Recurrent evolution of small body size and loss of the sword ornament in Northern swordtail fish.

Across the tree of life, species have repeatedly evolved similar phenotypes. While well-studied for ecological traits, there is also evidence for recurrent evolution of sexually selected traits. Swordtail fish (Xiphophorus) are a classic model system for studying sexual selection, and female Xiphophorus exhibit strong mate preferences for large male body size and a range of sexually dimorphic ornaments. Interestingly, sexually selected traits have also been lost multiple times in the genus. However, there has been uncertainty over the number of losses of ornamentation and large body size because phylogenetic relationships between species in this group have historically been controversial, partially due to prevalent gene flow. Here, we use whole-genome sequencing approaches to re-examine phylogenetic relationships within a Xiphophorus clade that varies in the presence and absence of sexually selected traits. Using wild-caught individuals, we determine the phylogenetic placement of a small, unornamented species, X. continens, confirming an additional loss of ornamentation and large body size in the clade. With these revised phylogenetic relationships, we analyze evidence for coevolution between body size and other sexually selected traits using phylogenetic comparative methods. These results provide insights into the evolutionary pressures driving the recurrent loss of suites of sexually selected traits.

Structural genomic variation and behavioral interactions underpin a balanced sexual mimicry polymorphism.

How phenotypic diversity originates and persists within populations are classic puzzles in evolutionary biology. While balanced polymorphisms segregate within many species, it remains rare for both the genetic basis and the selective forces to be known, leading to an incomplete understanding of many classes of traits under balancing selection. Here, we uncover the genetic architecture of a balanced sexual mimicry polymorphism and identify behavioral mechanisms that may be involved in its maintenance in the swordtail fish Xiphophorus birchmanni. We find that ∼40% of X. birchmanni males develop a "false gravid spot," a melanic pigmentation pattern that mimics the "pregnancy spot" associated with sexual maturity in female live-bearing fish. Using genome-wide association mapping, we detect a single intergenic region associated with variation in the false gravid spot phenotype, which is upstream of kitlga, a melanophore patterning gene. By performing long-read sequencing within and across populations, we identify complex structural rearrangements between alternate alleles at this locus. The false gravid spot haplotype drives increased allele-specific expression of kitlga, which provides a mechanistic explanation for the increased melanophore abundance that causes the spot. By studying social interactions in the laboratory and in nature, we find that males with the false gravid spot experience less aggression; however, they also receive increased attention from other males and are disdained by females. These behavioral interactions may contribute to the maintenance of this phenotypic polymorphism in natural populations. We speculate that structural variants affecting gene regulation may be an underappreciated driver of balanced polymorphisms across diverse species.

Swordtail fish hybrids reveal that genome evolution is surprisingly predictable after initial hybridization.

Over the past 2 decades, biologists have come to appreciate that hybridization, or genetic exchange between distinct lineages, is remarkably common-not just in particular lineages but in taxonomic groups across the tree of life. As a result, the genomes of many modern species harbor regions inherited from related species. This observation has raised fundamental questions about the degree to which the genomic outcomes of hybridization are repeatable and the degree to which natural selection drives such repeatability. However, a lack of appropriate systems to answer these questions has limited empirical progress in this area. Here, we leverage independently formed hybrid populations between the swordtail fish Xiphophorus birchmanni and X. cortezi to address this fundamental question. We find that local ancestry in one hybrid population is remarkably predictive of local ancestry in another, demographically independent hybrid population. Applying newly developed methods, we can attribute much of this repeatability to strong selection in the earliest generations after initial hybridization. We complement these analyses with time-series data that demonstrates that ancestry at regions under selection has remained stable over the past approximately 40 generations of evolution. Finally, we compare our results to the well-studied X. birchmanni × X. malinche hybrid populations and conclude that deeper evolutionary divergence has resulted in stronger selection and higher repeatability in patterns of local ancestry in hybrids between X. birchmanni and X. cortezi.

Phylogenomic analyses of all species of swordtail fishes (genus Xiphophorus) show that hybridization preceded speciation.

