ACTA BIOLOGICA SLOVENICA VOL. 54 [T. 2 LJUBLJANA 2011 prej/formerly BIOLO[KI VESTNIK ISSN 1408-3671 izdajatelj/publisher UDK 57(497.4) Dru{tvo biologov Slovenije Acta Biologica Slovenica Glasilo Društva biologov Slovenije – Journal of Biological Society of Slovenia Izdaja – Published by Društvo biologov Slovenije – Biological Society of Slovenia Glavna in odgovorna urednica – Editor in Chief Alenka Gaberšcik, e-mail: alenka.gaberscik@bf.uni-lj.si Tehnicni urednik – Managing Editor Gregor Zupancic, 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 Janžekovic (SLO), Branko Vreš (SLO), Boris Sket (SLO), Franc Batic (SLO), Georg A. Janauer (A), Doekele G. Stavenga (NL) Naslov uredništva – Adress of Editorial Office Acta Biologica Slovenica, Vecna 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: 2011 Tisk – Print: Tiskarna Pleško d.o.o., Ljubljana Naklada: 400 izvodov Cena letnika (dve številki): 15 € za posameznike, 42 € za ustanove Številka poslovnega racuna pri Ljubljanski banki: 02083-142508/30 Publikacijo je sofinancirala Javna agencija za knjigo Republike Slovenije Acta Biologica Slovenica je indeksirana v – is indexed in: CAB Abstracts, Ulrichsweb Obvestilo o smrti akademika Zvonimirja Devidéja Uredništvo revije Acta Biologica Slovenica obvešca svoje bralce, da je v Zagrebu, 10. septembra 2011, umrl akademik Zvonimir Devidé, priznani biolog in botanik. Ob njegovi 90-letnici rojstva je v prejšni številki naše revije prof. dr. Božidar Krajncic objavil obširen prispevek o njegovem življenju in delu. Spoštovanega rojaka bomo ohranili v trajnem spominu. Genetic background of uropathogenic Escherichia coli isolates from Slovenia in relation to fluoroquinolone and sulfamethoxazole/trimethoprim resistance Genetsko ozadje uropatogenih sevov bakterije Escherichia coli iz Slovenije v povezavi z odpornostjo proti fluorokinolonom in sulfametoksazol/trimetoprimu 1Marjanca Starcic Erjavec, 2Anja Palandacic, 1Darja Žgur-Bertok, 1Jerneja Ambrožic Avguštin* 1Department of Biology, Biotechnical Faculty, University of Ljubljana,Vecna pot 111, SI-1000 Ljubljana, Slovenia 2Department of Animal Science, Biotechnical Faculty, University of Ljubljana, Groblje 3, 1230 Domžale, Slovenia *correspondence: jerneja.ambrozic@bf.uni-lj.si Abstract: Atotal of 99 E. coli urinary tract isolates were investigated for phylo-genetic groups and 21 virulence related genes in relation to fluoroquinolone and sulfamethoxazole/trimethoprim resistance. We found that the B2 group was by far the most prevalent among susceptible isolates, while resistant isolates were more evenly distributed among groups A, B2 and D. Isolates from the B2 group exhibited the highest prevalence of virulence factors. Virulence genes hlyA, iroN and kpsMTII were statistically associated with fluoroquinolone susceptible isolates and picU with sulfamethoxazole/trimethoprim susceptible isolates. Fluoroquinolone susceptible isolates of the phylogenetic group Awere significantly associated with genes papGII, kpsMTII and iss and the susceptible group B2isolates with genes hra in iroN. Among isolates susceptible to sulfamethoxazole/trimethoprim the presence of the hra gene was statistically significantly associated with phylogenetic group B2, while among resistant isolates, papGII was associated with phylogenetic group D. Keywords: Escherichia coli, urinary tract, phylogenetic groups, virulence trait, fluoroquinolone resistance, sulfamethoxazole/trimethoprim resistance Izvlecek: Vnaši raziskavi smo 99 uropatogenih izolatov E. coli uvrstili v filoge­netske skupine in pri vsakem preverili prisotnost 21-ih genov povezanih z virulenco ter podatke analizirali v povezavi z odpornostjo izolata za fluorokinolone in sulfame­toksazol/trimetoprim. Ugotovili smo, da se izolati, ki so obcutljivi za fluorokinolone in /ali sulfametoksazol/trimetoprim uvršcajo predvsem v filogenetsko skupino B2, odporniizolatipavpribližnoenakihdeležihvskupineA,B2inD.Izolativfilogenetski skupiniB2soimelinajvecgenskihzapisovzavirulentnedejavnike.Izolatiobcutljiviza fluorokinolone so imeli statisticno znacilno pogosteje preucevane genske zapise hlyA, iroN in kpsMTII vprimerjavizodpornimiizolati,medtemkosoimeliizolatiobcutljivi za sulfametoksazol/trimetoprim v primerjavi z odpornimi izolati statisticno znacilno pogosteje genski zapis za picU.Priizolatih,kisobiliobcutljivizafluorokinolone,smo ugotovili statisticno znacilne povezave med prisotnostjo genov papGII, kpsMTII ter iss in uvrstitvijo izolata v filogenetsko skupino Ater genov hra in iroN ter uvrstitvijo izolata v filogenetsko skupino B2. Pri izolatih, ki so bili obcutljivi za sulfametoksazol/ trimetoprim, je bila statisticno znacilna povezava med prisotnostjo gena hra in uvr­stitvijo izolata v filogenetsko skupino B2, pri odpornih izolatih pa je bil gen papGII statisticno znacilno povezan z uvrstitvijo izolata v filogenetsko skupino D. Kljucne besede:Escherichia coli,secila,filogenetskeskupine,virulentnidejavniki, odpornost proti fluorokinolonom, odpornost proti trimetoprimu in sulfametoksazolu Introduction Urinary tract infections (UTIs) are one of the most frequent infectious diseases encountered in the developed world. Uropathogenic Escherichia coli (E. coli) strains (UPEC) are the major cause ofuncomplicatedUTIworldwide.InSlovenia E. coli causesapproximately80%ofallUTIs(Lindic 2005). In comparison to commensalE. coli strains, UPECpossessanarrayofvirulencefactorsnamely, adhesins,toxins,polysaccharidecoatings,invasins, ironuptakesystemsandsystemstoevadethehost immuneresponse(Oelschlaegeretal.2002).UPEC mainly belong to the B2 phylogenetic group and to a lesser extent to the D group, while commen­sal strains belong to groups Aand B1 (Picard et al. 1999). The most frequently prescribed drugs for the treatment of UTIs in general practices in Sloveniaaretrimethoprim/sulfamethoxazole(57% ofprescribedantibiotics)andthefluoroquinolones norfloxacinandciprofloxacin(38%ofprescribed antibiotics) (Car et al. 2003). However, a major problem in treatment of UTIs is the emergence of E. coli strains resistant to these first-line anti­microbials. To unravel the relationship between resistance and virulence, several studies have dealt with the characteristics of fluoroquinolone and/or trimethoprim/sulfamethoxazole resist­ant strains, including phylogenetic background (A, B1, B2 and D group) and virulence factors (Drews et al. 2005, Horcajada et al. 2005, John­son et al. 2003, Johnson et al. 2005, Johnson et al. 2009, Moreno et al. 2006, Piatti et al. 2008, Takahashi et al. 2009, Vila et al. 2002). Since no comparable data are available for UPEC isolates from Slovenia, we investigated the distribution ofvirulencegenesamongphylogeneticgroupsin relation to fluoroquinolone and sulfamethoxazole/ trimethoprim resistance. Material and methods Bacterial strains The UPEC isolates investigated in this study were collected and identified at the Institute of Public Health of the Republic of Slovenia (IVZ) between the years 2004–2007. The strains were isolated from urine of outpatients with cystitis, whosoughthelpatgeneralpractisesinSlovenian Community Health Centres. Only one isolate per patientwasincludedinourstudy.Arandomsample of45ciprofloxacinresistantand54ciprofloxacin susceptible isolates, as determined by the disk diffusion method and interpreted according to the CLSI standards (Clinical and Laboratory Standards Institute 2007), were included in the study.Additionallyallisolateswerealsotestedfor sulfamethoxazole/trimethoprim resistance. Detection of phylogenetic groups and virulence factors DNA to be PCR amplified for detection of phylogenetic groups and virulence factors was released from whole bacterial cells by boiling according to Le Bouguenec et al. (1992). For all isolatesthephylogeneticgroups(A,B1,B2andD) weredeterminedusingthetriplexPCRdescribed byClermontetal.(2000).Further,allisolateswere screenedforthepresenceof21urovirulencegenes, includingfimbriae/adhesins(fimH –type1-fimbrial adhesin, papGII – P-fimbrial adhesin II, sfaDE – S-fimbriae, bmaE – M-fimbrial adhesin, gafD – G-fimbrial adhesin, iha – non fimbrial adhesin Iha and hra – non fimbrial adhesin Hra), toxins/ autotransporters (hlyA – hemolysin A, hbp – hae­moglobin protease, sat – secreted autotransporter toxin, vat – vacuolating autotransporter toxin and picU – autotransporter involved in intestinal colo­nization PicU),invasins(ompA –outermembrane protein A, ibeA – invasion of brain endothelium and aslA – arylsulphatase-like protein), genes involved in iron acquisition (iucD – aerobactin synthesis, iroN – catecholate siderophore receptor and irp2 – yersiniabactin biosynthesis), capsule synthesis (kpsMTII), increased serum survival (iss) and uropathogenic specific protein (usp).The employed primers are available at http://www. bf.uni-lj.si/fileadmin/users/1/biologija/genetika/ Table-PCR-primers.pdf. Statistical analysis Thesignificanceoftheresultswasestablished using the Fisher’s exact test (2-tailed) available on-line on the web site http://www.langsrud.com/ fisher.htm.Thethresholdforstatisticalsignificance was set at a P value < 0.05. Results Prevalence of phylogenetic groups and virulence factors in relation to resistance phenotypes As seen from Table 1, the majority (30 out of 54, 56%) of the fluoroquinolone-susceptible strains were assigned to the phylogenetic group B2,followedbytheDgroupwith17strains(31%). Thefluoroquinolone-resistantstrainswereevenly distributedamongthephylogeneticgroupsA,B2 and D; 13 strains (29%) belonged to the Agroup, 14 strains (31%) to the B2 group and 16 strains (35%) to the D group. The differences between the prevalence of fluoroquinolone-susceptible and resistant strains in the A and B2 groups were statistically significant. Of the examined virulence genes 13 occurred with a higher preva­lenceamongfluoroquinolonesusceptibleisolates while the associations of hlyA, iroN and kpsMTII with susceptibility were statistically significant. In accordance with the higher prevalence of the majority of virulence genes, the average virulence score among susceptible strains was higher compared to resistant strains (7.76 versus 6.13). The majority (26 out of 48, 54%) of the sulfamethoxazole/trimethoprim-susceptiblestrains were assigned to the phylogenetic group B2, fol­lowed by the D group with 15 strains (31%). The sulfamethoxazole/trimethoprim-resistant strains were distributed more evenly among the phylo-genetic groups A(25%), B2 (35%) and D (35%). However, the differences between the number of sulfamethoxazole/trimethoprim-susceptible and resistant strains with regard to phylogenetic group was statistically not significant. Twelve of the examined virulence genes occurred with a higher prevalence among sulfamethoxazole/ trimethoprim-susceptible isolates (Table 1), but only the occurrence of picU was statistically significant. The average virulence score among sulfamethoxazole/trimethoprim susceptible and resistant isolates was 7.31 versus 6.75. Significant associations of virulence genes and phylogenetic groups in relation to resistance phenotypes Only virulence genes with a prevalence of =10% were selected for the analysis of associa­tions of virulence genes and phylogenetic groups in relation to resistance phenotypes (Table 2). While among fluoroquinolone and sulfa-methoxazole/trimethoprim-susceptiblestrainsthe majority of the virulence genes were detected in isolates of the B2 group, only the occurrence of hra and iroN wasstatisticallysignificant,thefirst among fluoroquinolone and sulfamethoxazole/ trimethoprim-susceptibleisolatesandthesecond only among sulfamethoxazole/trimethoprim-susceptible isolates (Table 2). Among the latter isolates papGII, kpsMTII and iss weresignificantly associated with phylogenetic group A. Among sulfamethoxazole/trimethoprim-resistantisolates only papGII wassignificantlyassociatedwiththe D group isolates (Table 2). Discussion This study showed that 56% of fluoroqui­nolone-susceptibleand 54% of sulfamethoxazole/ trimethoprim-susceptible isolates belonged to phylogeneticgroupB2,whilefluoroquinolone-and sulfamethoxazole/trimethoprim-resistantisolates wereevenlydistributedamonggroupsA,B2,and D. However, the percentage of fluoroquinolone-resistant isolates belonging to group B2 has been increasing in the last decade. Studies on E. coli isolatedfrom1998to2003reported12%(Johnson Table 1: Prevalence of phylogenetic groups and virulence genes in relation to resistance phenotypes among the studied E. coli isolates Tabela 1:Prevalencefilogenetskih skupin in genov za dejavnike virulence pri odpornih in obcutljivih izolatih E.coli Prevalence [N (%)] FQ SXT S R P S R P (N = 54) (N = 45) (N = 48) (N = 51) Phylogenetic group A 4 (7) 13 (29) 0.007 4 (8) 13 (25) ns B1 3 (6) 2 (4) ns 3 (6) 2 (4) ns B2 30 (56) 14 (31) 0.016 26 (54) 18 (35) ns D 17 (31) 16 (35) ns 15 (31) 18 (35) ns Virulence gene fimH 52 (96) 45 (100) ns 47 (98) 50 (98) ns papGII 25 (46) 17 (38) ns 19 (40) 23 (45) ns sfaDE 20 (37) 10 (22) ns 19 (40) 11 (22) ns bmaE 2 (4) 0 (0) ns 0 (0) 2 (4) ns gafD 0 (0) 0 (0) ns 0 (0) 0 (0) ns iha 21 (39) 18 (40) ns 14 (29) 25 (49) ns hra 16 (30) 7 (16) ns 14 (29) 9 (18) ns hlyA 10 (19) 0 (0) 0.002 6 (13) 4 (8) ns hbp 2 (4) 3 (7) ns 3 (6) 2 (4) ns sat 14 (26) 11 (24) ns 11 (23) 14 (27) ns vat 6 (11) 2 (4) ns 6 (13) 2 (4) ns picU 14 (26) 5 (11) ns 15 (31) 4 (8) 0.005 ompA 49 (91) 41 (91) ns 43 (90) 47 (92) ns ibeA 8 (15) 8 (18) ns 8 (17) 8 (16) ns aslA 0 (0) 0 (0) ns 0 (0) 0 (0) ns iucD 32 (59) 29 (64) ns 25 (52) 36 (71) ns iroN 37 (69) 17 (38) 0.003 29 (60) 25 (49) ns irp2 44 (81) 30 (67) ns 40 (83) 34 (67) ns kpsMTII 39 (72) 19 (42) 0.004 32 (67) 26 (51) ns iss 12 (22) 8 (18) ns 7 (15) 13 (25) ns usp 16 (30) 6 (13) ns 13 (27) 9 (18) ns AVS 7.76 6.13 7.31 6.75 FQ: Fluoroquinolone; Sxt: Sulfamethoxazole/Trimethoprim; S: Susceptible; R: Resistant; AVS: average virulence score (the average virulence score was calculated as the sum of all detected virulence associated genes divided with the number of isolates per group)/; ns – not statistically significant Table 2: Associations of virulence genes with phylogenetic groups in relation to resistance phenotypes among the studied E. coli isolates. Tabela 2:Povezava genov z zapisom za dejavnike virulence z uvrstitvijo v filogenetsko skupino in odpornostjo. Prevalence [no. (%)] FQ SXT S R P S R P Virulence Phylogenetic gene group fimH A 4 (100) 13 (100) ns 4 (100) 13 (100) ns B1 3 (100) 2 (100) ns 3 (100) 2 (100) ns B23 29 (97) 14 (100) ns 26 (100) 17 (94) ns D 16 (94) 16 (100) ns 14 (93) 18 (100) ns papGII A 3 (75) 0 (0) 0,006 2 (50) 1 (8) ns B1 1 (33) 0 (0) ns 1 (33) 0 (0) ns B23 13 (43) 5 (36) ns 11 (42) 7 (39) ns D 8 (47) 12 (75) ns 5 (33) 15 (83) 0,005 sfaDE A 1 (25) 1 (100) ns 0 (0) 2 (15) ns B1 0 (0) 0 (0) ns 0 (0) 0 (0) ns B23 15 (50) 6 (43) ns 15 (58) 6 (33) ns D 4 (24) 3 (19) ns 4 (27) 3 (21) ns bmaE A 0 (0) 0 (0) ns 0 (0) 0 (0) ns B1 1 (33) 0 (0) ns 0 (0) 1 (50) ns B23 0 (0) 0 (0) ns 0 (0) 0 (0) ns D 1 (11) 0 (0) ns 0 (0) 1 (7) ns iha A 1 (25) 4 (33) ns 0 (0) 5 (42) ns B1 0 (0) 0 (0) ns 0 (0) 0 (0) ns B23 12 (40) 8 (57) ns 9 (35) 11 (61) ns D 8 (47) 6 (38) ns 5 (33) 9 (50) ns hra A 0 (0) 4 (33) ns 0 (0) 4 (33) ns B1 0 (0) 0 (0) ns 0 (0) 0 (0) ns B23 13 (43) 1 (7) 0,019 12 (46) 2 (11) 0,021 D 3 (18) 2 (14) ns 2 (33) 3 (21) ns hlyA A 0 (0) 0 (0) ns 0 (0) 0 (0) ns B1 0 (0) 0 (0) ns 0 (0) 0 (0) ns B23 7 (23) 0 (0) ns 4 (15) 3 (17) ns D 3 (38) 0 (0) ns 2 (33) 1 (25) ns hbp A 0 (0) 1 (8) ns 1 (25) 0 (0) ns B1 0 (0) 1 (50) ns 0 (0) 1 (50) ns B23 2 (7) 0 (0) ns 1 (4) 1 (6) ns D 0 (0) 1 (7) ns 1 (11) 0 (0) ns sat A 1 (25) 0 (0) ns 0 (0) 1 (8) ns B1 0 (0) 0 (0) ns 0 (0) 0 (0) ns B23 8 (27) 7 (50) ns 7 (27) 8 (44) ns D 5 (29) 4 (29) ns 4 (44) 5 (28) ns 10 Acta Biologica Slovenica, 54 (2), 2011 Prevalence [no. (%)] FQ SXT S R P S R P Virulence Phylogenetic gene group vat A 0 (0) 1 (8) ns 0 (0) 1 (8) ns B1 0 (0) 0 (0) ns 0 (0) 0 (0) ns B23 6 (20) 1 (7) ns 6 (23) 1 (6) ns D 0 (0) 0 (0) ns 0 (0) 0 (0) ns picU A 2 (50) 1 (8) ns 2 (50) 1 (8) ns B1 0 (0) 0 (0) ns 0 (0) 0 (0) ns ompA ibeA iucD iroN irp2 kpsMTII B23 D A B1 B23 D A B1 B23 D A B1 B23 D A B1 B23 D A B1 B23 D A B1 9 (30) 4 3 (18) 0 3 (75) 10 1 (33) 1 28 (93) 14 17 (100) 16 0 (0) 1 0 (0) 2 7 (23) 2 1 (11) 3 4 (100) 7 2 (67) 1 17 (57) 12 9 (53) 9 3 (75) 4 2 (67) 1 25 (83) 7 7 (41) 5 4 (100) 5 2 (67) 1 27 (90) 14 11 (65) 10 2 (50) 0 0 (0) 1 (29) ns (0) ns (77) ns (50) ns (100) ns (100) ns (8) ns (100) ns (14) ns (21) ns (54) ns (50) ns (86) ns (56) ns (31) ns (50) ns (50) 0,032 (36) ns (42) ns (50) ns (100) ns (63) ns (100) 0,044 (50) ns 10 (38) 3 (20) 2 (50) 1 (33) 25 (96) 15 (100) 0 (0) 1 (33) 5 (19) 2 (22) 3 (75) 1 (33) 15 (58) 6 (40) 2 (50) 1 (33) 21 (81) 5 (33) 4 (100) 1 (33) 24 (92) 11 (73) 0 (0) 0 (0) 20 (77) 12 (80) 3 (75) 0 (0) 3 (12) 1 (11) 3 (17) 0 (0) 11 (85) 1 (50) 17 (96) 18 (100) 1 (8) 1 (50) 4 (22) 2 (14) 8 (62) 2 (100) 14 (78) 12 (67) 5 (38) 2 (100) 11 (61) 7 (39) 5 (42) 2 (100) 17 (94) 10 (56) 2 (17) 1 (50) 12 (67) 11 (61) 3 (25) 1 (50) 4 (22) 5 (28) ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns iss B23 D A B1 B23 D 24 (80) 8 13 (76) 10 4 (100) 2 1 (33) 0 6 (20) 1 1 (12) 5 (57) ns (63) ns (17) 0,006 (0) ns (7) ns (36) ns Starcic Erjavec et al.: Genetic background of uropathogenic Escherichia coli 11 Prevalence [no. (%)] FQ SXT S R P S R P Virulence Phylogenetic gene group usp A 1 (25) 0 (0) ns 0 (0) 1 (8) ns B1 0 (0) 0 (0) ns 0 (0) 0 (0) ns B23 13 (43) 6 (43) ns 11 (42) 8 (44) ns D 2 (25) 0 (0) ns 2 (33) 0 (0) ns Virulence traits with =10 % prevalence were included. FQ: Fluoroquinolone; SXT: sulfamethoxazole/trimethoprim; S: Susceptible; R: Resistant; et al. 2003), 0% (Johnson et al. 2005) and 11% (Morenoetal.2006)ofgroupB2fluoroquinolone resistant isolates, while among E. coli isolated from 2005 to 2007 the percentage raised upon 50% (Takahashi 2009) and 49% (Johnson et al. 2009). The relatively high percentage (31%) of fluoroquinolone-resistant isolates belonging to group B2 in our collection, comprising E. coli isolates from 2004–2007, is therefore in agree­ment with this trend. Theprevalenceofvirulencegenesamongthe studied UPEC isolates from Slovenia revealed that, resistant isolates possessed less virulence genes than susceptible isolates and vice versa, which is in accordance with results of similar studiesfromothercountries(Johnsonetal.2003, Johnson et al. 2005, Piatti et al. 2008). However, theexaminedUPECcollectionsexhibiteddistinct significant associations of fluoroquinolone and sulfamethoxazole/trimethoprimresistancepattern with particular virulence genes. For example, in our study we found that, the virulence genes hra and iroN were statistically significant among fluoroquinolone- susceptiblephylogeneticgroup B isolates, while Piatti et al. (2008) reported a significant association between gene iss and fluo­roquinolone susceptible group B isolates. To elucidate the basis of such differences further studies are needed, as for now we can onlyspeculatethatsamplesize,thecharacteristics of the studied isolates and their hosts might be relevant. In addition, the evolutionary origin of the association between possession of virulence factorsandsusceptibilityneedstobeclarified.To thisendseveralhypothesishavebeenpostulated: (i) acquisition/loss of pathogenicity islands, (ii) incompatibility of plasmids encoding virulence and resistance, (iii) less virulent strains are more prone to acquire resistance, (iv) acquisition of resistance promotes loss of virulence. To summarise, our results in comparison to studies performed before 2004 show a steep increase in the prevalence of fluoroquinolone­resistantstrainsbelongingtotheB2group.Itisof great concern that E. coli strains of the B2 group, whichareknowntoexhibitthegreatestvirulence potential, are readily acquiring resistance to fluo­roquinolones.Thesestrainsaddtionallyequipped withCTX-Mplasmidscarryingextended-spectrum beta-lactamases (ESBL) and plasmid-mediated quinolone resistance (PMQR) genes might be the source of highly resistant and virulent clonal groups, such as E. coli ST131. Povzetek Bakterije vrste Escherichia coli so sicer del normalne crevesne mikrobiote ljudi in živali s stalno telesno temperaturo, vendar med njimi obstajajotudi(potencialno)patogenisevi,kilahko povzrocijorazlicneokužbe.Odkomenzalnihsevov seobicajnolocijopoprisotnostištevilnihgenovz zapisi za dejavnike virulence, ki bakterijam med drugimomogocajopritrjanjenagostiteljskecelice, poškodbe gostiteljske celice, privzem železa in izogibanjeimunskemusistemu.Uropatogenisevi E. coli so poglavitni povzrocitelji nezapletenih okužb secil. Za zdravljenje teh okužb se najpog­osteje uporabljajo protimikrobne snovi sulfame­toksazol/trimetoprim in fluorokinoloni vendar je zaradi narašcanja odpornih sevov zdravljenje vse težje. Zato nas je zanimalo ali obstaja kakšna povezava med genetskim ozadjem uropatogenih sevov in odpornostjo proti sulfametoksazol/tri­metoprimuinfluorokinolonom.Vnašoraziskavo smo vkljucili 99, za protimikrobni ucinkovini obcutljivih in odpornih bakterij E. coli, ki so bile izolirane iz urina bolnikov z vnetjem secil. Vse seve smo uvrstili v filogenetske skupine in jih pregledalizaprisotnost21-ihgenovzzapisomza dejavnike virulence. Ugotovili smo, da je najvec obcutljivih izolatov iz filogenetske skupine B2, odporni izolati pa so enakomerno razporejeni v filogenetskih skupinah A, B2 in D in, da imajo izolati iz filogenetske skupini B2 najvec genskih zapisov za virulentne dejavnike. Poleg tega smo izsledili nekatere statisticno znacilne povezave med prisotnostjo genskega zapisa za virulentne dejavnike,zuvrstitvijosevavfilogenetskoskupino inodpornostjoprotisulfametoksazol/trimetoprimu in/ali fluorokinolonom. Zakljucki naše raziskave References so, da so sevi z vecjim naborom genov, ki pripo­morejokvirulenci,boljobculjivizaprotimikrobni ucinkoviniinobratnoter,dasouropatogeniizolati E. coli iz Slovenije po svojem naborugenskih zapi­sovzadejavnikevirulencepodobniuropatogenim izolatom iz drugih geografskih okolij. Razlika je le v znacilnih statisticnih povezavahposameznih genov s filogenetskimi skupinami. Acknowledgements ThisresearchwasfinancedbyGrantP1-0198 from the Slovenian Research Agency (ARRS). We also thank the OMM Department of The Institute of Public Health of Slovenia for the identified strains. Car,J.,Švab,I.,Kersnik,J.,Vegnuti,M,.2003.Managementoflowerurinarytractinfectioninwomen by Slovene GPs. Fam. Pract., 20, 452–6. Clermont, O., Bonacorsi. S., Bingen, E., 2000. Rapid and simple determination of the Escherichia coli phylogenetic group. Appl. Environ. Microbiol., 66, 4555–4558. Clinical and Laboratory StandardsInstitute, 2007. 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Takahashi,A.,Muratani,T.,Yasuda,M.,Takahashi,S.,Monden,K.,Ishikawa,K.,Ishikawa,K.,Kiyota, H.,Arakawa,S.,Matsumoto,T.,Shima,H.,Kurazono,H.,Yamamoto,S.,2009.Geneticprofilesof fluoroquinolone-resistant Escherichia coli isolatesobtainedfrompatientswithcystitis:Phylogeny, virulence factors, paiusp subtypes, and mutation patterns. J. Clin. Microbiol.,47, 791–5. Vila, J., Simon, K., Ruiz, J., Horcajada, J.P., Velasco, M., Barranco, M., Mensa, J., 2002. Are quinolone-resistant uropathogenic Escherichia coli less virulent? J. Infect. Dis., 186, 1039–42. Molecular modelling of FtsZ proteins based on their homology in Escherichia coli and Mycobacterium tuberculosis as the key stage of rational design of new antituberculous compounds Molekularno modeliranje proteinov FtsZ na osnovi njihove homologije v Escherichia coli in Mycobacterium tuberculosis kot kljucna stopnja racionalnega oblikovanja novih protituberkuloznih komponent Oleh Demchuk a, Pavel Karpov a, Peter Raspor b, Yaroslav Blume a* aDepartment of Genomics and Molecular Biotechnology, Institute of Food Biotechnology and Genomics, Natl. Academy of Sciences of Ukraine, Osipovskogo str, 2a, Kyiv, 04123, Ukraine; bBiotechnical Faculty, University of Ljubljana, Jamnikarjeva, 101, 1000 Ljubljana, Slovenia *correspondence: yablume@univ.kiev.ua Abstract: The analysis of the quality of X-ray structures from Mycobacterium tuberculosis FtsZ proteins, which are deposited in the ProteinDataBank, gave a possi­bility to select a 2Q1Y(Chain A) structure as a template for future in silico research. Also several spatial models of FtsZ protein from Escherichia coli were reconstructed with on-line servers »SWISS-MODEL Workspace« and I-TASSER, than the most appropriatestructurewasselected.Basingoncomplexbioinformaticstudy,themodel, whichwasrebuiltbySwissModelserverfrom2Q1Y(chainA)template,wassupposed as the most significant. Keywords: FtsZ, Escherichia coli, Mycobacterium tuberculosis, 3D-structure modelling and verification, in silico Izvlecek:Analiza struktur proteinov FtsZz X žarki izMycobacterium tuberculosis deponiranih v »ProteinDataBank« je dala možnost izbora strukture 2Q1Y(veriga A) kot matrice za nadaljno in silico raziskavo. Nekaj prostorskih modelov proteina FtsZ iz bakterije Escherichia coli je bilo rekonstruiranih na on-line serverju SwissModel in I-Tasser, kateremu je sledil izbor najprimernejše strukture. Na osnovi celovite bio-informacijske preverbe kaže, da je model narejen na platformi serverja SwissModel iz matrice 2Q1Y (veriga A) najbolj sprejemljiv za uporabo. Kljucne besede: FtsZ, Escherichia coli, Mycobacterium tuberculosis, 3D-struk­turno modeliranje in preverjanje, in silico Introduction and inadequate control programs have lead to the emergence of multidrug-resistant strains of Tuberculosis is the leading cause of death in M. tuberculosis (Raviglione 2000). Bacterial theworld fromasingleinfectious disease, claim-resistance to three or more ‘second-line’anti­ing over three million lives each year (Huang et biotics is classified as extremely drug-resistant al. 2006). Furthermore, poor patientcompliance tuberculosis. Therefore, there is an urgent need for the development of new anti-tuberculosis drugs with novel mechanism of action(s), which are active against drug-resistant as well as drug-sensitive M. tuberculosis strains.FtsZ(Filamentous temperature-sensitive protein Z) is an essential cell division protein has been shown to be a bac­terial homolog of the mammalian cytoskeleton proteintubulin(Kumaretal.2010).Accordingly, FtsZ protein is a very promising target for new antimicrobial drug development, and especially compounds effective against drug-resistant M. tuberculosis strains (Kumar et al. 2011). It is the perspective target for such numerous and diverse groups of low molecular weight compounds as benzimidazoles (Ohashi et al. 1999, Kumar et al. 2011), naphthalenesulfonates (Yu and Margolin 1998), azithromycins(Margalitetal.2004),ethyl carbamates(Whiteetal.2000,2002),diterpenoid phenols (Jaiswal et al. 2007) etc. At the same time, modern rational design of new compounds with antibacterial activity is impossible without stage of virtual screening, with application of accurate three-dimensional models of target FtsZ-proteins. The last is very important due to FtsZ – tubulin structure and function similarity, places particularly high demands on quality of 3D-models used for in silico molecular docking and virtual screening. At present, Worldwide ProteinDataBank (wwPDB – www.pdb.org: RCSB PDB (USA)/ PDBe(Europe)/PDBj(Japan))(Bermanetal.2003) containtherowofbacterialFtsZX-raystructures ofdifferentresolution:2R6R(1.70Ĺ)(Olivaetal. 2007) and 2R75 (1.40Ĺ) (Läppchenet al. 2008) from Aquifex aeolicus; 2VAM (2.50Ĺ) (Oliva et al. 2007), 2VXY(1.70 Ĺ) (Haydon et al. 2008), 2RHH (2.00Ĺ), 2RHJ (1.76 Ĺ), 2RHL(2.45 Ĺ) and 2RHO (2.45Ĺ) (Raymond et al. 2009) from Bacillus subtilis;1FSZ(2.80Ĺ)(LöweandAmos 1998), 1W5B (2.20 Ĺ), 1W5A (2.40 Ĺ), 1W58 (2.50Ĺ) and 1W59 (2.70 Ĺ) (Oliva et al. 2004) from Methanococcus jannaschii;1RLU(2.08Ĺ), 1RQ2 (1.86 Ĺ), 1RQ7 (2.60 Ĺ) (Leung et al. 2004), 2Q1X (2.35Ĺ) and 2Q1Y(2.30Ĺ) (Res-picio et al. database record) from M. tuberculosis; 1OFU (2.10Ĺ) (Cordell et al., 2003) and 2VAW (2.90 Ĺ) (Oliva et al. 2007) from Pseudomonas aeruginosa; 1W5F (2.00 Ĺ) (Oliva et al. 2004) from Thermotoga maritima.Mostofthesespecies belongtodifferentphylaandsubkingdomsofthe Bacteria kingdom, and one,M. jannaschii, to the phylum Euryarchaeota (subkingdom Archaea). Atthesametime,thereareanumberofdefectsin all deposited in the Protein Data Bank structures. (Höltjeetal.2008)MostofdepositedinPDBX-ray structures of bacterial FtsZs characterized by the loss of N- and C-terminal fragments (typically a few tens of residues), presence of gaps in protein globule,aswellasabsenceofcertainheavyatoms of side chains of amino acid residues. Unfortunately, until now, there are no more or less complete X-ray structures of E. coli FtsZ protein, model organism also plays an important role in modern biological engineering and indus­trialmicrobiology..urrentlyonlythe1F47(PDB) structure have the last 17 amino acid residues (Lys367-Asp383), forming a short unstructured region,endswithatwo-helixturnattheC-terminal end (Mosyak et al. 2000). However, we are also interested in complete structure of this protein, due to the fact that commercial analytical kits for in vitro binding experiments are more available for E. coli FtsZ protein analysis than analytical kits for M. tu­berculosis. In vitro and in silico modelling of interaction with low-molecular compounds of both, E. coli and M. tuberculosis FtsZ proteins, such as benzimidazole derivatives, allow us much accurate binding-site identification and analysis. Based on FtsZs structural homology, these experimentally confirmed binding site (or sites), can be extrapolated from E. coli protein to the structure of mycobacterial homolog. This allowusmoreaccuratepredictionofbindingsites of such new and promising anti-TB compounds as benzimidazoles. Thus, thepurposeof theresearchwas in silico modelling of three-dimensional structure of E. coli FtsZ protein, and qualitative reconstruction of M. tuberculosis FtsZ protein model based on com-prehensiveanalysisofX-raystructuresdeposited in the Protein Data Bank. Methods Analysis of Protein Data Bank structures of FtsZ M. tuberculosis Complete amino acid sequence of M. tubercu­losis FtsZ(P64170)(Coleetal.1998,Fleischmann etal. 2002) was downloaded fromUniProt(http:// www.uniprot.org/) database (The UniProt Con-sortium2008).Multiplealignmentsofaminoacid sequencesof M. tuberculosis FtsZPDB-structures and P64170were realized in ClustalX 2.0.5 with a set of BLOSSUMmatrices(http://www.clustal. org, Larkin et al. 2007). The PDB-structures of FtsZ M. tuberculosis proteinwereanalyzedusing »DeepView–Swiss-PdbViewer4.0.3«(Guexand Peitsch 1997; http://www.expasy.org/spdbv/). In the absence of heavy atoms in the side-chains the program generated a warning notice about the type and location of structural defects. Lack of amino acid residues was detected by using Ac-celrys Discovery Studio Visualizer 3.0 (Accelrys Software Inc. – http://accelrys.com/). Reconstruction of 3D model of E. coli FtsZ protein Complete amino acid sequence of E. coli O157:H7 FtsZ(P0A9A8) (Pernaet al. 2001) was downloaded from UniProt (http://www.uniprot. org/) database (The UniProt Consortium 2008). Athree-dimensional structural modelling was car­riedoutontheI-TASSERserver(Royetal.2010; http://zhanglab.ccmb.med.umich.edu/I-TASSER) and with »SwissModel Automatic Modelling Mode« of »SWISS-MODELWorkspace« server (http://swissmodel.expasy.org/) (Arnold et al. 2006).Bothserversrunninginautomaticmodeof PDBstructure(template)selection.Asaresultwe generated5modelsofthree-dimensionalstructures with I-TASSER, and and one more model with »SWISS-MODEL Workspace«. Additionally, with »SWISS-MODEL Workspace« server we constructedanothermodelbasedon2Q1Y(Chain A) template PDB structure which was specified in the manual mode. Root mean square deviations (RMSD) of the fitted 3-D structures were calculated using »molecule align« tool of PyMol 1.4 package (www.pymol.org). Estimation of protein model quality The 3-D structures quality was assessed by processing the models on the MolProbity server. (Chen et al. 2010, http://molprobity.biochem. duke.edu/) This study was performed to estimate the statistics of all-atom contacts (i.e. »all atoms Clashscore«) and protein geometry: defined percentages of poor rotamers, Ramachandran outliers, Ramachandran favoured, residues with bad bonds, residues with bad angles and defined Cß deviations >0.25Ĺ and MolProbity score. We used the Protein Structure and Model Assessment Tools available at »SWISS-MODEL Workspace«server(http://swissmodel.expasy.org) to assess the quality of the 3-D models based on Raw-scoreandZ-scoreofQMEAN6(Composite scoring function for model quality estimation) (Benkert et al. 2009) and global model quality estimation based on »DFire energy« (all-atom distance-dependent statistical potential) (Zhou and Zhou 2002). Results Selection and quality checking of X-ray Protein Data Bank structures of M. tuberculosis FtsZ Scanning the Worldwide Protein Data Bank (wwPDB) we revealed several crystal structures of the M. tuberculosis cell division protein FtsZ, determinedat1.86to2,60ĹbyX-raymethod.The followingPDBstructureshavebeenstudied:1RLU (Chains: A, B) and 2Q1Y(Chains: A, B) X-rays ofFtsZ-GTPgammaS(5’-guanosinediphosphate monothiophosphate) complexes (Chains: A, B), 1RQ2 (Chains: A, B) and 2Q1X (Chains: A, B) X-rays of FtsZ-citrate complexes (Chains: A, B) and 1RQ7 X-ray of FtsZ-GDPcomplex (Chains: A, B). Using pairwise sequence alignment of polypeptide chains A and B from X-ray PDB structureswithcompletesequencesfromUniProt we tested them on presence of gaps (meaning oc­currenceofdefectsinstructures).PDB-structures were verified on the presence of such artifacts as deficiency of heavy atoms (carbon, oxygen and nitrogen) in side chains of individual amino acid residues (using DeepView-Swiss-PdbViewer 4.0.3 software package). All gaps in polypeptide chains and residues with defective side chains were checked and represented in the Figure 1 and Table 1. As a result, ofthetenavailableX-raystructures (considering chains A and B) the only one chain of M. tuberculosis FtsZ protein was selected as a most complete experimentally proved structure and the base of subsequent work on its detailed 3-D reconstruction and in silico analysis. Such structure wasa chain Aof X-ray FtsZ-GTP-gamma-S complex from 2Q1Y(2.30 Ĺ, R-value=0.174, R-free=0.210) (DOI:10.2210/pdb2q1y/pdb). It has no just first 7 (N-end) and the last 66 (C-end) amino acid residues, but, as opposed to the same A-chainof1RLU(2.08Ĺ,R-value=0.182,R-free= 0.224)(Leungetal.,2004),hasacompleteatomic composition of all available amino acids. The 2Q1Y chain A were analyzed with MolProbity server,andthevaluesofevaluationfunctions(see Table 2), demonstrate its high quality for further in silico experimentsand modelling its interactions with low molecular weight compounds. Table 1: Features and identified defects in Protein Data Bank X-rey structures of Mycobacterium tuberculosis FtsZ. Tabela 1:Lastnosti in okvare struktur PDB v proteinu FTsZ iz bakterije Mycobacterium tuberculosis. PDB Strucrure 1RLU 1RQ2 Method X-Ray Diffraction X-Ray Diffraction Resolution, Ĺ 2.35 Ĺ 1.86 Ĺ Chain A B A B A Defective regions of molecules N-terminal Tubulin/FtsZ family, GTPase .