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MOLECULAR EVOLUTION dium channel blocker, evolved many times in toxic pufferfish, newts, and that feed on newts via various replacements at residues in NaV1proteinsthatinteractwithTTX Interacting amino acid replacements (17–19). Similarly, resistance to cardiac glycosides, which inhibit the sodium-potassium pump, has allow to evolve evolved at least 14 times in toxic insects and , as well as their predators, via amino resistance acid replacements in the cardiac-glycoside binding site (4, 20). Evolving epibatidine resistance involves dif- Rebecca D. Tarvin,1*† Cecilia M. Borghese,2† Wiebke Sachs,2,3‡ ferent strategies at the molecular level, as epi- Juan C. Santos,4 Ying Lu,1 Lauren A. O’Connell,5§ David C. Cannatella,1,6 batidine is an that shares a binding site R. Adron Harris,2 Harold H. Zakon1 with ACh, the endogenous ligand of nAChRs, whereas TTX and cardiac glycosides act on re- that wield face self-intoxication. Poison frogs have a diverse arsenal of ceptors that are not ligand-gated (21, 22). Re- defensive that target the . Among them is epibatidine, a nicotinic sistance to epibatidine thus requires decreased (nAChR) agonist that is lethal at microgram doses. Epibatidine sensitivity to epibatidine while preserving sen- shares a highly conserved binding site with acetylcholine, making it difficult to evolve sitivity to the ACh that resistance yet maintain nAChR function. Electrophysiological assays of and interacts with many of the same amino acids all nAChR revealed that one amino acid replacement, which evolved three times in poison Downloaded from without disrupting the normal receptor function. frogs, decreased epibatidine sensitivity but at a cost of acetylcholine sensitivity. However, receptor functionality was rescued by additional amino acid replacements that differed Phylogenetic identification of amino among poison frog lineages. Our results demonstrate how resistance to agonist toxins can acid replacements in the poison evolve and that such genetic changes propel organisms toward an adaptive peak of frog nAChR chemical defense. Based on what is known about the and

