6349

System. Appl. Microbiol. 12, 230-236 (1989)

Evolutionary Affinities of Heterobasidiomycetous 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. examined were Cystofilobasidium capitatum, 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 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 Fi/obasidiaceae.

Key words: Heterobasidiomycetous yeasts - Phylogeny - - 18S and 25S rRNA sequences­ Coenzyme Q system - Septal pore

Introduction

Considerable progress in taxonomy has been to the genera , 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, 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 . 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 electrophoresis. 1981; Mankin et aI., 1986). Nucleotide fragments generated in Sequencing reactions and sequences comparisons. Selected reg­ the chain elongation reactions were separated on 8% acrylamide ions of the 185 and 255 rRNA subunits were targeted with - 8M urea gels and visualized by autoradiography. Unrooted specific oligonucleotide primers and sequenced by the dideoxy­ trees were drawn from the analysis of sequence data using the nucleotide chain termination method as described by Lane et aJ. program DNAML, version 3.11, a phylogeny inference, max­ (1985). One region was sequenced from each of the large (255) imum likelihood program written by Joseph Felsenstein, Univer­ and the small (185) subunits. The large subunit primer and sity of Washington, Seattle. the first base of the rRNA sequence copied is 5'-

(277-310) S. polymorphum UCOA AAGCOAAAUA O'OGGCGAGAG ACCGAUAGCG AACAAGOACC GtlGAGGGAAA GAUGAAAAGC ACtltlOGGAAA GAGAGllUAAA CAGUACGUGA F. neo.t'ormans ••••••••••••••U.GA•••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• F. flori-forme .....u.... T. beigelii T. cutaneum T. pullulan.s •••••••••A P. rbodozyma ••••••N ••• c. capitatum C ••••••••• ••••••N ••• ...... c... M. furfur ••c.G••••• M. p4chydo..rmati.s ••c.G••••• R. toruloid• .s S. jobn.sonii. L. .scottii ••••A.CA••

(371-410) s. polymorpbUlll MO'OGtlUAAA AGGGAAACGA O'OGAAGtlCAG UCGtlGtl'CCA- UGGGGUOCAG CC--AGUtlCO GC-UGGUGUA -uucccc--u UGGACGGGtlC AACAUCAGW F. neoforman.s ••••••• G •••••••••••••••••••••••••••••u.u -...... - - ....•0'0-. -A•••••••••••••••••• F. floriforme ••••••• G ••••••••••••••••••••••••••• CO.Utl ••• -A••••••• --G•••••••C- •••••• C •••• tTCltlG- -A.UG••••••••••••••• T. beigelii ••••••• G ••••••••••••••••••••••••••••U.-U .U••A..... • .-•••• C •• C •••• -UtlG- -.A••••••••••••••••• T. cutanoum ••••••• G ••••••••••••••••••••••••••••U.-U .CA.A•••••• -U-G •••••••C-A•• C •• C ••• -UG-- •••A ••••••••••••••••• T. pullulan.s ••••••• G ••••••••••••••••••••••••A •• C1JUtlC C.A.AC ••••••--G •••••••C- •••••• C •••U.GGC. --UG.A••C ••G •••••••• P. rhodo%~ ••••••• G ••••••••••••••••••••••••A •• CGtlGC .N•• AC •••••UGGGU.C-. U.C.N•• C •• U •• - •• GGG. NCOG.A••••• G•••••••• c. capitatum ••••••• G ••••••••••••••••••••••••••• C.UGC COA.A..... ----•••••• U ••• UAGG-. -c.G.A••••• G•••••••• M. furfur ...... CG •.•••.•• G•• C C.A•• C.GCU CA •• AC.U ..•.COGc •• 0 ..•u U •••• OGGG. -A-G.OA••.• G••. OG .•. M. pachyde.rmati", •••••• CG •••••••• G •• C •••••••••• C.A•• CUGCU ••A.AC ••••••UtlGC •• U ••• U ••••••• U •••U.GGA. -A-G.UA.C ••G•••UG ••• R. toruloic:ht.$ ••••••• GG .•••..•••• C A.O.. CUOGc C ------.UOGC.-.------.U-o.A•• C..G. s. jobn.sonii ••••••• GG ••••••••••C •••••••••• A.U•• COAUtl C ••A.------.tlI1GC••• ------GA••C-G.A•• C. CG •••••••• L. 3cottii ••••••• GG •••••••••• C •••••••••• A.U•• CUAO'O C ••A...... • .M•••••• - ••• O'O.GA••U-G.A •• C ••G••••••••

