Large X-effects are absent in torrent frogs with nascent sex chromosomes.

Sex chromosomes are popularized as a special role in driving speciation. However, the empirical evidence from natural population processes has been limited to organisms with degenerated sex chromosomes, where hemizygosity is mainly considered to act as the driver of reproductive isolation. Here, we examined several hybrid zones of torrent frog Amolops mantzorum species complex, using an approach by mapping species-diagnostic loci onto the reference genome to compare sex-linked versus autosomal patterns of introgression. We find little support in sex-linked incompatibilities for large X-effects for these populations in hybrid zones with homomorphic sex chromosomes, due to the absence of the hemizygous effects. As expected, the large X-effects were not found in those with heteromorphic but newly evolved sex chromosomes, owing to the absence of strong genetic differences between X and Y chromosomes. The available data so far on amphibians suggest little role for sex-linked genes in speciation. The large X-effects in those with nascent sex chromosomes may not be as ubiquitous as presumed across the animal kingdom.

Sex chromosomes as supergenes of speciation: why amphibians defy the rules?

As reflected by the two rules of speciation (Haldane's rule and the large X-/Z-effect), sex chromosomes are expected to behave like supergenes of speciation: they recombine only in one sex (XX females or ZZ males), supposedly recruit sexually antagonistic genes and evolve faster than autosomes, which can all contribute to pre-zygotic and post-zygotic isolation. While this has been mainly studied in organisms with conserved sex-determining systems and highly differentiated (heteromorphic) sex chromosomes like mammals, birds and some insects, these expectations are less clear in organismal groups where sex chromosomes repeatedly change and remain mostly homomorphic, like amphibians. In this article, we review the proposed roles of sex-linked genes in isolating nascent lineages throughout the speciation continuum and discuss their support in amphibians given current knowledge of sex chromosome evolution and speciation modes. Given their frequent recombination and lack of differentiation, we argue that amphibian sex chromosomes are not expected to become supergenes of speciation, which is reflected by the rarity of empirical studies consistent with a 'large sex chromosome effect' in frogs and toads. The diversity of sex chromosome systems in amphibians has a high potential to disentangle the evolutionary mechanisms responsible for the emergence of sex-linked speciation genes in other organisms. This article is part of the theme issue 'Genomic architecture of supergenes: causes and evolutionary consequences'.

Hybrid Sterility, Genetic Conflict and Complex Speciation: Lessons From the Drosophila simulans Clade Species.

The three fruitfly species of the Drosophila simulans clade- D. simulans, D. mauritiana, and D. sechellia- have served as important models in speciation genetics for over 40 years. These species are reproductively isolated by geography, ecology, sexual signals, postmating-prezygotic interactions, and postzygotic genetic incompatibilities. All pairwise crosses between these species conform to Haldane's rule, producing fertile F1 hybrid females and sterile F1 hybrid males. The close phylogenetic proximity of the D. simulans clade species to the model organism, D. melanogaster, has empowered genetic analyses of their species differences, including reproductive incompatibilities. But perhaps no phenotype has been subject to more continuous and intensive genetic scrutiny than hybrid male sterility. Here we review the history, progress, and current state of our understanding of hybrid male sterility among the D. simulans clade species. Our aim is to integrate the available information from experimental and population genetics analyses bearing on the causes and consequences of hybrid male sterility. We highlight numerous conclusions that have emerged as well as issues that remain unresolved. We focus on the special role of sex chromosomes, the fine-scale genetic architecture of hybrid male sterility, and the history of gene flow between species. The biggest surprises to emerge from this work are that (i) genetic conflicts may be an important general force in the evolution of hybrid incompatibility, (ii) hybrid male sterility is polygenic with contributions of complex epistasis, and (iii) speciation, even among these geographically allopatric taxa, has involved the interplay of gene flow, negative selection, and positive selection. These three conclusions are marked departures from the classical views of speciation that emerged from the modern evolutionary synthesis.

Reduced Introgression of Sex Chromosome Markers in the Mexican Howler Monkey (Alouatta palliata × A. pigra) Hybrid Zone.

