PMC

Binding of RBPs to ape and human red blood cells. (A) Dot plots depict the binding of human P. vivax RBP2a and RBP2b proteins and chimpanzee P. vivax RBP2e and RBP3 proteins to human (first row), gorilla (second row), and chimpanzee (third row) red blood cells, respectively, along with antibody-only controls of human red blood cells (fourth row). RBP binding was detected using an RBP-specific polyclonal rabbit antibody and an anti-rabbit (Alexa Fluor 647-labeled) secondary antibody (y axis), and reticulocytes were identified by staining with TO (x axis). Flow cytometry gates separating normocytes from reticulocytes and protein binding from no protein binding are shown by vertical and horizontal lines, respectively. Numbers indicate the percentage of total cells within the respective gate. (B) Percentage of gorilla (green), chimpanzee (red), and human (black) normocytes bound by the respective RBP. (C) Percentage of gorilla, chimpanzee, and human reticulocytes bound by the respective RBP. Experiments were performed as three technical replicates with the background signal from the antibody-only control subtracted from each binding result.

Nucleotide sequence diversity in ape and human P. vivax. (A) The π calculated across a common set of 4,260 core genes for six chimpanzee and nine human P. vivax strains (as in , but three genes with fewer than 35 aligned sites were excluded). Median and mean (weighted by gene length) π values are indicated by solid and dashed lines, respectively; box and whiskers indicate the interquartile range and 99th percentiles, respectively. Plots including outliers are shown in SI Appendix, Fig. S1B. (B) Density plots of NI values shown on a log₂ scale for ape (red) and human (black) P. vivax genes. Values are shown for 1,585 genes with nonzero values of NI in both populations. (C) Site-frequency spectra of polymorphisms at fourfold degenerate (blue) and zerofold degenerate (orange) sites extracted from SNP data of human P. vivax samples from Southeast Asia ().

The rbp gene family in ape and human P. vivax. (A) A midpoint-rooted maximum-likelihood phylogenetic tree is shown depicting the relationships of human (black) and chimpanzee (PvSY56 and PvSY43, red) P. vivax rbp genes with their orthologs in P. knowlesi, P. cynomolgi, P. inui, P. fragile, and human P. malariae (purple). P. vivax, P. cynomolgi, and P. knowlesi genes are labeled according to their published names; genes from P. inui, P. fragile, and P. malariae are labeled according to the clade in which they are placed. Pseudogenes are indicated by yellow stars. The Inset shows the relationship of rbp1a sequences among representative human and three sequenced chimpanzee P. vivax strains, rooted using P. cynomolgi (see SI Appendix for details). (B) Locations of frameshift (purple) and premature stop (black) mutations in rbp2d, rbp2e, and rbp3 sequences assembled from published human P. vivax strains (, ), relative to the full-length coding sequence from chimpanzee P. vivax (light green). Each bar represents a set of mutations that occurred in two or more human P. vivax strains for which a full-length sequence was assembled (128, 162, and 227 sequences for rbp2d, rbp2e, and rbp3, respectively); the percentage of sequences containing the respective mutations is shown on the right, with “other” summarizing all mutations that occurred only once.

Evolutionary relationships of ape and human P. vivax strains. (A) An unrooted neighbor-joining tree constructed from a matrix of pairwise genetic distances from an alignment of 241 nuclear genes is shown for nine human (black) and six chimpanzee (red) P. vivax strains (the Inset shows the human P. vivax strains in greater detail). (B) As in A, but based on six nuclear genes with coverage in one gorilla P. vivax strain (green). The same human and chimpanzee P. vivax strains were included, except for PvSY81, which did not cover these genes. (C and D) Maximum-likelihood trees for fragments of nuclear genes PVP01_1418300 (C) and PVP01_1418500 (D) with P. cynomolgi and P. knowlesi included as outgroups. Sequences of P. carteri parasites are shown in blue. “Pv” denotes sequences from genome-wide analyses, shown in bold if generated by SWGA or derived from published data (SI Appendix, Table S2); all other sequences except for P. cynomolgi and P. knowlesi were generated by SGA and include a code identifying their geographic origin (SI Appendix, Fig. S5), ape subspecies (G.g.g., Gorilla gorilla gorilla; P.t.e., Pan troglodytes ellioti; P.t.t., Pan troglodytes troglodytes), and sample number (see SI Appendix, Table S4 for GenBank accession numbers). Bootstrap values ≥70 are shown for clades with two or more nonidentical tips. Fragment lengths in PvP01 are indicated above the trees. The scale bars indicate substitutions per site (see also SI Appendix, Fig. S3 for phylogenetic network representations).