Evolutionary mechanisms of intraspecies snake venom diversification: a systematic review of population-level genetic, regulatory, and functional evidence

1 Introduction

Snake venoms are complex adaptive traits shaped by evolutionary processes that influence prey immobilization, digestion, and defense. Venom phenotypes arise from coordinated action of multiple toxin families, and variations in venom composition can alter biological activity and ecological performance (Zancolli et al., 2019; Casewell et al., 2020). The evolution of venom systems therefore depends on mechanisms such as natural selection, gene duplication and loss, regulatory changes, and functional divergence acting on toxin genes and their expression (Calvete, 2013; Casewell et al., 2014). Evolutionary processes operate most directly at the population level, where selection, genetic drift, and gene flow shape phenotypic variation over ecological and contemporary evolutionary timescales (Aird et al., 2017; Margres et al., 2019). Therefore, population-level analyses are essential for understanding how venom traits evolve and diversify within species. Empirical studies have revealed substantial intraspecies venom variation associated with geography, prey composition, and population structure, indicating the possibility for different evolutionary trajectories to evolve within a single species (Daltry et al., 1996; Gibbs and Rossiter, 2008; Zancolli et al., 2019). These mechanisms include selection acting on toxin coding sequences, which can modify protein structure and function, and regulatory evolution, which can alter toxin abundance through gene expression changes. Structural variation such as gene duplication, loss, recombination, and copy number variation within toxin families further contributes to venom diversification. Studies in genetics and population genomics reveal that these mechanisms can operate simultaneously and with different relative importance across toxin families within the same species (Margres et al., 2017; Schield et al., 2022).

Venomics and molecular biology advancements have enabled increasingly detailed and quantitative characterization of venom phenotypes and have provided the empirical foundation for investigating these evolutionary mechanisms. Proteomic profiling through mass spectrometry has shown that venom composition is a structured and quantifiable phenotype, facilitating comparisons within and across different species (Calvete et al., 2007; Lomonte et al., 2014). Complementary transcriptomic analyses of venom glands have further revealed that regulation of gene expression is the major determinant of venom output, linking genomic variation to expressed venom phenotypes (Margres et al., 2014). Recently, an integrative study incorporating regulatory architecture and epigenomic features demonstrated that changes in venom composition across life stages can be driven by changes in gene regulation rather than coding sequence divergence alone, providing direct evidence that regulatory mechanisms contribute to intraspecies venom variation (Hogan et al., 2024). Despite the availability of population-focused venom proteomic, genomic, transcriptomic, and regulatory data, interpretation of the evolutionary mechanisms underlying venom diversification remains inconsistent. There are several studies that show variation in venoms without explicitly investigating whether the differences could be attributed to either adaptive evolution, neutral processes, or regulatory modulation, and analytical methods vary widely in their capacity to support robust evolutionary inference (Zancolli et al., 2019; Casewell et al., 2020; Schield et al., 2022). Differences in sampling design, population coverage, ontogeny representation, and methodological frameworks further limit direct comparison across studies (Casewell et al., 2020; Zancolli et al., 2019; Schield et al., 2022). Beyond evolutionary inference, population-level venom variation also presents significant translational implications, especially for the development of region-informed antivenoms and diagnostic strategies, which again contribute to the pressing need for robust mechanistic understanding of intraspecies venom diversification (Dingwoke et al., 2026).

Existing reviews of snake venom evolution and variation have synthesized evidence narratively and across broad taxonomic scales, providing valuable conceptual context but offering limited systematic evaluation of the evolutionary mechanisms operating within species (Tasoulis and Isbister, 2017; Casewell et al., 2020; Tan, 2022). Accordingly, empirical evidence on the evolutionary mechanisms driving intraspecies snake venom diversification remains fragmented and unevenly interpreted, and no systematic review has evaluated the strength of evidence supporting different mechanisms at the population level. This review addresses key questions concerning the mechanisms of intraspecies snake venom diversification and the consistency of the evolutionary evidence at the population level by synthesizing and classifying evidence according to evolutionary mechanisms and an inferential framework. To advance understanding of the evolutionary history of venoms at the population level, this review clarifies how venom evolves within species, evaluates the strength of evidence supporting proposed mechanisms, and provides a structured framework to guide future research in venom evolution biology. Figure 1 summarizes the population-level evolutionary pathways and mechanistic relationships reviewed here, integrating genomic, regulatory and functional processes that contribute to intraspecies snake venom diversity.

2 Methods

2.1 Review design and reporting standards

This study aimed to systematically review the empirical evidence on the evolutionary mechanisms driving intraspecies, population-level diversification of snake venom. It was developed and reported in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) 2020 statement (Page et al., 2021a). A review protocol was developed before the study to define the scope, eligibility criteria, and analytical framework.

2.2 Review questions

This systematic review was guided by a set of predefined review questions that structured study identification, data extraction, and synthesis of population-level evidence on snake venom evolution. The primary research question addressed in this review was: Which evolutionary mechanisms have been empirically demonstrated to drive the intraspecies, population-level diversification of snake venom, and how robust is the evidence supporting each mechanism? To address this overarching question, the following secondary questions were formulated: (a) Which evolutionary processes, including natural selection, gene duplication, and gene regulatory evolution, are most consistently supported by population-level studies of snake venom? (b) To what extent does venom diversification arise from coding sequence evolution compared to changes in gene expression and regulatory architecture? (c) How repeatable are population-level venom evolutionary patterns across geographically distinct populations within and among snake species? (d) What forms of evidence including genomic, transcriptomic, proteomic, and functional data provide the strongest support for the evolutionary mechanisms underlying venom variation within snake species? (e) Where do key methodological and inferential gaps persist that hinder robust inference in venom evolutionary biology? These guiding questions informed the eligibility assessment, data extraction, classification of evolutionary evidence, and synthesis of findings in this review.

2.3 Eligibility criteria

This review examined snake venom systems investigated at the intraspecies and population levels, including geographically distinct populations, subspecies complexes, and genetically differentiated populations within a species. Studies focusing exclusively on comparisons between different species, genera or higher taxonomic groups without explicit population-level analyses were excluded to maintain focus on evolutionary processes operating within species.

