Convergent Evolution of Toxin Resistance

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Title: Widespread convergence in toxin resistance by predictable molecular evolution

Authors: Beata Ujvari, Nicholas R. Casewell, Kartik Sunagar, Kevin Arbuckle, Wolfgang Wüster, Nathan Lo, Denis O’Meally, Christa Beckmann, Glenn F. King, Evelyne Deplazes and Thomas Madsen

Abstract: The question about whether evolution is unpredictable and stochastic or intermittently constrained along predictable pathways is the subject of a fundamental debate in biology, in which understanding convergent evolution plays a central role. At the molecular level, documented examples of convergence are rare and limited to occurring within specific taxonomic groups. Here we provide evidence of constrained convergent molecular evolution across the metazoan tree of life. We show that resistance to toxic cardiac glycosides produced by plants and bufonid toads is mediated by similar molecular changes to the sodium-potassium-pump (Na+/K+- ATPase) in insects, amphibians, and mammals. In toad-feeding reptiles, resistance is conferred by two point mutations that have evolved convergently on four occasions, whilst evidence of a molecular reversal back to the susceptible state in varanid lizards migrating to toad-free areas suggests that toxin resistance is maladaptive in the absence of selection. Importantly, resistance in all taxa is mediated by replacements of two of the 12 amino acids comprising the Na+/K+-ATPase H1-H2 extracellular domain that constitutes a core part of the cardiac glycoside binding site. We provide mechanistic insight into the basis of resistance by showing that these alterations perturb the interaction between the cardiac glycoside bufalin and the Na+/K+-ATPase. Thus, similar selection pressures have resulted in convergent evolution of the same molecular solution across the breadth of the kingdom, demonstrating how a scarcity of possible solutions to a selective challenge can lead to highly predictable evolutionary responses.

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SI Materials and Methods

Sequence data. Squamate sequence data for the H1-H2 domain (encoding the αM1- αM2 extracellular loop) of the α3 subunit of the Na+/K+-ATPase were generated as previously described (1). Genomic DNA was isolated by phenol-chloroform extraction. Using the primers previously detailed (2), DNA corresponding to the H1- H2 domain was subsequently amplified and sequenced on an ABI 3130xl Genetic Analyzer using a 121 BigDye Terminator Kit v.3.1 (Applied Biosystems). In total, we sampled 43 squamates, including the 18 varanid lizards previously reported (1). The DNA sequence data generated in this study have been submitted to GenBank with the accession numbers KP238131–KP238176. We supplemented these data with sequences generated from on going or completed genome sequencing projects for squamates (Burmese python, king cobra, green anole and bearded dragon) and non- squamate outgroups (American alligator, chicken, tuatara). A list of the sequenced taxa and their propensity to feed on bufonid toads is displayed in the SI Appendix, Table S1. Sequence data for the H1-H2 domain of the α subunit of the Na+/K+- ATPase from insects and anurans were obtained from previous studies (3, 4). The mammalian dataset was generated by isolating the previously reported sequence for the rat (5, 6) and undertaking BLAST similarity searches using the nucleotide, genome, EST and SRA databases of NCBI (http://www.ncbi.nlm.nih.gov) to identify homologous genes in related taxa.

Ancestral reconstructions and sequence evolution. For each group of taxa (squamates, insects, anurans and mammals), separate sequence alignments were generated using the MUSCLE algorithm (7). trees of the taxa represented in each dataset were then constructed from previously published studies (see Fig. 1 and SI Appendix, Figs. S4-S6 and S8 for details). This data was then used to reconstruct ancestral sequences at various nodes of the Na+/K+-ATPase phylogenies using the marginal sequence reconstruction method (8) implemented using the ASR algorithm on the Datamonkey web-server (9). The rate of evolution of Na+/K+-ATPase gene was estimated using the maximum-likelihood model (M8) of PAML package (10). The influence of episodic adaptive selection was assessed on each dataset using the state- of-the-art ‘mixed effects model of evolution’ (MEME) implemented in HyPhy (11). Coevolving amino acid sites were detected using the spidermonkey algorithm (12) in

