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Background  The merozoite surface protein-1 (MSP-1) is a candidate target for the development of blood stage vaccines against malaria. Polymorphism in MSP-1 can be useful as a genetic marker for strain differentiation in malarial parasites. Although sequence diversity in the MSP-1 locus has been extensively analyzed in field isolates of  Plasmodium falciparum  and  P. vivax , the extent of variation in its homologues in  P. ovale curtisi  and  P. ovale wallikeri , remains unknown.    Methodology/Principal Findings  Analysis of the mitochondrial cytochrome  b  sequences of 10  P. ovale  isolates from symptomatic malaria patients from diverse endemic areas of Thailand revealed co-existence of  P. ovale curtisi  (n = 5) and  P. ovale wallikeri  (n = 5). Direct sequencing of the PCR-amplified products encompassing the entire coding region of  MSP-1  of  P. ovale curtisi  ( PocMSP-1 ) and  P. ovale wallikeri  ( PowMSP-1 ) has identified 3 imperfect repeated segments in the former and one in the latter. Most amino acid differences between these proteins were located in the interspecies variable domains of malarial MSP-1. Synonymous nucleotide diversity (π S ) exceeded nonsynonymous nucleotide diversity (π N ) for both  PocMSP-1  and  PowMSP-1 , albeit at a non-significant level. However, when MSP-1 of both these species was considered together, π S  was significantly greater than π N  (p<0.0001), suggesting that purifying selection has shaped diversity at this locus prior to speciation. Phylogenetic analysis based on conserved domains has placed  PocMSP-1  and  PowMSP-1  in a distinct bifurcating branch that probably diverged from each other around 4.5 million years ago.    Conclusion/Significance  The MSP-1 sequences support that  P. ovale curtisi  and  P. ovale wallikeri  are distinct species. Both species are sympatric in Thailand. The low level of sequence diversity in  PocMSP-1  and  PowMSP-1  among Thai isolates could stem from persistent low prevalence of these species, limiting the chance of outcrossing at this locus.

Low Level of Sequence Diversity at Merozoite Surface Protein-1 Locus of Plasmodium ovale curtisi and P. ovale wallikeri from Thai Isolates