Studies were eligible for inclusion if they examined venom variation using genomic, population genetic, transcriptomic, proteomic, or functional data and provided explicit evolutionary interpretations. This criterion was applied to ensure that included studies contained analytically supported evolutionary inference rather than post hoc interpretation. Eligible genomic and population genetic studies included those assessing toxin level sequence variation, population structure and natural selection patterns. The review specifically considered evolutionary processes including natural selection acting on venom toxin genes, gene duplication and loss within toxin gene families, and regulatory and expression-level divergence interpreted as evolutionary changes. Transcriptomic and proteomic studies were included when expression-level divergence or compositional differences were analyzed within an evolutionary framework. Functional studies were eligible when experimental evidence demonstrated that population-level venom differences resulted in measurable biological effects relevant to venom function or ecological performance. Studies that reported venom variation descriptively without assessing, testing or inferring underlying evolutionary mechanisms were excluded. For the purposes of this study, explicit evolutionary interpretation referred to analyses that directly evaluated or tested predefined population-level evolutionary mechanisms, including natural selection, structural genomic variation, regulatory divergence, or functionally validated venom divergence. Consequently, studies primarily describing venom composition, genomic resources, phylogenetic relationships, or evolutionary history without directly evaluating these mechanisms were not eligible for inclusion, even when they contained relevant population-level biological data. This approach was adopted to minimize subjective reinterpretation of descriptive data and to maintain consistency in evaluating empirically supported evolutionary mechanisms. However, we acknowledge that this may exclude studies with relevant biological data that do not explicitly test evolutionary hypotheses. Overall, inclusion required clear empirical evidence demonstrating how evolutionary processes drive venom diversity at the population level.

2.4 Information sources and search strategy

A systematic literature search was conducted across PubMed, Web of Science, and Scopus to identify relevant population-level studies examining snake venom evolution. The initial searches were completed between 1 and 31 July 2025, and an updated search was subsequently conducted in August 2026 using the same databases and predefined eligibility criteria. Across the initial and updated searches, 286 records were identified and subsequently processed through the study selection workflow. The search strategy combined various controlled vocabulary terms, where applicable, with free text keywords related to snake venom, intraspecies and population-level variations, and evolutionary mechanisms.

The core search was executed using Boolean operators within the advanced search interfaces of each database, with strategies tailored to database-specific indexing and syntax requirements. Search terms were organized into three concept blocks: snake venom, population-level or within species variation, and evolutionary mechanisms. The primary search terms used were: (“snake venom” OR “venom evolution”) AND (“population structure” OR “population-level” OR “intraspecific variation”) AND (“natural selection” OR “gene duplication” OR “structural variation” OR “regulatory evolution” OR “gene expression” OR genomics OR transcriptomics OR proteomics). No restrictions were placed on year of publication; only studies published in English were retrieved. Following the initial search and study selection, data extraction was carried out between August 4 and 20, 2025 using a standardized extraction framework. It considered species identity, population scope, venom data type, analytical approach, evolutionary mechanisms, principal findings, and the strength of evolutionary inference. Formal synthesis and in-depth analysis of the evidence identified through the initial search were performed between August 25 and September 6, 2025, and the initial report was completed by September 30, 2025. An updated search was subsequently conducted in August 2026 using the same databases, search concepts, and predefined eligibility criteria. Records identified through the updated search were screened for eligibility, and eligible studies were extracted and incorporated into the final evidence synthesis before completion of the present manuscript. The search strategy was applied consistently across databases with minor syntax adaptations appropriate to each database.

2.5 Study selection

All records retrieved from the initial and updated database searches were imported into EndNote X9 reference management software, where duplicate records were identified and removed using the software’s automated duplicate detection function, followed by manual verification to ensure accuracy. Title and abstract screening and full-text eligibility assessment were performed independently by two authors, and any discrepancies were resolved through discussion and consensus. Full-text eligibility assessment was subsequently performed for records considered potentially relevant. The predefined eligibility criteria were applied consistently to records identified in both the initial and updated searches, including the requirement for population-level investigation of intraspecies venom variation and explicit evaluation or interpretation of an underlying evolutionary mechanism. Only primary population-level studies meeting all eligibility criteria were counted as included studies; studies identified during the updated search were not treated differently from those identified during the initial search. Background literature, review articles, and methodological references cited for contextual or conceptual purposes were not included in the PRISMA synthesis presented in Figure 2.

2.6 Data extraction

Data extraction was performed using a predefined and standardized data extraction framework developed before the study commenced to ensure consistent and systematic capture of information across all included studies. The framework consisted of a uniform set of extraction fields applied to each study, including species identity, population characteristics, geographic context, venom data type (genomic, transcriptomic, proteomic, or functional), evolutionary mechanisms examined, analytical approaches employed, and principal evolutionary findings. Each included study was reviewed in full, and relevant data were extracted according to these predefined fields to minimize subjective judgment and differences in data collection. The method facilitated systematic synthesis by enabling studies to be grouped and compared based on their common evolutionary mechanisms, data types and inferential approaches.

2.7 Classification of evolutionary evidence

Included studies were classified according to the strength of evolutionary inference supported by their data and analytical approaches. Classification was based on the type of evidence used to infer evolutionary mechanisms, rather than on study design alone. Studies were grouped into inferential categories ranging from descriptive reports of venom variation and correlational analyses linking venom variation to geographical and ecological factors, to investigations applying formal population genetic or statistical tests and those incorporating experimental or functional validation. Classification was guided by predefined criteria based on analytical approach and evidentiary strength. Evidence within eligible studies was categorized as descriptive when it documented venom variation without formal statistical or evolutionary testing, correlational when associations between venom variation and ecological or geographic factors were evaluated without direct testing of evolutionary mechanisms, population genetic when formal statistical or genomic tests of selection or population structure were applied, and functional validation when experimental assays demonstrated biological or ecological consequences of venom variation. Descriptive or correlational evidence was considered only when it occurred within studies that otherwise satisfied the predefined eligibility criteria and provided an explicit evolutionary interpretation. This classification system was applied consistently across all included studies and was used to structure the evidence synthesis and to evaluate the relative strength of support for different evolutionary mechanisms.

2.8 Qualitative methodological appraisal of included studies

A structured qualitative methodological appraisal was performed for all included studies to support interpretation of the overall evidence base. Given the heterogeneity in study designs, the appraisal considered predefined domains relevant to sampling adequacy, population representation, analytical approaches, evolutionary inference, and functional validation (Page et al., 2021b). The assessment considered key factors including sampling methods, population representation, analytical approaches, and the strength with which observed venom variation was linked to proposed evolutionary mechanisms. Additional domains included the adequacy of evolutionary inference methods and the presence or absence of functional validation. Rather than serving as a basis for study exclusion, this evaluation provided contextual insight into the reliability and interpretability of findings across studies applying diverse methodologies and data types. Consideration of methodological limitations supported balanced synthesis and cautioned the interpretation of evolutionary inferences in this review.

2.9 Data synthesis

The findings were synthesized using a narrative approach because the included studies varied significantly in taxa, study designs, data types, and analytical methods. The evidence was organized and interpreted based on the evolutionary mechanisms studied and the strength of evolutionary inference supported by each study. Meta-analysis was not performed because standardized outcome measures, comparable effect sizes, and sufficiently homogeneous study designs were not consistently reported in the literature. Narrative synthesis therefore provided the most appropriate framework for integrating evidence and comparing evolutionary mechanisms across diverse population-level investigations.

