Doublesexmediates the development of sex-specific pheromone organs inBicyclusbutterflies via multiple mechanisms

TheBicycluslineage of satyrid butterflies exhibits male-specific traits, the scent organ complex, used for chemical communication during courtship. This complex consists of tightly packed brush-like scales (hair-pencils) that rub against scent patches to disperse pheromones, but the evolution and molecular basis of the organ’s male-limited development remains unknown. Here, we examine the evolution of the number and location of the scent patches and hair-pencils within 53 species ofBicyclusbutterflies, and the involvement of the sex determinant genedoublesex (dsx)in scent organ development inBicyclus anynanausing CRISPR/Cas9. We show that scent patches and hair-pencils arose via multiple, independent gains, in a correlated manner. Further, an initially non-sex-specific Dsx protein expression pattern in developing wing discs becomes male-specific and spatially refined to areas that develop the scent organ complex over the course of development. Functional perturbations ofdsxshow that this gene is required for male patch development whereas hair-pencils can develop in both sexes without Dsx input. Dsx in females is, instead, required to repress hair-pencils. These findings suggest that the patches and hair-pencils evolve as correlated composite organs that are sex-limited via the spatial regulation ofdsx. Divergence in the function ofdsxisoforms occurs in both sexes, where the male isoform promotes patch development in males and the female isoform represses hair-pencil development in females, both leading to the development of male-limited traits. Furthermore, evolution in number and location of patches, but not of hair-pencils, appears to be regulated by spatial regulation ofdsx.