Charles T. Hanifin

Phenotypic mismatches reveal escape from arms-race coevolution

Because coevolution takes place across a broad scale of time and space, it is virtually impossible to understand its dynamics and trajectories by studying a single pair of interacting populations at one time. Comparing populations across a range of an interaction, especially for long-lived species, can provide insight into these features of coevolution by sampling across a diverse set of conditions and histories. We used measures of prey traits (tetrodotoxin toxicity in newts) and predator traits (tetrodotoxin resistance of snakes) to assess the degree of phenotypic mismatch across the range of their coevolutionary interaction. Geographic patterns of phenotypic exaggeration were similar in prey and predators, with most phenotypically elevated localities occurring along the central Oregon coast and central California. Contrary to expectations, however, these areas of elevated traits did not coincide with the most intense coevolutionary selection. Measures of functional trait mismatch revealed that over one-third of sampled localities were so mismatched that reciprocal selection could not occur given current trait distributions. Estimates of current locality-specific interaction selection gradients confirmed this interpretation. In every case of mismatch, predators were “ahead” of prey in the arms race; the converse escape of prey was never observed. The emergent pattern suggests a dynamic in which interacting species experience reciprocal selection that drives arms-race escalation of both prey and predator phenotypes at a subset of localities across the interaction. This coadaptation proceeds until the evolution of extreme phenotypes by predators, through genes of large effect, allows snakes to, at least temporarily, escape the arms race.

TOXICITY OF DANGEROUS PREY: VARIATION OF TETRODOTOXIN LEVELS WITHIN AND AMONG POPULATIONS OF THE NEWT Taricha granulosa

The ability to identify and accurately measure traits at the phenotypic interface of potential coevolutionary interactions is critical in documenting reciprocal evolutionary change between species. We quantify the defensive chemical trait of a prey species, the newt Taricha granulosa, thought to be part of a coevolutionary arms race. Variation in newt toxicity among populations results from variation in levels of the neurotoxin tetrodotoxin (TTX). Individual variation in TTX levels occurs within populations. Although TTX exists as a family of stereoisomers, only two of these (TTX and 6-epi-TTX) are likely to be sufficiently toxic and abundant to play a role in the defensive ecology of the newt.

The Chemical and Evolutionary Ecology of Tetrodotoxin (TTX) Toxicity in Terrestrial Vertebrates

Tetrodotoxin (TTX) is widely distributed in marine taxa, however in terrestrial taxa it is limited to a single class of vertebrates (Amphibia). Tetrodotoxin present in the skin and eggs of TTX-bearing amphibians primarily serves as an antipredator defense and these taxa have provided excellent models for the study of the evolution and chemical ecology of TTX toxicity. The origin of TTX present in terrestrial vertebrates is controversial. In marine organisms the accepted hypothesis is that the TTX present in metazoans results from either dietary uptake of bacterially produced TTX or symbiosis with TTX producing bacteria, but this hypothesis may not be applicable to TTX-bearing amphibians. Here I review the taxonomic distribution and evolutionary ecology of TTX in amphibians with some attention to the origin of TTX present in these taxa.

Tetrodotoxin levels of the rough-skin newt, Taricha granulosa, increase in long-term captivity.

We investigated the persistence of the neurotoxin tetrodotoxin (TTX) in individual captive newts (Taricha granulosa) from the Willamette Valley of Oregon using a non-lethal sampling technique. We found that the TTX levels of newts held in the laboratory for 1 yr increased. TTX stereoisomer-analog profiles were not affected by captive husbandry. Levels of TTX were high in newts from our study population and we observed substantial within population variation in quantitative levels of TTX. Females possessed more TTX than males, but the response of TTX levels to captivity did not differ between females and males. The stability of TTX toxicity in newts is consistent with other amphibian species where TTX is present and may indicate that exogenous factors play a less important role in TTX toxicity of newts than previously thought.

Parallel Arms Races between Garter Snakes and Newts Involving Tetrodotoxin as the Phenotypic Interface of Coevolution

Parallel “arms races” involving the same or similar phenotypic interfaces allow inference about selective forces driving coevolution, as well as the importance of phylogenetic and phenotypic constraints in coevolution. Here, we report the existence of apparent parallel arms races between species pairs of garter snakes and their toxic newt prey that indicate independent evolutionary origins of a key phenotype in the interface. In at least one area of sympatry, the aquatic garter snake, Thamnophis couchii, has evolved elevated resistance to the neurotoxin tetrodotoxin (TTX), present in the newt Taricha torosa. Previous studies have shown that a distantly related garter snake, Thamnophis sirtalis, has coevolved with another newt species that possesses TTX, Taricha granulosa. Patterns of within population variation and phenotypic tradeoffs between TTX resistance and sprint speed suggest that the mechanism of resistance is similar in both species of snake, yet phylogenetic evidence indicates the independent origins of elevated resistance to TTX.

