Research Article ISSN 2336-9744 (online) | ISSN 2337-0173 (print) The journal is available on line at www.biotaxa.org/em
A molecular genetic evidence of the occurrence of the freshwater snail Radix lagotis (Schrank, 1803) (Gastropoda: Lymnaeidae) in Bulgaria
KATRIN SCHNIEBS1*, DILIAN GEORGIEV2, PETER GLÖER3 & ANNA K. HUNDSDOERFER1
1Senckenberg Natural History Collections Dresden, Museum of Zoology, Königsbrücker Landstraße 159, D-1109 Dresden, Germany. E-mails: [email protected], [email protected] 2Department of Ecology and Environmental Conservation, University of Plovdiv, Tzar Assen Str. 24, BG-4000 Plovdiv, Bulgaria. E-mail: [email protected] 3Biodiversity Research Laboratory, Schulstraße 3, D-25491 Hetlingen, Germany. E-mail: [email protected]
*Corresponding author: E-mail: [email protected]
Received 5 August 2015 │ Accepted 19 August 2015 │ Published online 21 August 2015.
Abstract Radix lagotis (Schrank, 1803) was found in the Dragoman marshes in Bulgaria. By comparison of the mitochondrial cyt-b sequences (fragment of 329 bp) and sequence data obtained from the nuclear ITS-2 spacer these specimens fell into one cluster with sequences from R. lagotis from Germany, Austria and Czech Republic confirming the morphological determination and the first record of this species for Bulgaria.
Key words: Radix lagotis, Bulgaria, molecular genetics.
Introduction
Although Hubendick (1953) gave a very detailed morphological and anatomical description of Radix lagotis (Schrank, 1803), for a long time this species was not accepted as a distinct species by most European malacologists but referred to as forms of R. auricularia (Linnaeus, 1758) and R. peregra (O. F. Müller, 1774). However, it was generally accepted as species by Russian and Ukrainian malacologists (e. g. Kruglov & Starobogatov 1983, 1993; Stadnichenko 2004; Kruglov 2005). Using molecular genetic techniques Bargues et al. (2001) could distinguish R. lagotis from Czech Republic and Austria from the other Radix species occurring in Europe R. auricularia, R. balthica (Linnaeus, 1758), R. labiata (Rossmässler, 1835), and R. ampla (Hartmann, 1821) with certainty, and for the first time. The first evidences of the occurrence of R. lagotis in Germany were published by Oheimb et al. (2007) for Brandenburg, Glöer & Diercking (2009) for Hamburg, Schniebs et al. (2011) for Saxony, and Körnig et al. (2013) for Saxony-Anhalt. Morphological distinguishing characters between R. lagotis and other European Radix species were discussed by Schniebs et al. (2011, 2013) and Huňová et al. (2012). R. lagotis seems to be widely distributed in Europe and Western Siberia (Khokhutkin et al. 2003, 2009; Stadnichenko 2004; Kruglov 2005) as well as in Central Asia (Hubendick 1953; Stadnichenko 2004). But molecular genetically validated records are still published only from Austria (Bargues et al. 2001), Czech Republic (Bargues et al. 2001; Huňová et al. 2012), Germany (Schniebs et al. 2011, 2013; Körnig et al. 2013) and Kyrgyzstan (Glöer et al. 2014). Radix specimens and Radix species with an anatomy similar to
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R. lagotis have been described from the Palaearctic [Hubendick 1953: R. peregra from Siberia and R. ovata (Draparnaud, 1805) from the Kola Peninsula; Falniowski 1980: R. peregra from Poland, Figs. 10, 12, 15, 16, 17; R. fontinalis (Studer, 1820), R. intermedia (Lamarck, 1822), R. burnetti (Alder, 1848), R. tenera (Küster, 1862), R. persica (Issel, 1865), R. aberrans (Westerlund, 1897) R. middendorffi (Dybowski, 1903), R. jacutica (Starobogatov et Streletzkaja, 1967), R. amurensis (Kruglov, Moskvicheva et Starobogatov, 1984), R. kafanovi (Kruglov et Starobogatov, 1984), R. nogoonica (Kruglov et Starobogatov, 1983), R. novikovi (Kruglov