ligand, we hypothesized that epibatidine-bearing http://science.sciencemag.org/ cquiring chemicals from the environment oration, resulting in a complex phenotype or syn- frogs would have nAChRs that resist epibatidine and recycling them for antipredator defense drome known as (6). Theoretically, yet display normal ACh sensitivity and that the is a survival strategy that has evolved in aposematic and nonaposematic poison frogs rep- basis of resistance would involve genetic changes A nearly every major branch of life (1). Expo- resent alternative peaks on an adaptive landscape in the ligand-binding site. To test this hypothesis, sure to toxic chemicals may have high phys- that arose as a result of disruptive selection that we sequenced genes in poison frogs encoding iological costs, but it can also be an opportunity favored more extreme phenotypes over inter- the primary molecular target of epibatidine in for organisms to capitalize on these substances mediate ones (e.g., conspicuous but not well de- the , the a4b2 nAChR (chrna4 and chrnb2) as new resources. Organisms that accumulate fended, or defended but not aposematic) (7). The (23). Epibatidine has been detected in two dis- these chemicals risk self-intoxication unless they multiple origins of aposematism within dendro- tinct lineages of dendrobatids, and can resist their own defenses through compart- batids suggest that the switch from nonapose- (12), so we predicted two origins of mentalization, metabolic detoxification, or target- matic to aposematic phenotypes is easily attained resistance. Consequently, we sequenced these on September 21, 2017 site insensitivity—i.e., changes in the molecular within this group. Characterizing the evolution of genes from 9 species of these genera, as well as target of the toxin that affect its ability to bind (2). toxin resistance, a key step in this phenotypic 19 other species of poison frogs, including 8 species Many toxins target evolutionarily conserved pro- transition, may reveal pathways between these of Dendrobatinae ( and ), teins such as channels, which govern key adaptive peaks in which toxin resistance facili- a clade of chemically defended poison frogs nervous system functions. Thus, revealing the tates origins of toxin sequestration. lacking epibatidine, and 11 nondefended species mechanistic basis of toxin resistance deepens Chemically defended dendrobatids take up from (table S1) (24). our understanding of protein function and pro- their over 800 types of lipophilic alkaloids (8), Four sites in the b2 subunit (F106, S108, A110, vides insights into nervous system evolution (3, 4). many of which modulate nervous system function and I118, numeration of the mature human protein) Moreover, the physiology of toxin resistance is a (9). Their effects vary from benign to lethal (10), have unique amino acid replacements in the crucial aspect of chemical defense, and charac- but most are bitter-tasting and thus generally -sequestering dendrobatids Epipedobates, terizing the evolution of resistance might elucidate aversive to predators (11). Epibatidine, one of the Ameerega,andDendrobates [subgenus Oophaga how and why organisms acquire toxic defenses (5). best known of these alkaloids, was first isolated sensu (25)](Fig.1A),thelastofwhichisnotknown Neotropical poison frogs (Dendrobatidae) have from the Epipedobates to have epibatidine defenses. These replacements independently evolved chemical defenses at least anthonyi in 1974 (12). Epibatidine has an anal- are near the epibatidine-binding site in the a+–b– four times (6). The origins of chemical defense are gesic effect 200 times that of morphine, yet it interface: between loops A and E and in loop E usually accompanied by shifts toward bright col- targets a specific subset of nicotinic acetylcholine (Fig.1,BtoE)(21, 22). Each of these replacements receptors (nAChRs) rather than opioid receptors involves a single nucleotide change in the first or (13). Because of these qualities, epibatidine has second codon position (table S2) (24), suggesting 1 Department of Integrative Biology, University of Texas at inspired pharmacological innovations, although non-neutral evolution. Five additional sites in a4 Austin, Austin, TX 78712, USA. 2Waggoner Center for and Research, University of Texas at Austin, its has prohibited its successful develop- were found to have amino acid replacements Austin, TX 78712, USA. 3Department of Biology, University of ment as a pharmaceutical (14). unique to these poison frogs, but only one of these Konstanz, Konstanz, Germany 78457. 4Department of Toxic animals, including poison frogs, often (D176N) was near the epibatidine-binding site ’ Biological Sciences, St. John s University, Queens, NY 11439, evolve resistance to their toxins via amino acid (table S3 and fig. S1) (24). USA. 5Faculty of Arts and Sciences Center for Systems Biology, Harvard University, Cambridge, MA 02138, USA. 6Biodiversity replacements in toxin-binding sites (target-site Center, University of Texas at Austin, Austin, TX 78712, USA. insensitivity) (15, 16). The location of these re- Electrophysiology of amino acid *Corresponding author. Email: [email protected] †Authors placements is constrained by protein function, replacements in the poison frog nAChR ‡ contributed equally to the work. Present address: III. Medizinische leading to predictable and convergent mecha- The a4b2 nAChR is a pentameric protein that Klinik, Universitätsklinikum Hamburg-Eppendorf, Hamburg, Germany 20246. §Present address: Department of Biology, nisms of resistance (17). For example, resistance exhibits two different stoichiometries: a high- Stanford University, Stanford, CA 94305, USA. to (TTX), a NaV1 voltage-gated so- ACh sensitivity conformation (HS), (a4)2(b2)3,anda

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on September 21, 2017

Fig. 1. Amino acid replacements in b2 associated with alkaloid-defended are indicated to the left of the alignment (see Table 1). (B and C)Structure poison frogs. (A) Alignment of dendrobatid (black), nondendrobatid (gray), of the human (a4)2(b2)3 nAChR (22) (B) from the side and (C) from and outgroup (gray) b2 sequences (table S4). Yellow branches in the phylogeny extracellular space. a4 subunits are in light gray, b2 subunits are in gold, and [adapted from (25)] indicate alkaloid-defended lineages; asterisks indicate the ligand-binding sites are indicated by gray spheres. (D and E)Closer clades in which epibatidine has been detected; the unit of the scale bar is the view of the binding site from (D) extracellular space and (E) viewpoint number of expected substitutions per site. Focal species names are in bold indicated by labeled arrow in (C). Amino acid residues identified with gray and and colored by their amino acid replacement pattern. The amino acid replaced gold arrows are known to be involved in ACh and/or epibatidine binding only in Epipedobates poison frogs is in red (F106L); amino acids replaced (21, 28, 29). Single-letter abbreviations for the amino acid residues are as only in Ameerega are in purple (A110I and I118V); the convergently evolved follows:A,Ala;C,Cys;D,Asp;E,Glu;F,Phe;G,Gly;H,His;I,Ile;K,Lys;L,Leu; replacement is in cyan (S108C). Genotypes of clades with replacements M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.

