Systematics of basidiomycetous yeasts: a comparison oflarge subunit D1/D2 and internal transcribed spacer rDNA regions

Gloria Scorzetti a, J.W. Fell a;�, A. Fonseca b, Adele Statzell-Tallman a

a Rosenstiel School of Marine and Atmospheric Science, University of Miami, Miami, FL, USAb Faculty of Sciences and Technology, New University of Lisbon, Caparica, Portugal

Received 8 January 2002; received in revised form 15 May 2002; accepted 17 May 2002

First published online 12 July 2002

Abstract

Basidiomycetous yeasts in the Urediniomycetes and Hymenomycetes were examined by sequence analysis in two ribosomal DNAregions: the D1/D2 variable domains at the 5P end of the large subunit rRNA gene (D1/D2) and the internal transcribed spacers (ITS)1 and 2. Four major lineages were recognized in each class: Microbotryum, Sporidiobolus, Erythrobasidium and Agaricostilbum in theUrediniomycetes; Tremellales, Trichosporonales, Filobasidiales and Cystofilobasidiales in the Hymenomycetes. Bootstrap support formany of the clades within those lineages is weak; however, phylogenetic analysis provides a focal point for in-depth study of biologicalrelationships. Combined sequence analysis of the D1/D2 and ITS regions is recommended for species identification, while speciesdefinition requires classical biological information such as life cycles and phenotypic characterization.9 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Keywords: Yeast; Urediniomycete ; Hymenomycete; Phylogenetic analysis

1. Introduction

Molecular systematics of yeasts has emphasized eithercoding (D1/D2 variable domains of the large subunit orthe complete small subunit (SSU)) or non-coding (internaltranscribed spacers ITS1 and ITS2) regions of the ribo-somal DNA. As a consequence, an extensive D1/D2 data-base [1,2] and partial SSU (e.g. [3^8]) and ITS databases(e.g. [9^12]) are available for the classi¢cation and identi-¢cation of species. Due, in part, to the di¡erence in sizebetween the SSU (V1700 bases) and the ITS (V600bases) and D1/D2 (V600 bases), the latter two regionshave received the most attention for yeast systematics. Inviewing these two databases, the question arises : how ac-curate are either or both of these molecular regions for thedelineation of basidiomycetous species? Fell et al. [2] pro-vided a clue: they reported that the biologically distinctspecies Cryptococcus ater, Filobasidium elegans and Filo-basidium £oriforme, which could not be separated withD1/D2 analysis, were di¡erentiated with ITS analysis.Other examples of separation of biologically distinct spe-

cies requiring D1/D2 and/or ITS sequence analysis includeMrakia [13], Filobasidiella [2,14] and Pha⁄a/Xanthophyllo-myces [15]. The ITS region has also been recommended forthe identi¢cation of medically relevant yeasts [16,17].Based on this information, we sequenced the ITS regionfor 450 strains of 242 species of basidiomycetous yeasts inthe Urediniomycetes and Hymenomycetes. The followingdiscussion presents a comparative view of species separa-tions with the ITS and D1/D2 regions.

2. Materials and methods

Cultures were obtained from the Centraalbureau voorSchimmelcultures (CBS), Portuguese Yeast Culture Col-lection, New University of Lisbon (PYCC), U.S. Depart-ment of Agriculture, Peoria, IL, USA (NRRL Y), Amer-ican Type Culture Collection (ATCC), Brian Ste¡enson,North Dakota State University (KB) and Helen Vishniac,Oklahoma State University (Cryptococcus consortionis).Species names, strains and GenBank numbers are pro-vided in Tables 1 and 2. Strains and synonyms of speciesare indented under the type strain of the species. Taxo-nomic data of the described species and strains are avail-able in Kurtzman and Fell [18] and Barnett et al. [19] and

1567-1356 / 02 / $22.00 9 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.PII: S 1 5 6 7 - 1 3 5 6 ( 0 2 ) 0 0 1 2 8 - 9

* Corresponding author. Tel. : +1 (305) 361 4603.E-mail address: jfell@rsmas.miami.edu (J.W. Fell).

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www.fems-microbiology.org

Table 1List of hymenomycetous strains studied

Strain Strain number GenBank D1/D2 GenBank ITS Fig.

Bullera alba var. lactis CBS 7237 T AF363648 AF444665 1Bullera coprosmaensis CBS 8284 T AF363660 AF444485 1Bullera dendrophila CBS 6074 T AF189870 AF444443 1Bullera derxii CBS 7225 T AF189857 AF444405 1Bullera globispora CBS 6981 T AF075509 AF444407 1Bullera hannae CBS 8286 T AF363661 AF444486 1Bullera miyagiana CBS 7526 T AF189858 AF444409 1Bullera oryzae CBS 7194 T AF075511 AF444413 1Bullera penniseticola CBS 8623 T AF363649 AF444471 1Bullera pseudoalba CBS 7227 T AF075504 AF444399 1Bullera sinensis CBS 7238 T AF189884 AF444468 1Bullera unica CBS 8290 T AF075524 AF444441 1Bullera variabilis CBS 7347 T AF189855 AF444403 1Bullera sp. CBS 2013 AF444760 AF444666 1Bulleromyces albus mtB CBS 501 T AF075500 AF444368 1Bulleromyces albus mtA CBS 500 AF444757 AF444662 ^Bulleromyces albus3 anamorph CBS 502 AF444759 AF444664 ^Bulleromyces albus mtB CBS 6302 AF444758 AF444663 ^

Bulleromyces sp. mtA1 PYCC 5690 T AF444762 AF444668 1Bulleromyces sp. mtA1 PYCC 5691 AF444763 AF444669 ^Bulleromyces sp. mtA2 PYCC 5739 AF444764 AF444670 ^Bulleromyces sp. mtA2 PYCC 5740 AF444761 AF444667 ^

Cryptococcus adeliensis CBS 8351 T AF137603 AF145328 3Cryptococcus aerius CBS 155 T AF075486 AF145324 3*Torulopsis pseudoaeria CBS 4192 T AF181544 AF444376 ^

Cryptococcus albidisimilis CBS 7711 T AF137601 AF145325 3Cryptococcus albidisimilis3 ATCC 34633 AF137606 AF145331 ^

Cryptococcus albidus CBS 142 T AF075474 AF145321 3Cryptococcus albidus5 PYCC 2426 AF181514 AF444344 ^Cryptococcus albidus2 PYCC 4789 AF181531 AF444355 ^Cryptococcus albidus2 PYCC 4963 AF181509 AF444340 ^Cryptococcus albidusb PYCC 4990 AF181511 AF444342 ^*Cryptococcus genitalis5 CBS 5592 T AF181538 AF444370 ^*Torulopsis nadaensis5 CBS 969 T AF181516 AF444346 ^*Torulopsis rotundata5 CBS 945 T AF181517 AF444347 ^

Cryptococcus albidus var. kuetzingii CBS 1926 T AF137602 AF145327 3Cryptococcus albidus var. kuetzingii1 CBS 922 AF181504 AF444313 ^Cryptococcus albidus var. kuetzingii CBS 6086 AF181546 AF444378 ^

Cryptococcus albidus var. ovalis CBS 5810 T AF137605 AF145329 3Cryptococcus amylolentus CBS 6039 T AF105391 AF444306 1Cryptococcus antarcticus CBS 7687 T AF075488 AF145326 3Cryptococcus aquaticus CBS 5443 T AF075470 AF410469 4Cryptococcus arrabidensis CBS 8678 T AF181535 AF444362 3Cryptococcus bhutanensis CBS 6294 T AF137599 AF145317 3Cryptococcus cellulolyticus CBS 8294 T AF075525 AF444442 1Cryptococcus chernovii CBS 8679 T AF181530 AF444354 3Cryptococcus cylindricus CBS 8680 T AF181534 AF444360 3Cryptococcus cylindricus8 PYCC 5264 AF181533 AF444359 3

Cryptococcus curvatus CBS 570 T AF189834 AF410467 2Cryptococcus curvatus CBS 8126 AF444717 AF444458 ^

Cryptococcus dimennae CBS 5770 T AF075489 AF410473 1Cryptococcus di¥uens CBS 160 T AF075502 AF145330 3*Cryptococcus di¥uens var. uruguaiensis CBS 6436 T AF181543 AF444375 ^*Torulopsis albida var. japonica CBS 926 T AF181542 AF444374 ^

Cryptococcus £avus CBS 331 T AF075497 AF444338 1Cryptococcus friedmannii CBS 7160 T AF075478 AF145322 3Cryptococcus fuscescens CBS 7189 T AF075472 AF145319 3Cryptococcus gastricus CBS 2288 T AF137600 AF145323 3Cryptococcus gastricus5 CBS 1927 AF181501 AF444304 ^

Cryptococcus gilvescens CBS 7525 T AF181547 AF444380 3Cryptococcus heveanensis CBS 569 T AF075467 AF444301 1Cryptococcus huempii CBS 8186 T AF189844 AF444322 4

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Table 1 (Continued).

Strain Strain number GenBank D1/D2 GenBank ITS Fig.

