Phylogeny of tremellomycetous yeasts and related dimorphic and filamentous basidiomycetes reconstructed from multiple gene sequence analyses.

The Tremellomycetes (Basidiomycota) contains a large number of unicellular and dimorphic fungi with stable free-living unicellular states in their life cycles. These fungi have been conventionally classified as basidiomycetous yeasts based on physiological and biochemical characteristics. Many currently recognised genera of these yeasts are mainly defined based on phenotypical characters and are highly polyphyletic. Here we reconstructed the phylogeny of the majority of described anamorphic and teleomorphic tremellomycetous yeasts using Bayesian inference, maximum likelihood, and neighbour-joining analyses based on the sequences of seven genes, including three rRNA genes, namely the small subunit of the ribosomal DNA (rDNA), D1/D2 domains of the large subunit rDNA, and the internal transcribed spacer regions (ITS 1 and 2) of rDNA including 5.8S rDNA; and four protein-coding genes, namely the two subunits of the RNA polymerase II (RPB1 and RPB2), the translation elongation factor 1-α (TEF1) and the mitochondrial gene cytochrome b (CYTB). With the consideration of morphological, physiological and chemotaxonomic characters and the congruence of phylogenies inferred from analyses using different algorithms based on different data sets consisting of the combined seven genes, the three rRNA genes, and the individual protein-coding genes, five major lineages corresponding to the orders Cystofilobasidiales, Filobasidiales, Holtermanniales, Tremellales, and Trichosporonales were resolved. A total of 45 strongly supported monophyletic clades with multiple species and 23 single species clades were recognised. This phylogenetic framework will be the basis for the proposal of an updated taxonomic system of tremellomycetous yeasts that will be compatible with the current taxonomic system of filamentous basidiomycetes accommodating the 'one fungus, one name' principle.

Introduction

Unicellular basidiomycetes and dimorphic basidiomycetes with a stable free-living unicellular state during their life cycles are recognised as basidiomycetous yeasts (Boekhout ). They occur in all three subphyla of the Basidiomycota, namely Agaricomycotina, Pucciniomycotina and Ustilaginomycotina, which are presently recognised on the basis of molecular phylogenetic analyses (Fell et al., 2000, Scorzetti et al., 2002, James et al., 2006, Hibbett et al., 2007, Wuczkowski et al., 2011). Yeast taxa in the Agaricomycotina occur only in the basal Tremellomycetes lineage (Hibbett, 2006, Boekhout et al., 2011, Weiß et al., 2014). Phenotypic and molecular analyses revealed a close affiliation of basidiomycetous yeasts with various groups of filamentous basidiomycetes (Millanes ). However, yeasts and filamentous fungi have conventionally been studied by different scientific communities and classified using different criteria, resulting in the developments of hitherto independent taxonomic systems of the two groups of fungi. Recent molecular phylogenetic studies have shown the incompatibility between the taxonomic system of basidiomycetous yeasts and that of filamentous basidiomycetes. Furthermore many currently recognised genera of tremellomycetous yeasts, which are mainly defined based on phenotypic characters, are strikingly polyphyletic. For example, species of the genus Cryptococcus are located in all currently recognised orders of Tremellomycetes and occur intermingled with species of other genera, such as Bullera (Fell et al., 2000, Boekhout et al., 2011, Fonseca et al., 2011, Millanes et al., 2011, Weiß et al., 2014). The polyphyletic nature of the traditionally defined teleomorphic genus Tremella, which is usually dimorphic, is also remarkable. Several monophyletic clades have been recognised among Tremella species which occur interspersed with other teleomorphic and anamorphic genera (Boekhout et al., 2011, Millanes et al., 2011, Weiß et al., 2014). These problems existing in the current taxonomic systems of both yeasts and filamentous fungi in the Tremellomycetes remain to be resolved.

The high-level classification of Basidiomycota has been updated with results from the Assembling the Fungal Tree of Life (AFTOL) project that used a multigene sequence analysis approach (Lutzoni et al., 2004, James et al., 2006, Hibbett et al., 2007). However, only a limited number of basidiomycetous yeast taxa were included in that project, making it impossible to propose a corresponding revision of the taxonomic system of basidiomycetous yeasts. Consequently, the artificial classification system of these organisms largely remained in the latest edition of ‘The Yeasts, a Taxonomic Study’ [hereafter referred to as ‘The Yeasts’] (Kurtzman ), due to the lack of reliable multigene phylogenetic studies of these yeasts.

The requirement for revising the taxonomic system of tremellomycetous yeasts has been emphasised by recent progress in biodiversity studies of yeasts and by the change of fungal nomenclature adopting the ‘one fungus = one name’ concept (Hawksworth 2011). Molecular taxonomic studies have resulted in the availability of a comprehensive sequence database of the large subunit of the ribosomal RNA gene (LSU rDNA) D1/D2 domains and the ITS (including 5.8S) regions of rDNA for almost all known basidiomycetous yeast species (Fell et al., 2000, Scorzetti et al., 2002, Wang and Bai, 2008, Schoch et al., 2012). The rDNA sequence databases have become a molecular platform for rapid identification of yeasts, resulting in continued discovery of new taxa in recent years (Boekhout, 2005, Wang and Bai, 2008, Wuczkowski et al., 2011). The addition of these new species has contributed to the increase of the polyphyletic nature of many basidiomycetous yeast genera. For example, the distribution of Bullera species has expanded from Tremellales and Filobasidiales (Boekhout & Nakase 1998) to Trichosporonales (Nakase et al., 2002, Fungsin et al., 2006). With each new species being taxonomically misplaced, the chaos of the taxonomic system increases. Therefore, an updated taxonomic system is imperative for the correct placement of the vast amount of hidden yeast diversity.

Similar to filamentous fungi, many yeast species have separate teleomorphic and anamorphic names. As regulated by the new International Code of Nomenclature for algae, fungi and plants (Melbourne Code) (McNeill ), after January 1 2013, only one name is legitimate regardless of whether or not a sexual state exists. An updated taxonomic system, especially the redefinition of genera based on a robust multigene phylogeny, will be required for the name choices and to minimise the possibility of name changes in the future. The purpose of this study is to confidently resolve the phylogenetic relationships among tremellomycetous yeasts and dimorphic fungi based on multiple gene sequence analyses, resulting in a framework that allows us to update the taxonomic system of yeasts and related taxa in the Tremellomycetes.

Materials and methods

Taxon sampling

A total of 294 tremellomycetous yeast strains were included in this study, which covered the type strains of 286 currently recognised species and varieties, the type strains of six synonyms, and two additional strains with mating types opposite to those of the type strains (Table 1). From the 240 tremellomycetous yeast species and varieties included in the latest edition of ‘The Yeasts’ (Kurtzman ), 234 were included in this study. In addition, 52 tremellomycetous yeast species which were published too late for inclusion in the book were also employed in this study. The taxa sampled covered 16 teleomorphic and 19 anamorphic genera. The type strains of two pucciniomycetous and one ustilaginomycetous yeast species were employed as outgroup (Table 1).

Table 1

List of tremellomycetous yeasts and dimorphic taxa employed. The sequences with GenBank numbers in bold are determined in this study.

