VENTROSIA MARITIMA (MILASCHEWITSCH, 1916) AND V. VENTROSA (MONTAGU, 1803) IN GREECE: MOLECULAR DATA AS A SOURCE OF INFORMATION ABOUT SPECIES RANGES WITHIN THE HYDROBIINAE (CAENOGASTROPODA: TRUNCATELLOIDEA)

a bStract : Using molecular data (DNA sequences of mitochondrial COI and nuclear ribosomal 18SrRNA genes), we describe the occurrence of two species of Ventrosia Radoman, 1977: V. ventrosa (Montagu, 1803) and V. maritima (Milaschewitsch, 1916) in Greece. These species are found at two disjunct localities: V. ventrosa at the west coast of Peloponnese (Ionian Sea) and V. maritima on Milos Island in the Cyclades (Aegean Sea). Our findings expand the known ranges of both species: we provide the first molecularly confirmed record of V. ventrosa in Greece, and extend the range of the presumably Pontic V. maritima nearly 500 km SSW into the Aegean Sea. Our data confirm the species distinctness of V. maritima .


INTRODUCTION
Data on the Greek Hydrobiinae, inhabiting brackish waters, are less than scarce. Schütt (1980) did not list any species belonging to this subfamily in his monograph on the Greek Hydrobiidae. Although MuuS (1963MuuS ( , 1967 demonstrated that the morphology of the penis and the head pigmentation must be studied to determine a species, since the shell characters are too variable, many hydrobiologists and marine biologists report records of those species determined with the shell characters alone (e.g. koutSoubaS et al. 2000(e.g. koutSoubaS et al. , evagelopouloS et al. 2009). In fact, the differences shown by MuuS (1963MuuS ( , 1967, and confirmed by FalniowSki (1986FalniowSki ( , 1987FalniowSki ( , 1988, are good enough to distinguish between the genera: Hydrobia Hartmann, 1821, Peringia Paladilhe, 1874, andVentrosia Radoman, 1977, since morphostatic evolution, as defined by daviS (1992), is characteristic of the group. Non-adaptive radiation marked by the rapid proliferation of species without ecological differentiation (gittenberger 1991) (Vanatta, 1924) with European Ventrosia ventrosa (or, at least, that they are congeners) is not confirmed.
The present paper reports the occurrence of two species of Ventrosia in Greece, identified using molecular data.

