Phylogeography and species distinction in Bythinella (Caenogastropoda: Truncatelloidea: Bythinellidae) in Central Europe and the Balkans

Genus Bythinella Moquin-Tandon, 1856 is represented by over 250 nominal species of minute snails, inhabiting springs of Europe and Asia Minor. Wide variability and eco-plasticity of the shell, coupled with not necessarily justified assumptions of complete isolation of the populations inhabiting particular springs, has resulted in chaos in species-level systematics. The aim of the study is to complete and interpret these data, considering the populations from Central Europe and West Balkans not studied so far. Mitochondrial cytochrome c oxidase subunit I (COI) as well as five nuclear fragments: histone 3 (H3), ribosomal Internal Transcribed Spacer 1 (ITS-1), ribosomal Internal Transcribed Spacer 2 (ITS-2), 18S ribosomal RNA (18S) and 28S ribosomal RNA (28S) were sequenced. For COI the phylogenetic analysis included all the sequences from GenBank to infer the pattern of evolution as well as species distinction. The specimens were collected at 84 localities in Central and South Europe. For COI 151 specimens were sequenced, 92 of them also for the five nuclear fragments. Together with sequences obtained from GenBank, a total of 1,437 COI sequences from Bythinella were used. There were 206 polymorphic sites, and 360 haplotypes. Among the three techniques for species delimitation used, ABGD and ASAP gave the most conservative result: 86 ­mOTUs, grouped in 19 clades (A–T). New sequences were classified in nine clades. Sympatric occurrence of two ­mOTUs or even clades was found in as many as 16 springs and even of three ­mOTUs in two springs. The sympatric occurrence of Bythinella species is thus not exceptional. The morphology of clades was compared based on: shells, radulae, renal and pallial section of the female reproductive organs and penis with the tubular accessory gland. In general, the morphostatic character of evolution was confirmed.