Lineage/clade	Species	Strain	ITS (5.8S)	D1D2	SSU	RPB1	RPB2	TEF1	CYTB
Cystofilobasidiales
Cystofilobasidium	Cystofilobasidium bisporidii*	CBS 6346T	KF036597	EU085532	AB072225	KF036419	KF036832	KF037103	KF423238
	C. capitatum*	CBS 6358T	AF139627	AF075465	D12801	KF036420	KF036833	KF037104	/
	C. ferigula*	CBS 7202T	KF036598	CBS database	AB032628	/	/	KF037105	KF423239
	C. infirmominiatum*	CBS 323T	AF444400	AF075505	AB072226	/	KF036834	KF037106	KF423240
	C. lacus-mascardii*	CBS 10642T	EF613495	AY158642	KF036665	KF036421	KF036835	KF037107	KF423241
	C. macerans*	CBS 10757T	EU082231	EU082225	KF036666	KF036423	KF036837	KF037109	/
	C. macerans*	CBS 2206	AF444329	AF189848	AB032642	KF036422	KF036836	KF037108	KF423242
Guehomyces	Guehomyces pullulans	CBS 2532T	AF444417	EF551318	AB001766	KF036478	KF036892	KF037155	AF175778
	Tausonia pamirica	CBS 8428T	KF036600	EF118825	KF036692	/	/	/	/
huempii	Cryptococcus huempii	CBS 8186T	AF444322	AF189844	AB032636	KF036377	KF036790	KF037062	KF423200
	Mrakia curviuscula*	CBS 9136T	KF036599	EF118826	KF036684	KF036510	KF036924	KF037185	KF423313
Itersonilia	Itersonilia perplexans	CBS 363.85T	AB072233	AJ235274	AB072228	/	KF036900	/	KF423296
	Udeniomyces pannonicus	CBS 9123T	AB072229	AB077382	AB072227	KF036579	KF036996	KF037251	/
Mrakia	Mrakia blollopis*	CBS 8921T	AY038826	AY038814	KF036683	KF036509	KF036923	KF037184	/
	M. frigida*	CBS 5270T	AF144483	AF075463	D12802	KF036511	KF036925	KF037186	/
	M. nivalis*	CBS 5266T	AF144484	AF189849	DQ831017	KF036513	KF036927	KF037188	/
	M. gelida*	CBS 5272T	AF144485	AF189831	KF036685	KF036512	KF036926	/	KF423314
	M. stokesii*	CBS 5917T	AF144486	AF189830	KF036687	KF036515	KF036929	KF037190	/
	M. psychrophila*	CBS 10828T	EU224267	EU224266	/	/	/	/	/
	M. robertii*	CBS 8912T	AY038829	AY038811	KF036686	KF036514	KF036928	KF037189	KF423315
	Mrakiella aquatic	CBS 5443T	AF410469	AF075470	AB032621	KF036516	KF036930	KF037191	KF423316
	M. cryoconiti	CBS 10834T	AJ866976	GQ911524	/	/	/	KF037192	/
	M. niccombsii	CBS 8917T	AY029346	AY029345	KF036688	KF036517	KF036931	KF037193	KF423318
Udeniomyces	Udeniomyces megalosporus	CBS 7236T	AF444408	AF075510	D31657	KF036578	KF036995	/	/
	U. puniceus	CBS 5689T	AF444435	AF075519	D31658	KF036580	DQ836008	/	/
	U. pyricola	CBS 6754T	AF444402	AF075507	D31659	KF036581	KF036997	KF037252	/
Phaffia	Phaffia rhodozyma	CBS 5905T	AF139629	AF189871	KF036689	/	KF036933	KF037195	KF423320
	Xanthophyllomyces dendrorhous*	CBS 7918T	AF139628	AF075496	D31656	KF036582	KF036998	KF037253	KF423356
Filobasidiales
aerius	Cryptococcus aerius	CBS 155T	AF145324	AF075486	AB032614	KF036336	KF036748	KF037019	KF423160
	C. fuscescens	CBS 7189T	AF145319	AF075472	AB032631	KF036372	KF036784	KF037056	KF423195
	C. keelungensis	CBS 10876T	EF621565	EF621562	KF036637	/	KF036792	KF037064	KF423202
	C. phenolicus	CBS 8682T	AF444351	AF181523	KF036647	KF036394	KF036808	KF037079	KF423217
	C. terreus	CBS 1895T	AF444319	AF075479	AB032647	KF036409	KF036823	KF037094	KF423231
	C. elinovii	CBS 7051T	AF145318	AF137604	KF036631	KF036365	KF036777	KF037050	KF423189
	C. terricola	CBS 4517T	AF444350	AF181520	KF036659	KF036410	KF036824	KF037095	/
albidus	C. adeliensis	CBS 8351T	AF145328	AF137603	KF036610	KF036335	KF036747	KF037018	KF423159
	C. albidosimilis	CBS 7711T	AF145325	AF137601	KF036612	KF036338	KF036750	KF037021	KF423162
	C. albidus var. Albidus	CBS 142T	AF145321	AF075474	AB032616	/	KF036751	KF037022	KF423163
	C. albidus var. Kuetzingii	CBS 922	AF444313	AF181504	KF036613	KF036339	KF036752	KF037023	KF423164
	C. albidus var. Kuetzingii	CBS 1926T	AF145327	AF137602	AB032639	KF036340	KF036753	KF037024	KF423165
	C. albidus var. Ovalis	CBS 5810T	AF145329	AF137605	KF036614	/	KF036754	KF037025	KF423166
	C. antarcticus var.antarcticus	CBS 7687T	AF145326	AF075488	AB032620	KF036345	/	KF037030	KF423169
	C. antarcticus var. circumpolaris	CBS 7689T	KF036586	CBS database	KF036618	KF036346	KF036759	KF037031	KF423170
	C. bhutanensis	CBS 6294T	AF145317	AF137599	AB032623	KF036352	KF036765	KF037037	KF423176
	C. cerealis	CBS 10505T	FJ473371	FJ473376	KF036624	KF036356	/	KF037041	KF423180
	C. diffluens	CBS 160T	AF145330	AF075502	KF036630	KF036363	KF036775	KF037048	KF423187
	C. friedmannii	CBS 7160T	AF145322	AF075478	AB032630	KF036371	KF036783	KF037055	KF423194
	C. liquefaciens	CBS 968T	AF444345	AF181515	KF036638	KF036381	KF036794	KF037066	KF423203
	C. randhawai	CBS 10160T	AJ876528	AJ876599	KF036650	/	/	/	/
	C. saitoi	CBS 1975T	AF444372	AF181540	KF036651	KF036400	KF036814	KF037085	KF423222
	C. uzbekistanensis	CBS 8683T	AF444339	AF181508	KF036660	KF036412	KF036826	KF037096	KF423232
	C. vishniacii	CBS 7110T	AF145320	AF075473	AB032650	KF036414	/	KF037098	KF423234
cylindricus	Bullera taiwanensis	CBS 9813T	/	AB079065	AB072234	/	/	/	/
	Cryptococcus cylindricus	CBS 8680T	AF444360	AF181534	KF036628	KF036360	KF036772	KF037045	KF423184
	C. silvicola	CBS 10099T	AY898956	AY898955	KF036653	KF036402	KF036816	KF037087	KF423224
Filobasidium	C. chernovii	CBS 8679T	AF444354	AF181530	KF036625	KF036357	KF036769	KF037042	KF423181
	C. magnus	CBS 140T	AF190008	AF181851	AB032643	KF036383	KF036796	KF037068	KF423205
	C. oeirensis	CBS 8681T	AF444349	AF181519	KF036644	/	KF036804	KF037076	KF423213
	C. stepposus	CBS 10265T	DQ222455	DQ222456	KF036655	KF036405	KF036819	KF037090	KF423227
	C. wieringae	CBS 1937T	AF444373	AF181541	KF036663	KF036416	KF036829	KF037100	KF423236
	Filobasidium elegans*	CBS 7640EXT	AF190006	AF181548	KF036678	KF036474	KF036888	/	KF423285
	F. floriforme*	CBS 6241EXT	AF190007	AF075498	D13460	KF036475	KF036889	/	/
	F. globisporum*	CBS 7642EXT	AF444336	AF075495	AB075546	KF036476	KF036890	KF037153	KF423286
	F. uniguttulatum*	CBS 1730T	AF444302	AF075468	AB032664	KF036477	KF036891	KF037154	KF423287
gastricus	Cryptococcus aciditolerans	CBS 10872T	KF036583	AY731790	KF036609	/	KF036746	KF037017	KF423158
	C. agrionensis	CBS 10799T	KF036584	EU627786	KF036611	KF036337	KF036749	KF037020	KF423161
	C. gastricus	CBS 2288T	AF145323	AF137600	AB032633	KF036373	KF036785	KF037057	AB040652
	C. gilvescens	CBS 7525T	AF444380	AF181547	AB032634	KF036374	KF036786	KF037058	KF423196
	C. ibericus	CBS 10871T	KF036592	AY731791	KF036636	KF036379	KF036791	KF037063	KF423201
	C. metallitolerans	CBS 10873T	KF036594	AY731789	KF036639	KF036385	KF036798	KF037070	KF423207
single-species clade	C. arrabidensis	CBS 8678T	AF444362	AF181535	KF036621	KF036349	KF036762	KF037034	KF423173
	Filobasidium capsuligenum*	CBS 1906T	AF444381	AF363642	AB075544	KF036473	KF036887	KF037152	AB040654
Holtermanniales
Holtermanniella	Holtermanniella festucosa	CBS 10162T	AY462120	AY462119	KF036633	KF036367	KF036779	KF037052	KF423191
	H. mycelialis	CBS 7712T	AF408417	AJ311450	KF036641	KF036388	KF036801	KF037073	KF423210
	H. nyarrowii	CBS 8804T	AY006481	AY006480	KF036643	KF036390	KF036803	KF037075	KF423212
	H. wattica	CBS 9496T	FJ473373	AY138478	KF036662	KF036415	KF036828	KF037099	KF423235
	H. takashimae	CBS 11174T	FM246501	FM242574	KF036679	KF036486	/	KF037163	KF423295
Holtermannia	Holtermannia corniformis*	CBS 6979R	AF410472	AF189843	AF053718	KF036485	/	/	KF423294
Tremellales
amylolyticus	Cryptococcus amylolyticus	CBS 10048T	KF036585	AY562134	KF036616	KF036343	KF036757	KF037028	/
	C. armeniacus	CBS 10050T	KF036587	AY562140	KF036620	KF036348	KF036761	KF037033	KF423172
	C. bromeliarum	CBS 10424T	EU386359	DQ784566	KF036623	KF036353	KF036766	/	KF423177
	C. tibetensis	CBS 10456T	EF363146	EF363143	EF363152	KF036411	KF036825	/	/
aurantia	Tremella aurantia*	CBS 6965R	AF444315	AF189842	KF036693	KF036522	KF036937	KF037199	KF423323
	T. encephala*	CBS 8207R	AF042402	AF042220	KF036697	KF036526	KF036942	KF037204	KF423327
	T. encephala*	CBS 6968	AF410474	AF189867	KF036698	KF036525	KF036941	KF037203	/
	T. indecorata*	CBS 6976R	AF042432	AF042250	KF036704	KF036532	KF036948	KF037209	KF423333
aureus	Cryptococcus aureus	CBS 318T	AB035045	AB035041	AB085795	KF036350	KF036763	KF037035	KF423174
	C. flavescens	CBS 942T	KF036590	AB035042	AB085796	KF036368	KF036780	KF037053	KF423192
	C. terrestris	CBS 10810T	EU200782	EF370393	KF036658	KF036408	KF036822	KF037093	KF423230
Auriculibuller	Auriculibuller fuscus*	CBS 9648	AF444669	AF444763	KF036604	KF036314	KF036727	KF036999	KF423141
	Bullera japonica	CBS 2013T	AF444666	AF444760	/	/	/	/	/
	Cryptococcus taeanensis	CBS 9742T	AY686645	AY422719	KF036656	KF036406	KF036820	KF037091	KF423228
Bandoniozyma	Bandoniozyma glucofermentans	CBS 10381T	JN381033	AY520334	KF036605	KF036315	KF036728	KF037000	/
	B. noutii	CBS 8364T	AF444391	AF444700	KF036606	KF036316	KF036729	KF037001	KF423142
	B. complexa	CBS 11570T	GU321089	GU321090	KF036607	KF036317	KF036730	KF037002	KF423143
	B. tunnelae	CBS 6123T	AF444333	AF444687	KF036608	KF036318	KF036731	KF037003	/
Bulleribasidium	Bulleribasidium oberjochense*	CBS 9110T	GU327541	AF416646	GU327541	KF036333	KF036744	/	KF423157
	Mingxiaea begonia	CBS 10762T	AB118874	AB119462	AB118874	KF036500	KF036914	KF037177	KF423304
	M. foliicola	CBS 11407T	GQ438834	GQ438834	GQ438834	KF036501	KF036915	KF037178	KF423305
	M. hainanensis	CBS 11409T	GQ438828	GQ438828	GQ438828	KF036502	KF036916	KF037179	KF423306
	M. panici	CBS 9932T	AY188386	AY188387	AY188386	KF036503	KF036917	KF037180	KF423307
	M. pseudovariabilis	CBS 9609T	AF453288	AF544247	AF453290	KF036504	KF036918	KF037181	KF423308
	M. sanyaensis	CBS 11408T	GQ438831	GQ438831	GQ438831	KF036505	KF036919	KF037182	KF423309
	M. setariae	CBS 10763T	AB118875	AB119463	AB118875	KF036506	KF036920	KF037183	KF423310
	M. siamensis	CBS 9933T	AY188389	AY188388	AY188389	/	/	/	/
	M. variabilis	CBS 7347T	AF314965	AF189855	D31654	KF036507	KF036921	/	KF423311
	M. wuzhishanensis	CBS 11411T	GQ438830	GQ438830	GQ438830	KF036508	KF036922	/	KF423312
Bulleromyces	Bullera unica	CBS 8290T	AF444441	AF075524	D78330	KF036332	/	KF037015	KF423155
	Bulleromyces albus*	CBS 501T	AF444368	AF075500	X60179	KF036334	KF036745	KF037016	KF423156
Cryptococcus	Cryptococcus amylolentus*	CBS 6039T	AF444306	AF105391	AB032619	KF036342	KF036756	KF037027	KF423168
	Tsuchiyaea wingfieldii*	CBS 7118T	AF444327	AF177404	D64121	KF036577	KF036994	KF037250	AB040662
	Cryptococcus neoformans*	CBS 132T	AF444326	AF075484	HQ596559	KF036472	KF036886	KF037151	AB040655
	C. gattii*	CBS 6289T	AF444444	AF075526	KF036677	KF036470	KF036884	KF037149	KF423283
	Filobasidiella depauperata*	CBS 7841T	FJ534881	FJ534911	AJ568017	KF036471	KF036885	KF037150	KF423284
Derxomyces	Derxomyces anomala	CBS 9607T	AF453289	EF682504	AF453291	KF036424	KF036838	KF037110	KF423243
	D. boekhoutii	CBS 10824T	EU517057	EU517057	EU517057	KF036425	KF036839	KF037111	KF423244
	D. boninensis	CBS 9141T	AB022933	AY487568	AB022928	KF036426	KF036840	KF037112	KF423245
	D. cylindrical	CBS 9744T	AY487563	AY487563	EU517071	KF036427	KF036841	KF037113	KF423246
	D. hainanensis	CBS 10820T	EU517056	EU517056	EU517056	KF036428	KF036842	KF037114	KF423247
	D. hubeiensis	CBS 9747T	AY487567	AY487566	EU517069	KF036429	KF036843	KF037115	KF423248
	D. huiaensis	CBS 8287T	AB022931	AB118870	D78331	KF036430	KF036844	KF037116	KF423249
	D. komagatae	CBS 10153T	AF314977	AF544249	AF314995	KF036431	KF036845	KF037117	KF423250
	D. linzhiensis	CBS 10827T	EU517058	EU517058	EU517058	KF036432	KF036846	KF037118	KF423251
	D. mrakii	CBS 8288T	AB022932	AB118871	D78325	KF036433	KF036847	KF037119	KF423252
	D. nakasei	CBS 9746T	AY487565	AY487564	EU517070	KF036434	KF036848	KF037120	KF423253
	D. pseudocylindrica	CBS 10826T	EU517059	EU517059	EU517059	KF036435	KF036849	KF037121	KF423254
	D. pseudohuiaensis	CBS 7364T	AF314970	AF544250	AF314994	KF036436	KF036850	/	KF423255
	D. pseudoschimicola	CBS 7354T	AF314979	AF416647	AF314997	KF036437	KF036851	KF037122	KF423256
	D. qinlingensis	CBS 10818T	EU517060	EU517060	EU517060	KF036438	KF036852	KF037123	KF423257
	D. schimicola	CBS 9144T	AB022936	AY487570	AB022930	KF036439	KF036853	KF037124	KF423258
	D. simaoensis	CBS 10822T	EU517062	EU517062	EU517062	KF036440	KF036854	KF037125	KF423259
	D. waltii	CBS 9143T	AB022935	AY487569	AB022929	KF036441	KF036855	KF037126	KF423260
	D. wuzhishanensis	CBS 10825T	EU517063	EU517063	EU517063	KF036442	KF036856	KF037127	KF423261
	D. yunnanensis	CBS 10821T	EU517064	EU517064	EU517064	KF036443	KF036857	KF037128	KF423262
dimennae	Bullera globispora	CBS 6981T	AF444407	AF075509	D31650	KF036323	KF036736	KF037007	KF423148
	Cryptococcus carnescens	CBS 973T	KF036588	AB035054	AB085798	KF036354	KF036767	KF037039	KF423178
	C. dimennae	CBS 5770T	AF410473	AF075489	AB032627	KF036364	KF036776	KF037049	KF423188
	C. heimaeyensis	CBS 8933T	KF036591	DQ000317	KF036635	KF036376	KF036788	KF037060	KF423198
	C. peneaus	CBS 2409T	AB035047	AB035051	AB085799	KF036392	KF036806	KF037077	KF423215
	C. tephrensis	CBS 8935T	DQ000318	DQ000318	KF036657	KF036407	KF036821	KF037092	KF423229
	C. victoriae	CBS 8685T	AF444469	AF363647	KF036661	KF036413	KF036827	KF037097	KF423233
Dioszegia	Dioszegia Antarctica	CBS 10920T	DQ402529	FJ640575	KF036667	KF036444	KF036858	KF037129	KF423263
	D. athyri	CBS 10119T	EU070926	EU070931	KF036668	KF036445	KF036859	KF037130	KF423264
	D. aurantiaca	CBS 6980T	AB049613	AB104689	AB049615	KF036446	KF036860	KF037131	KF423265
	D. buhagiarii	CBS 10054T	AY885687	AY562151	EU517065	KF036447	KF036861	KF037132	KF423266
	D. butyracea	CBS 10122T	EU070924	EU070929	KF036669	KF036448	KF036862	KF037133	KF423267
	D. catarinonii	CBS 10051T	AY562154	AY562142	KF036670	KF036449	KF036863	/	KF423268
