Widespread convergence in toxin resistance by predictable molecular evolution

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, reptiles, and mammals. In toad-feeding reptiles, resistance is conferred by two point mutations that have evolved convergently on four occasions, whereas 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 2 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 animal kingdom, demonstrating how a scarcity of possible solutions to a selective challenge can lead to highly predictable evolutionary responses.

Keywords: bufotoxin cardenolide; constraint; genotype phenotype; ion transporters; parallelism.

Fig. 1.

Convergent molecular evolution of resistance to toad toxins in squamate reptiles and reversal to susceptibility in Australian varanid lizards. The timing of changes 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 clades of 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 being susceptible to toad toxins. Colored branches indicate the amino acid composition at key positions (susceptible, Q111 and G120; resistant, L111 and R120), and changes 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 with asterisks represent those falling beneath this threshold. Species tree was generated from refs. , .

Fig. 2.

Cross-phyla molecular convergence in the H1–H2 extracellular domain of the α Na⁺/K⁺-ATPase resulting in resistance to cardiac glycosides. Key amino acid residues found in the extracellular region are labeled by number. Resistance conferring residues are found grouped at position 111 at the N-terminal end and at positions 119, 120, and 122 at the C-terminal end. All individual amino acid changes have been demonstrated to contribute to resistance to cardiac glycosides in prior functional studies (, –17), with the exception of 111E proposed by Dobler et al. (9) and 119D proposed and validated here (Fig. 4). Convergent changes observed within listed taxa are indicated by boxes, with numbers reflecting the number of independent changes within that lineage (e.g., convergent changes from Q to L at 111 has occurred three times in snakes). Amino acid changes are highlighted 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⁺-ATPase was modified from ref. .

Fig. 3.

Convergent evolution of animal resistance to cardiac glycosides is mediated by changes in the isoelectric point of the H1–H2 extracellular domain of the 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, a 3D 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 axis reflect increases in isoelectric point, which are largely mediated by the replacement of amino acids with charged residues (Fig. 1 and SI Appendix, Table S2 and Figs. S4–S6 and S8). Animal pictures represent taxa found in each phylum that are resistant to cardiac glycosides. Note that the substantial decrease in isoelectric point observed in the squamate tree (red arrow) represents Australian varanid lizards, which have reverted back to the susceptible state.

Fig. 4.

Interactions between susceptible and resistant Na⁺/K⁺-ATPase and bufalin. (A) Chemical structure of bufalin. (B) Bufalin binding pocket in the crystal structure of bufalin bound to pig Na⁺/K⁺-ATPase-α1 (Sus scrofa; susceptible genotype; PDB ID code 4RES) (27). Bufalin wedges into a cavity formed by helices αM1–M2 (orange) (encoded by the H1–H2 extracellular domain), αM3–M4 (red), and αM4–M6 (gray). (C) The best structure from docking bufalin into a model of native (cardiac glycoside resistant) bufonid toad 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 best structure from docking bufalin into a model of bufonid toad Na⁺/K⁺-ATPase with substitutions R111Q and D119N, thereby forming the susceptible genotype. The β-surface of bufalin interacts with residues E117, E327, and T797, 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 a model of native (cardiac glycoside resistant) rat Na⁺/K⁺-ATPase. The β-surface of bufalin forms a hydrogen-bond with residues E327 and T797 but not D121. (F) The best structure from docking bufalin into a model of rat Na⁺/K⁺-ATPase with substitutions R111Q and D122N, thereby forming the susceptible genotype. The β-surface of bufalin forms hydrogen bonds with both T797 and D121 (H bonds < 3 Å). Predicted binding modes and ligand–protein interactions in resistant and susceptible genotypes of the Leptodactylus frog, hedgehog, and python can be found in the SI Appendix, Fig. S7 and Table S3.