Abstract
Mitochondria are essential components of eukaryotic cells, responsible for ATP production through oxidative phosphorylation. Despite their biological importance, unique challenges have hindered the adoption of automated mitochondrial genome (mitogenome) annotation methods, obstructing mitochondrial comparative genomics in a broad evolutionary context. Using Fungi as a study system and a Joint Genome Institute (JGI) annotated high-quality reference set, we observed broad patterns of mitochondrial evolution across the kingdom. We found that the median fungal mitogenome size is 58 kb and identified exceptionally large examples over 1 Mb in Pezizomycetes. All 14 expected oxidative phosphorylation protein-coding genes, plus rps3, were generally conserved. We found evidence of major evolutionary transitions within the Ascomycota, including the transfer of mitochondrially encoded atp8 and atp9 to the nuclear genomes across the Pezizomycotina and shifts in mitogenome tRNA patterns across the kingdom. We found substantial concordance between mitochondrial and nuclear evolution, enabling us to document 3131 total fungal mitogenomes from JGI-derived metagenomic datasets. We also identified 6467 total undeclared mitogenomes embedded in Genbank fungal nuclear assemblies. We provide interactive tools for mitogenome analysis through the JGI MycoCosm platform. Collectively, this work generated nearly 10 000 new fungal mitogenome annotations, providing a foundation and resources for future exploration of comparative fungal mitogenomics.