Montenegrinus

NORTH-WESTERN JOURNAL OF ZOOLOGY 8 (1): 164-171 Article No.: 121111 ©NwjZ, Oradea, Romania, 2012 www.herp-or.uv.ro/nwjz First record of Stagnicola montenegrinus Glöer & Pešić, 2009 (Mollusca: Gastropoda: Lymnaeidae) in Bulgaria and its taxonomic relationship to other European lymnaeids based on molecular analysis Katrin SCHNIEBS1,*, Peter GLÖER2, Dilian GEORGIEV3 and Anna K. HUNDSDOERFER1 1. Senckenberg Natural History Collections Dresden, Museum of Zoology, Königsbrücker Landstraße 159, D-1109 Dresden, Germany, E-mails: katrin.schniebs@senckenberg.de, anna.hundsdoerfer@senckenberg.de 2. Biodiversity Research Laboratory, Schulstraße 3, D-25491 Hetlingen, Germany, E-mail: gloeer@malaco.de 3. Department of Ecology and Environmental Conservation, University of Plovdiv, Tzar Assen Str. 24, BG-4000 Plovdiv, Bulgaria, E-mail: diliangeorgiev@abv.bg * Corresponding author, K. Schniebs, E-mail: katrin.schniebs@senckenberg.de Received: 24. October 2011 / Accepted: 19. February 2012 / Available online: 21. February 2012 / Printed: June 2012 Abstract. Stagnicola montenegrinus was found in the floodplain of the river Maritza in Plovdiv. By comparison of the cyt-b sequences (fragment of 329 bp) of one of the specimens with other European Stagnicola specimens, this specimen fell in a cluster together with two sequences of S. montenegrinus from Skadar Lake, confirming the morphological determination and the first record of this species for Bulgaria. S. montenegrinus is a species closely related to S. corvus. The species' occurrence is evidently not limited to the Skadar Lake region from where S. montenegrinus was originally described. Key words: Stagnicola montenegrinus, Bulgaria, Lymnaeidae, molecular genetics. Introduction For the freshwater snail fauna of Bulgaria currently, three species of the genus Stagnicola Jeffreys, 1830 are known: S. palustris (O. F. Müller, 1774), S. corvus (Gmelin, 1791), and S. turricula (Held, 1836) (Angelov 2000, Hubenov 2007). When S. montenegrinus was described as a new species of this genus, it was only known from three localities in Montenegro: the Skadar Lake, the Crnojevica River and the Humsko Blato near Vitoja (Glöer & Pešić 2009). Up to now no other records became known. In June 2010 three specimens of genus Stagnicola were collected in the floodplain of the river Maritza in Plovdiv (Bulgaria) and sent to the Senckenberg Natural History Collections Dresden, Museum of Zoology (SNSD) for determination and molecular genetics analyses. Anatomical examinations by Peter Glöer determined that these three specimens belong to S. montenegrinus. The aim of the study is to prove the determination based on shell and genital characters by molecular techniques. Material and methods The specimens of S. montenegrinus were found in shallow floods and pools on the north bank of Maritza River in Plovdiv city (Upper Thracian Lowland) near the VHVP- bridge (Fig. 1), N42° 09' 13.5'' E24° 43' 34.8'', leg. Dilian Georgiev, 09.06.2010. Figure 1. Habitat of S. montenegrinus on the northern banks of Maritza River in Plovdiv city (photo by Stanislava Vassileva). Dissections and measurements of genital organs and shells were carried out using stereo microscopes (ZEISS and OLYMPUS). Photographs were taken with a digital camera system (OLYMPUS DP10). For the taxonomy we followed the current European checklists (Falkner et al. 2001, Bank 2011). For outgroup comparison in the molecular genetic analyses we used Palaearctic specimens of the species Planorbarius corneus (Linnaeus 1758), Aplexa hypnorum (Linnaeus, 1758). Lymnaea stagnalis (Linnaeus, 1758), Galba truncatula (O. F. Müller, 1774), Omphiscola glabra (O. F. Müller, 1774), Radix auricularia (Linnaeus, 1758), R. balthica (Linnaeus, 1758), R. labiata (Rossmässler, 1835), Stagnicola montenegrinus in Bulgaria 165 Stagnicola palustris, S. fuscus (C. Pfeiffer, 1821), and S. corvus were used as ingroup. The snails were fixed in 70-80% ethanol. All specimens used in the study are listed in Ta- ble 1. They are stored in the Molluscan collection of the Senckenberg Natural History Collections Dresden, Museum of Zoology (SNSD). Table 1. Material used in the molecular genetic studies. ENA=European Nucleotide Archive. Code Collection Locality No. SNSD Planorbarius corneus (Linnaeus, 1758) Planorbarius corneus 1 Moll 52556 Planorbarius corneus 2 Moll 52557 Aplexa hypnorum (Linnaeus 1758) Aplexa hypnorum 1 Moll S348 Aplexa hypnorum 2 Moll S350 Galba truncatula (O. F. Müller 1774) Galba truncatula 1 Moll 52545 Galba truncatula 2 Moll 52546 Galba truncatula 3 Moll S1130 Galba truncatula 4 Moll S1131 Omphiscola glabra (O. F. Müller 1774) Omphiscola glabra 1 Moll S303 Omphiscola glabra 2 Moll S304 Omphiscola glabra 3 Moll S305 Lymnaea stagnalis (Linnaeus, 1758) Lymnaea stagnalis 1 Moll 53093 Lymnaea stagnalis 2 Moll 53094 ENA No. cyt-b ITS-2 Germany, Saxony, Linz, pond Goldgrubenteich, FR797880 FR797830 13°43'09"E 51°19'45"N FR797881 FR797831 Germany, Mecklenburg-Vorpommern, lake, Ne- FR797882 FR797832 bel, 12°42'02"E 53°15'32"N FR797883 FR797833 Germany, Saxony, Oelsnitz/Erzgebirge, former FR797892 FR797847 pond, 12°42'04"E 50°43'02"N FR797893 FR797848 Bulgaria, Osogovo Mountains, Smolichane FR797890 FR797845 Village, karst spring, 22°48'25.2"E 42°07'58.1"N FR797891 FR797846 Germany, Hamburg, Kollau, Mühlenau, 09°55'33"E 53°36'34"N Germany, Baden-Württemberg, lake Bodensee, peninsula Mettnau, north side, 09°00'04"E 47°43'52"N Lymnaea stagnalis 3 Moll 53108 Germany, Baden-Württemberg, Konstanz-Egg, Lymnaea stagnalis 4 Moll 53109 ditch Hockgraben, 9°11'34.2"E 47°40'57.3"N Stagnicola palustris (O. F. Müller 1774) Stagnicola palustris 1 Moll 48716 Germany, Saxony, wetland west of Burghausen, 12°14'44"E 51°21'33"N Stagnicola palustris 2 Moll 53095 Germany, Baden-Württemberg, lake Bodensee, Stagnicola palustris 3 Moll 53096 peninsula Mettnau, north side, 09°00'04"E 47°43'52"N Stagnicola palustris 4 Moll S1345 Germany, Mecklenburg-Vorpommern, lake Grosser Plaetschsee, south bank, 12°19'18"E 53°26'25"N Stagnicola fuscus (C. Pfeiffer 1821) Stagnicola fuscus 1 Moll 48550 Germany, Saxony, reservoir Lobstädt, north bank, 12°27'27"E 51°07'58"N Stagnicola fuscus 2 Moll 51794 Germany, Saxony, marsh wood near Raden, 13°29'57"E 51°22'23"N Stagnicola fuscus 3 Moll S2082 Germany, Saxony, nature reserve Alte See Grethen, marsh wood, 12°40'18"E 51°13'42"N Stagnicola fuscus 4 Moll S2199 Germany, Baden-Württemberg, nature reserve Erlich, marsh wood, R 3462394 H 5449072 Stagnicola fuscus 5 Moll S2946 Germany, Thuringia, alder marsh near Appenrode, 10°43'07"E 51°34'27"N Stagnicola corvus (Gmelin 1791) Stagnicola corvus 1 Moll 49821 Germany, Saxony, Niederspree, pond Großer Tiefzug, 14°53'38"E 51°24'20"N Stagnicola corvus 2 Moll 49872 Germany, Saxony, pond Vierteich near Freitelsdorf, 13°41'57"E 51°15'43"N Stagnicola corvus 3 Moll 52830 Germany, Saxony, Grethen, ditch on the west Stagnicola corvus 4 Moll 52831 side of the pond Kleiner Kirchenteich, Stagnicola corvus 5 Moll 52832 12°39'22"E 51°14'29"N Stagnicola montenegrinus Glöer & Pešić 2009 Stagnicola montenegrinus 1 Moll 51854 Montenergo: Skutari See, Vranjina 9°07'32.52"E Stagnicola montenegrinus 2 Moll 51855 42°16'37.37"N FR797887 FR797853 FR797888 FR797854 FR797889 FR797855 