A new genus and species of Hydrobiidae Stimpson, 1865 (Caenogastropoda, Truncatelloidea) from Peloponnese, Greece

Abstract Minute caenogastropod brackish-water gastropods, formerly classified as Hydrobia, are important elements of the brackish-water fauna and were objects of intensive study for many years. Until now, five genera have been distinguished, most of them represented by a number of species, but rather indistinguishable without molecular data (cytochrome oxidase subunit I – COI). In the eastern Mediterranean region, they are still poorly studied. In this paper, we present a new species of “Hydrobia” from the brackish Moustos spring, Arkadia, eastern Peloponnese, Greece. The shell, protoconch, radula, female reproductive organs, and penis are described and illustrated, together with the molecular (COI) relationships with other hydrobiids. All data confirm that these snails represent a distinct taxon, which must be classified as a new species belonging to a new genus. The formal descriptions are given. The closest, sister taxon is Salenthydrobia Wilke, 2003. The molecularly estimated time of divergence, 5.75 ± 0.49 Mya, coincides with 5.33 Mya, which is the time of the Oligocene flooding that terminated the Messinian salinity crisis. During the latter period, brackish “Lago-Mare” habitats were most probably suitable for the last common ancestor of Salenthydrobia and the newly described genus. Later, the Pliocene flooding isolated the Apennine and Peloponnese populations, promoting speciation.


Introduction
The typically brackish-water caenogastropod snails formerly known as Hydrobia inhabit estuaries and other brackish habitats around the northern Atlantic and adjacent seas; in many places, these small, tiny snails are numerous and their biomass is large. Thus, they form an important component of the brackish water fauna and have been studied for many aspects (for details see Muus 1967;Falniowski 1987;Fretter and Graham 1994). The simple shells show a set of plesiomorphic character states and are extremely variable, making species determination hardly possible (e.g., Muus 1963Muus , 1967Falniowski et al. 1977;Wilke and Falniowski 2001). Muus (1963Muus ( , 1967 demonstrated clear and stable differences in the penis morphology and snout and tentacle pigmentation between the three Baltic species, although the pigmentation was later found to be more variable but still useful for species determination (Falniowski 1986). However, later studies (e.g., Radoman 1973Radoman , 1983Giusti and Pezzoli 1984;Wilke and Pfenninger 2002;Wilke 2003), including those applying molecular data (mostly the partial sequences of mitochondrial cytochrome oxidase), confirmed these morphological differences, but as discriminating the genera. The discrimination of species within these genera was found possible only with molecular data. Currently, five genera are distinguished within Hydrobia s.l.
The genus Hydrobia Hartmann, 1821 (type species Cyclostoma acutum Draparnaud, 1805) is represented by several molecularly distinct species with rather restricted ranges, both Atlantic and Mediterranean. The nomenclatural problems (e.g., Bank et al. 1979;Falniowski and Szarowska 2002) Davis et al. (1989) suggested that North American Ecrobia truncata (Vanatta, 1924) was introduced from Europe and would then be a synonym of Ventrosia ventrosa. This was later confirmed by molecular data . Ecrobia (its type species, following the ICZN rules, Turbo ventrosus Montagu, 1803) is represented by a few molecularly distinguishable species Osikowski et al. 2016) known from the Baltic Sea to the Black Sea. The genus Adriohydrobia (type species Paludina gagatinella Küster, 1852 is known from the Adriatic Sea only; a few nominal spe-cies assigned earlier to Adriohydrobia are molecularly identical with A. gagatinella, and thus they became synonyms (Wilke and Falniowski 2001). The monotypic genus Salenthydrobia Wilke, 2003(type species Salenthydrobia ferreri Wilke, 2003, was found at three closely situated populations in the southernmost part of the Apennine Peninsula (Wilke 2003).
Reliable data are scarce on the group formerly known as Hydrobia in the eastern Mediterranean brackish habitats. The numerous records that exist of Peringia ulvae from the eastern Mediterranean are a good example, as this species does not occur in the Mediterranean Sea. Another example is the monograph on the Greek Hydrobiidae (Schütt 1980), which does not mention any representatives of "Hydrobia". Even though shells cannot be used for species determination, many hydrobiologists and marine biologists still record species determined by shell characters alone (e.g., Koutsoubas et al. 2000;Evagelopoulos et al. 2009). As stated above, the head pigmentation and penis morphology make identification of genera possible (Muus 1963(Muus , 1967Falniowski 1986Falniowski , 1987, while female reproductive organs are taxonomically less useful (Falniowski 1988). At species level, all morphological characters are hardly applicable because of morphostatic evolution (Davis 1992). Non-adaptive radiation, marked by the rapid proliferation of species without ecological differentiation (Gittenberger 1991), results in a flock of species that need not differ either morphologically or ecologically. Thus, at the species level in the Hydrobiinae, morphological characters cannot be used for species recognition alone, and molecular data are inevitably necessary to distinguish taxa.
So far, two species of Ecrobia have been recorded from six localities in Greece (Kevrekidis et al. 2005;Osikowski et al. 2016), one of them (E. ventrosa) at the western coast of the Peloponnese Peninsula. In summer 2009, a few specimens of "Hydrobia" were collected at the brackish Moustos spring at Arkadia, on the eastern coast of the Peloponnese. The aim of the present paper is to establish their phylogenetic position, applying morphological and molecular data.