Cryptococcus humicola CBS 571 T AF189836 AF410470 2Cryptococcus humicola5 CBS 8354 AF189851 AF444384 ^Cryptococcus humicola5 CBS 8371 AF189854 AF444398 ^

Cryptococcus laurentii CBS 139 T AF075469 AF410468 1Cryptococcus liquefaciens CBS 968 T AF181515 AF444345 1Cryptococcus liquefaciens PYCC 2406 AF181513 AF444343 ^Cryptococcus liquefaciens PYCC 2934 AF181518 AF444348 ^

Cryptococcus luteolus CBS 943 T AF075482 AF444323 1Cryptococcus macerans mta CBS 2206 T AF189848 AF444329 4Cryptococcus macerans mtK CBS 2425 AF075477 AF444317 4

Cryptococcus magnus CBS 140 T AF181851 AF190008 3Cryptococcus magnus CBS 8361 AF189852 AF444388 ^Cryptococcus magnus CBS 8362 AF189853 AF444389 ^Cryptococcus magnus CBS 8394 AF189872 AF444450 ^Cryptococcus magnus PYCC 4556 AF181528 AF444352 ^Cryptococcus magnus PYCC 4563 AF181529 AF444353 ^Cryptococcus magnus PYCC 4989 AF181510 AF444341 ^Cryptococcus magnus PYCC 5260 AF181532 AF444358 ^Cryptococcus magnus2 PYCC 5267 AF181536 AF444363 ^

Cryptococcus marinus CBS 5235 T AF189846 ^ 1Cryptococcus oeirensis CBS 8681 T AF181519 AF444349 3Cryptococcus oeirensis PYCC 5268 AF181537 AF444364 ^

Cryptococcus phenolicus CBS 8682 T AF181523 AF444351 3Cryptococcus podzolicus CBS 6819 T AF075481 AF444321 1Cryptococcus saitoi CBS 1975 T AF181540 AF444372 3*Naganishia globosa CBS 5106 T AF181539 AF444371 ^

Cryptococcus skinneri CBS 5029 T AF189835 AF444305 1Cryptococcus terreus CBS 1895 T AF075479 AF444319 3Cryptococcus terreus7 PYCC 2935 AF444692 AF444365 ^Cryptococcus terreus7 PYCC 4966 AF444694 AF444367 ^*Cryptococcus elinovii4 CBS 7051 T AF137604 AF145318 3*Cryptococcus himalayensis2 CBS 6293 T AF181502 AF410471 ^

Cryptococcus terricola CBS 4517 T AF181520 AF444350 3Cryptococcus terricola CBS 6435 AF181545 AF444377 ^

Cryptococcus uzbekistanensis CBS 8683 T AF181508 AF444339 3Cryptococcus victoriae CBS 8685 AF363647 AF444469 1Cryptococcus victoriae CBS 6550 AF444711 AF444447 ^Cryptococcus victoriae6 CBS 8884 AF444741 AF444645 ^

Cryptococcus vishniacii CBS 7110 T AF075473 AF145320 3*Cryptococcus asgardensis CBS 8141 T AF189839 AF444310 ^*Cryptococcus baldrensis CBS 8142 T AF189840 AF444311 ^*Cryptococcus consortionis A801-3aY92/20 T AF189880 AF444464 ^*Cryptococcus hemp£ingii CBS 8143 T AF189841 AF444312 ^*Cryptococcus lupi CBS 8100 T AF189860 AF444412*Cryptococcus socialis2 CBS 7158 T AF181503 AF444307 ^*Cryptococcus vishniacii var. asocialis CBS 8146 T AF189838 AF444309 ^*Cryptococcus wrightensis CBS 8145 T AF189837 AF444308 ^

Cryptococcus wieringae CBS 1937 T AF181541 AF444373 3Cryptococcus wieringae CBS 8353 AF181549 AF444383 ^

Cryptococcus sp. CBS 6984 AF444712 AF444448 4Cryptococcus sp. CBS 7712 AJ311450 AF408417 1Cryptococcus sp. CBS 7713 AJ311451 AF408418 ^Cryptococcus sp. CBS 7743 AJ311452 AF408419 ^

Cryptococcus sp. CBS 7890 AF075494 AF444332 1Cryptococcus sp. CBS 7944 AF444693 AF444366 3Cryptococcus sp. CBS 8024 AF444715 AF444453 1Cryptococcus sp.2 CBS 6024 AF444714 AF444452 ^Cryptococcus sp. CBS 6123 AF444687 AF444333 ^

Cryptococcus sp. CBS 8355 AF444696 AF444385 1Cryptococcus sp. CBS 8356 AF444697 AF444386 1Cryptococcus sp. CBS 8367 AF444703 AF444394 ^

Cryptococcus sp. CBS 8358 AF444698 AF444387 1Cryptococcus sp. CBS 8366 AF444702 AF444393 ^

Cryptococcus sp. CBS 8363 AF444699 AF444390 1

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Table 1 (Continued).

Strain Strain number GenBank D1/D2 GenBank ITS Fig.

Cryptococcus sp. CBS 8364 AF444700 AF444391 1Cryptococcus sp. CBS 8365 AF444701 AF444392 ^Cryptococcus sp. CBS 8368 AF444704 AF444395 ^

Cryptococcus sp. CBS 8372 AF444707 AF444410 1Cryptococcus sp. CBS 8369 AF444705 AF444396 1Cryptococcus sp. CBS 8507 AF444742 AF444646 1Cryptococcus sp. CBS 8804 AY006480 AY006481 1Cryptococcus sp. CBS 8924 AY029345 AY029346 4Cryptococcus sp. CBS 8925 AY029341 AY029342 1Cryptococcus sp. CBS 9089 AF444720 AF444487 3Cryptococcus sp. NRRL Y-17490 AF444713 AF444449 3Cryptococcus sp. PYCC 4949 AF444689 AF444356 3Cryptococcus sp. PYCC 4964 AF444690 AF444357 3Cryptococcus sp. PYCC 5266 AF444691 AF444361 3Cysto¢lobasidium bisporidii CBS 6347 AF075464 AF444299 4Cysto¢lobasidium capitatum CBS 6358 T AF075465 AF139627 4*Cysto¢lobasidium lari-marini CBS 7420 T AF075466 AF444300 ^

Cysto¢lobasidium ferigula CBS 7201 AF075487 AF444328 4Cysto¢lobasidium ferigula CBS 6954 AF444709 AF444445 ^

Cysto¢lobasidium in¢rmominiatum CBS 323 T AF075505 AF444400 4Dioszegia crocea CBS 6714 T AF075508 AF444406 1Dioszegia hungarica CBS 4214 T AF075503 AF444379 1*Bullera armeniaca CBS 7091 T AF189883 AF444467 ^

Fellomyces borneensis CBS 8282 T AF189877 AF444459 1Fellomyces chinensis CBS 8278 T AF189878 AF444460 1Fellomyces dystilii CBS 8545 T AF363652 AF444475 1Fellomyces fuzhouensis CBS 8243 T AF363659 AF444484 1Fellomyces fuzhouensis2 CBS 6133 AF075506 AF444401 ^

Fellomyces horovitziae CBS 7515 T AF189856 AF444404 1Fellomyces lichenicola CBS 8315 T AF363643 AF444462 1Fellomyces ogasawarensis CBS 8544 T AF363651 AF444474 1Fellomyces penicillatus CBS 5492 T AF177405 AF444337 1Fellomyces polyborus CBS 6072 T AF189859 AF444411 1Fellomyces sichuanensis CBS 8318 T AF189879 AF444461 1Fellomyces thailandicus CBS 8308 T AF363644 AF444463 1Fibulobasidium inconspicuum CBS 8237 AF363641 AF444318 1Filobasidiella neoformans CBS 132 T AF075484 AF444326 1Filobasidiella neoformans CBS 882 AF189845 AF444324 ^

Filobasidiella bacillispora CBS 6289 T AF075526 AF444444 1Filobasidium capsuligenum mta CBS 1906 T AF363642 AF444381 3Filobasidium capsuligenum mta CBS 4381 AF444695 AF444382 ^Filobasidium capsuligenum9 mtK CBS 4736 AF075501 AF444369 3Filobasidium capsuligenum10 mtK CBS 6219 AF181506 AF444334 ^Filobasidium capsuligenum CBS 8023 AF444688 AF444335 ^

Filobasidium elegans CBS 7640 AF181548 AF190006 3Filobasidium £oriforme CBS 6241 AF075498 AF190007 3Filobasidium globisporum CBS 7642 AF075495 AF444336 3Filobasidium uniguttulatum mta CBS 1730 T AF075468 AF444302 3Filobasidium uniguttulatum mtK CBS 1727 AF181500 AF444303 ^

Holtermannia corniformis CBS 6979 AF189843 AF410472 1Hyalodendron lignicola var. simplex CBS 220.34 T AF363657 AF444481 2Hyalodendron lignicola var. undulatum CBS 222.34 T AF363658 AF444482 2Kockovaella imperatae CBS 7554 T AF189862 AF444425 1Kockovaella machilophila CBS 8607 T AF363654 AF444477 1Kockovaella pha⁄i CBS 8608 T AF363655 AF444478 1Kockovaella sacchari CBS 8624 T AF363650 AF444472 1Kockovaella schimae CBS 8610 T AF363656 AF444479 1Kockovaella thailandica CBS 7552 T AF075516 AF444426 1Mrakia frigida CBS 5270 T AF075463 AF144483 4Mrakia gelida CBS 5272 T AF189831 AF144485 4Pha⁄a rhodozyma CBS 5905 T AF189871 AF139629 4Sirobasidium magnum CBS 6803 AF075475 AF444314 1Sirobasidium intermedium CBS 7805 AF075492 AF444330 1Sterigmatosporidium polymorphum CBS 8088 T AF075480 AF444320 1

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Table 1 (Continued).

Strain Strain number GenBank D1/D2 GenBank ITS Fig.

Tremella aurantia CBS 6965 AF189842 AF444315 1Tremella brasiliensis CBS 6966 AF189864 AF444429 1Tremella cinnabarina CBS 8234 AF189866 AF444430 1Tremella encephala CBS 6968 AF189867 AF410474 1Tremella foliacea CBS 6969 AF189868 AF444431 1Tremella fuciformis CBS 6970 AF075476 AF444316 1Tremella globispora CBS 6972 AF189869 AF444432 1Tremella mesenterica CBS 6973 AF075518 AF444433 1Tremella moriformis CBS 7810 AF075493 AF444331 1Trichosporon aquatile CBS 5973 T AF075520 AF410475 2Trichosporon asahii CBS 2479 T AF105393 AY055381 2Trichosporon asahii CBS 7137 AF189882 AF444466 ^Trichosporon asahii CBS 8520 AF189876 AF444457 ^Trichosporon asahii CBS 8640 AF189881 AF444465 ^

Trichosporon asteroides CBS 2481 T AF075513 AF444416 2Trichosporon brassicae CBS 6382 T AF075521 AF444436 2Trichosporon cutaneum CBS 2466 T AF075483 AF444325 2Trichosporon coremiiforme CBS 2482 T AF139983 AF444434 2Trichosporon coremiiforme CBS 2478 AF189863 AF444427 ^

Trichosporon domesticum CBS 8280 T AF075512 AF444414 2Trichosporon domesticum CBS 8111 AF189874 AF444454 ^

Trichosporon dulcitum CBS 8257 T AF075517 AF444428 2Trichosporon faecale CBS 4828 T AF105395 AF444419 2Trichosporon gracile CBS 8189 T AF105399 AF444440 2Trichosporon gracile CBS 8518 AF189875 AF444455 ^Trichosporon gracile CBS 8519 AF444716 AF444456 ^

Trichosporon guehoae CBS 8521 T AF105401 AF410476 2Trichosporon inkin CBS 5585 T AF105396 AF444420 2Trichosporon japonicum CBS 8641 T AF308657 AF444473 2Trichosporon jirovecii CBS 6864 T AF105398 AF444437 2Trichosporon laibachii CBS 5790 T AF075514 AF444421 2Trichosporon loubierii CBS 7065 T AF075522 AF444438 2Trichosporon moniliiforme CBS 2467 T AF105392 AF444415 2Trichosporon moniliiforme ATCC 46490 AF444719 AF444480 ^Trichosporon moniliiforme CBS 8400 AF189873 AF444451 ^

