Hybridization and gene expression: Beyond differentially expressed genes.

Gene expression has a key role in reproductive isolation, and studies of hybrid gene expression have identified mechanisms causing hybrid sterility. Here, we review the evidence for altered gene expression following hybridization and outline the mechanisms shown to contribute to altered gene expression in hybrids. Transgressive gene expression, transcending that of both parental species, is pervasive in early generation sterile hybrids, but also frequently observed in viable, fertile hybrids. We highlight studies showing that hybridization can result in transgressive gene expression, also in established hybrid lineages or species. Such extreme patterns of gene expression in stabilized hybrid taxa suggest that altered hybrid gene expression may result in hybridization-derived evolutionary novelty. We also conclude that while patterns of misexpression in hybrids are well documented, the understanding of the mechanisms causing misexpression is lagging. We argue that jointly assessing differences in cell composition and cell-specific changes in gene expression in hybrids, in addition to assessing changes in chromatin and methylation, will significantly advance our understanding of the basis of altered gene expression. Moreover, uncovering to what extent evolution of gene expression results in altered expression for individual genes, or entire networks of genes, will advance our understanding of how selection moulds gene expression. Finally, we argue that jointly studying the dual roles of altered hybrid gene expression, serving both as a mechanism for reproductive isolation and as a substrate for hybrid ecological adaptation, will lead to significant advances in our understanding of the evolution of gene expression.

Molecular Evolution across Mouse Spermatogenesis.

Genes involved in spermatogenesis tend to evolve rapidly, but we lack a clear understanding of how protein sequences and patterns of gene expression evolve across this complex developmental process. We used fluorescence-activated cell sorting (FACS) to generate expression data for early (meiotic) and late (postmeiotic) cell types across 13 inbred strains of mice (Mus) spanning ∼7 My of evolution. We used these comparative developmental data to investigate the evolution of lineage-specific expression, protein-coding sequences, and expression levels. We found increased lineage specificity and more rapid protein-coding and expression divergence during late spermatogenesis, suggesting that signatures of rapid testis molecular evolution are punctuated across sperm development. Despite strong overall developmental parallels in these components of molecular evolution, protein and expression divergences were only weakly correlated across genes. We detected more rapid protein evolution on the X chromosome relative to the autosomes, whereas X-linked gene expression tended to be relatively more conserved likely reflecting chromosome-specific regulatory constraints. Using allele-specific FACS expression data from crosses between four strains, we found that the relative contributions of different regulatory mechanisms also differed between cell types. Genes showing cis-regulatory changes were more common late in spermatogenesis, and tended to be associated with larger differences in expression levels and greater expression divergence between species. In contrast, genes with trans-acting changes were more common early and tended to be more conserved across species. Our findings advance understanding of gene evolution across spermatogenesis and underscore the fundamental importance of developmental context in molecular evolutionary studies.

Genetic divergence among threespine stickleback that differ in nuptial coloration.

Sexual signals are shaped by their intended and unintended receivers as well as the signalling environment. This interplay between sexual and natural selection can lead to divergence in signals in heterogeneous environments. Yet, the extent to which gene flow is restricted when signalling phenotypes vary across environments and over what spatial scales remains an outstanding question. In this study, we quantify gene flow between two colour morphs, red and black, of freshwater threespine stickleback fish (Gasterosteus aculeatus). We capitalize on the very recent divergence of signalling phenotypes in this system to characterize within-species and among-morph genetic variation and to test for levels of gene flow between colour morphs in Oregon and Washington. Despite limited evidence for assortative mating between allopatric red and black populations, we found that black populations are genetically distinct from nearby red populations and that the black morph appears to have evolved independently at least twice in Oregon and Washington. Surprisingly, we uncovered a group of stickleback in one small coastal stream, Connor Creek, which is genetically and morphologically distinct from the red and black colour morphs and from marine stickleback. Historically, both colour morphs have coexisted in this location and sometimes hybridized, raising new questions about the origins and history of these fish, which were first described as anadromous-black hybrids >50 years ago. Understanding how genetic variation is currently partitioned within and among populations and colour morphs in this system should prompt future studies to assess the relative roles of habitat, ecological and pre- and post-reproductive barriers in the genetic divergence and phenotypic patterns we observe in nature.

