Crotalus Durissus Ruruima: Current Knowledge on Natural History, Medical Importance, and Clinical Toxinology.

Crotalus durissus ruruima is a rattlesnake subspecies mainly found in Roraima, the northernmost state of Brazil. Envenomings caused by this subspecies lead to severe clinical manifestations (e.g. respiratory muscle paralysis, rhabdomyolysis, and acute renal failure) that can lead to the victim's death. In this review, we comprehensively describe C. d. ruruima biology and the challenges this subspecies poses for human health, including morphology, distribution, epidemiology, venom cocktail, clinical envenoming, and the current and future specific treatment of envenomings by this snake. Moreover, this review presents maps of the distribution of the snake subspecies and evidence that this species is responsible for some of the most severe envenomings in the country and causes the highest lethality rates. Finally, we also discuss the efficacy of the Brazilian horse-derived antivenoms to treat C. d. ruruima envenomings in Roraima state.

A Meta-Analysis of the Protein Components in Rattlesnake Venom.

The specificity and potency of venom components give them a unique advantage in developing various pharmaceutical drugs. Though venom is a cocktail of proteins, rarely are the synergy and association between various venom components studied. Understanding the relationship between various components of venom is critical in medical research. Using meta-analysis, we observed underlying patterns and associations in the appearance of the toxin families. For Crotalus, Dis has the most associations with the following toxins: PDE; BPP; CRL; CRiSP; LAAO; SVMP P-I and LAAO; SVMP P-III and LAAO. In Sistrurus venom, CTL and NGF have the most associations. These associations can predict the presence of proteins in novel venom and understand synergies between venom components for enhanced bioactivity. Using this approach, the need to revisit the classification of proteins as major components or minor components is highlighted. The revised classification of venom components is based on ubiquity, bioactivity, the number of associations, and synergies. The revised classification can be expected to trigger increased research on venom components, such as NGF, which have high biomedical significance. Using hierarchical clustering, we observed that the genera's venom compositions were similar, based on functional characteristics rather than phylogenetic relationships.

A Clot Twist: Extreme Variation in Coagulotoxicity Mechanisms in Mexican Neotropical Rattlesnake Venoms.

Rattlesnakes are a diverse clade of pit vipers (snake family Viperidae, subfamily Crotalinae) that consists of numerous medically significant species. We used validated in vitro assays measuring venom-induced clotting time and strength of any clots formed in human plasma and fibrinogen to assess the coagulotoxic activity of the four medically relevant Mexican rattlesnake species Crotalus culminatus, C. mictlantecuhtli, C. molossus, and C. tzabcan. We report the first evidence of true procoagulant activity by Neotropical rattlesnake venom in Crotalus culminatus. This species presented a strong ontogenetic coagulotoxicity dichotomy: neonates were strongly procoagulant via Factor X activation, whereas adults were pseudo-procoagulant in that they converted fibrinogen into weak, unstable fibrin clots that rapidly broke down, thereby likely contributing to net anticoagulation through fibrinogen depletion. The other species did not activate clotting factors or display an ontogenetic dichotomy, but depleted fibrinogen levels by cleaving fibrinogen either in a destructive (non-clotting) manner or via a pseudo-procoagulant mechanism. We also assessed the neutralization of these venoms by available antivenom and enzyme-inhibitors to provide knowledge for the design of evidence-based treatment strategies for envenomated patients. One of the most frequently used Mexican antivenoms (Bioclon Antivipmyn®) failed to neutralize the potent procoagulant toxic action of neonate C. culminatus venom, highlighting limitations in snakebite treatment for this species. However, the metalloprotease inhibitor Prinomastat substantially thwarted the procoagulant venom activity, while 2,3-dimercapto-1-propanesulfonic acid (DMPS) was much less effective. These results confirm that venom-induced Factor X activation (a procoagulant action) is driven by metalloproteases, while also suggesting Prinomastat as a more promising potential adjunct treatment than DMPS for this species (with the caveat that in vivo studies are necessary to confirm this potential clinical use). Conversely, the serine protease inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF) inhibited the direct fibrinogen cleaving actions of C. mictlantecuhtli venom, thereby revealing that the pseudo-procoagulant action is driven by kallikrein-type serine proteases. Thus, this differential ontogenetic variation in coagulotoxicity patterns poses intriguing questions. Our results underscore the need for further research into Mexican rattlesnake venom activity, and also highlights potential limitations of current antivenom treatments.

