Abstract
Fungal mitochondrial genomes are critical for understanding phylogenetics, evolution, and ecology of the Kingdom Fungi, yet they remain underrepresented in public databases. To address this, we developed a workflow to recover mitochondrial genomes from 12,902 fungal short read sequencing data housed in the Sequence Read Archive (SRA) records, assembling complete circular genomes from 2,695 species. This effort expanded fungal mitochondrial genome diversity by nearly 2.3X particularly in understudied phyla such as Mucoromycota (11X increase) and Zoopagomycota (8X increase). The new dataset contains novel yet undescribed mitochondrial genomes at numerous taxonomic levels, including 15 classes, 64 orders, 178 families, and 544 genera. Taxonomic analysis revealed broad ecological representation among the top-assembled species, including human pathogens (e.g., Cryptococcus tetragattii), plant pathogens (e.g., Melampsora larici-populina), edible mushrooms (e.g., Suillus luteus), and industrial fungi. By leveraging the not yet fully exploited SRA sequencing data, this study fills critical gaps in fungal mitochondrial genomics, tripling the currently known mitochondrial genome diversity of the Kingdom Fungi, and provides an extensive resource for phylogenetic and evolutionary research.