Sequence diversity patterns suggesting balancing selection in partially sex‐linked genes of the plant Silene latifolia are not generated by demographic history or gene flow

DNA sequence diversity in genes in the partially sex‐linked pseudoautosomal region (PAR) of the sex chromosomes of the plant Silene latifolia is higher than expected from within‐species diversity of other genes. This could be the footprint of sexually antagonistic (SA) alleles that are maintained by balancing selection in a PAR gene (or genes) and affect polymorphism in linked genome regions. SA selection is predicted to occur during sex chromosome evolution, but it is important to test whether the unexpectedly high sequence polymorphism could be explained without it, purely by the combined effects of partial linkage with the sex‐determining region and the population's demographic history, including possible introgression from Silene dioica. To test this, we applied approximate Bayesian computation‐based model choice to autosomal sequence diversity data, to find the most plausible scenario for the recent history of S. latifolia and then to estimate the posterior density of the most relevant parameters. We then used these densities to simulate variation to be expected at PAR genes. We conclude that an excess of variants at high frequencies at PAR genes should arise in S. latifolia populations only for genes with strong associations with fully sex‐linked genes, which requires closer linkage with the fully sex‐linked region than that estimated for the PAR genes where apparent deviations from neutrality were observed. These results support the need to invoke selection to explain the S. latifolia PAR gene diversity, and encourage further work to test the possibility of balancing selection due to sexual antagonism.     

Identifying new sex-linked genes through BAC sequencing in the dioecious plant Silene latifolia

Background

Silene latifolia represents one of the best-studied plant sex chromosome systems. A new approach using RNA-seq data has recently identified hundreds of new sex-linked genes in this species. However, this approach is expected to miss genes that are either not expressed or are expressed at low levels in the tissue(s) used for RNA-seq. Therefore other independent approaches are needed to discover such sex-linked genes.

Results

Here we used 10 well-characterized S. latifolia sex-linked genes and their homologs in Silene vulgaris, a species without sex chromosomes, to screen BAC libraries of both species. We isolated and sequenced 4 Mb of BAC clones of S. latifolia X and Y and S. vulgaris genomic regions, which yielded 59 new sex-linked genes (with S. vulgaris homologs for some of them). We assembled sequences that we believe represent the tip of the Xq arm. These sequences are clearly not pseudoautosomal, so we infer that the S. latifolia X has a single pseudoautosomal region (PAR) on the Xp arm. The estimated mean gene density in X BACs is 2.2 times lower than that in S. vulgaris BACs, agreeing with the genome size difference between these species. Gene density was estimated to be extremely low in the Y BAC clones. We compared our BAC-located genes with the sex-linked genes identified in previous RNA-seq studies, and found that about half of them (those with low expression in flower buds) were not identified as sex-linked in previous RNA-seq studies. We compiled a set of ~70 validated X/Y genes and X-hemizygous genes (without Y copies) from the literature, and used these genes to show that X-hemizygous genes have a higher probability of being undetected by the RNA-seq approach, compared with X/Y genes; we used this to estimate that about 30 % of our BAC-located genes must be X-hemizygous. The estimate is similar when we use BAC-located genes that have S. vulgaris homologs, which excludes genes that were gained by the X chromosome.

Conclusions

Our BAC sequencing identified 59 new sex-linked genes, and our analysis of these BAC-located genes, in combination with RNA-seq data suggests that gene losses from the S. latifolia Y chromosome could be as high as 30 %, higher than previous estimates of 10-20 %.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1698-7) contains supplementary material, which is available to authorized users.     

The potential for sexually antagonistic polymorphism in different genome regions

Sex differences in the fitness effects of alleles at a single locus (intralocus sexual antagonism, or SA) have several evolutionary consequences. Among the consequences of SA, polymorphisms at genes partially linked to the sex-determining region of the sex chromosome pair potentially drive the evolution of suppressed recombination between the sex chromosomes. Understanding the conditions under which SA polymorphism can exist at such pseudo-autosomal (or PAR) loci should increase understanding of the evolution of recombination between sex chromosome pairs, and can help predict when we may expect potentially empirically detectable allele frequency differences between the sexes. Models so far published have concluded that PAR genes can maintain SA polymorphisms over a wider range of selection coefficients than autosomal ones, but have used restrictive assumptions. We expand the modeling of SA alleles at a single locus with the full range of degrees of linkage to the male-specific region, to include strong or weak selection and the possibility of different dominance coefficients in the two sexes. We confirm the previous major conclusion that SA polymorphisms are generally maintained in a larger region of parameter space if the locus is in the PAR than if it is autosomal.     

