Snake Genomes Research Articles

New chromosome-scale genomes provide insights into marine adaptations of sea snakes (Hydrophis: Elapidae)

BackgroundSea snakes underwent a complete transition from land to sea within the last ~ 15 million years, yet they remain a conspicuous gap in molecular studies of marine adaptation in vertebrates.ResultsHere, we generate four new annotated sea snake genomes, three of these at chromosome-scale (Hydrophis major, H. ornatus and H. curtus), and perform detailed comparative genomic analyses of sea snakes and their closest terrestrial relatives. Phylogenomic analyses highlight the possibility of near-simultaneous speciation at the root of Hydrophis, and synteny maps show intra-chromosomal variations that will be important targets for future adaptation and speciation genomic studies of this system. We then used a strict screen for positive selection in sea snakes (against a background of seven terrestrial snake genomes) to identify genes over-represented in hypoxia adaptation, sensory perception, immune response and morphological development.ConclusionsWe provide the best reference genomes currently available for the prolific and medically important elapid snake radiation. Our analyses highlight the phylogenetic complexity and conserved genome structure within Hydrophis. Positively selected marine-associated genes provide promising candidates for future, functional studies linking genetic signatures to the marine phenotypes of sea snakes and other vertebrates.

Large-scale snake genome analyses provide insights into vertebrate development

Snakes are a remarkable squamate lineage with unique morphological adaptations, especially those related to the evolution of vertebrate skeletons, organs, and sensory systems. To clarify the genetic underpinnings of snake phenotypes, we assembled and analyzed 14 de novo genomes from 12 snake families. We also investigated the genetic basis of the morphological characteristics of snakes using functional experiments. We identified genes, regulatory elements, and structural variations that have potentially contributed to the evolution of limb loss, an elongated body plan, asymmetrical lungs, sensory systems, and digestive adaptations in snakes. We identified some of the genes and regulatory elements that might have shaped the evolution of vision, the skeletal system and diet in blind snakes, and thermoreception in infrared-sensitive snakes. Our study provides insights into the evolution and development of snakes and vertebrates.

Considering admixture when producing draft genomes: an example in North American ratsnakes (Pantherophis alleghaniensis/Pantherophis obsoletus).

The number of reference genomes of snakes lags behind several other vertebrate groups (e.g. birds and mammals). However, in the last two years, a concerted effort by researchers from around the world has produced new genomes of snakes representing members from several new families. Here, we present a high-quality, annotated genome of the central ratsnake (Pantherophis alleghaniensis), a member of the most diverse snake lineage, Colubroidea. Pantherophis alleghaniensis is found in the central part of the Nearctic, east of the Mississippi River. This genome was sequenced using 10X Chromium synthetic long reads and polished using Illumina short reads. The final genome assembly had an N50 of 21.82 Mb and an L50 of 22 scaffolds with a maximum scaffold length of 82.078 Mb. The genome is composed of 49.24% repeat elements dominated by long interspersed elements. We annotated this genome using transcriptome assemblies from 14 tissue types and recovered 28,368 predicted proteins. Finally, we estimated admixture proportions between two species of ratsnakes and discovered that this specimen is an admixed individual containing genomes from the western (Pantherophis obsoletus) and central ratsnakes (P. alleghaniensis). We discuss the importance of considering interspecific admixture in downstream approaches for inferring demography and phylogeny.

Conservation of Major Satellite DNAs in Snake Heterochromatin.

Repetitive DNA sequences constitute a sizeable portion of animal genomes, and tandemly organized satellite DNAs are a major part of them. They are usually located in constitutive heterochromatin clusters in or near the centromeres or telomeres, and less frequently in the interstitial parts of chromosome arms. They are also frequently accumulated in sex chromosomes. The function of these clusters is to sustain the architecture of the chromosomes and the nucleus, and to regulate chromosome behavior during mitosis and meiosis. The study of satellite DNA diversity is important for understanding sex chromosome evolution, interspecific hybridization, and speciation. In this work, we identified four satellite DNA families in the genomes of two snakes from different families: Daboia russelii (Viperidae) and Pantherophis guttatus (Colubridae) and determine their chromosomal localization. We found that one family is localized in the centromeres of both species, whereas the others form clusters in certain chromosomes or subsets of chromosomes. BLAST with snake genome assemblies showed the conservation of such clusters, as well as a subtle presence of the satellites in the interspersed manner outside the clusters. Overall, our results show high conservation of satellite DNA in snakes and confirm the "library" model of satellite DNA evolution.

