Description: 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 are important 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 utilize 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 . We find that the majority of male reproductive transcripts are testes-biased, accounting for ~15% of all annotated genes. Ejaculatory bulb (EB)-biased transcripts largely code for lipid metabolic enzymes, and contain orthologs of the D. melanogaster EB protein, Peb-me, which is involved in mating-plug formation. In addition, we identify 71 candidate SFPs, and show that this gene set has the highest rate of nonsynonymous codon substitution relative to testes- and EB-biased genes. Furthermore, we identify orthologs of 35 D. melanogaster SFPs that have conserved accessory gland expression in the virilis group. Finally, we show that several of the SFPs that have the highest rate of nonsynonymous codon substitution reside on chromosomal regions, which contributes to paternal gametic incompatibility between species. Our results show that SFPs rapidly diversify in the virilis group, and suggest that they likely play a role in PCSS and/or gametic isolation.

Description: The genetic basis of species differences remains understudied. Studies in insects have contributed significantly to our understanding of morphological evolution. Pigmentation traits in particular have received a great deal of attention and several genes in the insect pigmentation pathway have been implicated in inter- and intraspecific differences. Nonetheless, much remains unknown about many of the genes in this pathway and their potential role in understudied taxa. Here we genetically analyze the puparium color difference between members of the virilis group of Drosophila . The puparium of Drosophila virilis is black, while those of D. americana , D. novamexicana , and D. lummei are brown. We used a series of backcross hybrid populations between D. americana and D. virilis to map the genomic interval responsible for the difference between this species pair. First, we show that the pupal case color difference is caused by a single Mendelizing factor, which we ultimately map to an ~11-kb region on chromosome 5. The mapped interval includes only the first exon and regulatory region(s) of the dopamine N -acetyltransferase gene ( Dat ). This gene encodes an enzyme that is known to play a part in the insect pigmentation pathway. Second, we show that this gene is highly expressed at the onset of pupation in light brown taxa ( D. americana and D. novamexicana ) relative to D. virilis , but not in the dark brown D. lummei . Finally, we examine the role of Dat in adult pigmentation between D. americana (heavily melanized) and D. novamexicana (lightly melanized) and find no discernible effect of this gene in adults. Our results demonstrate that a single gene is entirely or almost entirely responsible for a morphological difference between species.

Description: Understanding the genetic basis of speciation is a central problem in evolutionary biology. Studies of reproductive isolation have provided several insights into the genetic causes of speciation, especially in taxa that lend themselves to detailed genetic scrutiny. Reproductive barriers have usually been divided into those that occur before zygote formation (prezygotic) and after (postzygotic), with the latter receiving a great deal of attention over several decades. Reproductive barriers that occur after mating but before zygote formation [postmating prezygotic (PMPZ)] are especially understudied at the genetic level. Here, I present a phenotypic and genetic analysis of a PMPZ reproductive barrier between two species of the Drosophila virilis group: D. americana and D. virilis . This species pair shows strong PMPZ isolation, especially when D. americana males mate with D. virilis females: ~99% of eggs laid after these heterospecific copulations are not fertilized. Previous work has shown that the paternal loci contributing to this incompatibility reside on two chromosomes, one of which (chromosome 5) likely carries multiple factors. The other (chromosome 2) is fixed for a paracentric inversion that encompasses nearly half the chromosome. Here, I present two results. First, I show that PMPZ in this species cross is largely due to defective sperm storage in heterospecific copulations. Second, using advanced intercross and backcross mapping approaches, I identify genomic regions that carry genes capable of rescuing heterospecific fertilization. I conclude that paternal incompatibility between D. americana males and D. virilis females is underlain by four or more genes on chromosomes 2 and 5.