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Background: Variation in snake venoms is well documented, both between and within species, with intraspecific venom variation often correlated with geographically distinct populations. The puff adder, Bitis arietans, is widely distributed across sub-Saharan Africa and into the Arabian Peninsula where it is considered a leading cause of the ~310,000 annual snakebites across the region, with its venom capable of causing substantial morbidity and mortality. Despite its medical importance and wide geographic distribution, there is little known about venom variation between different B. arietans populations and the potential implications of this variation on antivenom efficacy., Methodology: We applied a range of analyses, including venom gland transcriptomics, in vitro enzymatic assays and reverse phase chromatography to comparatively analyse B. arietans venoms originating from Nigeria, Tanzania, and South Africa. Immunological assays and in vitro enzymatic neutralisation assays were then applied to investigate the impact of venom variation on the potential efficacy of three antivenom products; SAIMR Polyvalent, EchiTAb-Plus and Fav-Afrique., Findings: Through the first comparison of venom gland transcriptomes of B. arietans from three geographically distinct regions (Nigeria, Tanzania, and South Africa), we identified substantial variation in toxin expression. Findings of venom variation were further supported by chromatographic venom profiling, and the application of enzymatic assays to quantify the activity of three pathologically relevant toxin families. However, the use of western blotting, ELISA, and in vitro enzymatic inhibition assays revealed that variation within B. arietans venom does not appear to substantially impact upon the efficacy of three African polyvalent antivenoms., Conclusions: The large distribution and medical importance of B. arietans makes this species ideal for understanding venom variation and the impact this has on therapeutic efficacy. The findings in this study highlight the likelihood for considerable venom toxin variation across the range of B. arietans, but that this may not dramatically impact upon the utility of treatment available in the region., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Dawson et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Despite decades of molecular research, phylogenetic relationships in Palearctic vipers (genus Vipera) still essentially rely on a few loci, such as mitochondrial barcoding genes. Here we examined the diversity and evolution of Vipera with ddRAD-seq data from 33 representative species and subspecies. Phylogenomic analyses of ∼ 1.1 Mb recovered nine major clades corresponding to known species/species complexes which are generally consistent with the mitochondrial phylogeny, albeit with a few deep discrepancies that highlight past hybridization events. The most spectacular case is the Italian-endemic V. walser, which is grouped with the alpine genetic diversity of V. berus in the nuclear tree despite carrying a divergent mitogenome related to the Caucasian V. kaznakovi complex. Clustering analyses of SNPs suggest potential admixture between diverged Iberian taxa (V. aspis zinnikeri and V. seoanei), and confirm that the Anatolian V. pontica corresponds to occasional hybrids between V. (ammodytes) meridionalis and V. kaznakovi. Finally, all analyzed lineages of the V. berus complex (including V. walser and V. barani) form vast areas of admixture and may be delimited as subspecies. Our study sets grounds for future taxonomic and phylogeographic surveys on Palearctic vipers, a group of prime interest for toxinological, ecological, biogeographic and conservation research., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Disintegrins, a family of snake venom protein, which are capable of modulating the activity of integrins that play a fundamental role in the regulation of many physiological and pathological processes. The main purpose of this study is to obtain the recombinant disintegrin (r-DI) and evaluate its biological activity. In this study, we explored a high-level expression prokaryotic system and purification strategy for r-DI. Then, r-DI was treated to assay effects on cell growth, migration, and invasion. The affinity for the interactions of r-DI with integrin was determined using Surface plasmon resonance (SPR) analyses. The r-DI can be expressed in Escherichia coli and purified by one-step chromatography. The r-DI can inhibit B16F10 cells proliferation, migration, and invasion. Also, we found that r-DI could interact with the integrin αIIbβ3 (GPIIb/IIIa). The r-DI can be expressed, purified, characterized through functional assays, and can also maintain strong biological activities. Thus, this study showed potential therapeutic effects of r-DI for further functional and structural studies., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Asian mock vipers of the genus Psammodynastes and African forest snakes of the genus Buhoma are two genera belonging to the snake superfamily Elapoidea. The phylogenetic placements of Psammodynastes and Buhoma within Elapoidea has been extremely unstable which has resulted in their uncertain and debated taxonomy. We used ultraconserved elements and traditional nuclear and mitochondrial markers to infer the phylogenetic relationships of these two genera with other elapoids. Psammodynastes, for which a reference genome has been sequenced, were found, with strong branch support, to be a relatively early diverging split within Elapoidea that is sister to a clade consisting of Elapidae, Micrelapidae and Lamprophiidae. Hence, we allocate Psammodynastes to its own family, Psammodynastidae new family. However, the phylogenetic position of Buhoma could not be resolved with a high degree of confidence. Attempts to identify the possible sources of conflict in the rapid radiation of elapoid snakes suggest that both hybridisation/introgression during the rapid diversification, including possible ghost introgression, as well as incomplete lineage sorting likely have had a confounding role. The usual practice of combining mitochondrial loci with nuclear genomic data appears to mislead phylogeny reconstructions in rapid radiation scenarios, especially in the absence of genome scale data., (© 2024. The Author(s).)

Background: Venom systems are ideal models to study genetic regulatory mechanisms that underpin evolutionary novelty. Snake venom glands are thought to share a common origin, but there are major distinctions between venom toxins from the medically significant snake families Elapidae and Viperidae, and toxin gene regulatory investigations in elapid snakes have been limited. Here, we used high-throughput RNA-sequencing to profile gene expression and microRNAs between active (milked) and resting (unmilked) venom glands in an elapid (Eastern Brown Snake, Pseudonaja textilis), in addition to comparative genomics, to identify cis- and trans-acting regulation of venom production in an elapid in comparison to viperids (Crotalus viridis and C. tigris)., Results: Although there is conservation in high-level mechanistic pathways regulating venom production (unfolded protein response, Notch signaling and cholesterol homeostasis), there are differences in the regulation of histone methylation enzymes, transcription factors, and microRNAs in venom glands from these two snake families. Histone methyltransferases and transcription factor (TF) specificity protein 1 (Sp1) were highly upregulated in the milked elapid venom gland in comparison to the viperids, whereas nuclear factor I (NFI) TFs were upregulated after viperid venom milking. Sp1 and NFI cis-regulatory elements were common to toxin gene promoter regions, but many unique elements were also present between elapid and viperid toxins. The presence of Sp1 binding sites across multiple elapid toxin gene promoter regions that have been experimentally determined to regulate expression, in addition to upregulation of Sp1 after venom milking, suggests this transcription factor is involved in elapid toxin expression. microRNA profiles were distinctive between milked and unmilked venom glands for both snake families, and microRNAs were predicted to target a diversity of toxin transcripts in the elapid P. textilis venom gland, but only snake venom metalloproteinase transcripts in the viperid C. viridis venom gland. These results suggest differences in toxin gene posttranscriptional regulation between the elapid P. textilis and viperid C. viridis., Conclusions: Our comparative transcriptomic and genomic analyses between toxin genes and isoforms in elapid and viperid snakes suggests independent toxin regulation between these two snake families, demonstrating multiple different regulatory mechanisms underpin a venomous phenotype., (© 2024. The Author(s).)

