Independent Recruitment of Different Types of Phospholipases A2 to the Venoms of Caenophidian Snakes: The Rise of PLA2-IIE within Pseudoboini (Dipsadidae).

Snake venoms harbor a wide and diverse array of enzymatic and nonenzymatic toxic components, allowing them to exert myriad effects on their prey. However, they appear to trend toward a few optimal compositional scaffolds, dominated by four major toxin classes: SVMPs, SVSPs, 3FTxs, and PLA2s. Nevertheless, the latter appears to be restricted to vipers and elapids, as it has never been reported as a major venom component in rear-fanged species. Here, by investigating the original transcriptomes from 19 species distributed in eight genera from the Pseudoboini tribe (Dipsadidae: Xenodontinae) and screening among seven additional tribes of Dipsadidae and three additional families of advanced snakes, we discovered that a novel type of venom PLA2, resembling a PLA2-IIE, has been recruited to the venom of some species of the Pseudoboini tribe, where it is a major component. Proteomic and functional analyses of these venoms further indicate that these PLA2s play a relevant role in the venoms from this tribe. Moreover, we reconstructed the phylogeny of PLA2s across different snake groups and show that different types of these toxins have been recruited in at least five independent events in caenophidian snakes. Additionally, we present the first compositional profiling of Pseudoboini venoms. Our results demonstrate how relevant phenotypic traits are convergently recruited by different means and from homologous and nonhomologous genes in phylogenetically and ecologically divergent snake groups, possibly optimizing venom composition to overcome diverse adaptative landscapes.

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.

Resistance Is Not Futile: Widespread Convergent Evolution of Resistance to Alpha-Neurotoxic Snake Venoms in Caecilians (Amphibia: Gymnophiona).

Predatory innovations impose reciprocal selection pressures upon prey. The evolution of snake venom alpha-neurotoxins has triggered the corresponding evolution of resistance in the post-synaptic nicotinic acetylcholine receptors of prey in a complex chemical arms race. All other things being equal, animals like caecilians (an Order of legless amphibians) are quite vulnerable to predation by fossorial elapid snakes and their powerful alpha-neurotoxic venoms; thus, they are under strong selective pressure. Here, we sequenced the nicotinic acetylcholine receptor alpha-1 subunit of 37 caecilian species, representing all currently known families of caecilians from across the Americas, Africa, and Asia, including species endemic to the Seychelles. Three types of resistance were identified: (1) steric hindrance from N-glycosylated asparagines; (2) secondary structural changes due to the replacement of proline by another amino acid; and (3) electrostatic charge repulsion of the positively charged neurotoxins, through the introduction of a positively charged amino acid into the toxin-binding site. We demonstrated that resistance to alpha-neurotoxins convergently evolved at least fifteen times across the caecilian tree (three times in Africa, seven times in the Americas, and five times in Asia). Additionally, as several species were shown to possess multiple resistance modifications acting synergistically, caecilians must have undergone at least 20 separate events involving the origin of toxin resistance. On the other hand, resistance in non-caecilian amphibians was found to be limited to five origins. Together, the mutations underlying resistance in caecilians constitute a robust signature of positive selection which strongly correlates with elapid presence through both space (sympatry with caecilian-eating elapids) and time (Cenozoic radiation of elapids). Our study demonstrates the extent of convergent evolution that can be expected when a single widespread predatory adaptation triggers parallel evolutionary arms races at a global scale.

First Insights into the Venom Composition of Two Ecuadorian Coral Snakes.