Spermatogenesis and the Evolution of Mammalian Sex Chromosomes.

Developmental constraint and sexual conflict shape the evolution of heteromorphic sex chromosomes. These contrasting forces are perhaps strongest during spermatogenesis in species with XY males. In this review, we consider how the unique regulatory environment and selective pressures of spermatogenesis interact to impact sex chromosome evolution in mammals. We explore how each developmental phase of spermatogenesis influences sex chromosome gene content, structure, and rate of molecular evolution, and how these attributes may contribute to speciation. We argue that a developmental context is fundamental to understanding sex chromosome evolution and that an evolutionary perspective can shed new light on our understanding of sperm development.

The effects of heterospecific mating frequency on the strength of cryptic reproductive barriers.

Heterospecific mating frequency is critical to hybrid zone dynamics and can directly impact the strength of reproductive barriers and patterns of introgression. The effectiveness of post-mating prezygotic (PMPZ) reproductive barriers, which include reduced fecundity via heterospecific matings and conspecific sperm precedence, may depend on the number, identity and order of mates. Studies of PMPZ barriers suggest that they may be important in many systems, but whether these barriers are effective at realistic heterospecific mating frequencies has not been tested. Here, we evaluate the strength of cryptic reproductive isolation in two leaf beetles (Chrysochus auratus and C. cobaltinus) in the context of a range of heterospecific mating frequencies observed in natural populations. We found both species benefited from multiple matings, but the benefits were greater in C. cobaltinus and extended to heterospecific matings. We found that PMPZ barriers greatly limited hybrid production by C. auratus females with moderate heterospecific mating frequencies, but that their effectiveness diminished at higher heterospecific mating frequencies. In contrast, there was no evidence for PMPZ barriers in C. cobaltinus females at any heterospecific mating frequency. We show that integrating realistic estimates of cryptic isolation with information on relative abundance and heterospecific mating frequency in the field substantially improves our understanding of the strong directional bias in F1 production previously documented in the Chrysochus hybrid zone. Our results demonstrate that heterospecific mating frequency is critical to understanding the impact of cryptic post-copulatory barriers on hybrid zone structure and dynamics, and that future studies of such barriers should incorporate field-relevant heterospecific mating frequencies.

The Composite Regulatory Basis of the Large X-Effect in Mouse Speciation.

The disruption of meiotic sex chromosome inactivation (MSCI) has been proposed to be a major developmental mechanism underlying the rapid evolution of hybrid male sterility. We tested this idea by analyzing cell-specific gene expression across spermatogenesis in two lineages of house mice and their sterile and fertile reciprocal hybrids. We found pervasive disruption of sex chromosome gene expression in sterile hybrids at every stage of spermatogenesis. Failure of MSCI was developmentally preceded by increased silencing of autosomal genes, supporting the hypothesis that divergence at the hybrid incompatibility gene, Prdm9, results in increased rates of autosomal asynapsis which in turn triggers widespread silencing of unsynapsed chromatin. We also detected opposite patterns of postmeiotic overexpression or hyper-repression of the sex chromosomes in reciprocal hybrids, supporting the hypothesis that genomic conflict has driven functional divergence that leads to deleterious X-Y dosage imbalances in hybrids. Our developmental timeline also exposed more subtle patterns of mitotic misregulation on the X chromosome, a previously undocumented stage of spermatogenic disruption in this cross. These results indicate that multiple hybrid incompatibilities have converged on a common regulatory phenotype, the disrupted expression of the sex chromosomes during spermatogenesis. Collectively, these data reveal a composite regulatory basis to hybrid male sterility in mice that helps resolve the mechanistic underpinnings of the well-documented large X-effect in mice speciation. We propose that the inherent sensitivity of spermatogenesis to X-linked regulatory disruption has the potential to be a major driver of reproductive isolation in species with chromosomal sex determination.

Heterogeneous genome divergence, differential introgression, and the origin and structure of hybrid zones.

