal products use in southeastern serbia 95 l at in n am e, s er bi an na m e an d fa m ily pa rt o f a pl an t fo rm ph ar m . f or m n am e of h m p a ll m ed ic in es us ed w ith h m p t he ra py gr ou ps pr es en t d is ea se s w he n h m p w as u se d v az ot al (1 0 m g) , v iv ac e (2 .5 m g) , x al at an so l c uc ur bi ta m os ch at a d uc he sn e, m us ka tn a tik va , c uc ur bi ta ce ae se m en ex tra ct ca ps ul e u ra sa n fo rte a m ar yl (2 m g) , c ar di op iri n (7 5 m g) , c on co r co r ( 1. 25 m g) , c or ne lin (1 0 m g) , c os op t s ol , d ak ta no l k re m , d ia ze pa m (5 m g) , m ol si do m in (4 m g) , m on op ril (1 0 m g) , n eb ile t ( 5 m g) , n itr og lic er in (0 .5 m g) , pa ra va no (2 0 m g) , p la vi x (7 5 m g) , si no de rm n , st ug er on fo rte , ta m so l ( 0. 4 m g) , te fo r ( 85 0 m g) , tr ita ce c om p (2 .5 m g + 12 .5 m g) , v az ot al (5 m g) , x al at an so l, zo fe ca rd (3 0 m g) a c e in hi bi to rs , ca lc iu m an ta go ni st s, an tia ng in al d ru g, an tih yp er te ns iv e dr ug , an tim yc ot ic dr ug , be nz od ia ze pi ne , be ta b lo ck er , de rm at ic a ge nt , hy po lip id em ic dr ug , a nt ip la te le t ag en t, or al an tid ia be tic dr ug , g la uc om a tre at m en t, be ni gn p ro st at ic hy pe rp la si a tre at m en t, ve rti go tre at m en t an gi na p ec to ris , de pr es si on , g la uc om a, hy pe rli pi da em ia , b en ig n pr os ta tic h yp er pl as ia , hy pe rte ns io n, c on ta ct de rm at iti s, ne ur as th en ia , pi ty ria si s, ca rd ia c in su ffi ci en cy , d ia be te s ty pe 2 c ur cu m a lo ng a l. , k ur ku m a, zi ng ib er ac ea e ra di x ex tra ct ca ps ul e k ur ku m a fo rte d ic lo fe na c du o (7 5 m g) no ns te ro id al an tiin fla m m at or y dr ug os te op or os is r ut ac ea e an tiin fla m m at ory d ru g c itr us x a ur an tiu m l. , g or ka n ar an dž a, r ut ac ea e pe ric ar pi um is ol at ed a ct iv e co m po ne nt s, es se nt ia l o il ca ps ul e, ta bl et d et ra le x, fl ux iv , g el om yt ro l fo rte , ph le bo di a (6 00 m g) , v en al ek , v en od ia p lu s a m lo di pi n (1 0 m g) , a zo pt so l, b et am sa l (0 .4 m g) , b en ep ro st (5 m g) , b is op ro lo l (2 .5 m g) , b ro m az ep am (3 m g) , b ru fe n (4 00 m g) , c ar di op iri n (1 00 m g) , c on co r co r ( 1. 25 m g) , c or as p (1 00 m g) , c or ne lin (2 0 m g) , d ia ze pa m (5 m g) , en al ap ril (1 0 m g) , en al ap ril (2 0 m g) , f ar in (5 m g) , f le ka ni d (1 00 m g) , h em op re s (5 0 m g + 5 m g) , h yp ol ip (2 0 m g) , la si x (4 0 m g) , m on iz ol (2 0 m g) , n ev ot en s ( 5 m g) , pr es ol ol (5 0 m g) , sp iro no la kt on (2 5 m g) , t en se c (5 m g) , tr im et ac or (3 5 m g) , a c e in hi bi to rs , ca lc iu m an ta go ni st s, an tia ng in al d ru g, an tia rr hy th m ic dr ug , be nz od ia ze pi ne , be ta b lo ck er , di ur et ic , hy po lip id em ic dr ug , a nt ip la te le t ag en t, no ns te ro id al an tiin fla m m at or y dr ug , o ra l an tic oa gu la nt , gl au co m a tre at m en t, be ni gn p ro st at ic hy pe rp la si a tre at m en t an gi na p ec to ris , de pr es si on , g la uc om a, hy pe rli pi da em ia , b en ig n pr os ta tic h yp er pl as ia , hy pe rte ns io n, ne ur as th en ia , s in us iti s, ca rd ia c in su ffi ci en cy , ve in th ro m bo si s biologica nyssana ● 12 (2) december 2021: 87-111 96 matejić et al. ● frequency of herbal medicinal products use in southeastern serbia l at in n am e, s er bi an na m e an d fa m ily pa rt o f a pl an t fo rm ph ar m . f or m n am e of h m p a ll m ed ic in es us ed w ith h m p t he ra py gr ou ps pr es en t d is ea se s w he n h m p w as u se d ec hi na ce a pu rp ur ea (l .) m oe nc h, e hi na ce a, a st er ac ea e he rb a ex tra ct eff er ve sc en t ta bl et b io gr ip in / / / eq ui se tu m a rv en se l ., ra st av ić , e qu is et ac ea e he rb a fr ag m en te d he rb al su bs ta nc e he rb al te a č aj p ro tiv br on hi tis a, u ro pr ot ek t č aj b ip re z (2 .5 m g) , c or ne lin (1 0 m g) , m ol ic or (2 m g) ca lc iu m an ta go ni st s, an tia ng in al d ru g, be ta b lo ck er an gi na p ec to ris , hy pe rte ns io n eu ca ly pt us g lo bu lu s la bi ll. , e uk al ip tu s, m yr ta ce ae fo liu m es se nt ia l o il ca ps ul e, c re am a lp sk a kr em a, g el om yt ro l fo rte , v ita lis sp or ts c re am b ru fe n (4 00 m g) no ns te ro id al an tiin fla m m at or y dr ug si nu si tis fo en ic ul um v ul ga re m ill ., m or ač , a pi ac ea e fr uc tu s ex tra ct , es se nt ia l o il, po w de re d he rb al su bs ta nc e , t in ct ur e, fr ag m en te d he rb al su bs ta nc e he rb al te a, oi nt m en t, po w de r, liq ui d, ta bl et a sa m c rv en i ko nj sk i ba lz am , č aj p ro tiv br on hi tis a, h ip p ča j za d oj ilj e, k ap i z a m rš av lje nj e, pl an tla x ča j za g rč ev e, te n he rb s, u ro bi tra t in st an t č aj b ip re z (2 .5 m g) , b ro m az ep am (3 m g) , c or ne lin (1 0 m g) , i nk on ta n (1 5 m g) , m ar oc en (5 00 m g) , m ol ic or (2 m g) , pa nr az ol (2 0 m g) , si da ta (5 0 m g) , v iv ac e (5 m g) , v iv ac e pl us a c e in hi bi to rs , ca lc iu m an ta go ni st s, an tia ng in al d ru g, an tid ep re ss an t dr ug , an tih yp er te ns iv e dr ug , be nz od ia ze pi ne , be ta b lo ck er , pr ot on p um p in hi bi to r, qu in ol on e an tib io tic , in co nt in en ce tre at m en t an gi na p ec to ris , de pr es si on , hy pe rte ns io n, ch ro ni c ob st ip at io n, in co nt in en ce fr an gu la p ur sh ia na c oo pe r, am er ič ka kr uš in a, r ha m na ce ae co rte x po w de re d he rb al su bs ta nc e ta bl et te n he rb s / / ch ro ni c ob st ip at io n fr an gu la a ln us m ill ., kr uš in a, r ha m na ce ae co rte x tin ct ur e, fr ag m en te d he rb al su bs ta nc e he rb al te a, li qu id k ap i z a m rš av lje nj e, k irk ol in a lu x ča j z a m rš av lje nj e / / / biologica nyssana ● 12 (2) december 2021: 87-111 matejić et al. ● frequency of herbal medicinal products use in southeastern serbia 97 l at in n am e, s er bi an na m e an d fa m ily pa rt o f a pl an t fo rm ph ar m . f or m n am e of h m p a ll m ed ic in es us ed w ith h m p t he ra py gr ou ps pr es en t d is ea se s w he n h m p w as u se d g en tia na lu te a l. , lin cu ra , g en tia na ce ae ra di x po w de re d he rb al su bs ta nc e , tin ct ur e sy ru p, ta bl et a m ar ap ha rm si ru p, t en he rb s / / ch ro ni c ob st ip at io n g er an iu m ro be rt ia nu m l. , z dr av ac , g er an ia ce ae he rb a ex tra ct liq ui d fe m is an a ka pi / / / g in kg o bi lo ba l ., gi nk o, g in kg oa ce ae fo liu m ex tra ct ca ps ul e, ta bl et b ilo bi l in te ns e, g in ko m ax , g yn kg ob il, ta na ka n a m lo di pi n (1 0 m g) , b ar io s (5 m g) , b ev ip le x, b ip re z (2 .5 m g) , c on co r c or (1 .2 5 m g) , d ila co r ( 0. 25 m g) , en al ap ril (2 0 m g) , i nd ap re s (1 .5 m g) , m ol si do m in (4 m g) , p ol iv it b , p re so lo l ( 10 0 m g) , s ko pr yl (1 0 m g) , s pi ro no la kt on (2 5 m g) , ta m so l ( 0. 4 m g) , tr ita ce c om p (5 m g + 25 m g) , v es tib o (1 6 m g) a c e in hi bi to rs , ca lc iu m an ta go ni st s, an tia ng in al d ru g, an tih yp er te ns iv e dr ug , b et a bl oc ke r, di ur et ic , ca rd io to ni c ag en t, be ni gn pr os ta tic hy pe rp la si a tre at m en t, ve rti go tre at m en t, vi ta m in be ni gn p ro st at ic hy pe rp la si a, hy pe rte ns io n, v oc al c or d an d la ry nx p ar al ys is , ca rd ia c in su ffi ci en cy , ve rti go a nd ti nn itu s g ly ci ne m ax (l .) m er r., so ja , f ab ac ea e se m en is ol at ed a ct iv e co m po ne nt s ca ps ul e es se nt ia le fo rte n (3 00 m g) fo lk is (5 m g) , h em ok vi n pl us , ly ric a (2 5 m g) , pr on is on (2 0 m g) , sa la zo py rin (5 00 m g) , t am so l (0 .4 m g) an tia ne m ic dr ug fo r m eg al ob la st ic an em ia , an tie pi le pt ic dr ug , an tih yp er te ns iv e dr ug , de pr es si on , b en ig n pr os ta tic h yp er pl as ia , hy pe rte ns io n, u lc er at iv e co lit is biologica nyssana ● 12 (2) december 2021: 87-111 matejić et al. ● frequency of herbal medicinal products use in southeastern serbia l at in n am e, s er bi an na m e an d fa m ily pa rt o f a pl an t fo rm ph ar m . f or m n am e of h m p a ll m ed ic in es us ed w ith h m p t he ra py gr ou ps pr es en t d is ea se s w he n h m p w as u se d gl uc oc or tic oi d, su lp ho na m id e, be ni gn p ro st at ic hy pe rp la si a tre at m en t g ly cy rr hi za g la br a l. , sl ad ić , f ab ac ea e ra di x po w de re d he rb al su bs ta nc e , fr ag m en te d he rb al su bs ta nc e he rb al te a, ta bl et č aj p ro tiv br on hi tis a, č aj sl at ko g ko re na , t en he rb s b ip re z (2 .5 m g) , c or ne lin (1 0 m g) , m ol ic or (2 m g) ca lc iu m an ta go ni st s, an tia ng in al d ru g, be ta b lo ck er an gi na p ec to ris , hy pe rte ns io n, c hr on ic ob st ip at io n, li ve r st ea to si s h ed er a he lix l ., br šl ja n, a ra lia ce ae fo liu m ex tra ct sy ru p, p ow de r, eff er ve sc en t ta bl et in tu si n pu lv is , pr os pa n si ru p, pr os pa n šu m eć e ta bl et e / / / h um ul us lu pu lu s l ., hm el j, c an na ba ce ae flo s ex tra ct oi nt m en t, liq ui d, ta bl et a sa m c rv en i ko nj sk i ba lz am , k ap i za u m ire nj e, r el ax b ro m az ep am (3 m g) , n ov om ix 30 f le xp en (1 00 ij/ m l), si da ta (5 0 m g) , v iv ac e (5 m g) , v iv ac e pl us a c e in hi bi to rs , an tid ep re ss an t dr ug , an tih yp er te ns iv e dr ug , be nz od ia ze pi ne , in su lin ce lia c di se as e, de pr es si on , hy pe rte ns io n, d ia be te s ty pe 1 h yp er ic um p er fo ra tu m l. , k an ta rio n, h yp er ic ac ea e flo s ex tra ct , es se nt ia l o il liq ui d, o il ex tra ct , v ag in al ta bl et h ip er va g in tim , k ap i z a um ire nj e, u lje od k an ta rio na za sp ol ja šn ju up ot re bu , v ag in al et e sa ka nt ar io no m b ar io s ( 5 m g) , c on co r c or (1 .2 5 m g) , c or ne lin (1 0 m g) ca lc iu m an ta go ni st s, be ta bl oc ke r hy pe rte ns io n la va nd ul a an gu st ifo lia m ill ., la va nd a, la m ia ce ae flo s es se nt ia l o il ge ll k es te n ge l / / / 98 biologica nyssana ● 12 (2) december 2021: 87-111 matejić et al. ● frequency of herbal medicinal products use in southeastern serbia l at in n am e, s er bi an na m e an d fa m ily pa rt o f a pl an t fo rm ph ar m . f or m n am e of h m p a ll m ed ic in es us ed w ith h m p t he ra py gr ou ps pr es en t d is ea se s w he n h m p w as u se d m al pi gh ia e m ar gi na ta d c ., ac er ol a, m al pi gh ia ce ae fr uc tu s ex tra ct eff er ve sc en t ta bl et b io gr ip in / / / m ar ru bi um v ul ga re l ., oč aj ni ca , l am ia ce ae he rb a po w de re d he rb al su bs ta nc e ta bl et te n he rb s / / ch ro ni c ob st ip at io n m at ri ca ri a ch am om ill a l. , k am ili ca , a st er ac ea e flo s ex tra ct ge ll, o in tm en t, sy ru p a sa m c rv en i ko nj sk i ba lz am , g in gi va l g el , h er bi ko si ru p be lo g sl ez a a m ar yl (2 m g) , a sp iri n pr ot ec t (1 00 m g) , b ro m az ep am (3 m g) , g lu co ph ag e (1 00 0 m g) , s id at a (5 0 m g) , v iv ac e (5 m g) , v iv ac e pl us a c e in hi bi to rs , an tid ep re ss an t dr ug , an tih yp er te ns iv e dr ug , be nz od ia ze pi ne , an tip la te le t ag en t, or al an tid ia be tic d ru g de pr es si on , hy pe rte ns io n, d ia be te s ty pe 2 m el is sa o ffi ci na lis l ., m at ič nj ak , l am ia ce ae fo liu m ex tra ct oi nt m en t, po w de r, liq ui d, ta bl et a sa m c rv en i ko nj sk i ba lz am , fe m is an b ka pi , h ip p ča j z a do jil je , k ap i z a um ire nj e, r el ax b ro m az ep am (3 m g) , n ov om ix 30 f le xp en (1 00 ij/ m l), s id at a (5 0 m g) , v iv ac e (5 m g) , v iv ac e pl us a c e in hi bi to rs , an tid ep re ss an t dr ug , an tih yp er te ns iv e dr ug , be nz od ia ze pi ne , in su lin ce lia c di se as e, de pr es si on , hy pe rte ns io n, d ia be te s ty pe 1 m en th a ar ve ns is l. , p ol js ka n an a, la m ia ce ae fo liu m es se nt ia l o il cr ea m a lp sk a kr em a, v ita lis sp or ts cr ea m / / / m en th a x pi pe ri ta l. , p ito m a na na , la m ia ce ae fo liu m es se nt ia l o il, fr ag m en te d he rb al su bs ta nc e he rb al te a, g el l č aj li st a na ne , g in gi va l g el a m ar yl (2 m g) , a sp iri n pr ot ec t (1 00 m g) , g lu co ph ag e (1 00 0 m g) an tip la te le t ag en t, or al an tid ia be tic d ru g hy pe rte ns io n, d ia be te s ty pe 2 99 biologica nyssana ● 12 (2) december 2021: 87-111 matejić et al. ● frequency of herbal medicinal products use in southeastern serbia l at in n am e, s er bi an na m e an d fa m ily pa rt o f a pl an t fo rm ph ar m . f or m n am e of h m p a ll m ed ic in es us ed w ith h m p t he ra py gr ou ps pr es en t d is ea se s w he n h m p w as u se d m yr tu s c om m un is l ., m irt a, m yr ta ce ae fo liu m es se nt ia l o il ca ps ul e, oi nt m en t g el om yt ro l fo rte , p av le ba lz am b ru fe n (4 00 m g) no ns te ro id al an tiin fla m m at or y dr ug si nu si tis o ci m um b as ili cu m l ., bo si lja k, l am ia ce ae fo liu m es se nt ia l o il liq ui d si nu so l m aj or an sp re j za n os a sp iri n pr ot ec t (1 00 m g) , b is op ro lo l (5 m g) , b ro m az ep am (3 m g) , d ic lo fe na c du o (7 5 m g) , h em om yc in (5 00 m g) be nz od ia ze pi ne , be ta b lo ck er , an tip la te le t ag en t, m ac ro lid e an tib io tic , no ns te ro id al an tiin fla m m at or y dr ug hy pe rte ns io n o en ot he ra b ie nn is l ., no ću ra k, o na gr ac ea e se m en es se nt ia l o il, oi l ca ps ul e, v ag in al ta bl et h ip er va g in tim , s ol ga r ul je n oć ur ka / / / o ri ga nu m v ul ga re l. , v ra ni lo va tr av a, la m ia ce ae fo liu m is ol at ed a ct iv e co m po ne nt s, fr ag m en te d he rb al su bs ta nc e he rb al te a, ca ps ul e pa lis ep t, u ro pr ot ek t č aj a ct aw el l (1 00 m g) , b in ev ol (5 m g) , b ro m az ep am (3 m g) , c or ne lin (2 0 m g) , e lic ea (1 0 m g) , i rb en id a pl us (1 50 m g + 12 .5 m g) , k ar vi le ks (1 2. 5 m g) , l ex ili um (3 m g) , l un at a (5 m g) , t ef or (5 00 m g) , t re si ba fl ex to uc h (2 00 ij/ m l), t rit ac e co m p (2 .5 m g + 12 .5 m g) ca lc iu m an ta go ni st s, an tid ep re ss an t dr ug , an tih yp er te ns iv e dr ug , an tip sy ch ot ic dr ug , be nz od ia ze pi ne , be ta b lo ck er , hy pn ot ic d ru g, in su lin , o ra l an tid ia be tic d ru g de pr es si on , hy pe rte ns io n, d ia be te s ty pe 1 100 biologica nyssana ● 12 (2) december 2021: 87-111 matejić et al. ● frequency of herbal medicinal products use in southeastern serbia l at in n am e, s er bi an na m e an d fa m ily pa rt o f a pl an t fo rm ph ar m . f or m n am e of h m p a ll m ed ic in es us ed w ith h m p t he ra py gr ou ps pr es en t d is ea se s w he n h m p w as u se d o ri ga nu m n m aj or an a l. , m aj or an , la m ia ce ae fo liu m es se nt ia l o il liq ui d si nu so l m aj or an sp re j za n os a sp iri n pr ot ec t (1 00 m g) , b is op ro lo l (5 m g) , b ro m az ep am (3 m g) , d ic lo fe na c du o (7 5 m g) , h em om yc in (5 00 m g) be nz od ia ze pi ne , be ta b lo ck er , an tip la te le t ag en t, m ac ro lid e an tib io tic , no ns te ro id al an tiin fla m m at or y dr ug hy pe rte ns io n o rt ho si ph on a ri st at us (b lu m e) m iq ., ja va č aj , la m ia ce ae fo liu m ex tra ct po w de r u ro bi tra t in st an t č aj in ko nt an (1 5 m g) , m ar oc en (5 00 m g) , pa nr az ol (2 0 m g) pr ot on p um p in hi bi to r, qu in ol on e an tib io tic , in co nt in en ce tre at m en t in co nt in en ce pa ss ifl or a ed ul is si m s, pa si flo ra , pa ss ifl or ac ea e he rb a ex tra ct ta bl et a lo ra u ltr a, sm ire la n oć c oa m le ss a (4 m g + 5 m g + 1. 25 m g) , eg lo ny l ( 50 m g) an tih yp er te ns iv e dr ug , an tip sy ch ot ic dr ug ga st ro -in te st in al ne ur os es , h yp er te ns io n pe tr os el in um c ri sp um (m ill .) fu ss , p er šu n, a pi ac ea e fo liu m ex tra ct , fr ag m en te d he rb al su bs ta nc e he rb al te a, li qu id fe m is an a k ap i, u ro pr ot ek t č aj / / / pi m pi ne lla a ni su m l ., an is , a pi ac ea e fr uc tu s ex tra ct po w de r h ip p ča j z a do jil je / / / pi nu s s yl ve st ri s l ., sr eb rn i b or , p in ac ea e fo liu m et ar sk o ul je oi nt m en t pa vl e ba lz am / / / pl an ta go la nc eo la ta l. , b ok vi ca , pl an ta gi na ce ae fo liu m ex tra ct , fr ag m en te d he rb al su bs ta nc e he rb al te a, li qu id č aj p ro tiv br on hi tis a, pr op ol is k ap i sa b ok vi co m b ip re z (2 .5 m g) , c or ne lin (1 0 m g) , m ol ic or (2 m g) an tia ng in al dr ug , c al ci um an ta go ni st s, be ta b lo ck er an gi na p ec to ris , hy pe rte ns io n po te nt ill a er ec ta (l .) r ae us ch ., sr če nj ak , r os ac ea e rh iz om a tin ct ur e ge ll g in gi va l g el a m ar yl (2 m g) , a sp iri n pr ot ec t (1 00 m g) , an tip la te le t ag en t, hy pe rte ns io n, d ia be te s ty pe 2 101 biologica nyssana ● 12 (2) december 2021: 87-111 matejić et al. ● frequency of herbal medicinal products use in southeastern serbia l at in n am e, s er bi an na m e an d fa m ily pa rt o f a pl an t fo rm ph ar m . f or m n am e of h m p a ll m ed ic in es us ed w ith h m p t he ra py gr ou ps pr es en t d is ea se s w he n h m p w as u se d g lu co ph ag e (1 00 0 m g) or al a nt id ia be tic dr ug pr im ul a ve ri s l. , j ag or če vi na , pr im ul ac ea e fo liu m , r ad ix ex tra ct , fr ag m en te d he rb al su bs ta nc e he rb al te a, sy ru p b ro nh o sa n ja go rč ev in a, tim ija n, di vi zm a, č aj p ro tiv br on hi tis a b ip re z (2 .5 m g) , c or ne lin (1 0 m g) , m ol ic or (2 m g) ca lc iu m an ta go ni st s, an tia ng in al dr ug , b et a bl oc ke r an gi na p ec to ris , hy pe rte ns io n rh am nu s a lp in a l. , že st ik a, r ha m na ce ae co rte x po w de re d he rb al su bs ta nc e ta bl et te n he rb s / / ch ro ni c ob st ip at io n rh eu m p al m at um l. , r ab ar ba ra , po ly go na ce ae ra di x po w de re d he rb al su bs ta nc e ta bl et te n he rb s / / ch ro ni c ob st ip at io n ri be s n ig ru m l. , c rn a rib iz la , g ro ss ul ar ia ce ae fr uc tu s ex tra ct pa st ill es is la c as si s b is op ro lo l (2 .5 m g) be ta b lo ck er hy pe rte ns io n ri ci nu s c om m un is l ., ric in us , e up ho rb ia ce ae se m en ex tra ct oi l e xt ra ct r ic in us ov o ul je / / / ro sa c an in a l. , ši pu ra k, r os ac ea e fr uc tu s ex tra ct , fr ag m en te d he rb al su bs ta nc e am po ul e, h er ba l te a, li qu id , s yr up c h a lp ha pl us , h er bi ko si ru p be lo g sl ez a, k ap i z a m rš av lje nj e, k irk ol in a lu x ča j z a m rš av lje nj e d ic lo fe na c du o (7 5 m g) no ns te ro id al an tiin fla m m at or y dr ug ar th rit is ro sm ar in us o ffi ci na lis l. , r uz m ar in , la m ia ce ae fo liu m ex tra ct , es se nt ia l o il ge ll, c re am , liq ui d a lp sk a kr em a, k ap i za u m ire nj e, k es te n ge l, v ita lis sp or ts cr ea m / / / 102 biologica nyssana ● 12 (2) december 2021: 87-111 matejić et al. ● frequency of herbal medicinal products use in southeastern serbia l at in n am e, s er bi an na m e an d fa m ily pa rt o f a pl an t fo rm ph ar m . f or m n am e of h m p a ll m ed ic in es us ed w ith h m p t he ra py gr ou ps pr es en t d is ea se s w he n h m p w as u se d ru bu s i da eu s l ., m al in a, r os ac ea e fo liu m ex tra ct liq ui d fe m is an a ka pi / / / ru sc us a cu le at us l ., ve pr in a, a sp ar ag ac ea e rh iz om a ex tra ct ta bl et er ba ve n re ta rd / / / sa lix a lb a l. , b el a vr ba , s al ic ac ea e co rte x ex tra ct eff er ve sc en t ta bl et b io gr ip in / / / sa tu re ja m on ta na l ., rta nj sk i č aj , l am ia ce ae he rb a fr ag m en te d he rb al su bs ta nc e he rb al te a r ta nj sk i č aj c on co r c or (1 .2 5 m g) be ta b lo ck er hy pe rte ns io n se nn a al ex an dr in a m ill ., se na , f ab ac ea e fo liu m po w de re d he rb al su bs ta nc e , fr ag m en te d he rb al su bs ta nc e he rb al te a, ta bl et č aj li st a se ne , k irk ol in a lu x ča j z a m rš av lje nj e, te n he rb s a m lo ga l (1 0 m g) , a nt ia gr ex (7 5 m g) , c ar di op iri n (7 5 m g) , c on co r (5 m g) , d ia ze pa m (5 m g ), g lu fo rm in (1 00 0 m g) , h lo rp ro m az in (2 5 m g) , m ic ar di s (8 0 m g) , p ril in da (5 m g) , t en se c (5 m g) , t re nt al (4 00 m g) , v as ili p (2 0 m g) a c e in hi bi to rs , ca lc iu m an ta go ni st s, an tih yp er te ns iv e dr ug , an tip sy ch ot ic dr ug , be nz od ia ze pi ne , be ta b lo ck er , hy po lip id em ic dr ug , an tip la te le t ag en t, or al an tid ia be tic dr ug , c irc ul at or y di so rd er s tre at m en t de pr es si on , hy pe rli pi da em ia , hy pe rte ns io n, c hr on ic ob st ip at io n, d ia be te s ty pe 2 se re no a re pe ns (w . b ar ta m ) s m al l, te st er as ta p al m a, a re ca ce ae fr uc tu s ex tra ct ca ps ul e pr os ta m ol un o, p ro st en al pe rf ec t a m ar yl (3 m g) , a nd ol (1 00 m g) , b er lit hi on 60 0 (6 00 m g) , b er od ua l n (0 .0 5 m g + 0. 02 1 m g) , c ar di pi ne (5 m g) , a c e in hi bi to rs , ca lc iu m an ta go ni st s, an tia ng in al dr ug , an tih yp er te ns iv e dr ug , an gi na p ec to ris , at ria l fi br ill at io n an d at ria l fl ut te r, be ni gn pr os ta tic h yp er pl as ia , hy pe rte ns io n, c hr on ic ob st ru ct iv e pu lm on ar y di se as e, n eu ra st he ni a, di ab et es ty pe 2 103 biologica nyssana ● 12 (2) december 2021: 87-111 matejić et al. ● frequency of herbal medicinal products use in southeastern serbia l at in n am e, s er bi an na m e an d fa m ily pa rt o f a pl an t fo rm ph ar m . f or m n am e of h m p a ll m ed ic in es us ed w ith h m p t he ra py gr ou ps pr es en t d is ea se s w he n h m p w as u se d c on co r c or (1 .2 5 m g) , c or ne lin (1 0 m g) , d ia ze pa m (5 m g) , d ur ofi lin (1 25 m g) , g lio ks an (8 0 m g) , g lu fo rm in (1 00 0 m g) , g lu co ph ag e (1 00 0 m g) , le xi liu m (3 m g) , m on os an (2 0 m g) , sk op ry l p lu s, sp iro no la kt on (2 5 m g) , t am so l (0 .4 m g) , t ef or (5 00 m g) , t en se c (5 m g) , t rit ac e (5 m g) be nz od ia ze pi ne , be ta b lo ck er , br on ch os pa sm ol yt ic a ge nt , di ur et ic , a nt ipl at el et a ge nt , or al a nt id ia be tic dr ug , d ia be tic po ly ne ur op ath y tre at m en t, be ni gn p ro sta tic h yp er pl as ia tre at m en t si ly bu m m ar ia nu m (l .) g ae rtn ., si ka vi ca , a st er ac ea e fr uc tu s ex tra ct ca ps ul e, ta bl et la go sa 1 50 , si ly m ar in fo lk is (5 m g) , h em ok vi n pl us , ly ric a (2 5 m g) , pr on is on (2 0 m g) , sa la zo py rin (5 00 m g) , t am so l (0 .4 m g) an tia ne m ic dr ug fo r m eg al ob la st ic an em ia , an tie pi le pt ic dr ug , an tih yp er te ns iv e dr ug , gl uc oc or tic oi d, su lp ho na m id e, be ni gn p ro st at ic hy pe rp la si a tre at m en t de pr es si on , b en ig n pr os ta tic h yp er pl as ia , hy pe rte ns io n, li ve r st ea to si s, ul ce ra tiv e co lit is so lid ag o vi rg aau re a l. , z la tn ic a, a st er ac ea e he rb a ex tra ct po w de r u ro bi tra t in st an t č aj in ko nt an (1 5 m g) , pr ot on p um p in hi bi to r, in co nt in en ce 104 biologica nyssana ● 12 (2) december 2021: 87-111 matejić et al. ● frequency of herbal medicinal products use in southeastern serbia l at in n am e, s er bi an na m e an d fa m ily pa rt o f a pl an t fo rm ph ar m . f or m n am e of h m p a ll m ed ic in es us ed w ith h m p t he ra py gr ou ps pr es en t d is ea se s w he n h m p w as u se d m ar oc en (5 00 m g) , pa nr az ol (2 0 m g) qu in ol on e an tib io tic , in co nt in en ce tre at m en t st yp hn ol ob iu m ja po ni cu m (l .) sc ho tt, ja pa ns ki b ag re m , fa ba ce ae flo s is ol at ed a ct iv e co m po ne nt s ta bl et er ba ve n re ta rd , f lu xi v c on co r c or (1 .2 5 m g) , fl ek an id (1 00 m g) an tia rr hy th m ic dr ug , b et a bl oc ke r ca rd ia c in su ffi ci en cy sy m ph yt um offi ci na le l ., ga ve z, b or ag in ac ea e ra di x ex tra ct ge ll b ei nw el l g av ez g el / / / ta ra xa cu m ca m py lo de s g .e .h ag lu nd , m as la ča k, c om po si ta e ra di x po w de re d he rb al su bs ta nc e ta bl et te n he rb s / / ch ro ni c ob st ip at io n th ym us se rp yl lu m l. , m aj ki na d uš ic a, la m ia ce ae he rb a fr ag m en te d he rb al su bs ta nc e he rb al te a č aj p ro tiv br on hi tis a b ip re z (2 .5 m g) , c or ne lin (1 0 m g) , m ol ic or (2 m g) ca lc iu m an ta go ni st s, an tia ng in al d ru g, be ta b lo ck er an gi na p ec to ris , hy pe rte ns io n th ym us v ul ga ri s l ., tim ija n, l am ia ce ae he rb a ex tra ct sy ru p b ro nh o sa n ja go rč ev in a, tim ija n, di vi zm a / / / tu ss ila go fa rf ar a l. , po db el , a st er ac ea e fo liu m fr ag m en te d he rb al su bs ta nc e he rb al te a č aj p ro tiv br on hi tis a b ip re z (2 .5 m g) , c or ne lin (1 0 m g) , m ol ic or (2 m g) ca lc iu m an ta go ni st s, an tia ng in al d ru g, be ta b lo ck er an gi na p ec to ris , hy pe rte ns io n u rt ic a di oi ca l ., ko pr iv a, u rti ca ce ae fo liu m , h er ba , ra di x ex tra ct ca ps ul e, p ow de r, ta bl et h ip p ča j za d oj ilj e, pr os te na l pe rf ec t, v en od ia p lu s a m ar yl (3 m g) , a nd ol (1 00 m g) , a zo pt so l, b er od ua l n (0 .0 5 m g + 0. 02 1 m g) , a c e in hi bi to rs , ca lc iu m a nt ag oni st s, an tia ng in al dr ug , b en zo di az ep in e, b et a bl oc ke r, an gi na p ec to ris , de pr es si on , gl au co m a, b en ig n pr os ta tic h yp er pl as ia , hy pe rte ns io n, c hr on ic ob st ru ct iv e pu lm on ar y di se as e, 105 biologica nyssana ● 12 (2) december 2021: 87-111 matejić et al. ● frequency of herbal medicinal products use in southeastern serbia l at in n am e, s er bi an na m e an d fa m ily pa rt o f a pl an t fo rm ph ar m . f or m n am e of h m p a ll m ed ic in es us ed w ith h m p t he ra py gr ou ps pr es en t d is ea se s w he n h m p w as u se d b is op ro lo l (2 .5 m g) , b ro m az ep am (3 m g) , c or as p (1 00 m g) , c or ne lin (1 0 m g) , c or ne lin (2 0 m g) , d ia ze pa m (5 m g) , en al ap ril (2 0m g) , fa rin (5 m g) , g lu co ph ag e (1 00 0 m g) , m on os an (2 0 m g) , p re so lo l (5 0 m g) , t am so l (0 .4 m g) ,t en se c (5 m g) , tr im et ac or (3 5 m g) , t rit ac e (5 m g) , v iv ac e (2 .5 m g) , x al at an so l br on ch os pa sm oly tic a ge nt , a ntip la te le t a ge nt , or al a nt id ia be tic dr ug , o ra l a nt ico ag ul an t, gl au co m a tre at m en t, be ni gn p ro sta tic h yp er pl as ia tre at m en t, ve in th ro m bo si s ne ur as th en ia , d ia be te s ty pe 2 , v ei n th ro m bo si s va cc in iu m m yr til lu s l. , b or ov ni ca , er ic ac ea e fo liu m ex tra ct ta bl et er ba ve n re ta rd , v en od ia p lu s a zo pt so l, b is op ro lo l (2 .5 m g) , b ro m az ep am (3 m g) , c or as p (1 00 m g) , c or ne lin (2 0 m g) , d ia ze pa m (5 m g) , en al ap ril (2 0m g) , fa rin (5 m g) , pr es ol ol (5 0 m g) , tr im et ac or (3 5 m g) , v iv ac e (2 .5 m g) , x al at an so l a c e in hi bi to rs , ca lc iu m an ta go ni st s, an tia ng in al d ru g, be nz od ia ze pi ne , be ta b lo ck er , an tip la te le t ag en t, or al an tic oa gu la nt , gl au co m a tre at m en t an gi na p ec to ris , de pr es si on , g la uc om a, hy pe rte ns io n, ne ur as th en ia , v ei n th ro m bo si s 106 biologica nyssana ● 12 (2) december 2021: 87-111 matejić et al. ● frequency of herbal medicinal products use in southeastern serbia l at in n am e, s er bi an na m e an d fa m ily pa rt o f a pl an t fo rm ph ar m . f or m n am e of h m p a ll m ed ic in es us ed w ith h m p t he ra py gr ou ps pr es en t d is ea se s w he n h m p w as u se d va cc in iu m m ac ro ca rpo n a ito n, a m er ič ka bo ro vn ic a, e ric ac ea e fr uc tu s fr ag m en te d he rb al su bs ta nc e he rb al te a č aj p lo da br us ni ce / / / va le ri an a offi ci na lis l. , v al er ija na , c ap rif ol ia ce ae ra di x ex tra ct oi nt m en t, liq ui d, ta bl et a sa m c rv en i ko nj sk i ba lz am , fe m is an b ka pi , k ap i za u m ire nj e, r el ax , sm ire la n oć , v al er ija na ka pi b ro m az ep am (3 m g) , c oa m le ss a (4 m g + 5m g + 1. 25 m g) , n ov om ix 3 0 fl ex pe n (1 00 ij/ m l), s id at a (5 0 m g) , v iv ac e (5 m g) , v iv ac e pl us a c e in hi bi to rs , an tid ep re ss an t dr ug , an tih yp er te ns iv e dr ug , be nz od ia ze pi ne , in su lin ce lia c di se as e, de pr es si on , hy pe rte ns io n, d ia be te s ty pe 1 ve rb as cu m p hl om oi de s l. , d iv iz m a, sc ro ph ul ar ia ce ae flo s ex tra ct sy ru p b ro nh o sa n ja go rč ev in a, tim ija , di vi zm a / / / vi sc um a lb um l ., im el a, s an ta la ce ae he rb a ex tra ct liq ui d fe m is an b ka pi / / / vi tis v in ife ra l ., gr ož đe , v ita ce ae fo liu m , s em en ex tra ct oi nt m en t, ta bl et a sa m c rv en i ko nj sk i ba lz am , er ba ve n re ta rd , s m ire la no ć b ro m az ep am (3 m g) , c oa m le ss a (4 m g + 5 m g + 1. 25 m g) , si da ta (5 0 m g) , v iv ac e (5 m g) , v iv ac e pl us a c e in hi bi to rs , an tid ep re ss an t dr ug , an tih yp er te ns iv e dr ug , be nz od ia ze pi ne de pr es si on , h yp er te ns io n ze a m ay s l ., ku ku ru z, po ac ea e st ig m a fr ag m en te d he rb al su bs ta nc e he rb al te a č aj k uk ur uz ne sv ile , k irk ol in a lu x ča j z a m rš av lje nj e, u ro pr ot ek t č aj c on co r ( 5 m g) , d ia ze pa m (5 m g) , g lu fo rm in (1 00 0 m g) , h lo rp ro m az in (2 5 m g) , p ril in da (5 m g) , t en se c (5 m g) , t re nt al (4 00 m g) a c e in hi bi to rs , an tip sy ch ot ic dr ug , be nz od ia ze pi ne , be ta b lo ck er , or al a nt id ia be tic dr ug , c irc ul at or y di so rd er s tre at m en t de pr es si on , hy pe rte ns io n, d ia be te s ty pe 2 107 the results showed that the respondents living in the municipality of aleksinac used more plant species for prevention and/or treatment of diseases compared to people from sokobanja and zaječar, while there was no difference between the respondents when it comes to the number of hmp (tab. 2). tab. 3 shows that women used more herbal species when it comes to prevention and/or treatment of the disease compared to men, while there was no difference between the subjects when it comes to the number of hmp. 108 biologica nyssana ● 12 (2) december 2021: 87-111 matejić et al. ● frequency of herbal medicinal products use in southeastern serbia location number of hmp per patient number of ps per patient aleksinac 1.05±0.22 3.33±2.48 sokobanja 1.12±0.33 1.95±1.87 zaječar 1.08±0.28 1.77±1.09 kruskal-wallis test chi-square=1.037; p=0.596 chi-square=9.749; p=0.008 mann-whitney u test post hoc / aleksinac vs. sokobanja, p=0.002 aleksinac vs. zaječar, p=0.038 sokobanja vs. zaječar, p=0.816 table 2. number of hmp and species per respondent in relation to location ps-plant species table 3. number of hmp and species per respondent in relation to location gender number of hmp per patient number of ps per patient male 1.97±0.25 1.67±1.73 female 1.13±0.34 2.69±2.12 mann-whitney u test z=-1.065; p=0.287 z=-3.210; p=0.001 age number of hmp per patient number of ps per patient ≤35 1.05±0.22 2.33±1.56 36-50 1.14±0.36 2.61±2.38 51-64 1.09±0.29 2.27±2.29 ≥ 65 1.10±0.31 1.72±1.62 kruskal-wallis test chi-square=1.223; p=0.747 chi-square=3.504; p=0.320 mann-whitney u test post hoc / / table 4. number of hmp and plant species per respondent in relation to age the analysis showed (tab. 4, tab. 5) there were no differences between the number of used hmp and plant species per subject in relation to age, and that they did not differ in the number of used hmp and plant species per subject in relation to the presence of chronic diseases. also, in our study it was concluded (tab. 6) that the subjects did not differ in the number of used hmp and herbal species per subject in relation to the number of conventional medicines per patient. respondents who did not have finished pharmaceutical products in therapy used hmp for the purpose of prevention in a higher percentage (tab. 7). table 5. number of hmp/species per respondent in relation to chronic diseases presence of chronic diseases number of hmp per patient number of ps per patient yes 1.12±0.33 2.04±2.06 no 1.08±0.28 2.41±1.95 mann-whitney u test z=-0.597; p=0.550 z=-1.141; p=0.254 in tab. 8, it can be seen that most finished pharmaceutical products and hmp were used for the same indications. as expected, in the group of patients who used hmp for an indication different from the one for which the respondents used conventional medicines, 63.5% of them did not use medicines at all (tab. 9). comparative analysis the area of southeastern serbia is known for its rich tradition of using herbal preparations and herbal medicines. the work by matejić et al. (2020), shows that in the villages of the timok and svrljig region, people often opt for the use of plants from nature. this is the first measure of prevention, and even treatment, while they later decide to visit the doctor. according to the results of this ethnobotanical research conducted from 2015 to 2017, the local population used a total of 195 species in traditional treatments, specifically 31 species were used by the population of the timok region, 77 both by the population of the timok and svrljig regions, and 87 by the population of the srvrljig region. 109 biologica nyssana ● 12 (2) december 2021: 87-111 matejić et al. ● frequency of herbal medicinal products use in southeastern serbia table 6. selection of indications for the use of hmp in relation to the number of medicines indication for the use of hp number of hmp per patient number of ps per patient 0 medicines 1.12±0.32 2.58±2.24 1-3 medicines 1.10±0.31 2.10±3.78 ≥ 4 medicines 1.07±0.26 1.64±1.42 kruskal-wallis test chi-square=0.387; p=0.824 chi-square=3.981; p=0.137 mann-whitney u test post hoc / / table 7. the purpose of the use of hmp in relation to the number of finished pharmaceutical products purpose of using hp 0 medicines 1-3 medicines ≥ 4 medicines prevention (n=79) 50 (63.29%) 11 (13.92%) 18 (22.78%) treatment (n=21) 2 (9.52%) 9 (42.86%) 10 (47.62%) χ2 test χ2=19.821; p<0.001 table 8. the most common indications for the use of conventional medicines and indications for the use of hmp indication n (%) indication n (%) cardiovascular diseases 37% cardiovascular diseases 18% benign prostatic hyperplasia 13% benign prostatic hyperplasia 19% mental disorders 12% mental disorders 11% diabetes 10% ocular disorders 6% other 24% patients without comorbidity 49 table 9. indications for the use of hmp in relation to the number of medicines indication for the use of hp 0 medicines 1-3 medicines ≥ 4 medicines same as in the case of finished pharmaceutical products (n=20) 1 (5%) 9 (45%) 10 (50%) different in relation to the indication of finished pharmaceutical products (n=80) 51 (63.75%) 11 (13.75%) 18 (22.50%) χ2 test χ2=19.821; p<0.001 the timok region has a total of 108 species that have been used in the traditional treatment of the population. people most often used the following: hypericum spp., matricaria chamomilla, mentha x piperita, urtica dioica, juglans regia and achillea millefolium. all plant species that were used in the timok region can be classified into a total of 47 families. among them, most species are members of the families lamiaceae, rosaceae, asteraceae, apiaceae and brassicaceae. depending on the type of plant, different parts of it were used to make herbal preparations, and in as many as 94.6% of cases the aerial parts, flower, fruit, leaf and root were used. the population most often used herbs to prepare tea, in the form of compresses, as a tincture, fresh and cooked. most of the plants were used internally (for internal use), 79.5% of the total number of plants, while a smaller number of plants were used externally (for external use), that is 20.5%. plants from the timok region were used in the treatment of the following systems: digestive, endocrine, metabolic and nutritional, integumentary, respiratory, circulatory and urinary, with 83.7% of all treated systems (matejić et al. 2020). the svrljig region is characterized by a total of 164 different plant species that the population used for traditional medical purposes. in the svrljig region, the most commonly used were: satureja montana, sambucus nigra, polygonum aviculare, marrubium vulgare and teucrium chamaedrys. all species are classified in 64 families, and the largest percentage of them belongs to the lamiaceae, rosaceae, asteraceae, polygonaceae and amaryllidaceae. the most used parts of plants were: aerial parts, leaf, flower, fruit and root. in 90.8% of cases, the plants were used in the form of tea, compresses, tinctures, for ritual purposes and fresh. in the largest percentage, the population used plants internally (for internal use), as much as 76.0%, while externally (for external use) 20.8% of the total number of plant species were used. however, 3.2% of species were used both externally and internally. the population used the plants in the treatment of various systems: digestive, endocrine, metabolic and nutritional, circulatory, urinary, integumentary, musculoskeletal and general and nonspecific, which represents 78.7% of all treated systems. the research by zlatković et al. (2014), conducted from 2011 to 2012, shows that 45 different species were in use on mount rtanj. the most commonly used was hypericum perforatum. the largest percentage of the used plants belonged to the families lamiaceae, rosaceae and asteraceae. they were used primarily in the treatment of the immune, respiratory and digestive systems. the work by jarić et al. (2015) showed that in the region of suva planina the population used 128 different plant species in the period from 2012 to 2014. most of them used the following species: achillea millefolium, allium cepa, allium sativum, arctostaphyllos uva-ursi, gentiana lutea, hypericum perforatum, juglans regia, matricaria chamomilla, mentha x piperita, plantago lanceolata, plantago major, salvia officinalis, sempervivum tectorum, tilia cordata and thymus serpyllum. all used species are classified according to families, and the most common families were: asteraceae, rosaceae, brassicaceae and alliaceae. these herbs were used to treat mainly the respiratory system, disorders of the genitourinary and the digestive system. the research by janaćković et al. (2019), realized during 2016, indicates that 37 wild species were used among the population of negotin valley, most often the following: matricaria chamomilla, urtica dioica, hypericum perforatum, salvia officinalis, plantago major, achillea millefolilium, calendula officinalis and taraxacum campylodes. the largest percentage belonged to the families lamiaceae, asteraceae and rosaceae, and they were primarily used to treat the immune, digestive and respiratory systems, as well as skin diseases. all these ethnopharmacological researches are related to the plants used in traditional medicine, while our study gives a review of the plant species and hmp which are ingredients of pharmacological products and are distributed in public pharmacies. the number of people who use these pharmacological products increase even in rural areas. conclusions obtained results reflect a high frequency of the use of hmp in southeastern serbia. residents of this area are more likely to opt for the use of various natural products than for conventional medicines. they use hmp to treat many diseases, and often in their prevention. the most commonly used preparation was urasan forte. the composition of this preparation includes nutmeg (cucurbita moschata duchesne), one of the most common plant species. it is used in prostate hyperplasia, which together with other diseases of the urinary system often occurs in the inhabitants of this area. the most common species was bitter orange (citrus x aurantium l.), an integral part of many hmp used in the treatment of hypertension, hyperlipidemia, venous thrombosis and sinusitis. most of the inhabitants suffered from hypertension, as confirmed by the relevant research conducted in this area. the results showed that the respondents from aleksinac used more species for prevention and/or treatment of diseases compared biologica nyssana ● 12 (2) december 2021: 87-111 110 matejić et al. ● frequency of herbal medicinal products use in southeastern serbia to people from sokobanja and zaječar, while there was no difference between the respondents when it comes to the number of hmp. respondents who did not have conventional medicines in therapy used hmp for prevention in a higher percentage. the local population prefers simple consumption of preparations in the form of capsules and tablets, but the traditional use of herbal teas is also very common. acknowledgements. the authors thank the ministry of education, science and technological development of the republic of serbia for financial support (grant no: 451-03-68/2020-14/ 200178, 451-03-9/2021-14/200113). we owe a special thanks to the adonis pharmacy (https:// www.adonisapoteka.rs), which made this research possible. references agency for drugs and medical devices of serbia 2021. national drug registry (belgrade). barnes, j. 2012: adverse drug reactions and pharmacovigilance of herbal medicines. in: talbot, j., aronson, j.k. (eds.), stephens’ detection and evaluation of adverse drug reacrions: principles and practice, willeyblackwell, chichester. dimitrijević, m., catić-đorđević, a., stefanović, n., pavlović, d., veličković-radovanović, r. 2014: self-medication in primary healthcare in the teritiry of the city of niš. acta medica medianae, 53(3): 19-24. heinrich, m., barnes, j., prietogracia, j., gibbons, s., williamson, e. 2018: fundamentals of pharmacognosy and phytotherapy. 3rd edition. elsevier. janaćković, p., gavrilović, m., savić, j., marin, p.d., dajić stevanović, z. 2019: traditional knowledge on plant use from negotin krajina (eastern serbia): an ethnobotanical study. indian journal of traditional knowledge, 18(1): 25–33. jarić, s., mačukanović-jocić, m., đurđević, l., mitrović, m., kostić, o., karadžić, b., pavlović, p. 2015: an ethnobotanical survey of traditionally used plants on suva planina mountain (southeastern serbia). journal of ethnopharmacology, 175: 93–108. luketina-šunjka, m., rančić, n., mihailović, n., radević, s., dagović, s., jakovljević, m. 2020: sealth self-evaluation of complementary and alternative medicine users in serbia: cross-sectional national study. acta medica medianae, 59(4): 3442. matejić, j., stefanović, n., ivković, m., živanović, n., marin, d.p., džamić, m.a. 2020: traditional uses of autochthonous medicinal and ritual plants and other remedies for health in eastern and southeastern serbia. journal of ethnopharmacology, 261: 113186. petrović, s., kukić-marković, j., pavlovićdrobac, m. 2012: biljni lekoviti proizvodi: uslovi za bezbednu primenu. archive of pharmacy, 62(2): 119-35. public health institute “timok” zaječar, 2020: analysis of the health status of the population of zaječar district in the period from 2015 to 2019 (zaječar). http://www.zavodzajecar.rs/images/pdf/ analizaokrugzajecar2015-2019.pdf public health institute niš, 2017: analysis of the health status of the population of the municipality of sokobanja in the period from 2012 to 2016 (niš). http://www.izjz-nis.org.rs/ download/informatika/2017%20analiza%20 zdravstvenog%20stanja%20stanovnistva%20 opstine%20sokobanja%20%202012-2016.pdf public health institute niš, 2017: analysis of the health status of the population of the municipality of aleksinac for 2015 (niš). sam, s. 2019: importance and effectiveness of herbal medicines. journal of pharmacognosy and phytochemistry, 8(2): 354-357. statistical office of the republic of serbia, 2016. municipalities and regions in the republic of serbia 2016 (belgrade). https://pod2.stat.gov.rs/ objavljenepublikacije/g2016/pdf/g20162020.pdf statistical office of the republic of serbia, 2017. municipalities and regions in the republic of serbia 2017 (belgrade). https://publikacije.stat.gov.rs/ g2017/pdf/g201713044.pdf statistical office of the republic of serbia, 2020. municipalities and regions in the republic of serbia 2020 (belgrade). https://publikacije.stat.gov.rs/ g2020/pdf/g202013047.pdf zhang, l. 2018: pharmacovigilance of herbal and traditional medicines. in: bate, a. (ed.), evidencebased pharmacovigilance clinical and quantitative aspects, springer nature, humana press. zlatković, b.k., bogosavljević, s.s., radivojević, a.r., pavlović, m.a., 2014: traditional use of the native medicinal plant resource of mt. rtanj (eastern serbia): ethnobotanical evaluation and comparison. journal of ethnopharmacology, 151: 704–713. 111 biologica nyssana ● 12 (2) december 2021: 87-111 matejić et al. ● frequency of herbal medicinal products use in southeastern serbia šabanović et al. 2023, biologica nyssana 14(1) 14 (1) june 2023: 7-13 doi: 10.5281/zenodo.8027050 orchis anthropophora (l.) all. (orchidaceae), a confirmed species for the flora of bosnia and herzegovina original article elvedin šabanović international university of brčko district, m. malića and i. džindića bb, brčko 76100, bosnia and herzegovina sabanovic2021@outlook.com (corresponding author) vladan djordjević faculty of biology, institute of botany and botanical garden, university of belgrade, takovska 43, 11000 belgrade, serbia đorđije milanović faculty of forestry, university of banja luka, stepe stepanovića 75a, banja luka 78000, bosnia and herzegovina dragana jenačković gocić department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, 18106 niš, serbia sanida bektić department of biology, faculty of sciences and mathematics, university of tuzla, tuzla, bosnia and herzegovina samira huseinović department of biology, faculty of sciences and mathematics, university of tuzla, tuzla, bosnia and herzegovina vladimir ranđelović† department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, 18106 niš, serbia received: december 09, 2022 revised: april 12, 2023 accepted: april 20, 2023 abstract: orchis anthropophora (l.) all. (orchidaceae) is a mediterranean-atlantic species distributed around the mediterranean region and in central and western europe. during a floristic survey in may 2022, this species was found at the ravno (trebimlja) locality (yh35 10×10 km utm grid cell), which is the first confirmed record of this species on the territory of bosnia and herzegovina. the population was found in rocky grassland, on limestone, at an altitude of 423 m. three individuals of o. anthropophora were found within an area of 100 m². based on the iucn red list categories and criteria, o. anthropophora is estimated as critically endangered in bosnia and herzegovina. key words: orchis anthropophora, orchid, flora, bosnia and herzegovina, balkan peninsula apstrakt: orchis anthropophora (l.) all. (orchidaceae), potvrđena vrsta za floru bosne i hercegovine orchis anthropophora (l.) all. (orchidaceae) je mediteransko-atlantska vrsta rasprostranjena u mediteranskom regionu, centralnoj i zapadnoj evropi. florističkim istraživanjem sprovedenim u maju 2022. godine, ova vrsta je pronađena na lokalitetu ravno (trebimlja) (yh35 10×10 km2 utm kvadrat), što je prvi potvrđeni nalaz ove vrste na području bosne i hercegovine. populacija je pronađena na travnom, karbonatnom kamenjaru, na nadmorskoj visini od 423 m. tri individue vrste o. anthropophora zabeležene su na površini od 100 m2. prema kategorijama i kriterijumima iucn crvene liste, o. anthropophora je procenjena kao krajnje ugrožena vrsta bosne i hercegovine. ključne reči: orchis anthropophora, orhideja, flora, bosna i hercegovina, balkansko poluostrvo introduction orchis tourn. ex l. is a genus in the orchid family (orchidaceae), occurring mainly in europe, north africa, and temperate asia, with the center of its diversity located in the mediterranean region (kretzschmar et al., 2007). this genus once included taxa of the genera anacamptis rich., dactylorhiza neck. ex nevski and gymnadenia r.no. in w.t. aiton (tyteca et al., 2012). molecular analyses have confirmed the polyphyletic status of the genus orchis s.l. and many taxa have been assigned to the expanded genera anacamptis rich. and neotinea rchb.f. (pridgeon et al., 1997; bateman et al., 1997, 2003). the genus orchis is divided into the subgenera orchis s.str. and masculae (kretzschmar et al., 2007). there is also a division of this genus into two genera: orchis, which includes all species and subspecies with a so-called ‘anthropomorphic’ lip (o. militaris group), and androrchis tyteca & klein, which includes all remaining ‘non-anthropomorphic’ species and subspecies (o. mascula group) (tyteca & klein, 2008, 2009). the genus orchis consists of 23 species and © 2023 šabanović et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 7 this paper is dedicated to professor vladimir ranđelović as a sign of gratitude for the provided knowledge, advice, support and the opportunity to be his collaborators. the authors 10 subspecies (wcsp, 2022). in bosnia and herzegovina, 12 taxa have been known until now: orchis anthropophora (l.) all., o. italica poir., o. mascula (l.) l. subsp. mascula, o. mascula (l.) l. subsp. speciosa (mutel) hegi, o. militaris l. subsp. militaris, o. pallens l., o. pauciflora ten., o. provincialis balb. ex lam. & dc, o. purpurea huds. subsp. purpurea, o. quadripunctata cirillo ex ten., o. simia lam. subsp. simia and o. spitzelii saut. ex w.d.j. koch subsp. spitzelii (šabanović et al., 2019, 2021; milanović et al., 2022). however, in the current „checklist of the orchidaceae of bosnia and herzegovina” (šabanović et al., 2021), the status of o. anthropophora in bosnia and herzegovina is marked ‘l’, which means that there are published data but no herbarium or photographic material confirming its finding. this study reports the first confirmed record of orchis anthropophora (l.) all. in bosnia and herzegovina. the objectives were: a) to provide its morphological description; b) to present its currently known distribution; c) to determine its habitat preferences; d) to determine its population size and e) to estimate the iucn threat status of this species in bosnia and herzegovina. materials and methods during a floristic survey conducted in may 2022 in the area of ravno (trebimlja), data were collected on the morphology, distribution, habitat preferences, and population size of orchis anthropophora (l.) all. the taxon was identified according to delforge (2006) and kretzschmar et al. (2007), while the nomenclature followed the world checklist of kew gardens (wcsp 2022). one specimen of o. anthropophora was collected for documentation purposes and deposited in the museum of the franciscan monastery in visoko–herbarium collection of fr. ivo radman. the present morphological description of the species is based on own observations and according to baumann et al. (2006), delforge (2006), kretzschmar et al. (2007) and fay & taylor (2015). the values of the measured traits of the specimen from bosnia and herzegovina are marked with bold numbers. the distribution of o. anthropophora in bosnia and herzegovina was mapped on a grid map with squares of 10×10 km, using the universal transverse mercator (utm) projection, grid zone 33t. geographic coordinates (longitude, latitude) and altitude were recorded using a garmin etrex 30 handheld gps device in the world geodetic system 84 (wgs 84). the boundaries of the regions on the distribution map of this species are given according to čadro et al. (2019). the vegetation units of the site with o. anthropophora were determined according to braun-blanquet (1964), whereas the nomenclature follows mucina et al. (2016). the habitat type was determined according to the eunis habitat classification (http://eunis.eea.europa.eu/). the geological substrate was determined in the field and from a 1:100,000 scale geologic map of the study area. abundance of o. anthropophora was determined by counting the total number of individuals. for the estimation of the threat status of o. anthropophora in bosnia and herzegovina, iucn (2012) red list categories and criteria were applied. results orchis anthropophora (l.) all., fl. pedem. 2: 148 (1785) synonyms: aceras anthropophorum (l.) w.t.aiton, arachnites anthropophorus (l.) f.w.schmidt, himantoglossum anthropophorum (l.) spreng., loroglossum anthropophorum (l.) rich., ophrys 8 biologica nyssana ● 14 (1) june 2023: 7-13 šabanović et al. ● orchis anthropophora (l.) all. (orchidaceae), a confirmed species for the flora of bosnia and herzegovina fig. 1. orchis anthropophora (l.) all. (bosnia and herzegovina, ravno, trebimlja, 09.05.2022, photo e. šabanović) biologica nyssana ● 14 (1) june 2023: 7-13 šabanović et al. ● orchis anthropophora (l.) all. (orchidaceae), a confirmed species for the flora of bosnia and herzegovina 9 anthropophora l., satyrium anthropophorum (l.) pers., serapias anthropophora (l.) jundz. (fig. 1). morphological characteristics perennial plant, 10–36.1–40 (–50) cm high, with two ovoid tubers. the stem is glabrous and cylindrical. there are 5–6–10 leaves, oblong-lanceolate, shiny above, bluish-green, not spotted, distinctly veined, 5–7.9–15 cm long and 0.5–2.4–4 cm wide. basal leaves are in a rosette, the middle leaves are erect, an upper leaf is bract-like, while 1–3 smaller sheathing leaves surround the lower part of the stem. the bracts are yellowish-green to pale green, acute, 5–7 mm long and 1.2–2.3 mm wide, shorter than the ovaries. the inflorescence is elongated, denser at the apex, 5–13.4–20 (–28) cm long, with 42–50 (–90) flowers. the flowers are ‘anthropomorphic’. sepals are oval, 5–8 (–11) mm long and 2.5-5 mm wide, green with reddish margins, forming a dense cap. petals are linear, hidden inside hood, 5–8 mm long and 1–1.7 mm wide, pale green. the lip is 3-lobed, without a spur, 10–12–16 mm long and 2.5–4 mm wide, dirty yellow, greenish yellow to orange with often darker reddish or brownish margins, with two shiny pale bosses at the base forming a nectariferous cup. the lateral lobes are linear and slender, forming the ‘arms’, 7 mm long. the middle lobe is 10 mm long, always longer than the lateral lobes and divided into two strap-shaped halves, forming the ‘legs’, sometimes separated by a tooth. the column is short, obtuse. the ovary is sessile, twisted. taxonomic notes the studied taxon changed its taxonomic position, starting from ophrys anthropophora l. in the broad definition of ophrys l. since then it has been classified in aceras r.br., arachnites f.w.schmidt, himantoglossum spreng., loroglossum rich., orchis l., satyrium l. and serapias l. (fay & taylor, 2015). since 1813, this taxon has been included in the monospecific genus aceras r.br. as aceras anthropophorum (l.) r.br (delforge, 2006). the only distinctive feature in which aceras differs from orchis is the absence of a spur. however, recent molecular studies have shown that this species actually belongs to the genus orchis s.s., excluding species placed in anacamptis rich. and neotinea rchb.f. by bateman et al. (1997). it is sister to the remainder of the genus (bateman et al., 2003) and actually belongs to the orchis militaris group and is one of the earliest differentiated species of the genus orchis. flowering time on may 9, 2022, three specimens of o. anthropophora at the trebimlja site were at peak flowering. this is consistent with the flowering time reported by fay & taylor (2015), who reported that this species flowers from march to july, with an optimal time from april to june. in greece, the peak flowering time of the species is around mid-april (tsiftsis & antonopoulos, 2017). general distribution orchis anthropophora is a mediterranean-atlantic species distributed around the mediterranean region and in central and western europe, occurring from england, belgium and the netherlands in the north to morocco and algeria in the south and from spain and england in the west to cyprus and rhodes in the east (delforge, 2006; kretzschmar et al., 2007; fay & taylor, 2015; wcsp, 2022). it is widespread in western europe, while rare in the eastern and northern parts of its range. according to published sources, it is distributed in albania, algeria, austria, the balearic islands, belgium, corsica, crete, croatia, cyprus, france, germany, great britain, greece, italy, lebanon-syria, montenegro, morocco, the netherlands, portugal, sardinia, sicily, spain, switzerland, tunisia, and turkey (delforge, 2006; kretzschmar et al., 2007; fay & taylor, 2015; tsiftsis & antonopoulos, 2017; wcsp, 2022). distribution in bosnia and herzegovina orchis anthropophora was found in the southern region of bosnia and herzegovina: ravno, trebimlja (near the trebimlja-čepikuće border crossing, on both sides of the road), n 42.8757111˚, e 17.8624806˚, mgrs 33t yh35, 423 m a.s.l., rocky grassland, limestone, 09 may 2022; coll. e. šabanović, det. e. šabanović, v. djordjević [the museum of the franciscan monastery in visoko – herbarium collection of fr. ivo radman 00315; photo documentation: e. šabanović] (fig. 2). the finding of o. anthropophora at the trebimlja locality (near the trebimlja-čepikuće border crossing) is the first confirmed record of this species on the territory of bosnia and herzegovina, representing the first record of this species in mgrs 33t yh35 10×10 km utm grid cell and southern region of bosnia and herzegovina. previously, this species was recorded in bosnia and herzegovina only by protić (1902: 29), who listed the species under the name aceras anthropophora r.br. and found it at the two localities (zelengora – utm cp00 and prijevor – utm cn19; fig. 2). these records have been mentioned for bosnia and herzegovina later by hayek (1933: 403) and šilić (1996: 360), but firstly by beck-mannagetta (1904: 512), who considered these records very doubtful. moreover, this researcher did not collect the plants during his botanical excursion in the mountains in se bosnia 10 biologica nyssana ● 14 (1) june 2023: 7-13 šabanović et al. ● orchis anthropophora (l.) all. (orchidaceae), a confirmed species for the flora of bosnia and herzegovina fig. 2. distribution of orchis anthropophora (l.) all. in bosnia and herzegovina fig. 3. habitat of orchis anthropophora (l.) all. (bosnia and herzegovina, ravno, trebimlja, 09.05.2022, photo e. šabanović) 11 biologica nyssana ● 14 (1) june 2023: 7-13 šabanović et al. ● orchis anthropophora (l.) all. (orchidaceae), a confirmed species for the flora of bosnia and herzegovina (fukarek, 1969), and there is no herbarium evidence or photographic material confirming these findings. since these records are located deep in the dinaric mountains, outside the climatic conditions and habitats suitable for this species, they must be treated as at least very doubtful or even erroneous (fig. 2). habitat and ecology orchis anthropophora was found on the locality trebimlja in submediterranean rocky grassland of the alliance chrysopogono grylli-koelerion splendentis horvatić 1973 within the ordo scorzoneretalia villosae kovačević 1959, partially overgrown by some individuals of fraxinus ornus l. and pistacia terebinthus l (fig. 3). the following accompanying taxa were recorded at the site with o. anthropophora: brachypodium retusum (pers.) p. beauv., koeleria splendens c. presl (aggr.), achnatherum bromoides (l.) p. beauv., micromeria juliana (l.) benth. ex rchb., salvia officinalis l., euphorbia spinosa l., tanacetum cinerariifolium (trevir.) sch. bip., genista sylvestris subsp. dalmatica (bartl.) h. lindb., inula verbascifolia (willd.) hausskn. and others. according to the eunis classification, this habitat type belongs to the eastern submediterranean dry grasslands (e1.55 code; https:// eunis.eea.europa.eu/habitats/997). the population of o. anthropophora was recorded on limestone, under full light regime, at an altitude of 423 m. the habitat preferences of this orchid recorded in bosnia and herzegovina correspond to its habitat preferences and ecological requirements in other parts of its range. namely, many authors pointed out that this species occurs on calcareous substrates, in full sun to mid-shade conditions, in xerophilous short grasslands, garrigue, scrub, forest edges, and more rarely in open forest habitats (delforge, 2006; kretzschmar et al., 2007; fay & taylor, 2015; tsiftsis & antonopoulos, 2017). this species has been found on soils with ph ranging from 5.5 to 8.6, but generally prefers alkaline soils (sundermann, 1980; wallenwein & saad, 2000; vakhrameeva et al., 2008; djordjević & tsiftsis, 2022). the recorded altitudinal range of this species from other countries is between 0 m and 1,700 m (baumann et al., 2006; delforge, 2006; tsiftsis & antonopoulos, 2017). population size only three individuals of o. anthropophora were found within an area of 100 m². therefore, its population size falls within the iucn category of fewer than 50 mature individuals. conservation status orchis anthropophora is protected by the convention on international trade in endangered species of wild fauna and flora (cites). the current status of this species in bosnia and herzegovina is estimated as critically endangered – cr d. the iucn status of the species in other countries is as follows: regionally extinct in cyprus, critically endangered in austria and bavaria, endangered in the united kingdom and luxembourg, vulnerable in switzerland, near threatened in croatia and last concern in spain and france (kull et al., 2016). monitoring of this species is needed to assess potential threats that may affect the species (e.g. road construction and habitat succession) and to monitor population dynamics. acknowledgements. the authors thank two anonymous reviewers and editor for their useful suggestions and comments on a previous version of the manuscript. phd elvedin šabanović was supported by the environmental fund of the federation of bosnia and herzegovina (www. fzofbih.org.ba). vladimir randjelović, phd was supported by the ministry of education, science and technological development of the republic of serbia (grant no. 45103-68/2022-14/200124); vladan djordjević, phd was supported by the ministry of education, science and technological development of the republic of serbia (grant no. 451-03-47/2023-01/200178). references bateman, r.m., pridgeon, a.m., & chase, m.w. 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(orchidaceae), a confirmed species for the flora of bosnia and herzegovina 13 ciftci, erol 2020, biologica nyssana 11(2) 11 (2) december 2020: 85-91 doi: 10.5281/zenodo.4393951 micromorphological studies on inflorescence and seeds of some plantain (plantago l.) taxa in turkey original article almila ciftci biology department, science faculty, istanbul university, istanbul, turkey almila.ciftci@istanbul.edu.tr (corresponding author) osman erol biology department, science faculty, istanbul university, istanbul, turkey erol@istanbul.edu.tr received: julz 14, 2020 revised: august 28, 2020 accepted: september 15, 2020 abstract: plantago l. (plantaginaceae) is one of the largest genera of the plantaginaceae family. there are relatively fewer morphological studies on the genus. seed surface micromorphology has become popular in plant taxonomy in recent years and found useful in most groups. the inflorescence and seed surface characteristics of 22 plantago taxa collected from turkey were examined using a scanning electron microscope (sem). cluster analysis performed in r. we observed that the micromorphology of the inflorescence parts and seeds of genus plantago are very variable, and do not support the classification based on general morphology. this study shows that these characteristics are highly variable and micromorphological characteristics of inflorescence parts are not good characters for making a specific distinction between different taxa. additionally, some groups have been to possess a stable seed surface characteristics. however, these characters are not reliable among all taxa, which means that they should be used carefully, especially when identifying widespread taxa. key words: inflorescence, micromorphology, plantago, plantaginaceae, seed apstract: mikromorfološka studija cvast i semena nekih taksona bokvica (plantago l.) u turskoj plantago l. (plantaginaceae) je jedan od najvećih rodova porodice plantaginaceae. postoji relativno malo morfoloških studija o ovom rodu. mikromorfologija površine semena postala je popularna u biljnoj taksonomiji poslednjih godina i smatra se korisnom u većini grupa. karakteristike cvasti i površine semena 22 taksona roda plantago sakupljenih iz turske ispitane su pomoću skenirajućeg elektronskog mikroskopa (sem). klaster analiza je izvršena u r. primetili smo da su mikromorfologija delova cvasti i semena roda plantago veoma promenljive i ne podržavaju klasifikaciju zasnovanu na opštoj morfologiji. ova studija pokazuje da su ove karakteristike veoma promenljive, a mikromorfološke karakteristike delova cvasti nisu dobri karakteri za pravljenje posebne razlike između različitih taksona. pored toga, neke grupe su pokazale da imaju stabilne karakteristike površine semena. međutim, ovi karakteri nisu pouzdani kod svih taksona, što znači da ih treba pažljivo koristiti, posebno pri identifikaciji rasprostranjenih vrsta. ključne reči: cvasti, mikromorfologija, plantago, plantaginaceae, seed introduction plantaginaceae is a very heterogeneous family with diverse evolutionary trends, and plantago l. is one of the greatest genera of the plantaginaceae family with 200 species, 56 subspecies, 188 varieties and 9 subvarieties according to albach et al. (2005). it is probably the most widespread genus of the family plantaginaceae (albach et al., 2005). plantago genus is divided into nineteen sections and two subgenera (pilger, 1937). 9 of these sections including 23 taxa, two of which are endemic to turkey, have been recorded in the flora of turkey and the east aegean islands (tutel, 1982). although rahn (1996) claimed that plantago can be divided into six genera, he divided the genus into six subgenera and twelve sections in order to prevent synonym pollution. these two classification suggestions for the genus are summarized in tab. 1. here in this study, we used pilger’s (1937) classifica© 2020 ciftci, erol. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 85 tion in order to be able to follow the ranking in the flora of turkey (tutel, 1982). plantago taxa are annual or perennial herbs or dwarf shrubs with alternate rosettes or opposite leaves. the inflorescence is a spike with 4-merous flowers. the sepals are imbricate and keeled. the flowers have bracts. the corolla is sympetalous and forms a cylindrical tube (tutel, 1993). even though various studies have been made with plantago taxa, morphological and systematic studies on plantago taxa are relatively limited (sagar & harper, 1964; sharma et al., 1990; tutel et al., 2005). micromorphological studies have been made in china (jun-zhe et al., 1992) and poland (klimko et al., 2004) which were both limited to the taxa in their region. the most extensive study on micromorphology of plantago seeds composed by shehata and loutfy (2006) included 31 taxa. seed surface micromorphology has become very popular in plant taxonomy in recent years. one of the main reasons for that is that structure of the seed surface seems to be the most stable character due to less contact with environment. flower elements are also one of the most stable characters in flowering plants (endress, 2001). for this reason, we decided to include the surface investigations of inflorescence elements in this study. considering the lack of the studies about plantago taxa, this study aims to clarify the relationships within plantago taxa in turkey; using seed and inflorescence features. moreover, sem was used to investigate further the micromorphology of the seed and the inflorescence surfaces of these taxa in turkey for the first time. materials and methods 22 turkish plantago taxa including four varieties, which were not presented in the flora of turkey and east aegean islands (tutel, 1982), are examined in this study (tab. 2). these taxa were obtained mostly from istf (istanbul university faculty of sciences herbarium) as well as from ank (ankara university faculty of science herbarium). we used 15 individuals’ inflorescence elements and seeds from each 86 sensu pilger sensu rahn subgen. sect. taxa sect. subgen. euplantago plantago plantago major subsp. major plantago plantago p. major subsp. intermedia gentianoides p. gentianoides lamprosantha p. media coronopus p. crassifolia coronopus coronopus p. coronopus p. weldenii p. holosteum p. maritima maritima oreades p. atrata montana albicans leucopsyllium p. albicans p. loeflingii arnoglossum p. lanceolata lanceifoliap. argentea p. lagopus hymenopsyllium p. cretica hymenopsyllium p. bellardii psyllium psyllium p. squarrosa psyllium psyllium p. afra p. scabra p. sempervirens p. euphratica table 1. classification of the turkish plantago taxa according to pilger [2] and rahn [4] biologica nyssana ● 11 (2) december 2020: 85-91 ciftci, erol ● micromorphological studies on inflorescence and seeds of some plantain (plantago l.) taxa in turkey 87 taxon when possible. apart from the other studies (jhun-zhe et al., 1992; klimko et al., 2004; shehata & loutfy, 2006) on plantago micromorphology, this study includes some other areas from the inflorescence parts (e.g. bract, anterior and posterior sepals, and corolla) (fig. 1). for the examination of the inflorescence characteristics, the lowest and the most mature flowers of the inflorescences have been chosen, and the macro morphological observations have been made using olympus szx7 stereomicroscope. the micromorphology of the inflorescence parts and seeds fig. 1. general view of a plantago flower (a). the observed areascorolla tube (1), petal (2), anterior sepal base (3), bract margin (4), posterior sepal carina (5) and margin (6), seed ventral (7) and dorsal (8) surfaces are shown with squares (b). scale bars 1 mm. table 2. the examined taxa in the study with voucher specimens taxa voucher no location p. major l. subsp. major istf 24301 rize p. major l. subsp. intermedia (gilib) lange istf 23423 i̇stanbul p. coronopus l. subsp. coronopus istf 557 i̇stanbul p. weldenii rchb. istf 2306 i̇stanbul p. crassifolia forsskal istf 40190 istanbul p. maritima l. istf 26779 kirklareli p. holosteum scop. istf 27341 ankara p. media l. istf 23998 erzurum p. atrata hoppe istf 23710 erzurum p. gentianoides sm. subsp. gentianoides istf 32242 bursa p. lanceolata l. istf 23372 istanbul p. argentea chaix istf 63 bursa p. lagopus l. istf 22271 istanbul p. albicans l. ank ankara p. loeflingii l. istf 35420 sanliurfa p. cretica l. istf 24743 antalya p. bellardii all. istf 27212 i̇stanbul p. squarrosa murray istf 9790 antalya p. scabra moench istf 32258 i̇stanbul p. afra l. istf 36362 mersin p. sempervirens crantz alm 27 tekirdağ p. euphratica decne. ex barnéoud istf 37227 malatya biologica nyssana ● 11 (2) december 2020: 85-91 ciftci, erol ● micromorphological studies on inflorescence and seeds of some plantain (plantago l.) taxa in turkey 88 have been observed and photographed by a jeol benchtop scanning electron microscope jcm-5000 under 10 kv of voltage. the nomenclature of the surface characteristics is adapted from stearn (1991) and barthlott et al. (1998). the categorical variables of surface characters in various anatomical compartments of plantago were used as variables in the cluster analysis. hierarchical cluster analysis of 26 categorical variables across 22 taxa was performed using a clustering algorithm via ward.d method. in brief, a mean distances matrix was generated and subjected to clustering with the ward.d method of the hclust function. following the clustering, the relationships between the sections were visualised as dendrograms using the tree functions in r v3.4.3. results the dissimilarity cluster analyses formed two main groups and four subgroups according to the surface characters (fig. 2). both of the groups included different taxa from different sections. the macro-morphological observations showed that the seed shapes and sizes of plantago taxa are very variable. the micromorphological aspects of the examined parts using sem are shown in tab. 3. the sem photos of all the examined areas are given in fig. 3. it can be seen from the fig. 3 that hairs, and epidermal cell arrangements are variable among the studied taxa. observations showed that the surface type of corolla tube was the most stable feature appearing to be lineate in all of the examined taxa but only differing in terms of cell size and presence of hairs (tab. 3, fig. 3). this result shows that the surfaces of the petals and the posterior sepals seem to have more or less stable features through sections (tab. 3, fig. 3). being the outermost part of a flower structure, bract has shown a great variety in terms of surface micromorphology among all the examined parts (tab. 3, fig. 3). in section coronopus bract surfaces are differing among the taxa whereas the seed surface characteristics are very stable. all taxa in this section has reticulate seed surface ornamentation and all but one taxa (p. holosteum) has undulated anticlinal walls. section coronopus also show stability in terms of inflorescence parts’ surface characters generally except anterior sepal and bract surfaces (tab. 3). similarly, taxa in section arnoglossum have very stable seed surface characteristics (smooth). the smooth seed surfaces have also been observed in some taxa of section psyllium. section psyllium, has the most variable seed surface characteristics. the inflorescence surface characters vary among these taxa, as well (tab. 3). sections lamprosantha, oreades fig. 2. cluster dendrogram of plantago taxa. the sections marked with different colors biologica nyssana ● 11 (2) december 2020: 85-91 ciftci, erol ● micromorphological studies on inflorescence and seeds of some plantain (plantago l.) taxa in turkey and gentianoides are represented by one taxon each, and these taxa have similar characteristics with each other, and in the terms of seed surface characters they resemble section plantago. in p. major subsp. major and in p. major subsp. intermedia seed surfaces are rugose. other examined parts (i.e. anterior sepal, posterior sepal carina, corolla tube, petal, and dorsal and ventral sides of seeds) show parallel characteristics within these two taxa except anterior sepals. the bases of anterior sepals are both rugose, but cell shapes and anticlinal wall nature is different (tab. 3). the seed surfaces are reticulate in section leucopsyllium and section hymenopsyllium. section leucopsyllium has diverse inflorescence surface characters. section hymenopsyllium taxa have more or less stable characters on micromorphological level with rugose posterior sepal carina surfaces, striate corolla tube, and striate petal surfaces but there is only two taxa available in this study. discussion the two subspecies of p. major appear in two different main groups. concurrently, the observation of the seed surfaces of p. major were controversial in previous studies focusing on the seed micromorphology of plantago taxa (klimko et al., 2004; shehata & loutfy, 2006; hoghoughi et al., 2016; verma & bharti, 2017). klimko et al. (2004) reported the seed surfaces of p. major as “not slightly rugose as it has been reported in previous studies” (rymkiewicz, 1979; rothmaler et al., 1984). on the contrary, other authors identified the same area as reticulate (shehata & loutfy, 2006), tuberculate (verma & bharti, 2017), and areolate with wrinkled surface (hog89 fig. 3. sem micrographs of studied areas. posterior sepal carina surfaces of (a) reticulate, plantago major subsp. major; (b) rugose, p. weldenii; (c) striate, p. gentianoides subsp. gentianoides; petal surfaces of (d) granulate, p. weldenii; (e) striate, p. gentianoides subsp. gentianoides; (f) rugose, p. sempervirens; anterior sepal base surfaces of (g) rugose, p. major subsp. major; (h) rugulose, p. coronopus subsp. coronopus; (i) granulate, p. albicans; bract margin surfaces of (j) reticulate, p. coronopus subsp. coronopus; (k) rugose, p. maritima; (l) smooth, p. argentea; p. squarrosa; (m) fissured, p. afra; (n) granulate, p. sempervirens; corolla tube surface of (o) lineat, p. gentianoides subsp. gentianoides; seed surfaces of (p) rugose, plantago major subsp. major; (q) reticulate, p. crassifolia; (r) smooth, p. lagopus. biologica nyssana ● 11 (2) december 2020: 85-91 ciftci, erol ● micromorphological studies on inflorescence and seeds of some plantain (plantago l.) taxa in turkey houghi et al, 2016). in this study, seed surfaces of both p. major subsp. major and p. major subsp. intermedia were identified as rugose. plantago lanceolata is another taxon which shows a great variety in the sense of seed micromorphology like p. major. verma et al. (2017) reported the seed surface of p. lanceolata as scalariform to reticulate with spindle shaped striations whereas hoghoughi et al. (2016) identifies it as negative reticulate, and the samples in this study showed no ornamentation (smooth surface). these taxa are widespread and adapted to a variety of habitats, therefore it is an expected outcome. similarly, p. maritima and p. gentianoides were reported as having regulate or areolate ornamentation and reticulum cristatum respectively (hoghoughi et al., 2016), but in this study they are identified as reticulate and rugose. it has been known that plantago taxa are very variable in morphology, and that this variation may be due to environmental differences (meudt, 2012). however, some taxa show very consistent surface characteristics. for instance, p. coronopus has found to be reticulate in both this study and others (hoghoughi et al., 2016). plantago major is a widespread species with capability to adapt in a variety of habitats, thus its positioning in the clustering may not be considered meaningful. section psyllium taxa have representatives in both main groups in dendrogram. some of these taxa have shown different seed surface characters than previously studied (shehata & loutfy, 2006). the comparison of these results shows that seed surfaces may show variation on an intraspecific level among sect. psyllium and also some other taxa (p. major and p. lanceolate) section coronopus taxa have a very distinct surface structure. all of the studied taxa in this section have shown undulated anticline membranes and ruminate seed surfaces. these findings are in accordance with the previous studies (klimko et al., 2004; shehata & loutfy, 2006). however, the classification of the coronopus section, including p. maritima, is problematic. rahn (1978); dietrich (1980) and klimko et al. (2004) have divided the section coronopus into two sections as section coronopus and section maritima. shehata & loutfy (2006) have separated the p. maritima taxon from subgenus coronopus. in this study, p. maritima emerged in the second main group with the other section coronopus (p. weldenii, p. crassifolia and p. holosteum) except p. coronopus. the dendrogram separated the taxa of sect. coronopus into four subgroups; p. maritima grouped with p. crassifolia; p. weldenii which was formerly assigned as a subspecies of p. coronopus. however, these findings don’t agree with the molecular data completely (serrano et al., 2017). in tutel et al. (2005), it has been suggested that hymenopsyllium section in subgenus euplantago is closer to subgenus psyllium based on morphological traits. in this study sect. hymenopsyllium is clustered completely in first main group, whereas most of the sect. psyllium taxa are placed in the second main group. therefore, this study does not support the initial statement. however, the results showed the latter section is very diverse in terms of micromorphologic traits and the study of molecular markers already showed p. bellardii and p. cretica are in the subgenus psyllium (ronsted et al., 2002). this shows the micromorphology of inflorescence parts of these taxa are not very reliable in this subgenus. the plantago taxa are known to be wind polinated but we observed that the flowers attract some insects (syrphidae) in field and alfred heilbronn botanical garden. by the reason of the fact that they do not have flashy flowers, it may be due to the uv reflection characteristics of the epicuticular waxes (whitney et al., 2009). some taxa having more ability to draw the insects (p. maritima, p. lanceolata, p. argentea) have granulate and striate petal surfaces. however, this finding needs further investigations. in the overall results of the examinations, we have pointed out that the inflorescence parts’ surfaces are variable among taxa except for the corolla tube, and the differences are probably not distinctive characteristics for these taxa. this may be due to its protective position. this work shows that hairs, epidermal cell arrangements and surface characters are very variable across the genus. therefore, analysis of inflorescence parts is not conclusive to make a specific distinction between different taxa. although some groups have very stable seed surface characteristics, they are not reliable among all taxa, which means it should be used with extreme care especially with the widespread taxa. further studies may be conducted to understand how variable the seed characteristics of the widespread taxa, and whether it is a result of different habitat types. acknowledgements. we would like to thank dr. vahap eldem for performing the data processing in r, and prof. dr. orhan kucuker for sharing his knowledge. this work was supported by the research fund of istanbul university, with project number 10413. references albach, d. c., meudt, h.m., oxelman, b. 2005: piecing together the “new” plantaginaceae. american journal of botany, 92: 297-315. barthlott, w., neinhuis, c., cutler, d., ditsch, f., meusel, i., theisen, i., wilhelmi, h. 1998: classification and terminology of plant epicuticular waxes. botanical journal of the linnean society, 126 90 biologica nyssana ● 11 (2) december 2020: 85-91 ciftci, erol ● micromorphological studies on inflorescence and seeds of some plantain (plantago l.) taxa in turkey (3): 237-260. dietrich, h. 1980: zytologische untersuchung innerhalb der familie der plantaginaceae. naturwissenschaften, 29: 559-587. endress, p.k. 2001: origins of flower morphology. journal of experimental zoology, 291 (2): 105-115. hoghoughi, t., jafari, a., mahmoodzadeh, h. 2016: palynological and seed micromorphological studies on plantago l. species in north eastern of iran. journal of biology and environmental science, 8 (1): 133-138. jun-zhe, l., qing-min, z., shu-hua, g., xingdi, z. 1992: seed morphology of plantago in china and its taxonomic significance. journal of systematics and evolution, 30 (2): 118-125. klimko, m., idzikowska, k., truchan, m., kreft, a. 2004: seed sculpture of polish species of the genus plantago l. acta societatis botanicorum poloniae, 73 (2): 103-111. meudt, h. m. 2012: a taxonomic revision of native new zealand plantago (plantaginaceae). new zealand journal of botany, 50 (2): 101-178. pilger, r. 1937: plantaginaceae. in: engler, a. 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(ed.), flora of turkey and the east aegean islands, 7: 504521, edinburgh university press. edinburgh. tutel, b. 1993: türkiye florası atlası (atlas flora turcicae) plantago 5-7. i̇. ü. yay. 3689 fen fak 225. i̇stanbul. tutel, b., kandemir, i., kus, s., kence, a. 2005: classification of turkish plantago l. species using numerical taxonomy. turkish journal of botany, 29 (1): 51-61. verma, a., kumar avinash bharti, n.g. 2017: macro and micro-morphological characteristics of plantago seeds and its implication for species identification. current botany 8: 159-163. whitney, h. m., kolle, m., andrew, p., chittka, l., steiner, u., glover, b. j. 2009: floral iridescence, produced by diffractive optics, acts as a cue for animal pollinators. science, 323: 130-133. 91 biologica nyssana ● 11 (2) december 2020: 85-91 ciftci, erol ● micromorphological studies on inflorescence and seeds of some plantain (plantago l.) taxa in turkey jenačković gocić et al. 2020, biologica nyssana 11(2) 11 (2) december 2020: 71-84 doi: 10.5281/zenodo.4393949 insight into the chorology of some endangered, rare and potentially invasive plant species in serbia original article dragana jenačković gocić department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, 18000 niš, serbia draganaj@pmf.ni.ac.rs (corresponding author) ljiljana bolbotinović department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, 18000 niš, serbia ljiljana.bolbotinovic@pmf.edu.rs marina jušković department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, 18000 niš, serbia marinaju@pmf.ni.ac.rs danijela nikolić department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, 18000 niš, serbia danid@pmf.ni.ac.rs vladimir ranđelović department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, 18000 niš, serbia vladar@pmf.ni.ac.rs received: december 07, 2020 revised: december 14, 2020 accepted: december 14, 2020 abstract: this paper presents the distribution of nine endangered, rare or potentially invasive plant species present in the serbian flora, based on field investigations, literature and herbarium data. the chorology of the following vascular plants: ranunculus lateriflorus, elatine alsinastrum, sedum stefco, galium boreale, ornithogalum narbonense, cyperus serotinus, cyperus odoratus, typha laxmannii and typha shuttleworthii, is shown on 10 x 10 km2 utm maps. the data on the distribution of these endangered and rare species provide a valuable base for estimating and revising their threatened status in serbia, as well as for conducting adequate conservation measures with regard to their habitats. chorological data on the cyperus odoratus species in serbia are significant for getting to know its naturalization history and spreading pathways in both serbia and its neighbouring countries. key words: distribution, endangered plants, flora, invasive species, new chorological data, rare species, serbia apstract: uvid u horologiju pojedinih ugroženih, retkih i potencijalno invazivnih biljnih vrsta u srbiji u ovom radu, prikazana je distribucija devet ugroženih, retkih i potencijalno invazivnih vrsta prisutnih u flori srbije na osnovu terenskih, literaturnih i herbarijumskih podataka. horologija sledećih vaskularnih biljaka: ranunculus lateriflorus, elatine alsinastrum, sedum stefco, galium boreale, ornithogalum narbonense, cyperus serotinus, cyperus odoratus, typha laxmannii i typha shuttleworthii, prikazana je na 10x10 km2 utm kartama. podaci o distribuciji ugroženih i retkih vrsta, koji su ovde prikazani, predstvaljaju dragocenu osnovu za procenu i reviziju njihovog statusa ugroženosti na području srbije, i sprovođenje adekvatnih mera zaštite njihovih staništa. podaci o distribuciji vrste cyperus odoratus u srbiji su od velikog značaja za upoznavanje istorije procesa naturalizacije i puteva širenja njenih individua u našoj i susednim zemljama. ključne reči: rasprostranjenje, ugrožene biljke, flora, invazivne vrste, novi horološki podaci, retke vrste, srbija introduction it is well-known that continuous monitoring of the floristic composition and abundance of plant species, especially those that are threatened, alien or weed, has enormous significances for biodiversity conservation. monitoring the population of threatened species has a significant role in conducting protection measures in order to prevent their eventual extinction, while collecting data on the presence and spreading pathways of new, mostly invasive species, is significant for controlling the size of their populations and protecting autochthonous flora. despite the intensive study of serbian flora over the last decades, new species are still being found (ranđelović et al., 2002; niketić et al., 2009; đorđević et al., 2010; đorđević et al., 2014; tomović et al., 2016; stojanović et al., 2017), as well as new localities for rare and endemic plant species (tomović et al., 2007; zlatković et al., 2007; niketić © 2020 jenačković gocić et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work noncommercially under the same license as the original. 71 & tomović, 2008; zlatković & bogosavljević, 2014; jenačković et al., 2015; đorđević et al., 2017), which supports the fact that the flora of serbia has not yet been, and probably never will be, fully explored. during intensive phytocoenological research conducted in the areas of ne, e and se serbia on diverse habitat types, including rivers, ponds, seasonal flooded habitats, wet meadows and rocks over the last three years, new chorological data on some threatened, rare, endemic and potentially invasive plant species have been collected. this research confirms the already known fact that the previously mentioned habitat types are refuges for numerous rare and endangered plants (stojanović et al., 1994), so they are significant for the conservation of genetic, species and ecosystem diversity. in the regions of ne, e and se serbia, they are, unfortunately, poorly investigated, both floristically (bogosavljević et al., 2007; petrović et al., 2007; stanković et al., 2009; jenačković et al., 2015) and phytocoenologically (ranđelovć et al., 2007; ranđelović & zlatković, 2010; jenačković et al., 2010; jenačković, 2017). generally, publishing new chorological data for the previously mentioned groups of plants enriches knowledge regarding their distribution and provides adequate material for writing and publishing new editions of flora of serbia, as well as red list and red books of the flora of serbia and the serbian list of invasive plant species. in this paper, the authors give detailed information on the distribution pattern of nine vascular plants in serbia: ranunculus lateriflorus dc., elatine alsinastrum l., sedum stefco stefanov, galium boreale l., ornithogalum narbonense l., cyperus serotinus rottb., cyperus odoratus l., typha laxmannii lepechin and typha shuttleworthii koch & sonder in koch. material and methods the distribution of each of the species presented in this study is showed on a 10 x 10 km2 utm grid based on field, literature and herbarium data. field investigation of different habitats was carried out in the period 2017-2020 in the areas of ne, e and se serbia. relevant floristic and phytocoenological literature published in the last seventy years was also used as an adequate source of chorological data. georeferenced data on the distribution of specimens deposited in the herbarium of the faculty of sciences and mathematics, department of biology and ecology, university of niš (hmn) and herbarium of the institute of botany and botanical garden “jevremovac”, university of belgrade (beou) are showed on utm grids, too. plant material collected during field investigation was determined by using the dichotomic identification keys (josifović, 1970 – 1980; tutin et al., 1964 – 1980). the nomenclature of the species recorded was uniformed to the flora europaea (tutin et al., 1964 – 1980). the threatened status for the species mentioned in the section above is considered in terms of iucn (2000) criteria and categories. results and discussion ranunculus lateriflorus dc. general distribution: r. lateriflorus has been recorded in europe (spain, france, italy, austria, slovakia, hungary, romania and all countries of the balkan peninsula except albania), north africa (morocco and algeria) and asia (countries of the western asia, ukraine and russia). distribution in serbia: most published data on r. lateriflorus distribution in serbia are for the province of vojvodina (diklić, 1992; purger, 1993; panjković et al., 2012; šturc, 2014) (fig. 1). other parts of serbia have been poorly investigated, especially the territory of nw, w, se and c serbia, pomoravlje and regions of kosovo. outside of vojvodina province, r. lateriflorus was recorded in the šumadija region: rakovica and grocka (diklić, 1992); ne serbia: negotin and kladovo (diklić, 1992); e serbia: pirot (nikolić et al., 1986; diklić, 1992) and s serbia: leskovac (diklić, 1992). unpublished chorological data: ne serbia: krivelj – ep88 (leg. pančić, j. 2327, 1876, beou); šumadija region: jovanovac – dp97 (leg. pančić, j. 2309, 1870, beou); banat region: melenci – dr44 (leg. niketić, m., tomović, g. 48059, 23.04.2016, beou); farkaždin – dr50 (leg. lakušić, d., tomović, g. 47302, 19.04.2016, beou; leg. lakušić, d., tomović, g., kuzmanović, n., kabaš, e., đurović, s., janković, i., milekić, t. 47375, 17.05.2016, beou). new chorological data: it has been found in the surroundings of donje međurovo village (e serbia – en69, 43°18’22’’ n, 21°50’25’’ e, 185 m a.s.l., leg./det. jenačković, d. 14367, 29.05.2018, hmn). at this study site, on a seasonally flooded and salted habitat that borders with a cultivated area, a small population of r. lateriflorus was recorded, which is under strict protection. it was made up of less than a hundred individuals. most of the individuals recorded are growing in association with eleocharis palustris and oenanthe silaifolia, and they are part of the composition of the halophilous community, with beckmannia eruciformis being dominant. habitat characteristics: r. lateriflorus is an annual therophyte that grows and blooms during the spring on wet, sandy and seasonally flooded habitats. iucn threatened status in the world: least concern (lc). iucn threatened status in serbia: endangered – vulnerable (en b1; b2b, c; c1; c2a/vu a1b, c, e; 72 biologica nyssana ● 11 (2) december 2020: 71-84 jenačković gocić et al. ● insight into the chorology of some endangered, rare and potentially invasive plant species in serbia 73 a2c, e; d1; d2). according to király (2007), r. lateriflorus is a legally protected species in hungary, where it is classified in the category nt (near threatened). generally, its habitats are exposed to strong negative anthropogenic pressure, which can be seen in the decreasing size of its population, so applying adequate conservation measure is needed not only in serbia, but also globally. elatine alsinastrum l. general distribution: e. alsinastrum is a eurosiberian sub-mediterranean species that grows in temporary water bodies in europe (from the iberian peninsula, north to france and belgium, east to poland and ukraine, and in almost all countries in the mediterranean basin), asia (russia, ukraine, georgia, armenia, w siberia and kazakhstan) and north africa morocco, algeria and tunisia (popiela et al., 2013). distribution in serbia: although blečić (1972) stated that the species is often found in serbia, literature sources and herbarium collections provide little data on e. alsinastrum chorology in serbia (fig. 2). until now, it has been documented that e. alsinastrum grows in ditches in the bačka (panjković, 2005) and srem regions (perić et al., 2016). unpublished chorological data: during the 19th century, josif pančić collected specimens of e. alsinastrum at a few locations south of vojvodina province. he found this species in the area surrounding belgrade (rakovica – dq55, leg. pančić, j. 3170, 1877, beou), sobovica (bele bare – dp88, leg. pančić, j. 3168, 1851, beou), bubanj – en69 (leg. pančić, j. 3167, 1885, beou) and in the valley of the kolubara river (leg. pančić, j. 14593, beou, this data is not shown on fig. 2). new chorological data: two years ago, in the area surrounding the village of donje međurovo (se serbia – utm, 43°18’18’’ n, 21°50’10’’ e, 183 m a.s.l.) in seasonally flooded habitats, a community was recorded, with e. alsinastrum dominance (fig. 2). the following species: eleocharis palustris, alisma plantago-aquatica, veronica anagallisaquatica, mentha pulegium and beckmannia eruciformis were part of the composition of the elatine alsinastrum community. specimens of e. alsinastrum are stored in hmn (leg./det. jenačković, d. 14370, 29.05.2018). more than a hundred years ago, josif pančić found e. alsinastrum in the vicinity of donje međurovo (bubanj – en69), but he did not publish that record. habitat characteristics: e. alsinastrum inhabits temporary water bodies or seasonally flooded zones of permanent water bodies. iucn threatened status in the world: near threatened (nt) with decreasing population trend. iucn threatened status in serbia: not threatened. fig 1. distribution map of ranunculus lateriflorus in serbia (a). the photographs show it growing on seasonally flooded habitats in the vicinity of donje međurovo village (b). biologica nyssana ● 11 (2) december 2020: 71-84 jenačković gocić et al. ● insight into the chorology of some endangered, rare and potentially invasive plant species in serbia 74 elatine triandra schkuhr is the only species from the genus elatine that is protected in serbia. based on literature sources and herbarium data, as well as the authors’ field experiences, it can be concluded that e. alsinastrum is a rare species of serbian flora which requires some kind of protection. its small populations inhabit sensitive habitat types that are threatened by diverse anthropogenic factors, such as the drainage of seasonally inundated land, stabilization of hydrological regimes, agricultural improvement etc. the same problem also exists in other european countries, so e. alsinastrum could be considered as rare species throughout most countries in central europe (schnittler & günther, 1999). elatine alsinastrum is classified as a critically endangered species in bulgaria (petrova & vladimirov, 2009), and near threatened in hungary (király, 2007). in croatia, it is rarely recorded, and known only from old literature and herbarium sources, so it is described as an insufficiently known plant species (dd – data deficient) (prlić, 2015). sedum stefco stefanov general distribution: it is an endemic species of the balkan peninsula that has been recorded in four countries (serbia, bulgaria, macedonia and greece) so far. distribution in serbia: the first data published on the presence of s. stefco in serbian flora date from the end of the 20th century and refer to radan mountain (zlatković et al., 1993; zlatković & ranđelović, 1995) and the pčinja river gorge (zlatković & ranđelović, 1995). by publishing data on the geographical position of population of the species s. stefco in e serbia (the temštica gorge) and s serbia (kozjak mountain – monastery prohor pčinjski), tomović et al. (2003) made the previous knowledge of its distribution more complete (fig. 3). new chorological data: two populations of the strictly protected species, s. stefco, were found on stara planina mountain at a locality called strejnica (e serbia – fp20, 43°21’20.24’’ n, 22°35’31.91’’ e, 1501 m a.s.l., leg./det. ranđelović, v., 10.07.2020; e serbia – fp20, 43°21’20.32’’ n, 22°35’31.70’’ e, 1550 m a.s.l., leg./det. ranđelović, v., 02.08.2020). individuals of the s. stefco species inhabit fragments of red sandstone with other species characteristic for rocky vegetation, including sedum annuum, sedum album, silene lerchenfeldiana, allium carinatum subsp. pulchellum, scleranthus perennis, anthemis carpatica, etc. habitat characteristics: in serbia, s. stefco is found on rocks (silicate and red sandstones), rock fragments, and dry, rocky ground at a wide range of altitudes, from 300 m to 1550 m a.s.l. iucn threatened status in the world: not threatened. iucn threatened status in serbia: near threatened – least concern (data deficient) (nt-lc fig 2. utm map of the distribution of elatine alsinastrum in serbia (a). the photographs present its populations that inhabit shallow, ephemeral depressions in the vicinity of donje međurovo village (b). biologica nyssana ● 11 (2) december 2020: 71-84 jenačković gocić et al. ● insight into the chorology of some endangered, rare and potentially invasive plant species in serbia (dd)). sedum stefco is classified as near threatened in the greek red data book (eleftheriadou et al., 2009), and vulnerable, and it is a legally protected species in the republic of bulgaria (petrova & vladimirov, 2010). galium boreale l. general distribution: it is distributed over the subarctic and temperate regions of the northern hemisphere. in europe, g. boreale is rare in the mediterranean region. distribution in serbia: g. boreale is a legally protected species in serbia. it is recorded in the bačka region (boža & vasić, 1986); nw serbia: maljen mountain (gajić, 1973; popović, 2005); w serbia: stolovi mountain (pavlović, 1974), tara mountain (gajić, 1988), zlatibor mountain (pavlović, 1951; gajić, 1973; novaković-vuković, 2015), the uvac river gorge (veljić et al., 2006); sw serbia: ozren mountain (pavlović, 1955; rakonjac, 2002) and pešter plateau (tatić, 1988; rakonjac, 2002) (fig. 4). new chorological data: while mapping herbaceous habitat types on vlasina plateau, individuals of g. boreale were recorded at two sites: murina valley (se serbia – fn12, 42°42’51.52’’ n, 22°21’39.09’’ e, 1264 m a.s.l., leg. jenačković gocić, d., det. ranđelović, v. 14383, 29.06.2020, hmn) and bratašnica (se serbia – fn02, 42°41’37.39’’ n, 22°19’17.48’’ e, 1226 m a.s.l., leg./det. ranđelović, v., 20.07.2020). they were a part of the meadow vegetation composition, together with individuals from the following species: sanguisorba officinalis, briza media, holcus lanatus, centaurea jacea, filipendula hexapetala, galium verum, hypericum maculatum subsp. immaculatum, potentilla erecta and campanula patula. habitat characteristics: g. boreale is a heliophilous species that prefers moist and well drained soils. it can tolerate partial shade and dry conditions. it is found in wet meadows and open woods. iucn threatened status in the world: not threatened. iucn threatened status in serbia: vulnerable – near threatened (data deficient) (vu-nt (dd)). galium boreale is a vulnerable species in the bulgarian flora (petrova & vladimirov, 2009), too. ornithogalum narbonense l. general distribution: it occurs in europe (portugal, spain, france, italy, romania and countries on the balkan peninsula), asia (turkey, azerbaijan, armenia, georgia, syria, lebanon, israel and jordan) and africa (egypt, libya, tunisia, algeria and morocco). distribution in serbia: according to diklić (1975), o. narbonense is a species with sporadic occurrence in serbia. data on its distribution in 75 fig 3. distribution of sedum stefco in serbia (a). photographs of sedum stefco specimens (b) and a mixed community dominated by succulent plant species sedum stefco and sedum album (c), developed on rocky fragments situated on stara planina mountain. biologica nyssana ● 11 (2) december 2020: 71-84 jenačković gocić et al. ● insight into the chorology of some endangered, rare and potentially invasive plant species in serbia serbia are poor. until now, o. narbonense has been recorded at two localities in the province of kosovo: podujevo and orahovac (nikolić et al., 1986) (fig. 5). ranđelović et al. (2005) stated that o. narbonense inhabits cultivated land in southeastern serbia, but did not give precise information on the geographical position of its populations. further field investigations are needed in order to determine whether o. narbonense is a rare or insufficiently investigated species in serbia. new chorological data: o. narbonense was recorded within meadow vegetation on vlasina biologica nyssana ● 11 (2) december 2020: 71-84 jenačković gocić et al. ● insight into the chorology of some endangered, rare and potentially invasive plant species in serbia 76 fig 4. utm map of the distribution of galium boreale in serbia (a) and photographs of individual specimens (b). fig 5. map of distribution of ornithogalum narbonense in serbia (a) and photographs of its inflorescence (b). biologica nyssana ● 11 (2) december 2020: 71-84 jenačković gocić et al. ● insight into the chorology of some endangered, rare and potentially invasive plant species in serbia plateau, in a place named jarčev potok (se serbia – fn02, 42°40’11.73’’ n, 22°20’12.35’’ e, 1234 m a.s.l., leg./det. ranđelović, v., 21.07.2020). agrostis capillaris, briza media, achillea millefolium, campanula patula, crepis conyzifolia, dianthus deltoides, galium verum, hypericum maculatum subsp. immaculatum, hypochaeris maculata, potentilla argentea and pastinaca hirsuta are the most abundant species at the site where these individuals of o. narbonense were found. habitat characteristics: it grows in sunny, grassy and dry places such as grasslands, roadsides, crops and disturbed ground. iucn threatened status in the world: not threatened. iucn threatened status in serbia: vulnerable – near threatened (data deficient) (vu-nt (dd)). cyperus serotinus rottb. general distribution: c. serotinus is a native species on the european and asian continents while it is naturalized in north america and australia. on the eurasian continent, c. serotinus is distributed from the iberia peninsula through the countries of central europe and the mediterranean basin, caucasus, kazakhstan, middle east and the himalayas to japan, most of china, taiwan and vietnam. distribution in serbia: c. serotinus is a rare and endangered species that is recorded in the banat region: deliblato sands (budak, 1983), labudovo okno (polić, 2006), žilovo islet, dolnice bay and stevan lowlands (stevanović et al., 2004); šumadija: on the banks of the danube river near belgrade (čanak, 1976), nw serbia: loznica (čanak, 1976), and c serbia: batušinac ponds (ranđelović et al., 2007) (fig. 6). unpublished chorological data: in the 21st century, individuals of the c. serotinus species were found at a few locations in the danube river valley, downstream from belgrade (malo bavanište embankment – eq05, leg. stevanović, v. 49867, 22.09.2002, beou; mali lap – eq16, leg. stevanović, v. 22335, 26.07.2003, beou; zatonje – eq35, leg. stevanović, v. 50093, 23.11.2013, beou; prahovo (bay kusjaka) – fq20, leg. stevanović, v. 50073, 23.07.2013, beou). new chorological data: one population of c. serotinus was recorded on the right bank of the danube river near tekija (ne serbia – fq14, 44°41’28’’ n, 22°24’56’’ e, leg. bolbotinović, lj., det. ranđelović, v. & jenačković, d. 14368, 25.09.2018, hmn). the first record (unpublished) for this toponymy dates from the 19th century (cyperus monti, tekija – fq14, leg. pančić j. 15359, 1877, beou; cyperus monti, tekija – fq14, leg. pančić j. 12347, 1878, beou), but it refers to “old” tekija, which was submerged after construction of the đerdap i hydropower dam. habitat characteristics: c. serotinus grows in seasonally flooded habitats, bays, banks of rivers and standing water bodies. 77 fig 6. utm maps of the distribution of cyperus serotinus (a) and a photograph of its inflorescence (b). biologica nyssana ● 11 (2) december 2020: 71-84 jenačković gocić et al. ● insight into the chorology of some endangered, rare and potentially invasive plant species in serbia iucn threatened status in the world: least concern (lc). iucn threatened status in serbia: near threatened – least concern (data deficient) (nt – lc (dd)). according to nikolić & topić (2005), c. serotinus is a vulnerable plant species in croatian flora. cyperus odoratus l. general distribution: c. odoratus is a pantropic species which has been naturalized in europe (spain, italy, serbia, bulgaria and romania) (verloove, 2014). distribution in serbia: for a long time, c. odoratus has been confused with c. strigosus and other species from the cyperus genus, so its exact naturalization history in serbia is not known. after the revision of herbarium specimens deposited in beou, verloove (2014) concluded that the first record of c. odoratus, under the erroneous binomial c. strigosus, was published at the beginning of the 21st century by stevanović et al. (2004, 2005). it refers to mali lap marsh situated near the village of dubovac (banat region – eq15) (fig. 7). stevanović et al. (2005) stated that c. strigosus grows in several places in the danube basin (km 1090 – 1075): žilovo islet, mali lap marsh, dolnice bay and the stevan lowlands. after the revision of herbarium material deposited in beou, the authors confirmed the presence of c. odoratus at žilovo islet – eq15 (cyperus sp., leg. stevanović, v., stevanović, b., sekulić, n., šinžar-sekulić, j. 49877, 08.09.2002, beou), while the presence of c. odoratus at dolnice bay and stevan lowlands remains unconfirmed. verloove (2014) stated that individuals from the c. odoratus species form small populations at numerous localities along the danube river, from kovin to mihajlovac near đerdap ii hydropower dam, but he did not give precise information on the localities. unpublished chorological data: after revision of specimens from the cyperus genus stored in beou, the authors established that c. odoratus grows in one more place in the banat region (malo bavanište – eq05, leg. stevanović, v. 49860, 22.09.2002, beou; leg. stevanović, v. 49866, 22.09.2002, beou). during 2020, individuals from the c. odoratus species were recorded at localities situated northwest of the places mentioned in earlier published papers (stevanović et al. 2005, verloove 2014): ada ciganlija (dq56 – 44°47’43.05” n, 20°25’31.15” e, leg. pantović, j., stevanoski, i., ćosić, m. 69151, 16.10.2019, beou) and pančevački rit (dq66 – 44°49’53.41” n, 20°30’14.35” e, leg. pantović, j., stevanoski, i., ćosić, m. 69160, 24.10.2019, beou) (fig. 7). new chorological data: c. odoratus was found at three locations (44°41’27’’ n, 22°24’56’’ e; 44°41’20’’ n, 22°24’47’’ e; 44°40’34’’ n, 22°24’70’’ e) on seasonally flooded habitat positioned in surrounding of tekija, near the danube river (ne serbia – fq14, leg. bolbotinović, lj., det. 78 fig 7. map of distribution of cyperus odoratus in serbia (a) and a photograph of individual specimens (b). jenačković, d. & ranđelović, v. 14369, 25.09.2018, hmn). habitat characteristics: in europe, c. odoratus is mostly found on mud flats, gravelly or sandy river banks, estuaries, and in riparian woodland, drainage channels and ditches (verloove, 2014). they form pure or mixed communities with other emergent macrophytes, such as c. serotinus. otherwise, c. odoratus occupies new pitches very quickly and behaves like an invasive environmental weed (verloove, 2014), so the knowledge of its distribution pattern and population status is important for determining how it spreads and how it can eventually be controlled. iucn threatened status in the world: not threatened. iucn threatened status in serbia: not threatened. typha laxmannii lepechin general distribution: t. laxmannii is distributed from europe through siberia, the caucasus, the middle east, kazakhastan and mongolia to the russian far east, china and japan. distribution in serbia: the first chorological data for t. laxmannii in serbia are from budak (1986), and they refer to the banat and bačka regions (fig. 8). radulović (2005) stated that individuals from the t. laxmannii species compose a community with typha latifolia dominance in carska bara special nature reserve. in the 21st century, a few authors have published data on t. laxmannii distribution outside the borders of vojvodina province. until now, it has been recorded in the following localities: batušinac ponds (ranđelović et al., 2007), šarpance village, vražji kamen, the vicinity of dimitrovgrad town, vranjska banja (zlatković et al., 2007), rgotina lake (zlatković & bogosavljević, 2014), smilovsko lake (jenačković, 2017) and zlatibor mountain (tomović et al., 2020) (fig.8). new chorological data: during the summer of 2020, a small population of t. laxmannii was recorded in a canal, positioned east of the dam, through which water enters vlasina lake (se serbia – fn03, 42°44’59.01’’n, 22°19’43.34’’e, 1222 m a.s.l., leg. nikolić, d., jušković, m., savić, a., det. ranđelović, v. 14388, 01.08.2020, hmn). at this site, t. laxmannii grows mainly in association with typha latifolia, juncus effusus, centaurium erythraea, etc. habitat characteristics: it prefers permanently flooded habitats with shallow waters or seasonally flooded habitats, such as the margins of ponds, pools, canals, rivers, streams and lakes. iucn threatened status in the world: least concern (lc). iucn threatened status in serbia: near threatened (nt). typha laxmannii is classified as a critically 79 biologica nyssana ● 11 (2) december 2020: 71-84 jenačković gocić et al. ● insight into the chorology of some endangered, rare and potentially invasive plant species in serbia fig 8. utm maps of the distribution of typha laxmannii (a) and a photograph of the population recorded at vlasina plateau (b). endangered species of croatian flora (nikolić & topić, 2005), and it was recorded during summer 2000 for the first time (topić & ozimec 2001). typha shuttleworthii koch & sonder in koch general distribution: it occurs in europe (from france east through the czech republic and hungary to romania and the ukraine), the caucasus and asia (turkey). distribution in serbia: t. shuttleworthii was considered to be a critically endangered species in serbia during the 20th century (ranđelović, 1999) because it was known at only a few localities: the spring of jabukovačka river on kukavica mountain, the area surrounding novi sad, kragujevac and sjenica (jovanović, 1986) (fig. 9). its status changed from critically endangered to endangered after establishing new sites in eastern serbia: crna reka gorge, svrljiški timok gorge and the environs of bosilegrad (topli dol and musulj) (tomović et al., 2009). the first data on t. shuttleworthii distribution in c serbia (radan mountain – gornji gajtan and kosmača) and w serbia (tara mountain – kurjačine and kozulja; golija mountain – izubra) were published this year (tomović et al., 2020). new chorological data: a small population of the strictly protected species t. shuttleworthii was recorded in a flooded depression on stara planina mountain, next to the road leading to tupavica waterfall (e serbia – fn49, 43°15’42’’ n, 22°46’26’’ e, 975 m a.s.l., leg./det. ranđelović, v. & jenačković, d. 14384, 22.07.2018, hmn). habitat characteristics: t. shuttleworthii usually builds small populations that cover a few square meters, mostly in anthropogenic habitats such as roadside ditches (tomović et al., 2020), river and stream valleys, swamps and wet meadows (kozłowska et al. 2011). iucn threatened status in the world: data deficient (dd). iucn threatened status in serbia: endangered (en b1 ab(i,ii,iii); b2 ab(i,ii,iii); c1) (tomović et al. 2009). there is a need for additional field investigation due to the possibility of its wider distribution in serbia. it is considered that its significant morphological similarity with t. latifolia is the main reason for the insufficient study of its distribution in serbia (tomović et al., 2020). the same problem exists in bulgaria and croatia, so t. shuttleworthii is classed as data deficient in these countries (petrova & vladimirov, 2009; nikolić & topić, 2005). király (2007) stated that t. shuttleworthii is extinct in hungary. conclusion the chorological data presented in this paper are a valuable base for better understanding the distribution patterns in serbia of the vascular plants analyzed in this study, as well as estimating and potentially reevaluating their threatened status, and controlling 80 fig 9. map of distribution of typha shuttleworthii in serbia (a) and a photograph of its inflorescence (b). biologica nyssana ● 11 (2) december 2020: 71-84 jenačković gocić et al. ● insight into the chorology of some endangered, rare and potentially invasive plant species in serbia 81 the size of their populations. the strictly protected and endangered plant species, r. lateriflorus, is poorly investigated outside the borders of vojvodina province, especially in w serbia and the province of kosovo. it mostly grows in shallow, seasonally flooded depressions that are affected by negative anthropogenic factors, so continuous monitoring of its population is needed due to a possible decrease in its population size. similar habitat types are preferred by e. alsinastrum, a globally near threatened species whose distribution in serbia has not been researched enough. although it is not recognized as threatened in serbia, the authors suggest that its status in this country should be reconsidered in accordance with the data provided in this research. according to existing floristic data, the balkan endemic species s. stefco is distributed in the southern and eastern parts of serbia. the border of its geographic range in serbia is expanding to the north by establishing its existing population on stara planina mountain. individuals from the legally protected and vulnerable species g. boreale were mainly recorded within wet meadows and open woods in western serbia. the borders of its geographic range have moved significantly to the east after establishing its presence on vlasina plateau. the poorly studied, vulnerable species of serbian flora, o. narbonense, requires additional investigation in order to collect information with regard to its presence in other parts of serbia (except the province of kosovo and vlasina plateau) and to reevaluate its threatened status. populations of the near threatened plant species c. serotinus are recorded at only a few sites in serbia, mainly in the valleys of the great lowland rivers including the danube, drina and južna morava. a recently recorded species in serbia, c. odoratus spreads quickly on gravelly and sandy substrates along the danube and sava rivers, by forming a mixed community with the autochthonous macrophyte species. it is considered important to monitor its populations due to its huge potential to inhabit new pitches. based on literature data and field experience, the authors consider that t. laxmanii has wider distribution in serbia than currently known. its habitats are insufficiently researched, especially south of vojvodina province, so additional field investigations are needed in order to adequately estimate its threatened status. during the 21st century, the amount of chorological data on the strictly protected species, t. shuttleworthii, has significantly increased, so its threatened status has changed from critically endangered to endangered in serbian flora. collecting data on its distribution and population size is important, not only for reevaluating its threatened status at a national level, but also for globally estimating the category of its vulnerability. acknowledgements. this study was supported by the project of ministry of science and technological development of republic of serbia (contract no. 451-0368/2020-14/200124). the authors would like to thank snežana vukojičić phd (curator at the beou herbarium) and marija marković phd (curator at hmn herbarium) for their help during the collection of chorological data for the species studied. references blečić, v. 1972: rod elatine l. in: josifović, m. 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(eds.): new floristic records in the balkans: 6. phytologia balcanica, 13 (3): 433455. zlatković, b., bogosavljević, s. 2014: report on the new floristic data from serbia. biologica nyssana, 5 (2): 123-129. 84 biologica nyssana ● 11 (2) december 2020: 71-84 jenačković gocić et al. ● insight into the chorology of some endangered, rare and potentially invasive plant species in serbia balci et al. 2025, biologica nyssana 16(1) 105 16 (1) june 2025: 105-110 doi: 10.46793/biolnyss.16.1.1b determination of pomological characteristics and biochemical contents of some pitaya varieties grown in muğla region original article alp gökhan balci isparta university of applied science, agriculture faculty, department of horticulture, isparta,türkiye, orcid: 0009-0007-9434-278x alpgokhanb@gmail.com (corresponding author) adnan nurhan yildirim isparta university of applied science, agriculture faculty, department of horticulture, isparta, türkiye, orcid: 0000-0003-2474-2116 ayşe vildan pepe isparta university of applied science, agriculture faculty, department of horticulture, isparta, türkiye, orcid: 0000-0002-4565-8602 received: october 18, 2024 revised: december 21, 2024 accepted: december 27, 2024 abstract: this study aimed to determine the fruit quality and antioxidant properties of „siam red” and „vietnam white” varieties grown in greenhouse conditions in muğla region. the fruit weight ranged from 203.55 to 232.20 g, with peel weight between 54.53 and 77.29 g, and flesh weight between 149.02 and 154.91 g. peel thickness varied from 2.69 to 3.32 mm, while flesh firmness ranged from 0.57 to 1.41 kg/cm². fruit width and length measured 65.63– 68.88 mm and 84.50–102.92 mm, respectively. regarding color parameters, the peel’s l* value ranged from 39.70 to 41.54, a* from 35.37 to 37.74, and b* from 11.03 to 13.35. the flesh color l* value varied between 33.67 and 74.62, a* between 10.22 and 45.73, and b* between -2.14 and 4.33. the total soluble solids (tss) content was 10.94–12.57%, ph ranged from 4.46 to 4.60, and titratable acidity (ta) was between 0.36 and 0.46%. the total phenolic content was 406.42–432.77 mg gae/100 g, total flavonoid content ranged from 20.10 to 22.99 mg catechin/100 g, and total antioxidant capacity was 73.74–79.40%. in this study, it was found that the pitaya cv. „vietnam white” produced larger, firmer, and brighter fruits with higher antioxidant content than the „siam red” variety. key words: pitaya, total phenolics, total flavonoids, antioxidant, pomological properties apstrakt: određivanje pomoloških karakteristika i biohemijskog sastava nekih sorti pitaje, gajenih u regionu mugla ova studija je imala za cilj utvrđivanje kvaliteta plodova i antioksidativnih svojstava sorti pitaje „siam red” i „wietnam white” gajenih u plastenicima regiona mugla. masa plodova se kretala od 203,55 do 232,20 g, masa kore od 54,53 do 77,29 g, a masa pulpe od 149,02 do 154,91 g. debljina kore varirala je od 2,69 do 3,32 mm, a čvrstoća pulpe od 0,57 do 1,41 kg (cm²)-1. širina i dužina plodova iznosila je 65,63 do 68,88 mm i 84,50 do 102,92 mm. što se tiče parametara boje, l* vrednost kore varirala je od 39,70 do 41,54, a* od 35,37 do 37,74, a b* od 11,03 do 13,35. l* vrednost boje mesa ploda kretala se između 33,67 i 74,62, a* između 10,22 i 45,73, a b* između -2,14 i 4,33. sadržaj ukupnih rastvorljivih čvrstih materija (tss) iznosio je 10,94– 12,57%, ph vrednost se kretala od 4,46 do 4,60, dok je titrabilna kiselost (ta) bila između 0,36 i 0,46%. ukupan sadržaj fenola bio je 406,42–432,77 mg gae/100 g, sadržaj ukupnih flavonoida varirao je od 20,10 do 22,99 mg katehina/100 g, dok je ukupni antioksidativni kapacitet bio u rasponu od 73,74 do 79,40%. u ovom istraživanju utvrđeno je da sorta pitaje „vietnam white” ima veće, čvršće, svetlije plodove i veći sadržaj antioksidanasa u poređenju sa sortom „siam red”. ključne reči: pitaja, ukupni fenoli, ukupni flavonoidi, antioksidans, pomološka svojstva introduction pitaya, also known as dragon fruit, is a type of fruit belonging to the genus hylocereus (a.berger) britton & rose. there are three species in the genus hylocereus. hylocereus undatus (haw.) britton & rose (colombia, mexico, south america), h. polyrhizus (f.a.c.weber) britton & rose (mexico) and h. megalanthus (k.schum. ex vaupel) ralf bauer (bolivia, peru, ecuador, colombia, venezuela) are the white fleshy ones with red rinds, red fleshy ones with red rinds and white fleshy ones with yellow rinds (zainoldin et al., 2009; nizamlıoğlu et al., 2021). pitaya originates from mexico, central © 2025 balci et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 106 and south america, but is also grown commercially in malaysia, vietnam and taiwan. in recent years, it has been cultivated in countries such as israel, the usa and australia (merten, 2003; attar et al., 2022). pitaya can be eaten raw and is also used in juice, wine, pate, desserts, and traditional medicine (sofowora et al., 2013). pitaya contains high levels of betalain. betalain is a water-soluble pigment with high protein and fiber content and high antioxidant activity (tze et al., 2012). in recent years, the use of betalain as a natural colorant in cosmetic products has become widespread. among the pitaya species, h. polyrhizus is considered to be the species with the highest betalain content (kamairudin et al., 2015). previous studies reported that the linoleic acid content of pitaya seeds was higher than that of canola, flax, sesame seeds, and grapes (ariffin et al., 2009). in addition, pitaya fruits are consumed as table food and used as food additives. it was stated that adding pitaya fruit pulp to yogurt increased lactic acid content, total phenols, and antioxidant activity (zainoldin et al., 2009). it has also been reported that fruit peel powder benefits dieters and can be used instead of fat, especially in ice cream (attar et al., 2022). pitaya fruits are reported to contain high levels of vitamin c, phenolic compounds, and organic acids. fruits are also rich in minerals such as potassium, calcium and magnesium (le bellec et al., 2006; tenore et al., 2012). in addition, it has been reported that it facilitates digestion, is good for asthma when consumed regularly, balances cholesterol levels, and reduces blood pressure (nomura et al., 2005). pitaya has attracted great attention from both growers and consumers in recent years due to its early yield, resistance to drought, and richness in nutrients and antioxidants. in fact, greenhouse cultivation has gained popularity in the aegean region in recent years. in this study, pomological and biochemical characteristics of „siam red” (h. polyrhizus) and „vietnam white” (h. undatus) varieties grown in greenhouse conditions in muğla were investigated. materials and methods this study was carried out on „siam red” (h. polyrhizus) and „vietnam white” (h. undatus) varieties grown in greenhouse conditions in muğla between 2023 and 2024. the study was carried out in 2 different varieties, with 3 replicates, 4 fruits in each replicate and 3 trees in each replicate according to the coincidence plots experimental design. measurement and analyses fruits were harvested from different plant parts for each variety at the commercial harvest time, and fruit weight was determined in g using a precision balance (sensitive to 0.01 g). fruit length and fruit width were measured in mm by a digital caliper with a precision of 0.01 mm. fruit peel color and fruit flesh color were measured from both sides of the fruit with the minolta cr-400 color meter and evaluated in terms of l*, a*, and b* (öztürk et al., 2012). the harvested fruits were cleaned and peeled, and the juices were filtered. afterward, the fruit juices were analyzed by a digital refractometer to determine the tss with the help of a digital ph meter. to determine the ta content of the juice, 10 ml of filtered fruit juice was added to 100 ml of distilled water and titrated with 0.1n naoh to 8.1. ta was calculated as % citric acid (öztürk et al., 2012). total phenolic content was determined using folin-ciocalteu’s chemical according to the method of singleton & rossi (1965). readings in the spectrophotometer were made at 750 nm wavelength. total flavonoid content was determined according to the method described by zhishen et al. (1999). readings in the spectrophotometer were made at 510 nm wavelength. total antioxidant capacity (dpph) (1,1-diphenyl-2picrylhydrazyl) was determined according to the method of kumaran & karunakaran (2006). readings in the spectrophotometer were made against methanol at a wavelength of 517 nm (çakır et al., 2021). statistical analysis the data obtained from the research were subjected to analysis of variance using the minitab package programme. tukey’s multiple comparison test was used to determine the significant differences between the varieties. results and discussion the pomological characteristics of the varieties are presented in tab. 1. statistically significant differences were found between the varieties at p≤0.05 level regarding fruit peel weight, fruit peel thickness, fruit flesh firmness and length. fruit weight ranged between 203.55 („siam red”) -232.20 g („vietnam white”), fruit peel weight 54.53 („siam red”) 77.29 g („vietnam white”), fruit flesh weight 149.02 („siam red”) 154.91 g („vietnam white”), fruit peel thickness 2.69 („siam red”) 3.32 mm („vietnam white”), fruit width 65.63 („siam red”) 68.88 mm („vietnam white”), fruit length 84.50 („siam red”) 102.92 mm („vietnam white”) and fruit flesh firmness 0.57 („siam red”) 1.41 kg (cm2)-1 („vietnam white”). the highest values in terms of all pomological characteristics were obtained in „vietnam white” variety. parmar & karetha (2020) determined the highest fruit weight as 265.86 g, the highest fruit biologica nyssana ● 16 (1) june 2025: 105-110 balci et al. ● determination of pomological characteristics and biochemical contents of some pitaya varieties grown in muğla region 107 biologica nyssana ● 16 (1) june 2025: 105-110 balci et al. ● determination of pomological characteristics and biochemical contents of some pitaya varieties grown in muğla region length as 9.51 cm, and the highest fruit width as 7.19 cm among the species. it has been reported that, in general, the varieties with white fruit flesh have higher values in terms of fruit weight, fruit peel weight, flesh weight, peel thickness, flesh firmness, width, and length than the varieties with red fruit flesh (parmar & karetha, 2020; altınkaya, 2024). (2024) determined the highest fruit peel l* value as (kbxvj) 51.59, the highest fruit flesh l* value as (vjxkb) 67.45, the highest fruit peel l* value as (rrxtk) 48.08, the highest fruit flesh l* value as (rrxrrr) 44.26 in the fruits obtained as a result of crossbreeding of varieties with white parents and red parents. in general, they found that the l* value of the varieties with white fruit flesh was higher than the varieties with red fruit flesh (demirkaplan, 2020; abirami et al., 2021; uğuz & gezici, 2021). in this study, the values of tss, ph, ta, total phenolic content, total flavonoid content, and total antioxidant capacity of the varieties are presented in tab. 3. there was no statistically significant difference between the varieties in terms of total antioxidant capacity (p≤0.05). the highest tss was determined in the „vietnam white” variety (12.57%), and the highest ph, tea, total phenolic content and total flavonoid content were determined in the „siam red” variety (4.60, 0.45%, 432.77 mg gae 100g-1, 22.99 mg ce 100g-1, respectively). the highest total antioxidant capacity was found in the „vietnam white” variety (79.40%). liaotrakoon (2013) compared the biochemical properties of red and white pitaya fruits and found that the amount of tss varied between 9.84% and 10.18%. esquivel et al. (2007) reported that the ph value of red pitaya fruit varied between 4.26 and 4.98 in their study with different pitaya varieties. table 1. pomological characteristics of pitaya varieties variety „vietnam white” „siam red” fruit weight (g) 232.20±20.30 203.55±0.97 fruit peel weight (g) 77.29±10.73a 54.53±2.49b fruit flesh weight (g) 154.91±7.54 149.02±68 fruit peel thickness (mm) 3.32±0.12a 2.69±0.25b fruit width (mm) 68.88±0.71 65.63±1.97 fruit length (mm) 102.92±4.60a 84.50±0.59b fruit flesh firmness (kg (cm2)-1) 1.41±0.29a 0.57±0.06b table 2. peel and fruit flesh colour values of pitaya varieties variety „vietnam white” „siam red” shell colour feature l* 41.54±0.80a 39.70±0.50b a* 35.37±0.27b 37.74±0.90a b* 11.03±0.49b 13.35±0.28a meat colour characteristic l* 74.62±1.17a 33.67±2.02b a* 10.22±0.46b 45.73±3.52a b* 4.33±0.18a -2.14±2.06b fruit color characteristics of the varieties are presented in tab. 2. statistically significant differences at p≤0.05 level were found between the varieties in terms of peel and flesh color l*, a*, and b* values. in the study, the highest l* value was obtained in the „vietnam white” variety (41.54) and the highest a* and b* values were obtained in the „siam red” variety (37.74, 13.35, respectively). colour and brightness properties in foods are one of the important sensory properties in terms of consumer preference (bilek, 2010). mahayothee et al. (2019) determined l* value as 29.56, a* value as 36.68, b* value as 5.19 of red pitaya in their study. uğuz & gezici (2021) determined the highest l* value (51.69), the highest a* value (33.06), and the highest b* value (6.33) in h. undatus. abirami et al. (2021) determined the highest l* value (51) in h. undatus, the lowest l* value in h. costariscensis, the highest a* value in h. costariscensis, the lowest a* value (-0.6) in h. undatus, the highest b* value (2.5) in h. undatus, the lowest b* value (-2.5) in h. costariscensis. altınkaya table 3. biochemical characteristics of pitaya varieties variety „vietnam white” „siam red” tss (%) 12.57±0.29a 10.94±0.80b ph 4.46±0.02b 4.60±0.00a ta (%) 0.36±0.01b 0.45±0.02a total phenolic content (mg gae 100g-1) 406.42±1.87b 432.77±10.80a total flavanoid content (mg catechin 100g-1) 20.10±0.70b 22.99±0.27a total antioxidant capacity (%) 79.40±3.65 73.74±11.57 108 biologica nyssana ● 16 (1) june 2025: 105-110 balci et al. ● determination of pomological characteristics and biochemical contents of some pitaya varieties grown in muğla region barcelon et al. (2015) analyzed the biochemical content of red pitaya fruit and found the ph value as 4.90. yılmaz et al. (2022) determined the total phenolic content of red pitaya fruit as 715 mg gae 100g-1. wu et al. (2006) determined the total phenolic content of fresh red pitaya pulp as 42.4 mg gae 100g-1. adnan et al. (2011) determined the phenolic content in red pitaya fruit seed oil as 1356 mg gae 100g-1. contreras-calderón et al. (2011) determined that the total phenolic content of seeds ranged between 61.5 and 1712 mg gae 100g-1 in a study of peel and seeds of 24 different exotic fruits. luu et al. (2021) reported that pitaya is an exotic tropical plant beneficial for human health due to its high nutritional value, bioactive molecules, and natural antioxidants. attar et al. (2022) reported that pitaya fruits with red-purple flesh had a higher total phenolic content than those with white flesh, and the total phenolic content of red-purple and whitefleshed pitaya species was 16.66 and 17.11 mg gae 100g-1 fw, respectively. abirami et al. (2021) found that the total phenolic content of different pitaya varieties varied between 32.5 and 42.5 mg gae 100g-1 fw. khatun et al. (2022) determined the antioxidant capacity of pitaya fruit to be 73.38%. attar et al. (2022) reported that pitaya fruits with red fruit flesh had higher antioxidant capacity than those with white fruit flesh. esquivel et al. (2007) reported that pitaya with purple flesh has a high antioxidant capacity and is rich in betalains. it was also reported that the antioxidant capacity of pitaya fruits with red and yellow flesh was higher than some other tropical fruits such as mango, lychee, longan, and papaya (chaves et al., 2004; wojdylo et al., 2007; beltránorozco et al., 2009). our research results showed differences from previous studies, which may be caused by a variety of factors such as location, maturity, cultural practices, and temperature (esquivel et al., 2007; tran et al., 2015; berk et al., 2022; çelik et al., 2024; pepe et al., 2024). conclusion in this study, some fruit quality characteristics and biochemical contents of „siam red’ and „vietnam white” pitaya varieties grown in greenhouse conditions in the aegean region were determined. as a result, it was found that the „vietnam white” variety produce the largest and brightest-colored fruits, with the highest fruit peel weight, fruit flesh weight, flesh firmness, width, length, tss, and total antioxidant capacity. the „siam red” variety was found to have the highest ph, ta, total phenolic content, and total flavonoid content. when evaluating the income obtained in recent years in our country, it is clear that there is a great interest in the cultivation of tropical fruits. moreover, due to its high antioxidant capacity, it is in high consumer demand. it is possible to say that it will be beneficial to carry out similar studies to increase the cultivation of tropical fruits in türkiye. this study will contribute to the literature on the determination of pomological and biochemical contents of pitaya varieties, which are newly known in türkiye, and will shed light on future studies. references abirami, k., swain, s., baskaran, v., venkatesan, k., sakthivel, k., & bommayasamy, n. 