Widespread convergence in toxin resistance bypredictable molecular evolutionBeata Ujvaria,b,1, Nicholas R. Casewellc,1,2, Kartik Sunagard,1, Kevin Arbucklee, Wolfgang Wüsterf, Nathan Log,Denis O’Meallyh, Christa Beckmanna, Glenn F. Kingi, Evelyne Deplazesi, and Thomas Madsena,j,k,2

aCentre for Integrative Ecology, School of Life and Environmental Sciences, Deakin University, Geelong, VIC 3216, Australia; bFaculty of Veterinary Science,University of Sydney, Sydney, NSW 2006, Australia; cAlistair Reid Venom Research Unit, Department of Parasitology, Liverpool School of Tropical Medicine,Liverpool L3 5QA, United Kingdom; dDepartment of Ecology, Evolution and Behavior, The Alexander Silberman Institute of Life Sciences, The HebrewUniversity of Jerusalem, Jerusalem 91904, Israel; eInstitute of Integrative Biology, University of Liverpool, Liverpool L69 7ZB, United Kingdom; fMolecularEcology and Fisheries Genetics Laboratory, School of Biological Sciences, Bangor University, Bangor LL57 2UW, United Kingdom; gSchool of BiologicalSciences, University of Sydney, Sydney, NSW 2006, Australia; hInstitute for Applied Ecology, University of Canberra, Bruce, ACT 2601, Australia; iInstitute forMolecular Bioscience, The University of Queensland, St Lucia, QLD 4072, Australia; jSchool of Molecular Biosciences, University of Sydney, Sydney, NSW 2006,Australia; and kSchool of Biological Sciences, University of Wollongong, Wollongong, NSW 2522, Australia

Edited by David M. Hillis, The University of Texas at Austin, Austin, TX, and approved July 27, 2015 (received for review June 17, 2015)

The question about whether evolution is unpredictable and stochas-tic or intermittently constrained along predictable pathways is thesubject of a fundamental debate in biology, in which understandingconvergent evolution plays a central role. At the molecular level,documented examples of convergence are rare and limited tooccurring within specific taxonomic groups. Here we provideevidence of constrained convergent molecular evolution acrossthe metazoan tree of life. We show that resistance to toxic cardiacglycosides produced by plants and bufonid toads is mediated bysimilar molecular changes to the sodium-potassium-pump (Na+/K+-ATPase) in insects, amphibians, reptiles, and mammals. In toad-feeding reptiles, resistance is conferred by two point mutationsthat have evolved convergently on four occasions, whereas evi-dence of a molecular reversal back to the susceptible state invaranid lizards migrating to toad-free areas suggests that toxinresistance is maladaptive in the absence of selection. Importantly,resistance in all taxa is mediated by replacements of 2 of the 12amino acids comprising the Na+/K+-ATPase H1–H2 extracellular do-main that constitutes a core part of the cardiac glycoside binding site.We provide mechanistic insight into the basis of resistance by show-ing that these alterations perturb the interaction between the car-diac glycoside bufalin and the Na+/K+-ATPase. Thus, similar selectionpressures have resulted in convergent evolution of the same molec-ular solution across the breadth of the animal kingdom, demonstrat-ing how a scarcity of possible solutions to a selective challenge canlead to highly predictable evolutionary responses.

constraint | parallelism | genotype phenotype | ion transporters |bufotoxin cardenolide

Convergent evolution is the process by which phenotypicsimilarities evolve independently among disparate species

