Venom Variation as a Window into the Ecology and Evolution of Snakes

Synopsis

Snake venoms are complex biochemical systems that function primarily in prey subjugation and defense, yet their composition varies extensively across individuals, populations, species, and environments. This variation provides a powerful framework for investigating ecological and evolutionary processes. Here, we offer a forward-looking synthesis of snake venom diversity that proposes new research directions and highlights how venom variation can illuminate eco-evolutionary dynamics across biological scales. We review evidence for 10 key contexts in which venom variation arises, including within-population differences, sexual dimorphism, geographic structuring, ontogenetic shifts, seasonal changes, interspecific divergence, hybridization, convergent evolution, prey specificity, and venom resistance. Together, these processes demonstrate that venom phenotypes are shaped by interacting selective pressures such as trophic ecology, predator–prey coevolution, environmental heterogeneity, and gene flow. While phylogenetic history establishes broad toxin composition patterns, ecological factors frequently drive rapid and repeated shifts in venom phenotype. We further outline the historical development of venom research, from early descriptive studies to modern integrative approaches enabled by advances in proteomics, transcriptomics, genomics, and functional assays. These methodological innovations increasingly allow venom composition to be linked directly to ecological performance and evolutionary outcomes. Despite this progress, major gaps remain, including limited taxonomic coverage, incomplete integration of ecological data, and insufficient experimental tests of adaptive hypotheses. Future research combining molecular, functional, and field-based approaches will be essential for resolving the mechanisms that generate and maintain venom diversity. As complex traits shaped by interacting ecological and evolutionary forces, snake venoms provide an exceptional model system for understanding how selection, constraint, and environmental context interact to produce phenotypic diversity across time and space.

Introduction

Venoms are complex biochemical innovations whose variation illuminates ecological and evolutionary processes across species and populations (Arbuckle 2015). In squamate reptiles (Fig. 1), venoms primarily facilitate prey capture and defence and consist of diverse mixtures of proteins, peptides, and other bioactive components produced and stored in specialised glands (Chan et al. 2016; Mackessy 2022). Venomous snakes occur across several lineages, spanning the families Elapidae, Viperidae, Atractaspididae, and Colubridae sensu lato (Fry et al. 2006; Quijada-Mascareñas and Wüster 2009; Zheng and Wiens 2016). Although this review focuses on snakes, venoms are also present in anguimorph lizards of the genus Heloderma (Fry et al. 2010; Sanggaard et al. 2015), which are discussed where relevant; their presence in Varanus remains debated (Fry et al. 2006; Hargreaves et al. 2014; Sweet 2016).

Fig. 1.

Venomous species exist across the squamate tree of life in multiple lineages. (A) Calloselasma rhodostoma is a pit viper distributed across Southeast Asia with venoms adapted for local prey communities, and the link between snake venom and diet was thoroughly investigated for the first time in this species (Daltry et al. 1996). (B) Naja nigricollis is an elapid snake from Sub-Saharan Africa with the ability to spit venom defensively (Kazandjian et al. 2021). (C) Leptophis ahaetulla is a venomous arboreal colubrid from South America with low toxicity toward mammals (Sánchez et al. 2018). (D) Heloderma suspectum is a venomous anguimorph lizard native to the warm deserts of North America, with venom used primarily for defensive purposes and serving secondary roles in prey incapacitation (Beck, 2005). Photos by Artur Tomaszek (A), Chad Keates (B), Damien Esquerré (C), and Bryan Hughes (D), and used with permission.

The extraordinary diversity of snake venom components is widely attributed to the “birth-and-death” model of gene evolution (Nei et al. 1997; Fry et al. 2003). Under this model, genes encoding physiological proteins undergo duplication, producing multiple copies that evolve under relaxed functional constraint. Although many duplicates accumulate deleterious mutations and become pseudogenes, others are retained and may become selectively expressed in the venom gland (Ohno 1970). These retained duplicates frequently undergo further rounds of duplication and divergence, producing large toxin gene families with substantial structural and functional diversity (Kordiš and Gubenšek 2000; Fry et al. 2003; Chang and Duda 2012; Casewell et al. 2013). Additional diversification can arise through mechanisms such as alternative and trans-splicing, particularly within toxin families including snake venom metalloproteinases (SVMPs), serine proteinases, and vascular endothelial growth factors (Shibata et al. 2018; Ogawa et al. 2019). Consistent with these dynamics, venom toxin genes commonly exhibit signatures of accelerated molecular evolution (Ohno et al. 2003; Ogawa et al. 2005; Doley et al. 2009) and positive selection (Gibbs and Rossiter 2008; Juarez et al. 2008; Rokyta et al. 2011), especially at surface-exposed residues that mediate protein–target interactions while preserving structural stability (Casewell et al. 2011; Sunagar and Moran 2015).

This evolutionary framework produces extensive venom variation across taxonomic scales. Differences in toxin composition and abundance reflect lineage-specific histories as well as ecological and selective pressures shaping toxin expression and deployment (Casewell et al. 2020). Diet is widely considered a major driver of venom evolution (Davies and Arbuckle 2019; Holding et al. 2021; Siqueira‐Silva et al. 2021) Species with highly specialized diets often possess relatively simple venoms; for example, many sea snakes (Elapidae) feed primarily on fish and typically express venoms dominated by three-finger α-neurotoxins and phospholipases A₂ (Mackessy and Tu 1993; Li et al. 2005; Wang et al. 2020). In contrast, many terrestrial front-fanged snakes exhibit biochemically complex venoms containing dozens of proteins and peptides from multiple toxin families (Oliveira et al. 2022). Variation in toxin composition and relative abundance can therefore substantially alter venom effects (Table 1), generating extensive phenotypic diversity within and among species.

