Cortex cis-regulatory switches establish scale colour identity and pattern diversity inHeliconius

InHeliconiusbutterflies, wing pattern diversity is controlled by a few genes of large effect that regulate colour pattern switches between morphs and species across a large mimetic radiation. One of these genes,cortex, has been repeatedly associated with colour pattern evolution in butterflies. Here we carried out CRISPR knock-outs in multipleHeliconiusspecies and show thatcortexis a major determinant of scale cell identity. Chromatin accessibility profiling and introgression scans identifiedcis-regulatory regions associated with discrete phenotypic switches. CRISPR perturbation of these regions in black hindwing genotypes recreated a yellow bar, revealing their spatially limited activity. In theH. melpomene/timaretalineage, the candidate CRE from yellow-barred phenotype morphs is interrupted by a transposable element, suggesting thatcis-regulatory structural variation underlies these mimetic adaptations. Our work shows thatcortexfunctionally controls scale colour fate and that itscis-regulatory regions control a phenotypic switch in a modular and pattern-specific fashion.