Wairimu, D. M.; Wilson, A. M.; Piombo, E.; Brandstrom Durling, M.; Broberg, M.; Jensen, B.; Ruffino, A.; Chaudhary, S.; De Fine Licht, H. H.; Dubey, M.; Karlsson, M.

Modes of reproduction and sexual strategies strongly influence the genetic diversity and evolutionary potential of a species. The ascomycete fungus Clonostachys rosea is reported to be homothallic (sexually self-fertile), although a rapid decay of genome-wide linkage disequilibrium is also reported, something that is not in line with an obligate homothallic mode of reproduction. To investigate this phenomenon, we identified the mating-type (MAT1) locus in 66 genome-sequenced C. rosea strains under the hypothesis that each strain contains genes from both MAT1 idiomorphs. Eleven strains indeed contained both MAT1-1 and MAT1-2 genes, suggesting homothallism. However, most strains harboured either MAT1-1 or MAT1-2 genes and co-existed in North America, Europe and China, suggesting heterothallism. The MAT1 locus of heterothallic strains was highly conserved, and the linkage disequilibrium half decay distance was 1050 bp, suggesting sexual outcrossing. The presence of conserved MAT1-1 or MAT1-2 idiomorphs in strains of other Clonostachys species shows that heterothallism is likely the ancestral state. A phylogenetic analysis of 2800 single-copy orthologous genes revealed that homothallic and heterothallic strains separated in two well-supported clades, indicating a single lineage of homothallic C. rosea, likely originating in South America, followed by intercontinental dispersal. Homothallic C. rosea strains displayed higher nucleotide diversity than heterothallic strains, indicating a lack of outcrossing. This unique case of both homothallic and heterothallic lineages within the same species provides an opportunity to study the genomic consequences of selfing in very closely related strains.