A butterfly pan-genome reveals that a large amount of structural variation underlies the evolution of chromatin accessibility.

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.

recolorize: An R package for flexible colour segmentation of biological images.

Colour pattern variation provides biological information in fields ranging from disease ecology to speciation dynamics. Comparing colour pattern geometries across images requires colour segmentation, where pixels in an image are assigned to one of a set of colour classes shared by all images. Manual methods for colour segmentation are slow and subjective, while automated methods can struggle with high technical variation in aggregate image sets. We present recolorize, an R package toolbox for human-subjective colour segmentation with functions for batch-processing low-variation image sets and additional tools for handling images from diverse (high-variation) sources. The package also includes export options for a variety of formats and colour analysis packages. This paper illustrates recolorize for three example datasets, including high variation, batch processing and combining with reflectance spectra, and demonstrates the downstream use of methods that rely on this output.

SARS-CoV-2 Omicron Replacement of Delta as Predominant Variant, Puerto Rico.

We reconstructed the SARS-CoV-2 epidemic caused by Omicron variant in Puerto Rico by sampling genomes collected during October 2021-May 2022. Our study revealed that Omicron BA.1 emerged and replaced Delta as the predominant variant in December 2021. Increased transmission rates and a dynamic landscape of Omicron sublineage infections followed.

Widespread Gene Expression Divergence in Butterfly Sensory Tissues Plays a Fundamental Role During Reproductive Isolation and Speciation.

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.

Selective sweeps on novel and introgressed variation shape mimicry loci in a butterfly adaptive radiation.

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 turn-over 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.

Divergence of chemosensing during the early stages of speciation.

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.

Parallel evolution of ancient, pleiotropic enhancers underlies butterfly wing pattern mimicry.

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.

Evolution at two time frames: Polymorphisms from an ancient singular divergence event fuel contemporary parallel evolution.

When environments change, populations may adapt surprisingly fast, repeatedly and even at microgeographic scales. There is increasing evidence that such cases of rapid parallel evolution are fueled by standing genetic variation, but the source of this genetic variation remains poorly understood. In the saltmarsh beetle Pogonus chalceus, short-winged 'tidal' and long-winged 'seasonal' ecotypes have diverged in response to contrasting hydrological regimes and can be repeatedly found along the Atlantic European coast. By analyzing genomic variation across the beetles' distribution, we reveal that alleles selected in the tidal ecotype are spread across the genome and evolved during a singular and, likely, geographically isolated divergence event, within the last 190 Kya. Due to subsequent admixture, the ancient and differentially selected alleles are currently polymorphic in most populations across its range, which could potentially allow for the fast evolution of one ecotype from a small number of random individuals, as low as 5 to 15, from a population of the other ecotype. Our results suggest that cases of fast parallel ecological divergence can be the result of evolution at two different time frames: divergence in the past, followed by repeated selection on the same divergently evolved alleles after admixture. These findings highlight the importance of an ancient and, likely, allopatric divergence event for driving the rate and direction of contemporary fast evolution under gene flow. This mechanism is potentially driven by periods of geographic isolation imposed by large-scale environmental changes such as glacial cycles.

Perfect mimicry between Heliconius butterflies is constrained by genetics and development.

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.

Genomics overrules mitochondrial DNA, siding with morphology on a controversial case of species delimitation.

Species delimitation is a major quest in biology and is essential for adequate management of the organismal diversity. A challenging example comprises the fish species of red snappers in the Western Atlantic. Red snappers have been traditionally recognized as two separate species based on morphology: Lutjanus campechanus (northern red snapper) and L. purpureus (southern red snapper). Recent genetic studies using mitochondrial markers, however, failed to delineate these nominal species, leading to the current lumping of the northern and southern populations into a single species ( L. campechanus). This decision carries broad implications for conservation and management as red snappers have been commercially over-exploited across the Western Atlantic and are currently listed as vulnerable. To address this conflict, we examine genome-wide data collected throughout the range of the two species. Population genomics, phylogenetic and coalescent analyses favour the existence of two independent evolutionary lineages, a result that confirms the morphology-based delimitation scenario in agreement with conventional taxonomy. Despite finding evidence of introgression in geographically neighbouring populations in northern South America, our genomic analyses strongly support isolation and differentiation of these species, suggesting that the northern and southern red snappers should be treated as distinct taxonomic entities.

