Past and present: Phylogeography of the Bufo gargarizans species complex inferred from multi-loci allele sequence and frequency data.

Using multi-locus DNA sequence and frequency data, we examined the phylogeographic patterns of the Asian toad Bufo gargarizans species complex. A total of 166 individuals from 56 sites were genotyped for one mitochondrial locus (516 base pairs, ND2 gene) and five nuclear intron loci (Sox9-2, Rho-3, CCNB2-3, UCH-2, DBI-2; 250-350 base pairs each). We conducted a phylogenetic analysis of the allele sequence data to construct gene trees for each individual locus using Bayesian inference, a multi-locus phylogeny based on all five nuclear loci using POFAD, and a phylogenetic network using the NeighborNet algorithm. Furthermore, we used population-genetic analysis of the allele frequency data to reveal ongoing processes, including an analysis of molecular variance (AMOVA), a Bayesian assignment analysis, and a non-equilibrium Bayesian method for estimating recent migration rate. Our phylogenetic analyses showed that the observed divergence in the B. gargarizans species complex likely dated back to 7-8 million years ago. Repeated range expansions during the inter-glacial periods of Pleistocene likely established the current distribution of genetic diversity, although historical vicariant events were still evidenced. Both the west and the southeast regions may have served as refugia during the glaciation, and the range expansion was in general from west to northeast. Additionally, we detected strong ongoing migration both from west to the other regions and from south to north, which likely represents a meta-population dynamic that has emerged over the past ∼10,000 years. The morphologically identified species Bufo tibetanus is not genetically diagnosable, and therefore should be considered a junior synonym of B. gargarizans.