3 Results

This section synthesizes evidence on intraspecies snake venom diversification according to the strength of evolutionary inference supported by different study designs and analytical approaches. To provide a structured interpretative framework, results were organized into hierarchical categories reflecting increasing inferential strength, ranging from descriptive and correlational studies to population genetics analyses and functional validation, based on predefined classification criteria described in Section 2.7. Figure 3 summarizes this hierarchy of evidence and is used to guide interpretation of the results.

3.1 Overview of included studies

The literature search and study selection process identified a body of empirical studies that examined intraspecies snake venom variation at the population level with explicit evolutionary interpretation. Following removal of duplicate records and application of predefined eligibility criteria, a subset of studies was retained for qualitative synthesis. The included literature spanned multiple snake families and genera, with a predominance of studies on viperid species, particularly rattlesnakes, reflecting their ecological diversity and the availability of genomic and venom datasets. This distribution also reflects the current evidence base, where population-level studies integrating evolutionary inference are disproportionately concentrated in a limited number of well-characterized viperid systems, with comparatively fewer studies available for elapid species and other medically important snake lineages. Included studies investigated different geographic populations within species, subspecies complexes or genetically structured populations. Population sampling designs varied widely, ranging from comparisons of a limited number of regional populations to broad geographic surveys encompassing multiple populations across a species’ range. Several studies incorporated ecological and environmental context, including prey composition and habitat differences, while others focused primarily on genetic and molecular divergence among populations.

The included studies encompassed a range of data types and analytical approaches used to investigate intraspecies snake venom diversity. Genomic and population genetic studies examined toxin gene sequence variation, patterns of natural selection and structural differences within venom gene families. Transcriptomic investigations analyzed venom gland gene expression to assess regulatory divergence among populations and across life stages. Proteomic studies used mass spectrometry-based approaches to characterize venom composition and were often integrated with transcriptomic data to link gene expression patterns to expressed venom phenotypes. A subset of studies incorporated functional assessments of venom evolution, including toxicity assays and prey-specific bioassays, to evaluate the biological consequences of population-level venom variation. Analytical approaches varied across studies: some applied formal population genetic tests or statistical models to infer evolutionary mechanisms, whereas others relied on correlational analyses linking venom variation to geographic or ecological factors. Integrated multi-omics investigations combining genomic, transcriptomic and proteomic data provided high mechanistic resolution but were comparatively less frequent. The study selection process and inclusion outcomes are summarized in the PRISMA 2020 flow diagram shown in Figure 2, and reporting of the review adheres to the PRISMA 2020 checklist provided in the Supplementary Material. The diversity of study designs, data types and analytical frameworks informed the classification of evidence and underpinned evaluation of the relative strength of evidence for different evolutionary mechanisms.

Table 1 summarizes population-level studies included in this systematic review, detailing the studied species, population sampling scope, data types used, evolutionary mechanisms studied, analytical approaches applied, and the resulting strength of evolutionary inference. Overall, these studies provide an empirical foundation for subsequent synthesis, enabling comparison of evolutionary mechanisms and evaluation of the relative strength of evidence across population-level studies.

Table 1. Summary of population-level studies included in the systematic review.

The strength of evolutionary inference reflects the highest level of inference supported by each study’s data and analytical approaches, recognizing that individual studies may employ multiple data types and contribute evidence across more than one inferential category. Classification follows predefined criteria described in Section 2.7, based on analytical approach and level of empirical support. Studies are presented in chronological order by publication year.

Sampling effort varied substantially across studies and analytical components. Several early transcriptomic and proteomic investigations relied on small numbers of individuals, including one or a few snakes per population, whereas later population-genomic studies generally incorporated larger range-wide samples. Sample sizes also frequently differed across genomic, transcriptomic, proteomic and functional components within individual studies, with molecularly intensive assays often conducted on subsets of the broader population sample (Table 1).

3.2 Evidence for natural selection on venom toxin genes

Population-level genomic and population genetic studies provide direct evidence that natural selection contributes to intraspecies venom diversification through effects on toxin gene coding sequences. This has focused on viperids, particularly rattlesnakes, which are well characterized in terms of population structure and venom variation. Population genomic studies of venom genes in rattlesnakes have revealed heterogeneous selective pressures acting across toxin gene families. In the Taiwan habu, venom genes showed evidence of positive selection and relaxed selective constraint, while genetic drift also contributed to population-level venom variation (Aird et al., 2017). Whole-genome analyses of rattlesnake venom genes have further identified signatures of balancing selection and elevated recombination, indicating that different toxin loci can experience distinct evolutionary pressures (Schield et al., 2022). Subsequent population genomic studies have similarly shown that different toxin gene families can experience distinct selective regimes, with some loci exhibiting signatures of selection while others are more strongly constrained (Schield et al., 2022). Comparative analyses across sympatric pit viper species further demonstrated that the relative contribution of selection and neutral processes varies among species, with selection providing stronger evidence of adaptive venom divergence in some specialists and neutral differentiation contributing more substantially in a generalist species (Margres et al., 2019). More recently, integrated whole-genome population genomic and transcriptomic analyses of geographically differentiated populations of the red diamond rattlesnake demonstrated that venom evolution is shaped by the combined effects of adaptive and neutral evolutionary processes, with environmental, ecological, and demographic factors jointly contributing to population-level venom diversification (Hirst et al., 2024). Collectively, these findings indicate that natural selection acts heterogeneously across venom gene repertoires within species and frequently interacts with other evolutionary processes to shape venom diversity.

Population-level whole-genome analyses have further strengthened evidence for balancing selection contributing to the maintenance of venom gene diversity within species (Schield et al., 2022). Venom gene regions in rattlesnake genomes are enriched for signatures of long-term balancing selection and elevated recombination, indicating that multiple functional toxin variants are maintained within populations rather than driven to fixation. Complementing these findings, it was demonstrated that geographic variation in venom composition is associated with both biotic and abiotic selective pressures (Smith et al., 2023), providing ecological, genomic, and functional evidence that local environmental conditions contribute to adaptive venom diversification among populations. Evidence from other population-level studies also indicates that selection is not consistently detectable across all venom systems. In the sidewinder rattlesnake, for example, limited evidence of directional selection and population differentiation suggested that sequence and expression variation contributed relatively little to venom divergence among populations (Rautsaw et al., 2019). Similarly, geographic venom variation in the Taiwanese bamboo viper was not adequately explained by phylogenetic relationships, leaving natural selection and founder effects as alternative explanations (Creer et al., 2003). Although evidence for population-specific balancing selection on individual toxin genes remains limited to a small number of studies meeting stringent population genetic criteria, the available evidence supports a role for natural selection in shaping intraspecies venom diversification. The form and intensity of selection vary across toxin families, populations, and ecological contexts, with evidence for positive, purifying, and balancing selection depending on functional constraints and environmental conditions. The strongest inferential support comes from studies applying formal population genetic models or integrating population genomic analyses with ecological and functional evidence, whereas studies based primarily on partial genetic divergence provide more limited evidence for evolutionary mechanisms.