(1–3). Although convergence is sometimes distinguished fromparallel evolution, both are part of a continuum, which often makesattempting to distinguish between them problematic and poten-tially misleading (4); here, we simply use the term “convergence”throughout. Convergence has strong bearing on a fundamental andheated debate on the predictability of evolution. Epitomized by thewritings of Gould (5) and Conway Morris (6), this debate centerson whether evolution is stochastic and unpredictable (5) or subjectto constraints that limit the available options for evolution, resultingin frequent convergence and a degree of predictability (6). However,evidence of convergence at the genetic level, where similar molec-ular changes confer the same change in phenotype, is currentlylimited to only a few taxonomically restricted examples (7–11).Cardiac glycosides offer an ideal model system to investigate the

extent to which evolution can be constrained to predictable changesthroughout the animal kingdom. These organic compounds arehighly toxic molecules that inhibit the sodium-potassium-pump(Na+/K+-ATPase), which disrupts ion transport and thereby

perturbs membrane potentials, often resulting in lethal car-diotoxicity (12, 13). Cardiac glycosides are produced independentlyby a number of plants and bufonid toads as secondary metabolitesand are used for defense against natural enemies. For example,milkweed, foxglove, and oleander plants produce “cardenolides”(e.g., ouabain) that protect against herbivorous insects (13), whereasbufonid toads secrete structurally and functionally similar de-fensive compounds, “bufotoxins” (e.g., bufalin), from their parotoidglands (14).Despite the toxicity of these molecules, natural resistance ex-

ists in many different herbivores and predators and is pre-dominately mediated by molecular alterations to the H1–H2extracellular domain of the Na+/K+-ATPase, resulting in target-site insensitivity to cardiac glycosides (9, 15, 16). For example,we recently showed that two amino acid replacements in theNa+/K+-ATPase α3 subunit (previously denoted α1) are responsible

Significance

Convergence has strong bearing on the fundamental debateabout whether evolution is stochastic and unpredictable orsubject to constraints. Here we show that, in certain circum-stances, evolution can be highly predictable. We demonstratethat several lineages of insects, amphibians, reptiles, andmammals have utilized the same molecular solution, via theprocess of convergence, to evolve resistance to toxic cardiacglycosides produced defensively by plants and bufonid toads.The repeatability of this process across the animal kingdomdemonstrates that evolution can be constrained to proceedalong highly predictable pathways at molecular and functionallevels. Our study has important implications for conservationbiology by providing a predictive framework for assessing thevulnerability of native fauna to the introduction of invasivetoxic toads.

Author contributions: B.U., N.R.C., and T.M. conceived and led the study; B.U., W.W., D.O.,C.B., and T.M. obtained samples; B.U. and T.M. generated DNA sequences; N.R.C. and K.S.performed evolutionary analyses; N.R.C. and K.A. performed isoelectric point and chargeanalyses; G.F.K. and E.D. performedmolecular modelling experiments; B.U., N.R.C., K.S., K.A.,W.W., N.L., G.F.K., E.D., and T.M. interpreted the resulting data; N.R.C. and T.M. wrote thepaper with input from all other authors.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Freely available online through the PNAS open access option.

Data deposition: The sequences reported in this paper have been deposited in the Gen-Bank database (accession nos. KP238131–KP238176).1B.U., N.R.C., and K.S. contributed equally to this work.2To whom correspondence may be addressed. Email: nicholas.casewell@lstmed.ac.uk ormadsen@uow.edu.au.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1511706112/-/DCSupplemental.

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for a 3,000-fold increased resistance to bufalin in toad-feedingAfrican and Asian varanid lizards, compared with toad toxin-susceptible Australian varanids (17). However, until now, com-parisons of the molecular basis of resistance and mechanisms ofaction across the diverse range of resistant taxa reported (9, 15, 17,18) have been lacking. Here we present an analysis of convergentmolecular changes to the Na+/K+-ATPase H1–H2 domain incardiac glycoside-resistant invertebrates and vertebrates. Our re-sults support the view that molecular evolution in the animalkingdom can be heavily constrained, resulting in convergent pro-cesses canalizing evolution along highly predictable pathways.