Background: European vipers (genus Vipera) are a well-studied taxonomic group, but the low resolution of nuclear sanger-sequenced regions has precluded thorough studies at systematic, ecological, evolutionary and conservation levels. In this study, we developed novel microsatellite markers for the three Iberian vipers, Vipera aspis, V. latastei and V. seoanei, and assessed their polymorphism in north-central Iberian populations., Methods and Results: Genomic libraries were developed for each species using an Illumina Miseq sequencing approach. From the 70 primer pairs initially tested, 48 amplified reliably and were polymorphic within species. Cross-species transferability was achieved for 31 microsatellites loci in the three target species and four additional loci that were transferable to one species only. The 48 loci amplified in average seven alleles, and detected average expected and observed heterozygosities of 0.7 and 0.55, in the three genotyped populations/species (26 V. aspis, 20 V. latastei and 10 V. seoanei)., Conclusions: Our study provides a selection of 48 polymorphic microsatellite markers that will contribute significantly to current knowledge on genetic diversity, gene flow, population structure, demographic dynamics, systematics, reproduction and heritability in these species, and potentially in other congeneric taxa., (© 2024. The Author(s).)

The desert vipers of the genus Cerastes are a small clade of medically important venomous snakes within the family Viperidae. According to published morphological and molecular studies, the group is comprised by four species: two morphologically similar and phylogenetically sister taxa, the African horned viper (Cerastes cerastes) and the Arabian horned viper (Cerastes gasperettii); a more distantly related species, the Saharan sand viper (Cerastes vipera), and the enigmatic Böhme's sand viper (Cerastes boehmei), only known from a single specimen in captivity allegedly captured in Central Tunisia. In this study, we sequenced one mitochondrial marker (COI) as well as genome-wide data (ddRAD sequencing) from 28 and 41 samples, respectively, covering the entire distribution range of the genus to explore the population genomics, phylogenomic relationships and introgression patterns within the genus Cerastes. Additionally, and to provide insights into the mode of diversification of the group, we carried out niche overlap analyses considering climatic and habitat variables. Both nuclear phylogenomic reconstructions and population structure analyses have unveiled an unexpected evolutionary history for the genus Cerastes, which sharply contradicts the morphological similarities and previously published mitochondrial approaches. Cerastes cerastes and C. vipera are recovered as sister taxa whilst C. gasperettii is a sister taxon to the clade formed by these two species. We found a relatively high niche overlap (OI > 0.7) in both climatic and habitat variables between C. cerastes and C. vipera, contradicting a potential scenario of sympatric speciation. These results are in line with the introgression found between the northwestern African populations of C. cerastes and C. vipera. Finally, our genomic data confirms the existence of a lineage of C. cerastes in Arabia. All these results highlight the importance of genome-wide data over few genetic markers to study the evolutionary history of species., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

This study presents the first complete mitogenome of the Brazilian Atlantic bushmaster Lachesis with insights into snake evolution. The total length was 17,177 bp, consisting of 13 PCGs, 22 tRNAs, two rRNAs and a duplicate control region (CRs). Almost all genes were encoded by the heavy-strand, except for the ND6 gene and eight tRNAs (tRNA-Gln, Ala, Asn, Cys, Tyr, Ser[TGA anticodon], Glu, Pro). Only ATG, ATA, and ATC were starting codons for protein-coding sequences. Stop codons mainly were TAA, AGA, AGG, and TAG; whereas ND1, ND3, and CYTB terminated with incomplete stop codons. Phylogeny retrieved Lachesis within the Crotalinae as the sister group of Agkistrodon; and the Lachesis+Agkistrodon clade as the sister group of (Sistrurus+Crotalus)+Bothrops. The tree supports Crotalinae, Viperinae, and Azemiopinae in the Viperidae family, being sister taxa of Colubridae+(Elapidae+Psammophiidae). The mean genetic distance across 15 snake families and 57 nucleotide sequences was 0.37. The overall mean value of genetic distance across the Crotalinae was 0.23, with Lachesis muta exhibiting the shortest distance of 0.2 with Agkistrodon piscivorus, Protobothrops dabieshanensis and P. flavoviridis and the greatest 0.25 with Gloydius blomhoffii, Trimeresurus albolabris, S. miliarius, and Deinagkistrodon acutus. The complete Atlantic L. muta mitogenome presented herein is only the third annotated mitogenome from more than 430 described Brazilian snake species.

Although Hepatozoon spp. remains the most prevalent intracellular protozoa infecting snakes, it was reported only in a few snake species of the Colubridae family in Türkiye. Moreover, studies on these hemoparasites are not available in venomous nose-horned vipers from Türkiye. In this study, we investigated Hepatozoon spp. in three individual Vipera ammodytes using morphological and molecular methods. Our results were positive for intraerythrocytic Hepatozoon spp. gamonts in all three snakes, exhibiting low parasitemia. The microscopic findings were further confirmed through molecular data. A genus-specific PCR assay targeting the 18S rRNA gene region of Hepatozoon spp., was performed using HemoF/HemoR and Hep300/Hep900 primers. The obtained sequences were concatenated and used in phylogenetic analyses in comparison with different Hepatozoon species. Although our (OP377741) isolate was separated into a different branch, it was clustered with the isolates of H. massardi (KC342526), H. cevapii (KC342525), and H. annulatum (ON262426) from Brazilian snakes. Moreover, gene similarity and pair-wise distance between our isolate and other Hepatozoon species infecting snakes were found to be 89.30-98.63% and 0.009-0.077, respectively. Hence, we reported a new species of Hepatozoon, namely Hepatozoon viperoi sp. nov. infecting V. ammodytes. Since the literature does not indicate the existence of such a Hepatozoon species in V. ammodytes in different countries, our data may contribute to the expanding knowledge of Hepatozoon species in snakes, providing new insights into the biodiversity of the haemogregarine protozoan parasite., (© 2023. The Author(s).)

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., (© 2023 Willi Hennig Society.)

Conservation genetic theory suggests that small and isolated populations should be subject to reduced genetic diversity i.e., heterozygosity and allelic diversity. Our 34 years study of an isolated island population of adders (Vipera berus) in southern Sweden challenges this notion. Despite a lack of gene flow and a yearly mean estimated reproductive adult population size of only 65 adult adders (range 12-171), the population maintains high levels of heterozygosity and allelic diversity similar to that observed in two mainland populations. Even a 14-year major "bottleneck" i.e., a reduction in adult adder numbers, encompassing at least four adder generations, did not result in any reduction in the island adders' heterozygosity and allelic diversity. Female adders are polyandrous, and fertilisation is non-random, which our empirical data and modelling suggest are underpinning the maintenance of the population's high level of heterozygosity. Our empirical results and subsequent modelling suggest that the positive genetic effects of polyandry in combination with non-random fertilisation, often overlooked in conservation genetic analyses, deserve greater consideration when predicting long-term survival of small and isolated populations., (© 2022. The Author(s), under exclusive licence to The Genetics Society.)

