Zoonotic origin of the human malaria parasite *Plasmodium malariae* from African apes

*Plasmodium malariae* is one of six Plasmodium species that commonly cause malaria in humans. Despite its widespread geographic distribution, it is the least well-characterized human malaria parasite, partly due to its typically low burden and understudied prevalence. While the origins of major human malaria parasites like *P. falciparum* and *P. vivax* have been traced to African apes, the evolutionary history of *P. malariae* remained unknown. Early observations noted a quartan parasite morphologically similar to *P. malariae* in chimpanzees, named *P. rodhaini*. Transmission studies showed apparent host flexibility, with *P. rodhaini* infecting humans and *P. malariae* infecting chimpanzees. This suggested a close relationship, but verification was hindered by a lack of maintained strains. More recent genetic studies have investigated *P. malariae* and its relatives. Studies of wild chimpanzees, gorillas, and bonobos revealed two distinct *P. malariae*-related lineages, one close to the human parasite and another more divergent. Genome sequences from chimpanzees showed a close relationship to *P. malariae* but were considered a separate species, possibly diverging over 3 million years ago. Another related lineage, *P. brasilianum*, infects New World monkeys and shows near-identity to *P. malariae* at limited loci, raising questions about the direction of host transfer. To resolve the species relationships and host ranges, this study used advanced techniques to generate new sequences from wild and captive apes and mined genomic databases. The goal was to determine the number of *P. malariae*-related species and trace their evolutionary history and host transitions.

Previous research has established the close relatives of *P. malariae* in non-human primates: *P. rodhaini* in African apes and *P. brasilianum* in New World monkeys. Early studies, largely based on morphological comparisons and limited transmission experiments, hinted at a close relationship between *P. malariae* and *P. rodhaini*, suggesting potential host flexibility or a common ancestor. However, the lack of robust genetic data hampered the clarification of their evolutionary relationship. More recent genetic analyses provided limited data sets mostly focusing on a few mitochondrial or nuclear genes. These studies pointed towards the existence of distinct *P. malariae*-related lineages in apes, but the precise number of species and the evolutionary relationships remained unresolved. Similarly, the directionality of host transfer between *P. malariae* and *P. brasilianum* has been contentious, with conflicting evidence suggesting both zoonotic transfer from apes to humans and anthroponotic transfer from humans to New World monkeys. The ambiguity highlights the need for comprehensive genomic analyses to resolve these outstanding questions.

This study employed a multi-faceted approach to investigate the evolutionary origins of *P. malariae*. First, new sequences were generated from fecal and blood samples of wild-living and captive chimpanzees and gorillas using limiting dilution PCR (LD-PCR). LD-PCR enhances the chances of obtaining parasite DNA when the parasite DNA is diluted in the host material. This approach overcame limitations associated with low parasite densities in ape samples. Second, a novel method was developed to identify *P. malariae*-related sequences within existing published genomic databases using data mining approaches. This greatly extended the number of sequences available for analysis and expanded the scope of phylogenetic reconstruction. The bioinformatics aspect involves several steps: initially, existing data resources like the NCBI GenBank and the European Nucleotide Archive were screened for suitable sequences. Then, sophisticated bioinformatic pipelines including read mapping, assembly, and genome annotation were employed to reconstruct the genomes of the discovered lineages. Once the genomes were assembled, phylogenetic trees and networks were constructed using suitable evolutionary models, aiming to depict the evolutionary relationships among different lineages. These combined approaches allowed for a more comprehensive understanding of the evolutionary history of *P. malariae* and related species. To assess genetic diversity within lineages, the researchers calculated nucleotide diversity statistics, examining the distribution of polymorphisms across the genome. They also used phylogenetic network approaches to visualize the relationships between lineages and to detect potential recombination events that can obfuscate phylogenetic signals. This multifaceted approach provided a robust framework for addressing the study's objectives.

Phylogenetic analyses of newly generated and previously published sequences revealed three distinct lineages within the *P. malariae*-related clade. One lineage (M2) is significantly divergent from *P. malariae* and includes sequences from gorillas, chimpanzees, and bonobos, as well as sequences from mosquitoes that had fed on apes. This lineage represents a previously unknown, distantly related species. A second lineage (M1-like) is much more closely related to *P. malariae*, the human infecting lineage, but lacks evidence of recent genetic exchange with it. This suggests that M1-like is another distinct ape-infecting species. *P. malariae* (M1) itself exhibits significantly lower genetic diversity than the ape-infecting lineages (M1-like and M2), indicating a recent bottleneck event consistent with a zoonotic transmission event. Importantly, *P. brasilianum* was found to cluster within the radiation of the human *P. malariae* lineages, providing evidence of anthroponosis (transmission from humans to New World monkeys). The finding that there is little evidence for recent genetic exchange between the ape-infecting lineages (M1-like and M2) and the human-infecting lineage (M1) strengthens the case for zoonotic origin. The significantly lower diversity observed in the human lineage further supports a recent cross-species transmission event, followed by a population expansion in the human host. A single region of elevated diversity on chromosome 10 in *P. malariae* suggests limited introgression from ape parasites after the initial zoonotic event.

The findings strongly support a zoonotic origin of *P. malariae* from an African ape ancestor. The low genetic diversity within *P. malariae* compared to its ape relatives indicates a recent transmission event followed by a population expansion within humans. The lack of substantial introgression from ape parasites supports the establishment of *P. malariae* as a distinct, human-adapted species. The parallel between the evolutionary history of *P. malariae* and *P. falciparum*, both showing recent zoonotic origins with limited subsequent gene flow from ape ancestors, highlights a common pattern in the emergence of human malaria parasites. The clear placement of *P. brasilianum* within the *P. malariae* radiation definitively resolves the long-standing debate regarding the directionality of host transfer, showing a clear anthroponotic origin. This work offers a more complete picture of the evolutionary dynamics of *P. malariae*, revealing new species and highlighting the crucial role of zoonotic transmission in the emergence of human malaria parasites.

This study definitively establishes the zoonotic origin of *P. malariae* from African apes. The identification of two new closely related ape-infecting species further illuminates the complexity of malaria parasite evolution. The findings emphasize the ongoing importance of monitoring ape populations for malaria parasites and underscores the potential for future zoonotic events. Future research could focus on further characterizing the newly discovered ape-infecting species, investigating the genetic mechanisms underlying host specificity, and examining the potential for future zoonotic transmission events.

The study is primarily based on genomic data. While comprehensive, the lack of extensive longitudinal studies may limit the certainty about the precise timing of the zoonotic event and the subsequent evolutionary trajectory of *P. malariae*. The reliance on existing genomic databases for some samples could introduce biases depending on sample collection and sequencing protocols. Furthermore, the study primarily focused on genetic data; functional studies would be needed to further explore the differences between the lineages at the phenotypic level.