Beruflich Dokumente
Kultur Dokumente
a
Department of Biology and Mountain Lake Biological Station, University of Virginia, Charlottesville, Va., and
b
Department of Biology, Utah State University, Logan, Utah, USA
00068977/15/08610048$39.50/0 Department of Biology and Mountain Lake Biological Station, University of Virginia
PO Box 400328
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E-Mail karger@karger.com
Charlottesville, VA 22904 (USA)
www.karger.com/bbe
E-Mail bbrodie@virginia.edu
adaptations [DePristo, et al., 2005; Weinreich et al., 2006; whereas closely related species of predators exploiting the
Stern and Orgogozo, 2008, 2009; Christin et al., 2010a]? same prey (with the same defense) might have a high
We know that when two populations or species experi- probability of parallel genetic evolution.
ence a common set of selection pressures, they often con- Neurophysiological phenotypes may be expected to
verge on the same phenotype [Losos, 1992; Joron et al., play an important role in adaptation in the predator-prey
2006; Arendt and Reznick, 2008; Rosenblum et al., 2010; interactions between snakes and amphibians that possess
Dobler et al., 2012]. What is less clear is whether such the poison tetrodotoxin (TTX). TTX binds to and blocks
convergent phenotypes are built by changes to the same voltage-gated sodium channels (NaV) in nerves and mus-
underlying mechanisms. The pan-adaptationist view cles, preventing Na+ movement across the membrane and
leans toward the expectation that selection will always resulting in arrest of action potentials (AP) [Hille, 2001;
find the best answer to the problem, leading to conver- Narahashi, 2008]. This simple but deadly effect of TTX
gence at the level of physiological and genetic mecha- makes it a powerful selection pressure that demands re-
nisms. The population genetic view tends to put more sponse or avoidance by predators. TTX has been found in
stock in the contingent sources of variation, expecting se- a wide variety of organisms, including many species of
lection to generate common phenotypes but perhaps newts and several other amphibian lineages [Hanifin,
through different genetic mechanisms. 2010]. TTX toxicity reaches its pinnacle in the North
The picture that is beginning to emerge from the ex- American newts of the genus Taricha, some of which pos-
ploration of genetic and genomic patterns of convergence sess enough TTX to kill 56 humans [Stokes et al., 2015].
is that adaptation is in fact predictable sometimes. In The origins and synthesis of TTX are not well understood
many cases of parallel (stemming from a recent common in any taxon. The diverse taxonomic distribution of the
ancestor) and convergent (occurring among distantly re- molecule, as well as its detection in free-living bacteria,
lated taxa) evolution, taxa are clearly reusing the same has led to the expectation that bacterial symbionts are re-
genes to solve evolutionary challenges [Arnegard et al., sponsible for TTX production in many taxa [Hanifin,
2010; Christin et al., 2010; Manceau et al., 2010; Rosen- 2010; Hanifin and Gilly, 2015].
blum et al., 2010]. One recent review suggests that the Around the world, snakes represent one of the most
probability of convergent evolution involving the same common groups of predators on amphibians. All snakes
gene lies somewhere between 0.3 and 0.5, with more swallow their prey whole, which means that snakes in-
closely related taxa having a higher likelihood of gene re- gest a full dose of a preys toxicity with each predation
use compared to more distantly related taxa [Conte et al., event. Probably as a result of this feeding behavior,
2012]. This picture primarily comes from studies of con- many snakes have evolved resistance to various toxic
vergence in morphological and color traits. Relatively lit- amphibians [Smith and White, 1955; Brodie, 1968; Bro-
tle is known about convergence and parallelism in the die et al., 1991; Ujvari et al., 2012; Mohammadi et al.,
genetic mechanisms involved with neurophysiological 2013]. In western North America, several species of gar-
traits. Even less is known about the predictability of con- ter snakes of the genus Thamnophis cooccur with the
vergence among gene paralogs that code for proteins ex- highly toxic Taricha and have evolved resistance to the
pressed in different tissues [Jost et al., 2008; McGlothlin TTX they deploy as a defense [Brodie and Brodie, 1991;
et al., 2014]. Brodie et al., 2005]. Moreover, populations of garter
Predator-prey interactions provide an exceptional snakes vary dramatically in their resistance to TTX.