Despite the medical significance to humans and important ecological roles filled by vipers, few high-quality genomic resources exist for these snakes outside of a few genera of pitvipers. Here we sequence, assemble, and annotate the genome of Fea's Viper (Azemiops feae). This taxon is distributed in East Asia and belongs to a monotypic subfamily, sister to the pitvipers. The newly sequenced genome resulted in a 1.56 Gb assembly, a contig N50 of 1.59 Mb, with 97.6% of the genome assembly in contigs >50 Kb, and a BUSCO completeness of 92.4%. We found that A. feae venom is primarily composed of phospholipase A2 (PLA2) proteins expressed by genes that likely arose from lineage-specific PLA2 gene duplications. Additionally, we show that renin, an enzyme associated with blood pressure regulation in mammals and known from the venoms of two viper species including A. feae, is expressed in the venom gland at comparative levels to known toxins and is present in the venom proteome. The cooption of this gene as a toxin may be more widespread in viperids than currently known. To investigate the historical population demographics of A. feae, we performed coalescent-based analyses and determined that the effective population size has remained stable over the last 100 kyr. This suggests Quaternary glacial cycles likely had minimal influence on the demographic history of A. feae. This newly assembled genome will be an important resource for studying the genomic basis of phenotypic evolution and understanding the diversification of venom toxin gene families., (© The Author(s) 2022. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

A new cryptic species of green pit viper is described from northeast India, based on specimens collected from the state of Mizoram and Meghalaya. The new species is a member of the subgenus Viridovipera and is sister to Trimeresurus medoensis based on molecular data for mitochondrial cytochrome b gene, whereas resembles Trimeresurus gumprechti morphologically. A combination of characters helps delimit the new species from its congeners. Description of the new species highlights the need for dedicated surveys across northeast India to document its reptilian diversity, as this represents the third new species of the genus to be described in the past three years., Competing Interests: The authors have declared that no competing interests exist.

This short essay pretends to make the reader reflect on the concept of biological mass and on the added value that the determination of this molecular property of a protein brings to the interpretation of evolutionary and translational snake venomics research. Starting from the premise that the amino acid sequence is the most distinctive primary molecular characteristics of any protein, the thesis underlying the first part of this essay is that the isotopic distribution of a protein's molecular mass serves to unambiguously differentiate it from any other of an organism's proteome. In the second part of the essay, we discuss examples of collaborative projects among our laboratories, where mass profiling of snake venom PLA2 across conspecific populations played a key role revealing dispersal routes that determined the current phylogeographic pattern of the species., (© 2021 The Author(s).)

The thermal environment can affect the evolution of morpho-behavioral adaptations of ectotherms. Heat is transferred from substrates to organisms by conduction and reflected radiation. Because brightness influences the degree of heat absorption, substrates could affect the evolution of integumentary optical properties. Here, we show that vipers (Squamata:Viperidae) inhabiting hot, highly radiative and superficially conductive substrates have evolved bright ventra for efficient heat transfer. We analyzed the brightness of 4161 publicly available images from 126 species, and we found that substrate type, alongside latitude and body mass, strongly influences ventral brightness. Substrate type also significantly affects dorsal brightness, but this is associated with different selective forces: activity-pattern and altitude. Ancestral estimation analysis suggests that the ancestral ventral condition was likely moderately bright and, following divergence events, some species convergently increased their brightness. Vipers diversified during the Miocene and the enhancement of ventral brightness may have facilitated the exploitation of arid grounds. We provide evidence that integument brightness can impact the behavioral ecology of ectotherms.

Colouration may endorse thermoregulatory and antipredatory functions in snakes. The thermal melanism hypothesis predicts that dark-coloured individuals are ecologically favoured in cool climates. However, the loss of aposematic and cryptic colourations may imply high predation for melanistic snakes. Here, we used the monophyletic group of Eurasian vipers (subfamily Viperinae) to test whether an increase in the extent of dark area inside the characteristic zigzag dorsal pattern is associated to colder environments. We measured two colouration traits in zigzag-patterned individuals (number of dorsal marks and weighted pigmentation index) and used a phylogenetic comparative approach to explore macroevolutionary patterns of dorsal pigmentation and test whether its extent is associated to ecogeographic characteristics of lineages' ranges. Phylogenetically-naïve and phylogenetically-informed analyses yielded a significant association between the degree of pigmentation of the zigzag pattern and environmental variables such as solar radiation, elevation and latitude. The degree of pigmentation of the zigzag pattern is highlighted as an adaptive trait that matches range attributes mirroring cold environments irrespective of the phylogeny. These results constitute the first large-scale evidence supporting the thermal melanism hypothesis in snakes, opening new avenues of inquiry for the mechanisms that shape the evolution of colour phenotypes.

Genetic factors are often overlooked in conservation planning, despite their importance in small isolated populations. We used mitochondrial and microsatellite markers to investigate population genetics of the adder (Vipera berus) in southern Britain, where numbers are declining. We found no evidence for loss of heterozygosity in any of the populations studied. Genetic diversity was comparable across sites, in line with published levels for mainland Europe. However, further analysis revealed a striking level of relatedness. Genetic networks constructed from inferred first degree relationships suggested a high proportion of individuals to be related at a level equivalent to that of half-siblings, with rare inferred full-sib dyads. These patterns of relatedness can be attributed to the high philopatry and low vagility of adders, which creates high local relatedness, in combination with the polyandrous breeding system in the adder, which may offset the risk of inbreeding in closed populations. We suggest that reliance on standard genetic indicators of inbreeding and diversity may underestimate demographic and genetic factors that make adder populations vulnerable to extirpation. We stress the importance of an integrated genetic and demographic approach in the conservation of adders, and other taxa of similar ecology., Competing Interests: The authors have declared that no competing interests exist.

The nose-horned viper, its nominotypical subspecies Vipera ammodytes ammodytes ( Vaa), in particular, is, medically, one of the most relevant snakes in Europe. The local and systemic clinical manifestations of poisoning by the venom of this snake are the result of the pathophysiological effects inflicted by enzymatic and nonenzymatic venom components acting, most prominently, on the blood, cardiovascular, and nerve systems. This venom is a very complex mixture of pharmacologically active proteins and peptides. To help improve the current antivenom therapy toward higher specificity and efficiency and to assist drug discovery, we have constructed, by combining transcriptomic and proteomic analyses, the most comprehensive library yet of the Vaa venom proteins and peptides. Sequence analysis of the venom gland cDNA library has revealed the presence of messages encoding 12 types of polypeptide precursors. The most abundant are those for metalloproteinase inhibitors (MPis), bradykinin-potentiating peptides (BPPs), and natriuretic peptides (NPs) (all three on a single precursor), snake C-type lectin-like proteins (snaclecs), serine proteases (SVSPs), P-II and P-III metalloproteinases (SVMPs), secreted phospholipases A 2 (sPLA 2 s), and disintegrins (Dis). These constitute >88% of the venom transcriptome. At the protein level, 57 venom proteins belonging to 16 different protein families have been identified and, with SVSPs, sPLA 2 s, snaclecs, and SVMPs, comprise ∼80% of all venom proteins. Peptides detected in the venom include NPs, BPPs, and inhibitors of SVSPs and SVMPs. Of particular interest, a transcript coding for a protein similar to P-III SVMPs but lacking the MP domain was also found at the protein level in the venom. The existence of such proteins, also supported by finding similar venom gland transcripts in related snake species, has been demonstrated for the first time, justifying the proposal of a new P-IIIe subclass of ancestral SVMP precursor-derived proteins.