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HyPhy: spidermonkey reconstructs the substitution history of the alignment using a maximum likelihood-based phylogenetic approach, followed by assessment of the joint distribution of substitution events using Bayesian graphical models to detect significant evolutionary associations among amino acid positions. Finally, the Fast, Unconstrained Bayesian AppRoximation (FUBAR) method (13) was utilised to detect sites in each dataset evolving under the pervasive influence of selection, whilst the Directional Evolution in Protein Sequences (DEPS) algorithm (14) was used for identifying sites that are the subject of directional evolution.

Phylogenetic analyses. To identify which α subunit of the multi-locus Na+/K+- ATPase gene family was the subject of resistance-conferring amino acid replacements in each animal lineage, we reconstructed the evolutionary history of these genes. Full- length sequence data were obtained for each α subunit by BLAST similarity searching representative taxonomic groups in the genome and protein databases of NCBI using various α subunit template sequences. The resulting amino acid sequence data were aligned using the MUSCLE algorithm (7) and then checked manually. For gene tree generation, we performed phylogenetic analysis using MrBayes v3.2 (15). First we selected an appropriate model of evolution favoured by the Akaike Information Criterion using ModelGenerator (16). The selected model (GTR + G) was implemented into Bayesian inference analysis using MrBayes on the CIPRES Science Gateway (www.phylo.org). The analysis was run in duplicate using four chains simultaneously (three heated and one cold) for 5x106 generations, sampling every 500th cycle from the chain and using default parameters in regards to priors. Tracer v1.5 (http://beast.bio.ed.ac.uk/tracer) was used to estimate effective sample sizes for all parameters (with all showing well in excess of the minimum accepted – 200) and to construct plots of ln(L) against generation to verify the point of convergence (burnin). Trees generated prior to the point of convergence were discarded through a conservative first 25% cutoff and a consensus gene tree was generated from the remaining trees sampled. The resulting gene tree was annotated with details of which α subunit confers resistance in the different animal lineages (see SI Appendix, Fig. S2).

Changes in isoelectric point and charged residues. The isoelectric point and changes in charge of the H1-H2 extracellular domain of the α Na+/K+-ATPase were

3 Convergent Evolution of Toxin Resistance calculated using the ProtParam tool (http://web.expasy.org/protparam) hosted at the ExPASy Bioinformatics Resource Portal. Both isoelectric points and the addition/loss of charged amino acid residues were calculated for all sequence data sourced from the susceptible and resistant taxa analysed in each taxonomic group (SI Appendix, Table S2). Statistical comparisons of changes in isoelectric point between resistant and susceptible taxa were performed using an unequal variance two-tailed t-test. We next investigated whether resistance was associated with a shift from neutral to charged amino acids using binomial tests in R v.3.1.0 (17). Binomial tests compare an observed proportion, in this case the proportion of resistance mutations that involved a neutral to charged amino acid shift, to an expected proportion. In effect, this test asks whether shifts to charged amino acids (as we observe) occur more frequently than shifts to other amino acids (that we do not observe). In order to ensure the robustness of our results, we used three different expected proportions. In the first, we simply used the proportion of all amino acids (excluding the ancestral one) that are charged. In the second, the expected proportion was as before except that amino acid shifts were weighted based on the number of codons that code for each one. This is likely to be more realistic as it accounts for the fact that, if mutations were random, it may be easier to shift to a new amino acid that has four possible codons than one which has only two. In the third, the expected proportion was based on a null model of equal nucleotide-base substitution, such that transitions were weighted based on the number of individual base changes required to shift from one amino acid to another. This is more realistic again as it accounts explicitly for silent mutations in the evolutionary process. Since all three analyses yielded qualitatively identical results (first version, P=6.86x10-5; second version, P=1.81x10-10; third version, P=6.90x10-7), we only report the third, most realistic version, in the main text of the paper.

