Abstract: The composition of snake venoms shows a high degree of variation at all taxonomic levels, and natural selection for diet has been implicated as a potential cause. Saw-scaled vipers (Echis) provide a good model for studying this phenomenon. The venoms of arthropod feeding species of Echis are significantly more toxic to natural scorpion prey than those of species which feed predominantly upon vertebrate prey. Although testing venom activity on natural prey is important for our understanding of the evolution of venom, natural prey species are often difficult to obtain in sufficient numbers for toxinological work. In order to test the viability of using cheaper and more easily available model organisms for toxicity assessments in evolutionary research, and the extent to which toxicity of arthropod-eating Echis venoms is increased to arthropods in general or targeted to certain groups, we conducted median lethal dosage (LD(50)) and time to death trials using the desert locust (Schistocerca gregaria) as a model arthropod, rarely consumed by wild Echis. The venoms of arthropod specialist Echis were found to be significantly more toxic to locusts than the venom of a vertebrate feeding outgroup (Bitis arietans), and one arthropod specialist venom was found to be more toxic than those species which feed upon arthropods infrequently or not at all. The venoms of arthropod specialists were also found to cause death and incapacitation faster than the vertebrate feeding outgroup. Despite some similarity of trends, there are considerable differences between the response of natural prey (scorpions) and a model arthropod (locust) to the venoms of Echis species. This suggests that when possible, natural prey rather than convenient model organisms should be used to gain an understanding of the functional significance of variation in venom composition in snakes.
Abstract: The proliferation of gene data from multiple loci of large multigene families has been greatly facilitated by considerable recent advances in sequence generation. The evolution of such gene families, which often undergo complex histories and different rates of change, combined with increases in sequence data, pose complex problems for traditional phylogenetic analyses, and in particular, those that aim to successfully recover species relationships from gene trees. Here, we implement gene tree parsimony analyses on multicopy gene family data sets of snake venom proteins for two separate groups of taxa, incorporating Bayesian posterior distributions as a rigorous strategy to account for the uncertainty present in gene trees. Gene tree parsimony largely failed to infer species trees congruent with each other or with species phylogenies derived from mitochondrial and single-copy nuclear sequences. Analysis of four toxin gene families from a large expressed sequence tag data set from the viper genus Echis failed to produce a consistent topology, and reanalysis of a previously published gene tree parsimony data set, from the family Elapidae, suggested that species tree topologies were predominantly unsupported. We suggest that gene tree parsimony failure in the family Elapidae is likely the result of unequal and/or incomplete sampling of paralogous genes and demonstrate that multiple parallel gene losses are likely responsible for the significant species tree conflict observed in the genus Echis. These results highlight the potential for gene tree parsimony analyses to be undermined by rapidly evolving multilocus gene families under strong natural selection.
Abstract: Gene duplication is a key mechanism for the adaptive evolution and neofunctionalization of gene families. Large multigene families often exhibit complex evolutionary histories as a result of frequent gene duplication acting in concordance with positive selection pressures. Alterations in the domain structure of genes, causing changes in the molecular scaffold of proteins, can also result in a complex evolutionary history and has been observed in functionally diverse multigene toxin families. Here, we investigate the role alterations in domain structure have on the tempo of evolution and neofunctionalization of multigene families using the snake venom metalloproteinases (SVMPs) as a model system. Our results reveal that the evolutionary history of viperid (Serpentes: Viperidae) SVMPs is repeatedly punctuated by domain loss, with the single loss of the cysteine-rich domain, facilitating the formation of P-II class SVMPs, occurring prior to the convergent loss of the disintegrin domain to form multiple P-I SVMP structures. Notably, the majority of phylogenetic branches where domain loss was inferred to have occurred exhibited highly significant evidence of positive selection in surface-exposed amino acid residues, resulting in the neofunctionalization of P-II and P-I SVMP classes. These results provide a valuable insight into the mechanisms by which complex gene families evolve and detail how the loss of domain structures can catalyze the accelerated evolution of novel gene paralogues. The ensuing generation of differing molecular scaffolds encoded by the same multigene family facilitates gene neofunctionalization while presenting an evolutionary advantage through the retention of multiple genes capable of encoding functionally distinct proteins.
Abstract: The development of snake antivenoms more than a century ago should have heralded effective treatment of the scourge of snakebite envenoming in impoverished, mostly rural populations around the world. That snakebite still exists today, as a widely untreated illness that maims, kills and terrifies men, women and children in vulnerable communities, is a cruel anachronism. Antivenom can be an effective, safe and affordable treatment for snakebites, but apathy, inaction and the politicisation of public health have marginalised both the problem (making snakebite arguably the most neglected of all neglected tropical diseases) and its solution. For lack of any coordinated approach, provision of antivenoms has been pushed off the public health agenda, leading to an incongruous decline in demand for these crucial antidotes, excused and fed by new priorities, an absence of epidemiological data, and a poor regulatory framework. These factors facilitated the infiltration of poor quality products that degrade user confidence and undermine legitimate producers. The result is that tens of thousands are denied an essential life-saving medicine, allowing a toll of human suffering that is a summation of many individual catastrophes. No strategy has been developed to address this problem and to overcome the intransigence and inaction responsible for the global tragedy of snakebite. Attempts to engage with the broader public health community through the World Health Organisation (WHO), GAVI, and other agencies have failed. Consequently, the toxinology community has taken on a leadership role in a new approach, the Global Snakebite Initiative, which seeks to mobilise the resources, skills and experience of scientists and clinicians for whom venoms, toxins, antivenoms, snakes and snakebites are already fields of interest. Proteomics is one such discipline, which has embraced the potential of using venoms in bio-discovery and systems biology. The fields of venomics and antivenomics have recently evolved from this discipline, offering fresh hope for the victims of snakebites by providing an exciting insight into the complexities, nature, fundamental properties and significance of venom constituents. Such a rational approach brings with it the potential to design new immunising mixtures from which to raise potent antivenoms with wider therapeutic ranges. This addresses a major practical limitation in antivenom use recognised since the beginning of the 20th century: the restriction of therapeutic effectiveness to the specific venom immunogen used in production. Antivenomic techniques enable the interactions between venoms and antivenoms to be examined in detail, and if combined with functional assays of specific activity and followed up by clinical trials of effectiveness and safety, can be powerful tools with which to evaluate the suitability of current and new antivenoms for meeting urgent regional needs. We propose two mechanisms through which the Global Snakebite Initiative might seek to end the antivenom drought in Africa and Asia: first by establishing a multidisciplinary, multicentre, international collaboration to evaluate currently available antivenoms against the venoms of medically important snakes from specific nations in Africa and Asia using a combination of proteomic, antivenomic and WHO-endorsed preclinical assessment protocols, to provide a validated evidence base for either recommending or rejecting individual products; and secondly by bringing the power of proteomics to bear on the design of new immunising mixtures to raise Pan-African and Pan-Asian polyvalent antivenoms of improved potency and quality. These products will be subject to rigorous clinical assessment. We propose radically to change the basis upon which antivenoms are produced and supplied for the developing world. Donor funding and strategic public health alliances will be sought to make it possible not only to sustain the financial viability of antivenom production partnerships, but also to ensure that patients are relieved of the costs of antivenom so that poverty is no longer a barrier to the treatment of this important, but grossly neglected public health emergency.
