From pathology to phylogeny: Highly rearranged mitochondrial genome of the emerging testudine intranuclear coccidium.

The testudine intranuclear coccidium (TINC) is an emerging, globally significant apicomplexan pathogen responsible for severe multisystemic disease in captive chelonians. In this study, samples were obtained from deceased captive Sri Lankan star tortoise (Geochelone elegans) in Taiwan and leopard tortoises (Stigmochelys pardalis) in Japan for comprehensive pathological and molecular analysis. Histopathological examination of three cases confirmed systemic infection characterized by intranuclear inclusions within epithelial cells of multiple organs, with lesions ranging from widespread coagulative necrosis to degenerative changes. Transmission electron microscopy further identified multiple intranuclear developmental stages, including trophozoites, meronts, and gamonts. Utilizing Oxford Nanopore long-read sequencing, we present the first complete mitochondrial genome of TINC, which exhibits a novel and highly rearranged architecture divergent from conserved patterns in the family Eimeriidae, featuring flanking direct and inverted repeats not previously reported in this family. Phylogenetic reconstructions using both 18S ribosomal RNA gene and concatenated mitochondrial protein-coding genes consistently place TINC within a robust, distinct clade situated between the families Eimeriidae and Sarcocystidae. Comparative sequence analysis of the partial cytochrome c oxidase subunit 1 gene confirmed genetic identity across TINC from different cases. These integrated pathological and genomic results define TINC as a genetically distinct lineage with unique molecular features, providing critical data for its taxonomic revision and new insights into mitochondrial genome evolution at the interface of two major apicomplexan families.