Trichosporon montevideense CBS 6721 T AF105397 AF444422 2Trichosporon mucoides CBS 7625 T AF075515 AF444423 2Trichosporon multisporum CBS 2495 T AF139984 AF414695 2Trichosporon ovoides CBS 7556 T AF075523 AF444439 2Trichosporon porosum CBS 2040 T AF189833 AF414694 2Trichosporon pullulans CBS 2532 T AF105394 AF444417 2Trichosporon pullulans CBS 2541 AF189861 AF444418 ^

Trichosporon sporotrichoides CBS 8246 T AF189885 AF444470 2Trichosporon veenhuisii CBS 7136 T AF105400 AF414693 2Trichosporon sp. CBS 5601 AF444710 AF444446 2Trichosporon sp. CBS 8245 AF444708 AF444424 2Trichosporon sp. CBS 8370 AF444706 AF444397 2Trichosporon sp. CBS 8381 AF444740 AF444644 2Trichosporon sp. CBS 9085 AF444743 AF444647 ^Trichosporon sp. CBS 9088 AF444744 AF444648 ^

Trichosporon sp. CBS 8686 AF444718 AF444476 2Trimorphomyces papilionaceus CBS 443.92 AF075491 AF444483 1Tsuchiyaea wing¢eldii CBS 7118 T AF177404 AF444327 1Udeniomyces megalosporus CBS 7236 T AF075510 AF444408 4Udeniomyces puniceus CBS 5689 T AF075519 AF444435 4Udeniomyces pyricola CBS 6754 T AF075507 AF444402 4Xanthophyllomyces dendrorhous CBS 7918 T AF075496 AF139628 4Xanthophyllomyces dendrorhous CBS 6938 AF444739 AF139632 ^

Xanthophyllomyces sp. CBS 9090 AF444721 AF444488 4

Compared to the type strain: 1a six-base deletion in D1D2, identical in ITS; 2identical in D1D2, one substitution in ITS; 3one substitution in D1D2,identical in ITS; 4one substitution in D1D2, one substitution in ITS; 5identical in D1D2, two substitutions in ITS; 6two substitutions in D1D2, identi-cal in ITS; 7three substitutions in D1D2, two substitutions in ITS; 8one substitution in D1D2, six substitutions in ITS; 9eight substitutions in D1D2,10 substitutions in ITS; 10eight substitutions in D1D2, 10 substitutions and a 13-base deletion in ITS.

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at the CBS web site (www2.cbs.knaw.nl/yeast/WebC.asp).Unidenti¢ed strains, potentially representing undescribedspecies, are included in the trees and tables. These strains,which are designated by genus and collection numbers,were included to illustrate the biodiversity within theclades. Formal description of these taxa will require astandard phenotypic study, which is not a part of thisreport.All strains in Table 1 were sequenced in the D1/D2 and

ITS regions, with the exception of the type strain of Cryp-tococcus marinus, which was not sequenced in the ITSregion due to technical problems. DNA extraction, PCRampli¢cation, puri¢cation, cycle sequencing and sequenceanalysis followed the protocol of Fell et al. [2]. Sequenceswere aligned with Megalign (DNAStar) and visually cor-rected. Phylogenetic analysis employed PAUP 4.0 usingparsimony analysis, neighbor-joining analysis as a startingtree and tree bisection-reconnection. Gaps were treated asmissing data. Bootstrap values were determined with

PAUP; values less than 50% were not recorded on thetree ¢gures.In addition to the strains sequenced in our laboratory,

the phylogenetic analyses included sequence data of Tre-mella by Chen [20] and Sporobolomyces by Vale¤rio et al.[21], Inacio, J. and Fonseca, A. (unpublished data) andTakashima and Nakase [22]. GenBank numbers for theseadditional strains, not reported in the tables, are listed inthe tree ¢gures.

3. Results

The basidiomycetous yeasts are distributed within threeclasses: Ustilaginomycetes, Urediniomycetes and Hyme-nomycetes [2,23^25]. The current report is directed tothe latter two classes. In Figs. 1 and 2, trees based onanalysis of the D1/D2 regions indicated the presence offour major lineages in each class, although some of this

Fig. 1. Hymenomycetes. Strict consensus of 100 equally parsimonious trees of the D1/D2 region of the large subunit rDNA (PAUP 4.0). Numbers onbranches represent bootstrap percentages (s 50%) from 100 full heuristic replications for the four lineages. Clades are signi¢ed to indicate their orienta-tion within the lineages. Number of ingroup taxa= 180, number of characters (NC) = 632, constant characters (CC)= 220, parsimony uninformativecharacters (PUC)= 53, parsimony informative characters (PIC) = 359, tree length (TL) = 3353, consistency index (CI) = 0.228, retention index(RI) = 0.790. Legend: (1) C. marinus, (2) C. humicola, (3) H. corniformis. Outgroup=members of the Agaricostilbum lineage.

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nomenclature must be regarded as provisional designa-tions. Statistical bootstrap support for these lineages var-ied. Within the Hymenomycetes class, the Tremellales(6 50%) may represent more than one lineage. Holterman-nia corniformis and related strains, included in this studyas members of the Tremellales (see Section 3.1.1), mayconstitute a separate lineage as suggested by the branchposition in Fig. 1. However, in Fig. 3 a strong bootstrapsupport (100%) links the Holtermannia clade to the Tre-mellales lineage in the ITS tree. The Trichosporonales lin-eage (84%) has a 64% branch (Cryptococcus humicola : seeSection 3.1.2), which may represent a sister lineage to theTrichosporonales (see Section 3.1.2). The Filobasidialeslineage has a strong support of 92%. The Cysto¢lobasi-diales lineage (74%) is composed of generally strong-sup-ported clades, which may represent multiple lineages (seeSection 3.1.4). Similar levels of support were found amongthe Urediniomycetes: Microbotryum (6 50%), Sporidiobo-

lus (73%), Erythrobasidium (100%) and Agaricostilbum(74%). The clades depicted in the Figs. 1 and 2 consensustrees agree with the parsimony trees (Figs. 3^10), with themajor exceptions of Cryptococcus marinus (see Section3.1.1), Sporobolomyces griseo£avus and Reniforma strues(Section 3.2.3).A major problem encountered in the presentation of

urediniomycetous and hymenomycetous ITS trees is thedissimilarity of sequences between phylogenetic groups;as a result, sequences from two or more phylogeneticgroups are di⁄cult to align. This sequence variabilitycan be illustrated by the number of base changes betweenclosely related species. For example, in the Microbotryumlineage (see Section 3.2.3) the number of nucleotide di¡er-ences in the D1/D2 region between Rhodotorula pustulaand Rhodotorula pilati is ¢ve out of 591 nucleotides exam-ined, whereas the ITS di¡erence between the two species is49/608. Another example: R. pilati and Sporobolomyces

Fig. 2. Urediniomycetes. Strict consensus of 100 equally parsimonious trees of the D1/D2 region of the large subunit rDNA (PAUP 4.0). Numbers onbranches represent bootstrap percentages (s 50%) from 100 full heuristic replications for the four lineages. Clades are signi¢ed to indicate their orienta-tion within the lineages. Number of ingroup taxa= 142, NC=632, CC=207, PUC=54, PIC=362, TL=2639, CI= 0.285, RI= 0.795. Legend: (1) S. gri-seo£avus, (2) R. strues. Outgroup=members of the Cysto¢lobasidium lineage.

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Table 2List of urediniomycetous strains studied

Strain Strain number GenBank D1/D2 GenBank ITS Fig.

Agaricostilbum hyphaenes CBS 7811 AF177406 AF444553 8Bensingtonia ciliata CBS 7514 T AF189887 AF444563 8Bensingtonia ingoldii CBS 7424 T AF189888 AF444519 8Bensingtonia miscanthi CBS 7282 T AF189891 AF444516 8Bensingtonia musae CBS 7965 T AF189892 AF444569 8Bensingtonia naganoensis CBS 7286 T AF189893 AF444558 8Bensingtonia phyllada CBS 7169 T AF189894 AF444514 8Bensingtonia sakaguchii CBS 8464 T AF363646 AF444626 8Bensingtonia subrosea CBS 7283 T AF189895 AF444565 8Bensingtonia yamatoana CBS 7243 T AF189896 AF444634 7Bensingtonia yuccicola CBS 7331 T AF189897 AF444518 8Bensingtonia sp. PYCC 5551 AF444770 AF444676 8Bensingtonia sp. PYCC 5562 AF444765 AF444671 8Bensingtonia sp. PYCC 5566 AF444766 AF444672 8Bensingtonia sp. PYCC 5547 AF444767 AF444673 ^

Chionosphaera apobasidialis CBS 7430 T AF177407 AF444599 8Colacogloea peniophorae PYCC 4285 AF189898 AF444591 7Erythrobasidium hasegawianum CBS 8253 T AF189899 AF444522 5Hyalopycnis blepharistoma CBS 591.93 AF189900 AF444555 7Kondoa aeria CBS 8352 T AF189901 AF444562 8Kondoa aeria4 anam. CBS 8378 AF189902 AF444595 ^Kondoa aeria4 PYCC 5549 AF204055 AF444675 ^Kondoa aeria4 PYCC 5565 AF204054 AF444674 ^

Kondoa malvinella CBS 6082 T AF189903 AF444498 8Kondoa sp. CBS 8379 AF189904 AF444596 8Kriegeria eriophori CBS 8387 AF189905 AF444602 7Kurtzmanomyces insolitus CBS 8377 T AF177408 AF444594 8Kurtzmanomyces nectairei CBS 6405 T AF177409 AF444494 8Kurtzmanomyces tardus CBS 7421 T AF177410 AF444566 8Leucosporidium antarcticum CBS 5942 T AF189906 AF444529 7Leucosporidium fellii CBS 7287 T AF189907 AF444508 7Leucosporidium scottii CBS 5930 T AF070419 AF444495 7Leucosporidium scottii1 CBS 5932 AF189908 AF444496 ^

Mastigobasidium intermedium mta CBS 7226 T AF189889 AF444564 7Mastigobasidium intermedium mtK CBS 7281 AF189890 AF444630 ^

Occultifur externus CBS 8732 T AF189909 AF444567 5Occultifur externus4 anam. PYCC 4557 AF189910 AF444643 ^Occultifur externus4 anam. PYCC 4823 AF189911 AF444642 ^

