VOL. 53 [T. 2 LJUBLJANA 2010 ACTA BIOLOGICA SLOVENICA prej/formerly BIOLO[KI VESTNIK ISSN 1408-3671 izdajatelj/publisher UDK 57(497.4) Dru{tvo biologov Slovenije ACTA BIOLOGICA SLOVENICA LJUBLJANA 2010 Vol. 53, [t. 1: 1–124 Acta Biologica Slovenica Glasilo Društva biologov Slovenije – Journal of Biological Society of Slovenia Izdaja – Published by Društvo biologov Slovenije – Biological Society of Slovenia Glavni in odgovorni urednik – Editor in Chief Alenka Gaberščik, e-mail: alenka.gaberscik@bf.uni-lj.si Tehnični urednik – Managing Editor Gregor Zupančič, e-mail: gregor.zupancic@bf.uni-lj.si Uredniški odbor – Editorial Board Robert Zorec (SLO), Matija Gogala (SLO), Nada Gogala (SLO), Alenka Malej (SLO), Livio Poldini (I), Mark Tester (AUS), Nejc Jogan (SLO), Mihael J. Toman (SLO), Franc Janžekovič (SLO), Branko Vreš (SLO), Boris Sket (SLO), Franc Batič (SLO), Georg A. Janauer (A), Doekele G. Stavenga (NL) Naslov uredništva – Adress of Editorial Offi ce Acta Biologica Slovenica, Večna pot 111, SI-1001 Ljubljana, Slovenija http://bijh.zrc-sazu.si/abs/ Oblikovanje – Design Žare Vrezec ISSN 1408-3671 UDK 57(497.4) Natisnjeno – Printed on: 2010 Tisk – Print: Tiskarna Pleško d.o.o., Ljubljana Naklada: 400 izvodov Cena letnika (dve številki): 15 € za posameznike, 42 € za ustanove Številka poslovnega računa pri Ljubljanski banki: 02083-142508/30 Publikacijo je sofi nancirala Javna agencija za knjigo Republike Slovenije. Acta Biologica Slovenica je indeksirana v – is indexed in: Biological Abstracts, Ulrichsweb. Za vedno je odšel Tone Wraber V letu, ko je Botanični vrt obhajal 200-letni- co delovanja, ko smo praznovali leto biodiverzi- tete, in v času, ki so ga vsako leto generacije štu- dentov biologije preživljale na terenskih vajah na Komni, nas je zapustil profesor dr. Tone Wraber. Kdorkoli je v zadnjih nekaj desetletjih vsaj malo pokukal med slovensko fl oristično litera- turo, mu je to ime gotovo ostalo v spominu, saj je bil Tone s svojim vsestranskim delovanjem in čez 1000 objavljenimi deli gotovo botanik, kakr- šnega slovenska narava še ni doživela. Tone se je rodil 4. marca 1938. Po materi Sa- bini, klasični fi lologinji, je podedoval ljubezen do jezikov, oče Maks, eden prvih slovenskih fi - tocenologov, pa mu je vcepil ljubezen do rastlin- stva in slovenskih gora. Taborništvo je spoznaval pri Pavlu Kunaverju, Sivem volku, in Tonetova pot med svečenike gorskega cvetja je bila s tem začrtana. A čeprav je vse življenje odločno in z ljubeznijo sledil tej poti, se ni mogel upreti niti številnim odcepom, vzporednicam in bližnjicam, ter je s svojo vsestransko aktivnostjo zaznamoval poleg botanike še kar nekaj drugih področij, bil je med utemeljitelji slovenskega naravovarstva, neumorno je preučeval zgodovino stroke in se uspešno spoprijemal z lingvističnimi izzivi. Končal je študij biologije na Univerzi v Lju- bljani in še kot študent sodeloval na tedanjem Zavodu za spomeniško varstvo. Z diplomskim delom »Rastlinstvo melišča pri Črnem jezeru nad Komarčo« pod mentorstvom prof. dr. Gabri- jela Tomažiča se je začelo prvo obdobje Toneto- ve fi tocenološke aktivnosti, ki se ji je posvečal intenzivneje do doktorata leta 1972 v Trstu pod mentorstvom prof. dr. Sandra Pignattija z delom »Contributo alla conoscenza della vegetazione pionere (Asplenietea rupestra e Thlaspeetea ro- tundifolii)«. Vzporedno s fi tocenološko aktivno- stjo je bil aktiven predvsem na področju fl oristike in taksonomije. Konec šestdesetih let se je tako pod njegovo koordinacijo pričelo sistematično kartiranje slovenske fl ore po vzorcu, ki je bil v rabi drugod v Srednji Evropi. To je po eni strani pomenilo ustrezno ureditev starejših fl orističnih podatkov in pripravo za vnos v računalnik, po drugi strani se je s pomočjo novih popisnih listov začelo s sistematičnim kartiranjem fl ore na tere- nu. V okviru teh aktivnosti je bilo pod Tonetovim mentorstvom izvedenih veliko diplomskih nalog, ki so ne le dale pomembne fl oristične rezultate iz dotlej manj raziskanih predelov Slovenije, ampak tudi izobrazile številne mlade poznavalce fl ore. Prva službena leta (1960-1970) je preživel v Prirodoslovnem muzeju in tu sodeloval s Cirilom Jegličem pri obnavljanju in urejanju alpskega bo- taničnega vrta Julijana v Trenti. Tudi kasneje je zavzeto skrbel za razvoj Julijane. Februarja 1968 je bil imenovan za asistenta v Botaničnem vrtu Univerze v Ljubljani in tedaj postal tudi kurator herbarijske zbirke LJU pri katedri za botaniko bi- ološkega oddelka. Na Oddelku za biologijo Bio- tehniške fakultete je bil zaposlen vse do upokoji- tve 2003 in se v teh letih od asistenta povzpel do rednega profesorja ter bil prav zadnje dni svojega življenja odlikovan z nazivom zaslužnega profe- sorja Univerze v Ljubljani. S svojimi bogato ilustriranimi predavanji je približal rastlinsko sistematiko in fl oro številnim študentom biologije. Predaval je predmete Siste- matska botanika, Osnove sistematske botanike, Biogeografi ja (rastlinski del), Osnove latinščine za biologe ter Pestrost in ogroženost rastlinstva. S posebnim žarom in predanostjo je predaval bo- tanično latinščino. Skrbno in ciljno izbrane teme predavanj so mladim generacijam pomagale ra- zumeti smisel in način uporabe njemu tako ljube latinščine v biologiji in tudi v vsakdanjem življe- nju. Predmet, čeprav neobvezen, je vsako leto pritegnil in navdušil mnoge študente biologije. Znanstvene članke je objavljal v domačih revijah, predvsem v Biološkem vestniku, reviji Acta biologica Slovenica in Hladnikia, pa tudi v tujih revijah, kot na primer Candollea in Acta botanica croatica. Med znanstvenimi in strokov- nimi monografi jami je gotovo strokovno najbolj opazno soavtorstvo pri vseh štirih izdajah Male fl ore Slovenije (1969, 1984, 1999 in 2007). S področja naravovarstva je najpomembnejše in najbolj vplivno delo Rdeči seznam praprotnic in semenk (1989) v soavtorstvu s Petrom Skober- netom. Pri oblikovanju temeljev varstva narave v Sloveniji je Tone sodeloval tudi z desetinami na- ravovarstvenih prispevkov, objavljenih v revijah Varstvo narave, Biološki vestnik in Proteus. Nje- gove ostale pomembnejše strokovne monografi je so Sto znamenitih rastlin na Slovenskem (1990), Rože na Slovenskem (besedila k fotografi jam Luke Pintarja, 1990), 2 x sto alpskih rastlin na Slovenskem (2006), Travniške rastline na Slo- venskem (skupaj z Andrejem Seliškarjem, 1986), s katerimi je dosegel in navdušil tudi bralce zunaj ozkega strokovnega kroga. Naravoslovno javnost je s strokovnimi in poljudno-strokovnimi članki redno seznanjal s fl orističnimi odkritji, opozarjal na naravovarst- vene probleme, podajal vtise s svojih botaničnih popotovanj, razpravljal o botaničnih zanimivo- stih pri nas in na tujem. S tem je širil naravoslov- no kulturo in vzpodbujal mlade k raziskovalne- mu delu, bistveno pa je tudi prispeval k razvoju in uveljavljanju slovenskega biološkega izrazja. Do narave je bil vedno iskreno in nesenti- mentalno pozoren. Na rastline ni gledal le kot na objekte preučevanja, bil je občudovalec nji- hove lepote, imenoval jih je 'cvetoče prijatelji- ce' in poskušal razumeti tudi njihovo vpetost v kulturno-zgodovinski okvir. Rad je pripovedoval njihove zgodbe in z veseljem smo mu prisluh- nili tako botaniki kot širša javnost. Večkrat je povedal in tudi napisal, da ima posebej rad ljudi, ki imajo radi rože, posebej, če to počno ob sicer drugačnem življenjskem poklicu. Ob vseh svojih obveznostih je za take ljubitelje vedno našel čas - s predavanji v lokalnih skupnostih, z vodenjem botaničnih izletov, aktivnim sodelovanjem v pla- ninskih društvih in podobno. S poljudnimi članki v revijah kot so npr. Moj mali svet in Naš vrt, s članki in diskusijami v dnevnem časopisju, in- tervjuji, televizijskimi in radijskimi oddajami ter številnimi predavanji v širši javnosti ob raznih priložnostih je izjemno prispeval k popularizaciji botanike in naravoslovja nasploh v širši javnosti. Kot področni urednik in s poldrugo stotino prispevkov je sodeloval pri nastanku Enciklope- dije Slovenije, ki jo je v letih 1987-2002 izdala založba Mladinska knjiga v sodelovanju s Slo- vensko akademijo znanosti in umetnosti. Poleg članstva v številnih domačih in tujih strokovnih združenjih, je bil ustanovni član Bota- ničnega društva Slovenije in kasneje tudi urednik glasila Hladnikia, predsednik društva in njegov častni član. Vsa leta pa je bil aktiven v Društvu biologov Slovenije, ki ga je vrsto let tudi vodil. Bil je tudi član uredniškega odbora društvenega glasila Acta Biologica Slovenica. Profesor dr. Tone Wraber je bil najbolj vpli- ven ter pisno daleč najbolj plodovit botanik delu- joč na območju Slovenije v 20. stoletju, v svojem delovanju pa je bil vsestranski. V pogovorih, do- pisovanjih, objavah in med skupnimi botanični- mi tereni je svoje bogato enciklopedično znanje in izkušnje vedno rad delil z nami, svojimi bota- ničnimi kolegi. Iskreno smo mu hvaležni. Vrzeli, ki jih je s svojim odhodom pustil med nami, še dolgo ne bodo zapolnjene. Requiescat in pace! N. Jogan in T. Bačič ACTA BIOLOGICA SLOVENICA LJUBLJANA 2010 Vol. 53, [t. 2: 5–12 fl owers. It is a Central European - Southern Siberian wet-meadow species (Pignatti 1982), distributed in C and E Europe, northwards to Latvia (Snogerup 1980). In Central Europe, the species is very rare and also listed among Cen- tral European vascular plants requiring priority Juncus atratus Krock. (Juncaceae) rediscovered in Slovenia Temnocvetni loček (Juncus atratus Krock., Juncaceae) ponovno najden v Sloveniji Tadej Lainščeka, Tinka Bačičb & Nejc Joganb,* aMotvarjevci 2C, 9207 Prosenjakovci, Slovenia bDepartment of Biology, Biotechnical Faculty, University of Ljubljana, Večna pot 111, SI-1000 Ljubljana, Slovenia *correspondence: nejc.jogan@bf.uni-lj.si Abstract: Juncus atratus Krock. of the Juncaceae family is a Central European- Southern Siberian wet-meadow species. In Central Europe, the species is very rare and threatened. In Slovenian Red Data List, it is listed among unsuffi ciently known species (K). The only record of the species in Slovenian territory (Prem near Ilirska Bistrica, SW Slovenia) is over 100 years old and has never been confi rmed afterwards. In May 2010, the species was found in Goričko (NE Slovenia), were it thrives on a wet meadow near Kobilje village. The article discusses the recent fi nding of the species in Slovenia in broader context, its distribution, habitat and threat factors. The aim of the study was also to provide bases for nature-conservation of the species in Slovenia. According to recent fi nding and the results of the study, the authors suggest Juncus atratus to be regarded as ‘endangered’ (E) in the national red data list. Key words: Juncus atratus, Juncaceae, nature-conservation, Slovenian fl ora, Goričko, Red Data List, wet meadows Izvleček: Temnocvetni loček (Juncus atratus Krock.) iz družine ločkovk (Juncaceae) je srednjeevropsko-južnosibirska vrsta vlažnih travnikov. V Srednji Evropi je vrsta zelo redka in ogrožena. Na slovenskem rdečem seznamu je navedena med nezadostno znanimi vrstami (K). Edini podatek o uspevanju vrste za Slovenijo (Prem pri Ilirski Bistrici, Jugozahodna Slovenija) je star več kot 100 let in ga kasnejše raziskave niso uspele potrditi. Maja 2010 je bila vrsta najdena na Goričkem (Severovzhodna Slovenija), kjer raste na vlažnem travniku pri vasi Kobilje. Članek obravnava nedavno najdbo vrste v Sloveniji v širšem kontekstu, razširjenost vrste, habitat in faktorje ogrožanja. Namen raziskave je postaviti temelje za ohranjanje vrste v Sloveniji. Glede na nedavno najdbo in rezultate naše raziskave, avtorji predlagajo, da se vrsto Juncus atratus v slovenskem rdečem seznamu uvrsti med prizadete vrste slovenske fl ore (E). Ključne besede: Juncus atratus, Juncaceae, naravovarstvo, slovenska fl ora, Goričko, rdeči seznam, vlažni travniki. Introduction Juncus atratus Krock. is a member of Jun- caceae family. It is 40-120 cm tall perennial with a creeping, sparingly branched rhizome with 7- to 11-angled leaves and infl orescence of 15-50 clusters, each with distinctly dark brown 6 Acta Biologica Slovenica, 53 (2), 2010 conservation measures (Schnittler and Günther 1999). Until recently, there was only one published (over 100 years old) record of the species from Slovenia of Pospichal (1897-99) from Submedi- terranean region (in upper valley of Reka river at Prem near Ilirska Bistrica, SW Slovenia) under synonim J. melananthos Rchb., with his remark, that the species probably thrives also in Brkini area nearby. The Pospichal’s record (in 0351/4 MTB quadrant) is the only dot in distribution map of Juncus atratus, published in ‘Materials for the Atlas of Flora of Slovenia’ (Jogan et al. 2001), which is a key reference for distributional data of Slovenian vascular plants and where the distribution maps of most taxa are shown. How- ever, the presence of the species on Pospichal’s locality was not confi rmed by other botanists afterwards, although the attempts were made to fi nd Juncus atratus in this area in the last decade (Rozman B., personal communication). As seen in general distribution map of this species in Figure 1: The locality of Juncus atratus near Kobilje (Goričko, Slovenia). Top righ corner: map of the vicinity of Kobilje with marked area, which is presented in bigger scale. Rest of the picture: aerial photo of the locality, depressions with J. atratus marked with white stars. Slika 1: Nahajališče temnocvetnega ločka Juncus atratus pri Kobilju (Goričko, Slovenija). Zgoraj desno: zem- ljevid okolice Kobilja z označenim izsekom, povečanim na letalskem posnetku. Ostali del slike: zračni posnetek nahajališča, populacije J. atratus označene z belimi zvezdicami. 7Lainšček et al.: Juncus atratus rediscovered in Slovenia Meusel et al. (1965), the mentioned Pospichal’s localities would lie on the western border of spe- cies’ distribution range. Populations here could have been destroyed by severe changes of wet- land habitat types in the last century but without available voucher material also a misdetermina- tion could not be excluded. During a fi eld excursion in Goričko (Sub- pannonian phytogeografi cal region), in May 2010 fi rst author of the present paper discovered the dark-fl owered Juncus plants, somewhat sim- ilar to Juncus articulatus, but distinctly taller, in wet-meadow near Kobilje village. Later, the plants were unambiguously determined as Jun- cus atratus. The article discusses the recent fi nding of the species in Slovenia. Since defi cient knowl- edge about distribution, ecology and threat fac- tors of species can cause serious problems in considerations and judgments when proposing nature-conservation strategies and composing red data lists, the article focuses on the issues mentioned above and discusses the broader con- text of the fi nding. The aim of the study was also to provide bases for nature conservation of the species in Slovenia. Methods The study included 3 fi eld-work days during vegetation season, on 30th May, 2nd July and 17th August 2010. The fi eld-work was carried out in Kobilje in Goričko region (Fig. 1). In the locality of Juncus atratus the approximate size of the population was assesed from number of fl owering shoots and vascular plants on the lo- cality were mapped. They were determined us- ing standard fl oristic literature (mostly Martinčič et al. 2007 and Fischer et al. 2008). After the fi eld work, revision of the avail- able herbarium material was done in LJU (De- partment of Biology, University of Ljubljana), which is the biggest herbarium collection with Slovenian plants. The revision included also re- lated Juncus taxa to detect possible misdetermi- nations. Where necessary, characters were ob- served and measured using a stereomicroscope. For the preparation of the distribution map we checked all available distribution data for Slovenia: literature records, herbarium data from LJU, unpublished data stored in the da- tabase ‘Flora Slovenije’ at the Centre for Car- tography of Fauna and Flora (CKFF), where also the distribution maps were produced. Dis- tribution patterns are presented in MTB grid (Niklfeld 1971). In distribution map, we used the biogeographic division of Slovenia by M. Wraber (1969), which was also adopted by the authors of the Slovenian fl oristic reference work Mala fl ora Slovenije (Martinčič et al. 2007). Results The newly discovered locality of J. atratus is situated about 700 m southwest from Kobilje vil- lage, 190 a.s.l.. It lies in Regional park Goričko. NE Goričko was recognized as important for the conservation of Natura 2000 habitat types with qualifi catory vegetion types of Molinion (6410) and Ranunculo-Alopecuretum (6510) (Jogan et al. 2004) and as such included in SI3000221 SCI with 7 qualifi catory habitat types including the mentioned two (Naravovarstveni atlas, http:// www.naravovarstveni-atlas.si/ISN2KJ/) The area of extensively maintained mead- ows at Kobilje is also ‘natural value of state im- portance’ (Kobilje - ekstenzivni travniki: 7298). Despite that, land owners’ management is not completely under nature-conservation control, which results in several negative impacts as dig- ging ditches, applying fertilizers, fi lling up wet depressions with rubbish, earth or waste build- ing materials. In the locality, the population thrives in 3 shallow depressions of the wet meadow, the larger beeing about 400 m2 large, while the two smaller depressions of cca. 200 m2. The maxi- mum depth of the depressions is cca. 0.5 m. The distances between the three depressions are ap- proximately 30 m. The depressions are much damper than the adjoining parts of the meadow and after rainfall the water can persist in them for several consequent days. The population of J. atratus in the largest depression counts about 30 - 40 fl owering indi- viduals, while the approximate number of fl ow- ering shoots in the two smaller depressions is up to 10. 8 Acta Biologica Slovenica, 53 (2), 2010 The meadow is mowed twice to three times in vegetation period, in June, August and Octo- ber. At the locality (Fig. 2), besides J. atratus, the following vascular plants were recorded: Agrostis stolonifera, Agrostis canina, Alopecu- rus pratensis, Carex vulpina, Eleocharis car- niolica, Gratiola offi cinalis, Holcus lanatus, Iris pseudacorus, Juncus articulatus, Juncus effusus, Lysimachia nummularia, Lythrum sali- caria, Myosotis scorpioides, Peplis portula, Polygonum minus, Ranunculus fl ammula. That type of vegetation fi ts well into Natura 2000 qualifying habitat type Ranunculo-Alopecure- tum (6510). For more precise description of the site ecological conditions a phyto-sociological analysis should be done, but that was beyond our scope. Of the above mentioned species, Eleocharis carniolica, Gratiola offi cinalis and Peplis por- tula are wetland species, considered vulnarable (V) in Slovenian Red Data List (Anon. 2002). From nature-conservation point of view, Eleo- charis carniolica is particularly important, as it is one of the Natura 2000 plants, listed among strictly protected plant species included in Ap- pendix 1 of the Bern Convention on the conser- vation of European wildlife and natural habitats (Bern, 19th September 1979) and in Annex 2 of Council Directive 92/43/EEC of 21 May 1992 on the conservation of natural habitats and of wild fauna and fl ora. The revision of Juncus material in LJU re- vealed that at present there is no available mate- rial for Juncus atratus in Slovenia. The distribu- tion map (Fig. 3) includes only two records for Figure 2: Juncus atratus in the recently found locality near Kobilje (Goričko, Slovenia). a: fl owering cluster detail with distinctly blackish tepals; b: compound infl orescence; c: locality with J. atratus. Photos: T. Lainšček. Slika 2: Temnocvetni loček (Juncus atratus) na novoodkritem nahajališču pri Kobilju (Goričko, Slovenija). a: delno socvetje z razločno črnorjavimi cvetnimi listi; b: celotno socvetje; c: rastišče J. atratus. Posnetki: T. Lainšček. 9Lainšček et al.: Juncus atratus rediscovered in Slovenia Slovenia: Pospichal’s record for Prem (0351/4) and fi rst author’s record for Goričko (9364/1). Discussion J. atratus is a well characterised species, but can be somewhat similar to J. articulatus, since the jointed rush is very variable in habit. In the fi eld, the two can be easily distinguished, since J. atratus is usually much higher, up to 120 cm, it has characteristically blackishbrown tepals (not just brown as in J. articulatus, but character not easily comparable and quite vari- able). Leaves are (5-) 7- to 11-angled and this character is apperent in fresh or dry state of the material. In contrary, the leaves of J. articulatus (as well as J. acutifl orus and J. alpinoarticula- tus) are not angled, just slightly ridged when dry. However, there is another character which should be taken into account: the leaves of J. ar- ticulatus, J. alpinoarticulatus and J. acutifl orus have very distinctive transverse septa, while in J. atratus leaves, the septa are present, but in- conspicuous. Descriptions followed Fischer et al. (2008) and Kirschner (2002). As mentioned in Introduction, in Central Europe, Juncus atratus is very rare and also endangered (Schnittler and Günther 1999). The species is threatened also in neighbouring Aus- tria. It is considered ‘vom Aussterben bedroht’ - ‘in risk of extinction’ by Niklfeld and Schratt- Ehrendorfer (1999) and is known to thrive in only one locality in Niederössterreich (March- feld) (Fischer et al. 2008). In Italy, the species is considered to be very rare, with only one known locality (near Verona) (Pignatti 1982). There are no records for Croatia. In Hungarian Red Data List the species is listed among ‘near threatened’ (NT) (Király 2007), distributed in several locali- ties, but scattered (Simon 2002). In Czech, J. at- ratus is considered ‘criticaly endangered taxon’ (CR) (Kubát, 2002) and in Germany ‘strongly endangered’ (Haeupler and Muer, 2000). Besides our record, there are no recent reports on the species for Slovenia and Pos- Figure 3: The known distribution of Juncus atratus in Slovenia. Slika 3: Znana razširjenost vrste Juncus atratus v Sloveniji. 10 Acta Biologica Slovenica, 53 (2), 2010 pichal’s observation remained unconfi rmed. The fact, that there are no other records of J. atra- tus in Slovenia can not be attributed to misde- terminations or neglect by botanists during the fi eldwork, as we saw, that the species is easily recognizable in the fi eld. Therefore we can con- clude, that J. atratus is indeed extremely rare in Slovene territory, as in Europe. According to Oberdorfer (1990) the species is charaster- istic of Cnidio-Violetum of Cnidion alliance, which is subcontinental vegetation of temporar- ily inundated meadows. In Slovenia this type of vegetation is not known, but we can expect its occurence in most continental parts of Slovenia, which is Goričko region. The habitat of Juncus atratus is endangered not only due to abandonment of mowing, which leads to gradual spontaneous reforestation, but particularly due to human infl uence on wet habi- tats, such as changing water regime, direct fi ll- ing of depression or fertilizing. Maintaining of this type of vegetation should encounter regular mowing at least one per year with removing of the mown grass and enabling of regular inunda- tion (Chytry et al. 2001). In the Slovene red data book, J. atratus is categorized as unsuffi ciently known species (K; Wraber and Skoberne 1989, Anonymous 2002), but Bačič (2006) suggested Juncus atratus to be recategorized in Ex?, since the only, over 100 years old record was never confi rmed, despite the attempts to fi nd the plant in the locality. Ac- cording to recent fi nding and the results of the study, the authors suggest Juncus atratus to be regarded as ‘endangered’ (E) in the national red list, since it is rare and its habitats are threatened because of human activity and natural succes- sion. Acknowledgement The authors are grateful to dr. Peter Schön- swetter and dr. Božo Frajman for confi rmation of the determination of Juncus plants and for the valuable remarks on the fi nding and to Centre for Cartography of Fauna and Flora for produc- ing the distribution map. Povzetek Temnocvetni loček Juncus atratus Krock. je predstavnik družine ločkovk (Juncaceae). Je 40 -120 cm visoka trajnica s plazečim, le malo razvejeno koreniko, s 7- to 11-robimi listi and socvetjem, sestavljnim iz 15-50 klobk z izrazi- to črnorjavimi cvetovi. Ta srednjeevropsko- južnosibirska vrsta vlažnih travnikov (Pignatti 1982) je razširjena v Srednji in Vzhodni Evropi, severno pa sega njen areal do Latvije (Snogerup 1980). V Srednji Evropi je vrsta zelo redka in navedena med ‘srednjeevropskimi vaskularnimi rastlinami, ki potrebujejo prednostna merila za ohranitev’ (Schnittler and Günther 1999). Do nedavnega edini podatek o uspevanju vrste v Sloveniji je star več kot 100 let. Gre za Pospichalovo (1897-99) navedbo za submed- iteransko fi togeografsko območje (zgornji del doline Reke pri Premu pri Ilirski Bistrici), av- tor pa piše, naj bi vrsta verjetno uspevala tudi v bližnjih Brkinih. Prisotnost vrste na tej lokaliteti botaniki kasneje kljub trudu niso uspeli potrditi (Rozman B., osebno posvetovanje). Med ter- ensko ekskurzijo na Goričko maja 2010 je prvi avtor na vlažnem travniku pri Kobiljem naletel na izrazito temnocvetne rastline ločka, ki so bile nekoliko podobne bleščečeplodnem ločku (J. articulatus), a precej višje. Kasneje so bile rast- line določene za J. atratus. Terenski del raziskave je potekal med veg- etacijsko sezono 2010, v maju, juliju in avgustu. Na nahajališču smo ocenili velikost populacije ter popisali najpogostejše rastline, ki uspevajo ob temnocvetnem ločku. Sledila je revizija her- barijskega materiala v herbarijski zbirki LJU na Oddelku za biologijo Biotehniške fakultete Uni- verze v Ljubljani. Novo odkrito nahajališče temnocvetnega ločka leži približno 700 m jugozahodno od vasi Kobilje na nadmorski višini 190 m. Območje je del Regijskega parka Goričko in predvsem del omrežja Natura 2000 (Goričko, SI5000009). Kljub temu pa upravljanje z zemljišči ni povsem pod nadzorom naravovarstva, kar ima lahko za posledico negativne vplive na habitate (kopanje izsuševalnih jarkov, uporaba gnojil, zasipanje mokrotnih depresij s smetmi, zemljo ali odpad- nim gradbenim materialom itd.). 11Lainšček et al.: Juncus atratus rediscovered in Slovenia Na lokaliteti uspeva populacija v treh plitvih, med seboj približno 30 m oddaljenih depresijah na vlažnem travniku. Največja depresija je ve- lika približno 400 m2, drugi dve pa sta manjši, obsegata približno 200 m2. Največja globina depresij je približno 0,5 m. Depresije so precej bolj mokrotne kot okolica in po deževjih v njih še nekaj dni zastaja voda. Populacija v največji depresiji šteje okoli 30-40 cvetočih primerkov, v manjših dveh pa do 10. Travnik kosijo dva do trikrat v vegetacijski sezoni, junija, avgusta in včasih tudi oktobra. Na nahajališču smo zabeležili poleg tem- nocvetnega ločka še naslednje vrste: Agrostis stolonifera, Agrostis canina, Alopecurus praten- sis, Carex vulpina, Eleocharis carniolica, Grati- ola offi cinalis, Holcus lanatus, Iris pseudacorus, Juncus articulatus, Juncus effusus, Lysimachia nummularia, Lythrum salicaria, Mysotis scor- pioides, Peplis portula, Polygonum minus, Ra- nunculus fl amula. Revizija ločkov v LJU je pokazala, da poleg naših primerkov zaenkrat ni na voljo nobenega herbarijskega materiala te vrste. Na terenu je temnocvetni loček načeloma lahko prepoznaven, le nekoliko podoben vrsti J. articulatus. Od te se dobro loči po višini (do 120 cm visok, J. articulatus je navadno znatno nižji), po izrazito in značilno črnorjavih perigonovih listih (pri J. articulatus so ti le svetlejše rjavi), v času cvetenja pa iz cvetov molijo značilno škrlatne brazde (pri J. articulatus so belkaste). Pomemben razlikovalni znak je tudi oglatost listov. Ti so (5) 7- do 11-oglati, kar je očitno tako pri svežih kot pri posušenih rastlinah. Listi vrste J. articulatus (kot tudi bližnjih, a redkejših J. acutifl orus in J. alpinoarticulatus) v svežem stanju niso robati in le nekoliko izbrazdani, ko so suhi. V listih vrste J. articulatus, J. alpinoar- ticulatus in J. acutifl orus so izrazite prečne pre- grade, vidne v posušenem stanju, ne da bi list vzdolžno prerezali. Tudi v listih J. atratus so prečne pregrade, vendar ne tako izrazite. Sled- nji znak je za opazovanje nekoliko neroden, če s temi vrstami nimamo predhodnih izkušenj. Temnocvetni loček je v Sredni Evropi izjem- no redek, še največ navedb je za Madžarsko (Simon 2002), a še tu se pojavlja le raztreseno. Kritično je ogrožen v sosednji Avstriji (Fischer et al. 2008), za Italijo obstala le ena sama naved- ba (Pignatti 1982), na Hrvaškem pa vrsta ne uspeva. V Sloveniji potrjeno uspeva le na enem samem nahajališču. Glede na to, da je vrsta dobro prepoznavna, je malo verjetno, da bi jo botanično oko na terenu spregledalo in bi bilo poznavanje njene razširjenosti iz tega razloga pomanjkljivo. Vrsta je kot kaže v resnici izjem- no redka, poleg tega pa so njeni habitati zelo ogroženi. Po eni strani jih ogroža naravna su- kcesija - spontano zaraščanje zaradi opuščanja košnje, posebej nevaren pa je vpliv človeka - spreminjanje vodnega režima, zasipanje vlažnih depresij in gnojenje. Za ohranjanje takšne veg- etacije je treba na območjih uvesti oz. ohraniti košnjo vsaj enkrat letno, z odstranjevanjem pokošenega materiala in dopustiti redno poplav- ljanje (Chytry et al. 2001). V slovenskem rdečem seznamu (Wraber in Skoberne 1989, Anonymous 2002) je tem- nocvetni loček obravnavan med nezadostno znanimi vrstami (K), pred leti pa je bila že pred- lagana uvrstitev med domnevno izumrle vrste - Ex? (Bačič 2006). Glede na nedavno najdbo in rezultate te raziskave, avtorji predlagajo, da se vrsto uvrsti v kategorijo ‚prizadeta vrsta’ (E). References Anonymous, 2002. Pravilnik o uvrstitvi ogroženih rastlinskih in živalskih vrst v rdeči seznam - Priloga 1. Uradni list RS 2/2002, pp. 1-16. Bačič, T., 2006. Nezadostno znane enokaličnice slovenskega Rdečega seznama [Insuffi ciently known monocots of the Slovene Red data list.]. Natura Sloveniae 8 (2), 5-54. Chytrý, M., Kučera, T., Kočí, M. (eds.), 2001. Katalog biotopů České republiky (Habitat Catalogue of the Czech Republic). Agentura ochrany přírody a krajiny ČR, Praha, p. 76. Fischer, M. A., Oswald, K., Adler, W., 2008. Exkursionsfl ora für Österreich, Liechtenstein und Südtirol. 3. Aufl age. Biologiezentrum der Oberösterreichischen Landesmuseen, Linz, 1392 pp. 12 Acta Biologica Slovenica, 53 (2), 2010 Haeupler, H., Muer, T., 2000. Bildatlas der Farn- und Blütenpfl anzen Deutschland. Verlag Eugen Ulmer Stuttgart, p. 590. Jogan, N., Bačič, T., Frajman, B., Leskovar, I., Naglič, D., Podobnik, A., Rozman, B., Strgulc Krajšek, S., Trčak, B., 2001. Gradivo za Atlas fl ore Slovenije. [Materials for the Atlas of fl ora of Slovenia]. Center za kartografi jo favne in fl ore, Miklavž na Dravskem polju, p. 209. Jogan, N., Kotarac, M., Lešnik A. (ed.), 2004. Opredelitev območij evropsko pomembnih negozdnih habitatnih tipov s pomočjo razširjenosti značilnih rastlinskih vrst: [Končno poročilo]. Naročnik: MOPE, ARSO, Ljubljana. CKFF, Miklavž na Dravskem polju. 961 pp. Digitalne priloge: http:// www.natura2000.gov.si/projektivec/koncno_porocilo_habitati.pdf Király, G. (ed.), 2007. Red list of the vascular fl ora of Hungary. Lővér print, Sopron, 73 pp. Kirschner, J., 2002. Part 7. Juncaceae 2: Juncus subg. Juncus. Species Plantarum, Flora of the World. Australian Biological Resources Study, Canberra. pp. 178-180. Kubát, K. (ed.), 2002. Klíč ke kvêtenê České republiky. Academia, Praha. p. 786. Martinčič, A., Wraber, T., Jogan, N., Podobnik, A., Turk, B., Vreš, B., Ravnik, V., Frajman, B., Strgulc Krajšek, S., Trčak, B., Bačič, T., Fischer, M. A., Eler, K., Surina, B., 2007. Mala fl ora Slovenije. Ključ za določanje praprotnic in semenk. Tehniška založba Slovenije, Ljubljana, 968 pp. Meusel, H., Jager, E., Weinert, E., 1965: Vergleichende Chorologie der zentraleuropäischen Flora, 1. Karten. Gustav Fischer Verlag, Jena. p. 85. Niklfeld, H., 1971. Bericht über die Kartierung der Flora Mitteleuropas. Taxon 20 (4): 545-571. Niklfeld, H., Schratt-Ehrendorfer, L. 1999. Rote Listen gefährdeter Pfl anzen Österreichs 2., neu be- arbeitete Aufl age - Farn- und Blütenpfl anzen. Grüne Reihe des Bundesministeriums für Umwelt, Jugend und Familie, Band 10. Verlag: Austria medienservice, Graz, 291 pp. Oberdorfer, E., 1990. Pfl anzensoziologische Exkursionsfl ora. Ulmer, Stuttgart, p. 150. Pignatti, S., 1982. Flora d‘Italia, vol. 3. Edagricole, Bologna, p. 441. Pospichal, E., 1897-1899. Flora des Österreichischen Küstenlandes 1-2. Franz Deuticke, Leipzig, Wien, p. 210. Schnittler, M., Günther, K. F. , 1999. Central European vascular plants requiring priority conservation measures - an analysis from national Red Lists and distribution maps. Biodiversity and Conser- vation 8, 891-925. Snogerup, S., 1980. Juncus L.. In: Tutin T. G. et al.: Flora Europaea Vol. 5. Cambridge University Press, Cambridge, pp. 102-111. Simon, T., 2002. A Magyarországi edényes fl óra határozója, harasztok - virágos növények. Nemzeti tankönyvkiadó, Budapest, p. 701. Wraber, M., 1969. Pfl anzengeographische Stellung und Gliederung Sloweniens. Plant Ecology 17: 176-199. Wraber, T., Skoberne, P., 1989. Rdeči seznam ogroženih praprotnic in semenk SR Slovenije. [Red data list of endangered vascular plants of SR Slovenia (Yugoslavia).]. Varstvo narave 14/15: 1-429. ACTA BIOLOGICA SLOVENICA LJUBLJANA 2010 Vol. 53, [t. 2: 13–21 Damage by Pests in Herbarium LJU Škoda zaradi herbarijskih škodljivcev v Herbariju LJU Tinka Bačiča, Branka Trčakb & Nejc Jogana* aUniversity of Ljubljana, Biotechnical Faculty, Department of Biology, Večna pot 111, 1001 Ljubljana, Slovenia bCentre for Cartography of Fauna and Flora, Klunova 3, SI-1000 Ljubljana *correspondence: nejc.jogan@bf.uni-lj.si Abstract: The article discusses the damage caused by herbarium pests in Herbarium LJU. The aim of the study was to determine the damage in the herbarium, to fi nd out, which herbarium-pests are present, and to investigate their food-preference by means of checking the extent of damage on a selection of plant families Alliaceae, Apiaceae, Araceae, Asteraceae, Brassicaceae, Chenopodiaceae, Cichoriaceae, Faba- ceae, Lamiaceae, Poaceae, Polypodiaceae, Ranunculaceae, Rosaceae and Scrophulariaceae. Since the Asteraceae and Cichoriaceae are known to be among the most attractive families for herbarium-pests, we examined them in detail. In the study about 7500 herbarium sheets were examined, which represents 5% of all the sheets in Herbarium LJU. In addition to the most frequent pest tobacco beetle (Lasioderma ser- ricorne), we also found beetles Stegobium paniceum and Attagenus piceus, booklice (Psocoptera), moulds and Pharaoh’s ants (cf. Monomorium pharaonis). Pest-damage was observed in 18 % of the examined herbarium sheets. The study confi rmed that the pests are prone to attack certain families over others: the greatest damage (about 25 % - 40 % of the damaged sheets) was observed in Apiaceae, Asteraceae s. lat., Brassicaceae, Fabaceae, Alliaceae, Araceae, Rosaceae and Chenopodiaceae, while the other investigated families ranked among less damaged (about less then 10 % of the damaged sheets). Among Asteraceae, tribus Cardueae proved to be the most attractive tribe for the pests. Keywords: Herbarium LJU, herbarium-pests, dry plants, Lasioderma serricorne, Stegobium paniceum, Attagenus piceus, Psocoptera. Izveček: Članek obravnava škodo, ki jo povzročajo herbarijski škodljivci v herbarijski zbirki LJU. Namen raziskave je bil ugotoviti obseg poškodovanosti rastlinskega materiala, določiti, kateri herbarijski škodljivci so prisotni v LJU in raziskati njihovo prehrambeno preferenco z ugotavljanjem obsega škode na izboru rastlinskih družin Alliaceae, Apiaceae, Araceae, Asteraceae, Brassicaceae, Chenopodiaceae, Cichoriaceae, Fabaceae, Lamiaceae, Poaceae, Polypodiaceae, Ranunculaceae, Rosaceae in Scrophulariaceae. Ker družini Asteraceae in Cichoriaceae veljata za najbolj privlačne za herbarijske škodljivce, smo ju še posebej podrobno obravnavali. V raziskavi smo pregledali okoli 7500 herbarijskih pol, kar predstavlja 5 % vseh pol v herbariju LJU. Ugotovili smo, da je v LJU najpogostejši škodljivec tobačni hrošč (Lasioderma serricorne), poleg njega pa smo naleteli tudi na hrošča Stegobium paniceum in Attagenus piceus, prašne uši (Psocoptera), molje and faraonske mravlje (cf. Monomorium pharaonis). Škodo zaradi herbarijskih škodljivcev smo opazili v 18 % pregledanih herbarijskih pol. Raziskava je potrdila, da herbarijski škodljivci jedo herbarijski material določenih rastlinskih družin raje kot drugih. Največjo poškodovanost herbariziranih rastlin (25 do 40 % pregledanih pol s poškodovanih materialom) smo tako opazili pri družinah Apiaceae, Aste- raceae s. lat., Brassicaceae, Fabaceae, Alliaceae, Araceae, Rosaceae in Chenopodiaceae. Ostale pregledane družine so bile znatno manj poškodovane (pod cca. 10 % poškodovanih pol). Znotraj družine Asteraceae se je kot najbolj privlačen za herbarijske škodljivce izkazal tribus Cardueae. Ključne besede: herbarjska zbirka LJU, herbarijski škodljivci, posušene rastline, Lasioderma serricorne, Stegobium paniceum, Attagenus piceus, Psocoptera. 