Interspecific hybridization allows the introgression or movement of alleles from one genome to another. While some genomic regions freely exchange alleles during hybridization, loci associated with reproductive isolation do not intermix. In many model organisms, the X chromosome displays limited introgression compared to autosomes owing to the presence of multiple loci associated with hybrid sterility or inviability (the "large X-effect"). Similarly, if hybrids are produced, the heterogametic sex is usually inviable or sterile, a pattern known as Haldane's rule. We analyzed the patterns of introgression of genetic markers located in the mitochondrial (control region) and nuclear (autosomal microsatellites and sex chromosome genes) genomes of two howler monkey species (Alouatta palliata and A. pigra) that form a natural hybrid zone in southern Mexico, to evaluate whether the large X-effect and Haldane's rule affect the outcomes of hybridization between these sister species. To identify the level of admixture of each individual in the hybrid zone (N = 254) we analyzed individuals sampled outside the hybrid zone (109 A. pigra and 39 A. palliata) to determine allele frequencies of parental species and estimated a hybrid index based on nuclear markers. We then performed a cline analysis using individuals in the hybrid zone to determine patterns of introgression for each locus. Our analyses show that although the hybrid zone is bimodal (with no known F1 s and few recent generation hybrids) and quite narrow, there has been extensive introgression in both directions, and there is a large array of admixed individuals in the hybrid zone. Mitochondrial and most autosomal markers showed bidirectional introgression, but some had biased introgression toward one species or the other. All markers on the sex chromosomes and a few autosomal markers showed highly restricted introgression. This pattern is consistent with the hypothesis that the sex chromosomes make a disproportionate contribution to reproductive isolation, and our results broaden the taxonomic representation of these patterns across animal taxa.

Introgression across Hybrid Zones Is Not Mediated by Large X-Effects in Green Toads with Undifferentiated Sex Chromosomes.

Divergence between incipient species remains an incompletely understood process. Hybrid zones provide great research potential, reflecting natural organismal genomic interactions and gene evolution in a variety of recombinants over generations. While sex chromosomes are known evolutionary drivers of reproductive isolation, empirical population genetics has mostly examined species with heteromorphic sex chromosomes. We recently reported restricted introgression at sex-linked markers in an amphibian system with homomorphic sex chromosomes (Hyla), consistent with a large X-effect, designating a greater role of sex chromosomes in driving hybrid incompatibilities. Here, using a similar approach, we examined two hybrid zones of Palearctic green toads (Bufo viridis subgroup), involving several lineages that arose at different times and form secondary contacts. We find no evidence for differential introgression of sex-linked versus autosomal markers across both zones. This absence of large X-effects in Bufo indicates that, unlike in Hyla, hybrid incompatibilities may not result from the faster-heterogametic sex and faster-male aspects of Haldane's rule. The recent suppression of XY recombination in Hyla but not in Bufo may have driven greater divergence between Hyla sex chromosomes, causing stronger reproductive isolation. Alternatively, stronger linkage among Hyla's sex-linked markers could restrict introgression. We hypothesize that the degree of sex-specific recombination may condition the importance of homomorphic sex chromosomes in speciation.

Evaluating genomic signatures of "the large X-effect" during complex speciation.

The ubiquity of the "two rules of speciation"-Haldane's rule and the large X-effect-implies a general, special role for sex chromosomes in the evolution of intrinsic postzygotic reproductive isolation. The recent proliferation of genome-scale analyses has revealed two further general observations: (a) complex speciation involving some form of gene flow is not uncommon, and (b) sex chromosomes in male- and in female-heterogametic taxa tend to show elevated differentiation relative to autosomes. Together, these observations are consistent with speciation histories in which population genetic differentiation at autosomal loci is reduced by gene flow while natural selection against hybrid incompatibilities renders sex chromosomes relatively refractory to gene flow. Here, I summarize multilocus population genetic and population genomic evidence for greater differentiation on the X (or Z) vs. the autosomes and consider the possible causes. I review common population genetic circumstances involving no selection and/or no interspecific gene flow that are nevertheless expected to elevate differentiation on sex chromosomes relative to autosomes. I then review theory for why large X-effects exist for hybrid incompatibilities and, more generally, for loci mediating local adaptation. The observed levels of sex chromosome vs. autosomal differentiation, in many cases, appear consistent with simple explanations requiring neither large X-effects nor gene flow. Discerning signatures of large X-effects during complex speciation will therefore require analyses that go beyond chromosome-scale summaries of population genetic differentiation, explicitly test for differential introgression, and/or integrate experimental genetic data.

