Causes and Consequences of Snake Venom Variation
Authors: Nicholas R. Casewell, Timothy N.W. Jackson, Andreas H. Laustsen, Kartik Sunagar
Journal: Trends in Pharmacological Sciences (2020)

Venom is an ecologically important functional trait in venomous snakes and its composition and activity often coevolve with the physiology of the prey animals it is deployed against.Variation in toxin venom components occurs both inter- and intraspecifically as the result of various processes, including gene duplication and the action of positive selection.The consequences of this variation is the generation of functionally diverse venoms that cause distinct pathologies in snakebite victims and which undermine the efficacy of antivenom treatments.Knowledge of the varying toxins found across medically important venoms is enabling the generation of new therapeutic approaches to circumvent venom variation to better treat the worlds 1.8 million annual snakebite victims. Snake venoms are mixtures of toxins that vary extensively between and within snake species. This variability has serious consequences for the management of the world's 1.8 million annual snakebite victims. Advances in 'omic' technologies have empowered toxinologists to comprehensively characterize snake venom compositions, unravel the molecular mechanisms that underpin venom variation, and elucidate the ensuing functional consequences. In this review, we describe how such mechanistic processes have resulted in suites of toxin isoforms that cause diverse pathologies in human snakebite victims and we detail how variation in venom composition can result in treatment failure. Finally, we outline current therapeutic approaches designed to circumvent venom variation and deliver next-generation treatments for the world's most lethal neglected tropical disease. Snake venoms are mixtures of toxins that vary extensively between and within snake species. This variability has serious consequences for the management of the world's 1.8 million annual snakebite victims. Advances in 'omic' technologies have empowered toxinologists to comprehensively characterize snake venom compositions, unravel the molecular mechanisms that underpin venom variation, and elucidate the ensuing functional consequences. In this review, we describe how such mechanistic processes have resulted in suites of toxin isoforms that cause diverse pathologies in human snakebite victims and we detail how variation in venom composition can result in treatment failure. Finally, we outline current therapeutic approaches designed to circumvent venom variation and deliver next-generation treatments for the world's most lethal neglected tropical disease. Venom is a remarkable evolutionary innovation found scattered across the animal tree of life [1.Schendel V. et al.The diversity of venom: the importance of behavior and venom system morphology in understanding its ecology and evolution.Toxins. 2019; 11: 666Crossref Scopus (92) Google Scholar]. Due to their diverse evolutionary histories and consequent variability, animal venoms have proven to be fascinating models for understanding a number of fundamental processes, including gene duplication, genotype–phenotype mapping, convergent evolution, and cell and tissue development [2.Dowell N.L. et al.The deep origin and recent loss of venom toxin genes in rattlesnakes.Curr. Biol. 2016; 26: 2434-2445Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 3.Post Y. et al.Snake venom gland organoids.Cell. 2020; 180: 233-247Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 4.Casewell N.R. et al.Solenodon genome reveals convergent evolution of venom in eulipotyphlan mammals.Proc. Natl. Acad. Sci. U. S. A. 2019; 116: 25745-25755Crossref PubMed Scopus (27) Google Scholar, 5.Sunagar K. et al.Cell type-specific expression profiling unravels the development and evolution of stinging cells in sea anemone.BMC Biol. 2018; 16: 108Crossref PubMed Scopus (34) Google Scholar, 6.Columbus-Shenkar Y.Y. et al.Dynamics of venom composition across a complex life cycle.Elife. 