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(lamiaceae) svetlana tošić*, sanja nikolić, marija jovanović, bojan zlatković, dragana stojičić university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia * e-mail: tosicsvetlana59@yahoo.com abstract: tošić, s., nikolić, s., jovanović, m., zlatković, b., stojičić, d.: micropropagation of micromeria juliana (l.) benth. ex rchb. (lamiaceae). biologica nyssana, 6 (1), september 2015: 17-23. micromeria juliana belongs to family lamiaceae, whose representatives are characterized by a significant level of essential oils and antioxidant components. several species of genus micromeria contain biologically active substances and are therefore used in folk medicine, food industry and cosmetic industry. methods of their tissue culture may provide rapid mass multiplication of plants for various purposes, including research on production, accumulation and metabolism of important secondary metabolites. the goal of this paper was to determine the protocol for regeneration of m. juliana plants through use of nodal explants on nutritive substrate with various growth regulators. the greatest number of axillary buds was formed in explants grown on ms nutritive medium with 3 μm benzyladenine (ba) and 0.57 μm indole-3-acetic acid (iaa). the explants grown at the medium without any growth regulators and the medium with auxin have shown spontaneous root formation. key words: axillary bud induction, shoot culture, biomass production apstrakt: tošić, s., nikolić, s., jovanović, m., zlatković, b., stojičić, d.: micropropagacija vrste micromeria juliana (l.) benth. ex rchb. (lamiaceae). biologica nyssana, 6 (1), septembar 2015: 17-23. micromeria juliana (l.) benth pripada familiji lamiaceae koja se odlikuje značajnim sadržajem etarskih ulja i antioksidativnih komponenti. vrste roda micromeria sadrže biološke aktivne supstance zbog čega se koriste u narodnoj medicini, kao i u prehrambenoj i kozmetičkoj industriji. metode kulture tkiva mogu da obezbede brzu i masovnu multiplikaciju biljaka za različite svrhe kao što je proučavanje produkcije, akumulacije i metabolizma značajnih sekundarnih metabolita. cilj ovog rada je da ustanovi protokol za regeneraciju biljaka m. juliana korišćenjem nodalnih eksplantata, na hranljivoj podlozi sa različitim regulatorima rastenja. najveći broj aksilarnih pupoljaka formiran je na eksplantatima gajenim na ms hranljivoj podlozi sa 3 μm benziladenina (ba) i 0,57 μm indol-3-sirćetne kiseline (iaa). eksplantati gajeni na podlozi bez regulatora rastenja i na podlozi sa auksinom su spontano ožiljavali. key words: indukcija aksilarnih pupoljaka, kultura izdanaka, produkcija biomase 6 (1) • september 2015: 17-23 biologica nyssana 6 (1)  september 2015: 17-23 tošić, s. et al.  micropropagation of micromeria juliana (l.) nenth. ex rchb.... 18 introduction in relation to the morphological characteristics and phylogenetic relationships, species of genus micromeria are grouped into three sections: cymularia, micromeria and pseudomelissa. the representatives of this genus in the flora of serbia (d i k l i ć , 1974) belong to micromeria (m. croatica, m. juliana, m. cristata and m. parviflora) and pseudomelissa (m. thymifolia, m. albanica and m. pulegium). m. juliana is comparatively rare species in serbia, recorded only in the vicinity of kačanik in kosovo and metohija province (d i k l i ć , 1974). according to c h a t e r & g u i n e a (1973) and š i l i ć (1979) the native range of the species includes countries of mediterranean region (montenegro, croatia, herzegovina, macedonia, greece, bulgaria, albania, france, portugal, italy, turkey, northwestern africa, as well as the western part of asia minor and crete). it inhabits dry, sunny sandbased habitats, stony ground and crevices in limestone rocks (fig. 1). this is a perennial, dwarf shrubby plant, with many erect, mostly simple stems, up to 40 cm in height. the leaves are narrowly ovate, upper linear-lanceolate, entire, with revolute margins and opposite. the root and the basal part of stems are somewhat fruticose. flowers are comparatively small, purple in sessile or shortly pedunculate verticillasters, organized into long, common inflorescences (d o r o s z e n k o , 1986). the representatives of genus micromeria are characterized by low morphological diversity and their growth and development are influenced by numerous ecological factors, leading to high diversity of their secondary metabolites. due to presence of secondary metabolites, species of genus micromeria show antioxidant (g ü l l ü c e et al., 2004; s t o j a n o v i ć & p a l i ć , 2008; v l a d i m i r k n e ž e v i ć et al., 2011;), antibacterial (t a b a n c a et al., 2001; d u r u et al., 2004; s a r a c & u g u r , 2007; s t o j a n o v i ć & p a l i ć , 2008), antifungal (m a r i n k o v i ć et al., 2003; a b o u j a w d a h et al., 2004; ö z c a n , 1999), antiphytoviral (b e z i ć et al., 2013), insecticidal (a s l a n et al., 2005), bioherbicidal (d u d a i et al., 1999) and allelopathic (d u d a i et al., 2009) biological activity. due to their pharmacological properties, plant species of genus micromeria are used in folk medicine for treating gastrointestinal and respiratory tract, skin infections and wounds, as well as for soothing pain and preventing insomnia (š a r i ć k u n d a l i ć et al., 2011). they are also used in cooking in order to improve taste and aroma of food, as additives and spices (t a b a n c a et al., 2001; g ü l l ü c e et al., 2004). fig. 1. micromeria juliana, canyon of river morača (montenegro) the high individual variability characterizing species of genus micromeria is a limiting factor for the intensity of their use in pharmaceutical purposes. the solution to this problem may include use of plant tissue cultures in vitro (s a h a et al., 2012). the methods of culture in vitro are important in reproduction of endemic and rare plants, contributing to preservation of biodiversity (f a y 1992; r e e d et al., 2011). growing in culture in vitro decreases the exploitation pressure on natural populations and in a short period of time may yield several thousand plants from a small amount of plant samples. the only species of genus micromeria previously grown in culture in vitro used to be m. pulegium (t o š i ć et al., 2015). material and methods plant material and source of explants the surface sterilization included treating seeds with 30% solution of na-hypochlorite with 4% of active chlorine for 25 minutes. after three rinsing in sterile distilled water, the seeds were treated with 5% solution of nystatin for 24 hours in order to eliminate possible fungal infections. next step included rinsing seeds in sterile distilled water three times, and then they were placed individually in test tubes on nutritive medium in aseptic conditions in order to germinate. after the seeds have germinated, the resulting plants were used for preparing explants – nodal segments. in order to introduce m. juliana into in vitro culture, seeds were biologica nyssana 6 (1)  september 2015: 17-23 tošić, s. et al.  micropropagation of micromeria juliana (l.) nenth. ex rchb.... 19 collected from plants in their natural habitat in canyon of river morača (montenegro). a voucher specimens, under the acquisition number 10850, are deposited at the herbarium collection of department of biology and ecology, faculty of science and mathematics in niš (hmn). culture medium and culture condition the nutritive medium used in culture in vitro was m u r a s h i g e & s k o o g (1962) – ms medium, including appropriate macroand micro mineral salts and organic additions. all medium have included 100 mg l-1 myo-inositol, 30 g l-1 sucrose and 7 g l-1 agar. the ph value of each medium was checked immediately before autoclaving and if necessary adjusted to ph 5.8 by addition of 0.1 n naoh. the sterilized nodal segments of m. juliana of approximately same size (5 mm) were placed on nutritive medium: without any growth regulators, with cytokinin ba (0.1, 0.3, 1, 3, 10 µm), with auxin iaa (0.57µm), and with combination of ba and iaa. each medium was used for 30 explants, 10 in each of three jars. the plants were maintained at the temperature 23˚c ± 3˚c and photoperiod of 16 hours of light and 8 hours of dark. the density of light flux was 47 µmol s-1m-2. measured parameters the explants were grown for four weeks until they formed axillary buds. the explants reacting positively to treatment were recorded and following parameters were measured: number of axillary buds per explant, length of each bud, fresh and dry mass of each explant. buds shorter than 1 mm were disregarded. after the fresh mass was measured, explants were dried in separate paper containers and dry mass was then measured by using the same analytic scales. shoot forming capacity (sfc) index was calculated according to martinez-pulido et al. (1992) as follows: sfc index = % explant with shoots x mean number of shoot per explants / 100. statistical methods data processing was performed by using the statistically-graphic package statgraphics, procedure anova and test lcd at the significance level p<0.05. results and discussion culture of m. juliana was formed by germinating seeds on ms nutritive medium. use of ms nutritive medium produces favorable results for in vitro germinating of numerous species (m o l i a & k w a p a t a , 2000), including many species of lamiaceae. nodal segments were isolated from in vitro germinated seedlings and placed in inductive medium. the initiation of the culture was achieved according to the same procedure, using nodal segments of seedlings, in clinopodium odorum (d i a z et al., 2012). use of nodal segments for regeneration of plants with the goal of mass production is considered a simple method for many species of lamiaceae, and at the same time it is the most reliable method for achieving uniform plant material (g e o r g e & s h e r r i n g t o n , 1984; d o d e et al., 2003). all explants on the ms nutritive medium without regulators (100%) have developed axillary buds (tab. 1). the smallest ratio of explants with axillary buds (63.3%) was recorded at the medium with 0.57 μm iaa. at the explants grown on medium without growth regulators there were on average 4.97 buds per explant. most buds were formed on explants grown on medium with 3 μm ba and 0.57 μm iaa. their number is statistically significantly greater than the number of buds formed on explants on all other medium. explants from this medium are characterized with the greatest multiplication index of 8.73. after three months, one explant may produce 3665 buds which potentially may produce new plants. low-concentration auxin combined with cytokinin often assists development of buds on explants (t e j a v a t h i & i n d i r a , 2011). the greatest average length of axillary buds was developed on explants grown at medium with 0.57 μm iaa (tab. 1). all concentrations of ba used in this study with or without auxin have inhibited elongation of axillary buds in m. juliana. the inhibitory effect of high cytokinin concentrations on elongation of buds was recorded in mentha piperita (g h a n t i et al., 2004) and melissa officinalis (t a v e r s et al., 1996). cytokinins may show inhibitory activity on elongation of axillary buds in spite of promoting their proliferation (v a n s t a d e n et al., 2008). the explants with the greatest average mass were grown on ms medium with 3 μm ba and 0.57 μm iaa (tab. 2). on that medium they formed the greatest number of buds, contributing to the greatest biomass. the explants from these medium were healthy, with good branching, green, quite uniform in stature, with shorter internodes and almost bushlike habitus (fig. 2). combination of cytokinin and auxin was used by numerous authors in order to induce bud formation. for example in agastache rugosa the greatest number and best quality of buds was achieved on ms medium with this particular combination of hormones (z i e l i n s k a et al., 2011). the comparison of explants of m. juliana biologica nyssana 6 (1)  september 2015: 17-23 tošić, s. et al.  micropropagation of micromeria juliana (l.) nenth. ex rchb.... 20 table 1. effect of plant growth regulators on micromeria juliana shoot proliferation, number of shoots per explants and shoot length after 28 days of culture. pgr (μm) explants producing shoots (%) shoots per explant shoot length (mm) sfc index ba iaa 100 4,97 ± 0,50abc 3,95 ± 0,31bc 5,86 0.1 96.7 5,86 ± 0,40bcd 3,25 ± 0,27ab 6,97 0.3 100 6,00 ± 0,36de 3,18 ± 0,24ab 5,72 1 96.7 7,21 ± 0,43e 3,19 ± 0,20ab 3,82 3 83.3 5,72 ± 0,38cd 2,72 ± 0,19a 4,14 10 70.0 3,95 ± 0,49ab 2,46 ± 0,23a 3,28 0.57 63.3 4,68 ± 0,33abcd 6,47 ± 0,79d 2,96 0.1 0.57 100 5,73 ± 0,42d 4,12 ± 0,37 c 5,73 0.3 0.57 93.3 3,68 ± 0,49a 4,18 ± 0,42 c 3,43 1 0.57 96.7 4,52 ± 0,42abc 3,94 ± 0,36 bc 4,37 3 0.57 93.3 9,39 ± 0,65f 3,84 ± 0,20 bc 8,73 10 0.57 100 4,37 ± 0,38ab 2,56 ± 0,18 a 4,37 the values are means ± standard error. means within the column of each factor followed by the same letter are not significantly different according to lsd multiple range test (p  0.05) table 2. effect of plant growth regulators on micromeria juliana biomass production (using nodal shoot segments as explants) after 28 days of culture. pgr (μm) biomass fresh weight per explant (mg) biomass dry weight per explant (mg) ba iaa 0.017 ± 0.0019a 0.0028 ± 0.00030a 0.1 0.022 ± 0.0016a 0.0031 ± 0.00019a 0.3 0.021 ± 0.0013a 0.0031 ± 0.00021a 1 0.022 ± 0.0016a 0.0034 ± 0.00024ab 3 0.015 ± 0.0010a 0.0022 ± 0.00015a 10 0.014 ± 0.0014a 0.0020 ± 0.00021a 0.57 0.046 ± 0.0079cd 0.0056 ± 0.00066bcd 0.1 0.57 0.027 ± 0.0019ab 0.0043 ± 0.00027abc 0.3 0.57 0.044 ± 0.0053c 0.0057 ± 0.00066cd 1 0.57 0.051 ± 0.0013cd 0.0060 ± 0.00057cd 3 0.57 0.061 ± 0.0042d 0.0070 ± 0.00041de 10 0.57 0.038 ± 0.0034bc 0.0085 ± 0.00074e the values are means ± standard error. means within the column of each factor followed by the same letter are not significantly different according to lsd multiple range test (p  0.05). fig. 2. m. juliana on ms medium with 0,57 µm iaa biologica nyssana 6 (1)  september 2015: 17-23 tošić, s. et al.  micropropagation of micromeria juliana (l.) nenth. ex rchb.... 21 grown on medium with ba and explants grown on medium with combination of cytokinin and auxin has shown an obvious prominent increase in biomass yield in medium supplemented with auxin. low-concentration auxin and ba have synergic effect contributing to formation of biomass in explants of m. juliana. ba and iaa also had positive effect on bud induction in species ocimum killimandscharicum l. (s h a r m a et al., 2013), ocimum citriodorum vis. (j a n a r t h a n a m & s u m a t h i , 2012), and in species mentha viridis l. (r a h m a n et al., 2013). medium with combination of auxin and cytokinin was effective for regeneration of ocimum basilicum (n i r m a l & s e h g a l , 1999) and salvia brachyodon (m i s i c et al., 2006). of the explants grown on medium without growth regulators, a small number has spontaneously developed adventive roots, but almost all explants grown on medium with auxin and without cytokinin have developed adventive roots, indicating strong role of auxin in development of adventive roots (fig. 2). conclusion micromeria juliana (l.) benth. ex rchb. was successfully introduced into in vitro culture. results of this study have shown that the maximum number of axillary buds (9.39) was formed in explants grown on nutritive medium with 3 μm ba and 0.57 μm iaa. the nutritive medium with 0.57 μm iaa was the most efficient in elongation of axillary buds, and the average bud length was 6.47 mm. combination of auxin and high concentrations of ba (3 and 10 μm, respectively) induced formation of the greatest fresh and dry mass, respectively. root formation in nodal explants took place in medium without growth regulators and in medium that contained both auxin and low concentration of ba. with the increase in concentration of ba there was a decrease of the number of explants developing adventive roots. this study has shown that the described micropropagation protocol enabled production of a substantial number of plantlets, starting with a small quantity of plant material. in addition, micropropagation of m. juliana may be considered as an alternative method for production of secondary metabolites. acknowledgements. the ministry of education, science and technological development of the republic of serbia (grant 173015, 173030) supported this research. references abou-jawdah, y., wardan, r., sobh, h., salameh, a. 2004: antifungal activities of extracts from selected lebanese wild plants against plant pathogenic fungi. phytopathologia mediterranea, 43: 377–386. aslan, i., calmasur, o., sahon, f., caglar, o. 2005: insecticidal effects of essential plant oils against ephestia kuehniella (zell.), lasioderma serricorne (f.) and sitophilus granarius (l.). journal of plant diseases and protection, 112 (3): 257–267. bezić, n., dunkić, v., vuko, e. 2013: antiphytoviral activity of essential oils of some lamiaceae species and there most important compounds on cmv and tmv. in: méndez-vilas, a. 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(eds.): plant propagation by tissue culture, (3rd edition). 1: 205–226. springer, bachground, dordrecht, netherlands. vladimir-knežević, s., blažeković, b., bival štefan, m., alegro, a., köszegy, t., petrik, j. 2011: antioxidant activities and polyphenolic contents of three selected micromeria species from croatia. molecules, 16: 1454-1470. zielinska, s., piatczak, e., kalemba, d., matkowski, a. 2011: influence of plant growth regulators on volatiles produced by in vitro grown shoots of agastache rugosa (fischer & c.a.meyer) o. kuntze. plant cell, tissue and organ culture, 107: 161–167. biologica nyssana 6 (1)  september 2015: 17-23 tošić, s. et al.  micropropagation of micromeria juliana (l.) nenth. ex rchb. 24 zlatić et al. 2025, biologica nyssana 16(1) 189 16 (1) june 2025: 189-197 doi: 10.46793/biolnyss.16.1.24z polyphenols and antioxidant capacity of selected compositae species original article nenad zlatić university of kragujevac, faculty of science, department of biology and ecology, kragujevac, serbia nenad.zlatic@pmf.kg.ac.rs (corresponding author) danilo stojanović university of belgrade, faculty of pharmacy, department of botany, belgrade, serbia milan stanković university of kragujevac, faculty of science, department of biology and ecology, kragujevac, serbia received: march 14, 2025 revised: may 16, 2025 accepted: may 16, 2025 abstract: species belonging to the compositae family are known for their rich content of phenolic compounds and flavonoids with notable antioxidant properties. given the increasing demand for natural antioxidants in various industries, this study aims to evaluate the total phenolic and flavonoid content alongside antioxidant activities of ethanolic extracts of six compositae species; to identify interspecific differences and highlight their potential for use in pharmaceutical, cosmetic, and food sectors. the investigated species include centaurea salonitana, c. atropurpurea, achillea atrata, a. clavennae, filago arvensis, and xeranthemum annuum. phenolic content was quantified utilizing the folin–ciocalteu method (gae), while flavonoid content was measured as rutin equivalent (rue). the antioxidant activity was determined via the dpph assay (ic50 values). significant interspecific variation was observed; notably, achillea atrata exhibited the highest phenolic content, whereas centaurea salonitana was distinguished by its elevated flavonoid content. key words: antioxidant activity, asteraceae, flavonoids, phenols, secondary metabolites apstrakt: polifenoli i antioksidativni kapacitet odabranih vrsta iz familije compositae vrste iz familije compositae poznate su po visokom sadržaju fenolnih jedinjenja i flavonoida, koji poseduju izražena antioksidativna svojstva. imajući u vidu sve veću potražnju za prirodnim antioksidansima u raznim industrijama, cilj ovog istraživanja je bio da se proceni ukupan sadržaj fenola i flavonoida, kao i antioksidativna aktivnost etanolnih ekstrakata šest vrsta familije compositae; da se identifikuju interspecijske razlike i istakne njihov potencijal za upotrebu u famaceutskom, kozmetičkom i prehrambenom sektoru. u okviru istraživanja ispitivani su etanolni ekstrakti sledećih vrsta: centaurea salonitana, c. atropurpurea, achillea atrata, a. clavennae, filago arvensis i xeranthemum annuum. ukupan sadržaj fenola određen je primenom folin–ciocalteu metode i izražen kao ekvivalent galne kiseline (gae), dok je sadržaj flavonoida izražen kao ekvivalent rutinu (rue). antioksidativna aktivnost određena je dpph metodom putem ic50 vrednosti. rezultati su pokazali značajne interspecijske varijacije, pri čemu je achillea atrata imala najviši sadržaj fenolnih jedinjenja, dok se centaurea salonitana istakla visokim sadržajem flavonoida. ključne reči: antioksidativna aktivnost, asteraceae, flavonoidi, fenoli, sekundarni metaboliti introduction the secondary metabolites produced by plants exhibit a diverse array of biological and pharmacological effects. among these, phenolic compounds are particularly notable due to their pronounced antioxidant properties, which play a vital role in the prevention of various oxidative stressrelated diseases, including cancer (dai & mumper, 2010). a highly diverse subset of polyphenolic compounds are the flavonoids, critical in processes ranging from plant–pathogen interaction, pollination and light screening. their accumulation is often enhanced in response to biotic and abiotic stressors associated with the increase in reactive oxygen species, emphasizing their protective, antioxidant function. in addition, flavonoids have demonstrated superior antioxidant capabilities in vitro compared to well-established antioxidants such as vitamin c and vitamin e, highlighting their potential for applications in products related to health preservation and enhancement (hernández et al. 2009). © 2025 zlatić et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 190 research has confirmed the presence of antioxidant polyphenols across various higher plants groups (lewis, 2017), and a correlation between phenolic content and antioxidant properties across different taxa (quettier et al. 2000; piluzza & bullitta, 2011). among the representatives of the highly diverse and numerous compositae family, the presence of phenolic compounds and antioxidant activity has been studied in several achillea species. these studies have shown a correlation between the high content of flavonoids detected in these species and their strong free radical scavenging activity (tuberoso et al. 2009; salomon et al. 2021). according to salomon et al. (2021), the ethyl acetate fraction of the extract from the aerial parts of a. atrata l. exhibited higher phenolic content and consequently greater antioxidant activity compared to a. millefolium l. recently, buzhala et al. (2022) have examined the total phenolic and flavonoid contents, along with antioxidant activities of several centaurea species from kosovo. specifically, c. atropurpurea waldst. & kit. (synonym for c. calocephala willd.) was found to possess the highest concentrations of these metabolites and exhibited the strongest antioxidant properties. according to carev et al. (2023), many species of the large and taxonomically complex genus centaurea are used in traditional medicine in croatia for the treatment of various conditions, including c. salonitana vis. which is known for its anti-inflammatory and wound-healing properties. however, this phytochemical study did not investigate the phenolic and flavonoid content of this species. prior research of immortelle (xeranthemum annuum l.) from southern serbia by stanković et al. (2011) identified it as a valuable natural source of biologically active compounds due to significant concentrations of polyphenols and flavonoids in extracts from the dried aerial parts, along with notable antioxidant potential. yet similar investigations into filago arvensis l. remain absent. all these findings incited this study which endeavors to conduct a comparative analysis of the total phenolic and flavonoid content and antioxidant activity of ethanol extracts from six species representing four genera within the compositae family that grow wild in serbia: centaurea salonitana vis., c. atropurpurea waldst. & kit., achillea atrata l., a. clavennae l., filago arvensis l. and xeranthemum annuum l. materials and methods chemicals ethanol was purchased from "zorka pharma" (šabac, serbia). standards of phenolic acids (gallic acid) and flavonoids (rutin hydrate), as well as chlorogenic acid and 2,2-diphenyl-1-picrylhydrazyl (dpph), were obtained from sigma chemicals co. (st. louis, mo, usa). the folin-ciocalteu reagent, 3-tert-butyl-4-hydroxyanisole (bha), and aluminum chloride (alcl₃) were sourced from fluka chemie ag (buchs, switzerland). all other solvents and reagents used in the study were of analytical grade. plant material the plant material was collected from central, southern, and southeastern serbia, as well as montenegro. aerial parts from ten representative individuals per species were selected as plant material. to preserve chemical integrity, these specimens were air-dried in darkness at room temperature and subsequently stored in sealed containers for further analysis. species identification was verified. voucher specimens were deposited in the herbarium of the department of biology and ecology, faculty of science, university of kragujevac, under the following numbers: centaurea salonitana (116/014-1), c. atropurpurea (116/014-2), achillea atrata (116/014-3), a. clavennae (116/014-4), filago arvensis (116/014-5), and xeranthemum annuum (116/014-6). preparation of plant extracts the air-dried plant material (10 g) was finely ground to obtain powdered material and subsequently extracted using 100 ml of 96% ethanol (plant material to solvent ratio 1:10, w/v) by maceration at room temperature for 24 hours. the mixture was filtered through whatman no. 1 filter paper, and the residue was re-extracted with an additional 100 ml of the same solvent under the same conditions. this step was repeated after 48 hours to ensure maximal extraction of bioactive compounds. the combined extracts were concentrated to dryness under reduced pressure at 40 °c using a rotary evaporator. dried extracts were stored in sterile sample tubes at 4 °c until further analysis. for spectrophotometric analysis, the dry extracts were dissolved in methanol to a final concentration of 1 mg/ml. determination of total phenolics in the plant extracts the total phenolic content was determined spectrophotometrically following the method outlined by singleton et al. (1999). a methanolic extract solution at a content of 1 mg/ml was used for the analysis. the reaction mixture was prepared by combining 0.5 ml of the methanolic plant extract solution, 2 ml of 7.5% sodium bicarbonate (nahco₃), and 2.5 ml of 10% folin-ciocalteu biologica nyssana ● 16 (1) june 2025: 189-197 zlatić et al. ● polyphenols and antioxidant capacity in six compositae species 191 biologica nyssana ● 16 (1) june 2025: 189-197 zlatić et al. ● polyphenols and antioxidant capacity in six compositae species reagent dissolved in water. a blank sample was prepared using 0.5 ml of ethanol, 2 ml of 7.5% nahco₃, and 2.5 ml of 10% folin-ciocalteu reagent. the prepared test tubes were incubated at 45 °c for 45 minutes. absorbance was measured at 765 nm using a spectrophotometer. all samples were analyzed in triplicate, and the mean absorbance value was recorded. a standard calibration curve was generated using gallic acid solutions, and the total phenolic content of the extracts was quantified based on the measured absorbance. results were expressed as milligrams of gallic acid equivalents per gram of extract (mg ga/g). determination of total flavonoids in the plant extracts the total flavonoid content in the analyzed herbal extracts was determined spectrophotometrically according to the method of quettier et al. (2000). the test sample was prepared by mixing 1 ml of a methanolic extract solution (1 mg/ml) with 1 ml of a 2% aluminum chloride (alcl₃) solution in methanol. the reaction mixture was incubated at room temperature (20°c) for 60 minutes. absorbance was measured at 415 nm using a spectrophotometer. each sample was analyzed in triplicate, and the mean absorbance value was recorded. a standard calibration curve was generated using rutin solutions, and the total flavonoid content of the extracts was quantified based on the measured absorbance. results were expressed as milligrams of rutin equivalents per gram of extract (mg ru/g). evaluation of antioxidant activity the ability of the plant extract to scavenge 1,1-diphenyl-2-picrylhydrazyl (dpph) free radicals was assessed using a standard method (takao et al., 1994). dpph was dissolved in methanol to a content of 80 μg/ml. the plant extract solution was prepared in methanol at a content of 1 μg/ml, followed by serial dilutions to obtain concentrations of 500, 250, 125, 62.5, 31.25, 15.62, 7.81, 3.90, 1.99, and 0.97 μg/ml. each diluted solution (1 ml) was mixed with 1 ml of dpph solution. after incubating the mixtures in the dark at room temperature (20 °c) for 30 minutes, absorbance was measured at 517 nm. the inhibition percentage was calculated using equation (1), and ic50 values were determined from the % inhibition versus content plot, using nonlinear regression analysis. data are presented as mean ± standard deviation (n=3). equation (1): dpph scavenging activity (%)=[(a0 a1) / a0] * 100 a0 is the absorbance of the control (blank) solution, and a1 is the absorbance of the sample solution, which contains both the antioxidant and dpph. statistical analysis experimental measurements were performed in triplicate and presented as the arithmetic mean of the three analyses ± standard deviation. to assess differences in total phenolic content (tpc), total flavonoid content (tfc), and antioxidant activity (aoa) among the six compositae species, oneway analysis of variance (anova) was conducted. when significant differences were detected (p<0.05), tukey’s hsd post-hoc test was applied to determine pairwise differences between species. statistical analysis was performed using the spss software package (spss for windows, version xii, 2008, chicago, il). results and discussion total phenolic content the total phenolic content of ethanolic extracts from the six compositae species was quantified using a spectrophotometric method with the folin–ciocalteu reagent. this method is based on the measurement of the redox potential of phenolic compounds in solution. upon dissolution, the phenols produce a proton and a phenoxide anion, which subsequently table 1. total amount of phenolic compounds (tpc), total amount of flavonoids (tfc) and total antioxidant activity (aoa) of compositae species tpc tfc aoa centaurea salonitana vis. 119.05 ± 9.71ab 81.28 ± 4.98b 107.00 ± 9.56b centaurea atropurpurea waldst. & kit. 97.11 ± 6.52bc 75.36 ± 4.55b 101.74 ± 8.52b achillea atrata l. 122.32 ± 10.06a 87.95 ± 5.62a 92.66 ± 7.27a achillea clavennae l. 100.91 ± 8.21bc 55.14 ± 3.21c 131.07 ± 10.34c filago arvensis l. 83.56 ± 4.23d 48.23 ± 2.11c 139.21 ± 10.89c xeranthemum annuum l. 94.60 ± 5.87c 62.44 ± 2.89c 143.12 ± 12.10c values sharing the same letter within a column are not significantly different according to tukey’s test (p≥0.05) 192 biologica nyssana ● 16 (1) june 2025: 189-197 zlatić et al. ● polyphenols and antioxidant capacity in six compositae species to a reduced capacity for scavenging free radicals compared to the other species. phenolic compounds are secondary metabolites found in plants, serving various biological roles, such as protecting against oxidative damage (dai & mumper, 2010). these findings emphasize the significant role of phenolic content in the antioxidant potential of different species within the compositae family (fig. 1). the results of this study indicate that species from both achillea and centaurea genera exhibit a notably high phenolic content consistent with previous research findings. studies have consistently reported the richness of these genera in phenolic compounds (tuberoso et al., 2009). specifically, centaurea saloniatana leaves contain 34.4 mg cae/g dw, while its inflorescences contain 21.9 mg cae/g dw (buzhala et al., 2022). a significantly higher content of phenolic compounds was observed in centaurea atropurpurea leaves (218.5 mg cae/g dw), whereas its inflorescences contained 21.4 mg cae/g dw (buzhala et al., 2022). based on the present study, both centaurea salonitana and c. atropurpurea exhibited a notably high phenolic content (119.05 mg gae/g and 97.11 mg gae/g respectively), which indicates a significant bioactive potential of these species and place them among the more phenolic-rich members of the genus centaurea. when compared to our other tested species, such as c. orientalis (19.38 mg gae/g), c. nigra (12.00 mg gae/g), and c. phrygia (8.08 mg gae/g), the differences are indicating strong interspecific variation in secondary metabolite content. previous studies have reported a wide range of phenolic concentrations within fig. 1. comparative bar chart of phenolic content and antioxidant capacity reduce the folin–ciocalteu reagent, resulting in the generation of a blue color. the intensity of this color is proportional to the content of phenolic compounds in the sample. the total phenolic content varied between the tested extracts, with notable differences between the species (tab. 1). the total phenolic content in the ethanolic extracts of the six compositae species ranged from 83.56 to 122.32 mg of gallic acid equivalents per gram (mg of ga/g). among the tested species, achillea atrata exhibited the highest content of total phenolics (122.32 mg of ga/g), while filago arvensis displayed the lowest content (83.56 mg of ga/g). the observed variability in the phenolic content across the studied species may be a function of genetic differences, environmental conditions, or developmental stages of the plants. based on the observed total phenolic contents, the six species can be classified into three distinct groups. the first group comprised two species with the highest phenolic contents: achillea atrata (122.32 mg of ga/g) and centaurea salonita (119.05 mg of ga/g), both showing notably high levels of phenolic compounds, indicative of substantial antioxidant potential. the second group includes achillea clavennae (100.91 mg of ga/g) and centaurea atropurpurea (97.11 mg of ga/g) which demonstrated intermediate phenolic levels, which may suggest a moderate potential for antioxidant activity. finally, the third group includes xeranthemum annum (94.60 mg of ga/g) and filago arvensis (83.56 mg of ga/g) representing species with the lowest phenolic contents, potentially linked 193 biologica nyssana ● 16 (1) june 2025: 189-197 zlatić et al. ● polyphenols and antioxidant capacity in six compositae species interspecific differences (tab. 1), and ranged from 48.23 to 87.95 mg of rutin equivalents per gram (mg of ru/g). the highest content of flavonoids was detected in the extract of achillea atrata (87.95 mg of ru/g), while filago arvensis showed the lowest content (48.23 mg of ru/g). these results indicate considerable variation in flavonoid levels across the studied species, which may reflect differences in their biochemical pathways, environmental conditions, or adaptive response strategies. based on the observed flavonoid contents, the six species can be classified into three distinct groups. the first group consists of species with the highest contents of flavonoids, notably achillea atrata (87.95 mg of ru/g). this species stands out due to its relatively high flavonoid content, which may suggest enhanced antioxidant properties. the second group includes species with moderate flavonoid contents: centaurea salonitana (81.28 mg of ru/g) and centaurea atropurpurea (75.36 mg of ru/g). these species exhibit intermediate flavonoid levels, which may contribute to moderate antioxidant activities, providing them with some degree of protection against oxidative stress. the third group includes species with the lowest flavonoid contents among the studied species: xeranthemum annum (62.44 mg of ru/g), achillea clavennae (55.14 mg of ru/g), and filago arvensis (48.23 mg of ru/g). these species show lower levels of flavonoids, which could be associated with a reduced capacity for scavenging free radicals compared to the other species. in addition to the achillea species, the present study confirms significant flavonoid content in centaurea salonitana and c. atropurpurea. the total flavonoid content in c. saloniatana leaves was 7.3 mg ce/g dw, while in its inflorescence, it reached 4.7 mg ce/g dw (buzhala et al., 2022). similarly, c. atropurpurea exhibited a high flavonoid content in leaves (56.1 mg ce/g dw) and a moderate level in inflorescences (5.7 mg ce/g dw) (buzhala et al., 2022). the present study showed that both centaurea salonitana and c. atropurpurea exhibited distinctly higher flavonoid contents, reaching 81.28 mg re/g and 75.36 mg re/g, respectively. when compared with the results obtained for other centaurea species, these values stand out as exceptionally high. for example, c. orientalis, the richest in flavonoids among our previously tested species, contained 40.57 mg qe/g, while c. nigra and c. phrygia showed significantly lower values of 28.57 mg qe/g and 24.92 mg qe/g, respectively (albayrak et al., 2017). even lower flavonoid levels were reported by albayrak et al. (2017) for c. aksoyi (5.83 mg qe/g) and c. amaena (7.69 mg qe/g). additionally, in the study conducted by aktumsek et al. (2013), where flavonoid content was expressed in rutin the genus centaurea, depending on species, plant parts, and extraction methods. kenny et al. (2014) observed total polyphenol content ranging from 61.7 to 173.2 mg gae/g in aqueous and ethanolic extracts of the aerial parts of c. nigra. similarly, albayrak et al. (2017) reported values between 82.27 and 175.40 mg gae/g for species such as c. kurdica, c. rigida, c. amanicola, c. cheirolopha, and c. ptosimopappoides. in contrast, lower concentrations were noted in c. aksoyi and c. amaena (3.28–12.92 mg gae/g) (aktumsek et al., 2011). these findings align with the current study, where both species were placed among those with moderate to high phenolic levels. however, differences in absolute values may be attributed to variations in extraction methods, plant part analyzed, or environmental conditions at the time of collection. likewise, the high phenolic content observed in achillea atrata in our study (122.32 mg ga/g) confirms earlier findings indicating the presence of bioactive flavonoids such as luteolin, apigenin, centaureidin, and nevadensin in its polar fractions (salomon et al., 2021). achillea clavennae, with a moderate phenolic level (100.91 mg ga/g), also showed the presence of pharmacologically active sesquiterpenes and monoterpenes, supporting its potential medicinal value. these findings support the significant pharmacological potential of achillea species. in contrast, present research identified lower phenolic contents in xeranthemum annuum and filago arvensis. this finding aligns only partially with the limited existing data: stanković et al. (2011) reported higher total phenolic contents in x. annuum (ranging from 101.33 to 159.48 mg ga/g), which may suggest that extraction methods and analyzed plant parts significantly influence the final phenolic yield. the differences in phenolic content between these species may be influenced by ecological factors as well, such as habitat, environmental conditions, and seasonal variations, all of which can affect the biosynthesis of secondary metabolites, including phenolic compounds. gaining insight into the distribution of phenolic compounds in these species is crucial for assessing their pharmacological potential and for their application in herbal medicine and other industries. flavonoids content the content of flavonoids in the ethanolic extracts of the observed six different compositae species was determined using a spectrophotometric method with alcl3, a process resulting in the formation of metal-flavonoid complexes, which can be measured spectrophotometrically. the total flavonoid content varied among the tested extracts, showing significant 194 biologica nyssana ● 16 (1) june 2025: 189-197 zlatić et al. ● polyphenols and antioxidant capacity in six compositae species equivalents (re), aqueous and methanolic extracts of centaurea antalyense yielded 30.76 mg re/g and 46.76 mg re/g, respectively. other studied species, such as c. polpodiifolia var. pseudobehen and c. pyrrhoblephara, showed values ranging from 40.38 to 45.94 mg re/g and 29.43 to 55.75 mg re/g, respectively. compared to these previously published data, the flavonoid concentrations found in c. salonitana and c. atropurpurea are notably higher, suggesting that these species possess a particularly rich profile of flavonoid compounds. moreover, the presence of specific flavonoids such as luteolin, apigenin, and centaureidin was confirmed in achillea atrata and achillea clavennae (salomon et al., 2021) while xeranthemum annuum was also identified as a source of flavonoids. the flavonoid contents in plant extracts analyzed in this study ranged from 22.25 to 62.42 mg ru/g, further demonstrating the variability in flavonoid content among species (stanković et al., 2011). these findings emphasize the significant role of flavonoids in the antioxidant potential of different species within the compositae family. however, further studies are needed to explore the specific flavonoid composition of different achillea species and their individual contributions to antioxidant activity, as well as to assess their bioavailability and long-term efficacy in practical applications. antioxidant potential the antioxidant activity (ic50) of the ethanolic extracts from six different compositae species was assessed and varied from 143.12 to 92.66 μg/ml (tab. 1). the ic50 values indicate the content of the extract required to inhibit 50% of free radical activity. the highest antioxidant activity was observed in achillea atrata (92.66 μg/ml), while the lowest antioxidant activity was recorded in xeranthemum annum (143.12 μg/ml). these results demonstrate considerable variation in the antioxidant potential of the studied species, likely influenced by the chemical composition of each species, particularly the presence and content of bioactive compounds such as phenolics and flavonoids. based on the observed antioxidant activity values, the six species can be categorized into three distinct groups. the first group includes the species with the highest antioxidant activity, achillea atrata (92.66 μg/ml), which stands out for its strong free radical scavenging ability. the second group consists of species such as centaurea atropurpurea (101.74 μg/ ml) and centaurea salonita (107.00 μg/ml) with moderate antioxidant activity, which may indicate a balanced ability to neutralize oxidative stress. the third group comprises species with the lowest antioxidant activity among the studied species, namely achillea clavennae (131.07 μg/ml), filago arvensis (139.21 μg/ml), and xeranthemum annum (143.12 μg/ml). these species show relatively lower antioxidant potential, which could be linked to their lower content of antioxidant compounds. the antioxidant potential of achillea atrata and a. clavennae, as assessed using the dpph assay in our study, revealed ic₅₀ values of 92.66 μg/ ml and 131.07 μg/ml, respectively. these values suggest a relatively moderate capacity for radical scavenging. in comparision to previously published data, significant differences in antioxidant strength become evident. gharibi et al. (2013) reported much lower ic₅₀ values for several achillea species: a. aucherii showed the weakest antioxidant activity with an ic₅₀ of 844 μg/ml, followed by a. kellalensis (518 μg/ml), and a. pachycephala, which demonstrated the strongest activity among them with an ic₅₀ of 248 μg/ml. these differences may be attributed to variations in extraction methods, the specific plant parts analyzed, ecological factors, or differences in phytochemical composition, particularly in the quantity and type of phenolic compounds and flavonoids. nevertheless, our results confirm that achillea species exhibit a broad range of antioxidant activities, and the moderate potency observed in a. atrata and a. clavennae may still hold pharmacological relevance, especially when considered in combination with other bioactive properties. the antioxidant activity observed in the extracts of achillea and other examined species, reflects the high flavonoid content present in their chemical composition. flavonoids, a subclass of phenolic compounds, are well-known for their ability to scavenge reactive oxygen species (ros) due to their phenolic hydroxyl groups. these groups play a key role in the neutralization of free radicals, which contributes to the observed strong antioxidant activity (nikolić et al., 2024). in particular, the flavonoids in achillea species have been shown to exhibit significant antiradical activity, largely due to their conjugated ring structure and the presence of hydroxyl groups, which facilitate hydrogen donation (tuberoso et al., 2009). this mechanism of action is further enhanced by the free hydroxyl groups, particularly the 3-oh group, which increases the reactivity of flavonoids with oxidants, stabilizing scavenger molecules and preventing oxidative damage (hernández et al., 2009). in achillea species, flavonoids such as apigenin, luteolin, and quercetin, which are frequently found in high contents, are particularly effective in scavenging free radicals. this strong antioxidant activity is a result of their ability to donate hydrogen atoms to reactive oxygen species, thereby stabilizing them and 195 biologica nyssana ● 16 (1) june 2025: 189-197 zlatić et al. ● polyphenols and antioxidant capacity in six compositae species preventing cellular damage. this dual functionality of flavonoids, both neutralizing free radicals and stabilizing scavenger molecules, contributes to their overall antioxidant efficacy (tuberoso et al., 2009; hernández et al., 2009). the antioxidant capacity assessed by the dpph assay in our study revealed that centaurea salonitana and c. atropurpurea exhibited strong radical scavenging activities, with ic₅₀ values of 107.00 μg/ml and 101.74 μg/ml, respectively. although a direct comparison with percentage-based radical scavenging data is limited due to differences in methodology and expression units, the results still highlight a notable contrast in antioxidant strength across species. for example, salachna et al. (2021) reported that among the analyzed species, c. orientalis demonstrated the highest dpph radical scavenging activity with 39.34%, while c. nigra and c. phrygia showed substantially lower activities of 10.47% and 9.43%, respectively. similarly, the abts radical scavenging potential was most pronounced in c. orientalis (91.69%), followed by c. nigra (44.62%) and c. phrygia (39.69%). compared to these values, the antioxidant potential of c. salonitana and c. atropurpurea appears significantly stronger when interpreted through the lens of their low ic₅₀ values (indicating higher activity), which suggests a more efficient free radical neutralization at lower extract concentrations. these findings not only underscore the remarkable antioxidant capacity of species but also correlate with their high flavonoid and polyphenol contents, which are likely key contributors to their bioactivity. such results highlight their potential pharmacological value and encourage further exploration of their chemical composition and possible therapeutic applications. comparatively, species such as xeranthemum annum and filago arvensis, which have lower phenolic and flavonoid contents, likely exhibit reduced antioxidant potential. the lower flavonoid content in these species suggests that they may not be as effective in neutralizing ros or stabilizing reactive molecules, thus indicating a potentially lower capacity for antioxidant defense (nikolić et al., 2024). stanković et al. (2011) reported that species with lower flavonoid content tend to show reduced antioxidant capacity, supporting our findings that the lower content of antioxidant compounds in these species correlates with a weaker ability to neutralize ros. the antioxidant activity of the examined species is closely related to their flavonoid content, which has been well-documented in the literature as a key factor in determining the antioxidant potential of plants (tuberoso et al., 2009). our study supports and expands upon this body of work, demonstrating that flavonoid-rich species, such as achillea atrata, exhibit strong antioxidant activity, while species with lower flavonoid content, like filago arvensis and xeranthemum annum, have reduced antioxidant capacity. the differences in antioxidant activity between observed species can be attributed to variations in the qualitative and quantitative composition of phenolic compounds and flavonoids, as these are crucial for antioxidant effectiveness. moreover, environmental factors such as habitat conditions, altitude, soil composition, and exposure to abiotic stressors also play a role in influencing the production of these secondary metabolites. this results in unique antioxidant profiles for each species. however, further studies are necessary to fully understand the phytochemical profiles of these species and their specific roles in mitigating oxidative stress. relationship between total phenolic content, total flavonoid content, and antioxidant activity pearson correlation analysis was conducted to explore the relationship between total phenolic content (tpc), total flavonoid content (tfc), and antioxidant activity (aoa) in the test plants. a strong positive correlation was observed between tpc and tfc (0.869) across all the tested plants, suggesting a significant association between the phenolic and flavonoid content in these species. in contrast, strong negative correlation was found between both tpc and antioxidant activity (aoa) (-0.766) and tfc and antioxidant activity (-0.904) in all the plants. these negative correlations indicate that higher contents of total phenolics and flavonoids do not inherently lead to increased antioxidant activity in this dataset. this outcome is expected, as the ic50 values, which represent the content required to inhibit 50% of the antioxidant activity, are inversely proportional to antioxidant potential, implying that a lower ic50 indicates higher antioxidant efficiency. our findings align with previous research, demonstrating a robust correlation between total phenolic content (tpc) and antioxidant activity (foo et al., 2017). tpc is recognized as a powerful antioxidant, commonly found in a wide range of medicinal plants, as well as fruits and vegetables (lewis, 2017). the antioxidant potential of phenolic compounds is primarily attributed to their oxidoreduction properties, which allow them to function as hydrogen donors, reducing agents, possible metal chelators, and quenchers of singlet oxygen (soobrattee et al., 2005). the presence of hydroxyl groups in phenolic compounds enables them to interact with reactive oxygen species (ros) and reactive nitrogen species, effectively terminating the radical chain reaction and preventing the formation of additional free radicals (valentão et 196 biologica nyssana ● 16 (1) june 2025: 189-197 zlatić et al. ● polyphenols and antioxidant capacity in six compositae species al., 2002). upon examining the relationship between the content of total phenolic compounds and antioxidant activity, it becomes evident that extracts with higher contents of phenolics tend to exhibit stronger antioxidant activity. this suggests that secondary metabolites, particularly those from the phenolic compound group, play a crucial role in driving the antioxidant properties observed in these plants. various studies have substantiated the link between the antioxidant activity of plant extracts and their total phenolic content, confirming the significant contribution of phenolic compounds to the overall antioxidant capacity of plants (piluzza & bullitta, 2011). conclusion the comprehensive evaluation of total phenolic and flavonoid content, and antioxidant potential of six species from the compositae family indicates significant variability in the contents of these bioactive compounds among the species, attributable to specific plant adaptations to ecological conditions, suggesting the possibility of chemical diversification depending on the environment. the highest contents of bioactive compounds were recorded in achillea atrata, which stands out as the species with the strongest antioxidant potential, high levels of phenols and flavonoids, making it a potential source for applications in industries dealing with natural products, such as the pharmaceutical, cosmetic, and food industries. on the other hand, species such as centaurea salonitana and c. atropurpurea exhibit moderate contents of bioactive compounds, which may also indicate their significance in the prevention of oxidative stress and other potential applications. these results highlight the need for further research that could provide deeper insights into the role of these plants in ecosystems, as well as their potential for the development of new biologically active products. therefore, the specific adaptations and biochemical profiles of these plants offer invaluable insights for future studies aimed at enhancing the utilization of plant extract in therapeutic and industrial contexts. this research underscores the potential of the analyzed compositae species as natural sources of antioxidants, with promising implications for product development in the pharmaceutical, cosmetic, and food sectors. acknowledgements. the research was supported by the ministry of science, technological development, and innovation of the republic of serbia [evidence numbers: 451-03-137/2025-03/200122; 451-03-137/2025-03/ 200161 and 451-03-137/2025-03/200161]. 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(2002). antioxidative properties of cardoon (cynara cardunculus l.) infusion against superoxide radical, hydroxyl radical, and hypochlorous acid. journal of agricultural and food chemistry, 50(17), 4989– 4993. https://doi.org/10.1021/jf020225o microsoft word bn-oa-0201-03 karalija, paric 29 original article ! the effect of ba and iba on the secondary metabolite production by shoot culture of thymus vulgaris l. erna karalija, adisa parić* faculty of science, university of sarajevo, zmaja od bosne 33-35, 71 000 sarajevo, bosnia and herzegovina * e-mail: adisacausevic@hotmail.com abstract: karalija, e., parić, a.: the effect of ba and iba on the secondary metabolite production by shoot culture of thymus vulgaris l.. biologica nyssana, 2 (1), september 2011: 29-35. in vitro shoots of the common thyme (thymus vulgaris l.) were established, and the effects of different concentrations of the cytokinin ba and the auxin iba on secondary metabolite production were investigated. the highest number of shoots was obtained through cultivation on the ms medium containing 2 mg/l ba and 0.1 mg/l iba and 4 mg/l ba and 0.1 mg/l iba (14.3 and 13.3 respectively). quantitative changes in chlorophyll a, chlorophyll b and carotenoid content were recorded in response to the effect of varying concentrations of growth regulators in the medium. furthermore, after addition of plant growth regulators (0.5 mg/l ba + 0.1 mg/l iba) to the medium, elicitation of phenolic compounds was recorded in plantlets. the concentration range of ba from 2 to 4 mg/l improved the production of flavonoids (0.61 and 0.64 mg/g fw). in contrast, plantlets cultivated on the same treatment, showed a decrease in monomeric anthocyanins . key words: common thyme, in vitro, micropropagation, secondary metabolite production introduction ! the common thyme or thymus vulgaris l., a member of the lamiaceae family, is an aromatic herb growing up to 30 cm in height and flowering from july to september (g r u e n w a l d et al., 2004). it is native to southern europe, where it is often cultivated as a culinary herb. thyme is used as spice and medicinal herb almost everywhere in the world (m o r a l e s , 2002). many pharmacological in vitro experiments carried out during the last decade revealed well defined pharmacological properties of both, the thyme essential oil and the plant extracts. the non-medicinal use of thyme is worthy of attention, because thyme is used in the food and aroma industries and it is widely used as a culinary ingredient and it serves as a preservative for foods especially due to its antioxidant effect (z a r z u e l o & c r e s p o , 2002). the active substances in the thyme essential oil are thymol, carvacrol, p-cymene, β pinene, γterpinene, β-caryophyllene, 1-borneol, 1, 8-cineole, etc (j u v e n et al., 1994). recent studies showed that thyme essential oils have strong antibacterial, antifungal, and antioxidative properties (c r u z et al., 1989; p i c c a g l i a et al., 1993; c o n s e n t i n o et al., 1999; k a r a m a n et al., 2001; v a r d a r et al., 2003, p r a b u s e e n i v a s a n et al., 2006; b u d k a & k h a n , 2010). essential oils of different species of genus thymus inhibit a broad spectrum of bacteria, with gram-positive bacteria generally being more susceptible than gram-negative bacteria (b l a k e w a y , 1986; f a r a g et al., 1986; d e a n s & r i t c h i e , 1987; k n o b l o c h et al., 1988). recently, the antibacterial property of thyme oil against some important food-borne pathogens, like salmonella enteritidis, escherichia coli, staphylococcus aureus, listeria monocytogenes, and 2 (1) • september 2011: 29-35 biologica nyssana 2 (1) september 2011: 29-35 karalija e., parić a. the effect of ba and iba on the secondary metabolite... 30 campylobacter jejuni, has been tested (s m i t h p a l m e r et al., 1998). several in vitro and in vivo screenings have shown that volatile oils may be used against fungal diseases, especially those from plants of the genus thymus (b l a k e w a y , 1986; f a r a g et al., 1986; d e a n s & r i t c h i e , 1987). a few studies demonstrate the antiviral effects of thyme extracts (h e r r m a n n & k u c e r a , 1967). micropropagation techniques play an important role in conservation and genetic improvement of pharmaceutical and medicinal plants. today, there is a tendency to develop in vitro systems of production as an alternative to field production of plants used as a source of active compounds. such system is not limited by sources of nutrients or climate changes and can thus be better controlled. for effective induction of secondary metabolite production, plant growth regulators (pgrs) are required (z h o n g et al., 1996; a l -s a n e ' et al., 2005; shilpashree & ravishankar, 2009), since they have significant effects on the metabolism of secondary metabolites. the quality, as well as the quantity, of plant growth regulators, plays a major role in the production capability of a given in vitro culture. pgrs affect cell multiplication and division, which increases production of secondary metabolites (k u r z & c o n s t a b e l , 1979; s t a b a , 1980). the effects of different pgrs on secondary metabolite production have previously been studied and confirmed in vrious plants (f u r u y a et al., 1971; i k u t a et al., 1975; i k e d a et al., 1976; i k u t a & i t o k a w a , 1982; n a i r et al., 1992; w e a t h e r s et al., 2005; k h a n et al., 2008). the aim of this study was to develop an efficient protocol for mass multiplication of t. vulgaris, through manipulation of different concentrations of plant growth regulators, and to examine the relationship between pgrs and the production and accumulation of antioxidants, polyphenols and chlorophyll. materials and methods plant material and treatments commercially purchased seeds of t. vulgaris l. (sjemenarna ljubljana d.d., slovenija) were surface-sterilised with 20% commercial bleach for 15 min followed by 3x5 min rinsing in sterile distilled water and were then germinated on a ms basal medium (m u r a s h i g e & s k o o g , 1962). shoot multiplication was investigated on the ms basal medium (bh; control) or on media supplemented with 0.5, 2, 4 mg/l ba and 0.1 mg/l iba (bi1; bi2 and bi4). the ph of all media was adjusted to 5.8 with 1 m koh or 1 m hcl before autoclaving at 121°c for 20 minutes. all cultures were incubated at 24±1°c under cool white fluorescent lights (3000 lux) and with the 16 h light photoperiod. the aerial parts of t. vulgaris were collected for analysis. photosynthetic pigments – extraction was undertaken in accordance to porra et al. (1989). quantification was done by spectrophotometric determination of absorbance on 663 nm (chlorophyll a), 646 nm (chlorophyll b) and 440 nm (carotenoids). the concentrations of photosynthetic pigments were expressed as mg of pigments per g of fresh weight (mg/g fw). secondary metabolite production – samples used for the analysis of concentrations of phenols, flavonoids and anthocyanins were frozen in liquid nitrogen and stored at -20°c. extraction of phenols and flavonoids was performed in 100% methanol. total phenol content was determined according to w o l f e et al. (2003), with folin-ciocalteu reagent through spectrophotometric readings of absorbance at 765 nm. total phenol content was expressed as mg of catechin per g of fw. total flavonoid content was analysed according to o r d o n et al. (2006) by absorbance readings at 415 nm. total flavonoid content was expressed as mg of ruthin per g of fw. monomeric anthocyanins were determined according to m a n c i n e l l i (1984). extraction was performed in acidified methanol (1% hcl in methanol), and the concentration was expressed as mg of cyanidin 3-glucoside per l of extract. observations and statistical analysis each treatment had 5 replicates containing 10 explants and all experiments were repeated three times. all analyses were done after 30 days of cultivation with no subcultivation. the data was analysed using spss 15.0 by employing parametric (pearson correlation, one tailed) and non-parametric (kruskal-walis analysis) tests. results and discussion shoot multiplication – the induction of shoot formation started after a week of culture cultivation with initiation of shoot meristemswhich was followed by growth of shoot buds. no multiplication was recorded on the ms medium without plant growth regulators (control) (fig. 1a), while a high rate of root induction was observed on the same medium. shoot apices grown in the presence of 0.5 mg/l ba and 0.1 mg/l iba, produced on average 8.2 axillary buds at the base of the first pair of leaves (tab. 1, fig. 1b). treatments with higher concentrations of ba (2 mg/l) increased the number of shoots, but shoots were much shorter and biologica nyssana 2 (1) september 2011: 29-35 karalija e., parić a. the effect of ba and iba on the secondary metabolite... 31 table 1. the induction of adventive shoots formation in thymus vulgaris l. treatment number of shoots per explant rate of regeneration (%) induction of root formation (%) bh 2.9 (±0,71) 100 81.25 bi1 8.2 (±0,56)* 100 0 bi2 14.3 (±0,82)* 100 0 bi4 13.3 (±0,84)* 100 0 the results represent mean values of three replications (±standard deviation) after 30 days on the ms basal medium with no plant growth regulators and with addition of different concentrations of ba and iba. *statistically important differences at 0.01 level with respect to control. bh 0 m g/l ba + 0 mg/l iba; bi1 0.5 mg/l ba + 0.1 mg/l iba; bi2 2 mg/l ba + 0.1 mg/l iba; bi4 4 mg/l ba + 0.1 mg/l iba; table 2. the effect of ba and iba on photosynthetic pigments in shoot cultures of t. vulgaris treatment chlorophyll a (mg/g) chlorophyll b (mg/g) total chlorophylls (mg/g) carotenoids (mg/g) bh 0.29 (±0.02) 0.16 (±0.01) 0.45 (±0.02) 0.51 (±0.03) bi1 0.27*(±0.01) 0.40* (±0.02) 1.25* (±0.03) 0.20* (±0.03) bi2 0.24*(±0.24) 0.39* (±0.01) 1.14* (±0.01) 0.22* (±0.01) bi4 0.10*(±0.10) 0.17* (±0.02) 0.48* (±0.01) 0.65* (±0.02) the results represent mean values of three replications (±standard deviation) after 30 days on the ms basal medium with no plant growth regulators and with addition of different concentrations of ba and iba. *statistically important differences at 0.01 level with respect to control. bh 0 m g/l ba + 0 mg/l iba; bi1 0.5 mg/l ba + 0.1 mg/l iba; bi2 2 mg/l ba + 0.1 mg/l iba; bi4 4 mg/l ba + 0.1 mg/l iba; table 3. the effect of ba and iba on secondary metabolite production in shoot cultures of t. vulgaris treatment phenols (mg/g) flavonoidis (mg/g) monomeric anthocynins (mg/l) bh 2.09 (±0.14) 0.50 (±0.01) 6.62 (±0.32) bi1 3.05* (±0.11) 0.55 (±0.02) 3.38*(±0.05) bi2 1.70* (±0.01) 0.61*(±0.01) 2.95*(±0.01) bi4 2.32 (±0.04) 0.64*(±0.01) 2.13* (±0.04) the results represent mean values of three replications (±standard deviation) after 30 days on the ms basal medium with no plant growth regulators and with addition of different concentrations of ba and iba. *statistically important differences at 0.01 level with respect to control. bh 0 m g/l ba + 0 mg/l iba; bi1 0.5 mg/l ba + 0.1 mg/l iba; bi2 2 mg/l ba + 0.1 mg/l iba; bi4 4 mg/l ba + 0.1 mg/l iba; had developed necrosis (fig. 1c). on the medium with 4 mg/l ba and 0.1 mg/l iba, an average of 13.3 shoots was obtained directly from the apical or the axillary buds (tab. 1, fig. 1d). similar results were observed in previous studies on the common thyme (b e h e s h t i & m o r t e z a , 2005). a high capability of explant multiplication was observed on the medium with the highest cytokinin/auxin ratio (2 and 4 / 0.1 mg/l) and leads us to consider these combinations as efficient promoters of t. vulgaris multiplication. photosynthetic pigments – the content of the total chlorophylls ranged from 0.45 mg/g fw for control to 1.25 mg/g fw for bi1 treatment (tab. 2). the increase in the total chlorophyll content was a result of a significant increase in chlorophyll b content, while chlorophyll a content decreased significantly when compared to the control. according to our results an increase in the cytokinin concentration in the medium (bi1 and bi2) significantly increases the amount of chlorophyll b produced, except for bi4 medium. on the other hand it shows reverse correlation with the chlorophyll a content. it was not possible to establish a direct link between concentrations of pgrs used and the content of photosynthetic pigments in all cases (s t e t l e r & l a e t s c h , 1965; e d e l m a n & h a n s o n , 1971). cytokinins as well as auxins can improve concentration of photosynthetic pigments (v e r m a & s e n , 2008; p a r s a e i m e h r et al., 2010; v a m i l et al., 2010). furthermore, it was shown that the biologica nyssana 2 (1) september 2011: 29-35 karalija e., parić a. the effect of ba and iba on the secondary metabolite... 32 biosynthesis pathway of chlorophyll is highly insensitive to light and cytokinins (y a r o n s k a y a et al., 2006). changes in the content of chlorophylls, caused by pgrs, could, most likely, be related to growth rate and primary and secondary metabolic activities (l i c h t e n t h a l e r , 1987). secondary metabolite production – the total phenol content was significantly higher on bi1 treatment, while total flavonoid content was higher on all treatments when compared with the control (tab. 3). it was previously reported that the maturation of cells always corresponds to an increase in secondary metabolites (s h e t t y & l a b b e , 1998; b i o n d i et al., 2002; p a r s a e i m e h r et al., 2010). on the other hand, the quality and the quantity of applied plant growth regulators have a significant effect on the metabolism of secondary metabolites (k h a n et al., 2008). in plant cell cultures the production of secondary metabolites supports cell multiplication and division, hence plant growth regulators are bound to have a major role in determining the production of potentiality of a given culture. during our research presence of higher concentrations of ba promoted shoot proliferation and flavonoid production. accumulation of flavonoids is usually correlated to morphogenesis during plant development (p a s q u a et al., 2003). however, no such positive influence of higher ba concentrations was observed for phenol content. only the ba concentration that tested the lowest (0.5 mg/l) induced phenol accumulation. the concentration of monomeric anthocyanins was significantly lower on all treatments in comparison to the control (tab. 3). anthocyanins exhibited positive correlation for chlorophyll a and carotenoids, but a negative correlation for flavonoids, with the significance at the 0.01 level. the reverse relation between the in vitro shoot proliferation and the production of anthocyanins production, which was found in this study, is a general complication found in in vitro secondary metabolites production. several studies dealing with this problem observed that 2.4-d or naa increase anthocyanin production and decrease growth rate. on the other hand some of the studies observed that ba or kin enhance production of anthocyanins, while inhibiting cell growth (m e y e r & v a n s t a d e n , 1995). the difficulties expressed through the reverse relationship between the production of anthocyanins and cell growth can cause problems in all secondary products induction media and additional research is needed to establish specific requirements of each plant species. a b c d figure 1. induction of adventive shoots formation on ms medium with ba and iba a – bh 0 m g/l ba + 0 mg/l iba; b – bi1 0.5 mg/l ba + 0.1 mg/l iba; c – bi2 2 mg/l ba + 0.1 mg/l iba; d – bi4 4 mg/l ba + 0.1 mg/l iba; conclusion our research shows that the shoot multiplication of common thyme is depended upon the treatment that is used. the highest number of adventive shoots was obtained on the ms media supplemented with 2 or 4 mg/l ba and 0.1 mg/l iba, which employed the highest concentration of cytokinins. an increase in the concentration of cytokinins above 2 mg/l was followed by a decrease in photosynthetic pigments content, and total monomeric anthocyanins content. cytokinins (0.5 mg/l) in combination with low auxin levels (0.1 mg/l) can enhance production of phenolic compounds, and higher concentrations of cytokinins can induce an increase in total biologica nyssana 2 (1) september 2011: 29-35 karalija e., parić a. the effect of ba and iba on the secondary metabolite... 