Butterfly wing patterns provide a rich comparative framework to study how morphological complexity develops and evolves. Here we used CRISPR/Cas9 somatic mutagenesis to test a patterning role for , a signaling ligand gene previously identified as a hotspot of shape-tuning alleles involved in wing mimicry. We show that loss-of-function causes multiple modifications of pattern elements in seven nymphalid butterfly species. In three butterflies with a conserved wing-pattern arrangement, is necessary for the induction of stripe-like patterns known as symmetry systems and acquired a novel eyespot activator role specific to forewings. In two species, specifies the boundaries between melanic fields and the light-color patterns that they contour. In the passionvine butterfly , removal shows opposite effects on adjacent pattern elements, revealing a dual role across the wing field. Finally, acquired a divergent role in the patterning of interveinous patterns in the monarch, a basal nymphalid butterfly that lacks stripe-like symmetry systems. These results identify as an instructive signal for the prepatterning of a biological system of exuberant diversity and illustrate how shifts in the deployment and effects of a single developmental gene underlie morphological change.

The gene has been implicated in butterfly wing pattern adaptation by genetic association, mapping, and expression studies. The actual developmental function of this gene has remained unclear, however. Here we used CRISPR/Cas9 genome editing to show that plays a fundamental role in nymphalid butterfly wing pattern development, where it is required for determination of all chromatic coloration. knockouts in four species show complete replacement of color pigments with melanins, with corresponding changes in pigment-related gene expression, resulting in black and gray butterflies. We also show that simultaneously acts as a switch gene for blue structural iridescence in some butterflies, demonstrating simple regulatory coordination of structural and pigmentary coloration. Remarkably, these knockouts phenocopy the recurring "black and blue" wing pattern archetype that has arisen on many independent occasions in butterflies. Here we demonstrate a simple genetic basis for structural coloration, and show that plays a deeply conserved role in butterfly wing pattern development.

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.

Developmental plasticity allows genomes to encode multiple distinct phenotypes that can be differentially manifested in response to environmental cues. Alternative plastic phenotypes can be selected through a process called genetic assimilation, although the mechanisms are still poorly understood. We assimilated a seasonal wing color phenotype in a naturally plastic population of butterflies (Junonia coenia) and characterized three responsible genes. Endocrine assays and chromatin accessibility and conformation analyses showed that the transition of wing coloration from an environmentally determined trait to a predominantly genetic trait occurred through selection for regulatory alleles of downstream wing-patterning genes. This mode of genetic evolution is likely favored by selection because it allows tissue- and trait-specific tuning of reaction norms without affecting core cue detection or transduction mechanisms.

Uncovering phylogenetic patterns of cis-regulatory evolution remains a fundamental goal for evolutionary and developmental biology. Here, we characterize the evolution of regulatory loci in butterflies and moths using chromatin immunoprecipitation sequencing (ChIP-seq) annotation of regulatory elements across three stages of head development. In the process we provide a high-quality, functionally annotated genome assembly for the butterfly, Heliconius erato. Comparing cis-regulatory element conservation across six lepidopteran genomes, we find that regulatory sequences evolve at a pace similar to that of protein-coding regions. We also observe that elements active at multiple developmental stages are markedly more conserved than elements with stage-specific activity. Surprisingly, we also find that stage-specific proximal and distal regulatory elements evolve at nearly identical rates. Our study provides a benchmark for genome-wide patterns of regulatory element evolution in insects, and it shows that developmental timing of activity strongly predicts patterns of regulatory sequence evolution.

BackgroundAmazona vittata is a critically endangered Puerto Rican endemic bird, the only surviving native parrot species in the United States territory, and the first parrot in the large Neotropical genus Amazona, to be studied on a genomic scale.FindingsIn a unique community-based funded project, DNA from an A. vittata female was sequenced using a HiSeq Illumina platform, resulting in a total of ~42.5 billion nucleotide bases. This provided approximately 26.89x average coverage depth at the completion of this funding phase. Filtering followed by assembly resulted in 259,423 contigs (N50 = 6,983 bp, longest = 75,003 bp), which was further scaffolded into 148,255 fragments (N50 = 19,470, longest = 206,462 bp). This provided ~76% coverage of the genome based on an estimated size of 1.58 Gb. The assembled scaffolds allowed basic genomic annotation and comparative analyses with other available avian whole-genome sequences.ConclusionsThe current data represents the first genomic information from and work carried out with a unique source of funding. This analysis further provides a means for directed training of young researchers in genetic and bioinformatics analyses and will facilitate progress towards a full assembly and annotation of the Puerto Rican parrot genome. It also adds extensive genomic data to a new branch of the avian tree, making it useful for comparative analyses with other avian species. Ultimately, the knowledge acquired from these data will contribute to an improved understanding of the overall population health of this species and aid in ongoing and future conservation efforts.

Butterfly wing patterns derive from a deeply conserved developmental ground plan yet are diverse and evolve rapidly. It is poorly understood how gene regulatory architectures can accommodate both deep homology and adaptive change. To address this, we characterized the cis-regulatory evolution of the color pattern gene WntA in nymphalid butterflies. Comparative assay for transposase-accessible chromatin using sequencing (ATAC-seq) and in vivo deletions spanning 46 cis-regulatory elements across five species revealed deep homology of ground plan–determining sequences, except in monarch butterflies. Furthermore, noncoding deletions displayed both positive and negative regulatory effects that were often broad in nature. Our results provide little support for models predicting rapid enhancer turnover and suggest that deeply ancestral, multifunctional noncoding elements can underlie rapidly evolving trait systems.

Heliconius butterflies, a speciose genus of Mullerian mimics, represent a classic example of an adaptive radiation involving 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, contrasting our understanding of how adaptive and neutral processes shaped genome evolution across their radiation. Here, we build new, highly-contiguous genome assemblies for nine new Heliconiini, reference-assembled genomes for 29 species, and improve 10 existing assemblies, to provide a major new dataset of annotated genomes for 63 species, including 58 species within the Heliconiini tribe. We provide a robust, dated heliconiine phylogeny, identify major patterns of introgression, explore the evolution of genome size, content, and the genomic basis of key innovations in this enigmatic group for the first time. We illustrate how dense genomic sampling improves our resolution of gene-phenotype links, and our understanding of how genomes evolve.