Archelosaurian Color Vision, Parietal Eye Loss, and the Crocodylian Nocturnal Bottleneck

Archelosaurian Color Vision, Parietal Eye Loss, and the Crocodylian Nocturnal Bottleneck Christopher A. Emerling*,1 1 Museum of Vertebrate Zoology, University of California Berkeley, Berkeley, CA *Corresponding author: E-mail: . Associate editor: Nicolas Vidal Abstract Key words: opsins, color vision, Crocodylia, Testudinata, parietal eye, nocturnal bottleneck. Introduction Article Vision is a critical sensory modality for most vertebrates, being important for foraging, predator avoidance, conspecific recognition, and migration. While aspects of the molecular basis for vision have been elucidated in many groups of vertebrates (Davies et al. 2012), Crocodylia and Testudinata (turtles) have largely been neglected. The currently species poor Crocodylia (24 extant spp.) originated 93 Ma (Oaks 2011) and is represented today by Alligatoridae, Crocodylidae, and Gavialidae. Despite being the sole descendants of a Triassic radiation of pseudosuchian archosaurs (Nesbitt 2011) and the closest living relatives of the frequently colorful and highly visual Aves, little is known about how their visual system has evolved. An important question about crocodylian vision stems from Walls (1942) seminal work on comparative ocular anatomy in vertebrates. In it, he states that crocodylian eyes “bear the stigmata of a long-continued nocturnality” (p. 613), including a rod-dominated retina, retinal regions containing cones that are “made as rod-like as possible” (Walls 1942, p. 616), a light collecting tapetum lucidum, and the absence of cone oil droplets, sclerotic rings (Nesbitt et al. 2013) and an annular pad of the lens (Walls 1942). Nagloo et al. (2016) also described a relatively large lens in crocodiles, typically associated with nocturnality, and retinal ganglion cell densities comparable to those of nocturnal squamates. Notably, many of these features are shared with mammals, which are thought to have undergone a long period of dim-light adaptation during the Mesozoic, termed a “nocturnal bottleneck” (Walls 1942; Gerkema et al. 2013). Recent studies have revealed that mammals reduced their genomic complement of light-associated genes, including both visual and nonvisual opsins (Gerkema et al. 2013) and enzymes that mitigate ultraviolet photo-oxidative damage (Kato et al. 1994; Osborn et al. 2015). If crocodylians did indeed experience a nocturnal bottleneck, a similar degree of light-associated gene inactivation and deletion should be reflected in crocodylian genomes. Testudinata is represented today by >300 species distributed across 14 families, with a fossil record that dates back to the Triassic (Joyce and Gauthier 2004; Li et al. 2008). A combination of recent genomic and fossil discoveries suggests that turtles are diapsid amniotes (Chiari et al. 2012; Schoch and Sues 2015), but the ecological origins of turtles are contentious: the very early stem testudine Odontochelys semitestacea was discovered in marine deposits (Li et al. 2008) and phylogeneticists frequently recover turtles as sister to the marine sauropterygians (Lee 2013), but various other stem turtles show evidence of terrestrial adaptations (Joyce and Gauthier 2004; Scheyer and Sander 2007; Anquetin 2011). Regardless of the precise timing of aquatic adaptation, the phylogenetic distribution of extant testudines unambiguously reconstructs the ancestor of crown turtles as a freshwater inhabitant (Joyce and Gauthier 2004). This, coupled with shifts to marine habitats (e.g., Chelonoidea), suggests that ß The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: 666 Mol. Biol. Evol. 34(3):666–676 doi:10.1093/molbev/msw265 Advance Access publication December 26, 2016 Vertebrate color vision has evolved partly through the modification of five ancestral visual opsin proteins via gene duplication, loss, and shifts in spectral sensitivity. While many vertebrates, particularly mammals, birds, and fishes, have had their visual opsin repertoires studied in great detail, testudines (turtles) and crocodylians have largely been neglected. Here I examine the genomic basis for color vision in four species of turtles and four species of crocodylians, and demonstrate that while turtles appear to vary in their number of visual opsins, crocodylians experienced a reduction in their color discrimination capacity after their divergence from Aves. Based on the opsin sequences present in their genomes and previous measurements of crocodylian cones, I provide evidence that crocodylians have co-opted the rod opsin (RH1) for cone function. This suggests that some crocodylians might have reinvented trichromatic color vision in a novel way, analogous to several primate lineages. The loss of visual opsins in crocodylians paralleled the loss of various anatomical features associated with photoreception, attributed to a “nocturnal bottleneck” similar to that hypothesized for Mesozoic mammals. I further queried crocodylian genomes for nonvisual opsins and genes associated with protection from ultraviolet light, and found evidence for gene inactivation or loss for several of these genes. Two genes, encoding parietopsin and parapinopsin, were additionally inactivated in birds and turtles, likely co-occurring with the loss of the parietal eye in these lineages. Genomic Basis for Color Vision in Turtles and Crocodylians . doi:10.1093/molbev/msw265 Results and Discussion I examined publically available genomes for species representing the three crocodylian families, Alligatoridae (Alligator mississippiensis [American alligator; 156 coverage], Alligator sinensis [Chinese alligator; 109]), Crocodylidae (Crocodylus porosus [saltwater crocodile; 74]), and Gavialidae (Gavialis gangeticus [Indian gharial; 81])(Wan et al. 2013; Green et al. 2014; Putnam et al. 2016) and cryptodiran turtles from the clades Testudinoidea (Chrysemys picta [Emydidae; painted turtle; 15, improved with cytogenetic mapping]), Americhelydia (Chelonia mydas [Cheloniidae; green sea turtle; 110]) and Trionychia (Trionychidae; Pelodiscus sinensis [Chinese softshell turtle; 105], Apalone spinifera [spiny softshell turtle; 33.4])(Wang et al. 2013; Badenhorst et al. 2015), along with outgroup taxa for comparison. I used a combination of gene predictions and BLAST searches against genomic contigs to determine the presence and functionality of 20 genes related to light-sensitivity (supplementary table S1 and dataset S1, Supplementary Material online). Turtle Visual Opsins Vertebrate phototransduction takes place in the rod and cone cells of the retina. Both cell types possess photosensitive pigments comprised of proteins called opsins covalently bound to retinoid chromophores. Upon absorbing light, these pigments initiate a phototransduction cascade that culminates in electrical signaling to the brain, resulting in vision. The ancestral vertebrate likely had (...truncated)