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.

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.

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.