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Rapid divergence in genital structures among nascent species has been posited to be an early-evolving cause of reproductive isolation, although evidence supporting this idea as a widespread phenomenon remains mixed. Using a collection of interspecific introgression lines between twoDrosophilaspecies that diverged ∼240,000 years ago, we tested the hypothesis that even modest divergence in genital morphology can result in substantial fitness losses. We studied the reproductive consequences of variation in the male epandrial posterior lobes betweenDrosophila mauritianaandD. sechelliaand found that divergence in posterior lobe morphology has significant fitness costs on several pre-fertilization and post-copulatory reproductive measures. Males with divergent posterior lobe morphology also significantly reduced the life span of their mates. Interestingly, one of the consequences of genital divergence was decreased oviposition and fertilization, which suggests that a sensory bias for posterior lobe morphology could exist in females, and thus posterior lobe morphology may be the target of cryptic female choice in these species. Our results provide evidence that divergence in genitalia can in fact give rise to substantial reproductive isolation early during species divergence, and they also reveal novel reproductive functions of the external male genitalia inDrosophila.

International audience; Animal terminalia represent some of the most diverse and rapidly evolving structures in the animal kingdom, and for this reason have been a mainstay in the taxonomic description of species. The terminalia of Drosophila melanogaster, with its wide range of experimental tools, have recently become the focus of increased interest in the fields of development, evolution, and behavior. However, studies from different disciplines have often used discrepant terminologies for the same anatomical structures. Consequently, the terminology of genital parts has become a barrier to integrating results from different fields, rendering it difficult to determine what parts are being referenced. We formed a consortium of researchers studying the genitalia of D. melanogaster to help establish a set of naming conventions. Here, we present a detailed visual anatomy of male genital parts, including a list of synonymous terms, and suggest practices to avoid confusion when referring to anatomical parts in future studies. The goal of this effort is to facilitate interdisciplinary communication and help newcomers orient themselves within the exciting field of Drosophila genitalia. ARTICLE HISTORY

External male reproductive structures have received considerable attention as a cause of reproductive isolation (RI), because the morphology of these structures often evolves rapidly between populations. This rapid evolution presents the potential for mechanical incompatibilities with heterospecific female structures during mating and could thus prevent interbreeding between nascent species. Although such mechanical incompatibilities have received little empirical support as a common cause of RI, the potential for mismatch of reproductive structures to cause RI due to incompatible species-specific tactile cues has not been tested. We tested the importance of mechanical and tactile incompatibilities in RI between Enallagma anna and E. carunculatum, two damselfly species that diverged within the past ∼250,000 years and currently hybridize in a sympatric region. We quantified 19 prezygotic and postzygotic RI barriers using both naturally occurring and laboratory-reared damselflies. We found incomplete mechanical isolation between the two pure species and between hybrid males and pure species females. Interestingly, in mating pairs for which mechanical isolation was incomplete, females showed greater resistance and refusal to mate with hybrid or heterospecific males compared to conspecific males. This observation suggests that tactile incompatibilities involving male reproductive structures can influence female mating decisions and form a strong barrier to gene flow in early stages of speciation.

The external genitalia are some of the most rapidly evolving morphological structures in insects. The posterior lobe of the male genital arch shows striking differences in both size and shape among closely related species of the Drosophila melanogaster species subgroup. Here, we dissect the genetic basis of posterior lobe morphology between D. mauritiana and D. sechellia, two island endemic species that last shared a common ancestor ∼300,000 years ago. We test a large collection of genome-wide homozygous D. mauritiana genetic introgressions, which collectively cover ∼50% of the genome, for their morphological effects when placed in a D. sechellia genetic background. We find several introgressions that have large effects on posterior lobe morphology and that posterior lobe size and posterior lobe shape can be separated genetically for some of the loci that specify morphology. Using next generation sequencing technology, we perform whole transcriptome gene expression analyses of the larval genital imaginal disc of D. mauritiana, D. sechellia, and two D. mauritiana–D. sechellia hybrid introgression genotypes that each have large effects on either posterior lobe size or posterior lobe shape. Many of the genes we identify as differentially expressed are expressed at levels similar to D. mauritiana in one introgression hybrid, but are expressed at levels similar to D. sechellia in the other introgression hybrid. However, we also find that both introgression hybrids express some of the same genes at levels similar to D. mauritiana, and notably, that both introgression hybrids possess genes in the insulin receptor signaling pathway, which are expressed at D. mauritiana expression levels. These results suggest the possibility that the insulin signaling pathway might integrate size and shape genetic inputs to establish differences in overall posterior lobe morphology between D. mauritiana and D. sechellia.

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.

