Where Does All the Poison Go? Investigating Toxicokinetics of Newt (Taricha) Tetrodotoxin (TTX) in Garter Snakes (Thamnophis).

Animals that consume toxic diets provide models for understanding the molecular and physiological adaptations to ecological challenges. Garter snakes (Thamnophis) in western North America prey on Pacific newts (Taricha), which employ tetrodotoxin (TTX) as an antipredator defense. These snakes possess mutations in voltage-gated sodium channels (Nav), the molecular targets of TTX, that decrease the binding ability of TTX to sodium channels (target-site resistance). However, genetic variation at these loci that cannot explain all the phenotypic variation in TTX resistance in Thamnophis. We explored a separate means of resistance, toxin metabolism, to determine if TTX-resistant snakes either rapidly remove TTX or sequester TTX. We examined the metabolism and distribution of TTX in the body (toxicokinetics), to determine differences between TTX-resistant and TTX-sensitive snakes in the rates at which TTX is eliminated from organs and the whole body (using TTX half-life as our metric). We assayed TTX half-life in snakes from TTX-resistant and TTX-sensitive populations of three garter snake species with a coevolutionary history with newts (T. atratus, T. couchii, T. sirtalis), as well as two non-resistant "outgroup" species (T. elegans, Pituophis catenifer) that seldom (if ever) engage newts. We found TTX half-life varied across species, populations, and tissues. Interestingly, TTX half-life was shortest in T. elegans and P. catenifer compared to all other snakes. Furthermore, TTX-resistant populations of T. couchii and T. sirtalis eliminated TTX faster (shorter TTX half-life) than their TTX-sensitive counterparts, while populations of TTX-resistant and TTX-sensitive T. atratus showed no difference rates of TTX removal (same TTX half-life). The ability to rapidly eliminate TTX may have permitted increased prey consumption, which may have promoted the evolution of additional resistance mechanisms. Finally, snakes still retain substantial amounts of TTX, and we projected that snakes could be dangerous to their own predators days to weeks following the ingestion of a single newt. Thus, aspects of toxin metabolism may have been key in driving predator-prey relationships, and important in determining other ecological interactions.

Repeated patterns of reptile diversification in Western North America supported by the Northern Alligator Lizard (Elgaria coerulea).

Understanding the processes that shape genetic diversity by either promoting or preventing population divergence can help identify geographic areas that either facilitate or limit gene flow. Furthermore, broadly distributed species allow us to understand how biogeographic and ecogeographic transitions affect gene flow. We investigated these processes using genomic data in the Northern Alligator Lizard (Elgaria coerulea), which is widely distributed in Western North America across diverse ecoregions (California Floristic Province and Pacific Northwest) and mountain ranges (Sierra Nevada, Coastal Ranges, and Cascades). We collected single-nucleotide polymorphism data from 120 samples of E. coerulea. Biogeographic analyses of squamate reptiles with similar distributions have identified several shared diversification patterns that provide testable predictions for E. coerulea, including deep genetic divisions in the Sierra Nevada, demographic stability of southern populations, and recent post-Pleistocene expansion into the Pacific Northwest. We use genomic data to test these predictions by estimating the structure, connectivity, and phylogenetic history of populations. At least 10 distinct populations are supported, with mixed-ancestry individuals situated at most population boundaries. A species tree analysis provides strong support for the early divergence of populations in the Sierra Nevada Mountains and recent diversification into the Pacific Northwest. Admixture and migration analyses detect gene flow among populations in the Lower Cascades and Northern California, and a spatial analysis of gene flow identified significant barriers to gene flow across both the Sierra Nevada and Coast Ranges. The distribution of genetic diversity in E. coerulea is uneven, patchy, and interconnected at population boundaries. The biogeographic patterns seen in E. coerulea are consistent with predictions from co-distributed species.

Coevolution with toxic prey produces functional trade-offs in sodium channels of predatory snakes.

