Macroevolutionary shifts of <i>WntA</i> function potentiate butterfly wing-pattern diversity

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 <i>WntA</i>, a signaling ligand gene previously identified as a hotspot of shape-tuning alleles involved in wing mimicry. We show that <i>WntA</i> loss-of-function causes multiple modifications of pattern elements in seven nymphalid butterfly species. In three butterflies with a conserved wing-pattern arrangement, <i>WntA</i> is necessary for the induction of stripe-like patterns known as symmetry systems and acquired a novel eyespot activator role specific to <i>Vanessa</i> forewings. In two <i>Heliconius</i> species, <i>WntA</i> specifies the boundaries between melanic fields and the light-color patterns that they contour. In the passionvine butterfly <i>Agraulis</i>, <i>WntA</i> removal shows opposite effects on adjacent pattern elements, revealing a dual role across the wing field. Finally, <i>WntA</i> 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 <i>WntA</i> 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.