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Hox gene clusters encode transcription factors that drive regional specialization during animal development: for example the Hox factor Ubx is expressed in the insect metathoracic (T3) wing appendages and differentiates them from T2 mesothoracic identities. Hox transcriptional regulation requires silencing activities that prevent spurious activation and regulatory crosstalks in the wrong tissues, but this has seldom been studied in insects other than Drosophila , which shows a derived Hox dislocation into two genomic clusters that disjoined Antennapedia ( Antp ) and Ultrabithorax ( Ubx ). Here, we investigated how Ubx is restricted to the hindwing in butterflies, amidst a contiguous Hox cluster. By analysing Hi-C and ATAC-seq data in the butterfly Junonia coenia , we show that a Topologically Associated Domain (TAD) maintains a hindwing-enriched profile of chromatin opening around Ubx . This TAD is bordered by a Boundary Element (BE) that separates it from a region of joined wing activity around the Antp locus. CRISPR mutational perturbation of this BE releases ectopic Ubx expression in forewings, inducing homeotic clones with hindwing identities. Further mutational interrogation of two non-coding RNA encoding regions and one putative cis- regulatory module within the Ubx TAD cause rare homeotic transformations in both directions, indicating the presence of both activating and repressing chromatin features. We also describe a series of spontaneous forewing homeotic phenotypes obtained in Heliconius butterflies, and discuss their possible mutational basis. By leveraging the extensive wing specialization found in butterflies, our initial exploration of Ubx regulation demonstrates the existence of silencing and insulating sequences that prevent its spurious expression in forewings.

The optix 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 optix plays a fundamental role in nymphalid butterfly wing pattern development, where it is required for determination of all chromatic coloration. optix 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 optix simultaneously acts as a switch gene for blue structural iridescence in some butterflies, demonstrating simple regulatory coordination of structural and pigmentary coloration. Remarkably, these optix 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 optix 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.

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. The diverse Heliconius butterflies have evolved key innovations, including pollen feeding, and are a quintessential example of adaptive radiation. Using comparative genomics, Cicconardi et al. identify targets of selection at coding and non-coding loci during major ecological transitions in Heliconius .

While piggyBac transposon-based transgenesis is widely used in various emerging model organisms, its relatively low transposition rate in butterflies and moths has hindered its use for routine genetic transformation in Lepidoptera. Here, we tested the suitability of a codon-optimized hyperactive piggyBac transposase ( hyPBase ) in mRNA form to deliver and integrate transgenic cassettes into the genome of the pantry moth Plodia interpunctella . Co-injection of hyPBase mRNA with donor plasmids successfully integrated 1.5–4.4 kb expression cassettes driving the fluorescent markers EGFP, DsRed, or EYFP in eyes and glia with the 3xP3 promoter. Somatic integration and expression of the transgene in the G 0 injected generation was detectable from 72-h embryos and onward in larvae, pupae and adults carrying a recessive white-eyed mutation. Overall, 2.5% of injected eggs survived into transgene-bearing adults with mosaic fluorescence. Subsequent outcrossing of fluorescent G 0 founders transmitted single-insertion copies of 3xP3::EGFP and 3xP3::EYFP and generated stable isogenic lines. Random in-crossing of a small cohort of G 0 founders expressing 3xP3::DsRed yielded a stable transgenic line segregating for more than one transgene insertion site. We discuss how hyPBase can be used to generate stable transgenic resources in Plodia and other moths.

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 WntA, a signaling ligand gene previously identified as a hotspot of shape-tuning alleles involved in wing mimicry. We show that WntA loss-of-function causes multiple modifications of pattern elements in seven nymphalid butterfly species. In three butterflies with a conserved wing-pattern arrangement, WntA is necessary for the induction of stripe-like patterns known as symmetry systems and acquired a novel eyespot activator role specific to Vanessa forewings. In two Heliconius species, WntA specifies the boundaries between melanic fields and the light-color patterns that they contour. In the passionvine butterfly Agraulis, WntA removal shows opposite effects on adjacent pattern elements, revealing a dual role across the wing field. Finally, WntA 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 WntA 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.

Hox gene clusters encode transcription factors that drive regional specialization during animal development: for example the Hox factor Ubx is expressed in the insect metathoracic (T3) wing appendages and differentiates them from T2 mesothoracic identities. Hox transcriptional regulation requires silencing activities that prevent spurious activation and regulatory crosstalks in the wrong tissues, but this has seldom been studied in insects other than Drosophila , which shows a derived Hox dislocation into two genomic clusters that disjoined Antennapedia ( Antp ) and Ultrabithorax ( Ubx ). Here, we investigated how Ubx is restricted to the hindwing in butterflies, amidst a contiguous Hox cluster. By analysing Hi-C and ATAC-seq data in the butterfly Junonia coenia , we show that a Topologically Associated Domain (TAD) maintains a hindwing-enriched profile of chromatin opening around Ubx . This TAD is bordered by a Boundary Element (BE) that separates it from a region of joined wing activity around the Antp locus. CRISPR mutational perturbation of this BE releases ectopic Ubx expression in forewings, inducing homeotic clones with hindwing identities. Further mutational interrogation of two non-coding RNA encoding regions and one putative cis- regulatory module within the Ubx TAD cause rare homeotic transformations in both directions, indicating the presence of both activating and repressing chromatin features. We also describe a series of spontaneous forewing homeotic phenotypes obtained in Heliconius butterflies, and discuss their possible mutational basis. By leveraging the extensive wing specialization found in butterflies, our initial exploration of Ubx regulation demonstrates the existence of silencing and insulating sequences that prevent its spurious expression in forewings.

