Selection and Evolvability of ALT-2.

<p>Values for selection (ω) and evolvability (E) were calculated for each amino acid residue in the ALT-2 sequence. The single E value was plotted against the ω value for pairwise comparisons between all species. Residues that were both significantly evolvable and under diversifying selection appear in the blue-shaded boxes. The two conserved ALT-2 sequences (<i>O</i>. <i>volvulus</i>, [filled circles] and <i>B malayi</i> [filled diamonds], <b>l A</b>) and the two diversified ALT-2 sequences (<i>L</i>. <i>loa</i> [open squares] and <i>W</i>. <i>bancrofti</i> [open triangles], <b>B</b>) served as positive and negative controls, respectively. Selection and evolvability of ALT-2 from <i>L</i>. <i>loa</i> followed a second-order polynomial distribution when compared to either <i>B</i>. <i>malayi</i> (<b>C</b>) or <i>O</i>. <i>volvulus</i> (<b>E</b>). Only a small number of residues in <i>W</i>. <i>bancrofti</i> were both diversified and evolvable when compared to <i>B</i>. <i>malayi</i> (<b>D</b>), but none were detected when compared to <i>O</i>. <i>volvulus</i> (<b>F</b>).</p

Phylogenetic Analysis.

<p>The evolutionary history of ALT-2 consensus sequences from <i>B</i>. <i>malayi</i>, <i>W</i>. <i>bancrofti</i>, <i>O</i>. <i>volvulus</i>, and <i>L</i>. <i>loa</i> was inferred using (<b>A</b>) the Neighbor-Joining method and (<b>B</b>) the Maximum Parsimony method (N = 500 bootstrap replicates each). Regardless of method, the branching order was the same and was consistent with ribosomal RNA-derived branching order.</p

Antigenicity and Selection Projection.

<p>Calculated ω values using the M8 model (Y axis, bar graph) are projected onto graphical measurements of antigenicity predictions (Z axis, line graph) across all sites in ALT-2 (X axis, marked by position number). Predicted antigenic epitopes are denoted with arrows. The cutoff ω value of 1 is depicted as a black line; values extended above the line represent residues under diversifying selection, and values below the line represent residues under purifying selection. Areas under diversifying selection in ALT-2 of <i>L</i>. <i>loa</i> and <i>W</i>. <i>bancrofti</i> have minimal overlap with predicted antigenicity.</p

Pairwise Evolvability (E) Analysis Design.

<p>Pairwise Evolvability (E) Analysis Design.</p

Evolutionary Selection Analysis.

<p>Calculated ω values are presented for each amino acid residue (X axis, marked by position number) for ALT-2 from the indicated species using the M8 model. The cutoff ω value of 1 is depicted as a black line; values extended above the line represent residues under diversifying selection, and values below the line represent residues under purifying selection. Statistically significant diversifying selection was detected globally in ALT-2 of <i>L</i>. <i>loa</i> (<i>P</i><0.001) and <i>W</i>. <i>bancrofti</i> (<i>P</i><0.05). Residues encompassing the signal peptide are beneath the solid lines, and those encompassing the VAD are beneath the dashed lines.</p

ALT-2 Evolvability in Filarial Parasites.

<p>Calculated evolvability (E) values (Y axis) are presented for each amino acid residue (X axis) for ALT-2 from the indicated pairs of species using the calculated ω values from each organism at each site. Cutoffs for significant deviations between conserved (normal distribution) and diversified sequences are shown as red lines (<i>P</i><0.05 or <i>P</i><0.001); values extended above the line represent residues that are significantly evolvable. Goodness-of-fit analyses between the two conserved ALT-2 sequences (<i>O</i>. <i>volvulus</i> and <i>B malayi</i>, <b>A</b>) and the two diversified ALT-2 sequences (<i>L</i>. <i>loa</i> and <i>W</i>. <i>bancrofti</i>, <b>B</b>) served as positive and negative controls, respectively. Multiple residues of <i>L</i>. <i>loa</i> were significantly evolvable when examined for deviation from <i>B</i>. <i>malayi</i> (<b>C</b>) or <i>O</i>. <i>volvulus</i> (<b>E</b>). Multiple residues of <i>W</i>. <i>bancrofti</i> were significantly evolvable when examined for deviation from <i>B</i>. <i>malayi</i> (<b>D</b>), but not <i>O</i>. <i>volvulus</i> (<b>F</b>).</p