A dense linkage map for a large repetitive genome: discovery of the sex-determining region in hybridizing fire-bellied toads (Bombina bombina and Bombina variegata).

Genomic analysis of hybrid zones offers unique insights into emerging reproductive isolation and the dynamics of introgression. Because hybrid genomes consist of blocks inherited from one or the other parental taxon, linkage information is essential. In most cases, the spectrum of local ancestry tracts can be efficiently uncovered from dense linkage maps. Here, we report the development of such a map for the hybridizing toads, Bombina bombina and Bombina variegata (Anura: Bombinatoridae). Faced with the challenge of a large (7-10 Gb), repetitive genome, we set out to identify a large number of Mendelian markers in the nonrepetitive portion of the genome that report B. bombina vs B. variegata ancestry with appropriately quantified statistical support. Bait sequences for targeted enrichment were selected from a draft genome assembly, after filtering highly repetitive sequences. We developed a novel approach to infer the most likely diplotype per sample and locus from the raw read mapping data, which is robust to over-merging and obviates arbitrary filtering thresholds. Validation of the resulting map with 4755 markers underscored the large-scale synteny between Bombina and Xenopus tropicalis. By assessing the sex of late-stage F2 tadpoles from histological sections, we identified the sex-determining region in the Bombina genome to 7 cM on LG5, which is homologous to X. tropicalis chromosome 5, and inferred male heterogamety. Interestingly, chromosome 5 has been repeatedly recruited as a sex chromosome in anurans with XY sex determination.

Complex Admixture Preceded and Followed the Extinction of Wisent in the Wild.

Retracing complex population processes that precede extreme bottlenecks may be impossible using data from living individuals. The wisent (Bison bonasus), Europe's largest terrestrial mammal, exemplifies such a population history, having gone extinct in the wild but subsequently restored by captive breeding efforts. Using low coverage genomic data from modern and historical individuals, we investigate population processes occurring before and after this extinction. Analysis of aligned genomes supports the division of wisent into two previously recognized subspecies, but almost half of the genomic alignment contradicts this population history as a result of incomplete lineage sorting and admixture. Admixture between subspecies populations occurred prior to extinction and subsequently during the captive breeding program. Admixture with the Bos cattle lineage is also widespread but results from ancient events rather than recent hybridization with domestics. Our study demonstrates the huge potential of historical genomes for both studying evolutionary histories and for guiding conservation strategies.

Para-allopatry in hybridizing fire-bellied toads (Bombina bombina and B. variegata): Inference from transcriptome-wide coalescence analyses.

Ancient origins, profound ecological divergence, and extensive hybridization make the fire-bellied toads Bombina bombina and B. variegata (Anura: Bombinatoridae) an intriguing test case of ecological speciation. Previous modeling has proposed that the narrow Bombina hybrid zones represent strong barriers to neutral introgression. We test this prediction by inferring the rate of gene exchange between pure populations on either side of the intensively studied Kraków transect. We developed a method to extract high confidence sets of orthologous genes from de novo transcriptome assemblies, fitted a range of divergence models to these data and assessed their relative support with analytic likelihood calculations. There was clear evidence for postdivergence gene flow, but, as expected, no perceptible signal of recent introgression via the nearby hybrid zone. The analysis of two additional Bombina taxa (B. v. scabra and B. orientalis) validated our parameter estimates against a larger set of prior expectations. Despite substantial cumulative introgression over millions of years, adaptive divergence of the hybridizing taxa is essentially unaffected by their lack of reproductive isolation. Extended distribution ranges also buffer them against small-scale environmental perturbations that have been shown to reverse the speciation process in other, more recent ecotypes.

Complete mitochondrial genome of the Greek marsh frog Pelophylax cretensis (Anura, Ranidae).

We sequenced the complete mitochondrial genome of the Greek marsh frog Pelophylax cretensis, a water frog species endemic to the island of Crete. The genome sequence was 17,829 bp in size, and the gene order and contents were identical to those of previously reported mitochondrial genomes of other water frog species. This is the first complete mitogenome (i.e. including control region) described for western Palaearctic water frogs.

Phylogenetic relationships among four new complete mitogenome sequences of Pelophylax (Amphibia: Anura) from the Balkans and Cyprus.