Table 1.

List of toxin families commonly found in squamate venoms, with major venom toxins bolded. Taxonomic clades possessing these toxins in large proportions are indicated (V = Viperidae, E = Elapidae, C = Colubridae, H = Heloderma) in addition to function of toxins and common biological effects. For a more complete review of toxins comprising squamate venoms and references, see Mackessy (2010a, 2021).

Toxin	Clades	Functions	Common biological effects	References
5′-nucleotidase (5N)	E, V	Nucleotide breakdown	Hypotension, reduced platelet aggregation, circulatory collapse contributing to immobilization	Dhananjaya and D’Souza (2010), Mackessy (2021)
Acetylcholinesterase (Ache)	C, E	Hydrolysis of acetylcholine	Depletion of neurotransmitters; tetanic paralysis	Mackessy (2010a, 2021), Frobert et al. (1997)
Aminopeptidase (AmPep)	E, V	Protein degradation	Degradation of regulatory peptides, tissue damage, interference with physiological signaling	Mackessy (2010a, 2021), Vaiyapuri et al. (2010)
Bradykinin-potentiating peptides (BPP)	V	Increases potency of bradykinin	Pain, hypotension, prey immobilization	Mackessy (2010a, 2021), Sciani and Pimenta (2017)
C-type lectins and C-type lectin-related proteins (CTL)	V	Bind to platelet and collagen receptor	Haemostasis disruption, platelet modulator	Mackessy (2010a, 2021), Morita (2005), Ogawa et al. (2005), Arlinghaus and Eble (2012)
Cobra venom factor (CVF)	E	Complement system activator that forms stable C3/C5 convertase	Complement depletion, immune disruption, increased inflammation	Kock et al. (2004), Vogel and Fritzinger (2010)
Cystatin (Cys)	E, V	Protease inhibition	Modulates proteolysis in venom; may protect other toxins and contribute to tissue disruption	Mackessy (2010a, 2021), Richards et al. (2011)
Cystein-rich secretory proteins (CRISP)	H, C, E, V	May induce hypothermia	Lethargy, paralysis, prey capture/immobilization	Mackessy (2010a, 2021), Tadokoro et al. (2020)
Disintegrins (Dis)	V	Inhibit binding of integrins to receptors	Anti-angiogenic and haemostasis-altering effects	Mackessy (2010a), Calvete et al. (2005, 2021), Calvete (2013), Almeida et al. (2023)
Hyaluronidase (Hyal)	H, E, V	Hydrolysis of interstitial hyaluronan	Decreased interstitial viscosity	Girish et al. (2002), Mackessy (2010a, 2021)
Kunitz-type protease inhibitor (Kun)	E, V	Protease inhibition, ion channel blocking	Neurotoxicity, paralysis, disruption of nerve signaling	Mackessy (2010a, 2021), Mukherjee et al. (2014)
L-amino-acid oxidase (LAAO)	E, V	Oxidative deamination of L-amino acids	Induces apoptosis, cell damage	Du and Clemetson (2002), Mackessy (2010a, 2021), Guo et al. (2012), Izidoro et al. (2014)
Natriuretic peptide (NP)	E, V	Cardiovascular regulation and vasodilation	Vasodilation, hypotension, disruption of blood pressure, rapid prey weakening	Schweitz et al. (1992), Mackessy (2010a, 2021), Vink et al. (2012)Ang et al. (2012)
Nerve growth factor (NGF)	E, V	Affects nerve cells and mast cell activation	Pain induction, inflammation, vascular permeability changes	Kostiza and Meier (1996), Mackessy (2010a, 2021), Trummal et al. (2011)
Ohanin/Vespryn (Oha-Vesp)	E	Disruption of nervous system and immobilization	Hypolocomotion, disorientation, reduced motor control	Pung, et al. (2005)
Peptide myotoxins (crotamine, myotoxin a, etc.)	V	Ion channel inhibitors	Rapid immobilization via tetanic hyperextension	Oguiura et al. (2005), Mackessy (2010a, 2021), Kerkis et al. (2014)
Phosphodiesterase (PDE)	C, E, V	Degrades phosphodiester bonds in nucleotides and nucleic acids	Hypotension, shock, disruption of cellular signaling, contributes to systemic toxicity	Dhananjaya and D’souza (2010), Mackessy (2010a, 2021)
Phospholipase A₂ (PLA₂)	H, C, E, V	Ca2+-dependent hydrolysis of 2-acyl groups in 3-sn-phosphoglycerides	Myotoxicity, myonecrosis, lipid membrane damage	Kini (2003), Mackessy (2010a, 2021)
PLA₂-based presynaptic neurotoxins	E, V	Blocks release of acetylcholine from axon terminus	Potent neurotoxicity, prey immobilization	Montecucco et al. (2008), Mackessy (2010a, 2021)
Phospholipase B (PLB)	E, V	Hydrolyzes phospholipids at multiple positions in membranes	Membrane disruption, hemolysis, tissue damage	Bernheimer, et al (1987), Ullah, 2020 and Masood (2020)
Phospholipase C (PLC)	E	Cleaves phospholipids to produce diacylglycerol and phosphorylated head groups	Cell membrane breakdown, inflammation, tissue necrosis	Huang, et al. (1995)
Serine proteases (SVSP)	H, C, E, V	Kallikrein-like; releases bradykinin, Thrombin like; catalysis of fibrinogen, and various others	Rapid hypotension, coagulopathy	Matsui et al. (2000), Kini (2005), Mackessy (2010a, 2021)
Snake venom metalloproteinases (SVMP)	C, V	Hydrolysis of structural proteins including basal lamina	Haemorrhage, necrosis and tissue destruction, possible prey predigestion	Gutiérrez (2000)Teixeira et al. (2005), Fox and Serrano (2008), Mackessy (2010a, 2021)
Three-finger toxins (3FTx)	C, E	Potent inhibitor of neuromuscular transmission, cytotoxicity, enzyme inhibiting effects, often taxon-specific effects	Flaccid paralysis, rapid prey immobilization/death	Mackessy (2010a, 2021), Sunagar et al. (2013), Utkin (2019)
Whey acidic protein motif protease inhibitor (Waprin)	E, V	Small secreted protease inhibitor with antimicrobial activity	May disrupt microbial defense or modulate proteolysis, minor direct toxicity	Nair, et al. (2007)

