Evolutionary history of a complex adaptation: Tetrodotoxin resistance in salamanders

Abstract

Understanding the processes that generate novel adaptive phenotypes is central to evolutionary biology. We used comparative analyses to reveal the history of tetrodotoxin (TTX) resistance in TTX-bearing salamanders. Resistance to TTX is a critical component of the ability to use TTX defensively but the origin of the TTX-bearing phenotype is unclear. Skeletal muscle of TTX-bearing salamanders (modern newts, family: Salamandridae) is unaffected by TTX at doses far in excess of those that block action potentials in muscle and nerve of other vertebrates. Skeletal muscle of non-TTX-bearing salamandrids is also resistant to TTX but at lower levels. Skeletal muscle TTX resistance in the Salamandridae results from the expression of TTX-resistant variants of the voltage-gated sodium channel NaV 1.4 (SCN4a). We identified four substitutions in the coding region of salSCN4a that are likely responsible for the TTX resistance measured in TTX-bearing salamanders and variation at one of these sites likely explains variation in TTX resistance among other lineages. Our results suggest that exaptation has played a role in the evolution of the TTX-bearing phenotype and provide empirical evidence that complex physiological adaptations can arise through the accumulation of beneficial mutations in the coding region of conserved proteins.

Is there more than one way to skin a newt? Convergent toxin resistance in snakes is not due to a common genetic mechanism

“…To date, the evolution of TTX resistance in all animal lineages examined appears to involve a predictable subset of replacements in the outer pore of the sodium channel proteins (Na v ) that interact directly and strongly with TTX (Hille, 2001;Fozzard and Lipkind, 2010). Remarkably, in H. platirhinos, the SCN4A gene (encoding Na v 1.4), a locus that appears to underlie a major portion of resistance in other vertebrates (Geffeny et al, 2005;Soong and Venkatesh, 2006;Jost et al, 2008;Feldman et al, 2012;Hanifin and Gilly, 2015), contains no allelic variation that would alter TTX ligation to the pore. Thamnophis with comparable levels of TTX resistance typically have two to four amino acid substitutions in this protein (Feldman et al, 2009(Feldman et al, , 2010 (Figure 3).…”

“…are known to contribute to TTX resistance in vertebrates (for example, Geffeny et al, 2005;Soong and Venkatesh, 2006;Jost et al, 2008;Hanifin and Gilly, 2015). We isolated and purified genomic DNA from muscle or liver tissue with the DNeasy Tissue Kit (Qiagen, Inc., Germantown, MD, USA).…”

“…In contrast, the allopatric H. platirhinos and H. nasicus showed only base levels of resistance, on par with other nonresistant snakes (Table 2) (Feldman et al, 2012). No H. platirhinos possessed derived SCN4A alleles found in the other TTX-resistant taxa or paralogs that have been examined thus far (Figure 3) (Geffeny et al, 2005;Soong and Venkatesh, 2006;Jost et al, 2008;Feldman et al, 2012;McGlothlin et al, 2014;Hanifin and Gilly, 2015). Thus, the resistance-conferring mutations in SCN4A known to reduce the binding affinity of TTX to the channel appear to be entirely lacking in TTX-resistant H. platirhinos.…”

“…Although TTX is found in a diverse array of taxa (Hanifin, 2010;Moczydlowski, 2013), only a few vertebrate groups appear to have evolved the ability to tolerate TTX, including tetraodontid fishes, newts, some frogs and a handful of snakes (for example, Soong and Venkatesh, 2006;Feldman et al, 2012;Hanifin and Gilly, 2015). Of these taxa, snakes are among the only vertebrates known to regularly consume TTX-defended prey (Feldman et al, 2012).…”

“…Given the highly specific action of TTX, one mechanism of TTX resistance seems obvious: functional changes to the outer pore of sodium channels that reduce the affinity of TTX to the protein. Indeed, all TTX-resistant vertebrates examined thus far have remarkably similar mutations that alter the structure of the outer pore and reduce TTX-binding affinity to the channel (Geffeny et al, 2005;Soong and Venkatesh, 2006;Jost et al, 2008;Feldman et al, 2012;McGlothlin et al, 2014;Hanifin and Gilly, 2015). In fact, this mechanism of TTX resistance appears nearly identical across several independent, coevolutionary arms races involving predatory snakes and their TTX-defended amphibian prey (Feldman et al, 2009(Feldman et al, , 2012, as well as across sodium channel paralogs within species (McGlothlin et al, 2014), suggesting that adaptation to TTX is highly predictable (Jost et al, 2008;Feldman et al, 2012;McGlothlin et al, 2014).…”