	D. changbaiensis	CBS 9608T	AY242817	AY242819	AY242817	KF036450	KF036864	KF037134	KF423269
	D. crocea	CBS 6714T	AB049612	AF075508	D31648	KF036451	KF036865	KF037135	AB040649
	D. cryoxerica	CBS 10919T	FJ640565	FJ640562	KF036671	KF036452	KF036866	KF037136	KF423270
	D. fristingensis	CBS 10052T	AY562158	AY562146	EU517066	KF036453	KF036867	/	KF423271
	D. hungarica	CBS 4214T	AB049614	AF075503	AB032638	KF036454	KF036868	KF037138	KF423272
	D. statzelliae	CBS 8925T	AY029342	AY029341	/	/	/	/	/
	D. takashimae	CBS 10053T	AY562160	AY562149	KF036672	KF036455	KF036869	/	KF423273
	D. xingshanensis	CBS 10120T	EU070923	EU070928	KF036673	KF036456	KF036870	KF037139	KF423274
	D. zsoltii	CBS 9127T	AF385445	AF544245	AF385443	KF036457	KF036871	KF037140	KF423275
Fellomyces	Fellomyces borneensis	CBS 8282T	AJ608642	AF189877	AB032659	KF036458	KF036872	KF037141	KF423276
	F. horovitziae	CBS 7515T	AF444404	AF189856	AB001033	KF036461	KF036875	KF037143	/
	F. penicillatus	CBS 5492T	AF444337	AF177405	AB001034	KF036464	KF036878	KF037144	/
	F. polyborus	CBS 6072T	AF444411	AF189859	KF036676	KF036465	KF036879	KF037145	/
Fibulobasidium	Fibulobasidium inconspicuum*	CBS 8237R	AF444318	AF363641	D64123	KF036468	KF036882	KF037147	KF423281
	F. murrhardtense*	CBS 9109T	GU327540	AF416648	GU327540	KF036469	KF036883	KF037148	KF423282
flavus	Cryptococcus flavus	CBS 331T	AF444338	AF075497	AB032629	KF036369	KF036781	/	KF423193
	C. paraflavus	CBS 10100T	AY395800	AY395799	KF036645	KF036391	KF036805	/	KF423214
	C. podzolicus	CBS 6819T	AF444321	AF075481	AB032645	KF036396	KF036810	KF037081	/
foliacea	C. fagi	CBS 9964T	DQ054534	DQ054535	KF036632	KF036366	KF036778	KF037051	KF423190
	C. skinneri	CBS 5029T	AF444305	AF189835	AB032646	KF036403	KF036817	KF037088	KF423225
	Tremella foliacea*	CBS 6969R	AF444431	AF189868	KF036700	KF036528	KF036944	KF037206	KF423329
	T. neofoliacea*	CBS 8475R	AF042415	AF042236	KF036706	/	/	/	/
hannae	Bullera hannae	CBS 8286T	AF444486	AF363661	D78327	KF036324	KF036737	KF037008	/
	B. penniseticola	CBS 8623T	AF444471	AF363649	AB005452	KF036329	KF036741	KF037012	KF423152
Hannaella	Hannaella coprosmaensis	CBS 8284T	AF444485	AF363660	D78326	KF036479	KF036893	KF037156	KF423288
	H. kunmingensis	CBS 8960T	AF325171	AB109558	AF325169	KF036480	KF036894	/	KF423289
	H. luteola	CBS 943T	AF444323	AF075482	AB032641	KF036481	KF036895	KF037158	KF423290
	H. oryzae	CBS 7194T	AF444413	AF075511	D31652	KF036482	KF036896	KF037159	KF423291
	H. sinensis	CBS 7238T	AF444468	AF189884	D78328	KF036483	KF036897	KF037160	KF423292
	H. surugaensis	CBS 9426T	AB100440	AB100440	AB100440	KF036484	KF036898	KF037161	KF423293
Kockovaella	Fellomyces chinensis	CBS 8278T	AF444460	AF189878	AB032660	KF036459	KF036873	KF037142	KF423277
	F. distylii	CBS 8545T	AF444475	AF363652	AB001036	/	/	/	/
	F. fuzhouensis	CBS 8243T	AF444484	AF363659	KF036674	KF036460	KF036874	/	KF423278
	F. lichenicola	CBS 8315T	AF444462	AF363643	AB032661	KF036462	KF036876	/	KF423279
	F. mexicanus	CBS 8279T	AJ608667	AJ627906	KF036675	KF036463	KF036877	/	/
	F. ogasawarensis	CBS 8544T	AF444474	AF363651	AB001035	/	/	/	KF423280
	F. sichuanensis	CBS 8318T	AF444461	AF189879	AB032662	KF036466	KF036880	/	/
	F. thailandicus	CBS 8308T	AJ608647	AF363644	AB044804	KF036467	KF036881	/	/
	Kockovaella barringtoniae	CBS 9811T	AB052631	AB292854	AB052631	KF036487	KF036901	KF037165	KF423297
	K. calophylli	CBS 8962T	AB042227	AB292852	AB042222	KF036488	KF036902	KF037166	/
	K. cucphuongensis	CBS 8959T	AB042225	AB292853	AB042220	KF036489	KF036903	KF037167	/
	K. imperatae	CBS 7554T	AB054091	AF189862	KF036680	KF036490	KF036904	KF037168	KF423298
	K. litseae	CBS 8964T	AB042223	AB292850	AB042218	KF036491	KF036905	KF037169	KF423299
	K. machilophila	CBS 8607T	AB054092	AF363654	AB005479	KF036492	KF036906	KF037170	/
	K. phaffii	CBS 8608T	AB054093	AF363655	AB005480	KF036493	KF036907	/	KF423300
	K. sacchari	CBS 8624T	AB054094	AF363650	AB005453	KF036494	KF036908	KF037171	/
	K. schimae	CBS 8610T	AB042228	AF363656	AB005482	KF036495	KF036909	KF037172	/
	K. thailandica	CBS 7552T	AB054095	AF075516	D64133	KF036496	KF036910	KF037173	KF423301
	K. vietnamensis	CBS 8963T	AB042226	AB292851	AB042221	KF036497	KF036911	KF037174	/
Kwoniella	Bullera dendrophila	CBS 6074T	AF444443	AF189870	D31649	KF036320	KF036733	KF037005	KF423145
	Cryptococcus bestiolae	CBS 10118T	FJ534873	FJ534903	KF036622	KF036351	KF036764	KF037036	KF423175
	C. dejecticola	CBS 10117T	AY917103	AY917102	KF036629	KF036362	KF036774	KF037047	KF423186
	C. heveanensis*	CBS 569T	AF444301	AF075467	AB032635	FJ534921	KF036789	KF037061	KF423199
	C. pinus	CBS 10737T	EF672246	EF672245	KF036648	KF036395	KF036809	KF037080	KF423218
	C. shivajii	CBS 11374T	FM212571	FM212446	KF036652	KF036401	KF036815	KF037086	KF423223
	Kwoniella mangroviensis*	CBS 8507T	AF444646	AF444742	KF036681	KF036498	KF036912	KF037175	KF423302
laurentii	Cryptococcus laurentii	CBS 139T	AF410468	AF075469	AB032640	KF036380	KF036793	KF037065	AB040653
	C. rajasthanensis	CBS 10406T	AM262325	AM262324	KF036649	KF036398	KF036812	KF037083	KF423220
melastomae	Bullera formosana	CBS 10306T	AB118873	AB119465	AB118873	KF036321	KF036734	/	KF423146
	B. melastomae	CBS 10305T	AB118872	AB119464	AB118872	KF036327	/	/	KF423150
moriformis	Tremella moriformis*	CBS 7810R	AF444331	AF075493	U00977	KF036534	KF036950	KF037211	KF423335
	T. nivalis*	CBS 8487R	AF042414	AF042232	KF036707	/	/	/	/
Papiliotrema	Cryptococcus nemorosus	CBS 9606T	AF472628	AF472625	KF036642	KF036389	KF036802	KF037074	KF423211
	C. perniciosus	CBS 9605T	AF472627	AF472624	KF036646	KF036393	KF036807	KF037078	KF423216
	Papiliotrema bandonii*	CBS 9107T	GU327539	AF416642	GU327539	KF036518	KF036932	KF037194	KF423319
pseudoalba	Bullera pseudoalba	CBS 7227T	AF444399	AF075504	D31660	KF036330	KF036742	KF037013	KF423153
	Cryptococcus cellulolyticus	CBS 8294T	AF444442	AF075525	AB032624	KF036355	KF036768	KF037040	KF423179
	C. anemochoreius	CBS 10258T	DQ830986	DQ384929	KF036617	KF036344	KF036758	KF037029	/
Tremella	Tremella brasiliensis*	CBS 6966R	AF444429	AF189864	KF036694	/	KF036938	KF037200	KF423324
	T. cinnabarina*	CBS 8234R	AF444430	AF189866	KF036695	KF036523	KF036939	KF037201	KF423325
	T. coalescens*	CBS 6967R	KF036601	AF189865	KF036696	KF036524	KF036940	KF037202	KF423326
	T. flava*	CBS 8471R	AF042403	AF042221	KF036699	KF036527	KF036943	KF037205	KF423328
	T. fuciformis*	CBS 6970R	AF444316	AF075476	KF036701	KF036529	/	KF037207	KF423330
	T. globispora*	CBS 6972R	AF444432	AF189869	KF036703	KF036531	KF036947	KF037208	KF423332
	T. mesenterica*	CBS 6973R	AF444433	AF075518	KF036705	KF036533	KF036949	KF037210	KF423334
	T. resupinata*	CBS 8488R	AF042421	AF042239	KF036708	KF036535	KF036951	KF037212	KF423336
	T. taiwanensis*	CBS 8479R	AF042412	AF042230	KF036709	KF036536	KF036952	KF037213	KF423337
	T. tropica*	CBS 8483R	AF042433	AF042251	KF036710	KF036537	KF036953	KF037214	/
single species clade	Bullera arundinariae	CBS 9931T	AF547662	AF547661	AF547660	KF036319	KF036732	KF037004	KF423144
	B. miyagiana	CBS 7526T	AF444409	AF189858	D31651	KF036328	KF036740	KF037011	KF423151
	B. sakaeratica	CBS 9934T	AY217651	AY211546	AY211544	KF036331	KF036743	KF037014	KF423154
	Cryptococcus allantoinivorans	CBS 9604T	AY315664	AY315662	KF036615	KF036341	KF036755	KF037026	KF423167
	C. cistialbidi	CBS 10049T	KF036589	AY562135	KF036626	KF036358	KF036770	KF037043	KF423182
	C. cuniculi	CBS 10309T	CBS database	DQ333885	KF036627	/	/	/	/
	C. mujuensis	CBS 10308T	KF036595	DQ333884	KF036640	KF036386	KF036799	KF037071	KF423208
	C. spencermartinsiae	CBS 10760T	EU249514	DQ513279	KF036654	KF036404	KF036818	KF037089	KF423226
	Cuniculitrema polymorpha*	CBS 9644T	KF036596	AY032662	KF036664	KF036418	KF036831	KF037102	/
	Sirobasidium intermedium*	CBS 7805	AF444330	AF075492	KF036690	KF036519	KF036934	KF037196	/
	S. magnum*	CBS 6803	AF444314	AF075475	KF036691	KF036520	KF036935	KF037197	KF423321
	Tremella giraffa*	CBS 8489R	AF042453	AF042271	KF036702	KF036530	KF036946	/	KF423331
	Trimorphomyces papilionaceus*	CBS 443.92	AF444483	AF075491	KF036726	KF036576	KF036993	KF037249	KF423355
Trichosporonales
cutaneum	Trichosporon cutaneum	CBS 2466T	AF444325	AF075483	KF036712	KF036545	KF036961	KF037221	AB175752
	T. debeurmannianum	CBS 1896T	AY143556	AY143554	KF036713	KF036546	KF036962	KF037222	KF423340
	T. dermatis	CBS 2043T	AY143557	AY143555	AB035585	KF036548	KF036964	KF037224	KF423342
	T. jirovecii	CBS 6864T	AF444437	AF105398	AB001758	/	KF036974	KF037234	AB175765
	T. moniliiforme	CBS 2467T	AF444415	AF105392	AB001761	KF036562	KF036979	KF037238	AB175772
	T. mucoides	CBS 7625T	AF444423	AF075515	AB001763	KF036564	KF036981	KF037240	AB040665
	T. smithiae	CBS 8370T	AF444397	AF444706	KF036720	KF036570	KF036987	KF037244	KF423350
	T. terricola	CBS 9546T	AB031517	AB086382	KF036722	KF036572	KF036989	/	KF423352
formosensis	Bullera formosensis	CBS 9812T	AY787859	AY787858	AB072235	KF036322	KF036735	KF037006	KF423147
	B. koratensis	CBS 10484T	AY919655	AY313006	AY863105	KF036325	KF036738	KF037009	KF423149
	B. lagerstroemiae	CBS 10483T	AY313033	AY313010	AY313033	KF036326	KF036739	KF037010	/
	Cryptococcus tepidarius	CBS 9427T	AB094045	AB094046	/	/	/	/	/
gracile/brassicae	Trichosporon dulcitum	CBS 8257T	AF444428	AF075517	AB001755	KF036551	KF036967	KF037227	AB175755
	T. gracile	CBS 8189T	AF444440	AF105399	AB001756	KF036554	KF036970	KF037230	AB175761
	T. laibachii	CBS 5790T	AF444421	AF075514	AB001760	KF036559	KF036976	KF037235	AB175769
	T. multisporum	CBS 2495T	AF414695	AF139984	AB001764	KF036565	KF036982	KF037241	AB175775
	T. loubieri	CBS 7065T	AF444438	AF075522	AB001759	KF036561	KF036978	KF037237	AB175771
	T. mycotoxinivorans	CBS 9756T	AJ601389	AJ601388	KF036718	KF036566	KF036983	KF037242	KF423347
	T. vadense	CBS 8901T	AY093425	AY093426	KF036723	KF036573	KF036990	KF037246	KF423353
	T. veenhuisii	CBS 7136T	AF414693	AF105400	KF036724	KF036574	KF036991	KF037247	AB175781
	T. brassicae	CBS 6382T	AF444436	AF075521	AB001731	KF036541	KF036957	KF037218	AB175750
	T. domesticum	CBS 8280T	AF444414	AF075512	AB001754	KF036550	KF036966	KF037226	AB175753
	T. montevideense	CBS 6721T	AF444422	AF105397	AB001762	KF036563	KF036980	KF037239	AB175774
	T. scarabaeorum	CBS 5601T	AF444446	AF444710	KF036719	KF036569	KF036986	KF037243	KF423349
haglerorum	Cryptococcus arboriformis	CBS 10441T	AB260936	AB260936	KF036619	KF036347	KF036760	KF037032	KF423171
	C. haglerorum	CBS 8902T	AY787857	AF407276	KF036634	KF036375	KF036787	KF037059	KF423197
porosum	Trichosporon dehoogii	CBS 8686T	AF444476	AF444718	KF036714	KF036547	KF036963	KF037223	KF423341
	T. gamsii	CBS 8245T	KF036602	AF444708	KF036716	KF036553	KF036969	KF037229	AB175780
	T. lignicola	CBS 219.34T	AY370684	AY370685	DQ836003	KF036560	KF036977	KF037236	KF423346
	T. porosum	CBS 2040T	AF414694	AF189833	AB051045	KF036568	KF036985	/	KF423348
	T. sporotrichoides	CBS 8246T	AF444470	AF189885	KF036721	KF036571	KF036988	KF037245	KF423351
	T. wieringae	CBS 8903T	AY315667	AY315666	KF036725	KF036575	KF036992	KF037248	KF423354
Trichosporon	T. aquatile	CBS 5973T	AF410475	AF075520	AB001730	KF036538	KF036954	KF037215	AB040664
	T. asahii	CBS 2479T	AY055381	AF105393	AB001726	KF036539	KF036955	KF037216	AB175744
	T. asteroids	CBS 2481T	AF444416	AF075513	AB001729	KF036540	KF036956	KF037217	AB175746
	T. caseorum	CBS 9052T	AJ319758	AJ319757	AJ319754	KF036542	KF036958	/	KF423338
	T. coremiiforme	CBS 2482T	AF444434	AF139983	AB001727	KF036544	KF036960	KF037220	AB175751
	T. dohaense	CBS 10761T	FJ228475	FJ228471	KF036715	KF036549	KF036965	KF037225	KF423343
	T. faecal	CBS 4828T	AF444419	AF105395	AB001728	KF036552	KF036968	KF037228	AB175758
	T. inkin	CBS 5585T	AF444420	AF105396	AB001757	KF036555	KF036971	KF037231	AB175764
	T. insectorum	CBS 10422T	KF036603	AY520383	AY520254	KF036556	KF036972	/	KF423344
	T. japonicum	CBS 8641T	AF444473	AF308657	AB001749	KF036557	KF036973	KF037233	KF423345
	T. lactis	CBS 9051T	AJ319759	AJ319756	AJ319755	KF036558	KF036975	/	/
	T. ovoides	CBS 7556T	AF444439	AF075523	AB001765	KF036567	KF036984	/	AB175776
Vanrija	Vanrija albida	CBS 2839T	AB035578	AB126584	AB039285	KF036399	KF036813	KF037084	KF423221
	V. humicola	CBS 571T	AF410470	AF189836	AB032637	KF036378	DQ645517	DQ645519	AB176693
	V. longa	CBS 5920T	AB035577	AB126589	AB035586	KF036382	KF036795	KF037067	KF423204
	V. musci	CBS 8899T	AB035579	AB126586	AB039378	KF036387	KF036800	KF037072	KF423209
	V. pseudolongus	CBS 8297T	AB051048	AB126587	AB051047	KF036397	KF036811	KF037082	KF423219
single species clade	Cryptococcus curvatus	CBS 570T	AF410467	AF189834	AB032626	KF036359	KF036771	KF037044	KF423183
	C. daszewskae	CBS 5123T	AB035580	AB126588	AB035582	KF036361	KF036773	KF037046	KF423185
	C. fragicola	CBS 8898T	AB035588	AB126585	AB035588	KF036370	KF036782	KF037054	/
	C. marinus	CBS 5235T	KF036593	AF189846	AB032644	KF036384	KF036797	KF037069	KF423206
	Cryptotrichosporon anacardii	CBS 9551T	AY549985	AY550002	DQ242636	KF036417	KF036830	KF037101	KF423237
	Trichosporon chiarellii	CBS 11177T	GQ338074	EU030272	KF036711	KF036543	KF036959	KF037219	KF423339
	T. guehoae	CBS 8521T	AF410476	AF105401	KF036717	/	/	/	AB175763
Pucciniomycotina
	Leucosporidium scottii	CBS 5930T	AF444495	AF070419	KF036682	KF036499	KF036913	KF037176	KF423303
	Sterigmatomyces halophilus	CBS 4609T	AF444556	AF177416	D64119	KF036521	KF036936	KF037198	KF423322
Ustilaginomycotina
	Ustilago maydis		AY854090	AF453938	X62396	XM754917	AY485636	AY885160	AB040663