et Starobogatov, 1983), R. manomaensis (Kruglov, Starobogatov et Zatravkin, 1984)] and it would be very interesting to revise the group using molecular genetic analyses to find out whether the alleged species actually belong to one widely distributed species or they can be validated as several species. For Bulgaria Wohlberedt (1911) reported R. lagotis from Panega River near Vratsa town and Hesse (1913, 1914) from deposits of Maritsa River near Plovdiv city. The species was included in the catalog of Angelov (2000), omitted by Hubenov (2007), and again listed by Georgiev & Hubenov (2013). In April 2011, Radix specimens were collected in the Dragoman marshes in Bulgaria and sent to the Senckenberg Natural History Collections Dresden, Museum of Zoology (SNSD) for molecular genetic analyses. Anatomical examinations showed that they are very similar to R. lagotis found in Germany. The aim of this study was to elucidate whether these individuals indeed belong to this species.
Material and Methods
Snails were fixed in 70-80% ethanol or isopropyl alcohol. Shell morphology, mantle pigmentation and anatomy were documented from the specimens studied. Dissections and measurements of genital organs were carried out using stereo microscope (Nikon SMZ18). Photographs were taken with a digital camera system (Nikon DS-Fi2). All specimens used for morphological and molecular genetic studies are listed in Table 1. They are stored in the mollusc collection of the Senckenberg Natural History Collections Dresden, Museum of Zoology (SNSD). For the taxonomy we followed the current European checklists (Falkner et al. 2001; Bank 2011).
Molecular techniques and phylogenetic analyses of sequences For outgroup comparison in the molecular genetic analyses we used Palaearctic specimens of the species Aplexa hypnorum (Linnaeus, 1758) from the freshwater gastropod family Physidae and from the lymnaeid species Lymnaea stagnalis (Linnaeus, 1758) and Stagnicola corvus (Gmelin, 1791). We included sequences of Radix auricularia (Linnaeus, 1758), R. balthica (Linnaeus, 1758), R. ampla (Hartmann, 1821) and R. labiata (Rossmässler, 1835) in the ingroup. In addition we used ITS-2 sequences of R. lagotis available from GenBank. Tissue samples taken from the foot were fixed in 100% ethanol. They were registered in the tissue collection of the SNSD with a tissue voucher number and the corresponding collection number in the mollusc collection of SNSD and stored at -80°C. All DNA-sequences have been placed in the European Nucleotide Archive (ENA, see http://www.ebi.ac.uk/ena/). For the molecular genetic analyses we obtained the sequence of the nuclear ITS-2 spacer (280 bp in A. hypnorum, up to 495 bp in L. stagnalis) and a 329 bp fragment of the cyt-b gene as a mitochondrial marker. For primers and protocols of DNA extraction, Polymerase Chain Reaction (PCR), purification of PCR products and DNA sequencing see Schniebs et al. (2011). Alignments were assembled using the sequence alignment editor BioEdit (Hall 1999). The ITS-2 alignment was obtained using the Clustal algorithm of MEGA version 4 (Tamura et al. 2007) and improved by eye. Phylogenetic analyses under the maximum parsimony (MP) criterion for the ITS-2 spacer and cyt-b fragment were carried out using PAUP (version 4.0b10; Swofford 2002; settings: gapmode = NewState, addseq = closest, the maximal number of trees with the setting of maxtree = 10000 did not have to be increased, since the number of best trees remained below 5000; number of bootstrap replicates = 10000). For presentation of the MP results for ITS-2 and cyt-b, one of the 4360 and 592 best trees respectively was chosen to be able to illustrate branch lengths (one showing the same overall topology as the majority rule consensus tree was chosen).