Given this diversity, snake venoms have long attracted scientific interest. Historically, much venom research has been motivated by clinical and human health considerations (e.g., Sintiprungrat et al. 2016). More recently, studies have increasingly examined venom variation through ecological and evolutionary perspectives (Barlow et al. 2009; Jackson et al. 2016; Sunagar et al. 2016; Zancolli et al. 2016; Strickland et al. 2018; Barua and Mikheyev 2019; Tasoulis et al. 2020; Smith et al. 2023a) Despite this progress, integrative eco-evolutionary analyses remain comparatively limited. Here, we provide a forward-looking perspective that synthesizes the current state of the field, highlighting ten ecological and evolutionary processes illuminated by snake venom variation. We emphasize that this framework is not exhaustive but rather intended as a conceptual springboard for future research. We also summarize methodological approaches used in venom research and outline promising directions for advancing this field.

Venoms as complex traits in the eco-evolutionary arena

Diet is a primary driver of snake venom evolution and variation (Daltry et al. 1996; Barlow et al. 2009; Holding et al. 2021). Because venom functions mainly in prey subjugation, its composition closely reflects a species’ trophic ecology and is expected to experience strong natural selection. Geographic variation may track differences in local prey communities (Holding et al. 2018), ontogenetic shifts often parallel age-related dietary changes (e.g., Avella et al. 2022a), and seasonal variation can reflect temporal prey fluctuations (Christian et al. 2007) Supporting this, multiple studies document increased prey-specific toxicity, enhancing foraging efficiency (Barlow et al. 2009; Gibbs and Mackessy 2009). Lineages that shifted to prey not requiring envenomation, such as eggs or molluscs, often reduce or lose venom components as seen in Aipysurus eydouxii (Mackessy and Tu 1993) and Dasypeltis scabra (Fry et al. 2008). While prey capture remains the main selective force shaping venom composition, it also functions defensively, as exemplified by venom-spitting in African and Asian elapid snakes (Naja spp. and Hemachatus spp.; Kazandjian et al. 2021). Furthermore, the evolution of venom resistance in predators of venomous snakes (Van Thiel et al. 2022), innate avoidance of characteristic warning color patterns (Smith 1977), and emergence of Batesian mimicry in some nonvenomous taxa (Davis Rabosky et al. 2016) collectively support a secondary defensive function of venom. These patterns show that venom evolution is shaped by trophic specialization, ecological context, and multifunctional selective pressures. Below, we outline 10 eco-evolutionary contexts linking venom variation to ecological and evolutionary processes in squamates (Fig. 2).

Fig. 2.

Snake venoms may show compositional variation in response to a variety of ecological and evolutionary processes. Indicated here are different processes that have been studied, and hold promise for future investigation. Photo of Bitis parviocula by Thor Håkonsen and used with permission.

Within-population variation

Increasing attention has focused on variation among individuals within single populations. In many rattlesnake species, venom composition appears relatively consistent among individuals (Mackessy et al. 2003; Smith et al. 2023a; Balchan et al. 2025b), although this pattern is not universal. In regions where divergent venom phenotypes meet, populations may harbor substantial individual diversity (Strickland et al. 2018; Smith et al. 2023a). High individual variation has also been documented in the absence of geographic transitions. For example, pygmy rattlesnakes (Sistrurus miliarius) from a Florida population show pronounced variation in venom toxicity toward lizard prey, suggesting adaptive significance at fine ecological scales (Smiley-Walters et al. 2019). Similarly, monocled cobras (Naja kaouthia) from West Bengal, India, exhibit striking interindividual differences in venom composition with likely functional consequences (Rashmi et al. 2021).

