Snakes (Colubridae: Erythrolamprus) With a Complex Toxic Diet Show Convergent yet Highly Heterogeneous Voltage-gated Sodium Channel Evolution.

Chemical defense has evolved convergently across multiple lineages and plays a crucial role in shaping ecological communities through selection for toxin resistance. Research on toxin resistance has been pivotal in understanding the genetic basis of trait evolution, as resistance can evolve through mutations in a few target genes, resulting in target-site resistance (TSR). However, in tropical ecosystems, multiple selective pressures from prey with different toxins create complex chemical scenarios for predators that require multiple resistance mechanisms. Royal ground snakes (Erythrolamprus spp.) are significant but understudied predators of poisonous frogs (families Bufonidae and Dendrobatidae), whose toxins affect voltage-gated sodium channels (VGSCs) and other neuromuscular system proteins. In this study, we investigated the evolution of TSR in VGSC genes in relation to toxic frog predation in Erythrolamprus snakes, tracing the phylogenetic origin and geographic distribution of TSR-conferring genotypes across six species in this group. Our findings reveal convergent yet highly heterogeneous TSR evolution in at least two species that evolved to predate poisonous frogs, and possibly in a third one. Amino acid changes at nine resistance-related positions across eight VGSC genes were identified, suggesting a shared evolutionary path across this gene family. Four of these changes are known to provide tetrodotoxin resistance in other animals. We observed polymorphism in resistance-related sites across species and VGSC paralogs, hinting at a complex evolutionary history of alleles at these loci. These findings offer new insights into adaptive mechanisms in predators with complex toxic diets and introduce Erythrolamprus as a model to understand variation in toxin resistance mechanisms.