SPECIES DISTINCTNESS OF BITHYNIA CETTINENSIS CLESSIN, 1887 AND B. ZETA GLÖER ET PEŠIĆ, 2007 (CAENOGASTROPODA:

a bStract : Shells of Bithynia : widely distributed B. tentaculata (Linnaeus), Balkan B. cettinensis Clessin from Cetina River in Croatia, and B. zeta Glöer et Pešić from Vitoja spring at Lake Skadar, Montenegro, as well as male genitalia of B. zeta and B. cettinensis were examined. The shells of all three taxa are similar, also the penes differ only slightly. The mean value of the length ratio of the tubular penial gland (measured along its curvature) to the penis right arm, measured along the curvature of its right margin, was 1.63 for B. zeta , and 1.33 for B. cettinensis . Those values differed slightly, especially compared to 5.0–5.9 for B. tentaculata . In the maximum likelihood (ML), as well as Bayesian (BI) trees, B. cettinensis and B. tentaculata were sister clades with p-distance of 0.007, and B. zeta was more distinct, with p-distance of 0.122 to B. cettinensis and 0.154 to B. tentaculata . The species distinctness of the three studied taxa was confirmed.


INTRODUCTION
The Bithyniidae Gray, 1857 inhabit fresh and brackish waters of Europe, Asia Minor, northern Asia, India, Indochina, China, part of Australia, western Africa, Lake Tanganyika, and some introduced localities in North America (falniowSki 1989). Apart from the "normal" feeding on detritus, they are also filter-feeders (lilly 1953(lilly , fretter & GraHam 1962. Bithynia tentaculata (Linnaeus, 1758) is one of the most widely distributed and most common freshwater species. Some other nominal species of Bithynia Leach, 1818, whose distinctness remains unclear, are known from southern Europe. cleSSin (1887) described B. cettinensis Clessin, 1887, from Cetina River in Croatia. Glöer & Pešić (2007) described three new species of Bithynia from Lake Skadar in Montenegro: B. zeta Glöer et Pešić, 2007, B. skadarskii Glöer et Pešić, 2007, and B. radomani Glöer et Pešić, 2007. The penis and penial gland morphology presented by Glöer & beran (2009) for B. cettinensis was based on only one penis, most probably somewhat damaged, and molecular data for the European Bithynia are known only for B. tentaculata. The aim of the present paper was to describe the penial morphology of B. cettinensis and to check the species distinctness of B. tentaculata, B. cet tinensis, and B. zeta, applying molecular data, namely cytochrome oxidase subunit I (COI) sequences.
For molecular study, the snails were washed twice in 80% ethanol and left to stand in it for ca. 12 hours. Afterwards, the ethanol was changed twice in 24 hours and finally, after a few days, the 80% solution was exchanged for a 96% one, stored at −20 °C.
The shells were photographed with a CANON EOS 50D digital camera, under a NIKON SMZ18 microscope with dark field. Dissection was done under NIKON SMZ18 microscope, and photographed with dark field. The measurements of the penis and tubular penial gland were taken on the photographs, with SILVA Map Measurer Plus curvimeter. The morphological terminology follows HerSHler & Ponder (1998).
The tissue was hydrated in TE buffer (3 × 10 min.); then total genomic DNA was extracted with the SHERLOCK extracting kit (A&A Biotechnology), and the final product was dissolved in 20 μl TE buffer.   (Hall 1999). The saturation test (xia 2000, xia et al. 2003) was performed using DAMBE (xia 2013). In the phylogenetic analysis additional sequences from GenBank were used (Table 1). The data were analysed using approaches based on Bayesian inference (BI) and maximum likelihood (ML). We applied the GTR model, whose parameters were estimated by the RaxML (StamatakiS 2014). The BI analyses were run using MrBayes v.

MORPHOLOGY
The shells of Bithynia tentaculata (Figs 3-6) and B. cettinensis (Figs 7-11) are slightly variable and similar: only smaller dimensions and somewhat lower spire distinguish B. cettinensis. The shells of B. zeta (Figs 12-18) are more variable, some of them with high spire (Fig. 15), some other resembling the ones of B. tentaculata (Figs 3-6). The penis is bifid in B. zeta (Figs 19-22), B. cettin ensis (Figs 23-29) and B. tentaculata (Fig. 30). Its right arm includes the terminal section of vas deferens. The left arm is known as flagellum, containing the outlet of tubular penial gland situated apically. The penial gland is long and coiled, proximally extending into the cephalic haemocoel. The mean length ratio of the tubular penial gland (measured along its curvature) to the penis right arm, measured along the curvature of its right margin (from point X to point Y in Fig. 30), is 1.63 for B. zeta, and 1.33 for B. cettin ensis (Table 2). PR -prostate; TE -testis; TPG -tubular penial gland; VD -vas deferens; X, Y -points between which the length of right arm, along its right margin, is measured)

MOLECULAR ANALYSIS
In total we analysed seven new sequences of cytochrome oxidase subunit I (COI) (552 bp, GenBank Accession Numbers see Table 1). The tests of xia et al. (2003) revealed no saturation. The topology of the trees obtained from BI and ML analyses was identical. The Bithynia sequences formed a distinct lineage with four clades (Fig. 31). B. cettinensis and B. tentaculata were sister clades with p-distance of 0.007. B. zeta was more distinct, with p-distance of 0.122 to B. cettinensis and 0.154 to B. tentaculata. The distances between the three studied species were not smaller than those between the other bithyniid species.

DISCUSSION
The anatomy of the reproductive organs of Bithynia was described by lilly (1953), fretter & GraHam (1962), falniowSki (1989, 1990), and SzarowSka (2006. The female reproductive organs are characteristic of the Bithyniidae, but can hardly be used in species distinction (falniowSki 1990). However, in some publications (e.g. falniowSki 1989, 1990, Glöer & Pešić 2007, Glöer & beran 2009) the term flagellum is used not for the left arm of the penis, but for the tubular penial gland.
falniowSki (1989,1990) found that in the Polish Bithynia the length ratio of the tubular penial gland (measured along its curvature) to the penis right arm, measured along the curvature of its right margin (from point X to point Y in Fig. 30), was the only anatomical character to allow for species discrimination. The value was 5.0-5.9 for B. tentaculata (Linnaeus, 1758), 2.5-3.25 for B. leachi (Sheppard, 1823), and ca. 4.3 for B. troscheli (Paasch, 1842). Later, this length ratio was considered in species delimitation in Bithynia (e.g. Glöer & yildirim 2006, Glöer & Pešić 2007, Glöer & rolán 2007). For the Bithynia from Lake Skadar, the ratio was: 1.5 for B. zeta, 2 for B. skadarskii, and 4 for B. ra domani. Considering the photographs in the present paper, as well as the drawings of falniowSki (1989,1990), compared with the ones of Glöer & yildirim (2006), Glöer & Pešić (2007), and Glöer & rolán (2007), it is clearly visible that the shape and proportions of the penis are more variable, and interspecific differences less marked. The same concerns the tubular gland/penis proportion, less constant in our measurements, but still confirming the determination of our Vitoja spring material as B. zeta.
Molecularly Bithynia zeta is markedly distinct from the other studied species, and -especially compared with the other sequences of the Bithyniidae from the GenBank -the molecular data confirm its distinctness as a species. On the other hand, the differences between B. cettinensis and B. tentaculata are not necessarily large enough to confirm the distinctness of those two species. Only somewhat smaller distances can be seen within B. siamensis (Lea, 1856), but also between the two nominal species of Gabbia Tryon, 1856.