A NEW SPECIES OF BYTHINELLA MOQUIN-TANDON, 1855 (CAENOGASTROPODA: TRUNCATELLOIDEA) FROM NAXOS ISLAND, GREECE

A bstrAct : A new species of Bythinella : B. walensae is described from Aria spring at Naxos Island, Greece. Cytochrome oxidase subunit I ( COI ) sequences of mtDNA, as well as internal transcribed spacer (ITS-1) of nuclear ribosomal DNA indicate distinctness of B. walensae . The shell, female reproductive organs and penis are described. The most characteristic of this species are: slender shell, narrow aperture, J-shaped bursa coupled with bulky and spherical receptaculum, and the length ratio of penis arms. Simple anatomy coupled with wide variation ranges overlapping between the species are emphasised.


INTRODUCTION
The genus Bythinella Moquin-Tandon, 1855 is a group of freshwater, dioecious, oviparous snails inhabiting springs and subterranean waters (Giusti & Pezzoli 1977, FAlniowski 1987. Its wide range extends from the Iberian Peninsula to western Asia, and from southern Poland to southern Greece. The diversity of Bythinella has mainly been studied in western, southern and central Europe (boeters 1973, 1998, Giusti & Pezzoli 1977, FAlniowski 1987, but the studies have mostly focussed on the external morphology and anatomy, initially of just the shell and, later, of the soft parts. It has been demonstrated, however, that morphology alone cannot be used for unequivocal species delimitation due to the limited number of taxonomically useful characters and their wide variation (FAlniowski 1987, 1992, szArowskA 2006, bicHAin et al. 2007a, b, FAlniowski et al. 2009a). More recent studies using molecular data have made it possible to distinguish several Bythinella species and also to synonymise a few nominal species (bicHAin et al. 2007a, b, HAAse et al. 2007, benke et al. 2009, FAlniowski et al. 2009a, b, 2012b, FAlniowski & szArowskA 2011, 2012. Several species of Bythinella have been described across Europe, mainly in western and central parts of the continent (e.g. rAdomAn 1976, 1983, bicHAin et al. 2007a. In contrast, relatively few Bythinella species have been identified in Greece and those that have been were initially identified based on morphological characters (rAdomAn 1976, 1983, scHütt 1980, reiscHütz et al. 2008). More recent research combining morphology and genetic markers, cytochrome oxidase subunit I (COI) and internal transcribed spacer (ITS-1), revealed eight putatively distinct species in continental Greece: two in the Peloponnese, one in the Attica and Parnassos Mts (Central Greece), one on Lefkas Island and four in northern Greece (FAlniowski & szArowskA 2011, 2012. During our research on the Aegean Bythinella (szArowskA et al. 2016) we found seven more molecularly distinct clades, of presumably species level.
The most distinct was the one inhabiting two springs at Naxos Island. For COI the p-distances between the clade inhabiting Naxos and the other clades were within 0.065-0.090, mean 0.080 for Naxos and the other Aegean clades, and 0.741 for Naxos and all the Greek Bythinella (szArowskA et al. 2016

MATERIAL AND METHODS
About ten specimens of Bythinella were collected at locality N01 ( Fig. 1): small spring at Aghio Kyriaki, 37°04'09.6"N, 25°26'48.9"E. About twenty specimens were collected at locality N02 ( Fig. 1), in a somewhat bigger spring Aria (Aria Pygi), 37°02'11.1"N, 25°29'37.8"E. Snails were collected by hand or sieve, and fixed with 80% ethanol. The shells and soft parts were photographed with a CANON EOS 50D digital camera, under a NIKON SMZ18 microscope with dark field and phase contrast. Five males and five females were dissected, the drawings were made from the photographs. A NIKON DS-5 digital camera measurement system was used to measure seven shell morphometric parameters ( The other ten paratypes destroyed for DNA extraction. Ten sequences of cytochrome oxidase subunit I (COI), GenBank numbers: KT353699-KT353700 from locality N01 (Aghio Kyriaki); KT353701-KT353708 from the type locality N02 (Aria Pygi).
Ten sequences of internal transcribed spacer ITS-1, GenBank numbers: KT353614-KT353615 from locality N01 (Aghio Kyriaki), and KT353616-KT353623 from the type locality N02 (Aria Pygi) (szArowskA et al. 2016). Diagnosis. Shell ovate-conical, with relatively high spire and simple outer lip, soft parts pigmented black, female reproductive organs with big J-shaped bursa copulatrix, big spherical receptaculum seminis, and long and narrow loop of renal oviduct, penis with flagellum and the arm containing vas deferens of similar length, narrow, especially the arm containing vas deferens. The most characteristic of this species are: slender shell, narrow aperture, J-shaped bursa coupled with bulky and spherical receptaculum, and the length ratio of penis arms. Description. Shell (Figs 2-13 Distribution and habitat. Known from the type locality (Aria spring), but also from another locality at Naxos Island (spring at Aghio Kyriaki).

DISCUSSION
Species boundaries in Bythinella are still unclear, despite hundreds of sequences now available in the GenBank (FAlniowski 1987, 1992, bicHAin et al. 2007a, b, szArowskA & FAlniowski 2008, benke et al. 2009, FAlniowski & szArowskA 2011, 2012, szArowskA et al. 2016. The simple and highly variable morphology is not sufficient even for species determination, thus molecular data are necessary. In Greece there are several molecularly discernible clades, most of them of species level. However, they have not been described so far, thus we cannot present formal differential diagnoses between B. walensae and its closest relatives. B. walensae is molecularly more distinct than the other still unnamed clades (Fig. 22).
The shell height, spire height and aperture height had the highest loadings in PC1, and the angle between the body whorl suture and the horizontal sur- clades. The same overlap is true of the female reproductive organs and penes. This is, however, a normal picture within the Truncatelloidea (FAlniowski 1987, 1992, FAlniowski et al. 2012a. One could either consider one or a few specimens of each species and, then, discover evident differences between the species, or study numerous specimens of each nominal taxon -and see the (nearly) continuous variation. Morphological characters within the Truncatelloidea usually lack the states which are unique, or characteristic at least, the ranges of variation are nearly always wide and overlap between the species or even genera (e.g. FAlniowski 1987, szArowskA & FAlniowski 2008, FAlniowski & szArowskA 2011, FAlniowski et al. 2012a, szArowskA et al. 2016). On the other hand, species identification with DNA sequences is always certain, although species boundaries remain disputable, especially with one locus and small divergence. In the case of B. walensae, however, there are two loci, and the p-distances are high (szArowskA et al. 2016).