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For maximum-likelihood analyses, including bootstrap support, we used RAxML (raxmlGUI 0.9 beta 2, Silvestro & Michalak 2010; Stamatakis et al. 2005). The settings were “ML+thorough bootstrap” with 100 (replicate) runs and 1000 (bootstrap) repetitions. All DNA-sequences have been placed in the European Nucleotide Archive (ENA, see http://www.ebi.ac.uk/ena/).
Table 1. Material used for the molecular genetic and morphological analyses. ENA=European Nucleotide Archive.
Code Collection No. Locality ENA No. SNSD cyt-b ITS-2 Aplexa hypnorum (Linnaeus, 1758) Aplexa hypnorum 1 Moll S348 Germany, Mecklenburg-Western Pomerania, lake Nebel N 53°15'32" FR797882 FR797832 E 12°42'02" Aplexa hypnorum 2 Moll S350 Germany, Mecklenburg-Western Pomerania, lake Nebel N 53°15'32" FR7978823 FR797833 E 12°42'02" Lymnaea stagnalis (Linnaeus, 1758) Lymnaea stagnalis 1 Moll 49239 Germany, Saxony, Dresden, old branch of River Elbe, N LN874260 HE573064 50°59’50.80" E 13°52’29.39" Lymnaea stagnalis 2 Moll 49835 Germany, Saxony, Niederspree, small pond, N 51°24'28" E 14°54'03" HE573103 HE573065 Lymnaea stagnalis 3 Moll 53108 Germany, Baden-Württemberg, Konstanz- Egg, ditch Hockgraben, N FR797894 FR797834 47°40'57.3" E 9°11'34.2" Lymnaea stagnalis 4 Moll 53109 Germany, Baden-Württemberg, Konstanz- Egg, ditch Hockgraben, N FR797895 FR797835 47°40'57.3" E 9°11'34.2" Lymnaea stagnalis 5 Moll S1760 Ukraine: province Zaporozhye, Zarechnoe village, Yushanly River HG932145 HG931962 Lymnaea stagnalis 6 Moll S2311 Bulgaria: Plovdiv, floodplain of the Mariza River, N 42° 09' 13.5'' E HG932147 HG931965 24° 43' 34.8'' Stagnicola corvus (Gmelin, 1791) Stagnicola corvus 1 Moll 49821 Germany, Saxony, Niederspree, pond Großer Tiefzug, N 51°24'20" E HE577659 HE577638 14°53'38" Stagnicola corvus 2 Moll 49872 Germany, Saxony, pond Vierteich near Freitelsdorf, N 51°15'43" E HE577660 HE577639 13°41'57" Stagnicola corvus 3 Moll 52830 Germany, Saxony, Grethen, ditch on the west side of the pond Kleiner HE577661 HE577640 Kirchenteich, N 51°14'29" E 12°39'22" Stagnicola corvus 4 Moll 52831 Germany, Saxony, Grethen, ditch on the west side of the pond Kleiner HE577662 HE577641 Kirchenteich, N 51°14'29" E 12°39'22" Radix auricularia (Linnaeus, 1748) Radix auricularia 1 Moll 50005 Germany, Saxony, Niederspree, pond Neuwiesenteich, N 51°24'19" E HE573104 HE573066 14°52'57" Radix auricularia 2 Moll 50079 Germany, Saxony, pond Vierteich near Freitelsdorf, N 51°15'43" E HE573105 HE573067 13°41'57" Radix auricularia 3 Moll 52857 Russia, Novosibirsk Region, Novosibirsk, Reservoir near Kirza River, HE557667 HE557647 N 54°14.224’ E 81°39.63114’ Radix auricularia 4 Moll 52859 Russia, Novosibirsk