14 Acta Biologica Slovenica, 53 (2), 2010 Introduction Herbaria are permanent collections only if preserved in dry, airy, dark place and protected against pests (Podobnik 1993). The problem of damage by pests is common problem of many herbaria. Most signifi cant herbarium pests are some insects, moulds and sometimes even some rodents. In temperate regions herbivore insects grazing plants in nature can not survive in heat- ed buildings and so they are not considered a threat to herbarium material. The infestation, caused by herbarium pests, occurs within store- houses, buildings, cabinets and herbaria (Skvor- cov 1977). Therefore fresh plant material is not infected by herbarium pests yet, and the other pests, brought with the fresh material from the fi eld, can not survive the drying procedure. There are two common features of herbari- um pests (Stein 1986): they feed on dry herbal material and they can survive under severe conditions of temperature and humidity in her- barium. Unlike the fresh-plant eating insects, being usually stenophagous, the herbarium pests are commonly euryphagous. They feed on the preserved plant material as well as on any other plant products like paper, textile, dry food etc. Provisionally deposed herbarium specimens in uncontrolled conditions are very likely to become infected (Skvorcov 1977). Most seri- ous threat to herbarium material is by insects, particularly some beetle species (Coleoptera). They can also be found in storehouses, caus- ing damage on cereals, fl our, tea, tobacco, as well as in museum collections. The most fre- quent pest, causing great damage in herbaria as well as in tobacco storehouses, is tobacco bee- tle Lasioderma serricorne (Anobiidae), which is distributed worldwide (Ryan 1995, Zuska 1994). Some other signifi cant beetle pests are: Stegobium paniceum (Anobiidae), Ptinus fur (Ptinidae), Anobium punctatum (Anobiidae), Hylopteres bajulus (Cerambycidae), Xestobium rufovilllosum (Anobiidae), Lyctus brunneus (Lyctidae), Mycrobium castaneum (Anobiidae), Attagenus piceus (Dermestidae) (Valentin 1993, Stein 1986). Some herbarium pests can also be found among other invertebrate groups: various booklices (Psocoptera) and silverfi sh (Lepisma saccharina, Thysanura). Long-lasting high humidity in herbarium room often results in fungal damage of plant material. Fungal attack causes decomposition of the plant tissues and may cause plant features to be obscured and unsuitable for study (Brid- son 1992). Even if the hyphae are destroyed, their spores may still persist for a longer peri- od of time and germinate, when the conditions change (Rode 1989). Naphthalene and lauryl pentachlorophenate (LPCP) are believed to have fungicidal properties, however, Thymol is quite effective as fungicide (Maden 2004). There are many different chemical methods for desinsec- tion and mould control in herbaria, but at present time there does not seem to be an ideal chemical to protect specimens from mould or insect at- tack (Clark 1986). Of the physical methods deep freezing for the incoming material is advisable (Clark 1986) and even microwave treatment is used in some herbaria (Hall 1981, Hill 1983). Interesting feature of herbarium pests is their food-preference: insect pests are known to be naturally attracted to herbarium mate- rial of certain plant families like Asteraceae s. lat., Brassicaceae, Capparaceae and petaloid monocotyledons (Liliaceae s. lat.). If Stego- bium paniceum is present, the damage occurs on Asteraceae s. lat., Apiaceae, Ericaceae and plant families that contain latex, e.g. Apocy- naceae, and Asclepiadaceae. (Bridson 1992). Families Betulaceae, Fagaceae, Caryophyl- laceae, Convolvulaceae, Poaceae and Polypo- diaceae are considered to be less attractive to pests and therefore less threatened (Skvorcov 1977). Since the important prevention measure against pests is a constant look-out for insects, Skvorcov (1977) recommends to bait the her- barium-pest traps with their favorite plant ma- terial to monitor the eventual infestation of the herbarium. He also suggests regular checks of Tragopogon, Angelica and Vicia faba herbari- um sheets. In the last decade, a considerable pest dam- age was observed in Herbarium LJU, at the De- partment of Biology, Biotechnical Faculty of University of Ljubljana. The aim of the study was to determine the damage in the herbarium, to fi nd out, which herbarium-pests are present, and to investigate their food-preference by 15Bačič et al.: Damage by Pests in Herbarium LJU means of checking the extent of damage on a set of certain plant families. The results of the research in Herbarium LJU should be compa- rable to other smaller herbaria in the temper- ate regions. They should also represent a basis for pest-control strategy in Herbarium LJU and comparable herbaria. Materials and Methods Herbarium LJU Herbarium LJU is a typical local herbarium, covering mostly Slovenian fl ora. It comprises about 160.000 herbarium sheets. Though it is relatively small, it represents the largest her- barium collection in Slovenia and has a history of over 100 years. It includes several important exsiccate collections, like Flora exsiccata Car- niolica and Flora exsiccata Styriaca, and rela- tively many type specimens. Before 1995 the herbarium was regularly fumigated with various gases, like carbon di- sulfi de and – later – hydrogen cyanide. In 1995 the herbarium was moved to a new location. The fi rst fumigation of the herbarium in the new locality was performed in 2000; phosphine gas was used. Until 2009 three additional fumiga- tions with phosphine gas were performed and re-infestation with insects has not been ob- served so far. A common practice for prevention of infestation in LJU is decontamination of the incoming dried material before it enters the her- barium room. The sheets are subjected to deep freezing (at least -18°C) for a few days. Until recently, the temperature and humid- ity in the herbarium were not controlled and the conditions somewhat vary during the year. Dur- ing our investigation, the temperature and hu- midity was measured. The air temperature was found to be 19°C and the humidity 42 %. Since the investigation was performed during heating season, the measured temperature was probably a bit higher then otherwise. The Examined Plant Material The following set of plant families was cho- sen for the examination: Alliaceae, Apiaceae, Araceae, Asteraceae, Cichoriaceae, Brassi- caceae, Chenopodiaceae, Fabaceae, Lamiace- ae, Poaceae, Polypodiaceae, Ranunculaceae, Rosaceae and Scrophulariaceae. For each of the studied families (with an exception of Asterales) in a random sample of genera a satisfactory amount of herbarium sheets has been chosen. In small plant families (Araceae, Alliaceae) a sample of over 30 her- barium sheets of various genera was chosen and in larger families (Apiaceae, Brassicaceae, Chenopodiaceae, Asteraceae, Cichoriaceae, Fabaceae, Lamiaceae, Poaceae, Rosaceae, Scrophulariaceae, Ranunculaceae and Polypo- diaceae) the size of the sample was over 100 sheets. The criterion for the random sampling of genera was set according to alphabetical order. For the inclusion of a certain genus in a sample, the genus had to be represented by a minimum of 10 sheets (smaller genera) or 30 sheets (larger genera) of at least one species: the examined genera and the sample sizes are listed in Table 1: Percentages of the damaged sheets in the examined genera. Tabela 1: Odstotek poškodovanih herbarijskih pol pri pregledanih rodovih. % damaged sheets inspected sheets Alliaceae Allium 24,1 170 Araceae Arum 25 24 Acorus 5 2 Apiaceae Aegopodium 28,6 21 Angelica 51,5 33 Anthriscus 34,0 50 Aethusa 33,3 21 Compositae Achilea 90,1 223 Adenostyles 13,9 36 Ambrosia 33,3 15 Antennaria 8,6 58 Anthemis 25,0 44 % damaged sheets inspected sheets 16 Acta Biologica Slovenica, 53 (2), 2010 Aposeris 11,8 34 Arctium 72,7 44 Arnica 42,9 35 Artemisia 46,8 79 Aster 17,4 167 Brassicaceae Aethionema 11,5 26 Alliaria 6,7 15 Alyssum 36,7 79 Arabidopsis 12,5 16 Arabis 22,7 255 Chenopodiaceae Atriplex 17,2 29 Fabaceae Anthyllis 41,5 130 Astragalus 1,0 103 Lamiaceae Acinos 2,3 130 Ajuga 0,0 96 Ballota 0,0 16 Betonica 3,8 79 Poaceae Achnatherum 0,0 20 Agropyron 0,0 62 Agrostis 0,0 39 Alopecurus 2,5 40 Anthoxanthum 1,8 56 Apera 16,7 12 Arrhenatherum 0,0 30 Polypodiaceae Adianthum 14,3 21 Asplenium 3,7 242 Athyrium 4,8 104 Telypteris 0,0 73 Pteridium 3,0 33 Ranunculaceae Actaea 5,0 40 Anemone 0,6 181 Batrachium 3,1 127 Pulsatilla 13,0 100 Rosaceae Agrimonia 0,0 39 Alchemilla 34,6 347 Aphanes 0,0 10 Aremonia 4,5 44 Aruncus 32,0 25 Amelanchir 25,0 44 Scrophulariaceae Bartsia 0,0 28 Digitalis 11,4 44 Scrophularia 40,9 44 % damaged sheets inspected sheets% damaged sheets inspected sheets Since ordo Asterales is known to be one of the most attractive families for herbarium pests, we examined it in detail, checking all the avail- able genera. In this separate analysis the follow- ing genera were included: Achilea, Adenostyles, Ambrosia, Anthemis, Antennaria, Aposeris, Arc- tium, Arnica, Artemisia, Aster, Bellis, Carduus, Carlina, Centaurea, Cirsium, Conyza, Crepis, Doronicum, Echinops, Erigeron, Eupatorium, Filago, Gnaphalium, Hieracium, Homogyne, Hypochoeris, Inula, Lactuca, Leontodon, Leu- canthemum, Petasites, Picris, Pulicaria, Saus- surea, Scorzonera, Senecio, Serratula, Solidago, Sonchus, Tanacetum, Taraxacum, Tragopogon and Tussilago. For each of the investigated gen- era all the available material was examined, with an exception of Hieracium, Crepis, Leontodon and Centaurea, where only a satisfactory large random sample was chosen. Very old sheets (over 100 years) were not taken into account, since most of the them were at that time protected with mercuric chloride, which provides a long-lasting immunity and may therefore blur the results. Non-slovenian material was also excluded from the examina- tion. Examining the Extent of Damage The examination of the extent of damage in- cluded visual inspection and counting the dam- aged and intact herbarium sheets. The counts obtained were used to calculate percentage of 17Bačič et al.: Damage by Pests in Herbarium LJU the damaged sheets for the examined families and genera. The sheets were characterized as ‘damaged’, if the following evidences of herbarium pests were present: – deposits of fi ne granular droppings on the plant specimens and paper, – missing parts of plants, holes in sheets, leaves, stems, roots and rhizomes, infl orescence and/ or fl owers as a result of larval forage, – ruined infl orescence and/or fl owers, – a presence of crawling or dead adult pests. Results and Discussion The herbarium pests In the last few years and during our exami- nation the following Coleoptera species were found: Lasioderma serricorne, Stegobium pan- iceum in Attagenus piceus. Most of the found in- dividuals were determined as Lasioderma serri- corne, while the last two species were represented each by only a single adult. All the adult beetles were found dead. The results partly correspond to the list of coleopteran pests, found in herbarium material in nature-history department of National museum in Ljubljana (Kos 1944). Among the listed species in Kos (1944) there are Attagenus piceus and Sitodrepa panicea (syn.: Stegobium paniceum), but Lasioderma serricorne, the main pest in LJU 50 years latter, was missing. The oth- er species from the museum herbarium are An- threnus verbasci, Ptinus fur, Cartodere fi liformis, C. fi lum (most common) (Kos 1944). Other observed pests belong to Psocoptera: the booklices were alive and very numerous. They are known to attack tenderer plant organs, like petals and anthers (Skvorcov 1977). The damage we observed was rather heavier: leaves, stems and peduncles were also attacked. Fungal damage was observed, too. Con- sidering the fact that in LJU there had been no humidity control, the fungal infection is not sur- prising. In the working room of herbarium, where a considerable amount of unsorted material is tem- Figure 1: Percentages of the damaged sheets in the examined families. Slika 1: Odstotek poškodovanih herbarijskih pol po družinah. 18 Acta Biologica Slovenica, 53 (2), 2010 porarily deposed and the temperature is a few degrees higher, Pharaoh’s ants (cf. Monomorium pharaonis) were found in some sheets. Bridson and Forman (1992) report that these pests are rather common in heated herbaria of temperate regions. They are also common pests in store- houses in Ljubljana (Hržič and Urek 1989). Figure 2: Percentages of the damaged sheets in the examined Asteraceae genera grouped by 2 subfamilies (upper graph: Asteroideae, lower graph: Cichorioideae). Tribus Cardueae shown by black coloumns. Slika 2: Odstotek poškodovanih herbarijskih pol pri košarnicah združenih po poddružinah (zgornji graf: Aster- oideae, spodnji graf: Cichorioideae), tribus Cardueae obarvan črno. 19Bačič et al.: Damage by Pests in Herbarium LJU The damage In the study there were about 7500 herbar- ium sheets examined, which roughly represent 5 % of all the sheets in Herbarium LJU. Pest- damage was observed in 18 % of the examined herbarium sheets. This percentage can not be ap- plied to all the material in LJU, since the family sample was not chosen randomly. The results fully confi rm the fact, that the pests are prone to attack certain families over oth- ers: in accordance with the literature ( Skvorcov 1977, Hall 1988, Bridson 1992, Nikolić 1996,) the greatest damage was observed in Apiaceae, Asteraceae, Cichoriaceae, Brassicaceae, Fa- baceae and Alliaceae (Fig. 1). The study showed approximately the same high level of damage in Araceae, Rosaceae and Chenopodiaceae (Fig.1). The high percentage of the damaged sheets in Chenopodiaceae was rather unexpected since Skvorcov (1977) reports the family to be among less threatened. However, the damage on Chenopodiaceae specimens was far less ex- treme than for instance on Apiaceae and Aster- aceae. In the column chart in Fig. 1 a gap be- tween the 10 % and 20 % of the damaged sheets can be observed: it represents the gap between the ‘threatened’ and the ‘less threatened’ fami- lies. Poaceae and Polypodiaceae proved to be among the latter. Family Lamiaceae, commonly reported to be attractive to pests (Skvorcov 1977), also appeared among the less damaged. Considering the great content of essential oils, which are known to act as repellent substances, this result can be explained. Families Ranun- culaceae and Scrophulariaceae also ranked among the less damaged. The two are known for their poisonous and repellent secondary substances. Tab. 1 shows the sample sizes and percentages of the damaged sheets in all of the examined genera. With over 20 000 species, family Asteraceae s.lat. is one of the largest plant families. Accord- ing to some authors (Cronquist 1981) one of the reasons for it’s evolutionary successfulness is also the presence of poisonous and repellent substances, for instance polyacetylene, sesquit- erpene lactones, alkaloids (Senecioneae), latex (Lactuceae) and further some malodoriferous essential substances (Heliantheae in Anthemi- deae). According to the study, the poisons and repellents are not very effective in the case of herbarium-pest attack. Among Asteraceae, Cardueae proved to be the most attractive tribe. The damage increases from about 50 % of the damaged sheets in gen- era Saussurea, Carlina, Cirsium and Carduus, reaches 70 % in genus Centaurea and the maxi- mum damage of 75 % in Echinops (Fig. 2). The damage is generally limited to the infl o- rescence, but sometimes leaves are also eaten. The tribe Cardueae is known for it’s ambigu- ous taxonomical status: it’s (sub)family alliance is yet uncertain (Heywood 1993). The position of the Senecioneae tribe, one of the less dam- aged, is also doubtful: some authors consider it as a third subfamily (Heywood 1993). As expected (Skvorcov 1977), among Cichoriace- ae, Tragopogon is the most attractive (almost 60 % of the damaged sheets). All the investi- gated genera belong to Lactuceae, which is also mentioned by Hall (1988) as one of the most attractive for the herbarium pests. The column chart of the two families in Fig. 2 reveals a gap approximately between the 15 % and 30 % of the damaged sheets, the ‘less threatened’ genera being under the lower value and the ‘threatened’ above the upper. Probably the pests eat the ‘less threatened’ material only by chance or when their population is suffi ciently large. Conclusions As a result of our study, a list of most threat- ened families and genera by herbarium pests is produced. For the herbaria with predominantly northern hemisphere temperate region plant ma- terial, keepers should regularly monitor sheets of Apiaceae, Asteraceae s. lat., Brassicaceae, Fabaceae, Alliaceae, Araceae, Rosaceae and Chenopodiaceae for the presence of common herbarium-pests. In addition to that, for Stego- bium paniceum a pheromone trap is commer- cially available and can be used specially in her- baria after infestation of Lasioderma serricorne to monitor effi ciency of pest control and early detect surviving population. 20 Acta Biologica Slovenica, 53 (2), 2010 Povzetek Težave s škodljivci imajo v mnogih her- barijskih zbirkah. Najbolj značilni herbarijski škodljivci so nekatere žuželke (posebej hrošči - Coleoptera, pa tudi prašne uši - Psocoptera in srebrne ribice - Thysanura), plesni in celo neka- teri glodalci. Herbarijski škodljivci se hranijo s posušenim rastlinskim materialom, kot tudi z izdelki iz rastlin, kot so papir, tkanine, suha hrana, tobak ... Žuželke v vlogi herbarijskih škodljivcev nar- avno privlačijo herbarizirane rastline določenih družin pred drugimi. Tako so na primer posebej ogrožene družine Asteraceae s. lat., Brassicace- ae, Capparaceae in petaloidne enokaličnice (Liliaceae s. lat.), po drugi strani pa so družine kot Betulaceae, Fagaceae, Caryophyllaceae, Convolvulaceae, Poaceae in Polypodiaceae škodljivcem manj privlačne (Skvorcov 1977). Herbarij LJU na Oddelku za biologijo Biotehniške fakultete Univerze v Ljubljani ob- sega približno 160 000 herbarijskih pol in kljub relativni majhnosti predstavlja največjo urejeno herbarijsko zbirko v Sloveniji. V zadnjem de- setletju je bila v herbariju LJU opažena znatna škoda zaradi harbarijskih škodljivcev. Namen raziskave je bil ugotoviti obseg škode, dognati, kateri herbarijski škodljivci se tu pojavljajo in ugotoviti njihovo prehrambeno preferenco s primerjanjem obsega poškodovanosti herbari- jskih pol različnih rastlinskih družin. Rezultati raziskave lahko služijo kot osnova za pripravo strategije nadzora nad herbarijskimi škodljivci tako v zbirki LJU, kot tudi v primerljivih manjših herbarijih v zmernem pasu. V raziskavo smo vključili naslednje rastlinske družine: Alliaceae, Apiaceae, Araceae, Asterace- ae, Cichoriaceae, Brassicaceae, Chenopodiaceae, Fabaceae, Lamiaceae, Poaceae, Polypodiaceae, Ranunculaceae, Rosaceae in Scrophulariaceae. Za vsako od raziskovanih družin (razen Asteral- ceae s. lat.) smo znotraj naključno izbranega niza rodov pregledovali herbarijske pole. Pri manjših družinah (Araceae, Alliaceae) je bil vzorec ve- lik preko 30 pol iz različnih rodov, pri velikih družinah (Apiaceae, Brassicaceae, Chenopo- diaceae, Asteraceae, Cichoriaceae, Fabaceae, Lamiaceae, Poaceae, Rosaceae, Scrophular- iaceae, Ranunculaceae in Polypodiaceae) pa je bilo število pregledanih pol čez 100. Košarnice (Asterales) smo pregledali podrobneje, in sicer v glavnem vse pole vseh razpoložljivih rodov: Achilea, Adenostyles, Ambrosia, Anthemis, Anten- naria, Aposeris, Arctium, Arnica, Artemisia, As- ter, Bellis, Carduus, Carlina, Centaurea, Cirsium, Conyza, Crepis, Doronicum, Echinops, Erigeron, Eupatorium, Filago, Gnaphalium, Hieracium, Homogyne, Hypochoeris, Inula, Lactuca, Leonto- don, Leucanthemum, Petasites, Picris, Pulicaria, Saussurea, Scorzonera, Senecio, Serratula, Solid- ago, Sonchus, Tanacetum, Taraxacum, Tragopo- gon in Tussilago. Za vsako polo smo ugotovili, ali herbar- izirane rastline kažejo znake poškodovanosti zaradi herbarijskih škodljivcev, kot so odloženi drobni zrnati iztrebki na rastlini ali papirju, požrti deli rastlin, luknje v papirju, listih, ste- blih, podzemnih delih, socvetju ali cvetovih kot posledica pašnje larv, uničenost socvetij ali cvetov in prisotnost živih ali mrtvih odras- lih škodljivcev. Poškodovane pole smo prešteli in iz števil poškodovanih in vseh pregledanih pol izračunali odstotek poškodovanih pol za posamezne družine oz. rodove. Opazili smo naslednje škodljivce: hrošče Lasioderma ser- ricorne, Stegobium paniceum in Attagenus piceus, prašne uši - Psocoptera in plesni, v her- barijskem predprostoru pa tudi faraonske mrav- lje (Monomorium pharaonis). Med hrošči nismo našli živih primerkov, prašne uši pa so bile žive in zelo številne. Napadajo predvsem nežnejše rastlinske dele, kot so cvetni listi in prašniki (Skvorcov 1977). Med raziskavo smo pregledali okoli 7500 pol, kar predstavlja približno 5 % vseh pol v LJU. Škodo zaradi herbarijskih škodljivcev smo opazili pri 18 % pregledanih pol. Rezultati povsem podpirajo navedbe, da imajo herbarijski škodljivci preferenco do določenih družin pred drugimi. Največja škoda je tako bila opažena pri družinah Apiaceae, Asteraceae, Cichoriaceae, Brassicaceae, Fa- baceae in Alliaceae (Fig. 1), ki jih tudi liter- atura navaja za posebej privlačne (Bridson 1992, Skvorcov 1977, Nikolić 1996, Hall 1988). Približno enak (visok) delež poškodovanosti pa je bil ugotovljen tudi za družine Araceae, Ro- saceae in Chenopodiaceae (Fig.1). Visoka stop- nja poškodovanost družine Chenopodiaceae je v 21Bačič et al.: Damage by Pests in Herbarium LJU nasprotju z literaturnimi navedbami, po katerih naj bi bila ta družina manj privlačna (Skvorcov, 1977). Družini Poaceae in Polypodiaceae sta se izkazali za manj privlačne. Tudi Lamiaceae, ki jih sicer navajajo za bolj privlačne (Skvor- cov 1977), so se v naši raziskavi uvrščale med manj poškodovane. To si lahko razlagamo z veliko vsebnostjo eteričnih olj, ki imajo repe- lentno delovanje. Tudi družini Ranunculaceae in Scrophulariaceae se uvrščata med manj privlačne, obe sta tudi znani po strupenih in re- pelentnih snoveh. Znotraj družine Asteraceae so se za najprivlačnejše izkazali predstavniki tribusa Cardueae. Odstotek poškodovanosti pol je naraščal od približno 50 % poškodovanih pol pri rodu Saussurea, Carlina, Cirsium in Carduus, dosegel 70 % pri rodu Centaurea, največja poškodovanost pol (75 %) pa se je iz- kazala pri rodu Echinops (Fig. 2). Najpogosteje so bila poškodovana socvetja, sem in tja pa tudi listi. Po pričakovanjih (glej Skvorcov 1977) je znotraj družine Cichoriaceae najprivlačnejši rod Tragopogon (skoraj 60 % poškodovanih pol). Acknowledgement The authors wish to thank Dr. Al Vrezec for determination of the Coleoptera species. We are also very grateful to Professor Dr. T. Wraber for all the support and helpful comments and to Ms. Jana Podakar at Tobačna tovarna Ljubljana for discussion and literature. References Bridson, D., Forman, L., 1992. The Herbarium Handbook. Royal Botanical Gardens, Kew, 303 pp. Clark, S.H., 1986. Preservation of herbarium specimens: an archive conservator’s approach. Taxon, 35, 675-683. Cronquist, A., 1981. An Integrated System of Classifi cation of Flowering Plants. Columbia University Press, New York, 1262 pp. Hall, D.W., 1981. Microwave: a method to control herbarium insects. Taxon, 30(4), 818-819. Hall, A.V., 1988. Pest Control in Herbaria. Taxon, 37(4), 885-907. Heywood, V.H., 1993. Flowering Plants of the World. Andromeda, Oxford, 335 pp. Hill, S.R., 1983. Microwave and the herbarium specimen: potential dangers. Taxon 32(4), 614-615. Hržič, A., Urek, G., 1989. Skladiščni škodljivci na območju Ljubljane. Sodobno kmetijstvo 22(3), 119-130. Kos, F., 1944. Postanek in razvoj Prirodoslovnega muzeja v Ljubljani. Prirodoslovna izvestja, knjiga 1, Prirodoslovni muzej Slovenije, Ljubljana, 199-219. Maden, K., 2004. Plant Collection and Herbarium Techniques, Our Nature, 2, 53-57. Nikolić, T., 1996. Herbarijski priručnik. Školska knjiga, Zagreb, 167 pp. Podobnik, A., 1993. Navodilo za izdelavo herbarija. VTOZD za biologijo, Biotehniška fakulteta, UEK v Ljubljani, 31 pp. Rode, J., 1989. Manj znana nevarnost za herbarijske zbirke. Proteus, 51(7), 277-278. Ryan, L., 1995. Post-harvest Tobacco Infestation Control. Chapman & Hall, London, 155 pp. Skvorcov, A.K., 1977. Gerbarij, posobie po metodike i tehnike. Nauka, Moskva. Stein, W., 1986. Vorratsschädlinge und Hausungziefer: Biologie, Ökologie, Gegenmaßnahmen. Ulmer, Stuttgart. Valentin, N., 1993. Comparative Analysis of Insect Control by Nitrogen, Argon and Carbon Dioxide in Museum, Archive and Herbarium Collections. International Biodeterioration & Biodegradation, 32, 263-278. Zuska, J., 1994. Haus- und Vorratschädlinge. Werner Dausien, Hanau, 192 pp. ACTA BIOLOGICA SLOVENICA LJUBLJANA 2010 Vol. 53, [t. 2: 23–31 Vegetation of the depressions with Eleocharis quinquefl ora in spring fens in Slovenia Vegetacija uleknin z vrsto Eleocharis quinquefl ora na povirnih barjih v Sloveniji Igor Zelnik1, Andrej Martinčič2, Branko Vreš3 1University of Ljubljana, Biotechnical Faculty, Department of Biology, Večna pot 111, SI-1000 Ljubljana, Slovenia. 2Zaloška 78a, SI-1000 Ljubljana, Slovenia. 3Institute of Biology, Scientifi c-Research centre of Slovenian Academy of Sciences and Arts, Novi trg 2, SI-1000 Ljubljana, Slovenia; *correspondence: igor.zelnik@bf.uni-lj.si Abstract: During the investigations of wetlands in Slovenia over the last decade specifi c plant communities in spring fens were found. Stands with species Eleocharis quinquefl ora oc- curring in depressions inundated with standing and/or running water were found in the Alpine, pre-Alpine and Dinaric phytogeographic regions of Slovenia. Standard Central European me- thod for vegetation research was used and multivariate analyses were performed using Syn-Tax program. Stands were classifi ed in two different species-poor, small-scale plant communities, most of them into association Eleocharitetum paucifl orae Lüdi 1921. This rare plant community occurs in the Alpine and Carpathian regions and in northern Europe and has not been recorded in Slovenia before. The association Eleocharitetum paucifl orae is a two-layered plant community of calcium-rich fens. It thrives in shallow temporary paddies and on the sandy or stony slopes with seeping water. Smaller group of relevés was classifi ed into association Scorpidio-Utricularietum minoris Ilschner ex T.Müller et Görs 1960. This association thrives in permanent paddies, where the water is deeper as in a case of the fi rst association. Since the dominating species Eleocharis quinquefl ora and Utricularia minor, respectively, have the status of a vulnerable species according to Red List of Slovenia, the stands of the studied communities, which represent vital populations, should be preserved as well as the corresponding habitat types. Key words: wetland, fen, plant community, Eleocharis quinquefl ora, Utricularia minor, vulnerable species. Izvleček: V sklopu preučevanja mokrišč v Sloveniji v zadnjem desetletju, smo v povirnih barjih našli specifi čne rastlinske združbe. V Alpskem, Predalpskem in Dinarskem fi togeograf- skem območju smo našli sestoje z vrsto Eleocharis quinquefl ora, ki se pojavljajo v ulekninah poplavljenih s stoječo ali tekočo vodo. Pri preučevanju vegetacije smo uporabili standardno srednjeevropsko metodo, multivariatne analize pa so bi le opravljene s programom Syn-Tax. Popise smo uvrstili v dve različni vrstnorevni miniaturni rastlinski združbi – večino v asociacijo Eleocharitetum paucifl orae Lüdi 1921. Ta redka rastlinska združba se pojavlja v Alpski in Kar- patski regiji ter v severni Evropi in v Sloveniji še ni bila popisana. Asociacija Eleocharitetum paucifl orae je dvoslojna rastlinska združba s kalcijem bogatih nizkih barij, uspeva v plitvih občasnih lužah in na peščenih ali kamnitih brežinah z mezečo vodo. Manjšo skupino popisov smo uvrstili v asosciacijo Scorpidio-Utricularietum minoris Ilschner ex T.Müller et Görs 1960. Ta asociacija uspeva v stalnih lužah, kjer je voda globlja kot pri prvi asociaciji. Ker imata domi- nantni vrsti Eleocharis quinquefl ora in Utricularia minor status ranljivih vrst v skladu z Rdečim seznamom Republike Slovenije, bi morali sestoje preučevanih združb, ki predstavljajo njihove vitalne populacije zavarovati, kakor tudi odgovarjajoč habitatni tip. 24 Acta Biologica Slovenica, 53 (2), 2010 Ključne besede: mokrišče, nizko barje, rastlinska združba, Eleocharis quinqu- efl ora, Utricularia minor, ranljive vrste. Introduction Almost 100 years ago Rübel (1911) recorded the stands of the characteristic species Eleocha- ris quinquefl ora on the sandy and wet bank of a dike on the altitude of 1715 m above the sea in Switzerland. Ten years later the association with the name Eleocharitetum paucifl orae Lüdi 1921 was described. The syntaxonomic classifi cation of the analysed type of vegetation is question- able (Martinčič and coworkers 1994, Pott 1995) and it depends on the authors. Koch (1926) and Tüxen (1937) treated com- munity Eleocharitetum paucifl orae as subas- sociation of the association Schoenetum nigri- cantis. Both authors classifi ed this community into class Scheuchzerio-Caricetea fuscae which unifi es plant communities of the fens. Passarge (1964) classifi ed this community named Triglo- chin-Eleocharis paucifl ora into the mentioned class and order Caricetalia davallianae, but into special alliance Eleocharition paucifl orae. Braun (1968) classifi ed this type of vegetation into class Utricularietea intermedio-minoris, order Utricularietalia intermedio-minoris and alliance Sphagno-Utricularion. Similarly Ober- dorfer (1977) classifi ed such stands into Scorpi- dio-Utricularietum minoris scorpidietosum var. Eleocharis quinquefl ora. Dierssen and Dierssen (1985), Steiner (1993), Pott (1995), Hájek and Háberová (2001) classifi ed this plant commu- nity into class Scheuchzerio-Caricetea fuscae, order Caricetalia davallianae, alliance Caricion davallianae and association Eleocharitetum paucifl orae Lüdi 1921. The classifi cation of vegetation with Utricu- laria minor is studied and explained in detail in Dite and coworkers (2006). The association Scorpidio-Utricularietum minoris Ilschner ex T.Müller et Görs 1960 is classifi ed into class Isoëto-Nanojuncetea (Hájek and Háberová 2001, Dite et al. 2006). Wetlands, especially fens are supposed to be highly endangered ecosystems and so are the fen plant-communities and specifi c plant taxa as well. Ecology and vegetation of fens were stud- ied in Slovenia by different authors: Leskovar 1990, 1996, Leskovar-Štamcar 1996, 1991, 1996, 2001, Martinčič and coworkers 1994, Zelnik. This type of plant communities, which thrives in inunda-ted depressions and shallow small lakes within different fen-vegetation types have not been studied before. Thus the aim of the paper is to present origi- nal data on fl oristic composition, the distribution and ecology of this type of vegetation in Slove- nia, to point out vulnerable species thriving in these stands and to emphasize the need to pro- tect the fens. Materials and methods The standard Central European phytoso- ciological method (Braun-Blanquet 1964) was used for vegetation sampling. Cover-abundance estimation values were used, which were trans- formed in accordance with van der Maarel (1979). Relevés were made in summer months, when the majority of plant species are in opti- mal phase and determinable. The size of the plots ranges from 1 to 15 m2 and depends on the microrelief. Multivariate statistical analyses of vegetation relevés were performed using SYN- TAX 2000 (Podani 2001) programme. We used ordination method - Principal Coordinates Anal- ysis (PCoA) and complement of Similarity ratio. The nomenclature of vascular plants fol- lows Martinčič et al. (2007); the nomenclature of mosses follows Martinčič (2003). The no- menclature of syntaxa is in accordance with Steiner (1993) and Valachovič and Ot’ahel’ová (2001). Results and discussion During the survey of fens in Slovenia the stands of these small-scale plant communities were also found and 16 stands were recorded. Analytical table (Tab. 1) shows the fl oristic composition of these species-poor stands. 25Zelnik et al.: Vegetation of the spring fens in Slovenia Table 1. Analytical table of associations: Eleocharitetum paucifl orae Lüdi 1921 and Scorpidio-Utricularietum minoris Ilschner ex. T.Müller et Görs 1960. V – vulnerable species, on the Red List of Ferns and Flower- ing plants in Republic Slovenia. Tabela 1. Analitična tabela asociacij Eleocharitetum paucifl orae Lüdi 1921 in Scorpidio-Utricularietum minoris Ilschner ex. T.Müller et Görs 1960. V – ranljiva vrsta, na Rdečem seznamu praprotnic in semenk republike Slovenije. 26 Acta Biologica Slovenica, 53 (2), 2010 On the base of the results of multivariate analysis of the relevés and their aggregation in two distinct groups (Fig. 1) these two relevé groups can be undoubtedly classifi ed in two mentioned plant communities. According to the presence and abundance of characteristic spe- cies bigger group was classifi ed into association Eleocharitetum paucifl orae Lüdi 1921, while the second group was classifi ed into association Scorpidio-Utricularietum minoris Ilschner ex T.Müller et Görs 1960. Floristic composition Beside the dominant species Eleocharis quinquefl ora ten other species have the status of vulnerable species according to Red list of republic of Slovenia (Wraber et al 2002) (Table 1). That means that over one third of present species of vascular plants (11 out of 32) are considered as vulnerable. The community Eleocharitetum paucifl o- rae (syn.: Scirpus paucifl orus ass. Osvald 1923, Eleocharitetum quinquefl orae (Zobrist 1935) Braun 1968 is a two-layered, species-poor, as- sociation of calcium-rich fens. Recorded rele- vés contain 6-18 species of vascular plants (Table 1). The association is defi ned by the absence of character species of other associations of the al- liance Caricion davallianae, but most of all, by dominance of Eleocharis quinquefl ora (Hájek and Háberová 2001). Steiner (1992, 1993) has defi ned the following diagnostic species com- bination: Character and dominant species: Eleocharis quinquefl ora Dominant and/or most common species: Drepanocladus revolvens agg. (dom.), Carex fl ava (subdom.), C. panicea, Eriophorum latifo- lium, E. angustifolium, Molinia caerulea, Par- nassia palustris, Pinguicula vulgaris, Tofi eldia calyculata. On the base of the presence and abundance of the cryptogams, we can divide the association Eleocharitetum paucifl orae Lüdi 1921 in two subassociations (Hájek and Háberová 2001). Steiner (1993) defi ned two subassociations - one with taxon Chara thriving on fl ooded sites and the other with Drepanocladus revolvens thriving on the calcium-rich stony or sandy sites with thin-layered peat. Since the species Drepanocladus revolvens thrives on acidic peat bogs, this subassociation could not be defi ned with the mentioned moss species but with spe- cies Drepanocladus cossonii, which was actu- ally found in our fens. Both of the subassocia- tions were found in the researched area (Tab. 1). Subassociation with Chara on average contains nine species of vascular plants, while the sub- association with Drepanocladus characteristic for less wet conditions contains 14 species on average. The reason is in anoxic conditions in inundated substrate, which often occur in the sites of the fi rst subassociation, while the sites of the second subassociations can dry up during the periods of summer droughts. On some of the sites of the same habitat type, but with slightly deeper and permanent water the community Scorpidio-Utricularietum minoris Ilschner ex T.Müller et Görs 1960 was found. This plant community is characterized, above all, by the dominance of free fl oating macrophyte Utricularia minor. Three charac- ter species occur in research area: Utricularia minor, Scorpidium scorpidioides and Triglochin palustre. Diferential species Eleocharis quin- quefl ora, which is subdominant in stands of this association (Valachovič and Ot’ahel’ová 2001) is present in all relevés (Tab. 1). Community Scorpidio-Utricularietum con- sists of 3-7 species on average only (Valachovič and Ot’ahel’ová 2001). Recorded relevés con- tain 4-6 species of vascular plants (Tab. 1), but 6-9 species in total, what is slightly higher than stands recorded in Slovakia. Possible reasons are warmer climate and a greater species pool of Dinaric fl oral province. Syntaxonomic position and distribution The distribution of the species Eleocharis quinquefl ora and the distribution of the associa- tion Eleocharitetum paucifl orae in Slovenia are shown on the map (Fig. 2). This rare association reaches here the southeast border of its distribu- tion. It was found in the Alpine, pre-Alpine and Dinaric phytogeographic regions of Slovenia in fens on dolomite limestone substrate in moun- tain belt. The altitude of those fens ranges be- tween 500 and 755 m above the sea level. 27Zelnik et al.: Vegetation of the spring fens in Slovenia Association Scorpidio-Utricularietum mi- noris has even narrower distribution in Slove- nia due to narrow distribution of its dominant species Utricularia minor. It was found on three localities only, namely on the Bloke plateau in Dinaric and near Hotedršica in pre-Alpine phy- togeographic region. In all three localities El- eocharitetum paucifl orae was also found. These stands were found within fens, the altitude ranged from 570 to 775 m. According to the fl oristic composition, pres- ence of the characteristic species and location within the communities from the mentioned fen vegetation types the association Eleocharitetum paucifl orae Lüdi 1921 was classifi ed into alli- ance Caricion davallianae, order Caricetalia davallianae and class Scheuchzerio-Caricetea fuscae. The mentioned order and a class unify the vegetation of fens. Association Eleocharitetum paucifl orae Lüdi 1921 occurs from SW France across the Alpine and Carpathian regions to the Northern Europe and Baltic region, respectively (Balátová- Tuláčková and Venanzoni 1990, Steiner 1992). Pott (1995) reports its occurring in pre-Alpine region, in fens and sites with seeping water in the mountains of the middle belt of Central Germany and on the shores of the islands in the North Sea. This community was also found in the alpine region in Austria, the closest in South Carinthia and Styria (Steiner 1992). Community is very rare in Slovakia and southern Poland (Hájek and Háberová 2001). We have classifi ed association Scorpidio- Utricularietum minoris Ilschner ex T.Müller et Görs 1960 into alliance Scorpidio-Utricularion minoris Pietsch 1964, order Utricularietalia in- termedio-minoris Pietsch 1965 and class Isoëto- Nanojuncetea Br.-Bl. et R.Tx. ex Westhoff et al. 1946, according to the fl oristic composition and presence of the characteristic species. The order Utricularietalia intermedio-minoris contains fl oat- ing plant communities of the fen and peat lakes. Stands of the association Scorpidio-Utric- Figure 1. Ordination diagram of the relevés obtained as a result of Principal Coordinate analysis. Relevés on the right-hand side (11, 12, 16 – in ellipse) were classifi ed into association Scorpidio-Utricularietum minoris. All other relevés belong to the association Eleocharitetum paucifl orae (in rectangle). Relevés 14 and 15 represent a transition since they contain species characteristic for both associations. Slika 1. Ordinacijski diagram popisov kot rezultat analize glavnih koordinat (PCoA). Popisi na desni strani (11, 12, 16 – v elipsi) so bili uvrščeni v asociacijo Scorpidio-Utricularietum minoris. Vsi ostali popisi spadajo v asociacijo Eleocharitetum paucifl orae (v pravokotniku). Popisa 14 in 15 predstavljata prehod, saj vse- bujeta značilne vrste obeh asociacij. 28 Acta Biologica Slovenica, 53 (2), 2010 ularietum minoris have the centre of distribu- tion in boreal-suboceanic region in Alpine re- gion stands are species-poorer (Valachovič and Ot’ahel’ová 2001). Association is common in Germany (Pott 1995) and in Austria (Wallnöfer 1993), but rarely found in Slovakia, Czech Re- public and southern Poland (Valachovič and Ot’ahel’ová 2001). The subassociation Scorpidio-Utricularie- tum minoris charetosum is very similar to El- eocharitetum paucifl orae charetosum. Ober- dorfer (1977) even treats both associations as Scorpidio-Utricularietum minoris. Some other authors also consider both associations as one. Ecology Both associations are pioneer plant commu- nities, which are sometimes only fragmentally developed (Valachovič and Ot’ahel’ová 2001) and we found them on disturbed sites within dif- ferent fen communities, mostly of the alliance Caricion davallianae. Eleocharitetum paucifl orae is a community of calcium-rich fens. It is always occurring in small patches on sites where the substrate is saturated with basic ions (Pott 1995). The per- meable sandy soil is wet due to seepage or high groundwater rich in carbonate (Steiner 1993). This community also thrives in coastal area in wet depressions of halophile grasslands (Pott 1995). In Slovenia this rare fen community can be found on limestone-rich sites. Dominant species Eleocharis quinquefl ora forms low and loose stands. These stands thrive on open and mostly wet sandy or silty substrate. This community thrives in spring areas in shallow paddies and on the sandy or stony slopes with seeping wa- ter. Water is alkaline and rich in Ca2+ (>20 mg/l), electric conductivity is always higher than 300 Figure 2. Distribution of the species Eleocharis quinquefl ora in Slovenia and of the association Eleocharitetum paucifl orae. Localities are presented in the grid of central European mapping. Slika 2. Razširjenost vrste Eleocharis quinquefl ora in asociacije Eleocharitetum paucifl orae v Slo- veniji. Lokalitete so predstavljene v mreži srednjeevropskega kartiranja. 29Zelnik et al.: Vegetation of the spring fens in Slovenia μS/cm (Martinčič 1994). In similar sites with slightly deeper water the community Scorpidio-Utricularietum mi- noris was also found. This community thrives mostly in paddies with 5-20 cm deep water. Water is mesotrophic to oligotrophic, pH being neutral to alkaline. Alkaline fens are classifi ed onto the List of a Decree on Habitat Types which are being pref- erentially maintained in good condition and are rare in Republic of Slovenia, vulnerable or they have small natural distribution area. Conclusions Both studied plant communities have been alternatively classifi ed as a subunit of the other one, by different authors. On the other hand they belong to different types of vegetation, even into different classes – Eleocharitetum paucifl orae belongs into the class of fens and Scorpidio- Utricularietum minoris belongs into the class of fen and/or peat lakes, that indicates the evident differences in ecological conditions and species composition. The most probable reason for such ambiguity is a low number of characteristic spe- cies in both associations, what could make clas- sifi cation diffi cult especially in the presence of species that both have in common. However the main difference in ecological conditions, which forms two vegetation types, is the duration of water phase. Water phase is more or less per- manent in case of the association with Utricu- laria (Scorpidio-Utricularietum minoris), while in the sites of other association (Eleocharitetum paucifl orae) the water dries up regularly. The common fact in both plant commu- nities is the high share of vulnerable Red List species and their rarity in Slovenia and in Cen- tral Europe. The presence of the studied plant communities would give additional reasons for protection of the fens, which have already been recognized as endangered habitat types. Stands with Eleocharis quinquefl ora are also vulnerable due to their dependence on hydrological regime in the landscape, which is changing nowadays. The studied stands are on the southeast margin of their distribution. Marginal areas are most sensitive to the changes, so these data represent important material for the estimation of conse- quences of the local as well as global changes. Povzetek Sestoji z vrsto Eleocharis quinquefl ora so bili popisani že pred skoraj 100 leti, vendar so še več desetletij po tem različni avtorji tovrstne sestoje uvrščali v različne vegetacijske tipe. Ne- kateri avtorji so sestoje s prevladujočima vrsta- ma Eleocharis quinquefl ora in Utricularia minor uvrstili v isto rastlinsko združbo. Vrstna sestava, ekologija in razširjenost preučevane vegetacije je v nekaterih evropskih državah dobro preučena, v Sloveniji pa o tem še ni bilo objavljenih del. V Sloveniji smo v povirnih barjih našli specifi čne rastlinske združbe. V Alpskem, Predalpskem in Dinarskem fi togeografskem območju smo našli sestoje z vrsto Eleocharis quinquefl ora, ki se po- javljajo v stalno ali občasno poplavljenih ulek- ninah. Pri preučevanju vegetacije smo uporabili standardno srednjeevropsko metodo, multiva- riatne analize pa so bile opravljene s programom Syn-Tax. Pri tem smo uporabili analizo glavnih koordinat (PCoA). Popise smo uvrstili v dve raz- lični vrstnorevni miniaturni rastlinski združbi – večino v asociacijo Eleocharitetum paucifl orae Lüdi 1921. Manjšo skupino popisov smo uvrstili v asosciacijo Scorpidio-Utricularietum minoris Ilschner ex T.Müller et Görs 1960. Asociacija Eleocharitetum paucifl orae je dvoslojna rastlinska združba s kalcijem bogatih nizkih barij, uspeva v plitvih občasnih lužah in na peščenih ali kamnitih brežinah z mezečo vodo. Je redka rastlinska združba, ki se pojavlja v Alpski in Karpatski regiji ter v severni Evropi. Asocia- cija je defi nirana predvsem z dominanco vrste Eleocharis quinquefl ora. Asociacija Scorpidio-Utricularietum minoris uspeva v stalnih lužah, kjer je voda globlja kot pri prvi asociaciji. V vodi na teh rastiščih je viso- ka koncentracija kalcija in ima bazično reakcijo. Asociacija je defi nirana predvsem z dominanco prosto plavajoče vrste Utricularia minor. Ker imata dominantni vrsti Eleocharis qu- inquefl ora in Utricularia minor status ranljivih vrst v skladu z Rdečim seznamom Republike Slovenije, bi morali sestoje preučevanih združb, 30 Acta Biologica Slovenica, 53 (2), 2010 ki predstavljajo njihove vitalne populacije zava- rovati, kakor tudi odgovarjajoč habitatni tip. Poleg omenjenih dominantnih vrst ima še devet drugih vrst status ranljive vrste in so uvr- ščene na Rdeči seznam RS, kar predstavlja več kot tretjino vseh popisanih vrst cvetnic. Na podlagi prisotnosti in pogostosti mahov in alg, lahko asociacijo Eleocharitetum paucif- lorae razdelimo na dve subasociaciji, ki smo jih tudi našli na preučevanem območju. Subasocia- cija s taksonom Chara uspeva na poplavljenih rastiščih, druga s taksonom Drepanocladus re- volvens, pa na kamnitih ali peščenih rastiščih. Ker vrsta Drepanocladus revolvens iz istoimen- skega agregata uspeva le na kislih visokih bar- jih, gre tukaj za vrsto Drepanocladus cossonii, ki smo jo v naših sestojih tudi določili in je zna- čilna za bazična rastišča. Izpostavljene so majhne okoljske razlike, ki pogojujejo razlike v vegetaciji. Sestoji z vrsto Eleocharis quinquefl ora so še posebno ranljivi, saj so odvisni od hidrološkega režima v krajini, ki pa se danes spreminja. Sestoji v Sloveniji se nahajajo na južni meji razširjenosti. Robni pre- deli arealov so najbolj občutljivi na spremembe, zato tovrstni zapisi predstavljajo pomemben do- kument, za ugotavljanje posledic, ne samo lo- kalnih, temveč tudi globalnih sprememb. Literature Balátová-Tuláčková E., Venanzoni R., 1990. 