33 flavonoid content. this study successfully established the optimal conditions for the in vitro growth of common thyme, with a potential for secondary metabolite production. further investigations are necessary in order to increase the production of secondary metabolites by in vitro cultures. references al-sane', k.o., shibli, r.a., freihat, n.m., hammouri, m.k. 2005: cell suspension culture and secondary metabolites production in african violet (saintpaulia ionantha wendl.). jordan journal of agricultural sciences, 1 (1): 84-92. beheshti, h.b., morteza, k. 2005: effects of media and growth regulators on garden thyme (thymus vulgaris l.) micropropagation. iranian journal of horticultural science and technology, 6 (2): 61-68. biondi, s., scaramagli, s., oksman-caldentey, k., poli, f. 2002: secondary metabolism in root and callus cultures of hyoscyamus muticus l.: the relationship between morphological organization and response to methyl jasmonate. plant science, 163 (2): 563−569. blakeway, j. 1986: the antimicrobial properties of essential oils. soap, perfumery & cosmetics, 59 (4): 201-203. budka, d., khan, n.a. 2010: the effect of ocimum basilicum, thymus vulgaris, origanum vulgare essential oils on bacillus cereus in rice-based foods. european journal of biological sciences, 2 (1): 17-20. consentino, s., tuberoso, c.i.g., pisano, b., satta, m., arzedi, e., palmas, f. 1999: in vitro antimicrobial activity and chemical composition of sardinian thymus essential oils. letters in applied microbiology, 29 (2): 130–135. cruz, t., cabo, m.p., cabo, m.m., jiménez, j., cabo j., ruiz, c. 1989: in vitro antibacterial effect of the essential oil of thymus longiflorus boiss. microbios, 60 (242): 59–61. deans, g.g., ritchie, g. 1987: antibacterial properties of plant essential oils. international journal of food microbiology, 5 (2): 165-180. edelman, j., hanson, a.d. 1971: sucrose suppression of chlorophyll synthesis in carrot callus culture. planta, 98: 150-156. farag, r.s., salem, h., badei, a.z.m.a., hassanein, d.e. 1986: biochemical studies on the essential oils of some medicinal plants. fette, seifen, anstrichmittel, 88 (2): 69-72. furuya, i., kojima, h., syono, k. 1971: regulation of nicotine biosynthesis by auxins in tobacco callus tissue. phytochemistry, 10: 1529-1532. gruenwald, j., bendler, t., jaenicke, c. 2004: pdr for herbal medicines. medical economics company. montvale (nj). herrmann, e.c., kucera, l.s. 1967: antiviral substances in plants of the mint family (labiatae) 111. peppermint (mentha piperita) and other mint plants. proceedings of the society for experimental biology and medicine, 124: 874-878. ikeda, t., matsumoto, t., noguchi, m. 1976: formation of ubiquinone by tobacco plant cells in suspension culture. phytochemistry, 15: 954959. ikuta, a., itokawa, h. 1982: berberine and other protoberberine alkaloids callus tissue of thalictrum minus. phytochemistry, 21: 14191421. ikuta, a., syono, k., furuya, t. 1975: alkaloids in plants regenerated from coptis callus cultures. phytochemistry, 14: 1209-1210. juven, b.j., kanner, j., schveol, f., weisslowicz, h. 1994: factors that interact with the antibacterial action of thyme essential oils and its active constituents. the journal of applied bacteriology, 76 (6): 626-631. karaman, s., digrak, m., ravid, v., iclim, a. 2001: antibacterial and antifungal activity of the essential oils of thymus revolutus celak from turkey. journal of ethnopharmacology, 76 (2): 183–186. khan, t., krupadanam, d., anwar, s.y. 2008: the role of phytohormone on the production of berberine in the calli cultures of an endangered medicinal plant, turmeric (coscinium fenestratum l.). african journal of biotechnology, 7 (18): 244-3246. knobloch, k., pauli, a., iberl, b., weis, n. weigand, h. 1988: mode of action of essential oil components on whole cell of bacteria and fungi in plate tests. in: schreier, p. 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(ed.), thyme 24: 278, taylor & francis, united kingdom. zhong, j.j., bai, y., wang, s.j. 1996: effects of plant growth regulators on cell growth and ginsenoside saponin production by suspension cultures of panax quinquefolium. journal of biotechnology, 45 (3): 227-234. stojanović et al. 2025, biologica nyssana 16(1) 75 16 (1) june 2025: 75-91 doi: 10.46793/biolnyss.16.1.7s floristic diversity of the rocky habitats of the stara planina mountains in serbia original article jovana stojanović university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18 000 niš, serbia jovanagreen6@gmail.com (corresponding author) bojan zlatković university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18 000 niš, serbia dragana jenačković gocić university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18 000 niš, serbia branko jotić university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18 000 niš, serbia marina jušković university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18 000 niš, serbia received: october 28, 2024 revised: february 15, 2025 accepted: february 19, 2025 abstract: the flora of rock outcrops in the stara planina mountains (serbia) was analyzed to assess its diversity, as well as its biological and chorological spectra. a total of 125 vascular plant species, representing 75 genera and 35 families, were identified. the dominant families were poaceae, caryophyllaceae, and asteraceae, while asplenium and sedum were the most species-rich genera. hemicryptophytes (48.8%) and chamaephytes (31.2%) were the predominant life forms, well-adapted to harsh rocky environments. the dominance of taxa from the eurasian mountain (32%) and eurasian (16%) area types reflects the montane-continental and temperate-continental climate characteristic of the study area. furthermore, the presence of glacial relict species highlights the role of high-altitude rocky habitats as microclimatic refugia, offering longterm protection against adverse climatic changes. this study underscores the substantial floristic diversity and biogeographical significance of rocky habitats in this part of the stara planina mts. key words: chasmophytes, the stara planina mts., biological spectrum, chorological spectrum, floristic diversity apstrakt: floristički diverzitet stenovitih staništa stare planine u srbiji flora stenovitih staništa stare planine (srbija) proučavana je sa ciljem utvrđivanja njenog diverziteta, biološkog i horološkog spektra. ukupno je identifikovano 125 vrsta vaskularnih biljaka iz 35 familija i 75 rodova. poaceae, caryophyllaceae i asteraceae su dominantne familije, a asplenium i sedum najzastupljeniji rodovi. hemikriptofite (48,8%) i hamefite (31,2%) predstavljaju dominantne životne forme, prilagođene stenovitim staništima. dominacija taksona koji pripadaju evroazijsko planinskom (32%) i evroazijskom (16%) areal tipu ukazuje na planinsko-kontinentalni i umerenokontinentalni tip klime, koji su karakteristični za istraživano područje. takođe, prisustvo glacijalnih reliktnih vrsta potvrđuje da stenovita staništa visokoplaninskih regiona imaju ulogu mikroklimatskih refugijuma, pružajući dugoročnu zaštitu od nepovoljnih klimatskih promena. ovo istraživanje ukazuje na znatan floristički diverzitet i biogeografski značaj stenovitih staništa ovog dela stare planine. ključne reči: hazmofite, stara planina, biološki spektar, horološki spektar, floristički diverzitet introduction due to their inaccessibility, rock outcrops are among the best-preserved terrestrial environments, providing refuge from grazing, competition, fire, anthropogenic pressures, and climatic changes (larson et al., 2000). the floristic composition of vegetation on these outcrops is shaped by geological and geomorphological characteristics (e.g., bedrock composition, structural heterogeneity, eluvial processes, and erosion), topographic features (e.g., exposure and slope), and microclimatic conditions (e.g., water availability, temperature, and insolation). in general, floristic diversity on rock outcrops tends to increase with higher humidity, greater rock stability, and an increased availability of space for seed germination (larson et al., 2000). chasmophytes are highly specialized plants adapted to endure the harsh conditions of the rock fissures they inhabit. many have evolved structural adaptations, such as bulbs, succulent leaves, aerial roots, vegetation mats (which facilitate the growth of other plants), and resurrection traits (ribeiro et al., 2007). chasmophytes share several functional © 2025 stojanović et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 76 traits: they are predominantly long-lived perennials, typically woody at the base, with an extended flowering period, high seed germination rates, and various mechanisms for long-distance seed dispersal (panitsa & kontopanou, 2017). the presence of vertical cliffs and rock outcrops leads to the fragmentation and isolation of open habitats, which support both endemic species and those at the edge of their distribution range (juvan et al., 2011; gwitira et al., 2013; gentili et al., 2015; zhao & gong, 2015; cutts et al., 2019; garcía et al., 2020). there is a strong link between chasmophyte ecology and endemism, as most endemic species are chasmophytes (panitsa & kontopanou, 2017). the occurrence of relict species within chasmophytic communities suggests that rock outcrops function as refugia from rapid climatic changes, allowing vegetation to persist as long as the rock remains intact (larson et al., 2000). these habitats support plant populations with highly stable demographic dynamics and low extinction risks (larson et al., 1999; picó & riba, 2002; garcía, 2003), as well as slow-growing, stable communities in a climax stage. although scattered records of species from the rocky habitats of the stara planina mts. in serbia exist in older literature (pančić, 1884; adamović, 1908-1911; josifović, 1970-1977), these habitats have remained largely understudied until recently. while recent studies have examined rock outcrop vegetation such as those by ranđelović et al. (2000, 2021) in serbia and szokala (2023) in bulgaria no comprehensive research has specifically addressed the floristic diversity, life-form spectra, or biogeographical patterns of the rock outcrop flora in this mountain range. this study represents the first detailed investigation of the diversity, biological spectra, and chorological patterns of rock outcrop flora in the serbian part of the stara planina mts. by filling this knowledge gap, our research enhances the broader understanding of biodiversity patterns and species specialization within these unique ecosystems. materials and methods the sampling area was discontinuous, covering only localities with rock outcrops that support chasmophytic communities (fig. 1), within the smaller western part of the stara planina mts. in serbia. the study area is bounded by the beli timok, trgoviški timok, and visočica rivers, as well as biologica nyssana ● 16 (1) june 2025: 75-91 stojanović et al. ● floristic diversity of the rocky habitats of the stara planina mountains in serbia fig. 1. map of sampling localities (1 babin zub peak; 2 crvene stene peak; 3 midžor peak; 4 pečka klisura gorge; 5 gorge between the villages of topli do and temska; 6 ponor plateau; 7 rosomački vrh peak; 8 rosomačka reka gorge; 9 tumba viewpoint; 10 kovani dol gorge; 11 tri čuke peak; 12 tupanar peak; 13 babin zub viewpoint; 14 visočica river gorge; and 15 žarkova čuka peak) 77 biologica nyssana ● 16 (1) june 2025: 75-91 stojanović et al. ● floristic diversity of the rocky habitats of the stara planina mountains in serbia the border with bulgaria. geologically, it is divided into northern pre-permian formations and southern permian to cretaceous sediments (lakušić & ćetković, 2007). soils on siliceous rock outcrops include silicate lithosols, regosols, and rankers, while calcareous rocks support calcaric leptosol. terrain variation creates diverse microclimates: the foothills have a temperate-continental climate with semi-dry summers and mild winters, whereas higher altitudes experience a montane-continental climate with long, cold winters. january temperatures range from -5.3 °c to 3.7 °c, and july temperatures from 11.8 °c to 28.8 °c (milovanović, 2010). precipitation during the growing season varies between 300 and 360 mm, peaking in may and june (lakušić & ćetković, 2007). the vertical vegetation profile of the stara planina mts. includes oak (3001100 m a.s.l.), beech (1100-1500 m a.s.l.), norway spruce (1500-1750 m a.s.l.), subalpine (1750-1900 m a.s.l.), and alpine (above 1900 m a.s.l.) altitudinal belts, with the timberline around 1550-1600 m a.s.l. (ranđelović et al., 2019). field research was conducted from june to september 2023, focusing on the flora of rock outcrops. collected specimens were deposited at the herbarium moesiacum niš (hmn). plant material was identified using the dichotomous keys of tutin et al. (1964-1980), josifović (1970-1977), sarić (1992), and sarić & diklić (1986). nomenclature follows the euro+med plantbase (2006+), except for asplenium × alternifolium wulfen (niketić & tomović, 2018), while classification aligns with wfo. plant life forms were determined following muellerdombois & ellenberg (1974), with adaptations for the flora of serbia by stevanović (1992a). taxa were classified into six life forms: phanerophytes (p), chamaephytes (ch), hemicryptophytes (h), geophytes (g), therophytes (t), and scandentophytes (s). chorotypes were identified according to meusel et al. (1965, 1978) and meusel & jäger (1992), as modified for serbia by stevanović (1992b). taxa were categorized into the following area types: arctic-alpine (aa), boreal (bor), central european (ce), cosmopolitan (cosm), eurasian (ea), eurasian mountain (eam), holarctic (hol), mediterranean-sub-mediterranean (med-smed), and pontic (pont). results and discussion a total of 125 vascular plant species, belonging to 35 families and 75 genera, were identified on the rock outcrops of the stara planina mts. in serbia (tab. 1). although rock outcrops host less than 10% of the total taxa reported for the vascular flora of the stara planina mts. in serbia (1,742 taxa) by lakušić & ćetković (2007), they display remarkable floristic diversity, especially considering the limited area table 1. list of recorded vascular plants on the rock outcrops of the stara planina mts. (serbia), including their life forms, area types, recorded localities, and bedrock types on which they were observed. abbreviations for life forms: phanerophytes (p), chamaephytes (ch), hemicryptophytes (h), geophytes (g), therophytes (t), and scandentophytes (s). abbreviations for area types: arctic-alpine (aa), boreal (bor), central european (ce), cosmopolitan (cosm), eurasian (ea), eurasian mountain (eam), holarctic (hol), mediterranean-sub-mediterranean (med-smed), and pontic (pont). locality numbers correspond to those in fig. 1. in the bedrock type column, 's' indicates siliceous rocks, 'l' indicates limestone, and 's/l' indicates both. for species found on both types of bedrock, the predominant type is bolded; if both types are equally represented, neither is bolded taxa life form area type locality bedrock type polypodiophyta fam: aspleniaceae asplenium adiantum-nigrum l. ch cosm 9, 10 s asplenium ceterach l. ch ea 4, 8 l asplenium ruta-muraria l. ch hol 1, 4, 5, 7, 8, 13, 14 s/l asplenium septentrionale (l.) hoffm. ch hol 2, 9, 10, 13, 15 s asplenium trichomanes l. ch cosm 1, 2, 5, 8, 9, 10, 12, 15 s/l asplenium viride huds. ch bor 1, 3, 6, 12 s asplenium × alternifolium wulfen ch hol 15 s fam: cystopteridaceae cystopteris fragilis (l.) bernh. ch cosm 1, 3, 5, 6, 8, 12, 14, 15 s/l 78 biologica nyssana ● 16 (1) june 2025: 75-91 stojanović et al. ● floristic diversity of the rocky habitats of the stara planina mountains in serbia fam. dryopteridaceae dryopteris expansa (c.presl) fraser-jenk. & jermy ch bor 1, 11 s dryopteris filix-mas (l.) schott ch hol 1 s fam. ophioglossaceae botrychium lunaria (l.) sw. g bor 3 s fam. polypodiaceae polypodium vulgare l. ch hol 1, 5 s fam. woodsiaceae woodsia alpina (bolton) gray ch aa 15 s pinophyta fam. cupressaceae juniperus communis subsp. nana syme p bor 1 s angiosperms fam. amaryllidaceae allium carinatum l. g medsmed 3 s allium carinatum subsp. pulchellum (g.don) bonnier & layens g medsmed 15 s allium flavum l. g medsmed 2, 5, 7 s/l allium schoenoprasum l. subsp. schoenoprasum g hol 15 s fam. apiaceae angelica sylvestris l. h bor 1 s seseli libanotis (l.) w.d.j.koch h ea 1 s fam. araliaceae hedera helix l. s ce 8 l fam. asparagaceae muscari comosum (l.) mill. g ea 9 s ornithogalum kochii parl. g medsmed 3, 13, 15 s fam. asteraceae anthemis cretica subsp. carpatica (willd.) grierson ch eam 1, 13 s aster alpinus l. h aa 1 s centaurea stoebe subsp. australis (a.kern.) greuter h pont 9 s centaurea stoebe subsp. serbica (prodan) ochsmann h pont 15 s cicerbita muralis (l.) wallr. h ea 8 l hieracium bifidum hornem. h bor 1 s leontodon crispus vill. h medsmed 2 s leucanthemum graminifolium (l.) lam. h eam 12 s 79 biologica nyssana ● 16 (1) june 2025: 75-91 stojanović et al. ● floristic diversity of the rocky habitats of the stara planina mountains in serbia pilosella alpicola (hoppe) f.w.schultz & sch. bip. h ce 9 s pilosella hoppeana (schult.) f.w.schultz & sch. bip. h eam 1 s senecio rupestris waldst. & kit. h eam 13 s solidago virgaurea l. h/g hol 9 s fam. brassicaceae arabis alpina subsp. caucasica (willd.) briq. h eam 7 l arabis sagittata (bertol.) dc. h ea 5 s aurinia saxatilis subsp. orientalis (ard.) t.r.dudley ch medsmed 5 l draba doerfleri wettst. h eam 1 s draba lasiocarpa rochel h eam 4 l rorippa lippizensis (wulfen) rchb. h eam 15 s fam. campanulaceae campanula rapunculoides l. h ea 14 l campanula rotundifolia l. h hol 1 s campanula sparsa friv. t ce 9 s campanula wanneri rochel h eam 1, 3, 5, 12, 13, 15 s fam. caryophyllaceae arenaria serpyllifolia l. t ea 4 l cerastium alpinum l. ch aa 3, 12 s cerastium banaticum (rochel) heuff. ch medsmed 15 s dianthus moesiacus vis. & pančić h eam 15 s dianthus petraeus waldst. & kit. subsp. petraeus ch eam 7 l heliosperma pusillum (waldst. & kit.) rchb. subsp. pusillum ch eam 1, 3, 12 s minuartia verna (l.) hiern ch ea 7, 12 s/l petrorhagia prolifera (l.) p.w.ball & heywood t medsmed 2 s petrorhagia saxifraga (l.) link ch medsmed 4, 5 l scleranthus perennis l. ch eam 13 s silene flavescens waldst. & kit. ch eam 4, 5, 14 l silene lerchenfeldiana baumg. ch eam 1, 9, 11, 13 s silene viscaria (l.) jess. h ea 5, 9 s fam. crassulaceae jovibarba heuffelii (schott) á.löve & d.löve ch eam 1, 15 s sedum album l. ch eam 2, 5, 7, 8, 10, 15 s/l sedum alpestre vill. ch eam 3, 12 s sedum annuum l. t aa 1, 2, 9, 12, 13, 15 s sedum cepaea l. t medsmed 9 s 80 biologica nyssana ● 16 (1) june 2025: 75-91 stojanović et al. ● floristic diversity of the rocky habitats of the stara planina mountains in serbia sedum dasyphyllum l. ch medsmed 8, 10 s/l sedum hispanicum l. h/t medsmed 2, 4 s/l sedum ochroleucum chaix ch medsmed 7 l sempervivum marmoreum griseb ch eam 3, 4, 15 s/l fam. cyperaceae carex kitaibeliana bech. h eam 15 s carex sempervirens vill. h eam 3, 12 s fam. ericaceae vaccinium myrtillus l. ch bor 1 s fam. gentianaceae gentiana asclepiadea l. h eam 1 s fam. geraniaceae geranium lucidum l. t ea 5 s geranium macrorrhizum l. g ea 1 s geranium purpureum vill. t medsmed 8, 14 l geranium rotundifolium l. t ea 4 l fam. hypericaceae hypericum maculatum crantz h eam 1, 12 s fam. juncaceae juncus trifidus l. h aa 3, 11, 12 s luzula luzuloides (lam.) dandy & wilmott h ce 1 s luzula spicata (l.) dc. h aa 3 s fam. lamiaceae clinopodium alpinum (l.) kuntze ch ea 2, 4, 15 s/l lamium garganicum l. h medsmed 1, 13 s satureja kitaibelii wierzb. ex heuff. ch medsmed 4 l stachys recta l. h ea 2 s thymus praecox subsp. jankae (čelak.) jalas ch medsmed 1, 3, 13, 15 s thymus praecox subsp. polytrichus (a.kern. ex borbás) jalas ch eam 1, 15 s fam. orobanchaceae euphrasia pectinata ten. t ea 15 s fam. oxalidaceae oxalis acetosella l. g ea 1 s fam. plantaginaceae plantago holosteum scop. h medsmed 15 s veronica barrelieri roem. & schult. h eam 15 s 81 biologica nyssana ● 16 (1) june 2025: 75-91 stojanović et al. ● floristic diversity of the rocky habitats of the stara planina mountains in serbia fam. poaceae agrostis capillaris l. h hol 1 s anisantha sterilis (l.) nevski t hol 2 s avenella flexuosa (l.) drejer h hol 9, 11 s bellardiochloa variegata (lam.) kerguélen h eam 11, 12 s festuca dalmatica (hack.) k.richt. h pont 1, 11, 12, 13 s festuca ovina subsp. supina (schur) oborny h ea 1, 3, 11, 12 s festuca panciciana (hack.) k.richt h eam 1, 7, 11, 13, 15 s/l festuca picturata pils h ce 12 s festuca rubra l. s.l. h hol 12 s melica transsilvanica schur h ea 2, 4 s/l poa alpina l. h aa 3, 15 s poa badensis haenke ex willd. h eam 7, 15 s/l poa laxa haenke h eam 1 s poa nemoralis l. h hol 1, 2, 5, 9, 10, 13 s sesleria coerulans friv. h eam 1, 3, 12 s sesleria filifolia hoppe h eam 7 l fam. polygonaceae rumex acetosella l. h hol 9 s fam. primulaceae primula minima l. h eam 3, 12 s fam. ranunculaceae clematis vitalba l. s ce 10 s ranunculus montanus willd. h eam 1 s thalictrum aquilegiifolium l. h ce 1 s fam. rosaceae drymocallis rupestris (l.) soják h ce 15 s rubus geniculatus kaltenb. p ce 9 s fam. rubiaceae asperula purpurea (l.) ehrend. ch eam 4 l galium album mill. h ce 5 l galium anisophyllon vill. h eam 4 l galium lucidum all. h medsmed 7 l galium odoratum (l.) scop. h ea 1 s fam. saxifragaceae saxifraga bryoides l. ch eam 3 s saxifraga moschata wulfen ch eam 3, 12 s saxifraga paniculata mill. ch aa 1, 3, 7, 12, 15 s/l saxifraga pedemontana subsp. cymosa engl. ch eam 1, 3 s saxifraga rotundifolia l. h eam 1, 5, 12 s saxifraga tridactylites l. t ea 3, 8, 10, 12 s/l 82 they occupy. moreover, several species documented in this study, such as poa laxa haenke (niketić et al., 2023), festuca picturata pils, rubus geniculatus kaltenb., and draba doerfleri wettst., are new records for the region, suggesting that the floristic diversity of the stara planina mts. is greater than previously recognized. however, it is important to note that the local endemic chasmophyte campanula calycialata v. randjel. & zlatković was not observed during the study, as expected, since it is considered extinct (ranđelović et al., 2019). the phylum polypodiophyta comprises six families, six genera, and 13 species, accounting for 10.4% of the total taxa recorded in the study area. the phylum pinophyta consists of a single family, represented by one species (0.8%). angiosperms are the most diverse, encompassing 28 families, 68 genera, and 111 species, constituting 88.8% of the total taxa identified. the dominant vascular plant families are poaceae barnhart, caryophyllaceae juss., and asteraceae giseke, represented by 16, 13, and 12 species, respectively (fig. 2), accounting for 12.8%, 10.4%, and 9.6% of the total species recorded. asplenium l. and sedum l. are the dominant genera, each contributing 5.6% of the total species, followed by saxifraga l. at 4.8% (fig. 2). certain genera, such as asplenium among ferns and sedum among herbaceous vascular plants, exhibit notable consistency in their global distribution on rock outcrops (larson et al., 2000). desiccation-tolerant plants, particularly poikilohydric species such as asplenium ruta-muraria l., a. ceterach l. (sádlo & chytrý, 2009), and succulent xerophytes from the genera sempervivum l. and sedum (oettli, 1905; lüth, 1993), are prevalent in these habitats. photographs of selected fern and succulent species found on rock outcrops in the stara planina mts. are presented in figs. 3 and 4, respectively. the species with the highest frequency within the study area are campanula wanneri rochel, heliosperma pusillum (waldst. & kit.) rchb. subsp. pusillum, and cystopteris fragilis (l.) bernh. the most dominant life forms on the rock outcrops are hemicryptophytes (48.8%) and chamaephytes (31.2%) (fig. 5a), which possess functional traits that enable them to endure the harsh conditions of these habitats (tomaselli et al., 2018). these plants have strong root systems that anchor them to the rock, allowing them to penetrate cracks in search of water and minerals (bacchetta et al., 2007). hemicryptophytes’ traits offer protection against harsh climatic conditions at higher altitudes, while enhancing survival at lower and mid-elevations, where the growing season is longer (carlsson et al., 1999; körner, 2003). furthermore, hemicryptophytes are dominant in the flora of serbia (diklić, 1984). in extreme cliff environments, the lignified underground and partially above-ground parts of chamaephytes help them withstand wind impacts and recover from harsh environmental conditions (georghiou & delipetrou, 2010). chamaephytes, more tolerant biologica nyssana ● 16 (1) june 2025: 75-91 stojanović et al. ● floristic diversity of the rocky habitats of the stara planina mountains in serbia fam. caprifoliaceae valeriana tripteris l. h eam 1 s fam. violaceae viola arvensis murray t ea 2 s fig. 2. a species and subspecies richness at the family level (families represented by one or two species are excluded); b species and subspecies richness at the generic level (genera represented by a single species are excluded) 83 biologica nyssana ● 16 (1) june 2025: 75-91 stojanović et al. ● floristic diversity of the rocky habitats of the stara planina mountains in serbia fig. 3. representative fern species inhabiting rock outcrops on the stara planina mts. (serbia) (a asplenium ceterach, b a. ruta-muraria, c cystopteris fragilis, d woodsia alpina, e asplenium trichomanes, and f a. septentrionale. photos by jovana stojanović 84 biologica nyssana ● 16 (1) june 2025: 75-91 stojanović et al. ● floristic diversity of the rocky habitats of the stara planina mountains in serbia fig. 4. representative succulent species inhabiting rock outcrops on the stara planina mts. (serbia) (a sedum dasyphyllum, b s. hispanicum, c jovibarba heuffelii, d sedum alpestre, and e s. album). photos by jovana stojanović 85 biologica nyssana ● 16 (1) june 2025: 75-91 stojanović et al. ● floristic diversity of the rocky habitats of the stara planina mountains in serbia of mountainous conditions than other life forms (lazarina et al., 2019), can complete their life cycle despite a short growing season, particularly at high altitudes (vogiatzakis et al., 2003). although the number of microhabitats suitable for colonization is limited (davis, 1951), therophytes are relatively well-represented (9.6%), which may indicate an increase in disturbances and habitat degradation (ranđelović et al., 2007). disturbances such as rockfalls and erosion expose bare soil, creating open habitats that therophytes can quickly exploit. their short life cycle allows for rapid reproduction, enabling them to establish before slower-growing species take root, thereby reducing competition. geophytes, though limited by the space available for underground storage organs (davis, 1951), are still present (7.2%), particularly in larger crevices or on ledges with greater soil accumulation. the diversity of life forms on rock outcrops can be attributed to the wide variety of microhabitats found in close proximity (oettli, 1905). fig. 5. a biological spectrum of the flora in the rocky habitats of the stara planina mts. in serbia (p phanerophytes; ch chamaephytes; h hemicryptophytes; g geophytes; t therophytes; and s scandentophytes); b chorological spectrum of the flora in the rocky habitats of the stara planina mts. in serbia (aa arctic-alpine; bor boreal; ce central european; cosm cosmopolitan; ea eurasian; eam eurasian mountain; hol holarctic; med-smed mediterranean-sub-mediterranean; and pont pontic) the phytogeographical analysis classified all recorded taxa into nine area types (fig. 5b). the eurasian mountain area type is predominant (32%), including taxa characteristic of chasmophytic communities on the stara planina mts. in serbia, such as campanula wanneri, heliosperma pusillum subsp. pusillum, silene flavescens waldst. & kit., s. lerchenfeldiana baumg., jovibarba heuffelii (schott) á. löve & d. löve, and saxifraga pedemontana subsp. cymosa engl. some species from the eurasian mountain area type are represented in fig. 6. the eurasian area type ranks second (16%), including species like asplenium ceterach, which are commonly found in rocky habitats. the predominance of taxa from these two area types suggests that the sampling localities exhibit montane-continental and temperate-continental characteristics, in line with the region's climate (lakušić & ćetković, 2007). the third most represented area type is mediterraneansub-mediterranean (15.2%), which includes species found exclusively on carbonate rocks within the study area, such as aurinia saxatilis subsp. orientalis (ard.) t.r.dudley, sedum ochroleucum chaix, and satureja kitaibelii wierzb. ex heuff. taxa belonging to this area type are typically associated with calcareous rocks and their presence in the study area can be attributed to the influence of the changed mediterranean climate from the aegean region, transmitted through river valleys and deep gorges, particularly the vardar valley and the južna morava river. this climate influence extends into the continental limestone areas of southeastern serbia (stevanović & stevanović, 1995). thermophilous and xerophilous habitats within the study area, especially low-elevation or south-facing carbonate rock formations, provide suitable conditions for species of the mediterranean-sub-mediterranean area type (jušković et al., 2010). additionally, the holarctic area type is well represented (11.2%), including chasmophytic ferns such as asplenium ruta-muraria and a. septentrionale (l.) hoffm. 86 biologica nyssana ● 16 (1) june 2025: 75-91 stojanović et al. ● floristic diversity of the rocky habitats of the stara planina mountains in serbia fig. 6. characteristic taxa of chasmophytic communities on the stara planina mts. (serbia) belonging to the eurasian mountain area type (a campanula wanneri, b heliosperma pusillum subsp. pusillum, c saxifraga pedemontana subsp. cymosa, d silene flavescens, and e s. lerchenfeldiana). photos by jovana stojanović this diverse composition underscores the balkan peninsula, particularly its central part, as a key convergence zone blending floristic elements from central europe, the boreal and arctic eurasian regions, as well as the eastern and central mediterranean (stevanović et al., 1995). the plant cover of rocky habitats is shaped by the bedrock composition, which influences the calcium carbonate levels in the soil. species that thrive on limestone are typically calcicolous, while those preferring siliceous substrates are generally calcifugous (kojić, 1984). the preference of 87 biologica nyssana ● 16 (1) june 2025: 75-91 stojanović et al. ● floristic diversity of the rocky habitats of the stara planina mountains in serbia the recorded taxa for different bedrock types is presented in tab. 1. the majority of taxa were found exclusively on siliceous rocks (90 species, 72% of all recorded taxa), followed by those documented only on limestone (20 species, 16%). a smaller proportion of taxa (15 species, 12%) were recorded on both bedrock types. among the prominent chasmophytes exclusive to siliceous rocks are campanula wanneri, silene lerchenfeldiana, asplenium septentrionale, and woodsia alpina (bolton) gray, while those confined to limestone include aurinia saxatilis subsp. orientalis and silene flavescens. some species, such as asplenium trichomanes l., cystopteris fragilis, saxifraga paniculata mill., and asplenium rutamuraria, were observed on both bedrock types. this is not surprising for asplenium trichomanes and cystopteris fragilis, as various subspecies of a. trichomanes are adapted to different substrates (e.g. a. trichomanes l. subsp. trichomanes prefers siliceous rocks, while a. trichomanes subsp. quadrivalens d.e.mey. is more suited to calcareous rocks). similarly, cystopteris fragilis demonstrates a broad ecological tolerance for different substrate ph levels (sádlo & chytrý, 2009). on the other hand, saxifraga paniculata and asplenium rutamuraria are generally considered calcicolous species (axmanová, 2022). in the study area, asplenium ruta-muraria is primarily found on calcareous rocks, while s. paniculata was more prevalent on siliceous rocks. this pattern can be explained by the presence of calcium compounds in acidic rocks, which may contribute to the formation of alkaline soil (oettli, 1905; lundquist, 1968). for example, silicate rocks like gneiss can contain calcium that accumulates in cracks, creating conditions conducive to the growth of calcicolous species (lüth, 1993). as a result, microhabitats on different rock types can exhibit similar soil ph values (larson et al., 2000), which in turn allows the development of the same plant species. rocky habitats are often regarded as microclimatic refugia (speziale & ezcurra, 2015; bátori et al., 2017; fitzsimons & michael, 2017), offering longterm shelter from harsh climatic conditions and enabling the survival of numerous relict species. these microhabitats act as buffers against extreme environmental conditions, making rock crevices vital refuges for arctic-alpine species, especially those found south of the arctic belt (garcía et al., 2020). the presence of arctic-alpine species in rock vegetation is a legacy of climatic fluctuations during glaciation and deglaciation, which confined their earlier distribution to mountainous regions and cliffs (larson et al., 2000). relict species thrive in lowcompetition microhabitats, such as crevices, where limited space restricts the growth of competing plants (crawford, 1989). in the study area, 9% of the recorded species are considered glacial relicts, including aster alpinus l., arabis alpina subsp. caucasica (willd.) briq., cerastium alpinum l., sedum annuum l., juncus trifidus l., luzula spicata (l.) dc., poa alpina l., p. laxa, primula minima l., saxifraga paniculata, and woodsia alpina. chasmophytic plant communities are of significant biogeographical importance due to the high number of endemic taxa they support. these endemic species flourish in steep, rocky environments with challenging soil conditions, where limited vegetation cover minimizes biotic interactions (panitsa & kontopanou, 2017). in this study, two balkan endemic taxa were identified: centaurea stoebe subsp. serbica (prodan) ochsmann and dianthus moesiacus vis. & pančić (tomović et al., 2014). several species recorded on the rock outcrops within the study area are designated as strictly protected or protected under national legislation (official gazette of the republic of serbia, no. 5/2010, 47/2011, 32/2016, and 98/2016). strictly protected species include campanula wanneri, dianthus moesiacus, and veronica barrelieri roem. & schult. protected species encompass silene lerchenfeldiana, juniperus communis subsp. nana syme, vaccinium myrtillus l., geranium macrorrhizum l., hypericum maculatum crantz, satureja kitaibelii, primula minima, drymocallis rupestris (l.) soják, galium odoratum (l.) scop., and saxifraga pedemontana subsp. cymosa. conclusion this study highlights the remarkable floristic diversity and ecological significance of rock outcrops in the stara planina mts. in serbia. it identifies a wide range of vascular plant species, including several new regional records. the adaptation of chasmophytes to harsh environmental conditions emphasizes their essential role in these unique ecosystems, which host a variety of endemic and relict species. the findings highlight the urgent need for conservation efforts, particularly for species protected by national legislation, in light of threats from habitat degradation and climate change. furthermore, the study calls for further research to fully assess the biodiversity of these habitats and to develop effective conservation strategies to preserve these valuable ecological resources. acknowledgements. this research was supported by the ministry of science, technological development and innovation of the republic of serbia (contract no. 451-03-136/2025-03/200124; 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(2015). diversity and conservation of plant species in dry valleys, southwest china. biodiversity and conservation, 24, 2611-2623. https://doi.org/10.1007/s10531-0150952-2 devetak, jakšić, 2019, biologica nyssana 10(1) 10 (1) september 2019: 35-41 doi: 10.5281/zenodo.3464002 lacewings (insecta: neuropterida: raphidioptera, megaloptera, neuroptera) collected in montenegro with checklist of species original article dušan devetak department of biology, faculty of natural sciences and mathematics, university of maribor, koroška cesta 160, 2000 maribor, slovenia dusan.devetak@guest.arnes.si (corresponding author) predrag jakšić čingrijina 14/25, zvezdara, 11000 beograd, serbia jaksicpredrag@gmail.com received: april 26, 2019 revised: july 1, 2019 accepted: august 13, 2019 abstract: seventeen species of neuropterida (raphidioptera, megaloptera, neuroptera) collected in montenegro during 2017-2018 are reported. an updated checklist of all known species from this country is presented. the list contains 75 species occurring in montenegro. of these, two species were reported for the first time for the country. one was a green lacewing chrysoperla lucasina, which has already been expected for the area. the second species new to montenegro, an owlfly libelloides longicornis, is particularly noteworthy, because its finding in the balkan peninsula was unexpected. key words: lacewings, neuropterida, neuroptera, montenegro, owlfly, libelloides longicornis, checklist apstract: mrežokrilci (insecta: neuropterida: raphidioptera, megaloptera, neuroptera) sakupljeni u crnoj gori, sa ček-listom vrsta dat je prikaz sedamnaest vrsta mrežokrilaca (raphidioptera, megaloptera, neuroptera) utvrđenih u crnoj gori tokom 2017. i 2018. godine. priložena je i inovirana ček-lista svih do sada utvrđenih vrsta u ovoj zemlji. tom listom potvrđeno je prisustvo 75 vrsta mrežokrilaca u crnoj gori. među njima dve vrste su po prvi put prikazane za crnu goru. jedna od njih je vrsta zlatooke chrysoperla lucasina, koja je i očekivana za to područje. druga nova vrsta za crnu goru je libelloides longicornis, čiji je nalaz značajan jer je prisustvo ove vrste na balkanskom poluostrvu neočekivano. ključne reči: mrežokrilci, neuropterida, neuroptera, crna gora, askalafide, libelloides longicornis, ček-lista introduction montenegro is a mountainous mediterranean country in the south-west part of the balkan peninsula, bordering the adriatic sea. despite the fact that montenegro is the second smallest country in the balkan peninsula, the diversity of the landscape, climate and soil created the conditions for high biodiversity. the research has been conducted in pre-selected areas – so-called key biodiversity areas (kbas) – during the field seasons 2017 and 2018 within the frame of the ipa project ‘establishment of natura 2000 network, montenegro’. the oldest known montenegrin record of lacewings (neuropterida: raphidioptera, megaloptera, neuroptera) dates back to 1908, when an owlfly, libelloides lacteus was mentioned in the country (van der weele, 1908). up to the second half of the 20th century, lacewings in the country have been insufficiently investigated, and based mainly on sporadic records (appendix 1). before the wwii, táborský in his studies of owlflies (ascalaphidae) presented the distribution for two ascalaphid species in the balkan countries, also in montenegro (táborský, 1936, 1939). in the sixties, a snakefly raphidia durmitorica was described as a new species (steinmann, 1964), but later this species name was recognized as a synonym of dichrostigma flavipes (aspöck & aspöck, 1966). in the second half of the 20th century, saure (1989) collected twelve lacewing species in montenegro. © 2019 devetak, jakšić. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 35 at the end of the 20th century, in the period from 1985 to 1988, slovenian and serbian entomologists collected lacewings in the durmitor national park, the results of their study is a faunal list that includes data for lacewings of the national park (devetak, 1991). later, a series of papers was published, containing very scattered information on the occurrence of these insects in the country (devetak, 1992, 1995, 1996, 1997; letardi and pantaleoni, 1996; devetak and devetak, 2004, popov, 2004; sziráki, 2014; devetak et al., 2015; devetak et al., 2016; devetak and zeqiri, 2018; appendix 1). until recently, 73 lacewing species were known for montenegro. however, in the preparation for a review of the fauna in selected areas in montenegro, efforts were made to determine whether other species of lacewings might be present in the country. the aim of the paper is to improve the knowledge on the occurrence of lacewings in montenegro. materials and methods lacewings were collected in selected sites during lepidopterological field work of the second author in 2017 and 2018. insects are preserved in the first author’s collection. we followed the nomenclature and taxonomy proposed by aspöck et al. (1980, 2001) and lacewing digital library (oswald, 2017). results in the period from 2017 to 2018, seventeen lacewing species were collected. two species – green lacewing chrysoperla lucasina and owlfly libelloides longicornis are new in montenegro. of these, the finding of the owlfly libelloides longicornis is particularly noteworthy. its finding represents the first documented record for the balkan peninsula. additionally, a checklist of neuropterida (raphidioptera, megaloptera, neuroptera) in montenegro is presented (appendix 1). list of species recorded in montenegro raphidioptera, snakeflies raphidiidae dichrostigma flavipes (stein, 1863) durmitor. sušica, vidikovac; 13/07/2018; p. jakšić leg.; 1 ♀. megaloptera sialidae, alderflies sialis lutaria (linnaeus, 1758) durmitor. zminičko jezero, 1295 m; 43°06’02.9”n, 19°14’54.9”e; 05/06/2017; p. jakšić leg.; 2 ♂ 5 ♀. durmitor. komarnica, nevidio; 05/08/2017; p. jakšić leg.; 1 ♂ 1 ♀. neuroptera osmylidae osmylus fulvicephalus (scopoli, 1763) bijelo polje, bistrica, đalovića klisura; 43°04’14.9”n, 19°54’04.6”e; 01/06/2017; p. jakšić leg.; 1 ♂ 1 ♀. chrysopidae, green lacewings nothochrysa fulviceps (stephens, 1836) durmitor mt., žabljak; 21/07/2017; p. jakšić leg.; 1 ♂ 1 ♀. chrysopa perla (linnaeus, 1758) bijelo polje, đalovića klisura, manastir sv. nikole; 780 m; 43°04’14.1”n, 19°54’12.3”e; 20/07/2014; p. jakšić leg.; 2 ♂. durmitor. bukovica, 999 m; 06/06/2017; p. jakšić leg.; 2 ♂. durmitor. kanjon sušice; 1180 m; 03/06/2017; p. jakšić leg.; 2 ♂ 2 ♀. durmitor. tepca; 880-920 m; 05/06/2017; p. jakšić leg.; 1 ♂. durmitor. zminičko jezero, 1322 m; 06/06/2017; p. jakšić leg.; 1 ♂. ljubišnja planina; 1068 m; 43°21’33”n, 19°06’39”e; p. jakšić leg.; 1 ♀. chrysopa walkeri mclachlan, 1893 komovi. šljivovički vis. 23/06/2018; p. jakšić leg.; 1 ♀. pseudomallada ventralis (curtis, 1834) komovi. šljivovički vis; 23/06/2018; p. jakšić leg.; 1 ♂. chrysoperla cf. carnea (stephens, 1836) bijelo polje, đalovića klisura, manastir sv. nikole; 780 m; 43°04’14.1”n 19°54’12.3”e; 20/07/2014; p. jakšić leg.; 1 ♀. komovi. šljivovički vis; 23/06/2018; p. jakšić leg.; 2 ♀. morača; 42°49’57”n 19°21’14”e; 507 m; 12/ vi/2018; p. jakšić leg.; 1 ♀. prokletije, gusinje: grbaja, 1143 m; 42°31’20”n, 19°47’12”e; 26/06/2018; p. jakšić leg.; 1 ♀. chrysoperla lucasina (lacroix, 1912) durmitor. žabljak; 21/07/2017; p. jakšić leg.; 3 ♀. new species for durmitor! komovi. šljivovički vis; 23/06/2018; p. jakšić leg.; 2 ♀. plužine, bioč. 43°11’02”n, 18°43’51”e; 10/07/2018; p. jakšić leg.; 2 ♀. prokletije, gusinje: grbaja, 1143 m; 42°31’20”n 19°47’12”e; 26/06/2018; p. jakšić leg.; 1 ♀. in the durmitor national park, individuals of a chrysoperla carnea (stephens)-complex of spe36 biologica nyssana ● 10 (1) september 2019: 35-41 devetak, jakšić ● lacewings (insecta: neuropterida: raphidioptera, megaloptera, neuroptera) collected in montenegro cies were found in eighties (devetak, 1991), but at that time it was not possible to separate different species within the complex. today, we are able to distinguish several species in europe (henry et al., 2013), and at least five in the balkan peninsula (e.g. henry et al., 1996, 1999, 2002, 2003; thierry and canard, 2015; devetak and rausch 2016). first record in montenegro! cunctochrysa albolineata (killington, 1935) durmitor. žabljak; 21/07/2017; p. jakšić leg.; 2 ♀. 37 hemerobiidae, brown lacewings megalomus tortricoides rambur, 1842 durmitor. žabljak; 21/07/2017; p. jakšić leg.; 1 ♂ 1 ♀. myrmeleontidae, antlions palpares libelluloides (linnaeus, 1764) ostrog. 10/07/2018; p. jakšić leg.; 1 ♂ (fig. 1-1). myrmecaelurus trigrammus (pallas, 1771) podgorica, cijevna, 38 m; 42°22’50.8”n 19°16’28.6”e; 02/06/2017; p. jakšić leg.; 6 larvae fig. 1. 1) antlion palpares libelluloides (linnaeus, 1764), male, ostrog. forewing length 52 mm. 2) antlion myrmecaelurus trigrammus (pallas, 1771), male. forewing length 35 mm. 3) antlion myrmeleon (myrmeleon) inconspicuus rambur, 1842, female, ulcinj. forewing length 29 mm. 4) antlion creoleon plumbeus (olivier, 1811), female. podgorica. forewing length 30 mm. 5) owlfly libelloides longicornis (linnaeus, 1764), female. đalovića klisura. forewing length 22 mm. 6) owlfly libelloides macaronius (scopoli, 1763), female. kom vasojevićki, trešnjevik. photo: 1-5: d. devetak; 6: p. jakšić. biologica nyssana ● 10 (1) september 2019: 35-41 devetak, jakšić ● lacewings (insecta: neuropterida: raphidioptera, megaloptera, neuroptera) collected in montenegro 38 (l3). adult is shown in fig. 1-2. myrmeleon (myrmeleon) inconspicuus rambur, 1842 podgorica, cijevna, 38 m; 42°22’50.8”n 19°16’28.6”e; 02/06/2017; p. jakšić leg.; 1 larva (l3). an adult from ulcinj is shown in fig. 1-3. creoleon plumbeus (olivier, 1811) podgorica, cijevna; 21/07/2017; p. jakšić leg.; 1 ♀ (fig. 1-4). ascalaphidae, owlflies libelloides longicornis (linnaeus, 1764) bijelo polje, đalovića klisura, manastir sv. nikole; 780 m; 43°04’14.1”n, 19°54’12.3”e; 20/07/2014; p. jakšić leg.; 1 ♀. (fig. 1-5). black spots on the wings of female are typical for l. longicornis (fig. 1-1). it is an extremely interesting finding – the first record in the balkan peninsula! this species occurs in central and western europe and italy (aspöck et al., 1980, 2001) and the finding in montenegro enlarges the knowledge on its occurrence in europe. libelloides macaronius (scopoli, 1763) podgorica, cijevna, 38 m; 42°22’50.8”n 19°16’28.6”e; 09/05/2017; p. jakšić leg.; 1 ♀. observations: bijelo polje, đalovića klisura, manastir sv. nikole; 780 m; 43°04’08.7”n, 19°54’09.4”e; 20/07/2017; 1 ♀ observed by p. jakšić. pivska planina,43°02’23.7”n, 18°48’12.4”e; 1451 m; 12/07/2018; 1 ♂observed by p. jakšić. kom vasojevićki, trešnjevik, 42°44’11.4”n, 19°41’04.2”e; 1303 m, 23/06/2018; 1 ♀ (fig. 1-6), observed and photographed by p. jakšić. discussion the paper presents lacewings collected in selected areas in montenegro during the years 2017 and 2018. with the new findings, the number of known neuropterida in montenegro has now raised to 75. the most spectacular was the finding of an owlfly, libelloides longicornis, which has not yet been recorded in the balkan peninsula. studies in future should confirm whether there is a stable population in montenegro. its known occurrence has ranged from western europe to italy and central europe (aspöck et al., 1980, 2001); in the light of the discovery of this species in montenegro, therefore we can expect more findings in the balkan peninsula in the future. acknowledgements the authors are grateful for helpful suggestions made by two anonymous reviewers on an earlier version of the manuscript. they have greatly helped to improve the manuscript. references aspöck, h., aspöck, u. 1966: studien an europäischen und kleinasiatischen arten des genus raphidia l. 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oswald, j.d. 2017: lacewing digital library. neuropterida species of the world. version 5.0. http:// lacewing.tamu.edu/speciescatalog/main. accessed on 16 december 2018 popov, a. 2004: the ascalaphidae (neuroptera) of the balkan peninsula. denisia, 13: 229-237. saure, c. 1989: beitrag zur kenntnis der neuropterenfauna jugoslawiens und griechenlands (insecta, planipennia). entomofauna, 10(4): 33-43. steinmann, h. 1964: raphidiopterological studies ii. new raphidia l. and raphidilla nav. species from europe and asia. acta zoologica hungarica, 10: 199-227. sziráki, g. 2014: data to the raphidioptera fauna of the balkan peninsula and crete. folia historiconaturalia musei matraensis, 38: 87-90. táborský, k. 1936: monografické zpracování druhu ascalaphus ottomanus germar. monographische bearbeitung der art ascalaphus ottomanus germar. sborník entomologického oddeleni národního musea v praze, praha, 14: 133-144. táborský, k. 1939: studie druhu ascalaphus macaronius scopoli. časopis národního musea v praze 113: 91-96. thierry, d., canard, m. 2015: contribution to the knowledge of green lacewings of croatia (insecta: neuropterida: chrysopidae). acta entomologica slovenica, 23:21-28. van der weele, h.w. 1908: ascalaphiden monographisch bearbeitet. collections zoologiques du baron edm. de selys longchamps, 8: 1-326. species / taxon literature records comments order r a p h i d i o p t e r a family raphidiidae raphidia (raphidia) ophiopsis ophiopsis linnaeus, 1758 devetak, 1991, 1992 dichrostigma flavipes (stein, 1863) steinmann, 1964; devetak, 1991, 1992; sziráki, 2014 this paper. steinmann (1964) described this species as raphidia durmitorica. phaeostigma (phaeostigma) galloitalicum (h. aspöck & u. aspöck, 1976) devetak, 1991, 1992; sziráki, 2014 appendix 1. checklist of lacewings (neuropterida) in montenegro biologica nyssana ● 10 (1) september 2019: 35-41 devetak, jakšić ● lacewings (insecta: neuropterida: raphidioptera, megaloptera, neuroptera) collected in montenegro 40 species / taxon literature records comment order m e g a l o p t e r a family sialidae sialis lutaria (linnaeus, 1758) devetak, 1991, 1992; devetak et al., 2016; this paper sialis fuliginosa pictet, 1836 devetak, 1991, 1992; devetak et al., 2016 order n e u r o p t e r a family osmylidae osmylus fulvicephalus (scopoli, 1763) saure, 1989; devetak, 1991; this paper family chrysopidae nothochrysa fulviceps (stephens, 1836) devetak, 1991, 1992 this paper nothochrysa capitata (fabricius, 1793) devetak, 1991, 1992 italochrysa italica (rossi, 1790) devetak, 1992 nineta flava (scopoli, 1763) devetak, 1991, 1992 chrysopidia (chrysotropia) ciliata (wesmael, 1841) devetak, 1991, 1992 chrysopa perla (linnaeus, 1758) devetak, 1991, 1992 this paper chrysopa walkeri mclachlan, 1893 devetak, 1991, 1992 this paper chrysopa dorsalis burmeister, 1839 devetak, 1991, 1992 chrysopa formosa brauer, 1851 devetak, 1991, 1992 chrysopa viridana schneider, 1845 devetak, 1991, 1992 chrysopa pallens (rambur, 1838) devetak et al., 2015 pseudomallada flavifrons flavifrons (brauer, 1851) saure, 1989; devetak, 1991, 1992 pseudomallada prasinus (burmeister, 1839) saure, 1989; devetak, 1991, 1992 pseudomallada ventralis (curtis, 1834) devetak, 1991, 1992 this paper pseudomallada clathratus (schneider, 1845) saure, 1989; devetak, 1992 chrysoperla cf. carnea (stephens, 1836) devetak, 1991, 1992 this paper chrysoperla lucasina (lacroix, 1912) this paper. first record in montenegro. cunctochrysa albolineata (killington, 1935) saure, 1989; devetak, 1991, 1992 this paper peyerimhoffina gracilis (schneider, 1851) devetak, 1991, 1992 family hemerobiidae hemerobius humulinus linnaeus, 1758 devetak, 1991, 1992 hemerobius stigma stephens, 1836 devetak, 1991, 1992 hemerobius pini stephens, 1836 devetak, 1991, 1992 hemerobius contumaxtjeder, 1932 devetak, 1991, 1992 hemerobius handschinitjeder, 1957 devetak, 1991, 1992 hemerobius micans olivier, 1793 devetak, 1991, 1992 hemerobius lutescens fabricius, 1793 devetak, 1991, 1992 hemerobius gilvus stein, 1863 devetak, 1991, 1992 hemerobius marginatus stephens, 1836 saure, 1989; devetak, 1991, 1992 wesmaelius concinnus (stephens, 1836) devetak, 1991, 1992 wesmaelius quadrifasciatus (reuter, 1894) devetak, 1991, 1992 wesmaelius nervosus (fabricius, 1793) devetak, 1991, 1992 wesmaelius malladai (navás, 1925) devetak, 1991, 1992 wesmaelius tjederi (kimmins, 1963) devetak, 1991, 1992 wesmaelius subnebulosus (stephens, 1836) devetak, 1991, 1992 sympherobius elegans (stephens, 1836) devetak, 1991, 1992 sympherobius fuscescens (wallengren, 1863) devetak, 1991, 1992 sympherobius pellucidus (walker, 1853) devetak, 1991, 1992 megalomus tortricoides rambur, 1842 devetak, 1991, 1992 this paper drepanepteryx phalaenoides (linnaeus, 1758) devetak, 1991, 1992 micromus variegatus (fabricius, 1793) devetak, 1991, 1992 micromus paganus (linnaeus, 1767) devetak, 1991, 1992 micromus lanosus (zelený, 1962) saure, 1989; devetak, 1991, 1992 family sisyridae sisyra nigra (retzius, 1783) devetak, 1991, 1992 biologica nyssana ● 10 (1) september 2019: 35-41 devetak, jakšić ● lacewings (insecta: neuropterida: raphidioptera, megaloptera, neuroptera) collected in montenegro species / taxon literature records comment family coniopterygidae aleuropteryx juniperi ohm, 1968 devetak, 1991, 1992 helicoconis (ohmopteryx) pseudolutea ohm, 1965 devetak, 1991, 1992 coniopteryx (c.) borealis tjeder, 1930 devetak, 1991, 1992 coniopteryx (c.) pygmaea enderlein, 1906 devetak, 1991, 1992 coniopteryx (c.) tineiformis curtis, 1834 devetak, 1991, 1992 coniopteryx (metaconiopteryx) esbenpeterseni tjeder, 1930 saure, 1989; devetak, 1991, 1992 coniopteryx (metaconiopteryx) arcuata kis, 1965 devetak, 1991, 1992 parasemidalis (parasemidalis) fuscipennis (reuter, 1894) devetak, 1991, 1992 semidalis aleyrodiformis (stephens, 1836) saure, 1989; devetak, 1991, 1992 conwentzia pineticola enderlein, 1905 devetak, 1991, 1992 family dilaridae dilar turcicus hagen, 1858 devetak, 1991, 1992 family mantispidae mantispa styriaca (poda, 1761) devetak, 1991, 1992 family myrmeleontidae palpares libelluloides (linnaeus, 1764) devetak, 1992, 1996 this paper myrmecaelurus trigrammus (pallas, 1771) devetak, 1992 this paper myrmeleon formicarius linnaeus, 1767 devetak, 1991, 1992 myrmeleon inconspicuus rambur, 1842 devetak, 1992 this paper euroleon nostras (geoffroy in fourcroy, 1785) devetak, 1991, 1992 macronemurus bilineatus brauer, 1868 devetak, 1992, 1997 neuroleon (neuroleon) microstenus (mclachlan, 1898) devetak, 1992; devetak and devetak, 2004 distoleon tetragrammicus (fabricius, 1798) devetak, 1991, 1992 creoleon plumbeus (olivier, 1811) devetak, 1992 this paper gymnocnemia variegata (schneider, 1845) letardi and pantaleoni, 1996 family ascalaphidae deleproctophylla australis (fabricius, 1787) devetak, 1992, 1995; popov, 2004 libelloides lacteus (brullé, 1832) van der weele, 1908;táborský, 1936; devetak, 1992; popov, 2004 libelloides longicornis (linnaeus, 1764) this paper. first record in montenegro and in the balkan peninsula. libelloides macaronius (scopoli, 1763) táborský,, 1939; saure, 1989; devetak, 1991, 1992; popov, 2004; devetak and zeqiri, 2018 this paper 41 biologica nyssana ● 10 (1) september 2019: 35-41 devetak, jakšić ● lacewings (insecta: neuropterida: raphidioptera, megaloptera, neuroptera) collected in montenegro 42 naceri et al. 2025, biologica nyssana 16(1) 145 16 (1) june 2025: 145-155 doi: 10.46793/biolnyss.16.1.16n foliar sugar microdoses as a biocontrol approach against the tomato leaf miner tuta absoluta: a sustainable pest management strategy original article ardjouna naceri department of agronomy, institute of veterinary and agronomic sciences, university of batna 1, 05000 batna, algeria; laboratory of cellular and molecular physiotoxicology-biomolecules, faculty of science of nature and life, university of batna 2, 05000 batna, algeria imene brahim department of agronomy, institute of veterinary and agronomic sciences, university of batna 1, 05000 batna, algeria; lapapeza laboratory, department of agricultural sciences, institute of veterinary and agricultural sciences, university of batna 1, 05000 batna, algeria smail chafaa department of ecology and environment, university of batna 2, 05000 batna, algeria; laboratory of cellular and molecular physiotoxicology-biomolecules, faculty of science of nature and life, university of batna 2, 05000 batna, algeria s.chafaa@univ-batna2.dz (corresponding author) nabil berttela institute of veterinary and agronomic sciences, department of agronomy, university of batna 1, 05000 batna, algeria fateh mimeche department of agricultural sciences, university of m’sila, 28000 m’sila, algeria received: march 02, 2025 revised: april 06, 2025 accepted: april 08, 2025 abstract: tomato crops are highly susceptible to pests like tuta absoluta, and current chemical control is detrimental to human health, the environment, and leads to resistance development. this study explores biocontrol by applying microdoses of sugars (fructose and sucrose) to tomato leaves to induce resistance against tuta absoluta larvae. tomato plants of the saada variety received 100 ppm and 1000 ppm of sucrose, fructose, and their mixture. it was noted that the 1000 ppm sucrose-fructose association (applied one after another) provided initial protection during the first three weeks, while fructose alone and the mixture were better in the last three weeks. fructose at 1000 ppm and the sucrose-fructose association maintained the infestation rate similar to the control. the mixture also inhibited the density of l1, l2, and l4 larvae, though l3 larvae were not inhibited. these observations underscore the promise of sugar-based therapies as biodegradable biocontrol agents, providing a green alternative to chemical pesticides. key words: solanum lycopersicum, tuta absoluta, sugar, induced resistance, exogenous apport apstrakt: mikrodoze šećera primenjene na lišće kao biološki pristup suzbijanju minera paradajzovog lista tuta absoluta: održiva strategija upravljanja štetočinama. usevi paradajza su veoma podložni štetočinama kao što je tuta absoluta, a trenutno hemijsko suzbijanje šteti ljudskom zdravlju, životnoj sredini i dovodi do razvoja otpornosti. ova studija istražuje biološko suzbijanje primenom mikrodoza šećera (fruktoze i saharoze) na listove paradajza kako bi se indukovala otpornost na larve tuta absoluta. biljke paradajza sorte saada tretirane su sa 100 ppm i 1000 ppm saharoze, fruktoze i njihove mešavine. primećeno je da su saharoza i fruktoza primenjene zajedno (jedna za drugom) u koncentraciji od 1000 ppm obezbedile početnu zaštitu tokom prve tri nedelje, dok su čista fruktoza i mešavina bile efikasnije tokom poslednje tri nedelje. fruktoza u koncentraciji od 1000 ppm i mešavina održavale su stopu infestacije sličnu kontroli. mešavina je takođe smanjila gustinu larvi l1, l2 i l4 stadijuma, dok larve l3 stadijuma nisu bile inhibirane. ova zapažanja ukazuju na potencijal šećernih tretmana kao biorazgradivih sredstava za biološko suzbijanje, pružajući ekološki prihvatljivu alternativu hemijskim pesticidima. ključne reči: solanum lycopersicum, tuta absoluta, šećer, indukovana otpornost, egzogeni doprinos introduction the micro lepidopteran, south american tomato borer, tuta absoluta (meyrick, 1917) (lepidoptera: gelechiidae), recently renamed phthorimaea absoluta (chang & metz, 2021), has rapidly spread, invading several afro-eurasian countries (oepp, 2021). the first outbreaks in algeria were observed in the spring of 2008 in protected tomato crops in the mostaganem region. it was later detected in various coastal areas, including the western, central, and parts of the eastern coast (oepp, 2021). in recent years, this pest has severely affected tomato cultivation in greenhouses and open fields, with damage ranging © 2025 naceri et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 146 from 50% to 100%. the south american tomato borer is characterized by a high reproductive potential, with several overlapping generations per year, a strong dispersal capacity, and significant damage caused by the destruction of vegetative and reproductive organs by its larvae; damage can reach up to 100% in the absence of effective management methods. it has been classified as a serious economic threat to solanaceous crops and, to a lesser extent, a pest of other economically important solanaceous crops, including potatoes, eggplants, peppers, and tobacco (biondi et al., 2018; mansour et al., 2018; guedes et al., 2019; verheggen & fontus, 2019; sawadogo et al., 2020; ponti et al., 2021). the management of tuta absoluta is highly dependent on chemical pesticides (illakwahhi & srivastava, 2017; han et al., 2019), which present serious hazards to human health and environmental safety via bioamplification and bioconcentration (hassaan & el nemr, 2020). this has led to the overuse of pesticides, hence the development of resistance by the insect t. absoluta to several classes of pesticides, especially organophosphates and pyrethroids (silva et al., 2016; haddi et al., 2017; zibaee et al., 2018). given these challenges and the ongoing efforts to develop better intervention strategies while preserving the natural environment in an ecologically sustainable manner, the search for alternative pest control methods and products remains a priority. this pursuit is typically carried out within the phytopharmaceutical industry, which focuses on crop protection and enhancing plant defences against harmful organisms through the use of macro and microorganisms, chemical mediators, and natural substances of mineral, plant, bacterial, or animal origin (isman, 2006; mamy & barriuso, 2022). a large quantity of trials has been carried out in several countries such as france, italy, greece, and algeria, which have suggested that foliar application of sucrose or fructose in low doses can serve as an alternative to pesticides for plant defence (arnault et al., 2015; 2016; brahim, 2022). these sugars have acquired critical regulatory roles and are increasingly being discovered as plant signalling molecules, modulating gene expression for metabolism, stress tolerance, and development. this also leaves open the possibility of priming-a physiological process that conditions plants for a faster and/or more intense defence response to subsequent stress at zero cost in terms of the entire metabolic cost of an induced defence response (arnault et al., 2015; ceusters et al., 2017). this new concept, "sweet immunity" or sugar-mediated defence (arnault et al., 2015), relies on foliar application of low-dose sugars to induce plant resistance to biotic stressors. the sweet immunity mechanism operates in a variety of phases: preattack, through the enhancement of basal resistance; during initial host recognition, through leaf surface interactions; upon pathogen challenge, through the induction of defence pathways (excluding insect responses) (arnault et al., 2021). this has been confirmed by derridj et al. (2011) and lambion & marollien (2016), who demonstrated that the exogenous application of these phytohormones in sub-doses provided resistance in plants against insect oviposition. this resistance phenomenon is achieved through two mechanisms: a local effect in which the modification of the chemical composition on the leaf surface interferes with the oviposition behaviour of females, and the other, systemic effect, wherein the sugars initiate signalling pathways leading to changes in gene expression that provides a generalized effect in the stimulation of natural defences (morkunas & ratajczak, 2014; formela-luboińska et al., 2020). indeed, the application of sugars has been considerably beneficial in several cases: apple and pear/codling moth (cydia pomonella l.), vine/ downy mildew (plasmopora viticola schwein.), corn/eastern corn borer (ostrinia nubulalis hbn.), melon/powdery mildew (oidium neolycopersici l. kiss), leek/thrips (thrips tabaci linde.), and tomato/powdery mildew (o. neolycopersici l. kiss) (ferré et al., 2008; derridj et al., 2012; arnault et al., 2021). however, regarding the effects of sugars on the tomato/leaf miner model, the field outcomes were variable after trials from 2016 to 2018 (arnault et al., 2021). given that t. absoluta is considered the most destructive pest of tomatoes, the findings from these trials should be considered preliminary. this study tests the hypothesis that sucrose and fructose treatments at different concentrations (at 100 and 1000 ppm) applied alone, combined, or as mixtures will influence tuta absoluta infestation levels by interfering with larval development and oviposition under laboratory conditions. materials and methods preparation of tomato seedlings and infestation laboratory experiments were conducted using tomato seedlings var—saada, obtained from f1 hybrid seeds. seeds were sown in plug trays filled with potting soil. after one week of germination, seedlings with two well-developed true leaves were transplanted into pots and kept under laboratory conditions; the plants became naturally infested by t. absoluta. biologica nyssana ● 16 (1) june 2025: 145-155 naceri et al. ● foliar sugar microdoses as a biocontrol approach against the tomato leaf miner tuta absoluta: a sustainable pest management strategy 147 biologica nyssana ● 16 (1) june 2025: 145-155 naceri et al. ● foliar sugar microdoses as a biocontrol approach against the tomato leaf miner tuta absoluta: a sustainable pest management strategy experimental design the experiment followed a completely randomized block design with three replications per treatment. each replication consisted of a single tomato plant in a pot, totaling 21 pots arranged randomly. two sugars, fructose and sucrose (both from fluka biochemika), were tested under the following conditions: 1. individually at doses of 100 ppm and 1000 ppm, 2. in a mixture and in association, at a dose of 1000 ppm. for the mixing modality, sucrose and fructose were each applied at a concentration of 1000 ppm and immediately in the same container after mixing them. in the case of the association modality, the sugars were applied one after the other at the same dose (1000 ppm) each. foliar spraying following the methodology described by derridj et al. (2009, 2011, 2012), brahim et al. (2018), arnault et al. (2015, 2021), and brahim (2022), the treatments were initiated just after the first mine apparition. early in the morning (at 6 a.m.), the foliar sprays were applied prior to the start of photosynthesis and when the sugar levels of the apoplastic intercellular spaces were low, with the manual sprayer (5 l). according to arnault et al. (2021), the applications were carried out every 15 days, from may 15, 2023, to july 3, 2023. the total number of applications made during our experiment was four: may 15, 2023; may 29, 2023; june 12, 2023; and june 26, 2023. arnault et al. (2021) indicated that trials conducted in his work in 2016 revealed that foliar application of sugars alone at a concentration of 100 ppm provides minimal or no protection. consequently, the authors recommended a higher concentration of approximately 1000 ppm. the solution was applied through foliar spraying in a non-dripping v-shaped jet across the entire plant to facilitate the absorption of sugar. the volume of the solution was adjusted to support vegetative growth, ensuring proper coverage of the foliage. the sugar foliar spraying was applied to the entire plant until runoff to maximize efficacy. these six experimental treatments were compared to a control. sampling and parameters studied in this study, we evaluated the effect of treatments on the infestation rate by the leaf miners on tomato leaflets and on the abundance of larval stages by sampling plants within our experimental design. each week during the experiment, we carefully checked the plants to count, enumerate, and identify the following parameters: monitoring infestation rates: the rate of infestation caused by leaf miner larvae on leaves was monitored throughout the experimental period. leaflets evaluation: the total number of healthy and infested leaflets was counted. in particular, three levels of leaves: basal, median, and apical were considered to gain insight into the overall health of the plants and the intensity of infestation pressure. identification of biological stages: the various biological stages of the leaf miner, including larval instars and pupae were recorded and documented. to ensure accuracy, plants were carefully reexamined using a handheld magnifying glass. assessment of treatment effectiveness: abbott's formula was applied to evaluate the final effectiveness of the treatments. t0: % total of leaflets affected in the control treatment; tt: % total of leaflets affected in the treated treatment. statistical analyses all statistical analyses were conducted using the statistica 8 software. the corresponding means were calculated for each variable studied, such as the average percentage of overall infestation, infestation by leaf levels, and the abundance of larval stages. a comparison of means was conducted using anova. beforehand, the conditions for using this test were verified (normality of the distribution through a shapiro-wilk test). a fisher lsd post hoc test was applied to identify significant differences between groups, with a significance level set at p<0.05. results dynamics of the infestation caused by tuta absoluta on tomato leaflets during the eight weeks of observation, the collected data revealed that the larval infestations of t. absoluta on tomato plants exhibited temporal variations. the initial weeks were marked by relatively low infestation levels ranging from 0 to 14.94, followed by a gradual increase after the initial two weeks of treatment (from 14.94 to 34.43). more specifically, during the first three weeks, the combination of sucrose and fructose at a concentration of 1000 ppm each proved to be the least affected, with an infestation rate of only 1.29%. in contrast, all other modalities experienced more significant attacks, exceeding the reference rate of the control, which was 4.33% (fig. 1). by the fourth week, all the infections showed a slight increase. the “association” and “mixing” modalities showed efficiency=100x((t0-tt)/t0) 148 particularly high infestation rates, reaching 28.04% and 34.43%, respectively, compared to 18.41% in the control group. however, from that date until the end of the trial (july 3rd), infestation levels declined in plants treated with a combination of sucrose and fructose at 1000 ppm and those treated only with fructose at 1000 ppm. these treatments ended up being the least affected, with final damage levels of 50% and 54.36% respectively. in contrast, the treatment using fructose alone at 100 ppm showed a continued increase in infestation, resulting in a maximum rate of 81.69%. sucrose at 100 ppm resulted in an infestation rate of 64.38%, while sucrose at 1000 ppm exhibited 61.34%. fructose at 1000 ppm led to 54.36%, and the sucrose-fructose association yielded the highest rate at 69.2%. these values are relatively close to that of the control, which reached 56.84% (fig. 1). average infestation rate foliar sprays of fructose at 1000 ppm, whether applied alone or in combination with sucrose at 1000 ppm, as well as sucrose at 1000 ppm alone or in combination with fructose at 1000 ppm, resulted in remarkably similar average infestation rates. specifically, the rates were 25.53±6.69%, 25.36±6.21%, 27.69±7.26%, and 27.43±9.39%, respectively. these values did not differ significantly from those observed in the control group (p>0.982). in contrast, the application of sucrose or fructose at 100 ppm (33.12±10.20% and 30.47±8.40%, in order), when used alone, did not significantly differ from the control, which had an infestation rate of 22.89±8.13%. furthermore, no statistically significant differences were observed between the different treatment modalities (fig. 2). infestation rate by layer foliar application of fructose at 1000 ppm demonstrated a marked increase in leaflet protection, with infestation rates decreasing at the apical layer (7.11%±3.99 vs. 8.14%±4.45 for the control) but increasing substantially at the basal layer (44.76%±10.14 vs. 35.52%±10.74 for the control). this pattern was even more pronounced in the case of treatment with sucrose at 1000 ppm alone (9.00%±4.93), where infestation levels at the apical layer were comparable to those of the control, but significantly higher infestations were recorded at the basal level (tab. 1). while basal infestation reached biologica nyssana ● 16 (1) june 2025: 145-155 naceri et al. ● foliar sugar microdoses as a biocontrol approach against the tomato leaf miner tuta absoluta: a sustainable pest management strategy fig. 1. evolution of the infestation rate on tomato leaflets 149 biologica nyssana ● 16 (1) june 2025: 145-155 naceri et al. ● foliar sugar microdoses as a biocontrol approach against the tomato leaf miner tuta absoluta: a sustainable pest management strategy fig. 2. average total infestation rate caused by the tuta absoluta larvae on tomato leaflets. anova, fisher's lsd tests, p<0.05; different letters show significant differences between treatments within each experiment table 1. average rate of vertical infestation modalities basal layer medium layer apical layer control 35.52%±10.74a 28.38%±9.80ab 8.14%±4.45b* sucrose (100 ppm) 42.73%±11.35a 35.74%±8.58a 17.12%±8.85a sucrose (1000 ppm) 39.05%±11.29a 38.47%±7.28a 9.00%±4.93b** fructose (100 ppm) 32.16%±11.90a 39.77%±8.88a 26.76%±10.49a fructose (1000 ppm) 43.24%±11.04a 31.53%±7.27a 7.11%±3.99b** mix (1000 ppm) 44.76%±10.14a 31.09%±7.26a 2.85%±1.57b** association (1000 ppm) 37.04%±12.92a 34.07%±10.86a 13.40%±5.80a 39.05%± 11.29 (vs. 35.52%± 10.74 for the control), in the case of the intermediate layer, only the fructose treatments applied at 1000 ppm (whether applied alone or in combination with sucrose at 1000 ppm), resulted in average infestation rates similar to the control (28.38%±9.80), with values of 31.53%±7.27 and 31.09%±7.26, respectively (tab. 1). the statistical analyses revealed significant, and in some cases, highly significant (p<0.01) differences among the various leaf levels examined across the seven tested conditions. however, the apical level showed significant differences in the control group and highly significant differences for the treatments with sucrose at 1000 ppm, fructose at 1000 ppm, and the mixture at 1000 ppm, compared to the other levels studied. anova, fisher's lsd tests, p<0.05; different letters show significant differences between treatments within each experiment.