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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. 

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. 

Although nearly phenotypically identical, few regulatory regions are conserved between two pairs of butterfly species. 

Abstract Neotropical Heliconius butterflies are well known for their intricate behaviors and multiple instances of incipient speciation. Chemosensing plays a fundamental role in the life history of these groups of butterflies and in the establishment of reproductive isolation. However, chemical communication involves synergistic sensory and accessory functions, and it remains challenging to investigate the molecular mechanisms underlying behavioral differences. Here, we examine the gene expression profiles and genomic divergence of three sensory tissues (antennae, legs, and mouthparts) between sexes (females and males) and life stages (different adult stages) in two hybridizing butterflies, Heliconius melpomene and Heliconius cydno. By integrating comparative transcriptomic and population genomic approaches, we found evidence of widespread gene expression divergence, supporting a crucial role of sensory tissues in the establishment of species barriers. We also show that sensory diversification increases in a manner consistent with evolutionary divergence based on comparison with the more distantly related species Heliconius charithonia. The findings of our study strongly support the unique chemosensory function of antennae in all three species, the importance of the Z chromosome in interspecific divergence, and the nonnegligible role of nonchemosensory genes in the divergence of chemosensory tissues. Collectively, our results provide a genome-wide illustration of diversification in the chemosensory system under incomplete reproductive isolation, revealing strong molecular separation in the early stage of speciation. Here, we provide a unique perspective and relevant view of the genetic architecture (sensory and accessory functions) of chemosensing beyond the classic chemosensory gene families, leading to a better understanding of the magnitude and complexity of molecular changes in sensory tissues that contribute to the establishment of reproductive isolation and speciation. 

Despite insertions and deletions being the most common structural variants (SVs) found across genomes, not much is known about how much these SVs vary within populations and between closely related species, nor their significance in evolution. To address these questions, we characterized the evolution of indel SVs using genome assemblies of three closely related Heliconius butterfly species. Over the relatively short evolutionary timescales investigated, up to 18.0% of the genome was composed of indels between two haplotypes of an individual H. charithonia butterfly and up to 62.7% included lineage-specific SVs between the genomes of the most distant species (11 Mya). Lineage-specific sequences were mostly characterized as transposable elements (TEs) inserted at random throughout the genome and their overall distribution was similarly affected by linked selection as single nucleotide substitutions. Using chromatin accessibility profiles (i.e., ATAC-seq) of head tissue in caterpillars to identify sequences with potential cis-regulatory function, we found that out of the 31,066 identified differences in chromatin accessibility between species, 30.4% were within lineage-specific SVs and 9.4% were characterized as TE insertions. These TE insertions were localized closer to gene transcription start sites than expected at random and were enriched for several transcription factor binding site candidates with known function in neuron development in Drosophila. We also identified 24 TE insertions with head-specific chromatin accessibility. Our results show high rates of structural genome evolution that were previously overlooked in comparative genomic studies and suggest a high potential for structural variation to serve as raw material for adaptive evolution. 

Heliconius butterflies have bright patterns on their wings that tell potential predators that they are toxic. As a result, predators learn to avoid eating them. Over time, unrelated species of butterflies have evolved similar patterns to avoid predation through a process known as Müllerian mimicry. Worldwide, there are over 180,000 species of butterflies and moths, most of which have different wing patterns. How do genes create this pattern diversity? And do butterflies use similar genes to create similar wing patterns? One of the genes involved in creating wing patterns is called cortex . This gene has a large region of DNA around it that does not code for proteins, but instead, controls whether cortex is on or off in different parts of the wing. Changes in this non-coding region can act like switches, turning regions of the wing into different colours and creating complex patterns, but it is unclear how these switches have evolved. Butterfly wings get their colour from tiny structures called scales, which each have their own unique set of pigments. In Heliconius butterflies, there are three types of scales: yellow/white scales, black scales, and red/orange/brown scales. Livraghi et al. used a DNA editing technique called CRISPR to find out whether the cortex gene affects scale type. First, Livraghi et al. confirmed that deleting cortex turned black and red scales yellow. Next, they used the same technique to manipulate the non-coding DNA around the cortex gene to see the effect on the wing pattern. This manipulation turned a black-winged butterfly into a butterfly with a yellow wing band, a pattern that occurs naturally in Heliconius butterflies. The next step was to find the mutation responsible for the appearance of yellow wing bands in nature. It turns out that a bit of extra genetic code, derived from so-called ‘jumping genes’, had inserted itself into the non-coding DNA around the cortex gene, ‘flipping’ the switch and leading to the appearance of the yellow scales. Genetic information contains the instructions to generate shape and form in most organisms. These instructions evolve over millions of years, creating everything from bacteria to blue whales. Butterfly wings are visual evidence of evolution, but the way their genes create new patterns isn't specific to butterflies. Understanding wing patterns can help researchers to learn how genetic switches control diversity across other species too. 

Characterizing the genetic complexity of adaptation and trait evolution is a major emphasis of evolutionary biology and genetics. Incongruent findings from genetic studies have resulted in conceptual models ranging from a few large-effect loci to massively polygenic architectures. Here, we combine chromatin immunoprecipitation sequencing, Hi-C, RNA sequencing, and 40 whole-genome sequences from Heliconius butterflies to show that red color pattern diversification occurred via many genomic loci. We find that the red wing pattern master regulatory transcription factor Optix binds dozens of loci also under selection, which frequently form three-dimensional adaptive hubs with selection acting on multiple physically interacting genes. Many Optix-bound genes under selection are tied to pigmentation and wing development, and these loci collectively maintain separation between adaptive red color pattern phenotypes in natural populations. We propose a model of trait evolution where functional connections between loci may resolve much of the disparity between large-effect and polygenic evolutionary models. 

Müllerian mimicry strongly exemplifies the power of natural selection. However, the exact measure of such adaptive phenotypic convergence and the possible causes of its imperfection often remain unidentified. Here, we first quantify wing colour pattern differences in the forewing region of 14 co-mimetic colour pattern morphs of the butterfly species Heliconius erato and Heliconius melpomene and measure the extent to which mimicking colour pattern morphs are not perfectly identical. Next, using gene-editing CRISPR/Cas9 KO experiments of the gene WntA , which has been mapped to colour pattern diversity in these butterflies, we explore the exact areas of the wings in which WntA affects colour pattern formation differently in H. erato and H. melpomene. We find that, while the relative size of the forewing pattern is generally nearly identical between co-mimics, the CRISPR/Cas9 KO results highlight divergent boundaries in the wing that prevent the co-mimics from achieving perfect mimicry. We suggest that this mismatch may be explained by divergence in the gene regulatory network that defines wing colour patterning in both species, thus constraining morphological evolution even between closely related species.