2018; 7e35014Crossref PubMed Scopus (50) Google Scholar], while the bioactivities of many toxins make them promising leads for the discovery of new human therapeutics [7.Clark G.C. et al.Friends or foes? Emerging impacts of biological toxins.Trends Biochem. Sci. 2019; 44: 365-379Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar]. The most well-studied venom systems are those of snakes. All 'advanced snakes' (superfamily: Colubroidea) have a pair of homologous oral venom glands located behind the eye on either side of the upper jaw [8.Jackson T.N.W. et al.Endless forms most beautiful: the evolution of ophidian oral glands, including the venom system, and the use of appropriate terminology for homologous structures.Zoomorphology. 2017; 136: 107-130Crossref Scopus (26) Google Scholar,9.Jackson K. The evolution of venom-delivery systems in snakes.Zool. J. Linnean Soc. 2003; 137: 337-354Crossref Scopus (70) Google Scholar]. These glands are connected to ducts that transfer the secreted venom to the base of morphologically diverse teeth that are often referred to as 'fangs'. For many snakes, including those of greatest medical importance, these fangs are found at the front of the mouth, contain an enclosed venom canal, and are a highly efficient mechanism that facilitates the rapid injection of a bolus of venom. Although venomous snakes predominantly use their venom to assist with the acquisition of prey, they may also deploy it in defensive bites to deter potential predators and aggressors, including people. The consequences of such human snakebites can be severe. Current estimates suggest that venomous snakes cause up to 138 000 deaths worldwide each year and perhaps as many as 500 000 additional cases of venom-induced morbidity [10.Gutiérrez J.M. et al.Snakebite envenoming.Nat. Rev. Dis. Primers. 2017; 3: 1-21Google Scholar]. Snakebite envenomings predominantly affect the rural impoverished populations of the tropics and consequently the World Health Organization (WHO) has listed snakebite as a priority neglected tropical disease (NTD) (see Glossary) [11.Chippaux J.-P. Snakebite envenomation turns again into a neglected tropical disease!.J. Venom. Anim. Toxins Incl. Trop. Dis. 2017; 23: 38Crossref PubMed Scopus (263) Google Scholar]. The pathological effects of snakebite are diverse and can include neuromuscular paralysis (neurotoxicity), hemorrhage and coagulopathy (hemotoxicity), and/or local swelling, blistering, and tissue necrosis (cytotoxicity) around the bite site [10.Gutiérrez J.M. et al.Snakebite envenoming.Nat. Rev. Dis. Primers. 2017; 3: 1-21Google Scholar]. These highly variable clinical signs are a direct consequence of variation in the toxin components found in venom; such variation can be extensive and occurs both inter- and intraspecifically [12.Massey D.J. et al.Venom variability and envenoming severity outcomes of the Crotalus scutulatus (Mojave rattlesnake) from Southern Arizona.J. Proteome. 2012; 75: 2576-2587Crossref PubMed Scopus (111) Google Scholar, 13.Casewell N.R. et al.Medically important differences in snake venom composition are dictated by distinct postgenomic mechanisms.Proc. Natl. Acad. Sci. U. S. A. 2014; 111: 9205-9210Crossref PubMed Scopus (182) Google Scholar, 14.Durban J. et al.Integrated venomics and venom gland transcriptome analysis of juvenile and adult Mexican rattlesnakes Crotalus simus, C. tzabcan, and C. culminatus revealed miRNA-modulated ontogenetic shifts.J. Proteome Res. 2017; 16: 3370-3390Crossref PubMed Scopus (75) Google Scholar, 15.Pla D. et al.Phylovenomics of Daboia russelii across the Indian subcontinent. Bioactivities and comparative in vivo neutralization and in vitro third-generation antivenomics of antivenoms against venoms from India, Bangladesh and Sri Lanka.J. Proteome. 2019; 207: 103443Crossref PubMed Scopus (53) Google Scholar, 16.Laxme R.S. et al.Beyond the 'big four': venom profiling of the medically important yet neglected Indian snakes reveals disturbing antivenom deficiencies.PLoS Negl. Trop. Dis. 2019; 13e0007899PubMed Google Scholar, 17.Jackson T.N. et al.Rapid radiations and the race to redundancy: an investigation of the evolution of Australian elapid snake venoms.Toxins (Basel). 2016; 8: 309Crossref Scopus (47) Google Scholar, 18.Jackson T.N. Fry B.G. A tricky trait: applying the fruits of the "function debate" in the philosophy of biology to the "venom debate" in the science of toxinology.Toxins (Basel). 