Seemingly unrelated traits often share the same underlying molecular mechanisms, potentially generating a pleiotropic relationship whereby selection shaping one trait can simultaneously compromise another. While such functional trade-offs are expected to influence evolutionary outcomes, their actual relevance in nature is masked by obscure links between genotype, phenotype, and fitness. Here, we describe functional trade-offs that likely govern a key adaptation and coevolutionary dynamics in a predator-prey system. Several garter snake (Thamnophis spp.) populations have evolved resistance to tetrodotoxin (TTX), a potent chemical defense in their prey, toxic newts (Taricha spp.). Snakes achieve TTX resistance through mutations occurring at toxin-binding sites in the pore of snake skeletal muscle voltage-gated sodium channels (NaV1.4). We hypothesized that these mutations impair basic NaV functions, producing molecular trade-offs that should ultimately scale up to compromised organismal performance. We investigate biophysical costs in two snake species with unique and independently evolved mutations that confer TTX resistance. We show electrophysiological evidence that skeletal muscle sodium channels encoded by toxin-resistant alleles are functionally compromised. Furthermore, skeletal muscles from snakes with resistance genotypes exhibit reduced mechanical performance. Lastly, modeling the molecular stability of these sodium channel variants partially explains the electrophysiological and muscle impairments. Ultimately, adaptive genetic changes favoring toxin resistance appear to negatively impact sodium channel function, skeletal muscle strength, and organismal performance. These functional trade-offs at the cellular and organ levels appear to underpin locomotor deficits observed in resistant snakes and may explain variation in the population-level success of toxin-resistant alleles across the landscape, ultimately shaping the trajectory of snake-newt coevolution.

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.

Preying dangerously: black widow spider venom resistance in sympatric lizards.

Lizards and spiders are natural adversaries, yet little is known of adaptations that lizards might possess for dealing with the venomous defences of spider prey. In the Western USA, two lizard species (Elgaria multicarinata and Sceloporus occidentalis) are sympatric with and predate western black widow spiders (Latrodectus hesperus). The consequences of black widow spider venom (BWSV) can be severe, and are well understood for mammals but unknown for reptiles. We evaluated potential resistance to BWSV in the lizards that consume black widows, and a potentially susceptible species (Uta stansburiana) known as prey of widows. We investigated BWSV effects on whole-animal performance (sprint) and muscle tissue at two venom doses compared with control injections. Sprint speed was not significantly decreased in E. multicarinata or S. occidentalis in any treatment, while U. stansburiana suffered significant performance reductions in response to BWSV. Furthermore, E. multicarinata showed minimal tissue damage and immune response, while S. occidentalis and U. stansburiana exhibited increased muscle damage and immune system infiltration in response to BWSV. Our data suggest predator-prey relationships between lizards and spiders are complex, possibly leading to physiological and molecular adaptations that allow some lizards to tolerate or overcome the dangerous defences of their arachnid prey.

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.

Every breath you take: assessing metabolic costs of toxin resistance in garter snakes (Thamnophis).

Trait specialization often comes at the expense of original trait function, potentially causing evolutionary tradeoffs that may render specialist populations vulnerable to extinction. However, many specialized adaptations evolve repeatedly, suggesting selection favors specialization in specific environments. Some garter snake (Thamnophis) populations possess specialized mutations in voltage-gated sodium channels that allow them to consume Pacific newts (Taricha) defended by a highly potent neurotoxin (tetrodotoxin). These mutations, however, also decrease protein and muscle function, suggesting garter snakes may suffer evolutionary tradeoffs. We measured a key physiological process, standard metabolic rate (SMR), to investigate whether specialized adaptations in toxin-resistant garter snakes affect baseline energy expenditure. In snakes, skeletal muscles influence metabolism and power ventilation, so inefficiencies of sodium channels in these muscles might impact whole-animal energy expenditure. Further, because sodium channels are membrane-bound proteins, inefficiencies of channel kinetics and performance might be exacerbated at suboptimal temperatures. We measured SMR in 2 species, Thamnophis atratus and Thamnophis sirtalis, that independently evolved tetrodotoxin resistance through unique mutations, providing replicate experiments with distinct underlying genetics and potential physiological costs. Despite our expectations, neither resistance phenotype nor sodium channel genotype affected metabolism and resistant snakes did not perform worse under suboptimal body temperature. Instead, T. atratus and T. sirtalis show nearly identical rates of mass-adjusted energy expenditure at both temperatures, despite differing eco-morphologies, life histories, and distant phylogenetic positions. These findings suggest SMR may be a conserved feature of Thamnophis, and that any organismal tradeoffs may be compensated to retain whole-animal function.

Phylogenomic analyses resolve relationships among garter snakes (Thamnophis: Natricinae: Colubridae) and elucidate biogeographic history and morphological evolution.

