Figure 1.
Phylogenetic relationships inferred from the TRPA1 and TRPV1 genes.
Phylogenetic relationships of nine snake families were reconstructed with the ML and Bayesian methods. Non-snake outgroups are not shown. The snake species with pit organs are highlighted by shading. Branches with ML bootstrap support >70% and Bayesian posterior probability >0.95 are indicated as asterisks. Note that in the TRPA1 tree, but not in the TRPV1 tree, the three ancestral branches leading to the three groups of pit-bearing snakes (bold lines) are considerably longer than their sister branches connecting to non-pit snake relatives.
Figure 2.
Sliding window analysis of the dN/dS ratios for six animal groups.
The estimates are based on the Nei–Gojobori method. The window size was set as 150 bp, and the step size was set as 15 bp. TM, transmembrane region; ANK, ankyrin repeat.
Table 1.
The Site Models Detecting Positive Selection in the TRPA1 Gene for Different Groups.
Figure 3.
Selective pressure variation of both TRPA1 and TRPV1 along branches of the tetrapod phylogeny.
The tree topology is inferred with ML for each gene. Branch lengths are drawn proportionally to the estimated numbers of nonsynonymous substitutions per codon. The trees are rooted by the frog sequence (not shown). The selective pressures of branches are assigned to dN/dS categories (tick marks along heat scale) by the GA-Branch method implemented in HyPhy. Branches assigned to a given dN/dS category share the same color. Deeper blue indicates a strong purifying selection, whereas hotter colors indicate elevated dN/dS ratios. For branches with letters along them, dN/dS values are re-estimated in PAML and subjected to a positive selection test by LRT.
Table 2.
Positive Selection Detection of the Snake TRPA1 Gene along Different Branches.
Figure 4.
Specific amino acid substitutions (TRPA1) accumulate in three important branches.
Specific amino acid substitutions (TRPA1) accumulate in branches (bold lines) leading to (A) the common ancestor of pit vipers, (B) the pit boa (Corallus hortulanus), and (C) the common ancestor of pythons. Specific amino acid substitutions for each branch are defined as all substitutions along the branch subtract those that also occur in the branch leading to the outgroup species (thin lines). These specific amino acid substitutions (indicated as vertical lines) are mapped above the TRPA1 domain organization schematic. Note that the substitution density of the transmembrane region (TM1 to TM6) is apparently lower than both the N-terminal region (ANK1 to TM1) and the C-terminal region (after TM6). TM, transmembrane region; ANK, ankyrin repeat.
Figure 5.
Amino acid sites of the TRPA1 protein that may be functionally important for infrared detection.
Amino acid residues that are highly conserved in all non-pit snakes, but divergent in the other three groups of pit-bearing snakes, are indicated by red circles. Amino acid residues that are separately conserved within pit vipers and the remaining snakes are indicated by green circles. These sites are numbered according to the full rattlesnake TRPA1 protein sequence. Positions conserved across all non-pit snakes and other non-snake vertebrates are indicated by black triangles below the sequences. These functionally important sites are mapped to the TRPA1 channel functional schematic (cyan blocks represent ankyrin repeats, and pink cylinders represent transmembrane domains). Note that most sites are distributed in the N-terminal domain (ANK1 to TM1), rather than the C-terminal domain (after TM6), suggesting that the N-terminal domain may be the responsible region for infrared detection capacity.
Table 3.
The species used in this study; newly generated sequences are in bold.
Table 4.
The primers used to amplify the TRPA1 and TRPV1 cDNA fragments.