1471-510) s. polymorpbUlll UGGACCGGCG GAAAAUGGCG GGAGGAAUGU AGCAG--CUC CGGUOGUGUO AOAGCCOCW GUCGtlAUACG O'OG-GtlCOGG ACUGAGGAAU GCAGCUCGC- F. neofonnan.s co.. U ••• U •••U •• G ••• U U ••••••••• G••• COCU•• G •• G •••••• ••••••••C ••••• C •••• A C •• - •• UG •• F. flori-form. .U•• U •• CU•••U •• A ••• U ••••••• C ••••U.CC- ••• G •••AAAC •• ••••••••C ••••AC •••• A G.-A•• UG •• ••••••••• C •••••A ••• ­ T. beigelii .U.U•••• U •••U ••A •• UA .U•••••••• GA.O'OC--•••••M ••••• •••••••A •• A ••AC ••••AC •• -.GtlGA• ••••••• •C• T. cutaneum • U.U•••• U •••U ••A •• UA .U•••••••• GA.U-C--••••••A ••••• •••••••A •• A.U.C ••••AC •• -.GUGA • ••••••• • C. T. pullulan.s .U.GG ••• U G.O'O••CG ••••••• G•••UC-••• - ••A •••••• ••••• • CGCN U •••A ••••A .C.-CC.G•• ••••••••• C •••••A ••• C P. rhodo::y:aa .C.GG ••• UN ••••• C •• GN •• GN ••• G •• G.N.UC-- •••••A •••••• •••••• CNNN U.U.G •• G.A .C.N.-UG•• ...... c ..... G ••• C c. capitatum ••• GAG •• O'O A.C •• G •• GA tlI1. ••••••• G•••AC-••• • ••••••AG. U •••••• UGA .C-O'OG •••• ••••••••• C •••• OG ••• C M. furfur •••• 0 •• 0 •••• G• • GCAtl'A .OC ••••••• G •• --e- - .. GC ••••• •••.••• t1C. A.O.G ••••• AC .. A-....A. •••A ••••• C ••••• --.-­ M. pachydezmatis C.AGtl •• U •••• G •• G •• t1A U ••• A ••••• G•••uc- - ..A •••••• .••.A •• OG. ACU.G .•.•• AC.A-.O.•• .UCA•.••• C ••.•0--.-­ R. toruloides •OCCGG.N••••0 A .O'OA •••••••••••0-••• - •• C •••..• •••• •0. OC. .CO.G...... C-COGGG •• ••••••••• C ••••• GU •• ­ S. jahn.sonii .O'OCGG •••••••••• C.1JA .AGA ••• G •••••••UU-•• - •• C •••••• .....u.uc. ACO.G •• U ••• C-COGGG •• •••••••••C •••••GU.-­ L. scottii .O'OGGG.O'O ••••••• C.UN .tlITO ••• G •••••••UU- •• - •• C •••••• •••• • U.UCC ACO.G ••U.A .C-UOCGG •• ••••••••• C •••• tJA •• --