3.3 Gene duplication, loss, and structural evolution of venom genes

Population-level genomic analyses demonstrate that diversification among populations arises not only from sequence variation but also from structural variation within the venom toxin gene repertoire (Nachtigall et al., 2025; Rokyta et al., 2015; Schield et al., 2022). Gene duplication, copy number variation, and recombination contribute to differences in venom gene family composition and potential functional capacity among populations, and can operate alongside selection on individual coding sequences (Figure 1). Detailed genomic characterization of the venom loci in the eastern diamondback rattlesnake revealed extensive duplication within major toxin gene families, with multiple paralogous copies present within individual genomes and variable representation across populations (Nachtigall et al., 2025; Schield et al., 2022). These findings indicate that venom gene families possess complex genomic architectures, including tandemly arrayed paralogs, and that such duplication events reflect standing structural variation within species rather than fixed species-level differences. Subsequent integrative genomics and transcriptomics analyses in the timber rattlesnake further showed that variation in venom composition among populations reflects differences in the presence, absence, and relative abundance of toxin gene copies within the genome (Rokyta et al., 2015). Significantly, these findings highlighted that structural differences in the venom gene repertoire can influence expressed venom phenotypes by determining which toxin genes are available for transcription, thereby shaping venom composition before any effects of regulatory divergence or sequence-level evolution.

Whole-genome population analyses further showed that venom gene regions are characterized by elevated structural complexity, including high levels of recombination and genomic rearrangement, consistent with ongoing duplication and turnover within toxin gene families (Schield et al., 2022). Similarly, it was demonstrated that integrating chromosome-level genomic resources with population genomic and transcriptomic analyses can resolve how structural genomic organization, together with demographic history and ecological factors, contributes to population-level venom diversification (Hirst et al., 2024). More recently, chromosome-level population genomic analyses identified a segregating structural variant associated with distinct venom phenotypes in geographically structured populations of the eastern diamondback rattlesnake, providing direct evidence that structural genomic variation contributes to population-level diversification (Nachtigall et al., 2025). Although these studies did not uniformly quantify gene duplication or loss across all toxin families, they collectively demonstrated that venom gene repertoires are dynamic genomic regions in which duplication, recombination, and structural reorganization generate population-level variation in gene content. These findings indicate that structural genomic variation provides an important substrate for venom diversification, while its evolutionary effects are shaped by interactions with regulatory, ecological, and demographic processes.

3.4 Regulatory evolution and expression-level divergence

Population-level transcriptomic studies provide evidence that intraspecies venom diversification can arise through regulatory evolution, manifested as differences in gene expression rather than alterations in toxin gene coding sequences (Margres et al., 2015, 2017; Rautsaw et al., 2019). These studies showed that variation in venom composition among populations and life stages is primarily driven by differential transcription of toxin genes that are otherwise highly conserved at the coding sequence level. Population-level analyses of venom expression in rattlesnakes demonstrated that regulatory divergence can occur among geographically differentiated populations, with differences in toxin gene expression contributing to population-level venom phenotypes (Margres et al., 2015). Comparative venom gland transcriptomics of geographically differentiated Naja kaouthia populations likewise identified population-level differences in toxin transcript profiles, demonstrating that expression divergence contributes to geographic venom variation in an elapid species (Tan et al., 2017). Consistent with these observations, transcriptomic and population genomics data were integrated to demonstrate that regulatory differences should be interpreted alongside genomic, ecological, and demographic processes when evaluating the mechanisms underlying population-level venom diversification (Hirst et al., 2024). In this study, differences in venom composition among populations were explained primarily by changes in relative toxin abundance driven by changes in gene expression, rather than by the presence of unique toxin genes. These findings provide evidence that regulatory mechanisms play a central role in shaping venom phenotype. In Bothrops atrox, comparative transcriptomic and proteomic analyses of genetically differentiated populations further identified substantial variation among toxin paralogues and individuals, with expression differences contributing to the observed venom phenotypes (Amazonas et al., 2018).

Population-level analyses further showed that regulatory evolution can proceed at different rates and along different trajectories, indicating that venom expression divergence is neither temporally uniform nor constrained to a single evolutionary mode (Margres et al., 2015). Subsequent population-focused analyses further demonstrated that regulatory divergence can produce distinct venom phenotypes within species, even in the absence of strong genetic differentiation among populations. Transcriptomic profiling across geographically separated populations revealed coordinated changes in the expression of multiple toxin families, consistent with regulatory evolution acting on the venom gland transcriptional program rather than on individual toxin genes alone (Margres et al., 2017). Strong evidence that adaptive venom divergence can occur predominantly through expression differentiation, with little corresponding toxin coding sequence divergence, was provided by population-level genomic and transcriptomic analyses (Margres et al., 2017). However, population-level expression divergence is not consistently pronounced across species; analyses of sidewinder rattlesnakes detected limited contributions of sequence and gene expression variation to population-level venom diversity (Rautsaw et al., 2019). In contrast, studies of ontogenetic venom variation have shown that age-related changes in venom composition can involve differential expression of specific toxin transcripts and produce functional differences in venom activity (Rokyta et al., 2017). More recent integrative work has linked such age-related changes to regulatory architecture and epigenomic state rather than coding sequence divergence alone (Hogan et al., 2024). Together, these studies provide useful mechanistic context for understanding regulatory processes underlying venom variation across life stages but do not constitute population-level evidence under the eligibility criteria of the present review. In contrast to these ontogenetic patterns, comparison of island and mainland populations of the eastern diamondback rattlesnake showed population differences in venom expression and ontogenetic trajectories, with evidence consistent with rapid local adaptation involving regulatory changes (Margres et al., 2026).

This study demonstrated that ontogenetic venom variability arises from dynamic changes in toxin gene expression mediated by regulatory architecture and epigenomic contexts, establishing regulatory evolution as a mechanistic contributor to intraspecies venom diversification. Collectively, these studies support regulatory evolution through differential gene expression and regulatory architecture as an important and recurrent contributor to venom phenotype diversification within some snake species and populations. Regulatory divergence and natural selection on toxin genes are often interconnected processes, as regulatory changes may be shaped by selective pressures and can interact with coding sequence evolution to influence venom phenotypes. Strength of inference is highest in studies integrating transcriptomic data with genomic context or regulatory information, whereas studies relying only on expression profiling without population-level replication offer more limited mechanistic resolution.

