UV photoreceptors and UV-yellow wing pigments in Heliconius butterflies allow a color signal to serve both mimicry and intraspecific communication.

Mimetic wing coloration evolves in butterflies in the context of predator confusion. Unless butterfly eyes have adaptations for discriminating mimetic color variation, mimicry also carries a risk of confusion for the butterflies themselves. Heliconius butterfly eyes, which express recently duplicated ultraviolet (UV) opsins, have such an adaptation. To examine bird and butterfly color vision as sources of selection on butterfly coloration, we studied yellow wing pigmentation in the tribe Heliconiini. We confirmed, using reflectance and mass spectrometry, that only Heliconius use 3-hydroxy-DL-kynurenine (3-OHK), which looks yellow to humans but reflects both UV- and long-wavelength light, whereas butterflies in related genera have chemically unknown yellow pigments mostly lacking UV reflectance. Modeling of these color signals reveals that the two UV photoreceptors of Heliconius are better suited to separating 3-OHK from non-3-OHK spectra compared with the photoreceptors of related genera or birds. The co-occurrence of potentially enhanced UV vision and a UV-reflecting yellow wing pigment could allow unpalatable Heliconius private intraspecific communication in the presence of mimics. Our results are the best available evidence for the correlated evolution of a color signal and color vision. They also suggest that predator visual systems are error prone in the context of mimicry.

Bybee SM ，Yuan F ，Ramstetter MD ，Llorente-Bousquets J ，Reed RD ，Osorio D ，Briscoe AD ... -  《-》

Positive selection of a duplicated UV-sensitive visual pigment coincides with wing pigment evolution in Heliconius butterflies.

The butterfly Heliconius erato can see from the UV to the red part of the light spectrum with color vision proven from 440 to 640 nm. Its eye is known to contain three visual pigments, rhodopsins, produced by an 11-cis-3-hydroxyretinal chromophore together with long wavelength (LWRh), blue (BRh) and UV (UVRh1) opsins. We now find that H. erato has a second UV opsin mRNA (UVRh2)-a previously undescribed duplication of this gene among Lepidoptera. To investigate its evolutionary origin, we screened eye cDNAs from 14 butterfly species in the subfamily Heliconiinae and found both copies only among Heliconius. Phylogeny-based tests of selection indicate positive selection of UVRh2 following duplication, and some of the positively selected sites correspond to vertebrate visual pigment spectral tuning residues. Epi-microspectrophotometry reveals two UV-absorbing rhodopsins in the H. erato eye with lambda(max) = 355 nm and 398 nm. Along with the additional UV opsin, Heliconius have also evolved 3-hydroxy-DL-kynurenine (3-OHK)-based yellow wing pigments not found in close relatives. Visual models of how butterflies perceive wing color variation indicate this has resulted in an expansion of the number of distinguishable yellow colors on Heliconius wings. Functional diversification of the UV-sensitive visual pigments may help explain why the yellow wing pigments of Heliconius are so colorful in the UV range compared to the yellow pigments of close relatives lacking the UV opsin duplicate.

Briscoe AD ，Bybee SM ，Bernard GD ，Yuan F ，Sison-Mangus MP ，Reed RD ，Warren AD ，Llorente-Bousquets J ，Chiao CC ... -  《-》

Ultraviolet and yellow reflectance but not fluorescence is important for visual discrimination of conspecifics by Heliconius erato.

Toxic butterflies have yellow hindwing bars that - unlike those of their closest relatives - reflect ultraviolet (UV) and long wavelength light, and also fluoresce. The pigment in the yellow scales is 3-hydroxy-dl-kynurenine (3-OHK), which is found in the hair and scales of a variety of animals. In other butterflies like pierids with color schemes characterized by independent sources of variation in UV and human-visible yellow/orange, behavioral experiments have generally implicated the UV component as most relevant to mate choice. This has not been addressed in butterflies, where variation exists in analogous color components, but moreover where fluorescence due to 3-OHK could also contribute to yellow wing coloration. In addition, the potential cost due to predator visibility is largely unknown for the analogous well-studied pierid butterfly species. In field studies with butterfly paper models, we show that both UV and 3-OHK yellow act as signals for when compared with models lacking UV or resembling ancestral yellow, respectively, but attack rates by birds do not differ significantly between the models. Furthermore, measurement of the quantum yield and reflectance spectra of 3-OHK indicates that fluorescence does not contribute to the visual signal under broad-spectrum illumination. Our results suggest that the use of 3-OHK pigmentation instead of ancestral yellow was driven by sexual selection rather than predation.

Finkbeiner SD ，Fishman DA ，Osorio D ，Briscoe AD ... -  《-》

Sexual Dimorphism and Retinal Mosaic Diversification following the Evolution of a Violet Receptor in Butterflies.

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.

McCulloch KJ ，Yuan F ，Zhen Y ，Aardema ML ，Smith G ，Llorente-Bousquets J ，Andolfatto P ，Briscoe AD ... -  《-》

Contrasting modes of evolution of the visual pigments in Heliconius butterflies.

The adult compound eyes of passion-vine butterflies in the genus Heliconius contain one more UV opsin than other butterflies. Together with an 11-cis-3-hydroxyretinal chromophore, their four opsin genes UVRh1, UVRh2, BRh, and LWRh produce four rhodopsins that are UV-, blue-, or long wavelength absorbing. One of the Heliconius UV opsin genes, UVRh2, was found to have evolved under positive selection following recent gene duplication, using the branch-site test of selection. Using a more conservative test, the small-sample method, we confirm our prior finding of positive selection of UVRh2 and provide new statistical evidence of episodic evolution, that is, positive selection followed by purifying selection. We also newly note that one of the positively selected amino acid sites contains substitutions with known spectral tuning effects in avian ultraviolet- and violet-sensitive visual pigments. As this is one of a handful of described examples of positive selection of any specific gene in any butterfly where functional variation between copies has been characterized, we were interested in examining the molecular and physiological context of this adaptive event by examining the UV opsin genes in contrast to the other visual pigment genes. We cloned BRh and LWRh from 13 heliconiine species and UVRh1 and UVRh2 from Heliconius elevatus. In parallel, we performed in vivo epi-microspectrophotometric experiments to estimate the wavelength of peak absorbance, λ(max), of several rhodopsins in seven heliconiine species. In contrast to UVRh2, we found both physiological and statistical evidence consistent with purifying selection on UVRh1, BRh, and LWRh along the branch leading to the common ancestor of Heliconius. These results underscore the utility of combining molecular and physiological experiments in a comparative context for strengthening evidence for adaptive evolution at the molecular level.

Yuan F ，Bernard GD ，Le J ，Briscoe AD ... -  《-》