Mitochondrial DNA variation in the hybridizing fire-bellied toads, Bombina bombina and B. variegata

Using five restriction enzymes, geographical variation of mitochondrial DNA (mtDNA) in Bombina bombina and B. variegata was studied in samples from 20 locations. Each restriction enzyme produced a species-specific fragment pattern. B. bombina haplotypes A and B were closely related to each other. In contrast, haplotypes A and B of B. variegata formed two distinct lineages. A very distinctive haplotype (C) was found in the Carpathian Mountains, whereas two other haplotypes, D and E (differing by a single AvaI site), were present in western Europe and the Balkans, respectively. Populations polymorphic for haplotypes D and E occurred in the central Balkans where the haplotypes could replace each other clinally. mtDNA sequence divergence between B. bombina and B. variegata was estimated as 6.0-8.1% and 4.7-5.2% between type C and types D/E of B. variegata. The latter divergence is contrary to allozyme and morphological data that place the western and Carpathian B. v. variegata together (Nei's D = 0.07) and separate them from the Balkan subspecies B. v. scabra (Nei's D = 0.18). Broad interspecific correlation among morphology, allozymes and mtDNA types in European fire-bellied toads argues that, despite continuous hybridization (interrupted perhaps during Pleistocene glacial maxima), little or no mtDNA introgression between the species has occurred outside the narrow hybrid zones that separate these parapatric species.     

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.     

Comparative Analysis of Mitochondrial Genomes in <Emphasis Type="Italic">Bombina</Emphasis> (Anura; Bombinatoridae)

The complete mitochondrial genomes of two basal anurans, Bombina bombina and B. variegata (Anura; Bombinatoridae), were sequenced. The gene order of their mitochondrial DNA (mtDNA) is identical to that of canonical vertebrate mtDNA. In contrast, we show that there are structural differences in regulatory regions and protein coding genes between the mtDNA of these two closely related species. Corrected sequence divergence between the mtDNA of B. bombina and B. variegata amounts to 8.7% (2.3% divergence in amino acids). Comparisons with two East Asian congeners show that the control region contains two repeat regions, LV1 and LV2, present in all species except for B. bombina, in which LV2 has been secondarily lost. The rRNAs and tRNAs are characterized by low nucleotide divergence. The protein coding genes are considerably more disparate, although functional constraint is high but variable among genes, as evidenced by dN/dS ratios. A mtDNA phylogeny established the distribution of autapomorphic nonsynonomous substitutions in the mitogenomes of B. bombina and B. variegata. Nine of 98 nonsynonomous substitutions led to radical amino acid replacements that may alter mitochondrial protein function. Most radical substitutions were found in ND2, ND4, or ND5, encoding mitochondrial subunits of complex I of the electron transport system. The extensive divergence between the mitogenomes of B. bombina and B. variegata is discussed in terms of its possible role in impeding gene flow in natural hybrid zones between these two species.     

Evolution of Bombina bombina and Bombina variegata (Anura: Discoglossidae) in the Carpathian Basin: a history of repeated mt-DNA introgression across species

The structure and geographic location of hybrid zones change through time. Current patterns result from present and historical population-environment interactions that act on each of the hybridizing taxa. This is particularly evident for species involved in complex hybrid zones, such as that formed by the toad species Bombina bombina and Bombina variegata (Anura: Discoglossidae), which interact along extensive areas in Central Europe. We used data on external morphology and partial sequences of the cytochrome oxidase I (cox1) and nicotinamide adenine dinucleotid dehydrogenase subunit 4 (nad4) mitochondrial DNA (mt-DNA) genes to analyze the current patterns of genetic structure shown by both species of Bombina along their contact zone in Hungary. Phylogenetic, phylogeographic, and historical demography analyses were applied to 1.5kb mt-DNA obtained from 119 individuals representing 24 populations from Hungary and additional specimens from Slovakia, Albania, and Bosnia-Herzegovina. We use these data to infer the evolutionary history of the isolated populations of B. variegata in Hungary and to discriminate between competing biogeographic scenarios accounting for the historical interactions between species in this region. Results from the inferred phylogenetic branching pattern and sequence divergence among species and populations support the following: (i) recent population expansion has occurred in Hungarian populations of B. bombina, which are genetically very homogeneous; (ii) the Hungarian populations of B. variegata correspond to two distinct mitochondrial lineages (Carpathian and Alpine, respectively); average maximum-likelihood-corrected sequence divergence between these lineages is 8.96% for cox1 and 10.85% for nad4; (iii) mt-DNA divergence among the three isolated western populations of B. variegata from Transdanubia is low, with four closely related haplotypes, which suggests that the isolation between these populations is the result of a recent process, possibly mediated by the invasion of B. bombina; and (iv) we have detected discordances between morphology and mt-DNA data in the Transdanubia region (Bakony Mountains, Mecsek Mountains, Orség area), suggesting mt-DNA introgression across species in this regions. These results are discussed with reference to previous biogeographic hypotheses.     

