Diversification of lepidopteran (moth and butterfly) color vision is due to gene duplication, gene loss, and sequence variation of opsin genes. How other vision-related genes are evolving in this group is not well characterized. This dissertation aims to survey the evolution and expression of candidate genes involved in lepidopteran vision. In my first study, I tested whether vision-related genes varied in expression between sexes and seasonal forms of a phenotypically plastic butterfly, Bicyclus anynana. B. anynana displays plasticity and sexual dimorphism in eye size. I identified eye development genes differentially expressed between seasonal forms, making them candidates underlying eye size differences. I found that more genes were differentially expressed between seasonal forms than sexes, with rearing temperature having a larger effect on the expression of vision-related genes in females. One of the genes that was differentially expressed between seasonal forms was annotated to have a CRAL-TRIO domain. Members of this gene family have a role in chromophore transport in vertebrates and Drosophila (pinta). This gene family has been shown to be evolving by linage-specific duplications in insects and has an expansion in Lepidoptera. For my next study, I used phylogenetics and transcriptomics to assess the evolution and expression patterns of members of this gene family in the butterfly Heliconius melpomene. Results indicated that a family member non-orthologous to Drosophila pinta takes on the role of chromophore binding. This brought upon the question: which phototransduction genes are conserved between Lepidoptera and Drosophila? For the last study of my dissertation, I used phylogenetics and transcriptomics to explore more gene families in order to identify which phototransduction cascade genes were conserved between Drosophila and Lepidoptera, and within Lepidoptera. Results suggested that moth and butterfly phototransduction cascades involve similar genes that vary from Drosophila cascades in instances where Lepidopteran-specific paralogs have a potential role in vision. This dissertation explored the role of plasticity in vision-related gene expression (chapter 1), provided the first evidence of visual function for a CRAL-TRIO domain gene in butterflies (chapter 2), and offered insights into the Lepidopteran phototransduction cascade (chapter 3).

Gene duplications sometimes result in large evolutionary shifts in adaptation, but often no phenotypic change occurs after duplication. In animals, molecular genetic changes, including duplications in opsin genes have resulted in highly diverse visual phenotypes. Unlike the diversity of vision-related phenotypes among orders and families, visual systems are mostly conserved among closely related species, and evidence is limited as to how visual traits initially diverge. The neotropical butterfly genus, Heliconius, is a good system for addressing these questions. Heliconius has duplicated UV opsin genes (UVRh1 and UVRh2) coincident with a UV-yellow wing pigment, a known sexual visual cue. I hypothesized that the Heliconius-specific opsin duplication has led to photoreceptor neofunctionalization in the genus, which might affect visual system evolution and divergence in the genus due to the importance of UV signals in Heliconius.

To understand the phenotypic effects of UV gene duplication in H. erato, I developed an intracellular recording technique in the lab that measures photoreceptor spectral sensitivities. I also compared differences in opsin expression and retinal mosaics across 23 species in the genus, using immunohistochemistry and RNA-Sequencing. Combining this data in a phylogenetic context, I analyzed the evolutionary history of the opsins and their expression patterns in Heliconius. Intracellular recordings confirm two distinct female-specific UV-sensitive cells in H. erato females, but males lack the UVRh1 cell. Unlike other groups with conserved eye design among closely-related species, I discovered a high diversity of both sex- and clade-specific retinal mosaics within the genus. I found three unique forms of sexual dimorphism, multiple independent pseudogenizations of UVRh2, and the independent evolution of a retinal mosaic in separate clades. The proportions of ommatidial types are tightly regulated within each species and sex. These patterns indicate that Heliconius species with only one UV cell subtype have higher proportions of blue cells, perhaps as a secondary method for detecting the genus-specific UV-yellow wing pigment.

The spectacular diversity in opsin expression represents the rarely-seen incipient stages of visual system divergence. Novel UV photoreceptor subtypes evolved in Heliconius via opsin gene duplication, and subsequent varying selection pressures have likely caused the increased diversity of retinal mosaics in Heliconius.