Patterns of Z chromosome divergence among Heliconius species highlight the importance of historical demography.

Sex chromosomes are disproportionately involved in reproductive isolation and adaptation. In support of such a "large-X" effect, genome scans between recently diverged populations and 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, that is "faster-X", 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 postzygotic 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 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.

Evolutionary history of a dispersal-associated locus across sympatric and allopatric divergent populations of a wing-polymorphic beetle across Atlantic Europe.

Studying the evolutionary history of trait divergence, in particular those related to dispersal capacity, is of major interest for the process of local adaptation and metapopulation dynamics. Here, we reconstruct the evolution of different alleles at the nuclear-encoded mitochondrial NADP(+)-dependent isocitrate dehydrogenase (mtIdh) locus of the ground beetle Pogonus chalceus that are differentially and repeatedly selected in short- and long-winged populations in response to different hydrological regimes at both allopatric and sympatric scales along the Atlantic European coasts. We sequenced 2788 bp of the mtIdh locus spanning a ~7-kb genome region and compared its variation with that of two supposedly neutral genes. mtIdh sequences show (i) monophyletic clustering of the short-winged associated mtIDH-DE haplotypes within the long-winged associated mtIDH-AB haplotypes, (ii) a more than tenfold lower haplotype diversity associated with the mtIDH-DE alleles compared to the mtIDH-AB alleles and (iii) a high number of fixed nucleotide differences between both mtIDH haplotype clusters. Coalescent simulations suggest that this observed sequence variation in the mtIdh locus is most consistent with a singular origin in a partially isolated subpopulation, followed by a relatively recent spread of the mtIDH-DE allele in short-winged populations along the Atlantic coast. These results demonstrate that even traits associated with decreased dispersal capacity can rapidly spread and that reuse of adaptive alleles plays an important role in the adaptive potential within this sympatric mosaic of P. chalceus populations.

Persistent inter- and intraspecific gene exchange within a parallel radiation of caterpillar hunter beetles (Calosoma sp.) from the Galápagos.

When environmental gradients are repeated on different islands within an archipelago, similar selection pressures may act within each island, resulting in the repeated occurrence of ecologically similar species on each island. The evolution of ecotypes within such radiations may either result from dispersal, that is each ecotype evolved once and dispersed to different islands where it colonized its habitat, or through repeated and parallel speciation within each island. However, it remains poorly understood how gene flow during the divergence process may shape such patterns. In the Galápagos islands, three phenotypically similar species of the beetle genus Calosoma occur at higher elevations of different islands, while lowlands are occupied by a fourth species. By genotyping all major populations within this radiation for two nuclear and three mitochondrial gene fragments and seven microsatellite markers, we found strong support that the oldest divergence separates the highland species of the oldest island from the remaining species. Despite their morphological distinctness, highland species of the remaining islands were genetically closely related to the lowland population on each island and within the same magnitude as lowland populations sampled at different islands. Repeated evolution of highland ecotypes out of the lowland species appears the most likely scenario and estimates of geneflow rates revealed extensive admixture among ecotypes within islands, as well as between islands. These findings indicate that gene exchange among the different populations and species may have shaped the phylogenetic relationships and the repeated evolution of these ecotypes.

A tight association in two genetically unlinked dispersal related traits in sympatric and allopatric salt marsh beetle populations.

