Postmating prezygotic isolation occurs at two levels of divergence in Drosophila recens and D. subquinaria.

Identifying the presence and strength of reproductive isolating barriers is necessary to understand how species form and then remain distinct in the face of ongoing gene flow. Here we study reproductive isolation at two stages of the speciation process in the closely related mushroom-feeding species Drosophila recens and D. subquinaria. We assess three isolating barriers that occur after mating, including the number of eggs laid, the proportion of eggs laid that hatched, and the number of adult offspring from a single mating. First, all three reproductive barriers are present between D. recens females and D. subquinaria males, which are at the late stages of speciation but still produce fertile daughters through which gene flow can occur. There is no evidence for geographic variation in any of these traits, concurrent with patterns of behavioral isolation. Second, all three of these reproductive barriers are strong between geographically distant conspecific populations of D. subquinaria, which are in the early stages of speciation and show genetic differentiation and asymmetric behavioral discrimination. The reduction in the number of eggs laid is asymmetric, consistent with patterns in behavioral isolation, and suggests the evolution of postmating prezygotic isolation due to cascade reinforcement against mating with D. recens. In summary, not only may postmating prezygotic reproductive barriers help maintain isolation between D. recens and D. subquinaria but they may also drive the earliest stages of isolation within D. subquinaria.