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.     

A new plant sex-linked gene with high sequence diversity and possible introgression of the X copy

We describe patterns of DNA sequence diversity in a newly identified sex-linked gene, SlX9/SlY9, in Silene latifolia (Caryophyllaceae). The copies on both sex chromosomes seem to be functional, and each maps close to the respective X- and Y-linked copy of another sex-linked gene pair, SlCypX/SlCypY. The Y-linked copy has low diversity, similar to what has been found for several other Y-linked genes in S. latifolia, and consistent with the theoretical expectations of hitch-hiking processes occurring on a non-recombining chromosome. However, SlX9 has higher diversity than other genes on the S. latifolia X chromosome. We evaluate the hypothesis of introgression from the closely related species S. dioica as an explanation for the high sequence diversity observed.     

THE EVOLUTIONARY DYNAMICS OF SEXUALLY ANTAGONISTIC MUTATIONS IN PSEUDOAUTOSOMAL REGIONS OF SEX CHROMOSOMES

Sex chromosomes can evolve gene contents that differ from the rest of the genome, as well as larger sex differences in gene expression compared with autosomes. This probably occurs because fully sex‐linked beneficial mutations substitute at different rates from autosomal ones, especially when fitness effects are sexually antagonistic (SA). The evolutionary properties of genes located in the recombining pseudoautosomal region (PAR) of a sex chromosome have not previously been modeled in detail. Such PAR genes differ from classical sex‐linked genes by having two alleles at a locus in both sexes; in contrast to autosomal genes, however, variants can become associated with gender. The evolutionary fates of PAR genes may therefore differ from those of either autosomal or fully sex‐linked genes. Here, we model their evolutionary dynamics by deriving expressions for the selective advantages of PAR gene mutations under different conditions. We show that, unless selection is very strong, the probability of invasion of a population by an SA mutation is usually similar to that of an autosomal mutation, unless there is close linkage to the sex‐determining region. Most PAR genes should thus evolve similarly to autosomal rather than sex‐linked genes, unless recombination is very rare in the PAR.     

Signatures of Sex-Antagonistic Selection on Recombining Sex Chromosomes

Sex-antagonistic (SA) selection has major evolutionary consequences: it can drive genomic change, constrain adaptation, and maintain genetic variation for fitness. The recombining (or pseudoautosomal) regions of sex chromosomes are a promising setting in which to study SA selection because they tend to accumulate SA polymorphisms and because recombination allows us to deploy the tools of molecular evolution to locate targets of SA selection and quantify evolutionary forces. Here we use coalescent models to characterize the patterns of polymorphism expected within and divergence between recombining X and Y (or Z and W) sex chromosomes. SA selection generates peaks of divergence between X and Y that can extend substantial distances away from the targets of selection. Linkage disequilibrium between neutral sites is also inflated. We show how the pattern of divergence is altered when the SA polymorphism or the sex-determining region was recently established. We use data from the flowering plant Silene latifolia to illustrate how the strength of SA selection might be quantified using molecular data from recombining sex chromosomes.     

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.     

DNA polymorphism in recombining and non-recombining mating-type-specific loci of the smut fungus Microbotryum

The population-genetic processes leading to the genetic degeneration of non-recombining regions have mainly been studied in animal and plant sex chromosomes. Here, we report population genetic analysis of the processes in the non-recombining mating-type-specific regions of the smut fungus Microbotryum violaceum. M. violaceum has A1 and A2 mating types, determined by mating-type-specific ‘sex chromosomes'' that contain 1–2 Mb long non-recombining regions. If genetic degeneration were occurring, then one would expect reduced DNA polymorphism in the non-recombining regions of this fungus. The analysis of DNA diversity among 19 M. violaceum strains, collected across Europe from Silene latifolia flowers, revealed that (i) DNA polymorphism is relatively low in all 20 studied loci (π∼0.15%), (ii) it is not significantly different between the two mating-type-specific chromosomes nor between the non-recombining and recombining regions, (iii) there is substantial population structure in M. violaceum populations, which resembles that of its host species, S. latifolia, and (iv) there is significant linkage disequilibrium, suggesting that widespread selfing in this species results in a reduction of the effective recombination rate across the genome. We hypothesise that selfing-related reduction of recombination across the M. violaceum genome negates the difference in the level of DNA polymorphism between the recombining and non-recombining regions, and may possibly lead to similar levels of genetic degeneration in the mating-type-specific regions of the non-recombining ‘sex chromosomes'' and elsewhere in the genome.     

