No evidence that Y-chromosome differentiation affects male fitness in a Swiss population of common frogs.

The canonical model of sex-chromosome evolution assigns a key role to sexually antagonistic (SA) genes on the arrest of recombination and ensuing degeneration of Y chromosomes. This assumption cannot be tested in organisms with highly differentiated sex chromosomes, such as mammals or birds, owing to the lack of polymorphism. Fixation of SA alleles, furthermore, might be the consequence rather than the cause of recombination arrest. Here we focus on a population of common frogs (Rana temporaria) where XY males with genetically differentiated Y chromosomes (nonrecombinant Y haplotypes) coexist with both XY° males with proto-Y chromosomes (only differentiated from X chromosomes in the immediate vicinity of the candidate sex-determining locus Dmrt1) and XX males with undifferentiated sex chromosomes (genetically identical to XX females). Our study finds no effect of sex-chromosome differentiation on male phenotype, mating success or fathering success. Our conclusions rejoin genomic studies that found no differences in gene expression between XY, XY° and XX males. Sexual dimorphism in common frogs might result more from the differential expression of autosomal genes than from sex-linked SA genes. Among-male variance in sex-chromosome differentiation seems better explained by a polymorphism in the penetrance of alleles at the sex locus, resulting in variable levels of sex reversal (and thus of X-Y recombination in XY females), independent of sex-linked SA genes.

Evolutionary and developmental dynamics of sex-biased gene expression in common frogs with proto-Y chromosomes.

BACKGROUND: The patterns of gene expression on highly differentiated sex chromosomes differ drastically from those on autosomes, due to sex-specific patterns of selection and inheritance. As a result, X chromosomes are often enriched in female-biased genes (feminization) and Z chromosomes in male-biased genes (masculinization). However, it is not known how quickly sexualization of gene expression and transcriptional degeneration evolve after sex-chromosome formation. Furthermore, little is known about how sex-biased gene expression varies throughout development. RESULTS: We sample a population of common frogs (Rana temporaria) with limited sex-chromosome differentiation (proto-sex chromosome), leaky genetic sex determination evidenced by the occurrence of XX males, and delayed gonadal development, meaning that XY individuals may first develop ovaries before switching to testes. Using high-throughput RNA sequencing, we investigate the dynamics of gene expression throughout development, spanning from early embryo to froglet stages. Our results show that sex-biased expression affects different genes at different developmental stages and increases during development, reaching highest levels in XX female froglets. Additionally, sex-biased gene expression depends on phenotypic, rather than genotypic sex, with similar expression in XX and XY males; correlates with gene evolutionary rates; and is not localized to the proto-sex chromosome nor near the candidate sex-determining gene Dmrt1. CONCLUSIONS: The proto-sex chromosome of common frogs does not show evidence of sexualization of gene expression, nor evidence for a faster rate of evolution. This challenges the notion that sexually antagonistic genes play a central role in the initial stages of sex-chromosome evolution.

A rapid rate of sex-chromosome turnover and non-random transitions in true frogs.

The canonical model of sex-chromosome evolution predicts that, as recombination is suppressed along sex chromosomes, gametologs will progressively differentiate, eventually becoming heteromorphic. However, there are numerous examples of homomorphic sex chromosomes across the tree of life. This homomorphy has been suggested to result from frequent sex-chromosome turnovers, yet we know little about which forces drive them. Here, we describe an extremely fast rate of turnover among 28 species of Ranidae. Transitions are not random, but converge on several chromosomes, potentially due to genes they harbour. Transitions also preserve the ancestral pattern of male heterogamety, in line with the 'hot-potato' model of sex-chromosome transitions, suggesting a key role for mutation-load accumulation in non-recombining genomic regions. The importance of mutation-load selection in frogs might result from the extreme heterochiasmy they exhibit, making frog sex chromosomes differentiate immediately from emergence and across their entire length.

Tissue Specificity and Dynamics of Sex-Biased Gene Expression in a Common Frog Population with Differentiated, Yet Homomorphic, Sex Chromosomes.

