Museum DNA reveals a new, potentially extinct species of rinkhals (Serpentes: Elapidae: Hemachatus) from the Eastern Highlands of Zimbabwe.

Genetic information plays a pivotal role in species recognition and delimitation, but rare or extinct animals can be difficult to obtain genetic samples from. While natural history wet collections have proven invaluable in the description of novel species, the use of these historical samples in genetic studies has been greatly impeded by DNA degradation, especially because of formalin-fixation prior to preservation. Here, we use recently developed museum genomics approaches to determine the status of an isolated population of the elapid snake genus Hemachatus from Zimbabwe. We used multiple digestion phases followed by single strand sequencing library construction and hybridisation capture to obtain 12S and 16S rDNA sequences from a poorly preserved tissue sample of this population. Phylogenetic and morphological analyses in an integrated taxonomic framework demonstrate that the Zimbabwean rinkhals population represents an old and highly distinct lineage, which we describe as a new species, Hemachatus nyangensis sp. nov. Our phylogenetic dating analysis is compatible with venom spitting having evolved in response to the threat posed by early hominins, although more data are required for a robust test of this hypothesis. This description demonstrates the power of museum genomics in revealing rare or even extinct species: Hemachatus from Zimbabwe are only known from a small area of the Eastern Highlands known for high endemism. No living specimens have been seen since the 1980s, most likely due to dramatic land-use changes in the Eastern Highlands, suggesting that the species could be extinct. In view of its recognition as a highly distinct lineage, urgent action is required to determine whether any populations survive, and to safeguard remaining habitat.

One Size Fits All-Venomics of the Iberian Adder (Vipera seoanei, Lataste 1878) Reveals Low Levels of Venom Variation across Its Distributional Range.

European vipers (genus Vipera) are medically important snakes displaying considerable venom variation, occurring at different levels in this group. The presence of intraspecific venom variation, however, remains understudied in several Vipera species. Vipera seoanei is a venomous snake endemic to the northern Iberian Peninsula and south-western France, presenting notable phenotypic variation and inhabiting several diverse habitats across its range. We analysed the venoms of 49 adult specimens of V. seoanei from 20 localities across the species' Iberian distribution. We used a pool of all individual venoms to generate a V. seoanei venom reference proteome, produced SDS-PAGE profiles of all venom samples, and visualised patterns of variation using NMDS. By applying linear regression, we then assessed presence and nature of venom variation between localities, and investigated the effect of 14 predictors (biological, eco-geographic, genetic) on its occurrence. The venom comprised at least 12 different toxin families, of which five (i.e., PLA2, svSP, DI, snaclec, svMP) accounted for about 75% of the whole proteome. The comparative analyses of the SDS-PAGE venom profiles showed them to be remarkably similar across the sampled localities, suggesting low geographic variability. The regression analyses suggested significant effects of biological and habitat predictors on the little variation we detected across the analysed V. seoanei venoms. Other factors were also significantly associated with the presence/absence of individual bands in the SDS-PAGE profiles. The low levels of venom variability we detected within V. seoanei might be the result of a recent population expansion, or of processes other than directional positive selection.

Medically important snakes and snakebite envenoming in Iran.

Snakebite is a relatively common health condition in Iran with a diverse snake fauna, especially in tropical southern and mountainous western areas of the country with a plethora of snake species. The list of medically important snakes, circumstances and effects of their bite, and necessary medical care require critical appraisal and should be updated regularly. This study aims to review and map the distributions of medically important snake species of Iran, re-evaluate their taxonomy, review their venomics, describe the clinical effects of envenoming, and discuss medical management and treatment, including the use of antivenom. Nearly 350 published articles and 26 textbooks with information on venomous and mildly venomous snake species and snakebites of Iran, were reviewed, many in Persian (Farsi) language, making them relatively inaccessible to an international readership. This has resulted in a revised updated list of Iran's medically important snake species, with taxonomic revisions of some, compilation of their morphological features, remapping of their geographical distributions, and description of species-specific clinical effects of envenoming. Moreover, the antivenom manufactured in Iran is discussed, together with treatment protocols that have been developed for the hospital management of envenomed patients.

Multilevel Comparison of Indian Naja Venoms and Their Cross-Reactivity with Indian Polyvalent Antivenoms.