Selective trade-offs and sex-chromosome evolution in Silene latifolia

Alleles of sexually antagonistic genes (i.e., genes with alleles affecting fitness in opposite directions in the two sexes) can avoid expression in the sex to which they are detrimental via two processes: they are subsumed into the nonrecombining, sex-determining portion of the sex chromosomes or they evolve sex-limited expression. The former is considered more likely and leads to Y-chromosome degeneration. We mapped quantitative trait loci of major effect for sexually dimorphic traits of Silene latifolia to the recombining portions of the sex chromosomes and found them to exhibit sex-specific expression, with the Y chromosome in males controlling a relatively larger proportion of genetic variance than the X in females and the average autosome. Both reproductive and ecophysiological traits map to the recombining region of the sex chromosomes. We argue that genetic correlations among traits maintain recombination and polymorphism for these genes because of balancing selection in males, whereas sex-limited expression represses detrimental alleles in females. Our data suggest that the Y chromosome of S. latifolia plays a major role in the control of key metabolic activities beyond reproductive functions.     

Nucleotide diversity in Silene latifolia autosomal and sex-linked genes

The plant Silene latifolia has separate sexes and sex chromosomes, and is of interest for studying the early stages of sex chromosome evolution, especially the evolution of non-recombining regions on the Y chromosome. Hitch-hiking processes associated with ongoing genetic degeneration of the non-recombining Y chromosome are predicted to reduce Y-linked genes'' effective population sizes, and S. latifolia Y-linked genes indeed have lower diversity than X-linked ones. We tested whether this represents a true diversity reduction on the Y, versus the alternative possibility, elevated diversity at X-linked genes, by collecting new data on nucleotide diversity for autosomal genes, which had previously been little studied. We find clear evidence that Y-linked genes have reduced diversity. However, another alternative explanation for a low Y effective size is a high variance in male reproductive success. Autosomal genes should then also have lower diversity than expected, relative to the X, but this is not found in our loci. Taking into account the higher mutation rate of Y-linked genes, their low sequence diversity indicates a strong effect of within-population hitch-hiking on the Y chromosome.     

Sexual antagonism and meiotic drive cause stable linkage disequilibrium and favour reduced recombination on the X chromosome

Sexual antagonism and meiotic drive are sex‐specific evolutionary forces with the potential to shape genomic architecture. Previous theory has found that pairing two sexually antagonistic loci or combining sexual antagonism with meiotic drive at linked autosomal loci augments genetic variation, produces stable linkage disequilibrium (LD) and favours reduced recombination. However, the influence of these two forces has not been examined on the X chromosome, which is thought to be enriched for sexual antagonism and meiotic drive. We investigate the evolution of the X chromosome under both sexual antagonism and meiotic drive with two models: in one, both loci experience sexual antagonism; in the other, we pair a meiotic drive locus with a sexually antagonistic locus. We find that LD arises between the two loci in both models, even when the two loci freely recombine in females and that driving haplotypes will be enriched for male‐beneficial alleles, further skewing sex ratios in these populations. We introduce a new measure of LD, , which accounts for population allele frequencies and is appropriate for instances where these are sex specific. Both models demonstrate that natural selection favours modifiers that reduce the recombination rate. These results inform observed patterns of congealment found on driving X chromosomes and have implications for patterns of natural variation and the evolution of recombination rates on the X chromosome.     