The asterisks indicate teleomorphic taxa; CBS database: sequences are available from the CBS database at http://www.cbs.knaw.nl/Collections/Biolomics.aspx?Table=CBS strain database.

DNA extraction, PCR, and sequencing

Genomic DNA was extracted from yeast cells actively growing on YPD medium using the method described in Bolano with minor modifications. The UltraClean® Microbial DNA Isolation Kit (MO BIO, CA) was used when high quality DNA templates were required for PCR amplification of some protein genes. A set of six genes was selected and sequenced based on previous studies of the Assembling the Fungal Tree of Life (AFTOL-1) project (James et al., 2006, Hibbett et al., 2007). These genes included three rRNA genes, namely the small subunit (SSU or 18S) of the ribosomal DNA (rDNA), D1/D2 domains of the large subunit (LSU or 26S) rDNA, and the internal transcribed spacer regions (ITS 1 and 2) of the rDNA, including the 5.8S rDNA; and three nuclear protein-coding genes, namely the two subunits of RNA polymerase II (RPB1 and RPB2) and translation elongation factor 1-α (TEF1). In addition, the mitochondrial gene cytochrome b (CYTB) was also included. The primers used for PCR amplification and sequencing of these genes are listed in Table 2. Because of the degenerate nature of the primers used for PCR amplification of the protein-coding genes, sometimes faint or multiple PCR bands were generated from PCR amplification or direct sequencing of amplicons failed. In these cases, amplicons were cloned using the pGEM®-T Easy Vector Systems (Promega Corporation, Madison) following the protocol of the kit. Positive colonies with an insert of expected size were chosen for sequencing.

Table 2

PCR and sequencing primers used in this study.

Primer name	Nucleotide sequence (5′–3′)	Reference
ITS and D1/D2
V9	TGC GTT GAT TAC GTC CCT GC→	Boekhout et al. 2003
RLR3R	←GGT CCG TGT TTC AAG AC	Boekhout et al. 2003
ITS4	←TCC TCC GCT TAT TGA TAT GC	White et al. 1990
NL1	GCA TAT CAA TAA GCG GAG GAA AAG→	O'Donnell 1993
SSU
NS1	GTA GTC ATA TGC TTG TCT→	White et al. 1990
NS24	←AAA CCT TGT TAC GAC TTT TA	Gargas & Taylor 1992
Oligo3	←GTA CAC ACC GCC CGT C	Hendriks et al. 1989
Oligo10	←TGG YRA ATG CTT TCG C	Hendriks et al. 1989
Oligo13	←ATA ACA GGT CTG TGA TGC CC	Hendriks et al. 1989
Oligo14	ATA ACA GGT CTG TGA TGC CC→	Hendriks et al. 1989
RPB1
RPB1-Af	GAR TGY CCD GGD CAY TTY GG→	Stiller & Hall 1997
RPB 1-Cr	←CCN GCD ATN TCR TTR TCC ATR TA	Matheny et al. 2002
RPB2
f RPB2-5F	GAY GAY MGW GAT CAY TTY GG→	Liu et al. 1999
RPB2-6F	TGG GGK WTG GTY TGY CCT GC→	Liu et al. 1999
RPB2-6R	←GCA GGR CAR ACC AWM CCC CA	Liu et al. 1999
RPB2-7R	←CCC ATW GCY TGC TTM CCC AT	Liu et al. 1999
bRPB2-7.1R	←CCC ATR GCY TGY TTM CCC ATD GC	Matheny 2005
TEF1
EF1-983F	GCY CCY GGH CAY CGT GAY TTY AT→	Rehner & Buckley 2005
EF1-2218R	←ATG ACA CCR ACR GCR ACR GTY TG	Rehner & Buckley 2005
EF1-2212R	←CCR ACR GCR ACR GTY YGT CTC AT	Rehner & Buckley 2005
1577F	CAR GAY GTB TAC AAG ATY GGT GG→	Rehner & Buckley 2005
1567R	←ACH GTR CCR ATA CCA CCR ATC TT	Rehner & Buckley 2005
CYTB
E1M4	TGR GGW GCW ACW GTT ATT ACT A→	Biswas et al. 2003
E2M4	←GGW ATA GMW SKT AAW AYA GCA TA	Biswas et al. 2003

Molecular phylogenetic analyses

Sequences were inspected and assembled using the SeqMan program in the Lasergene 7 software package (DNASTAR Inc., Madison) and were then aligned with Clustal X 1.83 (Thompson ). Spliceosomal intron regions were inferred from the insertions with canonical splice sites (GT-AG, GC-AG, AT-AC) (Babenko ) in the nucleotide sequence alignments between our data and reference cDNA sequences from GenBank. Exon sequences of the protein-encoding genes RPB1, RPB2, TEF1 and CYTB were manually aligned using MEGA 5 (Tamura ). Positions deemed ambiguous to align were excluded manually. Thereafter, multiple sequence alignments for ITS, D1/D2, SSU, RPB1, RPB2, TEF1, and CYTB were concatenated as a combined file.

