Individual and Population Age Impact Social Behavior and Network Structure in a Long-Lived Insect.

AbstractSocial behaviors vary among individuals, and social networks vary among groups. Understanding the causes of such variation is important for predicting or altering ecological processes such as infectious disease outbreaks. Here, we ask whether age contributes to variation in social behavior at multiple levels of organization: within individuals over time, among individuals of different ages, among local social environments, and among populations. We used experimental manipulations of captive populations and a longitudinal dataset to test whether social behavior is associated with age across these levels in a long-lived insect, the forked fungus beetle (Bolitotherus cornutus). In cross-sectional analyses, we found that older beetles were less connected in their social networks. Longitudinal data confirmed that this effect was due in part to changes in behavior over time; beetles became less social over 2 years, possibly because of increased social selectivity or reproductive investment. Beetles of different ages also occupied different local social neighborhoods. The effects of age on behavior scaled up: populations of older individuals had fewer interactions, fewer but more variable relationships, longer network path lengths, and lower clustering than populations of young individuals. Age therefore impacted not only individual sociality but also the network structures that mediate critical population processes.

Conspicuous coloration of toxin-resistant predators implicates additional trophic interactions in a predator-prey arms race.

Antagonistic coevolution between natural enemies can produce highly exaggerated traits, such as prey toxins and predator resistance. This reciprocal process of adaptation and counter-adaptation may also open doors to other evolutionary novelties not directly involved in the phenotypic interface of coevolution. We tested the hypothesis that predator-prey coevolution coincided with the evolution of conspicuous coloration on resistant predators that retain prey toxins. In western North America, common garter snakes (Thamnophis sirtalis) have evolved extreme resistance to tetrodotoxin (TTX) in the coevolutionary arms race with their deadly prey, Pacific newts (Taricha spp.). TTX-resistant snakes can retain large amounts of ingested TTX, which could serve as a deterrent against the snakes' own predators if TTX toxicity and resistance are coupled with a conspicuous warning signal. We evaluated whether arms race escalation covaries with bright red coloration in snake populations across the geographic mosaic of coevolution. Snake colour variation departs from the neutral expectations of population genetic structure and covaries with escalating clines of newt TTX and snake resistance at two coevolutionary hotspots. In the Pacific Northwest, bright red coloration fits an expected pattern of an aposematic warning to avian predators: TTX-resistant snakes that consume highly toxic newts also have relatively large, reddish-orange dorsal blotches. Snake coloration also seems to have evolved with the arms race in California, but overall patterns are less intuitively consistent with aposematism. These results suggest that interactions with additional trophic levels can generate novel traits as a cascading consequence of arms race coevolution across the geographic mosaic.

Conservation and Convergence of Genetic Architecture in the Adaptive Radiation of Anolis Lizards.

AbstractThe G matrix, which quantifies the genetic architecture of traits, is often viewed as an evolutionary constraint. However, G can evolve in response to selection and may also be viewed as a product of adaptive evolution. Convergent evolution of G in similar environments would suggest that G evolves adaptively, but it is difficult to disentangle such effects from phylogeny. Here, we use the adaptive radiation of Anolis lizards to ask whether convergence of G accompanies the repeated evolution of habitat specialists, or ecomorphs, across the Greater Antilles. We measured G in seven species representing three ecomorphs (trunk-crown, trunk-ground, and grass-bush). We found that the overall structure of G does not converge. Instead, the structure of G is well conserved and displays a phylogenetic signal consistent with Brownian motion. However, several elements of G showed signatures of convergence, indicating that some aspects of genetic architecture have been shaped by selection. Most notably, genetic correlations between limb traits and body traits were weaker in long-legged trunk-ground species, suggesting effects of recurrent selection on limb length. Our results demonstrate that common selection pressures may have subtle but consistent effects on the evolution of G, even as its overall structure remains conserved.

The road not taken: Evolution of tetrodotoxin resistance in the Sierra garter snake (Thamnophis couchii) by a path less travelled.