Limited mitochondrial DNA introgression in a Bombina hybrid zone

We analysed the variation at mitochondrial DNA (mtDNA) and six allozyme loci in two transects across a hybrid zone between the fire-bellied toads Bombina bombina and Bombina variegata in southern Poland. The mtDNA cline was narrower than allozyme clines in one transect (Przemyśl, β = 0.435, w  = 3.42 km) and shifted to the B .  bombina side in both (near Kraków by 1.32 km, α = 0.437, and near Przemyśl by 0.97 km, α = 0.319). Cytonuclear associations were weak. Narrowed mtDNA clines could be a by-product of female demography and lowered effective population size of mtDNA. Alternatively, restricted mtDNA introgression is a likely result of negative epistasis in recombinants augmented by environment-dependent selection because divergence of Bombina mitochondrial and nuclear genomes is large. The shift of the mtDNA cline, contrary to expectations from initial mating preferences and fecundity differences between the species, suggests that on the B .  bombina side of the zone hybrid females with B .  variegata mtDNA have a higher chance of leaving progeny.  © 2007 The Linnean Society of London, Biological Journal of the Linnean Society , 2007, 91 , 295–306.     

Reproductive biology of the fire-bellied toads, Bombina bombina and B. variegata (Anura: Discoglossidae): egg size, clutch size and larval period length differences

Clutch size, egg size, larval size and duration of larval period were estimated for samples of Bombina bombina and B. variegata from several populations. Bombina variegata lays fewer eggs per batch (mean 17.4) than B. bombina (mean 32.5). The volume of B. variegata eggs is 2.4 times greater and the dry weight 2.3 times greater than that of B. bombina. As a consequence, the volume of B. variegata hatchlings and their dry weight are respectively 1.9 times and 2.5 times greater than that of B. bombina. Analysis of variance showed that variation of egg size in B. variegata is significantly greater than in B. bombina. When reared under laboratory conditions freshly metamorphosed individuals of both species showed no difference in body size or weight. Although growth rates are equal, tadpoles of B. variegata reached metamorphosis 14 days earlier than B. bombina tadpoles because of differences in size of hatchlings. Differences in reproductive parameters found for the two European Bombina species can be interpreted as adaptations to contrasting environments in which they breed.     

Quantitative immunological studies of the albumins of several species of fire bellied toads, genus Bombina.

1. Rabbit antisera against purified serum albumin of Bombina bombina were used to study relationships between B. bombina, B. variegata, and B. orientalis. 2. Quantitative micro-complement fixation tests indicated the albumins of B. bombina from central Poland and Bulgaria were indistinguishable. The albumins from several populations of B. variegata differed very slightly from that of B. bombina. The albumin of B. orientalis was quite distinct from that of B. bombina. 3. Using albumin as a molecular clock, we estimated B. bombina and B. variegata diverged within the last million years, whereas the B. orientalis lineage diverged roughly 10-12 mil yr ago.     

Comparative analysis of the genetic structure of hybrid populations of European toads of the genus Bombina (Oken, 1816) from Ukraine and Croatia

The comparative analysis of hybrid populations of toads Bombina bombina and B. variegata from Ukraine and Croatia revealed the essential differences in their genetic structure. The population composition and the direction of genetic introgression are determined initially by the ecological factors (the locality type, the country relief). The clear predominance of the red-bellied toad Bombina bombina was found in two cases: in the lowland populations and in the big samples. The second parental species, B. variegata, predominates in the highlands and in the small samples, respectively.     