Reniforma strues CBS 8263 T AF189912 AF444573 7Rhodosporidium babjevae mtA1 CBS 7808 T AF070420 AF444542 6Rhodosporidium babjevae mtA2 CBS 9071 AF189913 AF444636 ^

Rhodosporidium diobovatum mtK CBS 6085 T AF070421 AF444502 6Rhodosporidium diobovatum5 mtK CBS 9076 AF444748 AF444653 ^Rhodosporidium diobovatum5 mta CBS 9081 AF444753 AF444658 6Rhodosporidium diobovatum5 CBS 9084 AF444756 AF444661 ^

Rhodosporidium £uviale CBS 6568 T AF189915 AY015432 6Rhodosporidium kratochvilovae CBS 7436 T AF071436 AF444520 6Rhodosporidium kratochvilovae mtA2 PYCC 4776 AF444774 AF444680 ^Rhodosporidium kratochvilovae mtA2 PYCC 4778 AF444772 AF444678 ^Rhodosporidium kratochvilovae1 mtA2 PYCC 4785 AF444773 AF444679 ^Rhodosporidium kratochvilovae anam. PYCC 4787 AF444777 AF444685 ^Rhodosporidium kratochvilovae mtA1 PYCC 4793 AF189918 AF444585 ^Rhodosporidium kratochvilovae mtA1 PYCC 4818 AF189916 AF444586 ^Rhodosporidium kratochvilovae mtA2 PYCC 4819 AF189917 AF444587 ^Rhodosporidium kratochvilovae mtA2 PYCC 4883 AF444775 AF444681 ^Rhodosporidium kratochvilovae mtA1 PYCC 5244 AF444776 AF444682 ^Rhodosporidium kratochvilovae s.s. PYCC 5579 AF444771 AF444677 ^Rhodosporidium kratochvilovae anam. PYCC 5580 AF444778 AF444686

Rhodosporidium lusitaniae CBS 7604 T AF070423 AY015430 6Rhodosporidium paludigenum mtA2 CBS 6566 T AF363640 AF444492 6Rhodosporidium paludigenum mtA1 CBS 6567 AF070424 AF444493 ^

Rhodosporidium sphaerocarpum mta CBS 5939 T AF070425 AF444499 6Rhodosporidium sphaerocarpum mta CBS 9075 AF444747 AF444652 ^

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Table 2 (Continued).

Strain Strain number GenBank D1/D2 GenBank ITS Fig.

Rhodosporidium sphaerocarpum mta CBS 9079 AF444751 AF444656 ^Rhodosporidium sphaerocarpum mta CBS 9080 AF444752 AF444657 ^Rhodosporidium sphaerocarpum mtA CBS 9082 AF444754 AF444659 ^

Rhodosporidium toruloides CBS 349 AF070426 AF444489 6Rhodosporidium toruloides CBS 9074 AF444746 AF444651 ^

Rhodotorula araucariae CBS 6031 T AF070427 AF444510 6Rhodotorula armeniaca CBS 8076 T AF189920 AF444523 5Rhodotorula aurantiaca CBS 317 T AF189921 AF444538 5Rhodotorula auriculariae CBS 6379 T AF189922 AF444507 7Rhodotorula bogoriensis CBS 4101 T AF189923 AF444536 7Rhodotorula bu¡onii CBS 2838 T AF189924 AF444526 7Rhodotorula creatinovora CBS 8620 T AF189925 AF444629 7Rhodotorula cresolica CBS 7998 T AF189926 AF444570 7Rhodotorula di¥uens CBS 5233 T AF075485 AF444533 7Rhodotorula ferulica CBS 7416 T AF363653 AF444632 7Rhodotorula ferulica CBS 7402 AF189927 AF444528 ^

Rhodotorula foliorum CBS 5234 T AF317804 AF444633 7Rhodotorula fragraria CBS 6254 T AF070428 AF444530 7Rhodotorula fujisanensis CBS 4551 T AF189928 AF444490 7Rhodotorula fujisanensis CBS 6371 AF189929 AF444574 ^Rhodotorula fujisanensis CBS 8264 AF189930 AF444572 ^*Rhodotorula futronensis CBS 8163 T AF189931 AF444525 7

Rhodotorula glutinis CBS 20 T AF070430 AF444539 6Rhodotorula dairenensis CBS 4406 T AF070429 AF444501 6Rhodotorula dairenensis PYCC 4784 AY033551 AF444683 ^Rhodotorula dairenensis PYCC 4897 AY033552 AF444684 ^

Rhodotorula graminis CBS 2826 T AF070431 AF444505 6Rhodotorula hylophila CBS 6226 T AF363645 AF444622 7Rhodotorula hordea CBS 6403 T AF189933 AF444524 7Rhodotorula ingeniosa CBS 4240 T AF189934 AF444534 7Rhodotorula javanica CBS 5236 T AF189935 AF444532 7Rhodotorula lactosa CBS 5826 T AF189936 AF444540 5Rhodotorula laryngis CBS 2221 T AF189937 AF190014 5Rhodotorula laryngis NRRL Y-17494 AF189939 AF444607 ^Rhodotorula laryngis NRRL Y-17503 AF189940 AF444616 ^Rhodotorula laryngis NRRL Y-17504 AF189941 AF444617 ^

Rhodotorula lignophila CBS 7109 T AF189943 AF444513 7Rhodotorula marina CBS 2365 T AF189944 AF444504 5Rhodotorula minuta CBS 319 T AF189945 AF190011 5Rhodotorula minuta2 CBS 4408 AF189946 AF444579 ^Rhodotorula minuta CBS 7296 AF189947 AF444620 ^

Rhodotorula mucilaginosa CBS 316 T AF070432 AF444541 6Rhodotorula mucilaginosa2 CBS 8383 AF189959 AF444649 ^Rhodotorula mucilaginosa6 CBS 9070 AF444738 AF444635 6Rhodotorula mucilaginosa2 CBS 9077 AF444749 AF444654 ^Rhodotorula mucilaginosa4 CBS 9078 AF444750 AF444655 ^Rhodotorula mucilaginosa4 CBS 9083 AF444755 AF444660 ^Rhodotorula mucilaginosa1 NRRL Y-17485 AF189952 AF444605 ^Rhodotorula mucilaginosa1 NRRL Y-17493 AF189953 AF444606 ^Rhodotorula mucilaginosa1 NRRL Y-17495 AF189954 AF444608 ^Rhodotorula mucilaginosa1 NRRL Y-17496 AF189955 AF444609 ^Rhodotorula mucilaginosa1 NRRL Y-17497 AF444725 AF444610 ^Rhodotorula mucilaginosa1 NRRL Y-17499 AF189956 AF444612 ^Rhodotorula mucilaginosa1 NRRL Y-17500 AF189957 AF444613 ^Rhodotorula mucilaginosa1 NRRL Y-17501 AF189958 AF444614 ^Rhodotorula mucilaginosa1 PYCC 4349 AF189951 AF444584 ^*Rhodotorula rubra CBS 17 T AF189960 AF444503 ^*Sporobolomyces alborubescens1 CBS 482 T AF189961 AF444497 ^

Rhodotorula muscorum CBS 6921 T AF070433 AF444527 7Rhodotorula nothofagi CBS 8166 T AF189950 AF444537 7Rhodotorula nothofagi1 CBS 9091 AF444736 AF444641 ^

Rhodotorula pallida CBS 320 T AF189962 AF444590 5Rhodotorula philyla CBS 6272 T AF075471 AF444506 7Rhodotorula pilati CBS 7039 T AF189963 AF444598 7

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Table 2 (Continued).

Strain Strain number GenBank D1/D2 GenBank ITS Fig.

Rhodotorula pustula CBS 6527 T AF189964 AF444531 7Rhodotorula sloo⁄ae CBS 5706 T AF189965 AF444627 5Rhodotorula sloo⁄ae4 CBS 7093 AF189966 AF444554 ^Rhodotorula sloo⁄ae4 CBS 7094 AF444722 AF444552 ^Rhodotorula sloo⁄ae4 CBS 7095 AF189967 AF444618 ^Rhodotorula sloo⁄ae3 PYCC 4887 AF189968 AF444589 ^

Rhodotorula sonckii CBS 6713 T AF189969 AF444601 6Rhodotorula vanillica CBS 7404 T AF189970 AF444575 7Rhodotorula yarrowii CBS 7417 T AF189971 AF444628 7Rhodotorula sp. CBS 6370 AF075499 AF444560 7Rhodotorula sp. CBS 6372 AF444728 AF444621 7Rhodotorula sp. CBS 8445 AF444729 AF444623 7Rhodotorula sp. CBS 8446 AF444730 AF444624 7Rhodotorula sp. CBS 8447 AF444731 AF444625 7Rhodotorula sp. CBS 8448 AF444732 AF444631 ^

Rhodotorula sp. CBS 8885 AF444745 AF444650 6Rhodotorula sp. CBS 8923 AY033643 AY033642 5Rhodotorula sp. CBS 8928 AY033640 AY033641 7Rhodotorula sp. CBS 8943 AY033636 AY033637 7Rhodotorula sp. CBS 8944 AY033639 AY033638 7Rhodotorula sp. CBS 9072 AF189932 AF444637 6Rhodotorula sp. CBS 9073 AF444733 AF444638 ^

Rhodotorula sp. CBS 9086 AF444734 AF444639 5Rhodotorula sp. CBS 9087 AF444735 AF444640 ^

Rhodotorula sp. PYCC 4689 AF444737 AF444588 5Rhodotorula sp. CBS 7295 AF444727 AF444619 ^

Rhodotorula sp. NRRL Y-17502 AF444726 AF444615 5Sakaguchia dacryoidea mtA1B1 CBS 6353 T AF189972 AF444597 5Sakaguchia dacryoidea mtA1B2 CBS 6356 AF189973 AF444500 ^Sakaguchia dacryoidea9 mtA2B1 CBS 7999 AF444723 AF444571 5

Sphacelotheca polygoni-persicariae PYCC 4293 AF189974 AF444593 7Sporidiobolus johnsonii HO CBS 5470 T AF070435 AY015431 6Sporidiobolus johnsonii8 (salmonicolor mtA2) CBS 2630 AF189976 AF444568 ^*Sporobolomyces holsaticus8 CBS 1522 T AF189975 AF444509 6

