MAT--gene structure and mating behavior of Hymenoscyphus fraxineus and Hymenoscyphus albidus.

Recently, different reproductive modes were proposed between the emerging forest pathogen Hymenoscyphus fraxineus and its closely related avirulent sister species, Hymenoscyphus albidus. In the present study, inter- and intraspecific crosses were performed to experimentally assess the reproduction barriers between the two species and to verify H. albidus' putative haploid-selfing reproductive mode. By means of H. fraxineus-specific microsatellite markers, no hybridization was observed in 29 apothecia that emerged from inter-specific crosses, suggesting reproduction barriers are well-established. In a similar experimental setup, we used two newly developed polymorphic H. albidus-specific microsatellites to show that haploid-selfing is H. albidus' only reproductive mode (N=17 apothecia). Further to this, the reproductive modes of both species were investigated under natural conditions. Microsatellite allele-segregation studies of H. fraxineus' single-spore progeny of apothecia (N=31) from field samples suggest that often more than two paternal nuclei are involved in mating. In contrast, analysis of single-spore progeny of field-collected H. albidus apothecia (N=21) confirmed the solely haploid-selfing reproductive mode detected in vitro. Furthermore, we present the complete mating type 1-1 locus of H. fraxineus and report the finding of three additional genes within this region; the as yet unobserved typical mating type gene MAT1-1-1, a DNA polymerase zeta catalytic subunit-like gene and a pre-mRNA-splicing factor SLU7-like gene. The same genes were also detected in the homothallic mating type locus of H. albidus. Further analysis confirmed the expression of all typical mating type genes (MAT1-2-1, MAT1-1-3, MAT1-1-1) in both species. Interestingly, the MAT1-1-3 gene of homothallic H. albidus is expressed despite three stop codons interrupting the coding sequence. Overall, our findings highlight vital differences in the reproduction systems of the two species and suggest that interspecific hybridization is not possible.