1571-626) S. polymorphum -CUUUAUGGC -CGGGGUOCG CCCACGUOCG AGCUOAGGAU GO'OGACAUAA UGGCUU F. neOrOrmatlS - ••••••••• - •••••••••••••••••••••••••••A •• F. .floriform·. - ••••••••• - ••••A •••• U ••••••••• U ••••••••• T. ~ig.lii - ••••••••• - ••• -C •••• GG •••••••••••••••••• T. cutan.um - •••• - •••• - ••• UC •••• GA •••••••••••••••••• T. pullula", U ••• GtIA•• U - ••• UC •••• GA ••••• C •• U ••••••••• •C •• G ••••• P.. rhodozym,8 UN.CACG •• NN •••UC.C •• GA ••••M •• C ••••••••• .C •• G••••• C.. a_pitae_ -.GCA.G•• U A-•• UC •••• GA ••• U •••• CA•••••••• •C •• G ••••• M.. rur~ur U.C ••••••• -G•• UC •••• GA ••• C •••• C ••••••••• •C••G.G••• M. paabyd.naati'" U.C •• C •••• -G••tIC •••• GA ••• C •••A CA•••••••• .C •• G.G ••• .R. eoruloidl.:l -.G-•••••• GA ••• C •••• Gtl.-.U•••AC ••••••••• •C •• GUGG •• S. joM6onii. - •••• U •• -. AU••• C •••• G••U-A.C.A C ••••••••• • CG.GtlGG •• L .. .scottii - •••• U •• -. A.A •• CUC ••• AG.-NN •••• UA •••••••• .C •• GtlGG •• Fig. 1. Alignment of 255 rRNA partial sequences (initiated at nucleotide position 635) from 13 heterobasidiomycetous yeasts listed in Table 1. Only the sequence of S. polymorphum is given in its entirety and serves as a reference for the other taxa. Dots in the other sequences indicate nucleotides to be identical to the reference species; dashes signify no bases present at those positions, and the letter "N" denotes an unidentified base. Heterobasidiomycerous YeaSts and rRNA Sequences 233

Results and Discussion In this work and in our comparison of the genera Sterig­ matomyces and Fellomyces (Gueho et a!., 1990), species The 255 and 185 rRNA panial sequences are presented seem to be characterized by unique nucleotides, and there in Figs. 1 and 2. The region sequenced from the large also appear to be nucleotide signatures for genera as well subunit (350 nucleotides) shows a rate of change much as higher orders of classification. For instance, they appear greater than the region sequenced from the small subunit especially well defined for the species pairs Trichosporoll (332 nucleotides), an observation common to other yeasts beigelii-T. cutaneum and Malassezia furfur-M. pachyder­ and all organisms for which sequences of these regions are matis. In contrast, there are no signature nucleotides known (Dams et a!', 1988; Gutell and Fox, 1988). Both characterizing the genus Trichosporon once T. pullulans is regions, appear quite different from those of ascomycetous added to the comparison. This confirms the present yeasts (Kurtzman, 1989; Peterson and Kurtzman, in pre­ heterogeneous nature of the genus even after exclusion of paration). For ascomycetes, base substitutions more com­ species with ascomycetous affinities (Gueho et a!., 1984). monly occur in first part of each sequence region while For instance, T. pullulans has the Q-9 ubiquinone system they are more scattered in heterobasidiomycetes. Further­ whereas T. beigelii is Q-9 or Q-lO, according to Yamada more, abundant insertions and deletions in the et al. (1982). DNA comparisons (E. Gueho, in prepara­ heterobasidiomycete large subunit, make alignment with tion) confirm the heterogeneity of the type species which the corresponding region of Saccharomyces cerevisiae am­ represents a complex of several species, including the two biguous. historically imponant names T. beigelii and T. cutaneum.

1284-1380) S. polymorphUlll GG1JGGAG OGAlltltlG1Je:tl GGOOAAooCC GAOAACGAAC GllGACCOOAA CCOGe:tlAAAO AGACAOGCCG GCOOOGGCOG GCOGCoo-GO CooCOOAGAG F. neot"o.naan" ...... O.. G llC.-..O.A . F. t"lqrit"o.noe ...... O.CG..O 0 oc -.A . T. ~Ji,- ...... O .. G O ..-.-C.A . T. cutWneum ...... c .. G c O ..-.-C.G .. T. pullulan.s ...... c .. G 0 O --C.G N. c. cepitatum ...... C.CG..O oc --.G . P. rbodozyma ...... C.CG.. O 0 0 --C.G N .. H. fur~ur ...... C.CA A O.A --NG N. M. pacbydemati.s ...... C.CA A O.A --.G . L. $cottii ...... CA.... • . R. toruloide.s •••••••••• •••••••••• •••••••••• •••••••••• •••••••••• ••••CA•••••••••••••A •••••••••• s.. john:sonii ...... CA......