Atractaspidines are poorly studied, fossorial snakes that are found throughout Africa and western Asia, including the Middle East. We employed concatenated gene-tree analyses and divergence dating approaches to investigate evolutionary relationships and biogeographic patterns of atractaspidines with a multi-locus data set consisting of three mitochondrial (16S, cyt b, and ND4) and two nuclear genes (c-mos and RAG1). We sampled 91 individuals from both atractaspidine genera (Atractaspis and Homoroselaps). Additionally, we used ancestral-state reconstructions to investigate fang and diet evolution within Atractaspidinae and its sister lineage (Aparallactinae). Our results indicated that current classification of atractaspidines underestimates diversity within the group. Diversification occurred predominantly between the Miocene and Pliocene. Ancestral-state reconstructions suggest that snake dentition in these taxa might be highly plastic within relatively short periods of time to facilitate adaptations to dynamic foraging and life-history strategies., Competing Interests: MFB is affiliated with Flora Fauna & Man, Ecological Services Ltd. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Scaling evolutionary trees to time is essential for understanding the origins of clades. Recently developed methods allow including the entire fossil record known for the group of interest and eliminated the need for specifying prior distributions for node ages. Here we apply the fossilized birth-death (FBD) approach to reconstruct the diversification timeline of the viperines (subfamily Viperinae). Viperinae are an Old World snake subfamily comprising 102 species from 13 genera. The fossil record of vipers is fairly rich and well assignable to clades due to the unique vertebral and fang morphology. We use an unprecedented sampling of 83 modern species and 13 genetic markers in combination with 197 fossils representing 28 extinct taxa to reconstruct a time-calibrated phylogeny of the Viperinae. Our results suggest a late Eocene-early Oligocene origin with several diversification events following soon after the group's establishment. The age estimates inferred with the FBD model correspond to those from previous studies that were based on node dating but FBD provides notably narrower credible intervals around the node ages. Viperines comprise two African and an Eurasian clade, but the ancestral origin of the subfamily is ambiguous. The most parsimonious scenarios require two transoceanic dispersals over the Tethys Sea during the Oligocene.

Understanding the influence of geographical events and climate changes on genetic diversity is essential in explaining current patterns of genetic structure and geographic distribution of organisms. We inferred phylogenetic relationships, investigated historical demography, explored the evolutionary history, and clarified intraspecific taxonomy of Protobothrops mucrosquamatus, which is one of the commonest and most wide-ranging Asian pitvipers. A total of 184 samples from 54 localities were sequenced and analyzed for two mitochondrial gene fragments and two nuclear genes. Phylogenetic reconstruction based on mtDNA sequences revealed the existence of a minimum of five geographically structured and well-supported lineages within P. mucrosquamatus. Based on the mtDNA gene tree, and the geographic relationship between populations allied by matrilineal lineages, a complex longitudinal and latitudinal diversification pattern was uncovered in P. mucrosquamatus. The estimated date of the origin of the species (about 5.3 Ma) and divergence of the intraspecific lineages match the rapid uplifting of Qinghai-Xizang Plateau, and is also consistent with those of some other co-distributed Asian pitvipers. Formation of the two island lineages (Taiwan and Hainan) was generally congruent with the first isolation of the islands, but the two lineages showed different relationships with the continental Asian populations in comparison with some other pitvipers. Population historical demographic analyses, based on three methods, showed that all lineages have experienced slight population expansion in and around the Dali Glacial. Tests of intraspecific taxonomy indicated that no cryptic taxon is present within this widely distributed snake., (Copyright © 2018 Elsevier Inc. All rights reserved.)

A proteomic and transcriptomic comparative analysis of the venoms of three Atheris species (A. squamigera, A. nitschei and A. chlorechis) was carried out by size exclusion liquid chromatography, gel electrophoresis, mass spectrometry, and mRNA sequencing. The improved proteomic profiling utilised in this work was combined with transcript studies, advancing our insights into venom composition, protein distribution and inter-species variation among the three bush vipers. Crude venoms of all three samples contained at least 10-20 protein components, ranging in size from ≤ 3 to > 98 kDa. Both approaches yielded converging overall information, pointing to phospholipases, disintegrins, serine proteases and metalloproteases as the major toxin classes, which are likely to explain the local and systemic symptoms observed in envenomation by Atheris genus. Being considered as the main factors involved in the distinct venom-induced pathologies, these identified snake venom proteins are of particular interest in terms of understanding their physiological and biological function as well as for their contribution in potential medical treatments.

Toxin genes in animals undergo accelerated evolution compared to non-toxin genes to be effective and competitive in prey capture, as well as to enhance their predator defense. Several mechanisms have been proposed to explain this unusual phenomenon. These include (a) frequent mutations in exons compared to introns and nonsynonymous substitutions in exons; (b) high frequency of point mutations are due to the presence of more unstable triplets in exons compared to introns; (c) Accelerated Segment Switch in Exons to alter Targeting (ASSET); (d) Rapid Accumulation of Variations in Exposed Residues (RAVERs); (e) alteration in intron-exon boundary; (f) deletion of exon; and (g) loss/gain of domains through recombination. By systematic analyses of snake venom disintegrin/metalloprotease genes, I describe a new mechanism in the evolution of these genes through exonization and intronization. In the evolution of RTS/KTS disintegrins, a new exon (10a) is formed in intron 10 of the disintegrin/metalloprotease gene. Unlike more than 90% new exons that are from repetitive elements in introns, exon 10a originated from a non-repetitive element. To incorporate exon 10a, part of the exon 11 is intronized to retain the open reading frame. This is the first case of simultaneous exonization and intronization within a single gene. This new mechanism alters the function of toxins through drastic changes to the molecular surface via insertion of new exons and deletion of exons., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Northern and western mountains of Iran are among the most important biodiversity and endemism hot spots for reptiles in the Middle East. Among herpetofauna, the montivipers represent an emblematic and fragmented endemic group for which estimating their level of genetic differentiation and defining conservation priorities is urgently needed. Here, we present the most comprehensive phylogenetic study on the Montivipera raddei species group comprising all 5 known taxa, among which 3 are endemic to Iran. Based on 2 mitochondrial genes, phylogenetic and phylogeographic analyses revealed 3 major lineages each presenting very contrasting distribution areas. The Iranian montivipers are highly structured in clades showing low genetic diversity and corresponding to high altitude summits. Molecular dating revealed the role of Quaternary paleo-climatic oscillations and altitudinal movements of montivipers in shaping genetic diversity and differentiation of these sky-island taxa. In addition, the best scenario of historical biogeography allowed identifying 3 possible refugial areas in Iran most likely arising by vicariance. Based on our mitochondrial results and pending additional data, we recognize 3 candidate species among the M. raddei complex: M. raddei, Montivipera latifii, and Montivipera kuhrangica that are coherent with their geographical distribution. We propose that the most appropriate evolutionary significant units for conservation of the montivipers are represented by 13 units among which 6 are recognized as high priority. Finally, we suggest some recommendations to the IUCN as well as to the Iranian conservation policies with respect to conservation prioritization.