The repeated evolution of tetrodotoxin (TTX) resistance provides a model for testing hypotheses about the mechanisms of convergent evolution. This poison is broadly employed as a potent antipredator defence, blocking voltage-gated sodium channels (Nav ) in muscles and nerves, paralysing and sometimes killing predators. Resistance in taxa bearing this neurotoxin and a few predators appears to come from convergent replacements in specific Nav residues that interact with TTX. This stereotyped genetic response suggests molecular and phenotypic evolution may be constrained and predictable. Here, we investigate the extent of mechanistic convergence in garter snakes (Thamnophis) that prey on TTX-bearing newts (Taricha) by examining the physiological and genetic basis of TTX resistance in the Sierra garter snake (Th. couchii). We characterize variation in this predatory adaptation across populations at several biological scales: whole-animal TTX resistance; skeletal muscle resistance; functional genetic variation in three Nav encoding loci; and levels of gene expression for one of these loci. We found Th. couchii possess extensive geographical variation in resistance at the whole-animal and skeletal muscle levels. As in other Thamnophis, resistance at both levels is highly correlated, suggesting convergence across the biological levels linking organism to organ. However, Th. couchii shows no functional variation in Nav loci among populations or difference in candidate gene expression. Local variation in TTX resistance in Th. couchii cannot be explained by the same relationship between genotype and phenotype seen in other taxa. Thus, historical contingencies may lead different species of Thamnophis down alternative routes to local adaptation.

Group and individual social network metrics are robust to changes in resource distribution in experimental populations of forked fungus beetles.

Social interactions drive many important ecological and evolutionary processes. It is therefore essential to understand the intrinsic and extrinsic factors that underlie social patterns. A central tenet of the field of behavioural ecology is the expectation that the distribution of resources shapes patterns of social interactions. We combined experimental manipulations with social network analyses to ask how patterns of resource distribution influence complex social interactions. We experimentally manipulated the distribution of an essential food and reproductive resource in semi-natural populations of forked fungus beetles Bolitotherus cornutus. We aggregated resources into discrete clumps in half of the populations and evenly dispersed resources in the other half. We then observed social interactions between individually marked beetles. Half-way through the experiment, we reversed the resource distribution in each population, allowing us to control any demographic or behavioural differences between our experimental populations. At the end of the experiment, we compared individual and group social network characteristics between the two resource distribution treatments. We found a statistically significant but quantitatively small effect of resource distribution on individual social network position and detected no effect on group social network structure. Individual connectivity (individual strength) and individual cliquishness (local clustering coefficient) increased in environments with clumped resources, but this difference explained very little of the variance in individual social network position. Individual centrality (individual betweenness) and measures of overall social structure (network density, average shortest path length and global clustering coefficient) did not differ between environments with dramatically different distributions of resources. Our results illustrate that the resource environment, despite being fundamental to our understanding of social systems, does not always play a central role in shaping social interactions. Instead, our results suggest that sex differences and temporally fluctuating environmental conditions may be more important in determining patterns of social interactions.

Group composition of individual personalities alters social network structure in experimental populations of forked fungus beetles.

Social network structure is a critical group character that mediates the flow of information, pathogens and resources among individuals in a population, yet little is known about what shapes social structures. In this study, we experimentally tested whether social network structure depends on the personalities of individual group members. Replicate groups of forked fungus beetles (Bolitotherus cornutus) were engineered to include only members previously assessed as either more social or less social. We found that individuals expressed consistent personalities across social contexts, exhibiting repeatable numbers of interactions and numbers of partners. Groups composed of more social individuals formed networks with higher interaction rates, higher tie density, higher global clustering and shorter average shortest paths than those composed of less social individuals. We highlight group composition of personalities as a source of variance in group traits and a potential mechanism by which networks could evolve.

Phenotypic Assortment Changes the Landscape of Selection.

Social interactions with conspecifics can dramatically affect an individual's fitness. The positive or negative consequences of interacting with social partners typically depend on the value of traits that they express. These pathways of social selection connect the traits and genes expressed in some individuals to the fitness realized by others, thereby altering the total phenotypic selection on and evolutionary response of traits across the multivariate phenotype. The downstream effects of social selection are mediated by the patterns of phenotypic assortment between focal individuals and their social partners (the interactant covariance, Cij', or the multivariate form, CI). Depending on the sign and magnitude of the interactant covariance, the direction of social selection can be reinforced, reversed, or erased. We report estimates of Cij' from a variety of studies of forked fungus beetles to address the largely unexplored questions of consistency and plasticity of phenotypic assortment in natural populations. We found that phenotypic assortment of male beetles based on body size or horn length was highly variable among subpopulations, but that those differences also were broadly consistent from year to year. At the same time, the strength and direction of Cij' changed quickly in response to experimental changes in resource distribution and social properties of populations. Generally, interactant covariances were more negative in contexts in which the number of social interactions was greater in both field and experimental situations. These results suggest that patterns of phenotypic assortment could be important contributors to variability in multilevel selection through their mediation of social selection gradients.