Within-population venom variation remains poorly studied in colubrid snakes. However, data from colubrine (Mackessy et al. 2006), dipsadine (Tioyama et al. 2023), and natricine snakes (Coppinger et al. 2025) suggest that substantial diversity remains unexplored. Understanding the ecological and genetic basis of individual-level venom variation will be essential for linking selection at fine spatial and temporal scales to broader patterns of venom evolution.

Sexual dimorphism

Empirical evidence for intersexual venom variation has historically been limited and inconsistent. Early studies generally reported little to no venom differences between sexes (Chippaux et al. 1991), suggesting broad conservation of venom composition. However, some biochemical studies hinted at potential sexual differences. An isoelectric focusing analysis of Calloselasma rhodostoma venoms detected a single unidentified protein band present in females but absent in males (Daltry et al. 1996). More recent proteomic studies have revealed subtle sex-associated differences in some species. In Bothrops jararaca, sex-specific protein bands were detected using one-dimensional electrophoresis, spot intensities differed on two-dimensional gels, and enzyme activities varied between sexes (Menezes et al. 2006; Pimenta et al. 2007). A MALDI-TOF mass spectrometry analysis further identified four bradykinin-potentiating peptides present exclusively in female venoms, although substantial individual variation and unclear functional significance complicate interpretation (Pimenta et al. 2007). Additional studies reported sexual differences in proteolytic activity toward fibrinogen and gelatin (Zelanis et al. 2016), as well as sex-specific variation in enzymatic and toxic activities (Furtado et al. 2006). An analysis of neonate Bothrops moojeni likewise found higher serine protease effects, hemorrhagic activity, and lethality in female venoms relative to males (Ferreira-Rodrigues et al. 2024). However, other studies have reported no sex-based venom differences in dimorphic pit viper species (Da Silva Aguiar et al. 2020; Gómez et al. 2021), including Tropidolaemus wagleri (Tan et al. 2017), despite its extreme sexual size dimorphism.

Sex-associated venom differences have also been reported in elapid snakes. In Naja atra, male and female venoms differed in acetylcholinesterase, nerve growth factor, and CRISPs based on electrophoretic and enzymatic analyses (Nie et al. 2022). In contrast, a recent integrative proteomic study found no evidence of sexual dimorphism in Vipera berus venoms (Schulte et al. 2026a). Overall, sexual dimorphism in venom composition appears subtle, species-specific, and far from universal. The ecological and evolutionary mechanisms underlying such variation—including sex-specific diets, reproductive roles, defensive behaviors, or hormonal regulation—remain poorly understood. Targeted, hypothesis-driven studies will be required to assess the prevalence and adaptive significance of venom sexual dimorphism across snakes.

Geographic variation

Although venom composition is relatively conserved in some snake species (Avella et al. 2023; Balchan et al. 2025b), geographically structured variation is widespread and represents the dominant pattern across snakes. Early investigations focused on the Mohave rattlesnake (Crotalus scutulatus), a species exhibiting multiple venom phenotypes with markedly different lethality. Initial work established the existence of these phenotypes (Glenn et al. 1983), and subsequent studies resolved their geographic structuring and environmental correlates (Zancolli et al. 2016; Strickland et al. 2018). Extensive geographic variation has since been documented in numerous rattlesnake species (Sunagar et al. 2014; Margres et al. 2021; Smith et al. 2023a), with geographically structured venom phenotypes representing a recurring pattern within the group (Mackessy 2008, 2010b).

Beyond rattlesnakes, geographic venom variation has been documented in other pit vipers (Crotalinae; Mora-Obando et al. 2020) and true vipers (Viperinae; Damm et al. 2024; Sarangi et al. 2025). Although fewer studies exist for elapid snakes, pronounced geographic variation has long been recognized in Asian cobras (Naja spp.; Chanda et al. 2018; Rashmi et al. 2021; Deka et al. 2023) and kraits (Bungarus spp.; Oh et al. 2019; Sunagar et al. 2021), where venom heterogeneity has important snakebite implications (Sintiprungrat et al. 2016). More recent studies have documented geographic variation in Australian (Hydrophiinae; Van Thiel et al. 2023), African (Elapinae; Hus et al. 2024), and Asian elapids (Elapinae; Tan et al. 2015). Geographic venom variation remains poorly studied in colubrid snakes and helodermatid lizards. Existing evidence suggests substantial variation in colubrine (Mackessy et al. 2006), dipsadine (Tioyama et al. 2023), and natricine snakes (Coppinger et al. 2025), whereas Heloderma venoms may be more conserved (Koludarov et al. 2014).

Recent work also highlights the potential role of abiotic environmental variables in shaping venom phenotypes (Strickland et al. 2018; Smith et al. 2023a; Sarangi et al. 2025). Although geographic patterns of venom variation are now well described for many species, the drivers underlying these patterns remain poorly resolved outside a few model systems. Identifying the biotic and abiotic forces structuring venom variation across landscapes remains a key frontier for understanding the eco-evolutionary dynamics of venom systems.

