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.

Functional Duplication of the Short-Wavelength-Sensitive Opsin in Sea Snakes: Evidence for Reexpanded Color Sensitivity Following Ancestral Regression.

Color vision is mediated by ancient and spectrally distinct cone opsins. Yet, while there have been multiple losses of opsin genes during the evolution of tetrapods, evidence for opsin gains via functional duplication is extremely scarce. Previous studies have shown that some secondarily marine elapid snakes have acquired expanded "UV-blue" sensitivity via changes at key spectral tuning amino acid sites of the Short-Wavelength Opsin 1 (SWS1) gene. Here, we use elapid reference genomes to show that the molecular origin of this adaptation involved repeated, proximal duplications of the SWS1 gene in the fully marine Hydrophis cyanocinctus. This species possesses four intact SWS1 genes; two of these genes have the ancestral UV sensitivity, and two have a derived sensitivity to the longer wavelengths that dominate marine habitats. We suggest that this remarkable expansion of the opsin repertoire of sea snakes functionally compensates for the ancestral losses of two middle-wavelength opsins in the earliest (dim-light adapted) snakes. This provides a striking contrast to the evolution of opsins during ecological transitions in mammals. Like snakes, early mammals lost two cone photopigments; however, lineages such as bats and cetaceans underwent further opsin losses during their adaptation to dim-light environments.

Macroevolution in axial morphospace: innovations accompanying the transition to marine environments in elapid snakes.

Sea snakes in the Hydrophis-Microcephalophis clade (Elapidae) show exceptional body shape variation along a continuum from similar forebody and hindbody girths, to dramatically reduced girths of the forebody relative to hindbody. The latter is associated with specializations on burrowing prey. This variation underpins high sympatric diversity and species richness and is not shared by other marine (or terrestrial) snakes. Here, we examined a hypothesis that macroevolutionary changes in axial development contribute to the propensity, at clade level, for body shape change. We quantified variation in the number and size of vertebrae in two body regions (pre- and post-apex of the heart) for approximately 94 terrestrial and marine elapids. We found Hydrophis-Microcephalophis exhibit increased rates of vertebral evolution in the pre- versus post-apex regions compared to all other Australasian elapids. Unlike other marine and terrestrial elapids, axial elongation in Hydrophis-Microcephalophis occurs via the preferential addition of vertebrae pre-heart apex, which is the region that undergoes concomitant shifts in vertebral number and size during transitions along the relative fore- to hindbody girth axis. We suggest that this macroevolutionary developmental change has potentially acted as a key innovation in Hydrophis-Microcephalophis by facilitating novel (especially burrowing) prey specializations that are not shared with other marine snakes.

First evidence of hemiclitores in snakes.

Female genitalia are conspicuously overlooked in comparison to their male counterparts, limiting our understanding of sexual reproduction across vertebrate lineages. This study is the first complete description of the clitoris (hemiclitores) in female snakes. We describe morphological variation in size and shape (n = 9 species, 4 families) that is potentially comparable to the male intromittent organs in squamate reptiles (hemipenes). Dissection, diffusible iodine contrast-enhanced micro-CT and histology revealed that, unlike lizard hemiclitores, the snake hemiclitores are non-eversible structures. The two individual hemiclitores are separated medially by connective tissue, forming a triangular structure that extends posteriorly. Histology of the hemiclitores in Australian death adders (Acanthophis antarcticus) showed erectile tissue and strands/bundles of nerves, but no spines (as is found in male hemipenes). These histological features suggest the snake hemiclitores have functional significance in mating and definitively show that the hemiclitores are not underdeveloped hemipenes or scent glands, which have been erroneously indicated in other studies. Our discovery supports that hemiclitores have been retained across squamates and provides preliminary evidence of differences in this structure among snake species, which can be used to further understand systematics, reproductive evolution and ecology across squamate reptiles.

A global reptile assessment highlights shared conservation needs of tetrapods.