Chromosome-scale genome assembly of the brown anole (Anolis sagrei), an emerging model species

Rapid technological improvements are democratizing access to high quality, chromosome-scale genome assemblies. No longer the domain of only the most highly studied model organisms, now non-traditional and emerging model species can be genome-enabled using a combination of sequencing technologies and assembly software. Consequently, old ideas built on sparse sampling across the tree of life have recently been amended in the face of genomic data drawn from a growing number of high-quality reference genomes. Arguably the most valuable are those long-studied species for which much is already known about their biology; what many term emerging model species. Here, we report a highly complete chromosome-scale genome assembly for the brown anole, Anolis sagrei – a lizard species widely studied across a variety of disciplines and for which a high-quality reference genome was long overdue. This assembly exceeds the vast majority of existing reptile and snake genomes in contiguity (N50 = 253.6 Mb) and annotation completeness. Through the analysis of this genome and population resequence data, we examine the history of repetitive element accumulation, identify the X chromosome, and propose a hypothesis for the evolutionary history of fusions between autosomes and the X that led to the sex chromosomes of A. sagrei.

Evolution of the Noncoding Features of Sea Snake Mitochondrial Genomes within Elapidae.

Mitochondrial genomes of four elapid snakes (three marine species [Emydocephalus ijimae, Hydrophis ornatus, and Hydrophis melanocephalus], and one terrestrial species [Sinomicrurus japonicus]) were completely sequenced by a combination of Sanger sequencing, next-generation sequencing and Nanopore sequencing. Nanopore sequencing was especially effective in accurately reading through long tandem repeats in these genomes. This led us to show that major noncoding regions in the mitochondrial genomes of those three sea snakes contain considerably long tandem duplications, unlike the mitochondrial genomes previously reported for same and other sea snake species. We also found a transposition of the light-strand replication origin within a tRNA gene cluster for the three sea snakes. This change can be explained by the Tandem Duplication—Random Loss model, which was further supported by remnant intervening sequences between tRNA genes. Mitochondrial genomes of true snakes (Alethinophidia) have been shown to contain duplicate major noncoding regions, each of which includes the control region necessary for regulating the heavy-strand replication and transcription from both strands. However, the control region completely disappeared from one of the two major noncoding regions for two Hydrophis sea snakes, posing evolutionary questions on the roles of duplicate control regions in snake mitochondrial genomes. The timing and molecular mechanisms for these changes are discussed based on the elapid phylogeny.

Reference genome of the California glossy snake, Arizona elegans occidentalis: A declining California Species of Special Concern.

The glossy snake (Arizona elegans) is a polytypic species broadly distributed across southwestern North America. The species occupies habitats ranging from California's coastal chaparral to the shortgrass prairies of Texas and southeastern Nebraska, to the extensive arid scrublands of central México. Three subspecies are currently recognized in California, one of which is afforded state-level protection based on the extensive loss and modification of its preferred alluvial coastal scrub and inland desert habitat. We report the first genome assembly of A. elegans occidentalis as part of the California Conservation Genomics Project (CCGP). Consistent with the reference genome strategy of the CCGP, we used Pacific Biosciences HiFi long reads and Hi-C chromatin-proximity sequencing technologies to produce a de novo assembled genome. The assembly comprises a total of 140 scaffolds spanning 1,842,602,218 base pairs, has a contig NG50 of 61 Mb, a scaffold NG50 of 136 Mb, and a BUSCO complete score of 95.9%, and is one of the most complete snake genome assemblies. The A. e. occidentalis genome will be a key tool for understanding the genomic diversity and the basis of adaptations within this species and close relatives within the hyperdiverse snake family Colubridae.

The rise of genomics in snake venom research: recent advances and future perspectives.