Genes encoding two three-finger toxins TFT-AF and TFT-VN, nucleotide sequences of which were earlier determined by cloning cDNA from venom glands of vipers Azemiops feae and Vipera nikolskii, respectively, were expressed for the first time in E. coli cells. The biological activity of these toxins was studied by electrophysiological techniques, calcium imaging, and radioligand analysis. It was shown for the first time that viper three-finger toxins are antagonists of nicotinic acetylcholine receptors of neuronal and muscle type.

Venoms are among the most biologically active secretions known, and are commonly believed to evolve under extreme positive selection. Many venom gene families, however, have undergone duplication, and are often deployed in doses vastly exceeding the LD50 for most prey species, which should reduce the strength of positive selection. Here, we contrast these selective regimes using snake venoms, which consist of rapidly evolving protein formulations. Though decades of extensive studies have found that snake venom proteins are subject to strong positive selection, the greater action of drift has been hypothesized, but never tested. Using a combination of de novo genome sequencing, population genomics, transcriptomics, and proteomics, we compare the two modes of evolution in the pitviper, Protobothrops mucrosquamatus. By partitioning selective constraints and adaptive evolution in a McDonald-Kreitman-type framework, we find support for both hypotheses: venom proteins indeed experience both stronger positive selection, and lower selective constraint than other genes in the genome. Furthermore, the strength of selection may be modulated by expression level, with more abundant proteins experiencing weaker selective constraint, leading to the accumulation of more deleterious mutations. These findings show that snake venoms evolve by a combination of adaptive and neutral mechanisms, both of which explain their extraordinarily high rates of molecular evolution. In addition to positive selection, which optimizes efficacy of the venom in the short term, relaxed selective constraints for deleterious mutations can lead to more rapid turnover of individual proteins, and potentially to exploration of a larger venom phenotypic space., (© The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

In our previous work, a thrombin-like enzyme (TLE), agkihpin, was successfully isolated, purified, cloned and named from the venom of Gloydius halys Pallas, having fibrinolytic, fibrinogenolytic and thrombosis-reduced activities, attenuating migration of liver cancer cell, and without bleeding risk. To explore the possibility of agkihpin as a thrombolytic and/or anti-metastasis agent in the future, in this study recombinant agkihpin was expressed and purified in Escherichia coli, and its biological activities investigated. Thus, r-agkihpin-2 was successfully expressed and purified and confirmed by Western blot and peptide mass fingerprinting. After purification and renaturation, 46 mg (399 U) of active r-agkihpin-2 was obtained from 1 L bacterial culture. The results of the arginine esterase activity assay, fibrin plate test fibrinogenolytic activity assay, thrombin-induced venous thrombosis assay, Scratch-Wound assay and bleeding assay showed that active r-agkihpin-2 had slightly lower TAME hydrolytic, fibrinolytic, fibrinogenolytic, thrombus-reduced and migration-attenuated activities than those of native agkihpin, and had no bleeding risk. These findings confirmed that, active r-agkihpin-2 could be further investigated for thrombolytic and/or anti-metastasis drug discovery in the future., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Objective: To design a specific polyclonal antibody against Deinagkistrodon acutus venom (DA-pAb) by immunizating New Zealand white rabbits., Results: The IgG fraction was purified by affinity chromatography, and specific antibodies were purified by immunoaffinity chromatography. Polyclonal antibodies were subjected to ELISA and western blotting to evaluate their immune reactivity. We identified the mimotopes by screening a phage display 12-mer peptide library against D. acutus venom. After three rounds of biopanning with DA-pAb, 30 positive clones were identified. Eighteen phage clones were sequenced, and their corresponding amino acid sequences were deduced. Additional immunoassays with the peptides and DA-pAb identified five sequences as possible epitopes. Recombinant antigens synthesized with the five epitopes were used for the immunization of BALB/c mice., Conclusion: The antibodies induced by these peptides recognized the recombinant antigen and D. acutus venom and protected mice against the hemorrhagic effects of the venom.

Identifying phylogenetically distinct lineages and understanding the evolutionary processes by which they have arisen are important goals of phylogeography. This information can also help define conservation units in endangered species. Such analyses are being transformed by the availability of genomic-scale data sets and novel analytical approaches for statistically comparing different historical scenarios as causes of phylogeographic patterns. Here, we use genomic-scale restriction-site-associated DNA sequencing (RADseq) data to test for distinct lineages in the endangered Eastern Massasauga Rattlesnake (Sistrurus catenatus). We then use coalescent-based modeling techniques to identify the evolutionary mechanisms responsible for the origin of the lineages in this species. We find equivocal evidence for distinct phylogenetic lineages within S. catenatus east of the Mississippi River, but strong support for a previously unrecognized lineage on the western edge of the range of this snake, represented by populations from Iowa, USA. Snakes from these populations show patterns of genetic admixture with a nearby non-threatened sister species (Sistrurus tergeminus). Tests of historical demographic models support the hypothesis that the genetic distinctiveness of Iowa snakes is due to a combination of isolation and historical introgression between S. catenatus and S. tergeminus. Our work provides an example of how model-based analysis of genomic-scale data can help identify conservation units in rare species.

Sinovipera sichuanensis is one of the Asian green pit vipers with less concern. It is endemic to China and only known in Hejiang, Sichuan Province and Jiangkou, Guizhou Province. In this study, we report the complete mitochondrial genome and characterize each partition. The complete mitochondrial genome is 17 225 bp in length containing 2 rRNAs, 13 protein-coding genes, 2 control regions and 22 tRNAs. We use Bayesian Inference (BI) and Maximum Likelihood (ML) methods to infer the phylogenetic relationship of S. sichuanensis. Both BI and ML analyses strongly support that S. sichuanensis is independent from the other two Asian green pit vipers.

Middle America is one of the most biodiverse regions in the world, harboring an exceptional number of rare and endemic species. This is especially true of Middle American cloud forests, where montane specialists occupy restricted, high-elevation ranges making them attractive candidates for investigating historical biogeography and speciation. One such highland-restricted species, the black speckled palm-pitviper (Bothriechis nigroviridis), occupies the Central, Tilarán, and Talamanca Cordilleras in Costa Rica and Panama. In this study, we investigate the genetic and morphological variation among populations of B. nigroviridis by inferring a multilocus phylogeny (21 individuals) and analyzing meristic scale characters with a principal component analysis (64 individuals). We find B. nigroviridis sensu stricto to be composed of two deeply divergent lineages, one with a restricted range in the northern and central Cordillera Talamanca and the other ranging throughout the Central, Tilarán, and Talamanca Cordilleras. Furthermore, these two lineages are morphologically distinct, with previously unrecognized differences in several characters allowing us to name and diagnose a new species B. nubestris sp. nov. We also examine the genetic and morphological variation within B. nigroviridis and discuss biogeographic hypotheses that may have led to the diversification of Bothriechis lineages.