**: p≤0.01 and*: p<0.05 150 biologica nyssana ● 16 (1) june 2025: 145-155 naceri et al. ● foliar sugar microdoses as a biocontrol approach against the tomato leaf miner tuta absoluta: a sustainable pest management strategy table 2. effects of low-doses sugar spraying on the abundance of larval instar. anova, fisher's lsd tests, p<0.05; different letters show significant differences between treatments within each experiment. *: p<0.05 modalities l1 l2 l3 l4 pupae control 0.37%±0.26a 0.58%±0.35a 0.33%±0.29a 1.33%±1.04a 0.13%±0.13a sucrose (100 ppm) 0.58%±0.30a 0.08%±0.08a 0.62%±0.35a 1.00%±0.91a 0.00a sucrose (1000 ppm) 0.28%±0.13ab 0.83%±0.53ab 0.58%±0.36ab 1.96%±1.32a 0.00b* fructose (100 ppm) 0.20%±0.14a 0.50%±0.46a 1.13%±0.79a 2.00%±1.48a 0.04%±0.04a fructose (1000 ppm) 0.33%±0.21ab 0.29%±0.16b* 0.62%±0.41ab 2.00%±1.24a 0.08%±0.05b* mix (1000 ppm) 0.21%±0.11a 0.25%±0.25a 0.87%±0.83a 0.54%±0.50a 0.00a association (1000 ppm) 0.45%±0.30ab 0.71%±0.42ab 0.54%±0.23ab 2.75%±1.91a 0.12%±0.06b* anova, fisher's lsd tests, p<0.05; different letters show significant differences between treatments within each experiment.*: p<0.05 abundance of larval instar and pupae according to tab. 2, almost all larval instars were present, though in variable abundance. an analysis of the average number of individuals at each developmental instar revealed that in the l1 instar, the lowest abundance of l1 larvae was observed when fructose alone was applied at 100 ppm, followed by application of mixture with respective values of 0.20±0.14 and 0.21±0.11.. in contrast, the highest abundance occurred after foliar spraying of sucrose alone at 100 ppm. in the case of l2 instar, the lowest l2 abundance was recorded in the sucrose at 100 ppm treatment (0.08±0.08), followed by fructose at 1000 ppm (0.29±0.16), and the mixture of the two sugars at 1000 ppm (0.25±0.25). on the other hand, the maximum average number of l2 was recorded in plants sprayed with sucrose alone at 1000 ppm (0.83±0.53), as well as in the association treatment at 1000 ppm. considering the l3 instar, all the treatments exhibited higher abundances than the control's (0.33±0.29), reaching a maximum of 0.87±0.8 in the sucrose–fructose mixture at 1000 ppm, but no statistically significant differences were observed. finally, in the l4 instar, the abundance was relatively low in the mixed treatment (0.54±0.50), while the other treatments showed l4 abundances that either exceeded or were close to those of the control. in the pupal stage, the average number of pupae forms was either zero or lower than that of the control for all treatments. statistical analysis of larval instar abundance revealed significant differences in specific treatments, particularly those with sucrose at 1000 ppm, fructose at 1000 ppm, and their combination at 1000 ppm. these differences allowed for the classification of treatments into three distinct statistical groups: "a," "b," and "ab." abbott efficacy of treatments based on the results of our study across different treatment modalities, we observed that at the apical level, the application of fructose at 1000 ppm, when applied alone, as well as in combination with sucrose at 1000 ppm, reduced infestation rate to 7.11% and 2.85%, respectively, compared to the control group, which exhibited an infestation rate of 8.14%. these reductions correspond to abbott efficacy gains of 12.65% and 64.98%. regarding the basal level, fructose used alone at 100 ppm decreases the infestation rate by 32.16% compared to the control modality, which has an infestation rate of 35.52%, resulting in an abbott efficacy of 9.45%. discussion several studies have actually proved that sugars such as sucrose and fructose, currently approved substances for controlling pests like the codling moth in apples and pears and the european corn borer can effectively enhance plant resistance. these sugars are believed to disrupt oviposition behaviour by modifying biochemical signals present on the leaf surface (arnault et al., 2016; brahim et al., 2018; arnault et al., 2021; brahim, 2022). our study demonstrated their vertical distribution efficacy vs. t. absoluta on tomatoes, highlighting apical leaf targeting as a novel strategy for sustainable management. however, this induced resistance may depend on various factors, including the type and concentration of sugar used, the developmental stage of the plant, the combination of sugars with other phytosanitary 151 biologica nyssana ● 16 (1) june 2025: 145-155 naceri et al. ● foliar sugar microdoses as a biocontrol approach against the tomato leaf miner tuta absoluta: a sustainable pest management strategy products, the timing of application, and the level of pest pressure or infestation (ferré et al., 2008; derridj et al., 2011; arnault et al., 2015; lambion & marollien, 2016; arnault et al., 2021; brahim, 2022). regarding the temporal progression of infestation, our results showed that during the first three weeks, the treatment consisting of a mixture of sucrose and fructose at 1000 ppm maintained the infestation level lower compared to those observed in the control group. the grab experiment on apples in 2017 (lambion & arnault, 2019), which tested the mixture of sucrose and fructose at 1000 ppm under field conditions, found that they provided moderate protection to fruits, particularly toward the end of the season. our controlled trials on tomatoes revealed a vertical stratification of efficacy, likely due to host-specific leaf traits (e.g., trichome density) and cation gradients (tiffrent, 2023). in a related study on maize, using three different sugars (glucose, sucrose, and fructose) at varying concentrations to mitigate borer-related injury, it was revealed that each elicited specific effects. for example, the application of sucrose at a concentration of 100 ppm was capable of reducing the development of botrytis on tomatoes, while the application of sucrose at the same concentration was found to be less effective against botrytis on beans (ondet, 2010). these findings emphasize that each phytosanitary bio-aggressor requires a specific sugar and dosage for optimal effectiveness. in general, our tests showed that sugarbased treatments did not significantly reduce the average total infestation rates on tomato leaflets independently of the modality tested. however, a vertical distribution effect was observed. at the basal level, fructose application at a concentration of 100 ppm caused a 9.45% reduction in the attack rate of leaf miner larvae, as measured by abbott efficacy. however, no statistically significant differences were found compared to the control. at the median level, all treatment modalities exhibited relatively uniform infestation rates, with no significant differences and generally lower values compared to the basal level. on the other hand, at the apical level, foliar application of fructose at 1000 ppm and the mixture treatment of sucrose and fructose at 1000 ppm showed a protective effect on leaflets, achieving the lowest infestation rates with abbott efficacy values of 12.65% and 64.98%, respectively. conversely, at the basal level, these same treatments were not effective. contrary to what has been demonstrated, female t. absoluta prefer the leaves of the apical part with lower calcium content for laying eggs rather than other parts of the tomato plant (leite et al., 1999; leite et al., 2004; proffit et al., 2011; cherif & verheggen, 2019). this contradiction could be explained by the effect of changes in the nutritional status of the leaves, particularly on the activity of the cations present on the leaf surface. in fact, tiffrent (2023) has demonstrated that spraying glucose and fructose on apple trees increases the concentration of calcium present on the surface of apple leaves. the apical efficacy of sugars aligns with t. absoluta’s preference for low-calcium leaves (leite et al., 2004), but our results suggest that sugars alter cation availability (e.g., ca2+) on treated organs (tiffrent, 2023). basal leaves, with thicker cuticles and lower sugar uptake, may resist this modulation. similarly, derridj and wu (1996) demonstrated that the corn borer lays its eggs on the leaf surfaces and levels of corn that are lowest in cations. also, the variability observed across leaf levels may be attributed to differences in the developmental stage of the leaves and the severity of pest pressure. indeed, apical leaflets, being younger, possess distinct morphological and chemical characteristics, such as the presence of trichomes and waxes, which influence the attraction or repulsion of biotic stressors compared to those of older layers. ondet (2010) demonstrated that sucrose application on maize at a very early stage reduces the number of egg deposits by the corn borer. conversely, when the plant reaches the reproductive stage, this same foliar application increases the number of eggs laid by the corn borer. lambion et al. (2018) reported through trials conducted in 2017 that the addition of fructose and sucrose to bacillus thuringiensis berliner at a concentration of 1 g/l enhanced its efficacy against t. absoluta under conditions of late and moderate attack. fiala et al. (1993) proposed that the carbohydrate content in the phylloplane is correlated with that of foliar tissues, with monosaccharides appearing at the junctions of epidermal cells above the apoplastic regions. the adult lepidopteran insect ostrinia nubilalis is sensitive to and responds to the detected sugar concentrations. these sugars are known to influence female host plant selection for oviposition, with fructose being the sugar most closely associated with female preference in host plant choice. according to ondet (2018), and following the experiments conducted within the framework of the usage (2012-2015) and sweet (2016-2019) projects aimed at controlling the apple codling moth, the efficacy of a mixture of fructose and sucrose at a concentration of 100 ppm was validated under conditions of pest pressure. similarly, the study conducted by brahim (2022) on the codling moth in algeria supports these findings. foliar sprays based on sugars also led to significant variations in the abundance of the 152 biologica nyssana ● 16 (1) june 2025: 145-155 naceri et al. ● foliar sugar microdoses as a biocontrol approach against the tomato leaf miner tuta absoluta: a sustainable pest management strategy different developmental stages of t. absoluta. analysis of the tested treatment modalities revealed notable differences in larval instar abundance. the association treatment at 1000 ppm proved particularly effective, showing a significant decrease in the abundance of the larval instars l1, l2, and l4 compared to the control and other treatments. these results align with field trials conducted between 2006 and 2008 on apple trees by anadiag, innophyt, and itcf, which evaluated the effectiveness of sucrose in combination with granulosis virus and in addition to classical phytosanitary products like imidan 50 wp. fructose alone at 100 ppm was only effective in reducing the abundance of l1, while sucrose sprayed alone at 100 ppm reduced the abundance of l2. these results support the idea that sugar-based treatments could be strategically applied to target the most vulnerable larval stages, particularly neonatal larvae, thereby optimizing plant protection timing (ferré et al., 2008; ondet, 2010). this is likely due to the very low number of chrysalises observed on the leaflets, which can be attributed to the generally low pupation rate of fourth-instar larvae on leaves. in fact, most pupation activity typically occurs in the soil. these findings are consistent with those reported by barrientos et al. (1998) and pereyra (2006), who found that pupation of the tomato leaf miner primarily takes place in the soil. the reduction in l1, l2, and l4 levels suggests that neonate larval development is interfered with, either through nutrient dilution (spann & schumann, 2010) or through induction of lignin/cellulose deposition (tiffrent, 2023). these biochemical alterations must be measured in the future. in summary, though sugars may boost plant immunity to certain herbivores and pathogens, they may also act as oviposition disruptors by altering leaf surface biochemical cues. this could also raise interest regarding the feeding behavior of larvae in the case of the tomato/t. absoluta pair. in the field, we recommended 1000 ppm sucrose and fructose mixtures applied during the vegetative stage on apical leaves where t. absoluta oviposition is the highest. the integration of sugars with b. thuringiensis in ipm programs could potentially improve larval control at the cost of pesticides. critical knowledge gaps are the effect of repeated sugar sprays on plant physiology (e.g., photosynthesis) and compatibility with t. absoluta natural enemies (e.g., trichogramma spp.). these findings make sugar sprays a sustainable control option for t. absoluta, as they are precise and have low environmental persistence. future research directions include optimal application timing and testing field-scale efficacy against fluctuating pest pressure. conclusion in this study, our findings demonstrate that even micro-doses of sugar, specifically a combination of sucrose and fructose at 1000 ppm each, can evoke a synergistic effect, resulting in a reduction in infestation levels by the end of the experiment. however, this effect provides only limited protection against leaf miner larvae. under field conditions, the efficacy of such treatments remains too low to justify their practical use as a standalone control method. therefore, we strongly discourage the use of exogenous sugar treatments as the sole strategy for plant protection. a more effective approach would involve combining sugar applications with other phytosanitary products, whether biological or chemical. more broadly, given the growing need to reduce pesticide use, we suggest that sugars could be integrated into pest management programs as long as all interacting factors, application timings, and environmental conditions are carefully considered to optimize their use within an integrated pest management framework. references abbott, w.s. 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(chenopodiaceae). biologica nyssana, 6 (1), september 2015: 11-16. environmental effects on morphological and physiological properties of offspring which occurs during development of mother plant are called maternal environmental effects. photoperiod is one of the crucial environmental factors according to which plants modify numerous physiological processes. maternal effect of photoperiod in a short day plant chenopodium rubrum extends through the whole life cycle of offspring and persist to the second generation, photoperiod during induction and evocation of flowering of mother plants showing the key influence. here we show that also non-inductive photoperiod preceding flowering induction of mother plants shows its maternal effect on offspring properties: seed size, seed germination and seed protein composition. presented data argues in favor of earlier suggested that relative amounts of seed proteins represent an “archive“ of photoperiods experienced by mother plants during their lives. key words: chenopodium rubrum, maternal effect of photoperiod, seed proteins, seed weight, seed germination apstrakt: mitrović, a., bogdanović pristov, j.: materinski efekat neprekidne svetlosti na karakteristike semena kratkodnevne biljke chenopodium rubrum l. (chenopodiaceae). biologica nyssana, 6 (1), septembar 2015: 11-16. faktori spoljašnje sredine koji utiču na morfološke i fiziološke karakteristike potomstva nazivaju se materinski efekti spoljašnje sredine. fotoperiod je jedan od ključnih faktora spoljašnje sredine prema kojem biljke modifikuju mnogobrojne fiziološke procese. materinski efekat fotoperioda proteže se kroz čitav životni ciklus potomstva i održava se i u sledećoj generaciji, pri čemu ključni uticaj ima fotoperiod kome su majke biljke bile izložene tokom indukcije i evokacije cvetanja. ovde pokazujemo da i neindukcioni fotoperiod koji prethodi indukciji cvetanja materinskih biljaka pokazuje materinski efekat na potomstvo: veličinu semena, klijanje semena i sastav proteina semena. prikazani rezultati govore u prilog ranije predloženog: relativna količina proteina semena predstavlja “arhivu” fotoperioda koju je majka biljka iskusila tokom svog životnog ciklusa. key words: chenopodium rubrum, materinski efekat fotoperioda, proteini semena, masa semena, klijanje semena 6 (1) • september 2015: 11-16 biologica nyssana 6 (1)  september 2015: 11-16 mitrović, a.. bogdanović pristov, j.  maternal effect of continuous light … 12 introduction annual plants experience a single environment during their life cycle, but they could be exposed to different environmental conditions through environmental changes. environmental effects on morphological and physiological properties of offspring which occurs during development of mother plant are called maternal environmental effects (g u t t e r m a n & e v e n a r i , 1972; g a l l o w a y , 2005). their expression depends on the offspring environment, they are expressed throughout the life cycle of the offspring and may persist for several generations. maternal environmental effects could be provoked by different environmental factors such as soil nutrients (s t r a t o n , 1989), temperature (l a c e y et al., 1997), photoperiod (c o o k , 1975; g u t t e r m a n , 1978; b e r t t e r o et al., 1999; m i t r o v i ć et al., 2010), co2 levels (s t e i n g e r et al., 2000). the day length (night length), i.e. photoperiod, is one of the most crucial environmental factors according to which plants modify numerous physiological processes, the transition to flowering being one of the most important turning points in mother plant life cycle in order to provide the success of its offspring. in species with strong maternal effects, maternal plants could be grown under appropriate environmental conditions to promote successful establishment of new populations. for example, in the case of assisted dispersal to habitats with longer day length, maternal plants can be grown under the appropriate day length so maternal effects are adaptive (s c h u l e r & o r r o c k , 2012). on the other hand, if the seeds disperse into a different habitat, maternal environmental effects may reduce the offspring fitness, but only for a single generation, since the response to the new habitat will induce new maternal effects (g a l l o w a y , 2005). chenopodium rubrum l. sel. 184 is a qualitative short day (sd) weedy annual (c u m m i n g , 1967) with well defined photoperiodic sensitivity. it is sensitive to the small changes in day length, with defined critical night length of 8 h (t s u c h i a & i s h i g u r i , 1981). c. rubrum is sensitive to photoperiodic stimulus for flowering as early as at cotyledonary stage (s e i d l o v á & o p a t r n á , 1978), when 6 adequate photoperiodic cycles are sufficient for photoperiodic flower induction. it is an early flowering species (c u m m i n g , 1967) which makes it a suitable model plant for studies of ontogenesis. under the suitable photoperiodic conditions in vitro it produces seeds in 10 weeks (m i t r o v i ć et al., 2007). c. rubrum plants modify their vegetative and reproductive development, in accordance with the photoperiod they are exposed to (c o o k , 1975; m i t r o v i ć et al., 2007). its response to photoperiod extends to the offspring and persists to the second generation, the suggested mechanism of maternal effect of photoperiod being through seed protein composition (m i t r o v i ć et al., 2010). in c. rubrum, as a sd plant able to receive photoperiodic flowering induction as early as at about 5 days of age, most of the data concerning maternal effect of photoperiod on offspring deal with photoperiods inductive for flowering and its alterations on the plants of the same age and early during their life (c o o k , 1975; m i t r o v i ć et al., 2010). hence, maternal effect of different photoperiods inductive for flowering on different phases of offspring ontogenesis (m i t r o v i ć et al., 2010) is defined. c. rubrum seed number and seed weight is determined by photoperiod mother plants are exposed to during induction and evocation of flowering (c o o k , 1975; m i t r o v i ć et al., 2007). seed germination and offspring growth is determined by photoperiod during flowering induction of mother plants, offspring flowering and seed maturation is determined by photoperiod their mothers experienced during induction and evocation of flowering, while photoperiod after the evocation of flowering of mother plants does not affect offspring ontogenesis significantly (m i t r o v i ć et al., 2010). on the other hand, in nature, c. rubrum plants flowers when summer days become shorter. this means that they receive photoperiodic induction for flowering later during their life and thus under the longest photoperiods physiologically possible (c o o k , 1975). in other words, in nature, transition from vegetative to reproductive development (flowering induction) occurs when night length becomes longer than its critical night length of 8 h (t s u c h i a & i s h i g u r i , 1981). in this regard, the information about timing in offspring life when photoperiod inductive for flowering is to be expected could be of great importance for offspring success. therefore, it is to be expected that besides photoperiod during induction and evocation of flowering of mother plants, also non-inductive photoperiod preceding flowering induction of mother plants and its duration shows its (maternal) effect on offspring. c u m m i n g (1967) observed significant decrease in seed size if sd plants from genus chenopodium were grown under non-inductive photoperiod preceding flowering induction compared to those grown continuously under inductive short days. but there is no more precise data of the effect of noninductive photoperiod preceding flowering induction on offspring properties. biologica nyssana 6 (1)  september 2015: 11-16 mitrović, a.. bogdanović pristov, j.  maternal effect of continuous light … 13 here we present preliminary data on maternal effect of non-inductive photoperiod preceding flowering induction on seed properties: seed size, seed germination and variation in seed protein pattern. in vitro culture of intact plants was selected because it enables precise control over environmental factors, as it was shown that other environmental factors, such as temperature, significantly affects maternal effect of photoperiod on different chenopodium species (b e r t e r o et al., 1999; m i t r o v i ć et al., 2007). as non-inductive photoperiod for obligatory sd plant c. rubrum, continuous light (cl) was selected as extreme long day (c u m m i n g , 1967; m i t r o v i ć et al., 2007). the duration of non-inductive photoperiod preceding flowering induction was chosen to be longer than the period necessary for c. rubrum plants to reach full flowering under inductive 14 h /10 h photoperiod in vitro (ž i v a n o v i ć et al., 1995). finally for photoperiod inductive for flowering 14 h /10 h was used, established as inductive in c. rubrum flowering research (k r e k u l e & s e i d l o v a , 1976; s e i d l o v á & o p a t r n á , 1978; ž i v a n o v i ć et al., 1995). material and methods plants in vitro intact c. rubrum plants were grown in vitro on ms (m u r a s h i g e & s k o o g , 1962) medium, as described in m i t r o v i ć et al. (2007). seedlings, 5 days old, were exposed to two different photoperiodic treatments: 65 days of a 14 h/10 h photoperiod or 17 days of continuous light followed by 43 days of 14 h/10 h (m i t r o v i ć et al., 2008). irradiance was about 70 μmol m-2 s-1. temperature in the growth chambers was 25 ± 2 ºc. seed collection matured seed were collected, dried for 1 month at room temperature, measured (4 replicates of 100 seeds) and capped on +8c until use for determination of seed protein content, for separation of seed proteins on sodium dodecylsulfate polyacrylamide gel electrophoresis (page), and for testing the effect of maternal photoperiod on germination. seed extraction samples of 0.03 g of dry seeds were imbibed 2.5 h in darkness at 32 c, and powdered in liquid nitrogen. proteins were extracted for 30 min at 4 ºc with 0.5 ml 0.05 m tris buffer (ph 7.4) containing 0.25 m sucrose and 1 mm edta, and centrifuged (4 ºc, 10000 × g 10 min). seed protein concentration protein concentration in seed samples derived from mother plants grown under different photoperiodic conditions was determined by b r a d f o r d (1976) method with bovine serum albumin as the standard. separation of seed proteins protein separation, in seed samples derived from mother plants grown under different photoperiodic conditions, was performed on sodium dodecilsulfate poliacrilamide gel electrophoresis (sds-page) (l a e m m l i , 1970). polyacrylamide gel electrophoresis was carried out under non/denaturing conditions in gels containing 10% polyacrylamide with a 4% stacking gel. a constant current of 25 (15) ma per gel was applied. equal volumes of all samples were loaded into the gels. proteins were visualized by coomassie blue staining. for sds-page separation all samples were run in triplicate. relative values of seed protein band intensities were determined in image master totallab 1.11. seed germination seeds were sown on moistened filter paper (5 ml distilled water) in petri dishes. germination was tested during 4 days (24 h dark at 32 °c, 24h dark at 10 °c and 48 h white light at 32 °c). every 24 h, 4 replicates of 100 seeds per treatment of mother plant were scored for germination. as a criterion of germination, radical protrusion by more than 2 mm was used. results and discussion for an obligatory sd plant, such as c. rubrum, extreme long day (cl) is non-inductive photoperiod, under which it grows continuously vegetative (c u m m i n g , 1967; m i t r o v i ć et al., 2007). analyzing literature concerning the effect of cl on plant growth and development s y s o e v a et al. (2010) showed that there are reports of cl both increasing plant developmental rate and inhibiting it. plant developmental response to cl depends on many factors including photoperiodic sensitivity, developmental stage and environmental conditions. in most long-day plants cl accelerated the reproductive cycle, while sdps responded to cl differently. on the other hand growing plants under cl is a way of economical crop production. it also provides better understanding of plant adaptations to the arctic polar 24 h photoperiod (s y s o e v a et al., 2010). biologica nyssana 6 (1)  september 2015: 11-16 mitrović, a.. bogdanović pristov, j.  maternal effect of continuous light … 14 maternal effect of non-inductive extreme long days, cl, preceding c. rubrum flowering induction on seed weight in previous work (m i t r o v i ć & b o g d a n o v i ć , 2008), we showed that about 5 times more seeds were collected from plants in which 17 non-inductive extreme long days (cl) preceded flowering induction by 14 h /10 h photoperiod, compared to those grown continuously under inductive 14 h /10 h photoperiod (m i t r o v i ć & b o g d a n o v i ć , 2008). as opposed, seed weight is for one third lower if collected from mother plants in which cl preceded flowering induction (fig. 1a). hence, we show significant difference in weight of seeds collected from mother plants induced for flowering under the same 14 h /10 h photoperiod, but with the time span of 17 days in their life cycle. this confirms c u m m i n g (1967) observation that sd plants from the genus chenopodium, grown under long days or cl before flowering induction, produces smaller seeds compared to seeds collected from plants grown continuously under the short days. on the other hand, on the basis of the experiments in which c. rubrum mother plants were grown continuously under different inductive photoperiods, it was shown that seed number and seed weight is determined by photoperiod mother plants experienced during induction and evocation of flowering (c o o k , 1975; m i t r o v i ć et al., 2007). from the abovementioned arises that seed size and weight is determined not only by the photoperiod under which induction and evocation of fig. 1. maternal effect of 17 days of continuous light (cl), extreme non-inductive photoperiod, preceding flowering induction on offspring properties in a qualitative short day plant chenopodium rubrum on: a) seed weight, b) seed germination, c) total seed protein content, d) width and intensity of seed protein bands (coomassie blue stained sds-page gel, rf values are marked for seed protein bands for which the difference in width and intensity are noticed between seed samples collected from mother plants grown continuously under inductive 14 h /10 h photoperiod and those in which 17 days of non-inductive cl preceded flowering induction by 14 h /10 h photoperiod), and e) relative intensities of seed protein bands (determined by image master totallab 1.11) biologica nyssana 6 (1)  september 2015: 11-16 mitrović, a.. bogdanović pristov, j.  maternal effect of continuous light … 15 flowering of mother plants occurred (c o o k 1975; m i t r o v i ć et al., 2007), it is also significantly affected by non-inductive photoperiod preceding flowering induction of mother plants. maternal effect of non-inductive extreme long days, cl, preceding c. rubrum flowering induction on seed germination fig. 1b shows that cl preceding flowering induction of mother plants also affect seed germination. germination of small seeds collected from mother plants in which 17 days of cl preceded flowering induction, is delayed and synchronized compared to germination of seeds collected from mother plants grown continuously under inductive 14 h /10 h photoperiod (fig. 1b). also, c u m m i n g (1967) showed that in small seeds, obtained from mother plants grown under non-inductive long days or cl before flowering induction were more dormant, the delay in germination happens due to thicker seed integument. at the same time, germination of seeds collected from mother plants grown continuously under different inductive photoperiods was determined by photoperiod experienced during flowering induction of mother plants (m i t r o v i ć et al., 2010). so, the same as seed size and weight, seed germination is, as well, determined not only by the photoperiod under which induction and evocation of flowering of mother plants occurred, but also is significantly affected by photoperiod preceding flowering induction of mother plants. maternal effect of non-inductive extreme long days, cl, preceding c. rubrum flowering induction, on seed proteins although seed storage proteins in chenopodium species are localized mostly in endosperm and embryo (p r e g o et al., 1998), while the difference in seed size are due to proportional difference in both the size of the embryo and endosperm in (c o o k , 1975), protein content (fig. 1c) was nearly twice higher in smaller seeds (fig. 1a) collected from mother plants in witch flowering induction was preceded by 17 days of cl. we suggested earlier that the mechanism of maternal effect of photoperiod can be through relative seed protein composition representing an “archive“ of photoperiods experienced by mother plants during their lives (m i t r o v i ć et al., 2010). b h a r g a v a et al. (2005) showed the difference in number, width and intensity of seed protein bands on sds-page gel, not only in seeds samples of different chenopodium species, but also in seed samples of the same species collected from widely separated localities (characterized by highly variable environmental factors). fig. 1d shows the presence of 33 protein bands on sds-page gel in both c. rubrum seed samples. the same number of protein bands was previously obtained (m i t r o v i ć et al., 2010) in seed samples collected from mother plants grown continuously under different inductive photoperiods in vitro. this confirmes that the difference in quantities of those 33 seed protein bands is only the result of different photoperiods mother plants were exposed to during their life cycle, as grown under precisely controlled environment in vitro. for 6 of them (fig. 1e), significant differences in width and intensity were noticed. protein bands with rf values 0.58, 0.64, 0.66, 0.80 and 0.83 showed about twice higher relative intensities in small seeds collected from mother plants in which flowering induction was preceded by 17 days of cl (fig. 1e). hence, in seeds collected from mother plants in witch flowering induction was preceded by cl, higher protein content (fig. 1c) is a result of increased amounts of those protein bands (with rf values 0.58, 0.64, 0.66, 0.80 and 0.83). at the same time, for those protein bands we showed earlier high correlations with the day length mother plants experienced during induction and evocation of flowering (m i t r o v i ć et al., 2010). therefore, this is the additional confirmation that relative amounts of those specific seed proteins are dependent on photoperiods experienced by mother plants during their lives. conclusion c. rubrum is a species with strong maternal effects. besides photoperiod during induction and evocation of flowering of mother plants (m i t r o v i ć et al., 2010), here we show that also non-inductive photoperiod preceding flowering induction of mother plants shows its maternal effect on offspring properties. maternal effect of noninductive, extreme long days, cl, preceding flowering induction of c. rubrum mother plants results in lowering seed size, enhancing seed dormancy, and increase in the amounts of some specific seed proteins. this goes in favor of earlier suggested that seed proteins represent an “archive“ of photoperiods experienced by mother plants during their lives. detailed analysis of altering noninductive photoperiods and its duration preceding c. rubrum flowering induction, as well as identification of seed proteins we found dependant on photoperiods experienced by mother plants, would contribute understanding the mechanism of maternal environmental effects. biologica nyssana 6 (1)  september 2015: 11-16 mitrović, a.. bogdanović pristov, j.  maternal effect of continuous light … 16 acknowledgements. this work was financed by the grants on173017 and iii43010 from the ministry of the 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2010: plants under continuous light: a review. plant stress, 4: 5-17. tsuchiya, t., ishiguri, y. 1981: role of the quality of light in the photoperiodic flowering response in four latitudinal ecotypes of chenopodium rubrum l. plant and cell physiology, 22: 525532. živanović, b., ćulafić, lj., filipović, a. 1995: the effects of hormones and saccharides on growth and flowering of green and herbicides-treated chenopodium rubrum l. plants. biologia plantarum, 37: 257-264. karaman et al. 2023, biologica nyssana 14(2) 14 (2) december 2023: 75-95 doi: 10.5281/zenodo.10203557 biodiversity of fungal species from tara mountain (serbia) original article maja karaman university of novi sad, faculty of sciences, department of biology and ecology, trg dositeja obradovića 2, 21000 novi sad, serbia maja.karaman@dbe.uns.ac.rs (corresponding author) ivan dudaš university of novi sad, faculty of sciences, department of biology and ecology, trg dositeja obradovića 2, 21000 novi sad, serbia eleonora čapelja university of novi sad, faculty of sciences, department of biology and ecology, trg dositeja obradovića 2, 21000 novi sad, serbia milana rakić university of novi sad, faculty of sciences, department of biology and ecology, trg dositeja obradovića 2, 21000 novi sad, serbia stevan baluban mycological society „novi sad”, bulevar mihajla pupina 26 i/3, 21000 novi sad, serbia miroslav marković university of novi sad, institute of lowland forestry and environment, antona čehova 13 d, novi sad, serbia ivana jovanović institute for nature conservation, japanska 35, 11070 new belgrade, serbia received: january 13, 2023 revised: may 29, 2023 accepted: september 22, 2023 abstract: the republic of serbia is one of the few european countries without a national database of recorded fungal species, so documenting and publishing data on fungal diversity is of outmost importance for our country. the presence of fungal specimens of tara mountain was examined in the following localities: mitrovac, predov krst, kaludjerske bare and zaovine at each site in summer and autumn during 2021 and 2022. a total of 320 taxa of fungi were recorded during this period, 10 of them are included in the serbian list of protected wild species of fungi: boletus edulis, b. reticulatus, cantharellus cibarius, craterellus cornucopioides, hydnum repandum, lactarius deliciosus, l. deterrimus, l. salmonicolor, marasmius oreades and russula cyanoxantha, while three species: hericium flagellum, h. coralloides and psilocybe serbica are listed in the serbian list of strictly protected wild specis of fungi. also, 38 species are indentified as an indicator species of old and preserved forests. the highest species diversity was founded in mitrovac. key words: mycological diversity, national park tara, fungi, indicator species apstrakt: biodiverzitet gljiva planine tare (srbija) republika srbija je jedna od retkih evropskih zemalja koja nema nacionalnu bazu podataka o evidentiranim vrstama gljiva, pa je dokumentovanje i objavljivanje podataka o raznovrsnosti gljiva od izuzetnog značaja za našu zemlju. istraživano je prisustvo plodnih tela gljiva planine tare u leto i jesen tokom 2021. i 2022. godine na sledećim lokalitetima: mitrovac, predov krst, kaluđerske bare i zaovine. u ovom periodu evidentirano je ukupno 320 taksona gljiva, od kojih je 10 na listi zaštićenih divljih vrsta gljiva u srbiji: boletus edulis, b. reticulatus, cantharellus cibarius, craterellus cornucopioides, hydnum repandum, lactarius deliciosus, l. deterrimus, l. salmonicolor, marasmius oreades, dok su vrste: hericium flagellum, h. coralloides i psilocybe serbica navedene u listi strogo zaštićenih divljih vrsta gljiva srbije. takođe, 38 vrsta je identifikovano kao indikatorske vrste starih i očuvanih šuma. najveći diverzitet vrsta zabeležen je na lokalitetu mitrovac. ključne reči: mikološki diverzitet, nacionalni park tara, gljive, indikatorske vrste introduction forests are among the most important terrestrial ecosystems, considering that they are the speciesrichest and are vital for sustaining life on earth (nilsson, 1992; rydin et al., 1997; gardenfors, 2000; bravo-oviedo et al., 2014). at the same time forests are one of the most endangered ecosystems on earth due to various human activities like unsustainable use of wood, pollution, poor management, climate factors, etc. (marchetti, 2004). fungi are a remarkably heterogeneous and diverse group of organisms present in nearly all ecosystems on our planet (hood, 2006). they are of special significance in forest ecosystems, where they fulfill an array of vital functions and constitute an important part of their total biodiversity (hawksworth, 2004). in forests, fungi are mainly present in soil where their mycelium makes up 89% of the total microbial biomass (frankland, 1982). however, one of the most important groups of forest fungi are decomposers of dead and decaying organic matter saprotrophs. this group of fungi is crucial for the functioning of forest ecosystems, as wood decomposition is decisive for nutrient cycling, carbon balance, and soil formation (lonsdale et al., 2008). a particularly significant group of fungi that decompose dead wood are lignicolous fungi that decompose lignocellulosic matter as an underutilized natural resource (karaman et al., 2012). mycorrhizal © 2023 karaman et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 75 fungi are another important group of forest fungi. they provide plants with easier absorption of mineral substances, which increases their resistance to different abiotic and biotic stresses (drought, pollutants, pathogens, etc.) (deacon, 2006). also, fungi occur on living organisms as pathogens, and regulate population dynamics of different species (fang et al., 2021; bahram & netherway, 2022). due to their sensitivity to human activities that reduce the naturalness and functionality of forest ecosystems, certain fungal species are used as indicators of old-growth forests, i.e. preserved, undisturbed, conservation-worthy forest habitats (christensen et al., 2004). from a conservation point of view, old-growth forests are crucial for the survival of valuable, rare, and endangered species (runnel & lõhmus, 2017). based on the presence of old-growth indicator fungal species, it is possible to prioritize protection and specific management of forest areas of high conservation value and at the same time to emphasize their importance and the importance of their protection and preservation (christensen et al., 2004). considering the importance of forest ecosystems and the lack of biodiversity data on fungal species in serbia, our research was focused on deepening the knowledge about the fungal diversity of mountain tara, which is located in western serbia and represents a part of the dinaric alps, especially in the area of national park tara. mt. so far, only a few papers related to the research on the diversity of macrofungal species in forest ecosystems of mt. tara have been published (čolić, 1967; karađić, 2006; rakić, 2019; jovanović et al., 2021; rakić et al., 2022). in addition to this, several lignicolous and mycorrhizal fungal taxa from tara mt. have been examined for accumulation of heavy metals and radionuclides (rakić et al., 2014). in our monitoring survey of fungal diversity of mt. tara, special four localities were selected. furthermore, the diversity of old-growth indicator species such as hericium coralloides (scop.) pers, ischnoderma benzoinum (wahlenb.) p. karst., mucidula mucida (schrad.) pat., pycnoporellus fulgens (fr.) donk, sparassis crispa (wulfen) fr., etc. have been detected. materials and methods study area the research was conducted in the national park tara, in four localities: mitrovac, kaludjerske bare, predov krst, and zaovine (fig. 1). site kaludjerske bare (43°54’22.4”n 19°31’30.8”e) is located in the southeastern part of tara and represents one of the most famous tourist hot spots on the mountain. in this locality, forests of black and white pine, fir, 76 biologica nyssana ● 14 (2) december 2023: 75-95 karaman et al. ● biodiversity of fungal species from tara mountain (serbia) fig. 1. map of four investigated sites on tara mt. biologica nyssana ● 14 (2) december 2023: 75-95 karaman et al. ● biodiversity of fungal species from tara mountain (serbia) 77 the nomenclature of the species names is in accordance with the database of index fungorum (www.indexfungorum.org), and mycobank (https:// www.mycobank.org/). representative specimens of fungal species were dried at 50 °c (scholtes fp 955.3, germany) and deposited within fungarium of the profungi laboratory (buns herbarium, department of biology and ecology, faculty of sciences, university of novi sad) (https://www.pmf. uns.ac.rs/en/research/groups/profungi/). all specimens were recorded using the inaturalist application (https://www.inaturalist.org/). results and discussion a total of 320 fungal taxa were observed in four localities on mt. tara. phylum ascomycota is represented by 13 species that belong to 9 genera and 8 families with xylaria as the most abundant genus represented by 3 species (tab. 1). phylum basidiomycota is represented by 329 taxa, belonging to 136 genera and 69 families. the most diverse family in phylum basidiomycota is russulaceae with 37 taxa. other representative families were: tricholomataceae (25), mycenaceae (21), agaricaceae (19), polyporaceae (17) and boletaceae (15) (fig. 2). the genus with the highest number of species is russula with 20 taxa determined. one specimen from phylum mucoromycotina was recorded spinellus fusiger (link) tiegh. (tab. 1). table 1. list of recorded fungal taxa and spruce grow on the limestone base. predov krst (43°56’28.4”n 19°18’34.3”e) is located in the most remote part of tara, in the northwest. it is represented by the ridges surrounded by the drina river. this is one of the best-preserved mountain areas in serbia, with nature reserves which are refugees and one of the last habitats of the tertiary relic plant species serbian spuce (picea omorika (pančić) purk). mitrovac (43°55’16.4”n 19°25’25.9”e) is located in the central part of mt. tara, with specific geological, climate, and hydrological characteristics. it has preserved numerous sinkholes, a unique post-glacial peat bog, and primeval-type vegetation with one of the few remaining habitats of serbian spuce. site zaovine (43°53’11.4”n 19°21’50.4”e) is located in the southern part of tara mountain, and represents area with mountainous character that includes the zaovine village, the zaovine lake and the canyon of river beli rzav „sklopovi”. data collection fungal diversity research was conducted during summer and autumn of 2021 and 2022. the fungal specimens were identified based on morphological (macroscopic and microscopic) descriptions on the microscope unit (kern, obn135). the identification of the species has been done according to specialized identification keys (breitenbach & kranzlin, 1984; knudsen & vesterholt, 2008; uzelac, 2009; aronsen & læssøe, 2016; læssøe & petersen, 2019) by using standard reagents (koh, feso4). taxa locality* indicator** functional group ascomycota discinaceae gyromitra infula (schaeff.) quél. kb, z sap gelatinodiscaceae ascocoryne cylichnium (tul.) korf m italy sap ascocoryne sarcoides (jacq.) j.w. groves & d.e. wilson m sap neobulgaria pura (pers.) petr. m, pk sap helvellaceae helvella atra j. könig m myc helvella crispa sowerby m, pk myc pezizaceae peziza saniosa schrad. pk myc pezizellaceae calycina citrina (hedw.) gray m sap xylariaceae 78 biologica nyssana ● 14 (2) december 2023: 75-95 karaman et al. ● biodiversity of fungal species from tara mountain (serbia) xylaria hypoxylon (l.) grev. m, pk sap xylaria longipes nitschke m sap xylaria polymorpha (pers.) grev. m, pk lithuania; italy; estonia sap basidiomycota agaricaceae agaricus essettei bon m, pk sap agaricus langei (f.h. møller) f.h. møller m, pk sap agaricus sylvicola (vittad.) peck kb, pk sap agaricus urinascens (jul. schäff. & f.h. møller) singer kb sap chlorophyllum olivieri (barla) vellinga m sap chlorophyllum rhacodes (vittad.) vellinga m sap crucibulum laeve (huds.) kambly kb sap cyathus striatus willd. pk sap cystodermella cinnabarina (alb. & schwein.) harmaja kb sap cystolepiota seminuda (lasch) bon kb sap echinoderma asperum (pers.) bon pk sap lepiota castanea quél. pk sap lepiota clypeolaria (bull.) p. kumm. kb, m, pk sap lepiota cristata (bolton) p. kumm. kb, m, pk sap lepiota ignivolvata bousset & joss. ex joss. pk, z sap lepiota magnispora murrill kb sap macrolepiota mastoidea (fr.) singer kb, z sap macrolepiota procera (scop.) singer kb, pk sap melanophyllum haematospermum (bull.) kreisel z sap amanitaceae amanita battarrae (boud.) bon kb myc amanita citrine pers. kb, m, pk myc amanita fulvoides neville & poumarat kb myc amanita gemmata (fr.) bertill. m myc amanita muscaria (l.) lam. kb, m, pk, z myc amanita pantherina (dc.) krombh. kb, m, pk, z myc amanita phalloides (vaill. ex fr.) link m myc amanita rubescens pers. kb, m, z myc amanita vaginata (bull.) lam. kb myc auriculariaceae auricularia auricula-judae (bull.) quél. pk sap biologica nyssana ● 14 (2) december 2023: 75-95 karaman et al. ● biodiversity of fungal species from tara mountain (serbia) 79 auricularia mesenterica (dicks.) pers. m eu sap auriscalpiaceae lentinellus flabelliformis (bolton) s. ito m, pk par/sap bankeraceae sarcodon imbricatus (l.) p. karst. m, pk estonia myc boletaceae boletus edulis bull. kb, m, pk myc boletus reticulates schaeff. m myc caloboletus calopus (pers.) vizzini kb, m, pk myc chalciporus piperatus (bull.) bataille z par imleria badia (fr.) vizzini kb, m myc leccinum albostipitatum den bakker & noordel. kb myc leccinum aurantiacum (bull.) gray kb, pk myc leccinum scabrum (bull.) gray z myc neoboletus luridiformis (rostk.) gelardi, simonini &vizzini kb, m, pk myc suillellus luridus (schaeff.) murrill pk myc suillellus queletii (schulzer) vizzini, simonini & gelardi pk myc xerocomellus chrysenteron (bull.) šutara kb, m, pk myc xerocomellus porosporus (imler ex watling) šutara m, pk myc xerocomellus pruinatus (fr.) šutara kb, m myc xerocomus subtomentosus (l.) quél. m myc clavariadelphaceae clavariadelphus pistillaris (l.) donk z estonia myc cortinariaceae cortinarius atrovirens kalchbr. kb, m myc cortinarius elegantior (fr.) fr. pk myc cortinarius flexipes (pers.) fr. kb, m myc cortinarius hercynicus (pers.) m.m. moser z myc cortinarius infractus (pers.) fr. m myc cortinarius ohlone bojantchev kb myc cortinarius olivaceofuscus kühner m, pk myc cortinarius pseudodaulnoyae rob. henry & ramm m myc cortinarius salor fr. m myc cortinarius sanguineus (wulfen) gray m myc cortinarius semisanguineus (fr.) gillet m myc cortinarius trivialis j.e. lange kb, z myc 80 biologica nyssana ● 14 (2) december 2023: 75-95 karaman et al. ● biodiversity of fungal species from tara mountain (serbia) cortinarius violaceus (l.) gray m myc crepidotaceae crepidotus applanatus (pers.) p. kumm. m, pk sap cyphellaceae baeospora myosura (fr.) singer z sap chondrostereum purpureum (pers.) pouzar m par/sap dacrymycetaceae calocera cornea (batsch) fr. m, z par/sap calocera furcata (fr.) fr. m par/sap calocera viscosa (pers.) fr. m, pk par/sap dacryobolaceae postia ptychogaster (f. ludw.) vesterh. kb par/sap echinodontiaceae amylostereum areolatum (chaillet ex fr.) boidin m par/sap entolomataceae clitopilus prunulus(scop.) p. kumm. kb, m, pk, z myc entocybe nitida (quél.) t.j. baroni, largent & v. hofst. m sap entoloma hebes (romagn.) trimbach kb sap entoloma lampropus (fr.) hesler pk sap exidiaceae guepinia helvelloides (dc.) fr. pk estonia sap pseudohydnum gelatinosum (scop.) p. karst. kb, m, pk sap fomitopsidaceae climacocystis borealis (fr.) kotl. & pouzar kb latvia par/sap fomitopsis betulina (bull.) b.k. cui, m.l. han & y.c. dai m par/sap fomitopsis pinicola (sw.) p. karst. kb, m, pk yugoslavia par/sap geastraceae geastrum fimbriatum fr. pk lithuania; estonia sap geastrum rufescens pers. kb lithuania; estonia sap geastrum striatum dc. pk lithuania; estonia sap geastrum triplex jungh. kb, m lithuania; estonia sap gloeophyllaceae gloeophyllum abietinum (bull.) p. karst. m par/sap gloeophyllum odoratum (wulfen) imazeki kb, m par/sap gloeophyllum sepiarium (wulfen) p. karst. m par/sap 81 biologica nyssana ● 14 (2) december 2023: 75-95 karaman et al. ● biodiversity of fungal species from tara mountain (serbia) gomphaceae phaeoclavulina flaccida (fr.) giachini kb sap ramaria aurea (schaeff.) quél. m, pk myc ramaria stricta (pers.) quél. pk sap gomphidiaceae chroogomphus rutilus (schaeff.) o.k. mill. kb, z par hericiaceae hericium clathroides (pall.) pers. m par/sap hericium coralloides (scop.) pers. m great britain; lithuania; central europe; europe; latvia; denmark; estonia; germany; yugoslavia; benelux par/sap hericium flagellum (scop.) pers. m central europe; yugoslavia par/sap hydnaceae cantharellus cibarius fr. m, pk myc clavulina coralloides (l.) j. schröt. m, z myc clavulina rugosa (bull.) j. schröt. m, z myc craterellus cornucopioides (l.) pers. m myc craterellus tubaeformis (fr.) quél. m myc hydnum repandum l. kb myc hydnum rufescens pers. m myc hydnangiaceae laccaria amethystine cooke kb, m, pk, z myc laccaria bicolor (maire) p.d. orton kb, z myc laccaria laccata (scop.) cooke kb, m, pk, z myc hygrophoraceae arrhenia spathulata (fr.) redhead kb sap chrysomphalina grossula (pers.) norvell, redhead & ammirati m czech republic sap cuphophyllus pratensis (pers.) bon kb sap hygrocybe chlorophana (fr.) wünsche kb myc hygrocybe coccinea (schaeff.) p. kumm. kb, m, z myc hygrophorus agathosmus (fr.) fr. kb myc hygrophorus capreolarius (kalchbr.) sacc. m myc hygrophorus chrysodon (batsch) fr. m myc hygrophorus cossus (sowerby) fr. pk myc hygrophorus eburneus (bull.) fr. kb, m, z myc hygrophorus latitabundus britzelm. kb myc 82 biologica nyssana ● 14 (2) december 2023: 75-95 karaman et al. ● biodiversity of fungal species from tara mountain (serbia) hygrophorus persoonii arnolds kb myc hygrophorus pudorinus (fr.) fr. m myc hygrophoropsidaceae hygrophoropsis aurantiaca (wulfen) maire kb, m, pk sap hymenochaetaceae phellinus pomaceus (pers.) maire kb, m, z par/sap hymenogastraceae galerina badipes (pers.) kühner m sap galerina graminea (velen.) kühner kb sap galerina hypnorum (schrank) kühner m sap galerina marginata (batsch) kühner kb, m sap gymnopilus penetrans (fr.) murrill kb, m, pk, z sap gymnopilus picreus (pers.) p. karst. m sap gymnopilus sapineus(fr.) murrill m sap hebeloma birrus (fr.) gillet m myc hebeloma crustuliniforme (bull.) quél. kb, m myc hebeloma sinapizans (paulet) gillet kb myc psilocybe coronilla (bull.) noordel. kb sap psilocybe serbica m.m. moser & e. horak kb, m sap incrustoporiaceae tyromyces chioneus (fr.) p. karst. m par/sap inocybaceae inocybe geophylla p. kumm. m myc inocybe griseolilacina j.e. lange z myc ischnodermataceae ischnoderma benzoinum (wahlenb.) p. karst. m, pk czech republic; yugoslavia par/sap laetiporaceae laetiporus sulphureus (bull.) murrill m par/sap lycoperdaceae apioperdon pyriforme (schaeff.) vizzini kb, m, pk italy sap bovista plumbea pers. kb sap bovistella utriformis (bull.) demoulin & rebriev kb sap lycoperdon echinatum pers. pk lithuania; estonia sap lycoperdon excipuliforme (scop.) pers. kb, pk, z sap lycoperdon molle pers. m sap lycoperdon perlatum pers. kb, m, pk sap lycoperdon pratense pers. kb sap biologica nyssana ● 14 (2) december 2023: 75-95 karaman et al. ● biodiversity of fungal species from tara mountain (serbia) 83 lyophyllaceae asterophora lycoperdoides (bull.) ditmar z sap lyophyllum decastes (fr.) singer pk sap tephrocybe rancida (fr.) donk kb, m sap macrocystidiaceae macrocystidia cucumis (pers.) joss. kb, m sap marasmiaceae crinipelliss cabella (alb. & schwein.) murrill kb sap marasmius oreades (bolton) fr. kb, pk sap marasmius rotula (scop.) fr. m, z sap marasmius torquescens quél. m, pk sap megacollybia platyphylla (pers.) kotl. & pouzar kb, m, pk sap meripilaceae meripilus giganteus (pers.) p. karst. pk germany par/sap meruliaceae phlebia tremellosa (schrad.) nakasone & burds. m par/sap mycenaceae hemimycena lactea (pers.) singer m sap hydropus marginellus (pers.) singer m central europe sap mycena aurantiomarginata (fr.) quél. m sap mycena crocata (schrad.) p. kumm. m, pk sap mycena epipterygia (scop.) gray kb, m, pk sap mycena galericulata (scop.) gray m, pk italy; european union sap mycena galopus (pers.) p. kumm. kb, m sap mycena haematopus (pers.) p. kumm. kb, m, pk sap mycena lammiensis harmaja pk sap mycena maculata p. karst. m germany sap mycena pelianthina (fr.) quél. kb sap mycena picta (fr.) harmaja m sap mycena pterigena (fr.) p. kumm. m sap mycena pura (pers.) p. kumm. kb, m, pk, z sap mycena rosea gramberg kb, m, pk sap mycena rosella (fr.) p. kumm. m sap mycena sanguinolenta (alb. & schwein.) p. kumm. m sap mycena stipata maas geest. & schwöbel kb, m, pk sap 84 biologica nyssana ● 14 (2) december 2023: 75-95 karaman et al. ● biodiversity of fungal species from tara mountain (serbia) mycena viridimarginata p. karst. m sap panellus stipticus (bull.) p. karst. kb, m, pk sap prunulus diosmus (krieglst. & schwöbel) c. hahn kb sap omphalotaceae collybiopsis confluens (pers.) r.h. petersen kb, m, pk sap collybiopsis peronata (bolton) r.h. petersen pk sap collybiopsis vaillantii (pers.) r.h. petersen kb sap gymnopusan drosaceus (l.) della magg. & trassin. m sap gymnopus dryophilus (bull.) murrill m sap gymnopus erythropus (pers.) antonín, halling & noordel. kb sap gymnopus hariolorum (bull.) antonín, halling & noordel. pk sap mycetinis alliaceus (jacq.) earle ex a.w. wilson & desjardin kb, m, pk, z sap mycetinis scorodonius (fr.) a.w. wilson & desjardin kb, m sap rhodocollybia butyracea (bull.) lennox kb, m, z sap rhodocollybia maculata (alb. & schwein.) singer kb sap rhodocollybia prolixa var. distorta (fr.) antonín, halling & noordel. m sap paxillaceae paxillus involutus (batsch) fr. kb, m, z myc phanerochaetaceae bjerkandera adusta (willd.) p. karst. kb, m, pk par/sap phyllotopsidaceae phyllotopsis nidulans (pers.) singer m great britain par/sap pleurocybella porrigens (pers.) singer m sap physalacriaceae armillaria gallica marxm. & romagn. kb, m par/sap hymenopelli sradicata (relhan) r.h. petersen kb, m, pk, z sap mucidula mucida (schrad.) pat. kb, m, pk germany par/sap xerula pudens (pers.) singer kb, pk, z myc pluteaceae pluteus cervinus (schaeff.) p. kumm. kb, m, pk sap pluteus petasatus (fr.) gillet pk sap biologica nyssana ● 14 (2) december 2023: 75-95 karaman et al. ● biodiversity of fungal species from tara mountain (serbia) 85 pluteus pouzarianus singer pk sap podoscyphaceae abortiporus biennis (bull.) singer m par/sap polyporaceae cerioporus varius (pers.) zmitr. & kovalenko pk par/sap coriolopsis gallica (fr.) ryvarden m great britain par/sap cyanosporus caesius (schrad.) mcginty kb, m, pk par/sap daedaleopsis confragosa (bolton) j. schröt. kb, pk par/sap daedaleopsis tricolor (bull.) bondartsev & singer m par/sap fomes fomentarius (l.) fr. kb, m, pk italy; germany; yugoslavia par/sap ganoderma applanatum (pers.) pat. m, pk european union; germany; yugoslavia par/sap ganoderma carnosum pat. m yugoslavia par/sap ganoderma resinaceum boud. m par/sap trametes cinnabarina (jacq.) fr. m par/saprothr trametes gibbosa (pers.) fr. m, z par/sap trametes hirsuta (wulfen) lloyd m, pk par/sap trametes ochracea (pers.) gilb. & ryvarden pk par/sap trametes pubescens (schumach.) pilát kb, m, pk italy par/sap trametes versicolor (l.) lloyd kb, m, pk par/sap trichaptum abietinum (pers. ex j.f. gmel.) ryvarden kb, m, pk par/sap trichaptum biforme (fr.) ryvarden m, pk par/sap psathyrellaceae candolleomyces candolleanus (fr.) d. wächt. & a. melzer (fr.) d. wächt. & a. melzer kb, m, pk sap coprinellus micaceus (bull.) vilgalys, hopple & jacq. johnson kb, m sap coprinopsis atramentaria (bull.) redhead, vilgalys & moncalvo m sap panaeolus acuminatus (p. kumm.) quél. kb sap pseudoclitocybaceae pseudoclitocybe cyathiformis (bull.) singer m czech republic sap pycnoporellaceae pycnoporellus fulgens (fr.) donk m, pk latvia; finland; estonia; yugoslavia par/sap rickenellaceae 86 biologica nyssana ● 14 (2) december 2023: 75-95 karaman et al. ● biodiversity of fungal species from tara mountain (serbia) rickenella fibula (bull.) raithelh. kb, m par rickenella swartzii (fr.) kuyper kb, m par russulaceae lactarius aurantiacus (pers.) gray kb, m, z myc lactarius blennius (fr.) fr. kb, m, pk myc lactarius controversus pers. kb myc lactarius deliciosus (l.) gray kb, m, pk myc lactarius deterrimus gröger kb, m myc lactarius luridus (pers.) gray m myc lactarius mairei malençon z myc lactarius pallidus pers. kb, pk myc lactarius picinus fr. pk myc lactarius rubrocinctus fr. kb myc lactarius rufus (scop.) fr. m, pk myc lactarius salmonicolor r. heim & leclair kb, m, z myc lactarius scrobiculatus (scop.) fr. kb, m myc lactariustorminosus (schaeff.) pers. kb myc lactarius uvidus (fr.) fr. kb, pk czech republic myc lactarius violascens (j. otto) fr. kb myc lactifluus vellereus (fr.) kuntze kb myc russula adusta (pers.) fr. m, z myc russula aeruginea lindblad ex fr. pk myc russula atropurpurea (krombh.) britzelm. kb, m myc russula badia quél. kb, m myc russula cessans a. pearson m myc russula chloroides (krombh.) bres. kb, m myc russula cyanoxantha (schaeff.) fr. kb, m, pk myc russula delica fr. m myc russula densifolia secr. ex gillet m myc russula fellea (fr.) fr. m myc russula fragilis fr. pk myc russula fragilis f. violascens (secr. ex gillet) sacc. m myc russula grata britzelm. m myc russula integra (l.) fr. kb, m, z myc russula mustelina fr. m myc russula nobilis velen. m myc russula ochroleuca fr. m myc russula sardonia fr. kb myc russulas olaris ferd. & winge kb myc russula torulosa bres. kb myc biologica nyssana ● 14 (2) december 2023: 75-95 karaman et al. ● biodiversity of fungal species from tara mountain (serbia) 87 sarcomyxaceae sarcomyxa serotina (pers.) v. papp m sap schizophyllaceae schizophyllum commune fr. m, pk, z sap sparassidaceae sparassis brevipes krombh. m yugoslavia sap sparassis crispa (wulfen) fr. m finland sap squamanitaceae cystoderma carcharias (pers.) fayod kb, m sap stereaceae stereum hirsutum (willd.) pers. m, pk par/sap stereum subtomentosum pouzar m, pk denmark; yugoslavia par/sap strophariaceae hypholoma capnoides (fr.) p. kumm. m sap hypholoma fasciculare (huds.) p. kumm. kb, m, pk sap hypholoma lateritium (schaeff.) p. kumm. kb, m sap kuehneromyces mutabilis (schaeff.) singer & a.h. sm. pk sap pholiota adiposa (batsch) p. kumm. kb sap pholiota aurivella (batsch) p. kumm. kb, m denmark; benelux sap pholiota lenta (pers.) singer m sap pholiota squarrosa (vahl) p. kumm. m sap stropharia aeruginosa (curtis) quél. kb, m, pk sap stropharia caerulea kreisel m, z sap suillaceae suilluscollinitus (fr.) kuntze kb myc suillus granulatus (l.) roussel kb, pk myc suillus luteus (l.) roussel z myc tremellaceae tremella mesenterica (schaeff.) pers. m par tricholomataceae clitocybe dealbata (sowerby) p. kumm. kb sap clitocybe fragrans (with.) p. kumm. kb, m sap clitocybe nebularis (batsch) p. kumm. kb, m sap clitocybe odora (bull.) p. kumm. kb, pk sap collybia cirrhata (schumach.) quél. m sap infundibulicybe geotropa (bull.) harmaja pk sap lepista nuda (bull.) cooke kb, m sap leucocortinarius bulbiger (alb. & schwein.) singer kb, pk myc 88 biologica nyssana ● 14 (2) december 2023: 75-95 karaman et al. ● biodiversity of fungal species from tara mountain (serbia) leucocybe connata (schumach.) vizzini, p. alvarado, g. moreno & consiglio m sap leucopaxillus gentianeus (quél.) kotl. kb, pk sap paralepista flaccida (sowerby) vizzini m, z sap tricholoma album (schaeff.) p. kumm. kb european union myc tricholoma bufonium (pers.) gillet m myc tricholom equestre (l.) p. kumm. z myc tricholoma evenosum (sacc.) rea kb myc tricholoma fracticum (britzelm.) kreisel kb myc tricholoma fulvum (dc.) bigeard & h. guill. kb myc tricholoma imbricatum (fr.) p. kumm. m myc tricholoma portentosum (fr.) quél. z myc tricholoma saponaceum (fr.) p. kumm. kb, m myc tricholoma saponaceum f. ardosiacum (bres.) bon m myc tricholoma sulphureum (bull.) p. kumm. kb, m, pk european union myc tricholoma terreum (schaeff.) p. kumm. kb, m myc tricholoma vaccinum (schaeff.) p. kumm. kb, m, z myc tricholomopsis rutilans (schaeff.) singer kb, m, pk sap mucoromycota phycomycetaceae spinellus fusiger (link) tiegh. m par * locality: kb – kaludjerske bare; m – mitrovac; pk – predov krst; z – zaovine. functional group: sap – saprotroph, par – parasite, par/sap – parasite/saprotroph, myc – mycorhizal ** regions in which a specific fungal species is listed as an indicator species of old-growth forests: benelux (walleyn & veerkamp, 2005); central europe (blaschke et al., 2009); czech republic (holec, 2003; dvořák et al., 2017); denmark (heilmann-clausen & christensen, 2000); estonia (parmasto, 2001; estonian ministry of environment, 2017); europe (christensen et al., 2004); european union (european commission, 2013); finland (kotiranta & niemelä, 1993; 1996); germany (schmid & helfer, 1999; müller et al., 2007; schmidt et al., 2012); great britain (ainsworth, 2004); italy (blasi et al., 2010); latvia (ek et al., 2002); lithuania (andersson & kriukelis, 2002); yugoslavia (tortić, 1998) the highest number of species was recorded on site mitrovac – 210 (fig. 3). the most represented genera on site mitrovac were mycena (15), russula (15), cortinarius (8) and tricholoma (7). of the total documented species diversity, 92 taxa were recorded only on site mitrovac. within the localities kaludjerske bare and predov krst, 152 and 113 taxa were recorded, respectively. the least number of taxa were observed on site zaovine – 48 taxa (tab. 1). the recorded taxa belong to four ecological categories. saprotrophic fungi (141 taxa) are the most represented ecological group, followed by mycorrhizal fungi (124 taxa), parasite/saprotrophs (47 taxa), parasites (6 taxa), and mutualist (2 taxa) (fig. 4). saprotrophic fungi were dominant on all localities except zaovine, where fungi from mycorrhizal ecological group stood out (tab. 1). ten recorded species are on the list of protected wild species of plants, animals, and fungi (annex ii of the „official gazette of rs”, no. 5/2010 and 47/2011) while three species are on the list of strictly protected wild species of plants, animals and fungi (annex i of the „official gazette of rs”, no. 5/2010 and 47/2011) (tab. 2). mitrovac is the most valuable locality for both protected (8 taxa: cantharellus cibarius, craterellus cornucopioides, lactarius salmonicolor, boletus edulis, lactarius deliciosus, lactarius deterrimus, russula cyanoxantha), and strictly protected 89 biologica nyssana ● 14 (2) december 2023: 75-95 karaman et al. ● biodiversity of fungal species from tara mountain (serbia) fig. 2. families of fungal species from the phylum basidiomycota recorded in the monitoring of four locations at mt. tara during 2021/2022 fig. 3. number (percent) of taxa per monitored locality 90 biologica nyssana ● 14 (2) december 2023: 75-95 karaman et al. ● biodiversity of fungal species from tara mountain (serbia) fig. 4. number (percent) of taxa per ecological category fungi (3 taxa: hericium flagellum, hericium coralloides, psilocybe serbica), and it’s followed by site kaludjerske bare with 7 protected (hydnum repandum, lactarius salmonicolor, boletus edulis, lactarius deliciosus, lactarius deterrimus, marasmius oreades, russula cyanoxantha) and 1 strictly protected species (psilocybe serbica). on sites predov krst and zaovine, no strictly protected species were recorded during the investigated period (tab. 2). also, 38 indicator fungal species of old-growth forests and valuable forest sites were observed, which is 12% of the total species number. recorded species were present on indicator lists of 14 different european regional units (tab. 1). highest number of recorded indicator species were from estonian indicator lists (estonian ministry of environment, 2017). hericium coralloides is species with the highest number of occurrences on indicator lists, it occurred on 10 out of 14 lists analyzed in this work (tab. 1). this species is considered as rare and as one of the representatives of undisturbed habitats with high conservation value (christensen et al., 2004; adamcik et al., 2007): austria (vulnerable vu), bulgaria (vulnerable vu), croatia (endangered en), denmark (near threatened nt), estonia (near threatened nt), germany (endangered en), latvia (vulnerable vu), macedonia (near threatened nt), netherland (endangered en), norway (near threatened nt), poland (vulnerable -vu), romania (near threatened nt), sweden (near threatened nt), switzerland (vulnerable vu) and ukraine (vulnerable vu) (http://iucn. ekoo.se/). in this research hericium coralloides was recorded only on site mitrovac, which is also confirmed previously by čolić (1967), rakić (2019), and jovanović et al. (2021). the highest number of indicator species belong to the saprotrophic ecological category (18), followed by parasite/saprotrophic (15), and mycorrhizal category (5). for the majority of saprotrophic, and all the parasite/saprotrophic indicator species, a prerequisite for survival is the presence of large amounts of dead wood of different quality (dimensions and decay stage) (holec et al., 2015). for fungi that are connected to woody substrate, it is generally considered that the most significant limiting factor for the survival of their populations is the presence of a suitable substrate (lonsdale et al., 2008). however, nordén et al. (2018) indicate that the connection of the habitat i.e. substrate (ecological continuity) is also a very important factor because for the species used as indicators, limiting factors for the survival of populations are also limited dispersion and establishment of individuals. the greatest number of indicator species was recorded on site mitrovac (28 of 210 taxa; 13,3%), but the greatest ratio between indicator and total recorded taxa was documented on site predov krst (18 of 113 taxa; 16%) (tab. 2). these results indicate 91 biologica nyssana ● 14 (2) december 2023: 75-95 karaman et al. ● biodiversity of fungal species from tara mountain (serbia) table 2. list of protected, strictly protected and indicator species kaludjerske bare mitrovac predov krst zaovine protected species boletus reticulatus schaeff. * * cantharellus cibarius fr. * craterellus cornucopioides (l.) pers. * hydnum repandum l. * lactarius salmonicolor r. heim &leclair * * * boletus edulis bull. * * * lactarius deliciosus (l.) gray * * * lactarius deterrimus gröger * * marasmius oreades (bolton) fr. * * russula cyanoxantha (schaeff.) fr. * * * total number: 7 8 5 1 strictly protected species hericium flagellum (scop.) pers. * hericium coralloides (scop.) pers. * psilocybe serbica m.m. moser & e. horak * * total number: 1 3 / / indicator species apioperdon pyriforme (schaeff.) vizzini * * * ascocoryne cylichnium (tul.) korf * auricularia mesenterica (dicks.) pers. * chrysomphalina grossula (pers.) norvell, redhead &ammirati * clavariadelphus pistillaris (l.) donk * climacocystis borealis (fr.) kotl. & pouzar * coriolopsis gallica (fr.) ryvarden * fomes fomentarius (l.) fr. * * * fomitopsis pinicola (sw.) p. karst. * * * ganoderma applanatum (pers.) pat. * * ganoderma carnosum pat. * geastrum fimbriatum fr. * geastrum rufescens pers. * geastrum striatum dc. * geastrum triplex jungh. * * guepinia helvelloides (dc.) fr. * hericium flagellum (scop.) pers. * hericium coralloides (scop.) pers. * hydropus marginellus (pers.) singer * ischnoderma benzoinum (wahlenb.) p. karst. * * biologica nyssana ● 14 (2) december 2023: 75-95 karaman et al. ● biodiversity of fungal species from tara mountain (serbia) 92 lactarius uvidus (fr.) fr. * * lycoperdon echinatum pers. * meripilus giganteus (pers.) p. karst. * mucidula mucida (schrad.) pat. * * mycena galericulata (scop.) gray * * mycena maculata p. karst. * pholiota aurivella (batsch) p. kumm. * * phyllotopsis nidulans (pers.) singer * pseudoclitocybe cyathiformis (bull.) singer * pseudoclitocybe cyathiformis (bull.) singer * pycnoporellus fulgens (fr.) donk * * sarcodon imbricatus (l.) p. karst. * * sparassis brevipes krombh * sparassis crispa (wulfen) fr. * stereum subtomentosum pouzar * * trametes pubescens (schumach.) pilát * * * tricholoma album (schaeff.) p. kumm. * tricholoma sulphureum (bull.) p. kumm. * * * xylaria polymorpha (pers.) grev. * * total number: 12 29 18 1 that mitrovac and predov krst are valuable preserved habitats for old growth indicator (endangered and rare) species of fungi. the importance of preserved forests in terms of the ecosystem services they provide is enormous. the long and undisturbed development of these forests, together with the natural dynamics of the renewal and destruction processes, contributes to the emergence of a very high structural complexity (franklin & van pelt, 2004). structural complexity is reflected in the presence of a large number of different microhabitats (kozák et al., 2018), which include dead wood, holes in wood, fruiting bodies of lignicolous fungi and other epiphytic structures, that at the same time represent a habitat for numerous different and important groups of organisms (paillet et al., 2010). the structural complexity of old, preserved forests enables the neutralization of summer forest soil temperatures (frey et al., 2016), thereby enabling the survival of species sensitive to climate change (betts et al., 2018). old preserved forests are more resilient, stable and adaptable than managed forests (alberto et al., 2013; watson et al., 2013). conclusion recent researches have shown the presence of a high species diversity on mt. tara, especially on the site mitrovac which contains a great number of rare and endangered taxa. it is of great importance to preserve those habitats since they can present fungal biodiversity hot spots. however, in order to make the overall diversity picture more complete, it is necessary to carry out long-term monitoring. acknowledgements. this research was supported by the joint project of national park tara with the faculty of sciences university of novi sad profungi laboratory (contracts no. 47/2021 and 43/2022), and the ministry of education, science and technological development of the republic of serbia (grant.no. 451-03-47/202301/200125). authors are grateful to the employees of the national park tara for logistical support in this research, but also to members of non-governmental societies: mycological society of novi sad, mushroom society „šumadija”, association of mushroom and nature lovers „vilino kolo” and mycological society of serbia for their contribution in the field research. this work is dedicated to the memory of the great naturalist prof. dr vladimir ranđelović. references adamcik, s., christensen, m., & heilmannclausen, j. 