Comprehensive assessments of species' extinction risks have documented the extinction crisis1 and underpinned strategies for reducing those risks2. Global assessments reveal that, among tetrapods, 40.7% of amphibians, 25.4% of mammals and 13.6% of birds are threatened with extinction3. Because global assessments have been lacking, reptiles have been omitted from conservation-prioritization analyses that encompass other tetrapods4-7. Reptiles are unusually diverse in arid regions, suggesting that they may have different conservation needs6. Here we provide a comprehensive extinction-risk assessment of reptiles and show that at least 1,829 out of 10,196 species (21.1%) are threatened-confirming a previous extrapolation8 and representing 15.6 billion years of phylogenetic diversity. Reptiles are threatened by the same major factors that threaten other tetrapods-agriculture, logging, urban development and invasive species-although the threat posed by climate change remains uncertain. Reptiles inhabiting forests, where these threats are strongest, are more threatened than those in arid habitats, contrary to our prediction. Birds, mammals and amphibians are unexpectedly good surrogates for the conservation of reptiles, although threatened reptiles with the smallest ranges tend to be isolated from other threatened tetrapods. Although some reptiles-including most species of crocodiles and turtles-require urgent, targeted action to prevent extinctions, efforts to protect other tetrapods, such as habitat preservation and control of trade and invasive species, will probably also benefit many reptiles.

Horizontal Transposon Transfer and Its Implications for the Ancestral Ecology of Hydrophiine Snakes.

Transposable elements (TEs), also known as jumping genes, are sequences able to move or copy themselves within a genome. As TEs move throughout genomes they often act as a source of genetic novelty, hence understanding TE evolution within lineages may help in understanding environmental adaptation. Studies into the TE content of lineages of mammals such as bats have uncovered horizontal transposon transfer (HTT) into these lineages, with squamates often also containing the same TEs. Despite the repeated finding of HTT into squamates, little comparative research has examined the evolution of TEs within squamates. Here we examine a diverse family of Australo-Melanesian snakes (Hydrophiinae) to examine if the previously identified, order-wide pattern of variable TE content and activity holds true on a smaller scale. Hydrophiinae diverged from Asian elapids ~30 Mya and have since rapidly diversified into six amphibious, ~60 marine and ~100 terrestrial species that fill a broad range of ecological niches. We find TE diversity and expansion differs between hydrophiines and their Asian relatives and identify multiple HTTs into Hydrophiinae, including three likely transferred into the ancestral hydrophiine from fish. These HTT events provide the first tangible evidence that Hydrophiinae reached Australia from Asia via a marine route.

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

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 (=alethinophidian) 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.

Horizontal transfer and subsequent explosive expansion of a DNA transposon in sea kraits (Laticauda).

Transposable elements (TEs) are self-replicating genetic sequences and are often described as important 'drivers of evolution'. This driving force is because TEs promote genomic novelty by enabling rearrangement, and through exaptation as coding and regulatory elements. However, most TE insertions potentially lead to neutral or harmful outcomes, therefore host genomes have evolved machinery to suppress TE expansion. Through horizontal transposon transfer (HTT) TEs can colonize new genomes, and since new hosts may not be able to regulate subsequent replication, these TEs may proliferate rapidly. Here, we describe HTT of the Harbinger-Snek DNA transposon into sea kraits (Laticauda), and its subsequent explosive expansion within Laticauda genomes. This HTT occurred following the divergence of Laticauda from terrestrial Australian elapids approximately 15-25 Mya. This has resulted in numerous insertions into introns and regulatory regions, with some insertions into exons which appear to have altered UTRs or added sequence to coding exons. Harbinger-Snek has rapidly expanded to make up 8-12% of Laticauda spp. genomes; this is the fastest known expansion of TEs in amniotes following HTT. Genomic changes caused by this rapid expansion may have contributed to adaptation to the amphibious-marine habitat.

Demographic analyses of marine and terrestrial snakes (Elapidae) using whole genome sequences.

