Domination Versus Sisterhoods in the Blood Microbiota of Migrating Birds: Patterns of Within- and Between-Individual Blood Parasite Diversity Revealed Through Metabarcoding.

Avian blood parasites of the genera Plasmodium, Haemoproteus, and Leucocytozoon are typically identified through Sanger sequencing of a partial cytochrome b fragment, the MalAvi barcoding region. This approach limits the detection of mixed infections and the relative frequencies of co-infecting parasites. In contrast, next-generation sequencing (NGS) can resolve these problems but has been underused for haemosporidian lineage identification in samples from the wild. We used an improved PCR protocol and sequencing with Illumina MiSeq to determine haemosporidian assemblages in wild birds captured at a migration stopover site in Bulgaria, Europe. From 406 samples obtained from 52 bird species, we detected 81 haemosporidian lineages in 131 infected samples from 32 species (32% prevalence). On average, individuals were infected with 2.4 lineages, with 59 birds infected by a single lineage, and 21 birds infected with 5-9 lineages. A subset of samples was Illumina- and Sanger-sequenced in parallel, finding mixed infections in 72 samples and 8× higher detection rate of mixed and co-infections through high-throughput sequencing. Both methods identified the same dominant (co-infecting) lineage (91%). Metabarcoding identified common mixed infections of sister lineage groups ("sisterhoods") known for prevalent lineages and morphospecies, including Plasmodium relictum p_SGS1, Haemoproteus motacillae h_YWT2, and Haemoproteus parabelopolskyi h_SYAT01. Some other lineages appeared consistently more dominant. Our study shows that in some host communities, metabarcoding can reveal a great diversity of mixed infections. This opens new horizons to the study of assemblages of haemosporidian parasites, their interactions within individual hosts, and co-evolution with other members of the blood microbiome and the hosts.