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In the last 240,000 years, males of the Drosophila simulans species clade have evolved striking differences in the morphology of their epandrial posterior lobes and claspers (surstyli). These appendages are used for grasping the female during mating and so their divergence is most likely driven by sexual selection. Mapping studies indicate a highly polygenic and generally additive genetic basis for these morphological differences. However, we have limited understanding of the gene regulatory networks that control the development of genital structures and how they evolved to result in this rapid phenotypic diversification. Here, we used new D. simulans/D. mauritiana introgression lines on chromosome arm 3L to generate higher resolution maps of posterior lobe and clasper differences between these species. We then carried out RNA-seq on the developing genitalia of both species to identify the expressed genes and those that are differentially expressed between the two species. This allowed us to test the function of expressed positional candidates during genital development in D. melanogaster. We identified several new genes involved in the development and possibly the evolution of these genital structures, including the transcription factors Hairy and Grunge. Furthermore, we discovered that during clasper development Hairy negatively regulates tartan (trn), a gene known to contribute to divergence in clasper morphology. Taken together, our results provide new insights into the regulation of genital development and how this has evolved between species.

The spatiotemporal regulation of gene expression is essential to ensure robust phenotypic outcomes. Pigmentation patterns in Drosophila are determined by pigments biosynthesized in the developing epidermis and the cis-regulatory elements of the genes involved in this process are well-characterized. Here, we report that the known primary epidermal enhancer is dispensable for the transcriptional activation of ebony (involved in light-colored pigment synthesis) in the developing epidermis of Drosophila melanogaster. The evidence was obtained by introducing an approximately 1 kbp deletion at the primary epidermal enhancer by genome editing. The effect of the primary epidermal enhancer deletion on pigmentation and on the endogenous expression pattern of a mCherry-fused ebony allele was examined in the abdomen. The expression levels of the mCherry-fused ebony in the primary epidermal enhancer-deleted strains were slightly higher than that of the control strain, indicating that the sequences outside the primary epidermal enhancer have an ability to drive an expression of this gene in the epidermis. Interestingly, the primary epidermal enhancer deletion resulted in a derepression of this gene in the dorsal midline of the abdominal tergites, where dark pigmentation is present in the wild-type individuals. This indicated that the primary epidermal enhancer fragment contains a silencer. Furthermore, the endogenous expression pattern of ebony in the 2 additional strains with partially deleted primary epidermal enhancer revealed that the silencer resides within a 351-bp fragment in the 5' portion of the primary epidermal enhancer. These results demonstrated that deletion assays combined with reporter assays are highly effective in detecting the presence of positively and negatively regulating sequences within and outside the focal cis-regulatory elements.

Significance The morphology of male genitalia evolves rapidly, probably driven by sexual selection. However, little is known about the genes underlying genitalia differences between species. Identifying these genes is key to understanding how sexual selection acts to produce rapid phenotypic change. We have found that the gene tartan underlies differences between male Drosophila mauritiana and Drosophila simulans in the size and bristle number of the claspers—genital projections that grasp the female during copulation. Moreover, since tartan encodes a protein that is involved in cell interactions, this may represent an alternative developmental mechanism for morphological change. Therefore, our study provides insights into the genetic and developmental bases for the rapid evolution of male genitalia and organ size more generally., Male genital structures are among the most rapidly evolving morphological traits and are often the only features that can distinguish closely related species. This process is thought to be driven by sexual selection and may reinforce species separation. However, while the genetic bases of many phenotypic differences have been identified, we still lack knowledge about the genes underlying evolutionary differences in male genital organs and organ size more generally. The claspers (surstyli) are periphallic structures that play an important role in copulation in insects. Here, we show that divergence in clasper size and bristle number between Drosophila mauritiana and Drosophila simulans is caused by evolutionary changes in tartan (trn), which encodes a transmembrane leucine-rich repeat domain protein that mediates cell–cell interactions and affinity. There are no fixed amino acid differences in trn between D. mauritiana and D. simulans, but differences in the expression of this gene in developing genitalia suggest that cis-regulatory changes in trn underlie the evolution of clasper morphology in these species. Finally, analyses of reciprocal hemizygotes that are genetically identical, except for the species from which the functional allele of trn originates, determined that the trn allele of D. mauritiana specifies larger claspers with more bristles than the allele of D. simulans. Therefore, we have identified a gene underlying evolutionary change in the size of a male genital organ, which will help to better understand not only the rapid diversification of these structures, but also the regulation and evolution of organ size more broadly.

Mechanical incompatibility of male and female genitalia is common in animals with internal fertilization. However, our knowledge regarding the precise mechanisms is limited. One key question regards the susceptibility of the match between male and female genitalia to morphological modification. To address this issue, we generated six different second-chromosome introgression lines possessing partially Drosophila mauritiana-like genital morphology in multiple structures in D. simulans background. Three of the six introgression males showed elevated mobility at some stages during copulation with D. simulans females; this was assumed to be an indication of genital mismatch. Notably, one of the introgression males with D. mauritiana-like enlarged anal plates showed occasional leakage of adhesive ejaculate on the body surface when mated with pure D. simulans females, suggesting apparent structural incompatibility in genital coupling. These observations suggested that both sexual and natural selection shape the anal plate morphology, highlighting the role of this structure as an important component of mechanical isolation. Partial replacement (introgression) by a sibling species genome can induce perturbations in genital coupling mechanics, suggesting that genital compatibility can be susceptible to subtle genomic changes at the early stages of divergence in these species.