3.5 Functional consequences of intraspecies venom evolution

A smaller subset of population-level studies directly evaluated whether intraspecies venom variation translates into functional differences in biological activity, moving beyond molecular or compositional divergence to test the effects on toxicity, prey specificity, and other functional outcomes relevant to ecological performance (Daltry et al., 1996; Skejić and Hodgson, 2013; Holding et al., 2016; Smiley-Walters et al., 2017; Margres et al., 2016; Smith et al., 2023). Functional evidence for population-level venom divergence was demonstrated through experimental analyses of venoms from geographically distinct populations of the Malayan pit viper. These experiments showed that differences in venom constituents were associated with variation in toxicity towards prey species, providing evidence that venom divergence within a single species can have measurable biological consequences (Daltry et al., 1996). Population divergence in venom bioactivity was also demonstrated in the eastern brown snake, where geographically distinct populations differed in venom procoagulant activity, linking population-level venom variation to differences in a measurable biological function (Skejić and Hodgson, 2013). Further resolution of population-level venom divergence emerged from integrative genomic and venom phenotyping studies. In the Mojave rattlesnake, geographic variation in venom composition was associated with structural genomic variation and environmental factors, providing evidence that population-level venom divergence can arise through interactions between genomic architecture and ecological conditions (Zancolli et al., 2019). Reciprocal assays of pygmy rattlesnake venoms and treefrog prey further provided evidence consistent with local adaptation, as the effects of venom depended on the geographic origin of both predator and prey (Smiley-Walters et al., 2017). Experimental evidence from the rattlesnake–squirrel system likewise showed population-level variation in venom function in relation to prey resistance, supporting the role of reciprocal venom–prey interactions in shaping venom phenotypes (Holding et al., 2016). More recently, venomics, population genomics, ecological analyses, and functional assays were integrated to demonstrate that adaptive venom diversification reflects the combined influence of ecological context and evolutionary processes (Smith et al., 2023).

Direct functional validation of expression-driven venom divergence within species was provided by experimental analyses demonstrating that population-level differences in venom gene expression translate into measurable differences in toxic activities (Margres et al., 2016). In this study, venoms from geographically distinct populations exhibited divergent toxic effects despite broadly similar toxin repertoires, confirming that regulatory divergence can produce functionally distinct venom phenotypes with ecological and biological consequences. However, direct functional validation remains limited to a relatively small number of population-level investigations, as many studies document venom variation without accompanying experimental assays. This limitation reflects practical and logistical challenges associated with conducting functional studies, including the need for controlled experimental systems, access to relevant prey or model organisms, and the complexity of linking venom variation to ecologically meaningful biological outcomes.

Future studies could address this gap by combining controlled venom activity assays with ecologically relevant experimental systems. Functional validation should not be restricted to lethality measures such as LD₅₀, particularly when the evolutionary question concerns prey capture, immobilization, or other ecologically relevant venom effects. Where appropriate, reciprocal assays using venom and prey from different geographic populations can provide more ecologically relevant evidence of local adaptation by testing whether venom effects depend on the geographic origin of both predator and prey, as demonstrated in pygmy rattlesnakes (Smiley-Walters et al., 2017). Similarly, assays incorporating prey resistance can provide complementary evidence for reciprocal venom–prey interactions, as demonstrated in the Northern Pacific rattlesnake–California ground squirrel system (Holding et al., 2016). Other controlled assays targeting relevant physiological or biochemical activities may also provide functional evidence when appropriate prey species are unavailable or impractical to use. Importantly, the choice of functional assay should be guided by the biological and ecological question being tested rather than by a single standardized endpoint. As such, the strongest evidence for functional consequences of venom evolution within species derives from studies that explicitly integrate population-level venom characterization with experimental testing of biological activities. Recent integrative studies combining functional assays with genomic and ecological analyses further strengthen this evidence by demonstrating that adaptive venom diversification is influenced by interactions between environmental variation, prey ecology, and population-level evolutionary processes (Smith et al., 2023).

3.6 Repeatability and parallelism in population-level venom evolution

Population-level studies examining multiple populations within species have produced mixed evidence regarding the repeatability of venom evolutionary trajectories. Rather than revealing a single pattern of venom evolution, these studies show that similar ecological pressures can lead to either parallel or divergent venom phenotypes, depending on the population history, ecological context, and the underlying genetic architecture (Zancolli et al., 2019; Strickland et al., 2018). Evidence for repeatable patterns of venom evolution has emerged from population-level functional and ecological analyses in the Malayan pit viper. Populations occupying similar dietary niches exhibited comparable venom toxicity profiles despite geographical separation, indicating that parallel ecological pressures can drive similar functional outcomes within species (Daltry et al., 1996). Consistent with this observation, it was shown that geographic variation in venom composition reflects the combined influence of biotic and abiotic selective pressures, indicating that similar ecological drivers can repeatedly shape venom phenotypes while also generating population-specific adaptive responses under different environmental conditions (Smith et al., 2023). Similarly, analyses of northern Pacific rattlesnake populations showed that venom divergence was associated with both genetic distance and local prey community composition, indicating that ecological and population history can jointly influence the extent and direction of venom differentiation among populations (Holding et al., 2018). Reciprocal functional tests in pygmy rattlesnakes also provided evidence consistent with local adaptation, with venom effects differing according to the geographic origin of predator and prey, although the pattern varied among measured functional outcomes (Smiley-Walters et al., 2017).

In contrast to these parallel patterns, population-level genomic and venomic analyses also revealed pronounced divergence among the populations existing under broadly similar environments. For instance, in a widely distributed rattlesnake species, venom phenotypes followed distinct evolutionary trajectories across populations, with differences in composition and toxicity reflecting historical contingency and genomic architecture rather than uniform ecological matching (Zancolli et al., 2019). Further evidence for non-uniform evolutionary outcomes was provided by population-level genomic analyses of Mojave rattlesnakes, where geographically structured populations exhibited distinct venom phenotypes associated with local selective pressures (Strickland et al., 2018). Conversely, analyses of sidewinder rattlesnake populations detected limited evidence of directional selection and population differentiation in venom sequence and expression, illustrating that substantial venom variation need not produce consistent divergence among populations (Rautsaw et al., 2019). These results demonstrated that venom evolution within species can follow markedly divergent paths across populations, even under broadly comparable ecological conditions. Similarly, it was shown that although ecological factors consistently influence venom diversification, the relative contributions of biotic and abiotic factors vary among populations, reinforcing that venom evolution reflects both repeatable responses to selection and context-dependent evolutionary trajectories (Smith et al., 2023). These studies indicate that population-level venom evolution reflects a balance between predictability and contingency. While parallel ecological pressures, such as diet, can promote repeated outcomes in some systems, venom evolutionary trajectories are not universally repeatable across populations. Historical population structure, genomic architecture, and the organization of venom gene repertoires modulate the extent to which parallel evolution occurs within species.

