Venom Composition of Neglected Bothropoid Snakes from the Amazon Rainforest: Ecological and Toxinological Implications.

Snake venoms have evolved in several families of Caenophidae, and their toxins have been assumed to be biochemical weapons with a role as a trophic adaptation. However, it remains unclear how venom contributes to the success of venomous species for adaptation to different environments. Here we compared the venoms from Bothrocophias hyoprora, Bothrops taeniatus, Bothrops bilineatus smaragdinus, Bothrops brazili, and Bothrops atrox collected in the Amazon Rainforest, aiming to understand the ecological and toxinological consequences of venom composition. Transcriptomic and proteomic analyses indicated that the venoms presented the same toxin groups characteristic from bothropoids, but with distinct isoforms with variable qualitative and quantitative abundances, contributing to distinct enzymatic and toxic effects. Despite the particularities of each venom, commercial Bothrops antivenom recognized the venom components and neutralized the lethality of all species. No clear features could be observed between venoms from arboreal and terrestrial habitats, nor in the dispersion of the species throughout the Amazon habitats, supporting the notion that venom composition may not shape the ecological or toxinological characteristics of these snake species and that other factors influence their foraging or dispersal in different ecological niches.

Comparing morphological and secretory aspects of cephalic glands among the New World coral snakes brings novel insights on their biological roles.

Oral and other cephalic glands have been surveyed by several studies with distinct purposes. Despite the wide diversity and medical relevance of the New World coral snakes, studies focusing on understanding the biological roles of the glands within this group are still scarce. Specifically, the venom glands of some coral snakes were previously investigated but all other cephalic glands remain uncharacterized. In this sense, performing morphological and molecular analysis of these glands may help better understand their biological role. Here, we studied the morphology of the venom, infralabial, rictal, and harderian glands of thirteen species of Micrurus and Micruroides euryxanthus. We also performed a molecular characterization of these glands from selected species of Micrurus using transcriptomic and proteomic approaches. We described substantial morphological variation in the cephalic glands of New World coral snakes and structural evidence for protein-secreting cells in the inferior rictal glands. Our molecular analysis revealed that the venom glands, as expected, are majorly devoted to toxin production, however, the infralabial and inferior rictal glands also expressed some toxin genes at low to medium levels, despite the marked morphological differences. On the other hand, the harderian glands were dominated by the expression of lipocalins, but do not produce toxins. Our integrative analysis, including the prediction of biological processes and pathways, helped decipher some important traits of cephalic glands and better understand their biology.

Patterns of morphological variation and ecological correlates in the skull of vipers (Serpentes: Viperidae).

The skull of vipers is a highly kinetic anatomical structure involved in envenomating and consuming of prey. Morphological knowledge about the viperid skull is based on studies on some groups of species, but information on its variation within the whole family and its functional morphology is still scarce. In this study, we aimed to explore variation in skull morphology among species of the three subfamilies of Viperidae, and test whether that variation correlates with macrohabitat and diet. We performed quantitative analyses of the viperid skull based on broad taxonomic sampling and two methodological approaches: linear and geometric morphometrics. The results of both approaches showed that much of the variation lies in differences of shape and relative size of the premaxilla, the nasals, the frontals, and the parietals. The results indicated that phylogeny and size influence the shape of the skull, but we also found evidence of morphological differentiation between arboreal and terrestrial species and in species with mammal specialist diet. Our findings imply that, besides evolutionary allometry and phylogenetic signal, demands of particular diets coupled with use of certain habitats have in part shaped morphological evolution of the viperid skull.

Venom phenotype conservation suggests integrated specialization in a lizard-eating snake.