Sex-biased genes are central to the study of sexual selection, sexual antagonism, and sex chromosome evolution. We describe a comprehensive de novo assembled transcriptome in the common frog Rana temporaria based on five developmental stages and three adult tissues from both sexes, obtained from a population with karyotypically homomorphic but genetically differentiated sex chromosomes. This allows the study of sex-biased gene expression throughout development, and its effect on the rate of gene evolution while accounting for pleiotropic expression, which is known to negatively correlate with the evolutionary rate. Overall, sex-biased genes had little overlap among developmental stages and adult tissues. Late developmental stages and gonad tissues had the highest numbers of stage- or tissue-specific genes. We find that pleiotropic gene expression is a better predictor than sex bias for the evolutionary rate of genes, though it often interacts with sex bias. Although genetically differentiated, the sex chromosomes were not enriched in sex-biased genes, possibly due to a very recent arrest of XY recombination. These results extend our understanding of the developmental dynamics, tissue specificity, and genomic localization of sex-biased genes.

Profound genetic divergence and asymmetric parental genome contributions as hallmarks of hybrid speciation in polyploid toads.

The evolutionary causes and consequences of allopolyploidization, an exceptional pathway to instant hybrid speciation, are poorly investigated in animals. In particular, when and why hybrid polyploids versus diploids are produced, and constraints on sources of paternal and maternal ancestors, remain underexplored. Using the Palearctic green toad radiation (including bisexually reproducing species of three ploidy levels) as model, we generate a range-wide multi-locus phylogeny of 15 taxa and present four new insights: (i) at least five (up to seven) distinct allotriploid and allotetraploid taxa have evolved in the Pleistocene; (ii) all maternal and paternal ancestors of hybrid polyploids stem from two deeply diverged nuclear clades (6 Mya, 3.1-9.6 Mya), with distinctly greater divergence than the parental species of diploid hybrids found at secondary contact zones; (iii) allotriploid taxa possess two conspecific genomes and a deeply diverged allospecific one, suggesting that genomic imbalance and divergence are causal for their partly clonal reproductive mode; (iv) maternal versus paternal genome contributions exhibit asymmetry, with the maternal nuclear (and mitochondrial) genome of polyploids always coming from the same clade, and the paternal genome from the other. We compare our findings with similar patterns in diploid/polyploid vertebrates, and suggest deep ancestral divergence as a precondition for successful allopolyploidization.

Identifying homomorphic sex chromosomes from wild-caught adults with limited genomic resources.

We demonstrate a genotyping-by-sequencing approach to identify homomorphic sex chromosomes and their homolog in a distantly related reference genome, based on noninvasive sampling of wild-caught individuals, in the moor frog Rana arvalis. Double-digest RADseq libraries were generated using buccal swabs from 30 males and 21 females from the same population. Search for sex-limited markers from the unfiltered data set (411 446 RAD tags) was more successful than searches from a filtered data set (33 073 RAD tags) for markers showing sex differences in heterozygosity or in allele frequencies. Altogether, we obtained 292 putatively sex-linked RAD loci, 98% of which point to male heterogamety. We could map 15 of them to the Xenopus tropicalis genome, all but one on chromosome pair 1, which seems regularly co-opted for sex determination among amphibians. The most efficient mapping strategy was a three-step hierarchical approach, where R. arvalis reads were first mapped to a low-coverage genome of Rana temporaria (17 My divergence), then the R. temporaria scaffolds to the Nanorana parkeri genome (90 My divergence), and finally the N. parkeri scaffolds to the X. tropicalis genome (210 My). We validated our conclusions with PCR primers amplifying part of Dmrt1, a candidate sex determination gene mapping to chromosome 1: a sex-diagnostic allele was present in all 30 males but in none of the 21 females. Our approach is likely to be productive in many situations where biological samples and/or genomic resources are limited.

Dmrt1 polymorphism covaries with sex-determination patterns in Rana temporaria.

Patterns of sex-chromosome differentiation and gonadal development have been shown to vary among populations of Rana temporaria along a latitudinal transect in Sweden. Frogs from the northern-boreal population of Ammarnäs displayed well-differentiated X and Y haplotypes, early gonadal differentiation, and a perfect match between phenotypic and genotypic sex. In contrast, no differentiated Y haplotypes could be detected in the southern population of Tvedöra, where juveniles furthermore showed delayed gonadal differentiation. Here, we show that Dmrt1, a gene that plays a key role in sex determination and sexual development across all metazoans, displays significant sex differentiation in Tvedöra, with a Y-specific haplotype distinct from Ammarnäs. The differential segment is not only much shorter in Tvedöra than in Ammarnäs, it is also less differentiated and associates with both delayed gonadal differentiation and imperfect match between phenotypic and genotypic sex. Whereas Tvedöra juveniles with a local Y haplotype tend to ultimately develop as males, those without it may nevertheless become functional XX males, but with strongly female-biased progeny. Our findings suggest that the variance in patterns of sex determination documented in common frogs might result from a genetic polymorphism within a small genomic region that contains Dmrt1. They also substantiate the view that recurrent convergences of sex determination toward a limited set of chromosome pairs may result from the co-option of small genomic regions that harbor key genes from the sex-determination pathway.

