Identifying the genomic changes that control morphological variation and understanding how they generate diversity is a major goal of evolutionary biology. In Heliconius butterflies, a small number of genes control the development of diverse wing color patterns. Here, we used full genome sequencing of individuals across the Heliconius erato radiation and closely related species to characterize genomic variation associated with wing pattern diversity. We show that variation around color pattern genes is highly modular, with narrow genomic intervals associated with specific differences in color and pattern. This modular architecture explains the diversity of color patterns and provides a flexible mechanism for rapid morphological diversification.

The wing patterns of butterflies and moths (Lepidoptera) are diverse and striking examples of evolutionary diversification by natural selection1,2. Lepidopteran wing colour patterns are a key innovation, consisting of arrays of coloured scales. We still lack a general understanding of how these patterns are controlled and if there is any commonality across the 160,000 moth and 17,000 butterfly species. Here, we identify a gene, cortex, through fine-scale mapping using population genomics and gene expression analyses, which regulates pattern switches in multiple species across the mimetic radiation in Heliconius butterflies. cortex belongs to a fast evolving subfamily of the otherwise highly conserved fizzy family of cell cycle regulators3, suggesting that it most likely regulates pigmentation patterning through regulation of scale cell development. In parallel with findings in the peppered moth (Biston betularia)4, our results suggest that this mechanism is common within Lepidoptera and that cortex has become a major target for natural selection acting on colour and pattern variation in this group of insects.

An important goal in evolutionary biology is to understand the genetic changes underlying novel morphological structures. We investigated the origins of a complex wing pattern found among Amazonian Heliconius butterflies. Genome sequence data from 142 individuals across 17 species identified narrow regions associated with two distinct red colour pattern elements, dennis and ray. We hypothesise that these modules in non-coding sequence represent distinct cis-regulatory loci that control expression of the transcription factor optix, which in turn controls red pattern variation across Heliconius. Phylogenetic analysis of the two elements demonstrated that they have distinct evolutionary histories and that novel adaptive morphological variation was created by shuffling these cis-regulatory modules through recombination between divergent lineages. In addition, recombination of modules into different combinations within species further contributes to diversity. Analysis of the timing of diversification in these two regions supports the hypothesis of introgression moving regulatory modules between species, rather than shared ancestral variation. The dennis phenotype introgressed into Heliconius melpomene at about the same time that ray originated in this group, while ray introgressed back into H. elevatus much more recently. We show that shuffling of existing enhancer elements both within and between species provides a mechanism for rapid diversification and generation of novel morphological combinations during adaptive radiation.

Research into Heliconius butterflies has made a significant contribution to evolutionary biology. Here, we review our understanding of the diversification of these butterflies, covering recent advances and a vast foundation of earlier work. Whereas no single group of organisms can be sufficient for understanding life's diversity, after years of intensive study, research into Heliconius has addressed a wide variety of evolutionary questions. We first discuss evidence for widespread gene flow between Heliconius species and what this reveals about the nature of species. We then address the evolution and diversity of warning patterns, both as the target of selection and with respect to their underlying genetic basis. The identification of major genes involved in mimetic shifts, and homology at these loci between distantly related taxa, has revealed a surprising predictability in the genetic basis of evolution. In the final sections, we consider the evolution of warning patterns, and Heliconius diversity more generally, within a broader context of ecological and sexual selection. We consider how different traits and modes of selection can interact and influence the evolution of reproductive isolation.