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Dissertation thesis, University of Ljubljana, Ljubljana, 196 pp. Appendix: List of the relevé localities (from Table 1): 1: 9556/1 Raduše, Smrčun; 2: 9556/1 Raduše, Smrčun; 3: 9556/1 Sele, Blatnik; 4: 9556/1 Sele, Blatnik; 5: 9556/1 Sele, Blatnik; 6: 9455/4 Kot pri Prevaljah; 7: 9455/4 Kot pri Prevaljah; 8: 9455/4 Kot pri Prevaljah; 9: 9455/4 Kot pri Prevaljah; 10: 9556/14 Raduše, Smrčun; 11: 0050/4 Hotedršica, Žejna dolina; 12: 0050/4 Hotedršica, Žejna dolina; 13: 0253/1 Volčje (Bloke); 14: 0050/2 Žibrše, Žejna dolina; 15: 0050/2 Žibrše, Žejna dolina; 16: 0252/2 Ulaka (Bloke); Environmental assessment and macrophytes of the watercourses Bloščica and Cerkniščica Okoljska ocena in makrofi ti vodotokov Bloščice in Cerkniščice Špela Mechora1, Urška Kuhar1, Mateja Germ1* 1Biotechnical Faculty, Department of Biology, Večna pot 111, 1000 Ljubljana, Slovenia *correspondence: mateja.germ@bf.uni-lj.si Abstract: The aim of the present work was to determine the abundance and distribution of macrophytes in streams Bloščica and Cerkniščica and to establish the relation between environ- ment characteristics and abundance of macrophytes. The environmental and macrophytes’ inven- tory was made on the whole length of the watercourse. We determined a presence, abundance and growth form of macrophytes and environmental parameters according to modifi ed RCE Inventory. Nineteen taxa were found in the watercourse Bloščica and 20 taxa in the watercourse Cerkniščica. Canonical correspondence analysis revealed that six environmental parameters signifi cantly affected macrophyte community, the most infl uential being bottom structure, the width of riparian zone, retention devices in a channel and the land use beyond the riparian zone. Key words: environmental assessment, macrophytes, watercourses Izvleček: V prispevku podajamo rezultate raziskav pojavljanja, razporeditve in pogostosti mak- rofi tov v vodotokih Bloščica in Cerkniščica ter ugotavljamo povezavo med okoljskimi razmerami in pojavljanjem makrofi tov. Makrofi te, njihovo pogostost, rastno obliko in stanje širšega vodnega okolja po prirejeni RCE metodi, smo popisali na celotni dolžini izbranih vodotokov. V vodotoku Bloščica smo popisali 19 taksonov, v vodotoku Cerkniščica pa 20 taksonov. Kanonična korespondenčna analiza je pokazala, da šest okoljskih parametrov značilno vpliva na pojavljanje in pogostost makrofi tov. Največji vpliv imajo struktura dna, širina obrežnega pasu, zadrževalne strukture v strugi in zaledje. Ključne besede: okoljska ocena, makrofi ti, vodotoki ACTA BIOLOGICA SLOVENICA LJUBLJANA 2010 Vol. 53, [t. 2: 33–43 Introduction Rivers are diverse and dynamic systems that play an important role in the complexity of the landscape (Chovanec et al. 2000). Macrophytes are fundamental to the structure and functioning of lowland river habitats (Baatrup-Pedersen and Riis 1999). Distribution and abundance of mac- rophytes are affected by several environmental and antropogenic factors and their interactions (Lacoul and Freedman 2006). Parameters exerting impact on macrophyte’s growth and abundance in running waters are the following: climate, hydrol- ogy, geomorphology, nutrients and other chemi- cal factors, biological interactions and human ac- tivities (Onaindia et al. 1996, Bernez et al. 2004, Hrivnàk et al. 2007). Ecological status of rivers is infl uenced by human activities that affect the physical properties of the riverbed, riparian vege- tation and land beyond the riparian zone (Petersen 1992). These changes worsened the conditions of the river ecosystem and water quality as well as altered communities of aquatic organisms, includ- ing macrophytes, which play important roles in energy fl ow, nutrient cycling and sedimentation processes (Holmes 1999, Gaberščik et al. 2003). Macrophytes improve water quality, both directly through oxygenation and nutrient recycling, and 34 Acta Biologica Slovenica, 53 (2), 2010 area. Almost half of the catchment of both wa- tercourses is covered by forest. The watercourse Bloščica is intermittent watercourse fl owing on Bloke plateau being a part of the watershed of the river Ljubljanica. Elevation of its fl ow ranged from 720 and 750 m. Catchment comprises of small tributaries (Runarščica, Blatni potok, Krajič, Ribjek) cov- ering about 25 km2. Due to its low slope, the watercourse Bloščica fl ows slowly and makes many meanders. It fl ows on dolomites fi rst 6 km of its length. At Velike Bloke it cut its bed into limestone and dolomite and fi nnaly sinks underground. The upper part of the watercourse Bloščica fl ows mainly through preserved land- scape, while the lower part from Ulake down- streams is more affected by human activity. Spring of the about 17 km long watercourse Cerkniščica is located in a hilly area of Sveti Vid and Cajnarji. The catchment comprises 50 km2. It is the biggest surface tributary of the lake Cerknica. The water level changes very quickly in the case of strong rain, so it can be designated as a torrent watercourse. The watercourse Cerkniščica indirectly by providing surface for water-puri- fying algae, fungi and bacteria (Holmes 1999). Species composition of macrophytes and their abundance refl ect the quality of an ecosystem as a whole. For that reason macrophytes are included in the EU Water Framework Directive (Council of the European Communities 2000), presenting one of the four indispensable biological elements, which determine the ecological status of rivers (Dodkins et al. 2005). The aims of the present study were to deter- mine the presence, abundance and distribution of macrophytes in watercourses Bloščica and Cerkniščica and to fi nd out the relation between the environmental parameters and the occur- rence of macrophytes. Materials and Methods Study area Watercourses Bloščica and Cerkniščica are located in Notranjska region (Slovenia) in karst Table 1: List of taxa, determined in the watercourse Bloščica. Tabela 1: Seznam vrst, prisotnih v Bloščici. Taxa Abbreviation Growth form Alisma plantago-aquatica L. Ali pla am Bryophyta Bryophy sa Chara sp. Cha sp. sp Equisetum palustre L. Equ pal he Lythrum salicaria L. Lyt sal he Mentha aquatica L. Men aqu am Mentha longifolia (L.) Hudson Men lon am Menyanthes trifoliata L. Men tri he Myosotis scorpioides L. Myo sco am Myriophyllum spicatum L. Myr spi sa Nasturtium offi cinale R. Br. In Aiton Nas off he Petasites hybridus (L.) Gaertner Pet hyb he Phragmithes australis (Cav.) Trin ex Steud. Phr aus he Plantago altissima L. Pla alt he Potamogeton lucens L. Pot luc sa Potamogeton nodosus Poir. Pot nod fl Schoenoplectus lacustris (L.) Palla Sch lac he Sparganium erectum L. Spa ere he, sa Typha latifolia L. Typ lat he Legend: ap = plants fl oating on the water surface, sp = submerged pleustophytes, sa = submerged anchored plants, fl = fl oating leaf rooted plants, am = amphiphytes, he = helophytes 35Mechora et al.: Environmental assessment and macrophytes fl ows in its upper part in narrow and deep valley. From Cajnarji to Begunje by Cerknica, its bottom becomes wider and steeper, covered by fl uvial de- posits. At Begunje by Cerknica it fl ows on karst area. The watercourse Cerkniščica is regulated in the settlements Cerknica and Dolenja vas. It sinks underground at Cerkniško polje. The catchments of both studied watercourses are part of the Natura 2000 network. Riparian, Channel, and Environment Inventory (RCE) Studied watercourses were divided to stretch- es from 360 to 1030 m long. The start of the new stretch was determined where presence or abun- dance of macrophytes changed, when we observed changes in land use type, channel characteristics or riparian zone. Every stretch was assessed accord- ing to the modifi ed RCE Inventory (Petersen 1992, Germ et al. 2003). RCE Inventory was developed for the assessment of physical condition of the riparian zone and the stream channel in lowland streams, fl owing through agricultural landscape. Modifi ed RCE Inventory consisted from 12 pa- rameters, each describing 4 levels of environmen- tal gradient. The parameters include land-use type beyond the riparian zone, characteristics of the riparian zone (width, completeness and vegeta- tion type), and morphology of the stream channel (channel structure, bank structure and undercut- ting, occurrence of retention structures and sedi- ment accumulation, type of stream bottom and de- tritus and dynamics of the fl ow). Figure 1: Distribution of macrophytes in the watercourse Bloščica. Stretch 10 was not surveyed. Slika 1: Razporeditev in pogostost makrofi tov v Bloščici. Odsek 10 ni bil pregledan. 36 Acta Biologica Slovenica, 53 (2), 2010 0 5 10 15 20 25 Phr aus Men aqu Men tri Lyt sal Myr spi Spa ere Cha sp. Myo sco Sch lac Pot nod Typ lat Pet hyb Pla alt Men lon Equ pal Pot luc Bryophy Ali pla Nas off RPM [%] Macrophyte survey The distribution and abundance of macro- phytes in studied watercourses were assessed from the source to the outfl ow, using a boat and a rake with hooks. The abundance was evaluated using a fi ve degree scale as follows (Kohler and Janauer 1995): 1 = very rare; 2 = infrequent; 3 = com- mon; 4 = frequent; 5 = abundant, predominant. Plants were identifi ed using the keys by Pres- ton (1995), Casper and Krausch (1980) and Martinčič et al. (1999). Statistical analysis On the basis of plant abundance, a relative plant mass was calculated (RPM) that is related to true biomass with function x3 (Pall and Janau- er 1995, Kohler and Janauer 1995). Canoni- Figure 2: Relative plant mass (RPM) of macrophytes in the watercourse Bloščica. Slika 2: Relativna rastlinska masa (RPM) makrofi tov v Bloščici. 0 0,5 1 Ali pla Bryophy Cha sp. Equ pal Lyt sal Men aqu Men lon Men tri Myo sco Myr spi Nas off Pet hyb Phr aus Pla alt Pot luc Pot nod Sch lac Spa ere Typ lat d Figure 3: The ratio of the length of the watercourse Bloščica overgrown by certain species of macrophytes »d« value; 0.5 for example means that 50 % of watercourse in owergrown with macrophytes. Slika 3: Delež dolžine vodotoka, poraslega z določeno vrsto makrofi tov »d« vrednost; 0,5 npr. pomeni, da je 50 % vototoka porastlega z določeno vrsto. cal correspondence analysis (CCA) (Canoco for Windows Version 4.5) was used to assess the relationship between the composition and abundance of macrophytes, and environmental parameters. Environmental parameters were coded numerically from 1 (the most modifi ed or degraded condition) to 4 (the natural or near natural condition). Results Presence and abundance of macrophytes In the watercourse Bloščica 19 taxa of mac- rophytes was detected on the 17.800 m length (Tab. 1 and Fig. 1). Three species found are list- ed on the Slovenian Red list of Pteridophyta and Spermatophyta (Ur. l. RS 82/2002) as vulnera- 37Mechora et al.: Environmental assessment and macrophytes Figure 4: Distribution of macrophytes in the watercourse Cerkniščica. Slika 4: Razporeditev in pogostost makrofi tov v Cerkniščici. ble (Menyanthes trifoliata, Potamogeton lucens and P. nodosus). The highest number of species was found in the stretches 18 (12) and 19 (12). The highest RPM reached Phragmites aus- tralis (20.9 %), followed by Mentha aquatica (17.6 %), Menyanthes trifoliata (12.4 %) and Lythrum salicaria (12.2 %) (Fig. 2). Lythrum salicaria and Mentha aquatica occured in more than 80 % of the watercourse, followed by Me- nyanthes trifoliata (d = 0.77) and Phragmithes australis (d = 0.73) (Fig. 3). The majority of spe- cies in the both watercourses had amphibious or helophitic growth form, while submerged spe- cies were relatively scarce. In the watercourse Cerkniščica 20 taxa of macrophytes were found (Tab. 2 and Fig. 4). Three of them are listed on the Slovenian Red list of Pteridophyta and Spermatophyta (Ur. l. RS 82/2002) determined as vulnerable: Nym- phaea alba, Potamogeton nodosus and Polygo- num amphibium. The highest RPM reached Petasites hybri- dus (17.5 %), followed by Cirsium oleraceum (13.5 %) and Equisetum palustre (12.4 %) (Fig. 5). Petasites hybridus occured in 85 % of the watercourse, followed by Lythrum salicaria (d = 0.67), Equisetum palustre and Cirsium olera- ceum (d = 0.62) (Fig. 6). 38 Acta Biologica Slovenica, 53 (2), 2010 Table 2: List of taxa, determined in the watercourse Cerkniščica. Tabela 2: Seznam vrst, prisotnih v Cerkniščici. Taxa Abbreviation Growth form Bryophyta Bryophy sa Caltha palustris L. Cal pal he Chara sp. Cha sp. sp Cirsium oleraceum (L.) Scop Cir ole he Equisetum palustre L. Equ pal he Lythrum salicaria L. Lyt sal he Mentha aquatica L. Men aqu am Mentha longifolia (L.) Hudson Men lon am Myosotis scorpioides L. Myo sco am Myriophyllum spicatum L. Myr spi sa Nymphaea alba L. Nym alb ap Petasites hybridus (L.) Gaertner Pet hyb he Phragmithes australis (Cav.) Trin ex Steud. Phr aus he Polygonum amphibium L. Pol amp am Potamogeton nodosus Poir. Pot nod fl Ranunculus sp. Ran sp. sa Schoenoplectus lacustris (L.) Palla Sch lac he Sparganium erectum L. Spa ere he, sa Typha latifolia L. Typ lat he Veronica anagallis-aquatica L. Ver ana sa Legend: ap = plants fl oating on the water surface, sp = submerged pleustophytes, sa = submerged anchored plants, fl = fl oating leaf rooted plants, am = amphiphytes, he = helophytes Environmental parameters and distribution of macrophytes Canonical correspondence analysis (CCA) (Canoco for Windows Version 4.5) was used to assess the relationship between environmental parameters and the composition and abundance of macrophytes in the watercourses Bloščica and Cerkniščica. Six examined parameters signifi cantly affected the variability within the macrophyte com- munity, the most infl uential were stream bottom type, width and completeness of the riparian zone, occurrence of retention devices, land use beyond the riparian zone and bank undercutting (Fig. 7). The stretches are arranged in the ordination diagram according to the characteristics of envi- ronmental parameters in individual stretch. The quality of environmental parameters increases in the direction of the arrows. The stretches of Bloščica and streaches of Cerkniščica were pres- ent at different parts of the ordination diagram. Rocky bottom was colonised by taxa Cir- sium oleraceum, Equisetum palustre and Pet- asites hibridus, while the mixture of slime and sand was overgrown by Menyanthes trifoliata, Mentha aquatica and Phragmites australis. The taxon Ranunculus sp. and the species V. anaga- lis-aquatica occurred in stretches surrounded by wetland and forests, while the species S. lacus- tris, P. lucens, Alisma plantago-aquatica and P. nodosus preferred open locations. Discussion The Riparian, Channel and Environmental (RCE) Inventory has been developed to assess the physical and biological conditions of small, lowland streams in agricultural areas (Petersen 1992). The modifi ed RCE inventory consists of twelve characteristics, which defi ne the struc- ture of the riparian zone, stream channel mor- 39Mechora et al.: Environmental assessment and macrophytes 0 5 10 15 20 Pet hyb Cir ole Equ pal Bryophy Men lon Spa ere Phr aus Lyt sal Cha sp. Cal pal Men aqu Myr spi Typ lat Nym alb Pot nod Pol amp Myo sco Ver ana Sch lac Ran sp. RPM [%] Figure 5: Relative plant mass (RPM) of macrophytes in the watercourse Cerkniščica. Slika 5: Relativna rastlinska masa (RPM) makrofi tov v Cerkniščici. phology and the biological condition in both habitats. Numerous agricultural point discharges from fi eld present a serious problem threat- ing a good ecological status of watercourses. Wide and complete riparian vegetation has key role in prevention of erosion and retention of organic and even toxic substances (Johnston et al. 1990). Prevailed land use type of the water- course Cerkniščica in upper part was forest and wet grassland and in lower parts agricultural and urban areas prevailed. Mosses were frequent, because of shading of the channel due to ripar- ian vegetation and water level fl uctuations. The river-bed of the watercourse Cerkniščica was channelized in the settlements Cerknica and Do- lenja vas and therefore riparian vegetation was scarce or absent. The bottom of the watercourse Bloščica consisted from fi ne, anaerobic sediment. Pre- vailing land use was wood and wet grassland. Riparian vegetation was removed at certain sites, that increased the vulnerability of the watercourse. This is also confi rmed with ca- nonical correspondence analysis that revealed that most infl uential environmental parameters shaping macrophyte community were bottom structure, width of riparian zone, retention de- vices in the channel and land use beyond the riparian zone. Macrophyte species diversity was relatively low in either of the studied watercourses. 19 taxa were detected in the watercourse Bloščica and 20 in the watercourse Cerkniščica. 14 taxa were pres- ent in both watercourses. Important parameter, affecting the growth of macrophytes is light (Hut- 0 0,5 1 Bryophy Cal pal Cha sp. Cir ole Equ pal Lyt sal Men aqu Men lon Myo sco Myr spi Nym alb Pet hyb Phr aus Pol amp Pot nod Ran sp. Sch lac Spa ere Typ lat Ver ana d Figure 6: The ratio of the length of the watercourse Cerkniščica overgrown by certain species of macrophytes »d« value; 0.5 for example means that 50 % of watercourse in owergrown with macrophytes. Slika 6: Delež dolžine vodotoka, poraslega z določeno vrsto makrofi tov »d« vrednost; 0,5 npr. pomeni, da je 50 % vototoka porastlega z določeno vrsto. 40 Acta Biologica Slovenica, 53 (2), 2010 cinson 1975). Shaded parts of the watercourses were scarcely colonised with vascular macro- phytes. Mosses were dominant group of macro- phytes in that stretches. Diversity and distribution of macrophytes in lowland rivers depend on the concentration of nutrients, current velocity and anthropogenic impact (Hrivnàk et al. 2007). Hu- man impact on water ecosystem was less evident in the case of the watercourse Bloščica, where wet grasslands colonising the areas along the water- course were less appropriate for agricultural use. Similarly, human impact was not prominent in the upper parts of watercourse Cerkniščica, since the area was covered by forests and wet meadows. Six species, listed on the Slovenian Red list (Ur. l. RS 82/2002) as vulnerable, thrive in the sites, where watercourses fl ow in the natural or little altered landscape with relatively wide riparian zone, and mixed silty and sandy sediment with organic matter as also reveals from previous re- searches (Kuhar et al. 2009). The decrease of heterogeneity of habitats in- duces lower diversity of macrophytes (O’Hare et al. 2006). In the watercourse Bloščica fi ne mate- -1.0 1.0 -1 .0 1 .0 LND USE R WIDTH R COMPL RET STR B UNDC BOTTOM Ali pla Bryophy Cal pal Cha sp. Cir ole Hip vul Lyt sal Men aqu Men lon Men tri Myo sco Myr spi Nas off Nym alb Pet hyb Phr aus Pla alt Pol amp Pot luc Pot nod Ran sp. Sch lac Spa ere Typ lat Ver ana Figure 7: CCA ordination diagram showing the relationship between the macrophytes and environmental parameters. Lnd use - land use pattern beyond the riparian zone; R width - width of riparian zone; R compl - com- pleteness of riparian zone; Ret str - retention structures; B undc - bank undercutting; Bottom - stream bottom; ● - Bloščica; ■ - Cerkniščica. Codes for macrophyte taxa are given in Table 1 and Table 2. Slika 7: CCA ordinacijski diagram s taksoni makrofi tov in spremenljivkami okolja. Lnd use - raba tal v zaledju struge; R width - širina obrežnega pasu; R compl - sklenjenost vegetacije v obrežnem pasu; Ret str - zadrževalne strukture v strugi; B undc - spodjedanje brega; Bottom - dno; ● - Bloščica; ■ - Cerkniščica. Oznake za taksone makrofi tov so v Tabeli 1 in Tabeli 2. 41Mechora et al.: Environmental assessment and macrophytes rial and detritus was the prevailing sediment type, banks predominantly consisted from fi ne inorgan- ic material. Lower number of macrophytes there- after refl ected the homogeneity of the habitat. Relatively homogeneous habitat was found also in the watercourse Cerkniščica. Flow velocity infl u- ences the occurrence of macrophytes (Janauer et al. 2010). Upper part of the watercourse Bloščica had higher slope and fast current velocity. Water- course became wider downstream, current veloc- ity was slower. In that part of the Bloščica, the variety of macrophytes species was higher. Riis et al. (2000) stated that water chemistry and different tolerance of species to nutrient load determine the distribution of macrophytes. Thus, on the basis on the presence of the certain spe- cies of macrophytes, the loading of watercourses with nutrients can be assessed. Acceleration of eutrophication is a consequence of human activ- ity, especially urbanization, agriculture and in- dustry (Germ et al. 2008). Potamogeton lucens grows in eutrophic, relatively deep and on cal- careous bedrock fl owing lowland streams (Pres- ton 1995) as holds true for the last part of the watercourse Bloščica. Genus Chara was found in both watercourses only in stretches located far from agricultural areas. Species richness of the genus Chara drops when amount of nutrient arises. Bornette and Arens (2002) stated that spe- cies in genus Chara are pioneer species in habi- tats, where disturbance appears very often. In the case of both watercourses water-level fl uctua- tions presented the main disturbance. Myriophil- lum spicatum avoids fast fl owing and oligotro- phic waters (Germ and Gaberščik 1999); it was found in the lower parts of studied watercourses. Conclusions 1. In the watercourse Bloščica 19 taxa of macro- phytes were detected. Three species found are listed on the Slovenian Red list of Pteridophyta and Spermatophyta as vulnerable namely Menyanthes trifoliata, Potamogeton lucens and P. nodosus. 2. In the watercourse Cerkniščica 20 taxa of macrophytes were found. Three of them are listed on the Slovenian Red list of Pteridophyta and Spermatophyta as vulnerable namely Nymphaea alba, Potamogeton nodosus and Polygonum amphibium. 3. The majority of species in the both watercourses had amphibious and helophytic growth form, while submerged species were relatively scarce. 4. Presence and abundance of macrophytes changed along the watercourses mainly due to land use type, width of the riparian zone and bottom properties. Abundance of macro- phytes in both watercourses was the highest in unshaded stretches with the middle current velocity. Povzetek Namen raziskave je bil ugotoviti pojavljanje, razporeditev in pogostost makrofi tov v Bloščici in Cerkniščici. Opisali smo stanje širšega vodnega okolja obeh vodotokov ter povezavo med okoljs- kimi razmerami in pojavljanjem makrofi tov. Vodotoka smo razdelili na odseke (24 odsekov na Bloščici, 23 odsekov na Cerkniščici), v katerih smo popisali makrofi te. Hkrati s popisom mak- rofi tov smo ocenili širše okolje s po Petersenu prirejeno RCE metodo (Germ et al. 2003) in habi- tatne parametre. V obeh vodotokih smo skupno popisali 25 taksonov makrofi tov. Večjo pestrost makrofi tov smo zasledili na mestih, kjer je vodni tok počasnejši in kjer je vpliv človeka zmanjšan (npr. urbanizirana območja, kmetijske površine). Vodotoka se razlikujeta v habitatnih parametrih in prisotnosti makrofi tov. Pomemben je tip sedimen- ta, saj se rastline lažje ukoreninjajo v bolj trdnem substratu kot v rahlih, fi nih delcih. V Bloščici se je večinoma pojavljal detrit, v Cerkniščici pa so se ob detritu pojavljali še pesek in skale. Kanonična korespondenčna analiza je pokazala, da šest okoljskih parametrov značilno vpliva na pojavljanje in pogostost makrofi tov. Največji vpliv imajo struktura dna, širina obrežnega pasu, zadrževalne strukture v strugi in zaledje. 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A hydro-acoustics approach of accessing macrophyte biomass data Hidro-akustično pridobivanje podatkov o biomasi makrofi tov Norbert Exler*, Georg Janauer Department of Limnology, University of Vienna, Department of Limnology, Althanstrasse 14, 1090 Vienna, Austria *correspondence: norbert.exler@univie.ac.at Abstract: Hydro-acoustic methods are commonly used to estimate the abundance and dis- tribution pattern of fi sh in aquatic environments, while studies on the assessment of the biomass of submerged macrophytes in the littoral zones are still rare. In the present study we provide fi rst results showing that indeed this method is a useful tool to estimate the aquatic plant stands in lakes. The aim of the recent presentation is to show an initial data evaluation by graphs describing hydro-acoustic signals at three distinct layers in a small shallow lake: the solid sediment, the fi ne or muddy sediment, and the ‘plant canopy’ of submerse macrophytes. The most diffi culties of data processing and assessment of biomass were for hydro-acoustic records close to the water surface where the echo-signal is interfered by refl ectance. Methodological details and progress in evaluating hydro-acoustic records will be discussed. Key words: aquatic macrophytes, biomass, hydro-acoustics Izvleček: Hidro-akustične metode se navadno uporabljajo za oceno pogostosti in zastopanosti rib v vodnem okolju, medtem ko so tovrstne raziskave biomase makrofi tov v litoralu razmeroma redke. Raziskava podaja rezultate, ki kažejo na uporabnost metode za oceno sestojev makrofi tov v jezerih. Namen raziskave je prikazati začetno vrednotenje podatkov hidro-akustičnih signalov na treh različnih plasteh majhnega plitvega jezera: trdnih usedlin, fi nih oziroma rahlih usedlin in sestojev potopljenih makrofi tov. Največja težava pri obdelavi podatkov in oceni biomase so bili signali v bližini vodne površine, ki so interferirali z odbojem. V prispevku je podan metodološki pristop in potek ocenitve hidro-akustičnih signalov. Ključne besede: vodni makrofi ti, biomasa, hidro-akustika ACTA BIOLOGICA SLOVENICA LJUBLJANA 2010 Vol. 53, [t. 2: 45–51 Introduction Assessing aquatic plant stands in freshwa- ter and marine systems might become of even more interest as macrophyte belts are known to be important to inhabit young fi sh, as they play an important role to share nutrient sources with planktonic primary producers by alternative sta- ble states and are hence of decisive importance for a good ecological status of an aquatic eco- system (Scheffer 2002). Beside the need of more quantitative information about macrophytes, studies about macrophyte records are still rare compared with other aquatic assemblages as e.g. studies of fi sh or plankton. One reason could be the effort used for recording macrophyte data. The state of art of assessing and processing biomass information of aquatic plants is based traditionally on harvesting methods, or on fi eld surveys focusing on visual plant mass estimates. The effort of estimating standing crop of mac- rophytes becomes even higher in case that re- cording can be done only by divers. Different from that approach is the application of hydro- acoustics. This method that is usually applied to fi sh-relevant research and commonly used in practice (Brandt 1996, Rakowitz et al. 2009), has also been used for macrophyte research for 46 Acta Biologica Slovenica, 53 (2), 2010 over two decades in some water bodies, with suffi cient success in freshwater and marine sci- ence (e.g. Komatsu et al. 2002), too. Some trials on macrophytes were done with dual frequency echo sounder (Dumfart and Pall 2003, Jäger et al. 2004), and some other trials detected biomass in an experimental set-up by means of horizon- tal echo sounding (Hohausová et al. 2008). Tri- als with the same low-cost instrument we were using have also been applied by the Portland State University (USA; Litz 2007). A widely applicable method for estimating macrophyte biomass, however, is missing. Echo sounding reveals only numerical infor- mation on depth. The advantage of the method, however, is, that many systems can store the original signal and can hence provide high reso- lution measurements by trials on both, temporal and spatial scales. Aside from calculating infor- mation on fi sh types, little effort has been put into differentiating a typical macrophyte signal, and a simplifi cation of data processing and dis- play of results is also needed. The aim of the study is to show graphically fi rst results of quan- titative recording of macrophyte stands by using a low-sophistication standard sonar equipment. This method allows to detect macrophyte stands and to calculate water plant-fi lled volumes lead- ing to a semi-automatic assessment of biomass. Materials and methods Our approach is based on low-sophistication standard sonar equipment normally used in fi sh- ery. The sonar generates a standard echogram with 256 bit resolution, which will be used for detection of ground, sediment and macrophyte height. This signal can be used for automatic height detection of macrophyte stands. The re- spective water volume covering the plants refers to a semiautomatic biomass estimate. LMS-480M Sonarviewer (Lowrance Inc.) was used in our research which is standard sonar Figure 1: Survey site “Dorfl acke”, a part of former side channels of the Danube River. Slika 1: Preiskovano območje “Dorfl acke”, del stare stranske struge reke Donave. 47Exler, Janauer: A hydro-acoustics approach of accessing macrophyte macrophyte assemblages at different height in the respective water layer. Further a yield index for specifi c macrophyte dominance pattern was developed (Janauer et Wychera 2000, Kohlbauer 2008). Such calibration data set and biomass calculations are not shown in the recent study for Dorfl acke. We focused here on the graphical presentation of hydro-acoustic measurements. The hydro-acoustics run was performed in zigzag pattern once during summer, on June 30, 2010. The track is shown in Fig. 1. Where plants reached the surface the transducer was lifted for a short moment to avoid getting entangled in plant stems. This procedure did not interfere with either the reception of the acoustic signal nor with the GPS reading. Study Area Dorfl acke is a relict part of the lower reach of the small river ‘Kleine Tulln’, which merged with Danube River side channels before the con- struction of the Greifenstein run-of-river hydro- electric power plant. Today this water body is located in parallel to the longitudinal levee of the power plant reservoir. The Dorfl acke receives most of its water from the groundwater and seep- age from the reservoir (Fig. 2). Its length and width is about 500 and 40 m, respectively. The maximum depth is 5.5 m. Figure 2: Track of hydro-acoustic run. Slika 2: Potek hidro-akustičnega pregleda dna. and usually used for detecting fi sh. This instru- ment features single frequency (50/200 kHz de- pending on the detected depth) with 500 watts of RMS power and displays depth from approxi- mately 50 cm to 400 m with a resolution of 5 cm on its 5” diagonal high resolution, 256-bit mono- chrome display. The high speed skimmer trans- ducer has a built-in temperature sensor. The in- strument is equipped with a 12 channel WAAS/ EGNOS compatible GPS receiver and provides accuracy up to 3 meters. The sonar has a built-in SDRAM card slot and stores the original sonar data for further processing in an internal raw data format. This internal data format was reverse en- gineered by means of Matlab version 7.4. This program was used for reading and processing the raw data and further post processing and visuali- sation. Google Earth (Google Inc.) and ArcGIS 9.3 (ESRI) are used for defi ning the shore line of the water body under survey. When using our equipment in areas under nature protection the instrument is used from a conventional rubber boat propelled by an electric outboard motor. Data processing for the calculation of mac- rophyte biomass yield is described in detail in Janauer and Wychera (2000). According to an empirical data set, the biomass yield was calcu- lated by the signifi cant relationship between the volume of the plant stands and the plant material harvested from patches of defi ned area of usually 0.25 to 0.5 m2. This relationship was tested for 48 Acta Biologica Slovenica, 53 (2), 2010 Results Results of data processing of hydro-acoustic records in shallow Dorfl acke are presented by graphical illustration shown in Figs. 3 and 4. Our measurements comprise a data set of 65 transversal transects and 3 longitudinal runs on the summer trial. The results of the summer macrophyte situation are exemplifi ed here by a single transversal transect (Fig. 3). This fi gure shows a typical echogram obtaining three zones of diffe-rent acoustic properties. The lowest line delineates the zone of the bottom layer at the ground and refers to solid sediment at the bot- tom of the lake. The area between the ground and the middle line indicates the zone of muddy sediment (fi ne sediment). The upper line refers to the ‘underwater canopy of macrophytes’. The area between the lines of fi ne sediment and of the canopy relates to water layers inhabiting macrophyte biomass. The dotted grey line in Fig. 3 might indicate the top layer where hydro- acoustic data do not provide reliable information on macrophyte biomass as explained in detail in the discussion. This shallow depth is electroni- cally attenuated and hence indicated a recorded signal which is invalid describing a hydro- acoustic pattern. The morphometry of the water basin of Dor- fl acke is shown in Fig. 4. This contour map is automatically generated by the sonar diagram and can be linked to transect information ex- emplifi ed in Fig. 4. The map shows that even a shallow water body can vary by spatial het- erogeneity. Even if most area is indicated by a depth of two meters only, some limited areas re- fer to a water depth deeper than 4-5 m. Further, this high resolution measurement shows that a littoral zone shallower than 1 m is more com- mon at the south shore line than compared to the northern part. Figure 3: Echogram of a transect, with ground line of solid sediment (white), sediment line of fi ne/muddy sediment (bold black), smoothed macrophyte line of plant canopy (thin black) and dotted grey line minimum depth of detection. Slika 3: Ehogram transekta z mejo trdne usedline (belo), mejo fi ne / rahle usedline (odebeljeno-črno), izravnano mejo makrofi tske vegetacije (tanka-bela) in minimalno mejo detekcije (pikčasta siva črta). 49Exler, Janauer: A hydro-acoustics approach of accessing macrophyte Discussion The results by our hydro-acoustic measure- ments sound promising as both, the water depth and the water plant canopy, were recorded with excellent accuracy. Based on this information a 3-D-model of the plant mass will be calculated for Dorfl acke in a further study relating to an es- timation of total macrophyte biomass. The analysis of the acoustic data shown in this study allowed an automatic separation and detection of three distinct ‘surfaces’ as regards fi ltering and edge detection of the echo sounding signal of solid sediment, the fi ne and/or muddy sediment, and the ‘plant canopy’ indicating the height of the aquatic plant stands. The graphi- cal presentation refers to a calculation of trian- gulated irregular networks which allow a three dimensional description required for estimating macrophyte biomass yield in water bodies. Figure 4: Depth contour map of Dorfl acke, colour coded. For better visualisation the y-axis has been stretched, banks are in white, some artefacts located outside the water body are due to mathematical interpolation. Both axes indicate the spatial distance in m. Slika 4: Globinske črte proste vode v vodnem telesu Dorfl acke (različno obarvano). Zaradi boljše preglednosti je merilo na y osi povečano, bregovi pa so belo obarvani. Artefakti zunaj vodnega telesa so posledica matematične interpolacije. 50 Acta Biologica Slovenica, 53 (2), 2010 A semiautomatic evaluation was carried out following the procedure described by Kohlbauer (2008), in a process of validating our approach. A problem in automatically assessing the total volume of plant stands is due a low reliability of data at the near surface layer. In the uppermost strata of the water body, usually at a range of less than 50 cm, the acoustic signal is interfered by the refl ectance of the water surface. As this shal- low layer at water surface provides an invalid hydro- acoustic signal, plant stands reaching all the way up to the water surface are not accu- rately recorded in these parts of the water body by standard data processing. Therefore data need to be corrected manually in an additional post processing step. The areas can be digitized by means of GIS and marked as ‘macrophytes-to- surface’ along the survey track or can be simply estimated by recording the perimeter of these ar- eas by means of normal GPS. In case of calculating biomass of submerse macrophytes, aside the hydro-acoustic data the information about the macrophyte composition along transects is needed. Therefore, a hydro- acoustic run might be accompanied by a botani- cal survey of the macrophyte beds in the fi eld. Processing the data in the lab, the signal of hy- dro-acoustics needs to be linked to the specifi c species composition at the respective measured area. In an earlier study by Janauer and Wichera (2000), a calibration data set for more than 20 macrophyte yield patterns is developed to pro- vide specifi c yield indices. Such index refers to the ratio of the macrophyte biomass and the water volume covered by the submerse plants for each macrophyte taxon. In a next step of data processing, the recent hydro-acoustic data of Dorfl acke presented in this study, will linked to this calibration index calculating fi nally the species-specifi c biomass. Conclusion Our results show fi rst promising results as graphical mapping provide in principle reliable data for macrophyte canopy and contour plots for water basin morphometry. In a next step a user-friendly access will be developed for digitizing the shoreline, and for a convenient user interface enabling post-process- ing the plant stand volumes, as well as the cal- culation of biomass will be developed. The au- tomatic differentiation of the different readings of solid and muddy sediment, and of the macro- phytes by means of Kalman fi lter as well as an automated detection of ‘macrophytes-to-surface’ areas will be included. Acknowledgement The authors acknowledge the valuable as- sistance of Wolfgang Mayerhofer, President of the Board of the Dorferneuerung Langenlebarn. We thank Dr. Michael Schabuss and Dr. Katrin Teubner for support during the fi eld work and discussing the ms. References Brandt S.B., 1996. Acoustic assessment of fi sh abundance and distribution. In: Murphy B.R.Willis D.W. (Eds.) Fisheries Techniques, 2nd edition, Am. Fish. Soc., 385-432. Dumfarth E., Pall, K., 2003. Die Erfassung der Unterwasservegetation mittels DGPS, Echsonde und Tauchkartierung. ICRA & Systema, Salzburg, www.icra.at/images/folder_makrophyten.pdf 2003 Hohausová, H., J. Kubecka, J., Frouzová J., Husák, S., Balk, H., 2008. Experimental Biomass Asses- sment of Three Species of Freshwater Aquatic Plants by Horizontal Acoustics, Journal Aquatic Plant Manage, 46, 82-88. Jäger, P., Pall, K., Dumfarth, E., 2004. A method of mapping macrophytes in large lakes with regard to the requirements of the Water Framework Directive. Limnologica, 34, 140-146. Janauer, G.A., Wychera, U., 2000. Biodiversity, succession and the functional role of macrophytes in the New Danube (Vienne, Austria). Archive of Hydrobiology, 135, 61-74. 