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.     

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

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 .     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Hybridization between the fire-bellied toads Bombina bombina and Bombina variegata in the karst regions of Slovakia and Hungary: morphological and allozyme evidence

Geographic variability and genetic interactions in the contact zone between the fire-bellied toads, Bombina bombina and B. variegata, were studied using analysis of morphological and genetic variation in sixteen samples from the Slovak Karst and Aggtelek Karst regions. Genotype frequencies at four marker loci (Ldh-1, Mdh-1, Adk, Hem) demonstrate the existence of a hybrid zone with highly variable population structures. While some samples appear to represent panmictic hybrid populations, other samples are very heterogeneous. Pure individuals of both species occurred together with hybrids at one site. Habitat segregation among genetically differentiated demes probably causes this heterogeneity of population structures. Increased frequencies of the allele Ldh-1^M, which is present in low proportion in B. bombina south of the contact zone, were found in some hybrid populations.     

Inheritance and linkage analysis of five enzyme loci in interspecific hybrids of toadlets,genus Bombina

Progeny produced from Bombina bombina, B. variegata, and field-collected interspecific hybrids have been analyzed for the inheritance of five enzyme loci, which are fixed for alternate alleles in the parental species. Lactate dehydrogenase (Ldh-1), NAD-dependent malate dehydrogenase (Mdh-1), creatine kinase (Ck), adenylate kinase (Ak), and glucosephosphate isomerase (Gpi) are all inherited in a Mendelian manner as codominant alleles at nuclear loci. Both parental alleles are equally functional in artificial F₁ hybrids (female B. bombina×male B. variegata) at each of the loci studied. No linkage between any pair of loci was observed. Discovery of this inherited biochemical variation combined with a technique for assaying individual genotypes without killing the animals makes feasible studies of hybrid population structure heretofore impossible.This work was partly supported by the Polish Academy of Sciences within the project MR-II/6.     

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.     

Early embryogenesis in discoglossoid frogs: a study of heterochrony at different taxonomic levels

We studied the timing of the early development of the cranial neural crest, neural tube differentiation, somite formation and body elongation in the closely related frog species Discoglossus montalenti , Discoglossus galganoi , Discoglossus pictus , Bombina orientalis , Bombina variegata and Bombina maxima . Despite their different egg and embryo sizes, event pairing shows that there are only very few heterochronies among the three Bombina species and the three Discoglossus species in early developmental events. Instead, differences become apparent at the genus level (interpreted as phylogenetic signal). For example, the separation into a mandibular and a hyoid portion of cranial neural crest could be seen earlier in the Discoglossus than in the Bombina species when compared with the development of the neural tube. The development of the cranial neural crest streams also shifts (relatively) forward in time when compared with body elongation and somite formation in the Discoglossus species in contrast to the Bombina species. Comparisons with other species show that attempts to correlate heterochronies in early development with life history changes should take into account the possibility of a rather high evolvability, i.e. a high probability of convergence in the timing of these early events.     

Genetic Differentiation Among Peripheral Populations of Bombina bombina from Thrace and Anatolia: An Allozyme Analysis

Genetic structures of Bombina bombina populations, located as peripheral isolates in Turkish Thrace and northwestern Anatolia, were analyzed by polyacrylamide gel electrophoresis using 20 allozyme loci, to investigate the populations’ current genetic variation and possible colonization history. Significant genetic variability was detected in most of the loci and all populations. Allozyme pairwise F _ST matrices and distribution of allele frequencies indicate their very close genetic relationships and relatively recent formation. Mean genetic distance values between Thracian and Anatolian populations indicate a Middle or Upper Pleistocene lineage separation before the formation of the Bosporus as an isolating geographic barrier. All the samples show substantial heterozygosity excess, and there was statistically significant evidence of recent bottlenecks. The extent and patterns of genetic divergence indicate that the Anatolian and Thracian populations have probably experienced bottlenecks, and incipient speciation may have occurred in Anatolian populations of B. bombina.     

Mating patterns in a hybrid zone of fire-bellied toads (Bombina): inferences from adult and full-sib genotypes

We present two novel methods to infer mating patterns from genetic data. They differ from existing statistical methods of parentage inference in that they apply to populations that deviate from Hardy-Weinberg and linkage equilibrium, and so are suited for the study of assortative mating in hybrid zones. The core data set consists of genotypes at several loci for a number of full-sib clutches of unknown parentage. Our inference is based throughout on estimates of allelic associations within and across loci, such as heterozygote deficit and pairwise linkage disequilibrium. In the first method, the most likely parents of a given clutch are determined from the genotypic distribution of the associated adult population, given an explicit model of nonrandom mating. This leads to estimates of the strength of assortment. The second approach is based solely on the offspring genotypes and relies on the fact that a linear relation exists between associations among the offspring and those in the population of breeding pairs. We apply both methods to a sample from the hybrid zone between the fire-bellied toads Bombina bombina and B. variegata (Anura: Disco glossidae) in Croatia. Consistently, both approaches provide no evidence for a departure from random mating, despite adequate statistical power. Instead, B. variegata-like individuals among the adults contributed disproportionately to the offspring cohort, consistent with their preference for the type of breeding habitat in which this study was conducted.     

Assessing the Threatened Status of Bumble Bee Species (Hymenoptera: Apidae) in Hungary,Central Europe

Decline in the populations of bumble bees and other pollinators stress the need for more knowledge about their conservation status. Only one of the 25 bumble bee species present in Hungary is included in the Hungarian Red List. We estimated the endangerment of the Hungarian bumble bee (Bombus Latr.) species using the available occurrence data from the last 50 years of the 20th century. Four of the 25 species were data deficient or extinct from Hungary. About 60% of species were considered rare or moderately rare. Changes in distribution and occurrence frequency indicated that 10 of the 21 native species showed a declining trend, while only three species increased in frequency of occurrence. According to the IUCN Red List categories, seven species (33% of the native fauna) should be labelled as critically endangered (CR) and 3 (14%) as endangered (EN). Our results stress an urgent need of protection plans for bumble bees in Hungary, and further underlines the validity of concern over bumble bees all over Europe.