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low-ACh sensitivity conformation (LS), (a4)3(b2)2 (26, 27). To determine experimentally whether the identified amino acid replacements provide resistance to epibatidine, we used site-directed mutagenesis to introduce poison frog amino acid replacements into human nAChRs. We then co- expressed the wild-type and mutated b2subunits with human a4 nAChR subunits in Xenopus laevis oocytes and measured acetylcholine and epibatidine concentration-response curves (CRCs) through two-electrode voltage clamp (parame- ters, results, and statistical analyses from all CRCs are shown in tables S5 to S16) (24). For each sub- unit combination, we injected different ratios of a4 and b2 transcripts to favor the formation of either HS or LS conformations (24). For brevity, we describe only HS nAChRs in the main text, because we found the same general pattern of channel sensitivity to ACh and epibatidine in both stoichiometries. For LS nAChR results, see fig. S2 and tables S5 to S7 (24). We also performed Downloaded from electrophysiology experiments to investigate whether the one replacement in the a4 subunit near the ligand-binding site (D176N) affected LS nAChR function, but we found no evidence for an effect (figs. S1 and S4 and table S8) (24).

For clarity, we denote all nAChR genotypes http://science.sciencemag.org/ with four letters indicating the amino acid res- idue at each of the four sites of interest (106, 108, 110, and 118) (see Table 1). Bold letters in each genotype indicate amino acid replacements introduced into a transcript via site-directed mutagenesis.

Human-to-frog mutants The Epipedobates and Ameerega replacement patterns (LCAI and FCVV genotypes) produced

by mutagenesis showed ACh CRCs identical on September 21, 2017 to that of the wild-type human FSAI genotype, whereas the subgenus Oophaga replacement pattern (FCAI) showed a decrease in sensitivity to ACh (Fig. 2A). All three nAChRs with poison frog amino acid replacement patterns (LCAI, FCVV,andFCAI) were less sensitive to epibati- dine than the wild-type receptor (Fig. 2B), in- dicating that these replacement patterns are sufficient to produce epibatidine-resistant phe- notypes (Table 1 and tables S5 to S7) (24). Inter- estingly, the ACh CRC is biphasic for the Oophaga replacement pattern, suggesting that in the hu- man genetic background the S108C replacement mayinduceassemblyofLSnAChRs.Becausethe LS stoichiometry possesses two kinds of binding sites, application of increasing concentration of ACh results in a biphasic curve that reflects activation of the two HS binding sites at low ACh Fig. 2. ACh and epibatidine concentration-response curves in high-sensitivity a4b2 nAChRs. concentrations and of the single LS binding site Left panels show responses to ACh, and right panels show responses to epibatidine. (A and at high ACh concentrations (24). Thus, resistance B) Human a4b2 nAChRs: wild-type genotype (FSAI) and receptors containing the amino acid to epibatidine conferred by the S108C replace- patterns identified in Epipedobates, Ameerega, and Dendrobates (Oophaga) poison frogs ment incurs a cost of ACh sensitivity in the (LCAI, FCVV, and FCAI genotypes, respectively). (C and D) Human a4b2 nAChRs: wild-type (FSAI) human b2subunit. and Ameerega genotypes (FCVV,FCAI, FSVI, and FSAV). (E and F) Human a4b2 nAChRs: We then characterized the physiological effect wild-type (FSAI) and Epipedobates genotypes (LCAI, LSAI, and FCAI). (G and H) Epipedobates a4b2 of each individual replacement in poison frogs by nAChRs: wild-type (LCAI) and human genotypes (FSAI, FCAI, and LSAI). Dotted lines correspond to generating human a4b2nAChRtranscriptswith human FSAI and LCAI curves from (C) and (D). Error bars smaller than the symbols single amino acid replacements (LSAI, FSVI, and are not visible. Data were fitted to either monophasic (solid line) or biphasic (dashed line) curves. (Inset) FSA V). As with the S108C replacement, human Schematic of HS a4b2 nAChR stoichiometry; ligand-binding sites are indicated by arrows. transcripts with the I118V replacement (FSAV,