3.7 Methodological and inferential gaps in the evidence base

Across the included literature, several recurring methodological and inferential limitations hindered robust interpretation of the evolutionary mechanisms underlying intraspecies venom diversity. The qualitative methodological appraisal indicated that the most common limitations across studies were limited population sampling, absence of formal evolutionary testing, and lack of functional validation. An important limitation is the taxonomic concentration of available studies, with a predominance of viperid species, particularly rattlesnakes, and limited representation of elapid species and other snake lineages from major biodiversity regions. A prominent gap was uneven population sampling, with many studies examining a limited number of populations or individuals within a population, thereby limiting the power to distinguish selection from neutral processes or historical contingency. Population genomic studies that achieved broader geographic sampling demonstrated substantial inferential resolution, highlighting the importance of dense population coverage for testing evolutionary hypotheses (Schield et al., 2022). In addition, the requirement for explicit evaluation of predefined population-level evolutionary mechanisms as an inclusion criterion may have led to exclusion of otherwise relevant descriptive, phylogenomic, or resource-generation studies that did not directly test those mechanisms. Another important limitation was the frequent reliance on descriptive venom variation without formal evolutionary tests. Several studies documented geographic or ecological differences in venom composition but did not apply population genetic models, explicit tests of selection, or replicated statistical frameworks capable of discriminating adaptive evolution from drift or regulatory modulation (Daltry et al., 1996; Zancolli et al., 2019). In contrast, studies that integrated population genetic or genome-wide data provided clearer mechanistic inference (Aird et al., 2017; Rautsaw et al., 2019; Schield et al., 2022; Hirst et al., 2024).

Limited integration across data types has also restricted inference in many studies. Investigations based solely on proteomic or transcriptomic profiles often lacked the genomic context necessary to determine whether observed patterns reflected structural genomic differences, regulatory evolution, or posttranscriptional processes. Integrated multi-omics studies combining genomic, transcriptomic, and proteomic data provided more robust insights into the mechanisms generating venom diversity but remain relatively rare in the population-level literature (Margres et al., 2015, 2017). Notably, studies that explicitly integrated genomic context and transcriptomic data provided substantially stronger evolutionary inference than descriptive or single omics approaches, highlighting the importance of regulatory-aware analytical frameworks (Margres et al., 2017). However, expression divergence without corresponding coding sequence divergence does not by itself establish adaptive causation, and stronger inference requires integration with population genetic, ecological, or functional evidence. Importantly, not all population-level transcriptomic studies detected strong regulatory divergence, as comparative analyses of multiple populations of the sidewinder rattlesnake revealed limited contributions of sequence and gene expression variation to venom diversity, indicating that expression divergence is not a universal outcome of population-level venom evolution (Rautsaw et al., 2019).

Functional validation represented a further bottleneck. Although several population-level studies demonstrated venom divergence at the molecular or compositional level, only a subset incorporated experimental assays to test biological activity, prey specificity, or ecological performance (Daltry et al., 1996; Skejić and Hodgson, 2013; Holding et al., 2016; Smiley-Walters et al., 2017; Margres et al., 2016; Smith et al., 2023). As a result, functional consequences of venom evolution within species remain underrepresented relative to molecular evidence, limiting the ability to draw strong conclusions about adaptive significance. Finally, emerging regulatory and epigenomic mechanisms remain sparsely explored at the population level. Recent integrative work has demonstrated that regulatory architecture and epigenomic state can contribute to venom variation across life stages (Hogan et al., 2024), but comparable analyses across geographically differentiated populations remain limited. This distinction highlights an important gap between evidence for regulatory mechanisms operating during ontogeny and evidence for population-level regulatory divergence. Collectively, these gaps indicate that while substantial progress has been made in documenting intraspecies venom variation, robust inference of evolutionary mechanisms remains uneven across the field and is not consistently supported across studies. Addressing these limitations may require broader population sampling, explicit evolutionary testing frameworks, increased integration of multi-omics data, and expanded use of functional assays to link molecular variation with biological performance. Table 2 synthesizes evidence across studies by summarizing the evolutionary mechanisms implicated in intraspecies venom diversification, the data types and analytical approaches supporting each mechanism, and the highest level of evolutionary inference achieved. This synthesis highlights both well supported mechanisms and areas where evidence remains limited or primarily descriptive.

Table 2. Strength of evidence supporting evolutionary mechanisms of intraspecies venom diversification.

Highest inferential strength reflects the strongest level of evidence demonstrated across population-level studies for each mechanism. Lack of consistent functional validation indicates that experimental testing of biological consequences has not been consistently performed, rather than evidence against functional relevance.

3.8 Towards a conceptual computational framework for integrative evidence synthesis

The evidence synthesized in this review highlights a recurring methodological challenge in population-level venom research. Although genomic, transcriptomic, proteomic, functional, ecological, and geographic studies provide valuable insights, they are often analyzed in isolation and with varying inferential strength. Differences in sampling design, analytical rigor, and methodological frameworks limit cross-study comparability (Section 3.7). This makes it difficult to identify broader evolutionary patterns or to evaluate how reliably different mechanisms are supported across populations. Building on the evidence synthesized in this review, a structured and integrative approach may help integrate mechanisms, data types, and evidentiary strength across studies. Computational and artificial intelligence (AI) methods offer supporting tools suited to this integrative aim. These approaches are well adapted to high-dimensional, multimodal, and non-linear biological datasets such as those generated in modern venom studies, although their effectiveness depends on the availability of sufficiently large and well-integrated datasets. These approaches do not replace evolutionary inference, but support it by synthesizing evidence, identifying recurring patterns, and highlighting mechanistic hypotheses for explicit testing (Hasin et al., 2017; Kitano, 2002).

A key application involves integrating multi-omics datasets across populations. Population-level studies increasingly generate matched genomic, transcriptomic, and proteomic data, yet interpretation often remains modality-specific. Non-causal computational methods such as clustering, dimensionality reduction, and network inference can detect combinations of structural genomic variation, regulatory divergence, and expression-level shifts that co-occur across populations. However, meaningful cross-study integration requires prior harmonization of data types, measurement scales, feature definitions, and analytical outputs to minimize variation introduced by differences in experimental and analytical methodologies. Where direct harmonization is not possible, analyses should be restricted to methodologically comparable datasets rather than treating heterogeneous measurements as directly equivalent. Such analysis may clarify when regulatory divergence operates independently of gene duplication or when structural variation constrains expression evolution, patterns that are currently inferred qualitatively across studies. Computational approaches also enable systematic examination of repeatability and contingency in venom evolution. By analyzing venom phenotypes, toxin gene architectures, ecological variables, and functional outcomes jointly, integrative computational frameworks may identify whether populations converge on similar venom phenotypes through shared mechanistic routes or reach comparable functional outcomes via distinct evolutionary trajectories. These analyses do not attribute adaptive causality but can guide targeted sampling and prioritize mechanisms for explicit testing. A further contribution lies in evaluating inferential strength across the literature. As highlighted in Figure 3 and Table 2, studies differ widely in evidentiary power. Computational frameworks can encode study attributes such as data type, analytical approach, sampling design, and the presence of functional validation. This enables structured comparison of evidence quality across mechanisms and populations and reduces the risk of misinterpreting descriptive variation as adaptive inference. Potential applications include: (a) systematic gap analyses identifying mechanism–data pairings with strong versus limited support; (b) pattern recognition in evolutionary routes using clustering and association detection, and (c) guidance for study design by identifying underexamined mechanisms, populations, and data types. A conceptual computational framework for integrating heterogeneous data streams in population-level venom evolution studies is depicted in Figure 4.