Snake venoms represent a danger to human health, but also a gold mine of bioactive proteins that can be harnessed for drug discovery purposes. The evolution of snakes and their venom has been studied for decades, particularly via traditional morphological and basic genetic methods alongside venom proteomics. However, while the field of genomics has matured rapidly over the past 2 decades, owing to the development of next-generation sequencing technologies, snake genomics remains in its infancy. Here, we provide an overview of the state of the art in snake genomics and discuss its potential implications for studying venom evolution and toxinology. On the basis of current knowledge, gene duplication and positive selection are key mechanisms in the neofunctionalization of snake venom proteins. This makes snake venoms important evolutionary drivers that explain the remarkable venom diversification and adaptive variation observed in these reptiles. Gene duplication and neofunctionalization have also generated a large number of repeat sequences in snake genomes that pose a significant challenge to DNA sequencing, resulting in the need for substantial computational resources and longer sequencing read length for high-quality genome assembly. Fortunately, owing to constantly improving sequencing technologies and computational tools, we are now able to explore the molecular mechanisms of snake venom evolution in unprecedented detail. Such novel insights have the potential to affect the design and development of antivenoms and possibly other drugs, as well as provide new fundamental knowledge on snake biology and evolution.

Two Reference-Quality Sea Snake Genomes Reveal Their Divergent Evolution of Adaptive Traits and Venom Systems.

True sea snakes (Hydrophiini) are among the last and most successful clades of vertebrates that show secondary marine adaptation, exhibiting diverse phenotypic traits and lethal venom systems. To better understand their evolution, we generated the first chromosome-level genomes of two representative Hydrophiini snakes, Hydrophis cyanocinctus and H. curtus. Through comparative genomics we identified a great expansion of the underwater olfaction-related V2R gene family, consisting of more than 1,000 copies in both snakes. A series of chromosome rearrangements and genomic structural variations were recognized, including large inversions longer than 30 megabase (Mb) on sex chromosomes which potentially affect key functional genes associated with differentiated phenotypes between the two species. By integrating multiomics we found a significant loss of the major weapon for elapid predation, three-finger toxin genes, which displayed a dosage effect in H. curtus. These genetic changes may imply mechanisms that drove the divergent evolution of adaptive traits including prey preferences between the two closely related snakes. Our reference-quality sea snake genomes also enrich the repositories for addressing important issues on the evolution of marine tetrapods, and provide a resource for discovering marine-derived biological products.

Impact of Repetitive DNA Elements on Snake Genome Biology and Evolution.

The distinctive biology and unique evolutionary features of snakes make them fascinating model systems to elucidate how genomes evolve and how variation at the genomic level is interlinked with phenotypic-level evolution. Similar to other eukaryotic genomes, large proportions of snake genomes contain repetitive DNA, including transposable elements (TEs) and satellite repeats. The importance of repetitive DNA and its structural and functional role in the snake genome, remain unclear. This review highlights the major types of repeats and their proportions in snake genomes, reflecting the high diversity and composition of snake repeats. We present snakes as an emerging and important model system for the study of repetitive DNA under the impact of sex and microchromosome evolution. We assemble evidence to show that certain repetitive elements in snakes are transcriptionally active and demonstrate highly dynamic lineage-specific patterns as repeat sequences. We hypothesize that particular TEs can trigger different genomic mechanisms that might contribute to driving adaptive evolution in snakes. Finally, we review emerging approaches that may be used to study the expression of repetitive elements in complex genomes, such as snakes. The specific aspects presented here will stimulate further discussion on the role of genomic repeats in shaping snake evolution.

Existence of Bov-B LINE Retrotransposons in Snake Lineages Reveals Recent Multiple Horizontal Gene Transfers with Copy Number Variation.