(1381-1480) s. polymorphUlll GGACtlOOOGG CGOOOAGCCA AAGGMGOOO GAGGCAAOAA CAGG1Je:tlG1JG AOGCCCOOAG AOGOOe:tlGGG COCACGCGCG COACAe:tlGAC UGAGCCAGCG F" neot"o.rman.s .....G.C C O.G .C .. F" flo.ri~o~. • ••••AACA OG 00...... • . ::%:~ .....AACA OG 00 . ;: •••• • G.C •••A.C•••••G .C ••••••••••••••••••••N••••••••••••••••••••••••••••••••••••••••••••••••••••••••• T. pullulan.s .....A.CA••A.C OO .0 . c. capitatum .....A.e:tl. O AG .0 A . P.. rhodoZ'ym4 ...... A.CA..A.C oo .0 N A .. M. furfur .....A N .0 N GA •. H. pachyde.r:m4t1.s •••••A •••••••••••••••0 •••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• G•••••• A •• L. scottii .....A.CA OG .0 N A .. R. toruloide3 •••••A.CA•••••••••00 .0•••••••••••••••••••••.••...•.••••••••.•••••••••..•••.•••••••.•••••.. A ••••••••• s. john.sonii .....A.CA 00 .0 A .

(1481-1580) s. polymorphUlll AGOOA-AOCA Ce:tlOGACCGA GAGG1Je:tlGGG OAAOC'ClOG'ClG AAACOCAG1JC G1JGCOOGGGA OAGAGCAooG CAAOOAooGC OCOOCAACGA GGAAOACCOA F" neot'ormans • ..e:tlO.C.G - C . F" floriforpe ....O-.e:tl GA G .. T. b •••J.il ....O-..AA ..O A ..O . r.. cut'an.um .. ..0- AG.O A.CO .. T. pullulan.s ...... 0-...... • GO A c u . c. cap.:itatum ....0--.00 GA 0 C 0 .. P.. rhodozyma .. ..OC-.OO GA O ..N NC 0 .. H. furfur • .. -.0-.00 O GAA 00 0 . H. pachyde,naati.s • .. -.C-.OO O GAA OC 0 . L. scottii ...-.-..A OG GA CA N NlL A A 0 . R. toruloido:r • ..CO-.C OG GA CA 0 A . S" john.sonii ...COO 0 OO GA CA 0 A A 0 ..

(1581-1622) s. polymorph"", G1JAAGCG1JGA GOCACCAGCO CGCGOOGAoo ACG'CICCCOGC CC F" neoforman.s F. .tlerrifom. .. ..0 ...... A ...... ~: :::t.~ T. pullulan.s ....0 .. c. capitatum ...... CA • ....0 0 .. P.. rhodozyma ...... CA. ....0 50 . M. furfur ....0 . M. pachydezmati.s ....0 . L• .scottii O O A . .R. toruloide.s O•••O•••A ••••••••••• S" john"onii O O A . Fig. 2. Alignment of 185 rRNA partial sequences (initiated at nucleotide position 1626) from 13 heterobasidiomycerous yeasts (Table 1) with S. polymorphum as the top-most sequence reference. This figure has the same characteristics than the Fig. 1. 234 E. Gueho, C. P. Kunzman, and S. W. Peterson