Sporidiobolus microsporus CBS 7041 T AF070436 AF444535 6Sporidiobolus pararoseus mtA2 CBS 491 T AF189977 AY015429 6*Sporobolomyces pararoseus7 mtA1 CBS 484 T AF070437 AF417115 6*Sporobolomyces ruber mtA2 CBS 4216 T AF189978 AF444604 ^

Sporidiobolus ruineniae CBS 5001 T AF070438 AY015433 6Sporidiobolus ruineniae var. coprophilus1 CBS 5811 T AF070434 AF444491 ^

Sporidiobolus salmonicolor mtA1 CBS 490 T AF070439 AY015434 6Sporidiobolus salmonicolor1 NRRL Y-17498 AF189979 AF444611 ^

Sporidiobolus sp. CBS 5541 AY015271 AY015435 6Sporobolomyces coprosmae CBS 7899 T AF189980 AF444577 5Sporobolomyces coprosmicola CBS 7897 T AF189981 AF444576 8Sporobolomyces dracophylli CBS 7900 T AF189982 AF444583 8Sporobolomyces elongatus CBS 8080 T AF189983 AF444561 5Sporobolomyces falcatus CBS 7368 T AF075490 AF444543 7Sporobolomyces foliicola CBS 8075 T AF189984 AF444521 5Sporobolomyces gracilis CBS 71 T AF189985 AF444578 5Sporobolomyces griseo£avus CBS 7284 T AF189986 AF444557 7Sporobolomyces inositophilus CBS 7310 T AF189987 AF444559 7Sporobolomyces kluyveri-nielii CBS 7168 T AF189988 AF444544 5Sporobolomyces lactophilus CBS 7527 T AF177411 AF444545 8Sporobolomyces linderae CBS 7893 T AF189989 AF444582 8Sporobolomyces marcillae CBS 4217 T AF070440 AY015437 6Sporobolomyces oryzicola CBS 7228 T AF189990 AF444546 5Sporobolomyces phyllomatis CBS 7198 T AF189991 AF444515 5Sporobolomyces roseus CBS 486 T AF070441 AY015438 6Sporobolomyces ruber CBS 7512 T AF189992 AF444550 8Sporobolomyces ruberrimus CBS 7500 T AF070442 AY015439 6*S. ruberrimus var. albus1 CBS 7501 T AF189993 AY015436 ^S. ruberrimus var. albus1 CBS 7253 AF189994 AF444581 ^

Sporobolomyces salicinus CBS 6983 T AF189995 AF444511 5

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tsugae D1/D2 region variation is 7/621, ITS is 81/652. Toavoid alignment di⁄culties between major phylogeneticgroups in the ITS tree, we followed the D1/D2 system ofclades (Figs. 1 and 2) and presented the data as individuallineages rather than as full trees.

3.1. Hymenomycetes

3.1.1. Tremellales (Fig. 3)As a preliminary exploration of the phylogenetic rela-

tionship of the traditional yeast genera with the dimorphicgenus Tremella, we included 20 species of Tremella in Fig.3. This number is insu⁄cient for a comprehensive study asBandoni [26] estimated that Tremella includes 120 or morespecies. Moreover, several other dimorphic genera are cur-rently recognized in the Tremellales [27]. Consequently,this incomplete sampling may account for the lack ofwell-supported clades and for the di¡erences in brancharrangements and species relationships in the ITS andD1/D2 Tremellales trees. For example, Bullera dendrophilaand Cryptococcus heveanensis appear related to Filobasi-diella in the ITS tree, but not in the D1/D2 tree. Similarly,Sterigmatosporidium polymorphum is related to Fellomycesand Kockovaella species in the D1/D2 and 18S [28] trees,but does not belong to a speci¢c clade in the ITS tree.Kirschner et al. [29] recently proposed the teleomorph ofSterigmatosporidium (Cuniculitrema polymorpha) based onthe observation of Tremella-type basidia in a dimorphicfungus associated with tunnels of bark beetles.Tremella spp. are generally separated from the yeasts

and appear, in the ITS tree, as four major clades sensuChen [20] : Foliacea, Fuciformis, Mesenterica and Auran-tia. In the D1/D2 tree, the Fuciformis clade is part of alarger Mesenterica clade. Cryptococcus skinneri (Foliaceaclade) is the only yeast species that appears to be relatedto Tremella spp. C. skinneri has been isolated from slime£uxes and insect frass associated with trees, which suggestsa similar habitat as the Tremella spp. The position of themembers of the Indecorata clade, which was indicated in

the trees of Chen [20] and Fell et al. [2], is di¡erent oneach tree, therefore systematic placement must await addi-tional data. Representatives of other dimorphic tremella-ceous fungi, Fibulobasidium, Sirobasidium and Trimorpho-myces, are scattered on both trees in isolated positions.H. corniformis, which appears related to two unnamedstrains of Cryptococcus (CBS 7712 and CBS 8804), is incontradictory positions in the D1/D2 and ITS trees. TheD1/D2 trees (Figs. 1 and 3) suggest that the Holtermanniaclade may represent a separate lineage in contrast to itsposition in the ITS Luteolus clade (bootstrap support100%).There are four distinguishable yeast clades in the Tre-

mellales D1/D2 and ITS trees: Fellomyces, Bulleromyces,Luteolus and Filobasidiella. The Fellomyces clade includesspecies of Fellomyces and Kockovaella. The presence ofconidia borne on elongate conidiophores is a commoncharacteristic in this clade. The two genera di¡er by theformation of ballistoconidia in Kockovaella. This morpho-logical characteristic is a useful taxonomic tool, howeverthe phylogenetic signi¢cance can be questioned.The Bulleromyces clade, which is weakly supported in

both trees, requires a detailed study of the biology andmolecular systematics of the included species and strains.Cryptococcus laurentii is a speci¢c example. Sugita et al.[10] reported genetic diversity in the ITS and D1/D2 re-gions among clinical strains of C. laurentii, suggesting ge-netically distinct species, which may or may not corre-spond to unstudied synonyms of C. laurentii [19]. Sexualcycles also deserve attention, as mating has been reportedbetween strains of C. laurentii [30]. Production of ballisto-conidia is variable within the clade. Moreover, Cryptococ-cus cellulolyticus and Bullera pseudoalba have identicalD1/D2 and ITS sequences, which would suggest conspeci-¢city and the loss of ballistoconidial formation by the typestrain of the former species.The Luteolus clade includes Cryptococcus luteolus, sev-

eral species of Bullera and the genus Dioszegia, which hasbeen resurrected by Takashima et al. [28]. The basis for

Table 2 (Continued).

Strain Strain number GenBank D1/D2 GenBank ITS Fig.

Sporobolomyces sasicola CBS 7285 T AF177412 AF444548 8Sporobolomyces singularis CBS 5109 T AF189996 AF444600 7Sporobolomyces subbrunneus CBS 7196 T AF189997 AF444549 8Sporobolomyces taupoensis CBS 7898 T AF177413 AF444592 8Sporobolomyces tsugae CBS 5038 T AF189998 AF444580 7Sporobolomyces xanthus CBS 7513 T AF177414 AF444547 8Sporobolomyces sp. CBS 6166 AF444724 AF444603Sterigmatomyces elviae CBS 5922 T AF177415 AF444551 8*Rhodotorula acuta CBS 7053 T AF189999 AF444512 ^*Rhodotorula dulciaminis1 CBS 7288 T AF190000 AF444517 ^

Sterigmatomyces halophilus CBS 4609 T AF177416 AF444556 8

Compared to the type strain: 1identical in D1D2, one substitution in ITS; 2one substitution in D1D2, identical in ITS; 3one substitution in D1D2, onesubstitution in ITS; 4identical in D1D2, two substitutions in ITS; 5one substitution in D1D2, two substitutions in ITS; 6one substitution in D1D2,three substitutions in ITS; 7two substitutions in D1D2, two substitutions in ITS; 8identical in D1D2, ¢ve substitutions in ITS; 9three substitutions inD1D2, 14 substitutions in ITS.

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the generic emendation is the occurrence of Bullera crocea,B. aurantiaca, B. armeniaca and Cryptococcus hungaricusin a separate clade in the ITS and 18S phylogenetic trees.Apart from the production of orange-pigmented colonies,the authors were unable to distinguish Dioszegia fromother anamorphic genera based on other phenotypic char-acteristics. Takashima et al. [28] dismissed ballistoconidialformation as a signi¢cant taxonomic characteristic at thegeneric and species levels ; they placed C. hungaricus andB. armeniaca in synonymy as Dioszegia hungarica. Despitethe morphological di¡erences, those two species have iden-tical sequences in the ITS and D1/D2 regions and 83%nDNA relatedness [28]. Ga¤cser et al. [31] reported, basedon D1/D2, ITS and partial 18S analyses, genetic diversityamong strains of C. hungaricus, which would suggest thepresence of additional taxa in the genus Dioszegia. Thismight also be the case with Cryptococcus sp. CBS 8925based on the D1/D2 and ITS trees (Fig. 3), which suggestsinclusion of that strain in Dioszegia.

Synonymy has been reported for Bullera derxii and Bul-lera sinensis, based on classical systematics and identicalD1/D2 sequences [32], and on 82^91% relative binding inDNA^DNA reassociation experiments [33]. In contrast,the two species di¡er by 12 bp in the SSU [33] and by6 bp (transitions:transversions (ti :tv) 4:2) in the ITS re-gion, which demonstrates the presence of genetic di¡er-ences between the two taxa. The latter authors have pro-posed the new combination B. sinensis var. lactis for thetype strain of Bullera alba var. lactis CBS 7237 based onintermediate values (40^50%) of DNA homology withB. sinensis and B. derxii ; nucleotide di¡erences betweenCBS 7237 and the type strains of the latter species wereone in D1/D2 and 11 or ¢ve in ITS.C. marinus, which is loosely attached to the Luteolus

clade in the D1/D2 tree, is known from a single marineisolate. Unfortunately we were unable to obtain an ITSsequence to ascertain the position in the ITS tree. TheD1/D2 consensus tree (Fig. 1) suggests that C. marinus is

Fig. 3. Tremellales. Phylogenetic analysis (PAUP 4 0b8) of the D1/D2 region of the large subunit rDNA. One of 100 equally parsimonious trees.NC=617, CC= 317, PUC=59, PIC=241, TL=1588, CI= 0.297, RI= 0.674. ITS: phylogenetic analysis (PAUP 4 0b8) of the ITS regions and 5.8S.Eleven equally parsimonious trees. NC=641, CC=208, PUC=34, PIC= 399, TL= 3179, CI = 0.297, RI= 0.688. Numbers on branches are the bootstrappercentages (s 50%) from 100 full heuristic replications. Trichosporon cutaneum, Trichosporon jirovecii and Trichosporon moniliiforme were used as theoutgroup. Due to space considerations the outgroup is not shown in this ¢gure. T= type strain.