FR797896 FR797836 FR797897 FR797837 FR797894 FR797834 FR797895 FR797835 FR797899 FR797841 HE577651 HE577631 HE577652 HE577632 HE577653 FR797838 HE577654 HE577633 HE577655 HE577634 HE577656 HE577635 HE577657 HE577636 HE577658 HE577637 HE577659 HE577638 HE577660 HE577639 HE577661 HE577640 HE577662 HE577641 HE577663 HE577642 HE577664 HE577643 HE577665 HE577644 Schniebs, K. et al. 166 Table 1. (continued) Code Stagnicola montenegrinus 3 Radix auricularia (Linnaeus 1758) Radix auricularia 1 Radix auricularia 2 Radix auricularia 3 Radix auricularia 4 Radix labiata (Rossmässler 1835) Radix labiata 1 Radix labiata 2 Radix labiata 3 Radix labiata 4 Radix balthica (Linnaeus 1758) Radix balthica 1 Radix balthica 2 Radix balthica 3 Radix balthica 4 Collection Locality No. SNSD Moll S2313 Bulgaria: floodplain of the Maritza river in Plovdiv, 24° 43' 34.8''E 42° 09' 13.5''N Moll 53070 Moll 53071 Moll 52857 Moll 52859 Germany, Bavaria, Weichering, pond in riverside forest, 11°19'23.6"E 48°43'34.1"N Moll 51275 Moll 51276 Moll 51696 Moll 51697 Germany, Saxony, pond near Langenberg, 12°51'21"E 50°33'09"N Moll 51281 Moll 51283 Moll 53111 Moll 53112 Switzerland, canton Basel-Landschaft, Liestal, Orishof, 07°43'03"E 47°28'22"N Russia, Novosibirsk Region, Novosibirsk Reservoir near Kirza River 81° 39.63114"E 54° 14.244”N Germany, Brandenburg, small lake near Wachow, 12°43'05"E 52°32'05"N Germany, Baden-Württemberg, Konstanz-Egg, pond near University, 09°11'29"E 47°41'09" N Molecular techniques Tissue samples were taken under a microscope from the feet of the snails and fixed in 100% ethanol. The samples were registered in the tissue collection of the SNSD with a new collection number and the collection number of the specimen in the molluscan collection of SNSD and stored at -80°C. For molecular genetic analyses we obtained sequence data of the complete nuclear ITS-2 marker (280 bp in A. hypnorum up to 491 bp in L. stagnalis) and a 329 bp fragment of the cyt-b gene as mitochondrial marker. DNA was extracted using DTAB (dodecyl trimethyl ammonium bromide) buffer (Gustincich et al. 1991). The tissue samples were washed with 100 µl TE buffer and subsequently incubated with 500 µl preheated DTAB for 30 min at 65°C. The incubation was continued after adding 10 µl Proteinase K (50 mg/ml) for 20-24 hours, followed by a short incubation with 10 µl RNase (10 mg/ml) for 30 min at 37°C. Remaining tissue fragments disintegrated after vortexing. For cleaning 550 µl chloroform/isoamyl alcohol (24/1) was used. The samples were vortexed for 20 sec and the phases subsequently separated again at 12 000 g for 3 min. With the upper aqueous phase the procedure was repeated. 100 µl 4M LiCl and 400 µl isopropanol were added to the aqueous phase for precipitation. The samples were cooled at -20°C for 30 min and subsequently the DNA was pelleted by centrifugation at 11 200 g for 20 min at 4°C. The liquid was disposed of and the pellets were dried by inverting the tubes on a paper towel. The pellets were cleaned twice with 200 µl ice-cold 70% ethanol. The DNA pellets were dried 10 min at 50°C and subsequently redissolved in 50 µl of TE buffer. The PCRs were carried out in a final volume of 20 µl with quantities of DNA from 0.5 to 5.0 µl depending on the concentration estimated by gel electrophoresis, 2 µl 10x PCR buffer (Bioron, incomplete), 1 µl MgCl2 (Bioron, ENA No. cyt-b ITS-2 HE577666 HE577645 FR797902 FR797903 HE577667 HE577668 FR797842 FR797843 HE577647 HE577646 HE573106 HE573107 HE577669 HE573108 HE573068 HE573069 HE577648 HE573070 HE577670 HE573133 HE573116 HE573117 HE577650 HE573082 HE573078 HE577649 0.055 µS/cm), 