Strains now confirmed to be T. cutaneum have Q-10 exhibited by species in the group. Each forms a primitive whereas those retypified as T. beigelii form Q-9 (G. Billon­ dolipore (pDP) classified as type 02P6 by Moore (1985). Grand, pers. comm.). Consequently, these data show This type is defined by a trilaminate septum (an electron Trichosporon to be heterogeneous for Coenzyme-Q. translucent laver between two electron dense lavers), a Further from our rRNA sequence data, it appears reason­ barrel-shaped 'pore crossed by dark bands (the dolipore able to exclude T. pullulans from the genus Trichosporon. itself) and no parenthesome (primitive DP), i. e., the dif­ In Fig. 3, species relationships are presented as a phy­ ferentiated elements of endoplasmic reticulum (ER) which logenetic tree resulting from analysis of the combined cap the pore on each side. Both species of Trichosporon large and small subunit partial rRNA sequences. This (Kreger-van Rij and Veenhuis, 1971a; M. Th. Smith, unrooted tree is divided into three major clusters. The unpublished data), F. neoformans (Kwon-Chung and Pop­ treatment of the 255 rRNA partial sequence alone gives a kin, 1976) and F. floriforme (Moore and Kreger-van Rij, similar tree with minor differences concerning the distan­ 1972) show organized ER above the pore and occasionally ces but not the branching. In contrast, the 185 partial (even in the same strain) a beginning of vesiculization of sequence alone shows a somewhat different arrangement those ER elements that makes the transition with the tre­ of species. From those differences, it seems apparent that mellaceous septum type 02P4' This latter type is com­ divergence of those two portions of the large and small pletely developed in the species Filobasidium cap­ unit molecules are not proceeding in parallel. suligenum and has parenthesomes formed of cone-shaped vesicles (Moore and Kreger-van Rij, 1972). Oberwinkler et al. (1983) discuss the heterogeneity within the family ~ S. polymorphum Filobasidiaceae and its relationship to the Tremella­ ....-:::-_ - F. neoformans les. In contrast, the relatedness of T. beigelii to the Filo­ /------F. floriforme basidiaceae is reinforced by an interesting antigenic simi­ / /(.--~beigelii larity between this species and , the anamorph of F. neoformans (McManus and Jones, / T. cutaneum 1985; Melcher et aI., 1988). The septal ultrastructure is / / / not yet known for S. polymorphum, but one might predict / / ~ T. pullulans it belongs to the type 02P6 (primitive dolipore). /L-- ~dOzyma Indeed, this species is, in accord with its coenzyme Q­ / C capitatum 10, cell wall xylose, ability to utilize inositol (Yamada, ~~------""':::-- M furfur 1989) and rRNA sequences (Gueho et aI., 1989) clearly '" ~M pachydermatis related to the anamorphic yeasts of the genus Fellomyces and to the Filobasidiaceae. The three species 1. scottii, S. johnsonii and R. tornloides represent another group that clusters when analyzed from either separate or combined subunit sequences. From the 255 and 185 rRNA sequences (Figs. 1 and 2), the taxa can be individually recognized by several L scottii single base substitutions and by a major deletion in the Fig. 3. Unrooted phylogenetic tree for the 13 heterobasidiomycet­ 255 subunit (Fig. 1). This deletion seems not to have ous yeasts listed in Table 1, calculated from the combined small affected estimates of distance in the phylogenetic tree and large subunit sequences given for each species in Figs. 1 and because omission of this area between bases 410-450 did 2, and using the maximum likelihood program DNAML, version not change the distance values (Fig. 3). These species have 3.11. The relative distances portrayed are proportional to the fucose instead of xylose in their cell walls (Sugiyama et aI., sum of all distances generated in the program analysis. Dashed 1985; von Arx and Weijman, 1979), do not assimilate lines designate uncertain branch points and branch lengths, based inositol and form septa perforated