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

Abstract

In <i>Heliconius</i> butterflies, wing colour pattern diversity and scale types are 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, <i>cortex</i>, has been repeatedly associated with colour pattern evolution in butterflies. Here we carried out CRISPR knockouts in multiple <i>Heliconius</i> species and show that <i>cortex</i> is a major determinant of scale cell identity. Chromatin accessibility profiling and introgression scans identified <i>cis</i>-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 the <i>H. melpomene/timareta</i> lineage, the candidate CRE from yellow-barred phenotype morphs is interrupted by a transposable element, suggesting that <i>cis</i>-regulatory structural variation underlies these mimetic adaptations. Our work shows that <i>cortex</i> functionally controls scale colour fate and that its <i>cis</i>-regulatory regions control a phenotypic switch in a modular and pattern-specific fashion.