Hybridization has been recognized to play important roles in evolution, however studies of the genetic consequence are still lagging behind in vertebrates due to the lack of appropriate experimental systems. Fish of the genus Xiphophorus are proposed to have evolved with multiple ancient and ongoing hybridization events. They have served as an informative research model in evolutionary biology and in biomedical research on human disease for more than a century. Here, we provide the complete genomic resource including annotations for all described 26 Xiphophorus species and three undescribed taxa and resolve all uncertain phylogenetic relationships. We investigate the molecular evolution of genes related to cancers such as melanoma and for the genetic control of puberty timing, focusing on genes that are predicted to be involved in pre-and postzygotic isolation and thus affect hybridization. We discovered dramatic size-variation of some gene families. These persisted despite reticulate evolution, rapid speciation and short divergence time. Finally, we clarify the hybridization history in the entire genus settling disputed hybridization history of two Southern swordtails. Our comparative genomic analyses revealed hybridization ancestries that are manifested in the mosaic fused genomes and show that hybridization often preceded speciation.

Hybridization in the Anthropocene - how pollution and climate change disrupt mate selection in freshwater fish.

Chemical pollutants and/or climate change have the potential to break down reproductive barriers between species and facilitate hybridization. Hybrid zones may arise in response to environmental gradients and secondary contact between formerly allopatric populations, or due to the introduction of non-native species. In freshwater ecosystems, field observations indicate that changes in water quality and chemistry, due to pollution and climate change, are correlated with an increased frequency of hybridization. Physical and chemical disturbances of water quality can alter the sensory environment, thereby affecting chemical and visual communication among fish. Moreover, multiple chemical compounds (e.g. pharmaceuticals, metals, pesticides, and industrial contaminants) may impair fish physiology, potentially affecting phenotypic traits relevant for mate selection (e.g. pheromone production, courtship, and coloration). Although warming waters have led to documented range shifts, and chemical pollution is ubiquitous in freshwater ecosystems, few studies have tested hypotheses about how these stressors may facilitate hybridization and what this means for biodiversity and species conservation. Through a systematic literature review across disciplines (i.e. ecotoxicology and evolutionary biology), we evaluate the biological interactions, toxic mechanisms, and roles of physical and chemical environmental stressors (i.e. chemical pollution and climate change) in disrupting mate preferences and inducing interspecific hybridization in freshwater fish. Our study indicates that climate change-driven changes in water quality and chemical pollution may impact visual and chemical communication crucial for mate choice and thus could facilitate hybridization among fishes in freshwater ecosystems. To inform future studies and conservation management, we emphasize the importance of further research to identify the chemical and physical stressors affecting mate choice, understand the mechanisms behind these interactions, determine the concentrations at which they occur, and assess their impact on individuals, populations, species, and biological diversity in the Anthropocene.

Pervasive gene flow despite strong and varied reproductive barriers in swordtails.

One of the mechanisms that can lead to the formation of new species occurs through the evolution of reproductive barriers. However, recent research has demonstrated that hybridization has been pervasive across the tree of life even in the presence of strong barriers. Swordtail fishes (genus Xiphophorus) are an emerging model system for studying the interface between these barriers and hybridization. We document overlapping mechanisms that act as barriers between closely related species, X. birchmanni and X. cortezi, by combining genomic sequencing from natural hybrid populations, artificial crosses, behavioral assays, sperm performance, and developmental studies. We show that strong assortative mating plays a key role in maintaining subpopulations with distinct ancestry in natural hybrid populations. Lab experiments demonstrate that artificial F1 crosses experience dysfunction: crosses with X. birchmanni females were largely inviable and crosses with X. cortezi females had a heavily skewed sex ratio. Using F2 hybrids we identify several genomic regions that strongly impact hybrid viability. Strikingly, two of these regions underlie genetic incompatibilities in hybrids between X. birchmanni and its sister species X. malinche. Our results demonstrate that ancient hybridization has played a role in the origin of this shared genetic incompatibility. Moreover, ancestry mismatch at these incompatible regions has remarkably similar consequences for phenotypes and hybrid survival in X. cortezi × X. birchmanni hybrids as in X. malinche × X. birchmanni hybrids. Our findings identify varied reproductive barriers that shape genetic exchange between naturally hybridizing species and highlight the complex evolutionary outcomes of hybridization.