2016; 8: 263Crossref Scopus (23) Google Scholar]. This variation also has a direct impact on the efficacy of snakebite treatments (antivenom), resulting in different antivenoms having to be manufactured against the venoms of distinct snake species [19.Williams D.J. et al.Ending the drought: new strategies for improving the flow of affordable, effective antivenoms in Asia and Africa.J. Proteome. 2011; 74: 1735-1767Crossref PubMed Scopus (177) Google Scholar]. Despite such complexity, recent advances in 'omic' technologies (e.g., proteomics, transcriptomics) have enabled the rapid characterization of the toxin components found in the venom of over 125 medically relevant species [20.Tasoulis T. Isbister G.K. A review and database of snake venom proteomes.Toxins. 2017; 9: 290Crossref Scopus (256) Google Scholar]. Here, we outline how this data has transformed our understanding of the processes that have generated snake venom variation and the consequences of such variation in the context of snakebite pathology and treatment. We also highlight how the rational application of venom composition data will enable the development of broadly effective therapies for snakebite envenoming, which is an essential step in mitigating the devastating effects this NTD inflicts upon the vulnerable victims of the tropics [21.Williams D.J. et al.Strategy for a globally coordinated response to a priority neglected tropical disease: snakebite envenoming.PLoS Negl. Trop. Dis. 2019; 13e0007059Crossref PubMed Scopus (173) Google Scholar]. Venom is a functional trait used by one organism to interfere with the homeostatic processes of another, generally to facilitate feeding or deter predators or competitors [18.Jackson T.N. Fry B.G. A tricky trait: applying the fruits of the "function debate" in the philosophy of biology to the "venom debate" in the science of toxinology.Toxins (Basel). 2016; 8: 263Crossref Scopus (23) Google Scholar]. Venom is therefore intrinsically ecological; a trait that mediates the outcome of interactions between two or more organisms [22.Jackson T.N. et al.Snake venom in context: neglected clades and concepts.Front. Ecol. Evol. 2019; 7: 332Crossref Scopus (36) Google Scholar]. 'Venomous' is not synonymous with 'dangerous', and the majority of venomous organisms, including snakes, pose no threat to humans, either because they rarely, if ever, envenom humans, or because the consequences of envenoming are trivial. Indeed, venom is a widespread trait amongst the 'advanced snakes', but almost all 'medically important' snakes (those capable of causing harm to humans via envenoming) are members of one of only three clades: the families Elapidae (cobras, mambas, sea snakes, taipans, and their relatives) and Viperidae (vipers and pit vipers, including adders, rattlesnakes, and their relatives) and the subfamily Atractaspidinae (mole vipers/stiletto snakes). As venom is an ecologically important functional trait for venomous snakes, its composition and activity coevolves with the physiology of the prey animals and, perhaps to a certain extent, the predators it is deployed against [23.Holding M.L. et al.Coevolution of venom function and venom resistance in a rattlesnake predator and its squirrel prey.Proc. R. Soc. B Biol. Sci. 2016; 283: 20152841Crossref PubMed Scopus (80) Google Scholar, 24.Ji X.H. et al.Receptor variability-driven evolution of snake toxins.Zool. Res. 2018; 39: 431-436PubMed Google Scholar, 25.Daltry J.C. et al.Diet and snake venom evolution.Nature. 1996; 379: 537-540Crossref PubMed Scopus (501) Google Scholar, 26.Jorge da Silva N. Jr and Aird, S.D. Prey specificity, comparative lethality and compositional differences of coral snake venoms.Comp. Biochem. Physiol. C Toxicol. Pharmacol. 