A Synthesis of Game Theory and Quantitative Genetic Models of Social Evolution.

Two popular approaches for modeling social evolution, evolutionary game theory and quantitative genetics, ask complementary questions but are rarely integrated. Game theory focuses on evolutionary outcomes, with models solving for evolutionarily stable equilibria, whereas quantitative genetics provides insight into evolutionary processes, with models predicting short-term responses to selection. Here we draw parallels between evolutionary game theory and interacting phenotypes theory, which is a quantitative genetic framework for understanding social evolution. First, we show how any evolutionary game may be translated into two quantitative genetic selection gradients, nonsocial and social selection, which may be used to predict evolutionary change from a single round of the game. We show that synergistic fitness effects may alter predicted selection gradients, causing changes in magnitude and sign as the population mean evolves. Second, we show how evolutionary games involving plastic behavioral responses to partners can be modeled using indirect genetic effects, which describe how trait expression changes in response to genes in the social environment. We demonstrate that repeated social interactions in models of reciprocity generate indirect effects and conversely, that estimates of parameters from indirect genetic effect models may be used to predict the evolution of reciprocity. We argue that a pluralistic view incorporating both theoretical approaches will benefit empiricists and theorists studying social evolution. We advocate the measurement of social selection and indirect genetic effects in natural populations to test the predictions from game theory and, in turn, the use of game theory models to aid in the interpretation of quantitative genetic estimates.

Sex linkage of the skeletal muscle sodium channel gene (SCN4A) explains apparent deviations from Hardy-Weinberg equilibrium of tetrodotoxin-resistance alleles in garter snakes (Thamnophis sirtalis).

The arms race between tetrodotoxin-bearing Pacific newts (Taricha) and their garter snake predators (Thamnophis) in western North America has become a classic example of coevolution, shedding light on predator-prey dynamics, the molecular basis of adaptation, and patterns of convergent evolution. Newts are defended by tetrodotoxin (TTX), a neurotoxin that binds to voltage-gated sodium channels (Nav proteins), arresting electrical activity in nerves and muscles and paralyzing would-be predators. However, populations of the common garter snake (T. sirtalis) have overcome this defense, largely through polymorphism at the locus SCN4A, which renders the encoded protein (Nav1.4) less vulnerable to TTX. Previous work suggests that SCN4A commonly shows extreme deviations from Hardy-Weinberg equilibrium (HWE) in these populations, which has been interpreted as the result of intense selection imposed by newts. Here we show that much of this apparent deviation can be attributed to sex linkage of SCN4A. Using genomic data and quantitative PCR, we show that SCN4A is on the Z chromosome in Thamnophis and other advanced snakes. Taking Z-linkage into account, we find that most apparent deviations from HWE can be explained by female hemizygosity rather than low heterozygosity. Sex linkage can affect mutation rates, selection, and drift, and our results suggest that Z-linkage of SCN4A may make significant contributions to the overall dynamics of the coevolutionary arms race between newts and snakes.

The geographic mosaic in parallel: Matching patterns of newt tetrodotoxin levels and snake resistance in multiple predator-prey pairs.

The Geographic Mosaic Theory of Coevolution predicts that coevolutionary arms races will vary over time and space because of the diverse ecological settings and population histories of interacting species across the landscape. Thus, understanding coevolution may require investigating broad sets of populations sampled across the range of the interaction. In addition, comparing coevolutionary dynamics between similar systems may reveal the importance of specific factors that structure coevolution. Here, we examine geographic patterns of prey traits and predator traits in the relatively unstudied interaction between the Sierra garter snake (Thamnophis couchii) and sympatric prey, the rough-skinned newt (Taricha granulosa), Sierra newt (Ta. sierrae) and California newt (Ta. torosa). This system parallels, in space and phenotypes, a classic example of coevolution between predatory common garter snakes (Th. sirtalis) and their toxic newt prey exhibiting hotspots of newt tetrodotoxin (TTX) levels and matching snake TTX resistance. We quantified prey and predator traits from hundreds of individuals across their distributions, and functional trait matching at sympatric sites. We show strong regional patterns of trait covariation across the shared ranges of Th. couchii and newt prey. Traits differ significantly among localities, with lower newt TTX levels and snake TTX resistance at the northern latitudes, and higher TTX levels and snake resistance at southern latitudes. Newts and snakes in northern populations show the highest degree of functional trait matching despite possessing the least extreme traits. Conversely, newts and snakes in southern populations show the greatest mismatch despite possessing exaggerated traits, with some snakes so resistant to TTX they would be unaffected by any sympatric newt. Nevertheless, individual variation was substantial, and appears to offer the opportunity for continued reciprocal selection in most populations. Overall, the three species of newts appear to be engaged in a TTX-mediated arms race with Th. couchii. These patterns are congruent with those seen between newts and Th. sirtalis, including the same latitudinal gradient in trait covariation, and the potential 'escape' from the arms race by snake predators. Such concordance in broad scale patterns across two distinct systems suggests common phenomena might structure geographic mosaics in similar ways.

