Abstract

Basidiomycetous yeasts in the Urediniomycetes and Hymenomycetes were examined by sequence analysis in two ribosomal DNA regions: the D1/D2 variable domains at the 5′ 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 the Urediniomycetes; Tremellales, Trichosporonales, Filobasidiales and Cystofilobasidiales in the Hymenomycetes. Bootstrap support for many of the clades within those lineages is weak; however, phylogenetic analysis provides a focal point for in-depth study of biological relationships. Combined sequence analysis of the D1/D2 and ITS regions is recommended for species identification, while species definition requires classical biological information such as life cycles and phenotypic characterization.

Introduction

Molecular systematics of yeasts has emphasized either coding (D1/D2 variable domains of the large subunit or the complete small subunit (SSU)) or non-coding (internal transcribed spacers ITS1 and ITS2) regions of the ribosomal DNA. As a consequence, an extensive D1/D2 database [1,2] and partial SSU (e.g. [3–8]) and ITS databases (e.g. [9–12]) are available for the classification and identification of species. Due, in part, to the difference in size between the SSU (∼1700 bases) and the ITS (∼600 bases) and D1/D2 (∼600 bases), the latter two regions have received the most attention for yeast systematics. In viewing these two databases, the question arises: how accurate are either or both of these molecular regions for the delineation of basidiomycetous species? Fell et al. [2] provided a clue: they reported that the biologically distinct species Cryptococcus ater, Filobasidium elegans and Filobasidium floriforme, which could not be separated with D1/D2 analysis, were differentiated with ITS analysis. Other examples of separation of biologically distinct species requiring D1/D2 and/or ITS sequence analysis include Mrakia[13], Filobasidiella[2,14] and Phaffia/Xanthophyllomyces[15]. The ITS region has also been recommended for the identification of medically relevant yeasts [16,17]. Based on this information, we sequenced the ITS region for 450 strains of 242 species of basidiomycetous yeasts in the Urediniomycetes and Hymenomycetes. The following discussion presents a comparative view of species separations with the ITS and D1/D2 regions.

Materials and methods

Cultures were obtained from the Centraalbureau voor Schimmelcultures (CBS), Portuguese Yeast Culture Collection, New University of Lisbon (PYCC), U. S. Department of Agriculture, Peoria, IL, USA (NRRL Y), American Type Culture Collection (ATCC), Brian Steffenson, North Dakota State University (KB) and Helen Vishniac, Oklahoma State University (Cryptococcus consortionis). Species names, strains and GenBank numbers are provided in Tables 1 and 2. Strains and synonyms of species are indented under the type strain of the species. Taxonomic data of the described species and strains are available in Kurtzman and Fell [18] and Barnett et al. [19] and at the CBS web site (http://www2.cbs.knaw.nl/yeast/WebC.asp). Unidentified strains, potentially representing undescribed species, are included in the trees and tables. These strains, which are designated by genus and collection numbers, were included to illustrate the biodiversity within the clades. Formal description of these taxa will require a standard phenotypic study, which is not a part of this report.