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Table 1. Effects of amino acid replacements on ligand responses in human and Epipedobates high-ACh sensitivity (HS) nAChRs. Relative fold

change in sensitivity induced by amino acid replacements was calculated as (mutant EC50)/(FSAI EC50) for each genetic background [i.e., (mutant)/ (reference); see tables S5 to S7] (24). Values greater than 1 indicate that relatively more ligand is required to elicit the same response; thus, higher values indicate lower sensitivity. ACh assessments for biphasic curves (>1) are qualitative, but both cases result in lowered sensitivity (see tables S5 and S6) (24). *P < 0.01; **P < 0.001 (two-way analysis of variance, corrected for multiple comparisons using Tukey’s test; see tables S9, S11, S13, and S14) (24).

Human genetic background Epipedobates genetic background

Amino acid Fold change Fold change in Amino acid Fold change Fold change Genotype Genotype replacement(s) in EC50 ACh EC50 epibatidine replacement(s) in EC50 ACh in EC50 epibatidine L106F FSAI (Wild-type) Reference Reference FSAI Reference Reference ...... C108S F106L LCAI 1 17** LCAI (Wild-type) 1 44** ...... S108C

...... LSAI F106L 1 1 LSAI C108S 1 1

...... FCAI S108C >1 49** FCAI L106F 2* 138** S108C FCVV A110V 1 6** –– – – Downloaded from

