Distinct regulatory networks control toxin gene expression in elapid and viperid snakes.

BACKGROUND: Venom systems are ideal models to study genetic regulatory mechanisms that underpin evolutionary novelty. Snake venom glands are thought to share a common origin, but there are major distinctions between venom toxins from the medically significant snake families Elapidae and Viperidae, and toxin gene regulatory investigations in elapid snakes have been limited. Here, we used high-throughput RNA-sequencing to profile gene expression and microRNAs between active (milked) and resting (unmilked) venom glands in an elapid (Eastern Brown Snake, Pseudonaja textilis), in addition to comparative genomics, to identify cis- and trans-acting regulation of venom production in an elapid in comparison to viperids (Crotalus viridis and C. tigris). RESULTS: Although there is conservation in high-level mechanistic pathways regulating venom production (unfolded protein response, Notch signaling and cholesterol homeostasis), there are differences in the regulation of histone methylation enzymes, transcription factors, and microRNAs in venom glands from these two snake families. Histone methyltransferases and transcription factor (TF) specificity protein 1 (Sp1) were highly upregulated in the milked elapid venom gland in comparison to the viperids, whereas nuclear factor I (NFI) TFs were upregulated after viperid venom milking. Sp1 and NFI cis-regulatory elements were common to toxin gene promoter regions, but many unique elements were also present between elapid and viperid toxins. The presence of Sp1 binding sites across multiple elapid toxin gene promoter regions that have been experimentally determined to regulate expression, in addition to upregulation of Sp1 after venom milking, suggests this transcription factor is involved in elapid toxin expression. microRNA profiles were distinctive between milked and unmilked venom glands for both snake families, and microRNAs were predicted to target a diversity of toxin transcripts in the elapid P. textilis venom gland, but only snake venom metalloproteinase transcripts in the viperid C. viridis venom gland. These results suggest differences in toxin gene posttranscriptional regulation between the elapid P. textilis and viperid C. viridis. CONCLUSIONS: Our comparative transcriptomic and genomic analyses between toxin genes and isoforms in elapid and viperid snakes suggests independent toxin regulation between these two snake families, demonstrating multiple different regulatory mechanisms underpin a venomous phenotype.

Genetic insights: mapping sex-specific loci in Siamese cobra (Naja kaouthia) sheds light on the putative sex determining region.

The location of female-specific/linked loci identified in Siamese cobra (Naja kaouthia) previously has been determined through in silico chromosome mapping of the Indian cobra genome (N. naja) as a reference genome. In the present study, we used in silico chromosome mapping to identify sex-specific and linked loci in Siamese cobra. Many sex-specific and sex-linked loci were successfully mapped on the Z sex chromosome, with 227 of the 475 specific loci frequently mapped in a region covering 57 Mb and positioned at 38,992,675-95,561,177 bp of the Indian cobra genome (N. naja). This suggested the existence of a putative sex-determining region (SDR), with one specific locus (PA100000600) homologous to the TOPBP1 gene. The involvement of TOPBP1 gene may lead to abnormal synaptonemal complexes and meiotic chromosomal defects, resulting in male infertility. These findings offer valuable insights into the genetic basis and functional aspects of sex-specific traits in the Siamese cobra, which will contribute to our understanding of snake genetics and evolutionary biology.

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.

Polyphyly of the Dinurinae Looss, 1907 (Digenea: Hemiuridae) and resurrection of the Mecoderinae Skrjabin & Guschanskaja, 1954 based on novel collection of Tubulovesicula laticaudi Parukhin, 1969 from marine elapid snakes in Sri Lanka.