Results and DiscussionTo examine the molecular mechanism of cardiac glycoside re-sistance, we sequenced the nucleotides coding the resistance-conferring 12 amino acids of the H1–H2 extracellular domain ofthe α3 subunit of the Na+/K+-ATPase in 47 squamate reptiles.These squamates represent 22 species that are known to be re-sistant to the toxic effects of the cardiac glycosides produced bybufonid toads, 18 that are susceptible and 7 that have not beenrecorded feeding on toads (SI Appendix, Table S1). Four aminoacid replacements were observed in the 47 taxa. Remarkably, alltoad toxin-resistant squamates have the same amino acid re-placements at two codons, leucine (L) at position 111 and arginine(R) at position 120, as those previously demonstrated to conferresistance to toad toxins in African and Asian varanid lizards (17).Furthermore, we find that squamates that are susceptible to toadtoxins (or that do not feed on toads) share the same amino acids atthese codons as susceptible Australian varanid lizards [glutamine(Q) at 111 and glycine (G) at 120]. The remaining two aminoacid positions (113 and 119) vary in composition across bothsusceptible and resistant squamates, strongly suggesting thatthese replacements do not affect toad toxin resistance.We next applied an evolutionary approach to test whether the

resistant genotypes resulted from homology or homoplasy by re-constructing the evolutionary history of sequence change in theNa+/K+-ATPase and estimating the ancestral character state across

the squamate tree. Our analyses demonstrate that ancestral squa-mates were susceptible to toad toxins and that resistance hasevolved convergently on at least four occasions, each governed by asingle, identical point mutation at two codons (111 and 120) (Fig.1). In addition to Afro-Asian varanids (17), we find convergentresistance-conferring amino acid changes in the phylogeneticallydistinct viperid, elapid, and natricine snake lineages (includingmembers of the toad toxin sequestering genus Rhabdophis) (19),representing convergent molecular evolution over ∼165 millionyears of separation (20). This constitutes compelling evidence ofadaptive molecular convergence underlying phenotypic conver-gence. Although 85% of the H1–H2 domain of the Na+/K+-ATPase remains highly conserved under the influence of strongnegative selection pressures [nonsynonymous to synonymousrate ratio (ω) of 0.07], one of the codons that governs resistance(position 111) was found to escape these evolutionary con-straints in toxin-consuming species and experience episodicbursts of diversifying selection (SI Appendix, Fig. S1). Fur-thermore, the two toxin resistance governing codons (111 and120) were found to be coevolving [posterior probability (pp) =0.83] and directionally evolving toward the amino acid targetsthat confer resistance to cardiac glycosides (L111 and R120).While basal Afro-Asian varanids possess the resistant genotype

(L111 and R120), the Australian varanids are characterized by thesusceptible genotype (Q111 and G120), indicating that the lattermay have reverted back to the ancestral state of “toad toxin sus-ceptibility” (Fig. 1). Although the alternative hypothesis, thatAsian and African varanids have independently evolved resistanceto toad toxins, appears similarly parsimonious (two gains versusone gain and one loss), our ancestral sequence reconstruction an-alyses support the reversal hypothesis. Indeed, a combination ofBayesian and maximum likelihood reconstruction methods pro-vided robust support (all key nodes pp > 0.95) for an early originof resistance in basal varanids followed by a reversal to thesusceptible genotype in varanids that migrated to the toad-freeAustralian continent (Fig. 1). Examples of such reversals areseemingly rare, as they usually relate to complex morphological

Fig. 1. Convergent molecular evolution of resistance to toad toxins in squamate reptiles and reversal to susceptibility in Australian varanid lizards. The timing ofchanges to resistant amino acids in the H1–H2 extracellular domain of the Na+/K+-ATPase gene correlates with taxa that feed on toads. Pictures of toads indicate cladesof taxa that are known to feed on toads without ill effects. The picture of a toad circled in red highlights that Australian varanid lizards have reverted back to beingsusceptible to toad toxins. Colored branches indicate the amino acid composition at key positions (susceptible, Q111 and G120; resistant, L111 and R120), andchanges in color represent the reconstructed timings of amino acid replacements. Sites 111 and 120 were found to be coevolving (pp = 0.83). The character state(resistant or susceptible) at all key nodes in the tree, including those relevant for timings of character change, are strongly supported (pp ≥ 0.95); nodes withasterisks represent those falling beneath this threshold. Species tree was generated from refs. 57, 58.

