Fungi are asexually and sexually reproducing organisms that can combine the evolutionary advantages of the two reproductive modes. However, for many fungi the sexual cycle has never been observed in the field or in vitro and it remains unclear whether sexual reproduction is absent or cryptic. Nevertheless, there are indirect approaches to assess the occurrence of sex in a species, such as population studies, expression analysis of genes involved in mating processes and analysis of their selective constraints. The members of the Phialocephala fortinii s. l. - Acephala applanata species complex (PAC) are ascomycetes and the predominant dark septate endophytes that colonize woody plant roots. Despite their abundance in many ecosystems of the northern hemisphere, no sexual state has been identified to date and little is known about their reproductive biology, and how it shaped their evolutionary history and contributes to their ecological role in forest ecosystems. We therefore aimed at assessing the importance of sexual reproduction by indirect approaches that included molecular analyses of the mating type (MAT) genes involved in reproductive processes.