Butterfly wing patterns provide a rich comparative framework to study how morphological complexity develops and evolves. Here we used CRISPR/Cas9 somatic mutagenesis to test a patterning role for , a signaling ligand gene previously identified as a hotspot of shape-tuning alleles involved in wing mimicry. We show that loss-of-function causes multiple modifications of pattern elements in seven nymphalid butterfly species. In three butterflies with a conserved wing-pattern arrangement, is necessary for the induction of stripe-like patterns known as symmetry systems and acquired a novel eyespot activator role specific to forewings. In two species, specifies the boundaries between melanic fields and the light-color patterns that they contour. In the passionvine butterfly , removal shows opposite effects on adjacent pattern elements, revealing a dual role across the wing field. Finally, acquired a divergent role in the patterning of interveinous patterns in the monarch, a basal nymphalid butterfly that lacks stripe-like symmetry systems. These results identify as an instructive signal for the prepatterning of a biological system of exuberant diversity and illustrate how shifts in the deployment and effects of a single developmental gene underlie morphological change.

The gene has been implicated in butterfly wing pattern adaptation by genetic association, mapping, and expression studies. The actual developmental function of this gene has remained unclear, however. Here we used CRISPR/Cas9 genome editing to show that plays a fundamental role in nymphalid butterfly wing pattern development, where it is required for determination of all chromatic coloration. knockouts in four species show complete replacement of color pigments with melanins, with corresponding changes in pigment-related gene expression, resulting in black and gray butterflies. We also show that simultaneously acts as a switch gene for blue structural iridescence in some butterflies, demonstrating simple regulatory coordination of structural and pigmentary coloration. Remarkably, these knockouts phenocopy the recurring "black and blue" wing pattern archetype that has arisen on many independent occasions in butterflies. Here we demonstrate a simple genetic basis for structural coloration, and show that plays a deeply conserved role in butterfly wing pattern development.

Butterfly eyespot colour patterns are a key example of how a novel trait can appear in association with the co-option of developmental patterning genes. Little is known, however, about how, or even whether, co-opted genes function in eyespot development. Here we use CRISPR/Cas9 genome editing to determine the roles of two co-opted transcription factors that are expressed during early eyespot determination. We found that deletions in a single gene, spalt, are sufficient to reduce or completely delete eyespot colour patterns, thus demonstrating a positive regulatory role for this gene in eyespot determination. Conversely, and contrary to previous predictions, deletions in Distal-less (Dll) result in an increase in the size and number of eyespots, illustrating a repressive role for this gene in eyespot development. Altogether our results show that the presence, absence and shape of butterfly eyespots can be controlled by the activity of two co-opted transcription factors.

Color pattern mimicry in Heliconius butterflies is a classic case study of complex trait adaptation via selection on a few large effect genes. Association studies have linked color pattern variation to a handful of noncoding regions, yet the presumptive cis-regulatory elements (CREs) that control color patterning remain unknown. Here we combine chromatin assays, DNA sequence associations, and genome editing to functionally characterize 5 cis-regulatory elements of the color pattern gene optix. We were surprised to find that the cis-regulatory architecture of optix is characterized by pleiotropy and regulatory fragility, where deletion of individual cis-regulatory elements has broad effects on both color pattern and wing vein development. Remarkably, we found orthologous cis-regulatory elements associate with wing pattern convergence of distantly related comimics, suggesting that parallel coevolution of ancestral elements facilitated pattern mimicry. Our results support a model of color pattern evolution in Heliconius where changes to ancient, multifunctional cis-regulatory elements underlie adaptive radiation.

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