The two "rules of speciation" in species with young sex chromosomes.

The two "rules of speciation," Haldane's rule (HR) and the large-X effect (LXE), are thought to be caused by recessive species incompatibilities exposed in the phenotype due to the hemizygosity of X-linked genes in the heterogametic sex. Thus, the reports of HR and the LXE in species with recently evolved non- or partially degenerate Y-chromosomes, such as Silene latifolia and its relatives, were surprising. Here, I argue that rapid species-specific degeneration of Y-linked genes and associated adjustment of expression of X-linked gametologs (dosage compensation) may lead to rapid evolution of sex-linked species incompatibilities. This process is likely to be too slow in species with old degenerate Y-chromosomes (e.g., in mammals), but Y-degeneration in species with young gene-rich sex chromosomes may be fast enough to play a significant role in speciation. To illustrate this point, I report the analysis of Y-degeneration and the associated evolution of gene expression on the X-chromosome of S. latifolia and Silene dioica, a close relative that shares the same recently evolved sex chromosomes. Despite the recent (≤1MY) divergence of the two species, ~7% of Y-linked genes have undergone degeneration in one but not the other species. This species-specific degeneration appears to drive faster expression divergence of X-linked genes, which may account for HR and the LXE reported for these species. Furthermore, I suggest that "exposure" of autosomal or sex-linked recessive species incompatibilities in the haploid plant gametophyte may mimic the presence of HR in plants. Both haploid expression and species-specific Y-degeneration need to receive more attention if we are to understand the role of these processes in speciation.

Patterns of Z chromosome divergence among Heliconius species highlight the importance of historical demography.

Sex chromosomes are disproportionately involved in reproductive isolation and adaptation. In support of such a "large-X" effect, genome scans between recently diverged populations and species pairs often identify distinct patterns of divergence on the sex chromosome compared to autosomes. When measures of divergence between populations are higher on the sex chromosome compared to autosomes, such patterns could be interpreted as evidence for faster divergence on the sex chromosome, that is "faster-X", barriers to gene flow on the sex chromosome. However, demographic changes can strongly skew divergence estimates and are not always taken into consideration. We used 224 whole-genome sequences representing 36 populations from two Heliconius butterfly clades (H. erato and H. melpomene) to explore patterns of Z chromosome divergence. We show that increased divergence compared to equilibrium expectations can in many cases be explained by demographic change. Among Heliconius erato populations, for instance, population size increase in the ancestral population can explain increased absolute divergence measures on the Z chromosome compared to the autosomes, as a result of increased ancestral Z chromosome genetic diversity. Nonetheless, we do identify increased divergence on the Z chromosome relative to the autosomes in parapatric or sympatric species comparisons that imply postzygotic reproductive barriers. Using simulations, we show that this is consistent with reduced gene flow on the Z chromosome, perhaps due to greater accumulation of incompatibilities. Our work demonstrates the importance of taking demography into account to interpret patterns of divergence on the Z chromosome, but nonetheless provides evidence to support the Z chromosome as a strong barrier to gene flow in incipient Heliconius butterfly species.

Selfish X chromosomes and speciation.