2001; 128: 425-456Crossref PubMed Google Scholar, 27.Barlow A. et al.Coevolution of diet and prey-specific venom activity supports the role of selection in snake venom evolution.Proc. Biol. Sci. 2009; 276: 2443-2449Crossref PubMed Scopus (238) Google Scholar]. Although primates have been predators of snakes since time immemorial and envenoming of humans by snakes is almost exclusively defensive, it is unlikely that humans have exerted any major defensive selective pressure on snake venoms. Rather, human envenomings are best viewed as collateral damage of the chemical arms race taking place between venomous snakes and their (mammalian) prey. Venom variation exists at multiple phylogenetic levels and is a consequence of both the contingent evolutionary histories of divergent lineages of venomous snakes and direct selection on the ecological deployment of specific toxins. At the deepest and most general level, the venoms of, for example, elapid and viperid snakes are different (Box 1); certain families of toxins have been recruited and utilized or have become central components of the venom of one lineage but not the other [28.Fry B.G. et al.The structural and functional diversification of the Toxicofera reptile venom system.Toxicon. 2012; 60: 434-448Crossref PubMed Scopus (100) Google Scholar]. Similarly, broad differences can exist in venom compositions between genera within each family and between species within each genus (e.g., [13.Casewell N.R. et al.Medically important differences in snake venom composition are dictated by distinct postgenomic mechanisms.Proc. Natl. Acad. Sci. U. S. A. 2014; 111: 9205-9210Crossref PubMed Scopus (182) Google Scholar,20.Tasoulis T. Isbister G.K. A review and database of snake venom proteomes.Toxins. 2017; 9: 290Crossref Scopus (256) Google Scholar,29.Ainsworth S. et al.The paraspecific neutralisation of snake venom induced coagulopathy by antivenoms.Commun. Biol. 2018; 1: 1-14Crossref PubMed Scopus (62) Google Scholar]). This much has long been understood and is why a number of antivenom manufacturers have developed multiple products for use in a given region (e.g., viper- and elapid-specific antivenoms).Box 1Toxin Gene Family Expansion and Coevolution with Delivery MechanismsVenom composition varies as a consequence of contingent evolutionary history and direct functional selection. For example, specific families of toxin genes have undergone extensive expansion and neofunctionalization in specific snake lineages. Three-finger toxins (3FTxs) are a major component of the venom of elapid snakes and this gene family has expanded considerably via lineage-specific duplications, which have facilitated the emergence of multiple novel functions in addition to the ancestral activity of neurotoxic antagonism of nicotinic acetylcholine receptors [52.Sunagar K. et al.Three-fingered RAVERs: rapid accumulation of variations in exposed residues of snake venom toxins.Toxins. 2013; 5: 2172-2208Crossref PubMed Scopus (83) Google Scholar,86.Kessler P. et al.The three-finger toxin fold: a multifunctional structural scaffold able to modulate cholinergic functions.J. Neurochem. 2017; 142: 7-18Crossref PubMed Scopus (59) Google Scholar]. Contrastingly, in viperid snakes, it is the snake venom metalloproteinases (SVMP) [17.Jackson T.N. et al.Rapid radiations and the race to redundancy: an investigation of the evolution of Australian elapid snake venoms.Toxins (Basel). 2016; 8: 309Crossref Scopus (47) Google Scholar] and group II phospholipase A2 (PLA2) [38.Koludarov I. et al.Family saga: reconstructing the evolutionary history of a functionally diverse gene family reveals complexity at the genetic origins of novelty.bioRxiv. 2019; (Published online March 20, 2019. https://doi.org/10.1101/583344)Google Scholar] toxin gene families that have undergone lineage-specific expansions, again facilitating the emergence of multiple novel activities. The multiple duplication events within these gene families result in redundant arrays of genes that form neofunctionalization hotspots [2.Dowell N.L. et al.The deep origin and recent loss of venom toxin genes in rattlesnakes.Curr. Biol. 2016; 26: 2434-2445Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar,17.Jackson T.N. et al.Rapid radiations and the race to redundancy: an investigation of the evolution of Australian elapid snake venoms.Toxins (Basel). 2016; 8: 309Crossref Scopus (47) Google Scholar,44.Giorgianni M.W. et al.The origin and diversification of a novel protein family in venomous snakes.Proc. Natl. Acad. Sci. U. S. A. 2020; 117: 10911-10920Crossref PubMed Scopus (37) Google Scholar,87.Suryamohan K. et al.The Indian cobra reference genome and transcriptome enables comprehensive identification of venom toxins.Nat. Genet. 