Hybrid zones have been promoted as windows on the evolutionary process and as laboratories for studying divergence and speciation. Patterns of divergence between hybridizing species can now be characterized on a genomewide scale, and recent genome scans have focused on the presence of 'islands' of divergence. Patterns of heterogeneous genomic divergence may reflect differential introgression following secondary contact and provide insights into which genome regions contribute to local adaptation, hybrid unfitness and positive assortative mating. However, heterogeneous genome divergence can also arise in the absence of any gene flow, as a result of variation in selection and recombination across the genome. We suggest that to understand hybrid zone origins and dynamics, it is essential to distinguish between genome regions that are divergent between pure parental populations and regions that show restricted introgression where these populations interact in hybrid zones. The latter, more so than the former, reveal the likely genetic architecture of reproductive isolation. Mosaic hybrid zones, because of their complex structure and multiple contacts, are particularly good subjects for distinguishing primary intergradation from secondary contact. Comparisons among independent hybrid zones or transects that involve the 'same' species pair can also help to distinguish between divergence with gene flow and secondary contact. However, data from replicate hybrid zones or replicate transects do not reveal consistent patterns; in a few cases, patterns of introgression are similar across independent transects, but for many taxa, there is distinct lack of concordance, presumably due to variation in environmental context and/or variation in the genetics of the interacting populations.

Barriers to gene exchange in hybridizing field crickets: the role of male courtship effort and cuticular hydrocarbons.

BACKGROUND: Pre-zygotic barriers often involve some form of sexual selection, usually interpreted as female choice, as females are typically the choosier sex. However, males typically show some mate preferences, which are increasingly reported. Here we document previously uncharacterized male courtship behavior (effort and song) and cuticular hydrocarbon (CHC) profiles in the hybridizing crickets Gryllus firmus and G. pennsylvanicus. These two species exhibit multiple barriers to gene exchange that act throughout their life history, including a behavioral barrier that results in increased time to mate in heterospecific pairs. RESULTS: We demonstrated that male mate choice (as courtship effort allocation) plays a more important role in the prezygotic behavioral barrier than previously recognized. In gryllids females ultimately decide whether or not to mate, yet we found males were selective by regulating courtship effort intensity toward the preferred (conspecific) females. Females were also selective by mating with more intensely courting males, which happened to be conspecifics. We report no differences in courtship song between the two species and suggest that the mechanism that allows males to act differentially towards conspecific and heterospecific females is the cuticular hydrocarbon (CHC) composition. CHC profiles differed between males and females of both species, and there were clear differences in CHC composition between female G. firmus and G. pennsylvanicus but not between the males of each species. CONCLUSION: Although many barriers to gene exchange are known in this system, the mechanism behind the mate recognition leading to reduced heterospecific mating remains unknown. The CHC profiles might be the phenotypic cue that allow males to identify conspecifics and thus to adjust their courtship intensity accordingly, leading to differential mating between species.

Gene flow and the maintenance of species boundaries.

Hybrid zones are regions where individuals from genetically differentiated populations meet and mate, resulting in at least some offspring of mixed ancestry. Patterns of gene flow (introgression) in hybrid zones vary across the genome, allowing assessment of the role of individual genes or genome regions in reproductive isolation. Here, we document patterns of introgression between two recently diverged species of field crickets. We sampled at a very fine spatial scale and genotyped crickets for 110 highly differentiated single nucleotide polymorphisms (SNPs) identified through transcriptome scans. Using both genomic and geographic cline analysis, we document remarkably abrupt transitions (<100 m) in allele frequencies for 50 loci, despite high levels of gene flow at other loci. These are among the steepest clines documented for any hybridizing taxa. Furthermore, the cricket hybrid zone provides one of the clearest examples of the semi-permeability of species boundaries. Comparisons between data from the fine-scale transect and data (for the same set of markers) from sampling a much larger area in a different region of the cricket hybrid zone reveal consistent patterns of introgression for individual loci. The consistency in patterns of introgression between these two distant and distinct regions of the hybrid zone suggests that strong selection is acting to maintain abrupt discontinuities within the hybrid zone and that genomic regions with restricted introgression likely include genes that contribute to nonecological prezygotic barriers.

Hybridization, introgression, and the nature of species boundaries.

