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Knowledge about subterranean aquatic gastropods is still limited and species inhabiting caves are typically only known from empty shells found at the surface. Phylogenetic analyses of such fauna are extremely rare. In the present work, we analysed seven species of five genera from 13 cave localities in Bulgaria, using molecular (mitochondrial COI and three nuclear markers: 18S rRNA, 28S rRNA and H3) techniques. All analysed DNA sequences from cave specimens formed one, highly supported, monophyletic lineage, divided into six subclades. According to molecular clock analyses, this subterranean clade originated 7-6.75 Mya. Our data support the “climatic-relict” hypothesis for the speciation of these taxa, assuming that surface ancestors colonized the caves and, after extinction of the surface population, cave populations evolved in allopatry. Extinction of the surface population was most probably caused by the climatic shift associated with the Messinian Salinity Crisis (5.96–5.33 Mya). A new model of climate-driven separation promoting speciation is proposed for the origin of the cave fauna.

Unique, Ancient Stygobiont Clade of Hydrobiidae (Truncatelloidea) in Bulgaria: the Origin of Cave Fauna