Congruence parameters.

<p>List of congruence parameters that support a growing phylogenetic signal and the presence of the “clonality threshold” in the species under study.</p

“Russian doll” model [10].

<p>When population genetic tests are performed with appropriate markers (of sufficient resolution) within each of the near-clades, a and b, that subdivide the species, A, under study (large tree, left part of the figure), they reveal within these near-clades a miniature picture of the whole species, with the two main PCE features, namely linkage LD and lesser near-clades (two small trees, a′ and b′, right part of the figure). This shows that PCE obtains also within the near-clades, and that these do not correspond to cryptic, potentially panmictic, biological species.</p

An extreme case of LD in <i>Trypanosoma cruzi</i>, the parasite responsible for Chagas disease.

<p>Top: two genetic loci (A and B) revealed by protein markers (multilocus enzyme electrophoresis [MLEE]); bottom: two genetic loci (C and D) revealed by DNA markers (Random Primed Amplified Polymorphic DNA). The four genetic loci are totally linked to each other: A 1–7 with B 1–7 with C 1–9 with D 1–9 on one hand, and A 8–12 with B 8–12 with C 10–14 with D 10–14 on the other. Cross genotypes (for example: A1 with D10, A2 with B8, or C3 with D13) have never been observed among more than 500 strains. The M lines in C and D are size markers (after [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005293#pntd.0005293.ref052" target="_blank">52</a>]).</p

The many different terms used in the pathogen population genetic literature to designate the same evolutionary entity (near-clade).

<p>The many different terms used in the pathogen population genetic literature to designate the same evolutionary entity (near-clade).</p

Evolutionary pattern of the semiclonal model [10].

<p>In a predominantly recombining species, occasional bouts of clonality generate “epidemic” clones (symbolized by dark lines), the lifetime of which is limited to at most a few years; their genetic makeup then vanishes in the common gene pool. If samples are surveyed at times A or B, the presence of repeated clonal genotypes will increase the level of LD of the population, although this population is a predominantly recombining one. Growing phylogenetic signal and clonality threshold (see text above) are lacking in this situation (see ref. [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005293#pntd.0005293.ref016" target="_blank">16</a>]).</p

“Russian doll” model.

<p>When population genetic tests are performed with adapted markers of sufficient resolution within each of the near-clades that subdivide the species under study (large tree, left part of the figure), they reveal a miniature picture of the whole species, with the two main PCE features, namely, LD and lesser near-clades (small tree, right part of the figure). This supports the hypothesis that the near-clades are not potentially panmictic, biological species and rather that they also undergo predominant clonal evolution.</p

Presence (green)/absence (red) distribution of VEP1 across the reference tree.

<p>The reference tree topology is based on information from various sources, including NCBI taxonomy <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022279#pone.0022279-Wheeler1" target="_blank">[49]</a>, ‘Tree of Life’ <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022279#pone.0022279-Maddison1" target="_blank">[50]</a>, ‘The All-Species Living Tree’ project <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022279#pone.0022279-Yarza1" target="_blank">[51]</a>, and TIMETREE <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022279#pone.0022279-Hedges1" target="_blank">[52]</a> (see the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022279#s2" target="_blank">Materials and Methods</a> section).</p

Comparative structural analysis of VEP1.

<p>a) Lesk-Hubbard plot of number of residue correspondences <i>vs</i>. RMSD for VEP1 and each of six least redundant extended SDR structures in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022279#pone-0022279-t001" target="_blank">Table 1</a>. Each color denotes a structure with PDB code and protein name as follows: red: <i>2v6g</i>-A, VEP1; dark blue: 2c20-A, UDP-glucose 4-epimerase; medium blue: 1bsv-A, GDP-fucose synthetase; light blue: 2pk3-A, GDP-6-deoxy-D-lyxo-4-hexulose reductase; dark green: 1orr-A, CDP-tyvelose 2-epimerase; medium green: 1rkx-C, CDP-glucose 4,6-dehydratase; and light green: 2c59-A, GDP-mannose 3,5-epimerase. b) Ribbon diagram of the VEP1 (<i>2v6g</i>) structure showing the distribution of residues scoring below and above the sieving RMSD in the Lesk-Hubbard plot. The conserved core is colored red (α helices) and green (β strands). The variable regions are colored in grey. The nucleotide cofactor (NADP) is drawn in ball-and-stick representation.</p

ML phylogenetic tree of VEP1.

<p>The tree was inferred from 239 amino acid characters using the empirical replacement matrix of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022279#pone.0022279-Le1" target="_blank">[61]</a>, setting amino acid frequencies as free parameters, gamma-distributed rates among sites (4 categories; <i>α</i> = 1.532), and a proportion of invariant sites (I = 0.060), referred to as LG+F+dG+I model. Non-parametric bootstrap (1000 replicates)/aLRT support scores greater than 50% are shown above the respective nodes. Light (right) and dark (left) background areas indicate, respectively, the sequences used for building the tree (identified using tBLASTn; >25% pairwise sequence identity), and the extant closest remote homologs of VEP1 (identified using remote homology searching methods), which were not used for tree building, but are shown to indicate this study's hypothesis about the evolutionary origin of VEP1. Subtrees subtending inferred bacteria-to-eukaryote LGT events are colored green (viridiplantae) and fucsia (fungi). Green and red dots next to the taxa labels indicate plant-associated non-phytopathogenic and phytopathogenic bacteria, respectively. α, β, γ, and δ denote Alpha-, Beta-, Gamma, and Epsilon-proteobacteria, respectively; Ac, Actinobacteria; Ba, Bacteroidetes; Ch, Chloroflexi; Fi, Firmicutes.</p

VEP1 (<i>2v6f</i>) amino acid primary sequence, secondary structural elements including α helices (arrows) and β strands (boxes), and motif logos for 10 structural/functional motifs (motifs 1–10) discussed in the text.

<p>In the primary sequence, motifs are colored red, and red residues outside motifs denote complete evolutionary conservation; the structurally conserved core in the MUSTANG-MR analysis is underlined; white/black backgrounds denote Rossmann dinucleotide-binding/substrate-binding domains, respectively. Secondary structural elements are labeled as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022279#pone.0022279-Thorn1" target="_blank">[47]</a>). Motif logos were derived from the 81 sequences MSA of this study. In motif logos, green denotes a polar residue, red a hydrophobic residue, cyan a basic residues, and blue an acidic residue; arrow points denote the direction of replacements at critical sites if VEP1 arose as depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022279#pone-0022279-g002" target="_blank">Figure 2</a>. Roman numerals next to motif logos denote I: embryophytes and bacterial cluster I; IIa: fungi, trebouxiophytes, and bacterial cluster IIa; and IIb: bacterial cluster IIb. Motifs 6 and 9 are newly described in this study.</p