Amazon parrots (Amazona spp.) colonized the islands of the Greater Antilles from the Central American mainland, but there has not been a consensus as to how and when this happened. Today, most of the five remaining island species are listed as endangered, threatened, or vulnerable as a consequence of human activity. We sequenced and annotated full mitochondrial genomes of all the extant Amazon parrot species from the Greater Antillean (A. leucocephala (Cuba), A. agilis, A. collaria (both from Jamaica), A. ventralis (Hispaniola), and A. vittata (Puerto Rico)), A. albifrons from mainland Central America, and A. rhodocorytha from the Atlantic Forest in Brazil. The assembled and annotated mitogenome maps provide information on sequence organization, variation, population diversity, and evolutionary history for the Caribbean species including the critically endangered A. vittata. Despite the larger number of available samples from the Puerto Rican Parrot Recovery Program, the sequence diversity of the A. vittata population in Puerto Rico was the lowest among all parrot species analyzed. Our data support the stepping-stone dispersal and speciation hypothesis that has started approximately 3.47 MYA when the ancestral population arrived from mainland Central America and led to diversification across the Greater Antilles, ultimately reaching the island of Puerto Rico 0.67 MYA. The results are presented and discussed in light of the geological history of the Caribbean and in the context of recent parrot evolution, island biogeography, and conservation. This analysis contributes to understating evolutionary history and empowers subsequent assessments of sequence variation and helps design future conservation efforts in the Caribbean.

While piggyBac transposon-based transgenesis is widely used in various emerging model organisms, its relatively low transposition rate in butterflies and moths has hindered its use for routine genetic transformation in Lepidoptera. Here, we tested the suitability of a codon-optimized hyperactive piggyBac transposase (hyPBase) in mRNA form to deliver and integrate transgenic cassettes into the genome of the pantry moth Plodia interpunctella. Co-injection of hyPBase mRNA with donor plasmids successfully integrated 1.5-4.4 kb expression cassettes driving the fluorescent markers EGFP, DsRed, or EYFP in eyes and glia with the 3xP3 promoter. Somatic integration and expression of the transgene in the G0 injected generation was detectable from 72-hr embryos and onward in larvae, pupae and adults carrying a recessive white-eyed mutation. Overall, 2.5% of injected eggs survived into transgene-bearing adults with mosaic fluorescence. Subsequent outcrossing of fluorescent G0 founders transmitted single-insertion copies of 3xP3::EGFP and 3xP3::EYFP and generated stable isogenic lines. Random in-crossing of a small cohort of G0 founders expressing 3xP3::DsRed yielded a stable transgenic line segregating for more than one transgene insertion site. We discuss how hyPBase can be used to generate stable transgenic resources in Plodia and other moths. Competing Interest Statement The authors have declared no competing interest.

Insects possess remarkable diversity in pigmentation across life stages, generated from a limited number of pigment precursors. ABCG transporters play central roles in pigmentation by mediating the intracellular transport of these precursors, yet their functions outside a few model systems remain poorly characterized. Here, we investigate the roles of the ABCG transporter genes white (w), scarlet (st), and oily kinshiryu (ok) across five lepidopteran species representing both butterflies and moths. CRISPR-Cas9 knockouts reveal conserved, tissue-specific functions of ABCG transporters throughout development, affecting larval and pupal integuments as well as adult eyes, but not scale coloration. Notably, our data indicate that both ommochrome and uric acid deposition underlie the coloration and opacity of Lepidoptera larvae and pupae, with the relative contributions of these pathways differing across species and stages. We also reveal that the vivid orange coloration of Gulf fritillary caterpillars arises from the interplay of ommochromes and urate granules, providing a clear example of how these pathways combine to produce diverse integument pigmentation. Loss of white was deleterious in four species examined, whereas scarlet knockouts produced viable individuals with easily detectable pigmentation phenotypes, identifying scarlet mutant backgrounds as a practical genetic marker for functional studies. Together, these findings show that conserved ABCG transporter complexes are differentially deployed across tissues and life stages, providing a mechanistic basis for the evolutionary diversification of pigmentation in Lepidoptera and enabling expanded functional genomics and transgenesis in emerging model species.Competing Interest StatementThe authors have declared no competing interest.Footnotes* https://doi.org/10.6084/m9.figshare.31395594Funder Information DeclaredInternational Human Frontier Science Program Organization, RGP0029NSF PRFP, 2109536