6349 System. Appl. Microbiol. 12, 230-236 (1989) Evolutionary Affinities of Heterobasidiomycetous Yeasts Estimated from 185 and 255 Ribosomal RNA Sequence Divergence 2 2 EVELINE GUEHO\ CLETUS P. KURTZMAN , and STEPHEN W. PETERSON J lnstitut Pasteur, Unite de Mycologie, 75724 Paris Cedex 15, France Northern Regional Research Center, U.S. Department of Agriculture, Peoria, IL 61604, USA Received May 20, 1989 Summary Phylogenetic relationships among heterobasidiomycetous yeasts, including anamorphic and teleomorphic taxa, have been compared from the sequence similarity of small (18S) and large (25S) subunit ribosomal RNA. Species examined were Cystofilobasidium capitatum, Filobasidiella neoformans, Fi/obasidium floriforme, Leucosporidium scottii, Malassezia furfur, M. pachydermatis, Phaffia rhodozyma, Rhodos­ poridium toru/oides, Sporidiobolus johnsonii, Sterigmatosporidium polymorphum, Trichosporon beige/ii, T. cutaneum and T. pullulans. The taxa cluster into three main groups. One group contains the non­ teliospore forming genera Sterigmatosporidium, Filobasidiella, Filobasidium and the anamorphic species T. beigeIii and T. cutaneum. The teliospore formers Leucosporidium, Rhodosporidium and Sporidiobo/us, all of which have simple septal pores, cluster into a single group, despite the heterogeneity of carotenoid formation. By contrast, C. capitatum appears separate, not only from Rhodosporidium, but also from the Filobasidiaceae with which it shares a primitive dolipore septum. The uniqueness of the genus Malassezia among yeasts is confirmed, and one might predict from nucleotide sequence similarity that teleomorphs of those lipophilic organisms would form teliospores whereas Trichosporon beigelii and T. cutaneum appear related to the family Fi/obasidiaceae. Key words: Heterobasidiomycetous yeasts - Phylogeny - Taxonomy - 18S and 25S rRNA sequences­ Coenzyme Q system - Septal pore Introduction Considerable progress in yeast taxonomy has been to the genera Cryptococcus, Rhodotorula or Vanri;a made during the last few years, particularly with the (Weijman et al., 1988; Moore, 1980 and 1987). Many extensive use of nuclear DNA comparisons. Unfortu­ generic separations and species assignments, however, still nately, DNA reassociation resolves only to the species level remain questionable. Knowledge of septal pore ultrastruc­ (Kurtzman, 1987), and the problem of correct generic ture has led to further refinements in classification. It can assignment of species becomes especially acute when sex­ confirm the filiation of taxa to the ascomycetes or ual states are unknown. Over the years, various workers basidiomycetes and it has helped define orders of the sub­ have emphasized such characters as physiology, class Heterobasidiomycetes (Moore, 1985). Unfortu­ diazonium blue B staining reaction, enzymatic sphaero­ nately, many yeasts are unable to product true hyphae. plast release, the coenzyme Q system, conidiogenesis, The possibility for a better understanding of phylogeny ultrastructure and carbohydrate composition of cell walls among fungi recently emerged from comparisons of as possible characters which allow a more natural classifi­ ribosomal RNA sequences. Ribosomal RNAs appear par­ cation (de Hoog et al., 1990). From these data, the imper­ ticularly well suited as general indicators of evolutionary fect yeast genera (blastomycetes) have been reclassified relationships because of their occurence in all species and along ascomycete/basidiomycete lines. For example, the their largely conservative structure and function (Fox et basidiomycetous species of Candida have been transferred al., 1980). Due to its small size, 5S rRNA can be easily Heterobasidiomycetous Yeasts and rRNA Sequences 231 sequenced, and therefore has been used first for phy­ Materials and Methods logenetic evaluations. From the comparison of Blanz and Unseld (1987) and the compilation of Wolters and Erd­ Yeast strains. The 13 species of heterobasidiomycetous yeasts mann (1988), about 80 55 rRNA sequences are now avail­ examined, which include anamorphs and teleomorphs, white and able from fungi (23 ascomycetes including yeasts and 56 red pigmented species, and teliospore and non-teliospore forms basidiomycetes). However, the precision of phylogenetic are listed in Table 1. All correspond to the type strain (T), or a mating type (MT) or an authentic (A) culture when the type was analysis using rRNA is restricted by the low informa­ 55 not available. Concerning the authentic strains, Ma/assezia tion content of the molecule (only 120 nucleotides long). pachydermatis NRRL Y-17165 (= GSU 8537) shares 95% DNA For instance, heterobasidiomycetes as different as homology with the type culture CBS 1879 (G/II§ho and Meyer. Filobasidium florifonne, Phaffia