Transition from heterothallism to homothallism is hypothesised to have facilitated speciation among emerging Botryosphaeriaceae wheat-pathogens.

White grain disorder (WGD) is a recently emerged wheat disease in Australia caused by three Botryosphaeriaceae fungi, from the genus Eutiarosporella. These species are E. tritici-australis, E. darliae, and E. pseudodarliae. Characterisation of the mating type genes for the WGD-species show that the genome sequence of a single E. darliae and E. pseudodarliae isolate both harbour MAT1-2-1 and MAT1-1-1, which suggests that these species are homothallic. However, unlike most other characterised mating-type loci from other homothallic Dothideomycetes, these species' MAT1-1-1 are located at a separate locus, inserted within the coding region of another gene. The sequenced strain of E. tritici-australis analysed did not harbour MAT1-1-1. Including the sequenced strain, we screened the mating type genes present in 16 E. tritici-australis individuals isolated from infected grain from fields in South Australia. Of these 16, 11 harbour MAT1-1-1 and the other five harbour MAT1-2-1. The genome of a MAT1-1-1 harbouring isolate was re-sequenced, which demonstrated that MAT1-1-1 was present at the MAT locus. We examined non-coding DNA surrounding the MAT1-1-1 gene in E. pseudodarliae and observed fragments of the MAT locus both up and downstream. These fragments and their orientation around MAT1-1-1 is similar to characterised heterothallic Botryosphaeriaceae. Based on these gene arrangements, we conclude that the new MAT1-1-1 containing locus likely originated from a cryptic DNA integration event between two heterothallic individuals. We hypothesise that this integration event led to the formation of a homothallic lineage, which is the common ancestor of E. darliae and E. pseudodarliae.