...... I118V –– – – ...... FSVI A110V 1 1 –– – – ...... FSAV I118V >1 75**

derived in Ameerega) provided moderate resistance genetic background ameliorate the large cost of ing site. Namely, the b2C108 residue directly con- http://science.sciencemag.org/ to epibatidine at a cost of ACh sensitivity, possibly this replacement in the human FCAI genotype tacts the side chain of a4W156, one of the main because this amino acid replacement also in- (Table 1). This difference may be explained by the determinants in stabilizing epibatidine binding duced assembly of LS nAChRs (Fig. 2, C and D, observation that the S108C replacement in human (Fig. 1, D and E) (21, 28). The sulfur-containing and Table 1). In contrast, human receptors with receptors appeared to induce formation of LS side chain of C108 is bulkier than that of serine, either F106L (Epipedobates)orA110V(Ameerega) nAChRs (Fig. 2A), which are less sensitive to ACh and it could modify the epibatidine-W156 inter- displayed no change in ACh and epibatidine sen- than HS nAChRs. However, the Epipedobates action. The I118V replacement in Ameerega,which sitivity, indicating that these replacements prob- nAChR never appeared to form the LS stoichi- also contributes to epibatidine resistance (Fig. 1, ably do not contribute to epibatidine resistance ometry, even when the injected complementary D and E), is next to F119, a residue that interacts (Fig. 2, E to H, and Table 1). Instead, these re- RNA subunit ratio favored its formation (compare with the epibatidine chloropyridine ring and placements appear to compensate for the decrease Fig. 2, G and H, to fig. S2, G and H) (24). Little is stabilizes the epibatidine atom through in ACh sensitivity incurred by the replacements known about the poison frog a4b2nAChR,but its backbone carboxyl group. Moreover, the A110V on September 21, 2017 that provided resistance (Table 1), because human the apparent absence of the LS stoichiometry in replacement is next to V111, another amino acid receptors with the LCAI genotype (Epipedobates) Epipedobates (evidenced by the lack of a bi- residue that interacts with epibatidine via van or the FCVV genotype (Ameerega)bothshowed phasic, right-shifted curve) lessens the cost of derWaalsforces(21, 28, 29). These replacements normal ACh response (Fig. 2, A and B, and the S108C replacement, and might be related are located in b sheets that are involved in epi- Table 1) (24). to epibatidine exposure and resistance. batidine binding but are less involved in ACh As predicted, the Epipedobates FCAI receptor binding (21, 28, 30). The b2− side of the binding Epipedobates-to-human mutants displayed a decrease in sensitivity to epibatidine pocket is further from ACh than is the a4+ side We synthesized and expressed the wild-type compared with FSAI (Table 1 and table S7) (24), and thus forms looser interactions with ACh, Epipedobates a4b2 nAChR (LCAI genotype) and confirming the role of S108C in epibatidine re- such that amino acid replacements in the b− a double mutant replicating the plesiomorphic sistance (Fig. 2, G and H). As with the human- region that allow changes in epibatidine binding human genotype (FSAI) in Xenopus laevis oocytes to-frog LSAI receptor, the Epipedobates-to-human may be less likely to affect ACh sensitivity. This and performed electrophysiology assays. The LSAI receptor affected neither ACh nor epibati- structure-function problem was apparently solved Epipedobates-to-human mutant (FSAI) showed dine sensitivities compared with FSAI (Table 1). via an identical genetic change three times greatly increased sensitivity to epibatidine but The LCAI genotype (wild-type in Epipedobates) within poison frogs and refined via different no change in sensitivity to ACh (Table 1, table S7, displayed normal responses to ACh and de- genetic changes at least twice in these lineages. and Fig. 2, G and H), indicating that the re- creased sensitivity to epibatidine (Table 1). Thus, placements in Epipedobates were necessary for as in the human receptor, C108 provides epi- Evolutionary pathways toward resistance. batidine resistance and L106 appears to com- epibatidine resistance To understand the contributions of each re- pensate by normalizing a4b2 receptor function Toxin resistance often evolves in response to re- placement when it occurs in the poison frog ge- in Epipedobates poison frogs. current exposure to toxins (2, 4, 31, 32); thus, pat- netic background, we expressed the single-mutant terns of resistance should reflect the evolutionary genotypes FCAI and LSAI in the Epipedobates Amino acid replacements in poison frog history of toxin exposure. The evolutionary pat- b2 subunit. Whereas S108C incurred a drastic nAChR are proximal to the epibatidine terns of amino acid replacements in the poison cost in ACh sensitivity in the human genetic back- binding site frog b2 nAChR subunit suggest that in each of ground (Fig. 2A; compare FSAI and FCAI), the We found that amino acid replacements in the the Epipedobates, Ameerega,andDendrobates Epipedobates-to-human FCAI mutant demon- poison frog b2subunit(Fig.1)altera4b2nAChR (Oophaga) clades (Fig. 1A), an ancestral species strated only a minor (but significant) decrease sensitivity to epibatidine (Fig. 2B). We propose was likely exposed to epibatidine, resulting in in sensitivity to ACh (compared to FSAI), sug- that this is in part due to the proximity of the selection for and evolution of epibatidine re- gesting that some other aspects of the poison frog amino acid replacements to the epibatidine bind- sistance about 5, 10, and 8 million years ago,