1

List of hymenomycetous strains studied

Strain Strain number GenBank D1/D2 GenBank ITS Fig. 
Bullera alba var. lactis CBS 7237 T AF363648 AF444665 
Bullera coprosmaensis CBS 8284 T AF363660 AF444485 
Bullera dendrophila CBS 6074 T AF189870 AF444443 
Bullera derxii CBS 7225 T AF189857 AF444405 
Bullera globispora CBS 6981 T AF075509 AF444407 
Bullera hannae CBS 8286 T AF363661 AF444486 
Bullera miyagiana CBS 7526 T AF189858 AF444409 
Bullera oryzae CBS 7194 T AF075511 AF444413 
Bullera penniseticola CBS 8623 T AF363649 AF444471 
Bullera pseudoalba CBS 7227 T AF075504 AF444399 
Bullera sinensis CBS 7238 T AF189884 AF444468 
Bullera unica CBS 8290 T AF075524 AF444441 
Bullera variabilis CBS 7347 T AF189855 AF444403 
Bullera sp. CBS 2013 AF444760 AF444666 
Bulleromyces albus mtB CBS 501 T AF075500 AF444368 
Bulleromyces 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 
Bulleromyces 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 
Cryptococcus aerius CBS 155 T AF075486 AF145324 
*Torulopsis pseudoaeria CBS 4192 T AF181544 AF444376 – 
Cryptococcus albidisimilis CBS 7711 T AF137601 AF145325 
Cryptococcus albidisimilis3 ATCC 34633 AF137606 AF145331 – 
Cryptococcus albidus CBS 142 T AF075474 AF145321 
Cryptococcus 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 
Cryptococcus albidus var. kuetzingii1 CBS 922 AF181504 AF444313 – 
Cryptococcus albidus var. kuetzingii CBS 6086 AF181546 AF444378 – 
Cryptococcus albidus var. ovalis CBS 5810 T AF137605 AF145329 
Cryptococcus amylolentus CBS 6039 T AF105391 AF444306 
Cryptococcus antarcticus CBS 7687 T AF075488 AF145326 
Cryptococcus aquaticus CBS 5443 T AF075470 AF410469 
Cryptococcus arrabidensis CBS 8678 T AF181535 AF444362 
Cryptococcus bhutanensis CBS 6294 T AF137599 AF145317 
Cryptococcus cellulolyticus CBS 8294 T AF075525 AF444442 
Cryptococcus chernovii CBS 8679 T AF181530 AF444354 
Cryptococcus cylindricus CBS 8680 T AF181534 AF444360 
Cryptococcus cylindricus8 PYCC 5264 AF181533 AF444359 
Cryptococcus curvatus CBS 570 T AF189834 AF410467 
Cryptococcus curvatus CBS 8126 AF444717 AF444458 – 
Cryptococcus dimennae CBS 5770 T AF075489 AF410473 
Cryptococcus diffluens CBS 160 T AF075502 AF145330 
*Cryptococcus diffluens var. uruguaiensis CBS 6436 T AF181543 AF444375 – 
*Torulopsis albida var. japonica CBS 926 T AF181542 AF444374 – 
Cryptococcus flavus CBS 331 T AF075497 AF444338 
Cryptococcus friedmannii CBS 7160 T AF075478 AF145322 
Cryptococcus fuscescens CBS 7189 T AF075472 AF145319 
Cryptococcus gastricus CBS 2288 T AF137600 AF145323 
Cryptococcus gastricus5 CBS 1927 AF181501 AF444304 – 
Cryptococcus gilvescens CBS 7525 T AF181547 AF444380 
Cryptococcus heveanensis CBS 569 T AF075467 AF444301 
Cryptococcus huempii CBS 8186 T AF189844 AF444322 
Cryptococcus humicola CBS 571 T AF189836 AF410470 
Cryptococcus humicola5 CBS 8354 AF189851 AF444384 – 
Cryptococcus humicola5 CBS 8371 AF189854 AF444398 – 
Cryptococcus laurentii CBS 139 T AF075469 AF410468 
Cryptococcus liquefaciens CBS 968 T AF181515 AF444345 
Cryptococcus liquefaciens PYCC 2406 AF181513 AF444343 – 
Cryptococcus liquefaciens PYCC 2934 AF181518 AF444348 – 
Cryptococcus luteolus CBS 943 T AF075482 AF444323 
Cryptococcus macerans mta CBS 2206 T AF189848 AF444329 
Cryptococcus macerans mtα CBS 2425 AF075477 AF444317 
Cryptococcus magnus CBS 140 T AF181851 AF190008 
Cryptococcus 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 – 
Cryptococcus oeirensis CBS 8681 T AF181519 AF444349 
Cryptococcus oeirensis PYCC 5268 AF181537 AF444364 – 
Cryptococcus phenolicus CBS 8682 T AF181523 AF444351 
Cryptococcus podzolicus CBS 6819 T AF075481 AF444321 
Cryptococcus saitoi CBS 1975 T AF181540 AF444372 
*Naganishia globosa CBS 5106 T AF181539 AF444371 – 
Cryptococcus skinneri CBS 5029 T AF189835 AF444305 
Cryptococcus terreus CBS 1895 T AF075479 AF444319 
Cryptococcus terreus7 PYCC 2935 AF444692 AF444365 – 
Cryptococcus terreus7 PYCC 4966 AF444694 AF444367 – 
*Cryptococcus elinovii4 CBS 7051 T AF137604 AF145318 
*Cryptococcus himalayensis2 CBS 6293 T AF181502 AF410471 – 
Cryptococcus terricola CBS 4517 T AF181520 AF444350 
Cryptococcus terricola CBS 6435 AF181545 AF444377 – 
Cryptococcus uzbekistanensis CBS 8683 T AF181508 AF444339 
Cryptococcus victoriae CBS 8685 AF363647 AF444469 
Cryptococcus victoriae CBS 6550 AF444711 AF444447 – 
Cryptococcus victoriae6 CBS 8884 AF444741 AF444645 – 
Cryptococcus vishniacii CBS 7110 T AF075473 AF145320 
*Cryptococcus asgardensis CBS 8141 T AF189839 AF444310 – 
*Cryptococcus baldrensis CBS 8142 T AF189840 AF444311 – 
*Cryptococcus consortionis A801-3aY92/20 T AF189880 AF444464 – 
*Cryptococcus hempflingii 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 
Cryptococcus wieringae CBS 8353 AF181549 AF444383 – 
Cryptococcus sp. CBS 6984 AF444712 AF444448 
Cryptococcus sp. CBS 7712 AJ311450 AF408417 
Cryptococcus sp. CBS 7713 AJ311451 AF408418 – 
Cryptococcus sp. CBS 7743 AJ311452 AF408419 – 
Cryptococcus sp. CBS 7890 AF075494 AF444332 
Cryptococcus sp. CBS 7944 AF444693 AF444366 
Cryptococcus sp. CBS 8024 AF444715 AF444453 
Cryptococcus sp. 2 CBS 6024 AF444714 AF444452 – 
Cryptococcus sp. CBS 6123 AF444687 AF444333 – 
Cryptococcus sp. CBS 8355 AF444696 AF444385 
Cryptococcus sp. CBS 8356 AF444697 AF444386 
Cryptococcus sp. CBS 8367 AF444703 AF444394 – 
Cryptococcus sp. CBS 8358 AF444698 AF444387 
Cryptococcus sp. CBS 8366 AF444702 AF444393 – 
Cryptococcus sp. CBS 8363 AF444699 AF444390 
Cryptococcus sp. CBS 8364 AF444700 AF444391 
Cryptococcus sp. CBS 8365 AF444701 AF444392 – 
Cryptococcus sp. CBS 8368 AF444704 AF444395 – 
Cryptococcus sp. CBS 8372 AF444707 AF444410 
Cryptococcus sp. CBS 8369 AF444705 AF444396 
Cryptococcus sp. CBS 8507 AF444742 AF444646 
Cryptococcus sp. CBS 8804 AY006480 AY006481 
Cryptococcus sp. CBS 8924 AY029345 AY029346 
Cryptococcus sp. CBS 8925 AY029341 AY029342 
Cryptococcus sp. CBS 9089 AF444720 AF444487 
Cryptococcus sp. NRRL Y-17490 AF444713 AF444449 
Cryptococcus sp. PYCC 4949 AF444689 AF444356 
Cryptococcus sp. PYCC 4964 AF444690 AF444357 
Cryptococcus sp. PYCC 5266 AF444691 AF444361 
Cystofilobasidium bisporidii CBS 6347 AF075464 AF444299 
Cystofilobasidium capitatum CBS 6358 T AF075465 AF139627 
*Cystofilobasidium lari-marini CBS 7420 T AF075466 AF444300 – 
Cystofilobasidium ferigula CBS 7201 AF075487 AF444328 
Cystofilobasidium ferigula CBS 6954 AF444709 AF444445 – 
Cystofilobasidium infirmominiatum CBS 323 T AF075505 AF444400 
Dioszegia crocea CBS 6714 T AF075508 AF444406 
Dioszegia hungarica CBS 4214 T AF075503 AF444379 
*Bullera armeniaca CBS 7091 T AF189883 AF444467 – 
Fellomyces borneensis CBS 8282 T AF189877 AF444459 
Fellomyces chinensis CBS 8278 T AF189878 AF444460 
Fellomyces dystilii CBS 8545 T AF363652 AF444475 
Fellomyces fuzhouensis CBS 8243 T AF363659 AF444484 
Fellomyces fuzhouensis2 CBS 6133 AF075506 AF444401 – 
Fellomyces horovitziae CBS 7515 T AF189856 AF444404 
Fellomyces lichenicola CBS 8315 T AF363643 AF444462 
Fellomyces ogasawarensis CBS 8544 T AF363651 AF444474 
Fellomyces penicillatus CBS 5492 T AF177405 AF444337 
Fellomyces polyborus CBS 6072 T AF189859 AF444411 
Fellomyces sichuanensis CBS 8318 T AF189879 AF444461 
Fellomyces thailandicus CBS 8308 T AF363644 AF444463 
Fibulobasidium inconspicuum CBS 8237 AF363641 AF444318 
Filobasidiella neoformans CBS 132 T AF075484 AF444326 
Filobasidiella neoformans CBS 882 AF189845 AF444324 – 
Filobasidiella bacillispora CBS 6289 T AF075526 AF444444 
Filobasidium capsuligenum mta CBS 1906 T AF363642 AF444381 
Filobasidium capsuligenum mta CBS 4381 AF444695 AF444382 – 
Filobasidium capsuligenum9 mtα CBS 4736 AF075501 AF444369 
Filobasidium capsuligenum10 mtα CBS 6219 AF181506 AF444334 – 
Filobasidium capsuligenum CBS 8023 AF444688 AF444335 – 
Filobasidium elegans CBS 7640 AF181548 AF190006 
Filobasidium floriforme CBS 6241 AF075498 AF190007 
Filobasidium globisporum CBS 7642 AF075495 AF444336 
Filobasidium uniguttulatum mta CBS 1730 T AF075468 AF444302 
Filobasidium uniguttulatum mtα CBS 1727 AF181500 AF444303 – 
Holtermannia corniformis CBS 6979 AF189843 AF410472 
Hyalodendron lignicola var. simplex CBS 220.34 T AF363657 AF444481 
Hyalodendron lignicola var. undulatum CBS 222.34 T AF363658 AF444482 
Kockovaella imperatae CBS 7554 T AF189862 AF444425 
Kockovaella machilophila CBS 8607 T AF363654 AF444477 
Kockovaella phaffii CBS 8608 T AF363655 AF444478 
Kockovaella sacchari CBS 8624 T AF363650 AF444472 
Kockovaella schimae CBS 8610 T AF363656 AF444479 
Kockovaella thailandica CBS 7552 T AF075516 AF444426 
Mrakia frigida CBS 5270 T AF075463 AF144483 
Mrakia gelida CBS 5272 T AF189831 AF144485 
Phaffia rhodozyma CBS 5905 T AF189871 AF139629 
Sirobasidium magnum CBS 6803 AF075475 AF444314 
Sirobasidium intermedium CBS 7805 AF075492 AF444330 
Sterigmatosporidium polymorphum CBS 8088 T AF075480 AF444320 
Tremella aurantia CBS 6965 AF189842 AF444315 
Tremella brasiliensis CBS 6966 AF189864 AF444429 
Tremella cinnabarina CBS 8234 AF189866 AF444430 
Tremella encephala CBS 6968 AF189867 AF410474 
Tremella foliacea CBS 6969 AF189868 AF444431 
Tremella fuciformis CBS 6970 AF075476 AF444316 
Tremella globispora CBS 6972 AF189869 AF444432 
Tremella mesenterica CBS 6973 AF075518 AF444433 
Tremella moriformis CBS 7810 AF075493 AF444331 
Trichosporon aquatile CBS 5973 T AF075520 AF410475 
Trichosporon asahii CBS 2479 T AF105393 AY055381 
Trichosporon asahii CBS 7137 AF189882 AF444466 – 
Trichosporon asahii CBS 8520 AF189876 AF444457 – 
Trichosporon asahii CBS 8640 AF189881 AF444465 – 
Trichosporon asteroides CBS 2481 T AF075513 AF444416 
Trichosporon brassicae CBS 6382 T AF075521 AF444436 
Trichosporon cutaneum CBS 2466 T AF075483 AF444325 
Trichosporon coremiiforme CBS 2482 T AF139983 AF444434 
Trichosporon coremiiforme CBS 2478 AF189863 AF444427 – 
Trichosporon domesticum CBS 8280 T AF075512 AF444414 
Trichosporon domesticum CBS 8111 AF189874 AF444454 – 
Trichosporon dulcitum CBS 8257 T AF075517 AF444428 
Trichosporon faecale CBS 4828 T AF105395 AF444419 
Trichosporon gracile CBS 8189 T AF105399 AF444440 
Trichosporon gracile CBS 8518 AF189875 AF444455 – 
Trichosporon gracile CBS 8519 AF444716 AF444456 – 
Trichosporon guehoae CBS 8521 T AF105401 AF410476 
Trichosporon inkin CBS 5585 T AF105396 AF444420 
Trichosporon japonicum CBS 8641 T AF308657 AF444473 
Trichosporon jirovecii CBS 6864 T AF105398 AF444437 
Trichosporon laibachii CBS 5790 T AF075514 AF444421 
Trichosporon loubierii CBS 7065 T AF075522 AF444438 
Trichosporon moniliiforme CBS 2467 T AF105392 AF444415 
Trichosporon moniliiforme ATCC 46490 AF444719 AF444480 – 
Trichosporon moniliiforme CBS 8400 AF189873 AF444451 – 
Trichosporon montevideense CBS 6721 T AF105397 AF444422 
Trichosporon mucoides CBS 7625 T AF075515 AF444423 
Trichosporon multisporum CBS 2495 T AF139984 AF414695 
Trichosporon ovoides CBS 7556 T AF075523 AF444439 
Trichosporon porosum CBS 2040 T AF189833 AF414694 
Trichosporon pullulans CBS 2532 T AF105394 AF444417 
Trichosporon pullulans CBS 2541 AF189861 AF444418 – 
Trichosporon sporotrichoides CBS 8246 T AF189885 AF444470 
Trichosporon veenhuisii CBS 7136 T AF105400 AF414693 
Trichosporon sp. CBS 5601 AF444710 AF444446 
Trichosporon sp. CBS 8245 AF444708 AF444424 
Trichosporon sp. CBS 8370 AF444706 AF444397 
Trichosporon sp. CBS 8381 AF444740 AF444644 
Trichosporon sp. CBS 9085 AF444743 AF444647 – 
Trichosporon sp. CBS 9088 AF444744 AF444648 – 
Trichosporon sp. CBS 8686 AF444718 AF444476 
Trimorphomyces papilionaceus CBS 443.92 AF075491 AF444483 
Tsuchiyaea wingfieldii CBS 7118 T AF177404 AF444327 
Udeniomyces megalosporus CBS 7236 T AF075510 AF444408 
Udeniomyces puniceus CBS 5689 T AF075519 AF444435 
Udeniomyces pyricola CBS 6754 T AF075507 AF444402 
Xanthophyllomyces dendrorhous CBS 7918 T AF075496 AF139628 
Xanthophyllomyces dendrorhous CBS 6938 AF444739 AF139632 – 
Xanthophyllomyces sp. CBS 9090 AF444721 AF444488 
