Geographic distributions of focal Thamnophis species. In California and Oregon, the garter snakes T. sirtalis (gray), T. atratus (blue), and T. couchii (green) broadly overlap with newts of the genus Taricha that possess the lethal neurotoxin TTX. Despite the potent effects of TTX, some populations of garter snakes are known to prey newts (red).

Two alternative models of adaptive molecular evolution in garter snakes in response to a common selective pressure, TTX poisoning, imposed by their newt prey. (A) In the first hypothesis (i), each population of newt-consuming garter snakes (black cones) has independently evolved resistance to TTX through convergent changes in Na_v1.4, the locus targeted by TTX (gray boxes and newts). Thus, a phylogeny of Na_v1.4 alleles should closely resemble the established garter snake phylogeny based on mtDNA (33). (B) In the second scenario (ii), adaptive variation in Na_v1.4 has a single origin, and the occurrence of elevated TTX resistance in separate garter snake lineages is due to either the unique sorting (recruitment) or introgression of this adaptive variation. If this hypothesis is correct, then we expect TTX-resistant garter snakes to form a monophyletic clade in a Na_v1.4 phylogeny, in contrast to the Thamnophis phylogeny.

Independent, adaptive molecular evolution in the skeletal muscle sodium channel (Na_v1.4). Amino acid replacements at sites important in TTX ligation in the P loops of Na_v1.4 are found only in TTX-resistant garter snakes and appear uniquely derived. (A) Structure of the α-subunit of Na_v1.4 showing the 4 domains (DI–DIV), their 6 transmembrane segments (S1–S6), and the linkers that connect segments. The 4 polypeptide chains that link S5 to S6 (bold) form the outer pore of the channel that allows selective permeation of Na⁺ ions; however, a number of residues that form the outer pore bind strongly to TTX, which occludes the pore and halts Na⁺ movement (see text for further discussion and citations). Approximate location of amino acid substitutions in DIII and DIV P loops (color-coded to species) discussed in text. (B) Phylogeny of Na_v1.4 alleles from Thamnophis and relatives based on 2.9 kb of Na_v1.4 sequence data (4.3-kb alignment), including the coding regions of all 4 P loops (1.0 kb) and portions of 3 linked introns (1.9 kb). The gene tree closely resembles independent estimates of garter snake phylogeny based on mtDNA (Fig. 2), and shows that elevated TTX resistance has evolved multiple times in garter snakes. Topology and nodal support values were estimated via Bayesian tree searches; some outgroups were pruned for simplicity; locality information is in Table S1. (C) Measures of whole-animal resistance to TTX (50% MAMU) alongside amino acid sequences of the DIII and DIV P loops (Table S1). Amino acid substitutions (arrows and replacements color-coded to species) occur at critical residues that change the structure and electrostatic environment of the pore and alter TTX binding affinity. Human sequence is given for comparison (M81758), but amino acid positions follow Na_v1.4 CDS from T. sirtalis AY851746; structures of the pore labeled below human sequence (*, selectivity filter; α, α-helix; β, β-strand).