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 ) 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, and , 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 crosses experience dysfunction: crosses with females were largely inviable and crosses with females had a heavily skewed sex ratio. Using F hybrids we identify several genomic regions that strongly impact hybrid viability. Strikingly, two of these regions underlie genetic incompatibilities in hybrids between and its sister species . 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 × hybrids as in hybrids. Our findings identify varied reproductive barriers that shape genetic exchange between naturally hybridizing species and highlight the complex evolutionary outcomes of hybridization.