Maximum likelihood (ML), neighbour-joining (NJ), and Bayesian analyses were conducted for separate and combined nucleotide data sets using RAxML v8.1.X (Stamatakis 2014), MEGA 5.0 (Tamura ) and MrBayes 3.2.1 (Ronquist ), respectively. ML analysis was implemented with the novel fast bootstrap algorithm with 100 replicates and a subsequent search for the best maximum-likelihood tree in conjunction with the GTRGAMMAI model approximation (Stamatakis 2014). NJ analysis was performed on the evolutionary distance data calculated from Kimura's two-parameter model (Kimura 1980). Bootstrap analyses (Felsenstein 1985) were performed from 1 000 random re-samplings in both ML and NJ analyses. A bootstrap proportion (BP) support above 70 % obtained from the ML and NJ analyses was considered as significant (Hillis & Bull 1993).

Bayesian analysis was implemented using heterogeneous models to the data set with seven unlinked partitions, one for each gene. The best-fit evolution model of each gene fragment in the data set was determined using the Bayesian Information Criterion (BIC) in jModeltest (Posada 2008). The ITS, D1D2, and SSU rDNA gene sequences were fitted to TPM3uf+G, TIM3+G, and TIM2+T+G models, respectively. The protein-coding genes RPB1 and CYTB both used the GTR+I+G model; whereas RPB2 and TEF1 used the TPM3uf+I+G and TPM1uf+G models, respectively. Six to fifty million generations were run with four Markov chains (three heated and one cold), sampling every 500 generations. The average standard deviation of split frequencies, below 0.01, was examined to identify the convergence of the two independent runs. Clades with posterior probabilities (PP) above 0.95 were considered as significantly supported (Larget & Simon 1999).

Results

Sequences generated and data sets constructed for phylogenetic analyses

A total of 1 147 new sequences were produced in this study, including 21 ITS, 123 SSU, 269 RPB1, 270 RPB2, 249 TEF1, and 215 CYTB sequences. In addition, a total of 777 previously published sequences of these genes from the type strains of tremellomycetous yeast taxa were retrieved from GenBank (Table 1). Different data sets consisting of the three rRNA genes (rDNA), the individual protein-coding genes, and the combined seven genes were constructed from the 1 924 sequences employed in this study (Table 3). In addition, a data set of 5.8S and LSU rDNA D1/D2 domain sequences was constructed to include more Tremella species whose sequences were determined from herbarium specimens (Millanes ).

Table 3

Nucleotide sequence data sets constructed for phylogenetic analyses.

Data set	No. of strains	No. of taxa	Length of alignment	Parsimony informative characters (%)
rDNA1	297	285	3 208	1 447 (45)
RPB1	271	262	758	615 (81)
RPB2	273	263	1 133	872 (77)
TEF1	249	238	909	498 (55)
CYTB	246	238	388	279 (71)
Seven-gene	281	269	6 298	3 623 (57)

The rDNA data set includes 296 ITS, 297 LSU D1/D2, and 292 SSU rDNA sequences.

These data sets were subjected to phylogenetic analyses using Bayesian, ML and NJ algorithms, respectively. The topologies of the trees obtained were compared visually to inspect the phylogenetic concordance among the taxa analysed, based on which monophyletic lineages and clades were recognised and defined (Table 4). As expected, among the trees drawn from different data sets analysed, the seven-gene trees exhibited the clearest resolution and strongest supports; and among the algorithms employed, the Bayesian analysis usually showed the most robust phylogeny (Table 4). Thus, the Bayesian tree constructed from the seven-gene data set was used as the primary basis for lineage and clade recognition and definition, and as the starting point for the subsequent comparison and discussion. The phylogenetic trees inferred from the rDNA data set containing all the taxa employed in this study were used as references to judge the phylogenetic positions of a minority of taxa which were absent in the seven-gene tree because of failure in sequencing of the protein coding genes.

Table 4

Monophyletic clades resolved in tremellomycetous yeasts and dimorphic taxa based on different data sets using different algorithms.

Lineage/Clade	RPB1	RPB2	TEF1	CYTB	rDNA	Seven-gene
PP/BP1/BP2	PP/BP1/BP2	PP/BP1/BP2	PP/BP1/BP2	PP/BP1/BP2	PP/BP1/BP2
Cystofilobasidiales	nm/nm/nm	1.0/100/99	.90/64/70	nm/nm/nm	1.0/87/100	1.0/100/100
Cystofilobasidium	1.0/100/100	1.0/100/99	nm/nm/nm	nm/nm/nm	1.0/100/100	1.0/100/100
Guehomyces	S	S	S	S	1.0/100/nm	S
huempii	1.0/100/100	1.0/100/100	1.0/100/100	1.0/100/99	1.0/100/100	1.0/100/100
Itersonilia	S	1.0/100/100	S	S	1.0/100/100	1.0/100/100
Mrakia	1.0/90/83	1.0/96/89	nm/nm/ns	nm/nm/nm	1.0/100/100	1.0/100/100
Udeniomyces	1.0/100/87	1.0/100/99	S	–	1.0/100/100	1.0/100/100
Phaffia	S	1.0/100/100	1.0/100/100	1.0/99/99	1.0/100/100	1.0/100/100
Filobasidiales	1.0/100/98	1.0/100/100	nm/nm/ns	nm/nm/nm	1.0/83/98	1.0/100/100
aerius	1.0/100/100	1.0/100/100	ns/nm/nm	nm/nm/nm	nm/nm/nm	1.0/100/85
albidus	1.0/100/100	1.0/100/100	1.0/90/99	nm/nm/nm	1.0/94/99	1.0/100/100
cylindricus	1.0/100/100	1.0/100/100	1.0/100/100	ns/55/ns	1.0/100/100	1.0/100/100
Filobasidium	1.0/99/100	1.0/100/100	1.0/97/92	nm/nm/nm	1.0/100/100	1.0/100/100
gastricus	1.0/100/99	1.0/100/98	nm/nm/nm	nm/nm/nm	1.0/99/91	1.0/100/100
Holtermanniales	1.0/100/99	1.0/100/100	1.0/100/100	.99/66/ns	1.0/100/100	1.0/100/56
Holtermanniella	1.0/62/78	1.0/100/100	1.0/100/100	nm/nm/nm	1.0/99/100	1.0/100/80
Tremellales/Trichosporonales	1.0/100/99	1.0/100/97	ns/ns/68	nm/nm/nm	.99/99/nm	1.0/100/55
Tremellales	nm/nm/nm	.95/72/nm	nm/nm/nm	nm/nm/nm	nm/nm/nm	ns/nm/nm
amylolyticus	1.0/83/84	1.0/100/100	1.0/97/88	1.0/99/99	1.0/100/100	1.0/100/100
aurantia	1.0/62//99	1.0/100/100	ns/ns/63	nm/nm/nm	nm/nm/nm	1.0/100/100
aureus	1.0/100/100	1.0/100/100	.99/85/98	nm/nm/nm	1.0/100/100	1.0/100/100
Auriculibuller	ns/59/nm	1.0/100/100	ns/ns/89	1.0/94/64	1.0/100/nm	1.0/100/100
Bandoniozyma	1.0/100/100	1.0/100/100	1.0/96/95	1.0/100/99	1.0/100/100	1.0/100/100
Bulleribasidium	.94/57/nm	1.0/67/56	nm/nm/nm	nm/nm/nm	1.0/100/100	1.0/100/100
Bulleromyces	1.0/100/100	S	1.0/94/98	1.0/99/99	1.0/100/100	1.0/100/100
Cryptococcus	1.0/98/80	1.0/100/99	nm/nm/nm	1.0/85/92	1.0/100/100	1.0/100/100
Derxomyces	.93/ns/nm	ns/ns/nm	nm/nm/nm	ns/ns/57	1.0/100/100	1.0/100/100
dimennae	1.0/96/93	1.0/99/100/	nm/nm/nm	nm/nm/nm	1.0/99/83	1.0/100/100
Dioszegia	1.0/96/89	1.0/93/99	nm/nm/nm	1.0/98/99	1.0/100/100	1.0/100/100
Fellomyces	nm/nm/nm	nm/nm/nm	1.0/69/63	S	1.0/87/98	1.0/64/nm
Fibulobasidium	1.0/100/100	1.0/100/100	1.0/99/100	1.0/100/99	1.0/100/100	1.0/100/100
flavus	nm/nm/nm	nm/nm/nm	S/S/S	1.0/96/99	.99/83/98	.97/72/82
foliacea	1.0/100/100	1.0/100/99	nm/nm/nm	nm/nm/nm	1.0/100/100	1.0/100/100
hannae	1.0/100/100	1.0/100/100	1.0/100/100	S	1.0/100/100	1.0/100/100
Hannaella	nm/nm/ns	1.0/99/95	nm/nm/nm	1.0/72/97	1.0/100/100	1.0/100/100
Kockovaella	1.0/62/74	nm/nm/nm	1.0/100/99	nm/nm/nm	1.0/100/100	1.0/96/96
Kwoniella	1.0/75/92	1.0/100/99	nm/nm/nm	nm/nm/nm	1.0/100/100	1.0/100/100
laurentii	1.0/100/99	1.0/100/69	.97/ns/nm	nm/nm/nm	1.0/100/92	1.0/100/100
melastomae	1.0/96/98	S	–	1.0/96/98	1.0/99/100	1.0/100/100
moriformis	S	S	S	S	1.0/100/95	S
Papiliotrema	1.0/100/100	1.0/100/100	1.0/95/97	1.0/99/99	1.0/100/nm	1.0/100/100
pseudoalba	.98/89/96	1.0/100/98	1.0/75/59	.97/100/99	.96/89/nm	1.0/100/100
Tremella	1.0/100/99	1.0/100/100	nm/nm/nm	nm/nm/nm	1.0/100/99	1.0/100/55
Trichosporonales	1.0/95/65	0.92/ns/nm	nm/nm/ns	nm/nm/nm	nm/nm/nm	1.0/100/100
gracile/brassicae	.90/61/nm	1.0/97/91	nm/nm/nm	ns/ns/nm	1.0/98/100	1.0/100/100
cutaneum	.96/ns/76	ns/ns/61	nm/60/86	nm/nm/nm	1.0/100/100	1.0/100/98
formosensis	1.0/100/99	1.0/100/95	1.0/92/66	1.0/100/99	1.0/100/100	1.0/100/100
Vanrija	1.0/100/100	1.0/100/100	1.0/100/100	1.0/98/99	1.0/97/77	1.0/100/100
haglerorum	nm/ns/nm	nm/ns/nm	nm/nm/ns	nm/nm/nm	1.0/91/100	1.0/78/91
porosum	ns/80/100	1.0/100/100	1.0/51/86	nm/nm/nm	1.0/100/100	1.0/100/100
Trichosporon	1.0/100/99	1.0/100/100	1.0/100/99	nm/nm/nm	1.0/100/100	1.0/100/100

Note. PP, Bayesian posterior probability; BP1 and BP2, bootstrap values from the maximum likelihood and neighbour-joining analyses, respectively; nm: not monophyletic; ns, not supported (PP < 0.9 or BP < 50 %); S: single species clade. Data sets that produce both significant PP (≥0.95) and BP (≥70 %) values have dark grey shaded cells; and data sets that produce either a significant PP or BP support value have light grey shaded cells.