Ontogenetic variation

Ontogenetic changes in venom composition are widespread and often align with ecological and body size shifts across an individual’s life history. Changes in habitat use, prey size, prey type, and predator exposure across developmental stages can impose stage-specific selective pressures on venom function (e.g., Mackessy et al. 2003). Ontogenetic venom variation is well documented in rattlesnakes (Mackessy 1988; Saviola et al. 2015; Borja et al. 2018; Mackessy et al. 2018) and other New World pit vipers (Guércio et al. 2006; Alape-Girón et al. 2008; Barlow et al. 2009; Machado Braga et al. 2020), where juveniles and adults often exhibit distinct venom compositions reflecting shifts from ectotherm- to endotherm-based diets. Two common trends include higher toxicity and reduced metalloproteinase content in neonate venoms relative to adults. These patterns likely reflect ontogenetic changes in prey communities (e.g., Andrade and Abe 1999), with venoms optimized for prey targeted at specific life stages. Comparable ontogenetic patterns occur in true vipers, including Daboia (Zdenek et al. 2022; Senji Laxme et al. 2024), Echis (Barker et al. 2025), and Vipera (Avella et al. 2022a; Qiao et al. 2024; Lakušić et al. 2025, 2025), though venom ontogeny remains unevenly studied across the group. Alternative patterns also occur; for example, juvenile Vipera venoms are often richer in SVMPs than those of adults (Schulte et al. 2026b; Avella et al. 2022a). In elapids, evidence for ontogenetic venom variation is mixed. Venoms of Naja naja appear largely consistent across developmental stages (Senji Laxme et al. 2024), whereas Naja kaouthia shows age-related differences in PLA₂s activity and isoforms (Modahl et al. 2016). In the Australian genus Pseudonaja, most species transition from noncoagulopathic venoms in juveniles to coagulopathic venoms in adults, likely reflecting shifts from reptilian to mammalian diets (Cipriani et al. 2017). Ontogenetic variation remains largely unexplored in helodermatid lizards (Dobson et al. 2024) and colubrid snakes (Mackessy et al. 2006), where diet and trophic ecology are poorly known for most species.

Seasonal variation

Because prey availability can change throughout the year, seasonality has been proposed as a potential driver of venom variation (Chippaux et al. 1991), although empirical support remains limited. A study on several rattlesnake species (Crotalus atrox, C. molossus, and C. oreganus helleri) detected no seasonal differences in venom composition over extended sampling periods (Gregory-Dwyer et al. 1986). Similarly, studies on C. durissus and C. molossus found no compositional changes but did detect seasonal variation in toxin activity (Macías-Rodríguez et al. 2014; Tasima et al. 2024). Early work suggested seasonal differences in Vipera ammodytes, with certain protein bands present in summer but absent in winter electrophoretic profiles (Gubenšek et al. 1974). However, venomic profiling of Pseudonaja textilis sampled over a 12-month period found no evidence of seasonal variation (Williams and White 1992). Overall, seasonal venom variation appears weakly supported and may be subtle, context-dependent, or expressed primarily at the level of toxin activity rather than composition. Future work may benefit from focusing on species inhabiting strongly seasonal environments, where fluctuating ecological conditions could impose differential selection on venom phenotypes.

Interspecific variation

A general expectation is that closely related taxa possess more similar venoms than phylogenetically distant relatives. At broad scales, the dominance and relative abundance of major toxin families follow clear phylogenetic patterns (Mackessy 2010a, 2021). Viperid venoms are typically dominated by higher-molecular-weight enzymatic toxins such as SVMPs and serine proteases (SVSPs), whereas elapid venoms are largely composed of smaller, nonenzymatic toxins, particularly three-finger toxins (3FTxs) and PLA₂s (Tasoulis and Isbister 2017; Barua and Mikheyev 2019). Colubrid snakes exhibit diverse venom phenotypes, most still poorly characterized, though several are rich in 3FTxs (Mackessy 2002; Junqueira-de-Azevedo et al. 2016; Modahl and Mackessy 2019).

Phylogenetic components of venom variation have been examined in several viperid (Mackessy 2010b; Gibbs et al. 2013; Holding et al. 2021; Zhao et al. 2023) and elapid clades (Cipriani et al. 2017; Sanz et al. 2019; Kazandjian et al. 2021; Van Thiel et al. 2023). Although these studies link clade-level venom differences to ecological drivers such as diet (Holding et al. 2021) and defense (Kazandjian et al. 2021), much of the phylogenetic structure underlying venom evolution remains unresolved. Despite expectations of phylogenetic signal, venom phenotypes often do not track evolutionary relationships within clades. A well-known example is the western rattlesnake complex (Crotalus viridis/C. oreganus), which exhibits two divergent venom phenotypes: a widespread proteolytic Type I venom, presumed ancestral, and a highly toxic Type II venom that has evolved repeatedly within the group (Mackessy 2008, 2010b). These phenotypes do not correspond to phylogenetic relationships and instead appear to have arisen multiple times independently. Similar patterns occur broadly across rattlesnakes (Crotalus spp.), where toxic phenotypes repeatedly evolve within clades otherwise dominated by proteolytic venoms (Colis-Torres et al. 2022; Borja et al. 2025).

In elapids, a PLA₂/3FTx dichotomy has been described in Micrurus coral snakes (Sanz et al. 2016, 2019) and Australo-Papuan elapids (Goldenberg et al. 2018; Van Thiel et al. 2023), where venoms are typically dominated by one of these toxin classes. At broader taxonomic scales, similar trade-offs between enzyme-rich and neurotoxin-dominated venoms may occur across venomous snakes, with venom proteomes tending toward either enzymatic or PLA₂/3FTx-dominated compositions (Barua and Mikheyev 2019). These patterns suggest ecological pressures may override phylogenetic inertia in shaping venom phenotypes. In contrast, venoms of Heloderma lizards appear highly conserved across phylogeny (Koludarov et al. 2014), possibly reflecting ecological similarity among species and a shared defensive function (Russell and Bogert 1981), although limited ecological data preclude firm conclusions.

