Wild bonobos host geographically restricted malaria parasites including a putative new Laverania species

Abstract

Malaria parasites, though widespread among wild chimpanzees and gorillas, have not been detected in bonobos. Here, we show that wild-living bonobos are endemically Plasmodium infected in the eastern-most part of their range. Testing 1556 faecal samples from 11 field sites, we identify high prevalence Laverania infections in the Tshuapa-Lomami-Lualaba (TL2) area, but not at other locations across the Congo. TL2 bonobos harbour P. gaboni, formerly only found in chimpanzees, as well as a potential new species, Plasmodium lomamiensis sp. nov. Rare co-infections with non-Laverania parasites were also observed. Phylogenetic relationships among Laverania species are consistent with co-divergence with their gorilla, chimpanzee and bonobo hosts, suggesting a timescale for their evolution. The absence of Plasmodium from most field sites could not be explained by parasite seasonality, nor by bonobo population structure, diet or gut microbiota. Thus, the geographic restriction of bonobo Plasmodium reflects still unidentified factors that likely influence parasite transmission.

Fig. 1

Plasmodium infections of wild-living bonobos. Ape study sites are shown in relation to the ranges of the bonobo (P. paniscus, dashed red) and the eastern chimpanzee (P. t. schweinfurthii, dashed blue), with white dots indicating sites where no Plasmodium infection was found (see Table 1 and Supplementary Table 3 for a list of all field sites and their code designation). The Tshuapa–Lomami–Lualaba (TL2) site where bonobos are endemically infected with multiple Plasmodium species, including a newly discovered Laverania species (B1), is shown in red with two dots indicating sampling on both sides of the Lomami River. Eastern chimpanzee field sites with endemic P. reichenowi, P. gaboni and/or P. billcollinsi infections are shown in yellow. A red circle highlights one bonobo (KR) and one chimpanzee (PA) field site where B1 parasite sequences were detected in a single faecal sample. Forested areas are shown in dark green, while arid or semiarid areas are depicted in brown. Major lakes and rivers are shown in blue. Dashed yellow lines indicate national boundaries. The scale bar indicates 200 km

Fig. 2

Relationship of bonobo parasites to ape Laverania species. A maximum likelihood tree of mitochondrial cytochrome B (cytB) sequences (956 bp) depicting the phylogenetic position of newly derived bonobo parasite sequences (magenta) is shown. Only distinct cytB haplotypes are depicted (the full set of SGA-derived bonobo parasite sequences is shown in Supplementary Fig. 1). Sequences are colour-coded, with capital letters indicating their field site of origin (see Fig. 1 for location of field sites) and lowercase letters denoting their host species and subspecies origin (ptt: P. t. troglodytes, red; pte: P. t. ellioti, orange; pts: P. t. schweinfurthii, blue; ggg: G. g. gorilla, green; pp: Pan paniscus, magenta). C1, C2 and C3 represent the chimpanzee parasites P. reichenowi, P. gaboni and P. billcollinsi; G1, G2 and G3 represent the gorilla parasites P. praefalciparum, P. adleri and P. blacklocki (the P. falciparum 3D7 reference sequence is shown in black). P. reichenowi (C1) and P. gaboni (C2) mitochondrial sequences are known to segregate into two geographically defined subclades according to their collection site in 'western' (W) or 'eastern' (E) Africa. Bonobo parasite sequences (magenta) cluster with P. gaboni from eastern chimpanzees (C2E), but also form a new clade, termed B1. The tree was constructed using PhyML with TIM2+I+G as the evolutionary model. Bootstrap values are shown for major nodes only (the scale bar represents 0.01 substitutions per site)