Local adaptation likely involves selection on multiple, genetically unlinked traits to increase fitness in divergent habitats. Conversely, recombination is expected to counteract local adaptation under gene flow by breaking down adaptive gene combinations. Western European populations of the salt marsh beetle Pogonus chalceus are characterized by large interpopulation variation at various geographical ranges in two traits related to dispersal ability, i.e. wing size and different allozymes of the mitochondrial NADP(+)-dependent isocitrate dehydrogenase (mtIdh) gene. In this study, we tested whether variation in wing length was as strongly genetically determined in locally adapted populations in a sympatric mosaic compared to allopatric populations, and if variation in mtIDH and wing size was genetically unlinked. We demonstrate that the genetic determination of wing size is very high (h (2) = 0.90) in sympatry and of comparable magnitude as geographically separated populations. Second, we show that, although frequencies of mtIDH allozymes are tightly associated with mean population wing size across Western European populations, the correlation is strongly reduced within some of the populations. These findings demonstrate that the divergence involves at least two traits under independent genetic control and that the genetically distinct ecotypes are retained at geographical distances with ample opportunity for gene flow.

Chromosomal inversions from an initial ecotypic divergence drive a gradual repeated radiation of Galápagos beetles.

Island faunas exhibit some of the most iconic examples where similar forms repeatedly evolve within different islands. Yet, whether these deterministic evolutionary trajectories within islands are driven by an initial, singular divergence and the subsequent exchange of individuals and adaptive genetic variation between islands remains unclear. Here, we study a gradual, repeated evolution of low-dispersive highland ecotypes from a dispersive lowland ecotype of Calosoma beetles along the island progression of the Galápagos. We show that repeated highland adaptation involved selection on multiple shared alleles within extensive chromosomal inversions that originated from an initial adaptation event on the oldest island. These highland inversions first spread through dispersal of highland individuals. Subsequent admixture with the lowland ecotype resulted in polymorphic dispersive populations from which the highland populations evolved on the youngest islands. Our findings emphasize the significance of an ancient divergence in driving repeated evolution and highlight how a mixed contribution of inter-island colonization and within-island evolution can shape parallel species communities.

Adaptive introgression of a visual preference gene.

Visual preferences are important drivers of mate choice and sexual selection, but little is known of how they evolve at the genetic level. In this study, we took advantage of the diversity of bright warning patterns displayed by Heliconius butterflies, which are also used during mate choice. Combining behavioral, population genomic, and expression analyses, we show that two Heliconius species have evolved the same preferences for red patterns by exchanging genetic material through hybridization. Neural expression of regucalcin1 correlates with visual preference across populations, and disruption of regucalcin1 with CRISPR-Cas9 impairs courtship toward conspecific females, providing a direct link between gene and behavior. Our results support a role for hybridization during behavioral evolution and show how visually guided behaviors contributing to adaptation and speciation are encoded within the genome.

Evidence for a selective link between cooperation and individual recognition.

The ability to recognize others is a frequent assumption of models of the evolution of cooperation. At the same time, cooperative behavior has been proposed as a selective agent favoring the evolution of individual recognition abilities. Although theory predicts that recognition and cooperation may co-evolve, data linking recognition abilities and cooperative behavior with evidence of selection are elusive. Here, we provide evidence of a selective link between individual recognition and cooperation in the paper wasp Polistes fuscatus through a combination of clinal, common garden, and population genomics analyses. We identified latitudinal clines in both rates of cooperative nesting and color pattern diversity, consistent with a selective link between recognition and cooperation. In behavioral experiments, we replicated previous results demonstrating individual recognition in cooperative and phenotypically diverse P. fuscatus from New York. In contrast, wasps from a less cooperative and phenotypically uniform Louisiana population showed no evidence of individual recognition. In a common garden experiment, groups of wasps from northern populations formed more stable and individually biased associations, indicating that recognition facilitates group stability. The strength of recent positive selection on cognition-associated loci likely to mediate individual recognition is substantially greater in northern compared with southern P. fuscatus populations. Collectively, these data suggest that individual recognition and cooperative nesting behavior have co-evolved in P. fuscatus because recognition helps stabilize social groups. This work provides evidence of a specific cognitive phenotype under selection because of social interactions, supporting the idea that social behavior can be a key driver of cognitive evolution.

Evolutionary dynamics of genome size and content during the adaptive radiation of Heliconiini butterflies.

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.