Resolving between novelty and homology in the rapidly evolving phallus of Drosophila.

The genitalia present some of the most rapidly evolving anatomical structures in the animal kingdom, possessing a variety of parts that can distinguish recently diverged species. In the Drosophila melanogaster group, the phallus is adorned with several processes, pointed outgrowths, that are similar in size and shape between species. However, the complex three-dimensional nature of the phallus can obscure the exact connection points of each process. Previous descriptions based upon adult morphology have primarily assigned phallic processes by their approximate positions in the phallus and have remained largely agnostic regarding their homology relationships. In the absence of clearly identified homology, it can be challenging to model when each structure first evolved. Here, we employ a comparative developmental analysis of these processes in eight members of the melanogaster species group to precisely identify the tissue from which each process forms. Our results indicate that adult phallic processes arise from three pupal primordia in all species. We found that in some cases the same primordia generate homologous structures whereas in other cases, different primordia produce phenotypically similar but remarkably non-homologous structures. This suggests that the same gene regulatory network may have been redeployed to different primordia to induce phenotypically similar traits. Our results highlight how traits diversify and can be redeployed, even at short evolutionary scales.

Modular tissue-specific regulation of doublesex underpins sexually dimorphic development in Drosophila.

The ability of a single genome to produce distinct and often dramatically different male and female forms is one of the wonders of animal development. In Drosophila melanogaster, most sexually dimorphic traits are controlled by sex-specific isoforms of the doublesex (dsx) transcription factor, and dsx expression is mostly limited to cells that give rise to sexually dimorphic traits. However, it is unknown how this mosaic of sexually dimorphic and monomorphic organs arises. Here, we characterize the cis-regulatory sequences that control dsx expression in the foreleg, which contains multiple types of sex-specific sensory organs. We find that separate modular enhancers are responsible for dsx expression in each sexually dimorphic organ. Expression of dsx in the sex comb is co-regulated by two enhancers with distinct spatial and temporal specificities that are separated by a genitalia-specific enhancer. The sex comb-specific enhancer from D. willistoni, a species that primitively lacks sex combs, is not active in the foreleg. Thus, the mosaic of sexually dimorphic and monomorphic organs depends on modular regulation of dsx transcription by dedicated cell type-specific enhancers.

Rapid Evolution of Gained Essential Developmental Functions of a Young Gene via Interactions with Other Essential Genes.

New genes are of recent origin and only present in a subset of species in a phylogeny. Accumulated evidence suggests that new genes, like old genes that are conserved across species, can also take on important functions and be essential for the survival and reproductive success of organisms. Although there are detailed analyses of the mechanisms underlying new genes' gaining fertility functions, how new genes rapidly become essential for viability remains unclear. We focused on a young retro-duplicated gene (CG7804, which we named Cocoon) in Drosophila that originated between 4 and 10 Ma. We found that, unlike its evolutionarily conserved parental gene, Cocoon has evolved under positive selection and accumulated many amino acid differences at functional sites from the parental gene. Despite its young age, Cocoon is essential for the survival of Drosophila melanogaster at multiple developmental stages, including the critical embryonic stage, and its expression is essential in different tissues from those of its parental gene. Functional genomic analyses found that Cocoon acquired unique DNA-binding sites and has a contrasting effect on gene expression to that of its parental gene. Importantly, Cocoon binding predominantly locates at genes that have other essential functions and/or have multiple gene-gene interactions, suggesting that Cocoon acquired novel essential function to survival through forming interactions that have large impacts on the gene interaction network. Our study is an important step toward deciphering the evolutionary trajectory by which new genes functionally diverge from parental genes and become essential.

Co-evolving wing spots and mating displays are genetically separable traits in Drosophila.

The evolution of sexual traits often involves correlated changes in morphology and behavior. For example, in Drosophila, divergent mating displays are often accompanied by divergent pigment patterns. To better understand how such traits co-evolve, we investigated the genetic basis of correlated divergence in wing pigmentation and mating display between the sibling species Drosophila elegans and Drosophila gunungcola. Drosophila elegans males have an area of black pigment on their wings known as a wing spot and appear to display this spot to females by extending their wings laterally during courtship. By contrast, D. gunungcola lost both of these traits. Using Multiplexed Shotgun Genotyping (MSG), we identified a ∼440 kb region on the X chromosome that behaves like a genetic switch controlling the presence or absence of male-specific wing spots. This region includes the candidate gene optomotor-blind (omb), which plays a critical role in patterning the Drosophila wing. The genetic basis of divergent wing display is more complex, with at least two loci on the X chromosome and two loci on autosomes contributing to its evolution. Introgressing the X-linked region affecting wing spot development from D. gunungcola into D. elegans reduced pigmentation in the wing spots but did not affect the wing display, indicating that these are genetically separable traits. Consistent with this observation, broader sampling of wild D. gunungcola populations confirmed that the wing spot and wing display are evolving independently: some D. gunungcola males performed wing displays similar to D. elegans despite lacking wing spots. These data suggest that correlated selection pressures rather than physical linkage or pleiotropy are responsible for the coevolution of these morphological and behavioral traits. They also suggest that the change in morphology evolved prior to the change in behavior.

An Atlas of Transcription Factors Expressed in Male Pupal Terminalia of Drosophila melanogaster.

During development, transcription factors and signaling molecules govern gene regulatory networks to direct the formation of unique morphologies. As changes in gene regulatory networks are often implicated in morphological evolution, mapping transcription factor landscapes is important, especially in tissues that undergo rapid evolutionary change. The terminalia (genital and anal structures) of Drosophila melanogaster and its close relatives exhibit dramatic changes in morphology between species. While previous studies have identified network components important for patterning the larval genital disc, the networks governing adult structures during pupal development have remained uncharted. Here, we performed RNA-seq in whole Drosophila melanogaster male terminalia followed by in situ hybridization for 100 highly expressed transcription factors during pupal development. We find that the male terminalia are highly patterned during pupal stages and that specific transcription factors mark separate structures and substructures. Our results are housed online in a searchable database (https://flyterminalia.pitt.edu/) as a resource for the community. This work lays a foundation for future investigations into the gene regulatory networks governing the development and evolution of Drosophila terminalia.

Effect of a refuge from persistent male courtship in the Drosophila laboratory environment.

The Drosophila melanogaster laboratory model has been used extensively in studies of sexual conflict because during the process of courtship and mating, males impose several costs upon females (e.g., reduced fecundity). One important difference between the laboratory and the wild is that females in the laboratory lack a spatial refuge from persistent male courtship. Here, we describe two experiments that examine the potential consequences of a spatial refuge for females. In the first experiment, we examined the influence of a spatial refuge on mating rate of females, and in the second one we examined its influence on females' lifetime fecundity. We found that females mated about 25% less often when a spatial refuge was available, but that the absence of a spatial refuge did not substantially increase the level of male-induced harm to females (i.e., sexual conflict).