-terminal tail tile domain aa:D313 – R379; aa:M1-Y7 ha: E29, K33, R64, L66, R181 ha: K236, E252 ..:R60-G70,G170-A173; aa:D313 – R379; aa:M1-H5 ha: N6, L8, E73, K77, K120, R140 ha: K236, D301 ..:R64-A69; aa:D313 – R379; aa:M1-Y7 ha: K33, M177 ha; K236 ..:R60-G70, D171-A173; aa:M1-Y7 aa:V314-R379 ha: K33, Q45, E73 .. R64-A69; aa:D313 -R379; aa:M1-Y7 ha: K33, L48 ha: K236, Q255 1RQ7 X-Ray Diffraction 2.60 Ĺ B ..:R60-G70, D171-A173; aa:V314-R379; aa:M1-H5 ha: K33, E73, K120, R140, S141, ha: K236, D313 E153 2Q1X X-Ray Diffraction 2.35 Ĺ A B aa:M1-Y7 ..:T63-G70 aa:D313 – R379 ..:R60-A71, R140­aa:M1-H5 aa:D313 – R379 N142,Q168-A173 2Q1Y X-Ray Diffraction 2.30 Ĺ A B aa:M1-Y7 -aa:D313 – R379 aa:M1-H5 ..:R60-G70, G170-A173 aa:D313 – R379 aa – lack of respective amino acid residues in the X-rey structure; ha – absence of heavy atoms (carbon, oxygen or nitrogen) in the side chains of respective amino acid residues. Figure 1: Numerous alignment of the full-length sequence of FtsZ protein (UniProtKB entry: P64170) from Mycobacterium tuberculosis and sequences of corresponding X-Ray Protein Data Bank structures: 1RLU, 1RQ2, 1RQ7, 2Q1X and 2Q1Y. Slika1: Razlicice sekvence proteina FtsZ (UniProtKB ( P64170)) iz bakterije Mycobacterium tuberculosis in sekvence pripadajocih struktur proteinov FtsZ z X žarki: 1RLU, 1RQ2, 1RQ7, 2Q1X and 2Q1Y. E. coli FtsZ protein spatial structure prediction DespitethegreatinterestinmycobacterialFtsZ asthetargetforantibacterialcompounds,majority of commercial analytical kits for in vitro binding experiments are more available for FtsZ protein from E. coli than its mycobacterial homolog. So, here we have a paradox situation, the presence of well proven three-dimensional structure of M. tuberculosis FtsZ protein on the one hand, and at the other hand, the fact that majority of the experimental toolstargetedE. coli FtsZ,forwhich there is a clear gap in 3D-structure research. So, now,wehaveonly1F47PDBstructurepresented only by last 17 amino acid residues (Lys367­Asp383), forming a short unstructured element, ending with two a-helix turns in C-end (Mosyak et al. 2000). In order to solve this problem we applied in silico homology modelling. Initially the sequence of E. coli FtsZ protein (UniProt: P0A9A6) has been sent to the »SWISS-MODELWorkspace« server, for model building (alignment).Withcompletelyautomaticmodelling of E. coli FtsZprotein,serverselectedthechainB of the 1OFU X-ray structure from the SulA-FtsZ complex (2.10Ĺ, R-value=0.216, R-free=0.255) from P. aeruginosa (Cordell et al., 2003) as the templatestructure.»SWISS-MODELWorkspace« servergeneratedonemodelfortargetFtsZprotein of 293 aminoacid residues in length (from Asn24 to Gly316 inclusive). The secondary and tertiary structures of model was completely similar to those in 1OFU except absence of small area in theN-end,coveringthefirstß-foldandsignificant part of the next a-helix (Fig.3a). In the structure of the matrix protein these elements are present (Ala11-Gly23 in FtsZ protein from E. coli, and Ala12-Ggly24 in FtsZ protein from P. aeruginosa). WhenthechainAof2Q1Ystructurewasassigned as a matrix (previously selected as the best X-ray structure of M. tuberculosis FtsZ protein, see above),»SWISS-MODELWorkspace«serverbuilt model of E. coli FtsZ protein, which contained in its structure above-mentioned region (Fig.2b) and was 305 amino acid residues in length (from Ala11 to Ile315 inclusive). Sequences of these structural areas in E. coli and M. tuberculosis FtsZ proteins have differences in amino acid residues: Ile16-Val14, Val18-Ile16 and G23-V20, respec­tively. Fitting of these two E. coli FtsZstructures, based on different modelling matrixes (Fig. 2c), demonstrate high level of structural similarity confirmed by root-mean-square deviation of Ca­atoms (RMSD=0.862 Ĺ) (Höltje et al. 2008). E. coli FtsZ protein spatial structure modelling performed by using I-TASSER server In parallel with using of classical template-based modelling, we have applied 3D-recon­struction using on-line I-TASSER server (http:// zhanglab.ccmb.med.umich.edu). I-TASSER 3D-models are built based on multiple-threading alignmentsbyLOMETS(WuandZhang2007)and iterativeTASSERassemblysimulations;function insightsarethenderivedbymatchingthepredicted models with protein function databases. (Roy et al. 2010) Following the sequence-to-structure-to-functionparadigm,theI-TASSERprocedure(Roy et al. 2010) for structure and function modelling involves four consecutive steps of: (a) template identificationbyLOMETS(WuandZhang2007); (b) fragment structure reassembly by replica-exchange Monte Carlo simulations (Zhang et al. 2002);(c)atomiclevelstructurerefinementusing REMO (Li et al. 2009) and FG-MD (Zhang et al. 2011); and (d) structure-based function interpreta­tions using COFACTOR (Roy et al. 2011). We submitted request of E. coli FtsZ-protein sequence (UniProt: P0A9A6) at the I-TASSER server using on default settings without manual assignment of template structure. As seen from Table 3, the server align our query sequence with a range of such template PDB structures as 2VAW (Chain A), 1FSZ (Chain A), 2VAM (Chain A), 2R6R (Chain A), 1W5F (Chain A) and 2RHL(Chain B). As a result, were generated five I-TASSER models of E. coli FtsZ protein of full length (from Met1 to Asp383) with different valuesofC-score(Table4,Fig.3).Model1hasthe highest value of C-score, indicating it as optimal model structure among generated by I-TASSER server. Based on server statistics Model 1 was also characterize by TM-score (template model-ling score) = 0.56±0.15 and root-mean-square deviation (RMSD)=9.6±4.6Ĺ. On theFigure4 wepresentresults of I-TASS­ER secondary structure and properties prediction for E. coli FtsZ protein. Confidence score values for the predicted structures are also indicating a Figure 2: Spatial structure models of FtsZ protein from Escherichia coli, built with the protein structure homology-modeling server »SWISS-MODEL Workspace« using X-rey template PDB structures of different origin: A– FtsZ from E. coli constructed on template 1OFU (Chain B) from P. aeruginosa; B – FtsZ from E. coli constructed on template 2Q1Y (Chain A) from M. tuberculosis, C – Fitting of constructed 3-D models of FtsZ protein from E. coli (light gray – based on 1OFU (Chain B) template, dark gray – based on 2Q1Y (Chain A)template).Proteinmodelspresentedassolid ribbondiagramwithside-chainatomsshownas lines. 3-D models were visualized in Accelrys Discovery Studio Visualizer. Slika 2: Tri dimenzionalni model FtsZ iz bakterije Escherichia coli narejen na serverju »SWISS-MODELWork-space« ob uporabi matric razlicnih izvorov: A– FtsZ iz E. coli narejen na matrici 1OFU_B (vir – P. aeruginosa), B – FtsZ iz E. coli narejen na matrici 2Q1Y_A(vir – M. tuberculosis), C – Prileganje modela FtsZ narejenega iz Escherichia coli (svetlo sivo – matrica 1OFU_B, temno siva – matrica 2Q1Y_A). Model proteina predstavljen kot polni trak s stranskimi verigami atomov (linije). Izdelan v Discovery Studio Visualizer. Figure 3: Spatial models of FtsZ Escherichia coli built on I-TASSER server. A– model1, B – model 2, C – model 3, D – model 4 and E – model 5. Protein models presented as solid ribbon diagram with side-chain atoms shown as lines. Boxes mark the C-terminal region that is absent in allcrystal structures of FtsZ proteins and was overbuilt by the server based on sequence similarity to the regions of other classes proteins. Generated in Discovery Studio Visualizer. Slika 3: Prostorski model FtsZ iz Escherichia coli narejen na I-TASSER serverju: A– model1, B – model 2, C – model 3, D – model 4 in E – model 5. Modeli proteinov so predstavljeni kot polni trak s stranskimi verigami atomov(linije). Okvir oznacuje C terminalno regijo, ki je odsotna v vseh strukturah kristala proteina FtsZ in je dograjena na serverju na osnovni podobnosti zaporedja v regijah pri ostalih proteinih. Izdelan v Discovery Studio Visualizer. ratherhighqualityofallbuiltmodels.Allthisalong Quality evaluation of E. coli FtsZ protein with a variety of conformational representations models ofC-terminalregionprovidedbyfiveserver-built models (Fig. 4) can not regard any from these All seven models of E. coli FtsZ were ex-structures as the quite probable and suitable for amined using the online MolProbity server and further bioinformatic research. »Protein Structure & Model Assessment Tools« of »SWISS-MODELWorkspace« server. Table 2: Results of evaluation of structure quality of chain 2Q1Y_A FtsZ Mycobacterium tuberculosis on a MolProbity server. Tabela 2:Rezultati vrednotenja kakovosti strukture verige 2Q1Y_AFtsZ iz bakterijeMycobacterium tuberculosis na serverju MolProbity. All-AtomContacts Clashscore for all atoms: 4.13 96th percentile* Poor rotamers 0.00% Goal: < 1% Ramachandran outliers 0.00% Goal: < 0.2% Ramachandran favored 100.00% Goal: > 98% ProteinGeometry Cß deviations > 0.25 Ĺ 0 Goal: 0 MolProbity score 1.20 99th percentile* Residues with bad bonds: 0.00% Goal: 0% Residues with bad angles: 0.00% Goal: < 0.1% Clashscore is the number of serious steric overlaps (> 0.4 Ĺ) per 1000 atoms. * 100th percentile is the best among structures of comparable resolution; 0th percentile is the worst. ^ MolProbity score is defined as the following: 0.42574*log(1+clashscore) + 0.32996*log(1+max(0,pctRotOut-1)) + 0.24979*log(1+max(0,100-pctRamaFavored-2)) + 0.5 Table 3: Top 10 template X-Ray structures selected by I-TASSER server for homology modelling of Escherichia coli FtsZ protein Tabela 3:Zgornjih 10 matric struktur z X žarki uporabljenih na I-TASSER-ju za modeliranje homolognosti FtsZ proteina bakterije Escherichia coli. Rank PDB Hit Ident1 Ident2 Cov. Norm. Z-score 1 2VAW (Chain A) 0.67 0.55 0.82 3.46 2 2VAW (Chain A) 0.67 0.55 0.82 5.88 3 1FSZ (Chain A) 0.44 0.40 0.86 6.38 4 2VAM (Chain A) 0.54 0.43 0.79 4.68 5 2R6R (Chain A) 0.46 0.39 0.84 6.20 6 2VAW (Chain A) 0.67 0.55 0.82 0.00 7 2VAW (Chain A) 0.67 0.55 0.82 13.50 8 1FSZ (Chain A) 0.48 0.26 0.53 5.77 9 1W5F (Chain A) 0.46 0.38 0.81 6.87 10 2RHL (Chain A) 0.52 0.43 0.82 5.00 (a) Rank of templates represents the top ten threading templates used by I-TASSER. (b) Ident1 is the percentage sequence identity of the templates in the threading aligned region with the query sequence. (c) Ident2 is the percentage sequence identity of the whole template chains with query sequence. (d) Cov. represents the coverage of the threading alignment and is equal to the number of aligned residues divided by the length of query protein. (e) Norm. Z-score is the normalized Z-score of the threading alignments. Alignment with a Normalized Z-score >1 mean a good alignment and vice versa. Table 4: MolProbity server and »Protein Structure and Model Assessment Tools« of »SWISSMODELWorkspace« server structure quality evaluation for 3-D models of Escherichia coli FtsZ protein. Tabela 4:Rezultati vrednotenja kakovosti strukture 3-D modela FtsZ iz bakterije Escherichia coli na serverju MolProbity in »Protein Structure and Model Assessment Tools« na serverju »SWISS-MODELWorkspace«. Models from I-TasserTemplate based models C-score from Swiss-Model Goal Verification Type Model Model Model Model Model 1OFU_B 2Q1Y_A 1-1.239 2-1.726 3-2.056 4-2.638 5-2.396 All-Atom 121.27(0th177.41(0th122.68(0th141.22(0th131.33(0th38.41(9th48.84(4th Clashscore, all atoms: Less is better contacts percentile*) percentile*) percentile*) percentile*) percentile*) percentile*) percentile*) Poor rotamers 1.36% 2.38% 2.38% 3.06% 2.72% 1.83% 0.00% <1% Ramachandran outliers 3.15% 2.89% 3.41% 3.94% 1.84% 0.34% 0.33% <0.2% Ramachandran favored 92.39% 89.76% 89.50% 89.76% 92.65% 95.53% 98.68% >98% Protein Cß deviations >0.25Ĺ 9 13 5 9 3 2 0 0 geometry 3.12(19th 3.55(8th3.40(11th3.54(8th3.37 (11th2.58(43rd2.16(67th MolProbity score^ Less is better percentile*) percentile*) percentile*) percentile*) percentile*) percentile*) percentile*) Residues with bad bonds: 0.00% 0.52% 0.00% 0.00% 0.00% 0.00% 0.00% 0% Residues with bad angles: 5.74% 6.27% 5.74% 4.18% 4.70% 0.00% 0.00% <0.1% DFire energy –494.67 –455.72 –471.66 –461.74 –471.66 –377.70 –412.32 Less is better QMEAN Raw score6 0.614 0.62 0.628 0.595 0.592 0.684 0.747 Higher value is better QMEAN Z-score 6 –1.79 –1.72 –1.62 –-2.01 –2.04 –0.96 –0.31 Less negative is better Clashscore is the number of serious steric overlaps (> 0.4 Ĺ) per 1000 atoms. * 100th percentile is the best among structures of comparable resolution; 0th percentile is the worst.^ MolProbity score isdefi ned asthe following: 0.42574*log(1+clashscore) + 0.32996*log(1+max(0,pctRotOut-1)) + 0.24979*log(1+max(0,100-pctRamaFavored-2)) + 0.5.DFire isan all-atom statistical potential based on a distance-scaled fi nite ideal-gasreference state. It’sused to assessnon-bonded atomic interactionsin the protein model.Alower energy indicates that a model is closer to the native conformation.QMEAN6 scoring function is a linear combination of six structural descriptors using statistical potentials: The local geometry is analysed by a torsion angle potential over three consecutive amino acids. Two distance-dependent interaction potentials are used to assess long-range interactions: the first is a residue-level implementation based on C-beta atoms only and the second an all-atom potential which is able to capture more details of the model. Asolvation potential investigates the burial status of the residues. Two additional terms describing the agreement of the predicted (from sequence) and the calculated secondary structure and solvent accessibility of the model. Figure 4: Results of secondary structure and properties prediction for I-TASSER model 1 of Escherichia coli FtsZ proteinSecondary structure elements are shown as »H« for a-helix, »S« for ß-sheet and »C« for coil. Conf.Score is confidence score values (higher values for better). Values range for predicted solvent accessibility (Solv.Acces.) vary from 0 (buried residue) to 9 (highly exposed residue). Bold and underlined are selected a sequences of FtsZ protein from E. coli models buildet on »SWISS-MODELWorkspace« server and based on 1OFU (Chain B) and 2Q1Y(Chain A) templates respectively. Slika 4: Napovedana sekundarna struktura in znacilnosti prvega modela FtsZ iz bakterije Escherichia coli na serverju I-TASSER.Sekundarni strukturni elementi so prikazani kot as »H« za alfa vijacnico, »S« za beta list in »C« za zanko. »Conf.Score« je merilo zaupanja ( višje vrednosti pomenibolje).Obmocjevrednostizanapovedno dostopnosttopila(Solv.Acces.) potekaod 0 (tesno vezan ostanek) do 9 ( visoko bremenjen ostanek). Odebeljenoin podcrtano so izbrana zaporedja modela FtsZ E. coli model narejenega na serverju »SWISS-MODELWorkspace« na osnovi matric 1OFU_B in 2Q1Y_A. Evaluation of all functions demonstrates a significantly higher quality of models, built with SWISS-MODEL server (Table 4). Evalua­tion of all-atom contacts, namely the number of serious steric overlaps of all atoms, allows us to determine model based on PDB matrix structure 1OFU (Chain B) as the best among all E. coli FtsZproteinmodels.Atthesametime,evaluation indexes of protein geometry, listed in Table 4, characterize another SWISS-MODELstructure, based on PDB template 2Q1Y(Chain A), as the most accurate. According to all-atom statistical potential DFire index, among I-TASSER models, the un­disputed leader are the Model 1 and an outsider – Model 4. At the same time, among SWISS­MODEL reconstructed models, structure, based on 2Q1Y (Chain A), demonstrate substantially superior quality in comparison with model based on Pseudomonas FtsZ structure. Evaluation in­dices »Raw score« and »Z-score« of QMEAN6, demonstrate undeniable superiority of E. coli FtsZ model based on 2Q1Y (Chain A) template. Discussion Choice of FtsZ M. tuberculosis model As shown in Figure 1, polypeptide chains of all FtsZ structures deposited in the database are lacking the first 5-7 a.a. residues of N-terminal region and 65-66 residues of C-terminal region (in fact, the last C-terminal region is absent at all). Also, eight chains, which are representing all five of the studied PDB-structures of FtsZ M. tuberculosis (in 1RLU and 2Q1Y – only Chains B), contain 5-10 residues gap within the loop, which corresponds to the loop between sheet S3 and helix H3 from 1FSZ – crystal structure of FtsZ protein from M. jannaschii (Oliva et al. 2004). B-chains of each structure are character­ized by the absence of 3-6 residues in the loop that extend to helixH7. Additionally, the chain B of 2Q1X has a short gap (3 a.a. residues) within the a-helix that corresponds to helix H5 of 1FSZ. Relative to the artifacts of residues, only 2Q1X and 2Q1Y crystals have all heavy atoms in all amino acid residues of structure (Table 1). Both of this two structures of FtsZ M. tuberculosis are characterized by complete composition of amino acid side chains, whereas the X-Rey structure obtained by Leung et al. 2004 (PDB: 1RQ7) are suffer from lack of some heavy atoms in at least three residues in each chain. Thus,ChainAfromthe2Q1Ystructure,which doesnotpossesstheseveninitial(N-end)andsixty sixC-endaminoacidresiduesbutincontrasttothe ChainAfrom1RLUhasfullatomcompositionof all available amino acid residues, in our opinion is a good three-dimensional model of FtsZ M. tuberculosis. This assumption was confirmed by the scores of all MolProbity quality evaluating functions for this structure (model) (Table 2). Construction and verification of FtsZ E. coli models The »SWISS-MODEL Workspace« is a web-based integrated service dedicated to pro­tein structure homology modelling. It assists in building protein homology models at different levels of complexity. Successful model building requiresatleastoneexperimentallydetermined3D structure (template) that shows significant amino acidsequencesimilaritywiththetargetsequence. Buildingahomologymodelcomprisesfourmain steps: identification of structural template(s), alig­nmentoftargetsequenceandtemplatestructure(s), model building, and model quality evaluation. These steps can be repeated until a satisfying modellingresultisachieved.Eachofthefoursteps requires specialized software and access to up-to-date protein sequence and structure databases. In fullautomaticmodeserverperformsallfoursteps itself and gives the complete three-dimensional model of the target protein with specified matrix structure. There is also a possibility to specify the necessary structure as the matrix manualy (Arnold et al. 2006). Thus, in full automatic modelling of FtsZ E. coli server used Chain B from 1OFU structure as a matrix. It should be noted that selection of this object as a matrix was based on the origin of these two bacterial species. Both of them are belonging to the class .-proteobacteria from phylum Proteobacteria. Although orders of these species are different, an affiliation to a common class is important enough to explain/suppose a higher level of similarity between sequence of FtsZ from E. coli and P. aeruginosa (Vaughan et al. 2004, Demchuk and Blume 2005), than with sequences from other bacterial species from dif­ferent phylums for which crystal structures are also presented in PDB database. However, such relatively close relationship between E. coli and P. aeruginosa was not suf­ficient to build a complete model of FtsZ E. coli. IncompleteN-terminalend of this modelstarting withtheAsn24,whichindicatestheabsenceoffirst beta-sheet and significant part of the next alpha-helix in the model structure (Fig. 2a), although in the structure of the matrix this elements are present (.la11-Gly23 in FtsZ from E.coli and .la12-Gly24 in FtsZ from P. aeruginosa). This artifact is surprising because of the fact that primary sequence of missing part of FtsZ E. coli is completely similar to that area in the template structure – FtsZ protein from P. aeruginosa. We can only assume some failure in the al­gorithmof»SWISS-MODELWorkspace«server taking note the fact that the using a chain 2Q1Y_A as specific template allowed server to generate FtsZ E. coli model, which containsthe mentioned area in its structure (Fig. 2b) and that is why it was stretched from the Ala11 to Ile315 inclusive. Ca-atom RMSD of obtained FtsZ E. coli models, whics were built on different matrices (Fig. 2c), is 0.862 Ĺ. This indicates a significant structural similarity of the obtained models (Gu andBourne2009).Anditcanbecomeanadditional demonstrationofthestructureconservatismofthis class of proteins (Erickson, 1998) and significant modelling accuracy while taking proteins from bacteria phylums class as a template. Minor dif­ferences observed in the turn of mainchain (Fig. 2c) are confined to the unstructured elements between ß-sheets and alpha-helices and fully capable of leveling through the lability of the secondarystructureelementsinthetime(Nyporko and Blume 2001). In parallel with the modelling on »SWISS-MODELWorkspace« server we have performed modelling of FtsZ E. coli onI-TASSERserver,an Internet service for protein structure and function predictions. It built 3-Dmodelsbased on multiple-threading alignments by LOMETS and iterative TASSERassemblysimulations;functioninsights arethenderivedbymatchingthepredictedmodels with protein function databases. I-TASSER (as ‘Zhang-Server’) was ranked as the No 1 server for protein structure prediction in recent CASP7, CASP8 and CASP9 experiments (http://predic­tioncenter.org/). It was also ranked as the best for function predictionin CASP9. CASP(or Critical Assessment of Techniques for Protein Structure Prediction) is a community-wide experiment for testing the state-of-the-art of protein structure predictions which takes place every two years since 1994. The experiment (often referred as a competition)isstrictlyblindbecausethestructures of testingproteins are unknown to the predictors (Roy et al. 2010, http://zhanglab.ccmb.med.umich. edu/I-TASSER/about.html). Asaresult,servergeneratedfivepotentialfull length models of FtsZ proteins from E. coli (Fig.3) with different values of C-score (see Table 4). C-scores a confidence score for estimating the quality of predicted models by I-TASSER. It is calculated based on the significance of thread­ing template alignments and the convergence parametersofthestructureassemblysimulations. C-scoreistypicallyintherangeof[-5,2],wherea C-score of higher value signifies a model with a highconfidenceandvice-versa(Royetal.2010). As it is shown in the Table 4 the best value of this parameter belongs to the model 1. I-TASSER server estimated accuracy for this model: 0.56±0.15 TM-score and 9.6±4.6 Ĺ RMSD.TM-scoreandRMSDareknownstandards of structural similarity between two structures which are usually used as measure of model accuracy when the native structure is known. In case where the native structure is not known, it becomes necessary to predict the quality of the modelling prediction, i.e. what is the distance between the predicted model and the native structures. TM-score is a recently proposed scale for measuring the structural similarity between two structures (Zhang and Skolnick 2004). The purpose of proposing TM-score is to solve the problem of RMSD which is sensitive to the local error.BecauseRMSDisanaveragedistanceofall residuepairsintwostructures,alocalerror(e.g.a misorientationofthetail)maycauseasignificant RMSDvaluealthoughtheglobaltopologymaybe correct.InTM-score,however,thesmalldistance is weighted stronger than the big distance which makes the score insensitive to the local model-ling error. ATM-score >0.5 indicates a model of correct topology and a TM-score <0.17 means a random similarity. This cutoff does not depend on the protein length (Roy et al. 2010). Thus, the quality of prediction of model 1 is quite satisfac­tory for these scores. OnFig.3wepresentedapredictedsecondary structures and properties of FtsZ protein from E. coli modelsgeneratedbyI-TASSER.Itshouldbe noted that the distribution of secondary structure elements(shownasHforalphahelixes,Sforbeta sheetsandCforcoils)matcheswiththatforcrystal structures of FtsZ (see figure 2 in Oliva et al., 2007). Confidence score values for the proposed structure also indicate a rather high quality of all predicted models. However, it should be noted that the value of this estimated parameter are at a slightly lower level for the C-tail region (which is not represented in any of the crystal structures ofFtsZproteins)incomparisonwiththeglobular N-andC-domains.We can speculate the possibil­ityofpredictedbyI-TASSERserverunstructured conformation of short N-terminal region in the structure of FtsZ E. coli because analogs are ob­servedinstructures2VAWand1FSZ.Butpredicted secondary structure of long and not structured C-terminal region between B10-sheet and final a-helix, provided in the FtsZ E. coli (Mosyak et al. 2000), does not inspire confidence in us. Low values of Confidence score and some crystal structures of FtsZ testified against coiled coil structure of this C-terminal region provided by I-TASSER server. Thus, in contrast to several crystalFtsZstructuresthatarestartingwithß-sheet B1 and breaking immediately after the ß-sheet B10 (1OFU, 1RQ7, 1RLU, 1RQ2, 2Q1X and 2Q1Y), FtsZ structures from 2R6R and 2R75 as well as 2VAP and 1FSZ have two additional ß-sheets B11 and B12. 1W5X structure has also a short a-helix between B10 and B11, which is terminated element of C-end region in structures 1W5F, 2RHH and 2RHJ. All together with a variety of conformational representations of this C-terminal region in five models provided by I-TASSER server (Fig. 4) can not regard any of these models as the most likely and suitable for further bioinformatics researches. For the final determination of the best poten­tial model of FtsZ E. coli among all constructed we have verified all seven candidates with the online server MolProbity and »Protein Structure & Model Assessment Tools« of »SWISS-MODEL Workspace«. MolProbityallowstoevaluatethequalityboth of all atoms contact and so the protein geometry of any three-dimensional biopolymer molecule. Evaluation of all-atom contacts, specifically the number of serious steric overlaps of all atoms, allows to choose model, built basing on template 1OFU_B, as a favourable. In this case second modelfrom»SWISS-MODELWorkspace«loses muchlessforthisparameterincomparetomodels ofI-TASSER.Ontheotherhand,estimationofall parameters of protein geometry (Table 4), allow ustoconsiderthe»SWISS-MODELWorkspace« model built based on template structure 2Q1Y (chain A), as the best one. However, on such pa-rametersas»poorrotamers«and»Ramachandran favoured« it’s not much better than the second model from this server and all models from I-TASSER.Asignificantbenefitofbothmodelsfrom »SWISS-MODELWorkspace«overmodelsfrom I-TASSERisobservedinratesof»Ramachandran outliers«, »Cß deviations«, »MolProbity score«. Significant differences are also observed in »Residueswithbadangles«parameter.Whiletwo modelsfrom»SWISS-MODELWorkspace«have no residues with bad angles, in structures from I-TASSER models the quantity of these residues reaches 4-6 %. It should be noted that residues withbadbondsobservedonlyinthecaseofmodel 2 among all seven analyzed models. Benefits of models built on the I-TASSER in all-atomstatisticalpotential»DFireenergy«dueto the bigger size of this models that are completely full unlike to the incomplete modelsfrom SWISS­MODEL server. Therefore, this parameter from »Protein structure & model assessment tools« we used to compare structures built only on the same server. Finally, for evaluating quality of three-dimensional structures, it was implemented QMEAN6 score (http://swissmodel.expasy.org/ qmean/cgi/index.cgi). QMEAN6 is a reliability score for the whole model which can be used in order to compare and rank alternative models of the same target. The quality estimate ranges between 0 and 1 with higher values for better models. Additionally, the pseudo energies of the four contributing statistical potential terms are provided as well as the percentage agreement between predicted and measured features from researches.Itlacksfirstsevenandlast66residues, the sequence and model, respectively. The com-but it is characterized by full atom composition parison of the differences of the terms among the of all available residues and excellent estimated models may help to understand the reason for the values of parameters from MolProbity. differences in the estimated model quality. In Also, among the seven potential FtsZ E. coli addition to the »Raw scores«, »Z-scores« of the models, built by I-TASSER server and »SWISS­QMEAN composite score as well as all terms are MODELWorkspace«, we have selected a model provided relating the quality estimates to scores fromthelastserver,basedonmatrix2Q1Y(chain obtained for high-resolution reference structures A). This model prevailed for qualitative evalu-solved experimentally by X-ray crystallography ation parameters not only full atom models of (Benkert et al. 2011). The QMEAN »Z-score« I-TASSER, but model, built on a matrix 1OFU representsanmeasureoftheabsolutequalityofa (chain B), which represents FtsZ structure from model by providing an estimate of the »degree of P. aeruginosa – bacterial species systemati­nativeness«ofthestructuralfeaturesobservedina cally much closer to E. coli (the same class of modelandbydescribingthelikelihoodthatagiven .