Proteins involved in post-copulatory interactions between males and females are among the fastest evolving genes in many species, usually attributed to their involvement in reproductive conflict. As a result, these proteins are thought to often be in the formation of postmating-prezygotic incompatibilities between species. The Drosophila dunni subgroup consists of a dozen recently diverged species found across the Caribbean islands with varying levels of hybrid incompatibility. We performed experimental crosses between species in the dunni group and see some evidence of hybrid incompatibilities. We also find evidence of reduced survival following hybrid mating, likely due to postmating-prezygotic incompatibilities. We assessed rates of evolution between these species genomes and find evidence of rapid evolution and divergence of some reproductive proteins, specifically the seminal fluid proteins. This work suggests the rapid evolution of seminal fluid proteins may be associated with postmating-prezygotic isolation, which acts as a barrier for gene flow between even the most closely related species.

1Proteins involved in post-copulatory interactions between males and females are among the fastest evolving 2 genes in many species and this has been attributed to reproductive conflict. Likely as a result, these proteins 3 are frequently involved in cases of post-mating-prezygotic isolation between species. The Drosophila dunni 4 subgroup consists of a dozen recently diverged species found across the Caribbean islands with varying 5 levels of hybrid incompatibility. We sought to examine how post-mating-prezygotic factors are involved in 6 isolation among members of this species group. We performed experimental crosses between species in the 7 dunni group and find evidence of hybrid inviability. We also find an insemination reaction-like response 8 preventing egg laying and leading to reduced female survival post-mating. To identify that genes may be 9 involved in these incompatibilities, we sequenced and assembled the genomes of four species in the dunni 10 subgroup and looked for signals of rapid evolution between species. Despite low levels of divergence, we 11

Background: Proteins involved in post-copulatory interactions between males and females are among the fastest evolving genes in many species and this has been attributed to reproductive conflict. Likely as a result, these proteins are frequently involved in cases of post-mating-prezygotic isolation between species. The Drosophila dunni subgroup consists of a dozen recently diverged species found across the Caribbean islands with varying levels of hybrid incompatibility.Results: We performed experimental crosses between species in the dunni group and find evidence of hybrid inviability likely due to post-mating-prezygotic incompatibilities. We next assessed rates of evolution between these species genomes and find evidence of rapid evolution and divergence of some reproductive proteins, specifically the seminal fluid proteins.Conclusions: This work suggests the rapid evolution of seminal fluid proteins can lead to post-mating-prezygotic isolation, which acts as a barrier for gene flow between even the most closely related species.