Micrurus is a medically relevant genus of venomous snakes composed of 85 species. Bites caused by coral snakes are rare, but they are usually associated with very severe and life-threatening clinical manifestations. Ecuador is a highly biodiverse country with a complex natural environment, which is home to approximately 20% of identified Micrurus species. Additionally, it is on the list of Latin American countries with the highest number of snakebites. However, there is no local antivenom available against the Ecuadorian snake venoms, and the biochemistry of these venoms has been poorly explored. Only a limited number of samples collected in the country from the Viperidae family were recently characterised. Therefore, this study addressed the compositional patterns of two coral snake venoms from Ecuador, M. helleri and M. mipartitus, using venomics strategies, integrating sample fractionation, gel electrophoresis, and mass spectrometry. Chromatographic and electrophoretic profiles of these snake venoms revealed interspecific variability, which was ascertained by mass spectrometry. The two venoms followed the recently recognised dichotomic toxin expression trends displayed by Micrurus species: M. helleri venom contains a high proportion (72%) of phospholipase A2, whereas M. mipartitus venom is dominated by three-finger toxins (63%). A few additional protein families were also detected in these venoms. Overall, these results provide the first comprehensive views on the composition of two Ecuadorian coral snake venoms and expand the knowledge of Micrurus venom phenotypes. These findings open novel perspectives to further research the functional aspects of these biological cocktails of PLA2s and 3FTxs and stress the need for the preclinical evaluation of the currently used antivenoms for therapeutic purposes in Ecuador.

Neotropics as a Cradle for Adaptive Radiations.

Neotropical ecosystems are renowned for numerous examples of adaptive radiation in both plants and animals resulting in high levels of biodiversity and endemism. However, we still lack a comprehensive review of the abiotic and biotic factors that contribute to these adaptive radiations. To fill this gap, we delve into the geological history of the region, including the role of tectonic events such as the Andean uplift, the formation of the Isthmus of Panama, and the emergence of the Guiana and Brazilian Shields. We also explore the role of ecological opportunities created by the emergence of new habitats, as well as the role of key innovations, such as novel feeding strategies or reproductive mechanisms. We discuss different examples of adaptive radiation, including classic ones like Darwin's finches and Anolis lizards, and more recent ones like bromeliads and lupines. Finally, we propose new examples of adaptive radiations mediated by ecological interactions in their geological context. By doing so, we provide insights into the complex interplay of factors that contributed to the remarkable diversity of life in the Neotropics and highlight the importance of this region in understanding the origins of biodiversity.

Path for recovery: an ecological overview of the Jambato Harlequin Toad (Bufonidae: Atelopus ignescens) in its last known locality, Angamarca Valley, Ecuador.

The Jambato Harlequin toad (Atelopus ignescens), a formerly abundant species in the Andes of Ecuador, faced a dramatic population decline in the 1980s, with its last recorded sighting in 1988. The species was considered Extinct by the IUCN until 2016, when a fortuitous discovery of one Jambato by a local boy reignited hope. In this study, we present findings from an investigation conducted in the Angamarca parish, focusing on distribution, abundance, habitat preferences, ecology, disease susceptibility, and dietary habits of the species. In one year we identified 71 individuals at different stages of development in various habitats, with a significant presence in agricultural mosaic areas and locations near water sources used for crop irrigation, demonstrating the persistence of the species in a complex landscape, with considerable human intervention. The dietary analysis based on fecal samples indicated a diverse prey selection, primarily comprising arthropods such as Acari, Coleoptera, and ants. Amphibian declines have been associated with diseases and climate change; notably, our study confirmed the presence of the pathogen Batrachochytrium dendrobatidis (Bd), but, surprisingly, none of the infected Jambatos displayed visible signs of illness. When analyzing climatic patterns, we found that there are climatic differences between historical localities and Angamarca; the temporal analysis also exposes a generalized warming trend. Finally, in collaboration with the local community, we developed a series of management recommendations for terrestrial and aquatic environments occupied by the Jambato.

Why Do Some Lineages Radiate While Others Do Not? Perspectives for Future Research on Adaptive Radiations.

