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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 myriad 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.Impact SummaryAs sequencing tools have advanced, we have found that barriers between animal species are more porous than once thought. Researchers have found evidence for hybridization between species throughout many branches of the tree of life. In some cases, these hybridization events can involve more than two species. Here, we develop a flexible and user-friendly tool that can be used to identify three-way hybrids and report the discovery of hybrids with ancestry from three swordtail (Xiphophorus) species from an anthropogenically impacted site on the Río Calnali in Hidalgo, Mexico. Researchers have studied hybrids between twoXiphophorusspecies along this river for decades, but this is the first documented case of hybridization involving three species. We explore hypotheses for what drove this hybridization event, including anthropogenic pollutants and reduced water quality.

Sea turtles represent an ancient lineage of marine vertebrates that evolved from terrestrial ancestors over 100 Mya. The genomic basis of the unique physiological and ecological traits enabling these species to thrive in diverse marine habitats remains largely unknown. Additionally, many populations have drastically declined due to anthropogenic activities over the past two centuries, and their recovery is a high global conservation priority. We generated and analyzed high-quality reference genomes for the leatherback (Dermochelys coriacea) and green (Chelonia mydas) turtles, representing the two extant sea turtle families. These genomes are highly syntenic and homologous, but localized regions of noncollinearity were associated with higher copy numbers of immune, zinc-finger, and olfactory receptor (OR) genes in green turtles, with ORs related to waterborne odorants greatly expanded in green turtles. Our findings suggest that divergent evolution of these key gene families may underlie immunological and sensory adaptations assisting navigation, occupancy of neritic versus pelagic environments, and diet specialization. Reduced collinearity was especially prevalent in microchromosomes, with greater gene content, heterozygosity, and genetic distances between species, supporting their critical role in vertebrate evolutionary adaptation. Finally, diversity and demographic histories starkly contrasted between species, indicating that leatherback turtles have had a low yet stable effective population size, exhibit extremely low diversity compared with other reptiles, and harbor a higher genetic load compared with green turtles, reinforcing concern over their persistence under future climate scenarios. These genomes provide invaluable resources for advancing our understanding of evolution and conservation best practices in an imperiled vertebrate lineage. This work was completed in part with resources provided by the University of Massachusetts' Green High Performance Computing Cluster (GHPCC). Funding was provided by the University of Massachusetts Amherst, NSF-IOS (grant #1904439 to L.M.K.), NOAA-Fisheries, National Research Council postdoctoral fellowship program to L.M.K., VGP, Rockefeller University, to E.D.J., HHMI to E.D.J., the Sanger Institute, Max-Planck-Gesellschaft, and grant contributions from Tom Gilbert, Paul Flicek, R.W.M., Karen A. Bjorndal, Alan B. Bolten, Ed Braun, N.J.G., T.M.-B., and A.F.S. We acknowledge CONICYT-DAAD for scholarship support to T.C.-V., the São Paulo Research Foundation to E.K.S.R.–FAPESP (grant #2020/10372-6). BeGenDiv is partially funded by the German Federal Ministry of Education and Research (BMbF, Förderkennzeichen 033W034A). The work of F.T.-N. and P.M. was supported by the Intramural Research Program of the National Library of Medicine, NIH. The work of M.P. was partially funded through the Federal Ministry of Education and Research (grant 01IS18026C). H.P. was supported by a Formació de Personal Investigador fellowship from Generalitat de Catalunya (FI_B100131). M.K. was supported by “la Caixa” Foundation (ID 100010434; code LCF/BQ/PR19/11700002), the Vienna Science and Technology Fund (WWTF), and the City of Vienna (VRG20-001). Funding for green turtle resequencing was provided by a Welsh Government Sêr Cymru II and the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No. 663830-BU115 and the Sea Turtle Conservancy, Florida Sea Turtle Grants Program (17-033R).

Natural hybridization events provide unique windows into the barriers that keep species apart as well as the consequences of their breakdown. Here, we characterize hybrid populations formed between the northern swordtail fish Xiphophorus cortezi and Xiphophorus birchmanni from collection sites on two rivers. We use simulations and new genetic reference panels to develop sensitive and accurate local ancestry calling in this novel system. Strikingly, we find that hybrid populations on both rivers consist of two genetically distinct subpopulations: a cluster of pure X. birchmanni individuals and one of phenotypically intermediate hybrids that derive ∼85-90% of their genome from X. cortezi. Simulations suggest that initial hybridization occurred ∼150 generations ago at both sites, with little evidence for contemporary gene flow between subpopulations. This population structure is consistent with strong assortative mating between individuals of similar ancestry. The patterns of population structure uncovered here mirror those seen in hybridization between X. birchmanni and its sister species, Xiphophorus malinche, indicating an important role for assortative mating in the evolution of hybrid populations. Future comparisons will provide a window into the shared mechanisms driving the outcomes of hybridization not only among independent hybridization events between the same species but also across distinct species pairs.

