Binary communication systems between signaling and signal perception are frequent targets of sexual selection and, by extension, hotspots in the evolution of sexually dimorphic traits. The selective forces and genetic changes underlying such phenotypes can be studied in systems where sex‐specific signaling and perception have emerged recently and show signs of coevolution. A promising model is found in Drosophila prolongata, which exhibits a species‐specific increase in the number of gustatory bristles on the male forelegs. In my dissertation research, I found that this transition coincides with a change in cuticular hydrocarbon (CHC) profiles: D. prolongata males show increased amounts of the long-chained 9‐pentacosene (9P) and 9‐heptacosene (9H), and a coincident decrease in the shorter-chained 9‐tricosene (9T), compared to females. This dimorphism sharply contrasts with the closest relatives of D. prolongata, in which these CHCs are sexually monomorphic. Perfuming D. prolongata females with 9P or 9H reduces copulation success, suggesting that they function as sex pheromones. To identify the genetic mechanism responsible for the evolutionary transition from sexually monomorphic to dimorphic production of these mono‐alkenes, I sequenced the oenocyte transcriptomes from males and females of D. prolongata and its relative D. carrolli. The top candidate gene that emerged from this analysis is elongase F (eloF), which encodes an enzyme known to be responsible for fatty acid elongation in D. melanogaster. Knocking out this gene in D. prolongata by CRISPR dramatically reduces the production of long-chain CHCs, and leads to a partial feminization of pheromone profiles in males. The lack of ELOF protein sequence divergence but a strikingly elevated expression of this locus in D. prolongata males argue in favor of regulatory instead of structural evolution underlying pheromone divergence. I tested this hypothesis using chimeric reporter constructs that carry a combination of D. prolongata and D. carrolli sequences. I found clear evidence that sexually dimorphic eloF expression has evolved in D. prolongata through cis-regulatory changes and appears to be associated with a species-specific insertion of a putative transposable element (TE) at this locus, which I name “honghaier.” My results suggest that pheromone synthesis may have coevolved with the chemosensory perception apparatus and reveals a possible molecular mechanism for the sexually dimorphic evolution of male-female communication.

The evolution of genes de novo from nongenic sequence has become more appreciated as a mechanism of gene origin. Most studies have used few genotypes or distantly related species to infer de novo genes, which may lead to undersampling short-lived or uncommon alleles. Here, we examine the messy beginnings of de novo genes as they arise in the population. We use a comparative transcriptomics approach with an expansive pool of tissue and genotype-specific sequences to capture the characteristics of rarer alleles. To do so, we used RNA-seq in accessory gland tissue from a core set of 29 D. melanogaster genotypes, as well as additional genotypes from two of its close relatives. We show the majority of unannotated genes are lowly transcribed and lack fixed expression within species. Some rare alleles can be found in multiple species, which is hard to reconcile with models of a discrete, single new allele origin and/or fast rates of loss. As a technical consideration, the rarity of many unannotated genes may inflate the de novo classification rate, as evidence of their expression in outgroups is likely to be missed. Next, we explored whether the location of these genes may suggest a mechanism for their origin. Surprisingly, we found within tissue-specific genes, gene age was inversely correlated with proximity to other tissue-specific genes. This does not support the idea that the youngest de novo genes simply reutilize the tissue-specific regulatory elements near annotated genes. Despite this, younger de novo genes showed an enrichment for some tissue-specific binding motifs, which could indicate convergent evolution of regulatory sequence elsewhere in intergenic space.