Species can be defined as populations that are diagnosably distinct, reproductively isolated, cohesive, or exclusive groups of organisms. Boundaries between species in sympatry are maintained by intrinsic barriers to gene exchange; these boundaries may not be uniform in space, in time, or across the genome. Here, we explore the nature of the species boundary, defined as the phenotypes/genes/genome regions that remain differentiated in the face of potential hybridization and introgression. We emphasize that species boundaries are semipermeable, with permeability (gene exchange) being a function of genome region. The early evidence for semipermeable species boundaries came from data on differential introgression in hybrid zones. This "genic view" of species was common in the hybrid zone literature even when few molecular markers were available to characterize genome-wide patterns of variation. Now, molecular tools allow detailed characterization of differentiation between diverging lineages and patterns of variation across natural hybrid zones, but the questions being asked by evolutionary biologists have remained much the same. Recent data (from DNA sequences and genotypes) reinforce earlier conclusions about the semipermeable nature of most species boundaries. However, debate persists over the nature and extent of genome divergence that accompanies speciation.

Quantitative Analyses of Coupling in Hybrid Zones.

In hybrid zones, whether barrier loci experience selection mostly independently or as a unit depends on the ratio of selection to recombination as captured by the coupling coefficient. Theory predicts a sharper transition between an uncoupled and coupled system when more loci affect hybrid fitness. However, the extent of coupling in hybrid zones has rarely been quantified. Here, we use simulations to characterize the relationship between the coupling coefficient and variance in clines across genetic loci. We then reanalyze 25 hybrid zone data sets and find that cline variances and estimated coupling coefficients form a smooth continuum from high variance and weak coupling to low variance and strong coupling. Our results are consistent with low rates of hybridization and a strong genome-wide barrier to gene flow when the coupling coefficient is much greater than 1, but also suggest that this boundary might be approached gradually and at a near constant rate over time.

Different complex regulatory phenotypes underlie hybrid male sterility in divergent rodent crosses.

Hybrid incompatibilities are a critical component of species barriers and may arise due to negative interactions between divergent regulatory elements in parental species. We used a comparative approach to identify common themes in the regulatory phenotypes associated with hybrid male sterility in two divergent rodent crosses, dwarf hamsters and house mice. We investigated three potential characteristic regulatory phenotypes in hybrids including the propensity towards over or underexpression relative to parental species, the influence of developmental stage on the extent of misexpression, and the role of the sex chromosomes on misexpression phenotypes. In contrast to near pervasive overexpression in hybrid house mice, we found that misexpression in hybrid dwarf hamsters was dependent on developmental stage. In both house mouse and dwarf hamster hybrids, however, misexpression increased with the progression of spermatogenesis, although to varying extents and with potentially different consequences. In both systems, we detected sex-chromosome specific overexpression in stages of spermatogenesis where inactivated X chromosome expression was expected, but the hybrid overexpression phenotypes were fundamentally different. Importantly, misexpression phenotypes support the presence of multiple histological blocks to spermatogenesis in dwarf hamster hybrids, including a potential role of meiotic stalling early in spermatogenesis. Collectively, we demonstrate that while there are some similarities in hybrid regulatory phenotypes of house mice and dwarf hamsters, there are also clear differences that point towards unique mechanisms underlying hybrid male sterility in each system. Our results highlight the potential of comparative approaches in helping to understand the importance of disrupted gene regulation in speciation.

Synthesis and Scope of the Role of Postmating Prezygotic Isolation in Speciation.

How barriers to gene flow arise and are maintained are key questions in evolutionary biology. Speciation research has mainly focused on barriers that occur either before mating or after zygote formation. In comparison, postmating prezygotic (PMPZ) isolation-a barrier that acts after gamete release but before zygote formation-is less frequently investigated but may hold a unique role in generating biodiversity. Here we discuss the distinctive features of PMPZ isolation, including the primary drivers and molecular mechanisms underpinning PMPZ isolation. We then present the first comprehensive survey of PMPZ isolation research, revealing that it is a widespread form of prezygotic isolation across eukaryotes. The survey also exposes obstacles in studying PMPZ isolation, in part attributable to the challenges involved in directly measuring PMPZ isolation and uncovering its causal mechanisms. Finally, we identify outstanding knowledge gaps and provide recommendations for improving future research on PMPZ isolation. This will allow us to better understand the nature of this often-neglected reproductive barrier and its contribution to speciation.

Post-mating prezygotic barriers to gene exchange between hybridizing field crickets.

