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.

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eTOC blurb Westerman, VanKuren et al. show that butterfly wing color maps to a putative cis-regulatory element adjacent to two aristaless genes. The genes are differentially expressed between white and yellow wings and CRISPR knockout of aristaless1 causes white wings to develop yellow. Both colors have been shared among species via hybridization.

Female-limited polymorphism occurs in multiple butterfly species with Batesian mimicry. While frequency-dependent selection is often argued as the driving force behind polymorphism in Batesian mimicry systems, male preference and alternative female mating strategies may also influence the maintenance of multiple female forms. Through a series of behavioural assays with the female-limited Batesian mimetic butterfly Papilio polytes, we show that males prefer stationary mimetic females over stationary non-mimetic females, but weigh female activity levels more heavily than female wing pattern when choosing between active mimetic and active non-mimetic females. Male preference for mimetic vs. non-mimetic females is independent of male genotype at the locus responsible for the female wing pattern, the autosomal gene doublesex. However male genotype does influence their response to active females. Male emphasis on female behaviour instead of appearance may reduce sexual selection pressures on female morphology, thereby facilitating frequency-dependent natural selection due to predation risk and toxic model abundance.

Some genes have repeatedly been found to control diverse adaptations in a wide variety of organisms. Such gene reuse reveals not only the diversity of phenotypes these unique genes control but also the composition of developmental gene networks and the genetic routes available to and taken by organisms during adaptation. However, the causes of gene reuse remain unclear. A small number of large-effect Mendelian loci control a huge diversity of mimetic butterfly wing color patterns, but reasons for their reuse are difficult to identify because the genetic basis of mimicry has primarily been studied in two systems with correlated factors: female-limited Batesian mimicry in Papilio swallowtails (Papilionidae) and non-sex-limited Müllerian mimicry in Heliconius longwings (Nymphalidae). Here, we break the correlation between phylogenetic relationship and sex-limited mimicry by identifying loci controlling female-limited mimicry polymorphism Hypolimnas misippus (Nymphalidae) and non-sex-limited mimicry polymorphism in Papilio clytia (Papilionidae). The Papilio clytia polymorphism is controlled by the genome region containing the gene cortex, the classic P supergene in Heliconius numata, and loci controlling color pattern variation across Lepidoptera. In contrast, female-limited mimicry polymorphism in Hypolimnas misippus is associated with a locus not previously implicated in color patterning. Thus, although many species repeatedly converged on cortex and its neighboring genes over 120 My of evolution of diverse color patterns, female-limited mimicry polymorphisms each evolved using a different gene. Our results support conclusions that gene reuse occurs mainly within ∼10 My and highlight the puzzling diversity of genes controlling seemingly complex female-limited mimicry polymorphisms.