Patterns of recombination in snakes reveal a tug-of-war between PRDM9 and promoter-like features.

In some mammals, notably humans, recombination occurs almost exclusively where the protein PRDM9 binds, whereas in vertebrates lacking an intact PRDM9, such as birds and canids, recombination rates are elevated near promoter-like features. To determine whether PRDM9 directs recombination in nonmammalian vertebrates, we focused on an exemplar species with a single, intact PRDM9 ortholog, the corn snake (Pantherophis guttatus). Analyzing historical recombination rates along the genome and crossovers in pedigrees, we found evidence that PRDM9 specifies the location of recombination events, but we also detected a separable effect of promoter-like features. These findings reveal that the uses of PRDM9 and promoter-like features need not be mutually exclusive and instead reflect a tug-of-war that is more even in some species than others.

A lethal mitonuclear incompatibility in complex I of natural hybrids.

The evolution of reproductive barriers is the first step in the formation of new species and can help us understand the diversification of life on Earth. These reproductive barriers often take the form of hybrid incompatibilities, in which alleles derived from two different species no longer interact properly in hybrids1-3. Theory predicts that hybrid incompatibilities may be more likely to arise at rapidly evolving genes4-6 and that incompatibilities involving multiple genes should be common7,8, but there has been sparse empirical data to evaluate these predictions. Here we describe a mitonuclear incompatibility involving three genes whose protein products are in physical contact within respiratory complex I of naturally hybridizing swordtail fish species. Individuals homozygous for mismatched protein combinations do not complete embryonic development or die as juveniles, whereas those heterozygous for the incompatibility have reduced complex I function and unbalanced representation of parental alleles in the mitochondrial proteome. We find that the effects of different genetic interactions on survival are non-additive, highlighting subtle complexity in the genetic architecture of hybrid incompatibilities. Finally, we document the evolutionary history of the genes involved, showing signals of accelerated evolution and evidence that an incompatibility has been transferred between species via hybridization.

Phylogenomics analyses of all species of Swordtails (Genus Xiphophorus ) highlights hybridization precedes speciation.

Hybridization has been recognized as an important driving force for evolution, however studies of the genetic consequence and its cause are still lagging behind in vertebrates due to the lack of appropriate experimental systems. Fish of the central American genus Xiphophorus were proposed to have evolved with multiple ancient and ongoing hybridization events, and served as a valuable research model in evolutionary biology and in biomedical research on human disease for more than a century. Here, we provide the complete genome resource and its annotation of all 26 Xiphophorus species. On this dataset we resolved the so far conflicting phylogeny. Through comparative genomic analyses we investigated the molecular evolution of genes related to melanoma, for a main sexually selected trait and for the genetic control of puberty timing, which are predicted to be involved in pre-and postzygotic isolation and thus to influence the probability of interspecific hybridization in Xiphophorus . We demonstrate dramatic size-variation of some gene families across species, despite the reticulate evolution and short divergence time. Finally, we clarify the hybridization history in the genus Xiphophorus genus, settle the long dispute on the hybridization origin of two Southern swordtails, highlight hybridizations precedes speciation, and reveal the distribution of hybridization ancestry remaining in the fused genome.

The Ecology of Hybrid Incompatibilities.

Ecologically mediated selection against hybrids, caused by hybrid phenotypes fitting poorly into available niches, is typically viewed as distinct from selection caused by epistatic Dobzhansky-Muller hybrid incompatibilities. Here, we show how selection against transgressive phenotypes in hybrids manifests as incompatibility. After outlining our logic, we summarize current approaches for studying ecology-based selection on hybrids. We then quantitatively review QTL-mapping studies and find traits differing between parent taxa are typically polygenic. Next, we describe how verbal models of selection on hybrids translate to phenotypic and genetic fitness landscapes, highlighting emerging approaches for detecting polygenic incompatibilities. Finally, in a synthesis of published data, we report that trait transgression-and thus possibly extrinsic hybrid incompatibility in hybrids-escalates with the phenotypic divergence between parents. We discuss conceptual implications and conclude that studying the ecological basis of hybrid incompatibility will facilitate new discoveries about mechanisms of speciation.