Molecular modelling. All docking simulations were performed with AutoDock vina 2.0 (18) using an approach similar to that described by Zhen et al. (19). To establish a docking protocol we first re-docked the cardiac glycoside bufalin into the crystal structure of bufalin bound to the pig Na+/K+-ATPase (PDB: 4RES) (20). The bufalin ligand was modelled with explicit polar hydrogens and torsional flexibility. The side chains of the ATPase residues Q111, E115, E116, E117, P118, D121, N122, L125, V322, A323, E327, E779, T797, I800 and D804 were treated as flexible while the remaining residues were held rigid. The ‘best’ structure from the re-docking

4 Convergent Evolution of Toxin Resistance experiment was defined as the structure from the top 10 highest affinity solutions that is closest to the coordinates of the bufalin ligand in the co-crystal structure (measured as heavy-atom Root Mean Square Deviation [RMSD] in Ångstrom). This best re- docking structure was used as a reference structure for docking runs using the Na+/K+- ATPase isolated from other taxa. We next modelled the binding of bufalin with wild- type and substitution mutants of the α1 Na+/K+-ATPase in the rat (Rattus norvegicus), hedgehog (Erinaceus europaeus), Leptodactylus frog (Leptodactylus latrans) and bufonid toad (Bufo marinus) and the α3 Na+/K+-ATPase in the python (Python bivittatus). To do so, we created homology models of the wild-type Na+/K+-ATPase protein for each taxa (all wild-types had the resistant genotype, except for the susceptible python) and the following H1-H2 domain (i.e. αM1-αM2 extracellular loop) substitution mutants: rat R111Q D122N, hedgehog R111Q D119Q, Leptodactylus frog R111Q D122N, bufonid toad R111Q D119N (all of which resulted in susceptible mutant genotypes) and python Q111L G120R (which resulted in a resistant mutant genotype). The following GenBank accession numbers were used for the wild-type sequence templates: rat, NP_036636.1; hedgehog, XP_007525565.1; python, XP_007437634.1. As full-length sequences for the α1 Na+/K+-ATPase of the Leptodactylus frog and bufonid toad were not available, we used the α1 Na+/K+- ATPase from Xenopus tropicalis (GenBank: NP_989407.1) and replaced the H1-H2 extracellular domain with the wild-type sequences from L. latrans and B. marinus used elsewhere in this study. All homology models were prepared with Modeller v9.10 (21) using the bufalin/pig Na+/K+-ATPase crystal structure (20) as a template. Model quality was checked using Swiss-PdbViewer (http://www.expasy.org/spdbv/). Bufalin was docked to each of the wild-type Na+/K+-ATPase and their substitution mutants. For each docking run, we calculated the RMSD between the best structure, defined as the top 10 high-affinity solutions closest to the coordinates of bufalin in the co-crystal structure, and the best structure from the re-docking of bufalin to the pig Na+/K+-ATPase/bufalin crystal structure. RMSD only provides information about the position of the ligand but not about the interactions between the ligand and the protein. In each model we thus determined the contacts between the ligand and the Na+/K+-ATPase and compared them to those in the co-crystal structure.