Extensive Divergence Between Mating-Type Chromosomes of the Anther-Smut Fungus

Genomic regions that determine mating compatibility are subject to distinct evolutionary forces that can lead to a cessation of meiotic recombination and the accumulation of structural changes between members of the homologous chromosome pair. The relatively recent discovery of dimorphic mating-type chromosomes in fungi can aid the understanding of sex chromosome evolution that is common to dioecious plants and animals. For the anther-smut fungus, Microbotryum lychnidis-dioicae (= M. violaceum isolated from Silene latifolia), the extent of recombination cessation on the dimorphic mating-type chromosomes has been conflictingly reported. Comparison of restriction digest optical maps for the two mating-type chromosomes shows that divergence extends over 90% of the chromosome lengths, flanked at either end by two pseudoautosomal regions. Evidence to support the expansion of recombination cessation in stages from the mating-type locus toward the pseudoautosomal regions was not found, but evidence of such expansion could be obscured by ongoing processes that affect genome structure. This study encourages the comparison of forces that may drive large-scale recombination suppression in fungi and other eukaryotes characterized by dimorphic chromosome pairs associated with sexual life cycles.     

THE EFFECTS OF SELECTION AND GENETIC DRIFT ON THE GENOMIC DISTRIBUTION OF SEXUALLY ANTAGONISTIC ALLELES

Sexual antagonism (SA) occurs when an allele that is beneficial to one sex, is detrimental to the other. This conflict can result in balancing, directional, or disruptive selection acting on SA alleles. A body of theory predicts the conditions under which sexually antagonistic mutants will invade and be maintained in stable polymorphism under balancing selection. There remains, however, considerable debate over the distribution of SA genetic variation across autosomes and sex chromosomes, with contradictory evidence coming from data and theory. In this article, we investigate how the interplay between selection and genetic drift will affect the genomic distribution of sexually antagonistic alleles. The effective population sizes can differ between the autosomes and the sex chromosomes due to a number of ecological factors and, consequently, the distribution of SA genetic variation in genomes. In general, we predict the interplay of SA selection and genetic drift should lead to the accumulation of SA alleles on the X in male heterogametic (XY) species and, on the autosomes in female heterogametic (ZW) species, especially when sexual competition is strong among males.     

Consequences of genetic linkage for the maintenance of sexually antagonistic polymorphism in hermaphrodites

When selection differs between males and females, pleiotropic effects among genes expressed by both sexes can result in sexually antagonistic selection (SA), where beneficial alleles for one sex are deleterious for the other. For hermaphrodites, alleles with opposing fitness effects through each sex function represent analogous genetic constraints on fitness. Recent theory based on single‐locus models predicts that the maintenance of SA genetic variation should be greatly reduced in partially selfing populations. However, selfing also reduces the effective rate of recombination, which should facilitate selection on linked allelic combinations and expand opportunities for balancing selection in a multilocus context. Here, I develop a two‐locus model of SA selection for simultaneous hermaphrodites, and explore the joint influence of linkage, self‐fertilization, and dominance on the maintainance of SA polymorphism. I find that the effective reduction in recombination caused by selfing significantly expands the parameter space where SA polymorphism can be maintained relative to single‐locus models. In particular, linkage facilitates the invasion of male‐beneficial alleles, partially compensating for the “female‐bias” in the net direction of selection created by selfing. I discuss the implications of accounting for linkage among SA loci for the maintenance of SA genetic variation and mixed mating systems in hermaphrodites.     

Heterochiasmy and the establishment of gsdf as a novel sex determining gene in Atlantic halibut

Atlantic Halibut (Hippoglossus hippoglossus) has a X/Y genetic sex determination system, but the sex determining factor is not known. We produced a high-quality genome assembly from a male and identified parts of chromosome 13 as the Y chromosome due to sequence divergence between sexes and segregation of sex genotypes in pedigrees. Linkage analysis revealed that all chromosomes exhibit heterochiasmy, i.e. male-only and female-only meiotic recombination regions (MRR/FRR). We show that FRR/MRR intervals differ in nucleotide diversity and repeat class content and that this is true also for other Pleuronectidae species. We further show that remnants of a Gypsy-like transposable element insertion on chr13 promotes early male specific expression of gonadal somatic cell derived factor (gsdf). Less than 4.5 MYA, this male-determining element evolved on an autosomal FRR segment featuring pre-existing male meiotic recombination barriers, thereby creating a Y chromosome. Our findings indicate that heterochiasmy may facilitate the evolution of genetic sex determination systems relying on linkage of sexually antagonistic loci to a sex-determining factor.     