Understanding the processes that drive phenotypic diversification and underpin speciation is key to elucidating how biodiversity has evolved. Although these processes have been studied across a wide array of clades, adaptive radiations (ARs), which are systems with multiple closely related species and broad phenotypic diversity, have been particularly fruitful for teasing apart the factors that drive and constrain diversification. As such, ARs have become popular candidate study systems for determining the extent to which ecological features, including aspects of organisms and the environment, and inter- and intraspecific interactions, led to evolutionary diversification. Despite substantial past empirical and theoretical work, understanding mechanistically how ARs evolve remains a major challenge. Here, we highlight a number of understudied components of the environment and of lineages themselves, which may help further our understanding of speciation and AR. We also outline some substantial remaining challenges to achieving a detailed understanding of adaptation, speciation, and the role of ecology in these processes. These major challenges include identifying factors that have a causative impact in promoting or constraining ARs, gaining a more holistic understanding of features of organisms and their environment that interact resulting in adaptation and speciation, and understanding whether the role of these organismal and environmental features varies throughout the radiation process. We conclude by providing perspectives on how future investigations into the AR process can overcome these challenges, allowing us to glean mechanistic insights into adaptation and speciation.

Online toolkits for collaborative and inclusive global research in urban evolutionary ecology.

Urban evolutionary ecology is inherently interdisciplinary. Moreover, it is a field with global significance. However, bringing researchers and resources together across fields and countries is challenging. Therefore, an online collaborative research hub, where common methods and best practices are shared among scientists from diverse geographic, ethnic, and career backgrounds would make research focused on urban evolutionary ecology more inclusive. Here, we describe a freely available online research hub for toolkits that facilitate global research in urban evolutionary ecology. We provide rationales and descriptions of toolkits for: (1) decolonizing urban evolutionary ecology; (2) identifying and fostering international collaborative partnerships; (3) common methods and freely-available datasets for trait mapping across cities; (4) common methods and freely-available datasets for cross-city evolutionary ecology experiments; and (5) best practices and freely available resources for public outreach and communication of research findings in urban evolutionary ecology. We outline how the toolkits can be accessed, archived, and modified over time in order to sustain long-term global research that will advance our understanding of urban evolutionary ecology.

Feeding ecology of the Terciopelo pit viper snake (Bothrops asper) in Ecuador.

Thoroughly documenting prey items and diet composition is crucial for understanding a predator's role in the ecosystem. In gape restricted predators, such as snakes, documenting and analyzing the type and size of the prey is important to interpret their ecological role. We describe the diet patterns of a species of venomous snake, the Terciopelo pit viper (Bothrops asper), from its Ecuadorian populations. Examining the gastrointestinal contents of museum specimens collected over an extensive area of the Pacific lowlands of Ecuador, we encountered 69 identifiable prey items from four major taxonomic groups (amphibians, centipedes, mammals, and reptiles). We evaluated the observed composition of prey to check for differences between sexes and size-classes. To complement our observations of the Terciopelo species complex throughout their distribution, we carried out a systematic literature review. Our data show an ontogenetic shift in diet, with a transition from more diverse diet in juveniles towards a mammal-specialized diet in adults, and distinct proportion of prey taxa between the sexes in the juvenile size class.

A retrospective study of clinical and epidemiological characteristics of snakebite in Napo Province, Ecuadorian Amazon.