Strain Strain number GenBank D1/D2 GenBank ITS Fig. 
Bullera alba var. lactis CBS 7237 T AF363648 AF444665 
Bullera coprosmaensis CBS 8284 T AF363660 AF444485 
Bullera dendrophila CBS 6074 T AF189870 AF444443 
Bullera derxii CBS 7225 T AF189857 AF444405 
Bullera globispora CBS 6981 T AF075509 AF444407 
Bullera hannae CBS 8286 T AF363661 AF444486 
Bullera miyagiana CBS 7526 T AF189858 AF444409 
Bullera oryzae CBS 7194 T AF075511 AF444413 
Bullera penniseticola CBS 8623 T AF363649 AF444471 
Bullera pseudoalba CBS 7227 T AF075504 AF444399 
Bullera sinensis CBS 7238 T AF189884 AF444468 
Bullera unica CBS 8290 T AF075524 AF444441 
Bullera variabilis CBS 7347 T AF189855 AF444403 
Bullera sp. CBS 2013 AF444760 AF444666 
Bulleromyces albus mtB CBS 501 T AF075500 AF444368 
Bulleromyces 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 
Bulleromyces 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 
Cryptococcus aerius CBS 155 T AF075486 AF145324 
*Torulopsis pseudoaeria CBS 4192 T AF181544 AF444376 – 
Cryptococcus albidisimilis CBS 7711 T AF137601 AF145325 
Cryptococcus albidisimilis3 ATCC 34633 AF137606 AF145331 – 
Cryptococcus albidus CBS 142 T AF075474 AF145321 
Cryptococcus 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 
Cryptococcus albidus var. kuetzingii1 CBS 922 AF181504 AF444313 – 
Cryptococcus albidus var. kuetzingii CBS 6086 AF181546 AF444378 – 
Cryptococcus albidus var. ovalis CBS 5810 T AF137605 AF145329 
Cryptococcus amylolentus CBS 6039 T AF105391 AF444306 
Cryptococcus antarcticus CBS 7687 T AF075488 AF145326 
Cryptococcus aquaticus CBS 5443 T AF075470 AF410469 
Cryptococcus arrabidensis CBS 8678 T AF181535 AF444362 
Cryptococcus bhutanensis CBS 6294 T AF137599 AF145317 
Cryptococcus cellulolyticus CBS 8294 T AF075525 AF444442 
Cryptococcus chernovii CBS 8679 T AF181530 AF444354 
Cryptococcus cylindricus CBS 8680 T AF181534 AF444360 
Cryptococcus cylindricus8 PYCC 5264 AF181533 AF444359 
Cryptococcus curvatus CBS 570 T AF189834 AF410467 
Cryptococcus curvatus CBS 8126 AF444717 AF444458 – 
Cryptococcus dimennae CBS 5770 T AF075489 AF410473 
Cryptococcus diffluens CBS 160 T AF075502 AF145330 
*Cryptococcus diffluens var. uruguaiensis CBS 6436 T AF181543 AF444375 – 
*Torulopsis albida var. japonica CBS 926 T AF181542 AF444374 – 
Cryptococcus flavus CBS 331 T AF075497 AF444338 
Cryptococcus friedmannii CBS 7160 T AF075478 AF145322 
Cryptococcus fuscescens CBS 7189 T AF075472 AF145319 
Cryptococcus gastricus CBS 2288 T AF137600 AF145323 
Cryptococcus gastricus5 CBS 1927 AF181501 AF444304 – 
Cryptococcus gilvescens CBS 7525 T AF181547 AF444380 
Cryptococcus heveanensis CBS 569 T AF075467 AF444301 
Cryptococcus huempii CBS 8186 T AF189844 AF444322 
Cryptococcus humicola CBS 571 T AF189836 AF410470 
Cryptococcus humicola5 CBS 8354 AF189851 AF444384 – 
Cryptococcus humicola5 CBS 8371 AF189854 AF444398 – 
Cryptococcus laurentii CBS 139 T AF075469 AF410468 
Cryptococcus liquefaciens CBS 968 T AF181515 AF444345 
Cryptococcus liquefaciens PYCC 2406 AF181513 AF444343 – 
Cryptococcus liquefaciens PYCC 2934 AF181518 AF444348 – 
Cryptococcus luteolus CBS 943 T AF075482 AF444323 
Cryptococcus macerans mta CBS 2206 T AF189848 AF444329 
Cryptococcus macerans mtα CBS 2425 AF075477 AF444317 
Cryptococcus magnus CBS 140 T AF181851 AF190008 
Cryptococcus 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 – 
Cryptococcus oeirensis CBS 8681 T AF181519 AF444349 
Cryptococcus oeirensis PYCC 5268 AF181537 AF444364 – 
Cryptococcus phenolicus CBS 8682 T AF181523 AF444351 
Cryptococcus podzolicus CBS 6819 T AF075481 AF444321 
Cryptococcus saitoi CBS 1975 T AF181540 AF444372 
*Naganishia globosa CBS 5106 T AF181539 AF444371 – 
Cryptococcus skinneri CBS 5029 T AF189835 AF444305 
Cryptococcus terreus CBS 1895 T AF075479 AF444319 
Cryptococcus terreus7 PYCC 2935 AF444692 AF444365 – 
Cryptococcus terreus7 PYCC 4966 AF444694 AF444367 – 
*Cryptococcus elinovii4 CBS 7051 T AF137604 AF145318 
*Cryptococcus himalayensis2 CBS 6293 T AF181502 AF410471 – 
Cryptococcus terricola CBS 4517 T AF181520 AF444350 
Cryptococcus terricola CBS 6435 AF181545 AF444377 – 
Cryptococcus uzbekistanensis CBS 8683 T AF181508 AF444339 
Cryptococcus victoriae CBS 8685 AF363647 AF444469 
Cryptococcus victoriae CBS 6550 AF444711 AF444447 – 
Cryptococcus victoriae6 CBS 8884 AF444741 AF444645 – 
Cryptococcus vishniacii CBS 7110 T AF075473 AF145320 
*Cryptococcus asgardensis CBS 8141 T AF189839 AF444310 – 
*Cryptococcus baldrensis CBS 8142 T AF189840 AF444311 – 
*Cryptococcus consortionis A801-3aY92/20 T AF189880 AF444464 – 
*Cryptococcus hempflingii 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 
Cryptococcus wieringae CBS 8353 AF181549 AF444383 – 
Cryptococcus sp. CBS 6984 AF444712 AF444448 
Cryptococcus sp. CBS 7712 AJ311450 AF408417 
Cryptococcus sp. CBS 7713 AJ311451 AF408418 – 
Cryptococcus sp. CBS 7743 AJ311452 AF408419 – 
Cryptococcus sp. CBS 7890 AF075494 AF444332 
Cryptococcus sp. CBS 7944 AF444693 AF444366 
Cryptococcus sp. CBS 8024 AF444715 AF444453 
Cryptococcus sp. 2 CBS 6024 AF444714 AF444452 – 
Cryptococcus sp. CBS 6123 AF444687 AF444333 – 
Cryptococcus sp. CBS 8355 AF444696 AF444385 
Cryptococcus sp. CBS 8356 AF444697 AF444386 
Cryptococcus sp. CBS 8367 AF444703 AF444394 – 
Cryptococcus sp. CBS 8358 AF444698 AF444387 
Cryptococcus sp. CBS 8366 AF444702 AF444393 – 
Cryptococcus sp. CBS 8363 AF444699 AF444390 
Cryptococcus sp. CBS 8364 AF444700 AF444391 
Cryptococcus sp. CBS 8365 AF444701 AF444392 – 
Cryptococcus sp. CBS 8368 AF444704 AF444395 – 
Cryptococcus sp. CBS 8372 AF444707 AF444410 
Cryptococcus sp. CBS 8369 AF444705 AF444396 
Cryptococcus sp. CBS 8507 AF444742 AF444646 
Cryptococcus sp. CBS 8804 AY006480 AY006481 
Cryptococcus sp. CBS 8924 AY029345 AY029346 
Cryptococcus sp. CBS 8925 AY029341 AY029342 
Cryptococcus sp. CBS 9089 AF444720 AF444487 
Cryptococcus sp. NRRL Y-17490 AF444713 AF444449 
Cryptococcus sp. PYCC 4949 AF444689 AF444356 
Cryptococcus sp. PYCC 4964 AF444690 AF444357 
Cryptococcus sp. PYCC 5266 AF444691 AF444361 
Cystofilobasidium bisporidii CBS 6347 AF075464 AF444299 
Cystofilobasidium capitatum CBS 6358 T AF075465 AF139627 
*Cystofilobasidium lari-marini CBS 7420 T AF075466 AF444300 – 
Cystofilobasidium ferigula CBS 7201 AF075487 AF444328 
Cystofilobasidium ferigula CBS 6954 AF444709 AF444445 – 
Cystofilobasidium infirmominiatum CBS 323 T AF075505 AF444400 
Dioszegia crocea CBS 6714 T AF075508 AF444406 
Dioszegia hungarica CBS 4214 T AF075503 AF444379 
*Bullera armeniaca CBS 7091 T AF189883 AF444467 – 
Fellomyces borneensis CBS 8282 T AF189877 AF444459 
Fellomyces chinensis CBS 8278 T AF189878 AF444460 
Fellomyces dystilii CBS 8545 T AF363652 AF444475 
Fellomyces fuzhouensis CBS 8243 T AF363659 AF444484 
Fellomyces fuzhouensis2 CBS 6133 AF075506 AF444401 – 
Fellomyces horovitziae CBS 7515 T AF189856 AF444404 
Fellomyces lichenicola CBS 8315 T AF363643 AF444462 
Fellomyces ogasawarensis CBS 8544 T AF363651 AF444474 
Fellomyces penicillatus CBS 5492 T AF177405 AF444337 
Fellomyces polyborus CBS 6072 T AF189859 AF444411 
Fellomyces sichuanensis CBS 8318 T AF189879 AF444461 
Fellomyces thailandicus CBS 8308 T AF363644 AF444463 
Fibulobasidium inconspicuum CBS 8237 AF363641 AF444318 
Filobasidiella neoformans CBS 132 T AF075484 AF444326 
Filobasidiella neoformans CBS 882 AF189845 AF444324 – 
Filobasidiella bacillispora CBS 6289 T AF075526 AF444444 
Filobasidium capsuligenum mta CBS 1906 T AF363642 AF444381 
Filobasidium capsuligenum mta CBS 4381 AF444695 AF444382 – 
Filobasidium capsuligenum9 mtα CBS 4736 AF075501 AF444369 
Filobasidium capsuligenum10 mtα CBS 6219 AF181506 AF444334 – 
Filobasidium capsuligenum CBS 8023 AF444688 AF444335 – 
Filobasidium elegans CBS 7640 AF181548 AF190006 
Filobasidium floriforme CBS 6241 AF075498 AF190007 
Filobasidium globisporum CBS 7642 AF075495 AF444336 
Filobasidium uniguttulatum mta CBS 1730 T AF075468 AF444302 
Filobasidium uniguttulatum mtα CBS 1727 AF181500 AF444303 – 
Holtermannia corniformis CBS 6979 AF189843 AF410472 
Hyalodendron lignicola var. simplex CBS 220.34 T AF363657 AF444481 
Hyalodendron lignicola var. undulatum CBS 222.34 T AF363658 AF444482 
Kockovaella imperatae CBS 7554 T AF189862 AF444425 
Kockovaella machilophila CBS 8607 T AF363654 AF444477 
Kockovaella phaffii CBS 8608 T AF363655 AF444478 
Kockovaella sacchari CBS 8624 T AF363650 AF444472 
Kockovaella schimae CBS 8610 T AF363656 AF444479 
Kockovaella thailandica CBS 7552 T AF075516 AF444426 
Mrakia frigida CBS 5270 T AF075463 AF144483 
Mrakia gelida CBS 5272 T AF189831 AF144485 
Phaffia rhodozyma CBS 5905 T AF189871 AF139629 
Sirobasidium magnum CBS 6803 AF075475 AF444314 
Sirobasidium intermedium CBS 7805 AF075492 AF444330 
Sterigmatosporidium polymorphum CBS 8088 T AF075480 AF444320 
Tremella aurantia CBS 6965 AF189842 AF444315 
Tremella brasiliensis CBS 6966 AF189864 AF444429 
Tremella cinnabarina CBS 8234 AF189866 AF444430 
Tremella encephala CBS 6968 AF189867 AF410474 
Tremella foliacea CBS 6969 AF189868 AF444431 
Tremella fuciformis CBS 6970 AF075476 AF444316 
Tremella globispora CBS 6972 AF189869 AF444432 
Tremella mesenterica CBS 6973 AF075518 AF444433 
Tremella moriformis CBS 7810 AF075493 AF444331 
Trichosporon aquatile CBS 5973 T AF075520 AF410475 
Trichosporon asahii CBS 2479 T AF105393 AY055381 
Trichosporon asahii CBS 7137 AF189882 AF444466 – 
Trichosporon asahii CBS 8520 AF189876 AF444457 – 
Trichosporon asahii CBS 8640 AF189881 AF444465 – 
Trichosporon asteroides CBS 2481 T AF075513 AF444416 
Trichosporon brassicae CBS 6382 T AF075521 AF444436 
Trichosporon cutaneum CBS 2466 T AF075483 AF444325 
Trichosporon coremiiforme CBS 2482 T AF139983 AF444434 
Trichosporon coremiiforme CBS 2478 AF189863 AF444427 – 
Trichosporon domesticum CBS 8280 T AF075512 AF444414 
Trichosporon domesticum CBS 8111 AF189874 AF444454 – 
Trichosporon dulcitum CBS 8257 T AF075517 AF444428 
Trichosporon faecale CBS 4828 T AF105395 AF444419 
Trichosporon gracile CBS 8189 T AF105399 AF444440 
Trichosporon gracile CBS 8518 AF189875 AF444455 – 
Trichosporon gracile CBS 8519 AF444716 AF444456 – 
Trichosporon guehoae CBS 8521 T AF105401 AF410476 
Trichosporon inkin CBS 5585 T AF105396 AF444420 
Trichosporon japonicum CBS 8641 T AF308657 AF444473 
Trichosporon jirovecii CBS 6864 T AF105398 AF444437 
Trichosporon laibachii CBS 5790 T AF075514 AF444421 
Trichosporon loubierii CBS 7065 T AF075522 AF444438 
Trichosporon moniliiforme CBS 2467 T AF105392 AF444415 
Trichosporon moniliiforme ATCC 46490 AF444719 AF444480 – 
Trichosporon moniliiforme CBS 8400 AF189873 AF444451 – 
Trichosporon montevideense CBS 6721 T AF105397 AF444422 
Trichosporon mucoides CBS 7625 T AF075515 AF444423 
Trichosporon multisporum CBS 2495 T AF139984 AF414695 
Trichosporon ovoides CBS 7556 T AF075523 AF444439 
Trichosporon porosum CBS 2040 T AF189833 AF414694 
Trichosporon pullulans CBS 2532 T AF105394 AF444417 
Trichosporon pullulans CBS 2541 AF189861 AF444418 – 
Trichosporon sporotrichoides CBS 8246 T AF189885 AF444470 
Trichosporon veenhuisii CBS 7136 T AF105400 AF414693 
Trichosporon sp. CBS 5601 AF444710 AF444446 
Trichosporon sp. CBS 8245 AF444708 AF444424 
Trichosporon sp. CBS 8370 AF444706 AF444397 
Trichosporon sp. CBS 8381 AF444740 AF444644 
Trichosporon sp. CBS 9085 AF444743 AF444647 – 
Trichosporon sp. CBS 9088 AF444744 AF444648 – 
Trichosporon sp. CBS 8686 AF444718 AF444476 
Trimorphomyces papilionaceus CBS 443.92 AF075491 AF444483 
Tsuchiyaea wingfieldii CBS 7118 T AF177404 AF444327 
Udeniomyces megalosporus CBS 7236 T AF075510 AF444408 
Udeniomyces puniceus CBS 5689 T AF075519 AF444435 
Udeniomyces pyricola CBS 6754 T AF075507 AF444402 
Xanthophyllomyces dendrorhous CBS 7918 T AF075496 AF139628 
Xanthophyllomyces dendrorhous CBS 6938 AF444739 AF139632 – 
Xanthophyllomyces sp. CBS 9090 AF444721 AF444488 