rhodozyma, Tremella 1989); Rhodosporidium torn/aides Y-6987 is a diploid strain mesenterica, and Itersonilia perplexans cluster with mem­ resulting from the conjugation of CBS 14 (mating type AI), the bers of the homobasidiomycetes. The availability of a rela­ holotype, and CBS 349 (mating type Al), the allotype; and tively quick and simple method for sequencing the larger Trichosporon beigelii Y-17147 is conspecific with the neotype rRNA species (16-185 and 25-285) (Sanger et al., 1977; culture (Gueho, manuscript in preparation). The cultures examined are maintained in the Agricultural Research Service Qu et aL, 1983; Lane et aL, 1985; Qu et aL, 1988) should Culture Collection (NRRL), Northern Regional Research Center give access to more accurate phylogenetic information. and in the Centraalbureau voor Schimmelculrures (CBS). The present work reports a comparison of 185 and 255 rRNA extraction and purification. Most strains were grown at rRNA subunit sequences from a range of 25°C in 100 ml of YM medium (0.3% veast extract, 0.3% malt heterobasidiomycetous yeasts in an effort to determine the extract, 0.5% peptone, 1% glucose) o~ a rotary shaker at 200 taxonomy and phylogeny of these species. rpm for ca. 24 h. Trichosporon pu//u/ans and Cystofi/obasidium Table 1. Selected phenotypic characteristics of the 13 heterobasidiomycetous species examined Species Strain N°' Source Type of Type of xylose assimila- NRRL CBS Ubiqui- septal in cell tion of noneb pore' wall d inositol Cystofilobasidium capitatum (Fell at al.) Y-10873T 6358 zooplancton Q8 pDP + Oberwinkler et Bandoni Filobasidie//a neoformans Kwon-Chung Y·170MT 882 equine Q10 pDP + + nasal tumor Filobasidium floriforme Olive Y_7453 MT 6241 dead leaves of Q10 pDP + + Erianthus giganteus Leucosporidium scottii Fell et al. Y-1497T 614 soil Q9 sP Malassezia furfur (Robin) Baillon Y-17157T 1878 scalp dandruff Q9 unknown unknown unknown (T of P. ovale) Malassezia pachydermatis (Weidman) Dodge Y-17165 A 8537 ear of dog Q9 unknown unknown unknown Phaffia rhodozyma Miller et al. Y-10921 T 5905 Fagus crenata Q10 unknown + Rhodosporidium tornloides Banno Y-6987A 315 air Q9 sP Sporidiobolus johnsonii Nyland Y-2559T 5470 Leaf of Rubus idaeus Q10 sP Sterigmatosporidium polymorphum Y-12762~lT 8089 water-logged planks Q10 unknown + + Kraepelin et Schulze in an old ore mine Trichosporon beigelii (Kiichenmeister et Rab.) Y-17147A 2936 white piedra of Q9 pDP + + Vuillemin monkey Trichosporon cutaneum (de Beurmann et al.) Y-1490T 2466 skin lesion Q10 pDP + + Ota Trichosporon pullulans (Lindner) Y-1522T 2532 air Q9 pDP + + Diddens et Lodder question- able 3 NRRL, ARS Culture Collection, Northern Regional Research Center, US Department of Agriculture, Peoria, Illinois, USA; CBS, Centraal­ bureau voor Schimmelculrures, Delft, the Netherlands. T, type culture; MT, mating type culture; A, authentic culture. M. pachydermatis NRRL = GSU 8537 from Georgia State University, Atlanta, USA b Coenzyme Q systems, from Sugiyama et al., 1985; Yamada and Kondo, 1973; Yamada et al., 1983; Yamada and Banno, 1984; Yamada et al., 1982. For M. furfur and M. pachydermatis, from Yamada, and T. beigelii, from Billon-Grand (unpublished data). , From Oberwinkler et al., 1983; Kwon-Chung and Popkin, 1976; Moore and Kreger-van Ri;, 1972; Kreger-van Ri; and Veenhuis, 1971b; Johnson-Reid and Moore, 1972; Kreger-van Rij and Veenhuis, 1971a. For Trichosporon spp., also from M. Th. Smith (unpublished data). pDP primitive dolipore; sP, simple pore d From Sugiyama et al., 1985; Weijman et al., 1988; von Arx and Weijman, 1979; Yamada, 1989; Wei;man, 1979 (referred to G. amycelicum CBS 186.38 for T. beigelii) 232 E. Gueho, C. P. Kunzman, and s. W.Peterson capitatum were grown at 15°C, Malassezia pachydermatis at GGTCCGTGTITCAAGACGG (635). The small subunit pnmer 30°C and M. furfur at 3rc with the medium enriched by 1% and the first base of the rRNA copied IS 5'­ olive oil. These four species were grown in shake culture for 48 h. ACGGGCGGTGTGTAC (1626). A second large subunit prim­ Cells were harvested by centrifugation, broken in a Braun er 5'-TIGGAGACCTGCTGCGG (1841) that we used for homogenizer (lg wet weightll0 ml4M guanidinium thiocyanate ascomycetes (Peterson and Kurtzman, in preparation) failed to buffer) and the rRNA was further purified according to the pro­ successfully prime all species in this srudy and has been omitted cedure described by Chirgwin et aJ. (1979). Purity of RNA samp­ from consideration. Alignment of both partial sequences les was estimated from spectrophotometric absorbance ratios examined were referenced to the primary structure of Sac­ 260/280 = 2.0D-2.15 and 230/260 S 0.5, and their integrity charomyces cerevisiae (Rubstov et aI., 1980; Georgie!1 et al.. assessed by non-denaturing agarose gel