Region, Novosibirsk, Reservoir near Kirza River, HE557668 HE557648 N 54°14.224’ E 81°39.63114’ Radix auricularia 5 Moll 53070 Germany, Bavaria, Weichering, pond in riverside forest, N FR797902 FR797842 11°19'23.6"E 48°43'34.1" Radix auricularia 6 Moll 53071 Germany, Bavaria, Weichering, pond in riverside forest, N FR797903 FR797843 11°19'23.6"E 48°43'34.1" Radix ampla (Hartmann, 1821) Radix ampla 1 Moll 51112 Russia, Chelyabinsk Region, river Miass near Dynamo village, N HE798470 HE798448 45°57' E 60°02' Radix ampla 2 Moll 51113 Russia, Chelyabinsk Region, river Miass near Dynamo village, N HE798471 HE798449 45°57' E 60°02' Radix ampla 3 Moll 53098 Germany, Bavaria, lake Ammersee, Stegen, N 48°04'32" E 11°08'07" HE573110 HE573072 Radix ampla 4 Moll 53099 Germany, Bavaria, lake Ammersee, Stegen, N 48°04'32" E 11°08'07" HE573111 HE573073
..…continued on the next page
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Table 1 (Continued)
Code Collection No. Locality ENA No. SNSD cyt-b ITS-2 Radix balthica (Linnaeus, 1758) Radix balthica 1 Moll 51283 Switzerland, canton Basel-Landschaft, Liestal, Orishof, N 47°28'22" HE573133 HE573082 E 07°43'03" Radix balthica 2 Moll 51292 Switzerland, canton Basel City, Riehen, Wiesengriener, N 47°35'21" HE573134 HE573083 E 07°38'32" Radix balthica 3 Moll 51834 Germany, Saxony, Dresden-Kleizschachwitz, river Elbe, N 51°00'03" HE573119 HE573079 E 13°52'21" Radix balthica 4 Moll 51860 Sweden, Øland, east shore near Lille Seby, N 56.345°E 16.565° HE573141 HE573090 Radix balthica 5 Moll 53111 Germany, Baden-Württemberg, Konstanz-Egg, pond near University, HE573116 HE573078 N 47°41'09" E 09°11'29" Radix balthica 6 Moll 53112 Germany, Baden-Württemberg, Konstanz-Egg, pond near University, HE573117 HE577649 N 47°41'09" E 09°11'29" Radix labiata (Rossmässler, 1835) Radix labiata 1 Moll 49708 Czech Republic, České Budějovice, Ponesice, N 49°06’00" E HE798473 HE798451 14°28’60”E Radix labiata 2 Moll 51276 Germany, Saxony, pond near Langenberg, N 50°33'09"E 12°51'21" HE798475 HE573069 Radix labiata 3 Moll 51696 Germany, Brandenburg, small lake near Wachow, N 52°32'05" E HE798477 HE577648 12°43'05" Radix labiata 4 Moll 51863 Montenegro, Žabljak, Black Lake, sand pools, N 43°08'50"E HE798480 HE798455 19°05'42" Radix labiata 5 Moll 51910 Germany, Saxony, Dehnitz, renatured swimming baths, N 51°20'56" HE798481 HE798456 E 12°44'24" Radix labiata 6 Moll 51964 Germany, Mecklenburg-Western Pomerania, Gressow, alder fenwod, HE798482 HE798454 N 53°41.898' E 11°18.592' Radix labiata 7 Moll 52415 Austria, Carinthia, Hermagor, N 46°37' E 13°22' HE798484 HE798457 Radix labiata 8 Moll 52427 Italy, Lombardy, Province of Brescia, Borno, lower Varicla Lake, N HE798486 HE798459 45°58’50” W 02°15’24” (Monte