Transposable elements (TEs) are dynamic elements present in all eukaryotic genomes. They can “jump” and amplify within the genome and promote segmental genome rearrangements on both autosomes and sex chromosomes by disruption of gene structures. The Bovine-B long interspersed nuclear element (Bov-B LINE) is among the most abundant TE-retrotransposon families in vertebrates due to horizontal transfer (HT) among vertebrate lineages. Recent studies have shown multiple HTs or the presence of diverse Bov-B LINE groups in the snake lineage. It is hypothesized that Bov-B LINEs are highly dynamic and that the diversity reflects multiple HTs in snake lineages. Partial sequences of Bov-B LINE from 23 snake species were characterized. Phylogenetic analysis resolved at least two Bov-B LINE groups that might correspond to henophidian and caenophidian snakes; however, the tree topology differed from that based on functional nuclear and mitochondrial gene sequences. Several Bov-B LINEs of snakes showed greater than 80% similarity to sequences obtained from insects, whereas the two Bov-B LINE groups as well as sequences from the same snake species classified in different Bov-B LINE groups showed sequence similarities of less than 80%. Calculation of estimated divergence time and pairwise divergence between all individual Bov-B LINE copies suggest invasion times ranging from 79.19 to 98.8 million years ago in snakes. Accumulation of elements in a lineage-specific fashion ranged from 9 × 10−6% to 5.63 × 10−2% per genome. The genomic proportion of Bov-B LINEs varied among snake species but was not directly associated with genome size or invasion time. No differentiation in Bov-B LINE copy number between males and females was observed in any of the snake species examined. Incongruence in tree topology between Bov-B LINEs and other snake phylogenies may reflect past HT events. Sequence divergence of Bov-B LINEs between copies suggests that recent multiple HTs occurred within the same evolutionary timeframe in the snake lineage. The proportion of Bov-B LINEs varies among species, reflecting species specificity in TE invasion. The rapid speciation of snakes, coinciding with Bov-B LINE invasion in snake genomes, leads us to better understand the effect of Bov-B LINEs on snake genome evolution.

Sensory Ecology: In Sea Snake Vision, One Plus One Makes Three

Snake genomes encode only two opsins for use in retinal cones, limiting their adaptive flexibility and color vision. Research now shows that, by using alternative opsin alleles, some sea snakes may add a third opsin spectral class to their retinas.

Discovery of the Gene Encoding a Novel Small Serum Protein (SSP) of Protobothrops flavoviridis and the Evolution of SSPs.

Small serum proteins (SSPs) are low-molecular-weight proteins in snake serum with affinities for various venom proteins. Five SSPs, PfSSP-1 through PfSSP-5, have been reported in Protobothrops flavoviridis (“habu”, Pf) serum so far. Recently, we reported that the five genes encoding these PfSSPs are arranged in tandem on a single chromosome. However, the physiological functions and evolutionary origins of the five SSPs remain poorly understood. In a detailed analysis of the habu draft genome, we found a gene encoding a novel SSP, SSP-6. Structural analysis of the genes encoding SSPs and their genomic arrangement revealed the following: (1) SSP-6 forms a third SSP subgroup; (2) SSP-5 and SSP-6 were present in all snake genomes before the divergence of non-venomous and venomous snakes, while SSP-4 was acquired only by venomous snakes; (3) the composition of paralogous SSP genes in snake genomes seems to reflect snake habitat differences; and (4) the evolutionary emergence of SSP genes is probably related to the physiological functions of SSPs, with an initial snake repertoire of SSP-6 and SSP-5. SSP-4 and its derivative, SSP-3, as well as SSP-1 and SSP-2, appear to be venom-related and were acquired later.

The Indian cobra reference genome and transcriptome enables comprehensive identification of venom toxins

Snakebite envenoming is a serious and neglected tropical disease that kills ~100,000 people annually. High-quality, genome-enabled comprehensive characterization of toxin genes will facilitate development of effective humanized recombinant antivenom. We report a de novo near-chromosomal genome assembly of Naja naja, the Indian cobra, a highly venomous, medically important snake. Our assembly has a scaffold N50 of 223.35 Mb, with 19 scaffolds containing 95% of the genome. Of the 23,248 predicted protein-coding genes, 12,346 venom-gland-expressed genes constitute the ‘venom-ome’ and this included 139 genes from 33 toxin families. Among the 139 toxin genes were 19 ‘venom-ome-specific toxins’ (VSTs) that showed venom-gland-specific expression, and these probably encode the minimal core venom effector proteins. Synthetic venom reconstituted through recombinant VST expression will aid in the rapid development of safe and effective synthetic antivenom. Additionally, our genome could serve as a reference for snake genomes, support evolutionary studies and enable venom-driven drug discovery.

