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. We sought to examine how post-mating-prezygotic factors are involved in isolation among members of this species group. We performed experimental crosses between species in the dunni group and find evidence of hybrid inviability. We also find an insemination reaction-like response preventing egg laying and leading to reduced female survival post-mating. To identify that genes may be involved in these incompatibilities, we sequenced and assembled the genomes of four species in the dunni subgroup and looked for signals of rapid evolution between species. Despite low levels of divergence, we found evidence of rapid evolution and divergence of some reproductive proteins, specifically the seminal fluid proteins. This suggests post-mating-prezygotic isolation as a barrier for gene flow between even the most closely related species in this group and seminal fluid proteins as a possible culprit. ### Competing Interest Statement The authors have declared no competing interest.
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