Sex-Specific Selection and the Evolution of Between-Sex Genetic Covariance.

Because the sexes share a genome, traits expressed in males are usually genetically correlated with the same traits expressed in females. On short timescales, between-sex genetic correlations (rmf) for shared traits may constrain the evolution of sexual dimorphism by preventing males and females from responding independently to sex-specific selection. However, over longer timescales, rmf may evolve, thereby facilitating the evolution of dimorphism. Although it has been suggested that sexually antagonistic selection may reduce rmf, we lack a general theory for the evolution of rmf and its multivariate analog, the between-sex genetic covariance matrix (B). Here, we derive a simple analytical model for the within-generation change in B due to sex-specific directional selection. We present a single-trait example demonstrating that sex-specific directional selection may either increase or decrease between-sex genetic covariance, depending on the relative strength of selection in each sex and on the current value of rmf. Although sexually antagonistic selection can reduce between-sex covariance, it will only do so when selection is much stronger in one sex than in the other. Counterintuitively, sexually antagonistic selection that is equal in strength in the 2 sexes will maintain positive between-sex covariance. Selection acting in the same direction on both sexes is predicted to reduce between-sex covariance in many cases. We illustrate our model numerically using empirical measures of sex-specific selection and between-sex genetic covariance from 2 populations of sexually dimorphic brown anole lizards (Anolis sagrei) and discuss its importance for understanding the resolution of intralocus sexual conflict.

Constraints Imposed by a Natural Landscape Override Offspring Fitness Effects to Shape Oviposition Decisions in Wild Forked Fungus Beetles.

Oviposition site decisions often maximize offspring fitness, but costs constraining choice can cause females to oviposit in poor developmental environments. It is unclear whether these constraints cumulatively outweigh offspring fitness to determine oviposition decisions in wild populations. Understanding how constraints shape oviposition in natural landscapes is a critical step toward revealing how maternal behavior influences fundamental phenomena like the evolution of specialization and the use of sink environments. Here, we used a genetic capture-recapture technique to reconstruct the oviposition decisions of individual females in a natural metapopulation of a beetle (Bolitotherus cornutus) that oviposits on three fungus species. We measured larval fitness-related traits (mass, development time, survival) on each fungus and compared the oviposition preferences of females in laboratory versus field tests. Larval fitness differed substantially among fungi, and females preferred a high-quality (high larval fitness) fungus in laboratory trials. However, females frequently laid eggs on the lowest-quality fungus in the wild. They preferred high-quality fungi when moving between oviposition sites, but this preference disappeared as the distance between sites increased and was inconsistent between study plots. Our results suggest that constraints on oviposition preferences in natural landscapes are sufficiently large to drive oviposition in poor developmental environments even when offspring fitness consequences are severe.

Corticosteroid responses of snakes to toxins from toads (bufadienolides) and plants (cardenolides) reflect differences in dietary specializations.

Toads are chemically defended by cardiotonic steroids known as bufadienolides. Resistance to the acute effects of bufadienolides in snakes that prey on toads is conferred by target-site insensitivity of the toxin's target enzyme, the Na+/K+-ATPase. Previous studies have focused largely on the molecular mechanisms of resistance but have not investigated the physiological mechanisms or consequences of exposure to the toxins. Adrenal enlargement in snakes often is associated with specialization on a diet of toads. These endocrine glands are partly composed of interrenal tissue, which produces the corticosteroids corticosterone and aldosterone. Corticosterone is the main hormone released in response to stress in reptiles, and aldosterone plays an important role in maintaining ion balance through upregulation of Na+/K+-ATPase. We tested the endocrine response of select species of snakes to acute cardiotonic steroid exposure by measuring circulating aldosterone and corticosterone concentrations. We found that Rhabdophis tigrinus, which specializes on a diet of toads, responds with lower corticosterone and higher aldosterone compared to other species that exhibit target-site resistance to the toxins but do not specialize on toads. We also found differences between sexes in R. tigrinus, with males generally responding with higher corticosterone and aldosterone than females. This study provides evidence of physiological adaptations, beyond target-site resistance, associated with tolerance of bufadienolides in a specialized toad-eating snake.

