Frequent genetic exchanges revealed by a pan-mitogenome graph of a fungal plant pathogen

Mitochondria are present in almost all eukaryotic lineages. The mitochondrial genomes (mitogenomes) evolve separately from nuclear genomes, and they can therefore provide relevant insights into the evolution of their host species. <i>Fusarium oxysporum</i> is a major fungal plant pathogen that is assumed to reproduce clonally. However, horizontal chromosome transfer between strains can occur through heterokaryon formation, and recently, signs of sexual recombination have been observed. Similarly, signs of recombination in <i>F. oxysporum</i> mitogenomes challenged the prevailing assumption of clonal reproduction in this species. Here, we construct, to our knowledge, the first fungal pan-mitogenome graph of nearly 500 <i>F</i>. <i>oxysporum</i> mitogenome assemblies to uncover the variation and evolution. In general, the gene order of fungal mitogenomes is not well conserved, yet the mitogenome of <i>F. oxysporum</i> and related species are highly colinear. We observed two strikingly contrasting regions in the <i>F. oxysporum</i> pan-mitogenome, comprising a highly conserved core mitogenome and a long variable region (6-16 kb in size), of which we identified three distinct types. The pan-mitogenome graph reveals that only five intron insertions occurred in the core mitogenome and that the long variable regions drive the difference between mitogenomes. Moreover, we observed that their evolution is neither concurrent with the core mitogenome nor with the nuclear genome. Our large-scale analysis of long variable regions uncovers frequent recombination between mitogenomes, even between strains that belong to different taxonomic clades. This challenges the common assumption of incompatibility between genetically diverse <i>F. oxysporum</i> strains and provides new insights into the evolution of this fungal species.IMPORTANCEInsights into plant pathogen evolution is essential for the understanding and management of disease. <i>Fusarium oxysporum</i> is a major fungal pathogen that can infect many economically important crops. Pathogenicity can be transferred between strains by the horizontal transfer of pathogenicity chromosomes. The fungus has been thought to evolve clonally, yet recent evidence suggests active sexual recombination between related isolates, which could at least partially explain the horizontal transfer of pathogenicity chromosomes. By constructing a pan-genome graph of nearly 500 mitochondrial genomes, we describe the genetic variation of mitochondria in unprecedented detail and demonstrate frequent mitochondrial recombination. Importantly, recombination can occur between genetically diverse isolates from distinct taxonomic clades and thus can shed light on genetic exchange between fungal strains.