Sex-linked gene traffic underlies the acquisition of sexually dimorphic UV color vision in Heliconius butterflies

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, some Heliconius butterflies 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 studied Heliconius 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 the H. charithonia genome, we discovered that UVRh1 is present on the W chromosome, making it obligately female-specific. By knocking out UVRh1, we show that UVRh1 protein expression is absent in mutant female eye tissue, as in wild-type male eyes. A PCR survey of UVRh1 sex-linkage across the genus shows that species with female-specific UVRh1 expression lack UVRh1 gDNA in males. Thus, acquisition of sex linkage is sufficient to achieve female-specific expression of UVRh1, though this does not preclude other mechanisms, like cis-regulatory evolution from also contributing. Moreover, both this event, and mutations leading to differential UV opsin sensitivity, occurred early in the history of Heliconius. 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).

Methods
Transcriptome fasta files were produced using Trinity and stringtie assemblies of RNA-seq data derived from antennae, heads, legs, and mouthparts of adult male and female Heliconius charithonia butterflies. Reference genome fasta file of DNA extracted from a single H. charithonia female pupae was produced using Falcon and Canu (v1.6) assemblies that were combined into a single fasta file with Quickmerge.

Behavioral data was collected by first training adult Heliconius charithonia butterflies to associate a sugar reward with 390 nm light. Butterflies were then given a choice between 380 nm and 390 nm lights at intensity ratios of 5:1, 1:1, and 1:5 where the first number indicates the rewarded light.