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characters (21, 22) rather than molecular changes to specificgenes. This apparent reversal suggests a level of constraint actingon the Na+/K+-ATPase, most likely in the form of a trade-offbetween reduced Na+/K+-pump efficiency in the resistant state(23) and the ability to feed on toxic toads. In this context, theseconstraints lend support to Conway Morris’ concept of few“engineering optima” for biological functions, which may result inpredictable evolution (6).Our analysis of the Na+/K+-pump in squamate reptiles demon-

strates the repeatability of molecular changes that underpin thegain and secondary loss of resistance to cardiac glycosides. We next

assessed whether convergent changes to the H1–H2 extracellulardomain occur in the diversity of other phyla reported to haveNa+/K+-pump–mediated resistance to cardiac glycosides, specifically(i) insects that feed on and sequester toxins from cardenolide-producing plants (9), (ii) muroid rodents that feed on thoseplants and insects (and in some cases bufonid toads) (24, 25),(iii) the frog Leptodactylus latrans (formally referred to asLeptodactylus ocellatus) that preys on bufonids (18), and (iv) bufonidtoads themselves (18) to prevent autotoxicity.First, we reconstructed the evolutionary history of the multilocus

Na+/K+-ATPase gene family and found that, in contrast to squamate

Fig. 2. Cross-phyla molecular convergence in theH1–H2 extracellular domain of the α Na+/K+-ATPaseresulting in resistance to cardiac glycosides. Keyamino acid residues found in the extracellular regionare labeled by number. Resistance conferring residuesare found grouped at position 111 at the N-terminalend and at positions 119, 120, and 122 at the C-ter-minal end. All individual amino acid changes havebeen demonstrated to contribute to resistance tocardiac glycosides in prior functional studies (9, 15–17), with the exception of 111E proposed by Dobleret al. (9) and 119D proposed and validated here (Fig.4). Convergent changes observed within listed taxaare indicated by boxes, with numbers reflecting thenumber of independent changes within that lineage(e.g., convergent changes from Q to L at 111 has oc-curred three times in snakes). Amino acid changes arehighlighted by letters (see also Fig. 1 and SI Appendix,Figs. S4–S6 and S8) and changes in charge by color(green, neutral charge; blue, positive charge; red,negative charge). Schematic of the Na+/K+-ATPasewas modified from ref. 15.

Fig. 3. Convergent evolution of animal resistance to cardiac glycosides is mediated by changes in the isoelectric point of the H1–H2 extracellular domain ofthe α Na+/K+-ATPase. A schematic tree of animal life (central) displays divergence times (in Myr) of major animal lineages based on paleontological constraints(26). Arrows on the tree represent the four major phyla analyzed here (insects, anuran amphibians, squamate reptiles, and mammals). For each phylum, a3D phylogenetic tree displays reconstructed changes in the isoelectric point of the H1–H2 extracellular domain of the α Na+/K+-ATPase. Increases in the z axisreflect increases in isoelectric point, which are largely mediated by the replacement of amino acids with charged residues (Fig. 1 and SI Appendix, Table S2 andFigs. S4–S6 and S8). Animal pictures represent taxa found in each phylum that are resistant to cardiac glycosides. Note that the substantial decrease inisoelectric point observed in the squamate tree (red arrow) represents Australian varanid lizards, which have reverted back to the susceptible state.

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reptiles, resistance in the above-described taxa is governed bychanges to the H1–H2 extracellular domain of the α1 subunit (SIAppendix, Fig. S2). Reconstructions of the evolutionary history ofα1 sequences revealed that, in all lineages, changes at two aminoacid positions (mediated by one or two point mutations per codon)of the H1–H2 domain confer toxin resistance (SI Appendix, Fig.S3–S6). Notably, one of these codons (111) is found consistentlyacross all taxa, whereas the second varies between phyla (119, 120,or 122) (Fig. 2). However, we find that previously describedchanges in the rat (Q111R and N122D) that synergistically confer