With one exception, the only known hemiurid trematodes that do not use teleost fishes as definitive hosts instead occur in marine elapid snakes. These comprise six species across four genera and three subfamilies, and so presumably indicate at least three independent invasions of marine snakes from teleost fishes. Here, one of these taxa, Tubulovesicula laticaudi Parukhin, 1969 (= T. orientalis Chattopadhyaya, 1970 n. syn.) is reported from Sri Lanka, collected from Shaw's sea snake Hydrophis curtus (Shaw) (Elapidae: Hydrophiinae: Hydrophinii), the annulated sea snake H. cyanocinctus Daudin and the yellow sea snake H. spiralis (Shaw) off Nayaru in the Bay of Bengal, and from H. spiralis in Portugal Bay, Gulf of Mannar. Novel molecular data, for COI mtDNA and ITS2 and 28S rDNA, are the first for a species of Tubulovesicula Yamaguti, 1934. Nominally, Tubulovesicula belongs in the Dinurinae Looss, 1907, but in phylogenetic analyses based on 28S rDNA, our sequences for T. laticaudi resolved relatively distant from that for representatives of Dinurus Looss, 1907, the type-genus, rendering the subfamily polyphyletic. Tubulovesicula laticaudi resolved closest to data for the type-species of the Plerurinae Gibson & Bray, 1979, but that subfamily is also polyphyletic. These findings lead us to re-evaluate an alternative classification considered by Gibson & Bray (1979). We propose restricting the Dinurinae for forms with a permanent sinus-organ (Dinurus, Ectenurus Looss, 1907; Erilepturus Woolcock, 1935; Paradinurus Vigueras, 1958; Qadriana Bilqees, 1971) and resurrect the Mecoderinae Skrjabin & Guschanskaja, 1954 for forms with a temporary sinus-organ (Mecoderus Manter, 1940, Allostomachicola Yamaguti, 1958, Stomachicola Yamaguti, 1934 and Tubulovesicula).

Whip it into shape: Revision of the Demansia psammophis (Schlegel, 1837) complex (Squamata: Elapidae), with a description of a new species from central Australia.

The genus Demansia Günther is the most diverse genus of Australian terrestrial elapids. A phylogenetic framework for the familiar but problematic taxa D. psammophis and D. reticulata (Gray) has been long overdue to ascertain interspecific relationships and resolve unclear taxonomic issues. Here we present an integrated molecular phylogenetic and morphological analyses to review species delineation, resulting in confirmation that both D. psammophis and D. reticulata are full species and that some populations referred to D. r. cupreiceps Storr are not distinguishable from more typical D. reticulata. We also find the widespread central Australian population (treated by most authors as part of cupreiceps) to be specifically distinct. We redescribe D. psammophis and D. reticulata to clarify morphological and geographical boundaries and describe D. cyanochasma sp. nov. based on a combination of molecular genetic markers, details of colour and pattern, adult total length and a few morphometric attributes. We also designate a lectotype for D. psammophis from the original syntype series and comment on the necessity for further taxonomic refinement of this distinctive group.

Highly Evolvable: Investigating Interspecific and Intraspecific Venom Variation in Taipans (Oxyuranus spp.) and Brown Snakes (Pseudonaja spp.).

Snake venoms are complex mixtures of toxins that differ on interspecific (between species) and intraspecific (within species) levels. Whether venom variation within a group of closely related species is explained by the presence, absence and/or relative abundances of venom toxins remains largely unknown. Taipans (Oxyuranus spp.) and brown snakes (Pseudonaja spp.) represent medically relevant species of snakes across the Australasian region and provide an excellent model clade for studying interspecific and intraspecific venom variation. Using liquid chromatography with ultraviolet and mass spectrometry detection, we analyzed a total of 31 venoms covering all species of this monophyletic clade, including widespread localities. Our results reveal major interspecific and intraspecific venom variation in Oxyuranus and Pseudonaja species, partially corresponding with their geographical regions and phylogenetic relationships. This extensive venom variability is generated by a combination of the absence/presence and differential abundance of venom toxins. Our study highlights that venom systems can be highly dynamical on the interspecific and intraspecific levels and underscores that the rapid toxin evolvability potentially causes major impacts on neglected tropical snakebites.

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.

The structural and functional divergence of a neglected three-finger toxin subfamily in lethal elapids.

Bungarus multicinctus is a widely distributed and medically important elapid snake that produces lethal neurotoxic venom. To study and enhance existing antivenom, we explore the complete repertoire of its toxin genes based on de novo chromosome-level assembly and multi-tissue transcriptome data. Comparative genomic analyses suggest that the three-finger toxin family (3FTX) may evolve through the neofunctionalization of flanking LY6E. A long-neglected 3FTX subfamily (i.e., MKA-3FTX) is also investigated. Only one MKA-3FTX gene, which evolves a different protein conformation, is under positive selection and actively transcribed in the venom gland, functioning as a major toxin effector together with MKT-3FTX subfamily homologs. Furthermore, this lethal snake may acquire self-resistance to its ß-bungarotoxin via amino acid replacements on fast-evolving KCNA2. This study provides valuable resources for further evolutionary and structure-function studies of snake toxins, which are fundamental for the development of effective antivenoms and drug candidates.