Patterns of recombination in snakes reveal a tug of war between PRDM9 and promoter-like features.

In vertebrates, there are two known mechanisms by which meiotic recombination is directed to the genome: in humans, mice, and other mammals, recombination occurs almost exclusively where the protein PRDM9 binds, while in species lacking an intact PRDM9, such as birds and canids, recombination rates are elevated near promoter-like features. To test if PRDM9 also directs recombination in non-mammalian vertebrates, we focused on an exemplar species, the corn snake (Pantherophis guttatus). Unlike birds, this species possesses a single, intact PRDM9 ortholog. By inferring historical recombination rates along the genome from patterns of linkage disequilibrium and identifying crossovers in pedigrees, we found that PRDM9 specifies the location of recombination events outside of mammals. However, we also detected an independent effect of promoter-like features on recombination, which is more pronounced on macro- than microchromosomes. Thus, our findings reveal that the uses of PRDM9 and promoter-like features are not mutually-exclusive, and instead reflect a tug of war, which varies in strength along the genome and is more lopsided in some species than others.

Individual variation in preference behavior in sailfin fish refines the neurotranscriptomic pathway for mate preference.

Social interactions can drive distinct gene expression profiles which may vary by social context. Here we use female sailfin molly fish (Poecilia latipinna) to identify genomic profiles associated with preference behavior in distinct social contexts: male interactions (mate choice) versus female interactions (shoaling partner preference). We measured the behavior of 15 females interacting in a non-contact environment with either two males or two females for 30 min followed by whole-brain transcriptomic profiling by RNA sequencing. We profiled females that exhibited high levels of social affiliation and great variation in preference behavior to identify an order of magnitude more differentially expressed genes associated with behavioral variation than by differences in social context. Using a linear model (limma), we took advantage of the individual variation in preference behavior to identify unique gene sets that exhibited distinct correlational patterns of expression with preference behavior in each social context. By combining limma and weighted gene co-expression network analyses (WGCNA) approaches we identified a refined set of 401 genes robustly associated with mate preference that is independent of shoaling partner preference or general social affiliation. While our refined gene set confirmed neural plasticity pathways involvement in moderating female preference behavior, we also identified a significant proportion of discovered that our preference-associated genes were enriched for 'immune system' gene ontology categories. We hypothesize that the association between mate preference and transcriptomic immune function is driven by the less well-known role of these genes in neural plasticity which is likely involved in higher-order learning and processing during mate choice decisions.

Genomes of two Extinct-in-the-Wild reptiles from Christmas Island reveal distinct evolutionary histories and conservation insights.

Genomics can play important roles in biodiversity conservation, especially for Extinct-in-the-Wild species where genetic factors greatly influence risk of total extinction and probability of successful reintroductions. The Christmas Island blue-tailed skink (Cryptoblepharus egeriae) and Lister's gecko (Lepidodactylus listeri) are two endemic reptile species that went extinct in the wild shortly after the introduction of a predatory snake. After a decade of management, captive populations have expanded from 66 skinks and 43 geckos to several thousand individuals; however, little is known about patterns of genetic variation in these species. Here, we use PacBio HiFi long-read and Hi-C sequencing to generate highly contiguous reference genomes for both reptiles, including the XY chromosome pair in the skink. We then analyse patterns of genetic diversity to infer ancient demography and more recent histories of inbreeding. We observe high genome-wide heterozygosity in the skink (0.007 heterozygous sites per base-pair) and gecko (0.005), consistent with large historical population sizes. However, nearly 10% of the blue-tailed skink reference genome falls within long (>1 Mb) runs of homozygosity (ROH), resulting in homozygosity at all major histocompatibility complex (MHC) loci. In contrast, we detect a single ROH in Lister's gecko. We infer from the ROH lengths that related skinks may have established the captive populations. Despite a shared recent extinction in the wild, our results suggest important differences in these species' histories and implications for management. We show how reference genomes can contribute evolutionary and conservation insights, and we provide resources for future population-level and comparative genomic studies in reptiles.