Biological specialization reduces the size of niche space while increasing efficiency in the use of available resources. Specialization often leads to phenotypic changes via natural selection aligning with niche space constraints. Commonly observed changes are in size, shape, behavior, and traits associated with feeding. One often selected trait for dietary specialization is venom, which, in snakes, often shows variation dependent on diet across and within species. The Neotropical Blunt-headed Treesnake (Imantodes cenchoa) is a highly specialized, rear-fanged, arboreal, lizard hunter that displays a long thin body, enlarged eyes, and a large Duvernoy's gland. However, toxin characterization of I. cenchoa has never been completed. Here, we use RNA-seq and mass spectrometry to assemble, annotate, and analyze the venom gland transcriptomes of four I. cenchoa from across their range. We find a lack of significant venom variation at the sequence and expression levels, suggesting venom conservation across the species. We propose this conservation provides evidence of a specialized venom repertoire, adapted to maximize efficiency of capturing and processing lizards. Importantly, this study provides the most complete venom gland transcriptomes of I. cenchoa and evidence of venom specialization in a rear-fanged snake, giving insight into selective pressures of venom across all snake species.

Total-evidence phylogeny and evolutionary morphology of New World pitvipers (Serpentes: Viperidae: Crotalinae).

Crotalines (pitvipers) in the Americas are distributed from southern Canada to southern Argentina, and are represented by 13 genera and 163 species that constitute a monophyletic group. Their phylogenetic relationships have been assessed mostly based on DNA sequences, while morphological data have scarcely been used for phylogenetic inquiry. We present a total-evidence phylogeny of New World pitvipers, the most taxon/character comprehensive phylogeny to date. Our analysis includes all genera, morphological data from external morphology, cranial osteology and hemipenial morphology, and DNA sequences from mitochondrial and nuclear genes. We performed analyses with parsimony as an optimality criterion, using different schemes for character weighting. We evaluated the contribution of the different sources of characters to the phylogeny through analyses of reduced datasets and calculation of weighted homoplasy and retention indexes. We performed a morphological character analysis to identify synapomorphies for the main clades. In terms of biogeography, our results support a single colonization event of the Americas by pitvipers, and a cladogenetic event into a Neotropical clade and a North American/Neotropical clade. The results also shed light on the previously unstable position of some taxa, although they could not sufficiently resolve the position of Bothrops lojanus, which may lead to the paraphyly of either Bothrops or Bothrocophias. The morphological character analyses demonstrated that an important phylogenetic signal is contained in characters related to head scalation, the jaws and the dorsum of the skull, and allowed us to detect morphological convergences in external morphology associated with arboreality.

Evolutionary allometry and ecological correlates of fang length evolution in vipers.

Traits for prey acquisition form the phenotypic interface of predator-prey interactions. In venomous predators, morphological variation in venom delivery apparatus like fangs and stingers may be optimized for dispatching prey. Here, we determine how a single dimension of venom injection systems evolves in response to variation in the size, climatic conditions and dietary ecology of viperid snakes. We measured fang length in more than 1900 museum specimens representing 199 viper species (55% of recognized species). We find both phylogenetic signal and within-clade variation in relative fang length across vipers suggesting both general taxonomic trends and potential adaptive divergence in fang length. We recover positive evolutionary allometry and little static allometry in fang length. Proportionally longer fangs have evolved in larger species, which may facilitate venom injection in more voluminous prey. Finally, we leverage climatic and diet data to assess the global correlates of fang length. We find that models of fang length evolution are improved through the inclusion of both temperature and diet, particularly the extent to which diets are mammal-heavy diets. These findings demonstrate how adaptive variation can emerge among components of complex prey capture systems.

The phylogenetic position of ridley's worm lizard reveals the complex biogeographic history of New World insular amphisbaenids.

