Bythinella is a minute dioecious caenogastropod that inhabits springs in central and southern Europe. In the Balkans, previous studies have addressed its morphological and genetic differentiation within Greece and Romania while the Bulgarian species have remained poorly known. The aim of the present paper has been to expand the knowledge on the subject in Bulgaria. Shell morphology and anatomy of the reproductive organs were examined, and a fragment of the mitochondrial cytochrome oxidase subunit I (COI) gene and the nuclear ribosomal Internal Transcribed Spacer 1 (ITS-1) were sequenced from 15 populations. Additional sequences from eight previously studied populations were included in our analyses. Phylogenetic analyses revealed five main mitochondrial DNA clades, which were partly confirmed by analyses of the ITS-1 sequences. The genetic differentiation between the clades was found to be in the range p=2.4-11.8%. Most of the populations belonged to clade I, representing Bythinella hansboetersi, and were distributed in SW Bulgaria. Clades II and III inhabit areas adjacent to clade I and were most closely related with the latter clade. Much more distinct were clade V, found at one locality in NW Bulgaria, and clade IV, found at one locality in SE Bulgaria, close to the sea. Four populations were found in caves, but only one of these represented a distinct clade. Considering the observed pattern of interpopulation differentiation of Bythinella in Bulgaria, we can suppose that isolation between clades I, II and III may have been caused by glaciations during the Pleistocene. The time of isolation between the above three clades and clade IV coincides with the Messinian Salinity Crisis, and the time of isolation between the clade V and the other four most probably reflects the isolation of the Rhodopes from western Balkan Mts by the seawater of the Dacic Basin.
Keywords: Balkans; Dacic Basin; Gastropoda; Messinian Salinity Crisis; phylogeography.