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Reproductive isolation reduces breeding between species. Traditionally, prezygotic and postzygotic barriers to reproduction have been broadly studied, but in recent years, attention has been brought to the existence of barriers that act after copulation but before fertilization. Here, we show that when D. virilis females from different geographic locations mate with D. novamexicana males, egg laying is normal, but fertilization rates are severely reduced, despite normal rates of sperm transfer. This reduction in fertilization is probably due to lower retention of heterospecific sperm in female storage organs one-to-two days after copulation. An inspection of egg hatchability in crosses between females and males from other virilis subgroup species reveals that isolation due to poor egg hatchability likely evolved during the diversification of D. virilis/D. lummei from species of the novamexicana-americana clade. Interestingly, the number of eggs laid by D. virilis females in heterospecific crosses was not different from the numbers of eggs laid in conspecific crosses, suggesting that females exert some form of cryptic control over the heterospecific ejaculate and that future studies should focus on how female and female-sperm interactions contribute to the loss or active exclusion of heterospecific sperm from storage

Male-Female Interactions and the Evolution of Postmating Prezygotic Reproductive Isolation among Species of the Virilis Subgroup