The molecular events underlying the evolution of the Snake Venom Metalloproteinase (SVMP) family from an A Disintegrin And Metalloproteinase (ADAM) ancestor remain poorly understood. Comparative genomics may provide decisive information to reconstruct the evolutionary history of this multi-locus toxin family. Here, we report the genomic organization of Echis ocellatus genes encoding SVMPs from the PII and PI classes. Comparisons between them and between these genes and the genomic structures of Anolis carolinensis ADAM28 and E. ocellatus PIII-SVMP EOC00089 suggest that insertions and deletions of intronic regions played key roles along the evolutionary pathway that shaped the current diversity within the multi-locus SVMP gene family. In particular, our data suggest that emergence of EOC00028-like PI-SVMP from an ancestral PII(e/d)-type SVMP involved splicing site mutations that abolished both the 3' splice AG acceptor site of intron 12* and the 5' splice GT donor site of intron 13*, and resulted in the intronization of exon 13* and the consequent destruction of the structural integrity of the PII-SVMP characteristic disintegrin domain.

The mitochondrial genome sequence of Ussuri mamushi is analyzed and presented publically for the first time. The genome is 17,208 bp in length and contains 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes and 2 control regions. The overall base composition is A (32.4%), C (28.4%), T (26.3%) and G (12.9%). The base compositions clearly presented the A-C skew, which is the most obvious in the protein-coding genes. Mitochondrial genome analyses based on MP, ML, NJ and Bayesian analyses yielded identical phylogenetic trees, indicating a close phylogenetic affinity of the 13 Crotalinae species. It appeared that no less than two major phyletic lineages were present in Crotalinae. The main clades within the Crotalinae include Protobothrops. A clade (G. brevicaudus, G. ussuriensis, G. intermedius, and G. sahatilis) with the Ovophis as the sister taxon to Protobothrops was supported by bootstrap values of 88%. The four Gloydius species formed a paraphyletic group with the high bootstrap value (100%) in all examinations.

Snake venoms are composed mainly of a mixture of proteins and peptides. Notably, all snake venom toxins have been assigned to a small number of protein families. Proteomic studies on snake venoms have recently identified the presence of Kazal-type inhibitor-like proteins in the neotropical arboreal snakes Bothriechis schlegelii and Bothriechis supraciliaris. In the present study, a Kazal-type component from B. schlegelii, named Kazal-type inhibitor-like protein (KTIL), has been completely sequenced and characterized for the first time. This protein, which contains 54 amino acid residues, shows sequence similarity to the third domain of the ovomucoid from avian species, which is a Kazal-like domain. KTIL did not inhibit the enzymatic activity of various serine proteinases at pH = 7.2 or pH = 8.0, but partially inhibited the activity of trypsin at pH = 5.4, and the only toxic effect in mice observed after different in vivo tests was the induction of footpad edema. KTIL was not lethal when injected in mice or chickens. The presence of Kazal-type proteins and mRNA only in species of the genus Bothriechis suggests a genus recruitment event in the early-Middle Miocene, the estimated time of emergence of this clade., (Copyright © 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Viridovipera stejnegeri is one of the most common pit vipers in Asia, with a wide distribution in southern China and Vietnam. We investigated historical demography and explored how the environment and climatic factors have shaped genetic diversity and the evolutionary history of this venomous snake. A total of 171 samples from 47 localities were sequenced and analysed for two mitochondrial gene fragments and three nuclear genes. Gene trees reveal the existence of two well-supported clades (Southwest China and Southeast China) with seven distinct and strongly supported, geographically structured subclades within V. stejnegeri. Estimation of divergence time and ancestral area suggests that V. stejnegeri originated at ~6.0 Ma in the late Miocene on the Yunnan-Guizhou Plateau. The estimated date of origin and divergence of the island populations of Taiwan and Hainan closely matches the geological origin of the both islands. The mtDNA gene tree reveals the presence of west-east diversification in V. stejnegeri populations. Complex orogenesis and heterogeneous habitats, as well as climate-mediated habitat differentiation including glacial cycles, all have influenced population structure and the distribution of this taxon. The validity of V. stejnegeri chenbihuii is questionable, and this subspecies most probably represents an invalid taxon., (© 2016 John Wiley & Sons Ltd.)

The Azemiops snakes are pit-less and phylogenetically located at the Crotalinae and Viperinae divergence. cDNAs encoding five Azemiops venom phospholipase (sPLA2) molecules were cloned and sequenced; their signal-peptides were similar to those of crotalid sPLA2s. Based on their calculated pI-values and residue-49 substitutions, they were designated as Af-E6, Af-N49a, Af-N49a1, Af-N49a2, and Af-N49b, respectively. The first three isoforms, comprising 3-4% of the venom proteins, were purified by reversed-phase HPLC. Af-E6 is catalytically active and has >80% sequence-similarity to other Glu(6)-PLA2 (a pitviper venom-marker). Results of phylogenetic analyses reveal that acidic Af-N49a and Af-N49a1 are rather unique and loosely linked with crotalid PLA2s, while Af-N49b is related to the viperid PLA2s with Ser(1) substitution. Notably, the Asn(49)-substitutions in these molecules imply catalytic-independent mechanisms. The 3D-models of Af-E6 and Af-N49a have surface electropotential maps similar to each other and to those of antiplatelet PLA2s, while the Af-N49b model is similar to basic and myotoxic sPLA2 molecules. From Azemiops feae and four other Viperidae, we cloned five novel Cys-rich secretory proteins (CRISPs). Azemiops CRISP and natriuretic-peptide precursors share more sequence similarities with those of crotalid venoms than with viperid venoms, further supporting the theory that Azemiops are sister taxons to pit vipers, especially Tropedolaemus., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Background: Studying patterns of introgression can illuminate the role of hybridization in speciation, and help guide decisions relevant to the conservation of rare taxa. Vipera magnifica and Vipera orlovi are small vipers that have high conservation status due to their rarity and restricted distributions in an area of the Caucasus region where two other related species are present - V. kaznakovi and V. renardi. Despite numerous observations of hybridization between different species of small vipers, and the potential of a hybrid origin for V. magnifica and V. orlovi based on their distribution with respect to V. kaznakovi and V. renardi, hypotheses of a hybrid origin have not been formally tested. Here we generate genomic-scale data by performing next generation sequencing of double digest restriction-site associated DNA libraries, and use these multilocus data to test whether these two species are of hybrid origin., Results: We generated over nine hundred loci for 38 specimens of six taxa, and analysed the dataset using Bayesian clustering and multivariate methods, as well as Patterson D-statistics, which can distinguish between incomplete lineage sorting and introgression as explanations for shared polymorphism. The results demonstrate a pattern of historical admixture in the two purported hybrids that is consistent with past gene flow from V. renardi into V. kaznakovi. The average admixture proportion in individuals was low (6.39 %) in the case of V. magnifica, but was higher in V. orlovi (19.02 %). We also show that the specific individual samples used in D-statistic tests can have a significant impact on inferences regarding the magnitude of introgression, suggesting the importance of including multiple individuals in these analyses., Conclusions: Our results support the conclusion that both V. orlovi and V. magnifica had formed through a hybridization event between V. kaznakovi and V. renardi. Given a low proportion of admixture and absence of clear ecological and morphological differences V. magnifica should be treated as a marginal population of V. kaznakovi. Further studies that include analyses of ecological segregation of V. orlovi from parental taxa and search for evolutionary consequences of hybridisation would clarify if V. orlovi is a distinct hybrid species. Until this we recommend preserving the current taxonomy and protection status of V. orlovi.