We present the full-length mitogenome sequences of four European water frog species: Pelophylax cypriensis, P. epeiroticus, P. kurtmuelleri and P. shqipericus. The mtDNA size varied from 17,363 to 17,895 bp, and its organization with the LPTF tRNA gene cluster preceding the 12 S rRNA gene displayed the typical Neobatrachian arrangement. Maximum likelihood and Bayesian inference revealed a well-resolved mtDNA phylogeny of seven European Pelophylax species. The uncorrected p-distance for among Pelophylax mitogenomes was 9.6 (range 0.01-0.13). Most divergent was the P. shqipericus mitogenome, clustering with the "P. lessonae" group, in contrast to the other three new Pelophylax mitogenomes related to the "P. bedriagae/ridibundus" lineage. The new mitogenomes resolve ambiguities of the phylogenetic placement of P. cretensis and P. epeiroticus.

Evolutionary trend for metamery reduction and gonad shortening in Anurans revealed by comparison of gonad development.

The gonads develop as the metameric mesodermal structures at the ventral surface of the mesonephroi. To study the evolutionary trends for anuran gonads, we performed comparative analysis of three species from the basal paraphyletic group - Archaeobatrachia (Bombina bombina, Xenopus laevis, Pelobates fuscus) and five species from more derived monophyletic Neobatrachia group (Hyla arborea, Bufotes viridis, Rana dalmatina, Rana arvalis, Rana temporaria). Light and scanning electron microscopy analysis showed that in Archaeobtrachia the undifferentiated gonads and ovaries, but not the testes, have pronounced external metamery. In contrast, Neobatrachia lacked external gonadal metamery and the internal metamery was well pronounced in the ovaries, but only rudimentary in the testes. There was also a difference in the site of genital ridge formation between these two groups. In Archaeobatrachia, the genital ridges developed along almost the entire length of mesonephroi, while in Neobatrachia they were limited to the anterior parts of the mesonephroi. A transient form was observed in P. fuscus, where the gonads formed along the anterior half of the mesonephroi. There was also an evolutionary trend for gradual postero-anterior shortening of the gonadal anlagen. In summary, our study indicates that among anurans there is an evolutionary trend for the gradual loss of metamery and shortening of the gonad.

Retinoic acid homeostasis regulates meiotic entry in developing anuran gonads and in Bidder's organ through Raldh2 and Cyp26b1 proteins.

The vitamin A (retinol) and its metabolites such as retinoic acid (RA) affect vertebrate gametogenesis. The level of RA in cells relies on the balance between its synthesis and degradation. The sex-dependent equilibrium is reached in different ways in various species. It is known that RA induces meiosis in developing gonads in mouse, chicken and urodel amphibians, but its role in anuran amphibians has not been studied. Here we show in six anuran species (Xenopus laevis, Bombina bombina, Hyla arborea, Bufo viridis, Rana arvalis and Rana temporaria) that cultured undifferentiated gonads were insensitive to RA treatment, but the RA induced ectopic meiosis in cultured larval testes. In larval testes of all studied species, the exogenous RA induced leptotene phase of I meiotic prophase in gonia, but only in H. arborea and B. viridis gonia progressed to zygotene phase. In the cultured developing ovaries, exogenous RA led to increase in the number of oocytes as compared to the control. Inhibition of either RA synthesis or RA-receptors prevented meiotic entry in larval gonads of all species. Exogenous RA rescued this inhibitory effect demonstrating that the balance in RA homeostasis plays a key role in meiotic entry in anuran gonads. The localization of two enzymes, Raldh2 and Cyp26b1, which antagonistically control RA levels and whose abundance suggests the sites of RA synthesis and degradation respectively, showed two distinct expression patterns specific for (i) X. laevis, H. arborea, R. arvalis, R. temporaria and (ii) B. bombina, B. viridis. Thus, RA, in correlation with specific expression patterns of Raldh2 and Cyp26b, induces meiosis during gonad development in anurans. In addition, in B. viridis, RA signalling seems important for development of the Bidder's organ containing oocytes both in males and females.

Complete mitochondrial genomes resolve phylogenetic relationships within Bombina (Anura: Bombinatoridae).

