Hybridization and postzygotic isolation promote reinforcement of male mating preferences in a diverse group of fishes with traditional sex roles.

Behavioral isolation is thought to arise early in speciation due to differential sexual and/or natural selection favoring different preferences and traits in different lineages. Instead, behavioral isolation can arise due to reinforcement favoring traits and preferences that prevent maladaptive hybridization. In darters, female preference for male coloration has been hypothesized to drive speciation, because behavioral isolation evolves before F1 inviability. However, as with many long-lived organisms, the fitness of second-generation hybrids has not been assessed because raising animals to adulthood in the laboratory is challenging. Of late, reinforcement of male preferences has been implicated in darters because male preference for conspecific females is high in sympatry but absent in allopatry in multiple species pairs. The hypothesis that reinforcement accounts for behavioral isolation in sympatry assumes that hybridization and postzygotic isolation are present. Here, we used genomic and morphological data to demonstrate that hybridization is ongoing between orangethroat and rainbow darters and used hybrids collected from nature to measure postzygotic barriers across two hybrid generations. We observed sex ratio distortion in adult F1s and a dramatic reduction in backcross survival. Our findings indicate that selection to avoid hybridization promotes the evolution of male-driven behavioral isolation via reinforcement in this system.