Multigene Assessment of the Species Boundaries and Sexual Status of the Basidiomycetous Yeasts <i>Cryptococcus flavescens</i> and <i>C</i>. <i>terrestris</i> (Tremellales)

<div><p><i>Cryptococcus flavescens</i> and <i>C</i>. <i>terrestris</i> are phenotypically indistinguishable sister species that belong to the order Tremellales (Tremellomycetes, Basidiomycota) and which may be mistaken for <i>C</i>. <i>laurentii</i> based on phenotype. Phylogenetic separation between <i>C</i>. <i>flavescens</i> and <i>C</i>. <i>terrestris</i> was based on rDNA sequence analyses, but very little is known on their intraspecific genetic variability or propensity for sexual reproduction. We studied 59 strains from different substrates and geographic locations, and used a multilocus sequencing (MLS) approach complemented with the sequencing of mating type (<i>MAT</i>) genes to assess genetic variation and reexamine the boundaries of the two species, as well as their sexual status. The following five loci were chosen for MLS: the rDNA ITS-LSU region, the rDNA IGS1 spacer, and fragments of the genes encoding the largest subunit of RNA polymerase II (<i>RPB1</i>), the translation elongation factor 1 alpha (<i>TEF1</i>) and the p21-activated protein kinase (<i>STE20</i>). Phylogenetic network analyses confirmed the genetic separation of the two species and revealed two additional cryptic species, for which the names <i>Cryptococcus baii</i> and <i>C</i>. <i>ruineniae</i> are proposed. Further analyses of the data revealed a high degree of genetic heterogeneity within <i>C</i>. <i>flavescens</i> as well as evidence for recombination between lineages detected for this species. Strains of <i>C</i>. <i>terrestris</i> displayed higher levels of similarity in all analysed genes and appear to make up a single recombining group. The two <i>MAT</i> genes (<i>STE3</i> and <i>SXI1/SXI2</i>) sequenced for <i>C</i>. <i>flavescens</i> strains confirmed the potential for sexual reproduction and suggest the presence of a tetrapolar mating system with a biallelic pheromone/receptor locus and a multiallelic HD locus. In <i>C</i>. <i>terrestris</i> we could only sequence <i>STE3</i>, which revealed a biallelic P/R locus. In spite of the strong evidence for sexual recombination in the two species, attempts at mating compatible strains of both species on culture media were unsuccessful.</p>