Material and methods
The snails were collected in 2009 by sieve at the Moustos spring ( Fig. 1), Arkadia, eastern Peloponnese, Greece (2 km N of Aghios Andreas, under the road from Astros to Korakovouni,37.3845,22.7444). The spring is situated about 500 m from the Aegean Sea. This large, brackish spring with sulphide content, rising from calcareous breccia, feeds a larger seashore lagoon called Limni Moustos with adjacent swamps hosting a bird reserve. The specimens were taken from the spring and the stony ridge towards the lagoon. No specimens were found in the lagoon.
The snails were fixed in 80% ethanol. The shells were photographed with a Canon EOS 50D digital camera, under a Nikon SMZ18 microscope with a dark field. The dissections were done under a Nikon SMZ18 microscope with a dark field, equipped with Nikon DS-5 digital camera, whose captured images were used to draw anatomical structures with a graphic tablet. Morphometric parameters of the shell were measured by one person using a Nikon DS-5 digital camera and ImageJ image analysis software (Rueden et al. 2017). The penes were photographed under Motic microscope with a dark field. The radulae were extracted with Clorox, applying the techniques described by Falniowski (1990), and examined and photographed using a HITACHI S-4700 scanning electron microscope.
Snails for molecular analysis were fixed in 80% ethanol. DNA was extracted from whole specimens; tissues were hydrated in TE buffer (3 × 10 min); then, total genomic DNA was extracted with the SHERLOCK extraction kit (A&A Biotechnology), and the final product was dissolved in 20 μl of tris-EDTA (TE) buffer. The extracted DNA was stored at −80 °C at the Department of Malacology, Institute of Zoology and Biomedical Research, Jagiellonian University in Kraków (Poland).
Mitochondrial cytochrome oxidase subunit I (COI) and nuclear histone 3 (H3) loci were sequenced. Details of PCR conditions, primers used, and sequencing were given by Szarowska et al. (2016a). Sequences were initially aligned in the MUSCLE (Edgar 2004) program in MEGA 7 (Kumar et al. 2016) and then checked in BI-OEDIT 7.1.3.0 (Hall 1999). Uncorrected p-distances were calculated in MEGA 7. The estimation of the proportion of invariant sites and the saturation test (Xia 2000;Xia et al. 2003) were performed using DAMBE (Xia 2013). In the phylogenetic analysis additional sequences from GenBank were used as reference (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 RaxML (Stamatakis 2014). The Bayesian analyses were run using MrBayes v. 3.2.3 (Ronquist et al. 2012) with defaults of most priors. Two simultaneous analyses were performed, each with 10,000,000 generations, with one cold chain and three heated chains, starting from random trees and sampling the trees every 1,000 generations. The first 25% of the trees were discarded as burn-in. The analyses were summarised as a 50% majority-rule tree. The ML analysis was conducted in RAxML v. 8.2.12 (Stamatakis 2014) using the RAxML-HPC v. 8 on XSEDE (8.2.12) tool via the CIPRES Science Gateway (Miller et al. 2010). To calibrate the molecular clock for COI, the divergence time between Peringia ulvae and Salenthydrobia ferrerii (Wilke 2003), with correction according to Falniowski et al. (2008), were used. The likelihoods for trees with and without the molecular clock assumption in a likelihood ratio test (LRT) (Nei and Kumar 2000) were calculated with PAUP. The relative rate test (RRT) (Tajima 1993) was performed in MEGA. As Tajima's RRTs and the LRT test rejected an equal evolutionary rate throughout the tree, time estimates were calculated using a penalized likelihood method (Sanderson 2002) in r8s v. 1.7 for Linux (Sanderson 2003).

GNHM
Goulandris Natural History Museum. Athens, Greece; HNHM Hungarian Natural History Museum, Budapest; NHMW Natural History Museum Vienna, Austria; NHMUK The Natural History Museum, London UK.

