Evolutionary dynamics of male reproductive genes in theDrosophila virilissubgroup

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.

The two “rules of speciation” in species with young sex chromosomes

“…Furthermore, D e is significantly lower for G expr compared to S expr genes (Figure a; Wilcoxon rank‐sum test p < 10 −6 ), indicating that stronger purifying selection at the haploid stage of the life cycle prevents expression divergence between species for most genes. In animals, the genes with functions related to male gametogenesis, such as accessory gland proteins in Drosophila, are often reported to show faster evolutionary rates, possibly due to positive selection fuelled by sexual conflict (e.g., Ahmed‐Braimah, Unckless, & Clark, ). The S. latifolia and S. dioica genes that are overexpressed in pollen (P expr ; the category includes genes with at least fivefold higher expression in pollen compared to male somatic tissues) also show accelerated expression divergence, compared to non‐overexpressed genes; in particular, the distribution of D e for P expr genes is significantly shifted upwards compared to G expr and S expr genes (Figure a; Wilcoxon rank‐sum test p < 10 −6 for both P expr :G expr and P expr :S expr comparisons).…”

Persistent postmating, prezygotic reproductive isolation between populations

“…() found no relationship between genetic distance and the strength of PMPZ isolation, suggesting divergence is not simply a result of isolation by distance. Instead, PMPZ isolation likely emerges as a by‐product of both sexual selection and sexual conflict which are important in shaping the rapid co‐evolution of ejaculate‐female reproductive tract interactions (Ahmed‐Braimah et al., ; Bono et al., ; Mendelson, Martin, & Flaxman, ; Pitnick et al., ). Given that D. montana males transfer and females store motile sperm for fertilization but (most of) these sperm do not penetrate eggs, incompatibility is likely because of mismatches between sperm and egg release.…”

“…A growing body of literature now shows PMPZ isolation is the primary or only barrier to gene flow in some closely related taxa, suggesting an important role in the early evolution of reproductive isolation (Ahmed‐Braimah, Unckless, & Clark, ; Bono, Matzkin, Hoang, & Brandsmeier, ; Cramer et al., ; Dean & Nachman, ; Soudi, Reinhold, & Engqvist, ; Turissini et al., ). However, the majority of research has focused on incompatibilities arising between species even though barriers that maintain reproductive isolation after divergence may not be the same barriers that were important in reducing gene flow during the initial stages of the speciation process (Butlin et al., ; Coyne & Orr, ; Turelli et al., ).…”

Seminal fluid protein divergence among populations exhibiting postmating prezygotic reproductive isolation

“…In polyandrous species, ejaculate × female reproductive tract interactions are subject to rapid coevolution and diversification, thought to result from postcopulatory sexual selection (sperm competition and cryptic female choice) and sexually antagonistic coevolution (Birkhead & Pizzari, 2002; Firman et al., 2017; Meslin et al., 2017; Sirot et al., 2015; but see Dapper & Wade, 2020). Thus, despite holding a central role in fertilization, ejaculate and female reproductive tract traits often show elevated rates of molecular and morphological evolution (Ahmed‐Braimah et al., 2017; McGeary & Findlay, 2020; Meslin et al., 2017; Rowe et al., 2015; Simmons & Fitzpatrick, 2019; VanKuren & Long, 2018). Divergence between populations in these traits is predicted to result in the early emergence of reproductive isolation that occurs after mating but before fertilization (postmating prezygotic; PMPZ) (Gavrilets, 2000; Lande, 1981; Panhuis et al., 2001).…”

“…When genetically manipulated, some Sfps elicit PMPZ‐like phenotypes such as sperm storage abnormalities or reduced oviposition rates (LaFlamme et al., 2012; Ravi Ram & Wolfner, 2007). Moreover, differences in Sfp expression between species, and abnormal gene expression profiles or changes in protein abundance in the female reproductive tract after mating with heterospecific vs. conspecific males are associated with PMPZ isolation (Ahmed‐Braimah et al., 2017; Bono et al., 2011; McCullough et al., 2020). Recently, ectopic injection of Sfps between populations of simultaneously hermaphroditic freshwater snails, Lymnaea stagnalis , that showed divergent gene expression patterns was shown to alter fecundity and sperm transfer (Nakadera et al., 2020).…”