Snake envenoming is caused by many biological species, rather than a single infectious agent, each with a multiplicity of toxins in their venom. Hence, developing effective treatments is challenging, especially in biodiverse and biogeographically complex countries such as India. The present study represents the first genus-wide proteomics analysis of venom composition across Naja species (N. naja, N. oxiana, and N. kaouthia) found in mainland India. Venom proteomes were consistent between individuals from the same localities in terms of the toxin families present, but not in the relative abundance of those in the venom. There appears to be more compositional variation among N. naja from different locations than among N. kaouthia. Immunoblotting and in vitro neutralization assays indicated cross-reactivity with Indian polyvalent antivenom, in which antibodies raised against N. naja are present. However, we observed ineffective neutralization of PLA2 activities of N. naja venoms from locations distant from the source of immunizing venoms. Antivenom immunoprofiling by antivenomics revealed differential antigenicity of venoms from N. kaouthia and N. oxiana, and poor reactivity towards 3FTxs and PLA2s. Moreover, there was considerable variation between antivenoms from different manufacturers. These data indicate that improvements to antivenom manufacturing in India are highly desirable.

Genomics reveals broad hybridization in deeply divergent Palearctic grass and water snakes (Natrix spp.).

Understanding speciation is one of the cornerstones of biological diversity research. Currently, speciation is often understood as a continuous process of divergence that continues until genetic or other incompatibilities minimize or prevent interbreeding. The Palearctic snake genus Natrix is an ideal group to study speciation, as it comprises taxa representing distinct stages of the speciation process, ranging from widely interbreeding parapatric taxa through parapatric species with very limited gene flow in narrow hybrid zones to widely sympatric species. To understand the evolution of reproductive isolation through time, we have sequenced the genomes of all five species within this genus and two additional subspecies. We used both long-read and short-read methods to sequence and de-novo-assemble two high-quality genomes (Natrix h. helvetica, Natrix n. natrix) to their 1.7 Gb length with a contig N50 of 4.6 Mbp and 1.5 Mbp, respectively, and used these as references to assemble the remaining short-read-based genomes. Our phylogenomic analyses yielded a well-supported dated phylogeny and evidence for a surprisingly complex history of interspecific gene flow, including between widely sympatric species. Furthermore, evidence for gene flow was also found for currently allopatric species pairs. Genetic exchange among these well-defined, distinct, and several million-year-old reptile species emphasizes that speciation and maintenance of species distinctness can occur despite continued genetic exchange.

Toxic Habits: An Analysis of General Trends and Biases in Snake Venom Research.

Biases in snake venom research have been partially identified but seldomly quantified. Using the Google Scholar web search engine, we collected a total of 267 articles published between 1964 and 2021, and reviewed them to assess the main trends in this field of study. We developed a 4-category classification of the harmful potential of each of the 298 snake species retrieved from the analysed publications, and tested whether taxonomy, realm of origin, and/or assigned hazard category could affect how often each of them appeared in the articles considered. Overall, viperids were significantly more represented than any other snake taxon retrieved. The Neotropics were the most represented biogeographic realm for number of studied species, whereas information about the country of origin of the analysed specimens was often incomplete. The vast majority of the publications focused on snake venom characterisation, whereas more ecology-related topics were rarely considered. Hazard category and biogeographic realm of origin of each species had a significant effect on the number of articles dedicated to it, suggesting that a snake's harmful potential and place of origin influence its popularity in venom studies. Our analysis showed an overall positive trend in the number of snake venom studies published yearly, but also underlined severe neglect of snake families of supposedly minor medical relevance (e.g., Atractaspididae), underrepresentation of some of the areas most impacted by snakebite (i.e., Indomalayan and Afrotropic realms), and limited interest in the ecological and functional context of snake venom.

The benefits of contributing to the citizen science platform iNaturalist as an identifier.

As the number of observations submitted to the citizen science platform iNaturalist continues to grow, it is increasingly important that these observations can be identified to the finest taxonomic level, maximizing their value for biodiversity research. Here, we explore the benefits of acting as an identifier on iNaturalist.

Bacterial Adaptation to Venom in Snakes and Arachnida.

