Animal genitalia are remarkably diverse, and long-standing attention has been paid for its biological causes and consequences since before Darwin. Sexual selection is currently recognized as a major driver of the evolution of male genital morphology, and evidence is growing for the role of natural selection owing to interspecific interaction and/or external environments. Coevolutionary diversification between male and female genital morphologies is found in some groups of organisms, but the evolution of female genital morphology is less understood. Recent advances in genetic (or genomic) analysis is becoming a powerful new tool for elucidating the cause of genital diversification.

Here we talk about these research frontiers in genital evolution with four case studies. Dr. Brian Langerhans will introduce functional and behavioral studies on fish genitalia in the context of natural and sexual selection and the role of genital divergence in speciation. Prof. Kensuke Nakata will report an intriguing mating behavior in spiders, female genital mutilation, and discuss its role in the evolution of female genitalia. Dr. John P. Masly will present a cutting-edge study of genes responsible for species-specific genital morphology in fruit flies. Dr. Yasuoki Takami will summarize functional aspects of ground beetle genitalia and show novel findings in recent genomic and developmental studies.

1. Genital divergence during adaptive radiation: natural selection, sexual selection, and reproductive isolation

R. Brian Langerhans (North Carolina State University)

The study of genital diversity has attracted considerable and growing interest in the past few decades. This is because at first glance genitalia seem to serve a simple purpose, but yet for animals with internal fertilization, genitalia are strikingly diverse and evolve more rapidly than perhaps any other type of trait. While much progress has been made in understanding the mechanisms that might explain this widespread pattern, here I investigate three heretofore underappreciated topics: 1) ecology's role in driving genital divergence, 2) the evolution of female genital morphology (males have received far more attention), and 3) the role of genital divergence in speciation. Examining an adaptive radiation of livebearing fishes (Gambusia hubbsi, Bahamas mosquitofish) inhabiting blue holes in The Bahamas, I find a strong role of ecology in altering both natural and sexual selection on genitalia: multiple aspects of both male and female genital morphology has repeatedly diverged between populations living with or without a major predatory fish species. Conducting laboratory experiments, we are now gaining a better understanding of how these genital morphologies function to influence mating success. Examining reproductive isolation between seven populations, we are uncovering the potential ways that rapid evolution of genital morphologies can influence speciation.

2. Female genital mutilation and its role in remating inhibition in spiders

Kensuke Nakata (Kyoto Women’s University)

In some spider species, females’ external genitalia are often found damaged. Details of this phenomenon has been examined in Cyclosa argenteoalba: a scape, a small projection on female genitalia which is essential for genital coupling, is frequently mutilated at her first mating, and mutilated females lose their re-mating ability. Such an external female genital mutilation (EFGM) may occur in about 80 other spider species, and some of them are in genus Cyclosa. A theoretical study suggests that EFGM could evolve because of the males' benefit of increased paternity share, even if EFGM imposes costs to males and females. To examine this hypothesis, I observed mating behavior in several Cyclosaspecies and compared mutilation rate, the frequency of females' second mating, and female behavior after copulation among them. Results showed that, although C. ginnagaand C. confusaconducted EFGM, mutilation rate was lower than that of C. argenteoalba. Surprisingly, although females of these two species did not mate with the second male, EFGM was not responsible for this in most cases: irrespective of being mutilated or not, mated females aggressively rejected the second male. These results suggest that securing paternity may not be important for the initial evolution of EFGM.

3. A novel gene specifies species-specific variation in a rapidly evolving genital structure

John P. Masly (University of Oklahoma)

Understanding how evolutionary forces like sexual selection and sexual conflict shape variation at the molecular level to direct rapid morphological change, such as that observed among the genitalia of internally-fertilizing species, is an important goal of evolutionary biology. Using a combination of recombination mapping and gene expression experiments, we identified a strong candidate gene for specifying species-specific morphological differences in a novel male genital structure within the Drosophila melanogaster species complex. CG14567has no known function, although its sequence features and protein domains suggest that it could be a signaling peptide. Our results from expression knock-down experiments in D. melanogasterand D. mauritianashow that CG14567possesses a similar function in both species and appears to be a negative regulator of growth. Interestingly, this gene appears to have evolved from a somewhat recent gene duplication event, and its expression level, expression pattern, and expression timing during development have diverged among species. CG14567also possesses several amino acid substitutions among the D. melanogaster complex species, although population genetic tests show no significant signal of positive natural selection in the protein-coding region. We are currently combining genome editing experiments with live-cell imaging approaches to identify how variation at CG14567gives rise to species-specific genital morphologies.

4. Causes and consequences of genital diversification in Ohomopterus ground beetles

Yasuoki Takami (Kobe University)

Remarkable diversity in genital morphology may result from combined effects of multiple selective factors. Genital morphologies of Ohomopterusground beetle species show coevolutionary diversification between the sexes. Male hook-like copulatory piece (CP) is inserted into the female vaginal appendix (VA) in copula, thereby male and female genitals are rigidly coupled. CP is used to displace rival spermatophores, and the size of CP covaries with proxies of sperm competition among species, indicating that sexual selection via sperm competition drives the diversification of CP. Sexually antagonistic selection also operates: the longer CP is more manipulative in female oviposition activitybut costly for the female due to egg dumping, while the longer VA is more resistant. Genomic analyses revealed that genes for interspecific variation in CP and VA reside in different locations within a same linkage group, implying that coevolution between the sexes is only loosely constrained and can respond to sexually antagonistic selection. As a consequence of genital diversification, coupling between divergent CP and VA inflicts fitness loss in contact zones, where reinforcing selection has resulted in further differentiation in CP and VA, i.e., reproductive character displacement. Thus, diversification of Ohomopterusgenitalia is driven by multiple selective factors, and facilitates species diversification.