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(2009). gljive srbije i zapadnog balkana. bgv logik. walleyn, r. & veerkamp, m. (2005). houtzwammen op beuk. kensoortenvoorsoor tenrijkebossen in belgieen nederland. natuur focus, 4: 82–88. watson, j.e.m., iwamura, t., & butt, n. (2013). mapping vulnerability and conservation adaptation strategies under climate change. nature climate change, 3: 989–994. http://iucn.ekoo.se/ https://www.inaturalist.org/ https://www.pmf.uns.ac.rs/en/research/groups/ profungi/ gospodinov, natcheva, 2020, biologica nyssana 11(1) 11 (1) september 2020: 31-33 doi: 10.5281/zenodo.4060289 chromosome studies of some thalloid liverworts in bulgaria original article galin gospodinov department of plant and fungal diversity and resources, institute of biodiversity and ecosystem research, bulgarian academy of sciences, sofia, bulgaria gospodinov.bryol@gmail.com (corresponding author) rayna natcheva department of plant and fungal diversity and resources, institute of biodiversity and ecosystem research, bulgarian academy of sciences, sofia, bulgaria renimoss@bio.bas.bg received: october 01, 2019 revised: january 22, 2020 accepted: january 26, 2020 abstract: here, we report for the first time chromosome counts for bryophytes from bulgaria. the mitotic chromosomes from gametophytes of aneura pinguis, pellia epiphylla, pellia neesiana and riccia fluitans were studied. all counts correspond to previous reports from other parts of the species’ range. key words: chromosome counts, liverworts, bulgaria apstract: studija hromozoma nekih taloidnih jetrenjača u bugarskoj ovde prvi put izveštavamo o broju hromozoma za briofite iz bugarske. ispitivani su mitotički hromozomi iz gametofita aneura pinguis, pellia epiphilla, pellia neesiana i riccia fluitans. svi brojevi odgovaraju prethodnim izveštajima iz drugih delova areala vrste. ključne reči: brojanje hromozoma, jetrenjače, bugarska introduction chromosome studies are a useful tool for taxonomists (fritsch, 1978). unfortunately, chromosome studies concerning bryophytes in europe are sporadic and represent chromosome numbers from a limited number of local populations. although chromosome numbers in bryophytes are fairly uniform and karyotype analyses are not often informative (newton, 1979), chromosome counts are a useful tool to distinguish morphologically similar haplodiploid species pairs. the knowledge on bryophyte chromosome counts was summarized by fritsch (1991). notably, information from south-eastern europe is missing. few other publications deal with chromosomes of certain genera of hepatics (berrie, 1960; boisselier-dubaylee & bischler, 1998; zheng & zhu, 2009; ochyra et al., 2016). the aim of this study was to report chromosome numbers of some thalloid liverworts from bulgaria. material and methods the liverworts were collected during different excursions and kept alive in a greenhouse, for the purpose of the research. after a period of adaptation a modified gomori’s haematoxylin staining method was used (melander & wingstrand, 1953). fresh actively growing thallus tips were placed in 0.01% solution of colchicine for 90 min. after washing in distilled h2o they were fixed in clarke’s solution (3:1 ethyl alcohol/glacial acetic acid) for 2 h at room temperature or in a fridge for 24 h. after washing in distilled h2o the thalli were placed in 1m hydrochloric acid for 40min at 60 °c followed by washing in distilled h2o. the samples were incubated in hydrochloric acid /ether in ratio 1:1 for 15 min at 60 °c, washed in distilled h2o the stained in gomori’s hematoxylin for 1.45/2 h at 60 °c. samples were squashed in 45% acetic acid and observed under a light microscope. © 2020 gospodinov, natcheva. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work noncommercially under the same license as the original. 31 results and discussion our results were in accordance with already published chromosome numbers for the respective species. for bulgaria this is the first chromosome report for bryophyte species. aneuraceae aneura pinguis (l.) dumort., n = 10 (fig. 1). only one exception in the a. pinguis chromosome number is known up to now (inoe, 1975) where the species shows chromosome number of n = 20. pelliaceae pellia epiphylla (l.) corda, n=9 (fig. 2). in europe, diploid number n=18 is present in pellia borealis lorb, an allopolyploid species with two haploid cryptic species within p. epiphylla as parental taxa (orzechowska et al., 2010). the presence of p. borealis in bulgaria was expected but could not be proven by the present study. pellia neesiana nees, n=9 (fig. 3) most previous studies for this species showed haploid chromosome count of n=9 in mitotic counts (fritsh, 1991) with a single old report of diploid number of n=18 (showalter, 1927). ricciaceae riccia fluitans l., n=8 (fig. 4) the species shows uniformity of chromosome numbers within its range as far as it is studied (fritsch 1991). the only polyploid reports most likely belong to its close relative riccia rhenana (heitz, 1927, lorbeer, 1934). acknowledgements. this research is part of project кп-06-н21/15/19.12.2018 „cryptic species in bulgarian flora – molecular species delimitation in the aneura pinguis complex” financed by the national science fund of bulgaria. references berrie, g. 1960: the chromosome numbers of liverworts (hepadcae and anthocerotae). transactions of the british bryological society, 3(5): 688-705. fritsch r. 1991: index to bryophyte chromosome counts. bryophyta bibliothek, 40: 1–352. fritsch r. 1978: chromosome numbers of some hungarian liverworts. abstracta botanica, vol v, suppl. 3. heitz e. 1927: über multiple und aberrante chromosomenzahlen. abhandlungen des naturwissenschaftlicher verein hamburg, 21(3-4): 47-57. lorbeer g. 1934: zytologie der lebermoose mit besonderer be-rücksichtigung allgemeiner chromosomenfragen. i. teil. jahrbücher für wissenschaftliche botanik, 80: 567-818. marie‐catherine, b., bischler, h. 1998: 32 biologica nyssana ● 11 (1) september 2020: 31-33 gospodinov, natcheva ● chromosome studies of some thalloid liverworts in bulgaria fig. 1. mitotic chromosomes of: a aneura pinguis, b pellia epiphylla, cpellia neesiana, d riccia fluitans 33 biologica nyssana ● 11 (1) september 2020: 31-33 gospodinov, natcheva ● chromosome studies of some thalloid liverworts in bulgaria allopolyploidy in the thalloid liverwort corsinia (marchantiales). botanica acta, 111: 490-496. melander y., wingstrand k.g. 1953: gomori’s haematoxylin as a chromosome stain. stain technology, 28: 217-223. newton m.e. 1979: chromosome morphology and bryophyte systematics. in: clarke g.c.s., duckett j. g. (eds.) bryophyte systematics. acad. press, london, new yourk, pp. 207 229. ochyra, r., przywara, l., kuta, e. 1982: karyological studies on some antarctic liverworts. journal of bryology, 12(2): 259-263. orzechowska, m., siwinska, d., maluszynska, j. 2010: molecular cytogenetic analyses of haploid and allopolyploid pellia species. journal of bryology 32, 113–121. showalter, a.m. 1927: hermaphroditism in a dioicous hepatic. pnas, 13(6): 369-372. zheng, m., zhu, r.-l. 2009: karyological studies on some species of radula (radulaceae, jungermanniopsida, marchantiophyta). nova hedwigia, 88: 229-244. ocokoljić et al. 2025, biologica nyssana 16(1) 93 16 (1) june 2025: 93-104 doi: 10.46793/biolnyss.16.1.30o the impact of climatic parameters in belgrade on the indigo himalayan species from the subtropical biome original article mirjana ocokoljić university of belgrade faculty of forestry, kneza viseslava 1, 11000 beograd, serbia djurdja petrov university of belgrade faculty of forestry, kneza viseslava 1, 11000 beograd, serbia djurdja.stojicic@sfb.bg.ac.rs (corresponding author) nevenka galečić university of belgrade faculty of forestry, kneza viseslava 1, 11000 beograd, serbia dragana skočajić university of belgrade faculty of forestry, kneza viseslava 1, 11000 beograd, serbia jelena čukanović university of novi sad faculty of agriculture, trg dositeja obradovića 8, 21000 novi sad, serbia dragan vujičić university of belgrade faculty of forestry, kneza viseslava 1, 11000 beograd, serbia isidora simović biosense institute, university of novi sad, 1 dr zorana dinđića, 21000 novi sad, serbia received: may 30, 2025 revised: june 19, 2025 accepted: june 19, 2025 abstract: understanding the impact of climatic parameters on vegetation is significant for understanding the potential of urban green spaces, which play a crucial role in the ecological protection of cities and adaptation to climate change, particularly in regulating ecosystem services. in future climatic conditions, exotic species must be taken into account for ecosystem preservation. therefore, for the first time, this study documents the differential attributes and phenology of indigofera heterantha wall. ex brandis in belgrade, thus increasing the floral diversity of serbia. through an integrative approach combining phenological and climatic data with biometric characteristics, indicators for the management and sustainability of indigo himalayan were identified, based on research conducted in 2024. the findings contribute to understanding urban biodiversity and strategies for adapting new exotics in belgrade. however, further research is necessary to predict the impact of climate change on indigo himalayan. key words: floristic list, exotic vegetation, biodiversity, phenology, morphology, landscape architecture, urban green spaces apstrakt: efekti klimatskih promena u beogradu na vrstu indigo himalayan iz suptropskog bioma poznavanje uticaja klimatskih parametara na vegetaciju je značajno za razumevanje potencijala urbanih zelenih površina koje imaju ključnu ulogu u ekološkoj zaštiti gradova i prilagođavanju klimatskim promenama, posebno u regulisanju usluga ekosistema. u budućim klimatskim uslovima u cilju očuvanja ekosistema moraju se uzeti u obzir egzotične vrste. stoga su u radu prvi put dokumentovani diferencijalni atributi i fenologija indigofera heterantha wall. ex brandis u beogradu, čime je povećan diverzitet flore srbije. integrativnim pristupom fenološkim i klimatskim podacima i biometrijskim osobinama, na osnovu istraživanja sprovedenih tokom 2024. godine, utvrdjeni su pokazatelji za upravljanje i održivost indigo himalayan. nalazi doprinose razumevanju urbanog biodiverziteta i strategija adaptacije nove egzote u beogradu. ipak, neophodna su dalja istraživanja za predikciju uticaja klimatskih promena na indigo himalayan. ključne reči: floristički spisak, egzotična vegetacija, biodiverzitet, fenologija, morfologija, pejzažna arhitektura © 2025 ocokoljić et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. introduction studies of floristic diversity help assess the plant wealth and potential values of a specific region and create floristic lists (shaheen et al., 2016). life forms are indicators of micro and macroclimates, which result from plant adaptations, and understanding their phenology is the most reliable bioindicator, as well as a source of knowledge about periodic biological events influenced by environmental conditions (ziter et al., 2019; čukanović et al., 2024). changes in the spring phenological key events of woody plants have been documented in research (bertin, 2008; petrov et al., 2024) and are more consistent in direction and magnitude than changes in summer and autumn phenophases (zheng et al., 2016; ocokoljić et al., 2023). zheng et al. (2016) and ocokoljić et al. (2023) analysed the bud burst, first flowering, full flowering, end of flowering, formation of the first leaf, full leafing, re-flowering, the beginning of leaf colour change, autumn colouration, the beginning of leaf fall, and the end of leaf fall in nine and twelve 15th symposium on the flora of southeastern serbia and neighboring regions 94 in situ phenomonitoring, within the established phenological network, indigofera heterantha wall. ex brandis was first identified during spring 2024 in the čukarica municipality, on banovo brdo, in a public green space. the research area is a plaza where an individual of indigo himalayan, newly planted in a parterre composition, is georeferenced with coordinates 44°47'01.22"n and 20°25'03.62"e, at an altitude of 112 m, with a northeastern orientation (ne aspect) and a terrain slope of 1.64° (level to gently sloping terrain). the soil is of the anthropogenised luvic chernozem type (anjos et al., 2015). climatic data data from the republic hydrometeorological institute of serbia (rhmz) (https://www.hidmet.gov. rs/ciril/meteorologija/klimatologija_godisnjaci.php and https://www.ogimet.com/synopsc.phtml.en, accessed on 14 february 2025) from the main meteorological station (mms) surčin (ϕ44º47'54.44"n and λ20º27'53.35"e, altitude 99 m) were used due to the similarity of environmental conditions. for the purposes of the study, time series of monthly and seasonal mean maximum and minimum air temperatures and precipitation were formed for the reference period (1991-2020) and the period 1991-2024, as well as daily maximum and minimum air temperature assessments for 2024, according to the respective percentiles. real–time field observations after reconnaissance during spring 2024, phenological observations for indigo himalayan were recorded daily until january 2025, using the bbch scale (meier, 1997: 19 first leaves fully expanded, 39 shoots about 90% of final length, 60 beginning of flowering (about 10% of flowers open), 65 full flowering (open at least 50% of flowers, first petal folling), 69 end of flowering (all petals follen), 87 fruit ripe for picking, 91 shoot growth completed (terminal bud developed; foliage still fully green), 95 leaves discolour (50% of leaves discoloured) i 97 all leaves fallen. during the full flowering phase and the development of fully matured leaves in 2024, samples of 10 fully developed compound leaves and 5 inflorescences were taken, and the length and width of the compound leaf, the number of leaflets per leaf, the length and width of the leaflets, the length and width of the inflorescences, the number of flowers per inflorescence, and the maximum diameter of individual flowers were determined. the leaves and inflorescences were taken from the lower third of the southern side of the canopy. the samples were herbarium-processed, and then the morphometric biologica nyssana ● 16 (1) june 2025: 93-104 ocokoljić et al. ● the impact of climatic parameters in belgrade on the indigo himalayan species from the subtropical biome woody taxa, respectively. the onset of spring and summer phenophases was recorded earlier, while autumn and winter phenophases occurred later compared to previous studies, showing significant correlations with air temperatures and accumulated heat sums. climate change can affect bud dormancy and cold resistance, which is important for the adaptation of woody plants and their survival during winter in the conditions of a moderately continental climate (lang et al., 1987). therefore, air temperatures are fundamental for regulating plant dormancy. in most woody plants of the temperate zone, growth cessation and bud dormancy are induced by short photoperiods in autumn, while low temperatures are required for breaking dormancy (cosmulescu & ionescu, 2018; ocokoljić et al., 2024). projected and current global warming in europe is a focus due to the detrimental impact of mild winters and high autumn temperatures on woody plants. concern is heightened for native species, as well as for nonnative species, including the exotic indigofera heterantha wall. ex brandis (first published in forest fl. n.w. india: 135, 1874), native to the subtropical biome from afghanistan to tibet and the himalayas (fop, 2025; powo, 2025). faced with incomplete knowledge of the effects of climate change and the impact of global warming on the growth, development, and phenology of indigo himalayan in belgrade’s public green spaces, a study was conducted to determine the impact of seasonal air temperatures during summer and autumn on phenological processes. the study also aimed to assess the impact of climate challenges on the cessation of growth, extension of the growing season, new growth, secondary flowering, and the morphological attributes of the newly identified exotic species in serbia. additionally, the aim of the work was to promote the application of phenological data in landscape architectural compositions to optimise maintenance costs, plant material selection for appropriate locations and purposes, considering that woody taxa are vital elements in these compositions. materials and methods study area in the territory of belgrade, a network of phenological stations was established over an area of 450 km² for the purposes of previous studies. in accordance with the recommendations of wmo (2009) and fhz (2013), the observation sites are representative of the broader surroundings based on land type, terrain appearance and position, aspect, slope, and coverage of the urban and suburban zones. through 95 biologica nyssana ● 16 (1) june 2025: 93-104 ocokoljić et al. ● the impact of climatic parameters in belgrade on the indigo himalayan species from the subtropical biome characteristics were determined using the uthscsa image tool, except for the flower diameter, which was measured with electronic calipers before herbarium processing. the abundance of the yield was assessed according to stilinović (1985) using the kaper scale from 0 to 5, where 0 – no yield (0% branches with fruits); 1 – very small yield (<20%); 2 – small yield (>20−<40%); 3 – moderate yield (>40−<60%); 4 – abundant yield (>60−<90%); and 5 – maximum yield (>90%). fruits were collected immediately after ripening in october, and their length and width were measured with high-precision (±0.001 mm) electronic calipers. the number of seeds in the fruits was also determined. the colour of the leaves, flowers, and fruits was defined using the rgb (red/green/blue) colour system. statistical analysis to determine the significance of trends in climate data series, the non-parametric mann-kendall test was used, and to quantify the magnitude of the identified trends in the climatic time series, the sen’s slope method was applied. growing degree days (gdd) for each of the key phenophases were determined using the method of lalić et al. (2021). relationships between air temperatures, primary and secondary flowering were analysed using the spearman rank test and regression. for the analysis of phenological changes, climate data and field observations were combined. phenological responses of indigo himalayan, even in the first year, were variable in relation to literature reports, so adequate non-parametric methods were applied in accordance with the complexity of climatic events. for the analysis of morphological, quantitative data of indigo himalayan, descriptive statistics and the spearman rank test were used. data processing was performed using the xlstat 2020 and past 4.11 software packages. results and discussion chronology of climate data on a monthly and annual basis, the average air temperatures, average maximum air temperatures, average minimum air temperatures, and total precipitation sums were calculated for the reference period (1991-2020), the period 2021-2023, and 2024 for the main meteorological station surčin (tab. 1). the results are in line with the predictions (ipcc, 2019) that global warming will reach 1.5 °c between 2030 and 2052. however, when comparing the period 2021-2023 to the reference period, it is observed that the average temperatures have already exceeded the stated value (2.6 °c), which is consistent with the findings that greater warming than the global average has been observed in many regions and that higher values are found over land (ipcc, 2019). spearman’s correlation coefficients were determined, with a probability of p<0.05 for the period 1991-2024, for average monthly maximum and minimum temperatures (0.979), average monthly maximum temperatures and total precipitation (0.706), and average monthly minimum temperatures and total precipitation (0.790). very strong positive correlations between average monthly maximum and minimum temperatures, as well as between average monthly minimum temperatures and total precipitation, were statistically significant and indicate their consistently increasing relationship. the correlation between average monthly maximum temperatures and total precipitation is moderate and positive. the results of the mann-kendall and sen’s slope tests for the parameters for which spearman’s correlation coefficients (ρ) were determined are summarized in tab. 2. a significant increase in average monthly maximum temperatures was recorded in april, july, august, and december, in average monthly minimum air temperatures in february and from june to december, while a significant increase in monthly precipitation sums was observed only in may. other trends were not statistically significant (tab. 2). the findings of the study are consistent with the reports of wmo (2021). impacts of warming seasons on phenology the required accumulated heat sum for the phenological phases of indigo himalayan during summer (fig. 1a) was 1354.3 °c 19bbch (first leaves fully expanded), 2119.7 °c 60bbch (beginning of flowering), 2481.3 °c 65bbch (full flowering), and 2797.23 °c 39bbch (shoots about 90% of final length). for the phenological phases during autumn (fig. 1b), it was 3162.3 °c 91bbch (shoot growth completed), 3262.7 °c 69bbch (end of flowering), 3348.8 °c – 19/2bbch (first leaves fully expanded), 3532.4 °c – 39/2bbch (shoots about 90% of final length), 3575.1 °c – 60/2bbch (beginning of flowering), 3621.0 °c 87bbch (fruit ripe for picking), 3729.5 °c – 65/2bbch (full flowering), and 3856.0 °c – 69/2bbch. the 97bbch phase all leaves fallen was recorded at the end of december on the first shoot growth, without the leaf discoloration phase (95bbch). secondary growth completely ceased on december 25th following two extremely rainy days, during which a total of 32.4mm of precipitation was recorded, including 14cm of snow (rhmz). the determined accumulated heat sums are consistent with those of ornamental taxa that flower in the summer and had secondary flowering during autumn 2022 in belgrade 96 biologica nyssana ● 16 (1) june 2025: 93-104 ocokoljić et al. ● the impact of climatic parameters in belgrade on the indigo himalayan species from the subtropical biome ta bl e 1. c lim at ic v ar ia bl es fo r t he re fe re nc e pe rio d 19 91 -2 02 0, 2 02 120 23 , 2 02 4 an d de vi at io ns fr om th e no rm fo r t em pe ra tu re s an d pr ec ip ita tio ns (% ) f or th e su rč in w ea th er s ta tio n ba se d on h ou rly a nd d ai ly d at a (s ou rc e r h m z) m ea n ai r te m pe ra tu re (° c ) m on th s p er io d i ii ii i iv v v i v ii v ii i ix x x i x ii t m ea n 19 91 /2 02 0 1 3 7. 5 12 .9 17 .6 21 .4 23 .2 23 .2 18 12 .8 7. 4 2. 2 12 .5 t m ea n 20 21 /2 02 3 3. 6 5. 3 7. 3 10 .8 17 .9 22 .9 25 .4 23 .8 19 .3 14 .3 8. 7 5. 3 13 .7 t m ea n 20 24 2. 9 10 .1 11 .6 15 .7 18 .6 24 .7 26 .8 27 .4 19 .9 14 .4 5. 7 3. 2 15 .1 d ev ia tio n t m ea n 20 24 fr om no rm al ity 1 99 120 20 1. 9 7. 1 4. 1 2. 8 1 3. 3 3. 6 4. 2 1. 9 1. 6 -1 .7 1 2. 6 d ev ia tio n t m ea n 20 24 fr om no rm al ity 2 02 120 23 -0 .7 4. 8 4. 3 4. 9 0. 7 1. 8 1. 4 3. 6 0. 6 0. 1 -3 -2 .1 1. 4 m ea n m ax im um a ir te m pe ra tu re (° c ) t m ea n 19 91 /2 02 0 4. 5 7. 4 12 .9 18 .4 23 .2 26 .9 29 29 .3 24 .1 18 .5 11 .9 5. 5 17 .6 t m ea n 20 21 /2 02 3 6. 9 10 .5 12 .7 16 23 .3 28 .3 31 .3 29 .8 25 .4 20 .1 12 .8 9. 1 18 .8 t m ea n 20 24 7. 1 15 .5 17 .2 21 .6 23 .7 29 .9 32 .2 33 .6 25 .9 20 .3 10 .4 5. 9 20 .3 d ev ia tio n t m ea n 20 24 fr om no rm al ity 1 99 120 20 2. 6 8. 1 4. 3 3. 2 0. 5 3 3. 2 4. 3 1. 8 1. 8 -1 .5 0. 4 2. 7 d ev ia tio n t m ea n 20 24 fr om no rm al ity 2 02 120 23 0. 2 5 4. 5 5. 6 0. 4 1. 6 0. 9 3. 8 0. 5 0. 2 -2 .4 -3 .2 1. 5 m ea n m in im um a ir te m pe ra tu re (° c ) t m ea n 19 91 /2 02 0 -2 .3 -1 2. 6 7. 1 11 .8 15 .4 16 .8 16 .9 12 .6 7. 9 3. 6 -0 .9 7. 5 t m ea n 20 21 /2 02 3 0. 7 0. 8 2 5. 6 12 .2 16 .6 18 .8 17 .8 13 .9 9. 3 5. 1 1. 9 8. 7 t m ea n 20 24 -0 .8 5. 2 6. 3 9. 3 13 .6 18 .4 20 .1 19 .8 14 .1 9. 3 1. 4 0. 7 9. 8 d ev ia tio n t m ea n 20 24 fr om no rm al ity 1 99 120 20 1. 5 6. 2 3. 7 2. 2 1. 8 3 3. 3 2. 9 1. 5 1. 4 -2 .2 1. 6 2. 3 d ev ia tio n t m ea n 20 24 fr om no rm al ity 2 02 120 23 -1 .5 4. 4 4. 3 3. 7 1. 4 1. 8 1. 3 2 0. 2 0 -3 .7 -1 .2 1. 1 t ot al a nd m ea n am ou nt s o f p re ci pi ta tio n (m m ) pe ri od i ii ii i iv v v i v ii v ii i ix x x i x ii ∑ t m ea n 19 91 /2 02 0 42 .4 34 41 .7 47 .4 68 .1 80 .1 58 .2 54 56 50 .7 45 .5 48 .3 62 6. 4 t m ea n 20 21 /2 02 3 48 35 .6 26 .8 59 .2 60 57 .5 65 .4 51 .9 72 .7 39 .9 10 3. 4 75 .4 69 5. 9 97 biologica nyssana ● 16 (1) june 2025: 93-104 ocokoljić et al. ● the impact of climatic parameters in belgrade on the indigo himalayan species from the subtropical biome t m ea n 20 24 37 .1 4. 8 27 .7 23 .3 99 .8 92 .5 69 .4 6. 5 86 .8 44 45 .6 63 .2 60 0. 7 d ev ia tio n t m ea n 20 24 fr om no rm al ity 1 99 120 20 87 .5 14 .1 66 .4 49 .1 14 6. 5 11 5. 4 11 9. 2 12 15 5 86 .8 10 0. 2 13 0. 8 95 .9 d ev ia tio n t m ea n 20 24 fr om no rm al ity 2 02 120 23 77 .2 13 .5 10 3. 5 39 .4 16 6. 3 16 0. 8 10 6. 1 12 .5 11 9. 4 11 0. 4 44 .1 83 .8 86 .3 ta bl e 2. r es ul ts o f t he m an nke nd al l t re nd a nd s en ’s s lo pe te st s fo r a ve ra ge m on th ly m ax im um a nd m in im um a ir te m pe ra tu re s an d m on th ly p re ci pi ta tio n su m s fo r t he p er io d 19 91 -2 02 4, b as ed o n da ta fr om m m s su rč in t es t m on th t m ea n m ax t m ea n m in pr ec ip ita tio n k en da ll' s ta u pva lu e se n' s sl op e k en da ll' s ta u pva lu e se n' s sl op e k en da ll' s ta u pva lu e se n' s sl op e i 0. 18 7 0. 12 3 0. 07 1 0. 23 0. 05 8 0. 07 2 0. 11 6 0. 34 3 0. 34 ii 0. 22 6 0. 06 2 0. 14 6 0. 30 1 0. 01 3 0. 10 9 0. 16 2 0. 18 2 0. 38 ii i 0. 12 3 0. 31 3 0. 05 2 0. 22 6 0. 06 2 0. 06 2 0. 03 4 0. 79 0. 11 5 iv 0. 24 3 0. 04 5* 0. 07 5 0. 11 6 0. 34 3 0. 02 9 -0 .0 36 0. 77 8 -0 .1 63 v -0 .0 41 0. 74 4 -0 .0 13 0. 16 6 0. 17 3 0. 03 2 0. 25 2 0. 03 8* 1. 01 1 v i 0. 21 9 0. 07 1 0. 06 0. 50 8 <0 .0 00 1* 0. 08 2 0. 04 1 0. 74 4 0. 23 5 v ii 0. 29 4 0. 01 5* 0. 07 2 0. 51 4 <0 .0 00 1* 0. 07 6 -0 .0 48 0. 7 -0 .2 83 v ii i 0. 25 5 0. 03 5* 0. 08 5 0. 46 9 0. 00 0* 0. 07 4 -0 .0 71 0. 56 3 -0 .3 31 ix 0. 19 4 0. 10 9 0. 06 9 0. 35 1 0. 00 4* 0. 07 6 -0 .0 23 0. 85 9 -0 .1 57 x 0. 19 1 0. 11 6 0. 06 8 0. 26 6 0. 02 8* 0. 05 5 -0 .0 37 0. 76 7 -0 .1 64 x i 0. 16 9 0. 16 3 0. 06 0. 24 4 0. 04 4* 0. 07 3 -0 .0 09 0. 95 3 -0 .0 07 x ii 0. 33 9 0. 00 5* 0. 14 1 0. 36 9 0. 00 2* 0. 10 3 0. 00 2 1 0. 00 8 *a s th e co m pu te d pva lu e is g re at er th an th e si gn ifi ca nc e le ve l 0 .0 5, o ne c an no t r ej ec t t he n ul l h yp ot he si s h 0. h 0: th er e is no tr en d in th e se rie s 98 biologica nyssana ● 16 (1) june 2025: 93-104 ocokoljić et al. ● the impact of climatic parameters in belgrade on the indigo himalayan species from the subtropical biome (ocokoljić et al., 2023). the studied elements of the phenological patterns suggest that indigo himalayan, due to its habitus and flowering, possesses high ornamental value at the end of summer and during fig. 1. phenogram of indigo himalayan in belgrade during 2024: a) 19bbch (first leaves fully expanded), 39bbch (shoots about 90% of final length), 60bbch (beginning of flowering), 65bbch (full flowering) and b) 69bbch (end of flowering), 87bbch (fruit ripe for picking), 91bbch (shoot growth completed), 19/2bbch (second first leaves fully expanded), 39/2bbch (second shoots about 90% of final length), 60/2bbch (second beginning of flowering), 65/2bbch (second full flowering) and 3856.0 °c – 69/2bbch (second end of flowering) autumn, which is why introduced species are important in the selection of plants for urban landscape design, as noted by khamraeva et al. (2024). according to wu & raven (2010), indigo 99 biologica nyssana ● 16 (1) june 2025: 93-104 ocokoljić et al. ● the impact of climatic parameters in belgrade on the indigo himalayan species from the subtropical biome himalayan flowers during the period of may-june, while according to brickell & kindersley (2008), flowering occurs during the summer, which is consistent with the pl@ntnet database (gbif, 2025) for the northern hemisphere and our research. however, secondary flowering, which followed on the new shoot growth (on average 27 cm) during september and october 2024 in belgrade (fig. 2), has not been previously recorded. the results require a comparative analysis of phenological and climatic data, considering the findings of büntgen et al. (2022) that the onset and longer duration of the growing season reflect the local climate. fig. 2. a) autumn shoots with secondary leafing (8 october 2024), and b) secondary flowering (with silver linden in the background in the leaf discoloration phase, 16 october 2024) of indigo himalayan on the square in belgrade considering that regular irrigation of the newly planted species was recorded during phenomonitoring, and that indigo himalayan is a drought-resistant species (agm, 2017), it is evident that air temperatures, primarily the maximum and minimum temperatures during summer and autumn, have the most significant impact on multiple phenological responses, as this is a species from the subtropical biome. therefore, to adequately understand the phenological responses to climate change, seasonal warming trends (summer and autumn) were determined based on data from mms surčin. the growth of t mean for summer 2024 (june-august) was 3.7 °c, for autumn (septemberfig. 3. graphical representation of sen's slope and p-value for seasonal mean monthly maximum and minimum air temperatures for summer (a and b) and autumn (c and d) for the period 1991-2024, based on data from mms surčin (values in red are slopes for statistically significant trends (p<0.05)) november) 0.6 °c, tmeanmax 3.5 °c and 0.7 °c, and tmeanmin 3.0 °c and 0.3 °c. the significance of trends in mean maximum and minimum air temperatures according to the sen's slope test is shown in fig. 3. a significant increase in mean maximum temperatures was observed only for july and august (summer). in contrast, tmeanmin shows significant increasing trends for both summer and autumn. the primary flowering lasted for 47 days, and after 28 days and a heatwave in september, secondary flowering began and lasted for 36 days until the first frost day (minimum temperature below 0 °c, rhmz). taking into account the aforementioned trends, and in order to assess the phenological responses based on daily maximum and minimum air temperatures, a regression analysis was conducted for primary and secondary flowering (fig. 4). a rising trend is evident for both variables during summer (fig. 4a and b) after approximating the isolated data, i.e., determining the regression line. however, the increase in daily maximum and minimum air temperatures did not affect primary flowering, which is expected given the species' origin. during secondary flowering (fig. 4c and d), the trend is decreasing for both variables, and the decline in minimum temperatures significantly 100 biologica nyssana ● 16 (1) june 2025: 93-104 ocokoljić et al. ● the impact of climatic parameters in belgrade on the indigo himalayan species from the subtropical biome fig. 4. scatter plots and predictions of the impact of daily maximum and minimum air temperatures during the primary (a and b) and secondary (c and d) flowering periods of indigo himalayan in belgrade affected secondary flowering. data analysis showed that for primary flowering of indigo himalayan, the daily maximum and minimum temperatures were in a ratio of 74.7%: with a maximum of 33.6 °c and a minimum of 19.6 °c; while for secondary flowering, in a ratio of 68.8%, the temperatures were 19.4 °c (max) and 8.3 °c (min). statistical significance of the conclusions, at p<0.0001, is confirmed by the results of the regression anova analysis (tab. 3). the research indicates that temperatures influence the trends of phenological phases in indigo himalayan in belgrade, which is consistent with documented variations globally (piao et al., 2019), in europe (menzel et al., 2020), and in serbia (čukanović et al., 2024, petrov et al., 2024). the findings also align with the statements by mack et al. (2000), suggesting that biological invaders thrive at the expense of the environment in natural areas, while exotic species in urban zones act as components of the landscape that improve environmental conditions. considering that, according to pérez–ramos et al. (2019), the phenology of plants is influenced by climate change differently depending on location and taxa, and given that the growth or decline of temperatures influences indigo himalayan to varying degrees („a tolerant species down to -15 °c that fully regenerates the following year even after freezing to the ground”, agm, 2017), it is possible to predict its adaptability, sustainability, ornamental value, as well as its ecological stability and ecosystem services. morphometric studies of the leaf, floral structures and fruits indigo himalayan is a deciduous shrub that can grow up to 3 meters in height (wu & raven 2010). in the studied location, by the end of 2024, it reached a height of 2.10 meters, with a long flowering period during the second half of summer (brickell & kindersley, 2008). considering that there are fewer such taxa in belgrade, and that the species was identified for the first time on a public green space, the study focused on the metric and morphological characteristics of the leaves, inflorescences, flowers, and fruits. the length of the leaves ranged from 16.5 cm to 26.8 cm, while the number of leaflets in the compound leaf ranged from 18 to 27. the length of the leaves reported in some earlier studies and 101 biologica nyssana ● 16 (1) june 2025: 93-104 ocokoljić et al. ● the impact of climatic parameters in belgrade on the indigo himalayan species from the subtropical biome table 3. anova results for the impact of daily maximum and minimum air temperatures on the flowering of indigo himalayan in belgrade, at a significance level of p<0.0001 parameter df ss ms f significance f summer tmax regression 1 3390.892 3390.892 3.407603 0.071481 residual 45 44779.32 995.096 total 46 48170.21 tmin regression 1 89.08413 89.08413 0.083375 0.7741 residual 45 48081.13 1068.47 total 46 48170.21 autumn tmax regression 1 1529.262 1529.262 1.641548 0.207679 residual 39 36332.3 931.5974 total 40 37861.56 tmin regression 1 6415.05 6415.05 7.955953 0.00001 residual 39 31446.51 806.3208 total 40 37861.56 research was considerably smaller than the findings in belgrade, as hammad et al. (2021) report a leaf length of 6 to 7 cm. the result for the number of leaflets is consistent with the fop (2025), which notes that a compound leaf contains between 13 and 31 leaflets. however, this study also observed that the leaves were both even and odd-pinnate (50:50%), a feature that has not been previously recorded in the literature available to the authors. the standard deviations were low (0.88 and 1.01), as were the coefficients of variation (13.43 and 13.44), indicating low variability for both parameters. the length of the leaflets ranged from 25.0 mm to 42.9 mm, and the width ranged from 11.8 mm to 19.2 mm, which exceeds the ranges of 5 to 25 mm for length and 2 to 15 mm for width reported by hammad et al. (2021) and fop (2025). the standard deviations were low (1.76 and 0.87), but the coefficients of variation were high (15.58 and 17.19), indicating higher variability. the spearman rank correlation coefficient, with a probability of p<0.05, between the length and width of the leaflets confirmed a strong positive correlation (0.98788), indicating that as the length of the leaflets increases, so does their width, as well as a moderate positive correlation between leaf length and the number of leaflets (0.66061), meaning that as the leaf length increases, the number of leaflets in the compound leaf also increases. other correlations were not significant. the upper side of the leaflets is dark green (rgb 49/76/21), and the underside is gray-green (rgb 125/149/117) the size of the inflorescence and flowers, as well as their color, are of particular importance for flowering ornamental species. the study results highlight the inflorescence length, which ranged between 4.0 and 4.8 cm, the inflorescence width, which was 4 cm for all analyzed inflorescences, the number of flowers per inflorescence, ranging from 22 to 24, and the largest flower diameter, which ranged from 0.8 to 1.1 cm (fig. 5a and b). the research findings for the inflorescence length align with the range of 4 to 8 cm reported by hammad et al. (2021). the available literature does not provide any information on the number of flowers per inflorescence, the width of the inflorescence, or the largest diameter of individual flowers. the small variability is confirmed by the low values of standard deviation (0.56, 0.1, 1.41, and 0.21) and coefficients of variation (12.85, 0.6, and 6.14) for the first three parameters, while a higher variability is observed for the largest flower diameter with a coefficient of variation of 22.32. the spearman rank correlation coefficient, with a probability of p<0.05, between the inflorescence length and the number of flowers, the inflorescence length and the flower diameter, and the number of flowers per inflorescence and the flower diameter is positive and complete, which is adequately confirmed by the results of descriptive statistics. other correlations were not significant. the visual aspect of the inflorescence and flower is shown in fig. 5a and b. the outer side of the petals is in shades of pinkish-purple (rgb 177/108/129 to 168/93/116), while the inner side is a light lavender color (rgb 168/135/144) with a yellow-brown or skin-like spot (rgb 188/168/177). the fruit yield was low (rating 1), and the seed production relative to the number of embryos was very low (9.09%). the fruits were elongated, linear pods in shades of brown ranging from rgb 100/70/46 to 86/64/50 (fig. 5c). the length of the pods ranged from 3.7 to 6.2 cm, the largest pod width from 6.5 102 biologica nyssana ● 16 (1) june 2025: 93-104 ocokoljić et al. ● the impact of climatic parameters in belgrade on the indigo himalayan species from the subtropical biome fig. 5. inflorescences a) and individual flower b) from the primary flowering (august 4, 2024) and c) mature fruits (october 16, 2024) of indigo himalayan in belgrade to 7 cm, and the number of seeds per pod ranged from 3 to 5. the length and width of the pods are in agreement with the findings of fop (2025), and the seed count is consistent with the results of wu & raven (2010). the standard deviation was low (1.30, 0.29, and 1.5), but the coefficients of variation for the length of the pods and seed count were high (27.57 and 31.49), indicating their higher variability. an exception is the largest pod width, where the coefficient of variation is low (4.33). the spearman rank correlation coefficients, with a probability of p<0.05, were not statistically significant for the analyzed fruit parameters. the pioneering research conducted in belgrade during 2024 and january 2025 contributes to the conservation of indigo himalayan as a flowering ornamental species from extinction, as predicted by bachman et al. (2024), although there are no predictions regarding the species' threat status (iucn, 2025). to make a reliable estimate of the future global vegetation development, it is essential to identify vegetation responses to climate change. in this sense, this quantitative review and analysis indicate acclimatization as an ecological process that enables the spread of indigo himalayan beyond the subtropical biome as a response to global warming. conclusion the study provides basic information on the effects of climate change on the phenology and morphology of the leaves, flowers, and fruits of indigo himalayan in an urban landscape, under the conditions of a moderately continental climate. however, it is essential that research continues in order to quantify all attributes of this new exotic species in serbia. future studies should focus on the aesthetics, functionality, identity, and ecology of urban landscapes where indigo himalayan serves as an element in landscape architectural compositions. this is because exotic species have advantages over native ones in terms of ecological performance and adaptive capacity, with the obligation to investigate their potential invasiveness when introduced to new habitats. the importance of further research confirms the concerns of researchers and designers regarding the potential negative impacts of exotic species and the advantages of using native species, which has 103 biologica nyssana ● 16 (1) june 2025: 93-104 ocokoljić et al. ● the impact of climatic parameters in belgrade on the indigo himalayan species from the subtropical biome led to the recommendation by scientists that native vegetation should be prioritized over exotic species, even in urban green spaces. acknowledgements. this scientific research conducted at the university of belgrade – faculty of forestry in 2025, under grant number 451-03-137/2025-03/200169 was supported by the ministry of science, technological development and innovation of the republic of serbia. references agm (award of garden merit plants). 2017. ornamental. retrieved from https://www.rhs.org. uk/plants/pdfs/agm-lists/agm-ornamentals.pdf anjos, l., gaistardo, c., deckers, j., dondeyne, s., eberhardt, e., gerasimova, m., harms, b., jones, a., krasilnikov, p., rein-sch, t., vargas, r., & zhang, g. 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(2019). scale-dependent interactions between tree canopy cover and impervious surfaces reduce daytime urban heat during summer. proceedings of the national academy of sciences usa, 116(15), 7575-7580. floristic investigation of the “grand park of tirana” with regard to urban indicators biologica nyssana 7 (2)  december 2016: 113-124 mesiti, a., dinga, l.  floristic investigation of the „grand park of tirana“… 113 original article received: 30 october 2016 revised: 01 december 2016 accepted: 07 december 2016 floristic investigation of the “grand park of tirana” with regard to urban indicators ajola mesiti, liri dinga university of tirana, faculty of natural science, research center of flora and fauna * e-mail: ajola_mesiti@yahoo.com abstract: mesiti, a., dinga, l.: floristic investigation of the “grand park of tirana” with regard to urban indicators. biologica nyssana, 7 (2), december 2016: 113-124. the paper presents a floristic investigation undertaken in the urban “grand park of tirana” in three years’ period (2012 – 2015). the purpose of this study is to give floristic data and distribution of spontaneous plant species which occur in “grand park of tirana” and determine and analyze their urban indicators. spontaneous flora of the park contains 240 species of vascular plants from 179 genera and 54 families. among the families the most dominants in species are the poaceae (30 species), fabaceae (24) and asteraceae (21). the analysis of life-form spectra shows that the dominant life form is the hemicryptophyte. the urban indicators analysis shows that the “grand park of tirana” is characterized by a high number of urbanophobic species, which mean that the park has still particular natural places. key words: urbanity, urban park, anthropotolerance, urban ecology apstrakt: mesiti, a., dinga, l.: floristička istraživanja “grand parka tirana” u odnosnu na urbane indikatore. biologica nyssana, 7 (2), decembar 2016: 113-124. u radu su prikazana floristička istraživanja sprovedena u urbanom “grand parku u tirani” i trogodišnjem period. cilj studije je bio da se daju floristički podaci i distribucija samoniklih biljnih vrsta koje se javljaju u “grand parku u tirani” i da se odredi i analizira njihova urbana indikacija. samonikla flora parka sadrži 240 vrsta vaskularnih biljaka iz 179 rodova i 54 familije. među familijama najdominantnije vrstama su poaceae (30 vrsta), fabaceae (24) i asteraceae (21). analiza spectra životnih formi je pokazala da je dominantna životna forma hemikriptofita. analiza urbanih indikatora pokazuje da je “grand park u tirani” okarakterisan velikim brojem urbanofobnih vrsta, što ukazuje da park još uvek ima prirodnih površina. key words: urbanizacija, urbani park, antropotolerancija, urbana ekologija introduction urban parks are defined as delineated open space areas, mostly dominated by vegetation and water, and generally reserved for public use. urban parks are usually locally defined (by authorities) as “parks” (k o n i j n e n d i j k et al., 2013). if they are well organized and maintained, they serve the city in different ways. the process of urbanization, has profoundly changed the landscape, and created new environmental conditions for plants (s u k o p p , 7 (2) • december 2016: 113-124 12th sfses • 16-19 june 2016, kopaonik mt doi: 10.5281/zenodo.200408 biologica nyssana 7 (2)  december 2016: 113-124 mesiti, a., dinga, l.  floristic investigation of the „grand park of tirana“… 114 1973; s u k o p p & w e r n e r , 1983). two-third of all europeans now reside in towns or cities (g i r a r d e t , 1992; eea, 1998) and recently aspects such as “amount of public green spaces per inhabitant”, “public parks” and “recreation areas” are often mentioned as important factors to make the city livable, pleasant and attractive for its citizens, which makes them one of the indicators of sustainable cities (c h i e s u r a , 2004). beside the direct impact on the environment such as noise reduction, air purification, microclimate stabilization, parks benefit the city life quality and fulfill other important human need. social capital is created in parks. the parks ensure a place where people can meet together, interact with each other in a shared environment (k u o et al., 1998). a well-organized park system contributes in the citizen’s health. certain areas for jogging, playing or bike lanes or areas to walk pets provide the possibilities to make an active lifestyle (s c h r o e d e r , 1991). natural capital is preserved in parks. the parks are not only recreation spaces, but natural sustainable structures which affect the evaluation of nature by inhabitants. urban parks are also attraction to tourists (k o n i j n e n d i j k et al., 2013), as an example we can mention the “central park of new york”. according to c r o m p t o n (2001), it is well known that urban parks affect the prices of the real estate and buildings around them, which is true in tirana for the areas nearby “grand park of tirana”. the arguments mentioned above, are strong reasons which have attracted scientists interest to study urban parks (c h i e s u r a , 2004; i g n a t i e v a & k o n e c h n a y a , 2004; n i e l s e n et al., 2014 etc.). the first comprehensive studies of flora of urban ecosystems started in the 1970′s (f a l i n s k i , 1971; s u k o p p , 1973 etc.), and then continued (s u k o p p & w e r n e r , 1983, w i t t i g et al., 1985; k o w a r i k , 1990). nowadays deep studies of flora on urban habitats, mostly in central and northern europe, are carried out (s u k o p p , 2002; k u h n et al., 2003; b o m a n o w s k a & w i t o s l a w s k i , 2008; m o r a c z e w s k i & s u d n i k w o j c i k o w s k a ,, 2007 etc.), more recently they are undertaken also in south europe, in italy (b i a n c o et al., 2003; c e l e s t i -g r a p o w et al., 2006; b u c c h e r i & m a r t i n i , 2015), croatia (m i l o v i ć & m i t i ć , 2012; a l e g r o et al., 2013), bosnia and herzegovina (m a s l o , 2014), serbia (j o v a n o v i ć , 1994; s t a v r e t o v i ć & j o v a n o v i ć , 2010), montenegro (s t e š e v i ć & j o v a n o v i ć , 2008; s t e š e v i ć et al., 2009, 2014) and greece (c h r o n o p o u l o s & c h r i s t o d o u l a k i s , 1996, 2000), and such interest is spread all over the world (m o o r e et al., 2003; z a n i n et al., 2005 loeb, 2006 etc.). in albania, this study in “grand park of tirana” represents the first scientific work on floristic investigations in urban parks. till now, the only publications related to the study area are some memories of agronomists who worked during and after the construction of the park (n i s h a n i , 2007; n i s h a n i & m ë z i u , 2013). the other publication is focused on natural ecosystems and they are very few, such as “flora of tirana” (p a p a r i s t o et al., 1962), phd thesis “vlerësimi i biodiversitetit floristik e fitosociologjik i parkut kombëtar (i propozuar) të dajtit” (k a l a j n x h i , 2006). the goal of this study was to give floristic data and distribution of native plant species which occur in “grand park of tirana”, and determine and analyze their urban indicators. if considered that “grand park of tirana” represents the only one of its kind in the country the study takes even more importance. additionally, current political developments give priorities to urban parks construction in other cities in the frame of urban development, thus this paper would support policies on further management of these areas. material and methods study area the grand park was built in the autumn of 1958 on the southern hills of the tirana city with a total surface of 350 ha (n i s h a n i & m ë z i u , 2013) and on existing area of vegetation. on the other hand, within this territory was also built the “artificial lake of tirana” with a surface of 48 ha. here were planted thousands of trees, shrubs and herbaceous ornamental plants, such as fraxinus ornus, acacia dealbata, ligustrum sp., cupressus sempervirens, laurus nobilis, pinus sp. etc. (n i s h a n i , 2007). it used to be the most important event of those years. inside the grand park are built many facilities like summer theater, memorial center, artworks which settled this area as true urban park. nearby the park, it is found also the “presidential palace park” which is surrounded by walls and it is allowed to be frequented by the city inhabitants. over the last decades, tirana’s urban areas have expanded, “swallowing” from farmland and forests. therefore, with the green areas decrease, the grand park of tirana has become the more frequented and consequently affected by the anthropogenic pressure, due to the lack of management and the over exploitation by its inhabitants. it is surrounded by roads with high traffic, inhabitant’s buildings, urban waste especially from restaurants and other recreational centers that biologica nyssana 7 (2)  december 2016: 113-124 mesiti, a., dinga, l.  floristic investigation of the „grand park of tirana“… 115 are around the park. nowadays, the “grand park of tirana” has an area of about 250 ha (fig. 1). the climatic conditions are related with the hilly lowland relief. the climate is classified as mediterranean climate with a wet winter and a warm and dry summer. of particular importance are the amplitudes of the higher annual average temperatures ranging from 15 to 22 0c in summer to below zero in winter (k a b o , 1990). vegetation of the study area the “grand park of tirana” is situated in an existing vegetation, which is still present nowadays. therefore, it makes this park a hybrid construction, composed by the native flora and the cultivated one. anyway the native vegetation where the park was build is the vegetation around the tirana city. in these areas mainly prevails the natural vegetation of mediterranean shrubs. it is represented by the alliance quercion ilicis br.-bl. ex molinier 1934 (ass. orno-quercetum ilicis wald.), but in the flysch slopes it degrades towards erico-arbutus unedo community (h o r v a t et al, 1974). on the upper altitudes, tirana city is surrounded by thermophile oak forests (quercetalia pubescentis br.