2020; 52: 106-117Crossref PubMed Scopus (70) Google Scholar,88.Schield D.R. et al.The origins and evolution of chromosomes, dosage compensation, and mechanisms underlying venom regulation in snakes.Genome Res. 2019; 29: 590-601Crossref PubMed Scopus (66) Google Scholar]. In both the elapid and viperid snake lineages, gene expansions and rapid sequence diversifications have occurred following the evolution of front-fanged, high-pressure delivery systems, highlighting the coevolutionary relationship that exists between toxin genes and venom delivery mechanisms [38.Koludarov I. et al.Family saga: reconstructing the evolutionary history of a functionally diverse gene family reveals complexity at the genetic origins of novelty.bioRxiv. 2019; (Published online March 20, 2019. https://doi.org/10.1101/583344)Google Scholar,52.Sunagar K. et al.Three-fingered RAVERs: rapid accumulation of variations in exposed residues of snake venom toxins.Toxins. 2013; 5: 2172-2208Crossref PubMed Scopus (83) Google Scholar]. Venom composition varies as a consequence of contingent evolutionary history and direct functional selection. For example, specific families of toxin genes have undergone extensive expansion and neofunctionalization in specific snake lineages. Three-finger toxins (3FTxs) are a major component of the venom of elapid snakes and this gene family has expanded considerably via lineage-specific duplications, which have facilitated the emergence of multiple novel functions in addition to the ancestral activity of neurotoxic antagonism of nicotinic acetylcholine receptors [52.Sunagar K. et al.Three-fingered RAVERs: rapid accumulation of variations in exposed residues of snake venom toxins.Toxins. 2013; 5: 2172-2208Crossref PubMed Scopus (83) Google Scholar,86.Kessler P. et al.The three-finger toxin fold: a multifunctional structural scaffold able to modulate cholinergic functions.J. Neurochem. 2017; 142: 7-18Crossref PubMed Scopus (59) Google Scholar]. Contrastingly, in viperid snakes, it is the snake venom metalloproteinases (SVMP) [17.Jackson T.N. et al.Rapid radiations and the race to redundancy: an investigation of the evolution of Australian elapid snake venoms.Toxins (Basel). 2016; 8: 309Crossref Scopus (47) Google Scholar] and group II phospholipase A2 (PLA2) [38.Koludarov I. et al.Family saga: reconstructing the evolutionary history of a functionally diverse gene family reveals complexity at the genetic origins of novelty.bioRxiv. 2019; (Published online March 20, 2019. https://doi.org/10.1101/583344)Google Scholar] toxin gene families that have undergone lineage-specific expansions, again facilitating the emergence of multiple novel activities. The multiple duplication events within these gene families result in redundant arrays of genes that form neofunctionalization hotspots [2.Dowell N.L. et al.The deep origin and recent loss of venom toxin genes in rattlesnakes.Curr. Biol. 2016; 26: 2434-2445Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar,17.Jackson T.N. et al.Rapid radiations and the race to redundancy: an investigation of the evolution of Australian elapid snake venoms.Toxins (Basel). 2016; 8: 309Crossref Scopus (47) Google Scholar,44.Giorgianni M.W. et al.The origin and diversification of a novel protein family in venomous snakes.Proc. Natl. Acad. Sci. U. S. A. 2020; 117: 10911-10920Crossref PubMed Scopus (37) Google Scholar,87.Suryamohan K. et al.The Indian cobra reference genome and transcriptome enables comprehensive identification of venom toxins.Nat. Genet. 2020; 52: 106-117Crossref PubMed Scopus (70) Google Scholar,88.Schield D.R. et al.The origins and evolution of chromosomes, dosage compensation, and mechanisms underlying venom regulation in snakes.Genome Res. 2019; 29: 590-601Crossref PubMed Scopus (66) Google Scholar]. In both the elapid and viperid snake lineages, gene expansions and rapid sequence diversifications have occurred following the evolution of front-fanged, high-pressure delivery systems, highlighting the coevolutionary relationship that exists between toxin genes and venom delivery mechanisms [38.Koludarov I. et al.Family saga: reconstructing the evolutionary history of a functionally diverse gene family reveals complexity at the genetic origins of novelty.bioRxiv. 2019; (Published online March 20, 2019. https://doi.org/10.1101/583344)Google Scholar,52.Sunagar K. et al.Three-fingered RAVERs: rapid accumulation of variations in exposed residues of snake venom toxins.Toxins. 