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The rapid development of sequencing technologies resulted in a wide expansion of genomics studies using venomous lineages. This facilitated research focusing on understanding the evolution of adaptive traits and the search for novel compounds that can be applied in agriculture and medicine. However, the toxin annotation of genomes is a laborious and time-consuming task, and no consensus pipeline is currently available. No computational tool currently exists to address the challenges specific to toxin annotation and to ensure the reproducibility of the process.

Here, we present ToxCodAn-Genome, the first software designed to perform automated toxin annotation in genomes of venomous lineages. This pipeline was designed to retrieve the full-length coding sequences of toxins and to allow the detection of novel truncated paralogs and pseudogenes. We tested ToxCodAn-Genome using 12 genomes of venomous lineages and achieved high performance on recovering their current toxin annotations. This tool can be easily customized to allow improvements in the final toxin annotation set and can be expanded to virtually any venomous lineage. ToxCodAn-Genome is fast, allowing it to run on any personal computer, but it can also be executed in multicore mode, taking advantage of large high-performance servers. In addition, we provide a guide to direct future research in the venomics field to ensure a confident toxin annotation in the genome being studied. As a case study, we sequenced and annotated the toxin repertoire of Bothrops alternatus, which may facilitate future evolutionary and biomedical studies using vipers as models.

ToxCodAn-Genome is suitable to perform toxin annotation in the genome of venomous species and may help to improve the reproducibility of further studies. ToxCodAn-Genome and the guide are freely available at https://github.com/pedronachtigall/ToxCodAn-Genome.

 

Abstract Background The explosive radiation and diversification of the advanced snakes (superfamily Colubroidea) was associated with changes in all aspects of the shared venom system. Morphological changes included the partitioning of the mixed ancestral glands into two discrete glands devoted for production of venom or mucous respectively, as well as changes in the location, size and structural elements of the venom-delivering teeth. Evidence also exists for homology among venom gland toxins expressed across the advanced snakes. However, despite the evolutionary novelty of snake venoms, in-depth toxin molecular evolutionary history reconstructions have been mostly limited to those types present in only two front-fanged snake families, Elapidae and Viperidae. To have a broader understanding of toxins shared among extant snakes, here we first sequenced the transcriptomes of eight taxonomically diverse rear-fanged species and four key viperid species and analysed major toxin types shared across the advanced snakes. Results Transcriptomes were constructed for the following families and species: Colubridae - Helicops leopardinus , Heterodon nasicus , Rhabdophis subminiatus ; Homalopsidae – Homalopsis buccata ; Lamprophiidae - Malpolon monspessulanus , Psammophis schokari , Psammophis subtaeniatus , Rhamphiophis oxyrhynchus ; and Viperidae – Bitis atropos , Pseudocerastes urarachnoides , Tropidolaeumus subannulatus , Vipera transcaucasiana . These sequences were combined with those from available databases of other species in order to facilitate a robust reconstruction of the molecular evolutionary history of the key toxin classes present in the venom of the last common ancestor of the advanced snakes, and thus present across the full diversity of colubroid snake venoms. In addition to differential rates of evolution in toxin classes between the snake lineages, these analyses revealed multiple instances of previously unknown instances of structural and functional convergences. Structural convergences included: the evolution of new cysteines to form heteromeric complexes, such as within kunitz peptides (the beta-bungarotoxin trait evolving on at least two occasions) and within SVMP enzymes (the P-IIId trait evolving on at least three occasions); and the C-terminal tail evolving on two separate occasions within the C-type natriuretic peptides, to create structural and functional analogues of the ANP/BNP tailed condition. Also shown was that the de novo evolution of new post-translationally liberated toxin families within the natriuretic peptide gene propeptide region occurred on at least five occasions, with novel functions ranging from induction of hypotension to post-synaptic neurotoxicity. Functional convergences included the following: multiple occasions of SVMP neofunctionalised in procoagulant venoms into activators of the clotting factors prothrombin and Factor X; multiple instances in procoagulant venoms where kunitz peptides were neofunctionalised into inhibitors of the clot destroying enzyme plasmin, thereby prolonging the half-life of the clots formed by the clotting activating enzymatic toxins; and multiple occasions of kunitz peptides neofunctionalised into neurotoxins acting on presynaptic targets, including twice just within Bungarus venoms. Conclusions We found novel convergences in both structural and functional evolution of snake toxins. These results provide a detailed roadmap for future work to elucidate predator–prey evolutionary arms races, ascertain differential clinical pathologies, as well as documenting rich biodiscovery resources for lead compounds in the drug design and discovery pipeline. 