Mario) Radix labiata 9 Moll 52580 Germany, Saxony, pond near Linz, N 51°20'51" E 13°43'33" HE798490 HE798463 Radix labiata 10 Moll S172 France, Bourgogne, Département Cote-d'Or HE798492 HE798465 Radix labiata 11 Moll S2904 Germany, Saxony, small brook north of Tharandt, N 51°00'08" E HE798496 HE798469 13°34'19" Radix lagotis (Schrank, 1803) Radix lagotis 1 GenBank Czech Republic, Kadov, Vasi and Podkadovsky pond − AJ319638 Radix lagotis 2 GenBank Austria, Schoenau, southeast of Vienna − AJ319639 Radix lagotis 3 Moll 49868 Germany, Saxony, pond Vierteich near Freitelsdorf, N 51°15'43" E HE573114 HE573076 13°41'57" Radix lagotis 4 Moll 50008 Germany, Saxony, Niederspree, pond Neuwiesenteich, R5491955 LN874261 LN874254 H5696706 Radix lagotis 5 Moll 51858 Germany, Brandenburg, Strodehne, N 51.74555° E 12.22396° LN874262 LN874255 Radix lagotis 6 Moll 52563 Germany, Saxony, pond Goldgrubenteich near Linz, N 51°19'46" E HE573115 HE573077 13°43'09" Radix lagotis 7 Moll 53239 Germany, Saxony, dam Doellnitzsee near Mutzschen, N 51°15'45" E HE573113 HE573075 12°55'18" Radix lagotis 8 Moll S2190 Germany, Mecklenburg-Western Pomerania, creek connecting the LN874263 LN874256 lakes Kirchstücker and Barnerstücker Radix lagotis 9 Moll S3765 Bulgaria. Dragoman marshland 40 km west of Sofia, N 42°56'11.7'' E LN874264 LN874257 22°57'9.3'' Radix lagotis 10 Moll S3766 Bulgaria. Dragoman marshland 40 km west of Sofia, N 42°56'11.7'' E LN874265 LN874258 22°57'9.3'' Radix lagotis 11 Moll S3770 Bulgaria. Dragoman marshland 40 km west of Sofia, N 42°56'11.7'' E LN874266 LN874259 22°57'9.3'' Radix lagotis 12 Moll S3767 Bulgaria. Dragoman marshland 40 km west of Sofia, N 42°56'11.7'' E − − 22°57'9.3''
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Figure 1. Hypothesis of the phylogenetic relationships of R. lagotis based on one of the 592 best maximum-parsimony trees of the mitochondrial marker cyt-b (fragment of 329 bp) (tree length=368, CI=0.6495, RI=0.9118). The overall topology corresponds to that of the strict consensus tree. Branch lengths are proportional to the number of substitutions. Bootstrap support values above 50% are reported below nodes. The R. lagotis specimens from Dragoman marshes are marked red.
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Figure 2. Hypothesis of the phylogenetic relationships of R. lagotis based on one of the best 4360 maximum- parsimony trees of the nuclear marker ITS-2 (tree length=997, CI=0.9057, RI=0.9829). The overall topology corresponds to that of the strict consensus tree. Branch lengths are proportional to the number of substitutions. Bootstrap support values above 50% are reported below nodes. The R. lagotis specimens from Dragoman marshes are marked red.