-bl. 1931), mainly of the alliance of quercion confertae, and in the degraded places is represented by the alliance carpinion orientalis horvat 1958 (k a l a j n x h i , 2006; h o r v a t et al., 1974). afforestation has influenced the park too. within the park is planted a small forest with pinus halepensis mill., and it is created the street of 1000 cypress trees (cupressus sempervirens l.). floristic analysis this analysis was based on application of the following procedures: 1) literature review, 2) collecting field data (fig. 2), 3) mapping of the species distribution using arcmap 10.1 (environmental systems research institute, new york, ny, usa); 4) analyzing components of flora, life form, chorology and urban indicators (urbanophobic, urbanoneutral, urbanophilic). our floristic investigation was conducted within the period 2012-2015 and the records were carried out for all the year (for some winter species), but most of the excursions were carried out during flowering seasons (from april to october). the main information of ecological plant groups (pine forest, oak forest, meadows, wetland) was derived by interpreting aerial photographs. the total area surface was divided into quadrants (300x300 m) (fig. 2). it was used as layers, topographic maps of tirana at a scale 1:10000 from 1957 and 1990 year, gis format shapefile map of the “grand park” at a scale 1:1250, and 9 aerophotogrammetric maps, which were obtained from the state archives building. the coordinative system used for the cartography was wgs, 1984. field data collection was mainly based on floristic registration, gps data recording with garmin software, photography and the unknown species were preserved in the herbarium. taxonomic identification was done by using the determination key books such as: “flora e shqipërisë” (p a p a r i s t o et al., 1988-2000), “flora ekskursioniste e shqipërisë” (d e m i r i , 1983), “flora europea” (t u t t i n et al., eds., 1964-1980), “udhëheqës fushor i florës së shqipërisë” (v a n g j e l i , 2003, 2015) and different international plant databases such as plant list and biolflor (www.theplantlist.org, www.biolflor.de). life form classification was defined according to the raunkiaer system (e l l e n b e r g & m u e l l e r -d o m b o i s , 1967) and for the conservation status of plants it is used the iucn proposed system (w a l t e r & g i l l e t ., 1998) and the national red list of wild flora and fauna (a n o n y m o u s , 2013). fig. 1. geographical position and the boundaries of the grand park of tirana biologica nyssana 7 (2)  december 2016: 113-124 mesiti, a., dinga, l.  floristic investigation of the „grand park of tirana“… 116 the plants species nomenclature was defined according the new system by euro+med plant database (ww2.bgbm.org). according to sukopp & werner (1983), the number of taxa of vascular plants in urban floras is determined by a series of factors: geographic positioning, climatic conditions and the possibility for the immigration of species. the flora of the park is analyzed based on confronting personal observation with literature data (w i t t i g et al., 1985; i g n a t i e v a & k o n e c h n a y a , 2004; b u c c h e r i & m a r t i n i , 2015; s t e š e v i ć et. al., 2014). it was studied according to the anthropotolerance and following urban indicators: a. urbanophilic plants (uphi)—species that prefer human-disturbed or human-altered habitats. they can be subdivided into extreme urbanophilic (uphi=5) and moderate urbanophilic (uphi=5) b. urbanoneutral plants (un)—species that can grow in undisturbed natural habitats as well as in human-disturbed habitats, c. urbanophobic plants (upho)—species that avoid human-altered urban habitats. they can be subdivided into extreme urbanophobic (upho=1) and moderate urbanophobic (upho=1) according to the theory of universal adaptive strategy (g r i m e & p i e r c e , 2012) we identified the ecological strategy of the park native species. results and discussion from our floristic investigations in this study, we could identify 240 species of spontaneous vascular plants in the “grand park of tirana” which contains from 179 genera and 54 families. this paper has not been analyzed the cultivated flora of the park, which makes 40% of the total number of plant species (m e s i t i et al., 2015), because it is quite difficult to analyze their urbanity indicators, since most of them are planted randomly in the area of study. it would need further studies to see the attitude of the cultivated flora in the future. also we excluded the species which we could not determine the urbanity indicators because of insufficient literature or recorded data. the entire study of park’s flora is an ongoing process, with determining unknown species and urban indicators. the spontaneous vascular flora of the park is 15% of the “flora of tirana” (p a p a r i s t o et al., 1962). the dominant families in species are poaceae (30 species), fabaceae (24 species), asteraceae (21 species), brassicaceae (15 species), lamiaceae (13 species), ranunculaceae (11 species), liliaceae (10 fig. 2. subdivision of the ecological groups of “grand park of tirana” into quadrants biologica nyssana 7 (2)  december 2016: 113-124 mesiti, a., dinga, l.  floristic investigation of the „grand park of tirana“… 117 table 1. presence of plant families in spontaneous flora of the “grand park of tirana” (n – number of species) family n family n poaceae 30 portulacaceae 2 fabaceae 24 oxalidaceae 2 asteraceae 21 ericaceae 2 brassicaceae 15 papaveraceae 2 lamiaceae 13 araceae 2 ranunculaceae 11 araliaceae 2 liliaceae 10 typhaceae 2 caryophyllaceae 8 urticaceae 2 scrophulariaceae 8 malvaceae 2 rosaceae 6 betulaceae 2 boraginaceae 6 hyppericaceae 1 rubiaceae 5 solanaceae 1 geraniaceae 5 chenopodiaceae 1 caprifoliaceae 4 cornaceae 1 fagaceae 4 apocynaceae 1 apiaceae 4 ulmaceae 1 convolvulaceae 4 rutaceae 1 euphorbiaceae 4 aristolochiaceae 1 aceraceae 3 plantaginaceae 1 poligonaceae 3 corylaceae 1 salicaceae 3 platanaceae 1 polypodiaceae 2 dioscoraceae 1 gentianaceae 2 equisetaceae 1 primulaceae 2 verbenaceae 1 oleaceae 2 violaceae 1 iridaceae 2 linaceae 1 orchidaceae 2 lythraceae 1 grand total 240 fig. 3. analysis of the urban indicators of species uphi=5; 9 no data; 9 uphi=4; 31 upho=1; 36 un=3; 71 upho=2; 84 0 10 20 30 40 50 60 70 80 90 uphi=5 no data uphi=4 upho=1 un=3 upho=2 species u rb an it y urban indicators of species biologica nyssana 7 (2)  december 2016: 113-124 mesiti, a., dinga, l.  floristic investigation of the „grand park of tirana“… 118 species). also there is a variety of families represented by small number of species (tab. 1). various genera in the study area give their contribution to the park natural floristic resource. among them, 142 genera are represented with only 1 species, 22 genera are represented with 2 species, 10 genera are represented with 3 species, 3 genera are represented with 4 species, 1 genera (geranium) is represented with 5 species and 1 another (trifolium) is represented with 7 species. by analyzing the floristic list, it is noticed in a high frequency the plants such as: plantago major l., trifolium repens l., poa annua l., which are indicators of a high human disturbance such as mowing, trampling and constructing. in order to analyze the urban impact in the plant list, it was carried out the urban indicators for the native plants species of the park, which is shown in (fig. 3). from analyzing the urban indicators, it is clearly shown in the table above that the moderate urbanophobic (upho=2) species prevails from the others and they are 84 species. the moderate urbanophobic species or mäßig urbanophobe arten, usually are placed externally to the urban area, but however, do not fail to penetrate toward the city, along rivers or green corridors (azonal distribution) (b u c c h e r i & m a r t i n i , 2015). the moderate urbanophobic species in the “grand park of tirana” are: anemone apennina l., anemone hortensis l., asparagus acutifolius l., briza maxima l., clematis flammula l., cyclamen hederifolium ait., marrubium vulgare l., pteridium aquilinum (l.) kuhn, typha latifolia l., etc. in addition, within the park exist a considerable amount of extreme urbanophobic species which are 36 species. one of the strong reason why the urbanophobic species are in a high number is because of the “presidential palace” park presence, which is forbidden for the public entrance therefore it serves as a shelter for most of the urbanophobic species (m e s i t i et al., 2015). some of the extreme urbanophobic species are: colchicum autumnale l., carex flacca schreb, muscari racemosum (l.) mill, ononis spinosa l., orchis laxiflora lam., teucrium chamaedrys l., etc. meanwhile, as it is shown in the map of distribution the urbanophobic plants (fig. 4), most of them are in the peripheral areas of the park, at the most spontaneous ecological groups such as: under oak forests, meadows, in the shrub community of maquis, hornbeam forests and on the lake shore. but it is clearly noticed that some spots are very near with the main roads of the park where the human influence is very high. this is due to the presence of some semi natural or hybrid habitats in different parts of the park. studying the ecological strategy of the urbanophobic species it results that almost 58.2% of them tend to adapt in semi natural habitats, because of their competitor feature. also, 40.8% of urbanophobic taxa are stress tolerant, which mean that they can leave in areas with high intensity of stress. they respond environmental stress with physiological variability (grime & pierce, 2012). such species are found in the park and we can mention: origanum vulgare l., phragmites australis (cav.) steud, rumex acetosella l., etc. as it was expected, the urbanoneutral is the second indicator richest in species, with 71 species and they are spread all over the park, without showing any special patterns for their distribution (fig. 5). these plants have a large specter of distribution, because of their ecological features such as: stress-tolerant species (21%) and competitors (33%). but, most of them are ruderal species (46%) and that is a valuable feature which permit them to adapt into various areas of the park. the urbaneutrale species maintain an indifferent attitude toward nutrients, water or sunlight. their presence clearly show that the park is under various forms of pressure. typical urbanoneutral species found during the floristic investigations are: achillea millefolium l., alopecurus utriculatus (l.) pers., capsella bursapastoris (l.) medik., dactylis glomerata l., echium vulgare l., euphorbia helioscopia l., fumaria officinalis l., geranium molle l., hedera helix l., lolium perenne l., myosotis arvensis (l.) hill., plantago lanceolata l., etc. the urbanophilic parameter is the third urban indicator that was analyzed. in the park 40 urbanophilic species were carried out. as it is presented in the distribution map (fig. 6), these species are found mostly on the park main street border. this street serves as promenade for the park and it the most frequented area, for different recreation activities. it starts from the northern part of the park and it crosses the entire park area, till southern part of the park. the urbanophile vascular plants found nearby this area, are competitors (37.5%), mostly ruderal plants (40.5%), and the rest are stress-tolerant plants (15%). these species gradually decline from the main street toward the periphery. any spot presented in the map is correlated with more than one urbanophilic species. some of the urbanophilic species found in the park are: cymbalaria muralis g. gaertn., b. mey. & scherb, rapistrum rugosum (l.) all., xanthium spinosum l., verbena officinalis l., mercurialis annua l., etc. during our determinations it resulted for some species the urban indicators differ from those one given in the literature. thus, according to the literature we can mention urbanophilic species such biologica nyssana 7 (2)  december 2016: 113-124 mesiti, a., dinga, l.  floristic investigation of the „grand park of tirana“… 119 fig. 4. distribution map of the urbanophobic species in the park fig. 5. distribution map of the urbanoneutral species in the park biologica nyssana 7 (2)  december 2016: 113-124 mesiti, a., dinga, l.  floristic investigation of the „grand park of tirana“… 120 as, bellis perennis l., malva sylvestris l., medicago arabica (l.) huds etc. that in the literature (s t e š e v i ć et al., 2014) are considered urbanoneutrale but in the park were found in altered habitats. this means that most of the park is very disturbed by various anthropogenic and urban factors so the species have been adapted to thus changes. but anyway, the study of urban indicators is an ongoing process. the management of the park is changing and now it is part of the agency of parks and recreation. therefore new analysis will be carried out for the spontaneous plants of this park. based on the park floristic list it was analyzed the plants life form. the analysis of the life forms shows that the dominant form is the hemicryptophyte (82 species), followed by the therophyte (72 species), phanerophyte (40 species), geophyte (37 species), chamephyte (8 species) and nanophanerophytes (1 species). the prevalence of the therophytes is due to the high urban disturbances in the park, and the presence of the geophytes in an urban area, is mentioned as a characteristic of mediterranean cities (s t e š e v i ć et al., 2014). the life form analysis demonstrates the plants ecological suitability, therefore it is given a correlation between the life form spectrum and urban indicators (tab. 2). for the study facility and to be understood better the correlation of the life forms and urban indicators, we have joined the urbanophobic subdivisions into one (upho=1+upho=2). the same was done for the urbanophilic subdivisions. table 2. the life form analysis of the species in “grand park of tirana” urban indicators life forms no data uphi un=3 upho total nanophanerophyte 1 1 chamephyte 2 6 8 geophyte 4 8 24 37 phanerophyte 1 9 12 18 40 therophyte 4 11 28 29 72 hemicryptophyte 4 14 21 44 82 grand total 9 40 71 120 240 it is clearly related that the large number of urbanophobic species is due to the large number in species of hemicryptophytes, therophytes and geophytes. the high number of hemicryptophyte urbanophobic species derives from the natural areas found in the southern-eastern part of the park such as: oak forest and hornbeam forest, where the urban fig. 6. distribution map of the urbanophilic species in the park biologica nyssana 7 (2)  december 2016: 113-124 mesiti, a., dinga, l.  floristic investigation of the „grand park of tirana“… 121 pressure is lower, and because of the presence of “presidential palace”, which we mention above, the anthropogenic presence is forbidden. the therophytes assign the urbanoneutral type. meanwhile the urbanophilic type is represented by almost all the life forms at the same way but mostly by hemicryptophytes and the therophyte. the phanerophyte type are very important for the park, because they compose the spontaneous forest of the park. most of them are urbanophobic and urbanoneutral species such as: quercus frainetto ten., carpinus orientalis mill., arbutus unedo l., erica arborea l., fraxinus ornus l., etc. table 3. correlation of the chorotypes and the urban indicators urban indicator chorotypes no data un=3 uphi upho total european-mediterranean 1 7 7 19 34 mediterranean 3 5 4 21 33 paleotemperated 6 3 12 21 circumboreal 5 1 7 13 eursiber 3 2 3 8 sub-mediterranean 0 1 1 subcosmopolitan 5 2 6 13 european 2 2 4 8 central-european 0 4 4 eurasian 5 2 9 16 north mediterranean 0 3 3 euroasian-subcosmopolitan 1 0 1 2 sub-balkanic 2 2 4 south east european 1 2 4 7 subendemic 1 0 1 2 south center european 0 1 1 european-caucasian 4 1 6 11 south european 1 3 4 paleotemperated and subtropical 3 0 0 3 east asian 1 0 0 1 pontic 1 0 0 1 south east european and pontic 1 0 1 2 mediterranean and central asian 2 1 0 3 south east european and south siberian 0 1 1 north american 1 1 0 2 balkanic 2 1 1 3 7 paleotemperated and cosmopolitan 1 0 0 1 central american 1 0 0 1 european and western asian 1 0 0 1 submediterranean and subatlantic 2 0 0 2 mediterranean and atlantic 1 1 2 n/a 1 1 0 0 2 east mediterranean 1 1 1 3 western european 1 0 0 1 north east mediterranean 1 0 0 1 center european and caucasian 0 1 1 east asian 0 1 1 east european and caucasian 1 0 0 1 paleotemperated and tropical 1 1 2 north american and east asian 1 0 0 1 asian 1 0 1 cosmopolitan 6 2 2 10 south east asian 1 0 0 1 artic 0 1 1 south american 1 0 1 euro mediterranean and pontic 0 1 1 grand total 9 71 40 120 240 biologica nyssana 7 (2)  december 2016: 113-124 mesiti, a., dinga, l.  floristic investigation of the „grand park of tirana“… 122 in addition to the floristic investigation it was analyzed also the chorology of the native vascular plants of the park. the park floristic values are enhanced by the presence of many species with distant bio geographical conditions such as: asian floristic elements (39 species) and european elements (68 species). in the total floristic elements of the park flora prevails the mediterranean and europeanmediterranean type with 33 species, which is an expected value, because of the geographical position, climatic conditions, and the native vegetation of the park. they are followed by paleotemperate with 21 species, eurasian with 16 species, 13 subcosmopolitan species and many other, such as: alpine, caucasian, eurasian, central and east european floristic elements. the most important is the presence of 11 balkan endemic and subendemic species. the entire spectrum of chorotypes is presented in the fig. 7. each of the floristic elements are very important for the park floristic values, but in order to give a general view of the floristic elements in the park, we joined the species with chorological affinity. therefore, from this analysis we carried out that the floristic elements that prevails is the mediterranean one with 82 species followed by the european element with 68 species. thus analysis is important and shows that the park is influenced by the mediterranean climatic. even for the chorology plan exists a correlation with the urban indicators (tab. 3). the europeanmediterranean type and the mediterranean types assign all of the urban indicators of the native plants species. besides them, the urbanoneutral type is characterized by a large specter of distribution, with paleotemperated, circumboreal, subcosmopolitan, and cosmopolitan species. the urbanophilic species are characterized, mostly by the paleotemperated type. the urbanophobic species are distinguished for being mostly european, mediterranean, paleotemperated, and eurasian type. it is very important and valuable that the park preservers some species with a specific status, important in national level and part of the albanian red list of flora (anonymous, 2013) such as colchicum autumnale l. (en a1b), hypericum perforatum l. (en a1b) and crocus boryi j. gay (lrcd) (fig. 8). in tearms of urbanity hypericum perforatum is an urbanoneutral type, but in national level its areal is very disturbed by the urban influence, since this plant is collected not properly for medicinal uses. the other two species, being geophytes and endangered are both urbanophobic type. conclusion the “grand park of tirana” is an urban park with different ecological groups, which are natural, seminatural and cultivated for ornamental purposes. it is the only one urban park for the city, and for the entire country. fig. 7. data on plant chorology fig. 8. distribution of hypericum perforatum, colchicum autumnale and crocus boryi in the park biologica nyssana 7 (2)  december 2016: 113-124 mesiti, a., dinga, l.  floristic investigation of the „grand park of tirana“… 123 the area is large enough and has the favorable geographical position and stable climatic conditions, to shelter a variety of plant native species. only in this study was carried out the analysis of 240 species from 179 genera and 54 families, most of them are hemicryptophytes and with mediterranean origin. in this park we found that 35% of the flora is urbanophobic, mostly ruderal and stress tolerant, which means that beside the urban pressure, that threatens the park continually, it is preserved well, and the natural habitats of the parks still prevails. it is strongly recommended that some areas of the park, especially the most natural one found in the southern part of the park, where the urbanophobic species prevails, must be better managed or become forbidden for the public frequentation. this site protection would prevent further deterioration of the native species and habitats in the park, and would give the possibility of damaged areas rehabilitation. even for the introduction of ornamental species within the park in the recreational areas, it is recommended to plant species from the native vegetation, which has ornamental values, and can adapt easily in urban areas such as the urbanoneutral species, such as: quercus frainetto ten., clematis vitalba l., colutea arborescens l. and gladiolus communis l.. also, beside this specific work, it is needed the knowledge of urban flora and especially the one that is inhabited and is in strong conflict with natural or semi natural habitats. urban indicators are an important feature to be considered in the future for the better management of the area. the “grand park of tirana” must be considered as a place of urban development and environmental education of its visitors. references alegro, a., bogdanović, s., rešetnik, i., boršić, i., cigić, p., & nikolić, t. 2013: flora of the seminatural marshland savica, part of the (sub) urban flora of the city of zagreb (croatia). natura croatica, 22 (1): 111-134. anonymous 2013: national red list of wild flora and fauna. ministry of environment of albania. bianco, p.m., fanelli, g., tescarollo, p., pignatti, s. 2003: ruderalization in a roman park as a result of changing management, urban habitats, 1 (1): 87-104. bomanowska, a., witoswlaski, p. 2008: selected aspects of diversity of synanthropic flora in the chosen cities of central poland. biodiversity research and conservation, 9-10: 35-42. buccheri, m., martini, f. 2015: piante urbanofobe, urbanoneutrali e urbanofile trieste. natural history science, 2 (1): 31-38. celesti grapow, l., pyšek, p., jarošik, v., blasi, c. 2006: determinants of native and alien species richness in the urban flora of rome. diversity and distributions, 12: 490–501. chiesura, a. 2004: the role of urban parks for the sustainable city. landscape and urban planning, 68: 129–138. chronopoulos, g., christodoulakis, d. 1996: contribution to the urban flora of greece: the flora of the city of patras and surrounding area. botanica helvetica, 106: 159-176. chronopoulos, g., christodoulakis, d. 2000: analysis of the adventive flora of a greek city: the example of patras. botanica helvetica, 110: 171189. crompton, j.l. 2001: the impact of parks on property values: a review of the empirical evidence. journal of leisure research, 33 (1): 1-31. demiri, m. 1983: flora ekskursioniste e shqipërisë. shtëpia botuese e librit shkollor. tiranë, 985 p. ellenberg, h., mueller-dombois, d. 1967: a key to raunkiaer plant life forms with reviesed subdivision. berichte des geobotanischen instituts der eth, stift., rübel, zürich, 37: 56-73. european environment agency (eea), 1998: state of the european environment. copenhagen. falinski, j.b. 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this review paper addresses this issue through specific examples. studies of some asteraceae taxa showed that morphological, micromorphological and anatomical data are extremely important in systematics. new opportunities for systematic morphology, micromorphology and anatomy in case of asteraceae taxonomy, but certainly also in other plant groups, that were not present in the premolecular era, are opening regarding synergistic multidisciplinary taxonomic, evolutionary and phylogenetic studies that combine molecular with morphological, anatomical and other analyses (e.g. chemophenetics describes a given taxon phenetically using specialized metabolites as phytochemical characters), keeping in the throne these “old fashioned” approaches. key words: morphology, taxonomy, characters, synergy apstract: mikromorfologija i anatomija u sistematici familije asteraceae. staromodan pristup? uporedna studija morfologije isprepletene sa anatomijom, uvek je bila osnova za sistematiku biljaka koja teži da objasni raznolikost, evoluciju i filogeniju biljaka. u molekularnom dobu, u sistematskim i filogenetskim studijama biljaka, neki autori umanjuju značaj morfologije, a posebno anatomije biljaka. međutim, da li su molekularne metode i podaci isključivo primaran i samodovoljan pristup u taksonomskom istraživanju biljaka? ovaj pregledni rad, kroz određene primere, daje odgovor na ovo pitanje. istraživanja određenih taksona iz familije asteraceae pokazala su da su morfološki, mikromorfološki i anatomski podaci izuzetno važni u sistematici. novi pristupi u sistematskoj morfologiji, mikromorfologiji i anatomiji u slučaju taksonomije asteraceae, ali sigurno i u slučaju taksonomije drugih grupa biljaka, koji nisu bili mogući u premolekularnom dobu, otvaraju se sada kao sinergija multidisciplinarnih taksonomskih, evolucionih i filogenetskih studija koje kombinuju molekularne sa morfološkim, anatomskim i drugim analizama i pristupima (npr. hemofenetika koja opisuje dati takson fenetički, upotrebom specijalizovanih metabolita kao fitohemijskih karaktera), držeći na prestolu ove „staromodne“ pristupe. ključne reči: morfologija, taksonomija, karakteri, sinergija introduction the comparative study of plant structure has always been the backbone of plant systematics, which strives to elucidate plant diversity, phylogeny and evolution (endress et al., 2000). plant taxonomic studies traditionally use morphological and karyological (stebbins, 1953), as well as micromorphological characters (hayat et al., 2009; bak & ozcan, 2018). micromorphological characters are of decisive importance in unring taxonomic and phylogenetic relationships of various plant groups and have been successfully used in plant systematic studies for decades (endress et al., 2000). for more than a © 2019 janaćković et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 77 13th symposium on the flora of southeastern serbia and neighboring regions century, comparative anatomy is used as a tool in the plant systematics. anatomical characters are very important in perceiving systematic and phylogenetic relationships of particular plant groups. indeed, anatomical features can provide useful characters which could help in identification of problematic plant taxa, as well as establishing their taxonomic relationships (metcalfe & chalk, 1957; scatena et al., 2005; makbul et al., 2011; sosa et al., 2014; karanović et al., 2015, gavrilović et al., 2019a, b; janaćković et al., 2019). on the other hand, the data provided by the phytochemists are extremely difficult to interpret in a cladistic context. even so, integration of phytochemical and macro-molecular characters can be of prominent benefit, and can help, for example, in the delineation of clades so far only supported by dna sequence data (enke et al., 2012). in order to overcome the confusion in the interpretation of phytochemical characters (specialized metabolites that actually describe the given taxon phenetically) as phylogenetic characters and “under the umbrella” of the term chemotaxonomy (which is mistakenly identified with chemosystematics), the new term chemophenetics has been proposed by zidorn, (2019). nevertheless, chemophenetic studies contribute to the phenetic description of taxa, similarly to anatomical, morphological and karyologycal approaches, which have already been recognized as of major importance for establishing “natural” systems and which continue to be of the highest importance for the description of organisms classified with the help of modern molecular methods (zidorn, 2019). asteraceae (compositae), habitually known as the daisy or sunflower family, represent one of three mega-diverse families which jointly count more than 25% of all extant angiosperm species (mandel et al., 2019). asteraceae, counting 25000–35000 species, comprise 10% of all flowering plant species. members of the sunflower family occur on every continent including antarctica (smith & richardson, 2011) and inhabit nearly every type of habitat on earth with the largest concentration of species in deserts, prairies, steppes, montane regions, and areas with mediterranean-like climates (mandel et al., 2019). according to newer fossil data and recent molecular clock dating, asteraceae likely originated during the late cretaceous: ∼83 mya (mandel et al., 2019). according to mandel et al. (2019) the family consists of 13 subfamilies and 47 tribes. asteraceae also includes species of wide economic interest, e.g. vegetables, sources of oil, medicinal plants, insecticides and many horticultural and garden ornamentals. however, some species of asteraceae constitute a big problem for agriculture as noxious weeds. as a one of the largest, natural (with a combination of several specialized morphological characteristics e.g., capitula, highly reduced and modified flowers, syngenesious anthers, inferior ovaries and very unique in the plant kingdom fruit cypsela) and economically most important families of flowering plants asteraceae has been researched for centuries. characters related to form, whether gross morphology, micromorphology, anatomy, embryology, palinology and so forth, regarded as morphologycal data, have larger impact on the cladistics and classification of the asteraceae than other characters e.g. chemical and molecular data (schönberger, 2002; stuessy, 2009). molecular data are significant but also insufficient, that is why, in some cases, better phylogenetic reconstructions of the asteraceae are obtained taking into account also a morphological data (pornpongrungrueng et al., 2007; gruenstaeudl et al., 2009; wang et al., 2013). more recent, modern-day studies of asteraceae taxa have shown that morphological, micromorphological and anatomical data are still extremely important in systematics of asteraceae family (makbul et al., 2011; wang et al., 2013; sosa et al., 2014; karanović et al., 2015; bombo et al., 2016; ginko et al., 2016; batista and de souza, 2017; gavrilović et al., 2017; gavrilović et al., 2018a, b; gavrilović et al., 2019a, b; janaćković et al., 2019). brief history and modern-day micromorphology and anatomy in systematics of asteraceae micromorphology a different sets of characters has been used to demarcate the asteraceae. certainly, one of the pioneer work regarding micromorpholgical approaches to asteraceae classification was done by cassini (1821). these summarize many of the microscopic traits on which cassini based his tribes. of these, pronate versus recurved mature style branches, stigmatic surfaces, truncate to enlarged style appendages, bases of the anther thecae with or without tails, shape of the anther collar and form of the corolla are most important. the stigmatic surfaces of many tribes (mutisieae, lactuceae, vernonieae, arctotideae, eremothamneae, cardueae) are consistently continuous over the inner surface of the style branch. in other tribes (eupatorieae, anthemideae, astereae, most inuleae, most heliantheae, most senecioneae) the stigmatic surface is divided into two lines. the nature of endothecial tissue of the stamens, which could be polarized or radial, showed to be good character in taxonomy within senecioneae (dormer, 1962). in subtribes senecioninae and othonninae, the endothecium is radial in all genera except dauresia, graphistylis, and perhaps synotis. in 78 biologica nyssana ● 10 (2) december 2019: 77-85 janaćković et al. ● micromorphology and anatomy in systematics of asteraceae. an old-fashioned approach? tussilagininae s.str. a polarized endothecium is the rule, but the radial type has been recorded in several genera (tephroseris, nemosenecio, psacaliopsis, psacalium, arnoglossum); in sinosenecio both types and an intermediate pattern seem to occur (jeffrey & chen, 1984). moreover, the anthers of cichorieae members vary considerably in length, but this variation probably occurs repeatedly within many genera and is therefore only of taxonomic relevance at the species level (kilian et al., 2009). in arctotideae morphological and micromorphological characters confirmed close relationships between the gorteria clade and berkheya clade (karis et al., 2009). microcharacters of involucral bracts are considered very helpful for delimitation in certain taxonomic groups of asteraceae (e.g., for subtribes of cardueae, with spiny pectinate-fimbriate appendages in cardopatiinae; usually spiny, innermost exappendiculate or with rudimentary appendages in carduinae; inner often conspicuous and coloured in carlininae; scarious, fimbriate, pectinate, spiny or unarmed appendage in centaureinae; and in many rows in echinopsinae (robinson, 2009; susanna & garcia-jacas, 2009). certain floral microcharacters (anther size, shape of the anther apical appendage, configuration of stigmatic areas on the inner surface of the style branch, and configuration of the endothecial thickenings and of the filament collar) of 36 taxa of sinosenecio showed that these floral characters are highly consistent with evidence from molecular systematics and cytology and provide the most important diagnostic characters in the tribe senecioneae, as in the family at large and strongly suggest a polyphyletic nature of this genus, as well as the need of a taxonomic change at generic level (liu & yang, 2011). still nowadays, micromorphological investigations of asteraceae could provide some novel characters (erbar & leins, 2015; gavrilović et al. 2017, 2019b). investigating style morphology of 395 species of 258 genera (covering all, in that time, 44 tribes of the asteraceae), erbar & leins (2015) found a new microstructural feature, namely, often conspicuous cuticular patterns on the stylar hairs (involved in secondary pollen presentation) and stylar appendages. they determined five different patterns of cuticular striation and when they put these patterns onto a generalized phylogenetic tree (based on molecular data), they concluded that there is considerable homoplasy in these features. nevertheless, cuticular patterns are still useful in characterizing some clades within the family. gavrilović et al. (2017) investigating involucral bract micromorphology found, for the first time, a large number of densely packed crystals on the involucral bract surface. also, the presence of nonglandular, curly trichomes and biologica nyssana ● 10 (2) december 2019: 77-85 janaćković et al. ● micromorphology and anatomy in systematics of asteraceae. an old-fashioned approach? 79 biseriate glandular trichomes on the bract surface, as well as the sylvite crystals on the petal surface of x. cylindraceum, clearly differentiates this species from x. annuum (gavrilović et al., 2017). comparative micromorphological analyses were conducted on five members of the xerantheminae, both perennial (amphoricarpos exsul and shangwua masarica) and annual (chardinia orientalis, siebera pungens and xeranthemum inapertum), showing that micromorphological traits link together perennial species, some link annual ones, some are species-specific, and some are common to all taxa (gavrilović et al., 2019b). we could conclude that morphology and micromorphology of florets (e.g., style base, anther appendages, trichomes on corollas), and inflorescence (involucral bracts characters, e.g., crystals and glandular and nonglandular trichomes on their wall) were used as major distinguishing features for subtribal and generic delimitation, even though these characters can sometimes be significant at the species level. anatomy at the beginning of the twentieth century col (18991901), in light of anatomy, reviewed in considerable detail distribution of laticiferous versus resiniferous tissue throughout the asteraceae. taxa with latex in canals or sacs occur in several tribes of the subfamily cichorioideae and consistently in the lactuceae, but very rarely in the asteroideae, where resin sacs and resin canals are common. carlquist (1966) investigated the basic plan of the wood anatomy of asteraceae (focusing on four tribes, anthemideae, ambrosieae, calenduleae, and arctotideae), which provided useful tribal characters and minor intertribal variation. even though carlquist (1966) stated that asteraceae members share a basically specialized wood plan and that wood anatomy is not likely to reward one with tribal or subtribal characters, certain characters are of systematic value within anthemideae, ambrosieae, calenduleae, and arctotideae (e.g., carbonized resins in intercellular spaces, secretory canals in rays, patterns of crystal occurrence are characters which may be of specific or generic value). metcalfe & chalk (1957) noted some particular anatomical traits, which showed to have taxonomic importance within the family, e.g., presence of secretory and laticiferous canals, types of nonglandular and glandular trichomes, occurrence of medullar and cortical vascular bundles and presence of anomalous secondary thickening. also, anatomical characteristics observable in asteraceae are: (a) presence of various types of glandular and non-glandular tri80 chomes; (b) papillae on the abaxial leaf epidermis; (c) anomocytic, anisocytic and rarely heliocytic stomata types; (d) presence of hydathodes; (e) presence of hypodermis; (f) homogeneous or heterogeneous mesophyll and (g) vascular bundles with a parenchymatic sheath composed of large cells (metcalfe & chalk, 1979). anatomy of several members of the tribe senecioneae showed that they possess resin ducts in stems, leaves and roots, sometimes also in floral parts, and even in cotyledons. the resin production is noted as stickiness and exudates on vegetative parts. this occurs in the tussilaginoid as well as in senecioid group (nordenstam et al., 2009). ginko et al. (2016) investigated suitability of anatomical characters of root and rhizome of 59 species belonging to 34 genera and 12 subtribes from tribes cardueae and cichorieae for taxonomic classification and phylogenetic reconstruction. in this case, anatomy is demonstrated as valuable to discriminate tribes and many species but not so for subtribes and genera. however, most anatomical traits seems to be homoplastic, which limits their application as phylogenetically informative characters. bombo et al. (2016) stated that anatomical features can help in resolving taxonomical problems within the genus aldama la llave, especially among brazilian members, which are difficult to identify taxonomically. on the basis of their findings, the authors concluded that anatomy is able to provide data which assist with the taxonomic problems within the four analysed species. the systematic value of leaf epidermal characters in asteraceae has been proven by numerous studies, as leaf surface is under strong genetic control (adedeji & jewoola, 2008; karanović et al., 2015). some leaf blade characters (e.g., epidermal anticlinal cell walls, epicuticular wax and trichome type) have shown to be diagnostic to separate aster l., galatella cass. and tripolium nees (karanović et al., 2015). a comparative study of the leaf epidermis in 12 species of asteraceae showed that the type and shape of trichomes, nature of cuticular striations and stomatal type are taxonomically important for the delimitation of species (adedeji & jewoola, 2008). qualitative anatomical characters (e.g., shape of the young stem and peduncle cross-sections, type of glandular trichomes and occurrence of cortical vascular bundles) were shown to be useful in delimitating x. annuum from x. cylindraceum (gavrilović et al., 2019a). moreover, some of the anatomical characters found in xeranthemum (secondary growth in roots and dorsiventral leaves) suggested that adaptation from mesophytic to xeric habitats (gavrilović et al., 2019a) are important for phylogenetic relationships within xerantheminae. anatomical data can also contribute in resolving complex taxonomy of certain genera, e.g. artemisia. in anatomical investigation of five artemisia species, janaćković et al. (2019) showed that some characters link together a. absinthium and a. arborescens from the same section; some other connect species belonging to different sections (a. campestris and a. arborescens; a. absinthium and a. judaica; a. judaica and a. herba-alba), while some could be considered as species-specific. we could summarize that certain anatomical characters, such us distribution of laticiferous versus resiniferous tissue is useful on subfamily an tribal level, while patterns of crystal occurrence might be significant at species or genus level. occurrence of cortical vascular bundles seems to be important on species level. root and rhizome anatomical traits have proven to be useful on tribal and species, but not on the genus level. leaf anatomical epidermal character are diagnostic and can be used for delimiting species. also, qualitative anatomical characters may have role in understanding and solving phylogenetic relationships, which are reflected in the systematics of given taxa. micromorphology and anatomy of cypsela the cypsela is a special form of dry indehiscent fruit in which the seed coat (testa) and fruit wall (pericarp) are tightly attached to one another and is exclusive characteristic of the family asteraceae (roth, 1977). as an exclusive fruit of the family cypsela and its features have been attracted by tournefort (1694), vaillant (1719), cassini (1819), lessing (1832), bentham (1873), hoffman (1894), cronquist (1955), robinson (1977), bremer (1994), nordenstam (1994), rao & datt (1996), robinson (1999), nordenstam et al. (2006), lack (2007) and mukherje & nordenstam (2004, 2010). cypsela morphology (macroand micromorphology) and anatomy have been widely used in illuminating taxonomic relationships in asteraceae and still represents a source of valid taxonomic characters (lavialle, 1912; stebbins, 1953; wagenitz, 1976; dittrich 1977; barthlott, 1984; singh & pandey, 1984; dittrich, 1985; bruhl & quinn, 1990; glynis, 1993; geng et al., 1994; blanca & díaz de la guardia, 1997; petit, 1997; häffner, 2000; zhu et al., 2006; garg & sharma, 2007; pandey & kumari, 2007; zarembo and boyko, 2008; abid & qaiser, 2009; abid & ali, 2010; inceer et al., 2012; ozcan & akinci, 2019). this is why micromorphology and anatomy of cypsela are separated herein. bremer (1987) stated the importance of cypselae characters at lower taxonomic levels but not at the tribal level. in asteraceae, the anatomy and micromorphology of biologica nyssana ● 10 (2) december 2019: 77-85 janaćković et al. ● micromorphology and anatomy in systematics of asteraceae. an old-fashioned approach? 81 biologica nyssana ● 10 (2) december 2019: 77-85 janaćković et al. ● micromorphology and anatomy in systematics of asteraceae. an old-fashioned approach? cypselas are taxonomically significant at both genus and species levels (abid & qaiser, 2009, kulkarni, 2013, hussein & eldemerdash, 2016; karanović et al., 2016; gavrilović et al., 2019b). cypselae size and shape, number of ribs, presence of prickles, ornamentation of the intercostal gaps, tapering (sharp or gradual) of the body/beak junction, shape of the beak, degree of swelling at its apex and pubescence of the annulus are diagnostic in tragopogon l. (blanca & díaz de la guardia, 1997). micromorphological characters of the cypselae (shape, surface, colour, size), pappus (stucture, shape, number, colour, size) and carpopodium (shape, position, diameter) in the tribes senecioneae and anthemidae are useful for assessing the relationship and delimitation at both generic and specific levels (abid & qaiser, 2009; abid & ali, 2010). as a result of morphological and anatomical investigations of the cypselae in east asian species of rhaponticum vaill., klasea cass., serratula l. and synurus iljin in the tribe cardueae s.l., zarembo & boyko (2008) clarified the following diagnostic traits at the species level: topography of epidermal cells of the pericarp, presence of phlobaphenes, occurrence, topography and localization of calcium oxalate crystals, and occurrence and location of secretory ducts in the mesocarp. karanović et al. (2016) showed that, besides receptacle characters, organisation of sclerenchymatous tissue in a fruit is a feature that tend to be diagnostic for genera inula, pulicaria, dittrichia and limbarda. also, some fruit features have been demonstrated to be especially useful in distinguishing certain similar species (e.g., inula britannica from i. oculus-christi). moreover, authors stated that i. helenium should be separated from the inula genus, as its authenticity is evident base on cypsela characters. silva et al. (2017) showed that features of the cypselae of the subtribe disynaphiinae such as the carpopodium, floral disc, pappus, outer mesocarp, sclerenchyma, phytomelanin layer, ribs and trichomes are valuable at both generic and specific levels. moreover, authors revealed the presence of a multiplicative pericarp only in a few symphyopappus spp., rare trait in asteraceae, which probably evolved independently in the family. cypsela structure also supports the exclusion of disynaphia praeficta from the subtribe, since this species posesses several different characters comparing with other representatives of disynaphiinae. characters of cypselae are shown to be very informative at generic level within xerantheminae (gavrilović et al., 2019). for instance, shangwua is distinguished from other genera in having glabrous cypsela, while chardinia only possesses papillose cypsela surface. moreover, cypsela features of xerantheminae taxa are significant for phylogeny of the subtribe (some characters share all members, while some characters separates annual from perennial genera). ozcan & akinci (2019) evaluated feasibility of cypsela characters as taxonomic markers investigating 21 taxa representing 12 genera of the tribe cardueae. they observed considerable variability in surface sculptures of pappus and cypselae, as well as in pericarp and testa structures. authors concluded that micromorphological and anatomical cypsela characters are distinct between the genera and are also useful for delimiting species. conclusion and future prospects the breakthrough of molecular tools in plant systematics and its contribution to phylogenetic frameworks was and it is still a tremendous stimulus for comparative morphology and anatomy. one should have in mind that the structure and biology of a majority of asteraceae members are far from sufficiently investigated, thus combining morpho-anatomical, phytochemical, and molecular studies are necessary to explore them. although this overview represents only a glimpse of a role of micromorphological and anatomical approaches to asteraceae systematics, it gives an valuable insight and perspective of this topic. thus, the accumulated knowledge and permanent investigation of asteraceae taxa using micromorphological and anatomical methods will put light on branching topologies of phylogenetic trees which molecular data established. new opportunities for systematic morphology, micromorphology and anatomy in case of asteraceae taxonomy, but certainly also in other plant groups, which were not present in the premolecular era, are now opening regarding synergistic multidisciplinary taxonomic, evolutionary and phylogenetic studies which combine molecular with morphological, anatomical and other approaches (e.g. chemophenetics), keeping in the throne these “old fashioned” approaches. acknowledgements. we acknowledge the financial support provided by the serbian ministry of education, science and technological development, project no. 173029. references abid, r., ali, n. 2010: cypsela morphology and its taxonomic significance for the tribe senecioneae (asteraceae) from pakistan. pakistan journal of botany, 42: 117–133. 82 abid, r. d., qaiser, m. 2009: taxonomic significance of the cypsela morphology in the tribe anthemideae (asteraceae) from pakistan and kashmir. pakistan journal of botany, 41: 555–579. adedeji, o., jewoola, o. a. 2008: importance of leaf epidermal characters in the asteraceae family. notulae botanicae horti agrobotanici cluj-napoca, 36:7–16 bak, f. e., ozcan, m. 2018: pollen morphology of endemic ne anatolian cirsium taxa (asteraceae). pakistan journal of botany, 50 (3): 1181–1185. barthlott, w. 1984: microstructural features of seed surfaces. in: heywood, v. h., moore, d. m. 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long-term ecological research sites, philippines dave p. buenavista department of biology, central mindanao university, university town, musuan, 8710 bukidnon, philippines (school of environment, natural resources and geography, bangor university, ll57 2ew, wales, united kingdom) * e-mail: davista.cmu@gmail.com abstract: buenavista, d.p.: contributions to the orchid flora of mindanao long-term ecological research sites, philippines. biologica nyssana, 8 (1), september 2017: 31-38. this contribution is based on the field studies on wild orchids conducted from september 2012 to november 2013 in one-hectare plots in five long-term ecological research sites in mindanao island, philippines. the family orchidaceae is the most threatened group of plants in the philippines however, it remains to be poorly known and understudied. this is the first preliminary report on the species richness and distribution of wild orchids in the identified research sites. a list is contains 79 orchid species belonging to 34 genera which represents approximately 7% of the known orchid species in the philippines. a total of 40 endemics and 3 threatened species were recorded in the sites. this list can be used as basis for future biodiversity assessment and conservation initiatives to identify and prioritize areas for immediate conservation of threatened and endemic orchid species as well as the vulnerable mountain ecosystems. key words: inventory, conservation, orchidaceae, mindanao, philippines apstrakt: buenavista, d.p.: prilog flori orhideja mesta dugoročnih ekoloških istraživanja mindanaa, filipini. biologica nyssana, 8 (1), septembar 2017: 31-38. ovaj prilog je zasnovan na terenskim istraživanjima divljih orhideja sprovedenim od septembra 2012 do novembra 2013 na pet površina od po jednog hektara u oblastima obuhvaćenim dugoročnim ekološkim istraživanjima na mindanao ostrvu, filipini. familija orchidaceae je najugroženija grupa biljaka na filipinima ali bez obzira na to, i dalje je slabo poznata i nedovoljno proučavana. ovo je prvi preliminarni izveštaj o bogatstvu vrsta i distribuciji divljih orhideja na identifikovanim lokalitetima istraživanja. lista sadrži 79 vrsta orhideja koje spadaju u 34 roda i predstavljaju približno 7% poznate flore orhideja na filipinima. ukupno 40 endemskih i 3 ugrožene vrste su pronađene na istraživanim lokalitetima. lista može poslužiti kao osnova za dalju procenu biodiverziteta i inicijativu konzervacione inicijative koje bi identifikovale i označile najbitnije lokalitete za hitnu i trenutnu konzervaciju ugroženih i endemičnih vrsta orhideja kao i ranjivih planinskih ekosistema. ključne reči: inventarizacija, konzervacija, orchidaceae, mindanao, filipini 8 (1) • september 2017: 31-38 doi: 10.5281/zenodo.963339 biologica nyssana 8 (1)  september 2017: 31-38 buenavista, d.p.  contributions to the orchid flora of mindenao… 32 introduction the philippines lies in the western pacific ocean and is geographically part of southeast asia, a region that occupies a mere three percent of the earth’s total surface, yet is home to 20 percent of all known species of plants and animals (m i t t e r m e i e r et al., 1999; m y e r s et al., 2000; a m b a l et al., 2012). it has one of the highest orchid floras in the world with approximately 141 genera and 1200 species (c o o t e s , 2001; a g o o et al., 2003; c o o t e s , 2011). about 85-90% of the philippine orchids are found nowhere else in the world (c o o t e s , 2011). by contrast to other countries, canada and the united states, including hawaii, puerto rico, and the virgin islands, have only 325 species among them (h e a n e y & r e g a l a d o , 1998). orchids have also been shown to be excellent indicators of overall biodiversity in an area (n a d k a r n i , 1992; s w a r t s & d i x o n , 2009). orchids are highly evolved with their pollinators and require a specific relationship with mycorrhizal fungi to germinate (l e a k e , 1994; r a s m u s s e n , 2002; o t e r o et al., 2005; m c c o r m i c k et al., 2006; s h e f f e r s o n et al., 2007; r a s m u s s e n & r a s m u s s e n , 2009); therefore they are intimately intertwined with the ecology of their habitat. it is therefore imperative that the conservation of habitat be considered if the orchids had to be conserved. however, habitat loss is one of the major factors that imperiled the philippine biodiversity (p o s a et al., 2008; s o d h i et al., 2010) losing over 80% of its original forest cover since the 16th century (fao, 2005; b a n k o f f , 2007). such threat had direct impact on the orchid flora considering that it has the highest number of threatened species (f e r n a n d o et al., 2008) compared to any plant groups in the philippines. besides the scanty number of orchid studies in the philippines, the assessment of orchid diversity and distributions is further limited by potential problems in nomenclature that are difficult to resolve because of the lack of access to reference specimens, digital imagery, and detailed data collection particularly in mindanao. species richness of endemic species richness is typically used to estimate the biodiversity value of a region (n a g e n d r a , 2002; c l e r g u e et al., 2005; o r m e et al., 2005). one of the most important tasks for conservationists is therefore to find objective methods for assessment of natural areas in terms of their conservation priority (j a n k o w s k i et al., 2009; k i n d l m a n n & v e r g a r a , 2009). defining conservation priorities is essential in minimizing biodiversity loss (b r o o k s et al., 2006; j a c q u e m y n et al., 2007) as it ensures that conservation action focuses on the species at the greatest risk of extinction and on the sites that are most important for their protection. the long-term fig. 1. geographical location of mindanao long-term ecological research sites biologica nyssana 8 (1)  september 2017: 31-38 buenavista, d.p.  contributions to the orchid flora of mindenao… 33 ecological research (lter) sites were established in five mountain regions in mindanao island in order to assess, conserve and protect the increasing number of endangered flora and fauna of the region. to date, knowledge on the orchid flora in the aforementioned lter sites has never been documented; hence, this study was undertaken. material and methods field inventory was conducted from september 2012 to november 2013 in the five long-term ecological research (lter) sites established in the different regions in mindanao island namely (fig. 1), mt. kitanglad, mt. apo, mt. malindang, mt. hamiguitan and mt. musuan. every lter site was delineated into one hectare permanent plot which was surveyed and inventoried. site 1 is mt. apo lter site is located within the vicinity of energy development company (edc), ilomavis, kidapawan city. the site was characterized as a montane forest located at 6°59´47´´n, 125°15´12´´e with an elevation of 19002000 meters above sea level (masl). the site has a close-canopy layer dominated by large trees and shrubs covered by bryophytes. site 2 is mt. hamiguitan lter site in davao oriental. the site is also a closed-canopy montane forest located at 6°43´58´´n, 126°9´58´´e with an elevation of 10001100 masl. site 3 is at the mt. kitanglad lter site situated 8°5´46´´n, 124°55´17´´e in lantapan, bukidnon. the permanent plot is within the montane forest with an elevation of 2100-2200 masl. site 4 is located at the upper montane forest of mt. malindang lter site situated 8°17´45´´n, 123°36´34´´e in misamis occidental at 1600-1700 masl elevation. the slightly open canopy layer of the forest was due to the overlapping crown of various types of trees, palm and shrubs. site 5 is mt. musuan lter site also in bukidnon. the permanent plot is situated at 7°52'36.02”n, 125°04'1.53”e in the lowland secondary, mixed agro-dipterocarp forest which is around 380-480 masl and has slightly to open canopy layer. plant collections were pressed and treated with denatured alcohol after the fieldwork and then further processed in the herbarium. the specimens were allowed to dry up to 75°c using a mechanical drier. since orchid taxonomy relies mainly on the characters of inflorescence, orchids with its flowers were collected whenever available during the field sampling. the inflorescence were dried and kept in separate herbarium pockets as it often wilts and deteriorates easily in the field. the collected plants were classified and identified using taxonomic keys from orchid floras, monographs and published articles of h o l t t u m (1954), d r e s s l e r & d o d s o n (1960); v a l m a y o r (1984); d e v o g e l (1988a,b); p e d e r s e n (1997); f e s s e l & b a l z e r (1999); a g o o et al. (2003); c o o t e s (2001, 2010; 2011) and s u a r e z (2010 & 2011). confirmation of the plant identification was done through direct communication with jim cootes, one of the authorities on philippine orchidaceae. all the collections were then deposited in the university museum of central mindanao university, bukidnon, philippines. results and discussion a total of 79 orchid species belonging to 34 genera was recorded in five lter sites in mindanao (tab. 1). taxonomic identification of the four species were undetermined due to the unavailability of the inflorescence during the survey. in terms of species richness per site, mt. apo had the highest number represented by 31 species belonging to 17 genera. this is followed by mt. kitanglad with 25 species from 16 genera; and mt. hamiguitan with a record of 19 species from 11 genera. the least record in was from mt. malindang with 18 species from 16 genera; and in mt. musuan with only 2 species from 2 genera (fig. 2). fig. 2. bar graph comparing the species richness of orchids in five mindanao lter sites the difference in the species richness between the 5 sites may be explained by the difference in many ecological factors such as elevation, microclimate conditions and presence of pollinators and mycorrhizal associates which have been reported to influence orchid diversity and distribution. j a c q u e m y n et al. (2005) reported that orchid species composition changed continuously with altitude, indicating turnover of species with increasing altitude. analogously, orchid breeding biologica nyssana 8 (1)  september 2017: 31-38 buenavista, d.p.  contributions to the orchid flora of mindenao… 34 table 1. list and distribution of wild orchids in mindanao lter sites species mt. apo mt. hamiguitan mt. kitanglad mt. malindang mt. musuan 1. agrostophyllum longivaginatum ames x x x 2. a. saccatilabium ames & quisumb. x x 3. appendicula laxifolia j.j.sm. x 4. a. malindangensis (ames) schltr. x x x 5. a. tembuyukenensis j.j.wood x 6. arundina graminifolia (d don) hochr. x 7. ascidieria cymbidifolia (ridl.) w.suarez & cootes x 8. bulbophyllum colubrimodum ames x 9. b. escritorii ames x 10. calanthe davaensis ames x 11. calanthe sp. x 12. cephalantheropsis longipes (hook.f.) ormerod x 13. c. obcordata (lindl.) ormerod x 14. ceratostylis latipetala ames x x x x 15. c. wenzelii ames x 16. c. subulata blume x x x 17. cheirostylis octodactyla ames x 18. coelogyne candoonensis ames x 19. c. salvaneraniana w.suarez x 20. crepidium quadridentatum (ames) szlach. x x 21. cryptostylis arachnites (blume) hassk. x x 22. cymbidium pubescens lindl. x 23. dendrobium auriculatum ames & quisumb. x 24. d. crumenatum sw. x 25. d. diffusum lo williams x 26. d. erosum (blume) lindl. x 27. d. phillipsii ames & quisumb. x 28. d. rhombeum lindl. x 29. d. tiongii cootes x 30. d. uniflorum griff. x 31. dendrochilum arachnites rchb.f. x x 32. d. cobbianum rchb.f. x 33. d. coccineum h.a.pedersen & gravend. x 34. d. elmeri ames x 35. d. glumaceum lindl. x x 36. d. kopfii lückel. x 37. d. longifolium rchb.f. x 38. d. macranthum schltr. x 39. d. mearnsii ames x 40. d. tenellum (nees & meyen) ames x 41. d. wenzelii ames 1915 x biologica nyssana 8 (1)  september 2017: 31-38 buenavista, d.p.  contributions to the orchid flora of mindenao… 35 42. epigeneium stella-silvae (loher & kraenzl.) summerh. x 43. e. treacherianum (reichb.f ex hook.f.) summerh. x 44. e. roseum (d.don) lindl. x 45. goodyera clausa (aa eaton ex ames) schltr. x 46. g. viridiflora (blume) blume x 47. habenaria stenopetala lindl. x 48. habenaria sp. x 49. hippeophyllum wenzelii ames x 50. lepidogyne longifolia (blume) blume x 51. liparis amesiana schltr. x 52. l. negrosiana ames x x 53. l. parviflora (blume) lindl. x 54. l. philippinensis (ames) schltr. x 55. mycaranthes candoonensis (ames) cootes & w.suarez x 56. m. gigantea (ames) cootes & w.suarez x x 57. oberonia hispidula ames x 58. octarrhena parvula thwaites x 59. paphiopedilum adductum asher x 60. p. ciliolare (rchb.f) stein x 61. phreatia listrophora ridl. x 62. p. sulcata (blume) j.j.sm. x 63. pinalia bractescens (lindl.) kuntze x 64. p. densa (ridl.) w.suarez & cootes x 65. p. macera (ames) w.suarez & cootes x 66. plocoglottis plicata (roxb.) ormerod 67. rhomboda sp. (lindl.) ormerod x 68. robiquetia cerina (rchb.f) garay x 69. robiquetia sp x 70. spathoglottis kimballiana hook.f. x 71. s. plicata blume x x 72. s. tomentosa lindl. x 73. thelasis pygmaea (griff.) lindl. x 74. trichoglottis latisepala ames x 75. trichotosia ramosii (leavitt) kraenzl x 76. undetermined sp. 1 x 77. undetermined sp. 2 x 78. undetermined sp. 3 x 79. undetermined sp. 4 x total 31 19 25 18 2 systems and floral traits also changed with altitude. relatively more auto-pollinating species were found at high altitudes compared with midand low-altitude sites where animal-pollinated species were most abundant. as such, at very extreme conditions, the absence of specific pollinators may therefore have a possible direct influence that limits their range of dispersal and distribution. furthermore, orchids are highly evolved with their pollinators and require a specific relationship with mycorrhizal fungi to biologica nyssana 8 (1)  september 2017: 00-00 buenavista, d.p.  contributions to the orchid flora of mindenao… 36 germinate (o t e r o et al., 2005, s h e f f e r s o n et al., 2007), therefore they are intimately intertwined with the ecology of their habitat. the findings of v a s q u e z et al. (2003) in bolivia also showed that orchid diversity was mainly concentrated in ecoregions with semi-humid to very humid montane rain forests between 1000 m to 3500 m elevation. the very low species richness observed in mt. musuan (380-480 masl) maybe due to the warm and dry climatic condition which is very unfavorable to some species of epiphytic orchids that heavily relies from air moisture and rainfall (b a r t h l o t t et al., 2001). aside from the fact that mt. musuan is already a secondary forest, the nearby cultivated agricultural areas suggests more anthropogenic disturbance in the area than the other four lter sites. such disturbance was likewise considered to negatively influence the species richness in an area (b a r t h l o t t et al., 2001). a total of 40 philippine endemic species were recorded in all the five lter sites. mt. apo harbors most of the endemics with 18 species, followed by mt. kitanglad with a record of 16 endemic species. the least was in mt. hamiguitan with only 9 endemic species then mt. malindang with 8 endemic species. mt. musuan had no endemic species. highly threatened species was also recorded in the field survey. based on iucn (2017), this includes the critically endangered paphiopedilum adductum asher (fig. 3) and the endangered paphiopedilum ciliolare (rchb.f) stein (fig. 4) from mt. hamiguitan, and the vulnerable epigeneium treacherianum (reichb.f ex hook.f.) summerh (fig. 5) from mt. apo. this reflects that the orchid family remains to be poorly known despite of their dominance in the philippine flora. the national list of threatened plants of f e r n a n d o et al. (2008) assessed only the 5% of philippine orchid flora and the remaining 95% remains to be data deficient or poorly studied. the lack of comprehensive ecological and taxonomic studies in this group of plants. conclusion the lter sites in mindanao harbors a number of endemic and understudied wild fig. 3. critically endangered paphiopedilum adductum asher fig. 4. endangered paphiopedilum ciliolare (rchb.f) stein fig. 5. vulnerable epigeneium treacherianum (reichb.f ex hook.f.) summerh. biologica nyssana 8 (1)  september 2017: 31-38 buenavista, d.p.  contributions to the orchid flora of mindenao… 37 orchids. the species composition in every site maybe influenced by various ecological factors such as elevation, forest types, micro-climate conditions and among others. this benchmark data presented from the lter sites in mindanao island, philippines can be utilized for future monitoring of orchid populations and conservation initiatives of threatened species. acknowledgements. the author would like to express his profound gratitude to jim cootes for validating the identification of specimens, dr. victor amoroso for the help and support, and to jerome ga-as for the company during the field work. references agoo, m.g., schuiteman, a., de vogel, e.f 2003: flora malesiana: orchids of the philippines vol. 1 world biodiversity database cd-rom series. national herbarium of the netherlands. ambal, r.g.r., duya, m.v., cruz, m.a., coroza, o.g., vergara, s.g., de silva, n., molinyawe, n., tabaranza, b. 2012: key biodiversity areas in the philippines: priorities for conservation. journal of threatened taxa, 4(8): 2788–2796. bankoff, g. 2007: one island too many: reappraising the extent of deforestation in the philippines prior to 1946. journal of historical geography, 33: 314-334. barthlott w., schmit-neuerburg, v. , nieder, j., engwald, s. 2001: diversity and abundance of vascular epiphytes: a comparison of secondary vegetation and primary montane rain forest in the venezuelan andes. plant ecology. 152: 145–156. brooks t.m., mittermeier, r.a., fonseca, g.a.b., gerlach, j., hoffmann, m., lamoreux, j.f., mittermeier, c.g., pilgrim, j.d., rodrigues, a.s.l. 2006: global biodiversity conservation priorities. science, 313: 58–61. clergue, b., amiaud, b., pervachon, f., lasserrejoulin, f., plantureux, s. 2005: biodiversity: function and assessment in agricultural areas: a review. agronomy for sustainable development, 25:1–15. cootes, j.e. 2001: the orchids of the philippines. timber press, inc. singapore. 231 p. cootes, j.e. 2011: philippine native orchid species. katha publishing co., inc. quezon city, philippines. 289 p. cootes, j.e. 2010: a new dendrochilum species from the philippines. orchideen journal, 3(3):114-115. de vogel, e. 1988a: orchid monographs vol. 3. revision in coleogyninae (orchidaceae) iii the genus pholidota, leiden, the netherlands. de vogel, e. 1988b: orchid monographs vol. 6. revision in coleogyninae, hortus botanicus, leiden, the netherlands. dressler, r. dodson, c. 1960: classification and phylogeny in orchidaceae. annals of missouri botanical garden, 47: 25-67. 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neuropathy original article nagarajan a. department of pharmaceutical chemistry, karpagam college of pharmacy, coimbatore – 641032, tamilnadu, india; the tamil nadu dr. m.g.r. medical university, chennai – 600032, tamilnadu, india nagarajan.a@kcp.edu.in (corresponding author) https://orcid.org/0000-0002-6845-934x vignesh c. department of biotechnology, sastra university, thanjavur, tamilnadu, india ramasamy t. department of biotechnology, ennam college of pharmacy, kinathukadavu taluk, coimbatore 641032 coimbatore – 641032, tamilnadu, india sasikala m. department of pharmaceutical analysis, karpagam college of pharmacy, coimbatore – 641032, tamilnadu, india; the tamil nadu dr. m.g.r. medical university, chennai – 600032, tamilnadu, india mohan s. department of pharmaceutics, karpagam college of pharmacy, coimbatore – 641032, tamilnadu, india; the tamil nadu dr. m.g.r. medical university, chennai – 600032, tamilnadu, india received: august 27, 2024 revised: september 25, 2024 accepted: september 30, 2024 abstract: aldose reductase enzyme is involved in key step controlling the polyol pathway and acknowledged as a possible target for diabetic neuropathy. the anionic region of the active site comprising residues his110 and tyr48 is involved in the detoxification mechanism that is considered important. this process involves eliminating toxicity through conversion of aldehydes such as methylglyoxal (meg) and 3-deoxyglucosone. so far, no drug has been developed that could selectively inhibit reduction of glucose to sorbitol without affecting the detoxification mechanism. to comprehend the critical interactions of the known aldose reductase inhibitors and the enzyme, molecular docking was conducted in this study. anionic region, specificity region and the active site are the three regions used separately for grid molecular docking studies to identify the possible inhibitors that selectively bind to specificity region. among the docked compounds, quercetin was found to bind to specificity region, so we chose reported quercetin analogue and built a pharmacophore model. subsequently, a collection of 82,000 natural substances from the ibs databank (interbioscreen) was searched using a 6-point pharmacophore (aaarrr) that was created. after the initial search returned 2,394 hits, a hierarchical docking approach was used to identify an intriguing hit molecule (mol_7921), which is quite close to a flavonoid compound. molecular dynamics simulation was used to confirm the hit molecule-aldose reductase complex’s structural stability. for its possible inhibitory effect, this molecule could be tested and used to treat diabetic neuropathy. key words: diabetic neuropathy, polyol pathway, methylglyoxal, quercetin, virtual screening, inhibitors apstrakt: iza granica: pionirska otkrića u inhibiciji aldoza reduktaze za dijabetičku neuropatiju enzim aldoza reduktaza je uključen u ključni korak kontrole poliolnog puta i prepoznat je kao moguća meta za dijabetičku neuropatiju. anjonski region aktivnog mesta, koji obuhvata ostatke his110 i tyr48, uključen je u mehanizam detoksikacije koji se smatra važnim. ovaj proces podrazumeva eliminaciju toksičnosti kroz konverziju aldehida, kao što su metil glioksal (meg) i 3-dezoksiglukozon. do sada nije razvijen nijedan lek koji bi mogao selektivno inhibirati redukciju glukoze u sorbitol bez uticaja na mehanizam detoksikacije. kako bismo razumeli ključne interakcije poznatih inhibitora aldoza reduktaze i enzima, sprovedeno je molekulsko dokovanje u ovoj studiji. anjonska regija, specifična regija i aktivno mesto su tri regije korišćene zasebno za dokovanje molekula kako bi se identifikovali mogući inhibitori koji se selektivno vezuju za specifičnu regiju. među dokovanim jedinjenjima, ustanovljeno je da se kvercetin vezuje za specifičnu regiju, pa smo odabrali prijavljeni analog kvercetina i izgradili farmakoforski model. nakon toga, pretraženo je 82.000 prirodnih supstanci iz ibs baze podataka (interbioscreen) koristeći farmakofor sa 6 tačaka (aaarrr). početna pretraga vratila je 2.394 pogodaka, nakon čega je korišćen hijerarhijski pristup dokovanju kako bi se identifikovalo ciljno jedinjenje (mol_7921), koje je veoma blisko flavonoidnom jedinjenju. simulacija molekulske dinamike je korišćena da potvrdi strukturnu stabilnost kompleksa ciljnog molekula i aldoza reduktaze. ovo jedinjenje bi moglo biti testirano i korišćeno za lečenje dijabetičke neuropatije zbog svog mogućeg inhibitornog efekta. ključne reči: dijabetička neuropatija, put poliola, metil glioksal, kvercetin, virtualni skrining, inhibitori © 2024 nagarajan et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 123 introduction diabetic neuropathy is a complication of diabetes attributed to the failure of peripheral nerves and reduction in myelinated nerve fiber density. diabetes treatment using herbal medicine has been explored and natural flavonoids such as quercetin find use in treating diabetes, as revealed by several studies (yagihashi et al., 2011; madić et al., 2017; jensen et al., 2021). it also depicts other symptoms such as nerve pinch, prolonged pain and loss of sensitivity (gronda et al., 2020). this disorder strikes the peripheral nerves that are present outside the brain, at the terminals of the spinal cord. they disrupt the nerve conduction mechanism in these peripheral nerves. in fact, the prevalence of diabetic neuropathy is 7% in the first year of diagnosis reaching up to 50% for those with more than 25 years (singh et al., 2021). loss of sensation in the lower limbs causes limb amputation in 1–2% of diabetic patients and requires high cost of treatment. making an early diagnosis and managing diabetic neuropathy are challenging, and currently, no cure is available. the polyol pathway converts glucose to sorbitol by reduction through enzyme aldose reductase which is the key factor behind this complication of diabetes. aldehyde reductase comes under the family of oxidoreductases. under normal conditions, aldose reductase enzyme doesn’t show significant affinity in binding to glucose. on the other hand, under high blood sugar conditions, the amount of glucose retained in the system without being converted into energy increases. such left out volumes of glucose easily pass through the membranes of nerve cells, kidney cells and retinal cells. after entrance, they bind to aldose reductase, as it has a higher affinity for them under such conditions (twarda-clapa et al., 2022). this doesn’t mean that the normal energy conversion process won’t take place, but the glucose being released in larger volumes cannot be incorporated into the normal process of energy conversion and moves into the next possible process, here being the polyol pathway. diabetic neuropathy is a complication associated with diabetes caused by an increase in glucose flux under hyperglycemic conditions leading to the accumulation of sorbitol in nerve cells (goto et al., 1995). this causes reduced myo-inositol production (goto et al., 1995) in addition to inducing the production of advanced glycation end products (age’s). the anionic region of the active site has residues his110 and tyr48 that are involved in the much important detoxification mechanism (klebe et al., 2004) wherein methylglyoxal and 3-deoxyglucosone are reduced to curb toxicity. several aldose reductase inhibitors (ari) emerged, but not successful at clinical owing to the adverse effects or inferior efficacies. epalrestat, licensed in japan is the single drug of this category (kumar et al., 2012). aldose reductase carries out a process of detoxifying the toxic aldehydes. all the synthetic and natural inhibitors studied so far interfere with the detoxification mechanism by occupying that region or binding to residues trp111, his110, and tyr48 (imran et al., 2022). this inner anionic region of the active site generally carries out this process of detoxification by binding to toxic aldehydes (sulaiman et al., 2022). when this process is affected, toxic aldehydes in the system glycate the nitrogen on collagen-like proteins, forming ages, which contribute to diabetic complications such as peripheral neuropathy, diabetic nephropathy, and diabetic angiopathy. therefore, finding a drug that would focus on selective inhibition of aldose reductase binding to glucose without affecting the detoxification mechanism would prove a good solution. the detoxification mechanism is a vital phenomenon that takes place with the assistance of aldose reductase enzyme, where the toxic aldehydes come and bind to the residues trp111, his 110, tyr48 present in the inner anionic region of the active site. glucose generally comes in and binds to the active site making interactions with tyr48 that donates a proton during the reduction mechanism (chen et al., 2022). therefore, finding an inhibitor that would block the entry of glucose (a bulkier group) while allowing toxic aldehydes, such as methylglyoxal and 3-deoxyglucosone (three-carbon group compounds), to enter the pocket helps us eliminate the reduction of glucose to sorbitol and does not affect the detoxification mechanism. sulaiman et al. (2022) reported the presence of markers related to antioxidant property and carbonic anhydrase level in correlation with diabetes complications (chen et al., 2022). advanced glycation end products were related to molecular mechanisms underlying the skin fibroblast growth parameters (ciccone et al., 2022). antioxidant utility in combination with aldose reductase inhibition has been evaluated with several benzaldoxime derivatives (zhang et al., 2022). the potential of aldose reductase inhibitory activity has been suggested to improve treating injury of medulla spinalis through neuronal differentiation of transplanted nscs (zheng et al., 2021). emodin has been evaluated as a potential candidate for treating metabolic diseases through its multifaceted role in inflammation and diabetes (saeed et al., 2022). these authors have identified 6-oxo-6,9-dihydro1h-purin-8-yl) thio) acetamide as potential aldose reductase inhibitor through pharmacophore-based 124 biologica nyssana ● 15 (2) december 2024: nagarajan et al. ● beyond boundaries: pioneering discoveries in aldose reductase inhibition for diabetic neuropathy biologica nyssana ● 15 (2) december 2024: nagarajan et al. ● beyond boundaries: pioneering discoveries in aldose reductase inhibition for diabetic neuropathy 125 search in nubbedb database (bakal et al., 2022). using molecular simulation and docking bakal et al. (2022) identified (z)-2-(5-((1-methoxynaphthalen2-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl) acetic acid derivative as aldose reductase inhibitor (fig. 1). recent reports on the identification of thiazolidin-dione through docking showed interactions with residues trp111, ala299, leu300 and phe122 located at the specificity pocket (balestri et al., 2022), and reviewed various natural and synthetic compounds that act as aldose reductase inhibitors, including cyclic imides, polyphenols, and carboxylic acid derivatives (friesner et al., 2006; zhu et al., 2022). zhu et al. (2022) revealed the purine derivatives (fig. 1) as a potential inhibitor of aldose reductase having an ic50 value of 0.038 μm. these reports suggest that there is still no selective inhibitor for aldose reductase warranting further search for a candidate molecule to address the diabetic complication affecting the eye. materials and methods overview of steps used in the present study is given in fig. 2. an initial docking analysis was carried out with set of natural compounds that were tested in vitro for inhibition of aldose reductase. we understood the pharmacophore requirements using the same set of compounds that were likely to bind to the target region i.e. the specificity region of our enzyme active site. then a pharmacophore model was developed using a set of analogues from the most likely compound that could be considered as a starting point for finding a perfect inhibitor. a 3d database search was carried out using the obtained pharmacophore model and a final docking analysis was carried out using the hits from the search to screen out fewer effective compounds. in silico tools such as virtual library screening, docking methods, ligprep, protein preparation and pharmacophore analysis were carried out using the gui maestro v9.1 of the schrödinger software (rajeswari et al., 2014). phase module of schrödinger software was used to build a six-point pharmacophore (verma & thareja, 2020). data set ligands for initial analysis the initial set of natural and synthetic ligands were obtained from different literature and deposited pdb structures, respectively. we collected diverse natural compounds (chaudhry et al., 1983) that have been found effective in vitro. ligands from co-crystallized pdb structures were obtained to confirm the reliability of our results from the initial docking analysis. also, methylglyoxal structure was obtained to confirm its binding to the anionic region of the binding pocket. the set of natural and synthetic compounds as described earlier were created using symyx draw v4.0. they were prepared using ligprep module of the gui maestro v9.1. docking was employed with extra-precision mode (xp mode) of glide software of the gui maestro v9.1. receptor protein the receptor protein was retrieved using the protein data bank which is a depository of protein structures. the structure of aldose reductase bound to two molecules of benzothiazepine inhibitor (pdb id: 3p2v) at the resolution of 1.69 ǻ was chosen as the receptor for all further studies (fig. 3). this is the structure of aldose reductase which is a 316aa long and co-crystallized with two benzothiazepine inhibitors in its active site. the compounds with structure shown in fig. 4 are taken for preliminary screening. results in our work, we docked methylglyoxal (a toxic aldehyde) and found it occupying the exact same anionic region. therefore, we used the docked fig. 1. structures of known aldose reductase inhibitors (z)-2-(5-((1-methoxynaphthalen-2-yl)methylene)-4oxo-2-thioxothiazolidin-3-yl)acetic acid, 2-(6-((4-bromo2-fluorobenzyl)amino)-9h-purin-9-yl)acetic acid (zhu et al., 2022) fig. 2. steps used in identification of hits 126 biologica nyssana ● 15 (2) december 2024: nagarajan et al. ● beyond boundaries: pioneering discoveries in aldose reductase inhibition for diabetic neuropathy fig. 3. structure of aldose reductase bound to novel benzothiazepine pdb id: 3p2v fig. 4. the quercetin scaffold used to derive analogues for pharmacophore model development receptor-ligand complex for further docking analysis with the set of natural and synthetic compounds. grids were prepared for the anionic region, important region and the active packet separately (fig. 5). quercetin analogues (fig. 6, tab. 1) with in vitro activity (kondhare et al., 2017) were used to build a qsar-based pharmacophore model and utilized this model to screen a library of natural compounds (82,000) obtained from ibs database in search of a selective inhibitor. these authors have compared the activity and stated that “improvement observed in the inhibition of glycosylated sugar myricitrin was higher than that observed for the aglycone myricetin (kondhare et al., 2017). the specificity pocket forms the entry part of the active site at the external surface. the docking fig. 5. the active site residues making up the two regions fig. 6. structures chosen for initial docking study analysis was carried out by setting grids for anion binding, region specific to binding and the active regio as a whole and the same set of compounds were docked into these three regions. the obtained results are presented in tab. 2. the reliability of this analysis was confirmed by the observations made with respect to the binding of the toxic aldehyde, methylglyoxal. methylglyoxal docked with the residues trp111, his110 and tyr48 of anionic region showed interaction which reflected the literature studies explaining the detoxification mechanism. also, the ligands obtained from pdb structures docked into the active site, making 80% of the interactions as in the deposited structures. in the case of rosmarinic acid, although it showed good interactions and better docking scores, its size and biologica nyssana ● 15 (2) december 2024: nagarajan et al. ● beyond boundaries: pioneering discoveries in aldose reductase inhibition for diabetic neuropathy 127 ta bl e 1. q ue rc et in a na lo gu es a nd th ei r a ct iv ity v al ue s s. n o. sc aff ol d su bs tit ue nt a ct iv ity [lo g( 1/ ic 50 )] µm s. n o. sc aff ol d su bs tit ue nt a ct iv ity [lo g( 1/ ic 50 )] µm 1 a 5, 7 , 3 ’, 4’ -o h ; 3 ,6 -o c h 3 7. 