Phylogeography of the fire-bellied toads Bombina: independent Pleistocene histories inferred from mitochondrial genomes.

The fire-bellied toads Bombina bombina and Bombina variegata, interbreed in a long, narrow zone maintained by a balance between selection and dispersal. Hybridization takes place between local, genetically differentiated groups. To quantify divergence between these groups and reconstruct their history and demography, we analysed nucleotide variation at the mitochondrial cytochrome b gene (1096 bp) in 364 individuals from 156 sites representing the entire range of both species. Three distinct clades with high sequence divergence (K2P = 8-11%) were distinguished. One clade grouped B. bombina haplotypes; the two other clades grouped B. variegata haplotypes. One B. variegata clade included only Carpathian individuals; the other represented B. variegata from the southwestern parts of its distribution: Southern and Western Europe (Balkano-Western lineage), Apennines, and the Rhodope Mountains. Differentiation between the Carpathian and Balkano-Western lineages, K2P approximately 8%, approached interspecific divergence. Deep divergence among European Bombina lineages suggests their preglacial origin, and implies long and largely independent evolutionary histories of the species. Multiple glacial refugia were identified in the lowlands adjoining the Black Sea, in the Carpathians, in the Balkans, and in the Apennines. The results of the nested clade and demographic analyses suggest drastic reductions of population sizes during the last glacial period, and significant demographic growth related to postglacial colonization. Inferred history, supported by fossil evidence, demonstrates that Bombina ranges underwent repeated contractions and expansions. Geographical concordance between morphology, allozymes, and mtDNA shows that previous episodes of interspecific hybridization have left no detectable mtDNA introgression. Either the admixed populations went extinct, or selection against hybrids hindered mtDNA gene flow in ancient hybrid zones.