Supergene evolution via gain of autoregulation

Development requires the coordinated action of many genes across space and time, yet numerous species have evolved the ability to develop multiple discrete, alternate phenotypes^1-5. Such polymorphisms are often controlled by supergenes, sets of tightly-linked loci that function together to control development of a polymorphic phenotype^6-10. Although theories of supergene evolution are well-established, the mutations that cause functional differences between supergene alleles have been difficult to identify. The doublesex gene is a master regulator of insect sexual differentiation but has been co-opted to function as a supergene in multiple Papilio swallowtail butterflies, where divergent dsx alleles control development of discrete non-mimetic or mimetic female wing shapes and color patterns^11-15. Here we demonstrate that the Papilio alphenor supergene evolved via recruitment of six new cis-regulatory elements (CREs) that control allele-specific dsx expression. Most dsx CREs, including four of the six new CREs, are bound by the DSX transcription factor itself. Our findings provide experimental support to classic supergene theory and suggest that autoregulation may provide a simple route to supergene origination and to the co-option of pleiotropic genes into new developmental roles.