Abstract: Snakebite is a significant cause of death and disability in subsistent farming populations of sub-Saharan Africa. Antivenom is the most effective treatment of envenoming and is manufactured from IgG of venom-immunised horses/sheep but, because of complex fiscal reasons, there is a paucity of antivenom in sub-Saharan Africa. To address the plight of thousands of snakebite victims in savannah Nigeria, the EchiTAb Study Group organised the production, testing and delivery of antivenoms designed to treat envenoming by the most medically-important snakes in the region. The Echis saw-scaled vipers have a wide African distribution and medical importance. In an effort to maximise the clinical utility of scarce antivenom resources in Africa, we aimed to ascertain, at the pre-clinical level, to what extent the E. ocellatus-specific EchiTAbG antivenom, which was designed specifically for Nigeria, neutralised the lethal activity of venom from two other African species, E. pyramidum leakeyi and E. coloratus.
Abstract: The genus Naja Laurenti, 1768, is partitioned into four subgenera. The typical form is restricted to 11 Asian species. The name Uraeus Wagler, 1830, is revived for a group of four non-spitting cobras inhabiting savannas and open formations of Africa and Arabia, while Boulengerina Dollo, 1886, is applied to four non-spitting African species of forest cobras, including terrestrial, aquatic and semi-fossorial forms. A new subgenus is erected for seven species of African spitting cobras. We recommend the subgenus rank as a way of maximising the phylogenetic information content of classifications while retaining nomenclatural stability.
Abstract: We use a combination of phylogenetic analysis of mtDNA sequences and multivariate morphometrics to investigate the phylogeography and systematics of the Egyptian cobra (Naja haje) species complex. Phylogenetic analysis of mitochondrial haplotypes reveals a highly distinct clade of haplotypes from the Sudano-Sahelian savanna belt of West Africa, and that the haplotypes of Naja haje arabica form the sister group of North and East African N. h. haje. Multivariate morphometrics confirm the distinctness of the Arabian populations, which are consequently recognised as a full species, Naja arabica Scortecci. The Sudano-Sahelian populations are also found to represent a morphologically distinct taxon, and thus a separate species, which we describe as Naja senegalensis sp. nov. The new species differs from all other members of the N. haje complex by a combination of colour pattern and scalation characteristics (especially higher numbers of scale rows around the neck), and the possession of a unique clade of mtDNA haplotypes. The distribution of the new species includes savanna areas of West Africa, from Senegal to western Niger and Nigeria.
Abstract: The processes that drive the evolution of snake venom variability, particularly the role of diet, have been a topic of intense recent research interest. Here, we test whether extensive variation in venom composition in the medically important viper genus Echis is associated with shifts in diet. Examination of stomach and hindgut contents revealed extreme variation between the major clades of Echis in the proportion of arthropod prey consumed. The toxicity (median lethal dose, LD(50)) of representative Echis venoms to a natural scorpion prey species was found to be strongly associated with the degree of arthropod feeding. Mapping the results onto a novel Echis phylogeny generated from nuclear and mitochondrial sequence data revealed two independent instances of coevolution of venom toxicity and diet. Unlike venom LD(50), the speed with which venoms incapacitated and killed scorpions was not associated with the degree of arthropod feeding. The prey-specific venom toxicity of arthropod-feeding Echis may thus be adaptive primarily by reducing venom expenditure. Overall, our results provide strong evidence that variation in snake venom composition results from adaptive evolution driven by natural selection for different diets, and underscores the need for a multi-faceted, integrative approach to the study of the causes of venom evolution.
Abstract: Venom variation occurs at all taxonomical levels and can impact significantly upon the clinical manifestations and efficacy of antivenom therapy following snakebite. Variation in snake venom composition is thought to be subject to strong natural selection as a result of adaptation towards specific diets. Members of the medically important genus Echis exhibit considerable variation in venom composition, which has been demonstrated to co-evolve with evolutionary shifts in diet. We adopt a venom gland transcriptome approach in order to investigate the diversity of toxins in the genus and elucidate the mechanisms which result in prey-specific adaptations of venom composition.
Abstract: We analyze the phylogeny of the medically important and taxonomically unresolved viper genus Echis using four mitochondrial gene fragments. The results show that the populations of the genus fall into four main clades: the Echis carinatus, E. coloratus, E. ocellatus and E. pyramidum groups. The E. pyramidum and E. coloratus groups are sister taxa but the interrelationships of this clade and the E. ocellatus and E. carinatus groups are unresolved. The initial divergence of the genus appears to coincide with the collision between Afro-Arabia and Eurasia, and that between the E. coloratus and E. pyramidum clades appears to be associated with the opening of the Red Sea. Later land connections between Africa and Arabia may have contributed to shaping the distribution of the E. pyramidum complex. The present distribution of E. carinatus may be the result of range expansion from southern India. Taxonomically, our results provide molecular evidence for the validity of Echis omanensis, E. khosatzkii, E. borkini and E. jogeri, for the presence of unsuspected genetic diversity within the E. pyramidum complex in eastern Africa, and for the conspecificity of E. carinatus and E. multisquamatus. The status of E. leucogaster remains to be confirmed.