Garter snakes (Thamnophis) are a successful group of natricines endemic to North America. They have become important natural models for ecological and evolutionary research, yet prior efforts to resolve phylogenetic relationships have resulted in conflicting topologies and weak support for certain relationships. Here, we use genomic data generated with a reduced representation double-digest RADseq approach to reassess evolutionary relationships across Thamnophis. We then use the resulting phylogeny to better understand how biogeography and feeding ecology have influenced lineage diversification and morphological evolution. We recovered highly congruent and strongly supported topologies from maximum likelihood and Bayesian analyses, but some discordance with a multispecies coalescent approach. All phylogenomic estimates split Thamnophis into two clades largely defined by northern and southern North American species. Divergence time estimates and biogeographic analyses indicate a mid-Miocene origin of Thamnophis in Mexico. In addition, historic vicariant events thought to explain biogeographic patterns in other lineages (e.g., Isthmus of Tehuantepec, Rocky Mountain Range, and Trans-Mexican Volcanic Belt) appear to have influenced patterns of diversification in Thamnophis as well. Analyses of morphological traits associated with feeding ecology showed moderate to strong phylogenetic signal. Nevertheless, phylogenetic ANOVA suggested significant differences in certain cranial morphologies between aquatic specialists and garter snakes that are terrestrial-aquatic generalists, independent of evolutionary history. Our new estimate of Thamnophis phylogeny yields an improved understanding of the biogeographic history and morphological evolution of garter snakes, and provides a robust framework for future research on these snakes.

The cost of travel: How dispersal ability limits local adaptation in host-parasite interactions.

Classical theory suggests that parasites will exhibit higher fitness in sympatric relative to allopatric host populations (local adaptation). However, evidence for local adaptation in natural host-parasite systems is often equivocal, emphasizing the need for infection experiments conducted over realistic geographic scales and comparisons among species with varied life history traits. Here, we used infection experiments to test how two trematode (flatworm) species (Paralechriorchis syntomentera and Ribeiroia ondatrae) with differing dispersal abilities varied in the strength of local adaptation to their amphibian hosts. Both parasites have complex life cycles involving sequential transmission among aquatic snails, larval amphibians and vertebrate definitive hosts that control dispersal across the landscape. By experimentally pairing 26 host-by-parasite population infection combinations from across the western USA with analyses of host and parasite spatial genetic structure, we found that increasing geographic distance-and corresponding increases in host population genetic distance-reduced infection success for P. syntomentera, which is dispersed by snake definitive hosts. For the avian-dispersed R. ondatrae, in contrast, the geographic distance between the parasite and host populations had no influence on infection success. Differences in local adaptation corresponded to parasite genetic structure; although populations of P. syntomentera exhibited ~10% mtDNA sequence divergence, those of R. ondatrae were nearly identical (<0.5%), even across a 900 km range. Taken together, these results offer empirical evidence that high levels of dispersal can limit opportunities for parasites to adapt to local host populations.

Ectoparasite Load Is Reduced in Side-Blotched Lizards (Uta stansburiana) at Wind Farms: Implications for Oxidative Stress.

AbstractWind-generated power is one of the fastest growing alternative energy strategies worldwide and will likely account for 20% of US energy production by 2030. The installation and maintenance of wind farms are associated with increased human activity and can generate noise pollution, disturb and fragment habitat, and even alter community composition and structure. These environmental and ecological changes can increase physiological stress for vertebrates and affect important life-history attributes, such as immune function. However, little is known about how wind farms influence physiology and disease or parasite resistance in nonvolant wildlife. Here, we test the notion that renewable wind farms increase physiological stress and correlated aspects of disease resistance (parasite load) in a common desert vertebrate, the side-blotched lizard (Uta stansburiana). We captured lizards from three wind farms and three undisturbed reference sites in the San Gorgonio Pass wind resource area in the Mojave Desert, California. We quantified individual external parasite loads and measured plasma antioxidant capacity and concentrations of reactive oxygen metabolites as a combined metric of oxidative stress. Contrary to our expectations, individuals at wind farm sites had significantly fewer external parasites than at undeveloped sites. Additionally, we found a slight positive correlation between parasite load and oxidative stress for individuals at wind farm sites but not at reference sites. Our results demonstrate a complex, potentially context-dependent relationship between stress physiology and disease resistance for lizards in anthropogenically disturbed environments. Understanding how wind farms affect the physiology and ecoimmunology of terrestrial fauna is necessary to mitigate the ecological costs of alternative energy development.

