Hidden Plasmodium Diversity revealed in southeastern Asian passerines using Amplicon Sequencing

Monitoring haemosporidian (Genus: Plasmodium) infections in passerine birds is essential for understanding the intricate dynamics of avian malaria and its implications for ecology and evolution of avian populations. In this study, we investigated the prevalence and diversity of malaria infections caused by Plasmodium species in three passerine species in Cat-Tien National Park, Vietnam. Using NGS amplicon sequencing and amplification of haemosporidian cytochrome b genes, we identified two known and ten novel Plasmodium lineages. Our genetic analysis revealed a high rate of Plasmodium infections in Little spiderhunter (Arachnothera longirostra; Nectariniidae), White-rumped shama (Copsychus malabaricus; Muscicapidae) and Blue-winged pitta (Pitta moluccensis; Pittidae). Species delimitation methods identified five distinct operational taxonomic units (OTU), with consistent results from ASAP, ABGD, and bPTP method, unlike the overestimated results by GMYC method. Each of the passerine species was infected with a specific subset of the total Plasmodium diversity. Phylogenetic analysis showed that sympatric Plasmodium OTU units are not closely related and possess overlapping host preferences. The findings suggest that differences in habitat use, such as the vertical strata occupied by different bird species, contribute to varying exposure levels to suitable vectors, thereby influencing infection rates and parasite diversity. Our findings corroborate the view that avian malaria parasites are not uniformly opportunistic; rather, their distribution is filtered by host identity and ecology. Understanding these dynamics is crucial for avian conservation and broader ecological studies, as avian malaria serves as a model for studying parasite-host co-evolution and the impact of environmental changes on disease dynamics.