In two papers published at about the same time almost thirty years ago, Frank (Evolution, 45, 1991a, 262) and Hurst and Pomiankowski (Genetics, 128, 1991, 841) independently suggested that divergence of meiotic drive systems-comprising genes that cheat meiosis and genes that suppress this cheating-might provide a general explanation for Haldane's rule and the large X-effect in interspecific hybrids. Although at the time, the idea was met with skepticism and a conspicuous absence of empirical support, the tide has since turned. Some of the clearest mechanistic explanations we have for hybrid male sterility involve meiotic drive systems, and several other cases of hybrid sterility are suggestive of a role for meiotic drive. In this article, I review these ideas and their descendants and catalog the current evidence for the meiotic drive model of speciation. In addition, I suggest that meiotic drive is not the only intragenomic conflict to involve the X chromosome and contribute to hybrid incompatibility. Sexually and parentally antagonistic selection pressures can also pit the X chromosome and autosomes against each other. The resulting intragenomic conflicts should lead to co-evolution within populations and divergence between them, thus increasing the likelihood of incompatibilities in hybrids. I provide a sketch of these ideas and interpret some empirical patterns in the light of these additional X-autosome conflicts.

Bounds to parapatric speciation: A Dobzhansky-Muller incompatibility model involving autosomes, X chromosomes, and mitochondria.

We investigate the conditions for the origin and maintenance of postzygotic isolation barriers, so called (Bateson-)Dobzhansky-Muller incompatibilities or DMIs, among populations that are connected by gene flow. Specifically, we compare the relative stability of pairwise DMIs among autosomes, X chromosomes, and mitochondrial genes. In an analytical approach based on a continent-island framework, we determine how the maximum permissible migration rates depend on the genomic architecture of the DMI, on sex bias in migration rates, and on sex-dependence of allelic and epistatic effects, such as dosage compensation. Our results show that X-linkage of DMIs can enlarge the migration bounds relative to autosomal DMIs or autosome-mitochondrial DMIs, in particular in the presence of dosage compensation. The effect is further strengthened with male-biased migration. This mechanism might contribute to a higher density of DMIs on the X chromosome (large X-effect) that has been observed in several species clades. Furthermore, our results agree with empirical findings of higher introgression rates of autosomal compared to X-linked loci.

The large X-effect on secondary sexual characters and the genetics of variation in sex comb tooth number in Drosophila subobscura.

Genetic studies of secondary sexual traits provide insights into whether and how selection drove their divergence among populations, and these studies often focus on the fraction of variation attributable to genes on the X-chromosome. However, such studies may sometimes misinterpret the amount of variation attributable to the X-chromosome if using only simple reciprocal F1 crosses, or they may presume sexual selection has affected the observed phenotypic variation. We examined the genetics of a secondary sexual trait, male sex comb size, in Drosophila subobscura. This species bears unusually large sex combs for its species group, and therefore, this trait may be a good candidate for having been affected by natural or sexual selection. We observed significant heritable variation in number of teeth of the distal sex comb across strains. While reciprocal F1 crosses seemed to implicate a disproportionate X-chromosome effect, further examination in the F2 progeny showed that transgressive autosomal effects inflated the estimate of variation associated with the X-chromosome in the F1. Instead, the X-chromosome appears to confer the smallest contribution of all major chromosomes to the observed phenotypic variation. Further, we failed to detect effects on copulation latency or duration associated with the observed phenotypic variation. Overall, this study presents an examination of the genetics underlying segregating phenotypic variation within species and illustrates two common pitfalls associated with some past studies of the genetic basis of secondary sexual traits.

The search for causal traits of speciation: Divergent female mate preferences target male courtship song, not pheromones, in Drosophila athabasca species complex.

Understanding speciation requires the identification of traits that cause reproductive isolation. This remains a major challenge since it is difficult to determine which of the many divergent traits actually caused speciation. To overcome this difficulty, we studied the sexual cue traits and behaviors associated with rapid speciation between EA and WN sympatric behavioral races of Drosophila athabasca that diverged only 16,000-20,000 years ago. First, we found that sexual isolation was essentially complete and driven primarily by divergent female mating preferences. To determine the target of female mate choice, we found that, unlike cuticular hydrocarbons (CHCs), male courtship song is highly divergent between EA and WN in both allopatry and sympatry and is not affected by latitudinal variation. We then used pheromone rub-off experiments to show no effect of CHCs on divergent female mate choice. In contrast, both male song differences and male mating success in hybrids exhibited a large X-effect and playback song experiments confirmed that male courtship song is indeed the target of sexual isolation. These results show that a single secondary sexual trait is a major driver of speciation and suggest that we may be overestimating the number of traits involved in speciation when we study older taxa.