The geographic mosaic of arms race coevolution is closely matched to prey population structure.

Reciprocal adaptation is the hallmark of arms race coevolution. Local coadaptation between natural enemies should generate a geographic mosaic pattern where both species have roughly matched abilities across their shared range. However, mosaic variation in ecologically relevant traits can also arise from processes unrelated to reciprocal selection, such as population structure or local environmental conditions. We tested whether these alternative processes can account for trait variation in the geographic mosaic of arms race coevolution between resistant garter snakes (Thamnophis sirtalis) and toxic newts (Taricha granulosa). We found that predator resistance and prey toxin levels are functionally matched in co-occurring populations, suggesting that mosaic variation in the armaments of both species results from the local pressures of reciprocal selection. By the same token, phenotypic and genetic variation in snake resistance deviates from neutral expectations of population genetic differentiation, showing a clear signature of adaptation to local toxin levels in newts. Contrastingly, newt toxin levels are best predicted by genetic differentiation among newt populations, and to a lesser extent, by the local environment and snake resistance. Exaggerated armaments suggest that coevolution occurs in certain hotspots, but prey population structure seems to be of particular influence on local phenotypic variation in both species throughout the geographic mosaic. Our results imply that processes other than reciprocal selection, like historical biogeography and environmental pressures, represent an important source of variation in the geographic mosaic of coevolution. Such a pattern supports the role of "trait remixing" in the geographic mosaic theory, the process by which non-adaptive forces dictate spatial variation in the interactions among species.

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.

Convergent adaptation to dangerous prey proceeds through the same first-step mutation in the garter snake Thamnophis sirtalis.

Convergent phenotypes often result from similar underlying genetics, but recent work suggests convergence may also occur in the historical order of substitutions en route to an adaptive outcome. We characterized convergence in the mutational steps to two independent outcomes of tetrodotoxin (TTX) resistance in separate geographic lineages of the common garter snake (Thamnophis sirtalis) that coevolved with toxic newts. Resistance is largely conferred by amino acid changes in the skeletal muscle sodium channel (NaV 1.4) that interfere with TTX-binding. We sampled variation in NaV 1.4 throughout western North America and found clear evidence that TTX-resistant changes in both lineages began with the same isoleucine-valine mutation (I1561V) within the outer pore of NaV 1.4. Other point mutations in the pore, shown to confer much greater resistance, accumulate later in the evolutionary progression and always occur together with the initial I1561V change. A gene tree of NaV 1.4 suggests the I1561V mutations in each lineage are not identical-by-decent, but rather they arose independently. Convergence in the evolution of channel resistance is likely the result of shared biases in the two lineages of T. sirtalis-only a few mutational routes can confer TTX resistance while maintaining the conserved function of voltage-gated sodium channels.

Historical Contingency in a Multigene Family Facilitates Adaptive Evolution of Toxin Resistance.

Novel adaptations must originate and function within an already established genome [1]. As a result, the ability of a species to adapt to new environmental challenges is predicted to be highly contingent on the evolutionary history of its lineage [2-6]. Despite a growing appreciation of the importance of historical contingency in the adaptive evolution of single proteins [7-11], we know surprisingly little about its role in shaping complex adaptations that require evolutionary change in multiple genes. One such adaptation, extreme resistance to tetrodotoxin (TTX), has arisen in several species of snakes through coevolutionary arms races with toxic amphibian prey, which select for TTX-resistant voltage-gated sodium channels (Nav) [12-16]. Here, we show that the relatively recent origins of extreme toxin resistance, which involve the skeletal muscle channel Nav1.4, were facilitated by ancient evolutionary changes in two other members of the same gene family. A substitution conferring TTX resistance to Nav1.7, a channel found in small peripheral neurons, arose in lizards ∼170 million years ago (mya) and was present in the common ancestor of all snakes. A second channel found in larger myelinated neurons, Nav1.6, subsequently evolved resistance in four different snake lineages beginning ∼38 mya. Extreme TTX resistance has evolved at least five times within the past 12 million years via changes in Nav1.4, but only within lineages that previously evolved resistant Nav1.6 and Nav1.7. Our results show that adaptive protein evolution may be contingent upon enabling substitutions elsewhere in the genome, in this case, in paralogs of the same gene family.

Do turtles follow the rules? Latitudinal gradients in species richness, body size, and geographic range area of the world's turtles.