A highly resolved and time-calibrated phylogeny based on nucleotide variation in 18 complete mitochondrial genomes is presented for all extant species and major lineages of fire-bellied toads of the genus Bombina (Bombinatoridae). Two sets of divergence time estimates are inferred by applying alternative fossil constraints as minima. Divergence time estimates from both analyses differed for the two oldest nodes. The earliest phylogenetic split occurred between small- and large-bodied Bombina (subgenera Bombina and Grobina, respectively) either in the Middle Oligocene or the Early Miocene. East Asian B. orientalis and European B. bombina+B. variegata diverged in the early or Middle Miocene. Divergence times inferred using the alternative fossil calibration strategies converged for the younger nodes, with broadly overlapping HPD intervals. The split between Bombina bombina and B. variegata occurred in the Late Miocene of Europe and somewhat preceded another deep mtDNA division between the Balkan B. v. scabra and B. v. variegata inhabiting the Carpathian Mts. Concurrently, the genetically distinct B. maxima diverged from other Grobina in southeast Asia in the Late Miocene or Pliocene. Our mtDNA phylogeny and a new species-tree analysis of published data (nuclear and mtDNA) suggest that B. fortinuptialis, B. lichuanensis and B. microdeladigitora may be conspecific geographic forms that separated due to Pleistocene climatic fluctuations in southeastern Asia. In the western Palearctic, the Late Pliocene to Pleistocene climatic vagaries most probably induced vicariant events in the evolutionary history of B. variegata that led to the formation of the two Balkan B. v. scabra lineages and the allopatric B. v. pachypus in the Apennine Peninsula. Divergence among B. bombina mtDNA lineages is low, with an Anatolian Turkey lineage as the sister group to the European mtDNA clades. In sum, Miocene diversification in the genus Bombina established six allopatrically distributed major mtDNA lineages that diversified during the Pliocene and Pleistocene and have survived until the present. The narrow habitat requirements of fire-bellied toads and extensive environmental changes throughout the Palearctic in the Neogene may have contributed to a putatively high extinction rate in these anurans resulting in the current east/west disjunction of their ranges.

Sexual dimorphism of AMH, DMRT1 and RSPO1 localization in the developing gonads of six anuran species.

In vertebrates, several genes which are differentially expressed in various species, have been implicated in sex determination and gonadal differentiation. We used immunolocalization to study the expression pattern of three proteins AMH, DMRT1, RSPO1 involved in the sexual differentiation of gonads. The pattern of AMH, DMRT1 and RSPO1 expression was analyzed in X. laevis and in five other divergent anuran species: Bombina bombina, Bufo viridis, Hyla arborea, Rana arvalis and Rana temporaria during gonadal development. The pattern of expression of AMH in the developing testes of six studied anuran species was similar to that described for other vertebrates. AMH was strongly expressed in differentiating Sertoli cells. Interestingly, in B. viridis, R. arvalis and R. temporaria, AMH was also expressed in ovaries. In all studied species, DMRT1 was highly expressed in the developing testes, in both the somatic and germ cells. It was also expressed at low level in ovaries in all studied species, with the exception of H. arborea. RSPO1 was expressed in the developing ovaries, especially in the somatic cells, and was almost undetectable in developing testes in all examined anurans. These developmental expression patterns strongly suggest an involvement of AMH and DMRT1 in the development of male gonads and of RSPO1 in the female gonads. The differences in the expression patterns of these proteins in the gonads of different species might reflect the diversity of gonadal development patterns in anurans resulting from long lasting and diverged paths of their evolution.

Differential effects of testosterone and 17ß-estradiol on gonadal development in five anuran species.

Sex hormones are essential for sexual differentiation and play a key role in the development of gonads in amphibians. The goal of this study was to evaluate the influence of exogenous sex steroids, testosterone, and 17ß-estradiol (E(2)) on development of gonads in five anuran species differing in their evolutionary positions, sex determination, and mode of gonadogenesis. We found that in two closely related species of fire-bellied toad, Bombina bombina and Bombina variegata, testosterone and E(2) exposure results in sex reversal as well as intersex and undifferentiated gonads. Similarly, sex reversal was observed in Hyla arborea after exposure to male or female sex steroids. Xenopus laevis was sensitive to E(2) but only moderately to testosterone. In Bufo viridis, treatment with either sex hormone provoked a developmental delay in gonads and Bidder's organs. Therefore, susceptibility to hormonal sex reversal appeared species dependent but unrelated to genetic sex determination and the type of gonadogenesis. We also found that the onset of sex steroid exposure influences gonad differentiation and the meiotic status of the germ cells depends on their location within the gonad. Our findings reveal differential sensitivity of amphibians to testosterone and E(2), establishing a hierarchy of sensitivity to these hormones among different anuran species.

Differential effects of busulfan on gonadal development in five divergent anuran species.