TETRODOTOXIN LEVELS IN EGGS OF THE ROUGH-SKIN NEWT, Taricha granulosa, ARE CORRELATED WITH FEMALE TOXICITY

We quantified the amount of the neurotoxin tetrodotoxin (TTX) present in females and newly deposited eggs of the rough-skin newt, Taricha granulosa, to examine the relationship between the toxicity of an individual female and the toxicity of her eggs. We found high levels of TTX in individual eggs as well as substantial variation among clutches. Variation in the amount of TTX per egg within individual clutches was extremely low. Female skin toxicity was positively correlated with the mean egg toxicity of her clutch. Neither egg volume nor female size was significantly correlated with egg TTX levels. Tetrodotoxin stereoisomer–analog profiles were identical for females and their eggs. The presence of high levels of TTX in individual eggs coupled with the relationship between levels of TTX in female skin and levels of TTX in her eggs suggests that the TTX present in eggs of T. granulosa is maternally derived. The lack of correlation between egg size and TTX levels in individual eggs, as well as the low levels of within clutch variation, may indicate that deposition of TTX in eggs of T. granulosa is not linked to the deposition of other egg resources (e.g., lipids or other yolk components).

Ontogeny of Tetrodotoxin Levels in Blue-ringed Octopuses: Maternal Investment and Apparent Independent Production in Offspring of Hapalochlaena lunulata

Many organisms provision offspring with antipredator chemicals. Adult blue-ringed octopuses (Hapalochlaena spp.) harbor tetrodotoxin (TTX), which may be produced by symbiotic bacteria. Regardless of the ultimate source, we find that females invest TTX into offspring and offspring TTX levels are significantly correlated with female TTX levels. Because diversion of TTX to offspring begins during the earliest stages of egg formation, when females are still actively foraging and looking for mates, females may face an evolutionary tradeoff between provisioning larger stores of TTX in eggs and retaining that TTX for their own defense and offense (venom). Given that total TTX levels appear to increase during development and that female TTX levels correlate with those of offspring, investment may be an active adaptive process. Even after eggs have been laid, TTX levels continue to increase, suggesting that offspring or their symbionts begin producing TTX independently. The maternal investment of TTX in offspring of Hapalochlaena spp. represents a rare examination of chemical defenses, excepting ink, in cephalopods.

Confirmation and Distribution of Tetrodotoxin for the First Time in Terrestrial Invertebrates: Two Terrestrial Flatworm Species ( Bipalium adventitium and Bipalium kewense )

The potent neurotoxin tetrodotoxin (TTX) is known from a diverse array of taxa, but is unknown in terrestrial invertebrates. Tetrodotoxin is a low molecular weight compound that acts by blocking voltage-gated sodium channels, inducing paralysis. However, the origins and ecological functions of TTX in most taxa remain mysterious. Here, we show that TTX is present in two species of terrestrial flatworm (Bipalium adventitium and Bipalium kewense) using a competitive inhibition enzymatic immunoassay to quantify the toxin and high phase liquid chromatography to confirm the presence. We also investigated the distribution of TTX throughout the bodies of the flatworms and provide evidence suggesting that TTX is used during predation to subdue large prey items. We also show that the egg capsules of B. adventitium have TTX, indicating a further role in defense. These data suggest a potential route for TTX bioaccumulation in terrestrial systems.

Evolutionary history of a complex adaptation: tetrodotoxin resistance in salamanders.

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.

Historical Contingency in a Multigene Family Facilitates Adaptive Evolution of Toxin Resistance

Novel adaptations must originate and function within an already established genome [1]. As a result, the ability of a species to adapt to new environmental challenges is predicted to be highly contingent on the evolutionary history of its lineage [2-6]. Despite a growing appreciation of the importance of historical contingency in the adaptive evolution of single proteins [7-11], we know surprisingly little about its role in shaping complex adaptations that require evolutionary change in multiple genes. One such adaptation, extreme resistance to tetrodotoxin (TTX), has arisen in several species of snakes through coevolutionary arms races with toxic amphibian prey, which select for TTX-resistant voltage-gated sodium channels (Nav) [12-16]. Here, we show that the relatively recent origins of extreme toxin resistance, which involve the skeletal muscle channel Nav1.4, were facilitated by ancient evolutionary changes in two other members of the same gene family. A substitution conferring TTX resistance to Nav1.7, a channel found in small peripheral neurons, arose in lizards ∼170 million years ago (mya) and was present in the common ancestor of all snakes. A second channel found in larger myelinated neurons, Nav1.6, subsequently evolved resistance in four different snake lineages beginning ∼38 mya. Extreme TTX resistance has evolved at least five times within the past 12 million years via changes in Nav1.4, but only within lineages that previously evolved resistant Nav1.6 and Nav1.7. Our results show that adaptive protein evolution may be contingent upon enabling substitutions elsewhere in the genome, in this case, in paralogs of the same gene family.