BACKGROUND: Snakebite envenoming remains a relevant public health problem in tropical and subtropical countries. In Ecuador, this is particularly true in an area of great diversity like the Amazon region. Nevertheless, there is scarce information about epidemiological and clinical characteristics of these accidents in this area. METHODS: This was a descriptive and retrospective study of snakebite cases treated at a tertiary hospital in the Napo Province, Ecuadorian Amazon, from 2015 to 2019. We collected sociodemographic and snakebite-related information, clinical aspects and the use of antivenom and antibiotics from medical records. RESULTS: Information from 133 snakebite accidents was reviewed in this time period. Reports of snakebite envenoming decreased over the years. In total, 67% of those bitten were from nearby indigenous communities, which were the most affected groups. When a species was identified, Bothrops atrox was responsible for the highest number of cases registered. Local clinical manifestations were more frequent than systemic signs, in keeping with the typical effects produced by bothropic venoms. Additionally, data showed that more antivenom vials were given than those suggested by the protocol of the Ecuadorian Ministry of Health, in proportion to the grade of severity. Finally, we identified a low incidence of adverse reactions with antivenom administration, as well as a frequent use of antibiotics. CONCLUSIONS: The profile of snakebite accidents in the Napo Province is very similar to that described for other localities in the Amazon region of Ecuador and neighboring countries, with its challenges and limitations. Such aspects underlie the importance of establishing a robust and science-based public health program to respond to this frequent, but neglected, tropical disease.

Unlocking the potential of snake venom-based molecules against the malaria, Chagas disease, and leishmaniasis triad.

Malaria, leishmaniasis and Chagas disease are vector-borne protozoal infections with a disproportionately high impact on the most fragile societies in the world, and despite malaria-focused research gained momentum in the past two decades, both trypanosomiases and leishmaniases remain neglected tropical diseases. Affordable effective drugs remain the mainstay of tackling this burden, but toxicicty, inneficiency against later stage disease, and drug resistance issues are serious shortcomings. One strategy to overcome these hurdles is to get new therapeutics or inspiration in nature. Indeed, snake venoms have been recognized as valuable sources of biomacromolecules, like peptides and proteins, with antiprotozoal activity. This review highlights major snake venom components active against at least one of the three aforementioned diseases, which include phospholipases A2, metalloproteases, L-amino acid oxidases, lectins, and oligopeptides. The relevance of this repertoire of biomacromolecules and the bottlenecks in their clinical translation are discussed considering approaches that should increase the success rate in this arduous task. Overall, this review underlines how venom-derived biomacromolecules could lead to pioneering antiprotozoal treatments and how the drug landscape for neglected diseases may be revolutionized by a closer look at venoms. Further investigations on poorly studied venoms is needed and could add new therapeutics to the pipeline.

A First Look at the Inhibitory Potential of Urospatha sagittifolia (Araceae) Ethanolic Extract for Bothrops atrox Snakebite Envenomation.

Bothrops atrox snakebites are a relevant problem in the Amazon basin. In this biodiverse region, the ethnomedicinal approach plays an important role as an alternative to antivenom therapy. Urospatha sagittifolia (Araceae) is a plant used for this purpose; however, its neutralizing properties have not been scientifically accessed. To fill this gap, we investigated the ability of U. sagittifolia to modulate the catalytic activity of Bothrops atrox venom, and their toxic consequences, such as local damage and lethality. The venom profile of B. atrox was assessed by chromatography and electrophoresis. Inhibition of the three main enzymatic and medically important toxins from the venom was evaluated using synthetic substrates and quantified by chromogenic activity assays. Additionally, the neutralization of lethality, hemorrhage and edema were investigated by in vivo assays. The possible interactions between venom proteins and plant molecules were visualized by polyacrylamide gel electrophoresis. Finally, the phytochemical constituents present in the ethanolic extract were determined by qualitative and quantitative analyses. The ethanolic extract reduced the activity of the three main enzymes of venom target, achieving ranges from 19% to 81% of inhibition. Our in vivo venom neuralizations assays showed a significant inhibition of edema (38.72%) and hemorrhage (42.90%). Additionally, lethality was remarkably counteracted. The highest extract ratio evaluated had a 75% survival rate. Our data support the biomedical value of U. sagittifolia as a source of natural enzyme inhibitors able to neutralize catalytically active B. atrox venom toxins and their toxic effects.

Bothrops atrox from Ecuadorian Amazon: Initial analyses of venoms from individuals.