context in which to explore convergence across wide ex- Snakes from localities where Taricha are highly toxic are
panses of evolutionary diversity. Although selection pres- highly resistant to TTX, but outside the range of Taricha
sures on predator and prey may differ because they are or where Taricha have low toxicity, garter snakes ex-
combating one another, both species also have to deal hibit ancestral levels of resistance [Brodie et al., 2002;
with some of the same weaponry, albeit from different Hanifin et al., 2008]. The physiological resistance of
sides of the battleground. Predators may adapt to resist a other snakes to TTX has not been measured, but it is fair
defensive compound used against them, but prey must to assume that, if a snake preys on a tetrodotoxic am-
also evolve mechanisms that allow them to possess and phibian, it must have some ability to tolerate the poison.
tolerate the same compound. Because predators and their Thus, snake predators represent a series of repeated
prey typically belong to vastly different phylogenetic lin- evolutionary experiments with which to test the predict-
eages, we would expect the probability that evolution in- ability of neurophysiological mechanisms during con-
volves common genes in both species to be especially low, vergent evolution.
148.228.86.95 - 3/21/2017 8:13:46 PM
DOI: 10.1159/000435905
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Color version available online
1.2 1,000
1.0
Average AP rise rate ratio
Whole-animal resistance
0.8 100
0.6
0.4 10
104 1.0
0.6
106
0.4
107
Willow Creek 0.2 Willow Creek
Benton Benton
Warrenton Warrenton
Bear Lake Bear Lake
108 0
108 107 106 105 104 1010 109 108 107 106 105 104
c Skeletal muscle Kd (M) d TTX concentration (M)
Fig. 1. The relationships between population differences in whole- pressed in Xenopus oocytes with skeletal muscle fiber Kd. d Con-
animal, muscle fiber, NaV1.4 and chimeric resistance of T. sirtalis. centration response curves of chimeric channels (human NaV1.4
a Population differences in skeletal muscle fiber response to TTX with only DIV amino acid replacements observed in each popula-
concentration for four populations. b Among-population correla- tion substituted). Colors denote the same populations in each pan-
tion of Kd of muscle fiber with whole-animal resistance measured el (colors refer to the online version only). Bars indicate standard
by racetrack locomotor bioassay (in mass-adjusted mouse units errors for each estimate; horizontal bars in d indicate standard er-
required to reduce a snake to 50% baseline crawl speed). c Among- rors around the interpolated 50% Kd. Drawn from data in Geffeney
population correlation of Kd of complete snake NaV1.4 protein ex- et al. [2002, 2005].
ter snakes, Geffeney et al. [2005] examined cDNA of snakes from each locality, with the most resistant snakes
NaV1.4 expressed in skeletal muscle. Although most of exhibiting four unique residues. The least resistant popu-
the outer pore of snake channels exhibited almost com- lation had only a single novel amino acid, a switch from
plete homology with rat and human channels, a limited Ile to Val in the pore helix (I1561V). A nonresistant pop-
number of unique amino acid substitutions were ob- ulation of snakes allopatric to toxic newts had an outer
served in the DIV P-loop. The number of substitutions pore structure identical to that of other vertebrates sus-
correlated roughly with the average level of resistance of ceptible to TTX.
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P M
L
V D Extracellular
E V N S
DI DII DIII T DIV A
Intracellular
NH2 COOH
L *
N A
D Extracellular
S T S
DI DII Y DIII T DIV
Intracellular
NH2 COOH
* N T Q Extracellular
D E A P M N
DI * DII DIII T DIV
G
Intracellular
NH2 COOH
Fig. 2. Convergent amino acid substitutions in NaV1.4 in predators vergent position substitutions shared by all three groups, orange
and prey. A schematic representation of the voltage-gated sodium for those shared by two groups, blue unique to snakes, pink unique
channel protein expressed in muscle is shown for snakes, newts to newts and purple unique to puffer fish. The derived amino acid
and puffer fish (including both NaV1.4a and 1.4b) with amino acid replacement is denoted by the single letter abbreviation in each
replacements in the outer pore noted. Substitutions include the ac- case. Where two or more substitutions are present at a convergent
cumulated derived changes seen in any of the species tested within site in a single group, side-by-side circles are shown; where more
each taxonomic group: snakes [Feldman et al., 2012], newts [Hani- than one substitution is observed at taxonomically unique sites, an
fin and Gilly, 2015] and puffer fish [Jost et al., 2008]. Not all of the asterisk is shown. Drawn from data of Jost et al. [2008], Feldman
substitutions illustrated are known to reduce TTX binding (e.g. DI et al. [2012] and Hanifin and Gilly [2015].