Abstract: Pseudonaja textilis is a widespread and common snake in eastern parts of Australia, but its distribution in New Guinea is poorly understood, and the origin of the New Guinea populations and its timing have been the subject of much speculation. Phylogenetic analysis of mitochondrial DNA sequences from three New Guinea populations of P. textilis indicates that New Guinea was colonised from two independent eastern and western migration routes most likely in the Pleistocene. One dispersal event from northern Queensland led to the populations in eastern New Guinea (Milne Bay, Oro and Central Provinces, Papua New Guinea), whereas another, from Arnhem Land to central southern New Guinea, led to the populations from the Merauke area, Indonesian Papua. The results are consistent with the effects of Pleistocene sea level changes on the physical geography of Australasia, and are thus suggestive of a natural rather than anthropogenic origin of the New Guinea populations. The taxonomic status of the New Guinean populations is discussed.
Abstract: Despite their medical interest, the phylogeny of the snake family Viperidae remains inadequately understood. Previous studies have generally focused either on the pitvipers (Crotalinae) or on the Old World vipers (Viperinae), but there has been no comprehensive molecular study of the Viperidae as a whole, leaving the affinities of key taxa unresolved. Here, we infer the phylogenetic relationships among the extant genera of the Viperidae from the sequences of four mitochondrial genes (cytochrome b, NADH subunit 4, 16S and 12S rRNA). The results confirm Azemiops as the sister group of the Crotalinae, whereas Causus is nested within the Viperinae, and thus not a basal viperid or viperine. Relationships among the major clades of Viperinae remain poorly resolved despite increased sequence information compared to previous studies. Bayesian molecular dating in conjunction with dispersal-vicariance analysis suggests an early Tertiary origin in Asia for the crown group Viperidae, and rejects suggestions of a relatively recent, early to mid-Tertiary origin of the Caenophidia.
Abstract: We describe a new species of giant spitting cobra, Naja ashei sp. nov., from eastern and north-eastern Africa. The species was previously regarded as a colour phase of the black-necked spitting cobra, N. nigricollis. However, mtDNA sequence data show it to be more closely related to N. mossambica than N. nigricollis. The new species is diagnosable from all other African spitting cobras by the possession of a unique clade of mtDNA haplotypes and a combination of colour pattern and scalation characteristics. Its distribution includes the dry lowlands of northern and eastern Kenya, north-eastern Uganda, southern Ethiopia and southern Somalia.
Abstract: Aim To investigate the phylogeography and execute a historical- demographic analysis of the Neotropical rattlesnake, Crotalus durissus, thereby testing the hypothesis of a Pleistocene central Amazon corridor of dry forest or savanna that partitioned the Amazonian rain forest into western and eastern portions. Location South America. Methods Using sequences of three mitochondrial genes, we estimated the phylogeography, gene and nucleotide diversity across the South American range of C. durissus. Tree topology tests were used to test alternative biogeographical hypotheses, and tests of population genetic structure and statistical parsimony networks and nested clade phylogeographic analysis (NCPA) were used to infer connectivity and historical population processes on both sides of the Amazon basin. Results Tree topology tests rejected the hypothesis of a coastal dispersal in favour of a central corridor scenario. Gene diversity was similar on both sides of the Amazon basin. Nucleotide diversity indicated that the populations from north of the Amazon basin represented ancestral populations. Analysis of molecular variance (AMOVA) showed that intra-population molecular variation was greater than between regions. Historical- demographic statistics showed significant population expansion south of the Amazon, and little differentiation in the north, indicating moderate past gene flow between north and south of the Amazon. The parsimony network connected clades from the Roraima and Guyana populations with Mato Grosso, suggesting an Amazonian central corridor, and NCPA supported allopatric fragmentation between north and south of the Amazon. Main conclusions The distribution of C. durissus on both sides of the Amazon basin is evidence of changes in the distribution of rain forest vegetation during the Pleistocene. Our results suggest a formerly continuous distribution of this rattlesnake along a central Amazonian corridor during the middle Pleistocene. Allopatric fragmentation inferred from NCPA is consistent with vicariance resulting from a subsequent closure of this habitat corridor. This study emphasizes the potential of trans-Amazonian open formation species to inform the debate on the past distribution of rain forests in the Amazon Basin.
Notes: Annual Meeting of the Association-for-Tropical-Biology-and-Conservation, Uberlandia, BRAZIL, 2005
Abstract: We use phylogenetic analysis of 1333 bp of mitochondrial DNA sequence to investigate the phylogeny and historical biogeography of the cobra-like elapid snakes, with special reference to the evolution of spitting and the phylogeography of the African spitting cobras, a radiation widespread in open vegetational formations throughout sub-Saharan Africa. Our results suggest that spitting adaptations appear to have evolved three times in cobras, but alternative scenarios cannot be rejected. The Asiatic Naja are monophyletic and originate from a single colonization of Asia from Africa. The radiation of the African spitting Naja appears to date back to the early Miocene and many speciation events in the group predate the Pliocene expansion of grasslands and the radiation of large grazing mammals in Africa. The cladogenic events in this complex appear to have been triggered by both ecological changes and tectonic events associated with the formation and expansion of the African Rift Valley. Taxonomically, our data confirm the inclusion of Boulengerina and Paranaja within Naja, and reveal a clade of African rainforest cobras including N. melanoleuca, Paranaja multifasciata and Boulengerina that constitutes the sister clade of the African open-formation non-spitting cobras. Naja nigricollis is polyphyletic, and we therefore recognize N. nigricincta as a separate species, more closely related to N. ashei and N. mossambica than to N. nigricollis.
Abstract: The Australian snake fauna is unique in harbouring more venomous species than non-venomous ones. Although taxonomic research on the elapid snakes of Australia goes back to the late 18th century, in stark contrast to other developed regions of the world (e.g. the continental USA), Australian snake taxonomy is very much in its infancy. Despite this, or perhaps because of this, the taxonomy of Australian snakes has been extraordinarily controversial, and many of the taxonomists involved correspondingly colourful. In this review, we explore the sometimes-tortured history of the taxonomic exploration of the venomous snake fauna of Australia, looking at some of the more colourful and notable contributors and highlighting systematic pitfalls that persist even today.