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phylogenetically distinct from other clades in the Tremel-lales.The Filobasidiella clade, which includes Filobasidiella

bacillispora, Filobasidiella neoformans, Cryptococcus amy-lolentus and Tsuchiyaea wing¢eldii, has strong support inthe ITS and D1/D2 trees. The latter two species havesimilar ecological habitats as they were collected from in-sect frass in plants in South Africa [34,35]. They haveidentical ITS sequences, di¡er by one transversion basechange in the D1/D2 region and are morphologicallydistinct. T. wing¢eldii produces conidia on stalk-like co-nidiophores on solid medium [35,36], a characteristic notreported for C. amylolentus [34]. However, the micromor-phological investigations of Sampaio et al. [37] demon-strated that the cellular outgrowths in T. wing¢eldii aredistinct from the typical stalks of species of Fellomyces,Kurtzmanomyces or Sterigmatomyces. Considering theclose phylogenetic relationship between the two species,their taxonomic status should be evaluated by DNA hy-bridizations. The other two species in the clade, F. neofor-mans and F. bacillispora, are closely related; they are thecausative agents of cryptococcosis in man, although themajority of the infections are attributed to F. neoformans.DNA relatedness between the two taxa is 55^63% [38] andbase position di¡erences between the type strains of thetwo taxa are: 1/593 nucleotides in the D1/D2 (ti :tv 1:0),4/529 in the ITS (ti :tv 3:1) and approximately 454/2057

(258:196) in the intergenic spacer (IGS) regions ([39],Diaz, M.R., personal communication).

3.1.2. Trichosporonales (Fig. 4)The genus Trichosporon, which is the major constituent

of the Trichosporonales, is characterized by the presenceof true mycelium that disarticulates to arthroconidia. Gue¤-ho et al. [40] recognized 19 species in the genus, a numberthat was expanded to 25 by Middelhoven et al. [41]. Sugitaand Nakase [6], based on SSU analysis of 20 taxa, re-ported the presence of three groups of species within thegenus Trichosporon. Our study, with an expanded number(33) of taxa and unidenti¢ed strains, concurs with theidentity of these three groups, which correspond to ourinformal designations of the Gracile (Sugita and Nakasegroup III), Cutaneum (group I) and Ovoides (group II)clades. We recognize a fourth clade, Hyalodendron, whichwas represented in the SSU tree of Sugita and Nakase as abranch separate from the three groups.The relationship of Hyalodendron to Trichosporon has

been recognized [42,43], although Hyalodendron is consid-ered to be a hyphomycete, due to the formation of drycolonies with true hyphae that may locally disarticulate.The morphology displayed by Hyalodendron may not beunusual in this clade. T. porosum, which belongs to thesame clade, was originally described in the genus Apiotri-chum. Investigation of the type strain of A. porosum (CBS

Fig. 4. Trichosporonales. D1D2: 88 equally parsimonious trees. NC=610, CC=441, PUC=52, PIC=117, TL=392, CI= 0.523, RI= 0.759. ITS: 100equally parsimonious trees. NC=589, CC=351, PUC=59, PIC=179, TL=530, CI= 0.655, RI= 0.853. 100 heuristic replications. Outgroup=C. amylo-lentus, F. neoformans var. neoformans and C. heveanensis. See Fig. 1 for abbreviations.

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2040) revealed few budding yeast-like cells, which is amorphological characteristic of Hyalodendron. Transferof A. porosum to Trichosporon was based on the discoveryby Middelhoven et al. [41] of yeast strains with typicalarthrosporulation and identical ITS and D1/D2 sequencesto A. porosum.Two species of Cryptococcus are among the Trichospo-

ronales: C. curvatus, which is weakly related to the Cuta-neum clade, and C. humicola. Sugita et al. [11] demon-strated that strains phenotypically similar to C. humicolarepresent a species complex, which (Figs. 1 and 4) mayconstitute a sister clade to the Trichosporonales.Comparison of the ITS and D1/D2 trees in the Tricho-

sporonales lineage (Fig. 4) demonstrated more variabilityin the D1/D2 region than in the ITS region. For example,no di¡erences were observed in ITS sequence alignmentsbetween Trichosporon laibachii and Trichosporon multispo-rum, with seven di¡erences (ti :tv 6:1) in the D1/D2 re-gion; Trichosporon montevideense and Trichosporon domes-ticum were identical in ITS and di¡ered at two positions(1:1) in D1/D2. SSU analyses also demonstrated nucleo-tide position di¡erences within those pairs of species [6].

3.1.3. Filobasidiales (Fig. 5)Physiological, biochemical and molecular (D1/D2) char-

acteristics of species within the Filobasidiales were pre-

sented by Fonseca et al. [44]; that reference should beconsulted for a detailed review. Our ITS and D1/D2data demonstrate four clades within the Filobasidiales lin-eage: Aerius, Floriforme (=Magnus clade in Fonseca etal. [44]), Albidus and Cylindricus.In the Aerius clade, Cryptococcus terreus, Cryptococcus

himalayensis, Cryptococcus elinovii, PYCC 2935 andPYCC 4966 (Table 1) appear to represent a single specieswhen compared on the basis of physiological propertiesand whole-cell protein patterns [44]. These strains, how-ever, di¡er from each other by one to three D1/D2 nucleo-tides and one ITS bp. This list of synonyms is provisional,pending further study of other genes and DNA hybridiza-tion experiments. Cryptococcus phenolicus, which di¡eredfrom C. terreus at three D1/D2 bp (ti :tv 3:0) and two ITSbp (0:2), has been designated as a separate species fromC. terreus based on the ability of C. phenolicus to utilizesucrose, melibiose and ra⁄nose [44]. Another strain, CBS7944, had ITS (1:2) and D1/D2 (2:0) di¡erences fromC. terreus ; however, there were no signi¢cant di¡erencesin assimilation tests between the two strains. The taxo-nomic status of CBS 7944 requires additional study. Com-parison of the Aerius clade in the ITS and D1/D2 trees(Fig. 5) indicates greater sequence variability in the D1/D2than in the ITS region; for example, C. terreus di¡ersfrom Cryptococcus fuscescens at 10 D1/D2 (10:0) nucleo-

Fig. 5. Filobasidiales. D1D2: 100 equally parsimonious trees. NC=611, CC=441, PUC=19, PIC=151, TL= 408, CI= 0.532, RI = 0.832. ITS: 100equally parsimonious trees. NC=694, CC= 317, PUC=36, PIC= 341, TL=1057, CI= 0.605, RI= 0.859. 100 heuristic replications. Outgroup=T. cuta-neum, T. jirovecii and T. moniliiforme. See Fig. 1 for abbreviations.

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tide positions and three ITS (1:2) positions. Di¡erences inphysiology between the two species amount to more than10 assimilation tests, e.g. Fonseca et al. [44].The Floriforme clade contains several species of the tele-

omorphic genus Filobasidium and anamorphic species inCryptococcus. As noted by Fell et al. [2], the biologicallydistinct species Cryptococcus magnus, F. £oriforme andF. elegans are indistinguishable in the D1/D2 region,whereas signi¢cant di¡erences are present in the ITS re-gion: C. magnus and F. elegans 21/627 (10:11); C. magnusand F. £oriforme 15/627 (9:6); and F. £oriforme andF. elegans 20/650 (12:8). All of the species of Filobasidiumare in this clade, except F. capsuligenum, which di¡ersfrom other species of Filobasidium by the ability to fer-ment glucose and possibly by morphology. Ultrastructuralstudies of F. capsuligenum demonstrated that the hyphalsepta have cone-shaped vesicular parenthesomes, whereasF. £oriforme hyphae lack parenthesomes [45,46].Strains in the Albidus clade are widely distributed in

nature and many of the species have been collected frompolar regions. Some species, such as Cryptococcus albidi-similis and Cryptococcus adeliensis, have been the subjectof cold-adapted enzyme research [47,48]. Sequence vari-ability in the D1/D2 region is, in general, greater than inthe ITS region. For example, the di¡erences betweenC. di¥uens and C. albidisimilis are D1/D2 9/612 (6:3),ITS 1/621 (0:1), Cryptococcus liquefaciens and C. albidisi-milis D1/D2 8/612 (7:1), ITS 0/621.The Cylindricus clade contains only one named species,

viz. Cryptococcus cylindricus and several undescribed

Cryptococcus species represented by single strains. Thesestrains have been isolated from lichens and fruiting bodiesof fungi and share some physiological characteristics withmembers of the Aerius and Floriforme clades. However,no particular features were found to separate these speciesfrom other members of the Filobasidiales. Therefore, adecision on their taxonomic position requires additionalbiological information.

3.1.4. Cysto¢lobasidiales (Fig. 6)The four bootstrap-supported clades in the Cysto¢loba-

sidiales lineage (Mrakia, Cysto¢lobasidium, Udeniomycesand Xanthophyllomyces) have extensive biological dif-ferences. The genera Mrakia and Cysto¢lobasidium formteliospores with holometabasidia. Udeniomyces is an ana-morphic ballistoconidia-forming genus of plant-associatedspecies that were originally described in the genus Bullera.Xanthophyllomyces produces an elongate holobasidiumfollowing cell-bud conjugation. The genus Pha⁄a repre-sents an anamorphic state of Xanthophyllomyces. Based onITS and IGS analyses [15], Pha⁄a rhodozyma is a speciesdistinct from Xanthophyllomyces dendrorhous.The species Cryptococcus aquaticus and Cryptococcus

macerans are included respectively in the Mrakia and Cys-to¢lobasidium clades. Mating between strains of C. ma-cerans has been reported but a complete sexual cycle (ex-pected to resemble Cysto¢lobasidium) was not observed[49]. Other anamorphic species among the Cysto¢lobasi-diales include Cryptococcus huempii and Trichosporon pul-lulans, which are weakly associated with the Mrakia and

Fig. 6. Cysto¢lobasidiales. D1D2: 78 equally parsimonious trees. NC=608, CC=428, PUC=28, PIC= 152, TL=355, CI = 0.704, RI = 0.864. ITS: twoequally parsimonious trees. NC=818, CC=388, PUC=64, PIC= 366, TL=1050, CI= 0.642, RI= 0.777. 100 full heuristic replications. Outgroup=C. fuscescens, Cryptococcus aerius and Cryptococcus terricola. See Fig. 1 for abbreviations.