2013; 5: 2172-2208Crossref PubMed Scopus (83) Google Scholar]. More recently, the extent of venom variation within species has begun to be recognized. Such variation exists between populations (i.e., regional variation) and between age/size classes [12.Massey D.J. et al.Venom variability and envenoming severity outcomes of the Crotalus scutulatus (Mojave rattlesnake) from Southern Arizona.J. Proteome. 2012; 75: 2576-2587Crossref PubMed Scopus (111) Google Scholar,14.Durban J. et al.Integrated venomics and venom gland transcriptome analysis of juvenile and adult Mexican rattlesnakes Crotalus simus, C. tzabcan, and C. culminatus revealed miRNA-modulated ontogenetic shifts.J. Proteome Res. 2017; 16: 3370-3390Crossref PubMed Scopus (75) Google Scholar, 15.Pla D. et al.Phylovenomics of Daboia russelii across the Indian subcontinent. Bioactivities and comparative in vivo neutralization and in vitro third-generation antivenomics of antivenoms against venoms from India, Bangladesh and Sri Lanka.J. Proteome. 2019; 207: 103443Crossref PubMed Scopus (53) Google Scholar, 16.Laxme R.S. et al.Beyond the 'big four': venom profiling of the medically important yet neglected Indian snakes reveals disturbing antivenom deficiencies.PLoS Negl. Trop. Dis. 2019; 13e0007899PubMed Google Scholar, 17.Jackson T.N. et al.Rapid radiations and the race to redundancy: an investigation of the evolution of Australian elapid snake venoms.Toxins (Basel). 2016; 8: 309Crossref Scopus (47) Google Scholar]. As venom is a dynamically evolving ecological trait, it stands to reason that whenever groups differ in their feeding ecology, there may be a corresponding difference in their venom composition. Juvenile snakes often consume different prey from adults of the same species and may also exhibit different foraging strategies and prey-handling behavior (e.g., juveniles may be nocturnal, whereas adults are more diurnal; juveniles may employ a bite-and-hold strategy, whereas adults may 'bite and release', etc.). The dynamism of venom evolution, which has been documented at the molecular level (see later), is further evidenced by the existence of regional variation, which may be linked both to ecological variance amongst populations and to neutral evolution, which may be pervasive in venom systems and work in tandem with positive selection [30.Aird S.D. et al.Population genomic analysis of a pitviper reveals microevolutionary forces underlying venom chemistry.Genome Biol. Evol. 2017; 9: 2640-2649Crossref PubMed Scopus (43) Google Scholar]. This dynamism itself generates another prediction based on evolutionary first principles: for a trait to evolve rapidly, there must be considerable heritable diversity within populations [31.Lewontin R.C. The units of selection.Annu. Rev. Ecol. Syst. 1970; 1: 1-18Crossref Google Scholar]. This prediction of variation in venom amongst adult members of a single population is only beginning to be investigated, but preliminary evidence suggests it will likely be confirmed [32.Smiley-Walters S.A. et al.High levels of functional divergence in toxicity towards prey among the venoms of individual pigmy rattlesnakes.Biol. Lett. 2019; 15: 20180876Crossref PubMed Scopus (12) Google Scholar,33.Petras D. et al.Intact protein mass spectrometry reveals intraspecies variations in venom composition of a local population of Vipera kaznakovi in Northeastern Turkey.J. Proteome. 2019; 199: 31-50Crossref PubMed Scopus (13) Google Scholar]. Venom toxin encoding genes originate from genes that code for endophysiological proteins (e.g., salivary, immunological, and pancreatic proteins, etc.) [34.Reyes-Velasco J. et al.Expression of venom gene homologs in diverse python tissues suggests a new model for the evolution of snake venom.Mol. Biol. Evol. 2015; 32: 173-183Crossref PubMed Scopus (60) Google Scholar]. Numerous mechanisms have been proposed to explain the origin and diversification of toxins and, thereby, the evolution of venom variability across snakes. These include gene duplication, domain loss, evolutionary tinkering of expression levels, alternative- and trans-splicing, and rapid evolution under positive Darwinian selection [35.Casewell N.R. et al.Complex cocktails: the evolutionary