We've heard the arguments about the relative importance of mutations in cis regulatory regions vs. coding sequences in evolution before — it's the idea that major transitions in evolution were accomplished more by changes in the timing and pattern of gene expression than by significant changes in the genes themselves. We developmental biologists tend to side with the cis-sies, because timing and pattern are what we're most interested in. But I have to admit that there are plenty of accounts of functional adaptation in populations that are well-founded in molecular evidence, and the cis regulatory element story is weaker in the practical sense that counts most in science (In large part, I think that's an artifact of the tools — we have better techniques for examining expressed sequences, while regulatory elements are hidden away in unexpressed regions of the genome. Give it time, the cis proponents will catch up!)

This morning, I was sent a nice paper that describes a pattern of functional change in an important molecule — there is absolutely no development in it. It's a classic example of an evolutionary arms race, though, so it's good that I mention this important and dominant side of the discipline of evolutionary biology — I know I leave the impression that all the cool stuff is in evo-devo, but there's even more exciting biology outside the scope of my tunnel vision. Also, this paper describes a situation and animals with which I am very familiar, and wondered about years ago.

When I was a graduate student in Oregon, I worked now and then with an emeritus faculty member named Jim Kezer — a great guy who was classically trained in natural history, and who would dazzle us benchies by taking us on field trips into the Oregon Cascades, where he could name every weed and insect we'd encounter, and he'd tell us all kinds of stories about these otherwise almost unnoticeable organisms. We made collecting trips up into a remote lake where we'd harvest rough-skinned newts, Taricha granulosa, for histology studies. This lake was swarming with newts — it was pretty much the only large animal you'd find there, and that was because they had a potent biochemical defense mechanism: they oozed a neurotoxin. These newts were not popular denizens of the lakes, because where they were found, the fish and frogs soon disappeared.

The toxin they secreted is called tetrodotoxin, or TTX. It's the same nasty substance that the pufferfish, fugu, contains — it binds the sodium channels of the nerves, blocking all electrical transmission. It's notoriously popular in sushi because at low doses it can cause a tingling sensation, similar to what you felt when the novocaine was wearing off after your visit to the dentist, and it also provides the titillating thrill of danger. Overdoses cause a flaccid paralysis, and can be lethal. More than a mild tingle, I suspect it's that entirely psychological frisson that this food might just kill you that lends fugu its culinary notoriety.

The newt has no other defenses. They don't have fangs or claws, they are as soft as noodles, and so these lakes are reduced to big bowls of squirmy delicate amphibian meat that is frustratingly untouchable by most predators because of the unfortunate fact that they are also using a nasty biotoxin in violation of all of the rules of the Geneva Convention. You might expect that if something…evolved…a countermeasure, this would be a situation ripe for exploitation.

And so it is. Some of the most successful predators of small amphibians are another herpetological marvel, the garter snakes, Thamnophis. Unfortunately, if you feed ordinary garter snakes a diet of rough-skinned newts, they tend to move more and more slowly as the innervation of their skeletal muscles undergoes a toxin blockade, and if they eat enough, they die. This is not a good thing from the snake's perspective, although the newts do get revenge and their relatives benefit from the subsequent reluctance of snakes to eat them. It also presents an evolutionary opportunity, in that resistance to TTX in snakes can be a real advantage, since they won't die and they'll be able to feast on squishy purplish-brown and orange tubes of meat.

This is happening right now. Populations of garter snakes, T. sirtalis, in California, Oregon, and Idaho are showing different degrees of resistance to TTX, and these differences are being traced right down to specific changes in the amino acid sequence of the snake sodium channel. It's happening repeatedly, too, with different populations independently acquiring different variations that confer differing degrees of resistance.

We know a lot about the structure and biophysics of the sodium channel — it's one of those universal proteins we find all over the animal kingdom. It's a protein that loops through the membrane multiple times, forming four cylindrical domains. These cylinders pack together, leaving a space at the center that is the pore proper; there are also regions of the protein that act as gates, opening to allow sodium to flow through and generate an electrical current, or closing to block it.

We also know how TTX works. It binds especially strongly to an aromatic amino acid on the outside of the cell, in domain I. In that place, it effectively blocks the pore, making the channel permanently closed so no current flows.