51Exler, Janauer: A hydro-acoustics approach of accessing macrophyte Kohlbauer, R., 2008. Saisonale Biomasseentwicklung von Makrophyten und deren Bezug zur Phyto- planktonentwicklung in den Gewässern der Lobau. MSc-Thesis, Vienna, 171 pp. Komatsu, T., Mikami, A., Sultana, S., Ishada, K. Hiraishi, T., Tatsukawa, K., 2003. Hydro-acoustic methods as a practical tool for cartography of seagrass beds. Otsuchi Marine Science, 28, 72-79. Lotz, J., 2007. Coral Fish Shoal Detection from Acoustic Echograms. Portland State University, NEAR- LAB, (http://nearlab.ece.pdx.edu/news.html). Rakowitz, G., Kubecka, J., Fesl, C., Keckeis, H., 2009. Intercalibration of hydroacoustic and mark– recapture methods for assessing the spawning population size of a threatened fi sh species. Journal of Fish Biology, 75, 1356–1370. Scheffer, M., 2002. Ecology of shallow lakes. In: Population and community series, 22. Kluwer Aca- demic Publisher. The Netherlands. pp 357. ACTA BIOLOGICA SLOVENICA LJUBLJANA 2010 Vol. 53, [t. 2: 53–60 In vitro propagation of Lilium martagon L. var. cattaniae Vis. and evaluation of genotoxic potential of its leaves and bulbs extracts In vitro razmnoževanje in ocena genotoksičnosti izvlečkov listov in čebul taksona Lilium martagon L. var. cattaniae Vis. Glamočlija Una1*, Haverić Sanin1, Čakar Jasmina1, Rahmanović Anisa1, Marjanović Damir1 1Institute for Genetic Engineering and Biotechnology, Gajev trg 4, 71000 Sarajevo, Bosnia and Herzegovina. *correspondence: una_buric@yahoo.com, una.glamoclija@gmail.com Abstract: Lilium martagon L. var. cattaniae Vis. (Liliaceae) is endemic plant of Dinaridi mountain. In this work we established protocol for fast in vitro propagation and multiplication of Lilium martagon var. cattaniae. The aim was to enable fast production of plant material as potential source of pharmaceutically valuable secondary metabolites. Seeds of L. martagon var. cattaniae were germinated on a Murashige and Skoog basal medium with a supplement of 0.15 mg/l gibberellic acid (GA3), and multiplication was performed on MS medium supplemented with 0.1 mg/l gibberellic acid (GA3), 0.2 mg/l indole-3-butyric acid (IBA) and 0.5 mg/l 6-ben- zylaminopurine (BAP). We used ultrasound assisted extraction to prepare extracts of leaves and bulbs of Lilium martagon var. cattaniae, which were evaluated for their genotoxic potential using Allium test and cytokinesis-block micronucleus test in human lymphocytes culture. There was statistically signifi cant difference between all used concentrations of lilium extracts and control on proliferation of cells of root tip of onion (Allium cepa). In cytokinesis-block micronucleus test no statistically signifi cant difference between frequencies of analyzed parameters in samples treated with tested concentrations of extracts and control was obtained. Keywords: in vitro culture, ultrasound assisted extraction, micronuclei, genotoxic, Lilium martagon L. var. cattaniae Vis. Izvleček: Lilium martagon L. var. cattaniae Vis. (Liliaceae) je endemični takson Dinaridov. Članek podaja načrt za hitro in vitro propagacijo in multiplikacijo taksona Lilium martagon L. var. cattaniae. Cilj je bil omogočiti hitro propagacijo rastlinskega materiala, ki je potencialni izvor metabolitov, pomembnih za farmacijo. Semena taksona Lilium martagon var. cattaniae smo kalili na Murashige in Skoog (MS) hranljivi podlagi, kateri smo dodali 0,15 mg/l giberelinske kisline (GA3). Multiplikacija je prav tako potekala na MS podlagi, ki smo ji dodali 0,1 mg/l gib- erelinske kisline (GA3), 0,2 mg/l indol-3-maslene kisline (IBA) in 0,5 mg/l 6-benzilaminopurina (BAP). Za pripravo izvlečkov listov in korenin taksona Lilium martagon var. cattaniae smo uporabili ultrazvočno ekstrakcijo. Za ugotavljanje genotoksičnega potenciala teh izvlečkov je bil uporabljen Allium test in citokinetski blok mikronukleus test na humanih limfocitih. Različne koncentracije izvlečkov lilij so imele značilen vpliv na proliferacijo celic korenin čebule (Allium cepa) glede na kontrolo. Pri citokinetskem blok mikronukleus testu ni bilo značilnih razlik med frekvencami analiziranih parametrov pri vzorcih obravnavanih z različnimi koncentracijami izvlečkov in kontrolo. Ključne besede: in vitro kultura, ultrazvočna ekstrakcija, mikronukleusi, genotoksičnost, Lilium martagon L. var. cattaniae Vis. 54 Acta Biologica Slovenica, 53 (2), 2010 Introduction Lilium martagon L. var. cattaniae Vis. Lil- iaceae is an endemic plant species of Bosnia and Herzegovina (Šilić 2007). According to Flora Europaea, (Matthews 1980) Turk’s-cap lilies in Europe are represented by: L. martagon L., L. chalcedonicum L., L. pomponium L., L. pyrenaicum Gouan. and L. carniolicum Bernh. ex Koch. In addition there are also endemic European taxa with unclear taxonomic status: L. albanicum Griseb., L. bosniacum (G. Beck) Beck ex Fritsch and L. jankae A. Kerner from the L. carniolicum complex, and L. cattaniae (Vis.) Vis. from the L. martagon complex (Mat- thews 1980). This species is named after Maria Cattani Selebam who showed differences between Lil- ium cattaniae Vis. (Vis.) and Lilium martagon L. to R. Visiani so he published in year 1872 that it is new species (Šilić 2007). This species is used as medicinal plant in Mediterranean area. It represents an important resource both for phyto- chemical and pharmacological research (Redžić 2010). Bulbs of its closest species Lilium mar- tagon L. posses cardiotonic properties and are used in the treatment of dysmenorrhoea (Khare 2007), liver diseases in both humans and animals in Northern Albania (Pieroni et al. 2005). Bulbs of Lilium martagon L. are used externaly for ul- cers (Khare 2007). There are studies confi rming presence of anticarcinogenic components, such as jatropham which is antileucemic agent, in Lil- ium martagon L. The presence of kaempferol, quercetin and isorhamnetin was identifi ed in L. martagon (Eisenreichová et al. 2004). HPLC analysis should be done to characterise crude leaf and bulb extracts of Lilium martagon L. var. cattaniae Vis. for major secondary metabolites. Culture in vitro is revolutionary method- ology useful in development of synthesis and accumulation of natural products and possible method for modifi cation of products (Reming- ton 2005). This is especially important when endemic and rare plants are used as a source of medically active substances. Bulbous plants, like lilies, have proved to be ideal for tissue culture, as their regeneration potential is usually high. Tissue culture has been applied to the propaga- tion of lilies since the late 1950’s. Nowadays, lil- ies are one of the most important bulbous crops produced in tissue culture also in an industrial scale. The advantage of this method is that it can ultimately provide a continuous, reliable source of natural products (Pelkonen 2005). When preparing herbal extracts, method of extraction plays very important role. In his- tory many different methods of extraction have been developed. Non-conventional extraction techniques have gained more attention recently, and one of these techniques is ultrasonically as- sisted extraction. In the area of inter-phase mass transfer, solid-liquid extractron appears to be most greatly enhanced by the application of ul- trasonic waves. The mechanism believed to be primarily responsible for the larger increases is the cell disruption brought about by cavitation. Cavitation can result when high-intensity acous- tic waves are passed through liquids producing small bubbles in the liquid. On collapse, the contents of the bubbles are compressed to very high temperatures and are capable of producing shock waves (Chendke et al. 1975). Ultrasonic extraction is simple, low cost in terms of solvent used and less time consumed. This method promotes better penetration of sol- vent into plant particles and uses low extraction temperature which affects the stability of active components (Rouhani et al. 2009). Ultrasonic processing is still in its infancy and requires a great deal of future research (Dolatowski et al. 2007). It is very important that herbal extracts used in traditional medicine, are not toxic, or that its toxicity is under defi ned limits. Remarkable as- pect of toxicity is genotoxicity. Numerous tests can be used in studying genotoxicity. In this study we used Allium test and cytokinesis-block micronucleus test in human lymphocytes culture, to evaluate genotoxicity potential of L. martagon var. cattaniae leaves and bulbs extracts. Materials and methods Plant material Seeds of Lilium martagon L. var. cattaniae Vis. used in this study as starting material were provided by dr.sci. Edina Muratovic. Seeds were collected on Borova Glava, 1100 m, Bosnia and 55Glamočlija et al.: Genotoxicity of Lilium martagon var cattaniae Herzegovina. Voucher specimens are deposit at Department of Biology, Faculty of Science, Uni- versity in Sarajevo. In vitro culture of Lilium martagon L. var. cattaniae Vis. For L. martagon var. cattaniae seed germi- nation, commercial MS (Murashige and Skoog, 1962) medium (Duchefa, Netherlands) was used. Medium pH was adjusted to 6.5. Seeds were sterilized and germinated according to pro- tocol given by Parić et al. 2008. Five to six cm long lilies explants were cut into smaller which contained 1-3 bulbs. Explants were inoculated on MS medium supplemented with 0.1mg/l GA3, 0.2 mg/l IBA (indole butiric acid) and 0.5 mg/l BAP (benzyl amino purine). Explants were cultivated for three weeks, subcultivated for three times and then collected for extraction. Extraction Bulbs and leaves were separated and plant material was dried in a fl ow of hot air, chopped and extracted with water. First, plant material was soaked in distilled water (1:20) for 2 hours. After that mixture was put in ultrasound bath (Iskra UZ4R) for 25 minutes. The mixture was put in a dark place for 22 hours with frequent shaking, fi ltrated and vacuum dried on tempera- ture 30 ºC and pressure 50 mbar. Dried extracts were held on room temperature above silica gel until they were used. Testing of genotoxic potential Allium test Fresh, healthy, equal-sized bul bs of a com- mercial variety of Allium cepa L. were selected. Just before use, the outer scales of the bulbs were carefully removed and the brownish bot- tom plates were scraped away without destroy- ing the root primordia. Four concentrations (0.1mg/ml, 0.5mg/ml, 1mg/ml and 5mg/ml) of dried extracts of leaves and bulbs were used in the experiment. For every concentration and for negative control two bulbs were taken. They were put in water on room temperature and in shadow for 48 hours. After that, bulbs were put in extracts for 24 hours (control bulbs were left in water). Then root tips were fi xated in ice-cold acetic acid: ethanol 1:3 on 4 ºC for few hours. Root tips were hydrolyzed with 1 N HCl at 60 ºC for 7 min and after that washed in distilled water. For every concentration two slides were prepared. Three root tips were taken for every slide, the meristematic cell region was removed by cutting 2 mm from the root cap, this section was set on a clean slide, macerated with metal stick, immersed in drop of 1% lacto-propion or- cein and squashed under a cover glass, excess color was removed with fi lter paper and after that, borders of cover glass were paraffi nated. Slides were observed under 40× magnifi cation. On every slide 1000 cells were analyzed for fre- quency of cells in interphase, prophase, meth- aphase, anaphase and telophase. Mitotic index (MI) was calculated as percentage of mitotic cells in all analyzed cells. Cytokinesis-block micronucleus test According to results of Allium test two con- centrations of extracts (0,1 mg/ml and 1 mg/ ml) were chosen for micronucleus test in human lymphocytes. Micronucleus test was performed on blood samples from four persons. Donors of blood were not on therapy with medicines and did not suffer any chronic disease in past 6 months, they were not smokers and age was between 24 and 27 years. Two donors were fe- male and two donors were male. The study was conducted in accordance to ethical principals. Cytokinesis-block micronucleus assay was performed according to protocol and scoring criteria given by Fenech and coworkers (2003) and Fenech (2000). Blood was cultivated for 72 hours on RPMI medium. After 24 hours of cul- tivation extracts were added. After 45 hours of cultivation citohalazin B was added (4.5 μg/ml). Estimation of lymphocytes proliferation was done by calculating nuclear division index (NDI) according to Eastmond and Tucker (1989). The calculation was made according a formula: NDI= [M1 + 2(M2) + 3(M3) + 4(M4)]/N, where M1-M4 represent cells with one to four nucleus, N is total number of viable cells analyzed. Statistical analysis Z-test and ANOVA followed by pair-wise comparisons with Newmans-Keuls Multiple 56 Acta Biologica Slovenica, 53 (2), 2010 comparison test were calculated for statistical analysis, using Winks 4.5 Professional software (TexaSoft, Cedar Hill, TX, USA). Level of sig- nifi cance was p≤0.05. Results and discussion In vitro propagation and multiplication of Lilium martagon L. var. cattaniae Vis. In vitro cultivation is very important teh- nique for production of signifi cant natural prod- ucts in pharmacy. One of the aims is to produce big amount of plant material in the shortest time. If the starting material is seed, germination is a very important factor, and it can be improved us- ing appropriate protocol for in vitro germination. Seeds of Lilium species generally have deep dor- mancy. Removing of seed coats and cutting seeds allowed germination, showing that dormancy of L. bosniacum and L. martagon var. cattaniae was induced by the presence of the testa (Parić et al. 2008). On different species of Lillium sp. it is determined that removal of seed testa in- creases germination. It is assumed that, besides it is physical barrier, testa contains physiological system that maintains dormancy of seed (Pelko- nen 2005). In this work sterilized seeds were ger- minated on MS medium with GA3. After 21 day of incubation all seeds germinated. Optimal combination and concentrations of growth regulators are necessary to achieve wanted processes on cellular level and wanted growth of the whole plant. There are many works about which combinations of GA3, BAP and IBA are good for growth and development of plants in vitro. For example Petrović and Jačimović-Plavšic (1992) proved that the best development and propagation of axillary buds of Aronia melanocarpa Elliot was achieved on MS medium supplemented with 0.1 mg/l GA3, 0.1 mg/l IBA and 0.5 mg/l BAP. In this work thirty days after germination plants were transferred to MS medium containing of GA3, IBA and BAP. Three multiplications lasting 20 days were per- formed and 86 g of fresh material was obtained. Selected method of extraction signifi cantly Figure 1: Mitotic index of cells of root tip of onion after treatment with extracts of L. martagon var. cattaniae and control. The data represent the means ± SD. Slika 1: Mitotski indeks celic korenin čebule po obravnavanju z izvlečki taksona L. martagon var. cattaniae v primerjavi s kontrolo. Rezultati so predstavljeni kot povprečje ± SD. Allium test mitotic index -2,00 0,00 2,00 4,00 6,00 8,00 10,00 12,00 14,00 0.0 0.1 0.5 1.0 5.0 Concentration of extract (mg/ml) M ito tic in de x (% ) control leaf extract bulb extract 57Glamočlija et al.: Genotoxicity of Lilium martagon var cattaniae Figure 2: Frequency of MN on 1000 BN cells after treatment with extracts of L. martagon var. cattaniae and control. The data represent the means ± SD. Slika 2: Frekvenca MN na 1000 BN celic po obravnavanju z izvlečki taksona L. martagon var. cattaniae v primerjavi z kontrolo. Rezultati so prikazani kot povprečje ± SD. Freqency of MN on 1000 BN cells for bulb and leaf extract 0,00 2,00 4,00 6,00 8,00 10,00 12,00 control 0.1 1.0 Concentration of extract (mg/ml) Fr eq en cy o f M N o n 10 00 B N c el ls control leaf extract bulb extract Figure 3: Nuclear division index after treatment with extracts of L. martagon var. cattaniae and control. The data represent the means ± SD. Slika 3: Indeks delitve nukleusa po obravnavanju z izvlečki taksona L. martagon var. cattaniae v primerjavi z kontrolo. Rezultati so predstavljeni kot povprečje ± SD. NDI for bulb and leaf extract 0,00 0,20 0,40 0,60 0,80 1,00 1,20 1,40 1,60 1,80 2,00 control 0.1 1.0 Concentration of extract (mg/ml) ND I ( nu cl ea r di vi si on in de x) control leaf extract bulb extract 58 Acta Biologica Slovenica, 53 (2), 2010 affects composition of obtained extracts. Ultra- sound assisted extraction attracts more attention recently because of many benefi ts, primarily better penetration of solvent into plant particles and low extraction temperature which affects the stability of active components (Rouhani et al. 2009). Weng and coworkers (2004) were doing ex- traction of loganin in wine on room temperature and they achieved concentration of 48 mg/l after 30 days, while using ultrasound assisted extrac- tion the concentration of 50 mg/l was achieved after 2 days. In this work total 9.85g of dried material was obtained (4.88g from leaves and 4.97g from bulbs). After extraction, 1.54 g of leaf powder and 1.12 g of bulbs powder was obtained. Allium test Four concentrations of extracts (0.1 mg/ml, 0.5 mg/ml, 1 mg/ml and 5 mg/ml) were exam- ined. Mitotic index (MI), presenting percentage of cells in mitosis in total number of analyzed cells, was determined (Figure 1) It was established that there were statistical- ly signifi cant differences between mitotic index in control and all tested samples. In vitro micronucleus test on human lym- phocytes Binucleated cells (BN) with one and two micronuclei (MN) were observed. Frequency of MN on 1000 BN cells are presented in Figure 2. Statistical evaluation of data using Z-test has shown that there is no statistically sig- nifi cant difference in frequency of micronuclei (MN) on 1000 binucleated (BN) cells among control and examined concentrations of leaf and bulb extracts. Results of calculating NDI for controls and treated cultures are presented in Figure 3. All extracts induced reduction of NDI comparing to control, except leaf extract with concentration 0.1 mg/ml which in two blood samples induced growth of NDI. After statisti- cal evaluation of data using Independent Group Analysis ANOVA test it was determined that there was no statistical signifi cance in NDI val- ues between control and examined concentra- tions of leaf and bulb extracts. Using Newman- Keuls multiple comparison it was determined that there was no signifi cant difference among all examined samples. Conclusions – Sterile germination of Lilium martagon var. cattaniae seed on MS medium with addition of 0.15 mg/l GA3 with removal of testa and border parts of endosperm was successful so in this work 100% germination was achieved. – In this work multiplication was done on MS medium with 0.1mg/l GA3, 0.2mg/l IBA and 0.5mg/l BAP and satisfying results were achieved. – Results of ultrasound assisted extraction were very good because from 4.88g of dried leaf 1.536g of dried extract was achieved, and from 4.97g of dried bulbs 1.117g of dried extract was achieved. – There was statistically signifi cant difference between all used concentrations of lilium extracts and negative control (water) on proliferation of cells of root tip of onion (Allium cepa). Leaf extract of 0.1 mg/ml signifi cantly increased proliferation was signifi cantly decreased. All concentrations of leaf extract signifi cantly decreased pro- liferation, and effect was bigger with higher concentrations. – Results of proliferation in micronucleus test on human lymphocytes are similar to those in allium test, but in this case there is no statistically signifi cant difference be- tween used extracts and control. To explain mechanisms with which extracts change proliferation it would be necessary to do chemical analysis of extracts and do some more investigations. – Used extracts didn’t show genotoxic properties under experimental conditions. Povzetek Lilium martagon L. var. cattaniae Vis. (Liliaceae) je endemični takson Dinaridov 59U. Glamočlija idr: Genotoxicity of Lilium martagon var cattaniae grown in vitro (Šilić 2007). Čebula se največ uporablja v ljudski medicini na območju Mediterana. Pomembna je za fi tokemijske in farmakološke raziskave. Čebula vrste Lilium martagon L. ima kardiotonične lastnosti in se uporablja pri obravnavanjih dismenoreje (Khare 2007) in za zdravljenje jetrenih bolezni pri ljudeh in živalih v Severni Albaniji (Pieroni et al. 2005). Čebula vrste Lilium martagon se uporablja za zunanje rane (Khare 2007), nekatere raziskave pa so pokazale prisotnost antikancerogenih kompo- nent (Eisenreichová et al. 2004). In vitro kultura se uporablja za masovno produkcijo in tako za ohranjanje endemičnih sort lilij. Kaljenje semen taksona L. martagon var. cattaniae je poteka- lo na MS hranljivi podlagi, kateri 0,15 mg/l giberelinske kisline (GA3). Kalitev je bila 100%. Za multiplikacijo smo uporabili MS podlago ter dodali 0,1 mg/l giberelinske kisline (GA3), 0,2 mg/l indol-3-maslene kisline (IBA) ter 0,5 mg/l 6-benzilaminopurina (BAP). Po treh ted- nih kultivacije smo rastline posušili in mate- rial uporabili za ekstracijo. Uporabljena je bila ultrazvočna ekstrakcija. Rezultati ultrazvočne ekstrakcije so bili odlični, saj smo iz 4,85 g suhih listov dobili 1,54 g suhega izvlečka, iz 4,9 g suhe čebule pa 1,12 g suhega izvlečka. Za ugo- tavljanje genotoksičnega potenciala izvlečkov lista in čebul taksona L. martagon var. cattaniae smo uporabili Allium test in citokinetski-blok mikronukleus test na humanih limfocitih in vitro. V primerjavi s kontrolo so vse koncentrac- ije izvlečkov lilij statistično značilno vplivale na proliferacijo celic korenin čebule (Allium cepa). Izvleček listov v koncentraciji 0,1 mg/ ml je značilno povečal proliferacijo, medtem ko so imele vse druge koncentracije negativne vplive. Učinek se je povečeval z višjanjem kon- centracije. Izračun NDI (nucelar division index) mikronukleus testa ni pokazal značilnih razlik. Acknowledgements This work was supported by Institute for Genetic Engineering and Biotechnology, Sara- jevo. We are thankful to dr. sci. Edina Muratovic for providing seeds of Lilium martagon L. var. cattaniae Vis. used in this study as starting ma- terial. Literature Chendke, P.K., Fogler, H.S., 1975. Macrosonics in industry: 4. Chemical processing. Ultrasonics, 13(1), 31-37. Dolatowski, Z.J., Stadnik, J., Stasiak, D., 2007. Applications of ultrasound in food technology. Acta Sci. Pol. Technol. Aliment., 6(3), 89-99. Eastmond, D.A., Tucker, J.D., 1989. Identifi cation of aneuplody-inducing agents using cytokinesis- blocked human lymphocytes and an antikinetochore antibody. Environ. Mol. Mutagen., 13, 34-43. Eisenreichová, E., Haladová, M., Mučaji, P., Grančai, D., 2004. The study of constituents of Lilium candidum L. Acta Facultatis Pharmaceuticae Comenianae, Department of Pharmacognosy and Botany, Faculty of Pharmacy,Comenius University, Bratislava, pp. 11. Fenech, M., Chang, W.P., Kirsch-Volders, M., Holland, N., Bonassi, S., Zeiger, E., 2003. HUMN project: detailed description of the scoring criteria for the cytokinesis-block micronucleus assay using isolated human lymphocyte cultures. Mutat. Res., 534, 65–75. Fenech, M., 2000. The in vitro micronucleus technique. Mutat. Res., 455, 81-95. Khare, C.P., 2007. Indian Medicinal Plants: An Illustrated Dictionary. Springer, pp. 374. Matthews, V., 1980. Lilium L. In: Tutin T. G., Heywood V.H., Burges N.A., Moore D.M., Valentine D.H., Walters S.M., Webb D.A. (eds.), Flora Europaea, Cambridge University Press., pp. 5-35. Murashige T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiologia Plantarum, 15, 473-479. Parić, A., Hindija, J., Muratović, E., Pojskić, N., Bajrović, K., 2008. Breaking dormancy of two en- demic Lilium species: Lilium bosniacum (G. Beck) Beck ex Fritsch and Lilium martagon L. var. cattaniae Vis. Seed science and technology, 36(3), 788-791. Pelkonen, V.P., 2005. Biotechnological approaches in lily (Lilium) production, Faculty of Science, Department of Biology, University of Oulu, Finland. Petrovic, D.M., Jacimovic-Plavšic, M.M. 1992: Aronia melancarpa and propagation in vitro. In: Da- miano, C., Read, P.E., Preece, J.E., Ladyman, J.A.R., Debergh, P. (eds.): ISHS Acta Horticulturae 300: In Vitro Culture, XXIII IHC. Firenze, pp. 133-136. Pieroni, A., Dibra, B., Grishaj, G., Grishaj, I., Maçai, S.G., 2005. Traditional phytotherapy of the Albanians of Lepushe, Northern Albanians Alps. Fitoterapia, 76, 379-399. Redžić, S., 2010. Wild medicinal plants and their usage in traditional human therapy (Southern Bosnia and Herzegovina, W. Balkan). Journal of Medicinal Plants Research 4(11), 1003-1027. Remington: The Science and Practice of Pharmacy. 2005: 21st ed. Lippincott Williams & Wilkins, Philadelphia, pp. 985-987. Rouhani, S., Alizadeh, N., Salimi, S., Haji-Ghasemi, T., 2009. Ultrasonic Assisted Extraction of Natural Pigments from Rhizomes of Curcuma Longa L. J.Prog. Color, Colorants, Coatings. PCCC-J-09-0119. Šilić, Č. 1990: Endemične biljke. 3rd edition. Svjetlost, Sarajevo. Weng, Y.M., Chuang, Y.C., Chen, W.L., Tseng, C.Y.. 2004. Ingredient extracting effi ciency and fun- ctional properties of Chinese medicinal herbal wines as affected by ultrasound-assisted extraction. 2004 IFT Annual Meeting and Food Expo. Las Vegas, Nevada 114F-20. 60 Acta Biologica Slovenica, 53 (2), 2010 ACTA BIOLOGICA SLOVENICA LJUBLJANA 2010 Vol. 53, [t. 2: 61–70 Respiration and ingestion rate of different sized Daphnia pulex fed on four algal species Dihanje in prehranjevanje različno velikih osebkov vrste Daphnia pulex s štirimi vrstami alg Tatjana Simčič National Institute of Biology, Večna pot 111, SI-1000 Ljubljana, Slovenia *correspondence: tatjana.simcic@nib.si Abstract: Respiration rate and ingestion rate for four different algal species (Scenedesmus quadricauda, Asterionella formosa, Aphanizomenon fl os-aquae and Planktotrix rubescens) of different sized Daphnia pulex were measured in the laboratory. Population of D. pulex grew maximally when it fed S. quadricauda, but the presence of P. rubescens and A. fl os-aquae caused negative population growth rate. Ingestion rates increased with increasing body size for all in- vestigated algae; the lowest b value was obtained for S. quadricauda and the highest one for P. rubescens. The amount of ingested carbon exceeded the required amount for standard metabolism in both small and large sized individuals fed all four algal species. Relatively higher amount of ingested A. fl os-aquae and P. rubescens in comparison with A. formosa and S. quadricauda and the results of the growth experiments indicate that the inhibitory effect of fi lamentous blue-green algae on D. pulex is more due to toxicity, low assimilation effi ciency or/and inadequate composi- tion than incapability of ingestion due to mechanical interference with fi laments. Key words: ingestion rate, respiration, Daphnia pulex, algae, growth scope Izvleček: V laboratoriju smo pri različno velikih osebkih Daphnia pulex merili dihanje in stopnjo hranjenja s štirimi različnimi vrstami alg (Scenedesmus quadricauda, Asterionella formosa, Aphanizomenon fl os-aquae and Planktotrix rubescens). Populacija D. pulex je najbolje uspevala pri hranjenju z algo S. quadricauda, v prisotnosti vrst A. fl os-aquae in P. rubescens pa smo opazili negativno rast populacije. Stopnja hranjenja se je povečevala z naraščajočo telesno velikostjo pri vseh vrstah alg; najnižjo vrednost b smo dobili pri hranjenju s S. quadricauda, najvišjo pa s P. rubescens. Količina zaužitega ogljika je presegala porabo za standardni metabo- lizem pri hranjenju z vsemi štirimi vrstami alg. Večje količine zaužitih vrst A. fl os-aquae in P. rubescens v primerjavi z vrstama A. formosa in S. quadricauda in rezultati rastnih poskusov kažejo, da je inhibitorni vpliv nitastih modro-zelenih alg na osebke vrste D. pulex bolj posledica strupenosti, nizke asimilacijske učinkovitosti ali/in neustrezne sestave kot pa nezmožnosti zaužitja zaradi težav, ki bi jih povzročala nitasta oblika alg. Ključne besede: stopnja hranjenja, dihanje, Daphnia pulex, alge, obseg rasti Introduction Herbivorous zooplankton is functionally important in aquatic webs. They constitute a link between primary producers and higher trophic levels. A number of studies have inves- tigated the effect of food quality (e.g. Knisley and Geller 1986, Fulton III 1988, Butler et al. 1989, Hawkins and Lampert 1989, Gulati and DeMott 1997, Kilham et al. 1997, Wagner and 62 Acta Biologica Slovenica, 53 (2), 2010 son 1983, Trabeau et al. 2004) where the effect of different blue-greens on respiration rate of adult stages of Daphnia in the presence of food was measured. However, information about the capability of different sized Daphnia to ingest enough food to meet their carbon demands, re- quired for standard metabolism, is still lacking. Such studies are important to obtain the basic information on growth scope of different sized animals. The aim of the present study was to deter- mine an ingestion rate of different sized individ- uals of D. pulex that were fed on four different algal species, i.e. Scenedesmus quadricauda, Asterionella formosa, Aphanizomenon fl os-aq- uae and Planktotrix rubescens. Respiration rates were measured in different sized developmental stages in order to estimate carbon demands for standard metabolism. It was hypothesized that ingestion rates of different algal species and res- piration rates differ in different sized animals. Growth experiments were carried out to test nutritional value of a single alga and possible toxicity of blue-green algae used in the experi- ments. Material and methods Cultures Daphnia pulex Leydig originated from a permanent laboratory culture in National Insti- tute of Biology (Ljubljana, Slovenia). Animals were kept in 10 L aquaria and some hundred specimens were there all the time. The water temperature was 24.0 ± 1.5 °C. The animals were fed every second day with suspension of Scenedesmus sp. and yeast. For feeding experi- ments adult females without eggs were selected, but for growth experiments ovigerous females of similar size were selected. Single animal were transferred using a narrow glass pipette. Algal cultures of Scenedesmus quadricau- da, Asterionella formosa, Aphanizomenon fl os- aquae and Planktotrix rubescens were obtained from the National Institute of Biology collec- tion (Ljubljana, Slovenia). Algae were cultured in Jaworski medium. Algal cultures were main- tained in log growth phase. Algal characteris- Kamjunke 2001) and food quantity (e.g. Porter et al. 1982, Urabe and Watanabe 1991) on feed- ing and/or on growth, survival and reproduction of Daphnia. As eutrophication often results in a prolif- eration of blue-green algae (Arnold 1971), the blue-green algae had been studied as food for Daphnia in several studies (e.g. Arnold 1971, Gliwicz 1977, Richman and Dodson 1983, Ful- ton III 1988, Gilbert and Durand 1990, Gliwicz 1990, DeMott 1999, Trabeau et al. 2004). Blue- greens are usually found to be an inadequate food for Daphnia due to mechanical interference of colonies of fi laments with food collection, low digestibility or poor nutritive quality. Many gen- era of blue-greens produce either hepatotoxic or neurotoxic secondary metabolites (Trabeau et al. 2004). In the recent years, food-quality research has increasingly focused on the biochemical nutrient requirements of Daphnia. It has been shown that fatty acid and phosphorous content of food affect the growth and reproduction of Daphnia (Sundbom and Vrede 1997, Park et al. 2002, Ferrão-Filho et al. 2003, Gladyshev et al. 2008, Martin-Creuzburg and Von Elert 2009). Daphnia is fi lter feeders, having appendages specialized for respiration and food gathering. Food is rejected when the collected amount is greater than it can be ingested, when it is physi- cally unacceptable (i.e., colonies or fi laments too large) or if it is chemically unacceptable (see Lampert 1987). The dependence of fi ltering or ingestion rate (IR) on the body length (L) of Daphnia can be described by power equation of the form IR= a Lb (Lampert 1987). One impor- tant factor infl uencing b is the size of the food. Although large particles can be better handled by large daphnids than by small ones (Lampert 1987), some studies showed that the feeding of larger Daphnia species and larger individuals of single species is more affected by the presence of fi laments than the feeding of smaller ones (Hawkins and Lampert 1989, Gilbert and Du- rand 1990). As fi lamentous and colonial algae are differently consumed by different body sized Daphnia (Hawkins and Lampert 1989), differ- ent ingestion of food particles in juveniles and adults was expected. The effect of food quality on both feeding and respiration of Daphnia were investigated in few studies (Richman and Dod- 63Simčič: Respiration and ingestion rate of Daphnia pulex tics are given in Table 1. Cells were counted and measured by Soft Imaging System, GmbH, analySIS 3.0, Münster, Germany. Suspension of a single alga was fi ltered through pre-weighted fi lter (glass microfi brile fi lter Whatman GF/C) and dried for 24 h at 60°C. Filters were weight- ed on 10 μg electrobalance (Sartorius). Dry weight of single cell or fi lament was calculated from the concentration and volume of fi ltered suspension. Population growth experiments Fifteen ovigerous females of similar size (2.20 ± 0.23 mm; average eggs number per female was 2.2) were placed in bottles, each containing 600 mL of synthetic medium (ISO standard) with algal concentration of 1*104 cells (or fi laments in the case of blue-green algae) per mL. These animals were collected from the same container, in order to assure that they had similar age and number of eggs. Three replicate bottles for each alga as food were started at the same time to avoid other factors that might af- fect on experimental conditions. Experimental bottles were kept at 25°C. Before feeding half of water was changed every second day. Relatively constant food level was kept during the experi- ments. Population growth experiments lasted for 14 days. At the end, animals were killed in for- malin solution. Body length was measured from the top of the helmet to the base of the spine us- ing Soft Imaging System, GmbH, analySIS 3.0, Münster, Germany. Population growth rates (r) were estimated as: r = (lnN2-lnN1)/(t2-t1), with N1 and N2 being the population sizes of sampling days t1 and t2. Feeding experiments At the beginning of the experiments, ani- mals were piped into tubes fi lled with 2 mL suspensions of algae. One animal was placed in each test tube. The initial algal cell concentra- tion was 1 * 104 cells mL-1. Animals were fed for 3 hours. Animals and algae were then killed with formalin solution. In each tube, the inges- tion rate (IR) were determined as IR = (c0 - c1) * V / t where c is the concentration of algae at the beginning (c0) and the end (c1) of the feeding time (t), and V is volume of suspension (2 mL). Animals were starved for 3 hours be- fore being used in feeding experiments. Respiration rate was estimated by the closed bottle method (Lampert 1984). 150 mL ground glass stoppered bottles were fi lled with synthetic medium and aerated water from the same, well- mixed, container. Ten bottles received animals (50 similar sized animals were placed in a single bottle), while three bottles served as fi nal con- Width of particles (μm) Length of particles (μm) Max dimension of colony (μm) Dry weight (μg cell -1) Aphanizomenon fl os-aquae 3.54 ± 0.73 (25) 42.47 ± 25.90 (64) - 5.33 * 10-4 Planktotrix rubescens 4.96 ± 0.53 (31) 103.96 ± 52.75 (31) - 1.07 * 10-3 Asterionella formosa 3.98 ± 0.67 (37) 46.96 ± 14.4 (43) 88.45 ± 25.1 (20) 2.36 * 10-4 Scenedesmus quadricauda 5.17 ± 0.59 (20) 8.70 ± 1.1 (20) 12.6 ± 3.51 (20) 6.83 * 10-5 Table 1: Morphological characteristics of algae used in experiments. Mean ± SD (n -number measured). Tabela 1: Morfološke značilnosti alg, uporabljenih v poskusih. Povprečje ± SD (n - število meritev). 64 Acta Biologica Slovenica, 53 (2), 2010 Mean ± SD Aphanizomenon fl os aquae -0.481 ± 0.052 Planktotrix rubescens -0.040 ± 0.021 Asterionella formosa 0.006 ± 0.001 Scenedesmus quadricauda 0.115 ± 0.010 Table 2: Population growth rates (d-1) for population of Daphnia pulex fed different species of algae (n = 3). Tabela 2: Stopnja rasti populacij (d-1) pri vrsti Daphnia pulex pri hranjenju z različnimi vrstami alg (n = 3). Figure 1: a) Number of individuals and b) percentage of populations for different size classes (mm) of Daphnia pulex fed Planktotrix rubescens (PLANK), Asterionella formosa (ASTER) and Scenedesmus quadricauda (SCEN). Slika 1: a) Število osebkov in b) odstotki populacij po velikostnih razredih (mm) pri vrsti Daphnia pulex, ki se je prehranjevala s Planktotrix rubescens (PLANK), Asterionella formosa (ASTER) in Scenedesmus quadricauda (SCEN). trols. All bottles were kept at 25°C. After 24 h the concentration of dissolved oxygen in the ex- perimental and control bottles was measured by polarographic oxygen electrode (OXI 96, WTW). The difference between the oxygen of each ex- perimental as well as mean oxygen concentration of control bottles was taken as the amount of oxy- gen consumed by animals. The amount of oxygen consumed was then converted to respiration rate per individum (μL O2 /ind/h). Respiratory carbon loss was calculated using the conversion factor 1 mL O2 = 0.5 mg Corg (Lampert 1984). In con- 65Simčič: Respiration and ingestion rate of Daphnia pulex verting respiration to carbon units, a respiratory quotient (RQ) of 1.0 was assumed. Growth scope was calculated as a subtraction of respiratory car- bon loss (Cres) from ingested carbon (Cing). Statistical analyses One-way analysis of variance (ANOVA) on population growth rates was performed to test differences between different algae. Linear regressions between body length and ingestion rate, and between body length and respiration rate were calculated using Microsoft Excel. Results and discussion Growth experiments Population growth experiments showed that population growth rates differed between food sources (ANOVA, p<0.001). Population of D. pulex grew maximally when it fed S. quadri- cauda (Table 2). Populations that fed A. formosa had positive growth rates, but the presence of P. rubescens caused negative population growth rate. Struc- ture of populations revealed that all individuals of populations that fed P. rubescens belonged to two the smallest size classes (Figure 1). Popu- lations that fed A. formosa had larger individu- als in comparison with P. rubescens as a food source, but the largest animals were observed in diet with S. quadricauda. Feeding on A. fl os- aquae resulted in the collapse of populations during six days. These results are in accord with those previ- ous studies where blue-greens are considered as poor-quality food for Daphnia, due to the inter- ference of the fi laments with the collection of Figure 2: Relationships between ingestion rates and body length of Daphnia pulex fed Aphanizomenon fl os-aquae (APHAN), Planktotrix rubescens (PLANK), Asterionella formosa (ASTER) and Scenedesmus quadri- cauda (SCEN). Slika 2: Razmerje med stopnjo hranjenja in telesno velikostjo osebkov Daphnia pulex pri prehranjevanju s Aphanizomenon fl os-aquae (APHAN), Planktotrix rubescens (PLANK), Asterionella formosa (ASTER) in Scenedesmus quadricauda (SCEN). APHAN 0 1 2 3 4 5 6 7 8 9 -1 -0.5 0 0.5 1 L n in g e s tio n r a te ( N o f il in d -1 h -1 ) Ln IR= 7.17 + 1.36 ln L r=0.81 p<0.001 PLANK 0 1 2 3 4 5 6 7 8 9 -1 -0.5 0 0.5 1 Ln IR= 6.39 + 2.35 ln L r=0.83 p<0.001 ASTER 0 1 2 3 4 5 6 7 8 9 -1 -0.5 0 0.5 1 Ln body length L n in g e s tio n r a te ( N o c e lls in d -1 h -1 ) Ln IR= 7.40 + 1.47 ln L r=0.90 p<0.001 SCEN 0 1 2 3 4 5 6 7 8 9 -1 -0.5 0 0.5 1 Ln body length Ln IR= 7.69 + 1.22 ln L r=0.81 p<0.001 66 Acta Biologica Slovenica, 53 (2), 2010 available food, toxicity, and a low nutritional quality (Arnold 1971, Gliwicz 1977, Richman and Dodson 1983, Fulton III, 1988, Gilbert and Durand 1990, Gliwicz 1990, DeMott 1999, Tra- beau et al. 2004). Arnold (1971) reported that there were differences among the blue-greens in their effects on animals. In the present study A. fl os-aquae showed toxicity towards D. pulex, but P. rubescens probably did not provide suf- fi cient nutrition to maintain a population of D. pulex (Table 2). DeMott (1999) reported that D. pulex exhibited stronger inhibition than D. magna, D. pulicaria, and D. galeata, when it fed a mixture of Scenedesmus acutus and Micro- cystis aeruginosa. Sharp decline in gross growth effi ciency showed on growth inhibition as a re- sult of both feeding inhibition and direct toxicity (DeMott 1999). The results of the present study revealed that S. quadricauda was high quality food for D. pulex as the number and body size of animals exceeded those of animals fed P. rubes- cens or A. formosa (Figure 1). High quality of Scenedesmus sp. was also reported by Hawkins and Lampert (1989) and Vijverberg (1989). Size of particles of this species is convenient to be high quality food for all life stages of the crus- taceans (Vijverberg 1989). As Planktotrix sp. has the lowest assimilation effi ciency among investigated algae and also, Asterionella sp. has lower assimilation effi ciency than Scenedesmus sp. (cited in Lampert 1987), dissimilar growth of populations could be probably partly explained by differences in assimilation rates. Feeding experiments Ingestion rate increased with body size for all investigated algae (Figure 2). Increasing of ingestion rates with increasing body length of cladocerans was also observed in DeMott (1982) and Mourelatos and Lacroix (1990). Regression showed that larger individuals con- sumed signifi cantly more food than smaller ones (p<0.001). The b value ranged from 1.22 for S. quadricauda to 2.35 for P. rubescens. One important factor infl uencing b is the size of the food. Large particles can be better handled by large daphnids than small ones. On the other hand, small daphnids have fi ner fi lters and can retain smaller particles (Lampert 1987). There- fore, low b values which were obtained in the presence of small food particles and high b val- ues for large particles (i.e. P. rubescens) are in accord with expectations. Previous studies showed that ingestion rate depends also on food concentration (DeMott Figure 3: Relationship between respiration rate and mean body length of Daphnia pulex present in single experi- mental bottle. Bars represent ± 1SD. Slika 3: Razmerje med stopnjo dihanja in povprečno velikostjo osebkov Daphnia pulex v posamezni poskusni steklenici. Odkloni predstavljajo ± 1SD. 