Results
The shells (Fig. 2) are broad, ovate-conic with a few flat whorls, rapidly growing and separated by moderately deep suture. Shell measurements are presented in Table 2. Nearly all adult shells have a corroded apex, and most display some injuries, and secondarily healed crevices, perhaps caused by the corrosion by sulphides. Very frequently there are scalariform shells, looking like they were secondarily formed after dissolution of the "normal" shell. The snout and tentacles are uniformly and intensively black pigmented. The female reproductive organs (Fig. 3) have a prominent spiral of black- Table 2. Shell measurements of Achaiohydrobia moreana; specimen symbols as in Fig. 2; measured variables: see Fig. 2 pigmented renal oviduct. The penis (Fig. 4) has a broad proximal part and long and narrow filament, without any outgrowth. The character states listed briefly above allow the classification to neither known species nor any known genus in the Hydrobiidae. We obtained two new sequences of COI (479 bp, GenBank Accession Numbers MW741741-MW741742), and two new sequences of H3 (309 bp, GenBank Accession Numbers MW776415-MW776416). The tests by Xia et al. (2003) for COI and H3 revealed no saturation. In all analyses, the topologies of the resulting phylograms were identical in both the ML and BI. The phylogram based on the cytochrome oxidase (Fig. 5) clearly show the position of this taxon within the "Hydrobia" as widely understood, and as a sister taxon (bootstrap support 75%) of Salenthydrobia ferreri, with an estimated divergence time of 5.75 ± 0.49 Mya. The pairwise p-distances (Table 3) calculated for the taxa shown in the phylogram (Fig. 5) are typical rather for inter-generic level.
Diagnosis. Shell broad, ovate-conic with a few flat whorls, rapidly growing and separated by a moderately deep suture; female reproductive organs with prominent, massive swelling of the spiral of the oviduct; oval bursa copulatrix with the duct longer than the bursa, receptaculum seminis prominent but smaller than the bursa, with the duct slightly distinguishable; penis tapering, widened at the base, without any outgrowths (nonglandular lobes) and without the distal papilla.
Derivatio nominis. The genus name refers to Achaia, one of the ancient names of Greece and the Greek people.
Remarks. The tapering penis with its broad base distinguishes Achaiohydrobia from Hydrobia and Peringia. The lack of any non-glandular outgrowths (lobes) distinguishes it from Hydrobia, Peringia, and Ventrosia. The lack of the distal papilla on the penis distinguishes it from Salenthydrobia. The massive swelling of the long spiral renal oviduct differentiates Achaiohydrobia from all other genera besides Hydrobia. The molecular divergence between Achaiohydrobia and the other genera (p = 0.109-0.167 for mitochondrial COI) is typical of the genus-level in Hydrobiidae. Holotype. GNHM 39589, leg. M. Szarowska and A. Falniowski, 16.07.2009; ethanol-fixed specimen ( Fig. 2A), brackish water of Moustos spring, 2 km N of Aghios Andreas, Arkadia, eastern Peloponnese, Greece (37.3845,22.7444), creeping on the stones and gravel.
Diagnosis. Shell broad, ovate-conic with a few flat whorls, rapidly growing and separated by a moderately deep suture; female reproductive organs with a prominent, massive swelling of the spiral of the oviduct; oval bursa copulatrix with the duct longer than the bursa, receptaculum seminis prominent but smaller than the bursa, with the duct slightly distinguishable; penis tapering, widened at the base, without any outgrowths (no glandular lobes) and without the distal papilla (diagnosis the same as for this monotypic genus).
Description. Shell (Fig. 2) broad, thick-walled, brownish, moderately translucent, up to 2.69 mm high and 1.34 mm broad, ovate-conic with about five flat whorls, growing rapidly and separated by moderately deep suture. Conical spire height about 0.25 of the shell height; body whorl prominent and broad. Aperture narrow, elongateelliptical; peristome complete and thin, in contact with the wall of the body whorl; umbilicus slit-like or completely covered by the parietal lip. Shell surface smooth, with growth lines hardly visible, often corroded.
Measurements of holotype and sequenced and illustrated shells: Table 2. Shell variability slight (Fig. 2).

− − −
The lateral tooth with 2 -1 -2 prominent and broad cusps, especially the bigger one. Inner marginal tooth with about 11, and outer marginal one with 16-19 cusps.
Soft parts morphology and anatomy. The mantle, snout, and tentacles intensively pigmented uniformly black. The female reproductive organs (Fig. 3) with a prominent, Figure 2. Shells of Achaiohydrobia moreana A holotype; shell measurements are shown: a = shell height, b = body whorl breadth, c = aperture height, d = spire height, e = aperture breadth, α = apex angle, β = angle between body whorl suture and horizontal surface. Scale bar: 1 mm.  massive swelling of the spiral of the intensively pigmented black renal oviduct. The bursa copulatrix oval, situated dextro-lateral to the style sac, with the duct longer than the bursa, the receptaculum seminis big, but smaller than the bursa, elongated, with the duct slightly distinguishable from the receptaculum. The penis (Fig. 4) tapering, widened at the base, without any nonglandular lobes, no papilla, its proximal section slightly broadened (if at all; see Fig. 4D).
Derivatio nominis. The specific epithet moreana refers to Morea, the medieval name of Peloponnese Peninsula.

