Sox21bunderlies the rapid diversification of a novel male genital structure betweenDrosophilaspecies

Summary The emergence and subsequent diversification of morphological novelties is a major feature of animal evolution 1–9 . However, in most cases little is known about the molecular basis of the evolution of novel structures and the genetic mechanisms underlying their diversification. The epandrial posterior lobes of the male genital arch is a novelty of some species of the Drosophila melanogaster subgroup 10–13 . The posterior lobes grasp the ovipositor of the female and then integrate between her abdominal tergites, and therefore these structures are important for copulation and species-recognition 10–12,14–17 . The posterior lobes evolved from co-option of a Hox regulated gene network from the posterior spiracles 10 and have since diversified in shape and size in the D. simulans clade in particular over the last 240,000 years driven by sexual selection 18–21 . The genetic basis of this diversification is highly polygenic but to the best of our knowledge none of the causative genes have yet been identified despite extensive mapping 22–30 . Identifying the genes underlying the diversification of these secondary sexual structures is essential to understanding the basis of changes in their morphology and the evolutionary impact on copulation and species recognition. Here, we show that the transcription factor encoded by Sox21b negatively regulates posterior lobe size during development. This is consistent with higher and expanded expression of Sox21b in D. mauritiana , which develops smaller posterior lobes compared to D. simulans . We tested this by generating reciprocal hemizygotes and confirmed that changes in Sox21b underlie posterior lobe evolution between these two species. Furthermore, we found that differences in posterior lobe size caused by the species-specific allele of Sox21b significantly affect the duration of copulation. 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 behaviour. Highlights Sox21b regulates the development of the epandrial posterior lobes, a recently evolved novel structure of some species of the Drosophila melanogaster subgroup, which has subsequently rapidly diversified in size and shape. D. mauritiana has smaller posterior lobes than D. simulans and more expansive expression of Sox21b in the developing genitalia. Using a reciprocal hemizygosity test, we show that variation in Sox21b underlies the diversification of epandrial posterior lobe size and shape between D. simulans and D. mauritiana . Behavioural tests show that the species allele of Sox21b causes differences in the duration of copulation in otherwise genetically identical backgrounds. Sox21b has evolved between D. simulans and D. mauritiana , and contributed to the divergence of a morphological novelty and copulatory behaviour between these two species.