Evolutionary lability in Hox cluster structure and gene expression in Anolis lizards.

Hox genes orchestrate development by patterning the embryonic axis. Vertebrate Hox genes are arranged in four compact clusters, and the spacing between genes is assumed to be crucial for their function. The genomes of squamate reptiles are unusually rich and variable in transposable elements (TEs), and it has been suggested that TE invasion is responsible for the Hox cluster expansion seen in snakes and lizards. Using de novo TE prediction on 17 genomes of lizards and snakes, I show that TE content of Hox clusters are generally 50% lower than genome‐wide TE levels. However, two distantly related lizards of the species‐rich genus Anolis have Hox clusters with a TE content that approaches genomic levels. The age distribution of TEs in Anolis lizards revealed that peaks of TE activity broadly coincide with speciation events. In accordance with theoretical models of Hox cluster regulation, I find that Anolis species with many TEs in their Hox clusters show aberrant Hox gene expression patterns, suggesting a functional link between TE accumulation and embryonic development. These results are consistent with the hypothesis that TEs play a role in developmental processes as well as in evolutionary diversifications.

Isolation and characterization of abundantly-expressed cDNAs from the Harderian gland of the garter snake (Thamnophis sirtalis: Colubridae)

The Harderian gland (HG) is an orbital structure whose proteinaceous secretions pass through the nasolacrimal duct to the vomeronasal organ (VNO). Though these three structures occur in many tetrapod vertebrates, the garter snake (Thamnophis sirtalis) is one of the few vertebrates in which the passage of the proteinaceous secretions have been experimentally shown. Secreted proteins from the HG may function as transporters for chemical signals to the VNO epithelium. To investigate the proteins being produced by the HG of the garter snake, cDNA libraries were constructed from HG mRNA, and several individual cDNAs were analyzed by sequencing, RT-qPCR, and PCR on genomic DNA. Two of the three cDNAs that were characterized are abundantly expressed only in the Harderian gland and contain putative signal sequences for secretion, which makes them candidates for transporter proteins secreted from the HG. One is a member of the large lipocalin family of proteins, based on its similarity to other members of that protein family. Many lipocalins are binding/carrier proteins for a variety of ligands. The other is a family of proteins, with five members identified so far, all of unknown structure and function and present in the garter snake genome but not in other squamate genomes.

Sirtilins – the new old members of the vitamin K‐dependent coagulation factor family

Essentials Blood coagulation is driven by vitamin K (VK)‐dependent proteases.We have identified and characterized ‘sirtilin’ as an additional VK‐dependent protease.Sirtilins emerged early in the evolution of the coagulation system of vertebrates.Ubiquitous occurrence might indicate an important functional role of sirtilins. SummaryBackgroundVitamin K (VK)‐dependent proteases are major players in blood coagulation, including both the initiation and the regulation of the cascade. Five different members of this protease family have been described, comprising the following coagulation factors: factor VII, FIX, FX, protein C (PC), and prothrombin (FII). FVII, FIX, FX and PC share a typical domain architecture, with an N‐terminal γ‐carboxyglutamate (Gla) domain, two epidermal growth factor‐like (EGF) domains, and a C‐terminal trypsin‐like serine protease (SP) domain.ObjectivesWe have identified uncharacterized proteins in snake genomes showing the typical Gla–EGF1–EGF2–SP domain architecture but relatively low sequence conservation compared to known VK‐dependent proteases. On the basis of sequence analysis, we hypothesized that these proteins are functional members of the VK‐dependent protease family.Methods/resultsUsing phylogenetic analyses, we confirmed the so‐called ‘sirtilins’ as an additional VK‐dependent protease class. These proteases were found in several vertebrates, including jawless fish, cartilaginous fish, bony fish, reptiles, birds, and marsupials, but not in other mammals. The recombinant zymogen form of Thamnophis sirtalis sirtilin was produced by in vitro renaturation, and was activated with human activated FXI. The activated form of sirtilin proteolytically cleaved peptide and protein substrates, including prothrombin. Mass spectrometry‐based substrate profiling of sirtilin revealed a narrower sequence specificity than those of FIX and FX.ConclusionsThe ubiquitous occurrence of sirtilins in many vertebrate classes might indicate an important functional role. Understanding the detailed functions of sirtilins might contribute to a deeper understanding of the evolution and function of the vertebrate coagulation system.

