Multigenomic Delineation of Plasmodium Species of the Laverania Subgenus Infecting Wild-Living Chimpanzees and Gorillas

Abstract

Plasmodium falciparum, the major cause of malaria morbidity and mortality worldwide, is only distantly related to other human malaria parasites and has thus been placed in a separate subgenus, termed Laverania Parasites morphologically similar to P. falciparum have been identified in African apes, but only one other Laverania species, Plasmodium reichenowi from chimpanzees, has been formally described. Although recent studies have pointed to the existence of additional Laverania species, their precise number and host associations remain uncertain, primarily because of limited sampling and a paucity of parasite sequences other than from mitochondrial DNA. To address this, we used limiting dilution polymerase chain reaction to amplify additional parasite sequences from a large number of chimpanzee and gorilla blood and fecal samples collected at two sanctuaries and 30 field sites across equatorial Africa. Phylogenetic analyses of more than 2,000 new sequences derived from the mitochondrial, nuclear, and apicoplast genomes revealed six divergent and well-supported clades within the Laverania parasite group. Although two of these clades exhibited deep subdivisions in phylogenies estimated from organelle gene sequences, these sublineages were geographically defined and not present in trees from four unlinked nuclear loci. This greatly expanded sequence data set thus confirms six, and not seven or more, ape Laverania species, of which P. reichenowi, Plasmodium gaboni, and Plasmodium billcollinsi only infect chimpanzees, whereas Plasmodium praefalciparum, Plasmodium adleri, and Pladmodium blacklocki only infect gorillas. The new sequence data also confirm the P. praefalciparum origin of human P. falciparum.

Keywords: Laverania; P. falciparum; Plasmodium parasites infecting chimpanzees and gorillas; cryptic Plasmodium species; single genome sequencing.

Fig. 1.

Locations of sites where Laverania-positive apes were sampled. The ranges of ape species and subspecies are indicated: Nigeria-Cameroonian chimpanzee (Pan t. ellioti) (magenta), central chimpanzees (Pan t. troglodytes) (red), eastern chimpanzees (Pan t. schweinfurthii) (blue), and western gorillas (G. g. gorilla) (yellow). Circles, squares, and octagons identify field sites where fecal samples were collected from chimpanzees, gorillas or both, respectively (supplementary table S1, Supplementary Material online); triangles indicate two chimpanzee sanctuaries. A red border indicates all field sites from which new Laverania sequences were generated for this study (supplementary table S2, Supplementary Material online). Country borders (white) and major rivers (blue) are indicated.

Fig. 2.

Maximum likelihood phylogeny of Laverania mitochondrial DNA sequences. A subset of 165 SGA-derived cytB sequences (956 bp) is shown; only distinct haplotypes per field site are shown (the full set of 709 SGA-derived cytB sequences is shown in supplementary fig. S1, Supplementary Material online). Sequences are color coded to indicate host: chimpanzees (Pan t. ellioti in orange, Pan t. troglodytes in red, and Pan t. schweinfurthi in blue), or gorillas (G. g. gorilla in green), with a two-letter code for the field site of origin (fig. 1). Reference sequences for human P. falciparum and chimpanzee P. reichenowi are in black. C1, C2, and C3 represent the chimpanzee parasite species P. reichenowi, P. gaboni, and P. billcollinsi; G1, G2, and G3 represent the gorilla parasite species P. praefalciparum, P. adleri, and P. blacklocki. Bootstrap values are shown for major nodes only (the scale bar represents 0.01 substitutions per site).

Fig. 3.

Maximum likelihood phylogenies of Laverania nuclear gene sequences. (A) ldh (772 bp), (B) eba175 (397 bp), (C) eba165 (790 bp), and (D) p47 (800 bp) gene sequences. Only distinct haplotypes per field site are shown (the full sets of sequences are shown in supplementary figs. S2–S5, Supplementary Material online). Sequences and clades are colored and labeled as in figure 2. Numbers on branches indicate percent bootstrap support. The scale bars indicate 0.01 substitutions per site.

Fig. 4.

Maximum likelihood phylogeny of Laverania apicoplast DNA sequences. A subset of 80 SGA-derived caseinolytic protease M (clpM) sequences (390 bp) is shown; only distinct haplotypes per field site are shown (the full set of 227 clpM sequences appears in supplementary fig. S6, Supplementary Material online). Sequences and clades are colored and labeled as in figure 2. Numbers on nodes indicate percent bootstrap support. The scale bar represents 0.01 substitutions per site.