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

6Parallel evolution of ecotypes occurs when selection independently drives the evolution of 7 similar traits across similar environments. The multiple origin of ecotypes is often inferred on 8 the basis of a phylogeny which clusters populations according to geographic location and not 9 by the environment they occupy. In contrast, when ecotypes arise once, expand their range 10 and colonise similar environments, their populations cluster by ecology and not geography. 11

Parallel evolution of ecotypes occurs when selection independently drives the evolution of similar traits across similar environments. The multiple origins of ecotypes are often inferred based on a phylogeny that clusters populations according to geographic location and not by the environment they occupy. However, the use of phylogenies to infer parallel evolution in closely related populations is problematic because gene flow and incomplete lineage sorting can uncouple the genetic structure at neutral markers from the colonization history of populations. Here, we demonstrate multiple origins within ecotypes of an Australian wildflower, Senecio lautus. We observed strong genetic structure as well as phylogenetic clustering by geography and show that this is unlikely due to gene flow between parapatric ecotypes, which was surprisingly low. We further confirm this analytically by demonstrating that phylogenetic distortion due to gene flow often requires higher levels of migration than those observed in S. lautus. Our results imply that selection can repeatedly create similar phenotypes despite the perceived homogenizing effects of gene flow.

22The wing patterns of butterflies are an excellent system with which to study phenotypic evolution. The 23 incredibly diverse patterns are generated from an array of pigmented scales on a largely two-24 dimensional surface, resulting in a visibly tractable system for studying the evolution of pigmentation. 25In Heliconius butterflies, much of this diversity is controlled by a few genes of large effect that regulate 26 pattern switches between races and species across a large mimetic radiation. One of these genescortex 27 -has been repeatedly mapped in association with colour pattern evolution in both Heliconius and other 28Lepidoptera, but we lack functional data supporting its role in modulating wing patterns. Here we 29 carried out CRISPR knock-outs in multiple Heliconius species and show that cortex is a major 30 determinant of scale cell identity. Mutant wing clones lacking cortex showed shifts in colour identity, 31 with melanic and red scales acquiring a yellow or white state. These homeotic transformations include 32 changes in both pigmentation and scale ultrastructure, suggesting that cortex acts during early stages of 33 scale cell fate specification rather than during the deployment of effector genes. In addition, mutant 34 clones were observed across the entire wing surface, contrasting with other known Heliconius mimicry 35 loci that act in specific patterns. Cortex is known as a cell-cycle regulator that modulates mitotic entry 36in Drosophila, and we found the Cortex protein to accumulate in the nuclei of the polyploid scale 37 building cells of the butterfly wing epithelium, speculatively suggesting a connection between scale cell 38 endocycling and colour identity. In summary, and while its molecular mode of action remains 39 mysterious, we conclude that cortex played key roles in the diversification of lepidopteran wing patterns 40 in part due to its switch-like effects in scale identity across the entire wing surface. 41 42 3

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