Fossorial but widespread: the phylogeography of the common spadefoot toad (Pelobates fuscus), and the role of the Po Valley as a major source of genetic variability.

Pelobates fuscus is a fossorial amphibian that inhabits much of the European plain areas. To unveil traces of expansion and contraction events of the species' range, we sequenced 702 bp of the mitochondrial cytochrome b gene. To infer the population history we applied phylogeographical methods, such as nested clade phylogeographical analysis (NCPA), and used summary statistics to analyse population structure under a neutral model of evolution. Populations were assigned to different drainage systems and we tested hypotheses of explicit refugial models using information from analysis of molecular variance, nucleotide diversity, effective population size estimation, NCPA, mismatch distribution and Bayesian dating. Coalescent simulations were used as post hoc tests for plausibility of derived or a priori assumed biogeographical hypotheses. Our combination of all approaches enabled the reconstruction of the colonization history and phylogeography of P. fuscus and confirmed a previous assumption of the existence of two major genetic lineages within P. fuscus. Using the Afro-European vicariance of Pelobates cultripes and Pelobates varaldii and applying Bayesian dating we estimated the divergence of these phylogeographical lineages to the Pliocene. We suggest the existence of three different glacial refugia: (i) the area between the Caspian and Black Seas as the origin for the expansion of the 'eastern lineage'; (ii) the Danube system as a centre of diversity for part of the 'western lineage'; (iii) the Po Valley, the largest centre of genetic variability. This fits the hypothesis that climatic fluctuation was a key event for differentiation processes in P. fuscus.