Fig. 3

A new Laverania species specific for bonobos. a, b Maximum likelihood phylogenetic trees are shown for nuclear gene fragments of the a erythrocyte-binding antigen 165 (eba165; 790 bp) and b the gametocyte surface protein P47 (p47; 800 bp) of Laverania parasites. Sequences are labelled and coloured as in Fig. 2 (identical sequences from different samples are shown; identical sequences from the same sample are excluded). C1, C2 and C3 represent the chimpanzee parasites P. reichenowi, P. gaboni and P. billcollinsi; G1, G2 and G3 represent the gorilla parasites P. praefalciparum, P. adleri and P. blacklocki (PrCDC and Pf3D7 reference sequences are shown in black). Bonobo parasite sequences cluster within P. gaboni (C2) or form a new distinct clade (B1), indicating a new Laverania species (see text for information on the single eba165 B1 sequence from an eastern chimpanzee). The trees were constructed using PhyML with TPM3uf+G (a) and GTR+G (b) as evolutionary models. Bootstrap values are shown for major nodes only (the scale bar represents 0.01 substitutions per site)

Fig. 4

Bonobo infections with non-Laverania parasites. Maximum likelihood phylogenetic trees are shown for mitochondrial and apicoplast gene sequences of non-Laverania parasites. Ape-derived a cytB (956 bp), b clpM (327 bp), c cox1 (296 bp) and d clpM (574 bp) sequences are labelled and coloured as in Fig. 2 (identical sequences from different samples are shown; identical sequences from the same sample are excluded). Human and monkey parasite reference sequences from the database are labelled by black squares and circles, respectively. Brackets indicate non-Laverania species, including P. malariae, P. vivax, P. ovale curtisi and P. ovale wallikeri (available sequences are too short to differentiate ape- and human-specific lineages) as well as the monkey parasites P. inui and P. hylobati. Newly identified bonobo parasite sequences are indicated by arrows, all of which are from the TL2 site. One TL2 cytB sequence clusters with a previously reported parasite sequence from a chimpanzee sample (DGptt540), forming a well-supported lineage that is only distantly related to human and ape P. malariae, and thus likely represents a new P. malariae-related species. The trees were constructed using PhyML with GTR+G (a), TRN+I (b, d) and TIM2+I (c) as evolutionary models. Bootstrap values over 70% are shown for major nodes only (the scale bar represents 0.01 substitutions per site)

Fig. 5

The Lomami River is not a barrier to Laverania parasite transmission. a Maximum likelihood phylogenetic tree of bonobo mitochondrial (D-loop) sequences. Haplotypes are labelled by field site (see Fig. 1 and refs. ^,,,, for their geographic location and code designation), with those identified at multiple field sites indicated (e.g., C/Wamba/KR/BN/IK/LA). Newly derived haplotypes from the TL2 site are shown in blue (previously reported mtDNA sequences are shown in black)^,,. Brackets highlight two clades that are exclusively comprised of mtDNA sequences from bonobos sampled east of the Lomami River. TL2 haplotypes that do not fall within these clades (denoted by arrows) were all sampled west of the Lomami River (TL2-W). The tree was constructed using PhyML with HKY+G as the evolutionary model. Bayesian posterior probability values ≥ 0.6 are shown (the scale bar represents 0.01 substitutions per site). b Locations of individual bonobo faecal samples collected at the TL2 site. Sampling locations west (TL2-W) and east (TL2-E and TL2-NE) of the Lomami River were plotted using GPS coordinates, with red and white dots indicating Laverania parasite positive and negative specimens, respectively. Samples that contained P. reichenowi, P. malariae-like, P. vivax-like and P. ovale-like parasites are also indicated. Forested areas are shown in green, while savannas are depicted in brown. The Lomami River is shown in blue. Local villages are denoted by black squares. The scale bar indicates 2 km

Fig. 6

Laverania infection of bonobos is not associated with particular faecal plant or microbiome constituents. A principal component analysis of unweighted UniFrac distances was used to visualise compositional differences of a, b plant (matK and rbcL) and c bacterial (16S rRNA) constituents in Laverania-positive (dark border) and -negative (light border) faecal samples from bonobos (blue) and chimpanzees (pink). The sample positions (shown for the first two components) do not indicate separate clustering of Laverania-positive and -negative samples