Author(s): Banerjee, Shreya M; Frey, Amy; Kurle, Carolyn M; Perrault, Justin R; Stewart, Kelly R | Abstract: Understanding species’ mating systems provides important information about their ecology, life history, and behavior. Direct observations of mating behaviors can be challenging, but molecular techniques can reveal information about mating systems and paternal identity in difficult-to-observe species such as sea turtles. Genetic markers can be used to assess the paternity of a clutch and to assign hatchlings to a father. Leatherback turtles Dermochelys coriacea sometimes mate with multiple individuals, resulting in clutches with mixed paternity; however, the effects of multiple paternity on hatchling quality are unclear. Leatherback hatchlings at Sandy Point National Wildlife Refuge, St. Croix, US Virgin Islands, exhibit visible variation in individual body size, sometimes within the same clutch. We collected morphometrics and tissue samples from hatchlings across multiple nesting seasons (2009, 2012, 2013, 2015, and 2016) and found that hatchlings exhibited small but statistically significant differences in morphometrics between years. We used maternal and hatchling microsatellite genotypes to reconstruct paternal genotypes, assigning fathers to each hatchling. We found multiple paternity in 5 of 17 clutches analyzed and compared differences in morphometrics between full-siblings with differences between half-siblings. We found no significant differences between morphometrics of hatchlings from the same mother but different fathers. We compared within-clutch variances in morphometrics for clutches with and without multiple paternity and found no significant difference in morphological variation between them. Therefore, we could not attribute differences in hatchling size within a clutch to paternal contribution. Understanding other factors affecting hatchling morphology, and other possible fitness metrics, may reveal insights into the benefits, or lack thereof, of polyandry in sea turtles.

Hybridization between species is widespread across the tree of life. As a result, many species, including our own, harbor regions of their genome derived from hybridization. Despite the recognition that this process is widespread, we understand little about how the genome stabilizes following hybridization, and whether the mechanisms driving this stabilization tend to be shared across species. Here, we dissect the drivers of variation in local ancestry across the genome in replicated hybridization events between two species pairs of swordtail fish: Xiphophorus birchmanni × X. cortezi and X. birchmanni × X. malinche. We find unexpectedly high levels of repeatability in local ancestry across the two types of hybrid populations. This repeatability is attributable in part to the fact that the recombination landscape and locations of functionally important elements play a major role in driving variation in local ancestry in both types of hybrid populations. Beyond these broad scale patterns, we identify dozens of regions of the genome where minor parent ancestry is unusually low or high across species pairs. Analysis of these regions points to shared sites under selection across species pairs, and in some cases, shared mechanisms of selection. We show that one such region is a previously unknown hybrid incompatibility that is shared across X. birchmanni × X. cortezi and X. birchmanni × X. malinche hybrid populations., Author summary We now know that hybridization, or the production of offspring between individuals of different species, happens frequently across many plant, animal, and fungi groups. As a result, the genomes of many contemporary species contain material derived from these hybridization events. At the same time, hybridization can have negative consequences on an organism’s ability to survive and reproduce. One major question is whether there are shared principles that determine which parts of the genome can move between species and which cannot. Here, we compare the genomes of several independently formed hybrid populations between the fish species pairs of Xiphophorus birchmanni and X. cortezi as well as X. birchmanni and X. malinche to begin to address this question. We find that across hybrid populations, regions of the genome with especially low recombination rates or especially high gene density are more likely to be derived from the parent species that contributed the majority of the genome to hybrids. Moreover, we identify regions of the genome that negatively interact in both types of hybrid population. Together our results demonstrate that genomic outcomes of hybridization between distinct species pairs are in part predictable.

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.