Animal venoms are considered sterile sources of antimicrobial compounds with strong membrane-disrupting activity against multidrug-resistant bacteria. However, venomous bite wound infections are common in developing nations. Investigating the envenomation organ and venom microbiota of five snake and two spider species, we observed venom community structures that depend on the host venomous animal species and evidenced recovery of viable microorganisms from black-necked spitting cobra (Naja nigricollis) and Indian ornamental tarantula (Poecilotheria regalis) venoms. Among the bacterial isolates recovered from N. nigricollis, we identified two venom-resistant, novel sequence types of Enterococcus faecalis whose genomes feature 16 virulence genes, indicating infectious potential, and 45 additional genes, nearly half of which improve bacterial membrane integrity. Our findings challenge the dogma of venom sterility and indicate an increased primary infection risk in the clinical management of venomous animal bite wounds. IMPORTANCE Notwithstanding their 3 to 5% mortality, the 2.7 million envenomation-related injuries occurring annually-predominantly across Africa, Asia, and Latin America-are also major causes of morbidity. Venom toxin-damaged tissue will develop infections in some 75% of envenomation victims, with E. faecalis being a common culprit of disease; however, such infections are generally considered to be independent of envenomation. Here, we provide evidence on venom microbiota across snakes and arachnida and report on the convergent evolution mechanisms that can facilitate adaptation to black-necked cobra venom in two independent E. faecalis strains, easily misidentified by biochemical diagnostics. Therefore, since inoculation with viable and virulence gene-harboring bacteria can occur during envenomation, acute infection risk management following envenomation is warranted, particularly for immunocompromised and malnourished victims in resource-limited settings. These results shed light on how bacteria evolve for survival in one of the most extreme environments on Earth and how venomous bites must be also treated for infections.

Convergent evolution of toxin resistance in animals.

Convergence is the phenomenon whereby similar phenotypes evolve independently in different lineages. One example is resistance to toxins in animals. Toxins have evolved many times throughout the tree of life. They disrupt molecular and physiological pathways in target species, thereby incapacitating prey or deterring a predator. In response, molecular resistance has evolved in many species exposed to toxins to counteract their harmful effects. Here, we review current knowledge on the convergence of toxin resistance using examples from a wide range of toxin families. We explore the evolutionary processes and molecular adaptations driving toxin resistance. However, resistance adaptations may carry a fitness cost if they disrupt the normal physiology of the resistant animal. Therefore, there is a trade-off between maintaining a functional molecular target and reducing toxin susceptibility. There are relatively few solutions that satisfy this trade-off. As a result, we see a small set of molecular adaptations appearing repeatedly in diverse animal lineages, a phenomenon that is consistent with models of deterministic evolution. Convergence may also explain what has been called 'autoresistance'. This is often thought to have evolved for self-protection, but we argue instead that it may be a consequence of poisonous animals feeding on toxic prey. Toxin resistance provides a unique and compelling model system for studying the interplay between trophic interactions, selection pressures and the molecular mechanisms underlying evolutionary novelties.

Interpopulational variation and ontogenetic shift in the venom composition of Lataste's viper (Vipera latastei, Boscá 1878) from northern Portugal.

