| Summary: | Hybridization is recognized as a notable driver of evolution and adaptation, which closely related species may exploit in the form of incomplete reproductive barriers. Three closely related species of <i>Ceratocystis</i> (i.e., <i>C. fimbriata</i>, <i>C. manginecans</i> and <i>C. eucalypticola</i>) have previously been shown to hybridize. In such studies, naturally occurring self-sterile strains were mated with an unusual laboratory-generated sterile isolate type, which could have impacted conclusions regarding the prevalence of hybridization and inheritance of mitochondria. In the current study, we investigated whether interspecific crosses between fertile isolates of these three species are possible and, if so, how mitochondria are inherited by the progeny. For this purpose, a PCR-RFLP method and a mitochondrial DNA-specific PCR technique were custom-made. These were applied in a novel approach of typing complete ascospore drops collected from the fruiting bodies in each cross to distinguish between self-fertilizations and potential hybridization. These markers showed hybridization between <i>C. fimbriata</i> and <i>C. eucalypticola</i> and between <i>C. fimbriata</i> and <i>C. manginecans</i>, while no hybridization was detected in the crosses involving <i>C. manginecans</i> and <i>C. eucalypticola</i>. In both sets of hybrid progeny, we detected biparental inheritance of mitochondria. This study was the first to successfully produce hybrids from a cross involving self-fertile isolates of <i>Ceratocystis</i> and also provided the first direct evidence of biparental mitochondrial inheritance in the <i>Ceratocystidaceae</i>. This work lays the foundation for further research focused on investigating the role of hybridization in the speciation of <i>Ceratocystis</i> species and if mitochondrial conflict could have influenced the process.
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