The question of whether spatial aspects of evolution differ in marine versus terrestrial realms has endured since Ernst Mayr's 1954 essay on marine speciation. Marine systems are often suggested to support larger and more highly connected populations, but quantitative comparisons with terrestrial systems have been lacking. Here, we compared the population histories of marine and terrestrial elapid snakes using the pairwise sequentially Markovian coalescent (PSMC) model to track historical fluctuations in species' effective population sizes (Ne ) from individual whole-genome sequences. To do this we generated a draft genome for the olive sea snake (Aiysurus laevis) and analysed this alongside six published elapid genomes and their sequence reads (marine species Hydrophis curtus, H. melanocephalus and Laticauda laticaudata; terrestrial species Pseudonaja textilis, Naja Naja and Notechis scutatus). Counter to the expectation that marine species should show higher overall Ne and less pronounced fluctuations in Ne , our analyses reveal demographic patterns that are highly variable among species and do not clearly correspond to major ecological divisions. At deeper time intervals, the four marine elapids appear to have experienced relatively stable Ne , while each terrestrial species shows a prominent upturn in Ne starting at ~4 million years ago (Ma) followed by an equally strong decline. However, over the last million years, all seven species show strong and divergent fluctuations. Estimates of Ne in the most recent intervals (~10 kya) are lowest in two of four marine species (H. melanocephalus and Laticauda), and do not correspond to contemporary range sizes in marine or terrestrial taxa.

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.

A new species of turtle-headed sea Snake (Emydocephalus: Elapidae) endemic to Western Australia.

We describe a new species of turtle-headed sea snake Emydocephalus orarius sp. nov. (Elapidae) from Western Australia's Coral Coast, Pilbara and Kimberley regions. Phylogenetic analysis of mitochondrial markers places the new species as the sister lineage to the two currently recognised species in Emydocephalus: E. annulatus from the Timor Sea reefs and Coral Sea, and E. ijimae from the Ryukyu Islands. Analysis of nuclear SNP data from the new species and E. annulatus from Australia and New Caledonia provides additional independent evidence of their evolutionary distinctiveness. The new taxon is usually morphologically diagnosable from its congeners using a combination of scalation and colour pattern characters, and appears to reach greater total lengths (>1 m in the new species versus typically ~80 cm in E. annulatus/E. ijimae). The new species is known largely from soft-bottomed trawl grounds, unlike E. annulatus and E.ijimae which usually inhabit coral reefs. The discovery of this new species brings the number of sea snake species endemic to Western Australia to six.

Patterns of intracolumnar size variation inform the heterochronic mechanisms underlying extreme body shape divergence in microcephalic sea snakes.

Sea snakes (Hydrophiinae) that specialize on burrowing eel prey have repeatedly evolved tiny heads and reduced forebody relative to hindbody girths. Previous research has found that these "microcephalic" forms have higher counts of precaudal vertebrae, and postnatal ontogenetic changes cause their hindbodies to reach greater girths relative to their forebodies. We examine variation in vertebral size along the precaudal axis of neonates and adults of three species. In the nonmicrocephalic Hydrophis curtus, these intracolumnar patterns take the form of symmetrical curved profiles, with longer vertebrae in the midbody (50% of body length) relative to distal regions. In contrast, intracolumnar profiles in the microcephalic H. macdowelli and H. obscurus are strongly asymmetrical curves (negative skewness) due to the presence of numerous, smaller-sized vertebrate in the forebody (anterior to the heart). Neonate and adult H. macdowelli and H. obscurus specimens all exhibit this pattern, implying an onset of fore- versus hindbody decoupling in the embryo stage. Based on this, we suggest plausible developmental mechanisms involving the presence and positioning of Hox boundaries and heterochronic changes in segmentation. Tests of our hypotheses would give new insights into the drivers of rapid convergent shifts in evolution, but will ultimately require studies of gene expression in the embryos of relevant taxa.

Novel vascular plexus in the head of a sea snake (Elapidae, Hydrophiinae) revealed by high-resolution computed tomography and histology.

Novel phenotypes are often linked to major ecological transitions during evolution. Here, we describe for the first time an unusual network of large blood vessels in the head of the sea snake Hydrophis cyanocinctus. MicroCT imaging and histology reveal an intricate modified cephalic vascular network (MCVN) that underlies a broad area of skin between the snout and the roof of the head. It is mostly composed of large veins and sinuses and converges posterodorsally into a large vein (sometimes paired) that penetrates the skull through the parietal bone. Endocranially, this blood vessel leads into the dorsal cerebral sinus, and from there, a pair of large veins depart ventrally to enter the brain. We compare the condition observed in H. cyanocinctus with that of other elapids and discuss the possible functions of this unusual vascular network. Sea snakes have low oxygen partial pressure in their arterial blood that facilitates cutaneous respiration, potentially limiting the availability of oxygen to the brain. We conclude that this novel vascular structure draining directly to the brain is a further elaboration of the sea snakes' cutaneous respiratory anatomy, the most likely function of which is to provide the brain with an additional supply of oxygen.