Interpretations of interspecific venom variation may also be complicated by discordance between venom gland transcriptomes and expressed proteomes (Sunagar et al. 2021), which may, for example, be driven by post-transcriptional regulation (Rokyta et al. 2015), alternative splicing (Ogawa et al. 2019), and/or post-translational modifications (Andrade-Silva et al. 2016). For example, genes encoding 3FTxs have been detected in venom gland transcriptomes (Junqueira-de-Azevedo et al. 2006; Pahari et al. 2007) despite the absence of corresponding toxins in venom proteomes from the same species (Sanz et al. 2006). Such incongruence highlights the importance of integrating transcriptomic, proteomic, and ecological data when assessing phylogenetic patterns of venom evolution (Davies and Arbuckle 2019; Schaeffer et al. 2023; Zhao et al. 2023; Modahl et al. 2024; Heptinstall et al. 2025).

Hybridization-induced variation

Hybridization is increasingly recognized as a contributor to phenotypic diversity across taxa (e.g., Myers et al. 2024), including venom phenotypes. Among venomous snakes, hybridization occurs both as isolated events (Neri-Castro et al. 2022) and within stable hybrid zones between species (Zancolli et al. 2016; Smith et al. 2023b). Laboratory studies show that interspecific hybridization can produce venom phenotypes intermediate to those of parental species (Aird et al. 1989; Smith and Mackessy 2016), and similar patterns occur in wild populations. Hybridization between the native Protobothrops flavoviridis and introduced P. elegans in the Ryukyu Islands produced individuals with intermediate venom phenotypes (Aird et al. 2015). Comparable results have been reported in several rattlesnake hybrid systems (Harrison et al. 2022; Neri-Castro et al. 2022; Roldán-Padrón et al. 2022; Smith et al. 2023b).

A prominent example occurs in a stable hybrid zone between Crotalus viridis and C. scutulatus in southwestern New Mexico, USA. C. viridis expresses a proteolytic Type I venom, whereas C. scutulatus produces a Type II venom characterized by the presynaptically neurotoxic PLA₂ Mojave toxin. Although pure C. scutulatus and admixed individuals within the contact zone possess Mojave toxin, it is absent from pure C. viridis populations outside the zone (Zancolli et al. 2016). This pattern suggests that hybridization can transfer potent toxins across species boundaries, while natural selection may constrain their introgression despite substantial gene flow.

Convergent evolution

Convergent evolution arises when similar selective pressures act on distantly related taxa and may occur through morphological (Muschick et al. 2012), behavioral (Blackledge and Gillespie 2004), or molecular mechanisms (Van Thiel et al. 2022). Although a wide diversity of toxins has evolved across the tree of life, there are relatively few biochemical pathways through which venoms incapacitate prey (e.g., membrane disruption or inhibition of neurotransmission). Consequently, striking cases of convergence among independently evolved or recruited toxins have been documented (Casewell et al. 2013). As a molecular component of the organismal phenotype, venom is therefore expected to exhibit convergent evolution under shared ecological pressures. However, few studies have explicitly examined venom convergence alongside convergent evolution in ecology, morphology, or behavior.

At broader scales, convergent patterns of toxin composition and abundance occur across diverse snake lineages. Venom proteomes frequently trend toward either PLA₂/3FTx-dominated or enzyme-rich configurations (Barua and Mikheyev 2019), suggesting similar functional demands may repeatedly shape venom phenotypes independent of shared ancestry.

Promising systems for investigating venom convergence include distantly related species with similar dietary specialization, as well as taxa that have independently transitioned into comparable ecological niches such as aquatic, arboreal, or fossorial habitats. Comparative analyses across these systems may help disentangle the relative roles of ecology and phylogeny in shaping venom evolution.

Prey specificity

Physiological differences among prey taxa impose distinct functional requirements on venom, favoring toxins optimized for subjugating specific prey types (Daltry et al. 1996). Diet was among the first ecological factors identified as a driver of venom variation, and regional differences in prey availability can exert strong selective pressures on venom proteomes.

Prey community variation drives venom divergence across numerous species (Daltry et al. 1996; Smith et al. 2023a) and appears widespread. In species with strong prey specificity, venoms may include taxon-specific toxins. Notable examples in colubrids include the bird-specific irditoxins A and B from Boiga irregularis (Pawlak et al. 2009), the lizard-specific fulgimotoxin from Oxybelis fulgidus (Heyborne and Mackessy 2021), and the lizard-specific sulditoxin and mammal-specific sulmotoxin 1 from Spilotes sulphureus (Modahl et al. 2018), all specialized 3FTxs. Among vipers, prey-specific toxins are formally characterized only in Crotalus viridis, where myotoxin a targets mammalian prey (Smith et al. 2023; Balchan et al. 2024). However, many vipers show prey-specific effects inferred from differential lethality across prey classes (Gibbs and Mackessy 2009; Richards et al. 2012; Healy et al. 2019; Lyons et al. 2020), suggesting prey specialization may be more widespread than currently documented, and underscoring a need for continued research.