The archipelago of Fernando de Noronha (FN) is located in the Equatorial South Atlantic Ocean, at 375 km off the northeastern coast of Brazil. Its endemic vertebrate land fauna is restricted to only six species, and three main hypotheses have been proposed to explain their presence in the archipelago. These hypotheses suggest FN had alternative biogeographic connections with: 1) the West Indies; 2) the South America mainland; or 3) Africa. Here, we evaluate for the first time the phylogenetic position of Amphisbaena ridleyi within the diversity of Amphisbaenia, and we infer the biogeographic processes that explain its presence in FN and its relationship with amphisbaenids from the West Indies. We analyzed a comprehensive multilocus dataset for Amphisbaenidae using maximum likelihood and time-calibrated Bayesian phylogenetic approaches. Based on our time-calibrated tree, we tested different biogeographic scenarios through historical biogeographic analyses. Our phylogenetic results for the high-level relationships of Amphisbaenia can be parenthetically summarized as (Rhineuridae, (Blanidae, (Bipedidae, (Cadeidae, (Trogonophidae, (Amphisbaenidae))))). Nine highly supported groups of species were recovered among the mainland South American amphisbaenids (SAA), whereas two phylogenetically distant groups of species were inferred for the West Indies: 1) WIC01, an Oligocene lineage present in Cuba and Hispaniola, which is the sister group of all other SAA groups; and 2) WIC02, a Miocene lineage that is restricted to southern Hispaniola and is closely related to Am. ridleyi. We estimated two events of transatlantic dispersal of amphisbaenians from Africa to West Indies: the dispersal of Cadeidae during the Middle Eocene, and the dispersal of the ancestor of Amphisbaena during the transition Eocene/Oligocene. These events were likely affected by the North Equatorial and South Equatorial currents, respectively, which have been flowing westwards since the Paleocene. The ancestral cladogenesis of Amphisbaena during the Late Oligocene is likely related to overwater dispersal events, or alternatively can be associated with the fragmentation of GAARlandia, when WIC01 was isolated in the West Indies, while the remaining groups of Amphisbaena diversified throughout the South American continent. During the Late Miocene, the ancestor of WIC02 dispersed from northern South America to the West Indies, while Am. ridleyi dispersed from the same region to FN. The overwater dispersal of WIC02 was driven by the North Brazilian Current and the dispersal of Am. ridleyi was likely influenced by the periodic shifts in direction and strength estimated for the North Equatorial Counter-Current during the Late Miocene.

Differences in PLA2 Constitution Distinguish the Venom of Two Endemic Brazilian Mountain Lanceheads, Bothrops cotiara and Bothrops fonsecai.

Interspecific differences in snake venom compositions can result from distinct regulatory mechanisms acting in each species. However, comparative analyses focusing on identifying regulatory elements and patterns that led to distinct venom composition are still scarce. Among venomous snakes, Bothrops cotiara and Bothrops fonsecai represent ideal models to complement our understanding of the regulatory mechanisms of venom production. These recently diverged species share a similar specialized diet, habitat, and natural history, but each presents a distinct venom phenotype. Here, we integrated data from the venom gland transcriptome and miRNome and the venom proteome of B. fonsecai and B. cotiara to better understand the regulatory mechanisms that may be acting to produce differing venom compositions. We detected not only the presence of similar toxin isoforms in both species but also distinct expression profiles of phospholipases A2 (PLA2) and some snake venom metalloproteinases (SVMPs) and snake venom serine proteinases (SVSPs) isoforms. We found evidence of modular expression regulation of several toxin isoforms implicated in venom divergence and observed correlated expression of several transcription factors. We did not find strong evidence for miRNAs shaping interspecific divergence of the venom phenotypes, but we identified a subset of toxin isoforms whose final expression may be fine-tuned by specific miRNAs. Sequence analysis on orthologous toxins showed a high rate of substitutions between PLA2s, which indicates that these toxins may be under strong positive selection or represent paralogous toxins in these species. Our results support other recent studies in suggesting that gene regulation is a principal mode of venom evolution across recent timescales, especially among species with conserved ecotypes.

When the tail shakes the snake: phylogenetic affinities and morphology of Atractus badius (Serpentes: Dipsadidae), reveals some current pitfalls on the snake's genomic age.

Atractus badius has a long and controversial nomenclatural history due to both its antiquity and the brevity of its original description. This species was described based on two syntypes from Java. Later, a lectotype was designated and the distribution range restricted to the Guiana Shield. Although this species has been repeatedly recorded throughout Amazonia and the Andes, these records have been erroneously assigned to A. badius because of a considerable level of confusion in the literature. We found 13 additional specimens of this poorly known snake, expanding our knowledge on its morphological variability, phylogenetic relationships and distribution. In this paper, we report new localities and data on meristic, morphometric, coloration, scales micro-ornamentation, osteology and hemipenial morphology to A. badius. We also determine the phylogenetic position of A. badius as nested in a composite Guiana Shield clade. In addition, we discuss recent advances on the systematics of Atractus and comment on some 'new arrangements' with respect to previously recognized species groups and available morphological evidence. Finally, we highlight the importance of accurate voucher identification before promoting taxonomic changes or implementing nomenclatural acts derived from new phylogenetic hypotheses, and notably propose the synonymy of the recently described A. pyroni with A. roulei.

