We used 20 de novo genome assemblies to probe the speciation history and architecture of gene flow in rapidly radiating Heliconius butterflies. Our tests to distinguish incomplete lineage sorting from introgression indicate that gene flow has obscured several ancient phylogenetic relationships in this group over large swathes of the genome. Introgressed loci are underrepresented in low-recombination and gene-rich regions, consistent with the purging of foreign alleles more tightly linked to incompatibility loci. Here, we identify a hitherto unknown inversion that traps a color pattern switch locus. We infer that this inversion was transferred between lineages by introgression and is convergent with a similar rearrangement in another part of the genus. These multiple de novo genome sequences enable improved understanding of the importance of introgression and selective processes in adaptive radiation.

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.

We introduce the concept of topology weighting, a method for quantifying relationships between taxa that are not necessarily monophyletic, and visualizing how these relationships change across the genome. A given set of taxa can be related in a limited number of ways, but if each taxon is represented by multiple sequences, the number of possible topologies becomes very large. Topology weighting reduces this complexity by quantifying the contribution of each taxon topology to the full tree. We describe our method for topology weighting by iterative sampling of subtrees (Twisst), and test it on both simulated and real genomic data. Overall, we show that this is an informative and versatile approach, suitable for exploring relationships in almost any genomic dataset. Scripts to implement the method described are available at http://github.com/simonhmartin/twisst.

We here pioneer a low-cost assembly strategy for 20 Heliconiini genomes to characterize the evolutionary history of the rapidly radiating genus Heliconius. A bifurcating tree provides a poor fit to the data, and we therefore explore a reticulate phylogeny for Heliconius. We probe the genomic architecture of gene flow, and develop a new method to distinguish incomplete lineage sorting from introgression. We find that most loci with non-canonical histories arose through introgression, and are strongly underrepresented in regions of low recombination and high gene density. This is expected if introgressed alleles are more likely to be purged in such regions due to tighter linkage with incompatibility loci. Finally, we identify a hitherto unrecognized inversion, and show it is a convergent structural rearrangement that captures a known color pattern switch locus within the genus. Our multi-genome assembly approach enables an improved understanding of adaptive radiation.

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.

Abstract1. The use of image data to quantify, study and compare variation in the colours and patterns of organisms requires the alignment of images to establish homology, followed by colour-based segmentation of images. Here, we describe an R package for image alignment and segmentation that has applications to quantify colour patterns in a wide range of organisms.2. patternize is an R package that quantifies variation in colour patterns obtained from image data. patternize first defines homology between pattern positions across specimens either through manually placed homologous landmarks or automated image registration. Pattern identification is performed by categorizing the distribution of colours using an RGB threshold, k-means clustering or watershed transformation.3. We demonstrate that patternize can be used for quantification of the colour patterns in a variety of organisms by analysing image data for butterflies, guppies, spiders and salamanders. Image data can be compared between sets of specimens, visualized as heatmaps and analysed using principal component analysis.4. patternize has potential applications for fine scale quantification of colour pattern phenotypes in population comparisons, genetic association studies and investigating the basis of colour pattern variation across a wide range of organisms. K E Y W O R D Scolour patterns, heatmap, image registration, image segmentation, landmarks | INTRODUCTIONNatural populations often harbour great phenotypic diversity. Variation in colour and pattern are of the more vivid examples of morphological variability in nature. Taxa as diverse as spiders (Cotoras et al., 2016;De Busschere, Baert, Van Belleghem, Dekoninck, & Hendrickx, 2012), insects (Katakura, Saitoh, Nakamura, & Abbas, 1994;Williams, 2007), fish (Endler, 1983;Houde, 1987), amphibians and reptiles (Allen, Baddeley, Scott-samuel, & Cuthill, 2013;Balogová & Uhrin, 2015;Calsbeek, Bonneaud, & Smith, 2008;Rabbani, Zacharczenko, Green, Abbani, & Acharczenko, 2015), mammals (Hoekstra, Hirschmann, Bundey, Insel, & Crossland, 2006;Nekaris & Jaffe, 2007) and plants (Clegg & Durbin, 2000;Mascó, Noy-Meir, & Sérsic, 2004) | ALIGNMENTSuperimposing colour patterns to quantify variation in their expression requires the homologous alignment of the anatomical structures they occur in. Image transformations for this alignment can be obtained from landmark based transformations or image registration techniques. | Landmark based transformationsLandmark based transformations use discrete anatomical points that are homologous among individuals in the analysis. Non-rigid, but uniform transformations from one set of "source" landmarks to a set of "target" landmarks such as affine transformations include translation, rotation, scaling and skewing (Hazewinkel, 2001). Additionally, nonuniform changes in shape between the source and target landmarks can be accounted for by storing the transformation as if it were "the bending of a thin sheet of metal," the so-called thin plate spline (TPS) transformation (Duchon, 1...

Sex chromosomes are disproportionately involved in reproductive isolation and adaptation. In support of such a ‘large-X’ effect, genome scans between recently diverged populations or species pairs often identify distinct patterns of divergence on the sex chromosome compared to autosomes. When measures of divergence between populations are higher on the sex chromosome compared to autosomes, such patterns could be interpreted as evidence for faster divergence on the sex chromosome, i.e. ‘faster-X’, or barriers to gene flow on the sex chromosome. However, demographic changes can strongly skew divergence estimates and are not always taken into consideration. We used 224 whole genome sequences representing 36 populations from two Heliconius butterfly clades (H. erato and H. melpomene) to explore patterns of Z chromosome divergence. We show that increased divergence compared to equilibrium expectations can in many cases be explained by demographic change. Among Heliconius erato populations, for instance, population size increase in the ancestral population can explain increased absolute divergence measures on the Z chromosome compared to the autosomes, as a result of increased ancestral Z chromosome genetic diversity. Nonetheless, we do identify increased divergence on the Z chromosome relative to the autosomes in parapatric or sympatric species comparisons that imply post-zygotic reproductive barriers. Using simulations, we show that this is consistent with reduced gene flow on the Z chromosome, perhaps due to greater accumulation of incompatibilities. Our work demonstrates the importance of taking demography into account in order to interpret patterns of divergence on the Z chromosome, but nonetheless provides evidence to support the Z chromosome as a strong barrier to gene flow in incipient Heliconius butterfly species.

Natural selection leaves distinct signatures in the genome that can reveal the targets and history of adaptive evolution. By analysing high-coverage genome sequence data from 4 major colour pattern loci sampled from nearly 600 individuals in 53 populations, we show pervasive selection on wing patterns in the Heliconius adaptive radiation. The strongest signatures correspond to loci with the greatest phenotypic effects, consistent with visual selection by predators, and are found in colour patterns with geographically restricted distributions. These recent sweeps are similar between co-mimics and indicate colour pattern turnover events despite strong stabilising selection. Using simulations, we compare sweep signatures expected under classic hard sweeps with those resulting from adaptive introgression, an important aspect of mimicry evolution in Heliconius butterflies. Simulated recipient populations show a distinct 'volcano' pattern with peaks of increased genetic diversity around the selected target, characteristic of sweeps of introgressed variation and consistent with diversity patterns found in some populations. Our genomic data reveal a surprisingly dynamic history of colour pattern selection and co-evolution in this adaptive radiation.