Evolution and genetics of accessory gland transcriptome divergence between Drosophila melanogaster and D. simulans.

Studies of allele-specific expression in interspecific hybrids have provided important insights into gene-regulatory divergence and hybrid incompatibilities. Many such investigations in Drosophila have used transcriptome data from complex mixtures of many tissues or from gonads, however, regulatory divergence may vary widely among species, sexes, and tissues. Thus, we lack sufficiently broad sampling to be confident about the general biological principles of regulatory divergence. Here, we seek to fill some of these gaps in the literature by characterizing regulatory evolution and hybrid misexpression in a somatic male sex organ, the accessory gland, in F1 hybrids between Drosophila melanogaster and D. simulans. The accessory gland produces seminal fluid proteins, which play an important role in male and female fertility and may be subject to adaptive divergence due to male-male or male-female interactions. We find that trans differences are relatively more abundant than cis, in contrast to most of the interspecific hybrid literature, though large effect-size trans differences are rare. Seminal fluid protein genes have significantly elevated levels of expression divergence and tend to be regulated through both cis and trans divergence. We find limited misexpression (over- or underexpression relative to both parents) in this organ compared to most other Drosophila studies. As in previous studies, male-biased genes are overrepresented among misexpressed genes and are much more likely to be underexpressed. ATAC-Seq data show that chromatin accessibility is correlated with expression differences among species and hybrid allele-specific expression. This work identifies unique regulatory evolution and hybrid misexpression properties of the accessory gland and suggests the importance of tissue-specific allele-specific expression studies.

Evolution of secondary cell number and position in the Drosophila accessory gland.

In animals with internal fertilization, males transfer gametes and seminal fluid during copulation, both of which are required for successful reproduction. In Drosophila and other insects, seminal fluid is produced in the paired accessory gland (AG), the ejaculatory duct, and the ejaculatory bulb. The D. melanogaster AG has emerged as an important model system for this component of male reproductive biology. Seminal fluid proteins produced in the Drosophila AG are required for proper storage and use of sperm by the females, and are also critical for establishing and maintaining a suite of short- and long-term postcopulatory female physiological responses that promote reproductive success. The Drosophila AG is composed of two main cell types. The majority of AG cells, which are referred to as main cells, are responsible for production of many seminal fluid proteins. A minority of cells, about 4%, are referred to as secondary cells. These cells, which are restricted to the distal tip of the D. melanogaster AG, may play an especially important role in the maintenance of the long-term female post-mating response. Many studies of Drosophila AG evolution have suggested that the proteins produced in the gland evolve quickly, as does the transcriptome. Here, we investigate the evolution of secondary cell number and position in the AG in a collection of eight species spanning the entire history of the Drosophila genus. We document a heretofore underappreciated rapid evolutionary rate for both number and position of these specialized AG cells, raising several questions about the developmental, functional, and evolutionary significance of this variation.

Population biology of accessory gland-expressed de novo genes in Drosophila melanogaster.

Early work on de novo gene discovery in Drosophila was consistent with the idea that many such genes have male-biased patterns of expression, including a large number expressed in the testis. However, there has been little formal analysis of variation in the abundance and properties of de novo genes expressed in different tissues. Here, we investigate the population biology of recently evolved de novo genes expressed in the Drosophila melanogaster accessory gland, a somatic male tissue that plays an important role in male and female fertility and the post mating response of females, using the same collection of inbred lines used previously to identify testis-expressed de novo genes, thus allowing for direct cross tissue comparisons of these genes in two tissues of male reproduction. Using RNA-seq data, we identify candidate de novo genes located in annotated intergenic and intronic sequence and determine the properties of these genes including chromosomal location, expression, abundance, and coding capacity. Generally, we find major differences between the tissues in terms of gene abundance and expression, though other properties such as transcript length and chromosomal distribution are more similar. We also explore differences between regulatory mechanisms of de novo genes in the two tissues and how such differences may interact with selection to produce differences in D. melanogaster de novo genes expressed in the two tissues.

Single-nucleus transcriptomes reveal evolutionary and functional properties of cell types in the Drosophila accessory gland.

Many traits responsible for male reproduction evolve quickly, including gene expression phenotypes in germline and somatic male reproductive tissues. Rapid male evolution in polyandrous species is thought to be driven by competition among males for fertilizations and conflicts between male and female fitness interests that manifest in postcopulatory phenotypes. In Drosophila, seminal fluid proteins secreted by three major cell types of the male accessory gland and ejaculatory duct are required for female sperm storage and use, and influence female postcopulatory traits. Recent work has shown that these cell types have overlapping but distinct effects on female postcopulatory biology, yet relatively little is known about their evolutionary properties. Here, we use single-nucleus RNA-Seq of the accessory gland and ejaculatory duct from Drosophila melanogaster and two closely related species to comprehensively describe the cell diversity of these tissues and their transcriptome evolution for the first time. We find that seminal fluid transcripts are strongly partitioned across the major cell types, and expression of many other genes additionally defines each cell type. We also report previously undocumented diversity in main cells. Transcriptome divergence was found to be heterogeneous across cell types and lineages, revealing a complex evolutionary process. Furthermore, protein adaptation varied across cell types, with potential consequences for our understanding of selection on male postcopulatory traits.

Polymorphism and Divergence of Novel Gene Expression Patterns in Drosophila melanogaster.

Transcriptomes may evolve by multiple mechanisms, including the evolution of novel genes, the evolution of transcript abundance, and the evolution of cell, tissue, or organ expression patterns. Here, we focus on the last of these mechanisms in an investigation of tissue and organ shifts in gene expression in Drosophila melanogaster. In contrast to most investigations of expression evolution, we seek to provide a framework for understanding the mechanisms of novel expression patterns on a short population genetic timescale. To do so, we generated population samples of D. melanogaster transcriptomes from five tissues: accessory gland, testis, larval salivary gland, female head, and first-instar larva. We combined these data with comparable data from two outgroups to characterize gains and losses of expression, both polymorphic and fixed, in D. melanogaster We observed a large number of gain- or loss-of-expression phenotypes, most of which were polymorphic within D. melanogaster Several polymorphic, novel expression phenotypes were strongly influenced by segregating cis-acting variants. In support of previous literature on the evolution of novelties functioning in male reproduction, we observed many more novel expression phenotypes in the testis and accessory gland than in other tissues. Additionally, genes showing novel expression phenotypes tend to exhibit greater tissue-specific expression. Finally, in addition to qualitatively novel expression phenotypes, we identified genes exhibiting major quantitative expression divergence in the D. melanogaster lineage.