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

BACKGROUND: Sea 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. RESULTS: Here, 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. CONCLUSIONS: We 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.

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.

The Genome of Shaw's Sea Snake (Hydrophis curtus) Reveals Secondary Adaptation to Its Marine Environment.

The transition of terrestrial snakes to marine life ∼10 Ma is ideal for exploring adaptive evolution. Sea snakes possess phenotype specializations including laterally compressed bodies, paddle-shaped tails, valvular nostrils, cutaneous respiration, elongated lungs, and salt glands, yet, knowledge on the genetic underpinnings of the transition remains limited. Herein, we report the first genome of Shaw's sea snake (Hydrophis curtus) and use it to investigate sea snake secondary marine adaptation. A hybrid assembly strategy obtains a high-quality genome. Gene family analyses date a pulsed coding-gene expansion to ∼20 Ma, and these genes associate strongly with adaptations to marine environments. Analyses of selection pressure and convergent evolution discover the rapid evolution of protein-coding genes, and some convergent features. Additionally, 108 conserved noncoding elements appear to have evolved quickly, and these may underpin the phenotypic changes. Transposon elements may contribute to adaptive specializations by inserting into genomic regions around functionally related coding genes. The integration of genomic and transcriptomic analyses indicates independent origins and different components in sea snake and terrestrial snake venom; the venom gland of the sea snake harbors the highest PLA2 (17.23%) expression in selected elapids and these genes may organize tandemly in the genome. These analyses provide insights into the genetic mechanisms that underlay the secondary adaptation to marine and venom production of this sea snake.

Visual adaptation of opsin genes to the aquatic environment in sea snakes.

BACKGROUND: Evolutionary transitions from terrestrial to aquatic life history cause drastic changes in sensory systems. Indeed, the drastic changes in vision have been reported in many aquatic amniotes, convergently. Recently, the opsin genes of the full-aquatic sea snakes have been reported. However, those of the amphibious sea snakes have not been examined in detail. RESULTS: Here, we investigated opsin genes and visual pigments of sea snakes. We determined the sequences of SWS1, LWS, and RH1 genes from one terrestrial, three amphibious and four fully-aquatic elapids. Amino acid replacements at four and one spectra-tuning positions were found in LWS and RH1, respectively. We measured or predicted absorption of LWS and RH1 pigments with A1-derived retinal. During their evolution, blue shifts of LWS pigments have occurred stepwise in amphibious sea snakes and convergently in both amphibious and fully-aquatic species. CONCLUSIONS: Blue shifted LWS pigments may have adapted to deep water or open water environments dominated by blue light. The evolution of opsins differs between marine mammals (cetaceans and pinnipeds) and sea snakes in two fundamental ways: (1) pseudogenization of opsins in marine mammals; and (2) large blue shifts of LWS pigments in sea snakes. It may be possible to explain these two differences at the level of photoreceptor cell composition given that cone and rod cells both exist in mammals whereas only cone cells exist in fully-aquatic sea snakes. We hypothesize that the differences in photoreceptor cell compositions may have differentially affected the evolution of opsins in divergent amniote lineages.

Vertebral evolution and ontogenetic allometry: The developmental basis of extreme body shape divergence in microcephalic sea snakes.

Snakes exhibit a diverse array of body shapes despite their characteristically simplified morphology. The most extreme shape changes along the precloacal axis are seen in fully aquatic sea snakes (Hydrophiinae): "microcephalic" sea snakes have tiny heads and dramatically reduced forebody girths that can be less than a third of the hindbody girth. This morphology has evolved repeatedly in sea snakes that specialize in hunting eels in burrows, but its developmental basis has not previously been examined. Here, we infer the developmental mechanisms underlying body shape changes in sea snakes by examining evolutionary patterns of changes in vertebral number and postnatal ontogenetic growth. Our results show that microcephalic species develop their characteristic shape via changes in both the embryonic and postnatal stages. Ontogenetic changes cause the hindbodies of microcephalic species to reach greater sizes relative to their forebodies in adulthood, suggesting heterochronic shifts that may be linked to homeotic effects (axial regionalization). However, microcephalic species also have greater numbers of vertebrae, especially in their forebodies, indicating that somitogenetic effects also contribute to evolutionary changes in body shape. Our findings highlight sea snakes as an excellent system for studying the development of segment number and regional identity in the snake precloacal axial skeleton.

Phototactic tails: Evolution and molecular basis of a novel sensory trait in sea snakes.