Complex hybridization between deeply diverged fish species in a disturbed ecosystem.

Over the past two decades researchers have documented the extent of natural hybridization between closely related species using genomic tools. Many species across the tree of life show evidence of past hybridization with their evolutionary relatives. In some cases, this hybridization is complex-involving gene flow between more than two species. While hybridization is common over evolutionary timescales, some researchers have proposed that it may be even more common in contemporary populations where anthropogenic disturbance has modified a myriad of aspects of the environments in which organisms live and reproduce. Here, we develop a flexible tool for local ancestry inference in hybrids derived from three source populations and describe a complex, recent hybridization event between distantly related swordtail fish lineages (Xiphophorus) and its potential links to anthropogenic disturbance.

Genome evolution is surprisingly predictable after initial hybridization.

Over the past two decades, evolutionary biologists have come to appreciate that hybridization, or genetic exchange between distinct lineages, is remarkably common - not just in particular lineages but in taxonomic groups across the tree of life. As a result, the genomes of many modern species harbor regions inherited from related species. This observation has raised fundamental questions about the degree to which the genomic outcomes of hybridization are repeatable and the degree to which natural selection drives such repeatability. However, a lack of appropriate systems to answer these questions has limited empirical progress in this area. Here, we leverage independently formed hybrid populations between the swordtail fish Xiphophorus birchmanni and X. cortezi to address this fundamental question. We find that local ancestry in one hybrid population is remarkably predictive of local ancestry in another, demographically independent hybrid population. Applying newly developed methods, we can attribute much of this repeatability to strong selection in the earliest generations after initial hybridization. We complement these analyses with time-series data that demonstrates that ancestry at regions under selection has remained stable over the past ~40 generations of evolution. Finally, we compare our results to the well-studied X. birchmanni×X. malinche hybrid populations and conclude that deeper evolutionary divergence has resulted in stronger selection and higher repeatability in patterns of local ancestry in hybrids between X. birchmanni and X. cortezi.

Introgression.

Biologists have forever sought to understand how species arise and persist. Historically, species that rarely interbreed, or are reproductively isolated, were considered the norm, while those with incomplete reproductive isolation were considered less common. Over the last few decades, advances in genomics have transformed our understanding of the frequency of gene flow between species and with it our ideas about reproductive isolation in nature. These advances have uncovered a rich and often complicated history of genetic exchange between species - demonstrating that such genetic introgression is an important evolutionary process widespread across the tree of life (Figure 1).

Genomic insights into variation in thermotolerance between hybridizing swordtail fishes.

Understanding how organisms adapt to changing environments is a core focus of research in evolutionary biology. One common mechanism is adaptive introgression, which has received increasing attention as a potential route to rapid adaptation in populations struggling in the face of ecological change, particularly global climate change. However, hybridization can also result in deleterious genetic interactions that may limit the benefits of adaptive introgression. Here, we used a combination of genome-wide quantitative trait locus mapping and differential gene expression analyses between the swordtail fish species Xiphophorus malinche and X. birchmanni to study the consequences of hybridization on thermotolerance. While these two species are adapted to different thermal environments, we document a complicated architecture of thermotolerance in hybrids. We identify a region of the genome that contributes to reduced thermotolerance in individuals heterozygous for X. malinche and X. birchmanni ancestry, as well as widespread misexpression in hybrids of genes that respond to thermal stress in the parental species, particularly in the circadian clock pathway. We also show that a previously mapped hybrid incompatibility between X. malinche and X. birchmanni contributes to reduced thermotolerance in hybrids. Together, our results highlight the challenges of understanding the impact of hybridization on complex ecological traits and its potential impact on adaptive introgression.

Imbalanced segregation of recombinant haplotypes in hybrid populations reveals inter- and intrachromosomal Dobzhansky-Muller incompatibilities.