1,000-fold resistance to the cardenolide ouabain (15) are present inadditional rodent taxa (SI Appendix, Fig. S4) and are identical tothose observed in the bufonid-eating frog L. latrans, thus demon-strating molecular convergence between amphibians and mammals(Fig. 2) after ∼330 million years of separation (26).To understand the functional significance of these mutations, we

analyzed the specific amino acid replacements underpinning re-sistance to cardiac glycosides across all phyla. We find that sig-nificant increases in the isoelectric point of the Na+/K+-ATPaseH1–H2 domain occur in resistant taxa compared with theirsusceptible counterparts (t test, P = 1.42 × 10−17; Fig. 3 and SIAppendix, Table S2). In all vertebrates and many insects, re-sistance to cardiac glycosides is mediated by the addition ofcharged amino acids to the ends of the H1–H2 extracellulardomain (Fig. 2). Indeed, compared with all possible amino acidreplacements, we find a significant excess of those that result inchanging the charge of the H1–H2 domain in species that areresistant to cardiac glycosides (binomial test, P = 6.9 × 10−7). Thisprovides very strong evidence that resistance is a consequence ofshifts from neutral amino acids to charged amino acids in thisparticular domain throughout the extremely divergent taxa in-vestigated herein.To elucidate how resistance is mediated by changes in charge,

we modeled the binding interaction between bufalin and the Na+/K+-pump of various animal taxa, using the pig Na+/K+-ATPase/bufalin crystal structure as our template (27). We compared theprotein–ligand interactions of resistant, wild-type Na+/K+-ATPase from the rat, Leptodactylus frog, and bufonid toad withmutants designed to reflect the ancestral, susceptible genotype.Due to the paucity of full-length sequences for squamates, weused the susceptible, wild-type Na+/K+-ATPase isolated from thegenome of the Burmese python (28) as our model and comparedit with mutants designed to confer the resistant genotype. In allcases, bufalin was found to bind in a similar orientation to thatobserved in the crystal structure of the pig Na+/K+-ATPase/bufalin complex (27); that is, bufalin wedges into an extracellular-facing cavity formed between helices αM1–M2 (encoded by theH1–H2 extracellular domain) and αM4–M6, with the lactonemoiety deeply buried in a hydrophobic funnel formed by αM4–M6(Fig. 4 and SI Appendix, Fig. S7). The β-surface of the steroid corein bufalin faces polar side chains in αM1–M2 (Fig. 4B and SI Ap-pendix, Table S3). However, there are critical differences in in-termolecular contacts between susceptible and resistant genotypesof the Na+/K+-ATPase (SI Appendix, Table S3). Most strikingly,hydrogen bonds between the OH14β group of bufalin (a substituentthat is conserved in all cardiac glycosides) and one or both of thecarbonyl oxygen atoms of D121 are present in all susceptiblemodels but absent from all of the resistant models, regardless of thenature of the proximal resistance mutation at positions 119, 120, or122. We predict that the net effect of these subtle alterations inintermolecular contacts will be decreased affinity of bufalin binding,leading to significantly reduced inhibition of Na+/K+-ATPase ac-tivity. Our data therefore provide mechanistic insight into theconvergent molecular evolution of cardiac glycoside resistance andvalidate mutagenesis studies describing specific amino acid changesthat confer resistance in rodents and squamates (15–17).Bufalin is only a moderately potent inhibitor of Na+/K+-ATPase

(Kd ∼43 nM for human Na+/K+-ATPase) (29), and therefore, evena 10-fold reduction in binding affinity is likely to significantly im-pair its ability to induce a pathological level of Na+/K+-ATPaseinhibition. This situation is comparable to kdr and superkdr mu-tations in arthropod voltage-gated sodium (NaV) channels thatconfer resistance to pyrethroid insecticides and DDT. Single pointmutations in domain II of arthropod NaV channels (mostly com-monly a semiconservative L1014F mutation in the pore-formingdomain II S6 helix) reduce deltamethrin affinity by only 17-fold,but this is sufficient to confer a high level of resistance to thispyrethroid (30). Analogous to bufalin resistance, a higher level of