Major lineages recognised among tremellomycetous yeasts

In the Bayesian tree constructed from the seven-gene data set, five lineages corresponding to the orders Tremellales, Trichosporonales, Filobasidiales and Cystofilobasidiales recognised by Boekhout and the order Holtermanniales proposed by Wuczkowski were resolved (Table 4, Fig. 1). The posterior probability for each of the Trichosporonales, Holtermanniales, Filobasidiales and Cystofilobasidiales lineages was 1.0. The support for the Tremellales was weak (PP = 0.51) when the basal foliacea clade of the lineage formed by Cryptococcus fagi, C. skinneri, C. spencermartinsiae, and Tremella foliacea was included. However, when this clade was not included, the Tremellales taxa formed a well-supported lineage with a PP value of 1.0 (Fig. 1).

Fig. 1

An outline of the phylogeny of tremellomycetous yeasts and dimorphic taxa inferred from a seven-gene data set including sequences of three rDNA genes, RPB1, RPB2, TEF1 and CYTB. The tree backbone is constructed using Bayesian analysis. Branch lengths are scaled in terms of expected numbers of nucleotide substitutions per site. The Bayesian posterior probabilities (PP) and bootstrap percentages (BP) of maximum likelihood and neighbour-joining analyses from 1 000 replicates are shown respectively from left to right on the deep and major branches resolved. Note: ns, not supported (PP < 0.9 or BP < 50 %); nm, not monophyletic.

The five lineages were also clearly recognised in the ML and NJ trees constructed from the seven-gene data set (Table 4, Fig. 1), though the statistic support values varied. The Cystofilobasidiales, Filobasidiales, and Trichosporonales lineages received 100 % bootstrap supports, while the Holtermanniales received a moderate bootstrap support (56 %) in the NJ tree, but a strong support (100 % BP) in the ML tree. In the ML tree, the foliacea clade was located basal to the Trichosporonales lineage. In the NJ tree, the foliacea clade was located basal to the Trichosporonales and Tremellales lineages, but the bootstrap support for this topology was weak (Fig. 1).

Cystofilobasidiales

The species of the Cystofilobasidiales clustered into seven well-supported clades in the Bayesian tree drawn from the seven-gene data set (Table 4, Fig. 2), being in agreement with Boekhout . Each of the clades was strongly supported with a posterior probability of 1.0. The clade contained all the Mrakia and Mrakiella species, except Mrakia curviuscula that formed a separated clade together with Cryptococcus huempii. Three of the four currently recognised Udeniomyces species formed the clade, while the other species of the genus, U. pannonicus, formed a clade together with Itersonilia perplexans. All six recognised Cystofilobasidium species clustered together in a single clade. Phaffia rhodozyma CBS 5905T and its proposed teleomorph, Xanthophyllomyces dendrorhous CBS 7918T, formed a well separated clade, but the type strains of the two taxa differ clearly in protein gene sequences, suggesting that they may represent different species. Previous studies showed that Guehomyces pullulans CBS 2532T and Tausonia pamirica CBS 8428T clustered together (Boekhout et al., 2011, Fell and Guého-Kellermann, 2011, Sampaio, 2011b). Unfortunately, due to the unsuccessful amplification and sequencing of the protein genes of T. pamirica CBS 8428T, this species was not included in the seven-gene data set. However, in the tree drawn from the rDNA data set, CBS 2532T and CBS 8428T formed a well-supported clade (Fig. 3). The seven clades were also all recognised and well-supported with bootstrap value of 100 % in the trees drawn from the ML and NJ analyses (Table 4).

Fig. 2

The phylogenetic relationships among species of the Cystofilobasidiales inferred from a seven-gene data set including sequences of three rDNA genes, RPB1, RPB2, TEF1 and CYTB. The tree backbone is constructed using Bayesian analysis. The Bayesian posterior probabilities (PP) and bootstrap percentages (BP) of maximum likelihood and neighbour-joining analyses from 1 000 replicates are shown respectively from left to right on the deep and major branches and clades resolved. Note: nm, not monophyletic; ns, not supported (PP < 0.9 or BP < 50 %).

Fig. 3

Phylogeny of tremellomycetous yeasts and dimorphic taxa based on the rDNA data set containing ITS, D1/D2, and SSU rDNA sequences. The tree backbone is constructed using Bayesian analysis. The Bayesian posterior probabilities (PP) and bootstrap percentages (BP) of maximum likelihood and neighbour-joining analyses from 1 000 replicates are shown respectively from left to right on the deep and major branches and in brackets following the clades resolved. Notes: nm, not monophyletic; ns, not supported (PP < 0.9 or BP < 50 %).

Filobasidiales

Bayesian analysis on the seven-gene data set recognised five strongly supported clades within the Filobasidiales, namely aerius, albidus, cylindricus, , and gastricus, being largely in agreement with Boekhout (Fig. 4). The albidus clade containing 17 Cryptococcus species and varieties was clearly separated from the rest of the Filobasidiales. The Filobasidium clade contained four teleomorphic species including the type species of the genus, F. floriforme, and five Cryptococcus species. The affinity of F. uniguttulatum to this clade was not supported in Boekhout and Weiß , but this study clearly showed that this species belongs to the clade with 1.0 posterior probability and 100 % bootstrap supports. This species was located in a basal branch of the clade together with C. wieringae (Fig. 4).

Fig. 4

The phylogenetic relationships among species of the Filobasidiales inferred from a seven-gene data set including sequences of three rDNA genes, RPB1, RPB2, TEF1 and CYTB sequences. The tree backbone is constructed using Bayesian analysis. The Bayesian posterior probabilities (PP) and bootstrap percentages (BP) of maximum likelihood and neighbour-joining analyses from 1 000 replicates are shown respectively from left to right on the deep and major branches and clades resolved. The branches ending with a filled cycle and a diamond represent single-species clades with a stable and unstable position, respectively. Note: nm, not monophyletic.

The gastricus clade contained six Cryptococcus species, including three species isolated from acid rock drainage (ARD) from a pyrite mine in Portugal. The three Cryptococcus species were recognised as the ARD ecoclade (Gadanho & Sampaio 2009). This ecoclade was supported by Bayesian and ML analyses based on the seven-gene data set, but not by NJ analysis. Therefore, we included this ecoclade in the gastricus clade.

The aerius and cylindricus clades contained seven and two Cryptococcus species in the seven gene tree, respectively (Fig. 4). Analyses based on the rDNA data set showed that Bullera taiwanensis, whose protein gene sequences were not successfully determined, also clustered in the cylindricus clade with strong statistical supports (PP = 1.0) (Table 4, Fig. 3). The two clades together with Filobasidium capsuligenum, which represent a separate monotypic clade, formed a well-supported lineage (PP = 1.0; BP > 95 %). Cryptococcus arrabidensis was not included in any of the clades recognised in the Filobasidiales and remained as a separate branch in the trees constructed using different algorithms (Fig. 4).

Holtermaniales

Five anamorphic Holtermanniella species proposed by Wuczkowski and the teleomorphic species Holtermannia corniformis were included in this small lineage (Fig. 5). This lineage was well separated from other groups of tremellomycetous yeasts and strongly supported in the seven-gene Bayesian and ML trees, though it was weakly supported in the NJ tree. Holtermannia corniformis was located as a basal branch in this lineage and its affinity with the Holtermanniella species was weakly supported by NJ analysis (Fig. 5), implying that this teleomorphic species may represent a distinct clade.

Fig. 5

The phylogenetic relationships among species of the Trichosporonales and Holtermaniales inferred from a seven-gene data set including sequences of three rDNA gene, RPB1, RPB2, TEF1 and CYTB sequences. The tree backbone is constructed using Bayesian analysis. The Bayesian posterior probabilities (PP) and bootstrap percentages (BP) of maximum likelihood and neighbour-joining analyses from 1 000 replicates are shown respectively from left to right on the deep and major branches resolved. The branches ending with filled cycles and diamonds represent single-species clades with a stable and unstable position, respectively. Note: nm, not monophyletic; ns, not supported (PP < 0.9 or BP < 50 %).

Trichosporonales

Seven well-supported clades with multiple species and seven single species clades were recognised in this order (Table 4, Fig. 5). The Trichosporon species were separated into four clades, namely cutaneum, gracile, porosum, and (also referred to as ovoides), supporting the classification of Middelhoven . The brassicae clade recognised in Sugita et al., 2004, Boekhout et al., 2011 and Sugita (2011) was also resolved in the Bayesian and ML trees based on the seven-gene data set, however, its separation from the gracile clade was only weakly supported in the seven-gene NJ tree. Furthermore, the distinction of the two clades was not supported in the Bayesian tree drawn from the rDNA data set. Therefore, we combined these two clades into a single clade. Among the Trichosporon species employed in this study, T. chiarellii could not be assigned to any clade. Trichosporon guehoae, whose protein gene sequences were not successfully amplified, was also located in a single branch in the tree drawn from the rDNA data set (Fig. 3).

In addition to the Trichosporon species, seven Cryptococcus, three Bullera and five Vanrija species and the monotypic genus Cryptotrichosporon (Okoli ) were included in the Trichosporonales lineage (Table 1, Fig. 5). The genus Vanrija which was recently reinstalled by Weiß for the five Cryptococcus species in the humicola clade recognised before (Boekhout et al., 2011, Fonseca et al., 2011) was confirmed to be a monophyletic group (Fig. 5). Two Cryptococcus species, C. arboriformis and C. haglerorum, formed the haglerorum clade which were resolved and well-supported in the seven-gene and the rDNA trees (Table 3, Fig. 3, Fig. 5). Other four Cryptococcus species, C. curvatus, C. daszewskae, C. fragicola, and C. marinus, occurred in single species branches. The three Bullera species formed a basal formosensis clade with strong statistical support (Table 4, Fig. 5). The thermotolerant species Cryptococcus tepidarius was located in this clade with a close relationship to B. lagerstroemiae based on rDNA sequence analysis (Fig. 3), being in agreement with Takashima . The protein gene sequences of C. tepidarius were not successfully determined. A close phylogenetic relationship of the formosensis clade with Cryptococcus marinus and Cryptotrichosporon anacardii was shown in the seven-gene Bayesian and ML trees, but the latter two species were located in separate clusters in the NJ tree (Fig. 5). In the trees drawn from the rDNA and single protein gene data sets, these two species did not cluster together, suggesting they represent different clades.

The affinity of Cryptococcus marinus within the Trichosporonales was strongly supported in the seven gene tree. It was located in a basal cluster of the order together with the formosensis clade and Cryptotrichosporon anacardii with strong support values from the Bayesian and ML analyses, but its phylogenetic position was not resolved by the NJ analysis (Fig. 5).

Tremellales

The majority of the taxa employed in this study belong to this lineage. Most of the clades recognised in Boekhout were confirmed here with improved resolution and stronger support values. While most of the species can be assigned into clear clades, some remained undetermined and the boundaries of some clades need to be examined further.

Twenty five well-supported clades were recognised among the 160 strains included in the Bayesian tree drawn from the seven-gene data set (Table 1, Table 4, Fig. 6). Five recently proposed or redefined genera based on molecular phylogenetic analyses were confirmed as monophyletic groups, including Bandoniozyma (Valente ), Bulleribasidium/Mingxiaea (Sampaio et al., 2002, Wang et al., 2011), Derxomyces, Dioszegia, and Hannaella (Takashima et al., 2001, Wang and Bai, 2008). Each of these clades received a posterior probability value of 1.0 in the Bayesian tree and bootstrap values of 100 % in the ML and NJ trees drawn from the seven-gene data set, respectively (Table 4, Fig. 6). These clades were also clearly resolved in the analyses using the rDNA and single protein gene data sets (Table 4).