by a simple pore (Kre­ on little differences exhibited between trees calculated from com­ ger-van Rij and Veenhuis, 1971b). This septum is typically bined or separate sequences. trilaminate with a light layer between two dark ones and its thickness decreases toward the narrow central pore. The ubiquinone system does not completely correlate with The species S. polymorphum, F. neoformans, F. other characteristics, suggesting that here it serves an floriforme, T. beigelii and T. cutaneum cluster whether the exclusionaty role in the definition of genera. Based on phylogenetic tree is constructed from the combined large coenzyme Q diversity (Sugiyama et aI., 1985), 1. scottii and small subunit data set or only from small subunit forms two groups; one, which includes the type culture sequences. The five species contain xylose in their cell has Q-9 but no xylose in cell walls, and the second, which walls (Yamada, 1989; Sugiyama et aI., 1985; Weijman, forms Q-10 and has cell walls containing xylose. From 1979), are able to grow on inositol as sole source of car­ this, the taxon 1. scottii seems to represent at least two bon and form Q-10 ubiquinone (Yamada and Banno, species and, Sugiyama et al. (1985) suggest for similar 1984; Sugiyama et aI., 1985; Yamada et aI., 1982) with reasons that the genera Leucosporidium and Rhodos­ the apparent exception of T. beigelii as discussed above. poridium, including their anamorphs Candida and Of greater interest is the similarity of septal ultrastructure Rhodotornla, should be revised taxonomically. Von Arx Heterobasidiomycetous Yeasts and rRNA Sequences 235 and Wei;man (1979) noted heterogeneity of cell wall com­ Dams, E., Hendriks, L., Van de Peer. Y., Neefs, j.-M.. Smits, G.. position in the genus Sporidiobolus, and it toO may be Vandenbempt, 1., De Wachter, R.: Compilation of small comprised of several phylogenetic lines. ribosomal subunit RNA sequences. Nucleic Acids Res. 16. Malassezia furfur and M. pachydermatis cluster with r87-r173 (1988) Fox, G. E., Stackebrandt, E., Hespe/l, R. B.. Gibson, j., Manilof!. one another and by virtue of numerous nucleotide sig­ ]., Dyer, T. A., Wolfe, R. 5., Balch, W. E., Tanner, R. 5.. natures seem well separated from other taxa. The rRNA Magrum, L. ]., Zablen, L. B., Blakemore, R., Gupta, R.. sequences confirm the unique nature of these organisms Bo~en, L., Lewis, B. ]., Stahl, D. A., Luehrsen. /\:. R.. Chen. /\:. which was already suspected from their unipolar budding N., and Woese, C. R.: The phylogeny of prokaryotes. Science on a broad site and from their unusual cell wall ultrastruc­ (Wash.) 209, 457-463 (1988) ture (Simmons and Ahearn, 1987). Both species have Q-9 Georgiev, 0.1., Nikolaev, M., Hadiiolov, A. A., Skrvabin. /\:. G.. ubiquinone (Yamada, personal communication), but they Zkharyev, V. M., Bayev, A. A.: The structures'of the yeast are separated by 11 % in nuclear DNA base composition ribosomal RNA genes. 4. complete sequence of the 25S rRNA (Gueho and Meyer, 1989) which suggests considerable gene from Saccharomyces cerevisiae. Nucleic Acids Res. 9. 6953-6958 (1981) divergence. Septal ultrastructure for these species is Gueho, E., Tredick, ]., Phaff, H. ].: DNA base composition and unknown. M. furfur forms filaments in the scales of pity­ DNA relatedness among species of Trichosporon Behrend. riasis versicolor (a skin disease) but they most likely cor­ Antonie v. Leeuwenhoek 50, 17-32 (1984) respond to pseudohyphae. From rRNA comparisons, one Gueho, E., Meyer, S. A.: A reevaluation of the genus MalassezlJ might expect the sexual state of Malassezia to include by means of genome comparison. Antonie v. Leeuwenhoek 55. teliospores. Phaffia rhodozyma and Cystofilobasidium 245-251 (1989) capitatum are another pair that cluster together in our Gueho, E., Kurtzman, C. P., Peterson, S. W.: Phylogenetic rela­ analysis. Oberwinkler et a1. (1983) created Cysto­ tionship among species of Sterigmatomyces and Fellomyces determined from ribosomal RNA sequences. Int. ]. System. fila