Comparative genomic investigation of high-elevation adaptation in ectothermic snakes Jia-Tang Lia,b,1,2, Yue-Dong Gaoc,1, Liang Xied,1, Cao Denge, Peng Shib,c, Meng-Long Guana, Song Huangf, Jin-Long Rena,g, Dong-Dong Wub,c, Li Dinga, Zi-Yan Huange, Hu Niee, Devon P. Humphreysh, David M. Hillish,2, Wen-Zhi Wangc,i,2, and Ya-Ping Zhangb,c,2 aCAS Key Laboratory of Mountain Ecological Restoration and Bioresource Utilization & Ecological Restoration and Biodiversity Conservation Key Laboratory of Sichuan Province, Chengdu Institute of Biology, Chinese Academy of Sciences, 610040 Chengdu, China; bCenter for Excellence in Animal Evolution and Genetics, Chinese Academy of Sciences, 650223 Kunming, China; cState Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, 650223 Kunming, China; dKey Laboratory of Birth Defects and Related Diseases of Women and Children, Ministry of Education, West China Second University Hospital, Sichuan University, 610041 Chengdu, China; eDepartment of Bioinformatics, DNA Stories Bioinformatics Center, 610041 Chengdu, China; fCollege of Life and Environment Sciences, Huangshan University, 245041 Huangshan, China; gUniversity of Chinese Academy of Sciences, 100049 Beijing, China; hDepartment of Integrative Biology and Biodiversity Center, University of Texas at Austin, Austin, TX 78712; and iGuizhou Academy of Testing and Analysis, 550014 Guiyang, China Contributed by David M. Hillis, June 22, 2018 (sent for review March 28, 2018; reviewed by T. Ryan Gregory and David D. Pollock) Several previous genomic studies have focused on adaptation to opportunity to study the genetics of local adaptation to extreme high elevations, but these investigations have been largely limited conditions in ectotherms. to endotherms. Snakes of the genus Thermophis are endemic to the Tibetan plateau and therefore present an opportunity to study Results and Discussion high-elevation adaptations in ectotherms. Here, we report the de Sequencing and Assembly of the Tibetan Hot-Spring Snake Genome. novo assembly of the genome of a Tibetan hot-spring snake (Ther- A total of ∼325 Gb of clean Illumina HiSeq pair-ended reads was mophis baileyi) and then compare its genome to the genomes of generated from a female T. baileyi, representing ∼185× coverage the other two species of Thermophis, as well as to the genomes of of the estimated 1.76-Gb genome (SI Appendix, Tables S1–S3). two related species of snakes that occur at lower elevations. We The resulting assembly is ∼1.74 Gb (98% of the estimated full identify 308 putative genes that appear to be under positive se- genome) with a scaffold N50 value of 2.41 Mb (SI Appendix, lection in Thermophis. We also identified genes with shared amino Table S4) and is larger than those of the other snake species with acid replacements in the high-elevation hot-spring snakes com- sequenced genomes (SI Appendix, Table S10). Approximately pared with snakes and lizards that live at low elevations, including 90% of the assembly was contained in the 889 longest scaffolds FEN1 the genes for proteins involved in DNA damage repair ( ) and (>363 kb), with the largest spanning 18.66 Mb. This assembly response to hypoxia (EPAS1). Functional assays of the FEN1 alleles reveal that the Thermophis allele is more stable under UV radia- Significance tion than is the ancestral allele found in low-elevation lizards and snakes. Functional assays of EPAS1 alleles suggest that the Ther- Thermophis mophis protein has lower transactivation activity than the low- Snakes of the genus are endemic to the Tibetan elevation forms. Our analysis identifies some convergent genetic plateau and occur at elevations over 3,500 m and present an mechanisms in high-elevation adaptation between endotherms opportunity to study the genetics mechanisms of adaptation to (based on studies of mammals) and ectotherms (based on our high-elevation conditions in ectotherms. Here, we provide a de Thermophis studies of Thermophis). novo genome of the Tibetan hot-spring snake, baileyi, and conduct a series of comparisons with other reptiles. snakes | de novo genome | comparative genomics | positive selection | We identify genes under positive selection and test properties high-elevation adaptation of allelic variants of proteins that are involved in DNA damage repair and responses to hypoxia. Functional assays reveal convergent genetic mechanisms that underlie high-elevation he Tibetan Plateau is the highest-elevation plateau on Earth, adaptation in both endotherms and ectotherms. Twith an average elevation of more than 4,000 m. The in- hospitality of its relatively extreme environment, including oxi- Author contributions: J.-T.L., D.M.H., and Y.-P.Z. designed research; J.-T.L., Y.-D.G., L.X., dative stress, UV radiation, and thermal extremes, has led to C.D., P.S., M.-L.G., D.-D.W., L.D., and Z.-Y.H. performed research; S.H. and J.