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SI Tables

Table S1. taxa previously demonstrated to be resistant or susceptible to toad toxins and those that have never been recorded feeding on toads. Resistant Susceptible Never recorded feeding on toads grass green tree snake Arafurae file snake (Natrix natrix) (Dendrelaphis punctulatus) (Achrochordus arafurae) dice snake brown tree snake mountain bronzeback (Natrix tesselata) (Boiga irregularis) (Dendrelaphis subocularis) salt marsh snake northern death adder water python (Nerodia clarkii) (Acanthophis praelongus) (Liasis fuscus) diamondback water snake marsh snake Burmese python (Nerodia rhombifera) (Hemiaspis signata) (Python molurus bivittatus) Leonard's Keelback king brown snake king cobra (Rhabdophis leonardi) (Pseudechis australis) (Ophiophagus hannah) red-necked keelback European adder green anole (Rhabdophis subminiatus) (Vipera berus) (Anolis carolinensis) checkered keelback carpet python bearded dragon (Xenochrophis piscator) (Morelia spilota) (Pogona vitticeps) shorthead blue-tonged lizard tuatara (Thamnophis brachystoma) (Tiliqua scincoides) (Sphenodon punctatus) blackbelly garter snake ridged-tailed monitor American alligator (Thamnophis melanogaster) (Varanus acanthurus) (Alligator mississippiensis) eastern ribbon snake pygmy desert monitor (Thamnophis sauritus) (Varanus eremius) false water cobra sand monitor (Hydrodynastes gigas) (Varanus gouldii) forest cobra Mertens' water monitor (Naja melanoleuca) (Varanus mertensi) Indian cobra Mitchells's water monitor (Naja naja) (Varanus mitchelli) puff adder yellow-spotted monitor (Bitis arietans) (Varanus panoptes) rhinocerous viper spotted tree monitor (Bitis nasicornis) (Varanus scalaris) Bengal monitor Storr's monitor (Varanus bengalensis) (Varanus storri) Dumeril's monitor black-headed monitor (Varanus dumerilii) (Varanus tristis) roughneck monitor lace monitor (Varanus rudicollis) (Varanus varius) water monitor (Varanus salvator) white-throated monitor (Varanus albigularis) savannah monitor (Varanus exanthematicus) Nile monitor (Varanus niloticus) Data used to classify the above described taxa taken from references (22-46).

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Table S2. Comparisons of the isoelectric point (pI) and the number of charged amino acid residues present in the H1-H2 extracellular domain of the α subunits of the Na+/K+-ATPase in resistant and susceptible taxa. Susceptible taxa Resistant taxa Phylum # charged residues # charged residues pI -ve +ve pI -ve +ve Squamates 3.37 4 0 3.84 4 1 3.37 4 0 3.84 4 1 3.37 4 0 3.84 4 1 3.37 4 0 3.84 4 1 3.37 4 0 3.84 4 1 3.37 4 0 3.84 4 1 3.37 4 0 3.84 4 1 3.37 4 0 3.84 4 1 3.37 4 0 3.84 4 1 3.37 4 0 3.84 4 1 3.37 4 0 3.84 4 1 3.37 4 0 3.84 4 1 3.37 4 0 3.92 4 1 3.37 4 0 3.84 4 1 3.37 4 0 3.84 4 1 3.37 4 0 3.92 4 1 3.37 4 0 3.92 4 1 3.37 4 0 3.84 4 1 3.37 4 0 3.84 4 1 3.37 4 0 3.84 4 1 3.37 4 0 3.84 4 1 3.37 4 0 3.84 4 1 3.37 4 0 3.37 4 0 3.37 4 0 3.37 4 0 3.37 4 0 3.43 4 0 3.67 2 0

Mammals 3.50 4 0 3.83 5 1 3.50 4 0 3.83 5 1 3.50 4 0 3.83 5 1 3.50 4 0 3.83 5 1 3.50 4 0 3.83 5 1 3.50 4 0 3.83 5 1 3.50 4 0 3.83 5 1 3.37 4 0 3.83 5 1 3.43 4 0 3.43 4 0 3.50 4 0 3.43 4 0 3.43 4 0

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Anurans 3.43 4 0 3.77 5 1 3.43 4 0 4.23 4 2 3.5 4 0 4.23 4 2 3.43 4 0 4.23 4 2 3.5 4 0 3.5 4 0 3.5 4 0 3.5 4 0 3.43 4 0 3.5 4 0 3.5 4 0 3.5 4 0 3.5 4 0 3.43 4 0