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, identical 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.

2

List of urediniomycetous strains studied

Strain Strain number GenBank D1/D2 GenBank ITS Fig. 
Agaricostilbum hyphaenes CBS 7811 AF177406 AF444553 
Bensingtonia ciliata CBS 7514 T AF189887 AF444563 
Bensingtonia ingoldii CBS 7424 T AF189888 AF444519 
Bensingtonia miscanthi CBS 7282 T AF189891 AF444516 
Bensingtonia musae CBS 7965 T AF189892 AF444569 
Bensingtonia naganoensis CBS 7286 T AF189893 AF444558 
Bensingtonia phyllada CBS 7169 T AF189894 AF444514 
Bensingtonia sakaguchii CBS 8464 T AF363646 AF444626 
Bensingtonia subrosea CBS 7283 T AF189895 AF444565 
Bensingtonia yamatoana CBS 7243 T AF189896 AF444634 
Bensingtonia yuccicola CBS 7331 T AF189897 AF444518 
Bensingtonia sp. PYCC 5551 AF444770 AF444676 
Bensingtonia sp. PYCC 5562 AF444765 AF444671 
Bensingtonia sp. PYCC 5566 AF444766 AF444672 
Bensingtonia sp. PYCC 5547 AF444767 AF444673 – 
Chionosphaera apobasidialis CBS 7430 T AF177407 AF444599 
Colacogloea peniophorae PYCC 4285 AF189898 AF444591 
Erythrobasidium hasegawianum CBS 8253 T AF189899 AF444522 
Hyalopycnis blepharistoma CBS 591.93 AF189900 AF444555 
Kondoa aeria CBS 8352 T AF189901 AF444562 
Kondoa 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 
Kondoa sp. CBS 8379 AF189904 AF444596 
Kriegeria eriophori CBS 8387 AF189905 AF444602 
Kurtzmanomyces insolitus CBS 8377 T AF177408 AF444594 
Kurtzmanomyces nectairei CBS 6405 T AF177409 AF444494 
Kurtzmanomyces tardus CBS 7421 T AF177410 AF444566 
Leucosporidium antarcticum CBS 5942 T AF189906 AF444529 
Leucosporidium fellii CBS 7287 T AF189907 AF444508 
Leucosporidium scottii CBS 5930 T AF070419 AF444495 
Leucosporidium scottii1 CBS 5932 AF189908 AF444496 – 
Mastigobasidium intermedium mta CBS 7226 T AF189889 AF444564 
Mastigobasidium intermedium mtα CBS 7281 AF189890 AF444630 – 
Occultifur externus CBS 8732 T AF189909 AF444567 
Occultifur externus4 anam. PYCC 4557 AF189910 AF444643 – 
Occultifur externus4 anam. PYCC 4823 AF189911 AF444642 – 
Reniforma strues CBS 8263 T AF189912 AF444573 
Rhodosporidium babjevae mtA1 CBS 7808 T AF070420 AF444542 
Rhodosporidium babjevae mtA2 CBS 9071 AF189913 AF444636 – 
Rhodosporidium diobovatum mtα CBS 6085 T AF070421 AF444502 
Rhodosporidium diobovatum5 mtα CBS 9076 AF444748 AF444653 – 
Rhodosporidium diobovatum5 mta CBS 9081 AF444753 AF444658 
Rhodosporidium diobovatum5 CBS 9084 AF444756 AF444661 – 
Rhodosporidium fluviale CBS 6568 T AF189915 AY015432 
Rhodosporidium kratochvilovae CBS 7436 T AF071436 AF444520 
Rhodosporidium 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 
Rhodosporidium paludigenum mtA2 CBS 6566 T AF363640 AF444492 
Rhodosporidium paludigenum mtA1 CBS 6567 AF070424 AF444493 – 
Rhodosporidium sphaerocarpum mta CBS 5939 T AF070425 AF444499 
Rhodosporidium sphaerocarpum mta CBS 9075 AF444747 AF444652 – 
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 
Rhodosporidium toruloides CBS 9074 AF444746 AF444651 – 
Rhodotorula araucariae CBS 6031 T AF070427 AF444510 
Rhodotorula armeniaca CBS 8076 T AF189920 AF444523 
Rhodotorula aurantiaca CBS 317 T AF189921 AF444538 
Rhodotorula auriculariae CBS 6379 T AF189922 AF444507 
Rhodotorula bogoriensis CBS 4101 T AF189923 AF444536 
Rhodotorula buffonii CBS 2838 T AF189924 AF444526 
Rhodotorula creatinovora CBS 8620 T AF189925 AF444629 
Rhodotorula cresolica CBS 7998 T AF189926 AF444570 
Rhodotorula diffluens CBS 5233 T AF075485 AF444533 
Rhodotorula ferulica CBS 7416 T AF363653 AF444632 
Rhodotorula ferulica CBS 7402 AF189927 AF444528 – 
Rhodotorula foliorum CBS 5234 T AF317804 AF444633 
Rhodotorula fragraria CBS 6254 T AF070428 AF444530 
Rhodotorula fujisanensis CBS 4551 T AF189928 AF444490 
Rhodotorula fujisanensis CBS 6371 AF189929 AF444574 – 
Rhodotorula fujisanensis CBS 8264 AF189930 AF444572 – 
*Rhodotorula futronensis CBS 8163 T AF189931 AF444525 
Rhodotorula glutinis CBS 20 T AF070430 AF444539 
Rhodotorula dairenensis CBS 4406 T AF070429 AF444501 
Rhodotorula dairenensis PYCC 4784 AY033551 AF444683 – 
Rhodotorula dairenensis PYCC 4897 AY033552 AF444684 – 
Rhodotorula graminis CBS 2826 T AF070431 AF444505 
Rhodotorula hylophila CBS 6226 T AF363645 AF444622 
Rhodotorula hordea CBS 6403 T AF189933 AF444524 
Rhodotorula ingeniosa CBS 4240 T AF189934 AF444534 
Rhodotorula javanica CBS 5236 T AF189935 AF444532 
Rhodotorula lactosa CBS 5826 T AF189936 AF444540 
Rhodotorula laryngis CBS 2221 T AF189937 AF190014 
Rhodotorula 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 
Rhodotorula marina CBS 2365 T AF189944 AF444504 
Rhodotorula minuta CBS 319 T AF189945 AF190011 
Rhodotorula minuta2 CBS 4408 AF189946 AF444579 – 
Rhodotorula minuta CBS 7296 AF189947 AF444620 – 
Rhodotorula mucilaginosa CBS 316 T AF070432 AF444541 
Rhodotorula mucilaginosa2 CBS 8383 AF189959 AF444649 – 
Rhodotorula mucilaginosa6 CBS 9070 AF444738 AF444635 
Rhodotorula 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 
Rhodotorula nothofagi CBS 8166 T AF189950 AF444537 
Rhodotorula nothofagi1 CBS 9091 AF444736 AF444641 – 
Rhodotorula pallida CBS 320 T AF189962 AF444590 
Rhodotorula philyla CBS 6272 T AF075471 AF444506 
Rhodotorula pilati CBS 7039 T AF189963 AF444598 
Rhodotorula pustula CBS 6527 T AF189964 AF444531 
Rhodotorula slooffiae CBS 5706 T AF189965 AF444627 
Rhodotorula slooffiae4 CBS 7093 AF189966 AF444554 – 
Rhodotorula slooffiae4 CBS 7094 AF444722 AF444552 – 
Rhodotorula slooffiae4 CBS 7095 AF189967 AF444618 – 
Rhodotorula slooffiae3 PYCC 4887 AF189968 AF444589 – 
Rhodotorula sonckii CBS 6713 T AF189969 AF444601 
Rhodotorula vanillica CBS 7404 T AF189970 AF444575 
Rhodotorula yarrowii CBS 7417 T AF189971 AF444628 
Rhodotorula sp. CBS 6370 AF075499 AF444560 
Rhodotorula sp. CBS 6372 AF444728 AF444621 
Rhodotorula sp. CBS 8445 AF444729 AF444623 
Rhodotorula sp. CBS 8446 AF444730 AF444624 
Rhodotorula sp. CBS 8447 AF444731 AF444625 
Rhodotorula sp. CBS 8448 AF444732 AF444631 – 
Rhodotorula sp. CBS 8885 AF444745 AF444650 
Rhodotorula sp. CBS 8923 AY033643 AY033642 
Rhodotorula sp. CBS 8928 AY033640 AY033641 
Rhodotorula sp. CBS 8943 AY033636 AY033637 
Rhodotorula sp. CBS 8944 AY033639 AY033638 
Rhodotorula sp. CBS 9072 AF189932 AF444637 
Rhodotorula sp. CBS 9073 AF444733 AF444638 – 
Rhodotorula sp. CBS 9086 AF444734 AF444639 
Rhodotorula sp. CBS 9087 AF444735 AF444640 – 
Rhodotorula sp. PYCC 4689 AF444737 AF444588 
Rhodotorula sp. CBS 7295 AF444727 AF444619 – 
Rhodotorula sp. NRRL Y-17502 AF444726 AF444615 
Sakaguchia dacryoidea mtA1B1 CBS 6353 T AF189972 AF444597 
Sakaguchia dacryoidea mtA1B2 CBS 6356 AF189973 AF444500 – 
Sakaguchia dacryoidea9 mtA2B1 CBS 7999 AF444723 AF444571 
Sphacelotheca polygoni-persicariae PYCC 4293 AF189974 AF444593 
Sporidiobolus johnsonii HO CBS 5470 T AF070435 AY015431 
Sporidiobolus johnsonii8 (salmonicolor mtA2) CBS 2630 AF189976 AF444568 – 
*Sporobolomyces holsaticus8 CBS 1522 T AF189975 AF444509 
Sporidiobolus microsporus CBS 7041 T AF070436 AF444535 
Sporidiobolus pararoseus mtA2 CBS 491 T AF189977 AY015429 
*Sporobolomyces pararoseus7 mtA1 CBS 484 T AF070437 AF417115 
*Sporobolomyces ruber mtA2 CBS 4216 T AF189978 AF444604 – 
Sporidiobolus ruineniae CBS 5001 T AF070438 AY015433 
Sporidiobolus ruineniae var. coprophilus1 CBS 5811 T AF070434 AF444491 – 
Sporidiobolus salmonicolor mtA1 CBS 490 T AF070439 AY015434 
Sporidiobolus salmonicolor1 NRRL Y-17498 AF189979 AF444611 – 
Sporidiobolus sp. CBS 5541 AY015271 AY015435 
Sporobolomyces coprosmae CBS 7899 T AF189980 AF444577 
Sporobolomyces coprosmicola CBS 7897 T AF189981 AF444576 
Sporobolomyces dracophylli CBS 7900 T AF189982 AF444583 
Sporobolomyces elongatus CBS 8080 T AF189983 AF444561 
Sporobolomyces falcatus CBS 7368 T AF075490 AF444543 
Sporobolomyces foliicola CBS 8075 T AF189984 AF444521 
Sporobolomyces gracilis CBS 71 T AF189985 AF444578 
Sporobolomyces griseoflavus CBS 7284 T AF189986 AF444557 
Sporobolomyces inositophilus CBS 7310 T AF189987 AF444559 
Sporobolomyces kluyveri-nielii CBS 7168 T AF189988 AF444544 
Sporobolomyces lactophilus CBS 7527 T AF177411 AF444545 
Sporobolomyces linderae CBS 7893 T AF189989 AF444582 
Sporobolomyces marcillae CBS 4217 T AF070440 AY015437 
Sporobolomyces oryzicola CBS 7228 T AF189990 AF444546 
Sporobolomyces phyllomatis CBS 7198 T AF189991 AF444515 
Sporobolomyces roseus CBS 486 T AF070441 AY015438 
Sporobolomyces ruber CBS 7512 T AF189992 AF444550 
Sporobolomyces ruberrimus CBS 7500 T AF070442 AY015439 
*S. ruberrimus var. albus1 CBS 7501 T AF189993 AY015436 – 
S. ruberrimus var. albus1 CBS 7253 AF189994 AF444581 – 
Sporobolomyces salicinus CBS 6983 T AF189995 AF444511 
Sporobolomyces sasicola CBS 7285 T AF177412 AF444548 
Sporobolomyces singularis CBS 5109 T AF189996 AF444600 
Sporobolomyces subbrunneus CBS 7196 T AF189997 AF444549 
Sporobolomyces taupoensis CBS 7898 T AF177413 AF444592 
Sporobolomyces tsugae CBS 5038 T AF189998 AF444580 
Sporobolomyces xanthus CBS 7513 T AF177414 AF444547 
Sporobolomyces sp. CBS 6166 AF444724 AF444603  
Sterigmatomyces elviae CBS 5922 T AF177415 AF444551 
*Rhodotorula acuta CBS 7053 T AF189999 AF444512 – 
*Rhodotorula dulciaminis1 CBS 7288 T AF190000 AF444517 – 