Despite the public health significance and potential applications to medical research, the evolutionary history of the Asian Lance-headed pitvipers (genus Protobothrops) remains inadequately studied. Previous research generally focused on a few selected species with no comprehensive molecular study of Protobothrops. Here, we conduct the first study to infer the phylogenetic relationships of all currently recognized species in this genus based on four mitochondrial DNA fragments and four nuclear genes. Bayesian Inference and Maximum Likelihood analyses show that within Protobothrops there are four strongly supported clades forming distinct subgroups. The first subgroup, which is sister to the other three, consists of three species, P. himalayanus, P. kaulbacki, and P. sieversorum. The second subgroup contains only P. mangshanensis. The final two subgroups, which are sister groups, include the other four and six Protobothrops species. Although our findings provide additional information on the phylogenetic relationships of the genus Protobothrops, the placement of P. dabieshanensis and P. elegans remains problematic. In addition, our molecular results indicate that P. trungkhanhensis, currently considered endemic to Vietnam, should be added to the species known from China. Our ancestral area estimation indicated that Protobothrops likely originated in southwestern China. This study improves our understanding of the evolutionary relationships among species of Asian Lance-headed pitvipers, providing a greater framework for future studies.

Phospholipases A2 are major components of snake venoms (svPLA2s) and are able to induce multiple local and systemic deleterious effects upon envenomation. Several snake species are provided with svPLA2 inhibitors (sbPLIs) in their circulating blood, which confer a natural resistance against the toxic components of homologous and heterologous venoms. The sbPLIs belong to any of three structural classes named α, β and γ. In the present study, we identified, characterized and performed structural and evolutionary analyses of sbαPLIs transcripts and the encoded proteins, in the most common Latin American pit vipers belonging to Crotalus, Bothrops and Lachesis genera. Mutation data indicated that sbαPLIs from Latin American snakes might have evolved in an accelerated manner, similarly to that reported for sbαPLIs from Asian snakes, and possibly co-evoluted with svPLA2s in response to the diversity of target enzymes. The importance of sbαPLI trimerization for the effective binding and inhibition of acidic svPLA2s is discussed and conserved cationic residues located at the central pore of the inhibitor trimer are suggested to be a significant part of the binding site of sbαPLIs to acidic svPLA2s. Our data contribute to the current body of knowledge on the structural and evolutionary characteristics of sbPLIs, in general, and may assist in the future development of selective inhibitors for secretory PLA2 from several sources., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Protein expression level is one of the strongest predictors of protein sequence evolutionary rate, with high-expression protein sequences evolving at slower rates than low-expression protein sequences largely because of constraints on protein folding and function. Expression evolutionary rates also have been shown to be negatively correlated with expression level across human and mouse orthologs over relatively long divergence times (i.e., ∼100 million years). Long-term evolutionary patterns, however, often cannot be extrapolated to microevolutionary processes (and vice versa), and whether this relationship holds for traits evolving under directional selection within a single species over ecological timescales (i.e., <5000 years) is unknown and not necessarily expected. Expression is a metabolically costly process, and the expression level of a particular protein is predicted to be a tradeoff between the benefit of its function and the costs of its expression. Selection should drive the expression level of all proteins close to values that maximize fitness, particularly for high-expression proteins because of the increased energetic cost of production. Therefore, stabilizing selection may reduce the amount of standing expression variation for high-expression proteins, and in combination with physiological constraints that may place an upper bound on the range of beneficial expression variation, these constraints could severely limit the availability of beneficial expression variants. To determine whether rapid-expression evolution was restricted to low-expression proteins owing to these constraints on highly expressed proteins over ecological timescales, we compared venom protein expression levels across mainland and island populations for three species of pit vipers. We detected significant differentiation in protein expression levels in two of the three species and found that rapid-expression differentiation was restricted to low-expression proteins. Our results suggest that various constraints on high-expression proteins reduce the availability of beneficial expression variants relative to low-expression proteins, enabling low-expression proteins to evolve and potentially lead to more rapid adaptation., (Copyright © 2016 by the Genetics Society of America.)

Protobothrops tokarensis (Pt), a Crotalinae snake, inhabits only Takarajima and Kodakarajima islands of the Tokara Islands located in the immediate north of Amami-Oshima island of Japan. Kodakarajima P. tokarensis venom gland cDNA library gave four types of phospholipase A2 (PLA2) cDNAs encoding neutral [Asp(49)]PLA2, basic [Asp(49)]PLA2, highly basic [Asp(49)]PLA2, and [Lys(49)]PLA2. As the amino acid sequences encoded by their open reading frames (ORFs) were identical to those of PLA2, PLA-B, PLA-N, and BPI (a [Lys(49)]PLA2), respectively, from Amami-Oshima P. flavoviridis (Pf) venom, they were named PtPLA2, PtPLA-B, PtPLA-N, and PtBPI. Chromatography of P. tokarensis venom gave three PLA2 isozymes, PtPLA2, PtPLA-B, and PtBPI. However, BPII and BPIII ([Lys(49)]PLA2s) expressed in Amami-Oshima P. flavoviridis venom were not found in P. tokarensis venom. Genomic polymerase chain reaction (PCR) for P. tokarensis liver DNAs with the unique primers gave PtBPI gene. Notably it was found that LINE (long interspersed nuclear element)-1 fragment is inserted into second intron of PtBPI gene. The LINE-1 fragment may prevent duplication of PtBPI gene and thus formation of plural [Lys(49)]PLA2 genes in P. tokarensis genome. The interisland variegation of venom [Lys(49)]PLA2 isozyme genes in Protobothrops genus snakes in the southwestern islands of Japan is discussed., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Using multiple markers and multiple analytical approaches is critical for establishing species boundaries reliably, especially so in the case of cryptic species. Despite development of new and powerful analytical methods, most studies continue to adopt a few, with the choice often being subjective. One such example is routine analysis of Amplified Fragment Length Polymorphism (AFLP) data using population genetic models despite disparity between method assumptions and data properties. The application of newly developed methods for analyzing this dominant marker may not be entirely clear in the context of species delimitation. In this study, we use AFLPs and mtDNA to investigate cryptic speciation in the Trimeresurus macrops complex that belongs to a taxonomically difficult lineage of Asian pitvipers. We analyze AFLPs using population genetic, phylogenetic, multivariate statistical, and Bayes Factor Delimitation methods. A gene tree from three mtDNA markers provided additional evidence. Our results show that the inferences about species boundaries that can be derived from population genetic analysis of AFLPs have certain limitations. In contrast, four multivariate statistical analyses produced clear clusters that are consistent with each other, as well as with Bayes Factor Delimitation results, and with mtDNA and total evidence phylogenies. Furthermore, our results concur with allopatric distributions and patterns of variation in individual morphological characters previously identified in the three proposed species: T. macrops sensu stricto, T. cardamomensis, and T. rubeus. Our study provides evidence for reproductive isolation and genetic distinctiveness that define these taxa as full species. In addition, we re-emphasize the importance of examining congruence of results from multiple methods of AFLP analysis for inferring species diversity., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Snake venom protein from Deinagkistrodon acutus (DA protein), one of the major venomous species in Taiwan, causes hemorrhagic symptoms that can lead to death. Although horse-derived antivenin is a major treatment, relatively strong and detrimental side effects are seen occasionally. In our study, yolk immunoglobulin (IgY) was purified from eggs, and DA protein was recognized using Western blotting and an enzyme-linked immunosorbent assay (ELISA), similar to therapeutic horse antivenin. The ELISA also indicated that specific IgY antibodies were elicited after the fifth booster, plateaued, and lasted for at least 3 months. To generate monoclonal single-chain variable fragment (scFv) antibodies, we used phage display technology to construct two libraries with short or long linkers, containing 6.24 × 10(8) and 5.28 × 10(8) transformants, respectively. After four rounds of biopanning, the eluted phage titer increased, and the phage-based ELISA indicated that the specific clones were enriched. Nucleotide sequences of 30 individual clones expressing scFv were analyzed and classified into four groups that all specifically recognized the DA venom protein. Furthermore, based on mass spectrometry, the scFv-bound protein was deduced to be snake venom metalloproteinase proteins. Most importantly, both IgY and mixed scFv inhibited the lethal effect in mice injected with the minimum lethal dosage of the DA protein. We suggest that together, these antibodies could be applied to the development of diagnostic agents or treatments for snakebite envenomation in the future., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Protobothrops xiangchengsis is the endangered snake which is only distributed in China. We determined the complete mitochondrial genome of P. xiangchengsis (HB-px20100722). The circle genome with the 17,240 bp total length contained 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes and 2 control regions. Overall base composition of the complete mtDNA was 33.16% A, 25.16% T, 29.17% C and 12.51% G. All the genes in P. xiangchengsis were distributed on the H-strand, except for the ND6 subunit gene and eight tRNA genes which were encoded on the L-strand.