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respectively (25). Although no clade of poison species sampled), nor in the closely related taxa 17. C. R. Feldman, E. D. J. Brodie Jr., E. D. I. Brodie 3rd, frogs that has epibatidine defense lacks amino Ameerega and (Fig. 1A). Thus, this M. E. Pfrender, Proc.Natl.Acad.Sci.U.S.A.109, 4556–4561 acid replacements in the b2nAChR,epibatidine second pathway, in which a novel genotype evolves (2012). 18. M. C. Jost et al., Mol. Biol. Evol. 25, 1016–1024 (2008). has only been detected in 2 of 3 sampled species without apparent cost, is not found in poison 19. J. W. McGlothlin et al., Mol. Biol. Evol. 31,2836–2846 (2014). of Epipedobates, in 2 of 12 sampled species of frogs. However, this pathway is known in the 20. S. Dobler, S. Dalla, V. Wagschal, A. A. Agrawal, Proc. Natl. Ameerega, and in none of 9 sampled species of brown -hopper (Nilaparvata lugens)(33), Acad.Sci.U.S.A.109, 13040–13045 (2012). Dendrobates (Oophaga)(9, 12). It is possible that in which two amino acid replacements confer 21. N. Kouvatsos, P. Giastas, D. Chroni-Tzartou, C. Poulopoulou, S. J. Tzartos, Proc. Natl. Acad. Sci. U.S.A. 113,9635–9640 (2016). some populations with epibatidine defense are ex- resistance to , a noncompetitive antagonist 22. C. L. Morales-Perez, C. M. Noviello, R. E. Hibbs, Nature 538, tinct or have not been detected or that the dietary of g-aminobutyric acid receptors. This occurs in 411–415 (2016). source of epibatidine, presumed to be an arthro- an apparently sequential process in which the 23. J. P. Sullivan et al., J. Pharmacol. Exp. Ther. 271,624–631 (1994). pod, is not as available as it was long ago (12). second amino acid change provides high re- 24. See the supplementary materials. 25. J. C. Santos et al., PLOS Biol. 7, e1000056 (2009). Although epibatidine resistance may have arisen sistance yet has a high fitness cost and never 26. M. Moroni, R. Zwart, E. Sher, B. K. Cassels, I. Bermudez, as a side effect of some other change to the pro- occurs without the first (33). It is unclear how Mol. Pharmacol. 70, 755–768 (2006). tein, mutations in the ligand-binding domain common such preexisting compensatory muta- 27. K. E. DeDominicis et al., Mol. Pharmacol. 92, 327–337 (2017). 28. S.-X. Li et al., Nat. Neurosci. 14, 1253–1259 (2011). are uncommon (Fig. 1A) and presumably evolve tions are, although it appears that mutations 29. A. K. Hamouda et al., Biochim. Biophys. Acta 1788, 1987–1995 under strong selective pressures. Regardless of providing incremental increases in resistance (2009). theapparentrarityofepibatidineinpoisonfrogs, are quite common. In Danainae butterflies, newts, 30. J. A. Olsen, T. Balle, M. Gajhede, P. K. Ahring, J. S. Kastrup, the epibatidine-resistant phenotype (determined garter snakes, and poison frogs, toxin resistance PLOS ONE 9, e91232 (2014). 31. X. Jiang, D. J. Lonsdale, C. J. Gobler, Limnol. Oceanogr. 56, by electrophysiology) does not appear to have tends to increase over evolutionary time via ad- 947–954 (2011). been lost in any resistant lineages (Oophaga, ditional amino acid replacements that occur in 32. C. R. Feldman, E. D. J. Brodie Jr., E. D. I. Brodie 3rd, Epipedobates,orAmeerega), suggesting that lack parallel with increased concentrations of chem- M. E. Pfrender, Proc. Natl. Acad. Sci. U.S.A. 106, 13415–13420 Downloaded from (2009). of resistance has a high cost, that reversion to a ical defenses (15, 16, 34, 35). It is possible that 33. Y. Zhang et al., Sci. Rep. 6, 32335 (2016). nonresistant phenotype is physiologically difficult, preadaptive mutations that allow resistance to 34. G. Petschenka et al., Evolution 67, 2753–2761 (2013). or that maintenance of epibatidine resistance is evolve with little cost are present in these orga- 35. S. L. Geffeney, E. Fujimoto, E. D. I. Brodie 3rd, E. D. J. Brodie Jr., – not costly. nisms and have not been identified. The pres- P. C. Ruben, Nature 434,759 763 (2005). 