Local and systemic effects caused by Crotalus durissus terrificus, Crotalus durissus collilineatus, and Crotalus durissus cascavella snake venoms in swiss mice.

INTRODUCTION: Crotalus envenomations cause serious complications and can be fatal without appropriate treatment. Venom isoforms present and inter/intraspecific variations in the venom composition can result in different symptoms presented by bites by snakes from the same species but from different geographical regions. We comparatively evaluated the local and systemic effects caused by Crotalus durissus terrificus (Cdt), C.d. collilineatus (Cdcolli), and C.d. cascavella (Cdcasc) envenomation. METHODS: Venom chromatography was performed. Proteolytic, phospholipase, and LAAO activities were analyzed. Edema, myotoxicity, hepatotoxicity, nephrotoxicity, and coagulation alterations were evaluated. RESULTS: The venom SDS-PAGE analyses found the presence of convulxin, gyroxin, crotoxin, and crotamine in Cdt and Cdcolli venoms. Crotamine was not present in the Cdcasc venom. Cdt, Cdcollli, and Cdcasc venoms had no proteolytic activity. Only Cdcasc and Cdt venoms had phospholipase activity. LAAO activity was observed in Cdcolli and Cdcasc venoms. Cdcolli and Cdcasc venoms caused 36.7% and 13.3% edema increases, respectively. Cdt venom caused a 10% edema induction compared to those by other venoms. All venoms increased TOTAL-CK, MB-CK, and LDH levels (indicating muscle injury) and ALT, AST, GGT, and ALP levels (markers of liver damage) and were able to induce a neuromuscular blockade. Urea and creatinine levels were also altered in both plasma and urine, indicating kidney damage. Only Cdcolli and Cdcasc venoms increased TAPP and TAP. CONCLUSIONS: Together, these results allow us to draw a distinction between local and systemic effects caused by Crotalus subspecies, highlighting the clinical and biochemical effects produced by their respective venoms.

Cryptic diversity in the Mexican highlands: Thousands of UCE loci help illuminate phylogenetic relationships, species limits and divergence times of montane rattlesnakes (Viperidae: Crotalus).

With the continued adoption of genome-scale data in evolutionary biology comes the challenge of adequately harnessing the information to make accurate phylogenetic inferences. Coalescent-based methods of species tree inference have become common, and concatenation has been shown in simulation to perform well, particularly when levels of incomplete lineage sorting are low. However, simulation conditions are often overly simplistic, leaving empiricists with uncertainty regarding analytical tools. We use a large ultraconserved element data set (>3,000 loci) from rattlesnakes of the Crotalus triseriatus group to delimit lineages and estimate species trees using concatenation and several coalescent-based methods. Unpartitioned and partitioned maximum likelihood and Bayesian analysis of the concatenated matrix yield a topology identical to coalescent analysis of a subset of the data in bpp. ASTRAL analysis on a subset of the more variable loci also results in a tree consistent with concatenation and bpp, whereas the SVDquartets phylogeny differs at additional nodes. The size of the concatenated matrix has a strong effect on species tree inference using SVDquartets, warranting additional investigation on optimal data characteristics for this method. Species delimitation analyses suggest up to 16 unique lineages may be present within the C. triseriatus group, with divergences occurring during the Neogene and Quaternary. Network analyses suggest hybridization within the group is relatively rare. Altogether, our results reaffirm the Mexican highlands as a biodiversity hotspot and suggest that coalescent-based species tree inference on data subsets can provide a strongly supported species tree consistent with concatenation of all loci with a large amount of missing data.