-proteobacteria), than M. tuberculosis. model is of comparable quality to experimental Thus, were have successfuly reconstructed structures. Models of low quality are expected 3-D models of the FtsZ proteins of E. coli and to have strongly negative QMEAN Z-scores (i.e. M. tuberculosis is of sufficient quality forfurther the model’s QMEAN score is several standard in silico studies such as molecular docking, mo-deviations lower than expected for experimental leculardymamicssimulationsandcomputational structures of similar size). 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The role of cell selection for pollen grain fertility after treatment of barley sprouts (Hordeum distichum L.) with UV-B irradiation Pomen izbora celic za plodnost pelodnih zrn po obravnavanju kalic jecmena (Hordeum distichum L.) z UV-B sevanjem Elena Kravets Department of Genomics and Molecular Biotechnology, Institute of Food Biotechnology and Genomics, National Academy of Sciences of Ukraine, 2a Osipovskogo Str., Kyiv 04123, Ukraine correspondence: kravetshelen@gmail.com Abstract: UV-B irradiation of barley sprouts within the range of 0.5-4.3 kJ/m2 induced an increase in the number of chromosome aberrations in the root meristem and pathologies in the reproductive system. Enhancement of cytomixis, increase of polymorphismandcytopathologyofpollengrainswereobservedinthemalegenerative system. The inverse trend was observed when intensity of cytomixis was compared to the pollen grain sterility. Damages induced by low doses of UV-B radiation were eliminated neither by DNA reparation nor by cell selection and were preserved in manycellgenerations.HighUV-Blevelledtotheactivationofcytomixisduetowhich the population of microsporocytes was released from the excess load. It is presumed that cytomixis present a form of cell selection which was induced by an excess of microsporocyte disturbances. Keywords: chromosomeaberrations,rootmeristem,microsporangium,cytomixis, pollen grain sterility, cell selection, UV-B radiation, Hordeum distichum L. Izvlecek: Obravnavanje kalic jecmena z UV-B sevanjem (od 0,5-4,3 kJ/m2) je povzrocilo nastanek številnih kromosomskih aberacij v rastnem meristemu korenin in motnje v reproduktivnem sistemu. Pri moških generativnih organih smo zasledili povecanje citomikse, polimorfizma in patoloških sprememb pelodnih zrn. Ugotovili smonegativnopovezanostmedjakostjocitomikseterpogostostjopatološkihsprememb pri tetradah mikrospor in sterilnostjopelodnih zrn. Poškodbe zaradi nizkih odmerkov UV-B sevanja s popravljanjem DNAin z izborom celic niso izginile in so se ohranile vec generacij celic. Visok odmerek je povzrocil citomikso, zaradi cesar se je sprostila populacija mikrosporocit. Predvidevamo, da citomiksa predstavlja nacin izbora celic, ki nastane zaradi vecjega obsega motenj mikrosporocit. Kljucne besede: kromosomske aberacije, koreniski meristem, mikrosporangij, citomiksa, sterilnost peloda, izbor celic, UV-B sevanje, Hordeum distichum L. Introduction Ultraviolet in the range of 280-320 nm affect all levels of plant organization and also signal, regulatory and energetic functions (Jordan 1996, Ziska et al. 1992, Ziska and Teramura 1992, Cald­well et al. 2003, 2007; Zhang et al. 2003, Koti et al. 2004 a, b, 2007, Hectorsetal.2010,Ballaréet al.2011,Krasylenkoetal.2011).Oneofthemost significant effects of enhanced UV-B radiation is an injury of the reproductive function of plants. It has been shown that additional UV-B radiation canexertgenotoxiceffectonthemeristem,inhibit growth and development, influence pollination, decrease the quantity of the produced pollen and seedproductionofplants(FlintandCaldwell1984, Conner and Neumeier 2002, Koti et al. 2004 a, b, 2007). Besides the direct action on generative organs, the main target of which is DNAof cells, UV-B radiation produces indirect effects which are realized by mechanisms connected with pho­toreception,transductionofsignalsandhormonal regulation (Tevini et al. 1981, Flint and Caldwell 1984, Santos et al. 1998; Caldwell et al., 2007, Demkura et al. 2010, Keller et al. 2011). The ef­fectofactiondependsinmanycasesongenotype, ecotype,thestageofontogenesisandotherreasons (Jordan1996,Torabinejadetal.1998,Caldwellet al. 2007, Li et al. 2010). The data on mechanisms of the effect of UV-B radiation on the generative organs of plants are not available. In this connec­tion, the main goal of the paper was to elucidate thecharacterofdamagesinthegenerativesystem induced by UV-B radiation, its dose dependence, and also to estimate the role of cell selection in normalization of the pollen grain fertility. Material and methods We used barley (Hordeum distichum L., 2n=14) of Scarlet variety of French selection. Three-day sprouts were irradiated by a 20 W Philips TLultraviolet lamp with filter cutting off theshort-waveregionoftheultravioletspectrum. Radiation doses were 0.5, 2.2 and 4.3 kJ/m2with the intensity 0.5 W/m2s1. One group of sprouts (about 100 plants) was fixed 24 and 48 hour after irradiation. The other group of sprouts (about 100 plants) was cultivated in soil to study the development of reproductive organs. The mate­rial was fixed with Navashin mixture; temporal slides were prepared according to the standard cytological protocol (Pausheva 1984). The fixa­tion of spike was made from the differentiation stage of microsporocytes to maturation of pollen grains. The slides were stained with acetoorcein followingenzymicmaceration(forrootmeristem) and either acetocarmin or Schiff’s reagent (Feul-genprotocol)formicrosporangium.Thequantity of anomalies in cell systems was counted and measurements were made. Thecalculationofthenumberofchromosomal aberrations was carried out by the ana- telophase method, the mitotic index (MI) was defined by percentageofmitoticdividingcells.Weestimated 10 roots per stage, 50-70 anthers with microspo­rocytes and 20 anthers for the analysis of pollen grains (PG) per stage. The data were statistically processed by the Microsoft Excel software. Results The effects of UV-B-radiation on the root me-ristem. In the first mitosis the quantity of chromo­some aberrations increased in proportion to the radiation dose, in the second mitosis the dose dependence varied: at high dose the level of chromosome aberrations decreased, while the number of degenerated cells increased (Table 1, Fig. 1). The cell degeneration occurred by the apoptosis type. It was particularly remarkable thatinindividualcasesundermaximalexposition of UVwe observed the phenomena of cytomixis connectedwithmigrationoftheinjuredchromatin alongtheplasmodesmalchannels.Therefore,the increasing of UV-B-radiation dose resulted in in-creasednumberofaberrationsinthefirstmitosis, while in the second, the induction dynamics of chromosomeaberrationsexhibitedbothdirectand inverse dose-dependence (Fig. 1). The effects of UV-B radiation on the reproductive system of plants. Microsporogenesis: Microsporogenesis was with the formation of tetrads of the isobilateral N/N Root meristem Microsporogenesis and the stage of the development of the pollen grainExperimental 24 h after irradiation 48 h after irradiation Microsporogenesis,Tetrads with Sterility of Sterility Sterility of the variant. MI, % Aberrant MI, % Aberrant degree anomalies, % microspores, of two-celled three-celled Radiation dose anaphases, % anaphases, % of cytomixis, % B % PG, % PG, % 1 Control 5.8 3.05±0.65 4.5 2.20±0.29 4.3 4.5±0.3 1.9±0.1 3.8±0.9 2.9±0.7 2 0.5 kJ/m2 6.2 2.61±0.73 5.2 5.75±0.96 7.3 6.0±0.6 2.1±0.3 9.0±1.7 11.3±1.4 3 2.2 kJ/m2 6.6 5.92±0.96 5.1 6.24±0.80 6.3 8.1±1.0 2.7±0.4 9.7±1.5 9.1±1.8 4 4.3 kJ/m2 5.8 8.82±1.10 5.0 4.52±0.82 19.7 8.5±0.7 3.7±0.4 6.8±1.5 3.8±0.8 Table1: The indices of cytogenetic disturbances in the root meristem of H. distichum sprouts and the generative sphere of the plants in the process of ontogenesis.Tabela 1:Indeksi citogenetskih motenj v meristemu korenin kalic vrste H. distichum in generativnih organih v procesu ontogeneze. structureispresentedinFigures3.and4..Under the influence of UV-B-radiation, cytomixis was themaintypeofpathologyinmicrosporogenesis. We consider that one should distinguish between weak (local), intensive and destructive (pathologi­cal) cytomixis. Local cytomixis is a physiologi­cal norm for barley. UV-B radiation, similar to other stress factors, intensifies the destructive character of cytomixis. In barley under maximal exposition of UV intensive cytomixis affected up to about 20% of microsporocytes (Fig. 3b). In this case stickenessand fluidity of chromatin increasedinmicrosporocytes.Weobservedakind of transitional chromatin (fragments of nuclei, chromosomes, micronuclei, bands of chromatin from cell to cell). Not all microsporangia were affected by cytomixis. Most of microsporocytes completed meiosis with the formation of normal, only rarely nonbalanced, tetrads of microspores (Figs. 4., b). In barley the bulk of »transitional« chromatin usually remained either in the com­position of cynticia or in the intracellular space. Destructive cytomixis usually occurred in less developed flowers and in immature spikes of the second growth and is, presumably, a way to eliminate the nonviable cell system. The dose dependence of cytomixis incorpo­ration into microsporogenesis was of nonlinear character (Fig. 1). In this case, low correlation was observed between the intensity of cytomixis and frequency of pathologies in tetrads of micro-spores.Thoughcytomixismightbethereasonfor formation of the unbalanced tetrads, both types of disturbances were, most likely, a result of the same reason associated with genetic instability (prolonged mutagenesis) induced by the effect of UV-B- radiation. The development ofpollengrain:Under nor­mal conditions, the development of male gameto­phyte in barley, similar to most of cereals, begins to release microspores from the microsporocyte envelope and includes the stages completing the formationofsporoderm,growthandpolarization of microspore. Then follows the first asymmetric mitosis,polarizationoftwo-celledpollengrain,the secondmitoticdivisionwhichareaccompaniedby the synthesis of cytoplasm, and then by the depo­sition of reserve substances in the vegetative cell cytoplasm(Batygina1974,Poddubnaya-Arnoldi 1976,Heslop-Harrison1979,Mascarenhas1989). 25 1. mitosis Frequency of disturbances (%) 20 15 10 5 0 2. mitosis m icros porocytes tetrades Figure 1: UV-B dose dependences of the number of cytogenetic disturbances in H. distichum sprouts in the first mitosis in the root meristem and microsporogenesis. Slika 1: Število citogenetskih motenj v odvisnosti od UV-B sevanja pri kalicah H. distichum pri prvi mitozi v koreninskem meristemu in mikrosporogenezi. 0 0,5 2,2 4,3 UV-B dose (kJ m-2) Figure 2: UV-B dose dependences of the number of cytogenetic disturbances of H. distichum in the phases of development of pollen grain. Slika 2: Število citogenetskih motenj pri vrsti H. distichum v fazah razvoja pelodnega zrna. Figure 3: Microsporogenesis: a – telophase of 2nd divisions of meiosis, control; b – telophase of 2nd divisions of meiosis, UV-B dose: 4.3 kJ\m2; intensive cytomixis. Slika 3: Mikrosporogeneza: a – telofaza druge delitve pri mejozi, kontrola; b – telofaza druge delitve pri mejozi, UV-B odmerek: 4,3 kJ\m2, intenzivna citomiksa. Figure 4: Formation of the tetrads: a – UV-B dose: 0.5 kJ\m2; b – UV-B dose: 4.3 kJ\m2. Slika 4: Nastanek tetrad: a – UV-B odmerek: 0,5 kJ\m2; b – UV-B odmerek: 4,3 kJ\m2. The mature pollen grain of barley has a pair of arrow-shapedspermsandavegetativecellnucleus, thecytoplasmofwhichisfilledwithamyloplasts. Ultraviolet-B radiation led to an enhancement of polymorphism and to disturbance of polarity in pollen grains, unsynchronized development, the increase of the frequency in the formation of oli­goplasmpollengrains(Fig.5.).Thelatterpresent an evidence for nonspecific character of gametic disturbancescausedbydifferentstressfactors.The appearance of oligoplasm pollen grains might be associated by either mutations of specific genes ofpollengrain,whoseexpressionintensifiesafter the first mitosis or by mutation which determines themalecytoplasmicsterility(Mascarenhas1990, Nirmala and Kaul 1994). Such pollen grains are late or interrupt in their development, and their sperms do not complete the cycle of their differ­entiation. According to the morphological traits degeneration of microspore nucleus, generative cell, sperms and nucleus of the vegetative cell in the pollen grain occur by apoptosis (Fig. 5b). In reality, the recent publication reports that UV-B irradiation can indeed initiate apoptotic processes in plant cells (Lytvyn et al. 2010). Thus,therangeofcytologicaldisturbancesin pollen sacs had a nonspecific character. Induction of disturbances in the course of pollen grain development correlated negatively withtheUV-Bradiationdoseandwiththedegree Figure 5: Mature three-celled pollen grains: a – normal and »oligoplasm« pollen grains, UV-B dose: 2.2 kJ\m2; b – apoptosis degradation of pollen grain nuclei, UV-B dose: 4.3 kJ\m2. Slika 5: Zrela tri-celicna pelodna zrna: a – normalna in »oligoplazemska« zrna, UV-B odmerek: 2,2 kJ\m2; b – apoptozni razpad jedra pri pelodnemu zrnu, UV-B odmerek: 4,3 kJ\m2. ofcytomixis(Table1,Fig.2).Anenhancementof the UV-B dose in first step increasedthe number of anomalous pollen grains, but further on it decreased. At the higher dose the level of pollen sterility was close to control. Discussion Thismightevidencedthethresholdcharacter of the effect and the induction of the recovery mechanisms. It is known that in response to increased level of cytogenetic disturbances the DNAreparationsystemsbecomemoreactiveand leading to apoptosis induction or to proliferative death of non-repaired cells via the cell selection (Calendo 2001). Due to cell selection, namely, to the haplontic cell selection (taking place at the ontogenesis haplophase) the cells with recessive lethalmutationsthatarenotsubjectedtotheaction of diplontic cell selection are eliminated (Gaul 1959).Theactionofhaplonticselectionisevident at the stage of the pollen grain maturation. Paradoxically, but disturbances induced by low doses of UV-B radiation were not eliminated neither by reparation nor by cell selection and are preserved in many cell generations. They remained beyond the reach even of the haplontic competitionresultinginrelativelyhighpercentage of the pollen grain sterility. Cytomixis began to act at higher UV-B-radiation dose, due to which the population of microsporocytes released from the excess of genetic load. Consequently, in the response of plants to radiation the threshold effect wasobservedwhichmaybecausedbydamagesof a number of DNAand other molecules initiating the reparation processes and cell selection (Calendo 2001).Theintensificationofcellselectionoccured in ontogenesis at the end of diplophase (micro- sporogenesis) and also at the end of haplophase (formationofgametes).Thusthelong-termeffects ofUVradiationonreproductivesystemarelikely causing the prolonged mutagenesis which was induced by irradiation. Decrease of disturbance number in reproductive tissues at the higher dose UVisprobablyconnectedwiththethresholdeffect and activation of restore processes. Cytomixisasaformofcellselection,occurring inmicrosporocytesbeforeandatthebeginningof meiosis,deservesspecialattention.Wepresumed that via cytomixis the population of microsporo­cytes regulated its excess, and simultaneously eliminatedfromthemutationalloadandsolvedthe problemsofnutritionalcharacter.Spacecontinuity anduniquenessofmicrosporocytesasacenocytic system of pollen sac were realized via cytomixis (Heslop-Harrison1966a,1966b,Welan1974,Guo andZheng2004).Cytomixismightplayaspecial role in provision of repairing processes in initials of male gametes. In reality in the case of local cytomixis the chromatin loops penetrate the intercellular chan­nels and united microsporocytes into groups in the early prophase of meiosis. Such contacts do not entail negative consequences for future meiosis. Vice versa, the microsporocytes that are not included in the network may fall out of the developmental program. They may be delayed in the prophase-metaphase of the first division of meiosis and undergo proliferative death. It is known the callose wall of microsporocytes is not an impermeable barrier and does not prevent the migrationofchromatinandorganellestopenetrate through wide intercellular channels along which notonlychromatinbutalsocytoplasmicorganelles, signal molecules and trophic factors may pass (Risueno et al. 1969, Zheng et al. 1987, Souza and Pagliarini 1997, Hecht 2000). It is believed that the intercellular contacts ensure not only the synchronization of meiosis but also the cellular population homogeneity in microsporocytes and equalize the qualitative state of pollen grains which is required for a rapid and successful pollination. Cellular selection in the population of mi-crosporocytes occurs through the so-called au­tonomousapoptosiswhichunlikemorphogenetic apoptosis, is not programmed but is initiated by the microsporocyte population itself. This assump­tion is supported by the irregularity of cytomixis, which does not occur simultaneously in all micro-sporangia or microsporocytes in the same anther. IntensivecytomixisisinducedbyUV-Bradiation, hybridization and other stress factors. Many researchers assume that the nature of intensive and destructive cytomixis is connected with the geneticdisbalance(disturbanceofhomeostasis)of polyploids,haploids,aneuploids,mutants,hybrids andapomicts(Poddubnaya-Arnoldi1976,Mantu and Sharma 1982, Bedi 1990, Orlova 1994). Stress factors, such as radiation, hybridization, chemical agents and herbicides usually enhance the destructive effect of cytomixis (Bobak and Herich 1978, Dwivedi et al. 1988, Bellucci et al. 2003). Usual environmental conditions and their seasonal fluctuations exert no marked effect on cytomixis, which is obviously under the genetic control (Mantu and Sharma 1982, Bellucci et al. 2003). The intensive and destructive (to a larger extent) cytomixis made the meiosis pattern more complicated and may led to serious genetic con­sequences. However, this is mainly peculiar to geneticallyunbalanced,sterileformsandinspikes of the second growth or nonviable individuals. Basingintheexperimentalmaterialanddatafrom literatureweassumedthatcytomixisreflectedthe mechanismsofthecellularselectionduringwhich cellular population limits the number of function-ingmicrosporocytes,thusregulatingredundancy, and eliminating the mutation load. Conclusion UV-B radiation of barley sprouts within the range of 0.5-4.3 kJ/m2 induced an increase in the number of chromosome aberrations in the root meristem and pathologies in the reproductive system. Enhancement of cytomixis, increased polymorphismandcytopathologyofpollengrains was observed in the male generative system. Inju­ries caused by UV-radiation were of nonspecific character. The negative correlation was observed betweentheintensityofcytomixisandfrequencies of pathologies in tetrads of microspores and also between the sterile level of pollen grains. The induction of disturbances during development of the pollen grain revealed negative dose depend­ence. Under the maximal exposition of UV-B radiation the index of pollen sterility approaches to that obtained at the control. Injuries induced by low doses of ultraviolet were not eliminated, either by DNAreparation either by cell selection and were preserved in many cell generations. An enhancement of the radiation dose led to the acti­vation of cytomixis due to which the population of microsporocytes was released from the excess load. We presume that cytomixis present a form of cell selection which is induced by an excess of the injuries a threshold level of microsporo­cytes. 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C0(2) Enhancement of Growth and Photosynthesis in Rice (Oryza sativa): Modification by Inceased Ultraviolet-B Radiation. Plant Physiology, 99 (2), 473-481. Defence responses of Norway spruce seedlings to elicitors of ectomycorrhizal fungus Pisolithus tinctorius and pathogen Heterobasidion annosum are affected by zeatin riboside Vpliv zeatin ribozida na obrambni odgovor kalic smreke po tretiranju z elicitorji mikorizne glive Pisolithus tinctorius in patogena Heterobasidion annosum Matevž Likar, Marjana Regvar Department of Biology, Biotechnical Faculty, University in Ljubljana, Vecna pot 111, 1000 Ljubljana, Slovenia *correspondence: matevz.likar@bf.uni-lj.si Abstract: Cytokinins are known to attenuate defence responses of plants after elicitor application or inoculation with fungi. To evaluate their role in the regulation of colonisation of Norway spruce (Picea abies) seedlings with mycorrhizal and pathogenic fungus, we analysed the effects of zeatin riboside (ZR) on: i) growth of ectomycorrhizal fungus Pisolithus tinctorius and pathogen Heterobasidion annosum in axenic cultures,ii) colonisation intensity of selected fungi on P. abies seedlings and iii) induction of defence reactions of spruce seedlings following elicitor treatment. Mycorrhizal fungus P. tinctorius showed increased growth at concentrations higher than 10-2 µM ZR that was accompanied by increased ergosterol concentrations. In contrast, decreased growth of pathogen H. annosum was observed at the highest ZR (10 µM) concentration. ZR treatment also increased colonisation of spruce seedlings with the mycorrhizal fungus. Application of cell wall preparations of both fungi incre­ased peroxidase (POD) activity in the roots of treated spruce seedlings, whereas only elicitors of H. annosum increased also phenylalanine ammonia-lyase (PAL) activity, levels of soluble phenolics and salicylic acis (SA) concentrations. Application of ZR negated the increased activity of POD that was observed in elicitor treated seedlings, accompanied by increased levels of soluble phenolics in the roots of elicitated seed­lings. In contrast, no effects of ZR treatment on PAL activity and SA accumulation could be observed. Our results suggest involvement of ZR in the regulation of cell wall modifications during the fungal colonisation with P. tinctorius and formation of ectomycorrhizae, by affecting the growth of fungal partner and non-specific defence reactions of the plant host. Keywords: cytokinins, peroxidases, phenolics, phenylalanine ammonia lyase, Picea abies Izvlecek: Iz literature je znano, da lahko citokinini vplivajo na obrambne reak­cije rastlin, ki se sprožijo po aplikaciji elicitorjev ali ob inokulaciji z živo glivo. Za ovrednotenje njihove vloge pri regulaciji kolonizacije kalic smreke (Picea abies) z mikoriznimi in patogenimi glivami, smo preverili vpliv zeatin ribozida (ZR): i) na rast ektomikorizne glive Pisolithus tintorius in patogena Heterobasidion annosum v aksenicni kulturi, ii) na stopnjo kolonizacije kalic smreke z obema glivama in iii) na aktivacijo obrambnih reakcij smreke po tretiranju z elicitorji. V aksenicni kulturi smo pri 10-2 µM koncentraciji ZR opazili pospešeno rast mikorizne glive P. tincto­rius, ki jo je spremljala povecana koncentracija ergosterola v miceliju. Nasprotno je bila rast patogene glive H. annosum pri najvišji koncentraciji ZR v gojišcu (10 µM) zavrta. Podobno kot v aksenicni kulturi je dodaten ZR pospešil kolonizacijo kalic smreke z ektomikorizno glivo, medtem ko na stopnjo kolonizacije s patogenom ni imel ucinka. Tretiranje smreke z elicitorji obeh gliv je povecalo aktivnost peroksidaz (POD) v koreninah kalic, samo elicitorji patogene glive H. annosum pa so povecali tudi aktivnost fenilalanin amonijeve liaze (PAL) in koncentracijo topnih fenolov ter proste salicilne kisline (SA). Dodatek ZR je znižal peroksidazno aktivnost v kalicah tretiranihzelicitorjiobehglivinpovecalkoncentracijotopnihfenolov.Nasprotno,ZR ni imel nobenega vpliva na aktivnost PALin akumulacijo salicilnekisline v koreninah smreke. Na podlagi naših rezultatov predvidevamo, da je ZR vpleten v regulacijo modifikacij celicne stene ob glivni kolonizaciji z ektomikorizno glivo P. tinctorius in vzpostavitev ektomikorize, preko delovanja na glivnega partnerja in nespecificne obrambne reakcije gostitelja. Kljucnebesede: citokinini, peroksidaze, fenilalanin amonijeva liaza, Picea abies Introduction Root exudate is a very diverse group of com-poundsreleasedfromtherootstotherhizosphere andcontainsalongwithaminoacids,organicacids, proteins and sugars also several plant growth regulators, including cytokinins (Neumann and Römhled 2000). Cytokinins play a crucial role in regulating proliferation and differentiation of plant cells, and also control various processes in plantgrowthanddevelopment(Sakakibara2006; Kyozuka 2007). Furthermore, cytokinins are known to influence the growth of fungi (Barker and Tagu 2000, Nasim and Rehman 2006). Colonisation of plants by different symbiotic and pathogenic fungi triggers the expression of defence related genes and induces local and systemic host responses (Ryals et al. 1996; Ham-merschmidt 1999). An almost ubiquitous feature of plant responses to fungal colonisation or elici-tors treatment is the activation of phenylalanine ammonia-lyase(PAL;EC4.3.1.5)andperoxidases (EC1.11.1.7),whichareassociatedwithphenolic chemistryand cellwallmodifications (Chitoor et al.1997).Itwasdemonstratedthatcytokininscan regulate activity and expression of peroxidases (Kadioglu and Durmus 1997, Limam et al. 1998) and can therefore affect the reactions leading to cell-wall reinforcements. When tobacco plants were wounded in the presence of the synthetic cytokinin, benzylaminopurine, production of jasmonicacid(JA)wasaccompaniedbysalicylic acid(SA)accumulation(Sanoetal.1996),whichis normallynotaccumulateduponwounding(Pieterse and Van Loon 1999). These results indicated that cytokinins may be involved in accumulation of JAandSA(Sanoetal.1996),whichtransmitsthe defence activationsignal from the point of infec­tion throughout the plant (Yalpani et al. 1993). Though majority of information concerning the role of PAL, peroxidases and SAin plant defence emanates from research on angiosperms, several studies suggest a similar role in host responses of gymnospermNorwayspruce(Asiegbuetal.1994, Kozlowski and Metraux 1998, Nagy et al. 2000, Nagy et al. 2004; Likar and Regvar 2008). In the present study we tested the effects of zeatin riboside (ZR) on the growth of the ec­tomycorrhizal fungus Pisolithus tinctorius and necrotrophic pathogen Heterobasidion annosum, as differential effects of cytokinin treatment on growth and colonisation of host roots with both fungal groups (ectomycorrhizal vs. necrotroph) was assumed. Ergosterol content in the fungal mycelia was tested in combination with the fun­gal growth studies, due to its importance in the formationofsterolrichdomains(Xuetal.2001), whichareinvolvedinseveralimportantprocesses such as endocytosis and hyphal growth (Alvarez et al. 2007). Further, the effects of ZR on the colonisation intensity and induction of defence reactions of spruce seedlings following elicitor treatment were examined, as cytokinins were already proven to affect activity and expression of some enzymes involved in the defence reac­tions (Kadioglu and Durmus 1997, Limamet al. 1998). Several differences in the spruce seedling responses after colonisation with P. tinctorius and H. annosum wereobservedpreviously(Likarand Regvar 2008) and are assumed to be at least in part influenced by cytokinins. Material and methods Effects of zeatin riboside on fungal growth Culturesof ectomycorrhizal fungus Pisolithus tinctorius (Mich.:Pers.)CokerandCouch(isolate DB49) andet al. necrotroph Heterobasidion an-nosum (Fr) Bref. (isolate DB75) are maintained in the fungal collection of Plant Physiology Lab (Biotechnical Faculty, University of Ljubljana). Sequences of ITS-rDNA region of both fungal species can be obtained from GenBank under accession numbers EU559631 and EU559632. Fungal cultures were grown on Melin-Norkrans­Marx(MNM)media(Marx1969),supplemented with zeatin riboside (ZR) at final concentrations: 0, 10-6, 10-4, 10-2, 1 and 10 µM. Due to different growth rates, growth of the individual fungus was monitored for two weeks in case of H. an-nosum and five weeks in case of P. tinctorius, after which mycelia were removed and frozen in liquid nitrogen, prior to freeze-drying (Christ, Alpha 2-4). Ergosterol analysis Twenty mg of dried fungal material were usedforergosterolextractionfollowingmodified protocol of Martin et al. (1990). For ergosterol quantification, 25 µl of ethanolic supernatant was injected into the high performance liquid chromatography (HPLC) system consisting of Waters 2960 separation module with Waters 996 PDA detector. UV absorbing compounds were separated by reverse-phase chromatography on a 5-µm Spherisorb C18, 250 mm x 4.6 mm column. The mobile phase was 100% methanol at 1 ml min-1. Ergosterol peak was identified by comparison of the retention time (absorbance at 280nm)andabsorbancespectrawiththepurified ergosterol standard (Nylund et al. 1992). Elicitation and inoculation experiments Seeds of Norway spruce (P. abies (L.) Karst. prov.Cermošnjice)weresurfacesterilisedin30% H2O2(105min),washedandsownonsterilepeat: vermiculite (1:3, V/V) mixture supplemented with Knop’s medium (Booth 1971). Seedlings were grown in growth chamber (16h of light, 23 °C) for three months, and then transferred to Petri dishes for the elicitation and colonisation experiments. For the elicitor treatment, fungal cell wall extracts of both fungal species were prepared as describedbySalzeretal.(1996).Forelicitationof spruceseedlings,elicitorswereaddedtotheMNM media at final concentration of 0.1 g l-1 (Likar and Regvar, 2008). To half of the seedlings ZR was added to MNM media at final concentration of 10-3µM(determinedasoptimalforspruceseedling growthpriortoexperiments),whilesteriledouble distilled water was used for the control. After ten days of growth the seedlings were collected and whole roots excised. Roots were freeze-dried (Christ, Alpha 2–4) and grinded to powder using liquid nitrogen prior to extraction of proteins and soluble phenolics. Fortheinoculationexperimentsthreemonths old spruce seedlings were transferred to petri dishes and covered with semi-circles of cloth without (control) or with the fungi, according to Chilvers et al. (1986). An additional semicircle of cotton soaked with half strength liquid MNM media without sugars was laid over the roots. Inoculated seedlings were grown for one month, after which colonisation of the spruce seedlings was estimated under a stereomicroscope (Leica, MZ8) by counting the short roots that had been entered by the hyphae. PAL and peroxidase activity Soluble proteins were extracted in 100mM phosphatebuffer(pH7.0,1:10=w/V)asdescribed byAlbrechetal.