Lataste's viper (Vipera latastei) is a venomous European viper endemic to the Iberian Peninsula, recognised as medically important by the World Health Organization. To date, no comprehensive characterisation of this species' venom has been reported. Here, we analysed the venoms of juvenile and adult specimens of V. latastei from two environmentally different populations from northern Portugal. Using bottom-up venomics, we produced six venom proteomes (three per population) from vipers belonging to both age classes (i.e., two juveniles and four adults), and RP-HPLC profiles of 54 venoms collected from wild specimens. Venoms from juveniles and adults differed in their chromatographic profiles and relative abundances of their toxins, suggesting the occurrence of ontogenetic changes in venom composition. Specifically, snake venom metalloproteinase (SVMP) was the most abundant toxin family in juvenile venoms, while snake venom serine proteinases (SVSPs), phospholipases A2 (PLA2s), and C-type lectin-like (CTLs) proteins were the main toxins comprising adult venoms. The RP-HPLC venom profiles were found to vary significantly between the two sampled localities, indicating geographic variability. Furthermore, the presence/absence of certain peaks in the venom chromatographic profiles appeared to be significantly correlated also to factors like body size and sex of the vipers. Our findings show that V. latastei venom is a variable phenotype. The intraspecific differences we detected in its composition likely mirror changes in the feeding ecology of this species, taking place during different life stages and under different environmental pressures. SIGNIFICANCE: Lataste's viper (Vipera latastei) is a medically important viper endemic to the Iberian Peninsula, inhabiting different habitats and undergoing a marked ontogenetic dietary shift. In the current study, we report the first proteomic analysis of V. latastei venom from two environmentally different localities in northern Portugal. Our bottom-up venomic analyses show that snake venom serine proteinases (SVSPs), phospholipases A2 (PLA2s), and C-type lectin-like (CTLs) proteins are the major components of adult V. latastei venom. The comparative analysis of young and adult venoms suggests the occurrence of ontogenetic shift in toxin abundances, with snake venom metalloproteinases (SVMPs) being the predominant toxins in juvenile venoms. Moreover, geographic venom variation between the two studied populations is also detected, with our statistical analyses suggesting that factors like body size and sex of the vipers are possibly at play in its determination. Our work represents the first assessment of the composition of V. latastei venom, and the first step towards a better understanding of the drivers behind its variability.

How do King Cobras move across a major highway? Unintentional wildlife crossing structures may facilitate movement.

Global road networks continue to expand, and the wildlife responses to these landscape-level changes need to be understood to advise long-term management decisions. Roads have high mortality risk to snakes because snakes typically move slowly and can be intentionally targeted by drivers.We investigated how radio-tracked King Cobras (Ophiophagus hannah) traverse a major highway in northeast Thailand, and if reproductive cycles were associated with road hazards.We surveyed a 15.3 km stretch of Highway 304 to determine if there were any locations where snakes could safely move across the road (e.g., culverts and bridges). We used recurse analyses to detect possible road-crossing events, and used dynamic Brownian Bridge Movement Models (dBBMMs) to show movement pathways association with possible unintentional crossing structures. We further used Integrated Step Selection Functions (ISSF) to assess seasonal differences in avoidance of major roads for adult King Cobras in relation to reproductive state.We discovered 32 unintentional wildlife crossing locations capable of facilitating King Cobra movement across the highway. While our dBBMMs broadly revealed underpasses as possible crossing points, they failed to identify specific underpasses used by telemetered individuals; however, the tracking locations pre- and post-crossing and photographs provided strong evidence of underpass use. Our ISSF suggested a lower avoidance of roads during the breeding season, although the results were inconclusive. With the high volume of traffic, large size of King Cobras, and a 98.8% success rate of crossing the road in our study (nine individuals: 84 crossing attempts with one fatality), we strongly suspect that individuals are using the unintentional crossing structures to safely traverse the road.Further research is needed to determine the extent of wildlife underpass use at our study site. We propose that more consistent integration of drainage culverts and bridges could help mitigate the impacts of roads on some terrestrial wildlife.

Mohave Rattlesnake (Crotalus scutulatus) Identification Revisited.

Crotalus scutulatus (Mohave rattlesnake) is a clinically significant pit viper broadly distributed across much of the arid southwestern United States and mainland Mexico. Identification of C scutulatus is a concern among emergency medical service and emergency department personnel owing to its reputation for severe envenomations and difficulty in visually differentiating between C scutulatus and other species, primarily Crotalus atrox (western diamond-backed rattlesnake). We contrast distinctive characteristics of C scutulatus, C atrox, and 3 other sympatric species: Crotalus molossus, Crotalus ornatus, and Crotalus viridis (western and eastern black-tailed rattlesnakes and prairie rattlesnake, respectively). Greenish coloration eliminates C atrox but does not confirm C scutulatus. Obvious coarse and fine speckling of the dorsal pattern and a pale postocular stripe intersecting the mouth characterize C atrox. Dorsal speckling is insignificant or absent in the other species, whereas the pale postocular stripe passes above the mouth in C scutulatus and C viridis and is absent in C molossus and C ornatus. Tails boldly ringed with alternating black and white or contrasting shades of gray are shared by C atrox and C scutulatus, respectively, but a lack of boldly ringed tails characterizes the other species. The proximal rattle segment is yellow and black, or entirely yellow, in C scutulatus but black in the others. The most reliable visual identifications are based on evaluations of multiple traits, all of which are variable to some extent. Traits such as tail ring width and the size and number of crown scales have frequently been overemphasized in the past.