Phylogenetically diverse diets favor more complex venoms in North American pitvipers.

The role of natural selection in the evolution of trait complexity can be characterized by testing hypothesized links between complex forms and their functions across species. Predatory venoms are composed of multiple proteins that collectively function to incapacitate prey. Venom complexity fluctuates over evolutionary timescales, with apparent increases and decreases in complexity, and yet the causes of this variation are unclear. We tested alternative hypotheses linking venom complexity and ecological sources of selection from diet in the largest clade of front-fanged venomous snakes in North America: the rattlesnakes, copperheads, cantils, and cottonmouths. We generated independent transcriptomic and proteomic measures of venom complexity and collated several natural history studies to quantify dietary variation. We then constructed genome-scale phylogenies for these snakes for comparative analyses. Strikingly, prey phylogenetic diversity was more strongly correlated to venom complexity than was overall prey species diversity, specifically implicating prey species' divergence, rather than the number of lineages alone, in the evolution of complexity. Prey phylogenetic diversity further predicted transcriptomic complexity of three of the four largest gene families in viper venom, showing that complexity evolution is a concerted response among many independent gene families. We suggest that the phylogenetic diversity of prey measures functionally relevant divergence in the targets of venom, a claim supported by sequence diversity in the coagulation cascade targets of venom. Our results support the general concept that the diversity of species in an ecological community is more important than their overall number in determining evolutionary patterns in predator trait complexity.

MITGARD: an automated pipeline for mitochondrial genome assembly in eukaryotic species using RNA-seq data.

MOTIVATION: Over the past decade, the field of next-generation sequencing (NGS) has seen dramatic advances in methods and a decrease in costs. Consequently, a large expansion of data has been generated by NGS, most of which have originated from RNA-sequencing (RNA-seq) experiments. Because mitochondrial genes are expressed in most eukaryotic cells, mitochondrial mRNA sequences are usually co-sequenced within the target transcriptome, generating data that are commonly underused or discarded. Here, we present MITGARD, an automated pipeline that reliably recovers the mitochondrial genome from RNA-seq data from various sources. The pipeline identifies mitochondrial sequence reads based on a phylogenetically related reference, assembles them into contigs, and extracts a complete mtDNA for the target species. RESULTS: We demonstrate that MITGARD can reconstruct the mitochondrial genomes of several species throughout the tree of life. We noticed that MITGARD can recover the mitogenomes in different sequencing schemes and even in a scenario of low-sequencing depth. Moreover, we showed that the use of references from congeneric species diverging up to 30 million years ago (MYA) from the target species is sufficient to recover the entire mitogenome, whereas the use of species diverging between 30 and 60 MYA allows the recovery of most mitochondrial genes. Additionally, we provide a case study with original data in which we estimate a phylogenetic tree of snakes from the genus Bothrops, further demonstrating that MITGARD is suitable for use on biodiversity projects. MITGARD is then a valuable tool to obtain high-quality information for studies focusing on the phylogenetic and evolutionary aspects of eukaryotes and provides data for easily identifying a sample using barcoding, and to check for cross-contamination using third-party tools.

Size Matters: An Evaluation of the Molecular Basis of Ontogenetic Modifications in the Composition of Bothrops jararacussu Snake Venom.