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Results
Molecular genetics The hypothesis of the phylogenetic relationships of R. lagotis based on one of the 592 best maximum parsimony (MP) trees of the cyt-b fragment is illustrated in Fig. 1 (tree length=368, CI=0.6495, RI=0.9118). It shows full or high support for basal branches. The species-clades received full or nearly full support in most cases except for R. ampla (82%). R. lagotis groups sister to R. balthica with very low support. The specimens identified as R. lagotis from the Dragoman marshland in Bulgaria fall into one cluster with full bootstrap support together with the R. lagotis specimens from Germany. In the RAxML tree of the cyt-b fragment (not shown) most of the basal branches show very low support. This very low support is expressed by a polytomy of R. auricularia, R. labiata, R. balthica, R. ampla, and R. lagotis. As in the MP tree the specimens identified as R. lagotis from the Dragoman marshland in Bulgaria fall into one cluster together with the R. lagotis specimens from Germany (bootstrap support 98%). In the hypothesis of the phylogenetic relationships of R. lagotis based on one of the 4360 best MP trees of the nuclear ITS-2 spacer (Fig. 2, tree length=997, CI=0.9057, RI=0.9829) all basal branches are fully or very highly supported. All species-clades have full support. R. lagotis groups sister to R. ampla with full support. The Radix specimens from the Dragoman marshland fall into one cluster with full bootstrap support together with the R. lagotis specimens from Germany, as well as with the sequences from R. lagotis specimens from Czech Republic and Austria from GenBank. In the RAxML tree of the nuclear ITS-2 spacer (not shown) the basal branches show high, low or very low support. Most of the species clades received low or very low support. This very low support is again expressed by a polytomy of R. balthica, R. ampla, and R. lagotis. But as in the MP tree the specimens identified as R. lagotis from the Dragoman marshland fall into one cluster with the R. lagotis specimens from Germany, as well as with the GenBank sequences from R. lagotis specimens from Czech Republic and Austria, however the bootstrap support in this case is only 58%.
Figure 3. Radix lagotis specimen from Dragoman marshland kept in aquaria.
Morphology The shells of the R. lagotis specimens from Dragoman marshland have a more slender conical egg-shaped form (Fig. 3, 4C, 5) than the specimens from Germany examined. The latter show a more rounder egg- shaped form (Fig. 4A, B). The shell height of the largest specimen examined from Dragoman marshland (Radix lagotis 10) is 14.02 mm with the number of whorls of 3.75. The largest specimen from Germany examined (Radix lagotis 3) has a shell height of 18.13 mm and it has 4 whorls. The mantle pigmentation of the Dragoman marshland specimens shows two types (Fig. 4C, 5): (1) mantle and mantle collar of a deep blue-black with roundish patches of lighter patches of gray-black on mantle, mantle edge bluish grey (Fig. 5A) and (2) mantle black or grey-yellow with numerous roundish distinct spots of white or grey-yellow, that
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Figure 4. Variability in Radix lagotis shells, mantle pigmentation, form and length of the bursa copulatrix and the length of the bursa duct: A) Germany, Saxony, pond Vierteich near Freitelsdorf (Radix lagotis 3, shell height 18.13 mm); B) Germany, Saxony, dam Doellnitzsee near Mutzschen (Radix lagotis 7, shell height 17.16 mm); C) Bulgaria, Dragoman marshland 40 km west of Sofia (Radix lagotis 9).
are very variable in size; mantle collar bluish white with numerous irregular small patches of black; mantle edge bluish white or yellowish (Fig. 4C, 5B). Thus the mantle pigmentation of the specimens of the Dragoman marshland type 2 differ from the mantle pigmentation of specimens from Germany examined, these shows a few small bluish distinct spots on a nearly black mantle and mantle edge (Fig. 4A) or a few
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blurred medium sized spots on a black mantle and a deep bluish mantle collar with numerous irregular small patches of black (Fig. 4B). In one specimen from Dragoman marshland that had a filled bursa the ratio of the length of the bursa duct to that of the bursa was of 1:1.44 (Fig. 4C). In two specimens from Germany that could be examined with filled bursas the ratios were of 1:2.0 (Fig. 4A) and 1:1.1 (Fig. 4B). In all specimens examined the bursa duct entered on the ventral side into the provagina above the female vent. The shape of the bursa is elongated egg-shaped or pear-shaped, if filled. Praeputium and penis sheath could be measured in two specimens from Dragoman marshland. The ratio of the length of the praeputium to that of the penial sheath varies from 1.20:1 to 1.93:1. In three specimens from Germany the following ratios of the length of the praeputium to that of the penial sheath were found: 0.94:1, 0.92:1, and 1.23:1.
