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.

Mating cues evolve rapidly and can contribute to species formation and maintenance. However, little is known about how sexual signals diverge and how this variation integrates with other barrier loci to shape the genomic landscape of reproductive isolation. Here, we elucidate the genetic basis of ultraviolet (UV) iridescence, a courtship signal that differentiates the males of Colias eurytheme butterflies from a sister species, allowing females to avoid costly heterospecific matings. Anthropogenic range expansion of the two incipient species established a large zone of secondary contact across the eastern United States with strong signatures of genomic admixtures spanning all autosomes. In contrast, Z chromosomes are highly differentiated between the two species, supporting a disproportionate role of sex chromosomes in speciation known as the large-X (or large-Z) effect. Within this chromosome-wide reproductive barrier, linkage mapping indicates that cis-regulatory variation of bric a brac (bab) underlies the male UV-iridescence polymorphism between the two species. Bab is expressed in all non-UV scales, and butterflies of either species or sex acquire widespread ectopic iridescence following its CRISPR knockout, demonstrating that Bab functions as a suppressor of UV-scale differentiation that potentiates mating cue divergence. These results highlight how a genetic switch can regulate a premating signal and integrate with other reproductive barriers during intermediate phases of speciation.

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

Butterfly color patterns provide visible and biodiverse phenotypic readouts of the patterning processes that occur in a developing epithelium. While the secreted ligand WntA was shown to instruct the color pattern formation in butterflies, its modes of reception and signal transduction remain elusive. Butterfly genomes encode four homologues of the Frizzled-family of Wnt receptors. Here we show that CRISPR mosaic knock-outs of frizzled2 (fz2) phenocopy the color pattern effects of WntA loss-of-function in multiple nymphalids. While WntA mosaic clones result in intermediate patterns of reduced size, consistently with a morphogen function, fz2 clones are cell-autonomous. Shifts in pupal expression in WntA crispants show that WntA and fz2 are under positive and negative feedback, respectively. Fz1 is required for Wnt-independent planar cell polarity (PCP) in the wing epithelium. Fz3 and Fz4 show phenotypes consistent with Wnt competitive-antagonist functions in vein formation (Fz3 and Fz4), wing margin specification (Fz3), and color patterning in the Discalis and Marginal Band Systems (Fz4). Overall, these data show that the WntA/Frizzled2 morphogen-receptor pair forms a signaling axis that instructs butterfly color patterning, and shed light on the functional diversity of insect Frizzled receptors.

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