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 F 1 crosses experience dysfunction: crosses with X. birchmanni females were largely inviable and crosses with X. cortezi females had a heavily skewed sex ratio. Using F 2 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 Ô X. birchmanni hybrids as in X. malinche Ô 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. Significance Statement: Biologists are fascinated by how the diverse species we see on Earth have arisen and been maintained. One driver of this process is the evolution of reproductive barriers between species. Despite the commonality of these barriers, many species still exchange genes through a process called hybridization. Here, we show that related species can have a striking array of reproductive barriers-from genetic interactions that harm hybrids to mate preferences that reduce hybridization in the first place. However, we also find that genetic exchange between these species is very common, and may itself play an important role in the evolution of reproductive barriers. Together, our work highlights the complex web of interactions that impact the origin and persistence of distinct species.

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.

Developmental environment contributes to rapid trait shifts among newly colonized subterranean habitats.

Recent colonization of extreme environments provides unique opportunities to study the early steps of adaptation and the potential for rapid convergent evolution. However, phenotypic shifts during recent colonization may also be due to plasticity in response to changes in the rearing environment. Here, we analyzed a suite of morphological and behavioral traits in paired surface, subterranean, and facultatively subterranean Mexican tetras ( Astyanax mexicanus) from recent introductions in two separate watersheds outside of their native range. We found a variety of phenotypic and behavioral shifts between subterranean and surface populations that are similar to those observed in relatively ancient populations in Mexico. Despite this rapid morphological divergence, we found that most of these trait differences were due to plasticity in response to rearing environments. While most trait assays in common-garden, lab-raised fish indicated that phenotypic shifts in wild fish were the result of plasticity, we also found evidence of genetic control in several traits present in subterranean populations. Interestingly, wall-following behavior, an important subterranean foraging behavior, was greater in lab-born subterranean fish than in lab-born surface fish, suggesting rapid divergence of this trait between subterranean and surface populations. Thus, this study sheds light on the early steps of subterranean evolution, identifies potential rapid behavioral evolution, and suggests that plasticity in traits involving exploratory behavior may facilitate subterranean colonization.