An exceptionally large, intron-rich mitochondrial genome and complex RNA processing in the desert-dwelling fungus Helvella bachu.

Fungal mitochondrial genomes exhibit exceptional variation in size and architecture, yet the genomic components and RNA-processing features associated with extreme expansion remain poorly understood. Here, we report the first complete mitochondrial genome for the genus Helvella, and characterize its architecture in H. bachu, an ectomycorrhizal ascomycete inhabiting hyper-arid desert ecosystems in western China. Using PacBio HiFi sequencing, Illumina polishing, and full-length Iso-Seq transcriptomics, we assembled a circular 587,425-bp mitogenome containing a canonical set of 15 protein-coding genes, 2 rRNAs, and 24 tRNAs. Its expanded size is associated with extensive non-coding sequence, including 103 introns within protein-coding and rRNA genes, totaling 372,582 bp and accounting for 63.4% of the mitogenome, as well as 121,658 bp of repetitive elements representing 20.7% of the genome. Most introns were classified as group I, and collectively harbored numerous open reading frames predicted to encode GIY-YIG or LAGLIDADG homing endonucleases. Full-length transcript data supported fully spliced major transcript models and revealed additional intron-retaining, alternative-splice, and polycistronic transcripts, highlighting substantial mitochondrial RNA-processing heterogeneity in H. bachu. Comparative analyses of 191 fungal mitogenomes showed that Pezizales encompasses multiple enlarged mitogenomes whose expansion is associated with varying contributions from intronic and intergenic sequences. Together, these findings define the structural basis of mitogenome expansion in H. bachu, reveal substantial heterogeneity in fungal mitochondrial RNA processing, and provide a comparative framework for investigating the evolution of giant mitogenomes in Pezizales.