Understanding the impact of postglacial recolonization on genetic diversity is essential in explaining current patterns of genetic variation. The central-marginal hypothesis (CMH) predicts a reduction in genetic diversity from the core of the distribution to peripheral populations, as well as reduced connectivity between peripheral populations. While the CMH has received considerable empirical support, its broad applicability is still debated and alternative hypotheses predict different spatial patterns of genetic diversity. Using microsatellite markers, we analysed the genetic diversity of the adder (Vipera berus) in western Europe to reconstruct postglacial recolonization. Approximate Bayesian Computation (ABC) analyses suggested a postglacial recolonization from two routes: a western route from the Atlantic Coast up to Belgium and a central route from the Massif Central to the Alps. This cold-adapted species likely used two isolated glacial refugia in southern France, in permafrost-free areas during the last glacial maximum. Adder populations further from putative glacial refugia had lower genetic diversity and reduced connectivity; therefore, our results support the predictions of the CMH. Our study also illustrates the utility of highly variable nuclear markers, such as microsatellites, and ABC to test competing recolonization hypotheses., (© 2015 John Wiley & Sons Ltd.)

A natural hybrid of Vipera ammodytes × Vipera berus was presented having low body weight, seizures and generalized swelling of the cephalic region. Based on the history of the case and clinical examination, hemorrhagic stomatitis of traumatic origin was diagnosed. The snake was kept in a terrarium with wood chips as a substrate, and the material had induced trauma in the oral mucosa which was further complicated with Salmonella Arizonae and Morganella morganii co-infection, abscessation and osteomyelitis. To the best of the authors' knowledge, this is the first reported case of bacterial infection in European snake hybrids and one of a few case reports in European snakes. Although wood chips are an inexpensive substrate, based on our findings, they should be avoided when keeping and breeding European vipers.

Background: The presence of intraspecific color polymorphism can have multiple impacts on the ecology of a species; as a consequence, particular color morphs may be strongly selected for in a given habitat type. For example, the asp viper (Vipera aspis) shows a high level of color polymorphism. A blotched morph (cryptic) is common throughout its range (central and western Europe), while a melanistic morph is frequently found in montane populations, presumably for thermoregulatory reasons. Besides, rare atypical uniformly colored individuals are known here and there. Nevertheless, we found in a restricted treeless area of the French Alps, a population containing a high proportion (>50%) of such specimens. The aim of the study is to bring insight into the presence and function of this color morph by (i) studying the genetic structure of these populations using nine microsatellite markers, and testing for (ii) a potential local diversifying selection and (iii) differences in dispersal capacity between blotched and non-blotched vipers., Results: Our genetic analyses support the occurrence of local diversifying selection for the non-blotched phenotype. In addition, we found significant color-biased dispersal, blotched individuals dispersing more than atypical individuals., Conclusion: We hypothesize that, in this population, the non-blotched phenotype possess an advantage over the typical one, a phenomenon possibly due to a better background matching ability in a more open habitat. In addition, color-biased dispersal might be partly associated with the observed local diversifying selection, as it can affect the genetic structure of populations, and hence the distribution of color morphs.

Protobothrops cornutus is the endangered snake which is distributed narrowly in southern China and other adjacent countries of Asia. We determined the complete mitochondrial genome of P. cornutus (HB-pc20090810). The circle genome with the 17,219 bp total length contained 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes and 2 control regions. Overall base composition of the complete mtDNA was 33.18% A, 24.80% T, 29.43% C and 12.58% G. All the genes in P. cornutus were distributed on the H-strand, except for the ND6 subunit gene and eight tRNA genes which were encoded on the L-strand.

phylin is a package for the r programming environment which offers different methods to spatially interpolate genetic information from phylogeographic data. These interpolations can be used to predict the spatial occurrence of different lineages within a phylogeny using a modified method of kriging, which allows the usage of a genetic distance matrix to derive a model of spatial dependence. phylin improves the available methods to generate interpolated surfaces from a phylogenetic trees by assessing the autocorrelation structure of the genetic information, interpolating the genetic data based on a statistical model, estimating the uncertainty of the predictions and identifying lineage occurrence and contact zones probability without projection of pairwise genetic distances into mid-points between sample locations. The package also includes methods to plot interpolation surfaces and provide summary tables from the generated data and models. We provide an example of the usefulness of this tool by inferring the spatial occurrence of distinct historical evolutionary lineages of the Lataste's viper (Vipera latastei Boscá, 1878) in the Iberian Peninsula and identifying potential contact areas. The maps of phylogenetic patterns obtained with these methods provide a spatial context to test hypotheses related to processes underlying the geographic distribution of genetic diversity and to inform conservation planning., (© 2014 John Wiley & Sons Ltd.)