Ontogenetic changes in venom composition have been described in Bothrops snakes, but only a few studies have attempted to identify the targeted paralogues or the molecular mechanisms involved in modifications of gene expression during ontogeny. In this study, we decoded B. jararacussu venom gland transcripts from six specimens of varying sizes and analyzed the variability in the composition of independent venom proteomes from 19 individuals. We identified 125 distinct putative toxin transcripts, and of these, 73 were detected in venom proteomes and only 10 were involved in the ontogenetic changes. Ontogenetic variability was linearly related to snake size and did not correspond to the maturation of the reproductive stage. Changes in the transcriptome were highly predictive of changes in the venom proteome. The basic myotoxic phospholipases A2 (PLA2s) were the most abundant components in larger snakes, while in venoms from smaller snakes, PIII-class SVMPs were the major components. The snake venom metalloproteinases (SVMPs) identified corresponded to novel sequences and conferred higher pro-coagulant and hemorrhagic functions to the venom of small snakes. The mechanisms modulating venom variability are predominantly related to transcriptional events and may consist of an advantage of higher hematotoxicity and more efficient predatory function in the venom from small snakes.

Phylogenomics, Biogeography, and Morphometrics Reveal Rapid Phenotypic Evolution in Pythons After Crossing Wallace's Line.

Ecological opportunities can be provided to organisms that cross stringent biogeographic barriers towards environments with new ecological niches. Wallace's and Lyddeker's lines are arguably the most famous biogeographic barriers, separating the Asian and Australo-Papuan biotas. One of the most ecomorphologically diverse groups of reptiles, the pythons, is distributed across these lines, and are remarkably more diverse in phenotype and ecology east of Lydekker's line in Australo-Papua. We used an anchored hybrid enrichment approach, with near complete taxon sampling, to extract mitochondrial genomes and 376 nuclear loci to resolve and date their phylogenetic history. Biogeographic reconstruction demonstrates that they originated in Asia around 38-45 Ma and then invaded Australo-Papua around 23 Ma. Australo-Papuan pythons display a sizeable expansion in morphological space, with shifts towards numerous new adaptive optima in head and body shape, coupled with the evolution of new micro-habitat preferences. We provide an updated taxonomy of pythons and our study also demonstrates how ecological opportunity following colonization of novel environments can promote morphological diversification in a formerly ecomorphologically conservative group. [Adaptive radiation; anchored hybrid enrichment; biogeography; morphometrics; snakes.].

Interrogating Genomic-Scale Data for Squamata (Lizards, Snakes, and Amphisbaenians) Shows no Support for Key Traditional Morphological Relationships.

Genomics is narrowing uncertainty in the phylogenetic structure for many amniote groups. For one of the most diverse and species-rich groups, the squamate reptiles (lizards, snakes, and amphisbaenians), an inverse correlation between the number of taxa and loci sampled still persists across all publications using DNA sequence data and reaching a consensus on the relationships among them has been highly problematic. In this study, we use high-throughput sequence data from 289 samples covering 75 families of squamates to address phylogenetic affinities, estimate divergence times, and characterize residual topological uncertainty in the presence of genome-scale data. Importantly, we address genomic support for the traditional taxonomic groupings Scleroglossa and Macrostomata using novel machine-learning techniques. We interrogate genes using various metrics inherent to these loci, including parsimony-informative sites (PIS), phylogenetic informativeness, length, gaps, number of substitutions, and site concordance to understand why certain loci fail to find previously well-supported molecular clades and how they fail to support species-tree estimates. We show that both incomplete lineage sorting and poor gene-tree estimation (due to a few undesirable gene properties, such as an insufficient number of PIS), may account for most gene and species-tree discordance. We find overwhelming signal for Toxicofera, and also show that none of the loci included in this study supports Scleroglossa or Macrostomata. We comment on the origins and diversification of Squamata throughout the Mesozoic and underscore remaining uncertainties that persist in both deeper parts of the tree (e.g., relationships between Dibamia, Gekkota, and remaining squamates; among the three toxicoferan clades Iguania, Serpentes, and Anguiformes) and within specific clades (e.g., affinities among gekkotan, pleurodont iguanians, and colubroid families).

A new species of Bothrops (Serpentes: Viperidae: Crotalinae) from Pampas del Heath, southeastern Peru, with comments on the systematics of the Bothrops neuwiedi species group.