novelty of venoms.Trends Ecol. Evol. 2013; 28: 219-229Abstract Full Text Full Text PDF PubMed Scopus (539) Google Scholar] (Figure 1). Gene duplication, which plays a key role in the evolution of phenotypic complexity and functional innovation, has been implicated in the diversification of venom. Venom protein encoding genes are theorized to undergo extensive duplications and evolve under a 'birth and death' model of evolution [36.Fry B.G. et al.Molecular evolution and phylogeny of elapid snake venom three-finger toxins.J. Mol. Evol. 2003; 57: 110-129Crossref PubMed Scopus (273) Google Scholar]. According to this model, repeated duplication events lead to the origin of new copies, most of which undergo pseudogenization into dysfunctional forms over time, while some subsequently evolve novel functions and are retained [37.Ohno S. Evolution by Gene Duplication. Springer-Verlag, 1970Crossref Google Scholar]. Although some assumptions of the original 'birth and death' model are questionable [38.Koludarov I. et al.Family saga: reconstructing the evolutionary history of a functionally diverse gene family reveals complexity at the genetic origins of novelty.bioRxiv. 2019; (Published online March 20, 2019. https://doi.org/10.1101/583344)Google Scholar], gene duplication has led to the formation of multi-locus toxin gene families with extraordinary structural and functional diversity [2.Dowell N.L. et al.The deep origin and recent loss of venom toxin genes in rattlesnakes.Curr. Biol. 2016; 26: 2434-2445Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar,29.Ainsworth S. et al.The paraspecific neutralisation of snake venom induced coagulopathy by antivenoms.Commun. Biol. 2018; 1: 1-14Crossref PubMed Scopus (62) Google Scholar,39.Vonk F.J. et al.The king cobra genome reveals dynamic gene evolution and adaptation in the snake venom system.Proc. Natl. Acad. Sci. U.S. A. 2013; 110: 20651-20656Crossref PubMed Scopus (322) Google Scholar] (see later). Gene duplication does not always introduce novelty though and can also underpin increased expression levels. Concerted evolution, by contrast to the 'birth and death' model, maintains high levels of sequence conservation amongst duplicates through recombination, as a strategy to increase expression levels of the encoded toxin [40.Moran Y. et al.Concerted evolution of sea anemone neurotoxin genes is revealed through analysis of the Nematostella vectensis genome.Mol. Biol. Evol. 2008; 25: 737-747Crossref PubMed Scopus (64) Google Scholar]. Although concerted evolution is yet to be noted in snakes, recurrent snake venom gene duplications do facilitate increased expression of the encoded toxin types [41.Margres M.J. et al.Selection to increase expression, not sequence diversity, precedes gene family origin and expansion in rattlesnake venom.Genetics. 2017; 206: 1569-1580Crossref PubMed Scopus (33) Google Scholar]. Since relative differences in the expression level of venom components can considerably alter the underlying toxicity [16.Laxme R.S. et al.Beyond the 'big four': venom profiling of the medically important yet neglected Indian snakes reveals disturbing antivenom deficiencies.PLoS Negl. Trop. Dis. 2019; 13e0007899PubMed Google Scholar], shifts in gene expression seem likely to also underpin evolutionary adaptations. Stochastic degeneration of genes has also been reported to result in significant evolutionary consequences [42.Shibata H. et al.The habu genome reveals accelerated evolution of venom protein genes.Sci. Rep. 2018; 8: 1-11Crossref PubMed Scopus (38) Google Scholar]. For example, several lineages of rattlesnakes have lost phospholipase A2 (PLA2) neurotoxin genes, which were once present in their common ancestor, and have shifted towards a more hemotoxic venom profile [2.Dowell N.L. et al.The deep origin and recent loss of venom toxin genes in rattlesnakes.Curr. Biol. 2016; 26: 2434-2445Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar]. Partial degeneration of gene segments have also been documented in venom protein encoding genes. Domain loss has been shown to mediate toxin neofunctionalization (Figure 1), with notable examples including truncations of snake venom metalloproteinases (SVMP) that led to the origin of structurally functionally diverse subclasses in