New Insights Into the Composition, Distribution and History of the Freshwater Isopod Species Complex Proasellus coxalis (Dollfus, 1892) in Europe and the Mediterranean Basin

1. Problematic taxa, often referred to as species complexes, are common among freshwater organisms. Understanding their species composition, distribution and history is key to integrating these taxa into many ecological disciplines. We investigated the taxonomy and biogeography of the Proasellus coxalis species complex (PCC), a diversified and functionally important group of asellid isopods (Pancrustacea) in freshwaters of the Mediterranean Basin and central Europe.
2. We used phylogenetic and phylogeographic analyses from specimens collected at 143 localities across the range of the PCC to document its diversity, phylogenetic position and historical biogeography. First, we used molecular species delimitation methods based on the mitochondrial cytochrome oxidase subunit I (COI) gene to identify molecular operational taxonomic units (MOTUs) representing putative species within that complex. Second, we used three additional genes—the 16S mitochondrial rDNA, FASTKD4 nuclear and 28S nuclear rDNA genes—to generate a four-gene dated phylogeny of Asellidae to test the monophyly and phylogenetic position of the PCC. Third, we applied statistical ancestral reconstruction methods to the four-gene dated phylogeny of the PCC to infer its biogeographic history. Fourth, we used Bayesian phylogeographic diffusion models based on the COI and 16S genes to reconstruct the range dynamics of the only widely distributed MOTU in this species complex.
3. We identified ≥ 25 MOTUs within the PCC. The eastern Mediterranean Basin, including the Pontic-Mediterranean and Italian peninsulas, contained numerous narrowly distributed MOTUs. In contrast, central Europe, northern Italy, the Dinarides, the Iberian peninsula and northwestern Africa were colonised by a single MOTU, which we term P. banyulensis. The four-gene phylogeny of the Asellidae clustered all PCC MOTUs into a well-supported monophyletic group, which we term the P. coxalis clade. Ancestral reconstruction methods indicated that the clade originated during the early Miocene in the southern Hellenides, at that time part of the Balkanian-Anatolian landmass. From there, it dispersed and diverged mainly during the Miocene, through the eastern Mediterranean Basin, including the Italian peninsula. Bayesian phylogeographic diffusion models revealed that central Europe and the western Mediterranean Basin were recently colonised from Italian populations of P. banyulensis. During the Pleistocene, multiple waves of colonisation of that species followed each other from central Italy, the most northerly (Germany, Sweden) and southerly (southern Spain, Morocco and Algeria) regions being colonised last.
4. We resolved major taxonomic, phylogenetic and biogeographic uncertainties that have marked more than a century of research on the PCC. Our updated understanding of this complex is that of a species-rich clade that has long diversified in the eastern Mediterranean Basin, but of which a single species—P. banyulensis—has recently colonised central Europe and the western Mediterranean Basin from Italian refugia. In central Europe, this species adds to two other widespread species—Asellus aquaticus from Pannonian refugia and P. meridianus, most probably from Iberian refugia—to produce mixed asellid communities.