Diversity of a-type and b-type VSG between <i>T. b. brucei</i> TREU 927/4 and <i>T. evansi</i> STIB805 compared.

<p>Histograms showing a-type VSG (A.) or b-type VSG (B.) distributions of strain-specific clade size in <i>T. b. brucei</i> (black bars) and <i>T. evansi</i> (red bars) as defined by the phylogeny (see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003404#pntd.0003404.s010" target="_blank">S10 Fig</a>.). Frequency distributions of a-type VSG (C.) or b-type VSG (D.) synonymous (<i>Ks</i>) and non-synonymous (<i>Ka</i>) substitution rates per site, and the ω (<i>Ka/Ks</i>) for orthologous pairs of <i>VSG</i> (a-type n = 151; b-type n = 112), as defined by the phylogeny, in relation to values for unambiguous non-VSG orthologous pairs (n = 6331).</p

Genes identified in <i>T. evansi de novo</i> assemblies.

<p>Genes identified in <i>T. evansi de novo</i> assemblies.</p

Bayesian phylogeny of <i>Trypanozoon</i> isolates based on the dihydrolipoamide dehydrogenase gene (LipDH; Tb927.11.16730).

<p>Panel A shows a mid-point rooted tree based on an alignment of 32 unique LipDH haplotypes, assembled from sequences derived from 13 <i>T. b. brucei</i> (Tbb), 3 <i>T. b. gambiense</i> type 1 (Tbg1), 4 <i>T. b. rhodesiense</i> (Tbr), 15 <i>T. evansi</i> (Tev) and 5 <i>T. equiperdum</i> (Teq) samples. Scale units for the phylogeny are substitutions per site. The chart illustrates the distribution of each haplotype among samples from each <i>Trypanozoon</i> taxon. Phylogenetic analysis grouped all but three of the haplotype sequences into one of five major clusters with strong support (posterior probabilities ≥0.9), which are referred to as clades V, W, X, Y and Z. Eight unique Tev/Teq genotypes were found, as summarized in panel B. Discounting minor sequence differences (1–2 SNPs) these were reduced to four major genotypes based on the position of haplotypes in the tree, which mirrored the type of mutation (A281Δ, M282L, A273P, WT) in the C-termini of the ATP synthase subunit γ in these the samples.</p

Pulse-field gel electrophoresis comparing chromosomes of <i>T. evansi</i> STIB805 (Tev) and <i>T. b. brucei</i> TREU 927/4 (927).

<p>While the sizes of megabase chromosomes are largely similar, differences in the intermediate and minichromosomes (825 kbp and smaller) are evident between <i>T. evansi</i> and <i>T. brucei</i>. Although <i>T. brucei</i> chromosomes I-XI were not unambiguously identified, labels to the left of the gel indicate bands consistent with the expected <i>T. brucei</i> chromosome sizes <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003404#pntd.0003404-Melville2" target="_blank">[46]</a>, as well as intermediate and minichromosomes. The signal at the top of the gel is from the well, as indicated.</p

Evaluation of the genetic differentiation between isolates of <i>T. evansi</i> and <i>T. equiperdum</i> and subspecies of <i>T. brucei</i> using principal component analysis (PCA) of microsatellite data.

<p>PCA was performed in R using the package adegenet. Within subspecies of <i>T. brucei</i>, the differentiation between temporally and geographically cohesive subgroups was estimated using DEST and calculated with the program smogd. Points representing individual genotypes are connected by a line to the centroid of an ellipse, which circumscribes a region encompassing 95% of the variance observed within six trypanosome subgroups identified: major <i>T. evansi</i> and <i>T. equiperdum</i> group (grey and pink), <i>T. b. rhodesiense</i> (red), <i>T. b. brucei</i> Kiboko (dark blue), <i>T. b. brucei</i> non-Kiboko (light blue), <i>T. b. gambiense</i> group 1 (dark green), <i>T. b. gambiense</i> group 2 (light green). <i>T. evansi</i> and <i>T. equiperdum</i> isolates that fell outside the major group are shown as black data points. The wide distribution of <i>T. evansi</i> and <i>T. equiperdum</i> isolates among distinct subgroups strongly supports multiple independent origins for these dyskinetoplastic strains. The first two principal components (PC1 and PC2) explain 29.2% and 8.4% of the total variance in the data, respectively. Data for isolates other than <i>T. evansi</i> and <i>T. equiperdum</i> had been published previously <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003404#pntd.0003404-Balmer1" target="_blank">[40]</a>.</p

Chromosomal breakdown of CDS found in TevSTIB805ra compared to Tb927 reference.

<p>Chromosomal breakdown of CDS found in TevSTIB805ra compared to Tb927 reference.</p

Proposed grouping of <i>T. evansi</i>/<i>T. equiperdum</i> isolates.

<p>Proposed grouping of <i>T. evansi</i>/<i>T. equiperdum</i> isolates.</p

Schematic of reads from <i>T. b. brucei</i> TREU 927/4 and <i>T. evansi</i> STIB805 mapped on to the procyclin loci of <i>T. brucei</i>.

<p>CDS are denoted by yellow arrows, with Tb927 GeneIDs above and gene names in parentheses. Reads that could be uniquely mapped to the reference are colored blue (intact paired reads), green (single forward reads) or red (single reverse reads). Reads that could be mapped to more than one position in the Tb927 reference were placed randomly and are colored yellow. Red asterisks denote mutated or absent CDS in <i>T. evansi</i>. Both PAG5 and PAG2* are missing in <i>T. evansi</i>, and mutations are present in all copies of EP2, PAG3, and GRESAG2.</p

Maximum parsimony haplotype network showing phylogenetic relationships among <i>Trypanozoon</i> haplotypes using cytochrome oxidase 1 (COX1).

<p>Relationships among lineages of <i>T. b. brucei (blue)</i>, <i>T. b. rhodesiense (red)</i>, <i>T. b. gambiense</i> group 1 (dark green) and <i>T. b. gambiense</i> group 2 (light green) are shown in circles as in reference <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003404#pntd.0003404-Balmer1" target="_blank">[40]</a>, with haplotypes new to this study shown as octagons, including two new haplotypes for <i>T. equiperdum</i> isolates (pink). The size of each circle or octagon is proportional to the frequency with which a particular haplotype was identified. Numbers in the circles and octagons correspond to haplotype ID. Empty circles indicate haplotypes that are inferred to exist but were not found. The light blue boxes correspond to the <i>T. brucei</i> clades defined in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003404#pntd.0003404-Balmer1" target="_blank">[40]</a>. Haplotypes for samples new to this study are as follows (from left to right): 8 =  Tbg14; 5 =  Tbr01/Tbr04; 20 =  Tbb49/Tbb50; 21 =  Tbb51; 3 =  Tbb20; 2 =  Tbb38; 23 =  Teq24; 14 =  Tbr02/Teq23; 22 =  Teq21/Teq22.</p

Mean average <i>Ka</i>/<i>Ks</i> estimates for orthologous CDS pairs between Tb927 and either <i>T. evansi</i> STIB805 (<i>Tev</i>) or <i>T. b. gambiense</i> DAL972 (<i>Tbg</i>).

<p>Mean average <i>Ka</i>/<i>Ks</i> estimates for orthologous CDS pairs between Tb927 and either <i>T. evansi</i> STIB805 (<i>Tev</i>) or <i>T. b. gambiense</i> DAL972 (<i>Tbg</i>).</p