(1994).Supernatantwasusedfor total protein quantification, as well as PAL and POD assays. A modified Lowry assay (Sander­mann and Strominger 1972) with bovine serum albuminasthestandardwasusedforquantification of total soluble proteins prior to enzyme assays. Concentrations of soluble proteins ranged from 1 to 2 mg ml-1. Peroxidaseactivitywasmeasuredasdescribed by Bashan et al. (1987), using guaiacol as sub-strateinanassaymixturecontaining:25µlofthe proteinextract,0.5ml100mMphosphatebuffer, 0.2 ml 1% guaiacol and 0.2 ml 10 mM H202. The enzyme activity was measured by monitoring the increase in absorbance at 470 nm (extinction coefficient of 26.6 mM-1cm-1) during the polym­ erization of guaiacol into tetraguaiacol (Chance and Maehly 1955). PAL was assayed spectrophotometrically following a modified method of Khan and Vai-dyanathan(1986).ThePALassaywasperformed at 37 °C for 1 h in an assay mixture containing: 100µl of the protein extract 450 µl 50 mM TRIS-HCl(pH8.8)and25µl100mML-phenylalanine. The PAL activity was measured by monitoring the increase in absorbance at 290 nm (extinc­tion coefficient of 16.6 mM-1 cm-1) during the production of t-cinnamic acid (Gomez-Vasquez et al. 2004). Total soluble phenolics Soluble phenolics in the roots of spruce seedlings were extracted subsequently in 90% methanol and 100% methanol (1:25, w/V). Total phenols in combined methanolic extracts were measured according to Marigó (1973). For this, 1 ml of 2% Na2CO3and75µlofFolin-Ciocalteau reagent(Kemika,Zagreb)wereaddedto100µlof phenolic extract. After 15 minutes of incubation at 25 °C in the dark, the absorbance at 750 nm was measured (HP 8452A spectrophotometer). Catechin (Sigma) was used as a standard. Analysis of salicylic acid (SA) SAwas extracted following the modified pro­cedure of Raskin et al. (1989). The initial extrac­tion procedure was the same as for the extraction of soluble phenolics. Pooled methanol extracts were dried and resuspended in 0.5 ml 5% (w/V) trichloroacetic acid. After 1 min of mixing by sonification(35kHz,Elma,Transonic460/H),the extracts were centrifuged for 10 min at 3,000xg (4 °C), and thesupernatants wereextracted twice with 0.5 ml of ethylacetate: cyclopentane (1:1, V/V). The organic phases were pooled and dried at 40 °C. The dried extracts were resuspended in 1.5 ml 100% methanol and filtered (0.22 µm) before injecting onto the HPLC. For the analysis of SA, we used the same HPLC system configuration as above with ad­ditional flourimetric 474 detector. SAwas identi­fied andquantified by monitoring fluorescenceat 407nm (the excitation wavelength was 305nm). The elution was carried out using: A – 20 mM sodium acetate buffer (pH 5.0) with 0.02% sodium azide (Sigma); and B – 100% methanol (Merck).Thegradientused was:0–4.5 min:75% A; 4.5–11min: 75%-30% A; 11-15 min: 30% A, at a flow rate of 1 ml min-1, with 100 µl of sample injected per run. Statistical analysis Experiments were repeated three times. As similar trends were observed in all experiments represented data is from a single experiment. Effects of elicitor and ZR treatment were determined by analysis of variance according to general linear model procedure. Differences among various treatment means were separated by Holm-Sidak post hoc test, while effects of ZR treatment on colonisation were evaluated using t-test. All analyses were performed at the 0.05 level of probability in SigmaStat (SPSS). Results Growth and ergosterol content of fungi in axenic cultures ZR treatment increased colony diameter and ergosterol concentration of mycorrhizal fungusP. tinctorius at concentrations higher than 10-2µMZR (Fig 1). In contrast, pathogen H. annosum showed a decrease in colony diameter at the highest ZR (10 µM ZR) concentration. Ergosterol concentra­tions in the mycelia of H. annosum showed only marginal effects of ZR, as we could observe an increase in the ergosterol concentration only at 10-2 µM concentration of ZR. Fungal colonisation One month after inoculation, colonisation of spruce seedlings with ectomycorrhizal fungus P. tinctorius reached40.2% ±5.3%, whilepathogen H. annosum colonised 58.9%±6.7% of the root tips.ZRtreatmentofspruceseedlingssignificantly increased (t-test, p < 0.05) the colonisation with mycorrhizal fungusP. tinctorius to73.4%±9.0% (a82%increaseincolonisationlevels).Noeffects of ZR treatment on the colonisation of spruce seedlings with the pathogen H. annosum were observed (colonisation of ZR treated seedlings was 63.0%±6.9%). Enzyme activity and phenolics Only treatment with elicitors from H. anno-sum increased the PAL activity in the roots of the treated seedlings (Fig. 2a), whereas no effects of Zeatin riboside concentration (µ M) elicitors of P. tinctorius or ZR on the PAL activity could be observed. Treatment with elicitors from both fungi induced POD activity in the roots of the treated seedlings(Fig.2b).ZRtreatmentdecreasedPOD activityinseedlingstreatedwithelicitorsfromboth fungi, whereas no effects of ZR on POD activity were observed in the control seedlings. Treatment with elicitors of necrotroph H. an-nosum increased root levels of soluble phenolics (Fig. 2c), whereas no increase in accumulation could be observed in seedlings treated with elicitors of P. tinctorius. ZR increased the levels of soluble phenolics for 20% in the roots of the seedlings treated with elicitors of H. annosum and for 28% in the seedlings treated with elicitors of P. tinctorius, but had no effect on the levels of soluble phenolics in the control seedlings. Treatment with elicitors of H. annosum increased also the root levels of free SA to 1.6 times the levels of the control (Fig. 2d), whereas elicitors of P. tinctorius had no effect on the root SAlevels.SimilarlytoPALactivity,ZRtreatment did not affect the SA concentrations in the elicitor treated nor control seedlings. Figure 1: Ergosterolconcentration(bars)andcolony diameter (.) of Pisolithus tinctorius and Heterobasidion annosum treatedwithzeatin riboside at 10-6 to 10 µM concentration (Means±SE,n=10forcolonydiameterand n=6forergosterolconcentrations).Letters depictstatisticallysignificantdifferenceof one-wayANOVAandHolm-Sidakposthoc test at p < 0.05. Slika 1: Koncentracijaergosterola(stolpci)inpremer kolonije (.) gliv Pisolithus tinctorius in Heterobasidion annosum rastocihnagojišcu z 10-6 µM – 10 µM koncentracijo zeatin ribozida(SV±SN,n=10zapremerkolonij inn=6zakoncentracijeergosterola).Crke predstavljajo statisticno znacilno razliko testa enosmerne ANOVA in Holm-Sidak post hoc testa pri p < 0,05. 40 10 2 0 0 Free SA content(ng g-1 FW) Soluble phenolics (nmol mg-1 FW) Peroxidase activity -1 -1 PAL activity (nmol HO min mg proteins) 22 -1 -1 (mmol CA minmg proteins) 12 10 8 30 20 6 4 500 400 300 40 200 20 100 0 0 Control P. tinctorius H. annosum Control P. tinctorius H. annosum Elicitor treatment Elicitor treatment Figure 2: Impact of elicitors of Pisolithus tinctorius or Heterobasidion annosum and zeatin riboside treatment on: a) phenylalanine ammonia lyase activity, b) peroxidase activity, c) soluble phenolics and d) free sali­cylic acid concentrations in roots of the spruce seedlings (Mean±SE, n = 10). Letters depict statistically significant difference of one-way ANOVA and Holm-Sidak post hoc test at p < 0.05. Slika 2: Vpliv tretiranja z elicitorji gliv Pisolithus tinctorius ali Heterobasidion annosum in zeatin ribozidom na: a) aktivnost fenilalanin amonijeve liaze, b) peroksidazna aktivnost, c) koncentracija topnih fenolov in d) koncentracija proste salicilne kisline v koreninah kalic smreke (SV±SN, n = 10). Crke predstavljajo statisticno znacilno razliko testa enosmerne ANOVA in Holm-Sidak post hoc testa pri p < 0,05. Discussion ZR treatment increased mycelial growth of ectomycorrhizal fungus P. tinctorius at concentra­tions, similar to the concentrations found in soil extracts (Van Staden and Dimalla 1976) and in vitro cultures of mycor­rhizal fungi (Wullschleger and Reid 1990, Kovac and Žel 1995). Stimulat­ing effects of cytokinins on growth of mycelia of ectomycorrhizalfungiwereobservedalsobyother authors (Gogala and Pohleven 1976, Pohleven 1988). Pohleven (1988) also observed increased fluidityoftheplasmamembranesofthehyphaeof Suillus variegatus aftercytokinintreatment,which is believed to contribute to increased growth of fungal mycelia. In our experiments ZR mediated increase in growth of P. tinctorius was accom­panied by increased ergosterol concentrations. Ergosterolis animportantconstituentofthelipid rafts and sterol rich domains (SRD), which have been implicated in fungi in important processes suchasendocytosis,virulenceandhyphalgrowth (Alvarez et al. 2007; Steinberg et al. 2007). Due to the importance of ergosterol as a constituent of SRDand itshigh ability to promote the forma­tion of SRDs (Xu et al. 2001), observed changes in ergosterol concentrations could be connected to morphological and physiological changes observed by Martin et al. (2001) and increased radial growth observed in our experiments. Posi­tive effects on radial growth of ectomycorrhizal fungus P. tinctorius couldsupporttheimportance ofcytokininsintheregulationoftheformationof the mycorrhiza, as wasalready pointed out by seve­ral authors (Gogala 1991; Barker and Tagu 2000; Martin et al. 2001). Indeed in our experiments increased colonisation with mycorrhizal fungus was observed in ZR treated spruce seedlings. In experimentsofMartinetal.(2001)treatmentwith zeatin changed the morphology of the hyphae of P. tinctorius and triggered enhanced accumula­tion of hyphaphorine, which plays an important role in the formation of the ectomycorrhizal root (Ditengou et al. 2000). Other studies have shown that cytokinins are able to supress host cell death in infectedtissues, therebyallowingfungal develop­ment and growth within healthy tissue (Murphy et al. 1997). Furthermore it was confirmed that biotrophic and hemibiotrophic fungal pathogens are able to releasethe active forms of cytokinins from the pool of inactive O-glucosides (Cooper andAshby1998),thusinfluencingthehostbalance of growth regulators (Walters and McRoberts 2006).Incontrasttobiotrophicandhemibiotrophic fungal pathogens, no production of cytokinins or O-glucoside cleaving enzymes was observed in fungal necrotrophs (Cooper and Ashby 1998). In our experiments thecolonisation levels of spruce seedlings with H. annosum were not affected by the applied ZR. Furthermore ZR treated H. an-nosum showed a decreased in colony growth at higher ZR concentrations, which seems to apply also to other necrotrophic fungi (Michniewicz et al. 1984). Treatment with elicitors from both fungi increased content of soluble proteins and POD activity in the roots of spruce seedlings, whereas increased PAL activity, accompanied by accu­mulation of soluble phenolics and free SA, was observed only in seedlings treated with elicitors of H. annosum. Increased PALand POD activity is often reported for Norway spruce treated with elicitors or after inoculation with ectomycor­rhizal and pathogenic fungi (Asiegbu et al. 1994; Mensen et al. 1998; Nagy et al. 2004, Likar and Regvar,2008)andisassociatedwithaccumulation of phenolics with function in wall strengthen­ing (Hammerschmidt 1999). In Norway spruce seedlings the inoculation with H. annosum and P. tinctorius increased activity of guiaicol POD and ferulic acid POD and induced expression of new POD isoforms (Likar and Regvar 2008). Increased POD activity and induction of POD isoforms was observed also after treatment with the cell preparations of both fungi, suggesting increased cell-wall strengthening (Cahill and McComb 1992), and possibly formation of POD-generated fungitoxic compounds. Inhibitory ef­ fects of ZR on the POD activity observed in our presentexperiment,couldthusseverelyaffectthe response of P. abies seedling to the colonisation bythetestedfungi.Indeed,BeckmanandIngram (1994) showed that exogenous kinetin is able to inhibit hypersensitive response in potato inoculated with Phytophtora infestans,whichcouldbedueto inhibition of the apoplastic peroxidases (Bolwell et al. 2002). Cytokinins are known to regulate the expression of acid peroxidases (Limam et al. 1998), which play an important role in cell wall strengthening (Chitoor et al. 1997; Chitoor et al. 1999). Increased levels of soluble phenolics in ZR treated spruce seedlings in our experiments in combination with decreased POD activity could suggest accumulation of phenolic precur­sors destined for polymerisation in the cell wall, thus leading to improved conditions for fungal penetration and formation of ectomycorrhizae. Production of cytokinins in several ectomycor­rhizal fungi (Kraigher et al. 1991) and improved mycorrhization of Norway spruce seedling after treatment wit ZR as observed by Gabrovšek and Gogala (1995) could support this hypothesis. In contrast to POD activity, PAL activity and free SA accumulation, was increased only in seedling treated with H. annosum cell wall preparation.Inadditiontosynthesisofprecursors ofseveralcellwallcompounds,PALcanalsoplay an important role in SAsynthesis (Mauch-Mani and Slusarenko 1996) and as such in spreading the defence activation signal throughout the plant (Ryalsetal.1996).Apositivecorrelationbetween SAand PALactivity, together with the accumula­tion of a SA precursors in the phenylpropanoid pathway was seen in H. annosum-inoculated spruce seedlings (Likar and Regvar 2008). As the synthesis of SA through the PAL pathway is linked to plant-cell death (Wildermuth et al. 2001), it was suggested that H. annosum exploits the plant-cell death for facilitation of its infec­tion (Likar and Regvar 2008), as was observed also for Botrytis cinerea-Arabidopsis thaliana interactions (Govrin and Levine 2000). In our experiments, ZR treatment did not affect either PAL activity or free SA accumulation after the treatmentwiththeelicitorsandthushadnoeffect ontheinductionofhypersensitivereaction.Based on our observations and the absence of cytokinin production in necrotrophic fungal pathogens, we assume that in contrast to biotrophic fungi, the cytokinins are not involved in the pathogenesis of the necrotrophs. In conclusion, stimulating effects of ZR on P. tinctorius (growth, ergosterol concentrations) and improved colonisation of spruce seedlings, suggest that cytokinins can successfully alter the growth and colonisation success of mycorrhizal fungal symbiont. Effects of ZR on cell-wall strengthening responses after elicitation with elicitors of both fungi point to involvement of cytokinins in non-specific defence responses. In combination cytokinins could play an important roleinregulationofcellwallmodificationsduring the fungal colonisation and formation of ecto­mycorrhizae, by simultaneously affecting fungal growthandphysiology,aswellasthenon-specific defencereactionsoftheplanthost.Incomparison with the ectomycorrhizal fungus P. tinctorius, elicitors of necrotroph H. annosum activated also PALactivityandtheSA-dependantsignalpathway, which showed no ZR-induced changes. Growth of the fungus in axenic culture and colonisation of spruce seedlings with the pathogen were not improved as in the case of the ectomycorrhizal fungus, suggesting a minor role of cytokinins in the pathogenesis of H. annosum on spruce. Povzetek Rastline s korenin sprošcajo kompleksno mešanico spojin, ki tvorijo koreninski eksudat. Pole aminokislin, organskih kislin, proteinov in sladkorje,koreninskieksudatvsebujetudištevilne rastneregulatorjekotsonpr.citokinini(Neumann in Römheld 2000). Citokinini igrajo pomembno vlogopriregulacijištevilnihprocesovtekomrasti in razvoja rastlin (Sakakibara 2006, Kyozuka 2007), hkrati pa lahko vplivajo tudi na rast gliv (Barker in Tagu 2000, Nasim in Rehman 2006). Iz literature je znano, da lahko citokinini vplivajo tudi na obrambne reakcije rastlin, ki se sprožijopoaplikacijielicitorjevaliobinokulaciji zživoglivo(KadiogluinDurmus1997,Limamet al. 1998, Sano et al. 1996). Vpricujoci raziskavi smo,zaovrednotenjenjihovevlogepriregulaciji kolonizacije kalic smreke (Picea abies) z miko­riznimi in patogenimi glivami, preverili vpliv zeatin ribozida (ZR): i) na rast ektomikorizne glive Pisolithus tintorius in patogena Heteroba­sidion annosum vaksenicnikulturi,ii)nastopnjo kolonizacije kalic smreke z obema glivama in iii) na aktivacijo obrambnih reakcij smreke po tretiranju z elicitorji. V aksenicni kulturi smo pri 10-2 µM kon­centraciji ZR opazili pospešeno rast mikorizne glive P. tinctorius, ki jo je spremljala povecana koncentracija ergosterola v miceliju. Nasprotno je bila rast patogene glive H. annosum pri najvišji koncentraciji ZR v gojišcu (10 µM) zavrta. Podob-no kot v aksenicnikulturi je dodatenZR pospešil kolonizacijokalicsmrekezektomikoriznoglivo, medtem ko na stopnjo kolonizacije s patogenom niimelucinka.Tretiranjesmrekezelicitorjiobeh gliv je povecalo aktivnost peroksidaz (POD) v koreninah kalic, samo elicitorji patogene glive H. annosum pa so povecali tudi aktivnost feni­lalanin amonijeve liaze (PAL) in koncentracijo topnih fenolov ter proste salicilne kisline (SA). Dodatek ZR je znižal peroksidazno aktivnost v kalicah tretiranih z elicitorji obeh gliv in povecal koncentracijo topnih fenolov. 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Yalpani, N., Leon, J., Lawton, M.A., Raskin I., 1993. Pathway of salicylic acid biosynthesis in healthy and virus-inoculated tobacco. Plant Physiol. 103, 315–321. Xu, X., Bittman, R., Duportail, G., Heissler, D., Vilcheze, C., London, E., 2001. Effects of the struc­ture of natural sterols and sphingolipids on the formation of ordered sphingolipid/sterol domains (rafts). The J. Biol. Chem. 36, 33540–33546. Wet meadows with Purple Moor-grass (Molinia caerulea) in Slovenia Mokrotni travniki z modro stožko (Molinia caerulea) v Sloveniji Igor Zelnik University of Ljubljana, Biotechnical Faculty, Department of Biology, Vecna pot 111, SI-1000 Ljubljana, Slovenia. *correspondence: igor.zelnik@bf.uni-lj.si Abstract: The paper presents wet meadow vegetation with taxon Molinia caerulea (L.) Moench subsp. caerulea in Slovenia. The main objective of this study was to examinetheplantspeciescompositionandplantspeciesrichnessofwetmeadowplant communitieswiththementioneddominatingorco-dominatingplanttaxon.Vegetation wasrecordedinaccordancewithstandardCentralEuropeanmethod.Vegetationtypes were classified by means of multivariate analysis. Four associations from the alliance Molinon Koch 1926 were identified and analysed: Plantagini altissimae-Molinietum caeruleae Marchiori & Sburlino 1982, Selino-Molinietum caeruleae Kuhn 1937, Carici davallianae-Molinietum caeruleae Špániková 1978 and Junco-Molinietum caeruleae Preising1951exKlapp1954.Ecologicalcharacteristics,plantspeciescompositionand richness of the delimited plant communities are presented, as well as their syntaxo­nomic position and distribution. For two of the mentioned communities relevés made in Slovenia are published here for the first time. Keywords: Molinia caerulea (L.) Moench subsp. caerulea, plant species compo­sition, vegetation ecology, wetlands Izvlecek: Vprispevkujepredstavljenavegetacijamokrotnihtravnikovstaksonom Molinia caerulea (L.) Moench subsp. caerulea v Sloveniji. Glavni cilj je bil preuciti floristicno sestavo in vrstno pestrost rastlinskih združb na mokrotnih travnikih z omenjenim dominantnim ali ko-dominantnim taksonom. Vegetacijo smo popisali po standardni srednjeevropski metodi. Vegetacijske tipe smo uvrstili v sintaksonomski sistem s pomocjo multivariatnih analiz. Dolocili in analizirali smo štiri asociacije iz zveze Molinon: Plantagini altissimae-Molinietum caeruleae Marchiori & Sburlino 1982, Selino-Molinietum caeruleae Kuhn 1937, Carici davallianae-Molinietum cae­ruleae Špániková 1978 in Junco-Molinietum caeruleae Preising 1951 ex Klapp 1954. Predstavljene so ekološke znacilnosti, floristicna sestava in vrstna pestrost navedenih rastlinskih združb kot tudi njihov sintaksonomski položaj in razširjenost. Za dve rast­linski združbi popise, narejene v Sloveniji, tukaj objavljamo prvic. Kljucne besede: Molinia caerulea (L.) Moench subsp. caerulea, floristicnasestava, vegetacijska ekologija, mokrišca Introduction Wetmeadowsandothersimilarwetland-types have been the object of several studies throughout Europe in the last decade (Hájek and Hájková 2004, Havlová et al. 2004, Hölzel and Otte 2004, Hrivnák2004,Grootjansetal.2005,Stancic2005, Zelnik 2005a,b, Hájková et al. 2006, Havlová 2006, Reznícková 2007, Janišová et al. 2007, Stancic 2008, Zelnik and Carni 2008a,b), since the threat to the biodiversity of these ecosystems is still increasing and numerous sites have been destroyed, respectively. Meadows of the alliance Molinion Koch1926 are found in nutrient-poor soils (Hölzel and Otte 2004), which may dry up during the summer (Botta-Dukát et al. 2005). They thrive on perma­nently to alternatively wet soils, which are acid, or alkaline (Ellmauer and Mucina 1993). These oligotrophic ecosystems need manage­ment, which should be neither excessive nor lack­ing in its intensity(Ellmauer and Mucina 1993). In the last decades a decrease in biodiversity of wet meadows due to intensified agricultural use, namely the use of fertilizers has been detected (EllmauerandMucina1993,Joyce2001,McCrea et al. 2001). Knowledge of the plant communities ena­bles us to forecast the likely changes in floristic composition after changes of site factors (Gre­villiot and Muller 2002). Nutrient availability and water regime are considered to be the most important factors determining the structure and properties of wet grassland vegetation (De Mars et al. 1996, van Duren and Pegtel 2000, Zelnik and Carni 2008a). Horvatic (1939) was the first researcher who systematically studied vegetation of the wet meadows in Slovenia. During the study of the vegetation of the lake Cerkniško jezero Ilijanic (1979) described new plant association Deschamp-sio-Plantaginetum altissimae, and classified it to the alliance Molinion.He recorded stands of this vegetation type in Planinsko polje as well. Vegeta­tion with Molinia caerulea inLjubljanamoorwas studiedbySeliškar(1986).Vegetationofthelake Cerkniško jezero was studied also by Martincic (1991, 2001), who described new plant associa­tions Schoeno nigricantis-Molinietum caeruleae Martincic 1991andSchoeno ferruginei-Molinietum caeruleae Martincic 2001 and classified them to the alliance Molinion. This type of vegetation on the Bloke plateau and in some other areas in Dinaric and Alpine regions was studied by Leskovar (1996). Kaligaric (1997) documented the thriving of the association Selino-Molinietum caeruleae Kuhn1937nearSlovenjGradec.Results ofthestudies ofthewetmeadows inSESlovenia (Krškobasin,BelaKrajina)arepublishedinZelnik (2005a), Zelnik and Carni (2008b). Vegetation ecology of some of the communities from the alliance Molinion in Slovenia is discussed in Zelnik and Carni (2008a). Besidethementionedpublicationsthatcover only some of the areas in Slovenia, wet meadow vegetation in major part of the country remained almostunknowntothescientificpublic,especially thepresence,structureanddistributionofseveral plant communities. One of the aims of this paper is to fill this gap and to present the characteristics and distribution of wet meadow plant communi­ties with Molinia caerulea (Purple Moor-grass) in Slovenia. The aims of this study were also to examine: – The structure and diversity of wet meadow plant communities with dominant taxon Molinia caerulea ssp. caerulea. – Relationshipsbetweenwetmeadowcommuni- ties of Molinon alliance in Slovenia and their distribution. Material and methods Study area The meadows were investigated across the majority of the state, in the continental part of SloveniafromthewesternborderofDinaricregion to the eastern border of the state, which is in the Pannonian region (from 14°10’to 16°20’E, from 45°40’to 46°50’N). There is a strong gradient in annual precipitation from the SW to NE part of the studied area. In the SW part the climate is more humid with an annual precipitation of 1500 mm, but in the NE part, which is the driest part of Slovenia the annual precipitation is 800 mm (Zupancic 1995). For this study vegetation plots fromNE,EandSEpartsofthestatewereexcluded from analysis due to absence of the taxon Molinia caerulea ssp. caerulea. Moreover, only stands with dominant or co-dominant mentioned taxon were considered for this study. There is also an altitudinalgradientwhichreflectsinmeanannual temperatures,assomeplotscanbefoundatabout 265 m and others 680 m a.s.l., but the majority is found between 300 and 500 m a.s.l. Vegetation analysis Vegetation was investigated according to the standard Central European method (Braun­Blanquet1964).Thecover-abundancevalueswere transformed according to van der Maarel (1979). Vegetation relevés (59) were made in the years 2003and2004.Thesizeofplotsvariesfrom15to 25 m2duetomicro-topography.Nomenclatureof plant taxafollows Ehrendorfer et al. (1973) with exception of taxa Centaurea macroptilon Borb., Centaurea carniolica Host. Similarity analyses of the relevés were car­ried out using the computer program SYN-TAX (Podani2001);anordinationmethod(PCoA)was performed.Dissimilarityofrelevéswasmeasured with Similarity ratio complement. Rare species were not excludedfrom the analysis. Clusters of relevés were classified into syntaxa according to Ellmauer and Mucina (1993) as well as local studies (Zelnik 2005a,b). Results and discussion Floristically defined wet meadow communities Sincetheobjectsofourstudywerestandswith abundant taxon Molinia caerulea ssp. caerulea, the classification to the alliance Molinion could be done without any doubt. Further classification of therelevéswasdoneonthebaseoftheordination diagram(Fig.1),whichshowsthegroupingofthe relevésaccordingtothesimilarityoftheirfloristic composition. The four groups corresponded to four wetmeadow plantassociations belonging to Molinion alliance and were classified according to Ellmauer and Mucina (1993), Sburlino et al. (1995),Zelnik(2005a,b),ZelnikandCarni(2008a): (1) Plantagini altissimae-Molinietum caeruleae Marchiori & Sburlino 1982, (2)Selino-Molinietum caeruleae Kuhn 1937, (3) Carici davallianae-Molinietum caeruleae Špániková 1978 and (4) Junco-Molinietum caeruleae Preising 1951 ex Klapp 1954. Despite the results of formalized classifica­tion of the stands with Molinia caerulea, that were obtained in several countries and are able to define only two different associations, we disagree with such simplification of this diverse vegetation in floristic and ecological sense that reflects in high species diversity and diversity of communities/ecosystems.Statisticallysignificant differences in many measured and/or calculated ecologicalparametersbetweentheseassociations were calculated and published (Zelnik and Carni 2008a),sotheseclearlydefinedassociationsfrom the alliance Molinion obviously exist. Syntaxonomical scheme of the studied vegetation: Molinio-Arrhenatheretea R.Tx. 1937 em. R. Tx. 1970 Molinietalia Koch 1926 Molinion Koch 1926 Plantagini altissimae-Molinietum caeruleae Marchiori & Sburlino 1982 Selino-Molinietum caeruleae Kuhn 1937 Carici davallianae-Molinietum caeruleae Špániková 1978 Junco-Molinietum caeruleae Preising 1951 ex Klapp 1954 Thesamplingplotsintheordinationdiagram weresegregatedaccordingtocommunities(Fig.1) and also correspond to the traditional method: – Plantagini-Molinietum withthehighestscores along the first axis (the most humid climate) and intermediate along the second axis; – Selino-Molinietum withintermediateposition that is in accordance with its central position within the alliance Molinion; – Carici-Molinietum with the highest scores along the first axis (the highest pH) and mo­derate humidity; – Junco-Molinietum withhighscoresalongfirst axis (humid sites – depressions); Their characteristic taxa are presented in Table 1, while their plant species composition is presentedinTables2and3.Intotal232planttaxa were found in 59 sampled plots, ranging from 14 to 69 plant taxa per plot. Figure 1: Ordinationdiagram of sampling plots based on analysis PCoA. Numbers correspond to the numbers of relevés in Tables 2-3 (complement of similarity ratio). Slika 1: Ordinacijski diagram popisov na osnovi metode PCoA. Številke so v skladu s številkami popisov v Tabelah 2-3 (komplement koeficienta podobnosti). • 1–8: Plantagini altissimae-Molinietum caeruleae, • 9–16: Junco-Molinietum caeruleae succiselletosum inflexae, . 17–21: Junco-Molinietum caeruleae typicum, • 22–38: Selino-Molinietum caeruleae, . 39–59: Carici davallianae-Molinietum caeruleae. Plantagini altissimae-Molinietum caeruleae Marchiori & Sburlino 1982 Thisassociationwasdescribedineasternparts of Po plain (Veneto, Friuli), where it thrives in helocrenic spring sites and in higher parts of the lowland(Sburlinoetal.1995).Standsoftenoccur indepressionswithinintensivelycultivatedareas, where the mineralization of soil organic matter is hindered due to high water content (Sburlino et al. 1995). The association Plantagini altissimae-Moli­nietum caeruleae Marchiori & Sburlino 1982 is found in the western part of studied area, where the climate is most humid and this is the main ecological difference with communities of the alliance Deschampsion Horvatic 1930, which thrive in the areas with more arid climate. Stands classified to this association were recorded in the valley of river Nanošcica. They thrive in bigger depressions, which do not lie Figure 2: OrdinationoftherelevésoftheassociationsPlantagini altissimae-Molinietum caeruleae and Deschampsio-Plantaginetum altissimae (PCoA, complement of similarity ratio): Slika 2: OrdinacijapopisovasociacijPlantagini altissimae-Molinietum caeruleae in Deschampsio-Plantaginetum altissimae (PCoA, komplement koeficienta podobnosti): 1–8:Zelnik, hoc loco, tab. 2 / 1–8 Plantagini altissimae-Molinietum caeruleae; • 9–25: Sburlino et al. (1995), tab. 1 / 1–17 Plantagini altissimae-Molinietum caeruleae; . 26–37: Ilijanic (1979), tab. 11 / 1–12 Deschampsio-Plantaginetum altissimae cirsietosum pannonici; • 38–54: Ilijanic (1979), tab. 11 / 13–29 Deschampsio-Plantaginetum altissimae typicum & filipenduletosum vulgaris; • 55–63: Zelnik, 2005b, tab. 6 / 1–9 Deschampsio-Plantaginetum altissimae caricetosum tomentosae; .