Abstract: Morphological differences between two semi-arboreal, bird-eating island pitvipers, Bothrops insularis and Gloydius shedaoensis, and their mainland relatives were investigated. Mitochondrial DNA sequence data for two genes show B. insularis to be rooted within B. jararaca. Bothrops insularis has a more anterior heart, a relatively longer tail, a longer head, and shorter fangs than B. jararaca. The greater head length is paralleled in the ecologically similar G. shedaoensis. Increased head size may represent an adaptation to the abundance of larger food items (migratory passerine birds), providing a selective advantage to snakes able to switch to larger prey at an earlier age. Furthermore, B. insularis and G. shedaoensis have converged on similar body sizes from opposite ancestral states. Other characters, including fang length, tail length and size of neonates do not show parallel variation in G. shedaoensis and B. insularis, suggesting that caution is required when interpreting character state shifts coinciding with ecological shifts in a single species only.
Abstract: Abstract Pleistocene fragmentation of the Amazonian rainforest has been hypothesized to be a major cause of Neotropical speciation and diversity. However, the role and even the reality of Pleistocene forest refugia have attracted much scepticism. In Amazonia, previous phylogeographical studies have focused mostly on organisms found in the forests themselves, and generally found speciation events to have predated the Pleistocene. However, molecular studies of open-formation taxa found both north and south of the Amazonian forests, probably because of vicariance resulting from expansion of the rainforests, may provide novel insights into the age of continuous forest cover across the Amazon basin. Here, we analyse three mitochondrial genes to infer the phylogeography of one such trans-Amazonian vicariant, the Neotropical rattlesnake (Crotalus durissus), which occupies primarily seasonal formations from Mexico to Argentina, but avoids the rainforests of Central and tropical South America. The phylogeographical pattern is consistent with gradual dispersal along the Central American Isthmus, followed by more rapid dispersal into and across South America after the uplift of the Isthmus of Panama. Low sequence divergence between populations from north and south of the Amazon rainforest is consistent with mid-Pleistocene divergence, approximately 1.1 million years ago (Ma). This suggests that the Amazonian rainforests must have become fragmented or at least shrunk considerably during that period, lending support to the Pleistocene refugia theory as an important cause of distribution patterns, if not necessarily speciation, in Amazonian forest organisms. These results highlight the potential of nonforest species to contribute to an understanding of the history of the Amazonian rainforests themselves.
Abstract: We analyze the phylogeny of three genera of Australasian elapid snakes (Acanthophis-death adders; Oxyuranus-taipans; Pseudechis-blacksnakes), using parsimony, maximum likelihood, and Bayesian analysis of sequences of the mitochondrial cytochrome b and ND4 genes. In Acanthophis and Pseudechis, we find evidence of multiple trans-Torresian sister-group relationships. Analyses of the timing of cladogenic events suggest crossings of the Torres Strait on several occasions between the late Miocene and the Pleistocene. These results support a hypothesis of repeated land connections between Australia and New Guinea in the late Cenozoic. Additionally, our results reveal undocumented genetic diversity in Acanthophis and Pseudechis, supporting the existence of more species than previously believed, and provide a phylogenetic framework for a reinterpretation of the systematics of these genera. In contrast, our Oxyuranus scutellatus samples from Queensland and two localities in New Guinea share a single haplotype, suggesting very recent (late Pleistocene) genetic exchange between New Guinean and Australian populations.
Abstract: Predator avoidance of noxious prey, aposematism and defensive mimicry are normally associated with bright, contrasting patterns and colours. However, noxious prey may be unable to evolve conspicuous coloration because of other selective constraints, such as the need to be inconspicuous to their own prey or to specialist predators. Many venomous snakes, particularly most vipers, display patterns that are apparently cryptic, but nevertheless highly characteristic, and appear to be mimicked by other, non-venomous snakes. However, predator avoidance of viper patterns has never been demonstrated experimentally. Here, the analysis of 813 avian attacks on 12,636 Plasticine snake models in the field shows that models bearing the characteristic zigzag band of the adder (Vipera berus) are attacked significantly less frequently than plain models. This suggests that predator avoidance of inconspicuously but characteristically patterned noxious prey is possible. Our findings emphasize the importance of mimicry in the ecological and morphological diversification of advanced snakes.
Abstract: We analyzed the origin and evolution of snake venom toxin families represented in both viperid and elapid snakes by means of phylogenetic analysis of the amino acid sequences of the toxins and related nonvenom proteins. Out of eight toxin families analyzed, five provided clear evidence of recruitment into the snake venom proteome before the diversification of the advanced snakes (Kunitz-type protease inhibitors, CRISP toxins, galactose-binding lectins, M12B peptidases, nerve growth factor toxins), and one was equivocal (cystatin toxins). In two others (phospholipase A(2) and natriuretic toxins), the nonmonophyly of venom toxins demonstrates that presence of these proteins in elapids and viperids results from independent recruitment events. The ANP/BNP natriuretic toxins are likely to be basal, whereas the CNP/BPP toxins are Viperidae only. Similarly, the lectins were recruited twice. In contrast to the basal recruitment of the galactose-binding lectins, the C-type lectins were shown to be Viperidae only, with the alpha-chains and beta-chains resulting from an early duplication event. These results provide strong additional evidence that venom evolved once, at the base of the advanced snake radiation, rather than multiple times in different lineages, with these toxins also present in the venoms of the "colubrid" snake families. Moreover, they provide a first insight into the composition of the earliest ophidian venoms and point the way toward a research program that could elucidate the functional context of the evolution of the snake venom proteome.
Abstract: A new species of spitting cobra Naja nubiae sp. nov. is described from north-eastern Africa. The distinctiveness of the new species is confirmed by multivariate analysis of pattern and scalation data. Phylogenetic analysis of mitochondrial DNA sequences reveals the new species to be the sister taxon of N. pallida, but with considerable levels of sequence divergence relative to that species. The populations concerned had previously been assigned to N. pallida. The new species differs from N. pallida principally in having more than one dark band across the neck and under the throat, as well as a pair of spots under the throat. It occupies a disjunct range across Egypt, the Sudan, Chad, Niger and Eritrea, where it seems to occupy primarily relatively mesic habitats. Naja mossambica is more closely related to N. nigricollis than to N. pallida and the new species. A key to the African species of Naja is presented.