We describe a new species of pitviper of the genus Bothrops from the Peruvian Pampas del Heath, in the Bahuaja-Sonene National Park. Pampas del Heath is an area of seasonally flooded savannas and a northwestern extension of the Gran Chaco Boliviano-Paraguayo. The new species is easily distinguished from its congeners by the exclusive combination of dorsal color pattern of body consisting of small C-shaped blotches, postocular stripe originating posteriorly to the eye, covering posterior supralabials, dorsum of the head with paired markings arranged symmetrically, venter cream heavily speckled with brown, prelacunal scale discrete in contact with second supralabial, three to five prefoveals, subfoveal single usually present, postfoveals absent to two, canthals two, seven intersupraoculars, one or two suboculars, two or three postoculars, seven or eight supralabials, nine to eleven infralabials, 26-27 interrictals, 23-25 middorsal scales, 172 ventrals in the female and 169-173 in males, 45 subcaudals in the female and 50 in males. We performed separate and combined phylogenetic analyses based on morphology and five mitochondrial genes and recovered the new species as a member of the Bothrops neuwiedi species group. All lineages of this clade inhabit the South American dry diagonal. This novel species of pitviper increases the known diversity of the genus Bothrops and adds to the number of described taxa from the unique and scarcely known ecosystem of Pampas del Heath.

Correction: Large-scale molecular phylogeny, morphology, divergence-time estimation, and the fossil record of advanced caenophidian snakes (Squamata: Serpentes).

[This corrects the article DOI: 10.1371/journal.pone.0216148.].

Large-scale molecular phylogeny, morphology, divergence-time estimation, and the fossil record of advanced caenophidian snakes (Squamata: Serpentes).

Caenophidian snakes include the file snake genus Acrochordus and advanced colubroidean snakes that radiated mainly during the Neogene. Although caenophidian snakes are a well-supported clade, their inferred affinities, based either on molecular or morphological data, remain poorly known or controversial. Here, we provide an expanded molecular phylogenetic analysis of Caenophidia and use three non-parametric measures of support-Shimodaira-Hasegawa-Like test (SHL), Felsentein (FBP) and transfer (TBE) bootstrap measures-to evaluate the robustness of each clade in the molecular tree. That very different alternative support values are common suggests that results based on only one support value should be viewed with caution. Using a scheme to combine support values, we find 20.9% of the 1265 clades comprising the inferred caenophidian tree are unambiguously supported by both SHL and FBP values, while almost 37% are unsupported or ambiguously supported, revealing the substantial extent of phylogenetic problems within Caenophidia. Combined FBP/TBE support values show similar results, while SHL/TBE result in slightly higher combined values. We consider key morphological attributes of colubroidean cranial, vertebral and hemipenial anatomy and provide additional morphological evidence supporting the clades Colubroides, Colubriformes, and Endoglyptodonta. We review and revise the relevant caenophidian fossil record and provide a time-calibrated tree derived from our molecular data to discuss the main cladogenetic events that resulted in present-day patterns of caenophidian diversification. Our results suggest that all extant families of Colubroidea and Elapoidea composing the present-day endoglyptodont fauna originated rapidly within the early Oligocene-between approximately 33 and 28 Mya-following the major terrestrial faunal turnover known as the "Grande Coupure" and associated with the overall climate shift at the Eocene-Oligocene boundary. Our results further suggest that the caenophidian radiation originated within the Caenozoic, with the divergence between Colubroides and Acrochordidae occurring in the early Eocene, at ~ 56 Mya.

Molecular mechanisms underlying intraspecific variation in snake venom.