Abstract Understanding the joint roles of protein sequence variation and differential expression during adaptive evolution is a fundamental, yet largely unrealized goal of evolutionary biology. Here, we use phylogenetic path analysis to analyze a comprehensive venom-gland transcriptome dataset spanning three genera of pitvipers to identify the functional genetic basis of a key adaptation (venom complexity) linked to diet breadth (DB). The analysis of gene-family-specific patterns reveals that, for genes encoding two of the most important venom proteins (snake venom metalloproteases and snake venom serine proteases), there are direct, positive relationships between sequence diversity (SD), expression diversity (ED), and increased DB. Further analysis of gene-family diversification for these proteins showed no constraint on how individual lineages achieved toxin gene SD in terms of the patterns of paralog diversification. In contrast, another major venom protein family (PLA2s) showed no relationship between venom molecular diversity and DB. Additional analyses suggest that other molecular mechanisms—such as higher absolute levels of expression—are responsible for diet adaptation involving these venom proteins. Broadly, our findings argue that functional diversity generated through sequence and expression variations jointly determine adaptation in the key components of pitviper venoms, which mediate complex molecular interactions between the snakes and their prey. 

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

Abstract 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. 

Ecologically divergent selection can lead to the evolution of reproductive isolation through the process of ecological speciation, but the balance of responsible evolutionary forces is often obscured by an inadequate assessment of demographic history and the genetics of traits under selection. Snake venoms have emerged as a system for studying the genetic basis of adaptation because of their genetic tractability and contributions to fitness, and speciation in venomous snakes can be associated with ecological diversification such as dietary shifts and corresponding venom changes. Here, we explored the neurotoxic (type A)–hemotoxic (type B) venom dichotomy and the potential for ecological speciation among Timber Rattlesnake (Crotalus horridus) populations. Previous work identified the genetic basis of this phenotypic difference, enabling us to characterize the roles geography, history, ecology, selection, and chance play in determining when and why new species emerge or are absorbed. We identified significant genetic, proteomic, morphological, and ecological/environmental differences at smaller spatial scales, suggestive of incipient ecological speciation between type A and type B C. horridus. Range-wide analyses, however, rejected the reciprocal monophyly of venom type, indicative of varying intensities of introgression and a lack of reproductive isolation across the range. Given that we have now established the phenotypic distributions and ecological niche models of type A and B populations, genome-wide data are needed and capable of determining whether type A and type B C. horridus represent distinct, reproductively isolated lineages due to incipient ecological speciation or differentiated populations within a single species. 

The genus Calliophis is the most basal branch of the family Elapidae and several species in it have developed highly elongated venom glands. Recent research has shown that C. bivirgatus has evolved a seemingly unique toxin (calliotoxin) that produces spastic paralysis in their prey by acting on the voltage-gated sodium (NaV) channels. We assembled a transcriptome from C. bivirgatus to investigate the molecular characteristics of these toxins and the venom as a whole. We find strong confirmation that this genus produces the classic elapid eight-cysteine three-finger toxins, that δδ-elapitoxins (toxins that resemble calliotoxin) are responsible for a substantial portion of the venom composition, and that these toxins form a distinct clade within a larger, more diverse clade of C. bivirgatus three-finger toxins. This broader clade of C. bivirgatus toxins also contains the previously named maticotoxins and is somewhat closely related to cytotoxins from other elapids. However, the toxins from this clade that have been characterized are not themselves cytotoxic. No other toxins show clear relationships to toxins of known function from other species.