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Udeniomyces clades. Little is known about C. huempii,other than the description of a single strain collected ina rain forest in Chile [50]. T. pullulans has distinct mor-phology (lack of septal pores and enteroblastic budding)and physiology (low optimum temperature of growth at17‡C, assimilation of nitrate and absence of growth withlactate) from other species in the genus Trichosporon [40].Sequence variability between species of the Cysto¢loba-

sidiales is, in general, greater in the ITS than D1/D2 re-gions. For example, Mrakia frigida and Mrakia gelida areidentical in the D1/D2 region and di¡er by 15/64 (ti :tv11:4) in ITS. X. dendrorhous and P. rhodozyma di¡er by1/636 (1:0) in D1/D2 and 8/712 (6:2) in ITS.

3.2. Urediniomycetes

3.2.1. Erythrobasidium (Fig. 7)The Erythrobasidium lineage includes genera with three

distinct life cycles. Occultifur externus, isolated from plantlitter, is a homothallic species that produces four-celledtransversely septate basidia, with forcibly discharged basi-diospores, directly from generative hyphae [51]. Sakagu-chia dacryoidea, which has been isolated from such dispa-rate locales as the open ocean and a brewery in France,has a heterothallic, bifactorial incompatibility system thatincludes the formation of teliospores and phragmometa-basidia following mating [52]. Erythrobasidium hasegawia-num was described from a single strain collected from anold beer yeast culture. The sexual state is reported as ho-mokaryotic, with a holobasidium that arises directly on

clamped, true mycelium [53]. However, this life cycle re-quires further examination in view of the Sampaio et al.[51] report that the mycelium has incomplete clamp con-nections and the interpretation that the alleged holobasi-dia are conidiogenous cells. All members of this lineageproduce deeply pigmented orange or red colonies. Perrieret al. [54] found qualitative di¡erences in the carotenoidspresent in Rhodotorula spp. belonging to either the Ery-throbasidium or the Sporidiobolus clades. Another distin-guishing feature of members of the Erythrobasidium line-age has been found by Takashima et al. [55] who reportedthe absence of fucose in the cell walls of all taxa inthis lineage when compared to other urediniomycetousyeasts.The Erythrobasidium lineage includes three major

clades, Occultifur, Aurantiaca and Gracilis, which are sup-ported by bootstrap analysis in the ITS and D1/D2 re-gions. O. externus is the only sexual species speci¢callyassociated with one of these clades. The other two cladesare composed of species of Rhodotorula and Sporobolomy-ces. Separation of the majority of the species in the Ery-throbasidium lineage can be accomplished with either ITSor D1/D2 analyses. An exception is S. oryzicola and S. co-prosmae, which require ITS analysis for identi¢cation(D1/D2: 0/629, ITS: 4/599 (1:3)). The two species di¡erin their ability to utilize 2-keto-D-gluconate, which is con-sidered by Boekhout and Nakase [56] to be a signi¢canttaxonomic character. DNA homology between the twospecies was found to be ca. 50% [57], which suggeststhat the two taxa are sibling species.

Fig. 7. Erythrobasidium. D1D2: one parsimony tree. NC=589, CC=387, PUC=21, PIC= 181, TL=415, CI= 0.639, RI= 0.840. ITS: two equally parsi-monious trees. NC=721, CC=360, PUC=88, PIC=273, TL=847, CI = 0.616, RI= 0.734. 100 heuristic replications. Outgroup=S. xanthus, S. taupoen-sis and Sporobolomyces sasicola. See Fig. 1 for abbreviations.

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3.2.2. Sporidiobolus (Fig. 8)The Sporidiobolus lineage includes species of the te-

liosporic genera Rhodosporidium and Sporidiobolus andtheir anamorphic counterparts in the genera Rhodotorulaand Sporobolomyces. All the species usually produce pig-mented colonies. The lineage is divided into four sup-ported clades Glutinis, Sphaerocarpum, Ruineniae andJohnsonii. Members of the Sporidiobolus clade havebeen reviewed by Gadanho and Sampaio [58], Gadanhoet al. [59] and Vale¤rio et al. [21]. The consensus of thisinformation demonstrated that the Glutinis clade, sensuGadanho and Sampaio [58] consisted of the two cladesthat we designated as Glutinis and Sphaerocarpum. OurD1/D2 tree concurs with the Gadanho and Sampaio con-cept, however the di¡erence in branch arrangements be-tween the ITS and D1/D2 trees prompted additional no-menclature.The Glutinis and Sphaerocarpum clades consist of spe-

cies of Rhodosporidium and Rhodotorula, the Johnsoniiclade includes species of the ballistoconidia-forming gen-era Sporidiobolus and Sporobolomyces, whereas the Rui-neniae clade is mixed. This general separation lends sup-port to the continued use of ballistoconidia as ataxonomic tool as argued by Gadanho et al. [59], althoughquestioned in the separation of species of Cryptococcusand Bullera in the Tremellales (see above).Some species within the Sporidiobolus lineage are closely

related based on sequence analysis. For example, Rhodo-torula glutinis and Rhodotorula graminis are identical inthe ITS region and di¡er by one transverse substitution

in the D1/D2 region. The two species have been shown tobe distinct on standard physiological properties [60] andon the basis of 30% nDNA relatedness [58,61]. The typestrains of Rhodosporidium babjevae and R. glutinis di¡er inthe ITS by 4/609 (ti :tv 3:1) and D1/D2 by 2/604 (2:0) ;nDNA hybridizations of strains of these two species dem-onstrate 25^61% relatedness [58]. In apparent contrast,Sporobolomyces holsaticus and Sporidiobolus johnsonii areidentical in D1/D2, di¡er at 5 bp (4:1) in ITS and register93% DNA hybridization [62]. Consequently, in that line-age, both the ITS and D1/D2 regions are required forspecies identi¢cation.

3.2.3. Microbotryum (Fig. 9)The Microbotryum ITS and D1/D2 trees are notable for

their lack of structured bootstrap-supported clades. Onereason is an incomplete sampling of taxa, namely the di-morphic plant parasites of the Microbotryales, sensuBauer et al. [63] for which ITS sequences are not available.For the purpose of orientation, we labeled two weakclades Colacogloea and Leucosporidium. There are twosexual states among yeasts in this lineage, speci¢cally inthe teliospore-forming genera Leucosporidium and Masti-gobasidium. The distinguishing features of Mastigobasidi-um are the formation of ballistoconidia and the mecha-nism of teliospore germination, in which elongatehyphae with curved phragmometabasidia form [64]. Sep-tate metabasidia in Leucosporidium are generally notcurved and develop directly from the teliospore or onshort hyphal extensions [65].

Fig. 8. Sporidiobolus. D1D2: 24 equally parsimonious trees. NC=599, CC= 426, PUC=44, PIC=129, TL= 386, CI= 0.560, RI= 0.837. ITS: ¢veequally parsimonious trees. NC=607, CC=326, PUC=54, PIC=227, TL=741, CI= 0.571, RI= 0.806. 100 full heuristic replications. Outgroup=R. bu¡onii, R. pustula and Rhodotorula bogoriensis. See Fig. 1 for abbreviations.

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Yeast species in the Microbotryum lineage include rep-resentatives of the anamorphic genera Rhodotorula, Spo-robolomyces, Reniforma and Bensingtonia. The vegetativecells of Reniforma are unique among the yeasts; they arekidney-shaped with an elaborate skirt that circles the edgeof the cell [66]. R. strues appears on a long branch withHeterogastridium pycnidiodeum (teleomorph name, ana-morphic name=Hyalopycnis blepharistoma), a relationshipthat may be arti¢cial due to long branch attraction. Thisrelationship is not con¢rmed in the Fig. 1 consensus tree,which suggests that R. strues may represent a separatelineage. Similarly, S. griseo£avus, which is unsupportedin any of the ITS and D1/D2 clades, may represent aseparate lineage as suggested by the Fig. 2 consensus tree.In the Microbotryum lineage, species can be separated

with either the ITS or D1/D2 region, however greater se-quence variability was found in the ITS region, for exam-ple Sporobolomyces singularis and Rhodotorula lignophiladi¡erences are: D1/D2 1/619 (ti :tv 1:0) and ITS 12/628(9:3) ; R. pustula and Rhodotorula bu¡oni D1/D2 3/592(1:2) and ITS 31/617 (17:14). Synonymy between Rhodo-torula fujisanensis, Rhodotorula futronensis and Rhodoto-rula nothofagi has been suggested by Sampaio and Fon-seca [67] based on similarity of phenotypic data. Thathypothesis is corroborated for R. fujisanensis and R. fu-tronensis on the basis of identical D1/D2 and ITS sequen-ces, but not for R. nothofagi (D1/D2 2:1; ITS 6:1). Aformal taxonomic proposal requires con¢rmation byDNA hybridization experiments and additional informa-tion on the sexual cycle of R. fujisanensis [60]. Anotherstrain in this lineage, CBS 6370, is currently included in

Rhodotorula foliorum (CBS Yeast Database), althoughD1/D2 and ITS sequence data suggest that CBS 6370 rep-resents a distinct species, as demonstrated with the proteindata of Vancanneyt et al. [68].

3.2.4. Agaricostilbum (Fig. 10)The Agaricostilbum lineage includes small, bootstrap-

supported clades, which we informally labeled as Kondoa,Agaricostilbum, Sasicola, Kurtzmanomyces and Subbrun-neus. A general morphological feature in this lineage is thepresence of blastoconidia on denticles or on short to longconidiophores that can develop sympodially, particularlyamong some species of Bensingtonia and Sporobolomyces.Sterigmatomyces produces stalk-like conidiophores thatseparate in the mid-region for bud release. In contrast,Kurtzmanomyces bud release is at the terminus of thestalk. Ballistoconidia are present in Kurtzmanomyces inso-litus [37] and species of Bensingtonia and Sporobolomyces.Separation of the latter two genera is based on the majorubiquinone (CoQ) present: CoQ9 in Bensingtonia andCoQ10 in Sporobolomyces. Analysis of the trees depictedin Fig. 10 shows that there is no phylogenetic basis for thisseparation. Furthermore, two other species of Bensingtoniabelong to the Microbotryum lineage, viz. Bensingtonia ya-matoana and Bensingtonia intermedia (anamorph of Mas-tigobasidium intermedium). A taxonomic rearrangement ofthose two genera that re£ects their phylogeny is hamperedby the ambiguous connection to other genera as revealedby the sequence data. The solution to this problem willrequire additional biological information, namely the ob-servation of sexual cycles in these clades.

