New reference genomes to distinguish the sympatric malaria parasites, Plasmodium ovale curtisi and Plasmodium ovale wallikeri

Despite Plasmodium ovale curtisi ( Poc ) and wallikeri ( Pow ) being important human-infecting malaria parasites that are widespread across Africa and Asia, little is known about their genome diversity. Morphologically identical, Poc and Pow are indistinguishable and commonly misidentified . Recent rises in the incidence of Poc / Pow infections have renewed efforts to address fundamental knowledge gaps in their biology, and to develop diagnostic tools to understand their epidemiological dynamics and malaria burden. A major roadblock has been the incompleteness of available reference assemblies (PocGH01, PowCR01; ~ 33.5 Mbp). Here, we applied multiple sequencing platforms and advanced bioinformatics tools to generate new reference genomes, Poc221 (South Sudan; 36.0 Mbp) and Pow222 (Nigeria; 34.3 Mbp), with improved nuclear genome contiguity (> 4.2 Mbp), annotation and completeness (> 99% Plasmodium spp., single copy orthologs). Subsequent sequencing of 6 Poc and 15 Pow isolates from Africa revealed a total of 22,517 and 43,855 high-quality core genome SNPs, respectively. Genome-wide levels of nucleotide diversity were determined to be 2.98 × 10 –4 ( Poc ) and 3.43 × 10 –4 ( Pow ), comparable to estimates for other Plasmodium species. Overall, the new reference genomes provide a robust foundation for dissecting the biology of Poc/Pow , their population structure and evolution, and will contribute to uncovering the recombination barrier separating these species.