Bothrops atrox is the most clinically relevant snake species within the Amazon region, which includes Ecuadorian territories. It comprises a large distribution, which could contribute to the genetic and venomic variation identified in the species. The high variability and protein isoform diversity of its venom are of medical interest, since it can influence the clinical manifestations caused by envenomation and its treatment. However, in Ecuador there is insufficient information on the diversity of venomic phenotypes, even of relevant species such as B. atrox. Here, we characterized the biochemical and toxicological profiles of the venom of six B. atrox individuals from the Ecuadorian Amazon. Differences in catalytic activities of toxins, elution profiles in liquid chromatography, electrophoretic patterns, and toxic effects among the analyzed samples were identified. Nonetheless, in the preclinical testing of antivenom, two samples from Mera (Pastaza) required a higher dose to achieve total neutralization of lethality and hemorrhage. Taken together, these data highlight the importance of analyzing individual venoms in studies focused on the outcomes of envenoming.

Venom variation in Bothrops asper lineages from North-Western South America.

Bothrops asper is a venomous pitviper that is widely distributed and of clinical importance in Mesoamerica and northern South America, where it is responsible for 50-80% of all envenomations by Viperidae species. Previous work suggests that B. asper has a complex phylogeographic structure, with the existence of multiple evolutionarily distinct lineages, particularly in the inter-Andean valleys of north South America. To explore the impact of the evolutionary history of B. asper on venom composition, we have investigated geographic variation in the venom proteome of this species from the populations from the Pacific side of Ecuador and south-western Colombia. Among the 21 classes of venom components identified, proteins from mainly four major toxin families, snake venom metalloproteases (PI- and PII-SVMP), phospholipases A2 (K49- and D49-PLA2s), serine proteinases (SVSP), and C-type lectins-like (CTL) proteins are major contributors to the geographic variability in venom. Principal component analyses demonstrate significant differences in venom composition between B. asper lineages previously identified through combination of molecular, morphological and geographical data, and provide additional insights into the selection pressures modulating venom phenotypes on a geographic scale. In particular, altitudinal zonation within the Andean mountain range stands out as a key ecological factor promoting diversification in venom. In addition, the pattern of distribution of PLA2 molecules among B. asper venoms complements phylogenetic analysis in the reconstruction of the dispersal events that account for the current biogeographic distribution of the present-day species' phylogroups. Ontogenic variation was also evident among venoms from some Ecuadorian lineages, although this age-related variation was less extreme than reported in B. asper venoms from Costa Rica. The results of our study demonstrate a significant impact of phylogenetic history on venom composition in a pitviper and show how analyses of this variation can illuminate the timing of the cladogenesis and ecological events that shaped the current distribution of B. asper lineages. BIOLOGICAL SIGNIFICANCE: Bothrops asper, called "the ultimate pitviper" due to its defensive behavior, large body size, and medical importance, represents a species complex that is widely distributed from southern México southwards across north-western South America to north-western Perú. This work reports the characterization of the venom proteomes of B. asper lineages from the Pacific sides of Ecuador and south-western Colombia. Multivariate analyses indicate that variability in venom composition among the B. asper lineages is driven by proteins from four major toxin families, presumably in response to selection pressures created by recent and historical ecological conditions created by geological and climatic events from the Pliocene-Pleistocene to the present along the Central and South American Continental Divide. The emerging biogeographic pattern of venom variation, interpreted in the context of the current phylogenetic hypotheses, support and complement previously proposed evolutionary Plio-Pleistocene dispersal events that shaped the present-day distribution range of B. asper lineages. In addition, our venomics data indicate the occurrence of genetic exchange between Colombian and Pacific Costa Rican populations, which may have occurred during the second wave of B. asper migration into Mesoamerica. Our work represents a foundation for a future broader sampling and more complete "-omics" analyses to deepen our understanding of the patterns and causes of venom variation in this medically important pitviper.

Evaluating the utility of camera traps in field studies of predation.