Insects 3.43 4 0 3.91 4 0 3.43 4 0 3.83 5 0 3.37 4 0 3.39 5 1 3.37 4 0 3.91 4 1 3.43 4 0 3.43 4 0 3.43 4 0 3.91 4 1 3.43 4 0 3.43 4 0 3.43 4 0 3.91 4 1 3.43 4 0 3.43 4 0 3.43 4 0 3.43 4 0 3.43 4 0 3.43 4 0 3.43 4 0 3.43 4 0 3.43 4 0 3.43 4 0 3.91 4 1 3.43 4 0

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Table S3. Protein-ligand interactions found by docking bufalin to the wild-type (native) Na+/K+-ATPase of rat, hedgehog, bufonid toad, Leptodactylus frog and python and their substitution mutants, in comparison to interactions found in the pig Na+/K+-ATPase/bufalin crystal structure (20). Cardiac RMSD from Contacts formed Species Substitutions glycoside best pig re- by the β-surface of sensitivity docking the steroid core in model (Å)* bufalin (< 4 Å)# BUF – E117:O Pig None (WT) Susceptible - BUF – D121:O BUF – T797:O BUF – E327:O Rat None (WT) Resistant 1.4 BUF – T797:O BUF – E327:O Rat R111Q, D122N Susceptible 1.5 BUF – D121:O BUF – T797:O Hedgehog None (WT) Resistant 1.3 BUF – E327:O

Hedgehog R111Q, D119Q Susceptible 1.4 BUF – D121:O BUF – T797:O BUF – Q111:O Bufonid None (WT) Resistant 1.9 BUF – E117:O BUF – T797:O BUF – E327:O BUF – E117:O Bufonid R111Q, D119N Susceptible 1.4 BUF – D121:O (2) BUF – T797:O Frog BUF – Q111:O (Leptodactylus) None (WT) Resistant 1.2 BUF – T797:O BUF – E327:O BUF – E117:O Frog R111Q, D122N Susceptible 0.9 BUF – D121:O (Leptodactylus) BUF – T797:O BUF – E327:O Python None (WT) Susceptible 0.6 BUF – D121:O BUF – T797:O Python Q111L, G120R Resistant 1.1 BUF – T797:O BUF – E327:O * Heavy-atom RMSD between the best structure from re-docking of bufalin to the pig ATPase/bufalin crystal structure (4RES.pdb) and best structure from docking of bufalin to the other species (where best is the structure among the top 10 high-affinity structures that is closest to the coordinates of bufalin in the co-crystal structure). # In addition to the listed interactions formed by the β-surface of the steroid core and residues in αM1-M2 and αM4 of ATPase, there are a number of van der Waals interactions between the α-surface of the steroid core and residues in αM5-M6. These interactions are not listed separately as they were mostly unaffected by the mutations.

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SI Figures

Fig. S1. Sequence alignment of the H1-H2 extracellular domain of the α3 Na+/K+- ATPase gene in squamate reptiles demonstrating the amino acid sites that are the target for directional selection and episodic diversification. A representative gene tree (see Fig. 1) is displayed alongside the sequence alignment, with taxa resistant to cardiac glycosides shaded in red. Amino acid residues previously demonstrated to confer resistance to cardiac glycosides (at positions 111 and 120) (1) are highlighted by arrows.

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Fig. S2. The evolutionary history of the α subunits of the Na+/K+-ATPase and the evolution of resistance to cardiac glycosides in . Boxed orange tip labels indicate the gene that taxa (or related taxa) have modified to evolve resistance to cardiac glycosides – α1: rodents, hedgehogs, leptodactylid frogs and bufonid toads; α3: and varanid lizards. Note that the python (which is susceptible to toad toxins) is used as a proxy for resistant squamates because there is no full length sequence information available for the Na+/K+-ATPase for any resistant squamate reptile at this time. Circles on nodes indicate the support for the tree topology displayed: black circles, Bayesian posterior probabilities of 1.00; grey circles, Bayesian posterior probabilities of >0.95.

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Fig. S3. Sequence alignment of the H1-H2 extracellular domain of the α subunit of the Na+/K+-ATPase gene of representative resistant and susceptible animal taxa. The alignment displays key amino acid residues that confer changes in charge and are associated with the resistant (red) and susceptible (green) genotype in different taxa (see also Fig. 2).