Sex-Chromosome Homomorphy in Palearctic Tree Frogs Results from Both Turnovers and X-Y Recombination.

Contrasting with birds and mammals, poikilothermic vertebrates often have homomorphic sex chromosomes, possibly resulting from high rates of sex-chromosome turnovers and/or occasional X-Y recombination. Strong support for the latter mechanism was provided by four species of European tree frogs, which inherited from a common ancestor (∼ 5 Ma) the same pair of homomorphic sex chromosomes (linkage group 1, LG1), harboring the candidate sex-determining gene Dmrt1. Here, we test sex linkage of LG1 across six additional species of the Eurasian Hyla radiation with divergence times ranging from 6 to 40 Ma. LG1 turns out to be sex linked in six of nine resolved cases. Mapping the patterns of sex linkage to the Hyla phylogeny reveals several transitions in sex-determination systems within the last 10 My, including one switch in heterogamety. Phylogenetic trees of DNA sequences along LG1 are consistent with occasional X-Y recombination in all species where LG1 is sex linked. These patterns argue against one of the main potential causes for turnovers, namely the accumulation of deleterious mutations on nonrecombining chromosomes. Sibship analyses show that LG1 recombination is strongly reduced in males from most species investigated, including some in which it is autosomal. Intrinsically low male recombination might facilitate the evolution of male heterogamety, and the presence of important genes from the sex-determination cascade might predispose LG1 to become a sex chromosome.

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.

Ever-young sex chromosomes in European tree frogs.

Non-recombining sex chromosomes are expected to undergo evolutionary decay, ending up genetically degenerated, as has happened in birds and mammals. Why are then sex chromosomes so often homomorphic in cold-blooded vertebrates? One possible explanation is a high rate of turnover events, replacing master sex-determining genes by new ones on other chromosomes. An alternative is that X-Y similarity is maintained by occasional recombination events, occurring in sex-reversed XY females. Based on mitochondrial and nuclear gene sequences, we estimated the divergence times between European tree frogs (Hyla arborea, H. intermedia, and H. molleri) to the upper Miocene, about 5.4-7.1 million years ago. Sibship analyses of microsatellite polymorphisms revealed that all three species have the same pair of sex chromosomes, with complete absence of X-Y recombination in males. Despite this, sequences of sex-linked loci show no divergence between the X and Y chromosomes. In the phylogeny, the X and Y alleles cluster according to species, not in groups of gametologs. We conclude that sex-chromosome homomorphy in these tree frogs does not result from a recent turnover but is maintained over evolutionary timescales by occasional X-Y recombination. Seemingly young sex chromosomes may thus carry old-established sex-determining genes, a result at odds with the view that sex chromosomes necessarily decay until they are replaced. This raises intriguing perspectives regarding the evolutionary dynamics of sexually antagonistic genes and the mechanisms that control X-Y recombination.

Description of microsatellite markers and genotyping performances using feathers and buccal swabs for the Ivory gull (Pagophila eburnea).

We report 22 new polymorphic microsatellites for the Ivory gull (Pagophila eburnea), and we describe how they can be efficiently co-amplified using multiplexed polymerase chain reactions. In addition, we report DNA concentration, amplification success, rates of genotyping errors and the number of genotyping repetitions required to obtain reliable data with three types of noninvasive or nondestructive samples: shed feathers collected in colonies, feathers plucked from living individuals and buccal swabs. In two populations from Greenland (n=21) and Russia (Severnaya Zemlya Archipelago, n=21), the number of alleles per locus varied between 2 and 17, and expected heterozygosity per population ranged from 0.18 to 0.92. Twenty of the markers conformed to Hardy-Weinberg and linkage equilibrium expectations. Most markers were easily amplified and highly reliable when analysed from buccal swabs and plucked feathers, showing that buccal swabbing is a very efficient approach allowing good quality DNA retrieval. Although DNA amplification success using single shed feathers was generally high, the genotypes obtained from this type of samples were prone to error and thus need to be amplified several times. The set of microsatellite markers described here together with multiplex amplification conditions and genotyping error rates will be useful for population genetic studies of the Ivory gull.