Abstract: The ubiquity of venom variation in snakes poses special problems for the manufacture of antivenom and has undermined the commercial attractiveness of this class of therapeutic agent. In particular, it has been amply documented that both interspecific and intraspecific variation in venom composition can affect the neutralisation capacity of antivenoms. This may be exacerbated by the selective use of tests of venom toxicity and antivenom efficacy, such. as the lethal dose and ED50, resulting in inadequate neutralisation of time, rather than dose, dependent toxins, particularly enzymes involved in defibrinogenating, haemorrhagic and necrotising venom activities. The clinical consequences can be reduced efficacy against some important venom activities or even complete treatment failure in critical envenomations. All these factors, combined with the ongoing reduction in the number of antivenom manufacturers world-wide, and concomitant contraction in the range of available antivenoms, present significant. challenges for the treatment of snakebite in the 21st century.
Abstract: Animal venom components are of considerable interest to researchers across a wide variety of disciplines, including molecular biology, biochemistry, medicine, and evolutionary genetics. The three-finger family of snake venom peptides is a particularly interesting and biochemically complex group of venom peptides, because they are encoded by a large multigene family and display a diverse array of functional activities. In addition, understanding how this complex and highly varied multigene family evolved is an interesting question to researchers investigating the biochemical diversity of these peptides and their impact on human health. Therefore, the purpose of our study was to investigate the long-term evolutionary patterns exhibited by these snake venom toxins to understand the mechanisms by which they diversified into a large, biochemically diverse, multigene family. Our results show a much greater diversity of family members than was previously known, including a number of subfamilies that did not fall within any previously identified groups with characterized activities. In addition, we found that the long-term evolutionary processes that gave rise to the diversity of three-finger toxins are consistent with the birth-and-death model of multigene family evolution. It is anticipated that this "three-finger toxin toolkit" will prove to be useful in providing a clearer picture of the diversity of investigational ligands or potential therapeutics available within this important family.
Abstract: We have demonstrated previously that antisera from mice immunised with DNA encoding the carboxy-terminal domain (JD9) of a potent haemorrhagic metalloproteinase, jararhagin, neutralised over 70% of the haemorrhagic activity of the whole Bothrops jararaca venom. Here, we demonstrate that the JD9-specific antibody possesses extensive immunological reactivity to venom components in snakes of distinct species and genera. The polyspecific immunological reactivity of the antibody showed a correlation with amino acid sequence identity and with predicted antigenic domains of JD9-analogues in venoms of snakes with closest phylogenetic links to B. jararaca. This study further promotes the potential of DNA immunisation to generate toxin-specific antibodies with polyspecific cover. An analysis of the reactivity of the JD9-specific antisera to B. atrox complex venoms that exhibited intraspecific variation in the venom proteome revealed, however, that the toxin-specific approach to antivenom development requires a more in-depth knowledge of the target molecules than is required for conventional antivenoms.
Abstract: The evolution of the venomous function of snakes and the diversification of the toxins has been of tremendous research interest and considerable debate. It has become recently evident that the evolution of the toxins in the advanced snakes (Colubroidea) predated the evolution of the advanced, front-fanged delivery mechanisms. Historically, the venoms of snakes lacking front-fanged venom-delivery systems (conventionally grouped into the paraphyletic family Colubridae) have been largely neglected. In this study we used liquid chromatography with mass spectrometry (LC/MS) to analyze a large number of venoms from a wide array of species representing the major advanced snake clades Atractaspididae, Colubrinae, Elapidae, Homalopsinae, Natricinae, Psammophiinae, Pseudoxyrhophiinae, Xenodontinae, and Viperidae. We also present the first sequences of toxins from Azemiops feae as well as additional toxin sequences from the Colubrinae. The large body of data on molecular masses and retention times thus assembled demonstrates a hitherto unsuspected diversity of toxins in all lineages, having implications ranging from clinical management of envenomings to venom evolution to the use of isolated toxins as leads for drug design and development. Although definitive assignment of a toxin to a protein family can only be done through demonstrated structural studies such as N-terminal sequencing, the molecular mass data complemented by LC retention information, presented here, do permit formulation of reasonable hypotheses concerning snake venom evolution and potential clinical effects to a degree not possible till now, and some hypotheses of this kind are proposed here. The data will also be useful in biodiscovery.
Abstract: The evolution of venom in advanced snakes has been a focus of long-standing interest. Here we provide the first complete amino acid sequence of a colubrid toxin, which we have called alpha-colubritoxin, isolated from the Asian ratsnake Coelognathus radiatus (formerly known as Elaphe radiata), an archetypal nonvenomous snake as sold in pet stores. This potent postsynaptic neurotoxin displays readily reversible, competitive antagonism at the nicotinic receptor. The toxin is homologous with, and phylogenetically rooted within, the three-finger toxins, previously thought unique to elapids, suggesting that this toxin family was recruited into the chemical arsenal of advanced snakes early in their evolutionary history. LC-MS analysis of venoms from most other advanced snake lineages revealed the widespread presence of components of the same molecular weight class, suggesting the ubiquity of three-finger toxins across advanced snakes, with the exclusion of Viperidae. These results support the role of venom as a key evolutionary innovation in the early diversification of advanced snakes and provide evidence that forces a fundamental rethink of the very concept of nonvenomous snake.
Abstract: Death adders (genus Acanthophis) are unique among elapid snakes in both morphology and venom composition. Despite this genus being among the most divergent of all elapids, the venom has been historically regarded as relatively quite simple. In this study, liquid chromatography/mass spectrometry (LC/MS) analysis has revealed a much greater diversity in venom composition, including the presence of molecules of novel molecular weights that may represent a new class of venom component. Furthermore, significant variation exists between species and populations, which allow for the LC/MS fingerprinting of each species. Mass profiling of Acanthophis venoms clearly demonstrates the effectiveness of this technique which underpins fundamental studies ranging from chemotaxonomy to drug design.
