Inventing an arsenal: adaptive evolution and neofunctionalization of snake venom phospholipase A2 genes

Abstract

Background: Gene duplication followed by functional divergence has long been hypothesized to be the main source of molecular novelty. Convincing examples of neofunctionalization, however, remain rare. Snake venom phospholipase A2 genes are members of large multigene families with many diverse functions, thus they are excellent models to study the emergence of novel functions after gene duplications.

Results: Here, I show that positive Darwinian selection and neofunctionalization is common in snake venom phospholipase A2 genes. The pattern of gene duplication and positive selection indicates that adaptive molecular evolution occurs immediately after duplication events as novel functions emerge and continues as gene families diversify and are refined. Surprisingly, adaptive evolution of group-I phospholipases in elapids is also associated with speciation events, suggesting adaptation of the phospholipase arsenal to novel prey species after niche shifts. Mapping the location of sites under positive selection onto the crystal structure of phospholipase A2 identified regions evolving under diversifying selection are located on the molecular surface and are likely protein-protein interactions sites essential for toxin functions.

Conclusion: These data show that increases in genomic complexity (through gene duplications) can lead to phenotypic complexity (venom composition) and that positive Darwinian selection is a common evolutionary force in snake venoms. Finally, regions identified under selection on the surface of phospholipase A2 enzymes are potential candidate sites for structure based antivenin design.

Figure 1

Molecular phylogeny of group-I phospholipase A₂ genes. (A) Bayesian phylogeny, branch lengths are given as number of substitutions per codon. (B) Evolution of group-I genes. Numbers above the branches are Bayesian posterior probability values (BP) followed by the d_N/d_S ratio (ω) or the number of nonsynonymous substitutions (N) if no synonymous substitutions (S) occurred along that branch (BP | ω or N/S). Branches in red were inferred to be under positive selection. Genes are labeled according the species they were identified from followed by the GenBank GI number for that gene. Pharmacological effects and higher order classifications are given to the right of clades. Pan, nontoxic pancreatic isoforms. AtC, anticoagulent. Ctx, cardiotoxic. PrC, procoagulent.

Figure 2

Molecular phylogeny of group-II (B) phospholipase A₂ genes. (A) Bayesian phylogeny, branch lengths are given as number of substitutions per codon. (B) Evolution of group-II genes. Numbers above the branches are Bayesian posterior probability values (BP) followed by the d_N/d_S ratio (ω) or the number of nonsynonymous substitutions (N) if no synonymous substitutions (S) occurred along that branch (BP | ω or N/S). Branches in red were inferred to be under positive selection. Genes are labeled according the species they were identified from followed by the GenBank GI number for that gene. Pharmacological effects and higher order classifications are given to the right of clades. AtC, anticoagulent. Chp/Chaperone, neurotoxin chaperone. Ntx, neurotoxic.

Figure 3

Molecular phylogeny of group-I phospholipase A₂ genes. Amino acid dataset with Bayesian posterior probabilities shown along branches.

Figure 4

Molecular phylogeny of group-II phospholipase A₂ genes. Amino acid dataset with Bayesian posterior probabilities shown along branches

Figure 5

The structure of group-I (A-C) and group-II (D-F) phospholipase A₂ proteins. The structures are represented by ribbons in A and D with disulfide bonds and catalytic residues shown as sticks and as molecular surfaces rendered in 3D in B, C, E and F. Residues are colored coded according to their approximate posterior mean ω (scale shown between rows) calculated under model M3 (discrete). B and E are in the same orientation as A and D, respectively, while C and F are rotated 180° about a horizontal axis through the molecule.

Figure 6

Ancestral sequence mapping for hydrophiinae group genes. The upper left panel shows the generalized phylogeny while each additional panel shows the cumulative amino acid changes that occurred for that lineage. Amino acids in panels A-E are colored by the lineage they changed in. For example, amino acid changes that occurred in the stem-lineage of neurotoxic genes (lineage C, panel C) are colored X; amino acid changes that occurred in ancestral lineages of neutrotoxins are colored Y and Z. In each panel the top two structures are shown with the molecular surface and the bottom structures are ribbons. Structures on left and right are rotated about a central axis 180°. Cystienes are shown in yellow

Figure 7

Ancestral sequence mapping for elapinae group genes. Organization follows Figure 6.

Figure 8

Ancestral sequence mapping for group-II genes. Organization follows Figure 6. Antipla., antiplatelet. Chp., chaperone. UnChar., genes with uncharacterized pharmacological effects.