BackgroundTranscriptomic data has demonstrated utility to advance the study of physiological diversity and organisms’ responses to environmental stressors. However, a lack of genomic resources and challenges associated with collecting high-quality RNA can limit its application for many wild populations. Minimally invasive blood sampling combined with de novo transcriptomic approaches has great potential to alleviate these barriers. Here, we advance these goals for marine turtles by generating high quality de novo blood transcriptome assemblies to characterize functional diversity and compare global transcriptional profiles between tissues, species, and foraging aggregations.ResultsWe generated high quality blood transcriptome assemblies for hawksbill (Eretmochelys imbricata),loggerhead (Caretta caretta), green (Chelonia mydas), and leatherback (Dermochelys coriacea) turtles. The functional diversity in assembled blood transcriptomes was comparable to those from more traditionally sampled tissues. A total of 31.3% of orthogroups identified were present in all four species, representing a core set of conserved genes expressed in blood and shared across marine turtle species. We observed strong species-specific expression of these genes, as well as distinct transcriptomic profiles between green turtle foraging aggregations that inhabit areas of greater or lesser anthropogenic disturbance.ConclusionsObtaining global gene expression data through non-lethal, minimally invasive sampling can greatly expand the applications of RNA-sequencing in protected long-lived species such as marine turtles. The distinct differences in gene expression signatures between species and foraging aggregations provide insight into the functional genomics underlying the diversity in this ancient vertebrate lineage. The transcriptomic resources generated here can be used in further studies examining the evolutionary ecology and anthropogenic impacts on marine turtles.

Natural hybridization events provide unique windows into the barriers that keep species apart as well as the consequences of their breakdown. Here we characterize hybrid populations formed between the northern swordtail fishXiphophorus corteziandX. birchmannifrom collection sites on two rivers. We develop sensitive and accurate local ancestry calling for this system based on low coverage whole genome sequencing. Strikingly, we find that hybrid populations on both rivers consist of two genetically distinct subpopulations: a cluster of nearly pureX. birchmanniindividuals and one of phenotypically intermediate hybrids that derive ~85-90% of their genome fromX. cortezi. Simulations and empirical data suggest that at both sites initial hybridization occurred ~150 generations ago, with little evidence for contemporary gene flow between subpopulations, likely due to strong assortative mating. The patterns of population structure uncovered here mirror those seen in hybridization betweenX. birchmanniand its sister species,X. malinche. Future comparisons will provide a window into the repeatability of the outcomes of hybridization not only across independent hybridization events between the same species but also across distinct species pairs.

Biologists since Darwin have been fascinated by the evolution of sexually selected ornaments, particularly those that reduce viability. Uncovering the genetic architecture of these traits is key to understanding how they evolve and are maintained. Here, we investigate the genetic architecture of a sexually selected ornament, the “sword” fin extension that characterizes many species of swordtail fish (Xiphophorus). Using sworded and swordless sister species ofXiphophorus, we generated a mapping population and show that the sword ornament is polygenic – with ancestry across the genome explaining substantial variation in the trait. After accounting for the impacts of genome-wide ancestry, we identify one major effect QTL that explains ∼5% of the overall variation in the trait. Using a series of approaches, we narrow this large QTL interval to a handful of likely candidate genes, including the genesp8. Notably,sp8plays a regulatory role in fin regeneration and harbors several derived substitutions that are predicted to impact protein function in the species that has lost the sword ornament. Furthermore, we find evidence of selection on ancestry atsp8in four natural hybrid populations, consistent with selection against the sword in these populations.

Mapping vertebrate incompatibility alleles Deleterious gene interactions may underlie the observed hybrid incompatibilities. However, few genes underlying hybrid incompatibilities have been identified, and most of these involve species that do not hybridize in natural conditions. Powell et al. used genome sequencing to map genes likely responsible for incompatibilities that reduce fitness in naturally occurring hybrid swordtail fish. These gene combinations result in malignant melanoma, which is found in naturally hybridizing populations but is not present in the parental populations (see the Perspective by Dagilis and Matute). Using genome and population resequencing, the authors performed a genome-wide association study to identify potentially causative mutations. Using an admixture mapping approach that assessed introgression between multiple swordtail fish species, the authors suggest that lineages carry different genes that interact with the same candidate gene, resulting in the observed melanomas and providing insight into convergent hybrid incompatibles that arise between species. Science , this issue p. 731 ; see also p. 710

Biologists since Darwin have been fascinated by the evolution of sexually selected ornaments, particularly those that reduce viability. Uncovering the genetic architecture of these traits is key to understanding how they evolve and are maintained. Here, we investigate the genetic architecture and evolutionary loss of a sexually selected ornament, the "sword" fin extension that characterizes many species of swordtail fish (Xiphophorus). Using sworded and swordless sister species of Xiphophorus, we generated a mapping population and show that the sword ornament is polygenic-with ancestry across the genome explaining substantial variation in the trait. After accounting for the impacts of genome-wide ancestry, we identify one major-effect quantitative trait locus (QTL) that explains ~5% of the overall variation in the trait. Using a series of approaches, we narrow this large QTL interval to several likely candidate genes, including genes involved in fin regeneration and growth. Furthermore, we find evidence of selection on ancestry at one of these candidates in four natural hybrid populations, consistent with selection against the sword in these populations.