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Hoxgene 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.Hoxtranscriptional regulation requires silencing activities that prevent spurious activation and regulatory crosstalks in the wrong tissues, but this has seldom been studied in insects other thanDrosophila, which shows a derivedHoxdislocation into two genomic clusters that disjoinedAntennapedia(Antp) andUltrabithorax(Ubx). Here, we investigated howUbxis restricted to the hindwing in butterflies, amidst a contiguousHoxcluster. By analysing Hi-C and ATAC-seq data in the butterflyJunonia coenia, we show that a Topologically Associated Domain (TAD) maintains a hindwing-enriched profile of chromatin opening aroundUbx. This TAD is bordered by a Boundary Element (BE) that separates it from a region of joined wing activity around theAntplocus. CRISPR mutational perturbation of this BE releases ectopicUbxexpression in forewings, inducing homeotic clones with hindwing identities. Further mutational interrogation of two non-coding RNA encoding regions and one putativecis-regulatory module within theUbxTAD 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 inHeliconiusbutterflies, and discuss their possible mutational basis. By leveraging the extensive wing specialization found in butterflies, our initial exploration ofUbxregulation demonstrates the existence of silencing and insulating sequences that prevent its spurious expression in forewings. 

The acquisition of novel sexually dimorphic traits poses an evolutionary puzzle: How do new traits arise and become sex-limited? Recently acquired color vision, sexually dimorphic in animals like primates and butterflies, presents a compelling model for understanding how traits become sex-biased. For example, someHeliconiusbutterflies uniquely possess UV (ultraviolet) color vision, which correlates with the expression of two differentially tuned UV-sensitive rhodopsins, UVRh1 and UVRh2. To discover how such traits become sexually dimorphic, we studiedHeliconius charithonia, which exhibits female-specific UVRh1 expression. We demonstrate that females, but not males, discriminate different UV wavelengths. Through whole-genome shotgun sequencing and assembly of theH. charithoniagenome, we discovered thatUVRh1is present on the W chromosome, making it obligately female-specific. By knocking outUVRh1, we show that UVRh1 protein expression is absent in mutant female eye tissue, as in wild-type male eyes. A PCR survey ofUVRh1sex-linkage across the genus shows that species with female-specific UVRh1 expression lackUVRh1gDNA in males. Thus, acquisition of sex linkage is sufficient to achieve female-specific expression ofUVRh1, though this does not preclude other mechanisms, likecis-regulatory evolution from also contributing. Moreover, both this event, and mutations leading to differential UV opsin sensitivity, occurred early in the history ofHeliconius. These results suggest a path for acquiring sexual dimorphism distinct from existing mechanistic models. We propose a model where gene traffic to heterosomes (the W or the Y) genetically partitions a trait by sex before a phenotype shifts (spectral tuning of UV sensitivity). 

Abstract As the genetic basis of natural and domesticated variation has been described in recent years, a number of hotspot genes have been repeatedly identified as the targets of selection, Heliconius butterflies display a spectacular diversity of pattern variants in the wild and the genetic basis of these patterns has been well-described. Here, we sought to identify the mechanism behind an unusual pattern variant that is instead found in captivity, the ivory mutant, in which all scales on both the wings and body become white or yellow. Using a combination of autozygosity mapping and coverage analysis from 37 captive individuals, we identify a 78-kb deletion at the cortex wing patterning locus, a gene which has been associated with wing pattern evolution in H. melpomene and 10 divergent lepidopteran species. This deletion is undetected among 458 wild Heliconius genomes samples, and its dosage explains both homozygous and heterozygous ivory phenotypes found in captivity. The deletion spans a large 5′ region of the cortex gene that includes a facultative 5′UTR exon detected in larval wing disk transcriptomes. CRISPR mutagenesis of this exon replicates the wing phenotypes from coding knock-outs of cortex, consistent with a functional role of ivory-deleted elements in establishing scale color fate. Population demographics reveal that the stock giving rise to the ivory mutant has a mixed origin from across the wild range of H. melpomene, and supports a scenario where the ivory mutation occurred after the introduction of cortex haplotypes from Ecuador. Homozygotes for the ivory deletion are inviable while heterozygotes are the targets of artificial selection, joining 40 other examples of allelic variants that provide heterozygous advantage in animal populations under artificial selection by fanciers and breeders. Finally, our results highlight the promise of autozygosity and association mapping for identifying the genetic basis of aberrant mutations in captive insect populations.