Elucidating the molecular mechanisms underlying snake venom variability provides important clues for understanding how the biological functions of this powerful toxic arsenal evolve. We analyzed in detail individual transcripts and venom protein isoforms produced by five specimens of a venomous snake (Bothrops atrox) from two nearby but genetically distinct populations from the Brazilian Amazon rainforest which show functional similarities in venom properties. Individual variation was observed among the venoms of these specimens, but the overall abundance of each general toxin family was conserved both in transcript and in venom protein levels. However, when expression of independent paralogues was analyzed, remarkable differences were observed within and among each toxin group, both between individuals and between populations. Transcripts for functionally essential venom proteins ("core function" proteins) were highly expressed in all specimens and showed similar transcription/translation rates. In contrast, other paralogues ("adaptive" proteins) showed lower expression levels and the toxins they coded for varied among different individuals. These results provide support for the inferences that (a) expression and translational differences play a greater role in defining adaptive variation in venom phenotypes than does sequence variation in protein coding genes and (b) convergent adaptive venom phenotypes can be generated through different molecular mechanisms. SIGNIFICANCE: Analysis of individual transcripts and venom protein isoforms produced by specimens of a venomous snake (Bothrops atrox), from the Brazilian Amazon rainforest, revealed that transcriptional and translational mechanisms contribute to venom phenotypic variation. Our finding of evidence for high expression of toxin proteins with conserved function supports the hypothesis that the venom phenotype consists of two kinds of proteins: conserved "core function" proteins that provide essential functional activities with broader relevance and less conserved "adaptive" proteins that vary in expression and may permit customization of protein function. These observations allowed us to suggest that genetic mechanisms controlling venom variability are not restricted to selection of gene copies or mutations in structural genes but also to selection of the mechanisms controlling gene expression, contributing to the plasticity of this important phenotype for venomous snakes.

Diversification in vipers: Phylogenetic relationships, time of divergence and shifts in speciation rates.

Snakes of the cosmopolitan family Viperidae comprise around 329 venomous species showing a striking heterogeneity in species richness among lineages. While the subfamily Azemiopinae comprises only two species, 70% of all viper species are arranged in the subfamily Crotalinae or the "pit vipers". The radiation of the pit vipers was marked by the evolution of the heat-sensing pits, which has been suggested to be a key innovation for the successful diversification of the group. Additionally, only crotalines were able to successfully colonize the New World. Here, we present the most complete molecular phylogeny for the family to date that comprises sequences from nuclear and mitochondrial genes representing 79% of all living vipers. We also investigated the time of divergence between lineages, using six fossils to calibrate the tree, and explored the hypothesis that crotalines have undergone an explosive radiation. Our phylogenetic analyses retrieved high support values for the monophyly of the family Viperidae, subfamilies Viperinae and Crotalinae, and 22 out of 27 genera, as well as well-supported intergeneric relationships throughout the family. We were able to recover a strongly supported sister clade to the New World pit vipers that comprises Gloydius, Ovophis, Protobothrops and Trimeresurus gracilis. Our results agree in many aspects with other studies focusing on the phylogenetics of vipers, but we recover new relationships as well. Despite the addition of new sequences we were not able to resolve some of the poor supported relationships previously suggested. Time of divergence estimates suggested that vipers started to radiate around the late Paleocene to middle Eocene with subfamilies most likely dating back to the Eocene. The invasion of the New World might have taken place sometime close to the Oligocene/Miocene boundary. Diversification analyses suggested a shift in speciation rates during the radiation of a sub-clade of pit vipers where speciation rates rapidly increased but slowed down toward the present. Thus, the evolution of the loreal pits alone does not seem to explain their explosive speciation rates. We suggest that climatic and geological changes in Asia and the invasion of the New World may have also contributed to the speciation shift found in vipers.

Another new and threatened species of lancehead genus Bothrops (Serpentes, Viperidae) from Ilha dos Franceses, Southeastern Brazil.

A new insular species of the genus Bothrops is described from Ilha dos Franceses, a small island off the coast of Espírito Santo State, in southeastern Brazil. The new species differs from mainland populations of B. jararaca mainly by its small size, relative longer tail, relative smaller head length, and relative larger eyes. The new species is distinguished from B. alcatraz, B. insularis and B. otavioi by the higher number of ventral and subcaudal scales, relative longer tail and smaller head. The new species is highly abundant on the island, being nocturnal, semiarboreal, and feeding on small lizards and centipeds. Due its unique and restricted area of occurrence, declining quality of habitat, and constant use of the island for tourism, the new species may be considered as critically endangered.