-L.R. contrib- various adaptive responses in a variety of species. The biology of uted new reagents/analytic tools; J.-T.L., Y.-D.G., L.X., P.S., M.-L.G., D.-D.W., L.D., Z.-Y.H., physiological responses to high-elevation stresses has been the H.N., D.P.H., D.M.H., and W.-Z.W. analyzed data; and J.-T.L., C.D., and D.M.H. wrote subject of over a century of research (1). Recent advances in ge- the paper. nomic technologies have opened up new opportunities to explore Reviewers: T.R.G., University of Guelph; and D.D.P., University of Colorado Health Sciences Center. the genetic basis of adaptation to extreme habitats. Consequently, many recent studies have focused on genetic adaptations to high The authors declare no conflict of interest. elevations, but most of these studies have been limited to endo- Published under the PNAS license. therms (2–8), with only one genomic study of a high-elevation Data deposition: The hot-spring snakes raw reads and genome assembly reported in this paper have been deposited in the NCBI database (Bioproject no. PRJNA473624). The species of ectotherms, the frog Nanorana parkeri (9). transcriptome and the whole-genome resequencing raw reads reported in this paper The genus Thermophis includes three closely related species have been deposited in the NCBI Sequence Read Archive database (SRA accession no. (10): Thermophis baileyi, the Tibetan hot-spring snake; Thermo- SRP150039). This whole-genome shotgun project has been deposited in the GenBank phis zhaoermii, the Sichuan hot-spring snake; and Thermophis database (accession no. QLTV00000000). The version described in this paper is version shangrila, the Shangri-La hot-spring snake. All three species are QLTV01000000. endemic to the Tibetan plateau and occur at elevations over 1J.-T.L., Y.-D.G., and L.X. contributed equally to this work. 3,500 m (Fig. 1A). Although they are found around hot springs, 2To whom correspondence may be addressed. Email: [email protected], [email protected]. these species still experience extreme environmental conditions, edu, [email protected], or [email protected]. including low concentrations of molecular oxygen, high levels of This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. UV radiation, and relatively dramatic fluctuations in tempera- 1073/pnas.1805348115/-/DCSupplemental. ture on a daily basis. Therefore, these species present an ideal Published online July 31, 2018. 8406–8411 | PNAS | August 14, 2018 | vol. 115 | no. 33 www.pnas.org/cgi/doi/10.1073/pnas.1805348115 Downloaded by guest on October 2, 2021 AB60.23 +28 -5 Gallus gallus Thermophis baileyi +13 -4 ~3.6km 78.18 Anas platyrhynchos +3/-28 Gene families +30 -7 Taeniopygia guttata 60.93 Expansion 236.73 +17 -15 Contraction Nipponia nippon 7.32 +94 -32 Alligator sinensis Archosauromorpha 253.34 +26/-0 +30 -76 Alligator mississippiensis +81 -6 27.89 Chrysemys picta bellii +13 -52 Chelonia mydas +52/-0 56.68 Sauropsida +50 -3 Pelodiscus sinensis 278.54 28.27 +48 -14 Thamnophis sirtalis 35.56 +74 -9 Thermophis baileyi Pseudoxenodon macrops 71.13 +12 -30 Ophiophagus hannah ~0.5km Lepidosauria +4/-13 +49 -0 160.18 Python bivittatus +42 -7 Pogona vitticeps -59 Pseudoxenodon bambusicola / 118.58 ~0.7km 314.9 +4 +43 -7 Anolis carolinensis 143.49 +18 -27 Ophisaurus gracilis 71.08 +105 -11 Mus musculus 87.61 +23 -24 Homo sapiens 348.87 170.83 Mammalia +16 -19 Canis familiaris 222.82 +55 -5 Monodelphis domestica +16/-4 384.59 +25 -6 Ornithorhynchus anatinus 202.36 +33 -18 Xenopus tropicalis +41/-9 +40 -13 Nanorana parkeri Thermophis zhaoermii Thermophis shangrila +31 -7 Latimeria chalumnae ~4km ~3.5km 450 400 300 200 100 0 Fig. 1. History of Thermophis snakes. (A) Geographical distribution and sampling locations of the snakes. (B) Phylogenetic position of Tibetan spring snake relative to other vertebrates. The branch lengths of the phylogenetic tree are scaled to estimated divergence time. Tree topology is supported by posterior probabilities of 1.0 for all nodes. The blue bars on the nodes indicate the 95% credibility intervals of the estimated posterior distributions of the divergence times. The red circles indicate the fossil calibration times used for setting the upper and lower bounds of the estimates. The number of significantly expanded EVOLUTION (orange) and contracted (blue) gene families is designated on each branch. captures more than 94% of the core eukaryotic genes (SI Ap- evolution (7, 13). Using OrthoMCL (14), we detected 92 gene pendix, Table S5). We identified ∼791 Mb of repetitive se- clusters (including 354 genes) that were specific to the Tibetan quences, which are predominantly made up of LTRs and other hot-spring snake. These species-specific genes were significantly unknown transposable elements (TEs) (SI Appendix, Table S12), overrepresented in three major molecular functional categories comprising 45.28% of the T. baileyi genome assembly.