Underwater hearing in sea snakes (Hydrophiinae): first evidence of auditory evoked potential thresholds.

The viviparous sea snakes (Hydrophiinae) are a secondarily aquatic radiation of more than 60 species that possess many phenotypic adaptations to marine life. However, virtually nothing is known of the role and sensitivity of hearing in sea snakes. This study investigated the hearing sensitivity of the fully marine sea snake Hydrophis stokesii by measuring auditory evoked potential (AEP) audiograms for two individuals. AEPs were recorded from 40 Hz (the lowest frequency tested) up to 600 Hz, with a peak in sensitivity identified at 60 Hz (163.5 dB re. 1 µPa or 123 dB re. 1 µm s-2). Our data suggest that sea snakes are sensitive to low-frequency sounds but have relatively low sensitivity compared with bony fishes and marine turtles. Additional studies are required to understand the role of sound in sea snake life history and further assess these species' vulnerability to anthropogenic noise.

Ultrastructural evidence of a mechanosensory function of scale organs (sensilla) in sea snakes (Hydrophiinae).

The evolution of epidermal scales was a major innovation in lepidosaurs, providing a barrier to dehydration and physical stress, while functioning as a sensitive interface for detecting mechanical stimuli in the environment. In snakes, mechanoreception involves tiny scale organs (sensilla) that are concentrated on the surface of the head. The fully marine sea snakes (Hydrophiinae) are closely related to terrestrial hydrophiine snakes but have substantially more protruding (dome-shaped) scale organs that often cover a larger portion of the scale surface. Various divergent selection pressures in the marine environment could account for this morphological variation relating to detection of mechanical stimuli from direct contact with stimuli and/or indirect contact via water motion (i.e. 'hydrodynamic reception'), or co-option for alternate sensory or non-sensory functions. We addressed these hypotheses using immunohistochemistry, and light and electron microscopy, to describe the cells and nerve connections underlying scale organs in two sea snakes, Aipysurus laevis and Hydrophis stokesii. Our results show ultrastructural features in the cephalic scale organs of both marine species that closely resemble the mechanosensitive Meissner-like corpuscles that underlie terrestrial snake scale organs. We conclude that the scale organs of marine hydrophiines have retained a mechanosensory function, but future studies are needed to examine whether they are sensitive to hydrodynamic stimuli.

Heterochronic Shifts Mediate Ecomorphological Convergence in Skull Shape of Microcephalic Sea Snakes.

Morphological variation among the viviparous sea snakes (Hydrophiinae), a clade of fully aquatic elapid snakes, includes an extreme "microcephalic" ecomorph that has a very small head atop a narrow forebody, while the hind body is much thicker (up to three times the forebody girth). Previous research has demonstrated that this morphology has evolved at least nine times as a consequence of dietary specialization on burrowing eels, and has also examined morphological changes to the vertebral column underlying this body shape. The question addressed in this study is what happens to the skull during this extreme evolutionary change? Here we use X-ray micro-computed tomography and geometric morphometric methods to characterize cranial shape variation in 30 species of sea snakes. We investigate ontogenetic and evolutionary patterns of cranial shape diversity to understand whether cranial shape is predicted by dietary specialization, and examine whether cranial shape of microcephalic species may be a result of heterochronic processes. We show that the diminutive cranial size of microcephalic species has a convergent shape that is correlated with trophic specialization to burrowing prey. Furthermore, their cranial shape is predictable for their size and very similar to that of juvenile individuals of closely related but non-microcephalic sea snakes. Our findings suggest that heterochronic changes (resulting in pedomorphosis) have driven cranial shape convergence in response to dietary specializations in sea snakes.

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.