The aim of this paper was to investigate the effects of germ cell depletion on the sexual differentiation of gonads in five anuran species. We used busulfan to eliminate the germ cells. Our results indicate that germ cells are not required for gonadal ridge formation or the development of the undifferentiated gonads. We observed a gradual degeneration of gonads in studied species and the transdifferentiation of the whole gonads into large fat bodies in Xenopus laevis. In the latter the sexual differentiation of gonads or seminiferous tubules were not impaired in the absence of germ cells. Thus, the X. laevis may serve as a model to study the human Del Castillo syndrome. Our study shows that in anuran amphibians the germ cells are not necessary for the formation of the testis, but they are crucial for development of the ovaries and are required for the maintenance of the gonadal structure.

Mitochondrial genome organization and divergence in hybridizing central European waterfrogs of the Pelophylax esculentus complex (Anura, Ranidae).

Natural transfer of mitochondrial DNA has occurred between three western Palaearctic waterfrog taxa: Pelophylax lessonae, Pelophylax ridibundus and their hybridogenetic hybrid, Pelophylax kl. esculentus. The transfer is asymmetric with most P. kl. esculentus and approximately one third of all central European P. ridibundus having mtDNA derived from P. lessonae (L-mtDNA). We obtained complete nucleotide sequences of multiple mitochondrial genomes (15,376-78 bp without control regions) from all 3 taxa, including a P. ridibundus frog with introgressed L-mtDNA. The gene content and organization of the mitogenomes correspond to those typical of neobatrachians. Divergence between the mtDNAs of P. lessonae and P. ridibundus is high with an uncorrected p-distance of 11.9% across the entire mitogenome. However, the rate of nucleotide substitution depends on the degree of functional constraint with up to 30-fold differences in levels of divergence. In general, mitochondrial genes encoding the translational machinery evolve very slowly, whereas genes encoding polypeptides of the electron transport system, especially the ND genes, evolve rapidly. Only 25 of 211-213 observed amino acid replacements could be classified as radical and are therefore more likely to be exposed to selection. A disproportionately high number of amino acid substitutions has occurred in the ND4, ND4L and cytb genes of the P. lessonae lineage (including 36% of all radical changes). In contrast to the interspecific divergence, nucleotide polymorphism within L- and R-mtDNA is very low: L-mtDNA haplotypes differed on average by only 19 nucleotides, while there was no variation within two mtDNAs derived from P. ridibundus. This is an expected finding considering that we have sampled a post-glacial expansion area. Moreover, the introgressed L-mtDNA on a P. ridibundus background differed from other L-mtDNAs by only a few substitutions, indicative of a very recent introgression event. We discuss our findings in the context of natural selection acting on L-mtDNA and its potential significance in cytonuclear epistasis.

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.

Differentiation and development of gonads in the yellow-bellied toad, Bombina variegata L., 1758 (Amphibia: Anura: Bombinatoridae).

The aim of this study was to investigate consecutive stages of gonadal development of the yellow-bellied toad (Bombina variegata) with particular emphasis on the origin of somatic and germ cell lineages as well as the timing of gonial cell migration. Changes in gonadal basal lamina distribution helped to explain the exceptional mode of gonadal differentiation in this species. Atypical and rapid differentiation of the male gonad in B. variegata is the result of the ability of gonial cells to migrate into the center of the gonad relatively early. Thus, the testis medulla contains germ cells from the onset of gonadal differentiation into cortex and medulla, whereas in other anurans a sterile medulla is characteristic of both future testes and ovaries; germ cells translocate into the medulla during the subsequent stage of testis development. This atypical testiculogenesis is probably the result of an acceleration of the sex determination period, indicating a contribution of sex determination heterochrony to the course of gonadogenesis. The results also suggest that medullar cells are derived from proliferating coelomic epithelial cells. Moreover, Sertoli cells constitute an integral part of the germinal epithelium in B. variegata, as in other vertebrates. Spermatids do not contact Sertoli cells just before spermiation and do not form bundles.

Comparative analysis of mitochondrial genomes in Bombina (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.

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.

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

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.

VARIATION IN MATING CALL ACROSS THE HYBRID ZONE BETWEEN THE FIRE-BELLIED TOADS BOMBINA BOMBINA AND B. VARIEGATA.