Artificial prey techniques-wherein synthetic replicas of real organisms are placed in natural habitats-are widely used to study predation in the field. We investigated the extent to which videography could provide additional information to such studies. As a part of studies on aposematism and mimicry of coral snakes (Micrurus) and their mimics, observational data from 109 artificial snake prey were collected from video-recording camera traps in three locations in the Americas (terra firme forest, Tiputini Biodiversity Station, Ecuador; premontane wet forest, Nahá Reserve, Mexico; longleaf pine forest, Southeastern Coastal Plain, North Carolina, USA). During 1,536 camera days, a total of 268 observations of 20 putative snake predator species were recorded in the vicinity of artificial prey. Predators were observed to detect artificial prey 52 times, but only 21 attacks were recorded. Mammals were the most commonly recorded group of predators near replicas (243) and were responsible for most detections (48) and attacks (20). There was no difference between avian or mammalian predators in their probability of detecting replicas nor in their probability of attacking replicas after detecting them. Bite and beak marks left on clay replicas registered a higher ratio of avian:mammalian attacks than videos registered. Approximately 61.5% of artificial prey monitored with cameras remained undetected by predators throughout the duration of the experiments. Observational data collected from videos could provide more robust inferences on the relative fitness of different prey phenotypes, predator behavior, and the relative contribution of different predator species to selection on prey. However, we estimate that the level of predator activity necessary for the benefit of additional information that videos provide to be worth their financial costs is achieved in fewer than 20% of published artificial prey studies. Although we suggest future predation studies employing artificial prey to consider using videography as a tool to inspire new, more focused inquiry, the investment in camera traps is unlikely to be worth the expense for most artificial prey studies until the cost:benefit ratio decreases.

A new species of pitviper of the genus Bothrops (Serpentes: Viperidae: Crotalinae) from the Central Andes of South America.

We describe a new species of montane pitviper of the genus Bothrops from the Cordillera Oriental of the Central Andes, distributed from southern Peru to central Bolivia. The new species can be distinguished from its congeners by the characteristic combination of a dorsal body color pattern consisting of triangular or subtriangular dark brown dorsal blotches, paired dark brown parallel occipital stripes, a conspicuous dark brown postocular stripe, the presence of canthorostrals in some specimens, prelacunal fused or partially fused with second supralabial, one scale usually separating internasals, rostral trapezoidal, two canthals oval to rounded, similar size or slightly larger than internasals, three or four medial intercanthals, eight to twelve intersupraoculars, intercanthals and intersupraoculars keeled and frequently slightly keeled, supraoculars oval, one to three suboculars, two to three postoculars, loreal subtriangular, two to six prefoveals, subfoveals absent, two or none postfoveals, one or two scales between suboculars and fourth supralabial, seven or eight supralabials, nine or eleven infralabials, 23-25 middorsal scales, 189-195 ventrals in females and 182-190 in males, 48-58 subcaudals in females and 54-63 in males, exceptionally undivided. The new species is apparently restricted to areas within Andean montane forests that are less humid and devoid of large trees.

Real-time DNA barcoding in a rainforest using nanopore sequencing: opportunities for rapid biodiversity assessments and local capacity building.