Fig. 4. Interactions between susceptible and resistant Na+/K+-ATPase andbufalin. (A) Chemical structure of bufalin. (B) Bufalin binding pocket in thecrystal structure of bufalin bound to pig Na+/K+-ATPase-α1 (Sus scrofa; sus-ceptible genotype; PDB ID code 4RES) (27). Bufalin wedges into a cavityformed by helices αM1–M2 (orange) (encoded by the H1–H2 extracellulardomain), αM3–M4 (red), and αM4–M6 (gray). (C) The best structure fromdocking bufalin into a model of native (cardiac glycoside resistant) bufonidtoad Na+/K+-ATPase. The β-surface of bufalin interacts with residues Q111,E117, E327, and T797 but makes no interactions <4 Å with D121. (D) The beststructure from docking bufalin into a model of bufonid toad Na+/K+-ATPasewith substitutions R111Q and D119N, thereby forming the susceptible ge-notype. The β-surface of bufalin interacts with residues E117, E327, andT797, and the OH14β group forms two hydrogen bonds (<3 Å) with the side-chain carboxyl of D121. (E) The best structure from docking bufalin into amodel of native (cardiac glycoside resistant) rat Na+/K+-ATPase. The β-sur-face of bufalin forms a hydrogen-bond with residues E327 and T797 but notD121. (F) The best structure from docking bufalin into a model of rat Na+/K+-ATPase with substitutions R111Q and D122N, thereby forming the suscep-tible genotype. The β-surface of bufalin forms hydrogen bonds with bothT797 and D121 (H bonds < 3 Å). Predicted binding modes and ligand–proteininteractions in resistant and susceptible genotypes of the Leptodactylus frog,hedgehog, and python can be found in the SI Appendix, Fig. S7 and Table S3.

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insecticide resistance is conferred by the combination of just twoNaV channel mutations in superkdr mutants (31).Because the evolution of resistance to cardiac glycosides ap-

pears to be highly predictable, we next interrogated Na+/K+-ATPase sequence data from a previously unstudied taxon, theEuropean hedgehog (Erinaceus europaeus), to establish whetherthese predictions hold in general. European hedgehogs fre-quently feed on bufonid toads and even anoint themselves withtoad toxins for antipredator defense (32, 33). Our analyses showthat the European hedgehog has the same resistance-conferringamino acid replacements in the H1–H2 extracellular domainof the α1 subunit (R and D) as those observed in rodents (SIAppendix, Fig. S8). Remarkably, these changes add identicalcharged residues at the same amino acid positions (111 and 119)as those found in bufonid toads (Figs. 2 and 3) and hence disruptcardiac glycoside binding interactions in a highly similar manner(SI Appendix, Fig. S7). These results further support strong mo-lecular convergence between amphibians and mammals andagain highlight the predictability of evolution in this system.As a result of the repeatability of mechanisms of resistance de-

scribed herein, the molecular composition of the Na+/K+-ATPasecould be used as a predictive tool to anticipate the ecological impactof invasive species harboring cardiac glycoside defenses on naïve andpotentially vulnerable animal populations. This is important, becausethe introduction of the cane toad (Bufo marinus) to the toad-freeAustralian continent in 1935 has resulted in severe declines of nu-merous naïve predators due to the toad’s high toxicity (34–36).Currently, toads have invaded, or are close to invading, other toad-free biodiversity hotspots, such as Madagascar and parts of Indo-nesia (37, 38), where they are also likely to threaten native faunasusceptible to cardiac glycosides, such as the world’s largest andmost iconic lizard, the Komodo dragon (Varanus komodoensis) (39).In summary, although evolutionary processes are likely to be