To stress or not to stress: Physiological responses to tetrodotoxin in resistant gartersnakes vary by sex.

The activation of the hypothalamic pituitary adrenal (HPA) axis is one of the most important physiological processes in coping with any deviation in an organism's homeostasis. This activation and the secretion of glucocorticoids, such as corticosterone, allow organisms to cope with perturbations and return to optimal physiological functioning as quickly as possible. In this study, we examined the HPA axis activation in common gartersnakes (Thamnophis sirtalis) as a response to a natural toxin, tetrodotoxin (TTX). This neurotoxin is found in high levels in the Rough-skinned Newt (Taricha granulosa), which is a prey item for these snakes. To consume this toxic prey, these snakes have evolved variable resistance. We hypothesized that the more resistant individuals would show a lower HPA axis response than less resistant individuals, as measured by corticosterone (CORT) and bactericidal ability, which is a functional downstream measurement of CORT's activity. We determined "resistance level" for tetrodotoxin from each individual snake by determining the dose which reduced race speed by 50%. Individuals were injected them with an increasing amount of tetrodotoxin (10, 25, and 50 MAMUs) to determine this value. Thirty minutes after every injection, we gathered blood samples from each snake. Our results show that, while there were no significant differences among individual CORT levels in a dose-dependent manner, female snakes did have a larger stress response when compared to both males and juveniles. Different life-histories could explain why females were able to mount a higher HPA axis response. However, TTX had no downstream effects on bactericidal ability, although juveniles had consistently lower values than adults. Our research shows a possible dichotomy between how each sex manages tetrodotoxin and gives way for a more comprehensive analysis of tetrodotoxin in an ecological context.

Evolutionary response when selection and genetic variation covary across environments.

Although models of evolution usually assume that the strength of selection on a trait and the expression of genetic variation in that trait are independent, whenever the same ecological factor impacts both parameters, a correlation between the two may arise that accelerates trait evolution in some environments and slows it in others. Here, we address the evolutionary consequences and ecological causes of a correlation between selection and expressed genetic variation. Using a simple analytical model, we show that the correlation has a modest effect on the mean evolutionary response and a large effect on its variance, increasing among-population or among-generation variation in the response when positive, and diminishing variation when negative. We performed a literature review to identify the ecological factors that influence selection and expressed genetic variation across traits. We found that some factors - temperature and competition - are unlikely to generate the correlation because they affected one parameter more than the other, and identified others - most notably, environmental novelty - that merit further investigation because little is known about their impact on one of the two parameters. We argue that the correlation between selection and genetic variation deserves attention alongside other factors that promote or constrain evolution in heterogeneous landscapes.

Multilevel and kin selection in a connected world.

Wild et al. argue that the evolution of reduced virulence can be understood from the perspective of inclusive fitness, obviating the need to evoke group selection as a contributing causal factor. Although they acknowledge the mathematical equivalence of the inclusive fitness and multilevel selection approaches, they conclude that reduced virulence can be viewed entirely as an individual-level adaptation by the parasite. Here we show that their model is a well-known special case of the more general theory of multilevel selection, and that the cause of reduced virulence resides in the opposition of two processes: within-group and among-group selection. This distinction is important in light of the current controversy among evolutionary biologists in which some continue to affirm that natural selection centres only and always at the level of the individual organism or gene, despite mathematical demonstrations that evolutionary dynamics must be described by selection at various levels in the hierarchy of biological organization.

Parallel evolution of tetrodotoxin resistance in three voltage-gated sodium channel genes in the garter snake Thamnophis sirtalis.