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5 0 0.5 1 1.5 2 2.5 3 Body length (mm) R e s p ir a ti o n r a te ( µ L O 2 i n d -1 h -1 ) R= -2.29 L1.52 r=0.96, p<0.001 67Simčič: Respiration and ingestion rate of Daphnia pulex 1982, Porter et al. 1982, Gilbert and Durand 1990, Mourelatos and Lacroix 1990). In gen- eral, ingestion rates increased with increasing food concentration until incipient limiting level (ILL) was reached. Above the ILL the inges- tion rate remains constant. Animals control the ingestion rate by rejecting of superfl uous food from the food groove with abdominal claw. The rate of rejection remains constant below the ILL, but it increases at high concentration (Porter et al. 1982). In the present study, both growth and feeding experiments were performed at concen- tration that was considered as ILL (i.e., 1*104 cell mL-1) (Porter et al. 1982). This food con- centration should provide optimal feeding con- ditions for animals. Respiration rate measurements Respiration rates (R) increased with increas- ing body length (L) of D. pulex according re- gression equation: ln R= -2.28 + 1.52 ln L (r= 0.96; p<0.001) (Figure 3). In the present experi- ments, the standard metabolism and expenditure on locomotion were measured. The expenditure on feeding and specifi c dynamic action (SDA) was minimal (Philippova and Postnov 1988) because the animals were not fed just prior to or during the experiments. Therefore, measured respiration rates represent minimal maintenance costs of metabolism in different sized animals. Feeding and processing of the food increase res- piration rates (Philippova and Postnov 1988) so higher respiratory carbon demands can be ex- pected in the presence of food. Relation between respiratory carbon demands and ingested amount of food The amount of ingested carbon exceeded the amount of that required for standard metabolism and locomotion in both small and large sized individuals for all four algal species (Figure 4). Therefore, animals of all sizes were capable of consumption suffi cient amount of S. quadricau- da and A. formosa as well as fi lamenous algae A. Figure 4: Relationship between the growth scope and body length of Daphnia pulex fed Aphanizomenon fl os- aquae (APHAN), Planktotrix rubescens (PLANK), Asterionella formosa (ASTER) and Scenedesmus quadricauda (SCEN). Slika 4: Razmerje med obsegom rasti in velikostjo osebkov Daphnia pulex pri prehranjevanju s Aphanizomenon fl os-aquae (APHAN), Planktotrix rubescens (PLANK), Asterionella formosa (ASTER) in Scenedesmus quadricauda (SCEN). 68 Acta Biologica Slovenica, 53 (2), 2010 fl os-aquae and P. rubescens to meet needs related to minimal metabolic demands. The amount of ingested carbon was the lowest for S. quadricau- da, while the highest values were observed for P. rubescens. The reason is probably different sized algal particles that were offered to animals, as a passive fi ltering of similar number of particles re- sulted in different amount of collected food. The results of the present study indicate that although P. rubescens can be ingested by D. pulex, it cannot provide suffi cient nutri- tion to support a population that does not have other food available. Arnold (1971) also found that some blue-green algae are inadequate food source for D. pulex. Animals can ingest food in large amounts but assimilate it poorly, or allo- cate most of the quantity assimilated to mainte- nance costs. Thus, those animals which fed on low quality food are unable to increase or even maintain the existent population. Conclusions It is concluded that both juvenile and adult D. pulex can ingest relatively large amount of blue-green algae as well as green algae and diatoms. Thus, these results indicate that the inhibitory effect of fi lamentous blue-green al- gae A. fl os-aquae and P. rubescens is more due to toxicity, low assimilation effi ciency or/and inadequate composition than incapability of ingestion due to mechanical interference with fi laments. Also, relatively small amount of in- gested S. quadricauda showed that this alga is high quality food for D. pulex. A. formosa probably should be considered as an adequate, but less qualitative food source in comparison with S. quadricauda. Acknowledgments Author is grateful to Milijan Šiško for his help with the algae and two reviewers for help- ful comments. This study was partly supported by Communities, relations and communica- tions in the ecosystems Research Programme (P1―0255), fi nanced by The Slovenian Re- search Agency. Povzetek Rastlinojedi zooplankton ima v prehran- jevalni mreži ključno vlogo, saj predstav- lja povezavo med primarnimi producenti in višjimi trofi čnimi nivoji. Številni raziskovalci, ki so preučevali vpliv količine in kvalitete hrane na prehranjevanje, rast, preživetje in razmnoževanje osebkov iz rodu Daphnia, poročajo, da so modro-zelene alge zaradi ve- likosti kolonij, slabe prebavljivosti, nizke hranilne vrednost in celo strupenosti pog- osto neustrezna hrana. V večini raziskav so preučevali vpliv hrane na odrasle osebke, manj pa je znanega o prehranjevanju mladičev oz. različno velikih osebkov. Ker je količina zaužite hrane odvisna od velikosti osebkov in velikosti delcev hrane, smo ugotavljali, ali različno ve- liki osebki lahko zaužijejo zadostno količino hrane, ki je potrebna za vzdrževanje osnovnih fi zioloških potreb. Tako smo ugotavljali stopnjo hranjenja pri različno velikih osebkih Daphnia pulex, ki smo jih hranili s štirimi različnimi vr- stami alg (Scenedesmus quadricauda, Asteri- onella formosa, Aphanizomenon fl os-aquae in Planktotrix rubescens). Z merjenjem dihanja pri različno velikih osebkih smo ocenili potrebo po energiji za standardni metabolizem. Hranil- no vrednost in strupenost posamezne vrste alge smo ugotavljali z rastnimi poskusi. Največja je bila rast populacije D. pulex pri hranjenju s S. quadricauda, nato sledi A. formosa. V pris- otnosti P. rubescens smo opazili negativno rast populacije, pri A. fl os-aquae pa je prišlo do njenega propada že v nekaj dneh. Rezultati rastnih poskusov so pokazali strupenost vrste A. fl os-aquae, nizko hranilno vrednost pri vrsti P. rubescens in najvišjo kvaliteto za vrsto S. quad- ricauda. V skladu s pričakovanji se je stopnja hranjenja povečevala z naraščajočo telesno ve- likostjo pri vseh vrstah alg. Najnižjo vrednost b smo dobili pri hranjenju vodnih bolh s S. quad- ricauda, najvišjo pa s P. rubescens. Količina zaužitega ogljika je presegala potrebe po en- ergiji za standardni metabolizem pri hranjenju z vsemi štirimi vrstami alg. Raziskava je poka- zala, da tako mladiči, kot tudi odrasli osebki D. pulex lahko zaužijejo dokaj veliko količino modrozelenih, zelenih in kremenastih alg. To pomeni, da je inhibitorni vpliv nitastih modro- 69Tatjana Simčič: Respiration and ingestion rate of Daphnia pulex zelenih alg vrste A. fl os-aquae in P. rubescens na D. pulex bolj posledica strupenosti, nizke asimi- lacijske učinkovitosti ali/in neustrezne sestave, kot pa nezmožnosti zaužitja zaradi težav, ki bi jih povzročala nitasta oblika alg. Vrsta A. for- mosa se je sicer izkazala kot zadosten vir hrane, a je bila v primerjavi z vrsto S. quadricauda manj hranilna. Literature Arnold, D. E., 1971. Ingestion, assimilation, survival, and reproduction by Daphnia pulex fed seven species of blue-green algae. Limnol. Oceanogr., 16 (6), 906-920. Butler, N. M., Suttle, C. A., Neill, W. E. 1989. Discrimination by freshwater zooplankton between single algal cells differing in nutritional status. Oecologia, 78 (3), 368-372. DeMott, W. R. 1982. Feeding selectivities and relative ingestion rates of Daphnia and Bosmina. Limnol. Oceanogr., 27 (3), 518-527. DeMott, W. R. 1999. Foraging strategies and growth inhibition in fi ve daphnids feeding on mixtures of a toxic cyanobacterium and a green algae. Freshwater Biol., 42 (2), 263-274. Ferrão-Filho, A. S., Fileto, C., Lopes N. P., Arcifa, M. S., 2003. Effect of essential fatty acids and N and P-limited algae on the growth rate of tropical cladocerans. Freshwater Biol., 48 (5), 758-767. Fulton III, R. S. 1988. Grazing on fi lamentous algae by herbivorous zooplankton. Freshwater Biol., 20 (2), 263-271. Gilbert, J. J., Durand, M. W., 1990. Effect of Anabaena fl os-aquae on the abilities of Daphnia and Keratella to feed and reproduce on unicellular algae. Freshwater Biol., 24 (3), 577-596. Gladyshev, M. I., Sushchik, N. N., Dubovskaya, O. P., Makhutova, O. N., Kalachova, G. S., 2008. Growth rate of Daphnia feeding on seston in a Siberian reservoir: the role of essential fatty acid. Aquat. Ecol., 42,617–627. Gliwicz, Z. M., 1977. Food size selection and seasonal succession of fi lter feeding zooplankton in an eutrophic lake. Ekol. Pol., 25(2), 179-225. Gliwicz, Z. M., 1990. Daphnia growth at different concentration of blue-green fi laments. Arch. Hydrobiol., 120 (1), 51-65. Gulati, R. D., DeMott, W. R., 1997. The role of food quality for zooplankton: remarks on the state- of-the-art, perspectives and priorities. Freshwater Biol., 38 (3), 753-768. Hawkins, P., Lampert, W., 1989. The effect of Daphnia body size on fi ltering rate inhibition in the presence of a fi lamentous cyanobacterium. Limnol. Oceanogr., 34 (6), 1084-1089. ISO-standard 6341:1996 (E). Determination of the inhibition of the mobility of Daphnia magna Straus (Cladocera, Crustacea)-Acute toxicity test - Third edition International Organization for Standardization, Geneve, 1996. Kilham, S. S., Kreeger, D. A, Goulden, C. E., Lynn, S. G., 1997. Effects of algal food quality on fecundity and population growth rates of Daphnia. Freshwater Biol., 38 (3), 639-647. Knisely, K., Geller, W., 1986. Selective feeding of four zooplankton species on natural lake phyto- plankton. Oecologia (Berlin), 69 (1), 86-94. Lampert, W., 1984. The measurement of respiration. In: Downing, J. A., Rigler, F. H. (eds), A manual on methods for the assessment of secondary productivity in fresh water. IPB Handbook 17, second edition, Blackwell Scientifi c Publications, pp. 413-468. Lampert, W., 1987. Feeding and nutrition in Daphnia, In: Peters, R. H., de Bernardi, R. (Eds), Daphnia, Memorie dell’Istituto Italiano di Idrobiologia. Verbania Pallanza, vol. 45, pp. 143-192. Martin-Creuzburg, D., von Elert, E., 2009. Good food versus bad food: the role of sterols and polyun- saturated fatty acids in determining growth and reproduction of Daphnia magna. Aquat. Ecol., 43, 943–950. Mourelatos, S., Lacroix, G., 1990. In situ fi ltering rates of Cladocera: Effect of body length, tempe- rature, and food concentration. Limnol. Oceanogr., 35 (5), 1101-1111. Park, S., Brett, M. T., Müller-Navarra, D. C., Goldman, C. R., 2002. Essential fatty acid content and the phosphorus to carbon ratio in cultured algae as indicators of food quality for Daphnia. Fre- shwater Biol., 47 (8), 1377-1390. Philippova, T. G., Postnov, A. L., 1988. The effect of food quality on feeding and metabolic expenditure in Cladocera. Int. Revue ges Hydrobiol., 73 (6), 601-615. Porter, G. K., Gerritsen, J., Orcutt Jr., J. D. 1982., The effect of food concentration on swimming pat- terns, feeding behaviour, ingestion, assimilation and respiration by Daphnia. Limnol. Oceanogr., 27 (5), 935-949. Richman, S., Dodson, S. I., 1983. The effect of food quality on feeding and respiration by Daphnia and Diaptomus. Limnol. Oceanogr., 28 (5), 948-956. Sundbom. M., Vrede, T., 1997. Effects of fatty acid and phosphorous content of food on the growth, survival and reproduction of Daphnia. Freshwater Biol., 38 (3), 665-674. Trabeau, M., Bruhn-Keup, R., McDermott, C., Keomany, M., Millsaps, A., Emery, A., de Stasio Jr., B., 2004. Midsummer decline of a Daphnia population attributed in part to cyanobacterial capsule production. J. Plankton Res., 26 (8), 949-961. Urabe, J., Watanabe, Y., 1991. Effect of food concentration on the assimilation and production effi - ciencies of Daphnia galeata G.O. Sars (Crustacea: Cladocera). Functional Ecology, 5(5), 635–641. Vijverberg, J., 1989. Culture techniques for studies on growth, development and reproduction of copepods and cladocerans under laboratory and in situ conditions: a review. Freshwater Biol., 21 (3), 317-373. Wagner, A., Kamjunke, N., 2001. Reduction of the fi ltration of Daphnia galeata by dissolved photo- synthetic products of edible phytoplankton. Hydrobiologia, 442 (1-3), 165-176. 70 Acta Biologica Slovenica, 53 (2), 2010 ACTA BIOLOGICA SLOVENICA LJUBLJANA 2010 Vol. 53, [t. 2: 71–97 Conservation assessment of the butterfl y fauna along the River Sava between Krško and the state border Naravovarstveno vrednotenje favne dnevnih metuljev ob reki Savi med Krškim in državno mejo Tatjana Čelik Jovan Hadži Institute of Biology, Scientifi c Research Centre of the Slovenian Academy of Science and Arts, Novi trg 2, P. O. Box 306, SI-1001 Ljubljana, Slovenia *correspondence: tcelik@zrc-sazu.si Abstract: An inventory of butterfl y fauna was carried out in 2008 within the southern part of the Ecological Important Area »the Sava River between Radeče and the state border with Croatia« with the aim of evaluating the most important areas for butterfl ies. Butterfl y fauna was surveyed within a study area of 32 km2, using the transect method. Twenty-one combined habitat types were included in the transect lines, with a total length of 19.2 km. The following parameters were used to evaluate the conservation importance of the combined habitat types: species richness of the combined habitat type, population density of species in combined habitat type, total population density of combined habitat type, maximum population density of species in combined habitat type, number of species with maximum population density in combined habitat type, number of threatened species in combined habitat type and number of threatened species with maximum population density in combined habitat type. A total of 69 species of butterfl ies (38% of Slovene butterfl y fauna) were recorded, 10 of which are threatened on national or European level. The most important combined habitat types for butterfl ies are extensively managed dry grasslands, abandoned dry grasslands, some types of semi-intensively used grasslands, some types of ruderal communities, and dry woodland rides and edges. On the basis of the distribution of the most important combined habitat types in the study area, four important areas for butterfl ies were designated, with a total area of 6.6 km2. They are important for preserving threatened species, ecological specialists and other rare or locally distributed species in the sub-pannonian part of SE Slovenia. Key words: the River Sava between Krško and the state border, Ecological Important Area, butterfl ies, population density, species richness, index of distribution, threatened species, Habitats Directive Izvleček: V južnem delu Ekološko pomembnega območja »Sava od Radeč do državne meje« (EPO Id: 63700), med Krškim in državno mejo s Hrvaško, smo v letu 2008 izvedli in- ventarizacijo favne dnevnih metuljev z namenom opredeliti naravovarstveno najpomembnejša območja za dnevne metulje v EPO. V raziskovanem območju s površino 32 km2 smo na transektih dolžine 19,2 km z metodo transektnega popisa inventarizirali relativno številčnost vrst v 21 zbirnih habitatnih tipih. Za naravovarstveno ovrednotenje zbirnih habitatnih tipov smo uporabili naslednje parametre: vrstno bogastvo zbirnega habitatnega tipa, populacijska gostota vrste v zbirnem habitatnem tipu, celokupna populacijska gostota v zbirnem habitatnem tipu, maksimalna populacijska gostota vrste v zbirnem habitatnem tipu, število vrst z maksimalno populacijsko gostoto v zbirnem habitatnem tipu in število ogroženih vrst v zbirnem habitatnem tipu. Zabeležili smo 69 vrst dnevnih metuljev (38 % favne dnevnih metuljev Slovenije), med njimi 10 v nacionalnem ali evropskem merilu ogroženih vrst. Naravovarstveno najpomembnejši habitatni tipi za dnevne metulje so ekstenzivno gospodarjeni suhi travniki, zaraščajoča suha travišča, nekateri polintenzivno gospodarjeni travniki, nekatere ruderalne združbe ter gozdne poti 72 Acta Biologica Slovenica, 53 (2), 2010 Introduction Butterfl ies are one of the most important invertebrate bioindicator groups (see, e.g., Ku- drna 1986, Oostermeijer and Van Swaay 1998, Ricketts et al. 2002; Maes and Van Dyck 2005, Thomas 2005, Settele et al. 2009). Data on their distribution and numbers have been collected in Europe at least 20 years within the frame- work of the project »Butterfl y Monitoring Schemes« (Van Sway and Van Strien 2008, Van Sway et al. 2008). The method for monitoring butterfl y populations is well described, exten- sively tested and scientifi cally sound (Pollard and Yates 1993). Butterfl ies are the only inver- tebrate taxon for which it is currently possible to estimate rates of decline among terrestrial in- sects (De Heer et al. 2005, Thomas 2005). They are also representative indicators of trends ob- served in most other terrestrial insects, which together represent approximately two-thirds of the world’s species (Thomas 2005). In 2007 as a result of the project »Streamlining European 2010 Biodiversity Indicators«, the European Environment Agency proposed 26 indicators for inclusion in the set of European biodiver- sity indicators (i.e. SEBI 2010 Indicators) (Van Sway and Van Strien 2008, Van Sway 2010). The biodiversity indicators offer a quick and easy tool for presenting general trends on the state of biodiversity (European Commission 2010). Butterfl ies were proposed as indicators for assessment of the state and trends in Euro- pean grasslands (European Grassland Butterfl y Indicator), which are the most important habitat of European species of butterfl ies (Van Sway 2010), since they are occupied by more than half of European species (280 species, 57%) (Van Sway et al. 2006). The main goal of this research was inven- tory of butterfl y fauna and, on the basis of the distribution and relative numbers of adults, to defi ne from a nature conservation point of view important populations, habitat types and areas for butterfl ies along the lower course of the River Sava between Krško and the state border, i.e., in the southern part of the ecologi- cally important area of the »Sava from Radeče to the state border« (EPO Id: 63700, http:// www.naravovarstveni-atlas.si). In the analysis, we included other habitat types, such as those on the basis of which EPO was defi ned (i.e., river with gravel bars, river branches, oxbows, fl oodplain groves, fragments of fl oodplain forest; http://www.naravovarstveni-atlas.si), above all various types of grassland, woodland edges, tall herb communities and ruderal areas. We thus at the same time supplemented the cur- rently knowledge of the fauna of EPO »Sava from Radeče to the state border«. Review of existing data There is relatively little published data about the butterfl y fauna of the study area. The only planned research of butterfl y fauna in the area of the lower Sava downstream from Krško was done by Rebeušek (2001). Older publications on the presence of some species of butterfl ies refer to the surroundings of Krško (e.g., Hafner 1909, Lorković 1993, 1996) and Bregana (e.g., Lorković and Mladinov 1971, Čelik et al. 2004). More numerous data have been published for the wider hinterland of the research area in the sub-Pannonian zoogeographic region (e.g., Kostanjevica, Novo Mesto, Tolsti vrh, Gorjanci, Kozje etc.) (e.g., Hafner 1909 – manuscript, Lorković 1927, Carnelutti and Michieli 1955, 1960, Sijarić 1991, Čelik et al. 2004, Čelik et al. 2005). in robovi na suhih rastiščih. Na podlagi razširjenosti najpomembnejših zbirnih habitatnih tipov v raziskovanem območju smo opredelili štiri naravovarstveno pomembna območja za dnevne metulje s skupno površino 6,6 km2. Območja so pomembna za ohranjanje ogroženih in drugih ekološko specializiranih, redkih ali lokalno razširjenih vrst v območju subpanonske JV Slovenije. Ključne besede: reka Sava med Krškim in državno mejo s Hrvaško, ekološko pomembno območje, dnevni metulji, populacijska gostota, vrstno bogastvo, indeks razširjenosti, ogrožene vrste, Direktiva o habitatih 73Čelik: Conservation assessment of the butterfl y fauna along the River Sava Material and Methods Study area The study area is situated in the south-east- ern part of Slovenia (45° 54' 35" N, 15° 34' 32" E; WGS 84), at an altitude of 150 m. It belongs to the sub-Pannonian region (Carnelutti 1992), the northern part borders on the pre-Alpine re- gion. It embraces the lowlands on both banks of the River Sava, from Krško in the north to the state border with Croatia by the settlement of Ribnica in the south; in the east it extends to the road Krško–Zgornji Obrež–Brežice–Dobova, and in the west to the road Žadovinek–Mrt- vice–Krška vas–Čatež ob Savi, at fi rst towards the south to the motorway Čatež ob Savi– Podgračeno (Fig. 1). It extends over an area of 32 km2. The study area is mainly agricultural landscape. It is consisted of fi ve landscape types (Denac et al. 2009): agricultural landscape (66%; arable fi elds, differently managed grasslands), mosaic of hedges, scrub, small farmland areas and small forest fragments where no habitat type predominates (14%), woodland (10%; mainly fragments of fl oodplain forest), river (8%; river- bed with banks, river branches, gravel bars) and other water bodies (2%; gravel pits, oxbows). In agricultural landscape, where arable fi elds and intensively used grasslands prevail (46% of the study area; cf. Trčak et al. 2008), the abandoned dry grasslands in early succession stages and the Figure 1: Study area and transect lines (TR) for inventory of butterfl ies along the River Sava between Krško and state border with Croatia in 2008. Slika 1: Raziskovano območje in transekti (TR) za inventarizacijo dnevnih metuljev ob reki Savi med Krškim in državno mejo s Hrvaško v letu 2008. 74 Acta Biologica Slovenica, 53 (2), 2010 extensively managed dry grasslands represent only a small part of the area (7%; cf. Trčak et al. 2008). The most extensive areas of these two types of grasslands extend in the NW part of the study area on the right bank of the River Sava. Minor areas are also in the northern part of the study area on the left bank of the River Sava, and in the SE part of the study area on the right and left bank of the River Sava. The important characteristic of the study area is also the pres- ence of invasive allochthonous plant species (e.g. Solidago gigantea, Solidago canadensis, Impatiens glandulifera, Rudbeckia laciniata, Echinocystis lobata, Fallopia japonica, Robina pseudoacacia, Ailanthus altissima), widely dis- tributed on moist ground of woodland areas, on river banks and near other water bodies. As pure stands they represent 2% of the study area, but in combination with other habitat types they ac- tually extend over a much larger area (Trčak et al. 2008). Field methods We surveyed the butterfl y fauna of the study area from March to September 2008, at 12 sampling sites (Fig. 1). At each sampling site, butterfl ies were monitored using the line tran- sect method (Pollard and Yates 1993, Thomas 2005) – 1 transect line/sampling site. Transect counts were conducted at a time interval of ap- proximately 14 days (1 count in April, 2 counts in May, 1 count in June, 2 counts in July, 1 count in September). The entire length of the transect lines was 19,157 metres. Each transect line was divided into sections, each section represented a specifi c habitat type. The habitat types were identifi ed exactly, to the 3rd and 4th levels of the methodology determined by Jogan et al. (2004). The 12 transect lines contained 53 sections. In the fi eld, it is not possible to distinguish between Colias alfacariensis Ribbe, 1905 and Colias hyale (Linnaeus, 1758), and also between Leptidea sinapis (Linnaeus, 1758) and Leptidea reali Reissinger, 1989. Therefore, the both Co- lias species were treated as a complex C. alfa- cariensis/hyale, and both Leptidea species as a complex L. sinapis/reali for the analysis. Data analysis Because of the large number of habitat types (40), we merged the 40 habitat types into 21 combined habitat types (Tab. 1) that we used in further analysis. A combined habitat type is a higher and more general defi ned category, which includes habitat types that are similar in terms of fl oristic composition, structure of vegetation and management. Therefore, the length of the com- bined habitat type is the sum of the lengths of all transect sections with habitat type belonging to those combined habitat type. For data analysis we used the following pa- rameters: population density, total population density, maximum population density, species richness, index of distribution of species in the study area, number of species which had maxi- mum population density in specifi c combined habitat type and number of threatened species in specifi c combined habitat type. The population density of each butterfl y species in each combined habitat type was cal- culated according to Kitahara et al. (2008) as follows. The monthly count was determined as the mean of twice-monthly counts conducted in May-July or as the value of single counts in April, June and September. The mean monthly count over the season was then calculated us- ing only those months when the species was observed to minimize the effect of variable volt- inism between species. Finally, the population density (number of adults/month/100 m of com- bined habitat type) was obtained by dividing the mean monthly count by the length of the com- bined habitat type (in metres x 100). The popula- tion density of each threatened species in each combined habitat type in proposed important areas for butterfl ies was calculated in the same manner, except that we included in the length of the combined habitat type only sections in tran- sects that lay within the proposed important area. The total population density in each com- bined habitat type was the sum of population densities of all species observed in each com- bined habitat type. The maximum population density of a species was the population density of species in the combined habitat type in which it was most abundant. 75Čelik: Conservation assessment of the butterfl y fauna along the River Sava The species richness in each combined habitat type/important area for butterfl ies was the total number of butterfl y species observed in each combined habitat type/important area for butterfl ies during the study period. The index of distribution of a species in the study area was expressed as the number of transect lines in which the species was observed. We treated as a generally widespread species, those species with an index of distribution > 9 (> 75% of all transect lines), as rare/locally distrib- uted species those with an index of distribution < 4 (< 25% of all transect lines). Kendall rank correlation coeffi cient was used to test whether a relationship exists be- tween the length of the combined habitat type and the following parameters: species richness of the combined habitat type, total popula- tion density, number of species with maximum population density in the combined habitat type, number of threatened species in the combined habitat type and number of threatened species with maximum population density in the com- bined habitat type. In the interpretation of spe- cies richness of the combined habitat types in relation to the length of the combined habitat type, we treated as “not different in terms of the length” those combined habitat types that differ in length by less than 50 m, and as “not differ- ent in terms of species richness” those combined habitat types that differ in number of species by less than 3. To defi ne the important areas for butter- fl ies in the study area, fi rst the conservation importance of the combined habitat types for butterfl ies was assessed. For the conservation evaluation of each combined habitat type, six parameters were used: species richness, median of population densities of species observed in the treated combined habitat type, total popula- tion density, number of species with maximum population density in the treated combined habitat type, number of threatened species in the combined habitat type and number of threatened species with maximum population density in the treated combined habitat type. The non-param- teric pairwise correlations were used to test the independence between parameters. Because of the small data set (21 combined habitat types) with a large number of tied ranks, Kendall’s tau was applied. In the cases where the signifi cant relationship between parameters was establis- hed, the actual values of Kendall’s correlation coefi cient shown only weak or moderate corre- lation. Therefore, all six parameters were used in the conservation assesment. They were applied as criteria in further conservation evaluation of the combined habitat types. For each criterion the combined habitat types were ranked according to parameter value. The rank 1 was assigned to the combined habitat type with the highest parame- ter value, and the rank 21 (in the case of data set without tied ranks) to the combined habitat type with the lowest value of parameter. The nature conservation value of each combined habitat type was determined as the median of ranks assigned to each combined habitat type within the scope off all criteria. The combined habitat types with the median value lower then 10,5 were defi ned as the most important for butterfl ies in the study area from the conservation point of view. Finally, the important areas for butterfl y species were ou- tlined on the base of the distribution of the most important combined habitat types within the study area. The distribution of all habitat types which were similar, in terms of fl oristic composi- tion, structure of vegetation and management to the combined habitat types of transect lines was established from the map of habitat types of the study area (Trčak et al. 2008). Statistical calculations were performed by SPSS 13.0 (SPSS Inc. 1989–2004). We used the program Arc Map 9.2 (ESRI Inc. 1999–2006) for outlining transects and important areas for but- terfl y species. The nomenclature of species of butterfl ies is taken from Van Sway et al. (2010), plant species from Martinčič et al. (2007), the ty- pology of habitat types according to Jogan et al. (2004), threatened species according to the Red list of Lepidoptera of Slovenia (Uradni list RS 82/2002), the Decree on protected wild animal species (Uradni list RS 46/2004), the European Red list of Butterfl ies (Van Swaay et al. 2010), the Directive on the conservation of natural habi- tats and of wild fauna and fl ora (Directive 92/43/ EEC) (hereinafter: Habitats Directive) and the Convention on the Conservation of European Wildlife and Natural Habitats (Uradni list RS 17/1999) (hereinafter: Bern Convention). 76 Acta Biologica Slovenica, 53 (2), 2010 Results and discussion Species richness and distribution of species in the study area In the period from March to September 2008, we recorded 7396 individuals from 69 species in the transect lines (Tab. 1), which represents 38% of all butterfl y species living in Slovenia. In the only previous planned inventory of butterfl ies, which was carried out in the study area in 2001, 60 species were recorded (Rebeušek 2001). They included four species that we did not record in 2008: Cyaniris semi- argus (Rottemburg, 1775), Argynnis niobe (Lin- naeus, 1758), Brentis ino (Rottemburg, 1775) and Neptis sappho (Pallas, 1771). In view of the fact that all four species were observed in 2001 in areas in which we carried out transect counts, the reasons that we did not register these spe- cies in 2008 could be: (i) very low numbers of the populations, which reduces the probability of detection of the species in a transect line; (ii) the species do not live in the areas of the transect lines. In 2008, we found 13 species that had not been observed in 2001: Aphantopus hyperantus (Linnaeus, 1758), Argynnis paphia (Linnaeus, 1758), Carcharodus alceae (Esper, 1780), Coe- nonympha arcania (Linnaeus, 1761), Leptotes pirithous (Linnaeus, 1767), Limenitis populi (Linnaeus, 1758), Lycaena hippothoe (Linnae- us, 1761), Melitaea didyma (Esper, 1778), Pieris mannii (Mayer, 1851), Satyrium acaciae (Fabri- cius, 1787), S. spini (Dennis & Schiffermüller, 1775), S. w-album (Knoch, 1782) and Thymeli- cus lineola (Ochsenheimer, 1808). Mostly intensively managed anthropogenic landscape of the study area is refl ected in the species richness of butterfl y fauna (69 species/32 km2). It is lower than in Natura 2000 sites and some other important conservation areas in Slo- venia, in which inventories of butterfl y fauna are already performed. It is evident from the comparisons with the species richness of the fol- lowing areas: Škocjanske jame Regional Park: 90 species/4 km2 (Čelik 2004); Natura 2000 site “Radensko polje” – SI3000171: 68 species/7 km2 (Rebeušek and Verovnik 2000); Natura 2000 site “Planinsko polje” – SI5000016: 78 species/10 km2 (Čelik 2007); Natura 2000 site “Banjšice” – SI3000034: 78 species/12 km2 (Čelik 2009). Generally widespread species (= index of distribution > 9) in the study area are species that are not ecologically specialised, of which we recorded 19: Aglais io (Linnaeus, 1758), Aricia agestis (Dennis & Schiffermüller, 1775), Boloria dia (Linnaeus, 1767), Brenthis daphne (Bergssträsser, 1780), Coenonympha glycerion (Borkhausen, 1788), C. pamphilus (Linnaeus, 1758), Colias crocea (Geoffroy, 1785), Cupido argiades (Pallas, 1771), Erynnis tages (Lin- naeus, 1758), Gonepteryx rhamni (Linnaeus, 1758), Leptidea sinapis/reali (Linnaeus, 1758/ Reissinger, 1989), Maniola jurtina (Linnaeus, 1758), Melitaea athalia (Rottemburg, 1775), M. phoebe (Dennis & Schiffermüller, 1775), Ochlodes sylvanus (Esper, 1777), Pieris napi (Linnaeus, 1758), P. rapae (Linnaeus, 1758), Plebejus argus (Linnaeus, 1758) and Polyom- matus icarus (Rottemburg, 1775) (Tab. 1). Rare/ locally distributed species (index of distribution < 4; 21 species) occupy woodland rides (11 spe- cies), abandoned dry and semi-dry grasslands (9 species), extensively managed dry grasslands (8 species), semi-intensively managed grasslands (7 species), woodland edges (7 species), ruderal areas (5 species) and scrub (1 species) (Tab. 1). Species richness of the combined habitat types The species richest combined habitat types in the study area are extensive dry grasslands with erect brome (48 species), abandoned dry and semi-dry grasslands (STzS: 46, STzT: 41 species), semi-intensively managed dry grass- lands with erect brome and tall oat-grass (40 species) and woodland rides on moist grund overgrown with autochthonous and allochtho- nous plant species (36 species) (Tab. 1). The signifi cant strong positive correla- tion exists between the length of the combined habitat type and its species richness (τ = 0.657, P<0.001). From a comparison between com- bined habitat types that differ in length by less than 50 m and in number of species by more than 2 (Fig. 2a) and combined habitat types of different lengths that do not differ in terms of the number of species (Fig. 2b), we can conclude 77Čelik: Conservation assessment of the butterfl y fauna along the River Sava Ta bl e 1: L is t o f r ec or de d sp ec ie s w ith p op ul at io n de ns iti es in c om bi ne d ha bi ta t t yp es in w hi ch a sp ec ie s a pp ea re d an d in de x of d is tri bu tio n of sp ec ie s re co rd ed in tr an se ct li ne s a lo ng th e R iv er S av a be tw ee n K rš ko a nd th e st at e bo rd er in 2 00 8. Ta be la 1 : Se zn am re gi str ira ni h vr st s p op ul ac ijs ki m i g os to ta m i v z bi rn ih h ab ita tn ih ti pi h in in de ks ra zš irj en os ti vr st, z ab el ež en ih n a tra ns ek tih o b re ki S av i m ed K rš ki m in d rž av no m ej o v le tu 2 00 8. C om bi ne d ha bi ta t t yp e G M vT G Ps S G Ps T G Pv T G Pv V s G R sS G R sT G R vT G R vV s M Ti A M Tp A N R g R gm R gm z ST eB ST pA ST pB A ST zS ST zT TV Tp Index of distribution Le ng th o f c om bi ne d ha bi ta t t yp e (m ) 54 9 50 1 38 1 20 68 68 6 20 2 64 2 49 2 12 46 19 99 12 43 50 44 8 47 6 28 7 28 38 97 2 10 57 19 23 92 8 16 9 Sp ec ie s Po pu la tio n de ns ity Ag la is io 0, 05 0, 05 0, 33 0, 02 0, 12 0, 20 0, 17 0, 04 0, 02 0, 05 0, 17 0, 01 0, 05 0, 01 0, 03 0, 07 12 Ag la is u rt ic ae 0, 07 0, 04 0, 05 0, 01 0, 04 0, 07 0, 07 0, 01 0, 02 8 An th oc ha ri s c ar da m in es 0, 20 0, 13 0, 16 0, 07 0, 08 0, 05 0, 04 0, 03 0, 11 0, 16 0, 35 0, 03 0, 04 0, 30 9 Ap at ur a ili a 0, 08 0, 14 0, 04 0, 04 0, 23 3 Ap ha nt op us h yp er an tu s 0, 05 0, 10 0, 46 0, 01 0, 03 0, 03 0, 49 0, 24 5 Ar as ch ni a le va na 0, 07 0, 09 0, 04 0, 05 0, 01 0, 11 0, 26 5 Ar gy nn is p ap hi a 0, 10 0, 01 0, 03 0, 02 4 Ar ic ia a ge st is 0, 02 0, 01 0, 19 0, 03 0, 06 0, 08 0, 09 0, 22 0, 03 0, 20 0, 40 0, 44 11 Bo lo ri a di a 0, 02 0, 01 0, 12 0, 28 0, 28 0, 48 0, 72 0, 56 0, 46 0, 10 10 Br en th is d ap hn e 0, 18 0, 15 0, 44 0, 16 0, 41 0, 72 0, 16 0, 21 0, 35 0, 47 0, 05 0, 22 10 C al lo ph ry s r ub i 0, 13 0, 05 0, 11 3 C ar ch ar od us a lc ea e 0, 05 0, 09 0, 01 3 C el as tr in a ar gi ol us 0, 07 0, 05 0, 05 5 C oe no ny m ph a ar ca ni a 0, 59 0, 02 0, 08 0, 05 0, 10 0, 07 1, 39 3, 18 5 C oe no ny m ph a gl yc er io n 0, 39 0, 01 0, 37 0, 21 0, 03 0, 14 0, 27 0, 44 0, 50 0, 60 0, 45 0, 93 1, 14 0, 84 11 C oe no ny m ph a pa m ph ilu s 0, 66 0, 02 0, 09 0, 43 0, 31 0, 03 0, 27 0, 42 0, 40 1, 00 0, 03 1, 70 0, 60 0, 78 0, 48 0, 57 0, 32 0, 15 12 C ol ia s a lfa ca ri en si s/ hy al e 0, 02 0, 03 0, 02 0, 01 0, 06 0, 02 0, 13 0, 05 0, 02 8 C ol ia s c ro ca 0, 39 0, 22 0, 03 0, 04 0, 07 0, 25 0, 16 0, 30 0, 13 0, 31 0, 83 0, 75 0, 81 0, 18 2, 31 0, 88 0, 32 0, 79 0, 42 0, 40 12 C up id o ar gi ad es 0, 87 0, 22 0, 39 0, 05 0, 02 0, 23 0, 28 0, 08 0, 10 0, 24 0, 95 0, 18 1, 31 0, 36 0, 15 0, 19 0, 45 0, 36 12 C up id o m in im us 0, 02 0, 05 2 78 Acta Biologica Slovenica, 53 (2), 2010 Er yn ni s t ag es 0, 09 0, 07 0, 26 0, 10 0, 05 0, 07 0, 03 0, 03 0, 17 0, 21 0, 02 0, 27 0, 29 0, 45 10 G la uc op sy ch e al ex is 0, 13 0, 02 0, 05 3 G on ep te ry x rh am ni 0, 09 0, 05 0, 56 0, 10 0, 02 0, 06 0, 27 0, 03 0, 03 0, 01 0, 02 0, 03 0, 03 0, 13 0, 04 0, 03 0, 09 0, 14 0, 08 0, 15 12 H es pe ri a co m m a 0, 04 0, 14 0, 07 0, 05 4 H et er op te ru s m or ph eu s 0, 02 0, 04 0, 09 0, 18 0, 65 4 Ip hi cl id es p od al ir iu s 0, 04 0, 02 0, 02 3 Is so ri a la th on ia 0, 01 0, 02 0, 03 0, 02 0, 02 5 La si om m at a m eg er a 0, 12 0, 02 0, 08 0, 05 0, 01 4 Le pt id ea si na pi s/ re al i 0, 11 0, 20 0, 45 0, 16 0, 16 0, 20 0, 34 0, 22 0, 26 0, 19 0, 39 0, 40 0, 04 0, 04 0, 21 0, 22 0, 22 0, 33 0, 58 0, 74 0, 53 12 Le pt ot es p ir ith ou s 0, 08 1 Li m en iti s p op ul i 0, 07 1 Li m en iti s r ed uc ta 0, 03 0, 31 0, 01 0, 05 0, 02 0, 01 0, 05 0, 10 5 Ly ca en a di sp ar 0, 15 0, 06 0, 03 2 Ly ca en a hi pp ot ho e 0, 04 1 Ly ca en a ph la ea s 0, 16 0, 08 0, 09 0, 10 4 Ly ca en a tit yr us 0, 03 0, 17 0, 01 0, 04 0, 01 0, 02 0, 03 0, 02 0, 02 6 M an io la ju rt in a 0, 32 0, 37 2, 26 0, 18 2, 72 1, 21 0, 10 0, 10 0, 66 0, 30 0, 17 0, 86 0, 27 1, 40 1, 20 0, 70 0, 07 12 M el an ar gi a ga la th ea 0, 05 0, 50 0, 39 3, 47 4, 91 0, 26 0, 04 0, 11 3, 10 0, 15 4, 92 8, 07 3, 61 9 M el ita ea a th al ia 0, 06 0, 05 0, 02 0, 31 0, 51 0, 21 1, 73 0, 41 0, 38 0, 82 0, 65 0, 09 0, 49 10 M el ita ea a ur el ia 0, 13 0, 50 0, 08 0, 10 1, 39 0, 05 0, 19 0, 59 0, 38 7 M el ita ea b ri to m ar tis 0, 07 0, 05 0, 07 4 M el ita ea c in xi a 0, 12 0, 19 0, 19 4 M el ita ea d id ym a 0, 01 0, 11 0, 01 3 M el ita ea p ho eb e 0, 02 0, 08 0, 02 0, 04 0, 22 0, 35 0, 11 0, 05 0, 14 0, 03 10 M in oi s d ry as 0, 40 0, 59 0, 05 1, 13 0, 50 0, 24 0, 03 0, 26 2, 65 2, 45 5 O ch lo de s s yl va nu s 0, 03 0, 03 0, 22 0, 03 0, 05 0, 17 0, 13 0, 17 0, 10 0, 12 0, 04 0, 06 0, 04 0, 10 0, 17 0, 10 0, 07 0, 69 12 Pa pi lio m ac ha on 0, 01 0, 06 0, 09 0, 02 0, 06 0, 02 8 Pa ra rg e ae ge ri a 0, 15 0, 21 2 Pi er is b ra ss ic ae 0, 02 0, 05 0, 05 0, 03 0, 03 0, 03 0, 02 8 C om bi ne d ha bi ta t t yp e G M vT G Ps S G Ps T G Pv T G Pv V s G R sS G R sT G R vT G R vV s M Ti A M Tp A N R g R gm R gm z ST eB ST pA ST pB A ST zS ST zT TV Tp Index of distribution Le ng th o f c om bi ne d ha bi ta t t yp e (m ) 54 9 50 1 38 1 20 68 68 6 20 2 64 2 49 2 12 46 19 99 12 43 50 44 8 47 6 28 7 28 38 97 2 10 57 19 23 92 8 16 9 Sp ec ie s Po pu la tio n de ns ity 79Čelik: Conservation assessment of the butterfl y fauna along the River Sava Pi er is m an ni i 0, 05 0, 12 0, 05 0, 03 5 Pi er is n ap i 0, 02 0, 10 0, 31 0, 20 0, 09 0, 20 0, 03 0, 04 0, 05 0, 07 0, 02 0, 02 0, 29 0, 42 0, 00 0, 01 0, 04 0, 08 12 Pi er is ra pa e 0, 22 0, 36 0, 39 0, 13 0, 28 0, 59 0, 56 0, 26 0, 14 0, 36 0, 47 0, 40 1, 05 0, 32 0, 38 0, 54 0, 27 0, 43 0, 22 0, 22 0, 12 12 Pl eb ej us a rg us 0, 20 0, 12 0, 01 0, 23 0, 03 0, 33 0, 19 1, 01 0, 04 0, 23 0, 84 0, 88 0, 44 0, 32 0, 15 10 Pl eb ej us a rg yr og no m on 0, 04 1 Pl eb ej us id as 0, 07 0, 05 0, 16 0, 05 3 Po ly go ni a c- al bu m 0, 14 0, 05 0, 04 0, 02 0, 02 0, 06 0, 03 7 Po ly om m at us b el la rg us 0, 02 0, 05 0, 16 3 Po ly om m at us ic ar us 0, 55 0, 02 0, 02 0, 04 0, 31 0, 30 0, 01 0, 22 0, 32 0, 59 0, 18 1, 22 0, 71 0, 37 0, 53 0, 57 0, 61 12 Po nt ia d ap lid ic e 0, 39 0, 03 0, 03 3 Py rg us m al va e 0, 11 0, 05 0, 01 0, 03 0, 03 0, 01 0, 02 0, 14 0, 07 8 Sa ty ri um a ca ci ae 0, 34 0, 16 0, 04 0, 28 0, 31 0, 43 3 Sa ty ri um p ru ni 0, 10 0, 20 0, 05 3 Sa ty ri um sp in i 0, 66 0, 08 0, 02 2 Sa ty ri um w -a lb um 1, 05 0, 01 0, 08 2 Th ym el ic us li ne ol a 1, 64 0, 48 2, 03 0, 16 0, 40 0, 48 2, 44 0, 21 1, 03 1, 14 0, 47 0, 54 9 Th ym el ic us sy lv es tr is 0, 26 0, 16 0, 16 0, 32 2 Va ne ss a at al an ta 0, 04 0, 08 0, 00 0, 03 0, 02 0, 07 0, 08 0, 00 0, 01 0, 01 0, 06 8 Va ne ss a ca rd ui 0, 01 0, 04 0, 03 0, 00 0, 01 0, 01 6 Ze ry nt hi a po ly xe na 0, 05 0, 58 0, 02 0, 08 0, 05 0, 08 0, 02 0, 13 0, 22 7 N um be r o f s pe ci es 25 23 29 36 18 15 34 21 30 30 28 6 15 20 24 48 34 40 46 41 12 To ta l p op ul at io n de ns ity 6, 32 3, 53 10 ,5 9 2, 64 1, 74 9, 51 12 ,5 5 4, 69 3, 73 4, 42 8, 06 3, 55 4, 43 2, 41 13 ,0 0 12 ,4 7 7, 13 16 ,3 9 22 ,5 3 18 ,6 7 2, 88 M ed ia n of p op ul at io n de ns iti es 0, 25 0, 15 0. 