1 µl of each primer (10 pmol/µl), 0.5 µl dNTP (10 mM), 0.2 µl Taq DNA polymerase (DFS-Taq Bioron) and the corresponding volume of sterile H2O. From the cyt-b gene a region of circa 370 bp was amplified with the primers UCytb151F and UCytb270R (Merritt et al. 1998) and a temperature profile of 94°C for 4 min, 40 cycles of 94°C for 40 s, 48°C for 40 s, 72°C for 1.15 min, followed by an extension at 72°C for 6 min and hold at 8°C, was used. The primers used for ITS-2 were LT1 (Bargues et al. 2001) and ITS2-Rixo (Almeyda-Artigas et al. 2000). The temperature profile used was the following: 94°C for 4 min, 40 cycles of 94°C for 30 s, 50°C for 30 s, 72°C for 1 min, followed by 7 min at 72°C and 8°C hold. PCR products were purified with 0.1 µl Exo Sap-It plus 4 µl ddH2O and incubated for 30 min at 37°C, followed by deactivation for 15 min at 80°C. The primers used for the cycle sequencing were UCytb151F for cyt-b and LT1 for ITS-2. The quantity of PCR product used for cycle-sequencing ranged from 0.5 to 5.0 µl depending on the concentration estimated by gel electrophoresis. 0.5 µl BigDye T-Mix (ABI, Applied Biosystems), 2.25 µl BigDye buffer (5x), 0.5 µl primer (10pmol), and sterile H2O were added to a total volume of 10 µl. The following temperature profile was used: 25 cycles of 96°C for 10 s, 50°C for 5 s, 60°C for 4 min and 8°C hold. The products were purified by adding 1 µl 3M NaAc (pH 4.6) and 25 µl EtOH (100%), centrifuging at 13 000 g for 15 min, inverting the tubes on a paper towel and washing with 200 µl 70% EtOH. After removing the EtOH the pellets were dried for 10 min at 50°C. Samples were sequenced on an ABI 3130 xl (Applied Biosystems). 1 Data analysis For maximum-likelihood analyses, including bootstrap support, we used raxmlGUI 0.9 beta 2 (RAxML) (Silvestro & Michalak 2010, Stamatakis et al. 2005). The settings were “ML+thorough bootstrap” with 100 (replicate) runs and 1000 (bootstrap) repetitions. Stagnicola montenegrinus in Bulgaria 167 Maximum-parsimony (MP) trees were reconstructed using PAUP (version 4.0b10; Swofford 2002; settings: gapmode=NewState, addseq=closest). For presentation of MP results, one of the best trees was chosen to be able to illustrate branch lengths (one showing the same overall topology as the majority rule consensus tree was chosen). Genetic distances of the cyt-b were calculated using MEGA version 4 (Tamura et al. 2007). Results Habitat and associated freshwater molluscs S. montenegrinus occurs in pools on the northern banks of Maritza River in Plovdiv city fed by high waters of the main river and a small tributary coming from north. The substrate is sand and mud, water is running very slow or standing depending on the water levels of the river Maritza. Bank vegetation is dominated by Salix spp., Typha spp., Phragmites australis (Cavanilles, 1799) Trinius ex Steudel, 1840, and water plants by Ceratophyllum demersum Linnaeus, 1753 and Elodea canadensis Michaux, 1803. Other molluscs found in the locality are: Unio pictorum (Linnaeus, 1758), Planorbis planorbis (Linnaeus, 1758), Physella acuta (Draparnaud, 1805), Radix auricularia (Linnaeus, 1758), Valvata piscinalis (O. F. Müller, 1774), Lymnaea stagnalis (Linnaeus, 1758), Planorbarius corneus (Linnaeus, 1758), Galba truncatula (O. F. Müller, 1774) and Anisus vortex (Linnaeus, 1758). Molecular genetics Distance analyses. Genetic distances (p-distance) from pair-wise comparisons of cyt-b sequences (fragment of 329 bp) are shown in Table 2. Distances between species of different families (Planorbidae, Physidae and Lymnaeidae) (outgroup comparison) ranged between 31.2% and 25.5%. Differences