This framework organizes population-level venom research into five interconnected layers. Layer 1 encodes data modalities used in venom research (genomic, population genetic, transcriptomic, proteomic, functional, ecological and geographic). Layer 2 maps each study to one or more evolutionary mechanisms or biological processes (coding sequence evolution, gene duplication and copy number variation, structural genomic reorganization, regulatory and expression divergence, and, where relevant, ontogenetic regulatory shifts). Functional divergence is treated as an outcome of these processes rather than as an evolutionary mechanism. Layer 3 encodes inferential strength (descriptive, correlational, population genetic testing, functional validation), following the hierarchy established in this review (Figure 3). Layer 4 applies computational and AI methods to integrate information from Layers 1–3 and identify patterns such as recurrent mechanism combinations or trajectories of venom evolution. Layer 5 highlights hypotheses, mechanisms lacking functional validation, underrepresented populations, and key knowledge gaps, thereby informing future population-level venom research. Such integrative approaches may be particularly useful for identifying clinically relevant venom variation and supporting region-specific antivenom strategies in snakebite-endemic settings. This framework is a prospective conceptual and assistive framework for evidence synthesis and hypothesis generation. Its purpose is evidence synthesis and hypothesis generation, not autonomous evolutionary inference. However, its applicability is constrained by the limited number of studies included in this review and the heterogeneity of available data types, study designs, and taxonomic representation. As such, patterns identified through such integrative approaches should be interpreted cautiously and viewed as hypothesis-generating rather than confirmatory, requiring validation through expanded datasets and targeted empirical studies. Successful implementation may require collaboration among evolutionary biologists, bioinformaticians, and computational scientists, alongside careful attention to interpretability and biological plausibility. As population-level venom datasets continue to grow, carefully applied integrative frameworks may help synthesize evidence, guide research prioritization, and support a more unified mechanistic understanding of venom evolution within species.

4 Discussion

4.1 An integrated view of evolutionary mechanisms driving intraspecies venom diversification

This systematic review synthesizes population-level evidence indicating that intraspecies snake venom diversification arises through multiple evolutionary mechanisms operating at different biological levels, including coding sequence evolution, structural genomic evolution, and regulatory divergence, with functional studies providing evidence for the biological consequences of these processes, consistent with broader conceptual frameworks of venom evolution described in the literature (Fry, 2005; Casewell et al., 2020). However, interpretation of these patterns is considered in the context of the available evidence base, which is disproportionately derived from a limited number of well-studied viperid systems. Instead, the relative contribution of each mechanism varies across snake taxa, toxin families, population histories, and ecological contexts. As summarized in Table 2, robust inference is strongest where population genetic or genomic data are integrated with transcriptomic, ecological, and functional analyses, whereas studies relying on descriptive or correlational evidence alone provide more limited mechanistic resolution. This integrated view reinforces the importance of population-level analyses for distinguishing adaptive evolutionary processes from neutral variation or regulatory modulation in venom systems. To help address this challenge, we outlined a computational framework (Section 3.8, Figure 4) that integrates heterogeneous data types and levels of inferential strength across studies, thereby supporting comparative analysis and structured hypothesis prioritization.

4.2 Regulatory divergence and coding sequence evolution represent the two most consistently supported mechanisms

Across the population-level literature reviewed in this study, natural selection acting on toxin gene coding sequences and regulatory divergence manifested as differential gene expression emerged as recurrently supported contributors to intraspecies venom diversification. These mechanisms are not independent processes; rather, they can operate in a coordinated and interdependent manner, with selection acting on both coding sequences and regulatory elements, and with regulatory changes influencing the functional effects of genetic variation (Talavera et al., 2025). Population genetic and genomic studies of rattlesnakes provide evidence that selection acts heterogeneously on venom toxin genes, with different toxin families exhibiting signatures of positive, purifying, or balancing selection across evolutionary contexts (Rokyta et al., 2015; Schield et al., 2022). Population genomic analysis of the Taiwan habu similarly identified heterogeneous evolutionary pressures among venom genes, including evidence of positive selection and relaxed selective constraint, while also indicating contributions from genetic drift (Aird et al., 2017). These findings demonstrate that coding sequence evolution contributes to venom diversification within species, but that its effects are neither uniform across the venom repertoires nor consistent across populations. Moreover, comparative analyses across pit viper species indicate that the relative contribution of selection and neutral processes can vary among species and populations (Margres et al., 2019).

Simultaneously, population-level transcriptomics and integrated multi-omics studies indicate that regulatory evolution frequently contributes to venom divergence among populations (Margres et al., 2015, 2017). Comparative venom gland transcriptomics in Naja kaouthia identified population-level differences in toxin transcript profiles between geographically differentiated populations, providing additional evidence that expression divergence contributes to venom variation beyond viperid systems (Tan et al., 2017). Similarly, transcriptomic and proteomic analyses of genetically differentiated Bothrops atrox populations identified variation in toxin paralogues and expression patterns, supporting a contribution of regulatory processes to population-level venom variation (Amazonas et al., 2018). Recent integrative population genomic and transcriptomic analyses further suggest that regulatory divergence should be interpreted alongside genomic architecture, demographic history, and ecological processes when evaluating population-level venom diversification (Hirst et al., 2024). In several well-characterized systems, regulatory divergence has been shown to produce distinct venom phenotypes despite minimal coding sequence differentiation, providing direct evidence that expression-level evolution can contribute substantially to venom divergence without extensive coding sequence change. However, regulatory and sequence divergence are not consistently pronounced across species; for example, sidewinder rattlesnake populations showed limited contributions of sequence and gene expression variation to overall venom diversity (Rautsaw et al., 2019). While these findings do not imply that regulatory mechanisms dominate venom evolution in all species, they collectively support regulatory divergence as a recurrent and empirically supported pathway for population-level venom diversification.