Venom resistance

Prey species may experience strong selection to evolve physiological mechanisms that neutralize the venoms of their predators (Holding et al. 2016a, 2016b). This has been intensively studied in snake–rodent systems, notably the California ground squirrel (Otospermophilus beecheyi) and Pacific rattlesnake (Crotalus oreganus). Beyond behavioral defenses (Rundus et al. 2007), squirrels possess biochemical mechanisms for detoxifying rattlesnake venoms (Poran et al. 1987; Robinson et al. 2021). Resistance varies geographically in this system, highlighting its evolutionary lability.

Venom resistance also occurs in numerous other snake–prey systems (Heatwole and Powell 1998; Chandrasekara et al. 2024) across North America (Pomento et al. 2016; Robinson et al. 2021; Balchan et al. 2024), South America (Voss 2013), Africa (Phillips et al. 2012), and Australia (Chandrasekara et al. 2024, 2025). The taxonomic and geographic breadth of resistance emphasizes the role of reciprocal selection and suggests coevolutionary arms races as key drivers of venom diversification.

Predators that frequently consume venomous snakes also show selection for resistance, including opossums (Didelphidae; Jansa and Voss 2011; Voss and Jansa 2012), mongooses (Herpestidae; Bdolah et al. 1997), and honey badgers (Mellivora capensis; Drabeck et al. 2015). However, relatively few studies have examined resistance in these predators, highlighting an important gap and a promising avenue for future research.

Methods and mindsets: Past, present and future

Research questions in venom biology have evolved over time, as have the methods used to address them. These methods target multiple levels of biological organization (Fig. 3), each providing distinct insights into venom complexity. Integrating data across levels is essential for linking genotype, phenotype, and extended phenotype, enabling a more comprehensive understanding of venom function and evolution.

Fig. 3.

Conceptual representation of the layered methods of studying venom variation. The five layers described here may be used in venomic investigations either singly or in combination and may address questions at different levels. Bridging these layers (e.g., via proteogenomics) may offer novel insights requiring integration across levels and datasets. Photo of Vipera berus by Thor Håkonsen and used with permission.

Fig. 4.

The natural history specimen represents a largely untapped resource from which to study venom variation. In addition to phenotypic data that may be pulled from the organism itself, the preserved venom gland represents a source of venom that may be used in addressing questions with geographic or temporal themes. Photo of Sistrurus miliarius specimen taken by Owen M. Edwards and used with permission.

Past

The earliest experimental studies of snake venom, beginning in the seventeenth century, were largely exploratory. Redi (1664) confirmed venom toxicity using whole-organism assays, and Fontana (1781) examined potency, dose dependence, and physiological effects. These studies advanced understanding of venom effects but treated venom as a static substance rather than a dynamic trait shaped by ecology or evolution.

In the nineteenth and early twentieth centuries, methodological advances allowed more detailed characterization of venom composition. Researchers catalogued components, quantified relative abundances, and contextualized venom systems. Major toxin families were first described during this period: PLA₂s (“hemolysins”) in the 1940s (De 1944), SVSPs (“reptilase”) in the 1950s (Bruck and Salem 1954), 3FTxs in the 1960s (Chang and Lee 1963), and SVMPs in the 1990s (Bjarnason and Fox 1994).

As resolution improved, venoms were shown to vary within species. Individual-level variation (Jímenez-Porras 1964; Glenn and Straight 1977), population differences (Aragón and Gubenšek 1981), and geographic structuring of venom phenotypes (Glenn and Straight 1978; Glenn et al. 1983; Minton and Weinstein 1986) were documented. Hybridization and introgression were shown to reshape venom phenotypes using immunochemical and functional assays, lethality tests, electrophoresis, and HPLC (Glenn and Straight 1989, 1990).

Parallel work on venom resistance demonstrated that prey could evolve physiological mechanisms to neutralize venoms. Rodents (Perez et al. 1978a, 1978b; 1979; De Wit 1982; Poran et al. 1987), opossums (Kilmon 1976; Werner and Vick 1977), and other taxa (Bdolah et al. 1997; Heatwole et al. 1999) were shown to detoxify venom using whole-organism and inhibition-based assays, measuring median lethal doses and venom neutralization capacities of various species. Diet also emerged as a major driver of venom evolution (Daltry et al. 1996), reinforced by prey-specific susceptibility (Jorge da Silva and Aird 2001) and ontogenetic shifts in diet and venom composition (Mackessy 1988).

Despite methodological and research biases, such as taxonomic skews toward viperids, geographic skews for Neotropical taxa, and an absence of integrated ecological data (Avella et al. 2022b), these early studies established enduring themes in venom research—variation, adaptation, and ecological interaction—while leaving the mechanisms largely unresolved.

Present

Contemporary research has transformed venoms from descriptive biochemical curiosities into tractable model systems for studying eco-evolutionary dynamics. This shift reflects both conceptual advances and analytical innovations that allow venoms to be interrogated at unprecedented resolution.