Fig. 6

The phylogenetic relationships among species of the Tremellales inferred from a seven-gene data set including sequences of three rDNA genes, RPB1, RPB2, TEF1 and CYTB sequences. The tree backbone is constructed using Bayesian analysis. The Bayesian posterior probabilities (PP) and bootstrap percentages (BP) of maximum likelihood and neighbour-joining analyses from 1 000 replicates are shown respectively from left to right on the deep and major branches resolved. The branches ending with filled cycles and diamonds represent single-species clades with a stable and unstable position, respectively. Note: nm, not monophyletic; ns, not supported (PP < 0.9 or BP < 50 %).

In addition to the monotypic teleomorphic genus Cuniculitrema, the Cuniculitremaceae designated by Kirschner contained Fellomyces and Kockovaella species. The species of the latter two anamorphic genera clustered into a well-supported cluster. However, two subclades represented by the type species of the two genera, F. polyborus and K. thailandica, respectively, could be recognised in the seven-gene Bayesian and ML trees (Fig. 6). The two subclades were also resolved in the NJ tree, with F. horovitziae being located as a basal branch to the two subclades. In the Bayesian and ML trees, this species was basal to the Fellomyces subclade with a PP and BP value of 1.0 and 64 %, respectively (Fig. 6).

The phylogenetic relationships among the species tentatively assigned to the Bulleromyces/Papiliotrema/Auriculibuller group by Boekhout were resolved in this study (Fig. 6). The teleomorphic species Bulleromyces albus and three anamorphic Bullera species occurred in a distinct group with two clades being recognised, namely the clade containing the anamorphic species Bullera unica, and the hannae clade formed by B. hannae and B. penniseticola. However, in the trees drawn from the rDNA data set, the close relationship of the two clades was not resolved (Fig. 3).

The monotypic teleomorphic genera Papiliotrema and Auriculibuller formed a well-supported group with one Bullera and 10 Cryptococcus species. This group showed a close relationship to the clade with strong support (Fig. 6). Five clades were recognised in this group (Table 4, Fig. 6). The clade contained two other Cryptococcus species, namely C. nemorosus and C. perniciosus; C. taeanensis showed a close affinity to the clade. The pseudoalba clade contained a Bullera species and two Cryptococcus species, C. anemochoreius and C. cellulolyticus. The laurentii and the aureus clades contained two and three Cryptococcus species, respectively.

Four recently described Cryptococcus species with orange coloured colonies (Inácio et al., 2005, Wang et al., 2007, Landell et al., 2009) clustered together in a well-supported amylolyticus clade. Two Bullera species described from Taiwan (Nakase ), which were assigned to the clade in Boekhout , formed a distinct melastomae clade closely related with the clade. Other clearly supported clades consisting of species with only or mainly yeast forms were the , dimennae, and clades. The dimennae clade, which was also resolved by Boekhout but was referred to as the victoriae clade by Fonseca , consisted of six Cryptococcus species and one Bullera species (B. globispora). In addition to the teleomorphic species Kwoniella mangroviensis, five Cryptococcus and one Bullera species were included in the clade. The opportunistically pathogenic species in the Cryptococcus neoformans complex and their teleomorphs were included in the clade together with Filobasidiella depauperata, C. amylolentus and Tsuchiyaea wingfieldii.

The Tremella species employed in the present study separated into different clades. Ten of them, including the type species of the genus, T. mesenterica, clustered in the clade. No species with mainly yeast forms in their life cycle were located in this clade. Three Tremella species formed the aurantia clade. Tremella moriformis was located in a group containing two Cryptococcus species (C. allantoinivorans and C. mujuensis) and Sirobasidium intermedium, a teleomorphic species. This group, which was tentatively included in the Bulleromyces/Papiliotrema/Auriculibuller group in Boekhout , was also resolved as a separate group in the ML and NJ trees with 93–98 % bootstrap supports (Fig. 6). However, C. mujuensis and S. intermedium were separated from the other species of this group in the tree drawn from the rDNA data set (Fig. 3, Fig. 7). With the consideration that the four species in this group exhibit quite different morphological characters from each other, they were regarded as representing four separate single species clades. Tremella nivalis and T. moriformis formed the moriformis clade with 1.0 PP and with over 95 % bootstrap support in the tree drawn from the rDNA sequence data set (Fig. 3). Another Sirobasidium species employed in this study, S. magnum, was located in a branch basal to the clade (Fig. 3, Fig. 6). Tremella foliacea and two Cryptococcus species (C. fagi and C. skinneri) clustered in the foliacea clade, which was located at the basal position of the Tremellales lineage in the seven-gene Bayesian tree (Fig. 5). The rDNA tree showed that T. neofoliacea was also located in this clade (Fig. 3).

Fig. 7

Phylogeny of tremellomycetous yeasts and dimorphic taxa based on 5.8S and LSU D1/D2 rDNA sequences from strains employed in this study and 26 more Tremella species employed in Millanes . The tree backbone is constructed using Bayesian analysis. The Bayesian posterior probabilities (PP) and bootstrap percentages (BP) of maximum likelihood and neighbour-joining analyses from 1 000 replicates are shown respectively from left to right on the deep and major branches and in brackets following the clades resolved. The species names in red represent fruiting-body forming taxa and those with a star superscript indicate that the sequences are from herbarium specimens of lichen-inhabiting species. Note: nm, not monophyletic; ns, not supported (PP < 0.9 or BP < 50 %).

Another group containing both yeast and filamentous taxa is the Trimorphomyces group. Two Bullera species and three Cryptococcus species were located in this group together with Trimorphomyces papilionaceus, a basidiocarp-forming species with a yeast state (Fig. 6). T. papilionaceus was regarded as representing a distinct clade because of its unique sexual reproductive structures (Bandoni and Boekhout, 2011, Boekhout et al., 2011). The three Cryptococcus species, C. flavus, C. paraflavus and C. podzolicus, were assigned to the flavus clade since they clustered together in the seven-gene and rDNA Bayesian trees with 0.97–0.99 PP supports (Fig. 3, Fig. 6). The two Bullera species in this group, B. sakaeratica and B. miyagiana, was separated by T. papilionaceus in the seven-gene and the rDNA trees (Fig. 3, Fig. 6). Therefore, they were regarded as representing two different single species clades.

The following species in the Tremellales lineage, Bullera arundinariae, Cryptococcus cistialbidi, Cryptococcus spencermatinsiae, Cuniculitrema polymorpha, and Tremella giraffe, could not be assigned to any recognised clade or group, because of their unstable or unresolved phylogenetic positions, or their unique phenotypic characters. Bullera arundinariae and C. cistialbidi were located as basal branches to the aurantia clade formed by four Tremella species in the seven-gene Bayesian tree (Fig. 6). While the close relationship of C. cistialbidi to the aurantia clade was consistent in different trees, B. arundinariae was located in different positions in the seven-gene NJ tree and the trees resulting from the rDNA data set (Fig. 3, Fig. 6). Cryptococcus spencermartinsiae was located in a branch basal to the foliacea clade with strong statistical support in the Bayesian and ML trees drawn from the seven-gene data set, but the species was located at a different position in the seven-gene NJ tree and the trees drawn from the rDNA data set (Fig. 3, Fig. 6). The teleomorphic species Cuniculitrema polymorpha (anamorph: Sterigmatosporidium polymorphum) was located in a branch basal to the Fellomyces/Kockovaella group. Tremella giraffa was located as a basal branch to the amylolyticus clade in the seven-gene tree with weak to moderate support values (Fig. 6), but its position was not resolved in the rDNA tree (Fig. 3).

In order to investigate further the relationships of yeasts with filamentous taxa in the Tremellomyetes, we retrieved the 5.8S and LSU rDNA sequences of 26 lichen-inhabiting Tremella species employed in Millanes that were absent in the current data set. These sequences were determined from herbarium specimens (Millanes ). The Bayesian tree obtained from the combined 5.8S and LSU D1/D2 rDNA sequence data set showed a largely identical topology with that obtained from the seven-gene data set and the five major lineages were also clearly resolved (Fig. 7). The majority of the additional 26 Tremella species were located in clades I, II and III as defined by Millanes which mainly contained lichen-inhibiting Tremella species; one in the aurantia clade containing Tremella taxa only; three in the foliacea clade containing both Tremella and Cryptococcus species; and one in the Trimorphomyces group (Fig. 7).

Discussion

In this study, we inferred the phylogeny of basidiomycetous yeasts and related dimorphic and filamentous basidiomycetes in the Tremellomycetes based on analyses of seven gene sequences using different phylogenetic algorithms. The majority of the yeast taxa and dimorphic basidiomycetes that have free-living unicellular states in their life cycles in the Agaricomycotina were employed. Five major lineages corresponding to the five orders currently recognised in the Tremellomycetes (Boekhout et al., 2011, Millanes et al., 2011, Weiß et al., 2014) were resolved. A total of 45 strongly supported monophyletic clades with multiple species and 23 single species clades were recognised. This phylogenetic framework will be the basis for an improved modern taxonomy unifying both yeast-like and filamentous species in the Tremellomycetes as well as anamorphs and teleomorphs occurring in this class. The result is also helpful for a better understanding of the evolution of characters and different life styles by integrating the phylogeny with biochemical, morphological and reproductive characteristics of unicellular, dimorphic and filamentous basidiomycetes in the Tremellomycetes.

Congruence of phylogenies inferred from analyses using different algorithms and data sets

Almost all currently recognised teleomorphic and anamorphic yeast species and dimorphic taxa in the Agaricomycotina were obtained from culture collections and revived for DNA isolation and PCR amplification in this study. Despite our best effort to obtain a complete sequence data set for all the genes and strains employed, the sequence of some genes, especially the nuclear protein-coding genes and the mitochondrial gene CYTB, could not be determined for a small percentage of strains because of failure in the PCR amplification or sequencing reactions. Specifically, 8.8 %, 8.1 %, 16.2 % and 17.2 % of the total 297 strains employed failed in the sequence determination of the RPB1, RPB2, TEF1 and CYTB genes, respectively. This problem is known from all groups of fungi (Schoch ). A previous study has shown that an inferred phylogeny is not sensitive to 25 % or even 50 % missing data for a sufficiently large alignments (e.g., ∼30 000 positions and 36 species) (Philippe ). Though the length of the seven-gene alignment in this study is only about 6 300 positions, the amount of missing data is also much less. Thus, we assume that the relative minor amount of missing data in our study will not significantly influence the reliability of the resulting phylogeny.

The phylogenies of the taxa compared in this study were inferred from analyses using different data sets and algorithms. The topologies of the trees constructed using different algorithms performed on different data sets were largely congruent as examined visually, which make the delimitation of major lineages and clades more clear and confident. In addition to the Clustal X, we also used the MAFFT program (Katoh & Standley 2013) to align the sequences and the alignments generated were subjected to ML analysis. The topologies of the trees obtained from the Clustal X and the MAFFT alignments were almost the same (data not shown). This further supports the notion that our inferred trees are reliable and not greatly influenced by the missing data as discussed above.

Bayesian analysis is usually believed to be more reliable compared to parsimony and neighbour-joining methods, especially for an extensive sampling with a high divergence occurring among the sequences (Alfaro et al., 2003, Holder and Lewis, 2003, James et al., 2006). As expected, the Bayesian analysis of the seven-gene data set showed the most robust phylogeny among the analyses performed (Table 4). However, analyses aiming at comparing Bayesian and ML supports have revealed that PP and BP values show significant correlation, but the strength of this correlation is highly variable and sometimes very low. ML BP values are generally lower than PP values, and thus, ML BP might be less prone to strongly supporting a wrong phylogenetic hypothesis (Douady ). Therefore, the boundaries of the lineages and clades recognised in this study were determined based not only on Bayesian analysis, but also on ML and NJ analyses, aiming to recognize reliable monophyletic groups.

