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The evolution of color vision is often studied through the lens of receptor gain relative to an ancestor with fewer spectral classes of photoreceptor. For instance, in Heliconius butterflies, a genus-specific UVRh opsin duplication led to the evolution of UV color discrimination in Heliconius erato females, a rare trait among butterflies. However, color vision evolution is not well understood in the context of loss. In Heliconius melpomene and Heliconius ismenius lineages, the UV2 receptor subtype has been lost, which limits female color vision in shorter wavelengths. Here, we compare the visual systems of butterflies that have either retained or lost the UV2 photoreceptor using intracellular recordings, ATAC-seq, and antibody staining. We identify several ways these butterflies modulate their color vision. In H. melpomene, chromatin reorganization has downregulated an otherwise intact UVRh2 gene, whereas in H. ismenius, pseudogenization has led to the truncation of UVRh2. In species that lack the UV2 receptor, the peak sensitivity of the remaining UV1 photoreceptor cell is shifted to longer wavelengths. Across Heliconius, we identify the widespread use of filtering pigments and co-expression of two opsins in the same photoreceptor cells. Multiple mechanisms of spectral tuning, including the molecular evolution of blue opsins, have led to the divergence of receptor sensitivities between species. The diversity of photoreceptor and ommatidial subtypes between species suggests that Heliconius visual systems are under varying selection pressures for color discrimination. Modulating the wavelengths of peak sensitivities of both the blue- and remaining UV-sensitive photoreceptor cells suggests that Heliconius species may have compensated for UV receptor loss.

Color vision modifications occur in animals via a process known as spectral tuning. In Heliconius butterflies, a genus-specific UVRh opsin duplication led to the evolution of UV color discrimination in Heliconius erato females, a rare trait among butterflies. In the H. melpomene and H. ismenius lineages, the UV2 receptor has been lost. Here we compare how loss of the UV2 photoreceptor has altered the visual system of these butterflies. We compare visual system evolution in three Heliconius butterfly species using a combination of intracellular recordings, ATAC-seq, and antibody staining. We identify several spectral tuning mechanisms including adaptive evolution of opsins, deployment of two types of filtering pigments, and co-expression of two distinct opsins in the same cell. Our data show that opsin gain and loss is driving rapid divergence in Heliconius visual systems via tuning of multiple spectral classes of photoreceptor in distinct lineages, potentially contributing to ongoing speciation in this genus.

Numerous animal lineages have expanded and diversified the opsin-based photoreceptors in their eyes underlying color vision behavior. However, the selective pressures giving rise to new photoreceptors and their spectral tuning remain mostly obscure. Previously, we identified a violet receptor (UV2) that is the result of a UV opsin gene duplication specific to Heliconius butterflies. At the same time the violet receptor evolved, Heliconius evolved UV-yellow coloration on their wings, due to the pigment 3-hydroxykynurenine (3-OHK) and the nanostructure architecture of the scale cells. In order to better understand the selective pressures giving rise to the violet receptor, we characterized opsin expression patterns using immunostaining (14 species) and RNA-Seq (18 species), and reconstructed evolutionary histories of visual traits in five major lineages within Heliconius and one species from the genus Eueides. Opsin expression patterns are hyperdiverse within Heliconius. We identified six unique retinal mosaics and three distinct forms of sexual dimorphism based on ommatidial types within the genus Heliconius. Additionally, phylogenetic analysis revealed independent losses of opsin expression, pseudogenization events, and relaxation of selection on UVRh2 in one lineage. Despite this diversity, the newly evolved violet receptor is retained across most species and sexes surveyed. Discriminability modeling of behaviorally preferred 3-OHK yellow wing coloration suggests that the violet receptor may facilitate Heliconius color vision in the context of conspecific recognition. Our observations give insights into the selective pressures underlying the origins of new visual receptors.

Intracellular recording is a powerful technique used to determine how a single cell may respond to a given stimulus. In vision research, intracellular recording has historically been a common technique used to study sensitivities of individual photoreceptor cells to different light stimuli that is still being used today. However, there remains a dearth of detailed methodology in the literature for researchers wishing to replicate intracellular recording experiments in the eye. Here we present the insect as a model for examining eye physiology more generally. Insect photoreceptor cells are located near the surface of the eye and are therefore easy to reach, and many of the mechanisms involved in vision are conserved across animal phyla. We describe the basic procedure for in vivo intracellular recording of photoreceptor cells in the eye of a butterfly, with the goal of making this technique more accessible to researchers with little prior experience in electrophysiology. We introduce the basic equipment needed, how to prepare a live butterfly for recording, how to insert a glass microelectrode into a single cell, and finally the recording procedure itself. We also explain the basic analysis of raw response data for determining spectral sensitivity of individual cell types. Although our protocol focuses on determining spectral sensitivity, other stimuli (e.g., polarized light) and variations of the method are applicable to this setup.

Most butterfly families expand the number of spectrally-distinct photoreceptors in their compound eye by opsin gene duplications together with lateral filter pigments, however most nymphalid genera have limited diversity, with only three or four spectral types of photoreceptor. Here we examine the spatial pattern of opsin expression and photoreceptor spectral sensitivities in Heliconius erato, a nymphalid with duplicate ultraviolet opsin genes, UVRh1 and UVRh2. We find that the H. erato compound eye is sexually dimorphic. Females express the two UV opsin proteins in separate photoreceptors, but males do not express UVRh1. Intracellular recordings confirmed that females have three short wavelength-sensitive photoreceptors (λmax=356 nm, ∼390 nm and 470 nm), while males have two (λmax=390 nm and ∼470 nm). We also found two long wavelength-sensitive photoreceptors (green, λmax ∼555 nm, and red, λmax ∼600 nm), which express the same LW opsin. The red cell's shifted sensitivity is probably due to perirhabdomal filtering pigments. Sexual dimorphism of the UV-absorbing rhodopsins may reflect the females' need to discriminate conspecifics from co-mimics. Red-green color vision may be used to detect differences in red coloration on Heliconius wings, or for host-plant identification. Among nymphalids so far investigated, only H. erato is known to possess five spectral classes of photoreceptor; sexual dimorphism of the eye via suppression of one class of opsin (here UVRh1 in males) has not—to our knowledge—been reported in any animal.