Unveiling hidden parasite diversity: Long-read mitogenomics in Eimeriidae and Haemosporida (Eukaryota: Apicomplexa)

Mitochondrial genomes of apicomplexan parasites exhibit remarkable structural diversity, ranging from highly reduced linear molecules to circular-mapping concatemers, yet their full characterization has been hindered by technical limitations in resolving complex infections. This study establishes a novel integrated workflow combining one-step PCR amplification with Oxford Nanopore Technologies (ONT) to sequence complete mitochondrial genomes from Eimeriidae and Haemosporida parasites. Successful assembly of 29 high-quality mitogenomes (12 Eimeriidae, 17 Haemosporida) from 15 samples, demonstrating the method's sensitivity. Comparative analyses revealed cryptic mixed/co-infections in 11 samples that were undetectable by Sanger sequencing, highlighting ONT's superior resolution for uncovering true parasite diversity. Phylogenomic reconstruction using the largest Eimeriidae dataset to date confirmed the monophyly of passeriform Isospora and identified a basal position for a novel Caryospora lineage from Ptyas major. In Haemosporida, analysis of 202 mitogenomes revealed non-monophyletic familial relationships. Selection analyses indicated predominant purifying selection in mitochondrial protein-coding genes of Eimeriidae. Our findings underscore the utility of long-read mitogenomics in elucidating complex infection dynamics and provide a scalable framework for biodiversity surveys of understudied apicomplexans parasites, with implications for understanding their evolutionary ecology and host-parasite interactions.