Molecular Adaptations for Sensing and Securing Prey and Insight into Amniote Genome Diversity from the Garter Snake Genome.

Colubridae represents the most phenotypically diverse and speciose family of snakes, yet no well-assembled and annotated genome exists for this lineage. Here, we report and analyze the genome of the garter snake, Thamnophis sirtalis, a colubrid snake that is an important model species for research in evolutionary biology, physiology, genomics, behavior, and the evolution of toxin resistance. Using the garter snake genome, we show how snakes have evolved numerous adaptations for sensing and securing prey, and identify features of snake genome structure that provide insight into the evolution of amniote genomes. Analyses of the garter snake and other squamate reptile genomes highlight shifts in repeat element abundance and expansion within snakes, uncover evidence of genes under positive selection, and provide revised neutral substitution rate estimates for squamates. Our identification of Z and W sex chromosome-specific scaffolds provides evidence for multiple origins of sex chromosome systems in snakes and demonstrates the value of this genome for studying sex chromosome evolution. Analysis of gene duplication and loss in visual and olfactory gene families supports a dim-light ancestral condition in snakes and indicates that olfactory receptor repertoires underwent an expansion early in snake evolution. Additionally, we provide some of the first links between secreted venom proteins, the genes that encode them, and their evolutionary origins in a rear-fanged colubrid snake, together with new genomic insight into the coevolutionary arms race between garter snakes and highly toxic newt prey that led to toxin resistance in garter snakes.

Squamate reptiles challenge paradigms of genomic repeat element evolution set by birds and mammals

Broad paradigms of vertebrate genomic repeat element evolution have been largely shaped by analyses of mammalian and avian genomes. Here, based on analyses of genomes sequenced from over 60 squamate reptiles (lizards and snakes), we show that patterns of genomic repeat landscape evolution in squamates challenge such paradigms. Despite low variance in genome size, squamate genomes exhibit surprisingly high variation among species in abundance (ca. 25–73% of the genome) and composition of identifiable repeat elements. We also demonstrate that snake genomes have experienced microsatellite seeding by transposable elements at a scale unparalleled among eukaryotes, leading to some snake genomes containing the highest microsatellite content of any known eukaryote. Our analyses of transposable element evolution across squamates also suggest that lineage-specific variation in mechanisms of transposable element activity and silencing, rather than variation in species-specific demography, may play a dominant role in driving variation in repeat element landscapes across squamate phylogeny.

Comparative analyses of putative toxin gene homologs from an Old World viper, Daboia russelii.

Availability of snake genome sequences has opened up exciting areas of research on comparative genomics and gene diversity. One of the challenges in studying snake genomes is the acquisition of biological material from live animals, especially from the venomous ones, making the process cumbersome and time-consuming. Here, we report comparative sequence analyses of putative toxin gene homologs from Russell’s viper (Daboia russelii) using whole-genome sequencing data obtained from shed skin. When compared with the major venom proteins in Russell’s viper studied previously, we found 45–100% sequence similarity between the venom proteins and their putative homologs in the skin. Additionally, comparative analyses of 20 putative toxin gene family homologs provided evidence of unique sequence motifs in nerve growth factor (NGF), platelet derived growth factor (PDGF), Kunitz/Bovine pancreatic trypsin inhibitor (Kunitz BPTI), cysteine-rich secretory proteins, antigen 5, andpathogenesis-related1 proteins (CAP) and cysteine-rich secretory protein (CRISP). In those derived proteins, we identified V11 and T35 in the NGF domain; F23 and A29 in the PDGF domain; N69, K2 and A5 in the CAP domain; and Q17 in the CRISP domain to be responsible for differences in the largest pockets across the protein domain structures in crotalines, viperines and elapids from the in silico structure-based analysis. Similarly, residues F10, Y11 and E20 appear to play an important role in the protein structures across the kunitz protein domain of viperids and elapids. Our study highlights the usefulness of shed skin in obtaining good quality high-molecular weight DNA for comparative genomic studies, and provides evidence towards the unique features and evolution of putative venom gene homologs in vipers.