Studies of sexual selection in speciation have traditionally focused on mate preference, with less attention given to traits that act between copulation and fertilization. However, recent work suggests that post-mating prezygotic barriers may play an important role in speciation. Here, we evaluate the role of such barriers in the field crickets, Gryllus firmus and Gryllus pennsylvanicus. Gryllus pennsylvanicus females mated with G. firmus males produce viable, fertile offspring, but when housed with both species produce offspring sired primarily by conspecifics. We evaluate patterns of sperm utilization in doubly mated G. pennsylvanicus females and find no evidence for conspecific sperm precedence. The reciprocal cross (G. firmus female × G. pennsylvanicus male) produces no progeny. Absence of progeny reflects a barrier to fertilization rather than reduced sperm transfer, storage or motility. We propose a classification scheme for mechanisms underlying post-mating prezygotic barriers similar to that used for premating barriers.

Unraveling patterns of disrupted gene expression across a complex tissue.

Whole tissue RNASeq is the standard approach for studying gene expression divergence in evolutionary biology and provides a snapshot of the comprehensive transcriptome for a given tissue. However, whole tissues consist of diverse cell types differing in expression profiles, and the cellular composition of these tissues can evolve across species. Here, we investigate the effects of different cellular composition on whole tissue expression profiles. We compared gene expression from whole testes and enriched spermatogenesis populations in two species of house mice, Mus musculus musculus and M. m. domesticus, and their sterile and fertile F1 hybrids, which differ in both cellular composition and regulatory dynamics. We found that cellular composition differences skewed expression profiles and differential gene expression in whole testes samples. Importantly, both approaches were able to detect large-scale patterns such as disrupted X chromosome expression, although whole testes sampling resulted in decreased power to detect differentially expressed genes. We encourage researchers to account for histology in RNASeq and consider methods that reduce sample complexity whenever feasible. Ultimately, we show that differences in cellular composition between tissues can modify expression profiles, potentially altering inferred gene ontological processes, insights into gene network evolution, and processes governing gene expression evolution.

Stage-specific disruption of X chromosome expression during spermatogenesis in sterile house mouse hybrids.

Hybrid sterility is a complex phenotype that can result from the breakdown of spermatogenesis at multiple developmental stages. Here, we disentangle two proposed hybrid male sterility mechanisms in the house mice, Mus musculus domesticus and M. m. musculus, by comparing patterns of gene expression in sterile F1 hybrids from a reciprocal cross. We found that hybrid males from both cross directions showed disrupted X chromosome expression during prophase of meiosis I consistent with a loss of meiotic sex chromosome inactivation (MSCI) and Prdm9-associated sterility, but that the degree of disruption was greater in mice with an M. m. musculus X chromosome consistent with previous studies. During postmeiotic development, gene expression on the X chromosome was only disrupted in one cross direction, suggesting that misexpression at this later stage was genotype-specific and not a simple downstream consequence of MSCI disruption which was observed in both reciprocal crosses. Instead, disrupted postmeiotic expression may depend on the magnitude of earlier disrupted MSCI, or the disruption of particular X-linked genes or gene networks. Alternatively, only hybrids with a potential deficit of Sly copies, a Y-linked ampliconic gene family, showed overexpression in postmeiotic cells, consistent with a previously proposed model of antagonistic coevolution between the X- and Y-linked ampliconic genes contributing to disrupted expression late in spermatogenesis. The relative contributions of these two regulatory mechanisms and their impact on sterility phenotypes await further study. Our results further support the hypothesis that X-linked hybrid sterility in house mice has a variable genetic basis, and that genotype-specific disruption of gene regulation contributes to overexpression of the X chromosome at different stages of development. Overall, these findings underscore the critical role of epigenetic regulation of the X chromosome during spermatogenesis and suggest that these processes are prone to disruption in hybrids.

The contribution of sex chromosome conflict to disrupted spermatogenesis in hybrid house mice.

