Evolutionary dynamics of male reproductive genes in the Drosophila virilis subgroup

Abstract

Postcopulatory sexual selection (PCSS) is a potent evolutionary force that can drive rapid changes of reproductive genes within species, and thus has the potential to generate reproductive incompatibilities between species. Male seminal fluid proteins (SFPs) are major players in postmating interactions, and likely the main targets of PCSS in males. The virilis subgroup of Drosophila exhibits strong interspecific gametic incompatibilities, and can serve as a model to study the genetic basis of PCSS and gametic isolation. However, reproductive genes in this group have not been characterized. Here we use short-read RNA sequencing of male reproductive organs to examine the evolutionary dynamics of reproductive genes in members of the virilis subgroup: D. americana, D. lummei, D. novamexicana, and D. virilis. For each of the three male reproductive organs (accessory glands, ejaculatory bulb, and testes), we identify genes that show strong expression bias in a given tissue relative to the remaining tissues. We find that the majority of male reproductive transcripts are testes-biased, accounting for ~15% of all annotated genes. Ejaculatory bulb-biased transcripts largely code for lipid metabolic enzymes, and contain orthologs of the D. melanogaster ejaculatory bulb protein, Peb-me, which is involved in mating-plug formation. In addition, we identify 71 candidate SFPs, and show that this set of genes has the highest rate of nonsynonymous codon substitution relative to testes- and ejaculatory bulb-biased genes. Furthermore, these SFPs are underrepresented on the X chromosome and are enriched for proteolytic enzymes, which is consistent with SFPs in other insect species. Surprisingly, we find 35 D. melanogaster SFPs with conserved accessory gland expression in the virilis group, suggesting these genes may have conserved reproductive roles in Drosophila. Finally, we show that several of the SFPs that have the highest rate of nonsynonymous codon substitutions reside on the centromeric half of chromosome 2, which contributes to paternal gametic incompatibility between species. Our results suggest that SFPs are under strong selection in the virilis group, and likely play a major role in PCSS and/or gametic isolation.