Background: Advancements in portable scientific instruments provide promising avenues to expedite field work in order to understand the diverse array of organisms that inhabit our planet. Here, we tested the feasibility for in situ molecular analyses of endemic fauna using a portable laboratory fitting within a single backpack in one of the world's most imperiled biodiversity hotspots, the Ecuadorian Chocó rainforest. We used portable equipment, including the MinION nanopore sequencer (Oxford Nanopore Technologies) and the miniPCR (miniPCR), to perform DNA extraction, polymerase chain reaction amplification, and real-time DNA barcoding of reptile specimens in the field. Findings: We demonstrate that nanopore sequencing can be implemented in a remote tropical forest to quickly and accurately identify species using DNA barcoding, as we generated consensus sequences for species resolution with an accuracy of >99% in less than 24 hours after collecting specimens. The flexibility of our mobile laboratory further allowed us to generate sequence information at the Universidad Tecnológica Indoamérica in Quito for rare, endangered, and undescribed species. This includes the recently rediscovered Jambato toad, which was thought to be extinct for 28 years. Sequences generated on the MinION required as few as 30 reads to achieve high accuracy relative to Sanger sequencing, and with further multiplexing of samples, nanopore sequencing can become a cost-effective approach for rapid and portable DNA barcoding. Conclusions: Overall, we establish how mobile laboratories and nanopore sequencing can help to accelerate species identification in remote areas to aid in conservation efforts and be applied to research facilities in developing countries. This opens up possibilities for biodiversity studies by promoting local research capacity building, teaching nonspecialists and students about the environment, tackling wildlife crime, and promoting conservation via research-focused ecotourism.

Independent Recruitment of Different Types of Phospholipases A2 to the Venoms of Caenophidian Snakes: The Rise of PLA2-IIE within Pseudoboini (Dipsadidae)

Snake venoms harbor a wide and diverse array of enzymatic and nonenzymatic toxic components, allowing them to exert myriad effects on their prey. However, they appear to trend toward a few optimal compositional scaffolds, dominated by four major toxin classes: SVMPs, SVSPs, 3FTxs, and PLA2s. Nevertheless, the latter appears to be restricted to vipers and elapids, as it has never been reported as a major venom component in rear-fanged species. Here, by investigating the original transcriptomes from 19 species distributed in eight genera from the Pseudoboini tribe (Dipsadidae: Xenodontinae) and screening among seven additional tribes of Dipsadidae and three additional families of advanced snakes, we discovered that a novel type of venom PLA2, resembling a PLA2-IIE, has been recruited to the venom of some species of the Pseudoboini tribe, where it is a major component. Proteomic and functional analyses of these venoms further indicate that these PLA2s play a relevant role in the venoms from this tribe. Moreover, we reconstructed the phylogeny of PLA2s across different snake groups and show that different types of these toxins have been recruited in at least five independent events in caenophidian snakes. Additionally, we present the first compositional profiling of Pseudoboini venoms. Our results demonstrate how relevant phenotypic traits are convergently recruited by different means and from homologous and nonhomologous genes in phylogenetically and ecologically divergent snake groups, possibly optimizing venom composition to overcome diverse adaptative landscapes.

Independent recruitment of different types of Phospholipases A2 to the venoms of Caenophidian snakes: the rise of PLA2-IIE within Pseudoboini (Dipsadidae)

Snake venoms harbor a wide and diverse array of enzymatic and nonenzymatic toxic components, allowing them to exert myriad effects on their prey. However, they appear to trend toward a few optimal compositional scaffolds, dominated by four major toxin classes: SVMPs, SVSPs, 3FTxs, and PLA2s. Nevertheless, the latter appears to be restricted to vipers and elapids, as it has never been reported as a major venom component in rear-fanged species. Here, by investigating the original transcriptomes from 19 species distributed in eight genera from the Pseudoboini tribe (Dipsadidae: Xenodontinae) and screening among seven additional tribes of Dipsadidae and three additional families of advanced snakes, we discovered that a novel type of venom PLA2, resembling a PLA2-IIE, has been recruited to the venom of some species of the Pseudoboini tribe, where it is a major component. Proteomic and functional analyses of these venoms further indicate that these PLA2s play a relevant role in the venoms from this tribe. Moreover, we reconstructed the phylogeny of PLA2s across different snake groups and show that different types of these toxins have been recruited in at least five independent events in caenophidian snakes. Additionally, we present the first compositional profiling of Pseudoboini venoms. Our results demonstrate how relevant phenotypic traits are convergently recruited by different means and from homologous and nonhomologous genes in phylogenetically and ecologically divergent snake groups, possibly optimizing venom composition to overcome diverse adaptative landscapes.

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.