Although several evolutionary forces have been proposed to contribute to genital morphological diversification, it is unclear which might act early during the evolution of novel structural traits. We test the hypothesis that mismatch between interacting male and female secondary sexual structures gives rise to increased harm to females, consistent with the outcome predicted from a history of sexual conflict. We mate Drosophila sechellia females to males from a collection of D. mauritiana-D. sechellia interspecific genetic introgression lines that possess quantitative morphological variation in the posterior lobe of the genital arch, an external genital structure that can cause wounds to the female abdomen during mating. We find that males with smaller posterior lobes, and those that possess lobes with similarities in shape to D. mauritiana, cause more severe wounding compared to either D. sechellia males with strain-specific morphologies or introgression males that possess larger lobes or lobes with more pronounced D. sechellia features. These results suggest a possible history of sexual conflict during the evolution of the posterior lobe in D. sechellia, but also suggest a potential contribution of divergence in sensory recognition mechanisms to posterior lobe evolution.

Postzygotic reproductive isolation is characterized by two striking empirical patterns. The first is Haldane's rule—the preferential inviability or sterility of species hybrids of the heterogametic (XY) sex. The second is the so-called large X effect—substitution of one species's X chromosome for another's has a disproportionately large effect on hybrid fitness compared to similar substitution of an autosome. Although the first rule has been well-established, the second rule remains controversial. Here, we dissect the genetic causes of these two rules using a genome-wide introgression analysis of Drosophila mauritiana chromosome segments in an otherwise D. sechellia genetic background. We find that recessive hybrid incompatibilities outnumber dominant ones and that hybrid male steriles outnumber all other types of incompatibility, consistent with the dominance and faster-male theories of Haldane's rule, respectively. We also find that, although X-linked and autosomal introgressions are of similar size, most X-linked introgressions cause hybrid male sterility (60%) whereas few autosomal introgressions do (18%). Our results thus confirm the large X effect and identify its proximate cause: incompatibilities causing hybrid male sterility have a higher density on the X chromosome than on the autosomes. We evaluate several hypotheses for the evolutionary cause of this excess of X-linked hybrid male sterility., Author Summary The evolution of reproductive isolation is a fundamental step in the origin of species. One kind of reproductive isolation, the sterility and inviability of species hybrids, is characterized by two of the strongest rules in evolutionary biology. The first is Haldane's rule: for species crosses in which just one hybrid sex is sterile or inviable, it tends to be the sex defined by having a pair of dissimilar sex chromosomes (e.g., the “XY” of males in humans). The second rule is the large X effect: the X chromosome has a disproportionately large effect on hybrid fitness. We dissected the genetic causes of these two rules of speciation by replacing many small chromosomal segments of the fruit fly Drosophila sechellia with those of a closely related species, D. mauritiana. Together, these segments cover 70% of the genome. We found that virtually all segments causing hybrid sterility or inviability act recessively and that hybrid male sterility is by far the most common type of hybrid incompatibility, confirming two leading theories about the causes of Haldane's rule. We also found that X-linked segments are more likely to cause hybrid male sterility than similarly sized autosomal segments. These results show that the large X effect is caused by a higher density of hybrid incompatibilities on the X chromosome., A genome-wide introgression analysis of Drosophila mauritiana chromosome segments in an otherwise D. sechellia genetic background confirms the large X effect, a cornerstone of speciation theory, and reveals its cause.

We describe reproductive isolation caused by a gene transposition. In certain Drosophila melanogaster – D. simulans hybrids, hybrid male sterility is caused by the lack of a single-copy gene essential for male fertility, JYAlpha . This gene is located on the fourth chromosome of D. melanogaster but on the third chromosome of D. simulans . Genomic and molecular analyses show that JYAlpha transposed to the third chromosome during the evolutionary history of the D. simulans lineage. Because of this transposition, a fraction of hybrids completely lack JYAlpha and are sterile, representing reproductive isolation without sequence evolution.

Interspecific hybrids and backcrossed organisms generally suffer from reduced viability and/or fertility. To identify and genetically map these defects, we introgressed regions of the Drosophila sechellia genome into the D. simulans genome. A female-biased sex ratio was observed in 24 of the 221 recombinant inbred lines, and subsequent tests attributed the skew to failure of Y-bearing sperm to fertilize the eggs. Apparently these introgressed lines fail to suppress a normally silent meiotic drive system. Using molecular markers we mapped two regions of the Drosophila genome that appear to exhibit differences between D. simulans and D. sechellia in their regulation of sex chromosome segregation distortion. The data indicate that the sex ratio phenotype results from an epistatic interaction between at least two factors. We discuss whether this observation is relevant to the meiotic drive theory of hybrid male sterility.