Abstract: We describe a new species of wolf snake of the genus Lycodom from the Cardamom Mountains of southwestern Cambodia. The new species differs from other species of Lycodom ill Southeast Asia in the following combination of characteristics: dorsal scales feebly keeled, in 17 rows at midbody; 12 well defined, broad white bands across black body and six across the tail; presence of both a single preocular and a loreal, causing absence of prefrontal-eve contact; three postoculars, two anterior temporals. Visually, the new species is most similar to Lycodon subcinctus, and specimens labeled as L. subcinctus from eastern Thailand and elsewhere in Cambodia should be checked against the diagnosis of the new species.
Abstract: We describe a new species of snake of the genus Drymarchon from Falcon State, north-western Venezuela. The distinctive nature of this species, compared to the two other South American mainland taxa of Drymarchon, is supported by principal components analysis of scalation and colour pattern characters. The taxa corais and melanurus, hitherto considered conspecific, are found to be highly distinct, but homogeneous throughout their considerable ranges; consequently, we consider melanurus to be a full species, separate from Drymarchon corais.
Abstract: Phylogeographic studies using mitochondrial DNA sequence information are frequently used as the principal source of evidence to infer species boundaries. However, a critical analysis of further evidence is essential to test whether different haplotype clades identify different species. We demonstrate a hypothesis-testing approach, using a combination of phylogeographic methods, multivariate morphometrics and matrix association tests, to investigate species boundaries in eastern Brazilian pitvipers conventionally assigned to the species Bothrops leucurus and B. pradoi. Two basal haplotype clades with partly overlapping geographical distributions are identified, which could either represent two partly sympatric species, or multiple haplotypes within one organismal lineage. We use partial Mantel matrix association tests to verify whether generalized morphology, or any of four Supposedly diagnostic characters for the two species, show any association with mtDNA variation. Negative results lead to the conclusion that the haplotype clades do not denote independently evolving organismal lineages, and do not constitute separate species under any criterion.
Abstract: We describe a new species of spitting cobra of the genus Naja from central Myanmar. Multivariate analyses of morphological characters and analyses of mtDNA sequences confirm the distinctiveness of the new species. Phylogenetic analysis of the mtDNA data indicate that, among the cobra species of the southeast Asian mainland, the new species is most closely related to the Thai spitting cobra, Naja siamensis. The new species is apparently endemic to an arid region in central Myanmar.
Abstract: We infer the phylogeography of the Western Rattlesnake (Crotalus viridis) using phylogenetic analysis of mitochondrial DNA sequences from 1345 bp of the genes for cytochrome b and NADH dehydrogenase subunit 4. Two main clades are revealed: one includes populations from east and south of the Rocky Mountains (conventionally referred to as Crotalus viridis viridis and C. v. nuntius), and the other consists of populations west of the Rocky Mountains. Within the western clade, a population from southern Arizona (C. v. cerberus) represents the sister taxon to the remaining western populations. The conventional subspecies recognized in this species do not fully correspond to the phylogenetic pattern, and a review of the systematic status of several populations is needed. Our data allow the inferences that small body size evolved twice and that the ability of one population (C. v. concolor) to secrete highly lethal toxins related to Mojave toxin arose within the complex. Our phylogeny should represent the basis for further studies on the causes of geographical variation in this complex.
Abstract: We used multivariate analysis to study patterns of geographic variation in morphology in the Aesculapian snake, Elaphe longissima, in Italy and other parts of its range, in order to evaluate the status of the southern Italian form, hitherto known as E. l. romana. Although that taxon was previously regarded as weakly differentiated, a recent study based on blood proteins showed a high level of differentiation, similar to that observed between full species of European Elaphe. Fourteen characters relating to external morphology were recorded from 104 adult specimens of Elaphe longissima from 52 localities, and subjected to multivariate analysis. The results show that southern Italian specimens are clearly distinct from those of other populations. The morphological divergence is coupled with a parapatric distribution pattern and a sharp transition at the contact zone in central Italy. We found the distribution limits of the two taxa to be situated about 100 km further south than previously suggested. Parapatry among morphologically clearly distinct forms, and differences in blood plasma suggesting considerable evolutionary divergence, provide reasons for considering the southern Italian form as a separate evolutionary species from E. longissima. The name Coluber romanus Suckow, 1798 is not available for the southern Italian species, for which the oldest available name is Callopeltis longissimus var. lineata Camerano, 1891, in the combination Elaphe lineata.
Abstract: We present recent findings in the systematics of venomous snakes, with emphasis on those which affect the nomenclature and our understanding of species limits in these animals. Changes in systematics reviewed here include particularly the genera Acanthophis, Elapsoidea, Bitis, Lachesis, Porthidium, Trimeresurus/Tropidolaemus and Vipera. Other new publications of more general interest to toxinologists are also presented.
Abstract: Two hundred twenty five prey items were identified from the stomach contents and faeces of museum specimens, roadkills, and live Calloselasma rhodostoma collected throughout Southeast Asia. Overall, this snake was found to eat a broad range of vertebrate and invertebrate prey, but exhibited strong intraspecific variation in its diet Ontogenetic variation was evident in all regions, typically expressed as a transition from predominantly ectothermic prey (arthropods, amphibians, and/or reptiles) to endothermic prey (mammals and birds), although the opposite trend occurred in parts of Thailand. Among snakes of similar size, significant geographic variation emerged in the proportions of different prey taxonomic classes constituting diet Sexual variation in prey type was also detected and feeding behavior changed according to reproductive status: gravid female pitvipers ceased feeding, and adult males appeared to fast during the mating season. Possible reasons for intraspecific variation in feeding ecology are discussed.
Abstract: Developments in our understanding of the systematics of venomous snakes since the beginning of 1996 are discussed and reviewed with special emphasis on their relevance and implications for toxinologists and clinicians. Groups of snakes affected by recent developments include the genera Elapomorphus, Rhabdophis, Vermicella, Atheris, Daboia, Agkistrodon/Gloydius, Bothrops/Bothriopsis and Trimeresurus. Other important publications on venomous snakes are noted.