Comparative study of carbohydrate chains released from the oviducal mucins of the two very closely related amphibian species Bombina bombina and Bombina variegata

The eggs of amphibians are surrounded by an extracellular matrix, termed jelly coat, which is mainly composed of hydrated mucin-type glycoproteins. These highly glycosylated molecules are synthesized by the oviduct and play an important role in the fertilization process. From a structural and chemical point of view, these oviducal mucins are very different from one species to another and they could be involved in the species-specificity of gamete interactions or could influence the parasite tropism. Bombina bombina and Bombina variegata are the two most closely related species within the genus, which hybridize readily in nature. Divergence occurred during geographic isolation estimated at 2-7 million years ago. The oviducal mucins of these species have been studied at the carbohydrate level, and the primary structures of 28 compounds have been established by NMR spectroscopy. The carbohydrate chains released from the oviducal mucins of the two species were similar and characterized by the common sequences GlcNAc(beta 1-3)[Fuc(alpha 1-4)]GlcNAc(beta 1-6) and GlcNAc(alpha 1-4)Gal(beta 1-4)Gal(beta 1-3) attached to GalNAc-ol (core 2). Nevertheless, some differences confirmed the strict species-specificity of amphibian oviducal carbohydrate chains observed previously. On the one hand, the presence of beta Gal 1,4-linked to beta GlcNAc in B. bombina, but not in B. variegata, can indicate that beta 4GalT: beta GlcNAc and beta 4GalT: beta Gal are two distinct glycosyltransferases. On the other hand, deaminoneuraminic acid (Kdn) is present in B. bombina, and N -glycolylneuraminic acid (NeuGc) in B. variegata. Although the enzymes involved in the biosynthesis of Kdn are not as well characterized, it can be suggested that at least one step of the biosynthetic pathway of NeuAc has been disrupted, leading the B. bombina oviducal NeuAc-9-synthase to use Man-6-P as a substrate, instead of ManNAc-6-P.     

A linkage map for the hybridising toads Bombina bombina and B. variegata (Anura: Discoglossidae)

Stable hybrid zones in which ecologically divergent taxa give rise to a range of recombinants are natural laboratories in which the genetic basis of adaptation and reproductive isolation can be unraveled. One such hybrid zone is formed by the fire-bellied toads Bombina bombina and B. variegata (Anura: Discoglossidae). Adaptations to permanent and ephemeral breeding habitats, respectively, have shaped numerous phenotypic differences between the taxa. All of these are, in principle, candidates for a genetic dissection via QTL mapping. We present here a linkage map of 28 codominant and 10 dominant markers in the Bombina genome. In an F2 cross, markers that were mainly microsatellites, SSCPs or allozymes were mapped to 20 linkage groups. Among the 40 isolated CA microsatellites, we noted a preponderance of compound and frequently interleaved CA-TA repeats as well as a striking polarity at the 5' end of the repeats.     

Hybridization of Bombina bombina and B. variegata (Anura, Discoglossidae) at a sharp ecotone in western Ukraine: comparisons across transects and over time

Abstract Bombina bombina and B. variegata are two anciently diverged toad taxa that have adapted to different breeding habitats yet hybridize freely in zones of overlap where their parapatric distributions meet. Here, we report on a joint genetic and ecological analysis of a hybrid zone in the vicinity of Stryi in western Ukraine. We used five unlinked allozyme loci, two nuclear single nucleotide polymorphisms and a mitochondrial DNA haplotype as genetic markers. Parallel allele frequency clines with a sharp central step occur across a sharp ecotone, where transitions in aquatic habitat, elevation, and terrestrial vegetation coincide. The width of the hybrid zone, estimated as the inverse of the maximum gradient in allele frequency, is 2.3 km. This is the smallest of four estimates derived from different clinal transects across Europe. We argue that the narrow cline near Stryi is mainly due to a combination of habitat distribution and habitat preference. Adult toads show a preference for either ponds ( B. bombina ) or puddles ( B. variegata ), which is known to affect the distribution of genotypes within the hybrid zones. At Stryi, it should cause a reduction of the dispersal rate across the ecotone and thus narrow the cline. A detailed comparison of all five intensively studied Bombina transects lends support to the hypothesis that habitat distribution plus habitat preference can jointly affect the structure of hybrid zones and, ultimately, the resulting barriers to gene flow between differentiated gene pools. This study also represents a resampling of an area that was last studied more than 70 years ago. Our allele-frequency clines largely coincide with those that were described then on the basis of morphological variation. However, we found asymmetrical introgression of B. variegata genes into B. bombina territory along the bank of a river.     