Sterigmatomyces halophilus CBS 4609 T AF177416 AF444556 
Strain Strain number GenBank D1/D2 GenBank ITS Fig. 
Agaricostilbum hyphaenes CBS 7811 AF177406 AF444553 
Bensingtonia ciliata CBS 7514 T AF189887 AF444563 
Bensingtonia ingoldii CBS 7424 T AF189888 AF444519 
Bensingtonia miscanthi CBS 7282 T AF189891 AF444516 
Bensingtonia musae CBS 7965 T AF189892 AF444569 
Bensingtonia naganoensis CBS 7286 T AF189893 AF444558 
Bensingtonia phyllada CBS 7169 T AF189894 AF444514 
Bensingtonia sakaguchii CBS 8464 T AF363646 AF444626 
Bensingtonia subrosea CBS 7283 T AF189895 AF444565 
Bensingtonia yamatoana CBS 7243 T AF189896 AF444634 
Bensingtonia yuccicola CBS 7331 T AF189897 AF444518 
Bensingtonia sp. PYCC 5551 AF444770 AF444676 
Bensingtonia sp. PYCC 5562 AF444765 AF444671 
Bensingtonia sp. PYCC 5566 AF444766 AF444672 
Bensingtonia sp. PYCC 5547 AF444767 AF444673 – 
Chionosphaera apobasidialis CBS 7430 T AF177407 AF444599 
Colacogloea peniophorae PYCC 4285 AF189898 AF444591 
Erythrobasidium hasegawianum CBS 8253 T AF189899 AF444522 
Hyalopycnis blepharistoma CBS 591.93 AF189900 AF444555 
Kondoa aeria CBS 8352 T AF189901 AF444562 
Kondoa 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 
Kondoa sp. CBS 8379 AF189904 AF444596 
Kriegeria eriophori CBS 8387 AF189905 AF444602 
Kurtzmanomyces insolitus CBS 8377 T AF177408 AF444594 
Kurtzmanomyces nectairei CBS 6405 T AF177409 AF444494 
Kurtzmanomyces tardus CBS 7421 T AF177410 AF444566 
Leucosporidium antarcticum CBS 5942 T AF189906 AF444529 
Leucosporidium fellii CBS 7287 T AF189907 AF444508 
Leucosporidium scottii CBS 5930 T AF070419 AF444495 
Leucosporidium scottii1 CBS 5932 AF189908 AF444496 – 
Mastigobasidium intermedium mta CBS 7226 T AF189889 AF444564 
Mastigobasidium intermedium mtα CBS 7281 AF189890 AF444630 – 
Occultifur externus CBS 8732 T AF189909 AF444567 
Occultifur externus4 anam. PYCC 4557 AF189910 AF444643 – 
Occultifur externus4 anam. PYCC 4823 AF189911 AF444642 – 
Reniforma strues CBS 8263 T AF189912 AF444573 
Rhodosporidium babjevae mtA1 CBS 7808 T AF070420 AF444542 
Rhodosporidium babjevae mtA2 CBS 9071 AF189913 AF444636 – 
Rhodosporidium diobovatum mtα CBS 6085 T AF070421 AF444502 
Rhodosporidium diobovatum5 mtα CBS 9076 AF444748 AF444653 – 
Rhodosporidium diobovatum5 mta CBS 9081 AF444753 AF444658 
Rhodosporidium diobovatum5 CBS 9084 AF444756 AF444661 – 
Rhodosporidium fluviale CBS 6568 T AF189915 AY015432 
Rhodosporidium kratochvilovae CBS 7436 T AF071436 AF444520 
Rhodosporidium 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 
Rhodosporidium paludigenum mtA2 CBS 6566 T AF363640 AF444492 
Rhodosporidium paludigenum mtA1 CBS 6567 AF070424 AF444493 – 
Rhodosporidium sphaerocarpum mta CBS 5939 T AF070425 AF444499 
Rhodosporidium sphaerocarpum mta CBS 9075 AF444747 AF444652 – 
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 
Rhodosporidium toruloides CBS 9074 AF444746 AF444651 – 
Rhodotorula araucariae CBS 6031 T AF070427 AF444510 
Rhodotorula armeniaca CBS 8076 T AF189920 AF444523 
Rhodotorula aurantiaca CBS 317 T AF189921 AF444538 
Rhodotorula auriculariae CBS 6379 T AF189922 AF444507 
Rhodotorula bogoriensis CBS 4101 T AF189923 AF444536 
Rhodotorula buffonii CBS 2838 T AF189924 AF444526 
Rhodotorula creatinovora CBS 8620 T AF189925 AF444629 
Rhodotorula cresolica CBS 7998 T AF189926 AF444570 
Rhodotorula diffluens CBS 5233 T AF075485 AF444533 
Rhodotorula ferulica CBS 7416 T AF363653 AF444632 
Rhodotorula ferulica CBS 7402 AF189927 AF444528 – 
Rhodotorula foliorum CBS 5234 T AF317804 AF444633 
Rhodotorula fragraria CBS 6254 T AF070428 AF444530 
Rhodotorula fujisanensis CBS 4551 T AF189928 AF444490 
Rhodotorula fujisanensis CBS 6371 AF189929 AF444574 – 
Rhodotorula fujisanensis CBS 8264 AF189930 AF444572 – 
*Rhodotorula futronensis CBS 8163 T AF189931 AF444525 
Rhodotorula glutinis CBS 20 T AF070430 AF444539 
Rhodotorula dairenensis CBS 4406 T AF070429 AF444501 
Rhodotorula dairenensis PYCC 4784 AY033551 AF444683 – 
Rhodotorula dairenensis PYCC 4897 AY033552 AF444684 – 
Rhodotorula graminis CBS 2826 T AF070431 AF444505 
Rhodotorula hylophila CBS 6226 T AF363645 AF444622 
Rhodotorula hordea CBS 6403 T AF189933 AF444524 
Rhodotorula ingeniosa CBS 4240 T AF189934 AF444534 
Rhodotorula javanica CBS 5236 T AF189935 AF444532 
Rhodotorula lactosa CBS 5826 T AF189936 AF444540 
Rhodotorula laryngis CBS 2221 T AF189937 AF190014 
Rhodotorula 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 
Rhodotorula marina CBS 2365 T AF189944 AF444504 
Rhodotorula minuta CBS 319 T AF189945 AF190011 
Rhodotorula minuta2 CBS 4408 AF189946 AF444579 – 
Rhodotorula minuta CBS 7296 AF189947 AF444620 – 
Rhodotorula mucilaginosa CBS 316 T AF070432 AF444541 
Rhodotorula mucilaginosa2 CBS 8383 AF189959 AF444649 – 
Rhodotorula mucilaginosa6 CBS 9070 AF444738 AF444635 
Rhodotorula 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 
Rhodotorula nothofagi CBS 8166 T AF189950 AF444537 
Rhodotorula nothofagi1 CBS 9091 AF444736 AF444641 – 
Rhodotorula pallida CBS 320 T AF189962 AF444590 
Rhodotorula philyla CBS 6272 T AF075471 AF444506 
Rhodotorula pilati CBS 7039 T AF189963 AF444598 
Rhodotorula pustula CBS 6527 T AF189964 AF444531 
Rhodotorula slooffiae CBS 5706 T AF189965 AF444627 
Rhodotorula slooffiae4 CBS 7093 AF189966 AF444554 – 
Rhodotorula slooffiae4 CBS 7094 AF444722 AF444552 – 
Rhodotorula slooffiae4 CBS 7095 AF189967 AF444618 – 
Rhodotorula slooffiae3 PYCC 4887 AF189968 AF444589 – 
Rhodotorula sonckii CBS 6713 T AF189969 AF444601 
Rhodotorula vanillica CBS 7404 T AF189970 AF444575 
Rhodotorula yarrowii CBS 7417 T AF189971 AF444628 
Rhodotorula sp. CBS 6370 AF075499 AF444560 
Rhodotorula sp. CBS 6372 AF444728 AF444621 
Rhodotorula sp. CBS 8445 AF444729 AF444623 
Rhodotorula sp. CBS 8446 AF444730 AF444624 
Rhodotorula sp. CBS 8447 AF444731 AF444625 
Rhodotorula sp. CBS 8448 AF444732 AF444631 – 
Rhodotorula sp. CBS 8885 AF444745 AF444650 
Rhodotorula sp. CBS 8923 AY033643 AY033642 
Rhodotorula sp. CBS 8928 AY033640 AY033641 
Rhodotorula sp. CBS 8943 AY033636 AY033637 
Rhodotorula sp. CBS 8944 AY033639 AY033638 
Rhodotorula sp. CBS 9072 AF189932 AF444637 
Rhodotorula sp. CBS 9073 AF444733 AF444638 – 
Rhodotorula sp. CBS 9086 AF444734 AF444639 
Rhodotorula sp. CBS 9087 AF444735 AF444640 – 
Rhodotorula sp. PYCC 4689 AF444737 AF444588 
Rhodotorula sp. CBS 7295 AF444727 AF444619 – 
Rhodotorula sp. NRRL Y-17502 AF444726 AF444615 
Sakaguchia dacryoidea mtA1B1 CBS 6353 T AF189972 AF444597 
Sakaguchia dacryoidea mtA1B2 CBS 6356 AF189973 AF444500 – 
Sakaguchia dacryoidea9 mtA2B1 CBS 7999 AF444723 AF444571 
Sphacelotheca polygoni-persicariae PYCC 4293 AF189974 AF444593 
Sporidiobolus johnsonii HO CBS 5470 T AF070435 AY015431 
Sporidiobolus johnsonii8 (salmonicolor mtA2) CBS 2630 AF189976 AF444568 – 
*Sporobolomyces holsaticus8 CBS 1522 T AF189975 AF444509 
Sporidiobolus microsporus CBS 7041 T AF070436 AF444535 
Sporidiobolus pararoseus mtA2 CBS 491 T AF189977 AY015429 
*Sporobolomyces pararoseus7 mtA1 CBS 484 T AF070437 AF417115 
*Sporobolomyces ruber mtA2 CBS 4216 T AF189978 AF444604 – 
Sporidiobolus ruineniae CBS 5001 T AF070438 AY015433 
Sporidiobolus ruineniae var. coprophilus1 CBS 5811 T AF070434 AF444491 – 
Sporidiobolus salmonicolor mtA1 CBS 490 T AF070439 AY015434 
Sporidiobolus salmonicolor1 NRRL Y-17498 AF189979 AF444611 – 
Sporidiobolus sp. CBS 5541 AY015271 AY015435 
Sporobolomyces coprosmae CBS 7899 T AF189980 AF444577 
Sporobolomyces coprosmicola CBS 7897 T AF189981 AF444576 
Sporobolomyces dracophylli CBS 7900 T AF189982 AF444583 
Sporobolomyces elongatus CBS 8080 T AF189983 AF444561 
Sporobolomyces falcatus CBS 7368 T AF075490 AF444543 
Sporobolomyces foliicola CBS 8075 T AF189984 AF444521 
Sporobolomyces gracilis CBS 71 T AF189985 AF444578 
Sporobolomyces griseoflavus CBS 7284 T AF189986 AF444557 
Sporobolomyces inositophilus CBS 7310 T AF189987 AF444559 
Sporobolomyces kluyveri-nielii CBS 7168 T AF189988 AF444544 
Sporobolomyces lactophilus CBS 7527 T AF177411 AF444545 
Sporobolomyces linderae CBS 7893 T AF189989 AF444582 
Sporobolomyces marcillae CBS 4217 T AF070440 AY015437 
Sporobolomyces oryzicola CBS 7228 T AF189990 AF444546 
Sporobolomyces phyllomatis CBS 7198 T AF189991 AF444515 
Sporobolomyces roseus CBS 486 T AF070441 AY015438 
Sporobolomyces ruber CBS 7512 T AF189992 AF444550 
Sporobolomyces ruberrimus CBS 7500 T AF070442 AY015439 
*S. ruberrimus var. albus1 CBS 7501 T AF189993 AY015436 – 
S. ruberrimus var. albus1 CBS 7253 AF189994 AF444581 – 
Sporobolomyces salicinus CBS 6983 T AF189995 AF444511 
Sporobolomyces sasicola CBS 7285 T AF177412 AF444548 
Sporobolomyces singularis CBS 5109 T AF189996 AF444600 
Sporobolomyces subbrunneus CBS 7196 T AF189997 AF444549 
Sporobolomyces taupoensis CBS 7898 T AF177413 AF444592 
Sporobolomyces tsugae CBS 5038 T AF189998 AF444580 
Sporobolomyces xanthus CBS 7513 T AF177414 AF444547 
Sporobolomyces sp. CBS 6166 AF444724 AF444603  
Sterigmatomyces elviae CBS 5922 T AF177415 AF444551 
*Rhodotorula acuta CBS 7053 T AF189999 AF444512 – 
*Rhodotorula dulciaminis1 CBS 7288 T AF190000 AF444517 – 
Sterigmatomyces halophilus CBS 4609 T AF177416 AF444556 