Promoting co-existence between humans and venomous snakes through increasing the herpetological knowledge base.

Snakebite incidence at least partly depends on the biology of the snakes involved. However, studies of snake biology have been largely neglected in favour of anthropic factors, with the exception of taxonomy, which has been recognised for some decades to affect the design of antivenoms. Despite this, within-species venom variation and the unpredictability of the correlation with antivenom cross-reactivity has continued to be problematic. Meanwhile, other aspects of snake biology, including behaviour, spatial ecology and activity patterns, distribution, and population demography, which can contribute to snakebite mitigation and prevention, remain underfunded and understudied. Here, we review the literature relevant to these aspects of snakebite and illustrate how demographic, spatial, and behavioural studies can improve our understanding of why snakebites occur and provide evidence for prevention strategies. We identify the large gaps that remain to be filled and urge that, in the future, data and relevant metadata be shared openly via public data repositories so that studies can be properly replicated and data used in future meta-analyses.

King or royal family? Testing for species boundaries in the King Cobra, Ophiophagus hannah (Cantor, 1836), using morphology and multilocus DNA analyses.

In widespread species, the diverse ecological conditions in which the populations occur, and the presence of many potential geographical barriers through their range are expected to have created ample opportunities for the evolution of distinct, often cryptic lineages. In this work, we tested for species boundaries in one such widespread species, the king cobra, Ophiophagus hannah (Cantor, 1836), a largely tropical elapid snake distributed across the Oriental realm. Based on extensive geographical sampling across most of the range of the species, we initially tested for candidate species (CS) using Maximum-Likelihood analysis of mitochondrial genes. We then tested the resulting CS using both morphological data and sequences of three single-copy nuclear genes. We used snapclust to determine the optimal number of clusters in the nuclear dataset, and Bayesian Phylogenetics and Phylogeography (BPP) to test for likely species status. We used non-metric multidimensional scaling (nMDS) analysis for discerning morphological separation. We recovered four independently evolving, geographically separated lineages that we consider Confirmed Candidate Species: (1) Western Ghats lineage; (2) Indo-Chinese lineage (3) Indo-Malayan lineage; (4) Luzon Island lineage, in the Philippine Archipelago. We discuss patterns of lineage divergence, particularly in the context of low morphological divergence, and the conservation implications of recognizing several endemic king cobra lineages.

Unexpected lack of specialisation in the flow properties of spitting cobra venom.

Venom spitting is a defence mechanism based on airborne venom delivery used by a number of different African and Asian elapid snake species ('spitting cobras'; Naja spp. and Hemachatus spp.). Adaptations underpinning venom spitting have been studied extensively at both behavioural and morphological level in cobras, but the role of the physical properties of venom itself in its effective projection remains largely unstudied. We hereby provide the first comparative study of the physical properties of venom in spitting and non-spitting cobras. We measured the viscosity, protein concentration and pH of the venom of 13 cobra species of the genus Naja from Africa and Asia, alongside the spitting elapid Hemachatus haemachatus and the non-spitting viper Bitis arietans. By using published microCT scans, we calculated the pressure required to eject venom through the fangs of a spitting and a non-spitting cobra. Despite the differences in the modes of venom delivery, we found no significant differences between spitters and non-spitters in the rheological and physical properties of the studied venoms. Furthermore, all analysed venoms showed a Newtonian flow behaviour, in contrast to previous reports. Although our results imply that the evolution of venom spitting did not significantly affect venom viscosity, our models of fang pressure suggests that the pressure requirements to eject venom are lower in spitting cobras than in non-spitting cobras.

Citizen science and online data: Opportunities and challenges for snake ecology and action against snakebite.