36. R. D. Tarvin, E. A. Powell, J. C. Santos, S. R. Ron, The evolutionary patterns underlying origins ence of such preadaptive mechanisms would D. C. Cannatella, Mol. Phylogenet. Evol. 109, 283–295 (2017). of epibatidine resistance in poison frogs reflect imply a shallow, “neutral” valley on the adaptive http://science.sciencemag.org/ an adaptive landscape with two peaks that max- landscape that facilitates the movement from ACKNOWLEDGMENTS imize fitness of alternative phenotypes: toxin- one adaptive peak to another. Data are available in the supplementary materials; sequences resistant and defended or toxin-sensitive and The Epipedobates, Ameerega,andDendrobates are archived in GenBank under accession numbers MF598757 to MF598762, MF580080 to MF580125, and MF619959 and undefended. Given that S108C provides epiba- (Oophaga) clades, which are evolutionarily young MF619960. R.D.T. is indebted to the many herpetological tidine resistance and thatitisfoundinallthree (6, 36), are an example of rapid and ongoing societies that funded this project: American Society of resistant clades, we argue that it provides a sub- diversification possibly driven by the evolution Ichthyologists and Herpetologists, Herpetologists’ League, stantial selective advantage. We suggest two pos- of resistance to antipredator toxins (15). We dem- Texas Herpetological Society, Minnesota Herpetological Society, Society for the Study of Amphibians and , Chicago sible evolutionary pathways for acquisition of toxin onstrate that resistance to epibatidine involves Herpetological Society, and North Carolina Herpetological Society, resistance. In the first, initial replacements may finely tuning a highly conserved binding site in addition to the National Geographic Society Young Explorer’s provide a small selective benefit of resistance yet without disrupting receptor function, providing Grant 9468-14, the Society for the Study of Evolution, the Society of Systematic Biologists, and NSF DEB-1404409. R.D.T. carrysomephysiologicalcostinreceptorfunction. insights into evolutionary pathways culminating on September 21, 2017 was supported by a Graduate School Continuing Fellowship at For example, the S108C replacement arose in- in chemical defenses. Thus, evolution, with mil- University of Texas at Austin and an NSF Graduate Research dependently in all three lineages and is sufficient lions of years and subjects, can solve complex Fellowship. J.C.S. was supported by start-up funds at St. John’s to produce an epibatidine-resistance phenotype. problems in systems biology that may otherwise University and by the National Science and Engineering Research However, it also incurs decreased sensitivity to seem impossible. Council Collaborative Research and Training Experience Training Program in Research at University of British Columbia. ACh in both the human and the Epipedobates J.C.S. also thanks J. W. Sites Jr., for his support as a postdoctoral backgrounds (Table 1), and the fitness cost of advisor at Brigham Young University with grants NSF EF-1241885 REFERENCES AND NOTES this replacement in living organisms is not clear. and EF-1241848. W.S. thanks D. R. Dietrich of University of 1. E. D. I. Brodie 3rd, Curr. Biol. 19, R931–R935 (2009). We speculate that yet unidentified mutants Konstanz for his support as a thesis advisor. L.A.O. was supported 2. L. Després, J.-P. David, C. Gallet, Trends Ecol. Evol. 22, by a Bauer Fellowship at Harvard University, a William F. Milton in the poison frog nAChR sustained receptor 298–307 (2007). grant from Harvard Medical School, and a L’Oreal For Women in functionality—i.e., by inducing nAChR expres- 3. J. W. McGlothlin et al., Curr. Biol. 26, 1616–1621 (2016). Science Fellowship. D.C.C. was supported by NSF DEB-1556967. – sion changes—until other replacements such as 4. B. Ujvari et al., Proc.Natl.Acad.Sci.U.S.A.112, 11911 11916 H.H.Z. was supported by NSF IOS-1557857 and PLR-1443637. C.M.B. (2015). F106L evolved to rescue receptor sensitivity to and R.A.H. were supported by the University of Texas at Austin 5. S. Dobler, Basic Appl. Ecol. 2,15–26 (2001). Waggoner Center for Alcohol and Addiction Research and NIH R01- ACh. Disruptive selection on populations with 6. J. C. Santos et al., Proc. Biol. Sci. 281, 20141761 (2014). AA006399. We thank the two anonymous reviewers and P. Andolfatto both genotypes may have propelled the popula- 7. S. Wright, Proc. Sixth Int. Congr. Genet. 