Local and systemic effects caused by Crotalus durissus terrificus, Crotalus durissus collilineatus, and Crotalus durissus cascavella snake venoms in swiss mice

Abstract INTRODUCTION: Crotalus envenomations cause serious complications and can be fatal without appropriate treatment. Venom isoforms present and inter/intraspecific variations in the venom composition can result in different symptoms presented by bites by snakes from the same species but from different geographical regions. We comparatively evaluated the local and systemic effects caused by Crotalus durissus terrificus (Cdt), C.d. collilineatus (Cdcolli), and C.d. cascavella (Cdcasc) envenomation. METHODS: Venom chromatography was performed. Proteolytic, phospholipase, and LAAO activities were analyzed. Edema, myotoxicity, hepatotoxicity, nephrotoxicity, and coagulation alterations were evaluated. RESULTS: The venom SDS-PAGE analyses found the presence of convulxin, gyroxin, crotoxin, and crotamine in Cdt and Cdcolli venoms. Crotamine was not present in the Cdcasc venom. Cdt, Cdcollli, and Cdcasc venoms had no proteolytic activity. Only Cdcasc and Cdt venoms had phospholipase activity. LAAO activity was observed in Cdcolli and Cdcasc venoms. Cdcolli and Cdcasc venoms caused 36.7% and 13.3% edema increases, respectively. Cdt venom caused a 10% edema induction compared to those by other venoms. All venoms increased TOTAL-CK, MB-CK, and LDH levels (indicating muscle injury) and ALT, AST, GGT, and ALP levels (markers of liver damage) and were able to induce a neuromuscular blockade. Urea and creatinine levels were also altered in both plasma and urine, indicating kidney damage. Only Cdcolli and Cdcasc venoms increased TAPP and TAP. CONCLUSIONS: Together, these results allow us to draw a distinction between local and systemic effects caused by Crotalus subspecies, highlighting the clinical and biochemical effects produced by their respective venoms.

Evidence for divergent patterns of local selection driving venom variation in Mojave Rattlesnakes (Crotalus scutulatus).

Snake venoms represent an enriched system for investigating the evolutionary processes that lead to complex and dynamic trophic adaptations. It has long been hypothesized that natural selection may drive geographic variation in venom composition, yet previous studies have lacked the population genetic context to examine these patterns. We leverage range-wide sampling of Mojave Rattlesnakes (Crotalus scutulatus) and use a combination of venom, morphological, phylogenetic, population genetic, and environmental data to characterize the striking dichotomy of neurotoxic (Type A) and hemorrhagic (Type B) venoms throughout the range of this species. We find that three of the four previously identified major lineages within C. scutulatus possess a combination of Type A, Type B, and a 'mixed' Type A + B venom phenotypes, and that fixation of the two main venom phenotypes occurs on a more fine geographic scale than previously appreciated. We also find that Type A + B individuals occur in regions of inferred introgression, and that this mixed phenotype is comparatively rare. Our results support strong directional local selection leading to fixation of alternative venom phenotypes on a fine geographic scale, and are inconsistent with balancing selection to maintain both phenotypes within a single population. Our comparisons to biotic and abiotic factors further indicate that venom phenotype correlates with fang morphology and climatic variables. We hypothesize that links to fang morphology may be indicative of co-evolution of venom and other trophic adaptations, and that climatic variables may be linked to prey distributions and/or physiology, which in turn impose selection pressures on snake venoms.

Arizona Ridge-nosed rattlesnake envenomation: Case report of a personal encounter with the official state reptile of Arizona, Crotalus willardi willardi.