Compared to the type strain: 1identical in D1D2, one substitution in ITS; 2one substitution in D1D2, identical in ITS; 3one substitution in D1D2, one substitution 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, five substitutions in ITS; 9three substitutions in D1D2, 14 substitutions in ITS.

All strains in Table 1 were sequenced in the D1/D2 and ITS regions, with the exception of the type strain of Cryptococcus marinus, which was not sequenced in the ITS region due to technical problems. DNA extraction, PCR amplification, purification, cycle sequencing and sequence analysis followed the protocol of Fell et al. [2]. Sequences were aligned with Megalign (DNAStar) and visually corrected. Phylogenetic analysis employed PAUP 4.0 using parsimony analysis, neighbor-joining analysis as a starting tree and tree bisection-reconnection. Gaps were treated as missing data. Bootstrap values were determined with PAUP; values less than 50% were not recorded on the tree figures.

In addition to the strains sequenced in our laboratory, the phylogenetic analyses included sequence data of Tremella by Chen [20] and Sporobolomyces by Valério et al. [21], Inacio, J. and Fonseca, A. (unpublished data) and Takashima and Nakase [22]. GenBank numbers for these additional strains, not reported in the tables, are listed in the tree figures.

Results

The basidiomycetous yeasts are distributed within three classes: Ustilaginomycetes, Urediniomycetes and Hymenomycetes [2,23–25]. The current report is directed to the latter two classes. In Figs. 1 and 2, trees based on analysis of the D1/D2 regions indicated the presence of four major lineages in each class, although some of this nomenclature must be regarded as provisional designations. Statistical bootstrap support for these lineages varied. Within the Hymenomycetes class, the Tremellales (<50%) may represent more than one lineage. Holtermannia corniformis and related strains, included in this study as members of the Tremellales (see Section 3.1.1), may constitute a separate lineage as suggested by the branch position in Fig. 1. However, in Fig. 3 a strong bootstrap support (100%) links the Holtermannia clade to the Tremellales lineage in the ITS tree. The Trichosporonales lineage (84%) has a 64% branch (Cryptococcus humicola: see Section 3.1.2), which may represent a sister lineage to the Trichosporonales (see Section 3.1.2). The Filobasidiales lineage has a strong support of 92%. The Cystofilobasidiales lineage (74%) is composed of generally strong-supported clades, which may represent multiple lineages (see Section 3.1.4). Similar levels of support were found among the Urediniomycetes: Microbotryum (<50%), Sporidiobolus (73%), Erythrobasidium (100%) and Agaricostilbum (74%). The clades depicted in the Figs. 1 and 2 consensus trees agree with the parsimony trees (Figs. 3–10), with the major exceptions of Cryptococcus marinus (see Section 3.1.1), Sporobolomyces griseoflavus and Reniforma strues (Section 3.2.3).

1

Hymenomycetes. Strict consensus of 100 equally parsimonious trees of the D1/D2 region of the large subunit rDNA (PAUP 4.0). Numbers on branches represent bootstrap percentages (>50%) from 100 full heuristic replications for the four lineages. Clades are signified to indicate their orientation within the lineages. Number of ingroup taxa=180, number of characters (NC)=632, constant characters (CC)=220, parsimony uninformative characters (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.

1

Hymenomycetes. Strict consensus of 100 equally parsimonious trees of the D1/D2 region of the large subunit rDNA (PAUP 4.0). Numbers on branches represent bootstrap percentages (>50%) from 100 full heuristic replications for the four lineages. Clades are signified to indicate their orientation within the lineages. Number of ingroup taxa=180, number of characters (NC)=632, constant characters (CC)=220, parsimony uninformative characters (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.

2

Urediniomycetes. Strict consensus of 100 equally parsimonious trees of the D1/D2 region of the large subunit rDNA (PAUP 4.0). Numbers on branches represent bootstrap percentages (>50%) from 100 full heuristic replications for the four lineages. Clades are signified to indicate their orientation 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. griseoflavus, (2) R. strues. Outgroup=members of the Cystofilobasidium lineage.

2

Urediniomycetes. Strict consensus of 100 equally parsimonious trees of the D1/D2 region of the large subunit rDNA (PAUP 4.0). Numbers on branches represent bootstrap percentages (>50%) from 100 full heuristic replications for the four lineages. Clades are signified to indicate their orientation 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. griseoflavus, (2) R. strues. Outgroup=members of the Cystofilobasidium lineage.

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 bootstrap percentages (>50%) from 100 full heuristic replications. Trichosporon cutaneum, Trichosporon jirovecii and Trichosporon moniliiforme were used as the outgroup. Due to space considerations the outgroup is not shown in this figure. T=type strain.

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 bootstrap percentages (>50%) from 100 full heuristic replications. Trichosporon cutaneum, Trichosporon jirovecii and Trichosporon moniliiforme were used as the outgroup. Due to space considerations the outgroup is not shown in this figure. T=type strain.

4

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

4

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

5

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

5

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

6

Cystofilobasidiales. D1D2: 78 equally parsimonious trees. NC=608, CC=428, PUC=28, PIC=152, TL=355, CI=0.704, RI=0.864. ITS: two equally 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.

6

Cystofilobasidiales. D1D2: 78 equally parsimonious trees. NC=608, CC=428, PUC=28, PIC=152, TL=355, CI=0.704, RI=0.864. ITS: two equally 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.

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 parsimonious trees. NC=721, CC=360, PUC=88, PIC=273, TL=847, CI=0.616, RI=0.734. 100 heuristic replications. Outgroup=S. xanthus, S. taupoensis and Sporobolomyces sasicola. See Fig. 1 for abbreviations.

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 parsimonious trees. NC=721, CC=360, PUC=88, PIC=273, TL=847, CI=0.616, RI=0.734. 100 heuristic replications. Outgroup=S. xanthus, S. taupoensis and Sporobolomyces sasicola. See Fig. 1 for abbreviations.

8

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

8

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

9

Microbotryum. D1D2: 100 equally parsimonious trees. NC=611, CC=362, PUC=78, PIC=171, TL=767, CI=0.454, RI=0.665. ITS: two equally parsimonious trees. NC=728, CC=244, PUC=95, PIC=389, TL=2711, CI=0. 362, RI=0. 571. 100 full heuristic replications. Outgroup=R. diobovatum, Rhodotorula araucariae and R. paludigenum. See Fig. 1 for abbreviations.

9

Microbotryum. D1D2: 100 equally parsimonious trees. NC=611, CC=362, PUC=78, PIC=171, TL=767, CI=0.454, RI=0.665. ITS: two equally parsimonious trees. NC=728, CC=244, PUC=95, PIC=389, TL=2711, CI=0. 362, RI=0. 571. 100 full heuristic replications. Outgroup=R. diobovatum, Rhodotorula araucariae and R. paludigenum. See Fig. 1 for abbreviations.

10

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

10

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

A major problem encountered in the presentation of urediniomycetous and hymenomycetous ITS trees is the dissimilarity of sequences between phylogenetic groups; as a result, sequences from two or more phylogenetic groups are difficult to align. This sequence variability can be illustrated by the number of base changes between closely related species. For example, in the Microbotryum lineage (see Section 3.2.3) the number of nucleotide differences in the D1/D2 region between Rhodotorula pustula and Rhodotorula pilati is five out of 591 nucleotides examined, whereas the ITS difference between the two species is 49/608. Another example: R. pilati and Sporobolomyces tsugae D1/D2 region variation is 7/621, ITS is 81/652. To avoid alignment difficulties between major phylogenetic groups in the ITS tree, we followed the D1/D2 system of clades (Figs. 1 and 2) and presented the data as individual lineages rather than as full trees.

Hymenomycetes

Tremellales (Fig. 3)

As a preliminary exploration of the phylogenetic relationship of the traditional yeast genera with the dimorphic genus Tremella, we included 20 species of Tremella in Fig. 3. This number is insufficient for a comprehensive study as Bandoni [26] estimated that Tremella includes 120 or more species. Moreover, several other dimorphic genera are currently recognized in the Tremellales [27]. Consequently, this incomplete sampling may account for the lack of well-supported clades and for the differences in branch arrangements and species relationships in the ITS and D1/D2 Tremellales trees. For example, Bullera dendrophila and Cryptococcus heveanensis appear related to Filobasidiella in the ITS tree, but not in the D1/D2 tree. Similarly, Sterigmatosporidium polymorphum is related to Fellomyces and Kockovaella species in the D1/D2 and 18S [28] trees, but does not belong to a specific clade in the ITS tree. Kirschner et al. [29] recently proposed the teleomorph of Sterigmatosporidium (Cuniculitrema polymorpha) based on the observation of Tremella-type basidia in a dimorphic fungus associated with tunnels of bark beetles.

Tremella spp. are generally separated from the yeasts and appear, in the ITS tree, as four major clades sensu Chen [20]: Foliacea, Fuciformis, Mesenterica and Aurantia. In the D1/D2 tree, the Fuciformis clade is part of a larger Mesenterica clade. Cryptococcus skinneri (Foliacea clade) is the only yeast species that appears to be related to Tremella spp. C. skinneri has been isolated from slime fluxes and insect frass associated with trees, which suggests a similar habitat as the Tremella spp. The position of the members of the Indecorata clade, which was indicated in the trees of Chen [20] and Fell et al. [2], is different on each tree, therefore systematic placement must await additional data. Representatives of other dimorphic tremellaceous fungi, Fibulobasidium, Sirobasidium and Trimorphomyces, are scattered on both trees in isolated positions. H. corniformis, which appears related to two unnamed strains of Cryptococcus (CBS 7712 and CBS 8804), is in contradictory positions in the D1/D2 and ITS trees. The D1/D2 trees (Figs. 1 and 3) suggest that the Holtermannia clade may represent a separate lineage in contrast to its position in the ITS Luteolus clade (bootstrap support 100%).

There are four distinguishable yeast clades in the Tremellales D1/D2 and ITS trees: Fellomyces, Bulleromyces, Luteolus and Filobasidiella. The Fellomyces clade includes species of Fellomyces and Kockovaella. The presence of conidia borne on elongate conidiophores is a common characteristic in this clade. The two genera differ by the formation of ballistoconidia in Kockovaella. This morphological characteristic is a useful taxonomic tool, however the phylogenetic significance can be questioned.

The Bulleromyces clade, which is weakly supported in both trees, requires a detailed study of the biology and molecular systematics of the included species and strains. Cryptococcus laurentii is a specific example. Sugita et al. [10] reported genetic diversity in the ITS and D1/D2 regions among clinical strains of C. laurentii, suggesting genetically distinct species, which may or may not correspond to unstudied synonyms of C. laurentii[19]. Sexual cycles also deserve attention, as mating has been reported between strains of C. laurentii[30]. Production of ballistoconidia is variable within the clade. Moreover, Cryptococcus cellulolyticus and Bullera pseudoalba have identical D1/D2 and ITS sequences, which would suggest conspecificity and the loss of ballistoconidial formation by the type strain of the former species.

The Luteolus clade includes Cryptococcus luteolus, several species of Bullera and the genus Dioszegia, which has been resurrected by Takashima et al. [28]. The basis for the generic emendation is the occurrence of Bullera crocea, B. aurantiaca, B. armeniaca and Cryptococcus hungaricus in 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 from other anamorphic genera based on other phenotypic characteristics. Takashima et al. [28] dismissed ballistoconidial formation as a significant taxonomic characteristic at the generic and species levels; they placed C. hungaricus and B. armeniaca in synonymy as Dioszegia hungarica. Despite the morphological differences, those two species have identical sequences in the ITS and D1/D2 regions and 83% nDNA relatedness [28]. Gácser et al. [31] reported, based on D1/D2, ITS and partial 18S analyses, genetic diversity among strains of C. hungaricus, which would suggest the presence of additional taxa in the genus Dioszegia. This might also be the case with Cryptococcus sp. CBS 8925 based on the D1/D2 and ITS trees (Fig. 3), which suggests inclusion of that strain in Dioszegia.

Synonymy has been reported for Bullera derxii and Bullera sinensis, based on classical systematics and identical D1/D2 sequences [32], and on 82–91% relative binding in DNA–DNA reassociation experiments [33]. In contrast, the two species differ by 12 bp in the SSU [33] and by 6 bp (transitions:transversions (ti:tv) 4:2) in the ITS region, which demonstrates the presence of genetic differences between the two taxa. The latter authors have proposed the new combination B. sinensis var. lactis for the type strain of Bullera alba var. lactis CBS 7237 based on intermediate values (40–50%) of DNA homology with B. sinensis and B. derxii; nucleotide differences between CBS 7237 and the type strains of the latter species were one in D1/D2 and 11 or five in ITS.

C. marinus, which is loosely attached to the Luteolus clade in the D1/D2 tree, is known from a single marine isolate. Unfortunately we were unable to obtain an ITS sequence to ascertain the position in the ITS tree. The D1/D2 consensus tree (Fig. 1) suggests that C. marinus is phylogenetically distinct from other clades in the Tremellales.

The Filobasidiella clade, which includes Filobasidiella bacillispora, Filobasidiella neoformans, Cryptococcus amylolentus and Tsuchiyaea wingfieldii, has strong support in the ITS and D1/D2 trees. The latter two species have similar ecological habitats as they were collected from insect frass in plants in South Africa [34,35]. They have identical ITS sequences, differ by one transversion base change in the D1/D2 region and are morphologically distinct. T. wingfieldii produces conidia on stalk-like conidiophores on solid medium [35,36], a characteristic not reported for C. amylolentus[34]. However, the micromorphological investigations of Sampaio et al. [37] demonstrated that the cellular outgrowths in T. wingfieldii are distinct from the typical stalks of species of Fellomyces, Kurtzmanomyces or Sterigmatomyces. Considering the close phylogenetic relationship between the two species, their taxonomic status should be evaluated by DNA hybridizations. The other two species in the clade, F. neoformans and F. bacillispora, are closely related; they are the causative agents of cryptococcosis in man, although the majority of the infections are attributed to F. neoformans. DNA relatedness between the two taxa is 55–63%[38] and base position differences between the type strains of the two 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).