37 0, 07 0, 10 0, 63 0, 37 0, 22 0, 12 0, 15 0, 29 0, 59 0, 30 0, 12 0, 54 0, 26 0, 21 0, 41 0, 49 0, 46 0, 24 N o. o f s pe ci es w ith m ax im um p op ul at io n de ns ity 1 2 10 0 1 2 4 0 2 0 4 0 4 2 12 5 2 2 7 8 1 C om bi ne d ha bi ta t t yp e G M vT G Ps S G Ps T G Pv T G Pv V s G R sS G R sT G R vT G R vV s M Ti A M Tp A N R g R gm R gm z ST eB ST pA ST pB A ST zS ST zT TV Tp Index of distribution Le ng th o f c om bi ne d ha bi ta t t yp e (m ) 54 9 50 1 38 1 20 68 68 6 20 2 64 2 49 2 12 46 19 99 12 43 50 44 8 47 6 28 7 28 38 97 2 10 57 19 23 92 8 16 9 Sp ec ie s Po pu la tio n de ns ity 80 Acta Biologica Slovenica, 53 (2), 2010 GMvT Small shrubs on moist ground; presence of allochtonous and autochtonous plant species GPsS Woodland ride on dry ground; autochtonous plant species predominate GPsT Woodland ride on dry ground; presence of allochtonous and autochtonous plant species GPvT Woodland ride on moist ground; presence of allochtonous and autochtonous plant species GPvVs Woodland ride with tall herb communities on moist ground; presence of allochtonous and autochtonous plant species GRsS Woodland edge on dry ground; autochtonous plant species predominate GRsT Woodland edge on dry ground; presence of allochtonous and autochtonous plant species GRvT Woodland edge on moist ground; presence of allochtonous and autochtonous plant species GRvVs Woodland edge with tall herb communities on moist ground; presence of allochtonous and autochtonous plant species MTiA Mesophilous mesotrophic and eutrophic grasslands with tall oatgrass (Arrhenathereum elatius L.); intensively managed MTpA Mesophilous mesotrophic and eutrophic grasslands with tall oatgrass (Arrhenathereum elatius L.); semi-intensively managed N Arable fi elds Rg Area with ruderal vegetation; mostly bare ground, only some herb species Rgm Area with ruderal vegetation; mostly shrub species Rgmz Area with ruderal vegetation; shrub and herb species STeB Dry and semi-dry grasslands with erect brome (Bromopsis erecta Huds.); extensively managed STpA Dry and semi-dry grasslands with tall oatgrass (Arrhenathereum elatius L.); semi-intensively managed STpBA Dry and semi-dry grasslands with erect brome (Bromopsis erecta Huds.) and tall oatgrass (Arrhenathereum elatius L.); semi-intensively managed STzS Abandoned dry and semi-dry grasslands; autochtonous shrub and tree species predominate STzT Abandoned dry and semi-dry grasslands; presence of allochtonous and autochtonous shrub and tree species TVTp Common reed bed along semi-intensively managed moist mesotrophic grassland Legend that the type of vegetation or/and type of man- agement in the combined habitat type are more important factors affecting species richness than the length of the combined habitat type. The in- fl uence of type of vegetation on the number of species in the combined habitat type is evident from the following comparisons: despite differ- ences in length < 50 m, (i) dry woodland edges in which autochtonous plant species predomi- nate (GRsS) are more species rich than common reed bed along semi-intensively managed moist mesotrophic grassland (TVTp) (Fig. 2a), and (ii) ruderal areas overgrown predominantly with shrubs (Rgm) are more species rich than mostly bare ruderal areas (Rg) (Fig. 2a), which are only a habitat for the thermoregulation of adults, and (iii) dry woodland edges (GRsT) are more spe- cies rich than woodland rides on moist ground overgrown with tall herb communities (GPvVs), among which predominate invasive allochtho- nous species that are not larval food plants or nectar plants of butterfl y species that live in Slo- venia (Fig. 2a); despite the shorter length (dif- ference in length is more than 50 m; see Data analysis), (iv) ruderal areas overgrown with shrub and herb species (Rgmz) not differ in spe- cies richness from scrub (GMvT) and dry wood- land rides (GPsS) (Fig. 2b), and (v) ruderal areas overgrown predominantly with shrub (Rgm) not differ in species richness from woodland rides on moist ground in which invasive allochtho- nous tall herb species predominate (GPvVs) (Fig. 2b), and (vi) dry woodland edges (GRsS) have the same number of species as bare ruderal 81Čelik: Conservation assessment of the butterfl y fauna along the River Sava areas (Rg) (Fig 2b). That the management of the combined habitat type is a more important fac- tor that infl uences species richness than its length is evident from the following comparisons: de- spite differences in length < 50 m, (i) abandoned dry grasslands in which autochtonous shrub and tree species predominate (STzS) are more spe- cies rich than intensively managed grasslands (MTiA) (Fig. 2a), and (ii) abandoned dry grass- lands with approximately the same proportion of allochtonous and autochtonous shrub and tree species (STzT) are more species rich than semi- intensively managed grasslands with tall oat- grass (STpA) (Fig. 2a); despite the shorter length (difference in length is more than 50 m; see Data analysis), (iii) abandoned dry grasslands with ap- proximately the same proportion of allochtonous and autochtonous shrub and tree species (STzT) not differ in species richness from semi-inten- sively managed dry grasslands with erect brome and tall oat-grass (STpBA) (Fig. 2b), and (iv) woodland edges (GRsT, GRvVs) and dry wood- land rides (GPsT) not differ in species richness from semi-intensively and intensively managed grasslands with tall oat-grass (STpA, MTpA, MTiA) (Fig. 2b). Namely, woodland edges and dry woodland rides overgrown with autochtho- nous richly fl owering species of herbs and shrubs are also feeding habitats of adults at the time when semi-intensively and intensively cultivated grasslands have already been mown. Total population density, median of population densities of species, maximum population density of species The total population density is not in correla- tion with the length of the combined habitat type (A) 82 Acta Biologica Slovenica, 53 (2), 2010 Figure 2: Species richness of the combined habitat type in relation to the length of the combined habitat type along the River Sava between Krško and the state border in 2008 (for abbreviations of the combined habitat types, see Tab. 1). (A) – Comparison between combined habitat types that differ in length by less than 50 m and in the number of species by more than two. (B) – Comparison between combined habitat types that differ in length by more than 50 m and do not differ in terms of the number of species (for explanation, see Data analysis).  - combined habitat types that refl ect the infl uence of type of vegetation,  - combined habitat types that refl ect the infl uence of management,  - combined habitat types that are not included in comparison Slika 2: Število vrst v zbirnih habitatnih tipih glede na dolžino zbirnega habitatnega tipa ob reki Savi med Krškim in državno mejo v letu 2008. (za razlago okrajšav zbirnih habitatnih tipov glej Tab. 1) (A) – Primerjava med zbirnimi habitatnimi tipi, ki se v dolžini razlikujejo za manj kot 50 m in v številu vrst za več kot dve. (B) – Primerjava med zbirnimi habitatnimi ttipi, ki se v dolžini razlikujejo za več kot 50 m in v številu vrst za manj kot 3 (za razlago glej poglavje Data analysis).  - zbirni habitatni tipi, ki odražajo vpliv tipa vegetacije,  - zbirni habitatni tipi, ki odražajo vpliv načina gospodarjenja,  - zbirni habitatni tipi, ki niso vključeni v primerjavo (τ = 0.124, P>0.4), despite the fact that there is a strong positive correlation between the species richness and the length of the combined habitat type (see above) and a moderate positive cor- relation between the species richness and the total population density (τ = 0.446, P<0.01). Concerning the fi nding that there is no correla- tion between the total population density and the length of combined habitat type, we can con- clude that the combined habitat types with the (B) 83Čelik: Conservation assessment of the butterfl y fauna along the River Sava highest total population densities are abandoned dry and semi-dry grasslands (STzS: 22,53; STzT: 18,67), semi-intensively managed dry grasslands with erect brome and tall oat-grass (STpBA: 16,39), shrub and herb-overgrown ru- deral areas (Rgmz: 13,00), dry woodland edges (GRsT: 12,55) and extensively managed dry grasslands (STeB: 12,47) (Tab. 1). In all other combined habitat types, the total population densities are at least twice (up to 13x in GPvVs) lower than in combined habitat type with the highest total population density (STzS). Total population density of the combined habitat type is also affected by the population densities of species that inhabit it, since the weak positive correlation between total population density and median value of population densi- ties of species that occupy the combined habitat type is signifi cant (τ = 0.313, P<0.01). There is also a signifi cant moderate positive correlation between total population density and the number of species with maximum population density in the combined habitat type (τ = 0.536, P<0.05) (Fig. 3). The actual values of Kendall’s correla- tion coefi cient show that the number of species with maximum population density in a combined habitat type is a more important factor infl uenc- ing total population density than the species rich- ness of the combined habitat type and population densities of species that inhabit it. Generally widespread grassland species (M. galathea, C. arcania, M. jurtina, M. dryas, T. lineola, C. crocea, M. athalia, C. pamphilus) have the highest population densities in the study area (Fig. 4), which are also often found to be markedly more abundant in grassland habitats (grasslands, abandoned grasslands) elsewhere in Slovenia than other species (own observa- tion); the species M. dryas is an exception. It is not widely distributed in Slovenia because it Figure 3: Total population density in relation to the number of species with the maximum population density in combined habitat type and the median value of population densities of species that inhabit combined habitat type in the study area along the River Sava between Krško and the state border in 2008 (median value of population densities of species is given by the diameter of the circle). (for abbreviations of the combined habitat types, see Tab. 1) Slika 3: Celokupna populacijska gostota zbirnega habitatnega tipa glede na število vrst z maksimalno populacijsko gostoto v zbirnem habitatnem tipu in mediano vrednostjo populacijskih gostot vrst, ki poseljujejo zbirni habitatni tip za zbirne habitatne tipe ob reki Savi med Krškim in državno mejo v letu 2008 (mediano vrednost populacijskih gostot vrst v zbirnem habitatnem tipu določa premer kroga). (za razlago okrajšav zbirnih habitatnih tipov glej Tab. 1) 84 Acta Biologica Slovenica, 53 (2), 2010 Figure 4: Comparison of maximum population densities of species recorded along the River Sava between Krško and the state border in 2008. The columns of threatened species are marked in black. Slika 4: Primerjava maksimalnih populacijskih gostot vrst, zabeleženih ob reki Savi med Krškim in državno mejo v letu 2008. Črno obarvani stolpci označujejo ogrožene vrste. 85Čelik: Conservation assessment of the butterfl y fauna along the River Sava mostly inhabits xerothermophilous grasslands. The caterpillars of the mentioned species feed on grasses (Poaceae), with the exception of the ubiquitous species C. crocea, of which the lar- val food plants are various low growing legume species (Fabaceae). It can be concluded from comparison of maximum population densities the relation between the sizes of populations of recorded species. The maximum population density of the most abundant species, M. galathea e.g., is 2.5–5x greater than the maximum population density of other of the aforementioned grassland species and 50–250x greater than a third of the species with the lowest maximum population densities. In the fi rst third of species (Fig. 4), two are threatened, Melitaea aurelia and Zeryn- thia polyxena, with maximum population densi- ties of 14 and 6 adults/month/1 km of combined habitat type, which means that their populations are relatively abundant in comparison with other species. The fi nding that ecological generalists predominate in the fi rst third of species, while the two threatened species are ecological spe- cialists relying on dry or semi-dry abandoned and extensively or semi-intensively managed grasslands and dry forest edges, also indicates the importance of the study area for both men- tioned species. Ecologically specialised species in the fi rst third include also the monofagous spe- cies Satyrium w-album, whose caterpillars feed on the leaves of elm (Ulmus spp.). This is an ar- boreal species, which means that, because of the behaviour of the adults (staying in the crowns), it is diffi cult to detect in a transect counts. The high assessed maximum population density of the species indicates that the study area, primar- ily dry woodland rides, is important for maintain- Figure 5: Distribution of the number of species with maximum population densities by combined habitat type for the species recorded along the River Sava between Krško and the state border in 2008. (for abbreviations of the combined habitat types, see Tab. 1) Slika 5: Porazdelitev števila vrst z maksimalnimi populacijskimi gostotami po zbirnih habitatnih tipih za vrste zabeležene ob reki Savi med Krškim in državno mejo v letu 2008. (za razlago okrajšav zbirnih habitatnih tipov glej Tab. 1) 86 Acta Biologica Slovenica, 53 (2), 2010 ing its populations. The main reason for high number of observed adults on the dry woodland rides were the extensive stands of annual fl ea- bane (Erigeron annus) serving as nectar plant. Three threatened species (Apatura ilia, Plebejus idas, Lycaena dispar), with maximum popula- tion densities of 1-2 adults/month/1 km of com- bined habitat type belong in the second third of recorded species, and 5 threatened species (Pieris mannii, Carcharodus alceae, Melitaea britomartis, Lycaena hippothoe, Plebejus argy- rognomon), with maximum population densities ≤ 1 adult/month/1 km of combined habitat type in the last third (Fig. 4). The number of species with maximum pop- ulation density in combined habitat type is not in correlation with the length of the combined habitat type (τ = 0.015, P>0.9). Considering this fi nding, the distribution of maximum population densities of species by combined habitat types (Fig. 5) directly indicates the importance of indi- vidual combined habitat types for the existence of populations of butterfl ies in the study area. The recorded species achieved the highest popu- lation densities in 17 combined habitat types. Four combined habitat types in which none of the recorded species had the highest density are intensively cultivated grasslands, arable fi elds and woodland edges and rides on moist ground (Tab. 1). On dry grasslands (extensive, semi- intensive, abandoned) 24 (35%) species had highest population densities, 18 (26 %) species in ruderal communities, 13 (18 %) species on woodland rides, 8 (12 %) species on woodland edges, 4 (6 %) of species on mesophilous semi- intensively managed grasslands and one species each on common reed bed along semi-intensi- vely managed moist mesotrophic grassland, on damp scrub and on woodland rides on moist ground overgrown with riverine shrubs and tall herb communities (Fig. 5). Threatened species We recorded 10 threatened species in the study area in 2008 (Tab. 2), which is 18% of the threatened butterfl y species in Slovenia. The species Lycaena dispar and Zerynthia polyxena are on the list of Annexes of the Habitats Direc- tive and the Bern Convention, which include 14 (Habitats Directive) and 13 (Bern Convention) of the butterfl y species of Slovenia; on the Red List of the Lepidoptera of Slovenia, they are classifi ed in the category of vulnerable species (36 butterfl y species); they are among protected animal spe- cies of Slovenia, whereby the animals (27 species of butterfl y) and their habitats (26 species of but- terfl y) are protected. The remaining eight threat- ened species on the Red List of the Lepidoptera of Slovenia are in the category of vulnerable spe- cies. Two (Melitaea aurelia, M. britomartis) of them are on the European Red List of Butterfl ies in the category of potentially threatened species (NT) and the remaining six among species for which there is a low risk of extinction (LC). The index of distribution indicates (Tab. 2) that 6 threatened species (A. ilia, C. alceae, L. dispar, L. hippothoe, P. argyrognomon, P. idas) are rare or very locally distributed in the study area. Threatened species occupy 17 combined habitat types (Fig. 6). Four combined habitat types in which none of the threatened species were recorded are arable fi elds, common red bed, woodland rides on moist ground overgrown with riverine shrubs and tall herb communities and dry woodland rides. The number of threatened species in com- bined habitat type is in signifi cant positive cor- relation with the length of the combine habitat type (τ = 0.406, P<0.05), the species richness of the combined habitat type (τ = 0.566, P<0.01), total population density (τ = 0.645, P<0.001) and the number of species with the maximum population density in the combined habitat type (τ = 0.479, P<0.01). The number of threatened species with maximum population densities in combined habitat type is not in correlation with the length of the combine habitat type (τ = 0.158, P>0.4) or the species richness of the combined habitat type (τ = 0.214, P>0.2). Seven threatened species were recorded on extensively managed dry grasslands (STeB) and on abandoned dry grasslands with tall oat- grass overgrown with autochthonous woody species (STzS). These are xerothermophilous or mesophilous thermophilous species: C. alceae, M. aurelia, M. britomartis, P. mannii, P. argy- rognomon, P. idas and Z. polyxena. Four xero- thermophilous species are the most abundant in these habitats (Tab. 2). The species M. aurelia 87Čelik: Conservation assessment of the butterfl y fauna along the River Sava Table 2: List of threatened species recorded in transect lines along the River Sava between Krško and the state border in 2008. Threat categories, number of habitat types in which species appeared, index of distribu- tion of species, maximum population density of species and combined habitat types in which a species achieves maximum population density are shown. (for abbreviations of the combined habitat types, see Tab. 1) Tabela 2: Seznam ogroženih vrst, zabeleženih na transektih ob reki Savi med Krškim in državno mejo v letu 2008. Prikazane so kategorije ogroženosti vrst, število zbirnih habitatnih tipov, v katerih je bila vrsta opažena, indeks razširjenosti vrst, maksimalna populacijska gostota vrst ter zbirni habitatni tip, v katerem vrsta dosega maksimalno populacijsko gostoto. (za razlago okrajšav zbirnih habitatnih tipov glej Tab. 1) Species (1) RS (2) RSE (3) UZŽV (4) FFH (5) BERN No. of (6) HT Index of distribution Maximum population density HT in which a species achieves maximum population density Apatura ilia V LC 5 3 0,23 Rgmz Carcharodus alceae V LC 3 3 0,09 Rgmz Lycaena dispar V LC 1, 2 II, IV II 3 2 0,15 Rg Lycaena hippothoe V LC 1 1 0,04 MTpA Melitaea aurelia V NT 9 7 1,39 STeB Melitaea britomartis V NT 3 4 0,07 STeB Pieris mannii V LC 4 5 0,12 MTpA Plebejus argyrognomon V LC 1 1 0,04 STeB Plebejus idas V LC 4 3 0,16 STzS Zerynthia polyxena V LC 1, 2 IV II 9 7 0,58 GRsT (1) RS = Red list of Lepidoptera of Slovenia (Uradni list RS 82/2002) (2) RSE = European Red list of Butterfl ies (Van Swaay s sod. 2010) (3) UZŽV = Decree on protected wild animal species (Uradni list RS 46/2004) (4) FFH = Council Directive 92/43/EEC on the Conservation of Natural Habitats and of Wild Fauna and Flora (5) BERN = Convention on the Conservation of European Wildlife and Natural Habitats (6) HT = combine habitat type has the most numerous populations on extensive, semi-intensive and abandoned dry grasslands with erect brome and dry woodland edges, where autochthonous plant species predominate. The food plants of the caterpillars are ribwort plantain (Plantago lanceolata), various species of speed- well (Veronica spp.) and cow-wheat (Melampy- rum spp.) (Beneš and Konvička 2002a). In the northern part of the study area (northeast of the settlement of Brege, southwest of the settlement of Zgornji Obrež), where there are extensive mo- saics of extensive and abandoned dry grasslands in early succession stages, there are very good living conditions for the species, so their popu- lations are relatively numerous in comparison with other butterfl y species (Fig. 4). The species M. britomartis appears mainly on extensive and abandoned dry grasslands with erect brome. The food plants of the caterpillars are large speedwell (Veronica teucrium), yellow rattle (Rhinantus minor) (Ebert and Rennwald 1993a, Beneš and Konvička 2002b) and ribwort plantain (Plantago 88 Acta Biologica Slovenica, 53 (2), 2010 lanceolata) (SBN 1991, Tolman and Lewing- ton 1997). The species P. argyrognomon was recorded in 2008 in only one location, in the southern part of the area, south of the settlement of Mihalovec, on extensive dry grassland with erect brome. The food plants of the caterpillars are crown vetch (Coronilla varia) and liquorice milkvetch (Astragalus glycyphyllos) (Ebert and Rennwald 1993b, Beneš 2002a). In research in 2001 (Rebeušek 2001), the species was found only in the northern part of the area, on the right bank of the Sava, east of the settlement of Brege. The species P. idas appears on extensive, semi- intensive and abandoned dry grasslands with erect brome. The caterpillars feed on various species of the legume family (Fabaceae), above all black medic (Medicago lupulina), red clover (Trifolium pratense), silky leaf woadwaxen (Ge- nista pilosa), bird’s-foot trefoil (Lotus cornicu- latus), honey clover (Melilotus albus), common kidney vetch (Anthyllis vulneraria), common broom (Sarothamnus scoparius) (Beneš 2002b), and common sea-buckthorn (Hippophaë rham- noides) from the oleaster family (Elaeagnaceae) (Huemer 2004). On semi-intensively managed grasslands seven threatened species were recorded, 2 of which achieve maximum population density on mesophilous grasslands (MTpA) (Tab. 2). The hygromesophilous species L. hippothoe was only recorded on mesophilous grassland with tall oat-grass south of the settlement of Loče. Figure 6: Importance of combined habitat types for threatened species recorded in transect lines along the River Sava between Krško and the state border in 2008. Combined habitat types inhabited by threatened spe- cies (white columns) and combined habitat types in which threatened species have maximum population densities (black columns) are shown. (for abbreviations of the combined habitat types, see Tab. 1) Slika 6: Število ogroženih vrst po zbirnih habitatnih tipih v transektnih linijah ob reki Savi med Krškim in državno mejo v letu 2008. Belo obarvani stolpci označujejo zbirne habitatne tipe, ki jih poseljujejo ogrožene vrste, črno obarvani stolpci pa zbirne habitatne tipe v katerih imajo ogrožene vrste maksimalne populacijske gostote. (za razlago okrajšav zbirnih habitatnih tipov glej Tab. 1) 89Čelik: Conservation assessment of the butterfl y fauna along the River Sava The food plants of the caterpillars are common sorrel (Rumex acetosa) (Ebert and Rennwald 1993b, Weidenhoffer and Fric 2002), sheep’s sorrel (R. acetosella) and bistort (Polygonum bistorta) (SBN 1991). The xerothermophilous species P. mannii appears on semi-intensive and abandoned dry grasslands and on mesophilous semi-intensive grasslands – such as are a mosaic of central European xerophilous lowland grass- lands on relatively dry soils with erect brome and intensively cultivated grasslands. The cat- erpillars feed on garlic cress (Peltaria alliacea), candytuft species (Iberis spp.) as well as other species from the crucifer family (Brassicaceae) (SBN 1991, Beneš 2002c, Huemer 2004). Ruderal areas are inhabited by 3 threatened species, which also achieve maximum population density in these habitats (Tab. 2). The mesophi- lous species A. ilia lives on light rides of riverine forests and on abandoned ruderal areas near the woody riverine vegetation. The food plants of the caterpillars are aspen (Populus tremula), black (P. nigra) and white (P. alba) poplar and willow, above all goat willow (Salix caprea) and white willow (S. alba) (Ebert and Rennwald 1993a, Vrabec 2002, Huemer 2004). The xerothermoph- ilous species C. alceae inhabits abandoned dry grasslands and ruderal areas that are overgrown with shrubs and herb species, among which are the food plants of caterpillars. These are species from the mallow genus, mainly greater musk- mallow (Malvea alcea), musk mallow (M. mos- chata) and common mallow (M. neglecta) and common hollyhock (Alcea rosea) (Ebert and Rennwald 1993b, Beneš 2002d). The hygrophi- lous species L. dispar appears mainly in ruderal areas that are either predominantly bare and only partially overgrown with tall herbs or overgrown with shrubs and herbs, and on semi-intensively managed dry grasslands with tall oat-grass. The food plants of the caterpillars are water dock (Ru- mex hydrolapathum), Scottish dock (R. aquati- cus), broad-leaved dock (R. obtusifolius), curled dock (R. crispus) and common sorrel (R. acetosa) (Ebert and Rennwald 1993b; SBN 1991). On woodland edges three threatened spe- cies were recorded, the species Z. polyxena has the most numerous populations in these habi- tats (Tab. 2). It inhabits sunny woodland edges on both dry and moist ground, abandoned dry grasslands, semi-intensive mesophilous grass- lands and extensive or semi-intensive dry grass- lands, in which there are sunny hedges, which are favourable habitats for the food plants of the caterpillars - birthwort (Aristolochia clematitis). Important areas for butterfl ies The conservation evaluation of each com- bined habitat type based on the ranking of the combined habitat types according to six criteria (i.e. species richness, median of population den- sities of species observed in the treated combined habitat type, total population density, number of species with maximum population density in the treated combined habitat type, number of threat- ened species in the combined habitat type, num- ber of threatened species with maximum popula- tion density in the treated combined habitat type) shows (Fig. 7) that 8 combined habitat types have median of ranks lower than 10,5. These com- bined habitat types are defi ned as most important for butterfl y species in the study area. The differ- ence in ranks between the combined habitat types with median of ranks lower than 10,5 and com- bined habitat with median of ranks higher than 10,5 is highly signifi cant (Mann–Whitney Z = –7.34; P<0.001). The most important combined habitat types for butterfl ies in the study area are extensively managed dry grasslands (STeB), abandoned dry grasslands in early succession stages (STzS, STzT), semi-intensively managed dry grasslands with erect brome (Bromopsis erecta) and tall oatgrass (Arrhenathereum ela- tius) (STpBA), semi-intensively managed me- sophilous grasslands with tall oatgrass (MTpA), ruderal areas overgrown with shrub and herb spe- cies (Rgmz) and dry woodland rides and edges in which allochtonous and autochtonous plant species are present (GPsT, GRsT). On the basis of the distribution of the most important combined habitat types we identifi ed four important areas for butterfl y species in the study area: (1) Žadovinek–Brege, (2) Čateške Toplice–Prilipe, (3) Loče–Mihalovec and (4) Zgornji Obrež (Fig. 8). The number of species in important areas ranges between 57% (Loče– Mihalovec) to 84% (Žadovinek–Brege) of the species richness of the study area (Tab. 3). Con- sidering the maximum population densities of 90 Acta Biologica Slovenica, 53 (2), 2010 threatened species, the area Žadovinek–Brege is most important for two species (M. aurelia, P. idas), the area Čateške Toplice–Prilipe for four species (A. ilia, C. alceae, P. mannii, L. dispar), the area Loče–Mihalovec for two species (L. hippothoe, P. argygnomon), and the area Zgornji Obrež also for two species (M. britomartis, Z. polyxena) (Tab. 3). Designated areas for butterfl ies are impor- tant (i) for preserving high butterfl y species rich- ness in the mainly intensively used agricultural landscape along the lower course of the River Sava between Krško and the state border with Croatia, and (ii) for preserving viable popula- tions of threatened and other ecologically spe- cialised, rare or locally distributed species (Tab. 3) and preventing their isolation in the region of sub-pannonian southeast Slovenia. Acknowledgement This research was a part of the project “Pre- gled živalskih in rastlinskih vrst, njihovih habi- tatov ter kartiranje habitatnih tipov s posebnim ozirom na evropsko pomembne vrste, ekološko pomembna območja, posebna varstvena obmo- čja, zavarovana območja in naravne vrednote na vplivnem območju predvidenih HE Brežice in HE Mokrice« fi nancially supported by the Hydro Power Plants on the lower Sava River (Hidroelektrarne na Spodnji Savi, d.o.o.). The author is grateful to Vesna Grobelnik for making the maps (Fig. 1 and 8), and to Marijan Govedič, Branko Vreš and two anonymous referees for notable improvement of the manuscript. Figure 7: Nature conservation value of each combined habitat type in the study area along the River Sava between Krško and the state border, based on the ranking of the combined habitat types according to six criteria. (for explanation, see Data analysis; for abbreviations of the combined habitat types, see Tab. 1) Slika 7: Naravovarstvena vrednost zbirnih habitatnih tipov v raziskovanem območju ob reki Savi med Krškim in državno mejo, ocenjena z rangiranjem zbirnih habitatnih tipov po šest kriterijih (za razlago glej poglavje Data analysis). (za razlago okrajšav zbirnih habitatnih tipov glej Tab. 1) 91Čelik: Conservation assessment of the butterfl y fauna along the River Sava Name of important area No. of species No. of threatened species Threatened species Population density Combined habitat type Rare / local distrubuted species / ecological specialists Žadovinek – Brege (3.9 km2) 58 6 Carcharodus alceae 0,02 STzS Iphiclides podalirius, Melitaea didyma, Satyrium pruni, S. acaciae, Glaucopsyche alexis, Polyommatus bellargus, Aphantopus hyperantus, Heteropterus morpheus Melitaea aurelia 1,70 STeB Melitaea aurelia 0,50 GRsS Melitaea aurelia 0,26 STzS Melitaea aurelia 0,09 STzT Melitaea aurelia 0,07 STpBA Melitaea britomartis 0,06 STeB Pieris mannii 0,03 STzS Plebeius idas 0,21 STzS Plebeius idas 0,09 STzT Plebeius idas 0,09 STeB Plebeius idas 0,07 STpBA Zerynthia polyxena 0,31 STzT Zerynthia polyxena 0,05 STeB Čateške Toplice - Prilipe (1.2 km2) 48 7 Apatura ilia 0,23 Rgmz Iphiclides podalirius, Melitaea didyma, Satyrium spini, S. w-album, S. acaciae Apatura ilia 0,15 GPvT Apatura ilia 0,14 GRvT Carcharodus alceae 0,09 Rgmz Carcharodus alceae 0,06 GMvT Lycaena dispar 0,07 STpA Lycaena dispar 0,06 Rgmz Melitaea aurelia 0,48 STpBA Melitaea aurelia 0,26 MTpA Melitaea aurelia 0,10 STpA Melitaea britomartis 0,10 STpA Pieris mannii 0,16 STpBA Pieris mannii 0,11 MTpA Zerynthia polyxena 0,16 MTpA Zerynthia polyxena 0,15 STpA Zerynthia polyxena 0,08 STpBA Zerynthia polyxena 0,06 GMvT Table 3: Important areas for butterfl y species along the River Sava between Krško and the state border with Croatia. Species richness, number of threatened species, population densities and combined habitat types of threatened species, and other rare/local distributed species or ecological specialists are shown for each important area. Bold text indicates the maximum population density of threatened species in the important area. (for abbreviations of the combined habitat types, see Tab. 1) Tabela 3: Vrstno bogastvo, število ogroženih vrst, populacijske gostote in zbirni habitatni tipi ogroženih vrst ter druge redke/lokalno razširjene/ekološko specializirane vrste v naravovarstveno pomembnih območjih za dnevne metulje ob reki Savi med Krškim in državno mejo s Hrvaško. S krepkim tiskom je označena maksimalna populacijska gostota ogrožene vrste v območju. (za razlago okrajšav zbirnih habitatnih tipov glej Tab. 1) 92 Acta Biologica Slovenica, 53 (2), 2010 Loče - Mihalovec (1.1 km2) 39 6 Lycaena dispar 0,02 MTpA Limenitis populi, Melitaea didyma Lycaena hippothoe 0,07 MTpA Melitaea britomartis 0,10 STeB Pieris mannii 0,13 MTpA Plebeius argyrognomon 0,19 STeB Zerynthia polyxena 0,05 STeB Zerynthia polyxena 0,03 MTpA Zerynthia polyxena 0,03 GRvVs Zgornji Obrež (0.4 km2) 49 3 Melitaea aurelia 1,54 STzS Iphiclides podalirius, Satyrium pruni, S. spini, S. w-album, Polyommatus bellargus, Aphantopus hyperantus, Heteropterus morpheus Melitaea aurelia 0,84 STzT Melitaea aurelia 0,17 GPsT Melitaea aurelia 0,08 GRsT Melitaea britomartis 0,26 STzS Zerynthia polyxena 0,58 GRsT Zerynthia polyxena 0,51 STzS Zerynthia polyxena 0,07 STzT Figure 8: Important areas for butterfl y species in the study area along the lower course of the River Sava between Krško and the state border with Croatia. Slika 8: Naravovarstveno pomembna območja za dnevne metulje v raziskovanem območju ob spodnjem toku reke Save med Krškim in državno mejo s Hrvaško. 93Čelik: Conservation assessment of the butterfl y fauna along the River Sava Povzetek Dnevni metulji so ena izmed najpo- membnejših nevretenčarskih bioindikatorskih skupin. So edini nevretenčarski takson za kate- rega je trenutno možno oceniti stopnjo upadanja številčnosti populacij in so reprezentativni in- dikatorji trendov opaženih pri večini kopenskih žuželk, ki predstavljajo približno dve tretjini vseh svetovnih vrst. Evropska okoljska agencija jih je, z namenom zmanjšanja upadanje biodiver- zitete do leta 2010, v okviru projekta »Stream- lining European 2010 Biodiversity Indicators« leta 2007 uvrstila med evropske biodiverzitetne indikatorje (SEBI 2010 Indicators), s katerimi se vrednoti splošne trende v stanju biodiverzitete v Evropi. Dnevni metulji so bili izbrani kot indika- torji za oceno stanja in trendov evropskih travišč (European Grassland Butterfl y Indicator), ki so najpomembnejši habitati evropskih vrst dnevnih metuljev, saj jih poseljuje več kot polovica (280 vrst, 57%) evropskih vrst. V južnem delu Ekološko pomembnega ob- močja »Sava od Radeč do državne meje« (EPO Id: 63700), med Krškim in državno mejo s Hr- vaško, smo v letu 2008 izvedli inventarizacijo favne dnevnih metuljev z namenom opredeliti naravovarstveno najpomembnejša območja za dnevne metulje v EPO. Raziskovano območje s površino 33 km2 je obsegalo približno 1 km ši- rok pas na obeh bregovih reke Save. Območje je pretežno kmetijska krajina, saj 66 % površine zavzemajo njive in različno gospodarjeni travni- ki. Prevladujejo njive in intenzivno gospodarjeni travniki (46 % površine raziskovanega območja), medtem ko ekstenzivno gospodarjeni suhi trav- niki in opuščena suha travišča v zgodnjih fazah zaraščanja zavzemajo le 7 % površine območja. Predvsem na vlažnih rastiščih (npr. gozdni robovi in poti, obrežja reke, gramoznic in mrtvic), so pri- ostne invazivne alohtone rastlinske vrste, ki kot samostojni sestoji predstavljajo kar 2 % površine območja, v kombinaciji z drugimi habitatnimi tipi po so razširjene na veliko večji površini. Favno dnevnih metuljev smo inventarizirali s standardizirano metodo transektnega popisa na 12 transektih. Na vsakem transektu smo šteli od- rasle osebke v časovnem intervalu pribl. 14 dni (1x v aprilu, 2x v maju, 1x v juniju, 2x v juliju, 1x v septembru). Vsak transekt je bil razdeljen na odseke, vsak odsek je predstavljal določen zbirni habitatni tip. Na transektih s skupno dolžino 19,2 km smo opredelili 21 zbirnih habitatnih tipov. Z indeksom razširjenosti smo identifi cirali splošno razširjene ter redke/lokalno razširjene vrste v raziskovanem območju. V procesu opredelitve naravovarstveno pomembnih območij za dnevne metulje smo najprej ovrednotili zbirne habitatne tipe v transektih. Za določitev naravovarstvene vrednosti vsakega zbirnega habitatnega tipa smo uporabili naslednje parametre: vrstno bogastvo zbirnega habitatnega tipa, populacijska gostota vrste v zbirnem habitatnem tipu, celokupna po- pulacijska gostota v zbirnem habitatnem tipu, število vrst z maksimalno populacijsko gostoto v zbirnem habitatnem tipu, število ogroženih vrst v zbirnem habitatnem tipu in število ogroženih vrst z maksimalmo populacijsko gostoto v zbirnem habitatnem tipu. S Kendallovim korelacijskim koefi cientom smo ugotavljali povezanost med uporabljenimi parametri. Parametre smo upora- bili kot kriterije v nadaljnem postopku evalvacije tako, da smo v okviru vsakega kriterija (parame- tra) zbirne habitatne tipe rangirali (od 1 do 21) glede na vrednost parametra. Rang 1 smo pripi- sali zbirnemu habitatnemu tipu z največjo in rang 21 (v primeru različnih rangov) zbirnemu habi- tatnemu tipu z najmanjšo vrednostjo parame- tra. Zbirne habitatne tipe z mediano vrednostjo rangov nižjo od 10, 5 smo defi nirali kot najpo- membnejše za dnevne metulje. Naravovarstveno pomembna območja za dnevne metulje smo nato opredelili na podlagi razširjenosti najpomemb- nejših zbirnih habitatnih tipov v raziskovanem območju, pri čemer smo vključili tudi habitatne tipe (podobne po fl oristični sestavi in strukturi vegetacije ter gospodarjenju najpomembnejšim zbirnim habitatnim tipom) izven transektnih li- nij. Zabeležili smo 69 vrst dnevnih metuljev (38 % favne dnevnih metuljev Slovenije), med njimi 10 v nacionalnem ali evropskem merilu ogroženih vrst (A. ilia, C. alceae, L. dispar, L. hippothoe, M. aurelia, M. britomartis, P. manni, P. argyrognomon, P. idas, Z. polyxena). 