••••••• • 64–65: Zelnik, 2005b, tab. 6 / 10–11 Deschampsio-Plantaginetum altissimae molinietosum arundinaceae; along the mentioned river on its floodplain, but along its tributaries that drain the areas on non-calcareous bedrock. Characteristic species and plant species composition: Sburlino et al. (1995) declared the species Plantago altissima as the only cha­racteristic species with sufficient constancy. Additionaly, species Centaurea carniolica is differential from other central European com­munities with Molinia. Beside the mentioned species we name the followingspeciesasdifferentialfromotherstands with Molinia (Tab. 2, relevés 1–8): Sanguisorba officinalis, Gratiola officinalis, Cirsium rivulare. From similar association Deschampsio-Plantaginetum altissimae this association could be told apart on the base of the Molinia caerulea, which is often dominant species here, but miss­ing in the mentioned. Besides, this association could be delimited from the mentioned due to presence of the following species: Serratula tinctoria, Carex nigra, Ranunculus flammula, Potentilla erecta. Syntaxonomic position and distribution in other countries:On the base of the comparison of the relevés we discovered the similarity of this association with the syntaxon Deschampsio-Plantaginetum altissimae cirsietosum pannonici Ilijanic 1979, which thrives on the Cerkniško jezero.Ilijanic(1979)hadalreadypointedoutthe possibility of delineation of this subassociation and definition as specific association. Multivari­ate analyses revealed relatively higher similarity of the mentioned subassociation with Plantagini altissimae-Molinietum caeruleae than other units of the Deschampsio-Plantaginetum altissimae (Fig. 2). According to these findings we classi­fiedmentionedsubassociationandourstandsinto association Plantagini altissimae-Molinietum caeruleae. However,standsfromnorthernItalyare richerinfenspecies,soonthebaseofourdataand additional research there is a possibility to describe a distinct subassociation in the future. The only report about the thriving of this association outside Po plain is given by Zelnik and Carni (2008a). Selino-Molinietum caeruleae Kuhn 1937 For this central association of the alliance Molinion many authors use the name Molini­etum caeruleae, or Molinietum medioeuropaeum Oberdorfer 1957, which are not clearly defined. For this reason Ellmauer and Mucina (1993) suggest the name Selino-Molinietum Kuhn 1937 for such stands. This is basophilic community that thrives in lowland and montane belt, mostly in less wet fensoils.Soilsarealmostequallywetthroughout the whole year. Stands are mown in late summer or early autumn once a year or biennially. This community is a stage in succession of aquatic ecosystems to terrestrial leading from tall-sedge communities of the alliance Magnocaricion elatae Koch 1926,low-sedges from the allianceCaricion davallianae Klika1934 to the community Selino-Molinietum (Ellmauer and Mucina 1993). This association can also develop on the edge of bog or with degradation of fens. First relevés from Slovenia that were classi­fied into this association are from the vicinity of Slovenj Gradec and were published by Kaligaric (1997). Our relevés from various sites were clas­sifiedintothisassociation,whichisinaccordance with its central position and wide distribution (Tab. 3, relevés 22–37). Characteristic species and plant species composition:This is a central community of the alliance Molinion that reflects in high constancy of characteristic species of this alliance. In accordance with other authors (Ellmauer andMucina1993,Kaligaric1997),wepointedout the following species as characteristic: Selinum carvifolia, Laserpitium prutenicum. Syntaxonomic position and distribution in other countries: Since this is a central association oftheallianceitsimilaritytotheotherassociations is relatively high. Community was described in southern Germany by Kuhn (1937), but many authors classified more or less similar stands to macro-association Molinietum caeruleae, or Mo-linietum medioeuropaeum, that prevented a review. Thereisconfusioninliterature,sincesomeauthors classify different associations or their parts into Selino-Molinietum (Pott1995).Eveninthelatest publications (Burkart et al. 2004, Havlová 2006, Janišová et al. 2007) this vegetationis classified as Molinietum Koch 1926. The reasons for this is the difficulty of clas­sification of the stands with Molinia caerulea,the lackofknowledgeoftheecologicalconditionsof thesesitesandtheabsenceofmultivariateanalyses that would enable comparisons of plant species composition with relevés of other associations. Another reason is the abandonment of mow­ing of the meadows, where stands with Molinia caerulea thrive that leads to the total dominance of this species, while others e.g. characteristic species of many associations disappear from the stands and makethesuitableclassification impos­sible. The consequence is the classification of such stands to the most intermediate association Selino-Molinietum. Distribution of this association is docu­mented in Germany (Kuhn 1937, Pott 1995), Austria (Balátová-Tulácková and Hübl 1985a, Ellmauer and Mucina 1993) and Italy (Sburlino et al. 1995). Carici davallianae-Molinietum caeruleae Špániková 1978 This community that represents a transition between wet meadows and fens is dominated by taxon Molinia caerulea ssp. caerulea and is often adjacent to the stands of Caricetum davallianae Dutoit 1924 (Špániková 1978), but its floristic composition is still richer in meadow species, which also dominate the stands. These stands are often found in helocrenic springs that are surrounded by mesic communi­ties. Soils are loamy and often with high share of organic matter. Soils are slightly acid to neutral, however,pHvaluesandhumidityarehigherthan in Selino-Molinietum. This association thrives in permanently wet sites rich in Ca2+ . These condi­tions reflect in higher share of the fen species. Since the soils are rarely dry enough, mowing with tractors is barely possible in these meadows thatwasevidentinthefield.Withtheloweringof waterlevelthevegetationturnstotheassociation Selino-Molinietum. In all studied sites the influence of base-rich groundwater was obvious. Moreover, this water drainsthelimestoneand/ordolomiteandsupplies these sites with high amounts of Ca2+ and Mg2+. These sites are never on plane, but on slight slopes (3–10°). Stands of this association were recorded in Alpine, Pre-Alpine, Dinaric and Pre-Dinaric phytogeographic regions of Slovenia, on the following locations: Dolic, Sopota, Tatinec, Nemška vas. Characteristic species and plant species composition:On the base of the comparison of our stands (Tab. 3, relevés 38–59) with stands of otherassociations,wedefined(inaccordancewith Dierschke 1994) the following species as diffe­rential: Carex davalliana, C. hostiana, Epipactis palustris, Koeleria pyramidata, Eriophorum latifolium. Mentioned species are basophilic and except Koeleria pyramidata are all fen species. Theymostlymatchwiththedifferentialspeciesof the subassociation Selino-Molinietum caricetosum davallianae Balátová-Tulácková & Hübl 1985 (Carex davalliana, C. flava, Eriophorum latifo­lium, Parnassia palustris), that was described in eastern limestone Alps by Balátová-Tulácková and Hübl (1985a). In the same publication these authors defined the same combination of the species (Carex davalliana, Eriophorum latifo­lium, Parnassia palustris) as differential for the subassociation Molinietum caeruleae caricetosum davallianae Görs 1951. Syntaxonomic position and distribution in other countries:Unclear classification of this basophilic vegetation in Slovenia was firstly pointed out by Kaligaric (1997). Authors (e.g. Seliškar 1986, Leskovar 1996) have classified such stands with stands of different other as­sociations (e.g. Selino-Molinietum, Plantagini altissimae-Molinietum caeruleae) into same syn-taxon Molinietum caeruleae s.l.Figure1presents clear delimitation of these stands from relevés of other associations. Studied association is similar to Succiso-Molinietum caeruleae (Kovacs 1962) Soó 1969 thatthrivesinPannonianregion,namelyinAustria (EllmauerandMucina1993)andHungary(Borhidi 2003).Incaseofincreasingmoistureofthosesites Wagner(1950)defineditstransitiontocommunity Schoenetum nigricantis Koch 1926. In our case increasing moisture of the sites led to the fen community Caricetum davallianae Dutoit 1924. Our relevés are very similar to the relevés published by Balátová-Tulácková and Hübl (1985a) that are recorded in the Lower-Austrian limestone Alps, where the bedrock is actually the sameasinourcases.Mentionedauthorsclassified those stands into Selino-Molinietum caricetosum davallianae Balátová-Tulácková&Hübl1985and Molinietum caeruleae caricetosum davallianae Görs 1951. Our relevés are also similar to the relevésfromnorthernItaly(Lombardy)published by Sburlino et al. (1995: Tab. 2 / 1–9). According to the mentioned facts and simi­larities we can conclude that this basophilic community is distributed over Slovenia and in other countries that reach into the southern and/ or northern belt of calcareous Alps, Carpathians and NW Dinaric mountains. Junco-Molinietum caeruleae Preising 1951 ex Klapp 1954 Soils in this community are of loamy texture and could be gleysols (Balátová-Tulácková and Hübl, 1985b) and/or peat-soils. These sites are rarely fertilized and mown only once a year (Ellmauer and Mucina 1993). Sites are mostly onintermittentlywetriverinesoilsorslopeswith helocrenic springs(hanging mires).Thisassocia­tion is common in areas with non-carboniferous bedrock and/or in wet places with thick layer of accumulated organic matter (peat). In Slovenia this association is most common in Ljubljana moor, where it was documented by Seliškar (1986). Stands of this association were recordedonthefollowinglocalities(Tab.2,relevés 9–21):Nemškavas,LogpriMokronogu,Selopri Bledu. Soils under the stands on the mentioned localities are influenced by the base-poor water that drains non-calcareous sediments and/or are in depressions where rainwater is stagnating for weeks. Characteristic species and plant species com-position:Associationispoorlyfloristicallydefined. Characteristic species of the order Molinietalia Koch 1926 aswell asof the alliance Molinion are relatively rare. Beside the mentionedcharacters, numerous species characteristic for the classes Scheuchzerio-Caricetea fuscae R.Tx. 1937 and Calluno-Ulicetea thatindicatenutrient-pooracidic soils, were found in these stands. Preising(1951),EllmauerandMucina(1993) defined species Juncus conglomeratus and J. ef­fusus as differential from other associations. Syntaxonomic position and distribution in other countries: After Ellmauer and Mucina (1993) this community is transitional towards following syntaxa: Calthion, Caricion fuscae (Scheuchzerio-Caricetea fuscae) and Violion caninae (Calluno-Ulicetea). Such stands are found in Austria on silicate bedrockonthealtitudesbetween525and660ma. s. l. (Balátová-Tulácková and Hübl 1985b). Asso­ciation thrives also in Hungary (Borhidi 1996), what is no longer evident in later publications (Borhidi 2003), since the name Nardo-Molinietum hungaricae is used for this type of vegetation. Asso­ciationisalsofoundinCzechRepublic(Blažková 1973,Havlová2006)andinGermany(Pott1995), althoughinlaterpublications(Burkartetal.2004) these stands are classified to community Juncus conglomeratus-Succisa pratensis. Division to lower syntaxa:Balátová-Tulácková and Hübl (1985b) classified relevés from Austria into subassociations scirpetosum sylvaticae Balátová-Tulácková & Hübl 1985 and typicum Preising 1951. Junco-Molinietum caeruleae typicum Preising 1951: Five our relevés, without differential spe­cies, were classified into typical subassociation (Tab.2,relevés17–21).Prevailingspeciesinthese stands are species of the order Molinietalia and class Molinio-Arrhenatheretea. We defined new subassociation succiselle­tosum inflexae due to dominance of the species Succisella inflexa (Tab. 2, relevés 9–16; holotipus: Tab. 2 / relevé 13). Junco-Molinietum caeruleae succiselletosum inflexae subass. nova hoc loco: On the bottoms of more distinctive depressions (> 0,2 m), beside the species Molinia caerulea, Succisella inflexa, that is characteristic species of the alliance De-schampsion Horvatic 1930, is very abundant. In such depressions soils are under influence of stagnating base-poor rainwater that makes the ecological conditions similar to the ones where communities of the alliance Deschampsion are found. Main difference in floristic composition is in presence and even dominance of the species Molinia caerulea. Because of the mentioned conditions lower herb layer is dominated by species Succisella inflexa and Carex panicea, while upper is domi­nated by Molinia caerulea. Other species only exceptionallyoccurwithhighercovervalues,their numberismuchlowerthaninothercommunities with Molinia caerulea.Fromotherclassesmarsh species of the class Phragmito-Magnocaricetea Klika 1941 can be found in these stands. Such stands were recorded in the area of Alpine-Dinaric mountain barrier where the precipitation amount is the highest in Slovenia. These localities were: Nemška vas near Ribnica, Selo pri Bledu. Thesestandsthriveinsitesthatarewetterthan sites of other communities likeJunco-Molinietum caeruleae typicum, Plantagini altissimae-Molini­etum, what reflects in plant species composition (Fig. 1). Further increasing of soil moisture on lower sites facilitates species of the classes Phragmito-Magnocaricetea and Scheuchzerio-Caricetea fuscae that become dominant. Conclusions Vegetation of wet meadows in the research area is very diverse. Our relevés with taxon Molinia caerulea subsp. caerulea wereclassified into alliance Molinion and further into four plant associations: Plantagini altissimae-Molinietum caeruleae Marchiori & Sburlino 1982, Carici davallianae-Molinietum caeruleae Špániková 1978, Selino-Molinietum caeruleae Kuhn1937, Junco-Molinietum caeruleae Preising1951.For the first two associations the relevés from Slov­eniaarepublishedforthefirsttimeinanalytical table in this paper. Beside the mentioned four associations another two vegetation types with dominantorco-dominantMoliniacaeruleaethat are transitional to fens are found in Slovenia, namely Schoeno nigricantis-Molinietum caer­uleae Martincic 1991 and Schoeno ferruginei-Molinietum caeruleae Martincic 2001. Since they occuronthemarginsofthefenswhichdistribution is very limited (Schoenetum nigricantis Koch 1926, Schoenetum ferruginei Du Rietz 1925) only,theyarenotwidelydistributedandarenot studied in this paper. The association Plantagini altissimae-Mo­linietum caeruleae Marchiori & Sburlino1982is found in the western part of studied area, where the climate is most humid and this is the main ecological difference with communities of the alliance Deschampsion, which thrive in the areas with more arid climate. This association is found also in northern Italy (Sburlino et al. 1995). The association Selino-Molinietum caeruleae Kuhn 1937 is the central association of the alli­ance Molinion andcorrespondstothecommunity Molinietum caeruleae Koch 1926 (Ellmauer and Mucina 1993, Zelnik 2005b), which is not ef­ficiently defined. This community is found all over central Europe. The association Carici davallianae-Molini­etum caeruleae Špániková 1978 is transitional to fen vegetation, but its floristic composition is still much richer in meadow species, which also dominate the stands. This association is found in central and north Slovenia (Zelnik 2005b). Apartfromtheotherthreeassociationswhich are more or less basophilic, the associationJunco-Molinietum caeruleae is acidophilic and is found in depressions with stagnating rainwater or on peat-soils. This association is found in central, southeast and east Slovenia (Zelnik 2005a,b). Mostspecies-richplantcommunitiesthrivein siteswithoutanyoutstandingparameterthatwould have a dominatinginfluence on the conditions. It is crucial that soils are nutrient-poor and flooded/ waterlogged for short periods only. Slovenia isin conjunction of Alpine,Mediter­ranean,DinaricandPannonianregionsthatreflects indiversityofwetmeadowplantcommunities.We found plant communities that were described in Germany, Italy and Slovakia, and are distributed alsoinAustria,Hungary,Croatia,CzechRepublic and Poland. Povzetek Mokrotnitravnikisobilivzadnjemdesetletju v Evropi pogosto predmet preucevanj, predvsem zaradi vedno vecje ogroženosti njihove biodiver­zitete, marsikje pa so ti ekosistemi že uniceni. Mokrotne travnikeiz zveze Molinion Koch 1926 najdemo na tleh, ki so revna s hranili in so stalno aliobcasnomokra.Zaohranjanjeteholigotrofnih ekosistemovjepotrebnogospodarjenje,kinesme biti prevec ali premalo intenzivno. Vegetacijo mokrotnih travnikov so v Slove­niji preucevali: Horvatic (1939), Ilijanic (1979), Seliškar(1986),Martincic(1991,2001),Leskovar (1996), Kaligaric (1997). Rezultati teh raziskav v zadnjem desetletju pa so objavljeni v: Zelnik (2005a), Zelnik in Carni (2008a,b). V Sloveniji je struktura rastlinskih združb mokrotnih travnikov podrobneje raziskana in objavljenapredvsemnaobmocjihkotjeCerkniško jezero, Ljubljansko barje, Bloke in Krška kot­lina, medtem ko je v ostalih obmocjih strokovni javnostinepoznana.Ciljprispevkajepredstavitev znacilnosti, floristicne sestave, vrstne pestrosti in razširjenosti rastlinskih združb mokrotnih travnikov z Molinia caerulea ssp. caerulea v Sloveniji. Vegetacijo smo popisali po standardni srednjeevropski metodi. Vegetacijske tipe smo uvrstili v sintaksonomski sistem s pomocjo multivariatnih analiz. Dolocili in analizirali smo štiri asociacije iz zveze Molinon: Plantagini altissimae-Molinietum caeruleae Marchiori & Sburlino 1982, Selino-Molinietum caeruleae Kuhn 1937, Carici davallianae-Molinietum caeruleae Špániková 1978 in Junco-Molinietum caeruleae Preising 1951 ex Klapp 1954. Plantagini altissimae-Molinietum caeruleae Marchiori & Sburlino 1982: Ta asociacija je bila opisana v vzhodnih predelih Padske nižine, kjer uspeva na povirnih mestih. Sestoje, ki smo jih uvrstili v to asociacijo smo popisali v dolini reke Nanošcice.Uspevajovvecjihulekninah,kipane ležijoneposrednoobNanošcicitemvecobnjenih pritokih,kiimajoprispevnaobmocjanapretežno nekarbonatnih kamninah. Vnašihpopisih(Tab.2,popisi1–8),sepoleg znacilnice Plantago altissima kot razlikovalnice od ostalih sestojev s stožko pojavljajo naslednje vrste: Sanguisorba officinalis, Gratiola officina­lis, Centaurea carniolica, Cirsium rivulare. Od podobne združbe Deschampsio-Plantaginetum altissimae se ta asociacija bistveno loci po vrsti Molinia caerulea,kijetukajpogostodominantna vrsta in po naslednjih vrstah: Serratula tinctoria, Carex nigra, Ranunculus flammula, Potentilla erecta. Selino-Molinietum caeruleae Kuhn 1937:Za to osrednjo združbo zveze Molinion mnogiavtorji uporabljajo ime Molinietum caeruleae, oziroma Molinietum medioeuropaeum Oberdorfer1957,ki pastapremalonatancnodefinirani.ZatoEllmauer & Mucina (1993) za tovrstne sestoje predlagata uporabo imena Selino-Molinietum Kuhn 1937. To je bazifilna združba, ki uspeva v nižinskem in montanskem pasu, vecinoma na manj vlažnih tleh.Tlasoprekocelegaletaenakomernovlažna. Prve popise iz Slovenije, ki so klasificirani v to asociacijo, je objavil Kaligaric (1997). Kotznacilnicesmoizpostavilinaslednjivrsti: Selinum carvifolia, Laserpitium prutenicum. Ker je to osrednja asociacija zveze Molinion, je zaradi svoje relativne zmernosti tudi najbolj podobna vsem ostalim združbam iz te zveze. Klasifikacijatehsestojevjepogostotežavnazaradi nepoznavanjarastišcnihrazmerinopušcanjakošnje tehpovršin,zaradicesarmodrastožkapopolnoma prevlada,ostalevrstemeddrugimtudiznacilnice, pa izginjajo iz sestojev. Carici davallianae-Molinietum caeruleae Špániková 1978: V tej združbi, ki predstavlja prehod med mokrotnimi travniki in nizkimi barji in je pogosto v stiku z asociacijo Caricetum daval­lianae, prevladuje vrsta Molinia caerulea.Sestoje te združbe vedno najdemo na povirnih mestih. Tla so ilovnata in pogosto z visokim deležem organske snovi (šotnata), reakcija tal je rahlo kisla do nevtralna, višja kot v asociaciji Selino-Molinietum. Na vseh rastišcih je bilo ocitno, da gre za vpliv z bazami bogate talne vode, oziroma mezece povirne vode. Ta rastišca so vedno na blagih pobocjih (3–10°). Na osnovi naših popisov smo kot dobre razliko­valnevrste,oziromaznacilnicedefiniralinaslednje bazifilne vrste: Carex davalliana, C. hostiana, Epipactis palustris, Koeleria pyramidata, Erio­phorum latifolium. V preteklosti so tovrstne popise razlicno klasificirali in jih združevali v sintakson Molini­etum caeruleae s.l., s popisi drugih sintaksonov z modro stožko. Na sliki 1 vidimo, da se popisi obravnavane asociacije jasno razlikujejo od osta­lih. Zelo podobni našim popisom, so sestoji, ki jihnavajataBalátová-Tulácková&Hübl(1985a) z avstrijskih apneniških Alp in sestoji iz severne Italije (Sburlino in sod. 1995). Junco-Molinietum caeruleae Preising1951ex Klapp 1954:Tlavtejzdružbisopogostooglejena, oziroma so šotnata. Ta asociacija je pogosta v obmocjih,kjerprevladujenekarbonatnapodlaga, oziroma na rastišcih, kjer je zaradi upocasnjene razgradnje nastala debela plast organskih snovi. V Sloveniji je ta asociacija pogosta predvsem na Ljubljanskem barju. Sestoje iz te asociacije smo popisali na lokacijah Nemška vas, Log pri Mokronogu in Selo pri Bledu, kjer so tla pod vplivom z bazami revne talne vode, oziroma zastajajoce deževnice. Združba je floristicno slabo definirana. Kot razlikovalniceodostalihstožkovijnavajamovrsti Juncus conglomeratus in J. effusus. V tipicno subasociacijo smo uvrstili pet popisov(Tabela2,popisi17–21),vkaterihnismo našli diferencialnih vrst. Zaradi dominance vrste Succisella inflexa vkotanjah,smodefiniralinovo subasociacijo succiselletosum inflexae. Junco-Molinietum caeruleae succiselletosum inflexae subass. nova hoc loco: Tabela 2, popisi 9–16; holotip: tabela 2 / popis 13. V izrazitejših ulekninah dlje casa zastaja deževnica, zato v teh sestojih z visoko pokrovnostjo uspeva tudi vrsta Succisella inflexa, ki je znacilnica zveze Des-champsion.Bistvenarazlikavfloristicnisestavije v uspevanju in dominanci vrste Molinia caerulea. Zaradiomenjenihekstremnihrazmervspodnjem zelišcnem sloju prevladujeta vrsti Succisella in-flexa in Carex panicea, v zgornjem pa dominira vrsta Molinia caerulea. Poleg teh, se ostale vrste le izjemoma pojavljajo z višjimi pokrovnostmi, število vrst pa je na splošno mnogo manjše kot v ostalih stožkovjih. Tovrstne sestoje smonašli na obmocju alpsko­dinarskepregrade,kjerjekolicinapadavinnajvecja v Sloveniji, in sicer na lokalitetah Nemška vas v Ribniški dolini in Selo pri Bledu. Slovenija je na sticišcu panonske, dinarske, alpske in sredozemske regije, kar se odraža tudi v pestrosti travniške vegetacije. VSlovenijismo References takonašliasociacije,kisobileopisanevNemciji, v Italiji in na Slovaškem, uspevajo pa tudi na Hrvaškem,naMadžarskem,vAvstriji,naCeškem, na Poljskem. Acknowledgments IwouldliketothankProf.Dr.AndrejMartincic and Assist.Prof.Dr. Andraž Carni for help and revision of this text, which originated as a part of my PhD. thesis. Balátová-Tulácková, E., Hübl, E., 1985a. 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Dissertation, University of Ljubljana (Slovenia), Ljubljana, 196 pp. Zelnik,I.,Carni,A.,2008a.WetmeadowsoftheallianceMolinionKoch1926andtheirenvironmental gradients in Slovenia. Biologia, 63, 187–196. Zelnik, I., Carni, A. 2008b. Distribution of plant communities, ecological strategy types and diversity along a moisture gradient. Community ecology, 9, 1–9. Zupancic,B.,1995.KlimatografijaSlovenije.Kolicinapadavin:obdobje1961–1990.Hidrometerološki zavod Slovenije, Ljubljana. Appendix: List of the relevé localities (from Tables 2 and 3): Plantagini altissimae-Molinietum: 1: 0251/1 Mrzlek; 2:0251/1 Mrzlek; 3:0251/1 Mrzlek; 4:0251/1 Zagon; 5: 0251/1 Mrzlek; 6:0251/1 Mrzlek; 7: 0251/1 Mrzlek; 8: 0251/1 Zagon. Junco-Molinietum caeruleae: 9: 0254/3Nemškavas; 10: 0254/3 Nemška vas; 11: 0254/3 Nemška vas; 12: 0254/3 Nemška vas; 13: 9650/4 Selo pri Bledu; 14: 9650/4 Selo pri Bledu;15: 9650/4 Selo pri Bledu; 16: 0254/3 Nemška vas; 17: 0254/3 Nemška vas; 18: 0254/3 Nemška vas; 19: 0254/3 Nemška vas; 20: 0056/4 Log pri Mo-kronogu; 21: 0056/4 Log pri Mokronogu. Selino-Molinietum caeruleae: 22: 0054/3 Radensko polje; 23: 0054/3 Radensko polje; 24: 0254/3 Nemška vas; 25:0054/3Radensko polje; 26: 9650/2 Podhom;27: 9955/2 Sopota;28: 0054/3 Radensko polje;29: Dobje; 30: 9556/1 Radoše; 31: 9650/2 Podhom; 32: 0054/3 Radensko polje; 33: 9752/1 Tatinec; 34:0054/3Radensko polje; 35: 0054/3 Radensko polje; 36: 0054/3 Radensko polje; 37: 0054/3 Radensko polje. Carici davallianae-Molinietum caeruleae: 38: 0054/3 Radensko polje; 39:9557/3 Dolic;40:9955/2 Sopota;41: 9557/3 Dolic;42:9557/3 Dolic; 43:9557/3 Dolic; 44:9955/2 Sopota; 45: 9955/2 Sopota; 46: 9955/2 Sopota; 47: 9955/2 Sopota; 48: 9955/2 Sopota; 49: 9955/2 Sopota; 50: 9955/2 Sopota; 51: 9557/3 Dolic; 52: 0056/3 Dol pri Trebnjem; 53: 9752/1 Tatinec; 54: 9752/1 Tatinec; 55: 9752/1 Tatinec; 56: 9752/1 Tatinec; 57: 9752/1 Tatinec; 58: 9752/1 Tatinec; 59: 9752/1 Tatinec. 66 Acta Biologica Slovenica, 54 (2), 2011 Community number Number of relevés Number of plant taxa 1 8 47 2 8 23 3 5 37 4 17 39 5 21 33 Plantagini altissimae-Molinietum caeruleae Sanguisorba officinalis 100 .... Plantago altissima 100 38.6 . Gratiola officinalis 100 622029 . Centaurea carniolica 100 . . 1819 Cirsium rivulare 75 12406 5 Serratula tinctoria 75 12 . 125 Ranunculus flammula 62 25206 . Carex nigra 50 .20. . Junco-Molinietum caeruleae succiselletosum inflexae subass. nova Succisella inflexa 25 100 . 29 . Junco-Molinietum caeruleae typicum Juncus conglomeratus 100 50 60 35 5 Juncus effusus 25 . 60 6. Selino-Molinietum caeruleae Selinum carvifolia 75 . 20 76 . Laserpitium prutenicum ... 71 . Carici davallianae-Molinietum caeruleae Carex davalliana . . . 12 95 Carex hostiana 12 50 20 71 90 Epipactis palustris . 12 . 6 90 Koeleria pyramidata . . . 24 76 Eriophorum latifolium . . . 6 52 Molinion Molinia caerulea subsp. caerulea 100 100 100 100 100 Succisa pratensis 75 12 60 94 95 Gentiana pneumonanthe 38 62 60 65 . Galium boreale 50 . 20 59 24 Carex distans 12 25 . 12 24 Molinia arundinacea . . 40 12 10 Inula salicina . 38 . . 5 Iris sibirica 12 . . 18 . Gladiolus palustris . . . 6 10 Carex tomentosa . . . 18 . Gentiana asclepiadea . . . 6 5 Polygala amarella . . . . 10 Ophioglossum vulgatum 12 . . . . Vicia tetrasperma . . 20 . . Molinietalia Filipendula ulmaria 88 88 100 53 19 Lythrum salicaria 62 100 100 29 33 Valeriana dioica 75 38 60 41 67 Lysimachia vulgaris 38 100 80 35 24 Equisetum palustre 38 88 40 18 62 Juncus acutiflorus 88 50 60 18 . Betonica officinalis 88 . 40 71 48 Angelica sylvestris 38 . 20 35 57 Deschampsia cespitosa 38 . 40 18 5 Genista tinctoria 12 . 20 53 29 Lychnis flos-cuculi 88 . 60 6 . Linum catharticum 25 . . 29 33 Thalictrum lucidum . 88 20 6 . Myosotis scorpioides 88 12 . 12 . Gymnadenia conopsea . . . 6 52 Lotus pedunculatus . 12 . 12 24 Senecio aquaticus agg. 50 38 . . . Cirsium palustre . . . . 29 Cirsium oleraceum . . . 29 33 Geum rivale . . . 6 24 Cardamine pratensis agg. 12 . 20 12 . Galium uliginosum . . . 6 14 Peucedanum coriaceum 38 . . . . Crepis paludosa 25 . . 6 . Dactylorhiza maculata 25 . . 6 . Colchicum autumnale 12 . . 12 . Veratrum album . . . 12 5 Hypericum tetrapterum . . 20 . 5 Scirpus sylvaticus . . . 6 5 Allium angulosum . . . 6 . Caltha palustris . . . 6 . Table 1: Shortened percentage synoptic table of the studied wet meadows plant communities with Molinia caerulea: 1 – Plantagini altissimae-Molinietum caeruleae, 2 – Junco-Molinietum caeruleae succiselletosum inflexae 3 – Junco-Molinietum caeruleae typicum, 4 – Selino-Molinietum caeruleae, 5 – Carici davallianae-Molinietum caeruleae. Tabela 1: Skrajšana sinopticna tabela (prisotnost v %) preucevanih rastlinskih združb mokrotnih travnikov z vrsto Molinia caerulea: Zelnik: Wet meadows with Molinia caerulea in Slovenia relevé number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 relevé code 163170162164168169165166 178173174175208209210180 181171172147148 year 04 04 04 04 04 04 04 04 04 04 04 04 04 04 04 04 04 04 04 04 04 month 7 7 7 7 7 7 7 7 8 8 8 8 8 8 8 8 8 8 8 6 6 day 1 29 1 1 6 29 1 1 12 3 3 3 20 24 24 12 12 3 3 11 11 plot size (m2) 15 0 24 18 20 24 18 18 24 20 20 24 20 18 20 20 20 24 20 20 18 altitude (m) 520520520520520520520520 482482482482425425425483 485490485240240 number of taxa 29 42 29 43 46 54 69 61 31 30 21 14 17 16 15 39 45 37 42 29 30 characteristic and differential taxa of the association Plantagini altissimae-Molinietum caeruleae Sanguisorba officinalis + 1 3 3 3 2 2 2 . . . . . . . . . . . . . Plantago altissima 3 3 3 + 2 4 + + + + . . . . . r . . . . . Gratiola officinalis + 1 2 + 3 3 + 1 3 3 + + . . . 1 2 . . . . Centaurea carniolica + + 1 + 1 1 2 + . . . . . . . . . . . . . Cirsium rivulare . . + + + + 1 + + . . . . . . . 1 . 1 . . Serratula tinctoria + + + + + + . . . + . . . . . . . . . . . Ranunculus flammula 2 4 + . + + . . . 3 . . . . . + . . . . 1 Carex nigra + 1 . . . + + . . . . . . . . . . . . . + characteristic species of the association Junco-Molinietum caeruleae Juncus conglomeratus + 3 1 + + 3 1 1 + + + . . . . + + 2 3 . . Juncus effusus . + . . . + . . . . . . . . . . . . + + 1 differential species of the subassociation succiselletosum inflexae subass. nova Succisella inflexa 4 . . . . . + . 4 3 3 1 3 2 3 3 . . . . . characteristic species of the association Selino-Molinietum caeruleae Selinum carvifolia . . 1 1 1 1 2 + . . . . . . . . . . 3 . . characteristic and differential species of the association Carici davallianae-Molinietum caeruleae Carex hostiana . + . . . . . . 1 . . . + + . 2 + . . . . Epipactis palustris . . . . . . . . . . . . . . . + . . . . . Molinion Molinia caerulea 1 2 4 5 5 3 4 3 4 4 5 5 4 5 5 4 4 4 4 5 4 Succisa pratensis . + . + + + 1 + + . . . . . . . 1 + 1 . . Gentiana pneumonanthe + . 1 . + . . . + + + + . . . + + + + . . Galium boreale . . + . 3 + + . . . . . . . . . . + . . . Carex distans . . . + . . . . + . . . . . . + . . . . . Molinia arundinacea . . . . . . . . . . . . . . . . . 1 + . . Inula salicina . . . . . . . . . . . . 2 + . + . . . . . Iris sibirica . . . . . . + . . . . . . . . . . . . . . Ophioglossum vulgatum . . . . . + . . . . . . . . . . . . . . . Vicia tetrasperma . . . . . . . . . . . . . . . . . . . + . Molinietalia Juncus acutiflorus 3 2 . + 2 2 + 1 + + 1 . . . . + 3 3 2 . . Valeriana dioica + 1 1 + + 1 . . 2 . + . . . . 2 2 1 . . + Filipendula ulmaria + . + 2 1 1 + + + + 1 2 . + + + + + 2 + + Betonica officinalis . + + 1 + + 3 3 . . . . . . . . + . + . . Lythrum salicaria 1 + . + . 1 + . 1 + + 1 + + 1 + + 2 1 + 1 Equisetum palustre . + . . + 1 . . . + + 1 1 3 2 + . . . + 2 Lysimachia vulgaris . 1 . + . + . . + + 1 1 1 2 2 + . + + 2 + Angelica sylvestris . . . . . + + + . . . . . . . . . . 1 . . Genista tinctoria . . . . + . . . . . . . . . . . + . . . . Linum catharticum . . . + + . . . . . . . . . . . . . . . . Lychnis flos-cuculi + + . + + + + + . . . . . . . . . + + + . Myosotis scorpioides + 1 + + + + + . . + . . . . . . . . . . . Deschampsia cespitosa . . . . . 1 + 1 . . . . . . . . + . . + . Thalictrum lucidum . . . . . . . . + . + + + + + + + . . . . Lotus pedunculatus . . . . . . . . . 2 . . . . . . . . . . . Senecio aquaticus agg. 1 + . + + . . . + 2 . . . . . 1 . . . . . Cardamine pratensis agg. . + . . . . . . . . . . . . . . . . + . . Peucedanum coriaceum . . 1 . + . + . . . . . . . . . . . . . . Crepis paludosa . . + + . . . . . . . . . . . . . . . . . Dactylorhiza maculata . . . + + . . . . . . . . . . . . . . . . Colchicum autumnale . . . . . . . + . . . . . . . . . . . . . Hypericum tetrapterum . . . . . . . . . . . . . . . . . . . + . Molinio-Arrhenatheretea Ranunculus acris 1 + 1 1 1 1 1 2 1 + + . . . . 2 2 + + 1 2 Prunella vulgaris 1 + . 1 + 3 3 1 2 3 . . + + . 1 + . . + . Lotus corniculatus + . . + 1 + + + + . . . + . . + 1 . . . . Leontodon danubialis . . . . 1 . . . . . . . . . . + 2 . . . . Centaurea jacea + . + . + . 1 + . . . . + . . . + . . . . Centaurea macroptilon . . . . + . + . . . . . . . . + + . . . + Holcus lanatus + + . + + 1 1 2 . . . . . . . . + + + 2 2 Lathyrus pratensis . . . 