Identification and molecular cloning of novel trypsin inhibitor analogs from the dermal venom of the Oriental fire-bellied toad (Bombina orientalis) and the European yellow-bellied toad (Bombina variegata)

The structural diversity of polypeptides in amphibian skin secretion probably reflects different roles in dermal regulation or in defense against predators. Here we report the structures of two novel trypsin inhibitor analogs, BOTI and BVTI, from the dermal venom of the toads, Bombina orientalis and Bombina variegata. Cloning of their respective precursors was achieved from lyophilized venom cDNA libraries for the first time. Amino acid alignment revealed that both deduced peptides, consisting of 60 amino acid residues, including 10 cysteines and the reactive center motif, -CDKKC-, can be affirmed as structural homologs of the trypsin inhibitor from Bombina bombina skin.     

Mitochondrial phylogeography of the Eurasian beaver Castor fiber L

Nucleotide variation in an approximately 490 bp fragment of the mitochondrial DNA control region (mtDNA CR) was used to describe the genetic variation and phylogeographical pattern in the Eurasian beaver (Castor fiber) over its entire range. The sampling effort was focused on the relict populations that survived a drastic population bottleneck, caused by overhunting, at the end of the 19th century. A total of 152 individuals grouped into eight populations representing all currently recognized subspecies were studied. Sixteen haplotypes were detected, none of them shared among populations. Intrapopulation sequence variation was very low, most likely a result of the severe bottleneck. Extreme genetic structure could result from human-mediated extinction of intermediate populations, but it could also be an effect of prior substantial structuring of the beaver populations with watersheds of major Eurasian rivers acting as barriers to gene flow. Phylogenetic analysis revealed the presence of two mtDNA lineages: eastern (Poland, Lithuania, Russia and Mongolia) and western (Germany, Norway and France), the former comprising more divergent haplotypes. The low level of sequence divergence of the entire cytochrome b gene among six individuals representing six subspecies suggests differentiation during the last glacial period and existence of multiple glacial refugia. At least two evolutionary significant units (ESU) can be identified, the western and the eastern haplogroup. The individual relict populations should be regarded as management units, the eastern subspecies possibly also as ESUs. Guidelines for future translocations and reintroductions are proposed.     

Comparative Phylogeography of the Atlantic Forest Endemic Sloth (Bradypus torquatus) and the Widespread Three-toed Sloth (Bradypus variegatus) (Bradypodidae, Xenarthra)

The comparative phylogeographic study of the maned sloth (Bradypus torquatus) and the three-toed sloth (Bradypus variegatus) was performed using a segment of mitochondrial DNA (mtDNA) control region. We examined 19 B. torquatus from two regions and 47 B. variegatus from three distant regions of Atlantic forest. This first characterization of molecular diversity indicates a great diversity (B. torquatus: h = 0.901 ± 0.039 and π = 0.012 ± 0.007; B. variegatus: h = 0.699 ± 0.039 and π = 0.010 ± 0.006) and very divergent mitochondrial lineages within each sloth species. The different sampled regions carry distinct and non-overlapping sets of mtDNA haplotypes and are genetically divergent. This phylogeographic pattern may be characteristic of sloth species. In addition, we infer that two main phylogeographic groups exist in the Atlantic forest representing a north and south distinct divergence.     

