Inferring the degree of incipient speciation in secondary contact zones of closely related lineages of Palearctic green toads (Bufo viridis subgroup)

Reproductive isolation between lineages is expected to accumulate with divergence time, but the time taken to speciate may strongly vary between different groups of organisms. In anuran amphibians, laboratory crosses can still produce viable hybrid offspring >20 My after separation, but the speed of speciation in closely related anuran lineages under natural conditions is poorly studied. Palearctic green toads (Bufo viridis subgroup) offer an excellent system to address this question, comprising several lineages that arose at different times and form secondary contact zones. Using mitochondrial and nuclear markers, we previously demonstrated that in Sicily, B. siculus and B. balearicus developed advanced reproductive isolation after Plio-Pleistocene divergence (2.6 My, 3.3–1.9), with limited historic mtDNA introgression, scarce nuclear admixture, but low, if any, current gene flow. Here, we study genetic interactions between younger lineages of early Pleistocene divergence (1.9 My, 2.5–1.3) in northeastern Italy (B. balearicus, B. viridis). We find significantly more, asymmetric nuclear and wider, differential mtDNA introgression. The population structure seems to be molded by geographic distance and barriers (rivers), much more than by intrinsic genomic incompatibilities. These differences of hybridization between zones may be partly explained by differences in the duration of previous isolation. Scattered research on other anurans suggests that wide hybrid zones with strong introgression may develop when secondary contacts occur <2 My after divergence, whereas narrower zones with restricted gene flow form when divergence exceeds 3 My. Our study strengthens support for this rule of thumb by comparing lineages with different divergence times within the same radiation.     

Dmrt1 polymorphism and sex‐chromosome differentiation in Rana temporaria

Sex‐determination mechanisms vary both within and among populations of common frogs, opening opportunities to investigate the molecular pathways and ultimate causes shaping their evolution. We investigated the association between sex‐chromosome differentiation (as assayed from microsatellites) and polymorphism at the candidate sex‐determining gene Dmrt1 in two Alpine populations. Both populations harboured a diversity of X‐linked and Y‐linked Dmrt1 haplotypes. Some males had fixed male‐specific alleles at all markers (“differentiated” Y chromosomes), others only at Dmrt1 (“proto‐” Y chromosomes), while still others were genetically indistinguishable from females (undifferentiated X chromosomes). Besides these XX males, we also found rare XY females. The several Dmrt1 Y haplotypes differed in the probability of association with a differentiated Y chromosome, which we interpret as a result of differences in the masculinizing effects of alleles at the sex‐determining locus. From our results, the polymorphism in sex‐chromosome differentiation and its association with Dmrt1, previously inferred from Swedish populations, are not just idiosyncratic features of peripheral populations, but also characterize highly diverged populations in the central range. This implies that an apparently unstable pattern has been maintained over long evolutionary times.     

Dobzhansky–Muller incompatibilities,dominance drive,and sex‐chromosome introgression at secondary contact zones: A simulation study

Dobzhansky–Muller (DM) incompatibilities involving sex chromosomes have been proposed to account for Haldane's rule (lowered fitness among hybrid offspring of the heterogametic sex) as well as Darwin's corollary (asymmetric fitness costs with respect to the direction of the cross). We performed simulation studies of a hybrid zone to investigate the effects of different types of DM incompatibilities on cline widths and positions of sex‐linked markers. From our simulations, X‐Y incompatibilities generate steep clines for both X‐linked and Y‐linked markers; random effects may produce strong noise in cline center positions when migration is high relative to fitness costs, but X‐ and Y‐centers always coincide strictly. X‐autosome and Y‐autosome incompatibilities also generate steep clines, but systematic shifts in cline centers occur when migration is high relative to selection, as a result of a dominance drive linked to Darwin's corollary. Interestingly, sex‐linked genes always show farther introgression than the associated autosomal genes. We discuss ways of disentangling the potentially confounding effects of sex biases in migration, we compare our results to those of a few documented contact zones, and we stress the need to study independent replicates of the same contact zone.     

HOMOLOGOUS SEX CHROMOSOMES IN THREE DEEPLY DIVERGENT ANURAN SPECIES

Comparative genomic studies are revealing that, in sharp contrast with the strong stability found in birds and mammals, sex determination mechanisms are surprisingly labile in cold‐blooded vertebrates, with frequent transitions between different pairs of sex chromosomes. It was recently suggested that, in context of this high turnover, some chromosome pairs might be more likely than others to be co‐opted as sex chromosomes. Empirical support, however, is still very limited. Here we show that sex‐linked markers from three highly divergent groups of anurans map to Xenopus tropicalis scaffold 1, a large part of which is homologous to the avian sex chromosome. Accordingly, the bird sex determination gene DMRT1, known to play a key role in sex differentiation across many animal lineages, is sex linked in all three groups. Our data provide strong support for the idea that some chromosome pairs are more likely than others to be co‐opted as sex chromosomes because they harbor key genes from the sex determination pathway.     

Polymorphism at a Sex-Linked Transcription Cofactor in European Tree Frogs (<Emphasis Type="Italic">Hyla arborea</Emphasis>): Sex-Antagonistic Selection or Neutral Processes?