Conflicts between phylogenies obtained from rDNA and protein-coding gene sequences have been observed in different studies on basidiomycetes (Matheny et al., 2002, Froslev et al., 2005, Matheny, 2005, Matheny et al., 2006, Matheny et al., 2007). However, in this study, the topologies of the trees and the clades resolved from the data sets of RPB1 and RPB2 were similar to those obtained from the rDNA data set (Table 4), except for the position of the Trichosporonales which was nested into the Tremellales in the RPB1-based phylogeny. Furthermore, RPB1 and RPB2 had an equivalent resolution power in the Cystofilobasidiales and Filobasidiales lineages. The Holtermanniales lineage was supported strongly (100 % BP) by the ML algorithm in the RPB2-based phylogeny but only received moderate support (62 % BP) in the RPB1-based phylogeny. The RPB1 and RPB2-based phylogenies constructed from Bayesian analysis supported the same number of clades in the Tremellales, while the RPB1-based phylogeny constructed from ML or NJ analyses resolved one more clade if compared to the RPB2-based phylogeny. The RPB1 and RPB2-based phylogenies drawn from Bayesian and ML analyses also resolved the same number of clades in the Trichosporonales. The TEF1 and CYTB sequences showed less parsimony-informative characters for the inference of phylogenetic relationship in the tremellomycetous yeasts compared to the RPB1 and RPB2 sequences. The TEF1 and CYTB data sets generated the lowest resolution across the Bayesian, ML and NJ trees, in which only 19 and 16 strongly supported clades were resolved with high BP and PP values, respectively (Table 4). The TEF1 and CYTB data sets were unable to resolve higher level taxonomic relationships, such as the five orders in the Tremellomycetes, and they did not show strong support to some clades, such as the , , foliacea, , , and clades, which were strongly supported by the analyses based on the other data sets. Our results suggest that RPB1 and RPB2 are more useful to infer reliable phylogeny of tremellomycetous yeasts than the TEF1 and CYTB genes. A previous study of basidiomycetes phylogeny also showed that the major clades at higher and lower taxonomic levels were more clearly resolved based on RPB2 than on TEF1 sequence data (Matheny ).

More robust topologies and higher resolution were achieved in this study than those obtained in previous studies based on the LSU rDNA D1/D2 domains or ITS-5.8S sequences (Fell et al., 2000, Scorzetti et al., 2002, Boekhout et al., 2011). The consensus is that the major groups recognised in the previous studies were confirmed in the present study. Fell studied 171 hymenomycetous yeast strains representing 116 species. They recognised four major lineages including the Cystofilobasidiales, Filobasidiales, Tremellales and Trichosporonales. However, the clades within each lineage were largely unresolved. In addition to the four major lineages, Scorzetti recognised clades within each lineage. Most of the clades recognised in the Cystofilobasidiales, Filobasidiales and Trichosporonales were in agreement to those recognised in this study. However, the fine phylogenetic relationships among the taxa in the Tremellales remained largely unresolved in the previous studies. Boekhout employed more strains and designated a fifth lineage containing the Holtermanniella clade and a teleomorphic species Holtermannia corniformis that was described as a separate order (Wuczkowski ). Our study confirmed this fifth lineage as a separate order Holtermanniales with 1.0 PP and 100 % ML BP supports. The phylogenetic position of Cryptococcus marinus has been debated. It was considered to belong to the Tremellales according to a phylogenetic analysis of SSU rDNA sequences (Takashima & Nakase 1999). The phylogenetic position in the LSU rDNA D1/D2 tree suggested that this species may represent a separate order within the Tremellomycetes (Scorzetti et al., 2002, Fonseca et al., 2011, Weiß et al., 2014). However, the affinity of this species with the Trichosporonales lineage was strongly supported in this study (Fig. 5).

The major lineages and clades recognised in this study are similar to those recognised in Millanes and Weiß , which sampled more teleomorphic and filamentous taxa in the Tremellomycetes. In their molecular phylogenetic study on the jelly fungi based on nuclear SSU, 5.8S and LSU rDNA sequences, Millanes employed three more teleomorphic genera Biatoropsis, Syzygospora and Tetragoniomyces, but limited yeast taxa. In addition to the teleomorphic genera employed in Millanes et al., 2011, Weiß et al., 2014 listed seven other teleomorphic genera that were not employed in our study, including Carcinomyces, Rhynchogastrema, Phyllogloea, Phragmoxenidium, Sigmogloea, Sirotrema, and Xenolachne in the Tremellomycetes. However, the latter five genera were not included in their phylogenetic analysis based on LSU D1/D2 sequences, because no DNA data were available from these genera. In the trees presented in Millanes and Weiß , the species of the teleomorphic and filamentous genera that were not included in this study were located in separated clades from those formed by yeast taxa.

Correlation between morphology, physiology and molecular phylogeny

Because of the morphological simplicity, it is not easy to find morphological characters that distinguish the five major lineages of tremellomycetous yeasts recognised by molecular phylogenetic analyses. Teleomorphic taxa belonging to the Tremellales usually form tremella-type basidia, e.g., phragmobasidia with longitudinal primary septa; whereas those of the Cystofilobasidiales and Filobasidiales are usually characterised by forming holobasidia (Wells and Bandoni, 2001, Boekhout et al., 2011). However, some species with holobasidia or transversely septate basidia, like Auriculibuller fuscus (Sampaio ), Papiliotrema bandonii (Sampaio ), Tremella fuciformis, T. hypogymniae (Millanes ) and Bulleribasidium oberjochense (Sampaio ) are also present in Tremellales. These observations show that different types of basidial septation can coexist in the same lineage. The sexual stage of the Trichosporonales species has not yet been observed. The majority of the species in this order are characterised by forming abundant true hyphae that disarticulate into arthroconidia. However, the filamentous species Tetragoniomyces uliginosus which was tentatively assigned to the Trichosporonales in Millanes and Weiß forms basidia in pustulate basidiocarps (Oberwinkler & Bandoni 1981).

The species in the genera Fellomyces and Kockovaella share a special morphological character of forming conidia on stalks (Nakase ). These species were located together in a cluster with strong PP and ML BP supports (Fig. 6). The affinity of F. horovitziae to the Fellomyces clade was weakly supported in ML analysis and not supported in NJ analysis. We tentatively assign F. horovitziae to the clade with the consideration of minimising name changes in the subsequent taxonomic treatment.

The ability to form ballistoconidia has since long been shown to be an unreliable phylogenetic marker (Nakase ). This observation is confirmed by the intermixture of species of the ballistoconidia-forming genera Bullera and Kockovaella with those of non ballistoconidia-forming genera Cryptococcus and Fellomyces. However, the morphology of ballistoconidia seems to be phylogenetically relevant. Ballistoconidia formed by species in the Cystofilobasidiales and Trichosporonales are usually bilaterally symmetrical, whereas those formed by species in the Tremellales and Filobasidiales are usually rotationally symmetrical (Boekhout ).

Within the Tremellales, some clades may be distinguished by colony morphology. For example, the anamorphic genera Derxomyces, Hannaella and Dioszegia are closely related, but are distinguishable by forming whitish to yellowish colonies with a butyrous texture, whitish colonies with a highly mucoid texture, and orange-coloured colonies with a butyrous texture, respectively (Wang & Bai 2008). The two Bullera species in the melastomae clade were assigned to the clade by Boekhout . However, they are morphologically different by forming yellowish to brownish colonies compared to the orange-coloured colonies of Dioszegia species (Takashima et al., 2001, Wang and Bai, 2008).

The physiological and biochemical differences among the major lineages are also quite elusive, though some trends have been observed (Sampaio and Fonseca, 1995, Sampaio, 2004). The majority of the Cystofilobasidiales and Filobasidiales species can utilise nitrate; whereas the Tremellales and Trichosporonales taxa are usually nitrate negative. The coenzyme Q (CoQ) system has been used as an important taxonomic criterion at the genus level in yeasts (Yamada & Kondo 1973). The major CoQ systems of the tremellomycetous yeasts are CoQ-8, CoQ-9 and CoQ-10 (Fell, 2011, Fell and Guého-Kellermann, 2011, Sampaio, 2011a, Sampaio, 2011b). The taxa with CoQ-8 are concentrated in the Cystofilobasidiales. The species within a strongly supported clade usually possess the same major CoQ type, which may be helpful to recognize and define homogenous clades. The species with the ability to ferment sugars, a rare trait among basidiomycetous yeasts, are concentrated in a few clades in the Cystofilobasidiales ( and /) and Tremellales (). One species in the Filobasidiales, Filobasidium capsuligenum, can also ferment glucose and maltose, while the other known Filobasidium species can not ferment glucose (Kwon-Chung 2011). F. capsuligenum was separated from the clade and located in a branch closely related with the cylindricus clade containing two Cryptococcus species with strong PP and BP supports (Fig. 4). Ultrastructurally, F. capsuligenum is also special by having cone-shaped vesicular parenthesomes (Moore & Kreger-van Rij 1972). Thus, we recognised this species as representing a distinct clade. Consequently, the cylindricus clade and the closely related aerius clade were recognised as separate clades.

Serological characteristics of Trichosporon species correspond to some extent with their phylogenetic clustering. Species in the cutaneum, Trichosporon and brassicae clades have serotypes I, II and III, respectively, while species in the gracile and porosum clades have serotype I-III, which is a serotype that reacts to both antisera I and III (Ikeda et al., 1996, Sugita and Nakase, 1998, Sugita et al., 2004, Sugita, 2011). However, the phylogenetic separation between the brassicae and gracile clades, which have different serotypes (III and I-III, respectively) was not supported in this study. The gracile and brassicae clades were recognised as separate clades based on D1/D2 rDNA sequence analyses and serological characteristics (Sugita et al., 2004, Boekhout et al., 2011, Sugita, 2011). However, both clades lacked bootstrap supports in the NJ trees drawn from D1/D2 sequences (Boekhout et al., 2011, Sugita, 2011). In this study, the monophyly of the gracile clades was not resolved and supported in the Bayesian tree drawn from the rDNA data set. Therefore, we combined the gracile and brassicae clades.

Life strategy evolution in Tremellomycetes

The multiple gene phylogeny of tremellomycetous yeasts is helpful for a better understanding on the evolution of different life styles and strategies. The tremellomycetous fungi present a high diversity of lifestyles, with many species being dimorphic, including both unicellular and filamentous growth forms (Bandoni, 1995, Sampaio, 2004, Boekhout et al., 2011). They are also nutritionally heterogeneous, comprising saprotrophs, animal parasites, and fungal-inhabiting (including lichen-inhabiting) species (Millanes et al., 2011, Weiß et al., 2014). A previous study on phylogeny and character evolution in tremellomycetous fungi based on three rDNA markers (nSSU, 5.8S and nLSU) showed that, in a broad sense, a specific life style or strategy is usually homoplastic; however, taxa with the same life strategy, for example, fungal- or lichen-inhabiting, usually form distinct clades (e.g., clades I, II and III in Millanes ). The results of this study also show that taxa with different life styles (e.g., dominated by unicellular and filamentous growth stages, respectively) usually form different clades, though clades with species having the same life styles may not be closely related.

This observation is also shown by the analysis based on an integrated 5.8S and LS D1/D2 sequence data set containing additional Tremella species as employed in Millanes . Though fruiting-body forming species were intermingled with yeast species throughout the Tremellales (Fig. 7), the former usually clustered into different groups from the latter. A few fruiting-body forming species, e.g., Papiliotrema bandonii, Tremella parmeliarum, T. polyporina, T. ramalinae, T. foliacea, and Trimorphomyces papilionaceus, were located in the same clusters together with some yeast taxa, but they usually formed distinct branches or clades. These results suggest that tremellomycetous fungi with the same life styles or nutritional strategies may be the result of convergent evolution as a result of early adaptation to different ecological niches or habitats.

Taxonomic consequences

As with many other groups of fungi, the taxonomic system of basidiomycetous yeasts needs to be updated to reflect the evolutionary relationships of the taxa concerned and to accommodate the requirements of the new nomenclatural code (McNeill ). Based on the results of this study, we will propose an updated taxonomic system for tremellomycetous yeasts which will have the best approximation of the molecular phylogeny and that will be compatible with the current taxonomic system of filamentous basidiomycetes. A considerable number of genera need to be redefined to include only the species in the monophyletic clades that contain the type species of those genera, and, secondly, many new genera need to be proposed to accommodate monophyletic clades that do not include any generic type species. The names of many species will be changed due to the proposal of new genera and adaptation of the ‘one fungus = one name’ principle at this stage. We believe that this updated taxonomic system based on a reliable phylogeny and extensive phenotypical comparisons will be relatively stable and minimise the necessity of future name changes.