Members of a gene family expressed in a single species often experience common selection pressures. Consequently, the molecular basis of complex adaptations may be expected to involve parallel evolutionary changes in multiple paralogs. Here, we use bacterial artificial chromosome library scans to investigate the evolution of the voltage-gated sodium channel (Nav) family in the garter snake Thamnophis sirtalis, a predator of highly toxic Taricha newts. Newts possess tetrodotoxin (TTX), which blocks Nav's, arresting action potentials in nerves and muscle. Some Thamnophis populations have evolved resistance to extremely high levels of TTX. Previous work has identified amino acid sites in the skeletal muscle sodium channel Nav1.4 that confer resistance to TTX and vary across populations. We identify parallel evolution of TTX resistance in two additional Nav paralogs, Nav1.6 and 1.7, which are known to be expressed in the peripheral nervous system and should thus be exposed to ingested TTX. Each paralog contains at least one TTX-resistant substitution identical to a substitution previously identified in Nav1.4. These sites are fixed across populations, suggesting that the resistant peripheral nerves antedate resistant muscle. In contrast, three sodium channels expressed solely in the central nervous system (Nav1.1-1.3) showed no evidence of TTX resistance, consistent with protection from toxins by the blood-brain barrier. We also report the exon-intron structure of six Nav paralogs, the first such analysis for snake genes. Our results demonstrate that the molecular basis of adaptation may be both repeatable across members of a gene family and predictable based on functional considerations.

Predictably Convergent Evolution of Sodium Channels in the Arms Race between Predators and Prey.

Evolution typically arrives at convergent phenotypic solutions to common challenges of natural selection. However, diverse molecular and physiological mechanisms may generate phenotypes that appear similar at the organismal level. How predictable are the molecular mechanisms of adaptation that underlie adaptive convergence? Interactions between toxic prey and their predators provide an excellent avenue to investigate the question of predictability because both taxa must adapt to the presence of defensive poisons. The evolution of resistance to tetrodotoxin (TTX), which binds to and blocks voltage-gated sodium channels (NaV1) in nerves and muscle, has been remarkably parallel across deep phylogenetic divides. In both predators and prey, representing three major vertebrate groups, TTX resistance has arisen through structural changes in NaV1 proteins. Fish, amphibians and reptiles, though they differ in the total number of NaV1 paralogs in their genomes, have each evolved common amino acid substitutions in the orthologous skeletal muscle NaV1.4. Many of these substitutions involve not only the same positions in the protein, but also the identical amino acid residues. Similarly, predictable convergence is observed across the family of sodium channel genes expressed in different tissues in puffer fish and in garter snakes. Trade-offs between the fundamental role of NaV1 proteins in selective permeability of Na+ and their ability to resist binding by TTX generate a highly constrained adaptive landscape at the level of the protein.

The role of corticosterone and toxicity in the antipredator behavior of the Rough-skinned Newt (Taricha granulosa).

A variety of mechanisms are responsible for enabling an organism to escape a predatory attack, including behavioral changes, alterations in hormone levels, and production and/or secretion of toxins. However, these mechanisms are rarely studied in conjunction with each other. The Rough-skinned Newt (Taricha granulosa) is an ideal organism to examine the relationships between these mechanisms because its behavioral displays and toxin secretion during a predator attack are well documented and readily characterized. While we found no direct relationship between antipredator behavior and endogenous levels of corticosterone (CORT), antipredator behavior was inhibited when exogenous CORT and adrenocorticotropic hormone (ACTH) were administered, resulting in high circulating concentrations of CORT, indicating that CORT may play a role in mediating the behavior. There was no correlation between the animal's toxicity and either CORT or behavior. The results of this study provide evidence that CORT plays an important, yet complex, role in the antipredator response of these amphibians.

Constraint shapes convergence in tetrodotoxin-resistant sodium channels of snakes.

Natural selection often produces convergent changes in unrelated lineages, but the degree to which such adaptations occur via predictable genetic paths is unknown. If only a limited subset of possible mutations is fixed in independent lineages, then it is clear that constraint in the production or function of molecular variants is an important determinant of adaptation. We demonstrate remarkably constrained convergence during the evolution of resistance to the lethal poison, tetrodotoxin, in six snake species representing three distinct lineages from around the globe. Resistance-conferring amino acid substitutions in a voltage-gated sodium channel, Na(v)1.4, are clustered in only two regions of the protein, and a majority of the replacements are confined to the same three positions. The observed changes represent only a small fraction of the experimentally validated mutations known to increase Na(v)1.4 resistance to tetrodotoxin. These results suggest that constraints resulting from functional tradeoffs between ion channel function and toxin resistance led to predictable patterns of evolutionary convergence at the molecular level. Our data are consistent with theoretical predictions and recent microcosm work that suggest a predictable path is followed during an adaptive walk along a mutational landscape, and that natural selection may be frequently constrained to produce similar genetic outcomes even when operating on independent lineages.