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

The incredibly diverse colour patterns in butterflies are generated from an array of pigmented scales on a largely two-dimensional surface, resulting in a visibly tractable system for studying phenotypic evolution. In Heliconius butterflies, much of this diversity is controlled by a few genes of large effect that regulate pattern switches between races and species across a large mimetic radiation. One of these genes - cortex - has been repeatedly mapped in association with colour pattern evolution in both Heliconius and other Lepidoptera. Here we carried out CRISPR knock-outs in multiple Heliconius species and races and show that cortex is a major determinant of scale cell identity. Mutant wing clones lacking cortex showed shifts in scale colour and ultrastructure, with melanic and red scales acquiring a yellow or white state. ATAC-seq chromatin accessibility profiles, phylogenetic scans of introgression, and 4C chromosome conformations identified cis-regulatory regions associated with variations in hindwing yellow bars, situated 3' of cortex. Remarkably, CRISPR perturbation of those sites in non-barred genotypes recreated a yellow bar, revealing a spatially limited activity of these sequences. In the H. melpomene/timareta lineage, the candidate CRE from yellow-barred phenotypes morphs is interrupted by an Helitron-like transposable element, suggesting that cis-regulatory structural variation underlies mimetic adaptations found throughout this radiation. While the molecular function of Cortex remains elusive, this work shows it functionally controls scale colour fate and that its cis-regulatory regions control a phenotypic switch in a modular and pattern-specific fashion.