Dolichoperoides macalpini (Nicoll, 1914) (Digenea: Dolichoperoididae) infecting venomous snakes (Elapidae) across Australia: molecular characterisation and infection parameters.

Specimens of Dolichoperoides macalpini (Nicoll, 1914) (Digenea: Dolichoperoididae) were collected from Australian venomous snakes (Elapidae): Notechis scutatus Peters, 1861 and Austrelaps superbus (Günther, 1858) from Tasmania and surrounding islands and N. s. occidentalis Glauert, 1948 from wetlands near Perth, Western Australia. Despite variation in morphological measurements, genetic analysis showed that the one species of digeneans infected the snakes from all locations. This study presents the first DNA sequences for D. macalpini (internal transcribed spacer, 18S, 28S), confirming its placement in a family separate from the Reniferidae and Telorchiidae. Analysis of the infection dynamics of infection in Western Australian snakes showed significant differences in levels of infection between wetland locations, season and year of collection. Infection of D. macalpini was reported in the gastrointestinal tract, including the mouth, in freshly euthanised snakes in Western Australia, and in the lung in Tasmanian snakes, consistent with earlier reports. Differences in morphology and site of infection are suggested to be due to a combination of season and maturity of the digenean, with infection potentially occurring early in the season, as the snakes emerge from torpor. The need for research on the seasonal dynamics of infection with this parasite is discussed.

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.

Dynamic genetic differentiation drives the widespread structural and functional convergent evolution of snake venom proteinaceous toxins.

BACKGROUND: The explosive radiation and diversification of the advanced snakes (superfamily Colubroidea) was associated with changes in all aspects of the shared venom system. Morphological changes included the partitioning of the mixed ancestral glands into two discrete glands devoted for production of venom or mucous respectively, as well as changes in the location, size and structural elements of the venom-delivering teeth. Evidence also exists for homology among venom gland toxins expressed across the advanced snakes. However, despite the evolutionary novelty of snake venoms, in-depth toxin molecular evolutionary history reconstructions have been mostly limited to those types present in only two front-fanged snake families, Elapidae and Viperidae. To have a broader understanding of toxins shared among extant snakes, here we first sequenced the transcriptomes of eight taxonomically diverse rear-fanged species and four key viperid species and analysed major toxin types shared across the advanced snakes. RESULTS: Transcriptomes were constructed for the following families and species: Colubridae - Helicops leopardinus, Heterodon nasicus, Rhabdophis subminiatus; Homalopsidae - Homalopsis buccata; Lamprophiidae - Malpolon monspessulanus, Psammophis schokari, Psammophis subtaeniatus, Rhamphiophis oxyrhynchus; and Viperidae - Bitis atropos, Pseudocerastes urarachnoides, Tropidolaeumus subannulatus, Vipera transcaucasiana. These sequences were combined with those from available databases of other species in order to facilitate a robust reconstruction of the molecular evolutionary history of the key toxin classes present in the venom of the last common ancestor of the advanced snakes, and thus present across the full diversity of colubroid snake venoms. In addition to differential rates of evolution in toxin classes between the snake lineages, these analyses revealed multiple instances of previously unknown instances of structural and functional convergences. Structural convergences included: the evolution of new cysteines to form heteromeric complexes, such as within kunitz peptides (the beta-bungarotoxin trait evolving on at least two occasions) and within SVMP enzymes (the P-IIId trait evolving on at least three occasions); and the C-terminal tail evolving on two separate occasions within the C-type natriuretic peptides, to create structural and functional analogues of the ANP/BNP tailed condition. Also shown was that the de novo evolution of new post-translationally liberated toxin families within the natriuretic peptide gene propeptide region occurred on at least five occasions, with novel functions ranging from induction of hypotension to post-synaptic neurotoxicity. Functional convergences included the following: multiple occasions of SVMP neofunctionalised in procoagulant venoms into activators of the clotting factors prothrombin and Factor X; multiple instances in procoagulant venoms where kunitz peptides were neofunctionalised into inhibitors of the clot destroying enzyme plasmin, thereby prolonging the half-life of the clots formed by the clotting activating enzymatic toxins; and multiple occasions of kunitz peptides neofunctionalised into neurotoxins acting on presynaptic targets, including twice just within Bungarus venoms. CONCLUSIONS: We found novel convergences in both structural and functional evolution of snake toxins. These results provide a detailed roadmap for future work to elucidate predator-prey evolutionary arms races, ascertain differential clinical pathologies, as well as documenting rich biodiscovery resources for lead compounds in the drug design and discovery pipeline.