This case report describes the effects of an envenomation from one of the most infrequently encountered species of rattlesnake in the United States, Crotalus willardi willardi (C. w. willardi), the Arizona Ridge-nosed Rattlesnake. A previously healthy 57-year-old male sustained a bite to his non-dominant hand from a C. w. willardi. The most pronounced effect from the envenomation was edema and progression of edema that extended from his hand to the mid bicep. He also experienced erythema and tenderness to palpation in the affected limb, and some diminished range of motion in the hand. He expressed only minimal pain. Other than a mildly positive D-Dimer and leukocytosis, he had no significant hematologic effects and no systemic effects. He was treated with standard doses of Crotalidae Polyvalent Immune Fab (Ovine). He reported complete recovery from the envenomation within three days of the bite. Although envenomation from rattlesnakes is somewhat common in Arizona, knowing the exact species of snake is not. Confirmed documentation is exceedingly rare as most people do not recognize the different rattlesnake species. In addition, some species of rattlesnake (such as C. w. willardi) are especially reclusive and found only in isolated mountainous regions. Being able to confirm an envenomation by C. w. willardi would require not only someone knowledgeable in herpetology, but also, preferably, photographic evidence. This case has both.

Cryptic genetic diversity, population structure, and gene flow in the Mojave rattlesnake (Crotalus scutulatus).

The Mojave rattlesnake (Crotalus scutulatus) inhabits deserts and arid grasslands of the western United States and Mexico. Despite considerable interest in its highly toxic venom and the recognition of two subspecies, no molecular studies have characterized range-wide genetic diversity and population structure or tested species limits within C. scutulatus. We used mitochondrial DNA and thousands of nuclear loci from double-digest restriction site associated DNA sequencing to infer population genetic structure throughout the range of C. scutulatus, and to evaluate divergence times and gene flow between populations. We find strong support for several divergent mitochondrial and nuclear clades of C. scutulatus, including splits coincident with two major phylogeographic barriers: the Continental Divide and the elevational increase associated with the Central Mexican Plateau. We apply Bayesian clustering, phylogenetic inference, and coalescent-based species delimitation to our nuclear genetic data to test hypotheses of population structure. We also performed demographic analyses to test hypotheses relating to population divergence and gene flow. Collectively, our results support the existence of four distinct lineages within C. scutulatus, and genetically defined populations do not correspond with currently recognized subspecies ranges. Finally, we use approximate Bayesian computation to test hypotheses of divergence among multiple rattlesnake species groups distributed across the Continental Divide, and find evidence for co-divergence at this boundary during the mid-Pleistocene.

Extremely Divergent Haplotypes in Two Toxin Gene Complexes Encode Alternative Venom Types within Rattlesnake Species.

Natural selection is generally expected to favor one form of a given trait within a population. The presence of multiple functional variants of traits involved in activities such as feeding, reproduction, or the defense against predators is relatively uncommon within animal species. The genetic architecture and evolutionary mechanisms underlying the origin and maintenance of such polymorphisms are of special interest. Among rattlesnakes, several instances of the production of biochemically distinct neurotoxic or hemorrhagic venom types within the same species are known. Here, we investigated the genetic basis of this phenomenon in three species and found that neurotoxic and hemorrhagic individuals of the same species possess markedly different haplotypes at two toxin gene complexes. For example, neurotoxic and hemorrhagic Crotalus scutulatus individuals differ by 5 genes at the phospholipase A2 (PLA2) toxin gene complex and by 11 genes at the metalloproteinase (MP) gene complex. A similar set of extremely divergent haplotypes also underlies alternate venom types within C. helleri and C. horridus. We further show that the MP and PLA2 haplotypes of neurotoxic C. helleri appear to have been acquired through hybridization with C. scutulatus-a rare example of the horizontal transfer of a potentially highly adaptive suite of genes. These large structural variants appear analogous to immunity gene complexes in host-pathogen arms races and may reflect the impact of balancing selection at the PLA2 and MP complexes for predation on different prey.