Discussion
The radula was the first internal structure considered in the gastropod systematics. In the snails formerly known as Hydrobia, whose shells especially lack taxonomically useful characters, the radulae were studied for a long time (Meyer and Möbius 1872;Woodward 1891;Kuhlgatz 1898;Krull 1935;Seifert 1935;Feliksiak 1938;Muus 1963Muus , 1967Bishop 1976;Falniowski et al. 1977;Bank and Butot 1984;Giusti and Pezzoli 1984;Wilke 2003). However, no constant differences were found to distinguish species, although some not quite stable differences in cusp basal cusp number, etc., were observed. The radula of our Achaiohydrobia also bears no unique character states.
The penis morphology of Achaiohydrobia is most similar to that of Adriohydrobia, but in the newly described genus it is more massive. Differentiating character states are the shape of the bursa copulatrix, its shorter duct and the massive swelling of the oviduct, traits not observable in Adriohydrobia. There are detailed data describing the differences in the female reproductive organs (for a summary see Wilke 2003). However, the examination of more numerous specimens (Falniowski 1988) has shown a much variability (also artefactual, physiological, etc.) which gives doubts to the usefulness of these character states for species or genus discriminations (Falniowski 1987(Falniowski , 1989(Falniowski , 1990. The broad base of the penis, listed by Wilke (2003) as one of the apomorphic traits of Salenthydrobia, can hardly be recognized as a synapomorphy characterizing all the specimens of our Achaiohydrobia. Only three specimens of Achaiohydrobia were photographed, and in one of them, no broad base was observed (Fig. 4D).
Brackish-water snails are considered to form isolated populations in suitable habitats, isolated by land, but also by the sea with its full salinity (e.g., Giusti and Pezzoli 1984;Falniowski 1987;Fretter and Graham 1994;Wilke 2003). Only P. ulvae, capable of living in full sea salinity and with lecithotrophic, although short-living, veliger larva, forms populations less affected by isolation. However, considering all data available (see Fretter and Graham 1994 for details), all "Hydrobia" taxa studied so far may be able to survive in open-sea salinity. Anyway, partial isolation promotes allopatric speciation in these gastropods. The type locality of Achaiohydrobia moreana is one of the springs fed by the complicated system of poljes and sink-holes in Arcadia (Higgins and Higgins 1996). In the geologic history of the region (e.g., Rögl 1998Rögl , 1999 there were many events, such as transgressions of the sea, that must have created conditions that would promote speciation. The estimated time of divergence between Achaiohydrobia and the phylogenetically close Salenthydrobia (5.75 ± 0.49 Mya) coincides with 5.33 Mya, which is the time of the Oligocene flooding that ended the Messinian salinity crisis; the estimate is comparable to the divergence time between Salenthydrobia and Peringia (Wilke 2003) and little older than the two species of Ecrobia ). An estimated 1.7% divergence per million years is comparable with other estimated times of divergence for Hydrobiidae (Wilke 2003;Osikowski et al. 2016).
The Messinian salinity crisis affected all the Recent basins of the Mediterranean (Krijgsman et al. 1999;McKenzie 1999). The uplift of the northern African and southern Iberian margins, probably due to the roll back of the Tethys oceanic lithosphere delaminating bands of lithospheric mantle from beneath the continental margin (Duggen et al. 2003), blocked the passage between the Atlantic and Mediterranean about 5.96 Mya. This resulted in the regression of the sea and a lowering of the water level by more than 1000 m. In the place of the Recent Mediterranean, there was a desert, crossed by the vast canyons of large rivers, with some water bodies too rich and others too poor in salt; thus, it was impossible for marine organisms to inhabit the area. There were at least 10 sea transgressions in the Mediterranean during the Messinian (Hsü 1983). The region of the Recent Sea of Marmara served as a gateway between the Paratethys and the Mediterranean. Frequent marine introgressions fed water bodies (basins) of a "Lago-Mare" character: large and deep, although with brackish habitats. Apart from brackish water bodies, there were also highly saline ones, but nowhere in the Mediterranean there were normal marine conditions. We should note, however, that such brackish water "Lago-Mare" basins were probably inhabitable for brackishwater snails. Later, 5.33 Mya at boundary between the Miocene from Pliocene, the abrupt catastrophic Pliocene transgression of water from the Atlantic, probably caused by gravity-induced slumping from the western margin of the Gibraltar arch into the Atlantic abyssal plains (Duggen et al. 2003), filled the Mediterranean basin with sea water again. The rapid, drastic changes formed barriers for the fresh-and brackishwater fauna, promoting speciation processes in many animals (e.g., Wilke 2003;Huyse et al. 2004;Falniowski et al. 2007).