Mitogenomics in Amanita, evolution, species delimitation and its utility at the species complex level

Mitochondrial genomes are among the most powerful sources for resolving evolutionary relationships in eukaryotes; however, their application in fungal phylogenetics remains underutilized. In this study, we employed comparative genomics and phylogenomics to evaluate the utility of the mitogenome within the genus Amanita , specifically at the species complex level. Our results demonstrate that mitochondrial coding regions provide robust species-level resolution, while structural features – including intron dynamics, synteny breaks and ORFs – reveal evolutionary trajectories often obscured by traditional nuclear markers. We identified highly variable gene synteny patterns and significant fluctuations in intron and ORF content, particularly within the cob and cox1 loci. Notably, these mitogenomic features varied considerably even among closely related species within the same section. Our findings underscore the dual utility of the mitochondrial genome for both interspecific inference and the identification of new cryptic lineages within established species complexes. Furthermore, we report a significant mitonuclear discordance, suggesting complex evolutionary histories such as ancestral hybridization or incomplete lineage sorting. These results highlight the necessity of integrating mitogenomics into fungal systematics to improve our understanding of species boundaries and evolutionary dynamics.