Understanding how and why biodiversity is structured across the globe has been central to ecology, evolution, and biogeography even before those disciplines took their modern forms. Three global-scale patterns in particular have been the focus of research and debate for decades: latitudinal gradients in species richness (richness decreases with increasing latitude), body size (body size increases with increasing latitude in endotherms; Bergmann's rule), and geographic range size (range size increases with increasing latitude; Rapoport's rule). Despite decades of study, the generality and robustness of these trends have been debated, as have their underlying causes. Here we investigate latitudinal gradients in species richness, body size, and range size in the world's turtles (Testudines), and add more evidence that these rules do not seem to apply across all taxa. We show that turtle diversity actually peaks at 25° north, a highly unusual global pattern. Turtles also fail to follow Bergmann's Rule, and may show the converse (larger at lower latitudes), though trends are weak. Turtles also show a complex relationship between latitude and range size that does not directly follow Rapoport's rule. Body size and geographic range size are significantly correlated, and multiple abiotic and biotic variables help explain the relationships between latitude and species diversity, body size, and range size. Although we show that turtles do not strictly follow some classic biogeographical rules, we also call for further in-depth research to investigate potential causal mechanisms for these atypical patterns.

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.

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.

Phylogeny, biogeography, and display evolution in the tree and brush lizard genus Urosaurus (Squamata: Phrynosomatidae).

The brush and tree lizards (Urosaurus) are a small clade of phrynosomatid lizards native to western North America. Though not as well known as their diverse sister clade, the spiny lizards (Sceloporus), some Urosaurus have nonetheless become model organisms in integrative biology. In particular, dramatic phenotypic and behavioral differences associated with specific mating strategies have been exploited to address a range of ecological and evolutionary questions. However, only two phylogenies have been proposed for the group, one of which is pre-cladistic and both based principally on morphological characters that might not provide robust support for relationships within the group. To help provide investigators working on Urosaurus with a robust phylogeny in which to frame ecological and evolutionary questions, we establish a molecular phylogeny for the group. We sampled three mitochondrial and three nuclear loci, and estimated phylogenetic relationships within Urosaurus using both maximum parsimony (MP) and Bayesian inference (BI), as well as a coalescent-based species tree approach. Finally, we used two methods of ancestral state reconstruction (ASR) to gain insight into the evolution of microhabitat preference and male display signals, traits that have been the focus of studies on Urosaurus. All reconstruction methods yield nearly the same ingroup topology that is concordant in most respects with the previous cladistic analysis of the group but with some significant differences; our data suggest the primary divergence in Urosaurus occurs between a clade endemic to the Pacific versant of Mexico and the lineages of Baja California and the southwestern US, rather than placing Urosaurus graciosus as the basal taxon and linking the Baja and Mexican endemics. We find support for a single transition to a saxicolous lifestyle within the group, and either the independent gain or loss of arboreality. The evolution of throat color patterns (i.e. dewlaps) appears complex, with multiple color morphs likely involving orange reconstructed as ancestral to the group and to most lineages, followed by a single transition to a fixed blue-throated morph in one clade. These results should provide a useful framework for additional comparative work with Urosaurus, and establish the phylogenetic context in which Urosaurus diversity arose.

Adaptive evolution of plastron shape in emydine turtles.

Morphology reflects ecological pressures, phylogeny, and genetic and biophysical constraints. Disentangling their influence is fundamental to understanding selection and trait evolution. Here, we assess the contributions of function, phylogeny, and habitat to patterns of plastron (ventral shell) shape variation in emydine turtles. We quantify shape variation using geometric morphometrics, and determine the influence of several variables on shape using path analysis. Factors influencing plastron shape variation are similar between emydine turtles and the more inclusive Testudinoidea. We evaluate the fit of various evolutionary models to the shape data to investigate the selective landscape responsible for the observed morphological patterns. The presence of a hinge on the plastron accounts for most morphological variance, but phylogeny and habitat also correlate with shape. The distribution of shape variance across emydine phylogeny is most consistent with an evolutionary model containing two adaptive zones--one for turtles with kinetic plastra, and one for turtles with rigid plastra. Models with more complex adaptive landscapes often fit the data only as well as the null model (purely stochastic evolution). The adaptive landscape of plastron shape in Emydinae may be relatively simple because plastral kinesis imposes overriding mechanical constraints on the evolution of form.