Sex‐antagonistic genes,XY recombination and feminized Y chromosomes

The canonical model of sex‐chromosome evolution predicts that sex‐antagonistic (SA) genes play an instrumental role in the arrest of XY recombination and ensuing Y chromosome degeneration. Although this model might account for the highly differentiated sex chromosomes of birds and mammals, it does not fit the situation of many lineages of fish, amphibians or nonavian reptiles, where sex chromosomes are maintained homomorphic through occasional XY recombination and/or high turnover rates. Such situations call for alternative explanatory frameworks. A crucial issue at stake is the effect of XY recombination on the dynamics of SA genes and deleterious mutations. Using individual‐based simulations, we show that a complete arrest of XY recombination actually benefits females, not males. Male fitness is maximized at different XY recombination rates depending on SA selection, but never at zero XY recombination. This should consistently favour some level of XY recombination, which in turn generates a recombination load at sex‐linked SA genes. Hill–Robertson interferences with deleterious mutations also impede the differentiation of sex‐linked SA genes, to the point that males may actually fix feminized phenotypes when SA selection and XY recombination are low. We argue that sex chromosomes might not be a good localization for SA genes, and sex conflicts seem better solved through the differential expression of autosomal genes.     

Chaos of Rearrangements in the Mating-Type Chromosomes of the Anther-Smut Fungus Microbotryum lychnidis-dioicae

Sex chromosomes in plants and animals and fungal mating-type chromosomes often show exceptional genome features, with extensive suppression of homologous recombination and cytological differentiation between members of the diploid chromosome pair. Despite strong interest in the genetics of these chromosomes, their large regions of suppressed recombination often are enriched in transposable elements and therefore can be challenging to assemble. Here we show that the latest improvements of the PacBio sequencing yield assembly of the whole genome of the anther-smut fungus, Microbotryum lychnidis-dioicae (the pathogenic fungus causing anther-smut disease of Silene latifolia), into finished chromosomes or chromosome arms, even for the repeat-rich mating-type chromosomes and centromeres. Suppressed recombination of the mating-type chromosomes is revealed to span nearly 90% of their lengths, with extreme levels of rearrangements, transposable element accumulation, and differentiation between the two mating types. We observed no correlation between allelic divergence and physical position in the nonrecombining regions of the mating-type chromosomes. This may result from gene conversion or from rearrangements of ancient evolutionary strata, i.e., successive steps of suppressed recombination. Centromeres were found to be composed mainly of copia-like transposable elements and to possess specific minisatellite repeats identical between the different chromosomes. We also identified subtelomeric motifs. In addition, extensive signs of degeneration were detected in the nonrecombining regions in the form of transposable element accumulation and of hundreds of gene losses on each mating-type chromosome. Furthermore, our study highlights the potential of the latest breakthrough PacBio chemistry to resolve complex genome architectures.     

Evidence of the accumulation of allele-specific non-synonymous substitutions in the young region of recombination suppression within the mating-type chromosomes of Neurospora tetrasperma

Currently, little is known about the origin and early evolution of sex chromosomes. This is largely due to the fact that ancient non-recombining sex chromosomes are highly degenerated, and thus provide little information about the early genomic events in their evolution. The Neurospora tetrasperma mating-type (mat) chromosomes contain a young (<6 Mya) and large region (>6.6 Mb) of suppressed recombination, thereby providing a model system to study early stages of sex chromosome evolution. Here, we examined alleles of 207 genes located on the N. tetrasperma mat a and mat A chromosomes to test for signs of genomic alterations at the protein level in the young region of recombination suppression. We report that the N. tetrasperma mat a and mat A chromosomes have each independently accumulated allele-specific non-synonymous codon substitutions in a time-dependent, and gene-specific manner in the recombinationally suppressed region. In addition, examination of the ratio (ω) of non-synonymous substitutions (dN) to synonymous substitutions (dS) using maximum likelihood analyses, indicates that such changes are associated with relaxed purifying selection, a finding consistent with genomic degeneration. We also reveal that sex specific biases in mutation rates or selection pressures are not necessary for genomic alterations in sex chromosomes, and that recombination suppression in itself is sufficient to explain these results. The present findings extend our current understanding of genomic events associated within the young region of recombination suppression in these fungal sex-regulating chromosomes.