predominately stochastic, in the present study we have outlined afascinating example of predictable convergent molecular evolu-tion. Our data further emphasize how the application of similar,strong, selective pressures appear to underpin the convergentevolution of resistance to both naturally occurring (e.g., cardiacglycosides, tetrodotoxin, venom toxins) (7–9, 40) and anthro-pogenically applied (e.g., insecticides, pesticides) (10, 30, 31, 41)toxic molecules. Interestingly, many of these toxic moleculesappear to interfere with essential components involved in thetransport of ions across cell membranes, typically by impingingupon ion channel or ion pump function (7–9, 30, 31, 40, 42). Inmany cases, convergent evolution of resistance to these toxins ismediated by a small number of similar molecular alterations tothe target molecule across unrelated taxa (Fig. 2) (7–9, 30, 40).These observations suggest that certain protein types appear tobe highly constrained in terms of the evolutionary solutions availableto overcome similar pressures, even when from highly divergentphylogenetic backgrounds. This concept is further reinforced bymultiple reports of ion transporters evolving via the process of con-vergence in other systems, including acid nociception in mammals(43) and electric communication in fish (44). It therefore appears thation transport proteins are heavily constrained and liable to canali-zation. Although it has been demonstrated that constraints can applysimultaneously to many proteins in complex systems (45), in the caseof ion transporters, it seems likely that their strict ion selectivity andnecessity for neurotransmission may result in most mutations beingdeleterious, thereby funneling molecular changes down the very few,similar, physiologically feasible pathways available in a repeatablemanner. Our findings with the animal Na+/K+-ATPase encapsulatethis process and prompt a general reconsideration of the pervasive-ness of constraint limiting the options available for evolution, therebypromoting predictable molecular convergence.

MethodsSI Appendix, SI Materials and Methods has additional information relatingto the methodologies described below.

Sequence Data. Sequence data for the H1–H2 domain of the α3 subunit of theNa+/K+-ATPase of 43 squamate reptiles were generated as previously de-scribed (17). We supplemented these data with sequences generated fromgenome sequencing projects for squamates and nonsquamate outgroups.A list of the sequenced taxa and their propensity to feed on bufonid toads isdisplayed in SI Appendix, Table S1, and the DNA sequence data generated inthis study have been submitted to GenBank with the accession numbersKP238131–KP238176. Sequence data for the H1–H2 domain of the α subunitof the Na+/K+-ATPase from insects and anurans were obtained from pre-vious studies (9, 18) and the mammalian dataset generated by similaritysearching various National Center for Biotechnology Information databases(www.ncbi.nlm.nih.gov) with the previously isolated sequence from the rat(15, 16) to identify homologous genes in related taxa.

Ancestral Sequence Reconstruction and Evolution. For each group of taxa(squamates, insects, anurans, and mammals), separate sequence alignmentswere generated using the Multiple Comparison by Log-Expectation (MUSCLE)algorithm (46). Species trees were constructed from previously publishedstudies (see Fig. 1 and SI Appendix, Figs. S4–S6 and S8 for details) and ancestralsequences reconstructed at various nodes of the Na+/K+-ATPase phylogeniesusing the marginal sequence reconstruction method (47) with the AncestralSequence Reconstruction (ASR) algorithm on the Datamonkey web server (48).The rate of evolution of the Na+/K+-ATPase gene was estimated using themaximum-likelihood model (M8) of PAML (phylogenetic analysis by maximumlikelihood) (49) and the influence of episodic adaptive selection assessed usingthe mixed effects model of evolution in HyPhy (50). Coevolving amino acidsites were detected using the spidermonkey algorithm (51) in HyPhy; the Fast,Unconstrained Bayesian AppRoximation method (52) was used to detectsites in each dataset evolving under the pervasive influence of selection;and the Directional Evolution in Protein Sequences algorithm (53) was usedfor identifying sites that are the subject of directional evolution.