2 + 1 1 2 r . . . . . . . + + 1 . . Festuca pratensis . + . + . + 2 2 . + . . . . . . + . . + + Ajuga reptans . + . . . + 1 1 . . . . . . . . . 2 1 + . Dactylis glomerata . . . . . + + 1 . . . . . . . . . . . . . Leucanthemum ircutianum . . + . + 1 1 + . . . . . . . . . . . . + Plantago lanceolata . . . . . . + + . . . . . . . . + . . . + Ranunculus repens 2 3 . 2 . + 3 + 2 2 + . . . . + + r + . + Trifolium pratense . . . + . 1 + + . . . . . . . . r . . . . Anthoxanthum odoratum . + . . . + + + . . . . . . . . . . + 2 2 Vicia cracca . . . + . . + + . . . . . + . . . 1 + . . Poa pratensis . . . . . + + + . . . . . . . . . + + . . Rumex acetosa . . . . . . + + . . . . . . . . . r + + + Agrostis stolonifera agg. . + . . . + . . . . . . . . 1 . . . . . . Mentha x verticillata . . . . . . . . 1 + + . . . . + + . . . . Galium mollugo agg. . . . . . . . + . . . . . . . . . + 1 1 . Cynosurus cristatus . . . . . . 1 + . . . . . . . . . . . . + Taraxacum officinale agg. . . . . . . . . . . . . . . . + . . . . . 68 Acta Biologica Slovenica, 54 (2), 2011 Trifolium repens . + . 1 + . . + . . . . . . . . . . . . . Trifolium dubium . + . . + . + . . . . . . . . . . . . . . Festuca rubra agg. . . . . . + + 1 . . . . . . . . + . . . . Cerastium holosteoides . . . . . . + + . . . . . . . . . . . 2 + Festuca nigrescens . . . . . . . . . . . . . . . . . . . 1 . Carex hirta . . . . . . . . . . . . . . . . . . 1 . . Achillea millefolium . . . . . . + + . . . . . . . . . . . . . Trifolium patens . . . + . . . . . + . . . . . . . . . . . Plantago major . . . . . + + . . . . . . . . . . . . . . Poa trivialis . . . . . . . . . . . . . . . . . . . 2 1 Lotus tenuis . . . . . . . . . . . . . . . + . . . . . Leucanthemum praecox . . . . . . + . . . . . . . . . . . . . . Alopecurus pratensis . . . . . . + . . . . . . . . . . . . . . Avenula pubescens . . . . . . + . . . . . . . . . . . . . . Stellaria graminea . . . . . . + . . . . . . . . . . . . . . Scheuchzerio-Caricetea fuscae Carex panicea 4 4 3 1 1 3 2 1 3 4 2 1 4 3 3 4 3 3 2 + 1 Parnassia palustris . . . . . . . . + . . . . . . + . . . . . Juncus articulatus . 2 . . . . . . . . . . . . . . . . . . . Agrostis canina . + + . + . 1 + . + . . . . . . . + . . . Carex flava . . + + . . + + . + . . . . . . . . . . + Taraxacum palustre . . . . + . . . . . . . . . . + . . . . . Dactylorhiza incarnata . . + . + + . + . . . . . . . + + . . . . Carex lepidocarpa . . . . + . . . . + . . . . . . . + . . + Orchis palustris . . . . . . . . . . . . . . . . . . . + 1 Festuco-Brometea Galium verum . . . . . . . . . . . . . . . . . . + . . Briza media . . . . . . + + . . . . . . . . + . . . . Filipendula vulgaris . . . . . . . + . . . . . . . . . . . . . Allium carinatum + . 1 + 1 1 + + . . . . . . . . . . . . . Carex flacca . . . . . . + . . . . . . . . . . . . . . Brachypodium pinnatum agg. . . . . . . . . . . . . . . . . . . 1 . . Trifolium montanum . . . . . . + 1 . . . . . . . . . . . . . Bromus erectus . . . . . . . + . . . . . . . . . . . . . Trifolium ochroleucon . . . . + . + . . . . . . . . . . . . . . Calluno-Ulicetea Potentilla erecta + + 3 2 3 2 1 2 1 + 1 + 1 . . 1 2 1 3 3 1 Danthonia decumbens . + . . . + 1 + . . . . . . . + + . . . . Festuca filiformis . . . . . . + + . . . . . . . . + . . . . Carex pallescens . . . + . . + + . . . . . . . . . . + . . Thymus pulegioides . . . . . . . + . . . . . . . . . . . . . Hieracium umbellatum . . . . . . . . . . . . . . . . + . + . . Luzula campestris agg. . . . . . . + + . . . . . . . . . . . . . Calluna vulgaris . . . . . . . . . . . . . . . . + . . . . Viola canina . . . . . . . . . . 1 . . . . . . . . . . Nardus stricta . . . . . . . . . . . . . . . . 1 . . . . Rhinanthus glacialis . . . . . . . + . . . . . . . . . . . . . Cuscuta epithymum . . . . . . + . . . . . . . . . . . . . . Phragmito-Magnocaricetea Galium palustre + 1 + 1 + + . + + + + + 2 1 1 1 + + + + 1 Carex acutiformis . 1 . . . + . . . . . . . . . . . + . . . Mentha aquatica + 1 + + + + . . . . . + + + 1 1 . . . 1 1 Carex acuta . . . + . . + + . . . . . . + . . . . 1 2 Lycopus europaeus . + . . . + + . + . 1 + . . . . . . . . . Phragmites australis . . . . . . . . . . . . 1 1 2 . . . . . . Carex elata . 2 . . . . . . . . . . . . . . . . . . . Iris pseudacorus . . . . . . . . . . . . . + . . . . . . . Carex otrubae . . . + . . . + . . . . . . . . . . . . . Scutellaria galericulata Late-successional taxa Veronica chamaedrys . . . . . . . . . . . . . + . + . . . . . . . . . . . . + . . . . . + + . . . Table 2: Analytical table 1 . . of the plant associations: Frangula alnus Cruciata glabra Alnus glutinosa . . . . . . . . . . . . . . . . . . . + . . + . r . . . . . + . . . . . . . . . . . . . . r . . r . r + . . + . . (1) – Plantagini altissimae-+ . . . . . Molinietum caeruleae, Quercus robur Solidago gigantea Salix cinerea . . . . . . . . . . . . . . . . . + . . . . . . . . . . . + . . . . . . . . r . . . . . + . . . . + r + . . + . . (2) – Junco-Molinietum . . . . . . caeruleae succiselletosum Salix repens / rosmarinifolia Anemone nemorosa Epilobium parviflorum . . . . . . . . . . . . . . . . . . . + . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 . . 1 . . + . . inflexae, (3) – Junco-Molini-. . . + . . etum caeruleae typicum. Veronica officinalis . . . . . . . . . . . . . . . . . + . . . Viburnum opulus Lathyrus linifolius . . . . . . . . . . . . . . . . r . . . . . . . . . . . . . . . . . 1 . . . . + . . Tabela 2: Analiticna tabela Tilia cordata Euonymus europaeus Other taxa . . . . . . . . . . . . . . . . . + . . . . . . . . . . . . . . . . + . + . . asociacij: (1) – Plantagini . . . altissimae-Molinietum Equisetum arvense . . . . . . . . . . . . . . . + . . . . . caeruleae, (2) – Junco-Mo- Erigeron annuus Elymus repens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + . + . . . . linietum caeruleae succisel- Calamagrostis epigejos . . . . . + . 1 . . . . . . . . . . . . . letosum inflexae, Rubus sp. Sisyrinchium bermudiana agg. . . . . . . . . . . . . . . . . . . . 1 . . . . . . . . . . . 3 . + + . . . . . . . (3) – Junco-Molinietum Equisetum ramosissimum . . . . . . . . . . . . . . . . . . . + 2 caeruleae typicum. Zelnik: Wet meadows with Molinia caerulea in Slovenia relevé number 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 relevé code 1061071799119923111721419811211372969798101 1352713313613725262829303132134867686970717475 year 03 03 04 03 04 03 03 03 04 04 03 03 03 03 03 03 03 04 03 04 04 04 03 03 03 03 03 03 03 04 03 03 03 03 03 03 03 03 month 8 8 8 7 8 8 7 8 8 8 7 7 6 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 6 6 6 6 6 6 6 6 day 18 18 12 2 20 21 2 25 5 20 2 2 30 12 12 13 13 5 21 5 5 5 21 21 21 27 27 27 27 5 27 30 30 30 30 30 30 30 plot size (m2) 18 24 24 24 20 18 20 15 18 20 20 24 24 20 20 18 16 25 20 20 24 24 16 18 20 16 16 20 20 20 24 24 20 24 20 24 20 30 322322altitude (m) 483323535685323350490540322322436323323323323 530684530530530680678680680678685685530265437436436436436435435 number of taxa 42 37 42 46 46 37 50 38 61 29 22 32 39 30 31 44 37 26 29 26 23 28 28 29 46 43 34 45 49 29 21 35 35 50 37 34 22 21 characteristic and differential taxa of the association Plantagini altissimae-Molinietum caeruleae Plantago altissima . . . . + . . . . . . . . . . . . Gratiola officinalis 3 4 1 3 . . + . . . . . . . . . . Centaurea carniolica . . . + . . + . . . . . . . . + . Cirsium rivulare . . 1 . . . . . . . . . . . . . . Serratula tinctoria . . . . . . . . . . . . . + + . . Ranunculus flammula . + . . . . . . . . . . . . . . . characteristic species of the association Junco-Molinietum caeruleae Juncus conglomeratus + + + . . 1 . . . . . + . . + . . Juncus effusus . . . . . + . . . . . . . . . . . differential species of the subassociation succiselletosum inflexae subass. nova Succisella inflexa + + . + . . . . . . . + . . . + . characteristic species of the association Selino-Molinietum caeruleae Selinum carvifolia + + 3 + . + + 2 3 . . + . 3 3 2 3 Laserpitium prutenicum + + . . r . . + 1 2 + + . 3 2 2 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + + + . . . . . . . . + . . . . . . + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . characteristic and differential species of the association Carici davallianae-Molinietum caeruleae Carex davalliana . . . . . . . 1 1 . . . . . . . . 4 2 3 3 3 + + 3 2 1 2 1 1 + 2 . + 1 3 5 5 Carex hostiana + + 1 + . . 1 + . 1 2 1 1 . + + . 1 3 3 2 2 . + + 1 . 2 1 1 4 3 4 2 2 3 1 2 Epipactis palustris . . . . + . . . . . . . . . . . . 2 + 1 + 1 + + 2 2 . + 1 r . + + + + 1 1 1 Koeleria pyramidata . . . . . . + . + . . . 1 . . + . . + + . . + + + + . + . + + 2 1 2 1 1 + + Eriophorum latifolium . . . . . . . . + . . . . . . . . + + . + + . . . + + + + . . + . . . 1 . + Molinion Molinia caerulea 4 5 4 3 4 5 4 5 4 4 5 5 5 4 4 4 4 4 4 4 4 4 5 4 5 5 4 4 5 5 4 4 4 5 5 5 4 5 Succisa pratensis 1 + 1 4 + 1 4 2 1 + + 1 + 2 1 . + 2 2 3 3 3 + 2 + 1 + . + 1 1 + + + + 1 + + Gentiana pneumonanthe + + 1 + . . + . . . 1 + . + + + + . . . . . . . . . . . . . . . . . . . . . Galium boreale + 2 + . 2 . . . + 3 . + + . . + + . . . . . . . . . . . . . . . 3 3 2 + + . Carex distans . . + . . . . . . . . . + . . . . . . . . . . . . . . + + . . 1 . + + . . . Molinia arundinacea . . . . + . . + . . . . . . . . . . . . . . . . . . . . + . . . . + . . . . Inula salicina . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + . . . Iris sibirica . . . . . . . . . . + . . . . + + . . . . . . . . . . . . . . . . . . . . . Gladiolus palustris . . . . . . . . . . . . + . . . . . . . . . . . . . . . . . . . + 1 . . . . Carex tomentosa . . + . . . . . . . . . . + . . 1 . . . . . . . . . . . . . . . . . . . . . Gentiana asclepiadea . . . . . . . . + . . . . . . . . . . . . . . . . . . . + . . . . . . . . . Polygala amarella . . . . . . . . . . . . . . . . . . . . . . . + + . . . . . . . . . . . . . Molinietalia Valeriana dioica 1 . 1 . 2 1 . + 1 . . . . . . + . 1 1 1 3 + + 1 1 2 3 1 2 + . . . . . . + . Filipendula ulmaria + + 2 + + . . . . . + + . . . + + r . . . + + . . . . . . + . . . . . . . . Betonica officinalis + + . 1 . . 2 . . 2 1 2 2 3 3 4 2 . . . . . . . + . . + . . + 1 1 3 2 + + + Lythrum salicaria . + + . + + . . + . . . . . . . . + + . . . . + . + . . + . . + . . . . + . Equisetum palustre . . . . . 1 . 2 3 . . . . . . . . + 2 2 4 3 2 2 + 2 3 + 3 + . . . . . . . . Lysimachia vulgaris + + + . r . . . . . . + . . . + . + . . . r . . . . + . + . . 1 . . . . . . Angelica sylvestris + + + . . 1 . . . . + + . . . . . r + + + . 1 + + + + 1 + 1 . . . . . . . . Genista tinctoria . . . 1 . . 2 . . + . 1 1 1 1 1 + . . . . . . . . . . . . . . + 2 + 1 1 . + Linum catharticum . . . + . . + . + + . . . + . . . . + + . . . + + + . + . . . + . . . . . . Cirsium oleraceum . . . . 2 2 . + + + . . . . . . . . . . . . . . . . . . + . . + + + 1 . + + Gymnadenia conopsea . . . . . . . . . . . . + . . . . . + + . + + 1 + + . + + . . . . + + . . . Lychnis flos-cuculi . . . . . . . . . . . . . . . + . . . . . . . . . . . . . . . . . . . . . . Myosotis scorpioides . . . . . + . + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Deschampsia cespitosa . . . 1 + . . . . . . . . . . + . . . . . . . . . . . . . . . . + . . . . . Thalictrum lucidum . . + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lotus pedunculatus . . . . + . . . . + . . . . . . . . . + . + . . . . . . . . . + . + . + . . Cirsium palustre . . . . . . . . . . . . . . . . . + . . + . . . . + + + . + . . . . . . . . Geum rivale . . . . . 3 . . . . . . . . . . . . . . . . . . 1 . + + + + . . . . . . . . Juncus acutiflorus + + 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cardamine pratensis agg. . . . . . + . . . . . . . . . + . . . . . . . . . . . . . . . . . . . . . . Galium uliginosum . . . . . . . . . . + . . . . . . + . . + + . . . . . . . . . . . . . . . . Crepis paludosa . . . . + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Dactylorhiza maculata . . . . . . . . + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Colchicum autumnale . . . . + + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Veratrum album . . . . . . . . . 1 . . + . . . . . . . . + . . . . . . . . . . . . . . . . Hypericum tetrapterum . . . . . . . . . . . . . . . . . . . . . . . . . . . . + . . . . . . . . . Scirpus sylvaticus . . . . . 1 . . . . . . . . . . . . . . . . . . + . . . . . . . . . . . . . Allium angulosum . . . + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Caltha palustris . . . . + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Molinio-Arrhenatheretea Ranunculus acris + + + 1 2 1 1 + 1 1 + + 1 1 + + + + 2 2 . 2 3 4 2 2 3 1 1 1 + 2 2 1 1 1 + 1 Prunella vulgaris . . + 1 + . 1 . 2 . . . 1 . . . + + + + . r + + 1 . . + + + . 1 1 . + + . . Lotus corniculatus + + + 1 . . 1 . + . . + 2 + + . + . + . . . 1 . + + . + 1 . + 2 1 1 1 1 + . Leontodon danubialis . + + 1 + . + . 1 1 . . 2 . . . . + + + + 2 . . + + 1 + + + 1 3 2 1 3 2 . + Centaurea jacea 1 + + 2 + + 1 + + r + + . . + 1 + . . . . . . . + + . + + . . . . . . . . . Centaurea macroptilon 1 + + 2 . . 2 . 1 . 1 1 . . + + + . . + . . . . + + . + + + + . . . . . . . Holcus lanatus + + . 1 + + 1 + . . + . . . . + . . . . . . . . . . . . . + . . . . . . . . Lathyrus pratensis . . + . 1 2 + + + . . . . . . . . . . . . . + 1 + . 2 + + + . . . . . . . . Festuca pratensis . . . 1 + + + . + . + . . . . . . . . + . . . . + . . . + + + + . . . . . . Ajuga reptans + + . . + . . . + . . . . + . . . . . . . . . . + + + 1 + . . + + . . . . . Dactylis glomerata . . . . + . + . + + . . . . + . + . . . . . . . + + . + + . + 1 + 1 1 + . . Leucanthemum ircutianum . . . + . . . . . . . . + . . . . . + . . . + + + + + + + . . . . + . + . . Acta Biologica Slovenica, 54 (2), 2011 Plantago lanceolata + . . . . . + . . . . . + . . . . . . . . . . + . + + . . + . + + + + + + + Ranunculus repens + . + . . 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Trifolium pratense . . . + + . + . + . . . + . . . . . . . . . . + + . . + . . . . . . . . . . Anthoxanthum odoratum . . . + . . 1 . . . . + . . . . . . . . . . . . . . + . . + . . . . . . . . Vicia cracca . . . . + + . + . . . . . . . . 1 . . . . . . . . . . . + . . . . . . . . . Poa pratensis . . + . . + . . + . . . . + . + + . . . . . . . . . . . . . . . . . . . . . Rumex acetosa . . . . . + . . . + . . . . . + . . . . . . . . . . + . . . . . . . . . . . Agrostis stolonifera agg. . . . . . . . . . . . . . + + + . . . . . . . . . . . . . . . . + + + . . . Mentha x verticillata 1 + + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Festuca rubra . + . 1 . . 2 . . . . 1 . + + + + . . . . . . . . . . . . . . . . . . . . . Galium mollugo agg. . . . . . + . + + . . . . . . . . . . . . . . . . . . . . 3 . . . . . . . . Cynosurus cristatus + + . + . . + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Leontodon hispidus . . . + . . + . + . . . . . . . . . . + + + . . . . + . . . . . . . . . . . Taraxacum officinale agg. . . . . + + . . . . . . . . . . . . . . . . + . . . . . . . . . . . . + . . Trifolium dubium . + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Festuca nigrescens . . . + . . + . . . . + . . . . . . . . . . . . . . . . . . . . . . . . . . Carex hirta . . . . . . + . . . . . . . . . + . . . . . . . . . . . . . . . . + . . . . Festuca arundinacea . . . . . + . . . . . . . . . . . . . . . . . . . . . . . . . + + + . . . . Galium album . . . . . . . . . . . . . . . . . . . . . . + . + + . 1 . . . . . . . . . . Euphrasia rostkoviana . . . . . . . . . . . . . . . . . . . . . . . . + + + + . . . . . . . . . . Achillea millefolium . . . . . + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Juncus inflexus . . . . . . . . + . . . . . . . . . . . . . . . . . . . + . . . . + . . . . Pimpinella major . . . . . . . . + . . . . . . . . . . . . . . . . . . . . + . . . . . . . . Rhinanthus minor . . . . . . . . + . . . . . . . . . . . . . . . . . . . . . . . + . . . . . Daucus carota + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Centaurea jacea agg. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + . . . . . . . . Arrhenatherum elatius . . . . . . . . + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Potentilla reptans . . . . + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Tragopogon orientalis . . . . . . . . . . . . + . . . . . . . . . . . . . . . . . . . . . . . . . Scheuchzerio-Caricetea fuscae Carex panicea 2 1 2 + 3 . 1 1 3 2 1 . + 1 + + . 3 1 2 3 3 1 1 1 2 2 2 1 2 + . . + + 1 + + Parnassia palustris . . + . + . . + . + . . . . . . . 2 2 2 2 3 + + + 1 + 2 1 1 . . . . . . + + Juncus articulatus . . . . . . . + + . . . . . . . . + + + + + . . . + + . + . 3 + . . . . . . Agrostis canina 1 2 . + . . 1 . . . + + . . . . . . . . . . . . . . . . . . . . . . . . . . Carex flava + . + . . 1 . + + . . . . . . . . . . . . . . . . . . . + . . . . . . . . . Taraxacum palustre . . . . . . . . . + . . . . . . . . . . . . . . . . . . . . . . 1 . 1 + 1 + Carex pulicaris + . . 2 . . 1 . . . 2 2 . + 1 + . . . . . . . . . . . . . . . . . . . . . . Carex lepidocarpa . . . . . . . . . . . . . . . . . . + . . . . . . . . . . . . . . . . . . . Orchis palustris . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + . . + . Juncus alpinoarticulatus . . . . . . . . . . . . . . . . . + . 1 + + . . . . . . . . . . . . . . . . Menyanthes trifoliata . . . . . . . . . . . . . . . . . + . . 3 + . . . . . . . . . . . . . . . . Epilobium palustre . . . . . . . . . . . . . . . . . . . . . . . . . . + . . . . . . . . . . . Carex echinata . . . . . . . + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Tofieldia calyculata . . . . . . . . + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Schoenus nigricans . . . . . . . . . 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . Primula farinosa . . . . . . . . . + . . . . . . . . . . . . . . . . . . . . . . . . . . . . Festuco-Brometea Galium verum + . + + + . 2 . + 1 . + 2 1 2 3 1 . . . . . . . 1 + . 1 . . . 1 2 2 . . . . Briza media . . . 1 . + 2 . + . . + . + + + + + + 1 + + . + + + + + + 1 + 1 + + + + . . Filipendula vulgaris . . . 1 . . 1 . . . . . 2 1 + + + . . . . . . . . . . . . . 1 + 2 1 + + . . Allium carinatum . . + + . . + . + . . . + . . . . . . . . . . . + . . . . . + + 1 + + + . + Centaurea pannonica . . . . . . . . . . . . 1 . . . . . . . . . . . . . . . . . . . + 2 + . . . Carex flacca . . . + . . . . . . . . 1 + + . 1 . . . . . . . . + . + . . + . + 1 + + + + Pimpinella saxifraga . . . . + . . . . . . . . . . . + . . . . . . . . . . . . . . . . . . . . . Brachypodium pinnatum agg. . . . . . . + . 1 2 . . . . . . . . . . . . . . + + . . + + . + + 1 + + . . Trifolium montanum . . . + . . + . + + + + + + + . + . . . . . . . . . . . . . + . . + + . . . Campanula glomerata . . . + . . . . . . . . + . . . . . . . . . . . . . . . . . . . . + . . . . Sedum sexangulare . . . . . . + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Plantago media . . . . r . . . . . . . . . . . . . + . . . . . + + . . . . . + . . + + . . Prunella grandiflora . . . . . . . . + 2 . . 1 . . . . . . . . . . . . . . . . . . . . 3 + + . . Gentiana verna . . . . . . . . . . . . . . . . . . . . . . . + + . . + . . . . . . . . . . Ononis spinosa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 + . . . Buphthalmum salicifolium . . . . . . . . . . . . + . . . . . . . . . . . . . . . . . . . . + . . . . Danthonia alpina . . . . . . . . . . . . 1 . . . . . . . . . . . . . . . . . . . . . . . . . Calluno-Ulicetea Potentilla erecta 2 . 3 3 3 1 2 3 3 3 2 2 2 3 3 4 3 1 3 3 3 3 3 3 3 3 1 3 3 3 2 3 3 2 2 3 2 3 Danthonia decumbens + + . . . . 3 . . + . + 1 + + + . . . . . . . . . . . . . . + . + + . . . . Festuca filiformis + . + 1 . . 1 . . . . 2 + 1 1 + + . . . . . . . . . . . . . . + . . . + . . Carex pallescens . . + + . + + + . . . . . + + + . . . . . . . . . . . . . . . . . . . . . . Thymus pulegioides . . . . + . . . + + . . + . . + . . . . . . . . . + . + + . . . . + . . . . Hieracium umbellatum + + . . . . . . . . . + . 1 + + 1 . . . . . . . . . . . . . . . . . . . . . Luzula campestris agg. + + . + . . + . . . . . . . . + . . . . . . . . . . . . . . . . . . . . . . Calluna vulgaris + . . . . . + . . . . + . + . + + . . . . . . . . . . . . . . . . . . . . . Viola canina . . 2 + . . + . . . . . + . . + . . . . . . . . . . . . . . . . . + . . . . Nardus stricta + . . 2 . . 2 . . . . . . . + 1 . . . . . . . . . . . . . . . . . . . . . . Polygala vulgaris . . . . . . + . . . . . . . . . . . . + . . . . . . + . . . . . . . . . . . Hieracium lactucella . . . . . . + . . . . . . . . . . . . . . . . + . . + . . . . . . . . . . . Rhinanthus glacialis . . . . . . . . . . . . . . . . . . . . . . . . . + . . . . . . . . . . . . Agrostis capillaris . . . . . . . . . . . . . . . . + . . . . . . . . . . . . . . . . . . . . . Phragmito-Magnocaricetea Galium palustre + + + . . + . + . . + . . + . + . . . . . . . . . . . . . . . . . . . . . . Mentha aquatica . . . . . . . + + . . . . . . . . 1 . . 2 . . . . + . . 1 . . . . . + + . 1 Carex acuta . . . . . . . + . . . . . . . . . . . . . + . . . . . + . . . . . . . . . . Lycopus europaeus . + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Carex vesicaria . . . . . . . 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Carex vulpina . + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Phragmites australis . . . . . . . 2 . + . . . . + . . . . . . . . . . . . . . . . 2 1 1 2 1 + 2 Peucedanum palustre . . . . . . . . . . . . 2 . . . . . . . . . . . . . . . . . . + + + + + . . Carex elata . . . . . . . . . . . . . . . . . . . . . . . . . + . . . . . . . . . . . . Zelnik: Wet meadows with Molinia caerulea in Slovenia Iris pseudacorus + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Carex paniculata . . . . . . . . + . . . . . . . . . . . . . . . . . . . + . . . . . . . . . Late-successional taxa Veronica chamaedrys . . . . . + . . . . . . . . . + . . . . . . . . . . + + . . . . . . . . . . Frangula alnus + + + . . . . + r . + + . . . . + . + . . . . . + . . . + . . . . . . . . . Cruciata glabra . . . . + + . + + . . . . . . . . . . . . . . . + + 1 + + . . . . . . . . . Alnus glutinosa . . + . . . . . + . . . . . . . . . . . . . 1 + + + + + + . . . . . . . + . Quercus robur . . . . + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . r . . Knautia drymeia . . . . . . . . + . . . . . . . + . . . . . + . + + 1 + 1 . . . . . . . . . Salix cinerea . . + . 1 . . . 1 . . . . . . . . . . . . + . . . . . . . . . . . . . . . . Salix repens / rosmarinifolia . . + . . . . . . . . . . . . . . . + . . 3 . . . + . . . . . . . . . . . . Eupatorium cannabinum . . . . . . . + + . . . . . . . . + + . + . . . . . . + . . . . . . . . . . Fraxinus excelsior . . . . r . . . + . . . . . . . . . . . r . . . . . . . . . . . . . . . . . Epilobium parviflorum . . . . . . . + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pinus sylvestris . . . . . . . + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Gymnadenia odoratissima . . . . . . . . . . . . . . . . . . . . . . + + . . . + . . . . + . . . . . Viburnum opulus . . . . . . . . . . . . . . . . . . . . . . . . . . . . + . . . . . . . . . Phyteuma spicatum . . . . + . . . + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Laserpitium latifolium . . . . . . . . . . . . . . . . . . . . . . + + + . . . . . . . . . . . . . Calamagrostis arundinacea . . . . . . . . . . . . . . . . . . + . . . . . . . . . + . . . . . . . . . Asarum europaeum . . . . . . . . r . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Carpinus betulus . . . . + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lathyrus linifolius . . . . . . . . . . . . . . . + . . . . . . . . . . . . . . . . . . . . . . Primula vulgaris . . . . + . . . + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Erica carnea . . . . . . . . . . . . . . . . . . . . . . . . . . . + + . . . . . . . . . Lathyrus latifolius . . . . . . . . . . . . . . . . + . . . . . . . . . . . . . . . . . . . . . Cornus sanguinea . . . . . . . . + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Solidago virgaurea . . . . . . . . . . . . . . . . . . . . . . 1 . . . . . . . . . . . . . . . Other taxa Equisetum arvense . . . . + . + . . . . . + . . . . . . . . . . . . . . . . . . . . + . . . . Chaerophyllum sp. . . . . . + . . + . . . . . . . . . . . . . . . + . . . . . . . . . . . . . Scabiosa triandra . . . . . . . . . . . . + . . . . . . . . . + . + 1 . + . . . . . + . . . . Betula pendula . . . . . . . + + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Picea abies . . . . . . . . . . . . . . . . + . . . . . + . . . + . . . . . . . . . . . Populus tremula . . . . . . . + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Euphorbia sp. . . . . . . . + . . . . + . . . . . . . . . . . . . . . . . . . . . . . . . Viola sp. . . . . . . . + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Alchemilla sp. . . . . + + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Danthonia sp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + + . . . . Salix purpurea . . . . . . . . + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Rosa sp. . . . . . . . . . . . . . . . . + . . . . . . . . . . . . . . . . . . . . . Corylus avellana . . . . . . . . . . . . . . . . . . . . . . . . . . . . + . . . . . . . . . Prunella sp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + . . . . Orobanche sp. . . . + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Table 3: Analyticalcomparable table of the plant associations: (4) – Selino-Molinietum caeruleae, (5) – Carici davallianae-Molinietum caeruleae. Tabela 3:Analiticna tabela asociacij: (4) – Selino-Molinietum caeruleae, (5) – Carici davallianae-Molinietum caeruleae. Status and distribution of the lynx (Lynx lynx) in the Swiss Alps 2005–2009 Status in razširjenost risa (Lynx lynx) v Švicarskih Alpah 2005–2009 *aFridolin Zimmermann, aAnja Molinari-Jobin, aAndreas Ryser, aChristine Breitenmoser-Wsten, aElias Pesenti, bUrs Breitenmoser aKORA,Thunstrasse 31, CH-3074 Muri, Switzerland Thunstrasse 31, CH-3074 Muri, Switzerland bInstitute of Veterinary Virology, University of Bern, Länggassstrasse 122, CH-3012 Bern, Switzerland *correspondence: f.zimmermann@kora.ch Abstract: We evaluated the status of lynx in the Swiss Alps for the period 2005– 2009. Even though the number of lynx presence signs remained almost stable between the present (2,068 signs) and previous pentad (2,091), there was a 7.6% increase in the areaoccupiedbythe5-kmcircularbuffersaroundtheconfirmedlynxsignsofpresence over the five years period (12,637 km2). The north-western Swiss Alps (VI) remained the compartment with the highest number of chance observations. It was followed by compartments central Switzerland west (III) and north-eastern Switzerland (II). These sub-populations acted as source in the current pentad, as signs of reproduction were reported almost every year. The translocation to north-eastern Switzerland is still the only significant contribution to the spatial increase of the lynx range in the last 10 years in the Swiss Alps. The small and vulnerable north-eastern Switzerland lynx sub-population plays an important role for the Alpine population. There is hope that in the future this sub-population could act as stepping stone to the eastern Alps and together with individuals dispersing from the central Switzerland west (III) sub­population would enable to found a new sub-population in central Switzerland east (IV). The status of the sub-population in the Valais (VII) is less clear. As only few signs of reproduction and mortalities were reported over the pentad, it acted more as sink than a source population. From the few signs of lynx presence reported in the remaining compartments (Grisons V, central Switzerland east IV and Ticino VIII) we concluded that only a few single lynx that did not yet establish the typical social organisation occur there. An occupancy-based population estimate from a parallel study resulted in about 111 (SE = 10) independent lynx for the period 2005–2009. This is higher than the 60–90 individuals estimated for the previous pentad. Keywords: Alps, distribution, Lynx lynx, monitoring, status, Switzerland Izvlecek:Prispevek ocenjuje stanje risa v Švicarskih alpah za obdobje 2005–2009. Ceprav je številcnost znakov prisotnosti risa med sedanjim (2068) in prejšnjim (2091) pet-letnim obdobjem ostala stabilna, se je obmocje skupaj s 5-km pufersko cono povecalo za 7,6 %. SZ Švicarske alpe (IV) tako ostajajo obmocje z najvišjim številom opažanj. Sledita obmocji osrednje Z Švice (III) in SV Švice (II). Omenjene sub-populacije so bile vir opažanj za zadnje pet-letno obdobje, saj so biliznaki repro­dukcije prisotni skoraj vsako leto. Širjenje na obmocje SVŠvice je edino prostorsko povecanjearealavŠvicarskihalpahvzadnjih10letih.MajhneinranljiveSVšvicarske sub-populacije risa imajo pomembno vlogo za vzdrževanje risa v Švicarskih alpah. Ostaja upanje, da bo ta populacija odigrala vlogo odskocne deske do VAlp in skupaj s posameznimi osebki, ki prihajajo iz osrednje švicarske »sub-populacije (III)« in bo omogocilanastaneknovesub-populacijevosrednjiVŠvici(IV).Stanjesub-populacije na obmocju Valais (VII) je manj jasno. Ker je za zadnje pet-letno obdobje znanih le malo znakov reprodukcije in smrtnosti, predstavlja bolj ponor kot vir. Na podlagi znakov risove prisotnosti na ostalih obmocjih (Grisons (V), osrednja V Švica (IV) in Ticino (VIII)) smo zakljucili, da se tam pojavljajo posamicni osebki, ki se še niso povezali v populacijsko strukturo. Po ocenah modela zasedenosti prostora (occupancy models) iz vzporedne študije ocenjujejo 111 (SE = 10) neodvisnih osebkov za obdobje 2005–2009. To je precej vec kot 60–90 osebkov ocenjenih za prejšnjo petletko. Kljucne besede: Alpe, razširjenost, Lynx lynx, monitoring, status, Švica Introduction Two lynx (Lynx lynx) populations are cur­rently present in the Alps originating from the reintroduction done in the 1970s. One lies in the western Alps in Switzerland and one in the Slovenian Alps, expanding into Italy and Austria. Switzerland harbours the most vital sub-population (Molinari-Jobin et al. 2010) and thus has a great responsibility regarding the conservation of the Alpine lynx population which has still to be considered as endangered according to the IUCN Red List criteria. Forty years after the first reintroduction, less than 20% of the whole suitable habitat in theAlps has been recolonized by the species, despite consid­erable efforts at the national and international level to expand the existing lynx areas. In Switzerland large parts of central Switzerland east, the Grisons, and Ticino are not yet colonized by lynx (Zim­mermann et al. 2010a). Lynx experts from theAlps considered illegal killing, habitat fragmentation and anthropogenic accidents to be the main reasons for this slow expansion (Molinari-Jobin et al. 2010). InSwitzerlandthemainconflictiswithhunters who compete with lynx for game, and who fear that high lynx densities diminish ungulate game (Breitenmoser et al. 2010). A side effect of this conflict is that the local authorities become reluc­tant to actively conserve lynx. Apossible solution proposed by hunters’ associations and wildlife managers, may be a controlled legal harvest to supress illegal killing. In parallel, this solution might increase the willingness of new cantons to actively reintroduce lynx as this new legislation would give them the rights to intervene under some circumstances. The Swiss Lynx Concept established in 2000 and updated in 