General characterization of the venoms from two species of rattlesnakes and an intergrade population (C. lepidus x aquilus) from Aguascalientes and Zacatecas, Mexico.

The venoms from two species of rock rattlesnakes and an intergrade population were studied. Differences were noted in SDS-PAGE and RP-HPLC profiles and only the venom from the intergrade population showed low procoagulant activity. Also, a Crotoxin-like neurotoxic PLA2 was identified in the venom of C. l. klauberi, the most toxic of the analyzed venoms. This is the first report of such a toxin in C. l. klauberi from Aguascalientes, Mexico.

Venom phenotypes of the Rock Rattlesnake (Crotalus lepidus) and the Ridge-nosed Rattlesnake (Crotalus willardi) from México and the United States.

Although the Mexican Highlands has the highest diversity of small-bodied rattlesnakes in the world, studies on the species found throughout this region have been relatively scarce. This has led to challenges with examining venom phenotypic characteristics, as well as species misidentifications and misclassifications. In the current study we investigated venom variation among four subspecies of Crotalus lepidus (C. l. klaluberi, C. l. lepidus, C. l. maculosus, C. l. morulus) and four subspecies of C. willardi (C. w. amabilis, C. w. obscurus, C. w. silus, and C. w. willardi) that inhabit regions of southwestern United States and central México. SDS-PAGE patterns show the presence of many of the major compounds found in other rattlesnake venoms, although minor variations in protein banding patterns and intensity are recognizable. Most notably, PI-metalloproteinase (SVMP) bands appear to be very faint to absent in northern C. l. lepidus and C. l. klauberi subspecies, but are fairly prominent in all other C. lepidus and C. willardi subspecies. Enzyme activity assays revealed that C. lepidus subspecies exhibit higher SVMP and thrombin-like activities when compared to C. willardi subspecies. Significant differences between subspecies were also observed for kallikrein-like serine protease, L-amino acid oxidase, and phosphodiesterase activities, although these differences appear to be random and fail to follow a geographical or phylogenetic trend. The same relationship was also observed for fibrinogenolytic and coagulation assays. Toxicity assays conducted on lab mice (Mus musculus), house geckos (Hemidactylus frenatus), and house crickets (Acheta domestica) revealed varying toxicities between subspecies, with C. l klauberi being the most toxic towards mice (LD50 = 1.36 µg/g) and house geckos (LD50 = 0.17 µg/g), and C. w. silus being most toxic to house crickets (LD50 = 1.94 µg/g). These results provide additional evidence that geographical isolation, natural selection, and adaptive evolution in response to diets may be driving forces contributing to population-level variation in venom composition.

Interfang Distances of Rattlesnakes: Sexual, Interspecific, and Body Size-related Variation, and Implications for Snakebite Research and Management.

OBJECTIVES: Snakebite severity corresponds to size of snake because the amount of venom a snake injects is positively associated with snake size. Because fang marks are often present on snakebite patients, we tested whether the relationship between snake length and distance between fang puncture wounds can be generalized for rattlesnakes of genus Crotalus. METHODS: We measured 2 interfang distances from 79 rattlesnakes of both sexes, 5 species, and varying body length: 1) distance between fang bases in anesthetized snakes, and 2) distance between fang punctures in a membrane-covered beaker bitten defensively. RESULTS: Statistical analyses supported our 2 hypotheses, that 1) body size-related fang divergence during fang protraction (ie, anterolateral movement during fang erection), and 2) the relationship between snake length and interfang distance are similar between the sexes and among different rattlesnake species. We therefore derived a general equation to estimate snake length based on distance between fang marks, and recommended 5 snake size categories: very small (<10 mm), small (10-15 mm), medium (15-20 mm), large (20-25 mm), and very large (>25 mm). CONCLUSIONS: The distance between fang marks on a snakebite patient may be used to estimate the size or size category of the offending snake, which in some cases may have predictive value for overall clinical severity of a given envenomation. Assessing interfang distance from puncture wounds can improve snakebite research and anticipation of snakebite severity.