Changes in Isoelectric Point and Charged Residues. The isoelectric point andchanges in charge of the H1–H2 extracellular domain of the αNa+/K+-ATPasewere calculated for all susceptible and resistant taxa in each dataset (SI Appendix,Table S2) using the ProtParam tool (web.expasy.org/protparam/). Statisticalcomparisons of changes in isoelectric point between resistant and susceptibletaxa were performed using an unequal variance two-tailed t test. We next in-vestigated whether resistance was associated with a shift from neutral tocharged amino acids using binomial tests in R v.3.1.0 (54). Binomial tests comparean observed proportion, in this case the proportion of resistance mutations thatinvolved a neutral to charged amino acid shift, to an expected proportion. Ineffect, this test asks whether shifts to charged amino acids (as we observe) occurmore frequently than shifts to other amino acids (that we do not observe). In ourtest, the expected proportion was based on a null model of equal nucleotide-base substitution, such that transitions were weighted based on the number ofindividual base changes required to shift from one amino acid to another,thereby accounting explicitly for silent mutations in the evolutionary process.

Molecular Modeling.All docking simulationswereperformedwithAutoDock vina2.0 (55) using an approach similar to that described by Zhen et al. (8). To establisha docking protocol we first redocked the cardiac glycoside bufalin into the crystalstructure of bufalin bound to the pig Na+/K+-ATPase [Protein Data Bank (PDB) IDcode 4RES] (27). The bufalin ligand was modeled with explicit polar hydrogensand torsional flexibility. The side chains of the ATPase residues Q111, E115, E116,E117, P118, D121, N122, L125, V322, A323, E327, E779, T797, I800, and D804weretreated as flexible, whereas the remaining residues were held rigid. The “best”structure from the redocking experiment was defined as the structure from thetop 10 highest affinity solutions that is closest to the coordinates of the bufalinligand in the cocrystal structure [measured as heavy-atom root mean square de-viation (RMSD) in Ångstrom]. This best redocking structure was used as a referencestructure for docking runs using the Na+/K+-ATPase isolated from other taxa. Wenext modeled the binding of bufalin with wild-type and H1–H2 extracellular do-main substitution mutants of the α1 Na+/K+-ATPase in the rat (mutants R111Q,D122N), hedgehog (R111Q D119Q), Leptodactylus frog (R111Q D122N), andbufonid toad (R111Q D119N) and the α3 Na+/K+-ATPase in the python (Q111LG120R). All homology models were prepared with Modeler v9.10 (56) using thebufalin/pig Na+/K+-ATPase crystal structure (27) as a template and model qualitychecked using Swiss-PdbViewer (spdbv.vital-it.ch). Bufalin was next docked to eachof the wild-type Na+/K+-ATPases and their substitution mutants. For each

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docking run, we calculated the RMSD between the best structure, defined as thetop 10 high-affinity solutions closest to the coordinates of bufalin in the cocrystalstructure, and the best structure from the redocking of bufalin to the pig Na+/K+-ATPase/bufalin crystal structure. RMSD only provides information about the po-sition of the ligand but not about the interactions between the ligand and theprotein. In each model, we thus determined the contacts between the ligandand the Na+/K+-ATPase and compared them to those in the cocrystal structure.

ACKNOWLEDGMENTS. We are grateful to P. Baverstock and J. Vindum forsupplying us with some of the samples used in the study. We thankA. Georges at the University of Canberra and G. Zhang and K. Lee at the

Beijing Genome Institute (Pogona genome project) for making available datafrom Pogona vitticeps. We thank E. Undheim, T. Jackson, and M. Berenbrink fordiscussions. The research was funded by theWhitehead Bequest (Conservation),Faculty of Veterinary Science, University of Sydney, and by the Ian Potter Foun-dation. B.U. was supported by a Deakin University Central Research GrantScheme, N.R.C. by a fellowship from the UK Natural Environment ResearchCouncil (NE/J018678/1), K.S. by a Marie Skłodowska-Curie Individual Fellowship(654294) from the European Commission, N.L. by a Queen Elizabeth II Fellowshipfrom the Australian Research Council, C.B. by an Alfred Deakin Postdoctoral Fel-lowship from Deakin University, G.F.K. by an Australian National Health andMedical Research Council (NHMRC) Principal Research Fellowship, and E.D. byan Early Career Research Fellowship from the NHMRC.

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