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Complete small ribosomal subunit RNA sequences were used to infer the relationship between several basidiomycetous yeasts, and to resolve the evolutionary position of the basidiomycetes among the fungi. The sequences were determined for Rhodosporidium toruloides (anamorph Rhodotorula glutinis), Filobasidiella neoformans (anamorph Cryptococcus neoformans), Trichosporon cutaneum, Bullera alba and Sporobolomyces roseus. The sequence of Leucosporidium scottii (anamorph formerly named Candida scottii) srRNA has already been published previously (Hendriks et al., J. Mol. Evol. 32, 167-177 (1991)). Using a tree construction program based on a distance matrix, a phylogenetic tree was constructed for all hitherto known fungal srRNA sequences, oomycetes and slime moulds not included. It showed the ascomycetes and the basidiomycetes to be sister groups, probably evolved from a zygomycete-like ancestor and diverged from each other about 840 Myr ago. Among the basidiomycetes, two clearly distinct groups can be recognized, one formed by the teliospore forming species (Rhodosporidium toruloides and Leucosporidium scottii), and the asexual yeast Sporobolomyces roseus, and the other formed by the non-teliospore forming species Filobasidiella neoformans and the asexual yeasts Bullera alba and Trichosporon cutaneum.

One-dimensional polyacrylamide gel electrophoretic whole-cell protein patterns of 112 isolates of 20 different yeast species, mainly belonging to the genus Candida, were compared by numerical analysis. This technique allowed to evaluate taxonomic relationships at the species level. Qualitative differences between the protein electrophoregrams of different species enabled to confirm anamorph/teleomorph relations and to detect synonymy between species. The high reproducibility allowed to evaluate quantitative differences found at the infraspecific level. Inter- and intrageneric relationships could be deduced after comparison with other taxonomic criteria. The construction of a database of protein fingerprints is therefore feasible and will be a useful tool for routine identification of yeasts.

The primary structure of the small ribosomal subunit RNA. (SSU rRNA) was determined for 13 species belonging to 10 ascomycete families and for the basidiomycetous anamorphic yeast Rhodotorula glutinis. The sequences were fitted into an alignment of all hitherto published complete or nearly complete eukaryotic small subunit rRNA sequences. The evolutionary relationships within the fungi were examined by construction of a tree from 87 SSU rRNA sequences, corresponding to 71 different species, by means of a distance matrix method and bootstrap analysis. It confirms the early divergence of the zygomycetes and the classical division of the higher fungi into basidiomycetes and ascomycetes. The basidiomycetes are divided into true basidiomycetes and ustomycetes. Within the ascomycetes, the major subdivisions hemiascomycetes and euascomycetes can be recognized. However, Schizosaccharomyces pombe does not belong to the cluster of the hemiascomycetes, to which it is assigned in classical taxonomic schemes, but forms a distinct lineage. Among the euascomycetes, the plectomycetes and the pyrenomycetes can be distinguished. Within the hemiascomycetes, the polyphyly of genera like Pichia or Candida and of families like the Dipodascaceae and the Saccharomycetaceae can be observed.

The primary structure of the small ribosomal subunit RNA (srRNA) molecule of the type strains of the ascosporogenous yeasts Debaryomcyes hansenii, Pichia anomala (synonym: Hansenula anomala), Pichia membranaefaciens, Schizosaccharomyces pombe, Zygosaccharomyces rouxii and Dekkera bruxellensis was determined. The srRNA sequences were aligned with previously published sequences from fungi, including those of 5 candida species, and an evolutionary tree was inferred The srRNA results were compared with chemotaxonomic criteria, e.g. the coenzyme Q system. The heterogeneity of the genera Candida and Pichia is clearly reflected by the srRNA analysis.

Summary Up to now the small ribosomal subunit RNA sequences of about 50 different eukaryotes have been published, of which only three belong to the fungi. We determined the complete srRNA sequence of the imperfect yeast Candida albicans. The sequence is 1788 nucleotides long and was determined at the DNA level using the dideoxy method with a set of primers specific for conserved sequences of small ribosomal subunit RNA. An evolutionary tree, comprising 58 organisms including C. albicans, was constructed. This tree shows a number of early diverging lineages such as a diplomonad, a microsporidian, an amoeba, slime molds, an euglenoid, kinetoplastids and sporozoans. Next within a relatively short time interval there is a radiation into a number of clusters composed of ciliates, metazoa, fungi and green plants. C. albicans was previously classified in the artificial taxon of imperfect fungi. The evolutionary tree presented in this paper clearly shows C. albicans to belong to the ascomycetous yeasts. An additional aim of this study was the refinement of the srRNA secondary structure model. Although the outline of this model is now well established, no consensus model exists in certain eukaryote-specific areas of high structural variability. The srRNA sequence of xC. albicans was fitted into the secondary structure model and the existence of a pseudoknot is proposed in one of these eukaryote-specific areas.