Nuclear-mitochondrial epistasis and drosophila aging: introgression of Drosophila simulans mtDNA modifies longevity in D. melanogaster nuclear backgrounds

Under the mitochondrial theory of aging, physiological decline with age results from the accumulated cellular damage produced by reactive oxygen species generated during electron transport in the mitochondrion. A large body of literature has documented age-specific declines in mitochondrial function that are consistent with this theory, but relatively few studies have been able to distinguish cause from consequence in the association between mitochondrial function and aging. Since mitochondrial function is jointly encoded by mitochondrial (mtDNA) and nuclear genes, the mitochondrial genetics of aging should be controlled by variation in (1) mtDNA, (2) nuclear genes, or (3) nuclear-mtDNA interactions. The goal of this study was to assess the relative contributions of these factors in causing variation in Drosophila longevity. We compared strains of flies carrying mtDNAs with varying levels of divergence: two strains from Zimbabwe (<20 bp substitutions between mtDNAs), strains from Crete and the United States (approximately 20-40 bp substitutions between mtDNAs), and introgression strains of Drosophila melanogaster carrying mtDNA from Drosophila simulans in a D. melanogaster Oregon-R chromosomal background (>500 silent and 80 amino acid substitutions between these mtDNAs). Longevity was studied in reciprocal cross genotypes between pairs of these strains to test for cytoplasmic (mtDNA) factors affecting aging. The intrapopulation crosses between Zimbabwe strains show no difference in longevity between mtDNAs; the interpopulation crosses between Crete and the United States show subtle but significant differences in longevity; and the interspecific introgression lines showed very significant differences between mtDNAs. However, the genotypes carrying the D. simulans mtDNA were not consistently short-lived, as might be predicted from the disruption of nuclear-mitochondrial coadaptation. Rather, the interspecific mtDNA strains showed a wide range of variation that flanked the longevities seen between intraspecific mtDNAs, resulting in very significant nuclear x mtDNA epistatic interaction effects. These results suggest that even "defective" mtDNA haplotypes could extend longevity in different nuclear allelic backgrounds, which could account for the variable effects attributable to mtDNA haplogroups in human aging.     

Comparative phylogeography of the two pink salmon broodlines: an analysis based on a mitochondrial DNA genealogy

Over most of their natural northern Pacific Ocean range, pink salmon (Oncorhynchus gorbuscha) spawn in a habitat that was repeatedly and profoundly affected by Pleistocene glacial advances. A strictly two-year life cycle of pink salmon has resulted in two reproductively isolated broodlines, which spawn in alternating years and evolved as temporal replicates of the same species. To study the influence of historical events on phylogeographical and population genetic structure of the two broodlines, we first reconstructed a fine-scale mtDNA haplotype genealogy from a sample of 80 individuals and then determined the geographical distribution of the major genealogical assemblages for 718 individuals sampled from nine Alaskan and eastern Asian even- and nine odd-year pink salmon populations. Analysis of restriction site states in seven polymerase chain reaction (PCR)-amplified mtDNA regions (comprising 97% of the mitochondrial genome) using 13 endonucleases resolved 38 haplotypes, which clustered into five genealogical lineages that differed from 0.065 to 0.225% in net sequence divergence. The lineage sorting between broodlines was incomplete, which suggests a recent common ancestry. Within each lineage, haplotypes exhibited star-like genealogies indicating recent population growth. The depth of the haplotype genealogy is shallow ( approximately 0.5% of nucleotide sequence divergence) and probably reflects repeated decreases in population size due to Pleistocene glacial advances. Nested clade analysis (NCA) of geographical distances showed that the geographical distribution observed for mitochondrial DNA (mtDNA) haplotypes resulted from alternating influences of historical range expansions and episodes of restricted dispersal. Analyses of molecular variance showed weak geographical structuring of mtDNA variation, except for the strong subdivision between Asian and Alaskan populations within the even-year broodline. The genetic similarities observed among and within geographical regions probably originated from postglacial recolonizations from common sources rather than extensive gene flow. The phylogeographical and population genetic structures differ substantally between broodlines. This can be explained by stochastic lineage sorting in glacial refugia and perhaps different recolonization routes in even- and odd-year broodlines.     