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Fig. S4. Evolution of cardiac glycoside resistance in rodents. The timing of the evolution of resistance to cardiac glycosides is indicated by the change in branch colours which represent specific amino acid changes previously demonstrated to confer resistance (5, 6). Genotypic resistance correlates with taxa previously known to feed on cardenolide producing plants, cardenolide-sequestering insects or bufonid toads (5, 6, 47-50) and the timing of this dietary change is indicated by the picture of foxglove. Species tree of representative mammals generated from references (51, 52).

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Fig. S5. Evolution of cardiac glycoside resistance in anuran amphibians. The timing of the evolution of resistance to cardiac glycosides is indicated by the change in branch colours which represent specific amino acid changes previously predicted to confer resistance (4). Genotypic resistance in Leptodactylus latrans (formerly known as L. ocellatus) correlates with known feeding on bufonids (53, 54) and the picture of a toad indicates the timing of this dietary change. Note that this resistance is mediated by amino acid replacements found in one gene copy resulting from a duplication event (4) (black circle). Resistance is found at positions 111 (Q to R) and 122 (N to D). Self-resistance to cardiac glycosides in bufonids appears to be mediated by the same amino acid replacements but found at positions 111 (Q to R) and 119 (N to D). Species tree of representative anurans generated from reference (4).

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Fig. S6. Evolution of cardiac glycoside resistance in insects. The timing of the evolution of resistance to cardiac glycosides is indicated by the change in branch colours which represent specific amino acid changes previously predicted to confer resistance (3). Amino acid changes correlate with the ability of taxa to feed on and, in some cases, sequester cardenolides produced by plants (3) and the pictures of foxglove indicate the reconstructed timing of this dietary shift. Note that some species within clades of taxa that feed on cardenolide-producing plants appear to have reverted back to not feeding on cardenolide-producing plants (3) – these species are indicated by asterisks. Species tree of representative insects generated from references (3, 55).

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Fig. S7. Results from modelling the interactions between bufalin and the Na+/K+- ATPase in resistant and susceptible genotypes of the Leptodactylus frog, hedgehog and python. (A, C, E) Best structure from docking bufalin into a model of the resistant Na+/K+-ATPase of Leptodactylus frog, hedgehog and python. Bufalin does not form

16 Convergent Evolution of Toxin Resistance hydrogen bonds with D121 (<3 Å) in any of these genotypes. (B, D, F) Best structure from docking bufalin into a model of the susceptible Na+/K+-ATPase of Leptodactylus frog, hedgehog and python. Bufalin forms interactions with D121 in all of these genotypes. All ligand-protein interactions formed by the β-surface of bufalin are listed in SI Appendix, Table S3. Wild-type and substitution mutants are described in the figure headings; for example, for the Leptodactylus frog, the wild-type sequence is resistant to bufalin, whereas the susceptible genotype was produced by generating a substitution mutant by replacing the charged amino acids at position 111 (R to Q) and 122 (D to N).

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Fig. S8. Evolution of cardiac glycoside resistance in the hedgehog. The timing of the evolution of resistance to cardiac glycosides is indicated by the change in branch colours, which represent specific charged amino acid changes. Genotypic resistance in the European hedgehog (Erinaceus europaeus) correlates with observations that this species feeds on toads and anoints itself with toad toxins (56, 57) – the timing of this dietary change is indicated by the picture of a toad. Rodents resistant to cardenolides (see SI Appendix, Fig. S4 for detail) are indicated by the picture of foxglove and are displayed in this tree to demonstrate that resistance to cardiac glycosides has evolved twice in mammals. Resistance in both rodents and hedgehogs are mediated by the same amino acid change to arginine at position 111, whilst the hedgehog has a replacement to aspartic acid at position 119, compared to rodents where the change to aspartic acid is found at position 122. Species tree of representative mammals generated from references (51, 52, 58).

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