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Evolutionary variation in the wing pigmentation of butterflies and moths offers striking examples of adaptation by crypsis and mimicry. Thecortexlocus has been independently mapped as the locus controlling color polymorphisms in 15 lepidopteran species, suggesting that it acts as a genomic hotspot for the diversification of wing patterns, but functional validation through protein-coding knockouts has proven difficult to obtain. Our study unveils the role of a long noncoding RNA (lncRNA) which we nameivory, transcribed from thecortexlocus, in modulating color patterning in butterflies. Strikingly,ivoryexpression prefigures most melanic patterns during pupal development, suggesting an early developmental role in specifying scale identity. To test this, we generated CRISPR mosaic knock-outs in five nymphalid butterfly species and show thativorymutagenesis yields transformations of dark pigmented scales into white or light-colored scales. Genotyping ofVanessa carduigermline mutants associates these phenotypes to small on-target deletions at the conserved first exon ofivory. In contrast,cortexgermline mutant butterflies with confirmed null alleles lack any wing phenotype and exclude a color patterning role for this adjacent gene. Overall, these results show that a lncRNA gene acts as a master switch of color pattern specification and played key roles in the adaptive diversification of wing patterns in butterflies. 

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. 

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

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 Heliconius 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 sites with significant resemblance to several transcription factor binding sites 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. 

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. 

Abstract Many animal species remain separate not because their individuals fail to produce viable hybrids but because they “choose” not to mate. However, we still know very little of the genetic mechanisms underlying changes in these mate preference behaviours. Heliconius butterflies display bright warning patterns, which they also use to recognize conspecifics. Here, we couple QTL for divergence in visual preference behaviours with population genomic and gene expression analyses of neural tissue (central brain, optic lobes and ommatidia) across development in two sympatric Heliconius species. Within a region containing 200 genes, we identify five genes that are strongly associated with divergent visual preferences. Three of these have previously been implicated in key components of neural signalling (specifically an ionotropic glutamate receptor and two regucalcins ), and overall our candidates suggest shifts in behaviour involve changes in visual integration or processing. This would allow preference evolution without altering perception of the wider environment. 

Chemosensory communication is essential to insect biology, playing indispensable roles during mate-finding, foraging, and oviposition behaviors. These traits are particularly important during speciation, where chemical perception may serve to establish species barriers. However, identifying genes associated with such complex behavioral traits remains a significant challenge. Through a combination of transcriptomic and genomic approaches, we characterize the genetic architecture of chemoperception and the role of chemosensing during speciation for a young species pair of Heliconius butterflies, Heliconius melpomene and Heliconius cydno . We provide a detailed description of chemosensory gene-expression profiles as they relate to sensory tissue (antennae, legs, and mouthparts), sex (male and female), and life stage (unmated and mated female butterflies). Our results untangle the potential role of chemical communication in establishing barriers during speciation and identify strong candidate genes for mate and host plant choice behaviors. Of the 252 chemosensory genes, HmOBP20 (involved in volatile detection) and HmGr56 (a putative synephrine-related receptor) emerge as strong candidates for divergence in pheromone detection and host plant discrimination, respectively. These two genes are not physically linked to wing-color pattern loci or other genomic regions associated with visual mate preference. Altogether, our results provide evidence for chemosensory divergence between H. melpomene and H. cydno , two rarely hybridizing butterflies with distinct mate and host plant preferences, a finding that supports a polygenic architecture of species boundaries. 

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. 

Abstract As the genetic basis of natural and domesticated variation has been described in recent years, a number of hotspot genes have been repeatedly identified as the targets of selection, Heliconius butterflies display a spectacular diversity of pattern variants in the wild and the genetic basis of these patterns has been well-described. Here, we sought to identify the mechanism behind an unusual pattern variant that is instead found in captivity, the ivory mutant, in which all scales on both the wings and body become white or yellow. Using a combination of autozygosity mapping and coverage analysis from 37 captive individuals, we identify a 78-kb deletion at the cortex wing patterning locus, a gene which has been associated with wing pattern evolution in H. melpomene and 10 divergent lepidopteran species. This deletion is undetected among 458 wild Heliconius genomes samples, and its dosage explains both homozygous and heterozygous ivory phenotypes found in captivity. The deletion spans a large 5′ region of the cortex gene that includes a facultative 5′UTR exon detected in larval wing disk transcriptomes. CRISPR mutagenesis of this exon replicates the wing phenotypes from coding knock-outs of cortex, consistent with a functional role of ivory-deleted elements in establishing scale color fate. Population demographics reveal that the stock giving rise to the ivory mutant has a mixed origin from across the wild range of H. melpomene, and supports a scenario where the ivory mutation occurred after the introduction of cortex haplotypes from Ecuador. Homozygotes for the ivory deletion are inviable while heterozygotes are the targets of artificial selection, joining 40 other examples of allelic variants that provide heterozygous advantage in animal populations under artificial selection by fanciers and breeders. Finally, our results highlight the promise of autozygosity and association mapping for identifying the genetic basis of aberrant mutations in captive insect populations.