Simultaneous Expression of UV and Violet SWS1 Opsins Expands the Visual Palette in a Group of Freshwater Snakes

Simultaneous Expression of UV and Violet SWS1 Opsins Expands the Visual Palette in a Group of Freshwater Snakes Einat Hauzman,*,1,2 Michele E.R. Pierotti,3 Nihar Bhattacharyya,4 Juliana H. Tashiro,1 Carola A.M. Yovanovich,†,3 Pollyanna F. Campos,5 Dora F. Ventura,1,2 and Belinda S.W. Chang4,6 1 Department of Experimental Psychology, Psychology Institute, University of S~ao Paulo, S~ao Paulo, Brazil Hospital Israelita Albert Einstein, S~ao Paulo, Brazil 3 Department of Zoology, Institute of Biosciences, University of S~ao Paulo, S~ao Paulo, Brazil 4 Department of Cell & Systems Biology, University of Toronto, Toronto, ON, Canada 5 Laborat orio de Toxinologia Aplicada, Center of Toxins, Immune-Response and Cell Signaling (CeTICS), Instituto Butantan, S~ao Paulo, Brazil 6 Department of Ecology & Evolutionary Biology, University of Toronto, Toronto, ON, Canada † Present address: School of Life Sciences, University of Sussex, Brighton, United Kingdom *Corresponding author: E-mail: . Associate editor: Anne Yoder 2 Key words: retina, visual pigments, opsins, SWS1, spectral tuning. Introduction (Wong 1989), and transmuted photoreceptors (Walls 1942), is consistent with such an evolutionary transition. In particular, the unique phenomenon of transmutation whereby the typical morphological and functional distinction between vertebrate cones and rods leaves way to transitional states and physiological interconversion between cones and rods (cone-like rods and rod-like cones) (Walls 1942; Sim~oes et al. 2015; Schott et al. 2016; Bhattacharyya et al. 2017; Hauzman et al. 2017) suggests the evolution of novel solutions in response to constraints (gene loss) brought about by past evolutionary history. The visual processing begins with the absorption of photons by a light-sensitive derivative of vitamin A, the chromophore, covalently bound to a G protein-coupled receptor, the opsin. Photoisomerization of the chromophore induces a ß The Author(s) 2021. 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Evol. 38(12):5225–5240 doi:10.1093/molbev/msab285 Advance Access publication September 25, 2021 5225 Fast Track The visual system of extant snakes is thought to be, at least in part, the result of an ancestral fossorial and nocturnal habitat (Walls 1942; Hsiang et al. 2015), resulting in the loss of many visual structures, including ocular ciliary muscles and scleral ossicles (Walls 1942; Ott 2006), photoreceptor types and organelles (e.g., double cones, oil droplets), and genes involved in visual processing, including two of the four vertebrate cone opsin classes (SWS2 and RH2), otherwise present in the lizards (Walls 1942; Underwood 1967; Davies et al. 2009; Sim~ oes et al. 2015; Emerling 2017). Although the nature of the ancestral snake habitat is still debated (e.g., Caprette et al. 2004; Hsiang et al. 2015; Lee et al. 2016), the unique origin of novel features in response to diurnality, such as an exclusive type of double cone (Walls 1942), cone microdroplets Article Snakes are known to express a rod visual opsin and two cone opsins, only (SWS1, LWS), a reduced palette resulting from their supposedly fossorial origins. Dipsadid snakes in the genus Helicops are highly visual predators that successfully invaded freshwater habitats from ancestral terrestrial-only habitats. Here, we report the first case of multiple SWS1 visual pigments in a vertebrate, simultaneously expressed in different photoreceptors and conferring both UV and violet sensitivity to Helicops snakes. Molecular analysis and in vitro expression confirmed the presence of two functional SWS1 opsins, likely the result of recent gene duplication. Evolutionary analyses indicate that each sws1 variant has undergone different evolutionary paths with strong purifying selection acting on the UV-sensitive copy and dN/dS 1 on the violet-sensitive copy. Site-directed mutagenesis points to the functional role of a single amino acid substitution, Phe86Val, in the large spectral shift between UV and violet opsins. In addition, higher densities of photoreceptors and SWS1 cones in the ventral retina suggest improved acuity in the upper visual field possibly correlated with visually guided behaviors. The expanded visual opsin repertoire and specialized retinal architecture are likely to improve photon uptake in underwater and terrestrial environments, and provide the neural substrate for a gain in chromatic discrimination, potentially conferring unique color vision in the UV–violet range. Our findings highlight the innovative solutions undertaken by a highly specialized lineage to tackle the challenges imposed by the invasion of novel photic environments and the extraordinary diversity of evolutionary trajectories taken by visual opsin-based perception in vertebrates. Abstract Hauzman et al. . doi:10.1093/molbev/msab285 5226 speculated that both alleles might be simultaneously expressed either in distinct visual cells or coexpressed in the same photoreceptor, potentially expanding the breadth of sensitivity in the UV–violet range. Similarly, in a genus of freshwater dipsadid snakes, Helicops, two studies found polymorphisms at the same SWS1 residue (Phe/Val86) (Sim~oes et al. 2016; Hauzman et al. 2017) and suggested a possible functional advantage associated with improved sensitivity in aquatic environments. Nevertheless, in both these independent lineages of aquatic snakes, it is, at present, not known whether both alleles are expressed in the same individual. In addition, if such condition were to be demonstrated, its functional importance would depend on their site of expression within the outer retina: if both opsins were simultaneously expressed in different photoreceptors, they would give rise to distinct sensitivity curves in the UV–violet range with the potential for expanded discrimination in the UV–violet range, but if they were coexpressed within the same cone cell, they would simply give rise to a single photoreceptor class intermediate in wavelength sensitivity between the Phe86 and the Val86 SWS1 opsin. Finally, we lack a functional characterization of the Phe/Val86 substitution and its effects on SWS1 wavelength sensitivity in any snake species. The aquatic Helicops snakes occupy a variety of lotic and lentic freshwater environments from very clear to murky waters (Lema et al. 1983; Martins and Oliveira 1998; De Aguiar and Di-Bernardo 2004). Freshwater habitats have the most variable and complex underwater light fields, where dissolved organic and inorganic matter affect scatter and the selective fil (...truncated)