The large-X effect in plants: increased species divergence and reduced gene flow on the Silene X-chromosome.

The disproportionately large involvement of the X-chromosome in the isolation of closely related species (the large-X effect) has been reported for many animals, where X-linked genes are mostly hemizygous in the heterogametic sex. The expression of deleterious recessive mutations is thought to drive the frequent involvement of the X-chromosome in hybrid sterility, as well as to reduce interspecific gene flow for X-linked genes. Here, we evaluate the role of the X-chromosome in the speciation of two closely related plant species - the white and red campions (Silene latifolia and S. dioica) - that hybridize widely across Europe. The two species evolved separate sexes and sex chromosomes relatively recently (~10(7)  years), and unlike most animal species, most X-linked genes have intact Y-linked homologs. We demonstrate that the X-linked genes show a very small and insignificant amount of interspecific gene flow, while gene flow involving autosomal loci is significant and sufficient to homogenize the gene pools of the two species. These findings are consistent with the hypothesis of the large-X effect in Silene and comprise the first report of this effect in plants. Nonhemizygosity of many X-linked genes in Silene males indicates that exposure of recessive mutations to selection may not be essential for the occurrence of the large-X effect. Several possible causes of the large-X effect in Silene are discussed.

Biased introgression of mitochondrial and nuclear genes: a comparison of diploid and haplodiploid systems.

Hybridization between recently diverged species, even if infrequent, can lead to the introgression of genes from one species into another. The rates of mitochondrial and nuclear introgression often differ, with some taxa showing biases for mitochondrial introgression and others for nuclear introgression. Several hypotheses exist to explain such biases, including adaptive introgression, sex differences in dispersal rates, sex-specific prezygotic isolation and sex-specific fitness of hybrids (e.g. Haldane's rule). We derive a simple population genetic model that permits an analysis of sex-specific demographic and fitness parameters and measures the relative rates of mitochondrial and nuclear introgression between hybridizing pairs. We do this separately for diploid and haplodiploid species. For diploid taxa, we recover results consistent with previous hypotheses: an excess of one sex among the hybridizing migrants or sex-specific prezygotic isolation causes a bias for one type of marker or the other; when Haldane's rule is obeyed, we find a mitochondrial bias in XY systems and a nuclear bias in ZW systems. For haplodiploid taxa, the model reveals that owing to their unique transmission genetics, they are seemingly assured of strong mitochondrial biases in introgression rates, unlike diploid taxa, where the relative fitness of male and female hybrids can tip the bias in either direction. This heretofore overlooked aspect of hybridization in haplodiploids provides what is perhaps the most likely explanation for differential introgression of mitochondrial and nuclear markers and raises concerns about the use of mitochondrial DNA barcodes for species delimitation in these taxa.

Mechanisms Underlying Mammalian Hybrid Sterility in Two Feline Interspecies Models.

The phenomenon of male sterility in interspecies hybrids has been observed for over a century, however, few genes influencing this recurrent phenotype have been identified. Genetic investigations have been primarily limited to a small number of model organisms, thus limiting our understanding of the underlying molecular basis of this well-documented "rule of speciation." We utilized two interspecies hybrid cat breeds in a genome-wide association study employing the Illumina 63 K single-nucleotide polymorphism array. Collectively, we identified eight autosomal genes/gene regions underlying associations with hybrid male sterility (HMS) involved in the function of the blood-testis barrier, gamete structural development, and transcriptional regulation. We also identified several candidate hybrid sterility regions on the X chromosome, with most residing in close proximity to complex duplicated regions. Differential gene expression analyses revealed significant chromosome-wide upregulation of X chromosome transcripts in testes of sterile hybrids, which were enriched for genes involved in chromatin regulation of gene expression. Our expression results parallel those reported in Mus hybrids, supporting the "Large X-Effect" in mammalian HMS and the potential epigenetic basis for this phenomenon. These results support the value of the interspecies feline model as a powerful tool for comparison to rodent models of HMS, demonstrating unique aspects and potential commonalities that underpin mammalian reproductive isolation.