19 vrst je splošno razširjenih (ekološko nespecializirane vrste), 21 vrst je redkih/lokalno razširjenih, med njimi je 6 ogroženih vrst. Vrstno najbogatejši zbirni habitatni tipi so ekstenzivno gospodarje- ni suhi travniki (48 vrst), opuščena suha travišča 94 Acta Biologica Slovenica, 53 (2), 2010 (46 oz. 41 vrst), polintenzivno gospodarjeni suhi travniki (40 vrst) in vlažne gozdne poti (36 vrst). Registrirane vrste imajo največje populacijske gostote na ekstenzivnih, polintenzivnih in za- raščajočih se suhih travnikih (24 vrst), na rude- ralnih površinah (18 vrst), na gozdnih poteh (13 vrst) in gozdnih robovih (8 vrst). Na intenzivno gospodarjenih travnikih, njivah ter vlažnih goz- dnih robovih in poteh nobena vrsta nima naj- številčnejših populacij. Največje populacijske gostote imajo splošno razširjene travniške vrste (M. galathea, C. arcania, M. jurtina, M. dryas, T. lineola, C. crocea, M. athalia, C. pamphilus). Primerjava maksimalnih populacijskih gostot med vrstami kaže, da v raziskovanem območju živijo pomembne populacije dveh ogroženih vrst, M. aurelia in Z. polyxena, ter ekološko specializirane vrste S. w-album. Ogrožene vrste imajo največje populacijske gostote na eksten- zivnih in zaraščajočih se suhih travnikih, mezo- fi lnih polintenzivno gospodarjenih travnikih, ru- deralnih združbah in na suhih gozdnih robovih. Osem zbirnih habitatnih tipov je z naravo- varstvenega vidika najpomembnejših za dnevne metulje v raziskovanem območju, to so: eksten- zivno gospodarjeni suhi travniki, opuščena suha travišča v zgodnjih fazah zaraščanja, polinten- zivno gospodarjeni travniki s pokončmim sto- klascem in visoko pahovko, polintenzivno go- spodarjeni mezofi lni travniki z visoko pahovko, ruderalne površine zaraščajoče se z grmovjem in zeliščnimi vrstami ter gozdni robovi in poti na suhih rastiščih. Na podlagi njihove razširje- nosti smo v raziskovanem območju opredelili 4 naravovarstveno pomembna območja za dnevne metulje (Žadovinek–Brege, Čateške Toplice– Prilipe, Loče–Mihalovec in Zgornji Obrež), s skupno površino 6,6 km2. Število vrst v pre- dlaganih območjih dosega 57 % (Loče–Miha- lovec) do 84 % (Žadovinek–Brege) vrstnega bogastva raziskovanega območja. Upoštevaje maksimalno populacijsko gostoto ogroženih vrst v predlaganih območjih, je območje Žado- vinek–Brege najpomembnejše za dve vrsti (M. aurelia, P. idas), območje Čateške Toplice–Pri- lipe za štiri vrste (A. ilia, C. alceae, P. mannii, L. dispar), območje Loče–Mihalovec za dve (L. hippothoe, P. argygnomon) in območje Zgornji Obrež za dve ogroženi vrsti (M. britomartis, Z. polyxena). 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Biological knowledge of Slovenian students in the living systems content area in PISA 2006 Znanje slovenskih učencev na vsebinskem področju Živi sistemi v raziskavi PISA 2006 Jelka Strgar University of Ljubljana, Biotechnical faculty, Department of Biology, Večna pot 111, SI-1000 Ljubljana, Slovenia *correspondence: jelka.strgar@bf.uni-lj.si Abstract: In the PISA 2006 program Slovenian students exceeded the international average of scientifi c achievements, while in the fi eld of living systems performed below the national average. The purpose of our analysis was to determine in which areas of biology Slovenian students are weak and in which they are strong, and to determine their biological knowledge in comparison to 23 other European countries, which, by various criteria, are the most comparable to Slovenia and interesting for us. We analysed 24 tasks that tested the biological knowledge in PISA 2006. We found that student achievement in Slovenia wasn’t poor in any of the tested biological top- ics. There are two factors that can explain lower achievements resolving individual tasks: (1) Question type: Students poorly answered the open-constructed response questions that require independent formulation of coherent responses. They were more successful with complex multiple choice questions and multiple choice questions, where they had to choose the correct answer from several given suggestions; (2) Diffi culty: Students didn’t perform as well with higher cognitive level tasks which required using knowledge. This suggests that in the period when these students were receiving elementary education, their biology teaching focused on developing knowledge of biological content (knowledge of science) and competence explaining phenomena scientifi cally, while the development of other knowledge (knowledge about science) and the competence to draw evidence-based conclusions (using knowledge) was inadequate. It is therefore in the hands of all in Slovenia involved in biological education, especially teachers of biology, to give students more opportunities for problem-solving, rather than only focusing on content. Key words: PISA 2006, biology, science, knowledge, competencies, Slovenia Izvleček: Slovenski učenci so v raziskavi PISA 2006 presegli mednarodno povprečje naravoslovnih dosežkov, vendar so bili na področju biologije slabši kot na drugih naravoslovnih področjih. Namen naše analize je bil ugotoviti, na katerih področjih biologije imajo slovenski učenci šibko in na katerih močno znanje ter kakšno je njihovo biološko znanje v primerjavi s 23 drugimi evropskimi državami, ki so po različnih kriterij najbolj primerljive s Slovenijo oziroma zanimive za nas. Analizirali smo 24 nalog, ki so v PISA 2006 preverjale biološko znanje. Ugo- tovili smo, da so bili dosežki učencev v Sloveniji pri vseh preverjenih bioloških temah dobri. Dejavnika, s katerima je bilo mogoče pojasniti slabše dosežke pri posameznih nalogah, sta bila tip naloge in njena težavnost. Učenci so slabše reševali naloge odprtega tipa, pri katerih so morali samostojno oblikovati smiseln odgovor. Uspešnejši so bili pri nalogah kompleksnega izbirnega tipa in izbirnega tipa, kjer so morali med danimi odgovori izbrati pravilnega. Učenci so slabše reševali naloge višje kognitivne stopnje, ki so zahtevale uporabo znanja. Iz tega sklepamo, da je bil v obdobju, ko so bili ti učenci vključeni v osnovnošolsko izobraževanje, pri biologiji poudarek na razvijanju poznavanja biološke vsebine (knowledge of science) in kompetence znanstveno razlaganje pojavov (explaining phenomena scientifi cally), pomanjkljivo pa je bilo ACTA BIOLOGICA SLOVENICA LJUBLJANA 2010 Vol. 53, [t. 2: 99–108 100 Acta Biologica Slovenica, 53 (2), 2010 Introduction In 2006 Slovenia participated for the fi rst time in the Program for International Student Assessment (PISA). The fi rst PISA study in 2000 focused on reading literacy, the second in 2003 on mathematical literacy, and the third in 2006 on scientifi c literacy, which means the pro- portion of science tasks was signifi cantly larger than the proportion of mathematical and read- ing tasks. The result was the fi rst extensive and complete collection of internationally compara- ble data on the science competencies of students in 57 countries from all over the world. The PISA differs from other assessments (for example from TIMSS, which is also interna- tional) in that it is not directly linked to the sci- ence curricula of any of the participating coun- tries, but collects data about the competences that students will need for effective functioning in their adult professional and personal life, and are important both for individuals and for the whole society. According to the OECD defi ni- tion (PISA 2006 2007), scientifi c literacy is “a capacity to use scientifi c knowledge, to identify questions, explain scientifi c phenomena, and to draw evidence-based conclusions in order to un- derstand and help make decisions about science- related issues.” The term ‘literacy’ indicates focusing on the application of knowledge and abilities (Bybee 2008). The tasks are structured in a manner that enables the assessment of the ability to solve tasks related to life situations, and hence not limited to knowledge of a specifi c subject (Štraus et al. 2007). The PISA includes students who, in most participating countries, are approaching the end of compulsory education. The age of participants is precisely specifi ed, ranging from 15 years and 3 months to 16 years and 2 months. This means that the PISA 2006 students were born in 1990. In the study 6,595 Slovenian students took part (Štraus et al. 2007). Science achievements of Slovenian students in PISA 2006 Slovenian students in PISA 2006 scored the average science achievement of 519 points (Tab. 1), therefore exceeding the average achievement of 500 points of all the participating students by 19 points (Achievements of students in PISA 2006). The PISA 2006 science assessment evalu- ated students’ knowledge in two areas: knowl- edge component and competency component. Knowledge component comprises two catego- ries - knowledge about science (which includes scientifi c inquiry and scientifi c explanations) and knowledge of science (which includes Earth and space systems, living systems, and physi- cal systems). The results of Slovenian students in the category of knowledge about science was as many as 9 points below the national average, while in the category of knowledge of science they showed a good knowledge in the areas of Earth and space systems (15 points above av- erage), and physical systems (12 points above average). In the area of living systems, cover- ing biology, Slovenian students ranked lower than the national average by 2 points (Tab. 1). On the international scale the biggest difference between knowledge about science and knowl- edge of science in PISA 2006 was 29 points. In the case of Slovenian students this difference was 17 points in favour of knowledge of sci- ence. These results are not a clear indicator of whether one or the other of these two categories of knowledge leads to higher overall scientifi c achievements (Štraus et al. 2007). In the competency component (explaining phenomena scientifi cally) our students achieved 4 points above the international average (Tab. 1). The other two competences are less devel- oped, but the differences are not signifi cant: in the competence of identifying scientifi c ques- tions they rank 2 points below the international razvijanje znanja o naravoslovnih znanostih (knowledge about science) ter kompetence uporaba naravoslovno-znanstvenih podatkov in preverjenih dejstev (using scientifi c evidence). V rokah vseh, ki se v Sloveniji ukvarjajo z biološkim izobraževanjem, predvsem pa učiteljev biologije, je, da usposobijo učence tudi s tega vidika, ne samo z vidika vsebinskega znanja. Ključne besede: PISA 2006, biologija, naravoslovje, znanje, kompetence, Slovenija 101Strgar: Biological knowledge of Slovenian students in living systems content average, and in the competence of drawing evidence-based conclusions 3 points below the international average. In many countries with the highest achievements the students are particular- ly successful in the competence of drawing evi- dence-based conclusions. It turns out that in all of the successful countries the students are quite strong in this competence. The conclusion is that the ability to draw evidence-based conclusions, i.e. interpret and apply scientifi c data and veri- fi ed facts, is particularly characteristic of highly developed science literacy (Štraus et al. 2007). Table 1: Comparison of the overall achievements of Slovenian students in science with the achievements of indi- vidual scientifi c competencies and in the various science areas (adapted from Štraus et al., p. 51). Tabela 1: Primerjava skupnega dosežka slovenskih učencev pri naravoslovju z dosežki pri posameznih naravoslovnih kompetencah in na posameznih naravoslovnih področjih (povzeto po Štraus et al., p. 51). Objective analysis The purpose of our analysis was to deter- mine the strong and weak areas of biological knowledge of Slovenian students, and where this knowledge ranks in comparison with other countries. These results allow us to extract the characteristics of biology teaching in Slovenian primary schools during the period when these students were in elementary school, and identify any necessary changes. Material and methods Tasks included in the analysis Of the total set of tasks in the area of living systems, i.e. tasks related to biological topics in PISA 2006, one part did not test the knowledge of (biology) science, but verifi ed other compo- nents of scientifi c literacy (knowledge about sci- ence, identifying scientifi c questions, explaining phenomena scientifi cally, drawing evidence- based conclusions) based on biological content. These tasks are not included in our analysis be- cause in this case biology was just a basis from which students demonstrated their knowledge and competencies not linked specifi cally to biol- ogy, but to any scientifi c topic. Students had to show knowledge of science in the content area of living systems in 27 tasks, of which 24 were used for our detailed analysis. Countries selected for comparison with Slovenian achievements There were 57 countries participating in PISA 2006, while for the purpose of our analy- Overall achieve- ment The difference between the overall achieve- ment and the achievements on individual rankings Science 519 Competency component 1. Identifying scientifi c questions 2. Explaining phenomena scientifi cally 3. Drawing evidence-based conclusions Knowledge component 1. Knowledge about science 2. Knowledge of science - Earth and space systems - Living systems - Physical systems -2 4 -3 -9 15 -2 12 102 Acta Biologica Slovenica, 53 (2), 2010 sis we presented the comparative performance of Slovenia and 23 other European countries, which are, according to various criteria (similar cultural background and history), most compa- rable with Slovenia and interesting for us. These were: Austria, Belgium, Switzerland, Czech Republic, Germany, Denmark, Spain, Estonia, Finland, France, Great Britain, Hungary, Ire- land, Iceland, Italy, Lithuania, Latvia, Neder- land, Norway, Poland, Portugal, Slovakia, and Sweden. Data analysis The overall achievements of the selected 23 countries in each of the 24 questions that tested the knowledge of (biology) science were calcu- lated. The signifi cant differences between the achievements of Slovenian students with regard to biological topic and the type of question were determined by one way ANOVA and Chi-square test (P < 0.05). Results and discussion 1. General achievements of Slovenian students in the area of living systems The achievements of Slovenian students in 24 biological questions ranged from 22.4% to 89.5% (Fig. 1). Within this, the majority of tasks placed between 45.0% and 77.9%. The upward deviation was demonstrated in one task with 89.5% and downward in a group of fi ve tasks where the achievement was less than 37%. 2. Achievements of Slovenian students in the area of living systems in terms of content (biological topic) The 24 analysed tasks tested the knowledge of six areas of biology. The tasks were classifi ed into four groups that contained 4 to 8 tasks each. We formed thematic groups: (1) ecology with 7 tasks; (2) physiology with 4 tasks; and (3) health with 8 tasks. The remaining three biological ar- Figure 1: Achievements of Slovenian students in 24 tasks testing competency knowledge of science based on the content area of living systems Slika 1: Dosežki slovenskih učencev pri 24 nalogah, ki so preverjale kompetenco znanje naravoslovja z vsebin- skega področja živi sistemi. 103Strgar: Biological knowledge of Slovenian students in living systems content Figure 2: Medians, quartiles, and extreme values of the achievements of Slovenian students in tasks testing the knowledge of science based on the content area of living systems. Slika 2: Mediane, kvartili in skrajne vrednosti dosežkov slovenskih učencev pri nalogah, ki so preverjale znanje naravoslovja z vsebinskega področja živi sistemi. Tematska področja: ekologija, fi ziologija, zdravje, drugo. eas were combined with the fourth group of var- ied content with 5 tasks. Such a division of tasks is not fully in line with the offi cial topics of the PISA study in the area of living systems, which are: Health, Natural Resources, Environmental Quality, Hazards, and Frontiers of Science and Technology (PISA 2006 2007), but it seemed sensible from the standpoint of biological con- tent, and, additionally, it gave us the minimum number of tasks for statistical data processing in each group. The achievements of Slovenian students are considerably dispersed (Fig. 2) in all three subject areas (ecology, physiology and health). In the fourth group, which includes tasks from various other areas of biology, the achievements are more level, which is attributed to the fact that all the tasks in this group were of the same type, in this case complex multiple choice ques- tions. Students gave the best answers in the ar- eas of physiology and health (median = 60.5 and 59.8), while the achievement in ecology is lower (median = 45.7), but the differences among the achievements in individual thematic areas are not statistically signifi cant (ANOVA, P > 0.05) 3. Achievements of Slovenian students in the area of living systems with regard to the type of question The 24 tasks used in our analysis were pre- sented in three different ways (8 tasks each): in the form of the complex multiple choice ques- tions (a set of options from which the student chooses one); in the form of multiple choice questions (4 or 5 suggested answers of which 104 Acta Biologica Slovenica, 53 (2), 2010 Figure 3: Medians, quartiles, and extreme values of the achievements of Slovenian students in tasks testing the knowledge of science based on the content area of living systems. CMCQ – complex multiple choice questions, MCQ – multiple choice questions, Open – open-constructed response questions. Slika 3: Mediane, kvartili in skrajne vrednosti dosežkov slovenskih učencev pri nalogah, ki so preverjale znanje naravoslovja z vsebinskega področja živi sistemi. Tipi nalog: C – naloge kompleksnega izbirnega tipa, M – naloge izbirnega tipa, O – naloge odprtega tipa. only one is correct); and in the form of open- constructed response questions (which require a longer written answer). Achievements of Slovenian students with regard to the type of task are not so dispersed (Fig. 3) as the achievement with regard to bio- logical topic. Students gave the best answers to multiple choice questions and slightly less appropriate answers to the complex multiple choice questions (median = 69.3 and 57.5), but this difference is not statistically signifi cant (ANOVA; P > 0.05). Students showed signifi - cantly lower achievements answering open- constructed response questions (median = 45.1; ANOVA; P = 0.004). Other research, for example National As- sessment of Knowledge in Slovenia, showed that students have diffi culties in formulating re- sponses to open-constructed response questions, which applies not only to biology, but to all areas (Jagodnik et al. 2009). Poor performance on these questions thus indicates lack of ability to formulate the knowledge into sensible short answers rather than lack of content knowledge. 4. Comparison of Slovenian students’ achieve- ments depending on topic and type, and devia- tions from the average of 24 countries A detailed analysis showed that the achieve- ments of Slovenian students were mainly infl u- 105Strgar: Biological knowledge of Slovenian students in living systems content Figure 4: Achievements o f Slovenian students in 24 tasks testing competency knowledge of science based on the content area of living systems, and relative deviations of these achievements compared to students of 23 European countries. Shown according to the relative deviation value. Slika 4: Dosežki slovenskih učencev pri 24 nalogah, ki so preverjale kompetenco znanje naravoslovja na vsebin- skem področju živi sistemi in relativno odstopanje dosežkov slovenskih učencev v primerjavi z učenci 23 evropskih držav. Prikaz po velikosti relativnega odstopanja. enced by the type of task, and not by the biologi- cal content that a task tested (χ2 test, P = 0.03). Some tasks deviated from these fi ndings, in other words, their result was infl uenced by biological content and not by the task type. Only from those tasks it is therefore possible to identify the strong and weak areas and other characteristics of bio- logical knowledge of Slovenian students. With regard to the biological topic, students were less successful in resolving four tasks: two in ecology, and one in physiology and health. The biological content of these tasks can be found in the Slovenian education curricula for primary school, which means that our students must have been exposed to the content. In our view the content should not have been too diffi cult for the students. But the diffi culty increased as a result of the way the questions were presented. All four questions the Slovenian students had trouble with required the utilisation of knowledge. We concluded that students in Slovenia don’t lack biological knowledge (knowledge of science), but other knowledge and competencies, namely knowledge about science and the competence to draw evidence-based conclusions. We also arrived at the same conclusion when we analysed the relative deviations of the achievements of Slovenian students com- paring to 23 European countries comparable to Slovenia for all 24 tasks testing the knowledge of science in the content area of living systems (Fig. 4). None of the tasks in which the Slov- enian students exceeded the average (right side of the graph) was, in our opinion, cognitively challenging. This means that upward devia- tions of achievements of Slovenian students can be largely explained by lower levels of cogni- tive diffi culty of tasks. On the other hand, it is our view that the tasks Slovenian students per- formed below average (left side of the graph) are cognitively more demanding, though one of them was impossible for Slovenian students to answer since Slovenian schools don’t address the content in the way the question anticipated. Our fi nding that students in Slovenia don’t lack biological knowledge (knowledge of science), 106 Acta Biologica Slovenica, 53 (2), 2010 but other knowledge and competencies, is con- sistent with what Štraus et al. (2007) wrote in the PISA 2006 National report for the entire area of science. They say that in order to effectively solve science tasks a sequence of three processes is required: (1) students must fi rst recognize the problem; (2) use their knowledge to explain phe- nomena scientifi cally; (3) and fi nally interpret and apply the results. Traditional teaching of sci- ence is often directed at another process, namely the scientifi c interpretation of phenomena or, in other words, the acquisition of key science knowledge. From the achievements of Slovenian students Štraus et al. (2007) concluded that the Slovenian elementary schools focus precisely on these work methods. Such teaching doesn’t lead to comprehensive science literacy because later in life the student’s ability to successfully use these data and arguments will be limited despite good command of the theory. In Slovenia, the current elementary school curriculum for the area of living systems, at least at the primary school level, considers the need for students to develop both content knowledge and the process of science, as they followed the recommendations in Benchmarks (American As- sociation for the Advancement of Science 1993). But the curricula for biology at higher levels of primary school do not. They are instead content- focused, with few process goals, and it is there- fore not surprising that our students in PISA 2006 showed relatively good content knowledge of biology (knowledge of science and compe- tence explaining phenomena scientifi cally), and, on the other hand, a considerable lack of other competences and knowledge about science. The PISA 2006 results not only offer insights into the current status, but also allow for predic- tions of how today’s 15 year olds will function as adults citizens who will need to use their knowledge and abilities in new situations and make decisions on issues related to, for exam- ple, the common good of humankind, wellness, the environment, human ecology, and other ap- plications of biology (Bybee 2008). Education in the 21st century should consider the changing circumstances. DeHard Hurd (2001) claims that today’s data is more qualitative than quantitative in nature. The intellectual skills required to do research in today’s biology are mostly those of problem-solving. It is therefore in the hands of all in Slovenia involved in biological education, especially teachers of biology, to give students opportunities to utilise their knowledge and not only focus on content. Conclusions The analysis of Slovenian students’ achieve- ments in 24 questions in the area of living sys- tems in PISA 2006 showed that: 1. The achievements of Slovenian students were good in all of the tested biological topics. 2. There are two factors that could explain the poor performance in specifi c tasks: – Question type: Students were less success- ful in answering open-constructed response questions where they had to independently formulate a coherent response. They were better with the complex multiple choice questions and multiple choice questions, where they had to pick a correct answer among several suggestions – Diffi culty: Students were less successful with higher cognitive level tasks that required the use of knowledge. This sug- gests that in the period, during which the students were given elementary educa- tion, biology teaching focused mainly on developing knowledge of biological content (knowledge of science) and competencies in explaining phenomena scientifi cally, and less on developing other knowledge (knowledge about science) and competences in drawing evidence-based conclusions (use of knowledge). The sample of tasks that we were able to in- clude in the analysis was predetermined by the PISA 2006 structure. Because it’s so small, our fi ndings should be viewed with caution. In ad- dition, due to a small number of available tasks the achievements of the students were analysed only with regard to the content and type of the task, not taking into account a third possible as- pect - profi ciency level. Each task was in fact classifi ed in one of six profi ciency levels on the basis of substantive considerations relating to the nature of the underlying competencies (PISA 2006 2007). 107Strgar: Biological knowledge of Slovenian students in living systems content From the standpoint of biology, a very impor- tant issue remains unresolved: what is the reason that Slovenian students achieved signifi cantly lower results in the area of living systems than in the areas of Earth and space systems, and physi- cal systems. This information could be obtained by an overall interdisciplinary analysis aimed at discovering the essential characteristics of biolo- gy, physics, and chemistry teaching in Slovenian elementary schools. A comparison would point to the aspects of teaching that strongly deviate in biology and are the likely reason for poorer performance of the students. Povzetek Slovenski učenci so v raziskavi PISA 2006 dosegli povprečni naravoslovni dosežek 519 točk, kar presega povprečje vseh sodelujočih držav (500 točk). Znotraj naravoslovja so bili slovenski učenci nadpovprečni na področju fi zike, kemije in geografi je, medtem ko so bili na področju biologije rahlo podpovprečni. Namen naše analize je bil ugotoviti, na katerih področjih biologije imajo slovenski učenci šibko in na katerih močno znanje ter kakšno je njihovo biološko znanje v primerjavi s 23 drugimi evrop- skimi državami, ki so po različnih kriterij najbolj primerljive s Slovenijo oziroma zanimive za nas. Iz celotnega nabora nalog, ki so bile v raziskavi PISA 2006 povezane z biološko tema- tiko, je bil del takih, ki so na bioloških vsebinah preverjale znanje o naravoslovnih znanostih (knowledge about science) in ne naravoslovnega znanja (knowledge of science) samega. Teh na- log nismo analizirali, ker je bila biologija v tem primeru samo tema, na kateri so morali učenci pokazati kompetenci znanstveno razlaganje (scientifi c explanations) ali znanstveno razisko- vanje (scientifi c enquiry), ki nista vezani samo na biologijo, temveč na katerokoli naravoslovno znanost. Kompetenco znanja (knowledge of sci- ence) na področju biologije so učenci morali pokazati pri 27 nalogah, od katerih smo jih 24 uporabili za našo analizo. Naloge smo razvrstili v tri tematske skupine: ekologija s 7 nalogami, fi ziologija s 4 nalogami in zdravje z 8 nalogami. Preostalih pet nalog smo združili v vsebinsko raznoliko četrto skupino. Ugotovili smo, da dosežki učencev v Slov- eniji pri vseh preverjenih bioloških temah dobri. Dejavnika, s katerima je bilo mogoče pojasniti slabše dosežke pri posameznih nalogah, sta bila tip naloge in njena težavnost. Učenci so slabše reševali naloge odprtega tipa, pri katerih so morali samostojno oblikovati smiseln odgovor. Uspešnejši so bili pri nalogah kompleksnega izbirnega tipa in izbirnega tipa, kjer so morali med danimi odgovori izbrati pravilnega. Učenci so slabše reševali naloge višje kognitivne stop- nje, ki so zahtevale uporabo znanja. Iz tega sk- lepamo, da je bil v obdobju, ko so bili ti učenci vključeni v osnovnošolsko izobraževanje, pri biologiji poudarek na razvijanju poznavanja biološke vsebine (knowledge of science) in kompetence znanstveno razlaganje pojavov (ex- plaining phenomena scientifi cally), pomanjk- ljivo pa je bilo razvijanje znanja o naravoslovnih znanostih (knowledge about science) ter kom- petence uporaba naravoslovno-znanstvenih po- datkov in preverjenih dejstev (using scientifi c evidence). V rokah vseh, ki se v Sloveniji uk- varjajo z biološkim izobraževanjem, predvsem pa učiteljev biologije, je, da usposobijo učence tudi s tega vidika, ne samo z vidika vsebinskega znanja. References American Association for the Advancement of Science, 1993. Benchmarks for Science Literacy. New York, Oxford University Press, 418 pp. Bybee, R.W., 2008. Scientifi c Literacy, Environmental Issues, and PISA 2006: The 200 Paul F-Bran- dwein Lecture. J Sci Educ Technol, 17, 566-585. DeHard Hurd, P., 2001. The Changing Image of Biology. The American Biology Teacher, 63 (4), 233–235. 108 Acta Biologica Slovenica, 53 (2), 2010 Dosežki učencev v raziskavi PISA 2006. http://www.pei.si/UserFilesUpload/fi le/raziskovalna_deja- vnost/PISA/PISA2006/Rezultati_raziskavePISA2006.pdf (15. 9. 2010) Jagodnik, A., Sopčič, B., Strgar, J., Volčini, D., Zupan, A., 2009. Analiza dosežkov nacionalnega preverjanja znanja iz biologije ob koncu tretjega obdobja. In: Rigler Šilc K. et Novak, M. (eds.): Nacionalno preverjanje znanja. Letno poročilo o izvedbi nacionalnega preverjanja znanja v šolskem letu 2008/2009. Ljubljana, Državni izpitni center, pp.179-191. http://www.ric.si/mma_bin.php/$fi leI/2009121612174940/$fi leN/Letno%20poročilo%202009%20 Tisk.pdf (15. 9. 2010) PISA 2006, 2007. Science Competencies for Tomorrow’s World. Vol. 1 – Analysis. OECD, 383 pp. Štraus, M., Repež, Maša, M., Štigl, Simona, (eds.), 2007. Nacionalno poročilo PISA 2006: naravo- slovni, bralni in matematični dosežki slovenskih učencev, Pedagoški inštitut, Ljubljana, 223 pp. Science goes to school: A new model for introduction of modern biology teaching strategies to Slovene schools Znanost gre v šolo: nov pristop k uvajanju sodobnih metod poučevanja bioloških vsebin v slovenske šole Barbara Vilhar*, Simona Strgulc Krajšek University of Ljubljana, Biotechnical Faculty, Department of Biology, Večna pot 111, SI-1000 Ljubljana, Slovenia, *correspondence: barbara.vilhar@bf.uni-lj.si Abstract: In the framework of the project Science Goes to School, we developed and tested a new model for introduction of modern biology teaching strategies to Slovene schools. The project focused around a close university-school partnership, bringing together the expertise of scientists from the University of Ljubljana and the experiences of teachers from 22 Slovene sec- ondary schools (grades 9-12, age of students 15-19). The project comprised three phases. Dur- ing the introductory workshop, project scientists and partner teachers identifi ed curriculum top- ics with an acute lack of good-quality teaching materials. During the second phase, university scientists developed new practical activities for students and prepared comprehensive teaching materials. Each new activity was tested in partner schools, with a scientist acting as a visiting teacher. Partner teachers were present in the class during testing and were hence trained in the authentic environment of their own classrooms. Both teachers and students contributed their comments and suggestions for improvement of new activities. The visiting scientist also acted as a role model motivating the students to consider science careers. During the third phase, the new teaching materials were published in a handbook for teachers and on the internet. In addi- tion, the new activities were presented to a wider community of teachers and school laboratory assistants during a training workshop. The project was favourably received among the teachers, the project scientists and the students in partner schools. To effi ciently improve biology educa- tion in Slovene schools, such activities require long-term, stable funding from national sources. Keywords: science education, biology, teaching, effective learning, university-school partnership Izvleček: V okviru projekta Znanost gre v šolo smo razvili in preizkusili nov pristop k uvajanju sodobnih metod poučevanja bioloških vsebin v slovenske šole. V središču projekta je bilo tesno partnerstvo univerze in šol, s katerim smo povezali strokovno znanje znanstvenikov z Univerze v Ljubljani in izkušnje učiteljev z 22 partnerskih srednjih šol. Projekt je obsegal tri faze. Med uvodno delavnico so znanstveniki in partnerski učitelji opredelili vsebine v učnem načrtu, pri katerih močno primanjkuje kakovostnih učnih gradiv. Med drugo fazo so znanstveniki razvijali nove praktične aktivnosti za pouk biologije in pripravili izčrpna učna gradiva. Vsako novo aktivnost smo preiz- kusili v partnerskih šolah, pri čemer je eden od znanstvenikov obiskal šolo kot gostujoči učitelj. Med testiranjem so bili partnerski učitelji prisotni v razredu in so se tako strokovno usposabljali v avtentičnem okolju lastnih učilnic. Tako učitelji kot dijaki so prispevali pripombe in predloge za izboljšanje novih aktivnosti. Gostujoči znanstvenik je bil hkrati vzornik pri spodbujanju dijakov, da bi se odločili za naravoslovne študije. Med tretjo fazo projekta smo nova učna gradiva objavili v priročniku za učitelje in na spletnih straneh. Poleg tega smo organizirali zaključno delavnico za učitelje, na kateri se je širši krog učiteljev in šolskih laborantov usposabljal za izvedbo novih aktivnosti. Projekt so z navdušenjem podprli učitelji, sodelujoči znanstveniki in dijaki s partnerskih ACTA BIOLOGICA SLOVENICA LJUBLJANA 2010 Vol. 53, [t. 2: 109–120 110 Acta Biologica Slovenica, 53 (2), 2010 šol. Za učinkovito izboljšanje kakovosti biološkega izobraževanja v slovenskih šolah bi morali tovrstne dejavnosti dolgoročno in stabilno fi nancirati iz nacionalnih virov. Ključne besede: naravoslovno izobraževanje, biologija, poučevanje, učinkovito učenje, partnerstvo univerz in šol Introduction Recently, biology became the most rapidly developing natural science. In addition, topics such as biodiversity, global warming, invasive species, genetically modifi ed organisms, stem cells and gene therapy have gained a high so- cial importance. Consequently, a modern citizen needs biological knowledge to cope with every- day problems, such as understanding the news in the media and deciding about health issues. For young people, the main source of up-to-date bio- logical knowledge is their biology teacher. In the context of the increasing importance of biological literacy for personal and social deci- sion-making, biology teachers face the challenge of updating the teaching content and changing their practices from teaching factual knowledge to conveying conceptual understanding of living systems. However, biology teachers have a high teaching load and have to cover a wide range of biology topics. They also lack time and expertise to convert important scientifi c discoveries into classroom activities. For the teacher to cope with emerging new bi- ology topics and the increasingly interdisciplinary and systemic approach to teaching biology, he/ she needs an excellent education and continuous in-service training (Moore 2003, 2007). In many schools, teaching lags far behind new scientifi c fi ndings. Many countries have reported problems with overloaded and outdated curricula, outdated textbooks, insuffi cient »real« practical work, the perception of biology as a »soft« scientifi c sub- ject, inappropriate pedagogy, lack of teacher and student enthusiasm and lack of continuous teacher training (Moore 2007, Tunniclife and Ueckert 2007). Introduction of new approaches to teaching science as an exciting and dynamic topic is a long- term process (Mervis 2002, Vilhar 2007). It com- prises development of new curricula and changes in teacher education, and has to be supported with new textbooks and teaching materials. One of the problems is development of scientifi c thinking in science class. Presently, the phylosophy of science is often wrongly presented as a collection of reci- pes for experiments (Mervis 2002, Bonner 2004, National Research Council 2002, 2005, Moore 2007). Empirical evidence shows that active learn- ing works (Michael 2006), motivating students to become active learners and problem solvers (Lujan and DiCarlo 2006). As stated in the review with a meaningful title Too much teaching, not enough learning: What is the solution, extensive curricular changes are required to achieve effective learning (Lujan and DiCarlo 2006). Modernisation of science teaching in schools has to be supported by active involvement of university scientists. Scientists are competent to select and suggest new science topics and help to develop new approaches to teaching biology in schools, conveying the true spirit of science (Bhat- tacharjee 2005, Moore 2007). However, these new ideas need to be adequately fi tted into the curricu- lum, since learning will only occur after teaching if students are given enough time to process the new information and connect it to their previous knowl- edge and conceptions (Tunniclife and Ueckert 2007). To achieve this, expertise of scientists has to be complemented with experience of teachers, who know well the capabilities of their students and real-life situations in the classroom (McDiar- mid et al. 1989, Tanner et al. 2003). The above mentioned problems are also present in Slovene schools. In the past, biology teachers frequently complained about the lack of systematic support from scientists. Students also felt that changes are needed in secondary school biology. More than 71% of students thought that the curriculum should be more connected to every- day life, 66% would like to have more experiments and 59% more excursions (fi eld work) during biol- ogy lessons (Gabršček et al. 2005; see Tab. 1). The project Science Goes to School connected scientists, teachers and students from secondary schools with the aim to improve science teaching. We tested a new model for introduction of mod- ern teaching strategies to our secondary schools. The project was a university-school partnership 111Vilhar, Strgulc Krajšek: A new model for improvement of biology teaching based on experiences of similar projects in other countries (e.g. Mervis 2002, Tanner et al. 2003), but taking into account the specifi c circumstances in Slovene schools and universities. Methods Survey of teachers’ perceptions of problems in biology education In order to investigate teachers’ perceptions of the major problems in biology education, we prepared a questionnaire for teachers in general secondary schools. The questionnaire was hand- ed out to teachers who participated at a training seminar in January 2006. Participation in the survey was voluntary. 