The secretive behavior and life history of snakes makes studying their biology, distribution, and the epidemiology of venomous snakebite challenging. One of the most useful, most versatile, and easiest to collect types of biological data are photographs, particularly those that are connected with geographic location and date-time metadata. Photos verify occurrence records, provide data on phenotypes and ecology, and are often used to illustrate new species descriptions, field guides and identification keys, as well as in training humans and computer vision algorithms to identify snakes. We scoured eleven online and two offline sources of snake photos in an attempt to collect as many photos of as many snake species as possible, and attempt to explain some of the inter-species variation in photograph quantity among global regions and taxonomic groups, and with regard to medical importance, human population density, and range size. We collected a total of 725,565 photos-between 1 and 48,696 photos of 3098 of the world's 3879 snake species (79.9%), leaving 781 "most wanted" species with no photos (20.1% of all currently-described species as of the December 2020 release of The Reptile Database). We provide a list of most wanted species sortable by family, continent, authority, and medical importance, and encourage snake photographers worldwide to submit photos and associated metadata, particularly of "missing" species, to the most permanent and useful online archives: The Reptile Database, iNaturalist, and HerpMapper.

An evaluation of the nomina for death adders (Acanthophis Daudin, 1803) proposed by Wells amp; Wellington (1985), and confirmation of A. cryptamydros Maddock et al., 2015 as the valid name for the Kimberley death adder.

We assess the availability of four names proposed by Wells Wellington (1985) for Australian death adders (Acanthophis). In agreement with previous literature, A. hawkei is an available name, whereas A. armstrongi, A. lancasteri, and A. schistos are not described in conformity with the requirements of Articles 13.1.1 or 13.1.2 of the International Code of Zoological Nomenclature and are therefore considered nomina nuda. Consequently, A. cryptamydros Maddock et al., 2015, is confirmed as the valid name for the Kimberley death adder of Western Australia. We comment on the need for greater clarity in the Code, and emphasise that the responsibility for establishing the availability of new nomina rests with their authors, not subsequent researchers.

Genome-wide data implicate terminal fusion automixis in king cobra facultative parthenogenesis.

Facultative parthenogenesis (FP) is widespread in the animal kingdom. In vertebrates it was first described in poultry nearly 70 years ago, and since then reports involving other taxa have increased considerably. In the last two decades, numerous reports of FP have emerged in elasmobranch fishes and squamate reptiles (lizards and snakes), including documentation in wild populations of both clades. When considered in concert with recent evidence of reproductive competence, the accumulating data suggest that the significance of FP in vertebrate evolution has been largely underestimated. Several fundamental questions regarding developmental mechanisms, nonetheless, remain unanswered. Specifically, what is the type of automixis that underlies the production of progeny and how does this impact the genomic diversity of the resulting parthenogens? Here, we addressed these questions through the application of next-generation sequencing to investigate a suspected case of parthenogenesis in a king cobra (Ophiophagus hannah). Our results provide the first evidence of FP in this species, and provide novel evidence that rejects gametic duplication and supports terminal fusion as a mechanism underlying parthenogenesis in snakes. Moreover, we precisely estimated heterozygosity in parthenogenetic offspring and found appreciable retained genetic diversity that suggests that FP in vertebrates has underappreciated evolutionary significance.

Unexpected lack of specialisation in the flow properties of spitting cobra venom.

Venom spitting is a defence mechanism based on airborne venom delivery used by a number of different African and Asian elapid snake species ('spitting cobras'; Naja spp. and Hemachatus spp.). Adaptations underpinning venom spitting have been studied extensively at both behavioural and morphological level in cobras, but the role of the physical properties of venom itself in its effective projection remains largely unstudied. We hereby provide the first comparative study of the physical properties of venom in spitting and non-spitting cobras. We measured the viscosity, protein concentration and pH of the venom of 13 cobra species of the genus Naja from Africa and Asia, alongside the spitting elapid Hemachatus haemachatus and the non-spitting viper Bitis arietans By using published microCT scans, we calculated the pressure required to eject venom through the fangs of a spitting and a non-spitting cobra. Despite the differences in the modes of venom delivery, we found no significant differences between spitters and non-spitters in the rheological and physical properties of the studied venoms. Furthermore, all analysed venoms showed a Newtonian flow behaviour, in contrast to previous reports. Although our results imply that the evolution of venom spitting did not significantly affect venom viscosity, our models of fang pressure suggests that the pressure requirements to eject venom are lower in spitting cobras than in non-spitting cobras.