Efficient Bayesian Species Tree Inference under the Multispecies Coalescent.

We develop a Bayesian method for inferring the species phylogeny under the multispecies coalescent (MSC) model. To improve the mixing properties of the Markov chain Monte Carlo (MCMC) algorithm that traverses the space of species trees, we implement two efficient MCMC proposals: the first is based on the Subtree Pruning and Regrafting (SPR) algorithm and the second is based on a node-slider algorithm. Like the Nearest-Neighbor Interchange (NNI) algorithm we implemented previously, both new algorithms propose changes to the species tree, while simultaneously altering the gene trees at multiple genetic loci to automatically avoid conflicts with the newly proposed species tree. The method integrates over gene trees, naturally taking account of the uncertainty of gene tree topology and branch lengths given the sequence data. A simulation study was performed to examine the statistical properties of the new method. The method was found to show excellent statistical performance, inferring the correct species tree with near certainty when 10 loci were included in the dataset. The prior on species trees has some impact, particularly for small numbers of loci. We analyzed several previously published datasets (both real and simulated) for rattlesnakes and Philippine shrews, in comparison with alternative methods. The results suggest that the Bayesian coalescent-based method is statistically more efficient than heuristic methods based on summary statistics, and that our implementation is computationally more efficient than alternative full-likelihood methods under the MSC. Parameter estimates for the rattlesnake data suggest drastically different evolutionary dynamics between the nuclear and mitochondrial loci, even though they support largely consistent species trees. We discuss the different challenges facing the marginal likelihood calculation and transmodel MCMC as alternative strategies for estimating posterior probabilities for species trees. [Bayes factor; Bayesian inference; MCMC; multispecies coalescent; nodeslider; species tree; SPR.].

The effects of hybridization on divergent venom phenotypes: Characterization of venom from Crotalus scutulatus scutulatus × Crotalus oreganus helleri hybrids.

Hybridization between divergent species can be analyzed to elucidate expression patterns of distinct parental characteristics, as well as to provide information about the extent of reproductive isolation between species. A known hybrid cross between two rattlesnakes with highly divergent venom phenotypes provided the opportunity to examine occurrence of parental venom characteristics in the F1 hybrids as well as ontogenetic shifts in the expression of these characters as the hybrids aged. Although venom phenotypes of adult rattlesnake venoms are known for many species, the effect of hybridization on phenotype inheritance is not well understood, and effects of hybridization on venom ontogeny have not yet been investigated. The current study investigates both phenomena resulting from the hybridization of a male snake with type I degradative venom, Crotalus oreganus helleri (Southern Pacific Rattlesnake), and a female snake with type II highly toxic venom, Crotalus scutulatus scutulatus (Mojave Rattlesnake). SDS-PAGE, enzymology, Western blot and reversed phase HPLC (RP-HPLC) were used to characterize the venom of the C. o. helleri male, the C. s. scutulatus female and their two hybrid offspring as they aged. In general, Crotalus o. helleri × C. s. scutulatus hybrid venoms appeared to exhibit overlapping parental venom profiles, and several different enzyme activity patterns. Both hybrids expressed C. o. helleri father-specific myotoxins as well as C. s. scutulatus mother-specific Mojave toxin. Snake venom metalloprotease activity displayed apparent sex-influenced expression patterns, while hybrid serine protease activities were intermediate to parental activities. The C. s. scutulatus × C. o. helleri hybrid male's venom profile provided the strongest evidence that type I and type II venom characteristics are expressed simultaneously in hybrid venoms, as this snake contained distinctive characteristics of both parental species. However, the possibility of sex-influenced development of metalloprotease activity, as seen in the ontogenetic shifts of the hybrid female, may influence the levels of expression of both type I and type II characteristics in hybrid venoms. Ultimately, the chronological analysis of this known hybrid system reveals the most distinct characteristics that can be used in determining successful hybridization between snakes that follow the type I-type II trend in rattlesnake venom composition, namely the presence of metalloprotease activity and Mojave toxin.