Bioindicator snake shows genomic signatures of natural and anthropogenic barriers to gene flow.

Urbanisation alters landscapes, introduces wildlife to novel stressors, and fragments habitats into remnant 'islands'. Within these islands, isolated wildlife populations can experience genetic drift and subsequently suffer from inbreeding depression and reduced adaptive potential. The Western tiger snake (Notechis scutatus occidentalis) is a predator of wetlands in the Swan Coastal Plain, a unique bioregion that has suffered substantial degradation through the development of the city of Perth, Western Australia. Within the urban matrix, tiger snakes now only persist in a handful of wetlands where they are known to bioaccumulate a suite of contaminants, and have recently been suggested as a relevant bioindicator of ecosystem health. Here, we used genome-wide single nucleotide polymorphism (SNP) data to explore the contemporary population genomics of seven tiger snake populations across the urban matrix. Specifically, we used population genomic structure and diversity, effective population sizes (Ne), and heterozygosity-fitness correlations to assess fitness of each population with respect to urbanisation. We found that population genomic structure was strongest across the northern and southern sides of a major river system, with the northern cluster of populations exhibiting lower heterozygosities than the southern cluster, likely due to a lack of historical gene flow. We also observed an increasing signal of inbreeding and genetic drift with increasing geographic isolation due to urbanisation. Effective population sizes (Ne) at most sites were small (< 100), with Ne appearing to reflect the area of available habitat rather than the degree of adjacent urbanisation. This suggests that ecosystem management and restoration may be the best method to buffer the further loss of genetic diversity in urban wetlands. If tiger snake populations continue to decline in urban areas, our results provide a baseline measure of genomic diversity, as well as highlighting which 'islands' of habitat are most in need of management and protection.

The omega-loop of cobra cytotoxins tolerates multiple amino acid substitutions.

Cobra cytotoxins (CTs), the three-fingered proteins, feature high amino acid sequence homology in the beta-strands and variations in the loop regions. We selected a pair of cytotoxins from Naja kaouthia crude venom to clarify the sequence-structure relationships. Using chromatography and mass spectroscopy, we separated and identified the mixture of cytotoxins 2 and 3, differentiated by the only Val 41/Ala 41 substitution. Here, using natural abundance 13C, 15N NMR-spectroscopy we performed chemical shift assignments of the signals of the both toxins in aqueous solution in the major and minor forms. Combining NOE and chemical shift data, the toxins' spatial structure was determined. Finally, we proved that the tip of the "finger"-2, or the loop-2 of cytotoxins adopts the shape of an omega-loop with a tightly-bound water molecule in its cavity. Comparison with other NMR and X-ray structures of cytotoxins possessing different amino acid sequences reveals spatial similarity in this family of proteins, including the loop-2 region, previously considered to be flexible.

Electric Blue: Molecular Evolution of Three-Finger Toxins in the Long-Glanded Coral Snake Species Calliophis bivirgatus.

The genus Calliophis is the most basal branch of the family Elapidae and several species in it have developed highly elongated venom glands. Recent research has shown that C. bivirgatus has evolved a seemingly unique toxin (calliotoxin) that produces spastic paralysis in their prey by acting on the voltage-gated sodium (NaV) channels. We assembled a transcriptome from C. bivirgatus to investigate the molecular characteristics of these toxins and the venom as a whole. We find strong confirmation that this genus produces the classic elapid eight-cysteine three-finger toxins, that δδ-elapitoxins (toxins that resemble calliotoxin) are responsible for a substantial portion of the venom composition, and that these toxins form a distinct clade within a larger, more diverse clade of C. bivirgatus three-finger toxins. This broader clade of C. bivirgatus toxins also contains the previously named maticotoxins and is somewhat closely related to cytotoxins from other elapids. However, the toxins from this clade that have been characterized are not themselves cytotoxic. No other toxins show clear relationships to toxins of known function from other species.

Convergent evolution of pain-inducing defensive venom components in spitting cobras.