4.3 Structural genomic variation provides evolutionary potential but limited direct functional inference

At the population level, structural genomic processes including gene duplication, copy number variation, recombination, and broader reorganization of venom loci have been documented as contributors to variation in venom gene repertoires (Rokyta et al., 2015; Schield et al., 2022; Nachtigall et al., 2025). Genomic analyses of viperid snakes have documented duplication and turnover within major toxin gene families, with these structural features associated with differences in gene content and genomic architecture among populations (Rokyta et al., 2015; Schield et al., 2022; Nachtigall et al., 2025). Recent evidence further demonstrates that segregating structural genomic variants can be associated with distinct venom phenotypes among populations, strengthening the evidence that structural genomic evolution contributes directly to population-level venom diversification (Nachtigall et al., 2025). These structural features provide a substrate for venom diversification by influencing which toxin genes are available for transcription and subsequent expression. Nevertheless, despite clear evidence for structural genomic complexity, direct links between gene duplication or copy number variation and population-level functional outcomes remain comparatively limited. Most studies on structural variation infer its evolutionary significance indirectly, without experimental validation of its effects on venom activity or ecological performance. As presented in Table 2, the highest inferential strength achieved for structural genomic mechanisms is typically population genetic testing, whereas direct experimental functional validation remains limited. This does not negate the importance of structural genomic evolution in venom systems; instead, it emphasizes the gap between genomic characterization and functional interpretation that constrains strong adaptive inference.

4.4 Functional validation remains the strongest but least common line of evidence

Experimental studies that directly assess the biological consequences of population-level divergence provide strong evidence that venom variation has measurable biological effects within species. Functional assays linking population-level venom variation to differences in biological activity or prey-related effects demonstrate that venom diversification can translate into measurable functional consequences (Daltry et al., 1996; Margres et al., 2016; Skejić and Hodgson, 2013; Holding et al., 2016; Smiley-Walters et al., 2017; Smith et al., 2023). Additional population-level functional studies have demonstrated divergence in venom bioactivity and prey-related effects, including differences in procoagulant activity among populations of Pseudonaja textilis (Skejić and Hodgson, 2013), population-level variation in venom function in relation to prey resistance (Holding et al., 2016), and reciprocal venom–prey effects consistent with local adaptation in pygmy rattlesnakes (Smiley-Walters et al., 2017). These studies demonstrate that population-level venom divergence at the molecular or compositional level can be associated with measurable differences in biological activity or prey-related performance, underscoring the limitations of compositional measurements alone for inferring biological effects. Integration of ecological, genomic, and functional evidence can further strengthen inference regarding the evolutionary processes associated with population-level venom diversification, although functional evidence alone does not establish adaptive causation (Smith et al., 2023). Despite their inferential strength, functional validation studies remain relatively scarce compared to genomics, transcriptomics, and proteomics investigations, in part due to the practical and experimental complexities involved in linking venom variation to biological and ecological outcomes. As a result, functional consequences of venom diversity are likely underrepresented in the literature relative to molecular evidence. Where functional assays have been integrated with expression or genomic data, they provide stronger evidence that regulatory and genetic divergence can be associated with ecologically relevant venom phenotypes (Margres et al., 2016). Expanding such integrative approaches may be essential for strengthening inferences regarding adaptive venom evolution at the population level.

5 Implications for future population-level venom research

The synthesis presented in this review highlights several priorities for advancing understanding of intraspecies venom evolution. First, broader and more taxonomically and geographically balanced sampling is needed to distinguish selection from drift and to assess the repeatability of venom evolutionary trajectories across geographic contexts. Sampling depth also remains an important limitation of the evidence base. Several studies inferred population-level venom divergence from relatively small numbers of individuals, while multi-omic investigations frequently applied transcriptomic or proteomic analyses to subsets of larger population samples. Future studies should therefore prioritize greater within-population replication, broader geographic coverage and, where feasible, matched genomic, transcriptomic, proteomic and functional measurements from the same individuals. Balanced sampling across populations, sexes, age classes and relevant ecological conditions would further reduce potential confounding and improve the ability to distinguish population-level divergence from individual or ontogenetic variation. Second, explicit evolutionary testing frameworks, including population genetic models and rigorous hypothesis testing, should be applied more consistently to avoid overinterpretation of descriptive venom variation. Third, greater integration of genomic, transcriptomic, ecological, and regulatory data is required to resolve the relative contributions of structural and regulatory mechanisms to venom diversification. Finally, increased incorporation of functional assays is vital for linking molecular divergence to biological performance and, when integrated with evolutionary and ecological evidence, evaluating potential adaptive significance.

In addition to strengthening evolutionary inference, improved resolution of population-level venom diversity has practical implications for regionally informed antivenom development and diagnostic strategies by clarifying the biological sources of venom variation (Dingwoke et al., 2026). Beyond these empirical priorities, integrative synthesis frameworks (such as the computational model outlined in Section 3.8) may also be useful in mapping evidence landscapes and identifying robust cross-study patterns. Addressing these priorities may help to enable more robust and mechanistically grounded inference of the evolutionary processes shaping snake venom diversity within species.

6 Conclusion

This systematic review synthesizes population-level evidence indicating that intraspecies venom diversification involves multiple interacting evolutionary mechanisms rather than a single universal pathway. These mechanisms include natural selection acting on toxin coding sequences, structural evolution of venom gene repertoires, and regulatory divergence in gene expression, with functional studies providing evidence for the biological consequences of this molecular and evolutionary variation. Regulatory divergence and heterogeneous selective pressures on toxin genes emerge as recurrently supported mechanisms at the population level, while structural genomic processes provide important evolutionary potential but are less frequently linked to functional outcomes. The strongest support for adaptive evolutionary interpretations arises from studies that integrate population genomics, transcriptomics, ecological evidence, and functional assays, although such integrative approaches remain comparatively rare. Many investigations document substantial population-level venom variation but rely on descriptive or correlational frameworks, limiting robust inference regarding evolutionary mechanisms. Consequently, functional validation and explicit evolutionary testing remain critical gaps in the current evidence base. By systematically evaluating the strength of evidence supporting different evolutionary mechanisms, this review clarifies which processes are well supported and where inference remains limited. Addressing these gaps may require broader population sampling, greater integration of multi-omics data and increased use of experimental assays, and careful integration and synthesis of heterogeneous evidence. Advancing such approaches may not only deepen understanding of venom evolutionary biology but also inform translational applications, including regionally informed antivenom development and diagnostic strategies. Collectively, these findings underscore the importance of population-level analyses for resolving the evolutionary processes shaping snake venom diversity within species.

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Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fevo.2026.1820178/full#supplementary-material

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Footnotes

Edited by: Chris Barratt, Wageningen University and Research, Netherlands

Reviewed by: Cassandra M. Modahl, Liverpool School of Tropical Medicine, United Kingdom

Senji Laxme R R, Indian Institute of Science (IISc), India