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One contribution of 17 to a theme issue 'Evo-devo in the genomics era, and the origins of morphological diversity'. A major challenge is to understand how conserved gene regulatory networks control the wonderful diversity of form that we see among animals and plants. Butterfly wing patterns are an excellent example of this diversity. Butterfly wings form as imaginal discs in the caterpillar and are constructed by a gene regulatory network, much of which is conserved across the holometabolous insects. Recent work in Heliconius butterflies takes advantage of genomic approaches and offers insights into how the diversification of wing patterns is overlaid onto this conserved network. WntA is a patterning morphogen that alters spatial information in the wing. Optix is a transcription factor that acts later in development to paint specific wing regions red. Both of these loci fit the paradigm of conserved protein-coding loci with diverse regulatory elements and developmental roles that have taken on novel derived functions in patterning wings. These discoveries offer insights into the 'Nymphalid Ground Plan', which offers a unifying hypothesis for pattern formation across nymphalid butterflies. These loci also represent 'hotspots' for morphological change that have been targeted repeatedly during evolution. Both convergent and divergent evolution of a great diversity of patterns is controlled by complex alleles at just a few genes. We suggest that evolutionary change has become focused on one or a few genetic loci for two reasons. First, pre-existing complex cis-regulatory loci that already interact with potentially relevant transcription factors are more likely to acquire novel functions in wing patterning. Second, the shape of wing regulatory networks may constrain evolutionary change to one or a few loci. Overall, genomic approaches that have identified wing patterning loci in these butterflies offer broad insight into how gene regulatory networks evolve to produce diversity.This article is part of the themed issue 'Evo-devo in the genomics era, and the origins of morphological diversity'.

Mating cues evolve rapidly and can contribute to species formation and maintenance. However, little is known about how sexual signals diverge and how this variation integrates with other barrier loci to shape the genomic landscape of reproductive isolation. Here, we elucidate the genetic basis of ultraviolet (UV) iridescence, a courtship signal that differentiates the males of Colias eurytheme butterflies from a sister species, allowing females to avoid costly heterospecific matings. Anthropogenic range expansion of the two incipient species established a large zone of secondary contact across the eastern United States with strong signatures of genomic admixtures spanning all autosomes. In contrast, Z chromosomes are highly differentiated between the two species, supporting a disproportionate role of sex chromosomes in speciation known as the large-X (or large-Z) effect. Within this chromosome-wide reproductive barrier, linkage mapping indicates that cis-regulatory variation of bric a brac (bab) underlies the male UV-iridescence polymorphism between the two species. Bab is expressed in all non-UV scales, and butterflies of either species or sex acquire widespread ectopic iridescence following its CRISPR knockout, demonstrating that Bab functions as a suppressor of UV-scale differentiation that potentiates mating cue divergence. These results highlight how a genetic switch can regulate a premating signal and integrate with other reproductive barriers during intermediate phases of speciation.

Background The color patterns that adorn lepidopteran wings are ideal for studying cell type diversity using a phenomics approach. Color patterns are made of chitinous scales that are each the product of a single precursor cell, offering a 2D system where phenotypic diversity can be studied cell by cell, both within and between species. Those scales reveal complex ultrastructures in the sub‐micrometer range that are often connected to a photonic function, including iridescent blues and greens, highly reflective whites, or light‐trapping blacks. Results We found that during scale development, Fascin immunostainings reveal punctate distributions consistent with a role in the control of actin patterning. We quantified the cytoskeleton regularity as well as its relationship to chitin deposition sites, and confirmed a role in the patterning of the ultrastructures of the adults scales. Then, in an attempt to characterize the range and variation in lepidopteran scale ultrastructures, we devised a high‐throughput method to quickly derive multiple morphological measurements from fluorescence images and scanning electron micrographs. We imaged a multicolor eyespot element from the butterfly Vanessa cardui (V. cardui), taking approximately 200 000 individual measurements from 1161 scales. Principal component analyses revealed that scale structural features cluster by color type, and detected the divergence of non‐reflective scales characterized by tighter cross‐rib distances and increased orderedness. Conclusion We developed descriptive methods that advance the potential of butterfly wing scales as a model system for studying how a single cell type can differentiate into a multifaceted spectrum of complex morphologies. Our data suggest that specific color scales undergo a tight regulation of their ultrastructures, and that this involves cytoskeletal dynamics during scale growth.