The aims of the study were (i) to reveal the pattern of phylogeny of Pseudamnicola inhabiting the Aegean Islands, (ii) to describe and analyse the variation of the morphology in 17 populations of Pseudamnicola from the springs on the Aegean Islands not studied so far and considering also another seven populations studied earlier and (iii) to find out which model is more applicable to the island Pseudamnicola populations: either a model in which a relict fauna rich in endemics is differentiated in a way that mainly reflects the geological history of the area or a model in which a relatively young fauna is composed of more or less widely distributed taxa, with relatively high levels of gene flow among the springs they inhabit. To address the above issues, the morphology and the mitochondrial genes—cytochrome oxidase subunit I (COI) and ribosomal 16S—and nuclear genes—ribosomal 18S, 28S and histone 3 (H3)—were analysed. COI and COI+16S rRNA+18S datasets gave trees with identical topology in both ML and Bayesian inference. The 24 studied populations of Pseudamnicola form 16 clades, each of them generally having low levels of intrapopulation genetic differentiation. The generalised mixed Yule coalescent (GMYC) procedure and the Automatic Barcode Gap Discovery (ABGD) analysis for COI identified 16 Pseudamnicola entities coinciding with clades of the ML tree based on 44 haplotypes and 189 sequences. The present pattern of diversity, together with dating of divergence time, reflects a short story of colonisation/recolonisation, supported by the Late Pleistocene land bridges, rather than the consequences of earlier geological events. The principal component analysis (PCA) on the shells of the molecularly distinct clades showed differences, although variability ranges often overlap. Female reproductive organs showed no differences between the clades, and penile characters differed only in some cases.

Belgrandiella A. J. Wagner, 1927 is a genus comprising minute snails with conic or turriform shells that are facultative stygobionts, inhabiting subterranean waters and springs. Few specimens or even empty shells of this taxon can be found, so the taxonomy is based mostly on the shell morphology and geographic distribution. Anatomy is known for a few taxa, and its usefulness is restricted. This, coupled with a popular idea that isolation is the first factor shaping divergence and speciation of the spring/cave fauna, resulted in dozens of nominal species of Belgrandiella, known from shells at a given locality. Herein we present shells, anatomy and four molecular markers (nuclear H3, 18S and 28S and mitochondrial cytochrome oxidase subunit I) in Belgrandiella from 16 localities in Slovenia, northern Croatia, and Bosnia and Herzegovina. Five sites are type localities of Boleana umbilicata, Belgrandiella kusceri, Belgrandiella krupensis, Belgrandiella zermanica and Belgrandiella robusta, respectively. Considering the shell and geographic distribution, we have determined B. cf. robusta Belgrandiella cf. croatica, Belgrandiella cf. fontinalis, Belgrandiella cf. kuesteri, Belgrandiella cf. pageti and Belgrandiella cf. koprivnensis The shells of all, as well as penes and female reproductive organs of two putative, species have been illustrated. Molecular analysis indicates four distinct clades, most probably of species rank for B. kusceri, B. cf. kuesteri, B. cf. fontinalis and B. cf. koprivnensis. Four nominal species (B. krupensis, B. robusta, B. umbilicata and B. zermanica) and one genus (Boleana) must be synonymised with Belgrandiella kusceri, and there are no more than four valid, biological species in the studied Belgrandiella. The molecular divergence is completely unmirrored by high morphological variability. Hence the level of endemism is markedly overestimated in these snails.