and DII substitutions unique to newts). Red circles indicate con-
transmit AP even when exposed to high TTX concentra- tution common in the DIV of garter snakes (I1561V;
tions. Primitive newts have slightly resistant muscle fibers alignment numbers differ among orthologs in different
compared to other salamanders, but not nearly as ex- taxa). Within the modern newts, three additional substi-
treme as those of their sister clade. As with garter snakes, tutions confer much greater resistance. In DIII, newts
this finding implicates NaV1.4 as a likely mechanism of share the same M1116T that is observed in T. couchii and
TTX resistance in prey. known from mutagenesis studies to reduce TTX binding
In salamanders, the evolutionary transition to with- 15-fold [Terlau et al., 1991; Feldman et al., 2012]. In DIV,
stand TTX and harbor large quantities of the poison for resistant newts have two charge-neutralizing replace-
defense appears to have occurred at the split between ments, D1431S and G1432D, in the identical P-loop posi-
primitive and modern newts. Amino acid replacements tion as the analogous substitutions (D1568N/S and
in the outer pore of NaV1.4 enabled this transition [Hani- G1569V/D) discovered in T. atratus, T. sirtalis and L. epi-
fin and Gilly, 2015]. Both clades of newts have a single nephelus, which are known to reduce TTX binding 300-
amino acid replacement, I1424S, in DIV that confers fold (fig.2) [Geffeney et al., 2005; Feldman et al., 2012;
slight reductions in binding affinity of TTX, consistent Hanifin and Gilly, 2015].
with the low-level muscle resistance measured in primi- Newts are not the only TTX-bearing prey. In fact, TTX
tive newts. This residue is analogous to the Ile-Val substi- was first identified from, and named for, tetraodontid
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NaV1 domains
NaV1.6a
IM DI DII
DIV DIII IV
arom- NaV1.4 DN
nonarom GV
NaV1.6b
NaV1.5
arom- Ancestrally
NaV1.1La nonarom NaV1.8
TTX insensitive
IM MT
NaV1.9
NaV1.1Lb IM MT
NaV1.6 IV
arom-
NaV1.4a nonarom
NaV1.3
IM MT CNS channels
NaV1.2 protected by the
blood-brain barrier
NaV1.1
NaV1.4b
DN
NaV1.7 GY
NaV1.5La
NaV1.5Lb Unknown
Fig. 3. Parallel evolution of TTX resistance in NaV1 paralogs with- across paralogs within each species are shown (unique replace-
in species. The gene trees of the family of paralogous NaV1 (SCNA ments and those found in paralogous genes in other taxa not
genes) are shown for one species of puffer fish (Tetraodon nigro- shown). In DI of puffer fish, arom-nonarom denotes several re-
viridis) and one species of garter snake (T. sirtalis). Boxes represent placements that alter a residue from an aromatic to a nonaromatic
the P-loop of each of the four domains (DIDIV) of the NaV1 pro- amino acid. Drawn from data in Jost et al. [2008] and McGlothlin
tein. Amino acid substitutions in these regions that are convergent et al. [2014].
This pattern of parallelism suggests dramatic con- that reduce TTX-binding affinity also reduce Na+ perme-
straints in the balance between the progressive evolution ability and selectivity [Feldman et al., 2012]. This funda-
of new ability (resistance) and the maintenance of critical mental trade-off suggests there are a limited number of
existing functions (selective Na+ permeability) that may switches that evolution can flip to respond to the selective
be somewhat unique to fundamental proteins such as challenge of TTX, resulting in a highly predictable set of
membrane ion channels. Because they perform such a molecular solutions to evolutionary problems.
critical and conserved role in AP propagation, there is lit- Although the available data point to a narrow and pre-
tle room for reduced functionality. The high sequence ho- dictable adaptive path that involves structural changes to
mology of the pore regions of NaV1 proteins suggests they SCNA genes, there is no reason to imagine that this is the
experience strong purifying selection, at least on the selec- only conceivable mechanism that could confer TTX re-
tivity filter of the channels where TTX binds. Site-directed sistance. Expression patterns of ancestrally resistant so-
mutagenesis studies show that amino acid substitutions dium channels, like the cardiac NaV1.5, could be altered
148.228.86.95 - 3/21/2017 8:13:46 PM
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