between different genera within the family Lymnaeidae ranged from 26.4% to 15.1%. Among the four Stagnicola species analysed, the values are between 19.4% and 14.7%. The highest difference between S. montenegrinus and the other three Stagnicola species is the difference to S. palustris (19.1%). The difference to S. corvus and S. fuscus are 17.5% and 14.7% respectively. The values of the three analysed Radix species ranged between 17.5% and 13.0%. Molecular phylogeny. The maximum-parsimony (MP) tree of the cyt-b sequences is illustrated in Fig. 2 (tree length = 532, consistency index = 0.5602, retention index = 0.8769). It shows low or very low support on basal branches. Omphiscola glabra is paraphyletic with respect to the other genera of Lymnaeidae analysed in this paper. Galba truncatula is paraphyletic with respect to Radix auricularia, R. labiata, and R. balthica. In contrast the clades of the species have full bootstrap support. Within analysed representatives of the genus Stagnicola, S. montenegrinus groups sister to S. fuscus with low support (66%) whereas S. palustris groups sister to S. corvus (bootstrap support 71%). The RAxML tree of the cyt-b sequences (not shown) shows similar results. The basal branches have low or very low support. This poor support is underlined by two polytomies: one between the S. fuscus      0.175 0.177 0.160 0.211 0.224 0.237 0.230       0.147 0.191 0.219 0.242 0.238 0.263        0.194 0.187 0.247 0.216 0.240                                    0.227   0.264 0.226  0.261 0.220 0.130 0.273 0.249 0.175 0.149 R. auricularia S. montenegrinus     0.214 0.237 0.214 0.258 0.218 0.192 0.207 0.209 R. balthiac S. corvus    0.202 0.230 0.241 0.232 0.259 0.224 0.151 0.158 0.161 R. labiata O. glabra   0.263 0.255 0.278 0.298 0.292 0.328 0.278 0.259 0.255 0.259 L. stagnalis G. truncatula  0.312 0.294 0.275 0.306 0.308 0.305 0.315 0.284 0.284 0.308 0.298 S. palustris A. hypnorum Planorbarius corneus Aplexa hypnorum Galba truncatula Omphiscola glabra Stagnicola corvus Stagnicola montenegrinus Stagnicola fuscus Stagnicola palustris Lymnaea stagnalis Radix labiata Radix balthica Radix auricularia P. corneus Table 2. Evolutionary distances (p-distance) of the cyt-b gene fragment (329 bp) calculated using MEGA version 4 (Tamura et al. 2007).             168 Figure 2. Hypothesis of the phylogenetic relationships of S. montenegrinus based on one of the 71 best maximumparsimony trees of the sequenced fragment of the mitochondrial marker cyt-b (329 bp; tree length = 532, consistency index = 0.5602, retention index = 0.8769). Branch lengths are proportional to the number of substitutions and the overall topology corresponds to that of the strict consensus tree. Bootstrap support values above 50% are reported below nodes. Radix-Galba cluster and the other genera of Lymnaeidae analysed, and the other within the RadixGalba group. S. montenegrinus groups sister to S. fuscus with low support (67%) too. S. palustris is the sister group to S. corvus (71%). The clades of all species have high bootstrap support. The maximum-parsimony (MP) tree of the nuclear marker ITS-2 (tree length = 1767, consistency index = 0.8087, retention index = 0.9650) (Fig. 3) is well-supported within the Lymnaeidae. Most of the basal branches have full bootstrap support. The tree shows the analysed representatives of the genus Radix as sister group to the other studied genera of Lymnaeidae. Within the latter G. truncatula groups sister to O. glabra, L. stagnalis and the Schniebs, K. et al. Figure 3. Hypothesis of the phylogenetic relationships of S. montenegrinus based on one of the 9 best maximumparsimony