35 out of 78 participants returned a fi lled-in questionnaire. The project Science Goes to School The idea for the project Science Goes to School was based on previous similar projects in other countries (e.g. Mervis 2002, Tanner et al. 2003), in particular the program Graduate STEM Fellows in K-12 Education in the USA (National Science Foundation 2010). This program sup- ports fellowships and training for graduate stu- dents in science, technology, engineering, and mathematics (STEM). We adapted the project activities to specifi c circumstances in Slovenia, as explained in the Results. The project duration was 1.5 years (project budget: 62 600 €). The project activities were evaluated us- ing questionnaires. The fi rst questionnaire was handed out to biology teachers participating at the introductory workshop in May 2006. 18 out of 20 teachers returned a fi lled-in questionnaire. The second questionnaire was handed out to 72 participants at the training workshop for teach- ers (53 biology teachers and 19 school laboratory assistants) in September 2007. 46 teachers and 13 laboratory assistants returned a fi lled-in ques- tionnaire. Statistical methods Standard statistical methods were used to analyse questionnaires. Data were analysed with the software package Prism 5 for Windows (Graph Pad Software). Average values are ex- pressed as mean ± standard error of the mean. Results Teachers’ perceptions of problems in biology education The questionnaire for teachers about the main problems in biolo gy education, which we Problem Fraction of teachers (%) N = 35 Fraction of students (%) N = 862 curriculum 71 71 in-service teacher training 69 --- laboratory and fi eld work 51 62 teaching materials (in Slovene) 46 --- textbooks and workbooks 40 19 general circumstances at school 37 --- Table 1: Perception of biology teachers and students about the main problems of biology education in secondary school. Teachers’ perceptions were evaluated in January 2006 on the basis of a questionnaire. The open- ended question was: List three main problems in biology education. Answers were grouped to categories. Data about students’ opinions are from Gaberšček et al. 2005. Tabela 1: Mnenja učiteljev biologije in bivših dijakov o problemih na področju biološkega izobraževanja v srednji šoli. Mnenja učiteljev so iz vprašalnika, ki so ga gimnazijski učitelji izpolnili januarja 2006. Vprašanje odprtega tipa je bilo: Napišite tri ključne probleme na področju biološkega izobraževanja. Odgovori so razvrščeni v kategorije. Podatki o mnenju bivših dijakov so iz Gaberšček in sod. 2005. 112 Acta Biologica Slovenica, 53 (2), 2010 Table 2: Expectations of biology teachers about activities of university scientists in the fi eld of biological education. Teachers’ perceptions were evaluated in January 2006 on the basis of a questionnaire. The open-ended question was: What are your expectations from the Department of Biology [Biotechnical Faculty, University of Ljubljana] in this fi eld? Answers were grouped to categories. Tabela 2: Pričakovanja učiteljev biologije o aktivnostih matične stroke na področju biološkega izobraževanja. Mnenja učiteljev so iz vprašalnika, ki so ga gimnazijski učitelji izpolnili januarja 2006. Prikazana je analiza odgovorov učiteljev biologije na vprašanje odprtega tipa: Kakšna so vaša pričakovanja od Oddelka za biologijo na tem področju? Odgovori so razvrščeni v kategorije. Activity Fraction of teachers (%)N = 35 introduction of novelties to school 77 in-service teacher training 71 collaboration with teachers 71 participation in development of curricula 29 increased impact in wider society 29 Figure 1: Teachers’ evaluation of activities in the fi eld of biological education. Teachers used “school” grades from 1 (very poor) to 5 (excellent) to evaluate activities. A - Involvement of scientists (2006): question- naire handed out at a workshop for biology teachers in secondary school in January 2006 (question: Use school grades from 1 to 5 to evaluate current activities of the Department of Biology [Biotechnical Faculty, University of Ljubljana] in the fi eld of biology education.) B - SGTS introductory workshop (2007): questionnaire handed out at the introductory workshop of the project Science Goes to School in May 2006 (question: Use school grades from 1 to 5 to evaluate today’s workshop.). C - SGTS fi nal workshop (2007): questionnaire handed out at the fi nal workshop of the project Science Goes to School in September 2007 (question: Use school grades from 1 to 5 to evaluate today’s workshop.). Slika 1: Mnenje učiteljev o dejavnostih na področju biološkega izobraževanja. Učitelji so dejavnosti ocenjevali s “šolskimi” ocenami od 1 do 5. A - Involvement of scientists (2006): anketa izvedena na seminarju za gimnazijske učitelje biologije januarja 2006 (vprašanje: Ocenite s šolsko oceno od 1 (nezadostno) do 5 (odlično) trenutno delovanje Oddelka za biologijo na področju biološkega izobraževanja.). B - SGTS introductory workshop (2007): anketa izvedena na uvodni delavnici projekta Znanost gre v šolo maja 2006 (vprašanje: S šolsko oceno od 1 do 5 ocenite delo na današnji delavnici.). C - SGTS fi nal workshop (2007): anketa izvedena na zaključni delavnici projekta Znanost gre v šolo septembra 2007 (vprašanje: S šolsko oceno od 1 do 5 ocenite današnji seminar.). 113Vilhar, Strgulc Krajšek: A new model for improvement of biology teaching prepared in January 2006, contained open-ended questions. Teachers’ answers were sorted into categories. The most frequently mentioned prob- lems were related to inappropriate and outdated curriculum, lack of good-quality teacher training seminars and workshops, problems with labo- ratory and fi eld work and lack of good-quality teaching materials in Slovene language (Tab. 1). We also investigated what teachers expected from university scientists working in the fi eld of biology. Teachers most frequently listed support in relation to introduction of novelties to school, involvement in in-service teacher training activi- ties and general collaboration with teachers (Tab. 2). Teachers were also asked to evaluate past ac- tivities of university scientists in the fi eld of biol- ogy education using the scale of school grades from 1 (very poor) to 5 (excellent). The average grade was 2.9 ± 0.2 (Fig. 1A). The project Science Goes to School The project Science Goes to School specifi - cally aimed to address three problems that teach- ers identifi ed as the main problems in biology education: lack of appropriate teacher training, lack of new materials for practical activities of students and lack of teaching materials in Slov- ene language (Tab. 1). The project was based on an intense partnership between university and schools, aiming to overcome the previous dis- content of teachers with insuffi cient involvement of university scientists in biology teaching in schools (Tab. 2, Fig. 1A). In particular, project scientists helped teachers introduce novelties to school and offered additional teacher training. The project collaborators included scientists from the Department of Biology, Biotechni- cal Faculty, University of Ljubljana and partner teachers from 22 secondary schools (grades 9-12, age of students 15-19). The project activities comprised three phases (Fig.2). During the one-day introductory work- shop, four project scientists and 20 partner teachers participated in brainstorming sessions, aiming to identify the topics in the secondary- school biology curriculum for which there was an acute lack of useful teaching materials. Possi- ble teaching strategies for these topics were also discussed. We decided to use new approaches to teaching biology that support effective teaching for long-lasting knowledge, such as experiment- based learning and educational games (Lujan and DiCarlo 2006). Teachers evaluated the introduc- tory workshop with an average grade 4.7 ± 0.1 (Fig. 1B). During the second phase, university scien- tists developed new activities and tested them in partner schools. The new practical activities for students were based on our own ideas or modi- fi ed from materials developed by other authors (Vilhar et al. 2007). Development of materials included testing of experiments in laboratories and writing supporting materials for teachers and students. We paid special attention to possible er- rors that students could do during execution of the practical activity at school and possible unex- pected results of experiments. We also reviewed relevant textbooks for secondary schools in Slov- ene language and exposed the main sources of misconceptions and problems with understand- ing of selected topics. We tested all new activities in partner schools. One of the project scientists came to the class- room during regular biology lessons and taught the subject using newly developed methods. The partner teacher was present in the classroom and was thus trained in the authentic environment of his/her own classroom. We collected the opin- ions about the new practical activity from the teachers and the students and used their ideas and comments to improve the teaching material. Partner teachers and students in visited schools thus actively contributed to the quality of newly developed practical activity. The improved ver- sion of the practical activity was tested again in another school. In some cases, new practical ac- tivities were tested prior to the fi rst visit to school with fi rst-year university students of biology and pre-service biology teachers, who volunteered to participate in the project. Within the framework of the project, we de- veloped and tested eight new activities, which can be directly used for teaching biology in Slov- ene secondary schools (Vilhar et al. 2007): – Diffusion and osmosis (experiment-based learning with educational role-playing game and a computer simulation; Strgulc Krajšek and Vilhar 2010) – Describing and naming in biology (discovery- 114 Acta Biologica Slovenica, 53 (2), 2010 based learning) – Determination keys (discovery-based learning) – How to grow fern gametophytes? (discovery- based learning) – Respiration (experiment-based learning using computer-linked measurement instruments) – Muscle fatigue (experiment-based learning using computer-linked measurement instru- ments) – The plant game (educational computer game) – Bio impro-league (educational card game). The prepared teaching materials include ref- erences to the relevant curriculum topics, dura- tion of the activity, theoretical background for teachers, detailed instructions for preparing and teaching the activity, worksheets for students with solutions and comments for teachers (in- cluding the expected results of experiments), links to websites with additional materials, lists of books and other relevant literature, safety warnings, explanations about common miscon- ceptions and how to overcome them, and some interesting stories linked to the topic that teach- ers can use to motivate the students. We paid special attention to include references to the history of science in the teaching activities (e.g. Strgulc Krajšek and Vilhar 2010), thus empha- sising the importance of the largely neglected aspect of science education, namely explaining to students the nature of science. During the third phase, at the end of the project, we made the new teaching activities available to a wider community of teachers. We published the teaching materials in a handbook for teachers (Vilhar et al. 2007), which was sent to all secondary schools. Supporting material was published on the project website (http:// znanost-gre-v-solo.biologija.org/). While some supporting material is publicly available, spe- cifi c comments for teachers are accessible with a password for registered teachers. The work- sheets for students were published in a format that allows teachers to modify the text and thus adapt the teaching materials to conform to their teaching strategies and the time they allocate to each activity. We also organised a one-day training work- shop for teachers and school laboratory assist- ants, where project scientists and partner teach- ers acted together as instructors. Participants (53 teachers and 19 school laboratory assistants) were divided into groups, so that each partici- pant was trained in three of the new activities. The seminar was closed with a general discus- sion of all participants and scientists, where impressions, questions and comments about the new activities were shared. Participants evaluated the quality of the training workshop with a questionnaire. Teach- ers evaluated the workshop with an average grade 4.7 ± 0.1 (Fig. 1C). They thought that the model for introduction of novelties to schools used in our project was very appropriate (aver- age grade 4.8 ± 0.1; Fig. 3A). They also evalu- ated fi ve of the eight new activities. The aver- age grades ranged from 4.3 to 4.8 (Fig. 3B-F). For the fi ve evaluated activities, the fraction of teachers who thought that they would use the new activity in school was 95% to 100%. Teach- ers also expressed a strong support for follow-up projects similar to the project Science Goes to School (average grade 4.91 ± 0.04; Fig. 3G). Discussion Our survey conducted in January 2006 clearly showed that teachers need and expect support from university scientists (Tab. 2; Fig. 1A). Prior to the project Science Goes to School, there was no close collaboration between scien- tists and teachers with the goal to develop new biology teaching strategies and introduce them to Slovene schools. While partner teachers were somewhat sceptical at the very beginning of the project Science Goes to School, the close univer- sity-school partnership later lead to enthusiasm among partner teachers and project scientists (Figs. 1B, 1C) and sparked valuable exchange of ideas and experiences. The cooperation of teachers and scientists thus turned out to be ben- efi cial for both partners. Laursen et al. (2007), who worked in the USA with K-12 students and their teachers, came to similar conclusions. The teachers liked the comprehensive ap- proach in the new teaching materials of the project Science Goes to School, with extensive theoretical background, explanation of common misconceptions, comments on possible mistakes 115Vilhar, Strgulc Krajšek: A new model for improvement of biology teaching that students can make in class and references to the history of science. The active involvement of university scientists reassured the teachers that the new teaching materials were of good qual- ity and contained correct scientifi c information. The teachers appreciated the fact that the new practical activities for students were focused around specifi c teaching goals (subject content knowledge) and were thus a constructive part of the overall learning process in biology class. Their support for the outcomes of the project is refl ected in their answers to questionnaires (Figs. 1B, 1C, 3). In addition, inclusion of school laboratory assistants in project training activities was perceived as important for improvement of biology education (analysis of questionnaires not shown). We paid special attention to tightly link the new activities to curriculum topics, and to instruct the teachers to give their students adequate guid- ance. Results of previous empirical investigations show that learning on the basis of students’ own previous experiences alone is less effi cient than teaching methods which include proper guidance of the students during the learning process (Klahr and Nigram 2004, Mayer 2004, Novak and Cañas 2006, Kirshner et al. 2006, Sweller et al. 2007). If students have no prior conceptual understand- Figure 2: Outline of activities of the project Science Goes to School. See the main text for details. Slika 2: Pregled aktivnosti v okviru projekta Znanost gre v šolo. Pri razvoju novih učnih gradiv za praktične aktivnosti so sodelovali univerzitetni znanstveniki, učitelji in dijaki s partnerskih šol. 116 Acta Biologica Slovenica, 53 (2), 2010 ing of a natural phenomenon that they are inves- tigating, they often acquire no new conceptual knowledge during their practical activities (No- vak and Cañas 2006). Likewise, development of skills such as learning to learn and searching for relevant information cannot replace the need for understanding science concepts, in particular not in the 21st century, when science knowledge needs to be upgradeable (Sweller et al. 2007). In this respect, the experiences in Norway are particularly interesting. After a curricular reform that focused on acquiring skills through various activities and neglected subject content knowl- edge, the achievements of students in math- ematics and science, evaluated in international studies PISA and TIMSS, dropped considerably Figure 3: Teachers’ evaluation of the training workshop of the project Science Goes to School. The question- naire was handed out at the fi nal training workshop for teachers in the framework of the project Science Goes to School in September 2007 The grading system is shown in the legend. A – question: Do you fi nd the approach to development of new activities which we used in the project Science Goes to School appropriate? B-F – general evaluation of the new school activity using school grading system from 1 to 5. F – question: Do you think it is reasonable to continue with activities similar to the project Science Goes to School? Slika 3: Mnenje učiteljev o zaključne delavnice za učitelje na projektu Znanost gre v šolo. Anketa je bila izvedena na zaključni delavnici za učitelje v okviru projekta Znanost gre v šolo septembra 2007. Način ocenjevanja je prikazan v legendi: A do F – “šolske ocene” od 1 do 5; G – 1 – sploh ne, 5 – zelo. A – Ali se vam zdi način razvoja aktivnosti, ki smo ga uporabili v projektu Znanost gre v šolo, primeren? B do F – Splošna ocena nove vaje: “šolska” ocena od 1 do 5. G – vprašanje: Ali se vam zdi smiselno nadaljevanje s programi, podobnimi projektu Znanost gre v šolo? 117Vilhar, Strgulc Krajšek: A new model for improvement of biology teaching (Institute for Teacher Education and School De- velopment 2006). These examples demonstrate that science teaching should focus around un- derstanding of science concepts, with a balanced use of different teaching methods A particular feature of the project Science Goes to School was a “science ambassador” – a scientist who visited schools during regular biol- ogy lessons. While the main goal of these visits was testing of new activities and teacher train- ing, the visiting scientist also served as a role model to increase students’ interest in science and motivate them to consider science careers. The students in partner schools responded with enthusiasm. Examples of their comments in questionnaires are: I wish we had more such les- sons, this way we learn more; I liked the lesson, it was fun and instructive; Come back again; Keep delivering such lessons in the future (see students’ comments in Strgulc Krajšek and Vil- har 2010). These experiences support previous observations that active learning works (Abra- hams and Millar 2008) and that universities can promote change towards more effi cient science teaching (Tanner et al. 2003). Different models of scientists visiting schools have been used in other countries, with encouraging results (e.g. Peplow 2004, Beck et al. 2006, Brodie 2006, Laursen 2007). The project Science Goes to School was a pilot project, introducing and testing a new mod- el for development of teaching materials and for teacher training in Slovenia. While the target subject of the project was biology, the same model is applicable for introduction of novelties in other science subjects (science, physics and chemistry). The strong support from the teach- ers (Fig. 3G) clearly shows that such activities should be continued on a regular basis. How- ever, these activities are only fi nanced in Slov- enia on a short-term basis (in particular with fi nancial support from the EU), which greatly diminishes the long-term impact on improve- ment of biology education. Notably, centres for biology teacher training with full-time staff exist in many EU countries, but there is no such cen- tre in Slovenia. In 2009, The European Network of Academies on Science Education stated that the use of limited EU seed funds must be fol- lowed up by substantial investments nationally, from ministries of education, Academies of Sci- ences, research institutions and industry (ALLEA 2009). The long-term strategy for improvement of science education in Slovenia should follow these guidelines. Conclusions – Biology teachers need and expect support from university scientists. – The new approach to university-school partner- ship developed and tested during the project Science Goes to School was favourably received among the teachers, the project scientists and the students in partner schools. – Long-term funding, in particular from the national sources, is needed for such activities to have a long-lasting effect on improvement of biology education in Slovenia. Povzetek Ob hitrem napredku bioznanosti ter naraš- čajočem pomenu biološkega znanja za osebne in družbene odločitve se precej spreminjajo tudi pristopi k biološkemu izobraževanju. Pri posodobitvi pouka učitelji biologije potrebujejo strokovno podporo in ustrezno strokovno iz- popolnjevanje (tab. 1), pri čemer pričakujejo tudi pomoč znanstvenikov z univerz (tab. 2, sl. 1A). V okviru projekta Znanost gre v šolo smo razvili in preizkusili nov pristop k uvajanju sodobnih metod poučevanja bioloških vsebin v slovenske šole. V središču projekta je bilo tesno partnerstvo univerze in šol, s katerim smo poveza- li strokovno znanje znanstvenikov z Univerze v Ljubljani in izkušnje učiteljev z 22 partnerskih srednjih šol. Zamisel za projekt smo oblikovali na temelju podobnih projektov v drugih državah (Mervis 2002, Tanner in sod. 2003, National Sci- ence Foundation 2010), pri čemer smo projektne dejavnosti prilagodili specifi čnim razmeram na slovenskih šolah in univerzah. Projekt je trajal leto in pol (vrednost projekta: 62 600 €). Projekt je obsegal tri faze (sl. 2). Med uvodno delavnico so znanstveniki in partnerski učitelji opredelili vsebine v učnem načrtu, pri katerih močno primanjkuje kakovostnih učnih gradiv, 118 Acta Biologica Slovenica, 53 (2), 2010 ter razpravljali o možnih didaktičnih pristopih k poučevanju teh tem. Med drugo fazo projekta so znanstveniki razvijali nove praktične aktivnosti za pouk bi- ologije in pripravili izčrpna učna gradiva. Vsako novo aktivnost smo preizkusili v partnerskih šolah, pri čemer je eden od znanstvenikov obis- kal šolo kot gostujoči učitelj. Med testiranjem so bili partnerski učitelji prisotni v razredu in so se tako strokovno usposabljali v avtentičnem okolju lastnih učilnic. Tako učitelji kot dijaki so prispevali pripombe in predloge za izboljšanje novih aktivnosti. Gostujoči znanstvenik je bil hkrati »znanstveni ambasador« - vzornik pri spodbujanju zanimanja dijakov za naravoslovne znanosti in naravoslovne študije. V okviru projekta smo pripravili gradiva za osem novih praktičnih aktivnosti (Vilhar in sod. 2007). Učna gradiva vsebujejo navedbo us- treznih tem in ciljev v učnem načrtu, trajanje ak- tivnosti, teoretično razlago za učitelje, navodila za pripravo in izvedbo aktivnosti, delovne liste za dijake z rešitvami in komentarji za učitelje (vključno s pričakovanimi rezultati poskusov), povezave na spletne strani z dodatnimi gradivi, seznam strokovne literature, varnostna opozo- rila, razlago o pogostih napačnih predstavah ter zanimivosti, s katerimi lahko učitelj motivira učence. V učna gradiva smo vključevali tudi zgodbe iz zgodovine znanosti, s katerimi lahko dijakom predstavimo naravo znanosti. Med tretjo fazo projekta smo nova učna gradiva objavili v priročniku za učitelje (Vil- har in sod. 2007) in na spletnih straneh (http:// znanost-gre-v-solo.biologija.org/). Organizirali smo tudi zaključno delavnico za učitelje, na kat- eri se je širši krog učiteljev in šolskih laborantov usposabljal za izvedbo novih aktivnosti. Projekt so z navdušenjem podprli učitelji (sl. 1B, 1C, 3), sodelujoči znanstveniki in dijaki s partnerskih šol. Projekt Znanost gre v šolo je bil pilotski projekt, s katerim smo v slovenski prostor uv- edli nov model za razvoj učnih gradiv in usposa- bljanje učiteljev. Čeprav je projekt obravnaval poučevanje biologije, je enak model uporaben tudi za posodobitev drugih naravoslovnih pred- metov (naravoslovja, kemije in fi zike). Za učinkovito izboljšanje kakovosti biološkega izobraževanja v slovenskih šolah bi morali tovrstne dejavnosti dolgoročno in stabilno fi nan- cirati iz nacionalnih virov (ALLEA 2009). Acknowledgements We thank Dr Gregor Zupančič and Dr Nejc Jogan for constructive discussions and collabo- ration on the project Science Goes to School / Znanost gre v šolo. The project was supported by the European Union (the European Social Fund) and The Ministry of Education and Sport of the Republic of Slovenia (http://znanost-gre- v-solo.biologija.org/). 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Lujan, H.L., DiCarlo, S.E., 2006. Too much teaching, not enough learning: what is the solution? Advances in Physiology Education, 30, 17-22. Mayer, R.E., 2004. Should there be a three-strikes rule against pure discovery learning? The case for guided methods of instruction. American Psychologist, 59, 14-19. McDiarmid, G.W., Ball, D.L., Anderson C.W., 1989. Why standing one chapter ahead doesn't really work: subject-specifi c pedagogy. In: Reynolds M.C. (ed.): Knowledge base for beginning teachers. Pergamon Press, Oxford, UK, pp. 193–205. Mervis, J., 2002. U.S. programs ask faculty to help improve schools. Science, 295, 265. Michael, J., 2006. Where's the evidence that active learning works? Advances in Physiology Educa- tion, 30,159-167. Moore, A., 2003. Breathing new life into the biology classroom. EMBO Reports, 4, 744-746. Moore, A., 2007. Biology education in a rapidly changing scientifi c and socio-economic context. In: Strgulc Krajšek, S., Popit T., Vičar M. (eds.): GENIalna prihodnost – genetika, determinizem in svoboda. Zbornik prispevkov posveta, Ljubljana, October 4-5, 2007. ZRSŠ in MŠŠ, pp. 224-228. National Research Council, 2002. Learning and understanding: Improving advanced study of math- ematics and science in U.S. high schools: Report of the Content Panel for Biology. Committee on Programs for Advanced Study of Mathematics and Science in American High Schools, National Research Council, USA, 66 p., ISBN 0-309-54195-6. National Research Council, 2005. America's lab report: Investigations in high school science. Sin- ger, S R., Hilton, M.L., Schweingruber, H.A. (eds.). Committee on High School Science, USA. National Science Foundation, 2010. NSF graduate STEM fellows in K-12 education (GK-12) pro- gram. http://www.gk12.org/ (October 10, 2010). Novak, J.D., Cañas, A.J., 2006. The theory underlying concept maps and how to construct and use them. 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In: Strgulc Krajšek, S., Popit, T., Vičar, M. (eds.): GENIalna prihodnost – genetika, determinizem in svoboda. Zbornik prispevkov posveta, October 4-5, 2007, Ljubljana, Slovenia. ZRSŠ in MŠŠ, pp. 229-238. Vilhar, B., Strgulc Krajšek, S., Zupančič, G., Jogan, N., 2007. Znanost gre v šolo: priročnik za 120 Acta Biologica Slovenica, 53 (2), 2010 izvedbo aktivnosti pri pouku biologije v gimnazijah in srednjih šolah. 1st ed. Univerza v Ljubljani, Biotehniška fakulteta, Oddelek za biologijo, Ljubljana, Slovenia, 124 p., ISBN = 978-961-90262-3-6. 121 INSTRUCTIONS FOR AUTHORS 1. Types of Articles a) SCIENTIFIC ARTICLES are comprehensive descriptions of original research and include a theoretical survey of the topic, a detailed presentation of results with discussion and conclusion, and a bibliography according to the IMRAD outline (Introduction, Methods, Results, and Discussion). In this category ABS also publishes methodological articles, in so far as they present an original method, which was not previously published elsewhere, or they present a new and original usage of an established method. The originality is judged by the editorial board if necessary after a consultation with the referees. The recommended length of an article including tables, graphs, and illustrations is up to fi fteen (15) pages; lines must be double-spaced. Scientifi c articles shall be subject to peer review by two ex- perts in the fi eld. b) REVIEW ARTICLES will be published in the journal after consultation between the edito- rial board and the author. Review articles may be longer than fi fteen (15) pages. c) BRIEF NOTES are original articles from various biological fi elds (systematics, biochem- istry, genetics, physiology, microbiology, ecology, etc.) that do not include a detailed theo- retical discussion. Their aim is to acquaint readers with preliminary or partial results of research. They should not be longer than fi ve (5) pages. Brief note articles shall be subject to peer review by one expert in the fi eld. d) CONGRESS NEWS acquaints readers with the content and conclusions of important con- gresses and seminars at home and abroad. e) ASSOCIATION NEWS reports on the work of Slovene biology associations. 2. Originality of Articles Manuscripts submitted for publication in Acta Biologica Slovenica should not contain previously published material and should not be under consideration for publication else- where. 3. Language Articles and notes should be submitted in English, or as an exception in Slovene if the topic is very local. As a rule, congress and association news will appear in Slovene. 4. Titles of Articles Title must be short, informative, and understandable. It must be written in English and in Slovene language. The title should be followed by the name and full address of the authors (and if possible, fax number and/or e-mail address). The affi liation and address of each author should be clearly marked as well as who is the corresponding author. 5. Abstract The abstract must give concise information about the objective, the methods used, the results obtained, and the conclusions. The suitable length for scientifi c articles is up to 250 words, and for brief note articles, 100 words. Article must have an abstract in both English and Slovene. 6. Keywords There should be no more than ten (10) keywords; they must refl ect the fi eld of research covered in the article. Authors must add keywords in English to articles written in Slovene. 122 Acta Biologica Slovenica, 53 (2), 2010 7. Running title This is a shorter version of the title that should contain no more than 60 characters with spaces. 8. Introduction The introduction must refer only to topics presented in the article or brief note. 9. Illustrations and Tables Articles should not contain more than ten (10) illustrations (graphs, dendrograms, pictures, photos etc.) and tables, and their positions in the article should be clearly indicated. All illustrative material should be provided in electronic form. Tables should be submitted on separate pages (only horizontal lines should be used in tables). Titles of tables and illustrations and their legends should be in both Slovene and English. Tables and illustra- tions should be cited shortly in the text (Tab. 1 or Tabs. 1-2, Fig. 1 or Figs. 1-2; Tab. 1 and Sl. 1). A full name is used in the legend title (e.g. Figure 1, Table 2 etc.), written bold, followed by a short title of the fi gure or table, also in bold. Subpanels of a fi gure have to be unambiguously indicated with capital letters (A, B, …). Explanations associated with subpanels are given alphabetically, each starting with bold capital letter (A), a hyphen and followed by the text. 10. The quality of graphic material Starting with the fi rst issue of the 53rd volume the ABS will be processing the graphic material only electronically. All the fi gures have to be submitted in the electronic form. The ABS publishes fi gures either in pure black and white or in halftones. The resolution should be 300 d.p.i. minimum for halftones and 600 d.p.i. for pure black and white. The smallest numbers and lettering on the fi gure should not be smaller than 8 points (2 mm height). The thickness of lines should not be smaller than 0.5 points. The permitted font families are Times, Times New Roman, Helvetica and Arial, whereby all fi gures in the same article should have the same font type. The fi gures should be prepared in TIFF, EPS or PDF format, whereby TIFF (ending *.tif) is the preferred type. When saving fi gures in TIFF format we recommend the use of LZW or ZIP compression in order to reduce the fi le sizes. The photographs can be submitted in JPEG format (ending *.jpg) with low compres- sion ratio. Before submitting a fi gure in EPS format make sure fi rst, that all the characters are rendered correctly (e.g. by opening the fi le fi rst in the programs Ghostview or GSview – depending on the operation system or in Adobe Photoshop). With PDF format make sure that lossless compression (LZW or ZIP) was used in the creation of the *.pdf fi le (JPEG, the default setting, is not suitable). Figures created in Microsoft Word, Excel, PowerPoint etc. will not be accepted without the conversion into one of the before mentioned formats. The same goes for graphics from other graphical programs (CorelDraw, Adobe Illustrator, etc.). The fi gures should be prepared in fi nal size, published in the magazine. The dimen- sions are 12.5 cm maximum width and 19 cm maximum height (width and height of the text on a page). 11. Conclusions Articles shall end with a summary of the main fi ndings which may be written in point form. 12. Summary Articles written in Slovene must contain a more extensive English summary. The reverse also applies. 123 13. Literature References shall be cited in the text. If a reference work by one author is cited, we write Allan (1995) or (Allan 1995); if a work by two authors is cited, (Trinajstić and Franjić 1994); if a work by three or more authors is cited, (Pullin et al. 1995); and if the reference appears in several works, (Honsig-Erlenburg et al. 1992, Ward 1994a, Allan 1995, Pullin et al. 1995). If several works by the same author published in the same year are cited, the individual works are indicated with the added letters a, b, c, etc.: (Ward 1994 a,b). The bibliography shall be arranged in alphabetical order beginning with the surname of the fi rst author, comma, the initials of the name(s) and continued in the same way with the rest of the authors, separated by commas. The names are followed by the year of publication, the title of the article, the international abbreviation for the journal (periodical), the volume, the number in parenthesis (optional), and the pages. Example: Mielke, M.S., Almeida, A.A.F., Gomes, F.P., Aguilar, M.A.G., Mangabeira, P.A.O., 2003. Leaf gas exchange, chlorophyll fl uorescence and growth responses of Genipa americana seedlings to soil fl ooding. Experimental Botany, 50 (1), 221-231. Books, chapters from books, reports, and congress anthologies use the following forms: Allan, J.D., 1995. Stream Ecology. Structure and Function of Running Waters, 1st ed. Chapman & Hall, London, 388 pp. Pullin, A.S., McLean, I.F.G., Webb, M.R., 1995. Ecology and Conservation of Lycaena dispar: British and European Perspectives. In: Pullin, A. S. (ed.): Ecology and Conserva- tion of Butterfl ies, 1st ed. Chapman & Hall, London, pp. 150-164. Toman, M.J., 1992. Mikrobiološke značilnosti bioloških čistilnih naprav. Zbornik referatov s posvetovanja DZVS, Gozd Martuljek, pp. 1-7. 14. Format and Form of Articles The manuscripts should be sent exclusively in electronic form. The format should be Mi- crosoft Word (*.doc) or Rich text format (*.rtf) using Times New Roman 12 font with double spacing, align left only and margins of 3 cm on all sides on A4 pages. Paragraphs should be separated by an empty line. The title and chapters should be written bold in font size 14, also Times New Roman. Possible sub-chapter titles should be written in italic. All scientifi c names must be properly italicized. Used nomenclature source should be cited in the Methods section. The text and graphic material should be sent to the editor-in-chief as an e-mail attachment. For the purpose of review the main *.doc or *.rtf fi le should contain fi gures and tables included (each on its own page). However, when submitting the manu- script the fi gures also have to be sent as separate attached fi les in the form described under paragraph 10. All the pages (including tables and fi gures) have to be numbered. All articles must be proofread for professional and language errors before submission. A manuscript element checklist (For a manuscript in Slovene language the same checklist is appropriately applied with a mirroring sequence of Slovene and English parts): 1. English title .................................................................................................................... (Times New Roman 14, bold) 2. Slovene title ................................................................................................................... (Times New Roman 14, bold) 3. Names of authors with clearly indicated addresses, affi liations and the name of the 124 Acta Biologica Slovenica, 53 (2), 2010 corresponding author ...................................................................................................... (Times New Roman 12) 4. Author(s) address(es) / institutional addresses ............................................................... (Times New Roman 12) 5. Fax and/or e-mail of the corresponding author .............................................................. (Times New Roman 12) 6. Keywords in English ...................................................................................................... (Times New Roman 12) 7. Keywords in Slovene ..................................................................................................... (Times New Roman 12) 8. Running title ................................................................................................................... (Times New Roman 12) 9. Abstract in English ......................................................................................................... (Times New Roman 12, title – Times New Roman 14 bold) 10. Abstract in Slovene ........................................................................................................ (Times New Roman 12, title – Times New Roman 14 bold) 11. Introduction .................................................................................................................... (Times New Roman 12, title – Times New Roman 14 bold) 12. Material and methods ..................................................................................................... (Times New Roman 12, title – Times New Roman 14 bold) 13. Results ............................................................................................................................ (Times New Roman 12, title – Times New Roman 14 bold) 14. Discussion ...................................................................................................................... (Times New Roman 12, title – Times New Roman 14 bold) 15. Summary in Slovene ...................................................................................................... (Times New Roman 12, title – Times New Roman 14 bold) 16. Figure captions; each in English and in Slovene ........................................................... (Times New Roman 12, fi gure designation and fi gure title – Times New Roman 12 bold) 17. Table captions; each in English and in Slovene ............................................................. (Times New Roman 12, table designation and table title – Times New Roman 12 bold) 18. Acknowledgements ........................................................................................................ (Times New Roman 12, title – Times New Roman 14 bold) 19. Literature ........................................................................................................................ (Times New Roman 12, title – Times New Roman 14 bold) 20. Figures, one per page; fi gure designation indicated top left .......................................... (Times New Roman 12 bold) 21. Tables, one per page; table designation indicated top left .............................................. (Times New Roman 12 bold) 22. Page numbering – bottom right ...................................................................................... (Times New Roman 12) 15. Peer Review All Scientifi c Articles shall be subject to peer review by two experts in the fi eld (one Slov- ene and one foreign) and Brief Note articles by one Slovene expert in the fi eld. With articles written in Slovene and dealing with a very local topic, both reviewers will be Slovene. In the compulsory accompanying letter to the editor the authors must nominate one foreign and one Slovene reviewer. However, the fi nal choice of referees is at the discretion of the Editorial Board. After publication the corresponding author will receive the *.pdf version of the paper.