Dobzhansky-Muller incompatibilities (DMIs) are a major component of reproductive isolation between species. DMIs imply negative epistasis and are exposed when two diverged populations hybridize. Mapping the locations of DMIs has largely relied on classical genetic mapping. Approaches to date are hampered by low power and the challenge of identifying DMI loci on the same chromosome, because strong initial linkage of parental haplotypes weakens statistical tests. Here, we propose new statistics to infer negative epistasis from haplotype frequencies in hybrid populations. When two divergent populations hybridize, the variance in heterozygosity at two loci decreases faster with time at DMI loci than at random pairs of loci. When two populations hybridize at near-even admixture proportions, the deviation of the observed variance from its expectation becomes negative for the DMI pair. This negative deviation enables us to detect intermediate to strong negative epistasis both within and between chromosomes. In practice, the detection window in hybrid populations depends on the demographic scenario, the recombination rate, and the strength of epistasis. When the initial proportion of the two parental populations is uneven, only strong DMIs can be detected with our method unless migration prevents parental haplotypes from being lost. We use the new statistics to infer candidate DMIs from three hybrid populations of swordtail fish. We identify numerous new DMI candidates, some of which are inferred to interact with several loci within and between chromosomes. Moreover, we discuss our results in the context of an expected enrichment in intrachromosomal over interchromosomal DMIs.

PRDM9 losses in vertebrates are coupled to those of paralogs ZCWPW1 and ZCWPW2.

In most mammals and likely throughout vertebrates, the gene PRDM9 specifies the locations of meiotic double strand breaks; in mice and humans at least, it also aids in their repair. For both roles, many of the molecular partners remain unknown. Here, we take a phylogenetic approach to identify genes that may be interacting with PRDM9 by leveraging the fact that PRDM9 arose before the origin of vertebrates but was lost many times, either partially or entirely-and with it, its role in recombination. As a first step, we characterize PRDM9 domain composition across 446 vertebrate species, inferring at least 13 independent losses. We then use the interdigitation of PRDM9 orthologs across vertebrates to test whether it coevolved with any of 241 candidate genes coexpressed with PRDM9 in mice or associated with recombination phenotypes in mammals. Accounting for the phylogenetic relationship among a subsample of 189 species, we find two genes whose presence and absence is unexpectedly coincident with that of PRDM9: ZCWPW1, which was recently shown to facilitate double strand break repair, and its paralog ZCWPW2, as well as, more tentatively, TEX15 and FBXO47ZCWPW2 is expected to be recruited to sites of PRDM9 binding; its tight coevolution with PRDM9 across vertebrates suggests that it is a key interactor within mammals and beyond, with a role either in recruiting the recombination machinery or in double strand break repair.

Analysis of ancestry heterozygosity suggests that hybrid incompatibilities in threespine stickleback are environment dependent.

Hybrid incompatibilities occur when interactions between opposite ancestry alleles at different loci reduce the fitness of hybrids. Most work on incompatibilities has focused on those that are "intrinsic," meaning they affect viability and sterility in the laboratory. Theory predicts that ecological selection can also underlie hybrid incompatibilities, but tests of this hypothesis using sequence data are scarce. In this article, we compiled genetic data for F2 hybrid crosses between divergent populations of threespine stickleback fish (Gasterosteus aculeatus L.) that were born and raised in either the field (seminatural experimental ponds) or the laboratory (aquaria). Because selection against incompatibilities results in elevated ancestry heterozygosity, we tested the prediction that ancestry heterozygosity will be higher in pond-raised fish compared to those raised in aquaria. We found that ancestry heterozygosity was elevated by approximately 3% in crosses raised in ponds compared to those raised in aquaria. Additional analyses support a phenotypic basis for incompatibility and suggest that environment-specific single-locus heterozygote advantage is not the cause of selection on ancestry heterozygosity. Our study provides evidence that, in stickleback, a coarse-albeit indirect-signal of environment-dependent hybrid incompatibility is reliably detectable and suggests that extrinsic incompatibilities can evolve before intrinsic incompatibilities.