Molecular and Genetic Evidence for a Tetrapolar Mating System in the Basidiomycetous Yeast Kwoniella mangrovensis and Two Novel Sibling Species

Kwoniella mangrovensis has been described as a sexual species with a bipolar mating system. Phylogenetic analysis of multiple genes places this species together with Kwoniella heveanensis in the Kwoniella clade, a sister clade to that containing two patho-genic species of global importance, Cryptococcus neoformans and Cryptococcus gattii, within the Tremellales. Recent studies defining the mating type loci (MAT) of species in these clades showed that, with the exception of C. neoformans and C. gattii, which are bipolar with a single biallelic multigeneMAT locus, several other species feature a tetrapolar mating system with two unlinked loci (homeodomain [HD] and pheromone/receptor [P/R] loci). We characterized several strains from the original study describing K. mangrovensis; twoMAT regions were amplified and sequenced: the STE20 gene (P/R locus) and the diver-gently transcribed SXI1 and SXI2 genes (HD locus). We identified five different mating types with different STE20/SXI allele combinations that together with results of mating experiments demonstrate that K. mangrovensis is not bipolar but instead has a tetrapolar mating system. Sequence and gene analysis for a 43-kb segment of the K. mangrovensis type strainMAT locus re-vealed remarkable synteny with the homologous K. heveanensis MAT P/R region, providing new insights into slower evolution ofMAT loci in theKwoniella compared to the Cryptococcus clade of the Tremellales. The study of additional isolates from plant substrates in Europe and Botswana using a combination of multilocus sequencing withMAT gene analysis revealed two novel sibling species that we name Kwoniella europaea and Kwoniella botswanensis and which appear to also have tetrapolar mating systems