2004 defines the general conservation and management goals, the co-operation between the FOEN and the can­tons. Besides the removal of stock raiders and the translocation from high density areas to areas not yet colonized to foster the spatial expansion of the lynx population, the Swiss Lynx Concept foresees that lynx are reduced through controlled hunting, if the impact of the lynx predation on roe deer and chamois is considered too strong. However this needs a revision of the hunting ordinance. The consultation of the hunting ordinance ended in October 10th 2011. By mid-2012 the Federal Council will adopt the report of the consultation and the ordinance. To counterbalance the slow expansion of the lynx population in Switzerland, six lynx were translocated in 2001 from the north-western Alps to north-eastern Switzerland. Another three from the Jura Mts. followed in 2003. All animals were fit with radio-collars in order to follow their move­ments, reproduction events and mortalities. In Switzerland the translocation of a total of 6 lynx to the north-east in 2001–2003 (Ryser et al. 2004) led to an increase of 7% of the lynx distribution range in the whole Alps (Molinari-Jobin et al. 2010). The monitoring conducted in winter 2005/06 in north-eastern Switzerland revealed that the lynx number was critically low (Ryser et al. 2006). Subsequently, in 2006 the north-eastern Swiss cantons and the FOEN based on recommendations from the program KORA decided to restock the north-eastern Switzerland lynx sub-population with additional 3–4 individuals that should mainly originate from the Jura Mts. to increase the chance of mixing up the genes of both meta-populations (Jura Mts. and Alps). In 2007, one male and one female lynx were translocated from the Jura Mts. and north-western Swiss Alps, respectively. Another female was translocated in 2008 from the Jura Mts. (KORA unpublished data). Similarly to the 2001 and 2003 translocations all individuals were fit with radio-collars to monitor the fate of individualsduringthefirstyearsaftertheirrelease. The translocation project ended in 2009 and was since then integrated into the national monitoring. The purpose of this report is to evaluate the status of lynx in the Swiss Alps for the period 2005–2009. Material and methods For organizational purposes, Switzerland was divided into 8 large carnivore management compartments, taking into account natural and artificial barriers to natural spread of lynx as well as political borders (Fig. 1). We used a stratified approach to monitor the lynx population (Breiten­moseral.2006)asfinancialresourcesarerestricted. There is a stratification in space (national level, compartments and smaller reference areas within compartments), in time (e.g. chance observations are gathered year round whereas systematic camera-trapping, which is very labor intensive, is conducted every 2 to 3 years in smaller reference areas) and in the datasets according to the type of observation and their validity (e.g. SCALP criteria; Molinari-Jobin et al. in press). On the national level questionnaires are sent on yearly bases to all game wardens of Switzerland (Capt et al. 1998). These questionnaires provide basic information about the detection/non detection of lynx, mortality, and reproduction as well as a subjective assessment of the trend of the lynx “population” within each game warden’s surveil­lance area over the whole Switzerland. Chance observations (sightings, tracks, wildlife killed) are gathered year round at the national and compart­ment level. Livestock killed by lynx need to be confirmed by trained people to be compensated, mainly game wardens. All damages to livestock reported are published online on our webpage. This allows an open review when permission for removal of an individual lynx as stockraider is issued by the cantons of the corresponding com­partment and the FOEN. Opportunistic camera-trapping, where camera-traps are set on ideal occasions principally at fresh kills, is conducted at the compartment level. At a smaller scale in reference areas (680–1,601 km2) within three large carnivore compartments (II, III and VI) we estimated the number of lynx using photographic capture-recapture models (e.g. Zimmermann et al. 2010b). These data are reported each year in our national large carnivore monitoring reports (e.g. Zimmermann et al. 2010a) to make this information available to the members of the lynx monitoring network, the decision makers, the NGOs and the general public. On the national level, five sources of infor­mation on the presence of lynx are available: (1) reports of lynx killed or found dead, or young orphaned lynx caught and put into captivity; (2) opportunistic camera trapping where camera-traps are set for ideal occasion, mainly at fresh kills; (3) samples confirmed by means of genetic analysis; (4) records of livestock killed by lynx; and (5) chance observations of wild prey remains, tracks, scats, sightings, and vocalisations. Three levels of reliability were distinguished according to the possibility to verify an observation (Molinari-Jobin et al. in press): Category 1 (C1) represent the hard facts (i.e. direct signs), e.g. all reports of lynx killed, found dead or removed from the wild as young orphaned lynx and put into captivity, as well as opportunistic photographs of lynx. We also include all samples that were identified to be lynx by means of genetic analysis in this category. All lynx photographs of one or more individuals taken at a kill were counted as a single detection. Lynx photographs taken at a given site along a trail were counted as single detection for each night even though several lynx were pictured the same night (this happened only on rare occasions). Category 2 (C2) represent all records of livestock killed,wildpreyremainsandtracksconfirmedby trained people, e.g. mainly game wardens. As all game wardens were instructed how to recognize signs of lynx presence, these records are mostly an objective proof of lynx presence, though both errors and even deception may occur. Category Results 3 (C3) represent chance observations of all wild prey remains and tracks reported by the public as The number of signs of presence recorded in well as all sightings, scats and vocalisations, e.g. theSwissAlpsfrom2005–2009(2,068)remained mainly indirect signs that can hardly be verified. stable compared to the previous pentad (2,091; The information about reproduction came from Molinari-Jobin et al. 2006). Signs of presence three different data sets: chance observations (C1–C3) were reported from all compartments, of juvenile lynx, photographs of juvenile lynx the fewest in the compartment Ticino (VIII) with during the opportunistic camera-trapping and 8 and the most in the north-western Swiss Alps juvenile lynx found dead or captured as orphans (VI) with 966 (Fig. 1). Intermediate values were for removal from the wild. found in the remaining compartments (345 in To be able to compare the spatial range of compartment II, 424 in III, 70 in IV, 68 in V, and the lynx population for the pentad 2005–2009 187 in VII). with those reported in previous status reports A total of 155 damages to livestock were (Molinari-Jobin et al. 2006), we computed two reported (Table 1), which is less than one fourth different measures of the spatial range: (1) the of the number reported for the previous pentad minimum convex polygon (MCP) encompassing (543). On the other hand, the number of wild prey all signs of presence belonging to category 2; and remains reported almost doubled (801 compared (2) a circular buffer of 5-km around the C2 signs to 449 in the previous pentad). of presence, resulting in an area of about 80 km˛ With 30 the number of lynx found dead or around each confirmed sign of presence. This removed from the wild remained almost stable area corresponds roughly to an average female compared to the previous pentad. Most losses oc-lynx home range size in the Alps (Breitenmoser-curredinthenorth-westernSwissAlps(16)andin Wsten et al., 2001). central Switzerland west (11), followed by north­ eastern Switzerland (2) and the Grisons (1). Table 1: Number of lynx records collected per year and category from The signs of reproduction CATEGORY 1 2005 2006 2007 2008 2009 Total showed almost the same pattern Photo Dead lynx Genetic sample Total CATEGORY 2 Livestock killed Wild prey remains Tracks 22 3 25 31 136 50 23 4 2 29 28 119 54 41 4 1 46 47 148 58 42 9 2 53 21 170 60 48 10 1 59 28 228 70 176 30 6 212 155 801 292 as the reported lynx mortali­ties (Fig. 2). The largest part of signs of reproduction came from compartments VI (32), II (32) and III (23). The remain­ing, all unconfirmed signs ex­cept one, came from the Valais (VII) with two, from central Switzerland east (IV) with one Total 217 201 253 251 326 1,248 and from the Grisons with two CATEGORY 3 of which one was a confirmed Wild prey remains Tracks 6 10 11 18 15 15 9 8 13 5 54 56 sign of reproduction, although it came from a juvenile lynx Sightings Vocalisations Scats Total 82 1 2 101 87 3 2 121 87 12 1 130 97 5 3 122 113 2 1 134 466 23 9 608 originating from north-eastern Switzerland (II) that died in Grisons during its dispersal. As in the previous pentad, 71% of the signs of presence be- Total all categories 343 351 429 426 519 2,068 long to the C1 and C2 category and thus have been confirmed. C1 signs of presence consider­ 2005–2009. ably increased in north-eastern Tabela 1: Število zbranih podatkov o znakih prisotnosti risa po letih in kategoriji v obdobju 2005–2009. and central Switzerland west Figure 1: Distribution of lynx signs of presence in Switzerland for the five-year period 2005 to 2009. (a) Category 1 data: dead lynx or lynx removed from the wild = stars, photos = squares and genetic proof = dots. (b) Category 2 data: killed livestock = dots, confirmed wild prey remains and tracks = triangles. (c) Category 3 data: unconfirmed wild prey remains and tracks, sightings, vocalisation and scats = triangles. The roman numbers refer to the management compartments (I = Jura Mountains, not considered here, II = North­eastern Switzerland, III = Central Switzerland west, IV = Central Switzerland east, V = Grisons, VI = North-western Swiss Alps, VII = Valais and VIII = Ticino). Slika 1: Razporeditev znakov prisotnosti risa v Švici za petletno obdobje 2005–2009. (a) Kategorija podatkov C1: mrtvi risi alirisi odvzeti iz narave = zvezde, fotografije = kvadrati in genetski dokazi = pike. (b) Kategorija podatkov C2: napadi na drobnico = pike, potrjeni ostanki naravnega plena in sledi = trikotniki. (c) Kategorija podatkov C3: ne potrjeni ostanki naravnega plena, sledi, opažanja, oglašanja in iztrebki risa = trikotniki. Rimske številke se nanašajo na upravljalske oddelke (I = Jura, tukaj ni obravnavano, II = severovzhodna Švica, III = osrednja Švica – zahod, IV = osrednja Švica – vzhod, V = Graubden, VI = severozahodne švicarske Alpe, VII = Valais in VIII = Ticino). compared to the previous pentad. C1 category data disappeared completely from western Grisons but in parallel some hard facts appeared for the first time in the northern part of the Grisons close to compartment II, where lynx were translocated. Category 2 signs of presence are more sparsely distributed in the western part of the Grisons comparedtothepreviouspentad.Forthefirsttime in central Switzerland west (III) some confirmed signs of presence (C2) were recorded South to the Napf region. The MCP encompassing all C2 signs of lynx presence increased from 20,166 km2 in 2000–2004 to 27,487 km2 in 2005–2009. The 5-km buffer around the C2 data resulted in a range estimate of 12,637 km2 compared to 11,736 km2 for the previous pentad (2000–2004). Discussion Development of lynx signs of presence About 47% of lynx signs of presence that were reported for this pentad stem from the north-western Swiss Alps (VI) although this compartment contains only about 18% of the suitable lynx habitat in the Swiss Alps (based on 10x10-km cells containing = 10% of suitable habitat fragment > 50 km2; Zimmermann et al. in prep.). It is followed by compartments central Switzerland west and north-eastern Switzerland with 20.5% and 17%, respectively although the suitably habitat in these compartments makes up only 8% and 11.7% of the suitable lynx habitat, respectively. Each of the remaining compartments Figure 2: Information about reproduction from three different sources: chance observations = tri­angles, dead lynx or lynx removed from the wild = stars and opportunistic camera-trapping = squares. Slika 2: Informacije o reprodukciji iz treh razlicnih virov: nakljucna opažanja = trikotniki, mrtvi risi ali risi odvzeti iz narave = zvezde in opor­ tunisticni posnetki s foto-pastmi = kvadrati. Table captions. (IV, V, VII and VIII) contained less than 10% of chance observations that were reported over the five year period although the suitable habitat within these compartments makes up 9% to 25% of the suitable lynx habitat. In the previous pentad (2000–2004) signs of reproduction were mainly reported in compart­ments north-western SwissAlps (VI) followed by central Switzerland west (III) and the Valais (VII) with very few signs. In north-eastern Switzerland (II), where lynx were translocated since 2001, signs of reproduction were only reported for the year 2003. In the current pentad (2005–2009) juvenile lynx were observed and reported every year in compartments north-western Swiss Alps (VI), north-eastern Switzerland (II) and central Swit­zerland west (III). In the Valais (VII) reproduction was only reported in 2006 and 2007. Even though a juvenile lynx was found dead in the Grisons in 2008, this lynx originated from north-eastern Switzerland (II) as revealed by genetic analyses (Breitenmoser-Würsten2009).In2009forthefirst time an isolated sighting of a juvenile lynx (C3) was reported in the southern Grisons (V) close to the border with the canton of Ticino (VIII). This needshowevertobeconfirmedinthenextpentad. In 2003 compartment central Switzerland east (IV) faced immigration of female AIKA that was translocated to north-eastern Switzerland (Ryser et al. 2004). Even though female AIKA was still present in this compartment in 2009, when she was photographed by a camera-trap set along a trail, no signs of reproduction were documented from 2005 to 2009 in the area known to be occu­pied by this female from the radio-telemetry and camera-trapping studies indicating a lack of males in this area. The only sign of reproduction that was reported in 2009 is an unconfirmed sighting of a juvenile lynx that was located at the south-western corner of the compartment nearby the border with central Switzerland west (III). Mortality showed almost the same spatial pattern as reproduction. In pentad 2000–2004 lynx found dead or removed from the popula­tion were reported every year only in compart­ment north-western Swiss Alps. In compartment north-eastern Switzerland they were reported in 2003 in 2004 and in the Valais only in 2004. In the current pentad, with the exception of the Valais from where no mortality was reported, mortality events were additionally reported in compartments central Switzerland west and the Grisons. Although the juvenile lynx found dead in the Grisons originated from the north-eastern Switzerland lynx sub-population (Breitenmoser-Wsten 2009). The damages to livestock in the current pentad make only one fourth of those reported in the pen-tad 2000–2004 and were as in the previous pentad mainly located in the north-western Swiss Alps. The possible reasons for this decrease are twofold. First, efficient prevention measures were imple­mented in the hot spots where damages occurred regularly in the past in the north-western Swiss Alps. Second the roe deer numbers have increased in the north-western Swiss Alps in recent years according to the observations reported by game wardens. Therefore lynx do not need to switch to sheep as they find enough wild ungulates to prey on (Breitenmoser et al. 2010). Even though the number of lynx presence signs slightly decreased between both pentads, the areas covered by the MCP encompassing all C2 data (27,487 km2) and the 5-km circular buffer around the C2 data (12,637 km2) increased by about 36% and 7.6%, respectively. The MCP of the C2 data is about two times larger than the 5-km buffered C2 lynx signs of presence. This discrepancy is due to the strong fragmentation of the Alps by both artificial and natural barriers. As a consequence the suitable lynx habitat in the Swiss Alps has a patchy distribution (Zimmermann 2004). The MCP approach is not suitable to measure the absolute spatial expansion of the lynx population in this fragmented mountain range as it contains large parts of unoccupied or unsuitable habitat that will never be occupied by lynx. Besides, our results highlighted that it is not suitable to measure the relative changes in the spatial distribution as well as it overestimated the rate of spatial change almost by a factor five. To get a more reliable estimation of the »real« area occupied by the lynx in the fragmented Alpine habitat we buffered the C2 data with a 5-km radius since the last status report (2000–2004; Molinari-Jobin et al. 2006). Although the area resulting from the buffered C2 data collected over a five years period is closer to the »real« spatial distribution of the Alpine lynx population compared to the MCP approach, it does neither take into account imperfect detection into the estimation of the area occupied by lynx nor any dynamic processes such as colonisation and extinction. To palliate these shortcomings we recently started to use a multiple season site occupancy approach to analyse our lynx presence data (Zimmermann et al. in prep.). Synthesis The north-western Swiss Alps is still the compartment with the highest number of reported lynx presence signs.As signs of reproduction and mortalities were reported every year we can con­clude that the sub-population is functioning well. This compartment is followed by compartments central Switzerland west (III) and north-eastern Switzerland (II) where signs of reproduction and mortalities increased in the last pentad. All three lynx sub-populations acted as source in the cur­rent pentad. The translocation to north-eastern Switzerlandisstilltheonlysignificantcontribution to the spatial increase of the lynx range in the last 10 years in the whole Alps (Molinari-Jobin et al. 2010). With about 8 independent lynx (KORA unpublished data), this small sub-population is however highly vulnerable. In the context of the Alpine population the north-eastern Switzerland sub-population is very important for the future expansion of the lynx, as it could act as stepping stone to the eastern Alps and could enable to fill the gap towards west (compartment IV). During the current pentad it was documented that at least two individuals already left the compartment: sub-adult male B132 showed the longest dispersal ever reported in theAlps and dispersed over more than 200 km to the Trentino (Haller 2009) and a juvenile lynx died while dispersing in the Grisons (V). However such spontaneous migrations are generally far too rare to allow the establishment of a population and these individuals, if they survive their dispersal, remain isolated for years. However when immigration from different directions is pos­sible – as it is currently the case for compartment central Switzerland West (IV) – the chances that several individuals settle down in a lynx-empty area and start to establish the classical social structure andfinallyreproduceareimproved(Zimmermann et al. 2007). The status of the sub-population in the Valais (VII) is less clear.As almost no signs of reproduction and mortalities were reported over the pentad it acted more as sink than a source. In the remaining compartments there are only a few single individuals that did not yet establish a social structure. An occupancy-based population estimate by Zimmermann et al. (in prep.) based on the ratio of population size estimated by means of photographic capture-recapture analyses and occupancy values of occupied range estimated that about 111 (SE=10) independent lynx lived in the Swiss Alps for the period 2005–2009. This is higher than the 60–90 individuals estimated for the previous period. Acknowledgements The KORA was supported by the Federal Of­ fice for the Environment (FOEN). We would like to thank all the state game wardens, hunters and nature lovers as well as the members of the lynx groups who have reported lynx observations for the monitoring program and/or helped with the setting and checking of the camera-traps during the systematic camera-trapping sessions. We are grateful to Hubert Potocnik for helpful comments on earlier version of this manuscript. Literature Breitenmoser, U., Breitenmoser-Wsten, C., Von Arx, M., Zimmermann, F., Ryser, A., Angst, C., Molinari-Jobin, A., Molinari, P., Linnell, J., Siegenthaler, A., Weber, J.-M., 2006. Guidelines for the Monitoring of the Lynx. KORA-Bericht, 33e, 1–31. Breitenmoser, U., Ryser, A., Molinari-Jobin, A., Zimmermann, F., Haller, H., Molinari, P., Breitenmoser- Würsten, Ch., 2010. The changing impact of predation as a source of conflict between hunters and reintroduced lynx in Switzerland. In: Macdonald, D. W, Loveridge, A. J., (eds): Biology and Conservation of Wild Felids, Oxford University Press, Oxford, UK, pp. 493–506. Breitenmoser-Wsten, C., Zimmermann, F., Ryser, A., Capt, S., Laass, J., Siegenthaler, A., Brei­tenmoser, U., 2001. Untersuchungen zur Luchspopulation in den Nordwestalpen der Schweiz 1997–2000. KORA-Bericht, 9d, 1–87. Breitenmoser-Wsten, Ch., 2009. Genetisch Analyse des in Landquart erfahrenen jungen Luch­sweibchens. Technischer Bericht des Projekts KORA, 4pp. Capt, S., Breitenmoser, U., Breitenmoser-Wsten, Ch., 1998. Monitoring of the lynx population in Switzerland. Environmental Encounters, 38, 105–108. Haller, H., 2009. Ein Jungluchs auf Reisen. Cratschla 1/2009, 4–13. Molinari-Jobin, A., Zimmermann, F., Angst, C., Breitenmoser-Wsten, Ch., Capt, S., Breitenmoser, U., 2006. Status and distribution of the lynx in the Swiss Alps 2000–2004. Acta Biologica Slo­venica, 49, 3–11. Molinari-Jobin, A., Marboutin, E., Wölfl, S., Wölfl, M., Molinari. P., Fasel. M., Kos, I., Blazic, M., Breitenmoser-Wrsten, Ch., Fuxjäger, Ch., Huber, T., Izotok, K., Breitenmoser, U., 2010. Recovery of the Alpine lynx Lynx lynx metapopulation. Oryx, 44 (2), 267–275. Molinari-Jobin, A., Kéry, M., Marboutin, E., Molinari, P., Koren, I., Fuxjäger, C., Breitenmoser-Würsten, Ch., Wölfl, S., Fasel, M., Kos, I., Wölfl, M., Breitenmoser, U., in press. Monitoring in the presence of species misidentification: the case of the Eurasian lynx in the Alps. Animal Conservation Ryser, A., Von Wattenwyl, K., Ryser-Degiorgis, M.-P., Willisch, Ch., Zimmermann, F., Breitenmoser, U., 2004. Luchsumsiedlung Nordostschweiz 2001–2003. KORA-Bericht, 22, 1–59. Ryser, A., Von Wattenwyl, K., Zimmermann, F., Breitenmoser, U., 2006. 2. Monitoringbericht LUNO2 Status Luchs Nordostschweiz Winter 2005/2006. KORA-Bericht, 34, 1–18. Zimmermann, F., 2004. Conservation of the Eurasian lynx (Lynx lynx) in a fragmented landscape – habitat models, dispersal, and potential distribution. PhD Thesis, Department of Ecology and Evolution, University of Lausanne, Switzerland. Zimmermann, F., Theus, M., Vogt, K., Ryser, A., Dirac, C., Breitenmoser-Wsten, Ch., Pesenti, E., Breitenmoser U., 2010b.Abundanz und Dichte des Luchses in den Nordwestalpen K-VI im Winter 2009/10. KORA-Bericht, 52, 1–15. Zimmermann, F., Weber, J.-M., Dirac, C., Ryser,A., Breitenmoser-Wrsten, Ch., Capt, S., Breitenmoser, U., 2010a. Monitoring der Raubtiere in der Schweiz 2009. KORA-Bericht, 53, 1–51. Zimmermann, F., Breitenmoser-Wsten, Ch., Breitenmoser, U., 2007. Importance of dispersal for the expansion of an Eurasian lynx (Lynx lynx) population in a fragmented landscape. Oryx, 41, 358–368. Status of the lynx (Lynx lynx) in the German Alps from 2005–2009 Status risa (Lynx lynx) v nemških Alpah v obdobju 2005–2009 Sybille Wölfla* and Manfred Wölflb* aLynx Project Bavaria, Trailling 1a, D – 93462 Lam bBavarian Conservation Agency, Referat 56 – Landschaftspflege und Wildtiermanagement, Hans-Högn-Straße 12, 95030 Hof/Saale *correspondence: info@luchs.bayern.de Abstract: We give a short overview ofthe monitoring results of lynx in the 5-year period 1995–2009. There is no confirmed evidence that there are lynx in the German Alps. Single individuals might have visited the area but signs occur only sporadically. In2008LargeCarnivoreNetworkhasbeenestablishedtoidentifyanddocumentsigns of lynx, wolf and bear. It is the first step to systemize the lynx monitoring. Anatural recolonization of the German Alps is not expected in the near future. Keywords: Lynx lynx, status, monitoring, German Alps Izvlecek:Podanjekratekpregledspremljanjastanjarisavpetihletih(1995–2009). Vtem casu ni potrjenih znakov prisotnosti risa v Nemških alpah. Posamezni primerki verjetno obiskujejo obmocje, vendar se znaki pojavljajo zelo razpršeno. Leta 2008 je bila vzpostavljena »Mreža velikih zveri« za identifikacijo znakov risa, volka in medveda. To je prvi korak k sistemskemu pristopu spremljanja stanja risa. V bližnji prihodnosti ne pricakujemo naravne rekolonizacije risa v Nemških alpah. Kljucne besede: Lynx lynx, status, monitoring, nemške Alpe Introduction Germany shares an area of around 5.000 km2 with the Alpine arc which extendsto 190.000 km2 in total.The nearest lynx(Lynx lynx)sub-populations to the Germans Alps are found in north-eastern Switzerland (distance 70 km) and in Slovenia (dis­tance180km).Anevaluationofapossiblenatural recolonisation of the German Alps concluded a very low probability for establishing a viable populationinthenextdecades(Molinari-Jobinet al. 2010). Even though single dispersers manage to migrate long distances and reach the German Alpsitwillneedaconstantflowoflynxdispersing from other sub-populations. Arecolonisation of the German Alps will be dependent on expand­ing sub-populations of Switzerland, Slovenia or Austria or on re-introduction efforts. Methods Since 2008 a so called Large Carnivore Net­work (LCN) is established to identify possible signs of lynx, wolf (Canis lupus) and bear (Ursus arctos) in the Bavarian Alps. The main focus of the training lies on a thorough documentation to allow verification of signs by experts with long­term experience. Data which cannot be verified (C3:sightings,allundocumentedreportsoftracks, kills, calls) are checked for plausibility and then included in or excluded from the data base and classified according to the SCALP categories (Molinari-Jobin et al. in press). To ensure a con-sistentvalidation,thedataverificationinBavaria is done by two lynx experts who independently evaluatethesigns.Thusthedataarecheckedtwice andtheprobabilityofamisclassificationisreduced. Results and Discussion The report comprised three 5-year periods of lynxmonitoringintheGermanAlps:1995–1999, 2000–2004 and 2005–2009 (Kaczensky 1998, Wölfl&Kaczensky2001,Wölfl2006,thisissue). In the last two periods very few possible lynx signs could be gathered and none of them could be verified or confirmed. Even an assignment to the SCALPcategory C3 seemed daring because of their very imprecise nature. During the 2005–2009 period we could only collect very few data as well (n=5, Table 1), all of them were sightings. Four of the chance observations occurred in the western part of the German Alps, Oberallgäu, in the years 2008 and 2009(Fig.1).Theywereaccompaniedbyrumours endof2009thatalynxhadbeenshotinthatarea. One observation stems from the eastern part of the German Alps, Berchtesgadener Land, and occurred in February 2009. Categories 1995–1999 2000–2004 2005–2009 C1 0 0 0 C2 0 0 0 C3 6 1 5 (all sightings) Total 6 1 5 Table 1: Number of lynx records collected per period percategory(C3:sightings,allundocumented reports of tracks, kills, calls). Tabela 1:Število zbranih podatkov o znakih prisot­nosti risa po obdobjih in po kategorijah (C3: videnja, nedokumentirana opažanja znakov, klicanje). Figure 1: Distribution of lynx signs of presence in the German Alps for the period 2005–2009. Slika 1: Razporeditev znakov prisotnosti risa v nemških Alpah vobdobju 2005–2009. It is noticeable that signs in all three periods (due to the presence of a wolf in 2009–2010) occurred either in the western part (Allgäu) or in couldberelatedtothe»increase«oflynxsigns. the eastern part (Berchtesgadener Land) of the Either this increase is only a function of raised German Alps. It is therefore possible that there awareness or is substantial, will be confirmed aresingledisperserscomingfromSwitzerlandor in the future. Austria/Slovenia. With the establishment and training of the LCN in 2008 we have a network of 25 persons Acknowledgements in the Bavarian Alps whose awareness is focused on large carnivore signs. This means much better The Lynx Project Bavaria is supported by conditions to take notice of signs by checking the Bavarian Ministry of Environment, Bund with local people or even actively looking for Naturschutz in Bayern e.V. (Bavarian society signs. Thus the probability of detecting even for the protection of nature), Landesbund für single lynx should be improved. This assump-VogelschutzinBayerne.V.