Abstract: Geographic variation of morphology and plumage colour was studied across a hybrid zone between allopatric populations of the carrion crow (Corvus corone corone) and the hooded crow (Corvus corone cornix) in northwestern Italy. Principal component analysis on plumage colour scores and canonical variate analysis on 37 morphometric variables indicated that a clinal geographic variation in plumage colour and morphology existed across the hybrid zone. Morphology and colour showed congruent (P < 0.005) variation. Plumage colour variation was strictly correlated with altitude (P < 0.001) and the same held true for morphology (P < 0.01). Parental allopatric populations of the carrion crow and the hooded crow inhabit different habitats in northern Italy. The hybrid zone corresponds to a piedmont belt, through which a steep ecological gradient exists. Present evidence could suggest that carrion and hooded crows have parapatrically diverged under the effects of ecological selective gradients. However, congruence in variation of different character systems argues in favour of a �phylogenetic� origin of this hybrid zone.
Abstract: Multivariate statistical methods were used to elucidate which environmental factors influence the activity patterns of free-living Malayan pit vipers, Calloselasma rhodostoma. Fourteen adult snakes were implanted with miniature radiotransmitters and located a total of 887 times in 5 months. The pit vipers usually remained coiled on the ground for several consecutive days before moving at night to a new site. Partial correlation tests revealed that the frequency and distance of movements to new sites by lagged snakes were highly positively correlated with ambient relative humidity, but not with rainfall, ambient temperature or the lunar cycle. This finding was corroborated by the frequency with which active non-tagged C. rhodostoma were encountered at night. In each site, the proportion of the snakes� bodies exposed to view was positively correlated with ambient humidity, and the snakes retreated to areas with deeper undergrowth when ambient humidity was low. Overt thermoregulatory behaviour was not observed, and implanted thermosensitive transmitters revealed that the snakes were passive thermoconformers. These findings seem to contradict much of the current literature which shows temperature to be the dominant abiotic factor affecting reptilian activity, but most herpetologists have considered only temperate forms. Ambient temperature in our tropical study site was warm and relatively constant throughout the year (mean daily range=24-33 degrees C), so the pit vipers could passively maintain body temperature within a fairly narrow range, with a daytime mean of 29.4 degrees C. Ambient relative humidity, on the other hand, was very variable, and confining exposure and activity to periods of high ambient humidity may be necessary to avoid dehydration.
Abstract: Changes to our understanding of venomous snake systematics, and the consequent changes in the nomenclature of these animals, have traditionally been a great source of confusion among biomedical researchers. This paper aims to facilitate access to the taxonomic literature by presenting a synopsis of the changes in venomous snake systematics that have taken place recently (primarily since 1992), together with some comments on the implications of these changes for toxinologists and clinicians. Some long-standing problems in venomous snake taxonomy receive special attention. This includes Asiatic Naja, Asiatic Agkistrodon/Gloydius, Bothrops and related genera, Trimeresurus, Echis, Daboia (including Daboia russellii) and Vipera. It is hoped that this synopsis will result in the use of a more up-to-date and interpretable nomenclature for venomous snakes in the toxinological literature.
Abstract: The Indochinese spitting cobra, Naja siamensis Laurenti, 1768, is redescribed In order to avoid future confusion with other Asiatic Naja species, and to fix this scientific name irrefutably, we designate a neotype for this species, and designate the same specimen as neotype for the junior synonym Naja naja isanensis Nutaphand, 1982. The pattern of geographic variation in this species is investigated by means of multivariate analysis of morphological characters. We summarize the literature concerning this species, with particular emphasis on separating information concerning N. siamensis from that relating to other Asiatic cobra species. This species is of considerable medical importance, and we present case histories of local envenoming and snake venom ophthalmia.
Abstract: We analyze the systematics of pitviper populations normally assigned to the species Bothrops atrox, B. moojeni, and B. marajoensis using multivariate analysis of morphological characters. Most populations of B. moojeni are clearly distinct from B. atrox, but there are phenotypically intermediate populations where the ranges of the two forms meet, suggesting the existence of a hybrid zone. The status of the populations currently assigned to B. marajoensis remains uncertain, as the populations assigned to this species are heterogeneous and poorly differentiated from B. atrox. This study confirms the results of previous workers, who found low levels of divergence within the B. atrox species complex.
Abstract: Until recently, all Asiatic cobra populations were regarded as belonging to one single species, Naja naja. Recent revisions have shown that there are in fact at least 10 full species of Asiatic Naja. In order to allow the existing literature to be reconciled with these recent discoveries, an interpretation of the older nomenclature is provided. Problematic areas, especially concerning the species N. sumatrana and N. siamensis, are highlighted.
Abstract: Venom composition within snake species can show considerable geographical variation, an important consideration because bites by conspecific populations may differ in symptomatology and require different treatments. The underlying causes of this phenomenon have never been explained. Here we present evidence that the variation in the venom of the pitviper Calloselasma rhodostoma (Serpentes: Viperidae) is closely associated with its diet. We also evaluated other possible causes of geographic variation in venom using partial Mantel tests and independent contrasts, but rejected both contemporary gene flow (estimated from geographical proximity) and the phylogenetic relationships (assessed by analysis of mitochondrial DNA) among populations as important influences upon venom evolution. As the primary function of viperid venom is to immobilize and digest prey and prey animals vary in their susceptibility to venom, we suggest that geographical variation in venom composition reflects natural selection for feeding on local prey.
Abstract: The Malayan pit viper (Calloselasma rhodostoma) is of major clinical significance both as a leading cause of snakebite and as the source of ancrod (Arvin). Although its venom has been extensively studied, the degree to which venom composition varies between individuals is poorly known. We individually analysed the venoms of over 100 C. rhodostoma using isoelectric focusing. In all populations, females produced an intense band that was absent from all males, and significant ontogenetic variation was detected. Principal components analysis of the banding profiles also revealed strong geographic variation, which was significantly congruent with variation in the biological activities of the venom (phosphodiesterase, alkalinephosphoesterase, L-amino acid oxidase, arginine ester hydrolase, 5'-nucleotidase, thrombin-like enzyme, haemorrhagic activity). Studies of captive-bred snakes indicate that the intraspecific variation in venom is genetically inherited rather than environmentally induced. The intraspecific variation in venom composition and biological activity could be of applied importance to snakebite therapy, both in correct diagnosis of the source of envenomation and in the development of a more effective antivenom. Greater attention should be given to the source of C. rhodostoma venom used in research to ensure reproducibility of results.