Nascent sex chromosomes offer a unique opportunity to investigate the evolutionary fate of genes recently trapped in non-recombining segments. A house-keeping gene (MED15) was recently shown to lie on the nascent sex-chromosomes of the European tree frog (Hyla arborea), with different alleles fixed on the X and the Y chromosomes. Here we document a polymorphism (glutamine deletion) in the X copy of the gene, and use population surveys and experimental crosses to test whether this polymorphism is neutral or maintained by sex-antagonistic selection. Tadpoles from parents of known genotypes revealed significant discrepancies from Mendelian inheritance, suggesting possible sex-antagonistic effects under laboratory conditions. Quantitatively, however, these effects did not meet the conditions for polymorphism maintenance. Furthermore, field estimates of female genotypic frequencies did not differ from Hardy–Weinberg equilibrium and allelic frequencies on the X chromosome did not differ between sexes. In conclusion, although sex-antagonistic effects cannot be excluded given the laboratory conditions, the X-linked polymorphism under study appears neutral in the wild. Alternatively, sex-antagonistic selection might still account for the fixation of a male-specific allele on the Y chromosome.     

Sex‐chromosome differentiation parallels postglacial range expansion in European tree frogs (Hyla arborea)

Occasional XY recombination is a proposed explanation for the sex‐chromosome homomorphy in European tree frogs. Numerous laboratory crosses, however, failed to detect any event of male recombination, and a detailed survey of NW‐European Hyla arborea populations identified male‐specific alleles at sex‐linked loci, pointing to the absence of XY recombination in their recent history. Here, we address this paradox in a phylogeographic framework by genotyping sex‐linked microsatellite markers in populations and sibships from the entire species range. Contrasting with postglacial populations of NW Europe, which display complete absence of XY recombination and strong sex‐chromosome differentiation, refugial populations of the southern Balkans and Adriatic coast show limited XY recombination and large overlaps in allele frequencies. Geographically and historically intermediate populations of the Pannonian Basin show intermediate patterns of XY differentiation. Even in populations where X and Y occasionally recombine, the genetic diversity of Y haplotypes is reduced below the levels expected from the fourfold drop in copy numbers. This study is the first in which X and Y haplotypes could be phased over the distribution range in a species with homomorphic sex chromosomes; it shows that XY‐recombination patterns may differ strikingly between conspecific populations, and that recombination arrest may evolve rapidly (<5000 generations).     

Cryptic diversity among Western Palearctic tree frogs: Postglacial range expansion, range limits, and secondary contacts of three European tree frog lineages (Hyla arborea group)

We characterize divergence times, intraspecific diversity and distributions for recently recognized lineages within the Hyla arborea species group, based on mitochondrial and nuclear sequences from 160 localities spanning its whole distribution. Lineages of H. arborea, H. orientalis, H. molleri have at least Pliocene age, supporting species level divergence. The genetically uniform Iberian H. molleri, although largely isolated by the Pyrenees, is parapatric to H. arborea, with evidence for successful hybridization in a small Aquitanian corridor (southwestern France), where the distribution also overlaps with H. meridionalis. The genetically uniform H. arborea, spread from Crete to Brittany, exhibits molecular signatures of a postglacial range expansion. It meets different mtDNA clades of H. orientalis in NE-Greece, along the Carpathians, and in Poland along the Vistula River (there including hybridization). The East-European H. orientalis is strongly structured genetically. Five geographic mitochondrial clades are recognized, with a molecular signature of postglacial range expansions for the clade that reached the most northern latitudes. Hybridization with H. savignyi is suggested in southwestern Turkey. Thus, cryptic diversity in these Pliocene Hyla lineages covers three extremes: a genetically poor, quasi-Iberian endemic (H. molleri), a more uniform species distributed from the Balkans to Western Europe (H. arborea), and a well-structured Asia Minor-Eastern European species (H. orientalis).     

High-density sex-specific linkage maps of a European tree frog (Hyla arborea) identify the sex chromosome without information on offspring sex

Identifying homology between sex chromosomes of different species is essential to understanding the evolution of sex determination. Here, we show that the identity of a homomorphic sex chromosome pair can be established using a linkage map, without information on offspring sex. By comparing sex-specific maps of the European tree frog Hyla arborea, we find that the sex chromosome (linkage group 1) shows a threefold difference in marker number between the male and female maps. In contrast, the number of markers on each autosome is similar between the two maps. We also find strongly conserved synteny between H. arborea and Xenopus tropicalis across 200 million years of evolution, suggesting that the rate of chromosomal rearrangement in anurans is low. Finally, we show that recombination in males is greatly reduced at the centers of large chromosomes, consistent with previous cytogenetic findings. Our research shows the importance of high-density linkage maps for studies of recombination, chromosomal rearrangement and the genetic architecture of ecologically or economically important traits.     

When Sex Chromosomes Recombine Only in the Heterogametic Sex: Heterochiasmy and Heterogamety in Hyla Tree Frogs

Sex chromosomes are classically predicted to stop recombining in the heterogametic sex, thereby enforcing linkage between sex-determining (SD) and sex-antagonistic (SA) genes. With the same rationale, a pre-existing sex asymmetry in recombination is expected to affect the evolution of heterogamety, for example, a low rate of male recombination might favor transitions to XY systems, by generating immediate linkage between SD and SA genes. Furthermore, the accumulation of deleterious mutations on nonrecombining Y chromosomes should favor XY-to-XY transitions (which discard the decayed Y), but disfavor XY-to-ZW transitions (which fix the decayed Y as an autosome). Like many anuran amphibians, Hyla tree frogs have been shown to display drastic heterochiasmy (males only recombine at chromosome tips) and are typically XY, which seems to fit the above expectations. Instead, here we demonstrate that two species, H. sarda and H. savignyi, share a common ZW system since at least 11 Ma. Surprisingly, the typical pattern of restricted male recombination has been maintained since then, despite female heterogamety. Hence, sex chromosomes recombine freely in ZW females, not in ZZ males. This suggests that heterochiasmy does not constrain heterogamety (and vice versa), and that the role of SA genes in the evolution of sex chromosomes might have been overemphasized.