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Abstract Heliconius butterflies, 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 the Heliconius stem. 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. 

While piggyBac transposon-based transgenesis is widely used in various emerging model organisms, its relatively low transposition rate in butterflies and moths has hindered its use for routine genetic transformation in Lepidoptera. Here, we tested the suitability of a codon-optimized hyperactive piggyBac transposase ( hyPBase ) in mRNA form to deliver and integrate transgenic cassettes into the genome of the pantry moth Plodia interpunctella . Co-injection of hyPBase mRNA with donor plasmids successfully integrated 1.5–4.4 kb expression cassettes driving the fluorescent markers EGFP, DsRed, or EYFP in eyes and glia with the 3xP3 promoter. Somatic integration and expression of the transgene in the G 0 injected generation was detectable from 72-h embryos and onward in larvae, pupae and adults carrying a recessive white-eyed mutation. Overall, 2.5% of injected eggs survived into transgene-bearing adults with mosaic fluorescence. Subsequent outcrossing of fluorescent G 0 founders transmitted single-insertion copies of 3xP3::EGFP and 3xP3::EYFP and generated stable isogenic lines. Random in-crossing of a small cohort of G 0 founders expressing 3xP3::DsRed yielded a stable transgenic line segregating for more than one transgene insertion site. We discuss how hyPBase can be used to generate stable transgenic resources in Plodia and other moths. 

Ancient multifunctional regulatory elements underlie the evolution of butterfly wing color patterns. 

Butterfly color patterns provide visible and biodiverse phenotypic readouts of the patterning processes. While the secreted ligand WntA was shown to instruct the color pattern formation in butterflies, its mode of reception remains 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, fz2 clones are cell-autonomous, consistent with a morphogen function. Shifts in expression of WntA and fz2 in WntA crispant pupae show that they 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.

 

Developmental plasticity allows genomes to encode multiple distinct phenotypes that can be differentially manifested in response to environmental cues. Alternative plastic phenotypes can be selected through a process called genetic assimilation, although the mechanisms are still poorly understood. We assimilated a seasonal wing color phenotype in a naturally plastic population of butterflies ( Junonia coenia ) and characterized three responsible genes. Endocrine assays and chromatin accessibility and conformation analyses showed that the transition of wing coloration from an environmentally determined trait to a predominantly genetic trait occurred through selection for regulatory alleles of downstream wing-patterning genes. This mode of genetic evolution is likely favored by selection because it allows tissue- and trait-specific tuning of reaction norms without affecting core cue detection or transduction mechanisms. 

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. 

Lepidopteran wing scales play important roles in a number of functions including color patterning and thermoregulation. Despite the importance of wing scales, however, we still have a limited understanding of the genetic mechanisms that underlie scale patterning, development, and coloration. Here, we explore the function of the phenoloxidase‐encoding genelaccase2in wing and scale development in the nymphalid butterflyVanessa cardui. Somatic deletion mosaics oflaccase2generated by CRISPR/Cas9 genome editing presented several distinct mutant phenotypes. Consistent with the work in other nonlepidopteran insect groups, we observed reductions in melanin pigmentation and defects in cuticle formation. We were also surprised, however, to see distinct effects on scale development including complete loss of wing scales. This study highlightslaccase2as a gene that plays multiple roles in wing and scale development and provides new insight into the evolution of lepidopteran wing coloration.