Females of many different species often mate with multiple males, creating opportunities for competition among their sperm. Although originally unappreciated, sperm competition is now considered a central form of post-copulatory male-male competition that biases fertilization. Assays of differences in sperm competitive ability between males, and interactions between females and males, have made it possible to infer some of the main mechanisms of sperm competition. Nevertheless, classical genetic approaches have encountered difficulties in identifying loci influencing sperm competitiveness while functional and comparative genomic methodologies, as well as genetic variant association studies, have uncovered some interesting candidate genes. We highlight how the systematic implementation of approaches that incorporate gene perturbation assays in experimental competitive settings, together with the monitoring of progeny output or sperm features and behavior, has allowed the identification of genes unambiguously linked to sperm competitiveness. The emerging portrait from 45 genes (33 from fruit flies, 8 from rodents, 2 from nematodes, and 2 from ants) is their remarkable breadth of biological roles exerted through males and females, the non-preponderance of sperm genes, and their overall pleiotropic nature.

Seminal fluid proteins (SFPs) are a group of reproductive proteins that are among the most evolutionarily divergent known. As SFPs can impact male and female fitness, these proteins have been proposed to evolve under postcopulatory sexual selection (PCSS). However, the fast change of the SFPs can also result from nonadaptive evolution, and the extent to which selective constraints prevent SFPs rapid evolution remains unknown. Using intra- and interspecific sequence information, along with genomics and functional data, we examine the molecular evolution of approximately 300 SFPs in Drosophila. We found that 50-57% of the SFP genes, depending on the population examined, are evolving under relaxed selection. Only 7-12% showed evidence of positive selection, with no evidence supporting other forms of PCSS, and 35-37% of the SFP genes were selectively constrained. Further, despite associations of positive selection with gene location on the X chromosome and protease activity, the analysis of additional genomic and functional features revealed their lack of influence on SFPs evolving under positive selection. Our results highlight a lack of sufficient evidence to claim that most SFPs are driven to evolve rapidly by PCSS while identifying genomic and functional attributes that influence different modes of SFPs evolution.

Speciation can occur through the presence of reproductive isolation barriers that impede mating, restrict cross-fertilization, or render inviable/sterile hybrid progeny. The D. willistoni subgroup is ideally suited for studies of speciation, with examples of both allopatry and sympatry, a range of isolation barriers, and the availability of one species complete genome sequence to facilitate genetic studies of divergence. D. w. willistoni has the largest geographic distribution among members of the Drosophila willistoni subgroup, spanning from Argentina to the southern United States, including the Caribbean islands. A subspecies of D. w. willistoni, D. w. quechua, is geographically separated by the Andes mountain range and has evolved unidirectional sterility, in that only male offspring of D. w. quechua females × D. w. willistoni males are sterile. Whether D. w. willistoni flies residing east of the Andes belong to one or more D. willistoni subspecies remains unresolved. Here we perform fecundity assays and show that F1 hybrid males produced from crosses between different strains found in Central America, North America, and northern Caribbean islands are reproductively isolated from South American and southern Caribbean island strains as a result of unidirectional hybrid male sterility. Our results show the existence of a reproductive isolation barrier between the northern and southern strains and suggest a subdivision of the previously identified D. willistoni willistoni species into 2 new subspecies.