Abstract: Bothrops xanthogrammus/asper, B. atrox and Lachesis muta are probably responsible for most cases of severe envenoming in Ecuador. In recent years, the most widely used antivenom ('Myn' Ronti, imported from Mexico) has proved clinically ineffective. There is an urgent need to identify an effective alternative for clinical testing. Five antivenoms with activity against Bothrops venoms were compared using standard World Health Organization rodent and in vitro assays: (i) 'Myn', Ronti Mexico SA ('B. atrox', 'Crotalus terrificus'), (ii) Instituto Butantan (Bothrops polyvalent, Brazil), (iii) Instituto Nacional de Hygiene y Medicina Tropical (Bothrops polyvalent, Ecuador), (iv) Instituto Nacional de Salud (B. asper, C. durissus and Lachesis muta, Colombia), and (v) Laboratorios Probiol (Bothrops, Lachesis and Crotalus, Colombia). The venoms against which these antivenoms were tested were Ecuadorian B. atrox, B. asper and B. xanthogrammus. Brazilian antivenom proved to be the most effective, followed by the Ecudorian and Colombian antivenoms. Mexican antivenom was completely ineffective in neutralizing the lethal effects of Ecuadorian Bothrops venoms. Monospecific Brazilian L. muta antivenom (Instituto Butantan) proved effective against Ecuadorian L. muta venom, but the Colombian polyspecific antivenoms did not. Clinical trials of Brazilian and Ecuadorian antivenoms are planned in the Amazon region of Ecuador in the near future.
Abstract: Observed patterns of within-species geographic variation may reflect phylogenetic history or ecogenetic adaptation, and it is frequently desirable to evaluate their relative contributions. Phylogenetic information can be recovered from relatively neutrally evolving molecular markers, but to what extent are they useful in understanding the causes of observed geographic patterns within species? In the lacertid lizard Gallotia galloti (Western Canary islands), it allows the colonization sequence to be hypothesized, giving a new perspective on the causes of morphological differentiation between island populations. In South-East Asian pit-vipers, venom evolution can be evaluated in relation to biogeographic processes and current ecology. At a finer geographic scale, alternative historical and geological hypotheses can be tested to explain within-island micropeographic variation in G. galloti in Tenerife. Similar work on Anolis oculatus within the island of Dominica, however, raises questions concerning some of the assumptions behind a molecular phylogenetic approach.
Abstract: We analyze the population systematics of Asiatic cobras in Indochina, China and the Andaman Islands by means of comparative sequencing of the cytochrome oxidase subunit I gene of the mitochondrial DNA molecule and multivariate analysis of morphological characters. Canonical variate analysis and mtDNA sequence information reveal that the cobras of this region comprise four distinct species: Naja atra from China and northern Vietnam, Naja kaouthia from Burma, central Thailand, Cambodia and southern Vietnam, Naja siamensis from Thailand, Cambodia and southern Vietnam, and Naja sagittifera from the Andaman Islands. The subspecies N. kaouthia suphanensis Nutaphand 1986 shows no mtDNA sequence difference from typical N. kaouthia from central Thailand, and multivariate analysis does not reveal differences in general phenotypic profile; the subspecies is therefore synonymised with Naja kaouthia. The cytochrome oxidase subunit I gene, little used in molecular taxonomy, is shown to be well suited for studies at the species level, as it shows taxonomically useful levels of interspecific divergence but low levels of intraspecific variation; this is particularly relevant for studies of rare species, where sample size is a problem. The combination of multivariate morphometrics and molecular systematics can be particularly powerful in resolving systematic problems in such cases.
Abstract: Because of possible variation in venom composition, an understanding of venomous snake systematics is of great importance for the optimization of antivenom treatment of snakebite patients. Intraspecific variation in the morphology of many venomous snakes complicates the definition and identification of some species when allopatric populations are involved. Selectively neutral or near-neutral mtDNA sequences can reveal evolutionary relationships obscured by ecogenetically-caused morphological variation. We use comparative sequencing of the cytochrome oxidase subunit 1 gene to reveal the existence of a widespread, cryptic species of spitting cobra from southeast Asia. This species, Naja siamensis, is widely sympatric with other Asiatic cobra species. This may be of considerable medical significance, and calls for further research into venom composition in Asiatic cobras.
Abstract: The population systematics of the cobras of the genus Naja in the Indian subcontinent and in central Asia were investigated using multivariate analysis of morphometric characters recorded from preserved material. The cobras from this region, formerly thought to belong to a single species, comprise three well-differentiated species: N. naja occurs throughout India, as well as in Pakistan, Nepal, and Bangladesh; N. oxiana is found in Soviet central Asia, northeastern Iran, Afghanistan, northern Pakistan, and northwestern India; N. kaouthia is found from Delhi east to Assam, and south to Vietnam and northern Malaysia. The pattern of intraspecific geographic variation in N. naja is investigated and discussed with reference to previously described subspecies, which are not recognized.
Abstract: We compare the fang morphology of the Asiatic cobra species, with particular reference to the occurrence of spitting behavior. The two nonspitting species, Naja naja and N. oxiana, have unmodified fangs with large venom discharge orifices; the other seven species, which have all been reported to spit, show varying degrees of reduction in discharge orifice size. There is considerable variation in the degree of fang modification among the species of spitting cobras, and only a small reduction in discharge orifice size seems to enable spitting. Some species show geographic variation in fang structure, and N. philippinensis shows sexual dimorphism in discharge orifice size.
Abstract: The population affinities of the Asiatic cobras of the genus Naja are investigated, using multivariate analysis of a range of morphological characters. This complex, which was formerly thought to be monospecific, consists of at least eight full species. In some cases, species whose bites require different antivenoms occur sympatrically. The new understanding of the systematics of the Asiatic cobra complex calls for a reappraisal of cobra antivenom use in Asia, and for more research into venom composition.
Abstract: A series of quantitative procedures (1 - pattern of anagenesis in putative phylogentic trees; 2 - <<correlation>> based tests comparing observed and hypothesized patterns: 3 - congruence assessed by random re-sampling) are tested against a series of <<known>> cases of ecogenetically or phylogenetically caused geographic variation to establish whether they can distinguish between these two causes. The former two procedures can distinguish between these causes in some circumstances, but the last procedure is not particularly useful for this purpose because independent morphological character systems can have congruent patterns of geographic variation even when they are ecogenetically caused.