trees of the nuclear marker ITS-2 (tree length = 1767, consistency index = 0.8087, retention index = 0.9650). Branch lengths are proportional to the number of substitutions and the overall topology corresponds to that of the strict consensus tree. Bootstrap support values above 50% are reported below nodes. Stagnicola species analysed. S. palustris groups sister to S. fuscus, S. corvus and S. montenegrinus. On the other hand, although not well supported (59%), S. fuscus groups as the sister group to a cluster consisting of S. corvus and S. montenegrinus that has full bootstrap support. The RAxML tree of the nuclear marker ITS-2 (not shown) shows G. truncatula as sister group to the other Lymnaeidae analysed. The three Radix Stagnicola montenegrinus in Bulgaria 169 species group sister to the genera Stagnicola, Omphiscola, and Lymnaea. In contrast to the MP tree there is no resolution within the genera Stagnicola, Omphiscola, and Lymnaea. S. montenegrinus forms a cluster together with S. corvus with bootstrap support of 95%. S. palustris groups sister to S. fuscus with bootstrap support of 66%. Morphology Two of the three specimens from the Maritza River floodplain have higher and wider shells (Table 3) than the specimens from Skadar Lake (shell height 10.6-21.5 mm, shell width 4.4-9 mm) (Glöer & Pešić 2009). The aperture is also a little higher than the spire, as defined as a differential characteristic between S. montenegrinus and S. corvus (Glöer & Pešić 2009). The mantle pigmentation is bluish black and lacks white spots as shown in Glöer & Pešić (2009, p. 55, Fig.2) for S. montenegrinus from Skadar Lake. The anatomy is very similar to that of the specimens from Montenegro. The prostate contains 3 folds and the bursa duct is also thickened at the distal end by entering the vagina (Fig. 4). Table 3. Shell measurements of S. montenegrinus from Maritza River in Plovdiv city. Shell height Shell width Aperture height SNSD Moll S2313 26.7 mm 10.3 mm 14.3 mm SNSD Moll S2314 25.0 mm 10.3 mm 14.1 mm SNSD Moll S2315 21.0 mm 9.6 mm 11.9 mm Figure 4. Stagnicola montenegrinus from Maritza River floodplain (Bulgaria). – 1: shell of SNSD Moll S2313; 2: shell of SNSD Moll S2314; 3: mantle pigmentation of SNSD Moll S2315; 4: cross-section through prostate gland SNSD Moll S2313; 5: male copulatory organ SNSD Moll S2313; 6: female sex tract. bc = bursa copulatrix, bd = bursa duct, cp = corpus pyriforme, pht = phallotheca, pr = prostate, = prp = praeputium, pvd = provaginal duct, vd = vas deferens. 170 Discussion The molecular distances of 17.5% between S. montenegrinus and S. corvus, 14.7% between S. montenegrinus and S. fuscus, and 19.1% between S. montenegrinus and S. palustris in the cyt-b fragment (about 329 bp) (Table 2) show similar values like the distances between S. palustris and S. fuscus as well as S. corvus and S. fuscus (19.4% and 17.7% respectively). The lowest value of 14.7% between S. montenegrinus and S. fuscus is even higher than the distance between the Radix species R. balthica and R. labiata (13.5%) as well as the distances between R. balthica and R. lagotis (Schrank, 1803) (9.0%), between R. ampla (Hartmann, 1821) and R. lagotis (9.2%) (Schniebs et al. 2011) or between Aenigmomphiscola europaea Kruglov and Starobogatov, 1981 and Ae. kazakhstanica Kruglov and Starobogatov, 1981 (9.0%) (Vinarski et al. 2011). Similar to other authors (e.g. Abramson 2009), we interpret the existing differences between the values of the molecular distances between species clearly distinguishable by morphological characteristics, as a consequence of different evolutionary speeds for the