Phylogeography of Bufo fowleri at its northern range limit

Many of the species that recolonized previously glaciated areas in the Great Lakes basin of North America over the past 10-12,000 years exhibit genetic evidence of multiple invasion routes and present-day secondary contact between deeply divergent lineages. With this in mind, we investigated the phylogeographical structure of genetic variability in Fowler's toads (Bufo fowleri) at the northern edge of its distribution where its range encircles the Lake Erie basin. Because B. fowleri is so closely tied to habitats along the Lake Erie shoreline, we would expect to find clear evidence of the number of invasions leading to the species' colonization of the northern shore. A 540 bp sequence from the mitochondrial control region was amplified and analysed for 158 individuals from 21 populations. Interpopulation sequence variation ranged from 0% to 6%. Phylogenetic analysis of p-distance using the neighbor-joining method revealed two deeply divergent (6% sequence divergence) mtDNA lineages (Phylogroup 1 and 2), possibly arising as a result of secondary contact of populations that entered the region from two separate glacial refugia. However, the phylogeographical pattern was not simple. The populations at Long Point, on the north shore of Lake Erie, clustered with the population from Indiana Dunes on Lake Michigan to form Phylogroup 2 whereas all other B. fowleri populations examined from both sides of Lake Erie constituted Phylogroup 1. Furthermore, mtDNA sequences from the related species Bufo americanus, obtained from populations outside the range of B. fowleri, clustered with mtDNA haplotypes of B. fowleri Phylogroup 1, indicating the possibility of partial introgression of mitochondria from one species to the other.     

The maintenance of reproductive isolation in a mosaic hybrid zone between the fire-bellied toads Bombina bombina and B. variegata

Abstract. Mosaic hybrid zones arise when ecologically differentiated taxa hybridize across a network of habitat patches. Frequent interbreeding across a small-scale patchwork can erode species differences that might have been preserved in a clinal hybrid zone. In particular, the rapid breakdown of neutral divergence sets an upper limit to the time for which differences at marker loci can persist. We present here a case study of a mosaic hybrid zone between the fire-bellied toads Bombina bombina and B. variegata (Anura: Discoglossidae) near Apahida in Romania. In our 20 × 20 km study area, we detected no evidence of a clinal transition but found a strong association between aquatic habitat and mean allele frequencies at four molecular markers. In particular, pure populations of B. bombina in ponds appear to cause massive introgression into the surrounding B. variegata gene pool found in temporary aquatic sites. Nevertheless, the genetic structure of these hybrid populations was remarkably similar to those of a previously studied transect near Pescenica (Croatia), which had both clinal and mosaic features: estimates of heterozygote deficit and linkage disequilibrium in each country are similar. In Apahida, the observed strong linkage disequilibria should stem from an imperfect habitat preference that guides most (but not all) adults into the habitats to which they are adapted. In the absence of a clinal structure, the inferred migration rate between habitats implies that associations between selected loci and neutral markers should break down rapidly. Although plausible selection strengths can maintain differentiation at those loci adapting the toads to either permanent or temporary breeding sites, the divergence at neutral markers must be transient. The hybrid zone may be approaching a state in which the gene pools are homogenized at all but the selected loci, not dissimilar from an early stage of sympatric divergence.     

Nuclear and mitochondrial phylogeography of the European fire-bellied toads Bombina bombina and Bombina variegata supports their independent histories

Exact location and number of glacial refugia still remain unclear for many European cold‐blooded terrestrial vertebrates. We performed a fine‐scaled multilocus phylogeographic analysis of two Bombina species combining mitochondrial variation of 950 toads from 385 sites and nuclear genes (Rag‐1, Ncx‐1) from a subset of samples to reconstruct their colonization and contemporary variation patterns. We identified the lowlands northwest of the Black Sea and the Carpathians to be important refugial areas for B. bombina and B. variegata, respectively. This result emphasizes the importance of Central European refugia for ectothermic terrestrial species, far north of the Mediterranean areas regarded as exclusive glacial refugia for the animals. Additional refugia for B. variegata have been located in the southern Apennines and Balkans. In contrast, no evidence for the importance of other east European plains as refugial regions has been found. The distribution of mtDNA and Ncx‐1 variation suggests the presence of local refugia near the Black Sea for B. bombina; however, coalescent simulations did not allow to distinguish whether one or two refugia were present in the region. Strong genetic drift apparently accompanied postglacial expansions reducing diversity in the colonization areas. Extended sampling, coupled with the multilocus isolation with migration analysis, revealed a limited and geographically restricted gene flow from the Balkan to Carpathian populations of B. variegata. However, despite proximity of inferred B. bombina and B. variegata refugia, gene exchange between them was not detected.