Is Hybridization a Source of Adaptive Venom Variation in Rattlesnakes? A Test, Using a Crotalus scutulatus × viridis Hybrid Zone in Southwestern New Mexico.

Venomous snakes often display extensive variation in venom composition both between and within species. However, the mechanisms underlying the distribution of different toxins and venom types among populations and taxa remain insufficiently known. Rattlesnakes (Crotalus, Sistrurus) display extreme inter- and intraspecific variation in venom composition, centered particularly on the presence or absence of presynaptically neurotoxic phospholipases A2 such as Mojave toxin (MTX). Interspecific hybridization has been invoked as a mechanism to explain the distribution of these toxins across rattlesnakes, with the implicit assumption that they are adaptively advantageous. Here, we test the potential of adaptive hybridization as a mechanism for venom evolution by assessing the distribution of genes encoding the acidic and basic subunits of Mojave toxin across a hybrid zone between MTX-positive Crotalus scutulatus and MTX-negative C. viridis in southwestern New Mexico, USA. Analyses of morphology, mitochondrial and single copy-nuclear genes document extensive admixture within a narrow hybrid zone. The genes encoding the two MTX subunits are strictly linked, and found in most hybrids and backcrossed individuals, but not in C. viridis away from the hybrid zone. Presence of the genes is invariably associated with presence of the corresponding toxin in the venom. We conclude that introgression of highly lethal neurotoxins through hybridization is not necessarily favored by natural selection in rattlesnakes, and that even extensive hybridization may not lead to introgression of these genes into another species.

Diversity-dependent cladogenesis throughout western Mexico: Evolutionary biogeography of rattlesnakes (Viperidae: Crotalinae: Crotalus and Sistrurus).

Rattlesnakes (Crotalus and Sistrurus) represent a radiation of approximately 42 species distributed throughout the New World from southern Canada to Argentina. Interest in this enigmatic group of snakes continues to accrue due, in part, to their ecomorphological diversity, contributions to global envenomations, and potential medicinal importance. Although the group has garnered substantial attention from systematists and evolutionary biologists for decades, little is still known regarding patterns of lineage diversification. In addition, few studies have statistically quantified broad-scale biogeographic patterns in rattlesnakes to ascertain how dispersal occurred throughout the New World, particularly among the different major biomes of the Americas. To examine diversification and biogeographic patterns in this group of snakes we assemble a multilocus data set consisting of over 6700bp encompassing three nuclear loci (NT-3, RAG-1, C-mos) and seven mitochondrial genes (12S, 16S, ATPase6, ATPase8, ND4, ND5, cytb). Fossil-calibrated phylogenetic and subsequent diversification rate analyses are implemented using maximum likelihood and Bayesian inference, to examine their evolutionary history and temporal dynamics of diversity. Based on ancestral area reconstructions we explore dispersal patterns throughout the New World. Cladogenesis occurred predominantly during the Miocene and Pliocene with only two divergences during the Pleistocene. Two different diversification rate models, advocating diversity-dependence, are strongly supported. These models indicate an early rapid radiation followed by a recent speciation rate decline. Biogeographic analyses suggest that the high elevation pine-oak forests of western Mexico served as a major speciation pump for the majority of lineages, with the desert biome of western North America colonized independently at least twice. All together, these results provide evidence for rapid diversification of rattlesnakes throughout the Mexican highlands during the Neogene, likely in response to continual orogenesis of Mexico's major mountain systems, followed by more recent dispersal into desert and tropical biomes.

Deconstructing a Species-Complex: Geometric Morphometric and Molecular Analyses Define Species in the Western Rattlesnake (Crotalus viridis).

