Sox21b underlies the rapid diversification of a novel male genital structure between Drosophila species.

The emergence and diversification of morphological novelties is a major feature of animal evolution.1,2,3,4,5,6,7,8,9 However, relatively little is known about the genetic basis of the evolution of novel structures and the mechanisms underlying their diversification. The epandrial posterior lobes of male genitalia are a novelty of particular Drosophila species.10,11,12,13 The lobes grasp the female ovipositor and insert between her abdominal tergites and, therefore, are important for copulation and species recognition.10,11,12,14,15,16,17 The posterior lobes likely evolved from co-option of a Hox-regulated gene network from the posterior spiracles10 and have since diversified in morphology in the D. simulans clade, in particular, over the last 240,000 years, driven by sexual selection.18,19,20,21 The genetic basis of this diversification is polygenic but, to the best of our knowledge, none of the causative genes have been identified.22,23,24,25,26,27,28,29,30 Identifying the genes underlying the diversification of these secondary sexual structures is essential to understanding the evolutionary impact on copulation and species recognition. Here, we show that Sox21b negatively regulates posterior lobe size. This is consistent with expanded Sox21b expression in D. mauritiana, which develops smaller posterior lobes than D. simulans. We tested this by generating reciprocal hemizygotes and confirmed that changes in Sox21b underlie posterior lobe evolution between these species. Furthermore, we found that posterior lobe size differences caused by the species-specific allele of Sox21b significantly affect copulation duration. Taken together, our study reveals the genetic basis for the sexual-selection-driven diversification of a novel morphological structure and its functional impact on copulatory behavior.

Unraveling the Genetic Basis for the Rapid Diversification of Male Genitalia between Drosophila Species.

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.