Cryptococcus nemorosus sp. nov. and Cryptococcus perniciosus sp. nov., related to Papiliotrema Sampaio et al. (Tremellales) Wladyslav I. Golubev, 1 Ma ´ rio Gadanho, 2 Jose ´ P. Sampaio 2 and Nikita W. Golubev 3 Correspondence Wladyslav I. Golubev wig@ibpm.serpukhov.su 1 Russia Collection of Microorganisms, Institute for Biochemistry and Physiology of Micro-organisms, Russian Academy of Sciences, Pushchino 142290, Russia 2 Centro de Recursos Microbiologicos, Secc ¸a ˜o Auto ´ noma de Biotecnologia, Faculdade de Cie ˆ ncias e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal 3 Mendeleev Chemical–Technological University, Moscow 125820, Russia Three mycocinogenic strains representing the genus Cryptococcus were isolated on glucuronate agar from plants and turf collected in the Prioksko-terrasny biosphere reserve (Russia). These isolates fit the standard description of Cryptococcus laurentii, but differ from its type strain in both their mycocin-sensitivity profiles and the killing patterns of their mycocins. Sequence analyses of the D1/D2 domain of the 26S rDNA and of the internal transcribed spacer region confirmed that these isolates represent two novel species, for which the names Cryptococcus nemorosus sp. nov. (type strain VKM Y-2906 T ) and Cryptococcus perniciosus sp. nov. (type strain VKM Y-2905 T ) are proposed. Morphological, physiological and biochemical characteristics, as well as mycocinotyping and molecular analysis, show a close affinity between these two novel anamorphic species and the teleomorphic species Papiliotrema bandonii (Tremellales). The distinctive feature of the vast majority of hymeno- mycetous yeasts is their ability to utilize a cyclitol, i-inositol, and/or D-glucuronate. Some fail to assimilate inositol but they retain the ability to utilize glucuronate, the first intermediate of inositol catabolism (Golubev, 1989). On the basis of these observations, glucuronate medium was pro- posed for estimation of the density and species composition of hymenomycetous yeasts in natural communities (Golubev, 2000). With glucuronate agar, both higher counts and a wider diversity of such yeasts were revealed during the study of yeast communities from the phyllosphere and soil of the Prioksko-terrasny biosphere reserve (Moscow region, Russia) compared with the more commonly used malt extract or glucose/yeast extract/peptone agar. Many of the isolates obtained with glucuronate agar were identified as Cryptococcus albidus (Saito) Skinner or Cryptococcus laurentii (Kufferath) Skinner according to taxonomic keys and criteria used for differentiation (Fell & Statzell-Tallman, 1998; Barnett et al., 2000). However, such identification results mainly from the large number of variable characteristics in the standard descriptions of these ubiquitous species. Yeast species that are easily identified usually exhibit specificity whereas, in many cases, yeasts that are considered ubiquitous are in fact heterogeneous assem- blages of species that are difficult to identify by conventional methods. The taxonomic heterogeneity of Cryptococcus albidus and Cryptococcus laurentii has been recognized in molecular biochemical studies (Vancanneyt et al., 1994; Fonseca et al., 2000; Sugita et al., 2000). These two species are also heterogeneous with respect to sensitivity to mycocins (zymocins, killer toxins) secreted by tremelloid killer yeasts (W. I. Golubev, unpublished data). Mycocin typing showed that most of the isolates from the Prioksko- terrasny reserve differed greatly from the Cryptococcus albidus and Cryptococcus laurentii type strains. Subsequent closer inspection led us to the conclusion that the cultures represented undescribed species. Strains Strains VKM Y-2905 T (isolate PTZ 59 T ) and VKM Y-2907 (isolate PTZ 441) were isolated, by plating on glucuronate agar, from turf and plants collected from a steppe plot with fescue/herbaceous vegetation in the Prioksko-terrasny bio- sphere reserve in September 1997 and 2000, respectively. Strain VKM Y-2906 T (isolate PTZ 75 T ) was also isolated, on this medium, from herbaceous plants collected in an oak forest in the same reserve in September 1997. All other The GenBank accession numbers for the 26S rDNA D1/D2 region sequences of C. perniciosus sp. nov. PYCC 5769 T and C. nemorosus sp. nov. PYCC 5768 T are AF472624 and AF472625. Published online ahead of print on 11 October 2002 as DOI 10.1099/ ijs.0.02374-0. 02374 G 2003 IUMS Printed in Great Britain 905 International Journal of Systematic and Evolutionary Microbiology (2003), 53, 905–911 DOI 10.1099/ijs.0.02374-0