Dermal phototaxis has been reported in a few aquatic vertebrate lineages spanning fish, amphibians and reptiles. These taxa respond to light on the skin of their elongate hind-bodies and tails by withdrawing under cover to avoid detection by predators. Here, we investigated tail phototaxis in sea snakes (Hydrophiinae), the only reptiles reported to exhibit this sensory behaviour. We conducted behavioural tests in 17 wild-caught sea snakes of eight species by illuminating the dorsal surface of the tail and midbody skin using cold white, violet, blue, green and red light. Our results confirmed phototactic tail withdrawal in the previously studied Aipysurus laevis, revealed this trait for the first time in A. duboisii and A. tenuis, and suggested that tail photoreceptors have peak spectral sensitivities between blue and green light (457-514 nm). Based on these results, and an absence of photoresponses in five Aipysurus and Hydrophis species, we tentatively infer that tail phototaxis evolved in the ancestor of a clade of six Aipysurus species (comprising 10% of all sea snakes). Quantifying tail damage, we found that the probability of sustaining tail injuries was not influenced by tail phototactic ability in snakes. Gene profiling showed that transcriptomes of both tail skin and body skin lacked visual opsins but contained melanopsin (opn4x) in addition to key genes of the retinal regeneration and phototransduction cascades. This work suggests that a nonvisual photoreceptor (e.g., Gq rhabdomeric) signalling pathway underlies tail phototaxis, and provides candidate gene targets for future studies of this unusual sensory innovation in reptiles.

Rates of population differentiation and speciation are decoupled in sea snakes.

Comparative phylogeography can inform many macroevolutionary questions, such as whether species diversification is limited by rates of geographical population differentiation. We examined the link between population genetic structure and species diversification in the fully aquatic sea snakes (Hydrophiinae) by comparing mitochondrial phylogeography across northern Australia in 16 species from two closely related clades that show contrasting diversification dynamics. Contrary to expectations from theory and several empirical studies, our results show that, at the geographical scale studied here, rates of population differentiation and speciation are not positively linked in sea snakes. The eight species sampled from the rapidly speciating Hydrophis clade have weak population differentiation that lacks geographical structure. By contrast, all eight sampled Aipysurus-Emydocephalus species show clear geographical patterns and many deep intraspecific splits, but have threefold slower speciation rates. Alternative factors, such as ecological specialization, species duration and geographical range size, may underlie rapid speciation in sea snakes.

Dynamic Expansions and Retinal Expression of Spectrally Distinct Short-Wavelength Opsin Genes in Sea Snakes.

The photopigment-encoding visual opsin genes that mediate color perception show great variation in copy number and adaptive function across vertebrates. An open question is how this variation has been shaped by the interaction of lineage-specific structural genomic architecture and ecological selection pressures. We contribute to this issue by investigating the expansion dynamics and expression of the duplicated Short-Wavelength-Sensitive-1 opsin (SWS1) in sea snakes (Elapidae). We generated one new genome, 45 resequencing datasets, 10 retinal transcriptomes, and 81 SWS1 exon sequences for sea snakes, and analyzed these alongside 16 existing genomes for sea snakes and their terrestrial relatives. Our analyses revealed multiple independent transitions in SWS1 copy number in the marine Hydrophis clade, with at least three lineages having multiple intact SWS1 genes: the previously studied Hydrophis cyanocinctus and at least two close relatives of this species; Hydrophis atriceps and Hydrophis fasciatus; and an individual Hydrophis curtus. In each lineage, gene copy divergence at a key spectral tuning site resulted in distinct UV and Violet/Blue-sensitive SWS1 subtypes. Both spectral variants were simultaneously expressed in the retinae of H. cyanocinctus and H. atriceps, providing the first evidence that these SWS1 expansions confer novel phenotypes. Finally, chromosome annotation for nine species revealed shared structural features in proximity to SWS1 regardless of copy number. If these features are associated with SWS1 duplication, expanded opsin complements could be more common in snakes than is currently recognized. Alternatively, selection pressures specific to aquatic environments could favor improved chromatic distinction in just some lineages.

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.

De Novo Venom-Gland Transcriptomics of Spine-Bellied Sea Snake (Hydrophis curtus) from Penang, Malaysia-Next-Generation Sequencing, Functional Annotation and Toxinological Correlation.

Envenomation resulted from sea snake bite is a highly lethal health hazard in Southeast Asia. Although commonly caused by sea snakes of Hydrophiinae, each species is evolutionarily distinct and thus, unveiling the toxin gene diversity within individual species is important. Applying next-generation sequencing, this study investigated the venom-gland transcriptome of Hydrophis curtus (spine-bellied sea snake) from Penang, West Malaysia. The transcriptome was de novo assembled, followed by gene annotation and sequence analyses. Transcripts with toxin annotation were only 96 in number but highly expressed, constituting 48.18% of total FPKM in the overall transcriptome. Of the 21 toxin families, three-finger toxins (3FTX) were the most abundantly expressed and functionally diverse, followed by phospholipases A2. Lh_FTX001 (short neurotoxin) and Lh_FTX013 (long neurotoxin) were the most dominant 3FTXs expressed, consistent with the pathophysiology of envenomation. Lh_FTX001 and Lh_FTX013 were variable in amino acid compositions and predicted epitopes, while Lh_FTX001 showed high sequence similarity with the short neurotoxin from Hydrophis schistosus, supporting cross-neutralization effect of Sea Snake Antivenom. Other toxins of low gene expression, for example, snake venom metalloproteinases and L-amino acid oxidases not commonly studied in sea snake venom were also identified, enriching the knowledgebase of sea snake toxins for future study.