Morphological data are a conduit for the recognition and description of species, and their acquisition has recently been broadened by geometric morphometric (GM) approaches that co-join the collection of digital data with exploratory 'big data' analytics. We employed this approach to dissect the Western Rattlesnake (Crotalus viridis) species-complex in North America, currently partitioned by mitochondrial (mt)DNA analyses into eastern and western lineages (two and seven subspecies, respectively). The GM data (i.e., 33 dorsal and 50 lateral head landmarks) were gleaned from 2,824 individuals located in 10 museum collections. We also downloaded and concatenated sequences for six mtDNA genes from the NCBI GenBank database. GM analyses revealed significant head shape differences attributable to size and subspecies-designation (but not their interactions). Pairwise shape distances among subspecies were significantly greater than those derived from ancestral character states via squared-change parsimony, with the greatest differences separating those most closely related. This, in turn, suggests the potential for historic character displacement as a diversifying force in the complex. All subspecies, save one, were significantly differentiated in a Bayesian discriminant function analysis (DFA), regardless of whether our priors were uniform or informative (i.e., mtDNA data). Finally, shape differences among sister-clades were significantly greater than expected by chance alone under a Brownian model of evolution, promoting the hypothesis that selection rather than drift was the driving force in the evolution of the complex. Lastly, we combine head shape and mtDNA data so as to derived an integrative taxonomy that produced robust boundaries for six OTUs (operational taxonomic units) of the C. viridis complex. We suggest these boundaries are concomitant with species-status and subsequently provide a relevant nomenclature for its recognition and representation.

Limitations of climatic data for inferring species boundaries: insights from speckled rattlesnakes.

Phenotypes, DNA, and measures of ecological differences are widely used in species delimitation. Although rarely defined in such studies, ecological divergence is almost always approximated using multivariate climatic data associated with sets of specimens (i.e., the "climatic niche"); the justification for this approach is that species-specific climatic envelopes act as surrogates for physiological tolerances. Using identical statistical procedures, we evaluated the usefulness and validity of the climate-as-proxy assumption by comparing performance of genetic (nDNA SNPs and mitochondrial DNA), phenotypic, and climatic data for objective species delimitation in the speckled rattlesnake (Crotalus mitchellii) complex. Ordination and clustering patterns were largely congruent among intrinsic (heritable) traits (nDNA, mtDNA, phenotype), and discordance is explained by biological processes (e.g., ontogeny, hybridization). In contrast, climatic data did not produce biologically meaningful clusters that were congruent with any intrinsic dataset, but rather corresponded to regional differences in atmospheric circulation and climate, indicating an absence of inherent taxonomic signal in these data. Surrogating climate for physiological tolerances adds artificial weight to evidence of species boundaries, as these data are irrelevant for that purpose. Based on the evidence from congruent clustering of intrinsic datasets, we recommend that three subspecies of C. mitchellii be recognized as species: C. angelensis, C. mitchellii, and C. Pyrrhus.

Isolation and characterisation of insulin-releasing compounds from Crotalus adamanteus, Crotalus vegrandis and Bitis nasicornis venom.

Crude venom from three venomous snakes, Crotalus adamanteus, Crotalus vegrandis and Bitis nasicornis was fractionated by gel filtration chromatography, and selected fractions screened for in-vitro insulinotropic activity using clonal pancreatic BRIN-BD11 cells. Nineteen fractions stimulated insulin secretion and the structural identity of bioactive compounds responsible was probed using MALDI-ToF MS and N-terminal Edman degradation sequencing. Partial N-terminal sequences were determined and their homology to existing sequences identified using BLAST searching. The main insulinotropic peptide families identified were made up of snake venom serine proteinases, phospholipases A2 (PLA2) and disintegrins. Snake venom constituents may have therapeutic potential for diabetes, with each of the three viper venoms tested providing insulinotropic compounds from a range of different toxin families.