MATERIAL AND METHODS
The material was collected with a sieve and found at two localities in Greece: (1) Ahivadolimni at Milos Island, Cyclades, Aegean Sea (Fig. 1), a saline lagoon, 36°41'17"N, 24°26'37"E, with no continuous contact with the sea, during the hot season in the form of several small, isolated water bodies, with rich submerged macrophyte vegetation (Fig. 2), and numerous snails on its bottom (Fig. 3). This is the locality described by pantazidou et al. (2006) in their paper on the euendolithic shell-boring cyanobacteria and chlorophytes; the authors reported the occurrence of Hydrobia acuta (Draparnaud, 1805) in this habitat.
(2) A big brackish lake West of Kato Xirokhórion ( Fig. 1), South of Argos, close to the West coast of Peloponnese (Peloponnisos), Ionian Sea, 37°30'30"N, 21°36'52"E, with rich vegetation of semi-submerged macrophytes. Snails were washed twice in 80% ethanol and left to stand in it for around 12 hours. The ethanol was changed twice more within 24 hours and finally, after a few days, the 80% solution was replaced with a 96% one, in which the samples were stored at -20°C. The shells were photographed with a CANON EOS 50D digital camera.
DNA was extracted from foot tissue. 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. The PCR reaction was performed with the following primers: LCO1490 (5'-GGTCAACAAATCATAAAGAT ATTGG-3') (FolMer et al. 1994) and COR722b (5'-TAAACTTCAGGGTGACCAAAAAATYA-3') (wilke & daviS 2000) for the cytochrome oxidase subunit I (COI) mitochondrial gene and SWAM18SF1 (5'-GAATGGCTCATTAAATCAGTCGAGGTTCCT TAGATGATCCAAATC-3'), and SWAM18SR1 (5'-ATCCTCGTTAAAGGGTTTAAAGTGTACTCATTCC AATTACGGAGC-3') for the 18S ribosomal RNA nuclear gene (paluMbi 1996). The PCR conditions were as follows: COI -initial denaturation step of 4 min at 94°C, followed by 35 cycles of 1 min at 94°C, 1 min at 55°C 2 min at 72°C, and a final extension of 4 min at 72°C; 18S -initial denaturation step of 4 min at 94°C, followed by 40 cycles of 45 s at 94°C, 45 s at 51°C, 2 min at 72°C and, after all cycles were completed, an additional elongation step of 4 min at 72°C was performed. The total volume of each PCR reaction mixture was 50 μl. To check the quality of the PCR products 10 μl of the PCR product was ran on 1% agarose gel. The PCR products were purified using Clean-Up columns (A&A Biotechnology) and were then sequenced in both directions (hilliS et al. 1996) using BigDye Terminator v3.1 (Applied Biosystems), following the manufacturer's protocol and with the primers described above. The sequencing reaction products were purified using ExTerminator Columns (A&A Biotechnology); DNA sequences then underwent electrophoresis on an ABI Prism sequencer. Four sequences of 18S, and eight of COI, were deposited in GenBank (Accession numbers: KJ406193-KJ406204). We also used the following COI sequences from GenBank: AY616139 for Ventrosia maritima (kevrekidiS et al. 2005), AF253077 for V. pontieuxini Radoman, 1973Radoman, (daviS et al. 1998, and AF118357 for V. ventrosa (Montagu, 1803) (wilke & daviS 2000). The sequences were aligned by eye using BioEdit 5.0.0 (hall 1999). MEGA5.10 (taMura et al. 2011) was applied to infer p-distances and phylogenetic relationships between the sequences, applying maximum likelihood technique. Nodal support was estimated using the bootstrap (BS) approach (FelSenStein 1985). Bootstrap values for ML trees were calculated using 10,000 bootstrap replicates, with MEGA5.10 and the same model parameters (HKY + Γ: Hasegawa, Kishino and Yano with Γ distribution) as for ML analysis.  (Table 1) were lower in the population from Milos (mean p-distance = 0.001) than in the population from Peloponnese (mean p-distance = 0.009). Mean p-distance in the group consisting of the Milos specimens and the ones from the GenBank: V. maritima from the Evros, Aegean Sea was 0.003, and, including the one of "B. pontieuxini" from the Black Sea was 0.006; in the group Figs 4-10. Shells of Ventrosia maritima from Ahivadolimni, scale bar equals 1 mm consisting of Peloponnese population and V. ventrosa from GenBank was 0.013; mean distance between the above two groups equaled 0.055.

RESULTS
In a maximum likelihood tree (Fig. 19), the four COI sequences from Milos form a clade with the ones of V. pontieuxini and V. maritima from the GenBank, and all four sequences from Peloponnese form a clade with the one of V. ventrosa from GenBank, with bootstrap support 100.

DISCUSSION
As could be seen in the photographs (Figs 4-18), shell variability of the two species overlaps, and species determination based on the shell characters is impossible.
Both p-distances, and the ML tree support the species assignment of the Milos populations to V. maritima, and the ones from Peloponnese to V. ventrosa. 100% bootstrap support, coupled with the level of intergroup p-distances, support species distinctness of V. maritima, sometimes questioned. The sequence of V. maritima from the Black Sea ("V. pontieuxini" in Fig. 19) differs more from the ones from Milos than the sequence from the Evros Delta, which confirms isolation by distance pattern of genetic differentiation in Ventrosia. Genetic differentiation within V. ventrosa is higher than within V. maritima, which could be expected in such a widely distributed species (e.g. FalniowSki 1987), but the intrapopulation diversity in the Peloponnese population was also high -somewhat higher than the one within all the specimens of V. maritima studied so far. radoMan (1983) reports V. ventrosa from the Adriatic Sea. bank (2012) does not report this spe-cies from Greece. To our knowledge, our study is the first molecularly confirmed report of the occurrence of V. ventrosa in this part of the Mediterranean Sea. radoMan (1973) described a new species V. pontieuxini, postulating species-level distinctness of the Black Sea Ventrosia, formerly classified as V. ventrosa. Later, V. pontieuxini Radoman, 1973 was considered a synonym of V. maritima (Milaschewitsch, 1916). As mentioned in the Introduction, kevrekidiS et al. (2005) found this species at the Evros Delta lagoons at the border of Greece and Turkey, East Aegean Sea. Thus, our finding expands the range of this presumably Pontic species 470 km SSW in the Aegean Sea. Last but not least, our study confirms that molecular data are very useful for information on the distribution and species ranges within the brackish water Hydrobiinae.