Figure 5. Variability in Radix lagotis shell and mantle pigmentation of two specimens from Dragoman marshland 40 km west of Sofia: A) Radix lagotis 10, shell height 14.02 mm; B) Radix lagotis 12, shell height 13.68 mm.
Discussion
Molecular genetics The results of our molecular genetic analyses of the nuclear ITS-2 spacer and the mitochondrial cyt-b fragment (329 bp) (Figs 1, 2) allow the conclusion that the Radix specimens from the Bulgarian Dragoman marshland identified morphological as R. lagotis indeed belong to this species. The different topology of branches within the MP and the RAxML trees based on cyt-b and ITS-2 can be explained with the fact that the maximum parsimony analyses use alignment gaps as additional distinguishing character (fifth base).
Morphology The shells and the mantle pigmentation of the R. lagotis specimens examined show a certain variability. Although the specimens from the Dragoman marshland attract attention by their more slender shells (Fig. 3, 4C, 5), it is already clear that R. lagotis cannot be differentiated from R. balthica by shell form and mantle pigmentation, because the same shell shapes and pattern of mantle pigmentation can be found in both species (see also Schniebs et al. 2011). The ratio of the length of the praeputium to that of the penial sheath varies from 0.92 to 1.93 (n=5). This lies within the limits of this character in R. labiata (0.58 to 2.53) (Schniebs et al. 2013) whereas variation in R. balthica is described as an interval between 0,4:1 and 1.6:1 by Meier-Brook in Glöer (2002). It is a first indication that this character is not really useful for differentiation between R. lagotis, R. balthica, R. labiata, R. ampla and R. auricularia. Hubendick (1953) described the relative length of the penis sheath as considerably shorter than the praeputium in some specimens and longer in others. Stadnichenko (2004) and Kruglov (2005) found the penial sheath in R. lagotis shorter than the praeputium and give a ratio of the length of the praeputium to that of the penial sheath of 1.30. According to Khokhutkin et al. (2009) as well as Andreeva et al. (2010), the penial sheath is one third shorter than the praeputium in R. lagotis. The ratio of the length of the bursa to the length of the bursa duct varies from 1:1.1 to 1:2.0 (n=3) when the bursa is filled. So the filled bursa could be two times longer than the bursa duct as described by
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Kruglov (2005), but both could also have nearly the same length or the bursa duct can reach to about 2/3 of the bursa (if filled) (see also Schniebs et al. 2011, 2013). Huňová et al. (2012) described the length of the bursa duct with “between half and full length of the bursa” and Stadnichenko (2004) with “half length of the bursa”. So this character also shows certain variability, but the longer bursa duct is still the best character for differentiation of R. lagotis from R. balthica and R. labiata. Further investigations are needed to analyse the intraspecific variability and distribution of R. lagotis.
Acknowledgements We would like to express our thanks to Prof. Dr Uwe Fritz (SNSD) for financial support for the greater part of the molecular analyses, Anke Müller (SNSD) for some sequences and the instructions of KS in lab work, as well as to Dr André Reimann (SNSD), Dr Michael L. Zettler (Leibnitz Institute for Baltic Sea Research Warnemünde), Prof. Dr ladimir Pe i (University of Montenegro), Robert Haldemann (Strausberg), Michael Korn (University of Konstanz, Limnological Institute), Christoph Oberer (Natural History Museum Basel), Holger Menzel-Harloff (Wismar), Willy de Mattia (Muggia), Dr Christian Albrecht (Justuts Liebig University Giessen), Dr Maxim V. Vinarski and Dr Alfried V. Karimov (Omsk State Pedagogical University), Dr Vitaliy Anistratenko (Institute of Zoology NAS of Ukraine, Kiev), Dr Nicole Schröder-Rogalla (Munich), Susanne Thiel (Munich), Kate ina Huňova (Prague), Andrea Pohl (Dresden), Gudrun Rutsch (Dresden) and Christa Schniebs (Oelsnitz) for the material collected and provided.
References
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