Eye-Transcriptome and Genome-Wide Sequencing for Scolecophidia: Implications for Inferring the Visual System of the Ancestral Snake

GBE Eye-Transcriptome and Genome-Wide Sequencing for Scolecophidia: Implications for Inferring the Visual System of the Ancestral Snake David J. Gower 1,*, James F. Fleming2,3, Davide Pisani 2,4, Freek J. Vonk5, Harald M. I. Kerkkamp6, Leo Peichl 7,8, Sonja Meimann7, Nicholas R. Casewell 9, Christiaan V. Henkel 6,10, ~es 2,11,12,* Michael K. Richardson 6, Kate L. Sanders11, and Bruno F. Simo Life Sciences, The Natural History Museum, London, United Kingdom 2 School of Life Sciences, University of Bristol, Bristol, United Kingdom 3 Institute for Advanced Biosciences, Keio University, Yamagata, Japan 4 School of Earth Sciences, University of Bristol, Bristol, United Kingdom 5 Naturalis Biodiversity Center, Leiden, The Netherlands 6 Institute of Biology, University of Leiden, Leiden, The Netherlands 7 Institute of Cellular and Molecular Anatomy, Dr. Senckenberg Anatomy, Goethe University Frankfurt, Frankfurt am Main, Germany 8 Institute of Clinical Neuroanatomy, Dr. Senckenberg Anatomy, Goethe University Frankfurt, Frankfurt am Main, Germany 9 Centre for Snakebite Research & Interventions, Liverpool School of Tropical Medicine, Liverpool, United Kingdom 10 Faculty of Veterinary Medicine, Norwegian University of Life Sciences, Ås, Norway 11 School of Biological Sciences, University of Adelaide, Adelaide, South Australia, Australia 12 School of Biological and Marine Sciences, University of Plymouth, Plymouth, United Kingdom *Corresponding authors. Email: ; . Accepted: 8 October 2021 Abstract Molecular genetic data have recently been incorporated in attempts to reconstruct the ecology of the ancestral snake, though this has been limited by a paucity of data for one of the two main extant snake taxa, the highly fossorial Scolecophidia. Here we present and analyze vision genes from the first eye-transcriptomic and genome-wide data for Scolecophidia, for Anilios bicolor, and A. bituberculatus, respectively. We also present immunohistochemistry data for retinal anatomy and visual opsin-gene expression in Anilios. Analyzed in the context of 19 lepidosaurian genomes and 12 eye transcriptomes, the new genome-wide and transcriptomic data provide evidence for a much more reduced visual system in Anilios than in non-scolecophidian (¼alethinophidian) snakes and in lizards. In Anilios, there is no evidence of the presence of 7 of the 12 genes associated with alethinophidian photopic (cone) phototransduction. This indicates extensive gene loss and many of these candidate gene losses occur also in highly fossorial mammals with reduced vision. Although recent phylogenetic studies have found evidence for scolecophidian paraphyly, the loss in Anilios of visual genes that are present in alethinophidians implies that the ancestral snake had a better-developed visual system than is known for any extant scolecophidian. Key words: gene loss, opsins, phylogeny, regressive evolution, Squamata, vision. Introduction The origin of major lineages (higher taxa) is a topic of broad interest in evolutionary biology (e.g., Darwin 1859; Smith and Szathmary 1997; Holland et al. 2008; Kemp 2015; Lane 2015; Eme et al. 2017), and the origin of snakes (Serpentes) is exemplary in this respect (e.g., Bellairs and Underwood ß The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (https://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact Genome Biol. Evol. 13(12) https://doi.org/10.1093/gbe/evab253 Advance Access publication 17 November 2021 1 1 GBE Gower et al. Significance The origin of snakes is subject to ongoing, high-profile debate. We present the first eye-transcriptomic and substantial genome-wide sequencing data for any scolecophidian snake (Scolecophidia are dedicated burrowers and form onehalf of the oldest divergence in the snake tree). Comparative analyses of the presence of functional vision genes among lizards and snakes strongly indicate that the visual system (especially that adapted for bright-light vision) of scolecophidians is too reduced to be a good model for the visual system of the ancestral snake, thus challenging the hypothesis that snakes evolved from extreme burrowers. 2 However, thus far these data are available only for a relatively small sample of alethinophidians, such that ascertaining whether inferred genomic changes occurred in the snake stem or the alethinophidian stem (within the snake crown group) has not been possible. Furthermore, interpretation of the few molecular genetic data that are available has been complicated by disagreement among phylogeneticists as to whether Scolecophidia is a monophyletic (i.e., comprising one half of the basal divergence among crown snakes: e.g., Lee et al. 2007) or paraphyletic (e.g., Wiens et al. 2012; Miralles et al. 2018; Burbrink et al. 2020) outgroup of all other crown snakes (Alethinophidia). Thus, the extent to which phenotypes of extant scolecophidians might represent highly derived adaptations to fossoriality and/or potentially plesiomorphic conditions present in the ancestral snake remains contentious. Here, we contribute to the debate by presenting analyses of vision-associated gene sequence data from the first eyetranscriptome and genome-wide sequence data and retinal immunohistochemistry data for Scolecophidia. The visual system has played a prominent role in debates about snake origins (Bellairs and Underwood 1951; Caprette et al. 2004; ~ es et al. 2015; Miralles et al. 2018), and we expect geSimo nomic data on vision genes to provide insight into the ecophenotype of the ancestral snake. For example, ancestral fossoriality can be predicted to have resulted in the last common ancestor of extant snakes having a proportionately greater loss of functionality in genes associated with brightlight (photopic) than dim-light (scotopic) vision, such as occurs, for example, in highly fossorial mammals (Emerling and Springer 2014). Results Vision-Gene Complements The presence or absence results for vision genes based on BLAST and phylogenetic analyses are summarized in table 1. Of the 48 vision-associated genes studied here, all but seven occur in snakes. These seven are the genes coding for the phosphodiesterase subunit PDE6A, the rhodopsin kinase GRK1, the transducin gamma subunit GNGT1, the solute carrier SLC24A1, the retinol dehydrogenase RDH11, and the Genome Biol. Evol. 13(12) https://doi.org/10.1093/gbe/evab253 Advance Access publication 17 November 2021 1951; Bellairs 1972; Rieppel 1988; Caprette et al. 2004; Lee ~es et al. 2015; 2005; Caldwell 2007; Hsiang et al. 2015; Simo Yi and Norell 2015; Emerling 2017; da Silva et al. 2018; Miralles et al. 2018). Res (...truncated)