59 26 a 5, 6, 3’ ,4 ’o h ; 7 -o c h 3 6. 52 2 a 3’ ,4 ’o h ; 5 ,6 ,7 ,8 -o c h 3 7. 49 27 a 6, 3’ ,4 ’o h ; 3 ,5 ,7 -o c h 3 6. 52 3 a 6, 3’ ,4 ’o h ; 5 ,7 ,8 -o c h 3 7. 44 28 a 5, 3’ ,4 ’o h ; 3 ,6 ,7 -o c h 3 6. 46 4 a 5, 7, 3’ ,4 ’o h ; 6 -o c h 3; 8c h 2p h 7. 47 29 a 5, 7, 4’ -o h ; 6 ,8 -o c h 3 6. 39 5 a 5, 3’ ,4 ’o h ; 6 ,7 ,8 -o c h 3 7. 41 30 a 5, 4’ -o h ; 6 ,7 ,8 -o c h 3 6. 27 6 a 3’ ,4 ’o h ; 5 ,7 ,8 -o c h 3 7. 35 31 a 5, 6, 3’ ,4 ’o h ; 3 ,7 -o c h 3 6. 09 7 a 5, 6, 7, 3’ ,4 ’o h ; 3 -o c h 3 7. 24 32 a 3, 5, 7, 3’ ,4 ’o h 6. 02 8 a 5, 6, 3’ ,4 ’o h ; 7 ,8 -o c h 3 7. 19 33 a 5, 6, 4’ -o h ; 7 ,8 -o c h 3 6. 07 9 a 7, 3’ ,4 ’o h ; 5 ,8 -o c h 3 7. 13 34 a 5, 6, 7, 4’ -o h ; 8 -o c h 3 5. 92 10 a 5, 3’ ,4 ’o h ; 7 ,8 -o c h 3 7. 11 35 a 5, 6, 7, 4’ -o h ; 8 ,3 ’o c h 3 5. 92 11 a 3, '4 ’o h ; 5 ,6 ,7 -o c h 3 7. 04 36 a 5, 4’ -o h ; 6 ,7 -o c h 3 5. 85 12 a 5, 6, 7, 3’ ,4 ’o h ; 8 -o c h 3 6. 92 37 a 5, 7, 3’ ,4 ’o h ; 3 -o -r h 6. 54 13 a 6, 3’ ,4 ’o h ; 5 ,7 -o c h 3 6. 85 38 a 5, 7, 4’ -o h ; 6 ,8 ,3 ’o c h 3 5. 35 14 a 4’ -o h ; 5 ,6 ,7 ,8 -o c h 3 6. 79 39 a 6, 4’ -o h ; 5 ,7 ,8 ,3 ’o c h 3 5. 2 15 a 8, 3’ ,4 ’o h ; 5 ,7 -o c h 3 6. 79 40 a 5, 4’ -o h ; 6 ,7 ,3 ’o c h 3 5. 17 16 a 3’ ,4 ’o h ; 3 ,5 ,7 ,8 -o c h 3 6. 77 41 a 5, 7o h ; 6 ,8 ,4 ’o c h 3 5. 14 17 a 5, 6, 7, 3’ ,4 ’o h 6. 69 42 a 5, 6, 7o h ; 8 -o c h 3 5. 09 18 a 5, 3’ ,4 ’o h ; 6 ,7 -o c h 3 6. 92 43 a 5, 6o h ; 7 ,8 -o c h 3 5. 08 19 a 5, 8, 3’ ,4 ’o h ; 7 -o c h 3 6. 64 44 a 3’ ,4 ’o h ; 5 ,6 ,7 -o c h 3; 3c o c h 3 5. 05 20 a 5, 7, 3’ ,4 ’o h ; 3 ,8 -o c h 3 6. 62 45 a 5, 3’ -o h ; 6 ,7 -o c h 3; 4’ -o -g lc 5. 09 21 a 6, 4’ -o h ; 5 ,7 ,8 -o c h 3 6. 6 46 a 5o h ; 6 ,7 ,3 ’o c h 3; 4’ -o -g lc 4. 6 22 a 3’ ,4 ’o h ; 5 ,6 ,7 -o c h 3 6. 57 47 a 5o h ; 6 ,7 -o c h 3; 4’ -o -g lc 4. 6 23 a 5, 7, 3’ ,4 ’o h ; 8 -o c h 3 6. 55 48 a 5, 7o h ; 6 ,8 ,3 ’o c h 3; 4’ -o -g lc 4. 33 24 a 7, 3’ ,4 ’o h ; 3 ,5 ,8 -o c h 3 6. 55 49 a 4’ -o h ; 5 ,6 ,7 ,8 ,3 ’o c h 3 4. 74 25 a 8o c h 3; 5, 6, 7, 3’ ,4 ’o c o c h 3 6. 52 50 a 5, 4’ -o h ; 6 ,8 ,3 ’o c h 3; 7o -g lc 4. 15 biologica nyssana ● 15 (2) december 2024: nagarajan et al. ● beyond boundaries: pioneering discoveries in aldose reductase inhibition for diabetic neuropathy table 2. quercetin analogues and their activity values compound interactions when docked into the specificity pocket (docking score) interactions when docked into the anionic region interactions when docked into the active site selectivity towards bis-coumarin leu300 [-11.74] leu300 [-12.62] trp111 [-11.10] intermediate epalrestat ala299, leu300 [-12.303] tyr48, his110, trp111 [-11.07] trp111 [-11.10] both regions sulfonic acid derivative leu301, ser302, trp111 [-11.04] trp111, tyr48 [-11.23] ala299, leu300, leu301, trp111 [-6.684] both quercetin leu300, leu301 [-12.981] leu300, leu301 [-12.719] ala299, leu301, his110 [-13.293] specificity region rosmarinic acid ser302(2), leu300, lys221 [-12.37] val47, tyr48, his110, trp111, ala299 [-14.904] ala299, leu301, nadp, his110 [-17.05] both regions fidarestat trp111, leu300 [-10.19] trp20, trp111, nadp [-10.10] leu300, trp111, his110, tyr48 [-10.45] both regions idd594 ala299, leu300, trp111 [-11.11] trp111, nadp, tyr48 [-13.13] trp111, his110, tyr48 [-6.457] both regions methylglyoxal (meg) trp111, his110 [-6.309] trp111, tyr48, his110 [-6.341] trp111, his110, tyr48 [-6.317] both regions * glide scores are enclosed in square brackets the volume that it occupies represented a drawback. as a part of the analysis for this process, we found quercetin selectively occupying the specificity pocket in all the three cases of docking (except for one case where it made interaction with the his110 when docked into the active site as a whole). quercetin, a natural ligand found in citrus fruits, has been identified as a potent inhibitor in various in vitro studies. however, it has exhibited some side effects, which have prevented its acceptance as a drug. therefore, with these observations we took over quercetin and proceeded for the next step of developing a pharmacophore model based on quercetin analogues. pharmacophore feature analysis we considered the compounds that docked into the specificity region and studied their pharmacophore features to understand the groups required for effective binding into that region. the compounds chosen were rosmarinic acid, sulfonic acid derivative, bis-coumarin, quercetin, fidarestat and idd594 (fig. 6). the ligands were prepared, and its conformers were generated as a part of the process. they were cleaned later to rule out bad, duplicated structures. respective activity values were specified and based fig. 7. adr features mapped over quercetin 128 biologica nyssana ● 15 (2) december 2024: nagarajan et al. ● beyond boundaries: pioneering discoveries in aldose reductase inhibition for diabetic neuropathy on the maximum hypothesis generated; a 3-point pharmacophore (fig. 7) was obtained with adr (acceptor, donor and ring) features. this provided us with an understanding that for a molecule to bind into the specificity region, it must have at least two of these three or a combination of such features. since our specificity comprises the residues ala299 and leu300-301, which possess hydrogen bond donor/acceptor functionalities, it facilitates interactions that form hydrogen bonds with incoming ligand molecules. provided that the ligand is appropriately sized, it will engage exclusively with the specificity region and will not intrude into the anionic region of the active site. although sulfonic acid derivative showed good fitness (3.000) and a better alignment score (0.000) than quercetin, it does not satisfy the necessary criteria and was found extending into the anionic region during the initial docking analysis. also, quercetin comparatively had better fitness (2.328) and a good alignment score (0.033). the size and the volume it occupies places quercetin ahead of sulfonic acid derivative to be taken for further studies. the specificity pocket has the property of adapting different inhibitors since the residues 297-300 show adaptations with respect to the inhibitor binding to it. as a smaller molecule than the sulfonic acid derivative, quercetin can create space for toxic aldehydes to enter upon binding. in contrast, the sulfonic acid derivative fits perfectly at the entry of the active site, preventing additional compounds from accessing this space. pharmacophore model development quercetin analogues were designed as described earlier in tab. 1 using symyx draw v4.0. these ligands were generated with ligprep of the maestro v9.1 gui interface. conformers were generated and their geometries were cleaned accordingly to rule out bad as well as duplicate structures. activity values (pic50) were specified to separate out the active from the inactive molecules. with quercetin in mind, a 6-point pharmacophore model was developed, achieving the highest score for this hypothesis. the model (fig. 8) had the features aaarrr (3 h-bond acceptors and 3 aromatic characteristics). the three ring features aligned with the core structure of a flavonoid. in our case, quercetin, the obtained model provides better chance for searching an optimum compound with the 6 features since it could result in finding a molecule which will have a smaller size and could make hydrogen bonds with the specificity pocket easily. this will help us finding a molecule with a similar core structure (3 ring structures) which is important for our study such that the functional groups would bind accordingly to the residues in the specificity region rather than entering the anionic region. virtual screening the 6–point pharmacophore (aaarrr) was used as the basis for search from a library of natural compounds obtained from ibs database (interbioscreen). a total of 82,000 compounds were taken from the database and a pharmacophore-based search was carried out. 2394 hits were obtained as a result of the initial search. these hits were stored as a separate entry and prepared using ligprep module of the gui maestro v9.1. ligprep helps in generating conformers and unique geometries. after preparation we had 5745 entries in hand. we proceeded with a stepwise docking-based screening process to screen out compounds and retain the best ones. the three stages included htvs docking, sp docking and xp docking. in high throughput virtual screening docking, the criteria were set to retain only 5% of the best posed molecule conformations. this brought down the number of hits to 287. in standard precision docking, the criteria were set to retain 10% of the best posed molecule conformations. thus, we ended up with 28 compounds for the final screening procedure. using extra precision docking, we initially selected the top 14 hits from the sp docking based on their scores and retained the best pose from this step. consequently, we identified a specific hit that docked as anticipated in the entry region of the specificity pocket. the final hit, mol_7921, exhibited a core structure similar to that of quercetin (fig. 9). it occupied the specificity pocket making strong hydrogen bond interaction with residues val297, ala299, leu300 (fig. 10a and fig. 10b) had a score fig. 8. model aaarrr (best fit a12) fig. 9. final hit (left) compared with quercetin 129 of -15.363 kcal/mol. it also did not interfere with the anionic region, as it docked into the active site entry region, which forms the pocket responsible for specificity. the molecule identified in this study effectively blocks glucose entry into the active site, covering almost the entire entry region while leaving a small space that prevents glucose from entering the anionic region. however, our final hit has a minor drawback: depending on its spatial orientation, it may or may not permit the entry of toxic aldehydes necessary for the detoxification mechanism. recent reports on the identification of thiazolidindione through docking showed interactions with residues trp111, ala299, leu300 and phe122 located at the specificity pocket, which also confirms our finding that the interactions with trp111 is critical for the inhibition of aldose reductase. toxicity prediction (fig. 11) for the hit compounds using protoxii indicates that the compound is safe for oral consumption as it falls under category 5. md simulation of hit using the gromacs molecular dynamics tool, a 100ns md simulation of the final hit compound complexed with aldose reductase enzyme was performed. the stability and compactness of the protein structure were determined by standard protocols involving “root mean square deviation” (rmsd), “root mean square fluctuations” (rmsf), and “radius of gyration” (rg). the average rmsd value is ~ 0.08 nm for the backbone protein (fig. 12a). higher rmsd values indicate that protein stability decreases, whereas a lower rmsd value indicates an increase in protein stability. rmsf calculates the fluctuations of individual residue in presence of the ligands. the macromolecule-ligand complex was maintained in a triclinic box with dimensions of 43:54:47 and solvated using the simple point charge (spc) water model, neutralized with a 0.15 m nacl salt concentration. the opls/aa force field was employed to parameterize the macromolecule and its ligands. energy minimization was performed using the steepest descent technique to eliminate steric disturbances between the macromolecule and the ligand. the leapfrog and two-phase ensemble equilibration algorithms were utilized for integration in the nvt and npt at fixed temperature (300 k) and pressure (1 bar). after initialization and equilibration, molecular dynamics simulations were conducted for 100 ns, with data collected to determine rmsd, rmsf, and hydrogen bonding. the rmsd and rmsf for the hit-aldose reductase complex (fig. 12b) showed an average rmsd of 0.1 nm during the period from 20 ns to 60 ns, indicating that the stability of the complex was biologica nyssana ● 15 (2) december 2024: nagarajan et al. ● beyond boundaries: pioneering discoveries in aldose reductase inhibition for diabetic neuropathy fig. 10. 2d interaction for the hit molecule and aldose reductase (a). different poses of hit molecule, occupying the specificity pocket (left) making interactions (left, right) with val297, ala299 and leu300 at distances of 2.2ǻ, 2.9ǻ and 2.0ǻ respectively (b) 130 biologica nyssana ● 15 (2) december 2024: nagarajan et al. ● beyond boundaries: pioneering discoveries in aldose reductase inhibition for diabetic neuropathy fig. 11. toxicity prediction for the hit molecule fig. 12. rmsd of protein with hit compound (a). rmsf of protein with hit compound (b) gradually maintained, with only slight deviations from the initial simulation (0.05 nm to 0.06 nm). the simulation results indicated that the complex remained stable from 25 ns to 100 ns, which is considered good stability throughout the simulation period. the rmsf analysis revealed an average fluctuation rate of 0.1 nm, confirming that the complex maintained structural stability with the ligand bound to it. a study by kondhare et al. (2022) describes the aldose reductase inhibitory activity of alkannin (fig. 13), a naphthoquinone-based compound from plants in the boraginaceae family. docking studies showed that alkannin formed hydrogen bonds with val297, ala299, leu300, and ser302, along with hydrophobic interactions (trp20, val47, fig. 13. alkannin 131 biologica nyssana ● 15 (2) december 2024: nagarajan et al. ● beyond boundaries: pioneering discoveries in aldose reductase inhibition for diabetic neuropathy tyr48, trp79, trp111, phe122, trp219, val297, cys298, ala299, leu300, and leu301). the results from in vitro studies show a potent dose-dependent inhibitory activity for aldose reductase at 22.77 μm. given the recent emphasis on identifying effective aldose reductase inhibitors, our results represent a starting point for the extensive research needed to achieve this goal. furthermore, the hit molecule identified here does not extend into the anionic region responsible for detoxifying toxic aldehydes produced by the lipid pathway. this is a crucial criterion for an aldose reductase inhibitor, as it prevents glucose from entering the active site. however, the binding orientation of this molecule may allow or restrict the entry of toxic aldehydes into the pocket. specifically, it may permit the entry of methylglyoxal due to its size and volume, while it does not allow glyceraldehyde, a four-carbon linear molecule, to enter. conclusion molecular ligand docking into three regions anionic region, specificity region, and active site region, allowed the identification of possible inhibitors that selectively bind to the specificity region. the hit molecule (mol_7921) bears much similarity to a flavonoid and can therefore be considered a good starting point for future studies. unlike the other compounds that we analyzed and studied in our earlier workflow, this molecule (mol_7921, fig. 9) does not extend into the anionic binding region or interfere with the residues tyr48, his110, and trp111, which are responsible for carrying out the detoxification mechanism of toxic aldehydes. this helped us fulfill the first objective of preventing glucose entry into the active site. however, close observations revealed that this molecule has bound in such a spatial orientation that it may or may not allow the entry of toxic aldehydes into the pocket. methylglyoxal, being a four-carbon linear molecule, might find it difficult to enter, while glyceraldehyde may be allowed to enter the pocket due to its size and volume. this is due to the presence of a sevenmembered ring at one end, which is responsible for this drawback. the results of the simulation showed the stability of the complex from 25 ns to 100 ns, indicating the intact nature of the bound form throughout the simulation period. the structural stability of the ligand-bound complex is further supported by rmsf, wherein the average fluctuation rate is 0.1 nm. however, the hit molecule needs to be tested to confirm the binding mode and its potential use for diabetic neuropathy. acknowledgements. the authors thank the management of karpagam charities trust, coimbatore and sastra university, thanjavur for their support throughout all aspects of our study. references bakal, r.l., jawarkar, r.d., manwar, j.v., jaiswal, m.s., ghosh, a., gandhi, a., zaki, m.e., al-hussain, s., samad, a., masand, v.h., & mukerjee, n. 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(2022). identification of 9h‐ purin‐6‐amine derivatives as novel aldose reductase inhibitors for the treatment of diabetic complications. archiv der pharmazie, 355(8), 2200043. 133 jušković et al. 2021, biologica nyssana 12(1) 12 (1) september 2021: 11-21 doi: 10.5281/zenodo.5522956 a comparative anatomical study on two closely related astragalus l. taxa (fabaceae) from the central part of the balkan peninsula original article marina jušković department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, niš, serbia marinaju@pmf.ni.ac.rs (corresponding author) dragana jenačković gocić department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, niš, serbia draganaj@pmf.ni.ac.rs danijela nikolić department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, niš, serbia danid@pmf.ni.ac.rs bojan zlatković department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, niš, serbia bojanzlat@yahoo.com vladimir ranđelović department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, niš, serbia vladar@pmf.ni.ac.rs received: april 29, 2021 revised: june 29, 2021 accepted: september 01, 2021 abstract: the present study involves species a. monspessulanus l. and a. spruneri boiss. section incani dc. from the area of central balkans. due to high polymorphism, this section is taxonomically the most problematic group and delimitations of its species are not completely clear. the main aims of the study were to determine the micro-morphological and anatomical variability of populations of these species based on quantitative characters of leaflet and leaf petiole, and also to show the potential degree of differentiation within species. additional goal was to determine anatomic parameters that may be used in taxonomy of studied species and their encompassing section. the results indicate high variability of analyzed characters and anatomical differentiation of populations of each species. results of statistical analyses indicate that characters which refer to petiole anatomy have higher taxonomic value than characters which refer to leaflet anatomy. anatomical features described in this paper mostly agree with characters found in previous studies of other astragalus species, and they have potential taxonomic significance. key words: astragalus monspessulanus, a. spruneri, anatomical characters, taxonomy apstract: uporedna anatomska istraživanja dva veoma srodna astragalus l. taksona sa centralnog dela balkanskog poluostrva u ovom radu su analizirane vrste a. monspessulanus l. i a. spruneri boiss., sekcije incani dc. sa područja centralnog balkana. sekcija incani dc. predstavlja taksonomski složenu sekciju, sa velikim brojem endemičnih vrsta, u okviru koje, zbog preklapanja morfoloških karaktera i velike fenotipske plastičnosti, odnosi između vrsta još uvek nisu u potpunosti razjašnjeni. glavni cilj rada bio je utvrđivanje mikro-morfološke i anatomske varijabilnosti populacija ovih vrsta na osnovu kvantitativnih karakteristika lamine i lisne drške, kao i prikaz potencijalnog stepena diferencijacije unutar vrsta. cilj je bio i otkriti anatomske karaktere koji se mogu koristiti u taksonomiji proučavanih vrsta, kao i sekcije kojoj one pripadaju. rezultati ukazuju na visoku varijabilnost analiziranih karaktera i anatomsku diferencijaciju populacija svake vrste. rezultati statističkih analiza takođe pokazuju da karakteristike koji se odnose na anatomiju lisne drške imaju veću taksonomsku vrednost od karakteristika koji se odnose na anatomiju lamine. anatomske karakteristike opisane u ovom radu uglavnom se slažu sa karakteristikama opisanim u prethodnim istraživanjima drugih vrsta roda astragalus i imaju potencijalni taksonomski značaj. ključne reči: astragalus monspessulanus, a. spruneri, anatomska diferencijacija, taxonomy introduction the genus astragalus l. is one of the largest genera of vascular plants, with 2500-3000 species (podlech, 1986; maassoumi, 1998; ranjbar & karamian, 2002). this genus is widespread, mostly in the northern hemisphere and in south america, with the diversity centers in arid and semiarid mountainous areas. the place of the greatest diversity and evolutionary differentiation is in southwestern asia (maassoumi & ranjbar, 1998). the region of southern europe, including balkan peninsula, contains a particularly large number of astragalus species (60). this is one of the taxonomically most interesting polymorphic genera of the balkan flora. there are 17 species in © 2021 jušković et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 11 flora of serbia according to diklić (1972). additional floristic research also provided evidence of species a. exscapus l. (boža, 1979), so with addition of the recently recorded species astragalus wilmottianus the genus astragalus is presently represented in serbia with 19 species (ranđelović et al., 2002). many of which are narrow-range endemics. in spite of numerous studies, genus astragalus is still characterized by insufficiently resolved taxonomic issues. the goal of the previous studies was to evaluate interspecies relationships within the genus astragalus, including both taxonomy and phylogeny and using morphological, phytogeographical, molecular systematics, palynological and karyological characters (podlech, 1986; sharawy et al., 2003; osaloo, 2003; taeb et al., 2007; mourad & sharawy, 2010; meher et al., 2012; el-sahhar et al., 2013; amini et al., 2018). this genus shows high morphological variability and complexity. certain characters, including characteristics of trichomes and leaves, were used in subgeneric classification of genus astragalus, introducing subgenera (podlech, 1982; zarre, 2000; zarre, 2003; meher et al., 2012). there is high significance of the overall shape of hairs and detailed micromorphological studies on hairs. zarre (2003) has shown that several characters of hairs may be used for phylogeny reconstruction in astragalus. the anatomy of vegetative organs of astragalus species has not been studied extensively. on the other hand, the anatomical parameters have been shown to greatly contribute to solving of significant taxonomic issues within various taxonomic groups (zarre et al., 2010; jušković et al., 2016, 2017; raca et al., 2017; stojanović et al., 2019). in the present paper, micro-morphological and anatomical variability of populations a. monspessulanus l. and a. spruneri boiss. were analyzed according to quantitative characters of leaflet and leaf petiole. intraspecies variability plays a key role in both long-term and shortterm responses of species toward variations in environmental factors. species a. monspessulanus and a. spruneri belongs to the section incani dc. this is one of the most species-rich sections of astragalus, with 140 species. due to extensive overlaps in morphological characters and high phenotype plasticity, section incani dc. represents a taxonomically interesting group. the selection of studied anatomical characters was based on previous research on astragalus taxa by haddad & barnett, 1989; zarre, 2003; pirani et al., 2006. the analyzed characters were sampled in three populations each of a. monspessulanus and a. spruneri from the central part of the balkan peninsula. the goal was to determine anatomic characters that may be used in determining taxonomy of the studied species as well as the section to which they belong. materials and methods the specimens of a. monspessulanus l. and a. spruneri boiss. were collected during the flowering season, from six native populations from the balkan peninsula. voucher specimens were deposited in the herbarium moesiacum niš, department of biology and ecology, faculty of sciences and mathematics, university of niš, serbia (hmn) (tab. 1). the collected plant material was either placed in the herbarium or fixed in 50% ethanol. the anatomical analysis of leaflet and leaf petiole was performed on permanent and temporary slides prepared by the standard histological method for light microscopy (ruzin, 1999). manual 12 biologica nyssana ● 12 (1) september 2021: 11-21 jušković et al. ● a comparative anatomical study on two closely related astragalus l. taxa from the central part of the balkan peninsula table 1. species analysed, with voucher numbers, and collection sites and dates taxon collection site collection date voucher number astragalus monspessulanus serbia: village petačinci 11-may 2012 hmn-7311 astragalus monspessulanus serbia: gorge of the river jerma, village vlasi 11-may 2012 hmn-7312 astragalus monspessulanus montenegro: morača river canyon, bioče 20-may 2012 hmn-7306 astragalus monspessulanus bulgaria: kyustendil, village staro selo 21-april 2012 hmn-7314 astragalus spruneri macedonia: matka canyon, monastery „matka“ 15-april 2012 hmn-7313 astragalus spruneri macedonia: mariovo, village zović 14-april 2012 hmn-7309 13 microtome (gligorijević & pejčinović, 1983) was utilized in order to make cross-sections. the cross sections of the petiole were taken in the middle, between the stem an lamina, following the observation by howard (1979). epidermal peels, for surface structure and stomata analyses, were prepared by using jefferson’s solution (10% nitric acid and 10% chrome-trioxide, 1:1), stained in safranin and alcian blue, and after the dehydration, mounted in canada balsam. the morphoanatomic measurements were performed on the microscope leica dm 2500-leica dfc490-leica qwin standard (leica microsystem, germany). in the present study, statistical analyses were carried out for 31 quantitative characters related to the leaflet and leaves petiole anatomy. statistical significance of differences between the species in regard to the analyzed characters was assessed by t-test for independent groups. the degree of variability and morpho-anatomical differentiation on level of population and species were established using principal component analysis (pca), canonical discriminant analysis (cda) and agglomerative hierarchical classification (single linkage method). these analyses were performed on the three data sets. one of them was included all characters analysed (leaflet and petiole anatomy), while the other two data sets included only the characters related to leaflet anatomy or petiole anatomy, respectively. all statistical analyses were done in statistical package statistica 8.0 (statsoft, 2007). results and discussion leaflet anatomy of a. monspessulanus the leaflets from all populations in species a. monspessulanus have shown similar anatomical patterns. internal leaflet tissues are organized in biologica nyssana ● 12 (1) september 2021: 11-21 jušković et al. ● a comparative anatomical study on two closely related astragalus l. taxa from the central part of the balkan peninsula fig. 1. cross sections of the leaflet midrib (left) and lamina (right) in investigated astragalus species. a – a. monspessulanus (vlasi), b – a. monspessulanus (petačinci), c – a. monspessulanus (bioče), d – a. spruneri (monastery „matka“), e – a. spruneri (staro selo), f – a. spruneri (zović). scale bars = 50 µm. abbreviations: ade – adaxial epidermis, abe – abaxial epidermis, ph – phloem, pp – palisade parenchyma, sp – spongy parenchyma, st – stomata, vb – vascular bundle, vs – vascular sheath, xy – xylem. 14 an isolateral structure (fig. 1). the leaflets are densely hairy, with bifurcate hairs with two equal or unequal arms, usually opposed in the same plane, tip pointed; arms with verrucose surface are observable particularly on the major vein surface (fig. 2). the presence of bifurcate hairs is a common character in the astragalus. number of hairs at the abaxial side of the leaflet is 0-12 and number on the adaxial side is 0-11. two epidermal layers develop on adaxial and abaxial surfaces of the leaflet. the cells of adaxial epidermis are visibly larger compared to the cells of abaxial epidermis. the outer walls are cutinized. cuticle is thin on both sides of leaf: 1.3-5.6 μm (fig. 1). the epidermal cells of the leaflet are polygonal or irregular in shape. the outline of the anticlinal walls has been described as straight, sinuous or sinuate (fig. 2). the stomata apparatus in astragalus species is present at both surfaces of leaflet, so it is called an amphistomatic leaf. the stomata are tiny, at the same level or slightly above the level of the other epidermal cells. stomata frequency varies, ranging from 57 to 181 per mm2 at the abaxial side and 0 to 161 per mm2 at the adaxial side. the greatest number of stomata (86-181 per mm2 at the abaxial and 0-161 per mm2 at the adaxial side) was recorded in plants from the village vlasi, while the smallest (57-161 per mm2 at the abaxial side and 60 – 128 per mm2 at the adaxial side) was observed in plants from bioče in montenegro (tab. 2). the dominant stomatal type in astragalus is anisocytic (fig. 2). the average recorded thickness of leaflet ranged from 255 μm (population at bioče and village vlasi) to 276 μm (petačinci population), with minimum recorded values of 178 μm and maximum recorded values of 329 μm (tab. 2). internal structure of leaflet does not differ significantly among the studied populations and species. the palisade parenchyma is 2-3 layered with cells arranged parallel to each other, where one layer is better developed. the spongy tissue is composed of 2-3 layers of chlorenchymatous cells with small intercellular spaces. there is a layer of atypical palisade tissue at the abaxial side in the biologica nyssana ● 12 (1) september 2021: 11-21 jušković et al. ● a comparative anatomical study on two closely related astragalus l. taxa from the central part of the balkan peninsula fig. 2. comparative overview of adaxial (left) and abaxial (right) epidermis (a-c; e-g) and trichomes on abaxial epidermis (d, h) in investigated astragalus species. a, d – a. monspessulanus (vlasi), b – a. monspessulanus (petačinci), c – a. monspessulanus bioče, e, h a. spruneri (monastery „matka“), f a. spruneri (staro selo), g – a. spruneri (zović). scale bars = 50 µm. 15 biologica nyssana ● 12 (1) september 2021: 11-21 jušković et al. ● a comparative anatomical study on two closely related astragalus l. taxa from the central part of the balkan peninsula characteristics species t-test p valuea. monsspesulanus a. sprunerii mean±sd mean ±sd leaf thickness (µm) 249.026±33.263 264.208±38.386 0.005 palisade tissue thickness (µm) 105.563±20.982 113.989±21.562 0.009 spongy tissue thickness (µm) 72.518±12.567 84.512±17.181 0.000 largest thickness of the leaf blade (µm) 328.854±31.797 325.899±41.043 0.590 height of adaxial epidermal cells (µm) 38.647±7.729 35.984±5.643 0.009 height of abaxial epidermal cells (µm) 35.894±6.347 34.354±4.939 0.071 cuticle thickness on the leaf adaxial epidermis (µm) 2.649±0.703 2.489±0.306 0.049 cuticle thickness on the leaf abaxial epidermis (µm) 2.475±0.7097 2.458±0.294 0.826 petiole surface area (μm²) 829225.244±184868.229 756978.071±229110.570 0.021 petiole central cylinder surface area (μm²) 484291.184±139401.760 404279.434±141626.392 0.000 petiole epidermis thickness (µm) 39.304±4.727 35.997±3.944 0.000 petiole cortex thickness (µm) 122.697±18.476 131.099±17.211 0.002 surface xylem area of the central vasculare bundle petiole (μm²) 37483.420±9592.423 21245.137±8586.570 0.000 surface phloem area of the central vasculare bundle petiole (μm²) 26875.076±7015.183 15728.974±6315.581 0.000 surface sclerenchym area of the central vasculare bundle of the petiole (μm²) 33323.145±9757.989 15173.440±5963.925 0.000 surface xylem area of the left vasculare bundle petiole (μm²) 9268.319±3833.309 6285.631±2333.143 0.000 surface phloem area of the left vasculare bundle petiole (μm²) 10338.462±4893.622 5889.551±2500.453 0.000 surface sclerenchym area of the left vasculare bundle of the petiole (μm²) 12358.751±4661.728 5589.495±2753.462 0.000 surface xylem area of the right vasculare bundle petiole (μm²) 8630.900±3309.513 6230.853±2189.006 0.000 surface phloem area of the right vasculare bundle petiole (μm²) 9294.905±2778.404 5772.199±2063.511 0.000 surface sclerenchym area of the right vasculare bundle of the petiole (μm²) 11416.189±4396.266 5320.995±2563.347 0.000 number of adaxial stomata (/mm2) 118.184±37.340 117.966±31.301 0.966 number of abaxial stomata (/mm2) 105.274±27.850 106.452±21.090 0.749 number of adaxial hair (/mm2) 3.354±2.619 5.110±2.585 0.000 number of abaxial hair (/mm2) 3.302±2.447 5.257±2.640 0.000 length of adaxial stomata (µm) 27.026±3.352 26.184±3.529 0.103 width of adaxial stomata (µm) 25.161±3.088 24.589±2.391 0.166 surface area of adaxial stomata (µm2) 538.831±122.565 510.127±113.029 0.104 length of abaxial stomata (µm) 27.614±3.633 25.787±3.199 0.000 width of abaxial stomata (µm) 25.418±3.265 24.250±2.293 0.006 surface area of abaxial stomata (µm2) 557.974±134.615 494.227±100.144 0.000 table 2. comparison of morphometric characteristics (mean ± sd and range) for a. monsspesulanus and a. spruneri mesophyll. individual cells or a group of cells match palisade parenchyma in shape and arrangement. intercellular spaces are present between cells in this part of mesophyll. the thickness of palisade tissue is 68-158 μm while thickness of spongy tissue is 48-118 μm. differences related to dimensions of palisade and spongy tissues correspond to differences in the total thickness of leaflets. mesophyll cells become rounded at median ribs (fig. 1). vascular bundles are abundant and surrounded by large cells of parenchyma. mechanical tissue is poorly represented. leaflet anatomy of a. spruneri the internal tissue in leaflets of species a. spruneri is organized in the same way as in the leaflets of a. monspessulanus (fig. 1). densely packed t-shaped hairs with equal, tip-pointed arms are present at both adaxial and abaxial sides of the leaflet, and they are particularly densely distributed at the main vein (fig. 2). differences in indumentum in comparison to species a. monspessulanus include density and distribution of trichomes. number of hairs varies in range of 1.7-12.4 at the abaxial side and 1.44-11.02 at the adaxial side. epidermis has single layers both on abaxial and adaxial sides, with thin cell walls and cuticle. cuticle on both sides is less developed and thinner (2-3.2 μm) than in populations of species a. monspessulanus. the outer epidermal cells are larger than the inner ones (fig. 1). the epidermal cells of leaflets are polygonal or irregular in shape. the outline of the anticlinal walls was described as straight, sinuous or sinuate (fig. 2). stomata are present both on abaxial and adaxial side (amphistomatic leaves). stomata frequency varies: 57-243 per mm2 at the abaxial side and 60161 per mm2 at the adaxial side. the greatest number of stomata (86-181 per mm2 at the abaxial side and 90-161 per mm2 at the adaxial side) was recorded in plants from the matka canyon, while the lowest number, 57-161 per mm2 at the abaxial side and 60 – 132 per mm2 at the adaxial side) was observed in plants from mariovo – zović (tab. 2). stomata are small and similar in size on both sides of epidermis. they are of anisocytic type. the mean thickness of the leaflets was 237 at kyustendil old village, 240 μm at mariovo-zović and 276 μm at matka canyon, with minimum at 187 μm and maximum at 336 μm (tab. 2). palisade tissue is usually composed of 1-2 layers of welldeveloped cells, and the spongy tissue has 2-3 cell layers. the thickness of the palisade tissue was 71159 μm and thickness of spongy tissue was 46-110 μm. lateral vascular bundles are present alongside the entire leaf. they are situated in the mesophyll, between spongy and palisade tissue (fig. 1). comparation between a. monspessulanus and a. spruneri according to the results of t-test (tab. 2), there is a significant statistical difference between a. monspessulanus and a. spruneri (p<0.05) in terms of the following leaf anatomy characters: leaf thickness, palisade tissue thickness, spongy tissue thickness, height of adaxial epidermal cells, thickness of cuticle at the adaxial side of the leaf, number of trichomes on abaxial and adaxial sides, length, width and surface area of stomata at the abaxial side of the leaf (tab. 2). although the results of t-test show statistically significant differences in 10 out of 18 characters related to leaf anatomy, results of pca and cda do not show clear separation of individuals of these two species on ordination diagrams (fig. 4, fig. 5). separation of individuals along the first pca ordination axis (surface area of stomata at the abaxial side of the leaf) and second pca ordination axis (leaf thickness) is statistically significantly contributed to only by a single character (stated in brackets). results of cda and classification analysis (fig. 6) indicate presence of clear differentiation in population of a. monspessulanus sampled in the canyon of morača valley in comparison to all other studied populations. the greatest recorded degree of similarity in leaflet anatomy was present between populations of a. spruneri sampled in north macedonia (matka canyon) and bulgaria (kyustendil) and between populations of species a. monspessulanus sampled in serbia. the anatomical results of this study are generally closely matching the referenced results for other species of genus astragalus (boughalleb et al., 2014). leaflets of species a. monspessulanus and a. spruneri are characterized by certain xeromorphic characteristics – relatively tiny and soft leaves with hairs and tiny stomata present both on adaxial and abaxial sides of the leaf, well-developed palisade tissue both on adaxial and abaxial sides of the leaf, and a high number of vascular bundles. the results of statistical analyses are generally indicating low utilitarian value of characters related to leaf anatomy for taxonomy purposes. according to literature sources, micromorphology of hairs is an important taxonomic character for genus astragalus and used in their systematics (taeb et al., 2007; zarre, 2000, 2003; zarre & podlech, 1996, 2001a,b,c; pirani et al., 2006). ghahremani-nejad (2004) examined value of trichome characteristics for separation of bifurcating-hair genus astragalus at the sectional level. the results point to a small range of hair character variation within the section incani dc. according to that author, in order to illuminate 16 biologica nyssana ● 12 (1) september 2021: 11-21 jušković et al. ● a comparative anatomical study on two closely related astragalus l. taxa from the central part of the balkan peninsula intersectional relationships, bifurcate hair characters should be studied more closely. petiole anatomy of a. monspessulanus in cross section, the outline of the petiole in species a. monspessulanus varies among populations from suborbicular to elliptic (fig. 3). the epidermis of all populations consists of a single layer of subcircular to subrectangular cells. cortex is situated below the epidermis, in form of a continuous ring composed of 3–5 layers of collenchymatous cells and parenchymatous cells. all populations have three prominent, primary vascular bundles, one dorsal median bundle and two ventral lateral bundles. the dorsal median bundle (dmb) is larger than the ventral lateral bundles. two to six smaller, secondary bundles accompany the three primary bundles. the vascular bundles are collateral and arranged in a circle, separated from one another by parenchymatic tissue. each vascular bundle is surrounded by a thick sclerenchymatous sheath composed of very thickwalled extraxylary fibers. the pith in all species is composed of large parenchyma cells (fig. 3). petiole anatomy of a. spruneri the outline of cross section of the petiole in species a. spruneri has a semi-triangular outline in which their abaxial sides were convex and their adaxial sides either flat or only slightly convex (fig. 3). just as in species a. monspessulanus, 2/3 of the petiole surface is composed of the central cylinder and 1/3 of the primary cortex. single–layered epidermis is covered with denser indumentum than the petiole epidermis of a. monspessulanus petiole. the cortex is composed of 2-3 layers of collenchyma and several layers of parenchyma cells. the central cylinder includes several vascular bundles, the largest main bundle in the center and two lateral, somewhat smaller vascular bundles. the number and size of vascular bundles varied between individuals of the same population as well as among populations: 5-11 bundles were recorded in different individuals. the vascular bundles are surrounded by well-developed sclerenchyma tissue. the central region is composed 17 biologica nyssana ● 12 (1) september 2021: 11-21 jušković et al. ● a comparative anatomical study on two closely related astragalus l. taxa from the central part of the balkan peninsula fig. 3. cross sections of the petiole in investigated astragalus species. a – a. monspessulanus (vlasi), b – a. monspessulanus (petačinci), c – a. monspessulanus (bioče), d – a. spruneri (monastery „matka“), e – a. spruneri (staro selo), f – a. spruneri (zović), g – dorsal median vascular bundle in a. monspessulanus (bioče), h, i – details of the spine petiole anatomy in a. spruneri (monastery „matka“). scale bars = 200 µm (a-f); 50 µm (g-i). abbreviations: co – cortex, dva – dorsal ventral axis, dmb dorsal median vascular bundle, e – epidermis, ph – phloem, pi – pith, sb – subsidiary vasculare bundle, sbs – sclerenchymatous bundle sheath, st – stomata, t – trichoma, va – ventral axis, vlb – ventral lateral vascular bundle, xy – xylem. biologica nyssana ● 12 (1) september 2021: 11-21 18 jušković et al. ● a comparative anatomical study on two closely related astragalus l. taxa from the central part of the balkan peninsula fig. 4. results of the principal component analysis (pca) and canonical discriminant analysis (cda) for populations of investigated astragalus species based on: all characters of leaflet and petiole anatomy, characters of leaflet anatomy and characters of petiole anatomy. fig. 5. results of the principal component analysis (pca) for populations of investigated astragalus species based on: all characters of leaflet and petiole anatomy, characters of leaflet anatomy and characters of petiole anatomy. of large, thin-walled parenchyma cells, just as in species a. monspessulanus (fig. 3). comparation between a. monspessulanus and a. spruneri according to the results of t-test, the studied species show statistically significant differences regarding the petiole anatomy (tab. 2). according to pca results, individuals of a. monspessulanus are almost completely differentiated from individuals of species a. spruneri along the first ordination axis (fig. 4, fig. 5). only very few individuals of a. monspessulanus sampled in the canyon of river morača show a more significant degree of similarity with individuals of species a. spruneri. on the other hand, a few individuals of a. spruneri sampled in bulgaria (kyustendil) show a significant degree of similarity with individuals of species a. monspessulanus (serbia: village vlasi). the list of characters making the highest contribution to differentiation along the first ordination axis of pca diagram includes all the analyzed characters except for thickness of petiole epidermis and thickness of primary cortex of petiole. cda diagram shows almost complete differentiation of species (fig. 5). individuals of species a. monspessulanus are concentrated in the positive part of cda diagram, while individuals of species a. spruneri are positioned in the negative part of the cda diagram. the results of agglomerative hierarchical classification support the results of other statistical analyses, i.e. existence of clear interspecies differentiation regarding the leaf stalk anatomy (fig. 6). although the statistical analysis of the dataset including all studied characters of leaflets and petioles has also supported interspecies differentiation, analysis of dataset including only the characters of petioles has provided more illustrative results. according to metcalfe & chalk (1950), the petiole has considerable taxonomic importance, as it is not heavily influenced by environmental changes. the general description of petiole cross-section is in accordance with previous data for other astragalus species (howard, 1979; haddad & barnett, 1989; pirani et al. 2006). research on european species of astragalus has shown that anatomic characteristics of the petiole have a limited taxonomic value, while characters of type of tissue in parenchyma of central region and amount of collenchyma tissue around the vascular bundles have high taxonomic importance for recognizing the two main species groups (haddad & barnett, 1989). species a. monspessulanus and a. spruneri belong to a group of taxa characterized by a relatively small number of thick-walled parenchyma cells in the central part of the petiole and a relatively high fig. 6. cluster analysis (single linkage method) for populations of investigated astragalus species based on: all characters of leaflet and petiole anatomy, characters of leaflet anatomy and characters of petiole anatomy. 19 biologica nyssana ● 12 (1) september 2021: 11-21 jušković et al. ● a comparative anatomical study on two closely related astragalus l. taxa from the central part of the balkan peninsula biologica nyssana ● 12 (1) september 2021: 11-21 20 jušković et al. ● a comparative anatomical study on two closely related astragalus l. taxa from the central part of the balkan peninsula amount of collenchyma tissue around the vascular bundles. conclusions our results show which anatomical and micromorphological characteristics can be used for taxonomic differentiation of species from the astragalus sect. incani. anatomical features described here largely agree with previous characters found in other astragalus species. anatomical and micromorphological characters described here have potential taxonomic and significance. results of statistical analyses indicate that characters which refer to petiole anatomy have higher taxonomic value than characters which refer to leaflet anatomy. acknowledgements. this study was supported by the project of ministry of science and technological development of republic of serbia (contract no. 451-039/2021-14/200124). references amini, e., kazempour‐osaloo, s., maassoumi a. a., zare‐maivan, h.. 2018: phylogeny, biogeography and divergence times of astragalus section incani dc. 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(liliaceae) from serbia and montenegro. biologica nyssana, 10(2): 125-133. taeb, f., zarre, s., podlech, d., tillich, j.h., kazempour, osaloo, s., maassoumi, a.a. 2007: a contribution to the phylogeny of annual species of astragalus (fabaceae) in the old world using hair micromorphology and other morphological characters. feddes repertorium, 118(5-6): 206-227. zarre, s. 2000. systematic revision of astragalus sect. adiaspastus, sect. macrophyllium and sect. pterophorus. englera, 18: 1–219. zarre, s. 2003: hair micromorphology and its phylogenetic application in thorny species of astragalus (fabaceae). botanical jurnal of the linnean society, 143: 323-330. zarre, s., podlech, d. 1996: taxonomic revision of astragalus l. sect. hymenostegis bunge (leguminosae). sendtnera, 3: 255−312. zarre, s., podlech, d. 2001a: a short contribution to genus astragalus l. (fabaceae) in turkey. pakistan journal of botany, 33(2): 153−155. zarre, s., podlech, d. 2001b: taxonomic revision of astragalus sect. acanthophace (fabaceae). sendtnera, 7: 233−255. zarre, s., podlech, d. 2001c: astragalus sect. semnanenses (fabaceae): a new monotypic section from iran. nordic journal of botany, 21(5): 485−491. 21 microsoft word bn-aa-0201-00 mitrovic et al biologica nyssana 2 (1) june 2011: 1-12 dimitrijević d. et al. the nature of the variability of the morphological characteristics… 1 review article ! lichens as source of versatile bioactive compounds tatjana mitrović*, slavisa stamenković, vladimir cvetković, miloš nikolić, svetlana tošić, dragana stojičić department of biology and ecology, faculty of sciences and mathematics, university of niš * e-mail: tatjanamitrovic@hotmail.com abstract: mitrović, t., stamenković, s., cvetković, v., nikolić, m., tošić, s., stojičić, d.: lichens as source of versatile bioactive compounds, biologica nyssana, 2 (1), september 2011: 1-6. lichens represent unique symbiosis of fungi (mycobionts) and algae (photobionts). living in extreme conditions they developed various compounds to survive. many of these original compounds have proven biological activities (antibiotic, antimycotic, antiviral, antitumor, antioxidant, etc) . this paper is synthesis of currently known data about lichens extracts and their potential use in pharmaceutics and medicine. key words: lichens, bioactive compounds, phytomedicine introduction ! pharmaceutical industry is forced to continual chase and development of new pharmacological active molecules. similar to higher plants, lichens are considered as potential source of novel biologically active compounds. lichens are complex symbiotic associations between a fungus (mycobiont) and an alga (photobiont) with unique characteristics in plant kingdom. it is estimated that there are approximately 25,000 species of lichens (c h a p m a n , 2009). they are proven as the earliest colonizers of terrestrial habitats on the earth with a worldwide distribution from artic to tropical regions and from the plains to the highest mountains (taylor et al., 1995). specific, even extreme, conditions of their existence, slow growth and long duration (age of several thousand years) are result of numerous protective compounds against different physical and biological influences (d e n t o n & k a r l e n , 1973). these metabolites are acters of lichen extracts’ bioactivity of various importance for modern pharmaceutics and medicine. short review of utilization of lichens back in time and modern investigation of lichen extracts and components and their application are given below. a brief history of lichen utilization throughout the ages, lichens are used in nutrition of many animals and humans during famine (for example, during leningrad siege) (r i c h a r d s o n , 1988; k a r a g o z et al., 2009). the desert species lecanora esculenta is considered as “biblical manna” (t r e a s e & e v a n s , 1978). since egyptians, lichens were used as dyes, perfumes and remedies in folk medicine (v a r t i a , 1973). it is known that romans dyed their togas with orchil, a purple pigment from roccella sp. and crottal, brown pigment from parmelia, ochrolechia and evernia sp. (m u g g i a et al., 2009). lichensdyed textile reached considerable economic importance in 18th century in some parts of the world as in the canary islands (m u g g i a et al., 2009). litmus, a blue coloring matter from lichen fermentation, was used as dye for textile and beverages (b e e c k e n et al., 1961). also, paper strips impregnated with litmus, a water extracted 2 (1) • september 2011: 1-6 biologica nyssana 2 (1) september 2011: 1-6 mitrović t. et al. lichens as source of versatile bioactive compounds 2 dyes from roccella sp., are used as ph indicators in laboratories from ancient times till present. extracts of some species of lichens, like evernia prunastri, are contents of perfumes (t r e a s e & e v a n s , 1978). naturally, the most important and studied application of lichens is the one in traditional medicine for treatment of animals and humans. for instance, new zealand maori traditionally use long, pendulous species of usnea for nappies and sanitary pads (p e r r y et al., 1999). also, usnea species have been used in asia, africa and europe for pain relief and fever control (o k u y a m a at al., 1995). usnea densirostra, known as “barba de la piedra” served as a cure for various disorders in argentina’s folk medicine (c o r r e c h e at al, 1998). two lichen species, parmelia caperata and umbilicaria sp. are reported in study of chilean traditional medicine (m u n o z et al., 1981). ramalina thrausta is used in finland for treatment of wounds, athlete’s foot or other skin diseases and taken to relieve sore throat and toothache (v a r t i a , 1973). cetraria islandica is ancient cough remedy known as “tonicum amarum” accepted as a mucilage drug (m u l l e r , 2001). various species worldwide are used in traditional medicine and said that they are able to cure dyspepsia, bleeding piles, diabetes, bronchitis, pulmonary tuberculosis, spermatorrhoea and other diseases of the blood and heart (r i c h a r d s o n , 1988). lichen metabolites generally, lichens metabolites can be divided into two groups: primary and secondary. primary metabolites are proteins, lipids, carbohydrates and other organic compounds involved in lichen’s metabolism and structure. secondary metabolites, also known as lichens substances, are produced mainly by the fungus and secreted onto the surface of the lichen’s hyphae either in amorphous forms or as crystals. they are in focus of this review.hhh lichen substances secondary metabolites are complex, but predominantly small molecules, which comprise up to 20% of lichen’s dry weight (m u g g i a et al., 2009). structures of more then 1000 different lichen substances are determined to date and many of them are pharmaceutically relevant (m u g g i a et al., 2009). secondary metabolites are products of polyketide pathway, mainly monocyclic and/or bicyclic phenols joined by an ester bond (depsides), both ester and ether bonds (depsidones) or furan heterocycle (dibenzofurans and usnic acid), antraquinones, xanthones, chromones and secondary aliphatic acids and esters (s t o j a n o v i ć et al., 2011). some of them are produced by the fungus or the alga per se, while others are exclusively produced by synergistic action of both partners in lichens. whole spectrum of lichen metabolites evolved for protective purposes against various physical and biological environmental factors (d e n t o n & k a r l e n , 1973). large amounts of phenolic compounds fungal melanins are synthesized and accumulated in the tallus in order to absorb uvb light and shelter the photobiont from excessive radiation (g a u s l a a & s o l h a u g , 2001). these photoprotectors have great antioxidant capacity (h i d a l g o et al., 1994, f e r n a n d e z et al., 1996) and can be used as preservatives in cosmetic products (m u l l e r , 2001). certain phenolic compounds protect lichens from herbivores (l a w r e y , 1989). theirs toxicity, feeding deterrence to insect larvae and nematocidal activity has been demonstrated (a h a d et al. 1991, e m m e r i c h et al., 1993). other lichen metabolites have antibiotic properties which prevent microbial degradation of the thallus (e m m e r i c h et al., 1993). some of lichen metabolites are involved in maintaining of the symbiotic equilibrium (h u n e c k , 1999), while others dissolved rocks for better attachment of lichens (s e a w a r d ,1997). biological activity of lichens lichen metabolites exert manifold biological activity. first, antibiotic properties of lichen extracts are known for decades. first study by burkholder originated from 1944. later, v a r t i a (1973) reported antimicrobial activity of several lichen species. according to wide screening of antimicrobial activity of lichen extracts, it seems that bacterial inhibitions can vary within the lichen extract, solvent used for extraction and bacteria tested. r a n k o v i c et al. (2007a; 2007b) tested aqueous, acetone and methanol extracts of cladonia furcata, parmelia caperata, parmelia pertusa, hypogimnia physodes, umbilicaria polyphylla, lasallia pustulata, parmelia sulcata, umbilicaria crustulosa and umbilicaria cylindrica from serbia on six species of bacteria and ten species of fungi. the strongest activity was observed with methanol extracts of parmelia pertusa and parmelia sulcata and the weakest activity was manifested by parmelia caperata and umbilicaria cylindrical. aqueous extracts of all tested lichen species were inactive. bacillus mycoides was the most sensitive bacterial species tested, whereas candida albicans was the most sensitive fungal species examined. other studies monitored ramalina farinacea and 69 species of lichens from new zealand and showed biologica nyssana 2 (1) september 2011: 1-6 mitrović t. et al. lichens as source of versatile bioactive compounds 3 their inhibitory effect against a lot of bacteria such as bacillus, pseudomonas, e.coli, streptococcus, staphylococcus, enterococcus, mycobacterium (e s i m o n e & a d i k w n , 1999; p e r r y et al., 1999). b e h e r a et al. (2005) reported that acetone, methanol and light petroleum extracts of lichen usnea ghattensis were effective against bacillus licheniformis, b. megaterium, b. subtilis and s. aureus. also, k a r a g o z et al. (2009) evaluated aqueous and ethanol extracts of 11 different species from turkey and determined potent antibacterial activity of aqueous extract of peltigera polydactyla and ethanol extract of ramalina farinacea. chemical identification of anti-gram-positive activities revealed evernic acid, vulpinic acid and hirtusneanoside as main acters (l a w r e y , 1986; r e z a n k a & s i g l e r , 2007). recently, m i t r o v i ć et al. (2011) studied antibacterial activity of methanol extracts of five lichen species (flavoparmelia caperata, evernia prunastri, hypogymnia physodes and cladonia foliacea). two lichen species were tested for the first time (evernia prunastri and cladonia foliacea). the analysis of their antibacterial potential were performed on 15 strains of bacteria and revealed the strongest inhibitory effect, especially on gram (+) bacteria, of hypogymnia physodes and cladonia foliacea. second, antifungal activities of certain lichen substances are also revealed. m a n o j l o v i c et al. (2005) reported antifungal activity of the anthraquinone parietin isolated from caloplaca cerina. two years later, antifungal properties were observed in extracts of the andean lichens protousnea poeppigii and usnea rigida, which contain divaricatinic acid, isodivaricatinic acid, usnic acid and 5-resorcinol (schmeda-hirschmann et al., 2008). also, m i t r o v i ć et al. (2011) determined strong antifungal effect of evernia prunastri and hypogymnia physodes. while evernia prunastri exerted the best effect on yeasts, hypogymnia physodes were better on filamentous fungi. third, antiviral properties has been attributed to specific lichen secondary metabolites. for instance, p e r r y et al. (1999) showed antiviral activity of usnic acid against herpes simplex type 1 and polio type 1 viruses. parietin extracted from teloschistes chrysophthalmus proved as virucidal for junin and tacaribe arenaviruses (f a z i o et al., 2007). lichenan, widely distributed in lichens, demonstrated inhibition of tobacco mosaic virus (s t u b l e r & b u c h e n a u e r , 1996). fourth, antitumor activities of lichens are of major importance as source of potential drugs for lethal malignant diseases. usnic acid, the most extensively studied lichen metabolite since its first isolation in 1844, exhibited an antiproliferative effect on human leukemia cells (k562) and endometrial carcinoma (hec-50) cells (c a r d e r e l l i et al., 1997, i n g o l f s d o t t i r , 2002, k r i s m u n d s d o t t i r et al., 2002). another lichen compound depsidone pannarin inhibited cell growth and induces apoptosis in human prostate carcinoma du-145 and human melanoma m14 cells (r u s s o et al. 2006, 2008). lichen polysaccharide cfp-2 reduced the viability of hl-60 and k562 cells (l i n et al., 2003). protolichesterinic acid isolated from cetraria islandica showed inhibition of growth of brest cancer cell lines and mitogenstymulated lymphocytes (o g m u n d s d o t t i r et al., 1998). inhibition of enzyme 5-lipoxygenase involved in inflammation, non-specific binding to dna polymerase β and dna ligase i are possible mechanisms of antitumor activity of protolichesterinic acid (m u l l e r , 2001). b u c a r et al. (2004) revealed inhibitory activities on 12(s)hete inside antiproliferative effect of several lichen substances in human platelets. furthermore, antipyretic and analgesic effects of lichen components were demonstrated on animal studies. for instance, usnic acid from usnea diffracta inhibited acetic-acid-induced writhing in mice and raised the pain threshold in dosedependent manner (o k u y a m a et al., 1995). finally, antioxidant properties, already mentioned previously concerned with phenolic content of lichens. j a y a p r a k a s h a and r a o (2000) examined antioxidant capacities of methyl orsellinate, atranorin, osellinic acid and lecanoric acid. b h a t t a r a i et al. (2008) noticed stronger antioxidant activities in lichens from antarctic that the one in lichens from native to temperate or tropical regions. m i t r o v i ć et al. (2011) compared the chemical content of lichen extracts (flavoparmelia caperata, evernia prunastri, hypogymnia physodes and cladonia foliacea) and their free radical scavenging ability. they observed strong correlation according to previous conclusions of r a n k o v i ć et al. (2010). hypogymnia physodes with the highest phenolic content showed the strongest antioxidant effect. conclusions lichens represent powerful source of new bioactive molecules for various pharmaceutical purposes. structure of more then 1000 lichen substances are available, but even more remain to be characterized, which is delayed by their natural occurrence in low concentrations. lichens were frequently ignored by pharmaceutical industries because of their slow growth. difficulties biologica nyssana 2 (1) september 2011: 1-6 mitrović t. et al. lichens as source of versatile bioactive compounds 4 encountered with collecting substantial amounts of plant materials may be circumvented by establishing lichen tissue-cultures that may be promising sources of novel biologically active compounds. isolation into pure culture was attempted on 1,183 species of lichen-forming and lichenicolous fungi (c r i t t e n d e n et al., 1995). however, it should be noted that lichen metabolites produced in mycobiontic cultures are not always identical to those produced from lichens themselves (m u l l e r , 2001). as an alternative method, the transfer of genes responsible for the production of lichen metabolites are suggested (h u n e c k , 1999). lichen tissue culture and gene transfer techniques may considerable enlarge the access to lichen derived substances and their excessive pharmaceutical screening. eventual drug-promising molecules from lichens can be improved with increased stabilities and reduced toxicities by modern computer-aided drug design programs and combinatorial chemistry (m u l l e r , 2001). acknowledgements. research study mentioned in this review was supported by the ministry of science and education of the republic serbia (project iii41018). references ahad, a.m., goto, y., kiuchi, f., tsuda, y., kondo, k., sato, t. 1991: nematocidal principles in “oakmoss absolute” and nematocidal activity of 2,4-dihydroxybenzoates. chem. pharm. bull., 39: 1043-1046. beecken, h., 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