Three components of mating call (pulse duration, cycle length, and fundamental frequency) were measured and six diagnostic enzyme loci scored across the hybrid zone between the toads Bombina bombina and B. variegata. All three call components differ significantly, but only cycle length is diagnostic. The clines in call coincide with those for enzymes, and have similar widths. This suggests that there is no strong selection on any of these characters. There are significant correlations between electrophoretic markers and call components, but these are no stronger than would be expected if the electrophoretic loci and the genes causing mating call were neutral. The selection differential on the call is no greater than 6% of the difference in mean cycle length between the two taxa. There is a substantial increase in the variance of cycle length in the center of the zone, suggesting that a small number of loci are involved (≈ three). Recombination between these loci will hinder the evolution of reinforcement and may partly be responsible for the lack of premating isolation between B. bombina and B. variegata.

THE GENETIC STRUCTURE OF THE HYBRID ZONE BETWEEN THE FIRE-BELLIED TOADS BOMBINA BOMBINA AND B. VARIEGATA: COMPARISONS BETWEEN TRANSECTS AND BETWEEN LOCI.

We compare the pattern of morphological and electrophoretic variation in the hybrid zone between Bombina bombina and B. variegata across two transects: one near Cracow and one 200 km away, near Przemysl in southeastern Poland. Morphological variation across the Przemysl transect had been surveyed more than 50 years ago; though we found a significant shift at one site, there is no evidence for gross movement over this period. Morphological and electrophoretic changes coincide, and the average shape of the clines is the same across both transects. At the center, most of the change in frequency of six diagnostic allozymes occurs within w = 6.05 km (2-unit support limits 5.56-6.54 km). These steep gradients are generated not by selection on the allozymes themselves, but by associations with other loci: though these markers are unlinked, they are in strong linkage disequilibrium with each other [R = D/√pquv = 0.22 (0.15-0.29) at the center]. Disequilibria are broken up as alleles diffuse away from the zone and flow into the new genetic background. The net barrier to the flow of genes from bombina into variegata, which is generated by these disequilibria, is B = 51 (22-81) km. The fitness of hybrids must be substantially reduced to produce such a barrier [W̄H /W̄P = 0.58 (0.54-0.68)], and this selection must be spread over many loci [N = 55 (26-88)]. Alleles introgress significantly less far than would be expected from the age of the zone and the estimated dispersal rate [σ = 0.99 (0.82-1.14) km gen.1/2 ]: this implies selection of se = 0.37 (0.15-0.58)% on the enzymes themselves. There is weak but significant linkage disequilibrium well away from the center of the zone; this, together with the presence of parental and F1 genotypes, suggests some long-range migration. However, such migration is not likely to cause significant introgression.

GENETIC ANALYSIS OF A HYBRID ZONE BETWEEN THE FIRE-BELLIED TOADS, BOMBINA BOMBINA AND B. VARIEGATA, NEAR CRACOW IN SOUTHERN POLAND.

The fire-bellied toads Bombina bombina and B. variegata differ extensively in biochemistry, morphology, and behavior. We use a survey of five diagnostic enzyme loci across the hybrid zone near Cracow in Southern Poland to estimate the dispersal rate, selection pressures, and numbers of loci which maintain this zone. The enzyme clines coincide closely with each other and with morphological and mitochondrial DNA clines. Although the zone lies on a broad transition between environments suitable for bombina and variegata, the close concordance of diverse characters, together with increased aberrations and mortality in hybrids, suggest that the zone is maintained largely by selection against hybrids. There are strong "linkage disequilibria" between each pair of (unlinked) enzyme loci (R̄ = 0.129 [2-unit support limits: 0.119-0.139]). These are probably caused by gene flow into the zone, and they give an estimate of dispersal (σ = 890 [790-940] m gen-½ ). The clines are sharply stepped, with most of the change occurring within 6.15 (5.45-6.45) km, but with long tails of introgression on either side. This implies that the effective selection pressure on each enzyme marker (due largely to disequilibrium with other loci) is s* = 0.17 (0.159-0.181) at the center but that the selection acting directly on the enzyme loci is weak or zero (se < 0.0038). The stepped pattern implies a barrier to gene flow of 220 (48-415) km. This would substantially delay neutral introgression but would have little effect on advantageous alleles; the two taxa need not evolve independently. Strong selection is needed to maintain such a barrier: hybrid populations must have their mean fitness reduced by a factor of 0.65 (0.60-0.77). This selection must be spread over a large number of loci to account for the concordant patterns and the observed cline widths (N = 300 [80-2,000]).