(BavarianSocietyfor tion is supported by the fact that all lynx signs the protection of birds) and the Wildland Trust of in the 2005–2009 period had been collected by the Bavarian Hunting Association. We thank all members of the LCN. members of the large carnivore network (LCN) However we have to keep in mind that the who have reported lynx observations for the presence of observers and a general raise of monitoring program. The manuscript benefited awarenessforlargecarnivoresbythepublic from a review by A. Molinari-Jobin. References Kaczensky, P., 1998. Present status and distribution of the lynx in the German Alps. Hystrix 10 (1), 39–42. Molinari-Jobin,A.,Kos,I.,Marboutin,E.,MolinariP.,Wölfl,S.,Fasel,M.,Breitenmoser-Würsten,C., Fuxjäger, C., Huber, T., Koren, I., Schmidt, K., Kusak, J., Valdmann, H., Zimmermann, F., Wölfl, M., Breitenmoser, U., 2010. Expansion of the lynx in the Alps. KORA Report No. 50, 17 pp. Molinari-Jobin, A. Kéry, M., Marboutin, E., Molinari, P., Koren, I., Fuxjäger, C., Breitenmoser-Würsten, Ch., Wölfl, S., Fasel, M., Kos, I., Wölfl, M. and Breitenmoser, U. (in press). Monitoring in the presence of species misidentification: the case of the Eurasian lynx in the Alps. Animal Conservation. Wölfl,M.,Kaczensky,P.,2001.PresentstatusanddistributionofthelynxintheGermanAlps.Hystrix 12(2), 39–41. Wölfl, M., 2006. Present status and distribution of the lynx in the German Alps 2000–2004. Acta Biologica Slovenica 49(1), 51–52. The importance of education of future elementary teachers about modern biotechnology issues Pomen izobraževanja bodocih uciteljev razrednega pouka o biotehnologiji Jana Ambrožic-Dolinšek1*, Andrej Šorgo2 1*University of Maribor, Faculty of Education, and Faculty of Natural Sciences and Mathematics, Koroška 160, 2000 Maribor, Slovenia 2University of Maribor, Faculty of Natural Sciences and Mathematics, Koroška 160, 2000 Maribor, Slovenia *correspondence: jana.ambrozic@uni-mb.si Abstract: Thetremendousdevelopmentofscienceandtechnologyhasinfluenced manyaspectsofoureverydaylives,societyandenvironment.Agoodexampleofsuch technology is biotechnology. However, besides its promise, this technology has also raised several controversial issues to which answers are not easily available. With increasing knowledge and applications on one side and controversy on the other the teaching of science is, anything but easy. Development of competencies for these issues, and questions like why, when, and how to integrate modern biotechnology into science education are becoming prominent in the near future. Nowadays, when we are confronted with issues of varying degrees of complexity and importance, it is necessary that teachers at all levels of education have the basic tools to cope with these issues. This is one of reason why we have attempted to establish what kind of knowledge, values and opinions about genetic engineering and genetically modified organisms (GMOs) are characteristic for the students, future Elementary Teachers, at threeSloveneFacultiesofEducation.Wecollectedanswersof360questionnairesfrom pre-service elementary school teachers and analysed their statements from the field of general and classical genetics, modern biotechnology, legislation and the acceptance of different kind of GMOs. Prospective teachers have some knowledge of general and classical genetics and less knowledge about the use of modern biotechnology. They have concerns and fears about different kind of GMOs, mostly negative attitudes towards different kinds of GMOs, or they hold no strong opinions about them. Micro­organisms and plants are generally more acceptable than GM animal. Furthermore, more knowledge does not mean that individual GMOs are more acceptable. Keywords: genetically modified organisms, GMO, students of elementary edu­ cation Abbreviations: GMO – genetically modified organism; GM – genetically mo­dified Izvlecek: Izjemen razvoj znanosti in tehnologije vpliva na številne vidike vsak­danjega življenja posameznika, družbe in okolja. Dober primer tovrstne tehnologije je biotehnologija. Poleg številnih obetov so s to tehnologijo povezana nekatera sporna vprašanja, na katera ni enostavnih odgovorov. Povecevanje znanj in uporabe na eni in polemik, na drugi strani, je razlog, da je poucevanje biotehnologije vse prej kot lahko. Kako usposobiti bodoce ucitelje za obravnavo takih in podobnih tem in zakaj, kdaj in kako vkljuciti sodobno biotehnologijo v izobraževanje postaja pomembno za bližnjo prihodnost.Zatojenujno,dabibiliuciteljinavsehravnehizobraževanjausposobljeni za obravnavo takih in podobnih tem. To je bil tudi eden od razlogov, zakaj smo želeli ugotoviti, kakšno je znanje, kakšne so vrednote in mnenja o genskem inženiringu in gensko spremenjenih organizmih (GSO) študentov, bodocih osnovnošolskih uciteljev treh slovenskih pedagoških fakultet (Univerze v Mariboru, Univerze v Ljubljani, Univerze na Primorskem). Zbrali smo odgovore anketnih vprašalnikov 360 bodocih uciteljev razrednega pouka, v katerihso se bodoci osnovnošolski ucitelji opredelili do trditev s podrocja splošne in klasicne genetike, moderne biotehnologije, zakonodaje ter sprejemanja razlicnih GSO. Bodoci ucitelji razrednega pouka imajo nekaj znanja o splošni in klasicni genetiki in manj znanja o uporabi moderne biotehnologije, veli­kokrat slabo sprejemajo razlicne GSO ali nimajo jasno izraženega mnenja o njih, pri cemer so mikroorganizmi in rastline v splošnem bolj sprejemljivi kot GS živali. Vec znanja nikakor ne pomeni, da so posamezni GSO bolj sprejemljivi. Kljucne besede: genetsko spremenjeni organizmi, GSO, študenti razrednega pouka Okrajšave: GSO – genetsko spremenjen organizem; GS – genetsko spremenjen Introduction The tremendous development of science and technology has influenced many aspects of our everyday lives, society and environment. Agood example of such technology is biotechnology. It is not a recent invention, and humans have used it for centuries. The making of wine, beer, yogurt, cheese and bread, for example, involve ancient biotechnology techniques that have enabled the progress of civilization. Increasing advances in this discipline, such as recombinant DNAtechnology and the manipulation of genes, as well as the introduction of genes into more or less related organism, the same or different plant and animal species or other organism, to obtain genetically modified organisms (GMOs), have produced many powerful applications and have great potential for future discoveries. However, besides its promise, this technology has also raised several controversial issues (food from GMOs, therapeutic and reproductive cloning, surrogate maternity, potential cloning of people, and the potentially harmful influence of GMOs on thehealth of people,animals, other organisms and the environment) to which answers are not easilyavailable.Theconsequenceofsuchissues, called socio-scientific issue (Sadler 2004, Sadler and Zeidler 2005a, Sadler and Zeidler 2005b), is that the transfer of biotechnology discoveries to crop production, industry or medicine is not restricted only by the technological limitations, underdeveloped scientific methods, or modes of scientific reasoning, but also by ethics, morals, faith,theeconomy,environmentalresponsibility, risks, politics, etc. (Christoph et al. 2008, Flores and Tobin 2002, Steward and McLean 2005, Yuntaetal.2005).Withincreasingknowledgeand applications on one side and controversy on the othertheteachingofscienceis,anythingbuteasy (Harms2002).Questionslikewhy,whenandhow to integrate biotechnology into science education will become prominent in the near future. The development of opinions and values is a lifelong process originated in early childhood and influenced by school practice; it is not im­mune to the values, opinions and knowledge of teachers. The formation of values in the case of socio-scientificissuesisnotatthecenterofteacher education,andfutureteachersoftenconstructtheir value system about these issues without relevant professionalfoundations(Ambrožic-Dolinšekand Šorgo2009).Nowadays,whenweareconfronted with issues of varying degrees of complexity and importance, it is necessary that teachers at all levels of education should have the basic tools to cope with them (Ambrozic-Dolinsek and Šorgo 2009, 2010).This is one of reason why we have attempted to establish what kind of knowledge, values and opinions about genetic engineering and genetically modified organisms (GMOs) are characteristic of students, future elementary teachers at three Slovene Faculties of Education: University of Maribor (PeFMb), University of Ljubljana (PeFLj) and University of Primorska (PeFKp).Ourresultscouldpotentiallybeincluded intheundergraduatecurriculumfortheeducation of future and current elementary teachers. Material and methods We collected 360 questionnaires from stu­dents,futureelementaryteachersatthreeSlovene Faculties of Education (University of Maribor (PeFMb), University of Ljubljana (PeFLj) and UniversityofPrimorska(PeFKp))intheacademic year 2007/2008. To find out student teachers’ knowledge and opinion about GMOs, a questionnaire was assembled. The questionnaire was divided into two parts: (1) knowledge, and (2) acceptance about GMO and was completed anonymously. Knowledge concerning genetics, biotechnology and GMO was evaluated through aquestionnaire consisting of 30 true–false statements (Table 1). Teachers had to choose among three options: yes; do not know; no.The correct answer on 17 statements was ‘yes’and on 13 statements ‘no’, a device which prevented guessing. The state­ments could be assigned to general and classical genetics, modern biotechnology and legislation. The reliability of the questionnaire, expressed as Cronbach’s alpha, was 0.827, which can be recognized as good. In Table 1 frequencies and percentagesofcorrect,incorrect,anddonotknow answers are reported. Furthermore we tried to establish the degree of acceptance of different kinds of GMO uses in possible reallifesituations, so weprovided state­ments about various GMOs – microorganisms, plantsandanimals(Table2).AcceptanceofGMOs wasevaluatedwithaclosedquestionnaire,where teachers were asked to choose among 17-items consistingofexistingorpotentially-existentGMOs and in such way to express their opinion about these. We provided three answers: 1- acceptable; 2 – don’t know, do not have an opinion; 3 – not acceptable. The reliability of the questionnaire, expressedasCronbach’salpha,was0.869,which can be recognized as good. Analysis of the results followed three tracks and the statistical package SPSS® 18.0wasusedfor data analysis. Chi-square (.2)statisticswereused to identify differences in frequencies of answers from two general fields: first from the statements from general genetics and the statements from classicandmodernbiotechnologyandlegislation andthesecondfromstatementsaboutacceptance of different kind of GMOs. To correlate their answers, the Pearson correlation coefficient was used. Symbols used in the figures are: ns denote statistically insignificant difference. Results and discussion Futureelementaryschoolteachersfromthree Slovenian universities (University of Maribor, University of Ljubljana, and University of Primorska) do have some basic knowledge of genetics (Table 1). They possess at least some knowledge about classical genetics and know something about genes, their structure, replica­tion, expression and mutations. The majority of them correctly determined 9 among 14 (64.3%) statements, incorrectly determined 2 among 14 (14.3%) statements and do not know 3 among 14 (21.4%) statements. However, we should not be satisfied with observed knowledge. For exam­ple, some of them believe that a cat can fertilize a female rabbit, and they do not know that the broad use of vegetative propagation in plants is a kind of cloning. Thepicturechangedwhentheyhadtochoose the correct statements in the areas of modern biotechnology and legislation. We observed deficiencies in their knowledge about current applications of modern biotechnology, such as transmission of genes between organisms, Table 1: Knowledge of future elementary teachers from three Slovene Faculties of Education about genetically modified organisms. The highest frequencies of answers for individual statement are in bold. Tabela 1:Znanje bodocih uciteljev razrednega pouka s treh Slovenskih pedagoških fakultet. Najvišje frekvence so oznacene s pisavo krepko. YES NO Do not Correct know/ Statement answer empty N% N%N% Knowledge about classical genetics 1 Bacteria have the ability to mutually exchange genes. Yes 52 15.2 46 13.5 243 71.3 3 Deoxyribonucleic acid (DNA) occurs only in genetically No 13 3.8 215 62.9 114 33.3 modified organisms. 4 Bacteria genes from yogurt that can be consumed can be No 45 13.2 119 34.8 178 52.0 incorporated into cells in the human organism. 5 Genes are sequences (of nucleotides) on chromosomes. Yes 183 53.5 42 12.3 117 34.2 6 Genes are not normally transmitted from species to Yes 87 25.4 166 48.5 88 25.8 species in nature. 10 A cat can fertilize a female rabbit; the resulting young No 10 2.9 227 66.4 105 30.7 rabbits have shorter ears. 11 Mutations are the result of cloning. No 105 30.7 58 46.2 79 23.1 12 Mutations are always inherited. No 60 17.5 185 54.1 97 28.4 13 Deoxyribonucleic acid (DNA) is a source of information Yes 190 55.4 15 4.5 132 39.2 for the synthesis of proteins. 18 Propagation of plants by cuttings is cloning. Yes 56 16.5 220 64.7 64 18.8 19 Recessive genes are never expressed. No 18 5.3 85 25.1 236 69.6 22 The sex of the child depends on male sex cells. Yes 223 65.2 79 23.1 40 11.7 25 All mutations are harmful. No 36 10.6 225 66.0 80 23.5 26 Bread rising is a biotechnological process. Yes 102 30.3 87 25.8 148 43.9 Knowledge about current applications of modern biotechnology 2 The vaccine against hepatitis B used to vaccinate all Yes 33 9.6 36 10.5 273 79.8 school children was produced with genetically modified yeast. 7 GM crops are cultivated in Slovenia. No 200 58.7 17 5.0 124 36.4 8 Insulin for treating human diabetes is produced from No 25 7.3 39 11.4 278 81.3 GM (genetically modified) pig and cow pancreata. 9 Products from GMO (genetically modified organisms) Yes 239 70.3 18 5.3 83 24.4 must be labeled as containing GM components. 14 Before application of GM (genetically modified) Yes 229 67.0 11 3.2 102 29.8 plants, it is obligatory to perform a risk assessment about possible harmful influences of GM plants on the health of people, animals (other organisms) and the environment. 15 Reproductive cloning from cells harvested from an No 183 53.5 22 6.4 137 40.1 adult produces an embryo from which develops a child genetically identical to this adult. Ambrožic-Dolinšek, Šorgo: Education of elementary teachers about biotechnology 89 YES NO Do not Statement Correct answer know/ empty N % N % N % 17 Therapeutic cloning from stem cells harvested from an Yes 98 28.7 20 5.8 224 65.5 adult produces several types of cells used for treating diseases or harmful tissues of the same person. 20 Ribonucleic acid (RNA) is a genetically modified form No 29 8.5 147 43.0 166 48.5 of deoxyribonucleic acid (DNA). 21 Slovenia has passed a law dealing with GMOs. Yes 51 8.5 31 43.0 258 48.5 23 Biogas methane from biogas reactors is produced by Yes 39 11.5 20 5.9 280 82.6 bacteria. 24 In Slovenia only GM corn is produced and marked as No 17 5.0 41 12.0 283 83.0 MON 810. 27 The cloning of genes and the cloning of organisms No 41 12.0 67 19.6 234 68.4 require the same methods of work. 28 Stem cells occur in adult humans. Yes 156 45.7 19 5.6 166 48.7 29 Cloning of human embryos is already possible. Yes 192 56.3 52 15.2 97 28.4 30 The transfer of animal genes to plants is possible. Yes 44 12.9 87 25.4 211 61.7 production of medicines with GMOs, cloning of organisms and about GMO legislation, and the cultivating of GM crops in Slovenia. The majority of them correctly determined 5 among 16 (31.0%) statements, incorrectly determined 2 among 16 (12.0%) statements and do not know 9 among 16 (56.2%) statements. Comparisonof»donotknow«with»yes«and »no« statements showed statistically significant higher number of »do not know« statements (.2 = 188.283, h = 4, p > 0.001) about current applica­tionsofmodernbiotechnology,thenaboutclassical genetics. The high percentages of »do not know« answers indicate that they are aware of their insuf­ficient knowledge about modern biotechnology. This could mean that future elementary teachers need additional more biotechnology topics in their education. Schoolpracticeisnotcompletelyimpervious to the knowledge, values, opinions and attitudes of teachers. In other words, teacher’s values, opinions and attitudes can play a certain role in the acceptance of biotechnology issues by school pupils by the whole vertical of compulsory educa-tion.Attitudestowardgeneticmodifiedorganisms among students, future elementary teachers at threeSloveneFacultiesofEducationwerealready evaluated and analysis of their answers reveals uncertainty, distrust and rejection (Ambrožic-Dolinšek and Šorgo 2009). The same is true for acceptanceofdifferentkindofGMOs.Among17 differentkindsofGMOs,only5areacceptableto more than 50% of students; students either find othersnotacceptableorhavenoopinion(Table2). This low level of acceptance again indicates that in most cases, the attitudes of future elementary schoolteachersfromthreeSlovenianuniversities toward GMOs are not positive or they hold no strong opinions about them. In dealing with acceptance, we were able to recognize two patterns. The first one is that GM microorganisms and plants are generally more acceptable than GM animals, which are actually unacceptable.Ourresultsconfirmthatacceptance ofonetypeofGMOdoesnotmeanthatsomeother GMOwillalsobeacceptable(StewardandMcLean 2005). The second pattern is that GMOs not used for food consumption are generally more accept­able if they or their parts cannot be used directly orindirectlyforconsumptionandiftheyproduce somethingrecognizedasusefulforpurposessuch as medicine, bio-fuel, or organic substances, and have the capacity to clean something, or to im­proveresistancetostressconditions.Adropinthe level of acceptance in pairs was observed, where plants tolerant to stress are acceptable to more than half the teachers, while plants manipulated to be tolerant to pests in food production are ac­ceptabletoonlyone-thirdofrespondents.Among plants,thelowestscoresweregiventoornamental plants, a result which can be connected with the level of perceived utility and benefit. Genetically manipulatedanimals,alwaysinthelowerranksof acceptability, are especially unacceptable if they have been manipulated for food consumption. The lowest scores in acceptability were given to geneticallymodifiedviruses.Wecanspeculatethat the answers somehow correlate with knowledge of and attitudes towards viruses as the cause of disease, which is never recognized as useful. In the uncertainty group (do not know; do not have an opinion), there occurred only microorganisms and viruses, which crossed the fifty percentages border. Students cannot decide whether or not manipulatedvirusesandmicroorganismsmodified for production of substances for the food industry andsynthesisoforganicsubstancesareacceptable. An interesting issue is their relation to health. It seemsthat,inthecaseofhealth,GMOplantsand microorganisms could become more acceptable. Whenhumanhealthisatissue,theacceptancelevel of GMOs appears higher, as has also been shown by other studies (Cavanagh et al. 2005). The correlation among knowledge and accept-ancelevelwascalculated.Therewasnocorrelation between knowledge and acceptance (r = 0,052ns). It seems that GMOs acceptance is not connected with more knowledge or more knowledge about geneticsdoesnotautomaticallymeanthatGMOs would be more accepted. Biotechnology is in broader sense the use of livingorganismstosolveproblemsandmakeuseful productsandapplications(ThiemanandPalladino 2009)andintendedtoimprovethequalityofhuman life.Currentlywearewitnessofpublicresistance and skepticism to science, especially to modern biotechnology.Someassignittothelowlevelsof knowledgeofscienceor»scientificallyilliterate« public (Allum et al. 2008) and the importance of introduction of biotechnology in the education at the whole vertical of undergraduate curriculum. Education should starts with introduction of the science behind simply everyday biotechnology practices as making of food stuff like cheese and breadandcontinueswithothermoresophisticated agronomy, food and drink producing practices later continuing with some modern biotechnol­ogy practices. Our study shows that there is no correlation between knowledge and acceptance of GMOs, andtheformerstudies(ŠorgoandAmbrožic2009, 2010) that there it is strong correlation between acceptanceandattitudesagainstGMOs,meaning that attitudes and not knowledge shaped the ac-ceptance.Sosimpleintroductionofbiotechnology, and science behind, by addition of new facts or teacher-provided explanations about ancient and current biotechnological processes does not influ­ence the acceptance. Public resistance and skepticism to science mean that modern biotechnology is not recog­nized only as something beneficial. Especially popular media sometimes present it as a threat, orcontroversialissue,causingconcernsinsociety (Šorgoetal.2011).Schoolsandteachers,asapart of society, mustbeprepared also for dealing with suchsocio-scientificissues and should betrained to developed competences based on active work of pupils such as critical thinking or scientific reasoning of pros and contra. Emotions are especially important part of ele­mentaryeducation(Cagranetal.2008)andcould be important factor in shaping attitudes toward different GMOs ant their acceptability (Šorgo et al. 2011). Emotions related to GMOs are usually negativeandhiddeninconcerns,risk,uncertainty, worry, anger and fear (Šorgo et al. 2011), and the samepatternwasobservedinemotionsexpressed by our future teachers. Negative emotions of future teacher against modern biotechnology, no matter of their origins, would not supported and leadtohigheracceptanceofthistechnology.This also supported the need for early introducing of biotechnology in education, development of posi­tive experiences with biotechnology and also the importance of education of competent future and current elementary teachers. Conclusions The students included in our study have concerns and fears aboutdifferentkind of GMOs and mostly negative attitudes towards different kinds of GMOs, or they hold no strong opinions about them. Only a few of GMOs are accepted by more than half the students. We also observed some knowledge (often severely flawed) about classicalgeneticsandlittleornoknowledgeabout currentapplicationsofmodernbiotechnologyand thelastisnotdifferingfromotherpublics(Allum et al. 2008). The early positive experiences with biotechnology are recommended. Schools and teachers, as a part of society, must be prepared also for dealing with socio-scientific issues. Povzetek Izjemen razvoj znanosti in tehnologije vpliva na številne vidike vsakdanjega življenja posameznika, družbe in okolja. Dober primer tovrstne tehnologije je biotehnologija. Poleg številnih obetov so s to tehnologijo povezana nekateraspornavprašanja,nakateranienostavnih odgovorov. Povecevanje znanja in uporabe na eni ter polemik, na drugi, dela poucevanje bio-tehnologije vse prej kot lahko. Kako usposobiti bodoce ucitelje za obravnavo takih tem in zakaj, kako in kdaj vkljuciti sodobno biotehnologijo v izobraževanje bo postalo pomembno v bližnji prihodnosti.Pomembnoje,dabibiliuciteljivseh ravni izobraževanja usposobljeni za obravnavo takih in podobnih tem. To je bil tudi eden od razlogov, zakaj smo želeli ugotoviti, kakšno je znanje, kakšne so vrednote in mnenja o genskem inženiringu in gensko spremenjenih organizmih (GSO)študentov,bodocihosnovnošolskihuciteljev treh slovenskih pedagoških fakultet (Univerze v Mariboru, Univerze v Ljubljani, Univerze na Primorskem). Zbrali smo odgovore anketnih Literature vprašalnikov 360 bodocih uciteljev razrednega pouka, v katerih so se bodoci osnovnošolski ucitelji opredelili do trditev s podrocja splošne in klasicne genetike, moderne biotehnologije, zakono­daje ter sprejemanja razlicnih vrst GSO. Bodoci ucitelji razrednegapouka imajo kar nekaj znanja osplošniinklasicnigenetiki,cepravzdoseženim ne moremo bitipovsem zadovoljni. Zelo šibko je njihovoznanjeouporabimodernebiotehnologije inznjopovezanozakonodajo.Bodociuciteljizelo slabosprejemajorazlicneGSOalinimajosvojega mnenja o njih. GS mikroorganizmi in rastline so v splošnem bolj sprejemljivi kot GS živali. Pri tem so še posebej nesprejemljive GS živali za hrano. Ko gre za zdravje so GS mikroorganizmi in rastline bolj sprejemljive. Med znanjem in sprejemanjem GSO ni korelacije, kar pomeni, da vecznanjanikakornepomeni,dabodoposamezni GSO bolj sprejemljivi. Acknowledgements WewouldliketothankDr.DarjaSkribeDimec (UniversityofLjubljana),ClaudioBattelliM.Sc. (University of Primorska), Dr. Alenka Lipovec, and Martina Rajšp (University of Maribor) for their help in collecting the data. This research was supported by the Slovene Ministry of Higher Education, Science and Technology within the Biodiversity Research Programme (Grant No. P1-0078). Allum,N.,Sturgis,P.,Tabourazi,D.,Brunton-Smith,I.,2008.Scienceknowledgeandattitudesacross cultures: a meta-analysis. Public Understanding of Science, 17, 35–54. Ambrožic-Dolinšek, J., Šorgo, A., 2009. Odnos študentov razrednega pouka do gensko spremenjenih organizmov (GSO). Acta Biologica Slovenica, 52(2), 21–31. Christoph,I.B.,Bruhn,M.,Roosen,J.,2008.Knowledge,attitudestowardsandacceptabilityofgenetic modification in Germany. Appetite, 51(1), 58–68. Cavanagh, H., Hood, J., Wilkinson, J., 2005. Riverina high school students’views of biotechnology. Electronic Journal of Biotechnology, 8(2), 121–127. [cited 19. 3. 2009]. Available from: http:// www.scielo.cl/pdf/ejb/v8n2/a01.pdf. ISSN: 0717-3458 Cagran, B., Grmek, Ivanuš, M., Štemberger, T., 2009. External differentiation and emotional-personal views of learning. Didactica Slovenica-Pedagoska Obzorja, 24(2), 3–19. Flores,V.S.,Tobin,A.J.,2002.Frankenfoods:Valuesaboutgeneticsembeddedinametaphor.American Biology Teacher, 64(8), 581–586. Harms, U., 2002. Biotechnology Education in Schools. Electronic Journal of Biotechnology [on line], 5(3), 205–211. Available from: http://www.ejbiotechnology.info/content/vol5/issue3/teaching/01/. Retrieved 24. 9. 08. Sadler,T.D., Zeidler, D.L., 2004. The morality of socioscientific issues: Construal and resolution of genetic engineering dilemmas. Science Education, 88(1), 4–27. Sadler,T.D., Zeidler, D.L., 2005a. The significance of content knowledge for informal reasoning re­garding socioscientific issues: Applying geneticknowledge to genetic engineering issues, Science Education, 89, 71–93. Sadler,T.D., Zeidler, D.L., 2005b. Patterns of informal reasoning in the context of socioscientific decision making. Journal of Research in Science Teaching, 42(1), 112–138. Stewart, P.A., McLean,W.P., 2005. Public opinion toward the first, second, and third generations of plant biotechnology. In vitro Cellular Developmental Biology Plant, 41(6), 718–724. Šorgo, A., Ambrožic-Dolinšek, J., 2009. The relationship among knowledge of, attitudes toward and acceptance of genetically modified organisms (GMOs) among Slovenian teachers. Electronic Journal of Biotechnology, 12(3) 1–13. Šorgo, A., Ambrožic-Dolinšek, J., 2010. Knowlege of, attitudes toward, and acceptance of genetically modified organisms among prospective teachers of biology, home economics, and grade school in Slovenia. Biochemistry and molecular biology education, 38(3) 141–150. Šorgo, A., Ambrožic-Dolinšek, J. Tomažic, I., Janžekovic, F., 2011. Emotions expressed toward ge­netically modified organisms among secondary school students and pre.service teachers. Journal of Baltic Science Education, 10(1), 53–64. Yunta,E.R.,Herrera,C.V.,Misseroni,A.,Milla,L.F.,Ooutomuro,D.,Lemus,I.S.,Lues,M.F.,Stepke, F.L., 2005. Attitudes towards Genomic Research in Four Latin American Countries. Electronic Journal of Biotechnology, 8(3), 238 – 248 [cited 24. 9. 2008]. Available from: http://www.ejbio-technology.info/content/vol8/issue3/full/9/BIP/. ISSN: 0717-3458. INSTRUCTIONS FOR AUTHORS 1. 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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 figures in the same article should have the same font type. The figures should be prepared in TIFF, EPS or PDF format, whereby TIFF (ending *.tif) is the preferred type. When saving figures in TIFF format we recommend the use of LZW or ZIP compression in order to reduce the file sizes. The photographs can be submitted in JPEG format (ending *.jpg) with low compression ratio. Editors reserve the right to reject the photos of poor quality. Before submitting a figure in EPS format make sure first, that all the characters are rendered correctly (e.g. by opening the file first 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 file (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 figures should be prepared in final size, published in the magazine. The dimensions 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 findings which may be written in point form. 12. Summary Articles written in Slovene must contain a more extensive English summary. The reverse also applies. 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, (Trinajstic and Franjic 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.:(Ward1994a,b).Ifdirectquotationsareused,thepagenumbersshouldbeincluded:Toman(1992: 5) or (Toman 1992: 5–6).The bibliography shall be arranged in alphabetical order beginning with the surname of the first 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 fluorescence and growth responses of Genipa americana seedlings to soil flood­ing. 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 Conservation of Butterflies, 1st ed. Chapman & Hall, London, pp. 150-164. Toman, M.J., 1992. Mikrobiološke znacilnosti bioloških cistilnih 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 Microsoft 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 scientific 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 file should contain figures and tables included (each on its own page). However, when submitting the manuscript the figures also have to be sent as separate attached files in the form described under paragraph 10. All the pages (including tables and figures) have to be numbered. All articles must be proofread for professional and language errors before submission. Amanuscript element checklist (For a manuscript in Slovene language the same checklist is appro­priately applied with a mirroring sequence of Slovene and English parts): English title – (Times New Roman 14, bold) Slovene title – (Times New Roman 14, bold) Names of authors with clearly indicated addresses, affiliations and the name of the corresponding author – (Times New Roman 12) Author(s) address(es) / institutional addresses – (Times New Roman 12) Fax and/or e-mail of the corresponding author – (Times New Roman 12) Keywords in English – (Times New Roman 12) Keywords in Slovene – (Times New Roman 12) Running title – (Times New Roman 12) Abstract in English (Times New Roman 12, title – Times New Roman 14 bold) Abstract in Slovene – (Times New Roman 12, title – Times New Roman 14 bold) Introduction – (Times New Roman 12, title – Times New Roman 14 bold) Material and methods – (Times New Roman 12, title – Times New Roman 14 bold) Results – (Times New Roman 12, title – Times New Roman 14 bold) Discussion – (Times New Roman 12, title – Times New Roman 14 bold) Summary in Slovene – (Times New Roman 12, title – Times New Roman 14 bold) Figure legends; each in English and in Slovene – (Times New Roman 12, title – Times New Roman 14 bold, figure designation and figure title – Times New Roman 12 bold) Table legends; each in English and in Slovene – (Times New Roman 12, title – Times New Roman 14 bold, table designation and table title – Times New Roman 12 bold) Acknowledgements – (Times New Roman 12, title – Times New Roman 14 bold) Literature – (Times New Roman 12, title – Times New Roman 14 bold) Figures, one per page; figure designation indicated top left – (Times New Roman 12 bold) Tables, one per page; table designation indicated top left – (Times New Roman 12 bold) Page numbering – bottom right – (Times New Roman 12) 15. Peer Review All Scientific Articles shall be subject to peer review by two experts in the field (one Slovene and one foreign) and Brief Note articles by one Slovene expert in the field. With articles written in Slovene and dealing with a very local topic, both reviewers will be Slovene. In the compulsory accompanying lettertotheeditortheauthorsmustnominateoneforeignandoneSlovenereviewer.However,thefinal choiceofrefereesisatthediscretionoftheEditorialBoard.Therefereeswillremainanonymoustothe author. The possible outcomes of the review are: 1. Fully acceptable in its present form, 2. Basically acceptable, but requires minor revision, 3. Basically acceptable, but requires important revision, 4. May be acceptable, but only after major revision, 5. Unacceptable in anything like its present form. In the case of marks 3 and 4 the reviewers that have requested revisions have to accept the suitability of the corrections made. In case of rejection the corresponding author will receive a written negative decision of the editor-in-chief. The original material will be erased from the ABS archives and can be returned to the submitting author on special request. After publication the corresponding author will receive the *.pdf version of the paper.