Molecular evolution of mating plug genes

Seminal fluid proteins (SPFs) in <em>Drosophila</em> show an unusually rapid molecular sequence evolution. SPFs are a major player in post-mating interactions and potential targets of postcopulatory sexual selection (PCSC). Mating plug (MP) proteins are a class of SPFs transferred to females along with the ejaculate. These proteins coagulate to form a plug in the female reproductive tract to delay the female remating. Female being polyandrous, stores males’ sperms and expels the rest of the ejaculate in order to remate. These different reproductive strategies of males and females can cause a potential conflict between the genes involves in this interaction and drive the evolution of proteins. Here, we report the molecular evolution of MP proteins <em>EbpI</em> and <em>EbpIII</em> present in <em>Drosophila</em> sub-genus. We focused on a young cluster of <em>Drosophila</em> species called <em>nasuta</em> subgroup, which offers the best model to study molecular evolution of fast-evolving proteins. We report <em>EbpI</em> duplication in the branch leading to <em>nasuta</em>-subgroup, which evolves under positive selection, while the duplicate does not. The positively selected sites are located in the structurally important region of <em>EbpI</em>. <em>EbpIII</em>, which belongs to a multi-gene family, showed evidence of birth and death evolution. We identified two functional copies and a pseudogenized copy of <em>EbpIII</em> across the <em>virilis</em> and <em>nasuta</em>-subgroups. Of the three functional copies, only <em>EbpIIIa</em> showed signature of positive selection in one of the selection tests. Our results suggest that the mating plug proteins rapidly diversify in <em>nasuta</em>-subgroup and potentially play a role in post-mating, prezygotic reproductive isolation.