1, 356–366 (1932). for comments that greatly improved the manuscript. We also thank tions with S108C toward a new adaptive peak. 8. J. W. Daly, T. F. Spande, H. M. Garraffo, J. Nat. Prod. 68, L. Coloma, A. Amézquita, M. Betancourth, S. Ron, and T. LaDuc 1556–1575 (2005). In the second possible trajectory, certain mu- for facilitating specimen collection and access to museum 9. J. C. Santos, R. D. Tarvin, L. A. O’Connell, in Chemical Signals in tissues. All specimens were collected under approved protocols tants already present in the gene pool provide a 13, B. A. Schulte, T. E. Goodwin, M. H. Ferkin, Eds. (Harvard University IACUC 12-10 and UT Austin IACUC 2012-00032 genetic background in which resistance arises (Springer International Publishing, Switzerland, 2016), and 2015-00205) and valid permits [001-13 IC-FAU-DNB/MA – without cost. For example, the artificial genotype pp. 305 337. and 001-11 IC-FAU-DNB/MA (Ecuador), IBD0359 Res 1177-2014 10. M. F. Roberts, M. Wink, Alkaloids: Biochemistry, Ecology and (Colombia), and SPR-1097-912 (USA)]. LSAI (F106L) shows no reduction in either ACh Medicinal Applications (Springer US, New York, 1998). or epibatidine sensitivity (Table 1). Thus, a frog 11. C. R. Darst, M. E. Cummings, Nature 440, 208–211 (2006). SUPPLEMENTARY MATERIALS 12. J. W. Daly, H. M. Garraffo, T. F. Spande, in Alkaloids: Chemical species with F106L has evolved a novel geno- www.sciencemag.org/content/357/6357/1261/suppl/DC1 and Biological Perspectives, S. W. Pelletier, Ed. (Pergamon, type (LSAI), intermediate between FSAI (ple- Materials and Methods New York, 1999), vol. 13, pp. 1–161. Supplementary Text siomorphic) and LCAI in Epipedobates,without 13. T. F. Spande et al., J. Am. Chem. Soc. 114, 3475–3478 (1992). Figs. S1 to S4 incurring a cost, which subsequently allows the 14. M. Dukat, R. A. Glennon, . Mol. Neurobiol. 23, 365–378 Tables S1 to S16 (2003). C108 replacement to also evolve without cost. Data S1 and S2 15.R.D.Tarvin,J.C.Santos,L.A.O’Connell, H. H. Zakon, However, the LSAI genotype does not exist in any References (37–51) D. C. Cannatella, Mol. Biol. Evol. 33,1068–1081 (2016). taxa we sampled. It is not present in Silverstoneia, 16. C. T. Hanifin, W. F. Gilly, Evolution 69,232–244 26 May 2017; accepted 24 August 2017 the sister group of Epipedobates (two of eight (2015). 10.1126/science.aan5061

Tarvin et al., Science 357, 1261–1266 (2017) 22 September 2017 5of5 Interacting amino acid replacements allow poison frogs to evolve epibatidine resistance Rebecca D. Tarvin, Cecilia M. Borghese, Wiebke Sachs, Juan C. Santos, Ying Lu, Lauren A. O'Connell, David C. Cannatella, R. Adron Harris and Harold H. Zakon

Science 357 (6357), 1261-1266. DOI: 10.1126/science.aan5061

Poison frogs resist their own chemical defense Poison frogs produce a neurotoxin that protects them from predation. The frogs, however, run the risk of

intoxicating themselves. Studying the frog neurotoxin epibatidine, which binds to acetylcholine receptors, Tarvin et al. Downloaded from found a single amino acid substitution. The substitution changes the configuration of the acetylcholine receptor, so that it decreases its sensitivity to the toxin. But acetylcholine signaling is essential for normal life. Expressing frog receptors in human cells revealed that different amino acid substitutions have occurred in different lineages that allow the frog to resist its own toxins while still letting target function effectively. Science, this issue p. 1261 http://science.sciencemag.org/

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