Revisiting the phylogeography of Asellus aquaticus in Europe: insights into cryptic diversity and spatiotemporal diversification

Pleistocene glaciations affected the present-day distribution and genetic diversity of animal species in Europe. Deep genetic subdivisions observed in European populations of the widespread freshwater isopod morphospecies, Asellus aquaticus, suggest the presence of putative cryptic species. We used the DNA barcodes of the cytochrome c oxidase subunit 1 (COI) gene combined with distance- and tree-based methods of species delimitation as a rapid tool for assessing the number of distinct operational taxonomic units (OTUs) representing potential cryptic species. The spatial and demographic aspect of A.aquaticus distribution was also analysed. We generated a tentative temporal framework for diversification within the morphospecies provided by the molecular clock approach. Altogether, our study included 603 COI sequences from 147 populations from all over Europe and Asia Minor, including the already published data deposited in GenBank. The mtDNA-based phylogenetic and OTU delimitation pattern was assessed with results of the nuclear data set analysis including the sequence data derived from this study and those previously submitted in GenBank. In total, 16 haplotypes of 28S rDNA were used representing all COI-based OTUs and 53 localities. Our results show that A.aquaticus is a conglomerate of genetically distinct COIOTUs. One of the OTUs seems to correspond to the nominative subspecies of A.aquaticus aquaticus, recently redescribed from Sweden, and another with the recently described A.kosswigi. Most of the OTUs are probably of pre-Pleistocene origin and have narrow ranges in southern Europe. A recent expansion, in both demographic and spatial terms, was revealed in one OTU, which is widely distributed in Europe and represents A.aquaticus aquaticus. This may be explained by the post-glacial recolonisation processes. According to our data, this OTU probably emerged and initially diversified in the west Balkans in the Middle/Late Pliocene with several lineages surviving and diversifying through the Pleistocene glaciations and expanding during the interglacials. In some cases, our 28S data support the COI-based OTUs and provide ample evidence for the existence of distinct OTUs, especially in mountainous and karst areas. However, other COIOTUs are not reciprocally monophyletic with respect to nuclear marker. This phylogenetic pattern can be interpreted predominantly as a result of incomplete sorting of nuclear lineages, potentially indicating an ongoing speciation process, but also as an effect of introgression resulting from secondary contact of formerly peripatric or allopatric mitochondrial lineages.