New Environment, New Invaders-Repeated Horizontal Transfer of LINEs to Sea Snakes.

Although numerous studies have found horizontal transposon transfer (HTT) to be widespread across metazoans, few have focused on HTT in marine ecosystems. To investigate potential recent HTTs into marine species, we searched for novel repetitive elements in sea snakes, a group of elapids which transitioned to a marine habitat at most 18 Ma. Our analysis uncovered repeated HTTs into sea snakes following their marine transition. The seven subfamilies of horizontally transferred LINE retrotransposons we identified in the olive sea snake (Aipysurus laevis) are transcribed, and hence are likely still active and expanding across the genome. A search of 600 metazoan genomes found all seven were absent from other amniotes, including terrestrial elapids, with the most similar LINEs present in fish and marine invertebrates. The one exception was a similar LINE found in sea kraits, a lineage of amphibious elapids which independently transitioned to a marine environment 25 Ma. Our finding of repeated horizontal transfer events into marine snakes greatly expands past findings that the marine environment promotes the transfer of transposons. Transposons are drivers of evolution as sources of genomic sequence and hence genomic novelty. We identified 13 candidate genes for HTT-induced adaptive change based on internal or neighboring HTT LINE insertions. One of these, ADCY4, is of particular interest as a part of the KEGG adaptation pathway "Circadian Entrainment." This provides evidence of the ecological interactions between species influencing evolution of metazoans not only through specific selection pressures, but also by contributing novel genomic material.

Spectral Diversification and Trans-Species Allelic Polymorphism during the Land-to-Sea Transition in Snakes.

Snakes are descended from highly visual lizards [1] but have limited (probably dichromatic) color vision attributed to a dim-light lifestyle of early snakes [2-4]. The living species of front-fanged elapids, however, are ecologically very diverse, with ∼300 terrestrial species (cobras, taipans, etc.) and ∼60 fully marine sea snakes, plus eight independently marine, amphibious sea kraits [1]. Here, we investigate the evolution of spectral sensitivity in elapids by analyzing their opsin genes (which are responsible for sensitivity to UV and visible light), retinal photoreceptors, and ocular lenses. We found that sea snakes underwent rapid adaptive diversification of their visual pigments when compared with their terrestrial and amphibious relatives. The three opsins present in snakes (SWS1, LWS, and RH1) have evolved under positive selection in elapids, and in sea snakes they have undergone multiple shifts in spectral sensitivity toward the longer wavelengths that dominate below the sea surface. Several relatively distantly related Hydrophis sea snakes are polymorphic for shortwave sensitive visual pigment encoded by alleles of SWS1. This spectral site polymorphism is expected to confer expanded "UV-blue" spectral sensitivity and is estimated to have persisted twice as long as the predicted survival time for selectively neutral nuclear alleles. We suggest that this polymorphism is adaptively maintained across Hydrophis species via balancing selection, similarly to the LWS polymorphism that confers allelic trichromacy in some primates. Diving sea snakes thus appear to share parallel mechanisms of color vision diversification with fruit-eating primates.

Venom gland transcriptomics and microRNA profiling of juvenile and adult yellow-bellied sea snake, Hydrophis platurus, from Playa del Coco (Guanacaste, Costa Rica).

With an extensive range in tropical and subtropical waters of Indo-Australian region and the Pacific coast of the American continent, the yellow-bellied sea snake, Hydrophis platurus, is the most broadly distributed snake species on our planet. We report a comparative analysis of the mRNA and microRNA transcriptional profiles of the venom glands of Costa Rican juvenile and adult yellow-bellied sea snakes, and correlate these datasets with the previously reported venom proteome gathered from an adult specimen of the same population of Playa del Coco. The transcriptomic profiles are qualitatively and quantitatively remarkably similar across both age classes, being overwhelmingly (>99%) dominated by only three toxin classes, three-finger toxins (3FTx, 88%), phospholipases A2 (PLA2, 10-11%), and cysteine-rich secretory proteins (CRISP, 1%). The only appreciable difference between the transcriptomes of juvenile and adult Costa Rican yellow-bellied sea snake lies in the relative abundance of short and long neurotoxic 3FTxs. Comparison of venom gland transcriptome and venom proteome datasets suggest that the venom arsenal of adult H. platurus from Playa del Coco (CR) is subjected to post-transcriptional modulation, involving repression of the translation of pelamitoxin a-encoding mRNAs and enhancement of the translational activity of PLA2 and CRISP mRNAs.