Trichosporonales (Fig. 4)

The genus Trichosporon, which is the major constituent of the Trichosporonales, is characterized by the presence of true mycelium that disarticulates to arthroconidia. Guého et al. [40] recognized 19 species in the genus, a number that was expanded to 25 by Middelhoven et al. [41]. Sugita and Nakase [6], based on SSU analysis of 20 taxa, reported the presence of three groups of species within the genus Trichosporon. Our study, with an expanded number (33) of taxa and unidentified strains, concurs with the identity of these three groups, which correspond to our informal designations of the Gracile (Sugita and Nakase group III), Cutaneum (group I) and Ovoides (group II) clades. We recognize a fourth clade, Hyalodendron, which was represented in the SSU tree of Sugita and Nakase as a branch separate from the three groups.

The relationship of Hyalodendron to Trichosporon has been recognized [42,43], although Hyalodendron is considered to be a hyphomycete, due to the formation of dry colonies with true hyphae that may locally disarticulate. The morphology displayed by Hyalodendron may not be unusual in this clade. T. porosum, which belongs to the same clade, was originally described in the genus Apiotrichum. Investigation of the type strain of A. porosum (CBS 2040) revealed few budding yeast-like cells, which is a morphological characteristic of Hyalodendron. Transfer of A. porosum to Trichosporon was based on the discovery by Middelhoven et al. [41] of yeast strains with typical arthrosporulation and identical ITS and D1/D2 sequences to A. porosum.

Two species of Cryptococcus are among the Trichosporonales: C. curvatus, which is weakly related to the Cutaneum clade, and C. humicola. Sugita et al. [11] demonstrated that strains phenotypically similar to C. humicola represent a species complex, which (Figs. 1 and 4) may constitute a sister clade to the Trichosporonales.

Comparison of the ITS and D1/D2 trees in the Trichosporonales lineage (Fig. 4) demonstrated more variability in the D1/D2 region than in the ITS region. For example, no differences were observed in ITS sequence alignments between Trichosporon laibachii and Trichosporon multisporum, with seven differences (ti:tv 6:1) in the D1/D2 region; Trichosporon montevideense and Trichosporon domesticum were identical in ITS and differed at two positions (1:1) in D1/D2. SSU analyses also demonstrated nucleotide position differences within those pairs of species [6].

Filobasidiales (Fig. 5)

Physiological, biochemical and molecular (D1/D2) characteristics of species within the Filobasidiales were presented by Fonseca et al. [44]; that reference should be consulted for a detailed review. Our ITS and D1/D2 data demonstrate four clades within the Filobasidiales lineage: Aerius, Floriforme (=Magnus clade in Fonseca et al. [44]), Albidus and Cylindricus.

In the Aerius clade, Cryptococcus terreus, Cryptococcus himalayensis, Cryptococcus elinovii, PYCC 2935 and PYCC 4966 (Table 1) appear to represent a single species when compared on the basis of physiological properties and whole-cell protein patterns [44]. These strains, however, differ from each other by one to three D1/D2 nucleotides and one ITS bp. This list of synonyms is provisional, pending further study of other genes and DNA hybridization experiments. Cryptococcus phenolicus, which differed from C. terreus at three D1/D2 bp (ti:tv 3:0) and two ITS bp (0:2), has been designated as a separate species from C. terreus based on the ability of C. phenolicus to utilize sucrose, melibiose and raffinose [44]. Another strain, CBS 7944, had ITS (1:2) and D1/D2 (2:0) differences from C. terreus; however, there were no significant differences in assimilation tests between the two strains. The taxonomic status of CBS 7944 requires additional study. Comparison of the Aerius clade in the ITS and D1/D2 trees (Fig. 5) indicates greater sequence variability in the D1/D2 than in the ITS region; for example, C. terreus differs from Cryptococcus fuscescens at 10 D1/D2 (10:0) nucleotide positions and three ITS (1:2) positions. Differences in physiology between the two species amount to more than 10 assimilation tests, e.g. Fonseca et al. [44].

The Floriforme clade contains several species of the teleomorphic genus Filobasidium and anamorphic species in Cryptococcus. As noted by Fell et al. [2], the biologically distinct species Cryptococcus magnus, F. floriforme and F. elegans are indistinguishable in the D1/D2 region, whereas significant differences are present in the ITS region: C. magnus and F. elegans 21/627 (10:11); C. magnus and F. floriforme 15/627 (9:6); and F. floriforme and F. elegans 20/650 (12:8). All of the species of Filobasidium are in this clade, except F. capsuligenum, which differs from other species of Filobasidium by the ability to ferment glucose and possibly by morphology. Ultrastructural studies of F. capsuligenum demonstrated that the hyphal septa have cone-shaped vesicular parenthesomes, whereas F. floriforme hyphae lack parenthesomes [45,46].

Strains in the Albidus clade are widely distributed in nature and many of the species have been collected from polar regions. Some species, such as Cryptococcus albidisimilis and Cryptococcus adeliensis, have been the subject of cold-adapted enzyme research [47,48]. Sequence variability in the D1/D2 region is, in general, greater than in the ITS region. For example, the differences between C. diffluens and C. albidisimilis are D1/D2 9/612 (6:3), ITS 1/621 (0:1), Cryptococcus liquefaciens and C. albidisimilis 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. These strains have been isolated from lichens and fruiting bodies of fungi and share some physiological characteristics with members of the Aerius and Floriforme clades. However, no particular features were found to separate these species from other members of the Filobasidiales. Therefore, a decision on their taxonomic position requires additional biological information.

Cystofilobasidiales (Fig. 6)

The four bootstrap-supported clades in the Cystofilobasidiales lineage (Mrakia, Cystofilobasidium, Udeniomyces and Xanthophyllomyces) have extensive biological differences. The genera Mrakia and Cystofilobasidium form teliospores with holometabasidia. Udeniomyces is an anamorphic ballistoconidia-forming genus of plant-associated species that were originally described in the genus Bullera. Xanthophyllomyces produces an elongate holobasidium following cell-bud conjugation. The genus Phaffia represents an anamorphic state of Xanthophyllomyces. Based on ITS and IGS analyses [15], Phaffia rhodozyma is a species distinct from Xanthophyllomyces dendrorhous.

The species Cryptococcus aquaticus and Cryptococcus macerans are included respectively in the Mrakia and Cystofilobasidium clades. Mating between strains of C. macerans has been reported but a complete sexual cycle (expected to resemble Cystofilobasidium) was not observed [49]. Other anamorphic species among the Cystofilobasidiales include Cryptococcus huempii and Trichosporon pullulans, which are weakly associated with the Mrakia and Udeniomyces clades. Little is known about C. huempii, other than the description of a single strain collected in a rain forest in Chile [50]. T. pullulans has distinct morphology (lack of septal pores and enteroblastic budding) and physiology (low optimum temperature of growth at 17°C, assimilation of nitrate and absence of growth with lactate) from other species in the genus Trichosporon[40].

Sequence variability between species of the Cystofilobasidiales is, in general, greater in the ITS than D1/D2 regions. For example, Mrakia frigida and Mrakia gelida are identical in the D1/D2 region and differ by 15/64 (ti:tv 11:4) in ITS. X. dendrorhous and P. rhodozyma differ by 1/636 (1:0) in D1/D2 and 8/712 (6:2) in ITS.

Urediniomycetes

Erythrobasidium (Fig. 7)

The Erythrobasidium lineage includes genera with three distinct life cycles. Occultifur externus, isolated from plant litter, is a homothallic species that produces four-celled transversely septate basidia, with forcibly discharged basidiospores, directly from generative hyphae [51]. Sakaguchia dacryoidea, which has been isolated from such disparate locales as the open ocean and a brewery in France, has a heterothallic, bifactorial incompatibility system that includes the formation of teliospores and phragmometabasidia following mating [52]. Erythrobasidium hasegawianum was described from a single strain collected from an old beer yeast culture. The sexual state is reported as homokaryotic, with a holobasidium that arises directly on clamped, true mycelium [53]. However, this life cycle requires further examination in view of the Sampaio et al. [51] report that the mycelium has incomplete clamp connections and the interpretation that the alleged holobasidia are conidiogenous cells. All members of this lineage produce deeply pigmented orange or red colonies. Perrier et al. [54] found qualitative differences in the carotenoids present in Rhodotorula spp. belonging to either the Erythrobasidium or the Sporidiobolus clades. Another distinguishing feature of members of the Erythrobasidium lineage has been found by Takashima et al. [55] who reported the absence of fucose in the cell walls of all taxa in this lineage when compared to other urediniomycetous yeasts.

The Erythrobasidium lineage includes three major clades, Occultifur, Aurantiaca and Gracilis, which are supported by bootstrap analysis in the ITS and D1/D2 regions. O. externus is the only sexual species specifically associated with one of these clades. The other two clades are composed of species of Rhodotorula and Sporobolomyces. Separation of the majority of the species in the Erythrobasidium lineage can be accomplished with either ITS or D1/D2 analyses. An exception is S. oryzicola and S. coprosmae, which require ITS analysis for identification (D1/D2: 0/629, ITS: 4/599 (1:3)). The two species differ in their ability to utilize 2-keto-d-gluconate, which is considered by Boekhout and Nakase [56] to be a significant taxonomic character. DNA homology between the two species was found to be ca. 50%[57], which suggests that the two taxa are sibling species.

Sporidiobolus (Fig. 8)

The Sporidiobolus lineage includes species of the teliosporic genera Rhodosporidium and Sporidiobolus and their anamorphic counterparts in the genera Rhodotorula and Sporobolomyces. All the species usually produce pigmented colonies. The lineage is divided into four supported clades Glutinis, Sphaerocarpum, Ruineniae and Johnsonii. Members of the Sporidiobolus clade have been reviewed by Gadanho and Sampaio [58], Gadanho et al. [59] and Valério et al. [21]. The consensus of this information demonstrated that the Glutinis clade, sensu Gadanho and Sampaio [58] consisted of the two clades that we designated as Glutinis and Sphaerocarpum. Our D1/D2 tree concurs with the Gadanho and Sampaio concept, however the difference in branch arrangements between the ITS and D1/D2 trees prompted additional nomenclature.

The Glutinis and Sphaerocarpum clades consist of species of Rhodosporidium and Rhodotorula, the Johnsonii clade includes species of the ballistoconidia-forming genera Sporidiobolus and Sporobolomyces, whereas the Ruineniae clade is mixed. This general separation lends support to the continued use of ballistoconidia as a taxonomic tool as argued by Gadanho et al. [59], although questioned in the separation of species of Cryptococcus and Bullera in the Tremellales (see above).

Some species within the Sporidiobolus lineage are closely related based on sequence analysis. For example, Rhodotorula glutinis and Rhodotorula graminis are identical in the ITS region and differ by one transverse substitution in the D1/D2 region. The two species have been shown to be distinct on standard physiological properties [60] and on the basis of 30% nDNA relatedness [58,61]. The type strains of Rhodosporidium babjevae and R. glutinis differ in the ITS by 4/609 (ti:tv 3:1) and D1/D2 by 2/604 (2:0); nDNA hybridizations of strains of these two species demonstrate 25–61% relatedness [58]. In apparent contrast, Sporobolomyces holsaticus and Sporidiobolus johnsonii are identical in D1/D2, differ at 5 bp (4:1) in ITS and register 93% DNA hybridization [62]. Consequently, in that lineage, both the ITS and D1/D2 regions are required for species identification.

Microbotryum (Fig. 9)

The Microbotryum ITS and D1/D2 trees are notable for their lack of structured bootstrap-supported clades. One reason is an incomplete sampling of taxa, namely the dimorphic plant parasites of the Microbotryales, sensu Bauer et al. [63] for which ITS sequences are not available. For the purpose of orientation, we labeled two weak clades Colacogloea and Leucosporidium. There are two sexual states among yeasts in this lineage, specifically in the teliospore-forming genera Leucosporidium and Mastigobasidium. The distinguishing features of Mastigobasidium are the formation of ballistoconidia and the mechanism of teliospore germination, in which elongate hyphae with curved phragmometabasidia form [64]. Septate metabasidia in Leucosporidium are generally not curved and develop directly from the teliospore or on short hyphal extensions [65].