SummarySegmental duplications are enriched within many eukaryote genomes, and their potential consequence is gene duplication. While previous theoretical studies of gene duplication have mainly focused on the gene silencing process after fixation, the process leading to fixation is even more important for segmental duplications, because the majority of duplications would be lost before reaching a significant frequency in a population. Here, by a series of computer simulations, we show that purifying selection against loss-of-function mutations increases the fixation probability of a new duplicate gene, especially when the gene is haplo-insufficient. Theoretically, the probability of simultaneous preservation of both duplicate genes becomes twice the loss-of-function mutation rate (uc) when the population size (N), the degree of dominance of mutations (h) and the recombination rate between the duplicate genes (c) are all sufficiently large (Nuc>1,h>0·1 andc>uc). The preservation probability declines rapidly withhand becomes 0 whenh=0 (haplo-sufficiency). We infer that masking deleterious loss-of-function mutations give duplicate genes an immediate selective advantage and, together with effects of increased gene dosage, would predominantly determine the fates of the duplicate genes in the early phase of their evolution.

Male sexual characters are often among the first traits to diverge between closely related species and identifying the genetic basis of such changes can contribute to our understanding of their evolutionary history. However, little is known about the genetic architecture or the specific genes underlying the evolution of male genitalia. The morphology of the claspers, posterior lobes, and anal plates exhibit striking differences between Drosophila mauritiana and D. simulans. Using QTL and introgression-based high-resolution mapping, we identified several small regions on chromosome arms 3L and 3R that contribute to differences in these traits. However, we found that the loci underlying the evolution of clasper differences between these two species are independent from those that contribute to posterior lobe and anal plate divergence. Furthermore, while most of the loci affect each trait in the same direction and act additively, we also found evidence for epistasis between loci for clasper bristle number. In addition, we conducted an RNAi screen in D. melanogaster to investigate if positional and expression candidate genes located on chromosome 3L, are also involved in genital development. We found that six of these genes, including components of Wnt signaling and male-specific lethal 3 (msl3), regulate the development of genital traits consistent with the effects of the introgressed regions where they are located and that thus represent promising candidate genes for the evolution these traits.

Cell death is a mechanism utilized by organisms to eliminate excess cells during development. Here, we describe a novel regulator of caspase-independent cell death, Mabiki (Mabi), that is involved in the repair of the head patterning defects caused by extra copies of bicoid in Drosophila melanogaster. Mabiki functions together with caspase-dependent cell death mechanisms to provide robustness during development.

Eye and head morphology vary considerably among insects and even between closely related species of Drosophila. Species of the D. melanogaster subgroup, and other Drosophila species, exhibit a negative correlation between eye size and face width (FW); for example, D. mauritiana generally has bigger eyes composed of larger ommatidia and conversely a narrower face than its sibling species. To better understand the evolution of eye and head morphology, we investigated the genetic and developmental basis of differences in eye size and FW between male D. mauritiana and D. simulans. QTL mapping of eye size and FW showed that the major loci responsible for the interspecific variation in these traits are localized to different genomic regions. Introgression of the largest effect QTL underlying the difference in eye size resulted in flies with larger eyes but no significant difference in FW. Moreover,introgression of a QTL region on the third chromosome that contributes to the FW difference between these species affected FW, but not eye size. We also observed that this difference in FW is detectable earlier in the development of the eye‐antennal disc than the difference in the size of the retinal field. Our results suggest that different loci that act at different developmental stages underlie changes in eye size and FW. Therefore, while there is a negative correlation between these traits in Drosophila, we show genetically that they also have the potential to evolve independently and this may help to explain the evolution of these traits in other insects. peerReviewed

Natural selection is assumed to act more strongly on X-linked loci than on autosomal loci because the fitness effect of a recessive mutation on the X chromosome is fully expressed in hemizygous males. Therefore, selection is expected to fix or remove recessive mutations on the X chromosome more efficiently than those on autosomes. However, the assumption that hemizygosity of the X chromosome selectively accelerates changes in allele frequency has not been confirmed directly. To examine this assumption, we investigated current natural selection on X-linked chemoreceptor genes in a natural population of Drosophila melanogaster by comparing nucleotide diversity, linkage disequilibrium (LD), and departure from the neutrality in 4 chemoreceptor genes on 100 X chromosomes each from female and male flies. The general pattern of nucleotide diversity and LD for the genes investigated was similar in females and males. In contrast, males harbored significantly fewer rare polymorphisms defined as singletons and doubletons. When all the gene sequences were concatenated, Tajima's D showed a significant departure from the neutrality in both females and males, whereas Fu and Li's F* value revealed departure only in males. These results suggest that some rare polymorphisms on the X chromosome from females are recessively deleterious and are removed by stronger purifying selection when transferred to hemizygous males.