High diversity and novel host associations of bat haemosporidian parasites in Cambodia: insights from a nationwide bat survey and multilocus analyses

Abstract Background Haemosporidian (“malaria”) parasites are a diverse group of vector-borne apicomplexan parasites that infect a wide range of vertebrate hosts, including humans. Bats harbour a remarkable diversity of haemosporidians and are considered important hosts in the evolution of mammalian haemosporidian lineages, yet parasite diversity, host associations, and evolutionary relationships remain poorly understood, particularly in biodiversity-rich Southeast Asia. Methods We conducted a nationwide molecular survey of 1180 bats representing 34 species across Cambodia and combined extensive sampling with multilocus phylogenetic and ecological analyses to investigate haemosporidian diversity, host associations, evolutionary relationships, and ecological correlates of infection. Results Haemosporidian infections were detected in 12.9% of the sampled bats and comprised diverse Polychromophilus and Hepatocystis taxa, including several novel host associations. Notably, Hepatocystis was detected in Emballonuridae for the first time and occurred across multiple bat families. We substantially expand sampling of a previously described Scotophilus -associated Polychromophilus lineage and demonstrate its remarkable genetic conservation across geographic regions. In contrast, Hepatocystis parasites exhibited substantial genetic diversity across multiple host taxa. Multi-gene analyses expanded phylogenetic representation of bat-associated Hepatocystis and revealed close relationships between some bat- and squirrel-associated lineages. These data further suggest considerable diversity among Asian Hepatocystis parasites and phylogenetic patterns consistent with diversification across Asian host groups. Ecological analyses indicated that the infection varied more among host species and sampling sites than with measured host traits or landscape attributes and revealed pronounced seasonal differences in infection probability together with lower infection rate in more densely populated landscapes. Conclusions Our findings expand current knowledge of bat haemosporidian diversity, host associations, and evolutionary relationships in Southeast Asia. Novel host associations, including the first record of Hepatocystis in Emballonuridae, highlight previously undocumented parasite diversity in regional bat communities. Infection was structured principally by host species and by conditions at individual roosting sites rather than by broader landscape composition, indicating that fine-scale roost ecology and vector biology are the more promising targets for understanding transmission in bats. Together, these findings contribute to a broader understanding of diversification and host associations in mammalian malaria parasites.