A major inflection point came with mass spectrometry-based analyses and the rise of the venomics framework in the early 2000s (Juárez et al. 2004; Calvete et al. 2007; Gutiérrez et al. 2009). By integrating reversed-phase high-performance liquid chromatography, gel electrophoresis, and mass spectrometry, venomics enabled comprehensive quantification of entire venom proteomes, reframing venoms as integrated systems rather than incomplete toxin inventories. Advances in liquid chromatography–tandem mass spectrometry and bioinformatic pipelines facilitated peptide-based identification from crude venoms, popularizing shotgun proteomics (OmPraba et al. 2010; Tan et al. 2016; Kunalan et al. 2018; Koua et al. 2022). Combined with traditional techniques such as gel electrophoresis, toxicity assays, and enzymatic activity assays (Lüddecke et al. 2024), these methods now permit fine-scale comparisons of venom composition, abundance, and function. Sequencing technologies have produced species-specific omics databases, including venom gland transcriptomes and genomes, allowing venom systems to be studied at the level of gene expression and regulatory architecture (Pahari et al. 2007; Casewell et al. 2009; Walker et al. 2020; Slagboom et al. 2022; Avella et al. 2024, 2025).

These methodological advances have coincided with a shift toward explicitly ecological and evolutionary questions. Venom datasets are increasingly integrated with phylogenetic (Holding et al. 2021; Kazandjian et al. 2021), environmental (Sarangi et al. 2025), and geographic data (Strickland et al. 2018; Avella et al. 2023; Smith et al. 2023). Studies of venom resistance now explore community-level dynamics (Robinson et al. 2021; Balchan et al. 2024, 2025a) and underlying molecular mechanisms (Jansa and Voss 2011; Gibbs, et al. 2020)

Ontogenetic venom variation is routinely examined across life stages (Mackessy et al. 2018), being increasingly contextualized within ecological and environmental frameworks (Mackessy et al. 2003; Lakušić et al. 2025). Individual-level variation within populations is now recognized as biologically meaningful, influencing foraging and prey capture (Smiley-Walters et al. 2019; Rashmi et al. 2021). Growing attention to predator–prey interactions continues to generate novel hypotheses about venom evolution, driving the development of new experimental and analytical approaches (Tian et al. 2020; Holding et al. 2021).

Future

Despite methodological and conceptual advances, the study of venom variation remains ripe for expansion. Future work should prioritize explicit tests of selective pressures, moving beyond correlations to experimentally assess how diet, prey physiology, or environmental factors shape venom phenotypes. Integrating molecular, organismal, and ecological data remains critical, with functional assays, reciprocal prey tests, and field manipulations linking venom composition to ecological performance. Combining genomics, transcriptomics, proteomics, functional assays, and field ecology can connect genotype, phenotype, and fitness, revealing mechanisms driving venom diversification (Drukewitz and Von Reumont 2019; Calvete et al. 2021; Lüddecke et al. 2023). Data sharing and centralized platforms will further accelerate these efforts (Zancolli et al. 2024; Castoe et al. 2025; Mehr et al. 2026).

Expanding taxonomic coverage beyond well-studied or medically relevant groups—particularly colubrids and helodermatid lizards—will help distinguish general patterns from lineage-specific traits. Fine-scale variation, including individual, sex-specific, seasonal, and microgeographic differences, warrants closer attention due to potential ecological and fitness consequences. Incorporating coevolutionary dynamics, such as venom resistance in prey and predators, will clarify reciprocal selection pressures shaping venom evolution.

Finally, venom systems provide a lens to study responses to rapid environmental change. Museum collections hold hundreds of thousands of venomous snake specimens (Fig. 4) spanning the past two centuries. Although venom in these preserved glands is largely denatured, protein identification and quantification may still be possible (Esquerré et al. 2026), enabling investigations of temporal and geographic variation otherwise inaccessible.

Conclusions

Snake venoms are dynamic, multifunctional traits shaped by a network of ecological and evolutionary pressures. Variation occurs across multiple scales—geographic, individual, ontogenetic, seasonal, interspecific, and sex-specific—reflecting interactions among diet, prey resistance, hybridization, and convergent adaptation. While phylogeny provides a framework for broad toxin patterns, ecological forces often override evolutionary inertia, generating remarkable phenotypic diversity. Advances in proteomics, transcriptomics, functional assays, and integrative field studies now allow direct links between venom composition, ecological performance, and evolutionary outcomes.

As complex traits, snake venoms offer an exceptional system for studying eco-evolutionary processes. Integrating existing and emerging methods with explicit ecological and evolutionary questions promises deeper insights into venom phenotypes. Expanding taxonomic breadth, incorporating fine-scale and coevolutionary dynamics, and leveraging new data sources will be critical for uncovering the drivers of venom diversification. Ultimately, venoms illustrate how selection, constraint, and ecological context interact to shape phenotypes across time and space.

Author contributions

N.R.B. and I.A. conceived the manuscript and conceptual framework with input from S.P.M., G.O.U.W., and D.E., and N.R.B. designed and created the figures, with input from all other authors. All authors reviewed and contributed to the final version of the manuscript.

Funding

This work was supported by the Natural Sciences and Engineering Research Council of Canada Postgraduate Scholarship—Doctoral [587615–2024] to N.R.B., the Australian Research Council DECRA Fellowship [DE230100003] to D.E., and the Deutsche Forschungsgemeinschaft [545040837] to I.A.

Data availability

No new data were generated or analysed in support of this research.