Background

One of the most striking features of avian vision is the variation in spectral sensitivity of the short wavelength sensitive (SWS1) opsins, which can be divided into two sub-types: violet- and UV- sensitive (VS & UVS). In birds, UVS has been found in both passerines and parrots, groups that were recently shown to be sister orders. While all parrots are thought to be UVS, recent evidence suggests some passerine lineages may also be VS. The great bowerbird (Chlamydera nuchalis) is a passerine notable for its courtship behaviours in which males build and decorate elaborate bower structures.

Results

The great bowerbird SWS1 sequence possesses an unusual residue combination at known spectral tuning sites that has not been previously investigated in mutagenesis experiments. In this study, the SWS1 opsin of C. nuchalis was expressed along with a series of spectral tuning mutants and ancestral passerine SWS1 pigments, allowing us to investigate spectral tuning mechanisms and explore the evolution of UV/violet sensitivity in early passerines and parrots. The expressed C. nuchalis SWS1 opsin was found to be a VS pigment, with a λmax of 403 nm. Bowerbird SWS1 mutants C86F, S90C, and C86S/S90C all shifted λmax into the UV, whereas C86S had no effect. Experimentally recreated ancestral passerine and parrot/passerine SWS1 pigments were both found to be VS, indicating that UV sensitivity evolved independently in passerines and parrots from a VS ancestor.

Conclusions

Our mutagenesis studies indicate that spectral tuning in C. nuchalis is mediated by mechanisms similar to those of other birds. Interestingly, our ancestral sequence reconstructions of SWS1 in landbird evolution suggest multiple transitions from VS to UVS, but no instances of the reverse. Our results not only provide a more precise prediction of where these spectral sensitivity shifts occurred, but also confirm the hypothesis that birds are an unusual exception among vertebrates where some descendants re-evolved UVS from a violet type ancestor. The re-evolution of UVS from a VS type pigment has not previously been predicted elsewhere in the vertebrate phylogeny.

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.

Rhodopsin is the visual pigment responsible for initiating the phototransduction cascade in vertebrate rod photoreceptors. Although well-characterized in a few model systems, comparative studies of rhodopsin function, particularly for nonmammalian vertebrates are comparatively lacking. Bowerbirds are rare among passerines in possessing a key substitution, D83N, at a site that is otherwise highly conserved among G protein-coupled receptors. While this substitution is present in some dim-light adapted vertebrates, often accompanying another unusual substitution, A292S, its functional relevance in birds is uncertain. To investigate functional effects associated with these two substitutions, we use the rhodopsin gene from the great bowerbird (Ptilonorhynchus nuchalis) as a background for site-directed mutagenesis, in vitro expression and functional characterization. We also mutated these sites in two additional rhodopsins that do not naturally possess N83, chicken and bovine, for comparison. Both sites were found to contribute to spectral blue-shifts, but had opposing effects on kinetic rates. Substitutions at site 83 were found to primarily affect the kinetics of light-activated rhodopsin, while substitutions at site 292 had a larger impact on spectral tuning. The contribution of substitutions at site 83 to spectral tuning in particular depended on genetic background, but overall, the effects of substitutions were otherwise surprisingly additive, and the magnitudes of functional shifts were roughly similar across all three genetic backgrounds. By employing a comparative approach with multiple species, our study provides new insight into the joint impact of sites 83 and 292 on rhodopsin structure-function as well as their evolutionary significance for dim-light vision across vertebrates.

Protein N-glycosylation is found in all domains of life and has a conserved role in glycoprotein folding and stability. In animals, glycoproteins transit through the Golgi where the N-glycans are trimmed and rebuilt with sequences that bind lectins, an innovation that greatly increases structural diversity and redundancy of glycoprotein-lectin interaction at the cell surface. Here we ask whether the natural tension between increasing diversity (glycan-protein interactions) and site multiplicity (backup and status quo) might be revealed by a phylogenic examination of glycoproteins and NXS/T(X ≠ P) N-glycosylation sites. Site loss is more likely by mutation at Asn encoded by two adenosine (A)-rich codons, while site gain is more probable by generating Ser or Thr downstream of an existing Asn. Thus mutations produce sites at novel positions more frequently than the reversal of recently lost sites, and therefore more paths though sequence space are made available to natural selection. An intra-species comparison of secretory and cytosolic proteins revealed a departure from equilibrium in sequences one-mutation-away from NXS/T and in (A) content, indicating strong selective pressures and exploration of N-glycosylation positions during vertebrate evolution. Furthermore, secretory proteins have evolved at rates proportional to N-glycosylation site number, indicating adaptive interactions between the N-glycans and underlying protein. Given the topology of the genetic code, mutation of (A) is more often nonsynonomous, and Lys, another target of many PTMs, is also encoded by two (A)-rich codons. An examination of acetyl-Lys sites in proteins indicated similar evolutionary dynamics, consistent with asymmetry of the target and recognition portions of modified sites. Our results suggest that encoding asymmetry is an ancient mechanism of evolvability that increases diversity and experimentation with PTM site positions. Strong selective pressures on PTMs may have contributed to the A+T → G+C shift in genome-wide nucleotide composition during metazoan radiation.