Incompatibilities on the sex chromosomes are important in the evolution of hybrid male sterility, but the evolutionary forces underlying this phenomenon are unclear. House mice (Mus musculus) lineages have provided powerful models for understanding the genetic basis of hybrid male sterility. X chromosome-autosome interactions cause strong incompatibilities in M. musculus F1 hybrids, but variation in sterility phenotypes suggests a more complex genetic basis. In addition, XY chromosome conflict has resulted in rapid expansions of ampliconic genes with dosage-dependent expression that is essential to spermatogenesis. Here, we evaluated the contribution of XY lineage mismatch to male fertility and stage-specific gene expression in hybrid mice. We performed backcrosses between two house mouse subspecies to generate reciprocal Y-introgression strains and used these strains to test the effects of XY mismatch in hybrids. Our transcriptome analyses of sorted spermatid cells revealed widespread overexpression of the X chromosome in sterile F1 hybrids independent of Y chromosome subspecies origin. Thus, postmeiotic overexpression of the X chromosome in sterile F1 mouse hybrids is likely a downstream consequence of disrupted meiotic X-inactivation rather than XY gene copy number imbalance. Y chromosome introgression did result in subfertility phenotypes and disrupted expression of several autosomal genes in mice with an otherwise nonhybrid genomic background, suggesting that Y-linked incompatibilities contribute to reproductive barriers, but likely not as a direct consequence of XY conflict. Collectively, these findings suggest that rapid sex chromosome gene family evolution driven by genomic conflict has not resulted in strong male reproductive barriers between these subspecies of house mice.

The Evolution of Widespread Recombination Suppression on the Dwarf Hamster (Phodopus) X Chromosome.

The X chromosome of therian mammals shows strong conservation among distantly related species, limiting insights into the distinct selective processes that have shaped sex chromosome evolution. We constructed a chromosome-scale de novo genome assembly for the Siberian dwarf hamster (Phodopus sungorus), a species reported to show extensive recombination suppression across an entire arm of the X chromosome. Combining a physical genome assembly based on shotgun and long-range proximity ligation sequencing with a dense genetic map, we detected widespread suppression of female recombination across ∼65% of the Phodopus X chromosome. This region of suppressed recombination likely corresponds to the Xp arm, which has previously been shown to be highly heterochromatic. Using additional sequencing data from two closely related species (P. campbelli and P. roborovskii), we show that recombination suppression on Xp appears to be independent of major structural rearrangements. The suppressed Xp arm was enriched for several transposable element families and de-enriched for genes primarily expressed in placenta, but otherwise showed similar gene densities, expression patterns, and rates of molecular evolution when compared to the recombinant Xq arm. Phodopus Xp gene content and order was also broadly conserved relative to the more distantly related rat X chromosome. These data suggest that widespread suppression of recombination has likely evolved through the transient induction of facultative heterochromatin on the Phodopus Xp arm without major changes in chromosome structure or genetic content. Thus, substantial changes in the recombination landscape have so far had relatively subtle influences on patterns of X-linked molecular evolution in these species.

Cryptic gametic interactions confer both conspecific and heterospecific advantages in the Chrysochus (Coleoptera: Chrysomelidae) hybrid zone.

Most species pairs are isolated through the collective action of a suite of barriers. Recent work has shown that cryptic barriers such as conspecific sperm precedence can be quite strong, suggesting that they evolve quickly. However, because the strength of multiple barriers has been formally quantified in very few systems, the relative speed with which conspecific sperm precedence evolves remains unclear. Here, we measure the strength of both conspecific sperm precedence and cryptic non-competitive isolation between the hybridizing sister species, Chrysochus auratus and C. cobaltinus (Coleoptera: Chrysomelidae), and compare the strength of those barriers to the strength of other known reproductive barriers in this system. Overall, cryptic barriers in this system are weaker than other barriers, indicating that they have not evolved rapidly. Furthermore, their evolution has been asymmetric. Non-competitive barriers substantially reduce the production of hybrid offspring by C. auratus females but not by C. cobaltinus females. In multiply-mated C. cobaltinus females, heterospecific sperm outcompete conspecific sperm, as evidenced by the fact that heterospecific males sired disproportionately more offspring than predicted from the results for singly-mated females. In C. auratus females, neither sperm type has a competitive advantage. Such asymmetries explain why nearly all F1 hybrids in the field are from crosses between C. cobaltinus females and C. auratus males. We discuss these findings in terms of understanding the cost of mating 'mistakes' in the Chrysochus hybrid zone. In addition, our discovery that 95% confidence intervals for commonly-used isolation statistics can be very wide has important implications for speciation research. Specifically, to avoid biases in the interpretation of such isolation metrics, we suggest that studies should routinely include error estimates in their analyses of reproductive isolation.