Mapping Bombina mitochondrial genomes: the conundrum of Carpathian Bombina variegata (Anura: Discoglossidae)

Abstract

Complete mitochondrial genomes (mtDNA) of five individuals representing two haplotypes of Bombina bombina and three of Bombina variegata were compared using restriction site maps. Phylogenetic analyses reveal three ancient mitochondrial lineages: (1) two very similar haplotypes A and B of B. bombina; (2) almost identical haplotypes D and E of B. variegata; and (3) haplotype C of B. variegata. Haplotype C is as different from haplotypes D/E as from A/B. These data are strikingly discordant with relationships based on morphology and allozymes. Haplotypes C and D/E represent a pre-Pleistocene mitochondrial divergence within B. variegata, nearly coincident with speciation between B. variegata and B. bombina. Geographical partitioning of the two divergent B. variegata mitochondrial lineages indicates repeated localization of the lineages in separate glacial refugia during the Pleistocene. That nuclear genes do not show a similar divergence, but rather indicate relatively free genetic exchange between populations with divergent mtDNAs, suggests that males dispersed much more widely than females during expansions from glacial refugia. Comparison of Bombina mtDNA maps with a restriction site map of Xenopus laevis mtDNA revealed 16 homologous sites; 12 of these may be nearly invariant across primitive anuran mtDNAs. Two distinct regions of heteroplasmy, representing two regions with variable numbers of sequence repeats [length variable (LV) regions], were characterized. Comparison with the Xenopus map places LV1, present in all five haplotypes, near the 5′-end of the control region, and LV2 present only in B. variegata, near the 3′-end. Although phylogenetic analyses did not group the two major B. variegata lineages together, presence of LV2 in both lineages supports placement of both within B. variegata.