Obviously, the animal that must most effectively resist the effects of TTX is the one that is producing the toxin. Species that make TTX, like fugu, typically replace that aromatic amino acid with one that doesn't bind TTX. It's a testimony to the hit-or-miss nature of mutations and evolutionary change that the snakes haven't stumbled onto that same change—they've instead made other small changes to the protein to reduce binding of TTX. Instead, they've tweaked the pore helix and β-strand from domain IV, which also reduces the effectiveness of TTX binding.

Here's a summary tree diagram of the differences found in these populations. We're looking at 5 different populations of snakes, named after their collection sites; Benton and Warrenton are in Oregon, Willow Creek is in California, and Bear Lake is in Idaho. Illinois represents the ancestral phenotypic state, a population from a state without TTX-secreting newts, and which has no TTX resistance.

TTX resistance is measured in MAMUs, or mass-adjusted mouse units — low numbers mean they have no particular resistance, while large numbers indicate increasing resistance. The Bear Lake and Illinois populations are sensitive, while the others have varying degrees of resistance.

The right side of the figure is the interesting bit: it shows the amino acid sequence of a small stretch of the protein in domain IV, and you can see the differences. All the resistant populations have a valine at position 1561, but notice that it is likely that these represent two independent origins. That valine alone only weakly improves resistance; the Benton population has an additional amino acid substitution that doubles the resistance. Willow Creek snakes have substantially greater resistance, and they also have 3 other different substitutions.



Amino-acid sequence differences for four snake populations. a, Phylogeographic relationships based on mitochondrial DNA analysis of 19 North American populations of Thamnophis sirtalis indicate separate origins of elevated resistance to TTX in the Willow Creek population compared with populations from Benton and Warrenton. Bear Lake is from a third lineage and is not resistant to TTX. Whole-animal TTX resistance for each population is reported in mass-adjusted mouse units (MAMU); branch colours reï¬ect statistically distinguishable levels of resistance. Whole-animal TTX resistance was measured as a mass-adjusted dose of TTX (MAMU) that produced an average of 50% decrease in snake sprint speed in each population. b, Amino-acid alignment of part of the domain IV S5-S6 linker that affects TTX binding from tsNa V 1.4. Green, pore α-helix; purple, β-strand; asterisk, selectivity ï¬lter. Dots indicate identical amino acids and grey shading highlights sequence differences between populations. Despite independent evolutionary histories, all resistant snakes share the substitution of valine for isoleucine at position 1,561. Amino-acid sequence differences for four snake populations. a, Phylogeographic relationships based on mitochondrial DNA analysis of 19 North American populations ofindicate separate origins of elevated resistance to TTX in the Willow Creek population compared with populations from Benton and Warrenton. Bear Lake is from a third lineage and is not resistant to TTX. Whole-animal TTX resistance for each population is reported in mass-adjusted mouse units (MAMU); branch colours reï¬ect statistically distinguishable levels of resistance. Whole-animal TTX resistance was measured as a mass-adjusted dose of TTX (MAMU) that produced an average of 50% decrease in snake sprint speed in each population. b, Amino-acid alignment of part of the domain IV S5-S6 linker that affects TTX binding from tsNa1.4. Green, pore α-helix; purple, β-strand; asterisk, selectivity ï¬lter. Dots indicate identical amino acids and grey shading highlights sequence differences between populations. Despite independent evolutionary histories, all resistant snakes share the substitution of valine for isoleucine at position 1,561.

There are other details — the proteins have been isolated, chimeric proteins generated to isolate specific regions, and they've been expressed in Xenopus oocytes, all demonstrating that these small changes are actually responsible for conferring TTX resistance. The meat of the story, though, is that we have concrete measurements of specific molecular changes that are responses to an evolutionary arms race, and we're seeing these differences emerge in different populations of a single species. This is evolution in action, and the observed appearance of new properties, traced right down to single changes in proteins.

Geffeney SL, Fujimoto E, Brodie ED III, Brodie ED Jr., Ruben PC (2005) Evolutionary diversification of TTX-resistant sodium channels in a predator-prey interaction, Nature 434:759-763.

Soong TW, Venkatesh B (2006) Adaptive evolution of tetrodotoxin resistance in animals. Trends Genet. 2006 Nov;22(11):621-6.