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

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.

 

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. 

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. 

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.