to accommodate Rhodosporidium capitatum Bact. (1990) (in press) which differs from the type species, R. toruloides, by hav­ Gutell, R. R., Fox, G. E.: Compilation of large subunit RNA ing aseptate basidia, the ability to utilize inositol, a Q-8 sequences presented in a structural format. Nucleic Acids Res. ubiquinone system and a primitive dolipore without vesic­ 16, r175-r269 (1988) les. This new genus was assigned to the Filobasidiaceae, de Haag, G. 5., Gueho, E., Boekhout, T.: Experimental fungal but it is clearly separated from other taxa in this family taxonomy and medical mycology. In: Handbook and applied because of the presence of teliospores, and by the absence Mycology, Vol. II, Humans, animals and insects (D. K. Arora, of clamp connections, xylose in the cell walls and Tre­ K. G. Mukerji and E. Drouhet, eds.). Marcel Dekker Inc., New mella-like haustoria. Cystofilobasidium may represent a York 1990 (in press) Johnson-Reid, J. A., Moore, R. T.: Some ultrastructure features transitional form between teliospore forming yeasts and of Rhodosporidium toruloides Banno. Antonie v. the Filobasidiaceae, whereas species such as Filobasidium Leeuwenhoek 38,417-437 (1972) capsuligenum may connect the Filobasidiaceae with the Kreger-van Rij, N.]. W., Veenhuis, M.: Septal pores in Trichos­ Tremel/aceae. By contrast, sexual reproduction is poron cutaneum. Sabouraudia 9, 36-38 (1971a) unknown for Phaffia rhodozyma, a fermentative species Kreger-van Rij, N. j. W., Veenhuis, M.: A comparative study of known only from its yeast phase. It appears basidiomycet­ the cell wall structure of basidiomycetous and related yeasts.]' ous from the presence of carotenoid pigments, coenzyme Gen. Microbiol. 69, 87-95 (1971b) Q-I0, xylose in cell walls and a positive diazonium blue B Kurtzman, C. P.: Prediction of biological relatedness among staining reaction (Miller et aI., 1976). The 25S and 18S yeasts from comparisons of nuclear DNA complementarity. Studies in Myco!. 30, 459-468 (1987) rRNA partial sequences from this study as well as the 5S Kurtzman, C. P.: Estimation of phylogenetic distances among rRNA (Walker, 1984) suggest that this yeast is probably ascomycetous yeasts from partial sequencing of ribosomal derived from an organism having a primitive dolipore in RNA. Yeast 5, S351-5354 (1989) its mycelial phase and a teleomorph similar to that of C. Kwon-Chung, K. ]., Popkin, T. ].: Ultrastructure of septal com­ capitatum. In contrast, the suggestion made by Miller et al. plex in Filobasidiella neoformans (Cryptococcus neoformans). (1976) to relate P. rhodozyma to is not sup­ J. Bact. 126, 524-528 (1976) ported by 25S and 18 rRNA sequence comparison. Lane, D. ]., Pace, B., Olsen, G. ]., Stahl, D. A., Sogin, M. L., Pace, N. R.: Rapid determination of 16S ribosomal RNA sequences for phylogenetic analyses. Proc. Nat!' Acad. Sci. USA 82, 6955-6959 (1985) Mankin, A. 5., Skryabin, K. G., Rubstov, P. M.: Identification of References ten additional nucleotides in the primary structure of yeast 185 rRNA. Gene 44, 143-145 (1986) Arx, ]. A., Von, Weijman, A. C. H.: Conidiation and carbohy­ McManus, E. j., Jones, ]. M.: Detection of a Trichosporon drate composition in Candida and Torulopsis. Antonie v. beigelii capsular polysaccharide in serum from a patient with Leeuwenhoeck 45, 547-555 (1979) disseminated Trichosporon infection. ]. Clin. Microbiol. 21, Blanz, P. A., Unseld, M.: Ribosomal RNA as a taxonomic tool in 681-685 (1985) mycology. Studies in Mycol. 30, 245-258 (1987) Melcher, G. P., Rinaldi, M. G., Frey, C. L., Drutz, D. 5.: Demon­ Chirgwin, ]. M., Przybyla, A. E., MacDonald, R. ]., Rutter, W. stration by immunoelectron-microscopy of a cell wall antigen 5.: Isolation of biologically active ribonucleic acid from sour­ in Trichosporon beigelii that cross-reacts with Cryptococcus ces enriched in ribonuclease. Biochemistry 18, 5294-5299 neoformans capsular polysaccharide. ]. Infect. Dis. 158, (1979) 901-902 (1988) 236 E. Gueho, C. P. Kurtzman, and S. W. Peterson