Convergent evolution provides insights into the selective drivers underlying evolutionary change. Snake venoms, with a direct genetic basis and clearly defined functional phenotype, provide a model system for exploring the repeated evolution of adaptations. While snakes use venom primarily for predation, and venom composition often reflects diet specificity, three lineages of cobras have independently evolved the ability to spit venom at adversaries. Using gene, protein, and functional analyses, we show that the three spitting lineages possess venoms characterized by an up-regulation of phospholipase A2 (PLA2) toxins, which potentiate the action of preexisting venom cytotoxins to activate mammalian sensory neurons and cause enhanced pain. These repeated independent changes provide a fascinating example of convergent evolution across multiple phenotypic levels driven by selection for defense.

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.

Systematics of Calliophis intestinalis with the Resurrection of Calliophis nigrotaeniatus (Elapidae, Serpentes).

The red-bellied form of Calliophis intestinalis (Laurenti, 1768) sensu lato was originally reported from Pahang, west Malaysia. To determine the taxonomic status of this form, we examined the type specimens of Elaps sumatranus Lidth De Jeude, 1890, Calliophis intestinalis everetti (Boulenger, 1896), and Callophis furcatus var. nigrotaeniatus Peters, 1863. The results indicated that the red-bellied form of C. intestinalis should be named as Calliophis nigrotaeniatus comb. nov., whose valid species status was based on morphological and molecular analyses. We designate a lectotype and redescribe the species, which is genetically close to Calliophis bilineatus (Peters, 1881) from the Philippines, and is clearly distinguishable from other congeners by possessing a pair of gray or dark blue lateral stripes and by being bright red on the ventrum. Elaps sumatranus and C. i. everetti are relegated to subjective junior synonyms of C. nigrotaeniatus.

Highlights of animal venom research on the geographical variations of toxin components, toxicities and envenomation therapy.

Geographical variation of animal venom is common among venomous animals. This kind of intraspecific variation based on geographical location mainly concerned from envenomation cases and brought new problems in animal venom studies, including venom components regulatory mechanisms, differentiation of venom activities, and clinical treatment methods. At present, food is considered as the most related factor influencing venom development. Related research defined the variational venomous animal species by the comparison of venom components and activities in snakes, jellyfish, scorpions, cone snails, ants, parasitoid wasps, spiders and toads. In snake venom studies, researchers found that antivenom effectiveness was variated to different located venom samples. As described in some snake venom research, developing region-specific antivenom is the development trend. The difficulties of developing region-specific antivenom and theoretical solutions have been discussed. This review summarized biological studies of animal venom geographical variation by species, compared venom components and major biological activities of the vary venom from the same species, and listed the basic methods in comparing venom protein compositions and major toxicity differences to provide a comprehensive reference.

Intra-Specific Venom Variation in the Australian Coastal Taipan Oxyuranus scutellatus.

Intra-specific venom variation has the potential to provide important insights into the evolution of snake venom, but remains a relatively neglected aspect of snake venom studies. We investigated the venom from 13 individual coastal taipans Oxyuranus scutellatus from four localities on the north-east coast of Australia, spanning a distance of 2000 km. The intra-specific variation in taipan venom was considerably less than the inter-specific variation between it and the other Australian elapids to which it was compared. The electrophoretic venom profile of O. scutellatus was visually different to six other genera of Australian elapids, but not to its congener inland taipan O. microlepidotus. There was minimal geographical variation in taipan venom, as the intra-population variation exceeded the inter-population variation for enzymatic activity, procoagulant activity, and the abundance of neurotoxins. The pre-synaptic neurotoxin (taipoxin) was more abundant than the post-synaptic neurotoxins (3FTx), with a median of 11.0% (interquartile range (IQR): 9.7% to 18.3%; range: 6.7% to 23.6%) vs. a median of 3.4% (IQR: 0.4% to 6.7%; range: 0% to 8.1%). Three taipan individuals almost completely lacked post-synaptic neurotoxins, which was not associated with geography and occurred within two populations. We found no evidence of sexual dimorphism in taipan venom. Our study provides a basis for evaluating the significance of intra-specific venom variation within a phylogenetic context by comparing it to the inter-specific and inter-generic variation. The considerable intra-population variation we observed supports the use of several unpooled individuals from each population when making inter-specific comparisons.

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.