Fig. 9. Microbotryum. D1D2: 100 equally parsimonious trees. NC=611, CC=362, PUC=78, PIC=171, TL=767, CI= 0.454, RI = 0.665. ITS: twoequally parsimonious trees. NC=728, CC= 244, PUC=95, PIC=389, TL=2711, CI = 0.362, RI= 0.571. 100 full heuristic replications. Outgroup=R. di-obovatum, Rhodotorula araucariae and R. paludigenum. See Fig. 1 for abbreviations.

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The Kondoa clade contains the only known yeast sexualcycle in the Agaricostilbum lineage. Kondoa spp. produceauricularioid basidia directly on the hyphae in the absenceof teliospores [69]. The namesake of the lineage, sensuSwann and Taylor [23^25], Agaricostilbum hyphaenes, re-sides in the Agaricostilbum clade. A. hyphaenes is a dimor-phic fungus that inhabits palms and produces synnemata-like basidiomata composed of long auricularioid basidiawith passively abstricted basidiospores. The other teleo-morphic genus in the lineage is Chionosphaera, which isalso dimorphic and loosely linked to the Kurtzmanomycesclade. The sexual state of Chionosphaera can arise on cul-ture media with formation of holobasidia of the Filobasi-dium-type.Species in the Agaricostilbum lineage can be identi¢ed

by either the D1/D2 or ITS regions, however di¡erencesare usually more pronounced in the ITS region. For ex-ample, Sporobolomyces xanthus and Sporobolomyces tau-poensis D1/D2 is 4/623 (ti :tv 4.0), ITS 16/578 (9:7) orKondoa aeria and PYCC 5566 D1/D2 1/638 (0:1), ITS34/635 (17:17). The latter represents an unnamed Bensing-tonia sp., which can be distinguished from K. aeria bythe presence of ballistoconidia, the absence of a sexualcycle and di¡erences in assimilation tests (Fonseca, A.and Inacio, J., unpublished data).

3.3. Sequence identity/variability between mating strains

Sequence variability between strains within a specieswas examined by comparison of ITS and D1/D2 sequences

of mating types for 14 species (Table 3). The results dem-onstrated identical ITS and D1/D2 sequences for matingstrains within Bulleromyces albus, Bulleromyces sp., Filo-basidium uniguttulatum, M. intermedium, R. babjevae, Rho-dosporidium sphaerocarpum and Rhodosporidium paludige-num. Sequence di¡erences between mating strains wereobserved ranging from one single base change in the ITSor D1/D2 regions to multiple base changes in both re-gions. For example, in Rhodosporidium kratochvilovaeone mating strain (PYCC 4785) di¡ered by one transver-sion in ITS. Single base di¡erences could be attributedto errors during strand synthesis or sequence analysis.However, PCR reactions, sequencing and analysis wererepeated to con¢rm the di¡erences. Several species dem-onstrated greater sequence di¡erences: C. macerans, F. cap-suligenum, Rhodosporidium diobovatum, S. dacryoidea,Sporidiobolus pararoseus and Sporidiobolus salmonicolor(Table 3). For example, mating strains of F. capsuligenumhad eight base changes in D1/D2 and 10 in ITS. Thenumber of transitions and transversions in the D1/D2and ITS regions for mating strains was found to be inthe same range as among closely related species. Strainsof S. salmonicolor (CBS 490) and S. johnsonii (CBS 2630)had a concentrated region of eight nucleotide changes andtwo nucleotide indels in a 10-bp region in the ITS2 region.These ‘hot spots’ of nucleotide changes were found infre-quently among closely related species and there did notappear to be a particular pattern for the location of the‘hot spots’ or other base di¡erences either among matingstrains or closely related species. Unfortunately, the ancil-

Fig. 10. Agaricostilbum. D1D2: six equally parsimonious trees. NC=640, CC=305, PUC=43, PIC=292, TL=1050, CI= 0.513, RI= 0.751. ITS: oneparsimony tree. NC=758, CC= 197, PUC=61, PIC=500, TL= 2388, CI= 0.469, RI= 0.627. 100 full heuristic replications. Outgroup=Sporobolomycessalicinus, Rhodotorula aurantiaca and Sporobolomyces kluyveri-nielii. See Fig. 1 for abbreviations.

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lary data (fertility of the progeny from sexual recombina-tions and DNA hybridization analyses), required to ascer-tain the phylogenetic status of these sequence-distinct mat-ing strains, are not available.

4. Discussion

Sequence analysis of either the D1/D2 or ITS regionscan be used for the identi¢cation of the majority of thespecies of basidiomycetous yeasts. Examples (C. magnusand F. elegans) have been presented where certain closelyrelated species could not be separated with the D1/D2region, but they could be di¡erentiated with the ITS re-gion. Other examples in the Filobasidiales and Trichospo-ronales lineages demonstrated the opposite situation, inspite of the expected greater variability in the ITS region,which is less constrained for mutations. As found betweenmating strains, the D1/D2 and ITS regions showed a con-siderable variation in the ti :tv and in location of thechanges, so that there did not appear to be distinct loca-tions within or between D1 and D2 or ITS1 and ITS2regions. Hotspot sites of mutation were found in the 3PD1/D2 region among some strains, but a trend was notobserved. Although ti/tv rate ratios were not provided inthis study, the rates are known to vary with genes andlineages. However, biological explanations for these varia-

tions in rates have not been determined [70]. As a conse-quence, a boundary between species (a prerequisite num-ber of nucleotide di¡erences in either regions to separatespecies) was not de¢ned by our study. The answer un-doubtedly lies not in the number of di¡erences, but ratherin the signi¢cance and function of the site mutations.Studies, such as secondary structure [71], would provideinsight into the question. Consequently, when examiningpure number di¡erences between di¡erent pairs of species,the answer varies. R. glutinis and R. graminis are identicalin the ITS region and di¡er at one transition site in D1D2;they are considered to be separate species based on 30%nDNA relatedness. In contrast, S. johnsonii and S. holsa-ticus are synonyms due to 93% nDNA relatedness; theyare identical in the D1/D2 region and di¡er by 5 bp inITS.The question of molecular characterization of species is

further complicated by di¡erences in the D1/D2 and ITSregions (Table 3) between mating strains. In some cases,the number and distribution of nucleotide changes suggestthat the strains represent distinct intermating species. Animportant avenue to discover is the level of relationshipbetween mating strains. As a model, the in-depth fertilityand genetic studies of F. neoformans and F. bacillisporahave concluded that the two taxa, which di¡er by ti :tv3:1 in the ITS1 region, are separate species [39,72]. Similarstudies with sequence-divergent mating strains (Table 3)

Table 3Comparison of ITS and D1/D2 sequences of mating types

Species Mating strains Number of di¡erent nucleotidesbetween groups(transitions:transversions)

Bulleromyces albus CBS 501 mtB, CBS 500 mtA, CBS 6302 mtB NdBulleromyces sp. PYCC 5740 mtA1, PYCC 5690 mtA1, PYCC 5691 mtA2,

PYCC 5739 mtA2Nd

Cryptococcus macerans CBS 2206 mta, CBS 2425 mtK D1D2 (3:0); ITS (6:5)Filobasidium capsuligenum CBS 4736 mtK, CBS 6219 mtK* Nd

CBS 1906 mta, CBS 4381 mta NdThe two groups di¡er by: D1D2(6:2); ITS (6:3)*(CBS 6219 is di¡erent from all theother strains by a 13-bp deletion)

Filobasidium uniguttulatum CBS 1730 mta, CBS 1727 mtK NdMastigobasidium intermedium CBS 7226 mta, CBS 7281 mtK NdRhodosporidium babjevae CBS 7808 mtA1, CBS 9071 mtA2 NdRhodosporidium diobovatum CBS 6085 mtK, CBS 9076 mta Nd

CBS 9081 mta D1D2 (1:0); ITS (2.0)Rhodosporidium kratochvilovae PYCC 4776 mtA2, PYCC 4778 mtA2, PYCC 4793 mtA1,

PYCC 4818 mtA1, PYCC 4819 mtA2, PYCC 4883 mtA2,PYCC 5244 mtA1

Nd

PYCC 4785 mtA2 ITS (0:1)Rhodosporidium paludigenum CBS 6566 mtA2 and CBS 6567 mtA1 NdRhodosporidium sphaerocarpum CBS 5939 mta, CBS 9082 mtA, CBS 9075 mta, CBS 9079 mta,

CBS 9080 mtaNd

Sakaguchia dacryoidea CBS 6353 mtA1B1, CBS 6356 mtA1B2 NdCBS 7999 mtA2B1 D1D2 (1:2); ITS (8:6)

Sporidiobolus pararoseus CBS 484 mtA1, CBS 491 mtA2 D1D2 (0:1); ITS(2:0)Sporidiobolus salmonicolor/johnsonii CBS 490 mtA1, CBS 2630 mtA2 D1D2 (7:0); ITS (20:6)

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o¡er an opportunity to examine the role of sequence anal-ysis in yeast systematics. The problem is exempli¢ed bymating strains of CBS 490 (S. salmonicolor) and CBS2630, which di¡er at seven positions in the D1/D2 and26 in the ITS. The sequence alignment of one of thesemating strains, CBS 2630, indicates a close relationshipto the homothallic species S. johnsonii (CBS 5470). Thelatter two strains are identical in D1/D2 and di¡er at¢ve positions in ITS. Mating between those strains hasnot been observed.The ITS and D1/D2 tree structures are not fully re-

solved as few clades show strong support, which resultsin insu⁄cient evidence for nomenclatural decisions, partic-ularly at the generic level. Consequently, clades have in-formal names and consist of species of di¡erent genera.However, the discovery of basidiomycetous yeast species isin a primordial phase as possibly only 1% of the species innature have been collected and described. These phyloge-netic trees are dynamic, therefore lineages and clades willsplit, and as new phylogenetic groups appear the branch-ing arrangements will change and generic identities andrelationships should become evident. At the present pointof discovery, the trees provided by ITS, D1/D2 and othergenes [73,74] provide focal points for biological and sys-tematic analysis of these yeasts. A point to recognize isthat the biology of the species must be the primary crite-rion in yeast systematics. A sequence, per se, does notdescribe a species and descriptions of new species shouldnot solely rely on nucleotide data.

Acknowledgements

This research was supported by funding from the OceanSciences Division, National Science Foundation and fromGenetic Vectors, Inc., Miami, FL, USA. David Yarrow,C.P. Kurtzman, Helen Vischniac and Brian Ste¡enson areacknowledged for providing strains and information re-garding these strains. J.P. Sampaio and T. Boekhoutalso provided critical information.

References

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