Significance: The optix gene is well known for its genetic association with wing pattern variation in butterflies; however, its actual function has never been directly confirmed. Using CRISPR genome editing in multiple butterfly species, we show that this gene plays a fundamental and deeply conserved role in the butterfly family Nymphalidae, where it acts as an activator of wing color. We were also surprised to discover that optix simultaneously controls blue iridescence in some species as well, providing an example of how a single gene can act as a switch to coordinate between structural and pigmentary coloration.

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

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.

Untangling the genetics of plasticity: The common buckeye butterfly, Junonia coenia , exhibits plastic coloration; it has two color morphs, light tan and dark red, that depend on day length and temperature. By selecting for more and less color plasticity, van der Burg et al. generated butterfly lines that were used to map the genetic variants that underlie differential coloration. Genome-wide analysis and RNA sequencing identified the genes most likely to be associated with the differences in color plasticity. Inactivation of genes with CRISPR–Cas9 identified three genes that affected the red phenotype, and other techniques identified cis-regulatory, noncoding genomic variants that were correlated with coloration. From these results, the authors were able to model how genetically encoded plasticity and assimilation of the plastic trait likely evolved. Science , this issue p. 721

Heliconiusbutterflies, a speciose genus of Müllerian mimics, represent a classic example of an adaptive radiation that includes a range of derived dietary, life history, physiological and neural traits. However, key lineages within the genus, and across the broader Heliconiini tribe, lack genomic resources, limiting our understanding of how adaptive and neutral processes shaped genome evolution during their radiation. Here, we generate highly contiguous genome assemblies for nine Heliconiini, 29 additional reference-assembled genomes, and improve 10 existing assemblies. Altogether, we provide a dataset of annotated genomes for a total of 63 species, including 58 species within the Heliconiini tribe. We use this extensive dataset to generate a robust and dated heliconiine phylogeny, describe major patterns of introgression, explore the evolution of genome architecture, and the genomic basis of key innovations in this enigmatic group, including an assessment of the evolution of putative regulatory regions at theHeliconiusstem. Our work illustrates how the increased resolution provided by such dense genomic sampling improves our power to generate and test gene-phenotype hypotheses, and precisely characterize how genomes evolve.

Butterfly wing patterns derive from a deeply conserved developmental ground plan yet are diverse and evolve rapidly. It is poorly understood how gene regulatory architectures can accommodate both deep homology and adaptive change. To address this, we characterized the cis-regulatory evolution of the color pattern gene WntA in nymphalid butterflies. Comparative assay for transposase-accessible chromatin using sequencing (ATAC-seq) and in vivo deletions spanning 46 cis-regulatory elements across five species revealed deep homology of ground plan–determining sequences, except in monarch butterflies. Furthermore, noncoding deletions displayed both positive and negative regulatory effects that were often broad in nature. Our results provide little support for models predicting rapid enhancer turnover and suggest that deeply ancestral, multifunctional noncoding elements can underlie rapidly evolving trait systems.