Yeast species in the Microbotryum lineage include representatives of the anamorphic genera Rhodotorula, Sporobolomyces, Reniforma and Bensingtonia. The vegetative cells of Reniforma are unique among the yeasts; they are kidney-shaped with an elaborate skirt that circles the edge of the cell [66]. R. strues appears on a long branch with Heterogastridium pycnidiodeum (teleomorph name, anamorphic name=Hyalopycnis blepharistoma), a relationship that may be artificial due to long branch attraction. This relationship is not confirmed in the Fig. 1 consensus tree, which suggests that R. strues may represent a separate lineage. Similarly, S. griseoflavus, which is unsupported in any of the ITS and D1/D2 clades, may represent a separate 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 sequence variability was found in the ITS region, for example Sporobolomyces singularis and Rhodotorula lignophila differences are: D1/D2 1/619 (ti:tv 1:0) and ITS 12/628 (9:3); R. pustula and Rhodotorula buffoni D1/D2 3/592 (1:2) and ITS 31/617 (17:14). Synonymy between Rhodotorula fujisanensis, Rhodotorula futronensis and Rhodotorula nothofagi has been suggested by Sampaio and Fonseca [67] based on similarity of phenotypic data. That hypothesis is corroborated for R. fujisanensis and R. futronensis on the basis of identical D1/D2 and ITS sequences, but not for R. nothofagi (D1/D2 2:1; ITS 6:1). A formal taxonomic proposal requires confirmation by DNA hybridization experiments and additional information on the sexual cycle of R. fujisanensis[60]. Another strain in this lineage, CBS 6370, is currently included in Rhodotorula foliorum (CBS Yeast Database), although D1/D2 and ITS sequence data suggest that CBS 6370 represents a distinct species, as demonstrated with the protein data of Vancanneyt et al. [68].

Agaricostilbum (Fig. 10)

The Agaricostilbum lineage includes small, bootstrap-supported clades, which we informally labeled as Kondoa, Agaricostilbum, Sasicola, Kurtzmanomyces and Subbrunneus. A general morphological feature in this lineage is the presence of blastoconidia on denticles or on short to long conidiophores that can develop sympodially, particularly among some species of Bensingtonia and Sporobolomyces. Sterigmatomyces produces stalk-like conidiophores that separate in the mid-region for bud release. In contrast, Kurtzmanomyces bud release is at the terminus of the stalk. Ballistoconidia are present in Kurtzmanomyces insolitus[37] and species of Bensingtonia and Sporobolomyces. Separation of the latter two genera is based on the major ubiquinone (CoQ) present: CoQ9 in Bensingtonia and CoQ10 in Sporobolomyces. Analysis of the trees depicted in Fig. 10 shows that there is no phylogenetic basis for this separation. Furthermore, two other species of Bensingtonia belong to the Microbotryum lineage, viz. Bensingtonia yamatoana and Bensingtonia intermedia (anamorph of Mastigobasidium intermedium). A taxonomic rearrangement of those two genera that reflects their phylogeny is hampered by the ambiguous connection to other genera as revealed by the sequence data. The solution to this problem will require additional biological information, namely the observation of sexual cycles in these clades.

The Kondoa clade contains the only known yeast sexual cycle in the Agaricostilbum lineage. Kondoa spp. produce auricularioid basidia directly on the hyphae in the absence of teliospores [69]. The namesake of the lineage, sensu Swann and Taylor [23–25], Agaricostilbum hyphaenes, resides in the Agaricostilbum clade. A. hyphaenes is a dimorphic fungus that inhabits palms and produces synnemata-like basidiomata composed of long auricularioid basidia with passively abstricted basidiospores. The other teleomorphic genus in the lineage is Chionosphaera, which is also dimorphic and loosely linked to the Kurtzmanomyces clade. The sexual state of Chionosphaera can arise on culture media with formation of holobasidia of the Filobasidium-type.

Species in the Agaricostilbum lineage can be identified by either the D1/D2 or ITS regions, however differences are usually more pronounced in the ITS region. For example, Sporobolomyces xanthus and Sporobolomyces taupoensis D1/D2 is 4/623 (ti:tv 4.0), ITS 16/578 (9:7) or Kondoa aeria and PYCC 5566 D1/D2 1/638 (0:1), ITS 34/635 (17:17). The latter represents an unnamed Bensingtonia sp., which can be distinguished from K. aeria by the presence of ballistoconidia, the absence of a sexual cycle and differences in assimilation tests (Fonseca, A. and Inacio, J., unpublished data).

Sequence identity/variability between mating strains

Sequence variability between strains within a species was examined by comparison of ITS and D1/D2 sequences of mating types for 14 species (Table 3). The results demonstrated identical ITS and D1/D2 sequences for mating strains within Bulleromyces albus, Bulleromyces sp., Filobasidium uniguttulatum, M. intermedium, R. babjevae, Rhodosporidium sphaerocarpum and Rhodosporidium paludigenum. Sequence differences between mating strains were observed ranging from one single base change in the ITS or D1/D2 regions to multiple base changes in both regions. For example, in Rhodosporidium kratochvilovae one mating strain (PYCC 4785) differed by one transversion in ITS. Single base differences could be attributed to errors during strand synthesis or sequence analysis. However, PCR reactions, sequencing and analysis were repeated to confirm the differences. Several species demonstrated greater sequence differences: C. macerans, F. capsuligenum, Rhodosporidium diobovatum, S. dacryoidea, Sporidiobolus pararoseus and Sporidiobolus salmonicolor (Table 3). For example, mating strains of F. capsuligenum had eight base changes in D1/D2 and 10 in ITS. The number of transitions and transversions in the D1/D2 and ITS regions for mating strains was found to be in the same range as among closely related species. Strains of S. salmonicolor (CBS 490) and S. johnsonii (CBS 2630) had a concentrated region of eight nucleotide changes and two nucleotide indels in a 10-bp region in the ITS2 region. These ‘hot spots’ of nucleotide changes were found infrequently among closely related species and there did not appear to be a particular pattern for the location of the ‘hot spots’ or other base differences either among mating strains or closely related species. Unfortunately, the ancillary data (fertility of the progeny from sexual recombinations and DNA hybridization analyses), required to ascertain the phylogenetic status of these sequence-distinct mating strains, are not available.

3

Comparison of ITS and D1/D2 sequences of mating types

Species Mating strains Number of different nucleotides between groups (transitions:transversions) 
Bulleromyces albus CBS 501 mtB, CBS 500 mtA, CBS 6302 mtB Nd 
Bulleromyces sp. PYCC 5740 mtA1, PYCC 5690 mtA1, PYCC 5691 mtA2, PYCC 5739 mtA2 Nd 
Cryptococcus macerans CBS 2206 mta, CBS 2425 mtα D1D2 (3:0); ITS (6:5) 
Filobasidium capsuligenum CBS 4736 mtα, CBS 6219 mtα* Nd 
 CBS 1906 mta, CBS 4381 mta Nd 
  The two groups differ by: D1D2 (6:2); ITS (6:3) 
  *(CBS 6219 is different from all the other strains by a 13-bp deletion) 
Filobasidium uniguttulatum CBS 1730 mta, CBS 1727 mtα Nd 
Mastigobasidium intermedium CBS 7226 mta, CBS 7281 mtα Nd 
Rhodosporidium babjevae CBS 7808 mtA1, CBS 9071 mtA2 Nd 
Rhodosporidium diobovatum CBS 6085 mtα, 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 Nd 
Rhodosporidium sphaerocarpum CBS 5939 mta, CBS 9082 mtA, CBS 9075 mta, CBS 9079 mta, CBS 9080 mta Nd 
Sakaguchia dacryoidea CBS 6353 mtA1B1, CBS 6356 mtA1B2 Nd 
 CBS 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) 
Species Mating strains Number of different nucleotides between groups (transitions:transversions) 
Bulleromyces albus CBS 501 mtB, CBS 500 mtA, CBS 6302 mtB Nd 
Bulleromyces sp. PYCC 5740 mtA1, PYCC 5690 mtA1, PYCC 5691 mtA2, PYCC 5739 mtA2 Nd 
Cryptococcus macerans CBS 2206 mta, CBS 2425 mtα D1D2 (3:0); ITS (6:5) 
Filobasidium capsuligenum CBS 4736 mtα, CBS 6219 mtα* Nd 
 CBS 1906 mta, CBS 4381 mta Nd 
  The two groups differ by: D1D2 (6:2); ITS (6:3) 
  *(CBS 6219 is different from all the other strains by a 13-bp deletion) 
Filobasidium uniguttulatum CBS 1730 mta, CBS 1727 mtα Nd 
Mastigobasidium intermedium CBS 7226 mta, CBS 7281 mtα Nd 
Rhodosporidium babjevae CBS 7808 mtA1, CBS 9071 mtA2 Nd 
Rhodosporidium diobovatum CBS 6085 mtα, 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 Nd 
Rhodosporidium sphaerocarpum CBS 5939 mta, CBS 9082 mtA, CBS 9075 mta, CBS 9079 mta, CBS 9080 mta Nd 
Sakaguchia dacryoidea CBS 6353 mtA1B1, CBS 6356 mtA1B2 Nd 
 CBS 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) 

Discussion

Sequence analysis of either the D1/D2 or ITS regions can be used for the identification of the majority of the species of basidiomycetous yeasts. Examples (C. magnus and F. elegans) have been presented where certain closely related species could not be separated with the D1/D2 region, but they could be differentiated with the ITS region. Other examples in the Filobasidiales and Trichosporonales lineages demonstrated the opposite situation, in spite of the expected greater variability in the ITS region, which is less constrained for mutations. As found between mating strains, the D1/D2 and ITS regions showed a considerable variation in the ti:tv and in location of the changes, so that there did not appear to be distinct locations within or between D1 and D2 or ITS1 and ITS2 regions. Hotspot sites of mutation were found in the 3′ D1/D2 region among some strains, but a trend was not observed. Although ti/tv rate ratios were not provided in this study, the rates are known to vary with genes and lineages. However, biological explanations for these variations in rates have not been determined [70]. As a consequence, a boundary between species (a prerequisite number of nucleotide differences in either regions to separate species) was not defined by our study. The answer undoubtedly lies not in the number of differences, but rather in the significance and function of the site mutations. Studies, such as secondary structure [71], would provide insight into the question. Consequently, when examining pure number differences between different pairs of species, the answer varies. R. glutinis and R. graminis are identical in the ITS region and differ at one transition site in D1D2; they are considered to be separate species based on 30% nDNA relatedness. In contrast, S. johnsonii and S. holsaticus are synonyms due to 93% nDNA relatedness; they are identical in the D1/D2 region and differ by 5 bp in ITS.

The question of molecular characterization of species is further complicated by differences in the D1/D2 and ITS regions (Table 3) between mating strains. In some cases, the number and distribution of nucleotide changes suggest that the strains represent distinct intermating species. An important avenue to discover is the level of relationship between mating strains. As a model, the in-depth fertility and genetic studies of F. neoformans and F. bacillispora have concluded that the two taxa, which differ by ti:tv 3:1 in the ITS1 region, are separate species [39,72]. Similar studies with sequence-divergent mating strains (Table 3) offer an opportunity to examine the role of sequence analysis in yeast systematics. The problem is exemplified by mating strains of CBS 490 (S. salmonicolor) and CBS 2630, which differ at seven positions in the D1/D2 and 26 in the ITS. The sequence alignment of one of these mating strains, CBS 2630, indicates a close relationship to the homothallic species S. johnsonii (CBS 5470). The latter two strains are identical in D1/D2 and differ at five positions in ITS. Mating between those strains has not been observed.

The ITS and D1/D2 tree structures are not fully resolved as few clades show strong support, which results in insufficient evidence for nomenclatural decisions, particularly at the generic level. Consequently, clades have informal names and consist of species of different genera. However, the discovery of basidiomycetous yeast species is in a primordial phase as possibly only 1% of the species in nature have been collected and described. These phylogenetic trees are dynamic, therefore lineages and clades will split, and as new phylogenetic groups appear the branching arrangements will change and generic identities and relationships should become evident. At the present point of discovery, the trees provided by ITS, D1/D2 and other genes [73,74] provide focal points for biological and systematic analysis of these yeasts. A point to recognize is that the biology of the species must be the primary criterion in yeast systematics. A sequence, per se, does not describe a species and descriptions of new species should not solely rely on nucleotide data.

Acknowledgements

This research was supported by funding from the Ocean Sciences Division, National Science Foundation and from Genetic Vectors, Inc., Miami, FL, USA. David Yarrow, C. P. Kurtzman, Helen Vischniac and Brian Steffenson are acknowledged for providing strains and information regarding these strains. J. P. Sampaio and T. Boekhout also provided critical information.

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