Gene expression evolution leads to much of the diversity observed among species. Expressiondivergence may be driven by regulatory evolution in cis loci and trans factors. Additionally, regulatory evolution may lead to hybrid incompatibilities in the form of misexpression linked to sterility. Many studies have shown that expression divergence varies widely among tissues, and male gonadal tissues tend to evolve particularly rapidly. There has been less work on somatic male reproductive tissues, and little work on the evolutionary properties of different cell types. Additionally, our understanding of how gene regulatory evolution and hybrid misexpression vary among tissues is especially limited. In this study I use the Drosophila male accessory gland and ejaculatory duct—organs that produce seminal fluid—as a for evolution of expression and regulation in cells and tissues. In Chapter I, I used single-nucleus RNA-Seq of the accessory gland and ejaculatory duct three Drosophila species to comprehensively describe the cell diversity of these tissues for the first time. I found that rates of transcriptome divergence were heterogenous among cell types and lineages, with ejaculatory duct cells evolving faster than the cells of the accessory gland. I also found that proteins characteristic of each cell type have variable rates of adaptive substitutions. In Chapter II, I used allele-specific expression in accessory glands and ejaculatory ducts of hybrids between D. melanogaster and D. simulans to estimate regulatory changes in cis and trans and quantify hybrid misexpression. I found an unexpected excess of trans-regulatory divergence in contrast to the prevailing expectation in the literature. I also found that the accessory gland has limited misexpression, potentially indicative of less hybrid dysgenesis in comparison to gonads. I integrated ATAC-Seq and RNA-Seq data to show that differences in chromatin accessibility correlate with divergence in both cis and trans. Taken together, these studies contribute to our knowledge of the unique evolution of the accessory gland and ejaculatory duct and underline the importance of tissue- and cell-type specific differences in expression and regulatory divergence.

The evolution of biological sexes is a fascinating field of study with much that remains to be learned. Decades of studies in Drosophila and in other animal models have made it clear that reproductive tissues in both sexes are some of the most rapidly evolving organs: They diverge more rapidly in gene expression relative to non- reproductive tissues, the genes that are biased in expression to reproductive tissues often show rapid divergence relative to non reproductive biased genes, and the reproductive organs themselves sometimes even show their capacity for physiological and behavioral changes, such as the ability of certain female reproductive organs to store sperm.

To better understand the genetic and genomic mechanisms that underlie the rapid evolution of reproductive tissues in response to local adaptation, and to study the extent of parallelism in gene expression in these tissues across species, I study the gene expression changes that underlie the accessory glands and testes across two geographic regions in three species, as seen in chapter one. Since much less is known about the female contributions to reproduction, and to begin to understand the rapid evolution of some of their tissues, I have begun to characterize the similarities and differences in gene expression across the seminal receptacle, the spermathecae, and the parovaria in eight species. This second chapter will help us better understand how certain female reproductive organs can gain and lose sperm storage functions across evolutionary time. Lastly, and to better understand how stem cell niches are maintained across species, I begin to characterize some of the gene expression differences across such niches in various species of Drosophila, and ask the question of whether such niches use similar or different gene products for the maintenance of healthy stem cells in these niches.

Studying the rapid evolution of reproductive organs helps us characterize the shared gene expression patterns that are core to reproduction across distantly related species, while also elucidating the divergence in gene expression that allows for the rise of new molecules, physiologies, and behaviors. My three chapters of studies contribute to our understanding of male and female reproductive system function and elaborate on our limited knowledge of the evolution of female reproductive tissues. Studying the diversity of genetic and phenotypic features of closely and distantly related species will eventually allow us to better understand the co-evolution of male and female sexes across species, and will allow us to study the extent of gene expression plasticity across sexes: If different gene products related to successful fertilization are made by both males and females, how amenable are such reproductive genes to switching expression across sexes, and how common are such switches? And what effects do sex-specific selection and local adaptation have on shaping content and expression of a genome?

Sperm competition is an important fitness component in many animal groups. Drosophila melanogaster males exhibit substantial genetic variation for sperm competitive ability and females show considerable genetic variation for first versus second male sperm use. Currently, the forces responsible for maintaining genetic variation in sperm competition related phenotypes are receiving much attention. While several candidate genes contributing to the variation seen in male competitive ability are known, genes involved in female sperm use remain largely undiscovered. Without knowledge of the underlying genes, it will be difficult to distinguish between different models of sexual selection such as cryptic female choice and sexual conflict. We used quantitative trait locus (QTL) mapping to identify regions of the genome contributing to female propensity to use first or second male sperm, female refractoriness to re-mating, and early-life fertility. The most well supported markers influencing the phenotypes include 33F/34A (P2), 57B (refractoriness) and 23F/24A (fertility). Between 10% and 15% of the phenotypic variance observed in these recombinant inbred lines was explained by these individual QTLs. More detailed investigation of the regions detected in this experiment may lead to the identification of genes responsible for the QTLs identified here.