cyt-b gene. The results of our molecular genetic analyses of the nuclear marker ITS-2 and the mitochondrial marker, the cyt-b fragment (329 bp) (Figs 2,3), are inconsistent and not congruent like in studies of Unionidae using the nuclear ribosomal internal transcribed spacer region in comparison to the mitochondrial genes 16S and COI (Källersjö et al. 2005). Whereas there is a significant difference in gene distances in the cyt-b fragment (about 329 bp) between all Stagnicola species analysed (Table 2) and S. montenegrinus groups sister to S. fuscus and S. corvus groups sister to S. palustris using this gene (both with only medium support of 66% and 71% respectively), the nuclear marker ITS-2 shows no sequence differences between the three specimens of S. corvus from Germany (collection No. SNSD Moll 49821, 49872 and 49872) and the specimens of S. montenegrinus from Skadar Lake and Bulgaria (Fig. 3). The result is the same if sequences of S. turricula from GenBank as well as own sequences from other Palaearctic Stagnicola species are included in the calculation (unpublished data). Although there is no difference between S. corvus and S. montenegrinus in nuclear marker ITS2 sequences we think that the parsimony tree (Fig. 3) reflects the most realistic relationships between the genera Omphiscola, Lymnaea, and Stagnicola, be- Schniebs, K. et al. cause own studies of the 18S rRNA gene (Vinarski et al. 2011) and analyses of Bargues & Mas-Coma (1997) as well as studies based on 16S, ITS-1 and ITS-2 spacers of other authors (Correa et al. 2010) have shown that these three genera are very closely related. Of the Stagnicola species analysed from Bulgaria, S. palustris is the only one with one prostate fold and it groups sister to the species with two and more folds in the ITS-2 parsimony tree. S. fuscus with two prostate folds groups sister to S. montenegrinus and S. corvus with three and more folds. So the ITS-2 tree (Fig. 3) appears also to reflect meaningful relations between the analysed species of Stagnicola. Anyway the clusters of the ITS-2 (Fig. 3) appear to reflect the subdivision into genera we used following the current European checklists (Falkner et al. 2001, Bank 2011). We thus have more confidence in this phylogenetic hypothesis based on the nuclear marker, than in the one based on the mitochondrial marker cyt-b. From anatomical differences important for differentiation of European Stagnicola species, as number of prostate folds and diameter of the bursa duct at the distal part, as well as from our molecular genetic analyses we conclude that S. montenegrinus is a species closely related to S. corvus. Its occurrence is not limited to the Skadar Lake region and needs further investigation. Acknowledgements. We would like to express our thanks to Prof. Dr. Uwe Fritz (SNSD) for financing the greatest part of the molecular analyses, Anke Müller (SNSD) for some sequences and the instruction of K.S. in lab work, as well as Prof. Dr. Vladimir Pešic (University of Montenegro), Michael Korn (University of Konstanz, Limnological Institute), R. Diercking (Hamburg), Dr. André Reimann (SNSD), Maxim V. Vinarski and Alfried V. Karimov (Omsk State Pedagogical University), Christoph Oberer (Natural History Museum Basel), Robert Haldemann (Strausberg), Dr. Nicole SchröderRogalla (Munich), Susanne Thiel (Munich), Andrea Pohl (Dresden), Gudrun Rutsch (Dresden) and Christa Schniebs (Oelsnitz) for the material collected and provided. We also thank the biologist Stanislava Vassileva (University of Plovdiv) who took the picture of the habitat. 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