Miller, H. W., Yoneyama, M., Soneda, M.: Phaffia, a new yeast Sugiyama, J., Fukagawa, M., Chiu, S. W., Komagata, K.: Cellular genus in the deuteromycotina (Blastomycetes). Int. J. System. carbohydrate composition, DNA base composition, ubi­ Bact. 26, 286-291 (1976) quinone systems, and diazonium blue Bcolor test in the genera Moore, R. T.: Taxonomic proposals for the classification of Rhodosporidium, Leucosporidium, Rhodotorula and related marine yeasts and other yeast-like fungi including the smuts. basidiomycetous yeasts. J. Gen. Appl. Microbiol. 31, 519-550 Botanica Marina 13, 361-373 (1980) (1985) Moore, R. T.: The challenge of the doliporelparenthesome sep­ Walker, W. F.: 55 rRNA sequences from Actractiellales and tum, pp 175-212. In: Developmental biology of higher fungi basidiomycetous yeasts and fungi imperfecti. System. Appl. (R. T. Moore et aI., eds.) Cambridge, Cambridge Univ. Press Microbiol. 5, 352-359 (1984) 1985 Weijman, A. C. M.: Carbohydrate composition and taxonomy of Moore, R. T.: Additions to the genus Vanr;;a. Bibliotheca Geotrichum, Trichosporon and allied genera. Antonie v. Mycologica 108, 161-173 (1987) Leeuwenhoek 45, 119-127 (1979) Moore, R. T., Kreger-van Rij, N. J. W.: Ultrastructure of We;;man, A. C. M., Rodrigues de Miranda, L., van der Walt, J. Filobasidium Olive. Can. J. Microbiol. 18, 1949-1951 (1972) P.: Redefinition of Candidq .J)erkhout and the consequent Oberwinkler, F., Bandoni, R., Blanz, P., Kisimova-Horovitz, L.: emendation of Cryptococc~\~dRhodotorula Harrison. Cystofilobasidium: a new genus in Filobasidiaceae. System. Antonie v. Leeuwenhoek 54, 545-553 (1988) Appl. Microbiol. 4, 114-122 (1983) Wolters, J., Erdmann, V. A.: compilation of 55 rRNA and 55 Qu, T. H., Michot, B., Bache//erie, J. P.: Improved methods for rRNA gene sequences. Nucleic Acids Res. 16, rl-r70 (1988) structure probing in large RNAs: a rapid "heterologous" Yamada, Y.: The coenzyme Q systems and new genera in Yeasts, sequencing approach is coupled to the direct mapping of nuc­ pp. 88-89. In: Current problems of opporrunistic fungal infec­ lease accessible sites. Application to the 5'terminal domain of tions. Proc. 4th Intern. Symp. Res. Ctr pathogenic fungi Mic­ eukaryotic 285 rRNA. Nucleic Acids Res. 11, 5903-5920 rob. Toxicoses (M. Miyaji, ed.). Chiba, Chiba University 1989 (1983) Yamada, Y., Banno, I.: The coenzyme Q system in strains of Qu, L. H., Nicoloso, M., Bache//erie, J. P.: Phylogenetic calibra­ species in the genus Sterigmatomyces (Cryptococcaceae) and tion of the 5'terminal domain of large rRNA achieved by its teleomorphic genus Sterigmatosporidium. Trans. Mycol. determining rwenty eurocaryotic sequences. J. Mol. Evol. 28, Soc. Japan 25, 455-460 (1984) 113-124 (1988) Yamada, Y., Kondo, K.: Coenzyme Q system in the classification Rubstov, P. M., Musakhanov, M. M., Zakharyev, V. M., Krayev, of the yeast genera Rhodotorula and Cryptococcus, and the A. S., Skryabin, K. G., Bayev, A. A.: The complete structure of yeastlike genera Sporobolomyces and Rhodosporidium. J. yeast ribosomal RNA genes. 1. The complete nucleotide sequ­ Gen. Appl. Microbiol. 19,59-77 (1973) ence of the 185 ribosomal RNAS gene from Saccharomyces Yamada, Y., Nakazawa, E., Kondo, K.: The coenzyme Qsystem cerevisiae. Nucleic Acids Res. 8, 5779-5794 (1980) in strains of Trichosporon species and related organisms. J. Sanger, F., Nicklen, S. Coulson, A. R.: DNA sequencing with Gen. Appl. Microbiol. 28, 355-358 (1982) chain terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, Yamada, Y., Ohishi,T., Kondo, K.: The coenzyme Q systems in 5463-5467 (1977) strains of some yeasts and yeast-like fungi. J. Gen. Appl. Mic­ Simmons, R. B., Ahearn, D. G.: Cell wall ultrastructure and robiol. 29, 51-57 (1983) diazonium blue B reaction of Sporopachydermia quercuum, tsugae and Ma/assezia spp. Mycologia 79, 38-43 (1987)

Dr. Eveline Gueho, lnstitut Pasteur, Unite de Mycologie, 25 rue du Dr. Roux, F-75724 Paris Cedex 15, France