Molecular taxonomy and systematics of yeasts and yeast llike fungi

Research project P 13876 Molecular taxonomy and systematics of yeasts and yeast like fungi Hansjörg PRILLINGER 28.06.1999 The project concentrates on two topics. The first topic will be the isolation and phenotypic and genotypic characterization of new yeast species or genera from the gut of invertebrates, especially, soil invertebrates and the aseptic fruiting body trama of mushrooms. For the characterization of new species and genera we have developed a new polyphasic approach using RAPD-PCR, cell wall sugars, ubiquinones, and partial ribosomal DNA sequencing. AFLP technology will be introduced and compared with RAPD-PCR. ITS sequences and sequences of the small subunit mitochondrial ribosomal DNA will be used in addition to differentiate species which could not be separated by partial sequencing of the D1/D2 region of the 26S rDNA. The second topic continues our systematic approach based originally on yeast cell wall sugars. Complete 18S rDNA sequences will be used: 1. To assign problematic species at the genus level (e.g. Eremothedum ashbyii, Hyphozyma roseonigra, H. variabilis, Nematospora coryli, Metschnikowia lunata, M. hawaiensis, Taphrina vestergrenii, Cryptococcus humicolus, C yarrowii), 2. To establish the new family the Nadsoniaceae (Nadsonia commutata, N. fulvescens, Schizoblastosporion starkeyi-henricii), 3. To corroboate that our cell wall sugar data are a reliable tool to assign the Basidiomycota at the class or order level (e.g. Exobasidium vaccinii, Rhamphospora nymphaeae, Schizonelia melanogramma; Kriegeria eriophori, Cryptococcus yarrowii), 4. Dimorphic Euascomycetes (Calyptrozyma, Hyphozyma, Lecythophora) will be used to confirm the new class of the Protomycetes which appears as a sister group of the Euascomycetes, and 5. For anamorphic genera like Schizoblastosporion, Hyphozyma, Lecythophora, and Cryptococcus complete 18S rDNA sequences in addition will help to overcome the artificial border of the Deuteromycetes.

Precise determination of yeasts is important for biotechnology and food technology, where yeasts found their main application in various fermentation processes, biochemically oriented studies and as starter cultures. Significant progress in yeast systematics has been made by the employment of molecular techniques which influenced our basic understanding of genetic structure and diversity of yeasts in natural habitats. Different approaches are used in delimitation of yeast taxa. Analysis of sequence polymorphisms in ribosomal RNA encoding genes has been successfully used for taxonomic investigations. The 18S rDNA are very useful for estimating phylogenetic relationships at the intergeneric and higher taxonomic level. On the other hand, the D1/D2 domain of the 26S rDNA and ITS1/ITS2 regions are used in the species delimitation. DNA-fingerprinting techniques like, RAPD (random amplified polymorphic DNA), PFGE (pulsed-field gel electrophoresis), RFLP (restriction fragment length polymorphisms) and AFLP (amplified fragment length polymorphism) are considered to be highly valuable tools for the species and subspecies discrimination. In the current project the 18S rDNA sequences of yeasts and dimorphic fungi were used to study phylogenetic relationships among the ascomycetous and basidiomycetous classes. Phylogenetic relationships of several genera with uncertain taxonomic position (Coniochaeta, Lecythophora, Hyphozyma, Candida, Rhodotorula, Fucotaphrina and Asterotremella) were investigated, to predict their closest phylogenetic relatives. The AFLP method is used for the estimation of genotypic relatedness among the strains of the anamorphic genus Fellomyces. Three approaches, the AFLP, the PCR-fingerprinting and the ITS1/ITS2 sequencing were correlated to study the potency of individual techniques in species delimitation.