<i>P</i>. <i>ovale</i> reticulocyte binding protein 2 (<i>rbp-2</i>) sequence alignment.

<p>Both <i>P</i>. <i>ovale curtisi</i> and <i>P</i>. <i>ovale wallikeri</i> sequences were aligned using Geneious software program in order to select a conserved region for the two <i>P</i>. <i>ovale</i> subspecies. Cytosine is labelled purple, adenine pink, guanine yellow and thymine green. The forward (PoRBP2FWD) and reverse (PoRBP2REV) primers are denoted in dark and light green boxes, respectively.</p

Oligonucleotide primer sequences used in this study.

<p>Oligonucleotide primer sequences used in this study.</p

Novel <i>P</i>. <i>ovale</i> primers only amplify <i>P</i>. <i>ovale</i> and not the other human-infecting species.

<p><i>P</i>. <i>falciparum</i>, <i>P</i>. <i>vivax</i>, <i>P</i>. <i>malariae</i>, <i>P</i>. <i>knowlesi</i>, <i>P</i>. <i>ovale curtisi</i>, <i>P</i>. <i>ovale wallikeri</i> DNA samples were utilized for primer specificity testing. Only the positive control (a known <i>P</i>. <i>ovale</i> sample), <i>P</i>. <i>ovale curtisi</i> and <i>P</i>. <i>ovale wallikeri</i> were amplified using our primers (amplification plots with Ct values of 25.57, 33.38 and 35.2 respectively). No amplification (flat lines) was noted for the other species and the no template control (NTC).</p

Summary of multi-locus genotyping using seven different markers (TA1, poly a, PfPK2, TA109, 2490, C2M34, and C3M69). DNW denotes loci for which there was no amplification after two attempts.

<p>Summary of multi-locus genotyping using seven different markers (TA1, poly a, PfPK2, TA109, 2490, C2M34, and C3M69). DNW denotes loci for which there was no amplification after two attempts.</p

Identification of recombination events in a single patient and general protocol used for identifying recombination in all patient samples using RDP4.

<p>(A) RDP4 generated recombination schematic indicating evidence of intra-segmental recombination. Regions of recombination events, and corresponding insertions from donor sequences are depicted. Significant sites including nucleotide position(s) involved in recombination are shown for the last recombinant (M_0608_H07). Significant nucleotide positions involved in recombination were checked in a similar fashion for all other recombinants. Recombination events were only considered if at least three recombination analysis methods were in agreement with the obtained results, a <i>p-</i>value of 0.05 or better was obtained, and the consensus recombination score was above the recommended 0.60 value. An example for recombinant M_0608_HO7 showing recombination signals identified by the (B) BOOTSCAN method, (C) RDP method, and (D) GENECONV method are shown. Lastly, the MAXCHI matrices tool when necessary was used to determine the optimal locations of breakpoint pairs, shown in (E). These graphically represent the probabilities of all potential breakpoint pairs that have the best associated <i>p-</i>values displayed by the color key beside the matrix. Dark red peaks indicate the most probable positions of breakpoint pairs, for recombinant M_0608_HO7 these were at nucleotide positions 438 and 733.</p

Summary of patient gravidity, total number of DBL3X sequences obtained, the number of clones sequenced and among these the number that were unique, and the nucleotide diversity.

<p>The value of π denotes the observed average pairwise nucleotide diversity.</p

VAR2CSA in placental parasites shows unprecendented diversity with no interindividual clustering.

<p>Maxiumum Liklihood Tree of 79 Unique DBL3X Sequences. Sequences were aligned by translation. Consensus tree shown, with 50% cutoff. Taxa colored by placental sample. Sequences from the same patient sample tend to group together in one to three clades with the exception of 0786-A09, denoted by an asterisk.</p

Novel motifs identified in Kenyan samples are associated with host immune status (gravidity).

<p>Motifs TKTK/PQQK are predominately found in primigravidae and TKQN in multigravidae. Sequence motifs (highlighted in grey) identified in Dahlback et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031565#pone.0031565-Dahlback2" target="_blank">[33]</a> show no strong parity association in Kenyan samples. * <i>P</i><0.001, by c² test.</p

Results from Tajima's Neutrality Test.

<p>The Tajima test statistic <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031565#pone.0031565-Dellicour1" target="_blank">[1]</a> was estimated using MEGA4 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031565#pone.0031565-Brabin1" target="_blank">[2]</a>. All positions containing gaps and missing data were eliminated from the dataset (Complete deletion option). The abbreviations used are as follows: m = number of sites, S = Number of segregating sites, p_s = S/m, Θ = p_s/a₁, and π = nucleotide diversity. D is the Tajima test statistic.</p

Toggling amino acids with gravidity-associated preferences.

<p>A total of nine sites were identified as varying significant between primigravidae (P) and multigravidae (M), shown here in relative proportion of toggling amino acids at each site (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031565#pone.0031565.s002" target="_blank">Table S1</a> for details and other toggling sites). The chi-square test (or Fisher's exact test when appropriate) was used; all sites, <i>P</i><0.05.</p